Activatable IL-18 Polypeptides

Abstract

The present disclosure relates to activatable IL-18 polypeptides, compositions comprising activatable IL-18 polypeptides, methods for their production, and methods of using activatable IL-18 polypeptides for the treatment of disease. In one aspect, the disclosure relates to the treatment of cancer using activatable IL-18 polypeptides.

Description

[Technical field]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 63 / 313,210, filed February 23, 2022, which is incorporated by reference in its entirety. [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 the treatment of cancer is to stimulate the immune system so that it recognizes and destroys tumors or other cancerous tissues. One way to activate 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 regulating the immune system. Some cytokine therapies utilize these properties of cytokines to boost a subject's immune system and kill cancer cells. Summary of the Invention

[0003] Provided herein is an activatable IL-18 (Act-IL-18) polypeptide that is activated in response to certain conditions or stimuli found within a target region of a subject after administration. In some cases, Act-IL-18 is administered in an inactivated form and later releases an active form of the IL-18 polypeptide upon contact with the condition or stimuli. In some cases, this allows the IL-18 polypeptide to preferentially modulate an immune response in a target region of a subject, such as a cancer or tumor microenvironment. In some cases, Act-IL-18 exhibits fewer side effects associated with systemic administration and distribution of a corresponding active IL-18 polypeptide. In some embodiments, the Act-IL-18 of the present disclosure includes an artificial terminal moiety that serves to inactivate the IL-18 polypeptide, such as a peptide attached to a terminus (e.g., N-terminus or C-terminus) of the IL-18 polypeptide. In some embodiments, the artificial terminal moiety is cleaved in one or more conditions associated with the desired target (e.g., tumor microenvironment), thereby releasing the active IL-18 polypeptide.

[0004] FIG. 1A illustrates an exemplary mechanism of action of an activatable IL-18 polypeptide provided herein, in which the IL-18 polypeptide includes an artificial terminal moiety that inactivates Act-IL-18, and upon cleavage of the artificial terminal moiety, an active IL-18 polypeptide is generated. FIG. 1B illustrates a similar concept of an artificial terminal moiety attached to the N-terminus of an IL-18 polypeptide. The illustrated artificial terminal moiety includes a linking peptide B that links a protease cleavage site having a specific cleavage site to the N-terminus of the IL-18 polypeptide. The artificial terminal moiety further includes a linking peptide A that links the protease cleavage site to a blocking moiety (labeled "mask" in the figure). Upon cleavage of the specific cleavage site, the mask and linking peptide A are released from the IL-18 polypeptide, but linking peptide B remains. This results in the formation of an active IL-18 polypeptide. FIG. 1C is similar to FIG. 1B, but the artificial terminal moiety is attached to the C-terminus of the IL-18 polypeptide.

[0005] In some embodiments, Act-IL-18 comprises an artificial terminal moiety comprising a blocking group linked to an IL-18 polypeptide. In some embodiments, the blocking moiety is located such that cleavage of the artificial terminal moiety releases the blocking moiety from the IL-18 polypeptide, thereby allowing the IL-18 polypeptide to interact with a receptor (or to interact with a receptor to a greater extent). In some embodiments, the blocking moiety comprises an IL-18 propeptide (e.g., the N-terminal portion of immature IL-18 that is endogenously cleaved by caspases to generate mature IL-18). In some embodiments, the IL-18 propeptide is linked to an IL-18 polypeptide-specific cleavage site that is cleaved by a protease other than a caspase (e.g., a protease that is upregulated in tumors or tumor microenvironments). In some embodiments, the IL-18 propeptide is linked to the N-terminus of the IL-18 polypeptide. In some embodiments, the blocking moiety comprises a domain of an IL-18 receptor subunit, such as the D3 domain of the IL-18 receptor alpha subunit (see, e.g., Tsutsumi et al., Nature Communications 5:5340 | DOI:10.1038 / ncomms6340 (published December 15, 2014) for a description of the IL-18 receptor domain structure).

[0006] In one aspect, provided herein is an activatable interleukin-18 (Act-IL-18) polypeptide comprising an artificial terminal moiety linked to an interleukin-18 (IL-18) polypeptide, the artificial terminal moiety comprising a specific cleavage site, wherein cleavage at the specific cleavage site converts the Act-IL-18 into an active IL-18 polypeptide. In some embodiments, when the artificial terminal moiety is intact, the Act-IL-18 is inactive.

[0007] In some embodiments, the specific cleavage site is preferentially cleaved at or near the target tissue of the subject, hi some embodiments, the specific cleavage site is preferentially cleaved within or near the tumor microenvironment.

[0008] In some embodiments, the specific cleavage site is specifically cleaved by a protease. In some embodiments, the protease is found in higher concentrations and / or exhibits higher proteolytic activity in the tumor microenvironment compared to non-tumor tissue. In some embodiments, the protease is selected from kallikrein, thrombin, chymase, carboxypeptidase A, elastase, proteinase 3 (PR-3), granzyme M, calpain, matrix metalloproteinase (MMP), a disintegrin and metalloproteinase (ADAM), fibroblast activation protein alpha (FAP), plasminogen activator, cathepsin, caspase, tryptase, and tumor cell surface proteases. In some embodiments, the artificial terminal portion comprises a peptide having a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a peptide sequence set forth in Table 2A. In some embodiments, the artificial terminal portion comprises a peptide having a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a peptide sequence set forth in Table 2B.

[0009] In some embodiments, the specific cleavage site is a redox-sensitive cleavage site. In some embodiments, the redox-sensitive cleavage site is preferentially cleaved in a reducing environment. In some embodiments, the specific cleavage site is a pH-sensitive cleavage site. In some embodiments, the pH-sensitive cleavage site is preferentially cleaved at a pH of less than 7.3, less than 7.2, less than 7.1, or less than 7.0.

[0010] In some embodiments, cleavage removes the entire artificial terminal moiety from the IL-18 polypeptide, hi some embodiments, cleavage leaves a portion of the artificial moiety attached to the IL-18 polypeptide.

[0011] In some embodiments, the artificial terminal moiety is a peptide. In some embodiments, cleavage of the artificial terminal moiety at the specific cleavage site leaves no amino acid residues of the peptide attached to the IL-18 polypeptide. In some embodiments, cleavage of the artificial terminal moiety at the specific cleavage site leaves at least one amino acid residue of the peptide attached to the IL-18 polypeptide. In some embodiments, cleavage of the artificial terminal moiety at the specific cleavage site leaves 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues of the peptide attached to the IL-18 polypeptide. In some embodiments, the peptide comprises multiple specific cleavage sites. In some embodiments, the peptide comprises 2, 3, or 4 specific cleavage sites. In some embodiments, the peptide is 2-35 amino acid residues in length. In some embodiments, the peptide is 8, 9, or 10 amino acid residues in length.

[0012] In some embodiments, the artificial terminal moiety is attached to the N-terminus or C-terminus of the IL-18 polypeptide.

[0013] In some embodiments, the artificial terminal portion is attached to the N-terminus of the IL-18 polypeptide. In some embodiments, the artificial terminal portion comprises a blocking portion. In some embodiments, the blocking portion is positioned such that cleavage at the specific cleavage site releases the blocking portion from the Act-IL-18 polypeptide. In some embodiments, the blocking portion comprises an IL-18 propeptide or a portion thereof, or a variant thereof. In some embodiments, the IL-18 propeptide is a human IL-18 propeptide or a variant thereof. In some embodiments, the IL-18 propeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence represented by SEQ ID NO:89. In some embodiments, the artificial terminal portion comprises a linking peptide between the specific cleavage site and the blocking portion.

[0014] In some embodiments, the artificial terminal portion is attached to the C-terminus of the IL-18 polypeptide. In some embodiments, the blocking portion is positioned such that cleavage at the specific cleavage site releases the blocking portion from the Act-IL-18 polypeptide. In some embodiments, the blocking portion comprises a domain of an IL-18 receptor subunit or a portion thereof, or a derivative thereof. In some embodiments, the domain of an IL-18 receptor subunit comprises the D3 domain of the IL-18 receptor alpha subunit, or a variant thereof. In some embodiments, the blocking portion comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence represented by SEQ ID NO:93. In some embodiments, the artificial terminal portion comprises a linking peptide between the specific cleavage site and the blocking portion. In some embodiments, the Act-IL-18 polypeptide comprises a linking peptide between the IL-18 polypeptide and the specific cleavage site.

[0015] In some embodiments, active IL-18 polypeptides exhibit reduced binding to IL-18 binding protein (IL-18BP) compared to WT IL-18. In some embodiments, active IL-18 polypeptides exhibit enhanced ability to bind to or activate IL-18R. In some embodiments, active IL-18 polypeptides exhibit up to a 100-fold reduced ability to bind to or activate IL-18R compared to WT IL-18.

[0016] In some embodiments, an IL-18 polypeptide comprises one or more modifications to the sequence set forth in SEQ ID NO: 1. In some embodiments, an IL-18 polypeptide comprises an amino acid sequence 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: 1. In some embodiments, an IL-18 polypeptide comprises at least one substitution at residues Y1, F2, E6, C38, K53, D54, S55, T63, C76, E85, M86, T95, D98, or C127, or any combination thereof. In some embodiments, the IL-18 polypeptide comprises a Y01G, F02A, E06K, V11I, C38S, C38A, K53A, D54A, S55A, T63A, C76S, C76A, E85C, M86C, T95C, D98C, C127S, or C127A amino acid substitution, or any combination thereof. In some embodiments, the IL-18 polypeptide comprises an E06K and a K53A amino acid substitution. In some embodiments, the IL-18 polypeptide comprises a T63A amino acid substitution. In some embodiments, the IL-18 polypeptide comprises a V11I amino acid substitution. In some embodiments, the IL-18 polypeptide comprises substitutions at one, two, three, or four residues selected from C38, C76, C98, and C127. In some embodiments, the IL-18 polypeptide comprises an amino acid sequence set forth in any one of SEQ ID NOs: 2-67. In some embodiments, the IL-18 polypeptide comprises the amino acid sequence represented by SEQ ID NO: 30. In some embodiments, the IL-18 polypeptide comprises the amino acid sequence represented by any one of SEQ ID NOs: 79-83.

[0017] In some embodiments, an Act IL-18 polypeptide has a half-maximal effective concentration (EC) that is at least 1,000-fold higher, 2,000-fold higher, 5,000-fold higher, 10,000-fold higher, 15,000-fold higher, or 20,000-fold higher than an activated IL-18 polypeptide for IL-18 receptor signaling activity.50 In some embodiments, the Act IL-18 polypeptide exhibits a half-maximal effective concentration (EC) that is about 10-fold to about 100-fold higher than an activated IL-18 polypeptide for IL-18 receptor signaling activity. 50 In some embodiments, an active IL-18 polypeptide exhibits a half maximal effective concentration (EC) within about 10-fold of an IL-18 polypeptide for IL-18 receptor signaling activity. 50 In some embodiments, the Act-IL-18 polypeptide exhibits a half-maximal effective concentration (EC) that is at least 10-fold higher than WT IL-18 for IL-18 receptor signaling activity. 50 ) is shown.

[0018] In some embodiments, the IL-18 polypeptide is synthetic.

[0019] In some embodiments, the polymer is attached to a residue of the IL-18 polypeptide.

[0020] In another aspect, a method of treating cancer in a subject in need thereof, comprising administering to the subject a pharma- ceutically effective amount of an Act-IL-18 polypeptide or pharmaceutical composition provided herein. 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, 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. In some embodiments, the solid cancer is a carcinoma or sarcoma. In some embodiments, the cancer is a blood cancer. In some embodiments, the blood 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 the Act-IL-18 polypeptide or pharmaceutical composition in lyophilized form.

[0021] Another aspect provides a method of making an Act-IL-18 polypeptide provided herein, the method comprising synthesizing two or more fragments of an Act-IL-18 polypeptide, ligating the fragments, and folding the ligated fragments.

[0022] Further aspects and advantages of the present disclosure will become readily apparent to those skilled in the art from the following detailed description, in which only exemplary embodiments of the present disclosure have been shown and described. As will be understood, the present disclosure is capable of other and different embodiments, and its several details are capable of modification 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.

[0023] 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 specification is intended to supersede and / or override any such conflicting material.

[0024] 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 description of the drawings]

[0025] [Figure 1A] FIG. 1 shows an illustration of an exemplary activation mechanism of Act-IL-18 provided herein. [Figure 1B] FIG. 1 shows an illustration of an exemplary activation mechanism of an Act-IL-18 polypeptide having a blocking moiety ("mask" in the figure) linked to the N-terminus of the IL-18 polypeptide. [Figure 1C] FIG. 1 shows an illustration of an exemplary activation mechanism of an Act-IL-18 polypeptide having a blocking moiety ("mask" in the figure) linked to the C-terminus of the IL-18 polypeptide. [Diagram 2] 1 shows the mechanism of action of IL-18 on IFNγ and IL-18BP production, as well as the mechanism of action of IL-18BP's IL-18 inhibitory activity. [Diagram 3] 1 shows coupling of dibenzocyclooctyne (DBCO) polyethylene glycol (PEG) to an azide-containing IL-18 polypeptide. [Figure 4] 1 shows binding of IL-18 polypeptides containing polymers to IL-18Rα. [Figure 5A]1 shows the IFNγ inducing ability of IL-18 polypeptides of the disclosure compared to wild-type IL-18 polypeptide. [Figure 5B] 1 shows IL-18BP inhibition of IL-18 polypeptides of the disclosure compared to wild-type IL-18 polypeptides. [Figure 6] 1 shows a schematic representation of the coupling of a bifunctional probe to an IL-18 polypeptide provided herein. [Figure 7] 1 shows a schematic representation of coupling of poly(ethylene glycol) moieties to bifunctional probe-activated IL-18 polypeptides. [Figure 8] 1 shows a conditionally activatable IL-18 with a terminal protease recognition sequence linked to a blocking moiety and an orthogonal conjugation handle. [Figure 9] 1 shows a conditionally activatable IL-18 polypeptide with an N-terminal protease. [Figure 10A] 1 shows a conditionally activatable IL-18 with a C-terminal protease recognition sequence and blocking site and an orthogonal conjugation handle. [Figure 10B] FIG. 1 shows a conditionally activatable IL-18 with a C-terminal protease recognition sequence and an orthogonal conjugation handle. [Figure 11] Illustrative examples of conditionally activatable, conjugable IL-18 polypeptides with the three N-terminal residues replaced with a protease recognition sequence and blocking site are shown. Upon proteolytic processing in the tumor microenvironment (TME), functional IL-18 muteins are revealed. [Figure 12] Illustrative examples of conditionally activatable, conjugable IL-18 polypeptides with the three C-terminal residues replaced with a protease recognition sequence and blocking site are shown, which upon proteolytic processing in the TME reveal functional IL-18 muteins. [Figure 13]Illustrative examples of conditionally activatable IL-18 polypeptides with the three N-terminal residues replaced with a protease recognition sequence and a conjugation handle are shown. Upon proteolytic processing in the TME, free functional IL-18 muteins are revealed. [Figure 14A] 1 shows an SDS-PAGE gel showing an activatable IL-18 polypeptide provided herein both before and after MMP treatment. [Figure 14B] 1 shows an SDS-PAGE gel showing an activatable IL-18 polypeptide provided herein both before and after MMP treatment. [Figure 14C] 1 shows an SDS-PAGE gel showing an activatable IL-18 polypeptide provided herein after MMP treatment and resin purification, FT indicates flow-through and E indicates eluate. [Figure 15A] 1 shows dose-response curves for IL-18 receptor activation of activatable IL-18 polypeptides provided herein. [Figure 15B] 1 shows dose-response curves for IL-18 receptor activation of activatable IL-18 polypeptides provided herein. [Figure 16A] 1 shows the sequence and peptide map of an activatable IL-18 polypeptide provided herein. [Figure 16B] 1 shows the sequence and peptide map of an activatable IL-18 polypeptide provided herein. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] 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, IFNγ, and the production of specific cytotoxic T lymphocytes that recognize specific tumor antigens. Th1 responses are an important part of the host defense against many microorganisms. However, Th1 responses are also associated with autoimmune diseases and organ transplant rejection.

[0027] 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.

[0028] 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 protozoal infections. IFNγ is also an important activator of macrophages and an inducer of class II major histocompatibility complex (MHC) molecule expression.

[0029] 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, the binding affinity of IL-18 to IL-18Rα is low. In cells expressing the co-receptor IL-18 receptor beta chain (IL-18Rβ), a high-affinity heterodimeric complex is formed, which then activates cell signaling.

[0030] The activity of IL-18 is antagonized by the presence of a high affinity naturally occurring IL-18 binding protein (IL-18BP). IL-18BP binds to IL-18 and neutralizes the biological activity of IL-18. 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 2 shows the mechanism 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 following stimulation of IFNγ production has limited the effectiveness of IL-18 as a therapeutic modality in previous attempts.

[0031] To counter this inactivation, variants of IL-18 with reduced binding to IL-18 BP are currently under investigation. Non-limiting examples of such variants include those with amino acid substitutions that inhibit binding to IL-18BP, such as those found in PCT Publication Nos. WO2019051015A1 and WO2002101049A2, each of which is incorporated herein by reference as if set forth in its entirety. However, high levels of IL-18 in serum can produce deleterious off-target effects through indiscriminate activation of IL-18 receptors in non-target tissues and cells. These off-target effects include lymphopenia, hyperglycemia, anemia, neutropenia, hypoalbuminemia, liver damage, elevated liver enzymes, lymphopenia, increased lymphocyte activation, and increased serum concentrations of creatinine, IFN-γ, and granulocyte-macrophage colony-stimulating factor. Importantly, high levels of circulating free IL-18, unbound by neutralizing IL-18BP, are involved in the pathogenesis of potentially life-threatening systemic autoinflammatory / autoimmune diseases such as adult-onset Still's disease (AOSD) and systemic juvenile idiopathic arthritis (sJIA), but also in the pathogenesis of the most severe complication, macrophage activation syndrome (MAS).

[0032] To prevent immune-related adverse events or minimize their off-target and systemic effects, provided herein is an activatable form of interleukin-18 (Act-IL-18). In some cases, Act-IL-18 is activated by conditions associated with target tissues, such as cancer or tumor microenvironment. In some cases, Act-IL-18 is preferentially activated in or near target tissues, thus resulting in increased local concentrations of active IL-18 compared to non-target tissues. This in turn minimizes the off-target effects of IL-18, but still allows IL-18 to regulate local immune responses in target tissues.

