Modified IL-18 Polypeptides

Abstract

The present disclosure relates to modified interleukin-18 (IL-18) polypeptides, methods for their production, and methods of using the modified IL-18 polypeptides for the treatment of diseases, including cancer. The modified IL-18 polypeptides may exhibit the ability to induce interferon gamma (IFNγ) production when contacted with a cell, may exhibit a reduced ability to be inhibited by IL-18 binding protein (IL-18BP) compared to wild-type IL-18 (WT IL-18), and may exhibit a ratio of half maximal inhibitory concentration (IC50) by IL-18BP to half maximal effective concentration (EC50) for inducing IFNγ production that is greater than that of WT IL-18.

Description

[Technical Field]

[0001] cross reference This application claims priority to U.S. Provisional Patent Application Nos. 63 / 313,210, filed February 23, 2022, 63 / 313,127, filed February 23, 2022, 63 / 313,222, filed February 23, 2022, 63 / 313,248, filed February 23, 2022, and 63 / 479,529, filed January 11, 2023, all of which are incorporated by reference in their entireties. [Background technology]

[0002] Immunotherapy utilizes a subject's immune system to aid in the treatment of disease. Immunotherapy can be designed to activate or suppress the immune system, depending on the nature of the disease being treated. The goal of immunotherapy for cancer treatment is to stimulate the immune system so that it recognizes and destroys tumors or other cancerous tissues. One method of activating the immune system to attack cancer cells in a subject's body is cytokine therapy. Cytokines are proteins produced in the body that are important in cell signaling and immune system regulation. Some cytokine therapies utilize these properties of cytokines to strengthen a subject's immune system and kill cancer cells. Summary of the Invention

[0003] In one aspect, provided herein are modified interleukin-18 (IL-18) polypeptides. The modified IL-18 polypeptides exhibit the ability to induce interferon gamma (IFNγ) production when contacted with cells. In some embodiments, the modified IL-18 polypeptides exhibit improved ability to induce IFNγ production in cells compared to wild-type IL-18 (WT IL-18). In some embodiments, the modified IL-18 polypeptides exhibit improved ability to signal through the IL-18 receptor compared to WT IL-18. In some embodiments, the modified IL-18 polypeptides exhibit a decreased ability to be inhibited by IL-18 binding protein (IL-18BP) compared to WT IL-18. In certain embodiments, the modified IL-18 polypeptides exhibit i) improved ability to induce IFNγ production in cells compared to WT IL-18, ii) improved ability to signal through the IL-18 receptor compared to WT IL-18, and iii) decreased ability to be inhibited by IL-18BP compared to WT IL-18.

[0004] In some embodiments, the modified IL-18 polypeptide has a ratio of half-maximal inhibitory concentration (IC50) by IL-18BP to half-maximal effective concentration (EC50) for inducing IFNγ production of at least 50,000. In some embodiments, the modified IL-18 polypeptide has a ratio of half-maximal inhibitory concentration (IC50) by IL-18BP to half-maximal effective concentration (EC50) for inducing IFNγ production of at least 298,000. In certain embodiments, for a modified IL-18BP, the ratio of IC50 by IL-18BP to EC50 for inducing IFNγ production is at least 50,000, at least 100,000, at least 200,000, at least 299,000, at least 300,000, at least 400,000, at least 500,000, at least 750,000, at least 1,000,000, at least 1,250,000, at least 1,500,000, or at least 1,750,000. In certain embodiments, for a modified IL-18BP, the ratio of IC50 by IL-18BP to EC50 for inducing IFNγ production is at least 50,000. In certain embodiments, for a modified IL-18BP, the ratio of the IC50 by IL-18BP to the EC50 for inducing IFNγ production is at least 100,000. In certain embodiments, for a modified IL-18BP, the ratio of the IC50 by IL-18BP to the EC50 for inducing IFNγ production is at least 200,000. In certain embodiments, for a modified IL-18BP, the ratio of the IC50 by IL-18BP to the EC50 for inducing IFNγ production is at least 299,000. In certain embodiments, for a modified IL-18BP, the ratio of the IC50 by IL-18BP to the EC50 for inducing IFNγ production is at least 300,000. In certain embodiments, for a modified IL-18BP, the ratio of the IC50 by IL-18BP to the EC50 for inducing IFNγ production is at least 400,000.In certain embodiments, for a modified IL-18BP, the ratio of the IC50 by IL-18BP to the EC50 for inducing IFNγ production is at least 500,000. In certain embodiments, for a modified IL-18BP, the ratio of the IC50 by IL-18BP to the EC50 for inducing IFNγ production is at least 750,000. In certain embodiments, for a modified IL-18BP, the ratio of the IC50 by IL-18BP to the EC50 for inducing IFNγ production is at least 1,000,000. In certain embodiments, for a modified IL-18BP, the ratio of the IC50 by IL-18BP to the EC50 for inducing IFNγ production is at least 1,250,000. In certain embodiments, for a modified IL-18BP, the ratio of the IC50 by IL-18BP to the EC50 for inducing IFNγ production is at least 1,500,000. In certain embodiments, for a modified IL-18BP, the ratio of IC50 by IL-18BP to EC50 for inducing IFNγ production is at least 1,750,000. The EC50 for inducing IFNγ production can be measured using an IFNγ induction NK-92 cell assay (e.g., as provided in the Examples herein), and the IC50 by IL-18BP can be measured by IL-18 binding protein-mediated inhibition of IFNγ secretion in an NK-92 cell assay (e.g., as provided in the Examples herein).

[0005] In some embodiments, the modified IL-18 polypeptide exhibits a half-maximal effective concentration (EC50) for inducing IFNγ production in cells that is at least 25-fold lower than the half-maximal effective concentration (EC50) of wild-type IL-18 (WT IL-18). In certain embodiments, the EC50 for inducing IFNγ production in cells for the modified IL-18 polypeptide is at least 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, or 80-fold lower than the EC50 for WT IL-18. In certain embodiments, the EC50 for inducing IFNγ production in cells for the modified IL-18 polypeptide is at least 30-fold lower than the EC50 for WT IL-18. In certain embodiments, the EC50 for inducing IFNγ production in cells for the modified IL-18 polypeptide is at least 40-fold lower than the EC50 for WT IL-18. In certain embodiments, the EC50 for inducing IFNγ production in cells for a modified IL-18 polypeptide is at least 50-fold lower than the EC50 for WT IL-18. In certain embodiments, the EC50 for inducing IFNγ production in cells for a modified IL-18 polypeptide is at least 60-fold lower than the EC50 for WT IL-18. In certain embodiments, the EC50 for inducing IFNγ production in cells for a modified IL-18 polypeptide is at least 70-fold lower than the EC50 for WT IL-18. In certain embodiments, the EC50 for inducing IFNγ production in cells for a modified IL-18 polypeptide is at least 80-fold lower than the EC50 for WT IL-18. The EC50 for inducing IFNγ production in cells can be measured using an IFNγ induction NK-92 cell assay (e.g., as provided in the Examples herein).

[0006] In some embodiments, the modified IL-18 polypeptide exhibits a half-maximal effective concentration (EC50) for signaling through the IL-18 receptor that is at least 4.6-fold lower than the EC50 of WT IL-18. In certain embodiments, the EC50 for signaling through the IL-18 receptor for the modified IL-18 polypeptide is at least 4.7-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, or 50-fold lower than the EC50 of WT IL-18. In certain embodiments, the EC50 for signaling through the IL-18 receptor for the modified IL-18 polypeptide is at least 4.7-fold lower than the EC50 of WT IL-18. In certain embodiments, the EC50 for signaling through the IL-18 receptor for the modified IL-18 polypeptide is at least 5-fold lower than the EC50 of WT IL-18. In certain embodiments, the EC50 for signaling through the IL-18 receptor for the modified IL-18 polypeptide is at least 10-fold lower than the EC50 of WT IL-18. In certain embodiments, the EC50 for signaling through the IL-18 receptor for the modified IL-18 polypeptide is at least 20-fold lower than the EC50 for WT IL-18. In certain embodiments, the EC50 for signaling through the IL-18 receptor for the modified IL-18 polypeptide is at least 30-fold lower than the EC50 for WT IL-18. In certain embodiments, the EC50 for signaling through the IL-18 receptor for the modified IL-18 polypeptide is at least 40-fold lower than the EC50 for WT IL-18. In certain embodiments, the EC50 for signaling through the IL-18 receptor for the modified IL-18 polypeptide is at least 50-fold lower than the EC50 for WT IL-18. The EC50 for signaling through the IL-18 receptor can be measured using a HEK-Blue IL18R reporter assay (e.g., as provided in the Examples herein).

[0007] In some embodiments, the modified IL-18 polypeptide has a dissociation constant (K D) at least 50-fold lower K with the IL-18 receptor alpha subunit D In certain embodiments, the modified IL-18 polypeptide has a K D K with the IL-18 receptor alpha subunit that is at least 52-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold lower than D In certain embodiments, the modified IL-18 polypeptide has a K D At least 52-fold lower K with IL-18 receptor alpha subunit D In certain embodiments, the modified IL-18 polypeptide has a K D K with IL-18 receptor alpha subunit at least 60-fold lower than D In certain embodiments, the modified IL-18 polypeptide has a K D At least 70-fold lower K with the IL-18 receptor alpha subunit D In certain embodiments, the modified IL-18 polypeptide has a K D K with IL-18 receptor alpha subunit at least 80-fold lower D In certain embodiments, the modified IL-18 polypeptide has a K D K with IL-18 receptor alpha subunit at least 90-fold lower than D In certain embodiments, the modified IL-18 polypeptide has a K D K with IL-18 receptor alpha subunit at least 100-fold lower D Shows.

[0008] In some embodiments, the modified IL-18 polypeptide has a dissociation constant (K) with IL-18 binding protein (IL-18BP) that is greater than that of wild-type IL-18. D In some embodiments, the modified IL-18 polypeptide exhibits a dissociation constant (K) with IL-18BP of at least 250 nM. DIn certain embodiments, the modified IL-18 polypeptide has a K D In certain embodiments, the K of the modified IL-18 polypeptide with IL-18BP is at least 2 nM, at least 5 nM, at least 10 nM, at least 20 nM, at least 50 nM, at least 100 nM, at least 200 nM, at least 300 nM, at least 400 nM, at least 500 nM, at least 750 nM, at least 1000 nM, at least 2500 nM, at least 5000 nM, or at least 10000 nM. D In certain embodiments, the K of the modified IL-18 polypeptide with IL-18BP is at least 2 nM. D In certain embodiments, the K of the modified IL-18 polypeptide with IL-18BP is at least 5 nM. D In certain embodiments, the K of the modified IL-18 polypeptide with IL-18BP is at least 10 nM. D In certain embodiments, the K of the modified IL-18 polypeptide with IL-18BP is at least 50 nM. D In certain embodiments, the K D In certain embodiments, the K of the modified IL-18 polypeptide with IL-18BP is at least 200 nM. D In certain embodiments, the K of the modified IL-18 polypeptide with IL-18BP is at least 300 nM. D In certain embodiments, the K of the modified IL-18 polypeptide with IL-18BP is at least 400 nM. D In certain embodiments, the K of the modified IL-18 polypeptide with IL-18BP is at least 500 nM. D In certain embodiments, the K of the modified IL-18 polypeptide with IL-18BP is at least 750 nM. DIn certain embodiments, the K of the modified IL-18 polypeptide with IL-18BP is at least 1000 nM. D In certain embodiments, the K of the modified IL-18 polypeptide with IL-18BP is at least 2500 nM. D In certain embodiments, the K of the modified IL-18 polypeptide with IL-18BP is at least 5000 nM. D is at least 10000 nM. The KD with IL-18BP can be measured using an IL-18BP binding alphaLISA assay (eg, as provided in the Examples herein).

[0009] In certain embodiments, the modified IL-18 polypeptide exhibits any one, any combination, or all of the following properties: i) a ratio of IC50 by IL-18BP to EC50 for inducing IFNγ production of at least 50,000, at least 100,000, at least 200,000, at least 298,000, at least 299,000, at least 300,000, at least 400,000, at least 500,000, at least 750,000, at least 1,000,000, at least 1,250,000, at least 1,500,000, or at least 1,750,000; ii) an EC50 for inducing IFNγ production in cells that is at least 25-fold, at least 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, or 80-fold lower than the EC50 of WT IL-18; iii) an EC50 for signaling through the IL-18 receptor that is at least 4.6-fold, at least 4.7-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, or 50-fold lower than the EC50 of WT IL-18; iv) a K with the IL-18 receptor alpha subunit that is at least 50-fold, at least 52-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold lower than wild-type IL-18 D , and v) a K with IL-18BP of at least 2 nM, at least 5 nM, at least 10 nM, at least 20 nM, at least 50 nM, at least 100 nM, at least 200 nM, at least 250 nM, at least 500 nM, at least 750 nM, at least 1000 nM, at least 2500 nM, at least 5000 nM, or at least 10000 nM D .

[0010] In certain embodiments, the modified IL-18 polypeptide comprises a substitution at residue V11. In certain embodiments, the modified IL-18 polypeptide comprises a substitution at residue V11I. Unless specifically stated otherwise, the numbering of residue positions provided in this paragraph and elsewhere in this disclosure is based on SEQ ID NO: 1 as the reference sequence. Unless specifically stated otherwise, the amino acid substitutions provided in this paragraph and elsewhere in this disclosure are with respect to SEQ ID NO: 1 as the reference sequence. In certain embodiments, the modified IL-18 polypeptide comprises a substitution at a residue selected from Y1, F2, E6, K8, S10, D17, T34, D35, S36, D37, D40, N41, I49, M51, K53, D54, S55, Q103, S105, G108, H109, ​​D110, and D132. In certain embodiments, the modified IL-18 polypeptide comprises a substitution at residue E6. In certain embodiments, the modified IL-18 polypeptide comprises an E6K substitution. In certain embodiments, the modified IL-18 polypeptide comprises a substitution at residue M51. In certain embodiments, the modified IL-18 polypeptide comprises an M51G substitution. In certain embodiments, the modified IL-18 polypeptide comprises a substitution at residue K53. In certain embodiments, the modified IL-18 polypeptide comprises a K53A substitution. In certain embodiments, the modified IL-18 polypeptide comprises a substitution at residue T63. In certain embodiments, the modified IL-18 polypeptide comprises a T63A substitution. In certain embodiments, the modified IL-18 polypeptide comprises i) a substitution at residue V11, and ii) substitutions at residues E6, M51, and / or K53. In certain embodiments, the modified IL-18 polypeptide comprises i) a V11I substitution, and ii) an E6K, M51G, and / or K53A substitution. In certain embodiments, the modified IL-18 polypeptide comprises substitutions at residues E6, V11, K53, and T63. In certain embodiments, the modified IL-18 polypeptide comprises E6K, V11I, K53A, and T63A substitutions. In certain embodiments, the modified IL-18 polypeptide comprises substitutions at residues V11, M51, K53. In certain embodiments, the modified IL-18 polypeptide comprises V11I, M51G, and K53A substitutions.In certain embodiments, the modified IL-18 polypeptide comprises substitutions at residues E6, V11, M51, K53, and T63. In certain embodiments, the modified IL-18 polypeptide comprises E6K, V11I, M51G, K53A, and T63A substitutions. In certain embodiments, the modified IL-18 polypeptide comprises substitutions at residues C38, C68, C76, and / or C127. In certain embodiments, the modified IL-18 polypeptide comprises a substitution at residue C38. In certain embodiments, the modified IL-18 polypeptide comprises a substitution at residue C68. In certain embodiments, the modified IL-18 polypeptide comprises a substitution at residue C76. In certain embodiments, the modified IL-18 polypeptide comprises a substitution at residue C127. In certain embodiments, the modified IL-18 polypeptide comprises a C38A, C38S, C68A, C68S, C76A, C76S, C127A, and / or C127S substitution. In certain embodiments, the modified IL-18 polypeptide comprises a C38A substitution. In certain embodiments, the modified IL-18 polypeptide comprises a C38S. In certain embodiments, the modified IL-18 polypeptide comprises a C68A substitution. In certain embodiments, the modified IL-18 polypeptide comprises a C68S. In certain embodiments, the modified IL-18 polypeptide comprises a C76A substitution. In certain embodiments, the modified IL-18 polypeptide comprises a C76S. In certain embodiments, the modified IL-18 polypeptide comprises a C127A substitution. In certain embodiments, the modified IL-18 polypeptide comprises a C127S. In certain embodiments, the modified IL-18 polypeptide comprises a substitution at each of residues C38, C76, and C127, wherein each of the substitutions at residues C38, C76, and C127 is to serine or alanine. In certain embodiments, the modified IL-18 polypeptide comprises a C38A, C76A, and C127A substitution. In certain embodiments, the modified IL-18 polypeptide comprises a C38S, C76S, and C127S substitution. In certain embodiments, the modified IL-18 polypeptide comprises a substitution at residues E6, V11, C38, K53, T63, C76, and C127.In certain embodiments, the modified IL-18 polypeptide comprises E6K, V11I, C38A, K53A, T63A, C76A, and C127A substitutions. In certain embodiments, the modified IL-18 polypeptide comprises substitutions at residues V11, C38, M51, K53, T63, C76, and C127. In certain embodiments, the modified IL-18 polypeptide comprises V11I, C38A, M51G, K53A, T63A, C76A, and C127A substitutions. In certain embodiments, the modified IL-18 polypeptide comprises substitutions at residues V11, C38, M51, K53, C76, and C127. In certain embodiments, the modified IL-18 polypeptide comprises V11I, C38A, M51G, K53A, C76A, and C127A substitutions. In certain embodiments, the modified IL-18 polypeptide comprises substitutions at residues E6, V11, C38, M51, K53, T63, C76, and C127. In certain embodiments, the modified IL-18 polypeptide comprises substitutions at E6K, V11I, C38A, M51G, K53A, T63A, C76A, and C127A. In certain embodiments, the modified IL-18 polypeptide comprises at least one glycine residue attached to the N-terminus of the IL-18 polypeptide. In certain embodiments, the modified IL-18 polypeptide comprises a chain of 1 to 10 glycine residues attached to the N-terminus of the IL-18 polypeptide. In certain embodiments, the modified IL-18 polypeptide comprises a chain of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 glycine residues, or any range therebetween, attached to the N-terminus of the IL-18 polypeptide. In certain embodiments, the modified IL-18 polypeptide comprises a glycine residue attached to the N-terminus of the IL-18 polypeptide. In certain embodiments, the modified IL-18 polypeptide comprises a chain of two glycine residues attached to the N-terminus of the IL-18 polypeptide. In certain embodiments, the modified IL-18 polypeptide comprises a chain of three glycine residues attached to the N-terminus of the IL-18 polypeptide. In certain embodiments, the modified IL-18 polypeptide comprises a chain of four glycine residues attached to the N-terminus of the IL-18 polypeptide.In certain embodiments, a modified IL-18 polypeptide comprises a chain of five glycine residues attached to the N-terminus of the IL-18 polypeptide. In certain embodiments, a modified IL-18 polypeptide comprises i) a chain of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or any range therebetween, glycine residues attached to the N-terminus of the IL-18 polypeptide, and ii) substitutions at residues E6, V11, C38, K53, T63, C76, and C127. In certain embodiments, a modified IL-18 polypeptide comprises i) a chain of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or any range therebetween, glycine residues attached to the N-terminus of the IL-18 polypeptide, and ii) E6K, V11I, C38A, K53A, T63A, C76A, and C127A substitutions. In certain embodiments, a modified IL-18 polypeptide comprises i) a glycine residue attached to the N-terminus of the IL-18 polypeptide, and ii) substitutions at residues E6, V11, C38, K53, T63, C76, and C127. In certain embodiments, a modified IL-18 polypeptide comprises i) a glycine residue attached to the N-terminus of the IL-18 polypeptide, and ii) E6K, V11I, C38A, K53A, T63A, C76A, and C127A substitutions. In certain embodiments, a modified IL-18 polypeptide comprises i) a chain of four glycine residues attached to the N-terminus of the IL-18 polypeptide, and ii) substitutions at residues E6, V11, C38, K53, T63, C76, and C127. In certain embodiments, the modified IL-18 polypeptide comprises i) a chain of four glycine residues attached to the N-terminus of the IL-18 polypeptide, and ii) E6K, V11I, C38A, K53A, T63A, C76A, and C127A substitutions. In certain embodiments, the modified IL-18 polypeptide comprises i) a chain of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 glycine residues, or any range therebetween, attached to the N-terminus of the IL-18 polypeptide, and ii) substitutions at residues E6, V11, C38, M51, K53, T63, C76, and C127.In certain embodiments, the modified IL-18 polypeptide comprises i) a chain of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 glycine residues, or any range therebetween, attached to the N-terminus of the IL-18 polypeptide, and ii) E6K, V11I, C38A, M51G, K53A, T63A, C76A, and C127A substitutions. In certain embodiments, the modified IL-18 polypeptide comprises i) a glycine residue attached to the N-terminus of the IL-18 polypeptide, and ii) substitutions at residues E6, V11, C38, M51, K53, T63, C76, and C127. In certain embodiments, the modified IL-18 polypeptide comprises i) a glycine residue attached to the N-terminus of the IL-18 polypeptide, and ii) E6K, V11I, C38A, M51G, K53A, T63A, C76A, and C127A substitutions.

[0011] In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, or 95% sequence identity to the sequence set forth in SEQ ID NO: 1. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 80% sequence identity to the sequence set forth in SEQ ID NO: 1. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 1. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 95% sequence identity to the sequence set forth in SEQ ID NO: 1. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the sequence set forth in any one of SEQ ID NOs: 2-33. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 80% sequence identity to the sequence set forth in any one of SEQ ID NOs: 2-33. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 90% sequence identity to the sequence set forth in any one of SEQ ID NOs: 2-33. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 95% sequence identity to the sequence set forth in any one of SEQ ID NOs: 2-33. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 98% sequence identity to the sequence set forth in any one of SEQ ID NOs: 2-33. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the sequence set forth in any one of SEQ ID NOs: 24-33. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 80% sequence identity to the sequence set forth in any one of SEQ ID NOs: 24-33.In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 90% sequence identity to the sequence set forth in any one of SEQ ID NOs: 24-33. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 95% sequence identity to the sequence set forth in any one of SEQ ID NOs: 24-33. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 98% sequence identity to the sequence set forth in any one of SEQ ID NOs: 24-33. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the sequence set forth in any one of SEQ ID NOs: 204-244. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 80% sequence identity to the sequence set forth in any one of SEQ ID NOs: 204-244. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 90% sequence identity to the sequence set forth in any one of SEQ ID NOs: 204-244. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 95% sequence identity to the sequence set forth in any one of SEQ ID NOs: 204-244. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 98% sequence identity to the sequence set forth in any one of SEQ ID NOs: 204-244. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 30. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 80% sequence identity to the sequence set forth in SEQ ID NO:30.In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 30. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 95% sequence identity to the sequence set forth in SEQ ID NO: 30. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 98% sequence identity to the sequence set forth in SEQ ID NO: 30. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 207. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 80% sequence identity to the sequence set forth in SEQ ID NO: 207. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 207. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 95% sequence identity to the sequence set forth in SEQ ID NO:30. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 98% sequence identity to the sequence set forth in SEQ ID NO:207. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO:239. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 80% sequence identity to the sequence set forth in SEQ ID NO:239. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO:239. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 95% sequence identity to the sequence set forth in SEQ ID NO:239.In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 98% sequence identity to the sequence set forth in SEQ ID NO: 239. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 239. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 241. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 80% sequence identity to the sequence set forth in SEQ ID NO: 241. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 241. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 95% sequence identity to the sequence set forth in SEQ ID NO: 241. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 98% sequence identity to the sequence set forth in SEQ ID NO: 241. In certain embodiments, the modified IL-18 polypeptide comprises the amino acid sequence set forth in SEQ ID NO:241. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO:242. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 80% sequence identity to the sequence set forth in SEQ ID NO:242. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO:242. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 95% sequence identity to the sequence set forth in SEQ ID NO:242. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 98% sequence identity to the sequence set forth in SEQ ID NO:242. In certain embodiments, the modified IL-18 polypeptide comprises the amino acid sequence set forth in SEQ ID NO:242.In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 244. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 80% sequence identity to the sequence set forth in SEQ ID NO: 244. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 244. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 95% sequence identity to the sequence set forth in SEQ ID NO: 244. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 98% sequence identity to the sequence set forth in SEQ ID NO: 244. In certain embodiments, the modified IL-18 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 244.

[0012] In one aspect, provided herein is a polymer-modified IL-18 polypeptide. The polymer-modified IL-18 polypeptide comprises a polymer covalently attached to a residue of the IL-18 polypeptide. In certain embodiments, the polymer is attached to a cysteine ​​of the modified IL-18 polypeptide. In certain embodiments, the polymer is attached at residue 68 of the modified IL-18 polypeptide, where the numbering of residue positions is based on SEQ ID NO: 1 as a reference sequence. In certain embodiments, the polymer is attached to any one of residues 79-120 of the modified IL-18 polypeptide, where the numbering of residue positions is based on SEQ ID NO: 1 as a reference sequence. In certain embodiments, the polymer is attached at residue 85. In certain embodiments, the polymer is attached at residue 86. In certain embodiments, the polymer is attached at residue 95. The polymer-modified IL-18 polypeptide exhibits a lower binding affinity (higher K D(resulting in a binding affinity) that is up to 8-fold lower than the binding affinity exhibited by the same modified IL-18 polypeptide without the polymer attached. In some embodiments, the polymer is covalently attached to a residue selected from residue 79, residue 80, residue 81, residue 82, residue 83, residue 84, residue 85, residue 86, residue 87, residue 88, residue 89, residue 90, residue 91, residue 92, residue 93, residue 94, residue 95, residue 96, residue 97, residue 98, residue 99, residue 100, residue 101, residue 102, residue 103, residue 104, residue 105, residue 106, residue 107, residue 108, residue 109, residue 110, residue 111, residue 112, residue 113, residue 114, residue 115, residue 116, residue 117, residue 118, residue 119, and residue 120. In some embodiments, the polymer is covalently attached at residue 85. In some embodiments, the polymer is covalently attached at residue 86. In some embodiments, the polymer is covalently attached at residue 95. In some embodiments, the polymer is covalently attached at residue 98. In some embodiments, the polymer is covalently attached at residue E85, E85C, E85D, E85Q, E85K, E85N, or E85Y. In some embodiments, the polymer is covalently attached at residue M86C, M86D, M86Q, M86K, M86N, M86E, or M86Y. In some embodiments, the polymer is covalently attached at residue T95C, T95D, T95Q, T95K, T95N, T95E, T95Y, D98C, D98Q, D98K, D98N, D98E, or D98Y.

[0013] In some embodiments, the polymer is attached to a natural amino acid residue. In some embodiments, the natural amino acid residue is selected from asparagine, aspartic acid, cysteine, glutamic acid, glutamine, lysine, and tyrosine. In some embodiments, the polymer is attached to a non-natural amino acid residue. In some embodiments, one of residues 79-120 is substituted with a cysteine. In some embodiments, the polymer comprises a conjugation handle or a reaction product of a conjugation handle and a complementary conjugation handle. In some embodiments, the conjugation handle or a reaction product of a conjugation handle and a complementary conjugation handle comprises an azide moiety, an alkyne moiety, or the reaction product of an azide-alkyne cycloaddition reaction.

[0014] In some embodiments, the polymer is a water-soluble polymer. In some embodiments, the water-soluble polymer comprises a poly(alkylene oxide), a polysaccharide, poly(vinylpyrrolidone), poly(vinyl alcohol), polyoxazoline, poly(acryloylmorpholine), or a combination thereof. In some embodiments, the water-soluble polymer comprises a poly(alkylene oxide). In some embodiments, the poly(alkylene oxide) is polyethylene glycol (PEG).

[0015] In some embodiments, the modified IL-18 polypeptide comprises one or more amino acid substitutions at residues Y1, F2, E6, V11, C38, K53, D54, S55, T63, C76, C127, or any combination thereof. In some embodiments, the modified IL-18 polypeptide comprises Y01G, F02A, E06K, V11I, C38S, C38A, C38Q, D54A, S55A, T63A, C76S, C76A, C127S, C127A amino acid substitutions, or any combination thereof. In some embodiments, the modified IL-18 polypeptide comprises a Y01G, F02A, E06K, V11I, C38S, C38A, C38Q, K53A, D54A, S55A, T63A, C76S, C76A, C127S, C127A amino acid substitution, or any combination thereof. In some embodiments, the modified IL-18 polypeptide comprises an E06K and a K53A amino acid substitution. In some embodiments, the modified IL-18 polypeptide comprises a V11I amino acid substitution. In some embodiments, the modified IL-18 polypeptide comprises a T63A amino acid substitution. In some embodiments, the modified IL-18 polypeptide comprises an N-terminal extension. In some embodiments, the modified IL-18 polypeptide comprises a polypeptide sequence having at least about 80% sequence identity to any one of SEQ ID NOs: 2-244. In some embodiments, the modified IL-18 polypeptide comprises a polypeptide sequence having at least about 80% sequence identity to any one of SEQ ID NOs: 2-67. In some embodiments, the polypeptide sequence is at least about 80% identical to SEQ ID NO:4 or SEQ ID NO:30. In some embodiments, the polypeptide sequence is at least about 80% identical to SEQ ID NO:4. In some embodiments, the polypeptide sequence is at least about 90% identical to SEQ ID NO:4. In some embodiments, the polypeptide sequence is at least about 95% identical to SEQ ID NO:4. In some embodiments, the polypeptide sequence is at least about 80% identical to SEQ ID NO:30. In some embodiments, the polypeptide sequence is at least about 90% identical to SEQ ID NO:30. In some embodiments, the polypeptide sequence is at least about 95% identical to SEQ ID NO:30.In some embodiments, the modified IL-18 polypeptide is recombinant.

[0016] In some embodiments, the modified IL-18 polypeptide contains (a) a homoserine residue located at any one of residues 26-36, (b) a homoserine residue located at any one of residues 45-67, (c) a homoserine residue located at any one of residues 70-80, (d) a homoserine residue located at any one of residues 100-130, (e) a norleucine or O-methyl-homoserine residue located at any one of residues 28-38, (f) a norleucine or O-methyl-homoserine residue located at any one of residues 46-56, (g) a norleucine or O-methyl-homoserine residue located at any one of residues 54-56, (h) a norleucine or O-methyl-homoserine residue located at any one of residues 54-56, (i) a norleucine or O-methyl-homoserine residue located at any one of residues 54-56, (j) a norleucine or O-methyl-homoserine residue located at any one of residues 54-56, (k ... (h) a norleucine or O-methyl-homoserine residue located at any one of residues 80 to 90; (i) a norleucine or O-methyl-homoserine residue located at any one of residues 108 to 118; and (j) a norleucine or O-methyl-homoserine residue located at any one of residues 145 to 155, wherein the numbering of residue positions in the modified IL-18 polypeptide is based on SEQ ID NO: 1 as a reference sequence. In some embodiments, the modified IL-18 polypeptide comprises one or more amino acid substitutions selected from homoserine (Hse)31, norleucine (Nle)33, O-methyl-homoserine (Omh)33, Hse50, Nle51, Omh51, Hse57, Nle60, Hse63, Omh60, Hse75, Nle86, Omh86, Hse116, Nle113, Omh113, Hse121, Nle150, and Omh150.

[0017] In some embodiments, the modified IL-18 polypeptides modulate IFNγ production, and the EC 50 (nM) is the EC 50 (nM) of the EC 50In some embodiments, the EC 200 of the ability of the modified IL-18 polypeptide to induce IFNγ is less than (nM). 50 (nM) is the EC of SEQ ID NO: 1 50 (nM) or less than 5-fold higher. In some embodiments, the EC 50 (nM) is the EC 50 In some embodiments, the EC 200 of the ability of the modified IL-18 polypeptide to induce IFNγ is less than (nM). 50 (nM) is the EC of SEQ ID NO: 1 50 (nM). In some embodiments, the modified IL-18 polypeptide has a K D and exhibiting less than 10-fold lower affinity, less than 5-fold lower affinity, or higher affinity for the IL-18 receptor alpha subunit (IL-18Rα) than for the IL-18 binding protein (IL-18BP) as measured by [K D IL-18Rα] / [K D In some embodiments, the modified IL-18 polypeptide binds to IL-18 receptor alpha (IL-18Rα). In some embodiments, the modified IL-18 polypeptide binds to IL-18Rα with a K of less than about 200 nM, less than about 100 nM, less than about 80 nM, less than about 70 nM, less than about 60 nM, or less than about 50 nM. D In some embodiments, the modified IL-18 polypeptide binds with a K of less than about 50 nM. D In some embodiments, the modified IL-18 polypeptide binds to the IL-18 receptor alpha / beta (IL-18Rα / β) heterodimer with a K of less than about 25 nM. D In some embodiments, the modified IL-18 polypeptide binds to the IL-18Rα / β heterodimer with a K of less than about 10 nM. D In some embodiments, the modified IL-18 polypeptide is conjugated to an additional peptide.

[0018] In one aspect, provided herein is a host cell comprising a modified IL-18 polypeptide provided herein. In some embodiments, provided herein is a method for producing a modified IL-18 polypeptide, the method comprising expressing the modified IL-18 polypeptide in a host cell. In some embodiments, the host cell is a prokaryotic or eukaryotic cell. In some embodiments, the host cell is a mammalian cell, an avian cell, a fungal cell, or an insect cell. In some embodiments, the host cell is a CHO cell, a COS cell, or a yeast cell.

