Il-18 fusion proteins and methods of producing il-18

Abstract

Fusion proteins are provided that consist of essential elements such as one or more proteins / polypeptides capable of transporting into the endoplasmic reticulum (ER) and fragments or variants of interleukin 18 (IL-18) or IL-18. The proteins / polypeptides capable of transporting into the ER include, but are not limited to, immune globular proteins (e.g., Fc regions, VHH antibodies, and scFv), and globular proteins (e.g., serum albumin). In some embodiments, the IL-18 may be a pro-IL-18 similar to a natural precursor cytokine, wherein IL-18 is fused to the C-terminus of a propeptide or a propeptide variant thereof. In some embodiments, the fusion protein may further comprise a cleavable peptide linker.

Description

Cross - Reference to Related Applications

[0001] This application claims priority under 35 U.S.C.§119(e) to U.S. Provisional Patent Application No. 63 / 395,476, filed on August 5, 2022, and U.S. Provisional Patent Application No. 63 / 463,505, filed on May 2, 2023, the entireties of which are incorporated herein by reference. Reference to Sequence Listing

[0002] This application contains a Sequence Listing submitted in computer - readable form and named "096034_000002WOPT_SequenceListing.xml", which is 366,973 bytes in size as measured in bytes and was created on August 3, 2023 (Creation Date). The information contained in this computer - readable form is incorporated herein by reference in its entirety. Technical Field

[0003] The present invention relates to new compositions that allow for enhanced production (including expression, secretion, and purification) of recombinantly produced IL - 18 cytokine, e.g., in mammalian cells, that allow for masking and unmasking of the biological activity of IL - 18, and / or that allow for improved binding specificity of the produced IL - 18 to its receptor (as compared to its binding protein) while at least maintaining the binding affinity to its receptor, as well as methods for preparing the compositions. Background Art

[0004] Members of the interleukin (IL) - 1 cytokine family play well - defined roles in host defense responses and inflammatory responses that lead to disease. IL - 18 belongs to the IL - 1 superfamily and is a pro - inflammatory cytokine that promotes type 1 immune responses. IL - 18, also known as interferon - γ - inducing factor, is encoded by the IL - 18 gene in humans. Similar to other IL - 1 family members, the IL - 18 gene lacks a signal peptide. The IL - 18 gene encodes a 193 - amino - acid precursor protein that is first synthesized as an inactive 24 kDa precursor without a signal peptide, which is cytoplasmic and accumulates in the cytoplasm. WO 97 / 24441 discloses a 193 - amino - acid protein corresponding to the IL - 18 precursor and encoding DNA. The IL - 18 precursor (also called Pro - IL - 18) is processed intracellularly (e.g., by caspase 1 (CASP1), chymotrypsin, and proteinase B) into its mature, biologically active 18 kDa molecule (157 amino acids; i.e., amino acid residues 37 - 193 of Uniprot ID Q14116). That is, after cleavage, the pro - peptide separates from the rest of the precursor, generating mature IL - 18 and the pro - peptide that originally inactivated the IL - 18 precursor.

[0005] Not wishing to be bound by a particular theory, mature forms of IL-18 are secreted and released into the extracellular environment by at least three unconventional pathways. This is in contrast to conventional secretion mediated by signal peptide-dependence and ER-Golgi trafficking. The unconventional pathways listed below are in no particular order of frequency. The first, called secretory autophagy, is the process involved in the secretion of cytosolic proteins that lack a signal peptide (leaderless cargoes). Here, IL-18 interacts with transmembrane emp24 domain-containing protein 10 (TMED10), a cargo receptor, and this interaction mediates trafficking from the cytoplasm into the endoplasmic reticulum-Golgi intermediate compartment (ERGIC), a compartment that provides membranes for the forming autophagosome, which serves as the mechanism for the secretory cargo to enter vesicles and thus be secreted. The second and third reported mechanisms for the release of IL-18 from cells include the rupture of dead cells undergoing apoptosis and / or gasdermin D-dependent plasma membrane permeability (see Tapia et al., IMMUNOLOGY, Volume 294, Issue 21, p8325-8335, 2019).

[0006] Mature IL-18 binds to the ligand receptor IL-18 receptor alpha (IL-18Rα), inducing the recruitment of IL-18Rβ (also known as IL-18 receptor accessory protein (IL-18RAP)) to form a high-affinity complex (approx. 18 nM, see Torigoe et al., Membranes and Bioenergetics, Volume 272, Issue 41, pp25737-25742, 1997), which signals through the toll / interleukin-1 receptor (TIR) domain. This signaling domain recruits the MyD88 adaptor protein, which activates the pro-inflammatory program and the NF-κB pathway. The activity of IL-18 can be inhibited by extracellular interleukin 18 binding protein (IL-18BP), which binds soluble IL-18 with a higher affinity (approx. 0.4 pM, see Kim et al., Proc Natl Acad Sci USA. 2000;97(3):1190-1195) than IL-18Rα, thus preventing IL-18 from binding to the IL-18 receptor.

[0007] Accordingly, it is an object of the present invention to provide compositions of matter that permit improved production (including expression, secretion, and purification) of recombinantly produced IL-18.

[0008] Another object of the present invention is to provide a substance composition that allows enhanced production of recombinantly produced IL-18, including a modified IL-18 or a fragment thereof, which maintains a similar binding affinity for IL-18Ra / b compared to native IL-18 and has a reduced binding affinity for IL-18BP compared to native IL-18 (i.e., binds to both at ~18 nM); or more preferably, for the produced IL-18 (or a fragment thereof), the binding affinity for IL-18BP is weaker than the binding affinity for IL-18Ra / b (i.e., the binding to IL-18BP < 18 nM and the binding to IL-18Ra / b is ~18 nM); and methods for preparing these compositions.

[0009] Another object of the present invention is to provide an IL-18 variant (or a fragment thereof).

[0010] Another object of the present invention is to provide a composition that allows masked activity in normal tissues and the circulation and specific release by proteases of IL-18 (or a fragment thereof) in the tumor microenvironment.

[0011] All publications herein are hereby incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The following description includes information that may be helpful in understanding the present invention. This does not represent an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, nor does it represent an admission that any of the publications specifically or implicitly referenced are prior art. Summary of the Invention

[0012] The following embodiments and aspects thereof are described and illustrated in connection with compositions and methods that are exemplary and illustrative, and not limiting in scope.

[0013] Various embodiments provide a fusion protein comprising: a first polypeptide or protein, or a fragment thereof, capable of being transported into the endoplasmic reticulum (ER); and interleukin 18 (IL-18), a fragment of IL-18, an IL-18 variant, or a fragment of an IL-18 variant, wherein the IL-18, the fragment of IL-18, the IL-18 variant, or the fragment of the IL-18 variant is located at the C-terminus of the fusion protein relative to the first polypeptide or protein capable of being transported into the ER.

[0014] In various embodiments, the IL-18 variant may have an amino acid sequence comprising amino acids 37-193 of SEQ ID NO: 250, which has one to five amino acid substitutions at positions E42, M87, K89, M96, and M149 of SEQ ID NO: 250.

[0015] In various embodiments, the IL-18 variant may have an amino acid sequence comprising positions 37-193 of SEQ ID NO: 251, which has one to five amino acid substitutions at positions E42, M87, K89, M96, and M149 of SEQ ID NO: 251.

[0016] In various embodiments, the IL-18 variant may have an amino acid sequence comprising positions 37-193 of SEQ ID NO: 251, which has one to five amino acid substitutions at positions E42, M87, K89, M96, and M149 of SEQ ID NO: 251 and one or more amino acid substitutions at positions C74, C104, C112, and C164.

[0017] In various embodiments, the amino acid substitutions at one or more of C74, C104, C112, and C164 may each independently be valine, alanine, or serine.

[0018] In various embodiments, the one to five amino acid substitutions may be one or more of the following: E42K, E42R, E42A, E42H, or E42Q; M87K or M87H; K89G, K89A, or K89E; M96L or M96I; or M149V or M149I.

[0019] In various embodiments, the one to five amino acid substitutions may be E42K, E42R, E42A, E42H, or E42Q; M87K or M87H; K89G, K89A, or K89E; M96L or M96I; and M149V or M149I.

[0020] In various embodiments, the IL-18, a fragment of IL-18, an IL-18 variant, or a fragment of an IL-18 variant further comprises its propeptide (PP) or a PP variant.

[0021] In various embodiments, the IL-18 propeptide variant comprises a polypeptide having AAEPVEDNX 1 INFVAMKFIDNTLYFIAEDDEN (SEQ ID NO: 238), wherein X 1 can be any amino acid other than cysteine.

[0022] In various embodiments, X 1 can be alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan (SEQ ID NO: 239). In various embodiments, X 1 can be valine (SEQ ID NO: 78). In various embodiments, X1 can be serine, threonine, asparagine or glutamine (SEQ ID NO: 240). In various embodiments, X 1 can be serine (SEQ ID NO: 76).

[0023] In various embodiments, the fusion protein does not contain a polypeptide consisting of the sequence X 1 -X 2 -X 3 -X 4 between the propeptide or propeptide variant and the mature IL-18 or mature IL-18 variant, where X 1 is L or absent, X 2 is E or absent, X 3 is S or absent, and X 4 is D or absent, and when X 1 、X 2 、X 3 and X 4 are present, the polypeptide consisting of the sequence X 1 -X 2 -X 3 -X 4 is LESD (SEQ ID NO: 253).

[0024] In various embodiments, the PP or PP variant can be located at the N-terminus relative to the IL-18, fragment of IL-18, IL-18 variant or fragment of IL-18 variant.

[0025] In various embodiments, the PP or PP variant serves as a masking domain.

[0026] In various embodiments, the fusion protein further comprises one or more protease cleavage sites.

[0027] In various embodiments, the one or more protease cleavage sites can be between the IL-18, fragment of IL-18, IL-18 variant or fragment of IL-18 variant and the first protein or its fragment capable of being transported into the endoplasmic reticulum (ER), or within the PP, between the PP or PP variant and the IL-18, fragment of IL-18, IL-18 variant or fragment of IL-18 variant, or within the PP, between the PP or PP variant and the first protein or its fragment capable of being transported into the endoplasmic reticulum (ER), or within the IL-18, fragment of IL-18, IL-18 variant or fragment of IL-18 variant, or within the PP, or a combination thereof.

[0028] In various embodiments, the fusion protein further comprises a second protein or a fragment thereof capable of being transported into the ER, wherein the second protein capable of being transported into the ER can be located at the C-terminus relative to interleukin-18 (IL-18), a fragment of IL-18, an IL-18 variant, or a fragment of an IL-18 variant.

[0029] In various embodiments, the fusion protein further comprises a protease cleavage site between the IL-18, a fragment of IL-18, an IL-18 variant, or a fragment of an IL-18 variant and the second protein or a fragment thereof capable of being transported into the endoplasmic reticulum (ER).

[0030] In various embodiments, the interleukin-18 (IL-18), a fragment of IL-18, an IL-18 variant, or a fragment of an IL-18 variant can be fused to the C-terminus of the first protein capable of being transported into the ER.

[0031] In various embodiments, the second protein or a fragment thereof capable of being transported into the ER can be fused to the C-terminus of the interleukin-18 (IL-18), a fragment of IL-18, an IL-18 variant, or a fragment of an IL-18 variant.

[0032] In various embodiments, compared to wild-type (wt) IL-18, the IL-18 variant can have a reduced binding to the IL-18 binding protein (IL-18BP). In various embodiments, the binding affinity of the IL-18 variant for the human IL-18 receptor (IL-18R) is within 30-fold of that of wild-type IL-18. In various embodiments, the ratio of the binding affinity of the fusion protein comprising the IL-18 variant for IL-18BP to the binding affinity of the fusion protein comprising the IL-18 variant for IL-18R can be no higher than 3:1.

[0033] In various embodiments, the first protein or a fragment thereof capable of being transported into the ER can be a globular protein, an immunoglobulin, or a fragment thereof, or can be a short polypeptide or protein engineered to have a signal peptide for transport into the ER, optionally, the short polypeptide or protein is about 2 kDa or no greater than 250 kDa.

[0034] In various embodiments, the first protein or a fragment thereof capable of being transported into the ER can be selected from the group consisting of: the crystallizable fragment (Fc) region, human serum albumin (HSA), β2-microglobulin, transferrin, the antigen-binding fragment region (Fab region), VHH antibody, single-chain variable fragment (scFV), anticalin, designed ankyrin repeat protein (DARPin), its binding domain, and fragments thereof.

[0035] In various embodiments, the first protein or fragment thereof capable of being transported into the ER of the fusion protein can be a type I transmembrane protein or fragment thereof, or a type II transmembrane protein or fragment thereof.

[0036] In various embodiments, the second protein or fragment thereof capable of being transported into the ER can be a globular protein, an immunoglobulin, or a fragment thereof, or can be a short polypeptide or protein engineered with a signal peptide for transport into the ER. Optionally, the short polypeptide or protein is about 2 kDa or not greater than 250 kDa.

[0037] In various embodiments, the second protein or fragment thereof capable of being transported into the ER can be selected from the group consisting of: the crystallizable fragment (Fc) region, human serum albumin (HSA), β2-microglobulin, transferrin, the antigen-binding fragment region (Fab region), VHH antibody, single-chain variable fragment (scFV), anticalin, designed ankyrin repeat protein (DARPin), its binding domain, and fragments thereof.

[0038] In various embodiments, the second protein or fragment thereof capable of being transported into the ER of the fusion protein can be a type I transmembrane protein or fragment thereof, or a type II transmembrane protein or fragment thereof.

[0039] In various embodiments, the Fc region can be the Fc region from IgA, IgM, IgG, or IgE. In various embodiments, the Fc region can be the Fc region from IgG4, KiH, or IgG1. In various embodiments, the Fc region can be the Fc region from Knob-in-hole, HA-TF, Xmab, ZW1, 7.8.60, Electrostatic Steering, DD-KK, EW-RVT, A107, or Duobody.

[0040] In various embodiments, one or more cysteines in the fusion protein can be modified. In various embodiments, one or more cysteines in the fusion protein can be replaced with natural or non-natural amino acids.

[0041] In various embodiments, one or more cysteines in the IL-18, fragment of IL-18, IL-18 variant, or fragment of IL-18 variant of the fusion protein can be modified or can be replaced with natural or non-natural amino acids.

[0042] In various embodiments, one or more cysteines in the PP or PP variant of the fusion protein can be modified or can be replaced with natural or non-natural amino acids.

[0043] In various embodiments, the natural amino acid can be charged, polar uncharged, or hydrophobic. In various embodiments, the natural amino acids can each independently be selected from serine and valine. In various embodiments, the natural amino acids can each independently be selected from threonine, asparagine, and glutamine.

[0044] In various embodiments, the natural amino acids can each independently be selected from alanine, isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan. In various embodiments, the natural amino acids can each independently be selected from phenylalanine, alanine, aspartic acid, and asparagine. In various embodiments, the natural amino acid can be valine. In various embodiments, the natural amino acids can each independently be selected from threonine, glutamine, aspartic acid, phenylalanine, isoleucine, and histidine.

[0045] In various embodiments, the protease can be selected from the group consisting of: EK, TEV, Adam17, cathepsin, MMP2, MMP9, MMP14, granzyme A, granzyme B, granzyme M, granzyme K, and combinations thereof.

[0046] In various embodiments, the fusion protein can have one or more sequences listed in any one of Table 1 and Table 4.

[0047] In various embodiments, the fusion protein can have polypeptide 1 and polypeptide 2 selected from Table 1 and optionally polypeptide 3. In various embodiments, the fusion protein can have polypeptide 1 and polypeptide 2 selected from Table 1 and optionally polypeptide 3, wherein polypeptide 1, polypeptide 2, and optionally polypeptide 3 can be from the same row of Table 1. In various embodiments, the fusion protein can have polypeptide 1 selected from Table 1, wherein polypeptide 1 contains HSA.

[0048] In various embodiments, the IL-18 variant can have an amino acid sequence selected from the mature IL-18 column in Table 1.

[0049] In various embodiments, the IL-18 variant can have an amino acid sequence selected from the mature IL-18 column in Table 1, and the propeptide can have an amino acid sequence selected from the propeptide column in Table 1, optionally from the same row.

[0050] Various embodiments provide an IL-18 propeptide variant that comprises a polypeptide having AAEPVEDNX 1 INFVAMKFIDNTLYFIAEDDEN, wherein X 1can be any amino acid other than cysteine (SEQ ID NO: 238).

[0051] In various embodiments, X 1 can be alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan (SEQ ID NO: 239). In various embodiments, X 1 can be valine (SEQ ID NO: 78). In various embodiments, X 1 can be serine, threonine, asparagine, or glutamine (SEQ ID NO: 240). In various embodiments, X 1 can be serine (SEQ ID NO: 76).

[0052] Various embodiments provide polynucleotides that encode any of the fusion proteins of the invention described herein. In various embodiments, the first protein capable of being transported into the ER can be encoded by a polynucleotide having one or more sequences shown in Table 2. In various embodiments, the polynucleotide can have one or more sequences from the same row as shown in Table 2.

[0053] Various embodiments provide expression vectors that contain a polynucleotide of any of the inventions described herein.

[0054] Various embodiments provide cells transfected with any of the expression vectors of the invention described herein. In various embodiments, the cells can be mammalian cells. In various embodiments, the cells can be bacterial cells.

[0055] Various embodiments provide methods for producing a fusion protein, including: culturing cells transfected with any of the expression vectors of the invention described herein in a cell culture medium to allow the fusion protein to be secreted into the cell culture medium.

[0056] In various embodiments, the method further includes isolating the fusion protein from the medium. In various embodiments, the method further includes purifying the fusion protein. In various embodiments, under transient transfection of CHO cells or HEK-293 cells, the fusion protein can be produced at greater than 135 mg / L.

[0057] Various embodiments provide methods for producing interleukin (IL-18), fragments thereof, IL-18 variants or fragments of IL-18 variants, including: culturing cells transfected with any of the expression vectors of the invention described herein in cell culture medium to allow production of the fusion protein and secretion into the extracellular space; and contacting the fusion protein with a protease to cleave the fusion protein, thereby producing the IL-18, its fragment, IL-18 variant or fragment of IL-18 variant.

[0058] In various embodiments, the method further includes isolating the fusion protein from the culture medium. In various embodiments, the method further includes purifying the fusion protein. In various embodiments, contacting the fusion protein with a protease includes including the protease in the cell culture medium.

[0059] In various embodiments, the protease is selected from the group consisting of: EK, TEV, Adam17, cathepsin, MMP2, MMP9, MMP14, granzyme A, granzyme B, granzyme M, and combinations thereof.

[0060] In various embodiments, the IL-18, its fragment, IL-18 variant or fragment of IL-18 variant can be produced at greater than 135 mg / L.

[0061] Other features and advantages of the present invention will become apparent from the following detailed description in conjunction with the accompanying drawings, which illustrate various features of embodiments of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Exemplary embodiments are illustrated in the figures referred to. The embodiments disclosed herein and the figures are intended to be illustrative rather than restrictive.

[0063] Figure 1 (Sub - figure A - Sub - figure E) depicts exemplary fusion proteins, where the N - terminus of pro - IL - 18 is fused to the C - terminus of the knob of the knobs - into - holes heterodimeric IgG1 protein; it has the following structure, which from the N - terminus to the C - terminus comprises a knobs - into - holes (KiH) Fc - pro - peptide (pp) - enterokinase - cleavable site (EK) - IL - 18 wild - type or its variant. This describes exemplary fusion proteins, such as ID: FUSE - 480, FUSE - 481, and FUSE - 442 in Table 1. pro - IL - 18 was further modified to reduce molecular aggregation, where each cysteine residue in both the pro - peptide and mature IL - 18 was replaced with serine (as in FUSE - 480, denoted as "IL - 18AS"), with alanine (as in FUSE - 481, denoted as "IL - 18AA") or with valine (as in FUSE - 442, denoted as "IL - 18AV"). Alternatively, the N - terminus of pro - IL - 18 can be fused to the C - terminus of the hole chain of the KiH heterodimeric IgG1 protein. The biological activity defined as EC50 - SEAP of each compound is shown in Sub - figure E.

[0064] Figure 2 (Sub - figure A - Sub - figure E) depicts exemplary fusion proteins, where the N - terminus of pro - IL - 18 is fused to the C - terminus of the IgG1 CH3 domain (which is also the knob chain of the knobs - into - holes heterodimeric IgG1 protein as in Figure 1), and pro - IL - 18 contains four amino - acid substitutions denoted as "pro - IL - 18mut2" that are hypothesized to reduce binding to IL - 18BP while maintaining wild - type binding to the IL - 18 receptor complex. These fusion proteins have the following structure, which from the N - terminus to the C - terminus comprises a knobs - into - holes (KiH) Fc - pro - peptide (PP) - enterokinase - cleavable site (EK) - IL - 18mut2. pro - IL - 18mut2 was further modified to reduce molecular aggregation, where each cysteine residue in both the pro - peptide and mature IL - 18mut2 was replaced with serine (denoted as "IL - 18mut2AS", as in FUSE - 422; Sub - figure B), with alanine (denoted as "IL - 18mut2AA", as in FUSE - 423; Sub - figure C) or with valine (denoted as "IL - 18mut2AV", as in FUSE - 424; Sub - figure D). The biological activity defined as EC50 - SEAP of each compound is shown in Sub - figure E.