[0033] In some embodiments, it has been observed herein that small moieties attached to the end of an IL-18 polypeptide (e.g., short peptide sequences or small linkers attached to the N-terminus or C-terminus of an IL-18 polypeptide, particularly the N-terminus) can functionally inhibit the activity of the IL-18 polypeptide. It was previously observed that endogenous IL-18 is initially expressed with an additional 36 amino acid segment at the N-terminus that is cleaved by caspases, but it was previously unclear whether other moieties (e.g., shorter peptide sequences) added to the end of mature IL-18 can inhibit IL-18 activity. Indeed, others have observed that IL-18 fusion proteins containing a 36 amino acid precursor peptide segment retain the signaling activity of the IL-18 receptor (see, e.g., PCT Publication No. WO2005014642A2, which is incorporated herein by reference as if set forth in its entirety).

[0034] In some embodiments, the Act-IL-18 polypeptides provided herein utilize a blocking moiety attached to the IL-18 polypeptide via a cleavable group to inhibit and / or reduce the activity of the IL-18 polypeptide. Such a blocking moiety may include the IL-18 propeptide or the D3 domain of the IL-18 receptor alpha subunit attached via a new cleavable group (e.g., not the endogenous cleavable group of full-length, immature IL-18). In such cases, cleavage of the specific cleavage group of the Act-IL-18 polypeptide releases the blocking moiety, thereby resulting in the activated form of the IL-18 polypeptide.

[0035] In one aspect, provided herein is an Act-IL-18 comprising an artificial terminal moiety that inactivates an IL-18 polypeptide at off-target locations but is cleaved to yield an active IL-18 polypeptide. In some embodiments, the artificial terminal moiety conditionally interferes with binding between an IL-18 receptor (IL-18Rαβ) and Act-IL-18. In some embodiments, conversion of the artificial terminal moiety by conditions in or near a target tissue (e.g., a diseased tissue such as a tumor microenvironment) weakens the receptor binding interference provided by the artificial terminal moiety. In some embodiments, the artificial terminal moiety interferes with binding between an IL-18 polypeptide and an IL-18 receptor in a non-target tissue. In some embodiments, conversion of the artificial terminal moiety (e.g., truncation of the artificial terminal moiety or other alteration of the artificial terminal moiety, e.g., oxidation / reduction or conformational change) leads to increased binding affinity and / or activation of the IL-18 receptor in the converted Act-IL-18 compared to unconverted Act-IL-18. In some embodiments, target tissue-specific conversion and activation of Act-IL-18 provides active IL-18 for the treatment of tumors with minimal active IL-18 in non-target tissues and cells. In some embodiments, active IL-18 polypeptides exhibit reduced ability to bind to and / or be neutralized by IL-18BP.

[0036] 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 there are many variations and modifications of the present disclosure, which are encompassed within the scope of the present disclosure.

[0037] Although various features of the 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 disclosure may be described herein for clarity in the context of separate embodiments, the disclosure may also be implemented in a single embodiment. The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0038] 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.

[0039] The following definitions are supplementary to those in the art and are directed to this application, and are not to be attributed to any related or unrelated cases, 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 testing of the present disclosure, preferred methods and materials are described herein. Therefore, the terms used herein are only for the purpose of describing specific embodiments, and are not intended to be limiting.

[0040] The terms used herein are for the purpose of describing particular instances only and are not intended to be limiting. In this specification, the use of the singular includes the plural unless expressly stated otherwise. As used herein, the singular forms "a," "an," and "the" are intended to include the plural unless the context clearly indicates otherwise.

[0041] In this application, the use of "or" means "and / or" unless otherwise stated. As used herein, the terms "and / or" and "any combination thereof" and their grammatical equivalents may be used interchangeably. These terms may convey that any combination is specifically contemplated. For illustrative purposes only, the following phrase "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.

[0042] 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, depending in part on the method of measuring or determining the value, i.e., the limitations of the measurement system. For example, "about" can mean within 1 or more than 1 standard deviation, 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 a particular value is described in this application and claims, the term "about" is deemed to mean within an acceptable error range for the particular value.

[0043] 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 including, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include"), or "containing" (and any form of including, such as "contains" and "contain") are inclusive or open 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 disclosure, and vice versa. Furthermore, the compositions of the disclosure can be used to achieve the methods of the disclosure.

[0044] References herein 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 disclosure, but not necessarily in all embodiments. To facilitate understanding of this disclosure, certain terms and phrases are defined below.

[0045] 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. The alpha-keto amino acids of the present disclosure have the structure shown in the following formula: [ka] , 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 in the L-orientation. 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 alpha-keto amino acid referred to corresponds to the above formula with the side chain of the amino acid referred to. When an alpha-keto amino acid residue is described 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 with 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.

[0046] Binding affinity refers to the strength of the binding interaction between a single molecule and its ligand / binding partner. A higher binding affinity refers to a stronger binding than a lower binding affinity. In some cases, binding affinity is determined by the dissociation constant (K D ) is measured by K D When comparing values, a binding interaction with a smaller value has a higher binding affinity than a binding interaction with a larger value. For protein-ligand interactions, K D is calculated according to the following formula:

number

[0047] In this specification, a certain amino acid sequence (e.g., a polypeptide sequence) is referred to as having a certain percent sequence identity to a reference sequence or referring to a residue at a position corresponding to a position of the reference sequence. Sequence identity is measured by the protein-protein BLAST algorithm using the following parameters: matrix BLOSUM62, gap cost presence: 11, extension: 1, and composition-adjusted conditional composition score matrix adjustment. This alignment algorithm is also used to evaluate whether residues are at "corresponding" positions through the analysis of the alignment of the two sequences being compared.

[0048] 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 the amino acid's side chain, as well as at other points on the amino acid) qualify as the same amino acid for purposes of sequence identity, particularly in the case of chemically synthesized polypeptides, as the "unprotected" forms. 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 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 (which are covalently modified), 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.

[0049] As used herein, reference is made to an amino acid or an amino acid sequence that appears "upstream" of another referenced amino acid sequence. The term "upstream" in this context means that the indicated amino acid or amino acid sequence is added to the N-terminal residue of the reference amino acid sequence (e.g., a methionine residue located upstream of the sequence "SDGTK" would have the sequence "MSDGTK"). In some cases, the numbering of the residues of the "upstream" amino acid or amino acid sequence uses a negative numbering system (e.g., reference to positions -1, -2, or -3 relative to the reference sequence). When such a numbering system is used, position -1 corresponds to the amino acid added to the N-terminus of the reference sequence, position -2 corresponds to the amino acid added to the N-terminus of position -1, etc. For example, a sequence of AM located upstream of the reference sequence SDGTK would result in the complete sequence of AMSDGTK, with M at position -1 and A at position -2 relative to the reference sequence.

[0050] The term "pharmaceutical acceptable" means approved or approvable by a regulatory agency of the Federal or state government, or listed in the United States Pharmacopeia or other generally recognized pharmacopoeia for use in animals, including humans.

[0051] The term "pharmaceutical 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.

[0052] A "pharmaceutically acceptable salt" suitable for this disclosure may be an acid or base salt generally considered in the art to be suitable for use in contact with human or animal tissues 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 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 acid, such as acetic acid, HOOC-(CH2) n Pharmaceutically acceptable salts include, but are not limited to, salts of acids such as -COOH (wherein n is 0, 2, 3, 4, or 4). Similarly, pharma- ceutically acceptable cations include, but are not limited to, sodium, potassium, calcium, aluminum, lithium, and ammonium. Those skilled in the art will recognize from this disclosure and knowledge in the art that further pharma- ceutical acceptable salts include those described in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, PA, p.1418 (1985). In general, pharma- ceutical acceptable acid salts or base salts can be synthesized from parent compounds that contain 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.

[0053] It is understood that the ranges provided herein are shorthand notations of all values ​​within the range.For example, the range of 1 to 50 includes any number, combination of numbers, or subranges 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, such as 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 opposite direction 50 to 40, 50 to 30, 50 to 20, and 50 to 10.

[0054] 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 non-human mammal (such as a non-human primate, cow, horse, dog, sheep, or cat).

[0055] 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.

[0056] 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 formula (1).

number

[0057] As used herein, the term "weight average molecular weight" (Mw) means the number defined by formula (2).

number

[0058] As used herein, "peak molecular weight" (Mp) means the molecular weight of the largest peak in a given analytical method (e.g., mass spectrometry, size exclusion chromatography, dynamic light scattering, analytical centrifugation, etc.).

[0059] As used herein, "conjugation handle" refers to a reactive group that can form a bond when contacted 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 described 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 increased potential for off-target conjugation. Conjugation Handles Table [Table 1]

[0060] 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 "PEG conjugation handle" is a conjugation handle attached to a PEG group (either directly or via a linker).

[0061] II. Activatable IL-18 Polypeptides The present disclosure relates to activatable IL-18 (Act-IL-18) polypeptides useful as therapeutic agents. The activatable IL-18 polypeptides provided herein can be used as immunotherapies or as part of other immunotherapeutic regimens.

[0062] In one aspect, the present disclosure relates to an artificial moiety attached to an IL-18 polypeptide. The artificial moiety may include peptides, amino acids, and other groups, such as polymers or spaces, that are used to link the artificial moiety to the IL-18 polypeptide. The artificial moiety includes a cleavable group that, upon cleavage, releases all or part of the terminal moiety. Upon cleavage, Act-IL-18 is converted to an active IL-18 polypeptide capable of carrying out IL-18 activity.

[0063] In one aspect, provided herein is an Act-IL-18 polypeptide comprising an artificial terminal moiety linked to an IL-18 polypeptide. In some embodiments, the artificial terminal moiety inhibits the Act-IL-18 polypeptide from interacting with an IL-18 receptor and / or inhibits signaling through the IL-18 receptor. In some embodiments, the artificial terminal moiety is capable of change in response to a condition or stimulus, resulting in conversion of the Act-IL-18 polypeptide to an active IL-18 polypeptide. In some embodiments, the change is cleavage of at least a portion of the artificial terminal moiety from the IL-18 polypeptide.

[0064] In one aspect, the Act-IL-18 of the present disclosure is activated in or near a target tissue of a subject. In some embodiments, Act-IL-18 is preferentially activated in or near a target tissue of a subject (e.g., activated at a higher rate in or near a target tissue compared to other tissues). In some embodiments, Act-IL-18 is preferentially activated in or near a target tissue of a subject, such that an area of ​​the target tissue or an area near the target tissue contains at least 2-fold, at least 4-fold, at least 8-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 200-fold, at least 500-fold, or at least 1000-fold higher concentration of active IL-18 polypeptide compared to non-target tissue. In some embodiments, the concentration compared is the peak concentration of active IL-18 polypeptide after administration of Act-IL-18. In some embodiments, Act-IL-18 is preferentially activated in or near a diseased tissue of a subject. In some embodiments, the Act-IL-18 polypeptide is preferentially activated in or near the cancer tissue of the subject. In some embodiments, the Act-IL-18 polypeptide is preferentially activated in or near the tumor microenvironment. In some embodiments, the Act-IL-18 is preferentially activated in the tumor microenvironment compared to non-tumor tissue.

[0065] In one aspect, provided herein is an activatable interleukin-18 (Act-IL-18) polypeptide comprising an artificial terminal moiety linked to an interleukin-18 (IL-18) polypeptide, the artificial terminal moiety comprising a specific cleavage site, where cleavage at the specific cleavage site converts the Act-IL-18 into an active IL-18 polypeptide.

[0066] artificial part In one aspect, provided herein are artificial moieties attached to IL-18 polypeptides. The artificial moieties provided herein function to attenuate the activity of IL-18 polypeptides while they are attached in their intact form. In some embodiments, cleavage of the artificial moiety functions to activate IL-18 polypeptides (e.g., allows IL-18 polypeptides to signal through IL-18Rαβ). The artificial moieties provided herein can be attached to any residue. In some embodiments, the artificial moiety is an artificial terminal moiety (e.g., attached to a terminal residue of IL-18 polypeptide).

[0067] In some embodiments, the artificial moiety can also have other functional groups attached (e.g., in addition to being attached to the IL-18 polypeptide, the artificial moiety is also attached to another group, such as an additional polypeptide (e.g., an antibody, a dummy receptor, another cytokine), a half-life extending polymer (e.g., poly(ethylene glycol) (PEG)), or another desired functional group. In some embodiments, cleavage of the artificial moiety also serves to cleave this additional group from the IL-18 polypeptide.

[0068] artificial end part In one aspect, the Act-IL-18 polypeptides provided herein include an artificial terminal moiety. In some embodiments, the artificial terminal moiety is a functional group, e.g., a peptide, small molecule, or other group, that is covalently attached to the IL-18 polypeptide. In some embodiments, the artificial terminal moiety is a peptide. When the artificial terminal moiety is a peptide, it is sometimes alternatively referred to herein as an "artificial terminal peptide." The artificial terminal moiety is a group that is not naturally attached to the terminus of a WT IL-18 polypeptide, such as the natural precursor 36 amino acid propeptide that is directly attached to WT IL-18. When the IL-18 polypeptide is a variant IL-18 polypeptide (e.g., having at least one amino acid substitution compared to WT IL-18), the artificial terminal moiety can be a natural propeptide. In some embodiments, the artificial terminal peptide is engineered to have the properties provided herein. In some embodiments, the artificial terminal moiety is fused to the IL-18 polypeptide (e.g., as a fusion protein). In some embodiments, the artificial end moiety is chemically attached to the IL-18 polypeptide or is incorporated into the IL-18 polypeptide by synthetic means (eg, during synthesis of the IL-18 polypeptide).

[0069] In some embodiments, the artificial end moieties provided herein inhibit at least one activity associated with an IL-18 polypeptide, such as the ability to bind to an IL-18 receptor or to effect signaling through the IL-18 receptor (IL-18Rαβ) (e.g., to induce production of IFNγ in immune cells). In some embodiments, when the artificial end moiety is intact, the Act-IL-18 polypeptide is in an inactive state (e.g., lacks or has a substantially reduced ability to bind to or to signal through IL-18Rαβ).

[0070] In some embodiments, the presence of an intact artificial terminal moiety on the IL-18 polypeptide causes the Act-IL-18 polypeptide to exhibit a binding affinity for IL-18Rαβ or IL-18R subunit that is at least 10-fold lower, at least 100-fold lower, at least 200-fold lower, at least 500-fold lower, or at least 1000-fold lower than WT IL-18. In some embodiments, the presence of an intact artificial terminal moiety on the IL-18 polypeptide causes the Act-IL-18 polypeptide to exhibit a binding affinity for IL-18Rαβ or IL-18R subunit that is at least 10-fold lower, at least 100-fold lower, at least 200-fold lower, at least 500-fold lower, or at least 1000-fold lower than an IL-18 polypeptide that does not have an artificial terminal moiety.

[0071] In some embodiments, the presence of an intact artificial terminal moiety on an IL-18 polypeptide causes an Act-IL-18 polypeptide to exhibit at least 10-fold less, at least 100-fold less, at least 200-fold less, at least 500-fold less, or at least 1000-fold less ability to induce IFNg production in a cell (e.g., an immune cell such as a NK cell) than WT IL-18. In some embodiments, the presence of an intact artificial terminal moiety on an IL-18 polypeptide causes an Act-IL-18 polypeptide to exhibit at least 10-fold less, at least 100-fold less, at least 200-fold less, at least 500-fold less, or at least 1000-fold less ability to induce IFNg production in a cell (e.g., an immune cell such as a NK cell) than an IL-18 polypeptide not having an artificial terminal moiety.

[0072] In some embodiments, the artificial terminal portion comprises a specific cleavage site. In some embodiments, the specific cleavage site is a site that is susceptible to cleavage under a particular specified or known condition. Non-limiting examples of specific cleavage sites include protease cleavage sites, sites that are susceptible to cleavage at a particular pH range (e.g., acid labile bonds), sites that are susceptible to cleavage via oxidation or reduction (e.g., disulfide bonds), photocleavable linkers, and the like.

[0073] In some embodiments, the specific cleavage site is selected to be preferentially cleaved (e.g., cleaved at a faster rate or to a greater extent) in a designated target tissue of a subject. In some embodiments, the specific cleavage site is preferentially located in or near the target tissue of a subject, such that the specific cleavage site is cleaved at least 2-fold, at least 4-fold, at least 8-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 200-fold, at least 500-fold, or at least 1000-fold faster than the cleavage of the specific cleavage site in a different tissue. In some embodiments, the target tissue is a diseased tissue of a subject. In some embodiments, the target tissue is a cancer tissue of a subject. In some embodiments, the target tissue is a tumor microenvironment. In some embodiments, the target tissue is a tumor.

[0074] In some embodiments, the specific cleavage site is positioned such that all or only a portion of the artificial end moiety is removed from the IL-18 polypeptide after cleavage. In some embodiments, none of the artificial end moiety is present on the IL-18 polypeptide after cleavage (e.g., only residues corresponding to residues in SEQ ID NO:1 are present after cleavage). In some embodiments, some of the artificial end moieties remain attached to the IL-18 polypeptide after cleavage.

[0075] The artificial terminal moiety may be attached to either the N-terminal or C-terminal residue of an IL-18 polypeptide. In some embodiments, the artificial terminal moiety is attached to the N-terminal residue. In some embodiments, the artificial terminal moiety is attached to the N-terminal amine of an IL-18 polypeptide. In some embodiments, the artificial terminal moiety is attached to the C-terminal residue. In some embodiments, the artificial terminal moiety is attached to the C-terminal carboxyl of an IL-18 polypeptide. In some embodiments, the N-terminal residue is the residue closest to residue position 1 of SEQ ID NO:1 present on an IL-18 polypeptide provided herein (e.g., the first residue of uncleaved SEQ ID NO:1). In some embodiments, the N-terminal residue of an IL-18 polypeptide is the residue at the position corresponding to position 1 of SEQ ID NO:1. In some embodiments, the N-terminal residue of an IL-18 polypeptide is Y1. In some embodiments, the N-terminal residue of an IL-18 polypeptide is Y1G. In some embodiments, the C-terminal residue is the residue at position 157 of SEQ ID NO:1. In some embodiments, the C-terminal residue is D157.

