Il-18 variant fusion proteins

Abstract

Described herein are IL-18 variants, and fusion proteins comprising IL-18 variants and anti-PD-1 antibodies or a fragment thereof. Also described herein are methods of using fusion proteins to treat cancer and other conditions.

Description

IL- 18 VARIANT FUSION PROTEINSCROSS REFERENCE

[0001] This application claims priority to U.S. Provisional Application No. 63 / 652,902 filed on May 29, 2024. and the U.S. Provisional Application No. 63 / 709.779 filed on October 21, 2024, and each of the disclosures are incorporated herein by reference in its entirety.SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML file format and is hereby incorporated by reference in its entirety. Said XML copy, created on May 28, 2025 is named JBI6997WOPCTl_SL.xml and is 329,283 bytes in size.BACKGROUND

[0003] All publications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The following description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

[0004] In order for the immune system to mount an effective anti-tumor response, two things must occur. T cells in the tumor environment must first engage antigenic tumor peptides presented by major histocompatibility complexes (MHC) on tumor cells. Next, the T cells must be induced by cytokines such as IL- 18, IL- 15 and IL-2 to produce costimulatory cytokines such as IFNγ. Recognition of tumor peptides alone in the absence of cytokine induction leads to T cells becoming anergic, thereby leading to tolerance. Accordingly, a very promising approach in cancer immunotherapy is cytokine-based treatments, such as IL- 18.

[0005] IL-18 is a cytokine that serves to promote the activity and survival of antigen presenting cells, NK cells and subsets of T cells. The cytokine is strongly regulated by a negative feedback loop that includes the induction of IL-18BP and IL-37, which act to bind IL-18 and IL-18Ra, respectively. In the tumor microenvironment (TME), the IL-18Rα / β complex is expressed on PD-U progenitor exhausted T cells that are responsive to PD-1 antagonism. IL-18has been proposed as an immunotherapeutic agent for the treatment of cancer, given its ability to stimulate anti-tumor immune cells. However, the clinical efficacy of IL- 18 has been limited due to its indiscriminate activity towards various cells.

[0006] Thus, there is a need for developing variants of IL- 18 which is attenuated for safety, but retains activity when brought into close proximity of the IL- 18 receptor complex.BRIEF SUMMARY

[0007] Disclosed herein are IL- 18 variants that bind to IL- 18 receptor (IL-18R) but exhibits decreased binding affinity to IL- 18 binding protein (IL-18BP) relative to the wild-type IL-18. In some embodiments, an IL-18 variant compnses an amino acid sequence of:wherein X1is Y or C;

[0008] In some embodiments, the IL- 18 variant further comprises an attenuator moiety at the N-terminus of the IL- 18 variant, wherein the attenuator moiety comprises from about one amino acid to about 70 amino acids in length. In some embodiments, the attenuator moiety is selected from any one of the attenuator sequences of Table 2.

[0009] Also disclosed herein are fusion proteins comprising an IL- 18 variant and a scaffold polypeptide, wherein the IL-18 variant comprises the amino acid sequence:wherein X1is Y or C;

[0010] In some embodiments, scaffold polypeptide is selected from an immunoglobulin, Fc region of an immunoglobulin, human serum albumin (HSA), beta2 microglobulin, transferrin, fragment antigen-binding region (Fab region), VHH antibody,single-chain variable fragment (scFv), anticalin, designed ankynn repeat protein (DARPin). type I transmembrane protein, or type II transmembrane protein or a fragment thereof.

[0011] In another embodiment, a fusion protein comprises an IL-18 variant and an anti-PDl antibody or a fragment thereof, wherein the IL-18 variant comprises an amino acid sequence selected from SEQ ID NOs: 2. 3, 4, 5, 6. 7, 8, 9. 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 242, 243, 244, 245, 246, 247, 248, or 183, and wherein the anti-PDl antibody or a fragment thereof comprises a variable heavy chain (VH) comprising an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 114, and a variable light chain (VL) comprising an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 115.

[0012] In another embodiment, a fusion protein comprises:(a) a first monomer comprising from N-terminus to C-terminus an IL-18 variant and a first Fc region;(b) a second monomer comprising from N-terminus to C-terminus a variable heavy chain (VH) and a second Fc region; and(c) a third monomer comprising a variable light (VL) chain, wherein the VH and VL form an antigen binding domain that binds to PD-1.

[0013] In a further embodiment, a method of treating a subject suffering from cancer comprises administering to the subject a composition comprising an IL-18 fusion protein, wherein the IL- 18 fusion protein comprises IL- 18 variant and an anti-PDl antibody or a fragment thereof, and wherein the IL-18 variant comprises an amino acid sequence selected from SEQ ID NOs: 2, 3, 4. 5, 6, 7. 8. 9, 10, 11, 12, 13. 14. 15. 16. 17. 18. 19, 20. 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 242, 243, 244, 245, 246, 247, 248, or 183, and wherein the anti- PDl antibody or a fragment thereof comprises a variable heavy chain (VH) comprising an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 114. and a variable light chain (VL) comprising an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 115.BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Exemplary embodiments are illustrated in referenced figures. It is intended that the embodiments and figures disclosed herein are to be considered illustrative rather than restrictive.

[0015] Figures 1A-1D depict illustrations of formats for IL- 18 variant fusion proteins. All proteins incorporate a human IgG scaffold. In the case of FIGs 1 A, IB and ID, the scaffold comprises a knob-in-hole (KIH) heterodimeric IgGl Fc. The IL-18 variant included in the fusion protein is fused (1) as a singular cassette to theN-terminal end of the hole (or knob) chain in FIGs 1 A and ID, (2) to both the N-terminal end of the hole (or knob) and the C-terminal end of the hole (or knob) in FIG. IB, or (3) to the N-terminal of the light chain (FIG. 1C).

[0016] Figure 2A-2F shows capacity of polypeptides ranging in length from 1 amino acid to 14 amino acids to impact the functional activity7of IL- 18. FIG. 2 A is a non-linear x-y graph of SEAP release (IL- 18 activity) relative to the concentration of test article. Human recombinant IL- 18 was used as the positive control (black squares) and human recombinant TNF-alpha as the negative control (grey square). A vast array of potency (EC50-SEAP) was observed whereby the N-terminal polypeptides attenuated IL-18 from 3 fold to >10,000 fold. We identified a relationship between the composition of the N-terminal polypeptide and the degree of attenuation. FIG. 2B is a regression analysis of N-terminal polypeptide length versus potency (EC50-SEAP), in which the R-squared (R) value was 0.25 indicative of a weak correlation. When we assessed the functional activity (FIG. 2C) and performed regression analysis (FIG. 2D) for non-charged N-terminal polypeptides consisting of glycine and serine residues, we observed a strong correlation with an R value of 0.88. Given that length N-terminal polypeptides only sometimes directly linked to the degree their capacity to attenuate IL- 18. we also examined hydrophobicity and charge density7. The results indicated a strong correlation with a R value of 0.8 (FIG. 2F). Summary' tables of potencies (EC50-SEAP) are shown beneath the non-linear x-y graphs for FIGs 2A and 2C.

[0017] Figures 3A-3B depict the functional activity as it relates to an IL- 18 variant targeted via fusion to a HER-2 specific antibody like protein in various formats to induce Antibody Dependent Cellular Cytotoxicity' (FIG. 3A) and IFNy release (FIG. 3B ) after 48 hours of co-culture of PBMC and HER2+ T47D tumor cells at an E:T ratio of 15: 1.

[0018] Figures 4A-4D show the capacity of an anti-PDl antibodies incorporating one of two exemplary IL-18 variants to induce T cell mediated tumor cell killing (FIGs 4A and 4C)and IFNy release (FIGs 4B and 4D). For FIGs 4A and 4C, the IL-18 variant was approximately 100 fold less potent than the IL-18 variant used for FIGs 4B and 4D.

[0019] Figure 5 shows the impact of exemplary IL-18 variants fused to anti-PDl antibodies on IFNy release mediated by PBMC activated with PeliCluster CD3 for 48 hours, washed and the re-exposed to a 10 fold lower concentration of PeliCluster CD3 for an additional 48 hours. PBMC was two different healthy human donors were assessed (donor 1 and donor 2 are shown in Figure 5 top and bottom plot, respectively). A summary table of EC50, Emax and AUC (area under the curve) values is shown below the x-y plots. Fuse 599 (IL-18m9-Fc) was used as the non -targeted benchmark with similar activity' to wild type IL- 18 (open grey circles, dashed line). Fusel 109 (black squares) and Fusel 177 (black diamonds) consisted of PD-1 targeted IL-18ml3 without and with a 100 fold N-terminal attenuator, respectively. Fuse 1229 (black downward facing triangles) and Fuse 1179 (black upward facing triangles) consisted of PD-1 targeted IL-18 ml3.1 and IL-18ml6, both containing a 100 fold N-terminal attenuator. Relative to Fuse599, Fusel 109 was about 100 fold attenuated (EC50) whereas Fusel 177, Fusel229, and Fusell79 were all greater than 3000 (donor 2) to greater than 1000 fold (donor 1) attenuated (EC50). Relative to Fuse599. the Emax for Fusel 177. Fusel229, and Fusel 179 was also reduced by >60%. The human IgGl antibody, anti-HEL (gray circle), was used as the isotype control and was associated with background IFNy release. A summary table of EC50, Emax and AUC (area under the curve) values is shown below the x-y plots.

[0020] Figures 6A-6C shows the capacity of IL-18 variants fused to anti-PDl antibodies to oppose functional exhaustion induced by sequential stimulation. On day 0, the cells were stimulated using the suboptimal concentration of a ROR1 x CD3 bsAb (Fuse608) equating 20% maximum killing and one of the following test articles at the lOx their EC99 of killing (i.e. on the killing plateau): Fuse 691 (open reverse triangle; anti-PDl), Fusel 109 (open circle; anti-PD-l-IL-18ml3 without a 100 fold N-terminal attenuator) or Fusel229 (closed circle; anti-PD-l-IL-18ml3.1 with a 100 fold N-terminal attenuator). In all cases, anti-HEL antibody (gray circle) was used as the isoty pe matched negative control. FIG. 6A, FIG. 6B and FIG. 6C are non-linear x-y plots of viable T cell numbers, viable tumor cell numbers and the concentration of IFNy in the supernatant as a function of time.

[0021] FIG.7 shows the ability of N-terminal amino acids to attenuate IL-18 activity. FIG. 7 A is a non-linear x-y graph of SEAP release (IL- 18 activity') relative to the concentration of test article. Human recombinant IL- 18 was used as the positive control (black squares) and human recombinant TNF-alpha as the negative control (grey square). A summary table ofpotency (EC50-SEAP) is shown beneath the non-linear x-y graphs as FIG 7B. FIG. 7C and 7D depict regression analysis plots.

[0022] FIG. 8 shows comparison of IL- 18 variant / nivolumab and IL- 18variant / cetrelimab fusion proteins. FIG. 8 A is a non-linear x-y graph of SEAP release (IL- 18 activity) relative to the concentration of test article. Human recombinant IL- 18 (rhIL18) was used as the positive control (black squares) and human recombinant TNF-alpha as the negative control (grey square). A summary table of potency (EC50-SEAP) is shown beneath the nonlinear x-y graph (FIG. 8B)

[0023] FIG. 9 shows the capacity of nivolumab and cetrelimab with or w ithout fusion to exemplary IL-18 variants to reduce binding between human PD-1 and human PD-L1. FIG. 9A and FIG. 9B are non-linear x-y graphs of PD-1 / PDL1 binding measured as the colorimertric intensity OD (450) of HRP oxidized 3,3',5,5'-tetramethylbenzidine relative to the concentration of test article. Anti-HEL was used as the negative control (gray squares).

[0024] FIG. 10 depicts binding affinity of nivolumab and cetrelimab with or without fusion to exemplary IL-18 variants to PD1 via flow cytometry. FIGs 10A and 10B are nonlinear line graphs of binding intensity' via MFI versus concentration of test article.

[0025] FIG. 11 depicts mouse xenograft studies using IL- 18 variant / nivolumab and IL-18 variant / cetrelimab fusion proteins.

[0026] FIG. 12 depicts functional activity' of various IL- 18 variants in HEK-Blue IL- 18 reporter cells. A non-linear x-y graph of SEAP release (IL-18 activity) relative to the concentration of test article is shown. Human recombinant wild type IL- 18 w as used as the positive control (black squares) and human recombinant TNF-alpha as the negative control (grey square). All test articles are annotated with symbols that are indicated within the legend. A summary table of potency (EC50-SEAP) with both fuse numbers and the associated IL-18 mutein designations is shown beneath the non-linear x-y graph.

[0027] FIG. 13 depicts activity of IL- 18 muteins containing the N-terminal lOOx attenuator peptide (GGGGS (SEQ ID NO: 32). termed “5L”) to induce non-targeted functional activity via activation of HEK Blue IL- 18. A non-linear x-y graph of SEAP release (IL-18 activity) relative to the concentration of test article. Human recombinant wild type IL- 18 was used as the positive control (black squares) and human recombinant TNF-alpha as the negative control (grey square). All test articles are annotated with symbols that are indicated within the legend. A summary table of potency (EC50-SEAP) with both fuse numbers and the associated 5L IL- 18 mutein designations is shown beneath the non-linear x-y graph.

[0028] FIG. 14 depicts IL-18 muteins 13. 19. or 9 containing different N-terminal attenuator peptides to induce non-targeted functional activity via activation of HEK Blue IL- 18. A non-linear x-y graph of SEAP release (IL- 18 activity ) relative to the concentration of test article. Human recombinant wild type IL- 18 was used as the positive control (black squares) and human recombinant TNF-alpha as the negative control (grey square). All test articles are annotated with symbols that are indicated within the legend. A summary table of potency (EC50-SEAP) with both fuse numbers and the associated IL-18 variant designations is shown beneath the non-linear x-y graph.

[0029] FIG. 15 depicts biophysical characterization of various IL-18 variants. FIG. 15 A is a non-linear x-y graph of SEAP release (IL- 18 activity) relative to the concentration of test article. Human recombinant wild type IL-18 was used as the positive control (black squares) and human recombinant TNF-alpha as the negative control (grey square). All test articles are annotated with symbols that are indicated within the legend. Melting curves for IL18mutl3AV, IL18mutl3.1AV, IL18mutl3.2AV. IL18mutl3.3AV, and IL18mutI3.4AV are shown in FIGs 15B, 15C, 15D, 15E and 15F, respectively.DETAILED DESCRIPTION

[0030] All references cited herein are incorporated by reference in their entirety as though fully set forth. Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0031] One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, the present invention is in no way limited to the methods and materials described. For purposes of the present invention, the following terms are defined.

[0032] Unless stated otherwise, the terms “a’' and ‘‘an’" and “the" and similar references used in the context of describing a particular embodiment of the application (especially in the context of claims) may be constmed to cover both the singular and the plural. The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein may be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context.The use of any and all examples, or exemplary language (for example, “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the application and does not pose a limitation on the scope of the application otherwise claimed. The abbreviation, “e.g.” is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation “e.g.” is synonymous with the term “for example.” No language in the specification should be construed as indicating any nonclaimed element essential to the practice of the application.

[0033] As used herein the term “about” when used in connection with a referenced numeric indication means the referenced numeric indication plus or minus up to 5% of that referenced numeric indication, unless otherwise specifically provided for herein. For example, the language “about 50%” covers the range of 45% to 55%. In various embodiments, the term “about” when used in connection with a referenced numeric indication can mean the referenced numeric indication plus or minus up to 4%, 3%, 2%, 1%, 0.5%, or 0.25% of that referenced numeric indication, if specifically provided for in the claims.

[0034] As used herein, the term “cis” in the context of interactions refers to the interaction between two molecules on the same cell, such as proteins expressed on the same cell. A nonlimiting example is the interaction between B7-1 and PD-L1. “Cis” interaction as used herein also includes multispecific proteins that bind two proteins on the same cell simultaneously.

[0035] As used herein, the term “gene expression” or “expression” of a fusion protein, is understood to mean the transcription of a DNA sequence, translation of the mRNA transcript, and secretion of a fusion protein product. In some embodiments, the expression process also includes or is followed by purification; for example, protein A affinity chromatography or other means such as size exclusion chromatography can be used for purification.

[0036] As used herein, “IL-18 fusion protein” refers to a fusion protein that includes wild-type IL- 18 or IL- 18 variants, unless specifically noted as only including the wild-type IL- 18, or only including the IL-18 variant. Thus, in particular embodiments, the “IL-18 fusion protein” only includes any one of the IL- 18 variants as described herein.

[0037] As used herein, the term “IL-18 variant” refers to IL-18 polypeptide that differs from that of mature wild type IL-18 by virtue of at least one modification. Nonlimiting examples of modifications include substitution, deletion, or insertion of one or more amino acid residues when compared to wild type mature IL- 18 polypeptide.

[0038] As used herein, the term “immunoglobulin heavy chain constant region” is used interchangeably with the term “Fc region” and is understood to mean the carboxyl- terminal portion of an immunoglobulin heavy chain constant region, or an analog or portion thereof capable of binding an Fc receptor. Each immunoglobulin heavy chain constant region comprises four or five domains. The domains are named sequentially as follows: CHl-hinge- CH2-CH3(-CH4). CH4 is present in IgM, which has no hinge region. In some embodiments, the immunoglobulin heavy chain constant region comprises an immunoglobulin hinge region, a CH2 domain and a CH3 domain.

[0039] As used herein, the term “immunoglobulin hinge region” is understood to mean an entire immunoglobulin hinge region or at least a portion of the immunoglobulin hinge region sufficient to form one or more disulfide bonds with a second immunoglobulin hinge region.

[0040] Percent (%) sequence identity with respect to a reference polypeptide sequence is the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary , to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are known for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Appropriate parameters for aligning sequences are able to be determined, including algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0041] As used herein, the term “subject” includes any human or non-human animal. “Non-human animal” includes all vertebrates, e.g., mammals and non-mammals. The term “mammal” as used herein, encompasses any mammal. Examples of mammals include, but are not limited to, non-human primates, cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys, humans, etc. The terms “subject” and “patient” can be used interchangeably herein. In some embodiments, the subject or patient is human.

[0042] As used herein, “Treat,” “treating,” or “treatment” of a disease or disorder such as cancer refers to accomplishing one or more of the following: reducing the severity and / or duration of the disorder, delaying the progression of the disorder, slowing the progression of the disorder, inhibiting worsening of symptoms characteristic of the disorder being treated, limiting or preventing recurrence of the disorder in subjects that have previouslyhad the disorder, or limiting or preventing recurrence of symptoms in subjects that were previously symptomatic for the disorder. As used herein, the terms “delaying the progression of’ or “slowing the progression of’ shall include (a) delaying or slowing the development of one or more symptoms or complications of the disease, condition or disorder; (b) delaying or slowing the development of one or more new / additional symptoms or complications of the disease, condition or disorder; and / or (c) delaying or slowing the progression of the disease, condition or disorder to a later stage or more serious form of said disease, condition or disorder.

[0043] As used herein, the term “vector” is understood to mean any nucleic acid comprising a nucleotide sequence competent to be incorporated into a host cell and to be recombined with and integrated into the host cell genome, or to replicate autonomously as an episome. Such vectors include linear nucleic acids, plasmids, phagemids, cosmids, RNA vectors, viral vectors and the like. Non-limiting examples of a viral vector include a retrovirus, an adenovirus and an adeno-associated virus.

[0044] This disclosure relates to the compositions of IL-18 variants and methods of use. In some embodiments, the disclosure comprises an IL- 18 variant that is mutated relative to wild type IL- 18 such that it can bind to one of IL- 18 natural binding partners, but not to the other (or has reduced binding to the other). For example, wild type IL- 18 binds to both IL-18R (to signal through the receptor) and IL- 18 binding protein (IL-18BP) which inhibits IL- 18 by preventing it from binding to IL-18R. In some embodiments, an IL- 18 variant disclosed herein binds to IL-18R but have reduced binding to (and in some cases do not bind to) IL-18BP. In some embodiments, the IL- 18 variants may further comprise short polypeptides at the N- terminus, called attenuator. The attenuator moiety may mask or inhibit the interaction of IL- 18 variant with its receptor IL-18R, thus regulating the IL- 18 variant function.

[0045] Also disclosed herein are IL- 18 fusion proteins. The fusion protein comprises wild ty pe or variant IL- 18 and an immunoglobulin or a fragment thereof. In some embodiments, the fusion protein comprises wild type or variant IL- 18 and an antibody that is targeted to proteins expressed on immune cells (e g., NK. cells, T cells, neutrophils, mast cells), tumor cells and / or cells in the tumor microenvironment.

[0046] As stated above, the IL- 18 variant may display reduced binding affinity' for its natural antagonist, IL-18BP, while maintaining the binding affinity to its receptor IL-18R. The activity of the IL- 18 variant may be attenuated or masked by the addition of attenuator moiety to the N-terminus of IL-18 variant. Without washing to be bound by theory, the attenuatormoiety may sterically inhibit binding of IL- 18 variant to its receptor 1L-18R. However, when the IL-18 variant with attenuator (attenuated IL-18 variant) is covalently linked to an antibody and delivered to a cell as a fusion protein, binding of the antibody to the cell surface results in positioning the attenuated IL-18 variant close to its receptor IL-18R and this close proximityincreases residence time for attenuated IL- 18 variant to bind to IL-18R and activate the IL- 18R in cis. Thus, one can control the activity of IL- 18 and deliver it to the cell of interest.

[0047] Disclosed herein are various IL-18 variants that display reduced binding affinity- for IL-18BP, while maintaining the binding affinity- to its receptor IL-18R.

[0048] In some embodiments. IL- 18 variant comprises an amino acid sequence as shown below:wherein X1is Y or C;

[0049] In some embodiments. IL- 18 variant comprises an amino acid sequence of SEQ ID NO: 1, wherein

[0050] In some embodiments, the IL- 18 variant is selected from one of the polypeptides of Table 1.Table 1: IL- 18 variants

[0051] In some embodiments, the IL-18 variant is selected from the group consisting

[0052] In some embodiments, the IL-18 variants disclosed herein further comprises “linker” or “attenuator” moiety which are about one amino acid to about 70 amino acids in length, attached to the N-terminus of the IL- 18 variant. The term “linker” or “attenuator” is used interchangeably. Examples of attenuator moieties include but are not limited to a single ammo acid, a dimer of two amino acids, a tri-mer of three amino acids, a 4-mer of four amino acids, a 5-mer of five amino acids, or an amino acid or a peptide selected from the group consisting of T, PT, MPT, S, GS, GGS, GGGS (SEQ ID NO: 184), and (GGGGXλ)n (SEQ ID NO: 185) wherein Xλis Q, A, E or S and n=l-5 or an integer larger than 5. In some embodiments, the attenuator moiety has the amino acid sequence of (GGGGS)n (SEQ ID NO: 32) where n is an integer between 1 and 5, thereby a polypeptide of 25 amino acids or shorter in length. Additional examples include (XλGGGG)n (SEQ ID NO: 186) wherein Xλis Q, A, E or S and n=l-5 or in some embodiments, an integer larger than 5, (GXλGGG)n (SEQ ID NO: 187) wherein Xλis Q. A, E or S and n=l-5 or in some embodiments, an integer larger than 5, (GGXλGG)n (SEQ ID NO: 188) wherein Xλis Q, A, E or S and n=l-5 or in some embodiments, an integer larger than 5, (GGGXλG)n (SEQ ID NO: 189) wherein Xλis Q, A, E or S and n=l-5 or in some embodiments, an integer larger than 5. Still additional examples include (XλGGG)n wherein Xλis Q, A, E or S and n=l-5 or in some embodiments, an integer larger than 5, (GXλGG)n wherein Xλis Q, A. E or S and n=l-5 or in some embodiments, an integer larger than 5, (GGXλG)n wherein Xλis Q, A, E or S and n=l-5 or in some embodiments, an integer larger than 5, (GGGXλ)n wherein Xλis Q, A, E or S and n=l-5 or in some embodiments, an integer larger than 5. Still additional examples include (XλGG)n wherein Xλis Q, A. E or S and n=l-5 or in some embodiments, an integer larger than 5. (GXλG)n wherein Xλis Q, A, E or S and n=l-5 or in some embodiments, an integer larger than 5, (GGXλ)n wherein Xλis Q, A, E or S and n=l-5 or in some embodiments. Still additional examples include (XλG)n wherein Xλis Q, A, E or S and n=l-5 or in some embodiments, an integer larger than 5, (GXλ)n wherein Xλis Q, A, E or S and n=l-5 or in some embodiments, an integer larger than 5. In some embodiments, the attenuator moiety is an IL- 18 propeptide or IL- 18 propeptide variant. In some embodiments, the attenuator moiety is afragment of an IL-18 propeptide or IL-18 propeptide variant; for example, about 30-36 amino acids in length, about 5-10, 11-20, 21-30, or 31-40 amino acids in length. As further examples, the attenuator moiety can be of 2, 3, or 4 amino acids in length, or flexible linkers or amino acid chain of 2-5, 6-10, 11-15, 16-20. 21-25, 26-30 amino acids. As further examples, the attenuator moiety is 1, 2, 3, or 4 amino acids, or flexible linkers or amino acid chain of 5, 6. 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, or 35 amino acids. As further examples, the attenuator moiety is 1-14 amino acids in length, 2-14 amino acids in length, 3-14 amino acids in length, 4-14 amino acids in length, 5-14 amino acids in length, 6-14 amino acids in length, 7-14 amino acids in length, 8-14 amino acids in length. 9-14 amino acids in length, 10- 14 amino acids in length, 11-14 amino acids in length, 12-14 amino acids in length, or 13-14 amino acids in length.

[0053] Nonlimiting examples of attenuator moiety sequences are described in Table 2 as shown below:Table 2: Attenuator Moiety Sequences

[0054] In various embodiments, the attenuator moiety is selected from the group consisting of S, GGGGS (SEQ ID NO: 32), EV, DG, LM, THKM (SEQ ID NO: 36), DWNN (SEQ ID NO: 37), NTHR (SEQ ID NO: 38), TVDWIT (SEQ ID NO: 39), KHVLFF (SEQ IDNO: 40), ASRHVQ (SEQ ID NO: 41), LSSKDKVD (SEQ ID NO: 42). GKMNLSWY (SEQ ID NO: 43), HMWQQMYN (SEQ ID NO: 44), EIVHAIIFHK (SEQ ID NO: 45), LERIQTRYIQ (SEQ ID NO: 46), NRSKMMSMIR (SEQ ID NO: 48), MHRNWVAKHGHL (SEQ ID NO: 49), HHNKWKHLDFSH (SEQ ID NO: 50), DVVQSSESNQEW (SEQ ID NO: 51), NFSLERHMNNRMYE (SEQ ID NO: 52), WYKWFFYSRMHSIL (SEQ ID NO: 53), WFRRHGTKHGQFVI (SEQ ID NO: 54), and AAEPVEDNVINFVAMKFIDNTLYFIAENDENGGGS (SEQ ID NO: 61).

[0055] These atenuator moieties contain representative residues and different combinations of amino acids with different atributes such as being positively charged, negatively charged, polar, non-polar, polar uncharged, hydrophobic, hydrophilic, small and flexible, rigid with a cyclic structure, having an aromatic ring, and having alkyl side chains or combinations thereof. These different combinations are fully representative to show that any peptide ranging from about 0.75 kDa to 250 kDa added to the N-terminus of mature IL-18 would act as an atenuator. In various embodiments, the atenuator moiety ranges from about 1 10 Da to 250 kDa. In various embodiments, the attenuator moiety ranges from about 1 kDa to 50 kDa, about 51 kDa to 100 kDa, 101 kDa to 150 kDa, 151 kDa to 200 kDa, 201 kDa to 250 kDa.

[0056] The amino acids in the atenuator moiety can comprise any natural amino acid or non-natural amino acids. Examples of non-natural amino acids include but are not limited to pyrrolysine, beta-amino acids, gamma-amino acids, homo amino acids, beta-homo- amino acids, N-methyl amino acids, alpha-methyl amino acids, D-amino acids, hydroxyproline (Hyp), beta-alanine, citrulline (Cit), ornithine (Om), norleucine (Nle). 3- mtrotyrosine, nitroarginine, pyroglutamic acid (Pyr). and naphtylalanine (Nal).

[0057] In some embodiments, the IL- 18 variant comprises the amino acid sequence of SEQ ID NO: 2 and further comprises any one of the atenuator moieties of Table 2 at the N- terminus. In some embodiments, the atenuator moiety is selected from of S, GGGGS (SEQ ID NO: 32). EV, DG. LM, THKM (SEQ ID NO: 36), DWNN (SEQ ID NO: 37), NTHR (SEQ ID NO: 38), TVDWIT (SEQ ID NO: 39), KHVLFF (SEQ ID NO: 40), ASRHVQ (SEQ ID NO: 41), LSSKDKVD (SEQ ID NO: 42), GKMNLSWY (SEQ ID NO: 43), HMWQQMYN (SEQ ID NO: 44), EIVHAIIFHK (SEQ ID NO: 45), LERIQTRYIQ (SEQ ID NO: 46), NRSKMMSMIR (SEQ ID NO: 48), MHRNWVAKHGHL (SEQ ID NO: 49). HHNKWKHLDFSH (SEQ ID NO: 50), DVVQSSESNQEW (SEQ ID NO: 51), NFSLERHMNNRMYE (SEQ ID NO: 52), WYKWFFYSRMHSIL (SEQ ID NO: 53),WFRRHGTKHGQFVI (SEQ ID NO: 54), and AAEPVEDNVINFVAMKFIDNTLYFIAENDENGGGS (SEQ ID NO: 61).

