HK40100300A - Interleukin-18 variants and methods of use - Google Patents

Description

Interleukin-18 variants and usage

Cross-references

This application claims the benefit of U.S. Provisional Application No. 63/108,794, filed November 2, 2020, which is incorporated herein by reference in its entirety.

Background Technology

Interleukin-18 (IL-18) is a pro-inflammatory cytokine that can stimulate T cells, NK cells, and myeloid cells. Given its ability to stimulate anti-tumor immune cells, IL-18 has been proposed as an immunotherapeutic agent for cancer treatment. However, the clinical efficacy of IL-18 is limited.

Therefore, there is a need for compositions and methods that provide effective IL-18 signaling activity for the treatment and prevention of cancer and other diseases and conditions. This disclosure fulfills these needs.

Incorporation

All publications, patents and patent applications mentioned in this specification are incorporated herein by reference as if each individual publication, patent or patent application were expressly and individually indicated to be incorporated by reference.

Incorporation of sequence lists

Also provided is the sequence list in the text file “SMCH-003PRV_SeqList_ST25.txt”, created on November 2, 2020, and 277KB in size. The contents of the text file are incorporated herein by reference in their entirety.

Summary of the Invention

Compositions comprising a variant (e.g., a stabilized) IL-18 polypeptide are provided. The stabilized IL-18 polypeptide is an IL-18 polypeptide comprising a ‘stabilizing mutation’, said mutation being a mutation of two cysteine residues (C38 and C68) relative to human wild-type IL-18 (SEQ ID NO:30). Surprisingly, it has been found that mutations of these two amino acids stabilize IL-18 better than all other possible combinations of cysteine mutations, see, for example, the working examples below. In some cases, the mutation is a C to S substitution, and therefore in some cases, the stabilized IL-18 polypeptide may comprise mutations C38S and C68S relative to human wild-type IL-18 (SEQ ID NO:30) (also referred to herein as “mutation pair C38S/C68S”). However, in some cases, the mutation at C68 results in an immunogenic epitope. Therefore, in some embodiments, the mutation at C68 is a non-immunogenic substitution. In some of these cases, the mutation is a C68 to G, A, V, D, E, or N mutation (in some cases, C68 to G, A, D, or N). Therefore, in some of these cases, the subject-stabilized IL-18 peptide contains the mutation pair C38S/C68S, C38S/C68G, C38S/C68A, C38S/C68V, C38S/C68D, C38S/C68E, or C38S/C68N. In some cases, the subject-stabilized IL-18 peptide contains the mutation pair C38S/C68G, C38S/C68A, C38S/C68V, C38S/C68D, C38S/C68E, or C38S/C68N. In some cases, the subject-stabilized IL-18 peptide contains mutant pairs C38S/C68S, C38S/C68G, C38S/C68A, C38S/C68D, or C38S/C68N.

In some cases, the subject-stabilized IL-18 peptide contains the mutation pair C38S/C68D. In some cases, the subject-stabilized IL-18 peptide contains the mutation pair C38S/C68G. In some cases, the subject-stabilized IL-18 peptide contains the mutation pair C38S/C68A. In some cases, the subject-stabilized IL-18 peptide contains the mutation pair C38S/C68N. In some cases, the subject-stabilized IL-18 peptide contains the mutation pair C38S/C68S.

In some cases, the subject-stabilized IL-18 peptide is a stabilized IL-18 variant peptide, i.e., an IL-18 variant peptide containing a stabilizing mutation (i.e., a mutation at positions C38 and C68 relative to human wild-type IL-18). In some such cases, the stabilized IL-18 variant peptide is a stabilized "decoy-resistant" (DR) IL-18 variant, and in other such cases, the stabilized IL-18 variant peptide is a stabilized "decoy-to-decoy" (D2D) IL-18 variant. In some embodiments, the stabilized IL-18 variant peptide has a binding affinity of less than 0.6 nM for IL-18BP.

In some embodiments, the subject-stabilized IL-18 peptide exhibits higher stability compared to IL-18 peptides with different sequences, wherein said higher stability results in less formation of dimers, multimers, aggregates, and/or degradation products after exposure to a stability assay. In some embodiments, the subject-stabilized IL-18 peptide exhibits higher stability than the wild-type IL-18 peptide. In some embodiments, the higher stability is measured by shelf-life stability. In some embodiments, the higher stability is determined by subjecting the modified IL-18 peptide or a formulation containing the modified IL-18 peptide disclosed herein to freeze-thaw cycles. In some embodiments, the higher stability is measured by subjecting the stabilized IL-18 variant peptide to agitation.

DR IL-18 variants are variants/mutants of IL-18 that bind to the IL-18 receptor (IL-18R), thereby inducing/enhancing/stimulating IL-18 signaling activity, but exhibit reduced (in some cases, almost no) binding to the inhibitory IL-18-binding protein (IL-18BP) (as opposed to the binding of WT IL-18 to IL-18BP). Therefore, DR IL-18 variants are IL-18R agonists resistant to the inhibition of IL-18BP. Examples of human DR IL-18 variants include, but are not limited to, those shown in SEQ ID NO:34-59, 73-91, and 191-193. Examples of mouse DR IL-18 variants include, but are not limited to, those shown in SEQ ID NO:60-72.

D2D IL-18 variants are variants/mutants of IL-18 that bind IL-18BP but exhibit reduced (in some cases, almost no) binding to IL-18R (as opposed to the binding of WT IL-18 to IL-18R). Therefore, D2D IL-18 variants bind IL-18BP, thereby antagonizing the effect of IL-18BP on IL-18. Thus, D2D IL-18 variants can be used to increase signaling via IL-18R because they act by inhibiting inhibitors. Examples of human D2D IL-18 variants include, but are not limited to, those shown in SEQ ID NO: 92-125. Examples of mouse D2D IL-18 variants include, but are not limited to, those shown in SEQ ID NO: 126-190.

In some cases, the stabilized DR IL-18 variant peptide contains amino acid positions M51 (e.g., M51E, M51R, M51K, M51T, M51D, or M51N), K53 (e.g., K53G, K53S, K53T, or K53R), Q56 (e.g., Q56G, Q56R, Q56L, Q56E, Q56A, Q56V, or Q56K), D110 (e.g., D110S, D110N, D110G, D110K, D110H, D110Q, or D110E), and N111 (e.g., N111G). Mutations at positions N111R, N111S, N111D, N111H, or N111Y, and stabilization mutations at positions C38 (e.g., C38S) and C68 (e.g., C68S, C68G, C68A, C68V, C68D, C68E, or C68N) (e.g., C38S/C68S, C38S/C68G, C38S/C68A, C38S/C68V, C38S/C68D, C38S/C68E, or C38S/C68N) (relative to human wild-type IL-18, SEQ ID NO:30). In some of these cases, the stabilized IL-18 variant peptide additionally contains a mutation at amino acid position S105 (e.g., S105D, S105A, S105N, S105R, S105D, or S105K); and in some cases, it also contains mutations at amino acid positions P57 (e.g., P57A, P57L, P57G, or P57K) and M60 (e.g., M60L, M60R, M60K, or M60Q).

In some cases, the stabilized DR IL-18 variant peptide contains amino acid positions M51 (e.g., M51E, M51R, M51K, M51T, M51D, or M51N), K53 (e.g., K53G, K53S, K53T, or K53R), Q56 (e.g., Q56G, Q56R, Q56L, Q56E, Q56A, Q56V, or Q56K), or D110 (e.g., D110S, D110N, or D110G). Mutations at D110K, D110H, D110Q or D110E) and N111 (e.g. N111G, N111R, N111S, N111D, N111H or N111Y) and stabilizing mutation pairs selected from the following: C38S/C68S, C38S/C68G, C38S/C68A, C38S/C68D and C38S/C68N (relative to human wild-type IL-18, SEQ ID NO:30). In some of these cases, the stabilized IL-18 variant peptide additionally contains a mutation at amino acid position S105 (e.g., S105D, S105A, S105N, S105R, S105D, or S105K); and in some cases, it also contains mutations at amino acid positions P57 (e.g., P57A, P57L, P57G, or P57K) and M60 (e.g., M60L, M60R, M60K, or M60Q).

In some cases, the stabilized DR IL-18 variant peptide contains amino acid positions M51 (e.g., M51E, M51R, M51K, M51T, M51D, or M51N), K53 (e.g., K53G, K53S, K53T, or K53R), Q56 (e.g., Q56G, Q56R, Q56L, Q56E, Q56A, Q56V, or Q56K), or D110 (e.g., D110S, D110N). Mutations at D110G, D110K, D110H, D110Q or D110E) and N111 (e.g. N111G, N111R, N111S, N111D, N111H or N111Y) and stabilizing mutation pairs selected from the following: C38S/C68G, C38S/C68A, C38S/C68D and C38S/C68N (relative to human wild-type IL-18, SEQ ID NO:30). In some of these cases, the stabilized IL-18 variant peptide additionally contains a mutation at amino acid position S105 (e.g., S105D, S105A, S105N, S105R, S105D, or S105K); and in some cases, it also contains mutations at amino acid positions P57 (e.g., P57A, P57L, P57G, or P57K) and M60 (e.g., M60L, M60R, M60K, or M60Q).

In some cases, the stabilized DRIL-18 variant peptide comprises an amino acid sequence having 85% or more identity with the amino acid sequence shown in any of SEQ ID NO:6 and 16-21.

In some cases, the stabilized DR IL-18 variant peptide comprises an amino acid sequence having 85% or more identity with the amino acid sequence shown in any one of SEQ ID NO: 6, 16, 17, 19, and 21.

In some embodiments, a stabilized IL-18 peptide (e.g., a stabilized DR IL-18 variant) is used to treat a disease or condition. In various embodiments, the disease or condition is cancer or an infectious disease, such as a poxvirus encoding an IL-18BP ortholog; a metabolic disease or condition (including obesity and diabetes); or macular degeneration (e.g., wet macular degeneration, such as wet age-related macular degeneration). For example, IL-18 protein can be used as an anti-angiogenic agent; as an illustrative example, in some cases, stabilized IL-18 protein can reduce choroidal neovascularization. Therefore, the provided methods include administering a stabilized IL-18 peptide (e.g., a stabilized DR IL-18 variant) to treat a disease or condition, such as, but not limited to, cancer, or an infectious disease, a metabolic disease or condition, or macular degeneration (e.g., wet macular degeneration, such as wet age-related macular degeneration).

In some embodiments, the method includes administering to a subject in need a composition comprising a stabilized IL-18 peptide (e.g., a stabilized DR IL-18 variant). In some embodiments, the method includes administering to a subject in need a composition comprising a stabilized IL-18 peptide (e.g., a stabilized DR IL-18 variant) and an adjuvant. In some such embodiments, the adjuvant includes immunotherapeutic agents, such as modified immune cells (e.g., T cells, such as chimeric antigen receptor T cells (CAR-T), armed CAR-T cells; NK cells, such as CAR-NK; tumor-infiltrating lymphocytes (TIL), TCR-T cells, etc.), viruses, antigens, vaccines, antibodies, immune checkpoint inhibitors, small molecules, chemotherapeutic agents, stem cells, etc. In some embodiments, the stabilized IL-18 peptide (e.g., a stabilized DR IL-18 variant) is used in methods of increasing immune system activity before, during, or after infection with bacteria, viruses, or other pathogens. In some implementations, a stabilized IL-18 peptide (e.g., a stabilized DR IL-18 variant) is used to increase the number and/or activity of immune cells (such as the number and/or activity of T cells, NK cells, and/or myeloid cells) in vitro, in vivo, or ex vivo.

The subject peptide can be administered in combination with immune checkpoint inhibitors and/or tumor modulatory antibodies, or in fusions with them. The subject peptide can be administered in combination with cell therapies, such as chimeric antigen receptor T cell (CAR-T cell), TCR-T cell, chimeric antigen receptor NK cell (CAR-NK cell), and tumor-infiltrating lymphocyte (TIL) therapy. The subject peptide can also be administered as part of a cell therapy, for example, it can be a protein secreted by cells (e.g., TRUCK cells (“T cells redirected to antigen-free cytokine-induced killing”) (which are in the form of CAR-T cells, CAR-NK cells, TIL cells, etc.)).

A method for administering an IL-18 peptide to an individual is also provided, wherein the method includes administering an IL-18 peptide (e.g., wild-type IL-18 or a variant, such as stabilized, DR, D2D, stabilized DR, or stabilized D2D IL-18 variant) to the individual, wherein the administration frequency is once a week or less.

Methods and compositions for generating IL-18 peptides are also provided. Such methods involve contacting an exogenously provided fusion protein with an exogenously provided SUMO (small ubiquitin-like modified) protease inside a bacterial cell, wherein the fusion protein comprises a peptide of interest fused to its N-terminus with a SUMO tag, wherein, within the bacterial cell, the SUMO protease cleaves the fusion protein, thereby removing the SUMO tag from the peptide of interest. In some cases, the peptide of interest is an IL-18 peptide (e.g., wild-type human IL-18 protein or IL-18 variants, such as stabilized, DR, D2D, stabilized DR, or stabilized D2D variants; see, for example, the stabilized and DR IL-18 variant peptides discussed above).

Formulations containing IL-18 peptides (e.g., wild-type human IL-18 protein or IL-18 variants, such as stabilized, DR, D2D, stabilized DR, or stabilized D2D variants, see, for example, the stabilized and DR IL-18 variant peptides discussed above). In some cases, in addition to IL-18 protein, the formulation also contains: a pH-maintaining buffer (e.g., between 6.2 and 6.8, such as His/His-HCl); a sugar (e.g., sucrose, such as 6-10%); a chelating agent (e.g., EDTA, such as 0.05-1.5 mM); an antioxidant (e.g., L-methionine, such as 4-6 mM); and an agent that prevents protein uptake (e.g., PS80, such as 0.01-0.03% w/v).

Attached Figure Description

The following detailed description of embodiments of the invention will be better understood when read in conjunction with the accompanying drawings. It should be understood that the invention is not limited to the precise arrangement and apparatus of the embodiments shown in the figures.

Figures 1A and 1B depict the results of exemplary experiments illustrating the IL-18 pathway as a target for tumor immunotherapy. Figure 1A provides a chart showing that the IL-18 pathway (including IL-18 and its receptor subunits) is upregulated in both activated and dysfunctional tumor T cell programs, as seen in RNA-seq expression analysis of cytokines and receptors in CD8+ TILs. As seen in the chart, genes were assigned “activated” and “dysfunctional” scores compared to naive T cells. Data points marked with diamonds and black text indicate IL-18 cytokines, IL-18R1 (Rα), and IL-18RAP (Rβ). Data adapted from Singer et al. (Singer, M. et al., 2016, Cell, 166:1500-1511, e1509). Figure 1B presents results of infection with LCMV (left; CD4) or VSV-OVA (right; CD8). The IL-18 receptor subunits IL-18Rα and IL-18Rβ are part of a gene expression program associated with long-term antigen exposure, as seen after infection with LCMV (left; CD4) or VSV-OVA (right; CD8). Data from the ImmGen database.

Figures 2A through 2C depict the results of exemplary experiments illustrating the characteristics of IL-18BP as a “soluble immune checkpoint.” Figure 2A shows how IL-18BP mediates an interferon-γ (IFN-γ)-driven IL-18 negative feedback loop, reminiscent of the immune checkpoint PD-L1. A schematic diagram of the IL-18/IFN-γ/IL-18BP feedback loop is depicted. Black arrows indicate stimulation, and dark gray circular lines indicate inhibition. Figure 2B shows the upregulation of IL-18BP in gastric and breast cancer using data from the TCGA and Oncomine databases. Figure 2C illustrates the close correlation between PD-1 and IL-18BP expression in a large number of breast and gastric cancer samples (from the TCGA database). R = 0.78 and 0.65, respectively.

Figures 3A through 3C depict the results of an exemplary experiment illustrating the use of yeast display to engineer human IL-18 variants independently of IL-18BP. Figure 3A provides a structure-guided library designed to randomize residues at the IL-18:IL-18BP interface and introduced into the yeast display system. Yeast clones were selected using magnetic and fluorescent cell sorting to achieve binding to IL-18Rα and deselected against IL-18BP. Figure 3B provides a summary of the directed evolution that generated IL-18BP-resistant IL-18 variants. The positive selection condition was hIL18RαSA-beads and hIL18Rα. The deselection condition was SA, hIL-18BP, and nIL-18BP tetramers alone. Figure 3C provides flow cytometry analysis of yeast-displayed WT IL-18 (left) or the directed-evolved variant (right). The Y-axis shows IL-18BP binding, and the X-axis shows IL-18Rα binding. After five rounds of directed evolution, the remaining clones showed a greater preference for IL-18Rα compared to IL-18BP.

Figure 4 depicts the results of an exemplary experiment, summarizing the sequences of the decoy-resistant human IL-18 (“DR-IL-18”, also known as “DR-18”) variant. The location of each mutation and the corresponding residue position in the mature form of wild-type human IL-18 are indicated at the top of the table. SEQ ID NO:38 to SEQ ID NO:58 represent sequences obtained after selection using directed evolution. SEQ ID NO:34-SEQ ID NO:37 are common sequences derived from the selected sequences. Shaded residues indicate the five most conserved mutations observed. The sequence in the top row (“WT hIL-18”) is shown as SEQ ID NO:30.

Figures 5A and 5B depict the results of exemplary experiments illustrating the biophysical characterization of human DR-IL-18 variants. Figure 5A provides binding isotherms for yeast-displayed DR-hIL-18 variants. As shown, yeast-displayed DR-IL-18 variants SEQ ID NO:34-37 and SEQ ID NO:39 are able to bind hIL-18Rα, with binding isotherms comparable to WT human IL-18 (left). In contrast, very little binding was observed with the same variants and hIL-18BP (right). Figure 5B shows a representative surface plasmon resonance sensor plot between immobilized biotinylated hIL-18BP and DR-IL-18 variants. Recombinant hIL-18 (left) binds IL-18BP with extremely high affinity, KD = 2.0 pM, while SEQ ID NO:34 (right) shows a much slower binding rate, dissociation rate, and KD = 15.2 nM. This data is summarized in Tables 6 and 7.

Figures 6A and 6B depict the results of exemplary experiments demonstrating that the human DR-IL-18 variants were not inhibited by IL-18BP. Figure 6A provides a graph showing that recombinant IL-18BP inhibited the binding of biotinylated IL-18Rα to yeast-displayed WT IL-18, but did not affect the DR-IL-18 variants SEQ ID NO:34-37 and SEQ ID NO:39 (shown in the graph on the left). In contrast, IL-18BP effectively neutralized the previously described IL-18 E42A, K89A, and E42A/K89A (Kim et al., 2001, Proc. Natl. Acad. Sci., 98(6):3304-3309) (shown in the graph on the right) (E42 and K89 of Kim et al. are E6 and K53 of SEQ ID NO:30, respectively). Biotinylated IL-18Rα was maintained at a fixed concentration of 100 nM in all samples. Figure 6B presents the results of the HEK Blue IL-18 signaling assay, showing that WT IL-18, along with SEQ ID NO:34, SEQ ID NO:36, and SEQ ID NO:37, stimulated IL-18 HEK-Blue reporter cells with comparable potency and efficacy (left). Wild-type IL-18 was highly sensitive to the application of recombinant IL-18BP in this assay (IC50 = 3 nM), while SEQ ID NO:34 and SEQ ID NO:36 were not inhibited by recombinant IL-18BP, even at a concentration of 1 μM (right). hIL-18 remained at a fixed concentration of 5 nM, and SEQ ID NO:34 and SEQ ID NO:36 remained at 2.5 nM.

Figures 7A through 7C depict the results of an exemplary experiment illustrating the use of yeast to engineer additional human IL-18 variants (version 2 variants) for independent IL-18BP production. Figure 7A provides a summary of the positions in the randomized human IL-18 in the version 2.0 library. Degenerate codons and the encoded amino acid set are given for each position. Figure 7B provides a summary of the directed evolution for generating version 2.0 IL-18BP resistant IL-18 variants. The positive selection condition was hIL18RαSA-beads and hIL18Rα. The anti-selection condition was SA, hIL-18BP, and hIL-18BP tetramers alone. Figure 7C provides a flow cytometry analysis of the progress in generating version 2.0 DR-IL-18 variants. Yeast obtained after rounds 1, 4, and 6 were simultaneously stained with 250 nM IL-18BP streptavidin-PE tetramer or 100 nM IL-18Rα directly labeled with Alexa Fluor 647. The Y-axis shows IL-18BP binding, and the x-axis shows IL-18Rα binding. After six rounds of directed evolution, the remaining clones showed a greater preference for IL-18Rα compared to IL-18BP.

Figure 8 depicts the results of an exemplary experiment, illustrating a summary of the sequences of the version 2.0 decoy-resistant human IL-18 (DR-IL-18) variant. The location of each mutation and the corresponding residue position in the mature form of wild-type human IL-18 are indicated at the top of the table. Shaded rows indicate recursive sequence variants obtained in rounds 5 and 6. The sequence in the top row (“WThIL-18”) is shown as SEQ ID NO:30.

Figure 9 depicts the results of exemplary experiments illustrating the biophysical characterization of the version 2.0 human DR-IL-18 variant. Figure 9A shows that the yeast-displayed version 2.0 DR-IL-18 variant can bind hIL-18Rα, with a binding isotherm comparable to WT human IL-18. Figure 9B shows, in contrast, very little binding was observed with the same variant and hIL-18BP. Figure 9C provides the thermostability of the version 2.0 DR-IL-18 variant, as assessed by heating the yeast-displayed variant for 15 minutes within a defined temperature range followed by hIL-18Rα staining. The thermostability of the version 2.0 DR-IL-18 variant is higher than that of WT IL-18 (Tm = 47.6°C) and the first-generation concordant sequence (Tm = 50.9 and 40.2 for SEQ ID NO: 24 and SEQ ID NO: 35, respectively). Figure 9D provides a summary of the receptor-binding properties and thermostability of the second-generation DR-IL-18 variant. NBD = No binding detected. N.D. = Undetermined value.

Figures 10A through 10C depict the results of an exemplary experiment illustrating the use of yeast to engineer murine IL-18 variants for independent IL-18BP development. Figure 10A provides a summary of the directed evolution that generated the IL-18BP-resistant murine IL-18 variant. The positive selection condition was mIL18RαSA-beads and mIL18Rα. The anti-selection condition was mIL-18BP and mIL-18BP tetramers. Figure 10B shows the results of flow cytometry analysis of the yeast-developed murine IL-18 variant after 5 rounds of directed evolution. The Y-axis shows IL-18BP binding, and the x-axis shows IL-18Rα binding. Figure 10C provides a summary of the sequences of the bait-resistant murine IL-18 (DR-IL-18) variant. The sequence in the top row (“mIL-18”) is shown as SEQ ID NO:31. The location of each mutation and the corresponding residue in the mature form of wild-type murine IL-18 is indicated at the top of the table. SEQ ID NO:62 to SEQ ID NO:72 represent sequences obtained after selection using directed evolution. SEQ ID NO:60 and SEQ ID NO:61 are common sequences derived from the selected sequences. Shaded residues indicate the five most conserved mutations observed.

Figures 11A and 11B depict the results of exemplary experiments illustrating the biophysical characterization of murine DR-IL-18 variants. Figure 11A shows that yeast-displayed DR-IL-18 variants SEQ ID NO:70, SEQ ID NO:67, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:60, and SEQ ID NO:61 are able to bind mIL-18Rα, with binding isotherms comparable to those of WT murine IL-18 (left). In contrast, very little binding was observed with the same variants and mIL-18BP (right). Figure 11B provides measurements of IL-18BP binding using a representative surface plasmon resonance sensor map of immobilized biotinylated mIL-18BP with murine DR-IL-18 variants. The recombinant mIL-18 (left) binds to mIL-18BP with high affinity (KD = 0.8 pM), while SEQ ID NO: 61 (right) shows significantly reduced binding (KD value > 10 μM). This data is summarized in Tables 8 and 9.

Figures 12A through 12D depict the results of an exemplary experiment illustrating the pharmacodynamics of DR-IL-18 administered to mice. Figure 12A is a schematic diagram of the study design. Mice were administered a mediator (PBS), mIL-18 (1 mg/kg), or the DR-IL-18 variant SEQ ID NO:61 (1 mg/kg) once daily for a total of seven doses (depicted as syringes). Blood samples were obtained two days prior to the experiment and five hours after injection on days 0, 3, and 6. Figure 12B shows peripheral blood cell counts of CD4, CD8, NK cells, and monocytes on days 0, 3, and 6. Both IL-18 and SEQ ID NO:61 increased NK cells and monocytes to similar extent by day 3. For each time point (day), the left bar represents PBS, the middle bar represents IL-18, and the right bar represents SEQ ID NO:61. Figure 12C shows CD69 expression on peripheral CD4, CD8, and NK cells. SEQ ID NO:61 stimulates CD69 expression on CD4 and CD8 cells, but IL-18 does not. Both IL-18 and SEQ ID NO:61 increase CD69 on NK cells, but SEQ ID NO:61 treatment results in sustained CD69 expression, which is evident on day 6, returning to baseline CD69 levels compared to IL-18. For each time point (day), the left bar represents PBS, the middle bar represents IL-18, and the right bar represents SEQ ID NO:61. Figure 12D provides serum cytokine levels of interferon-g (IFN-g), MIP-1b, and G-CSF. SEQ ID NO:61 treatment resulted in higher levels of IFN-g, MIP-1b, and G-CSF than mIL-18 treatment.

Figure 13 illustrates the results of an exemplary experiment demonstrating that DR-IL-18 treatment reduced body fat composition in mice. Body fat and lean body mass composition were measured every three days in mice treated with 0.01, 0.1, or 1 mg/kg of the DR-IL-18 variant SEQ ID NO:61 or 1 mg/kg WT mIL-18. SEQ ID NO:61 treatment produced a significant reduction in body fat as a percentage of total body weight (top panel). This is reflected in a decrease or stabilization of fat mass (left panel) and a consistent increase in lean body mass (right panel). Mice treated with the vector and mIL-18 showed an increase in body fat mass and stable lean body mass over the same treatment period.

Figures 14A and 14B depict the results of an exemplary experiment illustrating that DR-IL-18 is an effective immunotherapeutic agent in a melanoma model. Figure 14A provides a spider diagram of tumor growth in mice carrying Yummer1.7 melanoma tumors treated twice weekly with saline (control), WT IL-18 (0.32 mg/kg), DR-IL-18 variant SEQ ID NO:61 (0.32 mg/kg), anti-PD1 (8 mg/kg), IL-18 + anti-PD1, or SEQ ID NO:61 + anti-PD1. Figure 14B provides survival curves from the same group as in Figure 14A. As shown in Figure 14B, SEQ ID NO:61 is effective as a monotherapy in this model and is synergistic in combination with anti-PD1.

Figures 15A and 15B depict the results of exemplary experiments illustrating that the efficacy of DR-IL-18 in the melanoma model of Figure 14 depends on CD4 and CD8 lymphocytes as well as interferon-γ. Figure 15A provides a spider diagram of tumor growth in mice carrying Yummer1.7 melanoma tumors treated with saline (control), or DR-IL-18 variant SEQ ID NO:61 alone (0.32 mg/kg), or in combination with depleted antibodies against CD8, CD4, interferon-γ, or NK1.1. Figure 15B provides survival curves from the same group as in Figure 15A.

Figure 16 depicts the results of an exemplary experiment illustrating the dose-dependent efficacy of DR-IL-18 in an MC38 tumor model. The figure shows a spider diagram of tumor growth in mice carrying MC38 colon cancer tumors, treated every three days with PBS (control), 1.0 mg/kg WT IL-18, 1.0 mg/kg SEQ ID NO:61, 0.1 mg/kg SEQ ID NO:61, or 0.01 mg/kg SEQ ID NO:61. WT IL-18 was ineffective at 1 mg/kg, while SEQ ID NO:61 showed partial efficacy at 0.1 mg/kg and maximum efficacy at 1.0 mg/kg.

Figure 17 depicts the results of an exemplary experiment illustrating the efficacy of DR-IL-18 alone in combination with the immune checkpoint inhibitor anti-PD1 in an MC38 tumor model. A spider diagram of tumor growth is shown for mice carrying MC38 colon cancer tumors treated with PBS (control), 0.32 mg/kg WT IL-18, 0.32 mg/kg DR-IL-18 variant SEQ ID NO:61, 5 mg/kg anti-PD1, a combination of anti-PD1 and WT IL-18, or a combination of anti-PD1 and SEQ ID NO:61. All doses were administered intraperitoneally twice weekly for a total of up to six doses.

Figures 18A and 18B depict the results of an exemplary experiment investigating the antitumor mechanism of DR-IL-18 in mice carrying MC38 tumors. Figure 18A provides the results of a tumor immunophenotyping assay in mice treated with saline, WT IL-18, or the DR-IL-18 variant SEQ ID NO:61 twice weekly for a total of four doses. DR-IL-18 treatment resulted in an increase in the number of CD8 and NK cells per milligram of tumor (top left two figures) and increased expression of activation markers granzyme B and KLRG1 on CD8 and NK cells (top right two figures). Compared with saline treatment, DR-IL-18 treatment did not improve the CD8:Treg ratio, while WT IL-18 made the ratio less favorable. However, DR-IL-18 treatment increased the ratio of CD8 cells to the suppressive myeloid population, including tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells of monocyte and granulocyte origin (mMDSCs and gMDSCs) Figure 18B shows serum Luminex cytokine measurements from the same mice as in Figure 18A, obtained 24 hours after the fourth treatment dose. DR-IL-18 shows the significantly altered minor cytokine release profiles after treatment with WT IL-18, which significantly increased interferon-γ, IL-7, and IL-15 levels by more than 100-fold.

Figures 19A through 19C depict the results of an exemplary experiment illustrating the ability of DR-IL-18 to effectively treat immune checkpoint inhibitor-refractory tumors by impairing surface MHC class I expression. Figure 19A shows a spider plot of tumor growth in mice carrying B2m-deficient Yummer1.7 tumors treated with saline, anti-PD1 + anti-CTLA4, or the DR-IL-18 variant SEQ ID NO:61. Figure 19B includes a graph showing the survival percentage relative to days post-transplantation. DR-IL-18 exhibits robust efficacy regarding tumor growth and survival, curing 60% of treated mice in this model, which was even completely resistant to combination therapy with anti-CTLA4 + anti-PD1. This efficacy is NK cell dependent, as administration of DR-IL-18 with anti-NK1.1 (which depletes NK cells) negates the effect of SEQ ID NO:61 treatment. Figure 19C provides a graph comparing interferon-γ production and Ki67 levels in the tumor. NK cells isolated from the B2m-deficient Yummer1.7 were dysfunctional, exhibiting reduced proliferation (Ki67 staining) and function (interferon-γ secretion). However, treatment with DR-IL-18 reversed this phenotype, achieving robust proliferation and cytokine secretion.

Figures 20A through 20C depict exemplary experiments illustrating the engineering of human IL-18 variants into IL-18BP antagonists (or “decoy-decoy”, D2D) using yeast display. These variants bind to IL-18BP but do not signal, thus antagonizing the effect of IL-18BP on endogenous IL-18. Figure 20A is a summary of the locations of randomized human IL-18 in the D2D library. Degenerate codons and sets of encoded amino acids are given for each location. Figure 20B provides a summary of the directed evolution to generate D2D IL-18 variants that bind and neutralize IL-18BP but do not signal via IL-18R. The positive selection condition is mIL18BP. The anti-selection conditions are nIL18Rα, hIL18Rb, and mIL18Rα. (Figure 20C) Flow cytometry analysis of the progress in generating D2D hIL-18 variants. Yeast samples obtained after rounds 1-4 were stained with 1 nM mouse IL-18BP (left panel), 1 nM human IL-18BP (middle panel), or 1 μM IL18Rα plus 1 μM IL18Rβ. The selected variants showed enhanced IL-18BP binding in multiple rounds of selection, while IL18Rα or IL18Rβ binding was not increased.

Figure 21 depicts the results of an exemplary experiment, illustrating a summary of the sequences of the D2D human IL-18 variants. The location of each mutation and the corresponding residue position in the mature form of wild-type human IL-18 are indicated at the top of the table. The sequence in the top row (“WT hIL-18”) is shown as SEQ ID NO:30.

Figures 22A through 22C depict the results of exemplary experiments illustrating the biophysical characterization of human decoy-decoy (D2D) IL-28 variants. Figure 22A shows that yeast-displayed D2D IL-8 variants SEQ ID NO:120, SEQ ID NO:101, SEQ ID NO:94, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:123, SEQ ID NO:124, and SEQ ID NO:125 are able to bind hIL-18RBP, with binding isotherms comparable to WT human IL-18. Figure 22B shows, in contrast, very little binding was observed with the same variants and hIL-18Rα. Figure 22C provides a summary of the receptor-binding properties of the D2D IL-18 variants. NBD = No binding detected.

Figure 23 illustrates the results of an exemplary experiment, summarizing the sequences of the D2D murine IL-18 variants. The location of each mutation and the corresponding residue in the mature form of wild-type murine IL-18 are indicated at the top of the table. The sequence in the top row (“WT mIL-18”) is shown as SEQ ID NO:31.

Figure 24 depicts the results of biophysical affinity measurements (sensor maps) of the binding of second-generation DR-IL-18 variants to IL-18Rα and IL-18BP using surface plasmon resonance (SPR). Top row: Representative sensor maps of the indicated IL-18 variants (soluble analytes) to hIL-18Rα (immobilized ligands). Bottom row: Representative sensor maps of the indicated IL-18 variants to humans (hIL-18BP). The x-axis is time in seconds, and the y-axis is response units (RU). The curves are data over time observed at different concentrations (2-fold dilutions starting from 1 nM), superimposed with the best-fit curve of the assumed 1:1 Langmuir binding model.

Figures 25A and 25B depict data illustrating the efficacy of DR-IL-18 in a CT26 colorectal tumor model. 250,000 CT26 cells were subcutaneously implanted, and treatment began on day 7 once the tumor reached an average size of -60 ^mm³ . WT IL-18 and SEQ ID NO:61 were administered at 0.32 mg/kg twice weekly for a total of 5 doses. Anti-PD1 was administered at 10 mg/kg on the same schedule. Figure 25A is an overlay of the spider diagram, with black lines showing tumor growth in animals treated with saline (PBS) (circles), dark gray lines showing tumor growth in animals treated with WT IL-18 (squares), and gray lines showing tumor growth in animals treated with DR-IL-18 (SEQ ID NO:61) (triangles). Treatment with DR-IL-18 alone resulted in tumor growth inhibition and tumor clearance in a subgroup of animals, but not with WT IL-18. Figure 25B shows the survival curves of mice treated with anti-PD-1, WT IL-18, and DR-IL-18 (SEQ ID NO: 61). The number of complete responses is indicated in parentheses. DR-IL-18 resulted in 40% survival extension and tumor clearance in mice, which was superior to the checkpoint inhibitor anti-PD-1, but not WT IL-18.

Figures 26A and 26B depict data illustrating the efficacy of DR-IL-18 in a 4T1 breast cancer model and a B16-F10 melanoma model. Figure 26A shows tumor growth curves of 4T1 tumors transplanted into BALB/C mice after treatment with saline (PBS; black), WT IL-18 (dark gray), or the DR-IL-18 variant SEQ ID NO:61 (gray). Figure 26B shows tumor growth curves of B16-F10 tumors transplanted into C57BL/6 mice after treatment with saline (PBS; black), WT IL-18/TA99 (dark gray), or the DR-IL-18 variant SEQ ID NO:61 (gray). In both models, only DR-IL-18 resulted in tumor growth inhibition, but not WT IL-18. Treatment was administered after the tumor exceeded a mean volume of 50 ^mm³ , as indicated by the boxes marked with “t”.

Figures 27A and 27B depict data expanded from Figures 19A to 19C. The depicted data illustrate the efficacy of DR-IL-18 in treating additional MHC class I deficient tumor models resistant to immune checkpoint inhibitors. Figure 27A provides a graph of tumor volume in mice implanted with B2m-deficient MC38 cells relative to days post-implantation, prepared using CRISPR/Cas9-mediated deletion as described for B2m-deficient YUMMER cells. B2m-/-MC38 cells were subcutaneously implanted, and treatment was initiated on day 7 once the tumor reached an average of -65 ^mm³ . SEQ ID NO:61 was administered at 0.32 mg/kg twice weekly for 5 doses. Anti-PD1 and anti-CTLA4 were administered at 8 mg/kg on the same schedule. Figure 27B shows a graph of tumor volume in mice implanted with RMA/S cells relative to days post-implantation. RMA/S is a variant of the RMA lymphoma cell line containing a spontaneous mutation in Tapasin. The result was antigen-load deficiency, and consequently, reduced MHC class I surface expression. It is a C57BL/6 homolog and refractory to immune checkpoint inhibitors. Mice were subcutaneously implanted with 1,000,000 RMA/S cells, and treatment began on day 7. SEQ ID NO:61 was administered at 0.32 mg/kg twice weekly. Anti-PD1 was administered at 8 mg/kg on the same schedule. In both studies, treatment with the DR-18 variant SEQ ID NO:61 alone demonstrated antitumor efficacy, in the form of tumor growth inhibition (B2m ^-/- MC38) or tumor clearance (RMA/S).

Figure 28 illustrates the data illustrating the efficacy of DR-IL-18 in enhancing antibody-dependent cell-mediated cytotoxicity (ADCC). Ex vivo cytotoxicity studies used CFSE-labeled Raji (B-cell lymphoma) cells and isolated human peripheral blood mononuclear cells (PBMCs). PBMCs and labeled Raji cells were incubated together for 25 h at an effector:target (E:T) ratio of 1:10. Human DR-IL-18 variant hCS-1 (1 μM), rituximab (10 μg/mL), or a combination of both were applied to the samples, as indicated. Cytotoxicity was measured by flow cytometry and calculated as the fraction of CFSE cells that became DAPI-positive. DR-18, as a single agent, significantly stimulated tumor cell killing and significantly enhanced the killing effect of the therapeutic antibody rituximab. *p < 0.05, corrected for by two-factor ANOVA and Tukey's multiple comparisons.

Figures 29A and 29B depict data illustrating the antiviral efficacy of the DR-18 variant SEQ ID NO:61 in treating viral infections (e.g., in this case, in treating systemic vaccine viral infections). Figure 29A shows the experimental design. C57BL/6 mice were intraperitoneally (IP) infected with ^10⁶ PFU of vaccinia virus (VACV), and the IP administration included either 1 mg/kg WT mIL-18 or SEQ ID NO:61. Mice were sacrificed, and viral titers in blood and ovaries were measured by RT-PCR on day 3 post-infection. Figure 29B shows the quantification of VAV viral copies in the ovaries and blood of treated mice on day 3 post-infection. Treatment with SEQ ID NO:61 showed a significant decrease in viral titer, while WT IL-18 was ineffective. *p<0.05, **p<0.01, ***p<0.001.

Figure 30 illustrates the data demonstrating the activity of second-generation human DR-IL-18 variants. (Figure 30) WT IL-18 and human SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, and SEQ ID NO:87 stimulate IL-18HEK-Blue reporter cells. Compared to WT hIL-18, human SEQ ID NO:89, SEQ ID NO:90, and SEQ ID NO:91 showed enhanced potency, while SEQ ID NO:87 was equivalent to WT hIL-18 in potency. Therefore, the data demonstrate that all tested second-generation human DR-IL-18 variants actively signal via IL-18R.

Figure 31 depicts IL-18 variants with mutations at various cysteine positions, which were prepared and then run on an SDS/PAGE gel (visualized using Spyro-Ruby staining). R = reducing sample; N = non-reducing sample; MW = molecular weight. The numbers along the bottom refer to the SEQ ID number (see Table 13).

Figure 32 depicts the SDS/PAGE gel analysis of different C38S/C68 mutants with the potential T cell epitope removed from C68S. R = reduced sample. NR = non-reduced sample.

Figure 33 depicts size exclusion chromatography data from shelf-life stability studies of different C38S/C68 mutants with potential T-cell epitopes removed from C68S.

Figure 34 depicts the dose-response data using the HEK-Blue reporter cell line. All variants showed increased potency compared to WT IL-18, and the potency was similar to that of the parental variant SEQ ID NO:89.

Figure 35 depicts size exclusion chromatography data from SEQ ID NO:19 and SEQ ID NO:89 based on freeze-thaw studies.

Figure 36 depicts size exclusion chromatography data from SEQ ID NO:19 and SEQ ID NO:89 based on a stirring study.

Figures 37A and 37B depict the results of surface plasmon resonance experiments testing the affinity of SEQ ID NO:19 and wild-type human IL-18 for human and cynomolgus monkey IL-18Rα (Figure 37A) and IL-18BP (Figure 37B).

Figure 38 illustrates the results of the IL-18BP resistance assay. WT hIL-18 was effectively inhibited by the addition of IL-18BP, while SEQ ID NO:19 retained strong signal transduction ability at all IL-18BP concentrations.

Figure 39 depicts tumor growth data in mice based on a drug administration experiment (using mouse DR IL-18 variant SEQ ID NO:61).

Figure 40 depicts tumor growth data in mice based on an additional dosing experiment (using mouse DR IL-18 variant SEQ ID NO:61).

Figures 41A and 41B depict the drug tolerance experiment in primates (cynomolgus monkeys) using the DR IL-18 variant (SEQ ID NO:89 in this case).

Figures 42A and 42B depict the production and purification of the IL-18 peptide (in this case, DR IL-18 SEQ ID NO:89) using a cell-free SUMO protease that cleaves the N-terminal SUMO tag of the IL-18 protein.

Figures 43A and 43B depict the results of monitoring SUMO protease cleavage by RP-HPLC.

Figure 44 illustrates the production and purification of the IL-18 polypeptide (in this case, DRIL-18SEQ ID NO:19) using an exogenously provided SUMO protease that cleaves the N-terminal SUMO tag of the IL-18 protein inside a bacterial cell (in this case, Escherichia coli).

Figure 45 depicts RP-HPLC data for monitoring the SUMO protease cleavage reaction inside bacterial cells.

Figure 46 illustrates a schematic diagram of the secretion system of yeast (e.g., Pichia pastoris) that produces IL-18 variants.

Figure 47 depicts a representative reduced SDS-PAGE gel showing the expression of DR-18 (IL-18 variants, in this case, SEQ ID NO:89 and SEQ ID NO:87).

Figure 48 depicts a two-step chromatographic process (and associated data) that facilitates the label-free purification of IL-18 from a yeast secretory expression system (in this case, Pichia pastoris).

Detailed Implementation

Variant (e.g., stabilized) IL-18 peptides and methods of use (e.g., treating diseases or conditions) are provided. The subject-stabilized IL-18 peptide contains a mutation of two cysteine residues (C38 and C68) relative to human wild-type IL-18 (SEQ ID NO:30). In some cases, the mutation is a C to S substitution, and therefore the stabilized IL-18 peptide may contain mutations of C38S and C68S in some cases. In some cases, the subject-stabilized IL-18 peptide contains mutations of C38S/C68S, C38S/C68G, C38S/C68A, C38S/C68V, C38S/C68D, C38S/C68E, or C38S/C68N (e.g., in some cases, C38S/C68G, C38S/C68A, C38S/C68V, C38S/C68N, C38S/C68G, C38S/C68A, C38S/C68V, C38S/C68N, C38S/C68G, C38S/C68A, C38S/C68V, C38S/C68N, C38S/C68E ... 38S/C68D, C38S/C68E, or C38S/C68N; in some cases, C38S/C68S, C38S/C68G, C38S/C68A, C38S/C68D, or C38S/C68N; and in some cases, C38S/C68G, C38S/C68A, C38S/C68D, or C38S/C68N).

In some cases, the subject-stabilized IL-18 peptide is a stabilized IL-18 variant peptide (e.g., a stabilized “decoy-resistant” (DR) IL-18 variant or a stabilized “decoy-decoy” (D2D) IL-18 variant), that is, an IL-18 variant that contains additional mutations at positions C38 and C68 relative to human wild-type IL-18.

Methods are provided for administering an IL-18 peptide (e.g., wild-type IL-18 or a variant, such as stabilized, DR, D2D, stabilized DR, or stabilized D2D IL-18 variant) to an individual (e.g., subcutaneously), wherein the administration frequency is no more than once per week. Methods for producing/preparing the peptide are also provided. Some of these methods involve contacting an exogenously provided fusion protein inside a cell (e.g., a bacterial cell) with a SUMO protease, wherein the fusion protein includes a protein of interest fused to its N-terminus with a SUMO tag, whereby the SUMO protease cleaves the fusion protein to remove the SUMO tag from the protein of interest.

Before describing the invention in more detail, it should be understood that the invention is not limited to the specific embodiments described, as such embodiments are of course subject to variation. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting, as the scope of the invention will be limited only by the appended claims.

When a range of values is provided, it should be understood that each intervention value between the upper and lower limits of this range (accurate to one-tenth of the unit of the lower limit unless the context clearly indicates otherwise), and any other value or intervention value within this range, is covered by this invention. The upper and lower limits of these smaller ranges may be independently included within these smaller ranges and also covered by this invention, subject to any specific excluded limit value within the stated range. When the stated range includes one or two limits, the range excluding any one or both of those included limits is also included within this invention.

This document uses the term "approximately" before numerical values to provide certain ranges. The term "approximately" is used herein to provide textual support for the exact number that follows it, as well as numbers that are close to or approximate to the number following the term. In determining whether a numerical value is close to or approximate to a specifically listed numerical value, an unlisted numerical value that is close to or approximates can be a value that provides approximately equivalent to the specifically listed numerical value in the context in which it appears.

Unless otherwise defined, all 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 pertains. While any methods and materials similar to or equivalent to those described and used herein may also be used in the practice or testing of this invention, representative illustrative methods and materials are described hereafter.

All publications and patents referenced in this specification are incorporated herein by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and to disclose and describe the methods and/or materials combined in the referenced publication. Any reference to a publication refers to that prior to its filing date and should not be construed as an admission that the invention is not entitled to a prior publication due to prior art. Furthermore, the publication date provided may differ from the actual publication date, which may require independent verification.

It should be noted that, as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include multiple indicators unless the context clearly indicates otherwise. It should also be noted that claims may be drafted to exclude any optional elements. Therefore, this statement is intended as a prior basis for using exclusive terms such as “solely” or “only” in relation to the recitation of the elements of a claim, or for using a negative limitation.

Those skilled in the art will understand upon reading this disclosure that the individual embodiments described and illustrated herein have discrete components and features that can be readily separated from or combined with features of any other embodiments without departing from the scope or spirit of the invention. Any described method may be performed in the order of the described events or in any other logically possible order.

Although the apparatus and method have been or will be described in a functional interpretation for the sake of grammatical fluency, it should be clearly understood that the claims (unless expressly stated in 35 U.S.C. § 112) should not be construed as necessarily being limited in any way by the construction of “means” or “steps”, but should be given the full meaning and equivalents provided by the definition of the claims in accordance with the doctrine of judicial equivalents, and where the claims are expressly stated in 35 U.S.C. § 112, all legal equivalents should be given in accordance with 35 U.S.C. § 112.

definition

Unless otherwise defined, all 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 pertains. The following terminology will be used in describing and claiming protection for this invention.

It should also be understood that the terminology used herein is for the purpose of describing specific implementations only and is not intended to be restrictive.

The articles “a” and “an” are used in this text to refer to one or more of the grammatical objects of the article (i.e., at least one). By way of example, “element” means one or more elements.

As used herein, “about” when referring to measurable values such as quantity, duration, etc., means a non-limiting variation (where such variation is appropriate) covering ±40%, or ±20%, or ±10%, ±5%, ±1%, or ±0.1% of the specified value.

When used in the context of an organism, tissue, cell, or component thereof, the term "abnormal" refers to an organism, tissue, cell, or component thereof whose at least one observable or detectable characteristic (e.g., age, treatment, time of day) differs from those of organisms, tissues, cells, or components thereof that exhibit "normal" (expected) corresponding characteristics. A characteristic that is normal or expected for one cell or tissue type may be abnormal for different cell or tissue types.

As used herein, the term "antibody" refers to an immunoglobulin molecule capable of specifically binding to a particular epitope on an antigen. Antibodies can be complete immunoglobulins derived from natural or recombinant sources, and can be the immunoreactive portion of a complete immunoglobulin. The antibodies in this invention can exist in various forms, including, for example, polyclonal antibodies, monoclonal antibodies, intracellular antibodies (“internal antibodies”), Fv, Fab and F(ab)2, and single-chain antibodies (scFv), heavy-chain antibodies such as camel antibodies, synthetic antibodies, chimeric antibodies, and humanized antibodies (Harlow et al., 1999, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426).

As used in this article, “antibody heavy chain” refers to the larger of two types of polypeptide chains present in all antibody molecules in their naturally occurring conformation.

As used in this article, "antibody light chain" refers to the smaller of two types of polypeptide chains present in all antibody molecules in their naturally occurring conformation. κ and λ light chains refer to the two main isotypes of antibody light chains.

As used herein, the term "synthetic antibody" means an antibody generated using recombinant DNA technology, such as an antibody expressed by a bacteriophage as described herein. The term should be interpreted as referring to an antibody or specifying the amino acid sequence of said antibody, which is generated by synthesizing a DNA molecule encoding the antibody and which expresses an antibody protein, wherein said DNA or amino acid sequence is obtained using synthetic DNA or amino acid sequence techniques available and well known in the art.

As used in this article, “immunoassay” refers to any binding assay that uses antibodies capable of specifically binding to target molecules to detect and quantify the binding of target molecules.

As used herein, the term "specific binding," for example in relation to IL-18 variant peptides, refers to an IL-18 variant peptide that recognizes and binds to a specific molecule such as IL-18R or IL-18BP. For example, it can be said that wild-type IL-18 specifically binds to both IL-18R and IL-18BP. In some cases, IL-18 variant peptides exhibit significantly reduced binding to IL-18BP. For instance, an IL-18 variant peptide that specifically binds to a receptor from one species may also bind to receptors from one or more species. However, such cross-species reactivity itself does not alter the classification of IL-18 variant peptides as specific. In another instance, an IL-18 variant peptide that specifically binds to a receptor may also bind to different allelic forms of the receptor. However, such cross-reactivity itself does not alter the classification of IL-18 variant peptides as specific. In some contexts, the terms "specific binding" or "specifically binding" can be used to describe the interaction of an antibody, protein, or peptide with a chemical substance, implying that the interaction depends on the presence of a specific structure on the chemical substance (e.g., an antigenic determinant or epitope); for example, an IL-18 variant peptide recognizes and binds to a specific protein structure rather than binding to proteins broadly. Similarly, the term "target" (e.g., in the context of an antibody that "targets" a specific antigen) is used to refer to a specific binding partner of a given molecule. For example, an agent (e.g., an antibody) that "targets" a specific protein/antigen specifically binds to said protein/antigen, in a sense that it binds to said specific protein/antigen preferentially over binding to other proteins/antigens.

As used herein, the term "applicator" means any device used to administer the compositions of the present invention to a subject, including but not limited to hypodermic syringes, pipettes, iontophoresis devices, patches, etc.

As used herein, the term "coding sequence" refers to a nucleic acid sequence, its complementary sequence, or a portion thereof, that can be transcribed and/or translated to produce mRNA and/or polypeptides or fragments thereof. Coding sequences include exons in genomic DNA or immature primary RNA transcripts that bind together via cellular biochemical mechanisms to provide mature mRNA. The antisense strand is the complementary sequence of this nucleic acid, from which the coding sequence can be deduced. In contrast, as used herein, the term "non-coding sequence" refers to a nucleic acid sequence, its complementary sequence, or a portion thereof, that is not translated into amino acids in vivo or whose tRNAs do not interact to place or attempt to place amino acids. Non-coding sequences include intron sequences in genomic DNA or immature primary RNA transcripts and gene-associated sequences such as promoters, enhancers, silencers, etc.

As used herein, the terms "complementary" and "complementarity" refer to polynucleotides (i.e., nucleotide sequences) that are related by base pairing rules. For example, the sequence "A-G-T" is complementary to the sequence "T-C-A". Complementarity can be "partial," where only some nucleic acid bases match according to base pairing rules. Alternatively, there may be "complete" or "overall" complementarity between nucleic acids. The degree of complementarity between nucleic acid chains has a significant impact on the efficiency and strength of hybridization between nucleic acid chains. This is particularly important in amplification reactions and in detection methods that depend on the binding between nucleic acids.

"Disease" is a state of health in animals in which they are unable to maintain homeostasis, and if the disease is not treated, the animal's health will continue to deteriorate. In contrast, an animal's "symptom" is a state of health in which the animal can maintain homeostasis, but the animal's health is less favorable than when it is not symptom-related. Even without treatment, a symptom does not necessarily lead to a further decline in the animal's health.

As used in this article, “effective amount” means the amount that provides treatment, prevention, or other desired benefits.

"Encoding" refers to the inherent property of a specific sequence of nucleotides in a polynucleotide (e.g., gene, cDNA, or mRNA) to serve as a template for the synthesis of other polymers and macromolecules in biological processes. These polymers and macromolecules have defined nucleotide sequences (i.e., guide RNA, rRNA, tRNA, and mRNA) or defined amino acid sequences, and the resulting biological properties. Therefore, if the transcription and translation of mRNA corresponding to a gene produces a protein in a cell or other biological system, then the gene encodes that protein. Both the coding strand (whose nucleotide sequence is identical to the mRNA sequence and is typically provided in a sequence listing) and the non-coding strand (which serves as a template for gene or cDNA transcription) can be referred to as encoding the protein or other product of that gene or cDNA.

As used herein, and as applied to nucleic acids, the term "fragment" refers to a subsequence of a larger nucleic acid. The length of a "fragment" of nucleic acid can be at least about 15 nucleotides; for example, at least about 50 nucleotides to about 100 nucleotides, at least about 100 to about 500 nucleotides, at least about 500 to about 1000 nucleotides, at least about 1000 nucleotides to about 1500 nucleotides, about 1500 nucleotides to about 2500 nucleotides, or about 2500 nucleotides (and any integer values between them). As used herein, and as applied to proteins, polypeptides, or peptides, the term "fragment" refers to a subsequence of a larger protein, polypeptide, or peptide. The length of a "fragment" of a protein, polypeptide, or peptide can be at least about 5 amino acids; for example, at least about 10 amino acids, at least about 20 amino acids, at least about 50 amino acids, at least about 100 amino acids, at least about 200 amino acids, or at least about 300 amino acids (and any integer values between them).

The term "gene" refers to a nucleic acid (e.g., DNA) sequence containing the coding sequence necessary to produce a polypeptide, precursor, or RNA (e.g., mRNA). A polypeptide may be encoded by a full-length coding sequence or by any portion of the coding sequence, provided that the desired activity or functional properties (e.g., enzyme activity, receptor binding, signal transduction, immunogenicity, etc.) are preserved in the full length or fragment. The term also encompasses the coding region of a structural gene and the following sequences located adjacent to the coding region at the 5' and 3' ends, at a distance of approximately 2 kb or longer from either end, such that the gene corresponds to the length of the full-length mRNA and the length of the 5' regulatory sequence affecting the transcriptional properties of the gene. A sequence located at the 5' end of the coding region and present on the mRNA is called a 5'-untranslated sequence. 5'-untranslated sequences typically contain regulatory sequences. A sequence located at the 3' end or downstream of the coding region and present on the mRNA is called a 3'-untranslated sequence. The term "gene" encompasses both cDNA and the genomic form of a gene. The genomic form or clone of a gene contains coding regions interrupted by non-coding sequences called "introns," "insertion regions," or "insertion sequences." Introns are gene segments transcribed into nuclear RNA (hnRNA); introns can contain regulatory elements such as enhancers. Introns are removed or "cut out" from nuclear transcripts or primary transcripts; therefore, introns are absent from messenger RNA (mRNA) transcripts. mRNA functions during translation to specify the sequence or order of amino acids in the nascent polypeptide.

As used herein in the context of two or more nucleic acid or polypeptide sequences, the terms “homologous,” “identical,” or “identical” mean that the sequences have a specified percentage of identical residues in a specified region. The percentage can be calculated by: optimally aligning the two sequences; comparing the two sequences in a specified region; determining the number of positions of identical residues in the two sequences to obtain the number of matching positions; dividing the number of matching positions by the total number of positions in the specified region; and multiplying the result by 100 to obtain the percentage of sequence identity. In cases where the two sequences have different lengths or the alignment produces one or more staggered ends and the specified region being compared contains only a single sequence, the residues of the single sequence are included in the denominator, not the numerator, of the calculation. When comparing DNA and RNA, thymine (T) and uracil (U) can be considered equivalent. Identity can be determined manually or using computer sequencing algorithms such as BLAST or BLAST 2.0.

As used herein, the term "instructional material" includes publications, recordings, charts, or any other medium of expression that can be used to communicate the usefulness of the nucleic acids, peptides, polypeptides, and/or compounds of the present invention in the kit for the identification, mitigation, or treatment of the various diseases or conditions described herein. Optionally or alternatively, the instruction material may describe one or more methods for identifying or mitigating a disease or condition in the cells or tissues of a target. The instruction material of the kit may, for example, be attached to or shipped with the container containing the nucleic acids, polypeptides, and/or compounds of the present invention. Alternatively, the instruction material may be shipped separately from the container, with the intention of enabling the recipient to use the instruction material and the compound in a cooperative manner.

"Separated" means altered or removed from its natural state. For example, nucleic acids or peptides that are naturally present in living organisms are not "separated," but the same nucleic acids or peptides that are partially or completely separated from their native coexisting material are "separated." Separated nucleic acids or proteins can exist in a substantially purified form or in non-natural environments such as host cells.

As used herein, the terms "purified" and "purified" in the context of proteins refer to a level of purity that allows for the effective use of a protein, for example, in vitro, ex vivo, or in vivo. For a protein to be used in a given application, it should be substantially free of contaminants, other proteins, and/or chemicals that could interfere with the use of said protein in such application, or at least not intended to contain any chemicals included with the protein of interest. Such applications include the preparation of therapeutic compositions, the administration of proteins in therapeutic compositions, and other methods disclosed herein. Preferably, as mentioned herein, the “purified” protein is a protein that can be produced by any method (i.e., by direct purification from a natural source, in a recombinant manner, or in a synthetic manner), and it has been purified from other protein components such that the protein constitutes at least about 75% by weight of the total protein in the given composition, 80% by weight of the total protein in the given composition, and more preferably, at least about 85% by weight of the total protein in the given composition, and more preferably at least about 90% by weight of the total protein, and more preferably at least about 91% by weight of the total protein, and more preferably at least about 92% by weight of the total protein, and more preferably at least about 93% by weight of the total protein, and more preferably at least about 94% by weight of the total protein, and more preferably at least about 95% by weight of the total protein, and more preferably at least about 96% by weight of the total protein, and more preferably at least about 97% by weight of the total protein, and more preferably at least about 98% by weight of the total protein, and more preferably at least about 99% by weight of the total protein. For example, a purified polypeptide is one that has been separated from other components that might be associated with it in its natural state (e.g., when the protein is a naturally occurring protein) and from components that might be associated with it in the intracellular or extracellular environment. For example, in some cases, proteins can be purified from cell lysates (e.g., from bacterial cell lysates expressing exogenous proteins). As another example, proteins can be purified from extracellular culture media (e.g., from a medium in which cells (e.g., yeast cells) have already secreted proteins).

"Separated nucleic acid" refers to a nucleic acid chain or fragment that has been separated from the sequences flanking it in its naturally occurring state, for example, a DNA fragment that has been removed from its naturally adjacent sequences (e.g., sequences adjacent to the fragment in the genome where the fragment naturally exists). The term also applies to nucleic acids that have been purified substantially from other components that naturally accompany them (e.g., RNA, DNA, or proteins that naturally accompany them in cells). Therefore, the term includes, for example, recombinant DNA incorporated into vectors, autonomously replicating plasmids, or viruses, or as genomic DNA of prokaryotes or eukaryotes, or as a separate molecule independent of other sequences (e.g., as cDNA or as a fragment of genomic or cDNA produced by PCR or restriction enzyme digestion). It also includes recombinant DNA as part of a heterozygous gene encoding an additional polypeptide sequence.

The term "label" as used herein refers to a detectable compound or composition that is directly or indirectly conjugated to a probe to generate a "labeled" probe. The label itself may be detectable (e.g., radioisotope labeling or fluorescent labeling), or, in the case of enzyme labeling, may catalyze a chemical change in a detectable substrate compound or composition (e.g., avidin-biotin). In some cases, primers may be labeled to detect PCR products.

As used herein, the term “regulation” means a detectable increase or decrease in the activity and/or level of mRNA, peptide, or response in a subject compared to the activity and/or level of mRNA, peptide, or response in a subject without treatment or compound and/or compared to the activity and/or level of mRNA, peptide, or response in a otherwise identical but untreated subject. The term encompasses activating, inhibiting, and/or otherwise influencing natural signals or responses to mediate beneficial therapeutic, preventative, or other desired responses in a subject (e.g., a human). As used herein, “mutation,” “mutant,” or “variant” refers to a change in a nucleic acid or amino acid sequence relative to a reference sequence (which may be a naturally occurring normal/“wild-type” sequence) and includes translocations, deletions, insertions, and substitutions/point mutations. As used herein, “mutant” or “variant” refers to a nucleic acid or protein containing a mutation.

“Nucleic acid” refers to polynucleotides, including polynucleotides and polydeoxynucleotides. Nucleic acids according to the invention may comprise any polymer or oligomer of pyrimidine and purine bases, preferably cytosine, thymine, and uracil, and adenine and guanine, respectively. (See Albert L. Lehninger, Principles of Biochemistry, 793-800 (Worth Pub. 1982), which is incorporated herein in its entirety for all purposes). In fact, the invention contemplates any deoxyribonucleotide, ribonucleotide, or peptide nucleic acid component and any chemical variant thereof, such as methylated, hydroxymethylated, or glycosylated forms of these bases, etc. Polymers or oligomers may be heterogeneous or homogeneous in composition and may be isolated from naturally occurring sources or may be produced artificially or synthetically. Furthermore, nucleic acids may be DNA or RNA or mixtures thereof and may exist permanently or transitionally in single-stranded or double-stranded form, including homoduplex, heteroduplex, and hybrid states.

"Oligonucleotide" or "polynucleotide" is a nucleic acid of at least two, preferably at least eight, 15, or 25 nucleotides in length, but can be as long as 50, 100, 1000, or 5000 nucleotides, or a compound that specifically hybridizes with a polynucleotide. Polynucleotides include sequences of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) or their analogs, which can be isolated from natural sources, recombinantly produced, or artificially synthesized. Another example of a polynucleotide of the present invention is a peptide nucleic acid (PNA). (See U.S. Patent No. 6,156,501, which is incorporated herein by reference in its entirety.) The present invention also covers the presence of non-traditional base pairings, such as Hoogsteen base pairings, which have been identified in certain tRNA molecules and are presumed to be present in a triple helix. "Polynucleotide" and "oligonucleotide" are used interchangeably in this disclosure. It should be understood that when a nucleotide sequence is represented herein by a DNA sequence (e.g., A, T, G, and C), this also includes the corresponding RNA sequence (e.g., A, U, G, C), where "U" replaces "T".

The terms “individual,” “object,” “host,” and “patient” are used interchangeably in this document and refer to any mammalian object requiring diagnosis, treatment, or therapy, specifically, a human being.

As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably and refer to compounds containing amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that can constitute a protein or peptide sequence. A polypeptide includes any peptide or protein containing two or more amino acids linked together by peptide bonds. As used herein, the term refers to both short chains and longer chains, short chains being commonly referred to in the art, for example, as peptides, oligopeptides, and oligomers, and longer chains being commonly referred to in the art, as proteins, of which there are many types. “Polypeptide” includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, and so on. Polypeptides include natural peptides, recombinant peptides, synthetic peptides, mutant peptides, variant peptides, or combinations thereof.

As used herein, “polynucleotide” includes cDNA, RNA, DNA/RNA hybrids, antisense RNA, ribozymes, genomic DNA, synthetic forms and hybrid polymers, sense strands and antisense strands, and may be chemically or biochemically modified to express non-natural or derived, synthetic or semi-synthetic nucleotide bases. Furthermore, alterations to wild-type or synthetic genes are envisioned, including but not limited to the deletion, insertion, substitution, or fusion of one or more nucleotides with other polynucleotide sequences.

As used herein, “sample” or “biological sample” means biological material isolated from an object. A biological sample may contain any biological material suitable for detecting mRNA, peptides, or other markers of physiological or pathological processes in the object, and may include fluid, tissue, cellular, and/or non-cellular material obtained from an individual.

As used herein, the term "therapy" or "treatment regimen" refers to activities undertaken to prevent, treat, or modify a disease or condition, such as treatment procedures aimed at reducing or eliminating at least one sign or symptom of the disease or condition using pharmacological, surgical, dietary, and/or other techniques. A treatment regimen may include a prescribed dose of one or more compounds or surgical procedures. Therapies are generally beneficial and reduce or eliminate at least one sign or symptom of the condition or disease state; however, in some cases, the effects of a therapy may have undesirable effects or side effects. The effectiveness of a therapy is also affected by the subject's physiological condition, such as age, sex, genetics, weight, and other disease conditions.

The term "therapeuticly effective amount" refers to the amount of a subject compound or composition that will elicit the expected biological, physiological, clinical, or medical response in the cells, tissues, organs, systems, or objects being sought by researchers, veterinarians, physicians, or other clinicians. The term "therapeuticly effective amount" includes, when administered, an amount sufficient to treat one or more of the signs or symptoms of the condition or disease being treated. Therapeuticly effective amounts will vary depending on the compound or composition, the disease and its severity, and the age, weight, etc., of the subject being treated.

