CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63/401,400, filed August 26, 2022, U.S. Provisional Application No. 63/422,719, filed November 4, 2022, and U.S. Provisional Application No. 63/493,434, filed March 31, 2023, each of which is incorporated herein by reference in their entirety.

SEQUENCE LISTING

[0002] The present application contains a Sequence Listing which has been submitted in XML format via EFS-Web and is hereby incorporated by reference in its entirety. Said XML copy having been created August 23, 2023, is named “108843.00466. xml” and is 588,030 bytes in size.

FIELD

[0003] The present disclosure generally relates to IL-18 polypeptides and conjugates with one or more mutations, which illustrate improved effects (e.g., reduced binding protein interaction binding, reduced toxicity, and/or increased serum half-life). Also provided are pharmaceutical compositions, diagnostic compositions and kits containing the polypeptides and conjugates disclosed herein, nucleic acids and expression vectors encoding the polypeptides disclosed herein, cells comprising the same, and methods of using the polypeptides, nucleic acids, expression vectors, and cells for therapeutic, and diagnostic purposes.

BACKGROUND

[0004] Interleukin- 18 (IL- 18) is an immunostimulatory cytokine belonging to the IL-1 family. IL-18 plays an important role in immunity as it regulates both innate and adaptive immune responses. Expression of IL-18 leads to release of proinflammatory cytokines (e.g., IFNy) as well as NO and chemokines and has been shown to have antitumor activity in preclinical models. Unfortunately, however the clinical efficacy of IL- 18 monotherapies has been limited and substantial toxicides have also been observed. Thus, IL- 18 monotherapy has not fulfilled its promise [0005] Therefore, what are needed in the art are compositions and methods that provide effective IL-18 therapy for the treatment of cancer and other diseases. Fortunately, as will be apparent from the detailed description that follows, the present disclosure provides for these and other needs.

SUMMARY

[0006] Provided herein are human IL- 18 variants that exhibit higher IL- 18 receptor a (IL- 18Rα) binding affinity and activity than wild-type IL-18 and/or reduced binding to IL-18 binding protein (IL-18BP). In certain embodiments, the IL- 18 variant polypeptides provide reduced toxicity. In certain embodiments, the IL- 18 variant polypeptides provide increased stability, for instance increased serum stability.

[0007] In one aspect, the disclosure provides IL-18 variants capable of binding IL-18 receptor α (with wild-type affinity) and having a reduced IL-18 binding protein (1L-18BP) binding and/or response. In certain embodiments, the IL- 18 variants comprise at least one mutation at a position selected from the group consisting of: E6, N91, and K93. In certain embodiments, the positions are with reference to wild-type IL-18 (SEQ ID NO:1). In certain embodiments, the variants have an amino acid sequence at least 90% identical to SEQ ID NO: 1. In certain embodiments, the variants have an amino acid sequence at least 90% identical to SEQ ID NO: 3. In certain embodiments, the variants have an amino acid sequence at least 90% identical to SEQ ID NO: 4.

[0008] In one aspect, the disclosure provides IL-18 variants capable of binding IL-18 receptor α (with wild-type affinity) and having a reduced IL-18 binding protein (IL-18BP) binding and/or response. In certain embodiments, the IL- 18 variants comprise at least one mutation selected from the group consisting of: N14, SI 17, K4, 148, 171, E77, N78, K79, 180, S82, K84, E85, M86, N87, D90, D94, D98, Y120, E121, Y123, 1137, K140, and D157. In certain embodiments, the positions are with reference to wild-type IL-18 (SEQ ID NO:1). In certain embodiments, the variants have an amino acid sequence at least 90% identical to SEQ ID NO: 1. In certain embodiments, the variants have an amino acid sequence at least 90% identical to SEQ ID NO: 3. In certain embodiments, the variants have an amino acid sequence at least 90% identical to SEQ ID NO: 4. [0009] In another aspect, provided herein are fusion constructs comprising the IL-18 variants described herein and one or more additional polypeptides fused thereto. In another aspect, provided herein are conjugates comprising the IL-18 variants described herein linked to water- soluble polymers.

[00010] In another aspect, provided herein are polynucleotides encoding the IL-18 variants and/or fusion constructs described herein. In a further aspect, provided herein are expression vectors comprising the polynucleotides. In a further aspect, provided herein are cells comprising the polynucleotides or expression vectors. In some embodiments, the cells are selected from bacterial cells, fungal cells, and mammalian cells. In some embodiments, the cells are selected from E . coll cells, Saccharomyces cerevisiae cells, and CHO cells. In another aspect, provided herein are methods of making the IL- 18 variants and/or fusion constructs, for instance using the polynucleotides, expression vectors, and/or cells described herein.

[00011] In another aspect, provided herein are methods of treating, preventing, or diagnosing a disease or condition in a subject in need thereof, wherein the method includes administering to the subject an effective amount of the IL-18 variant, fusion construct, or conjugate of any of the foregoing embodiments, or a composition or a pharmaceutical composition containing the same. In some embodiments, the disease or condition is selected from a cancer, an autoimmune disease, an inflammatory disease, and an infection. In some embodiments, the effective amount is a therapeutically effective amount.

[00012] In another aspect, provided herein is the use of the IL- 18 variants, fusion constructs, or conjugates of any of the foregoing embodiments for treating, preventing, or diagnosing a disease or condition provided herein in a subject in need thereof. In another aspect, provided herein are IL-18 variants, fusion constructs, or conjugates of any of the foregoing embodiments for use in treating, preventing, or diagnosing a disease or condition provided herein in a subject in need thereof. In another aspect, provided herein are also IL- 18 variants, fusion constructs, or conjugates of any of the foregoing embodiments for use in the manufacture of a medicament for treating, preventing, or diagnosing a disease or condition provided herein in a subject in need thereof. In another aspect, provided herein are IL- 18 variants, fusion constructs, or conjugates of any of the foregoing embodiments for use in a method for treating or preventing any disease or disorder provided herein in a subject in need thereof. In another aspect, provided herein are IL- 18 variants, fusions constructs, or conjugates of any of the foregoing embodiments for use in the manufacture of a treatment for a disease or disorder provided herein. In some embodiments, the disease or condition is selected from a cancer, an autoimmune disease, an inflammatory disease, and an infection.

[00013] These and other embodiments along with many of its features are described in more detail in conjunction with the text below and attached figures. Other features, objects, and advantages will be apparent from the disclosure that follows.

BRIEF DESCRIPTION OF THE DRAWINGS

[00014] FIG. 1 shows an SDS-PAGE of IL- 18 PEGylated with DBCO-mPEG in different PEG sizes.

[00015] FIG. 2 shows PEG density of PEGylated IL-18 by RP-HPLC.

[00016] FIG. 3A shows that mCS2 variants conjugated at the M70 site to different PEG sizes ranging from 10K to 40K have extended PK profiles compared to unconjugated mCS2. For comparison, mCS2 variant conjugated at the SI 57 site to a 40K PEG is included. Balb/c mice were dosed with IV bolus administration of 1 mg/kg mCS2 variants. Plasma concentrations were determined by ELISA using an anti -mouse IL- 18 antibody. Data are presented as mean ± standard deviation (SD).

[00017] FIG. 3B shows that mCS2 variants conjugated at the S157 site to different PEG sizes ranging from 10K to 40K have extended PK profiles compared to unconjugated mCS2. For comparison, mCS2 variant conjugated at the M70 site to a 40K PEG (SP 10766) is included. Balb/c mice were dosed with IV bolus administration of 1 mg/kg mCS2 variants. Plasma concentrations were determined by ELISA using an anti -mouse IL- 18 antibody. Data are presented as mean ± standard deviation (SD).

[00018] FIG. 4A-FIG. 4B show certain IL-18 variants discovered from ribosome display library. FIG. 4A shows the results from an SPR based Biacore binding assay of certain IL-18 variants. FIG. 4B shows the results from a HEK Blue human IL- 18 reporter assay of certain IL-18 variants.

[00019] FIG. 5A-FIG. 5C show the results from a HEK Blue human IL- 18 reporter assay of certain IL-18 variants FIG. 5A shows certain IL-18 variants conjugated to a 30k PEG of at different conjugation sites. FIG. 5B shows certain IL-18 variants conjugated to PEGs of different sizes at the same conjugation sites. FIG. 5C shows certain IL-18 variants with cysteine mutations conjugated to a 30k PEG.

[00020] FIG. 6A and FIG. 6B show certain IL- 18 variant induced potent IFNγ release without the negative regulation of IL18BP when co-cultured with human PBMCs.

[00021] FIG. 7A-FIG. 7C show the PK profile in mice of certain human IL-18 variants. FIG. 7A shows certain IL- 18 variants conjugated to PEGs of different sizes. FIG.7B shows certain IL-18 variants conjugated to PEGs of different sizes at different conjugate sites. FIG. 7C shows certain IL-18 variants with cysteine mutations conjugated to a 40k PEG at D157 site.

[00022] FIG. 8A-FIG. 8C show A) SDS-PAGE gel of purification of nnAA-IL18. Left: captured Ulpl -cleaved nnAA-IL18, Right: PEGylated nnAA-IL18. B) Analytical SEC chromatogram of finalized PEGylated nnAA-IL18. C) Table describing titers, binding properties, and purity ofWT IL-18, HisSUMO-IL18, nnAA-IL18, and PEGylated nnAA-IL18. Titers were calculated based on the amount of SUMOylated protein that was purified. Purity % was assessed by analytical SEC, and PEG-to-protein ratio was calculated using SDS-PAGE gel densitometry.

[00023] FIG. 9A-FIG. 9D show A) SDS-PAGE gel analysis of lysates from high density fermentations producing nnAA-IL18 before (left lane) and after (right lane) induction with arabinose. The arrow indicates the presence of a band corresponding to the size of HisSUMO- nnAA-IL18 in the post-induction lysate that was not present in the pre-induction lysate. B) Deconvoluted intact LC-MS spectra of nnAA-ILl 8 before (top panel) and after (bottom panel) conjugation with a small molecule DBCO-amine. The mass shift of 1183 Da shows that the protein was conjugated with 1 DBCO-amine. C) and D) Biacore binding curves produced with a commercially-purchased WT IL-18 standard (C) and the PEGylated nnAA-IL18 (D). The y- axis represents signal intensity, while the x-axis shows the time in seconds. Parameters for each experiment are described in the table.

DETAILED DESCRIPTION

[00024] Provided herein are IL-18 variants, fusion constructs, and conjugates and compositions comprising the same, wherein the IL-18 variants comprise at least one amino acid substitution relative to a wild-type IL-18. As disclosed herein, the at least one amino acid substitution at a specific site can improve the characteristics of the IL-18 variant relative to a wild-type (i.e., parent) IL-18. For example, amino acid substitutions as disclosed herein can lead to reduced binding protein binding and/or reduced toxicity and/or increased stability, relative to a wild- type IL-18, which can contribute to favorable expression, thermal stability, and serum clearance profde. This can lead to advantages with respect to the use of the IL-18 variants, fusion constructs, or conjugates in therapy or diagnosis. In some embodiments, the variants provide mutations that facilitate conjugation, for instance to water-soluble polymers.

1.1. Definitions

[00025] Unless otherwise defined, all terms of art, notations and other scientific terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this invention pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a difference over what is generally understood in the art. The techniques and procedures described or referenced herein are generally well understood and commonly employed using conventional methodologies by those skilled in the art, such as, for example, the widely utilized molecular cloning methodologies described in Green & Sambrook, Molecular Cloning: A Laboratory Manual 4 ^th ed. (2012) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, or in Current Protocols in Molecular Biology (2022), Wiley Periodicals LLC. As appropriate, procedures involving the use of commercially available kits and reagents are generally carried out in accordance with manufacturer-defined protocols and conditions unless otherwise noted.

[00026] As used herein, the singular forms “a,” “an,” and “the,” include the plural referents unless the context clearly indicates otherwise.

[00027] The term “about,” as used herein, indicates and encompasses an indicated value and a range above and below that value. In certain embodiments, the term “about” indicates the designated value ± 10%, ± 5%, or ± 1%. In certain embodiments, the term “about” indicates the designated value ± one standard deviation of that value.

[00028] The term “combinations thereof,” as used herein, includes every possible combination of elements to which the term refers to.

[00029] Ranges: throughout this disclosure, various aspects of the disclosure can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[00030] The term “human interleukin-18,” or “human IL-18,” or “hIL-18,” as used herein, refers to a proinflammatory cytokine of the IL-1 family having an amino acid sequence according to amino acids 37-193 ofUniProt Accession No. Q14116 (SEQ ID NO:1). IL-18 is constitutively found as a precursor within the cytoplasm of a variety of cells including, e.g., macrophages and keratinocytes. The inactive IL-18 precursor is processed to its active form by caspase- 1, and is capable of stimulating IFNy production, and of regulating both T helper (Th) 1 and Th2 responses. In humans, IL-18 gene is located on chromosome 11. A representative active form IL-18 sequence is provided by SEQ ID NO: 1 :

YFGKLESKLS VIRNLNDQVL FIDQGNRPLF EDMTDSDCRD NAPRTI FI I S

MYKDSQPRGM AVTI SVKCEK I STLSCENKI I SFKEMNPPD NIKDTKSDI I FFQRSVPGHD NKMQFESSSY EGYFLACEKE RDLFKLILKK EDELGDRSIM FTVQNED .

[00031] The term “human interleukin- 18 receptor alpha” or “human IL-18Rα,” or “hIL-18 Rα,” as used herein, refers to a receptor for IL- 18 encoded by the IL- 18R1 gene. Representative IL- 18Rα sequences are provided by UniProt. Accession No. Q 13478.

[00032] The term “human interleukin- 18 receptor beta” or “human IL-18Rβ,” or “hIL-18 Rβ ,” as used herein, refers to IL-18 receptor accessory protein encoded by the IL-18RAP gene. Representative IL-18Rβ sequences are provided by UniProt. Accession No. 095256.

[00033] “IL- 18 binding protein,” or “IL-18BP” as used herein, refers to the protein encoded by the IL-18BP gene that is capable of binding and inhibiting IL-18. Representative IL-18BP sequences are provided by UniProt. Accession No. 095998.

[00034] The term “operably-linked,” as used herein, refers to a functional linkage between two elements, regardless of orientation or distance between the two elements, such that the function of one element is controlled or affected by the other element. For example, operable linkage with reference to a promoter and heterologous coding sequence means that the transcription of the heterologous coding sequence is under the control of, or driven by, the promoter. Tn another example, operable linkage with reference to an enhancer and promoter means that the enhancer increases the level of transcription driven by a promoter.

[00035] The term “isolated,” as used herein, refers to a substance that has been separated and/or recovered from its natural environment. For example, a naturally occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the co-existing materials in the natural system, is isolated. Such polynucleotide could be part of a vector and/or such polynucleotide or polypeptide could be part of a composition (e.g., a cell lysate), and still be isolated in that such vector or composition is not part of the natural environment for the nucleic acid or polypeptide.

[00036] An “isolated IL- 18 variant” is one that has been separated and/or recovered from a component of its natural environment. Components of the natural environment may include enzymes, hormones, and other proteinaceous or nonproteinaceous materials. In some embodiments, an isolated IL- 18 variant is purified to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence, for example by use of a spinning cup sequenator. In some embodiments, an isolated IL- 18 variant is purified to homogeneity by gel electrophoresis (e.g., SDS-PAGE) under reducing or nonreducing conditions, with detection by Coomassie blue or silver stain. In some aspects, an isolated IL-18 variant is prepared by at least one purification step.

[00037] The term “substantially pure” with respect to a composition comprising a variant IL- 18 refers to a composition that includes at least 80%, 85%, 90% or 95% by weight or, in certain embodiments, 95%, 98%, 99% or 100% by weight, e.g., dry weight, of the IL- 18 variant relative to the remaining portion of the composition. The weight percentage can be relative to the total weight of protein in the composition or relative to the total weight of IL-18 variant in the composition. Purity can be determined by techniques apparent to those of skill in the art, for instance SDS-PAGE.

[00038] In some embodiments, an isolated IL- 18 variant is purified to at least 80%, 85%, 90%, 95%, or 99% by weight. In some embodiments, an isolated IL-18 variant is purified to at least 80%, 85%, 90%, 95%, or 99% by volume. In some embodiments, an isolated IL-18 variant is provided as a solution comprising at least 85%, 90%, 95%, 98%, 99% to 100% by weight. In some embodiments, an isolated IL-18 variant is provided as a solution comprising at least 85%, 90%, 95%, 98%, 99% to 100% by volume.

[000391 “Affinity” refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., IL-18) and its binding partner (e.g., IL-18Rα). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity, which reflects a 1 : 1 interaction between members of a binding pair (e.g., IL-18 and IL-18Rα, or IL- 18 and IL-18BP). The affinity of a molecule X for its partner Y can be represented by the dissociation constant (KD). Affinity can be measured by common methods known in the art, including those described herein. Affinity can be determined, for example, using surface plasmon resonance (SPR) technology, such as a Biacore® instrument. In some embodiments, the affinity is determined at 25°C.

[00040] With regard to the binding of receptor to a target molecule (ligand), the terms “specific,” “specific binding,” “specifically binds to,” “specific for,” “selectively binds,” or “selective for,” as used herein, refers to a particular receptor or ligand that exhibits binding that is measurably different from a non-specific or non-selective interaction. Specific binding can be measured, for example, by determining binding of a molecule compared to binding of a control molecule. Specific binding can also be determined by competition with a control molecule that competes with the ligand for binding to the receptor. In that case, specific binding is indicated if the binding of the ligand to the receptor is competitively inhibited by the control molecule.

[00041] The term “k _d ” (sec ^-1 ), as used herein, refers to the dissociation rate constant of a particular receptor-ligand interaction. This value is also referred to as the k _o rr value.

[00042] The term “k _a ” (M ^-1 sec ^-1 ), as used herein, refers to the association rate constant of a particular receptor-ligand interaction. This value is also referred to as the k _on value.

[00043] The term “K _D ” (M), as used herein, refers to the dissociation equilibrium constant of a particular receptor-ligand interaction. KD = k _d /k _a .

[00044] The term “K _A ” (M ^-1 ), as used herein, refers to the association equilibrium constant of a particular receptor-ligand interaction. K _A = k _a / k _d .

[00045] The term “Tm” as used herein, has the meaning commonly understood in the art and refers to is the temperature at which the equilibrium between folded and unfolded forms of the enzyme is at its mid-point. [00046] The term “EC50” or “half maximal effective concentration” as used herein, has the meaning commonly understood in the art and refers to the concentration of a substance e.g., a drug, e.g., an IL-18 variant, which induces a response halfway between the baseline and maximum after a specified exposure time. Thus, EC50 can be defined as the concentration required to obtain a 50% effect and represents the concentration of a compound where 50% of its maximal effect is observed.

[00047] The term “half-life” or “t _1/2 ” as used herein refers to the amount of time required for the drug concentration measured in a sample to be reduced to half of its starting concentration or amount. The term “terminal t _1/2 ” as used herein, refers to the amount of time required for the drug concentration measured in a sample to be reduced to half of its pseudo-equilibrium concentration or amount.

[00048] The term “C ₀ ” as used herein, has the meaning commonly understood in the art and refers to the plasma concentration at the time of dosing (time 0).

[00049] The term “AUC” as used herein, has the meaning commonly understood in the art of pharmacokinetics, and refers to the area under the plasma drug concentration-time curve (AUC) and reflects the measure of how much drug reaches an individual’s bloodstream in a given period of time after a dose is given. AUC is dependent on the rate of elimination of the drug from the body and the dose administered. AUC is directly proportional to the dose when the drug follows linear kinetics and is inversely proportional to the clearance of the drug.

[00050] The term “AUC _0-last ” as used herein, has the meaning commonly understood in the art of pharmacokinetics, and refers to the AUC from dosing (time 0) to the last measurable concentration.

[00051] The term “ AUC _{0 -inf} ’ as used herein, has the meaning commonly understood in the art of pharmacokinetics, and refers to the AUC from dosing (time 0) extrapolated to infinity.

[00052] The term “clearance” as used herein, refers to the rate at which an active drug e.g., an IL-18 variant as disclosed herein, is removed from the body. “Clearance” is typically reported as the ratio of the elimination rate of a drug to the plasma drug concentration.

[00053] The term “V _ss ” as used herein, has the meaning commonly understood in the art and refers to the apparent volume of distribution at steady state which describes the physiological distribution of the drug candidate. [00054] The term “steady state” as used herein has the meaning commonly understood in the art of pharmacokinetics, and refers to the condition when the administration of a drug and the clearance are balanced, creating a plasma concentration that is unchanged by time.

[00055] The terms “protein,” “peptide,” and “polypeptide” are used interchangeably herein, and refer to a polymer of amino acid residues linked together by peptide (amide) bonds. Although the terms refer to a protein, peptide, or polypeptide of any size, structure, or function. Typically, a peptide will be at least three amino acids long and equal to or less than about 10 amino acids in length. A polypeptide is typically greater than 10 amino acids in length. A protein, peptide, or polypeptide may refer to an individual protein or a collection of proteins. One or more of the amino acids in a protein, peptide, or polypeptide may be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a hydroxyl group, a phosphate group, a farnesyl group, an isofarnesyl group, a fatty acid group, a linker for conjugation, functionalization, or other modification, etc., or may be substituted with a non- natural amino acid. A protein, peptide, or polypeptide may also be a single molecule or may be a multi-molecular complex. A protein, peptide, or polypeptide may be just a fragment of a naturally occurring protein or peptide. A protein, peptide, or polypeptide may be naturally occurring, recombinant, or synthetic, or any combination thereof. A protein may comprise different domains, for example, a protein binding domain and a catalytic domain. Any of the proteins provided herein may be produced by any method known in the art. For example, the proteins provided herein may be produced via recombinant protein expression and purification. Methods for recombinant protein expression and purification are well known, and include those described by Green and Sambrook, Molecular Cloning: A Laboratory Manual (4 ^th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)), the entire contents of which are incorporated herein by reference.

[00056] A “mutation” as used herein, refers to a change in nucleic acid or polypeptide sequence relative to a reference sequence. A mutation may comprise a substitution of a residue within a sequence, e.g., a nucleic acid or amino acid sequence, with another residue, or a deletion or insertion of one or more residues within a sequence. Mutations are typically described herein by identifying the original residue followed by the position of the residue within the sequence and by the identity of the newly substituted residue. Various methods for making the amino acid substitutions (mutations) provided herein are well known in the art, and are provided by, for example, Green and Sambrook, Molecular Cloning: A Laboratory Manual (4 ^th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)).

[00057] As noted above, a mutation can be a “substitution” mutation wherein the amino acid, or nucleotide at a particular position in a reference sequence is substituted with a different amino acid or nucleotide at that position in the amino acid or nucleic acid sequence. In some embodiments, a substitution replaces one amino acid at a specific location in a polypeptide or protein sequence for a different amino acid in that position of the polypeptide or protein sequence. In some embodiments, a “substitution” replaces a natural amino acid at a specific location in a polypeptide or protein sequence for a non-natural amino acid in that position of the polypeptide or protein sequence. Thus, the term “substitution” as used herein, refers to as “substitution” mutation as disclosed herein above.

[00058] The term “reversion mutation,” or “reversion” as used herein, refers to a particular type of substitution mutation wherein a polypeptide or nucleic acid sequence having a substitution mutation at a specific position in the sequence, acquires a mutation at that specific position that restores the original sequence. Thus, in some embodiments, a polypeptide sequence having a mutation at a specific position in the polypeptide sequence acquires a mutation that restores the amino acid at that specific position to the amino acid found in the reference sequence e.g., restores the wild-type sequence).

[00059] The term “wild-type” or “parent” refers to a naturally occurring gene or protein. These include a naturally occurring IL- 18 gene or protein.

[00060] The term “variant” or “mutant” as used herein, refers to a nucleic acid or polypeptide sequence having at least one mutation relative to a reference sequence. Accordingly, a “variant” or “mutant” typically has less than 100% sequence identity to a reference sequence.

[00061] The terms “identical,” or “percent identity,” in the context of two or more polypeptide or nucleic acid molecule sequences, means two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same over a specified region (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity), when compared and aligned for maximum correspondence over a comparison window, or designated region, as measured using methods known in the art, such as a sequence comparison algorithm, by manual alignment, or by visual inspection. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST (Altschul et al. Nucleic Acids Res. 2007, 25, 3389-3402), BLAST-2, ALIGN, MEGALIGN (DNASTAR), CLUSTALW, CLUSTAL OMEGA, or MUSCLE software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. Within the context of this disclosure, it is understood that where sequence analysis software is used for analysis, the results of the analysis are based on the “default values” of the program referenced. “Default values” mean any set of values or parameters which originally load with the software when first initialized.

[00062] As applied to polypeptides, the term “substantial similarity” or “substantially similar” means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity. In some aspects, residue positions, which are not identical, differ by conservative amino acid substitutions.

[00063] The term “amino acid,” as used herein, refers to the twenty common naturally occurring amino acids. Naturally occurring amino acids include alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine (Cys; C); glutamic acid (Glu; E), glutamine (Gin; Q), Glycine (Gly; G); histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Vai; V).

[00064] Naturally encoded amino acids are the proteinogenic amino acids known to those of skill in the art. They include the 20 common amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine) and the less common pyrrolysine and selenocysteine. Naturally encoded amino acids include post- translational variants of the 22 naturally occurring amino acids such as prenylated amino acids, isoprenylated amino acids, myristoylated amino acids, palmitoylated amino acids, N-linked glycosylated amino acids, O-linked glycosylated amino acids, phosphorylated amino acids and acylated amino acids. [00065] A “conservative substitution,” or a “conservative amino acid substitution,” as used herein, refers to the substitution of an amino acid with a chemically or functionally similar amino acid. Conservative substitution tables providing similar amino acids are well known in the art. Polypeptide sequences having such substitutions are known as “conservatively modified variants.” Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles. By way of example, the groups of amino acids provided in Tables 1-3 are, in some embodiments, considered conservative substitutions for one another.

Table 1. Selected groups of amino acids that are considered conservative substitutions for one another, in certain embodiments.

Table 2. Additional selected groups of amino acids that are considered conservative substitutions for one another, in certain embodiments. Table 3. Further selected groups of amino acids that are considered conservative substitutions for one another, in certain embodiments.

[00066] Additional conservative substitutions may be found, for example, in Creighton, Proteins: Structures and Molecular Properties 2nd ed. (1993) W. H. Freeman & Co., New York, NY. A polypeptide or protein generated by making one or more conservative substitutions of amino acid residues in a parent polypeptide or protein is referred to as a “conservatively modified variant ”

[00067] The term “non-natural amino acid” refers to an amino acid that is not a proteinogenic amino acid, or a post-translationally modified variant thereof. In particular, the term refers to an amino acid that is not one of the 20 common amino acids or pyrrolysine or selenocysteine, or post-translationally modified variants thereof. Exemplary non-natural amino acids include e.g., p-acetylphenylalanine (pAcF), azido-lysine (AzK), and p-azidomethyl-L -phenylalanine (pAMF). Non-natural amino acid such as pAcF, AzK, and pAMF provide side chains to which various secondary molecules e.g., polyethyleneglycol (PEG) can be conjugated/bound. In preferred embodiments, a non-natural amino acid is pAMF. pAMF is typically incorporated into proteins at the TAG amber codon using method known in the art (see e.g., Zimmerman, E. S. et al. Bioconjug. Chem. 25, 351-361 (2014)). pAMF incorporation provides an efficient approach for site-specific modification of the proteins and subsequent conjugation-site specific modification.