[0065] Figure 3(Sub - figures A - D) depict exemplary fusion proteins with (Sub - figure A) or without (Sub - figure B) a pro - peptide to examine the effect on masking the bioactivity of "IL - 18AV" (Sub - figure C), where Fc fusion variants were generated that contain "IL - 18AV" with a pro - peptide (Sub - figure A; FUSE - 442) or without a pro - peptide (Sub - figure B; FUSE - 505). The bioactivity of each compound, defined as EC50 - SEAP, is shown in Sub - figure D.

[0066] Figure 4 (Sub - figures A - D) depict exemplary fusion proteins with (Sub - figure A) or without (Sub - figure B) a pro - peptide to examine the effect of the pro - peptide on masking the bioactivity of "IL - 18mut2AV", where Fc fusion variants were generated that contain "IL - 18mut2AV" without a pro - peptide (thus, mature IL - 18 with a mutation, denoted as "matIL - 18mut2 - AV", see Sub - figure B; FUSE - 441) or with a pro - peptide (Sub - figure A; FUSE - 424). For the fusion protein without a pro - peptide, the EK cleavage site that replaces the cysteine protease 1 site was moved to a position directly between the CH3 domain of the stalk and mature IL - 18AV without adding a flexible linker. Sub - figure C depicts the activation readout obtained using HEK - Blue IL - 18AV reporter cells after titration with FUSE - 441 (Fc - EK - IL - 18AV) or FUSE - 424 (Fc - EKpp - IL - 18AV) with or without EK treatment. The bioactivity of each compound, defined as EC50 - SEAP, is shown in Sub - figure D.

[0067] Figure 5 (Sub - figures A - D) depict exemplary fusion proteins where the N - terminus of pro - IL - 18 is fused to the C - terminus of an IgG1 Fc protein or IgG4 Fc; they have the following structure that includes, from the N - terminus to the C - terminus, IgG1 Fc - pro - peptide (pp) - IL - 18AV (FUSE - 507; Sub - figure A) and IgG4 Fc - pro - peptide (PP) - IL - 18AV (FUSE - 509; Sub - figure B). Sub - figure C depicts the activation readout obtained by titrating cells with FUSE - 507 or FUSE - 509 with or without EK treatment and using HEK - Blue IL - 18AV reporter cells. The bioactivity of each compound, defined as EC50 - SEAP, is shown in Sub - figure D.

[0068] Figure 6(Subfigures A - E) describe exemplary fusion proteins, where the N - terminus of pro - IL - 18 is fused to the C - terminus of HSA with or without a pro - peptide (pp); it has the following structure, which from the N - terminus to the C - terminus includes HSA - pro - peptide (PP) - IL - 18AV (FUSE - 501; Subfigure A) and HSA - IL - 18AV (FUSE - 503; Subfigure B). Subfigures C and D describe activation readings. The biological activity of each compound defined as EC50 - SEAP is shown in Subfigure E.

[0069] Figure 7 (Subfigures A - E) describe exemplary fusion proteins, where the N - terminus of pro - IL - 18mut2 is fused to the C - terminus of HSA with or without a pro - peptide (PP); it has the following structure, which from the N - terminus to the C - terminus includes HSA - pro - peptide (PP) - IL - 18mut2AV (FUSE - 502; Subfigure A) and HSA - IL - 18mut2AV (FUSE - 504; Subfigure B). Subfigures C and D describe activation readings. The biological activity of each compound defined as EC50 - SEAP is shown in Subfigure E.

[0070] Figure 8 (Subfigures A - D) describes exemplary fusion proteins, where the C - terminus of pro - IL - 18 is fused to the N - terminus of the pestle of a mortar - and - pestle heterodimeric IgG1 protein with or without a pro - peptide (PP); it has the following structure, which from the N - terminus to the C - terminus includes pro - peptide (PP) - IL - 18AV - mortar - and - pestle (KiH) Fc (FUSE - 499; Subfigure A) and IL - 18AV - mortar - and - pestle (KiH) Fc (FUSE - 500; Subfigure B). Subfigure C describes activation readings. The biological activity of each compound defined as EC50 - SEAP in the presence or absence of cysteine protease 1 is shown in Subfigure D.

[0071] Figure 9A and Figure 9D describe exemplary fusion proteins having the following structure from the N - terminus to the C - terminus: Fc - ppMMP2 / 9 cleavage site - IL - 18 - AV (FUSE - 486), Fc - ppMMP9 / 2 cleavage site - IL - 18 - AV (FUSE - 487), where the cleavage site is specific for metalloproteinases MMP2 and MMP9, and the enzyme to the left of the slash is the preferred one; or FUSE - 485 (Fc - GzmBpp - IL - 18AV) and FUSE - 462 (Fc - GzmBpp - IL - 18mut2AV). Figure 9BDescribes activation readings related to MMP2-treated or untreated FUSE-486 and FUSE-487. The bioactivity of MMP2-treated or untreated FUSE-486 and FUSE-487, each defined as EC50-SEAP, is shown in Figure 9B as follows. Figure 9C It shows that the activity of FUSE587 is weakened by approximately 3000-fold relative to recombinant human IL-18. Interestingly, we observed that the IL-18AV variant released by cleavage of FUSE587 with MMP2 was still weakened by approximately 100-fold relative to recombinant IL-18. In contrast, the IL-18AV variant released by cleavage with granzyme B had activity similar to that of recombinant IL-18. Cleavage with granzyme B resulted in the release of mature IL-18AV without any N-terminal residues constituting the overhang; while after cleavage of FUSE486 and FUSE587 with MMP2, N-terminal polypeptide overhangs of 11 and 15 amino acids remained. We speculate that these overhangs may weaken the activity of IL-18AV, although to a lesser extent than the full-size variant propeptide. This phenomenon was further explored in Figure 11 and Figure 13 as follows. Figure 9D and Figure 9G also describe exemplary fusion proteins having the following structure from N-terminus to C-terminus: Fc-ppGb cleavage site-IL-18-AV (FUSE-485; Figure 9D ), or Fc-ppGb cleavage site-IL-18mut2-AV (FUSE-462; Figure 9D ), or Fab-cetuximab-Fc-ppGb cleavage site-IL-18-AV (FUSE-517; Figure 9G ), where the cleavage site is specific for granzyme B (Gb). Figure 9E Describes activation readings related to granzyme B-treated or untreated FUSE-462 and FUSE-485. Figure 9F Shows the bioactivity of granzyme B-treated or untreated FUSE-462 and FUSE-485, each defined as EC50-SEAP. Figure 9H Describes activation readings related to enzyme-treated or untreated FUSE-517. Figure 9I Shows the bioactivity of granzyme B-treated or untreated FUSE-517, defined as EC50-SEAP.

[0072] Figure 10 (panels A-G) depicts the effect of IL-18BP on the biological activity of recombinant human IL-18 (rhIL-18) and EK cleavage products of exemplary fusion proteins (Fc-ppEK-IL-18-AV (FUSE-442) and Fc-ppEK-IL-18mut2AV (FUSE-424)). The biological activity of each compound with and without the addition of IL-18BP (competition assay) defined as EC50-SEAP is shown in panels B, D and F, and the corresponding biological activity defined as EC50-SEAP is shown in panels C, E and G, respectively.

[0073] Figure 11 (sub-figure A) depicts a schematic representation of an exemplary fusion protein wherein the C-terminus of pro-IL-18 is fused to the N-terminus of the knob of a knob-hole heterodimeric IgG1 protein and having polypeptides of varying sizes fused to the N-terminus of mature IL-18. Figure 11 (sub-figure B) depicts the biological activity of each fusion protein using the IL-18 reporter cell line HEK-Blue IL-18.

[0074] Figure 12A , FIG. 12B(i), FIG. 12B(ii), Figure 12C , FIG. 12D(i), FIG. 12D(ii) and Figure 12E Engineered mutant fusion proteins of human IL-18 according to various embodiments of the present invention are described.

[0075] Figure 13 Shown is the effect of the size of the polypeptide fused to the N-terminus of mature IL-18 on the biological activity of a single IL-18AV fused to the N-terminus of IgG1 Fc.

[0076] Figures 14A - 14H Shown are the effects of replacing the cysteine ​​residues in the propeptide, which is fused together with mature IL-18 to form the pro-IL-18 variant cassette, and the cysteine ​​residues in mature IL-18 on the biological activity of each variant using the HEK Blue IL18 assay system.

[0077] Figures 15A - 15B The effect of targeting pro-IL18 into the immediate vicinity of its receptor complex (ie "cis-active") is shown. DETAILED DESCRIPTION

[0078] All references cited herein are incorporated by reference in their entirety as if fully set forth. Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton et al., Dictionary of Microbiology and Molecular Biology 3rd ed., rev. ed., J. Wiley & Sons (New York, NY 2006); March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 7th ed., J. Wiley & Sons (New York, NY 2013); and Sambrook and Russel, Molecular Cloning: A Laboratory Manual 4th ed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY 2012), provide general guidance to many of the terms used in this application to those skilled in the art. For references on how to prepare antibodies, see D. Lane, Antibodies: A Laboratory Manual 2nd ed., (Cold Spring Harbor Press, Cold Spring Harbor NY, 2013); Kohler and Milstein, (1976) Eur. J. Immunol. 6:511; Queen et al., U.S. Patent No. 5,585,089; and Riechmann et al., Nature 332:323 (1988); U.S. Patent No. 4,946,778; Bird, Science 242:423-42 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); Ward et al., Nature 334:544-54 (1989); Tomlinson I. and Holliger P. (2000) Methods Enzymol, 326, 461-479; Holliger P. (2005) Nat. Biotechnol. Sep; 23(9):1126-36).

[0079] Those skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which can be used in the practice of this invention. In fact, this invention is in no way limited to the methods and materials described. For the purposes of this invention, the following terms are defined as follows.

[0080] As used herein, unless otherwise specifically provided herein, when used in conjunction with a reference numeral indication, the term "about / approximately" means the reference numeral indication plus or minus up to 5% of the reference numeral indication. For example, the expression "about 50%" encompasses the range from 45% to 55%. In various embodiments, if specifically provided in the claims, when used in conjunction with a reference numeral indication, the term "about" may mean the reference numeral indication plus or minus up to 4%, 3%, 2%, 1%, 0.5%, or 0.25% of the reference numeral indication.

[0081] As used herein, the term "immunoglobulin heavy chain constant region" is used interchangeably with the term "Fc region" and is understood to mean the carboxyl-terminal portion of the immunoglobulin heavy chain constant region, or an analogue or portion thereof that is capable of binding to an Fc receptor. Each immunoglobulin heavy chain constant region contains four or five domains. These domains are named in sequence as follows: CH1 - hinge region - CH2 - CH3 (-CH4). CH4 is present in IgM, which does not have a hinge region. The immunoglobulin heavy chain constant region suitable for the present invention preferably contains an immunoglobulin hinge region and also preferably contains the CH3 domain. Most preferably, the immunoglobulin heavy chain constant region contains an immunoglobulin hinge region, a CH2 domain, and a CH3 domain.

[0082] As used herein, the term immunoglobulin "hinge region" is understood to mean the complete immunoglobulin hinge region, or at least a portion of the immunoglobulin hinge region that is sufficient to form one or more disulfide bonds with a second immunoglobulin hinge region.

[0083] As used herein, the term "vector" is understood to mean any nucleic acid that contains a nucleotide sequence capable of being integrated into a host cell and recombined with the host cell genome and integrated into the genome, or autonomously replicating as an episome. Such vectors include linear nucleic acids, plasmids, phagemids, cosmids, RNA vectors, viral vectors, etc. Non-limiting examples of viral vectors include retroviruses, adenoviruses, and adeno-associated viruses.

[0084] As used herein, the "gene expression" or "expression" of a fusion protein is understood to mean the transcription of a DNA sequence, the translation of the mRNA transcript, and the secretion of the fusion protein product. In some embodiments, the expression process also includes purification or post-purification; for example, purification can be performed using protein A affinity chromatography or other means (such as size exclusion chromatography).

[0085] As used herein, an "IL-18 fusion protein" refers to a fusion protein comprising wild-type IL-18 or an IL-18 variant, unless specifically noted as comprising only wild-type IL-18 or only an IL-18 variant. Thus, in certain embodiments, an "IL-18 fusion protein" comprises only any of the IL-18 variants described herein.

[0086] Regarding amino acid linkers in polypeptides, the term "linker" can be a short peptide, such as a dimer of two amino acids, a trimer of three amino acids, or a peptide selected from the group consisting of: T, PT, MPT, S, GS, GGS, GGGS (SEQ ID NO: 235), and (GGGGX λ (SEQ ID NO: 236)) n , wherein X λ is Q, A, E, or S, and n = 1 - 5 or an integer greater than 5. In some embodiments, the amino acid linker has the amino acid sequence of (GGGGS (SEQ ID NO: 237)) n , wherein n is an integer between 1 and 5, such that the length of the amino acid linker is 25 amino acids or shorter. In some embodiments, the amino acid linker is an IL-18 propeptide or an IL-18 propeptide variant. In some embodiments, the amino acid linker is a fragment of an IL-18 propeptide or an IL-18 propeptide variant; e.g., having a length of about 30 - 36 amino acids, a length of about 5 - 10 amino acids, 11 - 20 amino acids, 21 - 30 amino acids, or 31 - 40 amino acids. fusion protein

[0087] Various embodiments provide one or more fusion proteins, each fusion protein comprising: (i) IL-18, a fragment of IL-18, an IL-18 variant, or a fragment of an IL-18 variant; and (ii) a first protein capable of being transported into the endoplasmic reticulum (ER), or a fragment of said protein, including a cytoplasmic or nuclear protein engineered to be transported into the ER by adding a signal peptide / leader sequence to the N-terminus of such engineered protein. Preferably, the protein capable of being transported into the ER has an amino acid sequence that initiates the transmembrane transport of the initiating protein (e.g., IL-18 or a fragment, variant, or fragment of its variant) across the endoplasmic reticulum membrane. In various embodiments, the fusion protein further comprises an amino acid linker. For example, the amino acid linker can be located between (a) the protein capable of being transported into the endoplasmic reticulum (ER) and (b) the propeptide (or variant) or IL-18 (or variant).

[0088] In some embodiments, one or more fusion proteins do not contain the IL-18 propeptide or a variant thereof. In the present invention, "IL-18 propeptide", "propeptide", or "PP" are used interchangeably and describe the amino acid sequence that is linked to IL-18 or an IL-18 variant in the IL-18 precursor or IL-18 variant precursor and, upon removal, gives rise to mature IL-18 or a fragment thereof, or an IL-18 variant or a fragment thereof. For example, the IL-18 propeptide may have the sequence of amino acid residues 1-36 in Uniprot ID Q14116.

[0089] In some embodiments, one or more fusion proteins further contain a propeptide (PP) or a variant thereof. Examples of propeptide variants are provided herein, including those in Table 1. This can inactivate IL-18 or an IL-18 variant, and thus the propeptide is directly or indirectly linked to IL-18 or an IL-18 variant to form pro-IL-18 or a variant of pro-IL-18. Preferably, in the fusion protein, the PP or its variant is located at the N-terminus relative to IL-18 (or a fragment, variant, or fragment of a variant thereof).

[0090] In some embodiments, one or more fusion proteins further contain a cleavage site, which is preferably based on a peptide substrate sensitive to enzymatic / protease cleavage. The cleavage site may be within the PP, between the PP or its variant (if present) and IL-18 (or a fragment, variant, or fragment of a variant thereof); or may be between a protein capable of being transported into the ER and the PP (if present); or may be between a protein capable of being transported into the ER and IL-18 or a fragment, variant, or fragment of a variant thereof, particularly in the absence of a propeptide. In some embodiments, when the PP is present, the cleavage site is within the PP. In further embodiments, one or more fusion proteins comprise: (i) IL-18, a fragment of IL-18, an IL-18 variant, or a fragment of an IL-18 variant; (ii) a propeptide (PP) or a variant thereof that inactivates IL-18; and a cleavage site.

[0091] Examples of propeptide variants include polypeptides having AAEPVEDNX 1 INFVAMKFIDNTLYFIAEDDEN, where X 1 is any amino acid other than cysteine (SEQ ID NO: 238). In various embodiments, X 1 is alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan (SEQ ID NO: 239). In various embodiments, X 1 is valine (SEQ ID NO: 78). In various embodiments, X 1is serine, threonine, asparagine or glutamine (SEQ ID NO: 240). In various embodiments, X 1 is serine (SEQ ID NO: 76).

[0092] In various embodiments, the fusion protein does not comprise a sequence X between (i) the propeptide or propeptide variant and (ii) the mature IL-18 or mature IL-18 variant. 1 -X 2 -X 3 -X 4 A polypeptide composed of 1 L or not present, X 2 is E or does not exist, X 3 is S or does not exist, and X 4 is D or does not exist, when X 1 , X 2 , X 3 and X 4 When both are present, the sequence is LESD (SEQ ID NO: 253).

[0093] In various aspects of the fusion protein, IL-18 (or a fragment, variant, or fragment of a variant thereof) is linked to a first protein capable of transport into the ER via a polypeptide bond. The fusion protein can have a variety of configurations. Preferably, the N-terminus of IL-18 (or a fragment, variant, or fragment of a variant thereof) is directly or indirectly linked to the C-terminus of the first protein capable of transport into the ER via a polypeptide bond.

[0094] However, in other embodiments, the C-terminus of IL-18 (or a fragment, variant, or fragment of a variant thereof) is directly or indirectly linked to the N-terminus of a first protein capable of transport into the ER via a polypeptide bond. As a non-limiting example, an IL-18 variant (or IL-18, a fragment of IL-18, a fragment of an IL-18 variant) is fused to the N-terminus of the knob of a knob-to-hole heterodimeric IgG1 protein with or without a propeptide (pp).

[0095] In a further embodiment, the C-terminus of IL-18 is linked to the N-terminus of a first protein capable of trafficking into (or across) the ER, and a second protein capable of trafficking across the ER is typically fused to the N-terminus of IL-18 to mediate masking. It is contemplated that the fusion protein further comprises (iii) a second protein capable of trafficking into / across the ER, or a "scaffold" (such as a heat shock protein (HSP) which may not traffic across the ER). In some embodiments, if an HSP (nuclear protein) or a cytoplasmic protein is fused to the N-terminus of IL-18 to mediate masking, a signal peptide is typically fused to the N-terminus of the "scaffold" to mediate trafficking to the ER; and if the scaffold is fused to the C-terminus of IL-18 to stabilize the complex, then a second protein capable of trafficking across the ER is typically fused to the N-terminus of IL-18 to mediate masking. Thus, in some embodiments, IL-18 (or a fragment, variant, or fragment of a variant thereof) is located at the C-terminus of the fusion protein; in some embodiments, the N-terminus of IL-18 (or a fragment, variant, or fragment of a variant thereof) is C-terminal relative to the first protein capable of trafficking into the ER, and the C-terminus of IL-18 (or a fragment, variant, or fragment of a variant thereof) is N-terminal relative to the second protein capable of trafficking into the ER. The "first" or "second" protein capable of trafficking into the ER is used as a relative reference.

[0096] One or more exemplary amino acid sequences of the components of the fusion protein are shown in Tables 1 and 4.

