Constructs comprising il-18 components and uses thereof

Abstract

Provided are constructs comprising an interleukin 18 (IL-18) component associated with a binding moiety that specifically binds an antigen. In some cases, the IL-18 component has reduced binding affinity for an IL-18 receptor compared to a corresponding wild-type IL-18 polypeptide (e.g., SEQ ID NO: 1). In some cases, a single IL-18 component is associated with a multivalent antibody. Also provided are related nucleic acids, vectors, host cells, methods of making the constructs, pharmaceutical compositions comprising the constructs, and uses of such pharmaceutical compositions.

Description

Cross-references to related applications

[0001] This application claims priority to international application PCT / CN2024 / 080320, filed on March 6, 2024, the entire contents of which are incorporated herein by reference for all purposes. Reference to electronic sequence listing

[0002] The entire contents of the electronic serial number (334302000541SEQLIST.xml; size: 563,001 bytes; creation date: February 28, 2025) are incorporated herein by reference. Technical Field

[0003] This application relates to constructs comprising an interleukin-18 (IL-18) component associated with a binding portion of a specific binding antigen, said construct comprising an IL-18 polypeptide with reduced binding affinity compared to wild-type IL-18 (e.g., wild-type human IL-18); it also relates to methods for preparing such constructs, and methods for using such constructs in therapeutic applications. Background Technology

[0004] Interleukin-18 (IL-18, also known as interferon-γ inducible factor) is a pro-inflammatory cytokine that has been found to stimulate innate lymphocytes, myeloid cells, and antigen-stimulated non-naïve T cells (see, for example, Guo et al., (2012)). Trends Immunol. 33, 598-606) and natural killer cells (NK cells). In terms of treatment, recombinant IL-18 has been reported in preclinical models in combination with immune checkpoint inhibitors (ICIs) (Ma et al., (2016)). Clin Cancer Res 22: 2969-2980) and chimeric antigen receptor T (CAR-T) cells (Hu et al., (2017) Cell Rep (20, 3025-3033) synergistic effects. Studies have found that proteins in the interleukin-18 (IL-18) pathway are upregulated in tumor-infiltrating lymphocytes (TILs) (see, for example, Zhou et al., (2020)). Nature (583: 609-614), indicating that IL-18 treatment can enhance anti-tumor immunity. IL-18 has been administered to patients in clinical trials and has been found to be safe and well-tolerated (Robertson et al., (2006)). Clin Cancer Res 12, 4265-4273). However, clinical development of IL-18 has been limited due to its limited efficacy. IL-18BP is a high-affinity IL-18 inhibitor (K... D < 1 nM, see, for example, Dinarello et al., (2013) Front Immunol 4: 289, doi: 10.3389 / fimmu.2013.00289), which is frequently upregulated in various human and mouse tumors and is believed to limit the antitumor activity of IL-18 in preclinical models (e.g., mice) and clinical trials. Researchers have constructed IL-18 variant peptides that resist IL-18BP, retaining the signaling ability of IL-18 while being unaffected by IL-18BP inhibition (see, for example, Zhou et al., (2020)). Nature 583: 609-614).

[0005] Furthermore, because IL-18 modulates both innate and adaptive immunity, its dysregulation can lead to autoimmune or inflammatory diseases. For example, IL-18 plays a role in Th1-mediated immune responses by activating NK cells and Th1 cells, which participate in the host's defense against intracellular pathogen infection by producing IFNγ. IL-18 also induces the production of TNF and FasL, both of which are involved in cell growth, survival, and apoptosis. IL-18 is believed to drive Th2 immune responses by inducing the production of IL-4 and IL-13 from T cells, NK cells, mast cells, and basophils. Other mediators induced by IL-18 include inducible nitric oxide; cyclooxygenase-2 (Cox-2); pro-inflammatory cytokines IL-1β and IL-6; chemokines IL-8, MCP-1, and MIP-1α; intracellular adhesion molecule-1 (ICAM-1); and the growth factor GM-CSF. IL-18 also induces the production of cytokines such as IL-12 and IL-2. Given that IL-18 has pleiotropic effects, the regulation of IL-18 activity is crucial for preventing abnormal immune responses.

[0006] All publications, patents, patent applications, and the full disclosure of published patent applications cited in this article are incorporated herein by reference. Summary of the Invention

[0007] In one aspect, this application provides a construct comprising an IL-18 component associated with a binding moiety that specifically binds to an antigen, wherein the IL-18 component comprises an IL-18 polypeptide, wherein: 1) the IL-18 polypeptide comprises one or more modifications compared to a wild-type IL-18 polypeptide; 2) the IL-18 component further comprises a masking moiety, wherein the IL-18 polypeptide is linked to the masking moiety via a cleavable linker; or 3) the IL-18 component further comprises a peptide linker linking the IL-18 polypeptide to the binding moiety, wherein the peptide linker is no more than 15 amino acids in length. In some embodiments, the wild-type IL-18 polypeptide has the amino acid sequence shown in SEQ ID NO: 1. In some embodiments, the IL-18 component has a lower binding affinity to the IL-18 receptor than the wild-type IL-18 polypeptide. In some embodiments, the binding affinity is evaluated on cells expressing the IL-18 receptor but not the antigen. In some embodiments, the construct has a single IL-18 component.

[0008] In some embodiments of any of the above-described constructs, the binding portion comprises a small molecule.

[0009] In some implementations of any of the above-described constructs, the bonding portion does not include the Fc structural domain.

[0010] In some embodiments of any of the above-described constructs, the binding portion comprises an Fc domain. In some embodiments, the Fc domain comprises an IgG Fc domain. In some embodiments, the IL-18 component is fused to the C-terminus of the Fc domain. In some embodiments, the IL-18 component is a single IL-18 component fused to the C-terminus of the Fc domain in the construct.

[0011] In some embodiments of any of the above-described constructs, the binding portion comprises an antibody. In some embodiments, the antibody is a monovalent antibody. In some embodiments, the antibody is a multivalent antibody.

[0012] In another embodiment, this application provides a construct comprising a single IL-18 component associated with a binding portion of a multivalent antibody comprising a specific binding antigen, wherein the antibody comprises an Fc domain, the single IL-18 component comprises a single IL-18 polypeptide, the single IL-18 polypeptide comprising the wild-type IL-18 polypeptide or a variant thereof as shown in the amino acid sequence of SEQ ID NO: 1, and the single IL-18 component is fused to the Fc domain. In some embodiments, the IL-18 polypeptide comprises one or more modifications relative to the wild-type IL-18 polypeptide. In some embodiments, the IL-18 component further comprises a masking portion, wherein the IL-18 polypeptide is linked to the masking portion via a cleavable linker. In some embodiments, the IL-18 component further comprises a peptide linker linking the IL-18 polypeptide to the binding portion, wherein the peptide linker is no more than 15 amino acids in length. In some embodiments, the wild-type IL-18 polypeptide has the amino acid sequence shown in SEQ ID NO: 1. In some embodiments, the IL-18 component has a lower binding affinity to the IL-18 receptor than the wild-type IL-18 peptide. In some embodiments, the binding affinity is evaluated on cells expressing the IL-18 receptor but not the antigen.

[0013] In some embodiments of any of the above-described constructs involving antibodies, the antibody is a bivalent antibody.

[0014] In some embodiments involving any of the above-described constructs of the antibody, the antibody is selected from the group consisting of: full-length antibody, single-chain Fv (scFv), Fab, Fab', F(ab')2, Fv fragment, disulfide-stabilized Fv fragment (dsFv), (dsFv)2, nanobody (VHH), Fv-Fc fusion protein, scFv-Fc fusion protein, scFv-Fv fusion protein, diabody, tribody, and tetrabody. In some embodiments, the antibody comprises a full-length antibody, nanobody, Fab, or scFv. In some embodiments, the antibody is a full-length antibody comprising two heavy chains and two light chains. In some embodiments, the IL-18 component is fused to one or two heavy chains. In some embodiments, the IL-18 component is fused to the C-terminus of one or two heavy chains and / or one or two light chains of the full-length antibody. In some embodiments, the two heavy chains contain one or more modifications that facilitate the formation of a club-and-mortar structure.

[0015] In some embodiments of any of the above-described constructs, the IL-18 component is covalently conjugated to the binding moiety. In some embodiments, the IL-18 component is covalently conjugated to the binding moiety via a linker (e.g., a PEG linker). In some embodiments, the linker is a non-peptide linker with a length equivalent to a peptide linker of about 1-15 amino acids (e.g., about 1-5 amino acids, about 5-10 amino acids, about 10-15 amino acids, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids).

[0016] In some embodiments of any of the above-described constructs, the IL-18 component is fused to the binding moiety. In some embodiments, a linker exists between the IL-18 peptide and the binding moiety. In some embodiments, the linker is a peptide linker (e.g., a GS linker). In some embodiments, the linker is no more than about 35 amino acids long. In some embodiments, the linker is about 15-35 amino acids long. In some embodiments, the linker is about 1-15 amino acids long. In some embodiments, the linker is about 1-5 amino acids long. In some embodiments, the linker is about 5-10 amino acids long. In some embodiments, the linker is about 10-15 amino acids long. In some embodiments, the linker is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids long.

[0017] In some embodiments of any of the above-described constructs, the antigen is a tumor-specific antigen or a tumor-associated antigen. In some embodiments, the tumor is a solid tumor. In some embodiments, the antigen is a neoantigen.

[0018] In some embodiments of any of the above-described constructs, the antigen is an antigen expressed by immune cells. In some embodiments, the immune cells are tumor-infiltrating lymphocytes.

[0019] In some embodiments of any of the above-described constructs, the antigen is an immune checkpoint target expressed on T cells or NK cells. In some embodiments, the antigen is PD-1. In some embodiments, the binding domain comprises: a heavy chain variable region comprising VH-CDR1, VH-CDR2, and VH-CDR3; and a light chain variable region comprising VL-CDR1, VL-CDR2, and VL-CDR3. In some embodiments, VH-CDR1 comprises the amino acid sequence shown in SEQ ID NO: 470, VH-CDR2 comprises the amino acid sequence shown in SEQ ID NO: 471, VH-CDR3 comprises the amino acid sequence shown in SEQ ID NO: 472, VL-CDR1 comprises the amino acid sequence shown in SEQ ID NO: 473, VL-CDR2 comprises the amino acid sequence shown in SEQ ID NO: 474, and VL-CDR3 comprises the amino acid sequence shown in SEQ ID NO: 475. In some embodiments, VH-CDR1 contains the amino acid sequence shown in SEQ ID NO: 476, VH-CDR2 contains the amino acid sequence shown in SEQ ID NO: 477, VH-CDR3 contains the amino acid sequence shown in SEQ ID NO: 478, VL-CDR1 contains the amino acid sequence shown in SEQ ID NO: 479, VL-CDR2 contains the amino acid sequence shown in SEQ ID NO: 480, and VL-CDR3 contains the amino acid sequence shown in SEQ ID NO: 481.

[0020] In some embodiments, the IL-18 polypeptide is a wild-type IL-18 polypeptide containing the amino acid sequence shown in SEQ ID NO: 1.

[0021] In some embodiments, the IL-18 peptide comprises one or more modifications compared to the corresponding wild-type IL-18 peptide (e.g., SEQ ID NO: 1). In some embodiments, the IL-18 peptide exhibits a reduced binding affinity for IL-18Rβ compared to the corresponding wild-type IL-18 peptide (e.g., SEQ ID NO: 1). In some embodiments, the reduced binding affinity is: a) at least 50%, 60%, 70%, 80%, or 90%, or b) 50%-60%, 60%-70%, 70%-80%, 80%-90%, 90%-95%, 95%-96%, 96%-97%, 97%-98%, and / or 98%-99%. In some embodiments, the one or more modifications comprise amino acid substitutions selected from the group consisting of G108, H109, ​​K112, D110, R147, M150, G145, K79, D98, and K84, wherein the amino acid position is relative to the wild-type human IL-18 polypeptide shown in SEQ ID NO: 1. In some embodiments, the substitution is an alanine substitution. In some embodiments, the one or more modifications comprise amino acid substitutions selected from the group consisting of G108, H109, ​​K112, D110, R147, M150, G145, K79, and D98, wherein the amino acid position is relative to the wild-type human IL-18 polypeptide shown in SEQ ID NO: 1. In some embodiments, the substitution is an alanine substitution. In some embodiments, the IL-18 component exhibits a reduced binding affinity for IL-18Rα compared to the corresponding wild-type IL-18 polypeptide (e.g., SEQ ID NO: 1). In some embodiments, the binding affinity is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%. In some embodiments, the one or more modifications comprise amino acid substitutions selected from the group consisting of K4, L5, K8, R13, D17, E31, M33, T34, D35, D40, K53, R58, M60, K79, K84, D98, R104, and D132, wherein the amino acid position is relative to the wild-type human IL-18 polypeptide represented by SEQ ID NO: 1. In some embodiments, the substitution is an alanine substitution.

[0022] In some embodiments of any of the above-described constructs, the IL-18 component comprises an IL-18 polypeptide, the IL-18 polypeptide comprising two or more modifications. In some embodiments, the two or more modifications comprise two or more amino acid substitutions selected from the group consisting of G108, H109, ​​K112, D110, R147, M150, G145, K79, D98, K84, K4, L5, K8, R13, D17, E31, M33, T34, D35, D40, K53, R58, M60, R104, and D132, wherein the amino acid position is relative to the wild-type human IL-18 polypeptide shown in SEQ ID NO: 1. In some embodiments, the substitution is an alanine substitution.

[0023] In some embodiments of any of the above-described constructs, the IL-18 polypeptide comprises the amino acid sequence shown in any one of SEQ ID NO: 482-491.

[0024] In some embodiments of any of the above-described constructs, the IL-18 component further comprises a masking portion, wherein the IL-18 polypeptide is linked to the masking portion via a cleavable linker. In some embodiments, the masking portion comprises an IL-18 propeptide. In some embodiments, the IL-18 propeptide comprises a propeptide of human IL-18 (hIL-18). In some embodiments, the hIL-18 propeptide comprises the amino acid sequence shown in SEQ ID NO: 333. In some embodiments, the masking portion comprises a truncated propeptide of an IL-18 precursor. In some embodiments, the truncated propeptide of an IL-18 precursor comprises the amino acid sequence shown in SEQ ID NO: 336. In some embodiments, the masking portion comprises the extracellular domain of IL-18Rα, the extracellular domain of IL-18Rβ, an IL-18 binding protein, a fragment of any of the foregoing, or a variant of any of the foregoing. In some embodiments, the masking portion comprises the extracellular domain of IL-18Rα or a fragment thereof. In some embodiments, the fragment of the extracellular domain of the IL-18Rα comprises: a) a first and a second domain of the extracellular domain, or b) a third domain of the extracellular domain. In some embodiments, the masking portion comprises the amino acid sequence shown in any one of SEQ ID NO:353-356, or a functional variant thereof, the functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with any one of SEQ ID NO:353-356.

[0025] In some embodiments of any of the above-described constructs involving a cleavable linker, the cleavable linker comprises one or more sets of amino acid sequences recognized and cleaved by one or more proteases. In some embodiments, the one or more proteases are one or more tumor microenvironment (TME) proteases. In some embodiments, the one or more TME proteases are selected from the group consisting of: urokinase-type plasminogen activator, Matriptase, Legumin, prostate-specific antigen, dipeptidyl peptidase, Hepsin, matrix metalloproteinases, integrin-dissociating metalloproteinases, human leukocyte elastase, protease 3, prourokinase, plasminogen, staphylococcal kinase, cathepsin, tissue kallikrein, and kallikrein-related peptidase. In some embodiments, the one or more TME proteases are matrix metalloproteinases, and the matrix metalloproteinases are selected from the group consisting of: matrix metalloproteinase 1, matrix metalloproteinase 2, matrix metalloproteinase 3, matrix metalloproteinase 8, matrix metalloproteinase 9, matrix metalloproteinase 10, matrix metalloproteinase 12, and matrix metalloproteinase 14. In some embodiments, the cleavable linker comprises one or more amino acid sequences that are recognized and cleaved by one or more of matrix metalloproteinase 2, matrix metalloproteinase 9, matrix metalloproteinase 10, matrix metalloproteinase 14, urokinase-type plasminogen activator, Matriptase, and Legumain. In some embodiments, the cleavable linker comprises one or more amino acid sequences that are recognized and cleaved by at least two, three, or four proteases. In some embodiments, the one or more amino acid sequences are simultaneously recognized and cleaved by Legumain and one, two, or three of MMP9, MMP2, and MMP14. In some embodiments, the one or more amino acid sequences are simultaneously recognized and cleaved by uPA and one, two, or three of MMP9, MMP2, and MMP14. In some embodiments, the one or more amino acid sequences are recognized and cleaved by all of uPA, Legumain, MMP2, MMP14, and MMP9. In some embodiments, the cleavable linker comprises one or more amino acid sequences selected from the group consisting of SEQ ID NO: 337, SEQ ID NO: 338, SEQ ID NO: 339, and SEQ ID NO: 372-377. In some embodiments, the cleavable linker further comprises a spacer sequence. In some embodiments, the cleavable linker comprises two spacer sequences located flanking the N-terminus and C-terminus of an amino acid sequence recognized and cleaved by one or more proteases, respectively. In some embodiments, the cleavable linker comprises two or more groups of amino acid sequences recognized and cleaved by one or more proteases.In some embodiments, at least one set of the two or more sets of amino acid sequences recognized and cleaved by one or more proteases is side-joined with two spacer sequences in the cleavable linker, and each set of the two or more sets of amino acid sequences recognized and cleaved by one or more proteases is side-joined with two spacer sequences in the cleavable linker. In some embodiments, the spacer sequences comprise a GS linker. In some embodiments, the length of the GS linker is no more than about 10, 9, 8, 7, 6, or 5 amino acids. In some embodiments, the cleavable linker consists of no more than about 50, 40, 35, or 30 amino acids. In some embodiments, the cleavable linker consists of no less than about 10, 15, 20, or 25 amino acids. In some embodiments, the cleavable linker consists of about 25 to about 30 amino acids, and the masking portion comprises an extracellular domain of IL-18Rα. In some embodiments, the masking portion comprises the amino acid sequence shown in any one of SEQ ID NO: 353-356, or a functional variant thereof, said functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with any one of SEQ ID NO: 353-356. In some embodiments, the cleavable linker comprises the amino acid sequence shown in any one of SEQ ID NO: 399-407, SEQ ID NO: 492-493, and SEQ ID NO: 495.

[0026] In some embodiments, the IL-18 component comprises, from N-terminus to C-terminus,: a) a masking portion comprising a truncated propeptide of the IL-18 precursor, the truncated propeptide comprising the amino acid sequence shown in SEQ ID NO: 336; b) a cleavable linker; and c) an IL-18 polypeptide comprising the amino acid sequence shown in any one of SEQ ID NO: 1-299 and 307-318; optionally, the IL-18 polypeptide comprises the amino acid sequence shown in any one of SEQ ID NO: 39, 133, 311, 316, 465-469, 482-491, 27, 3, 105, 143, 144, 145, 43, 9, 17, 81, 117, 147, 149, and 150; optionally, the length of the cleavable linker is no more than about 50, 40, 30, 25, 20, 15, 12, or 10 amino acids.

[0027] In some embodiments, the IL-18 component comprises, from N-terminus to C-terminus, the following: a) a masking portion comprising the amino acid sequence shown in any one of SEQ ID NO: 353-356; b) a cleavable linker; and c) an IL-18 polypeptide comprising the amino acid sequence shown in any one of SEQ ID NO: 1-299 and 307-318; optionally, the IL-18 polypeptide comprises the amino acid sequence shown in any one of SEQ ID NO: 39, 133, 311, 316, 465-469, 482-491, 27, 3, 105, 143, 144, 145, 43, 9, 17, 81, 117, 147, 149, and 150; optionally, the cleavable linker is about 10-40, 10-30, 20-30, or 25-30 amino acids in length.

[0028] In some embodiments, the IL-18 component comprises, from N-terminus to C-terminus, the following: a) an IL-18 polypeptide comprising the amino acid sequence shown in any one of SEQ ID NO: 1-299 and 307-318; optionally, the IL-18 polypeptide comprises the amino acid sequence shown in any one of SEQ ID NO: 39, 133, 311, 316, 465-469, 482-491, 27, 3, 105, 143, 144, 145, 43, 9, 17, 81, 117, 147, 149, and 150; b) a cleavable linker; and c) a masking portion comprising the amino acid sequence shown in any one of SEQ ID NO: 353-356; optionally, the length of the cleavable linker is about 10-40, 10-30, 20-30, or 25-30 amino acids.

[0029] In some embodiments of any of the above-described constructs, the IL-18 component comprises: a) an amino acid sequence represented by any one of SEQ ID NO: 340-343, 345-352, and 496-500, or b) a functional variant thereof having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with any one of SEQ ID NO: 340-343, 345-352, and 496-500.

[0030] In some embodiments of any of the above-described constructs, the IL-18 peptide exhibits a reduced binding affinity for IL-18 binding protein (IL-18BP) compared to the corresponding wild-type IL-18 peptide (e.g., SEQ ID NO: 1); optionally, the reduced binding affinity is: a) at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, or b) about 10%-20%, 20%-30%, 30%-40%, 40%-50%, 50%-60%, 60%-70%, 70%-80%, 80%-90%, 90%-95%, 95%-96%, 96%-97%, 97%-98%, and / or 98%-99%.

[0031] In some embodiments of any of the above-described constructs, the IL-18 peptide contains at least three, four, five, or six mutations at G3, E6, D54, Q56, P57, N91, and R104 of the IL-18 peptide, wherein the amino acid positions are relative to the wild-type human IL-18 peptide shown in SEQ ID NO: 1.

[0032] In some embodiments of any of the above-described constructs, the IL-18 polypeptide contains a cysteine ​​residue at position 117 and at position 76, wherein the amino acid positions are relative to the wild-type IL-18 polypeptide shown in SEQ ID NO: 1. In some embodiments, the IL-18 polypeptide does not contain a cysteine ​​residue at position 38, and / or the IL-18 polypeptide does not contain a cysteine ​​residue at position 68. In some embodiments, the IL-18 polypeptide comprises: a) a cysteine ​​residue at position 117; b) a cysteine ​​residue at position 76; c) an isoleucine residue at position 38; and d) a serine residue at position 68, wherein the amino acid positions are relative to the wild-type IL-18 polypeptide shown in SEQ ID NO: 1.

[0033] In some embodiments of any of the above-described constructs, the IL-18 component comprises an amino acid sequence represented by any one of SEQ ID NO: 2-299, 307-318, 465-469, and 482-491; optionally, the IL-18 component comprises an amino acid sequence represented by any one of SEQ ID NO: 39, 133, 311, 316, 27, 3, 105, 143, 144, 145, 43, 9, 17, 81, 117, 147, 149, 150, 465-469, and 482-491.

[0034] On the other hand, this application provides one or more nucleic acids that encode any of the above-described constructs or a portion thereof.

[0035] On the other hand, this application provides one or more vectors containing one or more of the aforementioned nucleic acids.

[0036] On the other hand, this application provides a host cell comprising one or more of the aforementioned nucleic acids or one or more of the aforementioned vectors. In some embodiments, the host cell is a eukaryotic cell; optionally, the host cell is a CHO cell. In some embodiments, the host cell is a prokaryotic cell; optionally, the host cell is *Escherichia coli*.

[0037] In another aspect, this application provides a method for preparing any of the above-described constructs or a portion thereof, comprising: (a) culturing any of the above-described host cells under conditions expressing the construct or a portion thereof, and (b) recovering the construct or a portion thereof generated from the host cells.

[0038] On the other hand, this application provides a method for treating a disease or ailment, including applying any of the aforementioned constructs to an individual in need.

[0039] It should be understood that one, several, or all of the features described in the various embodiments herein can be combined with each other to form other embodiments of the invention. The above and other aspects of the invention will be apparent to those skilled in the art. The above and other embodiments of the invention will be further described in the following detailed description.

[0040] All publications, patents, patent applications, and the full disclosure of published patent applications cited in this article are incorporated herein by reference. Attached Figure Description

[0041] Figure 1A A schematic diagram of an exemplary construct is shown, the construct comprising: a binding portion (A) (e.g., a tissue-specific antigen targeting portion); an IL-18 component with reduced binding affinity to the IL-18 receptor, comprising an IL-18 polypeptide (C); and an optional half-life extension element (B), the components optionally fused via linkers (L1, L2).

[0042] Figure 1B A schematic diagram of another exemplary construct is shown, which includes: a binding portion (A) (e.g., a tissue-specific antigen targeting portion); an optional half-life extension element (B); an IL-18 peptide (C); a cleavable linker (L3); and a masking portion (D), wherein the above components are optionally fused by linkers (L1, L2).

[0043] Figure 2A A schematic diagram of different forms of immune cytokines is shown. These immune cytokines include IgG antibodies and different forms of IL-18 components.

[0044] Figure 2B This study demonstrates the activation of human IL-18 (hIL-18) receptor-mediated signaling by fusing anti-PD1 with different forms of the IL-18 peptide MM5.

[0045] Figure 2C A schematic diagram of an immune cytokine is shown, the immune cytokine comprising an anti-PD1 IgG antibody and an IL-18 component, the IL-18 component comprising an IL-18 polypeptide with reduced binding affinity to the IL-18 receptor.

[0046] Figure 2D It demonstrates the activation of hIL-18 receptor-mediated signaling by fusing anti-PD1 with the IL-18 variant MM5 (i.e., PD1-MM5) and by fusing anti-PD1 with an IL-18 component containing the IL-18 peptide MM5 with attenuated mutations (A1 to A7).

[0047] Figure 3A A schematic diagram of a HEK293 cell line overexpressing human PD1 is shown, which has an IL-18 reporter system (NFκB-luc / hIL-18RαRβ).

[0048] Figure 3B This study demonstrates the activation of hIL-18 receptor-mediated signaling in PD1-positive and PD1-negative cells by fusing anti-PD1 with an IL-18 component containing attenuated mutations (A1 to A3) in the IL-18 peptide.

[0049] Figure 4A It is demonstrated that hIL-18 receptor-mediated signaling is activated by fusing anti-PD1 with IL-18 components containing IL-18-activating peptides (M12 or MM5) (i.e., PD1-M12-R-L1, PD1-MM5-R-L1) and their activated forms (e.g., after removing the masking portion) (PD1-M12-R-L1 Cut, PD1-MM5-R-L1 Cut).

[0050] Figure 4B The study demonstrated that the release of hIFNγ from human PBMCs was induced by fusing anti-PD1 with IL-18 components containing IL-18-activating peptides (M12 or MM5) (i.e., PD1-M12-R-L1, PD1-MM5-R-L1) and their activated forms (e.g., after removing the masking portion) (PD1-M12-R-L1 Cut, PD1-MM5-R-L1 Cut).

[0051] Figure 4CThis study demonstrates the activation of hIL-18 receptor-mediated signaling by fusing anti-PD1 with an IL-18 component containing the IL-18-activating peptide M12 (PD1-M12-D12) and its activated form (e.g., after removing the masking portion) (PD1-M12-D12 Cut).

[0052] Figure 4D This study demonstrates the activation of hIL-18 receptor-mediated signaling by fusing anti-PD1 with an IL-18 component containing the IL-18-activating peptide MM5 (PD1-MM5-D12) and its activated form (e.g., after removing the masking portion) (PD1-MM5-D12 Cut).

[0053] Figure 5A This study demonstrates the activation of hIL-18 receptor-mediated signaling by fusing anti-PD1 with an IL-18 component containing either the IL-18-activating peptide M12 or the IL-18-activating variant MM5.

[0054] Figure 5B This study demonstrates how fusing anti-PD1 with an IL-18 component containing IL-18-activating peptides M12 or MM5 can induce the release of hIFNγ from human PBMCs.

[0055] Figure 5C This study demonstrates the activation of hIL-18 receptor-mediated signaling by fusing anti-PD1 with an IL-18 component containing the IL-18-activating peptide MM5 (PD1-MM5-R), wherein different linkers (L1 to L3) are used between the IL-18 peptide and the masking portion.

[0056] Figure 5D This study demonstrates the activation of hIL-18 receptor-mediated signaling by fusing anti-PD1 with an activatable IL-18 component containing the activatable IL-18 peptide MM5 (PD1-MM5-R), wherein different linkers (L1, L4) are used between the IL-18 peptide and the masking portion.

[0057] Figure 6 This study demonstrates the activation of hIL-18 receptor-mediated signaling in PD1-positive and PD1-negative cells by fusing anti-PD1 with an IL-18 component containing the IL-18 peptide MM5 (i.e., PD1-MM5), wherein the Fc domain is linked to the IL-18 peptide by a linker of different lengths.

[0058] Figure 7This study demonstrates the activation of hIL-18 receptor-mediated signaling in PD1-positive and PD1-negative cells by fusing anti-PD1 with a monovalent IL-18 component (e.g., a construct containing only one IL-18 component), wherein the IL-18 component comprises the IL-18 peptide MM5 (i.e., PD1-MM5-K).

[0059] Figure 8A This study demonstrates the activation of hIL-18 receptor-mediated signaling in PD1-positive and PD1-negative cells by fusing anti-PD1 with an activatable IL-18 component containing the IL-18 peptide MM5, wherein the IL-18 peptide MM5 further contains an amino acid mutation with reduced receptor binding capacity (i.e., PD1-MM5-A1-R).

[0060] Figure 8B This study demonstrates the activation of hIL-18 receptor-mediated signaling in PD1-positive and PD1-negative cells by fusing anti-PD1 with an activatable IL-18 component containing the IL-18 peptide MM5, wherein the IL-18 peptide MM5 has a mutation that weakens receptor binding ability (i.e., PD1-MM5-A2-R).

[0061] Figure 9A , Figure 9B and Figure 9C Schematic diagrams are shown illustrating the fusion of the IL-18 component with a full-length antibody, an antibody having an Fc domain and two scFv domains, and an antibody having an Fc domain and two VHH domains. The triangles in the diagrams represent a pestle-and-mortar structure. The IL-18 component can fuse with the C-terminus of any chain containing the Fc domain.

[0062] Figures 10A to 10G This study demonstrates the activation of hIL-18 receptor-mediated signaling in PD1-positive and PD1-negative cells by fusing anti-PD1 with the monovalent activatable IL-18 peptide MM5. Detailed Implementation

[0063] In one aspect, this application provides a construct comprising an IL-18 component associated with a binding moiety of a specifically binding antigen, wherein the IL-18 component comprises an IL-18 polypeptide, and the IL-18 component has a lower binding affinity for the IL-18 receptor than the wild-type IL-18 polypeptide. In another aspect, this application provides a construct comprising a single IL-18 component fused to a binding moiety of a multivalent antibody comprising a specifically binding antigen, wherein the single IL-18 component comprises a single IL-18 polypeptide, and the antibody comprises an Fc domain. In some embodiments, the IL-18 polypeptide comprises one or more modifications compared to the wild-type IL-18 polypeptide. In some embodiments, the IL-18 component further comprises a masking moiety, wherein the IL-18 polypeptide is linked to the masking moiety via a cleavable linker. In some embodiments, the IL-18 component further comprises a peptide linker linking the IL-18 polypeptide to the binding moiety, wherein the peptide linker is no more than 15 amino acids in length.

