IL-18 fusion proteins and methods for producing IL-18

Abstract

Fusion proteins are provided that are composed of one or more proteins / polypeptides capable of translocating into the endoplasmic reticulum (ER) and elements such as interleukin-18 (IL-18) or a fragment or variant of IL-18. Proteins / polypeptides capable of translocating into the ER include, but are not limited to, immunoglobular proteins such as Fc regions, VHH antibodies, and scFvs, as well as globular proteins such as serum albumin. The IL-18 may be precursor IL-18, which resembles a natural procytokine in which IL-18 is fused to the C-terminus of its propeptide or propeptide variant. In some embodiments, the fusion protein may also include a cleavable peptide linker.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 63 / 395,476, filed August 5, 2022, and U.S. Provisional Patent Application No. 63 / 463,505, filed May 2, 2023, both of which are incorporated by reference herein in their entireties.

[0002] Sequence Listing Reference This application contains a Sequence Listing that has been submitted in a computer readable form entitled "096034_000002WOPT_SequenceListing.xml," having a byte size of 366,973 bytes, created on August 3, 2023 (date of creation). The information contained in this computer readable form is incorporated herein by reference in its entirety.

[0003] FIELD OF THE INVENTION The present invention relates to novel compositions that allow for enhancing the production (including expression, secretion, and purification) of recombinantly produced IL-18 cytokine, for example in mammalian cells, masking and demasking the biological activity of IL-18, and / or improving the binding specificity of produced IL-18 to its receptor for its binding protein, while maintaining at least the binding affinity to its receptor, as well as methods for preparing said compositions. [Background technology]

[0004] background Members of the interleukin (IL)-1 cytokine family have established roles in host defense responses and inflammatory responses that contribute to disease. IL-18, a member of the IL-1 superfamily, is a pro-inflammatory cytokine that promotes type 1 immune responses. IL-18, also known as interferon gamma-inducing factor, is encoded in humans by the IL-18 gene. Like other IL-1 family members, the IL-18 gene lacks a signal peptide. The IL-18 gene encodes a 193-amino acid precursor protein, which is initially synthesized as an inactive 24 kDa precursor lacking the signal peptide, and which is cytosolic and accumulates in the cytoplasm. WO97 / 24441 (Patent Document 1) discloses a DNA-encoded 193-amino acid protein corresponding to the IL-18 precursor. The IL-18 precursor (also called pro-IL-18) is processed intracellularly (e.g., by caspase 1 (CASP1), chymase, and proteinase B) to its mature, biologically active molecule of 18 kDa (157 amino acids, i.e., amino acid residues 37-193 of Uniprot ID Q14116). Upon cleavage, the propeptide separates from the rest of the precursor, resulting in mature IL-18 and the propeptide that previously inactivated precursor IL-18.

[0005] Without intending to be bound by any particular theory, the mature form of IL-18 is secreted and released into the extracellular environment via at least three non-conventional pathways. This contrasts with conventional secretion, which is signal peptide-dependent and ER-Golgi transport-mediated. The non-conventional pathways listed below are not listed in any particular order of frequency. The first is called secretory autophagy, a process involved in the secretion of cytosolic proteins that do not contain a signal peptide (leaderless cargo). Here, IL-18 interacts with the cargo receptor transmembrane emp24 domain-containing protein 10 (TMED10). This interaction mediates its transport from the cytoplasm to the endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC), which provides membrane for forming autophagosomes and acts as a mechanism for the secretory cargo to enter the vesicles and thereby be secreted. The second and third reported mechanisms of IL-18 release from cells involve the rupture of dying cells that have undergone apoptosis and / or gasdermin D-dependent cell membrane permeabilization (see Tapia et al., IMMUNOLOGY, volume 294, issue 21, p8325-8335, 2019).

[0006] Mature IL-18 binds to its ligand receptor, IL-18 receptor alpha (IL-18Rα), inducing the recruitment of IL-18Rβ (also called IL-18 receptor accessory protein (IL-18RAP)) to form a high-affinity complex (approximately 18 nM; see Torigoe et al., Membranes and Bioenergetics, vol. 272, issue 41, pp. 25737-25742, 1997), which signals through the Toll / interleukin-1 receptor (TIR) domain. This signaling domain recruits the MyD88 adaptor protein, which activates the proinflammatory program and the NF-κB pathway. The activity of IL-18 can be inhibited by extracellular interleukin-18 binding protein (IL-18BP), which binds to soluble IL-18 with higher affinity than IL-18Rα (approximately 0.4 pM, see Kim et al., Proc Natl Acad Sci USA. 2000;97(3):1190-1195), thus preventing IL-18 binding to the IL-18 receptor. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] WO97 / 24441 [Non-patent literature]

[0008] [Non-Patent Document 1] Tapia et al., IMMUNOLOGY, volume 294, issue 21, p8325-8335, 2019 [Non-patent document 2] Torigoe et al., Membranes and Bioenergetics, vol.272, issue 41, pp25737-25742, 1997 [Non-patent document 3] Kim et al.,Proc Natl Acad Sci USA.2000;97(3):1190-1195 Summary of the Invention

[0009] It is therefore an object of the present invention to provide compositions of matter that allow for improved production (including expression, secretion, and purification) of recombinantly produced IL-18.

[0010] Another object of the present invention is to provide compositions of matter that allow for enhanced production of recombinantly produced IL-18, including modified IL-18 or fragments thereof, that maintain similar binding affinity for IL-18Ra / b compared to native IL-18 and have reduced binding affinity for IL-18BP compared to native IL-18 (i.e., binds to both at about 18 nM); or more preferably, produced IL-18 (or fragments thereof) that has weaker binding affinity for IL-18BP than for IL-18Ra / b (i.e., binding to IL-18BP is less than 18 nM and binding to IL-18Ra / b is about 18 nM); as well as methods for preparing these compositions.

[0011] Another object of the present invention is to provide variants of IL-18 (or fragments thereof).

[0012] Another object of the present invention is to provide compositions that allow for masked activity in normal tissues and circulation and specific release of IL-18 (or fragments thereof) by proteases in the tumor microenvironment.

[0013] All publications herein are 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 contains information that may be useful in understanding the present invention. No admission is made that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

[0014] Summary of the Invention The following embodiments and aspects thereof are described and illustrated in conjunction with compositions and methods that are intended to be exemplary and illustrative, not limiting in scope.

[0015] Various embodiments include: a fusion protein comprising a first polypeptide or protein or fragment thereof capable of translocating 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 C-terminal to the first polypeptide or protein capable of translocating into the ER. to provide.

[0016] In various embodiments, an IL-18 variant may have an amino acid sequence comprising amino acid positions 37-193 of SEQ ID NO:250, with one to five amino acid substitutions at positions E42, M87, K89, M96, and M149 of SEQ ID NO:250.

[0017] In various embodiments, an IL-18 variant may have an amino acid sequence comprising positions 37-193 of SEQ ID NO:251, with one to five amino acid substitutions at positions E42, M87, K89, M96, and M149 of SEQ ID NO:251.

[0018] In various embodiments, an IL-18 variant may have an amino acid sequence comprising positions 37-193 of SEQ ID NO: 251, with one or more amino acid substitutions at positions C74, C104, C112, and C164 of SEQ ID NO: 251, and one to five amino acid substitutions at positions E42, M87, K89, M96, and M149.

[0019] In various embodiments, the amino acid substitutions at one or more of C74, C104, C112, and C164 can each independently be valine, alanine, or serine.

[0020] In various embodiments, the one to five amino acid substitutions can be one or more of E42K, E42R, E42A, E42H, or E42Q; M87K, or M87H; K89G, K89A, or K89E; M96L, or M96I; or M149V or M149I.

[0021] In various embodiments, the one to five amino acid substitutions can be E42K, E42R, E42A, E42H, or E42Q; M87K or M87H; K89G, K89A, or K89E; M96L or M96I, and M149V or M149I.

[0022] In various embodiments, the IL-18, fragment of IL-18, IL-18 variant, or fragment of an IL-18 variant further comprises a propeptide (PP) or PP variant thereof.

[0023] In various embodiments, the IL-18 propeptide variant comprises a polypeptide having AAEPVEDNX1INFVAMKFIDNTLYFIAEDDEN (SEQ ID NO: 238), where X1 can be any amino acid except cysteine.

[0024] In various embodiments, X1 can be alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan (SEQ ID NO: 239). In various embodiments, X1 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, X1 can be serine (SEQ ID NO: 76).

[0025] In various embodiments, the fusion protein does not include a polypeptide consisting of the sequence X1-X2-X3-X4 between the propeptide or propeptide variant and the mature IL-18 or mature IL-18 variant, where X1 is L or absent, X2 is E or absent, X3 is S or absent, and X4 is D or absent; and when X1, X2, X3, and X4 are present, the polypeptide consisting of the sequence X1-X2-X3-X4 is a LESD (SEQ ID NO: 253).

[0026] In various embodiments, the PP or PP variant can be N-terminal to the IL-18, a fragment of IL-18, an IL-18 variant, or a fragment of an IL-18 variant.

[0027] In various embodiments, the PP or PP variant functions as a masking domain.

[0028] In various embodiments, the fusion protein further comprises one or more protease cleavage sites.

[0029] In various embodiments, one or more protease cleavage sites can be between the IL-18, fragment of IL-18, IL-18 variant, or fragment of an IL-18 variant and the first protein or fragment thereof capable of translocating into the endoplasmic reticulum (ER), or within the PP, between the PP or PP variant and the IL-18, fragment of IL-18, variant, or fragment of an IL-18 variant, or within the PP, between the PP or PP variant and the first protein or fragment thereof capable of translocating into the endoplasmic reticulum (ER), or within the IL-18, fragment of IL-18, variant, or fragment of an IL-18 variant, or within the PP, or combinations thereof.

[0030] In various embodiments, the fusion protein further comprises a second protein or fragment thereof capable of translocating into the ER, and the second protein capable of translocating into the ER can be C-terminal to interleukin-18 (IL-18), a fragment of IL-18, an IL-18 variant, or a fragment of an IL-18 variant.

[0031] In various embodiments, the fusion protein further comprises a protease cleavage site between the IL-18, fragment of IL-18, IL-18 variant, or fragment of an IL-18 variant and a second protein or fragment thereof capable of translocating into the endoplasmic reticulum (ER).

[0032] In various embodiments, 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 a first protein capable of translocating into the ER.

[0033] In various embodiments, the second protein or fragment thereof capable of translocating into the ER can be fused to the C-terminus of interleukin-18 (IL-18), a fragment of IL-18, an IL-18 variant, or a fragment of an IL-18 variant.

[0034] In various embodiments, the IL-18 variant may have reduced binding to IL-18 binding protein (IL-18BP) compared to wild-type (wt) IL-18. In various embodiments, the IL-18 variant has a binding affinity for the human IL-18 receptor (IL-18R) that 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 to IL-18BP:the binding affinity of the fusion protein comprising the IL-18 variant to IL-18R may be 3:1 or less.

[0035] In various embodiments, the first protein or fragment thereof capable of translocating into the ER can be a globular protein, an immunoglobular protein, or a fragment thereof, or can be a short polypeptide or protein engineered with a signal peptide for translocation into the ER, optionally the short polypeptide or protein is about 2 kDa or 250 kDa or less.

[0036] In various embodiments, the first protein or fragment thereof capable of translocating into the ER may be selected from the group consisting of a fragment crystallizable (Fc) region, human serum albumin (HSA), beta2 microglobulin, transferrin, a fragment antigen-binding 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.

[0037] In various embodiments, the first protein or fragment thereof of the fusion protein capable of translocating into the ER can be a type I transmembrane protein or fragment thereof, or a type II transmembrane protein or fragment thereof.

[0038] In various embodiments, the second protein or fragment thereof capable of translocating into the ER can be a globular protein, an immunoglobular protein, or a fragment thereof, or can be a short polypeptide or protein engineered with a signal peptide for translocation into the ER, optionally the short polypeptide or protein is about 2 kDa or 250 kDa or less.

[0039] In various embodiments, the second protein or fragment thereof capable of translocating into the ER may be selected from the group consisting of a fragment crystallizable (Fc) region, human serum albumin (HSA), beta2 microglobulin, transferrin, a fragment antigen-binding 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.

[0040] In various embodiments, the second protein or fragment thereof of the fusion protein capable of translocating into the ER can be a type I transmembrane protein or fragment thereof, or a type II transmembrane protein or fragment thereof.

[0041] In various embodiments, the Fc region may be an Fc region derived from IgA, IgM, IgG, or IgE. In various embodiments, the Fc region may be an Fc region derived from IgG4, KiH, or IgG1. In various embodiments, the Fc region may be an Fc region of knob-in-hole, HA-TF, Xmab, ZW1, 7.8.60, electrostatic steering, DD-KK, EW-RVT, A107, or Duobody.

[0042] 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 substituted with natural or unnatural amino acids.

[0043] In various embodiments, one or more cysteines in the IL-18, fragment of IL-18, IL-18 variant, or fragment of an IL-18 variant of the fusion protein may be modified or substituted with a natural or unnatural amino acid.

[0044] In various embodiments, one or more cysteines in the PP or PP variant of the fusion protein can be modified or substituted with a natural or unnatural amino acid.

[0045] In various embodiments, the naturally occurring amino acids may be charged, polar uncharged, or hydrophobic. In various embodiments, the naturally occurring amino acids may each independently be selected from serine and valine. In various embodiments, the naturally occurring amino acids may each independently be selected from threonine, asparagine, and glutamine.

[0046] In various embodiments, the natural amino acids can each be independently selected from alanine, isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan. In various embodiments, the natural amino acids can each be independently 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 be independently selected from threonine, glutamine, aspartic acid, phenylalanine, isoleucine, and histidine.

[0047] In various embodiments, the protease may 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.

[0048] In various embodiments, the fusion protein can have one or more sequences set forth in any one of Tables 1 and 4.

[0049] In various embodiments, the fusion protein may have polypeptide 1 and polypeptide 2, and optionally polypeptide 3, selected from Table 1. In various embodiments, the fusion protein may have polypeptide 1 and polypeptide 2, and optionally polypeptide 3, selected from Table 1, where polypeptide 1 and polypeptide 2, and optionally polypeptide 3, may be selected from the same row of Table 1. In various embodiments, the fusion protein may have polypeptide 1 selected from Table 1, where polypeptide 1 comprises HSA.

[0050] In various embodiments, the IL-18 variant may have an amino acid sequence selected from the mature IL-18 column in Table 1.

[0051] In various embodiments, the IL-18 variant may have an amino acid sequence selected from the mature IL-18 column of Table 1, and the propeptide may have an amino acid sequence selected from the propeptide column of Table 1, optionally from the same row.

[0052] Various embodiments provide an IL-18 propeptide variant comprising a polypeptide having AAEPVEDNX1INFVAMKFIDNTLYFIAEDDEN, where X1 can be any amino acid except cysteine (SEQ ID NO: 238).

[0053] In various embodiments, X1 can be alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan (SEQ ID NO: 239). In various embodiments, X1 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, X1 can be serine (SEQ ID NO: 76).

[0054] Various embodiments provide a polynucleotide encoding any one of the fusion proteins of the invention described herein. In various embodiments, the first protein capable of translocating into the ER can be encoded by a polynucleotide having one or more sequences set forth in Table 2. In various embodiments, the polynucleotide can have one or more sequences from the same row set forth in Table 2.

[0055] Various embodiments provide an expression vector comprising any one of the polynucleotides of the invention described herein.

[0056] Various embodiments provide a cell transfected with any one of the expression vectors of the invention described herein. In various embodiments, the cell can be a mammalian cell. In various embodiments, the cell can be a bacterial cell.

[0057] Various embodiments provide a method of producing a fusion protein, the method comprising culturing cells transfected with any one of the expression vectors of the invention described herein in cell culture medium such that the fusion protein is secreted into the cell culture medium.

[0058] In various embodiments, the method further comprises isolating the fusion protein from the culture medium. In various embodiments, the method further comprises purifying the fusion protein. In various embodiments, the fusion protein can be produced at greater than 135 mg / L under transient transfection in CHO cells or HEK-293 cells.

[0059] Various embodiments provide methods for producing interleukin-18 (IL-18), a fragment thereof, an IL-18 variant, or a fragment of an IL-18 variant, comprising culturing cells transfected with any one of the expression vectors of the invention described herein in cell culture medium such that a fusion protein is produced and secreted into the extracellular space; and contacting a protease with the fusion protein to cleave the fusion protein and produce IL-18, a fragment thereof, an IL-18 variant, or a fragment of an IL-18 variant.

[0060] In various embodiments, the method further comprises isolating the fusion protein from the culture medium. In various embodiments, the method further comprises purifying the fusion protein. In various embodiments, contacting the fusion protein with a protease comprises including the protease in the cell culture medium.

[0061] In various embodiments, the protease may be selected from the group consisting of EK, TEV, Adam17, cathepsin, MMP2, MMP9, MMP14, granzyme A, granzyme B, granzyme M, and combinations thereof.

[0062] In various embodiments, IL-18, a fragment thereof, an IL-18 variant, or a fragment of an IL-18 variant may be produced at greater than 135 mg / L.

[0063] Other features and advantages of the invention will become apparent from the following detailed description, taken in conjunction with the accompanying drawings, which illustrate, by way of example, various features of embodiments of the invention. [Brief explanation of the drawings]

[0064] Exemplary embodiments are illustrated in the referenced figures. It is intended that the embodiments and figures disclosed herein be considered illustrative and not restrictive.