[0076] In some embodiments, terminal residues of an IL-18 polypeptide are replaced such that the artificial terminal moiety is positioned such that the entire artificial terminal moiety is cleaved from the IL-18 polypeptide. For example, if it is intended to introduce a cleavage site at a position corresponding to residue 1 of SEQ ID NO:1, the protease cleavage sequence P1-P2-P3-P' 1- P' 2- P'3 can be selected (the cleavage site is between P3 and P'1), and residues 1, 2, and 3 of SEQ ID NO:1 can be replaced with P'1, P'2, and P'3, respectively, with P1-P2-P3- being added thereon. In this case, the artificial terminal portion is considered to comprise P1-P2-P3-, and P'1, P'2, and P'3 are included as part of the IL-18 polypeptide (the replaced residues).

[0077] Examples of artificial terminal moieties and their mechanism of action are shown in Figures 8-13. Figure 8 illustrates an artificial terminal peptide added to a terminal residue of an IL-18 polypeptide. The artificial moiety also has a blocking moiety attached that facilitates keeping the IL-18 polypeptide in an inactive state. When the artificial terminal moiety is cleaved (e.g., by tumor microenvironment proteases), the entire artificial terminal moiety is cleaved (as is the blocking moiety, revealing the terminal residue of the IL-18 polypeptide). The IL-18 in this example has a conjugation handle (which serves to attach additional groups (e.g., half-life extending polymers or additional polypeptides)) attached elsewhere on the IL-18 polypeptide, which may be attached before or after cleavage of the artificial terminal moiety.

[0078] Figure 9 shows a similar Act-IL-18 construct shown in the previous figure, but lacking the blocking site. In some cases, the presence of only a short peptide on the N-terminus can be sufficient to attenuate the activity of the IL-18 polypeptide, and activity can be restored upon cleavage.

[0079] FIG. 10A shows an Act-IL-18 construct similar to that shown in FIG. 8, but with an artificial terminal moiety attached to the C-terminus.

[0080] FIG. 10B shows an Act-IL-18 construct similar to that shown in FIG. 9, but with an artificial terminal moiety attached to the C-terminus.

[0081] FIG. 11 illustrates a construct similar to that shown in FIG. 8, but where residues 1, 2, and 3 of the IL-18 polypeptide are substituted such that the artificial terminal portion is completely cleaved (residues 1, 2, and 3, even though substituted, correspond to positions 1, 2, and 3 of SEQ ID NO:1). FIG. 12 is similar to FIG. 11, but where the artificial terminal portion is at the C-terminus. Instead of substituting residues 1, 2, and 3 of IL-18 illustrated in FIG. 11, a cleavable peptide can be selected such that native residues 1, 2, and 3 of the IL-18 polypeptide (e.g., Y, F, and G, respectively), or a subset of the native residues, form part of the cleavage recognition site for the relevant protease such that the entire artificial terminal portion is cleaved, thereby leaving an unmodified N-terminus of the IL-18 polypeptide after cleavage. For example, a sequence of -PLG- can be added to the N-terminal Y of the IL-18 polypeptide, allowing complete cleavage of the artificial terminal portion by a matrix metalloprotease. Such an approach can also be employed at the C-terminus of the IL-18 polypeptide.

[0082] Figure 13 illustrates an Act-IL-18 polypeptide comprising an artificial terminal moiety with a conjugation handle attached such that cleavage of the artificial terminal moiety also cleaves the conjugation handle (or anything attached to the conjugation handle) from the IL-18 polypeptide. As a non-limiting example, such a construct can be used to conjugate an antibody to an IL-18 polypeptide. Such a construct can be used to deliver an IL-18 polypeptide in a tissue-specific manner, and then activate it at a desired site via cleavage at the target tissue.

[0083] Peptide artificial terminal moiety In some embodiments, the artificial terminal portion comprises a peptide (also known as an "artificial terminal peptide"). In some embodiments, the artificial terminal portion consists of a peptide.

[0084] In some embodiments, cleavage of the specific cleavage site leaves no amino acid residues attached to the IL-18 polypeptide. In some embodiments, cleavage of the specific cleavage site leaves at least one amino acid residue attached to the IL-18 polypeptide. In some embodiments, cleavage of the specific cleavage site leaves up to 1, 2, 3, 4, or 5 amino acid residues attached to the IL-18 polypeptide. In some embodiments, cleavage of the specific cleavage site leaves 1, 2, 3, 4, or 5 amino acid residues attached to the IL-18 polypeptide. In some embodiments, cleavage of the specific cleavage site leaves 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid residues attached to the IL-18 polypeptide.

[0085] In some embodiments, Act-IL-18 may optionally include a tag used in the expression and / or purification of recombinant Act-IL-18 provided herein. The tag may be located upstream of the N-terminal artificial end peptide or downstream of the C-terminal artificial end peptide. The tag may be removed (e.g., enzymatically) prior to final formulation and / or administration to a subject. Exemplary tags include HIS6 (HHHHHH, SEQ ID NO: 84), Strep Tag (WSHPQFEK (SEQ ID NO: 85) or AWAHPQPGG (SEQ ID NO: 86), Or a chitin binding tag (TTNPGVSAWQVNTAYTAGQLVTYNGKTYKCLQPHTSLAGWEPSNVPALWQLQ, SEQ ID NO: 87).

[0086] Protease-cleavable artificial termini In some embodiments, the artificial end portion can be cleaved by a protease (e.g., the artificial end portion comprises a cleavable peptide). In some embodiments, the artificial end portion contains a cleavage site that can be specifically cleaved by one or more proteases. In some embodiments, the artificial end portion contains a cleavage site that can be cleaved at a site preferred by one or more proteases. In some embodiments, the specific cleavage site is a protease cleavage site.

[0087] In some embodiments, the protease is found in higher concentrations and / or exhibits higher proteolytic activity at or near a target tissue of a subject. In some embodiments, the target tissue is a diseased tissue. In some embodiments, the target tissue is a cancer. In some embodiments, the target tissue is a tumor microenvironment.

[0088] In some embodiments, the protease is found in higher concentrations and / or exhibits higher proteolytic activity in or near the tumor microenvironment compared to non-tumor tissue. In some embodiments, the protease is found in higher concentrations in or near the tumor microenvironment compared to non-tumor tissue. In some embodiments, the protease exhibits higher proteolytic activity in or near the tumor microenvironment compared to non-tumor tissue. In some embodiments, the protease is selected from kallikrein, thrombin, chymase, carboxypeptidase A, elastase, proteinase 3 (PR-3), granzyme M, calpain, matrix metalloproteinase (MMP), a disintegrin and metalloproteinase (ADAM), fibroblast activation protein alpha (FAP), plasminogen activator, cathepsin, caspase, tryptase, and tumor cell surface proteases. In some embodiments, the cleavable peptide is cleavable by a protease selected from kallikrein, thrombin, chymase, carboxypeptidase A, elastase, proteinase 3 (PR-3), granzyme M, calpain, matrix metalloproteinase (MMP), a disintegrin and metalloproteinase (ADAM), fibroblast activation protein alpha (FAP), plasminogen activator, cathepsin, caspase, tryptase, matriptase, and tumor cell surface proteases, or any combination thereof. In some embodiments, the protease is selected from kallikrein, thrombin, chymase, carboxypeptidase A, elastase, proteinase 3 (PR-3), granzyme M, urokinase plasminogen activator (uPA), calpain, matrix metalloproteinase (MMP), a disintegrin and metalloproteinase (ADAM), fibroblast activation protein alpha (FAP), matriptase, plasminogen activator, cathepsin, caspase, tryptase, and tumor cell surface proteases.In some embodiments, the cleavable peptide is cleavable by a protease selected from kallikrein, thrombin, chymase, carboxypeptidase A, elastase, proteinase 3 (PR-3), urokinase plasminogen activator (uPA), granzyme M, calpain, matrix metalloproteinase (MMP), a disintegrin and metalloproteinase (ADAM), fibroblast activation protein alpha (FAP), matriptase, plasminogen activator, cathepsin, caspase, tryptase, matriptase, and tumor cell surface proteases, or any combination thereof. In some embodiments, the protease is urokinase plasminogen activator (uPA) or matriptase. In some embodiments, the cleavable peptide is cleavable by urokinase plasminogen activator (uPA) or matriptase. In some embodiments, the protease is a protease selected from Table 1 or Table 2A. In some embodiments, the protease is a protease selected from Table 2B. [Table 2] TIFF2025508824000008.tif108159

[0089] In some embodiments, the protease cleavage site is contained within the recognition sequence recognized by the protease. In some embodiments, the recognition sequence is a natural peptide sequence that is incorporated into an artificial terminal portion. In some embodiments, the recognition sequence is a synthetic (e.g., artificial, designed, or engineered) sequence. In some embodiments, the recognition sequence comprises an amino acid 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 100% identical to a SEQ ID NO: in Table 2A. [Table 3]

[0090] In some embodiments, the recognition sequence comprises an amino acid 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 100% identical to a sequence number set forth in Table 2B. [Table 4] TIFF2025508824000011.tif240159TIFF2025508824000012.tif95159

[0091] In some embodiments, cleavage of the specific cleavage site leaves no amino acid residues attached to the IL-18 polypeptide. In such cases, the protease recognition sequence may be selected such that a portion of the IL-18 polypeptide constitutes or is compatible with the recognition sequence. For example, in some embodiments, the sequence PLG is added to the N-terminus of the IL-18 polypeptide (e.g., residue 1 of SEQ ID NO:1), such that the specific cleavage site is located between the G of PLG and the N-terminus of the IL-18 polypeptide, thereby resulting in an "intact" activated IL-18 polypeptide after cleavage.

[0092] In some embodiments, a portion of the protease recognition sequence that defines a specific cleavage site is included in the sequence of an IL-18 polypeptide (e.g., a portion of the protease recognition sequence is included at a position corresponding to a position in SEQ ID NO: 1). In some embodiments, a portion of the protease recognition sequence included in the sequence of an IL-18 polypeptide is substituted for the sequence represented by SEQ ID NO: 1. For example, in some embodiments, the last three amino acids of SEQ ID NO: 1 are substituted with -PLG to form part of the protease recognition site using an artificial terminal portion.

[0093] In some embodiments, cleavage of the specific cleavage site leaves at least one amino acid residue attached to the IL-18 polypeptide. In some embodiments, cleavage of the specific cleavage site leaves at most 1, 2, 3, 4, or 5 amino acid residues ... cleavage site leaves at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid residues attached to the IL-18 polypeptide. In some embodiments, cleavage of the specific cleavage site leaves at most 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue attached to the IL-18 polypeptide.

[0094] Additional exemplary cleavable peptide sequences that can be incorporated into the Act-IL-18 polypeptides provided herein are described in U.S. Patent Publications US2010 / 0189651, US2016 / 0289324, US2018 / 0125988, US2019 / 0153115, US2020 / 0385469, US2021 / 0260163, US2022 / 0048949, US2022 / 0267400, US2021 / 0115102, US2022 / 0002370, US2021 / 0163562, U S20200392235, US2021 / 0139553, US2021 / 0317177, US2020 / 0283489, US2021 / 0002343, US20 21 / 0292421, US2021 / 0284728, US2021 / 0269530, US2022 / 0054544, US2021 / 0355219, US2022 / 0073613, US2021 / 0047406, and / or Patent Cooperation Treaty Publications WO2021 / 202675, WO2021 / 062406, WO2021 / 142 471, WO2021 / 216468, WO2021 / 119516, WO2021 / 253360, WO2021 / 146455, WO2021 / 202678, WO2021 / 202673, WO2021 / 189139, WO2020 / 232303, WO2022 / 115865, WO2021 / 202678, and / or Chen et.al., J Bio Chem, 277, V6 P4485-4491 (2002).

[0095] Redox-cleavable artificial termini In some cases, different tissues of a subject may exhibit different redox potentials depending on the activity of the tissue. For example, tumors and the tumor microenvironment are associated with having substantially higher redox potentials than other healthy tissues. Thus, in some embodiments, the artificial end moieties provided herein take advantage of this property to allow preferential cleavage and activation of IL-18 polypeptides at tissue sites.

[0096] In some embodiments, the artificial end portion can be cleaved by a reduction or oxidation reaction. In some embodiments, the artificial end portion contains a cleavage site that can be specifically cleaved by a reduction or oxidation reaction. In some embodiments, the artificial end portion contains a cleavage site that can be cleaved at a site favored by a reduction or oxidation reaction. In some embodiments, the specific cleavage site is a redox sensitive cleavage site.

[0097] In some embodiments, the redox sensitive cleavage site is preferentially cleaved at or near the target tissue of interest, such that the specific cleavage site is cleaved at a rate that is at least 2-fold, at least 4-fold, at least 8-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 200-fold, at least 500-fold, or at least 1000-fold faster than the cleavage of the specific cleavage site in a different tissue. In some embodiments, the redox cleavage site is preferentially cleaved in a reducing environment. In some embodiments, the redox cleavage site is preferentially cleaved in a reducing environment associated with blood. In some embodiments, the redox cleavage site is preferentially cleaved in a reducing environment associated with interstitial fluid. In some embodiments, the redox cleavage site is preferentially cleaved in a reducing environment associated with lymphatic fluid. In some embodiments, the redox cleavage site is preferentially cleaved in a reducing environment of a tumor microenvironment.

[0098] pH-cleavable artificial termini The pH of circulating blood is buffered within a narrow range of 7.31-7.45. Variations outside this range usually result in acidosis or alkalosis, which are serious medical conditions. The tumor microenvironment is characteristically more acidic than the circulating blood pH due to a metabolic dysregulation in tumor cells known as the Warburg effect (proliferating tumor cells exhibit high rates of glycolysis followed by fermentation of pyruvate to lactate in the cytoplasm rather than oxidation of pyruvate in the mitochondrial TCA cycle). To maintain the pH of their cytoplasm, tumor cells transport hydrogen ions into the extracellular environment, resulting in an acidic tumor microenvironment.

[0099] In some embodiments, the specific cleavage site is a pH-sensitive cleavage site. In some embodiments, the pH-sensitive cleavage site is selected to cleave preferentially in a target tissue. In some embodiments, the target tissue is associated with a certain pH or pH difference compared to other local tissues.

[0100] In some embodiments, the pH-sensitive cleavage site is cleaved at a pH lower than physiological blood pH (e.g., less than about 7.3). In some embodiments, the pH-sensitive cleavage site is preferentially cleaved at a pH less than 7.3, less than 7.2, less than 7.1, or less than 7.0. In some embodiments, the pH-sensitive cleavage site is preferentially cleaved at an acidic pH. In some embodiments, the pH-sensitive cleavage site is preferentially cleaved at a pH less than 7, 6.5, 6, 5.5, or 5.

[0101] In some embodiments, the pH-sensitive cleavage site is preferentially cleaved at or near the target tissue of interest such that the specific cleavage site is cleaved at a rate that is at least 2-fold, at least 4-fold, at least 8-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 200-fold, at least 500-fold, or at least 1000-fold faster than cleavage of the specific cleavage site in a different tissue. In some embodiments, the tissue is the tumor microenvironment.

[0102] Blocking site In some embodiments, the artificial terminal moiety comprises a blocking moiety, which, when bound to an IL-18 polypeptide in an Act-IL-18 polypeptide, acts to prevent or inhibit binding of the IL-18 polypeptide to an IL-18 receptor or a subunit thereof (e.g., as measured by experiments designed to detect binding or by in vitro or in vivo activity assays of the Act-IL-18 polypeptide).

[0103] In some embodiments, the blocking moiety is a steric blocking group or a specific blocking group. In some embodiments, the blocking moiety is a steric blocking group. In some embodiments, the steric blocking group does not have a specific interaction with the IL-18 polypeptide, but its presence prevents the interaction of the Act-IL-18 polypeptide with the receptor due to its bulkiness. In some embodiments, the steric blocking group is a polymer (e.g., polyethylene glycol) or a polypeptide (e.g., albumin, Fc region, etc.).

[0104] In some embodiments, the blocking moiety is a specific blocking group. In some embodiments, the specific blocking group has a specific binding or other interaction with IL-18. Non-limiting examples of specific blocking groups may include IL-18 propeptide, an antibody or antigen-binding fragment that binds to IL-18, an IL-18 receptor subunit or domain or other fragments thereof, an IL-18 binding protein or fragment thereof, or other group capable of specifically binding to IL-18.

[0105] In some embodiments, the blocking moiety is the propeptide of IL-18. Endogenously, human IL-18 is expressed as an immature, inactive 193 amino acid fragment with the sequence MAAEPVEDNCINFVAMKFIDNTLYFIAEDDENLESDYFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 88), the first 36 amino acids being the propeptide that is cleaved by caspases to yield the mature, active form of IL-18 (SEQ ID NO: 1). In some embodiments, the IL-18 propeptide blocking moiety is attached to the N-terminus of an IL-18 polypeptide (e.g., via a cleavable peptide comprising a specific cleavage site and any optional linker peptide), and in some embodiments, this propeptide or a variant thereof is incorporated into Act-IL-18 as a blocking moiety. In some embodiments, the blocking moiety is a human IL-18 propeptide or a variant thereof. In some embodiments, the blocking moiety is an IL-18 propeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO:89. In some embodiments, the blocking moiety is an IL-18 propeptide comprising a sequence set forth in SEQ ID NO:89. In some embodiments, the blocking moiety is an IL-18 propeptide comprising the sequence set forth in SEQ ID NO:89 with a substitution of residue C10 (e.g., a C10S substitution (SEQ ID NO:90) or a C10A substitution (SEQ ID NO:91). In some embodiments, the IL-18 propeptide comprises one or more modifications (e.g., amino acid substitutions) that reduce the affinity of the IL-18 propeptide for the IL-18 polypeptide in the Act-IL-18 polypeptide.In some embodiments where the blocking moiety is an IL-18 propeptide, the specific cleavage site of the Act-IL-18 polypeptide is different from the specific cleavage site of the endogenous propeptide (i.e., the specific cleavage site is not the bond between D36 and Y37 of SEQ ID NO: 88). In some embodiments, the IL-18 propeptide that functions as the blocking moiety is connected to the N-terminus of the IL-18 polypeptide in the Act-IL-18 polypeptide via another protease recognition sequence (and optionally one or more linking peptides).

