WO2024031046A2 - Protéines de fusion d' il-18 et procédés de production d'il-18 - Google Patents

Protéines de fusion d' il-18 et procédés de production d'il-18 Download PDF

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WO2024031046A2
WO2024031046A2 PCT/US2023/071663 US2023071663W WO2024031046A2 WO 2024031046 A2 WO2024031046 A2 WO 2024031046A2 US 2023071663 W US2023071663 W US 2023071663W WO 2024031046 A2 WO2024031046 A2 WO 2024031046A2
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fragment
variant
fusion protein
protein
amino acid
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PCT/US2023/071663
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WO2024031046A3 (fr
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Xueyuan Zhou
Brian Rabinovich
Jeffrey TAKIMOTO
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Fuse Biotherapeutics Inc.
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/04—Fusion polypeptide containing a localisation/targetting motif containing an ER retention signal such as a C-terminal HDEL motif
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/50—Fusion polypeptide containing protease site

Definitions

  • This invention relates to new compositions allowing for enhanced production (including expression, secretion, and purification) of recombinantly produced IL-18 cytokine, for example in mammalian cells, for masking and de-masking of the biological activity of IL-18, and/or for improved binding specificity of produced IL-18 to its receptor over its binding protein, while at least maintaining the binding affinity to its receptor, as well as methods for preparing the compositions.
  • IL interleukin
  • IL-18 belongs to the IL-1 superfamily, and is a proinflammatory cytokine that facilitates type 1 immunity responses.
  • IL-18 also known as interferon-gamma inducing factor, is encoded in humans by the IL-18 gene.
  • IL-18 gene similar to other IL-1 family members, lacks a signal peptide.
  • the IL-18 gene encodes for a 193 amino acid precursor protein, first synthesized as an inactive 24 kDa precursor with no signal peptide, which is cytosolic and accumulates in cell cytoplasm.
  • WO 97/24441 discloses a 193 amino acid protein corresponding to IL-18 precursor and encoding DNA.
  • the IL-18 precursor (also referred to as Pro-IL-18) is processed intracellularly (e.g., by caspase 1 (CASP1), chymase, and proteinase B) into its mature biologically active molecule of 18 kDa (157 amino acids; i.e., amino acid residues 37-193 of Uniprot ID Q14116). That is, upon cleavage, the propeptide breaks away from the rest of the precursor, resulting in mature IL- 18 and the propeptide which formerly inactivated the precursor IL-18.
  • the mature form of IL-18 is secreted and released into the extracellular milieu via at least three unconventional pathways.
  • the unconventional pathways listed below are in no particular order of frequency.
  • the first is called secretory autophagy, a process involved in the secretion of cytosolic proteins without a signal peptide (leaderless cargoes).
  • IL-18 interacts with cargo receptor transmembrane emp24 domain-containing protein 10 (TMED10), and the interaction mediates the translocation from the cytoplasm into the endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC), a compartment contributing membranes to the forming autophagosome, which acts as a mechanism for secretory cargo entry into the vesicle, thereby secretion.
  • TMED10 cargo receptor transmembrane emp24 domain-containing protein 10
  • IL-18R ⁇ ligand receptor IL-18 receptor alpha
  • IL-18RAP IL-18 receptor accessory protein
  • IL-18 extracellular interleukin 18 binding protein (IL-18BP) that binds soluble IL-18 with a higher affinity (approximately 0.4 pM, see Kim et al., Proc Natl Acad Sci USA. 2000;97(3):1190-1195) than IL-18R ⁇ thus prevents IL-18 binding to IL-18 receptor.
  • IL-18BP extracellular interleukin 18 binding protein
  • compositions of matter that allow for improved production (including expression, secretion, and purification) of recombinantly produced IL-18.
  • compositions of matter that allow for enhanced production of recombinantly produced IL-18, including modified IL-18 or a fragment thereof, which maintains a similar binding affinity to IL-18Ra/b compared to natural IL-18, and has a reduced binding affinity to IL-18BP compared to natural IL-18 (i.e. binding to both at ⁇ 18 nM); or more preferably, the binding affinity to IL- 18BP is weaker than that to IL-18Ra/b (i.e.
  • a fusion protein comprising: a first polypeptide or protein capable of translocating into an endoplasmic reticulum (ER) or a fragment thereof; and an interleukin 18 (IL-18), a fragment of IL-18, an IL-18 variant, or a fragment of the IL-18 variant, wherein the IL-18, the fragment of IL-18, the IL-18 variant, or the fragment of the IL-18 variant is on the C-terminus end of the fusion protein relative to the first polypeptide or protein capable of translocating into the ER.
  • IL-18 interleukin 18
  • the IL-18 variant can 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.
  • the IL-18 variant can 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 [016] In various embodiments, the IL-18 variant can 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 and one to five amino acid substitutions at positions E42, M87, K89, M96, and M149, of SEQ ID NO:251 [017] In various embodiments, the amino acid substitutions at one or more of C74, C104, C112, and C164 can be each independently to valine, alanine or serine.
  • 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.
  • 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.
  • the IL-18, a fragment of IL-18, an IL-18 variant, or a fragment of the IL-18 variant further comprises its propeptide (PP) or a PP variant.
  • the IL-18 propeptide variant comprises a polypeptide having AAEPVEDNX 1 INFVAMKFIDNTLYFIAEDDEN (SEQ ID NO:238), wherein X 1 can be any amino acid except cysteine .
  • X 1 can be alanine, valine, isoleucine, leucin, methionine, phenylalanine, tyrosine or tryptophan (SEQ ID NO:239).
  • X 1 can be valine (SEQ ID NO:78). In various embodiments, X 1 can be serine, threonine, asparagine, or glutamine (SEQ ID NO:240). In various embodiments, X 1 can be serine (SEQ ID NO:76).
  • the fusion protein does not comprise a polypeptide consisting of the sequence X 1 -X 2 -X 3 -X 4 between the propeptide or propeptide variant, and the mature IL-18 or mature IL-18 variant, wherein X 1 is L or absent, X 2 is E or absent, X 3 is S or absent, and X 4 is D or absent, and when X 1 , X 2 , X 3 , and X 4 are present, the polypeptide consisting of the sequence X 1 -X 2 -X 3 -X 4 is LESD (SEQ ID NO:253).
  • the PP or the PP variant can be on the N-terminus end relative to the IL- 18, the fragment of IL-18, the IL-18 variant, or the fragment of the IL-18 variant. [025] In various embodiments, the PP or the PP variant serves as a masking domain. [026] In various embodiments, the fusion protein further comprises one or more protease cleavage sites.
  • the one or more protease cleavage sites can be between the IL-18, a fragment of IL-18, an IL-18 variant, or a fragment of the IL-18 variant and the first protein capable of translocating into an endoplasmic reticulum (ER) or fragment thereof, or within the PP, between PP or the PP variant and the IL-18, a fragment of IL-18, an IL-18 variant, or a fragment of the IL-18 variant, or within the PP, between the PP or the PP variant and the first protein capable of translocating into an endoplasmic reticulum (ER) or fragment thereof, or within the IL-18, a fragment of IL-18, an IL-18 variant, or a fragment of the IL-18 variant, or within the PP, or a combination thereof.
  • ER endoplasmic reticulum
  • the fusion protein further comprises a second protein capable of translocating into the ER or a fragment thereof, wherein the second protein capable of translocating into the ER can be on the C-terminus end relative to the interleukin 18 (IL-18), the fragment of the IL-18, the IL-18 variant, or the fragment of the IL-18 variant.
  • the fusion protein further comprises a protease cleavage site between the IL-18, a fragment of IL-18, an IL-18 variant, or a fragment of the IL-18 variant and the second protein capable of translocating into an endoplasmic reticulum (ER) or fragment thereof.
  • the interleukin 18 (IL-18), the fragment of the IL-18, the IL-18 variant, or the fragment of the IL-18 variant can be fused to the C-terminus of the first protein capable of translocating into the ER.
  • the second protein capable of translocating into the ER or a fragment thereof can be fused to the C-terminus of the interleukin 18 (IL-18), the fragment of the IL-18, the IL-18 variant, or the fragment of the IL-18 variant.
  • the IL-18 variant can have diminished binding to IL-18 binding protein (IL-18BP), as compared to a wild type (wt) IL-18.
  • the IL-18 variant having a binding affinity to the human IL-18 receptor (IL-18R) within 30-fold of the wild-type IL-18.
  • the ratio of 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 can be no higher than 3:1.
  • the first protein capable of translocating into the ER or a fragment thereof can be a globular protein, immunoglobular protein, or a fragment thereof, or can be a short polypeptide or protein engineered with a signal peptide for translocating into the ER, optionally the short polypeptide or protein being about 2kDa or no greater than 250 kDa.
  • the first protein capable of translocating into the ER or a fragment thereof can be selected from the group consisting of a fragment crystallizable (Fc) region, human serum albumin (HSA), beta2microglobulin, transferrin, fragment antigen-binding region (Fab region), VHH antibody, single- chain variable fragment (scFv), anticalin, designed ankyrin repeat protein (DARPin), a binding domain thereof, and a fragment thereof.
  • Fc fragment crystallizable region
  • HSA human serum albumin
  • Fab region fragment antigen-binding region
  • VHH antibody single- chain variable fragment
  • DARPin single-chain variable fragment
  • the first protein capable of translocating into the ER or a fragment thereof of the fusion protein can be a type I transmembrane protein or a fragment thereof or a type II transmembrane protein or a fragment thereof.
  • the second protein capable of translocating into the ER or a fragment thereof can be a globular protein, immunoglobular protein, or a fragment thereof, or can be a short polypeptide or protein engineered with a signal peptide for translocating into the ER, optionally the short polypeptide or protein being about 2kDa or no greater than 250 kDa.
  • the second protein capable of translocating into the ER or a fragment thereof can be selected from the group consisting of a fragment crystallizable (Fc) region, human serum albumin (HSA), beta2microglobulin, transferrin, fragment antigen-binding region (Fab region), VHH antibody, single- chain variable fragment (scFv), anticalin, designed ankyrin repeat protein (DARPin), a binding domain thereof, and a fragment thereof.
  • the second protein capable of translocating into the ER or a fragment thereof of the fusion protein can be a type I transmembrane protein or a fragment thereof, or a type II transmembrane protein or a fragment thereof.
  • the Fc region can be an Fc region from IgA, IgM, IgG, or IgE. In various embodiments, the Fc region can be an Fc region from IgG4, KiH, or IgG1. In various embodiments, the Fc region can be an Fc region from Knob-in-hole, HA-TF, Xmab, ZW1, 7.8.60, Electrostatic Steering, DD-KK, EW-RVT, A107, or Duobody. [040] In various embodiments, one or more cysteines in the fusion protein can be modified. In various embodiments, one or more cysteines in the fusion protein can be replaced with a natural or non-natural amino acid.
  • one or more cysteines in the IL-18, the fragment of IL-18, the IL-18 variant, or the fragment of the IL-18 variant of the fusion protein can be modified or can be replaced with a natural or non-natural amino acid.
  • the one or more cysteines in the PP or PP variant of the fusion protein can be modified or can be replaced with a natural or non-natural amino acid.
  • the natural amino acid can be charged, polar uncharged, or hydrophobic.
  • the natural amino acid can be each independently selected from serine and valine.
  • the natural amino acid can be each independently selected from threonine, asparagine, and glutamine.
  • the natural amino acid can be each independently selected from alanine, isoleucine, leucine methionine, phenylalanine, tyrosine, and tryptophan.
  • the natural amino acid the natural amino acid can be each independently selected from phenylalanine, alanine, aspartic acid, and asparagine.
  • the natural amino acid can be valine.
  • the natural amino acid can be each independently selected from threonine, glutamine, aspartic acid, phenylalanine, isoleucine and histidine.
  • the protease can be selected from the group consisting of EK, TEV, Adam17, cathepsin, MMP2, MMP9, MMP14, Granzyme A, Granzyme B, Granzyme M, Granzyme K, and combinations thereof.
  • the fusion protein can have one or more sequences as set forth in any one in Tables 1 and 4.
  • the fusion protein can have polypeptide 1 and polypeptide 2, and optionally polypeptide 3 selected from Table 1.
  • the fusion protein can have polypeptide 1 and polypeptide 2, and optionally polypeptide 3 selected from Table 1, wherein polypeptide 1 and polypeptide 2, and optionally polypeptide 3 can be selected from the same row of Table 1.
  • the fusion protein can have polypeptide 1 selected from Table 1, wherein polypeptide 1 comprises HSA.
  • the IL-18 variant can have an amino acid sequence selected from Mature IL-18 column in Table 1.
  • the IL-18 variant can have an amino acid sequence selected from Mature IL-18 column in Table 1
  • the propeptide can have an amino acid sequence selected from Propeptide column in Table 1, optionally the same from the same row.
  • an IL-18 propeptide variant comprising a polypeptide having AAEPVEDNX 1 INFVAMKFIDNTLYFIAEDDEN, wherein X 1 can be any amino acid except cysteine (SEQ ID NO:238).
  • X 1 can be alanine, valine, isoleucine, leucin, methionine, phenylalanine, tyrosine or tryptophan (SEQ ID NO:239).
  • X 1 can be valine (SEQ ID NO:78).
  • X 1 can be serine, threonine, asparagine, or glutamine (SEQ ID NO:240).
  • X 1 can be serine (SEQ ID NO:76).
  • Various embodiments provide for a polynucleotide encoding any one of the fusion protein of the invention described herein.
  • the first protein capable of translocating into the ER can be encoded by a polynucleotide having one or more sequences as set forth in Table 2.
  • the polynucleotide can have one or more sequences from the same row as set forth in Table 2.