[0097] Some embodiments provide a first protein / polypeptide capable of trafficking into the ER, the protein / polypeptide comprising an immunoglobulin heavy chain constant region. In some embodiments, the immunoglobulin heavy chain constant region comprises an immunoglobulin heavy chain constant region domain selected from the group consisting of a CH2 domain, a CH3 domain, and a CH4 domain, or a combination thereof. In some embodiments, the immunoglobulin heavy chain constant region comprises a CH2 domain and a CH3 domain. In some embodiments, the immunoglobulin heavy chain constant region lacks at least the CH1 domain. In some embodiments, the immunoglobulin heavy chain constant region is a human immunoglobulin heavy chain constant region. In some embodiments, the immunoglobulin heavy chain constant region is an immunoglobulin heavy chain constant region present in the same species as IL-18. In other embodiments, the immunoglobulin heavy chain constant region is an immunoglobulin heavy chain constant region present in the same species as the organism that is transformed or transfected with the nucleic acid molecule encoding the fusion protein or precursor of the fusion protein. Further embodiments provide a fusion protein that lacks an immunoglobulin variable region (V H )

[0098] In various embodiments, IL-18 (or a fragment, variant, or fragment of a variant thereof) is (in terms of sequence) identical to human IL-18 (or a fragment, variant, or fragment of a variant thereof), and the immunoglobulin heavy chain constant region comprises a hinge region and a CH2 domain or a CH3 domain, and more preferably comprises both a hinge region and a CH2 domain and a CH3 domain. In various embodiments, IL-18 (or a fragment, variant, or fragment of a variant thereof) is at least 95%, 90%, or 85% identical to human IL-18 (or a fragment, variant, or fragment of a variant thereof) (in terms of sequence), but has amino acid substitutions or other modifications that reduce the affinity of IL-18 (or a fragment, variant, or fragment of a variant thereof) for IL-18BP. It is contemplated that the immunoglobulin heavy chain constant region suitable for the present invention can be derived from any one of the five immunoglobulin classes known in the art as IgA (Igα), IgD (Igδ), IgE (Igε), IgG (Igγ), and IgM (Igμ). However, immunoglobulin heavy chain constant regions from the IgG class are preferred. Additionally, the immunoglobulin heavy chain constant region can be derived from any one of the IgG antibody subtypes known in the art as IgG1, IgG2, IgG3, and IgG4. The immunoglobulin heavy chain constant region domains have cross-homology between the various immunoglobulin classes. For example, the CH2 domain of IgG is homologous to the CH2 domains of IgA and IgD, and is homologous to the CH3 domains of IgM and IgE. Preferred immunoglobulin heavy chain constant regions include protein domains corresponding to the CH2 and CH3 regions of IgG, or functional portions or derivatives thereof. Further description of the immunoglobulin heavy chain constant region is discussed in detail in U.S. Patent No. 5,541,087 and U.S. Patent No. 5,726,044, which are incorporated herein by reference.

[0099] In multiple embodiments, the protein / polypeptide to be fused with IL-18 (or a fragment, variant, or fragment of a variant thereof) is a dimer of two immunoglobulin heavy chain constant regions / chains, optionally cross-linked by a pair of disulfide bonds between cysteines on adjacent hinge regions. In some embodiments, the hinge region may have an upper hinge domain, a core hinge domain, and a lower hinge domain. In some embodiments, the upper part of the hinge domain may contain or remove cysteines known to form disulfide bonds with the light chain or fab, resulting in, for example, EPKSC (SEQ ID NO: 241) or EPKSS (SEQ ID NO: 242) or EPKSA (SEQ ID NO: 243). For example, in addition to FUSE-501, FUSE-503, and FUSE-509, fusion proteins containing IgG1-based ER transporters may have removed cysteines from the hinge region. For example, in addition to FUSE-507 (FUSE-507 has EPKSA (SEQ ID NO: 243) in the hinge region), EPKSS (SEQ ID NO: 242) in the IgG1-based ER transporter. The hinge region may also contain a core hinge domain, for example, containing the sequence CPPCP (SEQ ID NO: 244) or a variant in which the cysteines are replaced. The hinge region may further contain a lower hinge domain, for example, containing the sequence APELLGGP (SEQ ID NO: 245) or APEAAGGP (SEQ ID NO: 246). In another example, FUSE-509 has an IgG4-based ER transporter and uses a hinge region as described in Chiu et al., Antibodies 2019, 8(4), 55, 2019. As shown in the figures, although constructs containing immunoglobulin hinge regions are preferred, cross-linking at other positions may be selected as needed within the scope of the present invention. Additionally, in some cases, more than two monomers may be non-covalently associated to produce dimers or multimers. In each aspect where the protein / polypeptide is a dimer of two immunoglobulin heavy chain constant regions / chains, IL-18 (or a fragment, variant, or fragment of a variant thereof) is linked to one and only one of the two (or more) immunoglobulin heavy chain constant regions / chains. In the case of wild-type IgG-Fc forming a homodimer, in various situations, IL-18 is placed at the C-terminus of each monomer of the Fc, thus placing two IL-18s at the C-terminus of the Fc. In certain situations, when one wild-type Fc fused with one IL-18 is mixed with another wild-type Fc not fused with IL-18, a heterodimer may be formed (for example, during the purification step). In other aspects, IL-18 (or a fragment, variant, or fragment of a variant thereof) is linked to each of the two (or more) immunoglobulin heavy chain constant regions / chains in the fusion protein.

[0100] In some embodiments, the two arms (or chains) of the immunoglobulin heavy chain constant region (e.g., Fc polypeptide) can be heterodimerized by creating a "knobs-into-holes" (KiH) mutation in the CH3 domain. This structural feature in the polypeptide arms allows the assembly of two half-antibodies (e.g., Fc heterodimers; and VH-CH and VL-CL domains). For example, a heteromultimer (including a heterodimer) can comprise a first polypeptide and a second polypeptide, each comprising a CH3 domain, wherein the polypeptides meet at an engineered interface within the CH3 domain, and the first polypeptide comprises an engineered protrusion ("knob") at the interface, wherein at least one contacting residue is replaced with an introduced residue having a larger side-chain volume than the original residue, and the second polypeptide comprises an engineered depression ("hole") at the interface, wherein at least one contacting residue is replaced with an introduced residue having a smaller side-chain volume than the original residue. In some embodiments, the engineered interface of the heteromultimer comprises at least two knob-hole mutation pairs. The volume and accessible surface area of each amino acid are described in A.A. Zamyatnin, Prog. Biophys. Mol. Biol. 24:107-123, 1972 and C. Chothia, J. Mol. Biol. 105:1-14, 1975. For example, the introduced residues for forming the knob can be arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W); and preferably, the original residue for forming the knob has a smaller side-chain volume, such as alanine, asparagine, aspartic acid, glycine, serine, threonine, or valine. As another example, the introduced residues for forming the hole can be alanine (A), serine (S), threonine (T), and valine (V); and preferably, the original residue for forming the hole has a larger side-chain volume, such as tyrosine, arginine, phenylalanine, or tryptophan. For example, the T366W mutation in the CH3 domain of the "knob" / protrusion chain, and the T366S / L368A / Y407V mutations in the CH3 domain of the "hole" / depression chain. Additionally, the KiH configuration can be accompanied by further mutations to allow S-S disulfide bond linkage between the two chains. In various aspects of the heterodimer in which the protein / polypeptide is in the KiH configuration, IL-18 (or a fragment, variant, or fragment of a variant thereof) is linked to only one of the two (or more) immunoglobulin heavy chain constant regions / chains (i.e., knob or hole).

[0101] In some embodiments, two or more arms (or chains) of an immunoglobulin heavy chain constant region (e.g., an Fc polypeptide) may comprise another symmetric-asymmetric spatial complementarity design (e.g., HA-TF, ZW1), charge-charge exchange interaction (DD-KK), charge-spatial complementarity exchange plus additional long-range electrostatic interactions (e.g., EW-RVT), or isotype chain exchange design (e.g., chain exchange engineered domain (SEED)), or Xmab, 7.8.60, electrostatic steering, A107, or Duobody, thereby forming a heterodimer / heteromultimer. Further description of these configurations and exemplary mutations / residues can be found in Front Immunol. 2016;7:394.

[0102] In additional embodiments, a suitable protein or fragment thereof capable of being transported into the ER in a fusion protein is a globular protein, an immunoglobulin, or a fragment thereof. In various embodiments, a suitable protein or fragment thereof capable of being transported into the ER in a fusion protein is a short polypeptide or protein engineered with a signal peptide for transport into the ER. For example, the short polypeptide or protein is about 2 kDa or no greater than 250 kDa. In additional instances, the short polypeptide or protein is about 2-5 kDa, about 6-10 kDa, about 11-20 kDa, about 21-30 kDa, about 31-40 kDa, about 41-50 kDa, about 51-75 kDa, about 76-100 kDa, about 101-125 kDa, about 126-150 kDa, about 151-175 kDa, about 176-200 kDa, about 201-225 kDa, or about 256-250 kDa.

[0103] In additional embodiments, a suitable protein capable of being transported into the ER can be a globular protein, human serum albumin (HSA), β2-microglobulin, transferrin, antigen-binding fragment region (Fab region), VHH antibody, single-chain variable fragment (scFv), anticalin, designed ankyrin repeat protein (DARPin), its binding domain, and its fragments. Additional suitable proteins capable of being transported into the ER may include a type I transmembrane protein or a fragment thereof, or a type II transmembrane protein or a fragment thereof.

[0104] In various embodiments, a fusion protein comprising a short polypeptide or protein and IL-18 or an IL-18 variant, or a fragment thereof, further comprises a second protein or a fragment thereof capable of being transported into the ER. The second protein or a fragment thereof capable of being transported into the ER can be an Fc domain, or HSA, β2-microglobulin, transferrin, an antigen-binding fragment region (Fab region), a VHH antibody, a single-chain variable fragment (scFv), an anticalin, a designed ankyrin repeat protein (DARPin), a binding domain thereof and fragments thereof, or a type I transmembrane protein or a fragment thereof, or a type II transmembrane protein or a fragment thereof. Figure 8A and Figure 11A (The first three from left to right) are non-limiting examples of such fusion proteins.

[0105] In other embodiments, the fusion protein further comprises a protein that cannot be naturally transported into the ER, such as a nuclear protein or a cytoplasmic protein fused to the N-terminus of IL-18. For such proteins, the following will be fused to the N-terminus of the non-ER-transporting protein: a signal peptide (also referred to as a leader sequence), such as the Ig-κ leader sequence in FUSE-499 (e.g., METDTLLLWVLLLWVPGSTG (SEQ ID NO: 247)), or one or more other signal peptides including but not limited to those derived from human albumin and human azurocidin, see Kober et al., Biotechnol Bioeng. 2013 Apr;110(4):1164-73. For example, the signal peptide can be located at the N-terminus of the propeptide or IL-18 (or a fragment, variant, or fragment of a variant thereof). Further examples of proteins capable of being transported into / entering / across the ER can be proteins engineered with a signal peptide (e.g., at the N-terminus). As an example, Hsp70 is a nuclear protein, but when a signal peptide is fused or linked to the N-terminus of Hsp70, it can be engineered to be an ER-transporting protein. In various embodiments, the addition of an N-terminal signal peptide (e.g., the Ig-κ leader sequence) is to replace Fc, a globular protein, or HSS, which would otherwise be present in the fusion proteins disclosed herein.

[0106] In some embodiments, the fusion protein (e.g., masked IL-18) further comprises a tumor-targeting fragment, e.g., a fragment targeting a cell surface protein (including but not limited to a tumor-associated antigen (TAA)). For example, as Figure 9G shown, FUSE-517 is a masked IL-18 fusion protein that also comprises an anti-EGFR antibody fragment, e.g., the Fab of cetuximab. One or more antigen-targeting (preferably tumor antigen-targeting) fragments of known antibodies are considered to be compatible with the fusion protein systems disclosed herein.

[0107] In some embodiments, the fusion protein (e.g., masked IL-18) comprises an activating receptor targeting fragment, e.g., a fragment that targets an activating receptor on the cell surface, including but not limited to CD16 on the surface of natural killer cells. Activating receptors include immunoreceptor tyrosine-based activation motif (ITAM)-associated receptors such as CD16 and NKp46. Activating receptors also include those receptors involved in spontaneous NK cell activation such as NKp46 (CD335), NKp30 (CD337), NKp44 (CD336), NKG2D (CD314), DNAM-1 (CD226), 2B4 (CD244), LFA-1 (CD11a-CD18), and CD2. In some embodiments, the fusion protein (e.g., masked IL-18) comprises both an activating receptor targeting fragment and a tumor targeting fragment. Examples of anti-CD16 fragments include but are not limited to the CH2 domain of IgG1, the CH2 domain of IgG4. In some embodiments, the fusion protein (e.g., masked IL-18) comprises a polypeptide fragment that targets an immune checkpoint, e.g., a fragment that targets an immune checkpoint expressed on T cells. For example, as Figure 4 shown, FUSE-694 is a masked IL-18 fusion protein that also comprises an anti-PD-1 fragment. Examples of immune checkpoints include but are not limited to PD-1, PD-L1, CTLA-4, LAG-3. One or more immune checkpoint targeting fragments of known antibodies are considered to be compatible with the fusion protein systems disclosed herein. Examples of anti-PD-1 fragments include fragments (e.g., Fab, Fv) from pembrolizumab, nivolumab, pidilizumab, AMP-514, spartalizumab, cemiplimab, AK105, BCD-100, BI 754091, JS001, LZM009, MGA012, Sym021, TSR-042, MGD013, AK104, XmAb20717, tislelizumab, or PF-06801591.

[0108] In some embodiments, the fusion protein (e.g., masked IL-18) comprises a targeting polypeptide, wherein the targeting polypeptide targets a protein on the same surface as IL-18RC. Examples of such proteins include, but are not limited to, CD16, γ9 TCR, δ2 TCR, or δ1 TCR, NKp46, CD137, CD40, or NKG2D. In some embodiments, the fusion protein (e.g., masked IL-18) comprises a targeting polypeptide, wherein the targeting polypeptide targets a protein on a cell that does not contain IL-18RC. In these embodiments, for cis or density effects, the IL-18 fusion protein needs to be delivered close to the IL-18R complex, thereby causing an interaction between the IL-18 fusion protein and the IL-18R complex. For example, an IL-18 fusion protein targeting a TAA can interact with the IL-18R complex on a T cell if (a) the fusion protein bridges the T cell and the TAA+ cell, or (b) the fusion protein is combined with another protein that bridges the T cell and the TAA+ cell, or (c) the fusion protein binds to the TAA+ cell that naturally interacts with the T cell by a secondary means (e.g., TCR / MHC interaction). In other examples, the fusion protein can be delivered to fibroblasts or other accessory cells in the tumor microenvironment and released by proteases such that it can act on IL-18R+ T or NK cells at a distance.

[0109] Exemplary targeting polypeptides include those identified in Table 5 or fragments thereof. Among those listed as antigen-binding antibodies, their VHH, Fab regions, or single-chain variable fragments (scFv) can be used as the antigen-binding sites of the multispecific antibodies disclosed herein.

[0110] In some embodiments, enterokinase is used for site-specific cleavage of a recombinant fusion protein containing an accessible enterokinase recognition site. For example, enterokinase can specifically cleave after the lysine residue at the C-terminus of its cleavage site Asp-Asp-Asp-Asp-Lys (SEQ ID NO: 87). Thus, the fragment resulting from this cleavage reaction does not inherit any residues from the DDDDK (SEQ ID NO: 87) recognition sequence. Additionally, DDDDK (SEQ ID NO: 87) is part of the octapeptide FLAG tag (DYKDDDDK, SEQ ID NO: 248), which can be used as a fusion tag for recognition by an antibody, for detection of the fusion protein by Western blot analysis of proteins, and for purification of the fusion protein by anti-FLAG affinity chromatography.

[0111] Preferably, the cleavage site can be based on peptide substrates sensitive to other enzymes, particularly proteases highly expressed in the tumor microenvironment, such as granzyme B, granzyme A, granzyme M, granzyme K, matrix metalloproteinases (MMP) 1 / 2 / 9 / 14 or other MMPs. It should be noted that granzymes are usually only upregulated in inflammatory tumors. For example, the substrate sequence of granzyme B can be Ile-Glu-Xaa-Asp↓Xaa-Gly (SEQ ID NO: 249), which is cleaved at the Asp↓Xaa peptide bond. Alternatively, the substrate sequence of granzyme B can also be Ile-Glu-Xaa-Asp↓, which is cleaved at the C-terminus of Asp, and Xaa can be Gln (Gln, SEQ ID NO: 88) or another amino acid.

[0112] Several immune cells can release granzymes, such as T cells, NK cells, neutrophils, and mast cells. In several embodiments, the fusion protein comprises (a) a polypeptide fragment targeting an immune checkpoint expressed on immune cells and / or a polypeptide fragment targeting an activating receptor on NK cells, and (b) a tumor-targeting fragment, and the fusion protein is effective for bringing immune cells (such as T cells, NK cells) to the tumor, which can cause the release of granzymes that release IL-18. For example, an IL-18 fusion protein comprising a polypeptide fragment targeting an immune checkpoint protein can reverse the exhaustion of NK cells and / or T cells (which can then release more granzymes).

[0113] In additional embodiments, the fusion protein comprises a cleavage site recognized by a serine protease, cysteine protease, aspartic protease, threonine protease, glutamic protease, metalloprotease, gelatinase, or asparaginyl peptide lyase. In some embodiments, the protease cleavage site is recognized by the following enzymes: cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin K, cathepsin L, kallikrein, hKl, hK10, hK15, plasmin, collagenase, type IV collagenase, stromelysin, Factor Xa, chymotrypsin-like protease, trypsin-like protease, elastase-like protease, subtilisin-like protease, actinidain, bromelain, calpain, cysteine protease, caspase-3, Mir 1-CP, papain, HIV-1 protease, HSV protease, CMV protease, rennin, pepsin, matriptase, legumain, plasmepsin, nepenthesin, metalloexopeptidase, metalloendopeptidase, matrix metalloproteinase (MMP), MMP1, MMP2, MMP3, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, ADAM10, ADAM17, ADAM12, urokinase-type plasminogen activator (uPA), enterokinase, prostate-specific antigen (PSA, hK3), interleukin-1β converting enzyme, thrombin, FAP (FAP-α), dipeptidyl peptidase or dipeptidyl peptidase IV (DPPIV / CD26), type II transmembrane serine protease (TTSP), neutrophil elastase, cathepsin G, proteinase 3, neutrophil serine protease 4, mast cell chymase, mast cell tryptase, dipeptidyl peptidase, and dipeptidyl peptidase IV (DPPIV / CD26). Table 1 includes non-limiting examples of cleavage sites. As a specific example, IEQD (SEQ ID NO: 88) can be used.

[0114] Contemplated are variants, fragments, or fragments of variants of IL-18 that are suitable for inclusion in a fusion protein and are preferred in some embodiments. For example, a variant of mature IL-18 can have one, two, three, four, five, or more amino acid substitutions compared to wild-type mature IL-18. For example, one or more cysteines in IL-18 or its propeptide are replaced with natural or non-natural amino acids, such as replacement of Cys with Ser, Ala, or Val, to reduce aggregation of the molecule in the fusion protein. Additional examples include replacement of cysteine with threonine, asparagine, or glutamine; replacement of cysteine with alanine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan; replacement of cysteine with phenylalanine, alanine, aspartic acid, or asparagine; or replacement of cysteine with threonine, glutamine, aspartic acid, phenylalanine, isoleucine, or histidine.

[0115] Variants of IL-18 can have 95%, 90%, 85%, 83%, 80%, 75%, 70%, 65% or at least 60% sequence identity with wild-type IL-18. In some embodiments, the variant of IL-18 can have at least 60% and at most 83% sequence identity with wild-type IL-18. In some embodiments, after cleavage at the cleavage site of the fusion protein, the IL-18 variant in the fusion protein is released as a functional fragment of about 15 kDa (e.g., as tested, on an electrophoresis gel). (It is contemplated that the released protein can be mature IL-18, which normally runs at 18 kDa but may appear at about 15 kDa, attributable to the molecular weight standards used or the specific percentage of polyacrylamide in the gel). Fragments of IL-18 can have 95%, 90%, 85%, 83%, 80%, 75%, 70%, 65% or at least 60% sequence identity (and / or length) with wild-type IL-18. In some embodiments, compared to native / wild-type mature IL-18, the IL-18 fragment produced by the fusion proteins disclosed herein, particularly after protease cleavage of the fusion protein, is less than 85% in size (e.g., about 83%, about 83%-80%, about 80%-75%, about 75%-70% or about 70%-65%); e.g., an IL-18 fragment of about 15 kDa in size, preferably has a binding affinity for IL-18Ra / b comparable to that of wild-type mature IL-18, the fragment is fused to a propeptide (or PP variant) and an ER transport protein (with or without mutations), and the fusion protein further comprises a protease cleavage site such that after protease cleavage, a small IL-18 fragment (e.g., about 15 kDa in size) is released. Preferably, this small IL-18 fragment retains the native binding affinity for IL-18Ra / b and has the same or lower binding affinity for IL-18BP relative to IL-18Ra / b. Preferably, the variant, fragment or fragment of the variant of IL-18 is capable of binding to IL-18R and forming a complex, thereby activating the pro-inflammatory program and / or the NF-κB pathway. In some embodiments, compared to wild-type IL-18, the variant, fragment or fragment of the variant of IL-18 can have an increased binding affinity (e.g., 150%, 140%, 130%, 120%, 110% or at least 100% relative to wild-type IL-18), and / or compared to wild-type IL-18, induce at least 150%, 140%, 130%, 120%, 110%, 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20% or 10% of the biological activity.In some embodiments, a variant, fragment, or fragment of a variant of IL-18 can have an increased binding affinity of 120%, 110%, or at least 100% relative to wild-type IL-18, and / or induce a biological activity of 120%, 110%, 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10% compared to wild-type IL-18. In additional embodiments, a variant, fragment, or fragment of a variant of IL-18 has a reduced binding to IL-18 binding protein (IL-18BP) compared to wild-type IL-18.