[00068] The term “disease,” or “disease or disorder” as used herein, refers any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ. Examples of diseases include neoplasia, pathogen infection of cell, etc.

[00069] As used herein, “treating” or “treatment” of any disease or disorder refers, in certain embodiments, to ameliorating a disease or disorder that exists in a subject. “Treating” or “treatment” includes ameliorating at least one physical parameter, which may be indiscernible by the subject. In yet another embodiment, “treating” or “treatment” includes modulating the disease or disorder, either physically (e.g., stabilization of a discernible symptom) or physiologically (e.g., stabilization of a physical parameter) or both. In yet another embodiment, “treating” or “treatment” includes delaying or preventing the onset of the disease or disorder. For example, in an exemplary embodiment, the phrase “treating cancer” refers to inhibition of cancer cell proliferation, inhibition of cancer spread (metastasis), inhibition of tumor growth, reduction of cancer cell number or tumor growth, decrease in the malignant grade of a cancer (e.g., increased differentiation), or improved cancer-related symptoms. Further, as used herein, “treatment” includes preventing or delaying the recurrence of the disease, delaying or slowing the progression of the disease, ameliorating the disease state, providing a remission (partial or total) of the disease, decreasing the dose of one or more other medications required to treat the disease, delaying the progression of the disease, increasing or improving the quality of life, increasing weight gain, and/or prolonging survival. Also encompassed by “treatment” is a reduction of pathological consequence of cancer.

[00070] As used herein, the term “therapeutically effective amount” or “effective amount” refers to an amount of a substance e.g., an IL-18 variant disclosed herein, or a composition comprising a substance, that when administered to a subject is effective to treat a disease or disorder. For example, in an exemplary embodiment, the phrase “effective amount” is used interchangeably with “therapeutically effective amount” or “therapeutically effective dose” and the like, and means an amount of a therapeutic agent that is effective to prevent or ameliorate a disease or the progression of the disease e.g., cancer, or result in amelioration of symptoms. Effective amounts of the compositions provided herein may vary according to factors such as the disease state, age, sex, weight of the animal or human.

[00071] The term “subject,” as used herein, refers to a mammalian subject. Exemplary subjects include, but are not limited to humans, monkeys, dogs, cats, mice, rats, cows, pigs, horses, camels, avians, goats, and sheep. In certain embodiments, the subject is a human. In some embodiments, the subject has a disease that can be treated with an IL-18 variant provided herein.

[00072] The term “therapeutically effective amount,” or “effective amount” as used herein, refers to the amount of the subject compound or composition that will elicit the biological, 1 0hysiologic, clinical, or medical response of a cell, tissue, organ, system, or subject that is being sought by the researcher, veterinarian, medical doctor, or other clinician. The term “therapeutically effective amount” refers to an amount of a compound e.g., an IL- 18 variant, or composition that, when administered, is sufficient to prevent development of, or treat at least to some extent, one or more of the signs or symptoms of the disorder or disease being treated. The therapeutically effective amount will vary depending on the compound or composition, the disease and its severity and the age, weight, etc., of the subject to be treated.

[00073] The term “pharmaceutical composition,” as used herein, refers to a composition that can be administrated to a subject in the context of treatment of a disease or disorder. In some embodiments, a pharmaceutical composition comprises an active ingredient, e.g., an IL-18 variant as disclosed herein, and a pharmaceutically acceptable excipient.

[00074] When referring to the compounds provided herein, the following terms have the following meanings unless indicated otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art. In the event that there is a plurality of definitions for a term herein, those in this section prevail unless stated otherwise.

[00075] The term “alkyl,” as used herein, unless otherwise specified, refers to a saturated straight or branched hydrocarbon. In certain embodiments, the alkyl group is a primary, secondary, or tertiary hydrocarbon. In certain embodiments, the alkyl group includes one to ten carbon atoms (i.e., C ₁ to C ₁₀ alkyl). In certain embodiments, the alkyl is a lower alkyl, for example, C _1-6 alkyl, and the like. In certain embodiments, the alkyl group is selected from the group consisting of methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, t-butyl, pentyl, isopentyl, neopentyl, hexyl, isohexyl, 3-methylpentyl, 2,2-dimethylbutyl, and 2,3- dimethylbutyl. In certain embodiments, “substituted alkyl” refers to an alkyl substituted with, for example, one, two, or three groups independently selected from a halogen (e.g., fluoro (F), chloro (Cl), bromo (Br), or iodo (I)), alkyl, -CN, -NO ₂ , amido, -C(O)-, -C(S)-, ester, carbamate, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, dialkylamino, haloalkyl, hydroxyl, amino, alkylamino, and alkoxy. In some embodiments, alkyl is unsubstituted.

[00076] The term “alkylene,” as used herein, unless otherwise specified, refers to a divalent alkyl group, as defined herein. “Substituted alkylene” refers to an alkylene group substituted as described herein for alkyl. In some embodiments, alkylene is unsubstituted. [00077] “Alkenyl” refers to an olefinically unsaturated hydrocarbon group, in certain embodiments, having up to about eleven carbon atoms or from two to six carbon atoms (e.g., “lower alkenyl”), which can be straight-chained or branched, and having at least one or from one to two sites of olefinic unsaturation. “Substituted alkenyl” refers to an alkenyl group substituted as described herein for alkyl.

[00078] The term “aryl,” as used herein, and unless otherwise specified, refers to phenyl, biphenyl, or naphthyl. In certain embodiments, the aryl group is unsubstituted. In certain embodiments, the an aryl group is substituted with one or more moieties (e.g., in some embodiments one, two, or three moieties) selected from the group consisting of halogen (e.g., fluoro (F), chloro (Cl), bromo (Br), or iodo (I)), alkyl, haloalkyl, hydroxyl, amino, alkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, sulfonic acid, sulfate, phosphonic acid, phosphate, and phosphonate, wherein each moiety is independently either unprotected, or protected as necessary, as would be appreciated by those skilled in the art (see, e.g., Greene, et al., Protective Groups in Organic Synthesis, John Wiley and Sons, Second Edition, 1991); and wherein the aryl in the arylamino and aryloxy substituents are not further substituted.

[00079] The term “arylene,” as used herein, and unless otherwise specified, refers to a divalent aryl group, as defined herein.

[00080] The term “heteroalkyl” refers to an alkyl, as defined herein, in which one or more carbon atoms are replaced by heteroatoms. As used herein, “heteroalkenyl” refers to an alkenyl, as defined herein, in which one or more carbon atoms are replaced by heteroatoms. As used herein, “heteroalkynyl” refers to an alkynyl, as defined herein, in which one or more carbon atoms are replaced by heteroatoms. Suitable heteroatoms include, but are not limited to, nitrogen (N), oxygen (O), and sulfur (S) atoms. Heteroalkyl, heteroalkenyl, and heteroalkynyl are optionally substituted. Examples of heteroalkyl moieties include, but are not limited to, aminoalkyl, sulfonylalkyl, and sulfinylalkyl. Examples of heteroalkyl moieties also include, but are not limited to, methylamino, methylsulfonyl, and methyl sulfinyl. “Substituted heteroalkyl” refers to heteroalkyl substituted with one, two, or three groups independently selected from halogen (e.g., fluoro (F), chloro (Cl), bromo (Br), or iodo (I)), alkyl, haloalkyl, hydroxyl, amino, alkylamino, and alkoxy. In some embodiments, a heteroalkyl group may comprise one, two, three, or four heteroatoms. Those of skill in the art will recognize that a 4- membered heteroalkyl may generally comprise one or two heteroatoms, a 5- or 6-membered heteroalkyl may generally comprise one, two, or three heteroatoms, and a 7- to 10-membered heteroalkyl may generally comprise one, two, three, or four heteroatoms.

[000811 The term “heteroalkylene,” as used herein, refers to a divalent heteroalkyl, as defined herein. “Substituted heteroalkylene” refers to a divalent heteroalkyl, as defined herein, substituted as described for heteroalkyl.

[00082] The term “heteroaryl” refers to a monovalent, monocyclic aromatic group and/or multicyclic aromatic group, wherein at least one aromatic ring contains one or more heteroatoms independently selected from oxygen, sulfur, and nitrogen within the ring. Each ring of a heteroaryl group can contain one or two oxygen atoms, one or two sulfur atoms, and/or one to four nitrogen atoms, provided that the total number of heteroatoms in each ring is four or less and each ring contains at least one carbon atom. In certain embodiments, the heteroaryl has from five to twenty, from five to fifteen, or from five to ten ring atoms. A heteroaryl may be attached to the rest of the molecule via a nitrogen or a carbon atom. In some embodiments, monocyclic heteroaryl groups include, but are not limited to, furanyl, imidazolyl, isothiazolyl, isoxazolyl, oxadiazolyl, oxazolyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl, pyrrolyl, triazolyl, thiadiazolyl, thiazolyl, thienyl, tetrazolyl, and triazinyl. Examples of bicyclic heteroaryl groups include, but are not limited to, benzofuranyl, benzimidazolyl, benzoisoxazolyl, benzopyranyl, benzothiadiazolyl, benzothiazolyl, benzothienyl, benzotriazolyl, benzoxazolyl, furopyridyl, imidazopyridinyl, imidazothiazolyl, indolizinyl, indolyl, indazolyl, isobenzofuranyl, isobenzothienyl, isoindolyl, isoquinolinyl, naphthyridinyl, oxazolopyridinyl, phthalazinyl, pteridinyl, purinyl, pyridopyridyl, pyrrol opyridyl, quinolinyl, quinoxalinyl, quinazolinyl, thiadiazolopyrimidyl, and thi enopyridyl. Examples of tricyclic heteroaryl groups include, but are not limited to, acridinyl, benzindolyl, carbazolyl, dibenzofuranyl, perimidinyl, phenanthrolinyl, phenanthridinyl, phenarsazinyl, phenazinyl, phenothiazinyl, phenoxazinyl, and xanthenyl. In certain embodiments, heteroaryl may also be optionally substituted as described herein. “Substituted heteroaryl” is a heteroaryl substituted as defined for aryl.

[00083] The term “heteroarylene” refers to a divalent heteroaryl group, as defined herein. “Substituted heteroarylene” is a heteroaiylene substituted as defined for aryl.

[00084] The term “substituted” refers to moieties having substituents replacing a hydrogen on one or more carbons or substitutable heteroatoms, e g., an NH or NEE of a compound. It will be understood that “substitution” or “substituted with” includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, i.e., a compound which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc. In certain embodiments, substituted refers to moieties having substituents replacing two hydrogen atoms on the same carbon atom, such as substituting the two hydrogen atoms on a single carbon with an oxo, imino or thioxo group. As used herein, the term “substituted” is contemplated to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic substituents of organic compounds. The permissible substituents can be one or more and the same or different for appropriate organic compounds.

[00085] In some embodiments, substituents may include any substituents described herein, for example: halogen, hydroxy, oxo (=0), thioxo (=S), cyano (-CN), nitro (-NO ₂ ), imino (=N-H), oximo (=N-0H), hydrazino (=N- NH ₂ ), -R ^b1 -OR ^a1 , -R ^b1 -OC(O)-R ^a1 , -R ^b1 -OC(O)-OR ^a1 , -R ^b1 - OC(O)-N(R ^a1 ) ₂ , -R ^b1 -N(R ^a ) ₂ , -R ^b1 -C(O)R ^a1 , -R ^b1 -C( O)OR ^a1 , -R ^b1 -C(O)N(R ^a1 ) ₂ , -R ^b1 -0-R ^c1 - C(O)N(R ^a1 ) ₂ , -R ^b1 -N(R ^a1 )C(O)OR ^a1 , -R ^b1 -N(R ^a )C(O)R ^a1 , -R ^b1 -N(R ^a1 )S( O) _t R ^a1 (where t is 1 or 2), -R ^b1 -S(O)tR ^a1 (where t is 1 or 2), -R ^b1 -S(O)tOR ^a1 (where t is 1 or 2), and -R ^b1 - S(O)tN(R ^a1 )2 (where t is 1 or 2); and alkyl, alkenyl, alkynyl, aryl, aralkyl, aralkenyl, aralkynyl, cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl, and heteroarylalkyl any of which may be optionally substituted by alkyl, alkenyl, alkynyl, halogen, haloalkyl, haloalkenyl, haloalkynyl, oxo (=0), thioxo (=S), cyano (-CN), nitro (-NO ₂ ), imino (=N- H), oximo (=N-0H), hydrazine (=N- NH ₂ ), -R ^b1 -OR ^a1 , -R ^b1 -OC(O)-R ^a1 , -R ^b1 -OC(O)- OR ^a1 , -R ^b1 -0C(0)-N(R ^a1 ) ₂ , -R ^b1 -N(R ^a ) ₂ , -R ^b1 -C(O)R ^a1 , -R ^b1 -C( O)OR ^a1 , -R ^b1 -C(0)N(R ^a1 ) ₂ , - R ^b1 -O-R ^c1 -C(0)N(R ^a1 ) ₂ , -R ^b1 -N(R ^a1 )C(0)0R ^a1 , -R ^b1 -N(R ^a1 )C(O)R ^a1 , -R ^b1 - N(R ^a1 )S(O)tR ^a1 (where t is 1 or 2), -R ^b1 -S(O)tR ^a1 (where t is 1 or 2), -R ^b1 -S(O)tOR ^a1 (where t is 1 or 2) and -R ^b1 -S(O)tN(R ^a1 )2 (where t is 1 or 2); wherein each R ^a1 is independently selected from hydrogen, alkyl, cycloalkyl, cycloalkylalkyl, aryl, aralkyl, heterocycloalkyl, heterocycloalkylalkyl, heteroaryl, or heteroarylalkyl, wherein each R ^a1 , valence permitting, may be optionally substituted with alkyl, alkenyl, alkynyl, halogen, haloalkyl, haloalkenyl, haloalkynyl, oxo (=0), thioxo (=S), cyano (-CN), nitro (-NO ₂ ), imino (=N-H), oximo (=N- OH), hydrazine (=N- NH ₂ ), -R ^b1 -OR ^a1 , -R ^b1 -OC(O)-R ^a1 , -R ^b1 -OC(O)-OR ^a1 , -R ^b1 -OC(O)- N(R ^a1 ) ₂ , -R ^b1 -N(R ^a1 ) ₂ , -R ^b1 -C(O)R ^a1 , -R ^b1 -C( O)OR ^a1 , -R ^b1 -C(O)N(R ^a1 ) ₂ , -R ^b1 -O-R ^c1 - C(O)N(R ^a1 ) ₂ ,-R ^b1 -N(R ^a1 )C(O)OR ^a1 ,-R ^b1 -N(R ^a1 )C(O)R ^a1 ,-R ^b1 -N(R ^a )S(O) _t R ^a1 (wheretis1or 2),-R ^b1 -S(O)tR ^a1 (wheretis 1 or2),-R ^b1 -S(O)tOR ^a1 (wheretis 1 or2)and-R ^b1 -S(O)tN(R ^a1 )2 (wheretis 1 or2); andwhereineachR ^b1 isindependently selectedfrom adirectbondora straightorbranched alkylene, alkenylene, oralkynylene chain, and eachR ^c is a straightor branchedalkylene,alkenyleneoralkynylenechain. [00086] Itwill be understoodby those skilled inthe artthat substituents canthemselves be substituted,ifappropriate. Introduction [00087] Interleukin-18(IL-18)isamemberoftheIL-1 familyofcytokines. IL-18isapotent inflammatory cytokine. As such, IL-18 is tightly regulated and synthesized as a precursor, which,inpermissiveconditions,iscleavedandreleasedasmatureIL- 18. [00088] TheIL-18 precursorproteinisprimarilyproducedbymacrophages andT cells. The inactiveIL-18precursorisprocessedtoitsactiveformbycaspase-1, andthematureformis capableofstimulatinginterferongamma(INFy)production,andofreg ulatingbothThelper1 (Thl)andTh2responses,therebyandparticipatinginbothinnateanda cquiredimmunity. [00089] Mature IL-18, in synergy with IL-12, is! associatedwith induction of cell-mediated immunityfollowinginfectionwithmicrobialproductssuchaslipopol ysaccharide(LPS).After stimulationwithIL-18,naturalkiller(NK)cellsandTcellsreleaset hecytokineINFywhich playsanimportantroleinactivatingmacrophagesandothercells.Tna dditiontoinducingINFy, IL-18isalsoinvolvedintheactivationofNF-kβ,Fasligandexpressi on,theinductionofboth CC and CXC chemokines, andhasbeenimplicatedbothinthepromotiontumorgrowthas wellastheanti-tumorimmuneresponse. [00090] Upon secretion, IL-18 is negatively regulated by a decoy receptor, IL-18 binding protein (IL-18BP) TheIL-18BPis asoluble, constitutively secretedprotein,which forms a complex with free IL-18, preventing its interaction with the IL-18 receptor, and thus neutralizingitsbiologicalactivity.TheaffinityofIL-18BPforIL- 18is-10,000timesgreater thanthatof IL-18Rα,and IL-18BPispresentintheserumofhealthyhumans at 20-foldmolar excesscomparedwithIL-18. [00091] The presence of inhibitors, such as IL-18BP, means that stimulating production of IL- 18 does not always lead to increased amounts of systemic or local IL-18. Furthermore, therapeutic compounds competing for IL-18 when bound to IL-18BP may disturb the delicate balance of free/active IL-18 and IL-18BP bound/inactive IL-18 existing in patients stricken with diseases/disorders characterized by IL-18 dis-regulation.

[00092] Developing therapeutic molecules for targets such as IL-18, which are regulated by natural inhibitors, is challenging. However, given the demonstrated an anti-tumorigenic function for IL- 18, numerous strategies are being implemented that aim to increase the level of IL-18 in the tumor microenvironment.

1.2. IL-18 Variants

[00093] Provided herein are IL-18 variants that comprise at least one amino acid substitution compared to a wild-type IL-18. In some embodiments, the IL- 18 variants comprise at least two amino acid substitutions. In some embodiments, the IL-18 variants comprise at least three, four, five, six, or more amino acid substitutions.

[00094] The at least one amino acid substitution can be made by standard techniques. In certain embodiments, the substitution is made by one or more mutations in the genetic sequence encoding the IL-18 variants.

[00095] In some embodiments, an IL-18 variant comprises an amino acid substitution in at least one amino acid position selected from the group consisting of E6, N91, and K93, and combinations thereof. In some embodiments, an IL-18 variant comprises two of the amino acid substitutions. In some embodiments, an IL-18 variant comprises three of the amino acid substitutions. In some embodiments, an IL-18 variant comprises an amino acid substitution at E6. In some embodiments, an IL-18 variant comprises an amino acid substitution at N91. In some embodiments, an IL-18 variant comprises an amino acid substitution atE6 and an amino acid substitution at N91. In some embodiments, an IL-18 variant comprises an amino acid substitution at K93. In some embodiments, the amino acid substitution position is according to the sequence of wild-type IL-18. In some embodiments, the amino acid substitution is with reference to SEQ ID NO: 1. In some embodiments, the IL-18 variant comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1 In some embodiments, the IL-18 variant comprises an amino acid sequence having at least 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 3. In some embodiments, the IL-18 variant comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 4.

[00096] In some embodiments, the IL-18 variant further comprises at least one substitution mutation at a position selected from the group consisting of: N14, C38, M51, K53, P57, M60, C76, C68, M86, Nl ll, SI 17, C127, and N155. In some embodiments, the IL-18 variant comprises an E6K substitution mutation and at least one substitution mutation selected from the group consisting of: N14C, C38S, M51Q, M51R, M51V, K53G, K53A, P57S, M60Y, C68S, C68D, C76A, M86V, N91K, N111R, N111K, S117C, C127A, and N155T. In some embodiments, the IL- 18 variant comprises an E6K substitution mutation and at least one substitution mutation selected from the group consisting of: N14C, C38S, M51Q, M51R, M51V, K53G, K53A, P57S, M60Y, C68S, C76A, M86V, N91K, N111R, N111K, S117C, C127A, and N155T.

[00097] In some embodiments, the IL- 18 variant comprises an amino acid sequence according to: SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 159, SEQ ID

NO: 161, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 172, SEQ ID

NO: 173, SEQ ID NO: 175, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID

NO: 186, SEQ ID NO: 187, SEQ ID NO: 189, SEQ ID NO: 192, SEQ ID NO: 194, SEQ ID

NO: 197, SEQ ID NO: 205, SEQ ID NO: 210, or SEQ ID NO: 213 [00098] In some embodiments, the IL-18 variant comprises an N91K substitution mutation. Tn some embodiments, the IL- 18 variant comprises an N91K substitution mutation and at least one substitution mutation selected from the group consisting of: E6K, N14C, C38S, C68D, M51Q, M51R, M51V, K53G, K53A, P57S, M60Y, C68S, C76A, M86V, N111R, N111K, S117C, C127A, and N155T. In some embodiments, the IL-18 variant comprises an N91K substitution mutation. In some embodiments, the IL-18 variant comprises an N91K substitution mutation and at least one substitution mutation selected from the group consisting of: E6K, N14C, C38S, M51Q, M51R, M51V, K53G, K53A, P57S, M60Y, C68S, C76A, M86V, N111R, N111K, S117C, C127A, and N155T.

[00099] In some embodiments, the IL- 18 variant comprises an amino acid sequence according to: SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 73, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 82, SEQ ID NO: 93, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 159, SEQ ID NO:

161, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 172, SEQ ID NO:

173, SEQ ID NO: 175, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO:

186, SEQ ID NO: 187, SEQ ID NO: 189, SEQ ID NO: 192, SEQ ID NO: 194, SEQ ID NO:

197, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 208, SEQ ID NO:

210, SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215, SEQ ID NO: 216, or SEQ ID NO: 218.

[000100] In some embodiments, the IL-18 variant comprises E6K, M51Q or M51R or M51V, K53G, P57S, M60Y, M86V, N91K, N111R, and N155T substitution mutations. In some embodiments, the IL-18 variant comprises E6K, M51V, K53G, P57S, M60Y, M86V, N91K, N1 11R, and N155T substitution mutations. Tn some embodiments, the IL-18 variant has an amino acid sequence according to SEQ ID NO: 3.

[000101] In some embodiments, the IL-18 variant comprises or further comprises an N111 residue. In some embodiments, in addition to any of the above mutations, the IL- 18 variant further comprises a K53A mutation. In some embodiments, in addition to any of the above mutations, the IL- 18 variant further comprises an N111 residue and a K53A mutation. In some embodiments, the IL-18 variant has an amino acid sequence amino acid sequence according to SEQ ID NO: 9.

[000102] In some embodiments, the IL-18 variant comprises or further comprises a mutation selected from the group consisting of: N111K, N111E, N111Q, N111T, N111I, N111L, N111P, N111 A, N111 V, N111M, and N111 W. In some embodiments, the IL-18 variant further comprises a deletion of D 110. In some embodiments, the IL-18 variant comprises an N111K mutation. In some embodiments, the IL- 18 variant has a sequence according to SEQ ID NO: 11.

[000103] In some embodiments, the IL-18 variant comprises or further comprises at least one wild-type residue selected from the group consisting of: M60, N155, and a combination thereof. In some embodiments, the IL- 18 variant has an amino acid sequence according to SEQ ID NO: 48 or SEQ ID NO: 50.

[000104] In some embodiments, the IL-18 variant comprises or further comprises a wild-type M86 residue. In some embodiments, the IL- 18 variant comprises a sequence according to SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 32, SEQ ID NO: 77.

[000105] In some embodiments, the IL-18 variant comprises or further comprises at least one wild-type residue selected from the group consisting of: M60, N155, and combinations thereof. In some embodiments, the IL-18 variant further comprises a Y60M mutation and a T155N wild-type residue or reversion mutation. In some embodiments, the IL-18 variant comprises a sequence according to SEQ ID NO: 82, SEQ ID NO: 93, SEQ ID NO: 159, SEQ ID NO: 161,

SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 172, SEQ ID NO: 173,

SEQ ID NO: 175, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO: 186,

SEQ ID NO: 187, SEQ ID NO: 189, SEQ ID NO: 192, SEQ ID NO: 194, SEQ ID NO: 197,

SEQ ID NO: 209, SEQ ID NO: 210, or SEQ ID NO: 213. [000106] In some embodiments, the IL-18 variant comprises or further comprises at least one substitution mutation selected from the group consisting of: N14C, C38S, C68S, C68D, C76A, SI 17C, C127A, and combinations thereof. In some embodiments, the IL-18 variant comprises or further comprises at least one substitution mutation selected from the group consisting of: N14C, C38S, C68S, C76A, SI 17C, C127A, and combinations thereof. In some embodiments, the IL-18 variant comprises a sequence according to: SEQ ID NO: 161, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 175, SEQ

ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO: 186, SEQ ID NO: 187, SEQ

ID NO: 189, SEQ ID NO: 192, SEQ ID NO: 194, SEQ ID NO: 197, SEQ ID NO: 209, SEQ

ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 212, or SEQ ID NO; 213. In some embodiments, the IL-18 variant comprises a sequence according to: SEQ ID NO: 161, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 175, SEQ

ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO: 186, SEQ ID NO: 187, SEQ

ID NO: 189, SEQ ID NO: 192, SEQ ID NO: 194, SEQ ID NO: 197, SEQ ID NO: 209, SEQ

ID NO: 210, or SEQ ID NO; 213. In some embodiments, the IL- 18 variant comprises or further comprises a C38S substitution mutation and a C68S substitution mutation. In some embodiments, the IL-18 variant comprises a sequence according to: SEQ ID NO: 168, SEQ ID NO: 172, or SEQ ID NO: 173. In some embodiments, the IL-18 variant comprises or further comprises a C38S substitution mutation and a C68D mutation. In some embodiments, the IL- 18 variant comprises a sequence according to: SEQ ID NO: 209, SEQ ID NO: 210, or SEQ ID NO: 213.

[000107] In some embodiments, the IL- 18 variant comprises an amino acid sequence having a substitution mutation at position N91, K93, or N91 and K93 according to SEQ ID NO: 1. In some embodiments, the IL-18 variant comprises or further comprises at least one mutation at a position selected from the group consisting of: Yl, E6, N14, C38, M51, S55, Q56, M60, C68, C76, D 110, N111, S117, and C 127. In some embodiments, the IL-18 variant comprises a K93N substitution mutation. In some embodiments, the IL-18 variant comprises a K93N substitution mutation and at least one substitution mutation selected from the group consisting of: Y1W, E6D, N14C, C38S, M51R, S55P, Q56N, M60I, C68S, C68D, C76A, M86V, N91R, D98E, DI 10N, N111H, SI 17C, and C127A substitution mutations. In some embodiments, the IL-18 variant comprises a K93N substitution mutation and at least one substitution mutation selected from the group consisting of: Y1W, E6D, N14C, C38S, M51R, S55P, Q56N, M60I, C68S, C76A, M86V, N91R, D98E, DI ION, N111H, S117C, and C127A substitution mutations. In some embodiments, the IL-18 variant has an amino acid sequence comprises an amino acid sequence according to: SEQ ID NO: 4, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 110, SEQ ID NO: 114, SEQ

ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ

ID NO: 158, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 169, SEQ

ID NO: 170, SEQ ID NO: 171, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ

ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 188, SEQ ID NO: 191, SEQ

ID NO: 193, SEQ ID NO: 196, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 206, SEQ

ID NO: 211, or SEQ ID NO: 212.