[0064] This application is based, at least in part, on the inventors' unique insight that by simultaneously reducing the binding affinity of the IL-18 component to the IL-18 receptor and associating the IL-18 component with the binding portion of a target antigen (e.g., a tissue-specific antigen), the IL-18 component can have a wider therapeutic window (i.e., a range of dosage concentrations between the minimum effective therapeutic concentration and the minimum toxic concentration) when specifically targeting cells (e.g., cells in tissues) that a) themselves simultaneously express the IL-18 receptor and the antigen (i.e., cis signaling), or b) co-express the IL-18 receptor and the antigen with neighboring cells (i.e., trans signaling, e.g., TILs expressing the IL-18 receptor and tumor cells expressing the antigen bound by the binding portion). This wider therapeutic window enables effective treatment with reduced or minimized toxicity.

[0065] As further detailed in the embodiments, the inventors have demonstrated three exemplary design schemes, each successfully reducing the binding affinity of the IL-18 component to the IL-18 receptor. These three exemplary methods include: a) modifying one or more amino acids (e.g., residues responsible for binding to IL-18Rα or IL-18Rβ) of the IL-18 peptide; b) linking the IL-18 peptide to a masking moiety via a cleavable linker; and c) linking the IL-18 peptide to a binding moiety via a linker (e.g., a peptide linker) and adjusting the linker length between the binding moiety and the IL-18 peptide. By conjugating such IL-18 components to exemplary binding moieties (e.g., anti-PD-1 antibodies), the therapeutic window can be increased by 100-fold to 10,000-fold. See, for example... Figure 3B , Figures 4A to 4B , Figures 5A to 5D , Figure 6 and Figure 7 .

[0066] This application is also based, at least in part, on another unique insight of the inventors, namely that constructs comprising a multivalent binding moiety (e.g., a bivalent antibody) and a single IL-18 component have advantages over corresponding constructs comprising two or more IL-18 components, including a wider therapeutic window, lower toxicity, and comparable efficacy. See, for example... Figure 7 Therefore, in another aspect, this application provides a construct comprising a single IL-18 component associated with a multivalent binding portion of a specific antigen (e.g., a multivalent antibody containing an Fc domain).

[0067] This application also provides a nucleic acid encoding the construct or a portion thereof, a vector and host cell containing the construct or a portion thereof, a method for preparing the construct or a portion thereof, and a method for using the construct (e.g., for treating cancer). definition

[0068] The term "antibody" is used in its broadest sense and encompasses a wide range of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies, and their antigen-binding fragments, as long as they exhibit the desired antigen-binding activity.

[0069] Full-length antibodies consist of two heavy chains and two light chains. The variable regions of the light and heavy chains are responsible for antigen binding. The variable domains of the heavy and light chains can be referred to as "V" and "V" domains, respectively. H " and "V L The variable regions of both chains typically contain three highly variable loop regions, called complementarity-determining regions (CDRs).

[0070] The term "single-domain antibody" or "sdAb" refers to a single antigen-binding polypeptide having three complementarity-determining regions (CDRs). An sdAb can bind to an antigen on its own without pairing with a corresponding CDR-containing polypeptide. In some cases, sdAbs are engineered from camel heavy chain antibodies (HCAbs), whose heavy chain variable domains are referred to herein as "V". H "Hs". Cameloid sdAbs are among the smallest known antigen-binding antibody fragments (see, for example, Hamers-Casterman et al.). Nature 363: 446-8 (1993); Greenberg et al., Nature 374: 168-73 (1995); Hassanzadeh-Ghassabeh et al., Nanomedicine (Lond) , 8: 1013-26 (2013)).

[0071] The “Fv” is the smallest antibody fragment containing a complete antigen recognition and binding site. This fragment consists of a dimer formed by a heavy chain variable region domain and a light chain variable region domain in a tight, non-covalently bound manner. These two domains fold to form six hypervariable rings (three from the heavy chain and three from the light chain), which provide the amino acid residues involved in antigen binding and confer antigen-binding specificity to the antibody. However, even a single variable domain (or half an Fv containing only three CDRs targeting a specific antigen) can recognize and bind to an antigen, although its affinity is lower than that of the entire binding site.

[0072] "Single-chain Fv", also abbreviated as "sFv" or "scFv", refers to a single-chain Fv. H and V L Antibody fragments are formed by linking antibody domains to create a single polypeptide chain. In some embodiments, the scFv polypeptide further comprises a domain located at V... H and V L The polypeptide linkers between the domains allow scFv to form the structures required for antigen binding. For a review of scFv, see Plückthun's work, published in [Journal Name]. The Pharmacology of Monoclonal Antibodies , vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0073] "Amino acid sequence identity percentage" or "homology," in the context of the peptide and antibody sequences described herein, is defined as the percentage of candidate sequences that share the same amino acid residues as the peptide to be compared, after sequence alignment and considering any conserved amino acid substitutions as part of sequence identity. Sequence alignment used to determine the amino acid sequence identity percentage can be performed by various methods known in the art, such as using publicly available computer software, such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR), or MUSCLE software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm required to achieve optimal alignment across the full length of the sequences to be compared. However, for the purposes of this document, the amino acid sequence identity percentage is generated using the MUSCLE sequence alignment computer program (Edgar, RC, Nucleic Acids Research 32(5): 1792-1797, 2004; Edgar, R.C., BMC Bioinformatics 5(1): 113, 2004).

[0074] As used herein, the terms “Fc domain,” “Fc region,” or “crystallizable fragment” refer to the C-terminal domain or region of the immunoglobulin heavy chain, including native sequence Fc regions and variant Fc regions. Although the boundaries of immunoglobulin heavy chain Fc regions may vary, the human IgG heavy chain Fc region is generally defined as extending from the amino acid residue at Cys226 or Pro230 to its carboxyl terminus. The C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) can be removed, for example, during antibody production or purification, or by recombinant engineering of the nucleic acid encoding the antibody heavy chain. Therefore, compositions of complete antibodies may comprise antibody populations with all K447 residues removed, antibody populations without any K447 residues removed, and antibody populations of mixtures that retain and remove K447 residues. Native sequence Fc regions suitable for the antibodies described herein include human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4 Fc regions.

[0075] "Bispecific" or "multispecific" antibodies can refer to antibodies that bind to two or more different antigens, or antibodies that bind to two or more different epitopes on the same antigen.

[0076] A "multivalent" antibody is an antibody with two or more antigen-binding moieties (excluding the IL-18 component), which can bind to the same antigen or different antigens. A "bivalent" antibody is an antibody with two or more antigen-binding moieties (excluding the IL-18 component). A monovalent antibody is an antibody with a single antigen-binding moieties (excluding the IL-18 component).

[0077] "Isolated" nucleic acid molecules encoding the polypeptides or cytokines described herein refer to nucleic acid molecules that have been identified and isolated from at least one contaminating nucleic acid molecule that is normally associated with them in their environment of origin. Preferably, the isolated nucleic acid is no longer associated with any component in its environment of origin. In some embodiments, the isolated nucleic acid molecules encoding the polypeptides or cytokines described herein exist in a form different from their native form or native environment.

[0078] As used in this paper, the term "vector" refers to a nucleic acid molecule capable of amplifying another nucleic acid linked to it. This term includes both vectors existing as self-replicating nucleic acid structures and vectors integrated into the host cell genome to which they are introduced. Some vectors can direct the expression of nucleic acids operatively linked to them. Such vectors are referred to herein as "expression vectors."

[0079] As used in this article, the terms "transfection," "transformation," or "transduction" refer to the process of transferring or introducing exogenous nucleic acids into host cells. "Transfected," "transformed," or "transduced" cells are cells that have been transfected, transformed, or transduced with exogenous nucleic acids. These cells include both the original recipient cells and their progeny cells.

[0080] The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably herein and refer to cells in which exogenous nucleic acids have been introduced, including their progeny cells. Host cells include “transformations” and “transformed cells,” which include the initial transformed cells and their derived progeny cells, without limitation on the number of passages. The nucleic acid content of progeny cells may not be exactly the same as that of the parent cells, and they may contain mutations. All mutant progeny cells that possess the same function or biological activity as the original transformed cells obtained through screening or selection are included within the scope of this document.

[0081] As used herein, “treatment / treating” refers to a method for achieving a beneficial or desired outcome (including clinical outcomes). For the purposes of this application, beneficial or desired clinical outcomes include, but are not limited to, one or more of the following: relief of one or more symptoms caused by the disease, reduction of the severity of the disease, stabilization of the disease state (e.g., prevention or delay of disease progression), prevention or delay of disease spread (e.g., metastasis), prevention or delay of disease recurrence, delay or slowing of disease progression, improvement of the disease state, achievement of disease remission (partial or complete remission), reduction of the dosage of one or more other medications required to treat the disease, delay of disease progression, improvement or enhancement of quality of life, weight gain, and / or prolonged survival. “Treatment” also includes reducing the pathological consequences of cancer (e.g., tumor volume). The methods of this application relate to any one or more of the above-mentioned aspects of treatment.

[0082] In the context of cancer, the term "treatment" includes any or all of the following: inhibiting cancer cell growth, inhibiting cancer cell replication, reducing the overall tumor burden, and improving one or more symptoms associated with the disease.

[0083] The term "inhibition" or "inhibit" refers to the reduction or cessation of any phenotypic trait, or the reduction or cessation of the incidence, degree, or likelihood of that phenotypic trait. "Reduction" or "inhibition" means reducing, decreasing, or stopping an activity, function, and / or quantity relative to a reference value. In some embodiments, "reduction" or "inhibit" means reducing the overall level by 20% or more. In another embodiment, "reduction" or "inhibit" means reducing the overall level by 50% or more. In yet another embodiment, "reduction" or "inhibit" means reducing the overall level by 75%, 85%, 90%, 95%, or more.

[0084] The terms “subject,” “individual,” and “patient” are used interchangeably herein and refer to mammals, including but not limited to humans, cattle, horses, cats, dogs, rodents, or primates. In some embodiments, the individual is a human.

[0085] It should be understood that the embodiments of this application described herein include embodiments that are “composed of” and / or “substantially composed of”.

[0086] When this article refers to "about" a value or parameter, it includes (and describes) changes to that value or parameter itself. For example, a description of "about X" includes a description of "X".

[0087] When this article mentions "not" a certain value or parameter, it usually means and describes something that is "different" from that value or parameter. For example, "This method is not used to treat type X cancer" means that this method is used to treat other types of cancer besides type X cancer.

[0088] The term “about XY” as used in this article has the same meaning as “about X to about Y”.

[0089] Unless the context clearly indicates otherwise, the singular forms “a,” “or,” and “the” used herein and in the appended claims include the plural objects referred to. Construct containing interleukin-18 (IL-18) components

[0090] In one respect, this article provides a construct comprising an IL-18 component associated with a binding portion of a specific binding antigen, wherein the IL-18 component has a reduced binding affinity for the IL-18 receptor compared to a corresponding wild-type IL-18 polypeptide (e.g., SEQ ID NO: 1).

[0091] On the other hand, this document provides a construct comprising an IL-18 component associated with a binding moiety that specifically binds to an antigen, wherein the IL-18 component comprises: 1) an IL-18 polypeptide comprising one or more modifications compared to a wild-type IL-18 polypeptide; 2) an IL-18 polypeptide and a masking moiety, wherein the IL-18 polypeptide is linked to the masking moiety via a cleavable linker; or 3) an IL-18 polypeptide and a peptide linker linking the IL-18 polypeptide to the binding moiety, wherein the peptide linker is no more than 15 amino acids in length, and the IL-18 component exhibits a reduced binding affinity to the IL-18 receptor compared to a corresponding wild-type IL-18 polypeptide (e.g., SEQ ID NO: 1). In some embodiments, the wild-type IL-18 polypeptide is human IL-18. In some embodiments, the wild-type IL-18 is human IL-18 comprising the amino acid sequence shown in SEQ ID NO: 1. YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 1)

[0092] In one aspect, this document provides a construct comprising an IL-18 component associated with a binding moiety of a specifically binding antigen, wherein the IL-18 component comprises an IL-18 polypeptide that includes one or more modifications compared to a wild-type IL-18 polypeptide, and wherein the IL-18 component exhibits reduced binding affinity to the IL-18 receptor compared to the corresponding wild-type IL-18 polypeptide (e.g., SEQ ID NO: 1). In some embodiments, the wild-type IL-18 polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1.

[0093] On the other hand, this document provides a construct comprising an IL-18 component associated with a binding moiety of a specifically binding antigen, wherein the IL-18 component comprises an IL-18 polypeptide comprising one or more modifications compared to a wild-type IL-18 polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1, and wherein the IL-18 component has a reduced binding affinity for the IL-18 receptor compared to the corresponding wild-type IL-18 polypeptide (e.g., SEQ ID NO: 1).

[0094] In one aspect, this document provides a construct comprising an IL-18 component associated with a binding moiety of a specifically binding antigen, wherein the IL-18 component comprises an IL-18 polypeptide and a masking moiety, the IL-18 polypeptide being linked to the masking moiety via a cleavable linker, and the IL-18 component exhibiting reduced binding affinity to the IL-18 receptor compared to a corresponding wild-type IL-18 polypeptide (e.g., SEQ ID NO: 1). In some embodiments, the IL-18 component comprises, from N-terminus to C-terminus,: a) a masking moiety; b) a cleavable linker; and c) an IL-18 polypeptide. In some embodiments, the masking moiety comprises a truncated propeptide of an IL-18 precursor. In some embodiments, the truncated propeptide comprises SEQ ID NO: 336.

[0095] In one aspect, this document provides a construct comprising an IL-18 component associated with a binding moiety of a specifically binding antigen, wherein the IL-18 component comprises an IL-18 polypeptide and a masking moiety, the IL-18 polypeptide being linked to the masking moiety via a cleavable linker, and the IL-18 component exhibiting reduced binding affinity to the IL-18 receptor compared to a corresponding wild-type IL-18 polypeptide (e.g., SEQ ID NO: 1). In some embodiments, the IL-18 component comprises, from N-terminus to C-terminus,: a) a masking moiety; b) a cleavable linker; and c) an IL-18 polypeptide. In some embodiments, the IL-18 component comprises, from N-terminus to C-terminus,: a) a masking portion comprising a truncated propeptide of the IL-18 precursor, the truncated propeptide comprising the amino acid sequence shown in SEQ ID NO: 336; b) a cleavable linker; and c) an IL-18 polypeptide comprising the amino acid sequence shown in any one of SEQ ID NO: 1-299, 307-318, 465-469, and 482-491. In some embodiments, the IL-18 component comprises, from N-terminus to C-terminus,: a) a masking portion comprising a truncated propeptide of the IL-18 precursor, the truncated propeptide comprising the amino acid sequence shown in SEQ ID NO: 336; b) a cleavable linker; and c) an IL-18 polypeptide comprising the amino acid sequence shown in any one of SEQ ID NO: 1-299, 307-318, 465-469, and 482-491, wherein the length of the cleavable linker is not more than about 50, about 40, about 30, about 25, about 20, about 15, about 12, or about 10 amino acids. In some embodiments, the IL-18 component comprises, from N-terminus to C-terminus,: a) a masking portion comprising a truncated propeptide of the IL-18 precursor, the truncated propeptide comprising the amino acid sequence shown in SEQ ID NO: 336; b) a cleavable linker; and c) an IL-18 polypeptide comprising the amino acid sequence shown in any one of SEQ ID NO: 1-299, 307-318, 465-469, and 482-491, wherein the length of the cleavable linker is not more than about 50, 40, 30, 25, 20, 15, 12, or 10 amino acids.

[0096] In one aspect, this document provides a construct comprising an IL-18 component associated with a binding moiety of a specifically binding antigen (e.g., PD-1), wherein the IL-18 component comprises an IL-18 polypeptide comprising amino acid substitutions selected from the group consisting of G108, H109, ​​K112, D110, R147, M150, G145, K79, D98, K84, K4, L5, K8, R13, D17, E31, M33, T34, D35, D40, K53, R58, M60, R104, and D132, wherein the amino acid positions are relative to the wild-type human IL-18 polypeptide shown in SEQ ID NO: 1. In some embodiments, the substitution is an alanine substitution. In some embodiments, the amino acid substitution is selected from the group consisting of G108, H109, ​​K112, D110, R147, M150, and G145. In some embodiments, the IL-18 component further comprises a masking portion fused to the IL-18 polypeptide via a cleavable linker. In some embodiments, the masking portion comprises an IL-18 propeptide (e.g., SEQ ID NO: 333 or 336). In some embodiments, the masking portion comprises an extracellular domain of IL-18Rα, an extracellular domain of IL-18Rβ, an IL-18 binding protein, a fragment of any of the foregoing, or a variant of any of the foregoing (e.g., an amino acid sequence shown in any of SEQ ID NO: 353-356, or a variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity thereto). In some embodiments, the cleavable linker comprises one or more amino acid sequences selected from the group consisting of: SEQ ID NO: 337, SEQ ID NO: 338, SEQ ID NO: 339, SEQ ID NO: 372-377, SEQ ID NO: 399-407, SEQ ID NO: 492-493, and 495. In some embodiments, the IL-18 polypeptide comprises G3P, E6K, D54G, Q56G, P57A, C38I, C68S, S117C, and N91T. In some embodiments, the IL-18 polypeptide comprises E6G, D54L, Q56T, P57E, N91G, C38I, C68S, S117C, and R104S. In some embodiments, the IL-18 polypeptide further comprises 76C and 127C. In some embodiments, the IL-18 polypeptide further comprises at least one of G108A, H109A, and K112A. In some embodiments, the IL-18 component is fused to the binding moiety via a peptide linker, wherein the peptide linker is no more than 15 amino acids in length (e.g., no more than about 12, 10, 8, or 5 amino acids).In some embodiments, the binding portion comprises an antibody containing an Fc domain, wherein the peptide linker is fused to the C-terminus of the Fc domain. In some embodiments, the antibody is a full-length antibody, scFv, or VHH.

[0097] In one aspect, this document provides a construct comprising an IL-18 component associated with a binding moiety of a specifically binding antigen (e.g., PD-1), wherein the IL-18 component comprises an IL-18 polypeptide and a masking moiety, the IL-18 polypeptide being linked to the masking moiety via a cleavable linker, the masking moiety comprising: a) a truncated propeptide of an IL-18 precursor (e.g., the amino acid sequence shown in SEQ ID NO: 336), or b) an extracellular domain of IL-18Rα or a fragment thereof (e.g., the amino acid sequence shown in any one of SEQ ID NO: 353-356). In some embodiments, the cleavable linker comprises two or more sets of amino acid sequences that are recognized and cleaved by a protease. In some embodiments, the cleavable linker comprises one or more sets of amino acid sequences that are recognized and cleaved by two or more proteases. In some embodiments, the one or more proteases comprise one or more tumor microenvironment (TME) proteases, and the one or more TME proteases are selected from the group consisting of: urokinase-type plasminogen activator, Matriptase, Legumain, prostate-specific antigen, dipeptidyl peptidase, hepsin, matrix metalloproteinases, integrin-dissociating metalloproteinases, human leukocyte elastase, protease 3, prourokinase, plasminogen, staphylococcal kinase, cathepsin, tissue kallikrein, and kallikrein-related peptidase. In some embodiments, the amino acid sequence is recognized and cleaved by a) Legumain and one, two, or three of MMP9, MMP2, and MMP14; b) uPA and one, two, or three of MMP9, MMP2, and MMP14; or c) all of uPA, Legumain, MMP2, MMP14, and MMP9. In some embodiments, the cleavable linker comprises one or more amino acid sequences selected from the group consisting of: SEQ ID NO: 337, SEQ ID NO: 338, SEQ ID NO: 339, SEQ ID NO: 372-377, SEQ ID NO: 399-407, SEQ ID NO: 492-493, and 495. In some embodiments, the IL-18 polypeptide comprises G3P, E6K, D54G, Q56G, P57A, C38I, C68S, S117C, and N91T. In some embodiments, the IL-18 polypeptide comprises E6G, D54L, Q56T, P57E, N91G, C38I, C68S, S117C, and R104S. In some embodiments, the IL-18 polypeptide further comprises 76C and 127C. In some embodiments, the IL-18 polypeptide further comprises at least one of G108A, H109A, and K112A.In some embodiments, the IL-18 component is fused to the binding moiety via a peptide linker, wherein the peptide linker is no more than 15 amino acids in length (e.g., no more than about 12, 10, 8, or 5 amino acids). In some embodiments, the binding moiety comprises an antibody containing an Fc domain, wherein the peptide linker is fused to the C-terminus of the Fc domain. In some embodiments, the antibody is a full-length antibody, scFv, or VHH. In some embodiments, the IL-18 component comprises, from N-terminus to C-terminus, a masking moiety, a cleavable linker, and an IL-18 polypeptide. In some embodiments, the IL-18 component comprises, from N-terminus to C-terminus, an IL-18 polypeptide, a cleavable linker, and a masking moiety. In some embodiments, the antibody is a full-length antibody.

[0098] In one aspect, this document provides a construct comprising an IL-18 component associated with a binding moiety of a specifically binding antigen (e.g., PD-1), wherein the IL-18 component comprises an IL-18 polypeptide and a peptide linker, the peptide linker connecting the IL-18 polypeptide to the binding moiety, and the peptide linker being no more than about 15 amino acids in length (e.g., no more than about 12, 10, 8, or 5 amino acids). In some embodiments, the binding moiety has an Fc domain, wherein the IL-18 component is fused to the C-terminus of the Fc domain. In some embodiments, the binding moiety comprises a full-length antibody comprising two heavy chains and two light chains. In some embodiments, the IL-18 component is fused to the C-terminus of one or both heavy chains. In some embodiments, the IL-18 component is fused to the C-terminus of one or two light chains. In some embodiments, the binding moiety comprises two polypeptides, each polypeptide comprising an scFv domain and an Fc region, the two Fc regions forming an Fc domain, and the IL-18 component being fused to the C-terminus of one or both polypeptides. In some embodiments, the binding portion comprises two polypeptides, each polypeptide comprising a VHH domain and an Fc region, the two Fc regions forming an Fc domain, and the IL-18 component is fused to the C-terminus of one or both polypeptides. In some embodiments, the IL-18 polypeptide comprises G3P, E6K, D54G, Q56G, P57A, C38I, C68S, S117C, and N91T. In some embodiments, the IL-18 polypeptide comprises E6G, D54L, Q56T, P57E, N91G, C38I, C68S, S117C, and R104S. In some embodiments, the IL-18 polypeptide further comprises 76C and 127C. In some embodiments, the IL-18 polypeptide further comprises at least one of G108A, H109A, and K112A.

[0099] In one aspect, this document provides a construct comprising an IL-18 component associated with a binding moiety of a specifically binding antigen, wherein the IL-18 component comprises an IL-18 polypeptide and a peptide linker connecting the IL-18 polypeptide to the binding moiety, the peptide linker being no more than 15 amino acids in length, and the IL-18 component exhibiting reduced binding affinity for the IL-18 receptor compared to a corresponding wild-type IL-18 polypeptide (e.g., SEQ ID NO: 1). An exemplary construct is shown below. Exemplary constructs containing the IL-18 component A heavy chain alternative that can be used in conjunction with a light chain.

[0100] In some embodiments, this document provides a construct comprising an IL-18 component associated with a binding moiety, wherein the binding moiety comprises a full-length antibody.

[0101] In some embodiments, the construct includes an IL-18 component associated with a binding domain, the binding domain comprising a full-length antibody that binds to an antigen, wherein the full-length antibody comprises two heavy chains and two light chains, and the IL-18 component is fused to the C-terminus of only one heavy chain.

[0102] In some embodiments, the construct comprises: a) first and second polypeptides, each comprising the amino acid sequence shown in SEQ ID NO: 419, or a functional variant thereof, the functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 419; and b) third and fourth polypeptides, each comprising the amino acid sequence shown in SEQ ID NO: 420, or a functional variant thereof, the functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 420.

[0103] In some embodiments, the construct comprises: a) first and second polypeptides, each comprising the amino acid sequence shown in SEQ ID NO: 421, or a functional variant thereof, the functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 421; and b) third and fourth polypeptides, each comprising the amino acid sequence shown in SEQ ID NO: 420, or a functional variant thereof, the functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 420.

[0104] In some embodiments, the construct comprises: a) first and second polypeptides, each comprising the amino acid sequence shown in SEQ ID NO: 422, or a functional variant thereof, the functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 422; and b) third and fourth polypeptides, each comprising the amino acid sequence shown in SEQ ID NO: 423, or a functional variant thereof, the functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 423.

[0105] In some embodiments, the construct comprises: a) first and second polypeptides, each comprising the amino acid sequence shown in SEQ ID NO: 424, or a functional variant thereof, the functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 424; and b) third and fourth polypeptides, each comprising the amino acid sequence shown in SEQ ID NO: 420, or a functional variant thereof, the functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 420.

[0106] In some embodiments, the construct comprises: a) first and second polypeptides, each comprising the amino acid sequence shown in SEQ ID NO: 425, or a functional variant thereof, the functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 425; and b) third and fourth polypeptides, each comprising the amino acid sequence shown in SEQ ID NO: 420, or a functional variant thereof, the functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 420.

[0107] In some embodiments, the construct comprises: a) first and second polypeptides, each comprising the amino acid sequence shown in SEQ ID NO: 426, or a functional variant thereof, the functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 426; and b) third and fourth polypeptides, each comprising the amino acid sequence shown in SEQ ID NO: 420, or a functional variant thereof, the functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 420.

[0108] In some embodiments, the construct comprises: a) first and second polypeptides, each comprising the amino acid sequence shown in SEQ ID NO: 427, or a functional variant thereof, the functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 427; and b) third and fourth polypeptides, each comprising the amino acid sequence shown in SEQ ID NO: 420, or a functional variant thereof, the functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 420.

[0109] In some embodiments, the construct comprises: a) first and second polypeptides, each comprising the amino acid sequence shown in SEQ ID NO: 428, or a functional variant thereof, the functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 428; and b) third and fourth polypeptides, each comprising the amino acid sequence shown in SEQ ID NO: 420, or a functional variant thereof, the functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 420.

[0110] In some embodiments, the construct comprises: a) first and second polypeptides, each comprising the amino acid sequence shown in SEQ ID NO: 429, or a functional variant thereof, the functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 429; and b) third and fourth polypeptides, each comprising the amino acid sequence shown in SEQ ID NO: 420, or a functional variant thereof, the functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 420.

[0111] In some embodiments, the construct comprises: a) first and second polypeptides, each comprising the amino acid sequence shown in SEQ ID NO: 430, or a functional variant thereof, the functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 430; and b) third and fourth polypeptides, each comprising the amino acid sequence shown in SEQ ID NO: 420, or a functional variant thereof, the functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 420.

[0112] In some embodiments, the construct comprises: a) first and second polypeptides, each comprising the amino acid sequence shown in SEQ ID NO: 431, or a functional variant thereof, the functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 431; and b) third and fourth polypeptides, each comprising the amino acid sequence shown in SEQ ID NO: 420, or a functional variant thereof, the functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 420.

[0113] In some embodiments, the construct comprises: a) first and second polypeptides, each comprising the amino acid sequence shown in SEQ ID NO: 432, or a functional variant thereof, the functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 432; and b) third and fourth polypeptides, each comprising the amino acid sequence shown in SEQ ID NO: 420, or a functional variant thereof, the functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 420.

[0114] In some embodiments, the construct comprises: a) first and second polypeptides, each comprising the amino acid sequence shown in SEQ ID NO: 433, or a functional variant thereof, the functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 433; and b) third and fourth polypeptides, each comprising the amino acid sequence shown in SEQ ID NO: 420, or a functional variant thereof, the functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 420.

[0115] In some embodiments, the construct comprises: a) first and second polypeptides, each comprising the amino acid sequence shown in SEQ ID NO: 434, or a functional variant thereof, the functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 434; and b) third and fourth polypeptides, each comprising the amino acid sequence shown in SEQ ID NO: 420, or a functional variant thereof, the functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 420.

[0116] In some embodiments, the construct comprises: a) first and second polypeptides, each comprising the amino acid sequence shown in SEQ ID NO: 435, or a functional variant thereof, the functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 435; and b) third and fourth polypeptides, each comprising the amino acid sequence shown in SEQ ID NO: 420, or a functional variant thereof, the functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 420.

[0117] In some embodiments, the construct comprises: a) first and second polypeptides, each comprising the amino acid sequence shown in SEQ ID NO: 436, or a functional variant thereof, the functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 436; and b) third and fourth polypeptides, each comprising the amino acid sequence shown in SEQ ID NO: 437, or a functional variant thereof, the functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 437.

[0118] In some embodiments, the construct comprises: a) first and second polypeptides, each comprising the amino acid sequence shown in SEQ ID NO: 439, or a functional variant thereof, the functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 439; and b) third and fourth polypeptides, each comprising the amino acid sequence shown in SEQ ID NO: 437, or a functional variant thereof, the functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 437.

[0119] In some embodiments, the construct comprises: a) first and second polypeptides, each comprising the amino acid sequence shown in SEQ ID NO: 438, or a functional variant thereof, the functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 438; and b) third and fourth polypeptides, each comprising the amino acid sequence shown in SEQ ID NO: 437, or a functional variant thereof, the functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 437.

[0120] In some embodiments, the construct comprises: a) first and second polypeptides, each comprising the amino acid sequence shown in SEQ ID NO: 440, or a functional variant thereof, the functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 440; and b) third and fourth polypeptides, each comprising the amino acid sequence shown in SEQ ID NO: 437, or a functional variant thereof, the functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 437.

[0121] In some embodiments, the construct comprises: a) first and second polypeptides, each comprising the amino acid sequence shown in SEQ ID NO: 441, or a functional variant thereof, the functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 441; and b) third and fourth polypeptides, each comprising the amino acid sequence shown in SEQ ID NO: 420, or a functional variant thereof, the functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 420.

[0122] In some embodiments, the construct comprises: a) first and second polypeptides, each comprising the amino acid sequence shown in SEQ ID NO: 442, or a functional variant thereof, the functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 442; and b) third and fourth polypeptides, each comprising the amino acid sequence shown in SEQ ID NO: 420, or a functional variant thereof, the functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 420.

[0123] In some embodiments, the construct comprises: a) first and second polypeptides, each comprising the amino acid sequence shown in SEQ ID NO: 443, or a functional variant thereof, the functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 443; and b) third and fourth polypeptides, each comprising the amino acid sequence shown in SEQ ID NO: 420, or a functional variant thereof, the functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 420.

[0124] In some embodiments, the construct comprises: a) first and second polypeptides, each comprising the amino acid sequence shown in SEQ ID NO: 444, or a functional variant thereof, the functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 444; and b) third and fourth polypeptides, each comprising the amino acid sequence shown in SEQ ID NO: 420, or a functional variant thereof, the functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 420.