  • Various embodiments provide for an expression vector comprising any one of the polynucleotides of the invention described herein.
  • Various embodiments provide for a cell transfected with any one of the expression vector of the invention described herein.
  • the cell can be a mammalian cell. In various embodiments, the cell can be a bacterial cell.
  • a method of producing a fusion protein comprising: culturing a cell transfected with any one of the expression vectors of the invention as described herein, in cell culture medium to allow the fusion protein to be secreted into the cell culture medium.
  • the method further comprises isolating the fusion protein from the culture medium. In various embodiments, the method further comprises purifying the fusion protein.
  • the fusion protein can be produced at greater than 135 mg/L under transient transfection in CHO cells or HEK-293 cells.
  • Various embodiments provide for a method of producing interleukin 18 (IL-18), a fragment thereof, an IL-18 variant, or a fragment of the IL-18 variant, comprising: culturing a cell transfected with any one of the expression vectors of the invention as described herein, in cell culture medium to allow a fusion protein to be produced and secreted into the extracellular space; and contacting a protease to the fusion protein to cleave the fusion protein to produce the IL-18, the fragment thereof, the IL-18 variant, or the fragment of the IL-18 variant.
  • the method further comprises isolating the fusion protein from the culture medium.
  • the method further comprises purifying the fusion protein.
  • contacting the protease to the fusion protein comprises including the protease in the cell culture medium.
  • the protease can be selected from the group consisting of EK, TEV, Adam17, cathepsin, MMP2, MMP9, MMP14, Granzyme A, Granzyme B, Granzyme M, and combinations thereof.
  • the IL-18, the fragment thereof, the IL-18 variant, or the fragment of the IL-18 variant can be produced at greater than 135mg/L.
  • FIG. 1 panels A-E depicts 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; which has a structure from N- to C-terminus comprising knobs-in-hole (KiH) Fc – propeptide (pp) – enterokinase-cleavable site (EK) – IL-18 wild type or its variants.
  • KiH knobs-in-hole
  • pp propeptide
  • EK enterokinase-cleavable site
  • fusion proteins such as IDs: FUSE-480, FUSE-481, and FUSE- 442 in Table 1.
  • Further modification was made to pro-IL-18 to reduce aggregation of the molecule, wherein each cysteine residue in both the pro-peptide and mature IL-18 was replaced with serine (as in FUSE-480, denoted as “IL-18AS”), with alanine (as in FUSE-481, denoted as “IL-18AA”), or with valine (as in FUSE-442, denoted as “IL-18AV”).
  • IL-18AS serine
  • IL-18AA alanine
  • IL-18AV valine
  • the N-terminus of pro-IL-18 can be fused to the C-terminus of the hole chain of a KiH heterodimeric IgG1 proteins.
  • FIG. 1 The biological activity defined as the EC50-SEAP for each compound is shown in panel E.
  • Figure 2 depicts exemplary fusion proteins in which the N-terminal of pro IL-18 was fused to the C-terminal of an IgG1 CH3 domain (which is also a knob chain of a knob-into-hole heterodimeric IgG1 protein as in Figure 1), and the pro IL-18 incorporated four amino acid substitutions hypothesized to reduce binding to IL-18BP while maintaining wild type binding to the IL-18 receptor complex, denoted as “pro-IL- 18mut2”.
  • fusion proteins have a structure from N- to C-terminus comprising knobs-in-hole (KiH) Fc – propeptide (PP) – enterokinase-cleavable site (EK) – IL-18mut2. Further modification was made to the pro-IL- 18mut2 to reduce aggregation of the molecule, wherein each cysteine residue in both the pro-peptide and mature IL-18mut2 was substituted with serine (denoted as “IL-18mut2AS”, as in FUSE-422; panel B), with alanine (denoted as “IL-18mut2AA”, as in FUSE-423; panel C), or with valine (denoted as “IL-18mut2AV”, as in FUSE- 424; panel D).
  • KH knobs-in-hole
  • PP propeptide
  • EK enterokinase-cleavable site
  • IL-18mut2 enterokinase-cleavable site
  • FIG. 1 The biological activity defined as the EC50-SEAP for each compound is shown in panel E.
  • Figure 3 depicts exemplary fusion proteins with (panel A) or without (panel B) the propeptide to examine the impact on masking of “IL-18AV” biological activity (panel C), wherein Fc fusion variants were generated incorporating “IL-18AV” with the propeptide (panel A; FUSE-442) or without (panel B; FUSE-505) the propeptide.
  • FIG. 1 The biological activity defined as the EC50-SEAP for each compound is shown in panel D.
  • FIG. 4 depicts exemplary fusion proteins with (panel A) or without (panel B) the propeptide to examine the impact of propeptide on masking of “IL-18mut2AV” biological activity, wherein Fc fusion variants were generated incorporating “IL-18mut2AV” without the propeptide (hence, a mature IL-18 with mutation, denoted as “matIL-18mut2-AV”, see panel B; FUSE-441) or with the propeptide (panel A; FUSE-424).
  • Panel C depicts the activation readout using the HEK-Blue IL-18AV reporter cell assay following exposure to a titration of FUSE-441 (Fc-EK-IL-18AV) or FUSE-424 (Fc-EKpp-IL-18AV) with or without treatment with EK.
  • the biological activity defined as the EC50-SEAP for each compound is shown in panel D.
  • FIG. 5 depicts exemplary fusion proteins in which the N-terminus of pro-IL-18 is fused to the C-terminus of IgG1 Fc protein or IgG4 Fc; which has a structure from N- to C-terminus comprising IgG1 Fc- propeptide (pp) - IL-18AV (FUSE-507; panel A) and IgG4 Fc- propeptide (PP) - IL-18AV (FUSE-509; panel B).
  • Panel C depicts the activation readout using the HEK-Blue IL-18AV reporter cell assay of exposing the cells to a titration of FUSE-507 or FUSE-509 with or without treatment with EK.
  • FIG. 1 The biological activity defined as the EC50-SEAP for each compound is shown in panel D.
  • FIG. 1 Panels A-E depicts exemplary fusion proteins in which the N-terminus of pro-IL-18 is fused to the C-terminus of HSA with or without the propeptide (pp); which has a structure from N- to C-terminus comprising HSA- propeptide (PP) - IL-18AV (FUSE-501; panel A) and HSA- IL-18AV (FUSE-503; panel B).
  • Panels C and D depict the activation readout.
  • the biological activity defined as the EC50-SEAP for each compound is shown in panel E.
  • FIG. 7 depicts exemplary fusion proteins in which the N-terminus of pro-IL- 18mut2 is fused to the C-terminus of HSA with or without the propeptide (PP); which has a structure from N- to C-terminus comprising HSA- propeptide (PP) - IL-18mut2AV (FUSE-502; panel A) and HSA- IL-18mut2AV (FUSE-504; panel B).
  • Panels C and D depict the activation readout.
  • the biological activity defined as the EC50- SEAP for each compound is shown in panel E.
  • FIG. 8 depicts 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 or without the propeptide (PP); which has a structure from N- to C-terminus comprising propeptide (PP) - IL-18AV - knobs-in-hole (KiH) Fc (FUSE-499; panel A) and IL-18AV - knobs-in-hole (KiH) Fc (FUSE-500; panel B).
  • Panel C depicts the activation readout.
  • FIG. 9A and 9D depicts exemplary fusion proteins with a structure from N- to C- terminus: Fc- ppMMP2/9-cleavage sites-IL-18-AV (FUSE-486), Fc-ppMMP9/2-cleavage sites-IL-18-AV (FUSE-487), in which the cleavage sites are specific for the metalloproteases, MMP2 and MMP9, with preferred enzyme to the left of the forward-slash, or FUSE-485 (Fc-GzmBpp-IL-18AV) and FUSE-462 (Fc-GzmBpp-IL-18mut2AV).
  • FIG. 9B depicts the activation readout relating to FUSE-486 and FUSE-487, with or without MMP2 treatment.
  • FIG. 9C shows that FUSE587 was about 3,000-fold attenuated relative to recombinant human IL-18.
  • cleavage of FUSE587 with MMP2 released and IL- 18AV variant that was still about 100-fold attenuated relative to recombinant IL-18.
  • cleavage with Granzyme B released an IL-18AV variant with activity similar activity as recombinant IL-18.
  • Figures 9D and 9G also depicts exemplary fusion proteins with a structure from N- to C- terminus: Fc-ppGb-cleavage sites-IL-18-AV (FUSE-485; 9D) or Fc-ppGb-cleavage sites-IL-18mut2-AV (FUSE-462; 9D) or Fab-Cetuximab-Fc-ppGb-cleavage sites-IL-18-AV (FUSE-517; 9G), in which the cleavage site is specific for granzyme B (Gb).
  • Figure 9E depicts the activation readout relating to FUSE-462 and FUSE-485, with or without granzyme B treatment.
  • Figure 9F shows the biological activity defined as the EC50-SEAP for each of FUSE-462 and FUSE-485, with or without granzyme B treatment.
  • Figure 9H depicts the activation readout relating to FUSE- 517, with or without enzyme treatment.
  • Figure 9I shows the biological activity defined as the EC50-SEAP for FUSE-517, with or without granzyme B treatment.
  • Figure 10 panels A-G depicts the impact of IL-18BP on the biological activity of recombinant human IL-18 (rhIL-18) and the EK cleavage products of the exemplary fusion proteins, Fc-ppEK-IL-18-AV (FUSE-442) and Fc-ppEK-IL-18mut2AV (FUSE-424).
  • FIG. 11 panel A) depicts diagrams 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 different size polypeptides fused to the N-terminus of mature IL-18.
  • Figure 12A, 12B(i), 12B(ii), 12C, 12D(i), 12D(ii) and 12E depict human IL-18 engineered mutant fusion proteins in accordance with various embodiments of the invention.
  • Figure 13 shows the impact of the size of polypeptides 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.
  • Figure 14A-14H shows the impact of the substituting the cysteine residue in the pro-peptide and cysteine residues in the mature IL18, which were fused together to form the pro-IL-18 variant cassette, on the biological activity of each variant using the HEK Blue IL18 assay system.
  • Figure 15A-15B shows the impact of targeting pro-IL18 to within close proximity of its receptor complex (i.e., “cis activity).
  • DESCRIPTION OF THE INVENTION [078] All references cited herein are incorporated by reference in their entirety as though fully set forth. Unless defined otherwise, 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.
  • the term “about” or “approximately” when used in connection with a referenced numeric indication means the referenced numeric indication plus or minus up to 5% of that referenced numeric indication, unless otherwise specifically provided for herein.
  • the language “about 50%” covers the range of 45% to 55%.
  • the term “about” when used in connection with a referenced numeric indication can mean the referenced numeric indication plus or minus up to 4%, 3%, 2%, 1%, 0.5%, or 0.25% of that referenced numeric indication, if specifically provided for in the claims.
  • 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 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 has no hinge region.
  • the immunoglobulin heavy chain constant region suitable for the invention preferably comprises an immunoglobulin hinge region, and preferably also includes a CH3 domain.
  • the immunoglobulin heavy chain constant region most preferably comprises an immunoglobulin hinge region, a CH2 domain and a CH3 domain.
  • immunoglobulin hinge region is understood to mean an entire immunoglobulin hinge region or at least a portion of the immunoglobulin hinge region sufficient to form one or more disulfide bonds with a second immunoglobulin hinge region.
  • vector is understood to mean any nucleic acid comprising a nucleotide sequence competent to be incorporated into a host cell and to be recombined with and integrated 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 and the like.
  • Non-limiting examples of a viral vector include a retrovirus, an adenovirus and an adeno-associated virus.
  • the term “gene expression” or “expression” of a fusion protein is understood to mean the transcription of a DNA sequence, translation of the mRNA transcript, and secretion of a fusion protein product.
  • the expression process also includes or is followed by purification; for example, protein A affinity chromatography or other means such as size exclusion chromatography can be used for purification.
  • IL-18 fusion protein refers to a fusion protein that includes wild-type IL-18 or IL-18 variants, unless specifically noted as only including the wild-type IL-18, or only including the IL-18 variant. Thus, in particular embodiments, the “IL-18 fusion protein” only includes any one of the IL-18 variants as described herein.
  • the amino acid linker has the amino acid sequence of (GGGGS (SEQ ID NO:237)) n where n is an integer between 1 and 5, thereby an amino acid linker of 25 amino acids or shorter in length.
  • the amino acid linker is an IL-18 propeptide or IL-18 propeptide variant.
  • the amino acid linker is a fragment of an IL-18 propeptide or IL-18 propeptide variant; for example, about 30-36 amino acids in length, about 5-10, 11-20, 21-30, or 31-40 amino acids in length.
  • Fusion proteins each comprising (i) an IL-18, a fragment of IL-18, an IL-18 variant, or a fragment of the IL-18 variant, and (ii) a first protein capable of translocating into an endoplasmic reticulum (ER) including a cytosolic or nuclear protein engineered to translocate into an endoplasmic reticulum (ER) by means of addition of signal peptide/leader sequence onto the N-terminus of such an engineered protein, or a fragment of said proteins.
  • ER endoplasmic reticulum
  • ER endoplasmic reticulum
  • ER cytosolic or nuclear protein engineered to translocate into an endoplasmic reticulum
  • the protein capable of translocating into an ER has an amino acid sequence which initiates transport of a protein (e.g., the IL-18 or its fragment, variant, or a fragment of its variant) across the membrane of the endoplasmic reticulum.
  • the fusion protein comprises further an amino acid linker.
  • the amino acid linker can be between (a) the protein capable of translocating into an endoplasmic reticulum (ER) and (b) the propeptide (or variant) or IL-18 (or variant).