[0065] [Figure 1-1]Figure 1 (Panels A-E) shows exemplary fusion proteins in which the N-terminus of pro-IL-18 is fused to the C-terminus of the knob of a knob-into-hole heterodimeric IgG1 protein, with an N- to C-terminal structure comprising knobs-in-hole (KiH) Fc-propeptide (pp)-enterokinase-cleavable site (EK)-IL-18 wild-type or its variant. This represents exemplary fusion proteins such as FUSE-480, FUSE-481, and FUSE-442 in Table 1. Further modifications were made to pro-IL-18 to reduce aggregation of the molecule, substituting each cysteine residue in both the propeptide and mature IL-18 with serine (designated "IL-18AS" in FUSE-480), alanine (designated "IL-18AA" in FUSE-481), or valine (designated "IL-18AV" in FUSE-442). 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 panel E. [Figure 1-2] See description of Figure 1-1.

[0066] [Figure 2-1]Figure 2 (Panels A-E) shows an exemplary fusion protein, designated "proIL-18mut2," in which the N-terminus of proIL-18 is fused to the C-terminus of an IgG1 CH3 domain (also the knob chain of the knobs-into-holes heterodimeric IgG1 protein, as in Figure 1), and proIL-18 incorporates four amino acid substitutions hypothesized to reduce binding to IL-18BP while maintaining wild-type binding to the IL-18 receptor complex. These fusion proteins have an N- to C-terminal structure containing knobs-in-holes (KiH) Fc-propeptide (PP)-enterokinase-cleavable site (EK)-IL-18mut2. Further modifications were made to pro-IL-18mut2 to reduce aggregation of the molecule, with each cysteine residue in both the propeptide and mature IL-18mut2 being replaced with serine (in FUSE-422, designated "IL-18mut2AS"; panel B), alanine (in FUSE-423, designated "IL-18mut2AA"; panel C), or valine (in FUSE-424, designated "IL-18mut2AV"; panel D). The biological activity, defined as EC50-SEAP, of each compound is shown in panel E. [Figure 2-2] See description of Figure 2-1.

[0067] [Figure 3] Figure 3 (Panels A-D) shows exemplary fusion proteins with (Panel A) or without (Panel B) the propeptide. The effect of masking on the biological activity of IL-18AV was investigated. Fc-fusion variants incorporating IL-18AV with (Panel A, FUSE-442) or without (Panel B, FUSE-505) the propeptide were generated. The biological activity, defined as the EC50-SEAP of each compound, is shown in Panel D.

[0068] [Figure 4]Figure 4 (Panels A-D) shows exemplary fusion proteins with (Panel A) or without (Panel B) the propeptide. To investigate the effect of the propeptide on masking the biological activity of "IL-18mut2AV," we generated Fc-fusion variants incorporating "IL-18mut2AV" without the propeptide (hence the mutated mature IL-18, denoted "matIL-18mut2-AV," see Panel B; FUSE-441) or with the propeptide (Panel A; FUSE-424). For fusion proteins lacking the propeptide, the EK cleavage site, which replaced the caspase 1 site, was moved directly between the CH3 domain of the knob and mature IL-18AV without the addition of a flexible linker. Panel C shows activation readout using the HEK-Blue IL-18AV reporter cell assay after exposure to titrations of FUSE-441 (Fc-EK-IL-18AV) or FUSE-424 (Fc-EKpp-IL-18AV), with or without treatment with EK. Biological activity, defined as EC50-SEAP for each compound, is shown in panel D.

[0069] [Figure 5] Figure 5 (Panels A-D) shows exemplary fusion proteins in which the N-terminus of pro-IL-18 is fused to the C-terminus of IgG1 Fc or IgG4 Fc, with N- to C-terminal structures including IgG1 Fc-propeptide (pp)-IL-18AV (FUSE-507, Panel A) and IgG4 Fc-propeptide (PP)-IL-18AV (FUSE-509, Panel B). Panel C shows activation readout using a HEK-Blue IL-18AV reporter cell assay when cells are exposed to titrations of FUSE-507 or FUSE-509 with or without treatment with EK. The biological activity, defined as EC50-SEAP, of each compound is shown in Panel D.

[0070] [Figure 6]Figure 6 (Panels A-E) shows exemplary fusion proteins in which the N-terminus of pro-IL-18, with or without the propeptide (pp), is fused to the C-terminus of HSA, with N- to C-terminal structures including HSA-propeptide (PP)-IL-18AV (FUSE-501, Panel A) and HSA-IL-18AV (FUSE-503, Panel B). Panels C and D show activation readouts. The biological activity, defined as EC50-SEAP for each compound, is shown in Panel E.

[0071] [Figure 7] Figure 7 (Panels A-E) shows exemplary fusion proteins in which the N-terminus of pro-IL-18mut2, with or without the propeptide (PP), is fused to the C-terminus of HSA, with N- to C-terminal structures including HSA-propeptide (PP)-IL-18mut2AV (FUSE-502; Panel A) and HSA-IL-18mut2AV (FUSE-504; Panel B). Panels C and D show activation readouts. The biological activity, defined as the EC50-SEAP for each compound, is shown in Panel E.

[0072] [Figure 8-1] Figure 8 (Panels A-D) shows exemplary fusion proteins in which the C-terminus of pro-IL-18, with or without the propeptide (PP), is fused to the N-terminus of the knob of a knob-into-hole heterodimeric IgG1 protein, with N- to C-terminal structures including propeptide (PP)-IL-18AV-knob-in-hole (KiH)Fc (FUSE-499, Panel A) and IL-18AV-knob-in-hole (KiH)Fc (FUSE-500, Panel B). Panel C shows the activation readout. For each compound, the biological activity, defined as EC50-SEAP with or without exposure to caspase-1, is shown in Panel D. [Figure 8-2] See description of Figure 8-1.

[0073] [Figure 9A]Figures 9A and 9D show exemplary fusion proteins with N- to C-terminal structures: Fc-ppMMP2 / 9 cleavage site-IL-18-AV (FUSE-486), Fc-ppMMP9 / 2 cleavage site-IL-18-AV (FUSE-487), where the cleavage sites are specific for metalloproteases, MMP2, and MMP9, with the preferred enzyme to the left of the forward slash; or FUSE-485 (Fc-GzmBpp-IL-18AV) and FUSE-462 (Fc-GzmBpp-IL-18mut2AV). Figure 9B shows the activation readout associated with FUSE-486 and FUSE-487, with or without MMP2 treatment. The biological activity, defined as EC50-SEAP, for each of FUSE-486 and FUSE-487, with or without MMP2 treatment, is shown in Figure 9B. Figure 9C shows that FUSE587 was approximately 3,000-fold attenuated compared to recombinant human IL-18. Interestingly, we observed that cleavage of FUSE587 with MMP2 released an IL-18AV variant that was still approximately 100-fold attenuated compared to recombinant IL-18. In contrast, cleavage with granzyme B released an IL-18AV variant with activity similar to that of recombinant IL-18. While cleavage with granzyme B results in the release of mature IL-18AV without any N-terminal residues comprising the overhang, cleavage of FUSE486 and FUSE587 with MMP2 leaves behind N-terminal polypeptide overhangs of 11 and 15 amino acids, respectively. We speculated that these overhangs may attenuate IL-18AV activity, albeit to a lesser extent than the full-size variant propeptide. This phenomenon was further investigated in Figures 11 and 13. Figures 9D and 9G show exemplary fusion proteins with N- to C-terminal structures. The Fc-ppGb cleavage site-IL-18-AV (FUSE-485; 9D), Fc-ppGb cleavage site-IL-18mut2-AV (FUSE-462; 9D), or Fab-cetuximab-Fc-ppGb cleavage site-IL-18-AV (FUSE-517; 9G) have granzyme B (Gb)-specific cleavage sites.Figure 9E shows the activation readout associated with FUSE-462 and FUSE-485, with or without granzyme B treatment. Figure 9F shows the biological activity, defined as EC50-SEAP, for each of FUSE-462 and FUSE-485, with or without granzyme B treatment. Figure 9H shows the activation readout associated with FUSE-517, with or without enzyme treatment. Figure 91 shows the biological activity, defined as EC50-SEAP, of FUSE-517, with or without granzyme B treatment. [Figure 9B] See legend to Figure 9A. [Figure 9C] See legend to Figure 9A. [Figure 9D] See legend to Figure 9A. [Figure 9E] See legend to Figure 9A. [Figure 9F] See legend to Figure 9A. [Figure 9G] See legend to Figure 9A. [Figure 9H] See legend to Figure 9A. [Figure 9I] See legend to Figure 9A.

[0074] [Figure 10-1] Figure 10 (panels A-G) shows the effect of IL-18BP on the biological activity of recombinant human IL-18 (rhIL-18) and the EK cleavage products of exemplary fusion proteins, Fc-ppEK-IL-18-AV (FUSE-442) and Fc-ppEK-IL-18mut2AV (FUSE-424). The biological activity (competition assay), defined as EC50-SEAP, for each compound with and without the addition of IL-18BP 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. [Figure 10-2] See description of Figure 10-1. [Figure 10-3] See description of Figure 10-1.

[0075] [Figure 11] Figure 11 (Panel A) shows a diagram of exemplary fusion proteins in which the C-terminus of pro-IL-18 is fused to the N-terminus of the knob of a knob-into-hole heterodimeric IgG1 protein with polypeptides of different sizes fused to the N-terminus of mature IL-18. Figure 11 (Panel B) shows the biological activity of each fusion protein using the IL-18 reporter cell line HEK-Blue IL-18.

[0076] [Figure 12-1] Figures 12A, 12B(i), 12B(ii), 12C, 12D(i), 12D(ii), and 12E show human IL-18 engineered mutant fusion proteins according to various embodiments of the present invention. [Figure 12-2] See description of Figure 12-1. [Figure 12-3] See description of Figure 12-1. [Figure 12-4] See description of Figure 12-1. [Figure 12-5] See description of Figure 12-1.

[0077] [Figure 13] FIG. 13 shows 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.

[0078] [Figure 14-1] Figures 14A-14H show the effect of substitution of cysteine residues in the propeptide, which are fused together to form the pro-IL-18 variant cassette, and in mature IL18, on the biological activity of each variant using the HEK Blue IL18 assay system. [Figure 14-2] See description of Figure 14-1. [Figure 14-3] See description of Figure 14-1.

[0079] [Figure 15] 15A-15B show the effect of targeting pro-IL18 to the close proximity of its receptor complex (ie, "cis-acting"). DETAILED DESCRIPTION OF THE INVENTION

[0080] Description of the Invention 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 3 rd ed., Revised, J. Wiley & Sons (New York, NY 2006); March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 7 th ed., J. Wiley & Sons (New York, NY 2013); and Sambrook and Russell, Molecular Cloning: A Laboratory Manual 4 thed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY 2012), provides those skilled in the art with a general guide to many of the terms used in this application. References for antibody preparation methods include D. Lane, Antibodies: A Laboratory Manual, 2nd Edition (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 See Enzymol, 326, 461-479; Holliger P. (2005) Nat. Biotechnol. Sep; 23(9): 1126-36).

[0081] One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, the present invention is in no way limited to the methods and materials described. For purposes of the present invention, the following terms are defined below.

[0082] As used herein, the term "about" or "approximately," when used in connection with a referenced numerical indication, means the referenced numerical indication plus or minus up to 5% of the referenced numerical indication, unless otherwise specifically stated herein. For example, the expression "about 50%" covers a range of 45% to 55%. In various embodiments, when used in connection with a referenced numerical indication, the term "about," when specifically provided in the claims, can mean the referenced numerical indication plus or minus up to 4%, 3%, 2%, 1%, 0.5%, or 0.25% of the referenced numerical indication.

[0083] 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 an immunoglobulin heavy chain constant region, or an analog or portion thereof capable of binding to an Fc receptor. Each immunoglobulin heavy chain constant region comprises four or five domains. The domains are named sequentially as follows: CH1-hinge-CH2-CH3 (-CH4). CH4 is present in IgM, which does not have a hinge region. An immunoglobulin heavy chain constant region suitable for the present invention preferably comprises an immunoglobulin hinge region, and preferably also comprises a CH3 domain. Most preferably, the immunoglobulin heavy chain constant region comprises an immunoglobulin hinge region, a CH2 domain, and a CH3 domain.

[0084] As used herein, the term immunoglobulin "hinge region" is understood to mean the entire immunoglobulin hinge region or at least a portion of an immunoglobulin hinge region sufficient to form one or more disulfide bonds with a second immunoglobulin hinge region.

[0085] As used herein, the term "vector" is understood to mean any nucleic acid comprising a nucleotide sequence that has the ability to be incorporated into a host cell and recombine and integrate into the host cell genome, or to replicate autonomously 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.

[0086] As used herein, the term "gene expression" or "expression" of a fusion protein is understood to mean the transcription of a DNA sequence, the translation of an mRNA transcript, and the secretion of the fusion protein product. In some embodiments, the expression process also includes or is followed by purification, and other means, such as, for example, protein A affinity chromatography, or size exclusion chromatography, can be used for purification.

[0087] As used herein, "IL-18 fusion protein" refers to a fusion protein that includes wild-type IL-18 or an IL-18 variant, unless specifically stated to include only wild-type IL-18 or only an IL-18 variant. Thus, in certain embodiments, an "IL-18 fusion protein" includes only any one of the IL-18 variants described herein.

[0088] The term "linker" with reference to an amino acid linker in a polypeptide includes a dimer of two amino acids, a trimer of three amino acids, or a group consisting of T, PT, MPT, S, GS, GGS, GGGS (SEQ ID NO: 235), and (GGGGXλ (SEQ ID NO: 236)). n where Xλ is Q, A, E, or S, and n=1 to 5, or an integer greater than 5. In some embodiments, the amino acid linker is (GGGGS (SEQ ID NO: 237)) nwherein n is an integer between 1 and 5, thereby resulting in an amino acid linker that is 25 amino acids or less in length. 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., about 30-36 amino acids in length, about 5-10, 11-20, 21-30, or 31-40 amino acids in length.

[0089] Fusion proteins Various embodiments provide one or more fusion proteins, each 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, or fragment of a protein, capable of translocating into the endoplasmic reticulum (ER), comprising a cytosolic or nuclear protein engineered to translocate into the ER by the addition of a signal peptide / leader sequence onto the N-terminus of such engineered protein. Preferably, the protein capable of translocating into the ER has an amino acid sequence that initiates transport of the protein (e.g., IL-18 or a fragment, variant, or fragment of a variant thereof) 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 between (a) the protein capable of translocating into the endoplasmic reticulum (ER) and (b) the propeptide (or variant) or IL-18 (or variant).

[0090] In some embodiments, one or more fusion proteins do not include an IL-18 propeptide or variant thereof. The terms "IL-18 propeptide," "propeptide," or "PP" of the present invention may also be used interchangeably to describe an amino acid sequence linked to an IL-18 or IL-18 variant in an IL-18 precursor or IL-18 variant precursor, which upon removal results in mature IL-18 or a fragment thereof, or an IL-18 variant or a fragment thereof. For example, an IL-18 propeptide may have the sequence of amino acid residues 1-36 of Uniprot ID Q14116.

[0091] In some embodiments, one or more fusion proteins also include a propeptide (PP) or a variant thereof. Examples of propeptide variants are provided herein, including those in Table 1. The propeptide is linked directly or indirectly to IL-18 or an IL-18 variant to form a precursor IL-18 or precursor IL-18 variant, which can inactivate IL-18 or an IL-18 variant. Preferably, the PP or variant thereof is N-terminal to IL-18 (or a fragment thereof, a variant, or a fragment of a variant thereof) in the fusion protein.

[0092] In some embodiments, the one or more fusion proteins also comprise a cleavage site, preferably based on a peptide substrate susceptible to enzyme / protease cleavage. The cleavage site may be located within the PP, between the PP or a variant thereof (if present) and IL-18 (or a fragment, variant, or fragment of a variant thereof), or between the protein capable of translocating into the ER and the PP (if present), or between the protein capable of translocating into the ER and IL-18 or a fragment, variant, or fragment of a variant thereof, particularly in the absence of the PP. In some embodiments, when the PP is present, the cleavage site is located within the PP. In further embodiments, the 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.

[0093] An example of a propeptide variant includes a polypeptide having AAEPVEDNX1INFVAMKFIDNTLYFIAEDDEN, where X1 is any amino acid except cysteine (SEQ ID NO: 238). In various embodiments, X1 is alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan (SEQ ID NO: 239). In various embodiments, X1 is valine (SEQ ID NO: 78). In various embodiments, X1 is serine, threonine, asparagine, or glutamine (SEQ ID NO: 240). In various embodiments, X1 is serine (SEQ ID NO: 76).

[0094] In various embodiments, the fusion protein does not include a polypeptide consisting of the sequence X1-X2-X3-X4 between (i) the propeptide or propeptide variant and (ii) the mature IL-18 or mature IL-18 variant, where X1 is L or absent, X2 is E or absent, X3 is S or absent, and X4 is D or absent, and when X1, X2, X3, and X4 are all present, the sequence is LESD (SEQ ID NO: 253).

[0095] In various embodiments of the fusion protein, IL-18 (or a fragment, variant, or fragment of a variant thereof) is linked by a polypeptide bond to a first protein capable of translocating into the ER. The fusion protein may have various configurations. Preferably, the N-terminus of IL-18 (or a fragment, variant, or fragment of a variant thereof) is linked, directly or indirectly, by a polypeptide bond to the C-terminus of the first protein capable of entering and translocating into the ER.

[0096] In yet other embodiments, the C-terminus of IL-18 (or a fragment, variant, or fragment of a variant thereof) is linked by a polypeptide bond, directly or indirectly, to the N-terminus of a first protein capable of entering and translocating to the ER. As a non-limiting example, an IL-18-variant (or IL-18, a fragment of IL-18, or a fragment of an IL-18 variant) is fused to the N-terminus of the knob of a knob-into-hole heterodimeric IgG1 protein, with or without a propeptide (pp).