[0125] In some embodiments, the construct comprises: a) first and second polypeptides, each comprising the amino acid sequence shown in SEQ ID NO: 445, or a functional variant thereof, the functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 445; and b) third and fourth polypeptides, each comprising the amino acid sequence shown in SEQ ID NO: 420, or a functional variant thereof, the functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 420.

[0126] In some embodiments, the construct comprises: a) first and second polypeptides, each comprising the amino acid sequence shown in SEQ ID NO: 448, or a functional variant thereof, the functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 448; and b) third and fourth polypeptides, each comprising the amino acid sequence shown in SEQ ID NO: 420, or a functional variant thereof, the functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 420.

[0127] In some embodiments, the construct comprises: a) first and second polypeptides, each comprising the amino acid sequence shown in SEQ ID NO: 449, or a functional variant thereof, the functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 449; and b) third and fourth polypeptides, each comprising the amino acid sequence shown in SEQ ID NO: 420, or a functional variant thereof, the functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 420.

[0128] In some embodiments, the construct comprises: a) a first polypeptide comprising the amino acid sequence shown in SEQ ID NO: 446, or a functional variant thereof, the functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 446; b) a second polypeptide comprising the amino acid sequence shown in SEQ ID NO: 447, or a functional variant thereof, the functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 447; and c) a third and a fourth polypeptide, each comprising the amino acid sequence shown in SEQ ID NO: 420, or a functional variant thereof, the functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 420.

[0129] In some embodiments, the construct comprises: a) a first polypeptide comprising the amino acid sequence shown in SEQ ID NO: 450, or a functional variant thereof, the functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 450; b) a second polypeptide comprising the amino acid sequence shown in SEQ ID NO: 451, or a functional variant thereof, the functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 451; and c) a third and a fourth polypeptide, each comprising the amino acid sequence shown in SEQ ID NO: 420, or a functional variant thereof, the functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 420.

[0130] In some embodiments, the construct comprises: a) a first polypeptide comprising the amino acid sequence shown in SEQ ID NO: 452, or a functional variant thereof, the functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 452; b) a second polypeptide comprising the amino acid sequence shown in SEQ ID NO: 451, or a functional variant thereof, the functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 451; and c) a third and a fourth polypeptide, each comprising the amino acid sequence shown in SEQ ID NO: 420, or a functional variant thereof, the functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 420.

[0131] In some embodiments, the construct comprises: a) a first polypeptide comprising the amino acid sequence shown in SEQ ID NO: 453, or a functional variant thereof, the functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 453; b) a second polypeptide comprising the amino acid sequence shown in SEQ ID NO: 451, or a functional variant thereof, the functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 451; and c) a third and a fourth polypeptide, each comprising the amino acid sequence shown in SEQ ID NO: 420, or a functional variant thereof, the functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 420.

[0132] In some embodiments, the construct comprises: a) a first polypeptide comprising the amino acid sequence shown in SEQ ID NO: 454, or a functional variant thereof, the functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 454; b) a second polypeptide comprising the amino acid sequence shown in SEQ ID NO: 451, or a functional variant thereof, the functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 451; and c) a third and a fourth polypeptide, each comprising the amino acid sequence shown in SEQ ID NO: 420, or a functional variant thereof, the functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 420.

[0133] In some embodiments, the construct comprises: a) a first polypeptide comprising the amino acid sequence shown in SEQ ID NO: 455, or a functional variant thereof, the functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 455; b) a second polypeptide comprising the amino acid sequence shown in SEQ ID NO: 451, or a functional variant thereof, the functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 451; and c) a third and a fourth polypeptide, each comprising the amino acid sequence shown in SEQ ID NO: 420, or a functional variant thereof, the functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 420.

[0134] In some embodiments, the construct comprises: a) a first polypeptide comprising the amino acid sequence shown in SEQ ID NO: 456, or a functional variant thereof, the functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 456; b) a second polypeptide comprising the amino acid sequence shown in SEQ ID NO: 451, or a functional variant thereof, the functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 451; and c) a third and a fourth polypeptide, each comprising the amino acid sequence shown in SEQ ID NO: 420, or a functional variant thereof, the functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 420.

[0135] In some embodiments, the construct comprises: a) a first polypeptide comprising the amino acid sequence shown in SEQ ID NO: 457, or a functional variant thereof, the functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 457; b) a second polypeptide comprising the amino acid sequence shown in SEQ ID NO: 451, or a functional variant thereof, the functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 451; and c) a third and a fourth polypeptide, each comprising the amino acid sequence shown in SEQ ID NO: 420, or a functional variant thereof, the functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 420.

[0136] In some embodiments, the construct comprises: a) a first polypeptide comprising the amino acid sequence shown in SEQ ID NO: 458, or a functional variant thereof, the functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 458; b) a second polypeptide comprising the amino acid sequence shown in SEQ ID NO: 451, or a functional variant thereof, the functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 451; and c) a third and a fourth polypeptide, each comprising the amino acid sequence shown in SEQ ID NO: 420, or a functional variant thereof, the functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 420.

[0137] In some embodiments, the construct comprises: a) a first polypeptide comprising the amino acid sequence shown in SEQ ID NO: 459, or a functional variant thereof, the functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 459; b) a second polypeptide comprising the amino acid sequence shown in SEQ ID NO: 451, or a functional variant thereof, the functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 451; and c) a third and a fourth polypeptide, each comprising the amino acid sequence shown in SEQ ID NO: 420, or a functional variant thereof, the functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 420.

[0138] In some embodiments, the construct comprises: a) a first polypeptide comprising the amino acid sequence shown in SEQ ID NO: 460, or a functional variant thereof, the functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 460; b) a second polypeptide comprising the amino acid sequence shown in SEQ ID NO: 451, or a functional variant thereof, the functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 451; and c) a third and a fourth polypeptide, each comprising the amino acid sequence shown in SEQ ID NO: 420, or a functional variant thereof, the functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 420.

[0139] In some embodiments, the construct comprises: a) a first polypeptide comprising the amino acid sequence shown in SEQ ID NO: 461, or a functional variant thereof, the functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 461; b) a second polypeptide comprising the amino acid sequence shown in SEQ ID NO: 451, or a functional variant thereof, the functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 451; and c) a third and a fourth polypeptide, each comprising the amino acid sequence shown in SEQ ID NO: 420, or a functional variant thereof, the functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 420.

[0140] In some embodiments, the construct comprises: a) a first polypeptide comprising the amino acid sequence shown in SEQ ID NO: 462, or a functional variant thereof, the functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 462; b) a second polypeptide comprising the amino acid sequence shown in SEQ ID NO: 451, or a functional variant thereof, the functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 451; and c) a third and a fourth polypeptide, each comprising the amino acid sequence shown in SEQ ID NO: 420, or a functional variant thereof, the functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 420.

[0141] In some embodiments, the construct comprises: a) a first polypeptide comprising the amino acid sequence shown in SEQ ID NO: 463, or a functional variant thereof, the functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 463; b) a second polypeptide comprising the amino acid sequence shown in SEQ ID NO: 451, or a functional variant thereof, the functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 451; and c) a third and a fourth polypeptide, each comprising the amino acid sequence shown in SEQ ID NO: 420, or a functional variant thereof, the functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 420.

[0142] In some embodiments, the construct comprises: a) a first polypeptide comprising the amino acid sequence shown in SEQ ID NO: 464, or a functional variant thereof, the functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 450; b) a second polypeptide comprising the amino acid sequence shown in SEQ ID NO: 464, or a functional variant thereof, the functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 451; and c) a third and a fourth polypeptide, each comprising the amino acid sequence shown in SEQ ID NO: 420, or a functional variant thereof, the functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 420.

[0143] In one aspect, this document provides a construct comprising an IL-18 component associated with a binding moiety of a specifically binding antigen, wherein the IL-18 component comprises an IL-18 polypeptide and a masking moiety, the IL-18 polypeptide being linked to the masking moiety via a cleavable linker, and the IL-18 component exhibiting a reduced binding affinity for the IL-18 receptor compared to a corresponding wild-type IL-18 polypeptide (e.g., SEQ ID NO: 1). In some embodiments, the IL-18 component comprises, from N-terminus to C-terminus,: a) a masking moiety; b) a cleavable linker; and c) an IL-18 polypeptide. In some embodiments, the IL-18 component comprises, from N-terminus to C-terminus,: a) a masking moiety comprising SEQ ID NO: 336; b) a cleavable linker comprising SEQ ID NO: 337; and c) an IL-18 polypeptide comprising SEQ ID NO: 133. In some embodiments, the IL-18 component comprises, from N-terminus to C-terminus,: a) a masking portion comprising SEQ ID NO: 336; b) a cleavable linker comprising SEQ ID NO: 337; and c) an IL-18 polypeptide comprising SEQ ID NO: 133, wherein the length of the cleavable linker is not more than about 50, about 40, about 30, about 25, about 20, about 15, about 12, or about 10 amino acids. In some embodiments, the IL-18 component comprises, from N-terminus to C-terminus,: a) a masking portion comprising SEQ ID NO: 336; b) a cleavable linker comprising SEQ ID NO: 337; and c) an IL-18 polypeptide comprising SEQ ID NO: 133, wherein the length of the cleavable linker is not more than about 50, 40, 30, 25, 20, 15, 12, or 10 amino acids.

[0144] In one aspect, this document provides a construct comprising an IL-18 component associated with a binding moiety of a specifically binding antigen, wherein the IL-18 component comprises an IL-18 polypeptide and a masking moiety, the IL-18 polypeptide being linked to the masking moiety via a cleavable linker, and the IL-18 component exhibiting reduced binding affinity to the IL-18 receptor compared to a corresponding wild-type IL-18 polypeptide (e.g., SEQ ID NO: 1). In some embodiments, the IL-18 component comprises, from N-terminus to C-terminus,: a) a masking moiety; b) a cleavable linker; and c) an IL-18 polypeptide. In some embodiments, the IL-18 component comprises, from N-terminus to C-terminus,: a) a masking portion comprising SEQ ID NO: 336; b) a cleavable linker comprising SEQ ID NO: 337; and c) an IL-18 polypeptide comprising one or more modifications, wherein the one or more modifications comprise amino acid substitutions selected from the group consisting of G108, H109, ​​K112, D110, R147, M150, G145, K79, D98, and K84, wherein the amino acid positions are relative to the wild-type human IL-18 polypeptide represented by SEQ ID NO: 1. In some embodiments, the IL-18 component comprises, from N-terminus to C-terminus,: a) a masking portion comprising SEQ ID NO: 336; b) a cleavable linker comprising SEQ ID NO: 337; and c) an IL-18 polypeptide comprising one or more modifications, wherein the one or more modifications comprise amino acid substitutions selected from the group consisting of G108, H109, ​​K112, D110, R147, M150, G145, K79, D98, and K84, wherein the amino acid positions are relative to the wild-type human IL-18 polypeptide shown in SEQ ID NO: 1, and the length of the cleavable linker is no more than about 50, about 40, about 30, about 25, about 20, about 15, about 12, or about 10 amino acids. In some embodiments, the IL-18 component comprises, from N-terminus to C-terminus,: a) a masking portion comprising SEQ ID NO: 336; b) a cleavable linker comprising SEQ ID NO: 337; and c) an IL-18 polypeptide comprising one or more modifications, wherein the one or more modifications comprise amino acid substitutions selected from the group consisting of G108, H109, ​​K112, D110, R147, M150, G145, K79, D98, and K84, wherein the amino acid positions are relative to the wild-type human IL-18 polypeptide shown in SEQ ID NO: 1, and the length of the cleavable linker does not exceed about 50, 40, 30, 25, 20, 15, 12, or 10 amino acids. In some embodiments, the substitution is an alanine substitution.

[0145] In some embodiments, the IL-18 component comprises, from N-terminus to C-terminus,: a) a masking portion comprising SEQ ID NO: 336; b) a cleavable linker comprising SEQ ID NO: 337; and c) an IL-18 polypeptide comprising an amino acid substitution at position G108, wherein the amino acid position is relative to the wild-type human IL-18 polypeptide shown in SEQ ID NO: 1. In some embodiments, the substitution is G108A. In some embodiments, the IL-18 polypeptide is referred to as “MM5 A1”.

[0146] In some embodiments, the IL-18 component comprises, from N-terminus to C-terminus,: a) a masking portion comprising SEQ ID NO: 336; b) a cleavable linker comprising SEQ ID NO: 337; and c) an IL-18 polypeptide comprising an amino acid substitution at position H109, ​​wherein the amino acid position is relative to the wild-type human IL-18 polypeptide shown in SEQ ID NO: 1. In some embodiments, the substitution is H109A. In some embodiments, the IL-18 polypeptide is referred to as “MM5 A2”.

[0147] In some embodiments, the IL-18 component comprises, from N-terminus to C-terminus,: a) a masking portion comprising SEQ ID NO: 336; b) a cleavable linker comprising SEQ ID NO: 337; and c) an IL-18 polypeptide comprising an amino acid substitution at position K112, wherein the amino acid position is relative to the wild-type human IL-18 polypeptide shown in SEQ ID NO: 1. In some embodiments, the substitution is K112A. In some embodiments, the IL-18 polypeptide is referred to as “MM5 A3”.

[0148] In some embodiments, the IL-18 component comprises, from N-terminus to C-terminus,: a) a masking portion comprising SEQ ID NO: 336; b) a cleavable linker comprising SEQ ID NO: 337; and c) an IL-18 polypeptide comprising an amino acid substitution at position D110, wherein the amino acid position is relative to the wild-type human IL-18 polypeptide shown in SEQ ID NO: 1. In some embodiments, the substitution is D110A. In some embodiments, the IL-18 polypeptide is referred to as “MM5 A4”.

[0149] In some embodiments, the IL-18 component comprises, from N-terminus to C-terminus,: a) a masking portion comprising SEQ ID NO: 336; b) a cleavable linker comprising SEQ ID NO: 337; and c) an IL-18 polypeptide comprising an amino acid substitution at position R147, wherein the amino acid position is relative to the wild-type human IL-18 polypeptide shown in SEQ ID NO: 1. In some embodiments, the substitution is R147A. In some embodiments, the IL-18 polypeptide is referred to as “MM5 A5”.

[0150] In some embodiments, the IL-18 component comprises, from N-terminus to C-terminus,: a) a masking portion comprising SEQ ID NO: 336; b) a cleavable linker comprising SEQ ID NO: 337; and c) an IL-18 polypeptide comprising an amino acid substitution at position M150, wherein the amino acid position is relative to the wild-type human IL-18 polypeptide shown in SEQ ID NO: 1. In some embodiments, the substitution is M150A. In some embodiments, the IL-18 polypeptide is referred to as “MM5 A6”.

[0151] In some embodiments, the IL-18 component comprises, from N-terminus to C-terminus,: a) a masking portion comprising SEQ ID NO: 336; b) a cleavable linker comprising SEQ ID NO: 337; and c) an IL-18 polypeptide comprising an amino acid substitution at position G145, wherein the amino acid position is relative to the wild-type human IL-18 polypeptide shown in SEQ ID NO: 1. In some embodiments, the substitution is G145A. In some embodiments, the IL-18 polypeptide is referred to as “MM5 A7”.

[0152] In one aspect, this document provides a construct comprising an IL-18 component associated with a binding moiety of a specifically binding antigen, wherein the IL-18 component comprises an IL-18 polypeptide and a masking moiety, the IL-18 polypeptide being linked to the masking moiety via a cleavable linker, and the IL-18 component exhibiting a reduced binding affinity for the IL-18 receptor compared to a corresponding wild-type IL-18 polypeptide (e.g., SEQ ID NO: 1). In some embodiments, the IL-18 component comprises, from N-terminus to C-terminus,: a) a masking moiety; b) a cleavable linker; and c) an IL-18 polypeptide. In some embodiments, the IL-18 component comprises, from N-terminus to C-terminus,: a) a masking moiety comprising SEQ ID NO: 336; b) a cleavable linker comprising SEQ ID NO: 337; and c) an IL-18 polypeptide comprising SEQ ID NO: 39. In some embodiments, the IL-18 component comprises, from N-terminus to C-terminus,: a) a masking portion comprising SEQ ID NO: 336; b) a cleavable linker comprising SEQ ID NO: 337; and c) an IL-18 polypeptide comprising SEQ ID NO: 39, wherein the length of the cleavable linker is not more than about 50, about 40, about 30, about 25, about 20, about 15, about 12, or about 10 amino acids. In some embodiments, the IL-18 component comprises, from N-terminus to C-terminus,: a) a masking portion comprising SEQ ID NO: 336; b) a cleavable linker comprising SEQ ID NO: 337; and c) an IL-18 polypeptide comprising SEQ ID NO: 39, wherein the length of the cleavable linker is not more than about 50, 40, 30, 25, 20, 15, 12, or 10 amino acids.

[0153] In some embodiments, this document provides a construct comprising an IL-18 component associated with a binding moiety of a specifically binding antigen, wherein the IL-18 component comprises an IL-18 polypeptide and a peptide linker linking the IL-18 polypeptide to the binding moiety, the peptide linker being no more than 15 amino acids in length, and the IL-18 component exhibiting reduced binding affinity to the IL-18 receptor compared to a corresponding wild-type IL-18 polypeptide (e.g., SEQ ID NO: 1).

[0154] In some embodiments, the IL-18 component comprises a peptide linker that prevents (e.g., inhibits) the IL-18 peptide from activating IL-18 receptor-mediated signaling (e.g., human IL-18 receptor-mediated signaling). In some embodiments, the IL-18 component has a peptide linker to prevent (e.g., inhibit) the IL-18 peptide from activating IL-18 receptor-mediated signaling (e.g., human IL-18 receptor-mediated signaling). In some embodiments, the IL-18 component has a peptide linker to prevent (e.g., inhibit) the IL-18 peptide from activating IL-18 receptor-mediated signaling (e.g., human IL-18 receptor-mediated signaling).

[0155] In some embodiments, the IL-18 peptide is fused to a peptide linker that prevents (e.g., inhibits) the IL-18 peptide from activating IL-18 receptor-mediated signaling (e.g., human IL-18 receptor-mediated signaling). In some embodiments, the IL-18 peptide is fused to a peptide linker to prevent (e.g., inhibit) the IL-18 peptide from activating IL-18 receptor-mediated signaling (e.g., human IL-18 receptor-mediated signaling). In some embodiments, the IL-18 peptide is fused to a peptide linker to prevent (e.g., inhibit) the IL-18 peptide from activating IL-18 receptor-mediated signaling (e.g., human IL-18 receptor-mediated signaling).

[0156] In some embodiments, the peptide linker comprises the amino acid sequence shown in any one of SEQ ID NO: 353-356, or a functional variant thereof, the functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with any one of SEQ ID NO: 353-356. In some embodiments, the cleavable linker comprises an amino acid sequence recognized and cleaved by a protease (e.g., a tumor microenvironment (TME) protease, such as MMP9, uPA, MMP2, or Leguminous). In some embodiments, the peptide linker comprises the amino acid sequence shown in any one of SEQ ID NO: 333, 336, and 353-356. In some embodiments, the peptide linker comprises two or more sets of the amino acid sequences shown in SEQ ID NO: 333, 336, and 353-356. In some embodiments, the length of the peptide linker is no more than about 35 amino acids. In some embodiments, the length of the peptide linker is about 15 to 35 amino acids. In some embodiments, the peptide linker reduces the binding affinity of the IL-18 peptide to IL-18 binding protein (IL-18BP) compared to the corresponding wild-type IL-18 peptide (e.g., SEQ ID NO: 1). In some embodiments, the binding affinity is reduced by: a) at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, or b) about 10%-20%, 20%-30%, 30%-40%, 40%-50%, 50%-60%, 60%-70%, 70%-80%, 80%-90%, 90%-95%, 95%-96%, 96%-97%, 97%-98%, and / or 98%-99%.

[0157] In one aspect, this document provides a construct comprising an IL-18 component associated with a binding moiety of a specifically binding antigen, wherein the IL-18 component comprises an IL-18 polypeptide and a masking moiety, the IL-18 polypeptide being linked to the masking moiety via a cleavable linker, and the IL-18 component exhibiting a reduced binding affinity for the IL-18 receptor compared to a corresponding wild-type IL-18 polypeptide (e.g., SEQ ID NO: 1). In some embodiments, the IL-18 component comprises, from N-terminus to C-terminus,: a) a masking moiety; b) a cleavable linker; and c) an IL-18 polypeptide. In some embodiments, the IL-18 component comprises, from N-terminus to C-terminus,: a) a masking moiety comprising SEQ ID NO: 336; b) a cleavable linker comprising SEQ ID NO: 337; and c) an IL-18 polypeptide comprising SEQ ID NO: 39. In some embodiments, the IL-18 component comprises, from N-terminus to C-terminus,: a) a masking portion comprising SEQ ID NO: 336; b) a cleavable linker comprising SEQ ID NO: 337; and c) an IL-18 polypeptide comprising SEQ ID NO: 39, wherein the length of the cleavable linker is not more than about 50, about 40, about 30, about 25, about 20, about 15, about 12, or about 10 amino acids. In some embodiments, the IL-18 component comprises, from N-terminus to C-terminus,: a) a masking portion comprising SEQ ID NO: 336; b) a cleavable linker comprising SEQ ID NO: 337; and c) an IL-18 polypeptide comprising SEQ ID NO: 39, wherein the length of the cleavable linker is not more than about 50, 40, 30, 25, 20, 15, 12, or 10 amino acids.

[0158] In some embodiments, the IL-18 peptide is a wild-type IL-18 peptide fused to a masking portion. In some embodiments, the IL-18 peptide is a wild-type IL-18 peptide fused to a peptide linker. In some embodiments, the IL-18 peptide is a wild-type IL-18 peptide fused to both a masking portion and a peptide linker.

[0159] In some embodiments, the IL-18 peptide contains at least one point mutation and is fused to a masking portion. In some embodiments, the IL-18 peptide contains at least one point mutation and is fused to a peptide linker. In some embodiments, the IL-18 peptide contains at least one point mutation and is fused to both a masking portion and a peptide linker.

[0160] In some embodiments, the IL-18 peptide contains at least two point mutations and is fused to a masking moiety. In some embodiments, the IL-18 peptide contains at least two point mutations and is fused to a peptide linker. In some embodiments, the IL-18 peptide contains at least two point mutations and is fused to both the masking moiety and the peptide linker.

[0161] In some embodiments, the IL-18 peptide contains at least three point mutations and is fused to a masking moiety. In some embodiments, the IL-18 peptide contains at least three point mutations and is fused to a peptide linker. In some embodiments, the IL-18 peptide contains at least three point mutations and is fused to both the masking moiety and the peptide linker.

[0162] In some embodiments, the IL-18 peptide contains at least three point mutations and is fused to a masking moiety. In some embodiments, the IL-18 peptide contains at least three point mutations and is fused to a peptide linker. In some embodiments, the IL-18 peptide contains at least three point mutations and is fused to both the masking moiety and the peptide linker.

[0163] In some embodiments, the IL-18 peptide contains at least four point mutations and is fused to a masking portion. In some embodiments, the IL-18 peptide contains at least four point mutations and is fused to a peptide linker. In some embodiments, the IL-18 peptide contains at least four point mutations and is fused to both the masking portion and the peptide linker.

[0164] In some embodiments, when the IL-18 receptor is expressed on cells that express the IL-18 receptor but not the antigen bound by the binding affinity (e.g., NFκB-luc / hIL-18RαRβ HEK293 cells as described in the examples), the constructs, IL-18 components, or IL-18 peptides described herein exhibit reduced binding affinity for the IL-18 receptor (e.g., wild-type human IL-18 receptor) compared to wild-type human IL-18 peptides. In some embodiments, via EC... 50 (e.g., as described in the embodiments) or K DThe assessed binding affinity to the IL-18 receptor was reduced by at least about 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% compared to the wild-type IL-18 peptide (e.g., a reduction of about 10%-20%, 20%-30%, 30%-40%, 40%-50%, 50%-60%, 60%-70%, 70%-80%, 80%-90%, 90%-95%, 95%-96%, 96%-97%, 97%-98%, and / or 98%-99%). In some embodiments, via EC... 50 The assessed binding affinity does not exceed one percent, one five-thousandth, one two-thousandth, or one-thousandth of the binding affinity of the wild-type IL-18 peptide shown in SEQ ID NO: 1.

[0165] In some implementations, when the IL-18 receptor is expressed in cells that express the IL-18 receptor but do not express the antigen bound by the binding affinity (e.g., NFκB-luc / hIL-18RαRβ HEK293 cells as described in the examples), EC 50 The binding affinity of the constructs, IL-18 components or IL-18 peptides described herein to the IL-18 receptor is evaluated to be at least 0.5, 1, 5, 10, 25, 50, 75, 100, 200, 500 or 1000 nM.

[0166] In some embodiments, when the IL-18 receptor is expressed in cells that simultaneously express the IL-18 receptor and an antigen bound by binding affinity (e.g., NFκB-luc / hIL-18RαRβHEK293 cells expressing representative antigens such as PD-1 as described in the examples), EC... 50 The binding affinity of the constructs, IL-18 components, or IL-18 peptides described herein to the IL-18 receptor is no higher than about 10 nM, 5 nM, 1 nM, 0.5 nM, or 0.1 nM.

[0167] In some implementations, ECs on cells that express the IL-18 receptor but not the antigen bound by the binding moiety are used. 50 ECs on cells that simultaneously express the IL-18 receptor and the antigen bound by the binding moiety 50The ratios are: a) at least approximately 10, 50, 100, 200, 250, 500, 750, 1000, 2000, 2500, 5000, 7500, 10000, 15000, 20000, 25000, 30000, or 35000, or b) approximately 10-50, 50-100, 100-200, 200-250, 250- 500, 500-750, 750-1000, 1000-2000, 2000-2500, 2500-5000, 5000-7500, 7500-10000, 10000-15000, 15000-20000, 20000-25000, 25000-30000 and / or 30000-35000. In some embodiments, the construct specifically binds to IL-18 receptor α (“IL-18Rα”), such as human IL-18Rα or “hIL-18α”, and has significantly reduced binding affinity to IL-18 binding proteins (“IL-18BP”) (e.g., human IL-18BP or “hIL-18BP”). In some embodiments, the construct exhibits a significantly reduced binding affinity for IL-18BP compared to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the construct has an affinity for IL-18Rα (e.g., hIL-18Rα) comparable to that of wild-type human IL-18 to IL-18Rα (e.g., hIL-18Rα) as shown in SEQ ID NO: 1. In some embodiments, the construct has an increased affinity for IL-18Rα (e.g., hIL-18Rα) compared to that of wild-type human IL-18 to IL-18Rα (e.g., hIL-18Rα) as shown in SEQ ID NO: 1.

[0168] In some implementations, the construct binds to IL-18Rα. D Less than approximately 5 × 10 -5 M, less than approximately 5 × 10 -6 M, less than approximately 5 × 10 -7 M, less than approximately 5 × 10 -8 M, less than approximately 5 × 10 -9 M, less than approximately 5 × 10 -10 M or less than approximately 5 × 10 -11 M, including any range between the values ​​mentioned above. In some embodiments, the combination of the construct with IL-18Rα K D Approximately 5 × 10 -5 To approximately 5 × 10 -11 M. In some embodiments, the binding of the construct to IL-18Rα K DApproximately 5 × 10 -7 To approximately 5 × 10 -11 M. In some embodiments, the binding of the construct to IL-18Rα K D Approximately 5 × 10 -7 To approximately 5 × 10 -10 M. In some embodiments, the binding of the construct to IL-18Rα K D Approximately 5 × 10 -7 To approximately 5 × 10 -9 M. In some embodiments, the binding of the construct to IL-18Rα K D Approximately 5 × 10 -8 To approximately 5 × 10 -11 M. In some embodiments, the binding of the construct to IL-18Rα K D Approximately 5 × 10 -9 To approximately 5 × 10 -11 M. In some embodiments, the binding of the construct to IL-18Rα K D Approximately 5 × 10 -8 To approximately 5 × 10 -10 M. In some embodiments, the binding of the construct to IL-18Rα K D Approximately 5 × 10 -7 To approximately 5 × 10 -10 M. In some embodiments, the binding of the construct to IL-18Rα K D Approximately 5 × 10 -9 To approximately 5 × 10 -10 M. In some embodiments, the binding of the construct to IL-18Rα K D Approximately 5 × 10 -7 To approximately 5 × 10 -9 M. In some embodiments, the binding of the construct to IL-18Rα K D Approximately 5 × 10 -8 To approximately 5 × 10 -9 M. In some embodiments, the construct binds to IL-18Rα (e.g., hIL-18Rα). K D Approximately 5 × 10 -5 Up to 5 × 10 -11 M, for example, approximately 5 × 10 -5 6 × 10 -5 7 × 10 -5 8 × 10 -5 9 × 10 -5 1 × 10-6 2 × 10 -6 3 × 10 -6 4 × 10 -6 5 × 10 -6 6 × 10 -6 7 × 10 -6 8 × 10 -6 9 × 10 -6 1 × 10 -7 2 × 10 -7 3 × 10 -7 4 × 10 -7 5 × 10 -7 6 × 10 -7 7 × 10 -7 8 × 10 -7 9 × 10 -7 1 × 10 -8 2 × 10 -8 3 × 10 -8 4 × 10 -8 5 × 10 -8 6 × 10 -8 7 × 10 -8 8 × 10 -8 9 × 10 -8 1 × 10 -9 2 × 10 -9 3 × 10 -9 4 × 10 -9 5 × 10 -9 6 × 10 -9 7 × 10 -9 8 × 10 -9 9 × 10 -9 1 × 10 -10 2 × 10 -10 3 × 10 -10 4 × 10 -10 5 × 10 -10 6 × 10 -10 7 × 10 -10 8 × 10 -10 9 × 10 -10 1 × 10 -11 2 × 10 -11 3 × 10 -11 4 × 10 -11 Or 5 × 10 -11M includes any range between the values ​​mentioned above. In some embodiments, the construct has a higher affinity for IL-18Rα (e.g., hIL-18Rα) than the wild-type human IL-18 to IL-18Rα (e.g., hIL-18Rα) shown in SEQ ID NO: 1. In some embodiments, the construct has an affinity for IL-18Rα (e.g., hIL-18Rα) comparable to (e.g., substantially the same) the wild-type human IL-18 to IL-18Rα (e.g., hIL-18Rα) shown in SEQ ID NO: 1.

[0169] In some embodiments, the construct exhibits a significantly reduced binding affinity to IL-18-binding protein (IL-18BP) compared to wild-type IL-18. In some embodiments, the binding affinity of the construct to IL-18BP is [not specified]. D Greater than 5 × 10 -9 M. In some implementations, the construct is combined with IL-18BP. K D Greater than 5 × 10 -8 M. In some implementations, the construct is combined with IL-18BP. K D Greater than 5 × 10 -7 M. In some implementations, the construct is combined with IL-18BP. K D Greater than 5 × 10 -6 M. In some implementations, the construct is combined with IL-18BP. K D Greater than 5 × 10 -5 M. In some embodiments, the construct does not bind to IL-18BP (e.g., hIL-18BP) (e.g., no detectable binding). In some embodiments, the construct that does not bind to IL-18BP (e.g., hIL-18BP) (e.g., no detectable binding) binds to IL-18BP (e.g., hIL-18BP). K D Greater than 10 -3 M.