  • the one or more fusion proteins do not comprise an IL-18 propeptide or its variant.
  • an “IL-18 propeptide”, or “propeptide” or “PP” in this invention may also be used interchangeably, which describes an amino acid sequence linked to IL-18 or IL-18 variant in an IL-18 precursor or IL-18 variant precursor, and which upon removal renders a mature IL-18 or its fragment, or IL-18 variant or its fragment thereof.
  • an IL-18 propeptide may have a sequence of amino acid residues 1-36 of Uniprot ID Q14116.
  • the one or more fusion proteins also include a propeptide (PP) or its variant. Examples of propeptide variants are provided herein, including those in Table 1.
  • the one or more fusion proteins also include a cleavage site, which is preferably based on a peptide substrate sensitive to enzymatic/protease cleavage.
  • the cleavage site may be positioned within the PP, between the PP or its variant (if present) and the IL-18 (or its fragment, variant, or a fragment of its variant); or may be positioned between the protein capable of translocating into an ER and the PP (if present); or may be positioned between the protein capable of translocating into an ER and the IL-18 or its fragment, variant, or a fragment of its variant, especially in the absence of a PP.
  • the cleavage site is positioned within the PP.
  • the one or more fusion proteins include (i) an IL-18, a fragment of IL-18, an IL-18 variant, or a fragment of the IL-18 variant, (ii), a propeptide (PP) or its variant, which inactivates IL-18, and a cleavage site.
  • propeptide variants include a polypeptide having AAEPVEDNX 1 INFVAMKFIDNTLYFIAEDDEN, wherein X 1 is any amino acid except cysteine (SEQ ID NO:238).
  • X 1 is alanine, valine, isoleucine, leucin, methionine, phenylalanine, tyrosine or tryptophan (SEQ ID NO:239). In various embodiments, X 1 is valine (SEQ ID NO:78). In various embodiments, X1 is serine, threonine, asparagine, or glutamine (SEQ ID NO:240). In various embodiments, X 1 is serine (SEQ ID NO:76).
  • the fusion protein does not comprise a polypeptide consisting of the sequence X 1 -X 2 -X 3 -X 4 between the (i) propeptide or propeptide variant, and (ii) the mature IL-18 or mature IL-18 variant, wherein X 1 is L or absent, X 2 is E or absent, X 3 is S or absent, and X 4 is D or absent, when X 1 , X 2 , X 3 , and X 4 are all present the sequence being LESD (SEQ ID NO:253).
  • the IL-18 (or its fragment, variant, or fragment of its variant) is linked by a polypeptide bond to the first protein capable of translocating into the ER.
  • the fusion proteins may have a variety of configurations.
  • the N-terminus of the IL-18 (or its fragment, variant, or a fragment of its variant) is linked by a polypeptide bond directly or indirectly to the C-terminus of the first protein capable of translocating in the ER.
  • the C-terminus of the IL-18 (or its fragment, variant, or a fragment of its variant) is linked by a polypeptide bond directly or indirectly to the N-terminus of the first protein capable of translocating in the ER.
  • the IL-18-variant (or IL-18, a fragment of IL-18, a fragment of the IL-18 variant) is fused to the N-terminus of a knob of a knob-into-hole heterodimeric IgG1 protein with or without a propeptide (pp).
  • a second protein capable of translocating through the ER is often fused to the N-terminus of the IL-18 to mediate masking.
  • a fusion protein further comprises (iii) a second protein capable of translocating into/through an ER, or a “scaffold” such as heat shock proteins (HSPs) that may not translocate through the ER.
  • HSP heat shock proteins
  • a second protein capable of translocating into/through an ER or a “scaffold” such as heat shock proteins (HSPs) that may not translocate through the ER.
  • HSP heat shock proteins
  • the IL-18 (or its fragment, variant, or a fragment of its variant) is at the C-terminus of the fusion protein; in some embodiments, the N-terminus of the IL-18 (or its fragment, variant, or a fragment of its variant) is on the C-terminus end relative to the first protein capable of translocating in the ER, and the C-terminus of the IL-18 (or its fragment, variant, or a fragment of its variant) is on the N-terminus end relative to the second protein capable of translocating in the ER.
  • the “first” or “second” protein capable of translocating in an ER is used as a relative reference.
  • the first protein/polypeptide capable of translocating into an ER comprises an immunoglobulin heavy chain constant region.
  • 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.
  • the immunoglobulin heavy chain constant region comprises a CH2 domain and a CH3 domain.
  • the immunoglobulin heavy chain constant region lacks at least a CH1 domain.
  • 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 the 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 with which a 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 lacks an immunoglobulin variable domain (V H ).
  • V H immunoglobulin variable domain
  • the IL-18 (or its fragment, variant, or fragment of its variant) is identical (in sequence) to that of a human origin, and the immunoglobulin heavy chain constant region comprises a hinge region, and a CH2 domain or a CH3 domain, and more preferably comprises a hinge region and both a CH2 domain and a CH3 domain.
  • the IL-18 (or its fragment, variant, or fragment of its variant) is at least 95%, 90%, or 85% identical (in sequence) to that of a human origin, but with amino acid substitutions or other modifications that reduces affinity of the IL-18 (or its fragment, variant, or fragment of its variant) for IL-18BP.
  • immunoglobulin heavy chain constant regions suitable for the invention may be derived from immunoglobulins belonging to any of the five immunoglobulin classes referred to in the art as IgA (Ig ⁇ ), IgD (Ig ⁇ ), IgE (Ig ⁇ ), IgG (Ig ⁇ ), and IgM (Ig ⁇ ).
  • immunoglobulin heavy chain constant regions from the IgG class are preferred.
  • the immunoglobulin heavy chain constant regions may be derived from any of the IgG antibody subclasses referred to in the art as IgG1, IgG2, IgG3, and IgG4. Immunoglobulin heavy chain constant region domains have cross-homology among the immunoglobulin classes.
  • the CH2 domain of IgG is homologous to the CH2 domain of IgA and IgD, and to the CH3 domain of IgM and IgE.
  • Preferred immunoglobulin heavy chain constant regions include protein domains corresponding to a CH2 region and a CH3 region of IgG, or functional portions or derivatives thereof. Further description of immunoglobulin heavy chain constant regions is discussed in detail in U.S. Pat. No. 5,541,087, and U.S. Pat. No. 5,726,044, which are incorporated by reference herein.
  • the protein/polypeptide to be fused with the IL-18 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.
  • a hinge region may have an upper hinge domain, a core hinge domain, and a lower hinge domain.
  • an upper portion of the hinge domain may include or remove the cysteine that is known to form a disulfide bond with the light chain or a fab, resulting in sequences such as EPKSC (SEQ ID NO:241) or EPKSS (SEQ ID NO:242) or EPKSA (SEQ ID NO:243).
  • fusion proteins including IgG1-based ER translocating protein, except FUSE-501, FUSE-503, and FUSE-509 may have removed cysteine from the hinge region, e.g., EPKSS (SEQ ID NO:242) in IgG1-based ER translocating protein, except for FUSE-507 (FUSE-507 has EPKSA (SEQ ID NO:243) in the hinge region).
  • a hinge region may also contain a core hinge domain, such as comprising a sequence CPPCP (SEQ ID NO:244) or a variant where the cysteine is replaced.
  • a hinge region may further include a lower hinge domain, such as comprising a sequence APELLGGP (SEQ ID NO:245) or APEAAGGP (SEQ ID NO:246).
  • FUSE-509 has an IgG4-based ER-translocating protein, using a hinge region as depicted in Chiu et al., Antibodies 2019, 8(4), 55, 2019. While constructs including immunoglobulin hinge regions are preferred, as depicted in the drawings, the invention contemplates that crosslinking at other positions may be chosen as desired.
  • two or more monomers may associate non-covalently to produce dimers or multimers.
  • the protein/polypeptide is a dimer of two immunoglobulin heavy chain constant regions/chains
  • the IL-18 (or its fragment, variant, or a fragment of its variant) is linked to one, and only one, of the two (or more) immunoglobulin heavy chain constant regions/chains.
  • a IL-18 is placed on the C-terminus of each monomer of the Fc, thereby having two IL-18 placed on the C-terminus of the Fc.
  • a heterodimer may form (e.g., in purification step) when one wild-type Fc fused to one IL-18 is mixed with another wild-type Fc not fused to IL-18.
  • an IL-18 (or its fragment, variant, or a fragment of its variant) is linked each of the two (or more) immunoglobulin heavy chain constant regions/chains in the fusion protein.
  • two arms (or chains) of immunoglobulin heavy chain constant regions e.g., Fc polypeptides
  • KiH knock-in-holes
  • a heteromultimer may comprise a first polypeptide and a second polypeptide each comprising a CH3 domain, wherein the polypeptides meet at an engineered interface within the CH3 domain, and the first polypeptide contains an engineered protuberance (“knob”) in the interface with at least one contact residue replaced with an import residue having a larger side chain volume than the original residue, and the second polypeptide contains an engineered cavity (“hole”) in the interface with at least one contact residue replaced with an import residue having a smaller side chain volume than the original residue.
  • knock engineered protuberance
  • hole engineered cavity
  • the engineered interface of a heteromultimer includes at least two protuberance-into-cavity mutant pairs. Volumes and accessible surface areas of each amino acid are described in A. A. Zamyatnin, Prog. Biophys. Mol. Biol.24: 107-123, 1972 and C. Chothia, J. Mol. Biol.105: 1-14, 1975.
  • import residues for the formation of a protuberance can be arginine (R), phenylalanine (F), tyrosine (Y) and tryptophan (W); and preferably the original residue for the formation of the protuberance has a small side chain volume, such as alanine, asparagine, aspartic acid, glycine, serine, threonine or valine.
  • import residues for the formation of a cavity can be alanine (A), serine (S), threonine (T) and valine (V); and preferably the original residue for the formation of the cavity has a large side chain volume, such as tyrosine, arginine, phenylalanine or tryptophan.
  • a T366W mutation in CH3 domain for the “knob”/protuberance chain and a T366S/L368A/Y407V mutation in CH3 domain for the “hole”/cavity chain.
  • the KiH configuration may be coupled further mutations to permit S-S disulfide linkage between the two chains.
  • the protein/polypeptide is a heterodimer of a the KiH configuration
  • the IL-18 or its fragment, variant, or a fragment of its variant
  • the two or more arms (or chains) of immunoglobulin heavy chain constant regions can contain another symmetric-to-asymmetric steric complementarity design (e.g., HA-TF, ZW1), a charge-to-charge swap interaction (DD-KK), a charge-to-steric complementarity swap plus additional long-range electrostatic interaction (e.g., EW-RVT), or an isotype strand swap design (e.g., strand- exchange engineered domain (SEED)), or Xmab, 7.8.60, Electrostatic Steering, A107, or Duobody, so as to form heterodimers/heteromultimers.
  • another symmetric-to-asymmetric steric complementarity design e.g., HA-TF, ZW1
  • DD-KK charge-to-charge swap interaction
  • EW-RVT charge-to-steric complementarity swap plus additional long-range electrostatic interaction
  • SEED isotype strand swap design
  • Xmab 7.8.
  • suitable proteins capable of translocating into the ER or a fragment thereof of the fusion protein is a globular protein, immunoglobular protein, or a fragment thereof.
  • suitable proteins capable of translocating into the ER or a fragment thereof of the fusion protein is a short polypeptide or protein engineered with a signal peptide for translocating into the ER.
  • the short polypeptide or protein being about 2 kDa or no greater than 250 kDa.
  • the short polypeptide or protein is about 2-5 kDa, about 6-10kDa, 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.
  • suitable proteins capable of translocating in an ER can be a globular protein, human serum albumin (HSA), beta2microglobulin, transferrin, fragment antigen-binding region (Fab region), VHH antibody, single-chain variable fragment (scFv), anticalin, designed ankyrin repeat protein (DARPin), a binding domain thereof, and a fragment thereof.
  • HSA human serum albumin
  • Fab region fragment antigen-binding region
  • VHH antibody single-chain variable fragment
  • DARPin single-chain variable fragment
  • Additional suitable proteins capable of translocating in an ER can include type I transmembrane proteins or a fragment thereof, or type II transmembrane proteins or a fragment thereof.
  • the fusion protein comprising the short polypeptide or protein and the IL-18 or IL-18 variant, or fragments thereof, further comprises a second proteins capable of translocating into the ER or a fragment thereof.
  • the second protein capable of translocating into the ER or a fragment thereof can be an Fc domain or HSA, beta2microglobulin, transferrin, fragment antigen-binding region (Fab region), VHH antibody, single-chain variable fragment (scFv), anticalin, designed ankyrin repeat protein (DARPin), a binding domain thereof, and a fragment thereof, or type I transmembrane proteins or a fragment thereof, or type II transmembrane proteins or a fragment thereof as described herein.
  • the fusion protein further comprises a protein that cannot naturally translocate into the ER such as nuclear or cytosolic proteins fused to the N-terminus of IL-18.
  • a signal peptide (which may be termed a leader sequence), such as Ig-kappa leader sequence (e.g., METDTLLLWVLLLWVPGSTG (SEQ ID NO:247)) in FUSE-499, 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.
  • the signal peptide may be on the N-terminus end of the propeptide or of the IL-18 (or its fragment, variant, or a variant of its fragment).
  • a further example of a protein capable of translocating in/into/through ER may be a protein engineered with a signal peptide, e.g., on the N-terminus end.
  • Hsp70 is a nuclear protein, but can be engineered to be an ER-translocating protein when fused or linked with a signal peptide on the N-terminus of Hsp70.
  • the addition of an N-terminal signal peptide, such as the Ig-kappa leader sequence, is in place of a Fc, globular protein, or HSS that’d otherwise be present in a fusion protein disclosed herein.