[0116] In some embodiments, IL-18 or a fragment or variant thereof cleaved from a fusion protein (especially one with a propeptide) has at least a 1000-fold, 2000-fold, 3000-fold, 5000-fold, 10000-fold, 30000-fold, 50000-fold, 70000-fold, 80000-fold, 90000-fold, or 100000-fold increase in biological activity (e.g., binding to IL-18R to form an IL-18 / IL-18Rα / β complex and inducing downstream signaling) compared to the uncleaved form in the fusion protein. In further embodiments, IL-18 or a fragment or variant thereof cleaved from a fusion protein has comparable biological activity or a difference in biological activity within about 10-fold, 20-fold, 30-fold, 40-fold, or 50-fold compared to recombinant human mature IL-18.

[0117] In some embodiments, the IL-18 or fragment or variant thereof cleaved from the fusion protein has a binding affinity for its IL-18R complex with an equilibrium dissociation constant (KD) of about 18 nM (e.g., 18 nM ± 0.3 nM, 18 nM ± 0.5 nM, 18 nM ± 1.0 nM). In some embodiments, the IL-18 or fragment or variant thereof cleaved from the fusion protein has an approximately the same binding affinity for its IL-18R complex as wild-type IL-18, or at least 100%, 95%, or 90%. In some embodiments, the IL-18 or fragment or variant thereof cleaved from the fusion protein has a higher binding affinity for its IL-18R complex than wild-type IL-18. For example, compared to wild-type IL-18, the binding affinity is at least 105%, 110%, or its KD value is at least 10% or 20% smaller than that of wild-type IL-18. Preferably, compared to wild-type IL-18, the IL-18 or fragment or variant thereof cleaved from the fusion protein has a reduced binding affinity for IL-18BP. For example, in some cases, the KD of the IL-18 or fragment or variant thereof cleaved from the fusion protein for IL-18BP is 18 nM or greater, such that it has a lower binding affinity for IL-18BP than for IL-18R. In some cases, the KD of the IL-18 or fragment or variant thereof cleaved from the fusion protein for IL-18BP is 18 nM or greater, while the KD of wild-type IL-18 for IL-18BP is about 0.4 nM. It is also contemplated that the KD may vary depending on instrument and protocol settings.

[0118] In various embodiments, the fusion protein comprises a first polypeptide or protein or a fragment thereof capable of being transported into the endoplasmic reticulum (ER); and interleukin 18 (IL-18), a fragment of IL-18, an IL-18 variant, or a fragment of an IL-18 variant, wherein the IL-18, fragment of IL-18, IL-18 variant, or fragment of an IL-18 variant is located at the C-terminus of the fusion protein relative to the first polypeptide or protein capable of being transported into the ER. In various embodiments, the first polypeptide is not the wild-type IL-18 propeptide. In various embodiments, the protein or fragment thereof capable of being transported into the endoplasmic reticulum (ER) is not the wild-type IL-18 propeptide. In addition to these features, additional features of the fusion protein are discussed herein.

[0119] In some embodiments, the fusion protein comprises an IL-18 variant. In various embodiments, the IL-18 variant has an amino acid sequence that comprises or consists of amino acids 37-193 of SEQ ID NO: 250 and has 1 to 5 amino acid substitutions at positions E42, M87, K89, M96, and M149 of MAAEPVEDNCI NFVAMKFIDNTLYFIAEDDENIEQDYFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDVRDNAPRTIFIISMYKDSQPRGMAVTISVKVEKISTLSVENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLAVEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 250). In various embodiments, the 1 to 5 amino acid substitutions are 1 amino acid substitution. In other embodiments, the 1 to 5 amino acid substitutions are 2 amino acid substitutions. In other embodiments, the 1 to 5 amino acid substitutions are 3 amino acid substitutions. In other embodiments, the 1 to 5 amino acid substitutions are 4 amino acid substitutions. In other embodiments, the 1 to 5 amino acid substitutions are 5 amino acid substitutions. In various embodiments, the IL-18 variant comprises no more than 5 amino acid substitutions other than cysteine substitutions.

[0120] In various embodiments, the IL-18 variant has an amino acid sequence that comprises or consists of amino acids 37-193 of SEQ ID NO: 251 and has 1 to 5 amino acid substitutions at positions E42, M87, K89, M96, and M149 of MAAEPVEDNCINFVAMKFIDNTLYFIAEDDENIEQDYFGKLESKLS VIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQPRGMAVTISV KCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGY FLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 251).

[0121] In various embodiments, the IL-18 variant has an amino acid sequence as follows, which sequence comprises or consists of amino acids 37-193 of SEQ ID NO: 251, and has one or more amino acid substitutions at positions C74, C104, C112 and C164 of SEQ ID NO: 251, and has 1 to 5 amino acid substitutions at positions E42, M87, K89, M96 and M149. In various embodiments, the amino acid substitutions at one or more of C74, C104, C112 and C164 are each independently substituted with valine, alanine or serine. In various embodiments, the amino acid substitutions at one or more of C74, C104, C112 and C164 are each substituted with alanine. In various embodiments, the amino acid substitutions at one or more of C74, C104, C112 and C164 are each substituted with serine.

[0122] In various embodiments, the 1 to 5 amino acid substitutions are one or more of the following substitutions: E42K, E42R, E42A, E42H or E42Q; M87K or M87H; K89G, K89A or K89E; M96L or M96I; or M149V or M149I. In various embodiments, the 1 to 5 amino acid substitutions are E42K, E42R, E42A, E42H or E42Q; M87K or M87H; K89G, K89A or K89E; M96L or M96I; and M149V or M149I.

[0123] In various embodiments, the fusion protein comprises an IL-18 variant selected from Table 1. In various embodiments, the fusion protein comprising an IL-18 variant selected from Table 1 further comprises a propeptide having an amino acid sequence selected from the "Propeptide" column of Table 1, and optionally from the same row as the IL-18 variant. In various embodiments, the fusion protein comprising an IL-18 variant selected from Table 1 and a propeptide selected from Table 1 further comprises a cleavage peptide selected from Table 1, and optionally from the same row as the IL-18 variant and the propeptide. As a specific example, IEQD (SEQ ID NO: 88) can be used.

[0124] In various embodiments, the IL-18 variant is an IL-18 variant disclosed in U.S. Patent No. 7,524,488, U.S. Patent Publication No. 2019 / 0070262, U.S. Patent Publication No. 2021 / 0015891 or PCT Publication No. WO 2022 / 038417, and the IL-18 variants and their sequences in each of these patents or publications are incorporated herein by reference as if fully set forth.

[0125] In various embodiments, the fusion protein further comprises a targeting polypeptide. In some embodiments, the targeting polypeptide targets a protein on the cell surface, where the cell surface also has IL-18RC, or the cell is capable of expressing IL-18RC. In various embodiments, the fusion protein binds to a cell having IL-18RC or capable of expressing IL-18RC upon cell activation, and activates the IL-18RC signal.

[0126] In other embodiments, the targeting polypeptide targets a protein on the cell surface that does not have IL-18RC, or the cell is not capable of expressing IL-18RC. A cell that does not have IL-18RC on its surface or is not capable of expressing IL-18RC is in close proximity to a cell that expresses IL-18RC or is capable of expressing IL-18RC. In other situations, the fusion protein can bring a cell that does not have IL-18RC on its surface or is not capable of expressing IL-18RC into close proximity to a cell that expresses IL-18RC or is capable of expressing IL-18RC.

[0127] In various embodiments, the targeting polypeptide comprises a tumor-associated antigen-binding domain.

[0128] In various embodiments, the fusion protein further comprises a binding domain for a protein expressed on an immune cell. In various embodiments, the fusion protein further comprises a binding domain for a protein expressed on an immune cell that expresses an IL-18 receptor complex or expresses an IL-18 receptor complex upon activation.

[0129] In various embodiments, the fusion protein further comprises an antibody or an antibody fragment, and the fusion protein binds to a tumor cell, or binds to stromal cells or immune cells in a tumor tissue. Examples of antibody fragments include Fc fragments, Fab fragments, Fv fragments, and other fragments discussed herein.

[0130] In various embodiments, the fusion protein further comprises a masking domain. In these embodiments, mature IL-18 or a mature IL-18 variant can be released from the masking domain by a protease. In various embodiments, the protease is a granzyme, which can be released from an immune cell. Examples of immune cells include, but are not limited to, NK cells, T cells, neutrophils, or mast cells. In various embodiments, the protease is a metalloprotease, which can be expressed in the tumor microenvironment. Further examples of granzyme types and proteases are described herein. In various embodiments, mature IL-18 enhances the activity of NK cells or T cells, and optionally, the activity is one or more of proliferation, survival, and cytotoxicity.

[0131] In various embodiments, the fusion protein further comprises a half-life extending molecule. Non-limiting examples of half-life extending molecules are half-life extending polypeptides, such as human serum albumin (HSA) or HSA binding fragments. In various embodiments, the fusion protein has reduced activity when not bound to cells having IL-18RC as compared to wild-type IL-18. In various embodiments, the reduced activity is at least a 75% reduction in activity as compared to wild-type IL-18.

[0132] In various embodiments, the fusion protein comprises polypeptide 1 and polypeptide 2 selected from Table 1. In various embodiments, the fusion protein further comprises polypeptide 3 selected from Table 1. In various embodiments, polypeptide 1, polypeptide 2, and optionally polypeptide 3 are selected from the same row of Table 1.

[0133] In various embodiments, the fusion protein comprises polypeptide 1 selected from Table 1, wherein polypeptide 1 comprises HSA.

[0134] In various embodiments, the fusion protein does not comprise an IL-18 variant disclosed in U.S. Patent No. 7,524,488, U.S. Patent Publication No. 2019 / 0070262, U.S. Patent Publication No. 2021 / 0015891, or PCT Publication No. WO2022 / 038417, the IL-18 variants in each of these patents or publications and their sequence mimics being incorporated herein by reference as if fully set forth. Propeptide variant

[0135] Various embodiments of the invention provide propeptide variants. In various embodiments, the propeptide variant has the following amino acid sequence: AAEPVEDNX 1 INFVAMKFIDNTLYFIAEDDEN, wherein X 1 is any amino acid other than cysteine (SEQ ID NO: 238). In various embodiments, X 1 is alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan (SEQ ID NO: 239). In various embodiments, X 1 is valine (SEQ ID NO: 78). In various embodiments, X 1 is serine, threonine, asparagine, or glutamine (SEQ ID NO: 240). In various embodiments, X 1 is serine (SEQ ID NO: 76). IL-18 variant

[0136] Various embodiments provide IL-18 variants.

[0137] In various embodiments, the IL-18 variant has an amino acid sequence as follows, which sequence comprises the amino acids at positions 37-193 of SEQ ID NO: 250 or consists of the amino acids at positions 37-193 of SEQ ID NO: 250, and has 1 to 5 amino acid substitutions at positions E42, M87, K89, M96, and M149 of MAAEPVEDNCINFVAMKFIDNTLYFIAEDDENIEQDYFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDVRDNAPRTIFIISMYKDSQPRGMAVTISVKVEKISTLSVENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLAVEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 250). In various embodiments, the 1 to 5 amino acid substitutions are 1 amino acid substitution. In other embodiments, the 1 to 5 amino acid substitutions are 2 amino acid substitutions. In other embodiments, the 1 to 5 amino acid substitutions are 3 amino acid substitutions. In other embodiments, the 1 to 5 amino acid substitutions are 4 amino acid substitutions. In other embodiments, the 1 to 5 amino acid substitutions are 5 amino acid substitutions. In various embodiments, except for cysteine substitutions, the IL-18 variant comprises no more than 5 amino acid substitutions.

[0138] In various embodiments, the IL-18 variant has an amino acid sequence as follows, which sequence comprises the amino acids at positions 37-193 of SEQ ID NO: 251 or consists of the amino acids at positions 37-193 of SEQ ID NO: 251, and has 1 to 5 amino acid substitutions at positions E42, M87, K89, M96, and M149 of MAAEPVEDNCINFVAMKFIDNTLYFIAEDDENIEQDYFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 251).

[0139] In various embodiments, the IL-18 variant has an amino acid sequence as follows, which sequence comprises or consists of amino acids 37-193 of SEQ ID NO: 251 and has one or more amino acid substitutions at positions C74, C104, C112 and C164 of SEQ ID NO: 251, and has 1 to 5 amino acid substitutions at positions E42, M87, K89, M96 and M149. In various embodiments, the amino acid substitutions at one or more of C74, C104, C112 and C164 are each independently substituted with valine, alanine or serine.

[0140] In various embodiments, the 1 to 5 amino acid substitutions are one or more of the following: E42K, E42R, E42A, E42H or E42Q; M87K or M87H; K89G, K89A or K89E; M96L or M96I; or M149V or M149I. In various embodiments, the 1 to 5 amino acid substitutions are E42K, E42R, E42A, E42H or E42Q; M87K or M87H; K89G, K89A or K89E; M96L or M96I; and M149V or M149I.

[0141] In various embodiments, the IL-18 variant is selected from the "mature IL18 variant" column in Table 1.

[0142] In various embodiments, the IL-18 variant further comprises an IL-18 propeptide or an IL-18 propeptide variant.

[0143] In various embodiments, the IL-18 variant further comprises an IL-18 propeptide variant having the following amino acid sequence: AAEPVEDNX 1 INFVAMKFIDNTLYFIAEDDEN, wherein X 1 is any amino acid other than cysteine (SEQ ID NO: 238). In various embodiments, X 1 is alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine or tryptophan (SEQ ID NO: 239). In various embodiments, X 1 is valine (SEQ ID NO: 78). In various embodiments, X 1 is serine, threonine, asparagine or glutamine (SEQ ID NO: 240). In various embodiments, X 1is serine (SEQ ID NO: 76). In various embodiments, the IL-18 variant is selected from the "Mature IL18 Variant" column of Table 1 and further comprises a propeptide having an amino acid sequence selected from the "Propeptide" column of Table 1 and optionally from the same row as the IL-18 variant.

[0144] In various embodiments, the IL-18 variant further comprises an IL-18 propeptide variant and a cleavage peptide. In various embodiments, the cleavage peptide is selected from Table 1. As a specific example, IEQD (SEQ ID NO: 88) can be used.

[0145] In various embodiments, the fusion protein comprises an IL-18 variant selected from Table 1, a propeptide selected from Table 1, and a cleavage peptide selected from Table 1, and optionally from the same row as the IL-18 variant and the propeptide.

[0146] In various embodiments, the IL-18 variant is not the IL-18 variant disclosed in U.S. Patent No. 7,524,488, U.S. Patent Publication No. 2019 / 0070262, U.S. Patent Publication No. 2021 / 0015891, or PCT Publication No. WO 2022 / 038417, and the IL-18 variants and their sequence mimics in each of these patents or publications are incorporated herein by reference as fully set forth. Polynucleotides, Vectors, and Cells

[0147] Various embodiments provide polynucleotides encoding the fusion proteins disclosed herein. For example, the polynucleotide can encode a first protein or polypeptide capable of being transported into the ER and IL-18 (or a fragment, variant, or fragment of a variant thereof) in the 5' to 3' direction. Non-limiting examples of such polynucleotides are in Table 2.

[0148] In addition, based on, for example, the following, the polynucleotide may optionally further comprise a "leader" or "signal" sequence: (1) a propeptide (PP) directly linked to IL-18 (or an IL-18 variant) as in FUSE499; or (2) an immunoglobulin light chain sequence directly fused to the hinge region of the immunoglobulin heavy chain constant region. In some embodiments, when the protein / polypeptide capable of being transported into the ER is based on an IgG sequence, the nucleic acid encodes at least an immunoglobulin hinge region (i.e., a hinge region comprising at least one cysteine amino acid capable of forming a disulfide bond with a second immunoglobulin hinge region sequence), an immunoglobulin CH2 domain, and a CH3 domain, and IL-18 (or a fragment, variant, or fragment of a variant thereof) in the 5' to 3' direction.

[0149] In various embodiments, the polynucleotide encoding the fusion protein can also be integrated into a replicable expression vector. Accordingly, there is also provided a vector encoding the fusion protein, which can express the fusion protein in, for example, a bacterial host, a target receptor, or both.

[0150] Additional embodiments provide cells transformed or transfected with one or more nucleic acid molecules (polynucleotides) encoding a fusion protein. The cells can be prokaryotic cells. Alternatively, the cells are eukaryotic cells, preferably mammalian cells, and more preferably human cells. Examples of mammalian cells include Chinese hamster ovary (CHO) cells, NS0 cells (a mouse myeloma cell line), PER.C6 cells, and human embryonic kidney cells (HEK cells).

[0151] In some embodiments, there are also provided non-human organisms transformed or transfected with one or more nucleic acid molecules encoding a fusion protein. Composition

[0152] Further embodiments provide a composition comprising a combination of two or more different fusion proteins, or a combination of nucleic acid sequences encoding a fusion protein. For example, there is provided a pharmaceutical composition, wherein the fusion protein or the nucleic acid molecule encoding the fusion protein is an active agent.

[0153] The pharmaceutical composition according to the present invention may further comprise any pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or vehicle that participates in carrying or transporting a compound of interest from one tissue, organ, or site of the body to another tissue, organ, or site of the body. For example, the carrier can be a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, or a combination thereof. Each component of the carrier must be "pharmaceutically acceptable" because it must be compatible with the other ingredients of the formulation. It must also be suitable for contact with any tissue or organ with which it may come into contact, meaning that it must not present a risk of toxicity, irritation, allergic reaction, immunogenicity, or any other complication that seriously outweighs its therapeutic benefits. The pharmaceutical composition according to the present invention can also be encapsulated, made into tablets, or prepared as an emulsion or syrup for oral administration. Pharmaceutically acceptable solid or liquid carriers can be added to enhance or stabilize the composition, or facilitate the preparation of the composition. Liquid carriers include syrups, peanut oil, olive oil, glycerol, physiological saline, alcohols, and water. Solid carriers include starch, lactose, calcium sulfate dihydrate, clay, magnesium stearate or stearic acid, talc, pectin, gum arabic, agar, or gelatin. The carrier can also include separate sustained-release materials (such as glyceryl monostearate or glyceryl distearate) or together with waxes. The pharmaceutical formulations are made by conventional pharmaceutical techniques as follows. For tablet form, it involves grinding, mixing, granulating, and tableting when necessary; for hard gelatin capsule form, it involves grinding, mixing, and filling. When a liquid carrier is used, the formulation will be in the form of a syrup, elixir, emulsion, or aqueous or non-aqueous suspension. Such liquid formulations can be administered orally directly or filled into soft gelatin capsules... The pharmaceutical composition according to the present invention can be delivered in a therapeutically effective amount. The precise therapeutically effective amount is the amount of the composition that will produce the most effective result with respect to the therapeutic effect in a given subject. This amount will vary depending on a variety of factors, including but not limited to the characteristics of the therapeutic compound (including activity, pharmacokinetics, pharmacodynamics, and bioavailability), the physiological condition of the subject (including age, gender, disease type and stage, general physical condition, responsiveness to a given dose, and type of drug), the nature of one or more pharmaceutically acceptable carriers in the formulation, and the route of administration. Those skilled in the clinical and pharmacological arts will be able to determine the therapeutically effective amount by routine experimentation, such as by monitoring the subject's response to the administration of the compound and adjusting the dose accordingly. For additional guidance, see Remington: The Science and Practice of Pharmacy (edited by Gennaro, 20th edition, Williams & Wilkins PA, USA) (2000). Production method

[0154] Also provided are methods for making a fusion protein or a nucleic acid encoding a fusion protein. Some embodiments provide for generating a fusion protein using conventional recombinant DNA methods. The fusion construct is preferably generated at the DNA level, and the resulting DNA is integrated into an expression vector and expressed to produce the fusion protein of the invention. Subsequently, the vector is expressed in a host cell to obtain the fusion protein; and optionally, the method further includes the step of recovering the fusion protein from the host cell culture. In some embodiments, a method for producing interleukin 18 (IL-18), a fragment thereof, an IL-18 variant or a fragment of an IL-18 variant includes culturing cells transfected with an expression vector comprising a nucleic acid encoding a fusion protein in cell culture medium such that the fusion protein can be produced and secreted into the extracellular space for purification; when the fusion protein is contacted with a protease, the fusion protein is cleaved to produce IL-18, a fragment thereof, an IL-18 variant or a fragment of an IL-18 variant. Exemplary nucleic acid molecules encoding the fusion protein are shown in Table 2. Other embodiments provide that chemical coupling using conventional chemical crosslinkers can be used to fuse multiple protein moieties.

[0155] In some embodiments, the nucleic acid molecule encoding the fusion protein is expressed in CHO cells or HEK-293 cells. Preferably, expressing the fusion protein in a host cell produces at least 135 mg / L of the secreted fusion protein recoverable from the host cell supernatant. In some embodiments, a fusion protein yield of at least 135 mg / L is obtained by transient transfection. In some embodiments, higher fusion protein yields are obtained by stable production cell clones or clone pools, such as at least 150 mg / L, 200 mg / L, 250 mg / L or 300 mg / L. In some embodiments, by transient transfection, the yield of the fusion protein is about 130 mg / L - 400 mg / L. In some embodiments, the fusion protein or IL-18, a fragment thereof, an IL-18 variant or a fragment of an IL-18 variant cleaved from the fusion protein is recovered from the host cell supernatant at greater than about 400 mg / L, between 350 mg / L - 400 mg / L, between 300 mg / L - 350 mg / L, between 200 mg / L - 300 mg / L, between 100 mg / L - 200 mg / L or at least 50 mg / L.