[0019] In another aspect, provided herein are pharmaceutical compositions comprising a modified IL-18 polypeptide provided herein and a pharmaceutically acceptable carrier or excipient. In one aspect, provided herein are pharmaceutical compositions comprising a polymer-modified IL-18 polypeptide provided herein and a pharmaceutically acceptable carrier or excipient. In some embodiments, the modified IL-18 polypeptide and / or pharmaceutical composition comprising the polymer-modified IL-18 polypeptide is in lyophilized form.

[0020] In another aspect, provided is a method of treating cancer in a subject in need thereof, comprising administering to the subject a pharmaceutically effective amount of a modified IL-18 polypeptide provided herein or a pharmaceutical composition comprising the modified IL-18 polypeptide. In one aspect, provided is a method of treating cancer in a subject in need thereof, comprising administering to the subject a pharmaceutically effective amount of a polymer-modified IL-18 polypeptide provided herein or a pharmaceutical composition comprising the polymer-modified IL-18 polypeptide. In some embodiments, the cancer is a solid cancer. In some embodiments, the solid cancer is adrenal gland cancer, anal cancer, bile duct cancer, bladder cancer, bone cancer, brain cancer, breast cancer, carcinoid cancer, cervical cancer, colorectal cancer, esophageal cancer, eye cancer, gallbladder cancer, gastrointestinal stromal tumor, germ cell cancer, head and neck cancer, kidney cancer, liver cancer, lung cancer, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, neuroendocrine cancer, oral cancer, oropharyngeal cancer, ovarian cancer, pancreatic cancer, pediatric cancer, penile cancer, pituitary cancer, prostate cancer, skin cancer, soft tissue cancer, spinal cancer, stomach cancer, testicular cancer, thymus cancer, thyroid cancer, ureter cancer, uterine cancer, vaginal cancer, or vulvar cancer. In some embodiments, the solid cancer is a carcinoma or sarcoma. In some embodiments, the cancer is a hematological cancer. In some embodiments, the hematological cancer is leukemia, non-Hodgkin's lymphoma, Hodgkin's lymphoma, AIDS-related lymphoma, multiple myeloma, plasmacytoma, post-transplant lymphoproliferative disorder, or Waldenstrom's macroglobulinemia. In some embodiments, the method comprises reconstituting a modified IL-18 polypeptide or pharmaceutical composition in lyophilized form.

[0021] Another aspect provides a method of making a modified IL-18 polypeptide provided herein, the method comprising synthesizing two or more fragments of a modified IL-18 polypeptide, ligating the fragments, and folding the ligated fragments. In some embodiments, at least one of the fragments of the IL-18 polypeptide comprises a conjugation handle. In some embodiments, the method comprises attaching a polymer to the folded, ligated fragment by reaction with the conjugation handle. In an aspect provided herein, a method of making a polymer-modified IL-18 polypeptide is provided, the method comprising synthesizing two or more fragments of a modified IL-18 polypeptide, ligating the fragments, folding the ligated fragments, and attaching a polymer to the folded, ligated fragment by reaction with a conjugation handle, wherein at least one of the fragments of the IL-18 polypeptide comprises a conjugation handle. In some embodiments, one of the fragments comprises a polymer.

[0022] Yet another embodiment herein provides a modified IL-18 polypeptide having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in any one of SEQ ID NOs: 2 to 33. An embodiment herein provides a modified IL-18 polypeptide having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in any one of SEQ ID NOs: 204 to 244.

[0023] Another embodiment provides a modified IL-18 polypeptide having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to a sequence set forth in any one of SEQ ID NOs: 24-33.

[0024] Another aspect provides a modified IL-18 polypeptide having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO: 30. In some embodiments, the IL-18 polypeptide comprises a covalently attached polymer. In some embodiments, the polymer is covalently attached at residue 68, 69, or 70. In some embodiments, the polymer is covalently attached at residue 68.

[0025]

[0013] Further aspects and advantages of the present disclosure will become readily apparent to those skilled in the art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be understood, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the present disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.

[0026] Incorporation by Reference All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent that the publications and patents or patent applications incorporated by reference conflict with the disclosure contained herein, the present specification is intended to supersede and / or override any such conflicting material.

[0027] The novel features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings. [Brief explanation of the drawings]

[0028] [Figure 1] 1 shows the mechanism of action of IL-18-induced IFNγ and IL-18BP production, and IL-18BP-mediated IL-18 inhibitory activity. [Figure 2] 1 shows the coupling of dibenzocyclooctyne (DBCO) polyethylene glycol (PEG) to an azide-containing modified IL-18 polypeptide. [Figure 3] 1 shows the binding of modified IL-18 polypeptides containing polymers to IL-18Rα. [Figure 4A] 1 shows the IFNγ-inducing ability of modified IL-18 polypeptides compared to wild-type IL-18 polypeptide. [Figure 4B] 1 shows the IL-18BP inhibition of modified IL-18 polypeptides compared to wild-type IL-18 polypeptide. [Figure 5] 1 shows a schematic representation of the coupling of a bifunctional probe to an IL-18 polypeptide provided herein. [Figure 6] 1 shows a schematic representation of coupling of poly(ethylene glycol) moieties to bifunctional probe-activated IL-18 polypeptides. [Figure 7] 1 shows a schematic representation of a synthetic route that can be used to synthesize a polymer-modified IL-18 polypeptide. [Figure 8] 1 shows further schematic diagrams of synthetic pathways that can be used to synthesize IL-18 polypeptides. [Figure 9] 1 shows further schematic diagrams of synthetic pathways that can be used to synthesize IL-18 polypeptides. [Figure 10] 1 shows further schematic diagrams of synthetic pathways that can be used to synthesize IL-18 polypeptides. [Figure 11] 1 shows exemplary purification results of the IL-18 polypeptides provided herein. [Figure 12] An exemplary analytical SEC-HPLC chromatogram of purified SEQ ID NO: 207 is shown (detection: 220 nm). [Figure 13] 1 shows an exemplary ESI-Q-TOF-HRMS spectrum of purified SEQ ID NO:207. [Figure 14] An exemplary analytical SEC-HPLC chromatogram of purified SEQ ID NO: 239 is shown (detection: 220 nm). [Figure 15] 1 shows an exemplary ESI-Q-TOF-HRMS spectrum of purified SEQ ID NO:239. [Figure 16] An exemplary analytical SEC-HPLC chromatogram of purified SEQ ID NO: 244 is shown (detection: 220 nm). [Figure 17] 1 shows an exemplary ESI-Q-TOF-HRMS spectrum of purified SEQ ID NO:244. [Figure 18] An exemplary analytical SEC-HPLC chromatogram of purified SEQ ID NO: 242 is shown (detection: 220 nm). [Figure 19] 1 shows an exemplary ESI-Q-TOF-HRMS spectrum of purified SEQ ID NO:242. [Figure 20] An exemplary analytical SEC-HPLC chromatogram of purified SEQ ID NO: 241 is shown (detection: 220 nm). [Figure 21] 1 shows an exemplary ESI-Q-TOF-HRMS spectrum of purified SEQ ID NO:242. [Figure 22] An exemplary analytical SEC-HPLC chromatogram of purified SEQ ID NO: 30 is shown (detection: 220 nm). [Figure 23] 1 shows an exemplary ESI-Q-TOF-HRMS spectrum of purified SEQ ID NO:30. [Figure 24]1 shows plots measuring the binding activity of wild-type IL-18 and modified IL-18 polypeptides to human IL-18 receptor alpha (CD218a) in surface plasmon resonance experiments, where the x-axis is time and the y-axis is relative response. The modified IL-18 polypeptides tested in these experiments were native wild-type IL-18 (SEQ ID NO: 01), SEQ ID NO: 57, SEQ ID NO: 59, and SEQ ID NO: 30. [Figure 25] 1 shows plots measuring the binding activity of wild-type IL-18 and modified IL-18 polypeptides to the heterodimeric human IL-18 receptor alpha (CD218a) and IL-18R accessory protein (IL-18RAP / CDw218b) in surface plasmon resonance experiments, where the x-axis is time and the y-axis is relative response. The modified IL-18 polypeptides tested in these experiments were native wild-type IL-18 (SEQ ID NO: 01), SEQ ID NO: 57, SEQ ID NO: 59, and SEQ ID NO: 30. [Figure 26] 1 shows plots measuring the binding activity of wild-type IL-18 and modified IL-18 polypeptides to human IL-18 binding protein (IL-18BP) in surface plasmon resonance experiments, where the x-axis is time and the y-axis is relative response. The modified IL-18 polypeptides tested in these experiments were native wild-type IL-18 (SEQ ID NO: 01), SEQ ID NO: 57, SEQ ID NO: 59, and SEQ ID NO: 30. [Figure 27]

[0033] Figure 1 shows plots measuring the ability of wild-type IL-18 and modified IL-18 polypeptides to bind to human IL-18 binding protein (IL-18BP). The figure shows the average free IL-18BP AlphaLISA signal on the y-axis and the dose of wild-type IL-18 and modified IL-18 polypeptides on the x-axis. The unconjugated IL-18 variants are native wild-type IL-18 (SEQ ID NO: 01), SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 30, SEQ ID NO: 207, SEQ ID NO: 239, SEQ ID NO: 241, SEQ ID NO: 242, and SEQ ID NO: 244. [Figure 28]1 shows plots measuring the ability of wild-type IL-18 and modified IL-18 polypeptides to stimulate IFN-gamma secretion by NK-92 cells. The figure shows the average IFNg alphaLISA signal on the y-axis and the dose of IL-18 polypeptide on the x-axis. The IL-18 polypeptides are native wild-type IL-18 (SEQ ID NO: 01), SEQ ID NO: 57, SEQ ID NO: 59, and SEQ ID NO: 30. [Figure 29] 1 shows plots measuring the ability of human IL-18 binding proteins to inhibit IFN-gamma secretion by NK92 cells stimulated with 2 nM wild-type IL-18 and modified IL-18 polypeptides. The figure shows the average IFNg alphaLISA signal on the y-axis and the dose of human IL-18 binding protein on the x-axis. The IL-18 polypeptides are native wild-type IL-18 (SEQ ID NO: 01), SEQ ID NO: 57, SEQ ID NO: 59, and SEQ ID NO: 30. [Figure 30] 1 shows a plot measuring the ability of wild-type IL-18 and modified IL-18 polypeptides to induce the NF-κB / AP-1-inducible secreted embryonic alkaline phosphatase (SEAP) reporter gene in Hek Blue cells expressing the IL-18 receptor. The figure shows the average SEAP reporter signal (OD620 nm) on the y-axis and the dose of IL-18 polypeptide on the x-axis. The IL-18 polypeptides are native wild-type IL-18 (SEQ ID NO: 01), SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 207, SEQ ID NO: 239, SEQ ID NO: 241, SEQ ID NO: 242, and SEQ ID NO: 244. [Figure 31] 1 shows plots measuring the ability of wild-type IL-18 and modified IL-18 polypeptides to stimulate IFN-gamma secretion by human peripheral blood mononuclear cells (PBMCs). The figure shows the average IFNg signal on the y-axis and the dose of IL-18 polypeptide on the x-axis. The IL-18 polypeptides are native wild-type IL-18 (SEQ ID NO: 01), SEQ ID NO: 57, SEQ ID NO: 59, and SEQ ID NO: 30. [Figure 32A]1 shows the effect of conjugating 30 kDa PEG to IL-18 variants on IL-18-mediated IFNγ secretion in human peripheral blood mononuclear cells. The EC50 values ​​for IL-18-mediated IFNγ secretion for IL-18 of SEQ ID NOs: 59, 4, and 9 are shown. [Figure 32B] 1 shows the effect of conjugating 30 kDa PEG to IL-18 variants on IL-18-mediated IFNγ secretion in human peripheral blood mononuclear cells. The EC50 values ​​for IL-18-mediated IFNγ secretion for IL-18 of SEQ ID NOs: 5 and 6 are shown. DETAILED DESCRIPTION OF THE INVENTION

[0029] The immune response to tumors is primarily a function of T helper type 1 (Th1) lymphocytes. Th1 responses include the secretion of cytokines IL-2, IL-12, IL-18, and IFNγ, and the production of specific cytotoxic T lymphocytes that recognize specific tumor antigens. Th1 responses are an important part of host defense against many microorganisms. However, Th1 responses are also associated with autoimmune diseases and organ transplant rejection.

[0030] Interleukin-18 (IL-18) is a proinflammatory cytokine that induces biological activities that initiate or promote host defense and inflammation after infection or injury. IL-18 has been implicated in autoimmune diseases, myocardial function, emphysema, metabolic syndrome, psoriasis, inflammatory bowel disease, hemophagocytic syndrome, macrophage activation syndrome, sepsis, and acute kidney injury. In some disease models, IL-18 plays a protective role.

[0031] IL-18 also plays a major role in the production of IFNγ from T cells and natural killer cells. IFNγ is a Th1 cytokine produced primarily by T cells, NK cells, and macrophages and is essential for innate and adaptive immunity against viral, some bacterial, and protozoan infections. IFNγ is also an important activator of macrophages and an inducer of class II major histocompatibility complex (MHC) molecule expression.

[0032] IL-18 forms a signaling complex by binding to the IL-18 alpha chain (IL-18Rα), the ligand-binding chain of mature IL-18. However, IL-18 binds to IL-18Rα with low affinity. In cells expressing the co-receptor IL-18 receptor beta chain (IL-18Rβ), a high-affinity heterodimeric complex is formed, which then activates cellular signaling.

[0033] To improve the therapeutic potential of IL-18, it may be desirable to add a half-life-extending polymer (e.g., polyethylene glycol (PEG)) to improve the pharmacokinetic (PK) or pharmacodynamic (PD) properties of IL-18. However, it has been observed that the addition of a polymer to certain residues of IL-18 results in a decrease in affinity for one or more subunits of the IL-18 receptor (e.g., the alpha subunit, the beta subunit, or the alpha / beta heterodimer), resulting in a concomitant decrease in the biological activity of IL-18 (e.g., a decrease in the potency to induce IFNγ production compared to WT IL-18). For example, PCT Publication No. WO2004091517, incorporated herein by reference in its entirety, shows that PEGylation of cysteine ​​residues (native or substituted) at positions 38, 68, 78, 121, 144, and 157 of IL-18 can, in some cases, result in a substantial loss of affinity for IL-18Rα and a substantial loss of ability to induce IFNγ production in immune cells, compared to both WT IL-18 and the unPEGylated form of the modified IL-18. Thus, identifying alternative sites for polymer attachment that reduce inactivation of IL-18 relative to other sites would provide substantial advantages.

[0034] To overcome these deficiencies, provided herein are polymer-modified IL-18 polypeptides that substantially retain IL-18R binding and biological activity. In some embodiments, the addition of a polymer to a residue of an IL-18 polypeptide provided herein allows for a half-life extended IL-18 with similar or slightly reduced IL-18R binding and / or biological activity compared to wild-type IL-18. In some embodiments, the addition of a polymer to a residue of an IL-18 polypeptide provided herein allows for a half-life extended IL-18 with similar or slightly reduced IL-18R binding and / or biological activity compared to IL-18 without the polymer attached. An exemplary diagram of a modified IL-18 polypeptide provided herein in which a polymer binds to the IL-18 receptor alpha subunit is shown in Figure 3.

[0035] While extending the half-life of IL-18 polypeptides using polymers in a manner that does not substantially reduce binding to IL-18R may provide advantages over wild-type and other variant IL-18, other considerations may also affect the therapeutic potential of modified IL-18 polypeptides. For example, the activity of IL-18 is balanced by the presence of the high-affinity, naturally occurring IL-18 binding protein (IL-18BP). IL-18BP binds to IL-18 and neutralizes its biological activity. Cell surface IL-18Rα competes with IL-18BP for IL-18 binding. Increased disease severity may be associated with an imbalance of IL-18 relative to IL-18BP, resulting in elevated levels of free IL-18 in the circulation. Figure 1 illustrates the mechanisms of action of IL-18, IFNγ production, IL-18BP production, and inhibition of IL-18 activity by IL-18BP. IL-18 induces IFNγ production, which in turn induces IL-18BP production. IL-18BP then competes with IL-18Rα to inhibit IL-18 activity. This feedback loop of IL-18BP production after IFNγ stimulation has limited the effectiveness of IL-18 as a therapeutic modality in previous attempts.

[0036] Thus, in some embodiments, IL-18 polypeptides modified with the polymers provided herein also exhibit reduced binding affinity for IL-18BP compared to wild-type IL-18. In some embodiments, the presence of the polymer results in reduced binding of the modified IL-18 polypeptide to IL-18BP. In some embodiments, the modified IL-18 polypeptide contains one or more amino acid substitutions that reduce the interaction of the modified IL-18 with IL-18BP. In some embodiments, the modified IL-18 polypeptide contains one or more amino acid substitutions that reduce the ability of IL-18 to be neutralized by IL-18BP and enhance the ability of IL-18 to bind to IL-18R. In some embodiments, the polymer-modified IL-18 polypeptides provided herein contain one or more amino acid substitutions that enable the IL-18 polypeptide to have an optimal combination of half-life, reduced neutralization of IL-18BP, and activity against IL-18R.

[0037] The following description and examples illustrate embodiments of the present disclosure. It should be understood that the present disclosure is not limited to the specific embodiments described herein and may therefore vary. Those skilled in the art will recognize that numerous variations and modifications of the present disclosure exist and are encompassed within the scope of the present disclosure.

[0038] While various features of the present disclosure may be described in the context of a single embodiment, the features may also be provided separately or in any suitable combination. Conversely, although the present disclosure may be described herein for clarity in the context of separate embodiments, the present disclosure may also be implemented in a single embodiment.

[0039] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0040] I. Definition All terms are intended to be understood as commonly understood by one of ordinary skill in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0041] The following definitions supplement those in the art and are directed to this application and are not to be construed as being in any related or unrelated context, such as any co-owned patents or applications. Although any methods and materials similar or equivalent to those described herein can be used in carrying out the tests of the present disclosure, preferred methods and materials are described herein. Therefore, the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0042] As used herein, the use of the singular includes the plural unless clearly stated otherwise. As used herein, the singular forms "a," "an," and "the" are intended to include the plural as well, unless the context clearly indicates otherwise.

[0043] In this application, the use of "or" means "and / or" unless stated otherwise. As used herein, the terms "and / or" and "any combination thereof," as well as their grammatical equivalents, may be used interchangeably. These terms may convey that any combination is specifically contemplated. For illustrative purposes only, the following phrases "A, B, and / or C" or "A, B, C, or any combination thereof" may mean "A individually, B individually, C individually, A and B, B and C, A and C, and A, B, and C." The term "or" may be used conjunctively or disjunctively unless the context specifically dictates disjunctive use.

[0044] The term "about" or "approximately" can mean within an acceptable error range for a particular value as determined by one of ordinary skill in the art, and will depend in part on the method for measuring or determining the value, i.e., the limitations of the measurement system. For example, "about" can mean within 1 or more standard deviations, as is customary in the art. Alternatively, "about" can mean a range of up to 20%, up to 15%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, within 5-fold, or within 2-fold of a value. Unless otherwise stated, when particular values ​​are described in this application and claims, the term "about" is deemed to mean within an acceptable error range for the particular value.

[0045] As used in this specification and claim(s), the words "comprising" (and any form of including, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include"), or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. It is contemplated that any embodiment discussed herein can be implemented with respect to any method or composition of the present disclosure, and vice versa. Furthermore, the compositions of the present disclosure can be used to achieve the methods of the present disclosure.

[0046] As used herein, references to "some embodiments," "embodiments," "one embodiment," or "other embodiments" mean that a particular feature, structure, or characteristic described in connection with an embodiment is included in at least some embodiments of the present disclosure, but not necessarily in all embodiments. To facilitate understanding of this disclosure, certain terms and phrases are defined below.

[0047] As used herein, reference is made to a polymer being "attached" or "covalently attached" to a residue of an IL-18 polypeptide. As used herein, "attached" or "covalently attached" means that the polymer is tethered to the indicated residue, and such tethering may include a linking group (i.e., a linker). Thus, when a polymer is "attached" or "covalently attached" to a residue, it is expressly intended that such linking groups are also encompassed.

[0048] As used herein, the term "alpha-keto amino acid" or "alpha-keto" before the name of an amino acid refers to an amino acid or amino acid derivative having a ketone functional group positioned between the carbon bearing the amino group and the carboxylic acid of the amino acid. Alpha-keto amino acids of the present disclosure have the structure shown in the following formula: TIFF2025508826000002.tif31135 where R is the side chain of any natural or unnatural amino acid. The R functional group can be either L- or D-oriented according to standard amino acid nomenclature. In a preferred embodiment, the alpha-keto amino acid is L-oriented. When the term "alpha-keto" is used before the name of a conventional natural amino acid (e.g., alpha-ketoleucine, alpha-ketophenylalanine, etc.) or a common unnatural amino acid (e.g., alpha-ketonorleucine, alpha-ketoO-methyl-homoserine, etc.), it is intended that the referenced alpha-keto amino acid corresponds to the above formula with the side chain of the referenced amino acid. Where an alpha-keto amino acid residue is recited in a peptide or polypeptide sequence herein, it is intended that protected forms of the relevant alpha-keto amino acid are also encompassed (e.g., for sequences terminating in a C-terminal alpha-keto amino acid, the terminal carboxylic acid group may be appropriately capped with a protecting group such as a tert-butyl group, or the ketone group with an acetal protecting group). Other protecting groups that are encompassed are well known in the art.

[0049] Binding affinity refers to the strength of the binding interaction between a single molecule and its ligand / binding partner. A higher binding affinity indicates a stronger binding than a lower binding affinity. In some cases, binding affinity is measured by the dissociation constant (KD) between two related molecules. When comparing KD values, a binding interaction with a lower value has a higher binding affinity than a binding interaction with a higher value. For protein-ligand interactions, KD is calculated according to the following formula: TIFF2025508826000003.tif12135In the formula, [L] is the concentration of the ligand, [P] is the concentration of the protein, and [LP] is the concentration of the ligand / protein complex.

[0050] As used herein, a particular amino acid sequence (e.g., a polypeptide sequence) is referred to as having a certain percent sequence identity with a reference sequence or as a residue at a position corresponding to a position in the reference sequence. Sequence identity is measured using the protein-protein BLAST algorithm, using the following parameters: matrix BLOSUM62, gap cost: 11, extension: 1, and composition-adjusted conditional composition score matrix adjustment. This alignment algorithm is also used to assess whether residues are at "corresponding" positions through an analysis of the alignment of the two sequences being compared.

[0051] As used herein, reference is made to a polymer being "attached" or "covalently attached" to a residue of an IL-18 polypeptide. As used herein, "attached" or "covalently attached" means that the polymer is tethered to the indicated residue, and such tethering may include a linking group (i.e., a linker). Thus, when a polymer is "attached" or "covalently attached" to a residue, it is expressly intended that such linking groups are also encompassed.

[0052] Unless otherwise specified, it is contemplated that "protected" forms of amino acids (e.g., those containing chemical protecting groups attached to the amino acid's functional group, particularly its side chain, as well as at other points on the amino acid) qualify as the same amino acid as the "unprotected" form for purposes of sequence identity, particularly in the case of chemically synthesized polypeptides. Such protected forms are also contemplated to be encompassed by the SEQ ID NOs provided herein. Non-limiting examples of protecting groups that may be included include fluorenylmethyloxycarbonyl (Fmoc), triphenylmethyl (trityl or trt), tert-butyloxycarbonyl (Boc), 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf), acetamidomethyl (Acm), tert-butyl (tBu or OtBu), 2,2-dimethyl-1-(4-methoxyphenyl)propane-1,3-diol ketal or acetal, and 2,2-dimethyl-1-(2-nitrophenyl)propane-1,3-diol ketal or acetal. Other protecting groups known in the art are also included. Similarly, modified forms of natural amino acids are also intended to qualify as the natural forms of the amino acids for purposes of sequence identity. For example, amino acids (which are covalently modified) that contain a side chain heteroatom that can be covalently modified (e.g., to add a conjugation handle, optionally via a linker), such as lysine, glutamine, glutamic acid, asparagine, aspartic acid, cysteine, or tyrosine, are counted as basic amino acids (see, e.g., Structure 2 below, which counts as a lysine for purposes of sequence identity and SEQ ID NO.) Similarly, amino acids that contain another group added to the C- or N-terminus are counted as basic amino acids.

[0053] In some cases, the peptides provided herein may be depsipeptides. For example, depsipeptide bonds result from certain ligation reactions (e.g., KAHA ligation) described herein during the synthesis of synthetic IL-18 and related precursor peptides. In particular, hydroxyl-containing amino acids (e.g., serine, threonine, and homoserine) form depsipeptide bonds with adjacent amino acids on the N-terminus. Thus, when a SEQ ID NO lists an amino acid sequence, it is intended that depsipeptide-type sequences are also encompassed, particularly at homoserine residues.

[0054] The term "pharmaceutically acceptable" means approved or approvable by a regulatory agency of the Federal or state government, or listed in the United States Pharmacopoeia or other generally recognized pharmacopoeias for use in animals, including humans.

[0055] The term "pharmaceutically acceptable excipient, carrier, or diluent" refers to an excipient, carrier, or diluent that can be administered to a subject together with a drug and that does not destroy its pharmacological activity and is non-toxic when administered in a dosage sufficient to deliver a therapeutic amount of the drug.

[0056] A "pharmaceutically acceptable salt" suitable for the present disclosure can be an acid or base salt generally considered in the art to be suitable for use in contact with the tissues of humans or animals without undue toxicity, irritation, allergic response, or other problem or impairment. Such salts include mineral and organic acid salts of basic residues such as amines, and alkali or organic salts of acidic residues such as carboxylic acids. Specific pharmaceutical salts include those of hydrochloric acid, phosphoric acid, hydrobromic acid, malic acid, glycolic acid, fumaric acid, sulfuric acid, sulfamic acid, sulfanilic acid, formic acid, toluenesulfonic acid, methanesulfonic acid, benzenesulfonic acid, ethanedisulfonic acid, 2-hydroxyethylsulfonic acid, nitric acid, benzoic acid, 2-acetoxybenzoic acid, citric acid, tartaric acid, lactic acid, stearic acid, salicylic acid, glutamic acid, ascorbic acid, pamoic acid, succinic acid, fumaric acid, maleic acid, propionic acid, hydroxymaleic acid, hydroiodic acid, phenylacetic acid, alkanoic acids, such as acetic acid, HOOC-(CH2) n- COOH (where n is 0, 2, 3, 4, or 4). Similarly, pharmaceutically acceptable cations include, but are not limited to, sodium, potassium, calcium, aluminum, lithium, and ammonium. One skilled in the art will recognize from this disclosure and knowledge in the art that additional pharmaceutically acceptable salts include those described in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, PA, p. 1418 (1985). In general, pharmaceutically acceptable acid or base salts can be synthesized from a parent compound containing a basic or acidic moiety by any conventional chemical method. Briefly, such salts can be prepared by reacting the free acid or base form of these compounds with a stoichiometric amount of an appropriate base or acid in a suitable solvent.

[0057] It is understood that ranges provided herein are shorthand notations for all values ​​within that range. For example, the range of 1 to 50 is understood to include any number, combination of numbers, or subrange from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50, as well as all intervening decimal points between the aforementioned integers, for example, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, and 1.9. For subranges, "nested subranges" extending from either end of the range are specifically contemplated. For example, nested subranges of the exemplary range 1 to 50 could include, in one direction, 1 to 10, 1 to 20, 1 to 30, and 1 to 40, or in the other direction, 50 to 40, 50 to 30, 50 to 20, and 50 to 10.

[0058] The term "subject" refers to an animal that is the object of treatment, observation, or experiment. By way of example, a subject includes a mammal, including, but not limited to, a human or a non-human mammal (such as a non-human primate, cow, horse, dog, sheep, or cat).

[0059] Certain formulas and other figures provided herein depict triazole reaction products resulting from azide-alkyne cycloaddition reactions. While such formulas generally depict only a single regioisomer of the resulting triazole formed in the reaction, these formulas are intended to encompass both resulting regioisomers. Thus, these formulas may be used to represent a single regioisomer (e.g., Only TIFF2025508826000004.tif16135 is illustrated, but the other positional isomer (e.g., TIFF2025508826000005.tif16135 is also intended to be encompassed.

[0060] The term "optional" or "optionally" means that the subsequently described event or circumstance may, but need not, occur, and that the description includes cases where the event or circumstance occurs and cases where it does not occur.

[0061] The term "moiety" refers to a specific segment or functional group of a molecule. A chemical moiety is a chemical entity that is often recognized as embedded in or attached to a molecule.

[0062] As used herein, "conjugation handle" refers to a reactive group capable of forming a bond upon contact with a complementary reactive group. In some cases, the conjugation handle preferably does not have substantial reactivity with other molecules that do not contain the intended complementary reactive group. Non-limiting examples of conjugation handles, their respective complementary conjugation handles, and corresponding reaction products can be found in the table below. Although the table headings place certain reactive groups under the heading of "conjugation handle" or "complementary conjugation handle," any reference to a conjugation handle is instead intended to include the complementary conjugation handle listed in the table (e.g., if trans-cyclooctene is the conjugation handle, tetrazine would be the complementary conjugation handle). In some cases, amine conjugation handles and conjugation handles complementary to amines are less preferred for use in biological systems due to the ubiquity of amines in biological systems and the increased likelihood of off-target conjugation. TIFF2025508826000006.tif111135

[0063] Throughout this application, prefix words are used before the term "conjugation handle" to indicate the functional group to which the conjugation handle is linked. For example, a "protein conjugation handle" is a conjugation handle attached to a protein (either directly or via a linker), an "antibody conjugation handle" is a conjugation handle attached to an antibody (either directly or via a linker), and a "linker conjugation handle" is a conjugation handle attached to a linker group (e.g., a bifunctional linker used to link a synthetic protein and an antibody).

[0064] As used herein, the term "number average molecular weight" (Mn) means the statistical average molecular weight of all individual units in a sample and is defined by equation (1): TIFF2025508826000007.tif17135, M i is the molecular weight of one molecule, and N i is the number of molecular weights.

[0065] As used herein, the term "weight average molecular weight" (Mw) means the number defined by formula (2). TIFF2025508826000008.tif17135, M i is the molecular weight of one molecule, and N i is the number of molecular weights.

[0066] As used herein, "peak molecular weight" (Mp) means the molecular weight of the highest peak in a given analytical method (e.g., mass spectrometry, size exclusion chromatography, dynamic light scattering, analytical centrifugation, etc.).

[0067] The term "alkyl" refers to a straight or branched hydrocarbon chain radical having from 1 to 20 carbon atoms and attached to the rest of the molecule by a single bond. An alkyl containing up to 10 carbon atoms is referred to as a C1-C10 alkyl; similarly, for example, an alkyl containing up to 6 carbon atoms is a C1-C6 alkyl. Alkyl groups containing other numbers of carbon atoms (and other moieties defined herein) are similarly represented. Alkyl groups include, but are not limited to, C1-C10 alkyl, C1-C9 alkyl, C1-C8 alkyl, C1-C7 alkyl, C1-C6 alkyl, C1-C5 alkyl, C1-C4 alkyl, C1-C3 alkyl, C1-C2 alkyl, C2-C8 alkyl, C3-C8 alkyl, and C4-C8 alkyl. Representative alkyl groups include, but are not limited to, methyl, ethyl, <<-propyl, 1-methylethyl ( / -propyl), <<-butyl, / -butyl, 5-butyl, <<-pentyl, 1,1-dimethylethyl ( / -butyl), 3-methylhexyl, 2-methylhexyl, 1-ethyl-propyl, and the like. In some embodiments, alkyl is methyl or ethyl. In some embodiments, alkyl is -CH(CH3)2 or -C(CH3)3. Unless stated otherwise in the specification, an alkyl group may be optionally substituted. "Alkylene" or "alkylene chain" refers to a straight or branched divalent hydrocarbon chain connecting the remainder of the molecule to a radical group. In some embodiments, alkylene is -CFF-, -CH2CH2-, or -CH2CH2CH2-. In some embodiments, alkylene is -CH2-. In some embodiments, alkylene is -CH2CH2-. In some embodiments, alkylene is -CH2CH2CH2-. Unless stated otherwise in the specification, an alkylene group may be optionally substituted.

[0068] The term "alkenylene" or "alkenylene chain" refers to a straight or branched divalent hydrocarbon chain having at least one carbon-carbon double bond connecting the rest of the molecule to a radical group. In some embodiments, alkenylene is -CH=CH-, -CEhCEUCH-, or -CH=CHCH-. In some embodiments, alkenylene is -CH=CH-. In some embodiments, alkenylene is -CHCH=CH-. In some embodiments, alkenylene is -CH=CHCH-. In some embodiments, alkenylene is -CH=CHCH-.

[0069] The term "alkynyl" refers to a type of alkyl group in which at least one carbon-carbon triple bond is present. In one embodiment, an alkenyl group has the formula -C≡CR X wherein R x refers to the remainder of the alkynyl group. In some embodiments, R x is H or alkyl. In some embodiments, alkynyl is selected from ethynyl, propynyl, butynyl, pentynyl, hexynyl, etc. Non-limiting examples of alkynyl groups include -C≡CH, -C≡CCH, -C≡CCHCH, and -CHC≡CH.

[0070] The term "alkynyl" refers to a type of alkyl group in which at least one carbon-carbon triple bond is present. In one embodiment, an alkenyl group has the formula -C≡CR X wherein R x refers to the remainder of the alkynyl group. In some embodiments, R x is H or alkyl. In some embodiments, alkynyl is selected from ethynyl, propynyl, butynyl, pentynyl, hexynyl, etc. Non-limiting examples of alkynyl groups include -C≡CH, -C≡CCH, -C≡CCHCH, and -CHC≡CH.