[0170] The affinity of the IL-18 peptides, IL-18 components, or constructs described herein for the IL-18 receptor, IL-18Rα, and / or IL-18BP can be experimentally determined using methods known in the art. Such methods include, but are not limited to: Western blot, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), electrochemiluminescence (ECL), immunoradioassay (IRMA), enzyme immunoassay (EIA), surface plasmon resonance (SPR), peptide scanning, and kinetic competitive screening based on fluorescence activated cell sorting (FACS). Combined part

[0171] The binding portion described herein can be any reagent that specifically binds to an antigen.

[0172] In some implementations, the binding portion comprises a small molecule.

[0173] In some implementations, the bonding portion does not have an Fc structural domain.

[0174] In some embodiments, the binding portion comprises an Fc domain. In some embodiments, the Fc domain is a human Fc domain or a variant thereof comprising one or more amino acid substitutions. In some embodiments, the Fc domain is a human IgG Fc domain or a variant thereof, such as a human IgG1, IgG2, or IgG4 Fc domain, or a variant of any of the foregoing. In some embodiments, the construct comprises, from N-terminus to C-terminus, the IL-18 peptide described herein and a human IgG1 Fc variant, the human IgG1 Fc variant comprising one or more modifications conducive to the formation of a club-and-socket structure. In some embodiments, the Fc domain is directly fused to the IL-18 component.

[0175] In some embodiments, the binding portion comprises an antibody. In some embodiments, the antibody is a monovalent antibody comprising an antigen-binding portion that specifically binds to an antigen. In some embodiments, two IL-18 components are fused to the monovalent antibody. In some embodiments, only one IL-18 component is fused to the monovalent antibody. In some embodiments, the antibody is a bivalent antibody comprising two antigen-binding portions that specifically bind to an antigen. In some embodiments, the bivalent antibody is fused to two IL-18 components. In some embodiments, the bivalent antibody is fused to only one IL-18 component.

[0176] In some embodiments, the antibody is selected from the group consisting of: full-length antibody, single-chain Fv (scFv), Fab, Fab', F(ab')2, Fv fragment, disulfide bond-stabilized Fv fragment (dsFv), (dsFv)2, nanobody (V H H), Fv-Fc fusion protein, scFv-Fc fusion protein, scFv-Fv fusion protein, biantibody, triantibody and tetraantibody.

[0177] In some embodiments, the binding moiety specifically binds to PD-1. In some embodiments, the binding moiety comprises: a heavy chain variable region comprising VH-CDR1, VH-CDR2, and VH-CDR3; and a light chain variable region comprising VL-CDR1, VL-CDR2, and VL-CDR3; wherein VH-CDR1 comprises the amino acid sequence shown in SEQ ID NO: 470, VH-CDR2 comprises the amino acid sequence shown in SEQ ID NO: 471, VH-CDR3 comprises the amino acid sequence shown in SEQ ID NO: 472, VL-CDR1 comprises the amino acid sequence shown in SEQ ID NO: 473, VL-CDR2 comprises the amino acid sequence shown in SEQ ID NO: 474, and VL-CDR3 comprises the amino acid sequence shown in SEQ ID NO: 475.

[0178] In some embodiments, the binding portion comprises: a heavy chain variable region comprising VH-CDR1, VH-CDR2, and VH-CDR3; and a light chain variable region comprising VL-CDR1, VL-CDR2, and VL-CDR3; wherein VH-CDR1 comprises the amino acid sequence shown in SEQ ID NO: 476, VH-CDR2 comprises the amino acid sequence shown in SEQ ID NO: 477, VH-CDR3 comprises the amino acid sequence shown in SEQ ID NO: 478, VL-CDR1 comprises the amino acid sequence shown in SEQ ID NO: 479, VL-CDR2 comprises the amino acid sequence shown in SEQ ID NO: 480, and VL-CDR3 comprises the amino acid sequence shown in SEQ ID NO: 481.

[0179] In some embodiments, the binding portion comprises a single-chain variable fragment (scFv) containing two heavy-chain variable regions and two light-chain variable regions. In some embodiments, the scFv has single specificity. In some embodiments, the scFv has dual specificity.

[0180] In some embodiments, the binding moiety comprises a single-domain antibody (nanobody; VHH) containing two heavy chain variable regions. In some embodiments, the binding moiety specifically binds to PD-1. In some embodiments, the binding moiety comprises a heavy chain variable region comprising VH-CDR1, VH-CDR2, and VH-CDR3, wherein VH-CDR1 comprises the amino acid sequence shown in SEQ ID NO: 470, VH-CDR2 comprises the amino acid sequence shown in SEQ ID NO: 471, and VH-CDR3 comprises the amino acid sequence shown in SEQ ID NO: 472. In some embodiments, the binding moiety comprises a heavy chain variable region comprising VH-CDR1, VH-CDR2, and VH-CDR3, wherein VH-CDR1 comprises the amino acid sequence shown in SEQ ID NO: 476, VH-CDR2 comprises the amino acid sequence shown in SEQ ID NO: 477, and VH-CDR3 comprises the amino acid sequence shown in SEQ ID NO: 478.

[0181] In some implementations, the bonding portion does not include the Fc structural domain.

[0182] In some implementations, the binding portion comprises a small molecule. antigen

[0183] In some embodiments, the antigen is a tumor-specific antigen or a tumor-associated antigen. Tumor-specific antigens or tumor-associated antigens may include: proteins that are normally highly isolated from the immune system; proteins that are normally produced at very low levels; proteins that are normally produced only at specific developmental stages; or proteins whose structure has been altered due to mutation.

[0184] Numerous tumor antigens are known in the art, and novel tumor antigens can be readily identified through screening. Non-limiting examples of tumor antigens include EGFR, Her2, EpCAM, CD20, CD30, CD33, CD47, CD52, CD133, CD73, CEA, gpA33, mucin, TAG-72, CIX, PSMA, folate-binding protein, GD2, GD3, GM2, VEGF, VEGFR, integrin, αVβ3, α5β1, ERBB2, ERBB3, MET, IGF1R, EPHA3, TRAILR1, TRAILR2, RANKL, FAP, and tendinin.

[0185] In some embodiments, the antigen is a tumor-associated antigen (e.g., a protein overexpressed on tumor cells relative to corresponding non-tumor cells). As used herein, “corresponding non-tumor cells” refers to non-tumor cells of the same cell type as tumor cells. It should be noted that such proteins are not necessarily different from tumor antigens. Non-limiting examples include: carcinoembryonic antigen (CEA), which is overexpressed in most colorectal, rectal, breast, lung, pancreatic, and gastrointestinal cancers; hyalin receptors (HER-2, neu, or c-erbB-2), which are commonly overexpressed in breast, ovarian, colorectal, lung, prostate, and cervical cancers; epidermal growth factor receptor (EGFR), which is highly expressed in a range of solid tumors including breast, head and neck, non-small cell lung cancer, and prostate cancer; desialylate glycoprotein receptor; transferrin receptor; Serine protease inhibitor enzyme complex receptor, expressed on the surface of hepatocytes; fibroblast growth factor receptor (FGFR), overexpressed in pancreatic ductal adenocarcinoma cells; vascular endothelial growth factor receptor (VEGFR), used for anti-angiogenic gene therapy; folic acid receptor, selectively overexpressed in 90% of non-mucinous ovarian cancers; cell surface glycocalyx; carbohydrate receptor; and polymerase receptor, which can be used to deliver genes to respiratory epithelial cells and is attractive for treating lung diseases such as cystic fibrosis.

[0186] In some implementations, the tumor is a solid tumor.

[0187] In some implementations, the antigen is a neoantigen.

[0188] In some embodiments, the antigen is an antigen expressed by immune cells. In some embodiments, the immune cells are tumor-infiltrating lymphocytes.

[0189] In some embodiments, the antigen is an immune checkpoint target expressed on T cells or NK cells. In some embodiments, the antigen is PD-1. In some embodiments, the antigen is CTLA-4. Association between the binding portion and the IL-18 component

[0190] The binding moiety described herein can be associated with IL-18 components or IL-18 peptides in various ways, as long as such association does not prevent the IL-18 component or binding moiety from binding to its target as described in this application.

[0191] In some embodiments, the IL-18 component is covalently conjugated to the binding moiety. In some embodiments, a linker (e.g., a PEG linker) exists between the IL-18 component or the IL-18 polypeptide and the binding moiety.

[0192] In some embodiments, the IL-18 component is fused to the binding moiety. In some embodiments, a linker exists between the IL-18 polypeptide and the binding moiety; optionally, the linker is a peptide linker containing no more than 35 amino acids.

[0193] In some embodiments, the binding portion comprises an Fc domain consisting of two peptides, and the IL-18 component is fused to the Fc domain. In some embodiments, the IL-18 component is fused to the C-terminus of the Fc domain. In some embodiments, a single IL-18 component is fused to the C-terminus of one of the two peptides in the Fc domain.

[0194] In some embodiments, the binding portion comprises an antibody, said antibody being a full-length antibody comprising two heavy chains and two light chains. In some embodiments, the IL-18 component is fused to one or two heavy chains. In some embodiments, the IL-18 component is fused to the C-terminus of one or two heavy chains and / or one or two light chains of the full-length antibody.

[0195] In some embodiments, the binding portion comprises a full-length antibody comprising two heavy chains and two light chains. In some embodiments, two or more IL-18 peptides or IL-18 components are fused to the antibody. In some embodiments, two IL-18 peptides or IL-18 components are fused to the C-terminus of each of the two heavy chains. In some embodiments, two IL-18 peptides or IL-18 components are fused to the C-terminus of each of the two light chains. In some embodiments, two IL-18 peptides or IL-18 components are fused to the N-terminus of each of the two heavy chains. In some embodiments, two IL-18 peptides or IL-18 components are fused to the C-terminus of each of the two light chains.

[0196] In some embodiments, the binding portion comprises a full-length antibody comprising two heavy chains and two light chains, and a single IL-18 peptide or a single IL-18 component is fused to the antibody. In some embodiments, the single IL-18 peptide or single IL-18 component is fused to the C-terminus of one of the heavy chains. In some embodiments, the single IL-18 peptide or single IL-18 component is fused to the C-terminus of one of the light chains. In some embodiments, the single IL-18 peptide or single IL-18 component is fused to the N-terminus of one of the heavy chains. In some embodiments, the single IL-18 peptide or single IL-18 component is fused to the N-terminus of one of the light chains.

[0197] In some embodiments, the binding portion comprises an antibody consisting of two peptides, each containing an scFv domain and an Fc region, the two Fc regions forming an Fc domain. In some embodiments, two or more IL-18 peptides or IL-18 components are fused to the antibody. In some embodiments, two IL-18 peptides or IL-18 components are fused to the C-terminus of two peptides, respectively. In some embodiments, two IL-18 peptides or IL-18 components are fused to the N-terminus of two peptides, respectively.

[0198] In some embodiments, the binding portion comprises an antibody consisting of two peptides, each containing an scFv domain and an Fc region, the two Fc regions forming an Fc domain, and a single IL-18 peptide or IL-18 component is fused to the antibody. In some embodiments, the single IL-18 peptide or IL-18 component is fused to the C-terminus of one of the two peptides. In some embodiments, the single IL-18 peptide or IL-18 component is fused to the N-terminus of one of the two peptides.

[0199] In some embodiments, the binding portion comprises an antibody consisting of two peptides, each containing a VHH domain and an Fc region, the two Fc regions forming an Fc domain. In some embodiments, two or more IL-18 peptides or IL-18 components are fused to the antibody. In some embodiments, two IL-18 peptides or IL-18 components are fused to the C-terminus of two peptides, respectively. In some embodiments, two IL-18 peptides or IL-18 components are fused to the N-terminus of two peptides, respectively.

[0200] In some embodiments, the binding portion comprises an antibody consisting of two peptides, each containing a VHH domain and an Fc region, the two Fc regions forming an Fc domain, and a single IL-18 peptide or IL-18 component is fused to the antibody. In some embodiments, the single IL-18 peptide or IL-18 component is fused to the C-terminus of one of the two peptides. In some embodiments, the single IL-18 peptide or IL-18 component is fused to the N-terminus of one of the two peptides. connector

[0201] In some embodiments, a linker exists between the binding portion and the IL-18 component or IL-18 peptide. The length, flexibility, and / or other properties of the linker used may affect the properties of the construct, including but not limited to affinity, specificity, or binding affinity to one or more specific antigens or epitopes. For example, a longer linker may be selected to ensure that there is no steric hindrance between two adjacent domains. In some embodiments, a shorter linker may be selected to suppress the binding affinity of the IL-18 peptide or IL-18 component to the IL-18 receptor. See, for example, the "Modulation of Linker Length" section below.

[0202] In some embodiments, the linker (e.g., a peptide linker) comprises flexible residues (e.g., glycine and serine), allowing adjacent domains to move freely relative to each other. For example, a glycine-serine dipeptide may be a suitable peptide linker. In some embodiments, the linker is a non-peptide linker. In some embodiments, the linker is a peptide linker. In some embodiments, the linker is an uncleavable linker. In some embodiments, the linker is a cleavable linker.

[0203] Other considerations regarding linkers include their impact on the physical or pharmacokinetic properties of the resulting compound, such as solubility, lipophilicity, hydrophilicity, hydrophobicity, stability (improving or decreasing stability and planned degradation), rigidity, flexibility, immunogenicity, regulation of antibody binding ability, and ability to be incorporated into micelles or liposomes. peptide linkers

[0204] Peptide linkers can have naturally occurring or non-natural sequences. For example, sequences derived from only the hinge region of a heavy chain antibody can be used as linkers. See, for example, WO1996 / 34103.

[0205] The peptide linker may have any suitable length. In some embodiments, the length of the peptide linker is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 75, 100 or more amino acids. In some embodiments, the length of the peptide linker is no more than about 100, 75, 50, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5 or fewer amino acids. In some embodiments, the length of the peptide linker is about 1 amino acid to about 10 amino acids, about 1 amino acid to about 20 amino acids, about 1 amino acid to about 30 amino acids, about 5 amino acids to about 15 amino acids, about 10 amino acids to about 25 amino acids, about 5 amino acids to about 30 amino acids, about 10 amino acids to about 30 amino acids, about 30 amino acids to about 50 amino acids, about 50 amino acids to about 100 amino acids, or about 1 amino acid to about 100 amino acids.

[0206] A fundamental technical feature of such peptide linkers is that they do not possess any polymerization activity. Characteristics of peptide linkers, including their lack of promotion of secondary structure formation, are well known in the art, for example, as described in Dall'Acqua et al. (Biochem. (1998) 37, 9266-9273), Cheadle et al. (Mol Immunol (1992) 29, 21-30), and Raag and Whitlow (FASEB (1995) 9(1), 73-80). For the purpose of "peptide linker," the particularly preferred amino acid is Gly. Furthermore, peptide linkers that do not promote any secondary structure formation are preferred. Interconnection between domains can be achieved, for example, through genetic engineering techniques. Methods for preparing fusion-type and operablely linked bispecific single-stranded constructs and expressing them in mammalian cells or bacteria are well known in the art (e.g., WO 99 / 54440; Ausubel, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, NY 1989 and 1994; or Sambrook et al., Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001).

[0207] The peptide linker may be a stable linker that cannot be cleaved by proteases, especially not by matrix metalloproteinases (MMPs).

[0208] The connector can also be a flexible connector (e.g., a GS connector). An exemplary flexible connector includes a glycine polymer (G). n Glycine-serine polymers (including, for example, (GS)) n ( SEQ ID NO: 501 (GSGGS) n ( SEQ ID NO: 502 (GGGGS) n ( SEQ ID NO: 503 ) and (GGGS) n ( SEQ ID NO: 504 (where n is an integer not less than 1), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers are relatively disordered and therefore can serve as neutral linking arms between different components. Glycine even occupies a significantly larger proportion than alanine. -ψ space, and its confinement is much less than that of residues with longer side chains (see Scheraga, Rev. Computational Chem. 11 173-142 (1992)). Those skilled in the art will recognize that antibody fusion proteins can be designed using fully flexible or partially flexible linkers, such that the linker may contain either a flexible linker portion or one or more portions that impart less flexibility, thereby forming the desired antibody fusion protein structure.

[0209] In addition, exemplary linkers also include amino acid sequences such as (GGGGS). n ( SEQ ID NO: 503 ), where n is an integer from 1 to 8, for example (GGGGS)3 ( SEQ ID NO: 363 It is referred to as "(G4S)3" or "GS3", or (GGGGS)6. SEQ ID NO: 505 (Referring to "(G4S)6" or "GS6"). In some embodiments, the peptide linker comprises the amino acid sequence (GSTSGSGKPGSGEGS). n ( SEQ ID NO: 506 ), where n is an integer from 1 to 3.

[0210] Non-peptide linkers

[0211] Coupling between two parts can be achieved through any chemical reaction capable of binding the two molecules, provided that both components retain their respective activities. Such linkages can include a variety of chemical mechanisms, such as covalent bonding, affinity bonding, intercalation bonding, coordination bonding, and complexation. In some embodiments, the bonding is covalent. Covalent bonding can be achieved through direct condensation between existing side chains or by introducing an external bridging molecule. In the art, a variety of divalent or multivalent linkers can be used for the coupling of protein molecules. For example, representative coupling agents can include organic compounds such as thioesters, carbodiimides, succinimides, diisocyanates, glutaraldehyde, diazobenzene, and hexamethylenediamine. This list is not intended to exhaustively describe all known coupling agents in the art, but rather to list some of the more common coupling agents (see Killen and Lindstrom, ...). Jour. Immun. 133: 1335-2549 (1984); Jansen et al., Immunological Reviews 62: 185-216 (1982); and Vitetta et al., Science 238: 1098 (1987)).

[0212] Linkers applicable to this application have been described in the literature (see, for example, Ramakrishnan, S. et al.). Cancer Res . 44: 201-208 (1984), which describes the application of MBS (M-maleimide benzoyl-N-hydroxysuccinimide ester). In some embodiments, the non-peptide linkers used herein include: (i) EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride; (ii) SMPT (4-succinimideoxycarbonyl-α-methyl-α-(2-pyridinedithio)toluene, Pierce Chem. Co., catalog number: 21558G); (iii) SPDP (succinimide-6-[3-(2-pyridinedithio)propionamide]hexanoate, Pierce Chem. Co., catalog number: 21651G); (iv) Sulfo-LC-SPDP (sulfosuccinimide-6-[3-(2-pyridinedithio)propionamide]hexanoate, Pierce Chem. Co., catalog number: 2165-G); and (v) Sulfo-NHS (N-hydroxysulfosuccinimide, Pierce Chem. Co. (product number: 24510) and EDC conjugate.

[0213] The linkers described above contain components with different properties, thus potentially resulting in bispecific antibodies with varying physicochemical properties. For example, Sulfo-NHS esters of alkyl carboxylic acids are more stable than those of aromatic carboxylic acids. Linkers containing NHS esters have lower solubility than those containing Sulfo-NHS esters. Furthermore, the linker SMPT contains sterically hindered disulfide bonds, thus enabling the formation of more stable antibody fusion proteins. Disulfide bonds are generally less stable than other bonds because they are cleaved in vitro, leading to a reduction in usable antibody fusion proteins. In particular, Sulfo-NHS can improve the stability of carbodiimide coupling reactions. When carbodiimides (e.g., EDC) are used in combination with Sulfo-NHS, the resulting esters are more resistant to hydrolysis than those using carbodiimides alone in coupling reactions. IL-18, IL-18 receptor (IL-R), and IL-18 binding protein (IL-18 BP)

[0214] Interleukin-18 (IL-18, also known as interferon-gamma inducible factor) is a protein encoded by the human IL-18 gene. IL-18 is a pro-inflammatory cytokine produced by both hematopoietic and non-hematopoietic cells. IL-18 regulates innate and adaptive immunity, and its dysregulation can lead to autoimmune or inflammatory diseases. IL-18 binds to IL-18R. IL-18 binding protein (IL-18BP) is a protein that binds to IL-18 and blocks the binding of IL-18 to IL-18R. IL-18 components and IL-18 peptides

[0215] The IL-18 component described herein comprises an IL-18 polypeptide. The IL-18 polypeptide may be a wild-type IL-18 polypeptide or an IL-18 variant polypeptide having one or more modifications compared to the wild-type human IL-18 polypeptide (e.g., SEQ ID NO: 1). In some embodiments, the IL-18 component further comprises a masking portion linked to the IL-18 polypeptide via a cleavable linker. In some embodiments, the IL-18 component further comprises a linker (e.g., any linker described in the "Linker" section above) for linking the IL-18 polypeptide to the binding portion.

[0216] In some embodiments, the IL-18 polypeptide is a wild-type IL-18 polypeptide. In some embodiments, the wild-type IL-18 polypeptide is human IL-18. In some embodiments, the wild-type IL-18 is human IL-18 comprising the amino acid sequence shown in SEQ ID NO: 1.

[0217] In some embodiments, the IL-18 component or IL-18 peptide has a reduced binding affinity for the IL-18 receptor (e.g., wild-type human IL-18 receptor) compared to the wild-type human IL-18 peptide shown in SEQ ID NO: 1. In some embodiments, when the IL-18 receptor is expressed in cells that express the IL-18 receptor but do not express the antigen bound by the binding affinity (e.g., NFκB-luc / hIL-18RαRβ HEK293 cells described in the examples), via EC... 50 The IL-18 peptides described herein were evaluated to have binding affinity for the IL-18 receptor of at least 0.5, 1, 5, 10, 25, 50, 75, 100, 200, 500, or 1000 nM. In some embodiments, via EC... 50 (e.g., as described in the embodiments) or K D The assessed binding affinity to the IL-18 receptor was reduced by at least about 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% (e.g., a reduction of about 10%-20%, 20%-30%, 30%-40%, 40%-50%, 50%-60%, 60%-70%, 70%-80%, 80%-90%, 90%-95%, 95%-96%, 96%-97%, 97%-98%, and / or 98%-99%) compared to the wild-type IL-18 peptide.

[0218] In some embodiments, the IL-18 component or IL-18 peptide has a reduced binding affinity for IL-18 binding proteins (e.g., wild-type human IL-18BP) compared to the wild-type human IL-18 peptide shown in SEQ ID NO: 1.

[0219] In some embodiments, the IL-18 component or IL-18 polypeptide, compared to the wild-type human IL-18 polypeptide shown in SEQ ID NO: 1, has: a) a reduced binding affinity for IL-18 binding proteins (e.g., wild-type human IL-18BP) and b) a reduced binding affinity for IL-18 receptors (e.g., wild-type human IL-18 receptor). Exemplary IL-18 components with reduced affinity for IL-18 receptors (e.g., wild-type human IL-18 receptor) A. Reducing the binding affinity of the IL-18 peptide to the IL-18 receptor by modifying it.

[0220] In some embodiments, the IL-18 peptide comprises one or more modifications relative to the corresponding wild-type IL-18 peptide (e.g., SEQ ID NO:1), wherein said one or more modifications reduce the binding affinity of the IL-18 peptide (or IL-18 component or construct) to the IL-18 receptor. See, for example... Nature Communications 5: 5340 (2014); Nature Structural & Molecular Biology 10: 966-971 (2003).

[0221] In some embodiments, the IL-18 peptide exhibits a reduced binding affinity for IL-18Rβ compared to the corresponding wild-type IL-18 peptide (e.g., SEQ ID NO: 1). In some embodiments, the reduced binding affinity is: a) at least about 50%, 60%, 70%, 80%, or 90%, or b) 50%-60%, 60%-70%, 70%-80%, 80%-90%, 90%-95%, 95%-96%, 96%-97%, 97%-98%, and / or 98%-99%.

[0222] In some embodiments, the one or more modifications comprise amino acid substitutions selected from the group consisting of G108, H109, ​​K112, D110, R147, M150, G145, K79, D98, and K84, wherein the amino acid position is relative to the wild-type human IL-18 polypeptide shown in SEQ ID NO: 1. In some embodiments, the substitution is an alanine substitution. In some embodiments, the one or more modifications comprise amino acid substitutions selected from the group consisting of G108A, H109A, K112A, D110A, R147A, M150A, G145A, K79A, D98A, and K84A. In some embodiments, the IL-18 peptide contains mutations at two or more (e.g., two, three, four or five) sites of G108, H109, ​​K112, D110, R147, M150, G145, K79, D98 and K84, wherein the amino acid positions are relative to the wild-type (WT) human IL-18 shown in SEQ ID NO: 1, for example, any combination of the above sites.

[0223] In some embodiments, the one or more modifications comprise amino acid substitutions selected from the group consisting of G108, H109, ​​K112, D110, R147, M150, G145, K79, and D98, wherein the amino acid position is relative to the wild-type human IL-18 polypeptide represented by SEQ ID NO: 1. In some embodiments, the substitution is an alanine substitution. In some embodiments, the one or more modifications comprise amino acid substitutions selected from the group consisting of G108A, H109A, K112A, D110A, R147A, M150A, G145A, K79A, and D98A. In some embodiments, the IL-18 peptide contains mutations at one or more (e.g., two, three, four, or five) sites of G108, H109, ​​K112, D110, R147, M150, G145, K79, and D98, wherein the amino acid positions are relative to the wild-type (WT) human IL-18 shown in SEQ ID NO: 1, for example, any combination of the above sites. Each of the above substitutions has been tested and demonstrated to have a reducing binding affinity effect.

[0224] In some embodiments, the one or more modifications include G108A substitution.

[0225] In some embodiments, the one or more modifications include H109A substitution.

[0226] In some embodiments, the one or more modifications include K112A substitution.

[0227] In some embodiments, the one or more modifications include D110A substitution.

[0228] In some embodiments, the one or more modifications include R147A substitution.

[0229] In some embodiments, the one or more modifications include M150A substitution.

[0230] In some embodiments, the one or more modifications include G145A substitution.

[0231] In some implementations, the one or more modifications include K79A substitution.

[0232] In some implementations, the one or more modifications include D98A substitution.

[0233] In some embodiments, the IL-18 component has a reduced binding affinity for IL-18Rα compared to the corresponding wild-type IL-18 polypeptide (e.g., SEQ ID NO: 1); optionally, the reduction in binding affinity is: a) at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, or b) about 10%-20%, 20%-30%, 30%-40%, 40%-50%, 50%-60%, 60%-70%, 70%-80%, 80%-90%, 90%-95%, 95%-96%, 96%-97%, 97%-98%, and / or 98%-99%.

[0234] In some embodiments, the one or more modifications comprise amino acid substitutions selected from the group consisting of K4, L5, K8, R13, D17, E31, M33, T34, D35, D40, K53, R58, M60, K79, K84, D98, R104, and D132, wherein the amino acid position is relative to the wild-type human IL-18 polypeptide represented by SEQ ID NO: 1. In some embodiments, the substitution is an alanine substitution. In some embodiments, the IL-18 peptide contains mutations at two or more (e.g., two, three, four, or five) sites of K4A, L5A, K8A, R13A, D17A, E31A, M33A, T34A, D35A, D40A, K53A, R58A, M60A, K79A, K84A, D98A, R104A, and D132A, wherein the amino acid positions are relative to the wild-type (WT) human IL-18 shown in SEQ ID NO: 1, for example, any combination of the above sites.

[0235] In some embodiments, the IL-18 component comprises an IL-18 polypeptide containing two or more (e.g., two, three, four, or five) modifications, said modifications including two or more (e.g., two, three, four, or five) amino acid substitutions selected from the group consisting of G108, H109, ​​K112, D110, R147, M150, G145, K79, D98, K84, K4, L5, K8, R13, D17, E31, M33, T34, D35, D40, K53, R58, M60, R104, and D132, wherein the amino acid position is relative to the wild-type human IL-18 polypeptide shown in SEQ ID NO: 1. In some embodiments, said substitution is an alanine substitution. In some embodiments, the IL-18 component comprises an IL-18 polypeptide containing two or more (e.g., two, three, four, or five) modifications, the modifications including two or more (e.g., two, three, four, or five) amino acid substitutions selected from the group consisting of G108A, H109A, K112A, D110A, R147A, M150A, G145A, K79A, D98A, K84A, K4A, L5A, K8A, R13A, D17A, E31A, M33A, T34A, D35A, D40A, K53A, R58A, M60A, R104A, and D132A.

[0236] In some embodiments, the IL-18 polypeptide comprises the amino acid sequence shown in any one of SEQ ID NO: 482-491. B. Concealed area

[0237] In some embodiments, the IL-18 component includes a masking portion, wherein the IL-18 polypeptide is linked to the masking portion via a cleavable linker.

[0238] In some embodiments, the masking portion comprises an IL-18 propeptide. In some embodiments, the IL-18 propeptide comprises a propeptide of human IL-18 (hIL-18). In some embodiments, the hIL-18 propeptide comprises the amino acid sequence shown in SEQ ID NO:333.

[0239] In some embodiments, the masking portion comprises a truncated propeptide of the IL-18 precursor. In some embodiments, the truncated propeptide of the IL-18 precursor comprises the amino acid sequence shown in SEQ ID NO: 336.

[0240] In some embodiments, the masking portion comprises the extracellular domain of IL-18Rα, the extracellular domain of IL-18Rβ, IL-18 binding protein, a fragment of any of the foregoing, or a variant of any of the foregoing.

[0241] In some embodiments, the masking portion comprises an extracellular domain of IL-18Rα or a fragment thereof. In some embodiments, the fragment of the extracellular domain of IL-18Rα comprises: a) a first and second domain of the extracellular domain, or b) a third domain of the extracellular domain. In some embodiments, the masking portion comprises an amino acid sequence represented by any one of SEQ ID NO: 353-356, or a functional variant thereof, the functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with any one of SEQ ID NO: 353-356.

[0242] In some embodiments, the IL-18 component comprises, from N-terminus to C-terminus,: a) a masking portion comprising a truncated propeptide of the IL-18 precursor, the truncated propeptide comprising the amino acid sequence shown in SEQ ID NO: 336; b) a cleavable linker; and c) an IL-18 polypeptide comprising the amino acid sequence shown in any one of SEQ ID NO: 1-299 and 307-318. In some embodiments, the IL-18 polypeptide comprises the amino acid sequence shown in any one of SEQ ID NO: 39, 133, 311, 316, 465-469, 482-491, 27, 3, 105, 143, 144, 145, 43, 9, 17, 81, 117, 147, 149, and 150. In some embodiments, the length of the cleavable linker is no more than about 50, 40, 30, 25, 20, 15, 12, or 10 amino acids.

[0243] In some embodiments, the IL-18 component comprises, from N-terminus to C-terminus,: a) a masking portion comprising the amino acid sequence shown in any one of SEQ ID NO: 353-356; b) a cleavable linker; and c) an IL-18 polypeptide comprising the amino acid sequence shown in any one of SEQ ID NO: 1-299 and 307-318. In some embodiments, the IL-18 polypeptide comprises the amino acid sequence shown in any one of SEQ ID NO: 39, 133, 311, 316, 465-469, 482-491, 27, 3, 105, 143, 144, 145, 43, 9, 17, 81, 117, 147, 149, and 150. In some embodiments, the cleavable linker is about 10-40, 10-30, 20-30, or 25-30 amino acids in length.