[000108] In some embodiments, the IL- 18 variant comprises an N91R substitution mutation. In some embodiments, the IL-18 variant comprises an N91R substitution mutation and at least one substitution mutation selected from the group consisting of: Y1W, E6D, N14C, C38S, M51R, S55P, Q56N, M60I, C68S, C68D, C76A, M86V, K93N, D98E, DI 10N, N111H, SI 17C, and C127A substitution mutations. In some embodiments, the IL-18 variant comprises an N91R substitution mutation and at least one substitution mutation selected from the group consisting of: Y1W, E6D, N14C, C38S, M51R, S55P, Q56N, M60I, C68S, C76A, M86V, K93N, D98E, DI 10N, N111H, S117C, and C127A substitution mutations. In some embodiments, the IL-18 variant has an amino acid sequence according to: SEQ ID NO: 4, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 11 1, SEQ ID NO: 115, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO:

147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 158, SEQ ID NO: 162, SEQ ID NO:

163, SEQ ID NO: 164, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO:

176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO:

185 SEQ ID NO: 188, SEQ ID NO: 191, SEQ ID NO: 193, SEQ ID NO: 196, SEQ ID NO:

199, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO:

211, SEQ ID NO: 212, or SEQ ID NO: 217.

[000109] In some embodiments, the IL-18 variant comprises or further comprises: E6D, M51R, S55P, Q56N, M60I, M86V, N91R, K93N, D98E, DI 10N, and N111H substitution mutations. In some embodiments, the IL-18 variant has a sequence according to SEQ ID NO: 4. In some embodiments, the IL- 18 variant comprises or further comprises a V86M mutation and a D98E mutation. In some embodiments, the IL-18 variant comprises or further comprises a substitution mutation selected from the group consisting of: N111M, N11 IT, N11 IP, N111F, N1 1 IL, N11 II, N111 V, N111 A, deletion of N110, and an N111 wild-type residue. In some embodiments, the IL-18 variant comprises or further comprises an DI 10 wild-type residue. In some embodiments, the IL- 18 variant comprises or further comprises an N111 wild-type residue. In some embodiments, the IL-18 variant comprises or further comprises a mutation at amino acid position M51, selected from the group consisting of M51Q and M51H. In some embodiments, the IL-18 variant comprises M51Q. In some embodiments, the IL-18 variant has an amino acid sequence according to SEQ ID NO: 57, SEQ ID NO: 145, SEQ ID NO: 148. In some embodiments, the IL-18 variant comprises or further comprises M51H. In some embodiments, the IL-18 variant has an amino acid sequence according to SEQ ID NO: 60. In some embodiments, the IL- 18 variant comprises or further comprises at least one wild-type residue selected from the group consisting of Yl, E6, Q56, N91, K93 and combinations thereof. In some embodiments, the IL-18 variant comprises or further comprises D6E.

[000110] In some embodiments, the IL- 18 variant has an amino acid sequence according to SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 149, SEQ ID NO: 158, SEQ ID NO: 162, SEQ ID

NO: 163, SEQ ID NO: 164, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID

NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID

NO: 185, SEQ ID NO: 188, SEQ ID NO: 191, SEQ ID NO: 193, SEQ ID NO: 196, SEQ ID

NO: 211, or SEQ ID NO: 212 [000111] In some embodiments, the IL-18 variant comprises or further comprises at least one substitution mutation selected from the group consisting of: N14C, C38S, C68S, C68D, C76A, SI 17C, C127A, and combinations thereof. In some embodiments, the IL-18 variant comprises or further comprises at least one substitution mutation selected from the group consisting of: N14C, C38S, C68S, C76A, SI 17C, C127A, and combinations thereof. In some embodiments, the IL-18 variant has a sequence according to: SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID

NO: 164, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO: 176, SEQ ID

NO: 177, SEQ ID NO: 178, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID

NO: 188, SEQ ID NO: 191, SEQ ID NO: 193, SEQ ID NO: 196, SEQ ID NO: 211, or SEQ ID

NO: 212.

[000112] In some embodiments, the IL-18 variant comprises or further comprises a C38S substitution mutation and a C68S substitution mutation. In some embodiments, the IL-18 variant has a sequence according to: SEQ ID NO: 169, SEQ ID NO: 170, or SEQ ID NO: 171. In some embodiments, the IL-18 variant comprises or further comprises a C38S substitution mutation and a C68D substitution mutation. In some embodiments, the IL- 18 variant has a sequence according to: SEQ ID NO: 211 or SEQ ID NO: 212.

[000113] In some embodiments, the IL-18 variant comprises or further comprises a wild-type Q56 residue. In some embodiments, the IL- 18 variant comprises or further comprises a Y1 wild-type residue.

[000114] In some embodiments, the IL-18 variant comprises or further comprises N11 IK and a mutation at amino acid position M51, selected from the group consisting of M51Y, M51L, M51V, M51I, M51H, M51S, M51A, M51Rand M51Q. In some embodiments, the IL-18 variant comprises or further comprises an M51Q mutation. In some embodiments, the IL-18 variant has an amino acid sequence according to SEQ ID NO: 65.

[000115] In some embodiments, the IL-18 variant comprises at least one mutation at a position selected from the group consisting of: N14, SI 17, K4, 148, 171, E77, N78, K79, 180, S82, K84, E85, M86, N87, D90, D94, D98, Y120, E121, Y123, 1137, K140, and DI 57 relative to SEQ ID NO: 1. In some embodiments, the IL-18 variant comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1. In some embodiments, the IL-18 variant comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 3. Tn some embodiments, the IL-18 variant comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 4.

[000116] In some embodiments, the IL-18 variant comprises or further comprises a mutation at position C68 of SEQ ID NO: 1. In some embodiments, the IL-18 variant comprises or further comprises an amino acid sequence having a substitution mutation at position N14 or SI 17. In some embodiments, the IL-18 variant comprises or further comprises an N14C mutation, a S117C mutation and combinations thereof In some embodiments, the IL-18 variant has a sequence according to SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, or SEQ ID NO: 197.

[000117] In some embodiments, the IL-18 variant comprises or further comprises at least three mutations selected from the group consisting of: C38S, C68S, C68D, C76A, and C127A relative to SEQ ID NO: 1. In some embodiments, the IL-18 variant comprises or further comprises at least three mutations selected from the group consisting of: C38S, C68S, C76A, and C127A relative to SEQ ID NO: 1. In some embodiments, the IL-18 variant has an amino acid sequence according to SEQ ID NO: 174, or SEQ ID NO: 181.

[000118] In some embodiments, the IL-18 variant comprises or further comprises a C38S substitution mutation and a C68D substitution mutation. In some embodiments, the IL-18 variant has an amino acid sequence according to SEQ ID NO: 209.

[000119] In some embodiments, the IL- 18 variant has at least 70% sequence identity to SEQ ID NO: 1. In some embodiments, the IL-18 variant has at least 75% sequence identity to SEQ ID

NO: 1. In some embodiments, the IL-18 variant has at least 80% sequence identity to SEQ ID

NO: 1. In some embodiments, the IL-18 variant has at least 85% sequence identity to SEQ ID

NO: 1. In some embodiments, the IL-18 variant has at least 90% sequence identity to SEQ ID

NO: 1. In some embodiments, the IL-18 variant has at least 95% sequence identity to SEQ ID

NO: 1. In some embodiments, the IL-18 variant has at least 96% sequence identity to SEQ ID

NO: 1. In some embodiments, the IL-18 variant has at least 97% sequence identity to SEQ ID

NO: 1. In some embodiments, the IL-18 variant has at least 98% sequence identity to SEQ ID

NO: 1. In some embodiments, the IL-18 variant has at least 99% sequence identity to SEQ ID

NO: L [000120] In some embodiments, the IL-18 variant has at least 70% sequence identity to SEQ ID

NO:3. In some embodiments, the IL-18 variant has at least 75% sequence identity to SEQ ID

NO:3 In some embodiments, the IL-18 variant has at least 80% sequence identity to SEQ ID

NO:3. In some embodiments, the IL-18 variant has at least 85% sequence identity to SEQ ID

NO:3. In some embodiments, the IL-18 variant has at least 90% sequence identity to SEQ ID

NO:3. In some embodiments, the IL-18 variant has at least 95% sequence identity to SEQ ID

NO:3. In some embodiments, the IL-18 variant has at least 96% sequence identity to SEQ ID

NO:3. In some embodiments, the IL-18 variant has at least 97% sequence identity to SEQ ID

NO:3 In some embodiments, the IL-18 variant has at least 98% sequence identity to SEQ ID

NO:3. In some embodiments, the IL-18 variant has at least 99% sequence identity to SEQ ID

NO:3

[000121] In some embodiments, the IL- 18 variant has at least 70% sequence identity to SEQ ID NO:4. In some embodiments, the IL-18 variant has at least 75% sequence identity to SEQ ID

NO:4. In some embodiments, the IL-18 variant has at least 80% sequence identity to SEQ ID

NO:4. In some embodiments, the IL-18 variant has at least 85% sequence identity to SEQ ID

NO:4. In some embodiments, the IL-18 variant has at least 90% sequence identity to SEQ ID

NO:4. In some embodiments, the IL-18 variant has at least 95% sequence identity to SEQ ID

NO:4. In some embodiments, the IL-18 variant has at least 96% sequence identity to SEQ ID

NO:4. In some embodiments, the IL-18 variant has at least 97% sequence identity to SEQ ID

NO:4. In some embodiments, the IL-18 variant has at least 98% sequence identity to SEQ ID

NO:4. In some embodiments, the IL-18 variant has at least 99% sequence identity to SEQ ID

NO:4.

[000122] Also within the scope are post-translationally modified variants of the IL- 18 variants disclosed herein. Any of the IL- 18 variants provided herein can be post-translationally modified in any manner recognized by those of skill in the art. Typical post-translational modifications for IL-18 variants include interchain disulfide bonding and glycosylation. The post-translational modification can occur during production, in vivo, in vitro, or otherwise. In some embodiments, the post-translational modification can be an intentional modification by a practitioner, for instance, using the methods provided herein.

[000123] Further included within the scope are IL-18 variants fused to further peptides or polypeptides. Exemplary fusions include, but are not limited to, e.g., a methionyl IL-18 variant in which a methionine is linked to the N-terminus of the IL-18 variant resulting from recombinant expression, fusions for the purpose of purification (including but not limited to, to poly-histidine or affinity epitopes), fusions with serum albumin binding peptides, and fusions with serum proteins such as serum albumin. The IL- 18 variants may comprise protease cleavage sequences, IL- 18 variant-binding domains (including but not limited to, FLAG or poly-His) or other affinity-based sequences (including but not limited to, FLAG, poly-His, GST, etc ). The IL- 18 variants may also comprise linked molecules that improve detection (including, but not limited to, GFP), purification, or other features of the IL-18 variant. In certain embodiments, the IL-18 variants comprise a C-terminal affinity sequence that facilitates purification of full-length IL- 18 variants. In certain embodiments, such C-terminal affinity sequence is a poly-His sequence, e.g., a 6-His sequence. In certain embodiments, the IL-18 variants comprise an N-terminal affinity sequence that facilitates purification of full- length IL-18 variants. In certain embodiments, such N-terminal affinity sequence is a poly-His sequence, e.g., a 6-His sequence. In certain embodiments, the IL- 18 variants are fused to a polypeptide sequence that facilitates expression or purification. In certain embodiments, the fusion polypeptide sequence is a small ubiquitin modifying protein (SUMO; Butt etal., 2009, Protein Expr Purif. 43(1): 1-9). In advantageous embodiments, the fusion protein can be cleaved from the IL- 18 variant during or after expression or purification. In some embodiments, the fused peptide or polypeptide specifically binds to a target molecule other than the target molecule bound by the IL-18 variant.

[000124] In some embodiments, the at least one amino acid substitution provides an IL-18 variant that has reduced IL-18BP binding. In some embodiments, the at least one amino acid substitution provides an IL-18 variant that has reduced toxicity. In some embodiments, the at least one amino acid substitution provides an IL-18 variant that has reduced IL-18BP binding and reduced toxicity.

[000125] In certain embodiments, the IL-18 variant has increased affinity for IL-18 receptor a (IL-I8Ra). In certain embodiments, the at least one mutation is on an IL-18Rα receptor contacting surface of the IL-18 variant. In certain embodiments, the at least one mutation in the IL-18 variant is located at an amino acid position that contacts IL-18Rα through hydrogen bonds and/or ionic bonds. In certain embodiments, the at least one mutation in the IL- 18 variant is at a position that contacts IL-18Rα through ionic bonds. In certain embodiments, one or more mutations increase binding of IL- 18 variant to IL-18Rα relative to an IL- 18 of the same sequence, other than the one or more mutations. In certain embodiments, one or more mutations increase binding of IL-18 variant to IL-18Rαby 10%, 20%, 25%, 50%, 75%, 100%, 125%, 150%, 200%, 250%, 300%, 400%, 500%, 1000%, 2000%, 3000%, or more.

[000126] In certain embodiments, the IL-18 variant has reduced affinity for IL- 18 binding protein (IL-18BP). In certain embodiments, the at least one mutation is on an IL-18BP receptor contacting surface of the IL-18 variant. In certain embodiments, the at least one mutation in the IL-18 variant is located at an amino acid position that contacts IL-18BP through hydrogen bonds and/or ionic bonds. In certain embodiments, the at least one mutation in the IL- 18 variant is at a position that contacts IL-18BP through ionic bonds. In certain embodiments, one or more mutations reduce binding of IL-18 variant to IL-18BP relative to an IL-18 of the same sequence, other than the one or more mutations. In certain embodiments, one or more mutations reduce binding of IL-18 variant to IL-18BP by 10%, 20%, 25%, 50%, 75%, 100%, 125%, 150%, 200%, 250%, 300%, 400%, 500%, 1000%, 2000%, 3000%, or more.

[000127] In certain embodiments, one or more mutations increase the stability of the IL-18 variant. In certain embodiments, one or more mutations increase the serum half-life of the IL- 18 variant. In certain embodiments, one or more mutations increase the serum half-life of the IL-18 variant relative to wild-type IL-18. In certain embodiments, one or more mutations increase the serum half-life of the IL-18 variant relative to an IL-18 of the same sequence, other than the one or more mutations. In certain embodiments, one or more mutations reduce the serum half-life of the IL- 18 variant by 10%, 20%, 25%, 50%, 75%, 100%, 125%, 150%, 200%, 250%, 300%, 400%, 500%, 1000%, 2000%, 3000%, or more.

1.3. IL-18 Conjugates

[000128] In some embodiments, provided herein are IL-18 conjugates comprising an IL-18 variant described herein. In some embodiments, at least one amino acid of the IL-18 conjugate is substituted with a non-natural amino acid conjugated to a water-soluble polymer by a linker. In certain embodiments, the IL-18 variant is linked to one payload, for instance a water-soluble polymer. In further embodiments, the IL- 18 variant is linked to more than one payload. In certain embodiments, the IL-18 variant is linked to two, three, four, five, six, seven, eight, nine, ten, or more payloads. The linker can be any linker capable of forming at least one bond to the IL-18 variant and at least one bond to a payload. Useful linkers are described the sections and examples below.

[0001291 In certain embodiments, the conjugate can be formed from an IL-18 variant that comprises one or more reactive groups. In certain embodiments, the conjugate can be formed from an IL-18 variant comprising all naturally encoded amino acids. Those of skill in the art will recognize that several naturally encoded amino acids include reactive groups capable of conjugation to a payload or to a linker. These reactive groups include cysteine side chains, lysine side chains, and amino-terminal groups. In these embodiments, the conjugate can comprise a payload or linker linked to the residue of a reactive group. In these embodiments, the payload precursor or linker precursor comprises a reactive group capable of forming a bond with a reactive group. Typical reactive groups include maleimide groups, activated carbonates (including but not limited to, p-nitrophenyl ester), activated esters (including but not limited to, N-hydroxysuccinimide, p-nitrophenyl ester, and aldehydes). Particularly useful reactive groups include maleimide and succinimide, for instance N-hydroxysuccinimide, for forming bonds to cysteine and lysine side chains. Additional reactive groups include alkynes, for example strained alkynes, and azides, for forming bonds to non-natural amino acids incorporated in IL-18 variant polypeptide chains. Further reactive groups are described in the sections and examples below.

[000130] In certain embodiments, the IL- 18 variant comprises one or more modified amino acids having a reactive group, as described herein. Typically, the modified amino acid is not a naturally encoded amino acid. These modified amino acids can comprise a reactive group useful for forming a covalent bond to a linker precursor or to a payload precursor. One of skill in the art can use the reactive group to link the IL- 18 variant to any molecular entity capable of forming a covalent bond to the modified amino acid. Thus, provided herein are conjugates comprising an IL-18 variant comprising a modified amino acid residue linked to a payload directly or indirectly via a linker. Exemplary modified amino acids are described in the sections below. Generally, the modified amino acids have reactive groups capable of forming bonds to linkers or payloads with complementary reactive groups.

[000131] The non-natural amino acids are positioned at select locations in a polypeptide chain of the IL-18 variant. These locations were identified as providing optimum sites for substitution with the non-natural amino acids. Each site is capable of bearing a non-natural amino acid with optimum structure, function and/or methods for producing the IL- 18 variant.

[0001321 In certain embodiments, a site-specific position for substitution provides an IL-18 variant that is stable. Stability can be measured by any technique apparent to those of skill in the art.

[000133] In certain embodiments, a site-specific position for substitution provides an IL-18 variant that has optimal functional properties. For instance, the IL-18 variant can show little or no loss of binding affinity for its target antigen compared to an IL- 18 variant without the site- specific non-natural amino acid. In certain embodiments, the IL-18 variant can show enhanced binding compared to an IL-18 variant without the site-specific non-natural amino acid.

[000134] In certain embodiments, a site-specific position for substitution provides an IL-18 variant that can be made advantageously. For instance, in certain embodiments, the IL-18 variant shows advantageous properties in its methods of synthesis, discussed below. In certain embodiments, the IL- 18 variant can show little or no loss in yield in production compared to an IL- 18 variant without the site-specific non-natural amino acid. In certain embodiments, the IL- 18 variant can show enhanced yield in production compared to an IL- 18 variant without the site-specific non-natural amino acid. In certain embodiments, the IL-18 variant can show little or no loss of tRNA suppression compared to an IL-18 variant without the site-specific non- natural amino acid. In certain embodiments, the IL-18 variant can show enhanced tRNA suppression in production compared to an IL- 18 variant without the site-specific non-natural amino acid.

[000135] In certain embodiments, a site-specific position for substitution provides an IL-18 variant that has advantageous solubility. In certain embodiments, the IL- 18 variant can show little or no loss in solubility compared to an IL- 18 variant without the site-specific non-natural amino acid. In certain embodiments, the IL- 18 variant can show enhanced solubility compared to an IL-18 variant without the site-specific non-natural amino acid.

[000136] In certain embodiments, a site-specific position for substitution provides an IL-18 variant that has advantageous expression. In certain embodiments, the IL-18 variant can show little or no loss in expression compared to an IL-18 variant without the site-specific non-natural amino acid. In certain embodiments, the IL- 18 variant can show enhanced expression compared to an IL- 18 variant without the site-specific non-natural amino acid. [000137] In certain embodiments, a site-specific position for substitution provides an IL-18 variant that has advantageous folding. In certain embodiments, the IL-18 variant can show little or no loss in proper folding compared to an IL- 18 variant without the site-specific non- natural amino acid. In certain embodiments, the IL- 18 variant can show enhanced folding compared to an IL- 18 variant without the site-specific non-natural amino acid.

[000138] In certain embodiments, a site-specific position for substitution provides an IL-18 variant that is capable of advantageous conjugation. As described below, several nonnatural amino acids have side chains or functional groups that facilitate conjugation of the IL-18 variant to a second agent, either directly or via a linker. In certain embodiments, the IL-18 variant can show enhanced conjugation efficiency compared to an IL-18 variant without the same or other non-natural amino acids at other positions. In certain embodiments, the IL-18 variant can show enhanced conjugation yield compared to an IL-18 variant without the same or other non-natural amino acids at other positions. In certain embodiments, the IL- 18 variant can show enhanced conjugation specificity compared to an IL- 18 variant without the same or other non-natural amino acids at other positions.

[000139] The one or more non-natural amino acids are located at selected site-specific positions in at least one polypeptide chain of the IL-18 variant. The polypeptide chain can be any polypeptide chain of the IL-18 variant without limitation.

[000140] In certain embodiments, the IL- 18 variants provided herein comprise one non-natural amino acid at a site-specific position. In certain embodiments, the IL- 18 variants provided herein comprise two non-natural amino acids at site-specific positions. In certain embodiments, the IL-18 variants provided herein comprise three non-natural amino acids at site-specific positions. In certain embodiments, the IL-18 variants provided herein comprise more than three non-natural amino acids at site-specific positions. In certain embodiments, the IL-18 variants provided herein comprise four non-natural amino acids at site-specific positions.

[000141] In certain embodiments, provided herein are conjugates according to Formula (C1) or (C2):

or a pharmaceutically acceptable salt, solvate, stereoisomer, regioisomer, or tautomer thereof, wherein:

COMP is a residue of an IL-18 variant;

POLY is a payload moiety, for instance a water-soluble polymer;

W ¹ and W ² are each independently a single bond, absent, or a divalent attaching group; each SG is a single bond, absent, or a divalent spacer group, or a trivalent spacer group;

R is hydrogen, a terminal conjugating group, or a divalent residue of a terminal conjugating group; and n is an integer from 1 to 10.

[000142] In some embodiments, each W ¹ and W ² is selected from the group consisting of a bond, -O-, -NH-, -C(O)-, -C(O)NH-, -NHC(O)-, and C ₁ -C ₈ alkylene. In some embodiments, each W ¹ and W ² is selected from the group consisting of a bond, -O-, -NH-, -C(O)-, -C(O)NH-, and - NHC(O)-. In some embodiments, each W ¹ and W ² is selected from the group consisting of a bond, -O-, and -NH-.

[000143] In some embodiments, a conjugate according to Formula (C1) or (C2) comprises n number of linked POLY moi eties, wherein n is an integer from 1 to 10. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is 5. In some embodiments, n is 6. In some embodiments, n is 7. In some embodiments, n is 8. In some embodiments, n is 10. 1.3.1. Spacer Groups

[000144] Spacer groups facilitate spacing of the conjugating group from the other groups of the compounds described herein. This spacing can lead to more efficient conjugation of the compounds described herein to an IL-18 variant. The spacer group can also stabilize the conjugating group and lead to improved overall IL-18 variant conjugate properties.

[000145] In certain embodiments, the spacer group is designated SP herein. Useful spacer groups include those described herein. In certain embodiments, the spacer group is:

[000146] In some embodiments, the SP is

[000147] In some embodiments, the SP is

[000148] In some embodiments, the spacer group is a diamine. In some embodiments, the spacer group is according to

1.3.2. Conjugating Groups and Residues Thereof

[000149] Conjugating groups facilitate conjugation of the payloads described herein to a second compound, such as an IL-18 variant described herein. In certain embodiments, the conjugating group is designated R herein. Conjugating groups can react via any suitable reaction mechanism known to those of skill in the art. In certain embodiments, a conjugating group reacts through a [3+2] alkyne-azide cycloaddition reaction, inverse-electron demand Diels- Alder ligation reaction, thiol-electrophile reaction, or carbonyl-oxyamine reaction, as described in detail herein. In certain embodiments, the conjugating group comprises an alkyne, strained alkyne, tetrazine, thiol, para-acetyl-phenylalanine residue, oxyamine, maleimide, or azide. In certain embodiments, the conjugating group is:

SH; wherein R ²⁰¹ is lower alkyl. In an embodiment, R ²⁰¹ is methyl, ethyl, or propyl. In an embodiment, R ²⁰¹ is methyl. Additional conjugating groups are described in, for example, U.S. Patent Publication No. 2014/0356385, U.S. Patent Publication No. 2013/0189287, U.S. Patent Publication No. 2013/0251783, U.S. Patent No. 8,703,936, U.S. Patent No. 9,145,361, U.S. Patent No. 9,222,940, and U.S. Patent No. 8,431,558.

[000150] After conjugation, a divalent residue of the conjugating group is formed and is bonded to the residue of an IL-18 variant. The structure of the divalent residue is determined by the type of conjugation reaction employed to form the conjugate.

[000151] In certain embodiments when a conjugate is formed through a [3+2] alkyne-azide cycloaddition reaction, the divalent residue of the conjugating group comprises a triazole ring or fused cyclic group comprising a triazole ring. In certain embodiment when a conjugate is formed through a strain-promoted [3+2] alkyne-azide cycloaddition (SPAAC) reaction, the divalent residue of the conjugating group is:

[000152] In certain embodiments when a conjugate is formed through a tetrazine inverse electron demand Diels- Alder ligation reaction, the divalent residue of the conjugating group comprises a fused bicyclic ring having at least two adjacent nitrogen atoms in the ring. In certain embodiments when a conjugate is formed through a tetrazine inverse electron demand Diels- Alder ligation reaction, the divalent residue of the conjugating group is:

[000153] In certain embodiments when a conjugate is formed through a thiol-mal eimide reaction, the divalent residue of the conjugating group comprises succinimidylene and a sulfur linkage. In certain embodiments when a conjugate is formed through a thiol-maleimide reaction, the divalent residue of the conjugating group is:

[000154] In certain embodiments, a conjugate is formed through a thiol-N-hydroxysuccinimide reaction using the following group:

[000155] The reaction involved for formation of the conjugate comprises the following step:

[000156] and the resulting divalent residue of the conjugating group is: [000157] In certain embodiments when a conjugate is formed through a carbonyl-oxyamine reaction, the divalent residue of the conjugating group comprises a divalent residue of a non- natural amino acid. In certain embodiments when a conjugate is formed through a carbonyl- oxyamine reaction, the divalent residue of the conjugating group is:

[000158] In certain embodiments when a conjugate is formed through a carbonyl-oxyamine reaction, the divalent residue of the conjugating group comprises an oxime linkage. In certain embodiments when a conjugate is formed through a carbonyl-oxyamine reaction, the divalent residue of the conjugating group is:

[000159] In an embodiment, provided herein is a conjugate according to Formula (C1) or (C2), or a pharmaceutically acceptable salt, solvate, stereoisomer, or tautomer thereof; wherein R comprises a triazole ring. In an embodiment, provided herein is a conjugate according to Formula (C1) or (C2), or a pharmaceutically acceptable salt, solvate, stereoisomer, or tautomer thereof; wherein R is a triazole ring or fused cyclic group comprising a triazole ring. In an embodiment, provided herein is a conjugate according to Formula (C1) or (C2), or a pharmaceutically acceptable salt, solvate, stereoisomer, or tautomer thereof; wherein R is:

[000160] In an embodiment, provided herein is a conjugate according to Formula (C1) or (C2), or a pharmaceutically acceptable salt, solvate, stereoisomer, or tautomer thereof; wherein R comprises a fused bicyclic ring having at least two adjacent nitrogen atoms in the ring. In an embodiment, provided herein is a conjugate according to Formula (C1) or (C2), or a pharmaceutically acceptable salt, solvate, stereoisomer, or tautomer thereof; wherein R is:

[000161] In an embodiment, provided herein is a conjugate according to Formula (C1) or (C2), or a pharmaceutically acceptable salt, solvate, stereoisomer, or tautomer thereof; wherein R comprises a sulfur linkage. In an embodiment, provided herein is a conjugate according to Formula (C1) or (C2), or a pharmaceutically acceptable salt, solvate, stereoisomer, or tautomer thereof; wherein R is:

[000162] In an embodiment, provided herein is a conjugate according to Formula (C1) or (C2), or a pharmaceutically acceptable salt, solvate, stereoisomer, or tautomer thereof; wherein R comprises a divalent residue of a non-natural amino acid. In an embodiment, provided herein is a conjugate according to Formula (C1) or (C2), or a pharmaceutically acceptable salt, solvate, stereoisomer, or tautomer thereof; wherein R is:

[000163] In an embodiment, provided herein is a conjugate according to Formula (C1) or (C2), or a pharmaceutically acceptable salt, solvate, stereoisomer, or tautomer thereof; wherein comprises an oxime linkage. In an embodiment, provided herein is a conjugate according to Formula (C1) or (C2), or a pharmaceutically acceptable salt, solvate, stereoisomer, or tautomer thereof; wherein R is:

[000164] In an embodiment, provided herein is a conjugate according to Formula (C1) or (C2), or a pharmaceutically acceptable salt, solvate, stereoisomer, or tautomer thereof; wherein comprises an oxime linkage. In an embodiment, provided herein is a conjugate according to Formula (C1) or (C2), or a pharmaceutically acceptable salt, solvate, stereoisomer, or tautomer thereof; wherein R is:

[000165] In an embodiment, provided herein is a conjugate according to Formula (C1) or (C2), or a pharmaceutically acceptable salt, solvate, stereoisomer, or tautomer thereof; wherein R is:

[000166] In an embodiment, provided herein is a compound according to Formula (C1) or (C2), or a pharmaceutically acceptable salt, solvate, stereoisomer, or tautomer thereof; wherein COMP is a residue of any compound known to be useful for conjugation to a payload, described herein, and an optional linker, described herein. In an embodiment, provided herein is a compound according to Formula (C1) or (C2), or a pharmaceutically acceptable salt, solvate, stereoisomer, or tautomer thereof; wherein COMP is a residue of an IL-18 variant chain.

