Activatable interleukin 18 polypeptides

Abstract

Provided are activatable interleukin 18 (IL-18) polypeptides 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, IL-18 variant polypeptide, or a fusion protein comprising a wild-type IL-18 or IL-18 variant polypeptide is linked to the masking moiety via a cleavable linker. Related nucleic acids, vectors, host cells, methods of making IL-18 variant polypeptides, pharmaceutical compositions comprising activatable IL-18 polypeptides, and therapeutic uses of such pharmaceutical compositions are also provided.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to International Application No. PCT / CN2023 / 072869, filed on January 18, 2023, International Application No. PCT / CN2023 / 072886, filed on January 18, 2023, and International Application No. PCT / CN2023 / 117197, filed on September 6, 2023, the entire contents of which are incorporated herein by reference in their entirety for all purposes.

[0003] Reference Electronic Sequence Listing

[0004] The contents of the electronic sequence listing (233002001641SEQLIST.xml; size: 445,962 bytes; creation date: January 17, 2024) are incorporated herein by reference in their entirety. Technical Field

[0005] The present application relates to activatable interleukin-18 (IL-18) polypeptides comprising (a) an IL-18 polypeptide (e.g., wild-type IL-18, a variant 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 polypeptide, a receptor, or a subdomain of a receptor). The present application also relates to methods of making such activatable IL-18 polypeptides, as well as methods of using such activatable IL-18 polypeptides in therapeutic applications. Background Art

[0006] Interleukin 18 (IL-18, also known as interferon-γ inducing factor) is a proinflammatory cytokine that has been found to stimulate innate lymphoid cells, myeloid cells, antigen-experienced non-naive T cells (see, for example, Guo et al. (2012) Trends Immunol. 33, 598–606), and antigen-experienced natural killer cells (NK cells). In terms of therapy, 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 in tumor-infiltrating lymphocytes (TILs) (see, e.g., Zhou et al. (2020) Nature 583:609–614), suggesting that IL-18 therapy may enhance anti-tumor 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, the clinical development of IL-18 has been limited by its limited efficacy. IL-18BP is a high-affinity IL-18 inhibitor (K D <1 nM, see, e.g., Dinarello et al. (2013) Front Immunol 4:289, doi:10.3389 / fimmu.2013.00289), which is frequently upregulated in a variety of human and murine tumors and is thought to limit the anti-tumor activity of IL-18 in preclinical models (e.g., mice) and clinical trials. IL-18BP-resistant IL-18 variant polypeptides, which retain signaling potential but are not affected by IL-18BP inhibition, have been engineered (see, e.g., Zhou et al. (2020) Nature 583:609-614).

[0007] 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 participate in host defense against intracellular pathogen infection by producing IFNγ. IL-18 also induces the production of TNF and FasL, both of which are involved in growth, survival, and apoptosis. IL-18 is thought to play a role in inducing the production of IL-4 and IL-13 by T cells, NK cells, mast cells, and basophils, thereby driving Th2 responses. Other mediators induced by IL-18 include inducible nitric oxide; cyclooxygenase (Cox-2); the proinflammatory cytokines IL-1β and IL-6; the 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, regulating IL-18 activity is crucial for preventing abnormal immune responses.

[0008] There is a need in the art for compositions and methods for activating IL-18 receptor-mediated signaling in an individual (e.g., by wild-type IL-18 or by IL-18BP-resistant IL-18 variant polypeptides) and / or stimulating antigen-experienced T cells or NK cells in an individual (e.g., activation at a tumor site) in a controlled manner to treat cancer and other IL-18-mediated diseases and disorders.

[0009] The disclosures of all publications, patents, patent applications, and published patent applications mentioned herein are incorporated by reference in their entirety. Summary of the Invention

[0010] In some embodiments, an interleukin 18 (IL-18) variant polypeptide is provided, comprising at least one mutation at a residue selected from the group consisting of F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and I149, wherein the amino acid positions are relative to wild-type human IL-18 as set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide comprises at least one mutation at a residue selected from the group consisting of G3, E6, D54, and N91, wherein the amino acid positions are relative to wild-type human IL-18 as set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide further comprises at least one mutation at a residue selected from the group consisting of Q56, P57, M60, Q103, R104, M113, and N155, wherein the amino acid positions are relative to wild-type human IL-18 as set forth in SEQ ID NO: 1. Exemplary IL-18 polypeptides can be found in, e.g., WO2020069398A1, WO2021097376A1, WO2023161853A1, WO2022038417A2, which are herein incorporated by reference in their entireties.

[0011] In some embodiments, the IL-18 polypeptide comprises at least one mutation at a residue selected from the group consisting of: G3, Q24, L29, Q56, P57, M60, A61, N91, K96, R104, R107, K140, N155, and I149, wherein the amino acid positions are relative to wild-type human IL-18 as set forth in SEQ ID NO: 1, optionally wherein the IL-18 polypeptide comprises a mutation at G3, E6, D54, and N91, further optionally wherein the variant polypeptide comprises a mutation at: (i) G3P; (ii) E6R or E6K; (iii) D54W, D54H, D54S, or D54Q; and (iv) N91V, N91A, N91G, or N91S. In some embodiments, the variant IL-18 polypeptide comprises 3P, 6K, 54G, 56G, 57A, and 91T. In some embodiments, the variant IL-18 polypeptide comprises 6G, 54L, 56T, 57E, 91G, and 104S.

[0012] In some embodiments, the IL-18 polypeptide comprises a substitution or a combination of substitutions selected from the group consisting of: 1) G3P, E6K, D54H, Q56D, P57R, N91V, and R104V; 2) G3S, E6K, D54W, Q56P, P57D, N91G, and R104T; 3) G3P, E6K, D54W, Q56H, P57V, N91A, and R104Y; 4) G3P, E6K, D54W, Q56G, P57V, N91V, and R104F; 5) G3E, E6T, D54W, Q56P, P57W, N91V, R104T, and N155K; 6) G3P, E6R, D54W, Q56T, N91V and R104T; 7) G3P, E6R, D54H, Q56T, P57A and N91A; 8) G3P, E6R, D54W, Q56P, P57A, N91A and R104L; 9) G3P, E6R, D54W, Q56R, P57A, N91S and R104S; 10) G3P, E6R, D54Q, Q56L, P57W, and N91S; 11) G3P, E6R, D54S, Q56R, P57N, N91G, and R104T; 12) G3P, E6K, D54G, Q56G, P57A, and N91T; 13) G3P, E6K, D54Q, Q56I, and P57W; 14) G3N, E6K, D54P, Q56S, P57S, N91R, and R104A; 15) G3P, E6R, D54S, Q56Y, P57T, and N91G; 16) G3P, E6R, D54S, Q56R, P57N, N91G, and R104S; 17) G3P, E6R, D54L, Q56T, P57A, and N91G ; 18) G3P, E6R, D54S, Q56R, P57R, N91G and R104S; 19) G3P, E6K, D54H, Q56E, P57Q and N91A; 20) G3P, E6R, D54S, Q56R, P57S, N91G and R104S; 21) G3P, E6R, D54S, Q56 S, P57T, N91G and R104S; 22) G3P, E6R, D54Y, Q56R, P57G, N91K and R104S; 23) G3P, E6R, D54Y, Q56T and P57R; 24) G3P, E6R, D54Y, Q56T and P57S; 25) G3P, E6R, D54L, Q 56T, P57T and N91R; 26) G3P, E6R, D54H, Q56D, P57K, N91V and R104Y; 27) G3P, E6R, D54H, Q56Y, P57T, N91V and R104Y; 28) G3P, E6A, D54W, Q56G, P57G, N91V and R104Y;29) G3P, E6M, D54F, Q56D, P57R, and N91P; 30) G3P, E6L, D54H, Q56T, P57V, and N91S; 31) G3P, E6R, D54H, Q56I, P57H, N91I, and R104Y; 32) G3P, E6G, D54S, Q56S, and P57R; 33) G3E, E6H, D54R, Q56T, and P57H; 34) G3P, E6R, D54H, Q56R, P57N, N91V, and R104E; 35) G3P, E6R, D54G, Q56 G, P57A and N91G; 36) G3P, E6S, D54A, Q56D, P57Q and N91G; 37) G3P, E6G, D54Q, Q56V and P57W; 38) G3P, E6S, D54W, Q56G, P57A, N91V and R104I; 39) G3P, E6R, D54W, Q56P, P57G, N91V and R104L; 40) G3D, E6K, D54P, Q56S, P57W and N91W; 41) G3P; 42) G3P, E6K, D54G, Q56G and P57A; 4 3) G3P, E6R, D54L, Q56G, P57S, and N91V; 44) G3P, E6R, V11I, D54G, Q56G, P57A, and N91G; 45) G3P, E6K, D54H, Q56Y, and P57S; 46) E6R, D54W, Q56S, and P57Q; 47) G3P, E6K, D54L, Q56T, P57Q, and N91V; 48) G3A, E6Y, D54R, Q56S, P57L, and N91G; 49) G3P, E6R, D54L, Q56T, P57I, and N91G; 5 0) E6G, D54L, Q56T, P57E, N91G, and R104S; 51) G3A, E6Y, D54R, Q56S, P57L, and N91A; 52) M60K and K96D; 53) G3P, E6R, and K96E; 54) M60K; 55) G3P and E6R; 56) G3P and E6K; 57) G3D, E6K, and N91S; 58) G3S and I149M; 59) G3P, E6R, and N91S; 60) E6R; 61) E6R and N91S; 62) V11I; and 63) G3S and K140R. ;

[0013] In some embodiments, the IL-18 variant polypeptide further comprises at least one mutation at a residue selected from the group consisting of: C38, C68, C76, D98, S117, and C127, wherein the amino acid positions are relative to wild-type human IL-18 as set forth in SEQ ID NO: 1. In some embodiments, the at least one mutation comprises a substitution at C38, a substitution at C68, and an S117C substitution. In some embodiments, the at least one mutation is selected from the group consisting of: C38S, C68S, C76S, and C127S. In some embodiments, the IL-18 variant polypeptide comprises (e.g., further comprises) a C38S, C68S, and C76S mutation. In some embodiments, the IL-18 variant polypeptide further comprises a set of mutations selected from the group consisting of: (a) C38I, C68S, and S117C; (b) C38V, C68I, S117C, and C127A; (c) C38S, C68I, S117C, and C127I; (d) C38I, C68I, C76V, and C127I; and (e) C38I, C68L, and C76Y. In some embodiments, the variant IL-18 polypeptide comprises 3P, 6K, 54G, 56G, 57A, and 91T. In some embodiments, the variant IL-18 polypeptide comprises 6G, 54L, 56T, 57E, 91G, and 104S.

[0014] In some embodiments, an interleukin-18 (IL-18) variant polypeptide is provided, comprising at least one mutation at a residue selected from the group consisting of C38, C68, C76, D98, S117, and C127, wherein the amino acid positions are relative to wild-type human IL-18 as set forth in SEQ ID NO: 1. In some embodiments, the at least one mutation comprises a substitution at C38, a substitution at C68, and an S117C substitution. In some embodiments, the at least one mutation is selected from the group consisting of C38S, C68S, C76S, and C127S. In some embodiments, the IL-18 variant comprises a C38S, C68S, and C76S mutation. In some embodiments, the IL-18 variant polypeptide comprises a set of mutations selected from the group consisting of: (a) C38I, C68S, and S117C; (b) C38V, C68I, S117C, and C127A; (c) C38S, C68I, S117C, and C127I; (d) C38I, C68I, C76V, and C127I; and (e) C38I, C68L, and C76Y. In some embodiments, the variant IL-18 polypeptide comprises 3P, 6K, 54G, 56G, 57A, and 91T. In some embodiments, the variant IL-18 polypeptide comprises 6G, 54L, 56T, 57E, 91G, and 104S.

[0015] In some embodiments, the wild-type IL-18 is human IL-18 comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide specifically binds to IL-18 receptor alpha (IL-18Rα) and exhibits reduced binding to IL-18 binding protein (IL-18BP) relative to wild-type IL-18. In some embodiments, the IL-18 variant polypeptide exhibits increased binding to IL-18Rα relative to wild-type IL-18. In some embodiments, the IL-18 variant polypeptide is expressed at a concentration of less than about 5×10 -5 M's K D In some embodiments, the IL-18 variant polypeptide binds to IL-18Rα at a concentration of about 5×10 -5 to about 5×10 -11 M's K D In some embodiments, the IL-18 variant polypeptide binds to IL-18Rα at a concentration greater than 5×10 -9 M's K D Binding to IL-18BP. In some embodiments, the IL-18 variant polypeptide does not exhibit binding to IL-18BP (eg, no detectable binding as measured by surface plasmon resonance).

[0016] 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.

[0017] 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.

[0018] 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.

[0019] 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.

[0020] In some embodiments, the variant polypeptide does not comprise a mutation at residue R104.

[0021] 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.

[0022] In some embodiments, the variant polypeptide does not comprise a mutation at residue Q56.

[0023] In some embodiments, the variant polypeptide comprises a mutation at residue P57, wherein the mutation is selected from the group consisting of: P57A, P57E, P57F, P57G, P57R, P57W, P57S, P57T, P57V, P57Q, P57H, P57I, P57K, P57L, P57N, P57Y, and P57D. In some embodiments, the mutation is selected from the group consisting of: P57A, P57G, P57R, P57W, P57S, P57T, and P57V.

[0024] In some embodiments, the variant polypeptide does not comprise a mutation at residue P57.

[0025] In some embodiments, the variant polypeptide comprises mutations at G3, E6, D54, and N91. In some embodiments, the variant polypeptide comprises mutations at: (i) G3P; (ii) E6R or E6K; (iii) D54W, D54H, D54S, or D54Q; and (iv) N91V, N91A, N91G, or N91S. In some embodiments, the variant polypeptide further comprises mutations at Q56 and P57.

[0026] In some embodiments, the variant polypeptide comprises the amino acid sequence set forth in any one of SEQ ID NOs: 2-299 and 307-318.

[0027] In another aspect, the present application provides a method for 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 as shown in SEQ ID NO: 1. In some embodiments, the variant IL-18 polypeptide comprises 3P, 6K, 54G, 56G, 57A, and 91T. In some embodiments, the variant IL-18 polypeptide comprises 6G, 54L, 56T, 57E, 91G, and 104S.

[0028] In another aspect, the present application provides a method for increasing the yield and / or purity of an IL-18 variant polypeptide, comprising engineering an IL-18 polypeptide by introducing cysteine ​​residues at positions 117 and / or 76 of the IL-18 polypeptide, thereby promoting disulfide bond formation between C117 and C76 of the variant polypeptide, wherein the amino acid positions of the IL-18 polypeptide are relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the variant IL-18 polypeptide comprises 3P, 6K, 54G, 56G, 57A, and 91T. In some embodiments, the variant IL-18 polypeptide comprises 6G, 54L, 56T, 57E, 91G, and 104S.

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

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

[0031] In some embodiments according to any of the methods above, introducing cysteine ​​at position 117 or 76 comprises substituting cysteine ​​for the amino acid at position 117 or 76. In some embodiments, the substitution of the amino acid at position 117 comprises an S117C substitution.

[0032] In some embodiments according to any of the above methods, the method further comprises, when the IL-18 polypeptide comprises cysteine ​​at one or both of positions 38 and 68, removing the cysteine ​​at one or both of positions 38 and / or 68. In some embodiments, removing the cysteine ​​at one or both of positions 38 and / or 68 comprises replacing the cysteine ​​at positions 38 and / or 68 with a different amino acid. In some embodiments, the substitution of cysteine ​​at position 38 comprises C38S, C38I, C38L, C38V, or C38M, optionally wherein the substitution of cysteine ​​at position 38 comprises C38S, C38I, and C38V. In some embodiments, the substitution of cysteine ​​at position 68 comprises C68S, C68I, C68V, C68L, or C68D, optionally wherein the substitution of cysteine ​​at position 38 comprises C68S, C68I, and C68L.

[0033] In some embodiments according to any of the above methods, the method further comprises retaining the cysteine ​​at position 127 when the IL-18 polypeptide comprises a cysteine ​​at position 127, or introducing a cysteine ​​at position 127 when the IL-18 polypeptide comprises a non-cysteine ​​at position 127.

[0034] In some embodiments according to any of the above methods, the method further comprises introducing an alanine or an amino acid without a hydrophobic side chain at position 127, optionally the amino acid without a hydrophobic side chain is selected from cysteine, serine, threonine, asparagine, glutamine, glycine, and proline, further optionally the amino acid without a hydrophobic side chain is selected from cysteine, serine, and threonine. In some embodiments, introducing an alanine or an amino acid without a hydrophobic side chain at position 127 comprises substituting the amino acid at position 127 of the IL-18 polypeptide with an alanine or an amino acid without a hydrophobic side chain.

[0035] In some embodiments according to any of the above methods, the IL-18 variant polypeptide comprises: a) I38, V38, or S38, b) I68, S68, or L68, c) C76, and d) C117. In some embodiments, the IL-18 variant polypeptide comprises I38, S68, C76, and C117. In some embodiments, the IL-18 variant polypeptide comprises V38, I68, C76, C117, optionally wherein the variant polypeptide further comprises A127. In some embodiments, the variant IL-18 polypeptide comprises 3P, 6K, 54G, 56G, 57A, and 91T. In some embodiments, the variant IL-18 polypeptide comprises 6G, 54L, 56T, 57E, 91G, and 104S.

[0036] In some embodiments according to any of the above methods, the IL-18 variant polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 311-312, 314-315, and 316-317, or a functional variant thereof having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 311-312, 314-315, and 316-317. A functional variant as used herein with respect to an IL-18 variant polypeptide is one that exhibits an IL-18 function comparable to that of a reference IL-18 polypeptide (e.g., at least 50%, 60%, 70%, 75%, or 80%), as measured, for example, by the methods described in Example 1.