[0097] In further embodiments, when the C-terminus of IL-18 is linked to the N-terminus of a first protein capable of entering (or translocating) the ER, a second protein capable of translocating through the ER is often fused to the N-terminus of IL-18 to mediate masking. It is contemplated that the fusion protein may further comprise (iii) a second protein capable of translocating into / through the ER, or a "scaffold" such as a heat shock protein (HSP) that may not translocate through the ER. In some embodiments, when an HSP (nuclear protein) or cytosolic protein is fused to the N-terminus of IL-18 to mediate masking, a signal peptide fused to the N-terminus of the "scaffold" is often required to mediate transport to the ER; however, when a scaffold is fused to the C-terminus of IL-18 and serves to stabilize the complex, a second protein capable of translocating through the ER is often 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 C-terminal to the fusion protein, and in some embodiments, IL-18 (or a fragment, variant, or fragment of a variant thereof) is C-terminal to a first protein that can enter and translocate to the ER, and IL-18 (or a fragment, variant, or fragment of a variant thereof) is N-terminal to a second protein that can enter and translocate to the ER. The "first" or "second" protein that can enter and translocate to the ER is used as a relative reference.

[0098] One or more exemplary amino acid sequences for each component of the fusion protein are shown in Tables 1 and 4.

[0099] In some embodiments, it is provided that the first protein / polypeptide capable of translocating into the ER comprises 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 a 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 an organism into which the nucleic acid molecule encoding the fusion protein or a precursor of the fusion protein is transformed or transfected. Further embodiments provide that the fusion protein comprises an immunoglobulin variable domain (V H ) is provided.

[0100] In various embodiments, the IL-18 (or fragment, variant, or fragment of a variant thereof) is identical (in sequence) to that of human origin, and the immunoglobulin heavy chain constant region comprises a hinge region, and a CH2 domain or a CH3 domain, more preferably a hinge region, and both a CH2 domain and a CH3 domain. In various embodiments, the IL-18 (or fragment, variant, or fragment of a variant thereof) is at least 95%, 90%, or 85% identical (in sequence) to that of human origin, but has amino acid substitutions or other modifications that reduce the affinity of IL-18 (or fragment, variant, or fragment of a variant thereof) for IL-18BP. It is contemplated that immunoglobulin heavy chain constant regions suitable for the present invention may be derived from immunoglobulins belonging to any of the five immunoglobulin classes referred to in the art as IgA (Igα), IgD (Igδ), IgE (Igε), IgG (Igγ), and IgM (Igμ). However, immunoglobulin heavy chain constant regions derived from the IgG class are preferred. Furthermore, the immunoglobulin heavy chain constant region can be derived from any of the IgG antibody subclasses referred to in the art as IgG1, IgG2, IgG3, and IgG4. The immunoglobulin heavy chain constant region domains have cross-homology between immunoglobulin classes. For example, the CH2 domain of IgG is homologous to the CH2 domain of IgA and IgD, and to the CH3 domain of IgM and IgE. A preferred immunoglobulin heavy chain constant region comprises protein domains corresponding to the CH2 and CH3 regions of IgG, or functional portions or derivatives thereof. Further descriptions of immunoglobulin heavy chain constant regions are provided in U.S. Patent Nos. 5,541,087 and 5,726,044, which are incorporated herein by reference.

[0101] In embodiments, the protein / polypeptide fused to 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 hinge domain may include or remove a cysteine known to form a disulfide bond with a light chain or Fab, resulting in a sequence such as EPKSC (SEQ ID NO: 241) or EPKSS (SEQ ID NO: 242) or EPKSA (SEQ ID NO: 243). For example, fusion proteins comprising IgG1-based ER translocation proteins, excluding FUSE-501, FUSE-503, and FUSE-509, may have a cysteine removed from the hinge region, e.g., EPKSS (SEQ ID NO: 242) in IgG1-based ER translocation proteins, excluding FUSE-507 (FUSE-507 has EPKSA (SEQ ID NO: 243) in the hinge region). The hinge region may also contain a core hinge domain, such as CPPCP (SEQ ID NO: 244) or a variant in which a cysteine is substituted. The hinge region may further include a lower hinge domain, such as APELLGGP (SEQ ID NO: 245) or APEAAGGP (SEQ ID NO: 246). In another example, FUSE-509 comprises an IgG4-based ER translocation protein using the hinge region illustrated in Chiu et al., Antibodies 2019, 8(4), 55, 2019. While constructs comprising an immunoglobulin hinge region are preferred, as shown in the figures, the present invention contemplates that cross-linking at other positions may be selected as desired. Additionally, in some cases, two or more monomers may non-covalently associate to form dimers or multimers. In various embodiments 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 that forms homodimers, in various examples, IL-18 is placed on the C-terminus of each monomer of Fc, thereby having two IL-18s placed on the C-terminus of the Fc. In some examples, heterodimers can form (e.g., during a purification process) when one wild-type Fc fused to one IL-18 is mixed with another wild-type Fc that is not fused to IL-18. In other embodiments, IL-18 (or a fragment, variant, or fragment of a variant thereof) is linked to each of two (or more) immunoglobulin heavy chain constant regions / chains in the fusion protein.

[0102] In some embodiments, two arms (or chains) of immunoglobulin heavy chain constant regions (e.g., Fc polypeptides) can be heterodimerized by generating "knob-in-hole" (KiH) mutations in the CH3 domain. This structural feature in the polypeptide arms allows for the assembly of two half antibodies (e.g., an Fc heterodimer, and a VH-CH domain and a VL-CL domain). For example, a heteromultimer (including a heterodimer) can include a first polypeptide and a second polypeptide, each comprising a CH3 domain, where the polypeptides associate at an engineered interface within the CH3 domain, with the first polypeptide containing an engineered protrusion ("knob") within the interface in which at least one contact residue is replaced with an import residue having a larger side chain volume than the original residue, and the second polypeptide containing an engineered cavity ("hole") within the interface in which at least one contact residue is replaced with an import residue having a smaller side chain volume than the original residue. In some embodiments, the engineered interface of the heteromultimer includes pairs of protrusion mutants that fit into at least two cavities. The volume and accessible surface area of each amino acid are described in A. A. Z. Samyatnin, Prog. Biophys. Mol. Biol. 24:107-123, 1972 and C. Chothia, J. Mol. Biol. 105:1-14, 1975. For example, the import residue for forming the protrusion may be arginine (R), phenylalanine (F), tyrosine (Y), or tryptophan (W), preferably, the original residue for forming the protrusion has a small side chain volume, such as alanine, asparagine, aspartic acid, glycine, serine, threonine, or valine. As another example, the import residue for forming the cavity may be alanine (A), serine (S), threonine (T), or valine (V), preferably, the original residue for forming the cavity has a large side chain volume, such as tyrosine, arginine, phenylalanine, or tryptophan. For example, the T366W mutation in the CH3 domain of the "knob" / protruding strand, and the T366S / L368A / Y407V mutation in the CH3 domain of the "hole" / cavity strand.Additionally, the KiH configuration can be coupled with additional mutations to allow for S-S disulfide bonds between the two chains. In various embodiments where the protein / polypeptide is a heterodimer of the KiH configuration, IL-18 (or a fragment, variant, or fragment of a variant thereof) is linked to one, and only one, of two (or more) immunoglobulin heavy chain constant regions / chains (i.e., knob or hole).

[0103] In some embodiments, two or more arms (or chains) of an immunoglobulin heavy chain constant region (e.g., an Fc polypeptide) can comprise alternative symmetric-asymmetric stereocomplementary designs (e.g., HA-TF, ZW1), charge-charge swap interactions (DD-KK), charge-steric complementarity swaps with additional long-range electrostatic interactions (e.g., EW-RVT), or isotype chain swap designs (e.g., strand exchange engineering domains (SEED)), or Xmab, 7.8.60, electrostatic steering, A107, or Duobody, to form heterodimers / heteromultimers. Further description of these constructs and exemplary mutations / residues can be found in Front Immunol. 2016;7:394.

[0104] In additional embodiments, the preferred protein or fragment thereof of the fusion protein that can translocate into the ER is a globular protein, an immunoglobular protein, or a fragment thereof. In various embodiments, the preferred protein or fragment thereof of the fusion protein that can translocate into the ER is a short polypeptide or protein engineered with a signal peptide for translocation into the ER. For example, the short polypeptide or protein is about 2 kDa or less than 250 kDa. As additional examples, short polypeptides or proteins are 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.

[0105] In additional embodiments, suitable proteins capable of entering and translocating to the ER may be globular proteins, human serum albumin (HSA), beta2 microglobulin, transferrin, fragment antigen-binding regions (Fab regions), VHH antibodies, single-chain variable fragments (scFv), anticalins, designed ankyrin repeat proteins (DARPins), binding domains thereof, and fragments thereof. Additional suitable proteins capable of entering and translocating to the ER may include type I transmembrane proteins or fragments thereof, or type II transmembrane proteins or fragments thereof.

[0106] In various embodiments, the 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 translocating into the ER. The second protein, or a fragment thereof, capable of translocating into the ER can be an Fc domain or HSA, beta2 microglobulin, transferrin, a fragment antigen-binding 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, as described herein. Figures 8A and 11A (first three from left to right) are non-limiting examples of such fusion proteins.

[0107] In yet other embodiments, the fusion protein further comprises a protein that cannot naturally translocate into the ER, such as a nuclear or cytosolic protein, fused to the N-terminus of IL-18. For such proteins, a signal peptide (sometimes referred to as a leader sequence), such as the Ig-kappa 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), is fused to the N-terminus of the non-ER-translocating protein. For example, the signal peptide can be on the N-terminus of the propeptide or IL-18 (or a fragment, variant, or variant of the fragment). Further examples of proteins that can translocate into / through / enter the ER may be, for example, proteins incorporating a signal peptide at their N-terminus. As an example, Hsp70 is a nuclear protein, but can be engineered to become an ER translocation protein when fused or linked to a signal peptide on the N-terminus of Hsp70. In various embodiments, the addition of an N-terminal signal peptide, such as an Ig-kappa leader sequence, is in place of the Fc, globular protein, or HSS present in the fusion proteins disclosed herein.

[0108] In some embodiments, the fusion protein (e.g., masked IL-18) further comprises a tumor-targeting fragment, e.g., a fragment that targets a cell surface protein, including, but not limited to, a tumor-associated antigen (TAA). As shown in Figure 9G, for example, 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 believed to be compatible with the fusion protein systems disclosed herein.

[0109] 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 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 and 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 shown in Figure 4, FUSE-694 is a masked IL-18 fusion protein that also contains an anti-PD1 fragment. Examples of immune checkpoints include, but are not limited to, PD-1, PD-L1, CTLA-4, and LAG-3. One or more immune checkpoint-targeting fragments of known antibodies are believed to be compatible with the fusion protein systems disclosed herein. Examples of anti-PD1 fragments include fragments (e.g., Fab, Fv) of 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.

[0110] In some embodiments, the fusion protein (e.g., masked IL-18) comprises a targeting polypeptide that 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 that targets a protein on a cell that does not contain IL-18RC. In these embodiments, the IL-18 fusion protein needs to be delivered near the IL-18R complex to produce a cis or density effect in the interaction between the IL-18 fusion protein and the IL-18R complex. For example, a TAA-targeted IL-18 fusion protein may interact with the IL-18R complex on a T cell if (a) the fusion protein crosslinks the T cell and the TAA+ cell, or (b) the fusion protein is combined with another protein that crosslinks the T cell and the TAA+ cell, or (c) the fusion protein binds to a TAA+ cell that naturally interacts with the T cell through secondary means (e.g., TCR / MHC interactions). In other examples, the fusion protein may be delivered to fibroblasts or other accessory cells in the tumor microenvironment and released by proteases, thereby acting at a distance on IL-18R+ T cells or NK cells.

[0111] Exemplary targeting polypeptides include those listed in Table 5, or fragments thereof. Of those listed as antigen-binding antibodies, their VHHs, Fab regions, or single-chain variable fragments (scFvs) can be used as the antigen-binding site of the multispecific antibodies disclosed herein.

[0112] In some embodiments, enterokinase is used for site-specific cleavage of recombinant fusion proteins containing accessible enterokinase recognition sites. For example, enterokinase can specifically cleave lysine residues C-terminal to and beyond its cleavage site, Asp-Asp-Asp-Asp-Lys (SEQ ID NO: 87). Thus, fragments produced from this cleavage reaction do not inherit residues from the DDDDK (SEQ ID NO: 87) recognition sequence. Furthermore, DDDDK (SEQ ID NO: 87) is part of an octapeptide FLAG tag (DYKDDDDK (SEQ ID NO: 248)), which can be utilized as a fusion tag for antibody recognition and detection of fusion proteins by Western blot analysis, as well as purification of fusion proteins by anti-FLAG affinity chromatography.

[0113] Preferably, the cleavage site may be based on a peptide substrate that is sensitive to other enzymes, particularly proteases highly expressed in the tumor microenvironment, such as granzyme B, granzyme A, granzyme M, granzyme K, matrix metalloproteinase (MMP) 1 / 2 / 9 / 14, or other MMPs. Notably, granzymes are typically upregulated only in inflamed tumors. For example, the substrate sequence for granzyme B may be Ile-Glu-Xaa-Asp↓Xaa-Gly (SEQ ID NO: 249), with cleavage at the Asp↓Xaa peptide bond. Alternatively, the substrate sequence for granzyme B may also be Ile-Glu-Xaa-Asp↓, with cleavage at the C-terminus of Asp, where Xaa may be Gln (SEQ ID NO: 88) or another amino acid.

[0114] Some immune cells can release granzymes, such as T cells, NK cells, neutrophils, and mast cells. In some embodiments, a fusion protein comprising (a) a polypeptide fragment that targets an immune checkpoint expressed on immune cells and / or a polypeptide fragment that targets an activating receptor on NK cells, and (b) a tumor-targeting fragment, can be effective in attracting immune cells (e.g., T cells, NK cells) to tumors, resulting in the release of granzymes that release IL-18. For example, an IL-18 fusion protein comprising a polypeptide fragment that targets an immune checkpoint protein can reverse the exhaustion of NK cells and / or T cells, subsequently releasing more granzymes.

[0115] In additional embodiments, the fusion protein comprises a cleavage site recognized by a serine protease, a cysteine protease, an aspartic acid protease, a threonine protease, a glutamic acid protease, a metalloproteinase, a gelatinase, or an asparagine peptide lyase. In some embodiments, the protease cleavage site is selected from the group consisting of cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin K, cathepsin L, kallikrein, ahKl, 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, caspase, caspase-3, Mir, and the like. 1-CP, papain, HIV-1 protease, HSV protease, CMV protease, chymosin, renin, pepsin, matriptase, legumain, plasmepsin, nepenthesin, metalloexopeptidase, metalloendopeptidase, matrix metalloproteinase (MMP), MMP1, MMP2, MMP3, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, ADAM10, ADAM17, ADAM12, urokinase plasminogen activator (uPA), en It is recognized by telokinase, prostate-specific target (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). Non-limiting examples of cleavage sites are included in Table 1. As a specific example, IEQD (SEQ ID NO: 88) can be used.

[0116] It is contemplated that variants, fragments, or fragments of variants of IL-18 are suitable for fusion protein compositions and are preferred in some embodiments. For example, variants of mature IL-18 may 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 may be substituted with natural or unnatural amino acids, such as Cys to Ser, Ala, or Val, to reduce aggregation of the molecule in the fusion protein. Additional examples include cysteine to threonine, asparagine, or glutamine; cysteine to alanine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan; cysteine to phenylalanine, alanine, aspartic acid, or asparagine; or cysteine to threonine, glutamine, aspartic acid, phenylalanine, isoleucine, or histidine.

[0117] The IL-18 variant may have 95%, 90%, 85%, 83%, 80%, 75%, 70%, 65%, or at least 60% sequence identity with wild-type IL-18. In some embodiments, the IL-18 variant may have at least 60% and up to 83% sequence identity with wild-type IL-18. In some embodiments, the IL-18 variant in the fusion protein is released (e.g., on a tested electrophoresis gel) as a functional fragment of about 15 kDa when cleaved at the fusion protein cleavage site. (The released protein is believed to be mature IL-18, which normally migrates at 18 kDa but may appear as approximately 15 kDa depending on the particular polyacrylamide ratio in the ladder or gel used.) Fragments of IL-18 may 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, the IL-18 fragment produced by the fusion proteins disclosed herein (particularly after protease cleavage of the fusion protein) is less than 85% (e.g., about 83%, about 83%-80%, about 80%-75%, about 75%-70%, or about 70%-65%) in size compared to native / wild-type mature IL-18. For example, an IL-18 fragment of about 15 kDa in size (preferably having binding affinity for IL-18Ra / b equivalent to that of wild-type mature IL-18) is fused to a propeptide (or PP variant) and an ER translocation protein (with or without mutations), and the fusion protein also contains a protease cleavage site, releasing a smaller IL-18 fragment (e.g., about 15 kDa in size) upon protease cleavage. Preferably, this smaller IL-18 fragment retains its native binding affinity for IL-18Ra / b and maintains binding affinity for IL-18BP equivalent to or less than that of IL-18Ra / b. Preferably, the variant, fragment, or fragment of a variant of IL-18 is capable of binding to IL-18R to form a complex, thereby activating a pro-inflammatory program and / or the NF-κB pathway.In some embodiments, the variant, fragment, or fragment of a variant of IL-18 has increased binding affinity compared to wild-type IL-18 (e.g., 150%, 140%, 130%, 120%, 110%, or at least 100% compared to wild-type IL-18) and / or is capable of inducing 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, the variant, fragment, or fragment of a variant of IL-18 has a 120%, 110%, or at least 100% increased binding affinity compared to wild-type IL-18 and / or is capable of inducing 120%, 110%, 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10% of the biological activity compared to wild-type IL-18. In additional embodiments, the variant, fragment, or fragment of a variant of IL-18 has reduced binding to IL-18 binding protein (IL-18BP) compared to wild-type IL-18.