[0244] In some embodiments, the IL-18 component comprises, from N-terminus to C-terminus,: a) an IL-18 polypeptide comprising the amino acid sequence shown in any one of SEQ ID NO: 1-299 and 307-318; b) a cleavable linker; and c) a masking portion comprising the amino acid sequence shown in any one of SEQ ID NO: 353-356. In some embodiments, the IL-18 polypeptide comprises the amino acid sequence shown in any one of SEQ ID NO: 39, 133, 311, 316, 465-469, 482-491, 27, 3, 105, 143, 144, 145, 43, 9, 17, 81, 117, 147, 149, and 150. In some embodiments, the cleavable linker is about 10-40, 10-30, 20-30, or 25-30 amino acids in length.

[0245] In some embodiments, the IL-18 component comprises: a) an amino acid sequence represented by any one of SEQ ID NO: 340-343, 345-352, and 496-500, or b) a functional variant thereof having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with any one of SEQ ID NO: 340-343, 345-352, and 496-500. Cuttable connector

[0246] In some embodiments, the cleavable linker comprises one or more sets of amino acid sequences that are recognized and cleaved by one or more proteases. Exemplary masking portion

[0247] In some embodiments, the masking portion comprises an amino acid sequence shown in any one of SEQ ID NO: 353-356, or a functional variant thereof, the functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with any one of SEQ ID NO: 353-356. In some embodiments, the cleavable linker comprises an amino acid sequence recognized and cleaved by a protease (e.g., a tumor microenvironment (TME) protease, such as MMP9, uPA, MMP2, or Leguminous). In some embodiments, the masking portion comprises an amino acid sequence shown in any one of SEQ ID NO: 333, 336, and 353-356. In some embodiments, the masking portion comprises two or more sets of amino acid sequences shown in SEQ ID NO: 333, 336, and 353-356. In some embodiments, the cleavable linker comprises at least one amino acid sequence recognized and cleaved by a protease. In some embodiments, the cleavable linker comprises two or more sequences recognized and cleaved by a protease. In some embodiments, the two or more sequences are recognized and cleaved by different proteases. In some embodiments, the two or more sequences are recognized and cleaved by the same protease. In some embodiments, the protease is a tumor microenvironment (TME) protease. In some embodiments, the TME protease is urokinase-type plasminogen activator (uPA), Matriptase (MTSP-1), Legumin, prostate-specific antigen (PSA), dipeptidyl peptidase (e.g., DPP4), hepsin, matrix metalloproteinases (including but not limited to: matrix metalloproteinase 1 (MMP1), matrix metalloproteinase 2 (MMP2), matrix metalloproteinase 3 (MMP3), matrix metalloproteinase 8 (MMP8), matrix metalloproteinase 9 (MMP9), etc.). Matrix metalloproteinase 10 (MMP10), matrix metalloproteinase 12 (MMP12) or matrix metalloproteinase 14 (MMP14)), integrin-dissociating metalloproteinases (ADAM10, ADAM17, etc.), human leukocyte elastase (HLE), protease 3 (PR3), prourokinase, plasminogen, staphylococcal kinase, serine protease, plasmin, cathepsins (B, L, S), tissue kallikrein and / or kallikrein-related peptidases (KLK1, KLK2, KLK3, KLK6, KLK7). In some embodiments, the cleavable linker comprises one or more amino acid sequences selected from the group consisting of: VLK, SEQ ID NO: 337, SEQ ID NO: 338, SEQ ID NO: 339, SEQ ID NO: 372-377, Kridel et al. (2002). J Biol Chem. The sequences described in 277(26): 23788-93, and Chen et al. (2002). J Biol Chem. The sequence described in 277(6):4485-4491.

[0248] In some embodiments, the cleavable linker comprises one or more amino acid sequences that are recognized and cleaved by one or more of matrix metalloproteinases 2, 9, 10, 14, urokinase-type plasminogen activator, Matriptase, and Legumin. In some embodiments, the cleavable linker may be recognized and cleaved by two or more proteases (e.g., ULM, LM9, see examples). In some embodiments, the cleavable linker has spacer sequences (GS linkers) flanking the amino acid sequences recognized by the proteases. In some embodiments, the cleavable linker is about 10-40, 15-40, 20-40, 20-30, or 25-30 amino acids in length. In some embodiments, the cleavable linker comprises one or more amino acid sequences selected from the group consisting of SEQ ID NO: 337, SEQ ID NO: 338, SEQ ID NO: 339, and SEQ ID NO: 372-377. In some embodiments, the cleavable linker further comprises spacer sequences; optionally, the cleavable linker comprises two spacer sequences flanking the N-terminus and C-terminus of an amino acid sequence recognized and cleaved by one or more proteases, respectively. In some embodiments, the cleavable linker comprises two or more sets of amino acid sequences recognized and cleaved by one or more proteases; optionally, at least one set of the two or more sets of amino acid sequences recognized and cleaved by one or more proteases is side-joined with the two spacer sequences of the cleavable linker, and each set of the two or more sets of amino acid sequences recognized and cleaved by one or more proteases is side-joined with the two spacer sequences of the cleavable linker. In some embodiments, the spacer sequence comprises a GS linker; optionally, the length of the GS linker does not exceed about 10, 9, 8, 7, 6, or 5 amino acids. In some embodiments, the cleavable linker consists of no more than about 50, 40, 35, or 30 amino acids. In some embodiments, the cleavable linker consists of no less than about 10, 15, 20, or 25 amino acids. In some embodiments, the cleavable linker consists of about 25 to about 30 amino acids, and the masking portion includes an extracellular domain of IL-18Rα; further optionally, the masking portion includes an amino acid sequence shown in any one of SEQ ID NO: 353-356, or a functional variant thereof, the functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with any one of SEQ ID NO: 353-356.In some embodiments, the cleavable linker comprises the amino acid sequence shown in any one of SEQ ID NO: 399-407, SEQ ID NO: 492-493, and SEQ ID NO: 495. In some embodiments, the IL-18 polypeptide is a wild-type IL-18 polypeptide. In some embodiments, the wild-type IL-18 polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1.

[0249] In some embodiments, the IL-18 component comprises, from N-terminus to C-terminus,: a) a masking portion; b) a cleavable linker; and c) an IL-18 polypeptide, wherein the masking portion comprises SEQ ID NO: 353.

[0250] In some embodiments, the IL-18 component comprises, from N-terminus to C-terminus,: a) an IL-18 polypeptide; b) a cleavable linker; and c) a masking portion. In some embodiments, the masking portion comprises SEQ ID NO: 353.

[0251] In some embodiments, the IL-18 component comprises, from N-terminus to C-terminus,: a) a masking portion; b) a cleavable linker; and c) an IL-18 polypeptide, wherein the masking portion comprises SEQ ID NO: 354.

[0252] In some embodiments, the IL-18 component comprises, from N-terminus to C-terminus,: a) an IL-18 polypeptide; b) a cleavable linker; and c) a masking portion. In some embodiments, the masking portion comprises SEQ ID NO: 354.

[0253] In some embodiments, the IL-18 component comprises, from N-terminus to C-terminus,: a) a masking portion; b) a cleavable linker; and c) an IL-18 polypeptide, wherein the masking portion comprises SEQ ID NO: 355.

[0254] In some embodiments, the IL-18 component comprises, from N-terminus to C-terminus,: a) an IL-18 polypeptide; b) a cleavable linker; and c) a masking portion. In some embodiments, the masking portion comprises SEQ ID NO: 355.

[0255] In some embodiments, the IL-18 component comprises, from N-terminus to C-terminus,: a) a masking portion; b) a cleavable linker; and c) an IL-18 polypeptide, wherein the masking portion comprises SEQ ID NO: 356.

[0256] In some embodiments, the IL-18 component comprises, from N-terminus to C-terminus,: a) an IL-18 polypeptide; b) a cleavable linker; and c) a masking portion. In some embodiments, the masking portion comprises SEQ ID NO: 356.

[0257] In some embodiments, the IL-18 component includes a masking portion that prevents (e.g., inhibits) the IL-18 peptide from activating IL-18 receptor-mediated signaling (e.g., human IL-18 receptor-mediated signaling). In some embodiments, the IL-18 peptide is masked to prevent (e.g., inhibit) the IL-18 peptide from activating IL-18 receptor-mediated signaling (e.g., human IL-18 receptor-mediated signaling). In some embodiments, the IL-18 peptide is masked to prevent (e.g., inhibit) the IL-18 peptide from activating IL-18 receptor-mediated signaling (e.g., human IL-18 receptor-mediated signaling). Exemplary linkers and proteases for cleavage are listed below. Exemplary linkers and proteases

[0258] In some embodiments, the cleavable linker comprises one or more amino acid sequences that are recognized and cleaved by at least two, three, or four proteases; optionally, the one or more amino acid sequences are recognized and cleaved by a) Legumain and one, two, or three of MMP9, MMP2, and MMP14; b) uPA and one, two, or three of MMP9, MMP2, and MMP14; or c) all of uPA, Legumain, MMP2, MMP14, and MMP9.

[0259] In some embodiments, the cleavable linker comprises one or more amino acid sequences selected from the group consisting of: SEQ ID NO: 337, SEQ ID NO: 338, SEQ ID NO: 339, and SEQ ID NO: 372-377.

[0260] In some embodiments, the cleavable linker further comprises spacer sequences. In some embodiments, the cleavable linker comprises two spacer sequences located on either side of the N-terminus and C-terminus of an amino acid sequence recognized and cleaved by one or more proteases.

[0261] In some embodiments, the cleavable linker comprises two or more sets of amino acid sequences that are recognized and cleaved by one or more proteases. In some embodiments, at least one set of the two or more sets of amino acid sequences recognized and cleaved by one or more proteases is side-joined with two spacer sequences in the cleavable linker, and each set of the two or more sets of amino acid sequences recognized and cleaved by one or more proteases is side-joined with two spacer sequences in the cleavable linker.

[0262] In some embodiments, the spacer sequence comprises a GS linker. In some embodiments, the length of the GS linker is no more than about 10, 9, 8, 7, 6, or 5 amino acids.

[0263] In some embodiments, the cleavable linker consists of no more than about 50, 40, 35, or 30 amino acids. In some embodiments, the cleavable linker consists of no less than about 10, 15, 20, or 25 amino acids. In some embodiments, the cleavable linker consists of about 25 to about 30 amino acids, and the masking portion includes an extracellular domain of IL-18Rα. In some embodiments, the masking portion includes the amino acid sequence shown in any one of SEQ ID NO: 353-356, or a functional variant thereof, the functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with any one of SEQ ID NO: 353-356. In some embodiments, the cleavable linker includes the amino acid sequence shown in any one of SEQ ID NO: 399-407, SEQ ID NO: 492-493, and SEQ ID NO: 495. C. Adjustment of the linker length between the binding portion and the IL-18 component

[0264] In some embodiments, the IL-18 component is fused to the binding portion. In some embodiments, the IL-18 component is covalently coupled to the binding portion. In some embodiments, a linker exists between the binding portion and the IL-18 component. In some embodiments, no linker exists between the binding portion and the IL-18 component.

[0265] In some embodiments, the linker is a non-peptide linker (e.g., a PEG linker). In some embodiments, the length of the non-peptide linker is equivalent to that of a peptide linker of about 1-15 amino acids (e.g., about 1-5 amino acids, about 5-10 amino acids, about 10-15 amino acids, or about or no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids).

[0266] In some embodiments, the linker is a peptide linker (e.g., a GS linker). In some embodiments, the linker is no more than about 35 amino acids long. In some embodiments, the linker is about 15-35 amino acids long. In some embodiments, the linker is about 1-15 amino acids long. In some embodiments, the linker is about 1-5 amino acids long. In some embodiments, the linker is about 5-10 amino acids long. In some embodiments, the linker is about 10-15 amino acids long. In some embodiments, the linker is no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids long. In some embodiments, the linker is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids long. IL-18 components or IL-18 peptides with reduced affinity for IL-18-binding proteins (such as wild-type human IL-18-binding protein).

[0267] In some embodiments, the IL-18 component or IL-18 peptide exhibits a reduced binding affinity for IL-18BP compared to the IL-18 peptide shown in SEQ ID NO: 1. In some embodiments, the reduced binding affinity is: a) at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, or b) about 10%-20%, 20%-30%, 30%-40%, 40%-50%, 50%-60%, 60%-70%, 70%-80%, 80%-90%, 90%-95%, 95%-96%, 96%-97%, 97%-98%, and / or 98%-99%, for example, through K as described below. D Determined by method.

[0268] In some embodiments, the one or more mutations are selected from the group consisting of G3, E6, D54, and N91. In some embodiments, the IL-18 peptide contains mutations at two or more sites among G3, E6, D54, and N91, for example, any combination of the aforementioned sites. In some embodiments, the two or more mutations are selected from the group consisting of G3, E6, D54, and N91. In some embodiments, the IL-18 peptide contains mutations at three or more sites among G3, E6, D54, and N91, for example, any combination of the aforementioned sites. In some embodiments, the three or more mutations are selected from the group consisting of G3, E6, D54, and N91. In some embodiments, the IL-18 peptide contains mutations at G3, E6, D54, and N91. In some embodiments, the IL-18 peptide does not contain mutations at sites other than G3, E6, D54, and / or N91. In some embodiments, the IL-18 peptide contains (or further contains) mutations at sites other than G3, E6, D54, and / or N91. In some embodiments, the IL-18 peptide contains one or more mutations (e.g., any 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mutations), wherein at least one, at least two, at least three, or at least four mutations are selected from the group consisting of G3, E6, D54, and N91. In some embodiments, the wild-type IL-18 is human IL-18 containing the amino acid sequence shown in SEQ ID NO: 1.

[0269] In some embodiments, the IL-18 polypeptide contains (e.g., also contains) at least one mutation at a residue selected from the group consisting of Q56, P57, M60, Q103, R104, M113 and N155, wherein the amino acid position is relative to the wild-type human IL-18 shown in SEQ ID NO: 1.

[0270] In some embodiments, the IL-18 polypeptide contains a mutation at the G3 residue, wherein the mutation is selected from the group consisting of G3P, G3D, G3E, G3N, and G3S. As used herein, “G3X” indicates that the wild-type amino acid G at position 3 of SEQ ID NO: 1 has been replaced with amino acid X. In some embodiments, the mutation is G3P, i.e., the wild-type G at position 3 of SEQ ID NO: 1 has been replaced with amino acid P.

[0271] In some embodiments, the variant polypeptide comprises (e.g., also comprises) a mutation at the E6 residue, wherein the mutation is selected from the group consisting of E6R, E6K, E6G, E6T, E6A, E6S, E6E, E6L, E6M, and E6N. In some embodiments, the mutation is selected from the group consisting of E6R, E6K, E6G, E6T, E6A, and E6S. In some embodiments, the mutation is selected from the group consisting of E6R and E6K.

[0272] In some embodiments, the variant polypeptide comprises (e.g., also comprises) a mutation at the D54 residue, wherein the mutation is selected from the group consisting of D54W, D54H, D54S, D54Q, D54L, D54Y, D54P, D54A, D54F, D54G, and D54T. In some embodiments, the mutation is selected from the group consisting of D54W, D54H, D54S, D54Q, D54L, and D54Y.

[0273] In some embodiments, the variant polypeptide comprises (e.g., also comprises) a mutation at the N91 residue, wherein the mutation is selected from the group consisting of N91V, N91A, N91G, N91S, N91I, N91P, N91R, N91T, N91C, N91K, and N91W. In some embodiments, the mutation is selected from the group consisting of N91V, N91A, N91G, and N91S.

[0274] In some embodiments, the variant further comprises a mutation at the R104 residue, wherein the mutation is selected from the group consisting of R104S, R104Y, R104T, R104L, R104V, R104A, R104F, R104H, R104I, and R104N. In some embodiments, the mutation is selected from the group consisting of R104S, R104Y, and R104T. In some embodiments, the IL-18 peptide does not contain a mutation at the R104 residue.

[0275] In some embodiments, the IL-18 peptide comprises (e.g., also comprises) a mutation at the Q56 residue, wherein the mutation is selected from the group consisting of Q56T, Q56G, Q56R, Q56S, Q56D, Q56P, Q56I, Q56Y, Q56H, Q56L, Q56E, Q56F, Q56N, and Q56V. In some embodiments, the mutation is selected from the group consisting of Q56T, Q56G, Q56R, Q56S, Q56D, Q56P, Q56I, and Q56Y.

[0276] In some embodiments, the IL-18 peptide comprises (for example, also comprises) a mutation at the p57 residue, wherein the mutation is selected from the group consisting of p57A, p57G, p57R, p57W, p57S, p57T, p57V, p57Q, p57H, p57K, p57N, p57Y, and p57D. In some embodiments, the mutation is selected from the group consisting of p57A, p57G, p57R, p57W, p57S, p57T, and p57V.

[0277] In some embodiments, the IL-18 peptide contains mutations at one or more sites among G3, E6, D54, and N91. In some embodiments, the IL-18 peptide contains mutations at two or more sites among G3, E6, D54, and N91. In some embodiments, the IL-18 peptide contains mutations at three or more sites among G3, E6, D54, and N91. In some embodiments, the IL-18 peptide contains mutations at G3, E6, D54, and N91. In some embodiments, the IL-18 peptide contains a G3P mutation. Additionally or alternatively, in some embodiments, the IL-18 peptide contains an E6R or E6K mutation. Additionally or alternatively, in some embodiments, the IL-18 peptide contains a D54W, D54H, D54S, or D54Q mutation. Additionally or alternatively, in some embodiments, the IL-18 peptide contains an N91V, N91A, N91G, or N91S mutation. In some embodiments, the IL-18 peptide does not contain mutations at sites other than G3, E6, D54, and / or N91. In some embodiments, the IL-18 peptide also contains mutations at sites other than G3, E6, D54, and / or N91. In some embodiments, the IL-18 peptide contains one or more mutations (e.g., any 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mutations), wherein at least one, at least two, at least three, or at least four mutations are selected from the group consisting of G3, E6, D54, and N91. In some embodiments, the IL-18 peptide also contains mutations at Q56 and / or P57. In some embodiments, the IL-18 peptide does not contain mutations at sites other than G3, E6, D54, N91, Q56, and / or P57. In some embodiments, the IL-18 peptide further contains mutations at sites other than G3, E6, D54, N91, Q56, and / or P57. In some embodiments, the IL-18 peptide contains one or more mutations (e.g., any 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mutations), wherein at least one, at least two, at least three, at least four, at least five, or at least six mutations are selected from the group consisting of G3, E6, D54, N91, Q56, and P57.

[0278] In some embodiments, the IL-18 peptide contains at least one (or two or three) mutations at residues selected from the group consisting of G3, E6, and M60, wherein the amino acid positions are relative to wild-type human IL-18 as shown in SEQ ID NO: 1. In some embodiments, the IL-18 peptide contains at least one of the following: a) G3P, G3S, G3E, G3N, G3D, or G3A; b) E6K, E6T, E6R, E6A, E6M, E6L, E6G, E6H, E6S, or E6Y; or c) M60K. In some embodiments, the IL-18 peptide further contains at least one or more (e.g., two, three, four, or five) mutations at residues selected from the group consisting of V11, D54, Q56, P57, N91, K96, R104, R140, I149, and N155. In some embodiments, the IL-18 peptide further comprises at least one of the following: a) D54H, D54W, D54Q, D54S, D54G, D54P, D54L, D54Y, D54F, D54R, or D54A; b) Q56D, Q56P, Q56H, Q56G, Q56T, Q56R, Q56L, Q56I, Q56S, Q56Y, Q56E, or Q56V; c) P57R, P57D, P57V, P57W, P57A, P57N, P57S, P57T, or P57V. 7Q, P57G, P57K, P57H, P57L, P57I or P57E; d) N91V, N91G, N91A, N91S, N91T, N91R, N91K, N91P, N91I or N91W; e) R104V, R104T, R104Y, R104F, R104T, R104L, R104S, R104A, R104E or R104I; f) N155K; g) V11I; h) K96D or K96E; i) I149M; or j) K140R.

[0279] In some embodiments, the IL-18 peptide contains mutations at two or more (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or thirteen) sites of F2, G3, E6, V11, Q24, L29, D54, A61, N91, K96, R107, K140, and I149, wherein the amino acid positions are relative to the wild-type (WT) human IL-18 shown in SEQ ID NO: 1, for example, any combination of the above sites.

[0280] In some embodiments, the IL-18 peptide comprises G3P, E6K, D54H, Q56D, P57R, N91V, and R104V. In some embodiments, the IL-18 peptide comprises G3S, E6K, D54W, Q56P, P57D, N91G, and R104T. In some embodiments, the IL-18 peptide comprises G3P, E6K, D54W, Q56H, P57V, N91A, and R104Y. In some embodiments, the IL-18 peptide comprises G3P, E6K, D54W, Q56G, P57V, N91V, and R104F. In some embodiments, the IL-18 peptide comprises G3E, E6T, D54W, Q56P, P57W, N91V, R104T, and N155K. In some embodiments, the IL-18 peptide comprises G3P, E6R, D54W, Q56T, N91V, and R104T. In some embodiments, the IL-18 peptide comprises G3P, E6R, D54H, Q56T, P57A, and N91A. In some embodiments, the IL-18 peptide comprises G3P, E6R, D54W, Q56P, P57A, N91A, and R104L. In some embodiments, the IL-18 peptide comprises G3P, E6R, D54W, Q56R, P57A, N91S, and R104S. In some embodiments, the IL-18 peptide comprises G3P, E6R, D54Q, Q56L, P57W, and N91S. In some embodiments, the IL-18 peptide comprises G3P, E6R, D54S, Q56R, P57N, N91G, and R104T. In some embodiments, the IL-18 peptide comprises G3P, E6K, D54G, Q56G, P57A, and N91T. In some embodiments, the IL-18 peptide comprises G3P, E6K, D54Q, Q56I, and P57W. In some embodiments, the IL-18 peptide comprises G3N, E6K, D54P, Q56S, P57S, N91R, and R104A. In some embodiments, the IL-18 peptide comprises G3P, E6R, D54S, Q56Y, P57T, and N91G. In some embodiments, the IL-18 peptide comprises G3P, E6R, D54S, Q56R, P57N, N91G, and R104S. In some embodiments, the IL-18 peptide comprises G3P, E6R, D54L, Q56T, P57A, and N91G. In some embodiments, the IL-18 peptide comprises G3P, E6R, D54S, Q56R, P57R, N91G, and R104S. In some embodiments, the IL-18 peptide comprises G3P, E6K, D54H, Q56E, P57Q, and N91A.In some embodiments, the IL-18 peptide comprises G3P, E6R, D54S, Q56R, P57S, N91G, and R104S. In some embodiments, the IL-18 peptide comprises G3P, E6R, D54S, Q56S, P57T, N91G, and R104S. In some embodiments, the IL-18 peptide comprises G3P, E6R, D54Y, Q56R, P57G, N91K, and R104S. In some embodiments, the IL-18 peptide comprises G3P, E6R, D54Y, Q56T, and P57R. In some embodiments, the IL-18 peptide comprises G3P, E6R, D54L, Q56T, P57T, and N91R. In some embodiments, the IL-18 peptide comprises G3P, E6R, D54H, Q56D, P57K, N91V, and R104Y. In some embodiments, the IL-18 peptide comprises G3P, E6R, D54H, Q56Y, P57T, N91V, and R104Y. In some embodiments, the IL-18 peptide comprises G3P, E6A, D54W, Q56G, P57G, N91V, and R104Y. In some embodiments, the IL-18 peptide comprises G3P, E6M, D54F, Q56D, P57R, and N91P. In some embodiments, the IL-18 peptide comprises G3P, E6L, D54H, Q56T, P57V, and N91S. In some embodiments, the IL-18 peptide comprises G3P, E6R, D54H, Q56I, P57H, N91I, and R104Y. In some embodiments, the IL-18 peptide comprises G3P, E6G, D54S, Q56S, and P57R. In some embodiments, the IL-18 peptide comprises G3E, E6H, D54R, Q56T, and P57H. In some embodiments, the IL-18 peptide comprises G3P, E6R, D54H, Q56R, P57N, N91V, and R104E. In some embodiments, the IL-18 peptide comprises G3P, E6R, D54G, Q56G, P57A, and N91G. In some embodiments, the IL-18 peptide comprises G3P, E6S, D54A, Q56D, P57Q, and N91G. In some embodiments, the IL-18 peptide comprises G3P, E6G, D54Q, Q56V, and P57W. In some embodiments, the IL-18 peptide comprises G3P, E6S, D54W, Q56G, P57A, N91V, and R104I. In some embodiments, the IL-18 peptide comprises G3P, E6R, D54W, Q56P, P57G, N91V, and R104L.In some embodiments, the IL-18 peptide comprises G3D, E6K, D54P, Q56S, P57W, and N91W. In some embodiments, the IL-18 peptide comprises a G3P-containing substitution. In some embodiments, the IL-18 peptide comprises G3P, E6K, D54G, Q56G, and P57A. In some embodiments, the IL-18 peptide comprises G3P, E6R, D54L, Q56G, P57S, and N91V. In some embodiments, the IL-18 peptide comprises G3P, E6R, V11I, D54G, Q56G, P57A, and N91G. In some embodiments, the IL-18 peptide comprises G3P, E6K, D54H, Q56Y, and P57S. In some embodiments, the IL-18 peptide comprises E6R, D54W, Q56S, and P57Q. In some embodiments, the IL-18 peptide comprises G3P, E6K, D54L, Q56T, P57Q, and N91V. In some embodiments, the IL-18 peptide comprises G3A, E6Y, D54R, Q56S, P57L, and N91G. In some embodiments, the IL-18 peptide comprises G3P, E6R, D54L, Q56T, P57I, and N91G. In some embodiments, the IL-18 peptide comprises E6G, D54L, Q56T, P57E, N91G, and R104S. In some embodiments, the IL-18 peptide comprises G3A, E6Y, D54R, Q56S, P57L, and N91A. In some embodiments, the IL-18 peptide comprises M60K and K96D. In some embodiments, the IL-18 peptide comprises G3P, E6R, and K96E. In some embodiments, the IL-18 peptide comprises a substitution containing M60K. In some embodiments, the IL-18 peptide comprises G3P and E6R. In some embodiments, the IL-18 peptide comprises G3P and E6K. In some embodiments, the IL-18 peptide comprises G3D, E6K, and N91S. In some embodiments, the IL-18 peptide comprises G3S and I149M. In some embodiments, the IL-18 peptide comprises G3P, E6R, and N91S. In some embodiments, the IL-18 peptide comprises a substitution containing E6R. In some embodiments, the IL-18 peptide comprises E6R and N91S. In some embodiments, the IL-18 peptide comprises a substitution containing V11I. In some embodiments, the IL-18 peptide comprises G3S and K140R.

[0281] The following lists exemplary IL-18 peptides that have been prepared and tested.

[0282] Table 1: Results of single-clonal FACS analysis of 117 clones

[0283] The affinity of the IL-18 variant peptide for hIL-18Rα was evaluated using a Protein A sensor coated with the hIL-18Rα ECD-Fc fusion protein via Octet RED96 biolayer interferometry (BLI), with WT hIL-18 as a control. The binding affinity of WT hIL-18 and the IL-18 variant peptide for hIL-18Rα ECD-Fc is shown in Table 2A. Table 2A: Affinity of IL-18 variant peptides to hIL-18Rα (Measured by BLI)

[0284] The mutation status of the above-mentioned IL-18 variant peptides (i.e., M1-M41, LM1-LM5, MM1-MM2, MM4-MM6, WM1-WM6, WM8 and WM10-WM14, corresponding to SEQ ID NO: 1-4, 8-9, 11-12, 17, 21-22, 26-28, 35, 38-39, 41, 43-44, 49-50, 54-56, 58-59, 65-66, 72-74, 81, 91, 94, 99, 104-105, 114, 116-117, 121-125, 131-134, 139-145, 147 and 149-153) is shown in Table 2B. Table 2B

[0285] As shown in Table 3, all tested IL-18 variant peptides were able to activate IL-18 receptor-mediated signaling in a dose-dependent manner. Unlike WT hIL-18 (whose activity was significantly reduced (approximately 80-fold) in the presence of 1 μg / mL hIL-18BP-hFc), the IL-18 variant peptides (i.e., M1-M7, M9-M41, LM1-LM5, MM1-MM2, MM4-MM6, WM1-WM6, WM8, and WM10-11, corresponding to SEQ ID NO: Methyl 2-4, 8-9, 11-12, 17, 21, 22, 26-28, 35, 38, 39, 41, 43, 44, 49, 50, 54-56, 58-59, 65-66, 72-74, 81, 91, 94, 99, 104-105, 114, 116-117, 121-125, 131-134, 139-145, 147, and 149-150 all exhibited anti-hIL-18BP activity, and their EC50 values ​​were... 50 The changes did not exceed 3-fold. These results indicate that these IL-18 variant peptides are significantly less affected by hIL-18BP in activating hIL-18 receptor-mediated signaling. The only exception is WM6, whose EC50 decreased significantly in the presence of 1 μg / mL hIL-18BP-hFc. 50 The activity was reduced by about 12-fold, but it was still significantly better than WT hIL-18. Table 3: EC5% of IL-18 variant peptides in hIL-18 reporter gene assay 50 (With or without 1 μg / mL hIL-18BP-hFc)

[0286] The ability of a variant IL-18 peptide to activate T / NK cells and induce hIFNγ release was investigated using PBMC-based experiments. Briefly, human PBMC cells were cultured at 5.0 × 10⁶ cells per well. 5 Cells were seeded at a density of 1000 μg / mL in 96-well plates and treated after 5 hours of culture. WT hIL-18 or its variant peptide was serially diluted with 1 ng / mL human IL-12 in (a) or (b) the presence or absence of 1 μg / mL hIL-18BP-hFc. The diluted solutions were then added to each well. After incubation at 37°C for 16 hours, the culture supernatant was collected from each well, and the hIFNγ concentration was determined by FRET assay using a kit (62HIFNGPEG, Cisbio) according to the manufacturer's instructions.

[0287] All tested IL-18 variant peptides were able to induce hIFNγ release from PBMCs in a dose-dependent manner. Furthermore, as shown in Table 4, the activity of WT hIL-18 was significantly reduced in the presence of 1 μg / mL hIL-18BP-hFc, and its EC50 was significantly decreased. 50 The decrease was approximately 35-fold. In contrast, IL-18 variant peptides (i.e., M11-M13, M15, M17, M21, M24, M29, M32-M33, M35-M36, MM5, WM4-WM6, WM8, and WM10-11, corresponding to SEQ ID NO: 3, 9, 17, 27, 39, 43, 49, 94, 105, 116-117, 133, and 143-150, respectively) all exhibited anti-hIL-18BP activity, with their EC50 values ​​being significantly lower than those of IL-18BP. 50 The changes did not exceed 2-fold. These results indicate that these IL-18 variant peptides are significantly less affected by hIL-18BP in terms of hIFNγ induction. Table 4: EC50 of IL-18 variant peptides in PBMC experiments 50 (With or without 1 μg / mL hIL-18BP) IL-18 peptides with one or more cysteine ​​substitutions

[0288] Studies have found that wild-type human IL-18 peptide is difficult to express in eukaryotic cells (such as CHO cells). As described below, the inventors discovered that by introducing one or more cysteine ​​substitutions into the IL-18 peptide, the yield and / or purity of the IL-18 peptide are significantly improved.