[0037] In another aspect, the present application provides a method for engineering an IL-18 variant polypeptide from an IL-18 polypeptide, comprising a) replacing the cysteine ​​at position 38 with another amino acid, b) replacing the cysteine ​​at position 68, and / or c) replacing the cysteine ​​at position 76, wherein the IL-18 variant polypeptide does not contain a free cysteine ​​at each of positions 38, 68, and 76. In some embodiments, the substituted amino acid in a), b), or c) is an amino acid having a hydrophobic side chain. In some embodiments, the amino acid with a hydrophobic side chain in a), b) or c) is selected from alanine, valine, isoleucine, leucine, methionine, aniline, tyrosine and tryptophan, optionally wherein the method comprises a) introducing any one of valine, isoleucine, leucine and methionine at position 38, b) introducing any one of valine, isoleucine and leucine at position 68, and / or c) introducing any one of valine and tyrosine at position 76, further optionally wherein introducing an amino acid with a hydrophobic side chain at position 38, 68 or 76 comprises substituting the amino acid at position 38, 68 or 76 with an amino acid with a hydrophobic side chain. In some embodiments, the method comprises a) introducing isoleucine at position 38, b) introducing isoleucine or leucine at position 68, and / or c) introducing any one of valine and tyrosine at position 76. In some embodiments, the IL-18 variant polypeptide comprises 38S, 38I, 38V, 38L, or 38M, optionally wherein the IL-18 variant polypeptide comprises 38S, 38I, or 38V, further optionally wherein the IL-18 variant polypeptide comprises 38I. In some embodiments, the IL-18 variant polypeptide comprises 68S, 68I, 68V, 68L, or 68D, optionally wherein the IL-18 variant polypeptide comprises 68S, 68I, or 68L, further optionally wherein the IL-18 variant polypeptide comprises 68S or 68L. In some embodiments, the IL-18 variant polypeptide comprises 76V or 76Y. In some embodiments, the IL-18 variant polypeptide comprises: a) 38I, b) 68I or 68L, and c) 76V or 76Y. In some embodiments, the IL-18 variant polypeptide does not comprise 117C. In some embodiments, the IL-18 variant polypeptide further comprises a cysteine, an alanine, or an amino acid without a hydrophobic side chain at position 127. In some embodiments, the IL-18 variant polypeptide comprises 38I, 68I, and 76V, optionally wherein the IL-18 variant polypeptide comprises 117S. In some embodiments, the IL-18 variant polypeptide further comprises 127I. In some embodiments, the IL-18 variant polypeptide comprises 38I, 68L, and 76Y, optionally wherein the IL-18 variant polypeptide comprises S117.In some embodiments according to any of the above methods, the IL-18 variant polypeptide comprises the amino acid sequence shown in any one of SEQ ID NOs: 314-315, or a functional variant thereof having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 314-315.

[0038] In some embodiments according to any of the above methods, the IL-18 polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 1-299, or a functional variant thereof having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 1-299, optionally wherein the IL-18 polypeptide comprises the amino acid sequence set forth in any one of SEQ ID NOs: 1, 3, 9, 17, 27, 39, 43, 49, 94, 105, 116-117, 133, and 143-150, or a functional variant thereof having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 1-299, NO: 1, 3, 9, 17, 27, 39, 43, 49, 94, 105, 116-117, 133 and 143-150 having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity thereof, further optionally wherein the IL-18 polypeptide comprises the amino acid sequence of any one of SEQ ID NO: 1, 3, 9, 17, 27, 39, 43, 49, 94, 105, 29 and 117, or a functional variant thereof having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity thereto, further optionally wherein the IL-18 polypeptide comprises the amino acid sequence of any one of SEQ ID NO: 1, 3, 9, 17, 27, 39, 43, 49, 94, 105, 29 and 117, The amino acid sequence shown in any one of NO:1, 39 and 133, or a functional variant thereof having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity with any one of SEQ ID NO:1, 39 and 133.

[0039] In another aspect, the present application provides an IL-18 variant polypeptide produced by any one of the above methods.

[0040] In another aspect, the present application provides an IL-18 variant polypeptide comprising the amino acid sequence shown in any one of SEQ ID NOs: 311-312, 314-315 and 316-317, or a functional variant thereof having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity with any one of SEQ ID NOs: 311-312, 314-315 and 316-317.

[0041] In another aspect, the present application provides an IL-18 variant polypeptide comprising a cysteine ​​at position 117 and a cysteine ​​at position 76, wherein the amino acid positions are relative to wild-type human IL-18 as set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide does not comprise a cysteine ​​at position 38, or the IL-18 variant polypeptide does not comprise a cysteine ​​at position 68. In some embodiments, the IL-18 variant polypeptide does not comprise a cysteine ​​at position 38, and the IL-18 variant polypeptide does not comprise a cysteine ​​at position 68. In some embodiments, the IL-18 variant polypeptide comprises 38S, 38I, 38V, 38L, or 38M, optionally wherein the IL-18 variant polypeptide comprises 38S, 38I, or 38V. In some embodiments, the IL-18 variant polypeptide comprises 68S, 68I, 68V, 68L, or 68D, optionally wherein the IL-18 variant polypeptide comprises 68S, 68I, or 68L. In some embodiments, the IL-18 variant polypeptide comprises 127C. In some embodiments, the IL-18 variant polypeptide comprises an alanine at position 127 or an amino acid without a hydrophobic side chain at position 127, optionally wherein the amino acid without a hydrophobic side chain is selected from cysteine, serine, threonine, asparagine, glutamine, glycine, and proline, further optionally wherein the amino acid without a hydrophobic side chain is selected from cysteine, serine, and threonine. In some embodiments, the IL-18 variant polypeptide comprises: a) I38, V38, or S38, b) I68, S68, or L68, c) C76, and d) C117. In some embodiments, the IL-18 variant polypeptide comprises I38, S68, C76, and C117. In some embodiments, the IL-18 variant polypeptide comprises V38, I68, C76, C117, optionally wherein the variant polypeptide further comprises A127. 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, 91G, and 104S.

[0042] In another aspect, the present application provides a fusion polypeptide comprising a) any one of the above-described IL-18 variant polypeptides, and b) a second portion. In some embodiments, the second portion comprises a half-life extending moiety, optionally wherein the half-life extending moiety is an albumin binding moiety or an Fc domain. In some embodiments, the half-life extending moiety comprises an Fc domain, optionally wherein the Fc domain is a human IgG Fc domain, further optionally wherein the human IgG Fc domain is a human IgG1 domain. In some embodiments, the Fc domain is a modified Fc domain with reduced effector function, optionally wherein the Fc domain comprises a human IgG1 Fc domain comprising an N297A mutation (EU numbering). In some embodiments, the second portion is fused to the N-terminus of the IL-18 variant polypeptide. In some embodiments, the second portion is fused to the C-terminus of the IL-18 variant polypeptide. In some embodiments, the fusion polypeptide further comprises a linker between the IL-18 variant polypeptide and the second portion, optionally wherein the linker is a peptide linker (e.g., a GS linker). In some embodiments, the fusion polypeptide comprises the amino acid sequence set forth in any one of SEQ ID NOs: 323-324, 326-328, 330-332, 340-341, 343-345, and 347, or a functional variant thereof having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 323-324, 326-328, 330-332, 340-341, 343-345, and 347.

[0043] In another aspect, the present application provides a dimer comprising two fusion polypeptides, wherein the fusion polypeptide is selected from any one or two of the fusion polypeptides described herein. In some embodiments, the dimer is a homodimer. In some embodiments, the dimer is a heterodimer.

[0044] In another aspect, the present application provides a nucleic acid encoding any one of the above-mentioned IL-18 variant polypeptides or any one of the fusion polypeptides.

[0045] In another aspect, the present 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, when the masking moiety is linked to the IL-18 polypeptide via a cleavable linker, the masking moiety inhibits the IL-18 polypeptide from activating IL-18 receptor-mediated signaling. In some embodiments, the masking moiety comprises an IL-18 propeptide, an extracellular domain of IL-18Rα, an extracellular domain of IL-18Rβ, an IL-18 binding protein, a fragment of any of the foregoing, or a variant of any 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.

[0046] In some embodiments, the cleavable linker comprises one or more amino acid sequences that can be 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 metalloproteinases, disintegrin and metalloproteinases, 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 metalloproteinases, wherein the matrix metalloproteinases are selected from the group consisting of: matrix metalloproteinase 1, matrix metalloproteinase 2, matrix metalloproteinase 3, matrix metalloproteinase 8, matrix metalloproteinase 9, matrix metalloproteinase 10, matrix metalloproteinase 12, and matrix metalloproteinase 14. In some embodiments, the cleavable linker comprises one or more amino acid sequences that can be recognized and cleaved by one or more of matrix metalloproteinase 9, matrix metalloproteinase 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.

[0047] 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.

[0048] In some embodiments, the IL-18 polypeptide comprises an IL-18 variant polypeptide. In some embodiments, the IL-18 variant polypeptide specifically binds to the IL-18 receptor alpha (IL-18Rα) and exhibits (i) substantially reduced binding to the IL-18 binding protein (IL-18BP) relative to wild-type IL-18, or (ii) no binding to IL-18BP. In some embodiments, the IL-18 variant polypeptide exhibits enhanced binding to IL-18Rα relative 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.

[0049] 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 a 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) relative 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 enhanced binding to IL-18Rα relative 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 a variant thereof of the fusion protein. In some embodiments, the C-terminus of the human IgG Fc domain or a 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.

[0050] 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.

[0051] In some embodiments, a dimer is provided comprising two activatable IL-18 polypeptides described herein. In some embodiments, the dimer is a homodimer. In some embodiments, the dimer is a heterodimer.

[0052] In some embodiments, a nucleic acid encoding an activatable IL-18 polypeptide described herein is provided. In some embodiments, a vector comprising a nucleic acid described herein is provided. In some embodiments, a host cell comprising a nucleic acid described herein or a vector described herein is provided. In some embodiments, a method for producing an activatable IL-18 polypeptide is provided, comprising: (a) culturing a host cell described herein under conditions that express an activatable IL-18 polypeptide, 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 HEK293 cell). In some embodiments, the method comprises (e.g., further comprises) purifying the activatable IL-18 polypeptide.

[0053] In some embodiments, a pharmaceutical composition is provided, comprising an activatable IL-18 polypeptide described herein, a nucleic acid described herein, or a vector described herein.

[0054] In some embodiments, a method of treating a disease in an individual is provided, comprising administering to the individual an effective amount of a pharmaceutical composition as described herein. In some embodiments, the disease is cancer. In some embodiments, a method of activating IL-18 receptor-mediated signaling in an individual is provided, comprising administering to the individual an effective amount of a pharmaceutical composition as described herein. In some embodiments, a method of stimulating immune cells that have experienced an antigen in an individual in need thereof is provided, comprising administering to the individual an effective amount of a pharmaceutical composition as described herein. In some embodiments, stimulation comprises increasing the activity and / or number of immune cells that have experienced the antigen.

[0055] In some embodiments, the individual is a human.

[0056] It should be understood that one, some or all of the characteristics of the various embodiments described herein can be combined to form other embodiments of the present invention. These and other aspects of the present invention will become apparent to those skilled in the art. These and other embodiments of the present invention will be further described by the following detailed description.

[0057] The disclosures of all publications, patents, patent applications, and published patent applications mentioned herein are incorporated by reference in their entirety. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1A Shown are the results of biolayer interferometry experiments performed to assess binding of WT hIL-18 and proIL-18 to hIL-18Rα.

[0059] Figure 1B Shown are activation of hIL-18 receptor-mediated signaling by proIL-18 before and after treatment of proIL-18 with caspase-1, and activation of hIL-18 receptor-mediated signaling by caspase-1-treated proIL-18 in the presence of hIL-18 binding protein.

[0060] Figure 2A Activation of hIL-18 receptor-mediated signaling by proIL-18-MMP9L before and after treatment of proIL-18-MMP9L with MMP9 is shown.

[0061] Figure 2B Activation of hIL-18 receptor-mediated signaling by proIL-18-MMP10L is shown.

[0062] Figure 2C Activation of hIL-18 receptor-mediated signaling by proIL-18-Legu L before and after treatment of proIL-18-Legu L with legumin is shown.

[0063] Figure 3A Size exclusion chromatography curves of ProM12, MMP9-cleaved ProM12 ("ProM12 Cut"), and M12 are shown.

[0064] Figure 3B Size exclusion chromatography curves of ProMM5, MMP9-cleaved ProMM5 ("ProMM5 Cut"), and MM5 are shown.

[0065] Figure 3C Activation of hIL-18 receptor-mediated signaling by ProM12, M12, ProM12 Cut, and ProM12 Cut + hIL-18 binding peptide (BP) is shown.

[0066] Figure 3D Activation of hIL-18 receptor-mediated signaling by ProMM5, MM5, ProMM5 Cut, and ProMM5 Cut + hIL-18 binding peptide (BP) is shown.

[0067] Figure 3E Activation of hIL-18 receptor-mediated signaling by ProM21, M21, and ProM21 Cut is shown.

[0068] Figure 3F Activation of hIL-18 receptor-mediated signaling by ProM13, M13, and ProM13 Cut is shown.

[0069] Figure 3G Activation of hIL-18 receptor-mediated signaling by ProM24, M24, and ProM24 Cut is shown.

[0070] Figure 3H Activation of hIL-18 receptor-mediated signaling by ProWM4, WM4, and ProWM4 Cut is shown.

[0071] Figure 3I Activation of hIL-18 receptor-mediated signaling by ProWM5, WM5, and ProWM5 Cut is shown.

[0072] Figure 3J Activation of hIL-18 receptor-mediated signaling by ProWM6, WM6, and ProWM6 Cut is shown.

[0073] Figure 3K Activation of hIL-18 receptor-mediated signaling by ProM12, M12, ProM12 digested with MMP9, and ProM12 digested with caspase-1 is shown.

[0074] Figure 4A Shown are the results of a PBMC-based assay performed to assess the ability of M12, ProM12, and ProM12 Cut to induce hIFNγ release.

[0075] Figure 4B Shown are the results of a PBMC-based assay performed to assess the ability of MM5, ProMM5, and ProMM5 Cut to induce hIFNγ release.

[0076] 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 Cut is shown.

[0077] Figure 5BActivation of hIL-18 receptor-mediated signaling by M12-DB6-Fc_N297A, D12-M12-DB6-Fc_N297A, and D12-M12-DB6-Fc_N297ACut is shown.

[0078] Figure 5C Activation of hIL-18 receptor-mediated signaling by M12-DB6-Fc_N297A, BPm-M12-DB6-Fc_N297A, and BPm-M12-DB6-Fc_N297ACut is shown.

[0079] Figure 6A Activation of hIL-18 receptor-mediated signaling by MM5-DB6-Fc_N297A, Ra-MM5-DB6-Fc_N297A, and Ra-MM5-DB6-Fc_N297A Cut is shown.

[0080] Figure 6B Activation of hIL-18 receptor-mediated signaling by MM5-DB6-Fc_N297A, D12-MM5-DB6-Fc_N297A, and D12-MM5-DB6-Fc_N297ACut is shown.

[0081] 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 Cut is shown.

[0082] 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 Cut is shown.

[0083] Figure 7 Activation of hIL-18 receptor-mediated signaling by Fc_N297A-M12-DB6, Fc_N297A-M12-DB6-Ra with different linker lengths, and Fc_N297A-M12-DB6-Ra Cut cleaved by MMP9 is shown.

[0084] Figure 8A 、 Figure 8B Activation of hIL-18 receptor-mediated signaling by Fc_N297A-M12-DB6, Fc_N297A-M12-DB6-Ra with a multi-protease recognition linker, and Fc_N297A-M12-DB6-Ra Cut cleaved by MMP9 are shown.

[0085] Figure 9A 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 Cut cleaved by MMP9, MMP14, or uPA, respectively, is shown.

[0086] Figure 9B 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 Cut cleaved by MMP9, MMP14, or uPA, respectively, is shown. DETAILED DESCRIPTION

[0087] Overview

[0088] The present application is based, in part, on applicants' identification of activatable IL-18 polypeptides 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 a site that is recognized and cleaved by, for example, 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, for example, after cleavage by a protease (e.g., a TME protease).

[0089] definition

[0090] As used herein, the terms "specific binding," "specific recognition," and "specifically for" refer to a measurable and reproducible interaction, such as the binding between a cytokine and its receptor, which determines the presence of a target in the presence of a heterogeneous population of molecules (including biomolecules). For example, a cytokine that specifically recognizes a receptor is a cytokine that binds to that receptor with greater affinity, avidity, ease, and / or longer duration than it binds to other targets. In some embodiments, the extent of binding of a cytokine to an unrelated target is less than about 10% of the binding of the cytokine to its receptor, as measured, for example, by radioimmunoassay (RIA). In some embodiments, a cytokine that specifically binds to its receptor has a specific binding affinity of ≤10-5 M, ≤10 -6 M, ≤10 -7 M, ≤10 -8 M, ≤10 -9 M, ≤10 -10 M, ≤10 -11 M or ≤10 -12 The dissociation constant (K D In some embodiments, the specific binding may include but does not require exclusive binding. The binding specificity of the cytokine can be determined experimentally by methods known in the art. Such methods include, but are not limited to, Western blotting, ELISA, RIA, ECL, IRMA, EIA, BIACORE TM -Testing and peptide scanning.

[0091] An "isolated" nucleic acid molecule encoding a polypeptide or cytokine as described herein is one that has been identified and separated from at least one contaminant nucleic acid molecule that is normally associated with the environment in which it was produced. Preferably, the isolated nucleic acid is free from all components associated with the production environment. In some embodiments, the isolated nucleic acid molecule encoding a polypeptide or cytokine as described herein is in a form that is different from the form or environment in which it is found in nature.

[0092] As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that are incorporated into the genome of a host cell into which they are introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors."

[0093] As used herein, the terms "transfection" or "transformation" or "transduction" refer to the process of transferring or introducing exogenous nucleic acid into a host cell. A "transfected" or "transformed" or "transduced" cell refers to a cell that has been transfected, transformed, or transduced with an exogenous nucleic acid. Cells include primary subject cells and their progeny.

[0094] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably to refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom, regardless of the number of passages. The nucleic acid composition of the progeny may not be completely identical to that of the parent cell and may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the original transformed cell are included herein.