[000167] In an aspect, provided herein is an IL-18 variant conjugate comprising payload, described herein, and an optional linker, described herein, linked to an IL- 18 variant, wherein COMP is a residue of the IL-18 variant. In an embodiment, provided herein is an IL-18 variant conjugate according to Formula (C1) or (C2), or a pharmaceutically acceptable salt, solvate, stereoisomer, or tautomer thereof, wherein: COMP is a residue of the IL-18 variant; and R comprises a triazole ring or fused cyclic group comprising a triazole ring. In an embodiment, provided herein is an IL-18 variant conjugate according to Formula (C1) or (C2), or a pharmaceutically acceptable salt, solvate, stereoisomer, or tautomer thereof, wherein: COMP is a residue of the IL-18 variant; and R is:

[000168] In an embodiment, provided herein is an IL- 18 variant conjugate according to Formula (C1) or (C2), or a pharmaceutically acceptable salt, solvate, stereoisomer, or tautomer thereof, wherein: COMP is a residue of the IL-18 variant; and R comprises a fused bicyclic ring, wherein the fused bicyclic ring has at least two adjacent nitrogen atoms in the ring. In an embodiment, provided herein is an IL-18 variant conjugate according to Formula (C1) or (C2), or a pharmaceutically acceptable salt, solvate, stereoisomer, or tautomer thereof, wherein: COMP is a residue of the IL- 18 variant; and R is:

[000169] In an embodiment, provided herein is an IL-18 variant conjugate according to Formula (C1) or (C2), or a pharmaceutically acceptable salt, solvate, stereoisomer, or tautomer thereof, wherein: COMP is a residue of the polypeptide; and R comprises a sulfur linkage. In an embodiment, provided herein is an IL-18 variant conjugate according to Formula (C1) or (C2), or a pharmaceutically acceptable salt, solvate, stereoisomer, or tautomer thereof, wherein: COMP is a residue of the polypeptide; and R is:

[000170] In an embodiment, provided herein is an IL-18 variant conjugate according to Formula (C1) or (C2), or a pharmaceutically acceptable salt, solvate, stereoisomer, or tautomer thereof, wherein: COMP is a residue of the polypeptide; and R comprises a divalent residue of a non- natural amino acid. In an embodiment, provided herein is an IL-18 variant conjugate according to Formula (C1) or (C2), or a pharmaceutically acceptable salt, solvate, stereoisomer, or tautomer thereof, wherein: COMP is a residue of the polypeptide; and R is:

[000171] In an embodiment, provided herein is an IL-18 variant conjugate according to Formula (C1) or (C2), or a pharmaceutically acceptable salt, solvate, stereoisomer, or tautomer thereof, wherein: COMP is a residue of the polypeptide; and R comprises an oxime linkage. In an embodiment, provided herein is an IL-18 variant conjugate according to Formula (C1) or (C2), or a pharmaceutically acceptable salt, solvate, stereoisomer, or tautomer thereof, wherein: COMP is a residue of the polypeptide; and R is:

[000172] In an embodiment, provided herein is an IL-18 variant conjugate according to Formula (C1) or (C2), or a pharmaceutically acceptable salt, solvate, stereoisomer, or tautomer thereof, wherein: COMP is a residue of the polypeptide; and R comprises an oxime linkage. In an embodiment, provided herein is an IL-18 variant conjugate according to Formula (C1) or (C2), or a pharmaceutically acceptable salt, solvate, stereoisomer, or tautomer thereof, wherein: COMP is a residue of the polypeptide; and R is:

[000173] In an aspect, provided herein is an IL-18 variant conjugate comprising a payload, described herein, and an optional linker, described herein, linked to an IL-18 variant according to Formula (C1) or (C2), or a pharmaceutically acceptable salt, solvate, stereoisomer, or tautomer thereof, wherein COMP is a residue of the IL-18 variant. In an embodiment, provided herein is an IL-18 variant conjugate according to Formula (C1) or (C2), or a pharmaceutically acceptable salt, solvate, stereoisomer, or tautomer thereof, wherein: COMP is a residue of the IL-18 variant; and R comprises a triazole ring or fused cyclic group comprising a triazole ring. In an embodiment, provided herein is an IL-18 variant conjugate according to Formula (C1) or (C2), or a pharmaceutically acceptable salt, solvate, stereoisomer, or tautomer thereof, wherein: COMP is a residue of the IL-18 variant; and R is:

[000174] In an embodiment, provided herein is an IL-18 variant conjugate according to Formula (C1) or (C2), or a pharmaceutically acceptable salt, solvate, stereoisomer, or tautomer thereof, wherein: COMP is a residue of the IL-18 variant; and R comprises a fused bicyclic ring, wherein the fused bicyclic ring has at least two adjacent nitrogen atoms in the ring. In an embodiment, provided herein is an IL-18 variant conjugate according to Formula (C1) or (C2), or a pharmaceutically acceptable salt, solvate, stereoisomer, or tautomer thereof, wherein: COMP is a residue of the IL- 18 variant; and R is:

[000175] In an embodiment, provided herein is an IL-18 variant conjugate according to Formula (C1) or (C2), or a pharmaceutically acceptable salt, solvate, stereoisomer, or tautomer thereof, wherein: COMP is a residue of the IL- 18 variant; and R comprises a sulfur linkage. In an embodiment, provided herein is an IL-18 variant conjugate according to Formula (C1) or (C2), or a pharmaceutically acceptable salt, solvate, stereoisomer, or tautomer thereof, wherein: COMP is a residue of the IL- 18 variant; and R is:

[000176] In an embodiment, provided herein is an IL-18 variant conjugate according to Formula (C1) or (C2), or a pharmaceutically acceptable salt, solvate, stereoisomer, or tautomer thereof, wherein: COMP is a residue of the IL-18 variant; and R comprises a divalent residue of a non- natural amino acid In an embodiment, provided herein is an IL-18 variant conjugate according to Formula (C1) or (C2), or a pharmaceutically acceptable salt, solvate, stereoisomer, or tautomer thereof, wherein: COMP is a residue of the IL-18 variant; and R is:

[000177] In an embodiment, provided herein is an IL-18 variant conjugate according to Formula (C1) or (C2), or a pharmaceutically acceptable salt, solvate, stereoisomer, or tautomer thereof, wherein: COMP is a residue of the IL-18 variant; and R comprises an oxime linkage. In an embodiment, provided herein is an IL-18 variant conjugate according to Formula (C1) or (C2), or a pharmaceutically acceptable salt, solvate, stereoisomer, or tautomer thereof, wherein: COMP is a residue of the IL- 18 variant; and R is:

[000178] In an embodiment, provided herein is an IL-18 variant conjugate according to Formula (C1) or (C2), or a pharmaceutically acceptable salt, solvate, stereoisomer, or tautomer thereof, wherein: COMP is a residue of the IL-18 variant; and R comprises an oxime linkage. In an embodiment, provided herein is an IL-18 variant conjugate according to Formula (C1) or (C2), or a pharmaceutically acceptable salt, solvate, stereoisomer, or tautomer thereof, wherein:

COMP is a residue of the IL- 18 variant; and R is:

[000179] In an aspect, provided herein is an IL-18 variant conjugate comprising a payload, described herein, and an optional linker, described herein, linked to an IL-18 variant chain according to Formula (C1) or (C2), or a pharmaceutically acceptable salt, solvate, stereoisomer, or tautomer thereof, wherein COMP is a residue of the IL-18 variant chain. In an embodiment, provided herein is an IL-18 variant conjugate according to Formula (C1) or (C2), or a pharmaceutically acceptable salt, solvate, stereoisomer, or tautomer thereof, wherein: COMP is a residue of the IL-18 variant chain; and R comprises a triazole ring or fused cyclic group comprising a triazole ring. In an embodiment, provided herein is an IL-18 variant conjugate according to Formula (C1) or (C2), or a pharmaceutically acceptable salt, solvate, stereoisomer, or tautomer thereof, wherein: COMP is a residue of the IL- 18 variant chain; and R is:

[000180] In an embodiment, provided herein is an IL-18 variant conjugate according to Formula (C1) or (C2), or a pharmaceutically acceptable salt, solvate, stereoisomer, or tautomer thereof, wherein: COMP is a residue of the IL-18 variant chain; and R comprises a fused bicyclic ring, wherein the fused bicyclic ring has at least two adjacent nitrogen atoms in the ring. In an embodiment, provided herein is an IL-18 variant conjugate according to Formula (C1) or (C2), or a pharmaceutically acceptable salt, solvate, stereoisomer, or tautomer thereof, wherein: COMP is a residue of the IL- 18 variant chain; and R is:

[000181] In an embodiment, provided herein is an IL-18 variant conjugate according to Formula (C1) or (C2), or a pharmaceutically acceptable salt, solvate, stereoisomer, or tautomer thereof, wherein: COMP is a residue of the IL- 18 variant chain; and R comprises a sulfur linkage. In an embodiment, provided herein is an IL-18 variant conjugate according to Formula (C1) or (C2), or a pharmaceutically acceptable salt, solvate, stereoisomer, or tautomer thereof, wherein: COMP is a residue of the IL- 18 variant chain; and R is:

[000182] In an embodiment, provided herein is an IL- 18 variant conjugate according to Formula (C1) or (C2), or a pharmaceutically acceptable salt, solvate, stereoisomer, or tautomer thereof, wherein: COMP is a residue of the IL- 18 variant chain; and R comprises a divalent residue of a non-natural amino acid. In an embodiment, provided herein is an IL- 18 variant conjugate according to Formula (C1) or (C2), or a pharmaceutically acceptable salt, solvate, stereoisomer, or tautomer thereof, wherein: COMP is a residue of the IL- 18 variant chain; and R is:

[000183] In an embodiment, provided herein is an IL- 18 variant conjugate according to Formula (C 1) or (C2), or a pharmaceutically acceptable salt, solvate, stereoisomer, or tautomer thereof, wherein: COMP is a residue of the IL- 18 variant chain; and R comprises an oxime linkage Tn an embodiment, provided herein is an IL-18 variant conjugate accordingto Formula (C1) or (C2), or a pharmaceutically acceptable salt, solvate, stereoisomer, or tautomer thereof, wherein: COMP is a residue of the IL- 18 variant chain; and R is:

[000184] In an embodiment, provided herein is an IL- 18 variant conjugate according to Formula (C 1) or (C2), or a pharmaceutically acceptable salt, solvate, stereoisomer, or tautomer thereof, wherein: COMP is a residue of the IL- 18 variant chain; and R comprises an oxime linkage. In an embodiment, provided herein is an IL- 18 variant conjugate accordingto Formula (C1) or (C2), or a pharmaceutically acceptable salt, solvate, stereoisomer, or tautomer thereof, wherein: COMP is a residue of the IL- 18 variant chain; and R is:

[000185] In an embodiment, provided herein is a conjugate according to any of the following formulas, where COMP indicates a residue of the IL-18 variant and POLY indicates a polymer moiety:

[000186] In any of the foregoing embodiments, the conjugate comprises n number of POLY moi eties, wherein n is an integer from 1 to 10. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is 5. In some embodiments, n is 6. In some embodiments, n is 7. In some embodiments, n is 8. In some embodiments, n is 9. In some embodiments, n is 10.

[000187] In certain embodiments, the conjugate is linked to one or more water-soluble polymers. A wide variety of macromolecular polymers and other molecules can be linked to the polypeptides described herein to modulate biological properties of the polypeptide, and/or provide new biological properties to the polypeptide. These macromolecular polymers can be linked to the polypeptide via a naturally encoded amino acid, via a non-naturally encoded amino acid, or any functional substituent of a natural or modified amino acid, or any substituent or functional group added to a natural or modified amino acid. The molecular weight of the polymer may be of a wide range, including but not limited to, between about 100 Da and about 100,000 Da or more.

[000188] The polymer selected may be water-soluble so that a protein to which it is attached does not precipitate in an aqueous environment, such as a physiological environment. The polymer may be branched or unbranched. Preferably, for therapeutic use of the end-product preparation, the polymer will be pharmaceutically acceptable. [000189] In certain embodiments, the proportion of polyethylene glycol molecules to polypeptide molecules will vary, as will their concentrations in the reaction mixture. In general, the optimum ratio (in terms of efficiency of reaction in that there is minimal excess unreacted protein or polymer) may be determined by the molecular weight of the polyethylene glycol selected and on the number of available reactive groups available. As relates to molecular weight, typically the higher the molecular weight of the polymer, the fewer number of polymer molecules which may be attached to the protein. Similarly, branching of the polymer should be taken into account when optimizing these parameters. Generally, the higher the molecular weight (or the more branches) the higher the polymer: protein ratio.

[000190] The water-soluble polymer may be any structural form including but not limited to linear, forked or branched. Typically, the water-soluble polymer is a poly(alkylene glycol), such as poly(ethylene glycol) (PEG), but other water-soluble polymers can also be employed. By way of example, PEG is used to describe certain embodiments.

[000191] PEG is a well-known, water-soluble polymer that is commercially available or can be prepared by ring-opening polymerization of ethylene glycol according to methods well known in the art (Sandler and Karo, Polymer Synthesis, Academic Press, New York, Vol. 3, pages 138-161). The term “PEG” is used broadly to encompass any polyethylene glycol molecule, without regard to size or to modification at an end of the PEG, and can be represented as linked to a polypeptide by the formula: XO-(CH ₂ CH ₂ O) _n -CH ₂ CH ₂ -Y where n is 2 to 10,000,X is H or a terminal modification, including but not limited to, a C1.4 alkyl, and Y is the attachment point to the polypeptide.

[000192] In some cases, a PEG terminates on one end with hydroxy or methoxy, i.e., X is H or CH ₃ (“methoxy PEG”). Alternatively, the PEG can terminate with a reactive group, thereby forming a bifunctional polymer. Typical reactive groups can include those reactive groups that are commonly used to react with the functional groups found in the 20 common amino acids (including but not limited to, maleimide groups, activated carbonates (including but not limited to, p-nitrophenyl ester), activated esters (including but not limited to, N-hydroxysuccinimide, p-nitrophenyl ester, and aldehydes) as well as functional groups that are inert to the 20 common amino acids but that react specifically with complementary functional groups present in non- naturally encoded amino acids (including but not limited to, azide groups, alkyne groups). It is noted that the other end of the PEG, which is shown in the above formula by Y, will attach either directly or indirectly to a polypeptide via a naturally occurring or non-naturally encoded amino acid. For instance, Y may be an amide, carbamate, or urea linkage to an amine group (including but not limited to, the epsilon amine of lysine or the N-terminus) of the polypeptide. Alternatively, Y may be a maleimide linkage to a thiol group (including but not limited to, the thiol group of cysteine). Alternatively, Y may be a linkage to a residue not commonly accessible via the 20 common amino acids. For example, an azide group on the PEG can be reacted with an alkyne group on the polypeptide to form a Huisgen [3+2] cycloaddition product. Alternatively, an alkyne group on the PEG can be reacted with an azide group present in a non-naturally encoded amino acid, such as the modified amino acids described herein, to form a similar product. In some embodiments, a strong nucleophile (including but not limited to, hydrazine, hydrazide, hydroxylamine, semicarbazide) can be reacted with an aldehyde or ketone group present in a non-naturally encoded amino acid to form a hydrazone, oxime or semicarbazone, as applicable, which in some cases can be further reduced by treatment with an appropriate reducing agent. Alternatively, the strong nucleophile can be incorporated into the polypeptide via a non-naturally encoded amino acid and used to react preferentially with a ketone or aldehyde group present in the water-soluble polymer.

[000193] Any molecular mass for a PEG can be used as practically desired, including but not limited to, from about 100 Daltons (Da) to 100,000 Da or more as desired (including but not limited to, sometimes 0.1-50 kDa or 10-40 kDa). Branched chain PEGs, including but not limited to, PEG molecules with each chain having a MW ranging from 1-100 kDa (including but not limited to, 150 kDa or 5-20 kDa) can also be used. A wide range of PEG molecules are described in, including but not limited to, the Shearwater Polymers, Inc. catalog, and the Nektar Therapeutics catalog, incorporated herein by reference.

[000194] Generally, at least one terminus of the PEG molecule is available for reaction with the IL-18 variant. For example, PEG derivatives bearing alkyne and azide moieties for reaction with amino acid side chains can be used to attach PEG to non-naturally encoded amino acids as described herein. If the non-naturally encoded amino acid comprises an azide, then the PEG will typically contain either an alkyne moiety to effect formation of the [3+2] cycloaddition product or an activated PEG species (i.e., ester, carbonate) containing a phosphine group to effect formation of the amide linkage. Alternatively, if the non-naturally encoded amino acid comprises an alkyne, then the PEG will typically contain an azide moiety to effect formation of the [3+2] Huisgen cycloaddition product. Tf the non-naturally encoded amino acid comprises a carbonyl group, the PEG will typically comprise a potent nucleophile (including but not limited to, a hydrazide, hydrazine, hydroxylamine, or semicarbazide functionality) in order to effect formation of corresponding hydrazone, oxime, and semicarbazone linkages, respectively. In other alternatives, a reverse of the orientation of the reactive groups described herein can be used, i.e., an azide moiety in the non-naturally encoded amino acid can be reacted with a PEG derivative containing an alkyne.

[000195] In some embodiments, the polypeptide variant with a PEG derivative contains a chemical functionality that is reactive with the chemical functionality present on the side chain of the non-naturally encoded amino acid.

[000196] In certain embodiments, the payload is an azide- or acetylene-containing polymer comprising a water-soluble polymer backbone having an average molecular weight from about 800 Da to about 100,000 Da. The polymer backbone of the water-soluble polymer can be poly(ethylene glycol). However, it should be understood that a wide variety of water-soluble polymers including but not limited to poly(ethylene)glycol and other related polymers, including poly(dextran) and poly(propylene glycol), are also suitable for use and that the use of the term PEG or poly(ethylene glycol) is intended to encompass and include all such molecules. The term PEG includes, but is not limited to, poly(ethylene glycol) in any of its forms, including bifunctional PEG, multiarmed PEG, derivatized PEG, forked PEG, branched PEG, pendent PEG (i.e., PEG or related polymers having one or more functional groups pendent to the polymer backbone), or PEG with degradable linkages therein.

[000197] The polymer backbone can be linear or branched. Branched polymer backbones are generally known in the art. Typically, a branched polymer has a central branch core moiety and a plurality of linear polymer chains linked to the central branch core. PEG is commonly used in branched forms that can be prepared by addition of ethylene oxide to various polyols, such as glycerol, glycerol oligomers, pentaerythritol and sorbitol. The central branch moiety can also be derived from several amino acids, such as lysine. The branched poly(ethylene glycol) can be represented in general form as R(-PEG-OH) _m in which R is derived from a core moiety, such as glycerol, glycerol oligomers, or pentaerythritol, and m represents the number of arms. Multi-armed PEG molecules, such as those described in U.S. Pat. Nos. 5,932,462 5,643,575; 5,229,490; 4,289,872; U.S. Pat. Appl. 2003/0143596; WO 96/21469; and WO 93/21259, each of which is incorporated by reference herein in its entirety, can also be used as the polymer backbone.

[0001981 Branched PEG can also be in the form of a forked PEG represented by PEG(YCHZ2)n, where Y is a linking group and Z is an activated terminal group linked to CH by a chain of atoms of defined length.

[000199] Yet another branched form, the pendant PEG, has reactive groups, such as carboxyl, along the PEG backbone rather than at the end of PEG chains.

[000200] In addition to these forms of PEG, the polymer can also be prepared with weak or degradable linkages in the backbone. For example, PEG can be prepared with ester linkages in the polymer backbone that are subject to hydrolysis. As shown herein, this hydrolysis results in cleavage of the polymer into fragments of lower molecular weight: PEG-CO ₂ -PEG- +H ₂ O→ PEG-CO ₂ H+HO-PEG- It is understood by those skilled in the art that the term poly(ethylene glycol) or PEG represents or includes all the forms known in the art including but not limited to those disclosed herein.

[000201] Many other polymers are also suitable for use. In some embodiments, polymer backbones that are water-soluble, with from 2 to about 300 termini, are particularly suitable. Examples of suitable polymers include, but are not limited to, other poly(alkylene glycols), such as poly(propylene glycol) (“PPG”), copolymers thereof (including but not limited to copolymers of ethylene glycol and propylene glycol), terpolymers thereof, mixtures thereof, and the like. Although the molecular weight of each chain of the polymer backbone can vary, it is typically in the range of from about 800 Da to about 100,000 Da, often from about 6,000 Da to about 80,000 Da.

[000202] Those of ordinary skill in the art will recognize that the foregoing list for substantially water-soluble backbones is by no means exhaustive and is merely illustrative, and that all polymeric materials having the qualities described herein are contemplated as being suitable for use.

[000203] In some embodiments the polymer derivatives are “multi-functional”, meaning that the polymer backbone has at least two termini, and possibly as many as about 300 termini, functionalized or activated with a functional group. Multifunctional polymer derivatives include, but are not limited to, linear polymers having two termini, each terminus being bonded to a functional group which may be the same or different. [000204] In certain embodiments, POLY is polyethylene glycol (PEG), methoxypolyethylene glycol (mPEG), polypropylene glycol) (PPG), copolymers of ethylene glycol and propylene glycol, poly(oxyethylated polyol), poly(olefinic alcohol), poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide), poly(hydroxyalkylmethacrylate), poly(saccharides), poly(oc-hydroxy acid), poly(vinyl alcohol), polyphosphazene, polyoxazolines (POZ), poly(N- acryloylmorpholine), polysarcosine, or a combination thereof. Tn some embodiments, POLY is polyethylene glycol (PEG). In some embodiments, POLY is methoxypolyethylene glycol (mPEG). In some embodiments, POLY is polypropylene glycol) (PPG). In some embodiments, POLY is copolymers of ethylene glycol and propylene glycol. In some embodiments, POLY is polypxyethylated polyol). In some embodiments, POLY is polyplefmic alcohol). In some embodiments, POLY is poly (vinylpyrrolidone). In some embodiments, POLY is poly(hydroxyalkylmethacrylamide). In some embodiments, POLY is poly(hydroxyalkylmethacrylate). In some embodiments, POLY is poly(saccharides). In some embodiments, POLY is poly(oc-hydroxy acid). In some embodiments, POLY is poly(vinyl alcohol). In some embodiments, POLY is polyphosphazene. In some embodiments, POLY is polyoxazolines (POZ). In some embodiments, POLY is poly(Y-acryloylmorpholine). In some embodiments, POLY is polysarcosine. In some embodiments, POLY is a nonpeptidic, water- soluble polymer. In certain embodiments, POLY includes a polyethylene glycol (PEG) or methoxypolyethylene glycol (mPEG). In certain embodiments, POLY is wherein represents attachment to the remainder of the compound, and wherein n1 is an integer from 1 to 10,000. In certain embodiments, n1 is an integer from 1 to 5,000. In certain embodiments, n1 is an integer from 1 to 2,500. In certain embodiments, n1 is an integer from 1 to 2,000. In certain embodiments, n1 is an integer from 1 to 1,000. In certain embodiments, n1 is an integer from 100 to 1,000. In certain embodiments, n1 is an integer from 100 to 500.

[000205] In certain embodiments, including any of the foregoing, POLY is a residue of a nonpeptidic, hydrophilic polymer. In certain embodiments, POLY is a residue of polyethylene glycol (PEG), methoxypolyethylene glycol (mPEG), polypropylene glycol) (PPG), copolymers of ethylene glycol and propylene glycol, polypxyethylated polyol), polyplefmic alcohol), poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide), poly(hydroxyalkylmethacrylate), poly(saccharides), poly(oc-hydroxy acid), poly(vinyl alcohol), polyphosphazene, polyoxazolines (POZ), poly(Y-acryloylmorpholine), polysarcosine, or a combination thereof. In certain embodiments, including any of the foregoing, POLY is a residue of polyethylene glycol (PEG), methoxypolyethylene glycol (mPEG), polypropylene glycol) (PPG), or a copolymer of ethylene glycol and propylene glycol. Tn certain embodiments, including any of the foregoing, POLY is a residue of methoxypolyethylene glycol (mPEG).

[0002061 In certain embodiments, including any of the foregoing, POLY is a residue of polyethylene glycol (PEG). In certain embodiments, including any of the foregoing, POLY is a residue of poly (propylene glycol) (PPG). In certain embodiments, including any of the foregoing, POLY is a residue of copolymers of ethylene glycol and propylene glycol. In certain embodiments, including any of the foregoing, POLY is a residue of poly(oxy ethylated polyol). In certain embodiments, including any of the foregoing, POLY is a residue of poly(olefinic alcohol). In certain embodiments, including any of the foregoing, POLY is a residue of poly(vinylpyrrolidone). In certain embodiments, including any of the foregoing, POLY is a residue of poly(hydroxyalkylmethacrylamide). In certain embodiments, including any of the foregoing, POLY is a residue of poly(hydroxyalkylmethacrylate). In certain embodiments, including any of the foregoing, POLY is a residue of poly(saccharides). In certain embodiments, including any of the foregoing, POLY is a residue of poly(a-hydroxy acid). In certain embodiments, including any of the foregoing, POLY is a residue of poly(vinyl alcohol). In certain embodiments, including any of the foregoing, POLY is a residue of polyphosphazene. In certain embodiments, including any of the foregoing, POLY is a residue of polyoxazolines (POZ). In certain embodiments, including any of the foregoing, POLY is a residue of poly(N-acryloylmorpholine). In certain embodiments, including any of the foregoing, POLY is a residue of polysarcosine.