  • the fusion protein e.g., a masked IL-18
  • the fusion protein further comprises a tumor targeting fragment, e.g., a fragment that targets cell surface proteins including but not limited to a tumor associated antigen (TAA).
  • TAA tumor associated antigen
  • FUSE-517 as shown in FIG.9G is a masked IL-18 fusion protein that also comprises an anti-EGFR antibody fragment, e.g., Fab of cetuximab.
  • the fusion protein e.g., a masked IL-18
  • an activation receptor targeting fragment e.g., a fragment that targets activation receptors on cell surface including but not limited to CD16 on natural killer cell surface.
  • Activation receptors include immunoreceptor tyrosine-based activation motif (ITAM)-associated receptors, such as CD16 and NKp46.
  • ITAM immunoreceptor tyrosine-based activation motif
  • Activation receptors also include those participating 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.
  • the fusion protein e.g., a masked IL-18
  • anti-CD16 fragments include but are not limited to CH2 domains of IgG1, CH2 domain of IgG4.
  • the fusion protein (e.g., a masked IL-18) comprises a polypeptide fragment that targets an immune checkpoint, e.g., fragment that targets an immune checkpoint expressed on T cell.
  • FUSE- 694 as shown in FIG. 4 is a masked IL-18 fusion protein that also comprises an anti-PD1 fragment.
  • Example immune checkpoints include but are not limited to PD-1, PD-L1, CTLA-4, LAG-3.
  • One or more immune checkpoint-targeting fragments of known antibodies are conceived to be compatible with the fusion protein system disclosed herein.
  • anti-PD1 fragments examples include fragments (e.g., Fab, Fv) from pembrolizumab, nivolumab, pidilizumab, AMP-514, spartalizumab, cemiplimab, AK105, BCD-100, BI 754091, JS001, LZM009, MGA012, Sym021, TSR-042, MGD013, AK104, XmAb20717, tislelizumab, or PF-06801591.
  • fragments e.g., Fab, Fv
  • the fusion protein (e.g., a masked IL-18) comprises a targeting polypeptide, wherein the targeting polypeptide targets a protein on the same surface as the IL-18 RC.
  • the targeting polypeptide targets a protein on the same surface as the IL-18 RC.
  • examples of such proteins include but are not limited to CD16, J9 TCR, G2 TCR or G1 TCR, NKp46, CD137, CD40 or NKG2D.
  • fusion protein (e.g., a masked IL-18) comprises a targeting polypeptide, wherein the targeting polypeptide targets a protein on a cell that does not contain an IL-18RC.
  • the IL-18 fusion protein will need to be delivered close to the IL-18R complex for a cis or density effect to result in the interaction between the IL-18 fusion protein and the IL-18R complex.
  • TAA targeted IL-18 fusion protein could get to interact with the IL-18R complex on T cells if (a) the fusion protein bridged T cells with TAA+ cells or (b) the fusion protein was combined with another protein that bridged T cells with TAA+ cells or (c) the fusion protein bound to a TAA+ cell that naturally interacted with T cell via a secondary means (e.g. TCR/MHC interaction).
  • the fusion protein could be delivered to fibroblasts or other accessory cells in the tumor microenvironment and released by proteases such that it can act on IL-18R+ T or NK cells at a distance.
  • Exemplary targeting polypeptides include those noted in Table 5 or fragments thereof. Of those listed as the antigen-binding antibodies, their VHH, Fab regions, or single-chain variable fragments (scFv) can be used as the antigen-binding site of the multispecific antibodies disclosed herein.
  • enterokinase is used for site-specific cleavage of recombinant fusion proteins containing an accessible enterokinase recognition site.
  • enterokinase can specifically cleave after the C-terminal end of the lysine residue at its cleavage site, Asp-Asp-Asp-Asp-Lys (SEQ ID NO:87). Therefore, the fragment produced from this cleavage reaction does not inherit any residues from the DDDDK (SEQ ID NO:87) recognition sequence.
  • DDDDK is a part of the octapeptide FLAG tag (DYKDDDDK (SEQ ID NO:248)), which can be utilized as a fusion tag for recognition by antibody, and for detection of fusion protein with Western blot analysis, as well as for purification of the fusion protein by Anti- FLAG affinity chromatography.
  • a cleavage site can be based on peptide substrates sensitive to other enzymes, especially proteases highly expressed in tumor microenvironment, such as granzyme B, granzyme A, granzyme M, granzyme K, matrix metalloproteinase (MMP) 1/2/9/14 or other MMPs.
  • granzymes are usually only upregulated in inflamed tumors.
  • a substrate sequence for granzyme B can be Ile–Glu–Xaa–Asp ⁇ Xaa– Gly (SEQ ID NO:249) with the cleavage at the Asp ⁇ Xaa peptide bond.
  • a substrate sequence for granzyme B can also be Ile–Glu–Xaa–Asp ⁇ , with the cleavage at the C-terminus end of Asp, and Xaa can be Gln (SEQ ID NO:88) or another amino acid.
  • Several immune cells can release granzyme, such as T cells, NK cells, neutrophils, and mast cells.
  • a fusion protein comprising (a) a polypeptide fragment that targets an immune checkpoint expressed on an immune cell and/or a polypeptide fragment that targets an activation receptor on NK cell, and (b) a tumor targeting fragment, is effective for bringing the immune cell (e.g., T cell, NK cell) to the tumor, which can result in the release of granzymes that release IL-18.
  • an IL-18 fusion protein comprising a polypeptide fragment targeting an immune checkpoint protein can reverse exhaustion of NK and/or T cells that then are capable of releasing more granzymes.
  • the fusion protein comprises a cleavage site recognized by a serine protease, a cysteine protease, an aspartate protease, a threonine protease, a glutamic acidprotease, a metalloproteinase, a gelatinase, or an asparagine peptide lyase.
  • the protease cleavage site is recognized by a Cathepsin B, a Cathepsin C, a Cathepsin D, a Cathepsin E, a Cathepsin K, a Cathepsin L, a kallikrein, ahKl, a hK10, a hK15, a plasmin, a collagenase, a Type IV collagenase, a stromelysin, a Factor Xa, a chymotrypsin-like protease, a trypsin-like protease, an elastase-like protease, a subtilisinlike protease, an actinidain, a bromelain, a calpain, a caspase, a caspase-3, a Mir 1-CP, a papain, a HIV-1 protease, a HSV proteas
  • Nonlimiting examples of cleavage sites are included in Table 1.
  • IEQD SEQ ID NO:88
  • a variant, fragment, or a fragment of a variant of the IL-18 is suitable, and in some embodiments preferred, for the composition of the fusion protein.
  • a variant of mature IL-18 can have one, two, three, four, five, or more amino acid substitutions compared to the wild type mature IL-18.
  • one or more cysteines in the IL-18 or its propeptide are replaced with a natural or non-natural amino acid, such as from Cys to Ser, Ala, or Val, so as to reduce aggregation of the molecule in the fusion protein.
  • cysteine to threonine, asparagine, or glutamine cysteine to alanine, isoleucine, leucine methionine, phenylalanine, tyrosine, or tryptophan
  • cysteine to phenylalanine, alanine, aspartic acid, or asparagine cysteine to threonine, glutamine, aspartic acid, phenylalanine, isoleucine or histidine.
  • a variant of IL-18 may have a 95%, 90%, 85%, 83%, 80%, 75%, 70%, 65% or at least 60% sequence identity to wild type IL-18.
  • a variant of IL-18 may have at least 60% and at most 83% sequence identity to wild type IL-18.
  • the variant of IL-18 in the fusion protein is released as an about 15 kDa functional fragment (e.g., on the electrophoresis gels as tested) when cleaved at a cleavage site of the fusion protein.
  • the released protein may be mature IL-18 which would normally run at 18 kDa but may appear as about 15 kDa due to the ladder being used or the specific polyacrylamide percentage in a gel.
  • a fragment of IL-18 may have a 95%, 90%, 85%, 83%, 80%, 75%, 70%, 65%, or at least 60% sequence identity (and/or length) to wild type IL-18.
  • an IL-18 fragment produced by the fusion protein disclosed herein, especially 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 natural/wild-type mature IL-18; for example, an IL-18 fragment of about 15 kDa in size, preferably having comparable binding affinity for IL-18Ra/b as the wild type mature IL-18, is fused to a propeptide (or PP variant) and an ER translocating protein (with or without mutations), and the fusion protein also includes a protease cleavage site, such that upon protease cleavage, a small IL-18 fragment (e.g., about 15 kDa in size), is released.
  • 85% e.g., about 83%, about 83%-80%, about 80%-75%, about 75%-70%, or about 70%-65%
  • this small IL-18 fragment maintains the natural binding affinity for IL-18Ra/b and an equal or lower binding affinity relative to IL-18Ra/b for IL-18BP.
  • a variant, fragment, or a fragment of a variant of the IL-18 is capable of binding IL-18R and forming complex, so as to activate proinflammatory programs and/or NF- ⁇ B pathway.
  • the variant, fragment, or a fragment of a variant of the IL-18 is capable of having an increased binding affinity (e.g., 150%, 140%, 130%, 120%, 110%, or at least 100% relative to wild type IL-18) and/or inducing the biological activity of at least 150%, 140%, 130%, 120%, 110%, 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10%, compared to wild type IL-18.
  • an increased binding affinity e.g., 150%, 140%, 130%, 120%, 110%, or at least 100% relative to wild type IL-18
  • the variant, fragment, or a fragment of a variant of the IL-18 is capable of having an increased binding affinity at 120%, 110%, or at least 100% relative to wild type IL-18, and/or inducing the biological activity of at 120%, 110%, 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10%, compared to wild type IL-18.
  • a variant, fragment, or a fragment of a variant of the IL-18 has diminished binding to IL-18 binding protein (IL-18BP), as compared to a wild type IL-18 [0116]
  • the IL-18 or its fragment or variant cleaved from the fusion protein has at least 1,000, 2,000, 3,000, 5,000, 10,000, 30,000, 50,000, 70,000, 80,000, 90,000, or 100,000-fold increase in biological activity (e.g., binding with IL-18R to form IL-18/IL-18RD/ ⁇ complex and induce downstream signaling), compared to an uncleaved form in
  • the IL-18 or its fragment or variant cleaved from the fusion protein has a comparable biological activity, or within about 10, 20, 30, 40, or 50-fold difference in the biological activity, compared to recombinant human mature IL- 18.
  • the IL-18 or its fragment or variant 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).
  • KD equilibrium dissociation constant
  • the IL-18 or its fragment or variant cleaved from the fusion protein has a binding affinity for its IL-18R complex which is about the same, or at least 100%, 95%, or 90%, compared to that of the wild type IL-18.
  • the IL-18 or its fragment or variant cleaved from the fusion protein has a binding affinity for its IL-18R complex which is greater than that of the wild type IL-18, e.g., a binding affinity that is at least 105%, 110% compared to that of the wild type IL-18, or having a KD value at least 10% or 20% smaller than that of wild type IL-18.
  • the IL-18 or its fragment or variant cleaved from the fusion protein has a reduced binding affinity for IL-18BP, compared to that of the wild type IL-18.
  • the IL-18 or its fragment or variant cleaved from the fusion protein has a KD with IL-18BP of 18 nM or greater, such that it has a lower binding affinity to IL-18BP than to IL-18R.
  • the IL-18 or its fragment or variant cleaved from the fusion protein has a KD with IL-18BP of 18 nM or greater, whereas the wild type IL-18 has a KD with IL-18BP of about 0.4 nM.
  • the fusion protein comprises a first polypeptide or protein capable of translocating into an endoplasmic reticulum (ER) or a fragment thereof; and an interleukin 18 (IL-18), a fragment of IL-18, an IL-18 variant, or a fragment of the IL-18 variant, wherein the IL-18, the fragment of IL-18, the IL-18 variant, or the fragment of the IL-18 variant is on the C-terminus end of the fusion protein relative to the first polypeptide or protein capable of translocating into the ER.
  • the first polypeptide is not the wild-type IL-18 propeptide.
  • the protein capable of translocating into an endoplasmic reticulum (ER) or a fragment thereof is not the wild-type IL-18 propeptide.
  • the fusion protein comprises an IL-18 variant.
  • 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 MAAEPVEDNCINFVAMKFIDNTLYFIAEDDENIEQDYFGKLESKLSVIRNLNDQVLFIDQGNRPLFED MTDSDVRDNAPRTIFIISMYKDSQPRGMAVTISVKVEKISTLSVENKIISFKEMNPPDNIKDTKSDIIFFQ RSVPGHDNKMQFESSSYEGYFLAVEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO:250).
  • the one to five amino acid substitutions is 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 other embodiments, the one to five amino acid substitutions are four amino acid substitutions. In other embodiments, the one to five amino acid substitutions are five amino acid substitutions. In various embodiments, the IL-18 variant comprises no more than five amino acid substitutions, with the exception of substituting cysteines.
  • 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 MAAEPVEDNCINFVAMKFIDNTLYFIAEDDENIEQDYFGKLESKLSVIRNLNDQVLFIDQGNRPLFED MTDSDCRDNAPRTIFIISMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQ RSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO:251).
  • 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 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.
  • the amino acid substitutions at one or more of C74, C104, C112, and C164 are each independently substituted to valine, alanine or serine.
  • the amino acid substitutions at one or more of C74, C104, C112, and C164 are each substituted to alanine.
  • the amino acid substitutions at one or more of C74, C104, C112, and C164 are each substituted to serine.
  • 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.
  • 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.
  • the fusion protein comprises an IL-18 variant selected from Table 1.
  • the fusion protein comprising the IL-18 variant selected from Table 1 further comprises a propeptide having an amino acid sequence selected from Propeptide column in Table 1, and optionally from the same row as the IL-18 variant.