[0118] In some embodiments, IL-18 or a fragment or variant thereof cleaved from a fusion protein has at least a 1,000-fold, 2,000-fold, 3,000-fold, 5,000-fold, 10,000-fold, 30,000-fold, 50,000-fold, 70,000-fold, 80,000-fold, 90,000-fold, or 100,000-fold increase in biological activity (e.g., binding to IL-18R to form an IL-18 / IL-18Rα / β complex and induce downstream signaling) compared to the uncleaved form, particularly in a fusion protein with the propeptide. In further embodiments, IL-18 or a fragment or variant thereof cleaved from a fusion protein has equivalent biological activity, or within about a 10-fold, 20-fold, 30-fold, 40-fold, or 50-fold difference in biological activity, compared to recombinant human mature IL-18.

[0119] In some embodiments, IL-18 or a 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, IL-18 or a fragment or variant thereof cleaved from the fusion protein has a binding affinity for the IL-18R complex that is about the same as, or at least 100%, 95%, or 90% of that of wild-type IL-18. In some embodiments, IL-18 or a fragment or variant thereof cleaved from the fusion protein has a binding affinity for its IL-18R complex that is greater than that of wild-type IL-18, e.g., a binding affinity that is at least 105%, 110%, or a KD value that is at least 10% or 20% less than that of wild-type IL-18. Preferably, the binding affinity of IL-18 or a fragment or variant thereof cleaved from the fusion protein to IL-18BP is reduced compared to that of wild-type IL-18. For example, in some examples, the KD of IL-18 or a fragment or variant thereof cleaved from the fusion protein with IL-18BP is 18 nM or greater, so that the binding affinity is lower for IL-18BP than for IL-18R. In some examples, the KD of IL-18 or a fragment or variant thereof cleaved from the fusion protein with IL-18BP is 18 nM or greater, while the KD of wild-type IL-18 with IL-18BP is about 0.4 nM. It is also contemplated that the KD may vary depending on the instrument and protocol settings.

[0120] In various embodiments, the fusion protein comprises a first polypeptide or protein, or fragment thereof, capable of translocating 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 C-terminal to the first polypeptide or protein capable of translocating into the ER. In various embodiments, the first polypeptide is not a wild-type IL-18 propeptide. In various embodiments, the protein, or fragment thereof, capable of translocating into the endoplasmic reticulum (ER), is not a wild-type IL-18 propeptide. In addition to these features, additional features of fusion proteins are discussed herein.

[0121] In some embodiments, the fusion protein comprises an IL-18 variant. In various embodiments, the IL-18 variant has an amino acid sequence comprising or consisting of amino acid positions 37-193 of SEQ ID NO: 250, with one to five amino acid substitutions at positions E42, M87, K89, M96, and M149 of MAAEPVEDNCINFVAMKFIDNTLYFIAEDDENIEQDYFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDVRDNAPRTIFIISMYKDSQPRGMAVTISVKVEKISTLSVENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLAVEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 250). In various embodiments, the one to five amino acid substitutions are one amino acid substitution. In other embodiments, the one to five amino acid substitutions are two amino acid substitutions. In another embodiment, the 1 to 5 amino acid substitutions are 3 amino acid substitutions. In another embodiment, the 1 to 5 amino acid substitutions are 4 amino acid substitutions. In another embodiment, the 1 to 5 amino acid substitutions are 5 amino acid substitutions. In various embodiments, the IL-18 variant contains 5 or fewer amino acid substitutions, excluding substituted cysteines.

[0122] In various embodiments, the IL-18 variant has an amino acid sequence comprising or consisting of amino acid positions 37-193 of SEQ ID NO: 251 with one to five amino acid substitutions at positions E42, M87, K89, M96, and M149 of MAAEPVEDNCINFVAMKFIDNTLYFIAEDDENIEQDYFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 251).

[0123] In various embodiments, the IL-18 variant has an amino acid sequence including or consisting of amino acid positions 37-193 of SEQ ID NO:251, and has one or more amino acid substitutions at positions C74, C104, C112, and C164, and one to five amino acid substitutions at positions E42, M87, K89, M96, and M149 of SEQ ID NO:251. 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.

[0124] In various embodiments, the one to five amino acid substitutions are one or more of E42K, E42R, E42A, E42H, or E42Q; M87K or M87H; K89G, K89A, or K89E; M96L or M96I; or M149V or M149I. In various embodiments, 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.

[0125] 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 an amino acid sequence selected 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 truncation peptide selected from Table 1 (optionally in the same row as the IL-18 variant and propeptide). As a particular example, IEQD (SEQ ID NO: 88) can be used.

[0126] 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. WO2022 / 038417, the IL-18 variants and sequences of each of these patents or publications being incorporated herein by reference as if fully set forth.

[0127] 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 that has IL-18RC or is capable of expressing IL-18RC upon cell activation and activates IL-18RC signaling.

[0128] In other embodiments, the targeting polypeptide targets proteins on the surface of cells that do not have IL-18RC or that are unable to express IL-18RC. Cells that do not have IL-18RC or that are unable to express IL-18RC on their surface are in close proximity to cells that express IL-18RC or that are able to express IL-18RC. In another example, the fusion protein can bring cells that do not have IL-18RC or that are unable to express IL-18RC on their surface into close proximity to cells that express IL-18RC or that are able to express IL-18RC.

[0129] In various embodiments, the targeting polypeptide comprises a tumor-associated antigen binding domain.

[0130] In various embodiments, the fusion protein further comprises a binding domain for a protein expressed on an immune cell, hi various embodiments, the fusion protein further comprises a binding domain for a protein expressed on an immune cell that expresses the IL-18 receptor complex, or on an immune cell that expresses the IL-18 receptor complex upon activation.

[0131] In various embodiments, the fusion protein further comprises an antibody or antibody fragment, wherein the fusion protein binds to tumor cells or immune or stromal cells in tumor tissue. Examples of antibody fragments include Fc fragments, Fab fragments, Fv fragments, and others discussed herein.

[0132] In various embodiments, the fusion protein further comprises a masking domain. In these embodiments, the mature IL-18 or mature IL-18 variant can be released from the masking domain by a protease. In various embodiments, the protease is a granzyme and 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, and metalloproteases can be expressed in the tumor microenvironment. Further examples of types of proteases and granzymes are described herein. In various embodiments, the mature IL-18 increases the activity of NK cells or T cells, optionally, the activity being one or more of proliferation, survival, and cytotoxicity.

[0133] In various embodiments, the fusion protein further comprises a half-life extender. Non-limiting examples of half-life extenders include half-life extending polypeptides, such as human serum albumin (HSA) or HSA-binding fragments. In various embodiments, the fusion protein has reduced activity compared to wild-type IL-18 when not bound to cells having IL-18RC. In various embodiments, the reduced activity is at least a 75% reduction in activity compared to wild-type IL-18.

[0134] 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 and polypeptide 2, and optionally polypeptide 3, are selected from the same row of Table 1.

[0135] In various embodiments, the fusion protein comprises a polypeptide 1 selected from Table 1, wherein polypeptide 1 comprises HSA.

[0136] In various embodiments, the fusion protein does not include 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 and sequences of each of these patents or publications are incorporated herein by reference as if fully set forth.

[0137] Propeptide variants Various embodiments of the present invention provide propeptide variants. In various embodiments, the propeptide variant has the following amino acid sequence: AAEPVEDNX1INFVAMKFIDNTLYFIAEDDEN, where X1 is any amino acid except cysteine (SEQ ID NO: 238). In various embodiments, X1 is alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan (SEQ ID NO: 239). In various embodiments, X1 is valine (SEQ ID NO: 78). In various embodiments, X1 is serine, threonine, asparagine, or glutamine (SEQ ID NO: 240). In various embodiments, X1 is serine (SEQ ID NO: 76).

[0138] IL-18 variants Various embodiments provide IL-18 variants.

[0139] In various embodiments, the IL-18 variant has an amino acid sequence comprising or consisting of amino acid positions 37-193 of SEQ ID NO: 250, with one to five amino acid substitutions at positions E42, M87, K89, M96, and M149 of MAAEPVEDNCINFVAMKFIDNTLYFIAEDDENIEQDYFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDVRDNAPRTIFIISMYKDSQPRGMAVTISVKVEKISTLSVENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLAVEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 250). In various embodiments, the one to five amino acid substitutions are one amino acid substitution. In other embodiments, the one to five amino acid substitutions are two amino acid substitutions. In other embodiments, the one to five amino acid substitutions are three amino acid substitutions. In another embodiment, the 1 to 5 amino acid substitutions are 4 amino acid substitutions. In another embodiment, the 1 to 5 amino acid substitutions are 5 amino acid substitutions. In various embodiments, the IL-18 variant contains 5 or fewer amino acid substitutions, excluding substituted cysteines.

[0140] In various embodiments, the IL-18 variant has an amino acid sequence comprising or consisting of amino acid positions 37-193 of SEQ ID NO: 251 with one to five amino acid substitutions at positions E42, M87, K89, M96, and M149 of MAAEPVEDNCINFVAMKFIDNTLYFIAEDDENIEQDYFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 251).

[0141] In various embodiments, the IL-18 variant has an amino acid sequence including or consisting of amino acid positions 37-193 of SEQ ID NO: 251, with one or more amino acid substitutions at positions C74, C104, C112, and C164, and one to five amino acid substitutions at positions E42, M87, K89, M96, and M149 of SEQ ID NO: 251. 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.

[0142] In various embodiments, the one to five amino acid substitutions are one or more of E42K, E42R, E42A, E42H, or E42Q; M87K or M87H; K89G, K89A, or K89E; M96L or M96I; or M149V or M149I. In various embodiments, 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.

[0143] In various embodiments, the IL-18 variant is selected from the "Mature IL18 Variant" column of Table 1.

[0144] In various embodiments, the IL-18 variant further comprises an IL-18 propeptide or an IL-18 propeptide variant.

[0145] In various embodiments, the IL-18 variant further comprises an IL-18 propeptide variant having the following amino acid sequence: AAEPVEDNX1INFVAMKFIDNTLYFIAEDDEN, where X1 is any amino acid except cysteine (SEQ ID NO: 238). In various embodiments, X1 is alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan (SEQ ID NO: 239). In various embodiments, X1 is valine (SEQ ID NO: 78). In various embodiments, X1 is serine, threonine, asparagine, or glutamine (SEQ ID NO: 240). In various embodiments, X1 is serine (SEQ ID NO: 76). In various embodiments, the IL-18 variant further comprises a propeptide having an amino acid sequence selected from the "Mature IL18 variant" column of Table 1, selected from the "Propeptide" column of Table 1, optionally selected from the same row as the IL-18 variant.

[0146] In various embodiments, the IL-18 variant further comprises an IL-18 propeptide variant and a truncation peptide. In various embodiments, the truncation peptide is selected from Table 1. As a specific example, IEQD (SEQ ID NO: 88) can be used.

[0147] In various embodiments, the fusion protein comprises an IL-18 variant selected from Table 1, a propeptide selected from Table 1, and a truncation peptide selected from Table 1 (optionally in the same row as the IL-18 variant and propeptide).

[0148] 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. WO2022 / 038417, the IL-18 variants and sequences of each of these patents or publications being incorporated herein by reference as if fully set forth.

[0149] Polynucleotides, vectors, and cells Various embodiments provide polynucleotides encoding the fusion proteins disclosed herein. For example, the polynucleotide may encode a first protein or polypeptide capable of entering and translocating into the ER and IL-18 (or a fragment, variant, or fragment of the variant) in a 5' to 3' direction. Non-limiting examples of such polynucleotides are listed in Table 2.

[0150] Additionally, the polynucleotide may also optionally comprise a "leader" or "signal" sequence, for example, based on (1) a propeptide (PP) directly linked to IL-18 (or an IL-18 variant), such as FUSE499, or (2) an immunoglobulin light chain sequence fused directly to the hinge region of an immunoglobulin heavy chain constant region. In some embodiments, when the protein / polypeptide capable of entering and translocating to the ER is based on an IgG sequence, the nucleic acid encodes, from 5' to 3', at least an immunoglobulin hinge region (i.e., a hinge region containing 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).

[0151] In various embodiments, a polynucleotide encoding a fusion protein may also be integrated into a replicable expression vector. Thus, a vector encoding the fusion protein is also provided, which can express the fusion protein in, for example, a bacterial host, an intended recipient, or both.

[0152] Additional embodiments provide cells transformed or transfected with one or more nucleic acid molecules (polynucleotides) encoding the fusion protein. The cells can be prokaryotic cells. Alternatively, the cells are eukaryotic cells, preferably mammalian cells, 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).

[0153] In some embodiments, non-human organisms transformed or transfected with one or more nucleic acid molecules encoding the fusion protein are also provided.

[0154] composition Further embodiments provide compositions comprising a combination of two or more different fusion proteins or combinations of nucleic acid sequences encoding fusion proteins, e.g., pharmaceutical compositions are provided in which the fusion proteins or nucleic acid molecules encoding the fusion proteins are the active agents.

[0155] Pharmaceutical compositions according to the present invention may also contain any pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or vehicle involved in carrying or transporting a compound of interest from one tissue, organ, or part of the body to another. For example, a carrier may 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" in that it must be compatible with the other components of the formulation. It must also be suitable for use in contact with tissues or organs with which it may come into contact, and must not pose a risk of toxicity, irritation, allergic reaction, immunogenicity, or other complications that unduly outweigh the therapeutic benefits. Pharmaceutical compositions according to the present invention may also be encapsulated, tableted, or prepared in an emulsion or syrup for oral administration. A pharmaceutically acceptable solid or liquid carrier may be added to enhance or stabilize the composition or to facilitate preparation of the composition. Liquid carriers include syrup, peanut oil, olive oil, glycerin, saline, alcohol, and water. Solid carriers include starch, lactose, calcium sulfate, dihydrate, terra alba, magnesium stearate or stearic acid, talc, pectin, acacia, agar, or gelatin. Carriers may also include sustained-release materials, such as glyceryl monostearate or glyceryl distearate, alone or with a wax. Pharmaceutical preparations are made according to conventional pharmaceutical techniques, including milling, mixing, granulating, and compressing, as needed, for tablet form, or milling, mixing, and filling for hard gelatin capsule form. When a liquid carrier is used, the preparation is in the form of a syrup, elixir, emulsion, or aqueous or non-aqueous suspension. Such liquid formulations may be administered directly or filled into soft gelatin capsules...Pharmaceutical compositions according to the present invention may be delivered in therapeutically effective amounts. The precise therapeutically effective amount is the amount of the composition that will produce the most effective results in terms of therapeutic efficacy in a given subject.This amount will vary depending on various factors, including, but not limited to, the characteristics of the therapeutic compound (activity, pharmacokinetics, pharmacodynamics, and bioavailability), the physiological condition of the subject (including age, sex, type and stage of disease, general physical condition, response to a given dose, and type of pharmaceutical), the nature of the pharmaceutically acceptable carrier in the formulation, and the route of administration. Those skilled in the clinical and pharmacological fields will be able to determine a therapeutically effective amount through routine experimentation, for example, by monitoring the subject's response to administration of the compound and adjusting the dosage accordingly. For additional guidance, see Remington: The Science and Practice of Pharmacy (Gennaro ed. 20th edition, Williams & Wilkins PA, USA) (2000).

[0156] Production method Methods for producing fusion proteins or nucleic acids encoding fusion proteins are also provided. Some embodiments provide that conventional recombinant DNA methodologies are utilized to generate the fusion proteins. The fusion construct is preferably generated at the DNA level, and the resulting DNA is incorporated into an expression vector and expressed to produce the fusion protein of the invention. The vector is then expressed in a host cell to obtain the fusion protein. Optionally, the method further comprises 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 comprises culturing cells transfected with an expression vector containing a nucleic acid encoding the fusion protein in a cell culture medium to produce the fusion protein and secrete it into the extracellular space for purification, which, upon contacting the fusion protein with a protease to cleave the fusion protein, produces IL-18, a fragment thereof, an IL-18 variant, or a fragment of an IL-18 variant. Exemplary nucleic acid molecules encoding fusion proteins are found in Table 2. Other embodiments provide that chemical conjugation using conventional chemical cross-linking agents can be used to fuse the protein moieties.

[0157] In some embodiments, the nucleic acid molecule encoding the fusion protein is expressed in CHO cells or HEK-293 cells. Preferably, expression of the fusion protein in the host cells results in at least 135 mg / L of secreted fusion protein recoverable from the host cell supernatant. In some embodiments, a fusion protein yield of at least 135 mg / L is obtained via transient transfection. In some embodiments, even higher yields of fusion protein, e.g., at least 150, 200, 250, or 300 mg / L, are obtained via stable producer cell clones or pools of clones. In some embodiments, transient transfection results in a fusion protein yield of about 130-400 mg / L. In some embodiments, the fusion protein, or IL-18, fragment thereof, IL-18 variant, or 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, 350-400 mg / L, 300-350 mg / L, 200-300 mg / L, 100-200 mg / L, or at least 50 mg / L.

[0158] In some embodiments, a Chinese hamster ovary (CHO) expression system is used to express the fusion protein by: (1) cell harvesting, which can be harvesting frozen CHO cells via a 37°C water bath; (2) cell subculture, which can be subcultured to achieve a cell density of 6 x 10 for transfection; 6 / ml; (3) Transfection and expression: Solution 1 (plasmid diluted in diluent) and Solution 2 (transfection reagent diluted in diluent) are used to mix Solution 1, Solution 2, and CHO cells, followed by incubating the mixture on a shaker at 32°C for 12 to 14 days to allow expression, and then collecting the culture supernatant after centrifugation.