As used herein, the term “treatment” for a disease or condition means reducing the frequency or severity of at least one sign or symptom of a disease or condition experienced by a subject. The terms “treatment,” “treating,” “treat,” etc., are used herein to generally refer to achieving a desired pharmacological and/or physiological effect. An effect may be preventative in relation to the complete or partial prevention of a disease or its symptoms, and/or therapeutic in relation to the partial or complete stabilization or cure of a disease and/or side effects attributable to it. The term “treatment” covers any treatment of a disease in mammals (specifically, humans) and includes: (a) preventing the occurrence of a disease or symptom in a subject who may be susceptible to it but has not yet been diagnosed with it; (b) suppressing the disease and/or symptoms, for example, slowing or halting their development (e.g., stopping tumor growth, slowing the rate of tumor growth, stopping the rate of cancer cell proliferation, etc.); or (c) alleviating disease symptoms, i.e., causing the remission of the disease and/or symptoms (e.g., causing a reduction in tumor size, a reduction in the number of present cancer cells, etc.). Those who need treatment include those who already have the disease (e.g., those with cancer, those with infections, those with metabolic disorders, those with macular degeneration, etc.) and those who need prevention (e.g., those with increased susceptibility to cancer, those with increased likelihood of infection, those suspected of having cancer, those suspected of harboring infections, those with increased susceptibility to metabolic diseases, those with increased susceptibility to macular degeneration, etc.).

As used herein, the term "wild-type" refers to a gene or gene product isolated from a naturally occurring source or having a naturally occurring sequence (e.g., a wild-type protein having a naturally occurring amino acid sequence may be isolated from a natural or synthetic source but is still considered a wild-type protein). In contrast, the terms "modified," "variant," or "mutant" refer to a gene or gene product that has modifications (i.e., altered characteristics) in sequence and/or functional properties compared to a wild-type gene or gene product.

Scope: Throughout this disclosure, multiple aspects may be presented in the form of scope. It should be understood that the description in scope form is for convenience and brevity only and should not be construed as immutably limiting the scope of the invention. Therefore, the description of scope should be considered to have all possible sub-scopes precisely disclosed, as well as individual numerical values within these scopes. For example, a description of a scope such as 1 to 6 should be considered to have precisely disclosed sub-scopes such as 1 to 2, 1 to 3, 1 to 4, 1 to 5, 2 to 3, 2 to 4, 2 to 5, 2 to 6, etc., as well as individual numbers within said scopes, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the scope.

Compositions and methods

As summarized above, in some embodiments, the compositions and methods of this disclosure comprise an IL-18 polypeptide having mutations at amino acid positions C38 and C68 relative to wild-type IL-18 (SEQ ID NO:30). Such variants are referred to herein as “stabilized” IL-18 variants.

In some embodiments, the compositions and methods of this disclosure comprise an IL-18 variant peptide that is mutated relative to wild-type IL-18, such that it binds to one of the natural binding partners of IL-18 but not to the other (or binds with reduced binding to the other). For example, wild-type IL-18 binds to IL-18R (which signals via a receptor) and IL-18BP (which inhibits IL-18 by preventing binding to IL-18R). In some cases, the IL-18 variant peptides discussed herein are “decoy-resistant IL-18” (“DR IL-18”) variants; such variants bind to IL-18R but bind with reduced binding to IL-18BP (and in some cases, not at all). In other cases, the IL-18 variant peptides discussed herein are “decoy-decoy IL-18” (“D2D IL-18”) variants; such variants bind to IL-18BP but bind with reduced binding to IL-18R (and in some cases, not at all). As shown in this disclosure, the IL-18 variant peptide can exhibit a binding affinity for IL-18Rα comparable to that of wild-type IL-18, while also exhibiting a reduced binding affinity for IL-18BP compared to wild-type IL-18.

In some cases, there is overlap between the terms used to describe various variants. For example, a DR IL-18 variant or a D2D IL-18 variant may include mutations at C38 and C68, and in such cases, the IL-18 variant would be a "stabilized" variant. Therefore, the term "stabilized" encompasses both stabilized wild-type IL-18 (WT IL-18 with C38 and C68 mutations) and stabilized variant proteins, such as DR IL-18 and D2D IL-18 variants with C38 and C68 mutations. Similarly, the term "DRIL-18 variant" encompasses both variants with and without C38/C68 mutations, and the term "D2D IL-18 variant" encompasses both variants with and without C38/C68 mutations. Therefore, the term "stabilized DR IL-18" variant can be used when discussing DR IL-18 variants with C38/C68 mutations, and the term "stabilized D2D IL-18" variant can be used when discussing D2D IL-18 variants with C38/C68 mutations.

Therefore, various implementation schemes of the "stabilized" IL-18 protein (various C38/C68 mutants) will be discussed below. This description applies equally to any type of IL-18 protein (e.g., wild-type, DR IL-18 variants, D2D IL-18 variants, etc.). C38/C68 mutations will be discussed first, followed by DR IL-18 variants and D2D IL-18 variants. All the variants discussed can be used in any of the methods described herein.

The stabilized IL-18 peptide is an IL-18 peptide containing a ‘stabilizing mutation’, which is a mutation of two cysteine residues (C38 and C68) relative to human wild-type IL-18 (SEQ ID NO:30). Surprisingly, it was found that mutations of these two amino acids stabilized IL-18 better than all other possible combinations of cysteine mutations, see, for example, the working examples below. In some cases, the mutation is a C to S substitution, and therefore in some cases, the stabilized IL-18 peptide may contain mutations C38S and C68S relative to human wild-type IL-18 (SEQ ID NO:30) (also referred to herein as the “mutation pair C38S/C68S”). In some cases, the subject IL-18 polypeptide comprises an amino acid sequence having 80% or more sequence identity with human wild-type IL-18 (SEQ ID NO:30) (e.g., 85% or more, 90% or more, 92% or more, 95% or more, 97% or more, 98% or more, or 98.5% or more sequence identity), and includes mutations at positions C38 and C68 relative to SEQ ID NO:30. In some cases, the subject IL-18 polypeptide comprises an amino acid sequence having 90% or more sequence identity with human wild-type IL-18 (SEQ ID NO:30) (e.g., 92% or more, 95% or more, 97% or more, 98% or more, or 98.5% or more sequence identity), and includes mutations at positions C38 and C68 relative to SEQ ID NO:30. Examples of such variant proteins include those shown in SEQ ID NO:6 and 16-21, but these sequences also contain additional mutations that make them DR IL-18 variants (see below), and such SEQ ID NO:6 and 16-21 are examples of stable DR IL-18 variants.

In some cases, the mutation is a C to S substitution, and therefore in some cases, the stabilized IL-18 peptide may contain mutations C38S and C68S (also referred to herein as “C38S/C68S”) relative to human wild-type IL-18 (SEQ ID NO: 30). In some cases, the mutation at C68 results in an immunogenic epitope. Therefore, in some embodiments, the mutation at C68 is a non-immunogenic substitution. In some such cases, the mutation is a C68 to G, A, V, D, E, or N mutation (in some cases, C68 to G, A, D, or N). Therefore, in some such cases, the subject-stabilized IL-18 peptide contains mutations C38S/C68S, C38S/C68G, C38S/C68A, C38S/C68V, C38S/C68D, C38S/C68E, or C38S/C68N. In some cases, the subject-stabilized IL-18 peptide contains mutations in C38S/C68G, C38S/C68A, C38S/C68V, C38S/C68D, C38S/C68E, or C38S/C68N. In some cases, the subject-stabilized IL-18 peptide contains mutations in C38S/C68S, C38S/C68G, C38S/C68A, C38S/C68D, or C38S/C68N. In some cases, the subject-stabilized IL-18 peptide contains a mutation in C38S/C68D. In some cases, the subject-stabilized IL-18 peptide contains a mutation in C38S/C68G. In some cases, the subject-stabilized IL-18 peptide contains the mutant C38S/C68A. In some cases, the subject-stabilized IL-18 peptide contains the mutant C38S/C68A. In some cases, the subject-stabilized IL-18 peptide contains the mutant C38S/C68N.

Therefore, in some embodiments, the subject IL-18 polypeptide comprises an amino acid sequence having 80% or more sequence identity with human wild-type IL-18 (SEQ ID NO:30) (e.g., 85% or more, 90% or more, 92% or more, 95% or more, 97% or more, 98% or more, or 98.5% or more sequence identity), and comprises mutant C38S/C68S, C38S/C68G, C38S/C68A, C38S/C68V, C38S/C68D, C38S/C68E, or C38S/C68N relative to SEQ ID NO:30. In some embodiments, the subject IL-18 polypeptide comprises an amino acid sequence having 80% or more sequence identity with human wild-type IL-18 (SEQ ID NO:30) (e.g., 85% or more, 90% or more, 92% or more, 95% or more, 97% or more, 98% or more, or 98.5% or more sequence identity), and comprises mutant C38S/C68G, C38S/C68A, C38S/C68V, C38S/C68D, C38S/C68E, or C38S/C68N relative to SEQ ID NO:30. In some embodiments, the subject IL-18 polypeptide comprises an amino acid sequence having 80% or more sequence identity with human wild-type IL-18 (SEQ ID NO:30) (e.g., 85% or more, 90% or more, 92% or more, 95% or more, 97% or more, 98% or more, or 98.5% or more sequence identity), and comprises mutant C38S/C68S, C38S/C68G, C38S/C68A, C38S/C68D, or C38S/C68N relative to SEQ ID NO:30. In some embodiments, the subject IL-18 polypeptide comprises an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 92% or more, 95% or more, 97% or more, 98% or more, or 98.5% or more sequence identity) with human wild-type IL-18 (SEQ ID NO:30), and comprises mutant C38S/C68G, C38S/C68A, C38S/C68D, or C38S/C68N relative to SEQ ID NO:30. In some embodiments, the subject IL-18 polypeptide comprises an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 92% or more, 95% or more, 97% or more, 98% or more, or 98.5% or more sequence identity) with human wild-type IL-18 (SEQ ID NO:30), and comprises mutant C38S/C68D relative to SEQ ID NO:30. In some embodiments, the subject IL-18 polypeptide comprises an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 92% or more, 95% or more, 97% or more, 98% or more, or 98.5% or more sequence identity) with human wild-type IL-18 (SEQ ID NO:30), and comprises the mutant C38S/C68G relative to SEQ ID NO:30. In some embodiments, the subject IL-18 polypeptide comprises an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 92% or more, 95% or more, 97% or more, 98% or more, or 98.5% or more sequence identity) with human wild-type IL-18 (SEQ ID NO:30), and comprises the mutant C38S/C68A relative to SEQ ID NO:30. In some embodiments, the subject IL-18 polypeptide comprises an amino acid sequence having 80% or more sequence identity with human wild-type IL-18 (SEQ ID NO:30) (e.g., 85% or more, 90% or more, 92% or more, 95% or more, 97% or more, 98% or more, or 98.5% or more sequence identity), and comprises the mutant C38S/C68N relative to SEQ ID NO:30.

In some embodiments, the subject IL-18 polypeptide comprises an amino acid sequence having 90% or more sequence identity with human wild-type IL-18 (SEQ ID NO:30) (e.g., 92% or more, 95% or more, 97% or more, 98% or more, or 98.5% or more sequence identity), and comprises mutant C38S/C68S, C38S/C68G, C38S/C68A, C38S/C68V, C38S/C68D, C38S/C68E, or C38S/C68N relative to SEQ ID NO:30. In some embodiments, the subject IL-18 polypeptide comprises an amino acid sequence having 90% or more sequence identity with human wild-type IL-18 (SEQ ID NO:30) (e.g., 92% or more, 95% or more, 97% or more, 98% or more, or 98.5% or more sequence identity), and comprises mutant C38S/C68G, C38S/C68A, C38S/C68V, C38S/C68D, C38S/C68E, or C38S/C68N relative to SEQ ID NO:30. In some embodiments, the subject IL-18 polypeptide comprises an amino acid sequence having 90% or more sequence identity with human wild-type IL-18 (SEQ ID NO:30) (e.g., 92% or more, 95% or more, 97% or more, 98% or more, or 98.5% or more sequence identity), and comprises mutant C38S/C68S, C38S/C68G, C38S/C68A, C38S/C68D, or C38S/C68N relative to SEQ ID NO:30. In some embodiments, the subject IL-18 polypeptide comprises an amino acid sequence having 90% or more sequence identity (e.g., 92% or more, 95% or more, 97% or more, 98% or more, or 98.5% or more sequence identity) with human wild-type IL-18 (SEQ ID NO:30), and comprises mutant C38S/C68G, C38S/C68A, C38S/C68D, or C38S/C68N relative to SEQ ID NO:30. In some embodiments, the subject IL-18 polypeptide comprises an amino acid sequence having 90% or more sequence identity (e.g., 92% or more, 95% or more, 97% or more, 98% or more, or 98.5% or more sequence identity) with human wild-type IL-18 (SEQ ID NO:30), and comprises mutant C38S/C68D relative to SEQ ID NO:30. In some embodiments, the subject IL-18 polypeptide comprises an amino acid sequence having 90% or more sequence identity (e.g., 92% or more, 95% or more, 97% or more, 98% or more, or 98.5% or more sequence identity) with human wild-type IL-18 (SEQ ID NO:30), and comprises the mutant C38S/C68G relative to SEQ ID NO:30. In some embodiments, the subject IL-18 polypeptide comprises an amino acid sequence having 90% or more sequence identity (e.g., 92% or more, 95% or more, 97% or more, 98% or more, or 98.5% or more sequence identity) with human wild-type IL-18 (SEQ ID NO:30), and comprises the mutant C38S/C68A relative to SEQ ID NO:30. In some embodiments, the subject IL-18 polypeptide comprises an amino acid sequence having 90% or more sequence identity with human wild-type IL-18 (SEQ ID NO:30) (e.g., 92% or more, 95% or more, 97% or more, 98% or more, or 98.5% or more sequence identity), and comprises the mutant C38S/C68N relative to SEQ ID NO:30.

Stabilized DR IL-18 or D2D IL-18 variant

In some cases, the subject-stabilized IL-18 peptide is a stabilized IL-18 variant peptide, i.e., an IL-18 variant peptide containing a stabilizing mutation (i.e., a mutation at positions C38 and C68 relative to human wild-type IL-18). In some such cases, the stabilized IL-18 variant peptide is a stabilized "decoy-resistant" (DR) IL-18 variant, and in other such cases, the stabilized IL-18 variant peptide is a stabilized "decoy-to-decoy" (D2D) IL-18 variant. Examples of human DR IL-18 variants include, but are not limited to, those shown in SEQ ID NO:34-59, 73-91, and 191-193, while examples of human D2D IL-18 variants include, but are not limited to, those shown in SEQ ID NO:92-125; see also the working examples below. DR-IL18 variants are described in detail below, and any of such variants may contain the C38/C68 mutation as described herein to become stabilized DR-IL18.

In some cases, the subject-stabilized DR IL-18 variants contain the amino acid sequence shown in SEQ ID NO:89, except that C38 and C68 are mutated (e.g., C38S/C68S, C38S/C68G, C38S/C68A, C38S/C68V, C38S/C68D, C38S/C68E, or C38S/C68N; in some cases, C38S/C68G, C38S/C68A, C38S/C68V, C38S/C68D, C38S/C68N). 68E or C38S/C68N; in some cases, C38S/C68S, C38S/C68G, C38S/C68A, C38S/C68D or C38S/C68N; in some cases, C38S/C68G, C38S/C68A, C38S/C68D or C38S/C68N; in some cases, C38S/C68D; in some cases, C38S/C68G; in some cases, C38S/C68A; in some cases, C38S/C68N). Examples of stable DR IL-18 variants include those shown in SEQ ID NO:6 and 16-21 (these are C38/C68 mutant forms of the DR-18 variant shown in SEQ ID NO:89). Those skilled in the art will readily recognize that for any desired IL-18 variant (see, for example, the working examples below) (e.g., human DR-IL variants as shown in SEQ ID NO:34-59, 73-91 and 191-193 (e.g., SEQ ID NO 87-91), or any of the human D2D IL-18 variants as shown in SEQ ID NO:92-125), equivalent stable variants can be readily produced.

Therefore, in some embodiments, the subject IL-18 polypeptide comprises an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 92% or more, 95% or more, 97% or more, 98% or more, or 98.5% or more sequence identity) with human wild-type IL-18 (SEQ ID NO:30) (or any one of SEQ ID NO:6 and 16-21, or any one of SEQ ID NO:34-59, 73-91 and 191-193, or any one of SEQ ID NO:87-91, or SEQ ID NO:89), and comprises mutant C38S/C68S, C38S/C68G, C38S/C68A, C38S/C68V, C38S/C68D, C38S/C68E or C38S/C68N relative to SEQ ID NO:30. In some embodiments, the subject IL-18 polypeptide comprises an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 92% or more, 95% or more, 97% or more, 98% or more, or 98.5% or more sequence identity) with human wild-type IL-18 (SEQ ID NO:30) (or any one of SEQ ID NO:6 and 16-21, or any one of SEQ ID NO:34-59, 73-91 and 191-193, or any one of SEQ ID NO:87-91, or SEQ ID NO:89), and comprises mutant C38S/C68G, C38S/C68A, C38S/C68V, C38S/C68D, C38S/C68E or C38S/C68N relative to SEQ ID NO:30. In some embodiments, the subject IL-18 polypeptide comprises an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 92% or more, 95% or more, 97% or more, 98% or more, or 98.5% or more sequence identity) with human wild-type IL-18 (SEQ ID NO:30) (or any one of SEQ ID NO:6 and 16-21, or any one of SEQ ID NO:34-59, 73-91 and 191-193, or any one of SEQ ID NO:87-91, or SEQ ID NO:89), and comprises mutant C38S/C68S, C38S/C68G, C38S/C68A, C38S/C68D or C38S/C68N relative to SEQ ID NO:30. In some embodiments, the subject IL-18 polypeptide comprises an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 92% or more, 95% or more, 97% or more, 98% or more, or 98.5% or more sequence identity) with human wild-type IL-18 (SEQ ID NO:30) (or any one of SEQ ID NO:6 and 16-21, or any one of SEQ ID NO:34-59, 73-91 and 191-193, or any one of SEQ ID NO:87-91, or SEQ ID NO:89), and comprises mutant C38S/C68G, C38S/C68A, C38S/C68D or C38S/C68N relative to SEQ ID NO:30. In some embodiments, the subject IL-18 polypeptide comprises an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 92% or more, 95% or more, 97% or more, 98% or more, or 98.5% or more sequence identity) with human wild-type IL-18 (SEQ ID NO:30) (or any one of SEQ ID NO:6 and 16-21, or any one of SEQ ID NO:34-59, 73-91 and 191-193, or any one of SEQ ID NO:87-91, or SEQ ID NO:89), and comprises the mutant C38S/C68D relative to SEQ ID NO:30.

In some embodiments, the subject IL-18 polypeptide comprises an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 92% or more, 95% or more, 97% or more, 98% or more, or 98.5% or more sequence identity) with human wild-type IL-18 (SEQ ID NO:30) (or any one of SEQ ID NO:6 and 16-21, or any one of SEQ ID NO:34-59, 73-91 and 191-193, or any one of SEQ ID NO:87-91, or SEQ ID NO:89), and comprises the mutant C38S/C68G relative to SEQ ID NO:30. In some embodiments, the subject IL-18 polypeptide comprises an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 92% or more, 95% or more, 97% or more, 98% or more, or 98.5% or more sequence identity) with human wild-type IL-18 (SEQ ID NO:30) (or any one of SEQ ID NO:6 and 16-21, or any one of SEQ ID NO:34-59, 73-91 and 191-193, or any one of SEQ ID NO:87-91, or SEQ ID NO:89), and comprises a mutant C38S/C68A relative to SEQ ID NO:30.

In some embodiments, the subject IL-18 polypeptide comprises an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 92% or more, 95% or more, 97% or more, 98% or more, or 98.5% or more sequence identity) with human wild-type IL-18 (SEQ ID NO:30) (or any one of SEQ ID NO:6 and 16-21, or any one of SEQ ID NO:34-59, 73-91 and 191-193, or any one of SEQ ID NO:87-91, or SEQ ID NO:89), and comprises a mutant C38S/C68N relative to SEQ ID NO:30. In some embodiments, the subject IL-18 polypeptide comprises an amino acid sequence having 90% or more sequence identity (e.g., 92% or more, 95% or more, 97% or more, 98% or more, or 98.5% or more sequence identity) with human wild-type IL-18 (SEQ ID NO:30) (or any one of SEQ ID NO:6 and 16-21, or any one of SEQ ID NO:34-59, 73-91 and 191-193, or any one of SEQ ID NO:87-91, or SEQ ID NO:89), and comprises mutant C38S/C68S, C38S/C68G, C38S/C68A, C38S/C68V, C38S/C68D, C38S/C68E or C38S/C68N relative to SEQ ID NO:30. In some embodiments, the subject IL-18 polypeptide comprises an amino acid sequence having 90% or more sequence identity (e.g., 92% or more, 95% or more, 97% or more, 98% or more, or 98.5% or more sequence identity) with human wild-type IL-18 (SEQ ID NO:30) (or any one of SEQ ID NO:6 and 16-21, or any one of SEQ ID NO:34-59, 73-91 and 191-193, or any one of SEQ ID NO:87-91, or SEQ ID NO:89), and comprises mutant C38S/C68G, C38S/C68A, C38S/C68V, C38S/C68D, C38S/C68E or C38S/C68N relative to SEQ ID NO:30.

In some embodiments, the subject IL-18 polypeptide comprises an amino acid sequence having 90% or more sequence identity (e.g., 92% or more, 95% or more, 97% or more, 98% or more, or 98.5% or more sequence identity) with human wild-type IL-18 (SEQ ID NO:30) (or any one of SEQ ID NO:6 and 16-21, or any one of SEQ ID NO:34-59, 73-91 and 191-193, or any one of SEQ ID NO:87-91, or SEQ ID NO:89), and comprises mutant C38S/C68S, C38S/C68G, C38S/C68A, C38S/C68D or C38S/C68N relative to SEQ ID NO:30. In some embodiments, the subject IL-18 polypeptide comprises an amino acid sequence having 90% or more sequence identity (e.g., 92% or more, 95% or more, 97% or more, 98% or more, or 98.5% or more sequence identity) with human wild-type IL-18 (SEQ ID NO:30) (or any one of SEQ ID NO:6 and 16-21, or any one of SEQ ID NO:34-59, 73-91 and 191-193, or any one of SEQ ID NO:87-91, or SEQ ID NO:89), and comprises mutant C38S/C68G, C38S/C68A, C38S/C68D or C38S/C68N relative to SEQ ID NO:30. In some embodiments, the subject IL-18 polypeptide comprises an amino acid sequence having 90% or more sequence identity (e.g., 92% or more, 95% or more, 97% or more, 98% or more, or 98.5% or more sequence identity) with human wild-type IL-18 (SEQ ID NO:30) (or any one of SEQ ID NO:6 and 16-21, or any one of SEQ ID NO:34-59, 73-91 and 191-193, or any one of SEQ ID NO:87-91, or SEQ ID NO:89), and comprises the mutant C38S/C68D relative to SEQ ID NO:30. In some embodiments, the subject IL-18 polypeptide comprises an amino acid sequence having 90% or more sequence identity (e.g., 92% or more, 95% or more, 97% or more, 98% or more, or 98.5% or more sequence identity) with human wild-type IL-18 (SEQ ID NO:30) (or any one of SEQ ID NO:6 and 16-21, or any one of SEQ ID NO:34-59, 73-91 and 191-193, or any one of SEQ ID NO:87-91, or SEQ ID NO:89), and comprises the mutant C38S/C68G relative to SEQ ID NO:30. In some embodiments, the subject IL-18 polypeptide comprises an amino acid sequence having 90% or more sequence identity (e.g., 92% or more, 95% or more, 97% or more, 98% or more, or 98.5% or more sequence identity) with human wild-type IL-18 (SEQ ID NO:30) (or any one of SEQ ID NO:6 and 16-21, or any one of SEQ ID NO:34-59, 73-91 and 191-193, or any one of SEQ ID NO:87-91, or SEQ ID NO:89), and comprises the mutant C38S/C68A relative to SEQ ID NO:30. In some embodiments, the subject IL-18 polypeptide comprises an amino acid sequence having 90% or more sequence identity (e.g., 92% or more, 95% or more, 97% or more, 98% or more, or 98.5% or more sequence identity) with human wild-type IL-18 (SEQ ID NO:30) (or any one of SEQ ID NO:6 and 16-21, or any one of SEQ ID NO:34-59, 73-91 and 191-193, or any one of SEQ ID NO:87-91, or SEQ ID NO:89), and comprises the mutant C38S/C68N relative to SEQ ID NO:30.

In some embodiments, the IL-18 variant peptide is a DR IL-18 variant (i.e., it binds IL-18R and exhibits reduced binding to IL-18BP). In some embodiments, the DR IL-18 variant binds IL-18BP with a binding affinity of 95% or less (e.g., 90% or less, 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, 5% or less, 2% or less, 1% or less, 0.05% or less, or 0.001% or less). In some implementations, the DRIL-18 variant binds to IL-18BP with a binding affinity of 10% or less (e.g., 5% or less, 2% or less, 1% or less, 0.05% or less, or 0.001% or less) of the binding affinity of wild-type IL-18 to IL-18BP.

In some embodiments, the _KD of the subject DR IL-18 variant to IL-18BP is 10 nM or greater (a higher _KD indicates a lower binding affinity). In some embodiments, the _KD of the subject DR-IL-18 variant peptide to IL-18BP is 20 nM or greater (e.g., 50 nM or greater, 100 nM or greater, 500 nM or greater, or 1 μM or greater).

IL-18 peptides (e.g., DR IL-18 peptides containing mutations at positions C38 and C68 relative to human wild-type IL-18) can exhibit enhanced stability compared to IL-18 peptides with different sequences (e.g., wild-type IL-18 sequence, or IL-18 sequence lacking C38 and C68 mutations but otherwise containing the same sequence as the subject IL-18 peptide).

In some embodiments, the stability of the IL-18 peptide disclosed herein (e.g., DR IL-18) can be assessed by subjecting the peptide or a formulation containing the peptide disclosed herein to freeze-thaw cycles, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, or at least 20 freeze-thaw cycles. Freeze-thaw cycles may include freezing the peptide or a formulation containing the peptide (e.g., to about -20°C or -80°C) and thawing the peptide or formulation (e.g., to about 20°C, 23°C, 25°C, or 37°C).

In some embodiments, the stability of the IL-18 peptide disclosed herein (e.g., DR IL-18) can be assessed by subjecting the peptide or a formulation containing the peptide disclosed herein to stirring. Stirring may include, for example, incubating the peptide or formulation at stirring speeds of about 100 rpm, about 150 rpm, about 200 rpm, or about 250 rpm (e.g., at about 20°C, about 23°C, about 25°C, about 30°C, about 37°C, or about 40°C). For example, samples may be tested after incubation at said temperature and stirring rate for about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 1 week, or about 2 weeks.

The stability of IL-18 peptides (e.g., DR IL-18) can be assessed by comparing the size of the major IL-18 peak as measured by size exclusion chromatography before and after the stability assays disclosed herein (e.g., before and after freeze-thaw cycles and/or stirring). In some embodiments, after exposure to the stability test conditions, the major peak remains at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 95.5%, at least about 96%, at least about 96.5%, at least about 97%, at least about 97.5%, at least about 98%, at least about 98.5%, at least about 99%, at least about 99.1%, at least about 99.1%, at least about 99.3%, at least about 99.4%, at least about 99.5%, at least about 99.6%, at least about 99.7%, at least about 99.8%, at least about 99.9%, at least about 99.95%, at least about 99.99%, or about 100% of the size of the major peak before exposure to the test conditions.

In some embodiments, after exposure to stability assay conditions, the retention of the major peak is at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, or at least about 50% greater than that of an IL-18 peptide with a different sequence (e.g., a wild-type IL-18 sequence, or an IL-18 sequence lacking C38 and C68 mutations but otherwise containing the same sequence as the subject IL-18 peptide).

The stability of an IL-18 peptide (e.g., DR IL-18) can be evaluated by determining the presence and magnitude of one or more peaks indicating the formation of dimers, polymers, aggregates, or degradation products, as measured by size exclusion chromatography, before and after the stability assays disclosed herein (e.g., before and after freeze-thaw cycles and/or stirring). In some embodiments, no or substantially no peaks indicating dimer formation are detected after exposing the IL-18 peptide (e.g., DR IL-18 in the formulations disclosed herein) to the stability assay conditions. In some embodiments, no or substantially no peaks indicating polymer formation are detected after exposing the IL-18 peptide (e.g., DR IL-18 in the formulations disclosed herein) to the stability assay conditions. In some embodiments, no or substantially no peaks indicating aggregate formation are detected after exposing the IL-18 peptide (e.g., DR IL-18 in the formulations disclosed herein) to the stability assay conditions. In some embodiments, after exposing the IL-18 peptide (e.g., DR IL-18 in the formulation disclosed herein) to stability assay conditions, no or substantially no peaks indicating the formation of degradation products were detected.

In some embodiments, after exposing an IL-18 peptide (e.g., DR IL-18 in the formulation disclosed herein) to stability assay conditions, the IL-18 peptide exhibits at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, less than 1% compared to an IL-18 peptide with a different sequence (e.g., a wild-type IL-18 sequence, or an IL-18 sequence lacking C38 and C68 mutations but otherwise containing the same sequence as the subject IL-18 peptide). At least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of dimers, polymers, aggregates, and/or degradation products are formed, for example, as determined by size exclusion chromatography.

In some embodiments, the disclosed DR IL-18 variants have a limit of detection ( _KD) of at least about 100 μM, at least about 10 μM, at least about 1 μM, at least about 900 nM, at least about 800 nM, at least about 700 nM, at least about 600 nM, at least about 500 nM, at least about 400 nM, at least about 300 nM, at least about 200 nM, at least about 150 nM, at least about 100 nM, at least about 90 nM, at least about 80 nM, at least about 70 nM, at least about 60 nM, at least about 50 nM, at least about 40 nM, at least about 30 nM, at least about 20 nM, or at least about 10 nM. In some embodiments, the disclosed DR IL-18 variants do not bind IL-18BP, substantially do not bind IL-18BP, or bind IL-18BP in a manner below the detection limit in the assays disclosed herein.

The DR IL-18 variants disclosed herein can exhibit binding to human IL-18R, for example, binding to human IL-18R with an affinity comparable to, or with a higher affinity than, that of wild-type human IL-18. In some embodiments, the DR IL-18 variants disclosed herein bind to human IL-18R with an affinity at least comparable to that exhibited by wild-type human IL-18 to human IL-18R.

In some implementations, the DR IL-18 variants disclosed herein bind to human IL-18Rα or human IL-18R, with _KD values of, for example, less than about 100 μM, less than about 10 μM, less than about 1 μM, less than about 900 nM, less than about 800 nM, less than about 700 nM, less than about 600 nM, less than about 500 nM, less than about 400 nM, less than about 300 nM, less than about 200 nM, less than about 150 nM, less than about 100 nM, less than about 90 nM, less than about 80 nM, less than about 70 nM, less than about 60 nM, less than about 50 nM, less than about 40 nM, less than about 30 nM, less than about 20 nM, less than about 10 nM, less than about 5 nM, less than about 4 nM, less than about 3 nM, less than about 2 nM, or less than about 1 nM.

In some embodiments, the DR IL-18 variants disclosed herein bind to human IL-18Rα or human IL-18R, with _KD values of, for example, about 1 pM to about 1 μM, about 1 pM to about 500 nM, about 1 pM to about 400 nM, about 1 pM to about 300 nM, about 1 pM to about 200 nM, about 1 pM to about 100 nM, about 1 pM to about 50 nM, about 100 pM to about 1 μM, about 100 pM to about 500 nM, about 100 pM to about 400 nM, about 100 pM to about 300 nM, and about 100 pM to about 200 nM. Approximately 100 pM to approximately 100 nM, approximately 100 pM to approximately 50 nM, approximately 1 nM to approximately 1 μM, approximately 1 nM to approximately 500 nM, approximately 1 nM to approximately 400 nM, approximately 1 nM to approximately 300 nM, approximately 1 nM to approximately 200 nM, approximately 1 nM to approximately 100 nM, approximately 1 nM to approximately 75 nM, approximately 1 nM to approximately 50 nM, approximately 1 nM to approximately 40 nM, approximately 1 nM to approximately 30 nM, or approximately 1 nM to approximately 10 nM.

In some embodiments, the IL-18BP/IL-18R dissociation constant ratio of the DR IL-18 variant is at least twice that of the wild-type IL-18 (note that an increased dissociation constant ratio implies a relative decrease in IL-18BP binding compared to IL-18R binding). In some embodiments, the IL-18BP/IL-18R dissociation constant ratio of the DR IL-18 variant is at least 20 times that of the wild-type IL-18. In some embodiments, the IL-18BP/IL-18R dissociation constant ratio of the DR IL-18 variant is at least 200 times that of the wild-type IL-18. In some embodiments, the IL-18BP/IL-18R dissociation constant ratio of the DR IL-18 variant is at least about 2,000 times that of the wild-type IL-18. In some embodiments, the IL-18BP/IL-18R dissociation constant ratio of the DR IL-18 variant is at least about 20,000 times that of the wild-type IL-18. In some embodiments, the IL-18BP/IL-18R dissociation constant ratio of the DR IL-18 variant is at least about 200,000 times that of the wild-type IL-18. In some embodiments, the IL-18BP/IL-18R dissociation constant ratio of the DR IL-18 variant is at least about 2,000,000 times that of the wild-type IL-18. In some embodiments, the IL-18BP/IL-18R dissociation constant ratio of the DR IL-18 variant is at least about 20,000,000 times that of the wild-type IL-18.

In some embodiments, the IL-18BP/IL-18R dissociation constant ratio of the DR IL-18 variant is at least about 3 times that of the wild-type IL-18. In some embodiments, the IL-18BP/IL-18R dissociation constant ratio of the DR IL-18 variant is at least about 30 times that of the wild-type IL-18. In some embodiments, the IL-18BP/IL-18R dissociation constant ratio of the DR IL-18 variant is at least about 300 times that of the wild-type IL-18. In some embodiments, the IL-18BP/IL-18R dissociation constant ratio of the DR IL-18 variant is at least about 3,000 times that of the wild-type IL-18. In some embodiments, the IL-18BP/IL-18R dissociation constant ratio of the DR IL-18 variant is at least about 30,000 times that of the wild-type IL-18. In some embodiments, the IL-18BP/IL-18R dissociation constant ratio of the DR IL-18 variant is at least about 300,000 times that of the wild-type IL-18. In some embodiments, the IL-18BP/IL-18R dissociation constant ratio of the DR IL-18 variant is at least about 3,000,000 times that of the wild-type IL-18. In some implementations, the IL-18BP/IL-18R dissociation constant ratio of the DR IL-18 variant is at least about 30,000,000 times that of the wild-type IL-18.

In some embodiments, the IL-18BP/IL-18R dissociation constant ratio of the DR IL-18 variant is at least about 10 times that of the wild-type IL-18. In some embodiments, the IL-18BP/IL-18R dissociation constant ratio of the DR IL-18 variant is at least about 100 times that of the wild-type IL-18. In some embodiments, the IL-18BP/IL-18R dissociation constant ratio of the DR IL-18 variant is at least about 1000 times that of the wild-type IL-18. In some embodiments, the IL-18BP/IL-18R dissociation constant ratio of the DR IL-18 variant is at least about 10,000 times that of the wild-type IL-18. In some embodiments, the IL-18BP/IL-18R dissociation constant ratio of the DR IL-18 variant is at least about 100,000 times that of the wild-type IL-18. In some embodiments, the IL-18BP/IL-18R dissociation constant ratio of the DR IL-18 variant is at least about 1,000,000 times that of the wild-type IL-18. In some embodiments, the IL-18BP/IL-18R dissociation constant ratio of the DR IL-18 variant is at least about 10,000,000 times that of the wild-type IL-18. In some implementations, the IL-18BP/IL-18R dissociation constant ratio of the DR IL-18 variant is at least about 100,000,000 times that of the wild-type IL-18.

In some embodiments, the inhibitory constant (Ki) of the DR-IL-18 variant against IL-18BP is greater than 3 nM (e.g., 5 nM or greater, 10 nM or greater, 50 nM or greater, 100 nM or greater, 500 nM or greater, 750 nM or greater, or 1 μM or greater). In some embodiments, the Ki of the DR-IL-18 variant peptide against IL-18BP is 500 nM or greater. In some embodiments, the Ki of the DR-IL-18 variant peptide against IL-18BP is 1 μM or greater.

In some embodiments, the subject IL-18 variant peptide (DR-IL-18) that binds to IL-15 18R and exhibits reduced binding to IL-18BP has a Ki of greater than 200 nM for IL-18BP (e.g., 500 nM or greater, 750 nM or greater, or 1 μM or greater). In some embodiments, the subject DR-IL-18 variant peptide has a Ki of 1 μM or greater for IL-18BP.

In some embodiments, the inhibitory constant (Ki) of the subject IL-18 variant peptide (DR-IL-18) that binds to IL-18R and exhibits substantially reduced binding to IL-18BP is at least twice that of the Ki of wild-type IL-18 to IL-18BP (i.e., the Ki of the subject IL-18 variant peptide to IL-18BP is at least twice that of the Ki of WT IL-18 to IL-18BP). For example, in some cases, the Ki of the subject DR-IL-18 variant peptide to IL-18BP is at least five times that of wild-type IL-18 to IL-18BP (e.g., at least 10 times, at least 50 times, at least 100 times, at least 200 times, at least 500 times, or at least 1000 times).

In some embodiments, the EC50 of the subject IL-18 variant peptide (DR-IL-18) that binds to IL-18R and exhibits substantially reduced binding to IL-18BP is at least twice that of wild-type IL-18 to IL-18BP (i.e., the EC50 of the subject IL-18 variant peptide to IL-18BP is at least twice that of WT IL-18 to IL-18BP). For example, in some cases, the EC50 of the subject DR-IL-18 variant peptide to IL-18BP is at least five times that of wild-type IL-18 to IL-18BP (e.g., at least 10 times, at least 50 times, at least 100 times, at least 200 times, at least 500 times, or at least 1000 times).

In any of the cases described above, the DR IL-18 variant may contain any of the C38/C68 mutations presented in the combinations described above (e.g., in some cases, C38S/C68S, C38S/C68G, C38S/C68A, C38S/C68A, C38S/C68V, C38S/C68D, C38S/C68E, or C38S/C68N; in some cases, C38S/C68S, C38S/ C68G, C38S/C68A, C38S/C68D, or C38S/C68N; in some cases, C38S/C68G, C38S/C68A, C38S/C68D, or C38S/C68N; C38S/C68D; in some cases, C38S/C68G; in some cases, C38S/C68A; in some cases, C38S/C68N; in some cases, C38S/C68S).

Similarly, for any of the following, the DR IL-18 variant may contain the C38/C68 mutation from any of the combinations presented above (e.g., in some cases, C38S/C68S, C38S/C68G, C38S/C68A, C38S/C68V, C38S/C68D, C38S/C68E, or C38S/C68N; in some cases, C38S/C68G, C38S/C68A, C38S/C68V, C38S/C68D, C38S/C68E, or C38S/C68N). S/C68N; in some cases, C38S/C68S, C38S/C68G, C38S/C68A, C38S/C68D or C38S/C68N; in some cases, C38S/C68G, C38S/C68A, C38S/C68D or C38S/C68N; C38S/C68D; in some cases, C38S/C68G; in some cases, C38S/C68A; in some cases, C38S/C68N; in some cases, C38S/C68S).

In some embodiments, the DR-IL-18 variant includes at least one mutation (e.g., at least two, at least three, at least four, at least five, or at least six mutations) at positions selected from the following amino acid positions: Y1, L5, K8, M51, K53, S55, Q56, P57, G59, M60, E77, Q103, S105, D110, N111, M113, V153, and N155. In various embodiments, the human IL-18 variant peptide includes at least four mutations at positions selected from the following amino acid positions: Y1, L5, K8, M51, K53, S55, Q56, P57, G59, M60, E77, Q103, S105, D110, N111, M113, V153, and N155. In various embodiments, the human IL-18 variant peptide comprises at least six mutations selected from the following amino acid positions: Y1, L5, K8, M51, K53, S55, Q56, P57, G59, M60, E77, Q103, S105, D110, N111, M113, V153, and N155. In various embodiments, the human IL-18 variant peptide comprises at least one mutation (e.g., at least two, at least three, at least four, at least five, or at least six mutations) selected from the following amino acid positions: Y1, L5, K8, S55, Q56, P57, G59, E77, Q103, S105, D110, N111, M113, V153, and N155. In some embodiments, the human IL-18 variant peptide includes at least one mutation (e.g., at least 2, at least 3, at least 4, at least 5, or at least 6 mutations) at positions selected from the following amino acid sites: Y1H, Y1R, L5H, L5I, L5Y, K8Q, K8R, M51T, M51K, M51D, M51N, M51E, M51R, K53R, K53G, K53S, K53T, S55K, S55R, Q56E, Q56A, Q56R, Q56V, Q56G, Q56K, Q56L, P57L, P57G, P57A, P57K, G59T, G 59A, M60K, M60Q, M60R, M60L, E77D, Q103E, Q103K, Q103P, Q103A, Q103R, S105R, S105D, S105K, S105N, S105A, D110H, D110K, D110N, D110Q, D110E, D110S, D110G, N111H, N111Y, N111D, N111R, N111S, N111G, M113V, M113R, M113T, M113K, V153I, V153T, V153A, N155K, and N155H.

In some embodiments, the DR IL-18 variant polypeptide comprises an amino acid sequence represented by any one of SEQ ID NO:34-59, 73-91, and 191-193. In some embodiments, the DR IL-18 variant polypeptide comprises an amino acid sequence having 80% or more (e.g., 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, or 100%) sequence identity with any one of SEQ ID NO:34-59, 73-91, and 191-193. In some embodiments, the DR IL-18 variant polypeptide comprises an amino acid sequence having 85% or more (e.g., 90% or more, 95% or more, 98% or more, 99% or more, or 100%) sequence identity with any one of SEQ ID NO:34-59, 73-91, and 191-193. In some embodiments, the DR IL-18 variant polypeptide comprises an amino acid sequence having 90% or more (e.g., 95% or more, 98% or more, 99% or more, or 100%) sequence identity with the amino acid sequence shown in any one of SEQ ID NO:34-59, 73-91, and 191-193.

In some cases, the DR-IL-18 variant relative to SEQ ID NO:30 contains at least one mutation (e.g., at least two, at least three, or at least four mutations) at amino acid positions selected from the following: M51, M60, S105, D110, and N111. In some cases, the DR-IL-18 variant relative to SEQ ID NO:30 contains at least three mutations at amino acid positions selected from the following: M51, M60, S105, D110, and N111. In some cases, the DR-IL-18 variant relative to SEQ ID NO:30 contains at least one mutation (e.g., at least two, at least three, or at least four mutations) at an amino acid position selected from M51, M60, S105, D110, and N111, wherein M51 is T, K, D, E, R, or N; M60 is K, Q, L, or R; S105 is R, D, K, A, or N; D110 is H, K, N, Q, E, N, S, or G; and N111 is H, D, Y, R, S, or G. In some cases, the DR-IL-18 variant relative to SEQ ID NO:30 contains at least three mutations (e.g., at least two, at least three, or at least four mutations) at amino acid positions selected from M51, M60, S105, D110, and N111, wherein M51 is T, K, D, E, R, or N; M60 is K, Q, L, or R; S105 is R, D, K, A, or N; D110 is H, K, N, Q, E, N, S, or G; and N111 is H, D, Y, R, S, or G. In some cases, the DR-IL-18 variant relative to SEQ ID NO:30 contains at least one mutation (e.g., at least two, at least three, or at least four mutations) at an amino acid position selected from M51, M60, S105, D110, and N111, wherein M51 is T or K; M60 is K or L; S105 is D, N, or A; D110 is K, N, S, or G; and N111 is H, Y, G, or R.

In some cases, the subject DR-IL-18 variant contains mutations at amino acid positions M51, M60, S105, D110, and N111 relative to SEQ ID NO:30. For example, in some cases, the subject DR-IL-18 variant contains mutations M51, M60, S105, D110, and N111 relative to SEQ ID NO:30, wherein M51 is T, K, D, E, R, or N; M60 is K, Q, L, or R; S105 is R, D, K, A, or N; D110 is H, K, N, Q, E, N, S, or G; and N111 is H, D, Y, R, S, or G. In some cases, the DR-IL-18 variant relative to SEQ ID NO:30 contains the mutations M51, M60, S105, D110, and N111, where M51 is T or K; M60 is K or L; S105 is D, N, or A; D110 is K, N, S, or G; and N111 is H, Y, G, or R. In other words, in some cases, the DR-IL-18 variant relative to SEQ ID NO:30 contains the mutations {M51T or M51K}; {M60K or M60L}; {S105D, S105N, S105A}; {D110K, D110N, D110S, or D110G}; and {N111H, N111Y, N111R, or N111G}.

In some cases, the subject DR-IL-18 variant relative to SEQ ID NO:30 contains at least one mutation (e.g., at least two, at least three, or at least four mutations) at amino acid positions selected from the following: M51, K53, Q56, S105, and N111. In some cases, the subject DR-IL-18 variant relative to SEQ ID NO:30 contains at least three mutations at amino acid positions selected from the following: M51, K53, Q56, S105, and N111. In some cases, the DR-IL-18 variant relative to SEQ ID NO:30 contains at least one mutation selected from M51, K53, Q56, S105, and N111 (e.g., at least two, at least three, or at least four mutations), wherein M51 is E, R, or K; K53 is G, S, or T; Q56 is E, A, R, V, G, K, or L; S105 is N, S, K, or G; and N111 is R, S, G, or D. In some cases, the DR-IL-18 variant relative to SEQ ID NO:30 contains at least three mutations selected from M51, K53, Q56, S105, and N111, wherein M51 is E, R, or K; K53 is G, S, or T; Q56 is E, A, R, V, G, K, or L; S105 is N, S, K, or G; and N111 is R, S, G, or D. In some cases, the DR-IL-18 variant relative to SEQ ID NO:30 contains at least one mutation selected from M51, K53, Q56, S105, and N111 (e.g., at least two, at least three, or at least four mutations), wherein M51 is K; K53 is G or S; Q56 is G, R, or L; S105 is S, N, or G; and N111 is G or R.

In some cases, the subject DR-IL-18 variant contains mutations at amino acid positions M51, K53, Q56, D110, and N111 relative to SEQ ID NO:30. For example, in some cases, the subject DR-IL-18 variant contains mutations M51, K53, Q56, D110, and N111 relative to SEQ ID NO:30, wherein M51 is E, R, or K; K53 is G, S, or T; Q56 is E, A, R, V, G, K, or L; D110 is S, N, G, or K; and N111 is R, S, G, or D. In some cases, the DR-IL-18 variant relative to SEQ ID NO:30 contains the mutations M51, K53, Q56, D110, and N111, where M51 is K; K53 is G or S; Q56 is G, R, or L; D110 is S, N, or G; and N111 is G or R. In other words, in some cases, the DR-IL-18 variant relative to SEQ ID NO:30 contains the mutations {M51K}; {K53G or K53S}; {Q56G, Q56R, or Q56L}; {D110S, D110N, or D110G}; and {N111R or N111G}.

In some cases, in addition to the mutations mentioned above, DR IL-18 variants also contain mutations at positions P57 and M60, and in some cases, a mutation at position S105. For example, in some cases, P57 is A, K, G, or L; M60 is L or R, and in some such cases, S105 is A, N, or D. In some cases, P57 is A (i.e., P57A) and M60 is L (i.e., M60L), and in some such cases, S105 is A, N, or D (in some cases, S105D).

In some cases, the subject DR-IL-18 variant contains an amino acid sequence that has 85% or more sequence identity with the wild-type human IL-18 amino acid sequence shown in SEQ ID NO:30 (e.g., 90% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity). Therefore, in some cases, the subject DR-IL-18 variant contains the following amino acid sequence, which (i) has 85% or more sequence identity with the wild-type human IL-18 amino acid sequence as shown in SEQ ID NO:30 (e.g., 90% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity); and (ii) contains at least one mutation relative to wild-type IL-18 (e.g., human IL-18) (e.g., at least 2, at least 3, at least 4, at least 5, or at least 6 mutations) (e.g., see all mutation combinations above).

In some cases, the DR-IL-18 variant contains an amino acid sequence that has 85% or more sequence identity (e.g., 90% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity) with any of the amino acid sequences shown in SEQ ID NO:34-59, 73-91, and 191-193. Therefore, in some cases, the subject DR-IL-18 variant contains the following amino acid sequences, which (i) have 85% or more sequence identity with the amino acid sequences shown in any one of SEQ ID NO:34-59, 73-91 and 191-193 (e.g., 90% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more or 99% or more sequence identity); and (ii) contain at least one mutation relative to wild-type IL-18 (e.g., human IL-18) (e.g., at least 2, at least 3, at least 4, at least 5 or at least 6 mutations).

In some cases, the subject DR-IL-18 variant contains the following amino acid sequence, which (i) has 85% or more sequence identity with the wild-type human IL-18 amino acid sequence shown in SEQ ID NO:30 (e.g., 90% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity); and (ii) contains at least one mutation (e.g., at least two, at least three, at least four, at least five, or at least six mutations) at the amino acid positions selected from Y1, L5, K8, M51, K53, S55, Q56, P57, G59, M60, E77, Q103, S105, D110, N111, M113, V153, and N155 relative to SEQ ID NO:30. In some cases, the subject DR-IL-18 variant contains the following amino acid sequences, which (i) have 85% or more sequence identity with the wild-type human IL-18 amino acid sequence shown in SEQ ID NO:30 (e.g., 90% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity); and (ii) contain at least four mutations at amino acid positions selected from Y1, L5, K8, M51, K53, S55, Q56, P57, G59, M60, E77, Q103, S105, D110, N111, M113, V153, and N155 relative to SEQ ID NO:30. In some cases, the subject DR-IL-18 variant contains the following amino acid sequences, which (i) have 85% or more sequence identity with the wild-type human IL-18 amino acid sequence shown in SEQ ID NO:30 (e.g., 90% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity); and (ii) contain at least six mutations at amino acid positions selected from Y1, L5, K8, M51, K53, S55, Q56, P57, G59, M60, E77, Q103, S105, D110, N111, M113, V153, and N155 relative to SEQ ID NO:30. In some cases, the subject DR-IL-18 variant contains the following amino acid sequence, which (i) has 85% or more sequence identity with the wild-type human IL-18 amino acid sequence shown in SEQ ID NO:30 (e.g., 90% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity); and (ii) contains at least one mutation (e.g., at least two, at least three, at least four, at least five, or at least six mutations) at the amino acid positions selected from Y1, L5, K8, S55, Q56, P57, G59, E77, Q103, S105, D110, N111, M113, V153, and N155 relative to SEQ ID NO:30.

In some cases, the subject DR-IL-18 variant contains the following amino acid sequence, which (i) has 85% or more sequence identity with the wild-type human IL-18 amino acid sequence shown in SEQ ID NO:30 (e.g., 90% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity); and (ii) contains at least one mutation (e.g., at least two, at least three, or at least four mutations) at an amino acid position selected from M51, M60, S105, D110, and N111 relative to SEQ ID NO:30. In some cases, the subject DR-IL-18 variant contains the following amino acid sequences, which (i) have 85% or more sequence identity with the wild-type human IL-18 amino acid sequence as shown in SEQ ID NO:30 (e.g., 90% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity); and (ii) contain at least three mutations at amino acid positions selected from M51, M60, S105, D110, and N111 relative to SEQ ID NO:30. In some cases, the subject DR-IL-18 variant contains the following amino acid sequence, which (i) has 85% or more sequence identity with the wild-type human IL-18 amino acid sequence shown in SEQ ID NO:30 (e.g., 90% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity); and (ii) contains at least one mutation selected from M51, M60, S105, D110, and N111 relative to SEQ ID NO:30 (e.g., at least two, at least three, or at least four mutations), wherein M51 is T, K, D, E, R, or N; M60 is K, Q, L, or R; S105 is R, D, K, A, or N; D110 is H, K, N, Q, E, N, S, or G; and N111 is H, D, Y, R, S, or G. In some cases, the subject DR-IL-18 variant contains the following amino acid sequence, which (i) has 85% or more sequence identity with the wild-type human IL-18 amino acid sequence shown in SEQ ID NO:30 (e.g., 90% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity); and (ii) contains at least three mutations at amino acid positions selected from M51, M60, S105, D110, and N111 relative to SEQ ID NO:30, wherein M51 is T, K, D, E, R, or N; M60 is K, Q, L, or R; S105 is R, D, K, A, or N; D110 is H, K, N, Q, E, N, S, or G; and N111 is H, D, Y, R, S, or G. In some cases, the subject DR-IL-18 variant contains the following amino acid sequence, which (i) has 85% or more sequence identity with the wild-type human IL-18 amino acid sequence shown in SEQ ID NO:30 (e.g., 90% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity); and (ii) contains at least one mutation (e.g., at least two, at least three, or at least four mutations) at an amino acid position selected from M51, M60, S105, D110, and N111 relative to SEQ ID NO:30, wherein M51 is T or K; M60 is K or L; S105 is D, N, or A; D110 is K, N, S, or G; and N111 is H, Y, G, or R.

In some cases, the subject DR-IL-18 variant contains the following amino acid sequences, which (i) have 85% or more sequence identity with the wild-type human IL-18 amino acid sequence shown in SEQ ID NO:30 (e.g., 90% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity); and (ii) contain mutations at positions M51, M60, S105, D110, and N111 relative to SEQ ID NO:30. In some cases, the subject DR-IL-18 variant contains the following amino acid sequences, which (i) have 85% or more sequence identity with the wild-type human IL-18 amino acid sequence shown in SEQ ID NO:30 (e.g., 90% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity); and (ii) contain the mutants M51, M60, S105, D110, and N111 relative to SEQ ID NO:30, wherein M51 is T, K, D, E, R, or N; M60 is K, Q, L, or R; S105 is R, D, K, A, or N; D110 is H, K, N, Q, E, N, S, or G; and N111 is H, D, Y, R, S, or G.

In some cases, the subject DR-IL-18 variant contains the following amino acid sequences, which (i) have 85% or more sequence identity with the wild-type human IL-18 amino acid sequence shown in SEQ ID NO:30 (e.g., 90% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity); and (ii) contain the mutants M51, M60, S105, D110, and N111 relative to SEQ ID NO:30, wherein M51 is T or K; M60 is K or L; S105 is D, N, or A; D110 is K, N, S, or G; and N111 is H, Y, G, or R.

In some cases, the subject DR-IL-18 variant contains the following amino acid sequence, which (i) has 85% or more sequence identity with the wild-type human IL-18 amino acid sequence shown in SEQ ID NO:30 (e.g., 90% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity); and (ii) contains at least one mutation (e.g., at least two, at least three, or at least four mutations) at positions selected from M51, K53, Q56, D110, and N111 relative to SEQ ID NO:30. In some cases, the subject DR-IL-18 variant contains the following amino acid sequences, which (i) have 85% or more sequence identity with the wild-type human IL-18 amino acid sequence shown in SEQ ID NO:30 (e.g., 90% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity); and (ii) contain at least three mutations at positions selected from M51, K53, Q56, D110, and N111 relative to SEQ ID NO:30. In some cases, the subject DR-IL-18 variant contains the following amino acid sequence, which (i) has 85% or more sequence identity with the wild-type human IL-18 amino acid sequence shown in SEQ ID NO:30 (e.g., 90% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity); and (ii) contains at least one mutation selected from M51, K53, Q56, D110, and N111 relative to SEQ ID NO:30 (e.g., at least two, at least three, or at least four mutations), wherein M51 is E, R, or K; K53 is G, S, or T; Q56 is E, A, R, V, G, K, or L; D110 is N, S, K, or G; and N111 is R, S, G, or D. In some cases, the subject DR-IL-18 variant contains the following amino acid sequence, which (i) has 85% or more sequence identity with the wild-type human IL-18 amino acid sequence shown in SEQ ID NO:30 (e.g., 90% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity); and (ii) contains at least three mutations selected from M51, K53, Q56, D110, and N111 relative to SEQ ID NO:30, wherein M51 is E, R, or K; K53 is G, S, or T; Q56 is E, A, R, V, G, K, or L; D110 is N, S, K, or G; and N111 is R, S, G, or D. In some cases, the subject DR-IL-18 variant contains the following amino acid sequence, which (i) has 85% or more sequence identity with the wild-type human IL-18 amino acid sequence as shown in SEQ ID NO:30 (e.g., 90% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity); and (ii) contains at least one mutation selected from M51, K53, Q56, D110, and N111 relative to SEQ ID NO:30 (e.g., at least two, at least three, or at least four mutations), wherein M51 is K; K53 is G or S; Q56 is G, R, or L; D110 is S, N, or G; and N111 is G or R.

In some cases, the subject DR-IL-18 variant contains the following amino acid sequences, which (i) have 85% or more sequence identity with the wild-type human IL-18 amino acid sequence shown in SEQ ID NO:30 (e.g., 90% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity); and (ii) contain mutations at positions M51, K53, Q56, D110, and N111 relative to SEQ ID NO:30. In some cases, the subject DR-IL-18 variant contains the following amino acid sequences, which (i) have 85% or more sequence identity with the wild-type human IL-18 amino acid sequence shown in SEQ ID NO:30 (e.g., 90% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity); and (ii) contain the mutants M51, K53, Q56, D110, and N111 relative to SEQ ID NO:30, wherein M51 is E, R, or K; K53 is G, S, or T; Q56 is E, A, R, V, G, K, or L; D110 is N, S, K, or G; and N111 is R, S, G, or D. In some cases, the subject DR-IL-18 variant contains the following amino acid sequences, which (i) have 85% or more sequence identity with the wild-type human IL-18 amino acid sequence as shown in SEQ ID NO:30 (e.g., 90% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity); and (ii) contain the mutants M51, K53, Q56, D110, and N111 relative to SEQ ID NO:30, wherein M51 is K; K53 is G or S; Q56 is G, R, or L; D110 is S, N, or G; and N111 is G or R.

In some cases, in addition to the mutations mentioned above, DR IL-18 variants also contain mutations at positions P57 and M60, and in some cases, a mutation at position S105. For example, in some cases, P57 is A, K, G, or L; M60 is L or R, and in some such cases, S105 is A, N, or D. In some cases, P57 is A (i.e., P57A) and M60 is L (i.e., M60L), and in some such cases, S105 is A, N, or D (in some cases, S105D).

Examples of murine IL-18 variant peptides (in this case, DR IL-18 variants) include, but are not limited to, mCS1 (SEQ ID NO:60), mCS2 (SEQ ID NO:61), mC1 (SEQ ID NO:62), mA12 (SEQ ID NO:63), mE8 (SEQ ID NO:64), mC10 (SEQ ID NO:65), mB7 (SEQ ID NO:66), mB1 (SEQ ID NO:67), mD1 (SEQ ID NO:68), mH7 (SEQ ID NO:69), mA7 (SEQ ID NO:70), mE1 (SEQ ID NO:71), and mH3 (SEQ ID NO:72).

As explained above, any IL-18 variant described in this paper can be ‘stabilized’ by further mutations of C38 and C68 relative to SEQ ID NO:30. As illustrative examples, in some cases, the subject-stabilized DR-IL-18 variants contain the following amino acid sequences: (i) having 85% or more sequence identity with the wild-type human IL-18 amino acid sequence as shown in SEQ ID NO:30 (e.g., 90% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity); (ii) containing mutations at positions M51, K53, Q56, D110, and N111 relative to SEQ ID NO:30 (or any one of SEQ ID NO:6 and 16-21, or any one of SEQ ID NO:34-59, 73-91, and 191-193, or any one of SEQ ID NO:87-91, or SEQ ID NO:89); and (iii) including mutations at C38. Mutations at C38 and C68 (e.g., C38S/C68S, C38S/C68G, C38S/C68A, C38S/C68V, C38S/C68D, C38S/C68E, or C38S/C68N; in some cases, C38S/C68G, C38S/C68A, C38S/C68V, C38S/C68D, C38S/C68E, or C38S/C68N; in some cases, C38S/ C68S, C38S/C68G, C38S/C68A, C38S/C68D, or C38S/C68N; in some cases, C38S/C68G, C38S/C68A, C38S/C68D, or C38S/C68N; in some cases, C38S/C68D; in some cases, C38S/C68G; in some cases, C38S/C68A; in some cases, C38S/C68N). In some cases, the DR-IL-18 variant contains the following amino acid sequence, which (i) has 85% or more sequence identity with the wild-type human IL-18 amino acid sequence shown in SEQ ID NO:30 (or any one of SEQ ID NO:6 and 16-21, or any one of SEQ ID NO:34-59, 73-91 and 191-193, or any one of SEQ ID NO:87-91, or SEQ ID NO:89). (e.g., 90% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity); (ii) containing mutations M51, K53, Q56, D110, and N111 relative to SEQ ID NO:30, wherein M51 is E, R, or K; K53 is G, S, or T; Q56 is E, A, R, V, G, K, or L; D110 is N, S, K, or G; and N 111 is R, S, G, or D; and (iii) includes mutations at C38 and C68 (e.g., C38S/C68S, C38S/C68G, C38S/C68A, C38S/C68V, C38S/C68D, C38S/C68E, or C38S/C68N; in some cases, C38S/C68G, C38S/C68A, C38S/C68V, C38S/C68D, C38S/C68E, or C38S/C68N). In some cases, C38S/C68S, C38S/C68G, C38S/C68A, C38S/C68D, or C38S/C68N; in some cases, C38S/C68G, C38S/C68A, C38S/C68D, or C38S/C68N; in some cases, C38S/C68D; in some cases, C38S/C68G; in some cases, C38S/C68A; in some cases, C38S/C68N). In some cases, the subject DR-IL-18 variant contains the following amino acid sequence, which (i) has 85% or more of the amino acid sequence of wild-type human IL-18 as shown in SEQ ID NO:30 (or any one of SEQ ID NO:6 and 16-21, or any one of SEQ ID NO:34-59, 73-91 and 191-193, or any one of SEQ ID NO:87-91, or SEQ ID NO:89). Multiple sequence identity (e.g., 90% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity); (ii) containing mutations M51, K53, Q56, D110, and N111 relative to SEQ ID NO:30, wherein M51 is K; K53 is G or S; Q56 is G, R, or L; D110 is S, N, or G; and N111 is G or R; and (iii) including mutations at C38 and C68 (e.g., C38S/C68S, C38S/C68G, C38S/C68A, C38S/C68V, C38S/C68D, C38S/C68E or C38S/C68N; in some cases, C38S/C68G, C38S/C68A, C38S/C68V, C38S/C68D, C38S/C68E or C38S/C68N; in some cases... In some cases, C38S/C68S, C38S/C68G, C38S/C68A, C38S/C68D, or C38S/C68N; in some cases, C38S/C68G, C38S/C68A, C38S/C68D, or C38S/C68N; in some cases, C38S/C68D; in some cases, C38S/C68G; in some cases, C38S/C68A; in some cases, C38S/C68N).

In some cases, in addition to the mutations mentioned above, DR IL-18 variants also contain mutations at positions P57 and M60, and in some cases, a mutation at position S105. For example, in some cases, P57 is A, K, G, or L; M60 is L or R, and in some such cases, S105 is A, N, or D. In some cases, P57 is A (i.e., P57A) and M60 is L (i.e., M60L), and in some such cases, S105 is A, N, or D (in some cases, S105D).

In some cases, the DR IL-18 variant (i) contains the mutations C38S/C68S, C38S/C68G, C38S/C68A, C38S/C68V, C38S/C68D, C38S/C68E, or C38S/C68N (relative to SEQ ID NO:30); (ii) has 90% or more sequence identity with the wild-type human IL-18 amino acid sequence as shown in SEQ ID NO:30 (e.g., 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity); and (iii) contains mutations at positions M51, K53, Q56, D110, and N111 relative to SEQ ID NO:30. In some such cases, the variant also contains mutations at positions P57 and M60. In some of these cases, the variant also includes a mutation at position S105.

In some cases, the subject DR IL-18 variant (i) contains the mutant C38S/C68S, C38S/C68G, C38S/C68A, C38S/C68V, C38S/C68D, C38S/C68E, or C38S/C68N (relative to SEQ ID NO:30); (ii) has 90% or more sequence identity with the wild-type human IL-18 amino acid sequence as shown in SEQ ID NO:30 (e.g. (iii) 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity); and (iii) mutations contained at positions M51, K53, Q56, D110, and N111 relative to SEQ ID NO:30, wherein M51 is K; K53 is G or S; Q56 is G, R, or L; D110 is S, N, or G; and N111 is G or R. In some such cases, the variant also contains a mutation at a position wherein P57 is A, K, G, or L; and M60 is L or R. In some such cases, the variant also contains a mutation at position S105, wherein S105 is A, N, or D.

In some cases, the DR IL-18 variant (i) contains the mutations C38S/C68G, C38S/C68A, C38S/C68V, C38S/C68D, C38S/C68E, or C38S/C68N (relative to SEQ ID NO:30); (ii) has 90% or more sequence identity with the wild-type human IL-18 amino acid sequence as shown in SEQ ID NO:30 (e.g., 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity); and (iii) contains mutations at positions M51, K53, Q56, D110, and N111 relative to SEQ ID NO:30. In some such cases, the variant also contains mutations at positions P57 and M60. In some such cases, the variant also contains a mutation at position S105.

In some cases, the subject DR IL-18 variant (i) contains the mutant C38S/C68G, C38S/C68A, C38S/C68V, C38S/C68D, C38S/C68E, or C38S/C68N (relative to SEQ ID NO:30); (ii) has 90% or more sequence identity with the wild-type human IL-18 amino acid sequence as shown in SEQ ID NO:30 (e.g., 92% or more). (iii) More, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity); and (iii) mutations contained at positions M51, K53, Q56, D110, and N111 relative to SEQ ID NO:30, wherein M51 is K; K53 is G or S; Q56 is G, R, or L; D110 is S, N, or G; and N111 is G or R. In some such cases, the variant also contains a mutation at a position wherein P57 is A, K, G, or L; and M60 is L or R. In some such cases, the variant also contains a mutation at position S105, wherein S105 is A, N, or D.

In some cases, the DR IL-18 variant (i) contains the mutations C38S/C68S, C38S/C68G, C38S/C68A, C38S/C68D, or C38S/C68N (relative to SEQ ID NO:30); (ii) has 90% or more sequence identity with the wild-type human IL-18 amino acid sequence as shown in SEQ ID NO:30 (e.g., 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity); and (iii) contains mutations at positions M51, K53, Q56, D110, and N111 relative to SEQ ID NO:30. In some such cases, the variant also contains mutations at positions P57 and M60. In some such cases, the variant also contains a mutation at position S105.