[0095] As used herein, " treatment " or " processing " is the method for obtaining the result (including clinical outcome) of benefit or expectation.For the purpose of the application, the clinical outcome of benefit or expectation includes but is not limited to following one or more: alleviate one or more symptoms caused by disease, alleviate the degree of disease, stabilize disease (for example, prevent or delay the deterioration of disease), prevent or delay the spread of disease (for example, metastasis), prevent or delay the recurrence of disease, delay or slow down the progress of disease, improve disease state, provide the alleviation (partial or complete) of disease, reduce the dosage of one or more other drugs needed for treating disease, delay the progress of disease, improve or improve quality of life, increase weight gain and / or prolong survival. " treatment " also encompasses the pathological consequences (for example, tumor volume) that reduce cancer. The method of the application contemplates any one or more of these aspects for the treatment of.

[0096] In the context of cancer, the term "treating" includes any or all of the following: inhibiting the growth of cancer cells, inhibiting the replication of cancer cells, reducing the overall tumor burden, and ameliorating one or more symptoms associated with the disease.

[0097] The term "inhibition" or "inhibit" refers to a reduction or cessation of any phenotypic characteristic, or a reduction or cessation of the incidence, extent, or likelihood of that characteristic. "Reduce" or "inhibit" refers to a decrease, reduction, or prevention of an activity, function, and / or amount as compared to a reference value. In certain embodiments, "reduce" or "inhibit" refers to the ability to cause an overall reduction of 20% or more. In another embodiment, "reduce" or "inhibit" refers to the ability to cause an overall reduction of 50% or more. In another embodiment, "reduce" or "inhibit" refers to the ability to cause an overall reduction of 75%, 85%, 90%, 95% or more.

[0098] The terms "subject," "individual," and "patient" are used interchangeably herein to refer to mammals, including but not limited to humans, cows, horses, cats, dogs, rodents, or primates. In some embodiments, the individual is a human.

[0099] It should be understood that the embodiments of the present application described herein include "consisting of" and / or "consisting essentially of" embodiments.

[0100] Reference herein to "about" a value or parameter includes (and describes) variations with respect to that value or parameter itself. For example, a description referring to "about X" includes a description of "X."

[0101] As used herein, the expression "not for" a certain value or parameter generally means and describes "except" a certain value or parameter. For example, a method is not used to treat type X cancer, which means that the method is used to treat other types of cancer other than type X.

[0102] As used herein, the term "about XY" has the same meaning as "about X to about Y."

[0103] As used herein and in the appended claims, the singular forms "a," "or," and "the" include plural referents unless the context clearly dictates otherwise.

[0104] Activatable interleukin-18 (IL-18) peptide

[0105] 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, IL-18 variant polypeptide, or a fusion protein comprising a wild-type IL-18 or IL-18 variant polypeptide. In some embodiments, when the masking moiety is linked to the IL-18 polypeptide via a cleavable linker, 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).

[0106] In some embodiments, the masking portion comprises or is derived from (i) an IL-18 propeptide (e.g., a human IL-18 propeptide), an extracellular domain of IL-18Rα (e.g., an extracellular domain of human IL-18Rα), an extracellular domain of IL-18Rβ (e.g., an extracellular domain of human IL-18Rβ), an IL-18 binding protein (e.g., a human IL-18 binding protein), a fragment of any of the foregoing, or a variant of any of the foregoing. In some embodiments, the masking portion comprises the amino acid sequence of any one of SEQ ID NOs: 333, 336, and 353-356. In some embodiments, the masking portion 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 can be recognized and cleaved by a protease. In some embodiments, the cleavable linker comprises two or more sequences that can be recognized and cleaved by a protease. In some embodiments, the two or more sequences can be recognized and cleaved by different proteases. In some embodiments, two or more sequences can be 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), membrane-type serine protease (matriptase, MTSP-1), legumin, prostate-specific antigen (PSA), dipeptidyl peptidase (e.g., DPP4), hepsin, matrix metalloproteinases (including but not limited to, e.g., matrix metalloproteinase 1 (MMP1), matrix metalloproteinase 2 (MMP2), matrix metalloproteinase 3 (MMP3), matrix metalloproteinase 8 (MMP8), matrix metalloproteinase 9 (MMP9), matrix metalloproteinase 10 (MMP10), matrix metalloproteinase 12 (MMP12), or matrix metalloproteinase 14 (MMP14)), a disintegrin and metalloproteinase (ADAM 10, 17, etc.), human leukocyte elastase (HLE), proteinase 3 (PR3), prourokinase, plasminogen, staphylokinase, serine proteases, plasmin, cathepsins (B, L, S), tissue kallikreins, and / or kallikrein-related peptidases (KLK 1, 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 NO: 372-377, the sequence described in Kridel et al. (2002) J Biol Chem. 277(26): 23788-93, and the sequence described in Chen et al. (2002) J Biol Chem. 277(6): 4485–4491.

[0107] 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 the IL-18 receptor alpha (IL-18Rα) and exhibits (i) substantially reduced binding to the IL-18 binding protein (IL-18BP) relative to wild-type IL-18, or (ii) no binding to IL-18BP. In some embodiments, the IL-18 variant polypeptide exhibits enhanced binding to IL-18Rα relative to wild-type IL-18. Exemplary IL-18 variant polypeptides that can 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 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, e.g., a variant of a human Fc domain comprising one or more amino acid insertions, deletions, or substitutions relative to a 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 variant of the human IgG1 Fc domain comprises an N297A substitution, wherein amino acid numbering is according to the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.In some embodiments, the variant of the human IgG1 Fc domain comprising the N297A substitution comprises the amino acid sequence of 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 variant of the human IgG1 Fc domain comprising the N297A substitution comprises the amino acid sequence of SEQ ID NO: 371 (see the sequence summary table).

[0108] 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 3 to 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 (e.g., glycine and serine). In some embodiments, the spacer sequence is or comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 363) or GGGGSGGGGSGSGGG (SEQ ID NO: 365).

[0109] 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 propeptide (i.e., pro-IL-18 propeptide), the extracellular domain of IL-18Rα, the extracellular domain of IL-18Rβ, an IL-18 binding protein, a fragment of any of the foregoing, or a variant of any of the foregoing. In some embodiments, the masking moiety comprises an IL-18 propeptide (i.e., pro-IL-18 propeptide), optionally wherein the IL-18 propeptide comprises a propeptide of human IL-18 (hIL-18), optionally wherein the propeptide 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 can be recognized and cleaved by a protease (e.g., a tumor microenvironment (TME) protease, such as MMP9, such as uPA, such as MMP2, such as legumin). In some embodiments, the cleavable linker is recognized and cleaved by two or more proteases (e.g., ULM, LM9, see Examples). In some embodiments, the cleavable linker has a spacer sequence (GS linker) that flanks the amino acid sequence recognized by the protease. In some embodiments, the cleavable linker is about 10 to 40, 15 to 40, 20 to 40, 20 to 30, or 25 to 30 amino acids in length. 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 cysteine. 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, optionally wherein 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, 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. In some embodiments, the IL-18 variant polypeptide comprises the amino acid sequence of SEQ ID NO: 311, 312, or 316.

[0110] In some embodiments, the activatable IL-18 polypeptide comprises, from N-terminus to C-terminus, (i) a masking moiety comprising a truncated propeptide 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, such as MMP9, such as uPA, such as MMP2, such as legumin). In some embodiments, the cleavable linker is recognized and cleaved by two or more proteases (e.g., ULM, LM9, see Examples). In some embodiments, the cleavable linker has a spacer sequence (GS linker) that flanks the amino acid sequence recognized by the protease. In some embodiments, the cleavable linker is about 10 to 40, 15 to 40, 20 to 40, 20 to 30, or 25 to 30 amino acids in length. 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 cysteine. 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, optionally wherein 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, 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. In some embodiments, the IL-18 variant polypeptide comprises the amino acid sequence of SEQ ID NOs: 311, 312, or 316.

[0111] 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 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., legumin). In some embodiments, the cleavable linker is recognized and cleaved by two or more proteases (e.g., ULM, LM9, see Examples). In some embodiments, the cleavable linker has a spacer sequence (GS linker) that flanks the amino acid sequence recognized by the protease. In some embodiments, the cleavable linker is about 10 to 40, 15 to 40, 20 to 40, 20 to 30, or 25 to 30 amino acids in length. 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 cysteine. 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, optionally wherein 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, 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. In some embodiments, the IL-18 variant polypeptide comprises the amino acid sequence of SEQ ID NOs: 311, 312, or 316.

[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, and (iii) a masking moiety. In some embodiments, the masking moiety comprises the 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 can be recognized and cleaved by a protease (e.g., a tumor microenvironment (TME) protease, e.g., MMP9, e.g., uPA, e.g., MMP2, e.g., legumin). In some embodiments, the cleavable linker can be recognized and cleaved by two or more proteases (e.g., ULM, LM9, see Examples). In some embodiments, the cleavable linker has a spacer sequence (GS linker) that flanks the amino acid sequence recognized by the protease. In some embodiments, the cleavable linker is about 10 to 40, 15 to 40, 20 to 40, 20 to 30, or 25 to 30 amino acids in length. 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 cysteine. 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, optionally wherein 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, 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. In some embodiments, the IL-18 variant polypeptide comprises the amino acid sequence of SEQ ID NOs: 311, 312, or 316.

[0113] 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 the 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 can be recognized and cleaved by a protease (e.g., a tumor microenvironment (TME) protease, e.g., MMP9, e.g., uPA, e.g., MMP2, e.g., legumin). In some embodiments, the cleavable linker is recognized and cleaved by two or more proteases (e.g., ULM, LM9, see Examples). In some embodiments, the cleavable linker has a spacer sequence (GS linker) that flanks the amino acid sequence recognized by the protease. In some embodiments, the cleavable linker is about 10 to 40, 15 to 40, 20 to 40, 20 to 30, or 25 to 30 amino acids in length. 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 cysteine. 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, optionally wherein 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, 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. 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.

[0114] 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 the 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 can be recognized and cleaved by a protease (e.g., a tumor microenvironment (TME) protease, e.g., MMP9, e.g., uPA, e.g., MMP2, e.g., legumin). In some embodiments, the cleavable linker is recognized and cleaved by two or more proteases (e.g., ULM, LM9, see Examples). In some embodiments, the cleavable linker has a spacer sequence (GS linker) that flanks the amino acid sequence recognized by the protease. In some embodiments, the cleavable linker is about 10 to 40, 15 to 40, 20 to 40, 20 to 30, or 25 to 30 amino acids in length. 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 cysteine. 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, optionally wherein 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, 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. 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.

[0115] 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 the 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 can be recognized and cleaved by a protease (e.g., a tumor microenvironment (TME) protease, e.g., MMP9, e.g., uPA, e.g., MMP2, e.g., legumin). In some embodiments, the cleavable linker is recognized and cleaved by two or more proteases (e.g., ULM, LM9, see Examples). In some embodiments, the cleavable linker has a spacer sequence (GS linker) that flanks the amino acid sequence recognized by the protease. In some embodiments, the cleavable linker is about 10 to 40, 15 to 40, 20 to 40, 20 to 30, or 25 to 30 amino acids in length. 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 cysteine. 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, optionally wherein 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, 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. 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.

[0116] 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 the 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 can be recognized and cleaved by a protease (e.g., a tumor microenvironment (TME) protease, e.g., MMP9, e.g., uPA, e.g., MMP2, e.g., legumin). In some embodiments, the cleavable linker is recognized and cleaved by two or more proteases (e.g., ULM, LM9, see Examples). In some embodiments, the cleavable linker has a spacer sequence (GS linker) that flanks the amino acid sequence recognized by the protease. In some embodiments, the cleavable linker is about 10 to 40, 15 to 40, 20 to 40, 20 to 30, or 25 to 30 amino acids in length. 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 cysteine. 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, optionally wherein 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, 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. 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.

[0117] 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 a 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 a 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 a variant thereof. 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 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.

[0118] In some embodiments, a dimer is provided comprising two activatable IL-18 polypeptides described herein. In some embodiments, the dimer is a homodimer. In some embodiments, the dimer is a heterodimer.

[0119] Interleukin-18 (IL-18) variant polypeptide

[0120] In some embodiments, the activatable IL-18 polypeptide comprises an IL-18 variant polypeptide. In some embodiments, the IL-18 variant polypeptide comprises a mutation at one or more of F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and I149, wherein the amino acid positions are relative to the wild-type (WT) human IL-18 shown in SEQ ID NO: 1. In some embodiments, the wild-type IL-18 is a human IL-18 comprising the amino acid sequence of SEQ ID NO: 1.

[0121] YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMY KDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGH DNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED(SEQ ID NO:1)

[0122] In some embodiments, the IL-18 variant polypeptide comprises a mutation at two or more of F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and 1149, wherein the amino acid positions are relative to wild-type (WT) human IL-18 as set forth in SEQ ID NO: 1, e.g., in any combination. In some embodiments, the two or more mutations are selected from the group consisting of: F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and 1149. In some embodiments, the IL-18 variant polypeptide comprises a mutation at three or more of F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and 1149, e.g., in any combination. In some embodiments, the three or more mutations are selected from the group consisting of: F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and 1149. In some embodiments, the IL-18 variant polypeptide comprises mutations at four or more of F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and 1149, e.g., in any combination. In some embodiments, the four or more mutations are selected from the group consisting of: F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and 1149. In some embodiments, the IL-18 variant polypeptide comprises a mutation at five or more of F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and 1149, e.g., in any combination. In some embodiments, the five or more mutations are selected from the group consisting of: F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and 1149. In some embodiments, the IL-18 variant polypeptide comprises a mutation at six or more of F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and 1149, e.g., in any combination. In some embodiments, six or more mutations are selected from the group consisting of: F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and 1149. In some embodiments, the IL-18 variant polypeptide comprises mutations at seven or more of F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and 1149, e.g., in any combination.In some embodiments, seven or more mutations are selected from the group consisting of: F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and 1149. In some embodiments, the IL-18 variant polypeptide comprises 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, eight or more mutations are selected from the group consisting of: F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and 1149. In some embodiments, the IL-18 variant polypeptide comprises a mutation at nine or more of F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and 1149, e.g., in any combination. In some embodiments, 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 a mutation at ten or more of F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and 1149, e.g., in any combination. In some embodiments, ten or more mutations are selected from the group consisting of: F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and 1149. In some embodiments, the IL-18 variant polypeptide comprises mutations at eleven or more of F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and 1149, e.g., in any combination. In some embodiments, the eleven or more mutations are selected from the group consisting of: F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and 1149. In some embodiments, the IL-18 variant polypeptide comprises mutations at twelve or more of F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and 1149, e.g., in any combination. In some embodiments, the twelve or more mutations are selected from the group consisting of: F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and 1149. In some embodiments, the IL-18 variant polypeptide comprises mutations at thirteen or more of F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and 1149, e.g., in any combination.In some embodiments, thirteen or more mutations are selected from the group consisting of: F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and 1149. In some embodiments, the IL-18 variant polypeptide comprises 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) a mutation at a position other than F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and I149. In some embodiments, the IL-18 variant polypeptide comprises one or more mutations (e.g., any of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mutations), wherein at least one, at least 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 I149. In some embodiments, the wild-type IL-18 is human IL-18 comprising the amino acid sequence of SEQ ID NO: 1.

[0123] In some embodiments, one or more mutations are selected from the group consisting of G3, E6, D54, and N91. In some embodiments, the IL-18 variant polypeptide comprises a mutation at two or more of G3, E6, D54, and N91, e.g., in any combination. In some embodiments, two or more mutations are selected from the group consisting of G3, E6, D54, and N91. In some embodiments, the IL-18 variant polypeptide comprises a mutation at three or more of G3, E6, D54, and N91, e.g., in any combination. In some embodiments, three or more mutations are selected from the group consisting of G3, E6, D54, and N91. In some embodiments, the IL-18 variant polypeptide comprises a mutation at G3, E6, D54, and N91. In some embodiments, the IL-18 variant polypeptide does not comprise a mutation at a position other than G3, E6, D54, and / or N91. In some embodiments, the IL-18 variant comprises (or further comprises) a mutation at a position other than G3, E6, D54, and / or N91. In some embodiments, the IL-18 variant polypeptide comprises one or more mutations (e.g., any of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mutations), wherein at least one, at least two, at least three, or at least four mutations are selected from 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.

[0124] In some embodiments, the IL-18 variant polypeptide comprises (e.g., further comprises) at least one mutation at a residue selected from Q56, P57, M60, Q103, R104, M113, and N155, wherein the amino acid positions are relative to wild-type human IL-18 set forth in SEQ ID NO: 1.

[0125] In some embodiments, the IL-18 variant polypeptide comprises (or further comprises) at least one, at least two, at least three, at least four, at least five, or six mutations at residues selected from C38, C68, C76, D98, S117, and C127, wherein the amino acid positions are relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide comprises (or further comprises) a substitution at C38 (i.e., wherein the wild-type cysteine ​​at position 38 is substituted with any amino acid), a substitution at C68 (i.e., wherein the wild-type cysteine ​​at position 68 is substituted with any amino acid), and an S117C substitution. In some embodiments, the IL-18 variant polypeptide comprises (or further comprises) a C38I, C38V, C38L, C38M, or C38S mutation. Additionally or alternatively, in some embodiments, the IL-18 variant polypeptide comprises (or further comprises) a C68S, C68I, C68D, C68V, or C68L mutation. Additionally or alternatively, in some embodiments, the IL-18 variant polypeptide comprises (or further comprises) a C76S, C76V, or C76Y mutation. Additionally or alternatively, in some embodiments, the IL-18 variant polypeptide comprises (or further comprises) an S117C mutation. Additionally or alternatively, in some embodiments, the IL-18 variant polypeptide comprises (or further comprises) a C127A, C127Y, C127F, C127L, or C127I mutation. In some embodiments, the IL-18 variant polypeptide comprises (or further comprises) at least one, at least two, at least three, or four mutations selected from C38S, C68S, C76S, and C127S, wherein the amino acid positions are relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide comprises (or further comprises) C38S, C68S, and C76S mutations. In some embodiments, the IL-18 variant polypeptide comprises (or further comprises or consists of) C38I, C68S, and S117C mutations. In some embodiments, the IL-18 variant polypeptide comprises (or further comprises or consists of) C38V, C68I, S117C, and C127A mutations. In some embodiments, the IL-18 variant polypeptide comprises (or further comprises or consists of) C38S, C68I, S117C, and C127I mutations. In some embodiments, the IL-18 variant polypeptide comprises (or further comprises or consists of) C38I, C68I, C76V, and C127I mutations. In some embodiments, the IL-18 variant polypeptide comprises (or further comprises or consists of) C38I, C68L, and C76Y mutations.