[0159] In some embodiments, a purification process is performed after expression of the fusion protein, including (1) washing the column with binding buffer (10 volumes) at a flow rate of 1 mL / min, (2) loading the column with the fusion protein-containing sample at a flow rate of 1 mL / min, (3) washing the column with 10 volumes of PBS buffer at a flow rate of 1 mL / min, (4) eluting the protein from the sodium with 40 mM sodium citrate (pH 3.4) (optionally, the eluted sample can be collected in a tube (1 ml / min) and the optical density (OD) measured at 280 nm using a NanoDrop), and (5) performing dialysis, for example, overnight against PBS buffer in a dialysis bag.

[0160] Table 1. Amino acid sequences of each component in an exemplary fusion protein ("FUSE"). (Most identical sequences are labeled with the same number next to the component. For example, the "polypeptide 1" sequence, i.e., the first polypeptide arm sequence, is 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-506, FUSE-507, FUSE-508, FUSE-509, FUSE-510, FUSE-511, FUSE-512, FUSE-513, FUSE-514, FUSE-515, FUSE-516, FUSE-517, FUSE-518, FUSE-519, FUSE-520, FUSE-521, FUSE-522, FUSE-523, FUSE-524, FUSE-525, FUSE-526, FUSE-527, FUSE-528, FUSE-529, FUSE-530, FUSE-531, FUSE-532, FUSE-533, FUSE-534, FUSE-535, FUSE-536, FUSE-537, FUSE-538, FUSE-539, FUSE-540, FUSE-541, FUSE-542, FUSE-543, FUSE-544, FUSE-545, FUSE-546, FUSE-547, FUSE-548, FUSE-549, FUSE-550, FUSE-551, FUSE-552, FUSE-553, FUSE-55 FUSE-516, FUSE-517, FUSE-545, FUSE-546, FUSE547, FUSE-556, FUSE-583-587, FUSE-599-602, FUSE-645, FUSE-686, FUSE-756-758, FUSE-775, FUSE-874-876, and FUSE-878-892 are identical and are labeled with a "0" next to each component. Residues in lowercase are amino acid positions that may be subject to mutation. TIFF2025527303000002.tif220121TIFF2025527303000003.tif220161TIFF2025527303000004.tif220161TIFF2025527303000005.tif220157TIFF2025527303000006.tif220161TIFF2025527303000007.tif220161TIFF2025527303000008.tif220161TIFF2025527303000009.tif220161TIFF2025527303000010.tif220161TIFF2025527303000011.tif220161TIFF2025527303000012.tif220157TIFF2025527303000013.tif220157TIFF2025527303000014.tif220161TIFF2025527303000015.tif220161TIFF2025527303000016.tif220161TIFF2025527303000017.tif220157TIFF2025527303000018.tif220161TIFF2025527303000019.tif220161TIFF2025527303000020.tif220157TIFF2025527303000021.tif220161TIFF2025527303000022.tif220161TIFF2025527303000023.tif220161TIFF2025527303000024.tif220157TIFF2025527303000025.tif220157TIFF2025527303000026.tif220161TIFF2025527303000027.tif220161TIFF2025527303000028.tif220161TIFF2025527303000029.tif220161TIFF2025527303000030.tif220161TIFF2025527303000031.tif220157TIFF2025527303000032.tif220161TIFF2025527303000033.tif220161TIFF2025527303000034.tif220161TIFF2025527303000035.tif220126.

[0161] Table 2: Nucleotide sequences of each polypeptide in Table 1 TIFF2025527303000036.tif223153TIFF2025527303000037.tif223143TIFF2025527303000038.tif223148TIFF2025527303000039.tif223138TIFF2025527303000040.tif223148TIFF2025527303000041.tif223148TIFF2025527303000042.tif223148TIFF2025527303000043.tif223148TIFF2025527303000044.tif223148TIFF2025527303000045.tif223148TIFF2025527303000046.tif223148TIFF2025527303000047.tif223148TIFF2025527303000048.tif223143TIFF2025527303000049.tif223133TIFF2025527303000050.tif223162TIFF2025527303000051.tif223143TIFF2025527303000052.tif223162TIFF2025527303000053.tif223143TIFF2025527303000054.tif223162TIFF2025527303000055.tif223133TIFF2025527303000056.tif223162TIFF2025527303000057.tif223133TIFF2025527303000058.tif223133TIFF2025527303000059.tif223157TIFF2025527303000060.tif223160TIFF2025527303000061.tif223162TIFF2025527303000062.tif223162TIFF2025527303000063.tif223158TIFF2025527303000064.tif223162TIFF2025527303000065.tif223162TIFF2025527303000066.tif223162TIFF2025527303000067.tif223162TIFF2025527303000068.tif223161TIFF2025527303000069.tif223113TIFF2025527303000070.tif223162TIFF2025527303000071.tif223162TIFF2025527303000072.tif223162TIFF2025527303000073.tif223161TIFF2025527303000074.tif223161TIFF2025527303000075.tif223161TIFF2025527303000076.tif223161TIFF2025527303000077.tif223161TIFF2025527303000078.tif223161TIFF2025527303000079.tif223161TIFF2025527303000080.tif223162TIFF2025527303000081.tif223162TIFF2025527303000082.tif223160TIFF2025527303000083.tif223162TIFF2025527303000084.tif223162TIFF2025527303000085.tif223162TIFF2025527303000086.tif223162TIFF2025527303000087.tif223123TIFF2025527303000088.tif223162TIFF2025527303000089.tif223161TIFF2025527303000090.tif223162TIFF2025527303000091.tif223157TIFF2025527303000092.tif223161TIFF2025527303000093.tif223161TIFF2025527303000094.tif223158TIFF2025527303000095.tif223162TIFF2025527303000096.tif223162TIFF2025527303000097.tif223162TIFF2025527303000098.tif223143TIFF2025527303000099.tif223162TIFF2025527303000100.tif223113TIFF2025527303000101.tif223162TIFF2025527303000102.tif223162TIFF2025527303000103.tif223162TIFF2025527303000104.tif223162TIFF202552 7303000105.tif223161TIFF2025527303000106.tif223161TIFF2025527303000107.tif223161TIFF2025527303000108.tif22335.

[0162] Table 3. Yields of exemplary fusion proteins TIFF2025527303000109.tif22383

[0163] (Table 4) Amino acid sequences for Fc heterodimer formation, including different versions of the knobs-into-holes of each polypeptide arm, which, in the case of an exemplary fusion protein, form (part of) a protein that can translocate to the ER. TIFF2025527303000110.tif223135TIFF2025527303000111.tif223156TIFF2025527303000112.tif223145TIFF2025527303000113.tif22387

[0164] (Table 5) The sequences in the references, publications, patent documents, web pages, antibodies and fragments thereof (e.g., VHH, Fab regions, or scFv) contained in this table are incorporated herein by reference as if fully set forth. TIFF2025527303000114.tif224147TIFF2025527303000115.tif224158TIFF202 5527303000116.tif224161TIFF2025527303000117.tif224159TIFF2025527303 000118.tif224157TIFF2025527303000119.tif224163TIFF2025527303000120. tif224157TIFF2025527303000121.tif224162TIFF2025527303000122.tif22426 [Example]

[0165] The following examples are provided to better illustrate the claimed invention and should not be construed as limiting the scope of the invention. Where specific materials are mentioned, they are for illustrative purposes only and are not intended to limit the invention. One skilled in the art may develop equivalent means or reactants without the exercise of inventive capacity and without departing from the scope of the invention.

[0166] Example 1 We engineered mammalian cell-expressible IL-18 variants by fusing the N-terminus of pro-IL-18 to the C-terminus of the IgG1 CH3 domain. Pro-IL-18 was fused to the knob of a knob-into-hole heterodimeric IgG1 protein. Further modifications were made to pro-IL-18 such that each cysteine residue in both the propeptide and mature IL-18 was replaced with serine ("IL-18AS" in FUSE-480), alanine ("IL-18AA" in FUSE-481), or valine ("IL-18AV" in FUSE-442) to reduce aggregation of the molecule. Diagrams of the three variants are shown in Figure 1, panel A.

[0167] Transient transfection in the ExpiCHO system yielded titers of 191, 214, and 198 mg / L for FUSE-480, FUSE-481, and FUSE-442, respectively. These Fc-proIL-18 fusion proteins were hypothesized to contain a "masked" form of IL-18, reducing the biological activity of the fused IL-18 until the propeptide was cleaved. To assess propeptide cleavage, an enterokinase (EK)-specific cleavage site was inserted into the propeptide (pp) upstream of the mature IL-18 sequence at the position of the endogenous caspase 1 site. EK was chosen for its robust protease activity and activity in phosphate-buffered saline. A reporter system was used to assess the biological activity of the Fc-proIL-18 fusion proteins before and after treatment with EK. Here, IL-18 activity was quantified using HEK-Blue-IL-18 cells. HEK-Blue IL-18 cells are generated 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. The cells are also engineered to be unresponsive 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 via a colorimetric assay. We observed that all three mutants induced >1000-fold and up to 100,000-fold lower biological activity compared to recombinant human mature IL-18. Upon demasking (enzymatic cleavage) by treatment with EK, the biological activity of the released proteins from the alanine-substituted mutant (FUSE-480) and valine-substituted mutant (FUSE-442) was not substantially different from that of recombinant human mature IL-18. However, after release of the serine mutant with EK(1B), biological activity was restored by over 30,000-fold, but it was approximately 50-fold less potent than recombinant human mature IL-18. Potency was measured as the concentration of test substance that induced maximal SEAP production (EC50-SEAP).

[0168] As shown in Figure 2, pro-IL-18mut2 was fused to the knob of a knob-into-hole heterodimeric IgG1 protein. IL-18mut2 incorporates four amino acid substitutions hypothesized to reduce binding to IL-18BP while maintaining wild-type binding to the IL-18 receptor complex. Further modifications were made to pro-IL-18mut2 such that each cysteine residue in both the propeptide and mature IL-18mut2 was replaced with serine (IL-18mut2AS), alanine (IL-18mut2AA), or valine (IL-18mut2AV) to reduce aggregation of the molecule. Diagrams of the three variants are shown in Figure 2, panel A. Biological activity was assessed using HEK-Blue as in Figure 1. All three variants induced at least 100,000-fold lower biological activity compared to recombinant human mature IL-18. Upon demasking (enzymatic cleavage) by treatment with EK, the biological activity of the released proteins from the alanine-substituted mutant (FUSE-423) and valine-substituted mutant (FUSE-424) was not substantially different from that of recombinant human mature IL-18. However, after release of the serine mutant with (FUSE422), biological activity was restored approximately 100,000-fold, but the potency was approximately 100-fold lower than that of recombinant human mature IL-18. Potency was measured as the concentration of test substance that induced maximal SEAP production (EC50-SEAP).

[0169] As shown in Figure 3, the effect of the propeptide on masking the biological activity of IL-18AV was examined in the context of Fc fusion variants incorporating IL-18AV on the C-terminus of the Fc with (Panel A, FUSE-442, Fc-EKpp-IL-18AV) or without (Panel B, FUSE-505, Fc-EK-IL-18AV) the propeptide. For fusions in which the propeptide was removed, the EK cleavage site, which replaced the caspase 1 site, was moved directly between the CH2 domain of the knob and mature IL-18AV without the addition of a flexible linker. Using the HEK-Blue IL-18AV reporter cell assay as a readout, these cells were exposed to titrations of FUSE-442 or FUSE-505 with or without treatment with EK. The biological activity of FUSE-442 and -505, measured as EC50-SEAP, was attenuated approximately 150-fold and 15,000-fold, respectively, compared to human recombinant IL-18AV (Panel C), with the incorporation of the propeptide contributing an additional approximately 100-fold attenuation compared to the Fc fusion alone. Treatment with EK, designed to cleave the IL-18AV fragment from FUSE-505 and FUSE-442, resulted in restoration of biological activity. In the case of FUSE-442, treatment with EK resulted in biological activity that was not substantially different from that of recombinant human mature IL-18AV. However, for FUSE-505, although biological activity was restored after treatment with EK, potency remained approximately 30-fold reduced compared to recombinant human mature IL-18AV. The EC50-SEAP for each compound is shown in Panel D. Considering that both FUSE-505 and FUSE-442 carry the same IL-18AV, the difference in activity after demasking with EK could not be explained by any differences in the IL-18AV variant. Rather, EK was observed to release IL-18AV from FUSE-505 (<10% cleavage efficiency) much less efficiently than FUSE-4442 (>95% cleavage efficiency). Therefore, the reduced biological activity observed from EK-cleaved FUSE-505 versus EK-cleaved 442 was the result of less IL-18AV being released by the former than the latter.We hypothesize that the lack of a flexible linker between the CH3 domain of the knob and IL-18AV in FUSE-505 results in little access to the EK cleavage site. In contrast, the propeptide incorporated into FUSE-442 allowed sufficient access of EK to the cleavage site for efficient release of IL-18AV. The data strongly suggest that in the context of IL-18AV Fc fusion proteins, the propeptide is not required but contributes to masking the biological activity of IL-18AV. In the absence of the propeptide, masking is likely the result of steric hindrance mediated by the protein fused to the N-terminus of IL-18AV.

[0170] As shown in Figure 4, the effect of the propeptide on masking the biological activity of IL-18mut2AV was examined in the context of Fc fusion variants incorporating IL-18AVmut2 at the C-terminus of the Fc, as shown in Figure 3, with or without the propeptide (Panel A of Figure 4, FUSE-424, Fc-EKpp-IL-18mut2AV) or without (Panel B, FUSE-441, Fc-EK-IL-18mut2AV). The biological activity of FUSE-441 and FUSE424 was significantly attenuated by more than 100,000-fold compared to human recombinant IL-18 (Panel C). Treatment with EK resulted in restoration of biological activity. In the case of FUSE-424, treatment with EK resulted in biological activity that was not substantially different from recombinant human mature IL-18. However, for FUSE-441, although most of the biological activity was restored after treatment with EK, potency remained approximately 10-fold reduced compared to recombinant human mature IL-18. The EC50-SEAP of each compound is shown in Panel D. Considering that both FUSE-441 and FUSE-442 possess the same IL-18mut2AV, the difference in activity after demasking with EK could not be explained by any differences in the IL-18 variants. Rather, EK was observed to release IL-18mut2AV from FUSE-441 (less than 20% cleavage efficiency) much less efficiently than FUSE-424 (more than 95% cleavage efficiency). Therefore, the reduced biological activity observed with EK-cleaved FUSE-441 versus EK-cleaved FUSE-424 was likely the result of less IL-18mut2AV being released by the former compared to the latter. Given the lack of a flexible linker between the CH3 domain of the knob and IL-18mut2AV in FUSE-441, we hypothesize that the EK cleavage site is largely inaccessible. For example, the flexible linker could be a propeptide, or the propeptide could behave as a flexible linker. In contrast, the propeptide incorporated into FUSE-424 allowed EKs sufficient access to its cleavage site for efficient release of IL-18mut2AV.The data strongly suggest that in the context of IL-18mut2AV Fc fusion proteins, the propeptide is not required for masking the biological activity of IL-18mut2AV, but rather is the result of steric hindrance mediated by the protein fused to the N-terminus of IL-18mut2AV. Therefore, 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, the Fc is approximately 28 kDa as a monomer, the HSA is approximately 66 kDa, and the VHH is approximately 14 kDa) can mask the biological activity of IL-18mut2AV. We hypothesize that an EK cleavage site containing a linker of sufficient size (e.g., 25 amino acids or larger) to allow access to EK when incorporated between the Fc or other N-terminal protein mask and IL-18mut2AV will substitute for the EK-containing propeptide. Furthermore, if such N-terminally masked proteins or fragments thereof are engineered or naturally transported through the endoplasmic reticulum (ER), expression yields from transient transfection of mammalian cells such as Expi-CHO will be acceptable for therapeutic development.

[0171] As shown in Figure 5, we investigated the effect of fusing the propeptide-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 formats, one molecule of propeptide-IL-18AV fusion was linked to each Fc-CH3 domain, resulting in two molecules of propeptide-IL-18AV fusion per IgG1 or IgG4 homodimer. As previously described, an EK-specific cleavage site was inserted between the propeptide and mature IL-18 sequence at the location of the endogenous caspase-I site. The HEK-Blue-IL-18 cell reporter system was used to assess the biological activity of the Fc-proIL-18 fusion protein before and after treatment with EK. Using this system as a readout, HEK-Blue-IL-18 cells were exposed to titrations of FUSE-507 (IgG1Fc-EKpp-IL-18AV) or FUSE-509 (IgG4Fc-EKpp-IL-18AV) with or without treatment with EK. The biological activity of FUSE-507 and -509, measured as EC50-SEAP, was significantly attenuated by >10,000-fold compared to human recombinant IL-18 (Panel C). Treatment with EK, designed to cleave the IL-18AV fragment from FUSE-507 and FUSE-509, resulted in a recovery of biological activity approximately twofold higher than that of hrIL-18. This difference is likely a result of the release of two IL-18AV molecules per IgG1 or IgG4 fusion upon complete cleavage of the IgG1 or IgG4 fusion proteins by EK, resulting in a molar ratio of IL-18AV to hrIL-18 of approximately 2:1. The EC50-SEAP for each compound is shown in panel D. Versions of FUSE-507 and FUSE-509 without the propeptide were also generated: IgG1Fc-EK-IL-18AV and IgG4Fc-EK-IL-18AV, respectively. These did not express well, likely due to the tendency of IL-18 to form homodimers and the absence of a flexible linker between the CH3 domains of IgG1 or IgG4 and mature IL-18AV (data not shown).The data show that in the context of propeptide-IL-18AV fusions, both expression of over 135 mg / L and masking / attenuation of IL-18AV biological activity are more than 100,000-fold higher (compare Figure 5 with Figures 1 and 3), regardless of whether IL-18AV is fused to the C-terminus of wild-type IgG1 or IgG4 (two molecules of IL-18AV) or to the IgG1 knob-into-hole heterodimer (one molecule of IL-18AV). Formats utilizing wild-type IgG1 or IgG4 demonstrate the ease of plug-and-play fusion of propeptide-IL-18 fusions, their variants, and fragments to a range of monoclonal antibodies, including commercially available ones such as avelumab (anti-PDL1), cetuximab (anti-EGFR), and trastuzumab (anti-HER2 / neu).