  • the fusion protein comprising the IL-18 variant selected from Table 1 and propeptide selected from Table 1, further comprises a cleavage peptide selected from Table 1, and optionally from the same row as the IL-18 variant and propeptide.
  • IEQD SEQ ID NO:88
  • IL-18 variant is an IL-18 variant disclosed in U.S. Patent 7,524,488, U.S. Patent Publication No.2019/0070262, U.S. Patent Publication No.2021/0015891, or PCT Publication No. WO 2022/038417, the IL-18 variants and sequences of each of these patent or publications of which are hereby incorporated by reference as though fully set forth.
  • the fusion protein further comprises a targeting polypeptide.
  • the targeting polypeptide targets a protein on a cell surface, wherein the cell surface also has an IL- 18 RC or the cell is capable of expressing the IL-18 RC.
  • the fusion protein binds to a cell having an IL-18 RC or capable of expressing the IL-18 RC upon activation of the cell, and activates the IL-18 RC signal.
  • the targeting polypeptide targets a protein on a cell surface that does not have an IL-18 RC or the cell is not capable of expressing the IL-18 RC.
  • the cell not having the IL-18 RC on its surface or not capable of expressing the IL-18 RC is in close proximity to a cell expressing the IL-18 RC or is capable of expressing the IL-18 RC.
  • the fusion protein can bring the cell not having the IL-18 RC on its surface or not capable of expressing the IL-18 RC into close proximity to a cell expressing the IL-18 RC or is capable of expressing the IL-18 RC.
  • the targeting polypeptide comprises a tumor associated antigen binding domain.
  • the fusion protein further comprises a binding domain for a protein expressed on immune cells.
  • the fusion protein further comprises a binding domain for a protein expressed on immune cells that express IL-18 receptor complex or on immune cells that upon activation express the IL-18 receptor complex.
  • the fusion protein further comprises an antibody or antibody fragment, and the fusion protein binds to a tumor cell, or to an immune cell or stromal cell in a tumor tissue. Examples of antibody fragments include Fc fragment, Fab fragment, Fv fragment as well as others discussed herein.
  • the fusion protein further comprises a masking domain. In these embodiments, a mature IL-18 or mature IL-18 variant can be released from a masking domain by a protease.
  • the protease is granzyme, which can be released from an immune cell.
  • immune cells include but are not limited to an NK cell, a T cell, a neutrophil, or a mast cell.
  • the protease is a metalloprotease, which the metalloprotease can be expressed in a tumor microenvironment. Further examples of protease and types of granzymes are described herein.
  • the mature IL-18 increases the activity of NK cells or T cells, and optionally the activity being one or more of proliferation, survival, and cytotoxicity.
  • the fusion protein further comprises half-life extending molecule.
  • a nonlimiting example of a half-life extending molecule is a half-life extending polypeptide; for example, human serum albumin (HSA) or an HSA-binding fragment.
  • the fusion protein has reduced activity as compared to wild-type IL-18 when not bound to a cell having the IL-18 RC. In various embodiments, the reduced activity is at least a 75% reduction in activity as compared to wild-type IL-18.
  • the fusion protein comprises polypeptide 1 and polypeptide 2 selected from Table 1.
  • the fusion protein further comprises polypeptide 3 selected from Table 1.
  • polypeptide 1 and polypeptide 2, and optionally polypeptide 3 is selected from the same row of Table 1.
  • the fusion protein comprises polypeptide 1 selected from Table 1, wherein polypeptide 1 comprises HSA.
  • the fusion protein does not comprise an IL-18 variant disclosed in U.S. Patent 7,524,488, U.S. Patent Publication No.2019/0070262, U.S. Patent Publication No.2021/0015891, or PCT Publication No. WO 2022/038417, the IL-18 variants and sequences of each of these patent or publications of which are hereby incorporated by reference as though fully set forth.
  • Propeptide Variants [0135] Various embodiments of the invention provide for propeptide variants.
  • the propeptide variant has the following amino acid sequence: AAEPVEDNX 1 INFVAMKFIDNTLYFIAEDDEN, wherein X 1 is any amino acid except cysteine (SEQ ID NO:238).
  • X 1 is alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine or tryptophan (SEQ ID NO:239).
  • X 1 is valine (SEQ ID NO:78).
  • X1 is serine, threonine, asparagine, or glutamine (SEQ ID NO:240).
  • X 1 is serine (SEQ ID NO:76).
  • 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 MAAEPVEDNCINFVAMKFIDNTLYFIAEDDENIEQDYFGKLESKLSVIRNLNDQVLFIDQGNRPLFED MTDSDVRDNAPRTIFIISMYKDSQPRGMAVTISVKVEKISTLSVENKIISFKEMNPPDNIKDTKSDIIFFQ RSVPGHDNKMQFESSSYEGYFLAVEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO:250).
  • the one to five amino acid substitutions is 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 other embodiments, the one to five amino acid substitutions are four amino acid substitutions. In other embodiments, the one to five amino acid substitutions are five amino acid substitutions. In various embodiments, the IL-18 variant comprises no more than five amino acid substitutions, with the exception of substituting cysteines.
  • 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 MAAEPVEDNCINFVAMKFIDNTLYFIAEDDENIEQDYFGKLESKLSVIRNLNDQVLFIDQGNRPLFED MTDSDCRDNAPRTIFIISMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQ RSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO:251).
  • 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 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.
  • the amino acid substitutions at one or more of C74, C104, C112, and C164 are each independently substituted to valine, alanine or serine.
  • 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.
  • 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.
  • the IL-18 variant is selected from the “Mature IL18 variant” column in Table 1.
  • the IL-18 variant further comprises an IL-18 propeptide or IL-18 propeptide variant.
  • the IL-18 variant further comprises an IL-18 propeptide variant having the following amino acid sequence: AAEPVEDNX 1 INFVAMKFIDNTLYFIAEDDEN, wherein X 1 is any amino acid except cysteine (SEQ ID NO:238).
  • X 1 is alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine or tryptophan (SEQ ID NO:239).
  • X 1 is valine (SEQ ID NO:78).
  • X 1 is serine, threonine, asparagine, or glutamine (SEQ ID NO:240). In various embodiments, X 1 is serine (SEQ ID NO:76).
  • the IL-18 variant is selected from the “Mature IL18 variant” column of Table 1, and further comprises a propeptide having an amino acid sequence selected from “Propeptide” column in Table 1, and optionally from the same row as the IL-18 variant. [0144] In various embodiments, the IL-18 variant further comprises an IL-18 propeptide variant, and a cleavage peptide. In various embodiments, the cleavage peptide selected from Table 1.
  • IEQD SEQ ID NO:88
  • a fusion protein comprising an IL-18 variant selected from Table 1, a propeptide selected from Table 1, and a cleavage peptide selected from Table 1, and optionally from the same row as the IL-18 variant and propeptide.
  • the IL-18 variant is not an IL-18 variant disclosed in U.S. Patent 7,524,488, U.S. Patent Publication No. 2019/0070262, U.S. Patent Publication No. 2021/0015891, or PCT Publication No. WO 2022/038417, the IL-18 variants and sequences of each of these patent or publications of which are hereby incorporated by reference as though fully set forth.
  • polynucleotides, vectors and cells [0147] Various embodiments provide polynucleotides encoding the fusion proteins disclosed herein.
  • the polynucleotides may encode in a 5’ to 3’ direction, a first protein or polypeptide capable of translocating in an ER and an IL-18 (or its fragment, variant, or a fragment of its variant).
  • Nonlimiting examples of such polynucleotides are in Table 2.
  • polynucleotides optionally may also include a “leader” or “signal” sequence based upon, for example, (1) a propeptide (PP) linked directly to IL-18 (or IL-18 variant) as in FUSE499 or (2) an immunoglobulin light chain sequence fused directly to a hinge region of the immunoglobulin heavy chain constant region.
  • a propeptide PP
  • IL-18 or IL-18 variant
  • an immunoglobulin light chain sequence fused directly to a hinge region of the immunoglobulin heavy chain constant region.
  • the nucleic acid encodes in a 5’ to 3’ direction, 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 an IL-18 (or its fragment, variant, or a fragment of its variant).
  • 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 an immunoglobulin CH2 domain and a CH3 domain
  • an IL-18 or its fragment, variant, or a fragment of its variant.
  • a polynucleotide encoding the fusion proteins may also be integrated within a replicable expression vector.
  • a vector encoding the fusion protein is also provided, which may express the fusion protein in, for example, a bacterial host, an intended recipient, or both.
  • Additional embodiments provide cells transformed or transfected with one or more nucleic acid molecules (polynucleotides) encoding the fusion protein.
  • the cell can be a prokaryotic cell.
  • the cell is a eukaryotic cell, preferably a mammalian cell, and more preferably a human cell.
  • 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).
  • compositions comprising combinations of two or more different fusion proteins, or combinations of the nucleic acid sequences encoding the fusion proteins.
  • a pharmaceutical composition is provided, wherein the fusion protein or a nucleic acid molecule encoding the fusion protein is an active agent.
  • the pharmaceutical compositions according to the invention can also contain any pharmaceutically acceptable carrier.
  • “Pharmaceutically acceptable carrier” refers to a pharmaceutically acceptable material, composition, or vehicle that is involved in carrying or transporting a compound of interest from one tissue, organ, or portion of the body to another tissue, organ, or portion of the body.
  • the 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 ingredients of the formulation.
  • compositions according to the invention can also be encapsulated, tableted or prepared in an emulsion or syrup for oral administration.
  • Pharmaceutically acceptable solid or liquid carriers 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, alcohols and water.
  • Solid carriers include starch, lactose, calcium sulfate, dihydrate, terra alba, magnesium stearate or stearic acid, talc, pectin, acacia, agar or gelatin.
  • the carrier may also include a sustained release material such as glyceryl monostearate or glyceryl distearate, alone or with a wax.
  • the pharmaceutical preparations are made following the conventional techniques of pharmacy involving milling, mixing, granulation, and compressing, when necessary, for tablet forms; or milling, mixing and filling for hard gelatin capsule forms. When a liquid carrier is used, the preparation will be in the form of a syrup, elixir, emulsion or an aqueous or non-aqueous suspension.
  • Such a liquid formulation may be administered directly p.o. or filled into a soft gelatin capsule.
  • the pharmaceutical compositions according to the invention may be delivered in a therapeutically effective amount.
  • the precise therapeutically effective amount is that amount of the composition that will yield the most effective results in terms of efficacy of treatment in a given subject. This amount will vary depending upon a variety of factors, including but not limited to the characteristics of the therapeutic compound (including activity, pharmacokinetics, pharmacodynamics, and bioavailability), the physiological condition of the subject (including age, sex, disease type and stage, general physical condition, responsiveness to a given dosage, and type of medication), the nature of the pharmaceutically acceptable carrier or carriers in the formulation, and the route of administration.
  • a method of producing interleukin 18 (IL-18), a fragment thereof, an IL-18 variant, or a fragment of the IL-18 variant comprises culturing a cell transfected with an expression vector comprising a nucleic acid encoding a fusion protein, in cell culture medium to allow a fusion protein to be produced and secreted into the extracellular space for purification; said protein, when contacting a protease to the fusion protein to cleave the fusion protein to produce the IL-18, the fragment thereof, the IL-18 variant, or the fragment of the IL-18 variant.
  • nucleic acid molecules encoding a fusion protein are seen in Table 2 Other embodiments provide that chemical conjugation using conventional chemical cross-linkers may be used to fuse protein moieties.
  • the nucleic acid molecules encoding the fusion proteins is expressed in CHO cells or HEK-293.
  • expressing the fusion proteins in the host cells results in a recoverable secreted fusion protein of at least 135 mg/L from supernatant of the host cells.
  • a yield of the fusion protein of at least 135 mg/L is obtained via transient transfection.
  • an even higher yield of the fusion protein e.g., at least 150, 200, 250, or 300 mg/L is obtained via stable producer cell clones, or pools of clones.
  • a yield of the fusion protein is about 130-400 mg/L.
  • a fusion protein, or the IL-18, the fragment thereof, the IL-18 variant, or the fragment of the IL-18 variant cleaved from the fusion protein is recovered in more than about 400 mg/L, between 350-400 mg/L, 300-350 mg/L, 200-300 mg/L, 100-200 mg/L, or at least 50 mg/L from supernatant of the host cells.
  • the fusion protein is produced via a process including the steps of: (1) cell recovery, which may be to recover frozen CHO cells via water bath at 37 ⁇ ; (2) cell subculturing, which may be to sub-culture the cells and adjust the cell density to 6 ⁇ 10 6 /ml for transfection; (3) transfection and expression, using a solution 1 (in which a plasmid is diluted with a diluting agent), a solution 2 (in which a transection reagent is diluted with a/the diluting agent), and then mixing the solution 1, the solution 2 and the CHO cells, followed by incubating the mixture at a shaker for expression for 12-14 days at 32 ⁇ to collect the supernatant of the culture after centrifuge.
  • cell recovery which may be to recover frozen CHO cells via water bath at 37 ⁇
  • cell subculturing which may be to sub-culture the cells and adjust the cell density to 6 ⁇ 10 6 /ml for transfection
  • transfection and expression using a solution 1 (in which a plasmid is diluted
  • a purification process is performed after the expression of the fusion protein.
  • a purification process includes the steps of: (1) washing a column with a binding buffer (10 times volume) at a flow rate of 1 mL/min; (2) loading a fusion-protein-containing sample into the column at a flow rate of 1 mL/min; (3) washing the column with 10x volumes of PBS buffer with a flow rate of 1 mL/min; (4) eluting the protein from the sodium with 40 mM sodium citrate (pH3.4); optionally the elution sample may be collected into tubes (1ml/min) and measured for optical density (OD) using NanoDrop at 280 nm; and (5) performing dialysis, e.g., against PBS buffer in a dialysis bag overnight.