[0126] In some embodiments, the IL-18 variant polypeptide further comprises a mutation at one or more of Q56, P57, and R104. In some embodiments, the IL-18 variant polypeptide further comprises a mutation at two or more of Q56, P57, and R104. In some embodiments, the IL-18 variant polypeptide further comprises a mutation at Q56, P57, and R104. In some embodiments, the IL-18 variant polypeptide does not comprise a mutation at a position other than G3, E6, D54, Q56, P57, N91, and / or R104. In some embodiments, the IL-18 variant comprises (or further comprises) a mutation at a position other than G3, E6, D54, Q56, P57, N91, and / or R104. In some embodiments, the IL-18 variant polypeptide comprises one or more mutations (e.g., any of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mutations), wherein at least one, at least two, at least three, at least four, at least five, at least six, or at least seven mutations are selected from the group consisting of: G3, E6, D54, Q56, P57, N91, and R104. In some embodiments, the IL-18 variant polypeptide does not comprise a substitution at Q56 and P57, wherein the amino acid positions are relative to SEQ ID NO: 1. In some embodiments, the wild-type IL-18 is human IL-18 comprising the amino acid sequence of SEQ ID NO: 1.

[0127] In some embodiments, the IL-18 variant polypeptide specifically binds to IL-18 receptor alpha ("IL-18Rα"), e.g., human IL-18Rα or "hIL-18α", and exhibits significantly reduced binding to IL-18 binding protein ("IL-18BP"), e.g., human IL-18 or "hIL-18BP". In some embodiments, the IL-18 variant polypeptide exhibits substantially reduced binding to IL-18BP relative to WT human IL-18 set forth in SEQ ID NO: 1. In some embodiments, the IL-18 variant polypeptide has an affinity for IL-18Rα (e.g., hIL-18Rα) that is comparable to the affinity of WT human IL-18 set forth in SEQ ID NO: 1 for IL-18Rα (e.g., hIL-18Rα). In some embodiments, the affinity of the IL-18 variant polypeptide for IL-18Rα (eg, hIL-18Rα) is increased compared to the affinity of WT human IL-18 shown in SEQ ID NO:1 for IL-18Rα (eg, hIL-18Rα).

[0128] In some embodiments, the IL-18 variant polypeptide binds to IL-18Rα, wherein K D Less than approximately 5x 10 -5 M, less than about 5x 10-6 M, less than about 5x 10 -7 M, less than about 5x 10 -8 M, less than about 5x 10 -9 M, less than about 5x 10 -10 M or less than about 5x10 -11 M, including any range between these values. In some embodiments, the IL-18 variant polypeptide binds to IL-18Rα, wherein K D About 5 x 10 -5 to about 5 x 10 -11 M. In some embodiments, the IL-18 variant polypeptide binds to IL-18Rα, wherein K D About 5 x 10 -7 to about 5 x 10 -11 M. In some embodiments, the IL-18 variant polypeptide binds to IL-18Rα, wherein K D About 5 x 10 -7 to about 5 x 10 -10 M. In some embodiments, the IL-18 variant polypeptide binds to IL-18Rα, wherein K D About 5x10 -7 to about 5 x 10 -9 M. In some embodiments, the IL-18 variant polypeptide binds to IL-18Rα, wherein K D About 5 x 10 -8 to about 5 x 10 -11 M. In some embodiments, the IL-18 variant polypeptide binds to IL-18Rα, wherein K D About 5 x 10 -9 to about 5 x 10 -11 M. In some embodiments, the IL-18 variant polypeptide binds to IL-18Rα, wherein K D About 5 x 10 -8 to about 5x10 -10 M. In some embodiments, the IL-18 variant polypeptide binds to IL-18Rα, wherein K D About 5 x 10 -7 to about 5 x 10 - 10 M. In some embodiments, the IL-18 variant polypeptide binds to IL-18Rα, wherein K D About 5 x 10 -9 About 5 x 10 -10 M. In some embodiments, the IL-18 variant polypeptide binds to IL-18Rα, wherein K D About 5 x 10 -7About 5 x 10 -9 M. In some embodiments, the IL-18 variant polypeptide binds to IL-18Rα, wherein K D About 5 x 10 -8 About 5 x 10 -9 M. In some embodiments, the IL-18 variant binds to IL-18Rα (e.g., hIL-18Rα), wherein K D About 5 x 10 -5 Up to 5x 10 -11 M, for example, about 5x 10 -5 , 6x 10 -5 , 7x 10 -5 , 8x 10 -5 , 9x 10 -5 , 1x 10 -6 , 2x 10 -6 , 3x 10 -6 , 4x 10 -6 , 5x 10 -6 , 6x10 -6 , 7x 10 -6 , 8x 10 -6 , 9x 10 -6 , 1x 10 -7 , 2x 10 -7 , 3x 10 -7 , 4x 10 -7 , 5x 10 -7 , 6x 10 -7 , 7x 10 -7 , 8x 10 -7 , 9x 10 -7 , 1x 10 -8 , 2x 10 -8 , 3x 10 -8 , 4x 10 -8 , 5x 10 -8 , 6x 10 -8 , 7x 10 -8 , 8x 10 -8 , 9x 10 -8 , 1x 10 -9 , 2x 10 -9 , 3x 10 -9 , 4x 10 -9 , 5x 10 -9 , 6x 10 -9 , 7x 10 -9 , 8x 10 -9 , 9x 10 -9 , 1x10 -10, 2x 10 -10 , 3x 10 -10 , 4x 10 -10 , 5x 10 -10 , 6x 10 -10 , 7x 10 -10 , 8x10 -10 , 9x 10 -10 , 1x 10 -11 , 2x 10 -11 , 3x 10 -11 , 4x 10 -11 or 5x 10 -11 M, including any range between these values. In some embodiments, the affinity of the IL-18 variant polypeptide for IL-18Rα (e.g., hIL-18Rα) is greater than the affinity of wild-type human IL-18 shown in SEQ ID NO: 1 for IL-18Rα (e.g., hIL-18Rα). In some embodiments, the affinity of the IL-18 variant polypeptide for IL-18Rα (e.g., hIL-18Rα) is comparable to (e.g., approximately the same as) the affinity of wild-type human IL-18 shown in SEQ ID NO: 1 for IL-18Rα (e.g., hIL-18Rα).

[0129] In some embodiments, the IL-18 variant polypeptide exhibits substantially reduced binding to IL-18 binding protein (IL-18BP) relative to wild-type IL-18. In some embodiments, the IL-18 variant polypeptide exhibits substantially reduced binding to IL-18 binding protein (IL-18BP) relative to wild-type IL-18. -9 M's K D In some embodiments, the IL-18 variant polypeptide binds to IL-18BP at a concentration of more than 5 x 10 -8 M's K D In some embodiments, the IL-18 variant polypeptide binds to IL-18BP at a concentration of more than 5 x 10 -7 M's K D In some embodiments, the IL-18 variant polypeptide binds to IL-18BP at a concentration of more than 5 x 10 -6 M's K D In some embodiments, the IL-18 variant polypeptide binds to IL-18BP at a concentration of more than 5x10 -5 M's K D In some embodiments, the IL-18 variant polypeptide does not exhibit binding (e.g., no detectable binding) to IL-18BP (e.g., hIL-18BP). In some embodiments, the IL-18 variant polypeptide that does not exhibit binding (e.g., no detectable binding) to IL-18BP (e.g., hIL-18BP) exhibits a binding affinity greater than 10-3 M's K D Binds to IL-18BP (e.g., hIL-18BP).

[0130] The affinity of IL-18 described herein for IL-18Rα and / or IL-18BP can be determined experimentally by methods known in the art. Such methods include, but are not limited to, for example, Western blotting, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), electrochemiluminescence (ECL) assay, immunoradiometric (IRMA) assay, enzyme immunoassay (EIA), surface plasmon resonance (SPR), peptide scanning, and kinetic competitive screening based on fluorescence activated cell sorting (FACS).

[0131] In some embodiments, the IL-18 variant polypeptide comprises a mutation at residue G3, wherein the mutation is selected from G3P, G3D, G3E, G3N, and G3S. As used herein, "G3X" means that G, the wild-type amino acid at position 3 in SEQ ID NO: 1, has been substituted with amino acid X. In some embodiments, the mutation is G3P, i.e., the wild-type G at position 3 in SEQ ID NO: 1 has been substituted with amino acid P.

[0132] In some embodiments, the variant polypeptide comprises (e.g., further comprises) a mutation at residue E6, wherein the mutation is selected from E6R, E6K, E6G, E6T, E6A, E6S, E6E, E6L, E6M, and E6N. In some embodiments, the mutation is selected from E6R, E6K, E6G, E6T, E6A, and E6S. In some embodiments, the mutation is selected from E6R and E6K.

[0133] 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.

[0134] In some embodiments, the variant polypeptide comprises (e.g., further comprises) a mutation at residue N91, wherein the mutation is selected from N91V, N91A, N91G, N91S, N91I, N91P, N91R, N91T, N91C, N91K, and N91W. In some embodiments, the mutation is selected from N91V, N91A, N91G, and N91S.

[0135] 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.

[0136] In some embodiments, wherein the IL-18 variant polypeptide comprises (e.g., further comprises) a mutation at residue Q56 selected from the group consisting of Q56T, Q56G, Q56R, Q56S, Q56D, Q56P, Q56I, Q56Y, Q56H, Q56L, Q56E, Q56F, Q56N, and Q56V. In some embodiments, the mutation is selected from the group consisting of Q56T, Q56G, Q56R, Q56S, Q56D, Q56P, Q56I, and Q56Y.

[0137] In some embodiments, wherein the IL-18 variant polypeptide comprises (e.g., further comprises) a mutation at residue P57 selected from the group consisting of P57A, P57G, P57R, P57W, P57S, P57T, P57V, P57Q, P57H, P57K, P57N, P57Y, and P57D, in some embodiments, the mutation is selected from the group consisting of P57A, P57G, P57R, P57W, P57S, P57T, and P57V.

[0138] In some embodiments, the IL-18 variant polypeptide comprises a mutation at one or more of G3, E6, D54, and N91. In some embodiments, the IL-18 variant polypeptide comprises a mutation at two or more of G3, E6, D54, and N91. In some embodiments, the IL-18 variant polypeptide comprises a mutation at three or more of G3, E6, D54, and N91. In some embodiments, the IL-18 variant polypeptide comprises a mutation at G3, E6, D54, and N91. In some embodiments, wherein the IL-18 variant polypeptide comprises a G3P mutation. Additionally or alternatively, in some embodiments, the IL-18 variant polypeptide comprises an E6R or E6K mutation. Additionally or alternatively, in some embodiments, the IL-18 variant polypeptide comprises a D54W, D54H, D54S, or D54Q mutation. Additionally or alternatively, in some embodiments, the IL-18 variant polypeptide comprises an N91V, N91A, N91G, or N91S mutation. In some embodiments, the IL-18 variant polypeptide does not contain mutations at positions other than G3, E6, D54, and / or N91. In some embodiments, the IL-18 variant further contains mutations at positions other than G3, E6, D54, and / or N91. In some embodiments, the IL-18 variant polypeptide contains one or more mutations (e.g., any of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mutations), wherein at least one, at least two, at least three, or at least four mutations are selected from G3, E6, D54, and N91. In some embodiments, the IL-18 variant polypeptide further contains mutations at Q56 and / or P57. In some embodiments, the IL-18 variant polypeptide does not contain mutations at positions other than G3, E6, D54, N91, Q56, and / or P57. In some embodiments, the IL-18 variant polypeptide further comprises a mutation at a position other than G3, E6, D54, N91, Q56, and / or P57. In some embodiments, the IL-18 variant polypeptide comprises one or more mutations (e.g., any of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mutations), wherein at least one, at least two, at least three, at least four, at least five, or at least six mutations are selected from G3, E6, D54, N91, Q56, and P57.

[0139] In some embodiments, the IL-18 variant polypeptide comprises an amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the 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 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 following the Examples.

[0140] Fusion peptide

[0141] In some embodiments, the activatable IL-18 polypeptides of the present disclosure comprise a fusion polypeptide comprising a wild-type IL-18 or IL-18 variant polypeptide described herein. In some embodiments, the fusion polypeptide comprises (1) a wild-type IL-18 or IL-18 variant polypeptide, and (2) a dimerization domain. In some embodiments, the fusion polypeptide comprises (1) two or more wild-type IL-18, two or more IL-18 variant polypeptides, or any combination of two or more wild-type IL-18 and IL-18 variant polypeptides, and (2) a dimerization domain. In some embodiments, the fusion polypeptide comprises a mask that prevents (e.g., inhibits) 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 wild-type IL-18 and / or IL-18 variant polypeptides in the fusion polypeptide are masked to prevent (e.g., inhibit) 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, each wild-type IL-18 and / or IL-18 variant polypeptide in the fusion polypeptide is masked to prevent (e.g., inhibit) 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, the dimerization domain is a leucine zipper (LZ) element, or comprises a leucine zipper element. Leucine zippers are generally identified as fragments of approximately 35 amino acids containing 4 to 5 leucine residues separated by six amino acids (Maniatis and Abel (1989) Nature 341: 24-25). Exemplary leucine zippers are present in a variety of eukaryotic DNA binding proteins, such as GCN4, C / EBP, c-Fos, c-Jun, c-Myc, and c-Max. In some embodiments, the dimerization domain is a helix-loop-helix domain, or comprises a helix-loop-helix domain (Murre, C. et al. (1989) Cell 58:537-544). The dimerization domain can also be selected from other proteins, such as retinoic acid receptors, thyroid hormone receptors, or other nuclear hormone receptors (Kurokawa et al. (1993) Genes Dev. 7:1423-1435), or yeast transcription factors GAL4 and HAP1 (Marmonstein et al. (1992) Nature 356:408-414; Zhang et al. (1993) Proc. Natl. Acad. Sci. USA 90:2851-2855). Dimerization domains are further described by Eisenman in US Pat. No. 5,624,818.In some embodiments, the dimerization domain is an antibody Fc domain.

[0142] In some embodiments, the fusion polypeptide comprises wild-type IL-18 (e.g., a wild-type IL-18 having an amino acid sequence set forth in SEQ ID NO: 1) and an antibody Fc domain. In some embodiments, the fusion polypeptide comprises an IL-18 variant polypeptide (e.g., an IL-18 variant polypeptide described herein) and an antibody Fc domain. In some embodiments, the C-terminus of wild-type IL-18 or the C-terminus of an IL-18 variant polypeptide is fused to the N-terminus of an antibody Fc domain. In some embodiments, the C-terminus of an antibody Fc domain is fused to the N-terminus of wild-type IL-18 or the N-terminus of an IL-18 variant polypeptide. In some embodiments, the Fc domain is a human Fc domain or a variant thereof comprising one or more amino acid substitutions. In some embodiments, the Fc domain is a human IgG Fc domain or a variant thereof, such as a human IgG1, IgG2, or IgG4 Fc domain or a variant of any of the foregoing. In some embodiments, the fusion polypeptide comprises (from N-terminus to C-terminus) an IL-18 variant polypeptide described herein and a human IgG1 Fc variant comprising an N297A mutation, wherein amino acid numbering is according to the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991. In some embodiments, the fusion polypeptide comprises (from N-terminus to C-terminus) wild-type IL-18 and a human IgG1 Fc variant comprising an N297A mutation. In some embodiments, the fusion polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 319-332 and 378-389.

[0143] Nucleic acids, vectors, host cells, and methods for producing activatable IL-18 polypeptides

[0144] Also contemplated are nucleic acid molecules encoding the activatable IL-18 polypeptides described herein. In some embodiments, nucleic acids encoding the activatable IL-18 polypeptides described herein are provided. Also provided are vectors into which the nucleic acids described herein are inserted.

[0145] Briefly, expression of the activatable IL-18 polypeptides described herein from natural or synthetic nucleic acids encoding activatable IL-18 polypeptides can be achieved by inserting the nucleic acid into an appropriate expression vector such that the nucleic acid is operably linked to 5' and 3' regulatory elements, including, for example, a promoter (e.g., a constitutive, regulatable, tissue-specific promoter) and a 3' untranslated region (UTR). The vector can be suitable for replication and integration in eukaryotic host cells. Typical cloning and expression vectors contain transcriptional and translational terminators, initiation sequences, and promoters useful for regulating expression of the desired nucleic acid sequence.

[0146] Nucleic acids can be cloned into various types of vectors. For example, nucleic acids can be cloned into vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0147] In addition, expression vectors can be provided to cells in the form of viral vectors. Viral vector technology is well known in the art, such as described in Sambrook et al. (2001, Molecular Cloning:A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals. The virus that can be used as a vector includes but is not limited to retrovirus, adenovirus, adeno-associated virus, herpes virus and slow virus. Generally speaking, suitable vectors contain a functional origin of replication, promoter sequence, convenient restriction endonuclease site and one or more selectable markers in at least one organism (see, for example, WO 01 / 96584; WO 01 / 29058; and U.S. Patent number 6,326,193).

[0148] Many virus-based systems have been developed for transferring genes into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. The selected gene can be inserted into a vector and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to the cells of the subject in vivo or in vitro. Many retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Many adenoviral vectors are known in the art. In some embodiments, lentiviral vectors are used. Vectors derived from retroviruses (e.g., lentiviruses) are suitable tools for achieving long-term gene transfer because they allow long-term, stable integration of transgenics and their propagation in daughter cells. Lentiviral vectors have additional advantages over vectors derived from oncorretroviruses (e.g., murine leukemia viruses) because they can transduce non-proliferating cells (e.g., hepatocytes). They also have the additional advantage of low immunogenicity.