[0172] To address the generality of masking of IL-18 and its variants by polypeptides fused to the N-terminus of the mature form of IL-18, we investigated the ability of N-terminal proteins (masks) distinct from the Fc domain of IgG to attenuate the biological activity of IL-18-AV, with or without the propeptide, as shown in Figure 6. The N-terminus of IL-18AV with (Panel A, FUSE-501) or without (Panel B, FUSE-503) the propeptide was fused to the C-terminus of human serum albumin (HSA). For fusions in which the propeptide was removed, an EK cleavage site, replacing the caspase 1 site, was transferred directly between the C-terminus of HSA and mature IL-18AV without the addition of a flexible linker. Using the HEK-Blue IL-18 reporter cell assay as a readout, these cells were exposed to titrations of FUSE-501 (HSA-EKpp-IL-18AV) or FUSE-503 (HSA-EK-IL-18AV), with or without treatment with EK. The biological activity, measured as EC50-SEAP, of FUSE-501 (Panel C) was significantly attenuated approximately 35,000-fold compared to human recombinant IL-18. FUSE-503 (Panel D), which does not contain the propeptide, was also attenuated approximately 3,500-fold. Treatment with EK, designed to cleave the IL-18AV fragment from FUSE-501 and FUSE-503, resulted in restoration of biological activity. In the case of FUSE-501, treatment with EK resulted in biological activity that was not substantially different from that of recombinant human mature IL-18. However, for FUSE-503, although biological activity increased with EK treatment, it remained approximately 40-fold weaker than human recombinant IL-18. The EC50-SEAP for each compound is shown in panel E. Given that both FUSE-501 and FUSE-503 possess the same IL-18AV, the difference in activity after demasking with EK could not be explained by any differences in the IL-18 variants. Rather, EK was observed to be much less efficient at releasing IL-18AV from FUSE-503 (less than 20% cleavage efficiency) than FUSE-501 (more than 95% cleavage efficiency).Therefore, the reduced biological activity observed from EK-cleaved FUSE-503 versus EK-cleaved -501 was the result of less IL-18AV released by the former than the latter. We hypothesized that the lack of a flexible linker between the C-terminus of HSA and IL-18AV in FUSE-503 renders the EK cleavage site less accessible. In contrast, the propeptide incorporated into FUSE-501 allowed sufficient access of EK to its cleavage site for efficient release of IL-18AV. Taken together with the data obtained from the Fc fusions, the results strongly indicate that the propeptide is not required for masking IL-18AV biological activity in the context of HSA-IL-18AV fusion proteins, but may contribute to greater attenuation when present in HSA-IL-18AV fusion proteins. For both FUSE-501 and -503, masking appears to be the result of steric hindrance mediated by the protein fused to the N-terminus of IL-18AV. Thus, this data further supports that any N-terminal protein of sufficient size can mask the biological activity of IL-18AV, and that an EK cleavage site containing a linker of sufficient size to allow access to EK when incorporated between Fc, HSA, or other N-terminal protein masks and IL-18AV will substitute for the EK-containing propeptide. Furthermore, if such N-terminal masking proteins or fragments thereof are engineered or naturally transported through the endoplasmic reticulum (ER), expression yields from transient transfection of mammalian cells such as Expi-CHO will be acceptable for therapeutic development.

[0173] To address the generality of masking of IL-18 and its variants by polypeptides fused to the N-terminus of the mature form of IL-18, we examined the ability of N-terminal proteins (masks) distinct from the Fc domain of IgG to attenuate the biological activity of IL-18-mut2AV, with or without the propeptide, as shown in Figure 7. The N-terminus of IL-18mut2AV with (Panel A, FUSE-502) or without (Panel B, FUSE-504) the propeptide was fused to the C-terminus of human serum albumin (HSA). For fusions in which the propeptide was removed, an EK cleavage site replacing the caspase 1 site was moved directly between the C-terminus of HSA and mature IL-18AV without the addition of a flexible linker. Using the HEK-Blue IL-18 reporter cell assay as a readout, these cells were exposed to titrations of FUSE-502 (HSA-EKpp-IL-18mu2AV) or FUSE-504 (HSA-EK-IL-18mut2AV), with or without treatment with EK. The biological activity of FUSE-502 (Panel C) and FUSE-504 (Panel D), measured as EC50-SEAP, was significantly attenuated by at least approximately 30,000-fold and approximately 50,000-fold, respectively, compared to human recombinant IL-18. Treatment with EK, designed to cleave the IL-18mut2AV fragment from FUSE-504 and FUSE-502, resulted in restoration of biological activity. In the case of FUSE-502, treatment with EK resulted in biological activity that was not substantially different from that of recombinant human mature IL-18 (Panel C). For FUSE-504, most of the biological activity was restored after treatment with EK, but potency remained approximately three-fold reduced compared to recombinant human mature IL-18 (Panel D). The EC50-SEAP for each compound is shown in Panel E. Given that both FUSE-504 and FUSE-502 possess the same IL-18mut2AV, the difference in activity after demasking with EK could not be explained by any differences in the IL-18 variant. Rather, EK was observed to be much less efficient at releasing IL-18mut2AV from FUSE-504 (less than 20% cleavage efficiency) than FUSE-502 (more than 95% cleavage efficiency).Therefore, the reduced biological activity observed with EK-cleaved FUSE-504 versus EK-cleaved FUSE-502 was the result of less IL-18mut2AV released by the former compared to the latter. We hypothesize that the lack of a flexible linker between the C-terminus of HAS and IL-18mut2AV in FUSE-504 renders the EK cleavage site largely inaccessible. In contrast, the propeptide incorporated into FUSE-502 allowed EK sufficient access to the cleavage site for efficient release of IL-18mut2AV. Taken together with the data obtained from the Fc fusions, the results strongly indicate that, in the context of the HSA-IL-18mut2AV fusion protein, the propeptide is not required to mask the biological activity of IL-18mut2AV, but rather is the result of steric hindrance mediated by the protein fused to the N-terminus of IL-18mut2AV. Thus, this data further supports that any N-terminal protein of sufficient size can mask the biological activity of IL-18mut2AV, and that an EK cleavage site containing a linker of sufficient size to allow access to EK when incorporated between Fc, HAS, or other N-terminal protein masks and IL-18mut2AV will substitute for the EK-containing propeptide. Furthermore, if such N-terminal masking proteins or fragments thereof are engineered or naturally transported through the endoplasmic reticulum (ER), expression yields from transient transfection of mammalian cells such as Expi-CHO will be acceptable for therapeutic development.

[0174] As shown in Figure 8, we investigated 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 show exemplary diagrams of IL-18AV fused to the knob of a knob-into-hole IgG1-Fc domain with (FUSE-499; Fc-ppIL-18AV; Panel A) or without (FUSE-500; Fc-IL-18AV; Panel B) the propeptide. In this case, we used the wild-type propeptide, in which all cysteine residues were replaced with valine but the caspase 1 site was maintained. To enable translocation of both constructs into the endoplasmic reticulum (ER) of mammalian cells and their expression / secretion, a signal peptide from the Ig kappa chain (IgK leader) was encoded upstream of either the propeptide in FUSE-499 or the IL-18AV in FUSE-500. Using the HEK-Blue IL-18 reporter cell assay as a readout, we investigated the ability of the C→V (AV) propeptide to mask IL-18AV (FUSE-499) and the ability of caspase-1 to demask / restore biological activity. The biological activity of FUSE-499, measured as EC50-SEAP, was significantly attenuated by >50,000-fold compared to human recombinant IL-18 (Panel C). In contrast, in the absence of the propeptide in FUSE-499, no reduction in biological activity was observed compared to human recombinant IL-18, indicating that the IL-18-Fc fusion protein is fully functional in the absence of the propeptide (or another polypeptide) attached to the N-terminus of mature IL-18. Importantly, demasking FUSE-499 with caspase-1 resulted in the restoration of biological activity nearly identical to that of human recombinant 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 biological activity. This data indicates that in a construct where IL-18AV is linked to the N-terminus of IgG, the propeptide is required for attenuation.Combined with previous data showing 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 without the presence of the propeptide, we hypothesize that any polypeptide of sufficient size fused to the N-terminus of mature IL-18 and / or its variants and fragments can mask the biological activity of IL-18. The smallest polypeptide tested was the propeptide (approximately 6 kDa), although polypeptides as small as 2 kDa are sufficient. In various embodiments, the short polypeptide or protein is approximately 2 kDa or 250 kDa or less. Furthermore, the propeptide in FUSE-499 does not naturally transport through the ER, but was engineered to do so by adding an IgK leader sequence upstream of it. This indicates that any polypeptide of sufficient size fused to the N-terminus of mature IL-18 and / or its variants and fragments is expected to attenuate the biological activity of IL-18. That is, the N-terminal polypeptide may naturally translocate into the endoplasmic reticulum (ER) or may be engineered to do so. In either case, transport through the ER is important for obtaining expression yields acceptable for therapeutic development from transient transfection of mammalian cells such as Expi-CHO.

[0175] As shown in Figures 9A-9I, we investigated whether proteases other than EK could be used to demask / activate pro-IL-18. Therefore, we selected (a) the matrix metalloproteinase (MMP), MMP2, which has been 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 accumulate in inflamed tumors. For MMP2, the EK cleavage site within the propeptide (pp) of FUSE-442 (Fc-EKpp-IL-18AV) was (a) replaced with the MMP2 / 9 cleavage sequence (GPLGVR (SEQ ID NO: 89)) to generate FUSE-486 (Fc-MMP2pp-IL-18AV) and (b) replaced with 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 forward slash preferentially cleaves the aforementioned peptide sequence. For granzyme B, the EK cleavage site within the propeptide (pp) of both FUSE-442 (Fc-EKpp-IL-18AV) and FUSE-424 (Fc-EKpp-IL-18mut2AV) was replaced with the prototypical cleavage site of granzyme B (IEQD (SEQ ID NO: 88)) to generate FUSE-485 (Fc-GzmBpp-IL-18AV) and FUSE-462 (Fc-GzmBpp-IL-18mut2AV). FUSE-486, FUSE-487, FUSE-485, and FUSE-462 are illustrated in Figures 9A and 9D. Using a HEK-Blue IL-18 reporter cell assay as a readout, these cells were exposed to titrations of (a) FUSE-486 or FUSE-487 with or without treatment with recombinant human MMP2 (9B), or (b) FUSE-485 or FUSE-462 (FUSE-485 and FUSE-462) with or without treatment with recombinant human granzyme B (see Figure 9D).The biological activity of the MMP prodrug fusions FUSE486 and FUSE487, measured as EC50-SEAP, was significantly attenuated by up to approximately 5,000-fold compared to rhIL-18. After treatment of FUSE-486 with MMP2, the biological activity of the demasked IL-18 AV fragment was restored to within 3-fold of rhIL-18. Next, a granzyme B cleavage site was added immediately C-terminal to the MMP site of FUSE486 to create FUSE587 (black triangles). As seen in Figure 9C, FUSE587 was attenuated by approximately 3,000-fold compared to recombinant human IL-18.

[0176] Interestingly, we observed that cleavage of FUSE587 with MMP2 still released an IL-18AV variant that was approximately 100-fold attenuated compared to recombinant IL-18. In contrast, cleavage with granzyme B released an IL-18AV variant with activity similar to that of recombinant IL-18. Granzyme B cleavage results in the release of mature IL-18AV without any N-terminal residues comprising the overhang, whereas MMP2 cleavage of FUSE486 and FUSE587 leaves behind 11- and 15-amino acid N-terminal polypeptide overhangs, respectively. We speculated that these overhangs may attenuate IL-18AV activity, albeit to a lesser extent than the full-size variant propeptide. This phenomenon was further investigated in Figures 11 and 13.

[0177] FUSE-485 and FUSE-462 were also significantly attenuated by >15,000-fold compared to human recombinant IL-18 (Figures 9E and 9F). Treatment with recombinant human granzyme B (rhGb), designed to cleave the IL-18AV and IL-18mut2AV fragments from FUSE-485 and FUSE-462, respectively, restored biological activity. In both cases, the masked IL-18 variants showed nearly identical attenuation, and the restored biological activity was approximately two-fold weaker than that of recombinant human mature IL-18. Furthermore, no significant difference was observed between the activities of demasked IL-18AV and IL-18mut2AV.

[0178] A summary table of the potency (EC50-SEAP) of each test substance is shown below in Figures 9A-9B and 9F.

[0179] Next, we investigated whether our findings could form the basis of 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). Therefore, we used cetuximab as a proof-of-concept TAA-targeting protein for pro-IL-18 fusion. Cetuximab is an EGFR-targeting monoclonal antibody commercially used to treat 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. Fusion of pp-Gb-IL-18AV to the CH3 domain of cetuximab, which forms a natural homodimer, was found to result in poor expression. This was presumably due to the presence of two Fabs, as the knob-into-hole IgG1 format, in which ppGb-IL-18AV was fused to the C-terminal knob (or hole) and an EGR-specific Fab was fused to the N-terminal knob (or hole), showed good expression. As shown in panel G, FUSE-517 (cetuximab_KiH_ppGb-IL-18-AV) consists of the Fab from 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, these cells were exposed to titrations of FUSE-517 (9H) with or without treatment with rhGb. Compared to rhIL-18, FUSE-517 was significantly attenuated by >250,000-fold. Treatment of FUSE-517 with rhGb, designed to cleave IL-18AV, restored biological activity to within 2-fold of rhIL-18.

[0180] Figure 9I is a summary table of potencies (EC50-SEAP) related to the data shown in Figure 9H. Overall, the data strongly indicate that both MMP2 and granzyme B can demask / activate IL-18AV (and IL-18mut2AV with granzyme B) in the context of a pp mask fused between the IgG CH3 domain and the mature IL-18AV fragment. Considering that other polypeptides that translocate through the ER can be successfully used as masks when fused to the N-terminus of several IL-18 variants, our observations strongly suggest that masked fusion proteins of IL-18, its variants, or its fragments can be engineered to be selectively activated by proteases found in tumors / inflammed tumors, including MMPs and granzymes. As shown in Figure 10, we examined the sensitivity of selected IL-18 variants to attenuation of their biological activity by their natural antagonist, IL-18BP. Panel A of Figure 10 shows the IL-18 fusion proteins examined. These variants are FUSE-442 (Fc-EKpp-IL-18AV) and FUSE-424 (Fc-EKpp-IL-18mut2AV). Both variants contain the same cysteine-to-valine substitution, but FUSE-424 has the mature IL-18AV (designated IL-18mut2AV) with the following mutations downstream of the polypeptide (pp): M51K, K53G, M60L, and M113V. These four residues within the mature 18-kDa fragment of IL-18 have previously been described as important for binding / masking wild-type IL-18. Therefore, we hypothesized that substituting these residues would reduce binding to IL-18BP, allowing IL-18AVmut2 to maintain biological activity in the presence of IL-18BP, which is often over-represented relative to IL-18 in the tumor microenvironment (TME). As in previous examples, HEK Blue IL-18 was used to assess MYD88-driven SEAP-mediated biological activity, and potency was reported as EC50-SEAP. FUSE-442 (Fc-EKpp-IL-18AV) and FUSE-424 (Fc-EKpp-IL-18mut2AV) were treated with EK to release mature IL-18AV or IL-18mut2AV, respectively.Each cleavage product was then titrated from 1 pg / ml to 1 μg / ml in medium alone or medium 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. Panels B (rhIL-18), D (FUSE-442), and F (FUSE-424) are nonlinear xy plots of SEAP release (IL-18R reporter activity) on the y-axis versus the concentration of test substance on the x-axis in the presence or absence of IL-18BP. A summary of the biological potency (EC50-SEAP) associated with each graph is shown in panels C, E, and G (below each xy plot). As has been well documented, we observed potent attenuation of rhIL-18 by rhIL-18BP, at least approximately 300-1000-fold. For IL-18AV released from FUSE422, we observed approximately 100-150-fold attenuation of biological activity (i.e., approximately 3-6-fold lower than rhIL-18), suggesting that the apparent affinity of IL-18AV for IL-18BP is weaker than that of rhIL-18. Importantly, IL-18mut2AV, a cleavage product of FUSE424, appeared to be resistant to biological attenuation by rhIL-18BP. As seen in panel F, no significant difference in biological activity was observed between IL-18mut2AV released from FUSE424 and the same molecule exposed to rhIL-18BP. This data strongly suggests that IL-18mut2AV binds to rhIL-18BP with much weaker apparent affinity than IL-18AV or rhIL-18. Thus, a masked form of IL-18mut2AV designed to be released by proteases present in the TME, including but not limited to MMP2, 9, and 14, and / or proteases released in inflamed tumors, including but not limited to granzymes A, B, and M, can be predicted to function in the presence of IL-18BP to promote antitumor activity through multiple pathways, including but not limited to IFNγ-mediated Th1 and Tc1 activity.