[0289] Wild-type IL-18 and its variant peptides M12 and MM5 were modified (or further modified) to include C38S, C68S, and C76S substitutions, respectively, to produce new variants containing amino acid sequences (see SEQ ID NO: 307-309).

[0290] Subsequently, the C-terminus of each of IL-18-SSS, M12-SSS, and M5-SSS was fused to the N-terminus of human IgG1 Fc containing an N297A substitution (EU number), thereby generating IL-18-SSS-Fc_N297A (SEQ ID NO: 319), M12-SSS_N297A (SEQ ID NO: 320), and M5-SSS_N-297A (SEQ ID NO: 321). The resulting fusion peptides were expressed in CHO cells and purified by protein A chromatography after harvesting. The yield of IL-18-SSS-Fc_N297A obtained by one-step protein A purification was 14.7 mg / L with a purity of 65%. In comparison, the yield of WT IL-18-Fc_N297A obtained by one-step protein A purification was approximately 16 mg / L with a purity of 11%. The yield of M12-SSS-Fc_N297A obtained after one-step protein A purification was 5.6 mg / mL with a purity of 72%. In contrast, no expression level of M12-Fc_N297A was detected in 100 mL of CHO host cells. The yield of MM5-SSS-Fc_N297A obtained after one-step protein A purification was 24.5 mg / L with a purity of 46%. After further gel filtration purification, the yield of MM5-SSS-Fc_N297A was 3.4 mg / L with a purity of 99%. In contrast, no expression level of MM5-Fc_N297A was detected in 100 mL of CHO host cells.

[0291] M12-SSS was further modified to include C127S substitution, thereby producing M12-SSSS (SEQ ID NO:322).

[0292] The C-terminus of M12-SSSS was fused to the N-terminus of a human IgG1 Fc variant containing an N297A substitution (EU number), resulting in M12-SSSS-Fc_N297A (SEQ ID NO: 322). The resulting M12-SSSS-Fc_N297A was expressed in CHO cells and purified by protein A chromatography after harvesting. The yield and purity of M12-SSSS-Fc_N297A were both reduced compared to M12-SSSS-Fc_N297A after the introduction of the C127S substitution (i.e., the fourth C→S substitution) (data not shown).

[0293] Subsequently, the activities of IL-18-SSS-Fc_N297A and M12-SSS-Fc_N297A in activating IL-18 receptor-mediated signaling were characterized. The activity of IL-18-SSS-Fc_N297A was comparable to that of wild-type IL-18 (data not shown), while the activity of M12-SSS-Fc_N297A was approximately 7-fold lower than that of M12 (data not shown). The signaling activity of IL-18-SSS-Fc_N297A was reduced in the presence of 1 μg / mL hIL-18BP-hFc (“BP”), while M12-SSS-Fc_N297A exhibited anti-hIL-18BP activity in the presence of 1 μg / mL hIL-18BP-hFc. The activity of MM5-SSS-Fc_N297A decreased by approximately 220-fold compared to MM5, but it exhibited anti-hIL-18BP activity in the presence of 1 μg / mL hIL-18BP-hFc. These results indicate that the three cysteine→serine substitutions do not affect IL-18BP tolerance. These data suggest that C38, C68, and C76 are crucial for improving the yield of IL-18 and IL-18 peptide-Fc fusion proteins during production and purification, resulting in both increased yield and purity. The half-life of MM5-SSS-Fc_N297A may be longer than that of MM5.

[0294] Computer screening based on molecular dynamics simulations, followed by AI-based stability assessments, identified mutation hotspots at four cysteine ​​residues (C38, C68, C76, and C127) in M12, as shown in Table 5. Furthermore, structure-guided design was employed to identify amino acids at specific spatial positions that, when replaced with Cys residues, could form novel non-natural disulfide bonds. Based on this, mutation strategies (i.e., DB6, DB7, DB8, DB9, and DB10) were proposed as shown in Table 5. Table 5: IL-18 peptide variants derived from M12

[0295] The mutation strategy shown in Table 5 was introduced into the IL-18 peptide variant M12, resulting in new variants M12-DB6, M12-DB7, M12-DB8, M12-DB9 and M12-DB10 (SEQ ID NO: 311-315).

[0296] The C-terminus of each of the M12-DB6, M12-DB7, M12-DB8, M12-DB9, and M12-DB10 variants was fused with a human IgG1 Fc variant containing an N297A substitution (EU number). The resulting fusion peptides, namely M12-DB6-Fc_N297A (SEQ ID NO: 323), M12-DB7-Fc_N297A (SEQ ID NO: 324), M12-DB8-Fc_N297A (SEQ ID NO: 325), M12-DB9-Fc_N297A (SEQ ID NO: 326), and M12-DB10-Fc_N297A (SEQ ID NO: 327), were expressed in CHO cells and purified by protein A chromatography after harvesting.

[0297] The expression yield and / or purity of M12-DB6-Fc_N297A, M12-DB7-Fc_N297A, M12-DB9-Fc_N297A, and M12-DB10-Fc_N297A were significantly superior to those of M12-Fc_N297A or M12-SSS-Fc_N297A. Among M12-DB6-Fc_N297A, M12-DB7-Fc_N297A, M12-DB9-Fc_N297A, and M12-DB10-Fc_N297A, M12-DB6-Fc_N297A showed the highest expression yield (313 mg / L) after one-step protein A purification, with a purity of 89.8% (as determined by SEC-HPLC). No expression level of M12-DB8-Fc_N297A was detected. The expression yield of M12-DB7-Fc_N297A was approximately 50% of that of M12-DB6-Fc_N297A. The purity of M12-DB7-Fc_N297A after protein A chromatography was comparable to that of M12-DB6-Fc_N297A.

[0298] The high yield and high purity (especially the high yield) of DB6 and DB7 are remarkable. The yield of the M12-DB6 form is approximately 60 times that of the SSS form. These data indicate that the strategy of removing cysteine ​​residues at positions 38 and 68 while introducing C117 to promote disulfide bond formation between C117 and C76 has significant advantages.

[0299] The low yield of DB7 and the undetectability of DB8 suggest that C127X substitution may not further increase peptide yield in mammalian cells. Mutations at the partially exposed C127 site require careful design. Based on the C38X+C68X+C76+S117C strategy, position 127 is more likely to be C than A, and the hydrophobic 127I significantly affects the correct folding of the IL-18 peptide. We further tested the corresponding IL-18-DB6 peptide variants, including C127N, C127T, and C127S, all of which showed expression levels comparable to DB6. Given the partially exposed nature of position 127 and the above results, the introduction of amino acids with hydrophobic side chains at position 127 should be avoided under this strategy.

[0300] Subsequently, the activities of M12-DB6-Fc_N297A, M12-DB7-Fc_N297A, M12-DB9-Fc_N297A, and M12-DB10-Fc_N297A in activating IL-18 receptor-mediated signaling were characterized. The activities of M12-DB6-Fc_N297A, M12-DB7-Fc_N297A, M12-DB9-Fc_N297A, and M12-DB10-Fc_N297A in activating IL-18 receptor-mediated signaling were significantly higher than those of WT IL-18. The signal transduction activities of M12-DB6-Fc_N297A, M12-DB7-Fc_N297A and M12-DB9-Fc_N297A were not inhibited in the presence of 1 μg / mL hIL-18BP-hFc.

[0301] Subsequently, “DB6” was replaced (i.e., C38I, C68S and S117C) and introduced into variant MM5, resulting in MM5-DB6 (see SEQ ID NO: 316).

[0302] The wild-type IL-18 was also replaced with “DB6”, resulting in WT IL18-DB6 (see SEQ ID NO: 317).

[0303] Another variant, WT-DBo (see SEQ ID NO:318), was designed by replacing S117C with wild-type IL-18.

[0304] The C-terminus of each of WT IL-18-DB6, WT IL-18-DBo, and MM5-DB6 was fused to the N-terminus of a human IgG1 Fc variant containing an N297A substitution (EU designation). The resulting fusion peptides, namely WT IL18-DB6-Fc_N297A (SEQ ID NO: 328), WT IL-18-DBo-Fc_N297A (SEQ ID NO: 329), and MM5-DB6-Fc_N297A (SEQ ID NO: 330), were expressed in CHO cells and purified by protein A chromatography after harvesting. The purified formulations were analyzed by SEC and SDS-PAGE (data not shown). After the same expression and purification process, no expression level of WT IL18-DBo-Fc_N297A was detected. After one-step protein A chromatography purification, the expression yield of WT IL18-DB6-Fc was 157 mg / L, and the purity of WT IL18-DB6-Fc_N297A was 91.6% (as determined by SEC-HPLC). These data confirm the significant advantage of the strategy of removing cysteine ​​residues at positions 38 and 68 while promoting disulfide bond formation between C76 and C117. These data also indicate that introducing a disulfide bond only between C76 and C117 may not be sufficient to increase the expression level of the fusion peptide in mammalian cells. By utilizing fusion peptides containing IL-18 variants, their expression levels in mammalian cells can be increased, wherein the IL-18 variants contain: (i) engineered C76-C117 disulfide bonds; (ii) C38 substitution (see Table 5); and (iii) C68 substitution (see Table 5). The expression yield and purity of MM5-DB6-Fc_N297A were consistently much higher than those of MM5-SSS-Fc_N297A. These data also indicate that the C38X+C68X+C76+S117C mutation strategy (e.g., "DB6" or "DB7" substitution) is suitable for improving the yield of WT IL-18 and IL-18 variant peptides during and after mammalian cell production.

[0305] Subsequently, fusion peptides were designed, in which the C-terminus of an N297A-substituted (EU number) human IgG1 Fc was fused to the N-terminus of each of the variants M12-DB6 and MM5-DB6. The resulting fusion peptides, Fc_N297A-MM5-DB6 (SEQ ID NO: 332) and Fc_N297A-M12-DB6 (SEQ ID NO: 331), were both expressed in CHO cells and purified by protein A chromatography after harvesting. The expression yield and purity of Fc_N297A-MM5-DB6 and Fc_N297A-M12-DB6 in CHO cells were comparable to those of M12-DB6-Fc_N297A or MM5-DB6-Fc_N297A. After one-step protein A chromatography purification, the purity of Fc_N297A-MM5-DB6 was approximately 97% (as determined by SEC-HPLC), while the purity of Fc_N297A-M12-DB6 was 89% (as determined by SEC-HPLC). These data indicate that the cysteine ​​mutation combined with disulfide bond introduction strategy is applicable to Fc fusions of any fusion sequence.

[0306] Subsequently, the activities of WT IL18-DB6-Fc_N297A, MM5-DB6-Fc_N297A, Fc_N297A-MM5-DB6, and Fc_N297A-M12-DB6 in activating IL-18 receptor-mediated signaling were characterized. WT IL18-DB6-Fc_N297A and MM5-DB6-Fc_N297A both showed stronger IL-18 receptor cell activation capacity compared to WT IL-18. The activity of Fc_N297A-MM5-DB6 was comparable to that of WT IL-18, while Fc_N297A-M12-DB6 showed significantly stronger IL-18 reporter cell activation capacity. In the presence of 1 μg / mL IL-18BP-hFc, the activity of WT IL18-DB6-Fc_N297A was significantly reduced, while the activity of MM5-DB6-Fc_N297A was not inhibited by IL-18BP. Both M12-DB6-Fc_N297A and MM5-DB6-Fc_N297A maintained their anti-IL-18BP activity, while the activity of WT IL18-DB6-Fc_N297A remained inhibited by IL-18BP. These results indicate that the "DB6" mutant combinations (i.e., C38I, C68S, and S117C, see Table 5) improved the expression profile of IL-18 and its variants without affecting their dependence on IL-18BP.

[0307] In summary, these results demonstrate that introducing the C38X+C68X+C76+S117C mutation into WT IL-18-Fc fusion peptides or IL-18 variant-Fc fusion peptides significantly increases the expression yield of the fusion peptides during mammalian cell production and improves the yield of the fusion peptides after chromatographic purification, without affecting anti-IL-18BP activity. Furthermore, WT IL-18 and IL-18 variants engineered to include the "DB6" mutation combination exhibit stronger activity in activating IL-18 receptor-mediated signal transduction. These results were observed in the following fusion peptides: fusion peptides in which the C-terminus of the Fc region is fused to the N-terminus of the IL-18 variant; and fusion peptides in which the C-terminus of the IL-18 variant is fused to the N-terminus of the Fc domain (see Table 6).

[0308] Table 6

[0309] In some embodiments, the IL-18 polypeptide comprises a variant polypeptide containing a cysteine ​​residue at position 117 and at position 76, wherein the amino acid positions are relative to the wild-type human IL-18 shown in SEQ ID NO: 1.

[0310] In some embodiments, the IL-18 variant peptide does not contain cysteine ​​at position 38, or the IL-18 variant peptide does not contain cysteine ​​at position 68.

[0311] In some embodiments, the IL-18 polypeptide comprises: a) cysteine ​​at position 117; b) cysteine ​​at position 76; c) isoleucine at position 38; and d) serine at position 68, wherein the amino acid positions are relative to the wild-type IL-18 polypeptide shown in SEQ ID NO: 1.

[0312] In some embodiments, the IL-18 variant peptide does not contain a cysteine ​​residue at position 38, and the IL-18 variant peptide does not contain a cysteine ​​residue at position 68. In some embodiments, the IL-18 variant peptide comprises 38S, 38I, 38V, 38L, or 38M; optionally, the IL-18 variant peptide comprises 38S, 38I, or 38V. In some embodiments, the IL-18 variant peptide comprises 68S, 68I, 68V, 68L, or 68D; optionally, the IL-18 variant peptide comprises 68S, 68I, or 68L. In some embodiments, the IL-18 variant peptide comprises 127C. In some embodiments, the IL-18 variant polypeptide comprises alanine or an amino acid without a hydrophobic side chain at position 127; optionally, the amino acid without a hydrophobic side chain is selected from the group consisting of cysteine, serine, threonine, asparagine, glutamine, glycine, and proline; more preferably, the amino acid without a hydrophobic side chain is selected from the group consisting of cysteine, serine, and threonine. In some embodiments, the IL-18 variant polypeptide comprises: a) I38, V38, or S38; b) I68, S68, or L68; c) C76; and d) C117. In some embodiments, the IL-18 variant polypeptide comprises I38, S68, C76, and C117. In some embodiments, the IL-18 variant polypeptide comprises V38, I68, C76, and C117; optionally, the variant polypeptide also comprises A127.

[0313] In some embodiments, the IL-18 polypeptide comprises G3P, E6K, D54G, Q56G, P57A, and N91T, wherein the amino acid positions are relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 polypeptide, compared to the wild-type human IL-18 polypeptide, a) has a reduced binding affinity for the IL-18 receptor, and / or b) has a similar or reduced binding affinity for the IL-18 binding protein. In some embodiments, the IL-18 polypeptide further comprises an S117C substitution. In some embodiments, the S117C substitution promotes the formation of a C76-C117 disulfide bond. In some embodiments, the IL-18 polypeptide further comprises a C38 substitution; optionally, the substitution is selected from the group consisting of C38I, C38V, C38L, and C38M; more optionally, the substitution is C38I or C38V. In some embodiments, the IL-18 polypeptide further comprises a C68 substitution; optionally, the substitution is selected from the group consisting of C68I, C68V, C68S, and C68D; further optionally, the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 polypeptide further comprises a C76 and / or C127 substitution; optionally, the C76 substitution is C76Y or C76V; optionally, the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L; further optionally, the C127 substitution is C127A or C127I. In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions; optionally, the variant polypeptide further comprises a C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant peptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant peptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant peptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant peptide further comprises C38I, C68L, and C76Y.

[0314] In some embodiments, the IL-18 peptide comprises G3P, E6K, D54G, Q56G, P57A, C38I, C68S, S117C, and N91T. In some embodiments, the IL-18 peptide further comprises 76C and 127C. In some embodiments, the IL-18 peptide further comprises at least one of G108A, H109A, and K112A.

[0315] In some embodiments, the IL-18 peptide comprises E6G, D54L, Q56T, P57E, N91G, and R104S, wherein the amino acid positions are relative to the wild-type human IL-18 shown in SEQ ID NO: 1. In some embodiments, the IL-18 peptide, compared to the wild-type human IL-18 peptide, a) has a reduced binding affinity for the IL-18 receptor, and / or b) has a similar or reduced binding affinity for the IL-18 binding protein. In some embodiments, the IL-18 peptide further comprises an S117C substitution. In some embodiments, the S117C substitution promotes the formation of a C76-C117 disulfide bond. In some embodiments, the IL-18 peptide further comprises a C38 substitution; optionally, the substitution is selected from the group consisting of C38I, C38V, C38L, and C38M; more preferably, the substitution is C38I or C38V. In some embodiments, the IL-18 polypeptide further comprises a C68 substitution; optionally, the substitution is selected from the group consisting of C68I, C68V, C68S, and C68D; further optionally, the substitution is C68I, C68L, or C38S. In some embodiments, the IL-18 polypeptide further comprises a C76 and / or C127 substitution; optionally, the C76 substitution is C76Y or C76V; optionally, the C127 substitution is selected from the group consisting of C127A, C127I, C127Y, C127F, and C127L; further optionally, the C127 substitution is C127A or C127I. In some embodiments, the variant polypeptide further comprises C38S, C68S, and C76S substitutions; optionally, the variant polypeptide further comprises a C127S substitution. In some embodiments, the variant polypeptide further comprises C38I, C68S, and S117C. In some embodiments, the variant peptide further comprises C38V, C68I, S117C, and C127A. In some embodiments, the variant peptide further comprises C38S, C68I, S117C, and C127I. In some embodiments, the variant peptide further comprises C38I, C68I, C76V, and C127I. In some embodiments, the variant peptide further comprises C38I, C68L, and C76Y.

[0316] In some embodiments, the IL-18 peptide comprises E6G, D54L, Q56T, P57E, N91G, C38I, C68S, S117C, and R104S. In some embodiments, the IL-18 peptide further comprises 76C and 127C. In some embodiments, the IL-18 peptide further comprises at least one of G108A, H109A, and K112A.

[0317] In some embodiments, the IL-18 polypeptide comprises an amino acid sequence represented by any one of SEQ ID NO: 1-299, 307-318, 465-469, and 482-491. In some embodiments, the IL-18 polypeptide comprises an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence represented by any one of SEQ ID NO: 1-299, 307-318, 465-469, and 482-491.

[0318] In some embodiments, the IL-18 polypeptide comprises the amino acid sequence shown in any one of SEQ ID NO: 1-299 and 307-318. In some embodiments, the IL-18 polypeptide comprises an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in any one of SEQ ID NO: 1-299 and 307-318. Methods for preparing nucleic acids, vectors, host cells, and constructs containing IL-18 components

[0319] This document also relates to nucleic acid molecules encoding constructs containing the IL-18 component described herein. In some embodiments, one or more nucleic acids are provided that encode a construct or a portion thereof containing the IL-18 component described herein (e.g., one or more polypeptides contained in the construct).

[0320] Vectors in which one or more nucleic acids described herein are inserted are also provided.

[0321] In short, the expression of the construct can be achieved by inserting a natural or synthetic nucleic acid encoding the IL-18 component described herein into a suitable expression vector, such that the nucleic acid is operatively linked to 5' and 3' regulatory elements, including, for example, promoters (e.g., constitutive promoters, regulated promoters, or tissue-specific promoters) and a 3' untranslated region (UTR). The vector is suitable for replication and integration in eukaryotic host cells. Typical cloning and expression vectors contain transcription and translation terminators, a start sequence, and a promoter for regulating the expression of the target nucleic acid sequence.

[0322] The nucleic acid can be cloned into various types of vectors. For example, the nucleic acid can be cloned into vectors, including but not limited to plasmids, phage particles, phage derivatives, animal viruses, and granules. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0323] Furthermore, the expression vector can be introduced into cells in the form of a viral vector. Viral vector technology is well known in the art, for example, as described in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory, New York) and other virology and molecular biology manuals. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. Typically, suitable vectors contain at least one functional origin of replication in an organism, a promoter sequence, an easily manipulated restriction endonuclease site, and one or more selection markers (see, for example, WO 01 / 96584; WO 01 / 29058; and U.S. Patent No. 6,326,193).

[0324] Various virus-based mammalian cell gene transfer systems have been developed. For example, retroviruses provide an easy-to-operate platform for gene delivery systems. Selected genes can be inserted into vectors using techniques known in the art, and packaged to form retroviral particles. Subsequently, the resulting recombinant virus can be isolated and delivered in vivo or in vitro to recipient cells. Various retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Various adenoviral vectors are known in the art. In some embodiments, lentiviral vectors are used. Vectors derived from retroviruses (e.g., lentiviruses) are suitable tools for achieving long-term gene transfer because they allow for long-term stable integration of transgenes and continuous inheritance in daughter cells. Compared to vectors derived from tumor retroviruses (e.g., murine leukemia virus), lentiviral vectors have the added advantage of being able to transduce non-proliferating cells, such as hepatocytes. Furthermore, lentiviral vectors have the added advantage of low immunogenicity.

[0325] Other promoter elements (such as enhancers) regulate the frequency of transcription initiation. These elements are typically located 30–110 base pairs (bp) upstream of the start site, although recent studies have shown that some promoters also contain functional elements downstream of the start site. The spacing between promoter elements is generally flexible, so promoter function is preserved even when these elements are inverted or shifted relative to each other. In the thymidine kinase (TK) promoter, the spacing between promoter elements can increase to 50 bp before its activity begins to decline.

[0326] An example of a suitable promoter is the cytomegalovirus (CMV) immediate early promoter sequence. This promoter sequence is a strongly constitutive promoter sequence capable of driving high-level expression of any polynucleotide sequence operatively linked to it. Another example of a suitable promoter is extension growth factor-1α (EF-1α). However, other constitutive promoter sequences may also be used, including but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukosis virus promoter, Epstein-Barr virus (EBV) immediate early promoter, Rous sarcoma virus promoter, and human gene promoters, such as actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter, but not limited thereto. Furthermore, the invention is not limited to constitutive promoters. The invention also relates to inducible promoters. The use of inducible promoters provides a molecular switch that enables the expression of a polynucleotide sequence operatively linked to it when expression is desired, and disables it when expression is not desired. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0327] In some embodiments, the expression of the nucleic acid encoding the construct containing the IL-18 component is inducible. In some embodiments, the nucleic acid encoding the construct is operatively linked to an inducible promoter, including any inducible promoter known in the art. In some embodiments, the nucleic acid encoding the construct containing the IL-18 component described herein is engineered to encode an epitope tag, for example, to facilitate the purification or detection of the peptide. Exemplary epitope tags include, but are not limited to: 6× His (also known as a His tag or hexahistine tag), FLAG, HA, Myc, V5, GFP (green fluorescent protein, such as enhanced green fluorescent protein or EGFP), SUMO (small ubiquitin-like modified protein), GST (glutathione S-transferase), β-GAL (β-galactosidase), luciferase, MBP (maltose-binding protein), RFP (red fluorescent protein), and VSV-G (vesicular stomatitis virus glycoprotein).

[0328] The constructs disclosed herein can be prepared by any means known in the art. Exemplary techniques for peptide preparation are described below; however, these exemplary techniques are for illustrative purposes only and are not intended to limit the invention.

[0329] The constructs containing the IL-18 component described herein can be prepared using recombinant methods. For the recombinant production of constructs containing IL-18 component peptides, the nucleic acid encoding the construct containing the IL-18 component is isolated and inserted into a reproducible vector for further cloning (DNA amplification) or expression. The DNA encoding the construct containing the IL-18 component can be readily isolated and sequenced using conventional methods (e.g., using oligonucleotide probes capable of specifically binding to the gene encoding the construct containing the IL-18 component). A variety of vectors are currently available. Vector components typically include, but are not limited to, one or more of the following: signal sequences, origin of replication, one or more marker genes, enhancer elements, promoters, and transcription termination sequences.

[0330] Both expression vectors and cloning vectors contain a nucleic acid sequence that enables the vector to replicate in one or more selected host cells, such as enabling the vector to replicate independently of host chromosomal DNA. This sequence may contain an origin of replication or an autonomous replication sequence. Such sequences are well known in the art for a wide variety of bacteria, yeasts, and viruses. Typically, mammalian expression vectors do not require an origin of replication component (an SV40 origin of replication can be used because it contains an early promoter). In some embodiments, a host cell is provided herein containing one or more nucleic acids encoding a construct containing the IL-18 component described herein, or one or more vectors containing one or more nucleic acids encoding a construct containing the IL-18 component described herein. In some embodiments, the host cell is a prokaryotic cell. In some embodiments, the prokaryotic cell is an *E. coli* cell. In some embodiments, the host cell is a eukaryotic cell. In some embodiments, the eukaryotic cell is a CHO cell.

[0331] Expression vectors and cloning vectors may contain selection genes or selection markers. Typical selection genes encode proteins that function to: (a) confer resistance to antibiotics or other toxins (e.g., ampicillin, neomycin, methotrexate, or tetracycline); (b) supplement auxotrophic deficiencies; or (c) provide critical nutrients unavailable in complex media. Examples of dominant selection include the use of drugs such as neomycin, mycophenolic acid, and hygromycin. Another example of selection markers applicable to mammalian cells is a marker that recognizes cells with the ability to take up nucleic acids encoding the construct, such as DHFR, glutamine synthase (GS), thymidine kinase, metallothionein type I and II (preferably primate metallothionein genes), adenosine deaminase, ornithine decarboxylase, etc. For example, for Chinese hamster ovary (CHO) cell lines lacking endogenous DHFR activity, selection is performed by culturing transformed cells in a medium containing methotrexate (Mtx) (a competitive antagonist of DHFR) after transfection with the DHFR gene.

[0332] Alternatively, after transfecting or co-transfecting the target construct's DNA sequence, the wild-type DHFR gene, and another selectable marker (such as aminoglycoside 3'-phosphotransferase (APH)) into host cells (especially wild-type host cells containing endogenous DHFR), selection can be performed by culturing the cells in a medium containing the selectable marker's corresponding selection agent (such as an aminoglycoside antibiotic, such as kanamycin, neomycin, or G418).

[0333] Expression vectors and cloning vectors typically contain a promoter recognized by the host organism, which is operatively linked to the nucleic acid encoding the construct. Promoters suitable for prokaryotic hosts include... pho The A promoter, β-lactamase and lactose promoter systems, alkaline phosphatase promoters, tryptophan (trp) promoter systems, and heterozygous promoters (e.g., the tac promoter) are all applicable. However, other known bacterial promoters are equally applicable. Eukaryotic promoter sequences are known. Yeast promoters are well-known in the art and can include inducible promoters / enhancers regulated by growth conditions. Almost all eukaryotic genes have an AT-rich region approximately 25 to 30 bases upstream of the transcription start site. Examples include, but are not limited to, promoters of 3-phosphoglycerate kinase or other glycolytic enzymes, such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triose phosphate isomerase, phosphoglucose isomerase, and glucokinase. In mammalian host cells, transcription of the vector-encoded IL-18 polypeptide or fusion polypeptide can be controlled, for example, by promoters derived from viral genomes. Early and late promoters of SV40 virus are readily available as fragments containing SV40 restriction endonucleases and also include the SV40 virus origin of replication. Immediate early promoters of human cytomegalovirus are readily available as fragments containing HindIII restriction endonucleases. Alternatively, long terminal repeat sequences of Rous sarcoma virus can be used as promoters.

[0334] In higher eukaryotes, transcription of DNA encoding the constructs described herein can often be enhanced by inserting enhancer sequences into vectors. Many enhancer sequences derived from mammalian genes (globin, elastase, albumin, alpha-fetoprotein, and insulin) are known. However, enhancers derived from eukaryotic viruses are commonly employed.

[0335] Expression vectors used for eukaryotic host cells (nucleated cells of yeast, fungi, insects, plants, animals, humans, or other multicellular organisms) should also contain sequences necessary for terminating transcription and stabilizing mRNA.

[0336] This document provides a method for preparing a construct or a portion thereof containing an IL-18 component. In some embodiments, the method includes culturing a host cell containing one or more nucleic acids and one or more vectors described herein under conditions expressing a construct or a portion thereof containing an IL-18 component. In some embodiments, the method includes recovering the construct or a portion thereof containing an IL-18 component generated from the host cell. This document also provides a method for preparing said construct, comprising: (a) culturing the host cell under conditions expressing a construct or a portion thereof containing an IL-18 component; and (b) recovering the construct or a portion thereof containing an IL-18 component generated from the host cell.

[0337] The host cells suitable for cloning or expressing the DNA in the vectors described herein are the aforementioned prokaryotic cells, yeast cells, or higher eukaryotic cells. Suitable prokaryotes include eubacteria, such as Gram-negative or Gram-positive bacteria, such as Enterobacteriaceae, such as Escherichia, and so on. E. coli ), Enterobacteriaceae ( Enterobacter Erwinia ( ) Erwinia ), Klebsiella spp. Klebsiella ), Proteus spp. Proteus Salmonella ( Salmonella For example, Salmonella typhimurium (Salmonella typhimurium) Salmonella typhimurium Serratia spp. Serratia For example, Serratia marcescens ( Serratia marcescans Shigella spp. Shigella Besides prokaryotes, eukaryotic microorganisms (such as filamentous fungi or yeast) are also suitable cloning or expression hosts for vectors encoding IL-18 peptide variants or fusion peptides. Saccharomyces cerevisiae (Saccharomyces cerevisiae) Saccharomyces cerevisiae Ordinary baker's yeast is the most commonly used lower eukaryotic host microorganism. Certain fungal and yeast strains that have achieved "humanization" of the glycosylation pathway can be selected to produce IL-18 peptides with partially or fully humanized glycosylation patterns. See, for example, Li et al. Nat. Biotech. 24: 210-215 (2006).

[0338] Plant cell cultures can also be used as hosts, including cotton, corn, potato, soybean, petunia, tomato, and duckweed. Leninaceae ), alfalfa M. truncatula ) and tobacco.