  • OD optical density
  • Example 1 The N-terminal of pro-IL-18 was fused to the C-terminal of an IgG1 CH3 domain so as to engineer a variant of IL-18 expressible in mammalian cells. Pro -IL-18 was fused to the knob of a knob into hole heterodimeric IgG1 protein.
  • pro-IL-18 to reduce aggregation of the molecule such that each cysteine residue in both the pro-peptide and mature IL-18 was substituted with serine (“IL-18AS”, as in FUSE-480), alanine (“IL-18AA”, as in FUSE-481), or valine (“IL-18AV”, as in FUSE-442). Illustrations of the three variants are shown in panel A of figure 1. [0160] Transient transfection in the ExpiCHO system resulted in titers of 191, 214, and 198 mg/L, for FUSE-480, FUSE-481, and FUSE-442, respectively.
  • Fc-pro-IL-18 fusion proteins were hypothesized as harboring a “masked” version of IL-18, where the biological activity of the fused IL-18 would be reduced until the propeptide is cleaved off.
  • a cleavage site specific for the enterokinase (EK) was inserted within the propeptide (“pp”) upstream of mature IL-18 sequence in the location of the endogenous Caspase 1 site. EK was chosen because of its robust protease activity and activity in phosphate buffered saline.
  • EK enterokinase
  • HEK-Blue-IL-18 cells were used to quantify IL-18 activity.
  • HEK-Blue IL-18 cells are made from HEK-293 engineered to express the human IL-18 receptor complex (IL-18RD/ ⁇ ) and an NF- ⁇ b/AP-1- inducible secreted embryonic alkaline phosphatase (SEAP) reporter gene. The cells are also engineered not to respond to human TNF- ⁇ and IL-1 ⁇ .
  • SEAP NF- ⁇ b/AP-1- inducible secreted embryonic alkaline phosphatase
  • IL-18 NF- ⁇ b/AP-1- inducible secreted embryonic alkaline phosphatase
  • SEAP embryonic alkaline phosphatase
  • 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 modification was made to pro-IL- 18mut2 to reduce aggregation of the molecule such that each cysteine residue in both the pro-peptide and mature IL-18mut2 was substituted with serine (IL-18mut2AS), alanine (IL-18mut2AA), or valine (IL-18mut2AV). Illustrations of the three variants are shown in panel A of figure 2. As in figure 1, the biological activity was assessed using HEK-Blue. Compared to recombinant human mature IL-18, all three mutants induced lower biological activity of at least 100,000 fold.
  • FUSE-441 and FUSE424 were highly attenuated compared to human recombinant IL-18 at the orders of >100,000 fold (panel C). Treatment with EK led to 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. For FUSE-441, however, although the vast majority of biological activity was restored following treatment with EK, potency remained reduced by ⁇ 10 fold compared to recombinant human mature IL-18.
  • the EC50-SEAP for each compound is shown in panel D.
  • a flexible linker can be the propeptide; or the propeptide behaves as a flexible linker.
  • the propeptide incorporated into FUSE-424 allowed for sufficient access of EK to its cleavage site for efficient release of IL-18mut2AV.
  • any N-terminal protein of sufficient size e.g., about 4 kDa or larger, e.g., the propeptide is about 4 kDa, Fc is about 28 kDa as a monomer, HSA is about 66 kDa, VHH is about 14 kDa
  • an EK cleavage site containing linker of sufficient size e.g., 25 amino acid or longer
  • N- terminal masking protein or fragment thereof be engineered or naturally be transported through the Endoplasmic Reticulum (ER), this would result in expression yields from transient transfection of mammalian cells such as Expi- CHO acceptable for therapeutic development.
  • ER Endoplasmic Reticulum
  • a cleavage site specific for EK was inserted between the propeptide and mature IL-18 sequence in the location of the endogenous Caspase-I site.
  • the HEK- Blue-IL-18 cell reporter system was used. Using this systems as a readout, we exposed HEK-Blue-IL-18 cells to a titration of FUSE-507 (IgG1Fc-EKpp-IL-18AV) or FUSE-509 (IgG4Fc-EKpp-IL-18AV) with or without treatment with EK.
  • the biological activity, measured as the EC50-SEAP, of FUSE-507 and -509 were highly attenuated compared to human recombinant IL-18 at the orders of >10,000 fold (panel C).
  • Treatment with EK, designed to cleave off the IL-18AV fragment from FUSE-507 and FUSE-509 led to restoration of biological activity that was approximately 2 fold greater than hrIL-18.
  • This difference was likely the result of two molecules of IL-18AV being release per IgG1 or IgG4 fusion resulting in about a 2:1 molar ratio of IL-18AV to hrIL-18 when the IgG1 or IgG4 fusion proteins were fully cleaved by EK.
  • the EC50-SEAP for each compound is shown in panel D.
  • the formats utilizing wild type IgG1 or IgG4 indicate allowance for straightforward plug and play fusion of propeptide-IL-18 fusions, their variants and fragments thereof to an array of monoclonal antibodies including commercialized ones such as Avelumab (anti-PDL1), Cetuximab (anti-EGFR), Trastuzumab (anti-HER2/neu), etc.
  • Avelumab anti-PDL1
  • cetuximab anti-EGFR
  • Trastuzumab anti-HER2/neu
  • IL-18AV 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).
  • HSA Human Serum Albumin
  • the EK cleavage site that replaced the Caspase 1 site was moved to a position directly in between the C- terminus of HSA and mature IL-18AV without the addition of a flexible linker.
  • HEK-Blue IL-18 reporter cell assay as a readout, we exposed these cells to a titration 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 the EC50-SEAP of FUSE-501 (panel C) was highly attenuated compared to human recombinant IL-18 at the order of ⁇ 35,000 fold.
  • FUSE-503 (panel D) which does not contain the propeptide, was also attenuated at the order of ⁇ 3,500 fold.
  • EK designed to cleave off the IL-18AV fragment from FUSE-501 and FUSE-503 led to restoration of biological activity.
  • treatment with EK resulted in biological activity that was not substantially different from recombinant human mature IL-18.
  • FUSE-503 although biological activity was increased by treatment with EK, it remained ⁇ 40 fold weaker than human recombinant IL-18.
  • N-terminal masking protein or fragment thereof be engineered or naturally be transported through the Endoplasmic Reticulum (ER), this would result in expression yields from transient transfection of mammalian cells such as Expi-CHO acceptable for therapeutic development.
  • ER Endoplasmic Reticulum
  • IL-18mut2AV 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).
  • HSA Human Serum Albumin
  • the EK cleavage site that replaced the Caspase 1 site was moved to a position directly in between the C-terminus of HSA and mature IL-18AV without the addition of a flexible linker.
  • FUSE-502 HSA-EKpp-IL-18mu2AV
  • FUSE-504 HSA-EK-IL-18mut2AV
  • the biological activity, measured as the EC50-SEAP, of FUSE-502 (panel C) and FUSE-504 (panel D) were highly attenuated compared to human recombinant IL-18 at the orders of at least ⁇ 30,000 fold and ⁇ 50,000 fold, respectively.
  • the propeptide incorporated into FUSE-502 allowed for sufficient access of EK to its cleavage site for efficient release of IL-18mut2AV.
  • the results indicate strongly that in the context of an HSA-IL-18mut2AV fusion protein, the propeptide is not required for masking of IL-18mut2AV biological activity but is rather the consequence of steric hindrance mediated by the protein fused to the N-terminal of IL-18mut2AV.
  • this data provides further support that any N-terminal protein of sufficient size would be capable of masking the biological activity of IL-18mut2AV and that an EK cleavage site containing linker of sufficient size to allow access to EK, when incorporated between the Fc, HAS or other N- terminal protein mask and IL-18mut2AV, would substitute for the EK containing propeptide.
  • N-terminal masking protein or fragment thereof be engineered or naturally be transported through the Endoplasmic Reticulum (ER), this would result in expression yields from transient transfection of mammalian cells such as Expi- CHO acceptable for therapeutic development.
  • FIG. 8 we examined the impact of the propeptide on the biological activity of a single IL-18AV fused to the N-terminus of IgG1 Fc.
  • Panels A and B are exemplary illustrations of IL-18AV fused on the knob of a knob into hole IgG1-Fc domain with the propeptide (FUSE-499; Fc-pp-IL-18AV; panel A) or without the propeptide (FUSE-500; Fc-IL-18AV; panel B).
  • FUSE-499 Fc-pp-IL-18AV
  • FUSE-500 Fc-IL-18AV
  • the signal peptide from the Ig Kappa chain was encoded upstream of either the propeptide of FUSE-499 or the IL-18AV of FUSE-500.
  • IgK leader the signal peptide from the Ig Kappa chain
  • the biological activity, measured as the EC50-SEAP, of FUSE499 was highly attenuated compared to human recombinant IL-18 at the order of >50,000 fold (panel C).
  • the propeptide in FUSE-500 there was no reduction in biological activity compared to human recombinant IL- 18, indicating that in the absence of a the propeptide (or another polypeptide) attached to the N-terminus of mature IL-18, the IL-18-Fc fusion protein is fully functional.
  • de-masking of FUSE-499 with Caspase 1 resulted in restoration of biological activity that was not appreciably different from to human recombinant IL-18.
  • the propeptide in FUSE-499 is not naturally transported through the ER but was engineered to do so via addition of an IgK leader sequence upstream to it.
  • IgK leader sequence upstream to it.
  • any polypeptide of sufficient size fused to the N-terminus of mature IL-18 and/or its variants and fragments thereof would be expected to attenuate the biological activity of IL-18. That is, the N-terminal polypeptide might naturally translocate into the Endoplasmic Reticulum (ER) or it may be engineered to do so. In both cases, transport through the ER is important for obtaining expression yields from transient transfection of mammalian cells such as Expi-CHO acceptable for therapeutic development.
  • MMP matrix metalloproteinase
  • Granzyme B which is released by cytotoxic lymphocytes including NK cells and CD8+ T cells and may therefore accumulate in inflamed tumors.
  • FUSE587 was about 3,000-fold attenuated relative to recombinant human IL-18.
  • cleavage of FUSE587 with MMP2 released and IL-18AV variant that was still about 100-fold attenuated relative to recombinant IL-18.
  • cleavage with Granzyme B released an IL-18AV variant with activity similar activity as recombinant IL-18.
  • Cleavage with Granzyme B results in release of mature IL-18AV without any N-terminal residues constituting an overhang, whereas 11 and 15 amino acid N-terminal polypeptide overhangs remain after cleavage of FUSE486 and FUSE587, respectively, with MMP2.
  • these overhangs might be attenuating IL-18AV activity, albeit to a lesser degree than the full size variant propeptide. This phenomenon was further investigated in FIG.11 and FIG.13.
  • FUSE-485 and FUSE-462 were also highly attenuated compared to human recombinant IL-18 at the orders of >15,000-fold (9E and 9F).
  • rhGb Granzyme B
  • the masked IL-18 variants displayed nearly identical attenuation and the restored biological activity was about 2-fold weaker than recombinant human mature IL-18. Further, we did not observe an appreciable difference between the activity of the demasked IL-18AV and IL- 18mut2AV.
  • FUSE-517 (Cetuximab_ KiH_ppGb-IL-18-AV), as illustrated in panel G 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.
  • HEK-Blue IL-18 reporter cell assay as a readout, we exposed these cells to a titration of FUSE-517 (9H) with or without treatment with rhGb. Relative to rhIL-18, FUSE-517 was highly attenuated at the order of >250,000-fold.
  • FIG. 9I is summary tables of potencies (EC50-SEAP) related to the data shown in FIG. 9H. Overall, the data indicates strongly that both MMP2 and granzyme B are capable of demasking/activating IL-18AV (and IL-18mut2AV with Granzyme B) in the context of a pp mask fused in-between an IgG CH3 domain and the mature IL-18AV fragment.
  • masked fusion proteins of IL-18 can be engineered to be selectively activated by proteases found in tumors/inflamed tumors including MMPs and Granzymes.
  • FIG 10 we examined the susceptibility of select IL-18 variants to attenuation of biological activity by its natural antagonist, IL-18 BP.
  • Panel A of figure 10 is an illustration of the IL-18 fusion proteins examined. That is, FUSE-442 (Fc-EKpp-IL-18AV) and FUSE-424 (Fc-EKpp-IL-18mut2AV).
  • FUSE-424 harbors a mature IL-18AV (termed IL-18mut2AV) downstream of the polypeptide (pp) that includes the following mutations: M51K, K53G, M60L and M113V.
  • IL-18mut2AV mature IL-18AV downstream of the polypeptide (pp) that includes the following mutations: M51K, K53G, M60L and M113V.
  • HEK Blue IL-18 was used to assess biological activity via MYD88 driven SEAP and potency reported as the EC50-SEAP.
  • FUSE-442 Fc-EKpp-IL-18AV
  • FUSE- 424 Fc-EKpp-IL-18mut2AV
  • Each cleavage product was then titrated from 1 pg/ml to 1 ⁇ g/ml in media alone or media containing 1.25 ⁇ g/ml human recombinant IL-18-BP (hrIL-18-BP).
  • IL-18 Human recombinant IL-18 (hrIL-18) was used as the 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 non-linear x-y plots of SEAP release (IL-18R reporter activity) on the y- axis versus the concentration of test article on the x-axis in the presence or absence of IL-18BP.
  • a summary of biological potencies (EC50-SEAP) relevant to each graph is shown as panels C, E, and G (beneath each x-y plot).