[0181] Example 2 As shown in Figure 11, we investigated 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 shows an exemplary diagram of IL-18AV fused onto the knob of a knob-into-hole IgG1-Fc domain with polypeptides of different sizes, ranging from the propeptide variant (FUSE-499, shown in Figure 18) to 35 amino acids (FUSE756) to 15 amino acids (FUSE757 and FUSE758). To enable translocation of all constructs into and expression / secretion by the endoplasmic reticulum (ER) of mammalian cells, a signal peptide from the Ig kappa chain (IgK leader) was encoded upstream of the polypeptide (and cleaved by signal peptidase in the ER). The FUSE756 and FUSE757 polypeptides consist of a series of glycine and serine residues, while the FUSE758 polypeptide is an overhanging sequence generated by MMP2-mediated cleavage of the MMP cleavage site KPLGLQARVVGGGG (SEQ ID NO: 252). In these three cases, the C-terminus of the N-terminal polypeptide also incorporated the granzyme B site IEQD (SEQ ID NO: 88). Using a 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, black squares) or rhIL-18 (black crosshatched "X"). All polypeptides fused to the N-terminus of mature IL-18AV reduced biological activity, measured as EC50-SEAP, by at least 100-fold. FUSE499 (black triangles) was the most attenuated (>10,000-fold). FUSE757 (black diamonds) and 758 (white inverted triangles), both of which contain an 11 amino acid polypeptide fused to the N-terminus of mature IL-18AV, were approximately 250-fold less biologically active than FUSE500 or rhIL-18. FUSE756 (white triangles), which contains one of the 31 amino acid polypeptides fused to the N-terminus of mature IL-18AV, was approximately 1000-fold less biologically active than FUSE500 or rhIL-18.Thus, all tested polypeptides, as well as larger proteins including HSA and fragments of IgG from previous examples, fused to the N-terminus of mature IL-18AV, regardless of size or amino acid composition, 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.

[0182] Example 3 Techniques and procedures HEK-Blue-IL-18 cell activation assay using IL-18 or FUSE protein Invivogen's HEK-BLUE™-IL-18 cells were maintained in culture medium (DMEM medium containing 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 selection reagent). HEK-BLUE™ IL-18 cells are engineered from the human embryonic kidney 293 (HEK293) cell line and stably express genes encoding the IL-18 receptor (IL-18R) and IL-18 receptor accessory protein (IL-18RAP), as well as the NF-κb / AP-1-inducible secreted embryonic alkaline phosphatase (SEAP) reporter gene. Thus, solutions such as QUANTIBLUE™ solution are useful for detecting bioactive IL-18 by monitoring activation of the NF-Kb and AP-1 pathways via quantitation of SEAP levels in the supernatant (produced upon activation of NF-Kb). Furthermore, responses to human TNF-α and IL-1β are blocked in HEK-BLUE™ IL-18 cells, and therefore HEK-BLUE™ IL-18 cells specifically respond to IL-18.

[0183] On the day of experimental setup, HEK-BLUE-IL-18 cells were gently rinsed twice with pre-warmed phosphate-buffered saline (PBS) and then detached in PBS by tapping the flask. Dissociated HEK-Blue-IL-18 cells were added at a concentration of 5 x 10 per mL.5 Cells were resuspended in prewarmed test medium (DMEM containing 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 100 μL. To seed the cells (50,000 cells per well), 100 μL of resuspended HEK-Blue-IL-18 cells were added to designated wells in a 96-well plate.

[0184] For protein preparation, FUSE protein was diluted in test medium by a 5-fold serial dilution from 500,000 pg / mL to 6.4 pg / mL (twice the final concentration). IL-18 was also diluted in test medium by a 5-fold serial dilution from 800 pg / mL to 1.28 pg / mL (twice the final concentration). To treat HEK-Blue-IL-18 cells, 100 μL of prepared IL-18 or FUSE protein was added to the designated wells of a 96-well plate containing 50,000 cells, and the protein was then gently mixed with the cells. The 96-well plate was then incubated at 37°C and 5% CO2 for 24 hours.

[0185] After 24 hours of activation, HEK-Blue-IL-18 cells released secreted alkaline phosphatase into the supernatant. 20 μL of HEK-Blue-IL-18 cell culture supernatant was transferred to a new 96-well plate. Meanwhile, Invivogen's QUANTI-Blue solution was prepared by adding 1 mL of QB reagent and 1 mL of QB buffer to 98 mL of sterile water. 180 μL of the prepared QUANTI-Blue solution was then added to the 96-well plate along with 20 μL of 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.

[0186] HEK-Blue-IL-18 cell activation assay using enterokinase (EK)-cleaved FUSE protein Invivogen HEK-Blue-IL-18 cells were maintained in culture medium. On the day of experimental setup, HEK-Blue-IL-18 cells were gently rinsed twice with pre-warmed phosphate-buffered saline (PBS) and then detached in PBS by tapping the flask. Dissociated HEK-Blue-IL-18 cells were cultured at 5 x 10 per mL. 5 The cells were resuspended in pre-warmed test medium at a density of 100 μL of resuspended HEK-Blue-IL-18 cells to seed the cells (50,000 cells per well) into designated wells in a 96-well plate.

[0187] For FUSE protein cleavage, 4 μg of FUSE protein was mixed with 80 ng of EK enzyme and 2 μL of 10x PBS in a tube. Sterile water was added to the tube to bring the total reaction volume to 20 μL. The tube was then incubated at 25°C for 40 minutes. After 40 minutes of incubation, the cleaved FUSE protein was diluted in test medium by a 5-fold serial dilution from 500,000 pg / mL to 6.4 pg / mL (twice the final concentration). IL-18 was also diluted in test medium by a 5-fold serial dilution from 800 pg / mL to 1.28 pg / mL (twice the final concentration). To treat HEK-Blue-IL-18 cells, 100 μL of prepared IL-18 or cleaved FUSE protein was added to the designated wells of a 96-well plate containing 50,000 cells, and the cells were then gently mixed with the protein. The 96-well plates were then incubated at 37°C, 5% CO2 for 24 hours.

[0188] 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 along with 20 μL of 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.

[0189] HEK-Blue-IL-18 cell activation assay using matrix metalloproteinase (MMP)-cleaved FUSE protein Invivogen HEK-Blue-IL-18 cells were maintained in culture medium. On the day of experimental setup, HEK-Blue-IL-18 cells were gently rinsed twice with pre-warmed phosphate-buffered saline (PBS) and then detached in PBS by tapping the flask. Dissociated HEK-Blue-IL-18 cells were cultured at 5 x 10 per mL. 5 The cells were resuspended in pre-warmed test medium at a density of 100 μL of resuspended HEK-Blue-IL-18 cells to seed the cells (50,000 cells per well) into designated wells in a 96-well plate.

[0190] 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 tube. Sterile water was added to the tube to bring the total reaction volume to 28 μL. The tube was then incubated at 37°C for 2 hours. After the 2-hour incubation, the cleaved FUSE protein was diluted in test medium by a 5-fold serial dilution from 500,000 pg / mL to 6.4 pg / mL (twice the final concentration). IL-18 was also diluted in test medium by a 5-fold serial dilution from 800 pg / mL to 1.28 pg / mL (twice the final concentration). To treat HEK-Blue-IL-18 cells, 100 μL of prepared IL-18 or cleaved FUSE protein was added to the designated wells of a 96-well plate containing 50,000 cells, and the cells were gently mixed with the protein. The 96-well plate was then incubated at 37°C, 5% CO 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, 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 along with 20 μL of 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.

[0192] HEK-Blue-IL-18 cell activation assay using caspase I-cleaved FUSE protein Invivogen HEK-Blue-IL-18 cells were maintained in culture medium. On the day of experimental setup, HEK-Blue-IL-18 cells were gently rinsed twice with pre-warmed phosphate-buffered saline (PBS) and then detached in PBS by tapping the flask. Dissociated HEK-Blue-IL-18 cells were cultured at 5 x 10 per mL. 5 The cells were resuspended in pre-warmed test medium at a density of 100 μL of resuspended HEK-Blue-IL-18 cells to seed the cells (50,000 cells per well) into designated wells in a 96-well plate.

[0193] For FUSE protein cleavage, 14 μg of FUSE protein was mixed with 0.5 units of caspase I enzyme and 2.8 μL of 10x assay buffer (500 mM Hepes, pH 7.2, 500 mM NaCl, 1% Chaps, 100 mM EDTA, 50% glycerol, and 100 mM DTT) in a tube. Sterile water was added to the tube to bring the total reaction volume to 28 μL. The tube was then incubated at 37°C for 2 hours. After the 2-hour incubation, the cleaved FUSE protein was diluted in test medium by a 5-fold serial dilution from 500,000 pg / mL to 6.4 pg / mL (twice the final concentration). IL-18 was also diluted in test medium by a 5-fold serial dilution from 800 pg / mL to 1.28 pg / mL (twice the final concentration). To treat HEK-Blue-IL-18 cells, 100 μL of prepared IL-18 or cleaved FUSE protein was added to the designated wells of a 96-well plate containing 50,000 cells, and the cells were gently mixed with the protein. The 96-well plate was then incubated at 37°C, 5% CO for 24 hours.

[0194] 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 along with 20 μL of 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.

[0195] HEK-Blue-IL-18 cell activation assay using granzyme B-cleaved FUSE protein Invivogen HEK-Blue-IL-18 cells were maintained in culture medium. On the day of experimental setup, HEK-Blue-IL-18 cells were gently rinsed twice with pre-warmed phosphate-buffered saline (PBS) and then detached in PBS by tapping the flask. Dissociated HEK-Blue-IL-18 cells were cultured at 5 x 10 per mL. 5 The cells were resuspended in pre-warmed test medium at a density of 100 μL of resuspended HEK-Blue-IL-18 cells to seed the cells (50,000 cells per well) into designated wells in a 96-well plate.

[0196] Mature 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 tube. Sterile water was added to the tube to bring the total reaction volume to 20 μL. The tube was then incubated at 25°C for 40 minutes. After the 40-minute incubation, activated human granzyme B was used to cleave FUSE protein. Briefly, 10 μg of fuse protein was mixed with 1 μg of activated human granzyme B in 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 the specified time indicated in each experiment.

[0197] Granzyme B-cleaved FUSE protein was then diluted in test medium by a 5-fold serial dilution to 500,000 pg / mL to 6.4 pg / mL (twice the final concentration). IL-18 was also diluted in test medium by a 5-fold serial dilution to 800 pg / mL to 1.28 pg / mL (twice the final concentration). To treat HEK-Blue-IL-18 cells, 100 μL of prepared IL-18 or cleaved FUSE protein was added to designated wells containing 50,000 cells in a 96-well plate, and the cells were then gently mixed with the protein. The 96-well plate was then incubated at 37°C and 5% CO2 for 24 hours.

[0198] 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 along with 20 μL of 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.

[0199] Blockade of IL-18 or FUSE protein-induced HEK-Blue-IL-18 cell activation by IL-18BP Invivogen HEK-Blue-IL-18 cells were maintained in culture medium. On the day of experimental setup, HEK-Blue-IL-18 cells were gently rinsed twice with pre-warmed phosphate-buffered saline (PBS) and then detached in PBS by tapping the flask. Dissociated HEK-Blue-IL-18 cells were cultured at 5 x 10 per mL. 5 The cells were resuspended in pre-warmed test medium at a density of 100 μL of resuspended HEK-Blue-IL-18 cells to seed the cells (50,000 cells per well) into designated wells in a 96-well plate.

[0200] Uncleaved or cleaved FUSE protein was prepared in test medium by 5-fold serial dilution from 1,000,000 pg / ml to 12.8 pg / ml (four times the final concentration). IL-18BP was diluted in test medium to a concentration of 5,000,000 pg / ml (four times the final concentration). To treat HEK-Blue-IL-18 cells, 50 μL of the prepared uncleaved or cleaved FUSE protein and 50 μL of the prepared IL-18BP were mixed and incubated for 1 hour, then added to designated wells in a 96-well plate with 50,000 cells. The cells were then gently mixed with the protein. The 96-well plate was then incubated at 37°C and 5% CO2 for 24 hours. Because our fusion protein exhibited reduced binding to the IL-18R complex, it is likely that it would also exhibit reduced binding to IL-18BP compared to the truncated FUSE protein, since both IL-18R and IL-18BP compete for binding to IL-18. Recent crystal structures of binary and ternary complexes of hIL-18 and its receptor indicate that IL-18BP binds to hIL-18 in direct competition with the hIL-18Rα D3 domain, 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).

[0201] 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 along with 20 μL of 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.

[0202] Biolayer Interferometry Binding Assay The coating protein was prepared in 1x 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 dilutions ranging from 400 nM to 1.6 nM) in 1x PBS containing 0.02% Tween-20. The biosensor was pre-wetted in 200 μL of 1x PBS containing 0.02% Tween-20 for 10 minutes. Meanwhile, the Octet BLI system (ForteBio) was pre-warmed for 30 minutes, with the flow rate set to 1000 rpm. The biosensor (capture biosensor) was immersed in 250 μL of 1x PBS containing 0.02% Tween-20 for 60 seconds at 30°C to obtain an initial baseline measurement. After the 60-second baseline reading, the biosensor was exposed to the coating protein for 300 seconds at 30°C to allow for association between the antibody and the biosensor (binding of the coating protein to the biosensor). Next, for the association reaction between the coating protein and the capture protein (association curve), the biosensor containing the coating protein was exposed to the capture protein in 250 μL of 1x PBS containing 0.02% Tween-20 for 300 seconds at 30 °C. After the 300-second association reaction between the coating protein and the capture protein, the biosensor containing the coating protein and the capture protein was exposed to 250 μL of 1x PBS containing 0.02% Tween-20 for 300 seconds at 30 °C. For the dissociation reaction between the coating protein and the capture protein (dissociation curve), the biosensor containing the coating protein and the capture protein was exposed to 250 μL of 1x PBS containing 0.02% Tween-20 for 300 seconds at 30 °C. For binding to IL18BP, each IL18 variant was coated onto an AHC biosensor chip 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 onto a nickel biosensor chip and probed with each recombinant IL18 variant at concentrations ranging from 400 nM to 1.6 nM. Binding affinities were calculated by built-in data fitting algorithms.

[0203] Example 4 As shown in Figure 12, 11 variants of human IL-18 (designated IL-18AV) were engineered and their ability to bind to recombinant human IL-18BP and recombinant human IL-18RA (also called IL-18Rα) was measured. All test substances were produced as Fc fusion proteins in which the IL-18 variant was fused to the N-terminus of the Fc (see Figure 11A). The positions and amino acid substitutions associated with each variant are shown in Figure 12A.

[0204] To assess binding to human IL18BP or human IL18Rα, kinetic binding graphs were generated via biolayer interferometry (BLI) using the Octet system (ForteBio). For binding to IL18BP, each IL18 mutant was coated onto an AHC biosensor chip 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 onto a nickel biosensor chip and probed with each recombinant IL18 mutant at concentrations ranging from 400 nM to 1.6 nM (Figure 12D). Summary tables of binding (binding affinity (KD), on-rate (k-on), and off-rate (k-dis)) to IL-18BP and IL18-RA are shown in Figures 12C and 12E, respectively.

[0205] All 11 IL-18 mutants bound to IL18BP with weaker affinity than FUSE500 (wild-type human IL-18-AV-Fc). Five mutants were not associated with significant binding: FUSE545, FUSE599, FUSE600, FUSE601, and FUSE602.

[0206] In contrast, all 11 mutants maintained significant binding to IL18RA, with affinities ranging from 11 nM to 30 nM, only 1.5- to 4-fold weaker than the affinity of FUSE500 for IL-18RA (7.4 nM).

[0207] Example 5 The binding affinities of 11 mutant variants of human IL-18 (denoted IL-18AV) to human IL18BP and human IL18Rα were tabulated as shown in the table below. Each IL-18 protein was produced as an Fc fusion protein, whereby the IL-18 variant was fused to the N-terminus of Fc (see Figure 11A). TIFF2025527303000123.tif115161

[0208] Example 6 As shown in Figure 13, 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 was examined.

[0209] Polypeptides of different sizes were investigated, ranging from a single N-terminal amino acid (FUSE874; inverted white triangle) and five amino acids (FUSE875) to the propeptide variant of FUSE-499 (upward-pointing black triangle; see Figure 11 for an illustration). FUSE500 was used as a full activity control without the N-terminal polypeptide.

[0210] To enable all constructs to translocate into, and be expressed / secreted by, the endoplasmic reticulum (ER) of mammalian cells, a signal peptide from the Ig kappa chain (IgK leader) was encoded upstream of the polypeptide (and cleaved by a signal peptidase in the ER).

[0211] The polypeptides of FUSE875 (5 residues; black star), FUSE876 (10 residues; white diamond), FUSE757 (15 residues; white circle), and FUSE756 (35 residues; black inverted triangle) consist of a series of glycine and serine residues. The N-terminal polypeptide related to FUSE758 (white triangle) is the sequence of the overhang generated by MMP2 cleavage of FUSE486 (see Figure 9). For FUSE756, FUSE757, and FUSE758, the last four amino acids of the N-terminal peptides consisted of IEQD (SEQ ID NO: 88), the granzyme B cleavage site (see Figure 11).

[0212] Using the HEK-Blue IL-18 reporter cell assay as a readout, we investigated the ability of polypeptides of different sizes to attenuate / mask IL-18AV-Fc (FUSE500, black squares) or rhIL-18 (black cross "X"). All polypeptides and single amino acids (serine) fused to the N-terminus of mature IL-18AV reduced biological activity, measured as EC50-SEAP, by at least 10-fold.

[0213] The degree of attenuation increased with increasing size of the polypeptide, resulting in a rank order of attenuation observed as follows: FUSE499>FUSE756, FUSE757, FUSE758>FUSE876, FUSE875>FUSE874. A summary table of potency (EC50-SEAP) is shown below the nonlinear xy graph.

[0214] Example 7 As shown in Figures 14A-14H, the effect of substitution of cysteine residues in the propeptide and mature IL-18, which were fused together to form the pro-IL-18 variant cassette, on the biological activity of each variant was examined using the HEK Blue IL-18 assay system. Unless otherwise noted, proteins were generated such that the propeptide-IL-18 variant was fused to the C-terminal knob or hole of knob-into-hole human IgG1 Fc, as previously shown in Figure 1A, which added a granzyme B cleavage site between the propeptide variant and mature IL-18 variant. The three variants evaluated in Figure 1 contained serine (FUSE480), alanine (FUSE481), or valine (FUSE442) substitutions for all cysteine residues in pro-IL-18. The mature IL-18 variants released from FUSE442 and FUSE481 were nearly as active as recombinant human IL-18, whereas the mature IL-18 released from FUSE480 (serine substitution) was approximately 100-fold attenuated compared to recombinant human IL-18 (Figures 1B-1D).