[0071] The term "aryl" refers to a radical containing at least one aromatic ring, where each atom forming the ring is a carbon atom. An aryl group can be optionally substituted. Examples of aryl groups include, but are not limited to, phenyl and naphthyl. In some embodiments, an aryl is phenyl. Depending on the structure, an aryl group can be a monoradical or a diradical (i.e., an arylene group). Unless otherwise specified herein, the term "aryl" or the prefix "ar-" (e.g., as in "aralkyl") is meant to include optionally substituted aryl radicals. In some embodiments, an aryl group includes a partially reduced cycloalkyl group as defined herein (e.g., 1,2-dihydronaphthalene). In some embodiments, an aryl group includes a fully reduced cycloalkyl group as defined herein (e.g., 1,2,3,4-tetrahydronaphthalene). When an aryl includes a cycloalkyl group, the aryl is attached to the remainder of the molecule via a carbon atom of the aromatic ring. The aryl radical can be a monocyclic or polycyclic (eg, bicyclic, tricyclic, or tetracyclic) ring system, which can include fused, spiro, or bridged ring systems.

[0072] The term "cycloalkyl" refers to a monocyclic or polycyclic non-aromatic radical in which each of the atoms forming the ring (i.e., skeletal atoms) is a carbon atom. In some embodiments, the cycloalkyl is saturated or partially unsaturated. In some embodiments, the cycloalkyl is a spirocyclic or bridged compound. In some embodiments, the cycloalkyl is fused to an aromatic ring (in which case the cycloalkyl is attached through a non-aromatic ring carbon atom). Cycloalkyl groups include groups having 3 to 10 ring atoms. Representative cycloalkyls include, but are not limited to, cycloalkyls having 3 to 10 carbon atoms, 3 to 8 carbon atoms, 3 to 6 carbon atoms, or 3 to 5 carbon atoms. Monocyclic cycloalkyl radicals include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. In some embodiments, the monocyclic cycloalkyl is cyclopentyl. In some embodiments, the monocyclic cycloalkyl is cyclopentenyl or cyclohexenyl. In some embodiments, the monocyclic cycloalkyl is cyclopentenyl. Polycyclic radicals include, for example, adamantyl, 1,2-dihydronaphthalenyl, 1,4-dihydronaphthalenyl, tetraynyl, decalinyl, 3,4-dihydronaphthalenyl-1(2H)-one, spiro[2.2]pentyl, norbornyl, and bicycle[1.1.1]pentyl. Unless stated otherwise in the specification, cycloalkyl groups may be optionally substituted.

[0073] The terms "heteroalkylene" or "heteroalkylene chain" refer to a straight or branched divalent heteroalkyl chain connecting the remainder of the molecule to a radical group. Unless stated otherwise in the specification, a heteroalkyl or heteroalkylene group may be optionally substituted as described below. Representative heteroalkylene groups include, but are not limited to, -CH2-O-CH2-, -CH2-N(alkyl)-CH2-, -CH2-N(aryl)-CH2-, -OCH2CHO-, -OCH2CHOCH2CHO-, or -OCH2CHOCH2CHOCH2CHO-.

[0074] The term "heterocycloalkyl" refers to a cycloalkyl group containing at least one heteroatom selected from nitrogen, oxygen, and sulfur. Unless otherwise specified in the specification, a heterocycloalkyl radical can be a monocyclic or bicyclic ring system, which can include fused (when fused to an aryl or heteroaryl ring, the heterocycloalkyl is attached through a non-aromatic ring atom) or bridged ring systems. The nitrogen, carbon, or sulfur atoms in the heterocyclyl radical can be optionally oxidized. The nitrogen atom can be optionally quaternized. The heterocycloalkyl radical can be partially or fully saturated. Examples of heterocycloalkyl radicals include, but are not limited to, dioxolanyl, thienyl[1,3]dithianyl, tetrahydroquinolyl, tetrahydroisoquinolyl, decahydroquinolyl, decahydroisoquinolyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, quinoclidinyl, thiazolidinyl, tetrahydrofuryl, trithianyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1-oxo-thiomorpholinyl, 1,1-dioxo-thiomorpholinyl. The term heterocycloalkyl also includes all ring forms of sugars, including, but not limited to, monosaccharides, disaccharides, and oligosaccharides. Unless otherwise noted, heterocycloalkyls have 2-12 carbons in the ring. In some embodiments, heterocycloalkyls have 2-10 carbons in the ring. In some embodiments, heterocycloalkyls have 2-10 carbons in the ring and contain 1 or 2 N atoms. In some embodiments, heterocycloalkyls have 2-10 carbons in the ring and contain 3 or 4 N atoms. In some embodiments, heterocycloalkyls have 2-12 carbons, 0-2 N atoms, 0-2 O atoms, 0-2 P atoms, and 0-1 S atoms in the ring.In some embodiments, a heterocycloalkyl has 2 to 12 carbons, 1 to 3 N atoms, 0 to 1 O atoms, and 0 to 1 S atoms within the ring. When referring to the number of carbon atoms in a heterocycloalkyl, it is understood that the number of carbon atoms in the heterocycloalkyl is not the same as the total number of atoms (including heteroatoms) comprising the heterocycloalkyl (i.e., the skeletal atoms of the heterocycloalkyl ring). Unless stated otherwise in the specification, a heterocycloalkyl group may be optionally substituted.

[0075] The term "heteroaryl" refers to an aryl group containing one or more ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, heteroaryl is monocyclic or bicyclic. Illustrative examples of monocyclic heteroaryls include pyridinyl, imidazolyl, pyrimidinyl, pyrazolyl, triazolyl, pyrazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxazolyl, isothiazolyl, pyrrolyl, pyridazinyl, triazinyl, oxadiazolyl, thiadiazolyl, furazanyl, indolizine, indole, benzofuran, benzothiophene, indazole, benzimidazole, purine, quinolizine, quinoline, isoquinoline, cinnoline, phthalazine, quinazoline, quinoxaline, 1,8-naphthyridine, and pteridine. Illustrative examples of monocyclic heteroaryls include pyridinyl, imidazolyl, pyrimidinyl, pyrazolyl, triazolyl, pyrazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxazolyl, isothiazolyl, pyrrolyl, pyridazinyl, triazinyl, oxadiazolyl, thiadiazolyl, and furazanyl. Illustrative examples of bicyclic heteroaryls include indolizine, indole, benzofuran, benzothiophene, indazole, benzimidazole, purine, quinolizine, quinoline, isoquinoline, cinnoline, phthalazine, quinazoline, quinoxaline, 1,8-naphthyridine, and pteridine. In some embodiments, heteroaryl is pyridinyl, pyrazinyl, pyrimidinyl, thiazolyl, thienyl, thiadiazolyl, or furyl. In some embodiments, heteroaryls contain 0 to 6 N atoms in the ring. In some embodiments, the heteroaryl contains 1-4 N atoms in the ring. In some embodiments, the heteroaryl contains 4-6 N atoms in the ring. In some embodiments, the heteroaryl contains 0-4 N atoms, 0-1 O atoms, 0-1 P atoms, and 0-1 S atoms in the ring. In some embodiments, the heteroaryl contains 1-4 N atoms, 0-1 O atoms, and 0-1 S atoms in the ring. In some embodiments, the heteroaryl is a C1-C9 heteroaryl.In some embodiments, the monocyclic heteroaryl is a C1-C5 heteroaryl. In some embodiments, the monocyclic heteroaryl is a 5- or 6-membered heteroaryl. In some embodiments, the bicyclic heteroaryl is a C6-C9 heteroaryl. In some embodiments, the heteroaryl group comprises a partially reduced cycloalkyl or heterocycloalkyl group as defined herein (e.g., 7,8-dihydroquinoline). In some embodiments, the heteroaryl group comprises a fully reduced cycloalkyl or heterocycloalkyl group as defined herein (e.g., 5,6,7,8-tetrahydroquinoline). When the heteroaryl comprises a cycloalkyl or heterocycloalkyl group, the heteroaryl is attached to the remainder of the molecule through a carbon or heteroatom of the heteroaromatic ring. The heteroaryl radical can be a monocyclic or polycyclic (e.g., bicyclic, tricyclic, or tetracyclic) ring system, which can include fused, spiro, or bridged ring systems.

[0076] The term "optionally substituted" or "substituted" means that the referenced group is optionally substituted with one or more additional groups individually and independently selected from D, halogen, -CN, -NH, -NH(alkyl), -N(alkyl), -OH, -COH, -COalkyl, -C(=O)NH, -C(=O)NH(alkyl), -C(=O)N(alkyl), -S(=O)NH, -S(=O)NH(alkyl), -S(=O)N(alkyl), alkyl, cycloalkyl, fluoroalkyl, heteroalkyl, alkoxy, fluoroalkoxy, heterocycloalkyl, aryl, heteroaryl, aryloxy, alkylthio, arylthio, alkylsulfoxide, arylsulfoxide, alkylsulfone, and arylsulfone. In some other embodiments, the optional substituents are independently selected from D, halogen, —CN, —NH, —NH(CH), —N(CH), —OH, —COH, —CO(C-C alkyl), —C(═O)NH, —C(═O)NH(C-C alkyl), —C(═O)N(C-C alkyl), —S(═O)NH, —S(═O)NH(C-C alkyl), —S(═O)N(C-C alkyl), C-C alkyl, C-C cycloalkyl, C-C fluoroalkyl, C-C heteroalkyl, C-C alkoxy, C-C fluoroalkoxy, —SC-C alkyl, —S(═O)C-C alkyl, and —S(═O)C-C alkyl. In some embodiments, optional substituents are independently selected from D, halogen, -CN, -NH, -OH, -NH(CH), -N(CH), -NH(cyclopropyl), -CH, -CHCH, -CF, -OCH, and -OCF. In some embodiments, substituents are substituted with one or two of the foregoing groups. In some embodiments, optional substituents on aliphatic carbon atoms (acyclic or cyclic) include oxo (=O).

[0077] II. Modified IL-18 Polypeptides Certain aspects of the present disclosure relate to modified IL-18 polypeptides useful as therapeutic agents. The modified IL-18 polypeptides provided herein can be used as immunotherapies or as part of other immunotherapeutic regimens. The modified IL-18 polypeptides provided herein exhibit reduced binding to IL-18 binding protein (IL-18BP) compared to WT IL-18 and / or improved IL-18 receptor (IL-18R) signaling activity in the presence of IL-18BP compared to WT IL-18. In certain embodiments, the modified IL-18 polypeptides provided herein exhibit reduced binding to IL-18 binding protein (IL-18BP) compared to WT IL-18 and improved IL-18 receptor (IL-18R) signaling activity in the presence of IL-18BP compared to WT IL-18.

[0078] IIa. Site-specific modification of modified IL-18 polypeptides In some embodiments, the modified IL-18 polypeptides described herein comprise one or more modifications at one or more amino acid residues. As used herein, the "modifications" of the modified IL-18 polypeptides are relative to SEQ ID NO: 1. In some embodiments, the numbering of residue positions in the modified IL-18 polypeptides is based on SEQ ID NO: 1 as the reference sequence.

[0079] Modifications of the polypeptides described herein include mutations, addition of various functional groups, deletion of amino acids, addition of amino acids, or any other alteration of the wild-type protein or protein fragment. Functional groups that can be added to the polypeptide include polymers, linkers, alkyl groups, detectable molecules such as chromophores or fluorophores, reactive functional groups, or any combination thereof. In some embodiments, functional groups are added to individual amino acids of the polypeptide. In some embodiments, functional groups are added site-specifically to the polypeptide.

[0080] In some embodiments, the modified IL-18 polypeptides described herein contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 modified amino acid residues.

[0081] In some embodiments, the modified IL-18 polypeptides described herein comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, or at least 9 amino acid substitutions, wherein the amino acid substitutions are relative to SEQ ID NO: 1. In some embodiments, the modified IL-18 polypeptides comprise between 1 and 9 amino acid substitutions. In some embodiments, the modified IL-18 polypeptide comprises 1 or 2 amino acid substitutions, 1 to 3 amino acid substitutions, 1 to 4 amino acid substitutions, 1 to 5 amino acid substitutions, 1 to 6 amino acid substitutions, 1 to 7 amino acid substitutions, 1 to 8 amino acid substitutions, 2 to 3 amino acid substitutions, 2 to 4 amino acid substitutions, 2 to 5 amino acid substitutions, 2 to 6 amino acid substitutions, 2 to 7 amino acid substitutions, 2 to 8 amino acid substitutions, 2 to 9 amino acid substitutions, 3 or 4 amino acid substitutions, 3 to 5 amino acid substitutions, 3 to 6 amino acid substitutions, 3 to 7 amino acid substitutions, 3 to 9 amino acid substitutions, 4 or 5 amino acid substitutions, 4 to 6 amino acid substitutions, 4 to 7 amino acid substitutions, 4 to 9 amino acid substitutions, 5 or 6 amino acid substitutions, 5 to 7 amino acid substitutions, 5 to 9 amino acid substitutions, 6 or 7 amino acid substitutions, 6 to 9 amino acid substitutions, or 7 to 9 amino acid substitutions. In some embodiments, the modified IL-18 polypeptide comprises 3 amino acid substitutions, 4 amino acid substitutions, 5 amino acid substitutions, 6 amino acid substitutions, 7 amino acid substitutions, or 9 amino acid substitutions, hi some embodiments, the modified IL-18 polypeptide comprises up to 4 amino acid substitutions, 5 amino acid substitutions, 6 amino acid substitutions, 7 amino acid substitutions, or 9 amino acid substitutions.

[0082] In some embodiments, the modified IL-18 polypeptides described herein comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, or at least 9 additional naturally occurring amino acid substitutions, wherein the naturally occurring amino acid substitutions are relative to SEQ ID NO: 1. In some embodiments, the modified IL-18 polypeptides comprise between 1 and 9 naturally occurring amino acid substitutions. In some embodiments, the modified IL-18 polypeptides include 1 or 2 naturally occurring amino acid substitutions, 1 to 3 naturally occurring amino acid substitutions, 1 to 4 naturally occurring amino acid substitutions, 1 to 5 naturally occurring amino acid substitutions, 1 to 6 naturally occurring amino acid substitutions, 1 to 7 naturally occurring amino acid substitutions, 1 to 8 naturally occurring amino acid substitutions, 2 to 3 naturally occurring amino acid substitutions, 2 to 4 naturally occurring amino acid substitutions, 2 to 5 naturally occurring amino acid substitutions, 2 to 6 naturally occurring amino acid substitutions, 2 to 7 naturally occurring amino acid substitutions, 2 to 8 naturally occurring amino acid substitutions, 2 to 9 naturally occurring amino acid substitutions, 3 or 4 naturally occurring amino acid substitutions, 3 to 5 naturally occurring amino acid substitutions, 3 to 6 naturally occurring amino acid substitutions, 3 to 7 naturally occurring amino acid substitutions, 3 to 9 naturally occurring amino acid substitutions, 4 or 5 naturally occurring amino acid substitutions, 4 to 6 naturally occurring amino acid substitutions, 4 to 7 amino acid substitutions, 4 to 9 naturally occurring amino acid substitutions, 5 or 6 naturally occurring amino acid substitutions, 5 to 7 amino acid substitutions, 5 to 9 naturally occurring amino acid substitutions, 6 or 7 naturally occurring amino acid substitutions, 6 to 9 naturally occurring amino acid substitutions, or 7 to 9 naturally occurring amino acid substitutions. In some embodiments, the modified IL-18 polypeptide comprises three naturally occurring amino acid substitutions, four naturally occurring amino acid substitutions, five amino acid substitutions, six naturally occurring amino acid substitutions, seven naturally occurring amino acid substitutions, or nine naturally occurring amino acid substitutions. In some embodiments, the modified IL-18 polypeptide comprises up to four naturally occurring amino acid substitutions, five naturally occurring amino acid substitutions, six naturally occurring amino acid substitutions, seven naturally occurring amino acid substitutions, or nine naturally occurring amino acid substitutions.

[0083] In some embodiments, the modified IL-18 polypeptide further comprises up to 10 non-canonical amino acid substitutions. In some embodiments, the modified IL-18 polypeptide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional non-natural amino acid substitutions.

[0084] In some embodiments, the modified IL-18 polypeptide further comprises unnatural amino acid substitutions at residues M33, M51, N60, M86, M113, and / or M150. In some embodiments, the unnatural amino acid residues replacing the methionine are each independently norleucine or O-methyl-homoserine. In some embodiments, the modified IL-18 polypeptide further comprises unnatural amino acid substitutions at residues 31, 75, and 116. In some embodiments, the modified IL-18 polypeptide further comprises homoserine (Hse)31, Hse75, and Hse116. In some embodiments, the modified IL-18 polypeptide further comprises unnatural amino acid substitutions at residues 31, 63, and 116. In some embodiments, the modified IL-18 polypeptide further comprises homoserine (Hse)31, Hse63, and Hse116. In some embodiments, the modified IL-18 polypeptide further comprises unnatural amino acid substitutions at residues 31, 63, 75, and 116. In some embodiments, the modified IL-18 polypeptide further comprises homoserine (Hse) 31, Hse 63, Hse 75, and Hse 116. In some embodiments, the modified IL-18 polypeptide further comprises unnatural amino acid substitutions at residues 31, 67, 75, and 116. In some embodiments, the modified IL-18 polypeptide further comprises homoserine (Hse) 31, Hse 67, Hse 75, and Hse 116. In some embodiments, the modified IL-18 polypeptide further comprises unnatural amino acid substitutions at residues 31, 57, 75, and 116. In some embodiments, the modified IL-18 polypeptide further comprises homoserine (Hse) 31, Hse 57, Hse 75, and Hse 116. In some embodiments, the modified IL-18 polypeptide further comprises unnatural amino acid substitutions at residues 31, 50, 75, and 116. In some embodiments, the modified IL-18 polypeptide further comprises homoserine (Hse) 31, Hse 50, Hse 75, and Hse 116. In some embodiments, the modified IL-18 polypeptide further comprises non-natural amino acid substitutions at residues 31, 50, 75, and 121.In some embodiments, the modified IL-18 polypeptide further comprises homoserine (Hse)31, Hse50, Hse75, and Hse121.

[0085] In certain embodiments, a modified IL-18 polypeptide comprises at least one glycine residue attached to the N-terminus of the IL-18 polypeptide. In certain embodiments, a modified IL-18 polypeptide comprises a chain of glycine residues attached to the N-terminus of the polypeptide, wherein the chain of glycine residues comprises 1 to 15 glycine residues. In certain embodiments, a modified IL-18 polypeptide comprises a chain of 1 to 10 glycine residues attached to the N-terminus of the IL-18 polypeptide. In certain embodiments, a modified IL-18 polypeptide comprises a chain of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 glycine residues, or any range therebetween, attached to the N-terminus of the IL-18 polypeptide. In certain embodiments, a modified IL-18 polypeptide comprises a glycine residue attached to the N-terminus of the IL-18 polypeptide. In certain embodiments, a modified IL-18 polypeptide comprises a chain of two glycine residues attached to the N-terminus of the IL-18 polypeptide. In certain embodiments, the modified IL-18 polypeptide comprises a chain of three glycine residues attached to the N-terminus of the IL-18 polypeptide. In certain embodiments, the modified IL-18 polypeptide comprises a chain of four glycine residues attached to the N-terminus of the IL-18 polypeptide. In certain embodiments, the modified IL-18 polypeptide comprises a chain of five glycine residues attached to the N-terminus of the IL-18 polypeptide. In certain embodiments, the modified IL-18 polypeptide comprises a chain of glycine residues attached to the N-terminus of the polypeptide, the chain of glycine residues being 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 1 to 12, 1 to 15, 2 to 3, 2 to 4, 2 to 5, 2 to 6, 2 to 7, 2 to 8, 2 to 9, 2 to 10, 2 to 12, 2 to 15, 3 to 4, 3 to 5, 3 to 6, 3 to 7, 3 to 8, 3 to 9, 3 to 10, 3 to 12 , 3-15, 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 4-12, 4-15, 5-6, 5-7, 5-8, 5-9, 5-10, 5-12, 5-15, 6-7, 6-8, 6-9, 6-10, 6-12, 6-15, 7-8, 7-9, 7-10, 7-12, 7-15, 8-9, 8-10, 8-12, 8-15, 9-10, 9-12, 9-15, 10-12, 10-15, or 12-15 glycine residues.In certain embodiments, a modified IL-18 polypeptide comprises a chain of glycine residues attached to the N-terminus of the polypeptide, wherein the chain of glycine residues comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, or 15 glycine residues. In certain embodiments, a modified IL-18 polypeptide comprises a chain of glycine residues attached to the N-terminus of the polypeptide, wherein the chain of glycine residues comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 12 glycine residues. In certain embodiments, a modified IL-18 polypeptide comprises a chain of glycine residues attached to the N-terminus of the polypeptide, wherein the chain of glycine residues comprises up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, or 15 glycine residues.

[0086] In certain embodiments, the modified IL-18 polypeptide comprises a substitution at residue Y1. In certain embodiments, the modified IL-18 polypeptide may comprise a Y1M substitution. Unless otherwise stated, the numbering of residue positions provided in this paragraph and elsewhere in this disclosure is based on SEQ ID NO: 1 as the reference sequence. Unless otherwise stated, the amino acid substitutions provided in this paragraph and elsewhere in this disclosure are with respect to SEQ ID NO: 1 as the reference sequence. In certain embodiments, the modified IL-18 polypeptide comprises a substitution at residue F2. In certain embodiments, the modified IL-18 polypeptide may comprise an F2A substitution. In certain embodiments, the modified IL-18 polypeptide comprises a substitution at residue E6. In certain embodiments, the modified IL-18 polypeptide comprises an E6K substitution. In certain embodiments, the modified IL-18 polypeptide comprises an E6R substitution. In certain embodiments, the modified IL-18 polypeptide comprises a substitution at residue K8. In certain embodiments, the modified IL-18 polypeptide comprises a K8L substitution. In certain embodiments, the modified IL-18 polypeptide comprises a K8E substitution. In certain embodiments, the modified IL-18 polypeptide comprises a K8R substitution. In certain embodiments, the modified IL-18 polypeptide comprises a substitution at residue V11. In certain embodiments, the modified IL-18 polypeptide may comprise a residue V11I substitution. In certain embodiments, the modified IL-18 polypeptide comprises a substitution at residue E31. In certain embodiments, the modified IL-18 polypeptide comprises a residue E31A substitution. In certain embodiments, the modified IL-18 polypeptide comprises a substitution at residue T34. In certain embodiments, the modified IL-18 polypeptide comprises a T34A substitution. In certain embodiments, the modified IL-18 polypeptide comprises a substitution at residue D35. In certain embodiments, the modified IL-18 polypeptide comprises a D35A substitution. In certain embodiments, the modified IL-18 polypeptide comprises a substitution at residue S36. In certain embodiments, the modified IL-18 polypeptide comprises an S36A substitution. In certain embodiments, the modified IL-18 polypeptide comprises a substitution at residue D37.In certain embodiments, the modified IL-18 polypeptide comprises a D37A substitution. In certain embodiments, the modified IL-18 polypeptide comprises a substitution at residue D40. In certain embodiments, the modified IL-18 polypeptide comprises a D40A substitution. In certain embodiments, the modified IL-18 polypeptide comprises a substitution at residue N41. In certain embodiments, the modified IL-18 polypeptide comprises an N41A substitution. In certain embodiments, the modified IL-18 polypeptide comprises a substitution at residue 149. In certain embodiments, the modified IL-18 polypeptide comprises a I49E substitution. In certain embodiments, the modified IL-18 polypeptide comprises a I49M substitution. In certain embodiments, the modified IL-18 polypeptide comprises a I49R substitution. In certain embodiments, the modified IL-18 polypeptide comprises a substitution at residue K53. In certain embodiments, the modified IL-18 polypeptide comprises a K53A substitution. In certain embodiments, the modified IL-18 polypeptide comprises a substitution at residue D54. In certain embodiments, the modified IL-18 polypeptide comprises a D54A substitution. In certain embodiments, the modified IL-18 polypeptide comprises a substitution at residue S55. In certain embodiments, the modified IL-18 polypeptide comprises a S55A substitution. In certain embodiments, the modified IL-18 polypeptide comprises a S55T substitution. In certain embodiments, the modified IL-18 polypeptide comprises a S55H substitution. In certain embodiments, the modified IL-18 polypeptide comprises a S55R substitution. In certain embodiments, the modified IL-18 polypeptide comprises a substitution at residue T63. In certain embodiments, the modified IL-18 polypeptide comprises a T63A substitution. In certain embodiments, the modified IL-18 polypeptide comprises a substitution at residue Q103. In certain embodiments, the modified IL-18 polypeptide comprises a Q103R substitution. In certain embodiments, the modified IL-18 polypeptide comprises a Q103E substitution. In certain embodiments, the modified IL-18 polypeptide comprises a Q103K substitution. In certain embodiments, the modified IL-18 polypeptide comprises a substitution at residue G108. In certain embodiments, the modified IL-18 polypeptide comprises a G108A substitution.In certain embodiments, the modified IL-18 polypeptide comprises a substitution at residue H109. In certain embodiments, the modified IL-18 polypeptide comprises an H109A substitution. In certain embodiments, the modified IL-18 polypeptide comprises a substitution at residue D110. In certain embodiments, the modified IL-18 polypeptide comprises a D110A substitution. In certain embodiments, the modified IL-18 polypeptide comprises a substitution at residue D132. In certain embodiments, the modified IL-18 polypeptide comprises a D132A substitution. In certain embodiments, the modified IL-18 polypeptide comprises a substitution at residue V153. In certain embodiments, the modified IL-18 polypeptide comprises a V153R substitution. In certain embodiments, the modified IL-18 polypeptide comprises a V153E substitution. In certain embodiments, the modified IL-18 polypeptide comprises a V153Y substitution. In certain embodiments, the modified IL-18 polypeptide comprises a substitution at residue C38. In certain embodiments, the modified IL-18 polypeptide comprises a C38A substitution. In certain embodiments, the modified IL-18 polypeptide comprises a C38S substitution. In certain embodiments, the modified IL-18 polypeptide comprises a substitution at residue C68. In certain embodiments, the modified IL-18 polypeptide comprises a C68A substitution. In certain embodiments, the modified IL-18 polypeptide comprises a C68S substitution. In certain embodiments, the modified IL-18 polypeptide comprises a substitution at residue C76. In certain embodiments, the modified IL-18 polypeptide comprises a C76A substitution. In certain embodiments, the modified IL-18 polypeptide comprises a C76S substitution. In certain embodiments, the modified IL-18 polypeptide comprises a substitution at residue C127. In certain embodiments, the modified IL-18 polypeptide comprises a C127A substitution. In certain embodiments, the modified IL-18 polypeptide comprises a C127S substitution. In certain embodiments, the modified IL-18 polypeptide comprises a substitution at residues C38, C68, C76, and / or C127. In certain embodiments, the modified IL-18 polypeptide comprises a C38A, C38S, C68A, C68S, C76A, C76S, C127A, and / or C127S substitution.In certain embodiments, the modified IL-18 polypeptide comprises C38A, C76A, and C127A substitutions. In certain embodiments, the modified IL-18 polypeptide comprises C38S, C76S, and C127S substitutions.