[0339] Suitable host cells for expressing glycosylated constructs are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. Numerous baculovirus strains and variants, along with corresponding susceptible insect host cells derived from the fall armyworm (Pterocarya stenoptera), have been identified. Spodoptera frugiperda Caterpillars), Aedes aegypti ( Aedes aegypti Mosquitoes), Aedes albopictus ( Aedes albopictus Mosquitoes), fruit flies (Drosophila melanogaster) Drosophila melanogaster Fruit flies), silkworms ( Bombyx mori (and other hosts)

[0340] Vertebrate cells can be used as hosts, which is a particular advantage of certain implementations described herein (see, for example, the section on "IL-18 peptides with one or more cysteine ​​substitutions"). Examples of useful mammalian host cell lines include: the monkey kidney CV1 cell line transformed with SV40 (COS-7, ATCC CRL 1651); the human embryonic kidney cell line (293 cells subcloned for suspension culture growth; Graham et al., J. Gen Virol. 36: 59 (1977)); juvenile hamster kidney cells (BHK, ATCC CCL 10); mouse Sertoli cells (TM4; Mather, Biol. Reprod. 23: 243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical cancer cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat hepatocytes (BRL 3A, ATCC CRL1442); human lung cells (W138, ATCC CCL 75); human hepatocytes (Hep G2, HB 8065); mouse mammary tumor cells (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals, NY Acad. Sci. 383: 44-68 (1982)); MRC 5 cells; FS4 cells; and the human hepatocellular carcinoma cell line (Hep G2). Other useful mammalian host cell lines include: Chinese hamster ovary (CHO) cells, including DHFR - CHO cells (Urlaub et al.) Proc. Natl. Acad. Sci. USA 77: 4216 (1980)); and myeloma cell lines, such as NS0 and Sp2 / 0. For reviews of certain mammalian host cell lines suitable for production, see, for example, Yazaki and Wu, Methods in Molecular BiologyVol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 255-268.

[0341] The host cells disclosed herein can be cultured in a variety of media. Commercially available media, such as Ham's F10 (Sigma), Minimal Essential Medium (MEM; Sigma), RPMI-1640 (Sigma), and DuPont Modified Eagle Medium (DMEM; Sigma), are all suitable for culturing host cells. Furthermore, Ham et al., Meth. Enz. 58:44 (1979); Barnes et al., Anal. Biochem. 102: 255 (1980); any of the culture media described in U.S. Patent Nos. 4,767,704, 4,657,866, 4,927,762, 4,560,655, or 5,122,469; WO 90 / 03430; WO 87 / 00195; or U.S. Reissue Patent No. 30,985 may be used as a culture medium for host cells. Any of the above culture media may be supplemented as needed with hormones and / or other growth factors (e.g., insulin, transferrin, or epidermal growth factor), salts (e.g., sodium chloride, calcium, magnesium, and phosphate), buffers (e.g., HEPES), nucleotides (e.g., adenosine and thymidine), and antibiotics (e.g., GENTAMYCIN). TM The formula includes drugs, trace elements (defined as inorganic compounds typically in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplementary components may also be added at appropriate concentrations known to those skilled in the art. Culture conditions (e.g., temperature, pH, etc.) are the same as those previously used for the selected expression host cells and will be obvious to those skilled in the art.

[0342] When using recombinant technology, constructs containing the IL-18 component can be generated intracellularly, in the periplasmic space, or directly secreted into the culture medium. If the construct containing the IL-18 component is generated intracellularly, particulate debris (host cell fragments or lysed fragments) is first removed, for example, by centrifugation or ultrafiltration. (Carter et al.) Bio / Technology 10: 163-167 (1992) describes a method for isolating antibodies secreted into the periplasmic space of Escherichia coli.

[0343] Cell-derived constructs containing IL-18 components can be purified using methods such as hydroxyapatite chromatography, hydrophobic interaction chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being one of the generally preferred purification steps. In some embodiments, the IL-18-containing constructs described herein include epitope tags (e.g., tags linked to the construct via cleavable linkers) to facilitate purification. Exemplary epitope tags include, but are not limited to: 6× His (also known as His tags or hexahistine tags), FLAG, HA, Myc, V5, GFP (green fluorescent protein, such as enhanced green fluorescent protein or EGFP), SUMO (small ubiquitin-like modified protein), GST (glutathione S-transferase), β-GAL (β-galactosidase), luciferase, MBP (maltose-binding protein), RFP (red fluorescent protein), and VSV-G (vesicular stomatitis virus glycoprotein). Treatment methods for diseases, methods to activate hIL-18 receptor-mediated signal transduction, and methods to stimulate antigen-empirical T cells or NK cells.

[0344] This document also provides methods for treating an individual disease or condition. The methods include administering to an individual suffering from the disease or condition a construct comprising an IL-18 component as described herein, a nucleic acid as described herein, a vector as described herein, and / or a pharmaceutical composition as described herein. In some embodiments, the disease or condition is a proliferative disease. In some embodiments, the proliferative disease is cancer. In some embodiments, the cancer is a solid tumor.

[0345] In some embodiments, this document provides methods for activating hIL-18 receptor-mediated signaling in an individual, the methods comprising administering to the individual a construct, a nucleic acid, a carrier, and / or a pharmaceutical composition as described herein. In some embodiments, this document provides methods for stimulating antigenic empirical T cells or NK cells in an individual, the methods comprising administering to the individual a construct containing an IL-18 component as described herein, a nucleic acid, a carrier, and / or a pharmaceutical composition as described herein. In some embodiments of any of the methods herein, the individual is a mammal (e.g., human, non-human primate, rat, mouse, cattle, horse, pig, sheep, goat, dog, cat, etc.). In some embodiments, the individual is a human. In some embodiments, the individual is a clinical patient, a clinical trial volunteer, an experimental animal, etc. Compositions, kits and products

[0346] This article also provides compositions (e.g., formulations) that comprise a construct, nucleic acid, vector, or host cell containing the IL-18 component described herein.

[0347] Suitable compositions can be prepared by mixing a construct, nucleic acid, vector, or host cell containing an IL-18 component of desired purity with an optional pharmaceutically acceptable carrier, excipient, or stabilizer. Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)).

[0348] This document also provides a kit comprising a construct, nucleic acid, or vector containing the IL-18 component described herein, or a host cell containing the nucleic acid or vector described herein. This kit may be used for any of the therapeutic methods described herein.

[0349] The kit described in this application is packaged in a suitable manner. Suitable packaging includes, but is not limited to, vials, bottles, wide-mouth bottles, flexible packaging (e.g., sealed polyester film bags or plastic bags), etc. The kit may optionally contain other components, such as buffer solutions and explanatory information.

[0350] Therefore, this application also provides articles of manufacture. The articles of manufacture may include a container and a label or packaging instructions affixed to or associated with the container. Suitable containers include vials (e.g., sealed vials), bottles, wide-mouth bottles, flexible packaging, etc. Typically, the container contains the composition and may have a sterile sampling port (e.g., the container may be an intravenous infusion bag, or a vial with a stopper for puncture by a subcutaneous needle).

[0351] Those skilled in the art will recognize that various embodiments are possible within the scope and spirit of this invention. The invention will now be described in more detail with reference to the following non-limiting embodiments. These embodiments are intended to further illustrate the invention and should not, of course, be construed as limiting its scope in any way. Exemplary Implementation

[0352] Implementation Scheme 1: A construct comprising an IL-18 component associated with a binding moiety that specifically binds to an antigen, wherein the IL-18 component comprises an IL-18 polypeptide, wherein: The IL-18 peptide contains one or more modifications compared to the wild-type IL-18 peptide; The IL-18 component further includes a masking portion, wherein the IL-18 peptide is linked to the masking portion via a cleavable linker; or The IL-18 component further includes a peptide linker that links the IL-18 polypeptide to the binding moiety, wherein the peptide linker is no more than 15 amino acids in length; and The IL-18 component has a lower binding affinity to the IL-18 receptor than the wild-type IL-18 polypeptide; optionally, the wild-type IL-18 polypeptide has the amino acid sequence shown in SEQ ID NO: 1; optionally, the binding affinity is evaluated on cells expressing the IL-18 receptor but not the antigen.

[0353] Implementation Scheme 2: The construct according to Implementation Scheme 1, wherein the construct has a single IL-18 component.

[0354] Implementation Scheme 3: The construct according to Implementation Scheme 1 or 2, wherein the binding portion comprises a small molecule.

[0355] Implementation Scheme 4: The construct according to any one of Implementation Schemes 1 to 3, wherein the bonding portion does not have an Fc domain.

[0356] Implementation Scheme 5: The construct according to any one of Implementation Schemes 1 to 3, wherein the bonding portion includes the Fc structural domain.

[0357] Implementation Scheme 6: The construct according to Implementation Scheme 5, wherein the Fc domain comprises an IgG Fc domain.

[0358] Implementation Scheme 7: The construct according to any one of Implementation Schemes 1 to 6, wherein the binding portion comprises an antibody.

[0359] Implementation Scheme 8: The construct according to Implementation Scheme 7, wherein the antibody is a monovalent antibody.

[0360] Implementation Scheme 9: The construct according to Implementation Scheme 7, wherein the antibody is a multivalent antibody.

[0361] Implementation Scheme 10: A construct comprising a single IL-18 component associated with a binding portion of a multivalent antibody containing a specific binding antigen, wherein the antibody contains an Fc domain, the single IL-18 component comprises a single IL-18 polypeptide, the single IL-18 polypeptide comprising a wild-type IL-18 polypeptide or a variant thereof; optionally, the wild-type IL-18 polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1, and the single IL-18 component is fused to the Fc domain.

[0362] Implementation Scheme 11: The construct according to Implementation Scheme 10, wherein: The IL-18 peptide contains one or more modifications compared to the wild-type IL-18 peptide; The IL-18 component further includes a masking portion, wherein the IL-18 peptide is linked to the masking portion via a cleavable linker; or The IL-18 component further includes a peptide linker that links the IL-18 polypeptide to the binding moiety, wherein the length of the peptide linker is no more than 15 amino acids. The wild-type IL-18 polypeptide has the amino acid sequence shown in SEQ ID NO: 1, and the IL-18 component has a lower binding affinity to the IL-18 receptor than the wild-type IL-18 polypeptide; optionally, the binding affinity is evaluated on cells expressing the IL-18 receptor but not the antigen.

[0363] Implementation Scheme 12: The construct according to any one of Implementation Schemes 9 to 11, wherein the antibody is a bivalent antibody.

[0364] Implementation Scheme 13: The construct according to any one of Implementation Schemes 7 to 12, wherein the antibody is selected from the group consisting of: full-length antibody, single-chain Fv (scFv), Fab, Fab', F(ab')2, Fv fragment, disulfide bond-stabilized Fv fragment (dsFv), (dsFv)2, nanobody (VHH), Fv-Fc fusion protein, scFv-Fc fusion protein, scFv-Fv fusion protein, biantibody, triantibody, and tetraantibody.

[0365] Implementation Scheme 14: The construct according to any one of Implementation Schemes 7 and 9 to 13, wherein the antibody is a full-length antibody comprising two heavy chains and two light chains; optionally, the two heavy chains comprise one or more modifications that facilitate the formation of a mortar and pestle structure.

[0366] Implementation Scheme 15: A construct according to any one of Implementation Schemes 1 to 14, wherein the IL-18 component is covalently conjugated to the binding moiety, optionally conjugated via a linker between the IL-18 polypeptide and the binding moiety.

[0367] Implementation Scheme 16: A construct according to any one of Implementation Schemes 1 to 14, wherein the IL-18 component is fused to the binding portion, optionally via a linker between the IL-18 polypeptide and the binding portion; optionally, the linker is a peptide linker containing no more than 35 amino acids.

[0368] Implementation Scheme 17: The construct according to Implementation Scheme 14 or 16, wherein the IL-18 component is fused with one or two heavy chains.

[0369] Implementation Scheme 18: A construct according to any one of Implementation Schemes 14 and 16 to 17, wherein the IL-18 component is fused to the C-terminus of one or two heavy chains and / or one or two light chains of the full-length antibody.

[0370] Implementation Scheme 19: The construct according to any one of Implementation Schemes 5 to 18, wherein the IL-18 component is fused to the C-terminus of the Fc domain.

[0371] Implementation Scheme 20: The construct according to Implementation Scheme 19, wherein the IL-18 component is a single IL-18 component in the construct; optionally, the antibody comprises a full-length antibody, a nanobody, a Fab or a scFv.

[0372] Implementation Scheme 21: The construct according to any one of Implementation Schemes 1 to 9 and 11 to 20, wherein the IL-18 component is fused to the binding portion via the peptide linker, and the length of the peptide linker is no more than 15 amino acids.

[0373] Implementation Scheme 22: The construct according to any one of Implementation Schemes 1 to 21, wherein the antigen is a tumor-specific antigen or a tumor-associated antigen.

[0374] Implementation Scheme 23: The construct according to Implementation Scheme 22, wherein the tumor is a solid tumor.

[0375] Implementation Scheme 24: The construct according to any one of Implementation Schemes 1 to 23, wherein the antigen is a neoantigen.

[0376] Implementation Scheme 25: A construct according to any one of Implementation Schemes 1 to 21, wherein the antigen is an antigen expressed by immune cells; optionally, the immune cells are tumor-infiltrating lymphocytes.

[0377] Implementation Scheme 26: A construct according to any one of Implementation Schemes 1 to 21, wherein the antigen is an immune checkpoint target expressed on T cells or NK cells.

[0378] Implementation Scheme 27: The construct according to Implementation Scheme 26, wherein the antigen is PD-1.

[0379] Implementation Scheme 28: The construct according to Implementation Scheme 27, wherein the binding domain comprises: a heavy chain variable region comprising VH-CDR1, VH-CDR2, and VH-CDR3; and a light chain variable region comprising VL-CDR1, VL-CDR2, and VL-CDR3, wherein: a) VH-CDR1 comprises the amino acid sequence shown in SEQ ID NO: 470, VH-CDR2 comprises the amino acid sequence shown in SEQ ID NO: 471, VH-CDR3 comprises the amino acid sequence shown in SEQ ID NO: 472, VL-CDR1 comprises the amino acid sequence shown in SEQ ID NO: 473, VL-CDR2 comprises the amino acid sequence shown in SEQ ID NO: 474, and VL-CDR3 comprises the amino acid sequence shown in SEQ ID NO: 475; or b) VH-CDR1 comprises the amino acid sequence shown in SEQ ID NO: 476, VH-CDR2 comprises the amino acid sequence shown in SEQ ID NO: 475, and VL-CDR3 comprises the amino acid sequence shown in SEQ ID NO: 476. The amino acid sequence shown in SEQ ID NO: 477, VH-CDR3 contains the amino acid sequence shown in SEQ ID NO: 478, VL-CDR1 contains the amino acid sequence shown in SEQ ID NO: 479, VL-CDR2 contains the amino acid sequence shown in SEQ ID NO: 480, and VL-CDR3 contains the amino acid sequence shown in SEQ ID NO: 481.

[0380] Implementation Scheme 29: The construct according to any one of Implementation Schemes 1 to 28, wherein the IL-18 polypeptide contains one or more modifications relative to the corresponding wild-type IL-18 polypeptide (e.g., SEQ ID NO: 1).

[0381] Implementation Scheme 30: The construct according to Implementation Scheme 29, wherein the IL-18 peptide has a reduced binding affinity for IL-18Rβ compared to the corresponding wild-type IL-18 peptide (e.g., SEQ ID NO: 1); optionally, the reduced binding affinity is: a) at least 50%, 60%, 70%, 80% or 90%, or b) 50%-60%, 60%-70%, 70%-80%, 80%-90%, 90%-95%, 95%-96%, 96%-97%, 97%-98% and / or 98%-99%.

[0382] Implementation Scheme 31: The construct according to Implementation Scheme 29 or 30, wherein the one or more modifications comprise amino acid substitutions selected from the group consisting of G108, H109, ​​K112, D110, R147, M150, G145, K79, D98 and K84, wherein the amino acid position is relative to the wild-type human IL-18 polypeptide shown in SEQ ID NO: 1; optionally, the substitution is an alanine substitution.

[0383] Implementation Scheme 32: The construct according to Implementation Scheme 31, wherein the one or more modifications comprise amino acid substitutions selected from the group consisting of G108, H109, ​​K112, D110, R147, M150, G145, K79 and D98, wherein the amino acid position is relative to the wild-type human IL-18 polypeptide shown in SEQ ID NO: 1; optionally, the substitution is an alanine substitution.

[0384] Implementation Scheme 33: The construct according to any one of Implementation Schemes 29 to 32, wherein the IL-18 component has a reduced binding affinity for IL-18Rα compared to the corresponding wild-type IL-18 polypeptide (e.g., SEQ ID NO: 1); optionally, the reduced binding affinity is: a) at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, or b) about 10%-20%, 20%-30%, 30%-40%, 40%-50%, 50%-60%, 60%-70%, 70%-80%, 80%-90%, 90%-95%, 95%-96%, 96%-97%, 97%-98%, and / or 98%-99%.

[0385] Implementation Scheme 34: The construct according to Implementation Scheme 33, wherein the one or more modifications comprise amino acid substitutions selected from the group consisting of K4, L5, K8, R13, D17, E31, M33, T34, D35, D40, K53, R58, M60, K79, K84, D98, R104, and D132, wherein the amino acid position is relative to the wild-type human IL-18 polypeptide shown in SEQ ID NO: 1; optionally, the substitution is an alanine substitution.

[0386] Implementation Scheme 35: A construct according to any one of Implementation Schemes 29 to 34, wherein the IL-18 component comprises an IL-18 polypeptide, the IL-18 polypeptide comprising two or more modifications, the two or more modifications comprising two or more amino acid substitutions selected from the group consisting of G108, H109, ​​K112, D110, R147, M150, G145, K79, D98, K84, K4, L5, K8, R13, D17, E31, M33, T34, D35, D40, K53, R58, M60, R104, and D132, wherein the amino acid position is relative to the wild-type human IL-18 polypeptide shown in SEQ ID NO: 1; optionally, the substitution is an alanine substitution.

[0387] Implementation Scheme 36: The construct according to any one of Implementation Schemes 1 to 35, wherein the IL-18 polypeptide comprises the amino acid sequence shown in any one of SEQ ID NO: 482-491.

[0388] Implementation Scheme 37: A construct according to any one of Implementation Schemes 1 to 9 and 10 to 36, wherein the IL-18 component further comprises a masking portion, and the IL-18 polypeptide is linked to the masking portion via a cleavable linker.

[0389] Implementation Scheme 38: The construct according to Implementation Scheme 37, wherein the masking portion comprises an IL-18 propeptide; optionally, the IL-18 propeptide comprises a propeptide of human IL-18 (hIL-18); optionally, the hIL-18 propeptide comprises the amino acid sequence shown in SEQ ID NO: 333.

[0390] Implementation Scheme 39: The construct according to Implementation Scheme 38, wherein the masking portion comprises a truncated propeptide of the IL-18 precursor; optionally, the truncated propeptide of the IL-18 precursor comprises the amino acid sequence shown in SEQ ID NO: 336.

[0391] Implementation Scheme 40: The construct according to Implementation Scheme 37, wherein the masking portion comprises the extracellular domain of IL-18Rα, the extracellular domain of IL-18Rβ, IL-18 binding protein, a fragment of any of the foregoing, or a variant of any of the foregoing.

[0392] Implementation Scheme 41: The construct according to Implementation Scheme 40, wherein the masking portion comprises an extracellular domain of IL-18Rα or a fragment thereof; optionally, the fragment of the extracellular domain of IL-18Rα comprises: a) a first and second domain of the extracellular domain, or b) a third domain of the extracellular domain; optionally, the masking portion comprises an amino acid sequence represented by any one of SEQ ID NO: 353-356, or a functional variant thereof, the functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with any one of SEQ ID NO: 353-356.

[0393] Implementation Scheme 42: A construct according to any one of Implementation Schemes 37 to 41, wherein the cleavable linker comprises one or more sets of amino acid sequences recognized and cleaved by one or more proteases; optionally, the one or more proteases are one or more tumor microenvironment (TME) proteases, and the one or more TME proteases are selected from the group consisting of: urokinase-type plasminogen activator, Matriptase, Legumin, prostate-specific antigen, dipeptidyl peptidase, Hepsin, matrix metalloproteinase, integrin metalloproteinase, human leukocyte elastase, protease 3, urokinase, plasminogen, staphylococcal kinase, cathepsin, tissue kallikrein, and kallikrein-related peptidase.

[0394] Implementation Scheme 43: The construct according to Implementation Scheme 42, wherein one or more TME proteases are matrix metalloproteinases, and the matrix metalloproteinases are selected from the group consisting of: matrix metalloproteinase 1, matrix metalloproteinase 2, matrix metalloproteinase 3, matrix metalloproteinase 8, matrix metalloproteinase 9, matrix metalloproteinase 10, matrix metalloproteinase 12 and matrix metalloproteinase 14.

[0395] Implementation Scheme 44: The construct according to Implementation Scheme 41 or 42, wherein the cleavable linker comprises one or more amino acid sequences that are recognized and cleaved by one or more of matrix metalloproteinase 2, matrix metalloproteinase 9, matrix metalloproteinase 10, matrix metalloproteinase 14, urokinase plasminogen activator, Matriptase, and Legumin.

[0396] Implementation Scheme 45: A construct according to any one of Implementation Schemes 37 to 44, wherein the cleavable linker comprises one or more amino acid sequences that are recognized and cleaved by at least two, three, or four proteases; optionally, the one or more amino acid sequences are recognized and cleaved by a) Legumain and one, two, or three of MMP9, MMP2, and MMP14, b) uPA and one, two, or three of MMP9, MMP2, and MMP14, or c) all of uPA, Legumain, MMP2, MMP14, and MMP9.

[0397] Implementation Scheme 46: The construct according to any one of Implementation Schemes 37 to 45, wherein the cleavable linker comprises one or more amino acid sequences selected from the group consisting of: SEQ ID NO: 337, SEQ ID NO: 338, SEQ ID NO: 339 and SEQ ID NO: 372-377.

[0398] Implementation Scheme 47: The construct according to any one of Implementation Schemes 37 to 46, wherein the cleavable linker further comprises a spacer sequence; optionally, the cleavable linker comprises two spacer sequences located on either side of the N-terminus and C-terminus of an amino acid sequence recognized and cleaved by one or more proteases.

[0399] Implementation Scheme 48: A construct according to any one of Implementation Schemes 37 to 47, wherein the cleavable linker comprises two or more sets of amino acid sequences recognized and cleaved by one or more proteases; optionally, at least one set of the two or more sets of amino acid sequences recognized and cleaved by one or more proteases is side-joined with two spacer sequences in the cleavable linker, and each set of the two or more sets of amino acid sequences recognized and cleaved by one or more proteases is side-joined with two spacer sequences in the cleavable linker.

[0400] Implementation Scheme 49: The construct according to Implementation Scheme 47 or 48, wherein the spacer sequence comprises a GS linker; optionally, the length of the GS linker is no more than about 10, 9, 8, 7, 6 or 5 amino acids.

[0401] Implementation Scheme 50: The construct according to any one of Implementation Schemes 37 to 49, wherein the cleavable linker consists of no more than about 50, 40, 35, or 30 amino acids; optionally: The cleavable linker consists of no less than about 10, 15, 20 or 25 amino acids; The cleavable linker consists of about 25 to about 30 amino acids, and the masking portion includes an extracellular domain of IL-18Rα; further optionally, the masking portion includes an amino acid sequence shown in any one of SEQ ID NO: 353-356, or a functional variant thereof, the functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with any one of SEQ ID NO: 353-356; or The cleavable linker comprises the amino acid sequence shown in any one of SEQ ID NO: 399-407, SEQ ID NO: 492-493, and SEQ ID NO: 495.

[0402] Implementation Scheme 51: The construct according to any one of Implementation Schemes 1 to 28 and 37 to 50, wherein the IL-18 polypeptide is a wild-type IL-18 polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1.

[0403] Implementation Scheme 52: A construct according to any one of Implementation Schemes 1 to 51, wherein the IL-18 component comprises, from the N-terminus to the C-terminus,: a) a masking portion comprising a truncated propeptide of an IL-18 precursor, the truncated propeptide comprising the amino acid sequence shown in SEQ ID NO: 336; b) a cleavable linker; and c) an IL-18 polypeptide comprising the amino acid sequence shown in any one of SEQ ID NO: 1-299 and 307-318; optionally, the IL-18 polypeptide comprises the amino acid sequence shown in SEQ ID NO: 336. The amino acid sequence represented by any one of 39, 133, 311, 316, 465-469, 482-491, 27, 3, 105, 143, 144, 145, 43, 9, 17, 81, 117, 147, 149, and 150; optionally, the length of the cleavable linker does not exceed about 50, 40, 30, 25, 20, 15, 12, or 10 amino acids.

[0404] Implementation Scheme 53: The construct according to any one of Implementation Schemes 1 to 51, wherein the IL-18 component comprises, from the N-terminus to the C-terminus, the following in sequence: a) a masking portion comprising the amino acid sequence shown in any one of SEQ ID NO: 353-356; b) a cleavable linker; and c) an IL-18 polypeptide comprising the amino acid sequence shown in any one of SEQ ID NO: 1-299 and 307-318; optionally, the IL-18 polypeptide comprises the amino acid sequence shown in any one of SEQ ID NO: 39, 133, 311, 316, 465-469, 482-491, 27, 3, 105, 143, 144, 145, 43, 9, 17, 81, 117, 147, 149, and 150; optionally, the length of the cleavable linker is about 10-40, 10-30, 20-30, or 25-30 amino acids.

[0405] Implementation Scheme 54: A construct according to any one of Implementation Schemes 1 to 51, wherein the IL-18 component comprises, from the N-terminus to the C-terminus, the following: a) an IL-18 polypeptide comprising the amino acid sequence shown in any one of SEQ ID NO: 1-299 and 307-318; optionally, the IL-18 polypeptide comprises the amino acid sequence shown in any one of SEQ ID NO: 39, 133, 311, 316, 465-469, 482-491, 27, 3, 105, 143, 144, 145, 43, 9, 17, 81, 117, 147, 149, and 150; b) a cleavable linker; and c) a masking portion comprising the amino acid sequence shown in any one of SEQ ID NO: 353-356; optionally, the length of the cleavable linker is about 10-40, 10-30, 20-30, or 25-30 amino acids.

[0406] Implementation Scheme 55: The construct according to any one of Implementation Schemes 1 to 54, wherein the IL-18 component comprises: a) an amino acid sequence represented by any one of SEQ ID NO: 340-343, 345-352 and 496-500, or b) a functional variant thereof having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity with any one of SEQ ID NO: 340-343, 345-352 and 496-500.

[0407] Implementation Scheme 56: A construct according to any one of Implementation Schemes 1 to 55, wherein the IL-18 polypeptide has a reduced binding affinity for IL-18 binding protein (IL-18BP) compared to the corresponding wild-type IL-18 polypeptide (e.g., SEQ ID NO: 1); optionally, the reduced binding affinity is: a) at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, or b) about 10%-20%, 20%-30%, 30%-40%, 40%-50%, 50%-60%, 60%-70%, 70%-80%, 80%-90%, 90%-95%, 95%-96%, 96%-97%, 97%-98%, and / or 98%-99%.

[0408] Implementation Scheme 57: A construct according to any one of Implementation Schemes 1 to 56, wherein the IL-18 peptide contains at least three, four, five or six mutations at G3, E6, D54, Q56, P57, N91 and R104 of the IL-18 peptide, wherein the amino acid positions are relative to the wild-type human IL-18 peptide shown in SEQ ID NO: 1.

[0409] Implementation Scheme 58: The construct according to any one of Implementation Schemes 1 to 57, wherein the IL-18 polypeptide contains a cysteine ​​residue at position 117 and at position 76, wherein the amino acid positions are relative to the wild-type IL-18 polypeptide shown in SEQ ID NO: 1.

[0410] Implementation Scheme 59: The construct according to Implementation Scheme 58, wherein the IL-18 polypeptide does not contain a cysteine ​​residue at position 38, and / or the IL-18 polypeptide does not contain a cysteine ​​residue at position 68.

[0411] Implementation Scheme 60: The construct according to Implementation Scheme 58 or 59, wherein the IL-18 polypeptide comprises: a) cysteine ​​at position 117; b) cysteine ​​at position 76; c) isoleucine at position 38; and d) serine at position 68, wherein the amino acid positions are relative to the wild-type IL-18 polypeptide shown in SEQ ID NO: 1.

[0412] Implementation Scheme 61: The construct according to any one of Implementation Schemes 1 to 60, wherein the IL-18 component comprises an amino acid sequence represented by any one of SEQ ID NO: 2-299, 307-318, 465-469, and 482-491; optionally, the IL-18 component comprises an amino acid sequence represented by any one of SEQ ID NO: 39, 133, 311, 316, 27, 3, 105, 143, 144, 145, 43, 9, 17, 81, 117, 147, 149, 150, 465-469, and 482-491.

[0413] Implementation Scheme 62: One or more nucleic acids that encode a construct or a portion thereof according to any one of Implementation Schemes 1 to 61.

[0414] Implementation Scheme 63: One or more vectors comprising one or more nucleic acids as described in Implementation Scheme 62.

[0415] Implementation Scheme 64: A host cell comprising one or more nucleic acids as described in Implementation Scheme 62, or one or more vectors as described in Implementation Scheme 63.

[0416] Implementation Scheme 65: The host cell according to Implementation Scheme 64, wherein the host cell is a eukaryotic cell; optionally, the host cell is a CHO cell.

[0417] Implementation Scheme 66: The host cell according to Implementation Scheme 64, wherein the host cell is a prokaryotic cell; optionally, the host cell is Escherichia coli.

[0418] Implementation Scheme 67: A method for preparing a construct or a portion thereof according to any one of Implementation Schemes 1 to 66, comprising: (a) Culturing host cells according to embodiment 64 under conditions expressing the construct or a portion thereof; and (b) Recycle the construct or a portion thereof produced by the host cell.

[0419] Implementation Scheme 68: A method for treating a disease or ailment, comprising administering to an individual in need the construct according to any one of Implementation Schemes 1 to 66.

[0420] Implementation Scheme 69: The method according to Implementation Scheme 68, wherein the disease or symptom is cancer; optionally, the cancer is a solid tumor. Example

[0421] The following embodiments are intended to provide a complete disclosure and description of the preparation and use methods of the present invention to those skilled in the art. These embodiments are not intended to limit the scope of protection that the inventors believe to fall within the present invention, nor are they intended to represent all or only the experiments conducted. Although every effort has been made to ensure the accuracy of the numerical values ​​used (e.g., quantities, temperatures, etc.), some experimental errors and deviations should be considered. The following embodiments are intended to illustrate this application only and should not be construed as limiting the scope of this application in any way. The following embodiments and detailed descriptions are for illustrative purposes only and not for limitation. method Expression and purification of constructs

[0422] Human IL-18 with a site mutation that weakens its binding affinity to the IL-18 receptor, or a peptide that can activate human IL-18, is fused to a tissue-specific antigen targeting moiety, which is fused to the human Fc region. For example, the N-terminus of the IL-18 peptide can be fused to the C-terminus of the Fc region of a tissue-specific antigen targeting antibody to construct an immune cytokine. The resulting fusion peptide is expressed in CHO cells, harvested, and purified by one-step protein A chromatography, with further purification performed if necessary. The purified formulation is analyzed by size exclusion chromatography and SDS-PAGE. Antibody fusion-mediated hIL-18 attenuated variants activate hIL-18 receptor-mediated signaling.