[0106] In some embodiments, the blocking moiety is a modified IL-18 propeptide (e.g., human IL-18 propeptide) directly attached to the N-terminus of the IL-18 polypeptide. In some embodiments, the modified IL-18 propeptide includes a modification that changes the native caspase cleavage site of SEQ ID NO:88 to a site that is cleaved by another protease (e.g., any of the proteases provided herein, such as a matrix metalloprotease). In some embodiments, the three C-terminal amino acids of the IL-18 propeptide are replaced with -PLG. In some embodiments, the IL-18 propeptide includes a -PLG replacement, Exemplary IL-18 Propeptide Sequences [Table 5]

[0107] In some embodiments, Act-IL-18 polypeptides comprising a human IL-18 propeptide or a variant thereof as a blocking moiety exhibit substantially reduced activity (e.g., substantially no activity or activity that is reduced by more than 1000-fold) compared to the IL-18 polypeptide itself or the activated form of the IL-18 polypeptide. In some embodiments, it may be desirable for the Act-IL-18 to which the IL-18 propeptide is bound to retain a higher activity prior to activation. To achieve this, in some embodiments, it may be advantageous to use an IL-18 propeptide derived from a non-human species. In some embodiments, the Act-IL-18 polypeptides provided herein comprise an IL-18 propeptide derived from a non-human species. In some embodiments, the non-human species is a mammal. In some embodiments, the non-human species is a primate, rodent, equine, bovine, ursine, porcine, equine, chiropteran, camelid, or other animal. In some embodiments, the non-human IL-18 propeptide has an amino acid sequence that is at least 50%, 60%, 70%, or 75% identical to the amino acid sequence of SEQ ID NO:89.

[0108] In some embodiments, the blocking moiety comprises a portion (e.g., a domain or portion thereof) of an IL-18 receptor subunit, or a variant thereof. In some embodiments, the portion of the IL-18 receptor subunit or a variant thereof is attached to the C-terminus of the IL-18 polypeptide in the Act-IL-18 polypeptide (e.g., via a cleavable peptide comprising a specific cleavage site and any optional linker peptide). In some embodiments, the blocking moiety comprises a portion of an IL-18 receptor alpha subunit or an IL-18 receptor beta subunit, or a variant thereof. In some embodiments, the blocking moiety comprises a portion of an IL-18 receptor alpha subunit or a variant thereof. In some embodiments, the blocking moiety comprises a domain of an IL-18 receptor alpha subunit or a variant thereof. In some embodiments, the blocking moiety comprises the extracellular domain of an IL-18 receptor alpha subunit, or a variant thereof. In some embodiments, the blocking moiety comprises the D1, D2, or D3 domain of an IL-18 receptor alpha subunit, or a variant thereof.

[0109] In some embodiments, the blocking site comprises the D3 domain of the IL-18 receptor alpha subunit, or a variant thereof. The sequence of the human D3 domain of the IL-18 receptor alpha subunit is set forth below in SEQ ID NO: 93. In some embodiments, the blocking site comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 93. In some embodiments, the blocking site comprises an amino acid sequence having at least 95% sequence identity to the sequence set forth in SEQ ID NO: 93. In some embodiments, the blocking site comprises a sequence set forth in SEQ ID NO: 93. In some embodiments, the blocking site D3 domain of the IL-18 receptor alpha subunit comprises a substitution that removes a glycosylation site from the D3 domain. In some embodiments, the blocking site comprises the amino acid sequence set forth in SEQ ID NO: 94 below. [Table 6]

[0110] In some embodiments, it may be desirable to introduce one or more amino acid substitutions into the sequence of the D3 domain of the IL-18 receptor alpha subunit when used as a blocking moiety to enhance or weaken binding of the Act-IL-18 polypeptide to the D3 domain of the IL-18 polypeptide. The full length human IL-18 receptor alpha subunit has the sequence (SEQ ID NO: 95) (UniProt ID: Q13478).

[0111] Linking peptide In some embodiments, the artificial terminal portion comprises one or more connecting peptides. In some embodiments, the connecting peptide of the artificial terminal portion is located between the specific cleavage site and the IL-18 polypeptide (i.e., the connecting peptide remains attached to the IL-18 polypeptide after cleavage), or between the specific cleavage site and the blocking site, or both (e.g., an Act-IL-18 polypeptide has two connecting peptides). In some embodiments, the connecting peptide comprises 1-50 amino acids, 1-40 amino acids, 1-30 amino acids, 1-25 amino acids, 1-20 amino acids, 1-15 amino acids, 1-10 amino acids, or 1-5 amino acids. In some embodiments, the connecting peptide comprises 1-15 amino acids. In some embodiments, the connecting peptide consists of the amino acids glycine and serine. In some embodiments, the connecting peptide consists of glycine. Non-limiting examples of connecting peptides include (GS) n (SEQ ID NO: 235), (GGS) n (SEQ ID NO: 236), (GGGS) n (SEQ ID NO: 237), (GGSG) n (SEQ ID NO: 238), or (GGSGG) n (SEQ ID NO: 239), (GGGGS) n (SEQ ID NO:240) where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. For example, the connecting peptide can be (GGGGS)3 (SEQ ID NO:241) or (GGGGS)4 (SEQ ID NO:242). Additional connecting peptides can include GGGGSGGGGSGGGG (SEQ ID NO:243). In embodiments where the artificial terminal portion includes multiple connecting peptides, each connecting peptide can be the same or different.

[0112] Molecular Orientation The Act-IL-18 polypeptides provided herein may have a variety of orientations. In some embodiments, the Act-IL-18 polypeptides provided herein have an orientation according to any one of the following formulas: (a) IL-18-CS, (b) IL-18-LP1-CS, (c) IL-18-LP1-CS-LP2-BM, (d) IL-18-CS-LP1-BM, (e) IL-18-CS-BM; (f) IL-18-LP1-CP-BM, (g) BM-CS-IL-18, (h) BM-LP1-CS-IL-18, (i) BM-LP1-CS-LP2-IL-18, (j) BM-CS-LP1-IL-18, (k) CS-LP1-IL-18, and (l) CS-IL-18, where IL-18 is the IL-18 polypeptide, CS is the portion containing the specific cleavage site (e.g., a protease recognition sequence), LP1 is the first connecting peptide, LP2 is the second connecting peptide, BM is the blocking moiety, and the orientation is shown from N-terminus to C-terminus.

[0113] In some embodiments, the Act-IL-18 polypeptide comprises an orientation of formula (a). In some embodiments, the Act-IL-18 polypeptide comprises an orientation of formula (b). In some embodiments, the Act-IL-18 polypeptide comprises an orientation of formula (c). In some embodiments, the Act-IL-18 polypeptide comprises an orientation of formula (d). In some embodiments, the Act-IL-18 polypeptide comprises an orientation of formula (e). In some embodiments, the Act-IL-18 polypeptide comprises an orientation of formula (f). In some embodiments, the Act-IL-18 polypeptide comprises an orientation of formula (g). In some embodiments, the Act-IL-18 polypeptide comprises an orientation of formula (h). In some embodiments, the Act-IL-18 polypeptide comprises an orientation of formula (i). In some embodiments, the Act-IL-18 polypeptide comprises an orientation of formula (j). In some embodiments, the Act-IL-18 polypeptide comprises an orientation of formula (k). In some embodiments, the Act-IL-18 polypeptide comprises an orientation of formula (l).

[0114] IL-18 Polypeptides The Act-IL-18 polypeptides herein include IL-18 polypeptides. The IL-18 polypeptides of the present disclosure may include some modifications relative to WT IL-18 (SEQ ID NO: 1), including, but not limited to, amino acid substitutions, deletions, additions, or attachments of polymer moieties.

[0115] In some embodiments, the IL-18 polypeptide comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:1. In some embodiments, the IL-18 polypeptide comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to a sequence represented by any one of SEQ ID NOs:2-67. In some embodiments, the IL-18 polypeptide comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to a sequence represented by any one of SEQ ID NOs:79-83. In some embodiments, the IL-18 polypeptide comprises an amino acid sequence represented by SEQ ID NO:30. In some embodiments, the IL-18 polypeptide comprises an amino acid sequence represented by SEQ ID NO:79. In some embodiments, the IL-18 polypeptide comprises the amino acid sequence represented by SEQ ID NO: 80. In some embodiments, the IL-18 polypeptide comprises the amino acid sequence represented by SEQ ID NO: 81. In some embodiments, the IL-18 polypeptide comprises the amino acid sequence represented by SEQ ID NO: 82. In some embodiments, the IL-18 polypeptide comprises the amino acid sequence represented by SEQ ID NO: 83.

[0116] Amino acid substitutions in IL-18 polypeptides In some embodiments, the IL-18 polypeptides of the Act-IL-18 polypeptides described herein include polypeptides that contain at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or more amino acid substitutions at one or more amino acid residues, where the positions of the substitutions are relative to the positions in IL-18 of SEQ ID NO: 1. In some embodiments, the IL-18 polypeptides contain from 1 to 9 amino acid substitutions. In some embodiments, the Act-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 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. In some embodiments, the IL-18 polypeptide further comprises additional substitutions of a synthetic IL-18 polypeptide (e.g., homoserine substitutions, norleucine substitutions, O-methyl-homoserine substitutions, other non-natural or modified amino acids). It is expressly contemplated that these substitutions may be included in addition to the substitutions provided in this section (e.g., a synthetic IL-18 polypeptide may comprise 1-9 amino acid substitutions discussed above and additional synthetic IL-18 amino acid substitutions).

[0117] In some embodiments, the 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, E69X, K70X, C76X, E85X, M86X, T95X, D98X, and C127X, wherein each X is independently a natural or unnatural amino acid. In some embodiments, the 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, E85C, M86C, T95C, D98C, C127A, and C127S.

[0118] In one aspect, described herein are Act-IL-18 polypeptides, including modified IL-18 polypeptides comprising E06K and K53A, where the numbering of residue positions of 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, where 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.

[0119] In some embodiments, the modified IL-18 peptide comprises at least one modification to the amino acid sequence of SEQ ID NO: 1, the modification being 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 to the amino acid sequence of SEQ ID NO: 1, the modifications being 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 to the amino acid sequence of SEQ ID NO: 1, the modifications being E06X, K53X, and S55X, or E06X, K53X, and T63X, where X is a natural or unnatural amino acid. In some embodiments, the 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, comprising E06X, K53X, S55X, and Y01X, comprising E06X, K53X, S55X, and F02X, comprising E06X, K53X, S55X, and D54X, 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.

[0120] 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, the modification being 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, the modifications comprising E06K and K53A, comprising E06K and S55A, comprising K53A and S55A, comprising E06K and T63A, or comprising 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, the modifications comprising E06K, K53A, and S55A, comprising E06K, V11I, and K53A, comprising E06K, C38A, and K53A, or comprising 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 include E06K, K53A, C38S, C68S, C76S, and C127S, or the modifications include K53A, T63A, C38S, C68S, C76S, and C127S. In some embodiments, the modified IL-18 polypeptide comprises at least seven modifications relative to the sequence of SEQ ID NO:1, the seven modifications include E6K, V11I, C38A, K53A, T63A, C76A, and C127A. 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 include Y01G, F02A, E06K, M51G, K53A, D54A, S55A, and T63A.In some embodiments, the modified IL-18 peptide comprises at least eight additional modifications to the amino acid sequence of SEQ ID NO:1, the modifications including Y01G, F02A, E06K, M51G, K53A, D54A, S55A, and T63A.

[0121] 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 that is 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 that is 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 one, two, three, four, five, or six 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, and 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.

[0122] The IL-18 polypeptide of the Act-IL-18 polypeptide described herein may include one or more non-standard amino acids. A "non-standard" amino acid can refer to an amino acid residue in D or L form that is not included among the 20 standard amino acids commonly incorporated into naturally occurring proteins. In some embodiments, one or more amino acids of an active Act-IL-18 polypeptide are substituted with one or more non-standard amino acids. Non-standard amino acids include, but are not limited to, N-alpha-(9-fluorenylmethyloxycarbonyl)-L-azidolysine (Fmoc-L-Lys(N3)-OH), N-alpha-(9-fluorenylmethyloxycarbonyl)-L-biphenylalanine (Fmoc-L-Bip-OH), and N-alpha-(9-fluorenylmethyloxycarbonyl)-O-benzyl-L-tyrosine (Fmoc-L-Tyr(Bzl)-OH.

[0123] Exemplary non-standard amino acids include azido-lysine (AzK), hydroxylysine, allo-hydroxylysine, ε-N,N,N-trimethyllysine, ε-N-acetyllysine, 5-hydroxylysine, Fmoc-Lys(Me,Boc), Fmoc-Lys(Me)3, Fmoc-Lys(palmitoyl), Fmoc-L-photo-lysine, DL-5-hydroxylysine, HL-photo-lysine, and / or other similar amino acids. Exemplary non-standard amino acids also include D-methionine, selenocysteine, and / or other similar amino acids.

[0124] Exemplary non-standard amino acids include p-acetyl-L-phenylalanine, p-iodo-L-phenylalanine, p-methoxyphenylalanine, O-methyl-L-tyrosine, p-propargyloxyphenylalanine, p-propargyl-phenylalanine, L-3-(2-naphthyl)alanine, 3-methyl-phenylalanine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, tri-O-acetyl-GlcNAcp-serine, L-dopa, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, p-boronophenylalanine, O-propargyltyrosine, L-phosphoserine, phosphonoserine, phosphonotyrosine, p-bromo Also included are phenylalanine, 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, alkyl, aryl, acyl, azido, cyano, halo, hydrazine, hydrazide, hydroxyl, alkenyl, alkynyl, ether, thiol, sulfonyl, seleno, ester, thioacid, boric acid, boronic acid, phospho, phosphono, phosphine, heterocyclic, enone, imine, aldehyde, hydroxylamine, keto, or amino substituted 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 non-standard amino acids are selected from β-amino acids, homoamino acids, cyclic amino acids, and amino acids with derivatized side chains. In some embodiments, the non-standard amino acid is β-alanine, β-aminopropionic acid, piperidinic acid, aminocaprioic acid, aminoheptanoic acid, aminopimelic acid, desmosine, diaminopimelic acid, N α -Ethylglycine, N α -Ethylaspargine, isodesmosine, allo-isoleucine, ω-methylarginine, N α -Methylglycine, Nα -Methylisoleucine, N α -Methylvaline, γ-carboxyglutamic acid, O-phosphoserine, N α -Acetylserine, N α -formylmethionine, 3-methylhistidine, and / or other similar amino acids.

[0125] In some embodiments, amino acid residues of an Act-IL-18 polypeptide are substituted with modified lysine residues. In some embodiments, the modified lysine residues include amino, azido, allyl, ester, and / or amide functional groups. In some embodiments, the modified lysine residues contain conjugation handles that can serve as useful anchor points for attaching additional moieties to the activated Act-IL-18 polypeptide. In some embodiments, the modified lysine residues have a structure constructed from the following precursor structure 1, structure 2, structure 3, or structure 4: [ka] In some embodiments, the Act-IL-18 polypeptide contains a substitution of a modified amino acid residue 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 Act-IL-18 polypeptide (e.g., polymers). The substitution can be with a naturally occurring amino acid (e.g., aspartic acid, cysteine, glutamic acid, lysine, serine, threonine, or tyrosine) that is more suitable for attachment of additional functional groups, a modified derivative of any naturally occurring amino acid, or any non-natural amino acid (e.g., an amino acid containing a desired reactive group, such as a click chemistry reagent such as an azide, an alkyne, etc.). Non-limiting examples of such modified amino acid residues include the modified lysine, glutamic acid, aspartic acid, and cysteine ​​provided below: [ka] , and [ka] where each n is an integer from 1 to 30. These non-limiting examples of modified amino acid residues may be used at any position where it is desirable to add an additional functionality (e.g., a polymer) to the Act-IL-18 polypeptide.

[0126] In some embodiments, any of structures 1-4 above, modified lysine, modified glutamic acid, modified aspartic acid, or modified cysteine ​​may be substituted at different residues of the Act-IL-18 polypeptide (e.g., either residues 68-70 or residues 80-100 using SEQ ID NO: 1 as a reference sequence) to allow for attachment at different sites on the IL-18 polypeptide. The azide functionality may also be replaced with another suitable conjugation handle.

[0127] 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 cycloadditions), Staudinger ligation, inverse electron demand Diels-Alder (IEDDA) reactions, "photoclick" chemistry, tetrazine cycloadditions with trans-cyclooctene, potassium acyltrifluoroborate (KAT) ligation, or metal-mediated processes such as olefin metathesis and Suzuki-Miyaura or Sonogashira cross-couplings.

[0128] 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]nona-4-yn-9-ylmethanol-containing reagents, dibenzocyclooctyne-amine reagents, difluorocyclooctyne, or derivatives thereof).

[0129] 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 moieties are provided 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.

[0130] In some embodiments, the group attached to the Act-IL-18 polypeptide (e.g., polymer moiety or additional polypeptide) comprises a conjugation handle or a reaction product of the conjugation handle and a complementary conjugation handle. In some embodiments, the reaction product of the conjugation handle and the complementary conjugation handle results from KAT ligation (reaction of potassium acyl trifluoroborate with hydroxylamine), Staudinger ligation (reaction of azide with phosphine), tetrazine cycloaddition (reaction of tetrazine with trans-cyclooctene), or Huisgen cycloaddition (reaction of alkyne with azide). In some embodiments, the group attached to the IL-18 polypeptide (e.g., polymer or additional polypeptide) will comprise a reaction product of the conjugation handle and the complementary conjugation handle used to attach the group to the Act-IL-18 polypeptide.

[0131] Polymers bound to Act-IL-18 polypeptides In one aspect, described herein is a polypeptide comprising an Act-IL-18 polypeptide, wherein the Act-IL-18 polypeptide comprises a covalently attached polymer. In some embodiments, described herein is an Act-IL-18 polypeptide comprising one or more polymers covalently attached to the Act-IL-18 polypeptide. In some embodiments, the described Act-IL-18 polypeptide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more polymers covalently attached to the Act-IL-18 polypeptide.