[0087] In certain embodiments, the modified IL-18 polypeptide comprises i) a substitution at residue V11 and ii) at least one additional substitution at a residue selected from Y1, F2, E6, K8, S10, D17, T34, D35, S36, D37, D40, N41, I49, M51, K53, D54, S55, Q103, S105, G108, H109, ​​D110, and D132. In certain embodiments, the modified IL-18 polypeptide comprises i) a substitution at residue V11 and ii) at least one additional substitution at a residue selected from Y1, F2, E6, K8, S10, D17, T34, D35, S36, D37, D40, N41, 149, M51, K53, D54, S55, Q103, S105, G108, H109, ​​D110, D132, and V153. In certain embodiments, the modified IL-18 polypeptide comprises i) a V11I substitution and ii) at least one additional substitution selected from Y1M, F2A, E6K, E6R, K8L, K8E, K8R, T34A, D35A, S36A, D37A, D40A, N41A, I49E, I49M, I49R, M51G, K53A, D54A, S55A, S55T, S55H, S55R, S55H, Q103R, Q103E, Q103K, S105K, S105I, G108A, H109A, D110A, and D132A. In certain embodiments, the modified IL-18 polypeptide comprises i) a V11I substitution and ii) at least one additional substitution selected from Y1M, F2A, E6K, E6R, K8L, K8E, K8R, T34A, D35A, S36A, D37A, D40A, N41A, I49E, I49M, I49R, M51G, K53A, D54A, S55A, S55T, S55H, S55R, S55H, Q103R, Q103E, Q103K, S105K, S105I, G108A, H109A, D110A, D132A, V153R, V153E, and V153Y. In certain embodiments, the modified IL-18 polypeptide comprises i) a substitution at residue V11, and ii) a substitution at residues E6, M51, and / or K53. In certain embodiments, the modified IL-18 polypeptide comprises i) a V11I substitution, and ii) an E6K, M51G, and / or K53A substitution.In certain embodiments, the modified IL-18 polypeptide comprises substitutions at residues E6, V11, K53, and T63. In certain embodiments, the modified IL-18 polypeptide comprises E6K, V11I, K53A, and T63A substitutions. In certain embodiments, the modified IL-18 polypeptide comprises substitutions at residues V11, M51, K53. In certain embodiments, the modified IL-18 polypeptide comprises V11I, M51G, and K53A substitutions. In certain embodiments, the modified IL-18 polypeptide comprises substitutions at residues E6, V11, M51, K53, and T63. In certain embodiments, the modified IL-18 polypeptide comprises E6K, V11I, M51G, K53A, and T63A substitutions. In certain embodiments, the modified IL-18 polypeptide comprises substitutions at residues E6, V11, C38, K53, T63, C76, and C127. In certain embodiments, the modified IL-18 polypeptide comprises E6K, V11I, C38A, K53A, T63A, C76A, and C127A substitutions. In certain embodiments, the modified IL-18 polypeptide comprises substitutions at residues V11, C38, M51, K53, T63, C76, and C127. In certain embodiments, the modified IL-18 polypeptide comprises V11I, C38A, M51G, K53A, T63A, C76A, and C127A substitutions. In certain embodiments, the modified IL-18 polypeptide comprises substitutions at residues V11, C38, M51, K53, C76, and C127. In certain embodiments, the modified IL-18 polypeptide comprises V11I, C38A, M51G, K53A, C76A, and C127A substitutions. In certain embodiments, the modified IL-18 polypeptide comprises substitutions at residues E6, V11, C38, M51, K53, T63, C76, and C127. In certain embodiments, the modified IL-18 polypeptide comprises substitutions at E6K, V11I, C38A, M51G, K53A, T63A, C76A, and C127A.In certain embodiments, the modified IL-18 polypeptide comprises i) a chain of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or any range therebetween, glycine residues attached to the N-terminus of the IL-18 polypeptide, and ii) substitutions at residues E6, V11, C38, K53, T63, C76, and C127. In certain embodiments, the modified IL-18 polypeptide comprises i) a chain of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or any range therebetween, glycine residues attached to the N-terminus of the IL-18 polypeptide, and ii) E6K, V11I, C38A, K53A, T63A, C76A, and C127A substitutions. In certain embodiments, a modified IL-18 polypeptide comprises i) a glycine residue attached to the N-terminus of the IL-18 polypeptide, and ii) substitutions at residues E6, V11, C38, K53, T63, C76, and C127. In certain embodiments, a modified IL-18 polypeptide comprises i) a glycine residue attached to the N-terminus of the IL-18 polypeptide, and ii) E6K, V11I, C38A, K53A, T63A, C76A, and C127A substitutions. In certain embodiments, a modified IL-18 polypeptide comprises i) a chain of four glycine residues attached to the N-terminus of the IL-18 polypeptide, and ii) substitutions at residues E6, V11, C38, K53, T63, C76, and C127. In certain embodiments, the modified IL-18 polypeptide comprises i) a chain of four glycine residues attached to the N-terminus of the IL-18 polypeptide, and ii) E6K, V11I, C38A, K53A, T63A, C76A, and C127A substitutions. In certain embodiments, the modified IL-18 polypeptide comprises i) a chain of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 glycine residues, or any range therebetween, attached to the N-terminus of the IL-18 polypeptide, and ii) substitutions at residues E6, V11, C38, M51, K53, T63, C76, and C127.In certain embodiments, the modified IL-18 polypeptide comprises i) a chain of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 glycine residues, or any range therebetween, attached to the N-terminus of the IL-18 polypeptide, and ii) E6K, V11I, C38A, M51G, K53A, T63A, C76A, and C127A substitutions. In certain embodiments, the modified IL-18 polypeptide comprises i) a glycine residue attached to the N-terminus of the IL-18 polypeptide, and ii) substitutions at residues E6, V11, C38, M51, K53, T63, C76, and C127. In certain embodiments, the modified IL-18 polypeptide comprises i) a glycine residue attached to the N-terminus of the IL-18 polypeptide, and ii) E6K, V11I, C38A, M51G, K53A, T63A, C76A, and C127A substitutions. In certain embodiments, the modified IL-18 polypeptide comprises substitutions at residues V11, C38, K53, C76, and C127. In certain embodiments, the modified IL-18 polypeptide comprises substitutions at residues V11, C38, K53, T63, C76, and C127. In certain embodiments, the modified IL-18 polypeptide comprises substitutions at residues V11, C38, K53, T63, C76, and C127. In certain embodiments, the modified IL-18 polypeptide comprises substitutions at residues V11, C38, K53, T63, C76, and C127. In certain embodiments, the modified IL-18 polypeptide comprises substitutions at V11I, C38A, K53A, T63A, C76A, and C127A substitutions. In certain embodiments, the modified IL-18 polypeptide comprises substitutions at residues V11, C38, K53, S55, C76, and C127. In certain embodiments, the modified IL-18 polypeptide comprises substitutions at V11I, C38A, K53A, S55A, C76A, and C127A. In certain embodiments, the modified IL-18 polypeptide comprises substitutions at residues V11, C38, K53, M51, C76, and C127. In certain embodiments, the modified IL-18 polypeptide comprises substitutions at V11I, C38A, K53A, M51G, C76A, and C127A. In certain embodiments, the modified IL-18 polypeptide comprises substitutions at residues V11, C38, K53, D54, C76, and C127. In certain embodiments, the modified IL-18 polypeptide comprises V11I, C38A, K53A, D54A, C76A, and C127A substitutions.In certain embodiments, the modified IL-18 polypeptide comprises substitutions at residues F2, V11, C38, K53, C76, and C127. In certain embodiments, the modified IL-18 polypeptide comprises substitutions at F2A, V11I, C38A, K53A, C76A, and C127A. In certain embodiments, the modified IL-18 polypeptide comprises substitutions at residues V11, E31, C38, K53, C76, and C127. In certain embodiments, the modified IL-18 polypeptide comprises substitutions at V11I, E31A, C38A, K53A, C76A, and C127A. In certain embodiments, the modified IL-18 polypeptide comprises substitutions at residues V11, T34, C38, K53, C76, and C127. In certain embodiments, the modified IL-18 polypeptide comprises V11I, T34A, C38A, K53A, C76A, and C127A substitutions. In certain embodiments, the modified IL-18 polypeptide comprises substitutions at residues V11, D35, C38, K53, C76, and C127. In certain embodiments, the modified IL-18 polypeptide comprises V11I, D35A, C38A, K53A, C76A, and C127A substitutions. In certain embodiments, the modified IL-18 polypeptide comprises substitutions at residues V11, S36, C38, K53, C76, and C127. In certain embodiments, the modified IL-18 polypeptide comprises V11I, S36A, C38A, K53A, C76A, and C127A substitutions. In certain embodiments, the modified IL-18 polypeptide comprises substitutions at residues V11, D37, C38, K53, C76, and C127. In certain embodiments, the modified IL-18 polypeptide comprises substitutions at V11I, D37A, C38A, K53A, C76A, and C127A. In certain embodiments, the modified IL-18 polypeptide comprises substitutions at residues V11, E31, D37, C38, K53, C76, and C127. In certain embodiments, the modified IL-18 polypeptide comprises substitutions at V11I, E31A, D37A, C38A, K53A, C76A, and C127A. In certain embodiments, the modified IL-18 polypeptide comprises substitutions at residues V11, C38, D40, K53, C76, and C127.In certain embodiments, the modified IL-18 polypeptide comprises V11I, C38A, D40A, K53A, C76A, and C127A substitutions. In certain embodiments, the modified IL-18 polypeptide comprises substitutions at residues V11, C38, N41, K53, C76, and C127. In certain embodiments, the modified IL-18 polypeptide comprises V11I, C38A, N41A, K53A, C76A, and C127A substitutions. In certain embodiments, the modified IL-18 polypeptide comprises substitutions at residues V11, C38, K53, C76, D132, and C127. In certain embodiments, the modified IL-18 polypeptide comprises V11I, C38A, K53A, C76A, D132A, and C127A substitutions. In certain embodiments, the modified IL-18 polypeptide comprises substitutions at residues V11, C38, K53, C76, G108, and C127. In certain embodiments, the modified IL-18 polypeptide comprises substitutions at residues V11, C38A, K53A, C76A, G108A, and C127A. In certain embodiments, the modified IL-18 polypeptide comprises substitutions at residues V11, C38, K53, In certain embodiments, the modified IL-18 polypeptide comprises substitutions at residues V11, C38, K53, C76, D110, and C127. In certain embodiments, the modified IL-18 polypeptide comprises substitutions at residues V11, C38, K53, C76, D110, and C127. In certain embodiments, the modified IL-18 polypeptide comprises substitutions at residues K8, V11, C38, C76, Q103, and C127. In certain embodiments, the modified IL-18 polypeptide comprises substitutions at residues K8R, V11I, C38A, C76A, Q103E, and C127A. In certain embodiments, the modified IL-18 polypeptide comprises K8E, V11I, C38A, C76A, Q103R, and C127A substitutions. In certain embodiments, the modified IL-18 polypeptide comprises substitutions at residues V11, C38, C76, Q103, and C127. In certain embodiments, the modified IL-18 polypeptide comprises V11I, C38A, C76A, Q103K, and C127A substitutions. In certain embodiments, the modified IL-18 polypeptide comprises substitutions at residues V11, C38, S55, C76, and C127. In certain embodiments, the modified IL-18 polypeptide comprises V11I, C38A, S55H, C76A, and C127A substitutions. In certain embodiments, the modified IL-18 polypeptide comprises V11I, C38A, S55R, C76A, and C127A substitutions. In certain embodiments, the modified IL-18 polypeptide comprises V11I, C38A, S55T, C76A, and C127A substitutions. In certain embodiments, the modified IL-18 polypeptide comprises substitutions at residues V11, C38, C76, S105, and C127. In certain embodiments, the modified IL-18 polypeptide comprises V11I, C38A, C76A, S105I, and C127A substitutions. In certain embodiments, the modified IL-18 polypeptide comprises V11I, C38A, C76A, S105K, and C127A substitutions.In certain embodiments, the modified IL-18 polypeptide comprises substitutions at residues E6, V11, C38, K53, T63, C76, and C127. In certain embodiments, the modified IL-18 polypeptide comprises substitutions at E6K, V11I, C38A, K53A, T63A, C76A, and C127A. In certain embodiments, the modified IL-18 polypeptide comprises substitutions at residues E6, K8, V11, C38, K53, T63, C76, and C127. In certain embodiments, the modified IL-18 polypeptide comprises substitutions at residues E6, K8L, V11I, C38A, K53A, T63A, C76A, and C127A. In certain embodiments, the modified IL-18 polypeptide comprises substitutions at residues E6, V11, C38, I49, K53, T63, C76, and C127. In certain embodiments, the modified IL-18 polypeptide comprises E6K, V11I, C38A, I49E, K53A, T63A, C76A, and C127A substitutions. In certain embodiments, the modified IL-18 polypeptide comprises E6K, V11I, C38A, I49M, K53A, T63A, C76A, and C127A substitutions. In certain embodiments, the modified IL-18 polypeptide comprises E6K, V11I, C38A, I49R, K53A, T63A, C76A, and C127A substitutions. In certain embodiments, the modified IL-18 polypeptide comprises substitutions at residues E6, V11, C38, K53, T63, C76, Q103, and C127. In certain embodiments, the modified IL-18 polypeptide comprises E6K, V11I, C38A, K53A, T63A, C76A, Q103R, and C127A substitutions. In certain embodiments, the modified IL-18 polypeptide comprises substitutions at residues E6, K8, V11, C38, K53, T63, C76, Q103, and C127. In certain embodiments, the modified IL-18 polypeptide comprises E6K, K8E, V11I, C38A, K53A, T63A, C76A, Q103R, and C127A substitutions. In certain embodiments, the modified IL-18 polypeptide comprises substitutions at residues E6, V11, C38, K53, T63, C76, V153, and C127.In some embodiments, the modified IL-18 polypeptide comprises at least seven modifications relative to the sequence of SEQ ID NO: 1, wherein the seven modifications include E6K, V11I, C38A, K53A, T63A, C76A, and C127A.

[0088] In certain embodiments, the modified IL-18 polypeptide comprises E6K, V11I, C38A, K53A, T63A, 76A, V153R, and C127A substitutions. In certain embodiments, the modified IL-18 polypeptide comprises E6K, V11I, C38A, K53A, T63A, C76A, V153E, and C127A substitutions. In certain embodiments, the modified IL-18 polypeptide comprises E6K, V11I, C38A, K53A, T63A, C76A, V153Y, and C127A substitutions. In certain embodiments, the modified IL-18 polypeptide comprises substitutions at residues E6, V11, C38, M51, K53, T63, C76, and C127. In certain embodiments, the modified IL-18 polypeptide comprises E6K, V11I, C38A, M51G, K53A, T63A, C76A, and C127A substitutions. In certain embodiments, the modified IL-18 polypeptide comprises E6R, V11I, C38A, K53A, T63A, C76A, and C127A substitutions. In certain embodiments, the modified IL-18 polypeptide comprises substitutions at residues Y1, E6, V11, C38, K53, T63, C76, and C127. In certain embodiments, the modified IL-18 polypeptide comprises Y1M, E6K, V11I, C38A, K53A, T63A, C76A, and C127A substitutions.

[0089] In one aspect, provided herein is a modified IL-18 polypeptide, the modified IL-18 polypeptide comprising a polymer covalently attached to residue 68 of the IL-18 polypeptide, and further comprising the following amino acid substitutions: E6K, V11I, C38A, K53A, T63A, C76A, and C127A, where the numbering of residue positions is based on SEQ ID NO: 1 as a reference sequence. In some embodiments, the modified IL-18 polypeptide has an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, or at least 95% identical to the sequence set forth in SEQ ID NO: 1. In some embodiments, the modified IL-18 polypeptide comprises the sequence set forth in SEQ ID NO: 30.

[0090] In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, or 95% sequence identity to the sequence set forth in SEQ ID NO:1. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 80% sequence identity to the sequence set forth in SEQ ID NO:1. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 85% sequence identity to the sequence set forth in SEQ ID NO:1. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO:1. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 91% sequence identity to the sequence set forth in SEQ ID NO:1. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 92% sequence identity to the sequence set forth in SEQ ID NO:1. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 93% sequence identity to the sequence set forth in SEQ ID NO:1. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 94% sequence identity to the sequence set forth in SEQ ID NO: 1. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 95% sequence identity to the sequence set forth in SEQ ID NO: 1. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 2. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 3. In certain embodiments, the modified IL-18 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 3.In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 4. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 5. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 6. In certain embodiments, the modified IL-18 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 6. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 7. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 8. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 9. In certain embodiments, the modified IL-18 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 9. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO:10.In certain embodiments, the modified IL-18 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 10. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 11. In certain embodiments, the modified IL-18 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 11. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 12. In certain embodiments, the modified IL-18 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 12. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 13. In certain embodiments, the modified IL-18 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 13. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 14. In certain embodiments, the modified IL-18 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 14. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 15. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 16. In certain embodiments, the modified IL-18 polypeptide comprises the amino acid sequence set forth in SEQ ID NO:16.In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 17. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 18. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 19. In certain embodiments, the modified IL-18 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 19. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 20. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 21. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 22. In certain embodiments, the modified IL-18 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 22. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO:23.In certain embodiments, the modified IL-18 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 23. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 24. In certain embodiments, the modified IL-18 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 24. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 25. In certain embodiments, the modified IL-18 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 25. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 26. In certain embodiments, the modified IL-18 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 26. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 27. In certain embodiments, the modified IL-18 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 27. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 28. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 29. In certain embodiments, the modified IL-18 polypeptide comprises the amino acid sequence set forth in SEQ ID NO:29.In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 30. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 80% sequence identity to the sequence set forth in SEQ ID NO: 30. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 85% sequence identity to the sequence set forth in SEQ ID NO: 30. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 30. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 95% sequence identity to the sequence set forth in SEQ ID NO: 30. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 95% sequence identity to the sequence set forth in SEQ ID NO: 30. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 98% sequence identity to the sequence set forth in SEQ ID NO:30. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 99% sequence identity to the sequence set forth in SEQ ID NO:30. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO:31. In certain embodiments, the modified IL-18 polypeptide comprises the amino acid sequence set forth in SEQ ID NO:31. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO:32. In certain embodiments, the modified IL-18 polypeptide comprises the amino acid sequence set forth in SEQ ID NO:32. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 33. In certain embodiments, the modified IL-18 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 33.

[0091] In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 204. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 205. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 206. In certain embodiments, the modified IL-18 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 206. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 207. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 80% sequence identity to the sequence set forth in SEQ ID NO: 207. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 85% sequence identity to the sequence set forth in SEQ ID NO: 207. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 207. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 95% sequence identity to the sequence set forth in SEQ ID NO: 207. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 98% sequence identity to the sequence set forth in SEQ ID NO: 207. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 99% sequence identity to the sequence set forth in SEQ ID NO:207.In certain embodiments, the modified IL-18 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 207. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 208. In certain embodiments, the modified IL-18 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 208. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 209. In certain embodiments, the modified IL-18 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 209. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 210. In certain embodiments, the modified IL-18 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 210. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 211. In certain embodiments, the modified IL-18 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 211. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 212. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 213. In certain embodiments, the modified IL-18 polypeptide comprises the amino acid sequence set forth in SEQ ID NO:213.In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 214. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 215. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 216. In certain embodiments, the modified IL-18 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 216. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 217. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 218. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 219. In certain embodiments, the modified IL-18 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 219. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO:220.In certain embodiments, the modified IL-18 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 220. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 221. In certain embodiments, the modified IL-18 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 221. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 222. In certain embodiments, the modified IL-18 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 222. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 223. In certain embodiments, the modified IL-18 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 223. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 224. In certain embodiments, the modified IL-18 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 224. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 225. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 226. In certain embodiments, the modified IL-18 polypeptide comprises the amino acid sequence set forth in SEQ ID NO:226.In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 227. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 228. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 229. In certain embodiments, the modified IL-18 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 229. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 230. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 231. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 232. In certain embodiments, the modified IL-18 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 232. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO:233.In certain embodiments, the modified IL-18 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 233. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 234. In certain embodiments, the modified IL-18 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 234. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 235. In certain embodiments, the modified IL-18 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 235. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 236. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 237. In certain embodiments, the modified IL-18 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 237. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 238. In certain embodiments, the modified IL-18 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 238. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 239. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 80% sequence identity to the sequence set forth in SEQ ID NO: 239. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 85% sequence identity to the sequence set forth in SEQ ID NO: 239. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 239. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 95% sequence identity to the sequence set forth in SEQ ID NO: 239. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 98% sequence identity to the sequence set forth in SEQ ID NO: 239. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 99% sequence identity to the sequence set forth in SEQ ID NO:239.In certain embodiments, the modified IL-18 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 239. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 240. In certain embodiments, the modified IL-18 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 240. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 241. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 80% sequence identity to the sequence set forth in SEQ ID NO: 241. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 85% sequence identity to the sequence set forth in SEQ ID NO: 241. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 241. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 95% sequence identity to the sequence set forth in SEQ ID NO: 241. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 98% sequence identity to the sequence set forth in SEQ ID NO: 241. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 99% sequence identity to the sequence set forth in SEQ ID NO: 241. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 242. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 80% sequence identity to the sequence set forth in SEQ ID NO: 242. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 85% sequence identity to the sequence set forth in SEQ ID NO:242.In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 242. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 95% sequence identity to the sequence set forth in SEQ ID NO: 242. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 98% sequence identity to the sequence set forth in SEQ ID NO: 242. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 99% sequence identity to the sequence set forth in SEQ ID NO: 242. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 243. In certain embodiments, the modified IL-18 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 243. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO:244. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 80% sequence identity to the sequence set forth in SEQ ID NO:244. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 85% sequence identity to the sequence set forth in SEQ ID NO:244. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO:244. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 95% sequence identity to the sequence set forth in SEQ ID NO:244. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 98% sequence identity to the sequence set forth in SEQ ID NO:244. In certain embodiments, the modified IL-18 polypeptide comprises an amino acid sequence having at least 99% sequence identity to the sequence set forth in SEQ ID NO:244.In certain embodiments, the modified IL-18 polypeptide comprises the amino acid sequence set forth in SEQ ID NO:244.

[0092] In one aspect, provided herein is a modified interleukin-18 (IL-18) polypeptide comprising a polymer conjugated to any one of residues 68-70, wherein the numbering of residue positions in the modified IL-18 polypeptide is based on SEQ ID NO: 1 as a reference sequence. In one aspect, provided herein is a modified interleukin-18 (IL-18) polypeptide comprising a polymer conjugated to residue 68, wherein the numbering of residue positions in the modified IL-18 polypeptide is based on SEQ ID NO: 1 as a reference sequence. In some embodiments, a polymer provided herein can be conjugated to a residue selected from residues 68, 69, or 70. In some embodiments, the polymer is conjugated to residue 68. In some embodiments, the polymer is conjugated to residue 68, 69, or 70 of an IL-18 polypeptide comprising a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or identical to any one of SEQ ID NOs: 13-33. In some embodiments, the polymer is attached to residue 68, 69, or 70 of an IL-18 polypeptide comprising a sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or identical to any one of SEQ ID NOs: 24-33.

[0093] The modified IL-18 polypeptides described herein may contain one or more unnatural amino acids. An "unnatural" amino acid can refer to an amino acid residue in the D- or L-form that is not among the 20 standard amino acids commonly incorporated into naturally occurring proteins. In some embodiments, one or more amino acids of a modified IL-18 polypeptide are substituted with one or more unnatural amino acids. Unnatural amino acids include, but are not limited to, L-azidolysine, p-azido-L-phenylalanine, and L-biphenylalanine.

[0094] Exemplary unnatural amino acids include p-acetyl-L-phenylalanine, p-iodo-L-phenylalanine, p-propargyloxyphenylalanine, p-propargyl-phenylalanine, L-3-(2-naphthyl)alanine, 3-methyl-phenylalanine, tri-O-acetyl-GlcNAcp-serine, L-dopa, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, and the like. Also included are -phenylalanine, p-boronophenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, analogs of tyrosine amino acids, analogs of glutamine amino acids, analogs of phenylalanine amino acids, analogs of serine amino acids, analogs of threonine amino acids, β-amino acids, cyclic amino acids other than proline or histidine, aromatic amino acids other than phenylalanine, tyrosine, or tryptophan, or combinations thereof. In some embodiments, the unnatural amino acids are selected from β-amino acids, homoamino acids, and cyclic amino acids. In some embodiments, the unnatural amino acids are β-alanine, β-aminopropionic acid, piperidinic acid, aminocapric acid, aminoheptanoic acid, aminopimelic acid, desmosine, diaminopimelic acid, N-aminopropionic ... α -Ethylglycine, N α -Ethylasparagine, Isodesmosine, Allo-isoleucine, N α -Methylglycine, N α -methylisoleucine, N α-methylvaline, γ-carboxyglutamic acid, N α -Acetylserine, N α -formylmethionine, 3-methylhistidine, and / or other similar amino acids.

[0095] In some embodiments, an amino acid residue of a modified IL-18 polypeptide is substituted with a conjugation handle. In some embodiments, the amino acid residue comprises an amino, azido, allyl, ester, and / or amide functional group. In some embodiments, the conjugation handle can serve as a useful anchor point for attaching additional moieties to the modified IL-18 polypeptide. In some embodiments, the amino acid residue is selected from precursor Structure 1, Structure 2, Structure 3, or Structure 4: with structure constructed from TIFF2025508826000009.tif58135.

[0096] In some embodiments, the modified IL-18 polypeptides contain substitutions of modified natural amino acid residues that can be used for the attachment of additional functional groups that can be used to facilitate conjugation reactions or attachment of various payloads to the modified IL-18 polypeptides (e.g., polymers). The substitutions can be with naturally occurring amino acids that are more suitable for attachment of additional functional groups (e.g., aspartic acid / asparagine cysteine, glutamic acid / glutamine, lysine, serine, threonine, or tyrosine), modified derivatives of any naturally occurring amino acid, or any non-natural amino acid (e.g., amino acids containing a desired reactive group, such as click chemistry reagents such as azide, alkyne, etc.). Non-limiting examples of modified natural amino acid residues include modified lysine, glutamic acid, aspartic acid, and cysteine, as provided below: TIFF2025508826000010.tif66135 where each n is an integer from 1 to 30. Other examples of natural amino acids that can be similarly modified include those having a heteroatom (e.g., tyrosine, serine, threonine) that can readily form a bond with a group suitable for linking a polymer group to the amino acid. These non-limiting examples of modified amino acid residues can be used at any position where it is desirable to add an additional functional group (e.g., a polymer) to the modified IL-18 polypeptide.

[0097] In some embodiments, any of the modified lysines, glutamic acids, aspartic acids, or cysteines shown in structures 1-4 above can replace different residues in the modified IL-18 polypeptide to allow for conjugation at different sites on the IL-18 polypeptide. The azide functional group can also be replaced with another suitable conjugation handle. The conjugation handles provided herein can be any suitable reactive group capable of reacting with a complementary reactive group. In some embodiments, the conjugation handle includes reagents for Cu(I)-catalyzed or "copper-free" alkyne-azide triazole-forming reactions (e.g., strain-promoted cycloaddition), Staudinger ligation, inverse electron-demand Diels-Alder (IEDDA) reaction, "photoclick" chemistry, tetrazine cycloaddition using trans-cyclooctene, or metal-mediated processes such as olefin metathesis and Suzuki-Miyaura, O-substituted hydroxylamines, potassium acyltrifluoroborates, or Sonogashira cross-coupling.

[0098] In some embodiments, the conjugation handle comprises a reagent for a "copper-free" alkyne azide triazole-forming reaction, non-limiting examples of alkynes for the alkyne azide triazole-forming reaction include cyclooctyne reagents (e.g., (1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-ylmethanol-containing reagents, dibenzocyclooctyne-amine reagents, difluorocyclooctyne, or derivatives thereof).

[0099] In some embodiments, the conjugation handle comprises a reactive group selected from azides, alkynes, tetrazines, halides, sulfhydryls, disulfides, maleimides, activated esters, alkenes, aldehydes, ketones, imines, hydrazines, acyltrifluoroborates, hydroxylamines (e.g., O-substituted hydroxylamines), phosphines, trans-cyclooctene, and hydrazides. In some embodiments, the conjugation handle and the complementary conjugation handle comprise "click" chemistry reagents. Exemplary groups of click chemistry residues are shown in Hein et al., "Click Chemistry, A Powerful Tool for Pharmaceutical Sciences," Pharmaceutical Research, volume 25, pages 2216-2230 (2008), Thirumurugan et al., "Click Chemistry for Drug Development and Diverse Chemical-Biology Applications," Chem. Rev. 2013, 113, 7, 4905-4979, US20160107999A1, US10266502B2, and US20190204330A1, each of which is incorporated by reference in its entirety.

[0100] In some embodiments, the group attached to the modified IL-18 polypeptide (e.g., polymer moiety or additional polypeptide) comprises a conjugation handle or the reaction product of a conjugation handle and a complementary conjugation handle. In some embodiments, the reaction product of a conjugation handle and a complementary conjugation handle results from a KAT ligation (reaction of potassium acyltrifluoroborate with hydroxylamine), a Staudinger ligation (reaction of an azide with a phosphine), a tetrazine cycloaddition (reaction of tetrazine with trans-cyclooctene), or a Huisgen cycloaddition (reaction of an alkyne with an azide). In some embodiments, the group attached to the IL-18 polypeptide (e.g., polymer or additional polypeptide) will comprise the reaction product of a conjugation handle and a complementary conjugation handle used to attach the group to the modified IL-18 polypeptide.

[0101] III. Polymer-modified IL-18 polypeptides Certain aspects of the present disclosure relate to polymer-modified IL-18 polypeptides useful as therapeutic agents. The polymer-modified IL-18 polypeptides provided herein can be used as immunotherapies or as part of other immunotherapeutic regimens. In some embodiments, the polymer-modified IL-18 polypeptides provided herein exhibit improved serum half-life compared to wild-type IL-18 (WT IL-18), reduced binding to IL-18 binding protein (IL-18BP) compared to WT IL-18, and / or enhanced IL-18 receptor (IL-18R) signaling activity in the presence of IL-18BP compared to WT IL-18.

[0102] In one aspect, provided herein are polymer-modified IL-18 polypeptides comprising a polymer covalently attached at a residue that minimally affects binding of the modified IL-18 polypeptide to the IL-18 receptor (IL-18R). In some embodiments, the addition of the polymer to the modified IL-18 polypeptide results in a polymer-modified IL-18 polypeptide that has a 10-fold or less decrease in binding affinity for IL-18R compared to the modified IL-18 polypeptide without the polymer (e.g., up to a 10-fold, 9-fold, 8-fold, 7-fold, 6-fold, 5-fold, 4-fold, 3-fold, or 2-fold decrease in affinity, or substantially the same affinity). In some embodiments, the polymer-modified IL-18 polypeptide to which the polymer is attached exhibits enhanced binding affinity for IL-18R compared to WT IL-18, or a slightly decreased affinity for IL-18R compared to WT IL-18. In some embodiments, the polymer-modified IL-18 polypeptide has increased affinity for the IL-18Rα / β heterodimer compared to WT IL-18. In one aspect, the polymer-modified IL-18 polypeptides described herein do not have a significantly reduced affinity for the IL-18Rα / β heterodimer compared to WT IL-18.

[0103] Such polymer-modified IL-18 polypeptides may exhibit binding properties for IL-18 receptor subunits that differ from those of wild-type IL-18 (e.g., higher or slightly lower affinity for the IL-18 receptor alpha subunit (IL-18Rα) or the IL-18 receptor beta subunit (IL-18Rβ)). In some embodiments, the binding affinity between the polymer-modified IL-18 polypeptide and IL-18Rα is the same as or higher than the binding affinity between wild-type IL-18 and IL-18Rα. In some embodiments, the binding affinity between the polymer-modified IL-18 polypeptide and IL-18Rα is the same as or slightly lower than the binding affinity between wild-type IL-18 and IL-18Rα. In some embodiments, the binding affinity between the polymer-modified IL-18 polypeptide and IL-18Rβ is the same as or higher than the binding affinity between wild-type IL-18 and IL-18Rβ. In some embodiments, the binding affinity between the polymer-modified IL-18 polypeptide and IL-18Rβ is the same as or slightly lower than the binding affinity between wild-type IL-18 and IL-18Rβ.

[0104] In some embodiments, the polymer-modified IL-18 polypeptides provided herein exhibit the ability to induce interferon gamma (IFNγ) production after administration to a subject. In some embodiments, the ability of the polymer-modified IL-18 polypeptide to induce IFNγ is comparable to that of wild-type IL-18 (e.g., exhibits an EC50 for IFNγ induction that is within about 10-fold of that of wild-type IL-18). Exemplary IL-18 polypeptides provided herein that exhibit this characteristic are shown in Figure 4A, which shows a comparison of IFNγ production (ng / mL, y-axis) as a function of concentration (nM, x-axis) of wild-type versus modified IL-18 polypeptides (muteins).

[0105] In one aspect, provided herein is a polymer-modified interleukin-18 (IL-18) polypeptide, comprising a polymer attached to a residue of the modified IL-18 polypeptide, wherein the polymer-modified IL-18 polypeptide has a dissociation constant (K) for the IL-18 receptor alpha / beta heterodimer (IL-18Rα / β) that is up to 8-fold greater than the binding affinity exhibited by a corresponding modified IL-18 polypeptide without the polymer attached. D In some embodiments, the dissociation constant of the IL-18Rα / β interaction of a polymer-modified IL-18 polypeptide is up to 2-fold, 4-fold, or 6-fold greater than the dissociation constant exhibited by the corresponding modified IL-18 polypeptide without the polymer attached.

[0106] In one aspect, provided herein is a polymer-modified interleukin-18 (IL-18) polypeptide comprising a polymer attached to a residue of the modified IL-18 polypeptide, wherein the polymer-modified IL-18 polypeptide has a dissociation constant (K) for IL-18 receptor alpha (IL-18Rα) that is up to 9-fold greater than the dissociation constant exhibited by a corresponding modified IL-18 polypeptide without the polymer attached. D In some embodiments, the dissociation constant of a polymer-modified IL-18 polypeptide for IL-18Rα interaction is up to 2-fold, 4-fold, or 6-fold greater than the dissociation constant exhibited by the corresponding modified IL-18 polypeptide without the polymer attached.

[0107] In one aspect, provided herein is a polymer-modified interleukin-18 (IL-18) polypeptide, wherein the IL-18 polypeptide comprises a polymer attached to any one of residues 79-120, where the numbering of residue positions in the modified IL-18 polypeptide is based on SEQ ID NO: 1 as the reference sequence. In some embodiments, the polymer is attached to residue 85, 86, 95, or 98.

[0108] In some embodiments, the polymer-modified IL-18 polypeptides provided herein also exhibit low-binding IL-18 binding proteins (IL-18BPs). In some embodiments, the reduced binding to IL-18BPs is the result of an attached polymer and / or additional modifications (e.g., additional amino acid substitutions) to the modified IL-18 polypeptides. In some embodiments, the polymer-modified IL-18 polypeptides provided herein are capable of inducing IFNγ even in the presence of IL-18BPs (e.g., the ability of the modified IL-18 polypeptides to induce IFNγ is not substantially inhibited by the presence of IL-18BPs) (nM, x-axis). An example of an IL-18 polypeptide with this property compared to wild-type IL-18 is shown in Figure 4B, which shows IFNγ production (ng / mL, y-axis) as a function of IL-18BP concentration (nM, x-axis) in samples treated with the same levels of wild-type IL-18 (circles) or modified IL-18 polypeptides provided herein (inverted triangles). Notably, the modified IL-18 polypeptides provided herein did not exhibit inhibition of their ability to induce IFNγ in the presence of IL-18BP, whereas wild-type IL-18 exhibited a substantial reduction in this ability as the concentration of IL-18BP increased.

[0109] The modified IL-18 polypeptides described herein may contain one or more unnatural amino acids. An "unnatural" amino acid can refer to an amino acid residue in the D- or L-form that is not among the 20 standard amino acids commonly incorporated into naturally occurring proteins. In some embodiments, one or more amino acids of a modified IL-18 polypeptide are substituted with one or more unnatural amino acids. Unnatural amino acids include, but are not limited to, L-azidolysine, p-azido-L-phenylalanine, and L-biphenylalanine.

[0110] Exemplary unnatural amino acids include p-acetyl-L-phenylalanine, p-iodo-L-phenylalanine, p-propargyloxyphenylalanine, p-propargyl-phenylalanine, L-3-(2-naphthyl)alanine, 3-methyl-phenylalanine, tri-O-acetyl-GlcNAcp-serine, L-dopa, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, and the like. Also included are -phenylalanine, p-boronophenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, analogs of tyrosine amino acids, analogs of glutamine amino acids, analogs of phenylalanine amino acids, analogs of serine amino acids, analogs of threonine amino acids, β-amino acids, cyclic amino acids other than proline or histidine, aromatic amino acids other than phenylalanine, tyrosine, or tryptophan, or combinations thereof. In some embodiments, the unnatural amino acids are selected from β-amino acids, homoamino acids, and cyclic amino acids. In some embodiments, the unnatural amino acids are β-alanine, β-aminopropionic acid, piperidinic acid, aminocapric acid, aminoheptanoic acid, aminopimelic acid, desmosine, diaminopimelic acid, N-aminopropionic ... α -Ethylglycine, N α -Ethylasparagine, Isodesmosine, Allo-isoleucine, N α -Methylglycine, N α -methylisoleucine, N α -methylvaline, γ-carboxyglutamic acid, N α -Acetylserine, N α -formylmethionine, 3-methylhistidine, and / or other similar amino acids.

[0111] In some embodiments, an amino acid residue of a modified IL-18 polypeptide is substituted with a conjugation handle. In some embodiments, the amino acid residue comprises an amino, azido, allyl, ester, and / or amide functional group. In some embodiments, the conjugation handle can serve as a useful anchor point for attaching additional moieties to the modified IL-18 polypeptide. In some embodiments, the amino acid residue is selected from precursor Structure 1, Structure 2, Structure 3, or Structure 4: with structure constructed from TIFF2025508826000011.tif54135.

[0112] In some embodiments, the polymer-modified IL-18 polypeptide contains substitutions of modified natural amino acid residues that can be used for the attachment of additional functional groups that can be used to facilitate conjugation reactions or attachment of various payloads to the modified IL-18 polypeptide (e.g., polymers). The substitutions can be for naturally occurring amino acids more suitable for attachment of additional functional groups (e.g., aspartic acid / asparagine cysteine, glutamic acid / glutamine, lysine, serine, threonine, or tyrosine), modified derivatives of any naturally occurring amino acid, or any non-natural amino acid (e.g., amino acids containing a desired reactive group, such as click chemistry reagents such as azide, alkyne, etc.). Non-limiting examples of modified natural amino acid residues include the modified lysine, glutamic acid, aspartic acid, and cysteine ​​provided below: TIFF2025508826000012.tif70135 where each n is an integer from 1 to 30. Other examples of natural amino acids that can be similarly modified include those having a heteroatom (e.g., tyrosine, serine, threonine) that can readily form a bond with a group suitable for linking a polymer group to the amino acid. These non-limiting examples of modified amino acid residues can be used at any position where it is desirable to add an additional functional group (e.g., a polymer) to the modified IL-18 polypeptide.

[0113] In some embodiments, any of structures 1-4 above, modified lysine, modified glutamic acid, modified aspartic acid, or modified cysteine ​​may substitute for a different residue of the modified IL-18 polypeptide (e.g., any of residues 79-120 using SEQ ID NO: 1 as a reference sequence) to allow for conjugation at a different site on the IL-18 polypeptide. The azide functionality can also be replaced with another suitable conjugation handle.

[0114] The conjugation handles provided herein can be any suitable reactive group capable of reacting with a complementary reactive group. In some embodiments, the conjugation handles include reagents for Cu(I)-catalyzed or "copper-free" alkyne-azide triazole-forming reactions (e.g., strain-promoted cycloaddition), Staudinger ligation, inverse electron-demand Diels-Alder (IEDDA) reaction, "photoclick" chemistry, tetrazine cycloaddition with trans-cyclooctene, or metal-mediated processes such as olefin metathesis and Suzuki-Miyaura, O-substituted hydroxylamine, potassium acyltrifluoroborate, or Sonogashira cross-coupling.