[0156] In some embodiments, a Chinese hamster ovary (CHO) expression system is used to produce a fusion protein through a process comprising the following steps: (1) cell resuscitation, where cryopreserved CHO cells can be resuscitated by a 37°C water bath; (2) cell subculture, where the cells can be subcultured and the cell density adjusted to 6×106 cells / mL for transfection; (3) transfection and expression, using Solution 1 (wherein the plasmid is diluted with a diluent), Solution 2 (wherein the transfection reagent is diluted with a diluent), and subsequently mixing Solution 1, Solution 2, and CHO cells, and then incubating the mixture in a shaker at 32°C for 12 - 14 days for expression, and collecting the supernatant of the culture after centrifugation.

[0157] In some embodiments, a purification process is performed after the expression of the fusion protein. In some embodiments, the purification process comprises the following steps: (1) flushing the column with binding buffer (10 volumes) at a flow rate of 1 mL / min; (2) loading the sample containing the fusion protein onto the column at a flow rate of 1 mL / min; (3) flushing the column with 10 volumes of PBS buffer at a flow rate of 1 mL / min; (4) eluting the protein from the column with 40 mM sodium citrate (pH 3.4); optionally, the eluted sample can be collected into a tube (1 mL / min) and the optical density (OD) measured at 280 nm using a NanoDrop; and (5) performing dialysis, for example, dialyzing overnight with PBS buffer in a dialysis bag. Table 1. Amino acid sequences of the components in an exemplary fusion protein (“FUSE”). (Most identical sequences are marked with the same number beside the components. For example, in FUSE - 422, FUSE - 423, FUSE - 424, FUSE - 441, FUSE - 442, FUSE - 462, FUSE - 480, FUSE - 481, FUSE - 484, FUSE - 485, FUSE - 486, FUSE - 487, FUSE - 499, FUSE - 500, FUSE - 505, FUSE - 516, FUSE - 517, FUSE - 545, FUSE - 546, FUSE5 - 556, FUSE - 583 to 587, FUSE - 599 to 602, FUSE - 645, FUSE - 686, FUSE - 756 to 758, FUSE - 775, FUSE - 874 to 876, and FUSE - 878 to 892, the “polypeptide 1” sequence, i.e., the first polypeptide arm sequence, is the same; and it is marked with “0” beside each respective component.) Residues represented by lowercase letters are amino acid positions with potential mutations. Table 2. Nucleotide sequences of the polypeptides in Table 1. Table 3. Yields of exemplary fusion proteins. Yield Yield Yield Yield FUSE - 422 191 mg / L FUSE - 501 147 mg / L FUSE - 583 86 mg / L FUSE - 775 79 mg / L FUSE - 423 214 mg / L FUSE - 52 159 mg / L FUSE - 584 124 mg / L FUSE - 782 41 mg / L FUSE - 424 198 mg / L FUSE - 503 44 mg / L FUSE - 585 136 mg / L FUSE - 783 99 mg / L FUSE - 441 386 mg / L FUSE - 504 135 mg / L FUSE - 586 107 mg / L FUSE - 827 207 mg / L FUSE - 442 197 mg / L FUSE - 505 162 mg / L FUSE - 587 228 mg / L FUSE - 874 246 mg / L FUSE - 462 304 mg / L FUSE - 507 557 mg / L FUSE - 599 76 mg / L FUSE - 875 137 mg / L FUSE - 480 130 mg / l FUSE - 508 34 mg / L FUSF - 600 79 mg / L FUSE - 876 216 mg / L FUSE - 81 199 mg / L FUSE - 509 537 mg / L FUSE - 601 86 mg / L FUSE - 887 155 mg / L FUSE - 484 164 mg / L FUSE - 510 32 mg / L FUSE - 602 64 mg / L FUSE - 888 149 mg / L FUSE - 485 348 mg / L FUSE - 516 215 mg / L FUSE - 691 69 mg / L FUSE - 889 223 mg / L FUSE486 400 mg / L FUSE - 517 150 mg / L FUSE - 694 67 mg / L FUSE - 890 190 mg / L FUSE - 487 205 mg / L FUSE - 545 144 mg / L FUSE - 756 46 mg / L FUSE - 891 181 mg / L FUSE - 499 410 mg / L FUSE - 546 143 mg / L FUSE - 757 96 mg / L FUSE - 892 371 mg / L FUSE - 500 253 mg / L FUSE - 547 135 mg / L FUSE - 758 64 mg / L Table 4. Amino acid sequences for Fc heterodimerization of the different versions of the pestle and mortar structures including each polypeptide arm for exemplary fusion proteins, which form (parts of) proteins capable of being transported into the ER. Examples

[0158] The following examples are provided to better illustrate the claimed invention and should not be construed as limiting the scope of the invention. To the extent that specific materials are recited, this is for illustrative purposes only and is not intended to limit the invention. Those skilled in the art may develop equivalent means or reactants without exercising inventive faculty and without departing from the scope of the invention. Example 1

[0159] The N-terminus of pro-IL-18 was fused to the C-terminus of the IgG1 CH3 domain in order to engineer an IL-18 variant that can be expressed in mammalian cells. Pro-IL-18 was fused to the stapes of a mortise and tenon heterodimeric IgG1 protein. Pro-IL-18 was further modified to reduce molecular aggregation such that each cysteine residue in the propeptide and mature IL-18 was replaced with serine (such as “IL-18AS” in FUSE-480), alanine (such as “IL-18AA” in FUSE-481), or valine (such as “IL-18AV” in FUSE-442). The description of these three variants is shown in Subfigure A of FIG. 1.

[0160] For FUSE-480, FUSE-481, and FUSE-442, transient transfection in the ExpiCHO system produced titers of 191 mg / L, 214 mg / L, and 198 mg / L, respectively. These Fc-pro-IL-18 fusion proteins were hypothesized to harbor a "masked" version of IL-18, in which the biological activity of the fused IL-18 was reduced until the propeptide was cleaved. To assess propeptide cleavage, a cleavage site specific for enterokinase (EK) was inserted into the propeptide ("pp") upstream of the mature IL-18 sequence within the endogenous caspase-1 site. EK was chosen because of its robust protease activity and activity in phosphate-buffered saline. To evaluate the biological activity of the Fc-pro-IL-18 fusion protein before and after EK treatment, a reporter system was used. Here, HEK-Blue-IL-18 cells were used to quantify the activity of IL-18. HEK-Blue IL-18 cells were made from HEK-293 cells engineered to express the human IL-18 receptor complex (IL-18Rα / β) and the NF-κb / AP-1-inducible secreted embryonic alkaline phosphatase (SEAP) reporter gene. These cells were also engineered to be non-responsive to human TNF-α and IL-1β. Upon exposure to IL-18, HEK-Blue IL-18 produces SEAP in a dose-dependent manner, which can be quantified by colorimetric assay. We observed that all three mutants induced a greater than 1000-fold and up to 100,000-fold reduced biological activity compared to recombinant human mature IL-18. After de-masking (enzymatic cleavage) by EK treatment, the biological activity of the proteins released from the alanine substitution mutant (FUSE-480) and the valine substitution mutant (FUSE-442) was not substantially different from that of recombinant human mature IL-18. However, after releasing the serine mutant (1B) with EK, although the biological activity was restored by more than 30,000-fold, it was approximately 50-fold less potent than recombinant human mature IL-18. Potency was measured as the concentration of the test substance that induced the maximum SEAP production (EC50-SEAP).

[0161] As shown in Figure 2, pro-IL-18mut2 was fused to the staphylococcal protein A domain of the staphylococcal protein A / IgG1 heterodimer. IL-18mut2 contains four amino acid substitutions that are hypothesized to reduce its binding to IL-18BP while maintaining its binding to the wild-type IL-18 receptor complex. Pro-IL-18mut2 was further modified to reduce aggregation of the molecule such that each cysteine residue in the propeptide and mature IL-18mut2 was replaced with serine (IL-18mut2AS), alanine (IL-18mut2AA), or valine (IL-18mut2AV). The description of the three variants is shown in panel A of Figure 2. As in Figure 1, HEK-Blue was used to assess bioactivity. Compared to recombinant human mature IL-18, all three mutants induced at least a 100,000-fold reduction in bioactivity. After unmasking (enzymatic cleavage) by EK treatment, the bioactivity of the proteins released from the alanine substitution mutant (FUSE-423) and valine substitution mutant (FUSE-424) was not substantially different compared to recombinant human mature IL-18. However, after release of the serine mutant (FUSE422) with EK, although the bioactivity was restored approximately 100,000-fold, it was approximately 100-fold less potent than recombinant human mature IL-18. Potency was measured as the concentration of the test substance that induced the maximum SEAP production (EC50-SEAP).

[0162] As Figure 3As shown, in the context of Fc fusion variants with (subpanel A, FUSE-442; Fc-EKpp-IL-18AV) or without (subpanel B, FUSE-505; Fc-EK-IL-18AV) a propeptide, we examined the effect of the propeptide on the masking of IL-18AV bioactivity, which variants incorporated IL-18AV at the C-terminus of Fc. For the fusions lacking the propeptide, the EK cleavage site substituting the caspase 1 site was moved to a position directly between the CH2 domain of the stinger and mature IL-18AV, and no flexible linker was added. Using the HEK-Blue IL-18AV reporter cell assay as a readout, we exposed these cells to different titers of FUSE-442 or FUSE-505 with or without EK treatment. Compared to recombinant human IL-18AV, the bioactivity of FUSE-442 and FUSE-505 measured as EC50-SEAP was attenuated by approximately 150-fold and 15,000-fold, respectively (subpanel C), such that the incorporation of the propeptide led to an additional attenuation of approximately 100-fold compared to Fc fusion alone. EK treatment designed to cleave the IL-18AV fragment from FUSE-505 and FUSE-442 restored the bioactivity. In the case of FUSE-442, EK treatment produced a bioactivity not substantially different from recombinant human mature IL-18AV. However, for FUSE-505, although the bioactivity was restored after EK treatment, the potency was still reduced by approximately 30-fold compared to recombinant human mature IL-18AV. The EC50-SEAP of each compound is shown in subpanel D. Given that both FUSE-505 and FUSE-442 harbor the same IL-18AV, the difference in activity after EK de-masking cannot be explained by any differences in the IL-18AV variant. Instead, we observed that the efficiency of EK releasing IL-18AV from FUSE-505 (cleavage efficiency <10%) was much lower than that from FUSE-4442 (cleavage efficiency >95%). Thus, the reduced bioactivity observed for EK-cleaved FUSE-505 compared to FUSE-442 is due to less IL-18AV being released from the former. We hypothesize that the lack of a flexible linker between the CH3 domain of the stinger and IL-18AV in FUSE-505 renders the EK cleavage site mostly inaccessible. In contrast, the propeptide incorporated into FUSE-442 allows EK to access its cleavage site sufficiently to effectively release IL-18AV. The data strongly suggest that in the context of an IL-18AV Fc fusion protein, the propeptide is not essential but helps to mask the bioactivity of IL-18AV. In the absence of the propeptide, masking may be the result of steric hindrance mediated by the protein fused to the N-terminus of IL-18AV.

[0163] As Figure 4As shown, the same Figure 3 IL-18AV in Figure 4In the context of Fc fusion variants with a propeptide (Subfigure A, FUSE-424; Fc-EKpp-IL-18mut2AV) or without a propeptide (Subfigure B, FUSE-441; Fc-EK-IL-18mut2AV) that incorporate IL-18mut2AV at the C-terminus of Fc, we examined the effect of the propeptide on the masking of IL-18mut2AV bioactivity. Compared to recombinant human IL-18, the bioactivity of FUSE-441 and FUSE424 was substantially attenuated >100,000-fold (Subfigure C). Treatment with EK restored the bioactivity. In the case of FUSE-424, treatment with EK produced a bioactivity that was not substantially different from recombinant human mature IL-18. However, for FUSE-441, although the vast majority of the bioactivity was restored after treatment with EK, the residual potency was reduced by ~10-fold compared to recombinant human mature IL-18. The EC50-SEAP of each compound is shown in Subfigure D. Given that both FUSE-441 and FUSE-442 harbor the same IL-18mut2AV, the difference in activity after EK de-masking cannot be explained by any differences in the IL-18 variant. Instead, we observed that the efficiency of EK release of IL-18mut2AV from FUSE-441 (cleavage efficiency <20%) was much lower than that from FUSE-424 (cleavage efficiency >95%). Thus, the reduced bioactivity observed for EK-cleaved FUSE-441 compared to EK-cleaved FUSE-424 may be due to less IL-18mut2AV being released from the former compared to the latter. We hypothesize that the lack of a flexible linker between the CH3 domain of the pestle in FUSE-441 and IL-18mut2AV renders the EK cleavage site mostly inaccessible. For example, the flexible linker could be a propeptide; or the propeptide serves as a flexible linker. In contrast, the propeptide incorporated into FUSE-424 allows EK sufficient access to its cleavage site, thus effectively releasing IL-18mut2AV. The data strongly suggest that in the context of IL-18mut2AV Fc fusion proteins, masking of IL-18mut2AV bioactivity does not require a propeptide, but is the result of steric hindrance mediated by the protein fused to the N-terminus of IL-18mut2AV. Thus, we hypothesize that any N-terminal protein of sufficient size (e.g., approximately 4 kDa or larger, e.g., the propeptide is approximately 4 kDa, Fc as a monomer is approximately 28 kDa, HSA is approximately 66 kDa, VHH is approximately 14 kDa) is capable of masking the bioactivity of IL-18mut2AV; and that when incorporated between an Fc or other N-terminal protein mask and IL-18mut2AV, an EK cleavage site containing a linker of sufficient size (e.g., 25 amino acids or longer) to allow access to EK will replace the propeptide containing EK.In addition, if such N-terminal masking proteins or fragments thereof are engineered or naturally transported through the endoplasmic reticulum (ER), this will result in acceptable expression yields from transient transfection in mammalian cells (such as Expi-CHO) for therapeutic development.

[0164] As Figure 5As shown, we examined the effect of fusing the pro-IL-18AV fusion to the C-terminus of wild-type IgG1 (FUSE-507, panel A) and wild-type IgG4 (FUSE-509, panel B). In both forms, each Fc-CH3 domain was linked to one molecule of the pro-IL-18AV fusion, such that each IgG1 or IgG4 homodimer had two molecules of the pro-IL-18AV fusion. As described previously, a cleavage site specific for EK was inserted at the position of the endogenous cysteine protein-I site between the pro-peptide and the mature IL-18 sequence. To assess the bioactivity of the Fc-pro-IL-18 fusion proteins before and after EK treatment, the HEK-Blue-IL-18 cell reporter system was used. Using this system as a readout, we exposed HEK-Blue-IL-18 cells to different titers of FUSE-507 (IgG1Fc-EKpp-IL-18AV) or FUSE-509 (IgG4Fc-EKpp-IL-18AV) with or without EK treatment. Compared to recombinant human IL-18, the bioactivity of FUSE-507 and FUSE-509, measured as EC50-SEAP, was substantially reduced >10,000-fold (panel C). Treatment with EK, which was designed to cleave the IL-18AV fragment from FUSE-507 and FUSE-509, restored the bioactivity, which was approximately 2-fold higher than that of hrIL-18. This difference may be the result of the release of two molecules of IL-18AV per IgG1 or IgG4 fusion, such that when the IgG1 or IgG4 fusion protein is fully cleaved by EK, the molar ratio of IL-18AV to hrIL-18 is approximately 2:1. The EC50-SEAP for each compound is shown in panel D. We also generated pro-peptide-free versions of FUSE-507 and FUSE-509, IgG1Fc-EK-IL-18AV and IgG4Fc-EK-IL-18AV, respectively. These were poorly expressed, probably due to the tendency of IL-18 to form homodimers and the absence of a flexible linker between the CH3 domain of IgG1 or IgG4 and mature IL-18AV (data not shown). The data indicate that in the context of the pro-IL-182AV fusion, expression of >135 mg / L and masking / diminution of IL-18AV bioactivity of up to or greater than 100,000-fold may be achieved regardless of whether IL-18AV is fused to the C-terminus of wild-type IgG1 or IgG4 (two IL-18AV molecules), or to the IgG1 knobs-into-holes heterodimer (one IL-18AV molecule). Figure 5 Same as Figure 1 and Figure 3Comparison). The use of wild-type IgG1 or IgG4 formats demonstrates that propeptide-IL-18 fusions, variants thereof, and fragments thereof can be directly and plug-and-play incorporated into a range of monoclonal antibodies, including commercially available antibodies such as Avelumab (anti-PDL1), Cetuximab (anti-EGFR), Trastuzumab (anti-HER2 / neu), etc.

[0165] such as Figure 6As shown, to address the generality of the masking effect of polypeptides fused to the N-terminus of the mature form of IL-18 on IL-18 and its variants, we examined the ability of N-terminal proteins (maskers) different from the Fc domain of IgG to attenuate the bioactivity of IL-18-AV with or without a propeptide. The N-terminus of IL-18AV with a propeptide (subpanel A, FUSE-501) or without a propeptide (subpanel B, FUSE-503) was fused to the C-terminus of human serum albumin (HSA). For the fusion lacking the propeptide, the EK cleavage site that replaced the caspase-1 site was moved to a position directly between the C-terminus of HSA and mature IL-18AV without adding a flexible linker. Using the HEK-Blue IL-18 reporter cell assay as a readout, we exposed these cells to different titers of FUSE-501 (HSA-EKpp-IL-18AV) or FUSE-503 (HSA-EK-IL-18AV) with or without EK treatment. Compared to recombinant human IL-18, the bioactivity of FUSE-501 (subpanel C), measured as EC50-SEAP, was substantially attenuated by ~35,000-fold. FUSE-503 without a propeptide (subpanel D) was also attenuated by ~3,500-fold. Treatment with EK, which was designed to cleave the IL-18AV fragment from FUSE-501 and FUSE-503, restored the bioactivity. In the case of FUSE-501, treatment with EK produced a bioactivity not substantially different from that of recombinant human mature IL-18. However, for FUSE-503, although treatment with EK increased the bioactivity, it was still ~40-fold weaker than recombinant human IL-18. The EC50-SEAP of each compound is shown in subpanel E. Given that both FUSE-501 and FUSE-503 harbor the same IL-18AV, the difference in activity after EK de-masking cannot be explained by any differences in the IL-18 variants. Instead, we observed that the efficiency of EK to release IL-18AV from FUSE-503 (cleavage efficiency <20%) was much lower than that from FUSE-501 (cleavage efficiency >95%). Thus, the reduced bioactivity observed for EK-cleaved FUSE-503 compared to EK-cleaved FUSE-501 is due to less IL-18AV being released from the former compared to the latter. We hypothesize that the lack of a flexible linker between the C-terminus of HSA and IL-18AV in FUSE-503 renders the EK cleavage site mostly inaccessible. In contrast, the propeptide incorporated into FUSE-501 allows EK to access its cleavage site sufficiently to effectively release IL-18AV.Combined with the data obtained from the Fc fusions, the results strongly suggest that in the context of the HSA-IL-18AV fusion protein, the propeptide is not required to mask the bioactivity of IL-18AV, but when present in the HSA-IL-18 fusion protein, it may contribute to a greater attenuation. For both FUSE-501 and FUSE-503, the masking appears to be the result of steric hindrance mediated by the protein fused to the N-terminus of IL-18AV. Thus, the data provide further support that any N-terminal protein of sufficient size is capable of masking the bioactivity of IL-18AV, and that an EK cleavage site containing a linker of sufficient size to allow access to EK will displace the EK-containing propeptide when incorporated between an Fc, HSA, or other N-terminal protein mask and IL-18AV. Additionally, if such N-terminal masking proteins or fragments thereof are engineered or naturally trafficked through the endoplasmic reticulum (ER), this will result in acceptable expression yields from transient transfection of mammalian cells (e.g., Expi-CHO) for therapeutic development.