[0058] In some embodiments, the IL-18 variant comprises the amino acid sequence of SEQ ID NO: 9 and further comprises any one of the attenuator moieties of Table 2 at the N- terminus. In some embodiments, the attenuator moiety is selected from of S, GGGGS (SEQ ID NO: 32), EV, DG, LM, THKM (SEQ ID NO: 36), DWNN (SEQ ID NO: 37), NTHR (SEQ ID NO: 38), TVDWIT (SEQ ID NO: 39), KHVLFF (SEQ ID NO: 40), ASRHVQ (SEQ ID NO: 41), LSSKDKVD (SEQ ID NO: 42), GKMNLSWY (SEQ ID NO: 43), HMWQQMYN (SEQ ID NO: 44), EIVHAIIFHK (SEQ ID NO: 45), LERIQTRYIQ (SEQ ID NO: 46), NRSKMMSMIR (SEQ ID NO: 48), MHRNWVAKHGHL (SEQ ID NO: 49), HHNKWKHLDFSH (SEQ ID NO: 50), DVVQSSESNQEW (SEQ ID NO: 51), NFSLERHMNNRMYE (SEQ ID NO: 52), WYKWFFYSRMHSIL (SEQ ID NO: 53), WFRRHGTKHGQFVI (SEQ ID NO: 54), and AAEPVEDNVINFVAMKFIDNTLYFIAENDENGGGS (SEQ ID NO: 61).

[0059] In some embodiments, the IL-18 variant comprises the amino acid sequence of SEQ ID NO: 15 and further comprises any one of the attenuator moieties of Table 2 at the N-terminus. In some embodiments, the attenuator moiety is selected from of S, GGGGS (SEQ ID NO: 32). EV, DG, LM, THKM (SEQ ID NO: 36), DWNN (SEQ ID NO: 37), NTHR (SEQ ID NO: 38), TVDWIT (SEQ ID NO: 39), KHVLFF (SEQ ID NO: 40), ASRHVQ (SEQ ID NO: 41), LSSKDKVD (SEQ ID NO: 42), GKMNLSWY (SEQ ID NO: 43), HMWQQMYN (SEQ ID NO: 44), EIVHAIIFHK (SEQ ID NO: 45), LERIQTRYIQ (SEQ ID NO: 46), NRSKMMSMIR (SEQ ID NO: 48), MHRNWVAKHGHL (SEQ ID NO: 49). HHNKWKHLDFSH (SEQ ID NO: 50), DVVQSSESNQEW (SEQ ID NO: 51). NFSLERHMNNRMYE (SEQ ID NO: 52), WYKWFFYSRMHSIL (SEQ ID NO: 53), WFRRHGTKHGQFVI (SEQ ID NO: 54), and AAEPVEDNVINFVAMKFIDNTLYFIAENDENGGGS (SEQ ID NO: 61).

[0060] In some embodiments, the IL-18 variant comprises the amino acid sequence of SEQ ID NO: 16 and further comprises any one of the attenuator moieties of Table 2 at the N-terminus. In some embodiments, the attenuator moiety is selected from of S, GGGGS (SEQ ID NO: 32), EV, DG, LM, THKM (SEQ ID NO: 36), DWNN (SEQ ID NO: 37), NTHR (SEQ ID NO: 38). TVDWIT (SEQ ID NO: 39), KHVLFF (SEQ ID NO: 40), ASRHVQ (SEQ ID NO: 41), LSSKDKVD (SEQ ID NO: 42), GKMNLSWY (SEQ ID NO: 43), HMWQQMYN (SEQ ID NO: 44), EIVHAIIFHK (SEQ ID NO: 45), LERIQTRYIQ (SEQ ID NO: 46),NRSKMMSM1R (SEQ ID NO: 48), MHRNWVAKHGHL (SEQ ID NO: 49). HHNKWKHLDFSH (SEQ ID NO: 50), DVVQSSESNQEW (SEQ ID NO: 51), NFSLERHMNNRMYE (SEQ ID NO: 52), WYKWFFYSRMHSIL (SEQ ID NO: 53), WFRRHGTKHGQFVI (SEQ ID NO: 54), and AAEPVEDNVINFVAMKFIDNTLYFIAENDENGGGS (SEQ ID NO: 61).

[0061] In some embodiments, the IL-18 variant comprises the amino acid sequence of SEQ ID NO: 19 and further comprises any one of the attenuator moieties of Table 2 at the N-terminus. In some embodiments, the attenuator moiety is selected from of S, GGGGS (SEQ ID NO: 32). EV, DG, LM, THKM (SEQ ID NO: 36), DWNN (SEQ ID NO: 37), NTHR (SEQ ID NO: 38), TVDWIT (SEQ ID NO: 39), KHVLFF (SEQ ID NO: 40), ASRHVQ (SEQ ID NO: 41), LSSKDKVD (SEQ ID NO: 42), GKMNLSWY (SEQ ID NO: 43), HMWQQMYN (SEQ ID NO: 44), EIVHAIIFHK (SEQ ID NO: 45), LERIQTRYIQ (SEQ ID NO: 46), NRSKMMSMIR (SEQ ID NO: 48), MHRNWVAKHGHL (SEQ ID NO: 49), HHNKWKHLDFSH (SEQ ID NO: 50), DVVQSSESNQEW (SEQ ID NO: 51). NFSLERHMNNRMYE (SEQ ID NO: 52), WYKWFFYSRMHSIL (SEQ ID NO: 53), WFRRHGTKHGQFVI (SEQ ID NO: 54), and AAEPVEDNVINFVAMKFIDNTLYFIAENDENGGGS (SEQ ID NO: 61).

[0062] In some embodiments, the IL-18 variant comprises the amino acid sequence of SEQ ID NO: 29 and further comprises any one of the attenuator moieties of Table 2 at the N-terminus. In some embodiments, the attenuator moiety is selected from of S, GGGGS (SEQ ID NO: 32), EV, DG, LM, THKM (SEQ ID NO: 36), DWNN (SEQ ID NO: 37), NTHR (SEQ ID NO: 38). TVDWIT (SEQ ID NO: 39), KHVLFF (SEQ ID NO: 40), ASRHVQ (SEQ ID NO: 41), LSSKDKVD (SEQ ID NO: 42), GKMNLSWY (SEQ ID NO: 43). HMWQQMYN (SEQ ID NO: 44), EIVHAIIFHK (SEQ ID NO: 45), LERIQTRYIQ (SEQ ID NO: 46), NRSKMMSMIR (SEQ ID NO: 48), MHRNWVAKHGHL (SEQ ID NO: 49), HHNKWKHLDFSH (SEQ ID NO: 50), DVVQSSESNQEW (SEQ ID NO: 51). NFSLERHMNNRMYE (SEQ ID NO: 52). WYKWFFYSRMHSIL (SEQ ID NO: 53), WFRRHGTKHGQFVI (SEQ ID NO: 54), and AAEPVEDNVINFVAMKFIDNTLYFIAENDENGGGS (SEQ ID NO: 61).

[0063] In some embodiments, the IL-18 variant comprises the amino acid sequence of SEQ ID NO: 30 and further comprises any one of the attenuator moieties of Table 2 at the N-terminus. In some embodiments, the attenuator moiety is selected from of S, GGGGS (SEQ ID NO: 32), EV, DG, LM, THKM (SEQ ID NO: 36), DWNN (SEQ ID NO: 37), NTHR (SEQID NO: 38). TVDWIT (SEQ ID NO: 39), KHVLFF (SEQ ID NO: 40), ASRHVQ (SEQ ID NO: 41), LSSKDKVD (SEQ ID NO: 42), GKMNLSWY (SEQ ID NO: 43), HMWQQMYN (SEQ ID NO: 44), EIVHAIIFHK (SEQ ID NO: 45), LERIQTRYIQ (SEQ ID NO: 46), NRSKMMSMIR (SEQ ID NO: 48), MHRNWVAKHGHL (SEQ ID NO: 49), HHNKWKHLDFSH (SEQ ID NO: 50), DVVQSSESNQEW (SEQ ID NO: 51). NFSLERHMNNRMYE (SEQ ID NO: 52), WYKWFFYSRMHSIL (SEQ ID NO: 53), WFRRHGTKHGQFVI (SEQ ID NO: 54), and AAEPVEDNVINFVAMKFIDNTLYFIAENDENGGGS (SEQ ID NO: 61).

[0064] In some embodiments, the IL-18 variant comprises the amino acid sequence of SEQ ID NO: 31 and further comprises any one of the attenuator moieties of Table 2 at the N-terminus. In some embodiments, the attenuator moiety is selected from of S, GGGGS (SEQ ID NO: 32), EV, DG, LM, THKM (SEQ ID NO: 36), DWNN (SEQ ID NO: 37), NTHR (SEQ ID NO: 38). TVDWIT (SEQ ID NO: 39), KHVLFF (SEQ ID NO: 40), ASRHVQ (SEQ ID NO: 41), LSSKDKVD (SEQ ID NO: 42), GKMNLSWY (SEQ ID NO: 43). HMWQQMYN (SEQ ID NO: 44), EIVHAIIFHK (SEQ ID NO: 45), LERIQTRYIQ (SEQ ID NO: 46), NRSKMMSMIR (SEQ ID NO: 48), MHRNWVAKHGHL (SEQ ID NO: 49), HHNKWKHLDFSH (SEQ ID NO: 50), DVVQSSESNQEW (SEQ ID NO: 51), NFSLERHMNNRMYE (SEQ ID NO: 52). WYKWFFYSRMHSIL (SEQ ID NO: 53), WFRRHGTKHGQFVI (SEQ ID NO: 54), and AAEPVEDNVINFVAMKFIDNTLYFIAENDENGGGS (SEQ ID NO: 61).

[0065] In some embodiments, IL- 18 variant comprises a protease cleavage site between the attenuator moiety and the N-terminus of the IL-18 variant. The protease cleavage site may be a cleavage site for a serine protease, a cysteine protease, an aspartate protease, a threonine protease, a glutamic acid protease, a metalloproteinase (ADAM), a gelatinase, or an asparagine peptide lyase. In some embodiments, the protease cleavage site is recognized by granzyme B, granzyme A, granzyme M, granzyme K, cathepsin B, cathepsin C, cathepsin D, cathepsin E. cathepsin K, cathepsin L, cathepsin G, kallikrein, plasmin, a collagenase, type IV collagenase, a stromelysin, Factor Xa, a chymotrypsin-like protease, a trypsin-like protease, an elastase-like protease, a subtilisin like protease, bromelain, a calpain, a caspase, papain, a HIV-1 protease, aHSV protease, a CMV protease, a chymosin, renin, pepsin, a matrix metalloprotease (MMP), a MMP1, a MMP2, a MMP3. a MMP8, a MMP9, a MMP10, a MMP11, a MMP12, a MMP13, a MMP 14, an ADAM 10, an ADAM17, an ADAM12, an urokinase plasminogen activator (uPA), an enterokinase, a prostate-specific target (PSA,hK3), thrombin, a dipeptidyl peptidase, a type II transmembrane serine protease (T1SP), a neutrophil elastase, a mast cell chymase, a mast cell tryptase, a dipeptidyl peptidase, or a dipeptidyl peptidase IV (DPPIV / CD26).

[0066] Also disclosed herein are isolated nucleic acids encoding any one of the IL-18 variants described herein, vectors (e.g.. lentiviral vector) comprising such nucleic acids, and host cells (e.g., CHO cell) comprising such nucleic acids or vectors. The host cell can be a prokaryotic cell, a eukaryotic cell, a mammalian cell, or a human cell. Examples of mammalian cells include Chinese hamster ovary (CHO) cells, NSO cells (a mouse myeloma cell line), PER.C6® cells, and human embryonic kidney cells (HEK cells).IL-18 Fusion Proteins

[0067] Also disclosed herein are IL-18 fusion proteins wherein the IL-18 variants disclosed herein are covalently linked to another protein. In some embodiments, the protein may be scaffold polypeptide. In some embodiments, a fusion protein comprises an IL-18 variant and a scaffold polypeptide, wherein the IL- 18 variant comprises the amino acid sequence of SEQ ID NO: 1, whereinX1is Y or C;X18is N or C.

[0068] In some embodiments, the fusion protein comprises an IL- 18 variant comprising an amino acid sequence of SEQ ID NO: 1, whereinX1is Y or C;

[0069] In some embodiments, the fusion protein comprises an IL-18 variant wherein the IL-18 variant comprises the amino acid sequence selected from SEQ ID NOs 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12. 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28. 29, 30, 31, 242, 243. 244, 245, 246, 247, 248. or 183.

[0070] In some embodiments, the fusion protein comprises a scaffold polypeptide. Nonlimiting examples of scaffold polypeptide include an immunoglobulin or a fragment thereof, Fc region of an immunoglobulin or a fragment thereof, human serum albumin (HSA), beta2microglobulin, transferrin, fragment antigen-binding region (Fab region), VHH antibody, single-chain variable fragment (scFv), anticalin, designed ank rin repeat protein (DARPin), or a binding domain thereof, or a fragment thereof, type I transmembrane proteins or a fragmentthereof, or type II transmembrane proteins or a fragment thereof. In some embodiments, the scaffold polypeptide is an immunoglobulin or a fragment thereof or a Fc region of an immunoglobulin or a fragment thereof. In some embodiments, the Fc region is an Fc region from an IgG4. knob-in-hole (KiH) Fc, or IgGl.

[0071] In some embodiments, the scaffold polypeptide is an immunoglobulin comprising a Fc region and an antigen binding domain, such as a VH, a VL, a VH and a VL, a Fab, a Fab’, a F(ab')2 , a Fd and a Fv fragments, a disulfide stabilized Fv fragment (dsFv), a (dsFv)2. a bispecific dsFv (dsFv-dsFv'), a disulfide stabilized diabody (ds diabody), a singlechain antibody molecule (scFv), or a disulfide stabilized single-chain antibody molecule (spFv or stapled scFv), a single domain antibody (sdab) an scFv dimer (bivalent diabody), a multispecific antibody formed from a portion of an antibody comprising one or more CDRs, a camelized single domain antibody, a nanobody, a domain antibody, a domain antibody (dAb) consisting of one VH domain or one VL domain, a shark variable IgNAR domain, a camelized VH domain, or a VHH domain. In some embodiments, the scaffold polypeptide may comprise the antigen binding domain without Fc region.

[0072] In some embodiments, the fusion protein further comprises an attenuator moiety at the N-terminus of the IL- 18 variant, wherein the attenuator moiety comprises from about one amino acid to about 70 amino acids in length. In some embodiments, the attenuator moiety may be any one of the attenuator sequences of Table 2. In some embodiments, the attenuator moiety is selected from of S, GGGGS (SEQ ID NO: 32), EV, DG, LM, THKM (SEQ ID NO: 36), DWNN (SEQ ID NO: 37), NTHR (SEQ ID NO: 38), TVDWIT (SEQ ID NO: 39), KHVLFF (SEQ ID NO: 40), ASRHVQ (SEQ ID NO: 41), LSSKDKVD (SEQ ID NO: 42), GKMNLSWY (SEQ ID NO: 43). HMWQQMYN (SEQ ID NO: 44), E1VHA1IFHK (SEQ ID NO: 45), LERIQTRYIQ (SEQ ID NO: 46), NRSKMMSMIR (SEQ ID NO: 48), MHRNWVAKHGHL (SEQ ID NO: 49), HHNKWKHLDFSH (SEQ ID NO: 50), DVVQSSESNQEW (SEQ ID NO: 51). NFSLERHMNNRMYE (SEQ ID NO: 52), WYKWFFYSRMHSIL (SEQ ID NO: 53), WFRRHGTKHGQFVI (SEQ ID NO: 54). and AAEPVEDNVINFVAMKFIDNTLYFIAENDENGGGS (SEQ ID NO: 61).

[0073] In some embodiments, the fusion protein further comprises a protease cleavage site between the attenuator moiety and the N-terminus of the IL- 18 variant. Nonlimiting examples of protease cleavage sites include cleavage sites recognized by granzyme B, granzyme A, granz me M, granzyme K, cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin K, cathepsin L, cathepsin G, kallikrein, plasmin, a collagenase, type IVcollagenase. a stromelysin, Factor Xa, a chymotrypsin-like protease, a trypsin-like protease, an elastase-like protease, a subtilisin like protease, bromelain, a calpain, a caspase, papain, a HIV-1 protease, a HSV protease, a CMV protease, a chymosin, renin, pepsin, a matrix metalloprotease (MMP), or a metalloproteinase (ADAM).Fusion Proteins Comprising IL-18 Variants and Anti-PD-1 Antibodies

[0074] Disclosed herein are various IL-18 fusion proteins that are covalently linked to antibodies. In some embodiments, the antibody is an anti-PDl antibody (or a fragment thereof) or an anti-PD-Ll antibody (or a fragment thereof). Nonlimiting examples of anti-PDl antibody include cetrelimab, pembrolizumab, nivolumab, pidilizumab, AMP -224, AMP-514, spartalizumab, cemiplimab, penpulimab (AK105), prolgolimab (BCD- 100), ezabenlimab (BI 754091), toripalimab (JS001), lipustobart (LZM009), retifanlimab (MGA012), Sym021, dostarlimab (TSR-042). tebotelimab (MGD013), cadonilimab (AK104), vudalimab (XmAb20717), tislelizumab, and PF-06801591. Examples of anti-PD-Ll antibodies include garivulimab (BGB-A333), cosibelimab (CK-301), FAZ053, envafolimab (KN035), MDX- 1105, betifisolimab (MSB2311), adebrelimab (SHR-1316), atezolizumab, avelumab, durvalumab, BMS-936559, CK-301, and M7824.

[0075] In some embodiments, a fusion protein comprises an IL- 18 variant and an anti-PDl antibody or a fragment thereof, wherein the IL- 18 variant comprises the amino acid sequence of:wherein X1is Y or C;

[0076] In some embodiments, the fusion protein comprises an IL-18 variant and an anti-PDl antibody or a fragment thereof, wherein the IL-18 variant comprises the amino acid sequence of SEQ ID NO: 1, whereinX1is Y or C;

[0077] In some embodiments, the fusion protein comprises an IL- 18 variant and an anti-PDl antibody or a fragment thereof wherein the IL- 18 variant comprises the amino acidsequence selected from SEQ ID NOs 2, 3, 4. 5. 6, 7, 8. 9, 10, 11. 12. 13. 14. 15. 16. 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 242, 243, 244, 245, 246, 247, 248, or 183.

[0078] In some embodiments, the anti-PDl antibody is cetrelimab. Cetrelimab is a fully human immunoglobulin G4 (IgG4) kappa monoclonal antibody (mAb) containing the hinge-stabilizing S228P mutation. Cetrelimab binds to PD-1 with high affinity and specificity, blocks binding to the programmed-cell death ligands 1 and 2 (PD-L1 and PD-L2), enhances pro-inflammatory cytokine production from ex-vivo stimulated T cells, and reduces tumor volume in human PD-1 knock-in (hPD-lKI) mice bearing MC38 murine colon carcinoma tumors. Cetrelimab is being investigated in clinical studies for the treatment of bladder cancer, prostate cancer, melanoma, and multiple myeloma as part of a combination treatment.

[0079] In some embodiments, the anti-PDl antibody or a fragment thereof comprises the CDRs and VH / VL sequences of cetrelimab as shown in Table 3. Further embodiments of anti-PDl antibody are disclosed in U.S. Patent No. 10,894,830 which is incorporated herein by reference in its entirety.Table 3: CDRs and VH / VL Sequences of cetrelimab

[0080] In some embodiments, the anti-PDl antibody or a fragment thereof comprises a variable heavy chain (VH) comprising an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 114, and a variable light chain (VL) comprising an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 115.

[0081] In some embodiments, the fusion protein comprises an IL-18 variant and an anti-PDl antibody or a fragment thereof, wherein the IL-18 variant comprises an amino acid sequence selected from SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23. 24. 25. 26, 27, 28, 29, 30, 31, 242, 243, 244, 245, 246, 247. 248, or 183, and wherein the anti-PDl antibody comprises a variable heavy chain (VH) comprising an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 114, and a variable light chain (VL) comprising an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 115.

[0082] In some embodiments, the IL-18 variant comprises an amino acid sequence of SEQ ID NO: 2, and the anti-PDl antibody or a fragment thereof comprises a variable heavy chain (VH) comprising an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 114, and a variable light chain (VL) comprising an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 115.

[0083] In some embodiments, the IL-18 variant comprises an amino acid sequence of SEQ ID NO: 3, and the anti-PDl antibody or a fragment thereof comprises a variable heavychain (VH) comprising an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 114, and a variable light chain (VL) comprising an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 115.

[0084] In some embodiments, the IL-18 variant comprises an amino acid sequence of SEQ ID NO: 4, and the anti-PDl antibody or a fragment thereof comprises a variable heavychain (VH) comprising an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 114, and a variable light chain (VL) comprising an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 115.

[0085] In some embodiments, the IL- 18 variant comprises an amino acid sequence of SEQ ID NO: 5, and the anti-PDl antibody or a fragment thereof comprises a variable heavy chain (VH) comprising an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 114, and a variable light chain (VL) comprising an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 115.

[0086] In some embodiments, the IL-18 variant comprises an amino acid sequence of SEQ ID NO: 6, and the anti-PDl antibody or a fragment thereof comprises a variable heavy chain (VH) comprising an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 114, and a variable light chain (VL) comprising an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 115.

[0087] In some embodiments, the IL-18 variant comprises an amino acid sequence of SEQ ID NO: 7, and the anti-PDl antibody or a fragment thereof comprises a variable heavychain (VH) comprising an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 114, and a variable light chain (VL) comprising an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 115.

[0088] In some embodiments, the IL-18 variant comprises an amino acid sequence of SEQ ID NO: 8, and the anti-PDl antibody or a fragment thereof comprises a variable heavychain (VH) comprising an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 114, and a variable light chain (VL) comprising an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 115.

[0089] In some embodiments, the IL-18 variant comprises an amino acid sequence of SEQ ID NO: 9, and the anti-PDl antibody or a fragment thereof comprises a variable heavychain (VH) comprising an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 114, and a variable light chain (VL) comprising an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 115.

[0090] In some embodiments, the IL-18 variant comprises an amino acid sequence of SEQ ID NO: 10, and the anti-PDl antibody or a fragment thereof comprises a variable heavy chain (VH) comprising an amino acid sequence that is at least 90%, at least 95%, at least 98%,at least 99%, or 100% identical to SEQ ID NO: 114. and a variable light chain (VL) comprising an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 115.

[0091] In some embodiments, the IL-18 variant comprises an amino acid sequence of SEQ ID NO: 11, and the anti-PDl antibody or a fragment thereof comprises a variable heavy chain (VH) comprising an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 114, and a variable light chain (VL) comprising an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 115.

[0092] In some embodiments, the IL-18 variant comprises an amino acid sequence of SEQ ID NO: 12, and the anti-PDl antibody or a fragment thereof comprises a variable heavy chain (VH) comprising an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 114, and a variable light chain (VL) comprising an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 115.

[0093] In some embodiments, the IL-18 variant comprises an amino acid sequence of SEQ ID NO: 13, and the anti-PDl antibody or a fragment thereof comprises a variable heavy chain (VH) comprising an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 114, and a variable light chain (VL) comprising an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 115.

[0094] In some embodiments, the IL-18 variant comprises an amino acid sequence of SEQ ID NO: 14, and the anti-PDl antibody or a fragment thereof comprises a variable heavy chain (VH) comprising an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 114, and a variable light chain (VL) comprising an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 115.

[0095] In some embodiments, the IL-18 variant comprises an amino acid sequence of SEQ ID NO: 15, and the anti-PDl antibody or a fragment thereof comprises a variable heavy chain (VH) comprising an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 114, and a variable light chain (VL) comprising an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 115.

[0096] In some embodiments, the IL- 18 variant comprises an amino acid sequence of SEQ ID NO: 16, and the anti-PDl antibody or a fragment thereof comprises a variable heavy chain (VH) comprising an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 114, and a variable light chain (VL) comprising an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 115.

[0097] In some embodiments, the IL-18 variant comprises an amino acid sequence of SEQ ID NO: 17, and the anti-PDl antibody or a fragment thereof comprises a variable heavy chain (VH) comprising an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 114, and a variable light chain (VL) comprising an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 115.

[0098] In some embodiments, the IL-18 variant comprises an amino acid sequence of SEQ ID NO: 18, and the anti-PDl antibody or a fragment thereof comprises a variable heavy chain (VH) comprising an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 114, and a variable light chain (VL) comprising an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 115.

[0099] In some embodiments, the IL-18 variant comprises an amino acid sequence of SEQ ID NO: 19, and the anti-PDl antibody or a fragment thereof comprises a variable heavy chain (VH) comprising an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 114, and a variable light chain (VL) comprising an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 115.

[0100] In some embodiments, the IL-18 variant comprises an amino acid sequence of SEQ ID NO: 20. and the anti-PDl antibody or a fragment thereof comprises a variable heavy chain (VH) comprising an amino acid sequence that is at least 90%, at least 95%. at least 98%, at least 99%, or 100% identical to SEQ ID NO: 1 14, and a variable light chain (VL) comprising an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 115.

[0101] In some embodiments, the IL- 18 variant comprises an amino acid sequence of SEQ ID NO: 21, and the anti-PDl antibody or a fragment thereof comprises a variable heavy chain (VH) comprising an amino acid sequence that is at least 90%, at least 95%, atleast 98%. at least 99%. or 100% identical to SEQ ID NO: 114, and a variable light chain (VL) comprising an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 115.

[0102] In some embodiments, the IL-18 variant comprises an amino acid sequence of SEQ ID NO: 22. and the anti-PDl antibody or a fragment thereof comprises a variable heavy chain (VH) comprising an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 114, and a variable light chain (VL) comprising an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 115.

[0103] In some embodiments, the IL-18 variant comprises an amino acid sequence of SEQ ID NO: 23, and the anti-PDl antibody or a fragment thereof comprises a variable heavy chain (VH) comprising an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%. or 100% identical to SEQ ID NO: 114, and a variable light chain (VL) comprising an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 115.

[0104] In some embodiments, the IL-18 variant comprises an amino acid sequence of SEQ ID NO: 24, and the anti-PDl antibody or a fragment thereof comprises a variable heavy chain (VH) comprising an amino acid sequence that is at least 90%, at least 95%. at least 98%, at least 99%, or 100% identical to SEQ ID NO: 1 14, and a variable light chain (VL) comprising an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 115.

[0105] In some embodiments, the IL- 18 variant comprises an amino acid sequence of SEQ ID NO: 25. and the anti-PDl antibody or a fragment thereof comprises a variable heavy chain (VH) comprising an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 114, and a variable light chain (VL) comprising an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 115.

[0106] In some embodiments, the IL- 18 variant comprises an amino acid sequence of SEQ ID NO: 26, and the anti-PDl antibody or a fragment thereof comprises a variable heavy chain (VH) comprising an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%. or 100% identical to SEQ ID NO: 114, and a variable light chain (VL) comprising an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 115.

[0107] In some embodiments, the IL-18 variant comprises an amino acid sequence of SEQ ID NO: 27, and the anti-PDl antibody or a fragment thereof comprises a variable heavy chain (VH) comprising an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%. or 100% identical to SEQ ID NO: 114, and a variable light chain (VL) comprising an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 115.

[0108] In some embodiments, the IL-18 variant comprises an amino acid sequence of SEQ ID NO: 28, and the anti-PDl antibody or a fragment thereof comprises a variable heavy chain (VH) comprising an amino acid sequence that is at least 90%, at least 95%. at least 98%, at least 99%, or 100% identical to SEQ ID NO: 114, and a variable light chain (VL) comprising an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 115.

[0109] In some embodiments, the IL- 18 variant comprises an amino acid sequence of SEQ ID NO: 29. and the anti-PDl antibody comprises a variable heavy chain (VH) comprising an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 114, and a variable light chain (VL) comprising an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 115.

[0110] In some embodiments, the IL-18 variant comprises an amino acid sequence of SEQ ID NO: 30, and the anti-PDl antibody or a fragment thereof comprises a variable heavy chain (VH) comprising an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%. or 100% identical to SEQ ID NO: 114, and a variable light chain (VL) comprising an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 115.

[0111] In some embodiments, the IL-18 variant comprises an amino acid sequence of SEQ ID NO: 31. and the anti-PDl antibody or a fragment thereof comprises a variable heavy chain (VH) comprising an amino acid sequence that is at least 90%, at least 95%. at least 98%, at least 99%, or 100% identical to SEQ ID NO: 1 14, and a variable light chain (VL) comprising an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 115.

[0112] In some embodiments, the IL- 18 variant comprises an amino acid sequence of SEQ ID NO: 242, and the anti-PDl antibody or a fragment thereof comprises a variable heavy chain (VH) comprising an amino acid sequence that is at least 90%, at least 95%, atleast 98%. at least 99%. or 100% identical to SEQ ID NO: 114, and a variable light chain (VL) comprising an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 115.

[0113] In some embodiments, the IL-18 variant comprises an amino acid sequence of SEQ ID NO: 243, and the anti-PDl antibody or a fragment thereof comprises a variable heavy chain (VH) comprising an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 114, and a variable light chain (VL) comprising an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 115.