[0115] In some embodiments, the conjugation handle comprises a reagent for a "copper-free" alkyne azide triazole-forming reaction, non-limiting examples of alkynes for the alkyne azide triazole-forming reaction include cyclooctyne reagents (e.g., (1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-ylmethanol-containing reagents, dibenzocyclooctyne-amine reagents, difluorocyclooctyne, or derivatives thereof).

[0116] In some embodiments, the conjugation handle comprises a reactive group selected from azides, alkynes, tetrazines, halides, sulfhydryls, disulfides, maleimides, activated esters, alkenes, aldehydes, ketones, imines, hydrazines, acyltrifluoroborates, hydroxylamines (e.g., O-substituted hydroxylamines), phosphines, trans-cyclooctene, and hydrazides. In some embodiments, the conjugation handle and the complementary conjugation handle comprise "click" chemistry reagents. Exemplary groups of click chemistry residues are shown in Hein et al., "Click Chemistry, A Powerful Tool for Pharmaceutical Sciences," Pharmaceutical Research, volume 25, pages 2216-2230 (2008), Thirumurugan et al., "Click Chemistry for Drug Development and Diverse Chemical-Biology Applications," Chem. Rev. 2013, 113, 7, 4905-4979, US20160107999A1, US10266502B2, and US20190204330A1, each of which is incorporated by reference in its entirety.

[0117] In some embodiments, the group attached to the modified IL-18 polypeptide (e.g., polymer moiety or additional polypeptide) comprises a conjugation handle or the reaction product of a conjugation handle and a complementary conjugation handle. In some embodiments, the reaction product of a conjugation handle and a complementary conjugation handle results from a KAT ligation (reaction of potassium acyltrifluoroborate with hydroxylamine), a Staudinger ligation (reaction of an azide with a phosphine), a tetrazine cycloaddition (reaction of tetrazine with trans-cyclooctene), or a Huisgen cycloaddition (reaction of an alkyne with an azide). In some embodiments, the group attached to the IL-18 polypeptide (e.g., polymer or additional polypeptide) will comprise the reaction product of a conjugation handle and a complementary conjugation handle used to attach the group to the modified IL-18 polypeptide.

[0118] IIIa. Point of attachment of polymer moiety to modified IL-18 polypeptide The polymer-modified IL-18 polypeptides described herein comprise one or more polymers. In some embodiments, the polymer-modified IL-18 polypeptide is conjugated to one polymer moiety.

[0119] In some embodiments, the polymer-modified IL-18 polypeptide comprises a polymer covalently attached to a residue of a modified IL-18 polypeptide described herein. In some embodiments, the polymer is covalently attached to a residue within the region of residues 79-120. In some embodiments, the polymer is covalently attached to a residue within the region of residues 79-100. In some embodiments, the polymer is covalently attached to a residue within the region of residues 81-110, residues 83-100, or residues 85-98. In some embodiments, the polymer is covalently attached to a residue within the region of residues 83-88, residues 84-87, or residues 85-86. In some embodiments, the polymer is covalently attached to a residue within the region of residues 94-102, residues 95-101, residues 96-100, or residues 97-99. In some embodiments, the polymer is covalently attached at residues 85, residues 86, residues 95, or residue 98.

[0120] In some embodiments, the residue to which the polymer is attached is a naturally occurring amino acid residue. In some embodiments, the residue to which the polymer is covalently attached is selected from cysteine, aspartic acid, asparagine, glutamic acid, glutamine, serine, threonine, lysine, and tyrosine. In some embodiments, the residue to which the polymer is covalently attached is selected from asparagine, aspartic acid, cysteine, glutamic acid, glutamine, lysine, and tyrosine. In some embodiments, the polymer is covalently attached to a cysteine. In some embodiments, the polymer is covalently attached to a lysine. In some embodiments, the polymer is covalently attached to a glutamine. In some embodiments, the polymer is covalently attached to an asparagine. In some embodiments, the residue to which the polymer is attached is the naturally occurring amino acid at that position in SEQ ID NO: 1. In some embodiments, the polymer is attached to a different naturally occurring amino acid that is substituted at the relevant position. In some embodiments, the polymer is site-specifically covalently attached to the naturally occurring amino acid.

[0121] In some embodiments, the polymer is attached at a non-natural amino acid residue. In some embodiments, the non-natural amino acid residue comprises a conjugation handle. In some embodiments, the conjugation handle facilitates the addition of the polymer to the modified IL-18 polypeptide. The conjugation handle can be any of the conjugation handles provided herein. In some embodiments, the polymer is site-specifically covalently attached to the non-natural amino acid.

[0122] In some embodiments, the polymer is covalently attached to a residue within the region of residues 79 to 120. In some embodiments, the polymer is covalently attached at residue 79, residue 80, residue 81, residue 82, residue 83, residue 84, residue 85, residue 86, residue 87, residue 88, residue 89, residue 90, residue 91, residue 92, residue 93, residue 94, residue 95, residue 96, residue 97, residue 98, residue 99, residue 100, residue 101, residue 102, residue 103, residue 104, residue 105, residue 106, residue 107, residue 108, residue 109, residue 110, residue 111, residue 112, residue 113, residue 114, residue 115, residue 116, residue 117, residue 118, residue 119, or residue 120. In some embodiments, the polymer is covalently attached at residue 85, residue 86, residue 95, or residue 98.

[0123] In some embodiments, the polymer is covalently attached at residue 85. In some embodiments, the polymer is covalently attached at residue E85, E85C, E85D, E85Q, E85K, E85N, or E85Y. In some embodiments, the polymer is covalently attached at residue E85. In some embodiments, the polymer is covalently attached at residue E85C. In some embodiments, the polymer is covalently attached to the unnatural amino acid at residue 85.

[0124] In some embodiments, the polymer is covalently attached at residue 86. In some embodiments, the polymer is covalently attached at residue M86C, M86D, M86Q, M86K, M86N, M86E, or M86Y. In some embodiments, the polymer is covalently attached at M86C. In some embodiments, the polymer is covalently attached to the unnatural amino acid at residue 86.

[0125] In some embodiments, the polymer is covalently attached at residue 95. In some embodiments, the polymer is covalently attached at residue T95, T95C, T95D, T95Q, T95K, T95N, T95E, or T95Y. In some embodiments, the polymer is covalently attached at residue T95C, T95D, T95Q, T95K, T95N, T95E, or T95Y. In some embodiments, the polymer is covalently attached at residue T95C. In some embodiments, the polymer is covalently attached to the unnatural amino acid at residue 95.

[0126] In some embodiments, the polymer is covalently attached at residue 98. In some embodiments, the polymer is covalently attached at residue D98, D98C, D98Q, D98K, D98N, D98E, or D98Y. In some embodiments, the polymer is covalently attached at residue D98C. In some embodiments, the polymer is covalently attached to the unnatural amino acid at residue 98.

[0127] In some embodiments, the polymer is covalently attached via the modified amino acid α. In some embodiments, the modified amino acid α is an amino acid-PEG-azide group. In some embodiments, the modified amino acid α is a glutamic acid, aspartic acid, lysine, cysteine, or tyrosine modified to incorporate an azide group linked to the amino acid via a PEG spacer. In some embodiments, the modified amino acid α has a structure selected from the following: TIFF2025508826000013.tif68135, wherein each n is independently an integer of 1 to 30. In some embodiments, n is an integer of 1 to 20, 1 to 10, 2 to 30, 2 to 20, 2 to 10, 5 to 30, 5 to 20, or 5 to 10. In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30. In some embodiments, n is 10. In some embodiments, n is 8. In some embodiments, n is 6. In some embodiments, n is 12.

[0128] In some embodiments, the modified amino acid α is located at a position on the modified IL-18 polypeptide in the region of residues 79-120. In some embodiments, the modified amino acid α is located at a position on the modified IL-18 polypeptide selected from residue 85, residue 86, or residue 98. In some embodiments, the modified amino acid α is located at residue 85 of the modified IL-18 polypeptide. In some embodiments, the modified amino acid α is located at residue 86 of the modified IL-18 polypeptide. In some embodiments, the modified amino acid α is located at residue 95 of the modified IL-18 polypeptide. In some embodiments, the modified amino acid α is located at residue 98 of the modified IL-18 polypeptide.

[0129] In some embodiments, the modified IL-18 polypeptide comprises a polymer covalently attached to a modified lysine residue. In some embodiments, the modified lysine residue comprises a conjugation handle. In some embodiments, the modified lysine residue comprises an azide. In some embodiments, the modified lysine residue has the structure of Structure B, wherein Structure B is TIFF2025508826000014.tif39135, wherein each n is independently an integer between 1 and 30. In some embodiments, n is an integer between 1 and 20, between 1 and 10, between 2 and 30, between 2 and 20, between 2 and 10, between 5 and 30, between 5 and 20, or between 5 and 10. In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30. In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, or 8. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is 5. In some embodiments, n is 6. In some embodiments, n is 8. In some embodiments, n is 10. In some embodiments, n is 12.

[0130] In some embodiments, the modified lysine of structure B is located at a position on the modified IL-18 polypeptide in the region of residues 79-120. In some embodiments, the modified lysine of structure B is located at a position on the modified IL-18 polypeptide selected from residues 85, 86, and 98. In some embodiments, the modified lysine of structure B is located at residue 85 of the modified IL-18 polypeptide. In some embodiments, the modified lysine of structure B is located at residue 86 of the modified IL-18 polypeptide. In some embodiments, the modified lysine of structure B is located at residue 95 of the modified IL-18 polypeptide. In some embodiments, the modified lysine of structure B is located at residue 98 of the modified IL-18 polypeptide.

[0131] In some embodiments, the modified IL-18 polypeptide comprises multiple polymers covalently attached thereto. In some embodiments, the modified IL-18 polypeptide comprises a second polymer covalently attached thereto. In some embodiments, the modified IL-18 polypeptide comprises a second polymer covalently attached at a residue provided herein.

[0132] IIIb. Polymers conjugated to modified IL-18 polypeptides In one aspect, described herein are polymer-modified polypeptides comprising modified IL-18 polypeptides, wherein the polymer-modified IL-18 polypeptides comprise a covalently attached polymer. In some embodiments, described herein are polymer-modified IL-18 polypeptides comprising one or more polymers covalently attached to the modified IL-18 polypeptide. In some embodiments, the polymer-modified IL-18 polypeptides comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more polymers covalently attached to the modified IL-18 polypeptide.

[0133] In some embodiments, the polymer comprises a conjugation handle that can be used to further attach additional moieties to the polymer-modified IL-18 polypeptide (e.g., the addition of additional polypeptides, e.g., antibodies). Any suitable reactive group capable of reacting with a complementary reactive group attached to another moiety can be used as the conjugation handle. In some embodiments, the conjugation handle comprises Cu(I)-catalyzed or "copper-free" reagents for alkyne-azide triazole formation (e.g., strain-promoted cycloaddition), Staudinger ligation, inverse electron-demand Diels-Alder (IEDDA) reaction, "photoclick" chemistry, tetrazine cycloaddition with trans-cyclooctene, potassium acyltrifluoroborate ligation (e.g., with O-substituted hydroxylamines), or metal-mediated processes such as olefin metathesis and Suzuki-Miyaura or Sonogashira cross-coupling.

[0134] In some embodiments, the conjugation handle comprises a reagent for a "copper-free" alkyne azide triazole-forming reaction, non-limiting examples of alkynes for the alkyne azide triazole-forming reaction include cyclooctyne reagents (e.g., (1R,8S,9s)-bicyclo[6.1.0]non-4-yn-9-ylmethanol-containing reagents, dibenzocyclooctyne-amine reagents, difluorocyclooctyne, or derivatives thereof).

[0135] In some embodiments, the conjugation handle comprises a reactive group selected from azides, alkynes, tetrazines, halides, sulfhydryls, disulfides, maleimides, activated esters, alkenes, aldehydes, ketones, imines, hydrazines, acyltrifluoroborates, hydroxylamines, phosphines, trans-cyclooctene, and hydrazides. In some embodiments, the conjugation handle and the complementary conjugation handle comprise "click" chemistry reagents. Exemplary groups of click chemistry residues are shown in Hein et al., "Click Chemistry, A Powerful Tool for Pharmaceutical Sciences," Pharmaceutical Research, volume 25, pages 2216-2230 (2008), Thirumurugan et al., "Click Chemistry for Drug Development and Diverse Chemical-Biology Applications," Chem. Rev. 2013, 113, 7, 4905-4979, US20160107999A1, US10266502B2, and US20190204330A1, each of which is incorporated by reference in its entirety.

[0136] In some embodiments, the polymer comprises a conjugation handle or the reaction product of a conjugation handle and a complementary conjugation handle. In some embodiments, the reaction product of a conjugation handle and a complementary conjugation handle results from a KAT ligation (reaction of potassium acyltrifluoroborate with hydroxylamine), a Staudinger ligation (reaction of an azide with a phosphine), a tetrazine cycloaddition (reaction of tetrazine with trans-cyclooctene), or a Huisgen cycloaddition (reaction of an alkyne with an azide). In some embodiments, the polymer comprises the reaction product of a conjugation handle and a complementary conjugation handle used to attach the polymer to a modified IL-18 polypeptide.

[0137] In some embodiments, the polymer comprises an azide moiety. In some embodiments, the polymer comprises an alkyne moiety. In some embodiments, the polymer comprises an azide moiety, an alkyne moiety, or the reaction product of an azide-alkyne cycloaddition reaction. In some embodiments, the reaction product of an azide-alkyne cycloaddition reaction is a 1,2,3-triazole.

[0138] In some embodiments, the polymer is attached to the modified IL-18 polypeptide via the use of a bifunctional linker. In some embodiments, the bifunctional linker reacts with a reactive group (e.g., a cysteine ​​sulfhydryl) of an amino acid residue on the modified IL-18 polypeptide to form a covalent bond. In some embodiments, in a second step, a second reactive group (e.g., a conjugation handle such as an azide or alkyne) of the bifunctional linker is used to attach a second moiety, such as a polymer.

[0139] In some embodiments, the polymer is a water-soluble polymer. In some embodiments, the water-soluble polymer comprises a poly(alkylene oxide), a polysaccharide, poly(vinylpyrrolidone), poly(vinyl alcohol), polyoxazoline, poly(acryloylmorpholine), or a combination thereof. In some embodiments, the water-soluble polymer is a polysaccharide. In some embodiments, the water-soluble polymer comprises a poly(alkylene oxide). In some embodiments, the poly(alkylene oxide) is polyethylene glycol (PEG).

[0140] In some embodiments, the polyethylene glycol has a weight-average molecular weight of about 0.1 kDa to about 50 kDa. In some embodiments, the polyethylene glycol has a weight-average molecular weight of about 0.5 kDa to about 50 kDa. In some embodiments, the polyethylene glycol has a weight-average molecular weight of about 10 kDa, about 20 kDa, or about 30 kDa. In some embodiments, the polyethylene glycol has a weight-average molecular weight of about 30 kDa, about 20 kDa to about 40 kDa, about 20 kDa to about 50 kDa, or about 40 kDa to about 50 kDa. In some embodiments, the polyethylene glycol has a weight-average molecular weight of about 0.5 kDa, about 1 kDa, about 5 kDa, about 10 kDa, about 20 kDa, about 40 kDa, or about 50 kDa. In some embodiments, the polyethylene glycol has a weight average molecular weight of at least about 0.1 kDa, about 0.5 kDa, about 1 kDa, about 5 kDa, about 10 kDa, about 20 kDa, or about 40 kDa, hi some embodiments, the polyethylene glycol has a weight average molecular weight of at most about 5 kDa, about 10 kDa, about 20 kDa, about 40 kDa, or about 50 kDa.

[0141] In some embodiments, the conjugated polymer has a weight average molecular weight of about 6,000 daltons to about 50,000 daltons. In some embodiments, the polymer has a weight average molecular weight of about 6,000 daltons to about 10,000 daltons, about 6,000 daltons to about 25,000 daltons, about 6,000 daltons to about 50,000 daltons, about 10,000 daltons to about 25,000 daltons, about 10,000 daltons to about 50,000 daltons, or about 25,000 daltons to about 50,000 daltons. In some embodiments, the polymer has a weight average molecular weight of about 6,000 daltons, about 10,000 daltons, about 25,000 daltons, or about 50,000 daltons. In some embodiments, the polymer has a weight average molecular weight of at least about 6,000 daltons, about 10,000 daltons, or about 25,000 daltons, hi some embodiments, the polymer has a weight average molecular weight of up to about 10,000 daltons, about 25,000 daltons, or about 50,000 daltons.

[0142] In some embodiments, the attached polymer has a weight average molecular weight of about 120 daltons to about 1,000 daltons. In some embodiments, the polymer has a weight average molecular weight of about 100 daltons to about 250 daltons, about 100 daltons to about 300 daltons, about 100 daltons to about 400 daltons, about 100 daltons to about 500 daltons, about 100 daltons to about 1,000 daltons, about 250 daltons to about 300 daltons, about 250 daltons to about 400 daltons, about 250 daltons to about 500 daltons, about 250 daltons to about 1,000 daltons, about 300 daltons to about 400 daltons, about 300 daltons to about 500 daltons, about 300 daltons to about 1,000 daltons, about 400 daltons to about 500 daltons, about 400 daltons to about 1,000 daltons, or about 500 daltons to about 1,000 daltons. In some embodiments, the polymer has a weight average molecular weight of about 100 daltons, about 250 daltons, about 300 daltons, about 400 daltons, about 500 daltons, or 1,000 daltons. In some embodiments, the polymer has a weight average molecular weight of at least about 100 daltons, about 250 daltons, about 300 daltons, about 400 daltons, or about 500 daltons. In some embodiments, the polymer has a weight average molecular weight of up to about 250 daltons, about 300 daltons, about 400 daltons, about 500 daltons, or about 1,000 daltons.

[0143] In some embodiments, the polymer comprises one or more linker groups. In some embodiments, a linker group is used to attach the polymer portion of the polymer to the modified IL-18 polypeptide. In some embodiments, the one or more linker groups comprise a bifunctional linker, e.g., an amide group, an ester group, an ether group, a thioether group, a carbonyl group, etc. In some embodiments, the one or more linker groups comprise an amide linker group. In some embodiments, the modified poly(alkylene oxide) comprises one or more spacer groups. In some embodiments, the spacer group comprises a substituted or unsubstituted C1-C6 alkylene group. In some embodiments, the spacer group comprises -CH2-, -CH2CH2-, or -CH2CH2CH2-. In some embodiments, the linker group is the product of a bioorthogonal reaction (e.g., a biocompatible and selective reaction). In some embodiments, the bioorthogonal reaction is a metal-mediated process such as Cu(I)-catalyzed or "copper-free" alkyne-azide triazole-forming reaction, Staudinger ligation, inverse electron-demand Diels-Alder (IEDDA) reaction, alkyne-nitrone cycloaddition chemistry, or olefin metathesis and Suzuki-Miyaura or Sonogashira cross-coupling. In some embodiments, the first polymer is attached to the IL-18 polypeptide via click chemistry.

[0144] In some embodiments, the polymer has the structure of formula (X): TIFF2025508826000015.tif15135, L 1 , L 2 , L 3 , L 4 , L 5 , L 6 , L 7 、 L 8 , and L 9 each independently represents -O-, -NR L -, -(C1-C6 alkylene)NR L -, -NR L (C1-C6 alkylene)-, -N(R L)2 + -, -(C1-C6 alkylene)N(R L )2 + -, -N(R L )2 + -(C1-C6 alkylene)-, -OP(=O)(OR L )O-, -S-, -(C1-C6 alkylene)S-, -S(C1-C6 alkylene)-, -S(=O)-, -S(=O)2-, -C(=O)-, -(C1-C6 alkylene)C(=O)-, -C(=O)(C1-C6 alkylene)-, -C(=O)O-, -OC(=O)-, -OC(=O)O-, -C(=O)NR L -, -C(=O)NR L (C1-C6 alkylene)-, -(C1-C6 alkylene)C(=O)NR L -, -NR L C(=O)-, -(C1-C6 alkylene)NR L C(=O)-, -NR L C(=O)(C1-C6 alkylene)-, -OC(=O)NR L -, -NR L C(=O)O-, -NR L C(=O)NR L -, -NR L C(=S)NR L -, -CR L =N-, -N=CR L , -NR L S(=O)2-, -S(=O)2NR L -, -C(=O)NR L S(=O)2-, -S(=O)2NR L C(=O)-, substituted or unsubstituted C1-C6 alkylene, substituted or unsubstituted C1-C6 heteroalkylene, substituted or unsubstituted C2-C6 alkenylene, substituted or unsubstituted C2-C6 alkynylene, substituted or unsubstituted C6-C 20 Arylene, substituted or unsubstituted C-C 20 Heteroarylene, -(CH2-CH2-O) qa -, -(O-CH2-CH2) qb -, -(CH2-CH(CH3)-O) qc -, -(O-CH(CH3)-CH2) qd-, the reaction product of a conjugation handle and a complementary conjugation handle, or absent; (C1-C6 alkylene) Each R L are independently hydrogen, substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C1-C4 heteroalkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C5 alkynyl, substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted C2-C7 heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; Each of qa, qb, qc, and qd independently represents an integer of 1 to 100; each TIFF2025508826000016.tif14135 includes a linker containing structure that is the point of attachment of the modified IL-18 polypeptide or polymer to the polymer portion.

[0145] In some embodiments, the polymer has the structure of formula (X'): TIFF2025508826000017.tif16135In the formula, each L' is independently -O-, -NR L -, -(C1-C6 alkylene)NR L -, -NR L (C1-C6 alkylene)-, -N(R L )2 + -, -(C1-C6 alkylene)N(R L )2 + -, -N(R L )2 + -(C1-C6 alkylene)-, -OP(=O)(OR L )O-, -S-, -(C1-C6 alkylene)S-, -S(C1-C6 alkylene)-, -S(=O)-, -S(=O)2-, -C(=O)-, -(C1-C6 alkylene)C(=O)-, -C(=O)(C1-C6 alkylene)-, -C(=O)O-, -OC(=O)-, -OC(=O)O-, -C(=O)NR L -, -C(=O)NR L (C1-C6 alkylene)-, -(C1-C6 alkylene)C(=O)NR L-, -NR L C(=O)-, -(C1-C6 alkylene)NR L C(=O)-, -NR L C(=O)(C1-C6 alkylene)-, -OC(=O)NR L -, -NR L C(=O)O-, -NR L C(=O)NR L -, -NR L C(=S)NR L -, -CR L =N-, -N=CR L , -NR L S(=O)2-, -S(=O)2NR L -, -C(=O)NR L S(=O)2-, -S(=O)2NR L C(=O)-, substituted or unsubstituted C1-C6 alkylene, substituted or unsubstituted C1-C6 heteroalkylene, substituted or unsubstituted C2-C6 alkenylene, substituted or unsubstituted C2-C6 alkynylene, substituted or unsubstituted C6-C 20 Arylene, substituted or unsubstituted C-C 20 Heteroarylene, -(CH2-CH2-O) qa -, -(O-CH2-CH2) qb -, -(CH2-CH(CH3)-O) qc -, -(O-CH(CH3)-CH2) qd -, the reaction product of a conjugation handle and a complementary conjugation handle, or absent; (C1-C6 alkylene) Each R L are independently hydrogen, substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C1-C4 heteroalkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C5 alkynyl, substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted C2-C7 heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; Each of qa, qb, qc, and qd independently represents an integer of 1 to 100; g is an integer from 1 to 100, each TIFF2025508826000018.tif12135 includes a linker containing structure that is the point of attachment of the modified IL-18 polypeptide or polymer to the polymer portion.

[0146] In some embodiments, the polymer-modified IL-18 polypeptides provided herein comprise a polymer comprising a linker selected from Table 1. In Table 1, each TIFF2025508826000019.tif9135 is the point of attachment to either the modified IL-18 polypeptide (eg, an amino group of the modified IL-18 polypeptide) or to the polymer portion of the polymer. TIFF2025508826000020.tif178135

[0147] In some embodiments, the water-soluble polymer is linear or branched. In some embodiments, the water-soluble polymer is branched and comprises multiple polyethylene glycol chains. In some embodiments, the water-soluble polymer comprises 1 to 10 polyethylene glycol chains. In some embodiments, the water-soluble polymer comprises 1 to 2 polyethylene glycol chains, 1 to 4 polyethylene glycol chains, 1 to 6 polyethylene glycol chains, 1 to 10 polyethylene glycol chains, 2 to 4 polyethylene glycol chains, 2 to 6 polyethylene glycol chains, 2 to 10 polyethylene glycol chains, 4 to 6 polyethylene glycol chains, 4 to 10 polyethylene glycol chains, or 6 to 10 polyethylene glycol chains.

[0148] In some embodiments, the polymer attached to the modified IL-18 polypeptide comprises a conjugation handle. In some embodiments, the conjugation handle attached to the polymer can be used to link additional moieties (e.g., additional polypeptides, e.g., antibodies or additional cytokines, or larger polymers) to the polymer-modified IL-18 polypeptide to form a conjugate composition. In some embodiments, the polymer comprises an alkyne or azide conjugation handle. In some embodiments, the conjugation handle is attached to a terminal atom of the polymer. A non-limiting example of a polymer having an attached conjugation handle is shown above in modified amino acid α. In some embodiments, the polymer has a structure of formula (I): TIFF2025508826000021.tif11135, wherein n is an integer from 2 to 30. In some embodiments, n is an integer from 2 to 10. In some embodiments, n is 2, 4, 6, 8, or 10. In some embodiments, n is 8.

[0149] In some embodiments, the addition of a polymer to a modified IL-18 polypeptide increases the stability of the modified IL-18 polypeptide in vivo or in vitro. In some embodiments, the addition of a polymer increases one or more pharmacokinetic (PK) properties of the modified IL-18 polypeptide. In some embodiments, a polymer-modified IL-18 polypeptide of the present disclosure comprises a covalently attached polymer to extend its plasma or serum half-life. In some embodiments, the plasma or serum half-life of a polymer-modified IL-18 polypeptide of the present disclosure is at least 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold longer than the plasma or serum half-life of a wild-type IL-18 polypeptide. In some embodiments, the plasma or serum half-life of a polymer-modified IL-18 polypeptide of the present disclosure is 1.5-fold to 10-fold longer than the plasma or serum half-life of a wild-type IL-18 polypeptide. In some embodiments, the half-life of a polymer-modified IL-18 polypeptide is at least 10% longer than the half-life of a wild-type IL-18 polypeptide. In some embodiments, the half-life of the polymer-modified IL-18 polypeptide is at least 30% longer than the half-life of the wild-type IL-18 polypeptide.

[0150] In some embodiments, the plasma or serum half-life of a polymer-modified IL-18 polypeptide described herein is at least 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold longer than the plasma or serum half-life of a modified IL-18 polypeptide to which no polymer is attached. In some embodiments, the plasma or serum half-life of a polymer-modified IL-18 polypeptide of the present disclosure is 1.5-fold to 10-fold longer than the plasma or serum half-life of a modified IL-18 polypeptide to which no polymer is attached. In some embodiments, the half-life of a polymer-modified IL-18 polypeptide is at least 10% longer than the half-life of a modified IL-18 polypeptide without a polymer. In some embodiments, the half-life of a polymer-modified IL-18 polypeptide is at least 30% longer than the half-life of a modified IL-18 polypeptide without a polymer.

[0151] IIIc. Additional Site-Specific Modifications of Modified IL-18 Polypeptides In some embodiments, the polymer-modified IL-18 polypeptides described herein contain one or more additional modifications at one or more amino acid residues. As used herein, an "additional modification" is a modification to an IL-18 polypeptide in addition to the attachment of the polymer to the residue and / or the substitution of the residue to which the polymer is attached. Similarly, an "additional substitution" is a substitution at a residue other than the residue to which the polymer is attached, which may or may not be substituted relative to SEQ ID NO: 1. In some embodiments, the additional modification is at a residue to which the polymer is not attached. In some embodiments, the numbering of residue positions in the modified IL-18 polypeptide is based on SEQ ID NO: 1 as the reference sequence.

[0152] Additional modifications to the polypeptides described herein include mutations, addition of various functional groups, deletion of amino acids, addition of amino acids, or any other alteration of the wild-type protein or protein fragment. Functional groups that can be added to the polypeptide include polymers, linkers, alkyl groups, detectable molecules such as chromophores or fluorophores, reactive functional groups, or any combination thereof. In some embodiments, functional groups are added to individual amino acids of the polypeptide. In some embodiments, functional groups are added site-specifically to the polypeptide.

[0153] In some embodiments, the modified IL-18 polypeptides described herein contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional modified amino acid residues.

[0154] In some embodiments, the modified IL-18 polypeptides provided herein comprise the amino acid sequence of any one of SEQ ID NOs: 2-203 provided herein. In some embodiments, the modified IL-18 polypeptides comprise an amino acid sequence that is at least 85% identical to any one of SEQ ID NOs: 2-203. In some embodiments, the modified IL-18 polypeptides provided herein comprise the amino acid sequence of any one of SEQ ID NOs: 2-33 provided herein. In some embodiments, the modified IL-18 polypeptides comprise an amino acid sequence that is at least 85% identical to any one of SEQ ID NOs: 2-33. In some embodiments, the modified IL-18 polypeptides provided herein comprise the amino acid sequence of any one of SEQ ID NOs: 68-163 provided herein. In some embodiments, the modified IL-18 polypeptides comprise an amino acid sequence that is at least 85% identical to any one of SEQ ID NOs: 68-163. In some embodiments, the modified IL-18 polypeptides comprise the amino acid sequence of SEQ ID NO: 30. In some embodiments, the modified IL-18 polypeptides comprise an amino acid sequence that is at least 85% identical to the sequence of SEQ ID NO: 30. In some embodiments, the modified IL-18 polypeptide comprises the amino acid sequence of SEQ ID NO: 59. In some embodiments, the modified IL-18 polypeptide comprises an amino acid sequence that is at least 85% identical to the sequence of SEQ ID NO: 59. In some embodiments, the modified IL-18 polypeptide comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, the modified IL-18 polypeptide comprises an amino acid sequence that is at least 85% identical to the sequence of SEQ ID NO: 2.

[0155] In some embodiments, a polymer-modified IL-18 polypeptide comprising a polymer described herein comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, or at least 9 additional amino acid substitutions, wherein the amino acid substitutions are relative to SEQ ID NO: 1. In some embodiments, the modified IL-18 polypeptide comprises between 1 and 9 amino acid substitutions. In some embodiments, the modified IL-18 polypeptide comprises 1 or 2 amino acid substitutions, 1 to 3 amino acid substitutions, 1 to 4 amino acid substitutions, 1 to 5 amino acid substitutions, 1 to 6 amino acid substitutions, 1 to 7 amino acid substitutions, 1 to 8 amino acid substitutions, 2 to 3 amino acid substitutions, 2 to 4 amino acid substitutions, 2 to 5 amino acid substitutions, 2 to 6 amino acid substitutions, 2 to 7 amino acid substitutions, 2 to 8 amino acid substitutions, 2 to 9 amino acid substitutions, 3 or 4 amino acid substitutions, 3 to 5 amino acid substitutions, 3 to 6 amino acid substitutions, 3 to 7 amino acid substitutions, 3 to 9 amino acid substitutions, 4 or 5 amino acid substitutions, 4 to 6 amino acid substitutions, 4 to 7 amino acid substitutions, 4 to 9 amino acid substitutions, 5 or 6 amino acid substitutions, 5 to 7 amino acid substitutions, 5 to 9 amino acid substitutions, 6 or 7 amino acid substitutions, 6 to 9 amino acid substitutions, or 7 to 9 amino acid substitutions. In some embodiments, the modified IL-18 polypeptide comprises 3 amino acid substitutions, 4 amino acid substitutions, 5 amino acid substitutions, 6 amino acid substitutions, 7 amino acid substitutions, or 9 amino acid substitutions, hi some embodiments, the modified IL-18 polypeptide comprises up to 4 amino acid substitutions, 5 amino acid substitutions, 6 amino acid substitutions, 7 amino acid substitutions, or 9 amino acid substitutions.