In some cases, the subject DR IL-18 variant (i) contains the mutations C38S/C68S, C38S/C68G, C38S/C68A, C38S/C68D, or C38S/C68N (relative to SEQ ID NO:30); (ii) has 90% or more sequence identity with the amino acid sequence of wild-type human IL-18 as shown in SEQ ID NO:30 (e.g., 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity); and (iii) contains mutations at positions M51, K53, Q56, D110, and N111 relative to SEQ ID NO:30, wherein M51 is K; K53 is G or S; Q56 is G, R, or L; D110 is S, N, or G; and N111 is G or R. In some of these cases, the variant also includes a mutation at a position where P57 is A, K, G, or L; and M60 is L or R. In some of these cases, the variant also includes a mutation at position S105, where S105 is A, N, or D.

In some cases, the DR IL-18 variant (i) contains the mutations C38S/C68G, C38S/C68A, C38S/C68D, or C38S/C68N (relative to SEQ ID NO:30); (ii) has 90% or more sequence identity with the wild-type human IL-18 amino acid sequence as shown in SEQ ID NO:30 (e.g., 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity); and (iii) contains mutations at positions M51, K53, Q56, D110, and N111 relative to SEQ ID NO:30. In some such cases, the variant also contains mutations at positions P57 and M60. In some such cases, the variant also contains a mutation at position S105.

In some cases, the subject DR IL-18 variant (i) contains the mutations C38S/C68G, C38S/C68A, C38S/C68D, or C38S/C68N (relative to SEQ ID NO:30); (ii) has 90% or more sequence identity with the amino acid sequence of wild-type human IL-18 as shown in SEQ ID NO:30 (e.g., 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity); and (iii) contains mutations at positions M51, K53, Q56, D110, and N111 relative to SEQ ID NO:30, wherein M51 is K; K53 is G or S; Q56 is G, R, or L; D110 is S, N, or G; and N111 is G or R. In some of these cases, the variant also includes a mutation at a position where P57 is A, K, G, or L; and M60 is L or R. In some of these cases, the variant also includes a mutation at position S105, where S105 is A, N, or D.

In some cases, the DR IL-18 variant (i) contains the mutation C38S/C68D (relative to SEQ ID NO:30); (ii) has 90% or more sequence identity with the wild-type human IL-18 amino acid sequence as shown in SEQ ID NO:30 (e.g., 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity); and (iii) contains mutations at positions M51, K53, Q56, D110, and N111 relative to SEQ ID NO:30. In some such cases, the variant also contains mutations at positions P57 and M60. In some such cases, the variant also contains a mutation at position S105.

In some cases, the DR IL-18 variant (i) contains the mutation C38S/C68D (relative to SEQ ID NO:30); (ii) has 90% or more sequence identity with the wild-type human IL-18 amino acid sequence as shown in SEQ ID NO:30 (e.g., 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity); and (iii) contains mutations at positions M51, K53, Q56, D110, and N111 relative to SEQ ID NO:30, where M51 is K; K53 is G or S; Q56 is G, R, or L; D110 is S, N, or G; and N111 is G or R. In some such cases, the variant also contains mutations at positions where P57 is A, K, G, or L; and M60 is L or R. In some of these cases, the variant also includes a mutation at position S105, where S105 is A, N, or D.

In some cases, the DR IL-18 variant (i) contains the mutation C38S/C68G (relative to SEQ ID NO:30); (ii) has 90% or more sequence identity with the wild-type human IL-18 amino acid sequence as shown in SEQ ID NO:30 (e.g., 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity); and (iii) contains mutations at positions M51, K53, Q56, D110, and N111 relative to SEQ ID NO:30. In some such cases, the variant also contains mutations at positions P57 and M60. In some such cases, the variant also contains a mutation at position S105.

In some cases, the DR IL-18 variant (i) contains the mutation C38S/C68G (relative to SEQ ID NO:30); (ii) has 90% or more sequence identity with the wild-type human IL-18 amino acid sequence as shown in SEQ ID NO:30 (e.g., 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity); and (iii) contains mutations at positions M51, K53, Q56, D110, and N111 relative to SEQ ID NO:30, where M51 is K; K53 is G or S; Q56 is G, R, or L; D110 is S, N, or G; and N111 is G or R. In some such cases, the variant also contains mutations at positions where P57 is A, K, G, or L; and M60 is L or R. In some of these cases, the variant also includes a mutation at position S105, where S105 is A, N, or D.

In some cases, the DR IL-18 variant (i) contains the mutation C38S/C68A (relative to SEQ ID NO:30); (ii) has 90% or more sequence identity with the wild-type human IL-18 amino acid sequence as shown in SEQ ID NO:30 (e.g., 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity); and (iii) contains mutations at positions M51, K53, Q56, D110, and N111 relative to SEQ ID NO:30. In some such cases, the variant also contains mutations at positions P57 and M60. In some such cases, the variant also contains a mutation at position S105.

In some cases, the DR IL-18 variant (i) contains the mutation C38S/C68A (relative to SEQ ID NO:30); (ii) has 90% or more sequence identity with the wild-type human IL-18 amino acid sequence as shown in SEQ ID NO:30 (e.g., 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity); and (iii) contains mutations at positions M51, K53, Q56, D110, and N111 relative to SEQ ID NO:30, where M51 is K; K53 is G or S; Q56 is G, R, or L; D110 is S, N, or G; and N111 is G or R. In some such cases, the variant also contains mutations at positions where P57 is A, K, G, or L; and M60 is L or R. In some of these cases, the variant also includes a mutation at position S105, where S105 is A, N, or D.

In some cases, the DR IL-18 variant (i) contains the mutation C38S/C68N (relative to SEQ ID NO:30); (ii) has 90% or more sequence identity with the wild-type human IL-18 amino acid sequence as shown in SEQ ID NO:30 (e.g., 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity); and (iii) contains mutations at positions M51, K53, Q56, D110, and N111 relative to SEQ ID NO:30. In some such cases, the variant also contains mutations at positions P57 and M60. In some such cases, the variant also contains a mutation at position S105.

In some cases, the DR IL-18 variant (i) contains the mutation C38S/C68N (relative to SEQ ID NO:30); (ii) has 90% or more sequence identity with the wild-type human IL-18 amino acid sequence as shown in SEQ ID NO:30 (e.g., 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity); and (iii) contains mutations at positions M51, K53, Q56, D110, and N111 relative to SEQ ID NO:30, where M51 is K; K53 is G or S; Q56 is G, R, or L; D110 is S, N, or G; and N111 is G or R. In some such cases, the variant also contains mutations at positions where P57 is A, K, G, or L; and M60 is L or R. In some of these cases, the variant also includes a mutation at position S105, where S105 is A, N, or D.

In some embodiments, the IL-18 peptide disclosed herein (e.g., DR IL-18) does not contain a cysteine residue at residue 78 (e.g., relative to SEQ ID NO: 30, 89, and/or 19). In some embodiments, the IL-18 peptide disclosed herein (e.g., DR IL-18) does not contain a mutation at residue N78 (e.g., relative to SEQ ID NO: 30, 89, and/or 19).

In some embodiments, the IL-18 peptide disclosed herein (e.g., DR IL-18) does not contain a cysteine residue at residue 121 (e.g., relative to SEQ ID NO: 30, 89, and/or 19). In some embodiments, the IL-18 peptide disclosed herein (e.g., DR IL-18) does not contain a mutation at residue E121 (e.g., relative to SEQ ID NO: 30, 89, and/or 19).

In some embodiments, the IL-18 peptide disclosed herein (e.g., DR IL-18) does not contain a cysteine residue at residue 144 (e.g., relative to SEQ ID NO: 30, 89, and/or 19). In some embodiments, the IL-18 peptide disclosed herein (e.g., DR IL-18) does not contain a mutation at residue L144 (e.g., relative to SEQ ID NO: 30, 89, and/or 19).

In some embodiments, the IL-18 peptide disclosed herein (e.g., DR IL-18) does not contain a cysteine residue at residue 157 (e.g., relative to SEQ ID NO: 30, 89, and/or 19). In some embodiments, the IL-18 peptide disclosed herein (e.g., DR IL-18) does not contain a mutation at residue D157 (e.g., relative to SEQ ID NO: 30, 89, and/or 19).

In some embodiments, the IL-18 peptide disclosed herein (e.g., DR IL-18) does not contain a cysteine residue at residue 78, residue 121, residue 144, or residue 157 (e.g., relative to SEQ ID NO: 30, 89, and/or 19). In some embodiments, the IL-18 peptide disclosed herein (e.g., DR IL-18) does not contain a mutation at residue N78, residue E121, residue L144, or residue D157 (e.g., relative to SEQ ID NO: 30, 89, and/or 19).

The IL-18 peptides disclosed herein (e.g., DR IL-18) may contain mutations at C38 and C68 as disclosed herein, and may lack any mutations that introduce non-natural cysteine residues into the peptide sequence. For example, in some embodiments, the IL-18 peptide (e.g., DR IL-18) contains only cysteine residues at positions 76 and/or 127 (e.g., relative to SEQ ID NO: 30, 89, or 19). In some embodiments, the IL-18 peptide (e.g., DR IL-18) does not contain any cysteine residues other than those at positions 76 and/or 127 (e.g., relative to SEQ ID NO: 30, 89, or 19). The absence of non-natural cysteine residues may contribute to the advantageous properties of the disclosed IL-18 peptides, for example, because cysteine residues may negatively affect the stability and/or biological activity of IL-18, for example, by allowing the formation of intramolecular or intermolecular disulfide bonds, dimers, aggregates, etc.

The IL-18 peptides disclosed herein (e.g., DR IL-18) may lack or substantially lack surface-exposed or solvent-exposed cysteine residues. The lack of surface-exposed or solvent-exposed cysteine residues may contribute to the advantageous properties of the disclosed IL-18 peptides, for example, because surface-exposed or solvent-exposed cysteine residues may negatively affect the stability and/or biological activity of IL-18, for example, by allowing the formation of intramolecular or intermolecular disulfide bonds, dimers, aggregates, etc. The IL-18 peptides disclosed herein (e.g., DR IL-18) may lack or substantially lack surface-exposed or solvent-exposed cysteine residues, as determined by, for example, structural modeling or assays detecting free thiols (such as Ellman titration). The IL-18 peptides disclosed herein (e.g., DR IL-18) may lack or substantially lack free or surface-exposed thiols.

The IL-18 peptides disclosed herein (e.g., DR IL-18) can be characterized by their molecular weight. For example, the molecular weight of an IL-18 peptide may be about 18 kDa. In some embodiments, the molecular weight of the IL-18 peptides disclosed herein is about 13 kDa, about 14 kDa, about 15 kDa, about 16 kDa, about 17 kDa, about 18 kDa, about 19 kDa, about 20 kDa, about 21 kDa, about 22 kDa, or about 23 kDa. In some embodiments, the molecular weight of the IL-18 peptides disclosed herein is at least about 10 kDa, at least about 11 kDa, at least about 12 kDa, at least about 13 kDa, at least about 14 kDa, at least about 15 kDa, at least about 16 kDa, at least about 17 kDa, at least about 18 kDa, at least about 19 kDa, or at least about 20 kDa. In some embodiments, the molecular weight of the IL-18 peptide disclosed herein is up to about 17 kDa, up to about 18 kDa, up to about 19 kDa, up to about 20 kDa, up to about 21 kDa, up to about 22 kDa, up to about 23 kDa, up to about 24 kDa, up to about 25 kDa, up to about 30 kDa, up to about 40 kDa, up to about 50 kDa, or up to about 100 kDa. In some embodiments, the molecular weight of the IL-18 peptide disclosed herein is about 18.15153 kDa, about 18.1515 kDa, about 18.152 kDa, about 18.15 kDa, about 18.2 kDa, or about 18 kDa.

The IL-18 polypeptide disclosed herein (e.g., DR IL-18) may contain, for example, about 130, about 135, about 140, about 145, about 150, about 151, about 152, about 153, about 154, about 155, about 156, about 157, about 158, about 159, about 160, about 161, about 162, about 163, about 1645, about 165, about 170, about 175, about 180, about 185, or about 190 amino acids.

In some embodiments, the IL-18 polypeptide disclosed herein (e.g., DR IL-18) comprises at least about 130, at least about 135, at least about 140, at least about 145, at least about 150, at least about 151, at least about 152, at least about 153, at least about 154, at least about 155, at least about 156, at least about 157, at least about 158, at least about 159, or at least about 160 amino acids. In some embodiments, the IL-18 polypeptide disclosed herein (e.g., DR IL-18) comprises up to about 155, up to about 156, up to about 157, up to about 158, up to about 159, up to about 160, up to about 161, up to about 162, up to about 163, up to about 1645, up to about 165, up to about 170, up to about 175, up to about 180, up to about 185, or up to about 190 amino acids. In some embodiments, the IL-18 polypeptide disclosed herein (e.g., DR IL-18) comprises about 150 to about 170, about 152 to about 162, about 155 to about 159, about 156 to about 158, or 157 amino acids.

The level of biological activity or potency of the IL-18 peptides disclosed herein (e.g., DR IL-18, such as DR IL-18 in the formulations disclosed herein) can be measured by an assay for biological activity. A non-limiting example of an assay that can be used to measure the level of potency or biological activity of the IL-18 peptides disclosed herein is the IL-18 HEK-Blue potency assay. HEK-Blue IL-18 cells are designed to detect biologically active IL-18 by monitoring the activation of the NFκβ and AP-1 pathways. These reporter cells are generated by stably transfecting HEK293-derived cells with genes encoding IL-18Rα and IL-18Rβ. The response to human TNF-α and IL-1β is also blocked, so the cells have a specific response to IL-18. These cells express a secretory embryonic alkaline phosphatase (SEAP) reporter gene under the control of an IFN-β minimal promoter fused to five NFκβ and five AP-1 binding sites. The binding of bioactive IL-18 to the heterodimeric IL-18 receptor on the cell surface of HEK-Blue IL-18 triggers a signal transduction cascade, leading to the production of SEAP, which can be quantified by colorimetric assay.

The biological activity or potency of the IL-18 peptide disclosed herein (e.g., DR IL-18, such as DR IL-18 in formulations disclosed herein) can be compared with a reference standard (e.g., wild-type IL-18) or a previously generated and characterized IL-18 peptide. In some embodiments, the biological activity or potency (e.g., EC50) of the IL-18 peptide disclosed herein (e.g., DR IL-18, such as DR IL-18 in formulations disclosed herein) is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the reference value. In some embodiments, the biological activity or potency (e.g., EC50) of the IL-18 peptide disclosed herein (e.g., DR IL-18, such as DR IL-18 in formulations of this disclosure) is at most 105%, at most 110%, at most 115%, at most 120%, at most 125%, at most 130%, at most 135%, at most 140%, at most 145%, at most 150%, at most 155%, at most 160%, at most 165%, at most 170%, at most 175%, at most 180%, at most 185%, at most 190%, at most 195%, at most 200%, at most 250%, or at most 300% of the reference values. In some embodiments, the biological activity or potency (e.g., EC50) of the IL-18 peptide disclosed herein (e.g., DR IL-18, such as DR IL-18 in formulations of this disclosure) is about 30% to about 500%, about 30% to about 250%, about 30% to about 200%, about 30% to about 175%, about 30% to about 150%, about 30% to about 140%, about 30% to about 130%, about 30% to about 500% of reference values. % to about 120%, about 30% to about 110%, about 30% to about 105%, about 50% to about 500%, about 50% to about 250%, about 50% to about 200%, about 50% to about 175%, about 50% to about 150%, about 50% to about 140%, about 50% to about 130%, about 50% to about 120%, about 50% to about 110%, about 50% to about 105%, about 60% to about 500%, about 60% to about 260%, about 60% to about 200%, about 60% to about 175%, about 60% to about 160%, about 60% to about 140%, about 60% to about 130%, about 60% to about 120%, about 60% to about 110%, about 60% to about 105%, about 70% to about 500%, about 70% to about 250%, about 70% to about 200%, about 70% to about 175%, about 70% to about 150%, about 70% to about 140%. 0%, about 70% to about 130%, about 70% to about 120%, about 70% to about 110%, about 70% to about 105%, about 90% to about 500%, about 90% to about 250%, about 90% to about 200%, about 90% to about 175%, about 90% to about 150%, about 90% to about 140%, about 90% to about 130%, about 90% to about 120%, about 90% to about 110%, or about 90% to about 105%. In some embodiments, the biological activity or potency (e.g., EC50) is at least 60% and at most 140% of the reference value.

In some embodiments, the bioactivity or potency (e.g., EC50) of the IL-18 peptide disclosed herein (e.g., DR IL-18, such as DR IL-18 in formulations of this disclosure) is at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%, at least 97.5%, at least 100%, at least 102.5%, at least 105%, at least 110%, at least 115%, at least 120%, at least 125%, at least 130%, at least 140%, at least 150%, or at least 200%, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 8 times, at least 9 times, or at least 10 times, as determined by the IL-18HEK-Blue potency assay.

In some embodiments, the IL-18 peptide disclosed herein exhibits values of less than about 100 μM, less than about 10 μM, less than about 1 μM, less than about 900 nM, less than about 800 nM, less than about 700 nM, less than about 600 nM, less than about 500 nM, less than about 400 nM, less than about 300 nM, less than about 200 nM, less than about 100 nM, less than about 50 nM, less than about 30 nM, less than about 10 nM, and less than about 5 nM. EC50 at approximately 4 nM, less than approximately 3 nM, less than approximately 2 nM, less than approximately 1 nM, less than approximately 900 pM, less than approximately 800 pM, less than approximately 700 pM, less than approximately 600 pM, less than approximately 500 pM, less than approximately 400 pM, less than approximately 300 pM, less than approximately 200 pM, less than approximately 100 pM, less than approximately 50 pM, less than approximately 10 pM, or less than approximately 1 pM, for example, as determined by the IL-18HEK-Blue potency assay.

In some embodiments, the IL-18 peptide disclosed herein exhibits values from about 1 pM to about 1 μM, from about 1 pM to about 500 nM, from about 1 pM to about 400 nM, from about 1 pM to about 300 nM, from about 1 pM to about 200 nM, from about 1 pM to about 100 nM, from about 1 pM to about 50 nM, from about 1 pM to about 10 pM, from about 10 pM to about 1 μM, from about 10 pM to about 500 nM, from about 10 pM to about 400 nM, from about 10 pM to about 300 nM, from about 10 pM to about 200 nM, and from about 10 pM to about 100 nM. EC50 of about nM, about 10 pM to about 50 nM, about 50 pM to about 1 μM, about 50 pM to about 500 nM, about 50 pM to about 400 nM, about 50 pM to about 300 nM, about 50 pM to about 200 nM, about 50 pM to about 100 nM, about 100 pM to about 1 μM, about 100 pM to about 500 nM, about 100 pM to about 400 nM, about 100 pM to about 300 nM, or about 100 pM to about 200 nM, for example, as determined by the IL-18HEK-Blue potency assay.

Nucleic acid

This document also provides nucleic acids encoding any of the proteins described herein (e.g., IL-18 proteins with mutants at C38 and C68). Such nucleic acids can take any convenient form (e.g., PCR products, vectors (such as viral vectors), plasmids, granules, microcircles, etc.). In some cases, the nucleotide sequence encoding the subject IL-18 protein is operatively linked to a promoter (under its control) (e.g., functional in prokaryotic cells, or in some cases, functional in eukaryotic cells such as yeast or immune cells such as T cells, NK cells, TILs, etc.).

This document also provides cells containing such nucleic acids and/or containing the subject IL-18 protein (e.g., a stabilized protein). In some cases, the cells are prokaryotic cells, such as bacterial cells (e.g., for the purpose of propagating nucleic acids or for the purpose of producing the subject protein); in some cases, the cells are eukaryotic cells, such as yeast cells (e.g., for the purpose of producing the subject protein); and in some cases, the cells are immune cells, such as T cells (e.g., CAR-T cells, such as TRUCK cells), TILs, TCR-T cells (T cells with engineered TCRs, e.g., engineered to bind target antigens with increased affinity compared to natural TCRs) or NK cells (e.g., CAR-NK cells), for example, such cells may be engineered to express (e.g., secrete) the subject IL-18 protein (e.g., a stabilized protein, such as SEQ ID NO:19).

Cytokine inhibitors

In some embodiments, the compositions of this disclosure comprise inhibitors of one or more cytokines. In some embodiments, the inhibitors of one or more cytokines include compounds, proteins, peptides, peptide mimics, antibodies, ribozymes, small molecule compounds, or antisense nucleic acid molecules (e.g., siRNA, miRNA, etc.) that inhibit the expression, activity, or both of one or more cytokines. In some embodiments, the inhibitor inhibits the expression, activity, or both of IL-17, IL-5, or IL-3. In some embodiments, the cytokine inhibitor reduces toxicity. In some embodiments, the cytokine inhibitor increases the amount of administered IL-18 variant peptide (e.g., stabilized IL-18 variant, DR IL-18 variant, D2D IL-18 variant, stabilized DRIL-18 variant, and stabilized D2D IL-18 variant).

treat

In various embodiments, this disclosure includes compositions comprising activators of IL-18 activity (e.g., stabilized IL-18 variants, DR IL-18 variants, D2D IL-18 variants, stabilized DR IL-18 variants, and stabilized D2D IL-18 variants) used in methods such as stimulating signaling activity in cells, tissues, organs, systems, or objects of interest with at least one IL-18R. In various embodiments, activators and treatment methods of the IL-18 active compositions of the present invention increase the amount of IL-18R signaling, the amount of immune cell activity, or both. In various embodiments, diseases and conditions for which increased IL-18R signaling can improve treatment outcomes include, but are not limited to, cancer, infectious diseases, macular degeneration, and metabolic diseases or conditions.

Various IL-18 peptides (e.g., IL-18 peptides with C38/C68 mutations) have been discussed above and will not be repeated here, as any protein discussed above can be used in the methods discussed herein. For example, in some cases, stabilized IL-18 peptides (e.g., stabilized IL-18 variant peptides, such as stabilized DR IL-18 variants and stabilized D2D IL-18 variants) are used to treat diseases or conditions such as cancer or infections (e.g., cancer resistant to immune checkpoint inhibitors, cancer associated with reduced or absent expression of MHC class I, cancer associated with high levels of IL-18BP (e.g., circulating or tumor expression), cancer associated with tumors expressing IL-18R on infiltrating T cells or NK cells, metabolic diseases or conditions, infectious diseases, viral infections (such as HIV), etc.).

In some cases, the applied peptide is a wild-type IL-18 peptide (e.g., wild-type human IL-18 protein). In some cases, the applied peptide is a subject-stabilized IL-18 peptide (e.g., a peptide containing amino acid mutations at positions C38 and C68 relative to SEQ ID NO:30). In some cases, the applied peptide is an IL-18 variant peptide, such as a DRIL-18 variant or a D2D IL-18 variant. In some cases, the applied peptide is a stabilized IL-18 variant peptide (e.g., a stabilized DR IL-18 variant or a stabilized D2D IL-18 variant, see, for example, the stabilized DRIL-18 variant peptide discussed above).

Fusion/Assembly

In some embodiments, the IL-18 peptide of this disclosure (e.g., stabilized IL-18, such as a stabilized DRIL-18 variant or a stabilized D2D IL-18 variant, etc.) (e.g., SEQ ID NO: 19) is conjugated to another molecule (e.g., fused to another protein), that is, the IL-18 variant peptide may be fused co-framed with a second peptide (fusion partner). In some embodiments, the second peptide (fusion partner) is capable of increasing the overall size of the fusion protein, for example, such that the fusion protein will not be rapidly cleared from circulation. In some cases, the IL-18 peptide (e.g., stabilized IL-18, such as a stabilized DRIL-18 variant or a stabilized D2D IL-18 variant, etc.) (e.g., SEQ ID NO: 19) is not fused with the second peptide.

In some embodiments, the second polypeptide (a fusion partner of an IL-18 variant polypeptide or a fragment thereof) is part or all of the immunoglobulin Fc region (i.e., the antibody Fc sequence). In other embodiments, the second polypeptide is any suitable polypeptide substantially similar to the Fc (e.g., providing increased size, polymerizing domains, and/or additional binding or interaction with Ig molecules). In some embodiments, the second polypeptide is part or all of human serum albumin (HSA). In some embodiments, the second polypeptide is part or all of an antibody, antibody fragment, camel antibody, or “nanobody,” or other affinity reagent that binds to or interacts with HSA. These fusion proteins can facilitate purification, polymerization, and exhibit increased in vivo half-life. In some cases, fusion proteins with disulfide-linked multimeric structures can also bind and neutralize other molecules more effectively.

When fused with a heterologous peptide, the portion corresponding to the IL-18 peptide (e.g., stabilized IL-18, such as a stabilized DRIL-18 variant or a stabilized D2D IL-18 variant, etc.) (e.g., SEQ ID NO:19) may be referred to as the “IL-18 variant peptide portion” of the subject IL-18 variant peptide.

In some cases, the second peptide is a marker sequence (e.g., an affinity tag), such as a peptide that facilitates the purification of the fusion peptide. For example, the marker amino acid sequence can be a hexahistine peptide, such as the tags provided in the pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), many of which are commercially available. As described by Gentz et al., Proc. Natl. Acad. Sci. USA 86:821-824, 1989, for example, hexahistine facilitates the purification of fusion proteins. Another peptide tag that can be used for purification is the "HA" tag, corresponding to an epitope derived from influenza hemagglutinin protein. Wilson et al., Cell 37:767, 1984. Adding peptide moieties to facilitate peptide processing is a well-known and routine technique in the art. The subject IL-18 peptide (e.g., stabilized IL-18, such as stabilized DR IL-18 variants or stabilized D2DIL-18 variants, etc.) (e.g., SEQ ID NO: 19) can be modified, for example, by conjugation/conjugation with a wide variety of other oligopeptides, proteins, and/or non-protein molecules to achieve a variety of purposes. For example, post-translational modifications such as isopentylation, acetylation, amidation, carboxylation, glycosylation, PEGylation (covalent attachment of a polyethylene glycol (PEG) polymer chain) are performed. Such modifications also include glycosylation modifications, such as those performed by modifying the glycosylated form of the peptide during its synthesis and processing or in further processing steps; for example, those performed by exposing the peptide to enzymes that affect glycosylation (such as mammalian glycosylationases or deglycosylationases). In some embodiments, the subject IL-18 variant peptide has one or more phosphorylated amino acid residues, such as phosphotyrosine, phosphotyserine, or phosphotythreonine.

In some embodiments, the IL-18 peptide disclosed herein is glycosylated. In some embodiments, the IL-18 peptide disclosed herein is non-glycosylated.

In some embodiments, the IL-18 peptide disclosed herein is conjugated with a water-soluble polymer. In some embodiments, the IL-18 peptide disclosed herein is polyethylene glycol-modified. In some embodiments, the IL-18 peptide disclosed herein is conjugated with: polyethylene glycol homopolymer, polyethylene glycol copolymer, polypropylene glycol homopolymer, poly(N-vinylpyrrolidone), poly(vinyl alcohol), poly(ethylene glycol-co-propylene glycol), poly(N-2-(hydroxypropyl)methacrylamide), poly(sialic acid), poly(N-acryloylmorpholine), or dextran.

In some embodiments, the IL-18 peptide disclosed herein is not conjugated with a water-soluble polymer. In some embodiments, the IL-18 peptide disclosed herein is not PEGylated. In some embodiments, the IL-18 peptide disclosed herein is not conjugated with: polyethylene glycol homopolymer, polyethylene glycol copolymer, polypropylene glycol homopolymer, poly(N-vinylpyrrolidone), poly(vinyl alcohol), poly(ethylene glycol-co-propylene glycol), poly(N-2-(hydroxypropyl)methacrylamide), poly(sialic acid), poly(N-acryloylmorpholine), or dextran.

In some other embodiments, the IL-18 peptides of this disclosure (e.g., stabilized IL-18, such as stabilized DR IL-18 variants or stabilized D2D IL-18 variants, etc.) (e.g., SEQ ID NO: 19) include agents that further modify them to improve their resistance to proteolytic degradation, optimize solubility, or make them more suitable for use as therapeutic agents. For example, variants of this disclosure also include analogs containing residues other than naturally occurring L-amino acids, such as D-amino acids or non-naturally occurring synthetic amino acids. The D-amino acids may replace some or all of the amino acid residues.

Co-administration and multispecific IL-18 variant peptides

In some cases, the subject IL-18 peptide (e.g., stabilized IL-18, such as stabilized DR IL-18 variants or stabilized D2D IL-18 variants, etc.) (e.g., SEQ ID NO: 19) is administered together with an additional agent. The terms “co-administration,” “co-administer,” and “combination” encompass the simultaneous, parallel, or sequential administration of two or more therapeutic agents (e.g., subject-stabilized IL-18) without specific time constraints. In some embodiments, the agents are simultaneously present in the cells or subject or exert their biological or therapeutic effects simultaneously. In some embodiments, the therapeutic agents are in the same composition or unit dosage form. In other embodiments, the therapeutic agents are in separate compositions or unit dosage forms. In some implementations, the first dose may be administered before (e.g., several minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks prior to the administration of the second therapeutic dose), simultaneously with, or after (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks later).

In some cases, the subject IL-18 peptide (e.g., stabilized IL-18, such as stabilized DR IL-18 variants or stabilized D2D IL-18 variants, etc.) (e.g., SEQ ID NO: 19) (e.g., formulated as a pharmaceutical composition) is co-administered with a cancer therapeutic agent, a therapeutic agent for treating an infection, or a cancer-directed antibody. Such administration may involve concurrent (i.e., simultaneous), prior, or subsequent administration of the drug/antibody relative to one or more agents of this disclosure. Those skilled in the art will readily determine the appropriate timing, sequence, and dosage of administration for the specific drugs and compositions of this disclosure.

In some implementations, treatment is accomplished by administering a combination of the subject IL-18 peptide (e.g., stabilized IL-18, such as a stabilized DR IL-18 variant or a stabilized D2D IL-18 variant, etc.) (e.g., SEQ ID NO:19) with another agent (e.g., an immunostimulant, an agent for treating chronic infections, a cytotoxic agent, an anticancer agent, etc.). One exemplary class of cytotoxic agents that can be used is chemotherapeutic agents. Exemplary chemotherapeutic agents include, but are not limited to, aldesleukin, altretamine, aifostine, asparaginase, bleomycin, capecitabine, carboplatin, carmustine, cladribine, cisapride, cisplatin, cyclophosphamide, cytarabine, dacarbazine (DTIC), dactinomycin, docetaxel, doxorubicin, dronabinol, duocarmycin, etoposide, and filgrastim. rastim), fludarabine, fluorouracil, gemcitabine, granisetron, hydroxyurea, idarubicin, ifosfamide, interferon alpha, irinotecan, lansoprazole, levamisole, leucovorin, megestrol, mesna, methotrexate, metoclopramide, mitomycin, mitotane, mitoxantrone, omeprazole, ondansetron, paclitaxel (Taxol) ^TM ), pilocarpine, prochloroperazine, rituximab, saproin, tamoxifen, taxol, topotecan hydrochloride, trastuzumab, vinblastine, vincristine, and vinorelbine tartrate.

The subject IL-18 peptide (e.g., stabilized IL-18, such as stabilized DR IL-18 variants or stabilized D2D IL-18 variants, etc.) (e.g., SEQ ID NO:19) is not necessarily but optionally formulated with one or more agents that enhance activity or otherwise increase therapeutic effect. In some embodiments, treatment is accomplished by administering the subject IL-18 peptide (e.g., stabilized IL-18, such as stabilized DR IL-18 variants or stabilized D2D IL-18 variants, etc.) (e.g., SEQ ID NO:19) in combination with an agent that modulates target cells (co-administration). Therefore, compositions comprising (and methods of using the compositions) are also contemplated herein: (a) the subject IL-18 peptide (e.g., stabilized IL-18, such as stabilized DR IL-18 variants or stabilized D2D IL-18 variants, etc.) (e.g., SEQ ID NO:19); and (b) an agent that modulates target cells. In some cases, the agent that modulates target cells is rituximab. In some cases, the agent used to modulate target cells is cetuximab.

An "opsonization agent" is any agent that can bind to target cells (e.g., cancer cells, cells carrying intracellular pathogens, etc.) and opsonize the target cells (e.g., labeling cells to achieve phagocytosis and/or to achieve antibody-dependent cell-mediated cytotoxicity (ADCC)). For example, any antibody that can bind to target cells (e.g., cancer cells, such as tumor cells), wherein the antibody has an FC region, is considered an opsonization agent. In some cases, opsonization agents are antibodies that bind to target cells (e.g., antitumor antibodies, anticancer antibodies, anti-infective antibodies, etc.).

For example, antibodies selective for tumor cell markers, radiation, surgery, and/or hormone deprivation, see Kwon et al., Proc. Natl. Acad. Sci U.S.A., 96:15074-9, 1999.

Angiogenesis inhibitors can also be combined with the methods of this invention. Many antibodies are currently used clinically to treat cancer, while others are in different stages of clinical development. For example, there are many antigens and corresponding monoclonal antibodies used to treat B-cell malignancies. One target antigen is CD20. Rituximab is a chimeric unconjugated monoclonal antibody targeting the CD20 antigen. CD20 plays an important functional role in B-cell activation, proliferation, and differentiation. The CD52 antigen is targeted by the monoclonal antibody alemtuzumab, which is indicated for the treatment of chronic lymphocytic leukemia. CD22 is targeted by many antibodies and has recently demonstrated efficacy in combination with toxins in chemotherapy-resistant hairy cell leukemia. Two novel monoclonal antibodies targeting CD20, tositumomab and ibritumomab, have been submitted to the U.S. Food and Drug Administration (FDA). These antibodies are conjugated with radioisotopes. Alemtuzumab (Campath) is used to treat chronic lymphocytic leukemia; Gemtuzumab (Mylotarg) is used to treat acute myeloid leukemia; Zevalin is used to treat non-Hodgkin's lymphoma; and Panitumumab (Vectibix) is used to treat colon cancer.

Monoclonal antibodies that can be used in the methods of this disclosure and are already used for solid tumors include, but are not limited to, edrecolomab and trastuzumab (herceptin). Edrecolomab targets the 17-1A antigen found in colorectal and rectal cancers and has been approved for these indications in Europe. Trastuzumab targets the HER-2/neu antigen. Cetuximab (erbitux) can also be used in the methods of this disclosure. These antibodies bind to the EGF receptor (EGFR) and have been used to treat solid tumors, including colorectal cancer and squamous cell carcinoma of the head and neck (SCCHN).

The subject IL-18 peptide (e.g., stabilized IL-18, such as stabilized DR IL-18 variants or stabilized D2D IL-18 variants, etc.) can be used with any of the agents mentioned above (e.g., agents like antibodies that opsonize target cells). Therefore, in some cases, the subject IL-18 peptide (e.g., stabilized IL-18, such as stabilized DR IL-18 variants or stabilized D2D IL-18 variants, etc.) is used in combination therapy (co-administered with) one or more opsonizers that are selective for cancer cells (e.g., tumor cells). In some cases, the subject IL-18 peptide (e.g., stabilized IL-18, such as stabilized DR IL-18 variants or stabilized D2D IL-18 variants, etc.) is used in combination therapy (with co-administered) with one or more of the following: cetuximab (bound to EGFR), panitumumab (bound to EGFR), rituximab (bound to CD20), trastuzumab (bound to HER2), pertuzumab (bound to HER2), alemtuzumab (bound to CD52), brentuximab (bound to CD30), tositumomab, ibritumomab, gemtuzumab, ibritumomab, and edrecolomab (bound to 17-1A) or combinations thereof.

In some cases, the subject IL-18 peptide (e.g., stabilized IL-18, such as stabilized DR IL-18 variants or stabilized D2D IL-18 variants, etc.) (e.g., SEQ ID NO:19) is combined with cancer cell opsonizers (e.g., those containing antigen-binding regions targeting: CD19, CD20, CD22, CD24, CD25, CD30, CD33, CD37, CD38, CD44, CD45, CD47, CD51, CD52, CD56, CD62L, CD70, CD74, CD79, CD80, CD96, CD97, CD99, CD123, CD134, CD138, CD152 (CTLA-4), CD200, CD213A2, CD221, CD248, CD276 (B7H3), B7H4, CD279 (PD-1), CD274). (PD-L1), CD319, SIRPa, EGFR, EPCAM, 17-1A, HER1, HER2, HER3, CD117, C-Met, HGFR, PDGFRA, AXL, TWEAKR, PTHR2, HAVCR2 (TIM3), GD2 gangliosides, MUC1, mucin CanAg, mesothelin, endothelial glycoprotein, Lewis-Y antigen, CEA, CEACAM1, CEACAM5, CA-125, PSMA, BAFF, FGFR2, TAG-72, gelatinase B, phosphatidylinositol proteoglycan 3, integrin-4, BCMA, CSF1R, SLAMF7, integrin α _vβ3 , TYRP1, GPNMB, CLDN18.2, FORR1, CCR4, CXCR4, MICA, _C242 antigen, DLL3, DLL4, EGFL7, vimentin, fibronectin extra domain-B, TROP-2, LRRC15, FAP, SLITRK6, NOTCH2, NOTCH3, tendinin-3, STEAP1 or NRP1 or any combination thereof) are administered together.

In some cases, the subject IL-18 peptide (e.g., stabilized IL-18, such as stabilized DR IL-18 variants or stabilized D2D IL-18 variants, etc.) (e.g., SEQ ID NO:19) is co-administered with an agent that targets one or more antigens selected from the following: CD19, CD20, CD22, CD24, CD25, CD30, CD33, CD38, CD44, CD47, SIRPA, CD52, CD56, CD70, CD96, CD97, CD99, CD123, CD279 (PD-1), CD274 (PD-L1), EGFR, 17-1A, HER2, CD117, C-Met, PTHR2, and HAVCR2 (TIM3).

In some cases, the subject IL-18 peptide (e.g., stabilized IL-18, such as stabilized DR IL-18 variants or stabilized D2D IL-18 variants, etc.) (e.g., SEQ ID NO: 19) is used in combination therapy (co-administered with) a convenient immunomodulator (e.g., an immune checkpoint inhibitor or an immune agonist). Examples of targets for immune checkpoint inhibitors, as described above, include, but are not limited to: PD-L1, PD1, CTLA4, TIM3, TIGIT, LAG3, B7H3, B7H4, VISTA, BTLA, CD47, SIRPα, CD48, CD155, CD160, TREM2, IDO1, adenosine 2A receptor, aryl hydrocarbon receptor, KIR, and LILRB2. Therefore, examples of immune checkpoint inhibitors include agents (e.g., antibodies) that inhibit proteins such as PD-L1, PD1, CTLA4, TIM3, TIGIT, LAG3, B7H3, B7H4, VISTA, BTLA, CD47, SIRPα, CD48, CD155, CD160, TREM2, IDO1, adenosine 2A receptor, aryl hydrocarbon receptor, KIR, and LILRB2. In some cases, a subject-stabilized IL-18 variant peptide (e.g., DR-IL-18 variant, D2D-IL-18 variant) (e.g., SEQ ID NO:19) is co-administered with an immune checkpoint inhibitor (e.g., an antibody) that inhibits the following: PD-L1, PD1, CTLA4, TIM3, TIGIT, LAG3, B7H3, B7H4, VISTA, BTLA, CD47, SIRPα, CD48, CD155, CD160, TREM2, IDO1, adenosine 2A receptor, aryl hydrocarbon receptor, KIR, LILRB2, or any combination thereof.

In some cases, topic-stabilized IL-18 variant peptides (e.g., DR-IL-18 variant, D2D-IL-18 variant) (e.g., SEQ ID NO:19) are co-administered with immune agonists. Examples of immune agonists include those that agonize proteins such as: tumor necrosis factor receptor superfamily (TNFRSF) proteins (e.g., GITR, 41BB, OX40, CD27, CD40, HVEM); immunoglobulin superfamily (IgSF) proteins (e.g., CD28, ICOS, CD226, NKG2D); TLRs (e.g., TLR2, TLR4, TLR5, TLR7, TLR9); and nucleic acid sensors (e.g., STING, cGAS, RIG-I (DDX58)). Examples of immune agonists also include, but are not limited to: inflammasome activators (agonists), T-cell binders (e.g., multispecific agents that crosslink CD3 and/or CD28 with tumor antigens, such as BiTE), and cytokines or cytokine variants (e.g., IL-2, IL-7, IL-10, IL-12, IL-15, IL-21, IL-23, IL-27, IL-33, TNF, TL1A, IFNA, IFNB, IFNG).

Therefore, in some cases, topic-stabilized IL-18 variant peptides (e.g., DR-IL-18 variant, D2D-IL-18 variant) (e.g., SEQ ID NO:19) are co-administered with immune agonists such as: agents that activate tumor necrosis factor receptor superfamily (TNFRSF) proteins (e.g., GITR, 41BB, OX40, CD27, CD40, HVEM) (e.g., antibodies); agents that activate immunoglobulin superfamily (IgSF) proteins (e.g., CD28, ICOS, CD226, NKG2D) (e.g., antibodies); agents that activate TLRs (e.g., T... Agents of LR2, TLR4, TLR5, TLR7, TLR9; agents that stimulate nucleic acid sensors (e.g., STING, cGAS, RIG-I (DDX58)); inflammasome activators; T cell binders (e.g., multispecific agents that crosslink CD3 and/or CD28 with tumor antigens, such as BiTE); cytokines or cytokine variants (e.g., IL-2, IL-7, IL-10, IL-12, IL-15, IL-21, IL-23, IL-27, IL-33, TNF, TL1A, IFNA, IFNB, IFNG); or any combination thereof.

In some cases, a subject-stabilized IL-18 variant peptide (e.g., DR-IL-18 variant, D2D-IL-18 variant) (e.g., SEQ ID DO:19) is co-administered with: TGFβ antagonists, such as anti-TGFβ antibodies; interferons, such as interferon α, interferon β, or interferon γ; TNF; TRAIL; lymphotoxin; LIGHT/TNSF14; and so on.

In some cases, the subject IL-18 peptide (e.g., stabilized IL-18, such as stabilized DR IL-18 variants or stabilized D2D IL-18 variants, etc.) (e.g., SEQ ID NO: 19) is used in combination therapy (co-administered with) an inhibitor of BTLA and/or CD160. In other cases, the subject IL-18 peptide (e.g., stabilized IL-18, such as stabilized DR IL-18 variants or stabilized D2D IL-18 variants, etc.) (e.g., SEQ ID NO: 19) is used in combination therapy (co-administered with) an anti-CD47/SIRPA agent (e.g., anti-CD47, anti-SIRPA, high-affinity CD47 binder, high-affinity SIRPA binder, etc.). In some cases, the subject IL-18 peptide (e.g., stabilized IL-18, such as stabilized DR IL-18 variants or stabilized D2D IL-18 variants, etc.) (e.g., SEQ ID NO: 19) is used in combination therapy (co-administered with) an inhibitor of TIM3 and/or CEACAM1.

In some cases, the subject IL-18 peptide (e.g., a stabilized IL-18, such as a stabilized DR IL-18 variant or a stabilized D2D IL-18 variant, etc.) (e.g., SEQ ID NO: 19) is fused with another protein (i.e., a "fusion partner," a "second peptide"). In some embodiments, the second peptide (the fusion partner of the subject IL-18 variant peptide) specifically binds to target molecules other than those bound by the IL-18 variant peptide portion of the fusion protein (e.g., IL-18R other than the variant that binds IL-18R; or IL-18BP other than the variant that binds IL-18BP).

Therefore, in some embodiments, the subject IL-18 peptide (e.g., stabilized IL-18, such as a stabilized DRIL-18 variant or a stabilized D2D IL-18 variant, etc.) (e.g., SEQ ID NO: 19) is multispecific (e.g., bispecific). The terms "multispecific" or "bispecific" are generally used when referring to an agent (e.g., a ligand or antibody) that recognizes two or more distinct antigens by possessing at least one region specific to a first target (e.g., the Fab of a ligand or first antibody) and at least one second region specific to a second target (e.g., the Fab of a ligand or second antibody). A bispecific agent specifically binds to two targets and is therefore a type of multispecific agent.

In some embodiments, the subject IL-18 peptide (e.g., stabilized IL-18, such as a stabilized DR IL-18 variant or a stabilized D2D IL-18 variant, etc.) (e.g., SEQ ID NO: 19) is multispecific (e.g., bispecific), such that a first region of the peptide contains the subject IL-18 variant peptide sequence (i.e., the first region contains the IL-18 variant peptide) and a second region specifically binds to another target molecule (e.g., an antigen). For example, in some cases, the IL-18 peptide (e.g., stabilized IL-18, such as a stabilized DR IL-18 variant or a stabilized D2D IL-18 variant, etc.) (e.g., SEQ ID NO: 19) is fused to a second peptide that specifically binds to a target molecule other than the target molecule bound by the IL-18 variant peptide.

In a co-administration context, any of the agents discussed above may be conjugated to the subject IL-18 peptide (e.g., stabilized IL-18, such as a stabilized DR IL-18 variant or a stabilized D2D IL-18 variant, etc.) (e.g., SEQ ID NO: 19). As used herein, the term "co-administration" is intended to cover compounds with such conjugations. For example, when agent 1 is co-administered with agent 2, the term is intended to cover embodiments in which agent 1 and agent 2 are not conjugated to each other, and also to cover embodiments in which agent 1 and agent 2 are conjugated to each other (e.g., where agent 1 and agent 2 are both proteins, and agent 1 is fused with agent 2).

In some cases, the second region of a multispecific IL-18 peptide (e.g., stabilized IL-18, such as a stabilized DR IL-18 variant or a stabilized D2D IL-18 variant, etc.) (e.g., SEQ ID NO: 19) contains a checkpoint inhibitor. In some cases, the second region inhibitor of the multispecific IL-18 variant peptide is selected from one or more proteins including PD-L1, PD1, CTLA4, TIM3, TIGIT, LAG3, B7H3, B7H4, VISTA, BTLA, CD47, SIRPα, CD48, CD155, CD160, TREM2, IDO1, adenosine 2A receptor, aryl hydrocarbon receptor, KIR, and LILRB2.

In some cases, the second region of a multispecific IL-18 peptide (e.g., stabilized IL-18, such as a stabilized DR IL-18 variant or a stabilized D2D IL-18 variant, etc.) (e.g., SEQ ID NO:19) contains an immune agonist. In some cases, the second region of the multispecific IL-18 variant peptide is activated by one or more proteins selected from the following: ICOS, GITR, 41BB, OX40, and CD40. Additional examples of immunostimulants that can be used as a second region (or part thereof) of a multispecific IL-18 peptide (e.g., stabilized IL-18, such as a stabilized DR IL-18 variant or a stabilized D2D IL-18 variant, etc.) (e.g., SEQ ID NO: 19) include, but are not limited to: agents that activate tumor necrosis factor receptor superfamily (TNFRSF) proteins (e.g., GITR, 41BB, OX40, CD27, CD40, HVEM) (e.g., antibodies or their antigen-binding regions); agents that activate immunoglobulin superfamily (IgSF) proteins (e.g., CD28, ICOS, CD226, NKG2D) (e.g., antibodies or their antigen-binding regions); and agents that activate immunoglobulin superfamily (IgSF) proteins (e.g., CD28, ICOS, CD226, NKG2D). Examples include antibodies or their antigen-binding regions; agents that activate TLRs (e.g., TLR2, TLR4, TLR5, TLR7, TLR9); agents that activate nucleic acid sensors (e.g., STING, cGAS, RIG-I(D DX58)); inflammasome activators; T cell binders (e.g., multispecific agents that crosslink CD3 and/or CD28 with tumor antigens, such as BiTE); cytokines or cytokine variants (e.g., IL-2, IL-7, IL-10, IL-12, IL-15, IL-21, IL-23, IL-27, IL-33, TNF, TL1A, IFNA, IFNB, IFNG); and so on.

In some cases, the second region of a multispecific IL-18 peptide (e.g., stabilized IL-18, such as stabilized DR IL-18 variants or stabilized D2D IL-18, etc.) (e.g., SEQ ID NO: 19) is a cancer cell opsonizer. In some cases, the second region of a multispecific IL-18 variant peptide targets one or more proteins selected from the following: CD19, CD20, CD22, CD24, CD25, CD30, CD33, CD38, CD44, CD47, SIRPA, CD52, CD56, CD70, CD96, CD97, CD99, CD123, CD279 (PD-1), CD274 (PD-L1), EGFR, 17-1A, HER2, CD117, C-Met, PTHR2, and HAVCR2 (TIM3). In some cases, the second region of a multispecific IL-18 peptide (e.g., stabilized IL-18, such as stabilized DRIL-18 variants or stabilized D2D IL-18 variants, etc.) (e.g., SEQ ID NO: 19) is an opsonizer targeting one or more proteins selected from the following: CD19, CD20, CD22, CD24, CD25, CD30, CD33, CD38, CD44, CD47, SIRPA, CD52, CD56, CD70, CD96, CD97, CD99, CD123, CD279 (PD-1), CD274 (PD-L1), EGFR, 17-1A, HER2, CD117, C-Met, PTHR2, and HAVCR2 (TIM3).

For example, in some cases, the second region of a multispecific IL-18 peptide (e.g., stabilized IL-18, such as a stabilized DRIL-18 variant or a stabilized D2D IL-18 variant, etc.) (e.g., SEQ ID NO: 19) contains an extracellular domain, such as the extracellular domain of PD-1, PD-L1, CD47 (e.g., an affinity CD47 variant/peptide), or SIRPA (e.g., a high affinity SIRPA variant/peptide). In some cases, the second region of the multispecific IL-18 variant peptide specifically binds to antigens selected from: CTLA-4, Lag-3, BTLA, Tim-3, CD244, CD40, CD40L, CD47, SIRPA, PD-1, and PD-L1.

In some embodiments, the biological activity of the IL-18 variant peptide is increased relative to wild-type IL-18. In some embodiments, the IL-18 variant does not contain cysteine residues, unlike the cysteine residues naturally present in wild-type IL-18. In some embodiments, the IL-18 variant peptide comprises an amino acid sequence that does not contain PEGylated amino acids.

In some embodiments, the subject IL-18 variant peptide (e.g., stabilized IL-18) comprises a linker (e.g., a linker peptide). For example, in some embodiments, the subject IL-18 variant peptide and the fusion partner are separated by a linker (e.g., a linker peptide). The linker peptide can have any of a plurality of amino acid sequences. Proteins can be linked by the linker peptide, which can have flexible properties (e.g., a flexible linker peptide), but other chemical linkages are not excluded. Suitable linkers include peptides with a length between about 6 and about 40 amino acids or between about 6 and about 25 amino acids. These linkers can be generated by coupling a protein with a synthetic linker-encoded oligonucleotide. Peptide linkers with a degree of flexibility can be used. The linker peptide can have virtually any amino acid sequence, and it should be remembered that in some cases, the linker will have a sequence that produces a generally flexible peptide. Small amino acids (such as glycine and alanine) are used to produce flexible peptides. It is conventional for those skilled in the art to generate such sequences. A variety of different linkers are commercially available and considered suitable.

In some embodiments, the IL-18 variant peptide (e.g., stabilized IL-18) is co-administered with immune cells such as tumor-infiltrating lymphocytes (TILs), CAR-T cells, TCR-T cells (T cells with engineered TCRs (e.g., T cells engineered to bind target antigens with increased affinity compared to natural TCRs), CAR-NK cells, or T cells or NK cells transduced with engineered T cell receptors. In other embodiments, the IL-18 variant peptide is co-administered with an oncolytic virus.

In some implementations, the nucleic acid encoding an IL-18 variant peptide (e.g., stabilized IL-18) is contained within engineered (“altered”) immune cells, such as CAR-T, TCR-T, or CAR-NK cells, or T cells or NK cells transduced with engineered T cell receptors or tumor-infiltrating lymphocytes (TILs). In this context, the engineered cells (e.g., altered T cells, altered NK cells, altered TILs) secrete the IL-18 variant peptide. The ability to secrete the IL-18 variant peptide can be regulated in a contextual manner (e.g., within the tumor microenvironment), for example, by synthesizing the NOTCH receptor.

In some implementations, the nucleic acid encoding an IL-18 variant peptide (e.g., stabilized IL-18) is contained within an oncolytic virus. In this case, cells infected with the oncolytic virus secrete the IL-18 variant peptide.

In some embodiments, an IL-18 variant peptide (e.g., stabilized IL-18) is applied to cells, tissues, organs, systems, or objects to treat a disease or condition. In some embodiments, a human IL-18 variant peptide is applied to cells, tissues, organs, systems, or objects. In some embodiments, a nucleic acid (e.g., DNA, cDNA, mRNA, etc.) encoding at least one human IL-18 variant peptide (e.g., stabilized IL-18) is applied to cells, tissues, organs, systems, or objects.

In some embodiments, the compositions of the present invention are applied to rodent cells, tissues, organs, systems, or objects for the treatment or prevention of diseases or conditions. In some embodiments, rodent IL-18 variant polypeptides or fragments thereof are applied to cells, tissues, organs, systems, or objects (e.g., human cells, tissues, organs, systems, or objects). In some embodiments, nucleic acids (e.g., DNA, cDNA, mRNA, etc.) encoding at least one rodent IL-18 variant polypeptide are applied to cells, tissues, organs, systems, or objects. As described elsewhere herein, examples of rodent IL-18 variant polypeptides (in this case, DR IL-18 variants) include, but are not limited to, SEQ ID NO: 60-72. Also as noted elsewhere herein, examples of rodent IL-18 variants (in this case, D2D IL-18 variants, which bind IL-18BP but have reduced binding to IL-18R) include, but are not limited to, SEQ ID NO: 126-190.

In some embodiments, the method includes administering to a subject, cell, or tissue an isolated nucleic acid molecule encoding one or more of the IL-18 variant peptides described herein (e.g., stabilized IL-18). An increase in IL-18 signaling levels, including through the use of IL-18 variant peptides (e.g., stabilized IL-18), can be assessed using a variety of methods, including those disclosed herein, as well as methods known in the art or developed in the future. That is, a person skilled in the art will understand based on the disclosure provided herein that an increase in the level or activity of IL-18 signaling can be readily assessed using the evaluation of the levels of nucleic acids (e.g., mRNA) encoding IL-18 or IL-18 variant peptides or fragments thereof, the levels of IL-18 or IL-18 variant peptides or fragments thereof, and/or the levels of IL-18 or IL-18 variant peptide or fragment activity in biological samples obtained from the subject.

Those skilled in the art will understand, based on the disclosure provided herein, that the compositions of this disclosure (IL-18 variant peptides, such as stabilized IL-18) can be used to treat, in whole (e.g., systemically) or in part (e.g., locally, cellularly, in tissues, or in organs) a disease or condition for which an increase in IL-18 signaling activity would be beneficial. Those skilled in the art will understand, based on the teachings provided herein, that diseases and conditions treatable by the compositions and methods described herein include any disease or condition for which an increase in IL-18 signaling would promote positive biological, physiological, clinical, or therapeutic outcomes.

In some embodiments, a method includes administering to a subject in need a composition comprising at least one IL-18 peptide (e.g., stabilized IL-18, such as a stabilized DR IL-18 variant or a stabilized D2D IL-18 variant, etc.) (e.g., SEQ ID NO: 19) and administering to the subject a composition comprising an additional agent. In one such embodiment, the additional agent includes an immunotherapeutic agent comprising, but not limited to, at least one selected from, but not limited to, altered T cells, chimeric antigen receptor T cells (CAR-T), armed CAR-T cells, chimeric antigen receptor NK cells (CAR-NK), TCR-T cells, tumor-infiltrating lymphocytes (TIL), viruses, antigens, vaccines, antibodies, immune checkpoint inhibitors, small molecules, chemotherapeutic agents, and stem cells. In some embodiments, a composition comprising at least one IL-18 peptide (e.g., stabilized IL-18, such as a stabilized DR IL-18 variant or a stabilized D2D IL-18 variant, etc.) (e.g., SEQ ID NO: 19) is used for a method of increasing the activity of the immune system before, during, or after infection with bacteria, viruses, or other pathogens. In some embodiments, a composition comprising at least one IL-18 variant peptide is used as a method for increasing the number and/or activity of immune cells (such as the number and/or activity of T cells, NK cells, and/or myeloid cells) in vitro, in vivo, or ex vivo.

In some embodiments, the additional agent includes an inhibitor of one or more cytokines. In some embodiments, the inhibitor of one or more cytokines includes compounds, proteins, peptides, peptide mimics, antibodies, ribozymes, small molecule compounds, or antisense nucleic acid molecules (e.g., siRNA, miRNA, etc.) that inhibit the expression, activity, or both of one or more cytokines. In some embodiments, the inhibitor inhibits the expression, activity, or both of IL-17, IL-5, or IL-3. In some embodiments, administration of the cytokine inhibitor reduces toxicity. In some embodiments, administration of the cytokine inhibitor increases the efficacy of the administered IL-18 peptide (e.g., stabilized IL-18, such as a stabilized DR IL-18 variant or a stabilized D2D IL-18 variant, etc.) (e.g., SEQ ID NO:19).

As used herein, the term “pharmaceutically acceptable carrier” means a chemical composition that can incorporate a suitable IL-18 peptide (e.g., stabilized IL-18, such as a stabilized DR IL-18 variant or a stabilized D2D IL-18 variant, etc.) (e.g., SEQ ID NO:19), and after incorporation, it can be used to administer an IL-18 peptide (e.g., stabilized IL-18, such as a stabilized DR IL-18 variant or a stabilized D2D IL-18 variant, etc.) to a subject (e.g., SEQ ID NO:19).

The development of IL-18 variants

SUMO protease

Compositions and methods are provided for preparing polypeptides of interest (e.g., inactive proteins) by cleaving SUMO tags from polypeptides of interest using a SUMO (small ubiquitin-like modified) protease. The SUMO tag can be placed at the N-terminus of the protein of interest, and the SUMO protease (e.g., ULP1) can remove the SUMO tag without residue via a cleavage reaction. In some cases, cleavage will occur in cells (e.g., in bacterial cells, in yeast cells), while in others, cleavage will occur in vitro without cells.

For example, in some cases, a fusion protein (including a protein of interest fused to its N-terminus with a SUMO tag) is introduced into a bacterial cell (e.g., via nucleic acid encoding the fusion protein), and a SUMO protease is also introduced into the same cell (in such cases, both proteins can be referred to as exogenously introduced), and thus, the cleavage reaction will occur inside the cell. In some cases, the protease will be encoded by nucleic acid integrated into the cell's genome, while in others, the nucleic acid will not be integrated (e.g., it could be a plasmid). The SUMO protease can be expressed from a constitutive promoter or can be operatively linked to an inducible promoter (such as a rhamnose-inducible promoter) (under its control). Any convenient inducible promoter can be used. Therefore, in some such cases, contacting the fusion protein with the SUMO protease in the cell (e.g., a bacterial cell) can include inducing the expression of the SUMO protease.

In some cases, the fusion protein and the SUMO protease are encoded by the same nucleic acid (e.g., a plasmid). In some such cases, both proteins are operatively ligated to the same promoter (e.g., they can be translated from bicis-trans mRNA), while in other cases, the two proteins are operatively ligated to separate promoters.

In some cases, fusion proteins are contacted with SUMO proteases in a cell-free in vitro environment. For example, the fusion protein can be purified/isolated and then contacted with purified SUMO proteases (which are readily available).

When the peptide of interest is an IL-18 peptide (e.g., wild-type IL-18, stabilized IL-18 variants, DR IL-18 variants, D2D IL-18 variants, stabilized DR IL-18 variants, stabilized D2D IL-18 variants, etc.) (e.g., SEQ ID NO: 19), not all such proteins are active when the N-terminal amino acid is methionine (e.g., human wild-type IL-18 protein is typically cleaved by caspase-1 to expose the N-terminal tyrosine, thus yielding the active mature form). Therefore, the methods disclosed herein can be used to generate active IL-18 peptides. In such cases, a SUMO (small ubiquitin-like modifier) tag can be used to facilitate the production of a large number of fusion proteins (IL-18 tagged with a SUMO tag), followed by removal of the SUMO tag to expose the N-terminally activated IL-18 protein (e.g., a protein with a non-methionine, such as tyrosine). The SUMO tag can be removed by contacting the fusion protein with a SUMO protease (e.g., yeast ULP1), which cleaves the tag to expose the native N-terminal amino acid (Tyr in the case of human IL-18). In some cases, the contact takes place inside a cell (such as a bacterial cell). In some such cases, the resulting active protein can then be purified, for example, from cell lysates (see further details below).

Any convenient SUMO tag can be used, and one such example is shown in SEQ ID NO:26. In some cases, for example, for subsequent purification purposes (see below), it is necessary to include an additional tag (e.g., a 6XHis tag) on the SUMO tag. SEQ ID NO:27 provides a non-limiting example of such a tagged SUMO tag.

Purification steps

In some embodiments, the subject matter ‘Preparation Method’/‘Generation’ further includes purifying the peptide of interest (e.g., IL-18 peptide, such as stabilized IL-18 variants, DR IL-18 variants, D2D IL-18 variants, stabilized DR IL-18 variants, stabilized D2D IL-18 variants, etc.) after removing the SUMO tag (e.g., SEQ ID NO: 19). In some such cases, cleavage (SUMO tag removal) occurs cell-free in vitro, and purification may include the removal of the SUMO tag and the SUMO protease. For example, in some cases, the SUMO protease and/or the SUMO tag may be tagged (e.g., His tagging), which in some cases may facilitate the removal of these components after cleavage, for example, for the purification of the peptide of interest, for example, using immobilized metal affinity chromatography (IMAC), such as a nickel column.

In some cases, cleavage (SUMO tag removal) occurs in cells (e.g., bacterial cells as discussed above), and the peptide of interest is purified from the cell lysate. In other cases, the peptide of interest is secreted into a culture medium (e.g., in some cases, when yeast cells are used), and then purified from the medium. Any convenient steps or series of steps can be used to purify the protein from, for example, bacterial lysates or a culture medium that has already secreted the protein. In some cases (e.g., when the peptide of interest is an IL-18 peptide, such as a stabilized IL-18 variant, a DR IL-18 variant, a D2D IL-18 variant, a stabilized DR IL-18 variant, a stabilized D2D IL-18 variant, etc.) (e.g., SEQ ID NO: 19), purification includes ion exchange chromatography (e.g., Capto Q), IMAC (e.g., to remove any residual his-tagged SUMO, uncut proteins, and/or proteases), multimodal chromatography (e.g., Capto MMC chromatography, Capto MMC being a multimodal salt-resistant resin used for capturing and purifying proteins from bulk feeds via packed bed chromatography), and hydrophobic interaction chromatography (HIC) (e.g., phenyl agarose gel HIC in some cases). In some cases, high-performance Q chromatography is also performed. In some of these cases, ultrafiltration (UF) and percolation (DF) steps are performed prior to each chromatographic step. See Figure 48 as an example of a possible purification process.

Those skilled in the art will understand that, when incorporated with the methods detailed herein, the invention is not limited to treatment once a disease or symptom has been identified. In particular, the symptoms of the disease or symptom need not be manifested to a degree harmful to the subject; in fact, it is not necessary to detect the disease or symptom in the subject prior to the application of treatment. That is, significant pathology arising from the disease or symptom need not occur before the invention can provide benefit. Therefore, as described more fully herein, the invention includes methods for preventing diseases and symptoms in a subject, since activators of IL-18 activity, as discussed elsewhere herein, can be applied to the subject prior to the onset of the disease or symptom, thereby preventing its development.

The pharmaceutical compositions disclosed herein

Compositions comprising peptides, peptide fragments, or IL-18 peptides (e.g., stabilized IL-18, such as stabilized DR IL-18 variants or stabilized D2D IL-18 variants, etc.) (e.g., SEQ ID NO: 19) as described elsewhere herein may be formulated and applied to a subject, as now described. By way of non-limiting example, IL-18 peptides (e.g., stabilized IL-18, such as stabilized DR IL-18 variants or stabilized D2D IL-18 variants, etc.) (e.g., SEQ ID NO: 19) for the treatment and/or prevention of diseases or conditions may be formulated and applied to a subject in any convenient manner, for example, as now described.

This disclosure covers the preparation and use of pharmaceutical compositions comprising compositions that can be used to treat or prevent diseases or conditions, disclosed herein as an active ingredient (IL-18 polypeptide, e.g., stabilized IL-18, such as stabilized DR IL-18 variants or stabilized D2D IL-18 variants, etc.) (e.g., SEQ ID NO: 19). Such pharmaceutical compositions may consist solely of the active ingredient in a form suitable for administration to a subject, or the pharmaceutical composition may comprise the active ingredient and one or more pharmaceutically acceptable carriers, one or more additional ingredients, or some combination thereof. The active ingredient may be present in the pharmaceutical composition in the form of a physiologically acceptable ester or salt, such as in combination with physiologically acceptable cations or anions, as is well known in the art.

As used herein, the term “pharmaceutically acceptable carrier” means a chemical composition to which a suitable IL-18 peptide (e.g., a stabilized IL-18, such as a stabilized DR IL-18 variant or a stabilized D2D IL-18 variant, etc.) (e.g., SEQ ID NO:19) can be combined and, after combination, can be used to apply to the subject.

In some embodiments, the pharmaceutical composition may comprise large, slowly metabolizing macromolecules such as proteins, polysaccharides such as chitosan, polylactic acid, polyglycolic acid and copolymers (such as latex-functionalized Sepharose ^™ , agarose, cellulose, etc.), polymeric amino acids, amino acid copolymers and lipid aggregates (such as oil droplets or liposomes).

Pharmaceutical compositions used to implement the subject method (e.g., IL-18 peptides, such as wild-type IL-18, stabilized IL-18, DR IL-18, D2D IL-18, stabilized DR IL-18, stabilized D2D IL-18, etc.) (e.g., SEQ ID NO: 19) can be administered to deliver about 0.1 ng to 100 mg per kilogram (e.g., 1 ng to 100 mg, 100 ng to 100 mg, 1 μg to 100 mg, 100 μg to 100 mg, 1 mg to 100 mg, 25 mg to 100 mg, 50 mg per kilogram). Dosage ranges from 1 mg to 100 mg, 1 ng to 80 mg, 100 ng to 80 mg, 1 μg to 80 mg, 100 μg to 80 mg, 1 mg to 80 mg, 25 mg to 80 mg, 50 mg to 80 mg, 1 ng to 50 mg, 100 ng to 50 mg, 1 μg to 50 mg, 100 μg to 50 mg, 1 mg to 50 mg, 25 mg to 50 mg, 50 mg to 50 mg, 1 ng to 50 mg, 100 ng to 50 mg, 1 μg to 50 mg, 100 μg to 50 mg, 1 mg to 50 mg, or 25 mg to 50 mg. Dosage is described in more detail elsewhere in this document.