[0114] In some embodiments, the IL-18 variant comprises an amino acid sequence of SEQ ID NO: 244, and the anti-PDl antibody or a fragment thereof comprises a variable heavy chain (VH) comprising an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%. or 100% identical to SEQ ID NO: 114, and a variable light chain (VL) comprising an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 115.

[0115] In some embodiments, the IL-18 variant comprises an amino acid sequence of SEQ ID NO: 245, and the anti-PDl antibody or a fragment thereof comprises a variable heavy chain (VH) comprising an amino acid sequence that is at least 90%, at least 95%. at least 98%, at least 99%, or 100% identical to SEQ ID NO: 1 14, and a variable light chain (VL) comprising an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 115.

[0116] In some embodiments, the IL- 18 variant comprises an amino acid sequence of SEQ ID NO: 246, and the anti-PDl antibody or a fragment thereof comprises a variable heavy chain (VH) comprising an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 114, and a variable light chain (VL) comprising an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 115.

[0117] In some embodiments, the IL- 18 variant comprises an amino acid sequence of SEQ ID NO: 247, and the anti-PDl antibody or a fragment thereof comprises a variable heavy chain (VH) comprising an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%. or 100% identical to SEQ ID NO: 114, and a variable light chain (VL) comprising an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 115.

[0118] In some embodiments, the IL-18 variant comprises an amino acid sequence of SEQ ID NO: 248, and the anti-PDl antibody or a fragment thereof comprises a variable heavy chain (VH) comprising an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%. or 100% identical to SEQ ID NO: 114, and a variable light chain (VL) comprising an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 115.

[0119] In some embodiments, the antigen binding domain of the anti-PDl antibody is a diabody, a Fab, Fab’, a F(ab’)2, a variable fragment (Fv), a single chain variable fragment (scFv), a (SCFV)2, a Fd fragment, stapled scFv fragment (spFv), a disulfide stabilized Fv fragment (dsFv), or a disulfide stabilized diabody (ds diabody).

[0120] Any of the VH and the VL domains identified herein (e.g. those that bind to PD-1) may be engineered into scFv format in either VH-linker-VL or VL-linker-VH orientation. In some embodiments, the scFv format is, from the N- to C-terminus, in the VH- linker-VL orientation. In other embodiments, the scFv format is. from the N- to C-terminus, in the VL-linker-VH orientation.

[0121] In some embodiments, the VH and VL disclosed herein may be linked together via a synthetic linker to form a scFv may be stabilized by the introduction of two disulfide bonds between the linker and the variable domains of the scFv to form a stabilized scFv molecules (herein referred to as spFv (or stapled scFv). The generation of stabilized scFv molecules (herein referred to as spFv (or stapled scFv) is described in W02021 / 030657 which is incorporated herein by reference in its entirety.

[0122] In some embodiments, the IL- 18 variants disclosed in Table 1 are covalently linked to the N-terminal portion of the Fc region of anti-PDl antibody or a fragment thereof, as shown in Fig. 1A and ID. In some embodiments, the IL-18 variants disclosed herein are covalently linked to both N-terminal and C-terminal portions of the Fc region of anti-PDl antibody or or a fragment thereof, as shown in Fig. IB. In other embodiments, the IL-18 variants are covalently linked to the N-terminus of light chain of anti-PDl antibody or a fragment thereof, as shown in Fig. 1C.

[0123] In some embodiments, the anti-PDl antibody is a humanized antibody or an antibody fragment. In some embodiments, the anti-PDl antibody is an IgG, IgGl, IgG2, IgG3, or IgG4.

[0124] In some embodiments, the anti-PDl antibody comprises an Fc region with one or more of the following properties: (a) reduced effector function when compared to theparent Fc; (b) reduced affinity to FcyRl, FcyRlIa, FcyRllb. FcyRIlIb and / or FcyRlIla. (c) reduced affinity to FcyRl (d) reduced affinity to FcyRlIa (e) reduced affinity to FcyRIIb, (f) reduced affinity7to FcyRIIIb or (g) reduced affinity7to FcyRlIla.

[0125] In some embodiments, the anti-PDl antibody comprises mutations in the Fc region resulting in reduced fc-mediated effector functions. Non-limiting examples of one or more amino acid substitutions include F234A / L235A, L234A / L235A, L234A / L235A / D265S, V234A / G237A / P238S / H268A / V309L / A330S / P331S, F234A / L235A, S228P / F234A7 L235A, N297A, V234A / G237A, K214T / E233P / L234V / L235A / G236- deleted / A327G / P331A / D365E / L358M, H268Q / V309L / A330S / P331S, S267E / L328F, L234F / L235E / D265A, L234A / L235A / G237A / P238S / H268A / A330S / P331S, S228P / F234A / L235A / G237A / P238S, and S228P / F234A / L235A / G236-deleted / G237A / P238S mutations, wherein residue numbering is according to the EU index.

[0126] In some embodiments, the Fc region of anti-PDl antibody comprises knobin-hole mutations. Knob-in-hole mutations are disclosed for example in WO 1996 / 027011 and include mutations on the interface of CH3 region in which an amino acid with a small side chain (hole) is introduced into the first CH3 region and an amino acid with a large side chain (knob) is introduced into the second CH3 region, resulting in preferential interaction between the first CH3 region and the second CH3 region. Exemplary CH3 region mutations forming a knob and a hole are T366Y / F405A, T366W / F405W, F405W / Y407A, T394W / Y407T, T394S / Y407A, T366W / T394S, F405W / T394S and T366W / T366S_L368A_Y407V.

[0127] In some embodiments, the Fc domain may comprise ProAla mutations. Coexpression of unique polypeptides to make a multispecific antibody, such as a bispecific antibody may lead to multiple mis-paired species that are difficult to separate from a target multispecific antibody due to, for example their similar biophy sical properties. ProAla IgG designs address this risk and comprise a set of mutations which exploits a unique mechanism based on natural antibody folding. Included are ProAla heavy chains engineered to increase their folding energy barriers such that only the cognate light and heavy chains can for example induce folding, chaperone release, and secretion. Mis-paired polypeptides fail to secrete from the cell, resulting in unprecedented purity of secreted multispecific antibodies, such as the bispecific antibodies (bsAbs) of the present invention. In some embodiments, antibodies have an enhanced pairing between a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide each comprises an immunoglobulin heavy chain constant domain 3 (CH3 domain). In some embodiments, the Fc domain of anti-PDl antibodycomprises a P374 mutation (Eu numbering) in an immunoglobulin heavy chain constant domain 3 (CH3 domain) (e.g., an IgG CH3 domain) of each of the first polypeptide and the second polypeptide, and one or more heterodimerization mutations in the first and / or second polypeptides. In some embodiments, the anti-PDl antibodies further comprise a third polypeptide and a fourth polypeptide, each comprising an immunoglobulin light chain constant domain 1 (CL domain), wherein at least one of the third polypeptide and the fourth polypeptide comprises a P141 mutation (Eu numbering) in its respective CL domain. In some embodiments, the third and / or fourth polypeptide further comprise one or more heterodimerization mutations. In some embodiments, the anti-PDl antibody comprises a P151 mutation (Eu numbering) in an immunoglobulin heavy chain constant domain 1 (CHI domain) (e.g., an IgG CHI domain) of at least one of the first and the second polypeptide, and optionally comprises a P141 mutation in a paired immunoglobulin light chain constant (CL) domain (e.g., an IgG CL domain), and one or more heterodimerization mutations in a first and / or a second polypeptide(s). In some embodiments, the anti-PDl antibody comprises a P374 mutation in a CH3 domain of each of the first polypeptide and the second polypeptide, a P151 mutation in a CHI domain of at least one of the first polypeptide and the second polypeptide, and optionally a P141 mutation in a paired immunoglobulin light chain constant (CL) domain (e.g., an IgG CL domain), and one or more heterodimerization mutations in the first and / or the second polypeptides. In some embodiments, the heterodimerization mutations can be in a CH3 domain of the first polypeptide and / or in the CH3 domain of the second polypeptide and can enhance pairing of the first and second polypeptides. In some embodiments, the heterodimerization mutations can be in a first CHI domain and / or its paired immunoglobulin light chain constant (CL) domain (e.g., an IgG CL domain) and / or in a second CHI domain and its paired CL domain and can enhance specific pairing of the first CHI domain and its paired CL domain and / or the second CHI domain and its paired CL domain. In some embodiments, the one or more heterodimerization mutations can be (i) in a first CH3 domain and in a second CH3 domain and (ii) in a first CHI domain and / or its paired CL domain and / or in a second CHI domain and / or its paired CL domain. In some embodiments, one or more heavy chain domains of the multispecific molecules described herein are exchanged for one or more light chain domains on at least one of the first polypeptide and the second polypeptide, and / or one or more light chain domains are exchanged for one or more heavy chain domains on at least one of a third polypeptide and a fourth polypeptide. In some embodiments, the P374 mutation is an amino acid substitutionP374X, wherein X comprises any amino acid. In some embodiments, the P374 mutation is an amino acid substitution P374X, wherein X comprises A, G or Q. In some embodiments, the mutation at position P151 (Eu numbering) is an amino acid substitution P151X, wherein X comprises any amino acid. In some embodiments, the mutation at position P151 (Eu numbering) is an amino acid substitution Pl 5 IX. wherein X comprises A or G. The combination of the proline mutations, the heterodimerization mutations in the various domains, and other mutations as described herein provides, for example, a means for enhanced selective assembly of cognate chains in multiparatopic multispecific molecules. Accordingly, the description and teachings in PCT / IB2025 / 050810 are herein incorporated by reference in its entirely.

[0128] In some embodiments, the IL-18-anti-PDl antibody fusion protein further comprises an attenuator moiety' at the N-terminus of the IL-18 variant, wherein the attenuator moiety comprises from about one amino acid to about 70 amino acids in length. In some embodiments, the attenuator moiety may be any one of the attenuator sequences of Table 2. In some embodiments, the attenuator moiety is selected from of S, GGGGS (SEQ ID NO: 32), EV, DG, LM, THKM (SEQ ID NO: 36), DWNN (SEQ ID NO: 37), NTHR (SEQ ID NO: 38), TVDWIT (SEQ ID NO: 39), KHVLFF (SEQ ID NO: 40), ASRHVQ (SEQ ID NO: 41), LSSKDKVD (SEQ ID NO: 42), GKMNLSWY (SEQ ID NO: 43), HMWQQMYN (SEQ ID NO: 44), EIVHAIIFHK (SEQ ID NO: 45), LERIQTRYIQ (SEQ ID NO: 46), NRSKMMSMIR (SEQ ID NO: 48), MHRNWVAKHGHL (SEQ ID NO: 49), HHNKWKHLDFSH (SEQ ID NO: 50), DVVQSSESNQEW (SEQ ID NO: 51), NFSLERHMNNRMYE (SEQ ID NO: 52). WYKWFFYSRMHSIL (SEQ ID NO: 53), WFRRHGTKHGQFVI (SEQ ID NO: 54), and AAEPVEDNVINFVAMKFIDNTLYFIAENDENGGGS (SEQ ID NO: 61).

[0129] In some embodiments, the fusion protein further comprises a protease cleavage site between the attenuator moiety' and the N-terminus of the IL-18 variant, and / or a protease cleavage site between IL- 18 variant and the anti-PDl antibody. Nonlimiting examples of protease cleavage sites include cleavage sites recognized by granzyme B, granzyme A, granzy me M, granzyme K, cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin K, cathepsin L, cathepsin G, kallikrein, plasmin, a collagenase, type IV collagenase, a stromelysin. Factor Xa, a chymotrypsin-like protease, a trypsin-like protease, an elastase-like protease, a subtilisin like protease, bromelain, a calpain, a caspase, papain, a HIV-1 protease, aHSV protease, a CMV protease, a chymosin, renin, pepsin, a matrix metalloprotease (MMP). or a metalloproteinase (ADAM).

[0130] Polypeptides of various IL-18 fusion proteins are listed in Table 4.Table 4: Listed below are the amino acid sequences of IL-18 fusion proteins. IL-18 variant sequences are highlighted in bold and the attenuator sequences are underlinedFusion Proteins Comprising IL-18 Variants and Monovalent anti-PDl Antibodies

[0131] Disclosed herein are fusion proteins comprising monovalent anti-PDl antibodies. In some embodiments, the fusion protein comprises:(a) a first monomer comprising from N-terminus to C-terminus an IL- 18 variant and a first Fc region;(b) a second monomer comprising from N-terminus to C-terminus a variable heavychain (VH) and a second Fc region; and(c) a third monomer comprising a variable light (VL) chain, wherein the VH and VL form an antigen binding domain that binds to PD-1.

[0132] In some embodiments, the IL-18 variant comprises the amino acid sequence of:wherein X1is Y or C;

[0133] In some embodiments, the IL-18 variant comprises the amino acid sequence selected from SEQ ID NOs 2, 3, 4, 5. 6, 7, 8. 9, 10, 11. 12. 13. 14. 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 242, 243, 244, 245, 246, 247, 248, or 183.

[0134] In some embodiments, the antigen binding domain comprises a heavy chain complementarity determining region (HCDR) 1 having an amino acid sequence of SEQ ID NO: 108, a HCDR2 having an amino acid sequence of SEQ ID NO: 109, a HCDR3 having an ammo acid sequence of SEQ ID NO: 110, and a light chain complementarity determining region (LCDR) 1 having an amino acid sequence of SEQ ID NO: 111, a LCDR2 having an amino acid sequence of SEQ ID NO: 112, and a LCDR3 having an amino acid sequence of SEQ ID NO: 113.

[0135] In some embodiments, the VH comprises an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 1 14, and the VL comprises an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%. or 100% identical to SEQ ID NO: 115.

[0136] In some embodiments, the IL- 18 variant further comprises an attenuator moiety at the N-terminus, and wherein the attenuator moiety comprises from about one amino acid to about 70 amino acids in length. In some embodiments, the attenuator moiety is selected from any one of the attenuator sequences of Table 2. In some embodiments, the attenuator moiety' is selected from of S, GGGGS (SEQ ID NO: 32), EV, DG. LM, THKM (SEQ ID NO: 36). DWNN (SEQ ID NO: 37). NTHR (SEQ ID NO: 38), TVDW1T (SEQ ID NO: 39), KHVLFF (SEQ ID NO: 40), ASRHVQ (SEQ ID NO: 41), LSSKDKVD (SEQ ID NO: 42), GKMNLSWY (SEQ ID NO: 43), HMWQQMYN (SEQ ID NO: 44), EIVHAIIFHK (SEQ ID NO: 45), LERIQTRYIQ (SEQ ID NO: 46), NRSKMMSMIR (SEQ ID NO: 48), MHRNWVAKHGHL (SEQ ID NO: 49). HHNKWKHLDFSH (SEQ ID NO: 50). DVVQSSESNQEW (SEQ ID NO: 51), NFSLERHMNNRMYE (SEQ ID NO: 52), WYKWFFYSRMHSIL (SEQ ID NO: 53), WFRRHGTKHGQFVI (SEQ ID NO: 54), and AAEPVEDNVINFVAMKFIDNTLYFIAENDENGGGS (SEQ ID NO: 61).

[0137] The "knob-in-hole" strategy may be used to generate IL- 18 fusion proteins. In some embodiments, one of the first and the second Fc regions comprises an amino acid substitution T366W, and the other of the first and the second Fc regions comprises one ormore amino acid substitutions T366S / L368A / Y407V, wherein numbering is according to EU numbering.

[0138] Other strategies such as promoting heavy chain heterodimerization using electrostatic interactions by substituting positively charged residues at one CH3 surface and negatively charged residues at a second CH3 surface may be used, as described in US Pat. Publ. No. US2010 / 0015133; US Pat. Publ. No. US2009 / 0182127; US Pat. Publ. No. US2010 / 028637 or US Pat. Publ. No. US2011 / 0123532, which are incorporated herein by reference. In other strategies, heterodimerization may be promoted by the following substitutions (expressed as modified position in the first CH3 domain of the first heavy chain / modified position in the second CH3 domain of the second heavy chain): L351Y_F405AY407V / T394W, T366I_K392M_T394W / F405A_Y407V, T366L_K392M_T394W / F405 A_Y407V, L351 Y_Y407A / T366A_K409F, L351Y Y407A / T366V K409F Y407A / T366A_K409F, or T350V_L351Y_F405A Y407V / T350V_T366L_K392L_T394W as described in U.S. Pat. Publ. No. US2012 / 0149876 or U.S. Pat. Publ. No. US2013 / 0195849, which are incorporated herein by reference.

[0139] In some embodiments, first and the second Fc regions comprise one or more amino acid substitutions that reduce Fc-mediated effector functions. Non-limiting examples of such amino acid substitutions or modifications include E234A / E235A / D265S; H435R / Y436F; S364K / E357Q; L368D / K370S; S364K; L368D / K370S; S364K; L368E / K370S; D401K; T411E / K360E / Q362E; T366W; and T366S / L368A / Y407V, wherein numbering is according to EU numbering.

[0140] In some embodiments, the fusion protein comprises:(a) the first monomer is selected from the amino acid sequence represented by SEQ ID NOs: 137, 142, 148, 172, 173, 178, 179, 180, 181, 249, 250, 251, 252, 253, 254, and 255;(b) the second monomer is selected from the amino acid sequence represented by SEQ ID NOs: 146 and 116.(c) the third monomer is selected from the amino acid sequence represented by SEQ ID NO: 117.

[0141] In some embodiments, the fusion protein comprises the first monomer comprising the amino sequence of SEQ ID NO: 137. the second monomer comprising the ammo acid sequence of SEQ ID NO: 146, and the third monomer comprising the amino acid sequence of SEQID NO: 117.

[0142] In some embodiments, the fusion protein comprises the first monomer comprising the amino sequence of SEQ ID NO: 142, the second monomer comprising the amino acid sequence of SEQ ID NO: 146, and the third monomer comprising the amino acid sequence of SEQID NO: 117.

[0143] In some embodiments, the fusion protein comprises the first monomer comprising the amino sequence of SEQ ID NO: 148, the second monomer comprising the amino acid sequence of SEQ ID NO: 146, and the third monomer comprising the amino acid sequence of SEQID NO: 117.

[0144] In some embodiments, the fusion protein comprises the first monomer comprising the amino sequence of SEQ ID NO: 172, the second monomer comprising the amino acid sequence of SEQ ID NO: 146, and the third monomer comprising the amino acid sequence of SEQID NO: 117.

[0145] In some embodiments, the fusion protein comprises the first monomer comprising the amino sequence of SEQ ID NO: 173. the second monomer comprising the amino acid sequence of SEQ ID NO: 146, and the third monomer comprising the amino acid sequence of SEQID NO: 117.

[0146] In some embodiments, the fusion protein comprises the first monomer comprising the amino sequence of SEQ ID NO: 178. the second monomer comprising the amino acid sequence of SEQ ID NO: 116, and the third monomer comprising the amino acid sequence of SEQID NO: 117.

[0147] In some embodiments, the fusion protein comprises the first monomer comprising the amino sequence of SEQ ID NO: 179. the second monomer comprising the ammo acid sequence of SEQ ID NO: 116, and the third monomer comprising the amino acid sequence of SEQID NO: 117.

[0148] In some embodiments, the fusion protein comprises the first monomer comprising the amino sequence of SEQ ID NO: 180, the second monomer comprising the amino acid sequence of SEQ ID NO: 116, and the third monomer comprising the amino acid sequence of SEQID NO: 117.

[0149] In some embodiments, the fusion protein comprises the first monomer comprising the amino sequence of SEQ ID NO: 181, the second monomer comprising the amino acid sequence of SEQ ID NO: 116, and the third monomer comprising the amino acid sequence of SEQID NO: 117.

[0150] In some embodiments, the fusion protein comprises the first monomer comprising the amino sequence of SEQ ID NO: 249, the second monomer comprising the amino acid sequence of SEQ ID NO: 116, and the third monomer comprising the amino acid sequence of SEQID NO: 117.

[0151] In some embodiments, the fusion protein comprises the first monomer comprising the amino sequence of SEQ ID NO: 250, the second monomer comprising the amino acid sequence of SEQ ID NO: 116, and the third monomer comprising the amino acid sequence of SEQID NO: 117.

[0152] In some embodiments, the fusion protein comprises the first monomer comprising the amino sequence of SEQ ID NO: 251, the second monomer comprising the amino acid sequence of SEQ ID NO: 116, and the third monomer comprising the amino acid sequence of SEQID NO: 117.

[0153] In some embodiments, the fusion protein comprises the first monomer comprising the amino sequence of SEQ ID NO: 252. the second monomer comprising the amino acid sequence of SEQ ID NO: 1 1 , and the third monomer comprising the amino acid sequence of SEQID NO: 117.

[0154] In some embodiments, the fusion protein comprises the first monomer comprising the amino sequence of SEQ ID NO: 253. the second monomer comprising the amino acid sequence of SEQ ID NO: 116, and the third monomer comprising the amino acid sequence of SEQID NO: 117.

[0155] In some embodiments, the fusion protein comprises the first monomer comprising the amino sequence of SEQ ID NO: 254. the second monomer comprising the ammo acid sequence of SEQ ID NO: 116, and the third monomer comprising the amino acid sequence of SEQID NO: 117.

[0156] In some embodiments, the fusion protein comprises the first monomer comprising the amino sequence of SEQ ID NO: 255, the second monomer comprising the amino acid sequence of SEQ ID NO: 116, and the third monomer comprising the amino acid sequence of SEQID NO: 117.Other IL- 18 Fusion Proteins

[0157] Also disclosed here are fusion proteins comprising IL-18 variants and the antibodies and antigen binding domains disclosed in Table 5.Table 5Nucleotide Sequences and Cells

[0158] Also disclosed herein are isolated nucleic acids encoding any of the fusion proteins described herein, vectors (e.g., lentiviral vector) comprising such nucleic acids, and host cells (e g., CHO cell) comprising such nucleic acids or vectors.

[0159] Also provided are vectors comprising the nucleic acid sequences described herein. The vectors can be expression vectors. Recombinant expression vectors containing a sequence encoding a polypeptide of interest are thus contemplated as within the scope of this disclosure. The expression vector may contain one or more additional sequences such as but not limited to regulator)’ sequences (e.g., promoter, enhancer), a selection marker, and a poly adenylation signal. Vectors for transforming a wide variety of host cells are well known and include, but are not limited to, plasmids, phagemids, cosmids, baculoviruses, bacmids, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), as well as other bacterial, yeast and viral vectors.

[0160] Numerous techniques are known in the art for the introduction of foreign genes into cells and may be used to construct the recombinant cells for purposes of carrying out the expression of IL- 18 fusion proteins, in accordance with the various embodiments described and exemplified herein. The technique used should provide for the stable transfer of the heterologous gene sequence to the host cell, such that the heterologous gene sequence is heritable and expressible by the cell progeny, and so that the necessary development and physiological functions of the recipient cells are not disrupted. Techniques which may be used include but are not limited to chromosome transfer (e.g., cell fusion, chromosome mediated gene transfer, micro cell mediated gene transfer), physical methods (e.g., transfection, spheroplast fusion, microinjection, electroporation, liposome carrier), viral vector transfer (e.g., recombinant DNA viruses, recombinant RNA viruses) and the like. Calcium phosphate precipitation and polyethylene glycol (PEG)-induced fusion of bacterial protoplasts with mammalian cells may also be used to transform cells.

[0161] Cells suitable for use in the expression of the IL- 18 fusion proteins described herein are preferably eukaryotic cells, more preferably cells of plant, rodent, or human origin, for example but not limited to NSO, CHO, CHOK1, perC.6, Tk-tsl3, BHK, HEK293 cells, COS-7, T98G, CV-l / EBNA, L cells, C127, 3T3, HeLa, NS1, Sp2 / 0 myeloma cells, and BHK cell lines, among others.

[0162] Nucleotide sequences encoding the IL-18 fusion proteins are listed in Table6.Table 6: Nucleotide sequences of IL- 18 fusion proteins- Ill -

[0163] Methods for making the fusion proteins, or nucleic acids encoding the fusion proteins are also described herein. Some embodiments provide that conventional recombinant DNA methodologies are utilized for generating the fusion proteins. The fusion constructs preferably are generated at the DNA level, and the resulting DNAs integrated into expression vectors, and expressed to produce the fusion proteins of the invention. Subsequently, the vector is expressed in a host cell to obtain the fusion protein; and optionally the method further includes a step of recovering the fusion protein from the host cell culture. In some embodiments, a method of producing interleukin 18 (IL- 18). a fragment thereof, an IL- 18 variant, or a fragment of the IL- 18 variant, comprises culturing a cell transfected with an expression vector comprising a nucleic acid encoding a fusion protein, in cell culture medium to allow a fusion protein to be produced and secreted into the extracellular space for purification: said protein, when contacting a protease to the fusion protein to cleave the fusion protein to produce the IL- 18. the fragment thereof, the IL-18 variant, or the fragment of the IL-18 variant. Exemplary nucleic acid molecules encoding a fusion protein are disclosed in Table 6. Other embodiments provide that chemical conjugation using conventional chemical cross-linkers may be used to fuse protein moieties.

[0164] In some embodiments, the nucleic acid molecules encoding the fusion proteins is expressed in CHO cells or HEK-293. Preferably, expressing the fusion proteins in the host cells results in a recoverable secreted fusion protein of at least 135 mg / L from supernatant of the host cells. In some embodiments, a yield of the fusion protein of at least 135 mg / L is obtained via transient transfection. In some embodiments, an even higher yield of the fusion protein, e.g., at least 150, 200, 250, or 300 mg / L is obtained via stable producer cell clones, or pools of clones. In some embodiments, from a transient transfection, a yield of the fusion protein is about 130-400 mg / L. In some embodiments, a fusion protein, or the IL-18, the fragment thereof, the IL-18 variant, or the fragment of the IL-18 variant cleaved from the fusion protein, is recovered in more than about 400 mg / L, between 350-400 mg / L, 300-350 mg / L, 200-300 mg / L, 100-200 mg / L, or at least 50 mg / L from supernatant of the host cells.

[0165] In some embodiments, using a Chinese hamster ovary (CHO) expression system, the fusion protein is produced via a process including the steps of: (1) cell recovery, which may be to recover frozen CHO cells via water bath at 37°C; (2) cell subculturing, which may be to sub-culture the cells and adjust the cell densify to 6x106 / ml for transfection; (3)transfection and expression, using a solution 1 (in which a plasmid is diluted with a diluting agent), a solution 2 (in which a transection reagent is diluted with a / the diluting agent), and then mixing the solution 1 , the solution 2 and the CHO cells, followed by incubating the mixture at a shaker for expression for 12-14 days at 32°C to collect the supernatant of the culture after centrifuge.

[0166] In some embodiments, a purification process is performed after the expression of the fusion protein. In some embodiments, a purification process includes the steps of: (1) washing a column with a binding buffer (10 times volume) at a flow rate of 1 mL / min; (2) loading a fusion-protein-containing sample into the column at a flow rate of 1 mL / min; (3) washing the column with lOx volumes of PBS buffer with a flow rate of 1 mL / min; (4) eluting the protein from the column with 40 mM sodium citrate (pH3.4); optionally the elution sample may be collected into tubes (Iml / min) and measured for optical density (OD) using NanoDrop at 280 nm; and (5) performing dialysis, e g., against PBS buffer in a dialysis bag overnight.Compositions

[0167] In various embodiments, the present invention provides pharmaceutical compositions including a pharmaceutically acceptable excipient along with a therapeutically effective amount of any one of the fusion proteins disclosed herein. '‘Pharmaceutically acceptable excipient” means an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and desirable, and includes excipients that are acceptable for veterinary use as well as for human pharmaceutical use. Such excipients may be solid, liquid, semisolid, or. in the case of an aerosol composition, gaseous.

[0168] In various embodiments, the fusion proteins are in pharmaceutical compositions formulated for delivery via any route of administration. “Route of administration” may refer to any administration pathway known in the art, including but not limited to aerosol, nasal, oral, transmucosal, transdermal or parenteral. “Transdermal” administration may be accomplished using a topical cream or ointment or by means of a transdermal patch. “Parenteral” refers to a route of administration that is generally associated with injection, including intraorbital, infusion, intraarterial, intracapsular, intracardiac, intradermal, intramuscular, intraperitoneal, inlrapulmonary. intraspinal, intrastemal. intrathecal, intrauterine, intravenous, subarachnoid, subcapsular, subcutaneous, transmucosal, or transtracheal. Via the parenteral route, the compositions may be in the form of solutions orsuspensions for infusion or for injection, or as lyophilized powders. Via the enteral route, the pharmaceutical compositions can be in the form of tablets, gel capsules, sugar-coated tablets, syrups, suspensions, solutions, powders, granules, emulsions, microspheres or nanospheres or lipid vesicles or polymer vesicles allowing controlled release. Via the parenteral route, the compositions may be in the form of solutions or suspensions for infusion or for injection. Via the topical route, the pharmaceutical compositions based on compounds according to the invention may be formulated for treating the skin and mucous membranes and are in the form of ointments, creams, milks, salves, powders, impregnated pads, solutions, gels, sprays, lotions or suspensions. They can also be in the form of microspheres or nanospheres or lipid vesicles or polymer vesicles or polymer patches and hydrogels allowing controlled release. These topical-route compositions can be either in anhydrous form or in aqueous form depending on the clinical indication. Via the ocular route, they may be in the form of eye drops.