[0156] In some embodiments, a polymer-modified IL-18 polypeptide comprising a polymer described herein comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, or at least 9 additional naturally occurring amino acid substitutions, wherein the naturally occurring amino acid substitutions are relative to SEQ ID NO: 1. In some embodiments, the modified IL-18 polypeptide comprises between 1 and 9 naturally occurring amino acid substitutions. In some embodiments, the modified IL-18 polypeptides include 1 or 2 naturally occurring amino acid substitutions, 1 to 3 naturally occurring amino acid substitutions, 1 to 4 naturally occurring amino acid substitutions, 1 to 5 naturally occurring amino acid substitutions, 1 to 6 naturally occurring amino acid substitutions, 1 to 7 naturally occurring amino acid substitutions, 1 to 8 naturally occurring amino acid substitutions, 2 to 3 naturally occurring amino acid substitutions, 2 to 4 naturally occurring amino acid substitutions, 2 to 5 naturally occurring amino acid substitutions, 2 to 6 naturally occurring amino acid substitutions, 2 to 7 naturally occurring amino acid substitutions, 2 to 8 naturally occurring amino acid substitutions, 2 to 9 naturally occurring amino acid substitutions, 3 or 4 naturally occurring amino acid substitutions, 3 to 5 naturally occurring amino acid substitutions, 3 to 6 naturally occurring amino acid substitutions, 3 to 7 naturally occurring amino acid substitutions, 3 to 9 naturally occurring amino acid substitutions, 4 or 5 naturally occurring amino acid substitutions, 4 to 6 naturally occurring amino acid substitutions, 4 to 7 amino acid substitutions, 4 to 9 naturally occurring amino acid substitutions, 5 or 6 naturally occurring amino acid substitutions, 5 to 7 amino acid substitutions, 5 to 9 naturally occurring amino acid substitutions, 6 or 7 naturally occurring amino acid substitutions, 6 to 9 naturally occurring amino acid substitutions, or 7 to 9 naturally occurring amino acid substitutions. In some embodiments, the modified IL-18 polypeptide comprises three naturally occurring amino acid substitutions, four naturally occurring amino acid substitutions, five amino acid substitutions, six naturally occurring amino acid substitutions, seven naturally occurring amino acid substitutions, or nine naturally occurring amino acid substitutions. In some embodiments, the modified IL-18 polypeptide comprises up to four naturally occurring amino acid substitutions, five naturally occurring amino acid substitutions, six naturally occurring amino acid substitutions, seven naturally occurring amino acid substitutions, or nine naturally occurring amino acid substitutions. In some embodiments, the modified IL-18 polypeptide further comprises up to 10 non-standard amino acid substitutions. In some embodiments, the modified IL-18 polypeptide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional non-natural amino acid substitutions.In some embodiments, the modified IL-18 polypeptide further comprises unnatural amino acid substitutions at residues M33, M51, N60, M86, M113, and / or M150. In some embodiments, the unnatural amino acid residues replacing the methionine are each independently norleucine or O-methyl-homoserine. In some embodiments, the modified IL-18 polypeptide further comprises unnatural amino acid substitutions at residues 31, 75, and 116. In some embodiments, the modified IL-18 polypeptide further comprises homoserine (Hse)31, Hse75, and Hse116. In some embodiments, the modified IL-18 polypeptide further comprises unnatural amino acid substitutions at residues 31, 63, and 116. In some embodiments, the modified IL-18 polypeptide further comprises homoserine (Hse)31, Hse63, and Hse116. In some embodiments, the modified IL-18 polypeptide further comprises unnatural amino acid substitutions at residues 31, 63, 75, and 116. In some embodiments, the modified IL-18 polypeptide further comprises homoserine (Hse) 31, Hse 63, Hse 75, and Hse 116. In some embodiments, the modified IL-18 polypeptide further comprises unnatural amino acid substitutions at residues 31, 67, 75, and 116. In some embodiments, the modified IL-18 polypeptide further comprises homoserine (Hse) 31, Hse 67, Hse 75, and Hse 116. In some embodiments, the modified IL-18 polypeptide further comprises unnatural amino acid substitutions at residues 31, 57, 75, and 116. In some embodiments, the modified IL-18 polypeptide further comprises homoserine (Hse) 31, Hse 57, Hse 75, and Hse 116. In some embodiments, the modified IL-18 polypeptide further comprises unnatural amino acid substitutions at residues 31, 50, 75, and 116. In some embodiments, the modified IL-18 polypeptide further comprises homoserine (Hse) 31, Hse 50, Hse 75, and Hse 116. In some embodiments, the modified IL-18 polypeptide further comprises non-natural amino acid substitutions at residues 31, 50, 75, and 121.In some embodiments, the modified IL-18 polypeptide further comprises homoserine (Hse)31, Hse50, Hse75, and Hse121.

[0157] In some embodiments, a polymer-modified IL-18 polypeptide comprising a polymer described herein comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, or at least 9 additional amino acid substitutions, wherein the amino acid substitutions are relative to any one of SEQ ID NOs: 68, 92, 116, 140, or 170. In some embodiments, a polymer-modified IL-18 polypeptide comprising a polymer described herein comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, or at least 9 additional amino acid substitutions, wherein the amino acid substitutions are relative to SEQ ID NOs: 68, 92, 116, or 140. In some embodiments, the modified IL-18 polypeptide comprises between 1 and 9 amino acid substitutions. In some embodiments, the modified IL-18 polypeptide comprises 1 or 2 amino acid substitutions, 1 to 3 amino acid substitutions, 1 to 4 amino acid substitutions, 1 to 5 amino acid substitutions, 1 to 6 amino acid substitutions, 1 to 7 amino acid substitutions, 1 to 8 amino acid substitutions, 2 to 3 amino acid substitutions, 2 to 4 amino acid substitutions, 2 to 5 amino acid substitutions, 2 to 6 amino acid substitutions, 2 to 7 amino acid substitutions, 2 to 8 amino acid substitutions, 2 to 9 amino acid substitutions, 3 or 4 amino acid substitutions, 3 to 5 amino acid substitutions, 3 to 6 amino acid substitutions, 3 to 7 amino acid substitutions, 3 to 9 amino acid substitutions, 4 or 5 amino acid substitutions, 4 to 6 amino acid substitutions, 4 to 7 amino acid substitutions, 4 to 9 amino acid substitutions, 5 or 6 amino acid substitutions, 5 to 7 amino acid substitutions, 5 to 9 amino acid substitutions, 6 or 7 amino acid substitutions, 6 to 9 amino acid substitutions, or 7 to 9 amino acid substitutions. In some embodiments, the modified IL-18 polypeptide comprises 3 amino acid substitutions, 4 amino acid substitutions, 5 amino acid substitutions, 6 amino acid substitutions, 7 amino acid substitutions, or 9 amino acid substitutions, hi some embodiments, the modified IL-18 polypeptide comprises a maximum of 4 amino acid substitutions, 5 amino acid substitutions, 6 amino acid substitutions, 7 amino acid substitutions, or 9 amino acid substitutions.

[0158] In some embodiments, the polymer-modified IL-18 polypeptide comprises one or more modifications in addition to those required for polymer attachment (e.g., amino acid substitutions at residues to which the polymer is not attached). In some embodiments, the additional modifications are 37~193 (SEQ ID NO: 1). SEQ ID NO: 1 reflects the biologically active form of IL-18. Endogenously, IL-18 is initially expressed with an additional 36 amino acid segment at the N-terminus, which is cleaved by caspases to mediate biological activity. In some embodiments, one modification is within amino acid residues 6-63 based on SEQ ID NO: 1. In some embodiments, one modification is at amino acid residue 6. In some embodiments, one modification is within amino acid residues 53-63. In some embodiments, one modification is at amino acid residue 53. In some embodiments, one modification is at amino acid residue 63.

[0159] In some embodiments, the modified IL-18 polypeptide comprises at least one additional modification to the amino acid sequence of SEQ ID NO: 1 selected from Y01X, F02X, E06X, S10X, V11X, D17X, C38X, M51X, K53X, D54X, S55X, T63X, C68X, C76X, and C127X, wherein each X is independently a natural or unnatural amino acid. In some embodiments, the modified IL-18 polypeptide comprises at least one additional modification to the amino acid sequence of SEQ ID NO: 1 selected from Y01G, F02A, E06K, S10T, V11I, D17N, C38S, C38A, C38Q, M51G, K53A, D54A, S55A, T63A, C68S, C68A, E69C, K70C, C76S, C76A, C127A, and C127S.

[0160] In one aspect, described herein are modified interleukin-18 (IL-18) polypeptides having a polymer provided herein, including modified IL-18 polypeptides comprising E06K and K53A, wherein the numbering of residue positions in the modified IL-18 polypeptide is based on SEQ ID NO: 1 as a reference sequence. In some embodiments, the modified IL-18 polypeptide further comprises T63A. In some embodiments, the modified IL-18 polypeptide further comprises at least one of Y01X, S55X, F02X, D54X, C38X, C68X, E69X, K70X, C76X, or C127X, wherein each X is independently an amino acid or amino acid derivative. In some embodiments, the modified IL-18 polypeptide further comprises at least one of Y01G, S55A, F02A, D54A, C38S, C38A, C68S, C68A, K70C, C76S, C76A, C127S, or C127A.

[0161] In some embodiments, the modified IL-18 peptide comprises at least one additional modification relative to the amino acid sequence of SEQ ID NO: 1, wherein the modification is E06X, K53X, S55X, or T63X, where X is a natural or unnatural amino acid. In some embodiments, the modified IL-18 peptide comprises at least two additional modifications relative to the amino acid sequence of SEQ ID NO: 1, wherein the modifications include E06X and K53X, E06X and S55X, K53X and S55X, E06X and T63X, or K53X and T63X, where X is a natural or unnatural amino acid. In some embodiments, the modified IL-18 peptide comprises at least three additional modifications relative to the amino acid sequence of SEQ ID NO: 1, wherein the modifications include E06X, K53X, and S55X, or E06X, K53X, and T63X, where X is a natural or unnatural amino acid. In some embodiments, the modified IL-18 peptide comprises at least four additional modifications relative to the amino acid sequence of SEQ ID NO: 1, the modifications comprising E06X, K53X, S55X, and T63X, or comprising E06X, K53X, S55X, and Y01X, or comprising E06X, K53X, S55X, and F02X, or comprising E06X, K53X, S55X, and D54X, or comprising E06X, K53X, S55X, and M51X, or comprising C38X, C68X, C76X, and C127X, where X is a natural or unnatural amino acid. In each embodiment in which multiple amino acid residues are replaced with a natural or unnatural amino acid X, each X is independently the same or a different amino acid.

[0162] In some embodiments, the modified IL-18 peptide comprises at least one additional modification relative to the amino acid sequence of SEQ ID NO: 1, wherein the modification is E06K, V11I, K53A, S55A, or T63A. In some embodiments, the modified IL-18 peptide comprises at least two additional modifications relative to the amino acid sequence of SEQ ID NO: 1, wherein the modifications include E06K and K53A, or include E06K and S55A, or include K53A and S55A, or include E06K and T63A, or include K53A and T63A. In some embodiments, the modified IL-18 peptide comprises at least three additional modifications relative to the amino acid sequence of SEQ ID NO: 1, wherein the modifications include E06K, K53A, and S55A, or include E06K, V11I, and K53A, or include E06K, C38A, and K53A, or include E06K, K53A, and T63A. In some embodiments, the modified IL-18 peptide comprises at least four additional modifications relative to the amino acid sequence of SEQ ID NO: 1, the modifications comprising E06K, K53A, S55A, and T63A; comprising E06K, K53A, S55A, and Y01G; comprising E06K, K53A, S55A, and F02A; comprising E06K, K53A, S55A, and D54A; comprising E06K, K53A, S55A, and M51G; or comprising C38S, C68S, C76S, and C127S. In some embodiments, the modified IL-18 peptide comprises at least six modifications relative to the amino acid sequence of SEQ ID NO: 1, the modifications including E06K, K53A, C38S, C68S, C76S, and C127S, or the modifications including K53A, T63A, C38S, C68S, C76S, and C127S. In some embodiments, the modified IL-18 peptide comprises at least eight modifications relative to the amino acid sequence of SEQ ID NO: 1, the modifications including Y01G, F02A, E06K, M51G, K53A, D54A, S55A, and T63A. In some embodiments, the modified IL-18 peptide comprises at least eight additional modifications relative to the amino acid sequence of SEQ ID NO: 1, the modifications including Y01G, F02A, E06K, M51G, K53A, D54A, S55A, and T63A.

[0163] In one aspect, provided herein is a modified IL-18 polypeptide comprising a polymer provided herein (e.g., a polymer conjugated to a residue provided herein) and further comprising E06K and K53A, wherein the numbering of modified IL-18 polypeptide residue positions is based on SEQ ID NO: 1 as a reference sequence. In some embodiments, the modified IL-18 polypeptide comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical to the amino acid sequence of SEQ ID NO: 30. In some embodiments, the modified IL-18 polypeptide comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical to the amino acid sequence of SEQ ID NO: 59. In some embodiments, the modified IL-18 polypeptide further comprises an amino acid substitution at one or more cysteine ​​residues. In some embodiments, the modified IL-18 polypeptide comprises one or more cysteines substituted with either serine or alanine. In some embodiments, the modified IL-18 polypeptide comprises an amino acid substitution at each cysteine ​​residue of SEQ ID NO: 1. In some embodiments, each cysteine ​​residue is substituted with serine or alanine. In some embodiments, the modified IL-18 polypeptide comprises a polymer covalently attached to an amino acid residue. In some embodiments, the modified IL-18 polypeptide comprises amino acid substitutions at 1, 2, 3, 4, 5, or 6 methionine residues. In some embodiments, each substitution at a methionine residue is for an O-methyl-L-homoserine residue or a norleucine residue. In some embodiments, each methionine residue is substituted with an O-methyl-L-homoserine residue. In some embodiments, the modified IL-18 polypeptide comprises a homoserine residue at one of positions 31, 116, and 63 and 75. In some embodiments, the modified IL-18 polypeptide comprises a homoserine residue at one of positions 31, 116, 75, and 50, 57, 63, and 67.In some embodiments, the modified IL-18 polypeptide comprises a homoserine residue at one of positions 31, 121, 75, and 50, 57, 63, and 67.

[0164] In some embodiments, a modified IL-18 polypeptide provided herein comprises an amino acid sequence having an amino acid substitution at residue C68 of SEQ ID NO: 30. In some embodiments, the amino acid substitution is a C68S or C68A substitution. In some embodiments, the modified IL-18 polypeptide further comprises a polymer attached at a residue in the region of residues 79-120, such as residues 85, 86, 95, or 98, as provided herein. In some embodiments, the IL-18 polypeptide comprises a cysteine ​​at residue 85, 86, 95, or 98.

[0165] In some embodiments, a modified IL-18 polypeptide provided herein comprises an amino acid sequence having an amino acid substitution at residue C68 of SEQ ID NO: 59. In some embodiments, the amino acid substitution is a C68S or C68A substitution. In some embodiments, the modified IL-18 polypeptide further comprises a polymer attached at a residue in the region of residues 79-120, such as residues 85, 86, 95, or 98, as provided herein. In some embodiments, the IL-18 polypeptide comprises a cysteine ​​at residue 85, 86, 95, or 98.

[0166] In some embodiments, the modified IL-18 polypeptide comprises a polypeptide sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NOs: 2-12. In some embodiments, the modified IL-18 polypeptide comprises a polypeptide sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity to SEQ ID NOs: 13-23. In some embodiments, the modified IL-18 polypeptide comprises a polypeptide sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity to SEQ ID NOs:24-33, and optionally, at least two additional amino acid substitutions relative to the given sequence, wherein one of the amino acid substitutions is at residue C68 (e.g., C68A or C68S) and the other amino acid substitution is at the residue to which the polymer is attached (e.g., residues 85, 86, 95, or 98). In some embodiments, the polypeptide sequence is at least about 80% identical to SEQ ID NO:30 or SEQ ID NO:59. In some embodiments, the polypeptide sequence is at least about 80% identical to SEQ ID NO:30. In some embodiments, the polypeptide sequence is at least about 90% identical to SEQ ID NO:30. In some embodiments, the polypeptide sequence is at least about 95% identical to SEQ ID NO:30. In some embodiments, the polypeptide sequence is at least about 80% identical to SEQ ID NO:59. In some embodiments, the polypeptide sequence is at least about 90% identical to SEQ ID NO: 59. In some embodiments, the polypeptide sequence is at least about 95% identical to SEQ ID NO: 59. In some embodiments, the modified IL-18 polypeptide is recombinant.

[0167] In some embodiments, the modified IL-18 polypeptide is synthetic. In some embodiments, the modified IL-18 polypeptide contains any of the amino acid substitutions present in the synthetic IL-18 polypeptides provided herein (e.g., one or more homoserine, O-methyl-homoserine, or norleucine residues provided herein). Any recombinant modified IL-18 polypeptide provided herein can also be prepared as the corresponding synthetic IL-18 polypeptide.

[0168] In some embodiments, the modified IL-18 polypeptide contains (a) a homoserine residue located at any one of residues 26-36, (b) a homoserine residue located at any one of residues 50-70, (c) a homoserine residue located at any one of residues 70-80, (d) a homoserine residue located at any one of residues 110-125, (e) a norleucine or O-methyl-homoserine residue located at any one of residues 28-38, (f) a norleucine or O-methyl-homoserine residue located at any one of residues 46-56, (g) a norleucine or O-methyl-homoserine residue located at any one of residues 54-56, (h) a norleucine or O-methyl-homoserine residue located at any one of residues 56-56, (i) a norleucine or O-methyl-homoserine residue located at any one of residues 58-59, (j) a norleucine or O-methyl-homoserine residue located at any one of residues 59-60, (k) a norleucine or O-methyl-homoserine residue located at any one of residues 60-62, (k) a norleucine or O-methyl-homoserine residue located at any one of residues 62-64, (k) a norleucine or O-methyl-homoserine residue located at any one of residues 64-66, (k) a norleucine or O-methyl-homoserine residue located at any one of residues 64-68 ... (h) a norleucine or O-methyl-homoserine residue located at any one of residues 80 to 90; (i) a norleucine or O-methyl-homoserine residue located at any one of residues 108 to 118; and (j) a norleucine or O-methyl-homoserine residue located at any one of residues 145 to 155, wherein the numbering of residue positions in the modified IL-18 polypeptide is based on SEQ ID NO: 1 as a reference sequence.

[0169] In some embodiments, the modified IL-18 polypeptide contains (a) a homoserine residue located at any one of residues 26-36, (b) a homoserine residue located at any one of residues 50-70, (c) a homoserine residue located at any one of residues 60-80, (d) a homoserine residue located at any one of residues 110-125, (e) an O-methyl-homoserine residue located at any one of residues 28-38, or (f) an O-methyl-homoserine residue located at any one of residues 46-56. (g) an O-methyl-homoserine residue located at any one of residues 54 to 64; (h) an O-methyl-homoserine residue located at any one of residues 80 to 90; (i) an O-methyl-homoserine residue located at any one of residues 108 to 118; and (j) an O-methyl-homoserine residue located at any one of residues 145 to 155, wherein the numbering of residue positions in the modified IL-18 polypeptide is based on SEQ ID NO: 1 as a reference sequence.

[0170] In some embodiments, the modified IL-18 peptide comprises an amino acid substitution with O-methyl-L-homoserine. In some embodiments, the modified IL-18 peptide comprises an amino acid substitution with O-methyl-L-homoserine at Met33, Met51, Met60, Met86, Met113, or Met150. In some embodiments, the modified IL-18 polypeptide comprises one or more amino acid substitutions selected from homoserine (Hse)31, norleucine (Nle)33, O-methyl-homoserine (Omh)33, Hse50, Nle51, Omh51, Hse57, Nle60, Omh60, Hse63, Hse67, Hse75, Nle86, Omh86, Hse116, Nle113, Omh113, Hse121, Nle150, and Omh150. In some embodiments, each methionine except M86 is replaced with Nle or Omh, and residue 86 is attached to a polymer.

[0171] In some embodiments, the modified IL-18 polypeptides described herein contain a linker moiety. In some embodiments, the linker moiety includes, but is not limited to, a polymer, a linker, a spacer, or a combination thereof. When attached to a specific amino acid residue, the linker moiety can modulate the activity or other properties of the modified IL-18 polypeptide compared to wild-type IL-18.

[0172] In some embodiments, the modified IL-18 polypeptide is linked to an additional polypeptide. In some embodiments, the modified IL-18 polypeptide and the additional polypeptide form a fusion polypeptide. In some embodiments, the synthetic IL-18 polypeptide is linked to the additional polypeptide through a non-covalent interaction. In some embodiments, the non-covalent interaction is biotin interacting with streptavidin or avidin. In some embodiments, the modified IL-18 polypeptide and the additional polypeptide are conjugated together (e.g., using a chemical linker such as a polymer provided herein). In some embodiments, the additional polypeptide is an antibody, antibody fragment, single-chain variable fragment (ScFv), peptide aptamer, cyclic peptide, branched peptide, growth factor, peptide hormone, chemokine, or cytokine. In some embodiments, the additional polypeptide is attached to a polymer provided herein (e.g., a polymer attached in the region of residues 79-120). In some embodiments, the additional polypeptide comprises an antibody or binding fragment thereof. In some embodiments, the antibody comprises a humanized antibody, a murine antibody, a chimeric antibody, a bispecific antibody, any fragment thereof, or any combination thereof. In some embodiments, the antibody is a monoclonal antibody or any fragment thereof. In some embodiments, the additional polypeptide is a cytokine. In some embodiments, the additional polypeptide is a half-life extending polypeptide (e.g., albumin).

[0173] IV. Biological Activities of Modified IL-18 Polypeptides IVa. Binding affinity Affinity for the IL-18 receptor alpha / beta heterodimer (IL-18Rα / β) In some embodiments, the polymer-modified IL-18 polypeptide to which the polymer is attached exhibits at most a slightly decreased affinity for IL-18Rαβ compared to WT IL-18 (SEQ ID NO: 1). In some embodiments, the polymer-modified IL-18 polypeptide exhibits at most 2-fold lower, at most 3-fold lower, at most 4-fold lower, at most 5-fold lower, at most 10-fold lower, at most 15-fold lower, at most 20-fold lower, at most 30-fold lower, at most 40-fold lower, at most 50-fold lower, at most 75-fold lower, or at most 100-fold lower affinity for IL-18Rαβ compared to the affinity of WT IL-18 for IL-18Rαβ. In some embodiments, the modified IL-18 polypeptide exhibits at most 10-fold lower affinity for IL-18Rαβ compared to the affinity of WT IL-18 for IL-18Rαβ. In some embodiments, the polymer-modified IL-18 polypeptide exhibits up to a 20-fold lower affinity for IL-18Rαβ compared to the affinity of WT IL-18 for IL-18Rαβ. In some embodiments, the polymer-modified IL-18 polypeptide exhibits up to a 50-fold lower affinity for IL-18Rαβ compared to the affinity of WT IL-18 for IL-18Rαβ. In some embodiments, the polymer-modified IL-18 polypeptide exhibits up to a 100-fold lower affinity for IL-18Rαβ compared to the affinity of WT IL-18 for IL-18Rαβ.

[0174] In some embodiments, modified IL-18 polypeptides provided herein that are not conjugated with a polymer exhibit improved or slightly decreased affinity for IL-18Rαβ compared to WT IL-18. In some embodiments, modified IL-18 polypeptides that do not contain a polymer exhibit improved (e.g., at least 2-fold higher, at least 5-fold higher, at least 10-fold higher, or at least 20-fold higher) affinity for IL-18Rαβ compared to WT IL-18. In some embodiments, modified IL-18 polypeptides that are not conjugated with a polymer exhibit at least 30-fold higher, at least 40-fold higher, or at least 50-fold higher affinity. In some embodiments, modified IL-18 polypeptides provided herein exhibit improved or slightly decreased affinity for IL-18Rαβ compared to WT IL-18. In some embodiments, the modified IL-18 polypeptide exhibits improved affinity for IL-18Rαβ (e.g., at least 2-fold greater, at least 5-fold greater, at least 10-fold greater, at least 20-fold greater, at least 30-fold greater, at least 40-fold greater, or at least 50-fold greater affinity) compared to WT IL-18.

[0175] In some embodiments, the polymer-modified IL-18 polypeptides provided herein having a polymer attached thereto have a K D In some embodiments, the polymer-modified IL-18 polypeptide exhibits at most a slight reduction in binding to IL-18Rαβ as measured by K D In some embodiments, the polymer-modified IL-18 polypeptide has a K with IL-18Rαβ of up to about 20 nM. D In some embodiments, the polymer-modified IL-18 polypeptide has a K with IL-18Rαβ of up to about 30 nM. D In some embodiments, the polymer-modified IL-18 polypeptide has a K with IL-18Rαβ of up to about 40 nM. DIn some embodiments, the polymer-modified IL-18 polypeptide has a K with IL-18Rαβ of up to about 50 nM. D Shows.

[0176] In some embodiments, polymer-modified IL-18 polypeptides provided herein having polymers attached thereto (e.g., covalently attached to residues in the region of residues 79-120, such as residues 85, 86, or 98) exhibit at most a slightly decreased affinity for IL-18Rαβ compared to the corresponding modified IL-18 polypeptide without the polymer attached. In some embodiments, the polymer-modified IL-18 polypeptides exhibit at most 2-fold lower, at most 3-fold lower, at most 4-fold lower, at most 5-fold lower, at most 10-fold lower, at most 15-fold lower, at most 20-fold lower, at most 30-fold lower, at most 40-fold lower, at most 50-fold lower, at most 75-fold lower, or at most 100-fold lower affinity for IL-18Rαβ compared to the affinity of the corresponding modified IL-18 polypeptide without the polymer attached thereto. In some embodiments, the polymer-modified IL-18 polypeptide exhibits up to a 2-fold lower affinity for IL-18Rαβ compared to the affinity of the corresponding modified IL-18 polypeptide without the polymer attached. In some embodiments, the polymer-modified IL-18 polypeptide exhibits up to a 3-fold lower affinity for IL-18Rαβ compared to the affinity of the corresponding modified IL-18 polypeptide without the polymer attached. In some embodiments, the polymer-modified IL-18 polypeptide exhibits up to a 4-fold lower affinity for IL-18Rαβ compared to the affinity of the corresponding modified IL-18 polypeptide without the polymer attached. In some embodiments, the polymer-modified IL-18 polypeptide exhibits up to a 5-fold lower affinity for IL-18Rαβ compared to the affinity of the corresponding modified IL-18 polypeptide without the polymer attached. In some embodiments, the polymer-modified IL-18 polypeptide exhibits up to a 10-fold lower affinity for IL-18Rαβ compared to the affinity of the corresponding modified IL-18 polypeptide without the polymer attached.

[0177] Affinity for IL-18 receptor alpha (IL-18Rα) The modified IL-18 polypeptides provided herein may exhibit higher binding affinity to the IL-18 receptor alpha subunit compared to WT IL-18 (SEQ ID NO: 1). In certain embodiments, the modified IL-18 polypeptides provided herein exhibit at least 50-fold, at least 52-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, at least 100-fold, at least 120-fold, at least 150-fold, at least 200-fold, at least 300-fold, or at least 500-fold higher binding affinity to the IL-18 receptor alpha subunit compared to WT IL-18. The modified IL-18 polypeptides may exhibit a dissociation constant (K D ) at least 50-fold lower K with the IL-18 receptor alpha subunit D In certain embodiments, the modified IL-18 polypeptide may exhibit a K D K with the IL-18 receptor alpha subunit that is at least 52-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold lower than D In certain embodiments, the modified IL-18 polypeptide has a K D At least 52-fold lower K with IL-18 receptor alpha subunit D In certain embodiments, the modified IL-18 polypeptide has a K D K with IL-18 receptor alpha subunit at least 60-fold lower than D In certain embodiments, the modified IL-18 polypeptide has a K D K with IL-18 receptor alpha subunit at least 70-fold lower than D In certain embodiments, the modified IL-18 polypeptide has a K D K with IL-18 receptor alpha subunit at least 80-fold lower DIn certain embodiments, the modified IL-18 polypeptide has a K D K with IL-18 receptor alpha subunit at least 90-fold lower than D In certain embodiments, the modified IL-18 polypeptide has a K D K with IL-18 receptor alpha subunit at least 100-fold lower D In certain embodiments, the modified IL-18 polypeptide has a K D 50- to 500-fold lower K D In certain embodiments, the modified IL-18 polypeptide has a K D 50x to 52x, 50x to 60x, 50x to 70x, 50x to 80x, 50x to 90x, 50x to 100x, 50x to 120x, 50x to 150x, 50x to 200x, 50x to 300x, 50x to 500x, 52x to 60x, 52x to 70x, 52x to 80x, 52x to 90x, 52x to 100x, 52x to 120x, 52x to 150x, 5 2x~200x, 52x~300x, 52x~500x, 60x~70x, 60x~80x, 60x~90x, 60x~100x, 60x~120x, 60x~150x, 60x~200x, 60x~300x, 60x~500x, 70x~80x, 70x~90x, 70x~100x, 70x~120x, 70x~150x, 70x~200x , 70x~300x, 70x~500x, 80x~90x, 80x~100x, 80x~120x, 80x~150x, 80x~200x, 80x~300x, 80x~5 00x, 90x~100x, 90x~120x, 90x~150x, 90x~200x, 90x~300x, 90x~500x, 100x~120x, 100x~150x, 100x-200x, 100x-300x, 100x-500x, 120x-150x, 120x-200x, 120x-300x, 120x-500x, 150x-200x, 150x-300x, 150x-500x, 200x-300x, 200x-500x, or 300x-500x lower K with IL-18 receptor alpha subunit DIn certain embodiments, the modified IL-18 polypeptide has a K D 50-fold, 52-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 120-fold, 150-fold, 200-fold, 300-fold, or 500-fold lower K with IL-18 receptor alpha subunit D In certain embodiments, the modified IL-18 polypeptide has a K D at least 50-fold, 52-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 120-fold, 150-fold, 200-fold, or 300-fold lower K with the IL-18 receptor alpha subunit D Shows.

[0178] In some embodiments, the polymer-modified IL-18 polypeptide to which the polymer is attached exhibits at most a slightly reduced affinity for IL-18Rα compared to WT IL-18 (SEQ ID NO: 1). In some embodiments, the polymer-modified IL-18 polypeptide exhibits at most 2-fold lower, at most 3-fold lower, at most 4-fold lower, at most 5-fold lower, at most 10-fold lower, at most 15-fold lower, at most 20-fold lower, at most 30-fold lower, at most 40-fold lower, at most 50-fold lower, at most 75-fold lower, at most 100-fold lower, at most 200-fold lower, or at most 500-fold lower affinity for IL-18Rα compared to the affinity of WT IL-18 for IL-18Rα. In some embodiments, the polymer-modified IL-18 polypeptide exhibits at most 10-fold lower affinity for IL-18Rα compared to the affinity of WT IL-18 for IL-18Rα. In some embodiments, the polymer-modified IL-18 polypeptide exhibits up to a 20-fold lower affinity for IL-18Rα compared to the affinity of WT IL-18 for IL-18Rα. In some embodiments, the polymer-modified IL-18 polypeptide exhibits up to a 50-fold lower affinity for IL-18Rα compared to the affinity of WT IL-18 for IL-18Rα. In some embodiments, the polymer-modified IL-18 polypeptide exhibits up to a 100-fold lower affinity for IL-18Rα compared to the affinity of WT IL-18 for IL-18Rα.

[0179] In some embodiments, the polymer-modified IL-18 polypeptides provided herein are D In some embodiments, the polymer-modified IL-18 polypeptide exhibits only a slight reduction in binding to IL-18Rα compared to when measured by IL-18Rα. In some embodiments, the polymer-modified IL-18 polypeptide exhibits a K of IL-18Rα of up to about 20 nM, up to about 30 nM, up to about 50 nM, up to about 75 nM, up to about 100 nM, up to about 200 nM, up to about 500 nM, up to about 1000 nM, up to about 2000 nM, up to about 3000 nM, or up to about 5000 nM. D In some embodiments, the polymer-modified IL-18 polypeptide has a K of IL-18Rα of up to about 100 nM. D In some embodiments, the polymer-modified IL-18 polypeptide has a K of IL-18Rα of up to about 200 nM. D In some embodiments, the polymer-modified IL-18 polypeptide has a K of IL-18Rα of up to about 500 nM. D In some embodiments, the polymer-modified IL-18 polypeptide has a K of IL-18Rα of up to about 1000 nM. D In some embodiments, the polymer-modified IL-18 polypeptide has a K of IL-18Rα of up to about 5000 nM. D Shows.

[0180] In some embodiments, polymer-modified IL-18 polypeptides provided herein having polymers attached thereto (e.g., covalently attached to a residue in the region of residues 79-100, such as residues 85, 86, or 98) exhibit at most a slightly decreased affinity for IL-18Rα compared to the corresponding modified IL-18 polypeptide without the polymer attached. In some embodiments, the polymer-modified IL-18 polypeptides exhibit at most 2-fold lower, at most 3-fold lower, at most 4-fold lower, at most 5-fold lower, at most 10-fold lower, at most 15-fold lower, at most 20-fold lower, at most 30-fold lower, at most 40-fold lower, at most 50-fold lower, at most 75-fold lower, at most 100-fold lower, at most 200-fold lower, or at most 500-fold lower affinity for IL-18Rαβ compared to the affinity of the corresponding modified IL-18 polypeptide without the polymer attached thereto. In some embodiments, the polymer-modified IL-18 polypeptide exhibits up to a 2-fold lower affinity for IL-18Rα compared to the affinity of the corresponding modified IL-18 polypeptide without the polymer attached. In some embodiments, the polymer-modified IL-18 polypeptide exhibits up to a 3-fold lower affinity for IL-18Rα compared to the affinity of the corresponding modified IL-18 polypeptide without the polymer attached. In some embodiments, the polymer-modified IL-18 polypeptide exhibits up to a 4-fold lower affinity for IL-18Rα compared to the affinity of the corresponding modified IL-18 polypeptide without the polymer attached. In some embodiments, the polymer-modified IL-18 polypeptide exhibits up to a 5-fold lower affinity for IL-18Rα compared to the affinity of the corresponding modified IL-18 polypeptide without the polymer attached. In some embodiments, the polymer-modified IL-18 polypeptide exhibits up to a 10-fold lower affinity for IL-18Rα compared to the affinity of the corresponding modified IL-18 polypeptide without the polymer attached.