In various embodiments, pharmaceutical compositions that can be used in the methods of the present invention can be administered, for example, systemically, parenterally, subcutaneously, intraperitoneally, or locally, such as in oral formulations, inhaled formulations including solids or aerosols, and via local or other similar formulations, or intraocularly. In addition to suitable therapeutic compositions, such pharmaceutical compositions may contain pharmaceutically acceptable carriers and other ingredients known to enhance and facilitate drug administration. Other possible formulations, such as nanoparticles, liposomes, other formulations containing active ingredients, and suitable modulators for immunologically based systemic administration, can also be used according to the methods of the present invention.

The carrier can carry the subject agent (e.g., an IL-18 variant peptide) in a variety of ways, including direct or covalent binding via a linker group and non-covalent association. Suitable covalent carriers include proteins such as albumin, peptides, and polysaccharides such as glycosaminoglycans, each having multiple sites for linking the moiety. The carrier can also carry the IL-18 peptide (e.g., stabilized IL-18, such as stabilized DRIL-18 variants or stabilized D2D IL-18 variants, etc.) via non-covalent association (e.g., non-covalent bonding or encapsulation) (e.g., SEQ ID NO: 19). For the purposes of this invention, the carrier can be soluble or insoluble.

Acceptable carriers, excipients, or stabilizers are non-toxic to the recipient at the doses and concentrations used and include: buffers such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexamethyl diammonium chloride; benzalkonium chloride, benzyl chloride; phenol, butanol, or benzyl alcohol; alkyl esters of p-hydroxybenzoate, such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10). (10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., zinc-protein complexes); and/or nonionic surfactants, such as TWEEN ^™ , PLURONICS ^™ , or polyethylene glycol (PEG). Formulations intended for in vivo administration must be sterile. This is readily achieved through filtration using a sterile filter membrane.

Active ingredients can also be encapsulated in microcapsules prepared, for example, by coagulation techniques or by interfacial polymerization, such as hydroxymethyl cellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or in crude emulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th edition, ed. Osol, A. (1980).

The compositions can be formulated as injectable drugs, in the form of liquid solutions or suspensions; they can also be formulated in solid forms suitable for dissolving or suspending in a liquid medium prior to injection. As discussed above, formulations can also be emulsified or encapsulated in liposomes or microparticles, such as polylactide, polyglycolic acid, or copolymers, to achieve enhanced adjuvant action. (Langer, Science 249:1527, 1990 and Hanes, Advanced Drug Delivery Reviews 28:97119, 1997). The agents of the present invention can be administered in reservoir injection or implant formulations that allow for sustained or pulsatile release of the active ingredient. Pharmaceutical compositions are generally formulated as sterile, substantially isotonic, and fully compliant with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration.

As used herein, the term “physiologically acceptable” ester or salt means an ester or salt form of an active ingredient that is compatible with any other component of the pharmaceutical composition and is harmless to the object to which the composition is to be administered.

The formulations of the pharmaceutical compositions described herein can be prepared by any method known in or subsequently developed in the field of pharmacology. Generally, such preparation methods include the following steps: associating the active ingredient with a carrier or one or more other excipients, and then, if necessary or desired, shaping or packaging the product into the desired single-dose or multi-dose units.

While the descriptions of pharmaceutical compositions provided herein primarily pertain to those intended for ethical human administration, those skilled in the art will understand that such compositions are generally suitable for administration to all species of animals. It is well known that pharmaceutical compositions intended for human administration can be modified to make them suitable for administration to a wide variety of animals, and such modifications can be designed and performed by a general veterinary pharmacologist simply through routine experiments (if necessary).

Pharmaceutical compositions that can be used in the methods of the present invention can be prepared, packaged, or sold in the form of formulations suitable for oral, rectal, vaginal, parenteral, topical, pulmonary, intranasal, buccal, intravenous, transdermal, intralesional, subcutaneous, intramuscular, ocular, intrathecal, and other known routes of administration. Other envisioned formulations include planned nanoparticles, liposome formulations, other formulations containing active ingredients, and immunologically based formulations.

The pharmaceutical compositions of the present invention may be prepared, packaged, or sold in bulk, in single unit doses, or in multiple single unit doses. As used herein, a “unit dose” is a discrete amount of a pharmaceutical composition containing a predetermined amount of active ingredient. The amount of active ingredient is generally equal to the dose of active ingredient to be administered to a subject or a suitable fraction of such dose, such as half or one-third of such dose.

The relative amounts of the active ingredient, pharmaceutically acceptable carrier, and any additional ingredients in the pharmaceutical compositions of the present invention will vary depending on the identity, body size, and/or condition of the subject being treated and, more particularly, on the route of administration of the composition. By way of example, the composition may contain 0.1% to 100% (w/w) of the active ingredient.

In addition to the active ingredient, the pharmaceutical compositions of the present invention may also contain one or more additional pharmaceutical active agents.

Controlled-release or sustained-release formulations of the pharmaceutical compositions of the present invention can be prepared using conventional techniques.

Formulations of the pharmaceutical compositions of the present invention suitable for oral administration may be prepared, packaged, or sold in the form of discrete solid dosage units, including but not limited to tablets, hard or soft capsules, flat capsules, lozenges, or rhomboid lozenges, each containing a predetermined amount of the active ingredient. Other formulations suitable for oral administration include, but are not limited to, powdered or granular formulations, aqueous or oily suspensions, aqueous or oily solutions, or emulsions.

Pharmaceutically acceptable excipients for preparing pharmaceutical compositions include, but are not limited to, inert diluents, granulators and disintegrants, binders, and lubricants. Known dispersants include, but are not limited to, potato starch and sodium glycolate starch. Known surfactants include, but are not limited to, sodium lauryl sulfate. Known diluents include, but are not limited to, calcium carbonate, sodium carbonate, lactose, microcrystalline cellulose, calcium phosphate, calcium hydrogen phosphate, and sodium phosphate. Known granulators and disintegrants include, but are not limited to, corn starch and alginate. Known binders include, but are not limited to, gelatin, gum arabic, pre-gelled corn starch, polyvinylpyrrolidone, and hydroxypropyl methylcellulose. Known lubricants include, but are not limited to, magnesium stearate, stearic acid, silica, and talc.

Liquid formulations of the pharmaceutical compositions of the present invention may be prepared, packaged and sold in liquid form or as dry products, intended for reconstitution with water or another suitable medium prior to use.

Liquid suspensions can be prepared using conventional methods to suspend the active ingredient in an aqueous or oily medium. Aqueous media include, for example, water and isotonic saline. Oily media include, for example, almond oil, oily esters, ethanol, vegetable oils (such as peanut oil, olive oil, sesame oil, or coconut oil), graded vegetable oils, and mineral oils (such as liquid paraffin). Liquid suspensions may also contain one or more additional ingredients, including but not limited to suspending agents, dispersants or wetting agents, emulsifiers, sedatives, preservatives, buffers, salts, flavoring agents, coloring agents, and sweeteners. Oily suspensions may also contain thickeners.

Known suspending agents include, but are not limited to, sorbitol syrup, hydrogenated edible fats, sodium alginate, polyvinylpyrrolidone, gum arabic, gum arabic, and cellulose derivatives such as sodium carboxymethyl cellulose, methyl cellulose, and 15-hydroxypropyl methyl cellulose. Known dispersing or wetting agents include, but are not limited to, naturally occurring phospholipids (such as lecithin), oxidized alkenes with fatty acids, with long-chain fatty alcohols, with esters derived from fatty acids and hexitols, or with esters derived from fatty acids and hexitol anhydrides (e.g., polyoxyethylene stearate, heptadecanethoxylated cetyl alcohol, polyoxyethylene sorbitan monooleate, and polyoxyethylene dehydrated sorbitan monooleate, respectively). Known emulsifiers include, but are not limited to, lecithin and gum arabic. Known preservatives include, but are not limited to, methyl, ethyl, or n-propyl-p-hydroxybenzoates, ascorbic acid, and sorbic acid. Known sweeteners include, for example, glycerin, propylene glycol, sorbitol, sucrose, and saccharin. Known thickeners for oily suspensions include, for example, beeswax, paraffin wax, and cetyl alcohol.

Liquid solutions of the active ingredient in aqueous or oily solvents can be prepared in substantially the same manner as liquid suspensions, the main difference being that the active ingredient is dissolved rather than suspended in the solvent. Liquid solutions of the pharmaceutical compositions of the present invention may contain every component described with respect to liquid suspensions, and it should be understood that suspending agents do not necessarily contribute to the dissolution of the active ingredient in the solvent. Aqueous solvents include, for example, water and isotonic saline. Oily solvents include, for example, almond oil, oily esters, ethanol, vegetable oils (such as peanut oil, olive oil, sesame oil, or coconut oil), graded vegetable oils, and mineral oils (such as liquid paraffin).

Powder and granule formulations of the pharmaceutical preparations of the present invention can be prepared using known methods. Such formulations can be applied directly to a subject, for example, to form tablets, fill capsules, or to prepare aqueous or oily suspensions or solutions by adding an aqueous or oily medium thereto. Each of these formulations may also contain one or more of dispersants or wetting agents, suspending agents, and preservatives. Additional excipients, such as fillers and sweeteners, flavoring agents, or coloring agents, may also be included in these formulations.

The pharmaceutical compositions of the present invention can also be prepared, packaged, or sold as oil-in-water emulsions or water-in-oil emulsions. The oil phase can be a vegetable oil (such as olive oil or peanut oil), a mineral oil (such as liquid paraffin), or a combination thereof. Such compositions may also contain one or more emulsifiers, such as: naturally occurring gums, such as gum arabic or tragacanth; naturally occurring phospholipids, such as soybean or lecithin phospholipids; esters or metaesters derived from combinations of fatty acids and hexitin anhydrides, such as sorbitan monooleate; and condensation products of such metaesters with ethylene oxide, such as polyoxyethylene sorbitan monooleate. These emulsions may also contain additional ingredients, including, for example, sweeteners or flavorings.

Methods of impregnating or coating materials with chemical compositions are known in the art, and these methods include, but are not limited to, methods of depositing or incorporating chemical compositions onto a surface, methods of incorporating chemical compositions into the structure of a material (i.e., such as a biodegradable material) during the synthesis of the material, and methods of absorbing an aqueous or oily solution or suspension into an absorbent material, followed by drying or not drying.

As used herein, “parenteral administration” of a pharmaceutical composition includes any route of administration characterized by physically disrupting the tissue of the object and administering the pharmaceutical composition through fissures in the tissue. Therefore, parenteral administration includes, but is not limited to, administering the pharmaceutical composition by: injecting the composition; applying the composition through a surgical incision; applying the composition through a tissue-penetrating non-surgical wound; and so on. Specifically, parenteral administration is contemplated as including, but not limited to, injections via the skin, subcutaneous tissue, intraperitoneum, intravenous vein, intramuscular injection, intracisional injection, and renal dialysis infusion techniques. In some cases, the subject IL-18 polypeptide (e.g., stabilized IL-18, such as stabilized DR IL-18 variants or stabilized D2D IL-18 variants, etc.) (e.g., SEQ ID NO:19) is administered subcutaneously.

Pharmaceutical compositions suitable for parenteral administration contain an active ingredient bound to a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus or continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage forms, such as in ampoules or in multi-dose containers containing preservatives. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous media, pastes, and implantable sustained-release or biodegradable formulations. Such formulations may also contain one or more additional ingredients, including but not limited to suspending agents, stabilizers, or dispersants. In some embodiments of formulations for parenteral administration, the active ingredient is provided in a dry (i.e., powder or granule) form for reconstitution with a suitable media (e.g., sterile pyrogen-free water), after which the reconstituted composition is administered parenterally.

Pharmaceutical compositions can be prepared, packaged, or marketed as sterile, injectable aqueous or oily suspensions or solutions. These suspensions or solutions can be formulated according to known techniques and may contain additional ingredients besides the active ingredient, such as dispersants, wetting agents, or suspending agents as described herein. Such sterile injectable formulations can be prepared using non-toxic, parenterally acceptable diluents or solvents, such as water or 1,3-butanediol. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils such as synthetic monoglycerides or diglycerides. Other available parenterally applicable formulations include those containing the active ingredient in microcrystalline form, as liposomes, or as biodegradable polymer systems. Compositions for sustained release or implantation may contain pharmaceutically acceptable polymeric or hydrophobic materials, such as emulsions, ion exchange resins, slightly soluble polymers, or slightly soluble salts.

Formulations suitable for topical application include, but are not limited to, liquid or semi-liquid formulations such as liniments, lotions, oil-in-water or water-in-oil emulsions such as creams, ointments, pastes, solutions, or suspensions. Topically applicable formulations may, for example, contain from about 1% to about 10% (w/w) of the active ingredient, although the concentration of the active ingredient may be as high as the solubility limit of the active ingredient in the solvent. Formulations for topical application may further contain one or more additional ingredients described herein.

The pharmaceutical compositions of the present invention can be prepared, packaged, or marketed in a formulation suitable for oral administration to the lungs. Such formulations may comprise dried particles containing the active ingredient and having a diameter ranging from about 0.5 to about 7 nanometers, preferably from about 1 to about 6 nanometers. Such compositions are conveniently available as a dry powder for application, which is administered using a device including a dry powder reservoir that directs the flow of propellant to its dispersed powder, or using a self-propelled solvent/powder dispersion container, such as a device containing the active ingredient dissolved or suspended in a low-boiling-point propellant in a sealed container. Preferably, such powders comprise particles, wherein at least 98% by weight of the particles have a diameter greater than 0.5 nanometers and at least 95% by weight of the particles have a diameter less than 7 nanometers. More preferably, at least 95% by weight of the particles have a diameter greater than 1 nanometer and at least 90% by weight of the particles have a diameter less than 6 nanometers. The dry powder composition preferably comprises a solid fine powder diluent such as sugar and is conveniently provided in unit dosage forms.

Low-boiling-point propellants typically include liquid propellants with a boiling point below 65°F at atmospheric pressure. Typically, the propellant constitutes 50 to 99.9% (w/w) of the composition, and the active ingredient constitutes 0.1 to 20% (w/w) of the composition. The propellant may also contain additional components such as liquid nonionic or solid anionic surfactants or solid diluents (preferably with particle sizes on the same order of magnitude as the particles containing the active ingredient).

The pharmaceutical compositions of the present invention formulated for pulmonary delivery may also provide the active ingredient in the form of droplets as a solution or suspension. Such formulations may be prepared, packaged, or marketed as optionally sterile, aqueous or diluted alcoholic solutions or suspensions containing the active ingredient, and are conveniently applicable using any spray or nebulizer. Such formulations may also contain one or more additional ingredients, including but not limited to flavoring agents such as sodium saccharin, volatile oils, buffers, surfactants, or preservatives such as methylparaben. The average diameter of the droplets provided via this route of administration is preferably in the range of about 0.1 to about 200 nanometers. The formulations described herein, which are suitable for pulmonary delivery, can also be used for intranasal delivery of the pharmaceutical compositions of the present invention. Another formulation suitable for intranasal administration is a coarse powder containing the active ingredient and having an average particle size of about 0.2 to 500 micrometers.

Such formulations are administered by nasal inhalation, i.e., by rapid inhalation through the nasal passages from a powder container placed near the nose. Formulations suitable for nasal administration may, for example, contain as little as 0.1% (w/w) and as much as 100% (w/w) of the active ingredient, and may also contain one or more additional ingredients described herein.

The pharmaceutical compositions of the present invention can be prepared, packaged, or marketed in formulations suitable for buccal application. Such formulations may be available, for example, as tablets or rhomboid tablets prepared using conventional methods, and may contain, for example, 0.1 to 20% (w/w) of the active ingredient, the remainder comprising an orally soluble or biodegradable composition and optionally one or more additional ingredients described herein. Alternatively, formulations suitable for buccal application may comprise powders or aerosolized or nebulized solutions or suspensions containing the active ingredient. Such powdered, aerosolized, and/or aerosolized formulations preferably have an average particle or droplet size in the range of about 0.1 nanometers to about 200 nanometers when dispersed, and may also contain one or more additional ingredients described herein.

The pharmaceutical compositions of the present invention can be prepared, packaged, or marketed in formulations suitable for ophthalmic application. Such formulations may be, for example, in the form of eye drops, comprising, for example, a 0.1-1.0% (w/w) solution or suspension of the active ingredient in an aqueous or oily liquid carrier. Such drops may also contain buffers, salts, or one or more other additional ingredients described herein. Other ophthalmologically applicable formulations available include those containing the active ingredient in microcrystalline or liposomal form.

As used herein, “additional ingredients” include, but are not limited to, one or more of the following: excipients; surfactants; dispersants; inert diluents; granulators and disintegrants; binders; lubricants; sweeteners; flavorings; colorants; preservatives; physiologically degradable compositions such as gelatin; aqueous media and solvents; oily media and solvents; suspending agents; dispersants or wetting agents; emulsifiers; sedatives; buffers; salts; thickeners; fillers; emulsifiers; antioxidants; antibiotics; antifungals; stabilizers; and pharmaceutically acceptable polymers or hydrophobic materials. Other “additional ingredients” that may be included in the pharmaceutical compositions of the present invention are known in the art and described, for example, in Genaro, ed., 1985, Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., which are incorporated herein by reference.

Exemplary formulation

In some embodiments, the formulation comprises: an active ingredient containing an IL-18 peptide; one or more pH-controlling buffers; one or more stabilizers; one or more solvents; a surfactant; an antioxidant; and a stabilizer. In some embodiments, the formulation of the IL-18 peptide comprises one or more of the following: L-histidine (His), L-histidine hydrochloride (His-HCl), sucrose, polysorbate 80, L-methionine, and disodium EDTA.

An example of a suitable formulation of an experimentally obtained IL-18 peptide (e.g., stabilized IL-18, such as a stabilized DR IL-18 variant or a stabilized D2D IL-18 variant, etc.) (e.g., SEQ ID NO: 19) comprises: (1) an IL-18 peptide (e.g., wild-type IL-18, stabilized IL-18, DR IL-18, D2D IL-18, stabilized DR IL-18, stabilized D2D IL-18, etc.) (e.g., SEQ ID NO: 19); (2) maintaining a pH between 6.2 and 6.8 (e.g., In some cases, 6.5) buffers (e.g., His/His-HCl, 8-12 mM, e.g., 10 mM); (3) sugars (e.g., sucrose, e.g., 6-10%; in some cases, 8%); (4) chelating agents (e.g., EDTA, e.g., 0.05-1.5 mM, e.g., 0.1 mM in some cases); (5) antioxidants (e.g., L-methionine, e.g., 4-6 mM, e.g., 5 mM in some cases); and (6) agents that prevent protein adsorption (e.g., PS80, e.g., 0.01-0.03% w/v, e.g., 0.02%).

Therefore, in some cases, the formulation contains: (1) an IL-18 polypeptide (e.g., wild-type IL-18, stabilized IL-18, DR IL-18, D2D IL-18, stabilized DR IL-18, stabilized D2D IL-18, etc.) (e.g., SEQ ID NO: 19); (2) about 10 mM His/His-HCl; (3) about 8% sucrose; (4) about 0.1 mM EDTA; (5) about 5 mM L-methionine; and (6) about 0.02% (w/v) PS80.

In some of the embodiments described above (e.g., when the formulation is administered subcutaneously), the IL-18 peptide (e.g., wild-type IL-18, stabilized IL-18, DR IL-18, D2D IL-18, stabilized DR IL-18, stabilized D2D IL-18, etc.) (e.g., SEQ ID NO: 19) is administered at a concentration in the range of 10-100 mg/ml (e.g., 10-90, 10-75, 10-60 mg/ml). It is present in concentrations of 10-50, 10-40, 10-30, 25-100, 25-90, 25-75, 25-60, 25-50, 25-40, 25-30, 30-100, 30-90, 30-75, 30-60, 30-50, 30-40, 40-100, 40-90, 40-75, 40-60, 40-50, 50-100, 50-90, 50-75 or 50-60 mg/ml. In some of the embodiments described above (e.g., when the formulation is administered subcutaneously), the IL-18 peptide (e.g., wild-type IL-18, stabilized IL-18, DR IL-18, D2D IL-18, stabilized DR IL-18, stabilized D2D IL-18, etc.) (e.g., SEQ ID NO: 19) will be present at a concentration in the range of 50-60 mg/ml. In some of the embodiments described above (e.g., when the formulation is administered subcutaneously), the IL-18 peptide (e.g., wild-type IL-18, stabilized IL-18, DR IL-18, D2D IL-18, stabilized DR IL-18, stabilized D2D IL-18, etc.) (e.g., SEQ ID NO: 19) will be present at a concentration in the range of 20-30 mg/ml.

In some embodiments, the pharmaceutical product is formulated in batches. In some embodiments, each batch is 1.5 L. In other embodiments, each batch is 10.5 L. Each batch may contain, for example, the IL-18 peptide of SEQ ID NO:19, L-histidine, L-histidine hydrochloride, sucrose, polysorbate 80, L-methionine, disodium EDTA, and water for injection. In some embodiments, a batch contains: 40 to 50 g of SEQ ID NO:19; 1.00 to 2.00 g of L-histidine; 0.25 to 1.00 g of L-histidine hydrochloride; 100.00 to 150.00 g of sucrose; 0.01 to 0.60 g of polysorbate 80; 1 to 1.5 g of L-methionine; 0.005 to 0.1 g of disodium EDTA; and enough water to fill approximately 1.5 L. In some embodiments, the batch contains: about 45g SEQ ID NO:19; about 1.91g L-histidine; about 0.57g L-histidine hydrochloride; about 120.0g sucrose; about 0.30g polysorbate 80; about 1.13g L-methionine; about 0.06g disodium EDTA; and enough water to fill about 1.5L. In some embodiments, the 10.5L batch contains: 300 to 350g SEQ ID NO:19; 10.00 to 15.00g L-histidine; 3.50 to 4.50g L-histidine hydrochloride; 800.00 to 900.00g sucrose; 1.5 to 2.5g polysorbate 80; 7.00 to 8.00g L-methionine; 0.1 to 0.6g disodium EDTA; and enough water to make up about 10.5L. In some embodiments, the 10.5L batch contains: about 315g SEQ ID NO:19; about 13.34g L-histidine; about 3.99g L-histidine hydrochloride; about 840.0g sucrose; about 2.1g polysorbate 80; about 7.88g L-methionine; about 0.42g EDTA disodium salt; and enough water to fill about 1.5L.

The formulations disclosed herein can be maintained at a pH that, for example, helps stabilize or maintain the activity of the compounds or peptides disclosed herein. The pH of the disclosed formulations can be, for example, in the range of about 3 to about 12. The pH of the composition can be, for example, about 3 to about 4, about 4 to about 5, about 5 to about 6, about 6 to about 7, about 7 to about 8, about 8 to about 9, about 9 to about 10, about 10 to about 11, or about 11 to about 12 pH units. The pH of the composition can be, for example, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, or about 12 pH units. The pH of the composition can be, for example, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or at most 12 pH units.

The pH of the formulations disclosed herein can be from about 5.5 to about 8. For example, the pH of the formulations disclosed herein can be about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, or about 8.0. In some embodiments, the pH is 6.5 ± 0.6, 6.5 ± 0.5, 6.5 ± 0.4, 6.5 ± 0.3, 6.5 ± 0.2, 6.5 ± 0.1, about 6.5, or 6.5. In some embodiments, the pH is 6.2±0.6, 6.2±0.5, 6.2±0.4, 6.2±0.3, 6.2±0.2, 6.2±0.1, about 6.2, or 6.2. In some embodiments, the pH is 6.8±0.6, 6.8±0.5, 6.8±0.4, 6.8±0.3, 6.8±0.2, 6.8±0.1, about 6.8, or 6.8. In some embodiments, the pH is 7.4±0.6, 7.4±0.5, 7.4±0.4, 7.4±0.3, 7.4±0.2, 7.4±0.1, about 7.4, or 7.4. In some embodiments, the pH is about 6.2 to about 6.8. If the pH is outside the range required by the preparer, it can be adjusted by using sufficient pharmaceutically acceptable acids and bases.

In some embodiments, the formulations disclosed herein may contain a buffer. In some embodiments, the buffer is used to maintain a stable pH and to help stabilize the compounds or peptides disclosed herein. In some embodiments, the buffer system contains at least one buffer whose buffering range completely or partially overlaps with the pH range of 5.5–7.4. In some embodiments, the buffer has a pKa of about 6.5 ± 0.5 or 6.2 ± 0.5.

Dosage

Typically, the amount of IL-18 peptide (e.g., wild-type IL-18, stable IL-18 variants, DR IL-18 variants, D2D IL-18 variants, stable DR IL-18 variants, stable D2D IL-18 variants, etc.) (e.g., SEQ ID NO: 19) that can be administered to animals (preferably humans) ranges from 0.001 mg to about 10 mg/kg (mpk) of animal body weight. In some embodiments, the IL-18 peptide (e.g., wild-type IL-18, stable IL-18 variants, DR IL-18 variants, D2D IL-18 variants, stable DR IL-18 variants, stable D2D IL-18 variants, etc.) (e.g., SEQ ID NO: 19) is administered at a dose in the range of 0.01-4 mpk (e.g., 0.01-3.5, 0.01-3.3). 0.01-3, 0.01-1.5, 0.01-2, 0.01-1.5, 0.01-1, 0.01-0.5, 0.01-0.35, 0.025-4, 0.025-3.5, 0.025-3.3, 0.025-3, 0.025-1.5, 0.025-2, 0.025-1.5, 0.025-1, 0.0250.5, 0.025-0.35, 0 0.05-4, 0.05-3.5, 0.05-3.3, 0.05-3, 0.05-1.5, 0.05-2, 0.05-1.5, 0.05-1, 0.05-0.5, 0.05-0.35, 0.1-4, 0.1-3.5, 0.1-3.3, 0.1-3, 0.1-1.5, 0.1-2, 0.1-1.5, 0.1-1, 0.1-0.5, 0.1-0 0.35, 0.25-4, 0.25-3.5, 0.25-3.3, 0.25-3, 0.25-1.5, 0.25-2, 0.25-1.5, 0.25-1, 0.25-0.5, 0.25-0.35, 0.5-4, 0.5-3.5, 0.5-3.3, 0.5-3, 0.5-1.5, 0.5-2, 0.5-1.5 or 0.5-1 mpk) are applied to individuals. In some embodiments, the IL-18 peptide (e.g., wild-type IL-18, stabilized IL-18 variants, DR IL-18 variants, D2D IL-18 variants, stabilized DR IL-18 variants, stabilized D2D IL-18 variants, etc.) (e.g., SEQ ID NO: 19) is administered at doses in the range of 0.05-3.5 mpk (e.g., 0.05-3.3, 0.05-3, 0.05-1.5, 0.05-2, 0.05-1.5, 0.05-1, 0.05-0.5, 0.05-0.35). 0.1-3.5, 0.1-3.3, 0.1-3, 0.1-1.5, 0.1-2, 0.1-1.5, 0.1-1, 0.1-0.5, 0.1-0.35, 0.25-3.5, 0.25-3.3, 0.25-3, 0.25-1.5, 0.25-2, 0.25-1.5, 0.25-1, 0.25-0.5, 0.25-0.35, 0.5-3.5, 0.5-3.3, 0.5-3, 0.5-1.5, 0.5-2, 0.5-1.5 or 0.5-1mpk) are applied to individuals. In some cases, the compound can be administered to animals multiple times a day, or it can be administered at a lower frequency, such as once a day, once a week, once every two weeks, once a month, or even less frequently, such as once every few months or even once a year or less.

However, as explained above, in some cases it is delivered once a week or less.

Application method

Application frequency

Those skilled in the art will understand that the IL-18 proteins discussed herein can be administered for a short period (e.g., over a short time period) or a long period (e.g., over a long time period, such as several months or a year or longer). Those skilled in the art will understand that they can be administered alone or in any combination with other agents. Furthermore, the IL-18 proteins described herein can be administered alone or in any combination in a temporal sense, as they can be administered simultaneously, and/or before, and/or after each other. Based on the disclosure provided herein, those skilled in the art will understand that IL-18 protein compositions can be used to treat diseases or conditions in patients of need, and that such proteins can be used alone or in any combination with another agent that affects treatment outcomes.

As surprisingly demonstrated in the following working examples, in some cases, administration of IL-18 peptides (e.g., wild-type IL-18, stable IL-18 variants, DRIL-18 variants, D2D IL-18 variants, stable DR IL-18 variants, stable D2D IL-18 variants, etc.) (e.g., SEQ ID NO: 19) can be harmful (e.g., toxic, potentially causing anemia) if administered too frequently, e.g., more than once a week. For example, the following working examples show that administration of DR-18 twice a week in primates (monkeys) resulted in a dose-dependent reduction in hemoglobin levels compared to saline-treated monkeys. In contrast, weekly administration did not result in a decrease in hemoglobin levels compared to saline-treated monkeys.

Therefore, in some embodiments, the IL-18 peptide (e.g., wild-type IL-18, stable IL-18 variants, DRIL-18 variants, D2D IL-18 variants, stable DR IL-18 variants, stable D2D IL-18 variants, etc.) (e.g., SEQ ID NO: 19) is administered to the individual once a week (one dose per week) or less (e.g., once every two weeks or less, once a month or less). In some cases, the IL-18 peptide (e.g., wild-type IL-18, stable IL-18 variants, DR IL-18 variants, D2D IL-18 variants, stable DR IL-18 variants, stable D2D IL-18 variants, etc.) is administered to the individual at a frequency ranging from once a week to once every 6 months (e.g., once a week to once every 4 months, once a week to once every 2 months, or once a week to once a month). In some cases, IL-18 peptides (e.g., wild-type IL-18, stable IL-18 variants, DR IL-18 variants, D2D IL-18 variants, stable DR IL-18 variants, stable D2D IL-18 variants, etc.) (e.g., SEQ ID NO:19) are administered to individuals at a frequency ranging from once a week to once a month.

In some cases, the IL-18 peptide (e.g., wild-type IL-18, stable IL-18 variants, DR IL-18 variants, D2D IL-18 variants, stable DR IL-18 variants, stable D2D IL-18 variants, etc.) (e.g., SEQ ID NO: 19) is administered to the individual approximately once a week. In other cases, the IL-18 peptide (e.g., wild-type IL-18, stable IL-18 variants, DR IL-18 variants, D2D IL-18 variants, stable DR IL-18 variants, stable D2D IL-18 variants, etc.) (e.g., SEQ ID NO: 19) is administered to the individual approximately once every two weeks. In some cases, the IL-18 peptide (e.g., wild-type IL-18, stable IL-18 variants, DR IL-18 variants, D2D IL-18 variants, stable DR IL-18 variants, stable D2D IL-18 variants, etc.) (e.g., SEQ ID NO: 19) is administered to the individual approximately once a month. In other cases, the IL-18 peptide (e.g., wild-type IL-18, stable IL-18 variants, DR IL-18 variants, D2D IL-18 variants, stable DR IL-18 variants, stable D2D IL-18 variants, etc.) is administered to the individual no more than once a week.

disease

In some embodiments, the methods disclosed herein can be used to treat or prevent tumors or cancerous tumors that have lost surface expression of MHC class I; such as tumors that have lost B2m (MHC locus) or tumors that have mutations in other members of the antigen-presenting and/or antigen-loading complex (such as tapasin).

Metabolic diseases and conditions include a variety of metabolic and endocrine-related diseases and conditions. The following are non-limiting examples of metabolic and endocrine-related diseases and conditions that can be treated or prevented by the methods and compositions of the present invention: obesity, diabetes, prediabetes, type II diabetes, mature onset diabetes of the young (MODY), hyperglycemia, metabolic syndrome, dyslipidemia, hypertriglyceridemia, and hypercholesterolemia.

Non-limiting examples of other diseases and conditions that can be treated or prevented using the compositions and methods of the present invention include viral infections, bacterial infections, parasitic infections, and low immune activity. In some embodiments, the viral infection is at least one of poxvirus, smallpox virus, molluscum contagiosum, HPV infection, and warts caused by a virus. In some embodiments, the infection is a systemic infection. In some embodiments, the viral infection is vaccinia virus infection. In some embodiments, the viral infection is a systemic vaccinia virus infection. In some embodiments, the bacterial infection is sepsis. In some embodiments, low immune activity is neutropenia, for example, which may occur in the presence of chemotherapy.

Non-limiting examples of other diseases and conditions that can be treated or prevented using the compositions and methods of the present invention include macular degeneration. For example, in some cases, the disease or condition is wet macular degeneration, and in some cases, the disease or condition is wet age-related macular degeneration. In some such cases, IL-18 variant peptides (e.g., stabilized IL-18) can be used as anti-angiogenic agents. For example, in some cases, the subject IL-18 variant peptide (e.g., stabilized IL-18) can reduce choroidal neovascularization.

In some embodiments, an IL-18 peptide (e.g., a stabilized IL-18, such as a stabilized DR IL-18 variant or a stabilized D2D IL-18 variant, etc.) (e.g., SEQ ID NO: 19) can be used to treat or prevent a disease or condition. In various embodiments, the disease or condition is cancer or a metabolic disease or condition, including obesity and diabetes (e.g., the subject method can result in a reduction of body fat). Therefore, in some embodiments, the composition comprises at least one IL-18 peptide (e.g., a stabilized IL-18, such as a stabilized DR IL-18 variant or a stabilized D2D IL-18 variant, etc.) (e.g., SEQ ID NO: 19). In other embodiments, a method of administering at least one IL-18 peptide (e.g., a stabilized IL-18, such as a stabilized DR IL-18 variant or a stabilized D2D IL-18 variant, etc.) (e.g., SEQ ID NO: 19) is performed to treat a disease or condition, such as, but not limited to, cancer or a metabolic disease or condition.

The following are non-limiting examples of cancers that can be treated or prevented by the methods and compositions disclosed herein: acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, appendix cancer, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain and spinal cord tumors, brainstem glioma, and brain tumors. N-tumor, breast cancer, bronchial tumors, Burkitt lymphoma, carcinoid tumor, central nervous system atypical teratoid/rhabdoid tumor, central nervous system embryonal tumors, central nervous system lymphoma, cerebellar astrocytoma, astrocytoma/malignant glioma (cer The following cancers are listed: cerebral astrocytoma/malignant glioma, cervical cancer, childhood visual pathway tumors, chordoma, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disorders, colon cancer, and colorectal cancer. Rectal cancer, craniopharyngioma, cutaneous cancer, cutaneous T-cell lymphoma, endometrial cancer, ependymoblastoma, ependymoma, esophageal cancer, ewing family of tumors, extracranial cancer, extragonadal germ cell tumor, extrahepatic bile duct cancer Cancer, extrahepatic cancer, eye cancer, fungal infections, gallbladder cancer, gastric (stomach) cancer, gastrointestinal cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), germ cell tumor, gestational cancer, gestational trophoblastic tumor. (e.g., umor), glioblastoma, glioma, hairy cell leukemia, head and neck cancer, hepatocellular carcinoma, histiocytosis, Hodgkin lymphoma, hypopharyngeal cancer, hypothalamic and visual pathway glioma, hypothalamic tumor, intraocular cancer. Intraocular melanoma, islet cell tumors, Kaposi's sarcoma, kidney (renal cell) cancer, Langerhans cell cancer, Langerhans cell histiocytosis, laryngeal cancer, leukemia, lip and oral cavity cancer, liver cancer, lung cancer, lymphoma, macroglobulinemia. Globulinemia, malignant fibrous histiocytoma of bone and osteosarcoma, medulloblastoma, medulloepithelioma, melanoma, Merkel cell carcinoma, mesothelioma, metastatic squamous neck cancer with occult primary, mouth cancer, multiple endocrine adenomas. Neoplasia syndrome, multiple myeloma, mycosis, myelodysplastic syndromes, myelodysplastic/myeloproliferative diseases, myelogenous leukemia, myeloid leukemia, myeloma, myeloproliferative disorders, nasal cavity and paranasal sinus cancer, nasopharyngeal carcinoma. Neuroblastoma, non-Hodgkin's lymphoma, non-small cell lung cancer, oral cancer, oral cavity cancer, oropharyngeal cancer, osteosarcoma and malignant fibrous histiocytoma, osteosarcoma and malignant fibrous histiocytoma of bone, ovarian cancer Ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, ovarian low-malignant potential tumor, pancreatic cancer, papillomatosis, paraganglioma, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, moderately differentiated pineal parenchymal tumor. Pine blastoma and supratentorial primitive neuroectodermal tumors, pituitary tumor, plasma cell neoplasm, plasma cell neoplasm/multiple myeloma, pleuropulmonary blastoma, primary central nervous system cancer, and primary tumors of the central nervous system. Primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell (kidney) cancer, renal pelvis and ureter cancer, respiratory tract carcinoma involving the nut gene on chromosome 15, retinoblastoma, rhabdomyosarcoma, and salivary gland cancer. This list includes various cancers such as gland cancer, sarcoma, Sezary syndrome, skin cancer (melanoma), skin cancer (non-melanoma), skin cancer, small cell lung cancer, small intestinal cancer, soft tissue cancer, soft tissue sarcoma, squamous cell carcinoma, squamous neck cancer, and stomach cancer. (Ach (gastric) cancer), supratentorial primitive neuroectodermal tumors, supratentorial primitive neuroectodermal tumors and pinoblastoma, T-cell lymphoma, testicular cancer, throat cancer, thymoma and thymic carcinoma, thyroid cancer, transitional cell carcinoma Transitional cell carcinoma of the renal pelvis and ureter, trophoblastic tumor, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, visual pathway and hypothalamic glioma, vulvar cancer, Waldenstrom macroglobulinemia, and Wilms' tumor.

In some non-limiting instances, cancer is defined not by the histological tissue of origin, but by its molecular characteristics. For example, tumors with a high tumor mutational burden (TMB), tumors with microsatellite instability (MSI), tumors lacking or reducing surface expression of MHC class I (e.g., due to the absence of β2-microglobulin or Tapasin), or tumors expressing high levels of IL-18BP. In some cases, those molecular characteristics are determined by next-generation sequencing (e.g., using TMB/MSI). In some cases, those molecular characteristics are determined by immunohistochemistry, immunofluorescence, or flow cytometry. Therefore, non-limiting examples of cancers that can be treated or prevented by the methods and compositions of this disclosure include solid tumors, liquid cancers, hematologic malignancies, teratomas, sarcomas, and carcinomas.

In some embodiments, the compositions and methods of this disclosure can be used to treat tumors or cancers resistant to immune checkpoint inhibitors (ICIs). Examples of immune checkpoint inhibitors include, but are not limited to: anti-PD1 agents such as anti-PD1 antibodies (e.g., nivolumab), anti-PD-L1 agents such as anti-PD-L1 antibodies, anti-CTLA4 (e.g., ipilimumab), anti-TIM3, anti-TIGIT, anti-LAG3, anti-B7H3, anti-B7H4, anti-VISTA, anti-BTLA, anti-CD47, anti-SIRPα, anti-CD48, anti-CD155, anti-CD160, anti-TREM2, anti-IDO1, anti-adenosine 2A receptor, anti-aryl hydrocarbon receptor, anti-KIR, and anti-LILRB2. Examples of targets for immune checkpoint inhibitors include, but are not limited to: PD-L1, PD1, CTLA4, TIM3, TIGIT, LAG3, B7H3, B7H4, VISTA, BTLA, CD47, SIRPα, CD48, CD155, CD160, TREM2, IDO1, adenosine 2A receptor, aryl hydrocarbon receptor, KIR, and LILRB2.

Therefore, examples of immune checkpoint inhibitors include agents that inhibit proteins such as PD-L1, PD1, CTLA4, TIM3, TIGIT, LAG3, B7H3, B7H4, VISTA, BTLA, CD47, SIRPα, CD48, CD155, CD160, TREM2, IDO1, adenosine 2A receptor, aryl hydrocarbon receptor, KIR, and LILRB2. In some cases, a subject-stabilized IL-18 variant peptide (e.g., DR-IL-18 variant, D2D-IL-18 variant) (e.g., SEQ ID NO:19) is co-administered with an immune checkpoint inhibitor (e.g., an inhibitor of PD-L1, PD1, CTLA4, TIM3, TIGIT, LAG3, B7H3, B7H4, VISTA, BTLA, CD47, SIRPα, CD48, CD155, CD160, TREM2, IDO1, adenosine 2A receptor, aryl hydrocarbon receptor, KIR, LILRB2, or any combination thereof).

In some embodiments, the compositions and methods disclosed herein can be used to treat cancers associated with high levels of IL-18BP (e.g., circulating or expressed by tumors) or cancers associated with tumors expressing IL-18R on infiltrating T cells or NK cells.

Therefore, the present invention relates to the prevention and treatment of diseases or conditions by administering a therapeutically effective amount of a variant IL-18 polypeptide (e.g., stabilized IL-18), a recombinant IL-18 polypeptide, an active IL-18 polypeptide fragment (e.g., IL-18 polypeptide variant, etc.) to cells, tissues, organs, or subjects in need, so as to treat the disease or condition, or its associated signs, symptoms, or pathology. In one embodiment, the present invention provides a method of administering a therapeutically effective amount of a variant IL-18 polypeptide (e.g., stabilized IL-18), a recombinant IL-18 polypeptide, an active IL-18 polypeptide fragment (e.g., IL-18 polypeptide variant, etc.) to a subject suffering from, suspected of having, or at risk of suffering from, the disease or condition described herein. In one embodiment, the present invention provides a method for administering a therapeutically effective amount of an IL-18 variant polypeptide (e.g., stabilized IL-18), a recombinant IL-18 variant polypeptide, or an active IL-18 variant polypeptide fragment (e.g., IL-18 variant peptide, etc.) to cells, tissues, or organs of a person suffering from, suspected of having, or at risk of suffering from, the disease or condition described herein.

Reagent test kit

The present invention also relates to kits used in the methods of the present invention. Such kits include: various combinations of components that can be used in any of the methods described elsewhere herein, including, for example, IL-18 peptides (e.g., stabilized IL-18, such as stabilized DR IL-18 variants or stabilized D2D IL-18 variants, etc.) (e.g., SEQ ID NO: 19); and/or materials for the quantitative analysis of IL-18 variant peptides or IL-18 variant nucleic acids; and/or materials for assessing the activity of IL-18 variant peptides or IL-18 variant nucleic acids; and/or explanatory materials. For example, in some embodiments, the kit includes components that can be used to quantify IL-18 variant nucleic acids in biological samples. In another embodiment, the kit includes components that can be used to quantify IL-18 variant peptides in biological samples. In yet another embodiment, the kit includes components that can be used to assess the activity (e.g., enzyme activity, ligand binding activity, etc.) of IL-18 variant peptides in biological samples.

In another embodiment, the kit includes components for monitoring the effectiveness of treatment administered to a subject in need, containing explanatory materials and components for determining whether the level of IL-18 signaling in a biological sample obtained from the subject is modulated during or after treatment. In various embodiments, to determine whether the level of IL-18 signaling in a biological sample obtained from the subject is modulated, the level of IL-18 signaling is compared to the level of at least one comparative control contained in the kit, such as a positive control, negative control, historical control, historical standard, or the level of another reference molecule in the biological sample. In some embodiments, the ratio of IL-18 signaling to a reference molecule is determined to aid in monitoring treatment.

Example

The invention will be further described in detail with reference to the following experimental embodiments. These embodiments are provided for illustrative purposes only and are not intended to be limiting, unless otherwise indicated. Therefore, the invention should not be construed in any way as limited to the following embodiments, but should be construed as covering any and all variations that become apparent from the teachings provided herein.

Without further description, it is believed that those skilled in the art can prepare and utilize the invention and implement the claimed methods using the preceding description and the following illustrative examples. Therefore, the following working examples should not be construed as limiting the remainder of this disclosure in any way.

Example 1: IL-18 variant peptide

IL-18 is a pro-inflammatory cytokine that can stimulate T cells, NK cells, and myeloid cells. Given its ability to stimulate anti-tumor immune cells, it has been proposed as an immunotherapeutic agent against cancer. As demonstrated in this paper, the therapeutic efficacy of recombinant IL-18 therapy may be greatly limited by the upregulation of its naturally occurring endogenous soluble inhibitor, IL-18BP. This disclosure is partly based on the development of variants of human and mouse IL-18 that exhibit minimal binding to IL-18BP. These cytokine variants have shown a relative preference for receptors (IL-18Rα and IL-18BP) that has been altered by hundreds of thousands to over a million times. These variants have demonstrated potent anti-tumor activity in preclinical tumor models, both as monotherapy and in combination with immune checkpoint inhibitors such as anti-PD-1. As an additional application, IL-18 also has a recognized anti-obesity effect, and this paper demonstrates that administration of the variants significantly reduces body fat composition compared to WTIL-18 therapy. Therefore, in addition to tumor immunotherapy, the novel variants also have indications for endocrinology/metabolism/obesity.

This article also describes an additional group of IL-18 variants that act as IL-18BP antagonists by exclusively binding to IL-18BP, with no or significantly reduced binding to IL-18Rα. It is hypothesized that these proteins could be used to enhance the activity of endogenous IL-18 by neutralizing IL-18BP.

The materials and methods used in these experiments are now described.

Protein expression and purification

Human IL-18, mouse IL-18 (amino acids 1-157), and their variants were assembled into gBlocks (Integrated DNA Technologies, IDT) and cloned into the pET28a-smt vector for expression of a protein with an N-terminal sumo tag and a C-terminal hexahistine tag in *E. coli* BL21(DE3) Rosetta strain. Protein expression was induced at 16°C with 0.5 mM IPTG for 20 h. The fusion protein was first purified using Ni chelating resin, followed by cleavage of the sumo tag with sumo protease. The protein was then separated from the aggregates by sequential ammonium sulfate cleavage, with the aggregates precipitated in 20% ammonium sulfate and the target protein precipitated in 70% ammonium sulfate. The protein precipitate was resuspended and applied again to Ni chelating resin to remove the sumo tag, followed by endotoxin removal washing with 0.1% Triton X-114. Finally, the eluted protein was exchanged for PBS via a PD-10 column (GE Healthcare) buffer. Monodispersity of protein samples was tested by size exclusion chromatography using FPLC (Bio-Rad) and a SEC650 column (Bio-Rad).

Human IL-18Rα extracellular domain (amino acids 19-329), IL-18Rβ extracellular domain (amino acids 15-356), and IL-18BP (amino acids 31-194) were secreted and purified via a baculovirus expression system. Briefly, all construct sequences were cloned into the pAcBN-BH3 vector (BDBiosciences) with an N-terminal gp67 signal peptide and a C-terminal AviTag™ and hexahistine tag. Fall armyworm (Spodoptera frugiperda) (Sf9) insect cells cultured at 27°C in SF900 II SFM medium (Invitrogen) were transfected with the plasmid constructs to establish a high-titer recombinant virus, which was subsequently amplified. Trichopulsia ni (High-Five) insect cells grown at 27°C in Insect Xpress medium (Lonza) were infected with the virus to express the recombinant protein. Three days after infection, the protein was extracted by Ni-NTA (QIAGEN) affinity chromatography, concentrated, and purified to >98% homogeneity using a SEC650 size exclusion column (Bio-Rad) equilibrated in 10 mM HEPES (pH 7.5) and 150 mM NaCl.

Mouse IL-18Rα extracellular domain (amino acids 19-329) and IL-18BP (amino acids 31-194) were produced as secreted proteins using the Expi293 expression system (Thermo Fisher). Briefly, all construct sequences were cloned into the BacMam expression vector pEZT_D_Lux, which contains an N-terminal H7 signal peptide and a C-terminal AviTag™ and a six-histidine tag. Following the manufacturer's instructions, Expi293 cells cultured at 37°C in Expi293 expression medium (Thermo Fisher) were transfected with the plasmid using the ExpiFectamine 293 transfection kit (Thermo Fisher). Cells were harvested 3-5 days after transfection. The protein purification procedure was the same as for human proteins.

For protein biotinylation, the C-terminal biotin receptor peptide (AviTag)-GLNDIFEAQKIEWHE was fused to all IL-18 receptor constructs. Protein biotinylation was performed using a soluble BirA conjugase in 0.1 mM Bicine (pH 8.3), 10 mM ATP, 10 mM magnesium acetate, and 0.5 mM biotin (Sigma). Protein purification was achieved by size exclusion on an SEC650 column, as described above.

Yeast display of IL-18

Human and mouse IL-18 gene blocks (IDTs) were synthesized and cloned into the pYAL vector and displayed on *Saccharomyces cerevisiae* strain EBY100. Single colonies of IL-18 yeast were grown overnight at 30°C in SDCAA liquid medium and induced for 1 day at 20°C in SGCAA liquid medium. The display level of IL-18 on yeast was validated by flow cytometry using an anti-cMyc tag antibody (anti-myc-PE; CellSignaling Technologies). Receptor staining with biotinylated IL-18Rα (with or without IL-18Rβ) or biotinylated IL-18BP was performed on ice in PBS supplemented with 0.5% BSA and 2 mM EDTA (PBE). All analyses were performed on a Sony SA3800 flow cytometer.

Human IL-18 Library Construction and Selection

For the first human decoy-resistant IL-18 library, fourteen hIL-18Rα and hIL-18BP contact residues in hIL-18 were identified homologously by comparing the structure of the hIL-18/hIL-18Rα/hIL-18Rβ complex (Protein Database ID 3OW4) with that of IL-18/IL-18BP (PDB ID 3F62) (Table 1). A library with these residues randomized was constructed using assembly PCR with the degenerate primers listed in Table 2. The theoretical diversity of the library was approximately 1.96 × ^10¹¹ unique protein sequences. The PCR products were further amplified with primers homologous to the pYAL vector and co-electroplated into EBY100 yeast cells along with linearized pYAL. The resulting library contained 2.5 × ^10⁸ transformants. For the second V2.0 human decoy-resistant IL-18 library, eleven hIL-18Rα and hIL-18BP contact residues from hIL-18 were selected, with a theoretical diversity of 3.44 x ^10⁹ variants (described in Figure 7A). Using degenerate primers, an assembly PCR was used to construct a library with these residues randomized, and this library, along with pYAL, was co-electroplated into EBY100 yeast. The resulting library had a diversity of 6 x ^10⁸ transformants.

Table 1 shows the design of the first person's IL-18 library.

Table 2 shows the primers used for the first human IL-18 library assembly.

For both libraries, the transformed yeast was recovered and amplified at 30°C in liquid synthetic dextran medium (SDCAA) containing casein amino acids, and induced by diluting 1:10 in liquid synthetic galactose medium (SGCAA) containing casein amino acids and culturing at 20°C for 24 h. An appropriate number of induced yeasts were used in each round to ensure at least 10-fold coverage of the expected diversity of the library at each step, and no less than ^10⁸ cells. All selection steps were performed at 4°C using PBE buffer (PBS with 0.5% BSA and 2 mM EDTA). The selection reagents for each round for the first-generation library are listed in Table 3. For round 1, yeast was anti-selected using anti-Cy5/Alexa Fluor 647 beads (Miltenyi) and LSMACS columns (Miltenyi) to remove non-specific bead-binding agents. Yeast was labeled with 1 μM biotinylated hIL-18Rα at 4 °C for 1 h, followed by positive selection using magnetic selection with SA/Alexa Fluor 647 beads and LS MACS columns. For round 2, anti-selection was performed with 1 μM biotinylated IL-18BP, and positive selection was the same as in round 1. For rounds 3-5, selection was performed by incubating yeast with 100 nM (rounds 3-4) or 10 nM (round 5) biotinylated IL-18Rα and 250 nM pre-formed biotin-capped hIL-18BP/SA-PE tetramer. After competitive binding, the yeast was washed and labeled with SA Alexa Fluor 647 to detect IL-18Rα. Display levels were determined by staining with Alexa Fluor 488-conjugated anti-cMyc (Cell Signaling Technologies) and the top 1% of display-normalized IL-18Rα binders (in IL-18BP non-bindings) were separated using FACS with a Sony SA3800 cell sorter. After each round of selection, the recovered yeast was expanded overnight in SDCAA medium at 30°C, followed by induction for 24 hours at 20°C by a 1:10 dilution in SGCAA medium.

The V2.0 human DR-IL-18 library is selected in a similar manner, and the specific selection steps are shown in Figure 7B.

Mouse IL-18 library construction and selection

The library construction and selection procedures were similar to those for human IL-18, with the following changes. Library construction was based on information from computer-modeled mouse IL-18/receptor complex structures (predicted by Phyer2.0). Thirteen sites were selected for randomization using primers described in Table 4 (Table 3). A library of 4 × ^10⁸ transformants was generated by co-electroporation with pYAL. The selection reagents used in each round are listed in Table 5.

Table 3 shows the design of the mouse IL-18 library.

Table 4 shows the primers for mouse IL-18 library assembly.

Table 5 shows a summary of reagents selected for library studies.

Surface plasmon resonance

Experiments were performed using a Biacore T100 at 25°C. Biotinylated IL-18Rα or IL-18BP was immobilized onto a Biacore biotin capture chip (S-series CAP sensor chip, GE Healthcare), yielding an Rmax of approximately 50 RU (IL-18Rα) or approximately 10 RU (IL-18BP). Measurements were performed using serial dilutions of the IL-18 variant in HEPES-P+ buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 0.005% surfactant P20). Surface regeneration was achieved by three 60-second injections of regeneration buffer (3/4 (v/v) 8 M guanidine hydrochloride and 1/4 (v/v) 1 M sodium hydroxide). Experiments were performed simultaneously in multiple channels to enhance observation. All data were analyzed using a 1:1 Langmuir binding model with Biacore T100 evaluation software version 2.0.

cell lines

HEK-Blue IL-18-sensing cells (InvivoGen) were maintained in complete medium (DMEM containing 10% heat-inactivated FBS, 2 mM L-glutamine, 50 U/mL penicillin, and 50 μg/mL streptomycin) supplemented with 100 μg/mL Normocin, 30 μg/mL Blastidin, 180 μg/mL Zeocin, and 200 μg/mL Hygromycin). YUMMER1.7 melanoma cells were cultured and prepared as previously described (Wang et al., 2017, Pigment Cell Melanoma Res., 30(4):428-435).

HEK-Blue Cytokine Activity Assay

For cytokine activity assays, 50,000 HEK-Blue IL-18-sensing cells per well of a flat-bottomed 96-well plate were incubated with gradually decreasing concentrations of recombinant human IL-18 in 200 μL of complete culture medium. After incubation at 37°C and 5% _CO2 for 20–24 h, 30 μL of cell culture supernatant was mixed with 170 μL of QUANTI-Blue assay medium (InvivoGen) and incubated at 37°C and 5% CO2 until a detectable color change from pink to blue was achieved (0.5–4 h). Alkaline phosphatase levels were quantified at 655 nm using a spectrophotometer. Cytokine activity was determined by calculating the relative absorbance value (the percentage of the maximum absorbance value measured at 655 nm) for each cytokine in the assay.

For the IL-18BP blocking assay, a fixed concentration of recombinant human IL-18 was pre-incubated with a gradually decreasing concentration of recombinant human IL-18BP at 4°C for 1 hour. Subsequently, the protein mixture was added to HEK-Blue IL-18 sensor cells, and measurements were performed as described.

mice

C57BL/6 wild-type mice (6-9 weeks old) from Jackson Laboratory were used for in vivo mouse experiments. Experimental groups were matched by weight, sex, and age. All animal experiments were conducted in accordance with the approval of the Yale Institutional Animal Care and Use Committee.

In vivo pharmacodynamics and pharmacokinetic studies

Mice (n=9 per group) received intraperitoneal (i.p.) injections of 1 mg/kg recombinant IL-18 (WT or variant SEQ ID NO: 61) or PBS as a mediator control daily. On days 1, 4, and 7, three mice from each group were sacrificed 5 hours after injection for blood collection via cardiac puncture and subsequent analysis of plasma or leukocytes (see Mouse IL-18BP ELISA, Luminex-based multiplex immunoassay for mouse cytokine analysis, and immunophenotyping via flow cytometry). Body temperature was monitored daily throughout the 7-day experiment using a rodent thermometer BIO-TK8851 (Bioseb) and a RET 3 rectal probe (Braintree Scientific Inc.) for mice. Body weight was monitored daily.

Plasma preparation from whole blood

According to the manufacturer's instructions, plasma is prepared from whole blood using EDTA-coated Microtainer plasma separators (BD). The plasma sample is frozen once at -20°C before being used for analytical assays.

IFN-γ and IL-18BP ELISA

To measure the level of human IFN-γ in cell culture supernatant, the Human IFN-γ ELISA MAX Deluxe Kit (BioLegend) with a sensitivity of 4 pg/mL and a detection range of 7.8–500 pg/mL was used, according to the manufacturer's instructions. For the quantification of human IL-18BP in cell culture supernatant, the Quantikine Human IL-18BP Immunoassay (R&D Systems) with a sensitivity of 7.52 pg/mL and a detection range of 26.6–1,700 pg/mL was used. The level of mouse IL-18BP in plasma was quantified using the Mouse IL-18BP ELISA Kit (R&D Systems) with a sensitivity of 0.156 ng/mL and a detection range of 0.156–10 ng/mL. All assays, including sample preparation, were performed according to the manufacturer's instructions.

Luminex-based multiplex immunoassay for mouse cytokine analysis

To quantify the levels of multiple mouse cytokines in plasma, including IFN-γ and IL-12, a luminex-based Bio-Plex Pro multiplex immunoassay (Bio-Rad) was performed using the Bio-Plex 200 system (Bio-Rad). Cytokines of interest were analyzed using Bio-Plex Pro mouse cytokine standards 23-Plex (Group I) reconstituted in DMEM, following the manufacturer's instructions.

Immunophenotyping by flow cytometry

For leukocyte analysis, 100 μL of whole blood was collected into a microtainer plasma separator (BD) coated with EDTA and mixed by inverting the tube several times. Red blood cell lysis was performed by adding ACK lysis buffer (VWR) and incubating at room temperature for 3–5 minutes. After adding MACS buffer (2 mM EDTA, 2% FBS in PBS), leukocytes were collected by centrifugation (5 minutes, 400 × g, 4 °C) and aspiration of the supernatant. Leukocytes were washed once with cold MACS buffer and collected again as described. The cell pellet was resuspended in 200 μL of MACS buffer containing 10% (v/v) rat serum (STEMCELL Technologies Inc.) and a specific fluorescently labeled antibody for staining for subsequent flow cytometry analysis. The following antibodies were used for staining at 4°C for 30 minutes: αCD4-AF700 (BioLegend), αCD8-APC (BioLegend), B220-APC-Cy7 (BioLegend), CD11b-PB (BioLegend), NK1.1-PE (BioLegend), NKp46-PE (BioLegend), and CD69-FITC (BioLegend). Afterward, leukocytes were washed twice with MACS buffer as described previously. Finally, cells were resuspended in 100 μL of MACS buffer, and samples were collected using a flow cytometer (Sony SA3800). 10 μL aliquots were taken and cell counting was performed using an Invitrogen Countess II automated cell counter (Thermo Fisher Scientific). FlowJo v10.3 software was used for data analysis, with forward and side-scattering gating for leukocytes and individual events.

Tumor treatment experiment

0.5 × ^10⁶ YUMMER1.7 cells were subcutaneously implanted into C57BL/6J mice. Treatment was initiated 7 days post-implantation when the tumor size was approximately 50 mg. Mice were assigned to treatment cohorts, including: 1) a mediator (saline); 2) anti-PD-1 (rat clone RMP1-14, Bio X Cell, West Lebanon, New Hampshire, US); 3) wild-type IL-18; 4) SEQ ID NO:61; 5) wild-type IL-18 + anti-PD-1; and 6) SEQ ID NO:61 IL-18 + anti-PD-1. Anti-PD-1, wild-type IL-18, and SEQ ID NO:61 IL-18 were administered intraperitoneally twice weekly at doses of 8 mg/kg, 0.32 mg/kg, and 0.32 mg/kg, respectively. Clinical signs of toxicity were monitored in mice, and tumor growth was tracked twice weekly using calipers. Mice were euthanized when the maximum tumor diameter reached or exceeded 1.5 cm; this was considered the endpoint for survival analysis.

The B2m-deficient YUMMER1.7 study was conducted in a similar manner with minor variations. 1.0 x ^10⁶ cells were transplanted because the tumor grew more slowly than the parental strain. Treatment consisted of saline, anti-PD1 plus anti-CTLA4, and SEQ ID NO:61, with the same timeline and dosage as described in the previous study.

The experimental results will now be described.

IL-18 axis as a target for cancer immunotherapy

To identify potential signaling nodes for immunotherapy interventions, single-cell RNA-seq data from tumor-infiltrating lymphocytes were analyzed to target the expression of cytokine pathway components (Singer et al., 2016, Cell, 166:1500-1511, e1509). The receptor subunits of IL-18, IL-18Rα (i.e., IL-18R1) and IL-18Rβ (i.e., IL-18RAP), as well as IL-18 itself, were upregulated in both activated and dysfunctional lymphocyte programs (Figure 1A). Further analysis of the ImmGen database revealed that the expression of both IL-18 receptor subunits was associated with the expression of T cell “depletion” markers (including PD-1, Tim3, Lag3, and TIGIT) in CD4 and CD8 cells following prolonged antigen exposure, as shown in Figure 1B. These expression signatures suggest that the IL-18 pathway can be used to selectively stimulate activated and dysfunctional/depleted T cells within tumors as a paradigm for immunotherapy. IL-18 is a Th1 cytokine, originally named "interferon-γ inducible factor" (IGIF) because it strongly stimulates the release of interferon-γ (IFN-γ) from T cells and NK cells. Feedback inhibition of IL-18 is achieved through IFN-γ-driven induction of IL-18BP, a high-affinity secreted decoy receptor for IL-18 that spatially inhibits IL-18's ability to bind to and activate its receptor (Figure 2A). Without being bound by any particular theory, this mechanism is reminiscent of IFN-γ induction of PD-L1, suggesting that IL-18BP may act as a "soluble immune checkpoint." Consistent with this hypothesis, IL-18BP has been found to be upregulated in several types of cancer in the TCGA and Oncomine databases, most notably breast cancer, gastric cancer, and brain cancer (Figure 2B). Furthermore, IL-18BP expression was closely correlated with the expression of the key immune checkpoint PD-1 in tumors (r = 0.65 and 0.78 in gastric and breast cancer, respectively, Figure 2C), indicating that IL-18BP may also contribute to tumor immune escape and lymphocyte depletion.

Recombinant IL-18 has been administered to cancer patients in multiple clinical trials. It has been found to be well tolerated even at high doses of 2 mg/kg, with pharmacodynamic outputs including an expansion of activated CD69 ⁺ natural killer (NK) cells and a dramatic increase in serum IFN-γ levels. However, a phase II trial in melanoma patients was discontinued due to lack of efficacy. Examination of the reported pharmacodynamic results from these clinical trials indicates that the efficacy of rIL-18 diminishes with repeated dosing, with rapid immune responses observed regarding peripheral NK cell activation/expansion and cytokine release (including IFN-γ and GM-CSF). This diminished rIL-18 efficacy is consistent with a significant increase in serum IL-18BP levels, exceeding pre-treatment levels by more than two orders of magnitude and typically exceeding 100 ng/mL. Without being bound by any particular theory, it is hypothesized that IL-18BP limits the efficacy of rIL-18 therapy, and that IL-18 variants unaffected by IL-18BP inhibition may be effective tumor immunotherapies. Furthermore, inhibitors of IL-18BP may be effective for tumor immunotherapy.

Engineered IL-18 variant resistant to IL-18BP inhibition (human DR-IL-18 variant)

To obtain an IL-18 variant that can signal via IL-18Rα/IL-18Rf3 but is unaffected by the inhibition of IL-18BP, directed evolution using yeast surface display was employed. First, the structure of the human IL-18:IL-18Rα:IL-18Rf3 ternary signal transduction complex (PDB = 3OW4) was analyzed, and residues of IL-18 sharing an interface with the signal transduction complex and IL-18BP were identified (Figure 3A). Since the structure of hIL-18:hIL-18BP was not yet determined, a correlation complex between IL-18 and IL-18BP viral (exogenous viral) orthologs was used (PDB = 3F62). Degenerate oligonucleotide primers and assembly PCR were used to generate a combinatorial library that randomized this set of residues to the identified surrogate set (see Table 1). This library was electroporated into yeast along with the N-terminal yeast display vector pYAL to obtain a library with 2.5 × ^10⁸ transformants. Using this library, directed evolution was performed through several rounds of selection using magnetic and fluorescent cell sorting (FACS) with recombinant hIL-18Rα and anti-selection with hIL-18BP, as summarized in Figure 3B. After five rounds of selection, the vast majority of library clones completely exchanged their relative preferences for hIL-18BP and hIL-18Rα compared to WT hIL-18 (Figure 3C). These clones were designated as the “DR-hIL-18” variant, where “DR” stands for “decoy resistance”.

Sequencing of 96 clones from the fifth post-pool revealed 21 unique sequences, which were analyzed to yield four “common sequences,” SEQ ID NO: 34-37 (Figure 4). To estimate the binding affinity of these variants for hIL-18Rα and hIL-18BP, binding isotherms for hIL-18Rα and IL-18BP binding were established using yeast-displayed cytokine variants and flow cytometry. As seen in Figure 5A, the DR-hIL-18 variant binds hIL-18Rα with an affinity comparable to WT IL-18, but shows severely attenuated binding with hIL-18BP, with an apparent binding EC50 value significantly greater than 1 μM. To further characterize the receptor-binding activity of the DR-IL-18 variant, cytokine recombinant expression was performed, and surface plasmon resonances of IL-18Rα and IL-18BP were observed (see representative traces in Figure 5B). These results are summarized in Tables 6 and 7, and show that the DR-hIL-18 variant has a significantly reduced preference for IL-18BP compared to IL-18Rα by several orders of magnitude.

Table 6 illustrates the binding affinity of human IL-18 variants IL-18Rα and IL-18BP using yeast binding isotherms.

NBD, no binding detected (20 μM used for ratio calculation), ------ undetermined value

Table 7 illustrates the binding affinity of IL-18Rα and IL-18BP for human IL-18 variants using SPR.