[0132] In some embodiments, the polymer comprises a conjugation handle that can be used to further attach additional moieties to the Act-IL-18 polypeptide (e.g., adding additional polypeptides, e.g., antibodies). Any suitable reactive group that can react with a complementary reactive group attached to another moiety can be used as a conjugation handle. In some embodiments, the conjugation handle comprises Cu(I)-catalyzed or "copper-free" reagents for 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, potassium acyltrifluoroborate (KAT) ligation, or metal-mediated processes such as olefin metathesis and Suzuki-Miyaura or Sonogashira cross-coupling.

[0133] In some embodiments, the IL-18 polypeptide comprises a polymer attached to a residue of the IL-18 polypeptide. In some embodiments, the polymer is attached to any one of residues 30-157 of the IL-18 polypeptide. In some embodiments, the polymer is covalently attached to a residue selected from residues 38, 68, 69, 70, 76, 78, 85, 86, 95, 98, 121, 127, and 144 of the IL-18 polypeptide. In some embodiments, the polymer is covalently attached to a residue selected from residues 68, 69, 70, 85, 86, 95, and 98 of the IL-18 polypeptide. In some embodiments, the polymer is covalently attached at residue 68 of the IL-18 polypeptide. In some embodiments, the polymer is covalently attached at residue 68 of the IL-18 polypeptide. In some embodiments, the polymer is covalently attached at residue 69 of the pro-IL-18 polypeptide. In some embodiments, the polymer is covalently attached at residue 70 of the IL-18 polypeptide. In some embodiments, the polymer is covalently attached at residue 85 of the IL-18 polypeptide. In some embodiments, the polymer is covalently attached at residue 86 of the IL-18 polypeptide. In some embodiments, the polymer is covalently attached at residue 98 of the IL-18 polypeptide.

[0134] 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 non-natural amino acid at residue 85.

[0135] 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 non-natural amino acid at residue 86.

[0136] 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 non-natural amino acid at residue 95.

[0137] 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.

[0138] In one aspect, provided herein is an Act-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 residue positions of the Act-IL-18 polypeptide is based on SEQ ID NO: 1 as a reference sequence. In some embodiments, the Act-IL-18 polypeptide comprises an amino acid sequence that is 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 Act-IL-18 polypeptide comprises an amino acid sequence of SEQ ID NO: 30. In some embodiments, the Act-IL-18 polypeptide comprises an amino acid sequence that is 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 Act-IL-18 polypeptide comprises an amino acid sequence of SEQ ID NO: 59. In some embodiments, the Act-IL-18 polypeptide further comprises an amino acid substitution at one or more cysteine ​​residues. In some embodiments, the Act-IL-18 polypeptide comprises one or more cysteines substituted with either serine or alanine. In some embodiments, the Act-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 Act-IL-18 polypeptide comprises a polymer covalently attached to an amino acid residue.

[0139] Active IL-18 polypeptide In some embodiments, cleavage of the specific cleavage site of the artificial end portion converts Act-IL-18 into an active IL-18 polypeptide. As used herein, an active IL-18 polypeptide refers to a truncated form of an IL-18 polypeptide that has some or all of the activity associated with the IL-18 polypeptide that is inactivated by the artificial end portion. Furthermore, unless the context clearly indicates otherwise, a reference simply to an "IL-18 polypeptide" refers to an IL-18 polypeptide that was never prepared in an activatable form (e.g., it refers to the original IL-18 polypeptide on which the Act-IL-18 polypeptide is based). In some embodiments, an active IL-18 polypeptide includes a portion of the artificial end portion that is still attached to the IL-18 polypeptide (e.g., a subset of amino acid residues of the artificial end portion). In some embodiments, an active IL-18 polypeptide is the same as the IL-18 polypeptide (e.g., has the same amino acid sequence as the IL-18 polypeptide because the entire artificial end portion has been truncated).

[0140] In some embodiments, the activated IL-18 polypeptides provided herein exhibit reduced binding to IL-18 binding protein (IL-18BP) compared to WT-IL-18. The activated IL-18 polypeptides may also exhibit binding properties for IL-18Rαβ that differ from wild-type IL-18 (e.g., higher affinity for IL-18Rαβ heterodimer or lower affinity for IL-18Rαβ heterodimer). In preferred embodiments, the affinity of the activated IL-18 polypeptide for IL-18Rαβ is not substantially lower than that of WT IL-18 for IL-18Rαβ (e.g., the affinity of the activated Act-IL-18 polypeptide for IL-18Rαβ is about 500-fold lower than that of wild-type IL-18).

[0141] In some embodiments, an active IL-18 polypeptide exhibits increased affinity for the IL-18 receptor alpha subunit (IL-18Rα) or IL-18 receptor beta subunit (IL-18Rβ) compared to wild-type IL-18. In some embodiments, an active IL-18 polypeptide has increased affinity for the IL-18Rα / β heterodimer compared to IL-18 WT. In one aspect, an active IL-18 polypeptide described herein has reduced affinity for the IL-18Rα / β heterodimer compared to wild-type IL-18.

[0142] In some embodiments, the binding affinity between an active IL-18 polypeptide and IL-18Rα is the same as or lower than the binding affinity between wild-type IL-18 and IL-18Rα. In some embodiments, the binding affinity between an active 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 an active IL-18 polypeptide and IL-18Rβ is the same as or lower than the binding affinity between wild-type IL-18 and IL-18Rβ. In some embodiments, the binding affinity between an active 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 an active IL-18 polypeptide and IL-18Rα / β heterodimer is the same as or lower than the binding affinity between wild-type IL-18 and IL-18Rα / β heterodimer. In some embodiments, the binding affinity between an active IL-18 polypeptide and the IL-18Rα / β heterodimer is the same as or higher than the binding affinity between wild-type IL-18 and the IL-18Rβ heterodimer.

[0143] In some embodiments, the activated IL-18 polypeptides provided herein exhibit the ability to induce interferon gamma (IFNγ) production after administration to a subject. In some embodiments, the ability 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). An exemplary IL-18 polypeptide exhibiting this characteristic is shown in Figure 5A, which shows a comparison of IFNγ production (ng / mL, y-axis) as a function of concentration (nM, x-axis) of wild-type versus IL-18 polypeptide (mutein).

[0144] In some embodiments, the activated Act-IL-18 polypeptides provided herein also exhibit low-binding IL-18 binding protein (IL-18BP). In some embodiments, the activated IL-18 polypeptides provided herein can induce IFNγ even in the presence of IL-18BP (e.g., the ability of the activated Act-IL-18 polypeptide to induce IFNγ is not substantially inhibited by the presence of IL-18BP) (nM, x-axis). An example of an IL-18 polypeptide with this property compared to wild-type IL-18 is shown in FIG. 5B, which shows IFNγ production (ng / mL, y-axis) as a function of IL-18BP concentration (nM, x-axis) in samples treated with the same level of wild-type IL-18 (circles) or IL-18 polypeptide (inverted triangles). In some embodiments, the activated IL-18 polypeptides provided herein exhibit a similar or only slightly reduced ability to induce IFNγ production compared to wild-type IL-18.

[0145] In some embodiments, the activated IL-18 polypeptides provided herein exhibit a significantly reduced inhibition of the ability to induce IFNγ production in the presence of an IL-18BP compared to wild-type IL-18. In some embodiments, the activated IL-18 polypeptides provided herein exhibit a similar or only slightly reduced ability to induce IFNγ production compared to wild-type IL-18, and exhibit a significantly reduced inhibition of the ability to induce IFNγ production in the presence of an IL-18BP compared to wild-type IL-18.

[0146] Biological activities of Act-IL-18 and active IL-18 polypeptide binding affinity In some embodiments, Act-IL-18 exhibits at least 10-fold, at least 100-fold, at least 500-fold, or at least 1000-fold lower affinity for the IL-18 receptor compared to wild-type IL-18 or activated IL-18 polypeptides. In some embodiments, Act-IL-18 exhibits at least 5-fold, at least 10-fold, at least 100-fold, at least 500-fold, or at least 1000-fold higher increased EC for IFN-γ production compared to wild-type IL-18 or activated IL-18 polypeptides. 50 In some embodiments, Act-IL-18 exhibits an increased EC2 activity at least 5-fold, at least 10-fold, at least 100-fold, at least 500-fold, or at least 1000-fold higher for IFN-γ production compared to an activated IL-18 polypeptide. 50 In some embodiments, Act-IL-18 exhibits at least 5-fold, at least 10-fold, at least 100-fold, at least 500-fold, at least 1000-fold higher increased EC for IFN-γ production compared to IL-18 polypeptide. 50 Shows.

[0147] In some embodiments, the activated 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 activated 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 activated IL-18 polypeptides exhibit at least 10-fold lower affinity for IL-18BP compared to WT IL-18. In some embodiments, the activated IL-18 polypeptides exhibit at least 20-fold lower affinity for IL-18BP compared to WT IL-18. In some embodiments, the activated IL-18 polypeptides exhibit at least 50-fold lower affinity for IL-18BP compared to WT IL-18. In some embodiments, an active IL-18 polypeptide exhibits at least an 80-fold lower affinity for IL-18BP compared to WT IL-18. In some embodiments, an active IL-18 polypeptide exhibits at least a 100-fold lower affinity for IL-18BP compared to WT IL-18.

[0148] In some embodiments, the active IL-18 polypeptides provided herein are D In some embodiments, an active IL-18 polypeptide exhibits reduced binding to IL-18BP as measured by K D In some embodiments, an active IL-18 polypeptide has a K with IL-18BP of at least about 1 nM. DIn some embodiments, an active IL-18 polypeptide has a K with IL-18BP of at least about 5 nM. D In some embodiments, an active IL-18 polypeptide has a K with IL-18BP of at least about 50 nM. D In some embodiments, an active IL-18 polypeptide has a K with IL-18BP of at least about 100 nM. D In some embodiments, an active IL-18 polypeptide has a K with IL-18BP of at least about 500 nM. D Shows.

[0149] In some embodiments, an active IL-18 polypeptide exhibits at most a slightly decreased affinity for IL-18Rαβ compared to WT IL-18 (SEQ ID NO: 1). In some embodiments, an active 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, an active 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, an active IL-18 polypeptide exhibits up to 20-fold lower affinity for IL-18Rαβ compared to the affinity of WT IL-18 for IL-18Rαβ. In some embodiments, an active IL-18 polypeptide exhibits up to 50-fold lower affinity for IL-18Rαβ compared to the affinity of WT IL-18 for IL-18Rαβ. In some embodiments, an active IL-18 polypeptide exhibits up to 100-fold lower affinity for IL-18Rαβ compared to the affinity of WT IL-18 for IL-18Rαβ. In some embodiments, an active IL-18 polypeptide exhibits increased affinity for IL-18Rαβ compared to WT IL-18. In some embodiments, the affinity is increased by at least 2-fold, at least 4-fold, at least 6-fold, at least 8-fold, or at least 10-fold compared to WT IL-18.

[0150] In some embodiments, an active IL-18 polypeptide exhibits at most a slightly decreased affinity for IL-18Rαβ compared to a corresponding IL-18 polypeptide that does not include an artificial terminal moiety. In some embodiments, an active 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 a corresponding IL-18 polypeptide that does not include an artificial terminal moiety for IL-18Rαβ. In some embodiments, an active IL-18 polypeptide exhibits at most 2-fold lower affinity for IL-18Rαβ compared to the affinity of a corresponding IL-18 polypeptide that does not include an artificial terminal moiety for IL-18Rαβ. In some embodiments, an active IL-18 polypeptide exhibits up to a 3-fold lower affinity for IL-18Rαβ compared to the affinity of a corresponding IL-18 polypeptide that does not include an artificial terminal portion for IL-18Rαβ. In some embodiments, an active IL-18 polypeptide exhibits up to a 4-fold lower affinity for IL-18Rαβ compared to the affinity of a corresponding wild-type IL-18 polypeptide that does not include an N-terminal domain for IL-18Rαβ. In some embodiments, an active IL-18 polypeptide exhibits up to a 5-fold lower affinity for IL-18Rαβ compared to the affinity of a corresponding IL-18 polypeptide that does not include an artificial terminal portion for IL-18Rαβ. In some embodiments, an active IL-18 polypeptide exhibits up to a 10-fold lower affinity for IL-18Rαβ compared to the affinity of a corresponding IL-18 polypeptide that does not include an artificial terminal portion for IL-18Rαβ.

[0151] In some embodiments, the active IL-18 polypeptides provided herein are DIn some embodiments, an active IL-18 polypeptide exhibits at most a slight reduction in binding to IL-18Rαβ as measured by K D In some embodiments, an active IL-18 polypeptide has a K with IL-18Rαβ of up to about 20 nM. D In some embodiments, an active Act-IL-18 polypeptide has a K with IL-18Rαβ of up to about 30 nM. D In some embodiments, an active IL-18 polypeptide has a K with IL-18Rαβ of up to about 40 nM. D In some embodiments, an active IL-18 polypeptide has a K with IL-18Rαβ of up to about 50 nM. D In some embodiments, the active IL-18 polypeptide has a K D In some embodiments, an active IL-18 polypeptide exhibits increased binding to IL-18Rαβ compared to WT IL-18 as measured by K D is at most about 2 nM, at most about 1 nM, at most about 0.5 nM, or at most about 0.2 nM.

[0152] In some embodiments, an active IL-18 polypeptide exhibits a broad window in which an active IL-18 polypeptide binds to IL-18Rαβ even in the presence of IL-18BP. In some embodiments, this window is greater than or equal to the K D K of Act-IL-18 / IL-18BP interaction against Dand a higher number indicates a larger window during which an active Act-IL-18 polypeptide is predicted to be active in vivo. In some embodiments, an active 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 IL-18 / IL-18Rab interaction. D K of IL-18 / IL-18BP interaction against D In some embodiments, an active 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, an active 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, an active 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, an active 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, an active 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, an active 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, an active 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, an active 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 is shown.

[0153] Functional activity of active IL-18 polypeptides In some embodiments, the active IL-18 polypeptides provided herein exhibit one or more activities associated with WT IL-18. In some embodiments, the active IL-18 polypeptides exhibit a similar ability to signal through the IL-18 receptor (IL-18Rαβ), but lack or exhibit a reduced ability to be inhibited by IL-18BP. In some embodiments, the ability of the active IL-18 polypeptides to signal through IL-18Rαβ is only slightly reduced compared to WT IL-18.

[0154] In some embodiments, an active IL-18 polypeptide modulates IFNγ production when contacted with a cell (e.g., an immune cell such as a NK cell). In some embodiments, the ability of an active IL-18 polypeptide to modulate IFNγ production is measured by measuring the half maximal effective concentration (EC 50 In some embodiments, the ability of an active Act-IL-18 polypeptide to induce IFNγ is measured as the EC 50 (nM) is the EC 50 (nM) of the EC 50 In some embodiments, the EC value of the ability of an active Act-IL-18 polypeptide to induce IFNγ is less than (nM). 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 an active IL-18 polypeptide to induce IFNγ. 50 is measured by an IFNγ-induced cellular assay.

[0155] In some embodiments, the EC of an active IL-18 polypeptide for its ability to induce IFNγ production is 50 In some embodiments, the EC of an active IL-18 polypeptide 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 of an active IL-18 polypeptide for its ability to induce IFNγ production is less than about 100 nM. 50 In some embodiments, the EC of an active IL-18 polypeptide for its ability to induce IFNγ production is less than about 50 nM. 50 is less than about 10 nM.

[0156] In some embodiments, an active form of IL-18 exhibits a reduced ability to have its IFNγ-inducing activity inhibited by IL-18BP compared to WT IL-18. In some embodiments, an active form of IL-18 exhibits a reduced ability to have its IFNγ-inducing activity inhibited by IL-18BP compared to WT IL-18. 50At 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 the half-maximal inhibitory concentration (IC) of IL-18BP. 50 In some embodiments, active IL-18 is measured by the IC of inhibition of WT IL-18 by IL-18BP. 50 The half-maximal inhibitory concentration (IC) of IL-18BP was at least 100-fold higher than that of 50 In some embodiments, active IL-18 is measured by the IC of inhibition of WT IL-18 by IL-18BP. 50 The half-maximal inhibitory concentration (IC) of IL-18BP was at least 500 times higher than that of 50 In some embodiments, active IL-18 is measured by the IC of inhibition of WT IL-18 by IL-18BP. 50 The half-maximal inhibitory concentration (IC) of IL-18BP was at least 1000 times higher than that of 50 ) is shown.

[0157] In some embodiments, the active IL-18 polypeptide is at a half-maximal effective concentration (EC 50 ) by IL-18BP against 50 ) favorable ratio (IC 50 / EC 50 In some embodiments, the IC 50 / EC 50 The ratio is increased compared to WT IL-18. In some embodiments, the IC 50 / EC 50The 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. 50 / EC 50 The ratio is increased by at least about 10-fold compared to WT IL-18. In some embodiments, the IC 50 / EC 50 The ratio is increased by at least about 100-fold compared to WT IL-18. In some embodiments, the IC 50 / EC 50 The ratio is increased by at least about 500-fold compared to WT IL-18. In some embodiments, the IC 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.

[0158] In some embodiments, the active IL-18 polypeptide regulates IFNγ production and the EC of the active Act-IL-18 polypeptide on IFNγ. 50 (nM) is the EC 50 In some embodiments, the EC of an active Act-IL-18 polypeptide against IFNγ is lower than (nM). 50 (nM) is the EC 50 (nM). In some embodiments, the EC 50 (nM) is the EC 50 (nM). In some embodiments, the EC 50(nM) is the EC 50 Approximately 15-fold lower than (nM).

[0159] Relative activities of activatable and active forms of IL-18 In some embodiments, the activated IL-18 polypeptide (e.g., after cleavage of the specific cleavage site) exhibits enhanced IL-18-related activity compared to an Act-IL-18 polypeptide with the specific cleavage site intact. In some embodiments, the activated IL-18 polypeptide exhibits at least 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1000-fold, 2000-fold, 5000-fold, 10000-fold, 15000-fold, or 20,000-fold enhanced activity over the Act-IL-18 polypeptide. Such activity may include induction of IFNγ production in cells (e.g., immune cells such as NK cells), activation of signal transduction via the IL-18 receptor (e.g., in a reporter assay), or another in vitro or in vivo activity. In some embodiments, an active IL-18 polypeptide exhibits enhanced binding to an IL-18 receptor or a subunit thereof (e.g., an IL-18 receptor alpha subunit) compared to an Act-IL-18 polypeptide (e.g., at least a 10-fold, 20-fold, 50-fold, or 100-fold lower K D (Having).