[0181] Affinity for IL-18BP In some embodiments, the modified IL-18 polypeptides provided herein exhibit reduced affinity for IL-18 binding protein (IL-18BP) compared to WT IL-18 (SEQ ID NO: 1). In some embodiments, the modified IL-18 polypeptides exhibit at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, at least 100-fold, or at least 200-fold lower affinity for IL-18BP compared to WT IL-18. In some embodiments, the modified IL-18 polypeptides exhibit at least 2-fold lower affinity for IL-18BP compared to WT IL-18. In some embodiments, the modified IL-18 polypeptides exhibit at least 2-fold lower affinity for IL-18BP compared to WT IL-18. In some embodiments, the modified IL-18 polypeptides exhibit at least 5-fold lower affinity for IL-18BP compared to WT IL-18. In some embodiments, the modified IL-18 polypeptides exhibit at least 10-fold reduced affinity for IL-18BP compared to WT IL-18. In some embodiments, the modified IL-18 polypeptides exhibit at least 20-fold reduced affinity for IL-18BP compared to WT IL-18. In some embodiments, the modified IL-18 polypeptides exhibit at least 30-fold reduced affinity for IL-18BP compared to WT IL-18. In some embodiments, the modified IL-18 polypeptides exhibit at least 40-fold reduced affinity for IL-18BP compared to WT IL-18. In some embodiments, the modified IL-18 polypeptides exhibit at least 50-fold reduced affinity for IL-18BP compared to WT IL-18. In some embodiments, the modified IL-18 polypeptides exhibit at least 60-fold reduced affinity for IL-18BP compared to WT IL-18. In some embodiments, the modified IL-18 polypeptides exhibit at least 70-fold reduced affinity for IL-18BP compared to WT IL-18.In some embodiments, the modified IL-18 polypeptides exhibit at least an 80-fold lower affinity for IL-18BP compared to WT IL-18. In some embodiments, the modified IL-18 polypeptides exhibit at least a 90-fold lower affinity for IL-18BP compared to WT IL-18. In some embodiments, the modified IL-18 polypeptides exhibit at least a 100-fold lower affinity for IL-18BP compared to WT IL-18. In some embodiments, the modified IL-18 polypeptides exhibit at least a 200-fold lower affinity for IL-18BP compared to WT IL-18.

[0182] In some embodiments, the polymer-modified IL-18 polypeptides provided herein exhibit reduced affinity for IL-18 binding protein (IL-18BP) compared to WT IL-18 (SEQ ID NO: 1). In some embodiments, the polymer-modified IL-18 polypeptides exhibit at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, or at least 100-fold lower affinity for IL-18BP compared to WT IL-18. In some embodiments, the polymer-modified IL-18 polypeptides exhibit at least 10-fold lower affinity for IL-18BP compared to WT IL-18. In some embodiments, the polymer-modified IL-18 polypeptides exhibit at least 20-fold lower affinity for IL-18BP compared to WT IL-18. In some embodiments, the polymer-modified IL-18 polypeptides exhibit at least 50-fold lower affinity for IL-18BP compared to WT IL-18. In some embodiments, the polymer-modified IL-18 polypeptide exhibits at least an 80-fold lower affinity for IL-18BP compared to WT IL-18. In some embodiments, the polymer-modified IL-18 polypeptide exhibits at least a 100-fold lower affinity for IL-18BP compared to WT IL-18.

[0183] In some embodiments, the polymer-modified IL-18 polypeptides provided herein are D In some embodiments, the polymer-modified IL-18 polypeptide exhibits reduced binding to IL-18BP compared to WT IL-18, as measured by K (K = K) = K ... D In some embodiments, the polymer-modified IL-18 polypeptide has a K with IL-18BP of at least about 1 nM. D In some embodiments, the polymer-modified IL-18 polypeptide has a K with IL-18BP of at least about 5 nM. D In some embodiments, the polymer-modified IL-18 polypeptide has a K with IL-18BP of at least about 50 nM. D In some embodiments, the polymer-modified IL-18 polypeptide has a K with IL-18BP of at least about 100 nM. D In some embodiments, the polymer-modified IL-18 polypeptide has a K with IL-18BP of at least about 500 nM. D Shows.

[0184] Ratio of affinities for IL-18BP and various IL-18 receptors In some embodiments, the modified IL-18 polypeptide exhibits a wide window in which the modified IL-18 polypeptide binds to IL-18Rαβ even in the presence of IL-18BP. In some embodiments, this window is greater than the K of the IL-18 / IL-18Rαβ interaction. D K of IL-18 / IL-18BP interaction against Dand a higher number indicates a larger window over which the modified IL-18 polypeptide is expected to be active in vivo. In some embodiments, the modified IL-18 polypeptide has a K of at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 10, at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, or at least about 50 for the IL-18 / IL-18Rαβ interaction. D K of IL-18 / IL-18BP interaction against D In some embodiments, the modified IL-18 polypeptide has a K of IL-18 / IL-18Rαβ interaction of at least about 2. D K of IL-18 / IL-18BP interaction against D In some embodiments, the modified IL-18 polypeptide has a K of IL-18 / IL-18Rαβ interaction of at least about 5. D K of IL-18 / IL-18BP interaction against D In some embodiments, the modified IL-18 polypeptide has a K of IL-18 / IL-18Rαβ interaction of at least about 10. D K of IL-18 / IL-18BP interaction against D In some embodiments, the modified IL-18 polypeptide has a K of IL-18 / IL-18Rαβ interaction of at least about 25. D K of IL-18 / IL-18BP interaction against D In some embodiments, the modified IL-18 polypeptide has a K of IL-18 / IL-18Rαβ interaction of at least about 30. D K of IL-18 / IL-18BP interaction against D In some embodiments, the modified IL-18 polypeptide has a K of IL-18 / IL-18Rαβ interaction of at least about 40. D K of IL-18 / IL-18BP interaction against DIn some embodiments, the modified IL-18 polypeptide has a K of IL-18 / IL-18Rαβ interaction of at least about 45. D K of IL-18 / IL-18BP interaction against D In some embodiments, the modified IL-18 polypeptide has a K of IL-18 / IL-18Rαβ interaction of at least about 50. D K of IL-18 / IL-18BP interaction against D The ratio of

[0185] In some embodiments, the modified IL-18 polypeptide exhibits a wide window through which the polymer-modified IL-18 polypeptide binds to IL-18Rαβ even in the presence of IL-18 BP. In some embodiments, this window is greater than or equal to the K of the IL-18 / IL-18Rαβ interaction. D K of IL-18 / IL-18BP interaction against D where a higher number indicates a larger window over which the polymer-modified IL-18 polypeptide is expected to be active in vivo. In some embodiments, the polymer-modified IL-18 polypeptide has a K of at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 10, at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, or at least about 50 for the IL-18 / IL-18Rαβ interaction. D K of IL-18 / IL-18BP interaction against D In some embodiments, the polymer-modified IL-18 polypeptide has a K of IL-18 / IL-18Rαβ interaction of at least about 2. D K of IL-18 / IL-18BP interaction against D In some embodiments, the polymer-modified IL-18 polypeptide has a K of IL-18 / IL-18Rαβ interaction of at least about 5. D K of IL-18 / IL-18BP interaction against DIn some embodiments, the polymer-modified IL-18 polypeptide has a K of IL-18 / IL-18Rαβ interaction of at least about 10. D K of IL-18 / IL-18BP interaction against D In some embodiments, the polymer-modified IL-18 polypeptide has a K of IL-18 / IL-18Rαβ interaction of at least about 25. D K of IL-18 / IL-18BP interaction against D In some embodiments, the polymer-modified IL-18 polypeptide has a K of IL-18 / IL-18Rαβ interaction of at least about 30. D K of IL-18 / IL-18BP interaction against D In some embodiments, the polymer-modified IL-18 polypeptide has a K of IL-18 / IL-18Rαβ interaction of at least about 40. D K of IL-18 / IL-18BP interaction against D In some embodiments, the polymer-modified IL-18 polypeptide has a K of IL-18 / IL-18Rαβ interaction of at least about 45. D K of IL-18 / IL-18BP interaction against D In some embodiments, the polymer-modified IL-18 polypeptide has a K of IL-18 / IL-18Rαβ interaction of at least about 50. D K of IL-18 / IL-18BP interaction against D The ratio of

[0186] In some embodiments, the K of IL-18 / IL-18Rαβ for a polymer-modified IL-18 polypeptide comprising a polymer attached at a residue provided herein (e.g., covalently attached to a residue within the region of residues 79-120, such as residues 85, 86, or 98) is D K of IL-18 / IL-18BP against Dis increased compared to WT IL-18. In some embodiments, the ratio is increased by at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 15-fold, or at least 20-fold compared to the ratio of WT IL-18.

[0187] IVb. Functional activity The modified IL-18 polypeptides provided herein have an increased ability to signal through the IL-18 receptor (IL-18R) compared to WT IL-18. The modified IL-18 polypeptides also exhibit a reduced ability to be inhibited by IL-18BP compared to WT IL-18.

[0188] In some embodiments, the polymer-modified IL-18 polypeptides provided herein having a polymer attached (e.g., at one of residues 79-120) exhibit one or more activities associated with WT IL-18. In some embodiments, the polymer-modified IL-18 polypeptides exhibit equivalent (e.g., substantially the same or only slightly reduced) ability to signal through the IL-18 receptor (IL-18R) compared to a corresponding modified IL-18 polypeptide without a polymer attached. In some embodiments, the polymer-modified IL-18 polypeptides exhibit equivalent ability to signal through IL-18R compared to WT IL-18. In some embodiments, the ability of the polymer-modified IL-18 polypeptide to signal through IL-18R is only slightly reduced compared to WT IL-18.

[0189] In some embodiments, the polymer-modified IL-18 polypeptide also exhibits a reduced ability to be inhibited by IL-18BP compared to WT IL-18. In some embodiments, the polymer-modified IL-18 polypeptide to which a polymer is attached exhibits a reduced ability to be inhibited by IL-18BP compared to the corresponding modified IL-18 polypeptide to which the polymer is not attached.

[0190] IFNγ production stimulating activity (EC50 ) In some embodiments, the modified IL-18 polypeptide modulates IFNγ production when contacted with a cell (e.g., an immune cell such as an NK cell). In some embodiments, the ability of the modified IL-18 polypeptide to modulate IFNγ production is measured by measuring the half-maximal effective concentration (EC 50The EC50 for inducing IFNγ production in cells is measured as the EC50 of the modified IL-18 polypeptide. The modified IL-18 polypeptide has a higher ability to induce IFNγ production in cells compared to WT IL-18. In certain embodiments, the EC50 for inducing IFNγ production in cells for the modified IL-18 polypeptide is 25- to 200-fold lower than the EC50 of ... EC50 for IL-18: 25x-30x, 25x-40x, 25x-50x, 25x-60x, 25x-70x, 25x-80x, 25x-90x, 25x-100x, 25x-150x, 25x-200x, 30x-40x, 30x-50x, 30x-60x, 30x-70x, 30x-80x, 30x-90x, 30x-100x, 30x-150x, 30x-200x, 40x-50x, 40x-60x, 40x-70x, 40x-80x, 40x-90x, 40x-100x, 40x-150x, 40x-200x, 50x-60x, 50x-60x, 50x-60x, 50x-70x, 50x-80x, 50x-90x, 5 ... 0x~70x, 50x~80x, 50x~90x, 50x~100x, 50x~150x, 50x~200x, 60x~70x, 60x~ 80x, 60x~90x, 60x~100x, 60x~150x, 60x~200x, 70x~80x, 70x~90x, 70x~100x , 70-fold to 150-fold, 70-fold to 200-fold, 80-fold to 90-fold, 80-fold to 100-fold, 80-fold to 150-fold, 80-fold to 200-fold, 80-fold to 500-fold, 90-fold to 100-fold, 90-fold to 150-fold, 90-fold to 200-fold, 100-fold to 150-fold, 100-fold to 200-fold, or 150-fold to 200-fold lower. In certain embodiments, the EC50 for inducing IFNγ production in cells for the modified IL-18 polypeptide is 25-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 150-fold, or 200-fold lower than the EC50 for WT IL-18. In certain embodiments, the modified IL-18 polypeptide has an EC50 for inducing IFNγ production in a cell that is at least 25-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 150-fold, or 200-fold lower than the EC50 of WT IL-18.In certain embodiments, the EC50 for the modified IL-18 polypeptide to induce IFNγ production in a cell is at least 30-fold lower than the EC50 for WT IL-18. In certain embodiments, the EC50 for the modified IL-18 polypeptide to induce IFNγ production in a cell is at least 40-fold lower than the EC50 for WT IL-18. In certain embodiments, the EC50 for the modified IL-18 polypeptide to induce IFNγ production in a cell is at least 50-fold lower than the EC50 for WT IL-18. In certain embodiments, the EC50 for the modified IL-18 polypeptide to induce IFNγ production in a cell is at least 60-fold lower than the EC50 for WT IL-18. In certain embodiments, the EC50 for the modified IL-18 polypeptide to induce IFNγ production in a cell is at least 70-fold lower than the EC50 for WT IL-18. In certain embodiments, the modified IL-18 polypeptide has an EC50 for inducing IFNγ production in cells that is at least 80-fold lower than the EC50 of WT IL-18.

[0191] In certain embodiments, the modified IL-18 polypeptide has an EC50 for signaling through the IL-18 receptor that is at least 4.6- to 200-fold lower than the EC50 for WT IL-18. In certain embodiments, the modified IL-18 polypeptide has an EC50 for signaling through the IL-18 receptor that is at least 4.6- to 200-fold lower than the EC50 for WT IL-18. IL-18 EC50 of at least 4.6x-4.7x, 4.6x-5x, 4.6x-10x, 4.6x-20x, 4.6x-30x, 4.6x-40x, 4.6x-50x, 4.6x-75x, 4.6x-100x, 4.6x-200x, 4.7x-5x, 4.7x-10x, 4.7x-20x, 4.7x-30x, 4.7x-40x, 4.7x-50x, 4.7x-75x, 4.7x-100x, 4.7x-200x, 5x-10x, 5x-20x, 5x-30x, 5x-40x, 5x-50x, 5x-75x, 5x-100x, 5x-20x 0x, 10x-20x, 10x-30x, 10x-40x, 10x-50x, 10x-75x, 10x-100x, 10x-200x, 20x-30x, 20x-40x, 20x-50x, 20x-75x, 20x-100x, 20x-200x, 30x-40x, 30x-50x, 30x-75x, 30x-100x, 30x-200x, 40x-50x, 40x-75x, 40x-100x, 40x-200x, 50x-75x, 50x-100x, 50x-200x, 75x-100x, 75x-200x, or 100x-200x lower. In certain embodiments, the EC50 for signaling through the IL-18 receptor for the modified IL-18 polypeptide is 4.6-fold, 4.7-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 75-fold, 100-fold, or 200-fold lower than the EC50 for WT IL-18. In certain embodiments, the EC50 for signaling through the IL-18 receptor for the modified IL-18 polypeptide is at least 4.6-fold, 4.7-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 75-fold, 100-fold, or 200-fold lower than the EC50 for WT IL-18. In certain embodiments, the EC50 for signaling through the IL-18 receptor for the modified IL-18 polypeptide is at least 4.7-fold lower than the EC50 for WT IL-18.In certain embodiments, the EC50 for signaling through the IL-18 receptor for the modified IL-18 polypeptide is at least 5-fold lower than the EC50 for WT IL-18. In certain embodiments, the EC50 for signaling through the IL-18 receptor for the modified IL-18 polypeptide is at least 10-fold lower than the EC50 for WT IL-18. In certain embodiments, the EC50 for signaling through the IL-18 receptor for the modified IL-18 polypeptide is at least 20-fold lower than the EC50 for WT IL-18. In certain embodiments, the EC50 for signaling through the IL-18 receptor for the modified IL-18 polypeptide is at least 30-fold lower than the EC50 for WT IL-18. In certain embodiments, the EC50 for signaling through the IL-18 receptor for the modified IL-18 polypeptide is at least 40-fold lower than the EC50 for WT IL-18. In certain embodiments, the modified IL-18 polypeptide has an EC50 for signaling through the IL-18 receptor that is at least 50-fold lower than the EC50 for WT IL-18. The EC50 for signaling through the IL-18 receptor can be measured using a HEK-Blue IL18R reporter assay (e.g., as provided in the Examples herein).

[0192] In some embodiments, the EC20 value of the ability of the polymer-modified IL-18 polypeptide to induce IFNγ is 50 (nM) is the EC of the L-18 polypeptide of SEQ ID NO: 1 50 (nM) of the polymer-modified IL-18 polypeptide. 50 is the EC of the IL-18 polypeptide of SEQ ID NO: 1 50 (nM). In some embodiments, the EC 50 is the EC of the IL-18 polypeptide of SEQ ID NO: 1 50(nM). In some embodiments, the EC 50 is the EC of the IL-18 polypeptide of SEQ ID NO: 1 50 (nM). In some embodiments, the EC 50 is the EC of the IL-18 polypeptide of SEQ ID NO: 1 50 (nM) of the ability of the polymer-modified IL-18 polypeptide to induce IFNγ. 50 is measured by an IFNγ induction cell assay.

[0193] In some embodiments, the EC of the polymer-modified IL-18 polypeptide for its ability to induce IFNγ production is 50 In some embodiments, the EC 20 of the polymer-modified IL-18 polypeptide for its ability to induce IFNγ production is less than about 100 nM, less than about 75 nM, less than about 50 nM, less than about 40 nM, less than about 30 nM, less than about 20 nM, less than about 15 nM, or less than about 10 nM. 50 In some embodiments, the EC 20 of the polymer-modified IL-18 polypeptide to induce IFNγ production is less than about 100 nM. 50 In some embodiments, the EC 20 of the polymer-modified IL-18 polypeptide to induce IFNγ production is less than about 80 nM. 50 In some embodiments, the EC 20 of the polymer-modified IL-18 polypeptide to induce IFNγ production is less than about 50 nM. 50 is less than about 10 nM.

[0194] In some embodiments, polymer-modified IL-18 polypeptides comprising a polymer conjugated thereto (e.g., a polymer conjugated to a region of residues 79-120, such as residues 85, 86, or 98) provided herein exhibit a comparable or only slightly reduced ability to induce IFNγ production compared to a corresponding modified IL-18 polypeptide that does not have a polymer conjugated thereto. In some embodiments, the EC of IFNγ production of the polymer-modified IL-18 polypeptide to which the polymer is conjugated is 50 (nM) is the EC of the modified IL-18 polypeptide without polymer attachment 50 In some embodiments, the EC of IFNγ production of the polymer-modified IL-18 polypeptide to which the polymer is attached is up to about 100-fold higher, up to about 50-fold higher, up to about 10-fold higher, up to about 9-fold higher, up to about 8-fold higher, up to about 7-fold higher, up to about 6-fold higher, up to about 5-fold higher, up to about 4-fold higher, up to about 3-fold higher, or up to about 2-fold higher. 50 (nM) is the EC of the modified IL-18 polypeptide without polymer attachment 50 In some embodiments, the EC 200 of the polymer-modified IL-18 polypeptide to which the polymer is attached is up to about 10-fold higher than that of the EC 200 of the polymer-modified IL-18 polypeptide to which the polymer is attached. 50 (nM) is the EC of the modified IL-18 polypeptide without polymer attachment 50 In some embodiments, the EC 200 of the polymer-modified IL-18 polypeptide to which the polymer is attached is up to about 5-fold higher than that of the EC 200 of the polymer-modified IL-18 polypeptide to which the polymer is attached. 50 (nM) is the EC of the modified IL-18 polypeptide without polymer attachment 50 In some embodiments, the EC 200 of the polymer-modified IL-18 polypeptide to which the polymer is attached is up to about 4-fold higher than that of the EC 200 of the polymer-modified IL-18 polypeptide to which the polymer is attached. 50 (nM) is the EC of the modified IL-18 polypeptide without polymer attachment 50 In some embodiments, the EC 200 of the polymer-modified IL-18 polypeptide to which the polymer is attached is up to about 3-fold higher than that of the EC 200 of the polymer-modified IL-18 polypeptide to which the polymer is attached. 50 (nM) is the EC of the modified IL-18 polypeptide without polymer attachment 50 is approximately twice as high as that of

[0195] Inhibition by IL-18BP (IC 50 ) In some embodiments, the modified IL-18 exhibits a reduced ability to have its IFNγ-inducing activity inhibited by IL-18BP compared to WT IL-18. In some embodiments, the modified IL-18 exhibits a half-maximal inhibitory concentration (IC) of the inhibition of WT IL-18 by IL-18BP. 50 ), the IC by IL-18BP is at least about 10-fold higher, at least about 20-fold higher, at least about 50-fold higher, at least about 75-fold higher, at least about 100-fold higher, at least about 200-fold higher, at least about 300-fold higher, at least about 400-fold higher, at least about 500-fold higher, at least about 600-fold higher, at least about 700-fold higher, at least about 800-fold higher, at least about 900-fold higher, or at least about 1000-fold higher. 50 In some embodiments, the modified IL-18 exhibits an IC of IL-18BP inhibition of WT IL-18. 50 The half-maximal inhibitory concentration (IC) of IL-18BP was at least approximately 100-fold higher than that of 50 In some embodiments, the modified IL-18 exhibits an IC of IL-18BP inhibition of WT IL-18. 50 The half-maximal inhibitory concentration (IC) of IL-18BP was at least approximately 500 times higher than that of 50 In some embodiments, the modified IL-18 exhibits an IC of IL-18BP inhibition of WT IL-18. 50 The half-maximal inhibitory concentration (IC) of IL-18BP was at least approximately 1000 times higher than that of 50 ) is shown.

[0196] In some embodiments, the modified IL-18 exhibits a half-maximal inhibitory concentration (IC) by IL-18BP of about 10 nM, at least about 50 nM, at least about 100 nM, at least about 200 nM, at least about 300 nM, at least about 400 nM, at least about 500 nM, at least about 600 nM, at least about 700 nM, at least about 800 nM, at least about 900 nM, or at least about 1000 nM.50 In some embodiments, the modified IL-18 exhibits a half-maximal inhibitory concentration (IC) with IL-18BP of at least about 10 nM. 50 In some embodiments, the modified IL-18 exhibits a half-maximal inhibitory concentration (IC) by IL-18BP of at least about 100 nM. 50 In some embodiments, the modified IL-18 exhibits a half-maximal inhibitory concentration (IC) by IL-18BP of at least about 1000 nM. 50 ) is shown.

[0197] In certain embodiments, the modified IL-18 polypeptide is characterized by a dissociation constant (K D ) is 250 nM to 10,000 nM. In certain embodiments, the K Dare 2nM~10nM, 2nM~20nM, 2nM~50nM, 2nM~100nM, 2nM~200nM, 2nM~500nM, 10nM~50nM, 10nM~100nM, 10nM~500nM, 20nM~50nM, 20nM~100nM, 20nM~500nM, 2 50nM~500nM, 250nM~750nM, 250nM~1,000nM, 250nM~1,250nM, 250nM~1,500 nM, 250nM~2,000nM, 250nM~3,000nM, 250nM~4,000nM, 250nM~5,000nM, 250 nM~7,500nM, 250nM~10,000nM, 500nM~750nM, 500nM~1,000nM, 500nM~1,25 0nM, 500nM~1,500nM, 500nM~2,000nM, 500nM~3,000nM, 500nM~4,000nM, 50 0nM~5,000nM, 500nM~7,500nM, 500nM~10,000nM, 750nM~1,000nM, 750nM~1 ,250nM, 750nM~1,500nM, 750nM~2,000nM, 750nM~3,000nM, 750nM~4,000nM, 750nM~5,000nM, 750nM~7,500nM, 750nM~10,000nM, 1,000nM~1,250nM, 1,0 00nM~1,500nM, 1,000nM~2,000nM, 1,000nM~3,000nM, 1,000nM~4,000nM, 1 ,000nM~5,000nM, 1,000nM~7,500nM, 1,000nM~10,000nM, 1,250nM~1,500n M, 1,250nM~2,000nM, 1,250nM~3,000nM, 1,250nM~4,000nM, 1,250nM~5,00 0nM, 1,250nM~7,500nM, 1,250nM~10,000nM, 1,500nM~2,000nM, 1,500nM~3 ,000nM, 1,500nM~4,000nM, 1,500nM~5,000nM, 1,500nM~7,500nM, 1,500nM ~10,000nM, 2,000nM~3,000nM, 2,000nM~4,000nM, 2,000nM~5,000nM, 2,00 0nM~7,500nM, 2,000nM~10,000nM, 3,000nM~4,000nM, 3,000nM~5,000nM, 3,In certain embodiments, the K, of the modified IL-18 polypeptide with IL-18BP is 0,000 nM to 7,500 nM, 3,000 nM to 10,000 nM, 4,000 nM to 5,000 nM, 4,000 nM to 7,500 nM, 4,000 nM to 10,000 nM, 5,000 nM to 7,500 nM, 5,000 nM to 10,000 nM, or 7,500 nM to 10,000 nM. D In certain embodiments, the K of the modified IL-18 polypeptide with IL-18BP is 250 nM, 500 nM, 750 nM, 1,000 nM, 1,250 nM, 1,500 nM, 2,000 nM, 3,000 nM, 4,000 nM, 5,000 nM, 7,500 nM, or 10,000 nM. D In certain embodiments, the K of the modified IL-18 polypeptide with IL-18BP is at least 250 nM, 500 nM, 750 nM, 1,000 nM, 1,250 nM, 1,500 nM, 2,000 nM, 3,000 nM, 4,000 nM, 5,000 nM, or 7,500 nM. D In certain embodiments, the K of the modified IL-18 polypeptide with IL-18BP is at least 2 nM. D In certain embodiments, the K of the modified IL-18 polypeptide with IL-18BP is at least 5 nM. D In certain embodiments, the K of the modified IL-18 polypeptide with IL-18BP is at least 10 nM. D In certain embodiments, the K of the modified IL-18 polypeptide with IL-18BP is at least 20 nM. D In certain embodiments, the K of the modified IL-18 polypeptide with IL-18BP is at least 50 nM. D In certain embodiments, the K D In certain embodiments, the K of the modified IL-18 polypeptide with IL-18BP is at least 200 nM. DIn certain embodiments, the K of the modified IL-18 polypeptide with IL-18BP is at least 300 nM. D In certain embodiments, the K of the modified IL-18 polypeptide with IL-18BP is at least 400 nM. D In certain embodiments, the K of the modified IL-18 polypeptide with IL-18BP is at least 500 nM. D In certain embodiments, the K of the modified IL-18 polypeptide with IL-18BP is at least 750 nM. D In certain embodiments, the K of the modified IL-18 polypeptide with IL-18BP is at least 1000 nM. D In certain embodiments, the K of the modified IL-18 polypeptide with IL-18BP is at least 2500 nM. D In certain embodiments, the K of the modified IL-18 polypeptide with IL-18BP is at least 5000 nM. D The K between IL-18BP and the IL-18BP is at least 10,000 nM. D can be measured using an IL-18BP binding alphaLISA assay (eg, as provided in the Examples herein).

[0198] In some embodiments, the polymer-modified IL-18 exhibits a reduced ability to have its IFNγ-inducing activity inhibited by IL-18BP compared to WT IL-18. In some embodiments, the polymer-modified IL-18 exhibits an IC of inhibition of WT IL-18 by IL-18BP. 50 the half-maximal inhibitory concentration (IC) of IL-18BP is at least about 10-fold higher, at least about 20-fold higher, at least about 50-fold higher, at least about 75-fold higher, at least about 100-fold higher, at least about 200-fold higher, at least about 300-fold higher, at least about 400-fold higher, at least about 500-fold higher, at least about 600-fold higher, at least about 700-fold higher, at least about 800-fold higher, at least about 900-fold higher, or at least about 1000-fold higher than 50In some embodiments, the polymer-modified IL-18 exhibits an IC of IL-18BP inhibition of WT IL-18. 50 The half-maximal inhibitory concentration (IC) of IL-18BP was at least approximately 100-fold higher than that of 50 In some embodiments, the polymer-modified IL-18 exhibits an IC of IL-18BP inhibition of WT IL-18. 50 The half-maximal inhibitory concentration (IC) of IL-18BP was at least approximately 500 times higher than that of 50 In some embodiments, the polymer-modified IL-18 exhibits an IC of IL-18BP inhibition of WT IL-18. 50 The half-maximal inhibitory concentration (IC) of IL-18BP was at least approximately 1000 times higher than that of 50 ) is shown.

[0199] In some embodiments, the polymer-modified IL-18 exhibits a half-maximal inhibitory concentration (IC) by IL-18BP of about 10 nM, at least about 50 nM, at least about 100 nM, at least about 200 nM, at least about 300 nM, at least about 400 nM, at least about 500 nM, at least about 600 nM, at least about 700 nM, at least about 800 nM, at least about 900 nM, or at least about 1000 nM. 50 In some embodiments, the polymer-modified IL-18 exhibits a half-maximal inhibitory concentration (IC) by IL-18BP of at least about 10 nM. 50 In some embodiments, the polymer-modified IL-18 exhibits a half-maximal inhibitory concentration (IC) by IL-18BP of at least about 100 nM. 50 In some embodiments, the modified IL-18 exhibits a half-maximal inhibitory concentration (IC) by IL-18BP of at least about 1000 nM. 50 ) is shown.

[0200] I C 50 / EC 50 Ratio of In some embodiments, the modified IL-18 polypeptides are at a half-maximal effective concentration (EC 50) by IL-18BP against the half-maximal inhibitory concentration (IC 50 ) favorable ratio (IC 50 / EC 50 In some embodiments, the IC for the modified IL-18 polypeptide is 50 / EC 50 In some embodiments, the IC of the modified IL-18 polypeptide is increased compared to WT IL-18. 50 / EC 50 The ratio is increased by at least about 2-fold, at least about 5-fold, at least about 10-fold, at least about 50-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, at least about 600-fold, at least about 700-fold, at least about 800-fold, at least about 900-fold, or at least about 1000-fold compared to WT IL-18. In some embodiments, the IC for the modified IL-18 polypeptide is increased by at least about 2-fold, at least about 5-fold, at least about 10-fold, at least about 500-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, at least about 600-fold, at least 50 / EC 50 The IC ratio is increased by at least about 10-fold compared to WT IL-18. In some embodiments, the IC ratio for the modified IL-18 polypeptide is 50 / EC 50 The IC ratio is increased by at least about 100-fold compared to WT IL-18. In some embodiments, the IC ratio for the modified IL-18 polypeptide is 50 / EC 50 The ratio is increased by at least about 500-fold compared to WT IL-18.

[0201] In certain embodiments, the ratio of the half-maximal inhibitory concentration (IC50) of IL-18BP to the half-maximal effective concentration (EC50) for inducing IFNγ production for the modified IL-18BP is 150,000 to 3,000,000. In certain embodiments, the ratio of the half-maximal inhibitory concentration (IC50) of IL-18BP to the half-maximal effective concentration (EC50) for inducing IFNγ production for the modified IL-18BP is 50,000 to 3,000,000. In certain embodiments, for modified IL-18BP, the ratio of IC50 by IL-18BP to EC50 for inducing IFNγ production is 100,000 to 300,000, 100,000 to 400,000, 100,000 to 500,000, 100,000 to 725,000, 100,000 to 1,000,000, 100,000 to 1,250,000, 100,000 to 1,500,000, 100,000 to 1,750 ,000, 100,000~2,000,000, 100,000~3,000,000, 298,000~300,000, 298,000~400,000, 298,000~500,000, 298,000~725,000, 298,000~1,000,000, 298,000~1,250,000, 298,000~1,500,000, 298,000~1,750,000, 298,000~2,000,000, 29 8,000~3,000,000, 300,000~400,000, 300,000~500,000, 300,000~725,000, 300,000~1,000,000, 300,000~1,250,000, 300,000~1,500,000, 300,000~1,750,000, 300,000~2,000,000, 300,000~3,000,000, 400,000~500,000, 400,000~7 25,000, 400,000~1,000,000, 400,000~1,250,000, 400,000~1,500,000, 400,000~1,750,000, 400,000~2,000,000, 400,000~3,000,000, 500,000~725,000, 500,000~1,000,000, 500,000~1,250,000, 500,000~150,000, 500,000~1,750,000, 500,000-2,000,000, 500,000-3,000,000, 725,000-1,000,000, 725,000-1,250,000, 725,000-1,500,000, 725,000-1,750,000, 725,000-2,000,000, 725,000-3,000,000, 1,000,000-1,250,000, 1,000,000-1,500,000, 1,000,000-1,750,000, 1,000,000-2,000,000 , 1,000,000 to 3,000,000, 1,250,000 to 1,500,000, 1,250,000 to 1,750,000, 1,250,000 to 2,000,000, 1,250,000 to 3,000,000, 1,500,000 to 1,750,000, 1,500,000 to 2,000,000, 1,500,000 to 3,000,000, 1,750,000 to 2,000,000, 1,750,000 to 3,000,000, or 2,000,000 to 3,000,000. In certain embodiments, for the modified IL-18BP, the ratio of IC50 to EC50 for inducing IFNγ production by IL-18BP is 298,000, 300,000, 400,000, 500,000, 725,000, 1,000,000, 1,250,000, 150,000, 1,750,000, 2,000,000, or 3,000,000. In certain embodiments, for a modified IL-18BP, the ratio of IC50 by IL-18BP to EC50 for inducing IFNγ production is at least 50,000, 100,000, 200,000, 298,000, 300,000, 400,000, 500,000, 725,000, 1,000,000, 1,250,000, 150,000, 1,750,000, 2,000,000, or 3,000,000. In certain embodiments, for a modified IL-18BP, the ratio of IC50 by IL-18BP to EC50 for inducing IFNγ production is at least 50,000. In certain embodiments, for the modified IL-18BP, the ratio of IC50 to EC50 for inducing IFNγ production by IL-18BP is at least 100,In certain embodiments, for a modified IL-18BP, the ratio of the IC50 by IL-18BP to the EC50 for inducing IFNγ production is at least 200,000. In certain embodiments, for a modified IL-18BP, the ratio of the IC50 by IL-18BP to the EC50 for inducing IFNγ production is at least 299,000. In certain embodiments, for a modified IL-18BP, the ratio of the IC50 by IL-18BP to the EC50 for inducing IFNγ production is at least 300,000. In certain embodiments, for a modified IL-18BP, the ratio of the IC50 by IL-18BP to the EC50 for inducing IFNγ production is at least 400,000. In certain embodiments, for a modified IL-18BP, the ratio of the IC50 by IL-18BP to the EC50 for inducing IFNγ production is at least 500,000. In certain embodiments, for a modified IL-18BP, the ratio of the IC50 by IL-18BP to the EC50 for inducing IFNγ production is at least 750,000. In certain embodiments, for a modified IL-18BP, the ratio of the IC50 by IL-18BP to the EC50 for inducing IFNγ production is at least 1,000,000. In certain embodiments, for a modified IL-18BP, the ratio of the IC50 by IL-18BP to the EC50 for inducing IFNγ production is at least 1,250,000. In certain embodiments, for a modified IL-18BP, the ratio of the IC50 by IL-18BP to the EC50 for inducing IFNγ production is at least 1,500,000. In certain embodiments, for a modified IL-18BP, the ratio of the IC50 by IL-18BP to the EC50 for inducing IFNγ production is at least 1,750,000. The EC50 for inducing IFNγ production can be measured using an IFNγ induction NK-92 cell assay (e.g., as provided in the Examples herein), and the IC50 by IL-18BP can be measured by IL-18 binding protein-mediated inhibition of IFNγ secretion in an NK-92 cell assay.

