RU2797536C2 - Interleukin-18 options and methods for their use - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to new pharmaceutical compositions. The invention discloses a composition containing variants of an IL-18 activity enhancer that ensures the activity of the IL-18 signaling pathway even in the presence of an inhibitory molecule such as IL-18BP.

EFFECT: invention can be applied in the immunotherapy of various types of cancer, including those resistant to inhibitors of immune checkpoints.

21 cl, 30 dwg, 12 tbl, 6 ex

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Nos. 62/554,605, filed September 6, 2017, and 62/652,279, filed April 3, 2018, each of which is hereby incorporated by reference in its entirety.

INTRODUCTION

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

Therefore, there is a need for compositions and methods that can provide effective activation of the IL-18 signaling pathway for the treatment and prevention of cancer and other diseases and disorders. The present invention is directed to solving the problem associated with this unmet need.

SUMMARY OF THE INVENTION

In one aspect, the invention relates to a composition comprising a variant IL-18 polypeptide. In some embodiments, the IL-18 polypeptide variant binds specifically to the IL-18 receptor (IL-18R), but the IL-18 polypeptide variant exhibits significantly reduced binding to the IL-18 binding protein (IL-18BP).

In some embodiments, the variant IL-18 polypeptide comprises at least one mutation relative to wild-type IL-18 (WT IL-18). In some embodiments, WT IL-18 is human IL-18 comprising the amino acid sequence of SEQ ID NO: 30. In some embodiments, WT IL-18 is murine IL-18 comprising the amino acid sequence of SEQ ID NO: 31.

In some embodiments, the variant IL-18 polypeptide comprises at least one mutation selected from the group consisting of Y1X, L5X, K8X, M51X, K53X, S55X, Q56X, P57X, G59X, M60X, E77X, Q103X, S105X , D110X, N111X, M113X, V153X and N155X, relative to SEQ ID NO: 30.

In some embodiments, the variant IL-18 polypeptide comprises at least one mutation selected from the group consisting of Y1H, Y1R, L5H, L5I, L5Y, K8Q, K8R, M51T, M51K, M51D, M51N, M51E, M51R . 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, relative to SEQ ID NO: 30.

In some embodiments, the IL-18 polypeptide variant includes mutations M51X, M60X, S105X, D110X, and N111X relative to SEQ ID NO: 30.

In some embodiments, the variant IL-18 polypeptide comprises mutations M51X, K53X, Q56X, S105X N111X relative to SEQ ID NO: 30.

In some embodiments, the variant IL-18 polypeptide comprises the amino acid sequence represented by any one of SEQ ID NOs.: 34-59, 73-91, 191-193, or a fragment thereof.

In some embodiments, the variant IL-18 polypeptide comprises at least one mutation selected from the group consisting of N1X, M50X, Y51X, K52X, S54X, E55X, V56X, R57X, G58X, L59X, R104X, N109X, and L151X , relative to SEQ ID NO: 31.

In some embodiments, the variant IL-18 polypeptide comprises at least one mutation selected from the group consisting of N1H, N1Y, M50A, M50S, M50V, M50G, M50T, Y51R, K52V, K52S, K52T, K52G, K52A , S54R, S54K, S54G, S54N, E55R, E55H, E55N, E55D, E55G, V56L, V56M, V56R, V56A, V56S, V56Q, R57G, R57K, G58A, L59K, L59R, L59V, R104K, R104L, R10 4Q, R104S , N109D and L151V, relative to SEQ ID NO: 31.

In some embodiments, the variant IL-18 polypeptide comprises the amino acid sequence represented by any one of SEQ ID NOs.: 60-72, or a fragment thereof.

In one aspect, the invention relates to a composition comprising a nucleic acid encoding a variant IL-18 polypeptide.