[000207] In certain embodiments, including any of the foregoing, wherein R ⁵ is hydrogen or methyl, x is an integer from 1 to 10000, inclusive, and represents attachment to the remainder of the compound or conjugate. In certain embodiments, including any of the foregoing, x is an integer between 1 to 5000. In certain embodiments, including any of the foregoing, x is an integer between 1 to 2500. Tn certain embodiments, including any of the foregoing, x is an integer between 1 to 1500. In certain embodiments, including any of the foregoing, x is an integer between 100 to 1000. In certain embodiments, including any of the foregoing, x is an integer between 100 to 500.

[000208] In some embodiments, provided herein are IL-18 conjugates having the structure of any of conjugates in the table below. In some embodiments, n is an integer from 1 to 8. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is 6. In some embodiments, n is 8. The present disclosure encompasses each and every regioisomer of the conjugate structures depicted below:

[000209] In some embodiments, provided herein are IL-18 conjugates having the structure of any of conjugates in the table below. In some embodiments, n is an integer from 1 to 8. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is 6. In some embodiments, n is 8. The present disclosure encompasses each and every regioisomer of the conjugate structures depicted below:

[000210] In any of the foregoing embodiments, the bracketed structure can be covalently bonded to one or more non-natural amino acids of the IL- 18 variant, wherein the one or more non- natural amino acids are located at sites selected from the group consisting of: K4, 148, K70, T71 , E77, N78, K79, 180, S82, K84, E85, M86, N87, D90, D94, D98, Y120, El 21 , Y123, Il 37, K140, and D157 of SEQ ID NO: 1. In certain embodiments, the non-natural amino acid is located at amino acid position 171. In certain embodiments, the non-natural amino acid is located at amino acid position K70. In certain embodiments, the non-natural amino acid is located at amino acid position DI 57.

[000211] In certain embodiments, the IL-18 of the conjugate comprises one or more non-natural amino acids. In certain embodiments, one or more linkers and/or payloads are conjugated to the one or more non-natural amino acids. In certain embodiments, the non-natural amino acid residue comprises a residue of a moiety selected from the group consisting of amino, carboxy, acetyl, hydrazino, hydrazido, semi carb azido, sulfanyl, azido and alkynyl. In certain embodiments, the non-natural amino acid residue is selected from the group consisting of: p- acetyl-L-phenylalanine, O-methyl-L-tyrosine, 3-methyl-phenylalanine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L- phenylalanine, p-acetyl-L-phenylalanine, p-benzoyl-L -phenylalanine, p-iodo-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, p- propargyloxy-phenylalanine, and p-azidomethyl-L-phenylalanine residues. In certain embodiments, the non-natural amino acid residue is para-azido-L-phenylalanine. In certain embodiments, the non-natural amino acid is para-azidomethyl-L-phenylalanine (pAMF). In certain embodiments, the non-natural amino acid is located at an amino acid position selected from amino acid positions K4, 148, K70, 171, E77, N78, K79, 180, S82, K84, E85, M86, N87, D90, D94, D98, Y120, E121, Y123, 1137, K140, and D157 of SEQ ID NO: 1. In certain embodiments, the non-natural amino acid is located at amino acid position 171. In certain embodiments, the non-natural amino acid is located at amino acid position K70. In certain embodiments, the non-natural amino acid is located at amino acid position DI 57.

[000212] In certain embodiments, the water-soluble polymer is selected from the group consisting of is polyethylene glycol (PEG), poly(propylene glycol) (PPG), copolymers of ethylene glycol and propylene glycol, poly(oxyethylated polyol), poly(olefinic alcohol), poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide), poly(hydroxyalkylmethacrylate), poly(saccharides), poly(a-hydroxy acid), poly(vinyl alcohol), polyphosphazene, polyoxazolines (POZ), poly(N-acryloylmorpholine), and combinations thereof. In certain embodiments, the water-soluble polymer is PEG. In certain embodiments, the PEG has an average molecular weight of between about 5KDa and about 50 KDa. Tn certain embodiments, the PEG is selected from the group consisting of a linear or branched PEG molecule having an average molecular weight of lOKDa, 20KDa, 30KDa, or 40KDa. In certain embodiments, the PEG has an average molecular weight of 30KDa. In certain embodiments, the PEG has an average molecular weight of 40KDa. In certain embodiments, the conjugate has an extended half-life compared to an identical variant lacking the water-soluble polymer.

[000213] In certain embodiments, provided herein are IL- 18 variants comprising one or more non-natural amino acids. These non-natural amino acids can facilitate conjugation to a payload, polymer, or linker to form conjugates. In certain embodiments, the non-natural amino acid is at a position selected from the group consisting of K4, 148, 170, 171, E77, N78, K79, 180, S82, K84, M86, N87, D94, D98, Nll l, Y120, E121, Y123, 1137, K140, and D157. In certain embodiments, a polynucleotide is provided, encoding one of these IL-18 variants. In certain embodiments, the polynucleotide encodes a TAG codon to facilitate incorporation of a non- natural amino acid according to the expression techniques described herein. Any non-natural amino acid can be incorporated at the TAG position. In certain embodiments, the non-natural amino acid is one described herein. In certain embodiments, the non-natural amino acid is azidomethylphenylalanine.

[000214] In certain embodiments, the IL-18 conjugate has an amino acid sequence according to SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 85, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 142, SEQ ID NO: 145, SEQ ID NO: 146, or SEQ ID NO: 149, SEQ ID NO: 163, SEQ ID NO: 165, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO: 177, SEQ ID NO: 179, SEQ ID NO: 184, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 191, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 212, or SEQ ID NO: 213.

[000215] In certain embodiments, the IL-18 conjugate has an amino acid sequence selected from SEQ ID NO: 170, SEQ ID NO: 172. In certain embodiments, the IL-18 conjugate comprises a PEG having an average molecular weight of 30KDa or 40KDa. In certain embodiments, the IL-18 conjugate has non-natural amino acid is located at amino acid position D157.

[000216] In certain embodiments, the IL-18 conjugate has an amino acid sequence according to SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91 , SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO:

106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO:

111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO:

116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 124, SEQ ID NO:

147, SEQ ID NO: 148, SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO: 171, SEQ ID NO:

173, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO: 185, or SEQ ID NO: 187. In certain embodiments, the IL-18 conjugate has IL-18 conjugate has an amino acid sequence selected from to SEQ ID NO: 82, SEQ ID NO: 77, and SEQ ID NO: 84, SEQ ID NO: 173. In certain embodiments, the IL-18 conjugate has an amino acid sequence according to SEQ ID NO: 24, SEQ ID NO: 27, or SEQ ID NO: 29. In certain embodiments, the IL-18 conjugate has the non- natural amino acid is located at amino acid position K4, 148, 171, E77, N78, K79, 180, S82, K84, E85, M86, N87, D90, D94, D98, Y120, E121, Y123, 1137, K140, or D157. In certain embodiments, the IL-18 conjugate has an amino acid sequence according to SEQ ID NO: 213, SEQ ID NO: 212, SEQ ID NO: 144, SEQ ID NO: 143, SEQ ID NO: 142, SEQ ID NO: 141,

SEQ ID NO: 140, SEQ ID NO: 139, SEQ ID NO: 138, SEQ ID NO: 137, SEQ ID NO: 136,

SEQ ID NO: 135, SEQ ID NO: 134, SEQ ID NO: 133, SEQ ID NO: 132, SEQ ID NO: 131,

SEQ ID NO: 130, SEQ ID NO: 129, SEQ ID NO: 128, SEQ ID NO: 127, SEQ ID NO: 126,

SEQ ID NO: 125, or SEQ ID NO: 124.

[000217] In certain embodiments, the TAG (*) position of the above amino acid sequences indicates a non-natural amino acid. In certain embodiments, the non-natural amino acid is one described herein. In certain embodiments, the non-natural amino acid is p- azidomethylphenylalanine.

1.4. Vectors, Host Cells, and Recombinant Methods

[000218] Also provided are isolated nucleic acids encoding IL-18 variants, vectors and host cells comprising the nucleic acids, and recombinant techniques for the production of the IL-18 variants and cytokines.

[000219] For recombinant production of the IL-18 variants, the nucleic acid encoding it may be isolated and inserted into a replicable vector for further cloning (i.e., amplification of the DNA) or expression. Tn some aspects, the nucleic acid may be produced by homologous recombination, for example as described in U.S. Patent No. 5,204,244.

[0002201 Many different vectors are known in the art. The vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence, for example as described in U.S. Patent No. 5,534,615.

[000221] Illustrative examples of suitable host cells are provided below. These host cells are not meant to be limiting.

[000222] Suitable host cells include any prokaryotic (e.g., bacterial), lower eukaryotic (e.g., yeast), or higher eukaryotic (e.g., mammalian) cells. Suitable prokaryotes include eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterob acteriaceae such as Escherichia (E. coli), Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella (S. typhimurium), Serratia (S. marcescans), Shigella, Bacilli (B. subtilis and B. licheniformis), Pseudomonas (P. aeruginosa'), and Streptomyces . One useful E. coli cloning host is E. coli 294, although other strains such as E. coli B, E. coli XI 776, and E. coli W3110 are suitable.

[000223] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are also suitable cloning or expression hosts for IL-18 variant-encoding vectors. Saccharomyces cerevisiae, or common baker’s yeast, is a commonly used lower eukaryotic host microorganism. However, a number of other genera, species, and strains are available and useful, such as Schizosaccharomyces pombe, Kluyveromyces (K. lactis, K. fragilis, K. bulgaricus K. wickeramii, K. waltii, K. drosophilarum, K. thermotolerans, and K. marxianus), Yarrowia, Pichia pastoris, Candida (C. albicans), Trichoderma reesia, Neurospora crassa, Schwanniomyces (S. occidentalis), and fdamentous fungi such as, for example Penicillium, Tolypocladium, and Aspergillus (A. nidulans and A. niger).

[000224] Useful mammalian host cells include COS-7 cells, HEK293 cells; baby hamster kidney (BHK) cells; Chinese hamster ovary (CHO); mouse sertoli cells; African green monkey kidney cells (VERO-76), and the like.

[000225] The host cells used to produce the IL- 18 variants may be cultured in a variety of media. Commercially available media such as, for example, Ham’s F10, Minimal Essential Medium (MEM), RPMI-1640, and Dulbecco’s Modified Eagle’s Medium (DMEM) are suitable for culturing the host cells. In addition, any of the media described in Ham et al.,Meth. Enz., 1979, 58:44; Barnes et al., Anal. Biochem.. 1980, 102:255; and U.S. Patent Nos. 4,767,704, 4,657,866, 4,927,762, 4,560,655, and 5,122,469, or WO 90/03430 and WO 87/00195 may be used.

[000226] Any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics, trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art.

[000227] The culture conditions, such as temperature, pH, and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.

[000228] When using recombinant techniques, the IL- 18 variants can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the IL- 18 variant is produced intracellularly, as a first step, the particulate debris, either host cells or lysed fragments, is removed, for example, by centrifugation or ultrafiltration. For example, Carter et al. (Bio/Technology, 1992, 10: 163-167) describes a procedure for isolating polypeptides which are secreted to the periplasmic space of A. coli. Briefly, cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30 min. Cell debris can be removed by centrifugation.

[000229] In some embodiments, an IL- 18 variant is produced by a method comprising the step of culturing a host cell described herein. In certain embodiments, the host cell comprises a nucleic acid, vector, or expression vector described herein for producing the IL- 18 variant. In certain embodiments, the IL-18 variant comprises one or more non-natural amino acids as described herein. In certain embodiments, the host cell further comprises a nucleic acid, vector, or expression vector encoding an aminoacyl tRNA synthetase (RS) specific for the non-natural amino acid. In certain embodiments, the host cell further comprises a nucleic acid, vector, or expression vector encoding a tRNA specific for the non-natural amino acid. In certain embodiments, any or each nucleic acid, vector, or expression vector is codon optimized for the host cell. In certain embodiments, the non-natural amino acid is p-azidom ethylphenylalanine. In certain embodiments, the host cell is E. coli. [000230] In some embodiments, the IL-18 variant is produced in a cell-free system Tn some aspects, the cell-free system is an in vitro transcription and translation system as described in Yin et al., mAbs. 2012, 4:217-225, incorporated by reference in its entirety. In some aspects, the cell-free system utilizes a cell-free extract from a eukaryotic cell or from a prokaryotic cell. In some aspects, the prokaryotic cell is E. coli. Cell-free expression of the IL-18 variant may be useful, for example, where the IL-18 variant accumulates in a cell as an insoluble aggregate, or where yields from periplasmic expression are low.

[000231] Where the IL-18 variant is secreted into the medium, supernatants from such expression systems are generally first concentrated using a commercially available protein concentration filter, for example, an Amicon® or Millipore® Pellcon® ultrafiltration unit. A protease inhibitor such as PMSF may be included in any of the foregoing steps to inhibit proteolysis and antibiotics may be included to prevent the growth of adventitious contaminants.

[000232] The IL-18 variant composition prepared from the cells can be purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being a particularly useful purification technique.

[000233] The matrix to which the affinity ligand is attached is most often agarose, but other matrices are available. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose.

[000234] Other techniques for protein purification, such as fractionation on an ion-exchange column, ethanol precipitation, Reverse Phase HPLC, chromatography on silica, chromatography on heparin Sepharose®, chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are also available, and can be applied by one of skill in the art.

[000235] Following any preliminary purification step(s), the mixture comprising the IL-18 variant of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH between about 2.5-4.5, generally performed at low salt concentrations (e g., from about 0-0.25 M salt).

[000236] In some embodiments, the IL-18 variant is conjugated, for instance as described below. 7.5. Conjugation

[000237] The conjugates can be prepared by standard techniques. In certain embodiments, an IL- 18 is contacted with a payload precursor under conditions suitable for forming a bond from the IL-18 to the payload to form an IL-18-payload conjugate. In certain embodiments, an IL-18 is contacted with a linker precursor under conditions suitable for forming a bond from the IL-18 to the linker. The resulting IL-18-linker is contacted with a payload precursor under conditions suitable for forming a bond from the IL-18-linker to the payload to form an IL-18-linker- payload conjugate. In certain embodiments, a payload precursor is contacted with a linker precursor under conditions suitable for forming a bond from the payload to the linker. The resulting payload-linker is contacted with an IL- 18 under conditions suitable for forming a bond from the payload-linker to the IL-18 to form an IL-18-linker-payload conjugate. Suitable linkers for preparing the IL- 18 conjugates are disclosed herein, and exemplary conditions for conjugation are described in the Examples below.

[000238] In some embodiments, an IL-18 conjugate is prepared by contacting an IL-18 as disclosed herein with a linker precursor having a structure of any of LP1-LP6:

1.6. Pharmaceutical Compositions and Methods of Administration

[000239] The IL-18 variants or IL-18 variant cytokines provided herein can be formulated into pharmaceutical compositions using methods available in the art and those disclosed herein. Any of the IL-18 variants or IL-18 variant cytokines provided herein can be provided in the appropriate pharmaceutical composition and be administered by a suitable route of administration.

[000240] The methods provided herein encompass administering pharmaceutical compositions comprising at least one IL-18 variant or IL-18 variant cytokine provided herein and one or more compatible and pharmaceutically acceptable carriers. In this context, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term “carrier” includes a diluent, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water can be used as a carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Examples of suitable pharmaceutical carriers are described in Martin, E.W., Remington ’s Pharmaceutical Sciences. [000241] In clinical practice the pharmaceutical compositions or IL-18 variants or IL-18 variant cytokines provided herein may be administered by any route known in the art. In certain embodiments, a pharmaceutical composition or IL-18 variant or IL-18 variant cytokine provided herein is administered parenterally.

[000242] The compositions for parenteral administration can be emulsions or sterile solutions. Parenteral compositions may include, for example, propylene glycol, polyethylene glycol, vegetable oils, and injectable organic esters (e.g., ethyl oleate). These compositions can also contain wetting, isotonizing, emulsifying, dispersing and stabilizing agents. Sterilization can be carried out in several ways, for example using a bacteriological filter, by radiation or by heating. Parenteral compositions can also be prepared in the form of sterile solid compositions which can be dissolved at the time of use in sterile water or any other injectable sterile medium.

[000243] In certain embodiments, a composition provided herein is a pharmaceutical composition or a single unit dosage form. Pharmaceutical compositions and single unit dosage forms provided herein comprise a prophylactically or therapeutically effective amount of one or more prophylactic or therapeutic IL- 18 variants or IL-18 variant cytokines.

[000244] Typical pharmaceutical compositions and dosage forms comprise one or more excipients. Suitable excipients are well-known to those skilled in the art of pharmacy, and non- limiting examples of suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. Whether a particular excipient is suitable for incorporation into a pharmaceutical composition or dosage form depends on a variety of factors well known in the art including, but not limited to, the way in which the dosage form will be administered to a subject and the specific IL-18 variant or IL- 18 variant cytokine in the dosage form. The composition or single unit dosage form, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.

[000245] Lactose free compositions provided herein can comprise excipients that are well known in the art and are listed, for example, in the U.S. Pharmacopeia (USP) SP (XXI)/NF (XVI). In general, lactose free compositions comprise an active ingredient, abinder/filler, and a lubricant in pharmaceutically compatible and pharmaceutically acceptable amounts. Exemplary lactose free dosage forms comprise an active ingredient, microcrystalline cellulose, pre gelatinized starch, and magnesium stearate. [000246] Components of the pharmaceutical composition can be supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ample of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

[000247] In some embodiments, the pharmaceutical composition is supplied as a dry sterilized lyophilized powder that is capable of being reconstituted to the appropriate concentration for administration to a subject. In some embodiments, IL-18 variants or IL-18 variant cytokines are supplied as a water free concentrate.

[000248] In another embodiment, the pharmaceutical composition is supplied in liquid form. In some embodiments, the pharmaceutical composition is provided in liquid form and is substantially free of surfactants and/or inorganic salts.

[000249] In some embodiments, the pharmaceutical composition is formulated as a salt form. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.

[000250] Further encompassed herein are anhydrous pharmaceutical compositions and dosage forms comprising an IL- 18 variant or IL- 18 variant cytokine, since water can facilitate the degradation of some IL- 18 variants or IL- 18 variant cytokines.

[000251] Anhydrous pharmaceutical compositions and dosage forms provided herein can be prepared using anhydrous or low moisture containing ingredients and low moisture or low humidity conditions. Pharmaceutical compositions and dosage forms that comprise lactose and at least one active ingredient that comprises a primary or secondary amine can be anhydrous if substantial contact with moisture and/or humidity during manufacturing, packaging, and/or storage is expected.

[000252] An anhydrous pharmaceutical composition should be prepared and stored such that its anhydrous nature is maintained. Accordingly, anhydrous compositions can be packaged using materials known to prevent exposure to water such that they can be included in suitable formulary kits. Examples of suitable packaging include, but are not limited to, hermetically sealed foils, plastics, unit dose containers (e.g., vials), blister packs, and strip packs.

[0002531 Further provided are pharmaceutical compositions and dosage forms that comprise one or more excipients that reduce the rate by which an IL- 18 variant or IL- 18 variant cytokine will decompose. Such excipients, which are referred to herein as “stabilizers,” include, but are not limited to, antioxidants such as ascorbic acid, pH buffers, or salt buffers.

[000254] Parenteral Dosage Forms

[000255] In certain embodiments, provided are parenteral dosage forms. Parenteral dosage forms can be administered to subjects by various routes including, but not limited to, subcutaneous, intravenous (including bolus injection), intramuscular, and intraarterial. Because their administration typically bypasses subjects’ natural defenses against contaminants, parenteral dosage forms are typically, sterile, or capable of being sterilized prior to administration to a subject. Examples of parenteral dosage forms include, but are not limited to, solutions ready for injection, dry products ready to be dissolved or suspended in a pharmaceutically acceptable vehicle for injection, suspensions ready for injection, and emulsions.

[000256] Suitable vehicles that can be used to provide parenteral dosage forms are well known to those skilled in the art. Examples include, but are not limited to: Water for Injection USP; aqueous vehicles such as, but not limited to, Sodium Chloride Injection, Ringer’s Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, and Lactated Ringer’s Injection; water miscible vehicles such as, but not limited to, ethyl alcohol, polyethylene glycol, and polypropylene glycol; and non-aqueous vehicles such as, but not limited to, com oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.

[000257] Excipients that increase the solubility of one or more of the IL-18 variants disclosed herein can also be incorporated into the parenteral dosage forms.

1. 7. Dosage and Unit Dosage Forms

[000258] In human therapeutics, the doctor will determine the posology which he considers most appropriate according to a preventive or curative treatment and according to the age, weight, stage of the infection and other factors specific to the subject to be treated.

[000259] The amount of the IL- 18 variant or IL- 18 variant cytokine or composition which will be effective in the prevention or treatment of a disorder, or one or more symptoms thereof, will vary with the nature and severity of the disease or condition, and the route by which the IL-18 variant or IL- 18 variant cytokine is administered. The frequency and dosage will also vary according to factors specific for each subject depending on the specific therapy (e.g., therapeutic or prophylactic agents) administered, the severity of the disorder, disease, or condition, the route of administration, as well as age, body, weight, response, and the past medical history of the subject. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.

[000260] The dose can be administered according to a suitable schedule, for example, once, two times, three times, or for times weekly. It may be necessary to use dosages of the IL- 18 variant or IL-18 variant cytokine outside the ranges disclosed herein in some cases, as will be apparent to those of ordinary skill in the art. Furthermore, it is noted that the clinician or treating physician will know how and when to interrupt, adjust, or terminate therapy in conjunction with subject response.

[000261] Different therapeutically effective amounts may be applicable for different diseases and conditions, as will be readily known by those of ordinary skill in the art. Similarly, amounts sufficient to prevent, manage, treat, or ameliorate such disorders, but insufficient to cause, or sufficient to reduce, adverse effects associated with the IL-18 variant or IL- 18 variant cytokine provided herein are also encompassed by the herein described dosage amounts and dose frequency schedules. Further, when a subject is administered multiple dosages of a composition provided herein, not all of the dosages need be the same. For example, the dosage administered to the subject may be increased to improve the prophylactic or therapeutic effect of the composition or it may be decreased to reduce one or more side effects that a particular subject is experiencing.

[000262] In certain embodiments, treatment or prevention can be initiated with one or more loading doses of an IL- 18 variant or IL- 18 variant cytokine or composition provided herein followed by one or more maintenance doses.

[000263] In certain embodiments, a dose of an IL- 18 variant or IL- 18 variant cytokine or composition provided herein can be administered to achieve a steady-state concentration of the IL- 18 variant or IL- 18 variant cytokine in blood or serum of the subject. The steady-state concentration can be determined by measurement according to techniques available to those of skill or can be based on the physical characteristics of the subject such as height, weight, and age.

[0002641 Therapeutic Applications

[000265] For therapeutic applications, IL-18 variants or IL-18 variant cytokines disclosed herein are administered to a mammal, generally a human, in a pharmaceutically acceptable dosage form such as those known in the art and those discussed above. For example, the IL-18 variants or IL-18 variant cytokines disclosed herein may be administered to a human intravenously as a bolus or by continuous infusion over a period of time, by intravenous, intramuscular, intraperitoneal, intra-cerebrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, or intratumoral routes. The IL-18 variants or IL-18 variant cytokines also are suitably administered by peritumoral, intralesional, or perilesional routes, to exert local as well as systemic therapeutic effects.

[000266] A therapeutically effective amount of the IL- 18 variant or IL- 18 variant cytokine or composition is an amount that is effective to reduce the severity, the duration and/or the symptoms of a particular disease or condition. The amount of the IL-18 variant or IL-18 variant cytokine or composition that will be therapeutically effective in the prevention, management, treatment and/or amelioration of a particular disease can be determined by standard clinical techniques. The precise amount of the IL-18 variant or IL-18 variant cytokine or composition to be administered with depend, in part, on the route of administration, the seriousness of the particular disease or condition, and should be decided according to the judgment of the practitioner and each subject’s circumstances.

[000267] Combination Therapies

[000268] The IL- 18 variant or IL- 18 variant cytokine described herein or compositions thereof can be administered concurrently with, prior to, or subsequent to, one or more additional therapeutically active agents. In general, each agent will be administered at a dose and/or on a time schedule determined for that agent. In will further be appreciated that the additional therapeutically active agent utilized in this combination can be administered together in a single composition or administered separately in different compositions. The particular combination to employ in a regimen will take into account compatibility of the inventive compound with the additional therapeutically active agent and/or the desired therapeutic effect to be achieved In general, it is expected that additional therapeutically active agents utilized in combination be utilized at levels that do not exceed the levels at which they are utilized individually. In some embodiments, the levels utilized in combination will be lower than those utilized individually. Additional therapeutically active agents include, but are not limited to, small organic molecules such as drug compounds (e.g., compounds approved by the Food and Drugs Administration as provided in the Code of Federal Regulations (CFR)), peptides, proteins, carbohydrates, monosaccharides, oligosaccharides, polysaccharides, nucleoproteins, mucoproteins, lipoproteins, synthetic polypeptides or proteins, small molecules linked to proteins, glycoproteins, steroids, nucleic acids, DNAs, RNAs, nucleotides, nucleosides, oligonucleotides, antisense oligonucleotides, lipids, hormones, vitamins and cells. In certain embodiments, the additional therapeutically agent is a cancer agent (e.g., a biotherapeutic or chemo therapeutic cancer agent).

[000269] Chemotherapeutic agents that may be used in combination with the compounds or pharmaceutically acceptable salts described herein include abiraterone acetate, altretamine, anhydrovinblastine, auristatin, bexarotene, bicalutamide, BMS 184476, 2,3,4,5,6-pentafluoro- N-(3-fluoro-4-m ethoxyphenyl) benzene sulfonamide, bleomycin, N,N-dimethyl-L-valyl-L- valyl-N-methyl-L-valyl-L-prolyl- 1 -Lproline-t-butylamide, cachectin, cemadotin, chlorambucil, cyclophosphamide, 3',4'-didehydro-4'deoxy-8'-norvin-caleukoblastine, docetaxol, doxetaxel, cyclophosphamide, carboplatin, carmustine, cisplatin, cryptophycin, cyclophosphamide, cytarabine, dacarbazine (DTIC), dactinomycin, daunorubicin, decitabine dolastatin, doxorubicin (adriamycin), etoposide, 5-fluorouracil, finasteride, flutamide, hydroxyurea and hydroxyurea andtaxanes, ifosfamide, liarozole, lonidamine, lomustine (CCNU), MDV3100, mechl or ethamine (nitrogen mustard), melphalan, mivobulin isethionate, rhizoxin, sertenef, streptozocin, mitomycin, methotrexate, taxanes, nilutamide, nivolumab, onapristone, paclitaxel, pembrolizumab, prednimustine, procarbazine, RPR109881, stramustine phosphate, tamoxifen, tasonermin, taxol, tretinoin, vinblastine, vincristine, vindesine sulfate, and vinflunine. Such chemotherapeutic agents may be provided as a pharmaceutically acceptable salt, where appropriate. In one embodiment, the additional therapeutically active agent is avascular endothelial growth factor (VEGF) receptor inhibitors including, but are not limited to, bevacizumab (AVASTIN), axitinib, brivanib alaninate ((S)- ((R)-l-(4-(4-fluoro-2-methyl-lH-indol-5-yloxy)-5-methylpyrro lo[2, l-f][l, 2, 4]tri azin-6- yloxy) propan-2-yl)2-aminopropanoate, also known as BMS-582664), motesanib (N-(2,3- dihydro-3,3-dimethyl-lH-indol-6-yl)-2-[(4-pyridinylmethyl)am ino]-3-pyridinecarboxamide), pasireotide, and sunitinib (SUTENT), sorafenib (NEXAVAR).