[0169] The pharmaceutical compositions can also contain any pharmaceutically acceptable carrier. “Pharmaceutically acceptable carrier" as used herein refers to a pharmaceutically acceptable material, composition, or vehicle that is involved in carrying or transporting a compound of interest from one tissue, organ, or portion of the body to another tissue, organ, or portion of the body. For example, the carrier may be a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, or a combination thereof. Each component of the carrier must be “pharmaceutically acceptable” in that it must be compatible with the other ingredients of the formulation. It must also be suitable for use in contact with any tissues or organs with which it may come in contact, meaning that it must not carry a risk of toxicity, irritation, allergic response, immunogenicity, or any other complication that excessively outweighs its therapeutic benefits.

[0170] The pharmaceutical compositions can also be encapsulated, tableted or prepared in an emulsion or syrup for oral administration. Pharmaceutically acceptable solid or liquid carriers may be added to enhance or stabilize the composition, or to facilitate preparation of the composition. Liquid carriers include syrup, peanut oil, olive oil, glycerin, saline, alcohols and water. Solid carriers include starch, lactose, calcium sulfate, dihydrate, terra alba, magnesium stearate or stearic acid, talc, pectin, acacia, agar or gelatin. The carrier may also include a sustained release material such as glyceryl monostearate or glyceryl distearate, alone or with a wax.

[0171] The pharmaceutical preparations are made following the conventional techniques of pharmacy involving milling, mixing, granulation, and compressing, when necessary, for tablet forms; or milling, mixing and filling for hard gelatin capsule forms. When a liquid carrier is used, the preparation will be in the form of a syrup, elixir, emulsion or an aqueous or non-aqueous suspension. Such a liquid formulation may be administered directly p.o. or filled into a soft gelatin capsule.

[0172] The pharmaceutical compositions may be delivered in a therapeutically effective amount. The precise therapeutically effective amount is that amount of the composition that will yield the most effective results in terms of efficacy of treatment in a given subject. This amount will vary depending upon a variety of factors, including but not limited to the characteristics of the therapeutic compound (including activity, pharmacokinetics, pharmacodynamics, and bioavailability), the physiological condition of the subject (including age, sex, disease type and stage, general physical condition, responsiveness to a given dosage, and type of medication), the nature of the pharmaceutically acceptable carrier or carriers in the formulation, and the route of administration. One skilled in the clinical and pharmacological arts will be able to determine a therapeutically effective amount through routine experimentation, for instance, by monitoring a subject’s response to administration of a compound and adjusting the dosage accordingly.Method of Treatment

[0173] Also disclosed herein are methods for treating a subject suffering from cancer. In some embodiments, a method of treating a subject suffering from cancer comprises administering an IL-18 fusion protein comprising an IL-18 variant and an anti-PDl antibody or a fragment thereof, wherein the IL- 18 variant comprises the amino acid sequence of:wherein X1is Y or C;

[0174] In some embodiments, the fusion protein comprises an IL- 18 variant and an anti-PDl antibody or a fragment thereof, wherein the IL-18 variant comprises the amino acid sequence of SEQ ID NO: 1, whereinX1is Y or C;X18is N or C.

[0175] In some embodiments, the fusion protein comprises an IL-18 variant and an anti-PDl antibody or a fragment thereof, wherein the IL-18 variant comprises an amino acid sequence selected from SEQ ID NOs: 2, 3, 4, 5, 6. 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23. 24. 25. 26. 27, 28, 29, 30, 31, 242, 243, 244, 245. 246, 247. 248, or 183, and wherein the anti-PDl antibody comprises a variable heavy chain (VH) comprising an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 114. and a variable light chain (VL) comprising an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 115.

[0176] In some embodiments, the cancer is solid cancer. In some embodiments, the solid cancer is adrenal cancer, anal cancer, bile duct cancer, bladder cancer, bone cancer, brain cancer, breast cancer, carcinoid cancer, cervical cancer, colorectal cancer, esophageal cancer, eye cancer, gallbladder cancer, gastrointestinal stromal tumor, germ cell cancer, head and neck cancer, kidney cancer, liver cancer, lung cancer, nasal cavity and paranasal sinus cancer, nasophary ngeal cancer, neuroblastoma, neuroendocrine cancer, oral cancer, oropharyngeal cancer, ovarian cancer, pancreatic cancer, pediatric cancer, penile cancer, pituitary' cancer, prostate cancer, skin cancer, soft tissue cancer, spinal cord cancer, stomach cancer, testicular cancer, thymus cancer, thyroid cancer, ureteral cancer, uterine cancer, vaginal cancer, or vulvar cancer.

[0177] In some embodiments, a method of treating a subject suffering from cancer comprises administering to the subject an IL- 18 fusion protein comprising an IL- 18 variant and an anti-PDl antibody or a fragment thereof, the IL- 18 variant comprises an amino acid sequence of SEQ ID NO: 2, and the anti-PDl antibody or a fragment thereof comprises a variable heavy chain (VH) comprising an amino acid sequence of SEQ ID NO: 114, and a variable light chain (VL) comprising an amino acid sequence of SEQ ID NO: 115.

[0178] In some embodiments, a method of treating a subject suffering from cancer comprises administering to the subject an IL-18 fusion protein comprising an IL-18 variant and an anti-PDl antibody or a fragment thereof, the IL- 18 variant comprises an amino acid sequence of SEQ ID NO: 9, and the anti-PDl antibody or a fragment thereof comprises a variable heavy chain (VH) comprising an amino acid sequence of SEQ ID NO: 114, and a variable light chain (VL) comprising an amino acid sequence of SEQ ID NO: 115.

[0179] In some embodiments, a method of treating a subject suffering from cancer comprises administering to the subject an IL-18 fusion protein comprising an IL-18 variant and an anti-PDl antibody or a fragment thereof, the IL- 18 variant comprises an amino acid sequence of SEQ ID NO: 15, and the anti-PDl antibody or a fragment thereof comprises a variable heavy chain (VH) comprising an amino acid sequence of SEQ ID NO: 114, and a variable light chain (VL) comprising an amino acid sequence of SEQ ID NO: 115.

[0180] In some embodiments, a method of treating a subject suffering from cancer comprises administering to the subject an IL- 18 fusion protein comprising an IL- 18 variant and an anti-PDl antibody or a fragment thereof, the IL- 18 variant comprises an amino acid sequence of SEQ ID NO: 16, and the anti-PDl antibody or a fragment thereof comprises a variable heavy chain (VH) comprising an amino acid sequence of SEQ ID NO: 114, and a variable light chain (VL) comprising an amino acid sequence of SEQ ID NO: 115.

[0181] In some embodiments, a method of treating a subject suffering from cancer comprises administering to the subject an IL-18 fusion protein comprising an IL-18 variant and an anti-PDl antibody or a fragment thereof, the IL-18 variant comprises an amino acid sequence of SEQ ID NO: 19, and the anti-PDl antibody or a fragment thereof comprises a variable heavy chain (VH) comprising an amino acid sequence of SEQ ID NO: 114, and a variable light chain (VL) comprising an amino acid sequence of SEQ ID NO: 115.

[0182] In some embodiments, a method of treating a subject suffering from cancer comprises administering to the subject an IL-18 fusion protein comprising an IL-18 variant and an anti-PDl antibody or a fragment thereof, the IL- 18 variant comprises an amino acid sequence of SEQ ID NO: 29, and the anti-PDl antibody or a fragment thereof comprises a variable heavy chain (VH) comprising an amino acid sequence of SEQ ID NO: 114, and a variable light chain (VL) comprising an amino acid sequence of SEQ ID NO: 115.

[0183] In some embodiments, a method of treating a subject suffering from cancer comprises administering to the subject an IL- 18 fusion protein comprising an IL- 18 variant and an anti-PDl antibody or a fragment thereof, the IL- 18 variant comprises an amino acid sequence of SEQ ID NO: 30, and the anti-PDl antibody or a fragment thereof comprises a variable heavy chain (VH) comprising an amino acid sequence of SEQ ID NO: 114, and a variable light chain (VL) comprising an amino acid sequence of SEQ ID NO: 115.

[0184] In some embodiments, a method of treating a subject suffering from cancer comprises administering to the subject an IL- 18 fusion protein comprising an IL-18 variant and an anti-PDl antibody or a fragment thereof, the IL- 18 variant comprises an amino acidsequence of SEQ ID NO: 31. and the anti-PDl antibody or a fragment thereof comprises a variable heavy chain (VH) comprising an amino acid sequence of SEQ ID NO: 114, and a variable light chain (VL) comprising an amino acid sequence of SEQ ID NO: 115.

[0185] In some embodiments, a method of treating a subject suffering from cancer comprises administering to the subject an IL- 18 fusion protein comprising an IL- 18 variant and an anti-PDl antibody or a fragment thereof, the IL- 18 variant comprises an amino acid sequence of SEQ ID NO: 242, and the anti-PDl antibody or a fragment thereof comprises a variable heavy chain (VH) comprising an amino acid sequence of SEQ ID NO: 114, and a variable light chain (VL) comprising an amino acid sequence of SEQ ID NO: 115.

[0186] In some embodiments, a method of treating a subject suffering from cancer comprises administering to the subject an IL-18 fusion protein comprising:(a) the first monomer is selected from the amino acid sequence represented by SEQ ID NOs: 137, 142, 148, 172, 173, 178, 179. 180, 181, 249, 250, 251, 252, 253, 254, and 255;(b) the second monomer is selected from the amino acid sequence represented by SEQ ID NOs: 146 and 116; and(c) the third monomer is selected from the amino acid sequence represented by SEQ ID NO: 117.

[0187] In some embodiments, a method of treating a subject suffering from cancer comprises administering to the subject an IL- 18 fusion protein comprising the first monomer comprising the amino sequence of SEQ ID NO: 137, the second monomer comprising the amino acid sequence of SEQ ID NO: 146, and the third monomer comprising the amino acid sequence of SEQ1D NO: 117.

[0188] In some embodiments, a method of treating a subject suffering from cancer comprises administering to the subject an IL- 18 fusion protein comprising the first monomer comprising the amino sequence of SEQ ID NO: 142, the second monomer comprising the amino acid sequence of SEQ ID NO: 146, and the third monomer comprising the amino acid sequence of SEQID NO: 117.

[0189] In some embodiments, a method of treating a subject suffering from cancer comprises administering to the subject an IL- 18 fusion protein comprising the first monomer comprising the amino sequence of SEQ ID NO: 148. the second monomer comprising the ammo acid sequence of SEQ ID NO: 146, and the third monomer comprising the amino acid sequence of SEQID NO: 117.

[0190] In some embodiments, a method of treating a subject suffering from cancer comprises administering to the subject an IL- 18 fusion protein comprising the first monomer comprising the amino sequence of SEQ ID NO: 172, the second monomer comprising the amino acid sequence of SEQ ID NO: 146, and the third monomer comprising the amino acid sequence of SEQID NO: 117.

[0191] In some embodiments, a method of treating a subject suffering from cancer comprises administering to the subject an IL- 18 fusion protein comprising the first monomer comprising the amino sequence of SEQ ID NO: 173, the second monomer comprising the amino acid sequence of SEQ ID NO: 146, and the third monomer comprising the amino acid sequence of SEQID NO: 117.

[0192] In some embodiments, a method of treating a subject suffering from cancer comprises administering to the subject an IL- 18 fusion protein comprising first monomer comprising the amino sequence of SEQ ID NO: 178. the second monomer comprising the amino acid sequence of SEQ ID NO: 116, and the third monomer comprising the amino acid sequence of SEQID NO: 117.

[0193] In some embodiments, a method of treating a subject suffering from cancer comprises administering to the subject an IL- 18 fusion protein comprising the first monomer comprising the amino sequence of SEQ ID NO: 179. the second monomer comprising the amino acid sequence of SEQ ID NO: 116, and the third monomer comprising the amino acid sequence of SEQID NO: 117.

[0194] In some embodiments, a method of treating a subject suffering from cancer comprises administering to the subject an IL- 18 fusion protein comprising the first monomer comprising the amino sequence of SEQ ID NO: 180. the second monomer comprising the amino acid sequence of SEQ ID NO: 116, and the third monomer comprising the amino acid sequence of SEQID NO: 117.

[0195] In some embodiments, a method of treating a subject suffering from cancer comprises administering to the subject an IL- 18 fusion protein comprising the first monomer comprising the amino sequence of SEQ ID NO: 181, the second monomer comprising the amino acid sequence of SEQ ID NO: 116, and the third monomer comprising the amino acid sequence of SEQID NO: 117.

[0196] In some embodiments, a method of treating a subject suffering from cancer comprises administering to the subject an IL- 18 fusion protein comprising the first monomer comprising the amino sequence of SEQ ID NO: 249, the second monomer comprising thearmno acid sequence of SEQ ID NO: 116, and the third monomer comprising the amino acid sequence of SEQID NO: 117.

[0197] In some embodiments, a method of treating a subject suffering from cancer comprises administering to the subject an IL- 18 fusion protein comprising the first monomer comprising the amino sequence of SEQ ID NO: 250. the second monomer comprising the amino acid sequence of SEQ ID NO: 116, and the third monomer comprising the amino acid sequence of SEQID NO: 117.

[0198] In some embodiments, a method of treating a subject suffering from cancer comprises administering to the subject an IL- 18 fusion protein comprising the first monomer comprising the amino sequence of SEQ ID NO: 251, the second monomer comprising the amino acid sequence of SEQ ID NO: 116, and the third monomer comprising the amino acid sequence of SEQID NO: 117.

[0199] In some embodiments, a method of treating a subject suffering from cancer comprises administering to the subject an IL- 18 fusion protein comprising the first monomer comprising the amino sequence of SEQ ID NO: 252, the second monomer comprising the amino acid sequence of SEQ ID NO: 116, and the third monomer comprising the amino acid sequence of SEQID NO: 117.

[0200] Also disclosed herein are methods comprising administering to a subject in need thereof an IL-18 variant wherein the IL-18 variant specifically binds to IL-18 receptor (IL-18R) and exhibits decreased binding affinity to IL-18 binding protein (IL-18BP) relative to the wild-type IL-18. In some embodiments, the IL-18 variants are those described in Table 1. In some embodiments, the IL- 18 variants are part of a fusion protein, such as a fusion protein comprising an IL- 18 variant and an anti-PDl antibody or a fragment thereof.

[0201] The IL-18 variant and IL-18 fusion proteins provided herein may be administered to a subject, in accord with known methods, such as subcutaneous, intravenous administration, intramuscular, intradermal, or as a bolus administration or by continuous infusion over a period of time. Dosage regimens are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. Parenteral compositions may be formulated in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of fusionprotein calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.EMBODIMENTS