In some embodiments, the composition further comprises one or more drugs selected from (i) an immune checkpoint inhibitor; (ii) an agent that inhibits one or more proteins selected from PD-L1, PD1, CTLA4, TIM3, TIGIT, LAG3, B7H3, B7H4, VISTA, ICOS, GITR, 41BB, OX40 and CD40; (iii) a cancer cell opsonizing agent; and (iv) an agent that targets one or more antigens selected from 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 ganglioside, MUC1 , CanAg mucin, mesothelin, endoglin, Lewis Y antigen, CEA, CEACAM1, CEACAM5, CA-125, PSMA, BAFF, FGFR2, TAG-72, gelatinase B, glypican 3, nectin-4, BCMA, CSF1R, SLAMF7, integrin α _v β ₃ , TYRP1, GPNMB, CLDN18.2, FOLR1, CCR4, CXCR4, MICA, C242 antigen, DLL3, DLL4, EGFL7, vimentin, fibronectin extra domain-B, TROP-2, LRRC15, FAP, SLITRK6, NOTCH2, NOTCH3 , tanascin-3, STEAP1 and NRP1.

In one aspect, the invention relates to a method of treating or preventing a disease or disorder in a subject in need thereof, comprising administering to the subject a composition comprising an IL-18 polypeptide variant or a nucleic acid encoding an IL-18 polypeptide variant.

In some embodiments, the disease or disorder is cancer. In some embodiments, the cancer is a cancer that is resistant to immune checkpoint inhibitors (ICIs). In some embodiments, the cancer is associated with a tumor that lacks the expression of MHC class I molecules.

In some embodiments, the disease or disorder is a metabolic disease or disorder. In some embodiments, the disease or disorder is an infectious disease.

In some embodiments, the method includes administering to the subject an IL-18 polypeptide variant and at least one other agent. In some embodiments, at least one other agent comprises an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor is an agent that inhibits PD-L1, PD1, CTLA4, TIM3, TIGIT, LAG3, B7H3, B7H4, VISTA, ICOS, GITR, 41BB, OX40, or CD40, or any combination thereof. In some embodiments, at least one other agent includes an agent that opsonizes cancer cells. In some embodiments, at least one other agent targets one or more antigens selected from 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 ganglioside, MUC1, CanAg mucin, mesothelin, endoglin, Lewis Y antigen, CEA, CEACAM1, CEACAM5, CA-125, PSMA, BAFF, FGFR2, TAG-72, gelatinase B, glypican 3, nectin-4, BCMA, CSF1R , SLAMF7, α _v β ₃ integrin, TYRP1, GPNMB, CLDN18.2, FOLR1, CCR4, CXCR4, MICA, C242 antigen, DLL3, DLL4, EGFL7, vimentin, fibronectin extra domain-B, TROP-2, LRRC15, FAP, SLITRK6, NOTCH2, NOTCH3, tanascin-3, STEAP1 and NRP1. In some embodiments, at least one other agent is conjugated to a variant IL-18 polypeptide. In some embodiments, at least one other agent is altered T cells or NK cells. In some embodiments, at least one other agent is an oncolytic virus.

In one aspect, the invention provides a composition comprising an IL-18 binding protein (IL-18BP) inhibitor or IL-18BP antagonist, wherein the inhibitor or antagonist inhibits the ability of IL-18BP to neutralize endogenous IL-18. In some embodiments, the inhibitor or antagonist comprises at least one selected from the group consisting of a chemical compound, a polypeptide, a peptide, a peptidomimetic, an antibody, a ribozyme, a small molecule chemical compound, and an antisense nucleic acid molecule.

In some embodiments, the composition comprises an IL-18 polypeptide variant, wherein the IL-18 polypeptide variant specifically binds to IL-18BP and wherein the IL-18 polypeptide variant exhibits significantly reduced binding to the IL-18 receptor (IL-18R).

In some embodiments, the variant IL-18 polypeptide comprises at least one mutation relative to wild-type IL-18 (WT IL-18). In some embodiments, WT IL-18 is human IL-18 comprising the amino acid sequence of SEQ ID NO: 30. In some embodiments, WT IL-18 is murine IL-18 comprising the amino acid sequence of SEQ ID NO: 31.