[000270] In one embodiment, the additional therapeutically active agent is a topoisomerase II inhibitor, including, but are not limited to, etoposide (also known as VP- 16 and etoposide phosphate, sold under the tradenames TOPOSAR, VEPESID, and ETOPOPHOS), and teniposide (also known as VM-26, sold under the tradename VUMON).

[000271] In one embodiment, the additional therapeutically active agent is an alkylating agent, including, but are not limited to, 5-azacytidine (VIDAZA), decitabine (DECOGEN), temozolomide (TEMCAD, TEMODAR, and TEMODAL), dactinomycin (also known as actinomycin-D and sold under the tradename COSMEGEN), melphalan (also known as L- PAM, L-sarcolysin, and phenylalanine mustard, sold under the tradename ALKERAN), altretamine (also known as hexamethylmelamine (HMM), sold under the tradename HEXALEN), carmustine (BCNU), bendamustine (TREANDA), busulfan (BUSULFEX® and MYLERAN®), carboplatin (PARAPLATIN®), lomustine (also known as CCNU, sold under the tradename CEENU®), cisplatin (also known as CDDP, sold under the tradenames PLATINOL® and PLATINOL®-AQ), chlorambucil (LEUKERAN®), cyclophosphamide (CYTOXAN® and NEOSAR®), dacarbazine (also known as DTIC, DIC and imidazole carboxamide, sold under the tradename DTIC-DOME®), altretamine (also known as hexamethylmelamine (HMM) sold under the tradename HEXALEN®), ifosfamide (IFEX®), procarbazine (MATULANE®), mechlorethamine (also known as nitrogen mustard, mustine and mechloroethamine hydrochloride, sold under the tradename MUSTARGEN®), streptozocin (ZANOSAR®), thiotepa (also known as thiophosphoamide, TESPA and TSP A, and sold under the tradename THIOPLEX®. Such alkylating agents may be provided as a pharmaceutically acceptable salt, where appropriate.

[000272] Examples of anti-tumor antibiotics include, but are not limited to, doxorubicin (sold under the tradenames ADRIAMYCIN® and RUBEX®), bleomycin (sold under the tradename LENOXANE®), daunorubicin (also known as dauorubicin hydrochloride, daunomycin, and rubidomycin hydrochloride, sold under the tradename CERUBIDINE®), daunorubicin liposomal (daunorubicin citrate liposome, sold under the tradename DAUNOXOME®), mitoxantrone (also known as DHAD, sold under the tradename NOVANTRONE®), epirubicin (sold under the tradename ELLENCE™), idarubicin (sold under the tradenames TDAMYCTN®, TDAMYCTN PFS®), and mitomycin C (sold under the tradename MUTAMYCIN®). Such anti-tumor antibiotics may be provided as a pharmaceutically acceptable salt, where appropriate.

[000273] In one embodiment, the additional therapeutically active agent is an anti-metabolite including, but are not limited to, claribine (2-chlorodeoxyadenosine, LEUSTATIN®), 5- fluorouracil (ADRUCIL®), 6-thioguanine (PURINETHOL®), pemetrexed (ALIMTA®), cytarabine (also known as arabinosylcytosine (Ara-C), sold under the tradename CYTOSAR- U®), cytarabine liposomal (also known as Liposomal Ara-C, sold under the tradename DEPOCYT™), decitabine (DACOGEN®), hydroxyurea and (HYDREA®, DROXIA™ and MYLOCEL™), fludarabine (FLUDARA®), floxuridine FUDR®), cladribine (also known as 2-chlorodeoxyadenosine (2-CdA) sold under the tradename LEUSTATIN™), methotrexate (also known as amethopterin, methotrexate sodium (MTX), sold under the tradenames RHEUMATREX® and TREXALL™), and pentostatin (NIPENT®). Such anti-metabolites may be provided as a pharmaceutically acceptable salt, where appropriate.

[000274] In one embodiment, the additional therapeutically active agent is a retinoid including, but are not limited to, alitretinoin (PANRETIN®), tretinoin (all-trans retinoic acid, also known as ATRA, sold under the tradename VESANOID®), isotretinoin (13-c/s-retinoic acid, sold under the tradenames ACCUTANE®, AMNESTEEM®, CLARA VIS®, CLARUS®, DECUTAN®, ISOTANE®, IZOTECH®, ORATANE®, ISOTRET®, and SOTRET®), and bexarotene (TARGRETIN®).

[000275] In certain embodiments, the additional therapeutically active agent is aldesleukin, altretamine, amifostine, asparaginase, bleomycin, capecitabine, carboplatin, carmustine, cladribine, cisapride, cisplatin, cyclophosphamide, cytarabine, dacarbazine (DTIC), dactinomycin, docetaxel, doxorubicin, dronabinol, duocarmycin, etoposide, fdgrastim, fludarabine, fluorouracil, gemcitabine, granisetron, hydroxyurea, idarubicin, ifosfamide, interferon alpha, irinotecan, lansoprazole, levamisole, leucovorin, megestrol, mesna, methotrexate, metoclopramide, mitomycin, mitotane, mitoxantrone, omeprazole, ondansetron, paclitaxel (TaxolTm), pilocarpine, prochloroperazine, rituximab, saproin, tamoxifen, taxol, topotecan hydrochloride, trastuzumab, vinblastine, vincristine, vinorelbine tartrate, and combinations thereof. [000276] The amount of additional therapeutic agent present in the compositions of this disclosure will be no more than the amount that would normally be administered in a composition comprising that therapeutic agent as the only active agent. The amount of additional therapeutic agent in the presently disclosed compositions will range from about 50% to 100% of the amount normally present in a composition comprising that agent as the only therapeutically active agent.

/.8. Diagnostic Applications

[000277] In some embodiments, the IL-18 variants or IL-18 variant cytokines provided herein are used in diagnostic applications. For example, an IL- 18 variant or IL- 18 variant cytokine disclosed herein that is specific for a given receptor may be useful in assays for the given receptor. In some aspects, the IL-18 variant or IL-18 variant cytokine can be used to detect the expression of the given receptor in various cells and tissues. These assays may be useful, for example, diagnosing cancer, infection, and autoimmune disease.

[000278] In the methods, the formation of a complex between the IL- 18 variant or IL- 18 variant cytokine and receptor can be detected by any method known to those of skill in the art. Examples include assays that use secondary reagents for detection, ELISA’ s and immunoprecipitation and agglutination assays. A detailed description of these assays is, for example, given in Harlow and Lane, IL- 18 variants: A Laboratory Manual (Cold Spring Harbor Laboratory, New York 1988 555-612, WO 96/13590 to Maertens and Stuyver, Zrein et al. (1998) and WO 96/29605.

[000279] For in situ diagnosis, the IL-18 variant or IL-18 variant cytokine may be administered to a subject by methods known in the art such as, for example, intravenous, intranasal, intraperitoneal, intracerebral, intraarterial injection such that a specific binding between the IL- 18 variant or IL- 18 variant cytokine and receptor may occur. The IL- 18 variant or IL- 18 variant cytokine /receptor complex may conveniently be detected through a label attached to the IL- 18 variant or IL- 18 variant cytokine or any other art-known method of detection.

[000280] In some diagnostic applications, the IL-18 variant or IL-18 variant cytokine may be labeled with a detectable moiety. Suitable detectable moieties include, but are not limited to radioisotopes, fluorescent labels, and enzyme-substrate labels. 1.9. Kits

[000281] In some embodiments, an IL- 18 variant or IL- 18 variant cytokine as described herein can be provided in a kit, i.e., a packaged combination of reagents in predetermined amounts with instructions for performing a procedure. In some embodiments, the procedure is a diagnostic assay. In other embodiments, the procedure is a therapeutic procedure.

EXAMPLES

EXAMPLE 1

LIBRARY DESIGN

[000282] A library of IL-18 sequence variants was designed by identifying amino acid side chains of IL- 18 potentially oriented towards the IL- 18 receptor and binding protein (BP) interface. Crystal structures of viral homologs of human IL-18BP in complex with IL-18 and IL-18 receptor complexes were used to model residues at the interface of the desired protein- protein interactions (PyMOL; PBD IDs: 3WO4, 4EEE, 3F62). Residues diversified in constructed libraries were chosen from these sites in SEQ ID NO: 1 (wild-type human IL-18): Yl, L5, E6, K8, M51, K53, S55, Q56, P57, G59, M60, N91, K93, Q103, S105, D 110, N111, Ml 13, N155, and DI 57. Positions were soft randomized to allow sampling of any amino acid at each position, with a bias towards the parental sequence.

EXAMPLE 2

RIBOSOME DISPLAY SELECTIONS

[000283] A HisSUMO-IL-18 library was constructed using a standard overlap extension PCR protocol (Heckman, K. L. & Pease, L. R. Gene splicing and mutagenesis by PCR-driven overlap extension. Nat. Protoc. 2, 924-932, (2007)) with mutagenic primers targeting desired residues. Selections for novel IL-18 variants were performed using standard ribosome display protocols and published protocols (Hanes, J. & Pltickthun, A. In vitro selection and evolution of functional proteins by using ribosome display. Proc. Natl. Acad. Sci. U. S. A. 94, 4937- 4942 (1997); Stafford, R.L. et al, In vitro Fab display- A cell-free system for IgG discovery. Protein Eng. Des. Sei. 4, 97-109 (2014); and Dreier, B. & Pltickthun, A. Ribosome display- a technology for selecting and evolving proteins from large libraries. Methods Mol. Biol. Clifton NJ 687, 283-306 (2011)). Commercial Ulpl (Sigma-Aldrich, cat no. SAE0067) was added to the in vitro transcription-translation reaction to allow for generation of HisSUMO cleaved IL- 18 without an N-terminal tag for selection. After multiple rounds of selection, the DNA from RT-PCR output was cloned into an optimized vector for cell-free expression (Yin, G. et al.). Aglycosylated IL-18 variants and IL-18 variant fragments were produced in a scalable in vitro transcription-translation system (mAbs 4, 2012) using standard molecular biology techniques. All constructs were His-SUMO-tagged to streamline purification and testing during screening.

EXAMPLE 3

ADDITIONAL ENGINEERING OF RIBOSOME DISPLAY SELECTED IL- 18 VARIANTS

[000284] IL-18 variants with mutations outside of the diversified library sites (G85E, Y10S, V86M, E98D, G90D, A63T, V130E), which were added during selection amplification cycles, were removed to assess functional importance. Additional residue diversity at specific positions was also explored. At each step, mutations with preferred biophysical and biochemical properties were selected for further interrogation. The specific mutational scans are shown in Table 4 below.

Table 4: Scanned mutations in selected IL-18 variants

[000285] As shown in Table 4, SRP3047-C05 was selected from ribosome-displayed IL-18 library. Next, a V130E mutation was introduced into SRP3047-C05.

[000286] SRP3047-D07 was selected from ribosome-displayed IL-18 library. Next, a G90D mutation was introduced into SRP3047-D07.

[000287] SRP3047-E05 was selected from ribosome-displayed IL-18 library. Next, an A63T mutation was introduced into SRP3047-E05.

[000288] SRP3048-A02 was selected from ribosome-displayed IL-18 library. Next, Y10S/C105S mutations were introduced into SRP3048-A02.

[000289] R8N and R8K mutations were introduced into the variant SRP3048-A02 C105S in combination with K60Y and K60M mutations respectively.

[000290] SRP3048-D09 was selected from ribosome-displayed IL- 18 library. Next, G85E mutation was introduced into SRP3048-D09.

[000291] SRP3048-G01 (SEQ ID NO: 3) was selected from ribosome-displayed IL-18 library. Next, V86M mutation was introduced into SRP3048-G01.

[000292] G53A and G53K mutations were introduced into SRP3048-G01 in combination with R111F and R111N mutations respectively to provide SRP3048-G01 G53A, R111F (SEQ ID NO: 10), SRP3048-G01 G53K, R11 1 (1SEQ ID NO: 8), SRP3048-G01 G53K, R111N (SEQ ID NO: 7), and SRP3048-G01 G53A, R11 IN (SEQ ID NO: 9).

[0002931 R111 was mutated to K, E, Q, T, I, L, P, A, V, M, and W, in the variant of SRP3048- G01_G53A. R111 was also deleted in the variant of SRP3048-G01_G53A. R111N mutation was introduced into the variant of SRP3048-G01_G53A while D110 was deleted.

[000294] R111 was mutated to T, Q, K and E in the variant of SRP3048-G01 G53A, V86M. Then, K70TAG (amber stop codon) and 171 TAG (amber stop codon) mutations were introduced into these variants respectively.

[000295] K6E, V51M, A53K, S57P, Y60M, K91N and T155N mutations were introduced into the variant of SRP3048-G01_G53A, R111E respectively. K6E, V51M, A53K, S57P, Y60M, K91N and T155N mutations were also introduced into the variant of SRP3048-G01_G53A, R11 IK respectively. K6E, V51M, A53K, S57P, Y60M, K91N and T155N mutations were also introduced into the variant of SRP3048-G01_G53A, R11 IQ respectively. K6E, V51M, A53K, S57P, Y60M, K91N and T155N mutations were also introduced into the variant of SRP3048- G01 G53A, R111T respectively.

[000296] K70TAG and I71TAG (amber stop codon) mutations were introduced into the variant of SRP3048-G01_G53A,V86M, R111K respectively. K91N/T155N, V51M, V51M/T155N, V5 1M/K91N/T 155N, Y60M/T 155N, Y60M/K91N/T 155N mutations were introduced into the variant of SRP3048-G01_G53A, V86M, R111K respectively. Then, I71TAG (amber stop codon) mutation was introduced into the variants respectively. V51M, V51M/K91N/T155N, Y60M, Y60M/T155N, Y60M/K91N/T155N mutations were introduced into the variant of SRP3048-G01 G53A,V86M, R111K respectively. Then, D157TAG (amber stop codon) was introduced into the variants respectively.

[000297] SRP3047-D05 was selected from the ribosome-displayed IL-18 library. Next, V86M/E98D mutations were introduced into SRP3047-D05. Then, D6E/R91N/N93K (ENK) mutations were introduced into the variant. And, W1Y/D6E/P55S/N56Q/R91N/N93K mutations were introduced into the variant.

[000298] Hl 11 was mutated to M, T, P, F, L, I, V, A, and N respectively in the variants of SRP3047-D05_V86M, E98D, the N110 of which was also deleted.

[000299] R51 was mutated to Y, L, V, I, H, S, A and Q in the variant of SRP3047-D05 V86M, E98D, N110D, Hl 1 IK, and SRP3047-D05 V86M, E98D, N110D, H111N respectively. [000300] W1 Y, D6E, P55S, N56Q, T60M, R91N, and N93K mutations were introduced into the variant of SRP3047-D05_V86M, E98D, N110D, H 111N, R51Q respectively. D157TAG (amber stop codon) mutation was introduced into these variants respectively.

[000301] I71TAG (amber stop codon) was introduced into the variant of SRP3047-D05 V86M, E98D, N110D, H111N, R51Q and SRP3047-D05_V86M, E98D, N110D, H111N, R51H respectively. D157TAG (amber stop codon) was introduced into the variant of SRP3047- D05_V86M, E98D, N110D, H111N, R51Q, SRP3047-D05_V86M, E98D, N110D, H111N, R51H and SRP3047-D05_V86M, E98D, N110D, H111N, R5 IM respectively.

[000302] A complete matrix of all mutation combinations of W1Y, D6E, N56Q, R91N and N93K were introduced into the variant of SRP3047-D05_V86M, E98D, N110D, H111N, R5 IQ. D 157TAG (amber stop codon) mutation was introduced into these variants respectively.

[000303] I71TAG (amber stop codon) mutation was introduced into the variant of SRP3047- D05_V86M, E98D, N110D, H111N, R51Q-W1Y, SRP3047-D05_V86M, E98D, N110D, H111N, R51Q-D6E and SRP3047-D05 V86M, E98D, N110D, H111N, R51Q-D6E, N56Q respectively.

EXAMPLE 4

MUTATION OF UNPAIRED CYSTEINES IN IL- 18 AND ENGINEERED VARIANTS

[000304] IL-18 and variants of IL-18 were mutated with a single or any combination of alanine or serine mutations at residues: C38, C68, C76, and C127. Table 42 shows certain mutated variants.

[000305] C to S or C to D substitution mutants disclosed in Table 42 were evaluated by comparing expression, stability, kinetics, and in vitro activity of mutant hIL-18 vs wild-type hIL-18. Variants that show improved expression and thermal stability were selected. In vitro activity of the variants and variants conjugated with PEGs was evaluated using human PBMC or cyno PBMCs. PK of the unconjugated variants and variants conjugated with PEGs will be evaluated. EXAMPLE 5

SELECTION OF IL- 18 SITES FOR PARA- AZIDOMETHYLPHENYLALANINE (PAMF) INCORPORATION

[0003061 Individual IL- 18 variants were designed to incorporate non-natural pAMF residues in place of specific residues using Sutro’s XpressCF+® cell-free expression platform (Yin et al Sci Rep 2017, 7, 1, 3026). Sites were chosen for pAMF incorporation to enable the conjugation of polyethylene glycol (PEG) moieties via copper-catalyzed azide-alkyne cycloaddition (CuAAC) or a copper-free conjugation method, e.g., strain-promoted azide-alkyne cycloaddition (SPAAC) through dibenzocyclooctyne (DBCO or DIBO).

[000307] The co-crystal structure of IL-18 bound to IL-18Rot and ILRβ (also known as IL-18R1 and IL-18R2 or IL-18RacP, respectively) (Tsutsumi et al., 2014, Nature Communications 5:5340; pdb code 3WO3 and 3WO4) was analyzed using PyMOL to identify which residues have side-chains that point to the solvent. Such residues were chosen for pAMF incorporation to enable conjugation to PEG. Conjugation of PEG to any of these sites were selected to increase the half-life of IL-18, lower the dose requirements, and/or increase the overall exposure. IL- 18 variants were made using standard mutagenesis or gene synthesis techniques and the positions for incorporating pAMF are shown in Table 5 and Table 6. As described herein, the cleavable HisSUMO fusion facilitates expression and purification. The SEQ ID NOs refer to the IL- 18 portions of the sequences.

Table 5: pAMF site scan for human IL-18

NA = not applicable; ND = not determined

Table 6. pAMF site scan for mCS2

EXAMPLE 6

CELL-FREE EXPRESSION OF IL-18 VARIANTS WITH OR WITHOUT PAMF INCORPORATION IN HTS

[000308] IL-18 was modified with an amino-terminal HIS-SUMO sequence to facilitate purification of the IL-18 variants. These variants were expressed in XpressCF+® in an overnight reaction in the presence of ¹⁴ C-Leucine. The expressability of the IL- 18 variants was estimated by ¹⁴ C-incorporation (total yield), and the amount remaining in solution (soluble yield) was further measured following centrifugation at 14,000 x g for 10 minutes.

[000309] The IL-18 variants were expressed in an XpressCF+® reaction. The cell-free extracts were prepared from a mixture of four extracts derived from four engineered strains: (1) an OmpT sensitive RF1 attenuated E. coli strain engineered to overexpress E. coli DsbC and FkpA, (2) a similar RF1 attenuated E. coli strain engineered to produce an orthogonal CUA- encoding tRNA for non-natural amino acid insertion at an Amber Stop Codon, (3) a similar RF1 attenuated E. coli strain engineered to produce the pAMF-specific amino-acyl tRNA synthetase and (4) a similar RF1 attenuated E. coli strain engineered to produce T7 RNA polymerase. Cell-free extract (1) was treated with 50 pM iodoacetamide for 30 min at RT (20°C) and added to a premix containing all other components. The final concentration in the protein synthesis reaction was 30% (v/v) cell extract (1), 1% (v/v) cell extract (2) or 5 pM orthogonal CUA-encoding tRNA, 0.6% (v/v) cell extract (3) or 5pM engineered pAMF- specific amino-acyl tRNA synthetase, 0.5% (v/v) cell extract (4) or 100 nM T7 RNAP, 2 mM para-azidomethylphenylalanine (pAMF), 2 mM GSSG, 8 mM magnesium glutamate, 10 mM ammonium glutamate, 130 mM potassium glutamate, 35 mM sodium pyruvate, 1.2 mM AMP, 0.86 mM each of GMP, UMP, and CMP, 2 mM amino acids (except 0.5 mM for Tyrosine and Phenylalanine), 4 mM sodium oxalate, 1 mM putrescine, 1.5 mM spermidine, 15 mM potassium phosphate, and 2.5-5 μg/mL IL- 18 or variants DNA.

[000310] Cell-free reactions were performed at 20-30°C for 12 hours on a shaker at 650 rpm in 96-well plates at 100 pL scale, in 24-well flower plates at 1 mL scale, in 100 x 10 mm petri dish at 8 mL scale or in stirred tanks at larger scales. His-SUMO-tagged IL-18 variants were purified by immobilized metal affinity chromatography (IMAC) purification methods incorporating 3 washes (30mM HEPES, 500 mMNaCl, lOmM imidazole pH 7.2) and elution in a high salt buffer (30mM HEPES, 500 mMNaCl, 300mM imidazole pH 7.2). IL- 18 variants were then liberated from the His-SUMO N-terminal tag by enzymatic digestion with Ulpl protease using standard methods (Lee, et al. (2008) Protein Science 17(7): 1241-1248). After buffer exchange into 30mM HEPES, 500mM NaCl pH 7.2, the samples were then passed through Ni-IMAC resin to remove the His-SUMO fragment.

[000311] The IL-18 variants were conjugated to a non-releasable PEG to allow half-life extension. In this design, pAMF sites for PEG conjugation were evaluated to allow conjugation without interfering with IL-18Rα receptor binding. Overall, the extended half-life and reduced binding to the decoy receptor IL-18BP may allow a preferred dosing regimen and increased therapeutic index over IL-18 based therapies. Linear or branched mPEG (lOKDa, 20KDa, 30KDa, 40KDa) were linked to dibenzocyclooctyne (DBCO). A 5 mM stock solution of DBCO-mPEG was mixed with 1-50 mg/mL IL- 18 variants incorporated with pAMF at DBCO- mPEG to pAMF ratio of 2-50 for 8 hours to 5 days at 22-35°C.

EXAMPLE 7

CELL-FREE EXPRESSION OF IL-18 VARIANTS IN STIRRED TANK

[000312] Cell free protein synthesis reactions were carried using the XpressCF+® system as described previously (Zawada, J. F. et al. Microscale to manufacturing scale-up of cell-free cytokine production— a new approach for shortening protein production development timelines. Biotechnol. Bioeng. 108, 1570-1578 (2011)). Briefly, cell free reactions were prepared by the addition of 37.5% v/v S30 extract, 3 μg/mL plasmid and a supermix containing amino-acids, NMPs and small molecules for energy generation (Cai, Q. et al. A simplified and robust protocol for immunoglobulin expression in Escherichia coli cell-free protein synthesis systems. Biotechnol. Prog. 31, 823-831 (2015)). Four macromolecular reagents were individually over-expressed in E. coli and added to the XpressCF+® reaction as reagent lysates at <1% v/v each: T7 RNA polymerase, E. coli peptide deformylase (PDF), and the orthogonal tRNA synthetase / tRNA pair from M. jannaschii which have been engineered for incorporation of the non-natural amino acid para-azidomethyl-L-phenylalanine (pAMF) at the TAG amber codon (Zimmerman, E. S. et al. Production of site-specific IL-18 variant-drug conjugates using optimized non-natural amino acids in a cell-free expression system. Bioconjug. Chem. 25, 351-361 (2014)). Small scale reactions were carried out in a Greiner V- bottom 96 well plates at 25 °C overnight with the addition of 2% v/v L-[14C(U)]-Leucine (Perkin Elmer) and the titer was calculated by scintillation counting comparing the total counts in the reaction to counts in the acid precipitable fraction corresponding to protein synthesized. Large scale reactions were carried out in a DASbox stirred tank (Eppendorf) at 250 mL volume with pH, DO and temperature control. Reactions were run with a temperature of 25 °C, pH was controlled at 7.3 using 1 M citrate and 1 M KOH, and DO was maintained at 20%.

EXAMPLE 8 PURIFICATION OF IL-18 VARIANTS

Clarification and IMAC affinity capture

[000313] The XpressCF+® expression of IL-18 mouse and human variants were clarified by centrifugation at 10,000 rpm for 20 minutes (Beckman, JLA-10.500 rotor) and filtered through a 0.22-pm membrane filter. The clarified material was loaded onto a HisTrap Excel affinity column equilibrated with 15 mM Tris-acetate, 500 mM NaCl, ImM DTT, pH 7.5. After 20 column volumes was applied to wash unbound impurities, the bound proteins were eluted with 20mM Tris-acetate, 300mM imidazole, ImM DTT, pH 7.5. The eluted fractions were analyzed by 4-12% SDS-PAGE gel electrophoresis and protein concentrations were determined by measured absorbance at 280 nm. Removal of His SUMO tag and anion exchange purification

[000314] Ulpl protease was mixed with the purified protein and incubated at room temperature for 1 hour. The digested reaction was analyzed by 4-12% SDS-PAGE to verify full cleavage of the His SUMO tag prior to HiPrep Desalting column with Sephadex G-25 resin for rapid buffer exchange into 20mM Tris-acetate, 150mM NaCl, ImM DTT, pH 7.5.

IMAC affinity polish and buffer exchange

[000315] For the final purification step, the desalted IL-18 mouse and human variants were applied to a HisTrap Exel affinity column equilibrated with 15mM Tris-acetate, 150mM NaCl, ImM DTT pH 7.5 as a flow through chromatography process. The target IL-18 variants were eluted from the column without adsorption whereas the remaining contaminants were strongly bound. A 15 -column-volume wash with 15mM Tris-acetate, 150mM NaCl, ImM DTT pH 7.5 was applied and the collected flow through and wash fractions were pooled. An Amicon Ultra- 15, 3kD centrifugal filter was used to concentrate and buffer exchange the IL- 18 mouse and human variants into PBS buffer, 6% sucrose, ImM DTT, pH 7.2.

EXAMPLE 9

SYNTHESIS OF PEG-DBCO LINKERS

Synthesis ofLPl Lysine based 2x20kDa (40 kDa)-PEG DBCO linker: [000316] m-PEG2-NHS (2 x 20kda) (9.1 g) was dissolved in 25 mL of anhydrous DCM. DBCO- Amine (0.126 g) and triethylamine (0.033 mL) were added sequentially, and the reaction mixture was stirred at room temperature for 3 h under N2 atm. The progress of this reaction was followed by analytical ELSD-HPLC (Column: Agilent Eclipse Plus C8, 0.75 mL/min), the crude product was slowly added into MTBE (800 mL). The suspension was stirred for 1 h and fdtered. Solid was washed and transferred into RBF then dried on vacuum for 3 h. Second purification: The product after first purification was dissolved in DCM (35 mL) and slowly crashed into 500 mL of zPrOH. The white precipitate was centrifuged, washed with zPrOH (2 x 120 mL), MTBE (3 x 120 mL). The product was transferred into RBF, dried on rotovap followed by drying on high vacuum pump for 4-5 h. Compound LP1 was confirmed by NMR (CDCh), MALDI- TOF, and analytical ELSD-HPLC.