[0149] Additional promoter elements (e.g., enhancers) regulate the frequency of transcription initiation. Typically, they are located in the region 30-110 base pairs (bp) upstream of the start site, although recently many promoters have been found to also contain functional elements downstream of the start site. The spacing between promoter elements is usually flexible, so when elements are inverted or moved relative to each other, promoter function can still be retained. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased to 50 bp, and then activity will begin to decline.

[0150] An example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence that can drive the high-level expression of any polynucleotide sequence operably linked thereto. Another example of a suitable promoter is elongation growth factor-1α (EF-1α). However, other constitutive promoter sequences may also be used, including but not limited to simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukosis virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoters, such as but not limited to actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter. In addition, the present invention should not be limited to the use of constitutive promoters. Inducible promoters are also considered to be part of the present invention. The use of an inducible promoter provides a molecular switch that can turn on expression of an operably linked polynucleotide sequence when expression is desired, or turn off expression when expression is not desired. Examples of inducible promoters include, but are not limited to, the metallothionein promoter, the glucocorticoid promoter, the progesterone promoter, and the tetracycline promoter.

[0151] In some embodiments, expression of the nucleic acid encoding the activatable IL-18 polypeptide is inducible. In some embodiments, the nucleic acid encoding the activatable IL-18 polypeptide is operably linked to an inducible promoter, including any inducible promoter known in the art. In some embodiments, the nucleic acid encoding the activatable IL-18 polypeptide described herein has been engineered to encode an epitope tag, for example, to facilitate purification or detection of the polypeptide. Exemplary epitope tags include, but are not limited to, 6x His (also known as a His tag or hexahistidine tag), FLAG, HA, Myc, V5, GFP (green fluorescent protein, such as enhanced green fluorescent protein or EGFP), SUMO (small ubiquitin-like modifier), GST (glutathione-S-transferase), β-GAL (β-galactosidase), luciferase, MBP (maltose binding protein), RFP (red fluorescent protein), and VSV-G (vesicular stomatitis virus glycoprotein).

[0152] 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 for illustrative purposes only and are not intended to be limiting.

[0153] The activatable IL-18 polypeptides described herein can be produced using recombinant methods. For recombinant production of activatable IL-18 polypeptides, nucleic acid encoding the activatable IL-18 polypeptide is isolated and inserted into a replicable vector for further cloning (amplification of the DNA) or expression. DNA encoding the activatable IL-18 polypeptide can be readily isolated and sequenced using conventional methods (e.g., by using oligonucleotide probes capable of specifically binding to the gene encoding the activatable IL-18 polypeptide). A variety of vectors can be used. Vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.

[0154] Expression vectors and cloning vectors all contain nucleic acid sequences that enable the vector to replicate in one or more selected host cells, for example to allow the vector to replicate independently of the host chromosomal DNA. Such sequences may include an origin of replication or an autonomously replicating sequence. Such sequences are well known for various bacteria, yeasts, and viruses. Typically, mammalian expression vectors do not require an origin of replication component (the SV40 origin can be used because it contains an early promoter).

[0155] Expression and cloning vectors may contain a selection gene or selectable marker. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins (e.g., ampicillin, neomycin, methotrexate, or tetracycline), (b) complement nutritional deficiencies, or (c) provide key nutrients not available in complex culture media. Examples of dominant selection utilize the drugs neomycin, mycophenolic acid, and hygromycin. Another example of a suitable selectable marker for mammalian cells is one that identifies cells that have taken up an activatable IL-18 polypeptide-encoding nucleic acid, such as DHFR, glutamine synthetase (GS), thymidine kinase, metallothionein-I and -II, preferably primate metallothionein genes, adenosine deaminase, ornithine decarboxylase, and the like. For example, a Chinese hamster ovary (CHO) cell line lacking endogenous DHFR activity that has been transformed with a DHFR gene can be identified by culturing transformants in culture medium containing methotrexate (Mtx), a competitive antagonist of DHFR.

[0156] Alternatively, host cells (particularly wild-type hosts containing endogenous DHFR) 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), 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.

[0157] Expression and cloning vectors typically contain a promoter recognized by the host organism, operably linked to a nucleic acid encoding an activatable IL-18 polypeptide. Promoters suitable for prokaryotic hosts include the pho A promoter, β-lactamase and lactose promoter systems, alkaline phosphatase promoters, tryptophan (trp) promoter systems, and hybrid promoters, such as the tac promoter. However, other known bacterial promoters are also suitable. Promoter sequences are known for eukaryotic organisms. Yeast promoters are well known in the art and may include inducible promoters / enhancers that are regulated by growth conditions. Almost all eukaryotic genes have an AT-rich region located approximately 25 to 30 bases upstream of the transcription start site. Examples include, but are not limited to, promoters for 3-phosphoglycerate kinase or other glycolytic enzymes, such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. Transcription of the activatable IL-18 polypeptide from a vector in a mammalian host cell can be controlled by, for example, promoters obtained from viral genomes. The early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment, which 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.

[0158] Transcription of a DNA encoding an activatable IL-18 polypeptide described herein by higher eukaryotes is typically enhanced by inserting an enhancer sequence into the vector. Numerous enhancer sequences are known from mammalian genes (globin, elastase, albumin, alpha-fetoprotein, and insulin). However, enhancers from eukaryotic cell viruses are commonly used.

[0159] Expression vectors used in eukaryotic host cells (nucleated cells of yeast, fungi, insect, plant, animal, human or other multicellular organisms) will also contain sequences necessary for the termination of transcription and for stabilizing the mRNA.

[0160] Suitable host cells for cloning or expressing the DNA in the vectors herein are the prokaryotes, yeasts, or higher eukaryotic cells described above. Suitable prokaryotes for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, such as Enterobacteriaceae, such as Escherichia, such as Escherichia coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, such as Salmonella typhimurium, Serratia, such as Serratia marcescans, and Shigella. In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are also suitable cloning or expression hosts for IL-18 variant polypeptide-encoding vectors or fusion polypeptide-encoding vectors. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used of the lower eukaryotic host microorganisms. Certain fungal and yeast strains can be selected in which the glycosylation pathways have been "humanized," thereby producing activatable IL-18 polypeptides with partially or fully human glycosylation patterns. See, for example, Li et al., Nat. Biotech. 24:210-215 (2006).

[0161] Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, duckweed (Leninaceae), alfalfa (M. truncatula), and tobacco can also be used as hosts.

[0162] Suitable host cells for expressing glycosylated activatable IL-18 polypeptides are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Many baculovirus strains and variants have been identified, as well as corresponding permissive insect host cells, from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly), and Bombyx mori.

[0163] Vertebrate cells can be used as hosts, and propagation of vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 cells or 293 cells subcloned for growth in suspension culture, Graham et al., J. GenVirol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocytes (HepG2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and human hepatoma cell line (Hep G2). Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR - CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines, such as NS0 and Sp2 / 0. For a review of certain mammalian host cell lines suitable for production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKCLo, ed., Humana Press, Totowa, NJ, 2003), pp. 255-268.

[0164] Host cells of the present disclosure can be cultured in a variety of culture media. Commercially available culture media such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM), Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable for culturing host cells. In addition, any culture medium described in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), U.S. Patent Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO 90 / 03430; WO 87 / 00195; or U.S. Reissue Patent 30,985 can be used as a culture medium for host cells. Any of these media may be supplemented as needed with hormones and / or other growth factors (e.g., insulin, transferrin, or epidermal growth factor), salts (e.g., sodium chloride, calcium, magnesium, and phosphate), buffers (e.g., HEPES), nucleotides (e.g., adenosine and thymidine), antibiotics (e.g., GENTAMYCIN TM Drugs), trace elements (defined as inorganic compounds typically present at final micromolar concentrations), and glucose or an equivalent energy source. Any other necessary supplements known to those skilled in the art may also be added at appropriate concentrations. Culture conditions (e.g., temperature, pH, etc.) are those previously used with the host cell selected for expression and will be apparent to those skilled in the art.

[0165] When using recombinant techniques, the activatable IL-18 polypeptide can be produced intracellularly, in the periplasmic space, or directly secreted into the culture medium. If the polypeptide is produced intracellularly, the first step is to remove the particulate debris (host cell or lysed debris), for example by centrifugation or ultrafiltration. Carter et al., Bio / Technology 10:163-167 (1992) describe a method for isolating antibodies secreted into the periplasmic space of Escherichia coli.

[0166] The polypeptide composition produced by the cells can be purified using, for example, hydroxyapatite chromatography, hydrophobic interaction chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being one of the generally preferred purification steps. In some embodiments, the activatable IL-18 polypeptide described herein comprises 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, 6x His (also known as a His tag or hexahistidine tag), FLAG, HA, Myc, V5, GFP (green fluorescent protein, such as enhanced green fluorescent protein or EGFP), SUMO (small ubiquitin-like modifier), GST (glutathione-S-transferase), β-GAL (β-galactosidase), luciferase, MBP (maltose binding protein), RFP (red fluorescent protein), and VSV-G (vesicular stomatitis virus glycoprotein).

[0167] Methods for treating disease, activating hIL-18 receptor-mediated signaling, and stimulating T cells or NK cells that have experienced antigen

[0168] Also provided herein are methods of treating a disease or condition in a subject. The methods comprise administering to a subject suffering from a disease or condition an activatable IL-18 polypeptide as described herein, a nucleic acid as described herein, a vector as described herein, and / or a pharmaceutical composition as described herein. In some embodiments, the disease or condition is a proliferative disease. In some embodiments, the proliferative disease is cancer.

[0169] In some embodiments, a method of activating IL-18 receptor-mediated signaling (e.g., human IL-18 receptor-mediated signaling) in an individual (e.g., a human individual) is provided, comprising administering to the individual an activatable IL-18 polypeptide as described herein, a nucleic acid as described herein, a vector as described herein, and / or a pharmaceutical composition as described herein. In some embodiments, provided herein is a method of stimulating antigen-experienced T cells or NK cells in an individual, comprising administering to the individual an activatable IL-18 polypeptide as described herein, a nucleic acid as described herein, a vector as described herein, and / or a pharmaceutical composition as 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.

[0170] Compositions, kits and articles of manufacture

[0171] Also provided herein are compositions (eg, formulations) comprising an activatable IL-18 polypeptide, nucleic acid, vector, or host cell described herein.

[0172] Suitable compositions are obtained by mixing activatable IL-18 polypeptides, nucleic acids, vectors or host cells having the desired purity, and optionally pharmaceutically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)).

[0173] Also provided are kits comprising an activatable IL-18 polypeptide, nucleic acid, vector, or host cell comprising a nucleic acid or vector as described herein. The kits can be used in any of the therapeutic methods described herein.

[0174] The kit of the present application is packaged in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed polyester film or plastic bags), etc. The kit may optionally provide other components, such as buffers and descriptive information.

[0175] Therefore, the present application also provides an article of manufacture. The article of manufacture may comprise a container and a label or package insert on or associated with the container. Suitable containers include vials (e.g., sealed vials), bottles, jars, flexible packaging, and the like. Typically, the container holds the composition and may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic needle).

[0176] Those skilled in the art will recognize that, within the scope and spirit of the present invention, multiple embodiments can be arranged. The present invention will now be described in more detail with reference to the following non-limiting examples. The following examples further illustrate the present invention, but certainly should not be construed as limiting its scope in any way.

[0177] Exemplary embodiments

[0178] 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, optionally 1) b) is fused to the N-terminus of a), or 2) b) is fused to the C-terminus of a).

[0179] 2. The activatable IL-18 polypeptide of embodiment 1, wherein when the masking moiety is linked to the IL-18 polypeptide via a cleavable linker, the masking moiety inhibits the IL-18 polypeptide from activating IL-18 receptor-mediated signaling.

[0180] 3. The activatable IL-18 polypeptide of embodiment 1 or 2, wherein the masking moiety comprises an IL-18 propeptide (i.e., pro-IL-18 propeptide), the extracellular domain of IL-18Rα, the extracellular domain of IL-18Rβ, an IL-18 binding protein, a fragment of any of the foregoing, or a variant of any of the foregoing.

[0181] 4. The activatable IL-18 polypeptide of any one of embodiments 1 to 3, wherein the masking moiety comprises an IL-18 propeptide (i.e., pro-IL-18 propeptide), optionally wherein the IL-18 propeptide comprises the propeptide of human IL-18 (hIL-18), optionally wherein the propeptide of hIL-18 comprises the amino acid sequence of SEQ ID NO: 333.

[0182] 5. The activatable IL-18 polypeptide of embodiment 4, wherein the masking moiety comprises a truncated propeptide of pro-IL-18.

[0183] 6. The activatable IL-18 polypeptide of embodiment 5, wherein the truncated propeptide of pro-IL-18 comprises the amino acid sequence of SEQ ID NO: 336.

[0184] 7. The activatable IL-18 polypeptide of any one of embodiments 1 to 3, wherein the masking moiety comprises the extracellular domain of IL-18Rα or a fragment thereof, optionally wherein the fragment of the extracellular domain of IL-18Ra comprises a) the first and second domains of the extracellular domain, or b) the third domain of the extracellular domain.

[0185] 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.

[0186] 9. The activatable IL-18 polypeptide of any one of embodiments 1 to 8, wherein the cleavable linker comprises one or more amino acid sequences that are recognized and cleaved by one or more proteases, optionally wherein the one or more proteases are one or more tumor microenvironment (TME) proteases.

[0187] 10. The activatable IL-18 polypeptide of embodiment 9, wherein the one or more proteases are one or more tumor microenvironment (TME) proteases, and wherein the one or more TME proteases are selected from the group consisting of: urokinase-type plasminogen activator (uPA), membrane-type serine proteases, legumin, prostate-specific antigen, dipeptidyl peptidase, hepsin, matrix metalloproteinases (MMPs), disintegrins and metalloproteinases, human leukocyte elastase, proteinase 3, prourokinase, plasminogen, staphylokinase, cathepsins, tissue kallikreins, and kallikrein-related peptidase.

[0188] 11. The activatable IL-18 polypeptide of embodiment 10, wherein the one or more TME proteases are matrix metalloproteinases, and wherein the matrix metalloproteinases are selected from the group consisting of: matrix metalloproteinase 1, matrix metalloproteinase 2, matrix metalloproteinase 3, matrix metalloproteinase 8, matrix metalloproteinase 9, matrix metalloproteinase 10, matrix metalloproteinase 12, and matrix metalloproteinase 14.

[0189] 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 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, membrane-type serine protease, and legumin.

[0190] 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, optionally wherein the one or more amino acid sequences are recognized and cleaved by a) both legumin and MMP9 / MMP2 / MMP14, b) both uPA and MMP9 / MMP2 / MMP14, c) all of uPA, legumin, MMP2, MMP14 and MMP9.

[0191] 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: 372-377.

[0192] 15. The activatable IL-18 polypeptide of any one of embodiments 1 to 14, wherein the cleavable linker further comprises a spacer sequence, optionally wherein the cleavable linker comprises two spacer sequences, the two spacer sequences being located on the N-terminal and C-terminal sides of the amino acid sequence recognized and cleaved by one or more proteases.

[0193] 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.

[0194] 17. The activatable IL-18 polypeptide of embodiment 16, wherein at least one of the two or more groups of amino acid sequences recognized and cleaved by one or more proteases is sandwiched by two spacer sequences in the cleavable linker, and wherein each of the two or more groups of amino acid sequences recognized and cleaved by one or more proteases is sandwiched by two spacer sequences in the cleavable linker.

[0195] 18. The activatable IL-18 polypeptide of any one of embodiments 1 to 17, wherein the spacer sequence comprises a GS linker, optionally wherein the GS linker is no more than about 10, 9, 8, 7, 6 or 5 amino acids in length.

[0196] 19. The activatable IL-18 polypeptide of any one of embodiments 1 to 18, wherein the cleavable linker consists of no more than about 50, 40, 35 or 30 amino acids.

[0197] 20. The activatable IL-18 polypeptide of any one of embodiments 1 to 19, wherein the cleavable linker consists of no less than about 10, 15, 20 or 25 amino acids.

[0198] 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, 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.

[0199] 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: 399-407.

[0200] 23. The activatable IL-18 polypeptide of any one of embodiments 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.

[0201] 24. The activatable IL-18 polypeptide of any one of embodiments 1 to 23, wherein the IL-18 polypeptide comprises an IL-18 variant polypeptide.

[0202] 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) relative to wild-type IL-18, or (ii) no binding to IL-18BP.

[0203] 26. The activatable IL-18 polypeptide of embodiment 24 or 25, wherein the IL-18 variant polypeptide exhibits increased binding to IL-18Rα relative to wild-type IL-18.

[0204] 27. The activatable IL-18 polypeptide of any one of embodiments 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, wherein the amino acid positions are relative to wild-type (WT) human IL-18 shown in SEQ ID NO: 1.

[0205] 28. The activatable IL-18 polypeptide of any one of embodiments 24 to 27, wherein the IL-18 variant polypeptide comprises a cysteine ​​at position 117 and a cysteine ​​at position 76, wherein the amino acid positions are relative to wild-type human IL-18 as set forth in SEQ ID NO: 1, optionally wherein 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.

[0206] 29. The activatable IL-18 polypeptide of any one of embodiments 24 to 28, wherein the IL-18 variant polypeptide comprises any one of SEQ ID NOs: 2-299 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.

[0207] 30. The activatable IL-18 polypeptide of any one of embodiments 1 to 29, wherein the IL-18 polypeptide comprises, from N-terminus to C-terminus, a) a masking moiety comprising a truncated propeptide of pro-IL-18 comprising 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, optionally wherein the cleavable linker is no more than about 50, 40, 30, 25, 20, 15, 12, or 10 amino acids in length.

[0208] 31. The activatable IL-18 polypeptide of any one of embodiments 1 to 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, optionally wherein the cleavable linker is about 10-40, 10-30, 20-30, or 25-30 amino acids in length.

[0209] 32. The activatable IL-18 polypeptide of any one of embodiments 1 to 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, optionally wherein the cleavable linker is about 10-40, 10-30, 20-30, or 25-30 amino acids in length.

[0210] 33. The activatable IL-18 polypeptide of 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 b) an amino acid sequence functional variant thereof, said functional variant 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.