------Indication value not determined

Functional characterization of human DR-IL-18 variants

A previous report by Kim et al. (Kim et al., 2001, Proc Natl Acad Sci U S A98(6):3304-9) described three hIL-18 variants with enhanced activity that were allegedly reduced by inhibition of IL-18BP: E42A, K89A, and E42A/K89A. These cytokine variants were displayed on yeast, and the inhibition of IL-18BP binding by IL-18Rα was assessed by flow cytometry. As seen in Figure 6A, although the DR-hIL-18 variants were unaffected by the inhibition of hIL-18Rα binding by hIL-18BP, the Kim et al. variants showed hIL-18BP neutralization that was approximately equivalent to that of WT hIL-18. These results indicate that the DR-hIL-18 variants are independent of IL-18BP, while the Kim et al. variants are highly sensitive to IL-18BP inhibition, similar to WT hIL-18.

To confirm that DR-hIL-18 can generate effective signal transduction via the IL-18 receptor in a cellular setting, concentration-response experiments were performed using the HEK-blue IL-18 reporter cell line. In this system, IL-18R signal transduction was read out via the expression of secreted alkaline phosphatase (SEAP) downstream of the NFκb/AP1 promoter. In the absence of IL-18BP, the DR-hIL-18 variant produced signal transduction EC50 values commensurate with WT hIL-18. However, the DR-hIL-18 variant was not actually inhibited by hIL-18BP, showing no detectable inhibition at 1 μM IL-18BP (Figure 6B). In summary, these studies demonstrate that the DR-hIL-18 variant is biologically active and unaffected by IL-18BP neutralization in a cellular signal transduction setting.

Engineering of a second-generation human IL-18 variant resistant to IL-18BP inhibition (human v2.0 DR-IL-18 variant) and representation

To obtain additional, potentially enhanced human DR-IL-18 variants, a second library of human IL-18 randomized at 11 sites was designed (Fig. 7A) and transformed into yeast as described above. The resulting library, selected from ⁶ x 10⁸ transformants as shown in Fig. 7B, exhibited a robust preference for IL-18Rα compared to IL-18BP in successive selection steps (Fig. 7C). After 5–6 rounds of selection, 17 unique sequences were recovered (Fig. 8). As shown in Fig. 9A, clones SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, and SEQ ID NO:87 showed equal or slightly stronger binding to IL-18Rα compared to WT IL-18, as measured by the yeast binding isotherm of biotinylated IL-18Rα. However, as shown in Fig. 9B, these clones did not show any significant binding to IL-18BP. Figure 9C depicts measurements of thermal stability of clones applied over a range of temperatures by demonstration to yeast, showing that their thermal stability is 7–13 °C higher than that of WT IL-18. These results are summarized in Figure 9D. Engineered IL-18 variants resistant to IL-18BP inhibition (murre DR-IL-18 variant).

Because IL-18 exhibits poor cross-reactivity with IL-18Rα between humans and mice, a murine equivalent of the DR-IL-18 variant was developed for studies in mice. Similar to the approach taken for hIL-18 described above, combinatorial libraries of mIL-18 variants with randomized similar mIL-18Rα/mIL-18BP contact residue sets were generated (Table 3), resulting in a library of 4 × ^10⁸ transformants. This library underwent directed evolution, similar to that performed with the human IL-18 library; the selection strategy is summarized in Figure 10A. After six rounds of selection, the remaining clones showed an almost complete preference for mIL-18Rα over mIL-18BP (Figure 10B). Analysis of the 96 clones revealed 11 unique sequences, from which two common sequences, SEQ ID NO:60 and SEQ ID NO:61, were identified (Figure 10C). Yeast binding isotherm and surface plasmon resonance experiments confirmed that these DR-IL-18 clones were even more independent of IL-18BP than the human IL-18 variants described in this paper, with mIL-18BP binding KD much higher than 1 μM, and mIL-18Rα binding remaining approximately equal to WT mIL-18 (Fig. 11A, Fig. 11B, Tables 8 and 9).

Table 8 illustrates the binding affinity of mouse IL-18 variants IL-18Rα and IL-18BP using yeast binding isotherms.

NBD, no binding detected (10 μM used for ratio calculation)

Table 9 illustrates the binding affinity of mouse IL-18 variants for IL-18Rα and IL-18BP using SPR.

NBD, no binding detected (30 μM used for ratio calculation)

In vivo pharmacodynamic studies of DR-IL-18 variants

To evaluate the biological effects of in vivo administration of the DR-IL-18 variant, pharmacodynamic studies were conducted in mice, comparing WT mIL-18 with SEQ ID NO:61. In the first study, mice were treated with a mediator (PBS), mIL-18 (1 mg/kg/day), or SEQ ID NO:61 (1 mg/kg/day) for a total of seven injections (Figure 12A). Flow cytometry analysis of peripheral blood phenotypes showed that, compared with mediator treatment, both WT mIL-18 and SEQ ID NO:61 increased peripheral NK cell counts by more than tenfold and peripheral monocyte counts by more than fivefold; total CD4 and CD8 cell counts were not significantly affected (Figure 12B). Examination of cell activation status by CD69 induction showed that SEQ ID NO:61 treatment significantly increased CD69 levels on CD4 and CD8 cells compared with mIL-18 or the mediator treatment; the CD4 and CD8 subgroups achieved positivity rates of over 30% and over 50%, respectively (Figure 12C). Although both mIL-18 and SEQ ID NO:61 stimulated CD69 expression on peripheral NK cells, achieving over 20% positivity by day 3, the CD69 level of mIL-18 decreased to a non-significant level by day 6, while that of SEQ ID NO:61 treatment remained significantly elevated (Figure 12C). Peripheral cytokine levels were also measured using a multiplex Luminex kit. As shown in Figure 12D, both mIL-18 and SEQ ID NO:61 increased serum IFN-g, MIP1b, and G-CSF compared to mediator treatment. However, by day 6, SEQ ID NO:61 reached much higher levels for each of these cytokines than mIL-18, because mIL-18 exhibits rapid immunization, and in the case of subsequent administration, the induced cytokine levels tend to plateau or decrease.

Effect of SEQ ID NO:61 on body fat composition

To assess the effect of the DR-IL-18 variant on body fat composition, C57BL/6 mice were administered 1 mg/kg WT IL-18 or 0.01, 0.1, or 1 mg/kg SEQ ID NO:61 via intraperitoneal injection every three days. Body fat and lean body mass composition were monitored by echoMRI. By day 12, all tested doses of SEQ ID NO:61 (1 mg/kg, 0.1 mg/kg, and 0.01 mg/kg) resulted in a significant reduction in the overall percentage of body fat, while the overall fat composition of mice treated with the mediator and mIL-18 did not change significantly (Fig. 13, top). Specifically, during the experiment, the total fat mass level of mice treated with SEQ ID NO:61 decreased or remained stable (Fig. 13, bottom left), but their total lean body mass generally increased (Fig. 13, bottom right). These results indicate that SEQ ID NO:61 and other variants disclosed herein can be used to therapeutically reduce body fat composition (e.g., for the treatment of obesity, diabetes, and/or metabolic syndrome).

Anti-tumor efficacy of DR-IL-18 variant

The antitumor efficacy of DR-IL-18 (SEQ ID NO:61) was evaluated using a transplantable, syngeneic YUMMER1.7 malignant melanoma tumor model. WT mIL-18 and SEQ ID NO:61 were administered intraperitoneally to mice carrying YUMMER1.7 tumors at a dose of 0.32 mg/kg every two weeks, with or without co-administration with an anti-PD1 antibody (8 mg/kg/q3d). Consistent with previous reports on its use in mice and humans, WT IL-18 did not affect tumor growth or survival compared to the mediator (saline), and when administered in combination, it only slightly enhanced anti-PD1 efficacy. However, SEQ ID NO:61 as a monotherapy cured 27% of treated mice and produced a partial response in another 27% of mice, with efficacy comparable to anti-PD1 treatment. The combination of SEQ ID NO:61 and anti-PD1 cured 80% of treated mice (Figures 14A and 14B).

To determine the mechanism of action of DR-IL-18 against YUMMER1.7 tumors, cell depletion studies were conducted using antibodies against CD8, CD4, NK1.1, and interferon-γ. As shown in Figures 15A and 15B, depletion of CD8 cells or neutralization of interferon-γ completely eliminated the effectiveness of DR-IL-18. Depletion of CD4 cells did not affect the initial activity of DR-IL-18 in relation to tumor growth; however, in CD4-treated mice, the treatment response was not sustained, indicating the role of CD4 cells in supporting and maintaining anti-tumor immunity. Depletion of NK cells did not affect tumor growth or survival in YUMMER1.7 cells.

The activity of DR-IL-18 was further evaluated in an immunogenic MC38 colorectal tumor model. First, a dose-exploration study was conducted, administering saline, WT IL-18 (1 mg/kg, twice weekly), or a series of DR-IL-18 doses (0.01 mg/kg, 0.1 mg/kg, or 1 mg/kg, twice weekly). As shown in Figure 16, WT IL-18 had no effect on tumor growth, while DR-IL-18 (SEQ ID NO: 61) showed dose-dependent efficacy, slowing tumor growth at 0.1 mg/kg and producing tumor regression at 1 mg/kg. The cohort was then expanded, and potential synergy with immune checkpoint inhibition was evaluated. Again, WT IL-18 showed no effect as monotherapy and did not show enhanced anti-PD1 efficacy. In contrast, DR-IL-18 showed robust monotherapy activity, comparable to or superior to anti-PD1, and showed outstanding synergy when administered together with the other therapy, producing complete regression in all treated mice, as shown in Figure 17.

To further characterize the mechanism of DR-IL-18, flow cytometry was used to investigate the immune infiltration of MC38 tumors from mice treated with saline, WT IL-18, or DR-IL-18 (SEQ ID NO: 61). Compared to saline or WT IL-18, DR-IL-18 treatment increased CD8 and NK cell infiltration per milligram of tumor and additionally led to the upregulation of effector cell activation markers such as granzyme B and KLRG1 (Fig. 18A, top row). Unlike other cytokine therapies such as IL-2 or IL-15, DR-IL-18 did not increase the intratumoral CD8:Treg ratio compared to saline treatment. However, DR-IL-18 treatment resulted in a more favorable tumor immune microenvironment by increasing the ratio of CD8 cells to tumor-associated macrophages (TAMs) and monocyte and granulocyte myeloid-derived suppressor cells (MDSCs). Secondary cytokine release profiles were also measured from serum of the same mice using Luminex assays. As shown in Figure 18B, DR-IL-18 treatment increased systemic levels of interferon-γ, IL-7, and IL-15 by more than 100-fold compared to WT IL-18 treatment. Taken together, these results indicate that DR-IL-18 exerts its antitumor efficacy through a unique mechanism of action different from IL-2, IL-15, or WT IL-18 treatments.

Some minor cytokines induced by DR-IL-18 therapy are expected to contribute to toxicity and/or reduced efficacy. For example, IL-17 is upregulated >100-fold by DR-IL-18, leading to colitis and psoriasis, and additionally stimulating granulocytes, which can become immunosuppressive myeloid-derived suppressor cells. IL-5 and IL-13, type 2 cytokines, are also upregulated by DR-IL-18 and may cause allergies, asthma exacerbations, or anaphylactic reactions. Th2 T cells do not contribute to immunotherapy responses and may promote the development of immunosuppressive Tregs. Therefore, in some cases, the efficacy and safety of DR-IL-18 can be enhanced by selectively inhibiting unwanted minor cytokines such as IL-17, IL-5, and IL-13, for example, through neutralizing antibodies.

Many tumors are resistant to immune checkpoint inhibition, either at the onset (primary resistance) or after an initial response to treatment (secondary resistance). The most common cause of resistance to checkpoint inhibitors is the loss of antigen presentation via MHC class I. Loss of surface MHC class I antigens is often associated with NK cell-mediated cytolysis; however, NK cells can become depleted in MHC class I deficient tumors. Since NK cells express IL-18R and our previous results in MC38 indicated that NK cells are amplified and activated via DR-IL-18, we tested whether DR-IL-18 could stimulate NK cell attack against MHC class I deficient tumors. We used CRISPR/Cas9 to knock out B2m in the Yummer1.7 cell line and found that implanted B2m-deficient Yummer1.7 tumors were refractory even with combination therapy of anti-CTLA4 and anti-PD1 (Figures 19A and 19B), which typically cured nearly 100% of parental Yummer1.7 tumors. However, single-dose treatment with DR-IL-18 (SEQ ID NO: 61) cured 60% of B2m-deficient Yummer1.7 tumors in an NK cell-dependent manner, as the effect was eliminated by depletion of anti-NK1.1 (Figs. 19A and 19B). Experiments were conducted to understand the effect of DR-IL-18 on intratumoral NK cells in MHC class I deficient tumors. Immunophenotyping of B2m-deficient Yummer1.7 tumors from mice treated with saline or DR-IL-18 was performed by flow cytometry. Twenty-four hours after the third dose, mice were sacrificed, tumors were dissected, and cell suspensions were treated with PMA/ionomycin for four hours. Intracellular flow cytometry was then used to analyze the proliferation index and functional capacity of NK cells using Ki67 and interferon-γ. As seen in Fig. 19C, insufficient interferon-γ production and Ki67 levels in NK cells from saline-treated B2m-deficient Yummer1.7 tumors indicated phenotypic depletion. In contrast, NK cells from tumors treated with DR-IL-18 exhibited robust interferon-γ production and Ki67 levels, with the majority of NK cells being positive for both markers. Therefore, these results demonstrate the effectiveness of DR-IL-18 in treating MHC class I deficient tumors that are refractory to immune checkpoint blockade in an NK cell-dependent manner.

These results demonstrate DR-IL-18 as a highly promising tumor immunotherapy agent and provide strong evidence that IL-18BP significantly limits the effectiveness of IL-18 therapy, given the greatly improved activity of the SEQ ID NO:61 DR-IL-18 variant. Based on these results, other strategies, such as blocking IL-18BP with antibodies, small proteins, and/or small molecules, are expected to enhance IL-18 therapy and other immunotherapy regimens.

Efforts have been made to engineer IL-18BP antagonists by generating “decoy-decoy” (D2D) or IL-18 variants that specifically bind IL-18BP but not IL-18Rα and therefore do not signal. The potential advantage of such agents is that they can be used to neutralize IL-18BP and enhance the activity of endogenous IL-18 without systematically driving IL-18R signaling. Therefore, IL-18 was randomized at the contact site against IL-18Rα (Figure 20A), and a yeast-displayed library was prepared as previously described for human and mouse DR-IL-18. As indicated in Figure 20B, the resulting library of 3.9 x ^10⁸ transformants was selected for three rounds, selecting for retained IL-18BP binding while simultaneously performing anti-selection against IL-18Rα. As seen in Figure 20C, each round of selection conferred enrichment for binding to IL-18BP (human and mouse) but not for IL-18Rα binding. Ninety-six clones were sequenced, yielding 31 unique sequences, from which three common sequences, SEQ ID NO:123, SEQ ID NO:124, and SEQ ID NO:125, were identified (Figure 21). Biophysical characterization of the resulting clones indicated that they exhibited binding isotherms with IL-18BP similar to those of WT IL-18 (Figure 22A), but with significantly reduced/absent binding to IL-18Rα (Figure 22B). These data are summarized in Figure 22C. The same selection process was performed for murine IL-18, generating a library of 2.0 x ^10⁸ transformants, from which 51 unique sequences were obtained, summarized in Figure 23.

Example 2: Binding affinity measurement of second-generation variants

Biophysical affinity measurements of the second-generation DR-IL-18 variant (binding of IL-18R relative to IL-18BP) were performed using surface plasmon resonance (SPR). Experiments were conducted using a Biacore T100 instrument at 25°C. Biotinylated IL-18Rα or IL-18BP was immobilized onto a Biacore biotin capture chip (S-series CAP sensor chip, GE Healthcare), yielding an Rmax of approximately 50 RU (IL-18Rα) or approximately 10 RU (IL-18BP). Measurements were performed using serial dilutions of the IL-18 variant in HEPES-buffered saline-P+ buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 0.005% surfactant P20). Surface regeneration was achieved by three 60-second injections of regeneration buffer (3/4 (v/v) 8 M guanidine hydrochloride and 1/4 (v/v) 1 M sodium hydroxide). Experiments were performed simultaneously in multiple channels to enhance observation. All data were analyzed using Biacore T100 evaluation software version 2.0 and a 1:1 Langmuir combination model.

The results confirmed that the DR-IL-18 variant of this disclosure exhibits a significantly reduced affinity for IL-18BP and binds to IL-18Rα with an affinity at least comparable to WT IL-18 (see Figure 24 for the generated sensor plots, Table 10 for a summary of the measured kinetics, Table 11 for a summary of the affinity measurements, and Table 12 for a general summary, including results for the dissociation constant ratio for the second-generation DR-IL-18 variant).

Table 10 summarizes the SPR data (kinetics) for the second-generation hDR-IL-18 variant.

Table 11 summarizes the SPR data (affinity) for the second-generation hDR-IL-18 variant.

Table 12 summarizes the SPR affinity measurements of the second-generation hDR-IL-18 variants for IL-18Rα and IL-18BP. The IL-18BP:Rα dissociation constant ratio is the ratio of the KD for IL-18BP to the KD for IL-18Rα, normalized to the same ratio for WT IL-18. A higher ratio indicates a stronger preference for binding IL-18Rα by the IL-18 variants relative to IL-18BP compared to WT IL-18. *Average of 2 studies. k is a multiple of 1,000. m is a multiple of 1,000,000.

Example 3: Efficacy in cancer treatment

The efficacy of DR-IL-18 variants was tested using a variety of different cancer models, including colorectal cancer, breast cancer, melanoma, and MHC class I deficient tumors resistant to immune checkpoint inhibitors. Results showed that DR-IL-18 variants that preferentially bind to IL-18R rather than IL-18BP could be used to treat a wide range of cancers (not limited to those tested).

To demonstrate the efficacy of DR-IL-18 in a colorectal cancer model, 250,000 CT26 cells were subcutaneously implanted, and treatment began on day 7, once the tumor reached an average size of approximately 60 ^mm³ . WT IL-18 and SEQ ID NO:61 were administered subcutaneously at 0.32 mg/kg twice weekly for a total of 5 doses. Anti-PD1 was administered at 10 mg/kg on the same schedule. Treatment with DR-IL-18 alone resulted in tumor growth inhibition and tumor clearance in animal subgroups (Figure 25A, providing an overlay of the spider diagram showing tumor growth in animals treated with saline (PBS, circles), WT IL-18 (squares), and DR-IL-18 (SEQ ID NO:61, triangles), but not with WT IL-18. DR-IL-18 resulted in prolonged survival (Figure 25B, showing survival curves of mice treated with anti-PD-1, WT IL-18, or DR-IL-18 (SEQ ID NO:61); the number of complete responses is indicated in parentheses), but not with WT IL-18. 40% of mice treated with DR-IL-18 exhibited tumor clearance, a superior improvement compared to the checkpoint inhibitor anti-PD-1. Survival curves of mice treated with anti-PD-1, WT IL-18, and DR-IL-18 (SEQ ID NO:61) are provided. The number of complete responses is indicated in parentheses, and tumor clearance was observed in 40% of the mice, a superior improvement compared to the checkpoint inhibitor anti-PD-1.

In the 4T1 breast cancer model and the B16-F10 melanoma model, only DR-IL-18 resulted in tumor growth inhibition, but WT IL-18 did not. Treatment was administered after the tumor exceeded a mean volume of 50 ^mm³ , as indicated by the boxes marked with “t”.

The efficacy of DR-IL-18 for other cancer types was evaluated using a 4T1 breast cancer model and a B16-F10 melanoma model. 500,000 4T1 cells were subcutaneously implanted into BALB/C mice, and 500,000 B16-F10 cells were subcutaneously implanted into C57BL/6 mice. Treatment was administered after the tumor exceeded a mean volume of 50 ^mm³ . In both models, only DR-IL-18 resulted in tumor growth inhibition, but not WT IL-18, as shown in Figures 26A (4T1 tumor) and 26B (B16-F10). Treatment was administered after the tumor exceeded a mean volume of 50 ^mm³ , as indicated by boxes marked with “t”. The efficacy of DR-IL-18 was also evaluated in an MHC class I deficient tumor model. B2m-deficient (and therefore MHC class I-deficient) MC38 cells were prepared using CRISPR/Cas9-mediated deletion, as described for B2m-deficient YUMMER cells. B2m-/-MC38 cells were subcutaneously implanted, and treatment began on day ⁷ when the tumor was approximately 65 mm³ on average. SEQ ID NO:61 was administered at 0.32 mg/kg twice weekly for 5 weeks. Anti-PD1 and anti-CTLA4 were administered at 8 mg/kg on the same schedule. MHC class I-deficient tumors were resistant to immune checkpoint suppression with anti-PD1 and anti-CTLA4, but DR-IL-18 treatment resulted in tumor growth inhibition (Figure 27A).

RMA/S is a variant of the RMA lymphoma lineage containing a spontaneous mutation in Tapasin, resulting in a defective antigen load and consequently reduced MHC class I surface expression. One thousand 1,000,000 RMA/S cells were subcutaneously implanted into C57BL/6 mice, and treatment began on day 7. SEQ ID NO:61 was administered at 0.32 mg/kg twice weekly. Anti-PD-1 was administered at 8 mg/kg on the same schedule. RMA/S tumors were resistant to immune checkpoint inhibition with anti-PD-1, while treatment with DR-IL-18 resulted in tumor growth inhibition (Figure 27B).

Example 4: Promotion of cancer cell killing by DR-IL-18 alone or in combination with therapeutic antibodies

In vitro cytotoxicity studies were conducted to evaluate the ability of DR-IL-18 to promote cancer cell killing. CFSE-labeled Raji (B-cell lymphoma) cells were used as target cells. Human peripheral blood mononuclear cells (PBMCs) were isolated and incubated with CFSE-labeled Raji cells at an effector:target (E:T) ratio of 1:10 for 25 h. Human DR-IL-18 variant hCS-1 (1 μM), anti-CD20 antibody rituximab (10 μg/mL), or a combination of both were administered. Cytotoxicity was measured by flow cytometry and calculated as the fraction of CFSE-positive DAPI cells. DR-IL-18 treatment resulted in the killing of target cells, and the combination of DR-IL-18 and rituximab resulted in a higher level of cancer cell killing than either agent alone, indicating that DR-IL-18 can enhance antibody-dependent cell-mediated cytotoxicity (ADCC; Figure 28; *p<0.05, corrected by two-factor ANOVA and Tukey's multiple comparisons). These data suggest that DR-IL-18 can be used in combination with opsonizers, such as tumor-targeting antibodies, to enhance the killing of cancer cells.

Example 5: Efficacy of DR-IL18 against viral infection

This example demonstrates that DR-IL-18 can be used to treat infectious diseases, such as viral infections. C57BL/6 mice were intraperitoneally (IP) infected with ^10⁶ PFU of vaccinia virus (VACV), and the IP administration included either 1 mg/kg WT mIL-18 or DR-IL-18 (SEQ ID NO: 61), as summarized in Figure 29A. Mice were sacrificed on day 3 post-infection, and viral titers in blood and ovaries were measured by RT-PCR. Treatment with DR-IL-18 resulted in a significant reduction in viral load in blood and ovaries, which was not the case with wild-type mIL-18 (Figure 29B, *p<0.05, **p<0.01, ***p<0.001). The efficacy of DR-IL-18 in this systemic viral infection model suggests that DR-IL-18 can be used to control infectious diseases.

Example 6: Second-generation human DR-IL-18 variant induces IL-18R signaling

The ability of second-generation human DR-IL-18 variants SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, and SEQ ID NO:87 to induce signal transduction via the IL-18 receptor was evaluated using IL-18HEK-Blue reporter cells. The generation of these DR-IL-18 variants is described in Example 1, and their affinity for IL-18Rα and IL-18BP is described in Example 2.

HEK-Blue IL-18 sensor cells (InvivoGen) were maintained in complete medium (containing 10% heat-inactivated FBS, 2 mM L-glutamine, 50 U/mL penicillin, and 50 μg/mL streptomycin) supplemented with 100 μg/mL Normocin, 30 μg/mL Blasticidin, 180 μg/mL Zeocin, and 200 μg/mL Hygromycin in DMEM). For cytokine activity assays, 50,000 HEK-Blue IL-18 sensor cells per well in a flat-bottomed 96-well plate were incubated with gradually decreasing concentrations of recombinant human IL-18 or DR-IL-18 variants in a total volume of 200 μL of complete medium. After incubation at 37°C and 5% _CO2 for 24 hours, 30 μL of cell culture supernatant was mixed with 170 μL of QUANTI-Blue assay medium (InvivoGen) and incubated at 37°C and 5% _CO2 until a detectable color change from pink to blue was achieved (0.5–4 hours). The level of alkaline phosphatase was quantified at 655 nm using a spectrophotometer.

Human SEQ ID NO:89, SEQ ID NO:90, and SEQ ID NO:91 showed enhanced potency compared to WT hIL-18, while SEQ ID NO:87 exhibited approximately equivalent potency to WT hIL-18 (Figure 30). Therefore, the data suggest that all tested second-generation human DR-IL-18 variants actively signal via IL-18R.

Example 7: Cysteine mutations that enhance the stability of IL-18 variants

SEQ ID NO:89 is more stable than WT IL-18; however, it can form disulfide-linked dimers, as well as intramolecular disulfide bonds. Therefore, all combinations of monocysteine to serine mutations were generated to test whether mutations at these positions could stabilize the IL-18 molecule and to determine which mutation combinations provided optimal stability.

Systematic mutations were performed on all four cysteine residues in SEQ ID NO:89 to serine residues, as shown in Table 13.

Table 13 shows the cysteine variants of SEQ ID NO:89 that were tested.

Clones SEQ ID NO:1-15 (which are variants of SEQ ID NO:89) were synthesized via Twist and cloned into a Pichia pastoris expression vector containing an n-terminal aga2 leader sequence for secretory expression. The plasmids were transformed into Pichia pastoris and expressed by induction with methanol at 30°C for 60 hours. After induction, the supernatant was run on an SDS/PAGE gel and visualized using Spyro-Ruby staining.

Clones SEQ ID NO:5 and SEQ ID NO:6 showed optimal expression without evidence of intermolecular or intramolecular disulfide bond formation (Fig. 31; e.g., a dimer band of approximately 35 kDa and a lower biband below 18 kDa, respectively, in the non-reduced sample). These two clones contain a combination of C38S and C68S mutations. Any clone with the C127 mutation exhibited lower expression yields. In Fig. 31, R = reduced sample; N = non-reduced sample; MW = molecular weight. The numbers along the bottom refer to the SEQ ID number.

Conclusion: Mutations at positions C38 and C68 (see, for example, SEQ ID NO:6) provide the greatest degree of stabilization (e.g., in the case of the DR-IL-18 variant SEQ ID NO:89). SEQ ID NO:6 (C38S/C68S) in particular shows enhanced expression and significantly increased stability, but with slightly lower potency (though still acceptable).

Example 8: Removal of predicted T cell epitopes

The C68S mutation generated in Example 7 resulted in a predicted novel epitope (e.g., a T-cell epitope). Therefore, six additional non-immunogenic substitutions (SEQ ID NO: 16-21) were constructed at position C68, and their stability/potency was then tested.

Clones containing a substitution mutation (G, A, V, D, E, or N) at C68, SEQ ID NO:16-21, were synthesized using Twist and cloned into an *E. coli* cytoplasmic expression vector with an N-terminal 6xHis-SUMO tag. *E. coli* was grown in 100 mL Terrific Broth in shake flasks and induced with IPTG at 20 °C for 16 h. After induction, the protein was purified using an immobilized metal affinity chromatography (IMAC) column; the SUMO tag was removed by adding the his-tagged Ulp1 protease. Free his-tagged SUMO and his-tagged Ulp1 were then removed using a second IMAC column. Yield and purity were assessed by SDS/PAGE.

The variants were expressed at high titers (Table 14) and showed no evidence of disulfide bond formation by non-reducing SDS-PAGE gel analysis (Figure 32; R = reduced sample; NR = non-reduced sample).

Table 14 summarizes the additional mutations screened at positions 38 and 68, the corresponding sequences, and the protein yield of the DR-IL-18 variants.

As shown in Figure 33, an accelerated shelf-life stability study was conducted to further evaluate the stability of the DR-IL-18 variant. The protein formulation was prepared in PBS at 1 mg/mL and incubated at 51°C for 48 hours. Samples were obtained at several time points, filtered through a 0.45 μm filter, and analyzed by size exclusion chromatography (SEC). The DR-IL-18 variant exhibited a range of stability under these conditions, with SEQ ID NO:16, SEQ ID NO:19, and SEQ ID NO:17 showing the maximum residence time as a percentage of the main peak relative to t=0 (Figure 33). The activity of WT IL-18 relative to the DR-IL-18 variant was measured using an IL-18 reporter assay, in which IL-18 receptor signaling led to the expression of secretory alkaline phosphatase (HEK-Blue IL-18 from InVivoGen). Reporter cells were cultured, stimulated, and secretory alkaline phosphatase was measured as described in Example 6. Compared to WT IL-18, all variants showed increased potency in inducing IL-18R signaling, and the potency was similar to that of the parent variant SEQ ID NO:89 (Table 15 and Figure 34).

Table 15 shows the EC50 values of the DR-IL18 variant for inducing IL-18R signaling, as determined by HEK-Blue reporter cell line assays (see also Figure 34).

Table 16 summarizes the ranking of yield, potency, and stability of the DR-IL-18 variants determined by the measurements in this embodiment.

Example 9: Characterization of the stability, affinity and potency of DR-IL-18 of this disclosure

Based on its favorable potency, stability, and immunogenicity profile, SEQ ID NO:19 was selected for further characterization (see Examples 7 and 8). Additional assays were performed to compare the stability, affinity, and potency of SEQ ID NO:19 (C38S/C68D) with SEQ ID NO:89 (parental C38/C68 molecule) and wild-type human IL-18.

The effects of freeze-thaw cycles on SEQ ID NO:19 and SEQ ID NO:89 were tested. Proteins were formulated in 20 mM acetate at pH 5.0, 8% sucrose, 0.1 mM EDTA, and 0.02% polysorbate (PS) 80. The formulations were then frozen at -80°C and thawed five times at 25°C. The proteins were then analyzed by size exclusion chromatography. A reduction in the main peak of SEQ ID NO:89 was observed, along with the appearance of a dimer peak (indicated by arrows) (Figure 35, top). In contrast, no change or minimal change was detected for SEQ ID NO:19 (Figure 35, bottom), indicating excellent stability of this DR-IL-18 variant after exposure to freeze-thaw cycles.

Stability comparisons were performed using stirring studies after exposure to stirring at 37°C. SEQ ID NO:19 and SEQ ID NO:89 proteins were prepared in 20 mM acetate at pH 5.0, 8% sucrose, 0.1 mM EDTA, and 0.02% PS80. The samples were stirred and shaken at 37°C for 72 hours. Samples were obtained at t = 0, 24 (d1), and 72 (d3) hours and analyzed by size exclusion chromatography. A decrease in the main peak of SEQ ID NO:89 was observed, as indicated by the arrows, with a relative increase in the dimer/HMW (high molecular weight) content (Figure 36, top). In contrast, no change or minimal change was detected for SEQ ID NO:19. Figure 36 depicts the size exclusion chromatographic data for SEQ ID NO:19 and SEQ ID NO:89 based on stirring studies.

IL-18 receptor affinity. The affinity of human IL-18 and SEQ ID NO:19 for human and cynomolgus monkey IL-18Rα and IL-18BP was determined using surface plasmon resonance. Biotinylated receptor proteins were immobilized on a streptavidin capture chip, and either IL-18 or SEQ ID NO:19 was used as the free analyte. Binding was determined using single-cell kinetics. In this assay, WT hIL-18 and SEQ ID NO:19 showed similar binding affinity for IL-18Rα (Fig. 37A). However, WT IL-18 bound IL-18BP with extremely high affinity (<1 nM), while SEQ ID NO:19 did not show detectable binding to IL-18BP (Fig. 37B).

The activities of WT IL-18 and SEQ ID NO:19 in the presence of IL-18BP were evaluated using an IL-18 reporter molecule assay, in which IL-18 receptor signaling led to the expression of secretory alkaline phosphatase (HEK-Blue IL-18 from InVivoGen). Reporter cells were cultured and secretory alkaline phosphatase levels were measured, with WT IL-18 or SEQ ID NO:19 applied to HEK-BLUE IL-18 reporter cells at fixed concentrations of 1.0 or 0.1 ng/mL, respectively, except in the presence of IL-18BP concentrations ranging from ^10⁻⁶ to ^10⁻⁹ M. Downstream secretory embryonic alkaline phosphatase (SEAP) activity was measured according to the manufacturer's instructions. WT hIL-18 was effectively inhibited by the addition of IL-18BP, while SEQ ID NO:19 retained strong induction of IL-18R signaling at all IL-18BP concentrations (Figure 38).

Example 10: Administration of IL-18 peptide

The first dosing study was conducted to compare the efficacy and tolerability of DR-IL-18 administration once daily, every other day, twice weekly, and once weekly in a cancer model. This study was conducted in mice, and therefore SEQ ID NO:61, a mouse DR IL-18 variant peptide (i.e., mouse IL-18 that binds to and activates mouse IL-18R but is not inhibited by mouse IL-18BP), was used. Human variants were not used because human IL-18 protein does not cross-react with mouse IL-18R (i.e., it does not bind to and activate mouse IL-18R).

250,000 MC38 cells were subcutaneously implanted, and treatment began ^once the tumor reached 80 mm³. Dosing was administered subcutaneously for 2 weeks at qD (once daily), qoD (every other day), BiW (twice weekly), or qW (once weekly). All doses and schedules showed efficacy (Figure 39). Most regimens were well tolerated. However, daily dosing at 0.1 mpk (mg/kg) and higher showed toxicity. Tumor growth typically occurred after treatment discontinuation. Once the endpoint was reached, a tumor growth curve was plotted against the last point previously recorded. The curve terminated once 50% or more of the animals reached the endpoint. Results: Daily dosing at 0.1 mpk (mg/kg) and higher appeared toxic and was associated with weight loss. DR-IL-18 was effective in all other dosing schedules. Efficacy was superior to anti-PD-1 therapy, which was evident at all tested doses, even at once-weekly dosing.

A second dosing study was conducted to compare once-weekly and twice-weekly dosing in a cancer model. SEQ ID NO:61 is used as above because these studies were conducted in mice. 250,000 MC38 cells were subcutaneously implanted, and treatment began once the tumor reached 100 ^mm³ (day 7). Animals were administered four doses once-weekly (qW) or two doses every two weeks (q2W) via subcutaneous injection. A 100-fold range of doses (0.01 to 1.0 mg/kg) was tested. All doses and schedules showed efficacy, even at 0.01 mg/kg. The maximum effective dose (MaxED) was approximately 0.32 mg/kg at both qW and q2W dosing (Figure 40). Strong DR IL-18 efficacy was observed even with infrequent pulsed dosing. Even once-every-two-week dosing was more effective than twice-weekly anti-PD-1 therapy. This is quite surprising given the dosing requirements of other interleukins (e.g., IL-2/IL-15, IL-10) that require prolonged half-life or frequent dosing (e.g., TID dosing of IL-2).

Monkey tolerability assays were performed to determine the tolerability of DR-IL-18 at various doses and using once-weekly (qW) or twice-weekly (biW) dosing regimens. Cynomolgus monkeys were treated subcutaneously with DR-IL-18 (SEQ ID NO:89 or SEQ ID NO:19) weekly (qW) or twice-weekly (biW) at doses ranging from 0.001 mg/kg to 3.2 mg/kg. Blood samples were drawn periodically and hemoglobin levels were measured. Illustrative dosing and sampling protocols are provided in Figure 41A. Results showed that dosing frequencies exceeding 1x/week resulted in an undesirable decrease in hemoglobin concentrations in the study. Cynomolgus monkeys treated subcutaneously with SEQ ID NO:89 twice weekly showed a dose-dependent decrease in hemoglobin compared to monkeys treated with saline (Figure 41B, top). In contrast, weekly treatment with DR-18 (SEQ ID NO:89 or SEQ ID NO:19), even at doses as high as 3.2 mg/kg, did not result in a decrease in hemoglobin levels compared to control saline treatment (Figure 41B, top and bottom).

Example 11: Production of DR-IL-18 variant in bacteria (Escherichia coli)

Procedure 1: Cell-free in vitro cleavage using SUMO-protein enzyme

In this embodiment, DR-IL-18 (in this case, SEQ ID NO:89) is tagged with a His-tagged SUMO tag, expressed in E. coli, and the SUMO tag is cleaved using a His-tagged SUMO protease to produce active IL-18 protein.

DR-IL-18 was cloned into an E. coli cytoplasmic expression vector with an N-terminal 6xHis-SUMO tag. E. coli were grown in 100 mL Terrific Broth in shake flasks and induced with IPTG at 20°C for 16 h. The fusion protein (the fusion of the His-tagged SUMO tag and DR-IL-18) was initially captured from bacterial lysates using immobilized metal affinity chromatography (IMAC). This step was highly efficient, yielding high yields at >90% purity. After cleavage with the His-tagged Ulp1 SUMO protease to remove the SUMO tag, the His-tagged SUMO tag and the His-tagged SUMO protease were efficiently removed using IMAC in exhaustion mode. Yield and purity were assessed by SDS/PAGE. After this step, the resulting protein purity was >90% (typically >95%) (Figure 42A). Cleavage was efficient and could be performed at ratios of 1:500 or higher. Note: Expression at a lower temperature (21°C) significantly increases soluble yield (this specific study was conducted at 26°C). Figure 42B summarizes the processing and purification steps.

Figures 43A and 43B provide the results of the cleavage reaction, as monitored by RP-HPLC determination. RP-HPLC determination can also be used to calculate step yield and titer.

Process 2: Cleavage within bacterial cells using co-expressed SUMO-protease

This example demonstrates that SUMO-tagged IL-18 (in this case, the stabilized DR IL-18 SEQ ID NO: 19) and SUMO-protease can be co-expressed in bacteria (in this case, *E. coli*), resulting in the removal of the SUMO tag via cleavage within the bacterial cell. The SUMO protease is expressed under the control of a rhamnose-inducible promoter. Rhamnose induction of the protease allows for individual, timed, and tunable induction of the protease relative to IL-18 (e.g., DR-IL-18). *E. coli* expressing SUMO-tagged DR-IL-18 was treated with 0.1 to 2 mM L-rhamnose, resulting in the induction of SUMO protease expression and cleavage of the SUMO tag, releasing the untagged SEQ ID NO: 19 (Figure 44). Note: On a non-reducing gel, the SUMO tag ran faster than DR-18; some cleavage was observed without rhamnose treatment due to promoter "leakage".

The total expression of the cleaved product was comparable to that of the SUMO-fusion (after taking SUMO quality into account). In vivo (in bacterial cells) cleavage was monitored using RP-HPLC (Figure 45, showing SUMO tag cleavage in rhamnose-treated cultures). 0.2–0.5 mM L-rhamnose appeared to provide good results in these specific experiments.

Successful results were obtained by placing the SUMO-protease in a bicistronic form downstream of IL-18 (e.g., DR-IL-18) and by expressing the SUMO protease from a separate promoter (e.g., from a separate plasmid), for example, under an inducible promoter (such as a rhamnose-inducible promoter).

Downstream processes can be used to capture and purify the cleaved product (i.e., the active DR-IL-18 protein) (see, for example, Example 12).

In vivo (in bacterial cells) cleavage was monitored using RP-HPLC. Figure 45 provides a chromatogram of in vivo (in bacterial cells) cleavage. Note: "Leakage" from the rhamnose promoter produced sufficient SUMO protease to cleave most of the fusion protein. 0.2–0.5 mM L-rhamnose appears to be the optimal concentration range for these specific experiments.

Example 12: Production of IL-18 variants using yeast (Pichia pastoris)

A system for expressing and secreting DR-IL-18 was developed using Pichia pastoris (yeast). The signal peptide was added to DR-IL-18 and cloned into an expression vector (Figure 46, top). This system resulted in the secretion of DR-IL-18 and cleavage of the signal peptide by a signal peptidase/Kex2, avoiding the need for a tag-removing protease.

In this illustrative embodiment, DR-IL-18 variants SEQ ID NO:89 and SEQ ID NO:87 were tested. The DR-IL-18 variants were synthesized by Twist Biosciences and cloned into a Pichia pastoris expression vector containing an n-terminal aga2 leader sequence for secretory expression. The plasmid was transformed into Pichia pastoris and expressed by methanol induction at 30°C for 60 hours, resulting in the production of DR-IL-18 at high titers, including >1 g/L in some cases. DR-IL-18 with an active native N-terminus (tyrosine in the case of human IL-18) was directly secreted into the culture medium (Figure 47).

A two-step chromatographic process was used to purify IL-18 (in this case, DR IL-18 SEQ ID NO: 89) without label to a purity >97%, monodispersity 96%, and <2 EU/mg (Figures 46 and 48; UFDF = ultrafiltration (UF) and percolation (DF)). “Capto MMC” is a multi-mode salt-resistant resin used for capturing and intermediate purification of proteins from bulk feeds via packed-bed chromatography. HIC = hydrophobic interaction chromatography (e.g., phenyl agarose gel HIC is depicted in this figure).

DR-18 purification was performed. Figure 48 shows an SDS-PAGE gel illustrating the purification of DR-18 (e.g., from Pichia pastoris or from bacteria). The chromatographic steps facilitated the label-free purification of IL-18 (in this case, DR IL-18 SEQ ID NO: 89) to a purity >97%, monodispersity 96%, and <2 EU/mg. UFDF = Ultrafiltration (UF) and Percolation (DF). “CaptoMMC” is a multi-mode salt-resistant resin used for capturing and intermediate purification of proteins from bulk feeds via packed-bed chromatography. HIC = Hydrophobic Interaction Chromatography (e.g., phenyl agarose gel HIC is depicted in this figure).

Example 13: Generation of a cell library for producing DR IL-18 variants

This embodiment illustrates the generation of a plasmid system and cell bank for producing SEQ ID NO:19 (a variant of DRIL-18 of this disclosure with mutations at positions C38 and C68).

A dual plasmid system was generated for the expression of SEQ ID NO:19 in *E. coli*. For full activity, IL-18 (e.g., DR IL-18, such as SEQ ID NO:19) may require its native N-terminus instead of an N-terminal methionine. Therefore, a plasmid was designed using a small ubiquitin-associated modification (SUMO) expression system that utilizes a SUMO leader sequence from *Saccharomyces cerevisiae* at the N-terminus, such that the SUMO leader sequence can be cleaved by the SUMO protease ULP1.

Expression of DR IL-18 with the SUMO leader peptide was driven by the T7 promoter in a high-copy plasmid to maximize expression. The pD451-SR plasmid backbone was used. This plasmid contains the kanamycin resistance gene, the pUC origin of replication, and a T7 promoter, the presence of which is required to express the protein SEQ ID NO:19 after induction with isopropyl β-d-1-thiogalactoside (IPTG).

A second plasmid with a lower copy number was used to achieve expression of the SUMO protease driven by a rhamnose promoter, which was relatively weak, allowing expression to be tuned by varying the amount of rhamnose added to the culture. The SUMO protease gene was cloned into plasmid pD883-SR. This plasmid contains the chloramphenicol resistance gene, the p15a origin of replication, and has a rhamnose promoter as part of the rhaBAD regulatory sequence to achieve controlled expression of the SUMO protease after rhamnose induction.

Two plasmids were simultaneously transformed into the *E. coli* strain T7 Express, and clones containing the plasmids were selected using agar plates containing kanamycin and chloramphenicol. T7 Express is a strain that achieves very high gene expression using the T7 promoter after IPTG induction. It contains T7 polymerase in the lactose operon and does not contain λ prophage. The system of two plasmids with different origins of replication and different antibiotic resistance genes allows both plasmids to be maintained stably and tested independently. Both the SUMO protease and the SUMO leader peptide have N-terminal His tags, allowing them to be removed by immobilized metal affinity chromatography. This expression system was designed to allow very high expression of correctly processed SEQ ID NO:19, which has been correctly cleaved by, for example, 2–3 g/L of SUMO protease.

Transform *E. coli* strain T7 Express competent cells with 1 μL of each of the two plasmids. The transformed cells were then grown overnight at 37°C on LB agar plates supplemented with 50 μg/mL kanamycin (KAM) and 50 μg/mL chloramphenicol (CAM). Single, well-grown colonies were inoculated into 10 mL of medium and grown overnight at 30°C. A cell bank was prepared by mixing an equal volume of the overnight culture with 30% (v/v) sterile glycerol solution, and the mixture was then aliquoted into appropriate 200 μL vials and stored at -20°C.

Thaw a vial and subculture it onto a TBA (Terrific Broth Agar) plate containing 50 μg/mL KAM and 50 μg/mL CAM, and incubate at 30°C for 20 to 24 hours. Inoculate a single colony into 50 mL of medium and incubate overnight in a shaker at 30°C and 220 rpm. Inoculate this culture at 0.2 to 0.3 U at OD 600 nm and incubate in a shaker at 30°C and 220 rpm. The final culture OD 600 nm was 1.9 (before glycerol). Prepare a new cell bank by mixing 56 mL of cell culture with 8 mL of 80% sterile glycerol (10% final concentration) and aliquoting it into 50 frozen vials (1 mL each). Label the vials and store directly at -80°C. Test this cell bank before and after freezing to demonstrate plasmid stability and retention of high concentrations of live cells (Table 17).

Table 17: Viability, purity, and plasmid stability of cell bank samples before and after freezing

Thaw a vial gently at 2°C to 8°C for ≤30 min. After thawing, transfer 1 mL of cell suspension to a 0.5 L shake flask containing prepared TB medium supplemented with salts and antibiotics (kanamycin and chloramphenicol). Incubate this culture at 37°C and 300 rpm for 15 to 18 h until a target optical density of ≥5.0 UO is achieved. Then, transfer the required amount of cell suspension from the first growth to a 1 L shake flask for further culture, continuing for at least 3 to 4 h until the desired optical density of 1.50 UO to 3.50 UO is achieved. Combine and mix 160 mL of the second growth cell culture with 160 mL of 50% glycerol solution, and aliquot 1.0 mL of the cell suspension into sterile cryogenic vials and label appropriately. Freeze vials from this second cell bank at -80 ± 10°C. Controls during cell bank preparation include the purity of the culture after freezing, the total viable cell count after freezing, and the stability of the plasmids after freezing. Tests showed no phage contamination and a plasmid copy number of 288.15. The IL-18 encoding plasmid retained 100% sequence identity with the originally designed sequence, and restriction enzyme digestion yielded the desired plasmid fragment size, indicating no changes to the plasmid structure or inserted sequence.

Example 14: Ellman titration of free thiols

This embodiment demonstrates that the natural SEQ ID NO:19, the DRIL-18 variant of this disclosure with mutations at positions C38 and C68, contains very little free cysteine.

Ellman's reagent (5,5'-dithio-bis-[2-nitrobenzoic acid]/DTNB) was used to estimate the presence of thiohydrogen groups in the samples by comparison with a standard curve of compounds containing thiohydrogen groups. DTNB reacts with the free thiol of the protein to produce a colored product, 2-nitro-5-thiobenzoate (TNB), which has an absorbance at 412 nm. For every mole of thiol reacted with DTNB, one mole of TNB is produced, and the number of free cysteine residues present per mole of protein can be estimated by quantifying the amount of TNB. The results showed a very low presence of free cysteine in the native SEQ ID NO:19 (Table 18), consistent with the following: mutations at C38 and C68 to other residues; lack of solvent exposure for the two remaining residues in SEQ ID NO:19; and reduced intramolecular and intermolecular disulfide bond formation observed in the peptides with such mutations in Examples 7 and 8.

Table 18: Determination of free thiol groups in sample SEQ ID NO:19.

Example 15: Formulation Development

Formulation development is conducted to select active pharmaceutical ingredients (APIs) and pharmaceutical product formulations for toxicology and clinical studies. SEQ ID NO:19 The API formulation development process includes evaluating optimal pH, protein concentration, and excipient screening. The formulation is designed to support the following product attributes: liquid form, sterility, suitability for subcutaneous injection, and a shelf life of at least 24 months at temperatures ranging from 2°C to 8°C or -20°C. The formulation is evaluated through stability studies. Details of the stability studies are mentioned below:

1. To assess potential alterations in protein integrity at selected pH values, stress-exposed samples were compared to initial samples. Stress conditions were maintained at 40 ± 2 °C/25% RH for up to 3 weeks.

2. Freeze-thaw cycles were performed to assess the sensitivity of the protein to repeated freezing and thawing. Up to five freeze-thaw cycles were performed under the following conditions: -80°C for up to 16 hours, followed by 8 hours at room temperature.

3. Shaking and stirring were performed to evaluate protein degradation pathways and rates (at 150 rpm, 25°C/60% RH, for up to 2 weeks). The drug substance formulation development studies of SEQ ID NO:19 are summarized in Table 19.

Table 19

Abbreviations: AEX-HPLC = Anion Exchange Liquid Chromatography; MFI = Microfluidic Imaging; RP-HPLC = Reversed-Phase High Performance Liquid Chromatography; PS80 = Polysorbate 80; SDS-PAGE = Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis; SE-HPLC = Size Exclusion Liquid Chromatography.

During Study 1, six formulations with different concentrations and pH values were compared under thermal stress conditions. After 3 weeks at 40±2℃/25%RH, the formulations with lower pH values were less stable. Therefore, pH 6.5 and 7.0 were selected for Study 2. Furthermore, protein purity during Study 1 did not show a dependence on protein concentration. Therefore, protein concentrations of 20 mg/mL and 30 mg/mL were used in Study 2. During Study 2, additional excipients, ethylenediaminetetraacetic acid (EDTA) and L-methionine, were added to the formulation to maintain optimal protein stability and prevent its oxidation. No differences were observed between formulations under all tested conditions. Based on the formulation development results, the final selected composition was 30 mg/mL SEQ ID NO:19 protein in 10 mM L-histidine/L-histidine-HCl, 8% sucrose, and 0.02% (w/v) polysorbate 80 at pH 6.5.

Example 16: Determining the therapeutic dose for human administration

The primary objective of this study was to determine the median dose (MTD) and relapse-to-diet (RP2D) of SEQ ID NO:19 in patients with relapsed or refractory solid tumors. Secondary objectives included assessing the overall safety and tolerability of SEQ ID NO:19, and characterizing the pharmacokinetic (PK) profiles of a single-dose regimen. Researchers monitored study safety by reviewing the following: incidence of diabetic thromboembolic events (DLTs) and adverse events (AEs), changes in clinical laboratory parameters, vital signs, ECG tests and parameters, physical examination, and the incidence of anti-SEQ ID NO:19 antibodies. Researchers also monitored for the likelihood of patients developing a cytokine storm, as SEQ ID NO:19 is an immune agonist.

Patients with relapsed or refractory solid tumors were enrolled in dose escalation studies to determine the maximum tolerated dose (MTD) and recommended phase 2 dose (RP2D) of SEQ ID NO:19. Eligible patients were 18 years of age or older and diagnosed with a solid tumor that had progressed with standard therapy or for which prolonging survival was unavailable or unsuitable for standard care. Patients received a subcutaneous (SC) dose of SEQ ID NO:19 once weekly for 28 days (one cycle). The dose range could be from 30 μg SEQ ID NO:19/kg patient (μg/kg) to 1200 μg/kg. Further dose escalations could include a range from 1200 μg/kg to 1.5 mg/kg. The initial test dose was 30 μg/kg. Any dose within those two ranges could be tested in the study to determine the MTD and RP2D. Dose escalation followed an mTPI model with an initial cohort of 2 patients, and major dose-limiting toxicity (DLT) monitoring was conducted 28 days after the first dose. Patients were monitored for 7 days after the first dose of the new medication until they were ready to receive the new dose.

The mTPI model uses a simple β-binomial stratified model where the decision rule is based on calculating the unit probability mass (UPM) corresponding to three dosing intervals: underdose, appropriate, and overdose. The mTPI method calculates the UPM for the three dosing intervals, and the one with the largest UPM signifies the corresponding dose discovery decision, which provides the dose level for future patients. The target toxicity probability is 25%, and the acceptable DLT interval is 20–30%. If the initial two patients in the first cycle do not experience a DLT, enrollment in the next cohort begins at the next dose level in the next cycle. If one of the two initial patients experiences a DLT, the dose is reduced to the next lowest dose. If no additional DLT is observed at the lower dose level when the cohort expands to six patients, dose escalation is resumed. Once the first DLT is observed, only dose escalations of ≤33% are applied to all remaining cohorts.

At the end of the study, the MTD estimate was derived from order-preserving regression. Specifically, the MTD estimate was the dose level at which the order-preserving estimate of the DLT rate was less than or equal to 25%, and it was the dose level at which the order-preserving estimate of the DLT rate was closest to the target DLT rate. If the maximum sample size of 42 patients had been achieved, dose escalation was stopped, and 6 to 12 patients were enrolled to predict the MTD dose level, or all explored doses appeared too toxic and the MTD was uncertain.

Blood samples were collected from all patients for PK analysis, and researchers used these samples to calculate PK parameters. Blood samples were collected on days 1, 2, 3, 8, 15, and 22 of cycle 1, days 1 and 15 of cycle 2, and day 1 of alternating cycles starting from cycle 3, until the study was completed. Patients also provided blood samples 30 days after the last dose of SEQ ID NO:19 and before starting a new therapy. PK parameters _Cmax , _tmax , AUC _0-1 , AUC _τ , AUC _0-∞ , and t _1/2 were estimated from plasma concentration-time data using non-compartmental analysis (where possible). Additional PK parameters may be considered where appropriate. Researchers also used blood samples to monitor patients for the development of drug resistance antibodies.

Example 17: Efficacy test in patients with solid tumors.

The primary objective of this study is to evaluate the antitumor activity of SEQ ID NO:19 under RP2D in a cohort of recurrent solid tumors. The primary efficacy endpoint is confirmed objective response (CR) or partial response (PR) observed in patients according to RECIST 1.1. Secondary efficacy endpoints will include: best objective response, disease control rate (DCR) (CR, PR, or stable disease (SD) ≥12 weeks), duration of response (DOR), time to response (ToR), progression-free survival (PFS), and overall survival (OS).

This study enrolled patients with different types of solid tumors to evaluate the efficacy of SEQ ID NO:19 treatment in these tumor types. Eligible patients were 18 years of age or older and had one of the following solid tumor types: melanoma, renal cell carcinoma (RCC), triple-negative breast cancer (TNBC), non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (SCCHN), or microsatellite instability-high (MSI-Hi) tumors. Patients underwent a mandatory tumor biopsy within 28 days of the first dose. For RCC, TNBC, NSCLC, and MSI-Hi, up to 25 patients were enrolled for each tumor type; for metastatic melanoma and SCCHN, up to 28 patients were enrolled for each cancer type. If no objective response was observed in the cancer type cohort, no additional patients were enrolled in the cohort.

Patients received a single subcutaneous dose of SEQ ID NO:19, once weekly for 28 days (one cycle). This dose was derived from the RP2D dose escalation study in humans. During the study, patients underwent disease assessment every 8 or 12 weeks, which may include computed tomography (CT), magnetic resonance imaging (MRI), and/or positron emission tomography (PET) scans. Researchers evaluated all targeted and non-target lesions from scans using the Responsive Evaluation Criteria for Solid Tumors (RECIST 1.1). Patients underwent a second mandatory tumor biopsy prior to the start of cycle 2. Optional biopsies were available upon disease progression during the study.

Blood samples were collected from all patients for PK analysis, and researchers used these samples to calculate PK parameters. Blood samples were collected on days 1 and 15 of cycle 1, days 1 and 15 of cycle 2, and day 1 of alternating cycles starting from cycle 3, until the study was completed. Patients also provided blood samples 30 days after the last dose of SEQ ID NO:19 and before starting a new therapy. PK parameters _Cmax , _tmax , AUC _0-1 , AUC _τ , AUC _0-∞ , and t _1/2 were estimated from plasma concentration-time data using non-compartmental analysis (where possible). Additional PK parameters may be considered where appropriate. Researchers also used blood samples to monitor patients for the development of drug resistance antibodies.

amino acid sequence

Wild-type IL-18 amino acid sequence

Human interleukin-18 (mature form)

YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED(SEQ ID NO:30)

Mature form of mouse interleukin-18

NFGRLHCTTAVIRNINDQVLFVDKRQPVFEDMTDIDQSASEPQTRLIIYMYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFQKRVPGHNKMEFESSLYEGHFLACQKEDDAFKLILKKKDENGDKSVMFTLTNLHQS(SEQ ID NO:31)

Q14116|IL18_Human Interleukin-18 (Uncleaved Precursor)

MAAEPVEDNCINFVAMKFIDNTLYFIAEDDENLESDYFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED(SEQ ID NO:32)

P70380|IL18_Mouse Interleukin-18 (Uncleaved Precursor)

MAAMSEDSCVNFKEMMFIDNTLYFIPEENGDLESDNFGRLHCTTAVIRNINDQVLFVDKRQPVFEDMTDIDQSASEPQTRLIIYMYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFQKRVPGHNKMEFESSLYEGHFLACQKEDDAFKLILKKKDENGDKSVMFTLTNLHQS(SEQ ID NO:33)

The amino acid sequence of the first-generation human interleukin-18 bait resistance variant.

The amino acid sequence of the second-generation human interleukin-18 bait resistance variant

Amino acid sequence of mouse interleukin-18 bait resistance variant

Human-decoy-decoy (D2D) variant amino acid sequence

Mouse bait-bait (D2D) variant amino acid sequence

SEQ ID NO:89 (also referred to herein as "6-12")

YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISKYSDSLARGLAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRDVPGHSRKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED(SEQ ID NO:89)

SEQ ID NO:6

Variants of SEQ ID NO:89 ( C38S/C68S)

YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSD S RDNAPRTIFIISKYSDSLARGLAVTISVK S EKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRDVPGHSRKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED

(SEQ ID NO:6)

SEQ ID NO:16

Variants of SEQ ID NO:89 ( C38S/C68G)

YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSD S RDNAPRTIFIISKYSDSLARGLAVTISVK G EKISTLSCENKIISFKEMNPPDNIKDT

KSDIIFFQRDVPGHSRKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED(SEQ IDNO:16)

SEQ ID NO: 17

Variants of SEQ ID NO:89 ( C38S/C68A)

YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSD S RDNAPRTIFIISKYSDSLARGLAVTISVK A EKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRDVPGHSRKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED(SEQ ID NO:17)

SEQ ID NO:18

Variants of SEQ ID NO:89 ( C38S/C68V)

YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSD S RDNAPRTIFIISKYSDSLARGLAVTISVK V EKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRDVPGHSRKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED(SEQ ID NO:18)

SEQ ID NO:19

Variants of SEQ ID NO:89 ( C38S/C68D)

YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSD S RDNAPRTIFIISKYSDSLARGLAVTISVK D EKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRDVPGHSRKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED(SEQ ID NO:19)

SEQ ID NO: 20

Variants of SEQ ID NO:89 ( C38S/C68E)

YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSD S RDNAPRTIFIISKYSDSLARGLAVTISVK E EKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRDVPGHSRKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED(SEQ ID NO:20)

SEQ ID NO:21

Variants of SEQ ID NO:89 ( C38S/C68N)

YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSD S RDNAPRTIFIISKYSDSLARGLAVTISVK N EKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRDVPGHSRKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED(SEQ ID NO:21)

SEQ ID NO: 1

YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSD S RDNAPRTIFIISKYSDSLARGLAVTISVK S EKISTLS S ENKIISFKEMNPPDNIKDTKSDIIFFQRDVPGHSRKMQFESSSYEGYFLA S EKERDLFKLILKKEDELGDRSIMFTVQNED(SEQ ID NO:1)

SEQ ID NO: 2

C ​ ​ ​

SEQ ID NO: 3

YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSD S RDNAPRTIFIISKYSDSLARGLAVTISVK C EKISTLS S ENKIISFKEMNPPDNIKDTKSDIIFFQRDVPGHSRKMQFESSSYEGYFLA S EKERDLFKLILKKEDELGDRSIMFTVQNED(SEQ ID NO:3)

SEQ ID NO:4

YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSD S RDNAPRTIFIISKYSDSLARGLAVTISVK S EKISTLS C ENKIISFKEMNPPDNIKDTKSDIIFFQRDVPGHSRKMQFESSSYEGYFLA S EKERDLFKLILKKEDELGDRSIMFTVQNED(SEQ ID NO:4)

SEQ ID NO:5

YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSD S RDNAPRTIFIISKYSDSLARGLAVTISVK S EKISTLS S ENKIISFKEMNPPDNIKDTKSDIIFFQRDVPGHSRKMQFESSSYEGYFLA C EKERDLFKLILKKEDELGDRSIMFTVQNED(SEQ ID NO:5)

SEQ ID NO:7

YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSD S RDNAPRTIFIISKYSDSLARGLAVTISVK C EKISTLS S ENKIISFKEMNPPDNIKDTKSDIIFFQRDVPGHSRKMQFESSSYEGYFLA C EKERDLFKLILKKEDELGDRSIMFTVQNED(SEQ ID NO:7)

SEQ ID NO:8

YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSD S RDNAPRTIFIISKYSDSLARGLAVTISVK C EKISTLS C ENKIISFKEMNPPDNIKDTKSDIIFFQRDVPGHSRKMQFESSSYEGYFLA S EKERDLFKLILKKEDELGDRSIMFTVQNED(SEQ ID NO:8)

SEQ ID NO:9

YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSD C RDNAPRTIFIISKYSDSLARGLAVTISVK S EKISTLS S ENKIISFKEMNPPDNIKDTKSDIIFFQRDVPGHSRKMQFESSSYEGYFLA C EKERDLFKLILKKEDELGDRSIMFTVQNED(SEQ ID NO:9)

SEQ ID NO: 10

YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSD C RDNAPRTIFIISKYSDSLARGLAVTISVK S EKISTLS C ENKIISFKEMNPPDNIKDTKSDIIFFQRDVPGHSRKMQFESSSYEGYFLA S EKERDLFKLILKKEDELGDRSIMFTVQNED(SEQ ID NO:10)

SEQ ID NO: 11

YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSD C RDNAPRTIFIISKYSDSLARGLAVTISVK C EKISTLS S ENKIISFKEMNPPDNIKDTKSDIIFFQRDVPGHSRKMQFESSSYEGYFLA S EKERDLFKLILKKEDELGDRSIMFTVQNED(SEQ ID NO:11)

SEQ ID NO: 12

YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSD S RDNAPRTIFIISKYSDSLARGLAVTISVK C EKISTLS C ENKIISFKEMNPPDNIKDTKSDIIFFQRDVPGHSRKMQFESSSYEGYFLA C EKERDLFKLILKKEDELGDRSIMFTVQNED(SEQ ID NO:12)

SEQ ID NO: 13

YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSD C RDNAPRTIFIISKYSDSLARGLAVTISVK S EKISTLS C ENKIISFKEMNPPDNIKDTKSDIIFFQRDVPGHSRKMQFESSSYEGYFLA C EKERDLFKLILKKEDELGDRSIMFTVQNED(SEQ ID NO:13)

SEQ ID NO: 14

YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSD C RDNAPRTIFIISKYSDSLARGLAVTISVK C EKISTLS S ENKIISFKEMNPPDNIKDTKSDIIFFQRDVPGHSRKMQFESSSYEGYFLA C EKERDLFKLILKKEDELGDRSIMFTVQNED(SEQ ID NO:14)

SEQ ID NO:15

YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSD C RDNAPRTIFIISKYSDSLARGLAVTISVK C EKISTLS C ENKIISFKEMNPPDNIKDTKSDIIFFQRDVPGHSRKMQFESSSYEGYFLA S EKERDLFKLILKKEDELGDRSIMFTVQNED(SEQ ID NO:15)

Example SUMO label

DSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQAPEDLDMEDNDIIEAHREQIGG(SEQ ID NO:26)

Example of a SUMO tag with the His tag

MGHHHHHHGSLQDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQAPEDLDMEDNDIIEAHREQIGG(SEQ ID NO:27)

Exemplary SUMO protease

LVPELNEKDDDQVQKALASRENTQLMNRDNIEITVRDFKTLAPRRWLNDTIIEFFMKYIEKSTPNTVAFNSFFYTNLSERGYQGVRRWMKRKKTQIDKLDKIFTPINLNQSHWALG IIDLKKKTIGYVDSLSNGPNAMSFAILTDLQKYVMEESKHTIGEDFDLIHLDCPQQPNGYDCGIYVCMNTLYGSADAPLDFDYKDAIRMRRFIAHLILTDALK(SEQ ID NO:28)

Exemplary His-tagged SUMO protease

MGSSHHHHHHSSGSLVPELNEKDDDQVQKALASRENTQLMNRD

NIEITVRDFKTLAPRRWLNDTIIEFFMKYIEKSTPNTVAFNSFFYTNL

SERGYQGVRRWMKRKKTQIDKLDKIFTPINLNQSHWALGIIDLKK

KTIGYVDSLSNGPNAMSFAILTDLQKYVMEESKHTIGEDFDLIHLD

CPQQPNGYDCGIYVCMNTLYGSADAPLDFDYKDAIRMRRFIAHLI

LTDALK

(SEQ ID NO:29)

Exemplary non-limiting aspects of this disclosure

The aspects of the subject matter of the invention described above (including embodiments) may be beneficial individually or in combination with one or more other aspects or embodiments. Without limiting the foregoing description, certain non-limiting aspects of this disclosure are provided in Groups A and B below. It will be apparent to those skilled in the art upon reading this disclosure that each individually numbered aspect may be used or combined with any previously or subsequently individually numbered aspect. This is intended to support combinations of all such aspects and is not limited to combinations of aspects explicitly provided below. It will be apparent to those skilled in the art that various changes and modifications may be made without departing from the spirit or scope of the invention.