  • IL-18mut2AV binds with far weaker apparent affinity to rhIL-18BP compared to IL-18AV or rhIL-18.
  • a masked version of IL-18mut2AV designed to be released by proteases present in the TME including but not limited to MMP2, 9 and 14 and/or proteases released in inflamed tumors including but not limited to Granzymes A, B and M may be predicted to function in the presence of IL-18BP to promote anti-tumor activity via multiple pathways including but not limited to IFN ⁇ mediated Th1 and Tc1 activity.
  • Example 2 [0174] As shown in figure 11, we examined the impact of the size of polypeptides 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.
  • Panels A are exemplary illustrations of IL-18AV fused on the knob of a knob into hole IgG1-Fc domain with different size polypeptides ranging from a propeptide variant (FUSE-499; described in FIG. 18) to 35 amino acids (FUSE756) to 15 amino acids (FUSE757 and FUSE758).
  • the signal peptide from the Ig Kappa chain (IgK leader) was encoded upstream of the polypeptides (and cleaved off the signal peptidase in the ER).
  • the polypeptides of FUSE756 and FUSE757 are made up of a series of glycine and serine residues whereas that of FUSE758 is the sequence of the overhang generated by MMP2 mediated cleavage of the MMP cleavage site, KPLGLQARVVGGGG (SEQ ID NO:252).
  • the C-terminus of the N-terminal polypeptides also incorporated the Granzyme B site, IEQD (SEQ ID NO:88).
  • IEQD Granzyme B site
  • HEK-Blue IL-18 reporter cell assay as a readout, we examined the capacity of the different size polypeptides to attenuate/mask IL-18AV-Fc (FUSE500; closed squares) or rhIL-18 (black cross hatches “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 (closed triangles) was the most attenuated (>10,000-fold).
  • FUSE757 (closed diamonds) and 758 (open reverse triangle), both of which included 11 amino acid polypeptides fused to the N-terminus of mature IL-18AV, were about 250- fold less biologically active relative to FUSE500 or rhIL-18.
  • FUSE756 (open triangles), which contained a polypeptide of 31 amino acids polypeptides fused to the N-terminus of mature IL-18AV was approximately 1000- fold less biologically active relative to FUSE500 or rhIL-18.
  • HEK-Blue-IL-18 cell activation assay with IL-18 or FUSE proteins
  • HEK-BLUE TM -IL-18 cells from Invivogen were maintained in culture medium (DMEM medium with 4.5 g/L glucose, 2 mM L-Glutamine, 10% (v/v) heat-inactivated fetal bovine serum, 100 U/mL penicillin, 100 ⁇ g/mL streptomycin, 100 ⁇ g/mL Normocin and 1 ⁇ HEK-blue selection reagent).
  • HEK-BLUETM IL-18 cells are engineered from the human embryonic kidney 293 (HEK293) cell line to stably express genes encoding the IL-18 receptor (IL-18R) and IL-18 receptor accessory protein (IL-18RAP) and express an NF- ⁇ b/AP-1-inducible secreted embryonic alkaline phosphatase (SEAP) reporter gene, therefore being useful for detection of bioactive IL-18 by monitoring the activation of the NF- ⁇ b and AP-1 pathways via quantification in the supernatant of the SEAP level (which is produced upon activation of NF- ⁇ b) with a solution such as QUANTI-BLUETM Solution.
  • IL-18R IL-18 receptor
  • IL-18RAP IL-18 receptor accessory protein
  • SEAP NF- ⁇ b/AP-1-inducible secreted embryonic alkaline phosphatase reporter gene
  • HEK-BLUETM IL-18 cells were gently rinsed twice with pre- warmed phosphate buffered saline (PBS) and then detached in PBS by tapping the flask.
  • PBS pre- warmed phosphate buffered saline
  • HEK-Blue-IL- 18 cells were resuspended in pre-warmed testing medium (DMEM with 4.5 g/L glucose, 2 mM L-Glutamine, 10% (v/v) heat-inactivated FBS, 100 U/mL penicillin, and 100 ⁇ g/mL streptomycin) at the density of 5 ⁇ 10 5 cell per mL.
  • DMEM pre-warmed testing medium
  • FUSE proteins were diluted in testing medium from 500000 pg/mL to 6.4 pg/mL (2 ⁇ of final concentration) by 5-fold serial dilution.
  • IL-18 was also diluted in testing medium from 800 pg/mL to 1.28 pg/mL (2 ⁇ of final concentration) by 5-fold serial dilution.
  • 100 ⁇ L of prepared IL-18 or FUSE proteins were added to the designated wells with 50000 cells in the 96-well plate, and then the proteins were gently mixed the cells. The 96-well plate was then incubated at 37 ⁇ with 5% CO 2 for 24 hours.
  • HEK-Blue-IL-18 cells released secreted alkaline phosphatase in the supernatant.20 ⁇ L of HEK-Blue-IL-18 cell culture supernatant was transferred to a new 96-well plate.
  • QUANTI-Blue solution from Invivogen was prepared by adding 1 mL of QB reagent and 1 mL of QB buffer to 98 mL of sterile water.180 ⁇ L of prepared QUANTI-Blue solution was then added to the 96-well plate with 20 ⁇ L of HEK-Blue-IL-18 cell culture supernatant and incubated at 37 ⁇ for 4 hours. The level of secreted alkaline phosphatase related to HEK-Blue-IL-18 cell activation was detected by measuring the OD at 630 nm using a spectrophotometer.
  • HEK-Blue-IL-18 cell activation assay with enterokinase (EK)-cleaved FUSE proteins [0179] HEK-Blue-IL-18 cells from Invivogen were maintained in culture medium. On the day of experiment 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. Detached HEK-Blue-IL-18 cells were resuspended in pre- warmed testing medium at the density of 5 ⁇ 10 5 cell per mL. To seed the cells (50000 cells per well), 100 ⁇ L of resuspended HEK-Blue-IL-18 cells were added to designated wells in 96-well plate.
  • PBS pre-warmed phosphate buffered saline
  • FUSE protein cleavage 4 ⁇ g of FUSE protein was mixed with 80 ng of EK enzyme and 2 ⁇ L of 10 ⁇ PBS in a tube. Sterile water was added to the tube to bring the total reaction volume to 20 ⁇ L. The tube was then incubated at 25 ⁇ for 40 minutes. After 40-minute incubation, cleaved FUSE proteins were diluted in testing medium from 500000 pg/mL to 6.4 pg/mL (2 ⁇ of final concentration) by 5-fold serial dilution. IL-18 was also diluted in testing medium from 800 pg/mL to 1.28 pg/mL (2 ⁇ of final concentration) by 5-fold serial dilution.
  • HEK-Blue-IL-18 cells 100 ⁇ L of prepared IL-18 or cleaved FUSE proteins were added to the designated wells with 50000 cells in the 96-well plate and then gently mixed the cells with proteins. The 96-well plate was then incubated at 37 ⁇ with 5% CO 2 for 24 hours. [0181] After 24-hour activation, 20 ⁇ L of HEK-Blue-IL-18 cell culture supernatant with secreted alkaline phosphatase was transferred to a new 96-well plate.
  • 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 ⁇ L of prepared QUANTI-Blue solution was then added to the 96-well plate with 20 uL of HEK-Blue-IL-18 cell culture supernatant and incubated at 37 ⁇ for 4 hours. The level of secreted alkaline phosphatase related to HEK-Blue-IL-18 cell activation was detected by measuring the OD at 630 nm using a spectrophotometer.
  • HEK-Blue-IL-18 cell activation assay with matrix metalloproteinase (MMP)-cleaved FUSE proteins [0182] HEK-Blue-IL-18 cells from Invivogen were maintained in culture medium. On the day of experiment 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. Detached HEK-Blue-IL-18 cells were resuspended in pre- warmed testing medium at the density of 5 ⁇ 10 5 cell per mL.
  • PBS pre-warmed phosphate buffered saline
  • FUSE protein cleavage 1 ⁇ g of FUSE protein was mixed with 280 ng of MMP2 or MMP9 enzyme and 2.8 ⁇ L 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 ⁇ L. The tube was then incubated at 37 ⁇ for 2 hours.
  • 10 ⁇ assay buffer 500 mM Tris, 100 mM CaCl2, 1500 mM NaCl, 0.5% (w/v) Brij-35, pH 7.5
  • cleaved FUSE proteins were diluted in testing medium from 500000 pg/mL to 6.4 pg/mL (2 ⁇ of final concentration) by 5-fold serial dilution.
  • IL-18 was also diluted in testing medium from 800 pg/mL to 1.28 pg/mL (2 ⁇ of final concentration) by 5-fold serial dilution.
  • 100 ⁇ L of prepared IL-18 or cleaved FUSE proteins were added to the designated wells with 50000 cells in the 96-well plate and then gently mixed the cells with proteins. The 96-well plate was then incubated at 37 ⁇ with 5% CO 2 for 24 hours.
  • 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 ⁇ L of prepared QUANTI-Blue solution was then added to the 96-well plate with 20 ⁇ L of HEK-Blue-IL-18 cell culture supernatant and incubated at 37 ⁇ for 4 hours.
  • HEK-Blue-IL-18 cell activation assay with Caspase I-cleaved FUSE proteins [0185] HEK-Blue-IL-18 cells from Invivogen were maintained in culture medium. On the day of experiment 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.
  • PBS pre-warmed phosphate buffered saline
  • HEK-Blue-IL-18 cells were resuspended in pre- warmed testing medium at the density of 5 ⁇ 10 5 cell per mL. To seed the cells (50000 cells per well), 100 ⁇ L of resuspended HEK-Blue-IL-18 cells were added to designated wells in 96-well plate. [0186] For FUSE protein cleavage, 14 ⁇ g of FUSE protein was mixed with 0.5 unit of Caspase I enzyme and 2.8 ⁇ L of 10 ⁇ assay buffer (500 mM Hepes, pH 7.2, 500 mM NaCl, 1% Chaps, 100 mM EDTA, 50% Glycerol, and 100 mM DTT) in a tube.
  • 10 ⁇ assay buffer 500 mM Hepes, pH 7.2, 500 mM NaCl, 1% Chaps, 100 mM EDTA, 50% Glycerol, and 100 mM DTT
  • cleaved FUSE proteins were diluted in testing medium from 500000 pg/mL to 6.4 pg/mL (2 ⁇ of final concentration) by 5-fold serial dilution.
  • IL-18 was also diluted in testing medium from 800 pg/mL to 1.28 pg/mL (2 ⁇ of final concentration) by 5-fold serial dilution.
  • 100 ⁇ L of prepared IL-18 or cleaved FUSE proteins were added to the designated wells with 50000 cells in the 96-well plate and then gently mixed the cells with proteins.
  • 96-well plate was then incubated at 37 ⁇ with 5% CO 2 for 24 hours.
  • 20 ⁇ L of HEK-Blue-IL-18 cell culture supernatant with secreted alkaline phosphatase was transferred to a new 96-well plate.
  • 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 ⁇ L of prepared QUANTI-Blue solution was then added to the 96-well plate with 20 ⁇ L of HEK-Blue-IL-18 cell culture supernatant and incubated at 37 ⁇ for 4 hours.
  • HEK-Blue-IL-18 cell activation assay with granzyme B-cleaved FUSE proteins [0188] HEK-Blue-IL-18 cells from Invivogen were maintained in culture medium. On the day of experiment 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.
  • PBS pre-warmed phosphate buffered saline
  • HEK-Blue-IL-18 cells were resuspended in pre- warmed testing medium at the density of 5 ⁇ 10 5 cell per mL. To seed the cells (50000 cells per well), 100 ⁇ L of resuspended HEK-Blue-IL-18 cells were added to designated wells in 96-well plate.
  • Mature active human granzyme B was generated by cleaving human pro-granzyme B using EK enzyme. In brief, 4 ⁇ g of human pro-granzyme B was mixed with 40 ng of EK enzyme and 2 ⁇ L of 10 ⁇ PBS in a tube. Sterile water was added to the tube to bring the total reaction volume to 20 ⁇ L. The tube was then incubated at 25 ⁇ for 40 minutes.
  • activated human granzyme B was used to cleave FUSE proteins.
  • 10 ⁇ g of fuse proteins were mixed with 1 ug 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 for certain time indicated in each experiment at 37 ⁇ C.
  • assay buffer 50 mM HEPES (pH 7.4), 100 mM NaCl, 0.1% CHAPS, 1 mM EDTA, 10% Glycerol
  • Granzyme B-cleaved fuse proteins were then diluted in testing medium from 500000 pg/mL to 6.4 pg/mL (2 ⁇ of final concentration) by 5-fold serial dilution.
  • IL-18 was also diluted in testing medium from 800 pg/mL to 1.28 pg/mL (2 ⁇ of final concentration) by 5-fold serial dilution.
  • 100 ⁇ L of prepared IL-18 or cleaved FUSE proteins were added to the designated wells with 50000 cells in the 96-well plate and then gently mixed the cells with proteins. The 96-well plate was then incubated at 37 ⁇ with 5% CO 2 for 24 hours.
  • 20 ⁇ L of HEK-Blue-IL-18 cell culture supernatant with secreted alkaline phosphatase was transferred to a new 96-well plate.
  • 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 ⁇ L of prepared QUANTI-Blue solution was then added to the 96-well plate with 20 ⁇ L of HEK-Blue-IL-18 cell culture supernatant and incubated at 37 ⁇ for 4 hours. The level of secreted alkaline phosphatase related to HEK-Blue-IL-18 cell activation was detected by measuring the OD at 630 nm using a spectrophotometer.