[0215] In this example, a variant of proIL-18 in which all cysteines have been replaced with valines contains an N-terminal EGFR-specific VHH (FUSE516, black triangles), and a variant of proIL-18 in which all cysteines have been replaced with serine contains an N-terminal PD-1-specific Fab (FUSE694, black diamonds). The biological activity of proIL-18 test substances was assessed using HEK Blue IL-18, as shown in Figure 1. Each was tested as an intact, untreated protein or after exposure to recombinant human granzyme B.

[0216] Figure 14A shows the results for FUSE516 (black triangles), in which the cysteine residues in its propeptide variant have been replaced with valines and all cysteines in its mature IL18 variant have been replaced with valines. Intact FUSE516 is approximately 1000-fold attenuated compared to recombinant human IL18, and the mature IL18 variant (white triangles) released from FUSE516 by granzyme B is nearly as active as recombinant human IL18.

[0217] Figure 14B shows the results for FUSE694 (black diamonds), in which the cysteine residues in its propeptide variant have been replaced with serine and all cysteines in its mature IL18 variant have been replaced with serine. Intact FUSE694 is over 10,000-fold attenuated compared to recombinant human IL18, and the mature IL18 variant released from FUSE694 by granzyme B (white diamonds) is approximately 60-fold less active than recombinant human IL18.

[0218] Figure 14C shows the results for FUSE887 (black inverted triangles), in which the cysteine residues in its propeptide variant have been replaced with threonines and all cysteines in its mature IL18 variant have been replaced with serine. Intact FUSE887 is over 10,000-fold attenuated compared to recombinant human IL18, and the mature IL18 variant (white inverted triangles) released from FUSE887 by granzyme B is approximately 100-fold less active than recombinant human IL18.

[0219] Figure 14D shows the results for FUSE888 (black triangles), in which the cysteine residues in its propeptide variant have been replaced with glutamines and all cysteines in its mature IL18 variant have been replaced with serine. Intact FUSE888 is over 10,000-fold attenuated compared to recombinant human IL18, and the mature IL18 variant (open triangles) released from FUSE888 by granzyme B is approximately 100-fold less active than recombinant human IL18.

[0220] Figure 14E shows the results for FUSE889 (black squares), in which the cysteine residues in its propeptide variant have been replaced with aspartic acid and all cysteines in its mature IL18 variant have been replaced with alanines. Intact FUSE889 is approximately 10,000-fold attenuated compared to recombinant human IL18, and the mature IL18 variant (white squares) released from FUSE889 by granzyme B is approximately 100-fold less active than recombinant human IL18.

[0221] Figure 14F shows the results for FUSE890 (black inverted triangles), in which the cysteine residues in the propeptide variant have been replaced with phenylalanines and all cysteines in the mature IL18 variant have been replaced with alanines. Intact FUSE890 is approximately 10,000-fold attenuated compared to recombinant human IL18, and the mature IL18 variant (white inverted triangles) released from FUSE890 by granzyme B is approximately 100-fold less active than recombinant human IL18.

[0222] Figure 14G shows the results for FUSE891 (black triangles), in which the cysteine residues in its propeptide variant have been replaced with isoleucine and all cysteines in its mature IL18 variant have been replaced with valine. Intact FUSE891 is approximately 3,000-fold attenuated compared to recombinant human IL18, and the mature IL18 variant (white triangles) released from FUSE891 by granzyme B is approximately 100-fold less active than recombinant human IL18.

[0223] Figure 14H shows the results for FUSE892 (black inverted triangles), in which the cysteine residues in its propeptide variant have been replaced with histidines and all cysteines in its mature IL18 variant have been replaced with valines. Intact FUSE892 is approximately 3,000-fold attenuated compared to recombinant human IL18, and the mature IL18 variant released from FUSE892 by granzyme B (white inverted triangles) is approximately 100-fold less active than recombinant human IL18.

[0224] A summary table of potency (EC50-SEAP) is shown to the right of each non-linear xy plot.

[0225] Example 8 As shown in Figures 15A-15B, we investigated the effect of targeting pro-IL18 to its receptor complex (i.e., "cis-active"). The pro-IL18 variants tested were (a) those in which all cysteine residues were replaced with serine (pro-IL18AS, FUSE782, and FUSE827) and (b) those in which all cysteines were replaced with valine (pro-IL18AV, FUSE783, and FUSE785). Pro-IL18AS or pro-IL18AV was fused to the N-terminal knob or hole of the PD1-specific knob-into-hole antibody (FUSE782 and FUSE783). The Fab fragments of these fusion proteins were derived from nivolumab. To test whether targeting pro-IL18 variants to modified PD-1 on the same cells expressing the IL-18R complex (cis effect) enhanced biological activity compared to cells not modified with PD-1 (trans effect), we (1) used HEK Blue IL18 as an IL18R complex-positive reporter system and (2) either used cells unmodified or modified with the extracellular domain of PD-1 using a CD46-specific Fab (clone YS5) in the N-terminal hole and a bispecific antibody (FUSE986) containing the PD-1 extracellular domain in the N-terminal knob. CD46 was chosen because its expression has been reported in the parental HEK 293 cell line (jitc.bmj.com / content / 6 / 1 / 55) and confirmed with HEK Blue IL18 (not shown). We also generated pro-IL18AS (FUSE827) and pro-IL18AV (FUSE775), which are functionally untargeted and can function only in trans. FUSE827 does not contain a targeting domain (i.e., Fc only), and FUSE775 replaces the PD-1-specific Fab with EGFR-specific VHH, 9G8, whose ligand (EGFR) is not expressed on HEK Blue IL18 (data not shown). Figures 15A and 15B are nonlinear xy plots of IL18 biological activity versus the concentration of each test substance using HEK Blue IL18 (Figure 15A) and PD-1-modified HEK Blue IL18 (Figure 15B).As previously observed for proIL18AS and proIL18AV, serine substitutions result in greater attenuation than valine substitutions. Thus, when HEK Blue IL18 (FIG. 15A, transactivation only) was treated with (a) the valine mutants FUSE783 (open inverted triangles) or FUSE775 (closed triangles), we observed a 1000-fold attenuation compared to recombinant human IL18, and (b) the serine mutants FUSE782 (open triangles) and FUSE827 (closed inverted triangles), we observed a greater than 100,000-fold attenuation compared to recombinant human IL18.

[0226] With regard to cis activity (Figure 15B using PD-1-modified HEK Blue IL18), we observed increased biological activity from the PD-1-targeted versions pro-IL18AS (FUSE782; approximately 100-fold compared to non-targeted FUSE827) and pro-IL18AV (FUSE783; approximately 30-fold compared to non-targeted FUSE775).

[0227] FUSE691 (nivolumab) was used as a negative control antibody in both Figures 15A and 15B. No biological activity was observed from this test substance. Comparing Figures 15A and 15B, no significant differences were observed in the biological activity of recombinant human IL18 or the non-targeting test substances (FUSE775 and FUSE827). In this context, the PD-1-targeted versions pro-IL18AS (FUSE782) and pro-IL18AV (FUSE783) were approximately 100- and 30-fold more active than unmodified HEK Blue IL-18 when exposed to PD-1-modified HEK Blue IL-18.

[0228] A summary table of the potency of each pro-IL18 variant is shown below each graph.

[0229] Various embodiments of the present invention are described above in the detailed description. While these descriptions directly describe the above embodiments, it should be understood that those skilled in the art may conceive of modifications and / or variations to the specific embodiments shown and described herein. Any such modifications or variations that fall within the scope of this description are intended to be included therein. Unless otherwise specified, it is the inventors' intention that the words and phrases in this specification and claims be given their ordinary and accustomed meanings to those skilled in the art.

[0230] The foregoing description of various embodiments of the present invention known to applicant at the time of filing is presented and intended for purposes of illustration and description. This description is not intended to be exhaustive or to limit the invention to the precise form disclosed, as many modifications and variations are possible in light of the above teachings. The described embodiments serve to illustrate the principles of the present invention and its practical application, enabling those skilled in the art to utilize the invention in various embodiments and various modifications suited to the particular use intended. Therefore, it is intended that the invention not be limited to the particular embodiments disclosed for carrying out the invention.

[0231] While specific embodiments of the present 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 present invention and its broader aspects; therefore, the appended claims are intended to encompass within their scope all such changes and modifications as fall within the true spirit and scope of the present invention. As used herein, the terms "comprising" or "comprises" are used in reference to compositions, methods, and their respective components that may include unspecified elements, whether useful or not, that are useful in an embodiment. Those skilled in the art will generally understand that the terms used herein are generally intended as "open" terms (e.g., the term "including" should be interpreted as "including, but not limited to," the term "having" as "having at least," the term "includes" as "including, but not limited to," etc.). For purposes of describing and claiming the present invention, the open-ended term "comprising" is used synonymously with terms such as including, containing, or having, although the present invention or embodiments thereof may alternatively be described using other terms such as, for example, "consisting of" or "consisting essentially of."

[0232] Unless otherwise indicated, the terms "a," "an," and "the," and similar references, as used in the context of describing specific embodiments of the present application (particularly in the context of the claims), can be construed to cover both the singular and the plural. The recitation of ranges of values herein is merely intended to serve as a shorthand method of individually referring to each separate value falling within the range. Unless otherwise indicated herein, each separate value is incorporated herein as if it were individually listed herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context. The use of any and all examples, or illustrative language (e.g., "etc."), provided with respect to specific embodiments herein is intended merely to further illustrate the application and does not limit the scope of the application as claimed. The abbreviation "eg" is derived from the Latin "exempli gratia" and is used herein to indicate a non-limiting example. Thus, the abbreviation "eg" is synonymous with the term "for example." No language in the specification should be construed as indicating any non-claimed element essential to the practice of the application. "Optional" or "optionally" means that the circumstance described below may or may not occur, and the description therefore includes instances in which the circumstance occurs and instances in which it does not occur. Groupings of alternative elements or embodiments of the disclosure disclosed herein should not be construed as limitations. Each group member may be referenced and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group may be included in, or deleted from, a group for reasons of convenience and / or patentability. When such inclusion or deletion occurs, the specification is deemed to contain the modified group and, therefore, satisfies the description of all Markush groups used in the appended claims.

Claims

1. a first polypeptide or protein or fragment thereof capable of translocating into the endoplasmic reticulum (ER); Interleukin 18 (IL-18), a fragment of IL-18, an IL-18 variant, or a fragment of an IL-18 variant. A fusion protein comprising: the IL-18, fragment of IL-18, IL-18 variant, or fragment of an IL-18 variant is C-terminal to the fusion protein relative to the first polypeptide or protein capable of translocating into the ER; The fusion protein.

2. The IL-18 variant is an amino acid sequence comprising amino acid positions 37-193 of SEQ ID NO:250, with 1-5 amino acid substitutions at positions E42, M87, K89, M96, and M149 of SEQ ID NO:250; The fusion protein of claim 1 ,

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

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

5. The IL-18 variant is an amino acid sequence comprising positions 37-193 of SEQ ID NO:251 with 1-5 amino acid substitutions at positions E42, M87, K89, M96, and M149 of SEQ ID NO:251; The fusion protein of claim 1 ,

6. the 1 to 5 amino acid substitutions are E42K, E42R, E42A, E42H, or E42Q; M87K or M87H; K89G, K89A, or K89E; M96L, or M96I; or M149V or M149I The fusion protein according to any one of claims 2 to 5, wherein the fusion protein is one or more of:

7. 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 The fusion protein according to any one of claims 2 to 5,

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

9. The IL-18 propeptide variant is AAEPVEDNX １ INFVAMKFIDNTLYFIAEDDEN (SEQ ID NO: 238), wherein X １ is any amino acid except cysteine.

10. X １ is alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan (SEQ ID NO: 239).

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

12. The fusion protein of any one of claims 9 to 11, wherein the PP or the PP variant is N-terminal to the IL-18, fragment of IL-18, IL-18 variant, or fragment of an IL-18 variant.

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

14. The fusion protein of any one of claims 1 to 13, further comprising one or more protease cleavage sites.

15. the one or more protease cleavage sites between said IL-18, a fragment of IL-18, an IL-18 variant, or a fragment of an IL-18 variant and said first protein or a fragment thereof capable of translocating into the endoplasmic reticulum (ER); or Within the PP, between the PP or the PP variant and the IL-18, a fragment of IL-18, an IL-18 variant, or a fragment of an IL-18 variant, or within the PP, between the PP or the PP variant and the first protein or fragment thereof capable of translocating into the endoplasmic reticulum (ER), or within said IL-18, fragment of IL-18, IL-18 variant, or fragment of an IL-18 variant, or within said PP; or combinations of these The fusion protein of claim 14,

16. 16. The fusion protein of any one of claims 1 to 15, further comprising a second protein or a fragment thereof capable of translocating into the ER, wherein the second protein capable of translocating into the ER is C-terminal to the interleukin-18 (IL-18), fragment of IL-18, IL-18 variant, or fragment of an IL-18 variant.

17. 17. The fusion protein of claim 16, further comprising a protease cleavage site between the IL-18, fragment of IL-18, IL-18 variant, or fragment of an IL-18 variant and the second protein or fragment thereof capable of translocating into the endoplasmic reticulum (ER).

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

19. The fusion protein of any one of claims 16 to 18, wherein the second protein or a fragment thereof capable of translocating into the ER is 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.

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

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

22. Binding affinity of the fusion protein comprising the IL-18 variant to IL-18BP: Binding affinity of the fusion protein comprising the IL-18 variant to IL-18R The fusion protein according to any one of claims 1 to 19, wherein the ratio of

23. 23. The fusion protein of any one of claims 1 to 22, wherein the first protein or fragment thereof capable of translocating into the ER is a globular protein, an immunoglobular protein, or a fragment thereof, or a short polypeptide or protein engineered with a signal peptide for translocation into the ER, optionally wherein the short polypeptide or protein is about 2 kDa or 250 kDa or less.

24. 24. The fusion protein of any one of claims 1 to 23, wherein the first protein or fragment thereof capable of translocating into the ER is selected from the group consisting of a fragment crystallizable (Fc) region, human serum albumin (HSA), beta2 microglobulin, transferrin, a fragment antigen-binding region (Fab region), a VHH antibody, a single-chain variable fragment (scFv), anticalins, designed ankyrin repeat proteins (DARPins), binding domains thereof, and fragments thereof.

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

26. 26. The fusion protein of any one of claims 16 to 25, wherein the second protein or fragment thereof capable of translocating into the ER is a globular protein, an immunoglobular protein, or a fragment thereof, or a short polypeptide or protein engineered with a signal peptide for translocation into the ER, optionally wherein the short polypeptide or protein is about 2 kDa or 250 kDa or less.

27. 27. The fusion protein of any one of claims 16 to 26, wherein the second protein or fragment thereof capable of translocating into the ER is selected from the group consisting of a fragment crystallizable (Fc) region, human serum albumin (HSA), beta2 microglobulin, transferrin, a fragment antigen-binding region (Fab region), a VHH antibody, a single-chain variable fragment (scFv), anticalins, designed ankyrin repeat proteins (DARPins), binding domains thereof, and fragments thereof.

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

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

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

31. 28. The fusion protein of any one of claims 24 and 27, wherein the Fc region is an Fc region from knob-in-hole, HA-TF, Xmab, ZW1, 7.8.60, electrostatic steering, DD-KK, EW-RVT, A107, or Duobody.

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

33. 32. The fusion protein of any one of claims 1 to 31, wherein one or more cysteines in the fusion protein are substituted with a natural or unnatural amino acid.

34. 32. The fusion protein of any one of claims 1 to 31, wherein one or more cysteines in the IL-18, fragment of IL-18, IL-18 variant, or fragment of an IL-18 variant of the fusion protein are modified or substituted with a natural or unnatural amino acid.

35. 35. The fusion protein of any one of claims 8 to 34, wherein one or more cysteines in the PP or PP variant of the fusion protein are modified or substituted with a natural or unnatural amino acid.

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

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

38. 38. The fusion protein of any one of claims 17 to 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. 39. The fusion protein of any one of claims 1 to 38, having one or more sequences set out in any one of Tables 1 and 4.

40. 2. The fusion protein of claim 1, having polypeptide 1 and polypeptide 2 selected from Table 1, and optionally polypeptide 3.

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

42. 2. The fusion protein of claim 1, having a polypeptide 1 selected from Table 1, wherein polypeptide 1 comprises HSA.

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

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

45. AAEPVEDNX １ INFVAMKFIDNTLYFIAEDDEN, wherein X １ is any amino acid except cysteine (SEQ ID NO: 238).

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

47. 47. The polynucleotide of claim 46, wherein the first protein capable of translocating into the ER is encoded by a polynucleotide having one or more sequences set forth in Table 2.

48. 47. The polynucleotide of claim 46, having one or more sequences from the same row listed in Table 2.

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

50. A cell transfected with the expression vector of claim 49.

51. 1. A method for producing a fusion protein, comprising: Culturing cells transfected with the expression vector of claim 49 in cell culture medium to secrete the fusion protein into the cell culture medium. The method comprising:

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

53. 1. A method for producing interleukin-18 (IL-18), a fragment thereof, an IL-18 variant, or a fragment of an IL-18 variant, comprising: Culturing cells transfected with the expression vector of claim 49 in a cell culture medium to produce and secrete the fusion protein into the extracellular space; contacting the fusion protein with a protease to cleave the fusion protein to produce the IL-18, fragment thereof, IL-18 variant, or fragment of an IL-18 variant; The method comprising:

54. 54. The method of claim 53, further comprising isolating the fusion protein from the culture medium.

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

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

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