[0166] As Figure 7As shown, to address the generality of the masking effect of polypeptides fused to the N-terminus of the mature form of IL-18 on IL-18 and its variants, we examined the ability of N-terminal proteins (maskers) different from the Fc domain of IgG to attenuate the bioactivity of IL-18-mut2AV with or without a propeptide. The N-terminus of IL-18mut2AV with a propeptide (subpanel A, FUSE-502) or without a propeptide (subpanel B, FUSE-504) was fused to the C-terminus of human serum albumin (HSA). For the fusion lacking the propeptide, the EK cleavage site replacing the caspase-1 site was moved to a position directly between the C-terminus of HSA and mature IL-18AV without adding a flexible linker. Using the HEK-Blue IL-18 reporter cell assay as a readout, we exposed these cells to different titers of FUSE-502 (HSA-EKpp-IL-18mu2AV) or FUSE-504 (HSA-EK-IL-18mut2AV) with or without EK treatment. Compared to recombinant human IL-18, the bioactivities of FUSE-502 (subpanel C) and FUSE-504 (subpanel D), measured as EC50-SEAP, were attenuated by at least ~30,000-fold and ~50,000-fold, respectively. Treatment with EK, designed to cleave the IL-18mut2AV fragment from FUSE-504 and FUSE-502, restored the bioactivity. In the case of FUSE-502, treatment with EK produced a bioactivity not substantially different from that of recombinant human mature IL-18 (subpanel C). For FUSE-504, although the vast majority of the bioactivity was restored upon EK treatment, the remaining potency was reduced by ~3-fold compared to recombinant human mature IL-18 (subpanel D). The EC50-SEAP of each compound is shown in subpanel E. Given that both FUSE-504 and FUSE-502 contain the same IL-18mut2AV, the difference in activity after EK de-masking cannot be explained by any differences in the IL-18 variants. Instead, we observed that the efficiency of EK to release IL-18mut2AV from FUSE-504 (cleavage efficiency <20%) was much lower than that from FUSE-502 (cleavage efficiency >95%). Thus, the reduced bioactivity observed for EK-cleaved FUSE-504 compared to EK-cleaved FUSE-502 is due to less IL-18mut2AV being released from the former compared to the latter. We hypothesize that the lack of a flexible linker between the C-terminus of HAS in FUSE-504 and IL-18mut2AV renders the EK cleavage site mostly inaccessible. In contrast, the propeptide incorporated into FUSE-502 allows EK to access its cleavage site sufficiently to efficiently release IL-18mut2AV.Combined with the data obtained from the Fc fusions, the results strongly suggest that in the context of the HSA-IL-18mut2AV fusion protein, masking of IL-18mut2AV bioactivity does not require the propeptide and is instead the result of steric hindrance mediated by the protein fused to the N-terminus of IL-18mut2AV. Thus, the data provide further support that any N-terminal protein of sufficient size is capable of masking the bioactivity of IL-18mut2AV and that when incorporated between an Fc, HAS, or other N-terminal protein masker and IL-18mut2AV, an EK cleavage site containing a linker of sufficient size to permit access to EK will displace the EK-containing propeptide. Additionally, if such an N-terminal masking protein or fragment thereof is engineered or naturally traffics through the endoplasmic reticulum (ER), this will result in acceptable expression yields from transient transfection of mammalian cells (e.g., Expi-CHO) for therapeutic development.

[0167] As shown in Figure 8, we examined the effect of the propeptide on the biological activity of a single IL-18AV fused to the N-terminus of IgG1 Fc. Panels A and B are exemplary illustrations of IL-18AV on the stob of a staphylococcal protein A IgG1-Fc domain fused with (FUSE-499; Fc-pp-IL-18AV, Panel A) or without (FUSE-500; Fc-IL-18AV, Panel B) a propeptide. In this case, we used a wild-type propeptide in which all cysteine residues were substituted with valine, but the caspase-1 site was retained. To allow both constructs to be translocated into the endoplasmic reticulum (ER) of mammalian cells and thus expressed / secreted, a signal peptide of Igκ chain (IgK leader sequence) was encoded upstream of the propeptide of FUSE-499 or IL-18AV of FUSE-500. Using the HEK-Blue IL-18 reporter cell assay as a readout, we examined the ability of our C→V (AV) propeptide to mask IL-18AV (FUSE-499) and the ability of caspase-1 to de-mask / recover the biological activity. Compared to recombinant human IL-18, the biological activity of FUSE499 measured as EC50-SEAP was attenuated >50,000-fold (Panel C). In contrast, in the absence of the propeptide in FUSE-500, there was no reduction in its biological activity compared to recombinant human IL-18, indicating that the IL-18-Fc fusion protein is fully functional in the absence of a propeptide (or another polypeptide) linked to the N-terminus of mature IL-18. Importantly, de-masking of FUSE-499 with caspase-1 restored the biological activity, with no significant difference from recombinant human IL-18. Treatment of FUSE-500, which contains neither a masking domain nor a caspase-1 cleavage site, with caspase-1 served as a negative control and had virtually no effect on the biological activity. This data indicates that a propeptide is required to attenuate in the configuration where IL-18AV is linked to the N-terminus of IgG. Combining with our previous data that the CH3 domain or HSA attenuated the biological activity of IL-18AV and IL-18mut2AV when fused to the N-terminus of each IL-18 variant in the absence of a propeptide, we hypothesize that any polypeptide with a size sufficient to be fused to the N-terminus of mature IL-18 and / or its variants and their fragments is capable of masking the biological activity of IL-18. Although the smallest polypeptide tested was the propeptide (∼6 kDa), polypeptides as small as 2 kDa also had sufficient size. In various embodiments, the short polypeptide or protein is about 2 kDa or no greater than 250 kDa. Additionally, the propeptide in FUSE-499 is not naturally translocated through the endoplasmic reticulum but was engineered to achieve translocation by adding an IgK leader sequence upstream of it.This indicates that any polypeptide of a size sufficient to fuse to the N-terminus of mature IL-18 and / or its variants and fragments thereof is expected to attenuate the bioactivity of IL-18. That is, the N-terminal polypeptide may be naturally transported into the endoplasmic reticulum (ER), or it can be engineered to achieve transport. In both cases, transport through the ER is important for obtaining therapeutically developable expression yields from transient transfection of mammalian cells (e.g., Expi-CHO).

[0168] As Figures 9A - 9I shown, we examined whether proteases other than EK, as a proof of concept, could be used to unmask / activate pro-IL-18. Thus, we selected (a) matrix metalloproteinase (MMP), MMP2, which is reported to be preferentially overexpressed in the tumor microenvironment, and (b) granzyme B, which is released by cytotoxic lymphocytes including NK cells and CD8+ T cells and can thus accumulate in inflammatory tumors. For MMP2, we replaced the EK cleavage site within the propeptide (pp) of FUSE-442 (Fc-EKpp-IL-18AV) with (a) the MMP2 / 9 cleavage sequence (GPLGVR (SEQ ID NO: 89)) to generate FUSE-486 (Fc-MMP2pp-IL-18AV), and with (b) the MMP9 / 2 cleavage sequence (VHMPLGFLGP (SEQ ID NO: 90)) to generate FUSE-487 (Fc-MMP2 / 9pp-IL-18AV). (Desnoyers et al., Sci Transl Med. 2013 Oct 16; 5(207):207ra144.). In each case, the MMP to the left of the slash preferentially cleaves the above peptide sequence. For granzyme B, we replaced the EK cleavage site within the propeptide (pp) of FUSE-442 (Fc-EKpp-IL-18AV) and FUSE-424 (Fc-EKpp-IL-18mut2AV) with the typical cleavage site of granzyme B (IEQD (SEQ ID NO: 88)); thus, FUSE-485 (Fc-GzmBpp-IL-18AV) and FUSE-462 (Fc-GzmBpp-IL-18mut2AV) were generated. FUSE-486, FUSE-487, FUSE-485, and FUSE-462 are shown in Figure 9A and Figure 9D Using the HEK-Blue IL-18 reporter cell assay as a readout, we exposed these cells to different titers of (a) FUSE-486 or FUSE-487 with or without recombinant human MMP2 treatment Figure 9B) or (b) FUSE-485 or FUSE-462 (FUSE-485 and FUSE-462) with or without recombinant human granzyme B treatment (see Figure 9D ). Compared with rhIL18, the bioactivity of the MMP prodrug fusions, namely FUSE486 and FUSE487, measured as EC50-SEAP was significantly attenuated by up to approximately 5000-fold. After treatment of FUSE-486 with MMP2, the bioactivity of the unmasked IL-18AV fragment was restored to within 3-fold of rhIL-18. Next, we added a granzyme B cleavage site to the C-terminus of FUSE486 immediately adjacent to the MMP site to create FUSE587 (black triangle). As Figure 9C can be seen, FUSE587 was attenuated by approximately 3000-fold relative to recombinant human IL-18.

[0169] Interestingly, we observed that the IL-18AV variant released by cleavage of FUSE587 with MMP2 was still attenuated by approximately 100-fold relative to recombinant IL-18. In contrast, cleavage with granzyme B released an IL-18AV variant with activity similar to recombinant IL-18. Cleavage with granzyme B resulted in the release of mature IL-18AV, which did not require any N-terminal residues constituting the overhang, while after cleavage of FUSE486 and FUSE587 with MMP2, respectively, N-terminal polypeptide overhangs of 11 and 15 amino acids remained. We speculate that, although to a lesser extent than the full-size variant propeptide, these overhangs may attenuate the activity of IL-18AV. This phenomenon was further investigated in Figure 11 and Figure 13 .

[0170] Compared with recombinant human IL-18, FUSE-485 and FUSE-462 were also significantly attenuated by >15,000-fold ( Figure 9E and Figure 9F ). Treatment with recombinant human granzyme B (rhGb) designed to cleave the IL-18AV fragment and the IL-18mut2AV fragment from FUSE-485 and FUSE-462, respectively, restored the bioactivity. In both cases, the masked IL-18 variants showed almost the same attenuation, and the restored bioactivity was approximately 2-fold weaker than recombinant human mature IL-18. In addition, we did not observe a significant difference in the activity between the unmasked IL-18AV and IL-18mut2AV.

[0171] A summary table of the potency (EC50-SEAP) of each test substance is shown in Figures 9A - 9B and Figure 9F below.

[0172] We next examined whether our findings could form the basis for a plug-and-play masked IL-18 platform for targeting pro-IL-18 variants to cell surface proteins, including but not limited to tumor-associated antigens (TAAs). Thus, we used cetuximab as our proof-of-concept TAA-targeting protein for the pro-IL-18 fusion. Cetuximab is a monoclonal antibody targeting EGFR and is commercially used for the treatment of multiple cancer indications, including colorectal cancer and head and neck cancer. First, we fused granzyme B-cleavable pp-IL-18AV to the C-terminus of the Fc domain of cetuximab. We found that the fusion of pp-Gb-IL-18AV to the CH3 domain of cetuximab, which forms a natural homodimer, was poorly expressed. This was presumably due to the presence of two fabs, as the knobs-into-holes IgG1 format in which ppGb-IL-18AV was fused to the C-terminus of the knob (or hole) and the EGR-specific fab was fused to the N-terminus of the knob (or hole) was well-expressed. As shown in sub-panel G, FUSE-517 (Cetuximab_KiH_ppGb-IL-18-AV) consists of the fab of cetuximab fused to the N-terminus of the Fc-knob and ppGb-IL-18AV fused to the C-terminus of the Fc-knob. Using the HEK-Blue IL-18 reporter cell assay as a readout, we exposed these cells to different titers of FUSE-517 (9H) with or without rhGb treatment. Relative to rhIL-18, FUSE-517 was substantially attenuated >250,000-fold. Treatment of FUSE-517 with rhGb, which was designed to cleave off IL-18AV, restored bioactivity to within 2-fold of rhIL-18.

[0173] Figure 9I is associated with such as Figure 9HSummary table of data related potency (EC50-SEAP). Overall, the data strongly suggest that, in the context of fusing a pp masker between the IgG CH3 domain and the mature IL-18AV fragment, both MMP2 and granzyme B are able to de-mask / activate IL-18AV (and de-mask / activate IL-18mut2AV with granzyme B). Given that we were able to successfully use other polypeptides trafficking through the ER as maskers when fused to the N-terminus of various variants of IL-18, our observations strongly suggest that fusion proteins of masked IL-18, its variants or its fragments can be engineered to be selectively activated by proteases (including MMPs and granzymes) found in tumors / inflammatory tumors. As shown in Figure 10, we examined the susceptibility of selected IL-18 variants to the attenuation of bioactivity caused by its natural antagonist (IL-18BP). Sub-panel A of Figure 10 is an illustration of the IL-18 fusion proteins examined, namely, FUSE-442 (Fc-EKpp-IL-18AV) and FUSE-424 (Fc-EKpp-IL-18mut2AV). Both variants contain the same cysteine to valine substitution. However, FUSE-424 harbors mature IL-18AV (termed IL-18mut2AV) downstream of the polypeptide (pp), which contains the following mutations: M51K, K53G, M60L and M113V. These four residues in the mature 18 kDa fragment of IL-18 were previously described as important for the binding / masking of wild-type IL-18. Thus, we hypothesized that substitution of these residues would reduce IL-18BP binding, such that IL-18AVmut2 would retain bioactivity in the presence of IL-18BP, which is typically overexpressed relative to IL-18 in the tumor microenvironment (TME). As in previous examples, HEK Blue IL-18 was used to assess bioactivity via MYD88-driven SEAP, and potency was reported as EC50-SEAP. FUSE-442 (Fc-EKpp-IL-18AV) and FUSE-424 (Fc-EKpp-IL-18mut2AV) were treated with EK separately to release mature IL-18AV or IL-18mut2AV. Each cleavage product was then titrated from 1 pg / mL to 1 μg / mL in separate media or media containing 1.25 μg / mL human recombinant IL-18-BP (hrIL-18-BP). Human recombinant IL-18 (hrIL-18) was used as a reference molecule for wild-type inhibition of HEK Blue IL-18 reporter activity mediated by rhIL-18BP.Subplots B (rhIL-18), D (FUSE-442), and F (FUSE-424) are non-linear x-y plots of SEAP release (IL-18R reporter activity) on the y-axis versus test article concentration on the x-axis in the presence or absence of IL-18BP. Summaries of the biological potencies (EC50-SEAP) associated with each plot are shown in subplots C, E, and G (below each x-y plot). As well documented, we observed a strong attenuation of rhIL-18 by rhIL-18BP of approximately at least 300-fold - 1000-fold. For IL-18AV released from FUSE442, we observed a biological activity attenuation of approximately 100 to 150-fold (i.e., approximately 3 - 6-fold less than rhIL-18), indicating that the apparent affinity of IL-18AV for IL-18BP is weaker than that of rhIL-18 for IL-18BP. Importantly, the cleavage product of FUSE424, IL-18mut2AV, appears to be resistant to the biological attenuation caused by rhIL-18BP. As shown in subplot F, we observed no significant difference in biological activity between IL-18mut2AV released from FUSE424 and the same molecule exposed to rhIL-18BP. This data strongly indicates that IL-18mut2AV binds to rhIL-18BP with a very weak apparent binding force compared to IL-18AV or rhIL-18. Thus, a masked version of IL-18mut2AV designed to be released by proteases present in the TME (including but not limited to MMP2, 9, and 14) and / or by proteases released in inflammatory tumors (including but not limited to granzyme A, B, and M) can be expected to function in the presence of IL-18BP to promote anti-tumor activity through multiple pathways (including but not limited to IFNγ-mediated Th1 and Tc1 activities). Example 2

[0174] As shown in Figure 11, we examined the effect of the size of the polypeptide fused to the N-terminus of mature IL-18 on the biological activity of a single IL-18AV fused to the N-terminus of IgG1 Fc. Panel A is an exemplary illustration of IL-18AV fused to the pestle of the IgG1-Fc domain of the pestle and mortar structure, which has polypeptides of different sizes ranging from a propeptide variant (FUSE-499; described in Figure 18) to 35 amino acids (FUSE756) to 15 amino acids (FUSE757 and FUSE758). To allow all constructs to be transported into the endoplasmic reticulum (ER) of mammalian cells and thus expressed / secreted, a signal peptide from the Igκ chain (IgK leader sequence) is encoded upstream of the polypeptide (and is cleaved off by signal peptidase in the ER). The polypeptides of FUSE756 and FUSE757 consist of a series of glycine and serine residues, while the polypeptide of FUSE758 is the sequence of the protruding end generated by cleavage of the MMP cleavage site mediated by MMP2, KPLGLQARVVGGGG (SEQ ID NO: 252). In all three cases, a granzyme B site, IEQD (SEQ ID NO: 88), was also incorporated at the C-terminus of the N-terminal polypeptide. Using the HEK-Blue IL-18 reporter cell assay as a readout, we examined the ability of polypeptides of different sizes to attenuate / mask IL-18AV-Fc (FUSE500; solid squares) or rhIL-18 (black cross-hatched "X"). All polypeptides fused to the N-terminus of mature IL-18AV reduced the biological activity measured as EC50-SEAP by at least 100-fold. FUSE499 (solid triangles) attenuated maximally (>10,000-fold). Both FUSE757 (solid diamonds) and 758 (open inverted triangles) contain an 11-amino acid polypeptide fused to the N-terminus of mature IL-18AV and reduced the biological activity by approximately 250-fold relative to FUSE500 or rhIL-18. FUSE756 (open triangles) contains a 31-amino acid polypeptide fused to the N-terminus of mature IL-18AV and reduced the biological activity by approximately 1000-fold relative to FUSE500 or rhIL-18. Thus, regardless of size or amino acid composition, all of the larger proteins containing HSA and IgG fragments in the previous examples and the polypeptides tested that were fused to the N-terminus of mature IL-18AV attenuated the biological activity of mature IL-18. The degree of attenuation appears to be positively correlated with the size of the polypeptide / protein fused to the N-terminus of IL-18. Example 3 Techniques and Procedures HEK-Blue-IL-18 Cell Activation Assay with IL-18 or FUSE Proteins

[0175] HEK-BLUE from InvivogenTM -IL-18 cells are maintained in a medium (DMEM medium with 4.5 g / L glucose, 2 mM L-glutamine, 10% (v / v) heat-inactivated fetal bovine serum, 100 U / mL penicillin, 100 μg / mL streptomycin, 100 μg / mL Normocin, and 1×HEK-blue screening reagent). HEK-BLUE TM IL-18 cells are engineered from the human embryonic kidney 293 (HEK293) cell line to stably express genes encoding the IL-18 receptor (IL-18R) and the IL-18 receptor accessory protein (IL-18RAP), and to express the NF-Kb / AP-1-inducible secreted embryonic alkaline phosphatase (SEAP) reporter gene. Thus, activation of the NF-Kb and AP-1 pathways is monitored by quantifying in the supernatant at the level of SEAP (which is produced upon NF-Kb activation) using, for example, QUANTI-BLUE TM Solution, which is useful for the detection of biologically active IL-18. In addition, in HEK-BLUE TM IL-18 cells, the responses to human TNF-α and IL-1β have been blocked, and thus HEK-BLUE TM IL-18 cells respond specifically to IL-18.

[0176] On the day of the experiment setup, HEK-BLUE-IL-18 cells are gently rinsed twice with pre-warmed phosphate-buffered saline (PBS), and then detached in PBS by tapping the culture flask. The detached HEK-Blue-IL-18 cells are resuspended in pre-warmed test medium (DMEM medium with 4.5 g / L glucose, 2 mM L-glutamine, 10% (v / v) heat-inactivated FBS, 100 U / mL penicillin, and 100 μg / mL streptomycin) at a density of 5×10 5 cells per milliliter. For cell seeding (50,000 cells per well), 100 μL of the resuspended HEK-Blue-IL-18 cells is added to the designated wells in a 96-well plate.

[0177] For protein preparation, the FUSE protein was serially diluted 5-fold from 500,000 pg / mL to 6.4 pg / mL (2× of the final concentration) in the test medium. IL-18 was also serially diluted 5-fold from 800 pg / mL to 1.28 pg / mL (2× of the final concentration) in the test medium. To treat HEK-Blue-IL-18 cells, 100 μL of the prepared IL-18 or FUSE protein was added to the designated wells containing 50,000 cells in a 96-well plate, and then the protein was gently mixed with the cells. Then the 96-well plate was incubated at 37 °C, 5% CO 2 for 24 hours.

[0178] After 24 hours of activation, HEK-Blue-IL-18 cells released secreted alkaline phosphatase into the supernatant. 20 μL of the HEK-Blue-IL-18 cell culture supernatant was transferred to a new 96-well plate. Meanwhile, the QUANTI-Blue solution from Invivogen was prepared by adding 1 mL of the QB reagent and 1 mL of the QB buffer to 98 mL of sterile water. Then 180 μL of the prepared QUANTI-Blue solution was added to the 96-well plate containing 20 μL of the HEK-Blue-IL-18 cell culture supernatant and incubated at 37 °C for 4 hours. The level of secreted alkaline phosphatase associated with the activation of HEK-Blue-IL-18 cells was detected by measuring the OD at 630 nm using a spectrophotometer. HEK-Blue-IL-18 cell activation assay with enterokinase (EK)-cleaved FUSE protein

[0179] HEK-Blue-IL-18 cells from Invivogen were maintained in the medium. On the day of the experiment setup, the HEK-Blue-IL-18 cells were gently rinsed twice with pre-warmed phosphate-buffered saline (PBS), and then the culture flask was tapped to detach the cells in PBS. The detached HEK-Blue-IL-18 cells were resuspended in pre-warmed test medium at a density of 5×10 5 cells per milliliter. To seed the cells (50,000 cells per well), 100 μL of the resuspended HEK-Blue-IL-18 cells was added to the designated wells in a 96-well plate.