[0202] The disclosure is also directed to the following embodiments:Embodiment 1. An IL-18 variant comprising an amino acid sequence of:wherein X1is Y or C;Embodiment 2. The IL- 18 variant of embodiment 1, wherein:X1is Y or C;X2is E, K, Q, H, or Y;Embodiment 3. The IL- 18 variant of embodiment 1 or 2, wherein the IL- 18 variant is selected from any one of the IL- 18 variants listed in Table 1.Embodiment 4. The IL-18 variant of any one of embodiments 1-3, wherein the IL-18 variant is selected from the group consisting of:YFGKLkSKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDVRDNAPRTIFllS MYdDSQPRGkAVTISVKVEKISTLSVENKIISFKEMNPPDNIKDTKSDIIFF QRSVPGHDNKMQFESSSYEGYFLAVEKERDLFKLILKKEDELGDRSIMF TVQNED (IL-18ml7; SEQ ID NO: 17);YFGKLqSKLSVIRwLNDQVLFIDQGNRPLFEDMTDSDVRDNAPRTIFIIS MYdDSQPRGkAVTISVKVEKISTLSVENKIISFKEMNPPDNIKDTKSDIIFF QRSVPGHDNKMQFESSSYEGYFLAVEKERDLFKLILKKEDELGDRSIMF TVQNED (IL-18ml8; SEQ ID NO: 18)YFGKLqSKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDVRDNAPRTIFllS MYdDSQPRGkAVTISVKVEKISTLSVENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESS1YEGYFLAVEKERDLFKLILKKEDELGDRSIMF TVQNED (IL-18ml9 / 13.1; SEQ ID NO: 19);YFGKLqSKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDVRDNAPRTIFIIS MYdDSQPRGkAVTISVKVEKISTLSVENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLAeEKERDLFKLILKKEDELGDRSIMF TVQNED (IL-18m20; SEQ ID NO: 20);YFGKLqSKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDVRDNAPRTIFIIS MYdDSQPRGkAVTISVKVEKISTLSVENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLAVEKERDLFKLILKKEDELGyRSIMF TVQNED (IL-18m21; SEQ ID NO: 21);YFGKLqSKLcVIRNLNDQVLFIDQGNRPLFEDMTDSDVRDNAPRTIFIISMYdDSQPRGkAVTISVKVEKISTLSVENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLAVEKERDLFKLILKKEDELGDRSIMF TVQcED (IL-18m22; SEQ ID NO: 22);YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDVRDNAPRTIFIISMYdDSQPRGkAVTISVKVEKISTLSVENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLAVEKERDLFKL1LKKEDELGDRS1MF TVQNED (IL-18m23; SEQ ID NO: 23);YFGKLqSKLSVIRNLNDQVLFIDQGNRPLFqDMTDSDVRDNAPRTIFIISMYdDSQPRGkAVTISVKVEKISTLSVENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLAVEKERDLFKLILKKEDELGDRSIMF TVQNED (IL-18m24; SEQ ID NO: 24); cFGKLqSKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDVRDNAPRTIFIISMYdDcQPRGkAVTISVKVEKISTLSVENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLAVEKERDLFKL1LKKEDELGDRS1MFT VQNED (IL-18m25; SEQ ID NO: 25);YFGKLhSKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDVRDNAPRTIFIISMYdDSQPRGkAVTISVKVEKISTLSVENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLAVEKERDLFKLILKKEDELGDRSIMF TVQNED (IL-18m26; SEQ ID NO: 26);YFGKLySKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDVRDNAPRTIFIISMYdDSQPRGkAVTISVKVEKISTLSVENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLAVEKERDLFKLILKKEDELGDRSIMF TVQNED (IL-18m27; SEQ ID NO: 27);YFGKLqSKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDVRDNAPRTIFIISMYdDSQPRGkAVTISVKVEKISTLSVENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSmYEGYFLAVEKERDLFKLILKKEDELGDRSIM FTVQNED (IL-18m28; SEQ ID NO: 28);YFGKLqSKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDVRDNAPRTIFIISMYdDSQPRGkAVTISVKVEKIyTLSVENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLAVEKERDLFKLILKKEDELGDRSIMF TVQNED (IL-18m29; SEQ ID NO: 29);YFGKLqSKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDVRDNAPRTIFIISMYdDSQPRGkAVTISVKVEKIlTLSVENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLAVEKERDLFKLILKKEDELGDRSIMF TVQNED (IL-18m30; SEQ ID NO: 30);YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDVRDNAPRTIFIISKYGDSQPRGLAVTISVKVEKISTLSVENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHRNKVQFESSSYEGYFLAVEKERDLFKLILKKEDELGDRSIM FTVQNED (IL-18m31; SEQ ID NO: 31);YFGKLESKLSV1RNLNDQVLFIDQGNRPLFEDMTDSDVRDNAPRT1F1ISKYGDSQPRGLAVTISVKVEKILTLSVENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHRNKVQFESS1YEGYFLAVEKERDLFKLILKKEDELGDRSIMF TVQNED (IL-18m32; SEQ ID NO: 242);YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDVRDNAPRTIFIISHYADSQPRGIAVTISVKVEKISTLSVENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHRNKIQFESS1YEGYFLAVEKERDLFKLILKKEDELGDRSIMFT VQNED (IL-18m33; SEQ ID NO: 243);YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDVRDNAPRTIFIISHYADSQPRGIAVTISVKVEKILTLSVENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHRNKIQFESS1YEGYFLAVEKERDLFKLILKKEDELGDRSIMFT VQNED (IL-18m34; SEQ ID NO: 244);YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDVRDNAPRTIFIISHYGDSQPRGMAVTISVKVEKILTLSVENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESS1YEGYFLAVEKERDLFKLILKKEDELGDRSIM FTVQNED (IL-18m35; SEQ ID NO: 245);YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDVRDNAPRTIFIISKYGDSQPRGLAVTISVKVEKIYTLSVENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHRNKVQFESS1YEGYFLAVEKERDLFKL1LKKEDELGDRS1MF TVQNED (IL-18m36; SEQ ID NO: 246);YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDVRDNAPRTIFIISHYADSQPRGIAVTISVKVEKIYTLSVENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHRNKIQFESS1YEGYFLAVEKERDLFKLILKKEDELGDRSIMFTVQNED (IL-18m37; SEQ ID NO: 247); andYFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDVRDNAPRTIFIISHYGDSQPRGMAVTISVKVEKIYTLSVENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESS1YEGYFLAVEKERDLFKL1LKKEDELGDRS1MFTVQNED (IL-18m38; SEQ ID NO: 248).Embodiment 5. The IL- 18 variant of any one of embodiments 1-4, where in the IL- 18 variant isYFGKLqSKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDVRDNAPRTIFIIS MYdDSQPRGkAVTISVKVEKISTLSVENKIISFKEMNPPDNIKDTKSDIIFF QRSVPGHDNKMQFESS1YEGYFLAVEKERDLFKLILKKEDELGDRSIMF TVQNED (IL-18ml9 / 13.1: SEQ ID NO: 19).Embodiment 6. The IL-18 variant of any one of embodiments 1-5, wherein the IL-18 variant isYFGKLqSKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDVRDNAPRTIFIISMYdD SQPRGkAVTISVKVEKIyTLSVENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHD NKMQFESSSYEGYFLAVEKERDLFKLILKKEDELGDRSIMFTVQNED (IL- 18m29; SEQ ID NO: 29).Embodiment 7. The IL-18 variant of any one of embodiments 1-6, wherein the IL-18 variant isYFGKLqSKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDVRDNAPRTIFIISMYdD SQPRGkAVTISVKVEKIlTLSVENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHD NKMQFESSSYEGYFLAVEKERDLFKLILKKEDELGDRSIMFTVQNED (IL- 18m30; SEQ ID NO: 30).Embodiment 8. The IL-18 variant of any one of embodiments 1-7, wherein the IL-18 variant isYFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDVRDNAPRTIFIISKYGD SQPRGLAVTISVKVEKISTLSVENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHR NKVQFESSSYEGYFLAVEKERDLFKLILKKEDELGDRSIMFTVQNED (IL- 18m31; SEQ ID NO: 31).Embodiment 9. The IL- 18 variant of any one of embodiments 1-8, further comprising an attenuator moiety at the N-terminus of the IL- 18 variant, wherein the attenuator moiety comprises from about one amino acid to about 70 amino acids in length.Embodiment 10. The IL- 18 variant of any one of embodiments 1-9, wherein the attenuator moiety is selected from any one of the attenuator sequences of Table 2.Embodiment 11. The IL- 18 variant of any one of embodiments 1-10, wherein the attenuator moiety is selected from of S, GGGGS (SEQ ID NO: 32). EV, DG. LM, THKM (SEQ ID NO: 36), DWNN (SEQ ID NO: 37), NTHR (SEQ ID NO: 38), TVDWIT (SEQ ID NO: 39), KHVLFF (SEQ ID NO: 40), ASRHVQ (SEQ ID NO: 41), LSSKDKVD (SEQ ID NO: 42), GKMNLSWY (SEQ ID NO: 43), HMWQQMYN (SEQ ID NO: 44), EIVHAIIFHK (SEQ ID NO: 45), LERIQTRYIQ (SEQ ID NO: 46), NRSKMMSMIR (SEQ ID NO: 48), MHRNWVAKHGHL (SEQ ID NO: 49), HHNKWKHLDFSH (SEQ ID NO: 50), DVVQSSESNQEW (SEQ ID NO: 51 ), NFSLERHMNNRMYE (SEQ ID NO: 52), WYKWFFYSRMHSIL (SEQ ID NO: 53), WFRRHGTKHGQFVI (SEQ ID NO: 54), and AAEPVEDNVINFVAMKFIDNTLYFIAENDENGGGS (SEQ ID NO: 61).Embodiment 12. The IL- 18 variant of any one of embodiments 1-11, further comprising a protease cleavage site between the attenuator moiety and the N-terminus of the IL- 18 variant.Embodiment 13. The IL- 18 variant of any one of embodiments 1-12, wherein the protease cleavage site is selected from a cleavage site recognized by granzyme B, granz me A, granzyme M, granzyme K, cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin K, cathepsin L, cathepsin G, kallikrein, plasmin, a collagenase, type IV collagenase, a stromelysin, Factor Xa, a chymotrypsin-like protease, a trypsin-like protease, an elastase-like protease, a subtilisin like protease, bromelain, a calpain, a caspase, papain, a HIV-1 protease, a HSV protease, a CMV protease, a chymosin, renin, pepsin, a matrix metalloprotease (MMP), or a metalloproteinase (ADAM).Embodiment 14. A nucleic acid sequence encoding an IL- 18 variant of any one of embodiments 1-13.Embodiment 15. A pharmaceutical composition comprising an IL- 18 variant of any one of embodiments 1-14, and a pharmaceutically acceptable excipient.Embodiment 16. A fusion protein comprising an IL- 18 variant and a scaffold polypeptide, wherein the IL- 18 variant comprises the amino acid sequence:wherein X1is Y or C;Embodiment 17. The fusion protein of embodiment 16, wherein the IL-18 variant comprises the amino acid sequence of SEQ ID NO: 1. whereinX1is Y or C;X2is E, K, Q, H, or Y;X3is S or C;Embodiment 18. The fusion protein of any one of embodiments 16-17, wherein the IL- 18 variant comprises the amino acid sequence selected from SEQ ID NOs 2, 3. 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 242, 243, 244, 245, 246, 247, 248, or 183.Embodiment 19. The fusion protein of any one of embodiments 16-18, wherein the scaffold polypeptide is selected from an immunoglobulin. Fc region of an immunoglobulin, human serum albumin (HSA), beta2 microglobulin, transferrin, fragment antigen-binding region (Fab region), VHH antibody, single-chain variable fragment (scFv), anticalin, designed ankyrin repeat protein (DARPin). type I transmembrane protein, or type II transmembrane protein or a fragment thereof.Embodiment 20. The fusion protein of any one of embodiments 16-19, further comprising an attenuator moiety at the N-terminus of the IL- 18 variant, wherein the attenuator moiety comprises from about one amino acid to about 70 amino acids in length.Embodiment 21. The fusion protein of any one of embodiments 16-20. wherein the attenuator moiety is selected from any one of the attenuator sequences of Table 2.Embodiment 22. The fusion protein of any one of embodiments 16-21, wherein the attenuator moiety is selected from of S, GGGGS (SEQ ID NO: 32). EV, DG. LM, THKM (SEQ ID NO: 36), DWNN (SEQ ID NO: 37), NTHR (SEQ ID NO: 38), TVDWIT (SEQ ID NO: 39), KHVLFF (SEQ ID NO: 40), ASRHVQ (SEQ ID NO: 41), LSSKDKVD (SEQ ID NO: 42), GKMNLSWY (SEQ ID NO: 43), HMWQQMYN (SEQ ID NO: 44), EIVHAIIFHK (SEQ ID NO: 45), LERIQTRYIQ (SEQ ID NO: 46), NRSKMMSMIR (SEQ ID NO: 48), MHRNWVAKHGHL (SEQ ID NO: 49), HHNKWKHLDFSH (SEQ ID NO: 50), DVVQSSESNQEW (SEQ ID NO: 51), NFSLERHMNNRMYE (SEQ ID NO: 52), WYKWFFYSRMHSIL (SEQ ID NO: 53), WFRRHGTKHGQFVI (SEQ ID NO: 54), and AAEPVEDNVINFVAMKFIDNTLYFIAENDENGGGS (SEQ ID NO: 61).Embodiment 23. The fusion protein of any one of embodiments 16-22, further comprising a protease cleavage site between the attenuator moiety and the N-terminus of the IL- 18 variant.Embodiment 24. The fusion protein of any one of embodiments 16-23, wherein the protease cleavage site is selected from a cleavage site recognized by granzyme B, granz me A, granzyme M, granzyme K, cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin K, cathepsin L, cathepsin G, kallikrein, plasmin, a collagenase, type IV collagenase, a stromelysin, Factor Xa, a chymotrypsin-like protease, a trypsin-like protease, an elastase-like protease, a subtilisin like protease, bromelain, a calpain, a caspase, papain, a HIV-1 protease, a HSV protease, a CMV protease, a chymosin, renin, pepsin, a matrix metalloprotease (MMP), or a metalloproteinase (ADAM).Embodiment 25. A nucleic acid sequence encoding a fusion protein of any one of embodiments 16-24.Embodiment 26. A pharmaceutical composition comprising a fusion protein of any one of embodiments 16-25, and a pharmaceutically acceptable excipient.Embodiment 27. A fusion protein comprising an IL- 18 variant and an anti-PDl antibody or a fragment thereof, wherein the IL- 18 variant comprises the amino acid sequence of:wherein X1is Y or C;Embodiment 28. The fusion protein of embodiment 27, wherein the IL- 18 variant comprises the amino acid sequence of SEQ ID NO: 1. whereinX1is Y or C;Embodiment 29. The fusion protein of embodiment 27 or 28. wherein the IL-18 variant comprises the amino acid sequence selected from SEQ ID NOs 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 242, 243, 244, 245, 246, 247, 248, or 183.Embodiment 30. The fusion protein of any one of embodiments 27-29, wherein the anti- PD 1 antibody or a fragment thereof comprises an antigen binding domain comprising a heavy chain complementarity determining region (HCDR) 1 having an amino acid sequence of SEQ ID NO: 108, a HCDR2 having an amino acid sequence of SEQ ID NO: 109, a HCDR3 having an ammo acid sequence of SEQ ID NO: 110. and a light chain complementarity determining region (LCDR) 1 having an amino acid sequence of SEQ ID NO: 111, a LCDR2 having an amino acid sequence of SEQ ID NO: 112, and a LCDR3 having an amino acid sequence of SEQ ID NO: 113.Embodiment 31. The fusion protein of any one of embodiments 27-30, wherein the anti- PD1 antibody or a fragment thereof comprises a variable heavy chain (VH) comprising an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 114, and a variable light chain (VL) comprising an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 115.Embodiment 32. The fusion protein of any one of embodiments 27-31, wherein the IL-18 variant comprises an amino acid sequence selected from SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27. 28. 29, 30, 31, 242, 243, 244, 245, 246. 247, 248, or 183, and wherein the anti-PDl antibody or a fragment thereof comprises a variable heavy chain (VH) comprising an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 114, and a variable light chain (VL) comprising an amino acid sequence that is at least 90%. at least 95%. at least 98%. at least 99%. or 100% identical to SEQ ID NO: 1 15.Embodiment 33. The fusion protein of any one of embodiments 27-32, wherein the IL-18 variant comprises an amino acid sequence of SEQ ID NO: 2, and wherein the anti-PDl antibody or a fragment thereof comprises a variable heavy chain (VH) comprising an amino acid sequence of SEQ ID NO; 1 14, and a variable light chain (VL) comprising an amino acid sequence of SEQ ID NO: 115.Embodiment 34. The fusion protein of any one of embodiments 27-33, wherein the IL- 18 variant comprises an amino acid sequence of SEQ ID NO: 9, and wherein the anti-PDl antibody or a fragment thereof comprises a variable heavy chain (VH) comprising an amino acid sequence of SEQ ID NO; 114, and a variable light chain (VL) comprising an amino acid sequence of SEQ ID NO: 115.Embodiment 35. The fusion protein of any one of embodiments 27-34, wherein the IL-18 variant comprises an amino acid sequence of SEQ ID NO: 15, and wherein the anti-PDl antibody or a fragment thereof comprises a variable heavy chain (VH) comprising an amino acid sequence of SEQ ID NO; 114, and a variable light chain (VL) comprising an amino acid sequence of SEQ ID NO: 115.Embodiment 36. The fusion protein of any one of embodiments 27-35, wherein the IL-18 variant comprises an amino acid sequence of SEQ ID NO: 16, and wherein the anti-PDl antibody or a fragment thereof comprises a variable heavy chain (VH) comprising an aminoacid sequence of SEQ ID NO; 114, and a variable light chain (VL) comprising an amino acid sequence of SEQ ID NO: 115.Embodiment 37. The fusion protein of any one of embodiments 27-36, wherein the IL-18 variant comprises an amino acid sequence of SEQ ID NO: 19, and wherein the anti-PDl antibody or a fragment thereof comprises a variable heavy chain (VH) comprising an amino acid sequence of SEQ ID NO; 114, and a variable light chain (VL) comprising an amino acid sequence of SEQ ID NO: 115.Embodiment 38. The fusion protein of any one of embodiments 27-37, wherein the IL-18 variant comprises an amino acid sequence of SEQ ID NO: 29, and wherein the anti-PDl antibody or a fragment thereof comprises a variable heavy chain (VH) comprising an amino acid sequence of SEQ ID NO; 114, and a variable light chain (VL) comprising an amino acid sequence of SEQ ID NO: 115.Embodiment 39. The fusion protein of any one of embodiments 27-38, wherein the IL- 18 variant comprises an amino acid sequence of SEQ ID NO: 30, and wherein the anti-PDl antibody or a fragment thereof comprises a variable heavy chain (VH) comprising an amino acid sequence of SEQ ID NO; 1 14, and a variable light chain (VL) comprising an amino acid sequence of SEQ ID NO: 115.Embodiment 40. The fusion protein of any one of embodiments 27-39, wherein the IL-18 variant comprises an ammo acid sequence of SEQ ID NO: 31, and wherein the anti-PDl antibody or a fragment thereof comprises a variable heavy chain (VH) comprising an amino acid sequence of SEQ ID NO; 114, and a variable light chain (VL) comprising an amino acid sequence of SEQ ID NO: 115.Embodiment 41. The fusion protein of any one of embodiments 27-40, wherein the antigen binding domain of the anti-PDl antibody is a diabody, a Fab, Fab’, aF(ab’)2, a variable fragment (Fv), a single chain variable fragment (scFv), a (ScFv)2, a Fd fragment, stapled scFv fragment (spFv), a disulfide stabilized Fv fragment (dsFv), or a disulfide stabilized diabody (ds diabody).42. The fusion protein of any one of embodiments 27-41. wherein the anti-PDl antibody further comprises a Fc region and wherein the Fc region comprises one or more amino acid substitutions selected from L234A / L235A / D265S; H435R / Y436F; S364K / E357Q;L368D / K370S; S364K; L368D / K370S; S364K; L368E / K370S; D401K;T411E / K360E / Q362E; T366W; and T366S / L368A / Y407V, wherein numbering is according to EU numbering.Embodiment 43. The fusion protein of any one of embodiments 27-42, wherein the anti- PDl antibody or a fragment thereof is an IgG.44. The fusion protein of any one of embodiments 27-43, wherein the IL-18 variant is covalently linked to N-terminal portion of a Fc region of anti-PDl antibody.Embodiment 45. The fusion protein of any one of embodiments 27-44, wherein a first IL- 18 variant is covalently linked to N-terminal portion of a Fc region of anti-PDl antibody and a second IL- 18 variant is covalently linked to C-terminal portion of a Fc region of anti- PDl antibody.Embodiment 46. The fusion protein of any one of embodiments 27-45, wherein the IL- 18 variant is covalently linked to N-terminal portion of a VL of anti-PDl antibody.Embodiment 47. The fusion protein of any one of embodiments 27-46, further comprising an attenuator moiety at the N-terminus of the IL-18 variant, wherein the attenuator moiety comprises from about one amino acid to about 70 amino acids in length.Embodiment 48. The fusion protein of any one of embodiments 27-47, wherein the attenuator moiety is selected from any one of the attenuator sequences of Table 2.Embodiment 49. The fusion protein of any one of embodiments 27-48, wherein the attenuator moiety is selected from of S, GGGGS (SEQ ID NO: 32). EV, DG, LM, THKM (SEQ ID NO: 36), DWNN (SEQ ID NO: 37), NTHR (SEQ ID NO: 38), TVDWIT (SEQ ID NO: 39), KHVLFF (SEQ ID NO: 40), ASRHVQ (SEQ ID NO: 41), LSSKDKVD (SEQ ID NO: 42), GKMNLSWY (SEQ ID NO: 43), HMWQQMYN (SEQ ID NO: 44), EIVHAIIFHK(SEQ ID NO: 45), LER1QTRY1Q (SEQ ID NO: 46), NRSKMMSM1R (SEQ ID NO: 48), MHRNWVAKHGHL (SEQ ID NO: 49), HHNKWKHLDFSH (SEQ ID NO: 50), DVVQSSESNQEW (SEQ ID NO: 51), NFSLERHMNNRMYE (SEQ ID NO: 52), WYKWFFYSRMHSIL (SEQ ID NO: 53), WFRRHGTKHGQFVI (SEQ ID NO: 54), and AAEPVEDNVINFVAMKFIDNTLYFIAENDENGGGS (SEQ ID NO: 61).Embodiment 50. The fusion protein of any one of embodiments 27-49, further comprising a protease cleavage site between the attenuator moiety and the N-terminus of the IL- 18 variant.Embodiment 51. The fusion protein of any one of embodiments 27-50, further comprising a protease cleavage site between IL-18 variant and the anti-PDl antibody or a fragment thereof.Embodiment 52. The fusion protein of any one of embodiments 27-51, wherein the protease cleavage site is selected from a cleavage site recognized by granzyme B, granzyme A, granzyme M, granzyme K, cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin K, cathepsin L, cathepsin G, kallikrein, plasmin, a collagenase, type IV collagenase, a stromelysin, Factor Xa, a chymotrypsin-like protease, a trypsin-like protease, an elastase-like protease, a subtilisin like protease, bromelain, a calpain, a caspase, papain, a HIV-1 protease, a HSV protease, a CMV protease, a chymosin, renin, pepsin, a matrix metalloprotease (MMP), or a metalloproteinase (ADAM).Embodiment 53. A nucleic acid sequence encoding a fusion protein of any one of embodiments 27-52.Embodiment 54. A pharmaceutical composition comprising a fusion protein of any one of embodiments 27-53, and a pharmaceutically acceptable excipient.Embodiment 55. A fusion protein comprising:(a) a first monomer comprising from N-terminus to C-terminus an IL-18 variant and a first Fc region;(b) a second monomer comprising from N-terminus to C-terminus a variable heavy chain (VH) and a second Fc region; and(c) a third monomer comprising a variable light (VL) chain, wherein the VH and VL form an antigen binding domain that binds to PD-1.Embodiment 56. The fusion protein of embodiment 55, wherein the IL-18 variant comprises the amino acid sequence of:wherein X1is Y or C;Embodiment 57. The fusion protein of embodiment 55 or 56, wherein the IL-18 variant comprises the amino acid sequence selected from SEQ ID NOs 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21. 22. 23, 24, 25, 26, 27, 28, 29, 30, 31. 242, 243, 244, 245, 246, 247, 248, or 183.Embodiment 58. The fusion protein of any one of embodiments 55-57. wherein the antigen binding domain comprises a heavy chain complementarity determining region (HCDR) 1 having an amino acid sequence of SEQ ID NO: 108, a HCDR2 having an amino acid sequence of SEQ ID NO: 109, a HCDR3 having an amino acid sequence of SEQ ID NO: 110, and a light chain complementarity determining region (LCDR) 1 having an amino acid sequence of SEQ ID NO: 11 1, a LCDR2 having an amino acid sequence of SEQ ID NO: 112, and a LCDR3 having an amino acid sequence of SEQ ID NO: 113.Embodiment 59. The fusion protein of any one of embodiments 55-58. wherein the VH comprises an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 114, and the VL comprises an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 115.Embodiment 60. The fusion protein of any one of embodiments 55-59, wherein the IL- 18 variant further comprising an attenuator moiety at the N-terminus, and wherein the attenuator moiety comprises from about one amino acid to about 70 amino acids in length.Embodiment 61. The fusion protein of any one of embodiments 55-60, wherein the attenuator moiety is selected from any one of the attenuator sequences of Table 2.Embodiment 62. The fusion protein of any one of embodiments 55-61, wherein the attenuator moiety is selected from of S, GGGGS (SEQ ID NO: 32). EV, DG, LM, THKM (SEQ ID NO: 36), DWNN (SEQ ID NO: 37). NTHR (SEQ ID NO: 38), TVDW1T (SEQ ID NO: 39), KHVLFF (SEQ ID NO: 40), ASRHVQ (SEQ ID NO: 41), LSSKDKVD (SEQ ID NO: 42), GKMNLSWY (SEQ ID NO: 43), HMWQQMYN (SEQ ID NO: 44), EIVHAIIFHK (SEQ ID NO: 45), LERIQTRYIQ (SEQ ID NO: 46), NRSKMMSMIR (SEQ ID NO: 48), MHRNWVAKHGHL (SEQ ID NO: 49), HHNKWKHLDFSH (SEQ ID NO: 50), DVVQSSESNQEW (SEQ ID NO: 51), NFSLERHMNNRMYE (SEQ ID NO: 52), WYKWFFYSRMHSIL (SEQ ID NO: 53), WFRRHGTKHGQFVI (SEQ ID NO: 54), and AAEPVEDNVINFVAMKFIDNTLYFIAENDENGGGS (SEQ ID NO: 61).Embodiment 63. The fusion protein of any one of embodiments 55-62, wherein one of the first and the second Fc regions comprises an amino acid substitution T366W, and the otherof the first and the second Fc regions comprises one or more amino acid substitutions T366S / L368A / Y407V, wherein numbering is according to EU numbering.Embodiment 64. The fusion protein of any one of embodiments 55-63, wherein the first and the second Fc regions comprise one or more amino acid substitutions selected from L234A / L235A / D265S; H435R / Y436F; S364K / E357Q; L368D / K370S; S364K;L368D / K370S; S364K; L368E / K370S; D401K; T411E / K360E / Q362E; T366W; and T366S / L368A / Y407V, wherein numbering is according to EU numbering.Embodiment 65. The fusion protein of any one of embodiments 55-64, wherein:(a) the first monomer is selected from the amino acid sequence represented by SEQ ID NOs: 137, 142, 148, 172, 173, 178, 179, 180, 181, 249, 250, 251, 252, 253, 254, and 255;(b) the second monomer is selected from the amino acid sequence represented by SEQ ID NOs: 146 and 1 16.(c) the third monomer is selected from the amino acid sequence represented by SEQ ID NO: 117.Embodiment 66. The fusion protein of any one of embodiments 55-65, wherein the first monomer comprises the amino sequence of SEQ ID NO: 137, the second monomer comprises the amino acid sequence of SEQ ID NO: 146, and the third monomer comprises the amino acid sequence of SEQID NO: 117.Embodiment 67. The fusion protein of any one of embodiments 55-66, wherein the first monomer comprises the amino sequence of SEQ ID NO: 142, the second monomer comprises the amino acid sequence of SEQ ID NO: 146, and the third monomer comprises the amino acid sequence of SEQID NO: 117.Embodiment 68. The fusion protein of any one of embodiments 55-67, wherein the first monomer comprises the amino sequence of SEQ ID NO: 148, the second monomer comprises the amino acid sequence of SEQ ID NO: 146, and the third monomer comprises the amino acid sequence of SEQID NO: 1 17.Embodiment 69. The fusion protein of any one of embodiments 55-68. wherein the first monomer comprises the amino sequence of SEQ ID NO: 172, the second monomer comprises the amino acid sequence of SEQ ID NO: 146, and the third monomer comprises the amino acid sequence of SEQID NO: 117.Embodiment 70. The fusion protein of any one of embodiments 55-69, wherein the first monomer comprises the amino sequence of SEQ ID NO: 173, the second monomer comprises the amino acid sequence of SEQ ID NO: 146, and the third monomer comprises the amino acid sequence of SEQID NO: 117.Embodiment 71. The fusion protein of any one of embodiments 55-70, wherein the first monomer comprises the amino sequence of SEQ ID NO: 178, the second monomer comprises the amino acid sequence of SEQ ID NO: 116, and the third monomer comprises the amino acid sequence of SEQID NO: 1 17.Embodiment 72. The fusion protein of any one of embodiments 55-71, wherein the first monomer comprises the amino sequence of SEQ ID NO: 179, the second monomer comprises the amino acid sequence of SEQ ID NO: 116, and the third monomer comprises the amino acid sequence of SEQID NO: 1 17.Embodiment 73. The fusion protein of any one of embodiments 55-72, wherein the first monomer comprises the amino sequence of SEQ ID NO: 180, the second monomer comprises the amino acid sequence of SEQ ID NO: 116, and the third monomer comprises the amino acid sequence of SEQID NO: 1 17.Embodiment 74. The fusion protein of any one of embodiments 55-73, wherein the first monomer comprises the amino sequence of SEQ ID NO: 181, the second monomer comprises the amino acid sequence of SEQ ID NO: 116, and the third monomer comprises the amino acid sequence of SEQID NO: 117.Embodiment 75. A method of treating a subject suffering from cancer comprises administering to the subject a composition comprising an IL-18 fusion protein, wherein the IL-18 fusion protein comprises IL- 18 variant and an anti-PDl antibody or a fragment thereof, and wherein the IL- 18 variant comprises the amino acid sequence of:wherein X1is Y or C;Embodiment 76. The method of embodiment 75, wherein the IL-18 variant comprises the amino acid sequence of SEQ ID NO: 1, whereinX1is Y or C;Embodiment 77. The method of embodiment 75 or 76. wherein the IL-18 variant comprises an amino acid sequence selected from SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 242, 243, 244, 245, 246, 247, 248, or 183, and wherein the anti-PDl antibody comprises a variable heavy chain (VH) comprising an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 114, and a variable light chain (VL) comprising an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 115.Embodiment 78. The method of any one of embodiments 75-77. wherein the cancer is solid cancer.Embodiment 79. The method of any one of embodiments 75-78, wherein the solid cancer is selected from adrenal cancer, anal cancer, bile duct cancer, bladder cancer, bone cancer, brain cancer, breast cancer, carcinoid cancer, cervical cancer, colorectal cancer, esophageal cancer, eye cancer, gallbladder cancer, gastrointestinal stromal tumor, germ cell cancer, head and neck cancer, kidney cancer, liver cancer, lung cancer, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, neuroendocrine cancer, oral cancer, oropharyngeal cancer, ovarian cancer, pancreatic cancer, pediatric cancer, penile cancer, pituitary cancer, prostatecancer, skin cancer, soft tissue cancer, spinal cord cancer, stomach cancer, testicular cancer, thymus cancer, thyroid cancer, ureteral cancer, uterine cancer, vaginal cancer, or vulvar cancer.Embodiment 80. The method of any one of embodiments 75-79. wherein the IL- 18 variant comprises an amino acid sequence of SEQ ID NO: 2, and the anti-PDl antibody or a fragment thereof comprises a variable heavy chain (VH) comprising an amino acid sequence of SEQ ID NO: 114, and a variable light chain (VL) comprising an amino acid sequence of SEQ ID NO: 115.Embodiment 81 . The method of any one of embodiments 75-80, wherein the IL- 18 variant comprises an amino acid sequence of SEQ ID NO: 9, and the anti-PDl antibody or a fragment thereof comprises a variable heavy chain (VH) comprising an amino acid sequence of SEQ ID NO: 114, and a variable light chain (VL) comprising an amino acid sequence of SEQ ID NO: 1 15.Embodiment 82. The method of any one of embodiments 75-81, wherein the IL- 18 variant comprises an amino acid sequence of SEQ ID NO: 15, and the anti-PDl antibody or a fragment thereof comprises a variable heavy chain (VH) comprising an amino acid sequence of SEQ ID NO: 114, and a variable light chain (VL) comprising an amino acid sequence of SEQ ID NO: 115.Embodiment 83. The method of any one of embodiments 75-82. wherein the 1L- 18 variant comprises an amino acid sequence of SEQ ID NO: 16, and the anti-PDl antibody or a fragment thereof comprises a variable heavy chain (VH) comprising an amino acid sequence of SEQ ID NO: 114, and a variable light chain (VL) comprising an amino acid sequence of SEQ ID NO: 115.Embodiment 84. The method of any one of embodiments 75-83, wherein the IL- 18 variant comprises an amino acid sequence of SEQ ID NO: 19, and the anti-PDl antibody or a fragment thereof comprises a variable heavy chain (VH) comprising an amino acid sequence of SEQ ID NO: 114, and a vanable light chain (VL) comprising an amino acid sequence of SEQ ID NO: 115.Embodiment 85. The method of any one of embodiments 75-84, wherein the IL- 18 variant comprises an amino acid sequence of SEQ ID NO: 29, and the anti-PDl antibody or a fragment thereof comprises a variable heavy chain (VH) comprising an amino acid sequence of SEQ ID NO: 114, and a variable light chain (VL) comprising an amino acid sequence of SEQ ID NO: 115.Embodiment 86. The method of any one of embodiments 75-85, wherein the IL- 18 variant comprises an amino acid sequence of SEQ ID NO: 30, and the anti-PDl antibody or a fragment thereof comprises a variable heavy chain (VH) comprising an amino acid sequence of SEQ ID NO: 114, and a variable light chain (VL) comprising an amino acid sequence of SEQ ID NO: 115.Embodiment 87. The method of any one of embodiments 75-86. wherein the IL- 18 variant comprises an amino acid sequence of SEQ ID NO: 31 , and the anti-PDl antibody or a fragment thereof comprises a variable heavy chain (VH) comprising an amino acid sequence of SEQ ID NO: 114, and a variable light chain (VL) comprising an amino acid sequence of SEQ ID NO: 115.Embodiment 88. A method of treating a subject suffering from cancer comprises administering to the subject an IL- 18 fusion protein comprising:(a) the first monomer is selected from the amino acid sequence represented by SEQ ID NOs: 137, 142. 148, 172, 173. 178, 179. 180, 181, 249. 250, 251. 252. 253, 254, and 255;(b) the second monomer is selected from the amino acid sequence represented by SEQ ID NOs: 146 and 116; and(c) the third monomer is selected from the amino acid sequence represented by SEQ ID NO: 117.Embodiment 89. The method of embodiment 88, wherein the fusion protein comprises the first monomer comprising the amino sequence of SEQ ID NO: 137. the second monomer comprising the amino acid sequence of SEQ ID NO: 146, and the third monomer comprising the amino acid sequence of SEQID NO: 1 17.Embodiment 90. The method of embodiment 88 or 89, wherein the fusion protein comprises the first monomer comprising the amino sequence of SEQ ID NO: 142, the second monomer comprising the amino acid sequence of SEQ ID NO: 146, and the third monomer comprising the amino acid sequence of SEQID NO: 117.Embodiment 91. The method of any one of embodiments 88-90, wherein the fusion protein comprises the first monomer comprising the amino sequence of SEQ ID NO: 148, the second monomer comprising the amino acid sequence of SEQ ID NO: 146, and the third monomer comprising the amino acid sequence of SEQID NO: 117.Embodiment 92. The method of any one of embodiments 88-91, wherein the fusion protein comprises the first monomer comprising the amino sequence of SEQ ID NO: 172, the second monomer comprising the amino acid sequence of SEQ ID NO: 146, and the third monomer comprising the amino acid sequence of SEQID NO: 1 17.Embodiment 93. The method of any one of embodiments 88-92, wherein the fusion protein comprises the first monomer comprising the amino sequence of SEQ ID NO: 173, the second monomer comprising the amino acid sequence of SEQ ID NO: 146, and the third monomer comprising the amino acid sequence of SEQID NO: 117.Embodiment 94. The method of any one of embodiments 88-93, wherein the fusion protein comprises the first monomer comprising the amino sequence of SEQ ID NO: 178, the second monomer comprising the amino acid sequence of SEQ ID NO: 116, and the third monomer comprising the amino acid sequence of SEQID NO: 117.Embodiment 95. The method of any one of embodiments 88-94, wherein the fusion protein comprises the first monomer comprising the amino sequence of SEQ ID NO: 179, the second monomer comprising the amino acid sequence of SEQ ID NO: 116, and the third monomer comprising the amino acid sequence of SEQID NO: 117.Embodiment 96. The method of any one of embodiments 88-95, wherein the fusion protein comprises the first monomer comprising the amino sequence of SEQ ID NO: 180, thesecond monomer comprising the amino acid sequence of SEQ ID NO: 116, and the third monomer comprising the amino acid sequence of SEQID NO: 117.Embodiment 97. The method of any one of embodiments 88-96, wherein the fusion protein comprises the first monomer comprising the amino sequence of SEQ ID NO: 181, the second monomer comprising the amino acid sequence of SEQ ID NO: 116, and the third monomer comprising the amino acid sequence of SEQID NO: 117.EXAMPLES

[0203] The following examples are provided to better illustrate the claimed invention and are not to be interpreted as limiting the scope of the invention. To the extent that specific materials are mentioned, it is merely for purposes of illustration and is not intended to limit the invention. One skilled in the art may develop equivalent means or reactants without the exercise of inventive capacity and without departing from the scope of the invention.EXAMPLE 1

[0204] As shown in figure 2. we examined die capacity of polypeptides ranging in length from 1 amino acid to 14 amino acids to impact the functional activity of IL-18. These polypeptides were chosen for randomness to include maximum heterogeneity including the parameters of length, charge and hydrophobicity. The polypeptides were fused to the N-terminus of an exemplary mature IL-18, which was further part of a fusion protein including the anti-PDl fab from nivolumab. To assess the functional activity of the array of N-terminal polypeptide / IL-18 variants, we treated HEK-Blue IL-18 reporter cells with the library of the aforementioned test articles. HEK-Blue IL-18 reporter cells contain an NF-KB / AP -promoter element that drives the expression of secreted embryonic alkaline phosphatase (SEAP) in a dose dependent manner in the presence of IL-18. FIG. 2A is a non-linear x-y graph of SEAP release (IL-18 activity) relative to the concentration of test article. Human recombinant IL-18 was used as the positive control (black squares) and human recombinant TNF-alpha as the negative control (grey square). A vast array of potency (EC50- SEAP) was observed whereby the N-terminal polypeptides attenuated IL-18 from 3 fold to >10,000 fold. We identified a relationship between the composition of the N-terminal polypeptide and the degree of attenuation. FIG. 2B is a regression analysis of N-terminal polypeptide length versus potency (EC50-SEAP), in which die R-squared (R) value was 0.25 indicative of a weak correlation. When we assessed the functional activity (FIG. 2C) and performed regression analysis (FIG. 2D) for non-charged N-terminal polypeptides consisting of glycine and serine residues, wc observed a strong correlation with an R value of 0.88. Given that length N- tennmal polypeptides only sometimes directly linked to the degree their capacity to atenuate IL-18, we alsoexamined hydrophobicity and charge density. For charge density, regression analysis consisting of N- terminal charge density versus potency (EC50-SEAP) for the large array of N-terminal polypeptides resulted in the observation of a moderate correlation with an associated R value of 0.67 (FIG. 2E). Finally, we combined length and charge density using the equation:and perfonned regression analysis consisting of the combined values versus potency (EC50-SEAP) for the large array of N-tenninal polypeptides. The results indicated a strong correlation with a R value of 0.8 (FIG. 2F). Summary tables of potencies (EC50-SEAP) are shown beneath the non-linear x-y graphs for FIGs 2A and 2C.EXAMPLE 2

[0205] As shown in figure 3, we assessed the capacity of an IL-18 variant targeted via fusion to a HER-2 specific antibody like protein in various formats to induce Antibody Dependent Cellular Cytotoxicity (ADCC) and IFNy release. PBMC (source of NK cells) from a healthy donor were co-cultured with HER2+ T47D tumor cells transduced with eGFP and firefly luciferase. Bioluminescence was measured after 48 hours as a measure of tumor cell viability and associated PBMC (NK cell) mediated ADCC. FIG. 3A is a non-linear x-y plot of tumor cell killing as a function of test article concentration. FIG. 3B is a non-linear x-y plot of IFNy release as a function of test article concentration. The formats tested were (1) an exemplary IL- 18 variant fused to the N-terminus of the trastuzumab light chain in a monovalent version of FIG. 1C (FUSE-1144; black square), (2) an exemplary IL-18 variant fused to the N-terminus of the hole heavy chain and a trastuzumab fab on the N-terminus of the knob heavy chain as per FIG. 1 A (FUSE-1099; black circle), (3) an exemplary IL-18 variant fused to the N-terminus and C-terminus of the hole heavy chain and a trastuzumab fab on the N-terminus of the knob heavy chain as per FIG. IB (FUSE-1100; open circle), (4) an exemplary IL- 18 variant fused to the N-terminus of the knob- heavy chain and trastuzumab fabs on both the N-terminus and C-terminus of the knob heavy chain as per FIG. ID (FUSE- 1101 ; open triangle), and (5) an exemplary IL-18 variant fused to the C-terminus of the knob heavy chain and trastuzumab fabs on both the N-terminus knob and hole heavy chains (FUSE-1142; black triangle). All IL-18 variants included an N-terminal polypeptide shown to attenuate the cytokine by 100 fold. The benchmark antibody was a trastuzumab biosimilar (open square). The negative control was an anti-Hen Egg Lysozyme (HEL) antibody. The rank order for the magnitude (considers both EC50 and Emax) of tumor cell killing wasFUSE-1101, FUSE-1142, FUSE-1100, FUSE-1109, trastuzumab and FUSE-1144. The rank order for the magnitude of IFNy release was FUSE- 1101, FUSE-1142, FUSE-1100, FUSE- 1109, FUSE-1144 and trastuzumab. Summary tables of potencies (EC50-SEAP), Emax and Area Under the curve (AUC) are shown beneath the non-linear x-y graphs for FIGs 3A and 3B.EXAMPLE 3

[0206] As shown in figure 4, we assessed the capacity of anti-PDl antibodies incorporating one of two exemplary IL- 18 variants to induce T cell mediated tumor cell killing (FIGs 4A and 4C) and IFNy release (FIGs 4B and 4D). For FIGs 4A and 4C, the IL- 18 variant was approximately 100 fold less potent than the IL- 18 variant used for FIGs 4B and 4D. All IL- 18 variants included an N-terminal polypeptide shown to attenuate the cytokine by 100 fold. For the purposes on this example, any enhancement of tumor cell killing and IFNy release relative to the impact of anti-PDl alone is interpreted as the result of a cis based interaction between pro-IL-18 and the IL-18R complex expressed on PD-1+ activated T cells. The assay system used consisted of purified human T cells mixed with MDA-MB-231 (ROR1+) stably expressing firefly luciferase and eGFP at an E:T ratio of 10: 1 and 10 pM of a ROR1 x CD3 bsAb (Fuse608). Fuse608 served to create an artificial synapse between the T cells and ROR1+ tumor cells and deliver a suboptimal signal via the TCR / CD3 complex.