Sequence List

<110> Simcha IL-18 Company

Yale University

Aaron Lin

Tom Boone

<120> Interleukin-18 variants and usage

<130> SMCH-003PRV

<160>  222

<170> PatentIn version 3.5

<210>  1

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400>  1

Tyr Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

Asp Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Glu Asp

20 25 30

Met Thr Asp Ser Asp Ser Arg Asp Asn Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Lys Tyr Ser Asp Ser Leu Ala Arg Gly Leu Ala Val Thr Ile

50 55 60

Ser Val Lys Ser Glu Lys Ile Ser Thr Leu Ser Ser Glu Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Gln Arg Asp Val Pro Gly His Ser Arg Lys

100 105 110

Met Gln Phe Glu Ser Ser Ser Tyr Glu Gly Tyr Phe Leu Ala Ser Glu

115 120 125

Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu Asp

145 150 155

<210>  2

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400>  2

Tyr Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

Asp Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Glu Asp

20 25 30

Met Thr Asp Ser Asp Cys Arg Asp Asn Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Lys Tyr Ser Asp Ser Leu Ala Arg Gly Leu Ala Val Thr Ile

50 55 60

Ser Val Lys Ser Glu Lys Ile Ser Thr Leu Ser Ser Glu Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Gln Arg Asp Val Pro Gly His Ser Arg Lys

100 105 110

Met Gln Phe Glu Ser Ser Ser Tyr Glu Gly Tyr Phe Leu Ala Ser Glu

115 120 125

Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu Asp

145 150 155

<210> 3

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 3

Tyr Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

Asp Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Glu Asp

20 25 30

Met Thr Asp Ser Asp Ser Arg Asp Asn Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Lys Tyr Ser Asp Ser Leu Ala Arg Gly Leu Ala Val Thr Ile

50 55 60

Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Ser Glu Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Gln Arg Asp Val Pro Gly His Ser Arg Lys

100 105 110

Met Gln Phe Glu Ser Ser Ser Tyr Glu Gly Tyr Phe Leu Ala Ser Glu

115 120 125

Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu Asp

145 150 155

<210> 4

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400>  4

Tyr Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

Asp Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Glu Asp

20 25 30

Met Thr Asp Ser Asp Ser Arg Asp Asn Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Lys Tyr Ser Asp Ser Leu Ala Arg Gly Leu Ala Val Thr Ile

50 55 60

Ser Val Lys Ser Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Gln Arg Asp Val Pro Gly His Ser Arg Lys

100 105 110

Met Gln Phe Glu Ser Ser Ser Tyr Glu Gly Tyr Phe Leu Ala Ser Glu

115 120 125

Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu Asp

145 150 155

<210> 5

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 5

Tyr Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

Asp Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Glu Asp

20 25 30

Met Thr Asp Ser Asp Ser Arg Asp Asn Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Lys Tyr Ser Asp Ser Leu Ala Arg Gly Leu Ala Val Thr Ile

50 55 60

Ser Val Lys Ser Glu Lys Ile Ser Thr Leu Ser Ser Glu Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Gln Arg Asp Val Pro Gly His Ser Arg Lys

100 105 110

Met Gln Phe Glu Ser Ser Ser Tyr Tyr Glu Gly Tyr Phe Leu Ala Cys Glu

115 120 125

Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu Asp

145 150 155

<210> 6

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 6

Tyr Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

Asp Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Glu Asp

20 25 30

Met Thr Asp Ser Asp Ser Arg Asp Asn Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Lys Tyr Ser Asp Ser Leu Ala Arg Gly Leu Ala Val Thr Ile

50 55 60

Ser Val Lys Ser Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Gln Arg Asp Val Pro Gly His Ser Arg Lys

100 105 110

Met Gln Phe Glu Ser Ser Ser Tyr Tyr Glu Gly Tyr Phe Leu Ala Cys Glu

115 120 125

Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu Asp

145 150 155

<210> 7

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 7

Tyr Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

Asp Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Glu Asp

20 25 30

Met Thr Asp Ser Asp Ser Arg Asp Asn Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Lys Tyr Ser Asp Ser Leu Ala Arg Gly Leu Ala Val Thr Ile

50 55 60

Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Ser Glu Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Gln Arg Asp Val Pro Gly His Ser Arg Lys

100 105 110

Met Gln Phe Glu Ser Ser Ser Tyr Tyr Glu Gly Tyr Phe Leu Ala Cys Glu

115 120 125

Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu Asp

145 150 155

<210> 8

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 8

Tyr Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

Asp Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Glu Asp

20 25 30

Met Thr Asp Ser Asp Ser Arg Asp Asn Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Lys Tyr Ser Asp Ser Leu Ala Arg Gly Leu Ala Val Thr Ile

50 55 60

Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Gln Arg Asp Val Pro Gly His Ser Arg Lys

100 105 110

Met Gln Phe Glu Ser Ser Ser Tyr Glu Gly Tyr Phe Leu Ala Ser Glu

115 120 125

Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu Asp

145 150 155

<210> 9

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400>  9

Tyr Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

Asp Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Glu Asp

20 25 30

Met Thr Asp Ser Asp Cys Arg Asp Asn Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Lys Tyr Ser Asp Ser Leu Ala Arg Gly Leu Ala Val Thr Ile

50 55 60

Ser Val Lys Ser Glu Lys Ile Ser Thr Leu Ser Ser Glu Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Gln Arg Asp Val Pro Gly His Ser Arg Lys

100 105 110

Met Gln Phe Glu Ser Ser Ser Tyr Tyr Glu Gly Tyr Phe Leu Ala Cys Glu

115 120 125

Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu Asp

145 150 155

<210> 10

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 10

Tyr Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

Asp Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Glu Asp

20 25 30

Met Thr Asp Ser Asp Cys Arg Asp Asn Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Lys Tyr Ser Asp Ser Leu Ala Arg Gly Leu Ala Val Thr Ile

50 55 60

Ser Val Lys Ser Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Gln Arg Asp Val Pro Gly His Ser Arg Lys

100 105 110

Met Gln Phe Glu Ser Ser Ser Tyr Glu Gly Tyr Phe Leu Ala Ser Glu

115 120 125

Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu Asp

145 150 155

<210> 11

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 11

Tyr Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

Asp Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Glu Asp

20 25 30

Met Thr Asp Ser Asp Cys Arg Asp Asn Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Lys Tyr Ser Asp Ser Leu Ala Arg Gly Leu Ala Val Thr Ile

50 55 60

Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Ser Glu Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Gln Arg Asp Val Pro Gly His Ser Arg Lys

100 105 110

Met Gln Phe Glu Ser Ser Ser Tyr Glu Gly Tyr Phe Leu Ala Ser Glu

115 120 125

Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu Asp

145 150 155

<210> 12

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 12

Tyr Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

Asp Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Glu Asp

20 25 30

Met Thr Asp Ser Asp Ser Arg Asp Asn Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Lys Tyr Ser Asp Ser Leu Ala Arg Gly Leu Ala Val Thr Ile

50 55 60

Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Gln Arg Asp Val Pro Gly His Ser Arg Lys

100 105 110

Met Gln Phe Glu Ser Ser Ser Tyr Tyr Glu Gly Tyr Phe Leu Ala Cys Glu

115 120 125

Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu Asp

145 150 155

<210> 13

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 13

Tyr Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

Asp Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Glu Asp

20 25 30

Met Thr Asp Ser Asp Cys Arg Asp Asn Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Lys Tyr Ser Asp Ser Leu Ala Arg Gly Leu Ala Val Thr Ile

50 55 60

Ser Val Lys Ser Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Gln Arg Asp Val Pro Gly His Ser Arg Lys

100 105 110

Met Gln Phe Glu Ser Ser Ser Tyr Tyr Glu Gly Tyr Phe Leu Ala Cys Glu

115 120 125

Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu Asp

145 150 155

<210> 14

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 14

Tyr Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

Asp Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Glu Asp

20 25 30

Met Thr Asp Ser Asp Cys Arg Asp Asn Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Lys Tyr Ser Asp Ser Leu Ala Arg Gly Leu Ala Val Thr Ile

50 55 60

Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Ser Glu Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Gln Arg Asp Val Pro Gly His Ser Arg Lys

100 105 110

Met Gln Phe Glu Ser Ser Ser Tyr Tyr Glu Gly Tyr Phe Leu Ala Cys Glu

115 120 125

Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu Asp

145 150 155

<210> 15

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 15

Tyr Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

Asp Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Glu Asp

20 25 30

Met Thr Asp Ser Asp Cys Arg Asp Asn Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Lys Tyr Ser Asp Ser Leu Ala Arg Gly Leu Ala Val Thr Ile

50 55 60

Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Gln Arg Asp Val Pro Gly His Ser Arg Lys

100 105 110

Met Gln Phe Glu Ser Ser Ser Tyr Glu Gly Tyr Phe Leu Ala Ser Glu

115 120 125

Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu Asp

145 150 155

<210> 16

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 16

Tyr Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

Asp Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Glu Asp

20 25 30

Met Thr Asp Ser Asp Ser Arg Asp Asn Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Lys Tyr Ser Asp Ser Leu Ala Arg Gly Leu Ala Val Thr Ile

50 55 60

Ser Val Lys Gly Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Gln Arg Asp Val Pro Gly His Ser Arg Lys

100 105 110

Met Gln Phe Glu Ser Ser Ser Tyr Tyr Glu Gly Tyr Phe Leu Ala Cys Glu

115 120 125

Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu Asp

145 150 155

<210> 17

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 17

Tyr Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

Asp Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Glu Asp

20 25 30

Met Thr Asp Ser Asp Ser Arg Asp Asn Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Lys Tyr Ser Asp Ser Leu Ala Arg Gly Leu Ala Val Thr Ile

50 55 60

Ser Val Lys Ala Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Gln Arg Asp Val Pro Gly His Ser Arg Lys

100 105 110

Met Gln Phe Glu Ser Ser Ser Tyr Tyr Glu Gly Tyr Phe Leu Ala Cys Glu

115 120 125

Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu Asp

145 150 155

<210> 18

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 18

Tyr Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

Asp Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Glu Asp

20 25 30

Met Thr Asp Ser Asp Ser Arg Asp Asn Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Lys Tyr Ser Asp Ser Leu Ala Arg Gly Leu Ala Val Thr Ile

50 55 60

Ser Val Lys Val Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Gln Arg Asp Val Pro Gly His Ser Arg Lys

100 105 110

Met Gln Phe Glu Ser Ser Ser Tyr Tyr Glu Gly Tyr Phe Leu Ala Cys Glu

115 120 125

Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu Asp

145 150 155

<210> 19

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 19

Tyr Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

Asp Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Glu Asp

20 25 30

Met Thr Asp Ser Asp Ser Arg Asp Asn Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Lys Tyr Ser Asp Ser Leu Ala Arg Gly Leu Ala Val Thr Ile

50 55 60

Ser Val Lys Asp Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Gln Arg Asp Val Pro Gly His Ser Arg Lys

100 105 110

Met Gln Phe Glu Ser Ser Ser Tyr Tyr Glu Gly Tyr Phe Leu Ala Cys Glu

115 120 125

Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu Asp

145 150 155

<210>  20

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400>  20

Tyr Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

Asp Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Glu Asp

20 25 30

Met Thr Asp Ser Asp Ser Arg Asp Asn Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Lys Tyr Ser Asp Ser Leu Ala Arg Gly Leu Ala Val Thr Ile

50 55 60

Ser Val Lys Glu Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Gln Arg Asp Val Pro Gly His Ser Arg Lys

100 105 110

Met Gln Phe Glu Ser Ser Ser Tyr Tyr Glu Gly Tyr Phe Leu Ala Cys Glu

115 120 125

Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu Asp

145 150 155

<210>  21

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400>  21

Tyr Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

Asp Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Glu Asp

20 25 30

Met Thr Asp Ser Asp Ser Arg Asp Asn Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Lys Tyr Ser Asp Ser Leu Ala Arg Gly Leu Ala Val Thr Ile

50 55 60

Ser Val Lys Asn Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Gln Arg Asp Val Pro Gly His Ser Arg Lys

100 105 110

Met Gln Phe Glu Ser Ser Ser Tyr Tyr Glu Gly Tyr Phe Leu Ala Cys Glu

115 120 125

Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu Asp

145 150 155

<210>  22

<211> 4

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400>  22

Ala Ala Ala Ala

1

<210> 23

<211> 4

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 23

Ala Ala Ala Ala

1

<210>  24

<211> 4

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400>  24

Ala Ala Ala Ala

1

<210> 25

<211> 4

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 25

Ala Ala Ala Ala

1

<210> 26

<211> 96

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 26

Asp Ser Glu Val Asn Gln Glu Ala Lys Pro Glu Val Lys Pro Glu Val

1 5 10 15

Lys Pro Glu Thr His Ile Asn Leu Lys Val Ser Asp Gly Ser Ser Glu

20 25 30

Ile Phe Phe Lys Ile Lys Lys Thr Thr Pro Leu Arg Arg Leu Met Glu

35 40 45

Ala Phe Ala Lys Arg Gln Gly Lys Glu Met Asp Ser Leu Arg Phe Leu

50 55 60

Tyr Asp Gly Ile Arg Ile Gln Ala Asp Gln Ala Pro Glu Asp Leu Asp

65 70 75 80

Met Glu Asp Asn Asp Ile Ile Glu Ala His Arg Glu Gln Ile Gly Gly

85 90 95

<210>  27

<211> 108

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 27

Met Gly His His His Gly Ser Leu Gln Asp Ser Glu Val

1 5 10 15

Asn Gln Glu Ala Lys Pro Glu Val Lys Pro Glu Val Lys Pro Glu Thr

20 25 30

His Ile Asn Leu Lys Val Ser Asp Gly Ser Ser Ser Glu Ile Phe Phe Lys

35 40 45

Ile Lys Lys Thr Thr Pro Leu Arg Arg Leu Met Glu Ala Phe Ala Lys

50 55 60

Arg Gln Gly Lys Glu Met Asp Ser Leu Arg Phe Leu Tyr Asp Gly Ile

65 70 75 80

Arg Ile Gln Ala Asp Gln Ala Pro Glu Asp Leu Asp Met Glu Asp Asn

85 90 95

Asp Ile Ile Glu Ala His Arg Glu Gln Ile Gly Gly

100 105

<210>  28

<211>  219

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 28

Leu Val Pro Glu Leu Asn Glu Lys Asp Asp Asp Gln Val Gln Lys Ala

1 5 10 15

Leu Ala Ser Arg Glu Asn Thr Gln Leu Met Asn Arg Asp Asn Ile Glu

20 25 30

Ile Thr Val Arg Asp Phe Lys Thr Leu Ala Pro Arg Arg Trp Leu Asn

35 40 45

Asp Thr Ile Ile Glu Phe Phe Met Lys Tyr Ile Glu Lys Ser Thr Pro

50 55 60

Asn Thr Val Ala Phe Asn Ser Phe Phe Tyr Thr Asn Leu Ser Glu Arg

65 70 75 80

Gly Tyr Gln Gly Val Arg Arg Trp Met Lys Arg Lys Lys Thr Gln Ile

85 90 95

Asp Lys Leu Asp Lys Ile Phe Thr Pro Ile Asn Leu Asn Gln Ser His

100 105 110

Trp Ala Leu Gly Ile Ile Asp Leu Lys Lys Lys Thr Ile Gly Tyr Val

115 120 125

Asp Ser Leu Ser Asn Gly Pro Asn Ala Met Ser Phe Ala Ile Leu Thr

130 135 140

Asp Leu Gln Lys Tyr Val Met Glu Glu Ser Lys His Thr Ile Gly Glu

145 150 155 160

Asp Phe Asp Leu Ile His Leu Asp Cys Pro Gln Gln Pro Asn Gly Tyr

165 170 175

Asp Cys Gly Ile Tyr Val Cys Met Asn Thr Leu Tyr Gly Ser Ala Asp

180 185 190

Ala Pro Leu Asp Phe Asp Tyr Lys Asp Ala Ile Arg Met Arg Arg Phe

195 200 205

Ile Ala His Leu Ile Leu Thr Asp Ala Leu Lys

210 215

<210>  29

<211>  233

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400>  29

Met Gly Ser Ser His His His His His His Ser Gly Ser Leu Val

1 5 10 15

Pro Glu Leu Asn Glu Lys Asp Asp Asp Gln Val Gln Lys Ala Leu Ala

20 25 30

Ser Arg Glu Asn Thr Gln Leu Met Asn Arg Asp Asn Ile Glu Ile Thr

35 40 45

Val Arg Asp Phe Lys Thr Leu Ala Pro Arg Arg Trp Leu Asn Asp Thr

50 55 60

Ile Ile Glu Phe Phe Met Lys Tyr Ile Glu Lys Ser Thr Pro Asn Thr

65 70 75 80

Val Ala Phe Asn Ser Phe Phe Tyr Thr Asn Leu Ser Glu Arg Gly Tyr

85 90 95

Gln Gly Val Arg Arg Trp Met Lys Arg Lys Lys Thr Gln Ile Asp Lys

100 105 110

Leu Asp Lys Ile Phe Thr Pro Ile Asn Leu Asn Gln Ser His Trp Ala

115 120 125

Leu Gly Ile Ile Asp Leu Lys Lys Lys Thr Ile Gly Tyr Val Asp Ser

130 135 140

Leu Ser Asn Gly Pro Asn Ala Met Ser Phe Ala Ile Leu Thr Asp Leu

145 150 155 160

Gln Lys Tyr Val Met Glu Glu Ser Lys His Thr Ile Gly Glu Asp Phe

165 170 175

Asp Leu Ile His Leu Asp Cys Pro Gln Gln Pro Asn Gly Tyr Asp Cys

180 185 190

Gly Ile Tyr Val Cys Met Asn Thr Leu Tyr Gly Ser Ala Asp Ala Pro

195 200 205

Leu Asp Phe Asp Tyr Lys Asp Ala Ile Arg Met Arg Arg Phe Ile Ala

210 215 220

His Leu Ile Leu Thr Asp Ala Leu Lys

225 230

<210> 30

<211> 157

<212> PRT

<213> Homo sapiens

<400> 30

Tyr Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

Asp Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Glu Asp

20 25 30

Met Thr Asp Ser Asp Cys Arg Asp Asn Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Met Tyr Lys Asp Ser Gln Pro Arg Gly Met Ala Val Thr Ile

50 55 60

Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Gln Arg Ser Val Pro Gly His Asp Asn Lys

100 105 110

Met Gln Phe Glu Ser Ser Ser Tyr Tyr Glu Gly Tyr Phe Leu Ala Cys Glu

115 120 125

Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu Asp

145 150 155

<210> 31

<211> 157

<212> PRT

<213> House Mouse

<400> 31

Asn Phe Gly Arg Leu His Cys Thr Thr Ala Val Ile Arg Asn Ile Asn

1 5 10 15

Asp Gln Val Leu Phe Val Asp Lys Arg Gln Pro Val Phe Glu Asp Met

20 25 30

Thr Asp Ile Asp Gln Ser Ala Ser Glu Pro Gln Thr Arg Leu Ile Ile

35 40 45

Tyr Met Tyr Lys Asp Ser Glu Val Arg Gly Leu Ala Val Thr Leu Ser

50 55 60

Val Lys Asp Ser Lys Met Ser Thr Leu Ser Cys Lys Asn Lys Ile Ile

65 70 75 80

Ser Phe Glu Glu Met Asp Pro Pro Glu Asn Ile Asp Asp Ile Gln Ser

85 90 95

Asp Leu Ile Phe Phe Gln Lys Arg Val Pro Gly His Asn Lys Met Glu

100 105 110

Phe Glu Ser Ser Leu Tyr Glu Gly His Phe Leu Ala Cys Gln Lys Glu

115 120 125

Asp Asp Ala Phe Lys Leu Ile Leu Lys Lys Lys Asp Glu Asn Gly Asp

130 135 140

Lys Ser Val Met Phe Thr Leu Thr Asn Leu His Gln Ser

145 150 155

<210> 32

<211> 193

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 32

Met Ala Ala Glu Pro Val Glu Asp Asn Cys Ile Asn Phe Val Ala Met

1 5 10 15

Lys Phe Ile Asp Asn Thr Leu Tyr Phe Ile Ala Glu Asp Asp Glu Asn

20 25 30

Leu Glu Ser Asp Tyr Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile

35 40 45

Arg Asn Leu Asn Asp Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro

50 55 60

Leu Phe Glu Asp Met Thr Asp Ser Asp Cys Arg Asp Asn Ala Pro Arg

65 70 75 80

Thr Ile Phe Ile Ile Ser Met Tyr Lys Asp Ser Gln Pro Arg Gly Met

85 90 95

Ala Val Thr Ile Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys

100 105 110

Glu Asn Lys Ile Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile

115 120 125

Lys Asp Thr Lys Ser Asp Ile Ile Phe Phe Gln Arg Ser Val Pro Gly

130 135 140

His Asp Asn Lys Met Gln Phe Glu Ser Ser Ser Tyr Glu Gly Tyr Phe

145 150 155 160

Leu Ala Cys Glu Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys

165 170 175

Glu Asp Glu Leu Gly Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu

180 185 190

Asp

<210> 33

<211> 192

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 33

Met Ala Ala Met Ser Glu Asp Ser Cys Val Asn Phe Lys Glu Met Met

1 5 10 15

Phe Ile Asp Asn Thr Leu Tyr Phe Ile Pro Glu Glu Asn Gly Asp Leu

20 25 30

Glu Ser Asp Asn Phe Gly Arg Leu His Cys Thr Thr Ala Val Ile Arg

35 40 45

Asn Ile Asn Asp Gln Val Leu Phe Val Asp Lys Arg Gln Pro Val Phe

50 55 60

Glu Asp Met Thr Asp Ile Asp Gln Ser Ala Ser Glu Pro Gln Thr Arg

65 70 75 80

Leu Ile Ile Tyr Met Tyr Lys Asp Ser Glu Val Arg Gly Leu Ala Val

85 90 95

Thr Leu Ser Val Lys Asp Ser Lys Met Ser Thr Leu Ser Cys Lys Asn

100 105 110

Lys Ile Ile Ser Phe Glu Glu Met Asp Pro Pro Glu Asn Ile Asp Asp

115 120 125

Ile Gln Ser Asp Leu Ile Phe Phe Gln Lys Arg Val Pro Gly His Asn

130 135 140

Lys Met Glu Phe Glu Ser Ser Leu Tyr Glu Gly His Phe Leu Ala Cys

145 150 155 160

Gln Lys Glu Asp Asp Ala Phe Lys Leu Ile Leu Lys Lys Lys Asp Glu

165 170 175

Asn Gly Asp Lys Ser Val Met Phe Thr Leu Thr Asn Leu His Gln Ser

180 185 190

<210> 34

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 34

Tyr Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

Asp Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Glu Asp

20 25 30

Met Thr Asp Ser Asp Cys Arg Asp Asn Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Thr Tyr Lys Asp Ser Gln Pro Arg Gly Lys Ala Val Thr Ile

50 55 60

Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Gln Arg Asp Val Pro Gly His Lys His Lys

100 105 110

Met Gln Phe Glu Ser Ser Ser Tyr Tyr Glu Gly Tyr Phe Leu Ala Cys Glu

115 120 125

Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu Asp

145 150 155

<210> 35

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 35

Tyr Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

Asp Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Glu Asp

20 25 30

Met Thr Asp Ser Asp Cys Arg Asp Asn Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Thr Tyr Lys Asp Lys Gln Pro Arg Ala Lys Ala Val Thr Ile

50 55 60

Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Gln Arg Asp Val Pro Gly His Lys His Lys

100 105 110

Met Gln Phe Glu Ser Ser Ser Tyr Tyr Glu Gly Tyr Phe Leu Ala Cys Glu

115 120 125

Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Ile Gln Asn Glu Asp

145 150 155

<210> 36

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 36

Arg Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

Asp Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Glu Asp

20 25 30

Met Thr Asp Ser Asp Cys Arg Asp Asn Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Thr Tyr Lys Asp Ser Gln Pro Arg Gly Lys Ala Val Thr Ile

50 55 60

Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Gln Arg Asp Val Pro Gly His Lys His Lys

100 105 110

Met Gln Phe Glu Ser Ser Ser Tyr Tyr Glu Gly Tyr Phe Leu Ala Cys Glu

115 120 125

Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu Asp

145 150 155

<210> 37

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 37

Arg Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

Asp Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Glu Asp

20 25 30

Met Thr Asp Ser Asp Cys Arg Asp Asn Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Thr Tyr Arg Asp Ser Gln Pro Arg Gly Lys Ala Val Thr Ile

50 55 60

Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Gln Arg Asn Val Pro Gly His Lys Tyr Lys

100 105 110

Met Gln Phe Glu Ser Ser Ser Tyr Tyr Glu Gly Tyr Phe Leu Ala Cys Glu

115 120 125

Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu Asp

145 150 155

<210> 38

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 38

Tyr Phe Gly Lys Leu Glu Ser Gln Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

Asp Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Glu Asp

20 25 30

Met Thr Asp Ser Asp Cys Arg Asp Asn Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Thr Tyr Lys Asp Lys Gln Pro Arg Thr Lys Ala Val Thr Ile

50 55 60

Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Gln Arg Arg Val Pro Gly His His Asn Lys

100 105 110

Met Gln Phe Glu Ser Ser Ser Tyr Tyr Glu Gly Tyr Phe Leu Ala Cys Glu

115 120 125

Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Val Gln Lys Glu Asp

145 150 155

<210> 39

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 39

Tyr Phe Gly Lys Leu Glu Ser Arg Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

Asp Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Glu Asp

20 25 30

Met Thr Asp Ser Asp Cys Arg Asp Asn Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Lys Tyr Lys Asp Lys Gln Pro Arg Ala Gln Ala Val Thr Ile

50 55 60

Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Gln Arg Asp Val Pro Gly His Lys His Lys

100 105 110

Met Gln Phe Glu Ser Ser Ser Tyr Tyr Glu Gly Tyr Phe Leu Ala Cys Glu

115 120 125

Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Ile Gln Asn Glu Asp

145 150 155

<210> 40

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<220>

<221> misc_features

<222>  (60)..(60)

<223> Xaa can be any naturally occurring amino acid.

<400>  40

Tyr Phe Gly Lys Leu Glu Ser Arg Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

Asp Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Glu Asp

20 25 30

Met Thr Asp Ser Asp Cys Arg Asp Asn Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Asp Tyr Lys Asp Lys Gln Pro Arg Ala Xaa Ala Val Thr Ile

50 55 60

Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Gln Arg Asp Val Pro Gly His Lys His Lys

100 105 110

Met Gln Phe Glu Ser Ser Ser Tyr Tyr Glu Gly Tyr Phe Leu Ala Cys Glu

115 120 125

Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Ile Gln Asn Glu Asp

145 150 155

<210> 41

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400>  41

Tyr Phe Gly Lys His Glu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

Asp Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Glu Asp

20 25 30

Met Thr Asp Ser Asp Cys Arg Asp Asn Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Thr Tyr Arg Asp Ser Gln Pro Arg Gly Lys Ala Val Thr Ile

50 55 60

Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Gln Arg Asp Val Pro Gly His Asn Asn Lys

100 105 110

Met Gln Phe Glu Ser Ser Ser Tyr Tyr Glu Gly Tyr Phe Leu Ala Cys Glu

115 120 125

Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Thr Gln Asn Glu Asp

145 150 155

<210> 42

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400>  42

Tyr Phe Gly Lys Ile Glu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

Asp Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Glu Asp

20 25 30

Met Thr Asp Ser Asp Cys Arg Asp Asn Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Lys Tyr Lys Asp Lys Gln Pro Arg Ala Gln Ala Val Thr Ile

50 55 60

Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Gln Arg Lys Val Pro Gly His Gln His Lys

100 105 110

Met Gln Phe Glu Ser Ser Ser Tyr Tyr Glu Gly Tyr Phe Leu Ala Cys Glu

115 120 125

Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Val Gln Lys Glu Asp

145 150 155

<210> 43

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400>  43

Tyr Phe Gly Lys Ile Glu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

Asp Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Glu Asp

20 25 30

Met Thr Asp Ser Asp Cys Arg Asp Asn Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Thr Tyr Lys Asp Arg Gln Pro Arg Gly Lys Ala Val Thr Ile

50 55 60

Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Glu Arg Asp Val Pro Gly His His His Lys

100 105 110

Met Gln Phe Glu Ser Ser Ser Tyr Tyr Glu Gly Tyr Phe Leu Ala Cys Glu

115 120 125

Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Ile Gln Asn Glu Asp

145 150 155

<210> 44

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400>  44

Tyr Phe Gly Lys Ile Glu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

Asp Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Glu Asp

20 25 30

Met Thr Asp Ser Asp Cys Arg Asp Asn Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Thr Tyr Lys Asp Lys Gln Pro Arg Gly Lys Ala Val Thr Ile

50 55 60

Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Gln Arg Asp Val Pro Gly His Lys His Lys

100 105 110

Met Gln Phe Glu Ser Ser Ser Tyr Tyr Glu Gly Tyr Phe Leu Ala Cys Glu

115 120 125

Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Thr Gln His Glu Asp

145 150 155

<210> 45

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400>  45

Tyr Phe Gly Lys Ile Glu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

Asp Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Glu Asp

20 25 30

Met Thr Asp Ser Asp Cys Arg Asp Asn Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Thr Tyr Lys Asp Lys Gln Pro Arg Ala Lys Ala Val Thr Ile

50 55 60

Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Gln Arg Arg Val Pro Gly His His His Lys

100 105 110

Met Gln Phe Glu Ser Ser Ser Tyr Tyr Glu Gly Tyr Phe Leu Ala Cys Glu

115 120 125

Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Ile Gln Lys Glu Asp

145 150 155

<210> 46

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400>  46

Tyr Phe Gly Lys Ile Glu Ser Arg Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

Asp Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Glu Asp

20 25 30

Met Thr Asp Ser Asp Cys Arg Asp Asn Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Thr Tyr Lys Asp Lys Gln Pro Arg Gly Lys Ala Val Thr Ile

50 55 60

Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Gln Arg Asp Val Pro Gly His Asp Tyr Lys

100 105 110

Met Gln Phe Glu Ser Ser Ser Tyr Tyr Glu Gly Tyr Phe Leu Ala Cys Glu

115 120 125

Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Ile Gln Lys Glu Asp

145 150 155

<210> 47

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400>  47

Tyr Phe Gly Lys Tyr Glu Ser Arg Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

Asp Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Glu Asp

20 25 30

Met Thr Asp Ser Asp Cys Arg Asp Asn Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Thr Tyr Arg Asp Ser Gln Pro Arg Gly Lys Ala Val Thr Ile

50 55 60

Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Gln Arg Asp Val Pro Gly His Glu His Lys

100 105 110

Met Gln Phe Glu Ser Ser Ser Tyr Tyr Glu Gly Tyr Phe Leu Ala Cys Glu

115 120 125

Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Val Gln Lys Glu Asp

145 150 155

<210> 48

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400>  48

His Phe Gly Lys Tyr Glu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

Asp Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Glu Asp

20 25 30

Met Thr Asp Ser Asp Cys Arg Asp Asn Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Thr Tyr Arg Asp Ser Gln Pro Arg Gly Lys Ala Val Thr Ile

50 55 60

Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Gln Arg Asp Val Pro Gly His His Asn Lys

100 105 110

Met Gln Phe Glu Ser Ser Ser Tyr Tyr Glu Gly Tyr Phe Leu Ala Cys Glu

115 120 125

Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Val Gln Lys Glu Asp

145 150 155

<210> 49

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400>  49

Arg Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

Asp Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Glu Asp

20 25 30

Met Thr Asp Ser Asp Cys Arg Asp Asn Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Thr Tyr Arg Asp Ser Gln Pro Arg Ala Lys Ala Val Thr Ile

50 55 60

Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Gln Arg Asp Val Pro Gly His Gln His Lys

100 105 110

Met Gln Phe Glu Ser Ser Ser Tyr Tyr Glu Gly Tyr Phe Leu Ala Cys Glu

115 120 125

Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Ala Gln Lys Glu Asp

145 150 155

<210> 50

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 50

Arg Phe Gly Lys Leu Glu Ser Arg Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

Asp Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Glu Asp

20 25 30

Met Thr Asp Ser Asp Cys Arg Asp Asn Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Asp Tyr Arg Asp Ser Gln Pro Arg Gly Arg Ala Val Thr Ile

50 55 60

Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Lys Arg Asn Val Pro Gly His Lys Tyr Lys

100 105 110

Met Gln Phe Glu Ser Ser Ser Tyr Tyr Glu Gly Tyr Phe Leu Ala Cys Glu

115 120 125

Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Val Gln His Glu Asp

145 150 155

<210> 51

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 51

Arg Phe Gly Lys Leu Glu Ser Arg Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

Asp Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Glu Asp

20 25 30

Met Thr Asp Ser Asp Cys Arg Asp Asn Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Asn Tyr Arg Asp Ser Gln Pro Arg Gly Gln Ala Val Thr Ile

50 55 60

Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Lys Arg Arg Val Pro Gly His Asn His Lys

100 105 110

Met Gln Phe Glu Ser Ser Ser Tyr Tyr Glu Gly Tyr Phe Leu Ala Cys Glu

115 120 125

Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Val Gln Lys Glu Asp

145 150 155

<210> 52

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 52

Arg Phe Gly Lys Leu Glu Ser Arg Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

Asp Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Glu Asp

20 25 30

Met Thr Asp Ser Asp Cys Arg Asp Asn Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Thr Tyr Lys Asp Ser Gln Pro Arg Gly Lys Ala Val Thr Ile

50 55 60

Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Gln Arg Asp Val Pro Gly His Lys His Lys

100 105 110

Met Gln Phe Glu Ser Ser Ser Tyr Tyr Glu Gly Tyr Phe Leu Ala Cys Glu

115 120 125

Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu Asp

145 150 155

<210> 53

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 53

Arg Phe Gly Lys His Glu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

Asp Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Glu Asp

20 25 30

Met Thr Asp Ser Asp Cys Arg Asp Asn Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Thr Tyr Arg Asp Ser Gln Pro Arg Gly Lys Ala Val Thr Ile

50 55 60

Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Glu Arg Asn Val Pro Gly His Lys Tyr Lys

100 105 110

Met Gln Phe Glu Ser Ser Ser Tyr Tyr Glu Gly Tyr Phe Leu Ala Cys Glu

115 120 125

Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu Asp

145 150 155

<210> 54

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<220>

<221> misc_features

<222> (155)..(155)

<223> Xaa can be any naturally occurring amino acid.

<400> 54

Arg Phe Gly Lys Leu Glu Ser Arg Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

Asp Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Glu Asp

20 25 30

Met Thr Asp Ser Asp Cys Arg Asp Asn Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Thr Tyr Arg Asp Ser Gln Pro Arg Ala Lys Ala Val Thr Ile

50 55 60

Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Glu Arg Asp Val Pro Gly His Gln His Lys

100 105 110

Met Gln Phe Glu Ser Ser Ser Tyr Tyr Glu Gly Tyr Phe Leu Ala Cys Glu

115 120 125

Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Ile Gln Xaa Glu Asp

145 150 155

<210> 55

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 55

Arg Phe Gly Lys Leu Glu Ser Arg Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

Asp Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Glu Asp

20 25 30

Met Thr Asp Ser Asp Cys Arg Asp Asn Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Thr Tyr Arg Asp Ser Gln Pro Arg Thr Lys Ala Val Thr Ile

50 55 60

Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Gln Arg Asn Val Pro Gly His His Asp Lys

100 105 110

Met Gln Phe Glu Ser Ser Ser Tyr Tyr Glu Gly Tyr Phe Leu Ala Cys Glu

115 120 125

Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Val Gln His Glu Asp

145 150 155

<210> 56

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 56

Arg Phe Gly Lys Leu Glu Ser Arg Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

Asp Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Glu Asp

20 25 30

Met Thr Asp Ser Asp Cys Arg Asp Asn Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Thr Tyr Lys Asp Ser Gln Pro Arg Ala Lys Ala Val Thr Ile

50 55 60

Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Glu Arg Asp Val Pro Gly His Gln His Lys

100 105 110

Met Gln Phe Glu Ser Ser Ser Tyr Tyr Glu Gly Tyr Phe Leu Ala Cys Glu

115 120 125

Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Ile Gln Lys Glu Asp

145 150 155

<210> 57

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 57

Arg Phe Gly Lys His Glu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

Asp Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Glu Asp

20 25 30

Met Thr Asp Ser Asp Cys Arg Asp Asn Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Thr Tyr Arg Asp Ser Gln Pro Arg Gly Lys Ala Val Thr Ile

50 55 60

Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Glu Arg Asn Val Pro Gly His Lys Tyr Lys

100 105 110

Met Gln Phe Glu Ser Ser Ser Tyr Tyr Glu Gly Tyr Phe Leu Ala Cys Glu

115 120 125

Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu Asp

145 150 155

<210> 58

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 58

Arg Phe Gly Lys Tyr Glu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

Asp Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Glu Asp

20 25 30

Met Thr Asp Ser Asp Cys Arg Asp Asn Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Thr Tyr Lys Asp Ser Gln Pro Arg Thr Lys Ala Val Thr Ile

50 55 60

Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys Asp Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Gln Arg Asp Val Pro Gly His Lys His Lys

100 105 110

Met Gln Phe Glu Ser Ser Ser Tyr Tyr Glu Gly Tyr Phe Leu Ala Cys Glu

115 120 125

Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu Asp

145 150 155

<210> 59

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 59

Arg Phe Gly Lys Leu Glu Ser Arg Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

Asp Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Glu Asp

20 25 30

Met Thr Asp Ser Asp Cys Arg Asp Asn Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Thr Tyr Arg Asp Ser Gln Pro Arg Thr Lys Ala Val Thr Ile

50 55 60

Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Gln Arg Lys Val Pro Gly His Asn His Lys

100 105 110

Met Gln Phe Glu Ser Ser Ser Tyr Tyr Glu Gly Tyr Phe Leu Ala Cys Glu

115 120 125

Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Val Gln Lys Glu Asp

145 150 155

<210> 60

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 60

Asn Phe Gly Arg Leu His Cys Thr Thr Ala Val Ile Arg Asn Ile Asn

1 5 10 15

Asp Gln Val Leu Phe Val Asp Lys Arg Gln Pro Val Phe Glu Asp Met

20 25 30

Thr Asp Ile Asp Gln Ser Ala Ser Glu Pro Gln Thr Arg Leu Ile Ile

35 40 45

Tyr Gly Tyr Ala Asp Ser Arg Val Arg Gly Lys Ala Val Thr Leu Ser

50 55 60

Val Lys Asp Ser Lys Met Ser Thr Leu Ser Cys Lys Asn Lys Ile Ile

65 70 75 80

Ser Phe Glu Glu Met Asp Pro Pro Glu Asn Ile Asp Asp Ile Gln Ser

85 90 95

Asp Leu Ile Phe Phe Gln Lys Arg Val Pro Gly His Asn Lys Met Glu

100 105 110

Phe Glu Ser Ser Leu Tyr Glu Gly His Phe Leu Ala Cys Gln Lys Glu

115 120 125

Asp Asp Ala Phe Lys Leu Ile Leu Lys Lys Lys Asp Glu Asn Gly Asp

130 135 140

Lys Ser Val Met Phe Thr Leu Thr Asn Leu His Gln Ser

145 150 155

<210> 61

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 61

His Phe Gly Arg Leu His Cys Thr Thr Ala Val Ile Arg Asn Ile Asn

1 5 10 15

Asp Gln Val Leu Phe Val Asp Lys Arg Gln Pro Val Phe Glu Asp Met

20 25 30

Thr Asp Ile Asp Gln Ser Ala Ser Glu Pro Gln Thr Arg Leu Ile Ile

35 40 45

Tyr Ala Tyr Gly Asp Ser Arg Ala Arg Gly Lys Ala Val Thr Leu Ser

50 55 60

Val Lys Asp Ser Lys Met Ser Thr Leu Ser Cys Lys Asn Lys Ile Ile

65 70 75 80

Ser Phe Glu Glu Met Asp Pro Pro Glu Asn Ile Asp Asp Ile Gln Ser

85 90 95

Asp Leu Ile Phe Phe Gln Lys Arg Val Pro Gly His Asn Lys Met Glu

100 105 110

Phe Glu Ser Ser Leu Tyr Glu Gly His Phe Leu Ala Cys Gln Lys Glu

115 120 125

Asp Asp Ala Phe Lys Leu Ile Leu Lys Lys Lys Asp Glu Asn Gly Asp

130 135 140

Lys Ser Val Met Phe Thr Leu Thr Asn Leu His Gln Ser

145 150 155

<210> 62

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 62

Asn Phe Gly Arg Leu His Cys Thr Thr Ala Val Ile Arg Asn Ile Asn

1 5 10 15

Asp Gln Val Leu Phe Val Asp Lys Arg Gln Pro Val Phe Glu Asp Met

20 25 30

Thr Asp Ile Asp Gln Ser Ala Ser Glu Pro Gln Thr Arg Leu Ile Ile

35 40 45

Tyr Ala Tyr Val Asp Arg Arg Leu Arg Gly Lys Ala Val Thr Leu Ser

50 55 60

Val Lys Asp Ser Lys Met Ser Thr Leu Ser Cys Lys Asn Lys Ile Ile

65 70 75 80

Ser Phe Glu Glu Met Asp Pro Pro Glu Asn Ile Asp Asp Ile Gln Ser

85 90 95

Asp Leu Ile Phe Phe Gln Lys Lys Val Pro Gly His Asn Lys Met Glu

100 105 110

Phe Glu Ser Ser Leu Tyr Glu Gly His Phe Leu Ala Cys Gln Lys Glu

115 120 125

Asp Asp Ala Phe Lys Leu Ile Leu Lys Lys Lys Asp Glu Asn Gly Asp

130 135 140

Lys Ser Val Met Phe Thr Leu Thr Asn Leu His Gln Ser

145 150 155

<210> 63

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 63

Asn Phe Gly Arg Leu His Cys Thr Thr Ala Val Ile Arg Asn Ile Asn

1 5 10 15

Asp Gln Val Leu Phe Val Asp Lys Arg Gln Pro Val Phe Glu Asp Met

20 25 30

Thr Asp Ile Asp Gln Ser Ala Ser Glu Pro Gln Thr Arg Leu Ile Ile

35 40 45

Tyr Ser Tyr Ser Asp Lys His Met Arg Gly Lys Ala Val Thr Leu Ser

50 55 60

Val Lys Asp Ser Lys Met Ser Thr Leu Ser Cys Lys Asn Lys Ile Ile

65 70 75 80

Ser Phe Glu Glu Met Asp Pro Pro Glu Asn Ile Asp Asp Ile Gln Ser

85 90 95

Asp Leu Ile Phe Phe Gln Lys Leu Val Pro Gly His Asn Lys Met Glu

100 105 110

Phe Glu Ser Ser Leu Tyr Glu Gly His Phe Leu Ala Cys Gln Lys Glu

115 120 125

Asp Asp Ala Phe Lys Leu Ile Leu Lys Lys Lys Asp Glu Asn Gly Asp

130 135 140

Lys Ser Val Met Phe Thr Leu Thr Asn Leu His Gln Ser

145 150 155

<210> 64

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 64

Asn Phe Gly Arg Leu His Cys Thr Thr Ala Val Ile Arg Asn Ile Asn

1 5 10 15

Asp Gln Val Leu Phe Val Asp Lys Arg Gln Pro Val Phe Glu Asp Met

20 25 30

Thr Asp Ile Asp Gln Ser Ala Ser Glu Pro Gln Thr Arg Leu Ile Ile

35 40 45

Tyr Val Tyr Thr Asp Gly Arg Arg Arg Gly Lys Ala Val Thr Leu Ser

50 55 60

Val Lys Asp Ser Lys Met Ser Thr Leu Ser Cys Lys Asn Lys Ile Ile

65 70 75 80

Ser Phe Glu Glu Met Asp Pro Pro Glu Asn Ile Asp Asp Ile Gln Ser

85 90 95

Asp Leu Ile Phe Phe Gln Lys Lys Val Pro Gly His Asp Lys Met Glu

100 105 110

Phe Glu Ser Ser Leu Tyr Glu Gly His Phe Leu Ala Cys Gln Lys Glu

115 120 125

Asp Asp Ala Phe Lys Leu Ile Leu Lys Lys Lys Asp Glu Asn Gly Asp

130 135 140

Lys Ser Val Met Phe Thr Leu Thr Asn Leu His Gln Ser

145 150 155

<210> 65

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 65

His Phe Gly Arg Leu His Cys Thr Thr Ala Val Ile Arg Asn Ile Asn

1 5 10 15

Asp Gln Val Leu Phe Val Asp Lys Arg Gln Pro Val Phe Glu Asp Met

20 25 30

Thr Asp Ile Asp Gln Ser Ala Ser Glu Pro Gln Thr Arg Leu Ile Ile

35 40 45

Tyr Ala Tyr Gly Asp Ser His Met Arg Gly Lys Ala Val Thr Leu Ser

50 55 60

Val Lys Asp Ser Lys Met Ser Thr Leu Ser Cys Lys Asn Lys Ile Ile

65 70 75 80

Ser Phe Glu Glu Met Asp Pro Pro Glu Asn Ile Asp Asp Ile Gln Ser

85 90 95

Asp Leu Ile Phe Phe Gln Lys Gln Val Pro Gly His Asn Lys Met Glu

100 105 110

Phe Glu Ser Ser Leu Tyr Glu Gly His Phe Leu Ala Cys Gln Lys Glu

115 120 125

Asp Asp Ala Phe Lys Leu Ile Leu Lys Lys Lys Asp Glu Asn Gly Asp

130 135 140

Lys Ser Val Met Phe Thr Val Thr Asn Leu His Gln Ser

145 150 155

<210> 66

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 66

His Phe Gly Arg Leu His Cys Thr Thr Ala Val Ile Arg Asn Ile Asn

1 5 10 15

Asp Gln Val Leu Phe Val Asp Lys Arg Gln Pro Val Phe Glu Asp Met

20 25 30

Thr Asp Ile Asp Gln Ser Ala Ser Glu Pro Gln Thr Arg Leu Ile Ile

35 40 45

Tyr Ala Tyr Gly Asp Ser Asn Ala Gly Gly Arg Ala Val Thr Leu Ser

50 55 60

Val Lys Asp Ser Lys Met Ser Thr Leu Ser Cys Lys Asn Lys Ile Ile

65 70 75 80

Ser Phe Glu Glu Met Asp Pro Pro Glu Asn Ile Asp Asp Ile Gln Ser

85 90 95

Asp Leu Ile Phe Phe Gln Lys Lys Val Pro Gly His Asn Lys Met Glu

100 105 110

Phe Glu Ser Ser Leu Tyr Glu Gly His Phe Leu Ala Cys Gln Lys Glu

115 120 125

Asp Asp Ala Phe Lys Leu Ile Leu Lys Lys Lys Asp Glu Asn Gly Asp

130 135 140

Lys Ser Val Met Phe Thr Leu Thr Asn Leu His Gln Ser

145 150 155

<210> 67

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 67

His Phe Gly Arg Leu His Cys Thr Thr Ala Val Ile Arg Asn Ile Asn

1 5 10 15

Asp Gln Val Leu Phe Val Asp Lys Arg Gln Pro Val Phe Glu Asp Met

20 25 30

Thr Asp Ile Asp Gln Ser Ala Ser Glu Pro Gln Thr Arg Leu Ile Ile

35 40 45

Tyr Gly Tyr Ala Asp Ser Asp Ala Arg Ala Lys Ala Val Thr Leu Ser

50 55 60

Val Lys Asp Ser Lys Met Ser Thr Leu Ser Cys Lys Asn Lys Ile Ile

65 70 75 80

Ser Phe Glu Glu Met Asp Pro Pro Glu Asn Ile Asp Asp Ile Gln Ser

85 90 95

Asp Leu Ile Phe Phe Gln Lys Ser Val Pro Gly His Asn Lys Met Glu

100 105 110

Phe Glu Ser Ser Leu Tyr Glu Gly His Phe Leu Ala Cys Gln Lys Glu

115 120 125

Asp Asp Ala Phe Lys Leu Ile Leu Lys Lys Lys Asp Glu Asn Gly Asp

130 135 140

Lys Ser Val Met Phe Thr Val Thr Asn Leu His Gln Ser

145 150 155

<210> 68

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 68

His Phe Gly Arg Leu His Cys Thr Thr Ala Val Ile Arg Asn Ile Asn

1 5 10 15

Asp Gln Val Leu Phe Val Asp Lys Arg Gln Pro Val Phe Glu Asp Met

20 25 30

Thr Asp Ile Asp Gln Ser Ala Ser Glu Pro Gln Thr Arg Leu Ile Ile

35 40 45

Tyr Gly Tyr Ser Asp Arg Gly Ser Lys Gly Lys Ala Val Thr Leu Ser

50 55 60

Val Lys Asp Ser Lys Met Ser Thr Leu Ser Cys Lys Asn Lys Ile Ile

65 70 75 80

Ser Phe Glu Glu Met Asp Pro Pro Glu Asn Ile Asp Asp Ile Gln Ser

85 90 95

Asp Leu Ile Phe Phe Gln Lys Gln Val Pro Gly His Asn Lys Met Glu

100 105 110

Phe Glu Ser Ser Leu Tyr Glu Gly His Phe Leu Ala Cys Gln Lys Glu

115 120 125

Asp Asp Ala Phe Lys Leu Ile Leu Lys Lys Lys Asp Glu Asn Gly Asp

130 135 140

Lys Ser Val Met Phe Thr Leu Thr Asn Leu His Gln Ser

145 150 155

<210> 69

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 69

Tyr Phe Gly Arg Leu His Cys Thr Thr Ala Val Ile Arg Asn Ile Asn

1 5 10 15

Asp Gln Val Leu Phe Val Asp Lys Arg Gln Pro Val Phe Glu Asp Met

20 25 30

Thr Asp Ile Asp Gln Ser Ala Ser Glu Pro Gln Thr Arg Leu Ile Ile

35 40 45

Tyr Met Tyr Ala Asp Arg Arg Ala Arg Gly Lys Ala Val Thr Leu Ser

50 55 60

Val Lys Asp Ser Lys Met Ser Thr Leu Ser Cys Lys Asn Lys Ile Ile

65 70 75 80

Ser Phe Glu Glu Met Asp Pro Pro Glu Asn Ile Asp Asp Ile Gln Ser

85 90 95

Asp Leu Ile Phe Phe Gln Lys Lys Val Pro Gly His Asp Lys Met Glu

100 105 110

Phe Glu Ser Ser Leu Tyr Glu Gly His Phe Leu Ala Cys Gln Lys Glu

115 120 125

Asp Asp Ala Phe Lys Leu Ile Leu Lys Lys Lys Asp Glu Asn Gly Asp

130 135 140

Lys Ser Val Met Phe Thr Val Thr Asn Leu His Gln Ser

145 150 155

<210> 70

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 70

Tyr Phe Gly Arg Leu His Cys Thr Thr Ala Val Ile Arg Asn Ile Asn

1 5 10 15

Asp Gln Val Leu Phe Val Asp Lys Arg Gln Pro Val Phe Glu Asp Met

20 25 30

Thr Asp Ile Asp Gln Ser Ala Ser Glu Pro Gln Thr Arg Leu Ile Ile

35 40 45

Tyr Ala Tyr Gly Asp Asn Arg Val Arg Gly Lys Ala Val Thr Leu Ser

50 55 60

Val Lys Asp Ser Lys Met Ser Thr Leu Ser Cys Lys Asn Lys Ile Ile

65 70 75 80

Ser Phe Glu Glu Met Asp Pro Pro Glu Asn Ile Asp Asp Ile Gln Ser

85 90 95

Asp Leu Ile Phe Phe Gln Lys Arg Val Pro Gly His Asn Lys Met Glu

100 105 110

Phe Glu Ser Ser Leu Tyr Glu Gly His Phe Leu Ala Cys Gln Lys Glu

115 120 125

Asp Asp Ala Phe Lys Leu Ile Leu Lys Lys Lys Asp Glu Asn Gly Asp

130 135 140

Lys Ser Val Met Phe Thr Leu Thr Asn Leu His Gln Ser

145 150 155

<210> 71

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 71

Tyr Phe Gly Arg Leu His Cys Thr Thr Ala Val Ile Arg Asn Ile Asn

1 5 10 15

Asp Gln Val Leu Phe Val Asp Lys Arg Gln Pro Val Phe Glu Asp Met

20 25 30

Thr Asp Ile Asp Gln Ser Ala Ser Glu Pro Gln Thr Arg Leu Ile Ile

35 40 45

Tyr Gly Tyr Gly Asp Ser Glu Arg Gly Gly Arg Ala Val Thr Leu Ser

50 55 60

Val Lys Asp Ser Lys Met Ser Thr Leu Ser Cys Lys Asn Lys Ile Ile

65 70 75 80

Ser Phe Glu Glu Met Asp Pro Pro Glu Asn Ile Asp Asp Ile Gln Ser

85 90 95

Asp Leu Ile Phe Phe Gln Lys Arg Val Pro Gly His Asp Lys Met Glu

100 105 110

Phe Glu Ser Ser Leu Tyr Glu Gly His Phe Leu Ala Cys Gln Lys Glu

115 120 125

Asp Asp Ala Phe Lys Leu Ile Leu Lys Lys Lys Asp Glu Asn Gly Asp

130 135 140

Lys Ser Val Met Phe Thr Leu Thr Asn Leu His Gln Ser

145 150 155

<210> 72

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 72

Tyr Phe Gly Arg Leu His Cys Thr Thr Ala Val Ile Arg Asn Ile Asn

1 5 10 15

Asp Gln Val Leu Phe Val Asp Lys Arg Gln Pro Val Phe Glu Asp Met

20 25 30

Thr Asp Ile Asp Gln Ser Ala Ser Glu Pro Gln Thr Arg Leu Ile Ile

35 40 45

Tyr Thr Arg Thr Asp Gly Gly Gln Lys Gly Val Ala Val Thr Leu Ser

50 55 60

Val Lys Asp Ser Lys Met Ser Thr Leu Ser Cys Lys Asn Lys Ile Ile

65 70 75 80

Ser Phe Glu Glu Met Asp Pro Pro Glu Asn Ile Asp Asp Ile Gln Ser

85 90 95

Asp Leu Ile Phe Phe Gln Lys Arg Val Pro Gly His Asp Lys Met Glu

100 105 110

Phe Glu Ser Ser Leu Tyr Glu Gly His Phe Leu Ala Cys Gln Lys Glu

115 120 125

Asp Asp Ala Phe Lys Leu Ile Leu Lys Lys Lys Asp Glu Asn Gly Asp

130 135 140

Lys Ser Val Met Phe Thr Leu Thr Asn Leu His Gln Ser

145 150 155

<210> 73

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 73

Tyr Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

Asp Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Glu Asp

20 25 30

Met Thr Asp Ser Asp Cys Arg Asp Asn Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Glu Tyr Lys Asp Ser Glu Leu Arg Gly Arg Ala Val Thr Ile

50 55 60

Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Pro Arg Ala Val Pro Gly His Asn Arg Lys

100 105 110

Val Gln Phe Glu Ser Ser Ser Tyr Tyr Glu Gly Tyr Phe Leu Ala Cys Glu

115 120 125

Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu Asp

145 150 155

<210> 74

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 74

Tyr Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

Asp Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Glu Asp

20 25 30

Met Thr Asp Ser Asp Cys Arg Asp Asn Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Lys Tyr Lys Asp Ser Ala Gly Arg Gly Leu Ala Val Thr Ile

50 55 60

Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Glu Arg Asp Val Pro Gly His Ser Asn Lys

100 105 110

Val Gln Phe Glu Ser Ser Ser Tyr Tyr Glu Gly Tyr Phe Leu Ala Cys Glu

115 120 125

Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu Asp

145 150 155

<210> 75

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 75

Tyr Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

Asp Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Glu Asp

20 25 30

Met Thr Asp Ser Asp Cys Arg Asp Asn Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Lys Tyr Gly Asp Ser Ala Ala Arg Gly Leu Ala Val Thr Ile

50 55 60

Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Gln Arg Ser Val Pro Gly His Lys Arg Lys

100 105 110

Met Gln Phe Glu Ser Ser Ser Tyr Tyr Glu Gly Tyr Phe Leu Ala Cys Glu

115 120 125

Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu Asp

145 150 155

<210> 76

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 76

Tyr Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

Asp Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Glu Asp

20 25 30

Met Thr Asp Ser Asp Cys Arg Asp Asn Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Lys Tyr Gly Asp Ser Arg Gly Arg Gly Leu Ala Val Thr Ile

50 55 60

Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Glu Arg Asp Val Pro Gly His Asn Ser Lys

100 105 110

Arg Gln Phe Glu Ser Ser Ser Tyr Glu Gly Tyr Phe Leu Ala Cys Glu

115 120 125

Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu Asp

145 150 155

<210> 77

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 77

Tyr Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

Asp Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Glu Asp

20 25 30

Met Thr Asp Ser Asp Cys Arg Asp Asn Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Lys Tyr Gly Asp Ser Val Pro Arg Gly Leu Ala Val Thr Ile

50 55 60

Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Ala Arg Ala Val Pro Gly His Ser Arg Lys

100 105 110

Thr Gln Phe Glu Ser Ser Ser Tyr Tyr Glu Gly Tyr Phe Leu Ala Cys Glu

115 120 125

Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu Asp

145 150 155

<210> 78

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 78

Tyr Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

Asp Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Glu Asp

20 25 30

Met Thr Asp Ser Asp Cys Arg Asp Asn Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Lys Tyr Ser Asp Ser Gly Ala Arg Gly Leu Ala Val Thr Ile

50 55 60

Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Ala Arg Ala Val Pro Gly His Gly Arg Lys

100 105 110

Thr Gln Phe Glu Ser Ser Ser Tyr Tyr Glu Gly Tyr Phe Leu Ala Cys Glu

115 120 125

Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu Asp

145 150 155

<210> 79

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 79

Tyr Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

Asp Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Glu Asp

20 25 30

Met Thr Asp Ser Asp Cys Arg Asp Asn Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Lys Tyr Ser Asp Ser Lys Ala Arg Gly Met Ala Val Thr Ile

50 55 60

Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Ala Arg Asp Val Pro Gly His Ser Ser Lys

100 105 110

Arg Gln Phe Glu Ser Ser Ser Tyr Glu Gly Tyr Phe Leu Ala Cys Glu

115 120 125

Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu Asp

145 150 155

<210> 80

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 80

Tyr Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

Asp Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Glu Asp

20 25 30

Met Thr Asp Ser Asp Cys Arg Asp Asn Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Lys Tyr Ser Asp Ser Leu Ala Arg Gly Leu Ala Val Thr Ile

50 55 60

Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Gln Arg Asp Val Pro Gly His Ser Arg Lys

100 105 110

Met Gln Phe Glu Ser Ser Ser Tyr Tyr Glu Gly Tyr Phe Leu Ala Cys Glu

115 120 125

Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu Asp

145 150 155

<210> 81

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 81

Tyr Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

Asp Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Glu Asp

20 25 30

Met Thr Asp Ser Asp Cys Arg Asp Asn Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Lys Tyr Ser Asp Ser Arg Ala Arg Gly Leu Ala Val Thr Ile

50 55 60

Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Gln Arg Asn Val Pro Gly His Gly Arg Lys

100 105 110

Met Gln Phe Glu Ser Ser Ser Tyr Tyr Glu Gly Tyr Phe Leu Ala Cys Glu

115 120 125

Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu Asp

145 150 155

<210> 82

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 82

Tyr Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

Asp Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Glu Asp

20 25 30

Met Thr Asp Ser Asp Cys Arg Asp Asn Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Lys Tyr Ser Asp Ser Arg Ala Arg Gly Leu Ala Val Thr Ile

50 55 60

Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Ala Arg Ser Val Pro Gly His Gly Arg Lys

100 105 110

Thr Gln Phe Glu Ser Ser Ser Tyr Tyr Glu Gly Tyr Phe Leu Ala Cys Glu

115 120 125

Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu Asp

145 150 155

<210> 83

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 83

Tyr Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

Asp Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Glu Asp

20 25 30

Met Thr Asp Ser Asp Cys Arg Asp Asn Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Lys Tyr Ser Asp Ser Arg Ala Arg Gly Leu Ala Val Thr Ile

50 55 60

Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Ala Arg Asp Val Pro Gly His Ser Gly Lys

100 105 110

Arg Gln Phe Glu Ser Ser Ser Tyr Glu Gly Tyr Phe Leu Ala Cys Glu

115 120 125

Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu Asp

145 150 155

<210> 84

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 84

Tyr Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

Asp Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Glu Asp

20 25 30

Met Thr Asp Ser Asp Cys Arg Asp Asn Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Lys Tyr Thr Asp Ser Arg Pro Arg Gly Leu Ala Val Thr Ile

50 55 60

Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Glu Arg Asp Val Pro Gly His Ser Ser Lys

100 105 110

Lys Gln Phe Glu Ser Ser Ser Tyr Glu Gly Tyr Phe Leu Ala Cys Glu

115 120 125

Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu Asp

145 150 155

<210> 85

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 85

Tyr Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

Asp Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Glu Asp

20 25 30

Met Thr Asp Ser Asp Cys Arg Asp Asn Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Lys Tyr Thr Asp Ser Arg Ala Arg Gly Met Ala Val Thr Ile

50 55 60

Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Glu Arg Asp Val Pro Gly His Asn Asp Lys

100 105 110

Arg Gln Phe Glu Ser Ser Ser Tyr Glu Gly Tyr Phe Leu Ala Cys Glu

115 120 125

Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu Asp

145 150 155

<210> 86

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 86

Tyr Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

Asp Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Glu Asp

20 25 30

Met Thr Asp Ser Asp Cys Arg Asp Asn Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Arg Tyr Lys Asp Ser Gly Lys Arg Gly Leu Ala Val Thr Ile

50 55 60

Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Arg Arg Ser Val Pro Gly His Ser Arg Lys

100 105 110

Val Gln Phe Glu Ser Ser Ser Tyr Tyr Glu Gly Tyr Phe Leu Ala Cys Glu

115 120 125

Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu Asp

145 150 155

<210> 87

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 87

Tyr Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

Asp Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Glu Asp

20 25 30

Met Thr Asp Ser Asp Cys Arg Asp Asn Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Lys Tyr Gly Asp Ser Gly Ala Arg Gly Leu Ala Val Thr Ile

50 55 60

Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Glu Arg Asp Val Pro Gly His Ser Gly Lys

100 105 110

Val Gln Phe Glu Ser Ser Ser Tyr Tyr Glu Gly Tyr Phe Leu Ala Cys Glu

115 120 125

Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu Asp

145 150 155

<210> 88

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 88

Tyr Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

Asp Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Glu Asp

20 25 30

Met Thr Asp Ser Asp Cys Arg Asp Asn Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Lys Tyr Gly Asp Ser Arg Pro Arg Gly Met Ala Val Thr Ile

50 55 60

Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Gln Arg Ala Val Pro Gly His Asn Arg Lys

100 105 110

Met Gln Phe Glu Ser Ser Ser Tyr Tyr Glu Gly Tyr Phe Leu Ala Cys Glu

115 120 125

Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu Asp

145 150 155

<210> 89

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 89

Tyr Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

Asp Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Glu Asp

20 25 30

Met Thr Asp Ser Asp Cys Arg Asp Asn Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Lys Tyr Ser Asp Ser Leu Ala Arg Gly Leu Ala Val Thr Ile

50 55 60

Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Gln Arg Asp Val Pro Gly His Ser Arg Lys

100 105 110

Met Gln Phe Glu Ser Ser Ser Tyr Tyr Glu Gly Tyr Phe Leu Ala Cys Glu

115 120 125

Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu Asp

145 150 155

<210> 90

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 90

Tyr Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

Asp Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Glu Asp

20 25 30

Met Thr Asp Ser Asp Cys Arg Asp Asn Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Lys Tyr Ser Asp Ser Arg Ala Arg Gly Leu Ala Val Thr Ile

50 55 60

Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Ala Arg Ser Val Pro Gly His Gly Arg Lys

100 105 110

Thr Gln Phe Glu Ser Ser Ser Tyr Tyr Glu Gly Tyr Phe Leu Ala Cys Glu

115 120 125

Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu Asp

145 150 155

<210> 91

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 91

Tyr Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

Asp Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Glu Asp

20 25 30

Met Thr Asp Ser Asp Cys Arg Asp Asn Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Lys Tyr Ser Asp Ser Arg Ala Arg Gly Leu Ala Val Thr Ile

50 55 60

Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Gln Arg Asn Val Pro Gly His Gly Arg Lys

100 105 110

Met Gln Phe Glu Ser Ser Ser Tyr Tyr Glu Gly Tyr Phe Leu Ala Cys Glu

115 120 125

Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu Asp

145 150 155

<210> 92

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 92

His Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

Gly Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Lys Asp

20 25 30

Met Thr Ala Ser Asp Cys Arg Ala Asn Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Phe Tyr Lys Asp Ser Gln Pro Arg Gly Met Ala Val Thr Ile

50 55 60

Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Ile Arg Ser Val Pro Gly Ala Asp Asn Lys

100 105 110

Phe Gln Phe Glu Ser Ser Ser Tyr Glu Gly Tyr Phe Leu Ala Cys Glu

115 120 125

Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu Asp

145 150 155

<210> 93

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 93

Asp Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

Asp Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Ala Asp

20 25 30

Met Thr Asp Asn Pro Cys Arg Ser Asn Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Phe Tyr Lys Asp Ser Gln Pro Arg Gly Ile Ala Val Thr Ile

50 55 60

Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Leu Arg Ser Val Pro Gly Pro Asp Asn Lys

100 105 110

Met Gln Phe Glu Ser Ser Ser Tyr Tyr Glu Gly Tyr Phe Leu Ala Cys Glu

115 120 125

Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu Asp

145 150 155

<210> 94

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 94

His Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

Gly Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Ala Asp

20 25 30

Met Glu Ala Ser Pro Cys Arg Asp Asn Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Phe Tyr Lys Asp Ser Gln Pro Arg Gly Leu Ala Val Thr Ile

50 55 60

Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Leu Arg Ser Val Pro Gly His Asp Asn Lys

100 105 110

Met Gln Phe Glu Ser Ser Ser Tyr Tyr Glu Gly Tyr Phe Leu Ala Cys Glu

115 120 125

Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu Asp

145 150 155

<210> 95

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 95

Leu Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

Gly Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Ala Asp

20 25 30

Met Thr Ser Ser Pro Cys Arg Ser Arg Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Phe Tyr Lys Asp Ser Gln Pro Arg Gly Phe Ala Val Thr Ile

50 55 60

Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Ile Arg Ser Val Pro Gly His Asp Asn Lys

100 105 110

Ile Gln Phe Glu Ser Ser Ser Tyr Glu Gly Tyr Phe Leu Ala Cys Glu

115 120 125

Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu Asp

145 150 155

<210> 96

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 96

His Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

Asp Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Thr Asp

20 25 30

Met Glu Ser Lys Pro Cys Arg Asp Ser Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Met Tyr Lys Asp Ser Gln Pro Arg Gly Ile Ala Val Thr Ile

50 55 60

Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Ile Arg Ser Val Pro Gly His Asp Asn Lys