  • HEK-Blue-IL-18 cells from Invivogen were maintained in culture medium. On the day of experiment 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. Detached HEK-Blue-IL-18 cells were resuspended in pre- warmed testing medium at the density of 5 ⁇ 10 5 cell per mL. To seed the cells (50000 cells per well), 100 ⁇ L of resuspended HEK-Blue-IL-18 cells were added to designated wells in 96-well plate.
  • PBS pre-warmed phosphate buffered saline
  • Non-cleaved or cleaved FUSE proteins were prepared in testing medium from 1000000 pg/ml to 12.8 pg/ml (4 ⁇ of final concentration) by 5-fold serial dilution.
  • IL-18BP was diluted in testing medium at the concentration of 5,000,000 pg/ml (4 ⁇ of final concentration).
  • 50 ⁇ L of prepared non-cleaved or cleaved FUSE proteins and 50 ⁇ L of prepared IL-18BP were mixed, incubated for 1 hour, and then added to the designated wells with 50000 cells in the 96-well plate and then gently mixed the cells with proteins. The 96-well plate was then incubated at 37 ⁇ with 5% CO 2 for 24 hours.
  • 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 ⁇ L of prepared QUANTI-Blue solution was then added to the 96-well plate with 20 ⁇ L of HEK-Blue-IL-18 cell culture supernatant and incubated at 37 ⁇ for 4 hours.
  • the level of secreted alkaline phosphatase related to HEK-Blue-IL-18 cell activation was detected by measuring the OD at 630 nm using a spectrophotometer.
  • Binding assay by Biolayer Interferometry [0195] Coating proteins were prepared in 1 ⁇ PBS with 0.02% Tween-20 with the final concentration of 15 ug/ml. Capturing protein were also prepared, and serial diluted (4-fold dilution ranging from 400 nM to 1.6 nM) in 1 ⁇ PBS with 0.02% Tween-20. The biosensors were pre-moistened in 200 uL of 1 ⁇ PBS with 0.02% Tween-20 for 10 minutes.
  • Octet BLI system (ForteBio) was prewarmed for 30 minutes and the flow rate was set to 1000 rpm.
  • the biosensors Capture biosensor
  • the biosensors were soaked in 250 uL of 1 ⁇ PBS with 0.02% Tween-20 for 60 seconds at 30 ⁇ C to get an initial baseline reading.
  • the biosensors were exposed to coating proteins for 300 seconds at 30 ⁇ C for the association between antibody and the biosensors (coupling coating proteins with biosensor).
  • the biosensors with coating proteins were then exposed to capturing proteins in 250 uL of 1 ⁇ PBS with 0.02% Tween-20 at 30 ⁇ C for 300 seconds for the association reaction between the coating proteins and capturing proteins (association curve).
  • each IL18 mutant was coated onto AHC biosensor tips and probed with recombinant His tagged IL-18BP at concentrations ranging from 400 nM to 1.6 nM.
  • His tagged IL18R ⁇ was coated into nickel biosensor tips and probed with recombinant each IL18 mutant at concentrations ranging from 400 nM to 1.6 nM.
  • Example 4 As shown in figure 12, we engineered eleven mutants of human IL-18 (IL-18AV shown) and measured their ability to bind recombinant human IL-18BP and recombinant human IL-18RA (also termed IL- 18RD). All test articles were generated as Fc fusion proteins with the IL-18 variant fused to the N-terminus of the Fc (see FIG.11A). The locations and amino acid substations associated with each variant are depicted in FIG.12A. [0197] To assess binding to human IL18BP or human IL18RD, kinetic binding graphs were generated via Bio-Layer Interferometry (BLI) using an Octet system (ForteBio).
  • each IL18 mutant was coated onto AHC biosensor tips and probed with recombinant His tagged IL-18BP at concentrations ranging from 400 nM to 1.6 nM (FIG.12B).
  • His tagged IL18RD was coated into nickel biosensor tips and probed with recombinant each IL18 mutant at concentrations ranging from 400 nM to 1.6 nM (FIG.12D).
  • Summary tables for binding to IL-18BP and IL18-RA binding affinity (KD), on rate (k-on) and off rate (k-dis) are shown in FIGs 12C and 12E, respectively.
  • Example 5 As shown in the table below, we tabulated the binding affinities of eleven mutant variants of human IL-18 (IL-18AV shown) to human IL18BP and human IL18RD. Each IL-18 protein was generated as an Fc fusion proteins whereby the IL-18 variant was fused to the N-terminus of the Fc (see FIG.11A). Protein Mutant IL-BP Kd (nM) IL-18RD Kd (nM) IL18 4E-10*** 2E-08**** il A 2 1 * 4 ** www.pnas.org/do/ 0. 073/pnas.97.3.
  • the signal peptide from the Ig Kappa chain (IgK leader) was encoded upstream of the polypeptides (and cleaved off the signal peptidase in the ER).
  • the polypeptides of FUSE875 (5 residues; closed stars), FUSE876 (10 residues; open diamonds), FUSE757 (15 residues; open circles), FUSE756 (35 residues; downward closed triangles) are made up of a series of glycine and serine residues.
  • the N-terminal polypeptide associated with FUSE758 (open upward triangles) is the sequence of the overhang generated by MMP2 cleavage of FUSE486 (see FIG.9).
  • FUSE756, FUSE757 and FUSE758 the final four amino acids of the N-terminal peptide consisted of the Granzyme B cleavage site, IEQD (SEQ ID NO:88) (see FIG.11).
  • IEQD SEQ ID NO:88
  • Example 7 [0207] As shown in figure 14A-14H, we examined the impact of the substituting the cysteine residue in the pro-peptide and cysteine residues in the mature IL18, which were fused together to form the pro-IL-18 variant cassette, on the biological activity of each variant using the HEK Blue IL18 assay system. Unless otherwise stated, the proteins were generated such that the propeptide-IL-18 variant was fused to the C-terminal knob or hole of a knobs into holes human IgG1 Fc as previously depicted in FIG.1A with a Granzyme B cleavage site added between the pro-peptide variant and the mature IL18 variant. The three variants assessed in FIG.
  • FUSE480 serine substitutions
  • FUSE481 alanine substitutions
  • FUSE442 valine substitutions
  • Mature IL-18 variants released from FUSE442 and FUSE481 were about as active as recombinant human IL-18 whereas mature IL18 released from FUSE480 (serine substituted) was approximately 100-fold attenuated versus recombinant human IL-18 (see FIGs 1B-1D).
  • the variant of pro-IL-18 in which all the cysteines were substituted for valine included an N-terminus EGFR specific VHH (FUSE516; closed triangle) and the variant of pro-IL-18 in which all the cysteines were substituted for serine included an N-terminus PD-1 specific Fab (FUSE694; closed diamond).
  • biological activity of the pro-IL18 test articles were assessed using HEK Blue IL18. Each was tested as an intact untreated protein or following exposure to recombinant human Granzyme B.
  • FIG 14A shows the results with FUSE516 (closed triangle), in which the cysteine residue in its pro-peptide variant is replaced with valine and all the cysteines in its mature IL18 variant are replaced by valine.
  • Intact FUSE516 is about 1000-fold attenuated relative to recombinant human IL18 and that the mature IL18 variant released from FUSE516 (open triangle) by Granzyme B is approximately as active as recombinant human IL18.
  • FIG 14B shows the results with FUSE694 (closed diamond), in which the cysteine residue in its pro-peptide variant is replaced with serine and all of the cysteines in its mature IL18 variant are replaced by serines.
  • Intact FUSE694 is greater than 10,000-fold attenuated relative to recombinant human IL18 and that the mature IL18 variant released from FUSE694 (open diamond) by Granzyme B is approximately 60-fold less active than recombinant human IL18.
  • FIG 14C shows the results with FUSE887 (closed reverse triangle), in which the cysteine residue in its pro-peptide variant is replaced with threonine and all the cysteines in its mature IL18 variant are replaced by serines.
  • Intact FUSE887 is greater than 10,000-fold attenuated relative to recombinant human IL18 and that the mature IL18 variant released from FUSE887 by Granzyme B (open reverse triangle) is approximately 100-fold less active than recombinant human IL18.
  • FIG 14D shows the results with FUSE888 (closed triangle), in which the cysteine residue in its pro-peptide variant is replaced with glutamine and all the cysteines in its mature IL18 variant are replaced by serines.
  • Intact FUSE888 is greater than 10,000-fold attenuated relative to recombinant human IL18 and that the mature IL18 variant released from FUSE888 (open triangle) by Granzyme B is approximately 100-fold less active than recombinant human IL18.
  • FIG 14E shows the results with FUSE889 (closed square), in which the cysteine residue in its pro-peptide variant is replaced with aspartic acid and all the cysteines in its mature IL18 variant are replaced by alanines.
  • Intact FUSE889 is greater about 10,000-fold attenuated relative to recombinant human IL18 and that the mature IL18 variant released from FUSE889 by Granzyme B (open square) is approximately 100-fold less active than recombinant human IL18.
  • FIG 14F shows the results with FUSE890 (closed reverse triangle), in which the cysteine residue in its pro-peptide variant is replaced with phenylalanine and all of the cysteines in its mature IL18 variant are replaced by alanines.
  • Intact FUSE890 is about than 10,000-fold attenuated relative to recombinant human IL18 and that the mature IL18 variant released from FUSE890 by Granzyme B (open reverse triangle) is approximately 100-fold less active than recombinant human IL18.
  • FIG 14G shows the results with FUSE891 (closed triangle), in which the cysteine residue in its pro-peptide variant is replaced with isoleucine and all the cysteines in its mature IL18 variant are replaced by valines.
  • Intact FUSE891 is about 3,000-fold attenuated relative to recombinant human IL18 and that the mature IL18 variant released from FUSE891 (open triangle) by Granzyme B is approximately 100-fold less active than recombinant human IL18.
  • FIG 14H shows the results with FUSE892 (closed reverse triangle), in which the cysteine residue in its pro-peptide variant is replaced with histidine and all the cysteines in its mature IL18 variant are replaced by valines.
  • Intact FUSE892 is greater about 3,000-fold attenuated relative to recombinant human IL18 and that the mature IL18 variant released from FUSE892 (open reverse triangle) by Granzyme B is approximately 100-fold less active than recombinant human IL18.
  • Summary tables of potency (EC50-SEAP) are show to the right of each non-linear x-y plot.
  • Example 8 [0218] As shown in figure 15A-15B, we examined the impact of targeting pro-IL18 to within close proximity of its receptor complex (i.e., “cis activity”).
  • Pro-IL-18 variants tested were (a) that in which all the cysteine residues were substituted with serines (pro-IL18AS, FUSE782 and FUSE827) and (b) that in which all the cysteines were substituted with valines (pro-IL18AV, FUSE783 and FUSE785).
  • Pro-IL18AS or pro-IL18AV were fused to the N-terminal knob or hole of a PD1 specific knobs into holes antibody (FUSE782 and FUSE783).
  • the fab for these fusion proteins was derived from nivolumab.
  • CD46 was chosen because of its reported expression the parental HEK 293 cell line (jitc.bmj.com/content/6/1/55), which we confirmed on HEK Blue IL18 (not shown).
  • pro-IL18AS FUSE827)
  • pro-IL18AV FUSE775
  • FUSE827 contained no targeting domain (i.e., Fc only) and FUSE775 replaced the PD-1 specific fab with an EGFR specific VHH, 9G8, the ligand for which (EGFR) was not expressed on HEK Blue IL18 (data not shown).
  • FIG.15A and FG.15B are nonlinear x-y plots of IL18 biological activity versus the concentration of each test article using HEK Blue IL18 (FIG.15A) and PD-1 decorated HEK Blue IL18 (FIG. 15B).
  • HEK Blue IL18 FIG.15A
  • PD-1 decorated HEK Blue IL18 FIG. 15B
  • FUSE691 (nivolumab) was used as the negative control antibody in both FIG.15A and FIG.15B . No biological activity was observed from this test article. No appreciable difference was observed in the biological activities of recombinant human IL18 or the non targeted test articles (FUSE775 and FUSE827), allowing for comparison across FIG.15A and FIG.15B.
  • pro-IL18AS FUSE782
  • pro-IL18AV FUSE783
  • Summary tables of the potency of each pro-IL18 variant are shown below each graph.
  • Various embodiments of the invention are described above in the Detailed Description. While these descriptions directly describe the above embodiments, it is understood that those skilled in the art may conceive modifications and/or variations to the specific embodiments shown and described herein. Any such modifications or variations that fall within the purview of this description are intended to be included therein as well.

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Abstract

L'invention concerne des protéines de fusion, qui sont composées d'éléments tels qu'une ou plusieurs protéines/un ou plusieurs polypeptides capables de translocation dans un réticulum endoplasmique (ER) et d'une interleukine 18 (IL-18) ou d'un fragment ou variant d'IL-18. Les protéines/polypeptides capables de translocation dans le ER comprennent, sans s'y limiter, une protéine immunoglobulaire telle qu'une région Fc, un anticorps VHH et un fragment scFv, et une protéine globulaire telle que la sérum albumine. L'IL-18 peut être un précurseur d'IL-18 qui ressemble à la pro-cytokine naturelle dans laquelle l'IL-18 est fusionnée à l'extrémité C-terminale de son propeptide ou d'un variant de propeptide. Dans certains modes de réalisation, les protéines de fusion peuvent également comprendre un lieur peptidique clivable.
PCT/US2023/071663 2022-08-05 2023-08-04 Protéines de fusion d' il-18 et procédés de production d'il-18 WO2024031046A2 (fr)

Applications Claiming Priority (4)

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US202263395476P 2022-08-05 2022-08-05
US63/395,476 2022-08-05
US202363463505P 2023-05-02 2023-05-02
US63/463,505 2023-05-02

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