[0423] For off-target activation, the NFκB-luc / hIL-18RαRβ HEK293 cell line, which stably expresses hIL-18Rα, hIL-18Rβ and NFκB luciferase reporter genes, was used as reporter cells to determine the ability of IL-18 peptides to activate hIL-18 receptor-mediated signal transduction.

[0424] To target and activate the anti-PD1 antibody fused with the IL-18 component, a stable HEK293 cell line overexpressing full-length human PD1 was constructed.

[0425] In short, reporter cells were first introduced at a density of 3.0 × 10⁶ cells per well. 4 Cells were seeded at a density of 1,000 cells per well in white 96-well plates. WT hIL-18 or a construct (e.g., an antibody fused to an IL-18 component) was serially diluted in culture medium. After incubation at 37°C for 16 hours, the luminescence intensity of each well was measured. hIFNγ release experiment based on human PBMCs

[0426] The ability of a variant IL-18 peptide to activate T / NK cells and induce hIFNγ release was investigated using PBMC-based experiments. Briefly, human PBMC cells were cultured at 5.0 × 10⁶ cells per well. 5 Cells were seeded at a density of 1000 μg / mL in 96-well plates and treated after 5 hours of culture. Wild-type (WT) hIL-18 or IL-18 variant peptides were serially diluted with 1 ng / mL human IL-12 in (a) or (b) conditions without 1 μg / mL hIL-18BP-hFc. The dilutions were then added to each well. After incubation at 37°C for 16 hours, the culture supernatant was collected from each well, and the hIFNγ concentration was determined by FRET assay using a kit (62HIFNGPEG, Cisbio) according to the manufacturer's instructions. Example 1: Structure-guided site-directed mutagenesis mediates attenuated IL-18 activity fused with tissue-specific antigen-targeting antibodies.

[0427] To achieve tissue-specific targeting and modulated activity, an IL-18 peptide with weakened IL-18 receptor binding affinity is fused to a tissue-specific antigen-targeting antibody (Figure 1). Constructs containing the IL-18 component can comprise any full-length IgG antibody (e.g., any tissue-specific antigen-targeting antibody) and any IL-18 peptide (e.g., the IL-18 peptide disclosed herein). Various construct formats are available, such as... Figure 2A As shown. For example, three different forms of anti-PD1 antibodies fused with the IL-18 peptide MM5-DB6 were prepared, and their IL-18 binding activity was tested. Among these three constructs containing the IL-18 component, PD1-MM5, formed by fusing the IL-18 peptide with the C-terminus of the Fc domain, exhibited significantly lower IL-18 arm activity than the construct formed by fusing with the N-terminal IL-18 variant. Figure 2B (See Table E1). Based on the “Fab-IL18” fusion, activity-attenuated variants of the IL-18 peptide were further designed. Table E1: Binding Affinity of Different Fusion Forms

[0428] One strategy to reduce IL-18 activity is through structure-guided site-directed mutagenesis. We hypothesize that mutations at a single site or multiple amino acids involved in IL-18 binding to IL-18Rα or IL-18Rβ can lead to decreased IL-18 activity. Using PD1-MM5 as an example, seven MM5-DB6 variants were constructed, all of which exhibited a single-site alanine substitution at the IL-18Rβ binding site. Figure 2CConsistent with the structural analysis results, the IL-18 arm activity of all seven mutated MM5-DB6 fusion proteins with PD1 antibodies (PD1-MM5-A1 to PD1-MM5-A7) was significantly reduced. Figure 2D (See Table E2). For the key amino acids G108, H109, ​​and K112 involved in IL-18Rβ interaction, single-point mutations at each site significantly reduced the IL-18 signaling activity of the resulting IL-18 peptides. These results strongly support our hypothesis that structure-guided mutations at single or multiple sites can reduce IL-18 activity. Table E2: Construct Receptor Activation

[0429] Tissue-specific IL-18-attenuated antibody fusion proteins are expected to exhibit modulated IL-18 activity in specific tissues (e.g., tumor microenvironment (TME)), and this tissue-targeting IL-18 activity can be detected using antigen overexpression systems. For example, targeted activation of IL-18-attenuated variants of PD1 antibody fusions or PD1-dependent cis-signaling can be detected using a PD1-overexpressing IL-18 signaling reporter system. Figure 3A Compared to the IL-18 reporter system without PD1 overexpression (PD1-), the PD1+IL-18 reporter system showed significantly higher activity of IL-18 activity-attenuated variants (PD1-MM5-A1, PD1-MM5-A2, and PD1-MM5-A3) with PD1 antibody fusion than WT IL-18. Figure 3B (Table E3). Other at-site mutations in the PD1-MM5 fusion protein attenuated variants (PD1-MM5-A4 to PD1-MM5-A7) also showed enhanced activity in the PD1+ system (data not shown). The activity difference between PD1 antibody fusion IL-18 peptide variants in the PD1- and PD1+ reporter systems, i.e., EC50... 50 -2 / EC 50 -1 is defined as the therapeutic index (TI) of this IL-18 variant in a specific tissue. These results indicate that IL-18 attenuated variants fused with tissue-specific targeting antibodies significantly broaden the therapeutic window of IL-18 activity. Table E3: Calculation results of the therapeutic window of the anti-PD1 construct containing the IL-18 peptide Example 2: Masking strategy mediates attenuated IL-18 activity fused with tissue-specific antigen-targeting antibody

[0430] Attenuated IL-18 activity can also be achieved through a masking strategy, in which an IL-18 variant fused with an antibody is linked to a masking portion via a cleavable linker. For example, in the PD1-IL18 fusion form, the masking IL-18 variant fuses with the extracellular domain (R) or its first and second domains (D12) of the masking portion IL-18Rα, and can fuse with the C-terminus of IL-18 via a cleavable linker (L1). Figure 4A and Figure 4B As shown, IL-18 signaling activity was assessed using IL-18 reporter gene assays and human PBMC assays. The results showed that regardless of whether the PD1 antibody was fused with the IL-18 variant M12-DB6 (PD1-M12) or MM5-DB6 (PD1-MM5), the IL-18 arm activity was significantly reduced due to Rα masking (PD1-M12-R-L1, PD1-MM5-R-L1). Another advantage of the masking strategy is that IL-18 activity can be restored after the masked portion is removed. In in vitro studies, when the masked IL-18 fused with the PD1 antibody was treated with an enzyme that recognizes the L1 linker, both PD1-M12-R-L1 and PD1-MM5-R-L1 recovered their IL-18 activity. Figure 4A and Figure 4B (See Tables E4 and E5). By employing another masking portion D12, both the masked PD1-M12-D12 and the masked PD1-MM5-D12 exhibited significantly reduced activity compared to their respective unmasked counterparts. Figure 4C and Figure 4D (Tables E6 and E7). Table E4: Activation of IL-18 signal transduction in different fusion forms Table E5: Activation of IL-18 signal transduction in different fusion forms Table E6: Activation of IL-18 signal transduction in different fusion forms Table E7: Activation of IL-18 signal transduction in different fusion forms

[0431] The masking strategy was also tested by fusing another anti-PD1 antibody (denoted by "k") with M12 or MM5. Figure 5A and Figure 5BAs shown, when the IL-18 variant was masked by IL-18Rα (denoted by "R") fused with another antibody, attenuated IL-18 activity was observed (Tables E8 and E9). Various linkers between the IL-18 variant and the masking portion were also tested. In the PD1-MM5-R system, similar to the L1 linker shown in Figure 4, the PD1-MM5-R molecules formed by the fusion of three other linkers with different amino acid sequences still exhibited significantly reduced IL-18 arm activity (…). Figure 5C and Figure 5D (See Tables E10 and E11). Modification of the Fc fragment of PD1-MM5-R did not affect the effectiveness of the attenuation strategy (data not shown). The above results strongly demonstrate that when the IL-18 peptide is fused with the antibody, the masking strategy can effectively attenuate the activity of the IL-18 peptide, and that this attenuation is reversible when the masking portion is removed. Table E8: Activation of IL-18 signaling in different constructs Table E9: Activation of IL-18 signaling in different constructs Table E10: Activation of IL-18 signaling in different constructs Table E11: Activation of IL-18 signaling in different constructs Example 3: Reduced IL-18 activity mediated by variable linker length fusion with tissue-specific antigen-targeting antibodies

[0432] The N-terminal conformation of IL-18 affects the binding of IL-18 to its receptor and receptor-mediated IL-18 signaling. Therefore, we hypothesize that the length of the linker between the Fc domain and the N-terminus of IL-18 influences IL-18 binding to its receptor. In the PD1-MM5 construct, the 15-amino acid glycine-serine linker was deleted (L0) or shortened to 5 amino acids (L5). Shortening the linker length from 15 amino acids significantly reduced IL-18 receptor activation activity. Figure 6 (See Table E12). This result confirms our hypothesis that the shorter the linker between Fc and the IL-18 variant, the lower the activity of the IL-18 variant protein fused with the tissue-targeting antibody. PD1-MM5 with L5 or L0 exhibits a higher therapeutic index than PD1-MM5 with a 15-amino acid linker (L15). Table E12: Treatment Window for Different Construct Forms Example 4: Reduced activity of monovalent IL-18 fused with tissue-specific antigen-targeting antibodies

[0433] based on Figure 2C The illustrated construct also includes a monovalent construct containing an IL-18 peptide fused to an antibody in a heterodimer form. Taking PD1-MM5 as an example, the monovalent construct (PD1-MM5-K) exhibits significantly lower IL-18 reporter gene activity than PD1-MM5, thus showing a higher therapeutic index as detected by the PD1+ and PD1- reporter systems. Figure 7 (Table E13). Table E13: Therapeutic Window in Different Construct Forms Example 5: IL-18 activity-attenuated variant fused with tissue-specific antigen-targeting antibody

[0434] By combining the strategies described above (e.g., through masking or IL-18 mutation), IL-18 peptides with reduced binding affinity to the IL-18 receptor were also obtained. These peptides functioned effectively when fused with their binding moieties (e.g., antibodies). For example, when both mutation and masking strategies were employed, the reduced-activity PD1-MM5-A1-R and PD1-MM5-A2-R exhibited unexpectedly decreased activity in PD1-negative IL-18 reporter gene assays, failing to activate signal transduction at concentrations up to 1000 nM, while only minimal activation was observed in PD1-positive IL-18 reporter systems. These results indicate that combining the strategies described above can yield a series of IL-18 peptides that can be used for antibody fusion to obtain constructs with varying levels of receptor activation. Table E14: Activation of IL-18 signaling in different constructs Example 6: Screening of constructs for attenuated IL-18 variants fused with tissue-specific antigen-targeting antibodies

[0435] Monovalent constructs containing IL-18 peptides and tissue-specific antigen-targeting antibodies come in three forms, such as... Figures 9A to 9C As shown. Several IL-18 variants fused with PD1 antibodies were constructed, and their activation effects on both targeted and non-targeted IL-18 receptor pathways were tested. Fusion proteins of anti-PD1 antibodies with a (e.g., monovalent) IL-18-activating protein molecule, with or without variable linker length, structure-guided mutations, or both, were prepared and tested. Corresponding molecules without the masking portion were also constructed as controls. Figures 10A to 10G As shown in Tables E15 to E20, all anti-PD-1 antibodies fused with IL-18-activating peptides exhibited significantly reduced IL-18 activity and a substantially increased therapeutic index. The therapeutic index of the corresponding unmasked antibody-IL-18 fusion proteins also showed a substantial increase. These results strongly support the... Figures 9A to 9C The disclosed development of attenuated IL-18 variants suitable for fusion with tissue-specific antigen-targeting antibodies. Table E15: Therapeutic Window in Different Construct Forms Table E16: Therapeutic Window in Different Construct Forms Table E17: Treatment Window for Different Construct Forms Table E18: Treatment Window in Different Construct Forms Table E19: Treatment Window for Different Construct Forms

[0436] The scope of this disclosure is not limited to the specific embodiments described, which are merely illustrative of various aspects of this disclosure, and any functionally equivalent compositions or methods are also within the scope of this disclosure. It will be apparent to those skilled in the art that various modifications and variations can be made to the methods and compositions of this disclosure without departing from the spirit or scope of this disclosure. Therefore, this disclosure is intended to cover various modifications and variations of this disclosure, provided they fall within the scope of the appended claims and their equivalents.

[0437] All publications and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication or patent application were specifically and individually indicated to be incorporated herein by reference.

[0438] This invention has been described according to specific embodiments discovered or proposed by the inventors, including preferred embodiments for carrying out the invention. Those skilled in the art will understand that, based on this disclosure, numerous modifications and alterations can be made to the specific embodiments illustrated without departing from the intended scope of the invention. For example, the basic DNA sequence can be altered without affecting the protein sequence due to codon redundancy. Furthermore, the protein structure can be altered based on considerations of biological functional equivalence without affecting the nature or extent of the biological action. All such modifications are intended to be included within the scope of the appended claims. Sequence Summary Table 。

Claims

1. A construct comprising an IL-18 component associated with a binding moiety that specifically binds to an antigen, wherein the IL-18 component comprises an IL-18 polypeptide, wherein: 1) The IL-18 peptide contains one or more modifications compared to the wild-type IL-18 peptide; 2) The IL-18 component further comprises a masking portion, wherein the IL-18 polypeptide is linked to the masking portion via a cleavable linker; or 3) The IL-18 component further comprises a peptide linker that connects the IL-18 polypeptide to the binding moiety, wherein the length of the peptide linker does not exceed 15 amino acids; and The IL-18 component has a lower binding affinity to the IL-18 receptor than the wild-type IL-18 polypeptide; optionally, the wild-type IL-18 polypeptide has the amino acid sequence shown in SEQ ID NO: 1; optionally, the binding affinity is evaluated on cells expressing the IL-18 receptor but not the antigen.

2. The construct according to claim 1, wherein the construct has a single IL-18 component.

3. The construct according to claim 1 or 2, wherein the binding portion comprises: a) a small molecule, or b) an Fc domain; optionally, the binding portion comprises an Fc domain; further optionally, the Fc domain is an IgG Fc domain.

4. The construct according to any one of claims 1 to 3, wherein the binding portion comprises an antibody; optionally, the antibody is a monovalent antibody.

5. The construct according to claim 4, wherein the antibody is a multivalent antibody.

6. A construct comprising a single IL-18 component associated with a binding portion of a multivalent antibody containing a specifically binding antigen, wherein the antibody comprises an Fc domain, the single IL-18 component comprises a single IL-18 polypeptide, the single IL-18 polypeptide comprising a wild-type IL-18 polypeptide or a variant thereof; optionally, the wild-type IL-18 polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1, and the single IL-18 component is fused to the Fc domain.

7. The construct according to claim 6, wherein: 1) The IL-18 peptide contains one or more modifications compared to the wild-type IL-18 peptide; 2) The IL-18 component further comprises a masking portion, wherein the IL-18 polypeptide is linked to the masking portion via a cleavable linker; or 3) The IL-18 component further includes a peptide linker that links the IL-18 polypeptide to the binding moiety, wherein the length of the peptide linker does not exceed 15 amino acids; The wild-type IL-18 polypeptide has the amino acid sequence shown in SEQ ID NO: 1, and the IL-18 component has a lower binding affinity to the IL-18 receptor than the wild-type IL-18 polypeptide; optionally, the binding affinity is evaluated on cells expressing the IL-18 receptor but not the antigen.

8. The construct according to any one of claims 5 to 7, wherein the antibody is a bivalent antibody.

9. The construct according to any one of claims 4 to 8, wherein the antibody is selected from the group consisting of: full-length antibody, single-chain Fv (scFv), Fab, Fab', F(ab')2, Fv fragment, disulfide-stabilized Fv fragment (dsFv), (dsFv)2, nanobody (V H H), Fv-Fc fusion protein, scFv-Fc fusion protein, scFv-Fv fusion protein, biantibody, triantibody and tetraantibody.

10. The construct according to any one of claims 4 to 9, wherein the antibody is a full-length antibody comprising two heavy chains and two light chains; optionally, the two heavy chains comprise one or more modifications that facilitate the formation of a club-and-mortar structure.

11. The construct according to any one of claims 1 to 10, wherein the IL-18 component: a) is covalently conjugated to the binding moiety, optionally via a linker between the IL-18 polypeptide and the binding moiety, or b) is fused to the binding moiety, optionally via a linker between the IL-18 polypeptide and the binding moiety; optionally, the linker is a peptide linker containing no more than 35 amino acids.

12. The construct of claim 10, wherein the IL-18 component is fused to one or two heavy chains; optionally, the IL-18 component is fused to the C-terminus of one or two heavy chains and / or one or two light chains of the full-length antibody.

13. The construct according to any one of claims 3 to 12, wherein the IL-18 component is fused to the C-terminus of the Fc domain; optionally, the IL-18 component is a single IL-18 component in the construct; further optionally, the antibody comprises a full-length antibody, a nanobody, a Fab, or a scFv.

14. The construct according to any one of claims 1 to 13, wherein the IL-18 component is fused to the binding portion via the peptide linker, and the length of the peptide linker is no more than 15 amino acids.

15. The construct according to any one of claims 1 to 14, wherein the antigen is a tumor-specific antigen or a tumor-associated antigen; optionally, the tumor is a solid tumor; further optionally, the antigen is a neoantigen; further optionally, a) the antigen is an antigen expressed by immune cells; optionally, the immune cells are tumor-infiltrating lymphocytes; or b) the antigen is an immune checkpoint target expressed on T cells or NK cells; optionally, the antigen is PD-1; further optionally, the binding domain comprises: a heavy chain variable region comprising VH-CDR1, VH-CDR2, and VH-CDR3; and a light chain variable region comprising VL-CDR1, VL-CDR2, and VL-CDR3, wherein: a) The VH-CDR1 contains the amino acid sequence shown in SEQ ID NO: 470, the VH-CDR2 contains the amino acid sequence shown in SEQ ID NO: 471, the VH-CDR3 contains the amino acid sequence shown in SEQ ID NO: 472, the VL-CDR1 contains the amino acid sequence shown in SEQ ID NO: 473, the VL-CDR2 contains the amino acid sequence shown in SEQ ID NO: 474, and the VL-CDR3 contains the amino acid sequence shown in SEQ ID NO: 475; or b) The VH-CDR1 contains the amino acid sequence shown in SEQ ID NO: 476, the VH-CDR2 contains the amino acid sequence shown in SEQ ID NO: 477, the VH-CDR3 contains the amino acid sequence shown in SEQ ID NO: 478, the VL-CDR1 contains the amino acid sequence shown in SEQ ID NO: 479, the VL-CDR2 contains the amino acid sequence shown in SEQ ID NO: 480, and the VL-CDR3 contains the amino acid sequence shown in SEQ ID NO:

481.

16. The construct according to any one of claims 1 to 15, wherein the IL-18 polypeptide comprises one or more modifications relative to the corresponding wild-type IL-18 polypeptide (e.g., SEQ ID NO: 1), wherein: a) The IL-18 peptide exhibits a reduced binding affinity for IL-18Rβ compared to the corresponding wild-type IL-18 peptide (e.g., SEQ ID NO: 1); further optionally, the reduction in binding affinity is: a) at least 50%, 60%, 70%, 80%, or 90%, or b) 50%-60%, 60%-70%, 70%-80%, 80%-90%, or 90%-95%; b) The one or more modifications comprise amino acid substitutions selected from the group consisting of G108, H109, ​​K112, D110, R147, M150, G145, K79, D98, and K84, wherein the amino acid position is relative to the wild-type human IL-18 polypeptide shown in SEQ ID NO: 1; optionally, the substitution is an alanine substitution; further optionally, the one or more modifications comprise amino acid substitutions selected from the group consisting of G108, H109, ​​K112, D110, R147, M150, G145, K79, and D98, wherein the amino acid position is relative to the wild-type human IL-18 polypeptide shown in SEQ ID NO: 1; optionally, the substitution is an alanine substitution; c) The IL-18 component compared to the corresponding wild-type IL-18 peptide (e.g., SEQ ID NO: 1) Reduced binding affinity for IL-18Rα; optionally, the reduced binding affinity is: a) at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, or b) 10%-20%, 20%-30%, 30%-40%, 40%-50%, 50%-60%, 60%-70%, 70%-80%, 80%-90%, 90%-95%, 95%-96%, 96%-97%, 97%-98%, and / or 98%-99%; further optionally, the one or more modifications comprise amino acid substitutions selected from the group consisting of K4, L5, K8, R13, D17, E31, M33, T34, D35, D40, K53, R58, M60, K79, K84, D98, R104, and D132, wherein the amino acid position is relative to SEQ ID NO: 1 represents the wild-type human IL-18 polypeptide; optionally, the substitution is an alanine substitution; and / or d) The IL-18 component comprises an IL-18 polypeptide, the IL-18 polypeptide comprising two or more modifications, the two or more modifications comprising two or more amino acid substitutions selected from the group consisting of G108, H109, ​​K112, D110, R147, M150, G145, K79, D98, K84, K4, L5, K8, R13, D17, E31, M33, T34, D35, D40, K53, R58, M60, R104 and D132, wherein the amino acid position is relative to the wild-type human IL-18 polypeptide shown in SEQ ID NO: 1; optionally, the substitution is an alanine substitution.

17. The construct according to any one of claims 1 to 16, wherein the IL-18 polypeptide comprises the amino acid sequence shown in any one of SEQ ID NO:482-491.

18. The construct according to any one of claims 1 to 9 and 6 to 17, wherein the IL-18 component further comprises a masking portion, and the IL-18 polypeptide is linked to the masking portion via a cleavable linker; optionally: a) The masking portion comprises an IL-18 propeptide; optionally, the IL-18 propeptide comprises a propeptide of human IL-18 (hIL-18); further optionally, the hIL-18 propeptide comprises the amino acid sequence shown in SEQ ID NO: 333; further optionally, the masking portion comprises a truncated propeptide of an IL-18 precursor; further optionally, the truncated propeptide of an IL-18 precursor comprises the amino acid sequence shown in SEQ ID NO: 336; or b) The masking portion comprises the extracellular domain of IL-18Rα, the extracellular domain of IL-18Rβ, IL-18 binding protein, a fragment of any of the foregoing, or a variant of any of the foregoing; optionally, the masking portion comprises the extracellular domain of IL-18Rα or a fragment thereof; further optionally, the fragment of the extracellular domain of IL-18Rα comprises: 1) a first domain and a second domain of the extracellular domain, or 2) a third domain of the extracellular domain; further optionally, the masking portion comprises the amino acid sequence shown in any of SEQ ID NO: 353-356, or a functional variant thereof, the functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with any of SEQ ID NO: 353-356.

19. The construct of claim 18, wherein the cleavable linker comprises one or more sets of amino acid sequences recognized and cleaved by one or more proteases; optionally: a) The one or more proteases are one or more tumor microenvironment (TME) proteases; optionally, the one or more TME proteases are selected from the group consisting of: urokinase-type plasminogen activator, Matriptase, Legumin, prostate-specific antigen, dipeptidyl peptidase, Hepsin, matrix metalloproteinases, integrin-dissociating metalloproteinases, human leukocyte elastase, protease 3, urokinase, plasminogen, staphylococcal kinase, cathepsin, tissue kallikrein, and kallikrein-related peptidase; further optionally, the one or more TME proteases are matrix metalloproteinases; further optionally, the matrix metalloproteinases are selected from the group consisting of: matrix metalloproteinase 1, matrix metalloproteinase 2, matrix metalloproteinase 3, matrix metalloproteinase 8, matrix metalloproteinase 9, matrix metalloproteinase 10, matrix metalloproteinase 12, and matrix metalloproteinase 14. b) The cleavable linker comprises one or more amino acid sequences that are recognized and cleaved by one or more of matrix metalloproteinase 2, matrix metalloproteinase 9, matrix metalloproteinase 10, matrix metalloproteinase 14, urokinase plasminogen activator, Matriptase, and Legumin. c) The cleavable linker comprises one or more amino acid sequences that are recognized and cleaved by at least two, three, or four proteases; optionally, the one or more amino acid sequences are recognized and cleaved by a) Legumain and one, two, or three of MMP9, MMP2, and MMP14, b) uPA and one, two, or three of MMP9, MMP2, and MMP14, or c) all of uPA, Legumain, MMP2, MMP14, and MMP9. d) The cleavable linker comprises one or more amino acid sequences selected from the group consisting of: SEQ ID NO:337, SEQ ID NO:338, SEQ ID NO:339 and SEQ ID NO:372-377; e) The cleavable linker further comprises a spacer sequence; optionally, the cleavable linker comprises two spacer sequences located on either side of the N-terminus and C-terminus of an amino acid sequence recognized and cleaved by one or more proteases; further optionally, the spacer sequence comprises a GS linker; further optionally, the GS linker is no longer than about 10, 9, 8, 7, 6, or 5 amino acids; and / or f) The cleavable linker comprises two or more sets of amino acid sequences recognized and cleaved by one or more proteases; optionally, at least one set of the two or more sets of amino acid sequences recognized and cleaved by one or more proteases is side-joined with two spacer sequences in the cleavable linker; further optionally, each set of the two or more sets of amino acid sequences recognized and cleaved by one or more proteases is side-joined with two spacer sequences in the cleavable linker; further optionally, the spacer sequences comprise GS linkers; further optionally, the length of the GS linker does not exceed about 10, 9, 8, 7, 6 or 5 amino acids.

20. The construct of claim 19, wherein the cleavable linker consists of no more than about 50, 40, 35, or 30 amino acids; optionally: 1) The cleavable linker consists of not less than about 10, 15, 20 or 25 amino acids; 2) The cleavable linker consists of about 25 to about 30 amino acids, and the masking portion contains an extracellular domain of IL-18Rα; further optionally, the masking portion contains an amino acid sequence shown in any one of SEQ ID NO: 353-356, or a functional variant thereof, the functional variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with any one of SEQ ID NO: 353-356; or 3) The cleavable linker comprises the amino acid sequence shown in any one of SEQ ID NO: 399-407, SEQ ID NO: 492-493 and SEQ ID NO:

495.

21. The construct according to any one of claims 1 to 15 and 18 to 20, wherein the IL-18 polypeptide is a wild-type IL-18 polypeptide comprising the amino acid sequence shown in SEQ ID NO:

1.

22. The construct according to any one of claims 1 to 21, wherein the IL-18 component comprises, from the N-terminus to the C-terminus,: a) a masking portion comprising a truncated propeptide of an IL-18 precursor, the truncated propeptide comprising the amino acid sequence shown in SEQ ID NO: 336; b) a cleavable linker; and c) an IL-18 polypeptide comprising the amino acid sequence shown in any one of SEQ ID NO: 1-299 and 307-318; optionally, the IL-18 polypeptide comprises the amino acid sequence shown in any one of SEQ ID NO: 39, 133, 311, 316, 465-469, 482-491, 27, 3, 105, 143, 144, 145, 43, 9, 17, 81, 117, 147, 149, and 150; optionally, the length of the cleavable linker is no more than about 50, 40, 30, 25, 20, 15, 12, or 10 amino acids.

23. The construct according to any one of claims 1 to 21, wherein the IL-18 component comprises, from the N-terminus to the C-terminus,: a) a masking portion comprising the amino acid sequence shown in any one of SEQ ID NO: 353-356; b) a cleavable linker; and c) an IL-18 polypeptide comprising the amino acid sequence shown in any one of SEQ ID NO: 1-299 and 307-318; optionally, the IL-18 polypeptide comprises the amino acid sequence shown in any one of SEQ ID NO: 39, 133, 311, 316, 465-469, 482-491, 27, 3, 105, 143, 144, 145, 43, 9, 17, 81, 117, 147, 149, and 150; optionally, the length of the cleavable linker is about 10-40, 10-30, 20-30, or 25-30 amino acids.

24. The construct according to any one of claims 1 to 21, wherein the IL-18 component comprises, from the N-terminus to the C-terminus,: a) an IL-18 polypeptide comprising the amino acid sequence shown in any one of SEQ ID NO: 1-299 and 307-318; optionally, the IL-18 polypeptide comprises the amino acid sequence shown in any one of SEQ ID NO: 39, 133, 311, 316, 465-469, 482-491, 27, 3, 105, 143, 144, 145, 43, 9, 17, 81, 117, 147, 149, and 150; b) a cleavable linker; and c) a masking portion comprising the amino acid sequence shown in any one of SEQ ID NO: 353-356; optionally, the length of the cleavable linker is about 10-40, 10-30, 20-30, or 25-30 amino acids.

25. The construct according to any one of claims 1 to 24, wherein the IL-18 component comprises: a) an amino acid sequence represented by any one of SEQ ID NO: 340-343, 345-352, and 496-500, or b) a functional variant thereof having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with any one of SEQ ID NO: 340-343, 345-352, and 496-500.

26. The construct according to any one of claims 1 to 25, wherein the IL-18 polypeptide has a reduced binding affinity for IL-18 binding protein (IL-18BP) compared to the corresponding wild-type IL-18 polypeptide (e.g., SEQ ID NO: 1); optionally, the reduction in binding affinity is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%.

27. The construct according to any one of claims 1 to 26, wherein the IL-18 polypeptide contains at least three, four, five or six mutations at G3, E6, D54, Q56, P57, N91 and R104 of the IL-18 polypeptide, wherein the amino acid positions are relative to the wild-type human IL-18 polypeptide shown in SEQ ID NO:

1.

28. The construct according to any one of claims 1 to 27, wherein the IL-18 polypeptide comprises cysteine ​​at position 117 and position 76, wherein the amino acid positions are relative to the wild-type IL-18 polypeptide shown in SEQ ID NO: 1; optionally, the IL-18 polypeptide does not contain cysteine ​​at position 38, and / or the IL-18 polypeptide does not contain cysteine ​​at position 68; further optionally, the IL-18 polypeptide comprises: a) cysteine ​​at position 117; b) cysteine ​​at position 76; c) isoleucine at position 38; and d) serine at position 68, wherein the amino acid positions are relative to the wild-type IL-18 polypeptide shown in SEQ ID NO:

1.

29. The construct according to any one of claims 1 to 28, wherein the IL-18 component comprises an amino acid sequence shown in any one of SEQ ID NO: 2-299, 307-318, 465-469, and 482-491; optionally, the IL-18 component comprises an amino acid sequence shown in any one of SEQ ID NO: 39, 133, 311, 316, 27, 3, 105, 143, 144, 145, 43, 9, 17, 81, 117, 147, 149, 150, 465-469, and 482-491.

30. One or more nucleic acids encoding a construct or a portion thereof according to any one of claims 1 to 29.

31. One or more vectors comprising one or more nucleic acids according to claim 30.

32. A host cell comprising one or more nucleic acids according to claim 30, or one or more vectors according to claim 31; optionally: a) the host cell is a eukaryotic cell; further optionally, the host cell is a CHO cell; or b) the host cell is a prokaryotic cell; further optionally, the host cell is Escherichia coli.

33. A method for preparing a construct or a portion thereof according to any one of claims 1 to 29, comprising: (a) Culture the host cells according to claim 32 under conditions expressing the construct or a portion thereof; as well as (b) Recycle the construct or a portion thereof produced by the host cell.

34. A method of treating a disease or condition, comprising administering to an individual in need the construct according to any one of claims 1 to 29; optionally, the disease or condition is cancer; further optionally, the cancer is a solid tumor.

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