Synthesis ofDBCO PEG linkers:

LP6 Y shaped 2x20 kDa (40 kDa)-PEG DBCO linker

[000317] LP2, LP3, LP4, LP5, and LP6 DBCO PEG linkers were prepared as described below using m-PEG-amine (5 kDa, 10 kDa, 20 kDa) and DBCO-C6-NHS ester.

[000318] DBCO-C6-NHS ester (in about 10% excess), mPEG- Amine was dissolved in anhydrous DCM. and the reaction was stirred for 24 hours with monitoring of consumption of DBCO-C6-NHS ester. Purification - repeated crystallization from MTBE until no DBCO-C6- NHS ester was detected by HPLC. DBCO PEG compounds were confirmed by ¹ H NMR (CDCl ₃ ), MALDI-TOF, and analytical ELSD-HPLC. EXAMPLE 10

IL- 18 PEGYLATION

[000319] 5 mM stock solution of linear DBCO-mPEG or branched DBCO-mPEG of 5 kDa, 10 kDa, 20 kDa, 30 kDa or 40 kDa was mixed with a final concentration of 1-50 mg/mL protein incorporated with pAMF in IxPBS at DBCO to pAMF ratio of 2-50. The mixture was incubated at 22-35 °C for 2-24hr. The PEG density was measured by 4-12% Bis-tris SDS- PAGE gel or RP-HPLC

EXAMPLE 11

PURIFICATION OF IL- 18 PEG CONJUGATES

[000320] The reaction consisting of conjugated IL-18 and unreacted PEG was further processed by an anion exchange column packed with Capto Q resin (Cytiva). Dilution of the IL-18/PEG reaction prior to purification was performed with binding buffer (20 mM Tris-acetate, pH 7.0) and bound to the Capto Q column with a 2-minute residence time during the load. A linear gradient with elution buffer (20mM Tris-acetate, 200mM NaCl, pH 7.0) was performed over 30 CV and the target elution fractions were collected and buffer exchanged into PBS buffer, 6% sucrose, ImM DTT pH 7.2 by Amicon Ultra-15, 10kD.

SDS-PAGE 4-12%

[000321] Analysis of the unconjugated and conjugated IL-18 human and mouse variant proteins by SDS-PAGE showed a single protein band at the correct molecular weight.

Analytical HPLC SEC

[000322] Monomer percentage and presence of impurities were checked by HPLC-SEC, performed with Ultimate 3000 system and Sepax Zenix-C SEC-150 (7.8 x 300 mm) for the unconjugated IL-18 mouse and human variants, while the PEGylated IL-18 mouse and human variants were analyzed with Sepax SRT SEC-300. The unconjugated and PEGylated IL-18 proteins eluted as a single peak on the analytical size-exclusion chromatogram with a reported monomer content percentage of >95%. PEG density analysis using gel densitometry

[000323] Gel densitometry analysis was used to estimate PEG density. 1-4 ug of PEGylated IL- 18 was loaded on 4-12% Bis-tris SDS-PAGE (NuPAGE™ Invitrogen). The gel ran in lx NuPAGE™ MES SDS Running Buffer (Invitrogen) with constant voltage at 400 volts for 35 minutes. The gel image was scanned using Bio-Rad Gel DOC EZ Imager (as shown in Fig. 1) and exported for densitometry analysis using ImageQuant TL 7.0 (GE Health). The PEGylated IL-18 migrated slower than unpegylated IL-18. PEG density was calculated using Equation 1 :

The calculated PEG density was over 0.95.

PEG density analysis using RP-HPLC

[000324] More accurate PEG density was quantified using PR-HPLC. PEGylated IL- 18 (5-50ug) was injected on to Agilent PLRP column (1000 Å, 5 mM, 150x2.1mm) equilibrated at 80 °C. Mobile phase A was 0.1% trifluoro acetic acid, and mobile phase B was 0.1% acetonitrile. Mobile phase B ramped from 10% to 95% in 15 min at 0.5mg/mL flow rate. It was held at 95% from 15-17 min and then decreased to 10% at 18 min. The post run was 3 min with 10% mobile phase B. As shown in FIG. 2, the gradient was able to separate unPEGylated IL-18, PEGylated IL- 18, and free PEG in different sizes, and therefore the method was applicable to quantify PEG density with or without PEG removal. PEG density was calculated using Equation 2:

[000325] The calculated PEG density was over 0.95. EXAMPLE 12

DIFFERENTIAL SCANNING FLUORIMETRY (DSF)

[000326] A protein thermal shift assay was carried out by mixing the protein to be assayed with an environmentally sensitive dye (SYPRO Orange, Life Technologies Cat #S-6650) in a phosphate buffered solution (PBS), and monitoring the fluorescence of the mixture in real time as it underwent controlled thermal denaturation. Protein solutions between 0.2-2 mg/mL were mixed at a 1-1 volumetric ratio with a 1-500 PBS-diluted solution of SYPRO Orange (SYPRO Orange stock dye is 5000X in DMSO). 10 pL aliquots of the protein-dye mixture were dispensed in quadruplicate in a 384-well microplate (Bio-Rad Cat #MSP-3852, plates pre- heated for 30 minutes at 95°C), and the plate is sealed with an optically clear sealing film (Bio- Rad Cat #MSB-1001) and placed in a 384-well plate real-time thermocycler (Bio-Rad CFX384 Real Time System). The protein-dye mixture is heated from 25°C to 95°C, at increments of 0.1 °C per cycle (about 1.5°C per minute), allowing 3 seconds of equilibration at each temperature before taking a fluorescence measurement. At the end of the experiment, the transition melting temperature is determined using the Bio-Rad CFX manager software.

EXAMPLE 13 LABEL-FREE KINETIC ANALYSIS WITH SPR

[000327] This example describes methods to identify IL- 18 variants that maintain binding to IL- 18Rα but have substantially reduced binding to IL-18BP, a decoy receptor that competes with IL-18Rα for IL- 18 binding. Thus, the kinetic binding of IL- 18 variants to IL-18Rα and IL- 18BP were assessed. Additionally, the impact of IL-18 pegylation on IL-18Rα and IL-18BP binding were also assessed.

[000328] Anti -His polyclonal IL-18 variants (GE Life Sciences) were immobilized onto a CM4 chip (GE Life Sciences) using amine coupling chemistry (from Amine Coupling Kit, GE Life Sciences). The immobilization steps were carried out at a flow rate of 25 μL/minute in lx HBS- EP+ buffer (GE Life Sciences). The sensor surfaces were activated for 7 min with a mixture of NHS (0.05 M) and EDC (0.2 M). The anti-His IL-18 variants were injected over all flow cells used in the study at a concentration of 25 μg/mL in 10 mM sodium acetate, pH 4.5, for seven minutes. Ethanolamine (1 M, pH 8.5) was injected for seven minutes to block any remaining activated groups. An average of 4,500 response units (RU) of capture IL-18 variant was immobilized on each flow cell used in the study.

[000329] Kinetic binding experiments were performed at 25 °C using lx HBS-EP+ buffer. IL- 18Rα-6his or IL-18BP-6his (human and mouse, Sino Biological) were injected over the anti- His surface at concentrations of 15 μg/mL and 7.5 μg/mL for IL-18Rα-6his or IL-18BP-6his, respectively, for 15 seconds at a flow rate of 10 μL/minute on flow cells 2 and 3 respectively, followed by a stabilization period for 30 seconds at the same flow rate. Kinetic characterization of conjugated or unconjugated IL- 18 or variants was carried out in a range of concentrations from 0.25 to 125 nM and one injection of 0 nM analyte. After capturing ligand (IL-18Rα-6his or IL-18BP-6his) on the anti -His surface, the analyte (IL- 18 variant) contact time was 180 seconds, followed by a 180 second dissociation time at a flow rate of 30 μL/min. Between each ligand capture and analyte binding cycle, regeneration was carried out using one injection of 10 mM Glycine pH 1.5 for 60 seconds at 50 μL/minute and a 30 second stabilization period, followed by an injection of 10 mM Glycine pH 1.5 for 30 seconds at 50 μL/minute and a 300 second stabilization period that ends the cycle. The data were fit with the Biacore T200 Evaluation software Kinetic Screen using a global fit 1 : 1 binding model with double reference subtraction and RI=Ymax/5.

[000330] Steady State binding experiments were performed at 25°C using 1x HBS-EP+ buffer. IL-18BP-6his (human and mouse, Sino Biological) was injected over the anti-His surface at concentrations of 7.5 μg/mL for 15 seconds at a flow rate of 10 μL/minute on the flow cells used in the study, followed by a stabilization period for 30 seconds at the same flow rate. Affinity characterization of conjugated or unconjugated IL- 18 or variants was carried out in a range of concentrations from 125 to 1000 nM and one injection of 0 nM analyte. After capturing ligand (IL-18BP-6his) on the anti-His surface, the analyte (IL- 18 variant) contact time was 90 seconds, followed by a 90 second dissociation time at a flow rate of 30 μL/min. Between each ligand capture and analyte binding cycle, regeneration was carried out using one injection of 10 mM Glycine pH 1.5 for 60 seconds at 50 μL/minute and a 30 second stabilization period, followed by an injection of 10 mM Glycine pH 1.5 for 30 seconds at 50 μL/minute and a 300 second stabilization period that ends the cycle. The data were fit with the Biacore T200 Evaluation software Affinity Screen using a global fit steady state affinity binding model with double reference subtraction and RI=Ymax/5. EXAMPLE 14

HEK-BLUE IL-18 REPORTER ASSAY

[000331] HEK-Blue IL-18 Reporter Cells (Invivogen, Cat# hkb-hmIL-18) were maintained in complete DMEM/F-12 Media (Coming) with 100IU Penicillin/lOOug/mL Streptomycin (Coming), 2mM GlutaMax (Gibco), 10% h.i. FBS (Sigma), 100 μg/mL Normocin (Invivogen), and HEK-Blue Selection antibiotics mix (Invivogen). On assay day, cells were harvested with Accutase, counted, and resuspended at 0.5 x 10 ⁶ cells/mL in HEK-Blue Detection media (Invivogen). 25ul of cells were seeded for a total of 12,500 cells per well in 384-well clear- bottom plate. Cells were treated with 25ul of serial dilution of IL-18 samples (1 :8 serial dilution of 4nM starting concentration) with or without 20nM of hIL-18BP (Sino Biological) and then incubated at 37°C, 5% CO ₂ for 16 hours. The plates were then read on a SpectraMax M5 plate reader for absorbance 640nM. Cell-only control wells were used to subtract background absorbance from treated wells. Raw readings were converted to % relative signal using the 4nM wt IL- 18 treated cells as controls. Data was fitted with non-linear regression analysis, using log (agonist) vs. response, variable slope, 4-parameter fit equation using GraphPad Prism.

Example 15 Human/NHP PBMCs based hIFNγ Release Assay

[000332] Human/NHP Peripheral Blood Mononuclear Cells (PBMCs) were isolated from healthy blood donors by Leukosep tube (Greiner Bio-One) and Nycoprep 1.077 buffer (Progen) according to the manufacture’s recommendation and cryopreserved. The day before the assay, PBMCs were thawed and cultured in PBMC cell culture media (RPMI supplemented with 10% heat-inactivated fetal bovine serum (Hyclone), 1% Penicillin/Streptomycin and 2 mmol/L-glutamax). On the day of assay, 50,000 PBMCs in lOOul assay media (PBMC cell culture media with 2ng/ml of human IL2 and 2ng/ml of human IL 12) were seeded into each well in a 96-well clear bottom plate. l OOul of serial dilution of IL-18 samples (8-point, 6-fold dilutions starting at 20 nM in assay media) with or without 20nM hIL-18BP were then added into PBMCs. PBMCs in assay media only were used as controls. After 48hrs of incubation in a cell culture incubator, the plates were centrifuged at 500 g for 4 minutes at 4°C and assay supernatant were collected into a 96-well V-bottom plate.

[000333] To measure the IFN-γ secreted by human/NHP PBMCs after IL-18 treatment, an ELISA kit (MabTech Monkey IFN-γ ELISA) was used according to the manufacture’s recommendation. ELISA plate was coated with mAb MT126L (2 μg/ml in PBS, pH 7.4) overnight at 4-8°C and blocked by 200 pl/well of incubation buffer (PBS with 0.05% Tween 20 and 0.1% BSA) for 1 hour at RT. After washing, serial dilution of hlFN-y standard and supernatant collected from the IL- 18 treated PBMCs (diluted to fit within the range of standard curve, typical dilution 1 : 100) were added into the plate and incubated for 2 hours at RT. After another washing step, mAb 7-B6-1 -biotin at 1 μg/ml in incubation buffer were then added and incubated for another hour at RT. After another washing step, Streptavidin-HRP (1 :1000 in incubation buffer) were then added to the plate and incubated for 1 hour at RT. Finally, TMB substrate solution were added before reading on M5 plate reader at 640 nm after suitable developing time. Concentration (μg/ml) of IFN- y was interpolated using hlFN-y standards. Data was fitted with non-linear regression analysis, using log (agonist) vs. response, variable slope, 4-parameter fit equation using GraphPad Prism.

EXAMPLE 16

RESULTS FOR REVERSAL OF FRAMEWORK MUTATIONS

[000334] Table 8 shows the results from the kinetic binding experiments and reporter assays previously described.

[000335] In particular, Table 8 provides comparison data from kinetic binding experiments and Hek-blue reporter assays results between framework mutants (outside of the IL-18BP binding site) of3048-D09, 3048-G01, 3047-C05, 3047-D05, 3047-D07, 3047-E05 and their reversion mutants. As shown in FIG. 4A, certain IL-18 variants of the present disclosure maintained IL- 18Ra binding, but only showed trace binding to IL-18BP. FIG. 4B shows certain IL- 18 variants of the present disclosure maintained the ability of activating IL- 18 pathway in HEKblue IL-18 reporter assay. 20nM of IL18BP did not affect the ability of the variants to activate IL18 reporters, while WT IL-18 was greatly affected by the presence of IL-18BP. Table 8: Human IL-18 variants +/- framework reversions at sites outside of the library design

NC = not calculated

EXAMPLE 17

IN VITRO RESULTS FOR G01 VARIANTS

[000336] Tables 9, 10, 11, 12, 13, 14, 15, 16, and 17 below show the results from the kinetic binding experiments, HEK-blue reporter assay and human and cyno PBMCs IFN-γ release assay previously described.

[000337] Table 9 shows thermostability, kinetic binding, and HEK-blue reporter activity data for selected G01 (SEQ ID NO: 3) variants. The variants all comprise the mutation G53A, and differ from each other at amino acid position R111. The R111 variants tested were selected from R111K, R111E, R111Q, R111T, R111I, R111L, R111P, R111A, R111V, R111M, R111W and deletion of R111. Here R indicates the parent sequence residue at position 111 rather than the wild-type residue at that position.

[000338] Table 10 shows thermostability, kinetic binding, and HEK-blue reporter activity data for selected G01 variants wherein pAMF has been incorporated at either amino acid position 171 or K70. The variants tested differed from each other at amino acid position R111. R111 variants were selected from R11 IK, R11 IQ, R11 IT, and R11 IE. The data show that variants comprising a pAMF at amino acid 171, performed better than an otherwise identical variant comprising a pAMF at amino acid position K70.

[000339] Table 11 shows thermostability, kinetic binding, and HEK-blue reporter activity data for select G01 single site reversion mutants. The reversion mutants were selected for their improved thermal stability, (2) potential to lower immunogenicity risk, or (3) preserve unique mutations. Mutations at position 51, 60, 155 were not essential to maintain binding properties, and E6K mutation was shown to be important for 3048-G01 (SEQ ID NO: 3) binding properties and reducing IL-18 BP binding.

[000340] Table 12 shows thermostability, kinetic binding, and HEK-blue reporter activity data for G01 reversion mutants having a combination of reversion mutations (as indicated) in combination with a pAMF introduced at either amino acid position 171 or D157.

[000341] Table 13 shows thermostability, kinetic binding, and HEK-blue reporter activity data for a selected group of G01 reversion mutants having a combination of reversion mutations (as indicated) in combination with a pAMF introduced at either amino acid position 171 or D 157. [000342] Table 14 shows thermostability, kinetic binding, and HEK-blue reporter activity for a selected subgroup of G01 reversion mutants having a combination of reversion mutations (as indicated) in combination with a PEG conjugated at either amino acid position 171 or D157.

[000343] Table 15 shows the result for cyno PBMC IFN-γ release assay and the HEK-blue reporter assay of selected G01 variants conjugated to PEGs of different sizes at D157 TAG site. This data indicated that conjugation to PEG reduced the in vitro activity of G01 variants. Higher PEG size correlated to greater reduction in activity.

[000344] Table 16 shows human PBMC IFN-γ release assay and HEK-blue reporter activity for a selected subgroup of G01 reversion mutants having a combination of reversion mutations (as indicated) in combination with or without a PEG conjugated at either amino acid position 171 or D157.

[000345] Table 17 shows thermostability, kinetic binding, and HEK-blue reporter activity for a selected sub-group of G01 reversion mutants having a combination of cysteine mutations (as indicated) with or without a PEG conjugated at either amino acid position 171 or DI 57. FIG. 5C shows curves of HEK-blue assay for examples of G01 variants with cysteine mutations conjugated to a 40k PEG.

[000346] Table 18 shows HEK-blue reporter activity for SRP3048-G01 variants with or without a PEG conjugated at either amino acid position 171 or DI 57. Examples of the dose response curves of a SRP3048-G01 variant conjugated to a 30k PEG and a 40K PEG at D157 site are shown in FIG. 5B.

[000347] Table 19 shows results from human and cyno PBMC IFN-γ release assay for SRP3048- G01 variants with cysteine mutations conjugated to a 40k PEG at 171 site.

[000348] Table 20 shows results from human PBMC IFN-γ release assay for a SRP3048-G01 variant with or without a 30k PEG conjugated at 171 site. The dose response curves are shown in FIG. 6A. The result from this assay indicates that the SRP3048-G01 variant with cysteine mutation induced potent IFN release when co-cultured with human PBMCs, which is more potent than the wtIL-18 and not affected by the presence of IL18BP. Conjugation to PEG slightly reduced the activity, which is still more potent than wtIL-18 and escapes the negative regulation of IL-18BP.

[000349] Table 21 shows results from IFN-γ release assay using human PBMCs or mouse splenocytes for SRP3048-G01 variants expressed in CF or intact E. coli with or without a 30k PEG conjugated at T71 site. The result from this assay indicates that the SRP3048-G01 variants with cysteine mutation induced potent IFN release when co-cultured with human PBMCs or mouse splenocytes, which is more potent than the wtIL-18 and not affected by the presence of IL 18BP. Conjugation to PEG slightly reduced the activity, which is still more potent than wtlL- 18 and escapes the negative regulation of IL-18BP.

Table 9: 3048-G01 G53A, mutational scan at position 11 1

Table 10: 3048-G01 with framework mutations V86M, G53A, R111KQET, pAMF incorporation at position 70 and 71

Table 11: 3048-G01 G53A/R111KQET, evaluation of mutation reversions at position 6, 51, 57, 60, 91, 155

ND = not determined

Table 12: 3048-G01 V86M/G53A/R111K, combining mutation reversions at position 51, 60, 91, 155 in an I7pAMF or D157pAMF background

NC = not calculable

Table 13. Certain SRP3048-G01 variants with reversion mutations and pAMF incorporation at D157 or 171 (unconjugated)

NC = not calculable, NB = No Binding

Table 14. SRP3048-G01 variants with reversion mutations and pAMF incorporation at DI 57 or 171 (conjugated)

NC = not calculable, NB = No Binding, ND = Not Determined

Table 15. Results for cyno PBMC IFN-γ release assay and HEK-blue reporter assay for selected G01 variants conjugated to PEGs of different sizes at 171 TAG site.

Table 16. Results for human PBMC IFN-γ release assay and HEK-blue reporter assay for SRP3048-G01 variants with reversion mutations and pAMF incorporation at DI 57 or 171

Table 17. SRP3048-G01 variants with cysteine mutations and pAMF or pAMF-PEG incorporation at DI 57 or T71

Table 18. HEK-blue reporter assay results for SRP3048-G01 variants with pAMF or pAMF-PEG incorporation at D157 or 171

Table 19. Human and cyno PBMC IFN-γ release assay for SRP3048-G01 variants with pAMF-PEG incorporation at 171

Table 20. Human PBMC release assay SRP3048-G01 variants with pAMF or pAMF-PEG incorporation at 171

Table 21. IFN-γ release assay for SRP3048-G01 variants expressed in CF or E. coli with pAMF or pAMF-PEG incorporation at T71

ND= Not Determined; NA=Not Active

EXAMPLE 18

IN VITRO RESULTS FOR D05 VARIANTS

[000350] Tables 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 and 35 below show the results from the kinetic binding experiments, reporter assays, human and NHP PBMC based assays as well as mouse splenocyte based assay previously described.

[000351] Table 22 shows thermostability, kinetic binding, and HEK-blue reporter activity data for a selected D05 (SEQ ID NO: 4) variant having a deletion of N110 and an H111N reversion mutation. The binding properties are maintained after deletion of position 110 and reversion to N at position 111.

[000352] Table 23 shows the thermostability, kinetic binding, and HEK-blue reporter activity data for SEQ ID NO: 57 and SEQ ID NO: 65 wherein a mutational scan was conducted at amino acid position R51 such that R51 was substituted by Q, A, or S, or H, or I, or V, or L, or Y.

[000353] Table 24 shows thermostability, kinetic binding, and HEK-blue reporter activity data for select D05 variants having a 40K PEG covalently attached to a pAMF residue located at position 171 or D I 57.

[000354] Table 25 shows thermostability, kinetic binding, and HEK-blue reporter activity data for a selected D05 variant having a single reversion at residues 1, 6, 51, 55, 56, 60, 91, or 93 and having a pAMF introduced at amino acid position DI 57.

[000355] Table 26 shows thermostability, kinetic binding, and HEK-blue reporter activity data for a selected D05 variant having a combination of reversion mutations at residues 1, 6, 51, 56, 91, or 93 (as indicated) in combination with a pAMF introduced at amino acid position DI 57.

[000356] Table 27 shows thermostability, kinetic binding, and HEK-blue reporter activity data for a selected D05 variant comprising combinations of reversion mutations W1Y (Y), D6E (E), N56Q (Q), R91N (N), and/or N93K (K).

[000357] Table 28 shows the result for cyno PBMCs IFN-γ release assay and the HEK-blue reporter assay of selected D05 variants conjugated to PEGs of different sizes at I71 TAG site. This data indicated that conjugation to PEG reduced the in vitro activity of D05 variants. Higher PEG size correlated to greater reduction in activity. [000358] Table 29 shows the result for cyno PBMC IFN-γ release assay and the HEK-blue reporter assay of selected D05 variants conjugated to PEGs of different sizes at DI 57 TAG site. This data indicated that conjugation to PEG reduced the in vitro activity of D05 variants. Higher PEG size correlated to greater reduction in activity.

[000359] Table 30 shows the kinetic binding data to human, rhesus and cyno IL-18Ra and IL- 18BP and,

[000360] Table 31 shows the human PBMC IFN-γ release assay and the HEK-blue reporter assay result of selected D05 variants conjugated to LP5 (30k PEG) at 171 and D157 TAG sites. FIG. 5A shows the dose response curves of the HEK-blue reporter assay. Conjugation to a 30k PEG slightly reduced the binding affinity of D05 variants to IL-18Ra, reduced the in vitro activity on HEK-blue reporter assay and IFN-γ release from human PBMCs. But the PEG conjugates still escaped IL-18BP negative regulation.

[000361] Table 32 shows the HEK-blue reporter assay result of a selected D05 variant conjugated to LP1 (40k PEG) at D157 TAG site or LP5 (30k PEG) at I71TAG site.

[000362] Table 33 shows thermostability, kinetic binding, and HEK-blue reporter activity for a selected sub-group of D05 reversion mutants having a combination of cysteine mutations (as indicated) with or without a PEG conjugated at either amino acid position DI 57.

[000363] Table 34 shows results from human PBMC IFN-γ release assay for a SRP3047-D05 variant with cysteine mutations with or without a 30k PEG conjugated at D157 site. The dose response curves are shown in FIG. 6B. The result from this assay indicates that the SRP3048- D05 variant with cysteine mutation induced similar potent IFN release as the wtIL-18 and not affected by the presence of IL18BP. Conjugation to a 30k PEG only slightly reduced the activity, but still escaped the negative regulation of IL-18BP.

[000364] Table 35 shows results from IFN-γ release assay using human PBMCs and mouse splenocyte for SRP3047-D05 variants expressed in CF or intact E. coli with or without a 30k PEG conjugated at 171 site. The result from this assay indicates that the SRP3047-D05 variants with cysteine mutation induced potent IFN release when co-cultured with human PBMCs or mouse splenocytes, which is similar to the wtIL-18 and not affected by the presence of IL18BP. Conjugation to PEG slightly reduced the activity, which still escapes the negative regulation of IL-18BP. Table 22. SRP3047-D05 with deletion of position 1 10 and reversion to N at position 1 1 1

NC = not calculable

Table 23. SRP3047-D05 mutational scan at R51 in context of V86M/E98D/N110D and H111N/K

ND = not determined

Table 24. D05 variants with combination of mutations conjugated to a PEG at D157 or 171 sites

NC = not calculable

Table 25. Single site reversion scan of SEQ ID NO: 57 at residues 1, 6, 51, 55, 56, 60, 91, 93 with D157 pAMF

NC = not calculable

Table 26. Selected D05 variants with combination mutations at residues 1, 6, 51, 56, 91, or 93 with DI 57 pAMF

Table 27. Selected D05 variants with combination of mutations at residues 1 , 6, 51 , 56, 91 , or 93 with PEG conjugated at DI 57

Table 28. Results for cyno PBMC IFN-γ release assay and HEK-blue reporter assay for selected D05 variants conjugated to PEGs of different sizes at 171 TAG site.

Table 29. Results for cyno PBMC IFN-γ release assay and HEK-blue reporter assay for selected D05 variants conjugated to PEGs of different sizes at DI 57 TAG site.

Table 30. Kinetic binding assay for D05 variants conjugated to LP5 at sites 171 and D 157

Table 31. Results for human PBMC IFN-γ release assay and HEK-blue reporter assay for D05 variants conjugated to LP5 at sites T71 and D157

Table 32. Results for HEK-blue reporter assay for D05 variants conjugated to PEG at sites T71 and DI 57

Table 33. Results for HEK-blue reporter assay for D05 variants with Cysteine mutations with or without PEG conjugation

Table 34. Results for human PBMC IFN-γ release assay for D05 variants with cysteine mutations with or without PEG

Table 35. release assay for SRP3047-D05 variants expressed in CF or E.coli with pAMF or pAMF-PEG incorporation at T71

ND= Not Determined; NA=Not Active

EXAMPLE 19

BALB/C SPLENOCYTE BASED MIFNy RELEASE ASSAY

[000365] Mouse spleens were isolated from healthy Balb/c mice and macerated Large aggregates were eliminated through sieving over 70 um cell strainer and erythrocytes were eliminated by ACK lysis buffer (Lonza). After a few washes, splenocytes were cryopreserved. On the day of assay setup, splenocytes were thawed and resuspended in assay medium (RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 1% Penicillin/Streptomycin, 2 mmol/L-glutamax, 2ng/mL human IL -2 and 2ng/mL mouse IL-12). 36,000 splenocytes in 120ul assay medium were seeded into each well in a 96-well clear bottom plate. 120ul of serial dilution of mouse IL- 18 samples (9-point, 10-fold dilutions starting at 20 nM in assay media) with or without 20nM mouse IL-18BP were then added into the splenocytes on 96 well plates. Splenocytes in assay media only were used as controls. After 72hrs of incubation in a cell culture incubator, the plates were centrifuged at 500 g for 4 minutes at 4°C and assay supernatant were collected into a new 96-well V-bottom plate.