[0207] A titration of test articles was added to the aforementioned T cell / tumor cell co-culture system. Tumor cell killing and IFNy release were measured after 72 hours. The test articles assessed for the non-linear x-y plots, FIGs 4A and 4B were: (1) anti-PDl (FUSE-1041; black reverse triangle), (2) an exemplary IL-18 variant fused to the N-terminus of the anti-PDl light chains as per FIG. 1C (FUSE-1219; black circle), and (3) an exemplary IL- 18 variant fused to the N-terminus of the hole heavy chain and an anti-PDl fab on the N- terminus of the knob heavy chain as per FIG. 1A (FUSE-1177; open circle). The test articles assessed for the non-linear x-y plots, FIGs 4C and 4D were:(1) anti-PDl (FUSE-1210; open triangle), (2) an exemplary IL-18 variant fused to the N-terminus of the anti-PDl light chains as per FIG. 4C (FUSE-121 1 ; black triangle), and (3) an exemplary IL- 18 variant fused to the N-terminus of the hole heavy chain and an anti-PDl fab on the N-terminus of the knob heavy chain as per FIG. 4A (FUSE- 1213; black circle). In all cases, anti-HEL antibody (gray circle) was used as theisotype matched negative control. A summary table of EC50, Emax and AUC (area under the curve) is shown beneath each x-y plot.EXAMPLE 4

[0208] As shown in figure 5, we assessed the impact of exemplary anti-PDl-pro- IL-18 antibodies containing different IL-18 variants on IFNy release mediated by PBMC activated with PeliCluster CD3 for 48 hours, washed and the re-exposed to a 10 fold lower concentration of PeliCluster CD3 for an additional 48 hours. PBMC was two different healthy human donors were assessed. Fuse 599 (IL-18m9A-Fc) was used as the non-targeted benchmark with similar activity to wild type IL-18 (open grey circles, dashed line). Fusel 109 (black squares) and Fusel l77 (black diamonds) consisted of PD-1 targeted IL-18ml3A without and with a 100 fold N-terminal attenuator, respectively. Fuse 1229 (black downward facing triangles) and Fuse 1179 (black upward facing triangles) consisted of PD-1 targeted IL-18 ml3. 1A and IL-18ml6A , both containing a 100 fold N-terminal attenuator. Relative to Fuse599, Fusel 109 was about 100 fold attenuated (EC50) whereas Fusel 177, Fusel229, and Fusel 179 were all greater than 3000 (donor 2) to greater than 1000 fold (donor 1) attenuated (EC50). Relative to Fuse599, the Emax for Fusel 177. Fusel229, and Fusel 179 was also reduced by >60%. The human IgGl antibody. anti-HEL (gray circle), was used as the isotype control and was associated with background IFNy release. A summary table of EC50, Emax and AUC (area under the curve) values is shown below the x-y plots.EXAMPLE 5

[0209] As shown in figure 6, we assessed the capacity of anti-PDl-pro-IL-18 antibodies containing different IL- 18 variants to oppose functional exhaustion induced by sequential stimulation. To this aim, pan T cells from a healthy human donor were mixed with Jurkat T cells stably expressing eGFP and the TAA. ROR1. The effector the target ratio (E:T) was 10: 1. On day 0, the cells were stimulated using the suboptimal concentration of a ROR1 x CD3 bsAb (Fuse608) equating 20% maximum killing and one of the following test articles at the lOx their EC99 of killing (i.e. on the killing plateau): Fuse 691 (open reverse triangle; anti-PDl), Fusel 109 (open circle; anti-PD-l-IL-18ml3A without a 100 fold N-terminal attenuator) or Fusel229 (closed circle; anti-PD-l-IL-18ml3.1A with a 100 fold N-terminal attenuator). In all cases, anti-HEL antibody (gray circle) w as used as the isotype matched negative control. Every 72 hours, the number of viable GFP+ cells was counted as a measureof tumor cell killing, the number of viable GFP-negative cells was counted as a measure of T cell survival and supernatant was collected for measuring IFNy release. Additionally, the cells mixtures were washed, tumor cells added back to an E:T of 10: 1 and all the test articles added again to same concentrations used on day 0. FIG. 6A, FIG. 6B and FIG. 6C are nonlinear x-y plots of viable T cell numbers, viable tumor cell numbers and the concentration of IFNy in the supernatant as a function of time.EXAMPLE 6

[0210] As shown in figure 7, we examined the capacity of different single amino acids fused to the N-terminus of an exemplary' IL-18 variant to impact functional activity. These single amino acids (an expansion of only S, tested in FIG. 2), were chosen for randomness to include maximum heterogeneity including the parameters of charge and hydrophobicity. The single amino acids were fused to the N-terminus of an exemplary mature IL-18 (IL-18mut2A), which was further part of a fusion protein including the anti-PDl fab from nivolumab (or the anti-mouse PD1 fab from RMPL 14 for the 10,000 fold attenuated control, FUSE-1015). To assess the functional activity of the array of N-terminal amino acids, we treated HEK-Blue IL- 18 reporter cells with the library of the aforementioned test articles. HEK-Blue IL-18 reporter cells contain an NF-KB / AP-promoter element that drives the expression of secreted embryonic alkaline phosphatase (SEAP) in a dose dependent manner in the presence of IL-18. FIG. 7A is anon-linear x-y graph of SEAP release (IL-18 activity) relative to the concentration of test article. Human recombinant IL- 18 was used as the positive control (black squares) and human recombinant TNF-alpha as the negative control (grey square). A range of potency (EC50-SEAP) was observed whereby the N- terminal amino acid attenuated IL- 18 from -100 fold to -1000 fold (see test articles incorporating a dotted line) relative to human recombinant IL-18. As anticipated. N-terminal fusion of the propeptide variant. AAEPVEDNVINFVAMKFIDNTLYFIAENDENGGGS (SEQ ID NO: 61), to IL-18, induced -10,000 fold reduced activity (FUSE1015; open circles). A summary' table of potency (EC50-SEAP) is show n beneath the non-linear x-y graphs as FIG 7B. We added these IL-18 variants to the array from FIG. 2 to examine the relationship between the composition of the N-terminal amino acid or polypeptide variant and the degree of attenuation. The two best relationships were identified when flexibility' measured as pLDDT score and negative charge density were used in a two parameter equation (FIG. 7C), or when charge residues, charge position and flexibility (predicted as per Meng et Kurgan(https: / / doi.org / 10. 1093 / bioinformatics / btw280) (FIG. 7D) were used in a four parameter equation. The equations are:The use of two parameters for the equation in FIG 7C resulted in a R value of 0.69 w hereas the equation in FIG. 7D, which incorporated four parameters, resulted in a R value of 0.75.

[0211] The following parameters were analyzed and are shown in a Pearson correlation matrix in FIG 7E: Length: number of amino acids; 1 / length: inverse value of length; Positive charge: number of positive charged residues (K, R); Positive charge density: number of positive charged residues (K, R) divided by length; Negative charge: number of negative charged residues (D, E); Negative charge density: number of negative charged residues (D, E) divided by length; Charged residues: number of positive or negative charged residues (D, E, K, R); Charge density: number of positive or negative charged residues (D. E, K, R) divided by length; pl: theoretical isoelectric point; hydrophobicity: sum of hydropathy value for each residue; GRAVY: mean hydrophobicity; GRAVY / length: mean hydrophobicity divided by length; pl / length: theoretical isoelectric point divided by length; molecular weight: theoretical molecular weight; 1 / molecular weight: inverse value of theoretical molecular weight; Flexibility: theoretical mean flexibility; Flexibility sum: theoretical sum of flexibility for each residue; Charge position: sum of position of positive or negative charged residues (D, E, K, R); Positive charge position: sum of position of positive charged residues (K, R); Negative charge position: sum of position of negative charged residues (D, E); Charge position adjusted: sum of position of positive or negative charged residues (D, E, K, R) multi plicated by (+1) for positive charged residues and (-1) for negative charged residues; pLLDT score residue 1: pLDDT score based on AlphaFold3 simulation for residue 1 of mature protein; pLLDT score residue 2: pLDDT score based on AlphaFold3 simulation for residue 2 of mature protein; pLLDT score residue 0: pLDDT score based on AlphaFold3 simulation for residue 0 (residue before residue 1 of mature protein); pLLDT linker: sum of pLDDT scores for each residue; pLLDT linker mean: mean pLDDT score for residues; pLDDT overall: sum of pLLDT linker, pLLDT score residue 1, and pLLDT scoreresidue 2; pLDDT overall mean: pLLDT overall divided by length + 2; CABS-flex residue 1: Root Mean Square Fluctuation in Angstroms for residue 1 of mature protein; CABS-flex residue 2: Root Mean Square Fluctuation in Angstroms for residue 2 of mature protein; CABS-flex residue 0: Root Mean Square Fluctuation in Angstroms for residue 0 (residue before residue 1 of mature protein); CABS-flex linker: sum of Root Mean Square Fluctuation in Angstroms for each residue; CABS-flex linker mean: mean Root Mean Square Fluctuation in Angstroms for residues; CABS-flex overall: sum of CABS-flex linker, CABS-flex residue 1, and CABS-flex residue 2; CABS-flex overall mean: CABS-flex overall divided by length + 2; Cabs flex residue -n+1: Root Mean Square Fluctuation in Angstroms for residue -1 (residue two positions before residue 1 of mature protein).EXAMPLE 7

[0212] As shown in figure 8, we examined the capacity of exemplary IL-18 variants targeted to human PD-1 via the fab from nivolumab versus cetrelimab to activate HEK Blue IL- 18 expressing human PD-1. The exemplary mature IL- 18 variant was IL18mutl3 (Fusel233, Fusel l77) or IL18mutl3.1 (Fusel229). Both of these IL18 variants are about 100 fold attenuated relative to recombinant human IL-18 and are considered functionally equivalent. In all cases, the IL 18 variant contained an N-terminal peptide that mediated an additional approximately 100 fold attenuation. Fusel777 and Fusel229 are targeted to human PD-1 via the fab from nivolumab whereas Fusel 233 contains the fab from cetrelimab. To assess functional activity7, we treated HEK-Blue IL-18 reporter cells stably expressing human PD-1 with each of Fusel233, Fusel229 or Fusel 177. HEK-Blue IL-18 reporter cells contain an NF-KB / AP-promoter element that drives the expression of secreted embryonic alkaline phosphatase (SEAP) in a dose dependent manner in the presence of IL- 18. FIG. 8A is a non-linear x-y graph of SEAP release (IL-18 activity ) relative to the concentration of test article. Human recombinant IL- 18 (rhIL18) was used as the positive control (black squares) and human recombinant TNF-alpha as the negative control (grey square). Relative to rhIL18, Fusel 177 (black open triangle) and Fusel229 (black open circle), both of which are targeted to PD1 via the fab from nivolumab, demonstrate similar activity, indicating that IL18mutl3 and IL18mutl3.1 are functionally equivalent in this assay system. IL18mutl3 targeted to PD1 via the fab from cetrelimab (Fusel233; black open square) demonstrated about lOx stronger potency (EC50) than IL18mutl3.1 targeted to PD1via the fab from nivolumab (Fusel 229). A summary table of potency (EC50-SEAP) is shown beneath the non-linear x-y graph (FIG. 8B).EXAMPLE 8

[0213] As shown in figure 9, we examined the capacity of nivolumab and cetrelimab with or without fusion to exemplary IL- 18 to reduce binding between human PD-1 and human PD-L1. To do so, the PD-1 [Biotinylated] : PD-L1 Inhibitor Screening ELISA Kit (ACROBiosystems, Cat No: EP-101) was used whereby recombinant PDL1 is immobilized and test articles assessed for their capacity to block binding of soluble biotinylated PD1 its immobilized ligand. FIG. 9A and FIG. 9B are non-linear x-y graphs of PD-1 / PDL1 binding measured as the colorimertric intensity OD (450) of HRP oxidized 3, 3', 5,5'- tetramethylbenzidine relative to the concentration of test article. Anti-HEL was used as the negative control (gray squares). In FIG. 9A, a bivalent IgGl-LALA mutations (L234A and L235A in the Fc region) of cetrelimab (Fusel210; black squares) was compared to a bivalent IgGl -LALA mutations of nivolumab (Fuse691 ; black open squares). In FIG. 9B, the IgGl - LALA knob into hole construct containing an exemplary IL 18 on the hole and the fab from cetrelimab on the knob (Fusel213; black triangles) was compared to the IgGl-LALA knob into hole construct containing an exemplary IL 18 on the hole and the fab from nivolumab on the knob (Fusel229; black open triangles). We observed that (a) Fusel210 was about 2-3 times more effective at blocking the interaction between PD1 and PDL1 compared to Fuse691 in a dose dependent manner and (b) Fusel213 was approximately 10 times more effective at blocking the interaction between PD1 and PDL1 compared to Fuse 1229 in a dose dependent manner. The negative control had no impact on binding of PD1 to PDL1 at any concentration tested. As such, cetrelimab, especially as a monovalent moiety in the context of an IL- 18 variant fusion protein is a more effective inhibitor of PD1 / PDL1 binding than nivolumab. The IC50 values for each test article are shown within each graph.EXAMPLE 9

[0214] As shown in figure 10, we examined the apparent binding affinity of nivolumab and cetrelimab with or without fusion to exemplary IL-18 to PD1 via flow’ cytometry. FIGs 10A and 10B are non-linear line graphs of binding intensity via MFI versus concentration of test article. In FIG. 10A, a bivalent IgGl-LALA mutation of cetrelimab (Fusel210; black squares) was compared to the IgGl-LALA knob into hole constructcontaining an exemplary 1L18 with a 100 fold attenuating N-terminal peptide on the hole and the fab from cetrelimab (Fusel 213; black open squares). 293-T cells stably expressing human PD1 were used as the cellular binding target. Little difference was observed between bivalent Fusel210 and monovalent Fusel213, with apparent affinities ranging from 1.6 nM (Fusel210) to 2.3 nM (Fusel213). In FIG. 10B. a bivalent IgGl-LALA version of nivolumab (Fuse691; black triangles) was compared to the IgGl-LALA knob into hole construct containing an exemplary IL 18 IL 18 with a 100 fold attenuating N-terminal peptide on the hole and the fab from nivolumab (Fusel229; black open triangles). HEK-293 cells stably- expressing human PD1 were used as the cellular binding target. Bivalent Fuse691 had an apparent affinity for human PD1 of 2.8 nM, about 3-10 fold stronger than the apparent affinity of monovalent Fusel 229. Taken together, the observations suggest that bivalency plays a lesser role in contributing to the apparent affinity- of cetrelimab than nivolumab for PD1, which may help explain the finding that monovalent centrelimab-IL18 variant fusion has an apparent affinity for PD1 that is about 5-10 folder stronger than the monovalent nivolumab-ILl 8 variant fusion. The EC50 apparent affinity values for each test article are printed within each graph.EXAMPLE 10

[0215] As shown in figure 11, we assessed the capacity of an exemplary IL-18 variant targeted to human PD-1 via the fab from cetrelimab versus the fab from nivolumab to mediate tumor growth inhibition in aggressive MC38 bearing C57BL / 6 human PD1 knock in mice. One million fast growing, anti-PDl / PDLl resistant MC38i tumor cells were injected subcutaneously into the flank of hPDl KI C57BL / 6 mice (GemPharmatech). When tumor volume reached -100 mmA3 (day 0), treatment was administered on days 0, 4 and 7 at 15 mg / kg. The study was terminated on Day 21. FIGs 11 A (PBS), 1 IB (Fusel 114; IgGl-LALA knob into hole construct containing IL18mut2A with a 100 fold attenuating N-terminal peptide on the hole and the fab from nivolumab on the knob). 11C (Fuse691; IgGl-LALA version of nivolumab), and 1 ID (Fusel213; IgGl-LALA knob into hole construct containing IL18mut2A with a 100 fold attenuating N-terminal peptide on the hole and the fab from cetrelimab on the knob), are line graphs of tumor volume as measured via calipers as a function of days. Each line represents an individual mouse. Compared to PBS (gray circles), we did not observe an appreciable difference in tumor grow th in mice receiving Fuse691 (black open squares). On average, we observed about a 50% TGI mice receiving Fusel 114(black squares) and Fusel213 (black triangles). TGI was quite heterogenous in mice receiving Fusel 114 and much of the mean TGI resulted from two mice, one of which obtained a complete response. Interestingly, although a complete response was not observed in the group treated with Fusel213, TGI across the group was less heterogeneous, whereby four out of 5 mice experienced TGI of at least 50%. Overall, although the sample size was small, one may interpret the data as representing a trend towards superiority of Fusel213 relative to Fusel 114.EXAMPLE 11

[0216] As shown in figure 12, we examined the capacity of a set of IL-18 muteins to induce non-targeted functional activity via activation of HEK Blue IL-18. In this set of proteins, each IL- 18 mutein w as fused w ithout an N-terminal attenuating peptide to the N- terminus of a knobs into holes Fc. In some cases, a fab targeting human or mouse PD1 was included using the format shown in FIG 1 A but this arm did not impact the incorporated IL- 18 mutein’s activity because the HEK Blue IL18 reporter cell line used did not express human nor mouse PD1. All IL18 muteins except Fuse831 (IL18mut2AS, in which all cysteines substituted to serines) were engineered with all cysteines substituted to valines for (1) reduced binding to human IL-18BP (IL- 18 muteins 2-9) and (2) reduced activity relative to wild type IL-18 (IL18 muteins 10-16, 31) and / or (3) enhanced thermostability (IL 18 mutein 19, also termed 13.1). FIG. 12 is a non-linear x-y graph of SEAP release (IL-18 activity) relative to the concentration of test article. Human recombinant wild type IL-18 was used as the positive control (black squares) and human recombinant TNF-alpha as the negative control (grey square). All test articles are annotated with symbols that are indicated within the legend. A range of potency (EC50-SEAP) was observed w hereby IL 18 muteins 2, 3, 4, 5, 6, 8, 10, 12 w ere not appreciably different from recombinant IL-18, IL18 muteins 7 and 9 w ere ~5 fold more potent than recombinant IL- 18, IL 18 muteins 11 and 31 (and IL18mut2AS) were about 10 fold less potent than recombinant IL- 18, IL 18 muteins 13 and 13.1 were about 100 fold less potent than recombinant IL- 18, IL 18 muteins 15 and 16 were about 500 and 100 fold less potent than recombinant IL- 18, respectively, and IL 18 mutein 14 was about 100,000 fold less potent than recombinant IL- 18. Each IL 18 mutein is shown on the graph as a specific symbol. A summary table of potency (EC50-SEAP) with both fuse numbers and the associated IL- 18 mutein designations is shown beneath the non-linear x-y graph.EXAMPLE 12

[0217] As shown in figure 13, we examined the capacity of a set of IL-18 muteins containing the N-terminal lOOx attenuator peptide (GGGGS (SEQ ID NO: 32). termed “5L”) to induce non-targeted functional activity via activation of HEK Blue IL-18. In this set of proteins, each IL-18 variant was fused to the N-terminus of a knobs into holes Fc. In all cases, the fab from nivolumab targeting human PD1 was included using the format shown in FIG 1A but this arm did not impact the incorporated IL- 18 mutein’s activity' because the HEK Blue IL 18 reporter cell line used did not express human PD1. All IL 18 muteins were engineered with all cysteines substituted to valines. IL18 variants were engineered for (1) reduced binding to human IL-18BP and (2) reduced activity relative to wild type IL-18. FIG. 13 is a non-linear x-y graph of SEAP release (IL-18 activity) relative to the concentration of test article. Human recombinant wild type IL- 18 was used as the positive control (black squares) and human recombinant TNF-alpha as the negative control (grey square). All test articles are annotated with symbols that are indicated within the legend. A range of potency (EC50-SEAP) was observed whereby 5L IL18 mutein 9 and 5L IL18 mutein 2 were about 10 fold and 100 fold less potent than recombinant IL-18, respectively, with the remainder ranging from about 1000 fold (5L IL18 mutein 25) to approximately 50,000 fold (5L IL18 mutein 21) less potent than recombinant IL-18. A summary table of potency (EC50-SEAP) with both fuse numbers and the associated 5L IL-18 mutein designations is shown beneath the non-linear x-y graph.EXAMPLE 13

[0218] As shown in figure 14, we examined the capacity of either IL-18 mutein 13, 13.1 or 9 containing different N-terminal attenuator peptides to induce non-targeted functional activity via activation of HEK Blue IL-18. In this set of proteins, each IL-18 variant was fused to the N-terminus of a knobs into holes Fc. In all cases, the fab from cetrelimab targeting human PD1 was included using the format shown in FIG 1 A but this arm did not impact the incorporated IL- 18 mutein’s activity because the HEK Blue IL 18 reporter cell line used did not express human PD1. All IL 18 muteins w ere engineered w ith all cysteines substituted to valines. IL18 variants were engineered for (1) reduced binding to human IL-18BP and (2) reduced activity' relative to wild type IL- 18. FIG. 14 is anon-linear x-y graph of SEAP release (IL-18 activity) relative to the concentration of test article. Humanrecombinant wild type IL- 18 was used as the positive control (black squares) and human recombinant TNF-alpha as the negative control (grey square). All test articles are annotated with symbols that are indicated within the legend.

[0219] For constructs containing IL18mutl3. 1, adding the N-terminal peptide consisting of a serine ("lL), or GGGGS (SEQ ID NO: 32) ("5L" ) resulted in about 10 fold and 100 fold reduced activity compared to IL18mutl3. 1 without an N-terminal peptide. IL18mutl 3. 1 without an N-terminal peptide was about 100 fold less potent than recombinant IL-18, making lL-IL18mutl3.1 and 5L-IL18mutl3.1 about 1000 and 10,000 less potent than recombinant IL-18. 5L-IL18mutl3. the parent of the thermally stabilized 5L-IL18mutl9 (also termed 5L-IL18mutl3. 1) was observed to mediate HEK Blue IL18 activity that was not appreciably different from 5L-IL18mutl3.1. Finally, IL18mut9 containing the N-terminal peptide attenuator AAEPVEDNVINFVAMKFIDNTLYFIAENDENGGGS (propeptide variant termed "pp" ) was about 10,000 fold less potent than recombinant IL-18. A summary table of potency (EC50-SEAP) with both fuse numbers and the associated IL- 18 variant designations is shown beneath the non-linear x-y graph.EXAMPLE 14

[0220] As shown in figure 15, we examined the capacity of four mutein 13 family members without an N-terminal attenuator peptide to induce PD1 -targeted functional activity via activation of HEK Blue IL- 18 stably expressing human PD1. In this set of proteins, each IL-18 variant was fused to the N-terminus of a knobs into holes Fc and the fab from nivolumab targeting human PD1 was included using the format shown in FIG 1A. This !L18mutl3 family of variants was engineered for enhanced thermal stability relative to the parental IL18mutl3. We performed an in-silico biophysical characterization of IL-18mutl3 using ThermoMPNN that identified serine residues at positions 108 and 155 as potential thermal liabilities. We replaced these serine residues as such: S155L (IL18mutl9, also termed ml3.1), S155M (IL18mut28, also termed ml3.2), S108Y (IL18mut29. also termed ml3.3) and S108L (IL18mut30, also termed ml3.4). FIG. 15 A is a non-linear x-y graph of SEAP release (IL- 18 activity) relative to the concentration of test article. Human recombinant wild type IL- 18 was used as the positive control (black squares) and human recombinant TNF- alpha as the negative control (grey square). All test articles are annotated with symbols that are indicated within the legend. All four proteins in the IL18mutl3 family elicited similar functional activity that was not appreciably different from one another nor the parentalIL18mutl3. A summary table of potency (EC50-SEAP) with fuse numbers, the associated IL- 18 mutein designation and melting temperature is shown beneath the non-linear x-y graph. Detailed melting curves for IL18mutl3AV, IL18mutl3.1AV, IL18mutl3.2AV, IL18mutl3.3AV, and IL18mutl3.4AV are shown in FIGs 15B, 15C, 15D, 15E and 15F, respectively. The rank order of thermal stability was identified as IL18mutl3.4, IL18mutl3.3, IL18mutl3.1 followed by IL18mutl3.2 whereby the Tm of the IL18 variant (Tml) for the four muteins, IL18mutl3.1, IL18mutl3.3 and IL18mutl3.4 and IL18mutl3.2 was ~62°C, ~64°C, ~65°C, and ~60°C, respectively. The parental IL18mutl3 had a Tml of ~54°C.EXAMPLE 15Human peripheral blood mononuclear cell (PBMC) isolation

[0221] PBMCs were isolated from whole blood from healthy donors using Ficoll- Paque Plus medium. In brief, 35 mL of diluted whole blood (1 volume of whole blood vs 1 volume of PBS) was gently overlay ed on top of 15 mL FicolLPaque Plus medium without disturbing the interface in a 50-mL conical tube. After centrifuging for 40 minutes at 400 x g at room temperature without brake, the buffy coat (interface layer between Ficoll and serum) was collected and diluted in 5 volumes of PBS. After centrifuging for 5 minutes at 500 x g at room temperature, PBMCs were resuspended in PBS and washed once in PBS by centrifuging for 5 minutes at 500 x g at room temperature. PBMCs were then resuspended in 5 mL of ACK lysis buffer and incubated for 5 minutes at room temperature to remove red blood cell residues. After the 5-minute incubation period, 45 mL PBS was added to PBMCs and centrifuged for 15 minutes at 100 x g at room temperature. At last, PBMCs were resuspended in culture medium (RPMI1640 with 10% heat-inactivated FBS and 1% penicillin / streptomycin) for cytotoxicity and IFN gamma release assay set-up. To store PBMCs for further usage, PBMCs were pelleted down by centrifuging for 5 minutes at 500 x g at room temperature and then resuspended in freezing medium (90% heat-inactivated FBS and 10% dimethyl sulfoxide (DMSO)) for storage in liquid nitrogen.NK cell isolation and expansion

[0222] Fresh PBMCs were washed twice in PBS and resuspended in EasySep Buffer from StemCell at a density of 50 million cells per mL in 50-mL conical tube. 50 uL of isolation cocktail from the NK cell isolation kit from StemCell was added to 1 mL of cell suspension and incubated at room temperature for 5 minutes. After the 5-minute incubation period, 50 uL of RapidSpheres from NK cell isolation kit w as added to 1 mL of thecell / antibody cocktail mixture. The total volume of the cell / cocktail / bead mixture was brought up to 25 or 50 mL with Easy Sep Buffer and incubated at room temperature for 10 minutes on EASYSEP magnet. After the 10-minute incubation period, the solution was transferred to a new 50-mL tube by keeping the tube on the magnet and incubated at room temperature for 5 minutes on EASYSEP magnet. The solution with purified NK cell was collected. Freshly purified NK cells were used for expansion or frozen down and stored in liquid nitrogen for further usage.

[0223] To expand NK cells, lx 500 pL of ImmunoCult™ NK cell expansion coating material from ImmunoCult NK cell expansion kit from StemCell was added to a nontissue culture-treated 24-well plate and incubated at room temperature for 2 hours. During the 2-hour incubation, freshly isolated NK cells were resuspended in ImmunoCult NK cell expansion medium from ImmunoCult NK cell expansion kit from StemCell at the density of 1 million per mL. After the 2-hour incubation, the plate was washed twice with PBS and 500 pL of NK cell suspension (0.5 million NK cells) was added to one well of the coated 24-well plate. The plate was incubated at 37°C with 5% CO2 for 3 days and 500 pL of ImmunoCult NK cell expansion medium was added to the well with NK cells. The plate was further incubated for another 4 days. At day 7, NK cells were collected by centrifuge at 300 x g for 10 minutes and resuspended in ImmunoCult NK cell expansion medium from ImmunoCult NK cell expansion kit from StemCell at the density of 0.2 million per mL. 1 mL of collected NK cells (0.2 million NK cells) was added to a well of new- coated 24-well plate and incubated for 4 days at 37°C with 5% CO2. At day 11, NK cells were collected again bycentrifuge at 300 x g for 10 minutes and resuspended in ImmunoCult NK cell expansion medium from ImmunoCult NK cell expansion kit from StemCell at the density of 0.2 million per mL. 1 mL of collected NK cells (0.2 million NK cells) was added to a well of new coated 24-well plate and incubated for another 3 days at 37°C with 5% CO2. NK cells after 14-days’ expansion were collected and frozen down and stored in liquid nitrogen for further usage.Cytotoxicity assay

[0224] On the day before assay setting up, selective antibiotics were removed from target cell (MDA-MB-231 eGFP FLUC cells or T-47D eGFP FLUC cells). On the day of assay setting up, target cells were collected by brief Tr pLE treatment and then washed with culture medium by centrifuging at 500 x g for 5 minutes at room temperature. Target cell lines were then resuspended in culture medium to determine the cell viability by trypan blue exclusion on Cellometer. The viable cell density- was adjusted to 50,000 cells / mL in culturemedia. 100 uL target cell suspension (5.000 target cell) was carefully dispensed to each well of a 96-well black clear flat-bottom tissue culture plate using multichannel pipettor. The plate was then incubated for 4-5 hours in tissue culture incubator to ensure that the target cells have attached to the bottom of the 96-well plate.