[0211] 34. The activatable IL-18 polypeptide of any one of embodiments 1 to 33, wherein the IL-18 polypeptide comprises a fusion protein comprising (1) a wild-type IL-18 or IL-18 variant polypeptide, and (2) a half-life extending domain, optionally wherein the half-life extending domain comprises an albumin binding portion or an antibody Fc domain or a variant thereof, optionally wherein the half-life extending domain is a human IgG Fc domain (e.g., hIgG1 Fc).

[0212] 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 α (IL-18Rα) and exhibits (i) substantially reduced binding to IL-18 binding protein (IL-18BP) relative to wild-type IL-18, or (ii) no binding to IL-18BP.

[0213] 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 relative to wild-type IL-18.

[0214] 37. The activatable IL-18 polypeptide of any one of embodiments 34 to 36, wherein the human IgG1 Fc domain variant of the fusion protein comprises an N297A mutation (EU numbering), optionally wherein the human IgG1 Fc domain variant comprises the amino acid sequence of SEQ ID NO: 371 or 390.

[0215] 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 a variant thereof of the fusion protein.

[0216] 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.

[0217] 40. The activatable IL-18 polypeptide of any one of embodiments 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, 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.

[0218] 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.

[0219] 42. A dimer comprising two activatable IL-18 polypeptides according to any one of claims 1 to 41.

[0220] 43. The dimer of embodiment 42, wherein the dimer is a homodimer.

[0221] 44. The dimer of embodiment 42, wherein the dimer is a heterodimer.

[0222] 45. A nucleic acid encoding the activatable IL-18 polypeptide of any one of embodiments 1 to 41.

[0223] 46. ​​A vector comprising the nucleic acid of embodiment 45.

[0224] 47. A host cell comprising the nucleic acid of embodiment 45 or the vector of embodiment 46.

[0225] 48. A method for producing an activatable IL-18 polypeptide, comprising: (a) culturing the host cell of embodiment 47 under conditions whereby the activatable IL-18 polypeptide is expressed, and (b) recovering the activatable IL-18 polypeptide produced by the host cell.

[0226] 49. The method of embodiment 48, further comprising purifying the activatable IL-18 polypeptide.

[0227] 50. A pharmaceutical composition comprising the activatable IL-18 polypeptide of any one of embodiments 1 to 41, the nucleic acid of embodiment 45, or the vector of embodiment 46.

[0228] 51. A method of treating a disease in a subject, comprising administering to the subject an effective amount of the pharmaceutical composition of embodiment 50.

[0229] 52. The method according to embodiment 51, wherein the disease is cancer.

[0230] 53. A method of activating IL-18 receptor-mediated signaling in an individual, comprising administering to the individual an effective amount of the pharmaceutical composition of embodiment 50.

[0231] 54. A method of stimulating antigen-experienced immune cells in an individual in need thereof, comprising administering to the individual an effective amount of the pharmaceutical composition of embodiment 50.

[0232] 55. The method of embodiment 54, wherein stimulation comprises increasing the activity and / or number of immune cells that have experienced the antigen.

[0233] 56. The method of any one of embodiments 36 to 40, wherein the individual is a human.

[0234] Example

[0235] The following examples are proposed to provide complete disclosure and description about how to make and use the present invention to those of ordinary skill in the art, which are not intended to limit the scope of its invention that the inventor thinks, nor are they intended to represent that the following experiments are all or unique experiments carried out. Efforts have been made to ensure the accuracy of the numerals (such as amounts, temperature, etc.) used, but some experimental errors and deviations should be considered. The following examples are only intended to serve as examples of the application and are therefore not to be considered as limiting the application in any way. The following examples and detailed description are only intended to illustrate, not to limit.

[0236] Example 1: Materials and methods used in Examples 2 to 4

[0237] Expression and purification of recombinant WT hIL-18 and IL-18 variant polypeptides

[0238] Wild-type ("WT") hIL-18, IL18 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 comprising the above were expressed in an E. coli system. Briefly, pET30a plasmids containing codon-optimized sequences encoding WT hIL-18, IL18 variant polypeptides, and activatable hIL-18 polypeptides were transfected into E. coli BL21 (DE3) cells. The transfected E. coli cells were expanded in culture, 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.

[0239] Expression and purification of recombinant hIL-18 in CHO cells

[0240] Fusion polypeptides comprising wild-type ("WT") hIL-18, IL18 variant polypeptides, or activatable IL-18 polypeptides (described in the Examples below) were expressed in a mammalian cell (293F or CHO) system. Briefly, a plasmid containing a signal peptide and a codon-optimized sequence encoding the fusion polypeptides described in these Examples was transfected into CHO cells. The transfected CHO cells were expanded and cultured in culture for 4 days. Next, the cell culture medium containing the secreted fusion polypeptide was collected and purified via Protein A chromatography. Some of the eluate was subjected to further gel filtration or ion exchange steps.

[0241] Evaluation of IL-18 activity by reporter gene assay

[0242] The NFκB-luc / hIL18RαRβ HEK293 cell line stably expressing hIL-18Rα, hIL-18Rβ, and the NFκB luciferase reporter gene was used as a reporter cell 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) to activate hIL-18 receptor-mediated signaling.

[0243] Briefly, reporter cells were first seeded into the wells of a white 96-well plate with a white bottom at a density of 3.0 × 10 4 IL-18 was serially diluted in culture medium in the presence or absence of 1 μg / mL hIL-18BP-hFc (a) and (b). Luminescence intensity was measured after incubation at 37°C for 16 hours.

[0244] Human PBMC-based hIFNγ release assay

[0245] A PBMC-based assay was performed to test the ability of wild-type IL-18, activatable IL-18 peptides (before treatment with tumor microenvironment proteases), and activatable IL-18 peptides (after treatment with tumor microenvironment proteases) to activate T / NK cells and induce hIFNγ release. Briefly, human PBMCs were seeded into wells of a 96-well plate at a density of 5.0 × 10 5 Cells were added to each well and cultured for 5 hours before treatment. hIL-18 was serially diluted with 1 ng / mL human IL-12 in the presence or absence of (a) 1 μg / mL hIL-18BP-hFc and then added to the wells. After incubation at 37°C for 16 hours, the culture medium was collected from each well and hIFNγ concentration was measured via FRET using a kit (62HIFNGPEG, Cisbio) according to the manufacturer's instructions.

[0246] Mouse splenocyte-based mIFNγ release assay

[0247] The mouse splenocyte-based murine IFNγ ("mIFNγ") release assay was performed as follows. Briefly, freshly isolated mouse splenocytes were seeded into wells of a 96-well plate at a density of 6.5 × 10 5 WT hIL-18 or IL-18 variant polypeptides were serially diluted with 1 ng / mL mouse IL-12 in the presence or absence of (a) 1 μg / mL mouse IL-18BP-Fc and then added to the wells. After incubation at 37°C for 16 hours, the culture medium from each well was collected and the concentration of mIFNγ in the culture medium was measured via FRET using a kit (62MIFNGPEG, Cisbio) according to the manufacturer's instructions.

[0248] Digestion of activatable IL-18 peptides

[0249] The activatable IL-18 polypeptides described in Examples 2 and 3 were cleaved by caspase-1 or tumor microenvironment ("TME") proteases (i.e., matrix metalloproteinases MMP2, MMP9, MMP10, and MMP14) to assess the efficiency of cleavage and the activity of the resulting "cleaved" forms of the activatable IL-18 polypeptides. For caspase-1 cleavage reactions, the activatable IL-18 polypeptides were incubated with preactivated caspase-1 (Abcam catalog #ab39901) at a ratio of 100 to 500 μg protein to 1 unit of caspase at room temperature for 1 to 16 hours. For MMP2, MMP9, MMP10, and MMP14 cleavage reactions, the activatable IL-18 polypeptides were incubated with preactivated MMP2, MMP9, MMP10, or MMP14 at a weight ratio of 100:1 to 500:1 (μg polypeptide to μg protease) at room temperature or 37°C for 1 to 16 hours. MMP2, MMP9, MMP10 and MMP14 cleavage reactions were stopped by placing samples on ice or storing at -80°C.

[0250] Example 2: Activatable IL-18 polypeptides comprising a masking moiety derived from the native propeptide of proIL-18

[0251] The IL-18 precursor (or "proIL18") remains inactive in the cytosol until a signal (such as inflammasome activation) induces maturation. The inflammasome is a multiprotein assembly composed of three proteins (nucleotide-binding oligomerization domain (NOD)-like receptor, apoptosis-associated speck-like protein (ASC) containing a CARD, and caspase-1) that forms when pathogens or other harmful substances (such as reactive oxygen species and urate crystals) are detected in the cytosol. The inflammasome then catalyzes the activation of the cysteine ​​protease caspase-1, which removes the propeptide from the IL-18 precursor to produce mature IL-18. The native propeptide of human proIL-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 comprising the same 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 to 36 of SEQ ID NO: 333) was replaced with a protease recognition sequence for MMP-2, MMP-9, MMP-10, MMP-14, legumin, or other tumor microenvironment ("TME")-specific proteases, or with a combination of one or more of the foregoing recognition sequences.

[0252] 2.1 Proof of concept: The native propeptide of human proIL-18 blocks the binding of mature WT human IL-18 to IL-18Ra and activates IL-18 receptor-mediated signaling

[0253] As discussed above with respect to recombinant WT IL-18 (i.e., "mature" WT IL-18), full-length human proIL-18 (SEQ ID NO: 335) was produced in Escherichia coli. The ability of proIL-18 to bind to hIL-18Rα was assessed via biofilm interferometry (BLI), and the ability of proIL-18 to activate IL-18 receptor-mediated signaling was assessed using the reporter gene assay of Example 1. Signaling assay experiments were performed using caspase-1-treated proIL-18. (Caspase-1-treated proIL-18 is also referred to herein as "cleaved proIL-18." Cleaved proIL-18 has the same amino acid sequence as mature WT IL-18 (i.e., SEQ ID NO: 1). Figure 1AAs shown, proIL-18 did not exhibit detectable binding to hIL-18Rα under the experimental conditions, i.e., where the concentration of proIL-18 was 32 nM. proIL-18 was also unable to activate hIL-18 receptor-mediated signaling. Figure 1B In contrast, cleaved proIL-18 activated hIL-18 receptor-mediated signaling to the same extent as recombinant WT IL-18. Figure 1B In the presence of 1 μg / ml human IL-18 binding protein ("BP"), the signaling activity of cleaved proIL-18 was inhibited. Figure 1B .

[0254] 2.2 Design, Production, and Characterization of Exemplary Cleavable Linkers

[0255] Activatable IL-18 polypeptides were designed, each comprising amino acids 1 to 26 of the native propeptide of proIL-18 (i.e., MAAEPVEDNCINFVAMKFIDNTLYFI, SEQ ID NO: 336) from the N-terminus to the C-terminus, an amino acid linker sequence comprising a protease recognition site for caspase-1 or TME protease, and WT hIL-18. See Table 1.

[0256] Table 1. Masked WT hIL-18 polypeptides

[0257]

[0258]

[0259] ProIL18, ProIL18-MMP9L, ProIL18-MMP10L, ProIL18-MMP2L, and ProIL18-Legu L were expressed and purified. ProIL18-MMP2 / MMP14L was poorly purified. ProIL18, ProIL18-MMP9L, ProIL18-MMP10L, and ProIL18-Legu L were well expressed in E. coli and purified by SEC to >95% purity as determined by SDS PAGE and SEC (data not shown).

[0260] The hIL-18 reporter cell assay described in Example 1 was used to assess the potency of ProIL18-MMP9L, ProIL18-MMP10L, and ProIL18-Legu L in activating hIL-18 receptor-mediated signaling. Figures 2A to 2CAs shown, ProIL18-MMP9L, ProIL18-MMP10L and ProIL18-Legu L were found to be approximately 1000-fold less potent than WT hIL-18 (ie, SEQ ID NO: 1), with Cmax even reduced to 100 nM.

[0261] The potency of ProIL18-MMP9L and ProIL18-Legu L to activate hIL-18 receptor-mediated signaling was partially restored after treatment with MMP9 and legumin, respectively. Figure 2A and Figure 2C The potency of "cleaved" ProIL18-MMP9L (ie, after MMP9 treatment) (EC50: 2.46 nM) was approximately 16-fold lower than that of recombinant WT hIL-18 (EC50: 0.16 nM). Figure 2A .

[0262] Example 3: Design and Characterization of Exemplary IL-18 Variant Polypeptides Comprising a Masking Moiety Derived from the proIL-18 Propeptide and an MMP9 Cleavable Linker

[0263] Constructs comprising a masking moiety of SEQ ID NO: 344 comprising amino acids 1 to 26 of the native propeptide of proIL-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-termini of the IL-18 variant polypeptides M12 and MM5 to generate ProM12 (SEQ ID NO: 345) and ProMM5 (SEQ ID NO: 346).

[0264] ProM12

[0265] MAAEPVEDNCINFVAMKFIDNTLYFISGGPGPAGMKGLPGSYFPKLKSKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKGSGARGMAVTISVKCEKISTLSCENKIISFKEMNPPDTIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED

[0266] ProMM5

[0267] MAAEPVEDNCINFVAMKFIDNTLYFISGGPGPAGMKGLPGSYFGKLGSKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKLSTERGMAVTISVKCEKISTLSCENKIISFKEMNPPDGIKDTKSDIIFFQSSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED

[0268] Then, ProM12 and ProMM5 were expressed in Escherichia coli, harvested and purified to >95% purity as determined by SDS PAGE and SEC. Next, ProM12 and ProMM5 were treated with MMP9 (as described in Example 1). The cleavage efficiency of ProM12 and ProMM5 by MMP9 was evaluated by HPLC-SEC. In short, Figure 3A As shown, the retention time of ProM12 is shorter than that of M12 without a masking moiety. The retention time of ProM12 treated with MMP9 (i.e., "ProM12 Cut") is between ProM12 and M12. Similar results were observed for ProMM5, MM5, and ProMM5 treated with MMP9 (i.e., "ProMM5 Cut"). Figure 3B . Figure 3A and Figure 3B The results indicate that ProM12 and ProMM5 are completely cleaved by MMP9 (i.e., the masking moiety is removed). Without being bound by theory, the molecular weight differences between, for example, M12 and "ProM12Cut" and, for example, MM5 and "ProMM5Cut" may be due to amino acid residues in the MMP9 linker that remain attached to the N-terminus of M12 or MM5 after MMP9 treatment.

[0269] The hIL-18 reporter cell assay described in Example 1 was used to assess the potency of activatable IL-18 variant polypeptides to activate hIL-18 receptor-mediated signaling. The following groups of polypeptides were tested: (1) ProM12 (SEQ ID NO: 345), M12, and ProM12 Cut (i.e., MMP9-treated ProM12); (2) ProMM5 (SEQ ID NO: 346), MM5, and ProMM5 Cut (i.e., MMP9-treated ProMM5); (3) ProM21 (SEQ ID NO: 347), M21, and ProM21 Cut (i.e., MMP9-treated ProM21); (4) ProM13 (SEQ ID NO: 348), M13, and ProM13 Cut (i.e., MMP9-treated ProM13); (5) ProM24 (SEQ ID NO: 349), M24, and ProM24 Cut (i.e., MMP9-treated ProM24); (6) ProWM4 (SEQ ID NO: 350), WM4, and ProWM4 Cut (i.e., MMP9-treated WM4); (7) ProWM5 (SEQ ID NO: 351), WM5, and ProWM5 Cut (i.e., MMP9-treated WM5); NO:351), WM5 and ProWM5 Cut (i.e., MMP9-processed WM5); and (8) ProWM6 (SEQ ID NO:352), WM6 and ProWM6 Cut (i.e., MMP9-processed WM6). Figure 3C-3J As shown, all tested activatable IL-18 polypeptides had greatly reduced potency compared to the corresponding "unmasked" IL-18 variant polypeptides, with Cmax reduced to 100 nM. Figure 3C-3J Among the activatable IL-18 peptides shown, ProWM4, ProWM5, and ProWM6 showed minimal hIL-18 receptor-mediated signaling at 100 nM. Figure 3H-3J ProM12 Cut, ProMM5 Cut, ProM21 Cut, ProM13 Cut, ProM24 Cut, ProWM4 Cut, ProWM5 Cut, and ProWM6 Cut showed restored hIL-18 receptor-mediated signaling. ProM12 Cut, ProM21 Cut, and ProM13 Cut showed almost complete restoration of signaling. Figure 3C 、 Figure 3E and Figure 3F and Table 2. ProMM5 Cut, ProM24 Cut, ProWM4 Cut, ProWM5 Cut, and ProWM6 Cut showed only partial restoration of hIL-18 receptor-mediated signaling. Figure 3D 、 Figure 3G 、 Figure 3H 、 Figure 3I and Figure 3J and Table 2. Table 2. Masking efficacy of the MMP9 masking moiety on ProM12, ProMM5, ProM21, ProM13, ProM24, ProWM4, ProWM5, and ProWM6 before and after MMP9 treatment

[0270]

[0271] The effect of BP on hIL-18 receptor-mediated signaling of ProM12 Cut and ProMM5 Cut was also evaluated. Figure 3C and Figure 3D As shown, ProM12 Cut and ProMM5 Cut retained BP resistance and exhibited similar signaling activity in the presence and absence of 1 μg / mL human IL-18 binding protein ("BP").

[0272] To confirm that the MMP9 recognition linker-fused masking moiety MAAEPVEDNCINFVAMKFIDNTLYFISGGPGPAGMKGLPG (SEQ ID NO: 344) is specifically cleaved by MMP9 but not by caspase-1, ProM12 was treated with MMP9 or caspase-1 in vitro and evaluated in the hIL-18 reporter cell assay described in Example 1. Figure 3K As shown, ProM12 Cut after MMP9 treatment showed restored signaling activity, while ProM12 Cut after caspase-1 treatment showed signaling activity comparable to that of untreated ProM12. These results indicate that the masking portion (SEQ ID NO: 344) fused to the MMP9 recognition linker is no longer susceptible to cleavage by caspase-1.