In some embodiments, the variant IL-18 polypeptide comprises at least one mutation selected from the group consisting of Y1X, L5X, D17X, E31X, T34X, D35X, S36X, D37X, D40X, N41X, M51X, Q56X, M60X , Q103X, H109X, M113X and R131X, relative to SEQ ID NO: 30.

In some embodiments, the variant IL-18 polypeptide comprises at least one mutation selected from the group consisting of Y1D, Y1F, Y1H, Y1L, L5F, L5H, D17A, D17G, D17R, D17H, E31A, E31T, E31G , E31K, E31R, T34A, T34K T34E, D35S, D35A, D35Y, S36N, S36K, S36R, D37P, D37A, D37R, D37H, D37L, D37V, D40Y D40S, D40A, N41K, N41S, N41R, M51F, M5 1L, M51I , Q56H, M60L, M60F, M60I, Q103L, Q103I, H109A, H109P, H109D, M113L, M113I, M113F and R131S, relative to SEQ ID NO: 30.

In some embodiments, the variant IL-18 polypeptide comprises the amino acid sequence represented by any one of SEQ ID NOs.: 92-125, or a fragment thereof.

In some embodiments, the IL-18 polypeptide variant includes mutations D17X, E30X, and Q103X, relative to SEQ ID NO: 30.

In some embodiments, the IL-18 polypeptide variant includes mutations D17X, E30X, D35X, M51X, and Q103X, relative to SEQ ID NO: 30.

In some embodiments, the variant IL-18 polypeptide comprises at least one mutation selected from the group consisting of N1X, L5X, D17X, E30X, T33X, D34X, I35X, D36X, M50X, Q102X, R104, H108X, N109X , M111X, D129X and D130X, relative to SEQ ID NO: 31.

In some embodiments, the variant IL-18 polypeptide comprises at least one mutation selected from the group consisting of N1Y, N1D, N1H, N1L, N1F, N1V, N1I, L5Y, L5H, D17Q, D17G, D17A, D17E , D17S, D17N, E30A, E30R, E30K, E30T, E30G, T33G, T33A, T33E, T33R, T33K, D34Y, D34S, D34A, I35T, I35K, I35R, D36V, D36A, D36G, D36H, D36P, D36R, D36L , M50F,M50L,Q102L,Q102I,R104E,R104A,R104P,R104G,R104Q,R104H,H108D,H108A,N109R,N109S,N109T,N109I,M111L,M111I,D129A,D129F 29V, D129Y, D129S, D130E, D130T , D130G, D130N, D130R, D130S, D130Q and D130H, relative to SEQ ID NO: 31.

In some embodiments, the variant IL-18 polypeptide comprises the amino acid sequence represented by any one of SEQ ID NOs.: 126-190, or a fragment thereof.

In one aspect, the invention relates to a composition comprising a nucleic acid encoding a variant IL-18 polypeptide.

In one aspect, the composition further comprises one or more drugs selected from (i) an immune checkpoint inhibitor; (ii) an agent that inhibits one or more proteins selected from PD-L1, PD1, CTLA4, TIM3, TIGIT, LAG3, B7H3, B7H4, VISTA, ICOS, GITR, 41BB, OX40 and CD40; (iii) a cancer cell opsonizing agent; and (iv) an agent that targets one or more antigens selected from 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 ganglioside, MUC1 , CanAg mucin, mesothelin, endoglin, Lewis Y antigen, CEA, CEACAM1, CEACAM5, CA-125, PSMA, BAFF, FGFR2, TAG-72, gelatinase B, glypican 3, nectin-4, BCMA, CSF1R, SLAMF7, integrin α _v β ₃ , TYRP1, GPNMB, CLDN18.2, FOLR1, CCR4, CXCR4, MICA, C242 antigen, DLL3, DLL4, EGFL7, vimentin, fibronectin extra domain-B, TROP-2, LRRC15, FAP, SLITRK6, NOTCH2, NOTCH3 , tanascin-3, STEAP1 and NRP1.