[0180] For FUSE protein cleavage, 4 μg of FUSE protein was mixed with 80 ng of EK enzyme and 2 μL of 10× PBS in a test tube. Sterile water was added to the test tube to bring the total reaction volume to 20 μL. Then the test tube was incubated at 25 °C for 40 minutes. After 40 minutes of incubation, the cleaved FUSE protein was serially diluted 5-fold in the test medium from 500000 pg / mL to 6.4 pg / mL (2× of the final concentration). IL-18 was also serially diluted 5-fold in the test medium from 800 pg / mL to 1.28 pg / mL (2× of the final concentration). To treat HEK-Blue-IL-18 cells, 100 μL of the prepared IL-18 or cleaved FUSE protein was added to the designated wells containing 50000 cells in a 96-well plate, and then the cells were gently mixed with the protein. Subsequently, the 96-well plate was incubated at 37 °C, 5% CO 2 2. Incubate for 24 hours.

[0181] After 24 hours of activation, 20 μL of the HEK-Blue-IL-18 cell culture supernatant containing secreted alkaline phosphatase was transferred to a new 96-well plate. Meanwhile, QUANTI-Blue solution was prepared by adding 1 mL of QB reagent and 1 mL of QB buffer to 98 mL of sterile water. Then 180 μL of the prepared QUANTI-Blue solution was added to the 96-well plate containing 20 μL of the HEK-Blue-IL-18 cell culture supernatant and incubated at 37 °C for 4 hours. The level of secreted alkaline phosphatase associated with HEK-Blue-IL-18 cell activation was detected by measuring the OD at 630 nm using a spectrophotometer. HEK-Blue-IL-18 cell activation assay with FUSE protein cleaved by matrix metalloproteinase (MMP)

[0182] HEK-Blue-IL-18 cells from Invivogen were maintained in the medium. On the day of the experiment setup, the HEK-Blue-IL-18 cells were gently rinsed twice with pre-warmed phosphate-buffered saline (PBS), and then the culture flask was tapped to detach the cells in PBS. The detached HEK-Blue-IL-18 cells were resuspended in pre-warmed test medium at a density of 5×10 5 cells per milliliter. To seed the cells (50000 cells per well), 100 μL of the resuspended HEK-Blue-IL-18 cells was added to the designated wells in a 96-well plate.

[0183] For FUSE protein cleavage, 1 μg of FUSE protein was mixed with 280 ng of MMP2 or MMP9 enzyme and 2.8 μL of 10× assay buffer (500 mM Tris, 100 mM CaCl2, 1500 mM NaCl, 0.5% (w / v) Brij-35, pH 7.5) in a test tube. Sterile water was added to the test tube to bring the total reaction volume to 28 μL. The test tube was then incubated at 37 °C for 2 hours. After 2 hours of incubation, the cleaved FUSE protein was serially diluted 5-fold from 500000 pg / mL to 6.4 pg / mL (2× of the final concentration) in the test medium. IL-18 was also serially diluted 5-fold from 800 pg / mL to 1.28 pg / mL (2× of the final concentration) in the test medium. To treat HEK-Blue-IL-18 cells, 100 μL of the prepared IL-18 or cleaved FUSE protein was added to the designated wells containing 50000 cells in a 96-well plate, and the cells were then gently mixed with the protein. Subsequently, the 96-well plate was incubated at 37 °C, 5% CO 2 2. Incubate for 24 hours.

[0184] After 24 hours of activation, 20 μL of the HEK-Blue-IL-18 cell culture supernatant containing secreted alkaline phosphatase was transferred to a new 96-well plate. Meanwhile, QUANTI-Blue solution was prepared by adding 1 mL of QB reagent and 1 mL of QB buffer to 98 mL of sterile water. Then, 180 μL of the prepared QUANTI-Blue solution was added to the 96-well plate containing 20 μL of the HEK-Blue-IL-18 cell culture supernatant and incubated at 37 °C for 4 hours. The level of secreted alkaline phosphatase associated with HEK-Blue-IL-18 cell activation was detected by measuring the OD at 630 nm using a spectrophotometer. HEK-Blue-IL-18 cell activation assay with FUSE protein cleaved by caspase I

[0185] HEK-Blue-IL-18 cells from Invivogen were maintained in the medium. On the day of the experiment setup, the HEK-Blue-IL-18 cells were gently rinsed twice with pre-warmed phosphate-buffered saline (PBS), and then the culture flask was tapped to dislodge the cells in PBS. The dislodged HEK-Blue-IL-18 cells were resuspended in pre-warmed test medium at a density of 5×10 5 cells per milliliter. To seed the cells (50000 cells per well), 100 μL of the resuspended HEK-Blue-IL-18 cells was added to the designated wells in a 96-well plate.

[0186] For FUSE protein cleavage, 14 μg of FUSE protein was mixed with 0.5 unit of caspase I enzyme and 2.8 μL of 10× assay buffer (500 mM Hepes (pH 7.2), 500 mM NaCl, 1% Chaps, 100 mM EDTA, 50% glycerol, and 100 mM DTT) in a test tube. Sterile water was added to the test tube to bring the total reaction volume to 28 μL. The test tube was then incubated at 37 °C for 2 hours. After 2 hours of incubation, the cleaved FUSE protein was serially diluted 5-fold from 500,000 pg / mL to 6.4 pg / mL (2× of the final concentration) in the test medium. IL-18 was also serially diluted 5-fold from 800 pg / mL to 1.28 pg / mL (2× of the final concentration) in the test medium. To treat HEK-Blue-IL-18 cells, 100 μL of the prepared IL-18 or cleaved FUSE protein was added to the designated wells containing 50,000 cells in a 96-well plate, and then the cells were gently mixed with the protein. Subsequently, the 96-well plate was incubated at 37 °C, 5% CO 2 2. Incubate for 24 hours.

[0187] After 24 hours of activation, 20 μL of the HEK-Blue-IL-18 cell culture supernatant containing secreted alkaline phosphatase was transferred to a new 96-well plate. Meanwhile, a QUANTI-Blue solution was prepared by adding 1 mL of QB reagent and 1 mL of QB buffer to 98 mL of sterile water. Then, 180 μL of the prepared QUANTI-Blue solution was added to the 96-well plate containing 20 μL of the HEK-Blue-IL-18 cell culture supernatant and incubated at 37 °C for 4 hours. The level of secreted alkaline phosphatase associated with the activation of HEK-Blue-IL-18 cells was detected by measuring the OD at 630 nm using a spectrophotometer. HEK-Blue-IL-18 cell activation assay with granzyme B-cleaved FUSE protein

[0188] HEK-Blue-IL-18 cells from Invivogen were maintained in the medium. On the day of the experiment setup, the HEK-Blue-IL-18 cells were gently rinsed twice with pre-warmed phosphate-buffered saline (PBS), and then the culture flask was tapped to dislodge the cells in PBS. The dislodged HEK-Blue-IL-18 cells were resuspended in pre-warmed test medium at a density of 5×10 5 cells per milliliter. To seed the cells (50,000 cells per well), 100 μL of the resuspended HEK-Blue-IL-18 cells was added to the designated wells in a 96-well plate.

[0189] Mature and active human granzyme B was generated by cleaving human pro-granzyme B using EK enzyme. Briefly, 4 μg of human pro-granzyme B was mixed with 40 ng of EK enzyme and 2 μL of 10× PBS in a test tube. Sterile water was added to the test tube to make the total reaction volume reach 20 μL. Then the test tube was incubated at 25 °C for 40 minutes. After incubation for 40 minutes, the activated human granzyme B was used to cleave the FUSE protein. Specifically, 10 μg of FUSE protein was mixed with 1 μg of activated human granzyme B in the assay buffer (50 mM HEPES (pH 7.4), 100 mM NaCl, 0.1% CHAPS, 1 mM EDTA, 10% glycerol) and incubated at 37 °C for a specific time indicated for each experiment.

[0190] Then, the FUSE protein cleaved by granzyme B was serially diluted 5-fold from 500000 pg / mL to 6.4 pg / mL (2× of the final concentration) in the test medium. IL-18 was also serially diluted 5-fold from 800 pg / mL to 1.28 pg / mL (2× of the final concentration) in the test medium. To treat HEK-Blue-IL-18 cells, 100 μL of the prepared IL-18 or cleaved FUSE protein was added to the designated wells containing 50000 cells in a 96-well plate, and then the cells were gently mixed with the protein. Subsequently, the 96-well plate was incubated at 37 °C, 5% CO 2 2 for 24 hours.

[0191] After 24 hours of activation, 20 μL of the HEK-Blue-IL-18 cell culture supernatant containing secreted alkaline phosphatase was transferred to a new 96-well plate. Meanwhile, the QUANTI-Blue solution was prepared by adding 1 mL of QB reagent and 1 mL of QB buffer to 98 mL of sterile water. Then 180 μL of the prepared QUANTI-Blue solution was added to the 96-well plate containing 20 μL of the HEK-Blue-IL-18 cell culture supernatant and incubated at 37 °C for 4 hours. The level of secreted alkaline phosphatase associated with the activation of HEK-Blue-IL-18 cells was detected by measuring the OD at 630 nm using a spectrophotometer. IL-18BP blocks the activation of HEK-Blue-IL-18 cells induced by IL-18 or FUSE protein

[0192] Maintain HEK-Blue-IL-18 cells from Invivogen in the culture medium. On the day of the experiment setup, gently rinse the HEK-Blue-IL-18 cells twice with pre-warmed phosphate-buffered saline (PBS), and then tap the culture flask to detach the cells in PBS. Resuspend the detached HEK-Blue-IL-18 cells at a density of 5×10 5 cells per milliliter in pre-warmed test medium. To seed the cells (50,000 cells per well), add 100 μL of the resuspended HEK-Blue-IL-18 cells to the designated wells in a 96-well plate.

[0193] Prepare the uncut or cut FUSE proteins by 5-fold serial dilution in the test medium from 1,000,000 pg / mL to 12.8 pg / mL (4× the final concentration). Dilute IL-18BP in the test medium to a concentration of 5,000,000 pg / mL (4× the final concentration). To treat the HEK-Blue-IL-18 cells, mix 50 μL of the prepared uncut or cut FUSE proteins with 50 μL of the prepared IL-18BP, incubate for 1 hour, and then add to the designated wells in the 96-well plate containing 50,000 cells, and then gently mix the cells with the proteins. Then incubate the 96-well plate at 37 °C, 5% CO 2 conditions for 24 hours. Since the binding of our fusion proteins to the IL-18R complex is weakened, we believe that they will also show reduced binding to IL-18BP compared to the cut FUSE proteins, as both IL-18R and IL-8BP compete for binding to IL-18. Recent crystal structures of the binary and ternary complexes of hIL-18 with its receptor have shown that IL-18BP directly competes with the hIL-18Rα D3 domain for binding to hIL-18, overlapping with the previously identified hIL-18 binding site II (Krumm et al., Acta Crystallogr F Struct Biol Commun. 2015 Jun 1;71(Pt 6):710–717).

[0194] Twenty μL of the HEK-Blue-IL-18 cell culture supernatant containing secreted alkaline phosphatase was transferred to a new 96-well plate 24 hours after activation. Meanwhile, a QUANTI-Blue solution was prepared by adding 1 mL of QB reagent and 1 mL of QB buffer to 98 mL of sterile water. Then, 180 μL of the prepared QUANTI-Blue solution was added to the 96-well plate containing 20 μL of the HEK-Blue-IL-18 cell culture supernatant and incubated at 37 °C for 4 hours. The level of secreted alkaline phosphatase associated with the activation of HEK-Blue-IL-18 cells was detected by measuring the OD at 630 nm using a spectrophotometer. Binding assay by Biolayer Interferometry

[0195] The coating protein was prepared in 1×PBS containing 0.02% Tween-20 at a final concentration of 15 μg / mL. The capture protein was also prepared and serially diluted (4-fold dilution, ranging from 400 nM to 1.6 nM) in 1×PBS containing 0.02% Tween-20. The biosensor was pre-wetted in 200 μL of 1×PBS containing 0.02% Tween-20 for 10 minutes. Meanwhile, the Octet BLI system (ForteBio) was preheated for 30 minutes and the flow rate was set to 1000 rpm. The biosensor (capture biosensor) was immersed in 250 μL of 1×PBS containing 0.02% Tween-20 at 30 °C for 60 seconds to obtain an initial baseline reading. After the 60-second baseline reading, the biosensor was exposed to the coating protein at 30 °C for 300 seconds for the association between the antibody and the biosensor (coupling the coating protein to the biosensor). Then, the biosensor with the coating protein was exposed to the capture protein in 250 μL of 1×PBS containing 0.02% Tween-20 at 30 °C for 300 seconds for the association reaction between the coating protein and the capture protein (association curve). After the 300-second association reaction between the coating protein and the capture protein, the biosensor with the coating protein and the capture protein was exposed in 250 μL of 1×PBS containing 0.02% Tween-20 at 30 °C for 300 seconds for the dissociation reaction between the coating protein and the capture protein (dissociation curve). For binding to IL18BP, each IL18 mutant was coated onto the AHC biosensor tip and probed with recombinant His-tagged IL-18BP at concentrations ranging from 400 nM to 1.6 nM. For binding to IL18Rα, His-tagged IL18Rα was coated into the nickel biosensor tip and probed with each recombinant IL18 mutant at concentrations ranging from 400 nM to 1.6 nM. The binding affinity was calculated by the built-in data fitting algorithm. Example 4

[0196] As shown in Figure 12, we engineered 11 mutants of human IL-18 (IL-18AV as shown) and measured their ability to bind recombinant human IL-18BP and recombinant human IL-18RA (also referred to as IL-18Rα). All test substances were generated as Fc fusion proteins with the IL-18 variant fused to the N-terminus of Fc (see Figure 11A ). Figure 12A The positions and amino acid substitutions associated with each variant are described in

[0197] To assess binding to human IL18BP or human IL18Rα, kinetic binding profiles were generated by biolayer interferometry (BLI) using the Octet system (ForteBio). For binding to IL18BP, each IL18 mutant was coated onto an AHC biosensor tip and probed with recombinant His-tagged IL-18BP at concentrations ranging from 400 nM to 1.6 nM (Figure 12B). For binding to IL18Rα, His-tagged IL18Rα was coated into a nickel biosensor tip and probed with each recombinant IL18 mutant at concentrations ranging from 400 nM to 1.6 nM (Figure 12D). Summary tables of the binding to IL-18BP and IL18-RA (binding affinity (KD), association rate (k-on), and dissociation rate (k-dis)) are shown in Figure 12C and Figure 12E respectively.

[0198] All 11 IL-18 mutants bound to IL18BP with weaker affinity compared to FUSE500 (wild-type human IL-18-AV-Fc). Five mutants were associated with no significant binding, namely FUSE545, FUSE599, FUSE600, FUSE601, and FUSE602.

[0199] Conversely, all 11 mutants maintained significant binding to IL18RA. The affinity ranged from 11 nM to 30 nM, only 1.5 to 4 times weaker than the affinity of FUSE500 for IL-18RA (7.4 nM). Example 5

[0200] As shown in the table below, we tabulated the binding affinities of 11 mutant variants of human IL-18 (IL-18AV as shown) for human IL18BP and human IL18Rα. Each IL-18 protein was generated as an Fc fusion protein by fusing the IL-18 variant to the N-terminus of Fc (see Figure 11A ). *Mean of 11 measurements, **Mean of 9 measurements ***www.pnas.org / doi / 10.1073 / pnas.97.3.1190 ****arthritis-research.biomedcentral.com / articles / 10.1186 / ar3295 Example 6

[0201] As Figure 13 shown, we examined the effect of the size of the polypeptide fused to the N-terminus of mature IL-18 on the biological activity of a single IL-18AV fused to the N-terminus of IgG1 Fc.

[0202] The single N-terminal amino acid (FUSE874; downward open triangle), as well as polypeptides of different sizes ranging from five amino acids (FUSE875) to the FUSE-499 propeptide variant (upward solid triangle; description see Figure 11), were studied. FUSE500 was used as a fully active control without an N-terminal polypeptide.

[0203] To allow all constructs to be transported into the endoplasmic reticulum (ER) of mammalian cells and thus expressed / secreted, a signal peptide (IgK leader sequence) from the Igκ chain was encoded upstream of the polypeptide (and cleaved off by signal peptidase in the ER).

[0204] The polypeptides of FUSE875 (5 residues; solid star), FUSE876 (10 residues; open diamond), FUSE757 (15 residues; open circle), FUSE756 (35 residues; downward solid triangle) consist of a series of glycine and serine residues. The N-terminal polypeptide associated with FUSE758 (upward open triangle) is the protruding end sequence generated by MMP2 cleavage of FUSE486 (see Figure 9). For FUSE756, FUSE757, and FUSE758, the last four amino acids of the N-terminal peptide consist of the granzyme B cleavage site IEQD (SEQ ID NO: 88) (see Figure 11).

[0205] Using the HEK-Blue IL-18 reporter cell assay as a readout, we examined the ability of polypeptides of different sizes to attenuate / mask IL-18AV-Fc (FUSE500; solid square) or rhIL-18 (black cross "X"). All polypeptides and single amino acids (serine) fused to the N-terminus of mature IL-18AV reduced the biological activity measured as EC50-SEAP by at least 10-fold.

[0206] As the polypeptide size increases, the degree of attenuation increases, such that the order of attenuation is observed as FUSE499 > FUSE756, FUSE757, FUSE758 > FUSE876, FUSE875 > FUSE874. A summary table of potency (EC50 - SEAP) is shown below the non-linear x-y graph. Example 7

[0207] As Figures 14A - 14H shown, using the HEK Blue IL18 assay system, we examined the effect of substituting cysteine residues in the propeptide and in mature IL18 (fused together to form the pro-IL-18 variant cassette) on the biological activity of each variant. Unless otherwise stated, proteins were generated such that the propeptide-IL-18 variant was fused to the pestle or mortar of the C-terminus of human IgG1 Fc with a pestle and mortar structure as described previously Figure 1A and a granzyme B cleavage site was added between the propeptide variant and the mature IL18 variant. The three variants evaluated in Figure 1 contained serine substitutions (FUSE480), alanine substitutions (FUSE481), or valine substitutions (FUSE442) at all cysteine residues of pro-IL-18. The activity of the mature IL-18 variants released from FUSE442 and FUSE481 was comparable to that of recombinant human IL-18, while the mature IL18 released from FUSE480 (serine-substituted) was attenuated by approximately 100-fold compared to recombinant human IL-18 (see Figures 1B - 1D ).

[0208] For this example, the variant of pro-IL-18 in which all cysteines were replaced with valine contained an N-terminal EGFR-specific VHH (FUSE516; solid triangles), and the variant of pro-IL-18 in which all cysteines were replaced with serine contained an N-terminal PD-1-specific Fab (FUSE694; solid diamonds). As in Figure 1, the biological activity of the pro-IL18 test articles was evaluated using HEK Blue IL18. Each was tested as a full, untreated protein, or after exposure to recombinant human granzyme B.

[0209] Figure 14A The results of FUSE516 (solid triangles) are shown, in which the cysteine residues in its propeptide variant were replaced with valine and all cysteines in its mature IL18 variant were replaced with valine. Intact FUSE516 was attenuated by approximately 1000-fold compared to recombinant human IL18, and the activity of the mature IL18 variant (open triangles) released from FUSE516 by granzyme B was comparable to that of recombinant human IL18.

[0210] Figure 14BThe results of FUSE694 (solid rhombus) are shown, in which the cysteine residues in its propeptide variant are replaced with serine, and all cysteines in its mature IL18 variant are replaced with serine. The full-length FUSE694 is attenuated by more than 10,000-fold relative to recombinant human IL18, and the activity of the mature IL18 variant (open rhombus) released from FUSE694 by granzyme B is approximately 60-fold lower than that of recombinant human IL18.

[0211] Figure 14C The results of FUSE887 (solid inverted triangle) are shown, in which the cysteine residues in its propeptide variant are replaced with threonine, and all cysteines in its mature IL18 variant are replaced with serine. The full-length FUSE887 is attenuated by more than 10,000-fold relative to recombinant human IL18, and the activity of the mature IL18 variant (open inverted triangle) released from FUSE887 by granzyme B is approximately 100-fold lower than that of recombinant human IL18.

[0212] Figure 14D The results of FUSE888 (solid triangle) are shown, in which the cysteine residues in its propeptide variant are replaced with glutamine, and all cysteines in its mature IL18 variant are replaced with serine. The full-length FUSE888 is attenuated by more than 10,000-fold relative to recombinant human IL18, and the activity of the mature IL18 variant (open triangle) released from FUSE888 by granzyme B is approximately 100-fold lower than that of recombinant human IL18.