[0273] Next, a PBMC-based assay was performed to test the ability of ProM12 and ProMM5 (ie, before and after treatment with MMP9) to induce hIFNγ release. Figure 4A and Figure 4B showed that WT hIL-18 could induce the release of human IFNγ in the presence of IL-12. 50 The concentration of ProM12 Cut and ProMM5 Cut was about 0.1 nM, while ProM12 and ProMM5 were unable to induce human IFNγ release even at 10 nM. After treatment with MMP9, ProM12 Cut and ProMM5 Cut both showed hIFNγ release activity. Figure 4A and Figure 4BProM12 Cut showed similar activity to WT hIL-18 or M12, while unmasked ProMM5 (ProMM5 Cut) showed approximately 7-fold lower activity than WT hIL-18. See Table 3 below.

[0274] Table 3. EC50 of masked and unmasked IL-18 variant polypeptides in PBMC assay

[0275]

[0276] Example 4: Activatable IL-18 Polypeptides Comprising Masking Moieties Derived from IL-18Rα, IL-18Rα Subdomains, IL-18Rβ, IL-18BP, and IL-18BP Variants

[0277] 4.1 Design and Characterization of Activatable IL-18 Polypeptides Comprising Masking Moieties Derived from IL-18Rα, IL-18Rα Subdomains, IL-18Rβ, IL-18BP, and IL-18BP Variants

[0278] Other masking moieties derived from IL-18Rα, IL-18Rα subdomains, IL-18Rβ, IL-18BP, and IL-18BP variants were designed as follows:

[0279] IL-18Ra (also referred to herein as "18Ra") - the extracellular domain of IL-18Ra (AA 20-318)

[0280]

[0281] IL-18Rα D12 (also referred to herein as "D12") - the first two domains of IL-18Rα (AA 20-208)

[0282]

[0283] IL-18Ra D3 (also referred to herein as "D3") - the third domain of IL-18Ra (AA 210-318)

[0284]

[0285] IL-18BPm (also referred to herein as "BPm") - a modified form of IL-18BP that has been shown to bind to and inhibit IL-18BP Ability to inhibit the activity of IL18 variant polypeptides

[0286]

[0287] Each of the masking moieties shown above was 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 generate an activatable M12-DB6-Fc_N297A polypeptide, the sequence of which is shown below:

[0288] Ra-M12-DB6-Fc_N297A

[0289] IL-18Ra sequence with Underline , the cleavable linker is in italics, the M12-DB6 sequence is in bold, and the Fc-N297A sequence is in plain text.

[0290]

[0291]

[0292] D12-M12-DB6-Fc_N297A

[0293] IL-18Ra D12 sequence carries Underline , the cleavable linker is in italics, the M12-DB6 sequence is in bold, and the Fc-N297A sequence is in plain text.

[0294]

[0295]

[0296]

[0297] D3-M12-DB6-Fc_N297A

[0298] IL-18Ra D3 sequence carries Underline , the cleavable linker is in italics, the M12-DB6 sequence is in bold, and the Fc-N297A sequence is in plain text.

[0299]

[0300]

[0301] BPm-M12-DB6-Fc_N297A

[0302] IL-18BPm sequence with Underline , the cleavable linker is in italics, the M12-DB6 sequence is in bold, and the Fc-N297A sequence is in plain text.

[0303]

[0304]

[0305] M12-DB6-D3-Fc_N297A

[0306] IL-18Ra D3 sequence carries Underline , the cleavable linker is in italics, the M12-DB6 sequence is in bold, and the Fc-N297A sequence is in plain text.

[0307]

[0308]

[0309]

[0310] Fc_N29A-D3-M12-DB6

[0311] IL-18Ra D3 sequence carries Underline , the cleavable linker is in italics, the M12-DB6 sequence is in bold, and the Fc-N297A sequence is in plain text. The additional spacer sequence (GGGGSGGGGSGGGGS, SEQ ID NO: 363) is represented as .

[0312]

[0313] Fc_N297A-M12-DB6-D3

[0314] IL-18Ra D3 sequence carries Underline , the cleavable linker is in italics, the M12-DB6 sequence is in bold, and the Fc-N297A sequence is in plain text. The additional linker sequence (GGGGSGGGGSGSGGG, SEQ ID NO: 365) is represented as .

[0315]

[0316] 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 by Protein A chromatography. After one-step Protein A purification, the purity of these activatable IL-18 polypeptides was determined to be 74% to 96% by SEC-HPLC and approximately 80% to 95% by SDS-PAGE (data not shown).

[0317] 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 before and after MMP9 treatment was assessed 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 also assessed.

[0318] like Figures 5A-5CAs shown in Table 4 below, the IL-18Ra, IL-18RaD12, and IL-BPm masking moieties all 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 by 100 to 1790 fold compared to M12-DB6-Fc_N297A). The IL-18Ra masking moiety exhibited the highest masking ability (see Figure 5A and Table 4). After MMP9 treatment, Ra-M12-DB6-Fc_N297ACut, D12-M12-DB6-Fc_N297A Cut, and BPm-M12-DB6-Fc_N297A Cut exhibited comparable signaling activity to that of M12-DB6-Fc_N297A (see Figures 5A-5C and Table 4), where the EC50 values ​​varied within 3-fold (see Table 4). Table 4. Efficacy of IL-18Rα, IL-18RαD12, and IL-18BP masking moieties in inhibiting hIL-18 receptor-mediated signaling of M12-DB6-Fc_N297A

[0319]

[0320] 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) to generate an activatable MM5-DB6-Fc_N297A polypeptide, the sequence of which is shown below:

[0321] Ra-MM5-DB6-Fc_N297A

[0322] IL-18Ra sequence with Underline , the cleavable linker is in italics, the MM5-DB6 sequence is in bold, and the Fc-N297A sequence is in plain text.

[0323]

[0324]

[0325] D12-MM5-DB6-Fc_N297A

[0326] IL-18Ra D12 sequence carries Underline , the cleavable linker is in italics, the MM5-DB6 sequence is in bold, and the Fc-N297A sequence is in plain text.

[0327]

[0328]

[0329]

[0330] MM5-DB6-Ra-Fc_N297A

[0331] IL-18Ra sequence with Underline , the cleavable linker is in italics, the MM5-DB6 sequence is in bold, and the Fc-N297A sequence is in plain text.

[0332]

[0333]

[0334]

[0335] Fc_N297A-Ra-MM5-DB6

[0336] IL-18Ra sequence with Underline , the cleavable linker is in italics, the MM5-DB6 sequence is in bold, and the Fc-N297A sequence is pure The additional linker sequence GGGGSGGGGSGGGGS (SEQ ID NO: 363) is represented as .

[0337]

[0338] Fc_N297A-MM5-DB6-Ra

[0339] IL-18Ra sequence with Underline , the cleavable linker is in italics, the MM5-DB6 sequence is in bold, and the Fc-N297A sequence is in plain text. The additional linker sequence (GGGGSGGGGSGSGGG, SEQ ID NO: 365) is represented as .

[0340]

[0341]

[0342] The potency of Ra-MM5-DB6-Fc_N297A, D12-MM5-DB6-Fc_N297A, Fc_N297A-Ra-MM5-DB6, and Fc_N297A-MM5-DB6-Ra in activating hIL-18 receptor-mediated signaling before and after MMP9 treatment was assessed 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 also assessed.

[0343] like Figures 6A-6D As shown in Table 5 below, both IL-18Ra and IL-18Ra D12 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 >1000-fold compared to MM5-DB6-Fc_N297A). Regardless of whether the masking moiety was fused to the N-terminus or C-terminus of MM5-DB6, IL-18Ra was able to mask MM5-DB6 and inhibit hIL-18 receptor-mediated signaling. After MMP9 treatment, Ra-MM5-DB6-Fc_N297A Cut, D12-MM5-DB6-Fc_N297A Cut, Fc_N297A-Ra-MM5-DB6 Cut, and Fc_N297A-MM5-DB6-Ra Cut exhibited slightly lower signaling activity than MM5-DB6-Fc_N297A or Fc_N297A-MM5-DB6 (see Figures 6A-6D and Table 5), where the EC50 values ​​varied within 15-fold (see Table 5).

[0344] Table 5. Efficacy of IL-18Rα and IL-18RαD12 in inhibiting hIL-18 receptor-mediated signaling of MM5-DB6-Fc_N297A and Fc_N297A-MM5-DB6

[0345]

[0346]

[0347] Example 5A: Activatable IL-18 polypeptides comprising a masking moiety derived from IL-18Rα with variable linker lengths

[0348] For masked IL-18, based on the simulated structure, the distance between the C-terminus of IL-18 and the N-terminus of IL-18Ra in the Fc-IL18-Ra format is predicted to be 34 nm. A 15-AA linker may lead to conformational stretching of the IL-18 and Ra interface. Longer linkers of 25 to 30 AA were tested.

[0349] Specifically, as previously described, IL-18Ra masked Fc-M12-DB6 was produced in CHO cells with variable lengths of cleavable sites (M9 = 15AA, M91 = 30AA, M92 = 25AA). Figure 7 As shown, C-terminally fused IL-18Ra reduced the activity of Fc-M12-DB6 when linked to linkers of all three different lengths, while Fc-M12-DB6-Ra-M9 with a 15 AA linker showed lower activity than 30 AA or 25 AA.

[0350] After in vitro cleavage by the protease MMP9, all three Ra-masked Fc-M12-DB6s showed significant recovery of activity, while cleaved Fc-M12-DB6-Ra-M91 and Fc-M12-DB6-Ra-M92 showed comparable activity to unmasked Fc-M12-DB6, and MMP9-cleaved Fc-M12-DB6-Ra-M9 showed partial recovery of activity. These data confirm that cleavable linkers with lengths of 25 to 30 amino acids have more optimal activity recovery after protease treatment.

[0351] Example 5B: Activatable IL-18 polypeptides comprising a masking moiety derived from IL-18Rα with different cleavable linker combinations

[0352] Ra-masked Fc-M12-DB6 with different cleavable linker combinations were further designed and studied to increase the probability of cleavage in the TME. MMP protease recognition substrates were fused to uPA substrate sequences, legumin cleavable linkers, or both to generate UM (uPA+MMP), ULM (uPA+legumin+MMP), and LM (legumin+MMP) linkers. Figure 8A and Figure 8B As shown, for Fc-M12-DB6-Ra, all six groups of attempted combination linkers showed reduced activity comparable to that of Fc-M12-DB6-Ra-M92. In addition, after in vitro MMP9 treatment, the six masked Fc-M12-DB6 showed significantly restored activity. Figure 8A and Figure 8B The data demonstrate that a cleavable linker between IL-18 and the masking moiety can be designed to be sensitive to a variety of proteases without compromising masking efficacy or activity recovery.

[0353] We further designed another multi-protease recognition linker named UM-2 to connect IL-18 to the masking part. Both Fc-M12-DB6-Ra-UM2 and Fc-MM5-DB6-Ra-UM2 were produced by CHO cells. Compared with the corresponding unmasked molecules, the masked Fc-M12-DB6-Ra and Fc-MM5-DB6-Ra fused with the UM-2 linker showed significantly reduced activity. In addition to MMP cleavage, MMP-14 and uPA treatment were also performed to remove the masking part Ra. The results showed that all three proteases we tested could effectively remove the masking part and restore the activity of the IL-18 variant ( Figure 9A 、 Figure 9B ).

[0354] In summary, for masked IL-18 or masked IL-18 variants, cleavable linkers with a length of 15 to 30 AA and cleavable linkers with multiple protease recognition sequences effectively maintain the masking effect of the masking moiety. In addition, the appropriate linker length enables good exposure of the protease recognition sequence, thereby achieving efficient enzymatic cleavage and effectively restoring the activity of IL-18 or IL-18 variants.

[0355] Example 6A.

[0356] IL-18 variant polypeptides have been constructed to hIL-18Rα. The binding affinities of WT hIL-18 and IL-18 variant polypeptides to hIL-18RαECD-Fc are shown in Table 6A.

[0357] Table 6A. Affinity of IL-18 variant polypeptides for hIL-18Rα (determined by BLI)

[0358]

[0359]

[0360] Mutations of these IL-18 variant polypeptides (i.e., M1-M41, LM1-LM5, MM1-MM2, MM4-MM6, WM1-WM6, WM8, WM10-WM14, corresponding to SEQ ID NOs: 1-4, 8-9, 11-12, 17, 21-22, 26-28, 35, 38-39, 41, 43-44, 49-50, 54-56, 58-59, 65-66, 72-74, 81, 91, 94, 99, 104-105, 114, 116-117, 121-125, 131-134, 139-145, 147, 149-153) are shown in Table 6B.

[0361] Table 6B

[0362]

[0363]

[0364] Example 6B: Functional Characterization of IL-18 Variant Polypeptides

[0365] 6.1 Activation of hIL-18 Receptor-Mediated Signaling by IL-18 Variant Peptides

[0366] The ability of IL-18 variant polypeptides to activate hIL-18 receptor-mediated signaling was determined using the NFκB-luc / hIL-18RαRβ HEK293 cell line stably expressing hIL-18Rα, hIL-18Rβ, and the NFκB luciferase reporter gene as reporter cells.

[0367] Briefly, reporter cells were first seeded into the wells of a white 96-well plate at a density of 3.0 × 10 4 WT hIL-18 or IL-18 variant polypeptides were serially diluted in culture medium in the presence or absence of 1 μg / mL hIL-18BP-hFc (a) and (b). Luminescence intensity was measured after incubation at 37°C for 16 hours.

[0368] As shown in Table 7, all tested IL-18 variant polypeptides exhibited potency to activate IL-18 receptor-mediated signaling in a dose-dependent manner. Unlike WT hIL-18, whose activity was greatly reduced (approximately 80-fold) in the presence of 1 μg / mL hIL-18BP-hFc, the IL-18 variant polypeptides (i.e., M1-M7, M9-M41, LM1-LM5, MM1-MM2, MM4-MM6, WM1-WM6, WM8, WM10-11, corresponding to SEQ ID NO: 2-4, 8-9, 11-12, 17, 21, 22, 26-28, 35, 38, 39, 41, 43, 44, 49, 50, 54-56, 58-59, 65-66, 72-74, 81, 91, 94, 99, 104-105, 114, 116-117, 121-125, 131-134, 139-145, 147, 149-150) showed Hil-18 BP resistance activity, among which EC 50 The change in the expression of IL-18 variant polypeptides was less than 3-fold, indicating that the IL-18 variant polypeptides were significantly less affected by IL-18BP in terms of IL-18 receptor-mediated signaling activation. WM6 was an exception, and its EC 50showed an approximately 12-fold loss in potency but was still much superior to WT hIL-18.

[0369] Table 7. EC values ​​of IL-18 variant polypeptides in the hIL-18 reporter gene assay in the presence or absence of 1 μg / mL hIL-18BP-hFc 50

[0370]

[0371]

[0372] 6.2 Human PBMC-based hIFNγ release assay

[0373] A PBMC-based assay was performed to test the ability of IL-18 variant polypeptides to activate T / NK cells and induce hIFNγ release. Briefly, human PBMC cells were seeded into wells of a 96-well plate at a density of 5.0 × 10 5 Cells were added to each well and cultured for 5 hours before treatment. WT hIL-18 or IL-18 variant polypeptides were serially diluted with 1 ng / mL human IL-12 in the presence or absence of (a) 1 μg / mL hIL-18BP-hFc and then added to the wells. After incubation at 37°C for 16 hours, culture medium was collected from each well and hIFNγ concentration was measured via FRET using a kit (62HIFNGPEG, Cisbio) according to the manufacturer's instructions.

[0374] The tested IL-18 variant polypeptides showed the efficacy of inducing hIFNγ release from PBMC 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 hIL-18BP-hFc, where EC 50 In contrast, IL-18 variant polypeptides (i.e., M11-M13, M15, M17, M21, M24, M29, M32-M33, M35-M36, MM5, WM4-WM6, WM8, WM10-11, corresponding to SEQ ID NOs: 3, 9, 17, 27, 39, 43, 49, 94, 105, 116-117, 133, 143-150) exhibited hIL-18BP anti-tumor activity, wherein EC 50 The change in α was no more than 2-fold, indicating that the IL-18 variant polypeptide was significantly less affected by hIL-18BP in terms of hIFNγ induction.

[0375] Table 8. EC values ​​of IL-18 variant polypeptides in the PBMC assay in the presence or absence of 1 μg / mL hIL-18BP 50

[0376]

[0377] 6.3 mIFNγ Release Assay Based on Mouse Splenocytes

[0378] To evaluate the in vivo efficacy of the IL-18 variant polypeptides of the present application, a mouse splenocyte-based murine IFNγ ("mIFNγ") release assay was performed as follows.

[0379] Briefly, freshly isolated mouse splenocytes were seeded into wells of a 96-well plate at a density of 6.5 × 10 5 WT hIL-18 or IL-18 variant polypeptides were serially diluted with 1 ng / mL mouse IL-12 in the presence or absence of (a) 1 μg / mL mouse IL-18BP-Fc and then added to the wells. After incubation at 37°C for 16 hours, the culture medium from each well was collected and the concentration of mIFNγ in the culture medium was measured via FRET using a kit (62MIFNGPEG, Cisbio) according to the manufacturer's instructions.