100 105 110

Phe Gln Phe Glu Ser Ser Ser Tyr Glu Gly Tyr Phe Leu Ala Cys Glu

115 120 125

Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu Asp

145 150 155

<210> 97

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 97

Tyr Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

Arg Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Thr Asp

20 25 30

Met Thr Tyr Lys Asp Cys Arg Asp Asn Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Phe Tyr Lys Asp Ser Gln Pro Arg Gly Phe Ala Val Thr Ile

50 55 60

Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Ile Arg Ser Val Pro Gly Ala Asp Asn Lys

100 105 110

Ile Gln Phe Glu Ser Ser Ser Tyr Glu Gly Tyr Phe Leu Ala Cys Glu

115 120 125

Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu Asp

145 150 155

<210> 98

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 98

His Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

Gly Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Gly Asp

20 25 30

Met Glu Ala Ser Pro Cys Arg Asp Asn Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Phe Tyr Lys Asp Ser Gln Pro Arg Gly Met Ala Val Thr Ile

50 55 60

Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Ile Arg Ser Val Pro Gly Ala Asp Asn Lys

100 105 110

Leu Gln Phe Glu Ser Ser Ser Tyr Glu Gly Tyr Phe Leu Ala Cys Glu

115 120 125

Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu Asp

145 150 155

<210> 99

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 99

His Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

Gly Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Thr Asp

20 25 30

Met Thr Ser Ser Asp Cys Arg Asp Lys Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Phe Tyr Lys Asp Ser Gln Pro Arg Gly Leu Ala Val Thr Ile

50 55 60

Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Leu Arg Ser Val Pro Gly Pro Asp Asn Lys

100 105 110

Phe Gln Phe Glu Ser Ser Ser Tyr Glu Gly Tyr Phe Leu Ala Cys Glu

115 120 125

Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu Asp

145 150 155

<210> 100

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 100

His Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

Gly Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Ala Asp

20 25 30

Met Glu Ser Asn Arg Cys Arg Asp Ser Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Met Tyr Lys Asp Ser Gln Pro Arg Gly Phe Ala Val Thr Ile

50 55 60

Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Leu Arg Ser Val Pro Gly His Asp Asn Lys

100 105 110

Ile Gln Phe Glu Ser Ser Ser Tyr Glu Gly Tyr Phe Leu Ala Cys Glu

115 120 125

Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu Asp

145 150 155

<210> 101

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 101

Tyr Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

Gly Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Thr Asp

20 25 30

Met Thr Ala Ser Pro Cys Arg Asp Asn Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Phe Tyr Lys Asp Ser Gln Pro Arg Gly Leu Ala Val Thr Ile

50 55 60

Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Leu Arg Ser Val Pro Gly His Asp Asn Lys

100 105 110

Ile Gln Phe Glu Ser Ser Ser Tyr Glu Gly Tyr Phe Leu Ala Cys Glu

115 120 125

Lys Glu Arg Ser Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu Asp

145 150 155

<210> 102

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 102

Asp Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

Asp Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Ala Asp

20 25 30

Met Lys Ser Asn Val Cys Arg Ala Asn Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Met Tyr Lys Asp Ser Gln Pro Arg Gly Met Ala Val Thr Ile

50 55 60

Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Ile Arg Ser Val Pro Gly Pro Asp Asn Lys

100 105 110

Leu Gln Phe Glu Ser Ser Ser Tyr Glu Gly Tyr Phe Leu Ala Cys Glu

115 120 125

Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu Asp

145 150 155

<210> 103

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 103

His Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

Gly Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Gly Asp

20 25 30

Met Glu Ala Ser Pro Cys Arg Ala Lys Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Ile Tyr Lys Asp Ser Gln Pro Arg Gly Phe Ala Val Thr Ile

50 55 60

Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Leu Arg Ser Val Pro Gly His Asp Asn Lys

100 105 110

Phe Gln Phe Glu Ser Ser Ser Tyr Glu Gly Tyr Phe Leu Ala Cys Glu

115 120 125

Lys Glu Arg Ser Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu Asp

145 150 155

<210> 104

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 104

His Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

Gly Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Ala Asp

20 25 30

Met Ala Ser Asn Arg Cys Arg Ala Asn Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Met Tyr Lys Asp Ser Gln Pro Arg Gly Phe Ala Val Thr Ile

50 55 60

Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Ile Arg Ser Val Pro Gly Pro Asp Asn Lys

100 105 110

Phe Gln Phe Glu Ser Ser Ser Tyr Glu Gly Tyr Phe Leu Ala Cys Glu

115 120 125

Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu Asp

145 150 155

<210> 105

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 105

Tyr Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

Asp Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Ala Asp

20 25 30

Met Lys Ala Lys Ala Cys Arg Ser Asn Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Phe Tyr Lys Asp Ser Gln Pro Arg Gly Phe Ala Val Thr Ile

50 55 60

Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Leu Arg Ser Val Pro Gly Ala Asp Asn Lys

100 105 110

Ile Gln Phe Glu Ser Ser Ser Tyr Glu Gly Tyr Phe Leu Ala Cys Glu

115 120 125

Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu Asp

145 150 155

<210> 106

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 106

His Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

His Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Thr Asp

20 25 30

Met Ala Asp Asn Ala Cys Arg Asp Asn Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Phe Tyr Lys Asp Ser Gln Pro Arg Gly Leu Ala Val Thr Ile

50 55 60

Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Ile Arg Ser Val Pro Gly Asp Asp Asn Lys

100 105 110

Met Gln Phe Glu Ser Ser Ser Tyr Tyr Glu Gly Tyr Phe Leu Ala Cys Glu

115 120 125

Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu Asp

145 150 155

<210> 107

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 107

Tyr Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

Gly Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Thr Asp

20 25 30

Met Lys Ser Asn Leu Cys Arg Ser Asn Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Phe Tyr Lys Asp Ser Gln Pro Arg Gly Ile Ala Val Thr Ile

50 55 60

Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Ile Arg Ser Val Pro Gly Asp Asp Asn Lys

100 105 110

Ile Gln Phe Glu Ser Ser Ser Tyr Glu Gly Tyr Phe Leu Ala Cys Glu

115 120 125

Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu Asp

145 150 155

<210> 108

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 108

His Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

Gly Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Arg Asp

20 25 30

Met Ala Ala Ser His Cys Arg Asp Ser Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Ile Tyr Lys Asp Ser Gln Pro Arg Gly Phe Ala Val Thr Ile

50 55 60

Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Leu Arg Ser Val Pro Gly His Asp Asn Lys

100 105 110

Ile Gln Phe Glu Ser Ser Ser Tyr Glu Gly Tyr Phe Leu Ala Cys Glu

115 120 125

Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu Asp

145 150 155

<210> 109

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 109

Tyr Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

Asp Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Ala Asp

20 25 30

Met Ala Ser Asn Pro Cys Arg Tyr Lys Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Met Tyr Lys Asp Ser Gln Pro Arg Gly Leu Ala Val Thr Ile

50 55 60

Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Leu Arg Ser Val Pro Gly Ala Asp Asn Lys

100 105 110

Leu Gln Phe Glu Ser Ser Ser Tyr Glu Gly Tyr Phe Leu Ala Cys Glu

115 120 125

Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu Asp

145 150 155

<210>  110

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 110

His Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

Gly Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Thr Asp

20 25 30

Met Ala Ser Asn His Cys Arg Tyr Asn Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Met Tyr Lys Asp Ser Gln Pro Arg Gly Leu Ala Val Thr Ile

50 55 60

Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Leu Arg Ser Val Pro Gly Ala Asp Asn Lys

100 105 110

Ile Gln Phe Glu Ser Ser Ser Tyr Glu Gly Tyr Phe Leu Ala Cys Glu

115 120 125

Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu Asp

145 150 155

<210>  111

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 111

His Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

Gly Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Ala Asp

20 25 30

Met Thr Asp Asn Pro Cys Arg Ser Arg Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Phe Tyr Lys Asp Ser Gln Pro Arg Gly Met Ala Val Thr Ile

50 55 60

Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Ile Arg Ser Val Pro Gly His Asp Asn Lys

100 105 110

Phe Gln Phe Glu Ser Ser Ser Tyr Glu Gly Tyr Phe Leu Ala Cys Glu

115 120 125

Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu Asp

145 150 155

<210> 112

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 112

Tyr Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

Gly Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Thr Asp

20 25 30

Met Thr Ala Ser His Cys Arg Ser Ser Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Leu Tyr Lys Asp Ser Gln Pro Arg Gly Met Ala Val Thr Ile

50 55 60

Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Leu Arg Ser Val Pro Gly His Asp Asn Lys

100 105 110

Phe Gln Phe Glu Ser Ser Ser Tyr Glu Gly Tyr Phe Leu Ala Cys Glu

115 120 125

Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu Asp

145 150 155

<210> 113

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 113

Tyr Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

Gly Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Thr Asp

20 25 30

Met Glu Tyr Arg Leu Cys Arg Ala Asn Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Phe Tyr Lys Asp Ser His Pro Arg Gly Leu Ala Val Thr Ile

50 55 60

Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Leu Arg Ser Val Pro Gly Asp Asp Asn Lys

100 105 110

Leu Gln Phe Glu Ser Ser Ser Tyr Glu Gly Tyr Phe Leu Ala Cys Glu

115 120 125

Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu Asp

145 150 155

<210> 114

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 114

Tyr Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

Gly Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Thr Asp

20 25 30

Met Glu Ser Ser Leu Cys Arg Asp Asn Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Leu Tyr Lys Asp Ser Gln Pro Arg Gly Met Ala Val Thr Ile

50 55 60

Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Leu Arg Ser Val Pro Gly Ala Asp Asn Lys

100 105 110

Phe Gln Phe Glu Ser Ser Ser Tyr Glu Gly Tyr Phe Leu Ala Cys Glu

115 120 125

Lys Glu Arg Ser Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu Asp

145 150 155

<210> 115

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 115

Tyr Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

Gly Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Lys Asp

20 25 30

Met Glu Ala Asn Asp Cys Arg Ser Ser Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Ile Tyr Lys Asp Ser Gln Pro Arg Gly Leu Ala Val Thr Ile

50 55 60

Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Ile Arg Ser Val Pro Gly Ala Asp Asn Lys

100 105 110

Met Gln Phe Glu Ser Ser Ser Tyr Tyr Glu Gly Tyr Phe Leu Ala Cys Glu

115 120 125

Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu Asp

145 150 155

<210> 116

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 116

Asp Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

Asp Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Ala Asp

20 25 30

Met Lys Ala Ser Ala Cys Arg Ala Asn Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Met Tyr Lys Asp Ser Gln Pro Arg Gly Leu Ala Val Thr Ile

50 55 60

Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Leu Arg Ser Val Pro Gly His Asp Asn Lys

100 105 110

Phe Gln Phe Glu Ser Ser Ser Tyr Glu Gly Tyr Phe Leu Ala Cys Glu

115 120 125

Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu Asp

145 150 155

<210> 117

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 117

Tyr Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

Gly Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Gly Asp

20 25 30

Met Thr Ala Lys His Cys Arg Ala Arg Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Phe Tyr Lys Asp Ser Gln Pro Arg Gly Met Ala Val Thr Ile

50 55 60

Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Ile Arg Ser Val Pro Gly Ala Asp Asn Lys

100 105 110

Phe Gln Phe Glu Ser Ser Ser Tyr Glu Gly Tyr Phe Leu Ala Cys Glu

115 120 125

Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu Asp

145 150 155

<210> 118

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 118

Phe Phe Gly Lys Phe Glu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

Gly Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Thr Asp

20 25 30

Met Glu Ser Lys Asp Cys Arg Asp Arg Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Phe Tyr Lys Asp Ser Gln Pro Arg Gly Leu Ala Val Thr Ile

50 55 60

Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Leu Arg Ser Val Pro Gly His Asp Asn Lys

100 105 110

Leu Gln Phe Glu Ser Ser Ser Tyr Glu Gly Tyr Phe Leu Ala Cys Glu

115 120 125

Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu Asp

145 150 155

<210> 119

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 119

Phe Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

Gly Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Ala Asp

20 25 30

Met Ala Ser Asn His Cys Arg Ala Asn Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Leu Tyr Lys Asp Ser Gln Pro Arg Gly Leu Ala Val Thr Ile

50 55 60

Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Ile Arg Ser Val Pro Gly His Asp Asn Lys

100 105 110

Met Gln Phe Glu Ser Ser Ser Tyr Tyr Glu Gly Tyr Phe Leu Ala Cys Glu

115 120 125

Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu Asp

145 150 155

<210> 120

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 120

Tyr Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

Gly Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Ala Asp

20 25 30

Met Thr Ser Lys Arg Cys Arg Asp Asn Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Leu Tyr Lys Asp Ser Gln Pro Arg Gly Phe Ala Val Thr Ile

50 55 60

Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Ile Arg Ser Val Pro Gly His Asp Asn Lys

100 105 110

Ile Gln Phe Glu Ser Ser Ser Tyr Glu Gly Tyr Phe Leu Ala Cys Glu

115 120 125

Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu Asp

145 150 155

<210>  121

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 121

Leu Phe Gly Lys His Glu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

Gly Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Gly Asp

20 25 30

Met Glu Ser Ser Pro Cys Arg Tyr Asn Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Phe Tyr Lys Asp Ser Gln Pro Arg Gly Leu Ala Val Thr Ile

50 55 60

Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Ile Arg Ser Val Pro Gly His Asp Asn Lys

100 105 110

Met Gln Phe Glu Ser Ser Ser Tyr Tyr Glu Gly Tyr Phe Leu Ala Cys Glu

115 120 125

Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu Asp

145 150 155

<210> 122

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 122

Tyr Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

Ala Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Thr Asp

20 25 30

Met Thr Ala Ser Pro Cys Arg Ser Ser Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Leu Tyr Lys Asp Ser Gln Pro Arg Gly Leu Ala Val Thr Ile

50 55 60

Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Leu Arg Ser Val Pro Gly Pro Asp Asn Lys

100 105 110

Ile Gln Phe Glu Ser Ser Ser Tyr Glu Gly Tyr Phe Leu Ala Cys Glu

115 120 125

Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu Asp

145 150 155

<210> 123

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 123

Tyr Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

Gly Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Ala Asp

20 25 30

Met Thr Asp Ser Asp Cys Arg Asp Asn Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Met Tyr Lys Asp Ser Gln Pro Arg Gly Met Ala Val Thr Ile

50 55 60

Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Leu Arg Ser Val Pro Gly His Asp Asn Lys

100 105 110

Met Gln Phe Glu Ser Ser Ser Tyr Tyr Glu Gly Tyr Phe Leu Ala Cys Glu

115 120 125

Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu Asp

145 150 155

<210> 124

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 124

Tyr Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

Gly Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Ala Asp

20 25 30

Met Thr Ser Ser Asp Cys Arg Asp Asn Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Phe Tyr Lys Asp Ser Gln Pro Arg Gly Met Ala Val Thr Ile

50 55 60

Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Leu Arg Ser Val Pro Gly His Asp Asn Lys

100 105 110

Met Gln Phe Glu Ser Ser Ser Tyr Tyr Glu Gly Tyr Phe Leu Ala Cys Glu

115 120 125

Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu Asp

145 150 155

<210> 125

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 125

Tyr Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

Gly Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Ala Asp

20 25 30

Met Glu Ser Ser Asp Cys Arg Asp Asn Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Phe Tyr Lys Asp Ser Gln Pro Arg Gly Leu Ala Val Thr Ile

50 55 60

Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Leu Arg Ser Val Pro Gly His Asp Asn Lys