[0202] In some embodiments, the polymer-modified IL-18 polypeptide is at a half-maximal effective concentration (EC 50 ) by IL-18BP against the half-maximal inhibitory concentration (IC 50 ) favorable ratio (IC 50 / EC 50 In some embodiments, the IC for the polymer-modified IL-18 polypeptide is 50 / EC 50 In some embodiments, the IC of the polymer-modified IL-18 polypeptide is increased compared to WT IL-18. 50 / EC 50 The ratio is increased by at least about 2-fold, at least about 5-fold, at least about 10-fold, at least about 50-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, at least about 600-fold, at least about 700-fold, at least about 800-fold, at least about 900-fold, or at least about 1000-fold compared to WT IL-18. In some embodiments, the IC for the polymer-modified IL-18 polypeptide is increased by at least about 2-fold, at least about 5-fold, at least about 10-fold, at least about 500-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, at least about 600-fold 50 / EC 50 The IC ratio is increased by at least about 10-fold compared to WT IL-18. In some embodiments, the IC ratio for the polymer-modified IL-18 polypeptide is 50 / EC 50 The IC ratio is increased by at least about 100-fold compared to WT IL-18. In some embodiments, the IC ratio for the polymer-modified IL-18 polypeptide is 50 / EC 50 In some embodiments, the IC of the modified IL-18 polypeptide is increased by at least about 500-fold compared to WT IL-18. 50 / EC 50 The ratio is at least about 2, at least about 5, at least about 10, at least about 50, at least about 100, at least about 250, or at least about 500.

[0203] V. Pharmaceutical Compositions In one aspect, described herein is a pharmaceutical composition comprising a modified IL-18 polypeptide provided herein and a pharmaceutically acceptable carrier or excipient. In some embodiments, the pharmaceutical composition comprises a plurality of modified IL-18 polypeptides. In one aspect, described herein is a pharmaceutical composition comprising a polymer-modified IL-18 polypeptide described herein and a pharmaceutically acceptable carrier or excipient. In some embodiments, the pharmaceutical composition comprises a plurality of polymer-modified IL-18 polypeptides. In one aspect, described herein is a pharmaceutical composition comprising a modified IL-18 polypeptide described herein and / or a polymer-modified IL-18 polypeptide described herein and a pharmaceutically acceptable carrier or excipient. In some embodiments, the pharmaceutical composition comprises a plurality of modified IL-18 polypeptides and / or a plurality of polymer-modified IL-18 polypeptides. In some embodiments, the pharmaceutical composition further comprises one or more excipients selected from a sugar, an inorganic salt, an antioxidant, a surfactant, or a buffering agent.

[0204] In some embodiments, the pharmaceutical composition further comprises a sugar. In certain embodiments, the sugar is selected from the group consisting of fructose, maltose, galactose, glucose, D-mannose, sorbose, lactose, sucrose, trehalose, cellobiose, raffinose, melezitose, maltodextrin, dextran, starch, mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol (glucitol), pyranosyl sorbitol, myo-inositol, cyclodextrin, and combinations thereof.

[0205] In some embodiments, the pharmaceutical composition comprises an inorganic salt, hi certain embodiments, the inorganic salt is selected from the group consisting of sodium chloride, potassium chloride, magnesium chloride, calcium chloride, sodium phosphate, potassium phosphate, sodium sulfate, or a combination thereof.

[0206] In certain embodiments, the pharmaceutical composition comprises an antioxidant selected from the group consisting of ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, potassium metabisulfite, propyl gallate, sodium metabisulfite, sodium thiosulfate, vitamin E, 3,4-dihydroxybenzoic acid, and combinations thereof.

[0207] In certain embodiments, the pharmaceutical composition comprises a surfactant, hi certain embodiments, the surfactant is selected from the group consisting of polysorbates, sorbitan esters, lipids, phospholipids, phosphatidylethanolamines, fatty acids, fatty acid esters, steroids, EDTA, zinc, and combinations thereof.

[0208] In certain embodiments, the pharmaceutical composition comprises a buffering agent, hi certain embodiments, the buffering agent is selected from the group consisting of citric acid, sodium phosphate, potassium phosphate, acetic acid, ethanolamine, histidine, an amino acid, tartaric acid, succinic acid, fumaric acid, lactic acid, Tris, HEPES, CHAPS, or a combination thereof.

[0209] In some embodiments, the pharmaceutical composition is formulated for parenteral or enteral administration. In some embodiments, the pharmaceutical composition is formulated for intravenous or subcutaneous administration. In some embodiments, the pharmaceutical composition is in lyophilized form.

[0210] In one aspect, described herein is a liquid or lyophilized composition comprising a described modified IL-18 polypeptide. In some embodiments, the modified IL-18 polypeptide is a lyophilized powder. In one aspect, described herein is a liquid or lyophilized composition comprising a described polymer-modified IL-18 polypeptide. In some embodiments, the polymer-modified IL-18 polypeptide is a lyophilized powder. In some embodiments, the lyophilized powder is resuspended in a buffer solution. In some embodiments, the buffer comprises a buffering agent, a sugar, a salt, a surfactant, or any combination thereof. In some embodiments, the buffer comprises phosphate. In some embodiments, the phosphate is sodium NaHPO. In some embodiments, the salt is sodium chloride. In some embodiments, the buffer comprises phosphate-buffered saline. In some embodiments, the buffer comprises mannitol. In some embodiments, the lyophilized powder is suspended in a solution comprising phosphate-buffered saline (pH 7.4) containing 50 mg / mL mannitol. In some embodiments, the pharmaceutical composition is a lyophilized composition that is reconstituted immediately prior to administration to a subject.

[0211] The modified IL-18 polypeptides described herein may be in a variety of dosage forms. In some embodiments, the modified IL-18 polypeptide is administered as a lyophilized powder. In some embodiments, the modified IL-18 polypeptide is administered as a suspension. In some embodiments, the modified IL-18 polypeptide is administered as a solution. In some embodiments, the modified IL-18 polypeptide is administered as an injectable solution. In some embodiments, the modified IL-18 polypeptide is administered as an IV solution. The polymer-modified IL-18 polypeptides described herein may be in a variety of dosage forms. In some embodiments, the polymer-modified IL-18 polypeptide is administered as a lyophilized powder. In some embodiments, the polymer-modified IL-18 polypeptide is administered as a suspension. In some embodiments, the polymer-modified IL-18 polypeptide is administered as a solution. In some embodiments, the polymer-modified IL-18 polypeptide is administered as an injectable solution. In some embodiments, the polymer-modified IL-18 polypeptide is administered as an IV solution.

[0212] VI. Treatment Methods In one aspect, described herein is a method of treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of a modified IL-18 polypeptide provided herein or a pharmaceutical composition described herein.

[0213] In one aspect, described herein is a method of treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of a polymer-modified IL-18 polypeptide comprising a polymer provided herein or a pharmaceutical composition described herein.

[0214] In one aspect, described herein is a modified IL-18 polypeptide provided herein for use in treating cancer in a subject in need thereof. In another aspect, described herein is a modified IL-18 polypeptide provided herein for use in the manufacture of a medicament for treating cancer in a subject in need thereof.

[0215] In another aspect, described herein is a polymer-modified IL-18 polypeptide comprising a polymer provided herein for use in treating cancer in a subject in need thereof. In another aspect, described herein is a polymer-modified IL-18 polypeptide comprising a polymer provided herein for use in the manufacture of a medicament for the treatment of cancer in a subject in need thereof.

[0216] In some embodiments, the cancer is a solid cancer, hi some embodiments, the solid cancer is adrenal gland cancer, anal cancer, bile duct cancer, bladder cancer, bone cancer, brain cancer, breast cancer, carcinoid cancer, cervical cancer, colorectal cancer, esophageal cancer, eye cancer, gallbladder cancer, gastrointestinal stromal tumor, germ cell cancer, head and neck cancer, kidney cancer, liver cancer, lung cancer, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, neuroendocrine cancer, oral cancer, oropharyngeal cancer, ovarian cancer, pancreatic cancer, pediatric cancer, penile cancer, pituitary cancer, prostate cancer, skin cancer, soft tissue cancer, spinal cancer, stomach cancer, testicular cancer, thymus cancer, thyroid cancer, ureter cancer, uterine cancer, vaginal cancer, or vulvar cancer.

[0217] In some embodiments, the cancer is a hematological cancer, ie, leukemia, non-Hodgkin's lymphoma, Hodgkin's lymphoma, AIDS-related lymphoma, multiple myeloma, plasmacytoma, post-transplant lymphoproliferative disorder, or Waldenstrom's macroglobulinemia.

[0218] The modified IL-18 polypeptides described herein can be administered to a subject in one or more doses. In some embodiments, the modified IL-18 polypeptide is administered in a single dose of an effective amount of the modified IL-18 polypeptide, including further embodiments in which (i) the modified IL-18 polypeptide is administered once daily, or (ii) the modified IL-18 polypeptide is administered to a subject multiple times throughout the day. In some embodiments, the modified IL-18 polypeptide is administered daily, every other day, twice a week, three times a week, once a week, every two weeks, three weeks, four weeks, five weeks, six weeks, twelve weeks, every three days, every four days, every five days, every six days, twice a week, three times a week, four times a week, five times a week, six times a week, monthly, twice a month, three times a month, four times a month, every two months, every three months, every four months, every five months, or every six months. Administration includes, but is not limited to, injection by any suitable route (eg, parenteral, enteral, intravenous, subcutaneous, etc.).

[0219] The polymer-modified IL-18 polypeptides described herein can be administered to a subject in one or more doses. In some embodiments, the polymer-modified IL-18 polypeptide is administered in a single dose of an effective amount of the modified IL-18 polypeptide, including further embodiments in which (i) the polymer-modified IL-18 polypeptide is administered once daily, or (ii) the polymer-modified IL-18 polypeptide is administered to a subject multiple times throughout the day. In some embodiments, the polymer-modified IL-18 is administered daily, every other day, twice a week, three times a week, once a week, every two weeks, three weeks, four weeks, five weeks, six weeks, twelve weeks, every three days, every four days, every five days, every six days, twice a week, three times a week, four times a week, five times a week, six times a week, monthly, twice a month, three times a month, four times a month, every two months, every three months, every four months, every five months, or every six months. Administration includes, but is not limited to, injection by any suitable route (eg, parenteral, enteral, intravenous, subcutaneous, etc.).

[0220] An effective response is achieved when a subject experiences partial or complete alleviation or reduction of signs or symptoms of the disease, specifically including, but not limited to, prolonged survival. Expected progression-free survival can be measured in months to years, depending on prognostic factors, including the number of recurrences, the stage of the disease, and other factors. Prolonged survival includes, but is not limited to, periods of at least 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 6 months, at least about 1 year, at least about 2 years, at least about 3 years, at least about 4 years, at least about 5 years, etc. Overall survival or progression-free survival can also be measured in months to years. Alternatively, an effective response can be when a subject's symptoms remain static and do not worsen. Further indications for treatment of an indication are described in more detail below. In some cases, the cancer or tumor is shrunk by at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

[0221] In some embodiments, the method further comprises reconstituting the modified IL-18 polypeptide or pharmaceutical composition in lyophilized form. In some embodiments, the modified IL-18 polypeptide or pharmaceutical composition is reconstituted prior to administration. In some embodiments, the composition is reconstituted immediately before administration, up to about 5 minutes before administration, up to about 20 minutes before administration, up to about 40 minutes before administration, up to 1 hour before administration, or up to about 4 hours before administration.

[0222] In some embodiments, the method further comprises reconstituting the polymer-modified IL-18 polypeptide or pharmaceutical composition in lyophilized form. In some embodiments, the polymer-modified IL-18 polypeptide or pharmaceutical composition is reconstituted prior to administration. In some embodiments, the composition is reconstituted immediately before administration, up to about 5 minutes before administration, up to about 20 minutes before administration, up to about 40 minutes before administration, up to 1 hour before administration, or up to about 4 hours before administration.

[0223] VII. Preparation of Recombinant IL-18 Polypeptides In one aspect, described herein is a method of producing a modified IL-18 polypeptide, the method comprising expressing the modified IL-18 polypeptide in a host cell.

[0224] In one aspect, described herein is a host cell comprising a modified IL-18 polypeptide.

[0225] In some embodiments, the host cell is a prokaryotic or eukaryotic cell. In some embodiments, the host cell is a mammalian cell, an avian cell, or an insect cell. In some embodiments, the host cell is a mammalian cell, an avian cell, a fungal cell, or an insect cell. In some embodiments, the host cell is a CHO cell, a COS cell, or a yeast cell.

[0226] In some embodiments, producing a polymer-modified IL-18 polypeptide comprises attaching a polymer to the modified IL-18 polypeptide. In some embodiments, the polymer is attached directly to a residue of the modified IL-18 polypeptide, optionally via a linker attached to the polymer. In some embodiments, a heterobifunctional linker is first attached to the modified IL-18 polypeptide, followed by attachment of the polymer to the heterobifunctional linker.

[0227] VIII. Manufacturing (synthesis) method Also provided herein are methods for synthesizing the modified IL-18 polypeptides provided herein. Also provided herein are methods for synthesizing polymer-modified IL-18 polypeptides (e.g., modified IL-18 polypeptides conjugated with polymers provided herein). In some cases, the modified IL-18 polypeptide is chemically synthesized rather than recombinantly expressed. In some cases, multiple fragment peptide precursors of the modified IL-18 polypeptide are synthesized and then ligated together using a suitable ligation methodology (e.g., alpha-keto acid hydroxylamine (KAHA) ligation). In some cases, after ligation, the resulting modified IL-18 polypeptide is folded to produce a modified IL-18 polypeptide having substantially the same secondary and tertiary structure as that of the recombinant or wild-type IL-18 polypeptide. In some cases, multiple fragment peptide precursors of the polymer-modified IL-18 polypeptide are synthesized and then ligated together using a suitable ligation methodology (e.g., alpha-keto acid hydroxylamine (KAHA) ligation). Optionally, after ligation, the resulting polymer-modified IL-18 polypeptide is folded to produce a modified IL-18 polypeptide having a secondary and tertiary structure substantially identical to that of a recombinant or wild-type IL-18 polypeptide, and the folded polypeptide is conjugated to a polymer to produce the polymer-modified IL-18 polypeptide.

[0228] In some cases, methionine residues in the modified IL-18 polypeptide are substituted for stability and / or to aid in folding of the linear modified IL-18 polypeptide to produce the final modified IL-18 polypeptide. The methionine side chain is susceptible to oxidation during the synthetic process (e.g., peptide synthesis and protein folding) and therefore, in some cases, results in the final IL-18 polypeptide being of insufficient quality for a particular application due to lack of homogeneity.

[0229] In some cases, to address these limitations, all methionine residues in modified IL-18 polypeptides were replaced with norleucine residues. In some cases, linear peptides were successfully synthesized, but the resulting polypeptides exhibited signs of instability, such as increased hydrophobicity and tendency to precipitation, as well as inconsistent biological activity, which may be due to misfolding resulting in altered secondary / tertiary structure of the modified IL-18 polypeptides compared to wild-type or recombinant IL-18.

[0230] In some cases, modified IL-18 polypeptides were synthesized to incorporate oxidized methionine directly during synthesis of the precursor peptide in an attempt to create a homogeneous linear protein without the complex mixture of partial methionine oxidation. In some cases, modified linear IL-18 polypeptides were successfully synthesized, but difficulties were encountered in reducing the methionine back to the non-oxidized form.

[0231] To address these challenges, we designed novel modified IL-18 polypeptide variants in which one or more methionine residues were replaced with O-methyl-L-homoserine (Omh) residues. Omh is a structural analog of natural methionine, in which the sulfur atom of methionine is replaced with oxygen. Due to the lack of a sulfur atom, the Omh residue is predicted to be less susceptible to oxidation, thereby enhancing the stability of the modified IL-18 polypeptide and facilitating its synthesis / purification. Furthermore, the increased hydrophilicity of the Omh residue compared to norleucine residues, combined with the higher structural homology of Omh to natural methionine residues, is predicted to promote proper folding and enhance the stability of the modified IL-18 polypeptide compared to variants with norleucine residues instead of methionine. Therefore, chemically synthesized modified IL-18 polypeptides in which methionine residues are replaced with Omh residues are predicted to offer several advantages over other synthetically modified IL-18 polypeptides.

[0232] In one aspect, described herein is a method of making a modified IL-18 polypeptide. In another aspect, described herein is a method of making a modified IL-18 polypeptide, comprising synthesizing two or more fragments of a modified IL-18 polypeptide and ligating the fragments.

[0233] In another aspect, described herein is a method for making a polymer-modified IL-18 polypeptide, comprising: a. synthesizing two or more fragments of a modified IL-18 polypeptide; b. ligating the fragments; c. folding the ligated fragments; and d. attaching a polymer to the modified IL-18 polypeptide.

[0234] In another aspect, described herein is a method for making a polymer-modified IL-18 polypeptide, comprising synthesizing two or more fragments of the modified IL-18 polypeptide, ligating the fragments, and attaching a polymer to the modified IL-18 polypeptide.

[0235] In another aspect, described herein is a method of making a modified IL-18 polypeptide, comprising: a. providing two or more fragments of a modified IL-18 polypeptide; b. ligating the fragments; and c. folding the ligated fragments.

[0236] In another aspect, described herein is a method of making a modified IL-18 polypeptide, comprising ligating two or more fragments of a modified IL-18 polypeptide, wherein at least one of the two or more fragments of the modified IL-18 polypeptide is synthetic, and folding the ligated fragments.

[0237] In some embodiments, two or more fragments of a modified IL-18 polypeptide are chemically synthesized. In some embodiments, two or more fragments of a modified IL-18 polypeptide are synthesized by solid phase peptide synthesis. In some embodiments, two or more fragments of a modified IL-18 polypeptide are synthesized by an automated peptide synthesizer.

[0238] In some embodiments, the modified IL-18 polypeptide is ligated from 2, 3, 4, 5, 6, 7, 8, 9, 10, or more peptide fragments. In some embodiments, the modified peptide is ligated from 2 peptide fragments. I...

Claims

1. A modified interleukin 18 (IL-18) polypeptide, the modified IL-18 polypeptide comprising: a) exhibits the ability to induce interferon gamma (IFNγ) production when contacted with cells; b) exhibits a reduced ability to be inhibited by IL-18 binding protein (IL-18BP) compared to wild-type IL-18 (WT IL-18); c) the ratio of the half-maximal inhibitory concentration (IC50) to the half-maximal effective concentration (EC50) for inducing IFNγ production by IL-18BP is at least 50,000; The modified IL-18 polypeptide.

2. 1. A modified interleukin-18 (IL-18) polypeptide, wherein the modified IL-18 polypeptide has a half-maximal effective concentration (EC50) for inducing IFNγ production in a cell that is at least 30-fold lower than the EC50 of wild-type IL-18 (WT IL-18).

3. 1. A modified interleukin-18 (IL-18) polypeptide, wherein the modified IL-18 polypeptide has a half maximal effective concentration (EC50) for signaling through an IL-18 receptor that is at least 5-fold lower than the EC50 of wild-type IL-18 (WT IL-18).

4. A modified interleukin-18 (IL-18) polypeptide, the modified IL-18 polypeptide having a dissociation constant (K) with the IL-18 receptor alpha subunit that is at least 60-fold lower than that of wild-type IL-18. Ｄ The modified IL-18 polypeptide,

5. 1. A modified interleukin-18 (IL-18) polypeptide, comprising a substitution at residue V11 and at least one further substitution at a residue selected from Y1, F2, E6, K8, S10, D17, T34, D35, S36, D37, D40, N41, I49, K53, M51, D54, S55, Q103, S105, G108, H109, ​​D110, and D132, wherein the numbering of residue positions is based on SEQ ID NO:1 as a reference sequence.

6. 6. The modified IL-18 polypeptide of any one of claims 1 to 5, wherein the modified IL-18 polypeptide has a dissociation constant (K) with an IL-18 binding protein of at least 20 nM, at least 50 nM, at least 100 nM, at least 200 nM, at least 300 nM, at least 400 nM, at least 500 nM, at least 750 nM, at least 1000 nM, at least 2500 nM, at least 5000 nM, or at least 10000 nM. Ｄ The modified IL-18 polypeptide,

7. 7. The modified IL-18 polypeptide of any one of claims 1 to 6, wherein the ratio of half maximal inhibitory concentration (IC50) by IL-18BP to half maximal effective concentration (EC50) for inducing IFNγ production is at least 50,000, at least 100,000, at least 200,000, at least 300,000, at least 400,000, at least 500,000, at least 750,000, at least 1,000,000, at least 1,250,000, at least 1,500,000, or at least 1,750,000.

8. 8. The modified IL-18 polypeptide of any one of claims 1 to 7, wherein the modified IL-18 polypeptide has a half maximal effective concentration (EC50) for inducing IFNγ production in a cell that is at least 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, or 80-fold lower than the EC50 of wild-type IL-18 (WT IL-18).

9. 9. The modified IL-18 polypeptide of any one of claims 1 to 8, wherein the modified IL-18 polypeptide has a half maximal effective concentration (EC50) for signaling through the IL-18 receptor that is at least 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, or 50-fold lower than the EC50 of wild-type IL-18 (WT IL-18).

10. 10. The modified IL-18 polypeptide of any one of claims 1 to 9, wherein the modified IL-18 polypeptide has a binding affinity (KD) with the IL-18 receptor alpha subunit that is at least 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold lower than wild-type IL-18.

11. The modified IL-18 polypeptide of any one of claims 1 to 10, wherein the modified IL-18 polypeptide comprises substitutions at residues C38, C68, C76, and / or C127.

12. 12. The modified IL-18 polypeptide of any one of claims 1 to 11, wherein the modified IL-18 polypeptide comprises a C38A, C38S, C68A, C68S, C76A, C76S, C127A, and / or C127S substitution.

13. 13. The modified IL-18 polypeptide of any one of claims 1 to 12, wherein the modified IL-18 polypeptide comprises a substitution at each of residues C38, C76, and C127, each of the substitutions at residues C38, C76, and C127 to serine or alanine.

14. The modified IL-18 polypeptide of any one of claims 1 to 13, wherein the modified IL-18 polypeptide comprises i) C38A, C76A, and C127A or ii) C38S, C76S, and C127S substitutions.

15. The modified IL-18 polypeptide of any one of claims 1 to 14, wherein the modified IL-18 polypeptide comprises C38A, C76A, and C127A substitutions.

16. The modified IL-18 polypeptide of any one of claims 1 to 15, wherein the modified IL-18 polypeptide comprises a substitution at residue T63.

17. The modified IL-18 polypeptide of any one of claims 1 to 16, wherein the modified IL-18 polypeptide comprises a T63A substitution.

18. The modified IL-18 polypeptide of any one of claims 1 to 17, wherein the modified IL-18 polypeptide comprises a substitution at residue K53.

19. The modified IL-18 polypeptide of any one of claims 1 to 18, wherein the modified IL-18 polypeptide comprises a K53A substitution.

20. The modified IL-18 polypeptide of any one of claims 1 to 19, wherein the modified IL-18 polypeptide comprises a substitution at residue E6.

21. The modified IL-18 polypeptide of any one of claims 1 to 20, wherein the modified IL-18 polypeptide comprises an E6K substitution.

22. The modified IL-18 polypeptide of any one of claims 1 to 21, wherein the modified IL-18 polypeptide comprises a substitution at residue M51.

23. The modified IL-18 polypeptide of any one of claims 1 to 22, wherein the modified IL-18 polypeptide comprises an M51G substitution.

24. The modified IL-18 polypeptide of any one of claims 1 to 23, wherein the modified IL-18 polypeptide comprises a V11I substitution.

25. The modified IL-18 polypeptide of any one of claims 1 to 24, wherein the modified IL-18 polypeptide comprises E6K, V11I, K53A, and T63A substitutions.

26. The modified IL-18 polypeptide of any one of claims 1 to 25, wherein the modified IL-18 polypeptide comprises V11I, M51G, and K53A substitutions.

27. The modified IL-18 polypeptide of any one of claims 1 to 26, wherein the modified IL-18 polypeptide comprises E6K, V11I, M51G, K53A, and T63A substitutions.

28. 28. The modified IL-18 polypeptide of any one of claims 1 to 27, wherein the modified IL-18 polypeptide comprises E6K, V11I, C38A, K53A, T63A, C76A, and C127A substitutions.

29. 29. The modified IL-18 polypeptide of any one of claims 1 to 28, wherein the modified IL-18 polypeptide comprises V11I, C38A, K53A, T63A, C76A, C127A and M51G substitutions.

30. 30. The modified IL-18 polypeptide of any one of claims 1 to 29, wherein the modified IL-18 polypeptide comprises E6K, V11I, C38A, K53A, T63A, C76A, C127A and M51G substitutions.

31. The modified IL-18 polypeptide of any one of claims 1 to 30, further comprising at least one glycine residue attached to the N-terminus of the IL-18 polypeptide.

32. 32. The modified IL-18 polypeptide of any one of claims 1 to 31, further comprising one glycine residue or a chain of two, three, four, or five glycine residues attached to the N-terminus of the IL-18 polypeptide.

33. 33. The modified IL-18 polypeptide of any one of claims 1 to 32, wherein the modified IL-18 polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, or 95% sequence identity to the sequence set forth in SEQ ID NO:

1.

34. The modified IL-18 polypeptide of any one of claims 1 to 32, wherein the modified IL-18 polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO:

30.

35. The modified IL-18 polypeptide of any one of claims 1 to 32, wherein the modified IL-18 polypeptide comprises the sequence represented by SEQ ID NO:

30.

36. The modified IL-18 polypeptide of any one of claims 1 to 32, wherein the modified IL-18 polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO:

207.

37. The modified IL-18 polypeptide of any one of claims 1 to 32, wherein the modified IL-18 polypeptide comprises the sequence represented by SEQ ID NO:

207.

38. The modified IL-18 polypeptide of any one of claims 1 to 32, wherein the modified IL-18 polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO:

239.

39. The modified IL-18 polypeptide of any one of claims 1 to 32, wherein the modified IL-18 polypeptide comprises the sequence represented by SEQ ID NO:

239.

40. The modified IL-18 polypeptide of any one of claims 1 to 32, wherein the modified IL-18 polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO:

244.

41. The modified IL-18 polypeptide of any one of claims 1 to 32, wherein the modified IL-18 polypeptide comprises the sequence represented by SEQ ID NO:

244.

42. The modified IL-18 polypeptide of any one of claims 1 to 32, wherein the modified IL-18 polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO:

242.

43. The modified IL-18 polypeptide of any one of claims 1 to 32, wherein the modified IL-18 polypeptide comprises the sequence represented by SEQ ID NO:

242.

44. The modified IL-18 polypeptide of any one of claims 1 to 32, wherein the modified IL-18 polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO:

241.

45. The modified IL-18 polypeptide of any one of claims 1 to 32, wherein the modified IL-18 polypeptide comprises the sequence represented by SEQ ID NO:

241.

46. A modified IL-18 polypeptide having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:

30.

47. A modified IL-18 polypeptide comprising the sequence set forth in SEQ ID NO:

30.

48. A modified IL-18 polypeptide comprising the sequence represented by SEQ ID NO:

207.

49. A modified IL-18 polypeptide comprising the sequence represented by SEQ ID NO:

239.

50. A modified IL-18 polypeptide comprising the sequence represented by SEQ ID NO:

244.

51. A modified IL-18 polypeptide comprising the sequence represented by SEQ ID NO:

242.

52. A modified IL-18 polypeptide comprising the sequence represented by SEQ ID NO:

241.

53. A modified IL-18 polypeptide having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to a sequence set forth in any one of SEQ ID NOs:2-33 or 204-244.

54. A modified IL-18 polypeptide comprising an amino acid sequence represented by any one of SEQ ID NOs: 2-33 or 204-244.

55. A modified IL-18 polypeptide having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to a sequence set forth in any one of SEQ ID NOs:24-33.

56. A modified IL-18 polypeptide comprising an amino acid sequence represented by any one of SEQ ID NOs: 24 to 33.

57. 56. A polymer-modified IL-18 polypeptide comprising a polymer covalently attached to the modified IL-18 polypeptide of any one of claims 1-55.

58. 57. The polymer-modified IL-18 polypeptide of claim 56, wherein the polymer is attached at residue 68 of the IL-18 polypeptide, the numbering of the residue positions being based on SEQ ID NO: 1 as a reference sequence.

59. 1. A polymer-modified interleukin-18 (IL-18) polypeptide comprising a polymer attached to any one of residues 79-120, wherein the numbering of residue positions in said modified IL-18 polypeptide is based on SEQ ID NO:1 as a reference sequence.

60. 60. The polymer-modified IL-18 polypeptide of claim 59, wherein the polymer is attached at residue 85.

61. 60. The polymer-modified IL-18 polypeptide of claim 59, wherein the polymer is attached at residue 86.

62. 60. The polymer-modified IL-18 polypeptide of claim 59, wherein the polymer is attached at residue 95.

63. The polymer-modified IL-18 polypeptide of any one of claims 59 to 62, wherein the polymer is attached to a cysteine ​​residue.

64. 64. A pharmaceutical composition comprising: (a) an IL-18 polypeptide according to any one of claims 1 to 56 or a polymer-modified IL-18 polypeptide according to any one of claims 57 to 63; and (b) a pharma- ceutically acceptable carrier or excipient.

65. 65. The pharmaceutical composition of claim 64, wherein the pharmaceutical composition is in a lyophilized form.

66. A method of treating cancer in a subject in need thereof, comprising administering to the subject a pharma- ceutical effective amount of a modified IL-18 polypeptide of any one of embodiments 1-56, a polymer-modified IL-18 polypeptide of any one of claims 57-63, or a pharmaceutical composition of claim 64 or 65.

67. 67. The method of claim 66, wherein the cancer is a solid cancer.

68. 68. The method of claim 67, wherein the solid cancer is adrenal gland cancer, anal cancer, bile duct cancer, bladder cancer, bone cancer, brain cancer, breast cancer, carcinoid cancer, cervical cancer, colorectal cancer, esophageal cancer, eye cancer, gallbladder cancer, gastrointestinal stromal tumor, germ cell cancer, head and neck cancer, kidney cancer, liver cancer, lung cancer, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, neuroendocrine cancer, oral cancer, oropharyngeal cancer, ovarian cancer, pancreatic cancer, childhood cancer, penile cancer, pituitary cancer, prostate cancer, skin cancer, soft tissue cancer, spinal cancer, gastric cancer, testicular cancer, thymus cancer, thyroid cancer, ureter cancer, uterine cancer, vaginal cancer, or vulvar cancer.

69. 68. The method of claim 67, wherein the solid cancer is metastatic renal cell carcinoma or melanoma.

70. 68. The method of claim 67, wherein the solid cancer is a carcinoma or a sarcoma.

71. 67. The method of claim 66, wherein the cancer is a hematological cancer.

72. 72. The method of claim 71, wherein the hematological cancer is leukemia, non-Hodgkin's lymphoma, Hodgkin's lymphoma, AIDS-related lymphoma, multiple myeloma, plasmacytoma, post-transplant lymphoproliferative disorder, or Waldenstrom's macroglobulinemia.

73. 73. The method of any one of claims 66-72, further comprising reconstituting said modified IL-18 polypeptide, said polymer-modified IL-18 polypeptide, or said pharmaceutical composition in lyophilized form.

74. 1. A modified IL-18 polypeptide comprising E6K, V11I, C38A, K53A, T63A, C76A, and C127A amino acid substitutions, wherein numbering of residue positions is based on SEQ ID NO:1 as a reference sequence, and wherein the modified IL-18 polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:

30.

75. 75. The modified IL-18 polypeptide of claim 74, comprising the amino acid sequence represented by SEQ ID NO:

30.

76. 1. A polymer-modified IL-18 polypeptide comprising a polymer covalently attached to residue 68 of said IL-18 polypeptide, and further comprising the following amino acid substitutions: E6K, V11I, C38A, K53A, T63A, C76A, and C127A, wherein the numbering of residue positions is based on SEQ ID NO:1 as a reference sequence.

77. 77. The modified IL-18 polypeptide of claim 76, wherein the modified IL-18 polypeptide has an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, or at least 95% identical to the sequence set forth in SEQ ID NO:

1.

78. 78. The modified IL-18 polypeptide of claim 77, wherein the modified IL-18 polypeptide comprises the sequence represented by SEQ ID NO:30.

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