[0380] Example 7: Generation and Characterization of IL-18 Variant Polypeptides Comprising Serine→Cysteine ​​and / or Cysteine→Serine Substitutions

[0381] 7.1 Wild-type IL-18 or IL-18 polypeptide variants containing a cysteine→serine substitution

[0382] Although the powerful immunostimulatory activity of IL-18 has been reported, recombinant IL-18 is mainly expressed in the Escherichia coli system, which is not suitable for the production standards of good manufacturing practice (GMP), which mainly uses mammalian host cells. Human IL-18 has four unpaired cysteines and does not contain disulfide bonds. Among the four cysteines, C38, C68 and C76 are highly exposed to the solvent, while C127 is partially exposed. During expression, due to the exposed free cysteine, IL-18 is prone to form undesirable aggregates, resulting in reduced yield and / or purity. In order to improve the correct folding and stability of IL-18, that is, with higher yield and qualified purity as the goal, we engineered IL-18 variants by using cysteine ​​mutation strategy and disulfide bond introduction strategy. The following experiments were performed to evaluate the effect of Cys mutation to Ser ("C→S") on the drugability 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 are modified (or further modified) to include C38S, C68S, and C76S substitutions, thereby generating novel variants comprising the following amino acid sequences:

[0383] IL-18 variant comprising C38S, C68S, and C76S substitutions in the context of WT hIL-18 ("IL-18-SSS"):

[0384]

[0385] IL-18 variant comprising C38S, C68S, and C76S substitutions in an M12 background ("M12-SSS"):

[0386]

[0387] IL-18 variant comprising C38S, C68S and C76S substitutions in the MM5 background ("MM5-SSS"):

[0388]

[0389] Fc fusion cytokines can improve pharmacokinetics and are suitable for a wider range of production processes. Next, the C-termini of IL-18-SSS, M12-SSS, and M5-SSS were 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 by Protein A chromatography. The yield of IL-18-SSS-Fc_N297A obtained after one-step Protein A purification was 14.7 mg / L with a purity of 65%. In comparison, 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%. After a one-step Protein A purification, M12-SSS-Fc_N297A produced a yield of 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. After a one-step Protein A chromatography, MM5-SSS-Fc_N297A produced a yield of 24.5 mg / L with a purity of 46%. After a further purification step by gel filtration, MM5-SSS-Fc_N297 produced a yield of 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.

[0390] M12-SSS was further modified to include a C127S substitution to generate M12-SSSS:

[0391]

[0392]

[0393] The C-terminus of M12-SSSS was fused to the N-terminus of a human IgG1 Fc variant comprising an N297A substitution (EU numbering) to generate M12-SSSS-Fc_N297A (SEQ ID NO: 322). M12-SSSS-Fc_N297A was expressed in CHO cells, harvested, and purified via Protein A chromatography. The introduction of an additional C127S substitution (i.e., the fourth C→S substitution) resulted in higher expression yields and purity than M12-Fc_N297A, however, it reduced the yield and purity of M12-SSSS-Fc_N297A compared to those of M12-SSS-Fc_N297A (data not shown), suggesting that the C127 mutation may have compromised the conformational advantages of the first three cysteine ​​mutations.

[0394] Next, the in vitro assay described in Example 6B, Section 6.1, was used to characterize the effectiveness of IL-18-SSS-Fc_N297A and M12-SSS-Fc_N297A in activating IL-18 receptor-mediated signaling. The effectiveness of IL-18-SSS-Fc_N297A was comparable to that of wild-type IL-18, while M12-SSS-Fc_N297A showed approximately 7-fold loss of effectiveness compared to M12. The signaling activity of IL-18-SSS-Fc_N297A was reduced in the presence of 1 μg / ml hIL-18BP-hFc ("BP"), while M12-SSS-Fc_N297A exhibited hIL-18BP-hFc resistance activity in the presence of 1 μg / ml hIL-18BP-hFc. MM5-SSS-Fc_N297A showed a potency loss of approximately 220-fold compared to MM5, but exhibited hIL-18BP resistance activity in the presence of 1 μg / ml hIL-18BP-hFc. These results indicate that the three cysteine ​​→ serine substitutions do not affect the IL-18BP resistance characteristics. These data indicate that mutations at positions C38, C68, and C76 prevent the three exposed cysteines from forming undesirable intramolecular and intermolecular disulfide bonds and are crucial for improving the yield and purity of IL-18 and IL-18 polypeptide-Fc fusion proteins. In addition, Fc-fused IL-18 and IL-18 variants are expected to exhibit longer half-lives.

[0395] 7.2 IL-18 polypeptides or IL-18 polypeptide variants comprising amino acid substitutions that remove and / or introduce cysteine ​​residues

[0396] The following experiments were performed to evaluate the effects of removing intrinsic cysteine ​​(i.e., naturally occurring cysteine ​​in the polypeptide sequence) and / or introducing disulfide bond substitutions on the drugability, expression, and purification of IL-18 variant polypeptides. M12 was used as a representative variant for engineering strategy selection. First, computer screening was performed based on molecular dynamics simulations, followed by stability assessment based on artificial intelligence, and mutation hotspots on each of the four cysteines (C38, C68, C76, and C127) of M12 were determined and listed separately. See Table 5. In addition, structure-guided design was used to determine the amino acids in the spatial position that, when replaced by Cys residues, can form new non-natural disulfide bonds. The mutation strategies shown in Table 5 (i.e., DB6, DB7, DB8, DB9, and DB10) were proposed. It was assumed that introducing the S117C mutation while retaining C76 could promote the disulfide bond between C117 and C76.

[0397] Table 9. IL-18 polypeptide variants derived from M12

[0398]

[0399]

[0400] The mutation strategy outlined in Table 9 was applied to the IL-18 polypeptide variant M12 to generate the new variants M12-DB6, M12-DB7, M12-DB8, M12-DB9, and M12-DB10. Mutation hotspots at individual sites were identified through in silico screening. This in silico screening, based on molecular dynamics simulations and artificial intelligence-based stability assessments, identified mutation hotspots at each of the four cysteines (C38, C68, C76, and C127) of M12 and are listed separately.

[0401] M12-DB6

[0402]

[0403] M12-DB7

[0404]

[0405] M12-DB8

[0406]

[0407] M12-DB9

[0408]

[0409] M12-DB10

[0410]

[0411] The C-terminal ends of M12-DB6, M12-DB7, M12-DB8, M12-DB9, and M12-DB10 variants were fused to human IgG1 Fc variants comprising N297A substitutions (EU numbering). The resulting fusion polypeptides (i.e., M12-DB6-Fc_N297A (SEQ ID NO: 323), M12-DB7-Fc_N297A (SEQ ID NO: 324), M12-DB8-Fc_N297A (SEQ ID NO: 325), M12-DB9-Fc_N297A (SEQ ID NO: 326), and M12-DB10-Fc_N297A (SEQ ID NO: 327)) were expressed in CHO cells, harvested, and purified via one-step Protein A chromatography. The purified preparations were analyzed via SEC and SDS PAGE (data not shown). See Table 10 below.

[0412] Table 10. Expression and purification of M12-DB6, M12-DB7, M12-DB8, M12-DB9 and M12-DB10 Fc_N297A fusion proteins

[0413]

[0414] “ / ” indicates no detectable purified protein.

[0415] 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 compared to M12-Fc_N297A or M12-SSS-Fc_N297A (see Table 6). Among M12-DB6-Fc_N297A, M12-DB7-Fc_N297A, M12-DB9-Fc_N297A, and M12-DB10-Fc_N297A, M12-DB6-Fc_N297A showed the highest expression yield (313 mg / L) after one-step Protein A purification, with a purity of 89.8% (determined by SEC-HPLC). M12-DB8-Fc_N297A was not expressed at a detectable level. 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.

[0416] The high yield and purity of DB6 and DB7, particularly the high yield, are remarkable. The M12-DB6 form is approximately 60 times purer than the SSS form. These data suggest that the strategy of removing cysteines at positions 38 and 68 while simultaneously introducing C117 to promote a disulfide bond between C117 and C76 is highly advantageous.

[0417] The low yield of DB7 and the undetectable yield of DB8 suggest that the C127X substitution may not further improve peptide production in mammalian cells. Mutations at the partially exposed position C127 require careful design. Based on the C38X+C68X+C76+S117C strategy, position 127 favors C over A, and the hydrophobic 127I significantly impacts the proper folding of the IL-18 peptide. We further tested the corresponding variant IL-18-DB6 peptides with C127N, C127T, and C127S, all of which expressed at levels comparable to DB6. Given the partially exposed nature of position 127 and the results obtained, introducing amino acids with hydrophobic side chains at position 127 should be avoided using this strategy.

[0418] 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 in activating IL-18 receptor-mediated signaling than WT IL-18. In the presence of 1 μg / ml hIL-18BP-hFc, the signaling activities of M12-DB6-Fc_N297A, M12-DB7-Fc_N297A, and M12-DB9-Fc_N297A were not inhibited. See Table 11.

[0419] Table 11. EC50 values ​​of M12-DB6-Fc_N297A, M12-DB7-Fc_N297A, M12-DB9-Fc_N297A, and M12-DB10-Fc_N297A in in vitro IL-18 receptor-mediated signaling assays

[0420]

[0421] Next, the "DB6" substitutions (i.e., C38I, C68S, and S117C) were introduced into variant MM5 to generate MM5-DB6:

[0422] MM5-DB6

[0423]

[0424] A "DB6" substitution was also introduced into wild-type IL-18 to generate WT IL18-DB6:

[0425] WT IL18-DB6

[0426]

[0427] Another variant, WT-DBo, was designed by introducing the S117C substitution in wild-type IL-18:

[0428] WT IL-18-DBo

[0429]

[0430]

[0431] The C-termini of WT IL-18-DB6, WT IL-18-DBo, and MM5-DB6 were fused to the N-termini of human IgG1 Fc variants containing N297A substitutions (EU numbering). The resulting fusion polypeptides WT IL18-DB6-Fc_N297A (SEQ ID NO: 328), WT IL-18-DBo-Fc_N297A (SEQ ID NO: 329), and MM5-DB6-Fc_N297A (SEQ ID NO: 330) were expressed in CHO cells, harvested, and purified by Protein A chromatography. The purified preparations were analyzed by SEC and SDS PAGE (data not shown). As shown in Table 12 below, no WT IL18-DBo-Fc_N297A protein was detected after the same expression and purification steps. After one-step Protein A chromatography, the expression yield of WT IL18-DB6-Fc was 157 mg / L and the purity of WT IL18-DB6-Fc_N297A was 91.6% (determined by SEC-HPLC) (see Table 12). This data confirms that the strategy of promoting disulfide bond formation between C76 and C117 and removing cysteines at positions 38 and 68 is very advantageous. These data also indicate that introducing a disulfide bond between C76 and C117 alone may not be sufficient to improve fusion polypeptide expression in mammalian cells. Improved expression in mammalian cells was achieved using fusion polypeptides comprising IL-18 variants comprising (i) an engineered C76-C117 disulfide bond, (ii) a C38 substitution (see Table 9), and (iii) a C68 substitution (see Table 9). MM5-DB6-Fc_N297A consistently showed higher expression yields and purity than MM5-SSS-Fc_N297A (see Table 12). These data also indicate that the C38X+C68X+C76+S117C mutation strategy (e.g., "DB6" or "DB7" substitutions) is suitable for increasing the yield of WT IL-18 and IL-18 variant polypeptides during production and subsequent purification in mammalian cells.

[0432] 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-termini of variants M12-DB6 and MM5-DB6, respectively. 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 exhibited comparable expression yield and purity in CHO cells compared to M12-DB6-Fc_N297A or MM5-DB6-Fc_N297A, respectively. After one-step Protein A chromatography purification, the purity of Fc_N297A-MM5-DB6 was approximately 97% (determined by SEC-HPLC), and the purity of Fc_N297A-M12-DB6 was 89% (determined by SEC-HPLC). These data indicate that the cystine mutation plus disulfide bond introduction strategy is applicable to Fc fusions performed in any fusion order.

[0433] Table 12. Yield and purity of WT IL18-DB6-Fc_N297A, WT IL18-DBo-Fc_N297A, MM5-DB6-Fc_N297A, Fc_N297A-MM5-DB6, and Fc_N297A-M12-DB6 after expression in CHO cells and Protein A chromatography

[0434]

[0435]

[0436] Next, the in vitro assay described in Example 6B, Section 6.1, was used to characterize the effectiveness of WT IL18-DB6-Fc_N297A, MM5-DB6-Fc_N297A, Fc_N297A-MM5-DB6, and Fc_N297A-M12-DB6 in activating IL-18 receptor-mediated signaling. Both WT IL18-DB6-Fc_N297A and MM5-DB6-Fc_N297A exhibited potent IL-18 receptor cell activation signals, which were stronger than those of WT IL-18 (see Table 13). Fc_N297A-MM5-DB6 exhibited similar potency to WT IL-18, while Fc_N297A-M12-DB6 exhibited a stronger activation signal to 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 greatly reduced, while the activity of MM5-DB6-Fc_N297A was not inhibited 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 was reduced by IL-18BP, indicating that the "DB6" group of mutations (i.e., C38I, C68S and S117C, see Table 9) improved the expression profiles of IL-18 and IL-18 variants without affecting IL-18BP dependence.

[0437] In particular, these results demonstrate that the strategy of introducing C38X+C68X+C76+S117C mutations into either WT IL-18-Fc fusion polypeptides or IL-18 variant-Fc fusion polypeptides significantly increases the expression yield of the fusion polypeptides when produced in mammalian cells and increases the yield of the fusion polypeptides purified after chromatography without affecting IL-18BP-resistant activity. Furthermore, WT IL-18 and IL-18 variants engineered to include the "DB6" set of mutations exhibited increased potency in activating IL-18 receptor-mediated signaling. This effect was observed in both fusion polypeptides in which the C-terminus of the Fc region was fused to the N-terminus of the IL-18 variant and in fusion polypeptides in which the C-terminus of the IL-18 variant was fused to the N-terminus of the Fc domain (see Table 14).

[0438] Table 13. EC50 values ​​of WT IL-18-DB6-Fc_N297A, MM5-DB6-Fc_N297A, Fc_N297A-MM5-DB6, and -Fc_N297A-M12-DB6 in in vitro IL-18 receptor-mediated signaling assays

[0439]

[0440] See Table 14 below, which summarizes the above data.

[0441] Table 14.

[0442]

[0443] The scope of the present disclosure is not limited by the specific embodiments, which are intended to serve as independent illustrations of various aspects of the present disclosure, and any functionally equivalent compositions or methods are within the scope of the present disclosure. It will be understood by those skilled in the art that various modifications and variations can be made to the methods and compositions of the present disclosure without departing from the spirit or scope of the present disclosure. Therefore, the present disclosure is intended to encompass modifications and variations of the present disclosure as long as they are within the scope of the appended claims and their equivalents.

[0444] 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.

[0445] The present invention has been described with reference to specific embodiments discovered or proposed by the inventors, which include preferred modes for carrying out the present invention. Those skilled in the art will recognize that, in light of this disclosure, various modifications and improvements may be made to the illustrated specific embodiments without departing from the intended scope of the present invention. For example, due to codon redundancy, the following DNA sequence may be altered without affecting the protein sequence. Furthermore, due to considerations of biological functional equivalence, the protein structure may be altered without affecting the type or quantity of the biological effect. All such modifications are intended to be included within the scope of the appended claims.

[0446] Sequence Summary Table

[0447]

[0448]

[0449]

[0450]

[0451]

[0452]

[0453]

[0454]

[0455]

[0456]

[0457]

[0458]

[0459]

[0460]

[0461]

[0462]

[0463]

[0464]

[0465]

[0466]

[0467]

[0468]

[0469]

[0470]

[0471]

[0472]

[0473]

[0474]

[0475]

[0476]

[0477]

[0478]

[0479]

[0480]

[0481]

[0482]

[0483]

[0484]

[0485]

[0486]

[0487]

[0488]

[0489]

[0490]

[0491]

[0492]

[0493]

[0494]

[0495]

[0496]

Claims

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

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

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

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

333.

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

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

336.

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

8. The activatable IL-18 polypeptide according to claim 3 or 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. 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, optionally wherein the one or more proteases are one or more tumor microenvironment (TME) proteases.

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

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

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, membrane-type serine protease, and legumin.

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, optionally wherein the one or more amino acid sequences are recognized and cleaved by a) both legumin and MMP9 / MMP2 / MMP14, b) both uPA and MMP9 / MMP2 / MMP14, c) all of uPA, legumin, MMP2, MMP14, and MMP9.

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

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

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

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

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. The activatable IL-18 polypeptide of any one of claims 1 to 19, wherein the cleavable linker consists of no less than about 10, 15, 20, or 25 amino acids.

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, 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.

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. 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. 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. 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) relative to wild-type IL-18, or (ii) no binding to IL-18BP.

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

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, wherein the amino acid positions are relative to wild-type (WT) human IL-18 shown in SEQ ID NO:

1.

28. The activatable IL-18 polypeptide according to 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, wherein the amino acid positions are relative to wild-type human IL-18 as set forth in SEQ ID NO: 1, optionally wherein the IL-18 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. The activatable IL-18 polypeptide of any one of claims 24 to 28, wherein the IL-18 variant polypeptide comprises any one of SEQ ID NOs: 2-299 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.

30. The activatable IL-18 polypeptide of any one of claims 1 to 29, wherein the IL-18 polypeptide comprises, from N-terminus to C-terminus, a) a masking moiety comprising a truncated propeptide of pro-IL-18 comprising 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, optionally wherein the cleavable linker is no more than about 50, 40, 30, 25, 20, 15, 12, or 10 amino acids in length.

31. The activatable IL-18 polypeptide of any one of claims 1 to 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, optionally wherein the cleavable linker is about 10-40, 10-30, 20-30, or 25-30 amino acids in length.

32. The activatable IL-18 polypeptide of any one of claims 1 to 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, optionally wherein the cleavable linker is about 10-40, 10-30, 20-30, or 25-30 amino acids in length.

33. The activatable IL-18 polypeptide according to 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 b) an amino acid sequence functional variant thereof, said functional variant 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.

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 IL-18 variant polypeptide, and (2) a half-life extending domain, optionally wherein the half-life extending domain comprises an albumin binding portion or an antibody Fc domain or a variant thereof, optionally wherein the half-life extending domain is a human IgG Fc domain (e.g., hIgG1 Fc).

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) relative to wild-type IL-18, or (ii) no binding to IL-18BP.

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

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

38. The activatable IL-18 polypeptide according to 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 a variant thereof of the fusion protein.

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

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

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

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

46. ​​A vector comprising the nucleic acid according to claim 45.

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

48. A method for producing an activatable IL-18 polypeptide, comprising: (a) culturing the host cell according to claim 47 under conditions where the host cell expresses an activatable IL-18 polypeptide, and (b) recovering the activatable IL-18 polypeptide produced by the host cell.

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

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

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

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

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

54. A method of stimulating antigen-experienced immune cells in an individual in need thereof, comprising administering to the individual an effective amount of the pharmaceutical composition of claim 50.

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

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

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