100 105 110

Met Gln Phe Glu Ser Ser Ser Tyr Tyr Glu Gly Tyr Phe Leu Ala Cys Glu

115 120 125

Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu Asp

145 150 155

<210> 126

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 126

Tyr Phe Gly Arg Tyr His Cys Thr Thr Ala Val Ile Arg Asn Ile Asn

1 5 10 15

Gln Gln Val Leu Phe Val Asp Lys Arg Gln Pro Val Phe Ala Asp Met

20 25 30

Gly Tyr Thr Val Gln Ser Ala Ser Glu Pro Gln Thr Arg Leu Ile

35 40 45

Tyr Met Tyr Lys Asp Ser Glu Val Arg Gly Leu Ala Val Thr Leu Ser

50 55 60

Val Lys Asp Ser Lys Met Ser Thr Leu Ser Cys Lys Asn Lys Ile Ile

65 70 75 80

Ser Phe Glu Glu Met Asp Pro Pro Glu Asn Ile Asp Asp Ile Gln Ser

85 90 95

Asp Leu Ile Phe Phe Leu Lys Glu Val Pro Gly His Arg Lys Leu Glu

100 105 110

Phe Glu Ser Ser Leu Tyr Glu Gly His Phe Leu Ala Cys Gln Lys Glu

115 120 125

Asp Glu Ala Phe Lys Leu Ile Leu Lys Lys Lys Asp Glu Asn Gly Asp

130 135 140

Lys Ser Val Met Phe Thr Leu Thr Asn Leu His Gln Ser

145 150 155

<210> 127

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 127

Asp Phe Gly Arg Leu His Cys Thr Thr Ala Val Ile Arg Asn Ile Asn

1 5 10 15

Asp Gln Val Leu Phe Val Asp Lys Arg Gln Pro Val Phe Ala Asp Met

20 25 30

Gly Ser Ile Ala Gln Ser Ala Ser Glu Pro Gln Thr Arg Leu Ile Ile

35 40 45

Tyr Phe Tyr Lys Asp Ser Glu Val Arg Gly Leu Ala Val Thr Leu Ser

50 55 60

Val Lys Asp Ser Lys Met Tyr Thr Leu Ser Cys Lys Asn Lys Ile Ile

65 70 75 80

Ser Phe Glu Glu Met Asp Pro Pro Glu Asn Ile Asp Asp Ile Gln Ser

85 90 95

Asp Leu Ile Phe Phe Leu Lys Ala Val Pro Gly Asp Asn Lys Ile Glu

100 105 110

Phe Glu Ser Ser Leu Tyr Glu Gly His Phe Leu Ala Cys Gln Lys Glu

115 120 125

Ala Thr Ala Phe Lys Leu Ile Leu Lys Lys Lys Asp Glu Asn Gly Asp

130 135 140

Lys Ser Val Met Phe Thr Leu Thr Asn Leu His Gln Ser

145 150 155

<210> 128

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 128

Tyr Phe Gly Arg Leu His Cys Thr Thr Ala Val Ile Arg Asn Ile Asn

1 5 10 15

Gly Gln Val Leu Phe Val Asp Lys Arg Gln Pro Val Phe Arg Asp Met

20 25 30

Ala Asp Thr Val Gln Ser Ala Ser Glu Pro Gln Thr Arg Leu Ile

35 40 45

Tyr Phe Tyr Lys Asp Ser Glu Val Arg Gly Leu Ala Val Thr Leu Ser

50 55 60

Val Lys Asp Ser Lys Met Ser Thr Leu Ser Cys Lys Asn Lys Ile Ile

65 70 75 80

Ser Phe Glu Glu Met Asp Pro Pro Glu Asn Ile Asp Asp Ile Gln Ser

85 90 95

Asp Leu Ile Phe Phe Ile Lys Pro Val Pro Gly Ala Ser Lys Met Glu

100 105 110

Phe Glu Ser Ser Leu Tyr Glu Gly His Phe Leu Ala Cys Gln Lys Glu

115 120 125

Ala Gly Ala Phe Lys Leu Ile Leu Lys Lys Lys Asp Glu Asn Gly Asp

130 135 140

Lys Ser Val Met Phe Thr Leu Thr Asn Leu His Gln Ser

145 150 155

<210> 129

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 129

His Phe Gly Arg Leu His Cys Thr Thr Ala Val Ile Arg Asn Ile Asn

1 5 10 15

Asp Gln Val Leu Phe Val Asp Lys Arg Gln Pro Val Phe Lys Asp Met

20 25 30

Glu Tyr Thr Val Gln Ser Ala Ser Glu Pro Gln Thr Arg Leu Ile

35 40 45

Tyr Met Tyr Lys Asp Ser Glu Val Arg Gly Leu Ala Val Thr Leu Ser

50 55 60

Val Lys Asp Ser Lys Met Ser Thr Leu Ser Cys Lys Asn Lys Ile Ile

65 70 75 80

Ser Phe Glu Glu Met Asp Pro Pro Glu Asn Ile Asp Asp Ile Gln Ser

85 90 95

Asp Leu Ile Phe Phe Ile Lys Ala Val Pro Gly Asp Arg Lys Ile Glu

100 105 110

Phe Glu Ser Ser Leu Tyr Glu Gly His Phe Leu Ala Cys Gln Lys Glu

115 120 125

Asp Asn Ala Phe Lys Leu Ile Leu Lys Lys Lys Asp Glu Asn Gly Asp

130 135 140

Lys Ser Val Met Phe Thr Leu Thr Asn Leu His Gln Ser

145 150 155

<210> 130

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 130

Tyr Phe Gly Arg Leu His Cys Thr Thr Ala Val Ile Arg Asn Ile Asn

1 5 10 15

Ala Gln Val Leu Phe Val Asp Lys Arg Gln Pro Val Phe Ala Asp Met

20 25 30

Ala Asp Lys Gly Gln Ser Ala Ser Glu Pro Gln Thr Arg Leu Ile Ile

35 40 45

Tyr Met Tyr Lys Asp Ser Glu Val Arg Gly Leu Ala Val Thr Leu Ser

50 55 60

Val Lys Asp Ser Lys Met Ser Thr Leu Ser Cys Lys Asn Lys Ile Ile

65 70 75 80

Ser Phe Glu Glu Met Asp Pro Pro Glu Asn Ile Asp Asp Ile Gln Ser

85 90 95

Asp Leu Ile Phe Phe Leu Lys Pro Val Pro Gly Asp Thr Lys Met Glu

100 105 110

Phe Glu Ser Ser Leu Tyr Glu Gly His Phe Leu Ala Cys Gln Lys Glu

115 120 125

Phe Gly Ala Phe Lys Leu Ile Leu Lys Lys Lys Asp Glu Asn Gly Asp

130 135 140

Lys Ser Val Met Phe Thr Leu Thr Asn Leu His Gln Ser

145 150 155

<210> 131

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 131

Tyr Phe Gly Arg Leu His Cys Thr Thr Ala Val Ile Arg Asn Ile Asn

1 5 10 15

Glu Gln Val Leu Phe Val Asp Lys Arg Gln Pro Val Phe Ala Asp Met

20 25 30

Gly Asp Arg His Gln Ser Ala Ser Glu Pro Gln Thr Arg Leu Ile

35 40 45

Tyr Met Tyr Lys Asp Ser Glu Val Arg Gly Leu Ala Val Thr Leu Ser

50 55 60

Val Lys Asp Ser Lys Met Ser Thr Leu Ser Cys Lys Asn Lys Ile Ile

65 70 75 80

Ser Phe Glu Glu Met Asp Pro Pro Glu Asn Ile Asp Asp Ile Gln Ser

85 90 95

Asp Leu Ile Phe Phe Ile Lys Pro Val Pro Gly Ala Ser Lys Leu Glu

100 105 110

Phe Glu Ser Ser Leu Tyr Glu Gly His Phe Leu Ala Cys Gln Lys Glu

115 120 125

Asp Asp Ala Phe Lys Leu Ile Leu Lys Lys Lys Asp Glu Asn Gly Asp

130 135 140

Lys Ser Val Met Phe Thr Leu Thr Asn Leu His Gln Ser

145 150 155

<210> 132

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 132

His Phe Gly Arg Leu His Cys Thr Thr Ala Val Ile Arg Asn Ile Asn

1 5 10 15

Asp Gln Val Leu Phe Val Asp Lys Arg Gln Pro Val Phe Arg Asp Met

20 25 30

Gly Ala Ile Gly Gln Ser Ala Ser Glu Pro Gln Thr Arg Leu Ile Ile

35 40 45

Tyr Phe Tyr Lys Asp Ser Glu Val Arg Gly Leu Ala Val Thr Leu Ser

50 55 60

Val Lys Asp Ser Lys Met Ser Thr Leu Ser Cys Lys Asn Lys Ile Ile

65 70 75 80

Ser Phe Glu Glu Met Asp Pro Pro Glu Asn Ile Asp Asp Ile Gln Ser

85 90 95

Asp Leu Ile Phe Phe Ile Lys Pro Val Pro Gly Asp Ser Lys Leu Glu

100 105 110

Phe Glu Ser Ser Leu Tyr Glu Gly His Phe Leu Ala Cys Gln Lys Glu

115 120 125

Val Asp Ala Phe Lys Leu Ile Leu Lys Lys Lys Asp Glu Asn Gly Asp

130 135 140

Lys Ser Val Met Phe Thr Leu Thr Asn Leu His Gln Ser

145 150 155

<210> 133

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 133

His Phe Gly Arg Leu His Cys Thr Thr Ala Val Ile Arg Asn Ile Asn

1 5 10 15

Ser Gln Val Leu Phe Val Asp Lys Arg Gln Pro Val Phe Thr Asp Met

20 25 30

Gly Ser Ile Val Gln Ser Ala Ser Glu Pro Gln Thr Arg Leu Ile Ile

35 40 45

Tyr Met Tyr Lys Asp Ser Glu Val Arg Gly Leu Ala Val Thr Leu Ser

50 55 60

Val Lys Asp Ser Lys Met Ser Thr Leu Ser Cys Lys Asn Lys Ile Ile

65 70 75 80

Ser Phe Glu Glu Met Asp Pro Pro Glu Asn Ile Asp Asp Ile Gln Ser

85 90 95

Asp Leu Ile Phe Phe Ile Lys Gly Val Pro Gly Asp Asn Lys Ile Glu

100 105 110

Phe Glu Ser Ser Leu Tyr Glu Gly His Phe Leu Ala Cys Gln Lys Glu

115 120 125

Asp Arg Ala Phe Lys Leu Ile Leu Lys Lys Lys Asp Glu Asn Gly Asp

130 135 140

Lys Ser Val Met Phe Thr Leu Thr Asn Leu His Gln Ser

145 150 155

<210> 134

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 134

Tyr Phe Gly Arg Leu His Cys Thr Thr Ala Val Ile Arg Asn Ile Asn

1 5 10 15

Ser Gln Val Leu Phe Val Asp Lys Arg Gln Pro Val Phe Arg Asp Met

20 25 30

Glu Asp Thr Pro Gln Ser Ala Ser Glu Pro Gln Thr Arg Leu Ile

35 40 45

Tyr Phe Tyr Lys Asp Ser Glu Val Arg Gly Leu Ala Val Thr Leu Ser

50 55 60

Val Lys Asp Ser Lys Met Ser Thr Leu Ser Cys Lys Asn Lys Ile Ile

65 70 75 80

Ser Phe Glu Glu Met Asp Pro Pro Glu Asn Ile Asp Asp Ile Gln Ser

85 90 95

Asp Leu Ile Phe Phe Ile Lys Arg Val Pro Gly Asp Ser Lys Leu Glu

100 105 110

Phe Glu Ser Ser Leu Tyr Glu Gly His Phe Leu Ala Cys Gln Lys Glu

115 120 125

Phe Glu Ala Phe Lys Leu Ile Leu Lys Lys Lys Asp Glu Asn Gly Asp

130 135 140

Lys Ser Val Met Phe Thr Leu Thr Asn Leu His Gln Ser

145 150 155

<210> 135

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 135

His Phe Gly Arg Leu His Cys Thr Thr Ala Val Ile Arg Asn Ile Asn

1 5 10 15

Ala Gln Val Leu Phe Val Asp Lys Arg Gln Pro Val Phe Gly Asp Met

20 25 30

Thr Ala Thr Val Gln Ser Ala Ser Glu Pro Gln Thr Arg Leu Ile

35 40 45

Tyr Phe Tyr Lys Asp Ser Glu Val Arg Gly Leu Ala Val Thr Leu Ser

50 55 60

Val Lys Asp Ser Lys Met Ser Thr Leu Ser Cys Lys Asn Lys Ile Ile

65 70 75 80

Ser Phe Glu Glu Met Asp Pro Pro Glu Asn Ile Asp Asp Ile Gln Ser

85 90 95

Asp Leu Ile Phe Phe Ile Lys Pro Val Pro Gly Asp Ser Lys Leu Glu

100 105 110

Phe Glu Ser Ser Leu Tyr Glu Gly His Phe Leu Ala Cys Gln Lys Glu

115 120 125

Asp Asn Ala Phe Lys Leu Ile Leu Lys Lys Lys Asp Glu Asn Gly Asp

130 135 140

Lys Ser Val Met Phe Thr Leu Thr Asn Leu His Gln Ser

145 150 155

<210> 136

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 136

Asn Phe Gly Arg Leu His Cys Thr Thr Ala Val Ile Arg Asn Ile Asn

1 5 10 15

Asn Gln Val Leu Phe Val Asp Lys Arg Gln Pro Val Phe Lys Asp Met

20 25 30

Glu Tyr Thr Leu Gln Ser Ala Ser Glu Pro Gln Thr Arg Leu Ile

35 40 45

Tyr Phe Tyr Lys Asp Ser Glu Val Arg Gly Leu Ala Val Thr Leu Ser

50 55 60

Val Lys Asp Ser Lys Met Ser Thr Leu Ser Cys Lys Asn Lys Ile Ile

65 70 75 80

Ser Phe Glu Glu Met Asp Pro Pro Glu Asn Ile Asp Asp Ile Gln Ser

85 90 95

Asp Leu Ile Phe Phe Ile Lys Pro Val Pro Gly Asp Asn Lys Leu Glu

100 105 110

Phe Glu Ser Ser Leu Tyr Glu Gly His Phe Leu Ala Cys Gln Lys Glu

115 120 125

Tyr Glu Ala Phe Lys Leu Ile Leu Lys Lys Lys Asp Glu Asn Gly Asp

130 135 140

Lys Ser Val Met Phe Thr Leu Thr Asn Leu His Gln Ser

145 150 155

<210> 137

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 137

Tyr Phe Gly Arg Leu His Cys Thr Thr Ala Val Ile Arg Asn Ile Asn

1 5 10 15

Gly Gln Val Leu Phe Val Asp Lys Arg Gln Pro Val Phe Ala Asp Met

20 25 30

Glu Ala Thr Arg Gln Ser Ala Ser Glu Pro Gln Thr Arg Leu Ile

35 40 45

Tyr Phe Tyr Lys Asp Ser Glu Val Arg Gly Leu Ala Val Thr Leu Ser

50 55 60

Val Lys Asp Ser Lys Met Ser Thr Leu Ser Cys Lys Asn Lys Ile Ile

65 70 75 80

Ser Phe Glu Glu Met Asp Pro Pro Glu Asn Ile Asp Asp Ile Gln Ser

85 90 95

Asp Leu Ile Phe Phe Ile Lys Gly Val Pro Gly Ala Asn Lys Met Glu

100 105 110

Phe Glu Ser Ser Leu Tyr Glu Gly His Phe Leu Ala Cys Gln Lys Glu

115 120 125

Asp Gly Ala Phe Lys Leu Ile Leu Lys Lys Lys Asp Glu Asn Gly Asp

130 135 140

Asn Ser Val Met Phe Thr Leu Thr Asn Leu His Gln Ser

145 150 155

<210> 138

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 138

Asn Phe Gly Arg Leu His Cys Thr Thr Ala Val Ile Arg Asn Ile Asn

1 5 10 15

Gly Gln Val Leu Phe Val Asp Lys Arg Gln Pro Val Phe Ala Asp Met

20 25 30

Arg Ala Ile Leu Gln Ser Ala Ser Glu Pro Gln Thr Arg Leu Ile Ile

35 40 45

Tyr Phe Tyr Lys Asp Ser Glu Val Arg Gly Leu Ala Val Thr Leu Ser

50 55 60

Val Lys Asp Ser Lys Met Ser Thr Leu Ser Cys Lys Asn Lys Ile Ile

65 70 75 80

Ser Phe Glu Glu Met Asp Pro Pro Glu Asn Ile Asp Asp Ile Gln Ser

85 90 95

Asp Leu Ile Phe Phe Leu Lys Gly Val Pro Gly Asp Asn Lys Leu Glu

100 105 110

Phe Glu Ser Ser Leu Tyr Glu Gly His Phe Leu Ala Cys Gln Lys Glu

115 120 125

Asp Arg Ala Phe Lys Leu Ile Leu Lys Lys Lys Asp Glu Asn Gly Asp

130 135 140

Lys Ser Val Met Phe Thr Leu Thr Asn Leu His Gln Ser

145 150 155

<210> 139

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 139

Tyr Phe Gly Arg Leu His Cys Thr Thr Ala Val Ile Arg Asn Ile Asn

1 5 10 15

Ala Gln Val Leu Phe Val Asp Lys Arg Gln Pro Val Phe Ala Asp Met

20 25 30

Glu Ala Thr Ala Gln Ser Ala Ser Glu Pro Gln Thr Arg Leu Ile

35 40 45

Tyr Phe Tyr Lys Asp Ser Glu Val Arg Gly Leu Ala Val Thr Leu Ser

50 55 60

Val Lys Asp Ser Lys Met Ser Thr Leu Ser Cys Lys Asn Lys Ile Ile

65 70 75 80

Ser Phe Glu Glu Met Asp Pro Pro Glu Asn Ile Asp Asp Ile Gln Ser

85 90 95

Asp Leu Ile Phe Phe Ile Lys Gly Val Pro Gly Ala Ser Lys Met Glu

100 105 110

Phe Glu Ser Ser Leu Tyr Glu Gly His Phe Leu Ala Cys Gln Lys Glu

115 120 125

Asp Gly Ala Phe Lys Leu Ile Leu Lys Lys Lys Asp Glu Asn Gly Asp

130 135 140

Lys Ser Val Met Phe Thr Leu Thr Asn Leu His Gln Ser

145 150 155

<210> 140

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 140

Leu Phe Gly Arg Leu His Cys Thr Thr Ala Val Ile Arg Asn Ile Asn

1 5 10 15

Gly Gln Val Leu Phe Val Asp Lys Arg Gln Pro Val Phe Ala Asp Met

20 25 30

Gly Ala Thr Leu Gln Ser Ala Ser Glu Pro Gln Thr Arg Leu Ile

35 40 45

Tyr Met Tyr Lys Asp Ser Glu Val Arg Gly Leu Ala Val Thr Leu Ser

50 55 60

Val Lys Asp Ser Lys Met Ser Thr Leu Ser Cys Lys Asn Lys Ile Ile

65 70 75 80

Ser Phe Glu Glu Met Asp Pro Pro Glu Asn Ile Asp Asp Ile Gln Ser

85 90 95

Asp Leu Ile Phe Phe Leu Lys Pro Val Pro Gly Asp Thr Lys Met Glu

100 105 110

Phe Glu Ser Ser Leu Tyr Glu Gly His Phe Leu Ala Cys Gln Lys Glu

115 120 125

Ala Ser Ala Phe Lys Leu Ile Leu Lys Lys Lys Asp Glu Asn Gly Asp

130 135 140

Lys Ser Val Met Phe Thr Leu Thr Asn Leu His Gln Ser

145 150 155

<210>  141

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 141

Asn Phe Gly Arg Leu His Cys Thr Thr Ala Val Ile Arg Asn Ile Asn

1 5 10 15

Gly Gln Val Leu Phe Val Asp Lys Arg Gln Pro Val Phe Glu Asp Met

20 25 30

Ala Tyr Thr Val Gln Ser Ala Ser Glu Pro Gln Thr Arg Leu Ile

35 40 45

Tyr Phe Tyr Lys Asp Ser Glu Val Arg Gly Leu Ala Val Thr Leu Ser

50 55 60

Val Lys Asp Ser Lys Met Ser Thr Leu Ser Cys Lys Asn Lys Ile Ile

65 70 75 80

Ser Phe Glu Glu Met Asp Pro Pro Glu Asn Ile Asp Asp Ile Gln Ser

85 90 95

Asp Leu Ile Phe Phe Ile Lys Gly Val Pro Gly Asp Ser Lys Met Glu

100 105 110

Phe Glu Ser Ser Leu Tyr Glu Gly His Phe Leu Ala Cys Gln Lys Glu

115 120 125

Tyr Asp Ala Phe Lys Leu Ile Leu Lys Lys Lys Asp Glu Asn Gly Asp

130 135 140

Lys Ser Val Met Phe Thr Leu Thr Asn Leu His Gln Ser

145 150 155

<210> 142

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 142

Asp Phe Gly Arg Leu His Cys Thr Thr Ala Val Ile Arg Asn Ile Asn

1 5 10 15

Asp Gln Val Leu Phe Val Asp Lys Arg Gln Pro Val Phe Lys Asp Met

20 25 30

Glu Ser Lys Pro Gln Ser Ala Ser Glu Pro Gln Thr Arg Leu Ile

35 40 45

Tyr Phe Tyr Lys Asp Ser Glu Val Arg Gly Leu Ala Val Thr Leu Ser

50 55 60

Val Lys Asp Ser Lys Met Ser Thr Leu Ser Cys Lys Asn Lys Ile Ile

65 70 75 80

Ser Phe Glu Glu Met Asp Pro Pro Glu Asn Ile Asp Asp Ile Gln Ser

85 90 95

Asp Leu Ile Phe Phe Leu Lys Ala Val Pro Gly Ala Ser Lys Leu Glu

100 105 110

Phe Glu Ser Ser Leu Tyr Glu Gly His Phe Leu Ala Cys Gln Lys Glu

115 120 125

Ala Asn Ala Phe Lys Leu Ile Leu Lys Lys Lys Asp Glu Asn Gly Asp

130 135 140

Lys Ser Val Met Phe Thr Leu Thr Asn Leu His Gln Ser

145 150 155

<210> 143

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 143

Leu Phe Gly Arg Leu His Cys Thr Thr Ala Val Ile Arg Asn Ile Asn

1 5 10 15

Gly Gln Val Leu Phe Val Asp Lys Arg Gln Pro Val Phe Ala Asp Met

20 25 30

Gly Asp Lys Val Gln Ser Ala Ser Glu Pro Gln Thr Arg Leu Ile

35 40 45

Tyr Met Tyr Lys Asp Ser Glu Val Arg Gly Leu Ala Val Thr Leu Ser

50 55 60

Val Lys Asp Ser Lys Met Ser Thr Leu Ser Cys Lys Asn Lys Ile Ile

65 70 75 80

Ser Phe Glu Glu Met Asp Pro Pro Glu Asn Ile Asp Asp Ile Gln Ser

85 90 95

Asp Leu Ile Phe Phe Ile Lys Pro Val Pro Gly Asp Asn Lys Leu Glu

100 105 110

Phe Glu Ser Ser Leu Tyr Glu Gly His Phe Leu Ala Cys Gln Lys Glu

115 120 125

Asp Glu Ala Phe Lys Leu Ile Leu Lys Thr Lys Asp Glu Asn Gly Asp

130 135 140

Lys Ser Val Met Phe Thr Leu Thr Asn Leu His Gln Ser

145 150 155

<210> 144

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 144

Tyr Phe Gly Arg His His Cys Thr Thr Ala Val Ile Arg Asn Ile Asn

1 5 10 15

Gln Gln Val Leu Phe Val Asp Lys Arg Gln Pro Val Phe Arg Asp Met

20 25 30

Ala Ala Thr Arg Gln Ser Ala Ser Glu Pro Gln Thr Arg Leu Ile

35 40 45

Tyr Met Tyr Lys Asp Ser Glu Val Arg Gly Leu Ala Val Thr Leu Ser

50 55 60

Val Lys Asp Ser Lys Met Ser Thr Leu Ser Cys Lys Asn Lys Ile Ile

65 70 75 80

Ser Phe Glu Glu Met Asp Pro Pro Glu Asn Ile Asp Asp Ile Gln Ser

85 90 95

Asp Leu Ile Phe Phe Leu Lys Gly Val Pro Gly Asp Asn Lys Met Glu

100 105 110

Phe Glu Ser Ser Leu Tyr Glu Gly His Phe Leu Ala Cys Gln Lys Glu

115 120 125

Asp Asp Ala Phe Lys Leu Ile Leu Lys Lys Lys Asp Glu Asn Gly Asp

130 135 140

Lys Ser Val Met Phe Thr Leu Thr Asn Leu His Gln Ser

145 150 155

<210> 145

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 145

Asn Phe Gly Arg Leu His Cys Thr Thr Ala Val Ile Arg Asn Ile Asn

1 5 10 15

Gln Gln Val Leu Phe Val Asp Lys Arg Gln Pro Val Phe Thr Asp Met

20 25 30

Glu Ser Ile Gly Gln Ser Ala Ser Glu Pro Gln Thr Arg Leu Ile Ile

35 40 45

Tyr Phe Tyr Lys Asp Ser Glu Val Arg Gly Leu Ala Val Thr Leu Ser

50 55 60

Val Lys Asp Ser Lys Met Ser Thr Leu Ser Cys Lys Asn Lys Ile Ile

65 70 75 80

Ser Phe Glu Glu Met Asp Pro Pro Glu Asn Ile Asp Asp Ile Gln Ser

85 90 95

Asp Leu Ile Phe Phe Leu Lys Ala Val Pro Gly Ala Asn Lys Leu Glu

100 105 110

Phe Glu Ser Ser Leu Tyr Glu Gly His Phe Leu Ala Cys Gln Lys Glu

115 120 125

Asp Ser Ala Phe Lys Leu Ile Leu Lys Lys Lys Asp Glu Asn Gly Asp

130 135 140

Lys Ser Val Met Phe Thr Leu Thr Asn Leu His Gln Ser

145 150 155

<210> 146

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 146

Phe Phe Gly Arg His His Cys Thr Thr Ala Val Ile Arg Asn Ile Asn

1 5 10 15

Gly Gln Val Leu Phe Val Asp Lys Arg Gln Pro Val Phe Gly Asp Met

20 25 30

Gly Asp Arg Val Gln Ser Ala Ser Glu Pro Gln Thr Arg Leu Ile

35 40 45

Tyr Met Tyr Lys Asp Ser Glu Val Arg Gly Leu Ala Val Thr Leu Ser

50 55 60

Val Lys Asp Ser Lys Met Ser Thr Leu Ser Cys Lys Asn Lys Ile Ile

65 70 75 80

Ser Phe Glu Glu Met Asp Pro Pro Glu Asn Ile Asp Asp Ile Gln Ser

85 90 95

Asp Leu Ile Phe Phe Ile Lys Ala Val Pro Gly Asp Ser Lys Ile Glu

100 105 110

Phe Glu Ser Ser Leu Tyr Glu Gly His Phe Leu Ala Cys Gln Lys Glu

115 120 125

Asp Gly Ala Phe Lys Leu Ile Leu Lys Lys Lys Asp Glu Asn Gly Asp

130 135 140

Lys Ser Val Met Phe Thr Leu Thr Asn Leu His Gln Ser

145 150 155

<210> 147

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 147

Val Phe Gly Arg His His Cys Thr Thr Ala Val Ile Arg Asn Ile Asn

1 5 10 15

Gly Gln Val Leu Phe Val Asp Lys Arg Gln Pro Val Phe Lys Asp Met

20 25 30

Thr Tyr Ile Asp Gln Ser Ala Ser Glu Pro Gln Thr Arg Leu Ile Ile

35 40 45

Tyr Met Tyr Lys Asp Ser Glu Val Arg Gly Leu Ala Val Thr Leu Ser

50 55 60

Val Lys Asp Ser Lys Met Ser Thr Leu Ser Cys Lys Asn Lys Ile Ile

65 70 75 80

Ser Phe Glu Glu Met Asp Pro Pro Glu Asn Ile Asp Asp Ile Gln Ser

85 90 95

Asp Leu Ile Phe Phe Leu Lys Ala Val Pro Gly Asp Thr Lys Met Glu

100 105 110

Phe Glu Ser Ser Leu Tyr Glu Gly His Phe Leu Ala Cys Gln Lys Glu

115 120 125

Ala Gln Ala Phe Lys Leu Ile Leu Lys Lys Lys Asp Glu Ile Gly Asp

130 135 140

Lys Ser Val Met Phe Thr Leu Thr Asn Leu His Gln Ser

145 150 155

<210> 148

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 148

Asn Phe Gly Arg Leu His Cys Thr Thr Ala Val Ile Arg Asn Ile Asn

1 5 10 15

Gly Gln Val Leu Phe Val Asp Lys Arg Gln Pro Val Phe Ala Asp Met

20 25 30

Thr Ala Thr Arg Gln Ser Ala Ser Glu Pro Gln Thr Arg Leu Ile

35 40 45

Tyr Met Tyr Lys Asp Ser Glu Val Arg Gly Leu Ala Val Thr Leu Ser

50 55 60

Val Lys Asp Ser Lys Met Ser Thr Leu Ser Cys Lys Asn Lys Ile Ile

65 70 75 80

Ser Phe Glu Glu Met Asp Pro Pro Glu Asn Ile Asp Asp Ile Gln Ser

85 90 95

Asp Leu Ile Phe Phe Ile Lys Gln Val Pro Gly Ala Asn Lys Ile Glu

100 105 110

Phe Glu Ser Ser Leu Tyr Glu Gly His Phe Leu Ala Cys Gln Lys Glu

115 120 125

Phe Arg Ala Phe Lys Leu Ile Leu Lys Lys Lys Asp Glu Asn Gly Asp

130 135 140

Lys Ser Val Met Phe Thr Leu Thr Asn Leu His Gln Ser

145 150 155

<210> 149

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 149

Asp Phe Gly Arg Leu His Cys Thr Thr Ala Val Ile Arg Asn Ile Asn

1 5 10 15

Gly Gln Val Leu Phe Val Asp Lys Arg Gln Pro Val Phe Gly Asp Met

20 25 30

Ala Tyr Ile Gly Gln Ser Ala Ser Glu Pro Gln Thr Arg Leu Ile Ile

35 40 45

Tyr Phe Tyr Lys Asp Ser Glu Val Arg Gly Leu Ala Val Thr Leu Ser

50 55 60

Val Lys Asp Ser Lys Met Ser Thr Leu Ser Cys Lys Asn Lys Ile Ile

65 70 75 80

Ser Phe Glu Glu Met Asp Pro Pro Glu Asn Ile Asp Asp Ile Gln Ser

85 90 95

Asp Leu Ile Phe Phe Ile Lys Ala Val Pro Gly His Ser Lys Ile Glu

100 105 110

Phe Glu Ser Ser Leu Tyr Glu Gly His Phe Leu Ala Cys Gln Lys Glu

115 120 125

Ser Gly Ala Phe Lys Leu Ile Leu Lys Lys Lys Asp Glu Asn Gly Asp

130 135 140

Lys Ser Val Met Phe Thr Leu Thr Asn Leu His Gln Ser

145 150 155

<210> 150

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 150

Tyr Phe Gly Arg Leu His Cys Thr Thr Ala Val Ile Arg Asn Ile Asn

1 5 10 15

Asp Gln Val Leu Phe Val Asp Lys Arg Gln Pro Val Phe Arg Asp Met

20 25 30

Gly Ser Ile Ala Gln Ser Ala Ser Glu Pro Gln Thr Arg Leu Ile Ile

35 40 45

Tyr Met Tyr Lys Asp Ser Glu Val Arg Gly Leu Ala Val Thr Leu Ser

50 55 60

Val Lys Asp Ser Lys Met Ser Thr Leu Ser Cys Lys Asn Lys Ile Ile

65 70 75 80

Ser Phe Glu Glu Met Asp Pro Pro Glu Asn Ile Asp Asp Ile Gln Ser

85 90 95

Asp Leu Ile Phe Phe Ile Lys Pro Val Pro Gly Ala Thr Lys Leu Glu

100 105 110

Phe Glu Ser Ser Leu Tyr Glu Gly His Phe Leu Ala Cys Gln Lys Glu

115 120 125

Asp Gly Ala Phe Lys Leu Ile Leu Lys Lys Lys Asp Glu Asn Gly Asp

130 135 140

Asn Ser Val Met Phe Thr Leu Thr Asn Leu His Gln Ser

145 150 155

<210> 151

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 151

Tyr Phe Gly Arg Leu His Cys Thr Thr Ala Val Ile Arg Asn Ile Asn

1 5 10 15

Glu Gln Val Leu Phe Val Asp Lys Arg Gln Pro Val Phe Thr Asp Met

20 25 30

Glu Ala Ile Gly Gln Ser Ala Ser Glu Pro Gln Thr Arg Leu Ile Ile

35 40 45

Tyr Phe Tyr Lys Asp Ser Glu Val Arg Gly Leu Ala Val Thr Leu Ser

50 55 60

Val Lys Asp Ser Lys Met Ser Thr Leu Ser Cys Lys Asn Lys Ile Ile

65 70 75 80

Ser Phe Glu Glu Met Asp Pro Pro Glu Asn Ile Asp Asp Ile Gln Ser

85 90 95

Asp Leu Ile Phe Phe Ile Lys Gly Val Pro Gly Asp Arg Lys Met Glu

100 105 110

Phe Glu Ser Ser Leu Tyr Glu Gly His Phe Leu Ala Cys Gln Lys Glu

115 120 125

Asp Gly Ala Phe Lys Leu Ile Leu Lys Lys Lys Asp Glu Asn Gly Asp

130 135 140

Lys Ser Val Met Phe Thr Leu Thr Asn Leu His Gln Ser

145 150 155

<210> 152

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 152

Phe Phe Gly Arg Leu His Cys Thr Thr Ala Val Ile Arg Asn Ile Asn

1 5 10 15

Asn Gln Val Leu Phe Val Asp Lys Arg Gln Pro Val Phe Glu Asp Met

20 25 30

Glu Tyr Arg Leu Gln Ser Ala Ser Glu Pro Gln Thr Arg Leu Ile

35 40 45

Tyr Met Tyr Lys Asp Ser Glu Val Arg Gly Leu Ala Val Thr Leu Ser

50 55 60

Val Lys Asp Ser Lys Met Ser Thr Leu Ser Cys Lys Asn Lys Ile Ile

65 70 75 80

Ser Phe Glu Glu Met Asp Pro Pro Glu Asn Ile Asp Asp Ile Gln Ser

85 90 95

Asp Leu Ile Phe Phe Leu Lys Pro Val Pro Gly Ala Ser Lys Leu Glu

100 105 110

Phe Glu Ser Ser Leu Tyr Glu Gly His Phe Leu Ala Cys Gln Lys Glu

115 120 125

Ser Asp Ala Phe Lys Leu Ile Leu Lys Lys Lys Asp Glu Asn Gly Asp

130 135 140

Lys Ser Val Met Phe Thr Leu Thr Asn Leu His Gln Ser

145 150 155

<210> 153

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 153

Asn Phe Gly Arg Leu His Cys Thr Thr Ala Val Ile Arg Asn Ile Asn

1 5 10 15

Asn Gln Val Leu Phe Val Asp Lys Arg Gln Pro Val Phe Ala Asp Met

20 25 30

Glu Asp Arg Leu Gln Ser Ala Ser Glu Pro Gln Thr Arg Leu Ile

35 40 45

Tyr Met Tyr Lys Asp Ser Glu Val Arg Gly Leu Ala Val Thr Leu Ser

50 55 60

Val Lys Asp Ser Lys Met Ser Thr Leu Ser Cys Lys Asn Lys Ile Ile

65 70 75 80

Ser Phe Glu Glu Met Asp Pro Pro Glu Asn Ile Asp Asp Ile Gln Ser

85 90 95

Asp Leu Ile Phe Phe Leu Lys Gly Val Pro Gly Asp Asn Lys Met Glu

100 105 110

Phe Glu Ser Ser Leu Tyr Glu Gly His Phe Leu Ala Cys Gln Lys Glu

115 120 125

Asp His Ala Phe Lys Leu Ile Leu Lys Lys Lys Asp Glu Asn Gly Asp

130 135 140

Lys Ser Val Met Phe Thr Leu Thr Asn Leu His Gln Ser

145 150 155

<210> 154

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 154

Tyr Phe Gly Arg Leu His Cys Thr Thr Ala Val Ile Arg Asn Ile Asn

1 5 10 15

Ala Gln Val Leu Phe Val Asp Lys Arg Gln Pro Val Phe Arg Asp Met

20 25 30

Gly Tyr Ile Leu Gln Ser Ala Ser Glu Pro Gln Thr Arg Leu Ile Ile

35 40 45

Tyr Leu Tyr Lys Asp Ser Glu Val Arg Gly Leu Ala Val Thr Leu Ser

50 55 60

Val Lys Glu Ser Lys Met Ser Thr Leu Ser Cys Lys Asn Lys Ile Ile

65 70 75 80

Ser Phe Glu Glu Met Asp Pro Pro Glu Asn Ile Asp Asp Ile Gln Ser

85 90 95

Asp Leu Ile Phe Phe Leu Lys Pro Val Pro Gly Asp Thr Lys Ile Glu

100 105 110

Phe Glu Ser Ser Leu Tyr Glu Gly His Phe Leu Ala Cys Gln Lys Glu

115 120 125

Asp Asn Ala Phe Lys Leu Ile Leu Lys Lys Lys Asp Glu Asn Gly Asp

130 135 140

Lys Ser Val Met Phe Thr Leu Thr Asn Leu His Gln Ser

145 150 155

<210> 155

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 155

Tyr Phe Gly Arg Leu His Cys Thr Thr Ala Val Ile Arg Asn Ile Asn

1 5 10 15

Asp Gln Val Leu Phe Val Asp Lys Arg Gln Pro Val Phe Gly Asp Met

20 25 30

Ala Asp Thr Ala Gln Ser Ala Ser Glu Pro Gln Thr Arg Leu Ile Ile

35 40 45

Tyr Phe Tyr Lys Asp Ser Glu Val Arg Gly Leu Ala Val Thr Leu Ser

50 55 60

Val Lys Asp Ser Lys Met Ser Thr Leu Ser Cys Lys Asn Lys Ile Ile

65 70 75 80

Ser Phe Glu Glu Met Asp Pro Pro Glu Asn Ile Asp Asp Ile Gln Ser

85 90 95

Asp Leu Ile Phe Phe Ile Lys Pro Val Pro Gly Asp Ser Lys Met Glu

100 105 110

Phe Glu Ser Ser Leu Tyr Glu Gly His Phe Leu Ala Cys Gln Lys Glu

115 120 125

Ala Asp Ala Phe Lys Leu Ile Leu Lys Lys Lys Asp Glu Asn Gly Asp

130 135 140

Lys Ser Val Met Phe Thr Leu Thr Asn Leu His Gln Ser

145 150 155

<210> 156

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 156

Asp Phe Gly Arg Leu His Cys Thr Thr Ala Val Ile Arg Asn Ile Asn

1 5 10 15

Gly Gln Val Leu Phe Val Asp Lys Arg Gln Pro Val Phe Glu Asp Met

20 25 30

Ala Tyr Ile Ala Gln Ser Ala Ser Glu Pro Gln Thr Arg Leu Ile Ile

35 40 45

Tyr Phe Tyr Lys Asp Ser Glu Val Arg Gly Leu Ala Val Thr Leu Ser

50 55 60

Val Lys Asp Ser Lys Met Ser Thr Leu Ser Cys Lys Asn Lys Ile Ile

65 70 75 80

Ser Phe Glu Glu Met Asp Pro Pro Glu Asn Ile Asp Asp Ile Gln Ser

85 90 95

Asp Leu Ile Phe Phe Ile Lys Pro Val Pro Gly Asp Ser Lys Ile Glu

100 105 110

Phe Glu Ser Ser Leu Tyr Glu Gly His Phe Leu Ala Cys Gln Lys Glu

115 120 125

Ala Asp Ala Phe Lys Leu Ile Leu Lys Lys Lys Asp Glu Asn Gly Asp

130 135 140

Lys Ser Val Met Phe Thr Leu Thr Asn Leu His Gln Ser

145 150 155

<210> 157

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 157

Asn Phe Gly Arg Leu His Cys Thr Thr Ala Val Ile Arg Asn Ile Asn

1 5 10 15

Glu Gln Val Leu Ser Val Asp Lys Arg Gln Pro Val Phe Arg Asp Met

20 25 30

Lys Tyr Ile Leu Gln Ser Ala Ser Glu Pro Gln Thr Arg Leu Ile Ile

35 40 45

Tyr Phe Tyr Lys Asp Ser Glu Val Arg Gly Leu Ala Val Thr Leu Ser

50 55 60

Val Lys Asp Ser Lys Met Ser Thr Leu Ser Cys Lys Asn Lys Ile Ile

65 70 75 80

Ser Phe Glu Glu Met Asp Pro Pro Glu Asn Ile Asp Asp Ile Gln Ser

85 90 95

Asp Leu Ile Phe Phe Leu Lys Gly Val Pro Gly Asp Asn Lys Met Glu

100 105 110

Phe Glu Ser Ser Leu Tyr Glu Gly His Phe Leu Ala Cys Gln Lys Glu

115 120 125

Tyr Gly Ala Phe Lys Leu Ile Leu Lys Lys Lys Asp Glu Asn Gly Asp

130 135 140

Lys Ser Val Met Phe Thr Leu Thr Asn Leu His Gln Ser

145 150 155

<210> 158

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 158

Asp Phe Gly Arg Leu His Cys Thr Thr Ala Val Ile Arg Asn Ile Asn

1 5 10 15

Glu Gln Val Leu Phe Val Asp Lys Arg Gln Pro Val Phe Thr Asp Met

20 25 30

Ala Tyr Ile Leu Gln Ser Ala Ser Glu Pro Gln Thr Arg Leu Ile Ile

35 40 45

Tyr Phe Tyr Lys Asp Ser Glu Val Arg Gly Leu Ala Val Thr Leu Ser

50 55 60

Val Lys Glu Ser Lys Met Ser Thr Leu Ser Cys Lys Asn Lys Ile Ile

65 70 75 80

Ser Phe Glu Glu Met Asp Pro Pro Glu Asn Ile Asp Asp Ile Gln Ser

85 90 95

Asp Leu Ile Phe Phe Ile Lys Ala Val Pro Gly Asp Ser Lys Leu Glu

100 105 110

Phe Glu Ser Ser Leu Tyr Glu Gly His Phe Leu Ala Cys Gln Lys Glu

115 120 125

Asp Thr Ala Phe Lys Leu Ile Leu Lys Lys Lys Asp Glu Asn Gly Asp

130 135 140

Lys Ser Val Met Phe Thr Leu Thr Asn Leu His Gln Ser

145 150 155

<210> 159

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 159

Asp Phe Gly Arg Leu His Cys Thr Thr Ala Val Ile Arg Asn Ile Asn

1 5 10 15

Asn Gln Val Leu Phe Val Asp Lys Arg Gln Pro Val Phe Lys Asp Met

20 25 30

Glu Ser Thr Ala Gln Ser Ala Ser Glu Pro Gln Thr Arg Leu Ile

35 40 45

Tyr Met Tyr Lys Asp Ser Glu Val Arg Gly Leu Ala Val Thr Leu Ser

50 55 60

Val Lys Asp Ser Lys Met Ser Thr Leu Ser Cys Lys Asn Lys Ile Ile

65 70 75 80

Ser Phe Glu Glu Met Asp Pro Pro Glu Asn Ile Asp Asp Ile Gln Ser

85 90 95

Asp Leu Ile Phe Phe Leu Lys Gly Val Pro Gly Ala Ser Lys Leu Glu

100 105 110

Phe Glu Ser Ser Leu Tyr Glu Gly His Phe Leu Ala Cys Gln Lys Glu

115 120 125

Ala Gly Ala Phe Lys Leu Ile Leu Lys Lys Lys Asp Glu Asn Gly Asp

130 135 140

Lys Ser Val Met Phe Thr Leu Thr Asn Leu His Gln Ser

145 150 155

<210> 160

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 160

His Phe Gly Arg Leu His Cys Thr Thr Ala Val Ile Arg Asn Ile Asn

1 5 10 15

Glu Gln Val Leu Phe Val Asp Lys Arg Gln Pro Val Phe Ala Asp Met

20 25 30

Glu Ala Ile Gly Gln Ser Ala Ser Glu Pro Gln Thr Arg Leu Ile Ile

35 40 45

Tyr Phe Tyr Lys Asp Ser Glu Val Arg Gly Leu Ala Val Thr Leu Ser

50 55 60

Val Lys Glu Ser Lys Met Ser Thr Leu Ser Cys Lys Asn Lys Ile Ile

65 70 75 80

Ser Phe Glu Glu Met Asp Pro Pro Glu Asn Ile Asp Asp Ile Gln Ser

85 90 95

Asp Leu Ile Phe Phe Ile Lys Gly Val Pro Gly Asp Thr Lys Leu Glu

100 105 110

Phe Glu Ser Ser Leu Tyr Ala Gly His Phe Leu Ala Cys Gln Lys Glu

115 120 125

Asp Gly Ala Phe Lys Leu Ile Leu Lys Lys Lys Asp Glu Asn Gly Asp

130 135 140

Lys Ser Val Met Phe Thr Leu Thr Asn Leu His Gln Ser

145 150 155

<210> 161

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 161

Ile Phe Gly Arg Leu His Cys Thr Thr Ala Val Ile Arg Asn Ile Asn

1 5 10 15

Glu Gln Val Leu Phe Val Asp Lys Arg Gln Pro Val Phe Lys Asp Met

20 25 30

Arg Tyr Ile Val Gln Ser Ala Ser Glu Pro Gln Thr Arg Leu Ile Ile

35 40 45

Tyr Phe Tyr Lys Asp Ser Glu Val Arg Gly Leu Ala Val Thr Leu Ser

50 55 60

Val Lys Asp Ser Lys Met Ser Thr Leu Ser Cys Lys Asn Lys Ile Ile

65 70 75 80

Ser Phe Glu Glu Met Asp Pro Pro Glu Asn Ile Asp Asp Ile Gln Ser

85 90 95

Asp Leu Ile Phe Phe Ile Lys Glu Val Pro Gly Ala Ser Lys Leu Glu

100 105 110

Phe Glu Ser Ser Leu Tyr Glu Gly His Phe Leu Ala Cys Gln Lys Glu

115 120 125

Asp Glu Ala Phe Lys Leu Ile Leu Lys Lys Lys Asp Glu Asn Gly Asp

130 135 140

Lys Ser Val Met Phe Thr Leu Thr Asn Leu His Gln Ser

145 150 155

<210> 162

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 162

Tyr Phe Gly Arg Leu His Cys Thr Thr Ala Val Ile Arg Asn Ile Asn

1 5 10 15

Ala Gln Val Leu Phe Val Asp Lys Arg Gln Pro Val Phe Thr Asp Met

20 25 30

Gly Tyr Thr Leu Gln Ser Ala Ser Glu Pro Gln Thr Arg Leu Ile

35 40 45

Tyr Leu Tyr Lys Asp Ser Glu Val Arg Gly Leu Ala Val Thr Leu Ser

50 55 60

Val Lys Asp Ser Lys Met Ser Thr Leu Ser Cys Lys Asn Lys Ile Ile

65 70 75 80

Ser Phe Glu Glu Met Asp Pro Pro Glu Asn Ile Asp Asp Ile Gln Ser

85 90 95

Asp Leu Ile Phe Phe Ile Lys Pro Val Pro Gly His Asn Lys Ile Glu

100 105 110

Phe Glu Ser Ser Leu Tyr Glu Gly His Phe Leu Ala Cys Gln Lys Glu

115 120 125

Asp Arg Ala Phe Lys Leu Ile Leu Lys Lys Lys Asp Glu Asn Gly Asp

130 135 140

Lys Ser Val Met Phe Thr Leu Thr Asn Leu His Gln Ser

145 150 155

<210> 163

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 163

Asn Phe Gly Arg Leu His Cys Thr Thr Ala Val Ile Arg Asn Ile Asn

1 5 10 15

Asn Gln Val Leu Phe Val Asp Lys Arg Gln Pro Val Phe Arg Asp Met

20 25 30

Ala Ser Thr Ala Gln Ser Ala Ser Glu Pro Gln Thr Arg Leu Ile

35 40 45

Tyr Met Tyr Lys Asp Ser Glu Val Arg Gly Leu Ala Val Thr Leu Ser

50 55 60

Val Lys Asp Ser Lys Met Ser Thr Leu Ser Cys Lys Asn Lys Ile Ile

65 70 75 80

Ser Phe Glu Glu Met Asp Pro Pro Glu Asn Ile Asp Asp Ile Gln Ser

85 90 95

Asp Leu Ile Phe Phe Ile Lys Gly Val Pro Gly Ala Asn Lys Ile Glu

100 105 110

Phe Glu Ser Ser Leu Tyr Glu Gly His Phe Leu Ala Cys Gln Lys Glu

115 120 125

Asp Asp Ala Phe Lys Leu Ile Leu Lys Lys Lys Asp Glu Asn Gly Asp

130 135 140

Lys Ser Val Met Phe Thr Leu Thr Asn Leu His Gln Ser

145 150 155

<210> 164

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 164

Asp Phe Gly Arg Leu His Cys Thr Thr Ala Val Ile Arg Asn Ile Asn

1 5 10 15

Gly Gln Val Leu Phe Val Asp Lys Arg Gln Pro Val Phe Glu Asp Met

20 25 30

Lys Asp Arg Ala Gln Ser Ala Ser Glu Pro Gln Thr Arg Leu Ile

35 40 45

Tyr Phe Tyr Lys Asp Ser Glu Val Arg Gly Leu Ala Val Thr Leu Ser

50 55 60

Val Lys Asp Ser Lys Met Ser Thr Leu Ser Cys Lys Asn Lys Ile Ile

65 70 75 80

Ser Phe Glu Glu Met Asp Pro Pro Glu Asn Ile Asp Asp Ile Gln Ser

85 90 95

Asp Leu Ile Phe Phe Leu Lys Ala Val Pro Gly His Ser Lys Met Glu

100 105 110

Phe Glu Ser Ser Leu Tyr Glu Gly His Phe Leu Ala Cys Gln Lys Glu

115 120 125

Asp Glu Ala Phe Lys Leu Ile Leu Lys Lys Lys Asp Glu Asn Gly Asp

130 135 140

Lys Ser Val Met Phe Thr Leu Thr Asn Leu His Gln Ser

145 150 155

<210> 165

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 165

Asn Phe Gly Arg Leu His Cys Thr Thr Ala Val Ile Arg Asn Ile Asn

1 5 10 15

Glu Gln Val Leu Phe Val Asp Lys Arg Gln Pro Val Phe Ala Asp Met

20 25 30

Thr Asp Ile Ala Gln Ser Ala Ser Glu Pro Gln Thr Arg Leu Ile Ile

35 40 45

Tyr Met Tyr Lys Asp Ser Glu Val Arg Gly Leu Ala Val Thr Leu Ser

50 55 60

Val Lys Glu Ser Lys Met Ser Thr Leu Ser Cys Lys Asn Lys Ile Ile

65 70 75 80

Ser Phe Glu Glu Met Asp Pro Pro Glu Asn Ile Asp Asp Ile Gln Ser

85 90 95

Asp Leu Ile Phe Phe Leu Lys Pro Val Pro Gly Asp Ile Lys Met Glu

100 105 110

Phe Glu Ser Ser Leu Tyr Glu Gly His Phe Leu Ala Cys Gln Lys Glu

115 120 125

Tyr Gly Ala Phe Lys Leu Ile Leu Lys Lys Lys Asp Glu Asn Gly Asp

130 135 140

Asn Ser Val Met Phe Thr Leu Thr Asn Leu His Gln Ser

145 150 155

<210> 166

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 166

Tyr Phe Gly Arg Leu His Cys Thr Thr Ala Val Ile Arg Asn Ile Asn

1 5 10 15

Glu Gln Val Leu Phe Val Asp Lys Arg Gln Pro Val Phe Ala Asp Met

20 25 30

Thr Asp Thr Leu Gln Ser Ala Ser Glu Pro Gln Thr Arg Leu Ile

35 40 45

Tyr Phe Tyr Lys Asp Ser Glu Val Arg Gly Leu Ala Val Thr Leu Ser

50 55 60

Val Lys Asp Ser Lys Met Ser Thr Leu Ser Cys Lys Asn Lys Ile Ile

65 70 75 80

Ser Phe Glu Glu Met Asp Pro Pro Glu Asn Ile Asp Asp Ile Gln Ser

85 90 95

Asp Leu Ile Phe Phe Leu Lys Gly Val Pro Gly Asp Asn Lys Met Glu

100 105 110

Phe Glu Ser Ser Leu Tyr Glu Gly His Phe Leu Ala Cys Gln Lys Glu

115 120 125

Asp Thr Ala Phe Lys Leu Ile Leu Lys Lys Lys Asp Glu Asn Gly Asp

130 135 140

Lys Ser Val Met Phe Thr Leu Thr Asn Leu His Gln Ser

145 150 155

<210> 167

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 167

Tyr Phe Gly Arg Leu His Cys Thr Thr Ala Val Ile Arg Asn Ile Asn

1 5 10 15

Glu Gln Val Leu Phe Val Asp Lys Arg Gln Pro Val Phe Ala Asp Met

20 25 30

Thr Asp Thr Leu Gln Ser Ala Ser Glu Pro Gln Thr Arg Leu Ile

35 40 45

Tyr Phe Tyr Lys Asp Ser Glu Val Arg Gly Leu Ala Val Thr Leu Ser

50 55 60

Val Lys Asp Ser Lys Met Ser Thr Leu Ser Cys Lys Asn Lys Ile Ile

65 70 75 80

Ser Phe Glu Glu Met Asp Pro Pro Glu Asn Ile Asp Asp Ile Gln Ser

85 90 95

Asp Leu Ile Phe Phe Leu Lys Gly Val Pro Gly Asp Asn Lys Met Glu

100 105 110

Phe Glu Ser Ser Leu Tyr Glu Gly His Phe Leu Ala Cys Gln Lys Glu

115 120 125

Asp Thr Ala Phe Lys Leu Ile Leu Lys Lys Lys Asp Glu Asn Gly Asp

130 135 140

Lys Ser Val Met Phe Thr Leu Thr Asn Leu His Gln Ser

145 150 155

<210> 168

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 168

Tyr Phe Gly Arg Leu His Cys Thr Thr Ala Val Ile Arg Asn Ile Asn

1 5 10 15

Glu Gln Val Leu Phe Val Asp Lys Arg Gln Pro Val Phe Ala Asp Met

20 25 30

Thr Asp Thr Leu Gln Ser Ala Ser Glu Pro Gln Thr Arg Leu Ile

35 40 45

Tyr Phe Tyr Lys Asp Ser Glu Val Arg Gly Leu Ala Val Thr Leu Ser

50 55 60

Val Lys Asp Ser Lys Met Ser Thr Leu Ser Cys Lys Asn Lys Ile Ile

65 70 75 80

Ser Phe Glu Glu Met Asp Pro Pro Glu Asn Ile Asp Asp Ile Gln Ser

85 90 95

Asp Leu Ile Phe Phe Leu Lys Gly Val Pro Gly Asp Asn Lys Met Glu

100 105 110

Phe Glu Ser Ser Leu Tyr Glu Gly His Phe Leu Ala Cys Gln Lys Glu

115 120 125

Asp Thr Ala Phe Lys Leu Ile Leu Lys Lys Lys Asp Glu Asn Gly Asp

130 135 140

Lys Ser Val Met Phe Thr Leu Thr Asn Leu His Gln Ser

145 150 155

<210> 169

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 169

His Phe Gly Arg Leu His Cys Thr Thr Ala Val Ile Arg Asn Ile Asn

1 5 10 15

Gly Gln Val Leu Phe Val Asp Lys Arg Gln Pro Val Phe Lys Asp Met

20 25 30

Lys Tyr Ile Val Gln Ser Ala Ser Glu Pro Gln Thr Arg Leu Ile Ile

35 40 45

Tyr Met Tyr Lys Asp Ser Glu Val Arg Gly Leu Ala Val Thr Leu Ser

50 55 60

Val Lys Asp Ser Lys Met Ser Thr Leu Ser Cys Lys Asn Lys Ile Ile

65 70 75 80

Ser Phe Glu Glu Met Asp Pro Pro Glu Asn Ile Asp Asp Ile Gln Ser

85 90 95

Asp Leu Ile Phe Phe Leu Lys Ala Val Pro Gly His Ser Lys Ile Glu

100 105 110

Phe Glu Ser Ser Leu Tyr Glu Gly His Phe Leu Ala Cys Gln Lys Glu

115 120 125

Asp Ser Ala Phe Lys Leu Ile Leu Lys Lys Lys Asp Glu Asn Gly Asp

130 135 140

Lys Ser Val Met Phe Thr Leu Thr Asn Leu His Gln Ser

145 150 155

<210> 170

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 170

His Phe Gly Arg Leu His Cys Thr Thr Ala Val Ile Arg Asn Ile Asn

1 5 10 15

Gly Gln Val Leu Phe Val Asp Lys Arg Gln Pro Val Phe Lys Asp Met

20 25 30

Lys Tyr Ile Val Gln Ser Ala Ser Glu Pro Gln Thr Arg Leu Ile Ile

35 40 45

Tyr Met Tyr Lys Asp Ser Glu Val Arg Gly Leu Ala Val Thr Leu Ser

50 55 60

Val Lys Asp Ser Lys Met Ser Thr Leu Ser Cys Lys Asn Lys Ile Ile

65 70 75 80

Ser Phe Glu Glu Met Asp Pro Pro Glu Asn Ile Asp Asp Ile Gln Ser

85 90 95

Asp Leu Ile Phe Phe Leu Lys Ala Val Pro Gly His Ser Lys Ile Glu

100 105 110

Phe Glu Ser Ser Leu Tyr Glu Gly His Phe Leu Ala Cys Gln Lys Glu

115 120 125

Asp Ser Ala Phe Lys Leu Ile Leu Lys Lys Lys Asp Glu Asn Gly Asp

130 135 140

Lys Ser Val Met Phe Thr Leu Thr Asn Leu His Gln Ser

145 150 155

<210> 171

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 171

Tyr Phe Gly Arg Leu His Cys Thr Thr Ala Val Ile Arg Asn Ile Asn

1 5 10 15

Gly Gln Val Leu Phe Val Asp Lys Arg Gln Pro Val Phe Glu Asp Met

20 25 30

Lys Ala Lys Ala Gln Ser Ala Ser Glu Pro Gln Thr Arg Leu Ile Ile

35 40 45

Tyr Phe Tyr Lys Asp Ser Glu Val Arg Gly Leu Ala Val Thr Leu Ser

50 55 60

Val Lys Asp Ser Lys Met Ser Thr Leu Ser Cys Lys Asn Lys Ile Ile

65 70 75 80

Ser Phe Glu Glu Met Asp Pro Pro Glu Asn Ile Asp Asp Ile Gln Ser

85 90 95

Asp Leu Ile Phe Phe Ile Lys Pro Val Pro Gly Ala Ser Lys Met Glu

100 105 110

Phe Glu Ser Ser Leu Tyr Glu Gly His Phe Leu Ala Cys Gln Lys Glu

115 120 125

Asp Gly Ala Phe Lys Leu Ile Leu Lys Lys Lys Asp Glu Asn Gly Asp

130 135 140

Lys Ser Val Met Phe Thr Leu Thr Asn Leu His Gln Ser

145 150 155

<210> 172

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 172

Tyr Phe Gly Arg Leu His Cys Thr Thr Ala Val Ile Arg Asn Ile Asn

1 5 10 15

Gly Gln Val Leu Phe Val Asp Lys Arg Gln Pro Val Phe Glu Asp Met

20 25 30

Lys Ala Lys Ala Gln Ser Ala Ser Glu Pro Gln Thr Arg Leu Ile Ile

35 40 45

Tyr Phe Tyr Lys Asp Ser Glu Val Arg Gly Leu Ala Val Thr Leu Ser

50 55 60

Val Lys Asp Ser Lys Met Ser Thr Leu Ser Cys Lys Asn Lys Ile Ile

65 70 75 80

Ser Phe Glu Glu Met Asp Pro Pro Glu Asn Ile Asp Asp Ile Gln Ser

85 90 95

Asp Leu Ile Phe Phe Ile Lys Pro Val Pro Gly Ala Ser Lys Met Glu

100 105 110

Phe Glu Ser Ser Leu Tyr Glu Gly His Phe Leu Ala Cys Gln Lys Glu

115 120 125

Asp Gly Ala Phe Lys Leu Ile Leu Lys Lys Lys Asp Glu Asn Gly Asp

130 135 140

Lys Ser Val Met Phe Thr Leu Thr Asn Leu His Gln Ser

145 150 155

<210> 173

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 173

Tyr Phe Gly Arg Leu His Cys Thr Thr Ala Val Ile Arg Asn Ile Asn

1 5 10 15

Gly Gln Val Leu Phe Val Asp Lys Arg Gln Pro Val Phe Glu Asp Met

20 25 30

Lys Ala Lys Ala Gln Ser Ala Ser Glu Pro Gln Thr Arg Leu Ile Ile

35 40 45

Tyr Phe Tyr Lys Asp Ser Glu Val Arg Gly Leu Ala Val Thr Leu Ser

50 55 60

Val Lys Asp Ser Lys Met Ser Thr Leu Ser Cys Lys Asn Lys Ile Ile

65 70 75 80

Ser Phe Glu Glu Met Asp Pro Pro Glu Asn Ile Asp Asp Ile Gln Ser

85 90 95

Asp Leu Ile Phe Phe Ile Lys Pro Val Pro Gly Ala Ser Lys Met Glu

100 105 110

Phe Glu Ser Ser Leu Tyr Glu Gly His Phe Leu Ala Cys Gln Lys Glu

115 120 125

Asp Gly Ala Phe Lys Leu Ile Leu Lys Lys Lys Asp Glu Asn Gly Asp

130 135 140

Lys Ser Val Met Phe Thr Leu Thr Asn Leu His Gln Ser

145 150 155

<210> 174

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 174

Leu Phe Gly Arg Leu His Cys Thr Thr Ala Val Ile Arg Asn Ile Asn

1 5 10 15

Gly Gln Val Leu Phe Val Asp Lys Arg Gln Pro Val Phe Gly Asp Met

20 25 30

Gly Ser Ile Pro Gln Ser Ala Ser Glu Pro Gln Thr Arg Leu Ile Ile

35 40 45

Tyr Phe Tyr Lys Asp Ser Glu Val Arg Gly Leu Ala Val Thr Leu Ser

50 55 60

Val Lys Asp Ser Lys Met Ser Thr Leu Ser Cys Lys Asn Lys Ile Ile

65 70 75 80

Ser Phe Glu Glu Met Asp Pro Pro Glu Asn Ile Asp Asp Ile Gln Ser

85 90 95

Asp Leu Ile Phe Phe Ile Lys His Val Pro Gly Ala Thr Lys Met Glu

100 105 110

Phe Glu Ser Ser Leu Tyr Glu Gly His Phe Leu Ala Cys Gln Lys Glu

115 120 125

Asp Asn Ala Phe Lys Leu Ile Leu Lys Lys Lys Asp Glu Asn Gly Asp

130 135 140

Lys Ser Val Met Phe Thr Leu Thr Asn Leu His Gln Ser

145 150 155

<210> 175

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 175

Leu Phe Gly Arg Leu His Cys Thr Thr Ala Val Ile Arg Asn Ile Asn

1 5 10 15

Gly Gln Val Leu Phe Val Asp Lys Arg Gln Pro Val Phe Gly Asp Met

20 25 30

Gly Ser Ile Pro Gln Ser Ala Ser Glu Pro Gln Thr Arg Leu Ile Ile

35 40 45

Tyr Phe Tyr Lys Asp Ser Glu Val Arg Gly Leu Ala Val Thr Leu Ser

50 55 60

Val Lys Asp Ser Lys Met Ser Thr Leu Ser Cys Lys Asn Lys Ile Ile

65 70 75 80

Ser Phe Glu Glu Met Asp Pro Pro Glu Asn Ile Asp Asp Ile Gln Ser

85 90 95

Asp Leu Ile Phe Phe Ile Lys His Val Pro Gly Ala Thr Lys Met Glu

100 105 110

Phe Glu Ser Ser Leu Tyr Glu Gly His Phe Leu Ala Cys Gln Lys Glu

115 120 125

Asp Asn Ala Phe Lys Leu Ile Leu Lys Lys Lys Asp Glu Asn Gly Asp

130 135 140

Lys Ser Val Met Phe Thr Leu Thr Asn Leu His Gln Ser

145 150 155

<210> 176

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 176

Tyr Phe Gly Arg Leu His Cys Thr Thr Ala Val Ile Arg Asn Ile Asn

1 5 10 15

Ser Gln Val Leu Phe Val Asp Lys Arg Gln Pro Val Phe Thr Asp Met

20 25 30

Ala Tyr Thr Val Gln Ser Ala Ser Glu Pro Gln Thr Arg Leu Ile

35 40 45

Tyr Phe Tyr Lys Asp Ser Glu Val Arg Gly Leu Ala Val Thr Leu Ser

50 55 60

Val Lys Asp Ser Lys Met Ser Thr Leu Ser Cys Lys Asn Lys Ile Ile

65 70 75 80

Ser Phe Glu Glu Met Asp Pro Pro Glu Asn Ile Asp Asp Ile Gln Ser

85 90 95

Asp Leu Ile Phe Phe Ile Lys Ala Val Pro Gly Asp Ser Lys Leu Glu

100 105 110

Phe Glu Ser Ser Leu Tyr Glu Gly His Phe Leu Ala Cys Gln Lys Glu

115 120 125

Asp Asn Ala Phe Lys Leu Ile Leu Lys Lys Lys Asp Glu Asn Gly Asp

130 135 140

Lys Ser Val Met Phe Thr Leu Thr Asn Leu His Gln Ser

145 150 155

<210> 177

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 177

Tyr Phe Gly Arg Leu His Cys Thr Thr Ala Val Ile Arg Asn Ile Asn

1 5 10 15

Ser Gln Val Leu Phe Val Asp Lys Arg Gln Pro Val Phe Thr Asp Met

20 25 30

Ala Tyr Thr Val Gln Ser Ala Ser Glu Pro Gln Thr Arg Leu Ile

35 40 45

Tyr Phe Tyr Lys Asp Ser Glu Val Arg Gly Leu Ala Val Thr Leu Ser

50 55 60

Val Lys Asp Ser Lys Met Ser Thr Leu Ser Cys Lys Asn Lys Ile Ile

65 70 75 80

Ser Phe Glu Glu Met Asp Pro Pro Glu Asn Ile Asp Asp Ile Gln Ser

85 90 95

Asp Leu Ile Phe Phe Ile Lys Ala Val Pro Gly Asp Ser Lys Leu Glu

100 105 110

Phe Glu Ser Ser Leu Tyr Glu Gly His Phe Leu Ala Cys Gln Lys Glu

115 120 125

Asp Asn Ala Phe Lys Leu Ile Leu Lys Lys Lys Asp Glu Asn Gly Asp

130 135 140

Lys Ser Val Met Phe Thr Leu Thr Asn Leu His Gln Ser

145 150 155

<210> 178

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 178

Tyr Phe Gly Arg Leu His Cys Thr Thr Ala Val Ile Arg Asn Ile Asn

1 5 10 15

Gly Gln Val Leu Phe Val Asp Lys Arg Gln Pro Val Phe Thr Asp Met

20 25 30

Gly Ala Arg Val Gln Ser Ala Ser Glu Pro Gln Thr Arg Leu Ile

35 40 45

Tyr Phe Tyr Lys Asp Ser Glu Val Arg Gly Leu Ala Val Thr Leu Ser

50 55 60

Val Lys Asp Ser Lys Met Tyr Thr Leu Ser Cys Lys Asn Lys Ile Ile

65 70 75 80

Ser Phe Glu Glu Met Asp Pro Pro Glu Asn Ile Asp Asp Ile Gln Ser

85 90 95

Asp Leu Ile Phe Phe Leu Lys Pro Val Pro Gly Asp Asn Lys Leu Glu

100 105 110

Phe Glu Ser Ser Leu Tyr Glu Gly His Phe Leu Ala Cys Gln Lys Glu

115 120 125

Ser Gly Ala Phe Lys Leu Ile Leu Lys Lys Lys Asp Glu Asn Gly Asp

130 135 140

Lys Ser Val Met Phe Thr Leu Thr Asn Leu His Gln Ser

145 150 155

<210> 179

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 179

Tyr Phe Gly Arg Leu His Cys Thr Thr Ala Val Ile Arg Asn Ile Asn

1 5 10 15

Gly Gln Val Leu Phe Val Asp Lys Arg Gln Pro Val Phe Thr Asp Met

20 25 30

Gly Ala Arg Val Gln Ser Ala Ser Glu Pro Gln Thr Arg Leu Ile

35 40 45

Tyr Phe Tyr Lys Asp Ser Glu Val Arg Gly Leu Ala Val Thr Leu Ser

50 55 60

Val Lys Asp Ser Lys Met Tyr Thr Leu Ser Cys Lys Asn Lys Ile Ile

65 70 75 80

Ser Phe Glu Glu Met Asp Pro Pro Glu Asn Ile Asp Asp Ile Gln Ser

85 90 95

Asp Leu Ile Phe Phe Leu Lys Pro Val Pro Gly Asp Asn Lys Leu Glu

100 105 110

Phe Glu Ser Ser Leu Tyr Glu Gly His Phe Leu Ala Cys Gln Lys Glu

115 120 125

Ser Gly Ala Phe Lys Leu Ile Leu Lys Lys Lys Asp Glu Asn Gly Asp

130 135 140

Lys Ser Val Met Phe Thr Leu Thr Asn Leu His Gln Ser

145 150 155

<210> 180

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 180

Asp Phe Gly Arg Leu His Cys Thr Thr Ala Val Ile Arg Asn Ile Asn

1 5 10 15

Gly Gln Val Leu Phe Val Asp Lys Arg Gln Pro Val Phe Gly Asp Met

20 25 30

Lys Ala Thr Gly Gln Ser Ala Ser Glu Pro Gln Thr Arg Leu Ile

35 40 45

Tyr Phe Tyr Lys Asp Ser Glu Val Arg Gly Leu Ala Val Thr Leu Ser

50 55 60

Val Lys Asp Ser Lys Met Ser Thr Leu Ser Cys Lys Asn Lys Ile Ile

65 70 75 80

Ser Phe Glu Glu Met Asp Pro Pro Glu Asn Ile Asp Asp Ile Gln Ser

85 90 95

Asp Leu Ile Phe Phe Ile Lys Ala Val Pro Gly Ala Asn Lys Leu Glu

100 105 110

Phe Glu Ser Ser Leu Tyr Glu Gly His Phe Leu Ala Cys Gln Lys Glu

115 120 125

Ala Gly Ala Phe Lys Leu Ile Leu Lys Lys Lys Asp Glu Asn Gly Asp

130 135 140

Lys Ser Val Met Phe Thr Leu Thr Asn Leu His Gln Ser

145 150 155

<210>  181

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 181

Asp Phe Gly Arg Leu His Cys Thr Thr Ala Val Ile Arg Asn Ile Asn

1 5 10 15

Gly Gln Val Leu Phe Val Asp Lys Arg Gln Pro Val Phe Gly Asp Met

20 25 30

Lys Ala Thr Gly Gln Ser Ala Ser Glu Pro Gln Thr Arg Leu Ile

35 40 45

Tyr Phe Tyr Lys Asp Ser Glu Val Arg Gly Leu Ala Val Thr Leu Ser

50 55 60

Val Lys Asp Ser Lys Met Ser Thr Leu Ser Cys Lys Asn Lys Ile Ile

65 70 75 80

Ser Phe Glu Glu Met Asp Pro Pro Glu Asn Ile Asp Asp Ile Gln Ser

85 90 95

Asp Leu Ile Phe Phe Ile Lys Ala Val Pro Gly Ala Asn Lys Leu Glu

100 105 110

Phe Glu Ser Ser Leu Tyr Glu Gly His Phe Leu Ala Cys Gln Lys Glu

115 120 125

Ala Gly Ala Phe Lys Leu Ile Leu Lys Lys Lys Asp Glu Asn Gly Asp

130 135 140

Lys Ser Val Met Phe Thr Leu Thr Asn Leu His Gln Ser

145 150 155

<210> 182

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 182

Asp Phe Gly Arg Leu His Cys Thr Thr Ala Val Ile Arg Asn Ile Asn

1 5 10 15

Ser Gln Val Leu Phe Val Asp Lys Arg Gln Pro Val Phe Arg Asp Met

20 25 30

Gly Ser Ile His Gln Ser Ala Ser Glu Pro Gln Thr Arg Leu Ile Ile

35 40 45

Tyr Phe Tyr Lys Asp Ser Glu Val Arg Gly Leu Ala Val Thr Leu Ser

50 55 60

Val Lys Asp Ser Lys Met Ser Thr Leu Ser Cys Lys Asn Lys Ile Ile

65 70 75 80

Ser Phe Glu Glu Met Asp Pro Pro Glu Asn Ile Asp Asp Ile Gln Ser

85 90 95

Asp Leu Ile Phe Phe Leu Lys Ala Val Pro Gly Ala Asn Lys Leu Glu

100 105 110

Phe Glu Ser Ser Leu Tyr Glu Gly His Phe Leu Ala Cys Gln Lys Glu

115 120 125

Asp Gly Ala Phe Lys Leu Ile Leu Lys Lys Lys Asp Glu Asn Gly Asp

130 135 140

Lys Ser Val Met Phe Thr Leu Thr Asn Leu His Gln Ser

145 150 155

<210> 183

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 183

Asp Phe Gly Arg Leu His Cys Thr Thr Ala Val Ile Arg Asn Ile Asn

1 5 10 15

Ser Gln Val Leu Phe Val Asp Lys Arg Gln Pro Val Phe Arg Asp Met

20 25 30

Gly Ser Ile His Gln Ser Ala Ser Glu Pro Gln Thr Arg Leu Ile Ile

35 40 45

Tyr Phe Tyr Lys Asp Ser Glu Val Arg Gly Leu Ala Val Thr Leu Ser

50 55 60

Val Lys Asp Ser Lys Met Ser Thr Leu Ser Cys Lys Asn Lys Ile Ile

65 70 75 80

Ser Phe Glu Glu Met Asp Pro Pro Glu Asn Ile Asp Asp Ile Gln Ser

85 90 95

Asp Leu Ile Phe Phe Leu Lys Ala Val Pro Gly Ala Asn Lys Leu Glu

100 105 110

Phe Glu Ser Ser Leu Tyr Glu Gly His Phe Leu Ala Cys Gln Lys Glu

115 120 125

Asp Gly Ala Phe Lys Leu Ile Leu Lys Lys Lys Asp Glu Asn Gly Asp

130 135 140

Lys Ser Val Met Phe Thr Leu Thr Asn Leu His Gln Ser

145 150 155

<210> 184

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 184

Tyr Phe Gly Arg Leu His Cys Thr Thr Ala Val Ile Arg Asn Ile Asn

1 5 10 15

Glu Gln Val Leu Phe Val Asp Lys Arg Gln Pro Val Phe Lys Asp Met

20 25 30

Lys Asp Lys Leu Gln Ser Ala Ser Glu Pro Gln Thr Arg Leu Ile Ile

35 40 45

Tyr Phe Tyr Lys Asp Ser Glu Val Arg Gly Leu Ala Val Thr Leu Ser

50 55 60

Val Lys Asp Ser Lys Met Ser Thr Leu Ser Cys Lys Asn Lys Ile Ile

65 70 75 80

Ser Phe Glu Glu Met Asp Pro Pro Glu Asn Ile Asp Asp Ile Gln Ser

85 90 95

Asp Leu Ile Phe Phe Leu Lys Gly Val Pro Gly Asp Asn Lys Leu Glu

100 105 110

Phe Glu Ser Ser Leu Tyr Glu Gly His Phe Leu Ala Cys Gln Lys Glu

115 120 125

Phe Gly Ala Phe Lys Leu Ile Leu Lys Lys Lys Asp Glu Asn Gly Asp

130 135 140

Lys Ser Val Met Phe Thr Leu Thr Asn Leu His Gln Ser

145 150 155

<210> 185

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 185

Tyr Phe Gly Arg Leu His Cys Thr Thr Ala Val Ile Arg Asn Ile Asn

1 5 10 15

Glu Gln Val Leu Phe Val Asp Lys Arg Gln Pro Val Phe Lys Asp Met

20 25 30

Lys Asp Lys Leu Gln Ser Ala Ser Glu Pro Gln Thr Arg Leu Ile Ile

35 40 45

Tyr Phe Tyr Lys Asp Ser Glu Val Arg Gly Leu Ala Val Thr Leu Ser

50 55 60

Val Lys Asp Ser Lys Met Ser Thr Leu Ser Cys Lys Asn Lys Ile Ile

65 70 75 80

Ser Phe Glu Glu Met Asp Pro Pro Glu Asn Ile Asp Asp Ile Gln Ser

85 90 95

Asp Leu Ile Phe Phe Leu Lys Gly Val Pro Gly Asp Asn Lys Leu Glu

100 105 110

Phe Glu Ser Ser Leu Tyr Glu Gly His Phe Leu Ala Cys Gln Lys Glu

115 120 125

Phe Gly Ala Phe Lys Leu Ile Leu Lys Lys Lys Asp Glu Asn Gly Asp

130 135 140

Lys Ser Val Met Phe Thr Leu Thr Asn Leu His Gln Ser

145 150 155

<210> 186

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 186

Tyr Phe Gly Arg Leu His Cys Thr Thr Ala Val Ile Arg Asn Ile Asn

1 5 10 15

Gly Gln Val Leu Phe Val Asp Lys Arg Gln Pro Val Phe Ala Asp Met

20 25 30

Ala Ser Thr His Gln Ser Ala Ser Glu Pro Gln Thr Arg Leu Ile

35 40 45

Tyr Phe Tyr Lys Asp Ser Glu Val Arg Gly Leu Ala Val Thr Leu Ser

50 55 60

Val Lys Asp Ser Lys Met Ser Thr Leu Ser Cys Lys Asn Lys Ile Ile

65 70 75 80

Ser Phe Glu Glu Met Asp Pro Pro Glu Asn Ile Asp Asp Ile Gln Ser

85 90 95

Asp Leu Ile Phe Phe Leu Lys Gly Val Pro Gly Ala Asn Lys Ile Glu

100 105 110

Phe Glu Ser Ser Leu Tyr Glu Gly His Phe Leu Ala Cys Gln Lys Glu

115 120 125

Asp Asp Ala Phe Lys Leu Ile Leu Lys Lys Lys Asp Glu Asn Gly Asp

130 135 140

Lys Ser Val Met Phe Thr Leu Thr Asn Leu His Gln Ser

145 150 155

<210> 187

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 187

Tyr Phe Gly Arg Leu His Cys Thr Thr Ala Val Ile Arg Asn Ile Asn

1 5 10 15

Gly Gln Val Leu Phe Val Asp Lys Arg Gln Pro Val Phe Ala Asp Met

20 25 30

Ala Ser Thr His Gln Ser Ala Ser Glu Pro Gln Thr Arg Leu Ile

35 40 45

Tyr Phe Tyr Lys Asp Ser Glu Val Arg Gly Leu Ala Val Thr Leu Ser

50 55 60

Val Lys Asp Ser Lys Met Ser Thr Leu Ser Cys Lys Asn Lys Ile Ile

65 70 75 80

Ser Phe Glu Glu Met Asp Pro Pro Glu Asn Ile Asp Asp Ile Gln Ser

85 90 95

Asp Leu Ile Phe Phe Leu Lys Gly Val Pro Gly Ala Asn Lys Ile Glu

100 105 110

Phe Glu Ser Ser Leu Tyr Glu Gly His Phe Leu Ala Cys Gln Lys Glu

115 120 125

Asp Asp Ala Phe Lys Leu Ile Leu Lys Lys Lys Asp Glu Asn Gly Asp

130 135 140

Lys Ser Val Met Phe Thr Leu Thr Asn Leu His Gln Ser

145 150 155

<210> 188

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 188

Tyr Phe Gly Arg Leu His Cys Thr Thr Ala Val Ile Arg Asn Ile Asn

1 5 10 15

Gly Gln Val Leu Phe Val Asp Lys Arg Gln Pro Val Phe Ala Asp Met

20 25 30

Ala Ser Thr His Gln Ser Ala Ser Glu Pro Gln Thr Arg Leu Ile

35 40 45

Tyr Phe Tyr Lys Asp Ser Glu Val Arg Gly Leu Ala Val Thr Leu Ser

50 55 60

Val Lys Asp Ser Lys Met Ser Thr Leu Ser Cys Lys Asn Lys Ile Ile

65 70 75 80

Ser Phe Glu Glu Met Asp Pro Pro Glu Asn Ile Asp Asp Ile Gln Ser

85 90 95

Asp Leu Ile Phe Phe Leu Lys Gly Val Pro Gly Ala Asn Lys Ile Glu

100 105 110

Phe Glu Ser Ser Leu Tyr Glu Gly His Phe Leu Ala Cys Gln Lys Glu

115 120 125

Asp Asp Ala Phe Lys Leu Ile Leu Lys Lys Lys Asp Glu Asn Gly Asp

130 135 140

Lys Ser Val Met Phe Thr Leu Thr Asn Leu His Gln Ser

145 150 155

<210> 189

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 189

Tyr Phe Gly Arg Leu His Cys Thr Thr Ala Val Ile Arg Asn Ile Asn

1 5 10 15

Ser Gln Val Leu Phe Val Asp Lys Arg Gln Pro Val Phe Gly Asp Met

20 25 30

Lys Tyr Ile Val Gln Ser Ala Ser Glu Pro Gln Thr Arg Leu Ile Ile

35 40 45

Tyr Phe Tyr Lys Asp Ser Glu Val Arg Gly Leu Ala Val Thr Leu Ser

50 55 60

Val Lys Asp Ser Lys Met Ser Thr Leu Ser Cys Lys Asn Lys Ile Ile

65 70 75 80

Ser Phe Glu Glu Met Asp Pro Pro Glu Asn Ile Asp Asp Ile Gln Ser

85 90 95

Asp Leu Ile Phe Phe Leu Lys Gly Val Pro Gly Asp Thr Lys Met Glu

100 105 110

Phe Glu Ser Ser Leu Tyr Glu Gly His Phe Leu Ala Cys Gln Lys Glu

115 120 125

Ser Gly Ala Phe Lys Leu Ile Leu Lys Lys Lys Asp Glu Asn Gly Asp

130 135 140

Lys Ser Val Met Phe Thr Leu Thr Asn Leu His Gln Ser

145 150 155

<210> 190

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 190

Tyr Phe Gly Arg Leu His Cys Thr Thr Ala Val Ile Arg Asn Ile Asn

1 5 10 15

Ser Gln Val Leu Phe Val Asp Lys Arg Gln Pro Val Phe Gly Asp Met

20 25 30

Lys Tyr Ile Val Gln Ser Ala Ser Glu Pro Gln Thr Arg Leu Ile Ile

35 40 45

Tyr Phe Tyr Lys Asp Ser Glu Val Arg Gly Leu Ala Val Thr Leu Ser

50 55 60

Val Lys Asp Ser Lys Met Ser Thr Leu Ser Cys Lys Asn Lys Ile Ile

65 70 75 80

Ser Phe Glu Glu Met Asp Pro Pro Glu Asn Ile Asp Asp Ile Gln Ser

85 90 95

Asp Leu Ile Phe Phe Leu Lys Gly Val Pro Gly Asp Thr Lys Met Glu

100 105 110

Phe Glu Ser Ser Leu Tyr Glu Gly His Phe Leu Ala Cys Gln Lys Glu

115 120 125

Ser Gly Ala Phe Lys Leu Ile Leu Lys Lys Lys Asp Glu Asn Gly Asp

130 135 140

Lys Ser Val Met Phe Thr Leu Thr Asn Leu His Gln Ser

145 150 155

<210> 191

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 191

Tyr Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

Asp Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Glu Asp

20 25 30

Met Thr Asp Ser Asp Cys Arg Asp Asn Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Lys Tyr Gly Asp Ser Val Pro Arg Gly Leu Ala Val Thr Ile

50 55 60

Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Ala Arg Ala Val Pro Gly His Ser Arg Lys

100 105 110

Thr Gln Phe Glu Ser Ser Ser Tyr Tyr Glu Gly Tyr Phe Leu Ala Cys Glu

115 120 125

Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu Asp

145 150 155

<210> 192

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 192

Tyr Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

Asp Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Glu Asp

20 25 30

Met Thr Asp Ser Asp Cys Arg Asp Asn Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Lys Tyr Ser Asp Ser Arg Ala Arg Gly Leu Ala Val Thr Ile

50 55 60

Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Ala Arg Ser Val Pro Gly His Gly Arg Lys

100 105 110

Thr Gln Phe Glu Ser Ser Ser Tyr Tyr Glu Gly Tyr Phe Leu Ala Cys Glu

115 120 125

Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu Asp

145 150 155

<210> 193

<211> 157

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 193

Tyr Phe Gly Lys Leu Glu Ser Lys Leu Ser Val Ile Arg Asn Leu Asn

1 5 10 15

Asp Gln Val Leu Phe Ile Asp Gln Gly Asn Arg Pro Leu Phe Glu Asp

20 25 30

Met Thr Asp Ser Asp Cys Arg Asp Asn Ala Pro Arg Thr Ile Phe Ile

35 40 45

Ile Ser Lys Tyr Ser Asp Ser Arg Ala Arg Gly Leu Ala Val Thr Ile

50 55 60

Ser Val Lys Cys Glu Lys Ile Ser Thr Leu Ser Cys Glu Asn Lys Ile

65 70 75 80

Ile Ser Phe Lys Glu Met Asn Pro Pro Asp Asn Ile Lys Asp Thr Lys

85 90 95

Ser Asp Ile Ile Phe Phe Ala Arg Asp Val Pro Gly His Ser Gly Lys

100 105 110

Arg Gln Phe Glu Ser Ser Ser Tyr Glu Gly Tyr Phe Leu Ala Cys Glu

115 120 125

Lys Glu Arg Asp Leu Phe Lys Leu Ile Leu Lys Lys Glu Asp Glu Leu

130 135 140

Gly Asp Arg Ser Ile Met Phe Thr Val Gln Asn Glu Asp

145 150 155

<210> 194

<211> 58

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 194

cattttcatt aagatgcagt tacttcgctg tttttcaata ttttctgtta ttgctagc 58

<210> 195

<211> 80

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesized sequence

<220>

<221> misc_features

<222>  (31)..(31)

<223> n is a, c, g, or t

<220>

<221> misc_features

<222>  (42)..(42)

<223> n is a, c, g, or t

<400> 195

aattacggat gaccgaaagt ykggattcaw ncttgccgaa anrtgctaaa acgctagcaa 60

taacagaaaa tattgaaaaa                               80

<210> 196

<211> 51

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 196

actttcggtc atccgtaatt tgaacgacca agtccttttt attgaccagg g 51

<210> 197

<211> 55

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 197

actatccgtc atatcctcga ataagggacg attgccctgg tcaataaaaa ggact 55

<210> 198

<211> 44

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 198

cttattcgag gatatgacgg atagtgattg ccgtgacaac gccc 44

<210> 199

<211> 84

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesized sequence

<220>

<221> misc_features

<222>  (22)..(22)

<223> n is a, c, g, or t

<220>

<221> misc_features

<222>  (46)..(46)

<223> n is a, c, g, or t

<400> 199

actgagattg ttaccgcchb tnyacggggt tgwyyatcty tatasnyaga gatgatgaaa 60

attgtacgag gggcgttgtc acgg                                 84

<210> 200

<211> 52

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 200

ggcggtaaca atctcagtta agtgcgaaaa aatctcgaca ctttcttgtg aa 52

<210>  201

<211> 53

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 201

ggttcatttc cttgaacgaa atgatcttgt tttcacaaga aagtgtcgag att 53

<210>  202

<211> 62

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400>  202

catttcgttc aaggaaatga acccgccgga taatatcaag gatacaaaat cagatattat 60

tt

<210>  203

<211> 88

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesized sequence

<220>

<221> misc_features

<222>  (27)..(27)

<223> n is a, c, g, or t

<400> 203

tgatgagctc tcgaattgca tcttatnwtb gtgtccaggc acwyyacgwt bgaagaaaat 60

aatatctgat tttgtatcct tgatatta                     88

<210>  204

<211> 51

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400>  204

ataagatgca attcgagagc tcatcatacg aaggttactt tttagcctgc g 51

<210>  205

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 205

aattaactta aacaggtcgc gctccttctc gcaggctaaa aagtaacctt 50

<210>  206

<211> 49

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 206

gcgacctgtt taagttaatt cttaagaaag aagatgagtt gggggatcg 49

<210>  207

<211> 61

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 207

ccagaaccac cgtcctcwtb ctgadyggta aacatgatgc tacgatcccc caactcatct 60

t

<210>  208

<211> 52

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 208

gaggacggtg gttctggatc cgaacaaaag cttatctccg aagaagactt gg 52

<210>  209

<211>  41

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 209

ccaccagatc caccaccacc caagtcttct tcggagataa g 41

<210>  210

<211> 62

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 210

cattttcatt aagatgcagt tacttcgctg tttttcaata ttttctgtta ttgctagcgt 60

tt

<210>  211

<211> 55

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesized sequence

<220>

<221> misc_features

<222>  (25)..(25)

<223> n is a, c, g, or t

<400>  211

ttgtacagtg aagtcggcca aaawntgcta aaacgctagc aataacagaa aatat 55

<210>  212

<211> 59

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400>  212

gccgacttca ctgtacaacc gcagtaatac ggaatataaa tgaccaagtt ctcttcgtt 59

<210>  213

<211> 67

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 213

ttgatcaata tcagtcatat cctcgaacac aggctgtctt ttgtcaacga agagaacttg 60

gtcattt                                                                                

<210>  214

<211> 54

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400>  214

gtgttcgagg atatgactga tattgatcaa agtgccagtg aaccccagac caga 54

<210>  215

<211> 78

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesized sequence

<220>

<221> misc_features

<222>  (24)..(24)

<223> n is a, c, g, or t

<220>

<221> misc_features

<222>  (30)..(30)

<223> n is a, c, g, or t

<220>

<221> misc_features

<222>  (32)..(32)

<223> n is a, c, g, or t

<220>

<221> misc_features

<222>  (42)..(42)

<223> n is a, c, g, or t

<220>

<221> misc_features

<222>  (44)..(44)

<223> n is a, c, g, or t

<220>

<221> misc_features

<222>  (46)..(46)

<223> n is a, c, g, or t

<220>

<221> misc_features

<222>  (48)..(48)

<223> n is a, c, g, or t

<400> 215

tcacagagag ggtcacagcy hbtnywbybn bnybwyygtc snbnynsnyg tatattatca 60

gtctggtctg gggttcac                                   78

<210>  216

<211> 58

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 216

gctgtgaccc tctctgtgaa ggatagtaaa atgtctaccc tctcctgtaa gaacaaga 58

<210>  217

<211> 69

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 217

gtatatcatc aatattttca ggtggatcca tttcctcaaa ggaaatgatc ttgttcttac 60

aggagagggg

<210>  218

<211> 99

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesized sequence

<220>

<221> misc_features

<222>  (59)..(59)

<223> n is a, c, g, or t

<220>

<221> misc_features

<222>  (74)..(74)

<223> n is a, c, g, or t

<400> 218

aatggatcca cctgaaaata ttgatgatat acaaagtgat ctcatattct ttcagaaand 60

hgttccagga cacnataaga tggagtttga atcttcact 99

<210>  219

<211> 57

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 219

ccttttggca agcaagaaag tgtccttcat acagtgaaga ttcaaactcc atcttat 57

<210>  220

<211> 60

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 220

ctttcttgct tgccaaaagg aagatgatgc tttcaaactc attctgaaaa aaaaggatga 60

<210>  221

<211> 77

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400> 221

ccaccacttt gatgtaagtt agtrdbagtg aacattacag atttatcccc attttcatcc 60

ttttttttca gaatgag                                       77

<210>  222

<211> 59

<212> DNA

<213> Artificial sequence

<220>

<223> Synthesized sequence

<400>  222

actaacttac atcaaagtgg tggttctgga tccgaacaaa agcttatctc cgaagaaga 59

Claims (96)

1. A modified interleukin-18 (IL-18) polypeptide that preferentially binds to the IL-18 receptor (IL-18R) relative to the IL-18 binding protein (IL-18BP), said polypeptide comprising: (i) an amino acid sequence having 85% or more sequence identity with the wild-type (WT) IL-18 as shown in SEQ ID NO:30 or with the IL-18 variant amino sequence shown in any one of SEQ ID NO:6, 87-91 and 16-21; and (ii) a mutation at amino acid positions C38 and C68 relative to SEQ ID NO:30, wherein said modified IL-18 preferentially binds to IL-18R relative to IL-18BP. 2. The modified IL-18 polypeptide of claim 1, wherein the mutation at position C38 is C38S, and the mutation at position C68 is C68S, C68G, C68A, C68V, C68D, C68E, or C68N. 3. The modified IL-18 peptide of claim 1, wherein the mutation at position C38 is C38S, and the mutation at position C68 is C68S, C68G, C68A, C68D, or C68N. 4. The modified IL-18 peptide of claim 1, wherein the mutation at position C38 is C38S, and the mutation at position C68 is C68G, C68A, C68V, C68D, C68E, or C68N. 5. The modified IL-18 polypeptide of claim 1, wherein the mutation at position C38 is C38S, and the mutation at position C68 is C68G, C68A, C68D, or C68N. 6. The modified IL-18 polypeptide of claim 1, wherein the mutation at position C38 is C38S and the mutation at position C68 is C68S. 7. The modified IL-18 polypeptide of claim 1, wherein the mutation at position C38 is C38S and the mutation at position C68 is C68G. 8. The modified IL-18 peptide according to any one of claims 1-7, wherein the IL-18 peptide is a decoy-resistant (DR) IL-18 variant or a decoy-decoy (D2D) IL-18 variant. 9. The modified IL-18 polypeptide according to any one of claims 1-7, wherein the IL-18 polypeptide is DRIL-18, wherein the DRIL-18 variant comprises mutations at at least two of the following positions relative to SEQ ID NO:30: Y1, L5, K8, M51, K53, S55, Q56, P57, G59, M60, E77, Q103, S105, D110, N111, M113, V153, and N155. 10. The modified IL-18 peptide of claim 9, wherein the DRIL-18 variant comprises mutations at at least two of the following positions relative to SEQ ID NO:30: M51, K53, Q56, P57, M60, Q103, S105, D110, N111, and M113. 11. The modified IL-18 peptide of claim 9, wherein the DRIL-18 variant comprises mutations at at least five of the following positions relative to SEQ ID NO:30: M51, K53, Q56, P57, M60, Q103, S105, D110, N111, and M113. 12. The modified IL-18 peptide of claim 9, wherein the DRIL-18 variant comprises mutations at positions M51, K53, Q56, D110 and N111 relative to SEQ ID NO:30. 13. The modified IL-18 peptide of claim 12, wherein the DR IL-18 variant further comprises mutations at positions P57 and M60 relative to SEQ ID NO:30. 14. The modified IL-18 peptide of claim 13, wherein the DR IL-18 variant further comprises a mutation at position S10 relative to SEQ ID NO:30. 15. The modified IL-18 peptide of claim 9, wherein the DRIL-18 variant comprises, relative to SEQ ID NO:30, at least five of the following 18 mutations: (1) Y1H or Y1R; (2) L5H, L5I, or L5Y; (3) K8Q or K8R; (4) M51T, M51K, M51D, M51N, M51E, or M51R; (5) K53R, K53G, K53S, or K53T; (6) S55K or S55R; (7) Q56E, Q56A, Q56R, Q56V, Q56G, Q56K, or Q56L; (8) P57L, P57G, P57A, or P57K; (9) G59T or G59A; (10) M60K, M60Q, M60R or M60L; (11) E77D; (12) Q103E, Q103K, Q103P, Q103A or Q103R; (13) S105R, S105D, S105K, S105N or S105A; (14) D110H, D110K, D110N, D110Q, D110E, D110S or D110G; (15) N111H, N111Y, N111D, N111R, N111S or N111G; (16) M113V, M113R, M113T or M113K; (17) V153I, V153T or V153A; and (18) N155K or N155H. 16. The modified IL-18 peptide of claim 9, wherein the DRIL-18 variant comprises, relative to SEQ ID NO:30, at least 5 of the following 10 mutations: (1) M51T, M51K, M51D, M51N, M51E, or M51R; (2) K53R, K53G, K53S, or K53T; (3) Q56E, Q56A, Q56R, Q56V, Q56G, Q56K, or Q56L; (4) P57L, P57G, P57A, or P57K; (5) M60K, M... 60Q, M60R or M60L; (6) Q103E, Q103K, Q103P, Q103A or Q103R; (7) S105R, S105D, S105K, S105N or S105A; (8) D110H, D110K, D110N, D110Q, D110E, D110S or D110G; (9) N111H, N111Y, N111D, N111R, N111S or N111G; and (10) M113V, M113R, M113T or M113K. 17. The modified IL-18 peptide of claim 9, wherein the DRIL-18 variant comprises at least five of the following seven mutations relative to SEQ ID NO:30: (1) M51E, M51R, M51K, M51T, M51D or M51N; (2) K53G, K53S, K53T or K53R; (3) Q56G, Q56R, Q56L, Q56E, Q56A, Q56V or Q56K; (4) D110S, D110N, D110G, D110K, D110H, D110Q or D110E; (5) N111G, N111R, N111S, N111D, N111H or N111Y; (6) P57A, P57L, P57G, or P57K; and (7) M60L, M60R, M60K or M60Q. 18. The modified IL-18 peptide of claim 17, wherein the DR IL-18 variant further comprises, relative to SEQ ID NO:30, a mutation S105D, S105A, S105N, S105R, S105D, or S105K. 19. The modified IL-18 polypeptide of any one of claims 1-7, wherein the IL-18 polypeptide is a DR IL-18 variant that specifically binds to the IL-18 receptor (IL-18R) but exhibits reduced binding to the IL-18 binding protein (IL-18BP) compared to the WTIL-18, wherein the DR IL-18 variant comprises the mutants M51K, K53S, Q56L, D110S, and N111R relative to SEQ ID NO:30. 20. The modified IL-18 peptide of claim 19, wherein the DR IL-18 variant further comprises mutants P57A, M60L, and S105D. 21. The modified IL-18 polypeptide of claim 20, wherein the mutation at position C38 is C38S, and the mutation at position C68 is C68G, C68A, C68D, or C68N. 22. The modified IL-18 polypeptide of claim 21, wherein the mutation at position C38 is C38S and the mutation at position C68 is C68G. 23. The modified IL-18 polypeptide of claim 21, wherein the DR IL-18 variant comprises the amino acid sequence shown in any one of SEQ ID NO: 1-21. 24. The modified IL-18 polypeptide of claim 21, wherein the DR IL-18 variant comprises the amino acid sequence shown in any one of SEQ ID NO: 6 and 16-21. 25. The modified IL-18 polypeptide of claim 21, wherein the DR IL-18 variant comprises the amino acid sequence shown in any one of SEQ ID NO: 6, 16, 17, 19 and 21. 26. The modified IL-18 polypeptide of claim 21, wherein the DR IL-18 variant comprises an amino acid sequence having 85% or sequence identity with the amino acid sequence shown in SEQ ID NO:19. 27. The modified IL-18 polypeptide of claim 21, wherein the DR IL-18 variant comprises the amino acid sequence shown in SEQ ID NO:19. 28. The modified IL-18 polypeptide of claim 1, wherein the modified IL-18 polypeptide does not contain any non-natural cysteine residues of the IL-18 polypeptide. 29. The modified IL-18 polypeptide of claim 1, wherein the binding affinity of the IL-18 polypeptide to IL-18BP is less than 0.6 nM. 30. The modified IL-18 polypeptide of claim 1, wherein the modified IL-18 polypeptide exhibits higher stability compared to IL-18 polypeptides with different sequences. 31. The modified IL-18 peptide of claim 30, wherein the modified IL-18 peptide exhibits less formation of dimers, polymers, aggregates and/or degradation products after exposure to stability assays, freeze-thaw cycles or stirring. 32. The modified IL-18 polypeptide of claim 1, wherein the amino acid sequence having 85% or more sequence identity with WT IL-18 does not contain polyethylene glycol-modified amino acids. 33. The modified IL-18 polypeptide of claim 1, wherein the amino acid sequence having 85% or more sequence identity with WTIL-18 does not contain non-natural cysteine residues. 34. The modified IL-18 polypeptide of claim 1, wherein the amino acid sequence having 85% or more sequence identity with WTIL-18 does not contain polyethylene glycolated cysteine residues. 35. A pharmaceutical composition comprising the modified IL-18 polypeptide as described in any one of claims 1-34 and a pharmaceutically acceptable excipient. 36. The composition of claim 35, further comprising an immune checkpoint inhibitor that inhibits PD-L1, PD1, CTLA4, TIM3, TIGIT, LAG3, B7H3, B7H4, VISTA, BTLA, CD47, SIRPα, CD48, CD155, CD160, TREM2, IDO1, adenosine 2A receptor, aryl hydrocarbon receptor, KIR, LILRB2, or any combination thereof. 37. The composition of claim 35, further comprising an immune agonist selected from: agents that activate tumor necrosis factor receptor superfamily (TNFRSF) proteins (e.g., GITR, 41BB, OX40, CD27, CD40, HVEM); agents that activate immunoglobulin superfamily (IgSF) proteins (e.g., CD28, ICOS, CD226, NKG2D); agents that activate TLRs (e.g., TLR2, TLR4, TLR5, TLR7, TLR9); agents that activate nucleic acid sensors (e.g., STING, cGAS, RIG-I (DDX58)); inflammasome activators; T cell binders; cytokines or cytokine variants (e.g., IL-2, IL-7, IL-10, IL-12, IL-15, IL-21, IL-23, IL-27, IL-33, TNF, TL1A, IFNA, IFNB, IFNG); or any combination thereof. 38. The composition of claim 35, further comprising a cancer cell opsonizer targeting one or more antigens selected from the group consisting of: CD19, CD20, CD22, CD24, CD25, CD30, CD33, CD37, CD38, CD44, CD45, CD47, CD51, CD52, CD56, CD62L, CD70, CD74, CD79, CD80, CD96, CD97, CD99, CD123, CD134, CD138, CD152 (CTLA-4), CD200, CD213A2, CD221, CD248, CD276 (B7-H3), B7-H4, CD279 (PD-H3). -1), CD274 (PD-L1), CD319, EGFR, EPCAM, 17-1A, HER1, HER2, HER3, CD117, C-Met, HGFR, PDGFRA, AXL, TWEAKR, PTHR2, HAVCR2 (TIM3), GD2 gangliosides, MUC1, mucin CanAg, mesothelin, endothelial glycoprotein, Lewis-Y antigen, CEA, CEACAM1, CEACAM5, CA-125, PSMA, BAFF, FGFR2, TAG-72, gelatinase B, phosphatidylinositol proteoglycan 3, integrin-4, BCMA, CSF1R, SLAMF7, integrin α _vβ3 , TYRP1, GPNMB, CLDN18.2, FORR1, CCR4, CXCR4, MICA, _C242 antigen, DLL3, DLL4, EGFL7, vimentin, fibronectin extradomain-B, TROP-2, LRRC15, FAP, SLITRK6, NOTCH2, NOTCH3, tendinin-3, STEAP1, and NRP1. 39. The composition of claim 35, wherein the interleukin-18 (IL-18) polypeptide is conjugated with the immune checkpoint inhibitor, the immune agonist, or the cancer cell opsonizer. 40. The composition of any one of claims 35-39, further comprising engineered T cells or NK cells, chimeric antigen receptor T cells (CAR-T cells), engineered TCR-T cells, chimeric antigen receptor NK cells (CAR-NK cells), or tumor-infiltrating lymphocytes (TILs). 41. A pharmaceutical formulation comprising the modified IL-18 polypeptide as described in any one of claims 1-40. 42. The pharmaceutical formulation of claim 41, further comprising the IL-18 polypeptide at a concentration of 15-100 mg/mL. 43. The pharmaceutical preparation of claim 41 or claim 42, wherein the pharmaceutical preparation is formulated for subcutaneous injection. 44. The pharmaceutical preparation according to any one of claims 41-43, comprising: A buffer that maintains the pH between 6.2 and 6.8. sugar, Chelating agents Antioxidants, and Agents that prevent protein absorption. 45. A nucleic acid comprising a nucleotide sequence encoding the IL-18 polypeptide as described in any one of claims 1-34. 46. The nucleic acid of claim 45, wherein the nucleic acid is a plasmid or a viral vector. 47. The nucleic acid of claim 45 or claim 46, wherein the nucleotide sequence encoding the IL-18 polypeptide is immediately followed by a 3' fusion of a nucleotide sequence encoding a small ubiquitin-like modification (SUMO) tag and is framed with the nucleotide sequence, the small ubiquitin-like modification (SUMO) tag being cleavable by SUMO protease. 48. The nucleic acid of claim 47, wherein the SUMO protease is ULP1. 49. The nucleic acid of claim 48, wherein the SUMO tag comprises the amino acid sequence shown in SEQ ID NO: 26 or 27. 50. A cell comprising the nucleic acid as described in any one of claims 45-49. 51. The cell of claim 50, wherein it comprises a nucleic acid containing a nucleotide sequence encoding a SUMO protease. 52. The cell of claim 50 or claim 51, wherein the cell is a bacterial cell. 53. The cell of claim 50, wherein the cell is a yeast cell. 54. The cell of claim 50, wherein the cell is an engineered T cell or NK cell, a chimeric antigen receptor T cell (CAR-T cell), an engineered TCR-T cell, a chimeric antigen receptor NK cell (CAR-NK cell), or a tumor-infiltrating lymphocyte (TIL) that expresses an interleukin-18 (IL-18) polypeptide. 55. A treatment method comprising administering to an individual in need a modified IL-18 polypeptide as described in any one of claims 1-34, thereby treating the individual's disease or condition. 56. The method of claim 55, wherein the disease or condition is selected from: cancer; cancer resistant to immune checkpoint inhibitors (ICIs); cancer associated with tumors with reduced or absent surface expression of MHC class I; cancer associated with high levels of IL-18BP; cancer with high tumor mutational burden (TMB); cancer with tumors exhibiting microsatellite instability (MSI); cancer associated with tumors expressing IL-18R on infiltrating T cells or NK cells; metabolic diseases or conditions; and infectious diseases. 57. The method of claim 55 or claim 56, wherein the method comprises administering an immune checkpoint inhibitor and an immune agonist or cancer cell opsonizer to the individual. 58. The method of claim 55 or claim 56, wherein the method comprises administering to the individual an immune checkpoint inhibitor that inhibits PD-L1, PD1, CTLA4, TIM3, TIGIT, LAG3, B7H3, B7H4, VISTA, BTLA, CD47, SIRPα, CD48, CD155, CD160, TREM2, IDO1, adenosine 2A receptor, aryl hydrocarbon receptor, KIR, LILRB2, or any combination thereof. 59. The method of claim 55 or claim 56, wherein the method comprises administering to the individual an immune agonist selected from: agents that activate tumor necrosis factor receptor superfamily (TNFRSF) proteins (e.g., GITR, 41BB, OX40, CD27, CD40, HVEM); agents that activate immunoglobulin superfamily (IgSF) proteins (e.g., CD28, ICOS, CD226, NKG2D); agents that activate TLRs (e.g., TLR2, TLR4, TLR5, TLR7, TLR9); agents that activate nucleic acid sensors (e.g., STING, cGAS, RIG-I (DDX58)); inflammasome activators; T cell binders; cytokines or cytokine variants (e.g., IL-2, IL-7, IL-10, IL-12, IL-15, IL-21, IL-23, IL-27, IL-33, TNF, TL1A, IFNA, IFNB, IFNG); or any combination thereof. 60. The method of any one of claims 55-59, wherein the method comprises administering to the individual a cancer cell opsonizer targeting one or more antigens selected from the group consisting of: CD19, CD20, CD22, CD24, CD25, CD30, CD33, CD37, CD38, CD44, CD45, CD47, CD51, CD52, CD56, CD62L, CD70, CD74, CD79, CD80, CD96, CD97, CD99, CD123, CD134, CD138, CD152 (CTLA-4), CD200, CD213A2, CD221, CD248, CD276 (B7-H3), B7-H4, CD279 (PD-1), CD274 (PD-L1), CD319, EGFR, EPCAM, 17-1A, HER1, HER2, HER3, CD117, C-Met, HGFR, PDGFRA, AXL, TWEAKR, PTHR2, HAVCR2 (TIM3), GD2 gangliosides, MUC1, mucin CanAg, mesothelin, endothelial glycoprotein, Lewis-Y antigen, CEA, CEACAM1, CEACAM5, CA-125, PSMA, BAFF, FGFR2, TAG-72, gelatinase B, phosphatidylinositol proteoglycan 3, integrin-4, BCMA, CSF1R, SLAMF7, integrin α _vβ3 , TYRP1, GPNMB, CLDN18.2, FORR1, CCR4, CXCR4, MICA, _C242 antigen, DLL3, DLL4, EGFL7, vimentin, fibronectin extradomain-B, TROP-2, LRRC15, FAP, SLITRK6, NOTCH2, NOTCH3, tendinin-3, STEAP1, and NRP1. 61. The method of any one of claims 55-60, wherein the method comprises administering engineered T cells or NK cells, chimeric antigen receptor T cells (CAR-T cells), engineered TCR-T cells, chimeric antigen receptor NK cells (CAR-NK cells), or tumor-infiltrating lymphocytes (TILs) to the individual. 62. The method of any one of claims 55-61, wherein the method comprises administering an oncolytic virus to the individual. 63. A method for preparing an active polypeptide, the method comprising: Inside a bacterial cell, an exogenously provided fusion protein is contacted with an exogenously provided SUMO (small ubiquitin-like modified) protease, wherein the fusion protein contains a polypeptide of interest fused to an N-terminus with a SUMO tag. Within the bacterial cell, the SUMO protease cleaves the fusion protein to remove the SUMO tag from the polypeptide of interest, wherein the polypeptide of interest is the IL-18 polypeptide as described in any one of claims 1-34, the IL-18 polypeptide being activated by removing the SUMO tag. 64. The method of claim 63, wherein the N-terminal amino acid of the IL-18 polypeptide is tyrosine after cleavage by the SUMO protease. 65. The method of claim 64, wherein the IL-18 peptide is a decoy-resistant (DR) IL-18 variant or a decoy-decoy (D2D) IL-18 variant. 66. The method of any one of claims 63-65, wherein the SUMO protease is ULP1. 67. The method of any one of claims 63-66, wherein the SUMO tag comprises the amino acid sequence shown in SEQ ID NO: 26 or 27. 68. The method of any one of claims 63-67, wherein the contact comprises introducing a nucleic acid encoding the fusion protein into the bacterial cell. 69. The method of any one of claims 63-68, wherein the contact comprises introducing a nucleic acid encoding the SUMO protease into the bacterial cell. 70. The method of any one of claims 63-69, wherein the contact comprises inducing the expression of the SUMO protease from an inducible promoter in the bacterial cell. 71. The method of claim 70, wherein the inducible promoter is a rhamnose-inducible promoter. 72. The method of any one of claims 63-71, further comprising purifying the polypeptide of interest after removing the SUMO tag. 73. The method of any one of claims 64-65, further comprising purifying the IL-18 peptide after removing the SUMO tag, wherein the purification comprises ion exchange chromatography, immobilized metal affinity chromatography (IMAC), multimode chromatography (MMC), and hydrophobic interaction chromatography (HIC). 74. The method of any one of claims 63-73, wherein the contact occurs inside the cell. 75. The method of any one of claims 63-73, wherein the contact occurs in vitro without cells. 76. A treatment method comprising administering to an individual in need a modified IL-18 peptide as described in any one of claims 1-34, thereby treating the individual for a disease or condition, wherein the frequency of administration is once a week or less. 77. The method of claim 76, wherein the frequency of application is once every two weeks or less. 78. The method of claim 76, wherein the frequency of application is in the range of once a week to once a month. 79. The method of any one of claims 76-78, wherein the IL-18 polypeptide is the IL-18 polypeptide of any one of claims 1-34. 80. The method of any one of claims 76-78, wherein the IL-18 polypeptide is a decoy-resistant (DR) IL-18 variant or a decoy-decoy (D2D) IL-18 variant. 81. The method of any one of claims 76-80, wherein the application comprises subcutaneous application. 82. The method of any one of claims 76-80, wherein the administration comprises administering the IL-18 polypeptide at a dose in the range of 0.025-3.5 mg/kg. 83. The method of any one of claims 76-82, wherein the disease or condition is selected from: cancer; cancer resistant to immune checkpoint inhibitors (ICIs); cancer associated with tumors with reduced or absent surface expression of MHC class I; cancer associated with high levels of IL-18BP; cancer associated with tumors expressing IL-18R on infiltrating T cells or NK cells; metabolic diseases or conditions; and infectious diseases. 84. The method of any one of claims 74-81, wherein the method comprises administering an immune checkpoint inhibitor or a cancer cell opsonizer to the individual. 85. The method of any one of claims 74-81, wherein the method comprises administering to the individual an immune checkpoint inhibitor that inhibits PD-L1, PD1, CTLA4, TIM3, TIGIT, LAG3, B7H3, B7H4, VISTA, BTLA, CD47, SIRPα, CD48, CD155, CD160, TREM2, IDO1, adenosine 2A receptor, aryl hydrocarbon receptor, KIR, LILRB2, or any combination thereof. 86. The method of any one of claims 74-81, wherein the method comprises administering to the individual an immune agonist selected from: agents that activate tumor necrosis factor receptor superfamily (TNFRSF) proteins (e.g., GITR, 41BB, OX40, CD27, CD40, HVEM); agents that activate immunoglobulin superfamily (IgSF) proteins (e.g., CD28, ICOS, CD226, NKG2D); agents that activate TLRs (e.g., TLR2, TLR4, TLR5, TLR7, TLR9); agents that activate nucleic acid sensors (e.g., STING, cGAS, RIG-I (DDX58)); inflammasome activators; T cell binders; cytokines or cytokine variants (e.g., IL-2, IL-7, IL-10, IL-12, IL-15, IL-21, IL-23, IL-27, IL-33, TNF, TL1A, IFNA, IFNB, IFNG); or any combination thereof. 87. The method of any one of claims 74-81, wherein the method comprises administering to the individual a cancer cell opsonizer targeting one or more antigens selected from the group consisting of: CD19, CD20, CD22, CD24, CD25, CD30, CD33, CD37, CD38, CD44, CD45, CD47, CD51, CD52, CD56, CD62L, CD70, CD74, CD79, CD80, CD96, CD97, CD99, CD123, CD134, CD138, CD152 (CTLA-4), CD200, CD213A2, CD221, CD248, CD276 (B7-H3), B7-H4, CD279 (PD-1), CD274 (PD-L1), CD319, EGFR, EPCAM, 17-1A, HER1, HER2, HER3, CD117, C-Met, HGFR, PDGFRA, AXL, TWEAKR, PTHR2, HAVCR2 (TIM3), GD2 gangliosides, MUC1, mucin CanAg, mesothelin, endothelial glycoprotein, Lewis-Y antigen, CEA, CEACAM1, CEACAM5, CA-125, PSMA, BAFF, FGFR2, TAG-72, gelatinase B, phosphatidylinositol proteoglycan 3, integrin-4, BCMA, CSF1R, SLAMF7, integrin α _vβ3 , TYRP1, GPNMB, CLDN18.2, FORR1, CCR4, CXCR4, MICA, _C242 antigen, DLL3, DLL4, EGFL7, vimentin, fibronectin extradomain-B, TROP-2, LRRC15, FAP, SLITRK6, NOTCH2, NOTCH3, tendinin-3, STEAP1, and NRP1. 88. The method of any one of claims 74-85, wherein the method comprises administering engineered T cells or NK cells, chimeric antigen receptor T cells (CAR-T cells), engineered TCR-T cells, chimeric antigen receptor NK cells (CAR-NK cells), or tumor-infiltrating lymphocytes (TILs) to the individual. 89. The method of any one of claims 74-86, wherein the method comprises administering an oncolytic virus to the individual. 90. A pharmaceutical preparation comprising: The IL-18 polypeptide as described in any one of claims 1-34, Buffer, sugar, Chelating agents Antioxidants, and Agents that prevent protein absorption. 91. The pharmaceutical formulation of claim 90, wherein the buffer comprises His/His-HCl. 92. The pharmaceutical formulation of claim 91, wherein the buffer maintains the pH between 6.2 and 6.8. 93. The pharmaceutical preparation according to any one of claims 90 to 92, wherein the sugar comprises sucrose. 94. The pharmaceutical formulation of any one of claims 90 to 93, wherein the chelating agent comprises EDTA or a salt thereof. 95. The pharmaceutical formulation according to any one of claims 90 to 94, wherein the antioxidant is L-methionine. 96. The pharmaceutical formulation according to any one of claims 90 to 95, wherein the agent preventing protein absorption is polysorbate 80.