[0160] In some embodiments, an Act IL-18 polypeptide has a higher half-maximal effective concentration (EC) for IL-18 receptor signaling activity (e.g., in a HEK-Blue reporter assay) than an active IL-18 polypeptide. 50 In some embodiments, an Act IL-18 polypeptide exhibits an EC value at least 1,000-fold higher, 2,000-fold higher, 5,000-fold higher, 10,000-fold higher, 15,000-fold higher, or 20,000-fold higher for IL-18 receptor signaling activity than an activated IL-18 polypeptide. 50In some embodiments, the Act IL-18 polypeptide has an EC value at least 1,000-fold higher than an activated IL-18 polypeptide for IL-18 receptor signaling activity. 50 In some embodiments, the Act IL-18 polypeptide has an EC value at least 5,000-fold higher than an activated IL-18 polypeptide for IL-18 receptor signaling activity. 50 In some embodiments, the Act IL-18 polypeptide has an EC value at least 10,000-fold higher than an activated IL-18 polypeptide for IL-18 receptor signaling activity. 50 In some embodiments, the Act IL-18 polypeptide has an EC value at least 20,000-fold higher than an activated IL-18 polypeptide for IL-18 receptor signaling activity. 50 Shows.

[0161] In some embodiments, the Act-IL-18 polypeptide exhibits slightly reduced activity compared to an activated IL-18 polypeptide. In some embodiments, the Act-IL-18 polypeptide exhibits a half-maximal effective concentration (EC) that is about 10-fold to about 100-fold higher than an activated IL-18 polypeptide for IL-18 receptor signaling activity. 50 In some embodiments, the Act IL-18 polypeptide has an EC value that is about 10-fold to about 50-fold higher than that of an activated IL-18 polypeptide with respect to IL-18 receptor signaling activity. 50 Shows.

[0162] In some embodiments, an active IL-18 polypeptide has comparable activity compared to the activity of the IL-18 polypeptide from which the Act-IL-18 polypeptide is derived. In some embodiments, an active IL-18 polypeptide has a half maximal effective concentration (EC) within about 10-fold of an IL-18 polypeptide for IL-18 receptor signaling activity. 50 ) is shown.

[0163] III. Pharmaceutical Compositions In one aspect, described herein is a pharmaceutical composition comprising an activated Act-IL-18 polypeptide provided herein and a pharma- ceutically acceptable carrier or excipient. In some embodiments, the pharmaceutical composition comprises a plurality of Act-IL-18 polypeptides. In some embodiments, the pharmaceutical composition further comprises one or more excipients selected from sugars, inorganic salts, antioxidants, surfactants, or buffers.

[0164] 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.

[0165] In some embodiments, the pharmaceutical composition comprises an inorganic salt. In 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.

[0166] In certain embodiments, the pharmaceutical composition comprises an antioxidant, hi certain embodiments, the antioxidant is 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.

[0167] 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.

[0168] In certain embodiments, the pharmaceutical composition comprises a buffering agent. In certain embodiments, the buffering agent is selected from the group consisting of citric acid, sodium phosphate, potassium phosphate, acetic acid, ethanolamine, histidine, amino acids, tartaric acid, succinic acid, fumaric acid, lactic acid, Tris, HEPES, CHAPS, or combinations thereof.

[0169] 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.

[0170] In one aspect, described herein is a liquid or lyophilized composition comprising a described Act-IL-18 polypeptide. In some embodiments, the Act-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 a phosphate salt. In some embodiments, the phosphate salt is sodium Na2HPO4. In some embodiments, the salt is sodium chloride. In some embodiments, the buffer comprises a 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.

[0171] The Act-IL-18 polypeptides described herein may be in a variety of dosage forms. In some embodiments, the Act-IL-18 polypeptides are administered as a lyophilized powder. In some embodiments, the Act-IL-18 polypeptides are administered as a suspension. In some embodiments, the Act-IL-18 polypeptides are administered as a solution. In some embodiments, the Act-IL-18 polypeptides are administered as an injectable solution. In some embodiments, the Act-IL-18 polypeptides are administered as an IV solution.

[0172] IV.Host cells In one aspect, described herein is a host cell that expresses an Act-IL-18 polypeptide.

[0173] In one aspect, described herein is a method of producing an Act-IL-18 polypeptide, the method comprising expressing an Act-IL-18 polypeptide in a host cell.

[0174] 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.

[0175] V. 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 an Act-IL-18 polypeptide or a pharmaceutical composition described herein.

[0176] In another aspect, described herein is an Act-IL-18 polypeptide provided herein for use in treating cancer in a subject in need thereof. In another aspect, described herein is an Act-IL-18 polypeptide provided herein for the manufacture of a medicament for treating cancer in a subject in need thereof.

[0177] 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, childhood 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.

[0178] In some embodiments, the cancer is a blood cancer. In some embodiments, the cancer is a blood cancer. In some embodiments, the blood cancer is leukemia, non-Hodgkin's lymphoma, Hodgkin's lymphoma, AIDS-related lymphoma, multiple myeloma, plasmacytoma, post-transplant lymphoproliferative disorder, or Waldenstrom's macroglobulinemia.

[0179] In some embodiments, the Act-IL-18 polypeptide is administered in a single dose of an effective amount of the Act-IL-18 polypeptide, including further embodiments in which (i) the Act-IL-18 polypeptide is administered once a day, or (ii) the Act-IL-18 polypeptide is administered to the subject multiple times during the day. In some embodiments, the Act-IL-18 polypeptide is administered daily, every other day, 3 times a week, once a week, twice a week, every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, every 6 weeks, every 12 weeks, every 3 days, every 4 days, every 5 days, every 6 days, twice a week, 3 times a week, 4 times a week, 5 times a week, 6 times a week, once a month, twice a month, 3 times a month, 4 times a month, once every 2 months, once every 3 months, once every 4 months, once every 5 months, or once every 6 months.

[0180] In some embodiments, the method further comprises reconstituting the Act-IL-18 polypeptide or pharmaceutical composition in lyophilized form. In some embodiments, the Act-IL-18 polypeptide or pharmaceutical composition is reconstituted prior to administration. In some embodiments, the composition is reconstituted immediately prior to administration, up to about 5 minutes prior to administration, up to about 20 minutes prior to administration, up to about 40 minutes prior to administration, up to 1 hour prior to administration, or up to about 4 hours prior to administration.

[0181] Exemplary IL-18 Sequences The sequences provided in the table below represent exemplary IL-18 polypeptides that may be part of the Act-IL-18 polypeptides provided herein. In some embodiments, the IL-18 polypeptide of the Act-IL-18 polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence represented by any one of SEQ ID NOs: 1-67. In some embodiments, the IL-18 polypeptide of the Act-IL-18 polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence represented by any one of SEQ ID NOs: 79-83. In some embodiments, the IL-18 polypeptide of the Act-IL-18 polypeptide comprises an amino acid sequence represented by any one of SEQ ID NOs: 1-67. In some embodiments, the Act-IL-18 polypeptide comprises an amino acid sequence represented by SEQ ID NO: 30. In some embodiments, the IL-18 polypeptide of the Act-IL-18 polypeptide comprises an amino acid sequence represented by any one of SEQ ID NOs: 79-83. In some embodiments, the IL-18 polypeptide of the Act-IL-18 polypeptide comprises an amino acid sequence represented by SEQ ID NO: 79. In some embodiments, the IL-18 polypeptide of the Act-IL-18 polypeptide comprises an amino acid sequence represented by SEQ ID NO: 80. In some embodiments, the IL-18 polypeptide of the Act-IL-18 polypeptide comprises an amino acid sequence represented by SEQ ID NO: 81. In some embodiments, the IL-18 polypeptide of the Act-IL-18 polypeptide comprises an amino acid sequence represented by SEQ ID NO: 82. In some embodiments, the IL-18 polypeptide of the Act-IL-18 polypeptide comprises an amino acid sequence represented by SEQ ID NO: 83.

[0182] [Table 7] TIFF2025508824000019.tif236159TIFF2025508824000020.tif232159TIFF2025508824 000021.tif230159TIFF2025508824000022.tif233159TIFF2025508824000023.tif23615 9TIFF2025508824000024.tif228159TIFF2025508824000025.tif236159TIFF2025508824 000026.tif231159TIFF2025508824000027.tif231159TIFF2025508824000028.tif69159

[0183] Additional Exemplary IL-18 Constructs Also provided herein are IL-18 polypeptides that include modifications relative to SEQ ID NO:1 listed in the table below, each of which has been assigned a composition ID. In some embodiments, the IL-18 polypeptide of the Act-IL-18 polypeptide includes the set of amino acid substitutions shown for any one of the constructs depicted below. In the constructs depicted below, each of the substitutions is listed using SEQ ID NO:1 as a reference sequence. In some embodiments, the IL-18 polypeptide of the Act-IL-18 polypeptide includes only the substitutions shown for the constructs below relative to SEQ ID NO:1 (i.e., the IL-18 polypeptide has only the set of substitutions shown, with the remaining residues being those represented in SEQ ID NO:1).

[0184] [Table 8] [Table 9] TIFF2025508824000031.tif237159TIFF2025508824000032.tif237159TIFF2025508824000033.tif24159

[0185] Although the present disclosure and its advantages have been described in detail, it should be understood that various changes, substitutions, and alterations could be made therein without departing from the spirit and scope of the present disclosure, as defined in the appended claims.

[0186] The present disclosure is further illustrated in the following examples, which are provided for illustrative purposes and are not intended to limit the present disclosure in any way. EXAMPLES

[0187] Example 1 – Expression and purification of recombinant Act-IL18 The recombinant Act-IL-18 variants provided herein can be prepared according to the protocols provided below.

[0188] Soluble His-SUMO-IL18 variants E. coli BL21(DE3) harboring a plasmid encoding an N-His-SUMO tagged IL-18 variant fusion is inoculated into 3 L of LB medium and induced with 0.4 mM IPTG for 6 h at 30 °C. Cells are pelleted and cell lysis is performed by sonication in lysis buffer: PBS, pH 7.4. Soluble protein is purified with Ni-NTA beads 6FF (wash 1 with PBS, 20 mM imidazole, pH 7.4, wash 2 with PBS, 50 mM imidazole, pH 7.4, elution with PBS, 500 mM imidazole, pH 7.4). The fractions containing the protein are pooled and dialyzed into PBS pH 7.4, followed by SUMO digestion.The protein is then purified in two steps by Ni-NTA beads (continuing the flow-through sample) and gel filtration.The fractions containing the protein are pooled and QC is performed using analytical techniques such as SDS-PAGE and analytical SEC.

[0189] Non-soluble His-SUMO-IL18 variants E. coli BL21(DE3) harboring a plasmid encoding an N-His-SUMO tagged IL-18 variant fusion is inoculated into 10 L of LB medium and induced with 0.4 mM IPTG for 6 h at 30 °C. Cells are pelleted and cell lysis is performed by sonication in lysis buffer: PBS, 8 M urea, pH 7.4. Proteins are purified with Ni-NTA beads 6FF (wash 1 with PBS, 8 M urea, 20 mM imidazole, pH 7.4, wash 2 with PBS, 8 M urea, 50 mM imidazole, pH 7.4, elution with PBS, 8 M urea, 500 mM imidazole, pH 7.4).

[0190] The fractions containing the protein are pooled and dialyzed into PBS pH 7.4, followed by SUMO digestion.The protein is then purified with Ni-NTA beads (column equilibrated with PBS, 8M urea, pH 7.4, washed with PBS, 8M urea, pH 7.4, eluted with PBS, 8M urea, pH 7.4).The fractions containing the protein are pooled and dialyzed into PBS pH 7.4, and QC is performed using analytical techniques such as SDS-PAGE and analytical SEC.

[0191] Insoluble, untagged IL18 variant E. coli BL21(DE3) carrying a plasmid encoding mIL-18 are inoculated into 2 L of LB medium and induced with 0.4 mM IPTG for 6 h at 30° C. Cells were pelleted and cell lysis was performed by sonication in lysis buffer: 110 mM Tris, 1.1 M guanidine HCl, 5 mM DTT, pH 8.9. Protein purified via Q Sepharose FF (equilibration buffer 20 mM MES, pH 7.0, elution with an increasing gradient from 0 to 1 M NaCl).

[0192] Bicistronic system Use a single colony of E. coli BL21 containing the plasmid (e.g., SEQ ID NO:59) as an inoculum for 10 mL of LB containing 25 μg / mL kanamycin sulfate and incubate overnight at 37 °C and 200 rpm. Use 1 mL of the preculture to inoculate 1 L of autoinducing terrific broth containing 100 μg / mL kanamycin sulfate. Incubate the culture at 37 °C and 110 rpm for 4 h, then transfer to 15 °C and incubate for an additional 15 h. Resuspend the cells in 10-15 mL of lysis buffer (100 mM HEPES, 1 mM EDTA, 5 mM DTT, 20 μg / mL lysozyme, 0.1 mg / mL DNase I, 1 mM PMSF, pH 7.5) and gently shake at 4 °C for 1 h. The cells are then lysed by sonication and the soluble protein fraction is obtained by centrifugation (16'000 xg, 30 min, 4°C) and filtration (0.2 μm membrane).

[0193] The supernatant was adjusted to approximately pH 7 and loaded onto a tandem column system (2x SP CIEX + 1x HiPrep DEAE FF 16 / 10, all from cytiva) using a 50 mL superloop (loading less than 30 mL of lysate per run). The system was run with wash buffer (25 mM HEPES, 1 mM EDTA, 5 mM DTT, pH 7.0) and fractions containing protein (second main peak) were collected and pooled.

[0194] The tandem columns are separated into their respective types. The DEAE column was eluted with a step gradient of buffers E1 and E2 (25 mM Bis-Tris propane HCl, pH 9.5 and 25 mM Bis-Tris propane HCl, 1 M NaCl, pH 9.5, respectively). First, 100% E1 was run for 8 CV, followed by a gradient of 0% to 12% E2 in 5 CV, after which the gradient was maintained at 12% for another 10 CV. This was followed by a gradient of 12% to 40% E2 in 5 CV, maintaining the gradient at 40% for another 5 CV. The fractions containing the protein (second main peak) were collected and pooled with the previous fractions. The SP column was washed in the same way and discarded, as there should be no protein in this eluate.

[0195] The pooled sample is adjusted to pH 9.5 and loaded onto a Mono Q (small scale) or Hitrap Q (large scale) column. The buffers used are E2 and E3 (25 mM Bis-Tris propane HCl, 1.5 M ammonium sulfate, pH 9.5). The stepwise elution gradient starts with 8% E3 in 15 CV, increases to 16% E3 in 5 CV, and 50% E3 in 3 CV. The fractions containing the protein are found in the second main peak.

[0196] The fractions containing the target protein are pooled and concentrated by diafiltration (10 kDa MWCO, less than 3500×g, 4° C.). The concentrated sample is loaded onto a Superdex 75 equilibrated with buffer (20 mM potassium phosphate, 150 mM KCl, 1 mM DTT, pH 6.0). The fractions containing the target protein are collected, pooled and concentrated.

[0197] Example 1B – Expression and purification of selected activatable IL-18 polypeptides An activatable IL-18 candidate with an N-terminal propeptide attached was prepared according to the method provided below.

[0198] 1.1 - Expression of activatable IL-18 candidates Activatable IL-18 candidates were generated as N-terminal fusions to N-His-SUMO-IL18. The genes were synthesized and cloned into plasmids. The plasmids were transformed into E. coli BL21(DE3). Expression was performed in shake flasks containing TB medium. Cells were grown at 37°C until they reached an OD600 of approximately 1.2, after which they were induced with 0.1 mM IPTG and cultured at 18°C ​​for an additional 20 hours. Cells were harvested by centrifugation.

[0199] 1.2 - Purification of activatable IL-18 candidates 1.2.1 - Cell lysis Cells were resuspended in lysis buffer (20 mM Tris / HCl, pH 8.0, 0.15 M NaCl, 10 mM imidazole, and 1 tablet of EDTA-free complete protease inhibitor (Roche, COEDTAF-RO) per liter of production) at 100 mL of buffer per liter of culture and disrupted twice with a homogenizer at 1000 bar. Debris was removed by centrifugation twice at 40'000g for 45 min with flask changes between each centrifugation, followed by filtration through a 0.22 μm filter.

[0200] 1.2.2 - Affinity purification and endotoxin removal The lysate was loaded at 5 mL / min onto Ni NTA resin (Cytiva, 17524802) pre-equilibrated with 20 mM Tris / HCl, pH 8.0, 0.15 M NaCl, 10 mM imidazole, and washed with the same buffer for 5 CV. To remove endotoxins, the column was washed at 10 mL / min with 20 mM Tris / HCl, pH 8.0, 0.15 M NaCl, 10 mM imidazole, 0.1% Triton X-114 for 30 CV. The column was washed at 5 mL / min with 20 mM Tris / HCl, pH 8.0, 0.15 M NaCl, 10 mM imidazole for 5 CV, and the protein of interest was eluted by linearly increasing the imidazole concentration. The column was then regenerated with 0.5 M NaOH.

[0201] 1.2.3 - SUMO Digestion and Dialysis To cleave the SUMO tag, SUMO protease was added to the elution pool at a w / w ratio of 1:250 (protein:SUMO enzyme) and incubated for 18 h at 4° C. Concurrently, the protein was dialyzed (20 mM Tris, pH 8.0, 150 mM NaCl) to reduce the imidazole concentration.

[0202] 1.2.4 - Purification by reverse IMAC To remove the cleaved tag and SUMO protease, the digested protein was passed through a Ni NTA resin column pre-equilibrated with 20 mM Tris / HCl, pH 8.0, 0.15 M NaCl, 10 mM imidazole at 5 mL / min. The flow-through was collected.

[0203] The flow-through was concentrated to 2.6 mg / mL and buffer exchanged into either 20 mM HEPES, 150 mM NaCl, 0.5 mM TCEP, 10% glycerol, pH 7.5 or PBS, 10% glycerol, pH 7.4. Protein was stored at -70°C until further quality control.

[0204] Example 1C The Act-IL-18 polypeptide was prepared using a mammalian expression system using methods well known to those skilled in the art.