[0213] Figure 14E The results of FUSE889 (solid square) are shown, in which the cysteine residues in its propeptide variant are replaced with aspartic acid, and all cysteines in its mature IL18 variant are replaced with alanine. The full-length FUSE889 is attenuated by more than approximately 10,000-fold relative to recombinant human IL18, and the activity of the mature IL18 variant (open square) released from FUSE889 by granzyme B is approximately 100-fold lower than that of recombinant human IL18.

[0214] Figure 14F The results of FUSE890 (solid inverted triangle) are shown, in which the cysteine residues in its propeptide variant are replaced with phenylalanine, and all cysteines in its mature IL18 variant are replaced with alanine. The full-length FUSE890 is attenuated by approximately 10,000-fold relative to recombinant human IL18, and the activity of the mature IL18 variant (open inverted triangle) released from FUSE890 by granzyme B is approximately 100-fold lower than that of recombinant human IL18.

[0215] Figure 14GThe results of FUSE891 (solid triangles) are shown, in which the cysteine residues in its propeptide variant are replaced with isoleucine, and all cysteines in its mature IL18 variant are replaced with valine. The full-length FUSE891 is attenuated by approximately 3000-fold relative to recombinant human IL18, and the activity of the mature IL18 variant (open triangles) released from FUSE891 by granzyme B is approximately 100-fold lower than that of recombinant human IL18.

[0216] Figure 14H The results of FUSE892 (solid inverted triangles) are shown, in which the cysteine residues in its propeptide variant are replaced with histidine, and all cysteines in its mature IL18 variant are replaced with valine. The full-length FUSE892 is attenuated by more than approximately 3000-fold relative to recombinant human IL18, and the activity of the mature IL18 variant (open inverted triangles) released from FUSE892 by granzyme B is approximately 100-fold lower than that of recombinant human IL18.

[0217] The summary representation of the potency (EC50-SEAP) is presented on the right side of each non-linear x-y plot. Example 8

[0218] As Figures 15A - 15BAs shown, we examined the effect of targeting pro-IL-18 near its receptor complex (i.e., "cis-activity"). The pro-IL-18 variants tested were: (a) variants in which all cysteine residues were replaced with serine (pro-IL18AS, FUSE782, and FUSE827); and (b) variants in which all cysteines were replaced with valine (pro-IL18AV, FUSE783, and FUSE785). Pro-IL18AS or pro-IL18AV was fused to the N-terminal pestle or mortar of a PD1-specific mortise and tenon structure antibody (FUSE782 and FUSE783). The fabs of these fusion proteins were derived from nivolumab. To test whether targeting pro-IL-18 variants to PD1 modified on the same cells expressing the IL-18R complex (cis-effect) enhances biological activity compared to cells not modified with PD-1 (trans-effect), (1) we used HEK Blue IL18 as our IL18R complex positive reporter system; and (2) either unmodified cells or a bispecific antibody was used to modify the cell line with the extracellular domain of PD-1, which bispecific antibody contained a CD46-specific fab (clone YS5) at the N-terminal mortar and the extracellular domain of PD-1 (FUSE986) at the N-terminal pestle. CD46 was chosen because of its reported expression in the parental HEK293 cell line (jitc.bmj.com / content / 6 / 1 / 55), which we confirmed on HEK Blue IL18 cells (not shown). We also generated pro-IL18AS (FUSE827) and pro-IL18AV (FUSE775), which were functionally non-targeting in this regard and could only act in trans. FUSE827 contained a non-targeting domain (i.e., only Fc), and FUSE775 replaced the PD-1-specific fab with an EGFR-specific VHH (9G8), for which the ligand of (EGFR) was not expressed on HEK Blue IL18 (data not shown). Figure 15A and Figure 15B was a non-linear x-y plot of the concentration of each test substance using HEK Blue IL18 ( Figure 15A ) and PD-1-modified HEK Blue IL18 ( Figure 15B ) versus IL18 biological activity. As previously observed for pro-IL18AS and pro-IL18AV, serine substitution produced a greater attenuation than valine substitution. Thus, when HEK Blue IL18 ( Figure 15A; when only trans-active), we observed a ~1000-fold attenuation relative to recombinant human IL18; and when treated with (b) serine mutants FUSE782 (open triangles) and FUSE827 (solid inverted triangles), we observed a >100,000-fold attenuation relative to recombinant human IL18.

[0219] Regarding cis-activity ( Figure 15B , using PD-1-modified HEK Blue IL18), we observed increased biological activity of the PD-1-targeted versions of pro-IL18AS (FUSE782; ~100-fold relative to non-targeted FUSE827) and pro-IL18AV (FUSE783; ~30-fold relative to non-targeted FUSE775).

[0220] FUSE691 (nivolumab) was used as a negative control antibody in Figure 15A and Figure 15B No biological activity was observed from this test article. No significant difference was observed in the biological activity of recombinant human IL18 or non-targeted test articles (FUSE775 and FUSE827), allowing comparison between Figure 15A and Figure 15B In this context, the PD-1-targeted versions of pro-IL18AS (FUSE782) and pro-IL18AV (FUSE783) were ~100-fold and 30-fold more active when exposed to PD-1-modified HEK Blue IL-18 compared to unmodified HEK Blue L18.

[0221] A summary table of the potency of each pro-IL-18 variant is shown below each figure.

[0222] The various embodiments of the present invention are described above in the detailed description. Although these descriptions directly set forth the above embodiments, it will be understood that those skilled in the art may conceive of modifications and / or alterations to the specific embodiments shown and described herein. Any such modifications or alterations falling within the scope of this specification are equally intended to be included therein. Unless otherwise specified, the inventors intend for the words and phrases in the specification and claims to be given the ordinary and accustomed meaning as understood by those of ordinary skill in the art to which the invention pertains.

[0223] The foregoing description of the various embodiments of the invention known to the applicant at the time of filing this application has been given and is intended for purposes of illustration and description. This description is not intended to be exhaustive nor to limit the invention to the precise forms disclosed, and many modifications and variations are possible in light of the above teachings. The described embodiments are used to explain the principles of the invention and its practical application and to enable others skilled in the art to utilize the invention in various embodiments with various modifications as are suited to the particular use contemplated. Accordingly, the invention is not intended to be limited to the particular embodiments disclosed for carrying out the invention.

[0224] Although particular embodiments of the invention have been shown and described, it will be apparent to those skilled in the art that, based on the teachings herein, changes and modifications can be made without departing from the invention and its broader aspects, and accordingly, the appended claims are intended to cover all such changes and modifications that fall within the true spirit and scope of the invention. As used herein, the term "comprising / including / containing" is used to refer to a composition, method, and corresponding components useful in a particular embodiment, but also admits the inclusion of unspecified elements, whether useful or not. Those skilled in the art will understand that, generally, the terms used herein should be understood as "open-ended" terms (e.g., the term "including" should be interpreted as "including but not limited to", the term "having" should be interpreted as "having at least", the term "containing" should be interpreted as "containing but not limited to", etc.). Although the open-ended term "comprising" is used as a synonym for terms such as "including", "containing", or "having" to describe and claim the invention, the invention or embodiments thereof may alternatively be described using other terms such as "consisting of" or "consisting essentially of".

[0225] Unless otherwise indicated, the terms "a / an" and "the" and similar referents used in the context of describing particular embodiments of the present application (especially in the context of the claims) may be construed to cover both the singular and the plural. The recitation of a range of values herein is merely intended to serve as a convenient method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each separate value is incorporated into the specification as if it were individually recited herein. Unless otherwise stated herein or clearly contradicted by context, all methods described herein can be performed in any suitable order. The use of any and all examples or exemplary language (e.g., "such as") provided herein with respect to certain embodiments is merely intended to better illuminate the present application and does not pose a limitation on the scope of the present application that is otherwise claimed. The abbreviation "e.g." is derived from the Latin exempli gratia and is used herein to denote a non-limiting example. Thus, the abbreviation "e.g." is synonymous with the term "for example". Any language in the specification should not be construed as indicating any non-claimed element essential to the practice of the present application. "Optional" or "optionally" means that the subsequently described circumstance may or may not occur, and thus the description includes both the case where the circumstance occurs and the case where the circumstance does not occur. The grouping of alternative elements or embodiments of the present disclosure disclosed herein should not be construed as limiting. Each member of the group can be individually or in any combination with other members of the group or other elements found herein be mentioned and claimed. For reasons of convenience and / or patentability, one or more members of the group may be included in or deleted from the group. When any such inclusion or deletion occurs, the present specification is hereby considered to include the modified group, thereby satisfying the written description of all Markush groups used in the appended claims.

Claims

1. A fusion protein, the fusion protein comprising: A first polypeptide or protein capable of being transported into the endoplasmic reticulum (ER), or a fragment thereof; and Interleukin-18 (IL-18), a fragment of IL-18, an IL-18 variant or a fragment of an IL-18 variant, wherein, The IL-18, the fragment of IL-18, the IL-18 variant or the fragment of the IL-18 variant is located at the C-terminus of the fusion protein relative to the first polypeptide or protein capable of being transported into the ER.

2. The fusion protein according to claim 1, wherein, The IL-18 variant has an amino acid sequence comprising amino acids at positions 37-193 of SEQ ID NO: 250, and has one to five amino acid substitutions at positions E42, M87, K89, M96 and M149 of SEQ ID NO:

250.

3. The fusion protein according to claim 1, wherein, The IL-18 variant has an amino acid sequence comprising amino acids at positions 37-193 of SEQ ID NO: 251, and has one to five amino acid substitutions at positions E42, M87, K89, M96 and M149 of SEQ ID NO: 251 and one or more amino acid substitutions at positions C74, C104, C112 and C164.

4. The fusion protein according to claim 3, wherein, Each of the one or more amino acid substitutions at C74, C104, C112 and C164 is independently valine, alanine or serine.

5. The fusion protein according to claim 1, wherein, The IL-18 variant has an amino acid sequence comprising amino acids at positions 37-193 of SEQ ID NO: 251, and has one to five amino acid substitutions at positions E42, M87, K89, M96 and M149 of SEQ ID NO:

251.

6. The fusion protein according to any one of claims 2-5, wherein, The one to five amino acid substitutions are one or more of the following: E42K, E42R, E42A, E42H or E42Q; M87K or M87H; K89G, K89A or K89E; M96L or M96I; or M149V or M149I.

7. The fusion protein according to any one of claims 2-5, wherein, The one to five amino acid substitutions are: E42K, E42R, E42A, E42H or E42Q; M87K or M87H; K89G, K89A or K89E; M96L or M96I; and M149V or M149I.

8. The fusion protein according to any one of claims 1-7, wherein, The IL-18, the fragment of IL-18, the IL-18 variant or the fragment of the IL-18 variant further comprises its propeptide (PP) or a PP variant.

9. The fusion protein according to claim 8, wherein, The IL-18 propeptide variant comprises a polypeptide having AAEPVEDNX 1 INFVAMKFIDNTLYFIAEDDEN (SEQ ID NO: 238), wherein X 1 is any amino acid other than cysteine.

10. The fusion protein according to claim 9, wherein, X 1 is alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine or tryptophan (SEQ ID NO: 239).

11. The fusion protein according to any one of claims 8-10, wherein, The fusion protein does not contain a polypeptide consisting of sequences X 1 -X 2 -X 3 -X 4 between the propeptide or propeptide variant and the mature IL-18 or mature IL-18 variant, where X 1 is L or does not exist, X 2 is E or does not exist, X 3 is S or does not exist, and X 4 is D or does not exist, and when X 1 , X 2 , X 3 and X 4 exist, the polypeptide composed of the sequence X 1 -X 2 -X 3 -X 4 is LESD (SEQ ID NO: 253).

12. The fusion protein according to any one of claims 9-11, wherein, the PP or the PP variant is located at the N-terminus relative to the IL-18, the fragment of IL-18, the IL-18 variant or the fragment of the IL-18 variant.

13. The fusion protein according to any one of claims 9-11, wherein, the PP or the PP variant serves as a masking domain.

14. The fusion protein according to any one of claims 1-13, wherein the fusion protein further comprises one or more protease cleavage sites.

15. The fusion protein according to claim 14, wherein, the one or more protease cleavage sites: between the IL-18, the fragment of IL-18, the IL-18 variant or the fragment of the IL-18 variant and the first protein or its fragment capable of being transported into the endoplasmic reticulum (ER), or within the PP, between the PP or the PP variant and the IL-18, the fragment of IL-18, the IL-18 variant or the fragment of the IL-18 variant, or within the PP, between the PP or the PP variant and the first protein or its fragment capable of being transported into the endoplasmic reticulum (ER), or within the IL-18, the fragment of IL-18, the IL-18 variant or the fragment of the IL-18 variant, or within the PP, or a combination thereof.

16. The fusion protein according to any one of claims 1-15, wherein the fusion protein further comprises a second protein or its fragment capable of being transported into the ER, wherein, the second protein capable of being transported into the ER is located at the C-terminus relative to interleukin 18 (IL-18), the fragment of IL-18, the IL-18 variant or the fragment of the IL-18 variant.

17. The fusion protein according to claim 16, wherein the fusion protein further comprises a protease cleavage site between the IL-18, the fragment of IL-18, the IL-18 variant or the fragment of the IL-18 variant and the second protein or its fragment capable of being transported into the endoplasmic reticulum (ER).

18. The fusion protein according to any one of claims 1-17, wherein, the interleukin 18 (IL-18), the fragment of IL-18, the IL-18 variant or the fragment of the IL-18 variant is fused to the C-terminus of the first protein capable of being transported into the ER.

19. The fusion protein according to any one of claims 16-18, wherein, the second protein or its fragment capable of being transported into the ER is fused to the C-terminus of the interleukin 18 (IL-18), the fragment of IL-18, the IL-18 variant or the fragment of the IL-18 variant.

20. The fusion protein according to any one of claims 1-19, wherein, compared with wild-type (wt) IL-18, the IL-18 variant has a reduced binding to the IL-18 binding protein (IL-18BP).

21. The fusion protein according to any one of claims 1-19, wherein, The binding affinity of the IL-18 variant for the human IL-18 receptor (IL-18R) is within 30-fold that of wild-type IL-18.

22. The fusion protein according to any one of claims 1-19, wherein, the binding affinity of the fusion protein comprising the IL-18 variant for IL-18BP: the binding affinity of the fusion protein comprising the IL-18 variant for IL-18R is not higher than 3:

1.

23. The fusion protein according to any one of claims 1-22, wherein, the first protein or fragment thereof capable of being transported into the ER is a globular protein, an immunoglobulin, or a fragment thereof, or a short polypeptide or protein engineered with a signal peptide for transport into the ER, optionally, the short polypeptide or protein is about 2 kDa or not greater than 250 kDa.

24. The fusion protein according to any one of claims 1-23, wherein, the first protein or fragment thereof capable of being transported into the ER is selected from the group consisting of: the crystallizable fragment (Fc) region, human serum albumin (HSA), β2-microglobulin, transferrin, the antigen-binding fragment region (Fab region), VHH antibody, single-chain variable fragment (scFV), anticalin, designed ankyrin repeat protein (DARPin), its binding domain, and fragments thereof.

25. The fusion protein according to any one of claims 1-23, wherein, the first protein or fragment thereof capable of being transported into the ER of the fusion protein is a type I transmembrane protein or a fragment thereof, or a type II transmembrane protein or a fragment thereof.

26. The fusion protein according to any one of claims 16-25, wherein, the second protein or fragment thereof capable of being transported into the ER is a globular protein, an immunoglobulin, or a fragment thereof, or a short polypeptide or protein engineered with a signal peptide for transport into the ER, optionally, the short polypeptide or protein is about 2 kDa or not greater than 250 kDa.

27. The fusion protein according to any one of claims 16-26, wherein, the second protein or fragment thereof capable of being transported into the ER is selected from the group consisting of: the crystallizable fragment (Fc) region, human serum albumin (HSA), β2-microglobulin, transferrin, the antigen-binding fragment region (Fab region), VHH antibody, single-chain variable fragment (scFV), anticalin, designed ankyrin repeat protein (DARPin), its binding domain, and fragments thereof.

28. The fusion protein according to any one of claims 16-26, wherein, the second protein or fragment thereof capable of being transported into the ER of the fusion protein is a type I transmembrane protein or a fragment thereof, or a type II transmembrane protein or a fragment thereof.

29. The fusion protein according to any one of claims 24 and 27, wherein, the Fc region is the Fc region from IgA, IgM, IgG, or IgE.

30. The fusion protein according to any one of claims 24 and 27, wherein, The Fc region is the Fc region derived from IgG4, KiH or IgG1.

31. The fusion protein according to any one of claims 24 and 27, wherein, the Fc region is the Fc region derived from a knobs-into-holes structure, HA-TF, Xmab, ZW1, 7.8.60, electrostatic steering, DD-KK, EW-RVT, A107 or Duobody.

32. The fusion protein according to any one of claims 1-31, wherein, one or more cysteines in the fusion protein are modified.

33. The fusion protein according to any one of claims 1-31, wherein, one or more cysteines in the fusion protein are replaced with natural or non-natural amino acids.

34. The fusion protein according to any one of claims 1-31, wherein, one or more cysteines in the IL-18, fragment of IL-18, IL-18 variant or fragment of IL-18 variant of the fusion protein are modified or replaced with natural or non-natural amino acids.

35. The fusion protein according to any one of claims 8-34, wherein, one or more cysteines in the PP or PP variant of the fusion protein are modified or replaced with natural or non-natural amino acids.

36. The fusion protein according to any one of claims 33-35, wherein, the natural amino acids are each independently selected from serine and valine.

37. The fusion protein according to any one of claims 33-35, wherein, the natural amino acids are each independently selected from threonine, asparagine and glutamine.

38. The fusion protein according to any one of claims 17-37, wherein, the protease is selected from the group consisting of: EK, TEV, Adam17, cathepsin, MMP2, MMP9, MMP14, granzyme A, granzyme B, granzyme M, granzyme K and combinations thereof.

39. The fusion protein according to any one of claims 1-38, the fusion protein has one or more sequences listed in any one of Table 1 and Table 4.

40. The fusion protein according to claim 1, the fusion protein has polypeptide 1 and polypeptide 2 selected from Table 1 and optionally polypeptide 3.

41. The fusion protein according to claim 1, the fusion protein has polypeptide 1 and polypeptide 2 selected from Table 1 and optionally polypeptide 3, wherein, polypeptide 1, polypeptide 2 and optionally polypeptide 3 are selected from the same row of Table 1.

42. The fusion protein according to claim 1, the fusion protein has polypeptide 1 selected from Table 1, wherein, polypeptide 1 contains HSA.

43. The fusion protein according to any one of claims 1-42, wherein, the IL-18 variant has an amino acid sequence selected from the mature IL-18 column in Table 1.

44. The fusion protein according to any one of claims 8-42, wherein, The IL-18 variant has an amino acid sequence selected from the mature IL-18 column in Table 1, and the propeptide has an amino acid sequence selected from the propeptide column in Table 1, optionally from the same row.

45. An IL-18 propeptide variant, said variant comprising a polypeptide having AAEPVEDNX 1 INFVAMKFIDNT LYFIAEDDEN, Wherein, X 1 is any amino acid other than cysteine (SEQ ID NO: 238).

46. A polynucleotide encoding the fusion protein according to any one of claims 1-44 or the IL-18 propeptide variant according to claim 45.

47. The polynucleotide according to claim 46, wherein, the first protein capable of being transported into the ER is encoded by a polynucleotide having one or more sequences shown in Table 2.

48. The polynucleotide according to claim 46, the polynucleotide having one or more sequences from the same row shown in Table 2.

49. An expression vector comprising the polynucleotide according to any one of claims 46-48.

50. A cell transfected with the expression vector according to claim 49.

51. A method for producing a fusion protein, the method comprising: culturing the cells transfected with the expression vector according to claim 49 in a cell culture medium to allow the fusion protein to be secreted into the cell culture medium.

52. The method according to claim 51, wherein, under transient transfection of CHO cells or HEK-293 cells, the fusion protein is produced at a level greater than 135 mg / L.

53. A method for producing interleukin (IL-18), a fragment thereof, an IL-18 variant or a fragment of an IL-18 variant, the method comprising: culturing the cells transfected with the expression vector according to claim 49 in a cell culture medium to allow the fusion protein to be produced and secreted into the extracellular space; and contacting the fusion protein with a protease to cleave the fusion protein, thereby producing the IL-18, a fragment thereof, an IL-18 variant or a fragment of an IL-18 variant.

54. The method according to claim 53, the method further comprising separating the fusion protein from the culture medium.

55. The method according to claim 54, the method further comprising purifying the fusion protein.

56. The method according to claim 53, wherein, the protease is selected from the group consisting of: EK, TEV, Adam17, cathepsin, MMP2, MMP9, MMP14, granzyme A, granzyme B, granzyme M, and combinations thereof.

57. The method according to any one of claims 53-56, wherein, the IL-18, a fragment thereof, an IL-18 variant or a fragment of an IL-18 variant is produced at a level greater than 135 mg / L.

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