[0225] For the cytotoxicity assay using NK cells or PBMCs as effector cells, NK cells or PBMCs were pelleted down by centrifuge for 5 minutes at 500 * g at room temperature and resuspended in culture medium. The viability of cells was also determined via try pan blue exclusion and the viable NK or PBMC density' was adjusted to 3 million cells / mL in RPMI1640 medium with 10% FBS and 1% penicillin / streptomycin. After 4-5 hours of incubation, culture medium was carefully removed from 96-well plates with target cells. 50 uL of 3 million cells / mL NKs or PBMCs suspension (150,000 cells) was added to the designated well in the 96-well plate with target cell, which would result in the E:T ratio of 30: 1. To result in the E:T ratio of 10: 1, 5: 1, 2: 1 or 1: 1, 3 million cells / mL of NKs or PBMCs were further diluted with RPMI1640 medium with 10% FBS and 1% penicillin / streptomycin by 3, 6, 15, or 30 times. 50 uL of diluted NK cells or PBMCs suspension w as then added to the designated w ells in the 96-well plate with target cell.

[0226] For the cytotoxicity assay using activated T cells as effector cells, isolated T cells were pretreated with human CD3 / CD28 dynabeads (2 uL per million T cells) for 72 hours. After 72-hour activation, CD3 / CD28 dynabeads were removed from activated T cells on magnet. Activated T cells were then pelleted down by centrifuge for 5 minutes at 500 x g at room temperature and resuspended in culture medium. The viability of T cells was also determined via trypan blue exclusion and the viable T cell density was adjusted to 2 million cells / mL in RPMI1640 medium with 10% FBS and 1% penicillin / streptomycin. After 4-5 hours of incubation, culture medium was carefully removed from 96-well plates with target cells. 25 uL of 2 million cells / mL activated T cell suspension (50,000 cells) w as added to the designated well in the 96-well plate with target cell, which would result in the E:T ratio of 10: 1. Meanwhile, Fuse-608 was prepared in RPMI1640 medium with 10% FBS and 1% penicillin / streptomycin at the concentration of 40 pM. 25 uL of 40 pM prepared Fuse-608 was added to designated well in the 96-w ell plate with target cell and activated T cells.

[0227] FUSE proteins were prepared and serially diluted (5-fold serial dilution) in RPMI1640 medium with 10% FBS and 1% penicillin / streptomycin ranging from 200 nM to 2.56 pM. 50 uL of prepared FUSE proteins at different concentrations were then added to the designated w ells in the 96-w ell plate with PBMC and target cells and incubated for thededicated time period (24. 48 or 72 hours) at 37 °C with 5% CO2. At the end of the incubation period, the 96-well plates were centrifuged for 1 minute at 500 *g to transfer 50pL of supernatant to V-bottom storage plate using a multichannel pipettor for IFN gamma release, granzyme B release or HEK-BLUE-IL-18 cell activation assay. ONE-Glo luciferase Assay solution was brought to room temperature. 50 uL of One-Gio solution was then added to the designated well with cells and incubated for 2 minutes at room temperature. The bioluminescence (IFU) was measured on a plate reader with preset Bio-luminance protocol. Killing of target cell was calculated by using the formula [Killing percentage of testing sample = (IFU of sample with PBMC and target cell - IFU of testing sample) / (IFU of sample with PBMC and target cell - IFU of sample with PBMC alone)* 100],T cell isolation

[0228] Fresh PBMCs were washed twice in PBS and resuspended in EasySep Buffer from StemCell at the density of 50 million cells per mL in 50-mL conical tube. 50 uL of isolation cocktail from T cell isolation kit from StemCell was added to 1 mL of cell suspension and incubated at room temperature for 5 minutes. After the 5 -minutes’ incubation. 50 uL of RapidSpheres from T cell isolation kit w as added to 1 mL of the cell / antibody cocktail mixture. The total volume of the cell / cocktail / bead mixture w as brought up to 25 or 50 mL with EasySep Buffer and incubated at room temperature for 10 minutes on EASYSEP magnet. After the 10-minutes’ incubation, the solution was transferred to a new 50-mL tube by keeping the tube on the magnet and incubated at room temperature for 5 minutes on EASYSEP magnet. The solution with purified T cell was collected. Freshly purified T cells were frozen down and stored in liquid nitrogen for further usage.Dendritic cell generation

[0229] Fresh PBMCs were resuspended in AIM V medium at the density of 5 million per ml and 40 mL of PBMC suspension was added to a T-174 sterile cell culture flask. The flasks were incubated in a humidified incubator maintained at 37°C and 5% CO2 for 2 hours. After 2 hours’ incubation, the non-adherent cells were removed by firmly tapping the flask and completely aspirating off media in the flask. 40 mL of AIM-V medium was immediately added to the adherent cells with 800 U / ml GM-CSF and 500 U / ml IL-4 to each T-175 cell culture flask containing the adherent monocytes. The flasks w ere placed in a humidified incubator at 37°C and 5% CO2 for 7 days. After the 7-day culture period, DCs were harvested, frozen down and stored in liquid nitrogen for further usage.HEK-Blue-IL-18 cell activation assay with human granzyme B-cleaved FUSE proteins or supernatant from cytotoxicity assay

[0230] HEK-BLUE-IL- 18 cells from Invivogen were maintained in culture medium (DMEM medium with 4.5 g / L glucose, 2 mM L-Glutamine, 10% (v / v) heat- inactivated fetal bovine serum (FBS), 100 U / mL penicillin, 100 pg / mL streptomycin, 100 pg / mL Normocin and l x HEK-blue selection reagent). HEK-BLUE™ IL- 18 cells are engineered from the human embryonic kidney 293 (HEK293) cell line to stably express genes encoding the IL- 18 receptor (IL-18R) and IL- 18 receptor accessory protein (IL- 18RAP) and express an NF- KB / AP-l-inducible secreted embryonic alkaline phosphatase (SEAP) reporter gene, therefore being useful for detection of bioactive IL- 18 by monitoring the activation of the NF-KB and AP-1 pathways via quantification in the supernatant of the SEAP level (which is produced upon activation of NF-KB) with a solution such as QU ANTIBLUE™ Solution. In addition, the responses to human TNF-a and IL-1 [3 have been blocked in HEK-BLUE™ IL- 18 cells, and so the HEK-BLUE™ IL- 18 cells are responsive specifically to IL- 18.

[0231] On the day of experiment setup, HEK-BLUE-IL-18 cells were gently rinsed twice with pre-warmed phosphate buffered saline (PBS) and then detached in PBS by tapping the flask. Detached HEK-Blue-IL-18 cells were resuspended in pre-w armed testing medium (DMEM with 4.5 g / L glucose, 2 mM L-Glutamine, 10% (v / v) heat-inactivated FBS, 100 U / mL penicillin, and 100 pg / mL streptomycin) at the density of 3x 105cell per mL. To seed the cells (50,000 cells per well), 180 pL of resuspended HEK-Blue-IL-18 cells were added to designated wells in 96-well plate.

[0232] For FUSE protein cleavage with human granzyme B, human pro-granzyme B was activated with enterokinase (EK) by incubating 2 pg of human pro-granzyme B with 20 ng of EK in 1 x PBS for 90 minutes at 37C. To cleave FUSE protein, 20 pg of FUSE protein was incubated with 2 pg of EK-activated human granzy me B in assay buffer (50 mM HEPES (pH 7.4), 100 mM NaCl, 0.1% CHAPS, 1 mM EDTA, 10% Glycerol) for 10 minutes at 37 C. After the 10-minute incubation period, cleaved FUSE proteins were diluted in testing medium (DMEM with 4.5 g / L glucose, 2 mM L-Glutamine, 10% (v / v) heat-inactivated FBS, 100 U / mL penicillin, and 100 pg / mL streptomycin) from 12,500,000 pg / mL to 160 pg / rnL (10x of final concentration) by 5-fold serial dilution. IL- 18 was also diluted in testing medium from 4,000 pg / mL to 1.28 pg / mL (10x of final concentration) by 5-fold serial dilution. To treat HEK-Blue-IL-18 cells, 20 pL of prepared IL- 18 or cleaved FUSE proteinswere added to the designated wells with 50.000 cells in the 96-well plate and then gently mixed the cells with proteins. The 96-well plate was then incubated at 37°C with 5% CO2 for 24 hours.

[0233] To check the activity' of the supernatant from cytotoxicity' study in inducing the activation of HEK-Blue-IL-18 cell, 20 pL of supernatant from the cytotoxicity study was added to designated wells containing 50,000 cells in the 96-well plate and then gently mixed the cells with supernatant. The 96-well plate was then incubated at 37°C with 5% CO2 for 24 hours

[0234] After the 24-hour activation incubation period, 20 pL of HEK-Blue-IL-18 cell culture supernatant containing the secreted alkaline phosphatase was transferred to a new 96-well plate. Meanwhile, QUANTI-Blue solution was prepared by adding 1 mL of QB reagent and 1 mL of QB buffer to 98 mL of sterile water. 80 pL of the prepared QUANTI- Blue solution was then added to the 96-well plate with 20 pL of HEK-Blue-IL-18 cell culture supernatant and incubated at 37°C for 4 hours. The level of secreted alkaline phosphatase related to HEK-Blue-IL-18 cell activation was detected by measuring the absorbance at 630 nm using a spectrophotometer.T cell and dendritic cell co-culture(MLR) assay

[0235] Dendritic cells were generated from monocytes in PBMCs with 800 U / ml GM-CSF and 500 U / ml IL-4. Allogeneic T cells from PBMCs were mixed with dendritic cells at the ratio of 10: 1 in RPMI1640 medium with 10% FBS and1% penicillin / streptomycin. 100K T cells w ith 10K dendritic cells in 150 uL of medium w ere added to the wells in 96-well plate. Fuse proteins were prepared and serially diluted (5-fold serial dilution) in RPMI1640 medium with 10% FBS and 1% penicillin / streptomycin ranging from 200 nM to 2.56 pM. 150 uL of prepared FUSE proteins at different concentrations were then added to the designated w ells in the 96-well plate with T cell and dendritic cells and incubated for the dedicated time period (48, 72, 96 or 144 hours) at 37 °C with 5% CO2. At the end of the incubation period, the 96-well plates were centrifuged for 1 minute at 500 *g to transfer 250pL of supernatant to V-bottom storage plate using a multichannel pipettor for IFN gamma release detection.IFN gamma release detection

[0236] IFN gamma release from cytotoxicity assay was measured using human IFN gamma ELISA detection kit. In brief, coating antibody provided in the kit was diluted to suggested concentration by following the protocol provided by the kit manufacturer. 100 ul of diluted coating antibody was added to the Nunc MaxiSorp flat-bottom 96-well plate. Plates were sealed and incubated overnight at 4 °C. On the following day. the plates were then washed 4 times with the Wash Buffer. To block non-specific binding and reduce background, 200 pL 1 x Assay Diluent A was added, and the plates were incubated at room temperature for 1 hour on a plate shaker (400 rpm). After blocking, the plates were washed 4 times with Wash Buffer. 100 pL / well of standards (prepared with culture medium) or samples were then added to the appropriate wells and incubated at room temperature for 2 hours on a plate shaker (400 rpm). After the 2-hour incubation with samples or standards, the plates were washed 4 times with Wash Buffer and 100 pL of diluted Detection Antibody solution was added to incubate at room temperature for 1 hour on a plate shaker (400 rpm). After the 1 - hour incubation period with detection antibody, the plates were washed 4 times with Wash Buffer and 100 pL of diluted Avidin-HRP solution was added to incubate at room temperature for 30 minutes on a plate shaker (400 rpm). After 30-minute incubation with Avidin-HRP, the plates were washed 5 times with Wash Buffer and 100 pL of freshly mixed TMB Substrate Solution was added to incubate at room temperature for 20 minutes in the dark. 100 pL of Stop Solution was then added to each well to stop the reaction. The absorbance at 450 nm was measured on a plate reader and the concentration of IFN gamma in each sample was back-calculated using the standard curve generated with the absorbance of different concentration of standards.Granzyine B release detection

[0237] Granzyme B release from cytotoxicity assay was measured using human granzyme B ELISA detection kit. In brief, coating antibody provided in the kit was diluted to suggested concentration by following the protocol provided by the kit manufacturer. 100 ul of diluted coating antibody was added to the Nunc MaxiSorp flat-bottom 96-well plate. The plates were sealed and incubated overnight at 4 °C. The plates were then washed 3 times with the Wash Buffer. To block non-specific binding and reduce background, 200 pL 1 * Assay Diluent A was added, and the plates were incubated at room temperature for 1 hour on a plate shaker (400 rpm). After blocking, the plates were washed 3 times with the Wash Buffer. 100 pL / well of standards (prepared with culture medium) or samples were then added to the appropriate wells and incubated at room temperature for 2 hours on a plate shaker (400 rpm).After the 2-hour incubation penod with samples or standards, the plates were washed 3 times with Wash Buffer and 100 μL of diluted Detection Antibody solution was added to incubate at room temperature for 1 hour on a plate shaker (400 rpm). After the 1-hour incubation period with detection antibody, the plates were washed 3 times with Wash Buffer and 100 pL of diluted Avidin-HRP solution was added to incubate at room temperature for 30 minutes on a plate shaker (400 rpm). After 30-minute incubation with Avidin-HRP, the plates were washed 4 times with Wash Buffer and 100 pL of freshly mixed TMB Substrate Solution w as added to incubate at room temperature for 20 minutes in the dark. 100 pL of Stop Solution was then added to each well to stop the reaction. The absorbance at 450 nm was measured on a plate reader and the concentration of granzyme B in each sample was back-calculated by using the standard curve generated with the absorbance of different concentration of standards.Sequential killing assay

[0238] Pan T cells from PBMCs were prepared in RPMI1640 complete medium with 10% FBS and 1% penicillin / streptomycin at the density of 4 million per mL. Jurkat eGFP ROR1 cells were resuspended in RPMI1640 complete medium with 10% FBS and 1% penicillin / streptomycin at the density of 0.4 million per mL. 200K pan T cell (50 uL) and 20K Jurkat eGFP Flue cell (50 uL) were added to the designated wells in a V-bottom 96-well plate. Testing articles were prepared in RPMI1640 medium with 10% FBS and 1 % penicillin / streptomycin at the concentration of 500 nM (10 times of final concentration) while signal "1" priming antibody (Fuse-608) w as prepared in RPMI1640 medium with 10% FBS and 1% penicillin / streptomycin at the concentration of 50 pM (10 times of final concentration). Then, 30 uL of prepared testing articles (final concentration: 50 nM) and signal "1" priming antibody (final concentration: 5 pM) were added to the designated wells in the V-bottom 96-well plate and bring the total volume of each well to 300 uL with RPMI1640 complete medium. After 3-day incubation at 37 °C with 5% CO2, the cells were pelleted down to collect the supernatant for IFN gamma release detection while the cells were resuspended in 100uL RPMI1640 complete medium. Then. 10 uL of cell suspension were mixed with 10 uL of ReadyCount™ Red Dead Cell Stain to count the cell number in bright field (total cell number), TexasRed Field (dead cell number) and GFP field (Jurkat eGFP cell number). After obtainig the cell number, the cell number of Jurkat eGFP ROR1 cell were adjusted to 20K in each designated wells and newly prepared testing articles (final concentration: 50 nM) and signal "1" priming antibody (final concentration: 5 pM) w ereadded and adjusted the total volume of each well to 300 uL with RPM11640 complete medium. After another 3-day incubation at 37 °C with 5% CO2, repeat cell counting and resupply with Jurkat eGFP ROR1 cell (20K per well) and newly prepared testing articles and signal "1" priming antibody for sequential killing until the target cells grew out in all groups. If the target cell (Jurkat eGFP Flue cell) grew out in certain groups, only newly prepared testing articles (final concentration: 50 nM) and signal "1" priming antibody (final concentration: 5 pM) were added to the wells of these groups without adding more target cells and brought the total volume of each well to 300 uL with RPMI1640 complete medium.Pelicluster CD3 antibody-primed PBMC activation assay

[0239] PBMCs were cultured with 0.5 ug / mL Pelicluster CD3 antibody in RPMI1640 complete medium with 10% FBS and 1% penicillin / streptomycin at the density of 1 million per mL for 2 days at 37 °C with 5% CO2. After 48-hour priming with 0.5 ug / mL Pelicluster CD3 antibody, PBMCs were washed twice and resuspended in RPMI1640 complete medium with 10% FBS and 1% penicillin / streptomycin with 50 ng / mL Pelicluster CD3 antibody at the density of 1 million per mL. 150 uL of pnmed PBMC suspension was added to the designated wells in the V-bottom 96- well plate. Fuse proteins were prepared and serially diluted (5-fold serial dilution) in RPMI1640 medium with 10% FBS and 1% penicillin / streptomycin ranging from 200 nM to 2.56 pM. 150 uL of prepared FUSE proteins at different concentrations were then added to the designated wells in the 96-well plate with primed PBMCs and incubated for 48 hours at 37 °C with 5% CO2. At the end of the incubation period, the 96-well plates w ere centrifuged for 1 minute at 500 xg to transfer 250pL of supernatant to V-bottom storage plate using a multichannel pipettor for IFN gamma release detection.Calculation of Biophysical and Structural Properties of N-Terminally Fused Peptides

[0240] The theoretical isoelectric point (pl) and molecular weight of the protein or peptide were determined by application of the ProtParam tool available through the Expasy server. Hydrophobicity was determined by summing the hydropathy values assigned to each amino acid residue according to the Kyte-Doolittle scale. The Grand Average of Hydropathy (GRAVY) was calculated as the total hydropathy value divided by the number of residues in the sequence.

[0241] The theoretical flexibility was predicted by employing a method based on known procedures, such as described by Meng and Kurgan. Flexibility values were computed for each residue and aggregated across the sequence.

[0242] Charge-related properties were calculated as follows:• The overall charge position was determined according to the formula:• The positive charge position was determined according to• The negative charge position was determined according to• The adjusted charge position was determined according towhere s (residue) is defined as:

[0243] The pLDDT confidence score for the structure was obtained by applying the AlphaFold3 protein structure prediction system. Root Mean Square Fluctuation (RMSF) values, expressed in Angstroms, were determined based on standard dynamic simulation procedures as described by Kuriata et al.Multicomponent Analysis of Peptide Characteristics Affecting IL- 18 Biological Activity

[0244] To assess the effect of the properties of the N-terminally fused peptide on IL- 18 activity', a multivariate analysis was conducted using the GraphPad Prism software. A total of 36 peptide properties were selected and calculated for each peptide, including: length,reciprocal of length (1 / length). positive charge, positive charge density, negative charge, negative charge density, charge density, number of charged residues, isoelectric point (pl), hydrophobicity, Grand Average of Hydropathy (GRAVY), GRAVY divided by length (GRAVY / length), pl divided by length (pl / length), molecular weight, reciprocal of molecular weight (1 / MW), flexibility, sum of flexibility values, charge position, positive charge position, negative charge position, adjusted charge position, predicted Local Distance Difference Test (pLDDT) scores for residues 1, 2, and 0, pLDDT score for the linker region, mean pLDDT score for the linker, overall pLDDT score, mean overall pLDDT score, C-alpha backbone flexibility (Cabs flex) for residues 1, 2, and 0, Cabs flex for the linker region, mean Cabs flex for the linker, overall Cabs flex, mean overall Cabs flex, and Cabs flex for the residue at position -n+1. Multiple linear regression analysis was performed with an F-statistic calculated as the mean square of the regression (MS(Reg)) divided by the mean square of the residuals (MS(Res)). The regression was carried out using a significance level (alpha) of 0.01. an effect size of 0.39, and a statistical power of 0.8.PD1 / PDL1 interaction blocking assay

[0245] To assess the inhibition of PD-1 / PD-L1 interaction by PD-lxIL-18 fusion proteins, a PD-1 [biotinylated] :PD-L1 inhibitor screening ELISA Kit from ACRO Biosystems (Cat No: EP-130) was used by following the protocol provided with the kit. In brief, PD-L1 protein w as resuspended in distilled w ater and diluted in coating buffer at the concentration suggested by the manufacturer. Next, 100 uL of prepared PD-L1 protein was added to designated wells in the 96-well ELISA plate (provided by the manufacturer) to incubate at 4 °C overnight. After the overnight incubation, the plate was washed three times with Washing Buffer (provided by the manufacturer and prepared following the provided procedure) and blocked with 200 uL Blocking Buffer (provided by the manufacturer) at 37 °C for 1.5 hours. During the 1-5 -hour blocking, testing proteins and control neutralizing antibody provided by the manufacturer were prepared in Diluting Buffer (provided by the manufacturer and prepared following the provided procedure) at the concentration of 2-fold of final concentration. For biotinylated PD-1 protein preparation, biotinylated PD-1 protein w as resuspended in distilled water following the provided procedure and then diluted in Diluting Buffer at the concentration suggested by the manufacturer. Meanwhile. 1 volume of prepared biotinylated PD-1 protein was mixed with 1 volume of prepared testing proteins or control neutralizing antibody to incubate for 30 minutes at 37 °C. After 1.5-hour blocking, the platewas washed three times with Washing Buffer and 100 uL of prepared biotinylated PD-1 protein and Fuse proteins or control neutralizing antibody mixture after 30-minutes incubation was added to the designated wells to incubate for 1 hour at 37 °C. During the 1- hour incubation, streptavidin-HRP was resuspended in distilled water following the procedure provided by the manufacturer and then diluted in Diluting Buffer at the concentration suggested by the manufacturer. After the 1-hour incubation, the plate was washed three times with Washing Buffer and 100 uL of prepared streptavidin-HRP was added to the designated wells and incubate for 1 hour at 37 °C. Next, the plate was washed three times with Washing Buffer and 100 uL of Substrate (provided by the manufacturer) was added to incubate at room temperature for 20 minutes. The reaction was stopped by adding 50 uL Stop solution (provided by the manufacturer) to measure the OD at 450 nm on a spectrometer.

Claims

CLAIMSWhat is claimed:

1. An IL- 18 variant comprising an amino acid sequence of:wherein X1is Y or C;2. The IL- 18 variant of claim 1, wherein:X1is Y or C;3. The TL- 18 variant of claim 1 , wherein the IL-18 variant is selected from any one of the IL-18 variants listed in Table 1.

4. The IL- 18 variant of claim 1. wherein the IL- 18 variant is selected from the group consisting ofYFGKLkSKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDVRDNAPRTIFIIS MYdDSQPRGkAVTISVKVEKISTLSVENKIISFKEMNPPDNIKDTKSDIIFF QRSVPGHDNKMQFESSSYEGYFLAVEKERDLFKLILKKEDELGDRSIMF TVQNED (IL-18ml7; SEQ ID NO: 17);YFGKLqSKLSVIRwLNDQVLFIDQGNRPLFEDMTDSDVRDNAPRTIFIIS MYdDSQPRGkAVTISVKVEKISTLSVENKIISFKEMNPPDNIKDTKSDIIFF QRSVPGHDNKMQFESSSYEGYFLAVEKERDLFKLILKKEDELGDRSIMF TVQNED (IL-18ml8; SEQ ID NO: 18)YFGKLqSKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDVRDNAPRTIFIIS MYdDSQPRGkAVTISVKVEKISTLSVENKIISFKEMNPPDNIKDTKSDIIFF QRSVPGHDNKMQFESS1YEGYFLAVEKERDLFKLILKKEDELGDRSIMF TVQNED (IL-18ml9 / 13.1; SEQ ID NO: 19);YFGKLqSKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDVRDNAPRTIFIISMYdDSQPRGkAVTISVKVEKISTLSVENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLAeEKERDLFKLILKKEDELGDRSIMF TVQNED (IL-18m20; SEQ ID NO: 20);YFGKLqSKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDVRDNAPRTIFIISMYdDSQPRGkAVTISVKVEKISTLSVENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLAVEKERDLFKLILKKEDELGyRSIMF TVQNED (IL-18m21; SEQ ID NO: 21);YFGKLqSKLcVIRNLNDQVLFIDQGNRPLFEDMTDSDVRDNAPRTIFIISMYdDSQPRGkAVTISVKVEKISTLSVENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLAVEKERDLFKLILKKEDELGDRSIMF TVQcED (IL-18m22; SEQ ID NO: 22);YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDVRDNAPRTIFIISMYdDSQPRGkAVTISVKVEKISTLSVENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLAVEKERDLFKLILKKEDELGDRSIMF TVQNED (IL-18m23; SEQ ID NO: 23);YFGKLqSKLSVIRNLNDQVLFIDQGNRPLFqDMTDSDVRDNAPRTIFIISMYdDSQPRGkAVTISVKVEKlSTLSVENKllSFKEMNPPDNIKDTKSDIlFFQRSVPGHDNKMQFESSSYEGYFLAVEKERDLFKLILKKEDELGDRSIMFTVQNED (IL-18m24; SEQ ID NO: 24); cFGKLqSKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDVRDNAPRTIFIISMYdDcQPRGkAVTISVKVEKISTLSVENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLAVEKERDLFKLILKKEDELGDRSIMFTVQNED (IL-18m25; SEQ ID NO: 25);YFGKLhSKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDVRDNAPRTIFIISMYdDSQPRGkAVTISVKVEKISTLSVENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLAVEKERDLFKL1LKKEDELGDRS1MF TVQNED (IL-18m26; SEQ ID NO: 26);YFGKLySKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDVRDNAPRTIFIISMYdDSQPRGkAVTISVKVEKISTLSVENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLAVEKERDLFKLILKKEDELGDRSIMF TVQNED (IL-18m27; SEQ ID NO: 27);YFGKLqSKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDVRDNAPRTIFIISMYdDSQPRGkAVTISVKVEKISTLSVENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSmYEGYFLAVEKERDLFKLILKKEDELGDRSIM FTVQNED (IL-18m28; SEQ ID NO: 28);YFGKLqSKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDVRDNAPRTIFIISMYdDSQPRGkAVTISVKVEKIyTLSVENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLAVEKERDLFKLILKKEDELGDRSIMF TVQNED (IL-18m29; SEQ ID NO: 29);YFGKLqSKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDVRDNAPRTIFIISMYdDSQPRGkAVTISVKVEKIlTLSVENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLAVEKERDLFKLILKKEDELGDRSIMF TVQNED (IL-18m30; SEQ ID NO: 30);YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDVRDNAPRTIFIISKYGDSQPRGLAVTISVKVEKISTLSVENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHRNKVQFESSSYEGYFLAVEKERDLFKLILKKEDELGDRSIM FTVQNED (IL-18m31; SEQ ID NO: 31);YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDVRDNAPRTIFIISKYGDSQPRGLAVTISVKVEKILTLSVENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHRNKVQFESS1YEGYFLAVEKERDLFKLILKKEDELGDRSIMF TVQNED (IL-I8m32; SEQ ID NO: 242);YFGKLESKLSV1RNLNDQVLFIDQGNRPLFEDMTDSDVRDNAPRT1F1IS HYADSQPRGIAVTISVKVEKISTLSVENKIISFKEMNPPDNIKDTKSDIIFF QRSVPGHRNKIQFESS1YEGYFLAVEKERDLFKLILKKEDELGDRSIMFT VQNED (IL-18m33; SEQ ID NO: 243);YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDVRDNAPRTIFIIS HYADSQPRGIAVTISVKVEKILTLSVENKIISFKEMNPPDNIKDTKSDIIFF QRSVPGHRNKIQFESS1YEGYFLAVEKERDLFKLILKKEDELGDRSIMFT VQNED (IL-18m34; SEQ ID NO: 244);YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDVRDNAPRTIFIIS HYGDSQPRGMAVTISVKVEKILTLSVENKIISFKEMNPPDNIKDTKSDIIF FQRSVPGHDNKMQFESS1YEGYFLAVEKERDLFKLILKKEDELGDRSIM FTVQNED (IL-18m35; SEQ ID NO: 245);YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDVRDNAPRTIFIIS KYGDSQPRGLAVTISVKVEKIYTLSVENKIISFKEMNPPDNIKDTKSDIIF FQRSVPGHRNKVQFESS1YEGYFLAVEKERDLFKLILKKEDELGDRSIMF TVQNED (IL-18m36; SEQ ID NO: 246);YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDVRDNAPRTIFIIS HYADSQPRGIAVTISVKVEKIYTLSVENKIISFKEMNPPDNIKDTKSDIIFF QRSVPGHRNK1QFESS1YEGYFLAVEKERDLFKL1LKKEDELGDRSIMFT VQNED (IL-18m37; SEQ ID NO: 247); andYFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDVRDNAPRTIFIIS HYGDSQPRGMAVTISVKVEKIYTLSVENKIISFKEMNPPDNIKDTKSDIIF FQRSVPGHDNKMQFESS1YEGYFLAVEKERDLFKLILKKEDELGDRSIM FTVQNED (IL-18m38; SEQ ID NO: 248).

5. The IL-18 variant of claim 1, where in the IL-18 variant isYFGKLqSKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDVRDNAPRTIFIISMYdDSQPRGkAVTISVKVEKISTLSVENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESS1YEGYFLAVEKERDLFKLILKKEDELGDRSIMFTVQNED (IL-18ml9 / 13.1; SEQ ID NO: 19).