[0205] Example 2 – Cleavage of activatable IL-18 polypeptides Cleavage of activatable IL-18 polypeptides was performed according to the following protocol: IL-18 candidates (activatable or non-cleavable controls) were incubated with either MMP2 (SIGMA, PF023), MMP7 (SIGMA, CC1059), or MMP9 (SIGMA, PF024) diluted in MMP buffer MMP (25 mM TRIS, 10 mM CaCl2, 0.05% Brij25, pH 7.5) at a final concentration of 1 ug / ml for MMP2 and MMP9, and 10 ug / ml for MMP7. Protein controls without enzyme were incubated under the same conditions. After 20 hours of incubation, samples were collected for SDS gel analysis.

[0206] For candidates that displayed a C-terminal masking domain, an additional washing step with nickel beads was performed to remove the cleaved histidine-tagged masking domain and uncleaved protein. Briefly, the protein and enzyme solution was incubated with an excess of nickel beads (at least 10 uL of dry beads for every 40 ug of expected protein) for 30 min under shaking. The flow-through was collected and bound residues were eluted from the beads by incubation with 20 mM TRIS, 150 mM NaCl, 500 mM imidazole, pH 8.

[0207] Figures 14A-C show SDS-PAGE gels obtained from cleavage experiments performed on the indicated IL-18 molecules. Figure 14A shows the results of digestion of N-terminally masked candidates with MMP2, MMP7, and MMP9, where protease-treated samples are indicated with (+) and untreated samples with (-). Variant C127 showed efficient cleavage by each enzyme, C185 showed moderate cleavage by each enzyme, and C187 showed moderate cleavage by MMP2 and MMP7 but not by MMP9, while the original IL-18 C086 and the uncleavable control C190 remained intact in all conditions. Figure 14B shows the results of digestion (unpurified) of C-terminally masked candidates with MMP2, MMP7, and MMP9, where protease-treated samples are indicated with (+) and untreated samples with (-). Variant C136 showed no cleavage by any enzyme, C137 showed no cleavage by any enzyme, C172 showed efficient cleavage by all enzymes, C173 showed some cleavage by MMP7, C189 showed efficient cleavage by all enzymes, and C191 showed moderate cleavage by MMP2, efficient cleavage by mMP7, and no / minimal cleavage by MMP9. Figure 14C shows the results of digestion of nickel purified flow-through (FT) and eluate (E) of the C-terminally masked candidates (same samples as in Figure 14B).

[0208] Example 3A – HEK-Blue IL18R reporter assay An IL-18Rαβ positive HEK-Blue reporter cell line is used to determine binding of IL-18 variants to IL-18Rαβ and subsequent downstream signaling. A general protocol is outlined below.

[0209] 5 × 104 cells of HEK-Blue IL18R reporter cells (InvivoGen, #hkb-hmil18) are seeded into each well of a 96-well plate and stimulated with 0-100 nM of IL-18 polypeptide variants at 37 °C and 5% CO2. After 20 h of incubation, 20 μL of cell culture supernatant is then taken from each well and mixed with 180 μL of QUANTI-Blue medium in a 96-well plate and incubated for 1 h at 37 °C and 5% CO2. The absorbance signal at 620 nm is then measured in an Enspire plate reader using 680 and 615 nm as excitation and emission wavelengths, respectively. GraphPad PRISM software is used to calculate the half-maximal effective dose (EC50) based on variable slope and four-parameter analysis.

[0210] The Act-IL-18 polypeptides provided herein exhibit reduced or eliminated binding ability to stimulate IFNγ compared to WT IL-18 or IL-18 polypeptides without an artificial terminal portion. After cleavage, the ability to stimulate IFNγ is restored, but may be altered compared to WT IL-18.

[0211] Example 3B - HEK-Blue IL18R Reporter Assay - Results The HEK-Blue IL-18R reporter assay described above was performed on activatable and control IL-18 polypeptides before and after treatment with the indicated MMPs. Activity in the HEK-Blue IL18R assay is shown in the table below. [Table 10]

[0212] Example 3C The above HEK-Blue IL-18R reporter assay was also performed on additional IL-18 polypeptides that can be incorporated into the Act-IL-18 polypeptides provided herein. The IL-18 polypeptides provided below are expected to behave similarly to C086 (SEQ ID NO: 30) when converted to an Act-IL-18 polypeptide as otherwise provided herein. [Table 11] TIFF2025508824000036.tif193159

[0213] Example 4 – IL-18 stability assessment The stability of activatable and control IL-18 polypeptides was assessed using nanodifferential scanning fluorimetry (nanoDSF). Activatable IL-18 polypeptide constructs conjugated with either the propeptide or the IL-18 receptor D3 subunit showed improved stability compared to C086. Conversely, IL-18 polypeptides with short extension peptides attached to the N-terminus showed lower stability. [Table 12]

[0214] In some cases, IL-18 polypeptides were subjected to treatment with MMPs or in MMP buffer without MMPs according to the following general protocol: 100 uL of the indicated IL-18 sample at approximately 1 mg / mL was mixed with 100 uL of MMP-2 at 2 ug / mL in MMP assay buffer (25 mM TRIS, 10 mM CaCl2, 0.05% Brij 25, pH 7.5). Samples were incubated with shaking at 24°C for 16 hours. [Table 13]

[0215] Example 5A –Conjugation of Act-IL-18 Polypeptides The Act-IL-18 polypeptides provided herein are conjugated to a PEG functional group. In some cases, PEG is attached via a bifunctional linker, which is first attached to the desired residue of the Act-IL-18 polypeptide (e.g., E85C, or another suitable naturally occurring cysteine, e.g., C68), or a cysteine ​​residue that is incorporated at the desired site, such as residue 86 or 98). Once attached to the IL-18 polypeptide, the second functional group of the bifunctional linker is used to attach the PEG moiety. An exemplary schematic of such a process is shown in FIG. 6. In some cases, a bifunctional linker as shown in FIG. 6 is not required, since the IL-18 polypeptide already contains the desired conjugation handle for attachment of PEG or other groups. An exemplary protocol for the recombinant IL-18 variants provided herein is described below.

[0216] Conjugation - Recombinant IL-18 is stored at a concentration of 2.4 mg / mL in potassium phosphate buffer (pH 7.0) containing 50 mM KCl and 1 mM DTT at -80°C. The sample is thawed on ice to obtain a clear solution. The protein solution is diluted with PBS, pH 7.4. At a concentration of approximately 0.4 mg / mL, a clear solution is obtained.

[0217] The protein solution is dialyzed against PBS, pH 7.4 (2 times 2 h against 600 mL and 1 time 18 h against 800 mL). After dialysis, a clear solution with no signs of precipitation is obtained. Protein concentration is obtained by BCA protein assay using UV absorbance at 280 nm.

[0218] Prepare a stock solution of bifunctional probe (Bromoacetamido-PEG5-azide, CAS:1415800-37-1) in water at a concentration of 20 mM. Mix 500 μL of protein solution with 25 μL of probe solution. Adjust pH to 7.5 and react at 20 °C for 3 h.

[0219] The progress of the synthesis is monitored by reverse-phase HPLC on an Aeris WIDEPORE C18 200 Å column (3.6 μm, 150 × 4.6 mm) using a gradient of 5 to 30% (2.5 min) and 30 to 75% (7.5 min) of CH3CN containing 0.1% TFA (v / v) at a flow rate of 1 mL / min at 40 °C, and by MALDI-TOF MS.

[0220] Purification - In some cases, ion exchange chromatography is used to purify the conjugated protein. To remove excess probe, the reaction mixture (volume is approximately 500 μL) is loaded onto a Hi-Trap-G-FF-1 mL column with 25 mM Tris (pH 7.4) as buffer. The column is eluted with a linear gradient of 0 to 0.35 M NaCl in the same buffer. Fractions containing the target protein are collected, buffer exchanged (25 mM Tris, pH 7.4, 75 mM NaCl, 5% glycerol) and concentrated at 0.4 mg / mL. The concentration of the purified protein is determined by UV absorbance at 280 nm and BCA protein assay. The protein solution is kept at -80 °C.

[0221] Characterization - The purity and identity of recombinant and conjugated proteins from commercial sources are confirmed by analytical SEC, HPLC and MALDI-TOF MS.

[0222] The Act-IL-18 polypeptide may then be further conjugated to additional groups, such as a polymer or an additional polypeptide.

[0223] Example 5B – PEGylation of Act-IL-18 polypeptide Following conjugation of a bifunctional linker as described in Example 5A and shown in FIG. 7, the Act-IL-18 polypeptide may be covalently linked to a PEG group. An exemplary schematic of this process is shown in FIG. 7. An exemplary protocol for the conjugation reaction between PEG and a suitably activated IL-18 polypeptide is provided below. Additionally, the desired PEG group can be covalently linked to the Act-IL-18 polypeptide using the below protocol that incorporates a conjugation handle directly during the preparation of the Act-IL-18 polypeptide (e.g., during the synthesis of a synthetic IL-18 polypeptide). An exemplary schematic of such a process is shown in FIG. 3. The resulting polymer-linked Act-IL-18 polypeptide is shown in FIG. 4 and is predicted to have an extended half-life.

[0224] Conjugation - Recombinant Act-IL-18 polypeptide comprising SEQ ID NO:59 with an artificial terminal moiety as provided herein is stored at -80°C in PBS (pH 7.4) containing 75 mM NaCl and 5% (v / v) glycerol. Before the PEGylation reaction, the sample is thawed on ice to obtain a clear solution. 200 μL of protein solution (0.4 mg / mL) is mixed with 2.0 mg of DBCO-polyethylene glycol polymer. This is allowed to react overnight at 20°C.

[0225] The progress of the synthesis is monitored by reverse-phase HPLC on an Aeris WIDEPORE C4 200 Å column (3.6 μm, 150 × 4.6 mm) using a gradient of 5 to 30% (2.5 min) and 30 to 75% (7.5 min) of CH3CN containing 0.1% TFA (v / v) at a flow rate of 1 mL / min at 40 °C, and by MALDI-TOF MS.

[0226] Purification - To remove excess PEG, the reaction mixture is diluted with Tris buffer (25 mM, pH 7.4) and loaded onto a Hi-Trap-Q-FF column using 25 mM Tris (pH 7.4) as buffer. The column is eluted with a linear gradient of 0 to 0.35 M NaCl in the same buffer. Fractions containing the target protein are collected, buffer exchanged (25 mM Tris, pH 7.4, 75 mM NaCl, 5% glycerol) and concentrated at 0.04 mg / mL. The concentration of the purified protein is determined by BCA protein assay. The protein solution is kept at -80 °C.

[0227] Characterization - The purity and identity of the conjugated protein is confirmed by HPLC and MALDI-TOF MS.

[0228] Similar protocols can also be used to conjugate an Act-IL-18 polypeptide to another polypeptide, such as a suitable activating antibody.

[0229] Example 6 –Characterization of Act-IL-18 polypeptides The Act-IL-18 polypeptides provided herein are subjected to a series of analytical experiments to characterize the compositions. The Act-IL-18 polypeptides are analyzed by HPLC to determine the degree of homogeneity of the compositions. The Act-IL-18 polypeptide compositions are also analyzed by MALDI-MS to determine the MW and molecular weight distribution of the compositions. Additionally, the Act-IL-18 polypeptide compositions are analyzed by circular dichroism to compare the folding of the Act-IL-18 polypeptide compositions compared to wild-type IL-18.

[0230] Example 7 – Formulation of Act-IL-18 Polypeptides The lyophilized Act-IL-18 polypeptide is suspended in a solution containing 10-50 mM histidine buffer, 5-10% trehalose, and 0.02% tween. The lyophilized Act-IL-18 polypeptide can also be resuspended in other suitable or appropriate buffers, such as PBS (pH 7.4) containing mannitol (e.g., 50 mg / mL) and tween (e.g., 0.02%).

[0231] Example 8 –IL-18 SPR measurements The interaction of wild-type and Act-IL-18 polypeptides with human IL-18 receptor subunits is measured using surface plasmon resonance (SPR) technology. To capture 6 μg / mL of Fc-fused human IL-18Rα, 6 μg / mL of Fc-fused human IL-18Rβ, or 2 μg / mL of Fc-fused human IL-18BP isoform a (IL-18BPa), anti-human IgG antibodies are coupled onto a CM5 chip by amine coupling for 30 minutes before capture. In another setup, 6 μg / mL of alpha and beta IL-18 receptors are mixed and pre-incubated for 30 minutes before capture of the alpha / beta heterodimeric IL-18 receptor.

[0232] Kinetic binding of IL-18 analyte is measured using a Biacore 8K instrument with 2-fold serial dilutions starting at 1 μM down to 0.98 nM. Regeneration of the surface back to amine-coupled anti-IgG antibodies is performed for each concentration of analyte. To measure protein association to the receptor, samples are injected for 60 seconds at a flow rate of 50 μL / min, followed by 300 seconds of buffer only to detect dissociation. The running buffer used is 1xPBS with 0.05% Tween 20. Relative response units (RU, Y-axis) are plotted against time (sec, X-axis) and analyzed with a kinetic 1:1 binding model for monomeric receptor binding and binding to IL-18BP. A kinetic heterogeneous ligand fitting model is applied for alpha / beta heterodimer binding.

[0233] The Act-IL-18 polypeptides provided herein exhibit reduced or eliminated binding to IL-18Rαβ, IL-Rα, and / or IL-18Rβ compared to WT IL-18 or an IL-18 polypeptide without an artificial terminal portion. After cleavage, binding to these IL-18 receptor proteins is restored, but may be altered compared to WT IL-18.

[0234] Example 9 –IL-18BP binding alphaLISA assay To determine the binding affinity of each IL-18 variant to IL-18BP, a human IL-18BP AlphaLISA assay kit was used, which detects the presence of the free form of IL-18BP.

[0235] Sixteen 3-fold serial dilutions of IL-18 analyte are prepared in aMEM medium supplemented with 20% FCS, Glutamax, and 25 μM β-mercaptoethanol in the presence of 5 ng / mL His-tagged human IL-18BP. Final IL-18 analyte concentrations range from 2778 nM to 0.2 pM.

[0236] After 1 hour incubation at room temperature, free IL-18BP levels are measured using a human IFNγ AlphaLISA® assay kit. In a 384-well OPTI plate, 5 μL of 5X anti-IL-18BP acceptor beads are added to 7.5 μL of IL-18 / IL-18BP mix. After 30 minutes incubation at room temperature with shaking, 5 μL of biotinylated anti-IL-18BP antibody is added to each well. The plate is incubated for an additional hour at room temperature. Under reduced light, 12.5 μL of 2X streptavidin (SA) donor beads are pipetted into each well and the wells are incubated for an additional 30 minutes at room temperature with shaking. The AlphaLisa signal is then measured on an Enspire plate reader with excitation and emission wavelengths of 680 and 615 nm, respectively. Dissociation constants (KD) are calculated based on a variable slope, 4-parameter analysis using GraphPad PRISM software.

[0237] The Act-IL-18 polypeptides provided herein may exhibit reduced or eliminated binding to IL-18BPs to which artificial terminal moieties have been attached.

[0238] The above experiments were also performed with IL-18 polypeptides that can be incorporated into the Act-IL-18 polypeptides provided herein to assess their ability to bind IL-18BP. [Table 14] TIFF2025508824000040.tif254159TIFF2025508824000041.tif78159

[0239] Example 10 –IFNγ induction cell assay The ability of the IL-18 polypeptides provided herein is assessed for their ability to induce IFNγ in a cellular assay according to the following protocol.

[0240] The NK cell line NK-92 (ATCC® CRL-2407™), derived from a lymphoma patient, is cultured in aMEM medium supplemented with 20% FCS, Glutamax, 25 μM B-mercaptoethanol, and 100 IU / mL recombinant human IL-2.

[0241] On the day of the experiment, cells are harvested and washed with aMEM medium without IL-2 and containing 1 ng / mL recombinant human IL-12. After counting, cells are seeded at 100,000 cells / well in 384-well titer plates and incubated at 37°C / 5% CO2. Sixteen 4-fold serial dilutions of IL-18 analytes were prepared in aMEM medium and 1 ng / mL IL-12 was added to the NK-92 cells. Final IL-18 analyte concentrations ranged from 56 nM to 5x10-5 pM.

[0242] After incubating the cells for 16-20 hours at 37°C / 5% CO2, carefully transfer 5 μL of the supernatant into a 384-microwell OptiPlate. IFNγ levels are measured using the Human IFNγ AlphaLISA® Assay Kit. Briefly, 10 μL of 2.5X AlphaLISA anti-IFNγ acceptor beads and biotinylated antibody anti-IFNγ mix are added to 5 μL of NK-92 supernatant. The mixture is incubated for 1 hour at room temperature with shaking. Under subdued light, 2.5 μL of 2X streptavidin (SA) donor beads are pipetted into each well and the wells are incubated for 30 minutes at room temperature with shaking. The AlphaLISA signal is then measured in an EnSpire™ plate reader using 680 nm and 615 nm as excitation and emission wavelengths, respectively. GraphPad PRISM software is used to calculate the half-maximal effective concentration (EC50) based on variable slope and four-parameter analysis.

[0243] The Act-IL-18 polypeptides provided herein exhibit reduced or eliminated binding ability to stimulate IFNγ compared to WT IL-18 or IL-18 polypeptides without an artificial terminal portion. After cleavage, the ability to stimulate IFNγ is restored, but may be altered compared to WT IL-18.

[0244] Example 11 –IL-18 binding protein inhibition cell assay The NK cell line NK-92 (ATCC® CRL-2407™), derived from a lymphoma patient, was cultured in aMEM medium supplemented with 20% FCS-Glutamax™, 25 μM B-mercaptoethanol, and 100 IU / mL recombinant human IL-2.

[0245] On the day of the experiment, cells are harvested and washed with aMEM medium without IL-2 and containing 1 ng / mL recombinant human IL-12. After counting, cells are seeded at 100,000 cells / well in 384-well titer plates and incubated at 37°C / 5% CO2. Sixteen two-fold serial dilutions of Fc-fused human IL-18 binding protein isoform a (IL-18BPa) are prepared in aMEM medium. 1 ng / mL IL-12 containing 2 nM of each Act-IL-18 polypeptide variant is added to the NK-92 cells. The final IL-18 analyte concentration was 1 nM, and the final IL-18BPa concentrations ranged from 566 nM to 17 pM.