[000366] To measure the mouse IFN-γ secreted by mouse splenocytes after IL- 18 treatment, an ELISA kit (BD OptEIA Mouse IFN- y ELISA Set) was used according to the manufacture’s recommendation. ELISA plate was coated with 100 pL per well of Capture IL-18 variant diluted in Coating Buffer. After wash, the plate was blocked with Assay Diluent buffer. Standard (mIFNy) and samples were diluted in Assay Diluent and 100 pL of each standard, sample, and control was added into appropriate wells. After incubation for 2 hours at RT, the plates were washed and 100 pL of Working Detector (Detection IL-18 variant + Streptavidin- HRP reagent) was added to each well. After another incubation for 1 hour at RT and wash, 100 pL of KPL SureBlue Reserve TMB Microwell Peroxidase Substrate (CAT#5120-0083) was added to each well. Incubated plate (without plate sealer) for 15-30 minutes at room temperature in the dark before adding 100 pL of KPL TMB BlueSTOP Solution (CAT#5150- 0024) to each well. Read absorbance at 650 nm within 30 minutes of stopping reaction. Concentration (pg/ml) of IFN-γ was interpolated using mIFN-γ standard. Data was fitted with non-linear regression analysis, using log (agonist) vs. response, variable slope, 4-parameter fit equation using GraphPad Prism. [000367] The in vitro activity of mIL-18, mCS2 and mCS2 conjugated to LP5 was evaluated in the IFN- y release assay and the EC50 was summarized in Table 36. mCS2 was about 20 -fold more potent than wild-type mIL-18 and the activity was not affected by the presence of 20nM mIL-18BP. Conjugated of LP5 reduced the mCS activity for about 70-fold.

Table 36: Mouse splenocytes IFN- y release assay for mCS2 conjugated to a PEG at SI 57 site

EXAMPLE 20

IN VIVO PHARMACOKINETIC (PK) ASSESSMENT OF IL- 18 VARIANTS

[000368] Pharmacokinetic (PK) profdes of SP10539 (mIL-18 WT), SP10538 (surrogate mouse variant mCS2), and mCS2 variants conjugated to different non-releasable PEG chains (10K - 40K) at different sites (M70 or S157) were assessed in non-tumor bearing BALB/c animals. Mice received a single bolus IV injection of 1 mg/kg SP10539, SP10538, or mCS2 variants (n= 3 per sampling time). Blood was collected in vacutainer tubes and serum was harvested by centrifugation. All samples were stored at -80 °C until analysis. Samples were processed and analyzed by ELISA to determine serum concentrations of IL- 18 species. Analysis of PK parameters was conducted using Phoenix WinNonLin. The PEGylated mCS2 variants with prolonged half-life (T _1/2 ) and exposure (increased area under the curve, AUC) vs. unconjugated mCS2 are predicted to have greater therapeutic utility and can be administered with less frequency.

EXAMPLE 21 PHARMACOKINETIC PROFILE OF MCS2 IN BALB/C MICE

[000369] The following Example illustrates the PK profde of mCS2 variants conjugated to different non-releasable PEG chains (10K - 40K) at two different sites, M70 or S 157 (mouse sites corresponding to human sites I71 and DI 57, respectively). Sequence alignment of mouse IL-18 with human IL-18 indicates that amino acid positions M70 and S 157 in mouse IL-18, are equivalent to amino acid positions 171 and DI 57, in human IL- 18, respectively.

[000370] The PK profde of mCS2 variants conjugated to different non-releasable PEG chains (10K - 40K) at M70 or S157 was evaluated by total IL-18 variant levels following a single 1 mg/kg dose in non-tumor bearing Balb/c mice.

[000371] Table 37 shows the PK profde of mCS2 variants conjugated to PEG at M70 as well as SP10539 and SP10538. Table 38 shows the PK profde of mCS2 variants conjugated to PEG at SI 57.

[000372] Serum samples were collected at several time points up to seven days for PK analysis. The mean plasma concentration profdes of SP10539 and SP10538 are shown in FIG. 3A. As shown in Table 37, SP10539 has longer T1/2, lower clearance and higher exposure (increased area under the curve, AUC), while the SP10538 has fast clearance and is undetectable after 1 hour. This supports published literature which suggests that binding of IL-18BP to IL-18 stabilizes the inactive form of IL-18 in circulation (Harms, R.Z. et al. Interleukin (IL)-18 Binding Protein Deficiency Disrupts Natural Killer Cell Maturation and Diminishes Circulating IL- 18. Front. Immunol. 8, 1020 (2017)).

[000373] Site specific conjugation of non-releasable PEG chains to mCS2 at both M70 site (mCS2-M70-PEG) or S157 (mCS2-S157-PEG) site resulted in half-life extension compared to mCS2 (FIG. 3A and FIG. 3B, respectively). Increasing PEG chain lengths directly correlated with prolonged half-life (T1/2), higher exposure or AUC, and lower clearance (CL) for both mCS2-M70-PEG (Table 37) and mCS2-S157-PEG (Table 38) variants. The site of PEG conjugation also appears to influence PK profde. Higher exposures were observed for mCS2 variants with larger 30K or 40K PEG chains conjugated at SI 57 site compared to those conjugated at M70 site. Direct comparison in two independent studies showed approximately 5-10 fold higher exposure for SP10767 vs. SP10766 (Table 37 and Table 38).

[000374] As shown in FIG. 3A and FIG. 3B, mCS2 variants conjugated to different non- releasable PEG chains (10K - 40K) at different sites (M70 or SI 57) have extended PK profde compared to SP10538 (unconjugated mCS2). Table 37: Summary of mCS2-M70-PEG Variant PK Parameters in Balb/c Mice

*Insufficient data to calculate PK parameters

Table 38: Summary of mCS2-S157-PEG Variant PK Parameters in Balb/c Mice (Experiment 2)

* Insufficient data to calculate PK parameters

Table 39: Summary of PK Parameters in SCTD beige mice of G01 variants conjugated to PEGs at different TAG sites

Table 40: Summary of PK Parameters in Balb/c Mice of D05 variants conjugated to PEGs at different TAG sites

Table 41: Summary of PK Parameters in Balb/c Mice of a G01 variant with Cysteine mutations

EXAMPLE 22

PHARMACOKINETIC PROFILE OF HUMAN IL 18 VARIANTS IN MICE

[000375] Pharmacokinetic (PK) profiles of human IL-18 variants were assessed in non-tumor bearing SCID beige mice. Mice received a single bolus IV injection of 3 mg/kg (n= 3 per sampling time). Blood was collected in vacutainer tubes and serum was harvested by centrifugation. All samples were stored at -80 °C until analysis. Samples were processed and analyzed by ELISA to determine serum concentrations of IL- 18 species. Analysis of PK parameters was conducted using Phoenix WinNonLin.

[000376] The PK profile of a GO 1 variant and its PEG conjugates are shown in FIG. 7A and the PK parameters summarized in Table 39. Conjugation to PEGs significantly improved the PK profile of the G01 variant. Conjugation of a 40K PEG further improved the PK profile of the G01 variant.

[000377] The PK profile of a D05 variant and its PEG conjugates are shown in FIG. 7B and the PK parameters summarized in Table 40. Conjugation to PEGs significantly improved the PK profile of the D05 variant. Conjugation of a 30K PEG at D157 site shows better PK profile compared to conjugation at the I71 site. Conjugation to a 40K PEG only slightly improved the PK profile compared to 3 OK PEG.

[000378] The PK profile of a GO 1 variant with and without cysteine mutations are shown in FIG. 7C and the PK parameters summarized in Table 41. Single C68S mutation significantly improved the PK profile of the G01 variant. The combination of C38S and C68S mutations further improved the PK profile of the G01 variant.

EXAMPLE 23

PRODUCTION OF IL 18 171 PAMF IN E. COLI CELLS WITH HIGH DENSITY FERMENTATION

[000379] Interleukin- 18 (IL18) production was demonstrated in E. coli. Tn order to achieve efficient amber suppression sufficient for high titers, genes for non-natural amino acid incorporation were expressed on a first plasmid (RS plasmid), while genes for expression of the protein of interest were expressed on a second plasmid (product plasmid). [000380] To generate the RS plasmid, a gene having the coding sequence (CDS) for an aminoacyl tRNA synthetase (RS) specific for para-azidomethylphenylalanine (pAMF) was cloned into a medium copy (pl 5a origin) plasmid with a carbenicillin (Carb) selection cassette behind a constitutive promoter followed by an inducible T7 promoter (T7p) and a strong ribosome binding site (RBS). Three copies of a tRNA specific for para- azidomethylphenylalanine (pAMF) were cloned behind the pAMF RS CDS, with 23 nucleotide non-coding DNA spacers before each tRNA sequence. For non-natural amino acid incorporation in reducing A. coli strain SBDG175, the pJ411 product plasmid was used bearing the sequence of the protein of interest (POI) behind a T7 promoter (T7 pr.) and followed by a T7 terminator (T7 term ). The product plasmid is high copy (pUC origin of replication) and bears a kanamycin resistance gene (KanR) while the pJ434 RS plasmid bore the sequence of the pAMF RS behind a T7 promoter (T7 pr.) and constitutive PcO promoter (PcO pr ). The pAMF RS sequence is followed by one copy of the AS tRNA and a T7 terminator (T7 term.). The RS plasmid is medium copy (pl 5a origin of replication) and bears an ampicillin resistance gene (AmpR).

[000381] In order to incorporate nnAAs into recombinantly-expressed proteins in E. coli via amber suppression, three genetic elements are required: (1) a coding sequence for a protein of interest containing a TAG codon at the desired nnAA incorporation site, (2) an orthogonal RS that will recognize the nnAA of interest and load it onto a cognate tRNA (Zimmerman, E.S.; Heibeck, T.H.; Gill, A.; Li, X.; Murray, C.J.; Madlansacay, M R ; Tran, C.; Uter, N.T.; Yin, G.; Rivers, P.J.; et al. Production of Site-Specific Antibody-Drug Conjugates Using Optimized Non-Natural Amino Acids in a Cell-Free Expression System. Bioconjugate Chem. 2014, 25, 351-361, incorporated by reference herein in its entirety), and (3) a cognate AS tRNA not recognized by any of the native RSs that will be charged with the nnAA and associate with the TAG codon of the mRNA of interest (Wang, L.; Brock, A.; Herberich, B.; Schultz, P.G. Expanding the Genetic Code of Escherichia Coli. Science 2001, 292, 498-500, doi: 10.1126/science.1060077; and Guo, J.; Melançon, C.E.; Lee, H.S.; Groff, D.; Schultz, P.G. Evolution of Amber Suppressor TRNAs for Efficient Bacterial Production of Proteins Containing Nonnatural Amino Acids. Angew Chem Int Ed Engl 2009, 48, 9148-9151, each incorporated by reference herein in its entirety), causing the nnAA to be incorporated into a protein as it is translated by the ribosome. To achieve amber suppression in the strain, a second plasmid (deemed the RS plasmid) was designed that encoded pAMF RS and its cognate AS tRNA. This DNA was cloned into a medium copy pl 5a plasmid behind an inducible T7 promoter and a constitutive PcO promoter.

[000382] To generate the product plasmid, the coding sequence for IL18 with an N-terminal HisSUMO tag and with a TAG codon at the positions coding for amino acid 171 was codon optimized for E. coli. The construct was cloned behind a T7p and strong RBS into a high copy (pUC origin) plasmid with a kanamycin (Kan) selection cassette.

[000383] The E. coli strain for expression of IL18 was generated by transforming E. coli S175 strain with both the RS plasmid and product plasmid. Transformations were plated on LB agar containing 50 μg/mL kanamycin and 100 μg/mL carbenicillin. Single colonies were picked and transferred into culture tubes with 3 mL of TB media containing 50 μg/mL kanamycin and 100 μg/mL carbenicillin for overnight growth at 37° C. The culture tube was used to inoculate a shake flask with I17-SF shake flask media containing 50 μg/mL of kanamycin and 100 μg/mL of carbenicillin at 8% (v/v) seeding density. The shake flask was harvested once the culture achieved an OD 595 nm greater than 3. Glycerol was added to the shake flask to a final concentration of 16-20% (v/v). The cell bank was collected and aliquoted into 2 mL vials, flash frozen in liquid nitrogen and stored at -80°C.

EXAMPLE 24:

FED-BATCH RECOMBINANT PROTEIN EXPRESSION OF IL18 I71PAMF IN HDF

[000384] To generate the product plasmid, each product gene was synthesized and then cloned into pJ411. This vector has a kanamycin resistance marker and a pUC high copy origin of replication, and the expression cassette has a T7 promoter for high level transcription.

[000385] To generate the HisSUMO-nnAA-IL18 plasmid, the coding sequence for IL18 with an N-terminal HisSUMO tag and with a TAG codon at the positions coding for amino acid 171 was codon optimized for E. coli. The construct was cloned behind a T7p and strong RBS into a high copy (pUC origin) plasmid with a kanamycin (Kan) selection cassette.

[000386] For the incorporation of non-natural amino acids into proteins of interest, the selected codons in product genes where non-natural amino acids would be incorporated were substituted with the amber codon “TAG.” The gene for nnAA-IL18 was mutated to contain 1 TAG codon. [000387] Plasmid sequences were verified by sequencing.

[000388] The coding sequence for the pAMF RS was cloned into a medium copy pJ434 plasmid behind a constitutive PcO promoter (Groff, D.; Armstrong, S.; Rivers, P.J.; Zhang, J.; Yang, J.; Green, E.; Rozzelle, J.; Liang, S.; Kittle, J.D.; Steiner, A.R.; et al. Engineering toward a Bacterial “Endoplasmic Reticulum” for the Rapid Expression of Immunoglobulin Proteins. MAbs 2014, 6, 671-678, incorporated herein by reference in its entirety). One copy of the amber suppressor tRNA (AS tRNA) were included on the pJ434 plasmid 3’ to the pAMF RS coding sequence after a 20 base pair spacer sequence.

[000389] Unlike the other secreted mammalian proteins in this paper, IL- 18 must be maintained in a reduced state.

[000390] To generate a strain for the expression of this nnAA-IL18 protein, SBDG175 was transformed with both the RS plasmid and product plasmid which were maintained with kanamycin and carbenicillin.

[000391] All plasmids were transformed into the reducing E. coli strain SBDG175 (Hanson, J.; Groff, D.; Carlos, A.; Usman, H.; Fong, K.; Yu, A.; Armstrong, S.; Dwyer, A.; Masikat, M.R.; Yuan, D.; et al. An Integrated In Vivo/In Vitro Protein Production Platform for Site-Specific Antibody Drug Conjugates. Bioengineering 2023, 10, 304, incorporated herein by reference in its entirety), which has a functional thioredoxin pathway and is capable of reducing cytoplasm, and plated on selective media. For wild type proteins, only the high copy product plasmid was transformed. For nnAA-containing proteins, each product plasmid was co-transformed with an RS plasmid.

[000392] For initial screening experiments, protein expressions were performed in shake flasks. For each experiment, a single colony was picked from transformation plates into 2-3 mL of Terrific Broth (Teknova, Hollister, CA) containing appropriate antibiotic(s). After overnight incubation at 37°C in a shaking incubator at 250 RPM, cultures were inoculated into 50 mL of fresh Terrific Broth + antibiotics in a shake flask. Cultures were incubated at 37°C in a shaking incubator at 250 RPM until the optical density at 595 nm (OD595) reached 1.0-2.0. At this time, arabinose was added to induce expression of the protein(s) of interest. For cultures expressing nnAA proteins, pAMF was added at 2 mM. Cultures were transferred to 25°C for expression of all proteins except IL- 18 which was transferred to 20°C. After expression for 16-18 hours, cells were harvested by centrifugation for 5 minutes at ~7000g. Cells were resuspended in 10 mL per gram of wet cells in phosphate buffered saline (PBS) containing 0.1 mg/mL lysozyme and benzonase. After incubation on ice for 30 minutes, cells were lysed by sonication. Soluble lysates were isolated by centrifugation at >20, 000g for 30 minutes. After initial test expression to confirm nnAA-IL-18 production (data not shown), the process was scaled in 250 mL fermenters. Initially, for a fed batch process, at low OD595 values, this strain exhibited growth similar to the SBDG419 strains. However, during the fed-batch phase of high-density fermentation, the specific growth rate dropped when the OD595 reached higher than 50, leading to overfeeding and acetate production (data not shown). This was addressed by developing a modified feed program in which the target p was lowered stepwise after 10 hours. With these modifications, it was possible to maintain low acetate and glucose over the entire fermentation, leading to successful production of the nnAA-IL18 in 500 mL scale high-density fermentations. The procedure for such high-density fermentations was as follows:

[000393] To express IL-18 171 PAMF and other constructs, a shake flask with I17-SF media (as described in Hanson, I.; Groff, D.; Carlos, A.; Usman, H.; Fong, K.; Yu, A.; Armstrong, S.; Dwyer, A.; Masikat, M.R.; Yuan, D.; et al. An Integrated In Vivo/In Vitro Protein Production Platform for Site-Specific Antibody Drug Conjugates. Bioengineering 2023, 10, 304, which is incorporated herein by reference in its entirety) containing 50 μg/mL of kanamycin and 100 μg/mL of carbenicillin was inoculated with a 2 mL cell bank vial at a seeding density of 8% (v/v). Once the shake flask culture reached an OD 595 nm of 3-4, it was used to inoculate a 250 mL bioreactor at a seeding density of 8% (v/v) in batched media consisting of I17-SF shake flask media, 50 μg/mL of kanamycin, 100 μg/mL of carbenicillin, and 0.1% (v/v) A204 antifoam. The bioreactor temperature, dissolved oxygen and pH setpoints at inoculation were set to 37° C, 30% and 7, respectively.

[000394] Once the cells grew to an OD 595 nm between 3-5 during the batch phase of the fermentation, the fed batch phase began by feeding 5x 117 media at an exponential rate of 0.2 h-1. The exponential feed rate during the fed batch phase was altered to 0.175 h-1, 0.15 h-1, 0.135 h-1 and 0.12 h-1 at hours 10, 11, 14 and 15, respectively. After 18 hours total in the fed batch phase, the temperature was decreased to 20° C, and the exponential feed rate was decreased to 0.02 h-1.

[000395] One hour later, IL 18 induction began by adding pAMF to a target concentration of 4 mM and L-Arabinose to a target concentration of 4 g/L based on the culture volume in the bioreactor prior to induction. The induction phase took 48 hours before the bioreactor was harvested. At the end of the fermentation, the culture was collected and centrifuged at 18,592 xG and 2-8° C for 15 min in a floor centrifuge. The supernatant was discarded, and the cell pellets were resuspended with DPBS + ImM DTT at a concentration of 9.09% (w/w). The cell resuspension was then passed twice through an Avestin Homogenizer (EmulsiFlex-C5) at 17,000 Psi to disrupt the cells and generate the crude lysate. The crude lysate was clarified by centrifuging at 18,000-20,000 xG and 2-8° C for 30 minutes in a floor centrifuge. The supernatant (clarified lysate) was collected and aliquoted, flash frozen in liquid nitrogen and stored at -80°C.

[000396] Lysate supernatants were applied to Ni-NTA resin that had been pre-equilibrated with PBS. After application of the supernatant, the resin was washed with PBS containing 10 mM imidazole before the protein was eluted across several fractions with PBS containing 200 mM imidazole. The purest fractions were identified by analysis via SDS-PAGE then pooled and concentrated in 10 kDa MWCO Ami con centrifuge filters. Samples were quantified by adjusting the absorbance at 280 nm according to the calculated molar absorbance of the protein and considering the % purity calculated by gel densitometry analysis from an SDS-PAGE gel.

[000397] For purification of HisSUMO-IL18 and HisSUMO-nnAA-IL18, lysates were centrifuged at 30,000g for 30 minutes, and the supernatants were applied to Cytiva Ni Sepharose excel resin that had been pre-equilibrated with PBS. After application of the supernatant, the resin was washed with PBS containing 10 mM imidazole before the protein was eluted across several fractions with PBS containing 200 mM imidazole. Protein was digested with Ulpl (1:20 w/w ratio) for 1 hour. 1 mM DTT was added to lysis and elution buffers to maintain the protein in a reduced state.

[000398] After complete Ulpl digestion, proteins were polished with two additional column steps. Proteins were first buffer exchanged into 20 mM Tris, 300 mM sodium chloride, pH 7.5 with Cytiva Sephadex G-25 fine resin. Then they were applied back onto Cytiva Ni Sepharose excel resin and the flowthrough contained the target protein. The final pool was concentrated and buffer exchanged into PBS, 9% sucrose, pH 6 with Ami con centrifuge filters (10 kDa MWCO) for conjugation.

[000399] For PEGylation of IL-18, the reaction consisting of conjugated IL-18 and unreacted PEG was further processed by anion exchange column packed with Capto Q resin (Cytiva). Dilution of the IL-18/PEG reaction prior to purification was performed with binding buffer (20mM Tris-acetate, pH 7.0) and bound to the Capto Q column. A linear gradient with elution buffer (20mM Tris-acetate, 200mM NaCl, pH 7.0) was performed over 30 CV and the target elution fractions were collected and buffer exchanged into PBS buffer, 6% sucrose, 1 mM DTT, pH 7.2 in Amicon Ultra- 15, lOkD spin filters.

[000400] For intact LC-MS analysis, samples (10-50 pmol) were injected into an Agilent 1200 series system with a Binary SL pump. Mobile phases A was 0.1% formic acid in water and mobile phase B was 0.1% formic acid in acetonitrile. Proteins were separated using an Agilent PLRP-S HPLC column (2.1 x 50 mm, 1000 A, 5 micrometers) heated at 80°C in Agilent column oven with flow rate of 0.3 mL/min. The gradient started with 10% mobile phase B and was kept at 10% B for the first 2 min. and was then ramped up to 70% B in 8 min. Mobile phase B was then increased to 95% by 11 min. and was held for 3 min. before it was decreased to 10% by 14.5 min. Data was acquired on an Agilent 6520A Accurate Mass Q-TOF MS with mass detection range of 500 - 3200 m/z. The source gas temperature was at 325°C, drying gas flow at 8 L/min, nebulizer at 30 psig. Capillary voltage was set at 4000 V, fragmentor voltage at 250 V and skimmer 1 was set at 65 V. The instrument mode used was 2 GHz, Standard (3200 m/z).

[000401] Spectra from all peaks on the total ion chromatogram were extracted and deconvoluted using Maximum Entropy algorithm from MassHunter Qualitative (B6.00) from Agilent. A mass range of 10,000-100,000 Da was searched. Adduct use in the deconvolution setting was proton. Peaks were filtered by setting a signal -to-noise ratio of > 30.0. Top 90% of the peak height was used to calculate average mass.

[000402] For conjugation of nnAA-IL18 to DBCO amine, protein concentrations were brought to 1 mg/mL in DPBS. The DBCO-amine was added at a drug to pAMF ratio of 3: 1, and 500 mM NaCl was added to the reaction to improve DBCO-amine solubility. The conjugation reaction was incubated overnight at 30°C prior to LC-MS analysis.

[000403] For conjugation of nnAA-IL 18 with DBCO-PEG, proteins were dialyzed into lx DPBS + 9% Sucrose prior to conjugation. The PEG of interest was prepared in water as a 5mM stock solution. Targeting the final protein concentration at Img/ml, the protein was formulated in DPBS buffer, and 3 molar equivalents of PEG were added per mole of pAMF. Conjugation reactions were incubated at 30°C (nnAA-ILl 8) overnight in a Thermomixer (Fisher scientific, Allentown Pennsylvania) with agitation at 450rpm.

[0004041 For PEG-to-protein ratio calculation, after conjugate cleanup, PEGylated proteins were analyzed via SDS-PAGE. PEG-to-protein ratios were calculated by gel densitometry analysis using the Lane and Bands image analysis tools in the Image Lab software (version 5.2.1, Bio- Rad).

[000405] For kinetic receptor binding analysis of IL-18, kinetics analysis was performed using standard ligand capture techniques with a Biacore T200 instrument coupled with an anti- histidine antibody (Cytiva) on a CM4 chip. Human Cterm-6his tagged receptors (human IL18R1, Sino Biological) was used as a ligand. Capture of IL18Rl-6his was followed by the application of appropriate concentration ranges for sample cytokine analytes based on expected affinities at the receptor targets Analysis for hIL18Rl-6his kinetics was performed on double reference subtracted sensorgrams using a global 1: 1 binding model with RI equal to Ymax/5.

[000406] Lysates from the 500 mL nnAA-IL18 fermentation were initially analyzed via SDS- PAGE, showing the presence of a band at ~30 kDa (FIG. 9A) after induction with arabinose. To assess the quality and activity of the nnAA-IL18 produced in E. coli, the protein was captured using immobilized Ni chromatography, resulting in an initial capture titer of 490 mg/L (FIG. 8C). Following SUMO cleavage with Ulpl, the protease and His-SUMO were removed with another round ofNi chromatography (FIG. 8C). A small volume of the nnAA- IL18 protein was then conjugated with a small molecule DBCO-amine and analyzed via intact LC-MS to verify the identity of the resulting nnAA-IL18 and validate its conjugatability (FIG. 9B). A mass shift corresponding to labeling with a single DBCO-amine was observed, confirming the incorporation of a single pAMF nnAA. After confirming successful incorporation of pAMF, a large scale PEGylation reaction of nnAA-IL18 was performed with a DBCO-PEG molecule followed by anion exchange chromatography to remove the unconjugated PEG (FIG. 8B). Analytical SEC revealed that the final PEGylated material was more than 94% homogenous (FIGS. 8B-8C). Next, the KD for the IL-18 receptor was measured for the PEGylated nnAA-IL18 using a Biacore Surface Plasmon Resonance system. A WT IL- 18 standard had an affinity for the IL- 18 receptor of approximately 1 nM, while the KD for PEGylated IL-18 protein had a moderately lower affinity at 19.2 nM (FIG. 8C and FTGS. 9C-9D), demonstrating that PEGylation did not significantly disturb the protein’s biological activity.

EXAMPLE 25:

CYSTEINE MUTATION SEQUENCES

Table 42: Cysteine mutation sequences EXAMPLE 26

SEQUENCES

Table 43 provides sequences referred to herein.

[000407] The present disclosure provides the IL-18 variants in addition to HisSUMO fusions (SEQ ID NO: 198) of the variants.

[000408] All patents and patent publications referred to herein are hereby incorporated by reference. Certain modifications and improvements will occur to those skilled in the art upon a reading of the foregoing description. It should be understood that all such modifications and improvements have been deleted herein for the sake of conciseness and readability but are properly within the scope of the following claims.