6. The IL- 18 variant of claim 1, wherein the IL- 18 variant is YFGKLqSKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDVRDNAPRTIFIISMYdD SQPRGkAVTISVKVEKIyTLSVENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHD NKMQFESSSYEGYFLAVEKERDLFKLILKKEDELGDRSIMFTVQNED (IL- 18m29; SEQ ID NO: 29).

7. The IL- 18 variant of claim 1 , wherein the IL- 18 variant is YFGKLqSKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDVRDNAPRTIFIISMYdD SQPRGkAVTISVKVEKIlTLSVENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHD NKMQFESSSYEGYFLAVEKERDLFKLILKKEDELGDRSIMFTVQNED (IL- 18m30; SEQ ID NO: 30).

8. The IL- 18 variant of claim 1, wherein the IL- 18 variant is YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDVRDNAPRTIFIISKYGD SQPRGLAVTISVKVEKISTLSVENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHR NKVQFESSSYEGYFLAVEKERDLFKLILKKEDELGDRSIMFTVQNED (IL- 18m31; SEQ ID NO: 31).

9. The IL-18 variant of claim 1 , further comprising an attenuator moiety at the N-terminus of the IL- 18 variant, wherein the attenuator moiety comprises from about one amino acid to about 70 amino acids in length.

10. The IL-18 variant of claim 9, wherein the attenuator moiety is selected from any one of the attenuator sequences of Table 2.

11. The IL- 18 variant of claim 9, wherein the attenuator moiety is selected from of S, GGGGS (SEQ ID NO: 32), EV, DG, LM, THKM (SEQ ID NO: 36), DWNN (SEQ ID NO: 37), NTHR (SEQ ID NO: 38), TVDWIT (SEQ ID NO: 39). KHVLFF (SEQ ID NO: 40), ASRHVQ (SEQ ID NO: 41), LSSKDKVD (SEQ ID NO: 42), GKMNLSWY (SEQ ID NO: 43), HMWQQMYN (SEQ ID NO: 44), EIVHAIIFHK (SEQ ID NO: 45), LERIQTRYIQ(SEQ ID NO: 46). NRSKMMSM1R (SEQ ID NO: 48). MHRNWVAKHGHL (SEQ ID NO: 49), HHNKWKHLDFSH (SEQ ID NO: 50), DVVQSSESNQEW (SEQ ID NO: 51), NFSLERHMNNRMYE (SEQ ID NO: 52), WYKWFFYSRMHSIL (SEQ ID NO: 53), WFRRHGTKHGQFVI (SEQ ID NO: 54), and AAEPVEDNVINFVAMKFIDNTLYFIAENDENGGGS (SEQ ID NO: 61).

12. The IL-18 variant of claim 9, further comprising a protease cleavage site between the attenuator moiety and the N-terminus of the IL- 18 variant.

13. The IL-18 variant of claim 12, wherein the protease cleavage site is selected from a cleavage site recognized by granzyme B, granzyme A, granzyme M, granzyme K, cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin K, cathepsin L, cathepsin G, kallikrein, plasmin, a collagenase, type IV collagenase, a stromelysin, Factor Xa, a chymotrypsin-like protease, a trypsin-like protease, an elastase-like protease, a subtilisin like protease, bromelain, a calpain, a caspase, papain, a HIV-1 protease, a HSV protease, a CMV protease, a chymosin, renin, pepsin, a matrix metalloprotease (MMP), or a metalloproteinase (ADAM).

14. A nucleic acid sequence encoding an IL- 18 variant of claim 1.

15. A pharmaceutical composition comprising an IL- 18 variant of claim 1, and a pharmaceutically acceptable excipient.

16. A fusion protein comprising an IL- 18 variant and a scaffold polypeptide, wherein the IL- 18 variant comprises the amino acid sequence:wherein X1is Y or C;17. The fusion protein of claim 16, wherein the IL-18 variant comprises the amino acid sequence of SEQ ID NO: 1, whereinX1is Y or C;X18is N or C.

18. The fusion protein of claim 16, wherein the IL- 18 variant comprises the amino acid sequence selected from SEQ ID NOs 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15. 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 242, 243, 244. 245, 246. 247, 248, or 183.

19. The fusion protein of claim 16, wherein the scaffold polypeptide is selected from an immunoglobulin, Fc region of an immunoglobulin, human serum albumin (HSA), beta2 microglobulin, transferrin, fragment antigen-binding region (Fab region), VHH antibody, single-chain variable fragment (scFv), anticalin, designed ankyrin repeat protein (DARPin), type I transmembrane protein, or type II transmembrane protein or a fragment thereof.

20. The fusion protein of claim 16, further comprising an attenuator moiety at the N- terminus of the IL- 18 variant, wherein the attenuator moiety comprises from about one amino acid to about 70 amino acids in length.

21. The fusion protein of claim 20, wherein the attenuator moiety is selected from any one of the attenuator sequences of Table 2.

22. The fusion protein of claim 20, wherein the attenuator moiety is selected from of S, GGGGS (SEQ ID NO: 32), EV, DG, LM, THKM (SEQ ID NO: 36), DWNN (SEQ ID NO: 37), NTHR (SEQ ID NO: 38), TVDWIT (SEQ ID NO: 39). KHVLFF (SEQ ID NO: 40), ASRHVQ (SEQ ID NO: 41), LSSKDKVD (SEQ ID NO: 42), GKMNLSWY (SEQ ID NO: 43), HMWQQMYN (SEQ ID NO: 44), EIVHAIIFHK (SEQ ID NO: 45), LERIQTRYIQ (SEQ ID NO: 46), NRSKMMSMIR (SEQ ID NO: 48), MHRNWVAKHGHL (SEQ ID NO: 49), HHNKWKHLDFSH (SEQ ID NO: 50), DVVQSSESNQEW (SEQ ID NO: 51), NFSLERHMNNRMYE (SEQ ID NO: 52), WYKWFFYSRMHSIL (SEQ ID NO: 53). WFRRHGTKHGQFVI (SEQ ID NO: 54), and AAEPVEDNVINFVAMKFIDNTLYFIAENDENGGGS (SEQ ID NO: 61).

23. The fusion protein of claim 20. further comprising a protease cleavage site between the attenuator moiety and the N-terminus of the IL- 18 variant.

24. The fusion protein of claim 23. wherein the protease cleavage site is selected from a cleavage site recognized by granzyme B, granzyme A, granzyme M, granzyme K, cathepsin B, cathepsin C, cathepsin D. cathepsin E, cathepsin K, cathepsin L, cathepsin G, kallikrein, plasmin, a collagenase, type IV collagenase, a stromelysin, Factor Xa, a chymotrypsin-like protease, a trypsin-like protease, an elastase-like protease, a subtilisin like protease, bromelain, a calpain, a caspase, papain, a HIV-1 protease, a HSV protease, a CMV protease, a chymosin, renin, pepsin, a matrix metalloprotease (MMP), or a metalloproteinase (ADAM).

25. A nucleic acid sequence encoding a fusion protein of claim 16.

26. A pharmaceutical composition comprising a fusion protein of claim 16, and a pharmaceutically acceptable excipient.

27. A fusion protein comprising an IL- 18 variant and an anti-PDl antibody or a fragment thereof, wherein the IL-18 variant comprises the amino acid sequence of:wherein X1is Y or C;28. The fusion protein of claim 27, wherein the IL-18 variant comprises the amino acid sequence of SEQ ID NO: 1, whereinX1is Y or C;29. The fusion protein of claim 27. wherein the IL- 18 variant comprises the amino acid sequence selected from SEQ ID NOs 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 242, 243, 244, 245, 246, 247, 248, or 183.

30. The fusion protein of claim 27, wherein the anti-PDl antibody or a fragment thereof comprises an antigen binding domain comprising a heavy chain complementarity determining region (HCDR) 1 having an amino acid sequence of SEQ ID NO: 108, a HCDR2 having anamino acid sequence of SEQ ID NO: 109, a HCDR3 having an amino acid sequence of SEQ ID NO: 110, and a light chain complementarity determining region (LCDR) 1 having an amino acid sequence of SEQ ID NO: 111, a LCDR2 having an amino acid sequence of SEQ ID NO: 112, and a LCDR3 having an amino acid sequence of SEQ ID NO: 113.

31. The fusion protein of claim 27, wherein the anti-PDl antibody or a fragment thereof comprises a variable heavy chain (VH) comprising an amino acid sequence that is at least 90%, at least 95%. at least 98%, at least 99%, or 100% identical to SEQ ID NO: 114, and a variable light chain (VL) comprising an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 115.

32. The fusion protein of claim 27, wherein the IL- 18 variant comprises an amino acid sequence selected from SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19. 20, 21, 22, 23, 24, 25, 26, 27, 28.

29.

30. 31, 242, 243. 244, 245. 246, 247, 248, or 183, and wherein the anti-PDl antibody or a fragment thereof comprises a variable heavy chain (VH) comprising an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 114, and a variable light chain (VL) comprising an amino acid sequence that is at least 90%. at least 95%. at least 98%. at least 99%. or 100% identical to SEQ ID NO: 1 15.

33. The fusion protein of claim 27, wherein the IL- 18 variant comprises an amino acid sequence of SEQ ID NO: 2, and wherein the anti-PDl antibody or a fragment thereof comprises a variable heavy chain (VH) comprising an amino acid sequence of SEQ ID NO; 114, and a variable light chain (VL) comprising an amino acid sequence of SEQ ID NO: 1 15.

34. The fusion protein of claim 27, wherein the IL- 18 variant comprises an amino acid sequence of SEQ ID NO: 9, and wherein the anti-PDl antibody or a fragment thereof comprises a variable heavy chain (VH) comprising an amino acid sequence of SEQ ID NO; 114, and a variable light chain (VL) comprising an amino acid sequence of SEQ ID NO: 115.

35. The fusion protein of claim 27, wherein the IL- 18 variant comprises an amino acid sequence of SEQ ID NO: 15, and wherein the anti-PDl antibody or a fragment thereofcomprises a variable heavy chain (VH) comprising an amino acid sequence of SEQ ID NO;114, and a variable light chain (VL) comprising an amino acid sequence of SEQ ID NO: 1 15.

36. The fusion protein of claim 27, wherein the IL- 18 variant comprises an amino acid sequence of SEQ ID NO:

16. and wherein the anti-PDl antibody or a fragment thereof comprises a variable heavy chain (VH) comprising an amino acid sequence of SEQ ID NO; 114, and a variable light chain (VL) comprising an amino acid sequence of SEQ ID NO: 115.

37. The fusion protein of claim 27, wherein the IL- 18 variant comprises an amino acid sequence of SEQ ID NO: 19, and wherein the anti-PDl antibody or a fragment thereof comprises a variable heavy chain (VH) comprising an amino acid sequence of SEQ ID NO; 114, and a variable light chain (VL) comprising an amino acid sequence of SEQ ID NO: 115.

38. The fusion protein of claim 27. wherein the IL- 18 variant comprises an amino acid sequence of SEQ ID NO: 29, and wherein the anti-PDl antibody or a fragment thereof comprises a variable heavy chain (VH) comprising an amino acid sequence of SEQ ID NO; 114, and a variable light chain (VL) comprising an amino acid sequence of SEQ ID NO: 115.

39. The fusion protein of claim 27, wherein the IL-18 variant comprises an amino acid sequence of SEQ ID NO: 30, and wherein the anti-PDl antibody or a fragment thereof comprises a variable heavy chain (VH) comprising an amino acid sequence of SEQ ID NO; 114, and a variable light chain (VL) comprising an amino acid sequence of SEQ ID NO: 115.

40. The fusion protein of claim 27, wherein the IL- 18 variant comprises an amino acid sequence of SEQ ID NO: 31, and wherein the anti-PDl antibody or a fragment thereof comprises a variable heavy' chain (VH) comprising an amino acid sequence of SEQ ID NO; 114, and a variable light chain (VL) comprising an amino acid sequence of SEQ ID NO: 115.

41. The fusion protein of claim 27, wherein the antigen binding domain of the anti-PDl antibody or a fragment thereof is a diabody, a Fab, Fab', a F(ab’)2, a variable fragment (Fv), a single chain variable fragment (scFv), a (ScFv)2, a Fd fragment, stapled scFv fragment (spFv), a disulfide stabilized Fv fragment (dsFv), or a disulfide stabilized diabody (ds diabody).

42. The fusion protein of claim 27, wherein the anti-PDl antibody or a fragment thereof further comprises a Fc region and wherein the Fc region comprises one or more amino acid substitutions selected from L234A / L235A / D265S; H435R / Y436F; S364K / E357Q; L368D / K370S; S364K; L368D / K370S; S364K; L368E / K370S; D401K;T411E / K360E / Q362E; T366W; and T366S / L368A / Y407V, wherein numbering is according to EU numbering.

43. The fusion protein of claim 27, wherein the anti-PDl antibody or a fragment thereof is an IgG.

44. The fusion protein of claim 27, wherein the IL- 18 variant is covalently linked to N- terminal portion of a Fc region of anti-PDl antibody.

45. The fusion protein of claim 27, wherein a first IL-18 variant is covalently linked to N- terminal portion of a Fc region of anti-PD 1 antibody and a second IL-18 variant is covalently linked to C-terminal portion of a Fc region of anti-PDl antibody.

46. The fusion protein of claim 27, wherein the IL- 18 variant is covalently linked to N- terminal portion of a VL of anti-PDl antibody.

47. The fusion protein of claim 27, further comprising an attenuator moiety at the N- terminus of the IL- 18 variant, wherein the attenuator moiety comprises from about one amino acid to about 70 ammo acids in length.

48. The fusion protein of claim 47, wherein the attenuator moiety is selected from any one of the attenuator sequences of Table 2.

49. The fusion protein of claim 47, wherein the attenuator moiety is selected from of S, GGGGS (SEQ ID NO: 32), EV, DG, LM, THKM (SEQ ID NO: 36), DWNN (SEQ ID NO: 37), NTHR (SEQ ID NO: 38), TVDWIT (SEQ ID NO: 39), KHVLFF (SEQ ID NO: 40), ASRHVQ (SEQ ID NO: 41), LSSKDKVD (SEQ ID NO: 42), GKMNLSWY (SEQ ID NO: 43), HMWQQMYN (SEQ ID NO: 44), EIVHAIIFHK (SEQ ID NO: 45), LERIQTRYIQ (SEQ ID NO: 46), NRSKMMSMIR (SEQ ID NO: 48), MHRNWVAKHGHL (SEQ ID NO:49), HHNKWKHLDFSH (SEQ ID NO: 50), DVVQSSESNQEW (SEQ ID NO: 51). NFSLERHMNNRMYE (SEQ ID NO: 52), WYKWFFYSRMHSIL (SEQ ID NO: 53), WFRRHGTKHGQFVI (SEQ ID NO: 54), and AAEPVEDNVINFVAMKFIDNTLYFIAENDENGGGS (SEQ ID NO: 61).

50. The fusion protein of claim 47, further comprising a protease cleavage site between the attenuator moiety and the N-terminus of the IL- 18 variant.

51. The fusion protein of claim 27. further comprising a protease cleavage site between IL- 18 variant and the anti-PDl antibody or a fragment thereof.

52. The fusion protein of claim 51, wherein the protease cleavage site is selected from a cleavage site recognized by granzyme B, granzyme A. granzyme M, granzyme K, cathepsin B, cathepsin C. cathepsin D, cathepsin E, cathepsin K, cathepsin L, cathepsin G. kallikrein, plasmin, a collagenase, type IV collagenase, a stromelysin, Factor Xa, a chymotrypsin-like protease, a trypsin-like protease, an elastase-like protease, a subtilisin like protease, bromelain, a calpain, a caspase, papain, a HIV-1 protease, a HSV protease, a CMV protease, a chymosin, renin, pepsin, a matrix metalloprotease (MMP), or a metalloproteinase (ADAM).

53. A nucleic acid sequence encoding a fusion protein of claim 27.

54. A pharmaceutical composition comprising a fusion protein of claim 27. and a pharmaceutically acceptable excipient.

55. A fusion protein comprising:(a) a first monomer comprising from N-terminus to C-terminus an IL-18 variant and a first Fc region;(b) a second monomer comprising from N-terminus to C-terminus a variable heavy chain (VH) and a second Fc region; and(c) a third monomer comprising a variable light (VL) chain, wherein the VH and VL form an antigen binding domain that binds to PD-1.

56. The fusion protein of claim 55. wherein the IL-18 variant comprises the amino acid sequence of:wherein X1is Y or C;57. The fusion protein of claim 55, wherein the IL-18 variant comprises the amino acid sequence selected from SEQ ID NOs 2, 3. 4, 5, 6, 7. 8, 9, 10, 11, 12, 13, 14, 15.

16.

17.

18. 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 242, 243, 244, 245, 246, 247, 248, or 183.

58. The fusion protein of claim 55, wherein the antigen binding domain comprises a heavy chain complementarity determining region (HCDR) 1 having an amino acid sequence of SEQ ID NO: 108, a HCDR2 having an amino acid sequence of SEQ ID NO: 109, a HCDR3 having an amino acid sequence of SEQ ID NO: 110, and a light chain complementarity determiningregion (LCDR) 1 having an amino acid sequence of SEQ ID NO: 111, a LCDR2 having an amino acid sequence of SEQ ID NO: 1 12, and a LCDR3 having an amino acid sequence of SEQ ID NO: 113.

59. The fusion protein of claim 55. wherein the VH comprises an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 114, and the VL comprises an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 115.

60. The fusion protein of claim 55, wherein the IL- 18 variant further comprising an attenuator moiety at the N-terminus, and wherein the attenuator moiety comprises from about one amino acid to about 70 amino acids in length.

61. The fusion protein of claim 60, wherein the attenuator moiety is selected from any one of the attenuator sequences of Table 2.

62. The fusion protein of claim 60, wherein the attenuator moiety is selected from of S, GGGGS (SEQ ID NO: 32), EV, DG, LM, THKM (SEQ ID NO: 36), DWNN (SEQ ID NO: 37), NTHR (SEQ ID NO: 38), TVDWIT (SEQ ID NO: 39), KHVLFF (SEQ ID NO: 40), ASRHVQ (SEQ ID NO: 41), LSSKDKVD (SEQ ID NO: 42), GKMNLSWY (SEQ ID NO: 43), HMWQQMYN (SEQ ID NO: 44), EIVHAIIFHK (SEQ ID NO: 45), LERIQTRYIQ (SEQ ID NO: 46), NRSKMMSMIR (SEQ ID NO: 48), MHRNWVAKHGHL (SEQ ID NO: 49), HHNKWKHLDFSH (SEQ ID NO: 50), DVVQSSESNQEW (SEQ ID NO: 51). NFSLERHMNNRMYE (SEQ ID NO: 52), WYKWFFYSRMHSIL (SEQ ID NO: 53), WFRRHGTKHGQFVI (SEQ ID NO: 54), and AAEPVEDNVINFVAMKFIDNTLYFIAENDENGGGS (SEQ ID NO: 61).

63. The fusion protein of claim 55, wherein one of the first and the second Fc regions comprises an amino acid substitution T366W, and the other of the first and the second Fc regions comprises one or more amino acid substitutions T366S / L368A / Y407V, wherein numbering is according to EU numbering.

64. The fusion protein of claim 55, wherein the first and the second Fc regions comprise one or more amino acid substitutions selected from L234A / L235A / D265S; H435R / Y436F; S364K / E357Q; L368D / K370S; S364K; L368D / K370S; S364K; L368E / K370S; D401K; T411E / K360E / Q362E; T366W; and T366S / L368A / Y407V, wherein numbering is according to EU numbering.

65. The fusion protein of claim 55, wherein:(a) the first monomer is selected from the amino acid sequence represented by SEQ ID NOs: 137, 142, 148, 172, 173. 178, 179, 180. 181, 249, 250, 251, 252, 253, 254, and 255;(b) the second monomer is selected from the amino acid sequence represented by SEQ ID NOs: 146 and 116.(c) the third monomer is selected from the amino acid sequence represented by SEQ ID NO: 117.

66. The fusion protein of claim 55, wherein the first monomer comprises the amino sequence of SEQ ID NO: 137, the second monomer comprises the amino acid sequence of SEQ ID NO: 146, and the third monomer comprises the amino acid sequence of SEQID NO: 117.

67. The fusion protein of claim 55, wherein the first monomer comprises the amino sequence of SEQ ID NO: 142, the second monomer comprises the amino acid sequence of SEQ ID NO: 146, and the third monomer comprises the amino acid sequence of SEQID NO: 117.

68. The fusion protein of claim 55, wherein the first monomer comprises the amino sequence of SEQ ID NO: 148, the second monomer comprises the amino acid sequence of SEQ ID NO: 146, and the third monomer comprises the amino acid sequence of SEQID NO: 117.

69. The fusion protein of claim 55, wherein the first monomer comprises the amino sequence of SEQ ID NO: 172, the second monomer comprises the amino acid sequence of SEQ ID NO: 146, and the third monomer comprises the amino acid sequence of SEQID NO: 117.

70. The fusion protein of claim 55, wherein the first monomer comprises the amino sequence of SEQ ID NO: 173, the second monomer comprises the amino acid sequence of SEQ ID NO: 146, and the third monomer comprises the amino acid sequence of SEQID NO: 117.

71. The fusion protein of claim 55, wherein the first monomer comprises the amino sequence of SEQ ID NO: 178, the second monomer comprises the amino acid sequence of SEQ ID NO: 11 , and the third monomer comprises the amino acid sequence of SEQID NO: 117.

72. The fusion protein of claim 55, wherein the first monomer comprises the amino sequence of SEQ ID NO: 179, the second monomer comprises the amino acid sequence of SEQ ID NO: 116, and the third monomer comprises the amino acid sequence of SEQID NO: 117.

73. The fusion protein of claim 55, wherein the first monomer comprises the amino sequence of SEQ ID NO: 180, the second monomer comprises the amino acid sequence of SEQ ID NO: 116, and the third monomer comprises the amino acid sequence of SEQID NO: 1 17.

74. The fusion protein of claim 55, wherein the first monomer comprises the amino sequence of SEQ ID NO: 181, the second monomer comprises the amino acid sequence of SEQ ID NO: 116, and the third monomer comprises the amino acid sequence of SEQID NO: 117.

75. A method of treating a subject suffering from cancer comprises administering to the subject a composition comprising an IL- 18 fusion protein, wherein the IL- 18 fusion protein comprises IL-18 variant and an anti-PDl antibody or a fragment thereof, and wherein the IL- 18 variant comprises the amino acid sequence of:wherein X1is Y or C;76. The method of claim 75, wherein the IL-18 variant comprises the amino acid sequence of SEQ ID NO: 1, whereinX1is Y or C;X18is N or C.

77. The method of claim 75, wherein the IL-18 variant comprises an amino acid sequence selected from SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8. 9, 10, 11, 12, 13. 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 242, 243, 244. 245, 246, 247, 248, or 183, and wherein the anti-PDl antibody or a fragment thereof comprises a variable heavy chain (VH) comprising an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 114, and a variable light chain (VL) comprising an amino acid sequence that is at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 1 15.

78. The method of claim 75, wherein the cancer is solid cancer.

79. The method of claim 78. wherein the solid cancer is selected from adrenal cancer, anal cancer, bile duct cancer, bladder cancer, bone cancer, brain cancer, breast cancer, carcinoid cancer, cervical cancer, colorectal cancer, esophageal cancer, eye cancer, gallbladder cancer, gastrointestinal stromal tumor, germ cell cancer, head and neck cancer, kidney cancer, liver cancer, lung cancer, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, neuroendocrine cancer, oral cancer, oropharyngeal cancer, ovarian cancer, pancreatic cancer, pediatric cancer, penile cancer, pituitary cancer, prostate cancer, skin cancer, soft tissue cancer, spinal cord cancer, stomach cancer, testicular cancer, thymus cancer, thyroid cancer, ureteral cancer, uterine cancer, vaginal cancer, or vulvar cancer.

80. The method of claim 75, wherein the IL- 18 variant comprises an amino acid sequence of SEQ ID NO: 2, and the anti-PDl antibody or a fragment thereof comprises a variable heavy chain (VH) comprising an amino acid sequence of SEQ ID NO: 114, and a variable light chain (VL) comprising an amino acid sequence of SEQ ID NO: 115.

81. The method of claim 75, wherein the IL- 18 variant comprises an amino acid sequence of SEQ ID NO: 9, and the anti-PDl antibody or a fragment thereof comprises a variable heavy chain (VH) comprising an amino acid sequence of SEQ ID NO: 114, and a variable light chain (VL) comprising an amino acid sequence of SEQ ID NO: 115.

82. The method of claim 75, wherein the IL- 18 variant comprises an amino acid sequence of SEQ ID NO: 15, and the anti-PDl antibody or a fragment thereof comprises a variable heavy chain (VH) comprising an amino acid sequence of SEQ ID NO: 114, and a variable light chain (VL) comprising an amino acid sequence of SEQ ID NO: 115.

83. The method of claim 75, wherein the IL- 18 variant comprises an amino acid sequence of SEQ ID NO: 16, and the anti-PDl antibody or a fragment thereof comprises a variable heavy chain (VH) comprising an amino acid sequence of SEQ ID NO: 114, and a variable light chain (VL) comprising an amino acid sequence of SEQ ID NO: 115.

84. The method of claim 75, wherein the IL- 18 variant comprises an amino acid sequence of SEQ ID NO: 19, and the anti-PDl antibody or a fragment thereof comprises a variable heavy chain (VH) comprising an amino acid sequence of SEQ ID NO: 114, and a variable light chain (VL) comprising an amino acid sequence of SEQ ID NO: 1 15.

85. The method of claim 75, wherein the IL- 18 variant comprises an amino acid sequence of SEQ ID NO: 29, and the anti-PDl antibody or a fragment thereof comprises a variable heavy chain (VH) comprising an amino acid sequence of SEQ ID NO: 114, and a variable light chain (VL) comprising an amino acid sequence of SEQ ID NO: 115.

86. The method of claim 75, wherein the IL- 18 variant comprises an amino acid sequence of SEQ ID NO: 30, and the anti-PDl antibody or a fragment thereof comprises a variable heavy chain (VH) comprising an amino acid sequence of SEQ ID NO: 114, and a variable light chain (VL) comprising an amino acid sequence of SEQ ID NO: 115.

87. The method of claim 75, wherein the IL-18 variant comprises an amino acid sequence of SEQ ID NO: 31, and the anti-PDl antibody or a fragment thereof comprises a variable heavy chain (VH) comprising an amino acid sequence of SEQ ID NO: 114, and a variable light chain (VL) comprising an amino acid sequence of SEQ ID NO: 115.

88. A method of treating a subject suffering from cancer comprises administering to the subject an IL- 18 fusion protein comprising:(a) the first monomer is selected from the amino acid sequence represented by SEQ ID NOs: 137, 142, 148, 172, 173, 178, 179, 180, 181, 249, 250, 251, 252, 253, 254, and 255;(b) the second monomer is selected from the amino acid sequence represented by SEQ ID NOs: 146 and 116; and(c) the third monomer is selected from the amino acid sequence represented by SEQ ID NO: 117.

89. The method of claim 88, wherein the fusion protein comprises the first monomer comprising the amino sequence of SEQ ID NO: 137, the second monomer comprising the amino acid sequence of SEQ ID NO: 146, and the third monomer comprising the amino acid sequence of SEQID NO: 117.

90. The method of claim 88. wherein the fusion protein comprises the first monomer comprising the amino sequence of SEQ ID NO: 142, the second monomer comprising the amino acid sequence of SEQ ID NO: 146, and the third monomer comprising the amino acid sequence of SEQID NO: 117.

91. The method of claim 88, wherein the fusion protein comprises the first monomer comprising the amino sequence of SEQ ID NO: 148, the second monomer comprising the amino acid sequence of SEQ ID NO: 146, and the third monomer comprising the amino acid sequence of SEQID NO: 117.

92. The method of claim 88, wherein the fusion protein comprises the first monomer comprising the amino sequence of SEQ ID NO: 172, the second monomer comprising the amino acid sequence of SEQ ID NO: 146, and the third monomer comprising the amino acid sequence of SEQID NO: 117.

93. The method of claim 88, wherein the fusion protein comprises the first monomer comprising the amino sequence of SEQ ID NO: 173, the second monomer comprising the amino acid sequence of SEQ ID NO: 146, and the third monomer comprising the amino acid sequence of SEQID NO: 1 17.

94. The method of claim 88. wherein the fusion protein comprises the first monomer comprising the amino sequence of SEQ ID NO: 178, the second monomer comprising the amino acid sequence of SEQ ID NO: 116, and the third monomer comprising the amino acid sequence of SEQID NO: 117.

95. The method of claim 88, wherein the fusion protein comprises the first monomer comprising the amino sequence of SEQ ID NO: 179, the second monomer comprising the amino acid sequence of SEQ ID NO: 116, and the third monomer comprising the amino acid sequence of SEQID NO: 117.

96. The method of claim 88, wherein the fusion protein comprises the first monomer comprising the amino sequence of SEQ ID NO: 180, the second monomer comprising the amino acid sequence of SEQ ID NO: 116, and the third monomer comprising the amino acid sequence of SEQID NO: 1 17.

97. The method of claim 88, wherein the fusion protein comprises the first monomer comprising the amino sequence of SEQ ID NO: 181, the second monomer comprising the amino acid sequence of SEQ ID NO:

116. and the third monomer comprising the amino acid sequence of SEQID NO: 1 17.

Images