[0246] After incubating the cells for 16-20 hours at 37°C / 5% CO2, carefully transfer 5 μL of the supernatant into a 384-microwell OptiPlate. IFNγ levels are measured using the Human IFNγ AlphaLISA Assay Kit. Briefly, 10 μL of 2.5X AlphaLISA anti-IFNγ acceptor beads and biotinylated antibody anti-IFNγ mix are added to 5 μL of NK-92 supernatant. The mixture is incubated for 1 hour at room temperature with shaking. Under subdued light, 2.5 μL of 2X SA donor beads are pipetted into each well and the wells are incubated for 30 minutes at room temperature with shaking. The AlphaLISA signal is then measured in an EnSpire™ plate reader using 680 nm and 615 nm as excitation and emission wavelengths, respectively. GraphPad PRISM software is used to calculate half-maximal inhibitory concentrations (IC50) based on variable slope and 4-parameter analysis.

[0247] The Act-IL-18 variants of the present disclosure are active and capable of inducing IFNγ secretion in vitro following cleavage of the artificial terminal portion, but show reduced or no ability to induce IFNγ without cleavage.

[0248] To assess the activity of the modified IL-18 polypeptides and their suitability for transformation into Act-IL-18 polypeptides, the experiments described in Examples 10 and 11 were carried out with the modified IL-18 polypeptides. [Table 15] TIFF2025508824000043.tif191159

[0249] Example 12 –Pharmacokinetic and pharmacodynamic properties of Act-IL-18 polypeptide variants The pharmacokinetic (PK) and pharmacodynamic (PD) properties of selected IL-18 polypeptide variants are measured. Three C57BL / 6 mice are tested per group and time point. IL-18 variants are applied via a single intravenous injection. Mice are divided into four dose groups: 0.5 mg / kg, 0.1 mg / kg, 0.02 mg / kg, 0.004 mg / kg, and four time point groups: 5 min, 6 h, 24 h, 48 h. In a separate experiment, PD and PD are measured as provided for C57BL / 6 mice, but healthy mice are replaced with tumor model mice (e.g., mice bearing MC-38 tumor cell xenografts).

[0250] The immune-related effects of PD are determined by analyzing cytokine levels in plasma. The following plasma cytokines are measured: IFNγ, CXCL9, CXCL10, GM-CSF, IL-1a, FasL, and IL-18BP. The activation state of leukocytes is determined by monitoring surface markers (ICOS, PD-1, CD25, CD69, and Fas). Bioanalysis is performed by detecting the total amount of IL-18 variants (free and IL-18BP complexed). Corning high-binding half-area plates (Fisher Scientific, Reinach, Switzerland) are coated overnight at 4° C. with 25 μl of anti-IL18 monoclonal antibody (MBL, catalog number D043-3, clone 25-2G) (2 μg / ml in PBS). The plates are then washed four times with 100 μl of PBS-0.02% Tween 20. The plate surface is blocked with 25 μl of PBS-0.02% Tween20-1% BSA for 1 hour at 37° C. The plate is then washed 4 times with 100 μl of PBS-0.02% Tween20. 25 microliters of IL-18 variant (or mouse plasma) is added in PBS-0.02% Tween20-0.1% BSA in 8-fold serial dilutions starting at 50 nM and going down to 0.02 nM and incubated for 2 hours at 37° C. The plate is then washed 4 times with 100 μl of PBS-0.02% Tween20 and 25 μl of biotinylated anti-IL18 monoclonal antibody (MBL, catalog number D045-6, clone 159-12B) (2 μg / ml in PBS). The plates are incubated for 2 hours at 37°C and then washed four times with 100 μl of PBS-0.02% Tween 20. Twenty-five microliters of streptavidin-horseradish peroxidase (number RABHRP3, Merck, Buchs, Switzerland) diluted 1:500 in PBS-0.02% Tween 20-0.1% BSA are added to each well and incubated for 30 minutes at room temperature. The plates are then washed four times with 100 μl of PBS-0.02% Tween 20.50 microliters of TMB substrate reagent (number CL07, Merck, Buchs, Switzerland) is added to each well and incubated for 5 minutes at 37° C. After 5 minutes at 37° C., the horseradish peroxidase reaction is stopped by adding 50 μl / well of 0.5 M H2SO4 stop solution. The ELISA signal is then measured at 450 nm in an EnSpire plate reader from Perkin Elmer (Schwerzenbach, Switzerland).

[0251] PK and PD in healthy mice show little IL-18-related activity after administration of Act-IL-18 polypeptide due to the presence of the artificial terminal moiety, although slight effects can be measured due to the presence of endogenous proteases that can cleave the artificial terminal moiety at low background rates. The distribution of active IL-18 and IL-18 activity is not specific to any tissue.

[0252] Conversely, tumor model mice that received Act-IL-18 polypeptides with artificial terminal portions that are preferentially cleaved by tumor-associated proteases exhibit high local levels of both active IL-18 and signs of IL-18 activity within and around the tumor microenvironment, but within the tumor and just outside the area immediately surrounding the tumor.

[0253] Example 13 –PBMC stimulation assay The ability of IL-18 variants to stimulate peripheral blood mononuclear cells (PBMCs) is assessed according to the following protocol.

[0254] Lymphocyte isolation: Blood from buffy coats of healthy volunteers is diluted with an equal volume of PBS and slowly poured onto the top of a SepMate tube pre-filled with 15 mL of Histopaque-1077. The tube is centrifuged at 1200 g for 10 min, the upper layer is collected and washed three times with PBS containing 2% fetal bovine serum. PBMCs are counted and stored frozen as aliquots of 20 x 106 cells.

[0255] Cryopreserved PBMCs are thawed and stimulated with a gradient of human IL-18 variants ranging from 0.2 pM to 1 µM in RPMI containing 10% fetal bovine serum.

[0256] Cytokine production after 24 hours of stimulation is measured by Legendplex (Biolegend #740930) on a multicolor flow cytometer. GraphPad PRISM software is used to calculate the half-maximal effective concentration (EC50) of IFNγ released into the culture supernatant based on variable slope and four-parameter analysis.

[0257] Surface expression of FcγRIII on NK cells is measured by flow cytometry (mouse IgG1 clone 3G8) after 72 hours of stimulation.

[0258] The Act-IL-18 variants of the present disclosure are active and capable of inducing IFNγ secretion in vitro following cleavage of the artificial terminal portion, but show reduced or no ability to induce IFNγ without cleavage.

[0259] Although the present disclosure and its advantages have been described in detail, it should be understood that various changes, substitutions, and alterations could be made therein without departing from the spirit and scope of the present disclosure, as defined in the appended claims.

Claims

1. 1. An activatable interleukin-18 (Act-IL-18) polypeptide comprising: an artificial terminal moiety attached to an interleukin-18 (IL-18) polypeptide; the artificial end portion comprises a specific cleavage site; The Act-IL-18 polypeptide, wherein cleavage at the specific cleavage site converts the Act-IL-18 into an active form of the IL-18 polypeptide.

2. The Act-IL-18 polypeptide of claim 1, wherein the Act-IL-18 is inactive when the artificial terminal portion is intact.

3. The Act-IL-18 polypeptide of claim 1 or 2, wherein the specific cleavage site is preferentially cleaved at or near a target tissue of a subject.

4. The Act-IL-18 polypeptide of any one of claims 1 to 3, wherein the specific cleavage site is preferentially cleaved within or in the vicinity of a tumor microenvironment.

5. The Act-IL-18 polypeptide according to any one of claims 1 to 4, wherein the specific cleavage site is specifically cleaved by a protease.

6. The Act-IL-18 polypeptide of claim 5, wherein the protease is found in higher concentrations and / or exhibits higher proteolytic activity in the tumor microenvironment compared to non-tumor tissue.

7. 7. The Act-IL-18 polypeptide of claim 5 or 6, wherein the protease is selected from kallikrein, thrombin, chymase, carboxypeptidase A, elastase, proteinase 3 (PR-3), granzyme M, urokinase plasminogen activator (uPA), calpain, matrix metalloproteinases (MMPs), a disintegrin and metalloproteinase (ADAM), fibroblast activation protein alpha (FAP), matriptase, plasminogen activators, cathepsins, caspases, tryptases, and tumor cell surface proteases.

8. The Act-IL-18 polypeptide of any one of claims 5 to 7, wherein the artificial terminal portion comprises a peptide having a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a peptide sequence set forth in Table 2A.

9. The Act-IL-18 polypeptide of any one of claims 5 to 7, wherein the artificial terminal portion comprises a peptide having a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a peptide sequence set forth in Table 2B.

10. The Act-IL-18 polypeptide according to any one of claims 1 to 4, wherein the specific cleavage site is a redox-sensitive cleavage site.

11. The Act-IL-18 polypeptide of claim 9, wherein the redox-sensitive cleavage site is preferentially cleaved in a reducing environment.

12. The Act-IL-18 polypeptide of any one of claims 1 to 4, wherein the specific cleavage site is a pH-sensitive cleavage site.

13. 13. The Act-IL-18 polypeptide of claim 12, wherein the pH-sensitive cleavage site is preferentially cleaved at a pH of less than 7.3, less than 7.2, less than 7.1, or less than 7.

0.

14. The Act-IL-18 polypeptide of any one of claims 1 to 13, wherein the truncation removes the entire artificial terminal portion from the IL-18 polypeptide.

15. The Act-IL-18 polypeptide of any one of claims 1 to 13, wherein said cleavage leaves a portion of said artificial moiety attached to said IL-18 polypeptide.

16. The Act-IL-18 polypeptide of any one of claims 1 to 15, wherein the artificial terminal portion comprises a peptide.

17. 17. The Act-IL-18 polypeptide of claim 16, wherein upon cleavage of the artificial terminal portion at the specific cleavage site, no amino acid residues of the peptide remain attached to the IL-18 polypeptide.

18. 17. The Act-IL-18 polypeptide of claim 16, wherein cleavage of said artificial terminal portion at said specific cleavage site leaves at least one amino acid residue of said peptide attached to said IL-18 polypeptide.

19. 19. The Act-IL-18 polypeptide of claim 16 or 18, wherein cleavage of the artificial terminal portion at the specific cleavage site leaves 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues of the peptide attached to the IL-18 polypeptide.

20. The Act-IL-18 polypeptide of any one of claims 16 to 19, wherein the peptide comprises multiple specific cleavage sites.

21. The Act-IL-18 polypeptide of any one of claims 16 to 20, wherein the peptide comprises 2, 3, or 4 specific cleavage sites.

22. The Act-IL-18 polypeptide of any one of claims 1 to 21, wherein the artificial terminal moiety is attached to the N-terminus or C-terminus of the IL-18 polypeptide.

23. The Act-IL-18 polypeptide of claim 22, wherein the artificial terminal moiety is attached to the N-terminus of the IL-18 polypeptide.

24. The Act-IL-18 polypeptide of claim 23, wherein the artificial end portion comprises a blocking site.

25. 25. The Act-IL-18 polypeptide of claim 24, wherein the blocking moiety is positioned such that cleavage at the specific cleavage site releases the blocking moiety from the Act-IL-18 polypeptide.

26. The Act-IL-18 polypeptide of claim 24 or 25, wherein the blocking moiety comprises an IL-18 propeptide or a portion thereof, or a variant thereof.

27. The Act-IL-18 polypeptide of claim 26, wherein the IL-18 propeptide is a human IL-18 propeptide or a variant thereof.

28. 27. The Act-IL-18 polypeptide of claim 26, wherein the IL-18 propeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO:

89.

29. The Act-IL-18 polypeptide of any one of claims 24 to 28, wherein the artificial terminal portion comprises a linking peptide between the specific cleavage site and the blocking site.

30. 23. The Act-IL-18 polypeptide of claim 22, wherein the artificial terminal moiety is attached to the C-terminus of the IL-18 polypeptide.

31. The Act-IL-18 polypeptide of claim 30, wherein the artificial end portion comprises a blocking site.

32. 32. The Act-IL-18 polypeptide of claim 31, wherein the blocking moiety is positioned such that cleavage at the specific cleavage site releases the blocking moiety from the Act-IL-18 polypeptide.

33. The Act-IL-18 polypeptide of claim 31 or 32, wherein the blocking moiety comprises a domain of an IL-18 receptor subunit or a portion thereof, or a derivative thereof.

34. 34. The Act-IL-18 polypeptide of claim 33, wherein the domain of the IL-18 receptor subunit comprises the D3 domain of the IL-18 receptor alpha subunit, or a variant thereof.

35. The Act-IL-18 polypeptide of any one of claims 33 or 34, wherein the blocking moiety comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence represented by SEQ ID NO:

93.

36. The Act-IL-18 polypeptide of any one of claims 31 to 35, wherein the artificial terminal portion comprises a linking peptide between the specific cleavage site and the blocking site.

37. 37. The Act-IL-18 polypeptide of any one of claims 1 to 36, wherein the active form of the IL-18 polypeptide exhibits reduced binding to IL-18 binding protein (IL-18BP) compared to WT IL-18.

38. 38. The Act-IL-18 polypeptide of any one of claims 1 to 37, wherein the active form of the IL-18 polypeptide exhibits enhanced ability to bind to or activate IL-18R.

39. 38. The Act-IL-18 polypeptide of any one of claims 1 to 37, wherein the active form of the IL-18 polypeptide exhibits up to 100-fold reduced ability to bind to or activate IL-18R compared to WT IL-18.

40. The Act-IL-18 polypeptide of any one of claims 1 to 39, wherein the IL-18 polypeptide comprises one or more modifications relative to the sequence set forth in SEQ ID NO:

1.

41. 41. The Act-IL-18 polypeptide of claim 40, wherein the IL-18 polypeptide comprises an amino acid sequence 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:

1.

42. 42. The Act-IL-18 polypeptide of claim 40 or 41, wherein the IL-18 polypeptide comprises at least one substitution at residues Y1, F2, E6, C38, K53, D54, S55, T63, C76, E85, M86, T95, D98, or C127, or any combination thereof.

43. 43. The Act-IL-18 polypeptide of any one of claims 40-42, wherein the IL-18 polypeptide comprises a Y01G, F02A, E06K, V11I, C38S, C38A, K53A, D54A, S55A, T63A, C76S, C76A, E85C, M86C, T95C, D98C, C127S, or C127A amino acid substitution, or any combination thereof.

44. The Act-IL-18 polypeptide of any one of claims 40 to 43, wherein the IL-18 polypeptide comprises E06K and K53A amino acid substitutions.

45. The Act-IL-18 polypeptide of any one of claims 40 to 44, wherein the IL-18 polypeptide comprises a T63A amino acid substitution.

46. The Act-IL-18 polypeptide of any one of claims 40-45, wherein the IL-18 polypeptide comprises a V11I amino acid substitution.

47. 47. The Act-IL-18 polypeptide of any one of claims 40 to 46, wherein the IL-18 polypeptide comprises substitutions with one, two, three, or four residues selected from C38, C76, C98, and C127.

48. The Act IL-18 polypeptide of claim 40, wherein the IL-18 polypeptide comprises an amino acid sequence represented by any one of SEQ ID NOs: 2-67.

49. 41. The Act IL-18 polypeptide of claim 40, wherein the IL-18 polypeptide comprises the amino acid sequence set forth in SEQ ID NO:

30.

50. The Act-IL-18 polypeptide of claim 40, wherein the IL-18 polypeptide comprises an amino acid sequence represented by any one of SEQ ID NOs: 79 to 83.

51. The Act IL-18 polypeptide has a half maximal effective concentration (EC) that is at least 1,000-fold higher, 2,000-fold higher, 5,000-fold higher, 10,000-fold higher, 15,000-fold higher, or 20,000-fold higher than the activated form of the IL-18 polypeptide for IL-18 receptor signaling activity. ５０ The Act IL-18 polypeptide of any one of claims 1 to 50, wherein

52. The Act IL-18 polypeptide has a half maximal effective concentration (EC ) that is about 10 to about 100 times higher than the active form of the IL-18 polypeptide for IL-18 receptor signaling activity. ５０ The Act IL-18 polypeptide of any one of claims 1 to 50, wherein

53. The active form of the IL-18 polypeptide has a half maximal effective concentration (EC) within about 10-fold of the IL-18 polypeptide for IL-18 receptor signaling activity. ５０ The Act-IL-18 polypeptide of any one of claims 1 to 52, wherein

54. The Act-IL-18 polypeptide has a half-maximal effective concentration (EC ) that is at least 1000-fold higher than that of WT IL-18 for IL-18 receptor signaling activity. ５０ The Act-IL-18 polypeptide of any one of claims 1 to 53, wherein the Act-IL-18 polypeptide has the following structure:

55. The Act-IL-18 polypeptide has a half-maximal effective concentration (EC) that is at least 10-fold higher than that of WT IL-18 for IL-18 receptor signaling activity. ５０ The Act-IL-18 polypeptide of any one of claims 1 to 54, wherein said Act-IL-18 polypeptide has the following structure:

56. The Act-IL-18 polypeptide of any one of claims 1 to 55, wherein the IL-18 polypeptide is synthetic.

57. The Act-IL-18 polypeptide of any one of claims 1 to 56, comprising a polymer attached to a residue of the IL-18 polypeptide.

58. a) an Act-IL-18 polypeptide according to any one of claims 1 to 57, and b) a pharmaceutical composition comprising a pharma- ceutically acceptable carrier or excipient.

59. 1. A method of treating cancer in a subject in need thereof, comprising: The method comprising administering to the subject a pharma- ceutically effective amount of an Act-IL-18 polypeptide of any one of claims 1 to 57 or a pharmaceutical composition of claim 58.

60. 60. The method of claim 59, wherein the cancer is a solid cancer.

61. 61. The method of claim 60, 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.

62. 61. The method of claim 60, wherein the solid cancer is metastatic renal cell carcinoma or melanoma.

63. 61. The method of claim 60, wherein the solid cancer is a carcinoma or a sarcoma.

64. 60. The method of claim 59, wherein the cancer is a hematological cancer.

65. 65. The method of claim 64, 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.

66. 66. The method of any one of claims 59 to 65, further comprising reconstituting said Act-IL-18 polypeptide or said pharmaceutical composition in lyophilized form.

Images