In one aspect, the invention relates to a method of treating or preventing a disease or disorder in a subject in need thereof, comprising administering to the subject a composition comprising an IL-18 binding protein (IL-18BP) inhibitor or an IL-18BP antagonist.

In some embodiments, the disease or disorder is cancer. In some embodiments, the cancer is a cancer that is resistant to immune checkpoint inhibitors (ICIs). In some embodiments, the cancer is associated with a tumor that lacks the expression of class I MHC molecules.

In some embodiments, the disease or disorder is a metabolic disease or disorder. In some embodiments, the disease or disorder is an infectious disease.

In some embodiments, the method includes administering to the subject at least one other agent in addition to an IL-18BP inhibitor or IL-18BP antagonist. In some embodiments, at least one other agent comprises an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor is an agent that inhibits PD-L1, PD1, CTLA4, TIM3, TIGIT, LAG3, B7H3, B7H4, VISTA, ICOS, GITR, 41BB, OX40, or CD40, or any combination thereof. In some embodiments, at least one other agent includes an agent that opsonizes cancer cells. In some embodiments, at least one other agent targets one or more antigens selected from 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 ganglioside, MUC1, CanAg mucin, mesothelin, endoglin, Lewis Y antigen, CEA, CEACAM1, CEACAM5, CA-125, PSMA, BAFF, FGFR2, TAG-72, gelatinase B, glypican 3, nectin-4, BCMA, CSF1R , SLAMF7, α _v β ₃ integrin, TYRP1, GPNMB, CLDN18.2, FOLR1, CCR4, CXCR4, MICA, C242 antigen, DLL3, DLL4, EGFL7, vimentin, fibronectin extra domain-B, TROP-2, LRRC15, FAP, SLITRK6, NOTCH2, NOTCH3, tanascin-3, STEAP1 and NRP1. In some embodiments, the inhibitor or antagonist is an IL-18 polypeptide variant and at least one other agent is conjugated to an IL-18 polypeptide variant. In some embodiments, at least one other agent is altered T cells or NK cells. In some embodiments, at least one other agent is an oncolytic virus.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description of the embodiments of the invention will be better understood when read in conjunction with the accompanying drawings. It should be borne in mind that the invention is not limited to the specific means and tools of the embodiments shown in the drawings.

Figure 1A and Figure 1B present results from an exemplary study that demonstrates that the IL-18 signaling pathway is a target for tumor immunotherapy. Figure 1A shows the IL-18 signaling pathway (including IL-18 and its receptor subunits) that is overregulated in both activated and dysfunctional tumor T cell programs as seen from an analysis of cytokine and receptor RNAseq expression in CD8+TIL. . Genes are assigned scores for their "activation" and "dysfunction" compared to naive T cells. Yellow indicates cytokine IL-18, IL-18R1 (Rα) and IL-18RAP (Rβ). Data taken from Singer, M. et al., 2016, Cell, 166: 1500-1511, e1509. Figure 1B shows IL-18 subunit receptors IL-18Rα and IL-18Rβ that are part of a gene expression program associated with chronic antigen exposure as seen after infection with Lymphatic Choriomeningitis Virus (LCMV) (left, CD4) or recombinant Vesicular Vesicular Virus. stomatitis encoding ovalbumin (VSV-OVA) (right, CD8). Data taken from the ImmGen database.

Figures 2A - 2C present results from an exemplary study that demonstrates that IL-18BP has the function of a "soluble immune checkpoint form". Figure 2A shows IL-18BP that mediates interferon-γ (IFN-γ)-driven IL-18 negative feedback resembling the PD-L1 immune checkpoint. Schematic representation of the IL-18/ IFN-γ /IL-18BP feedback loop. Black arrows indicate stimulation, red circles indicate inhibition. Figure 2B shows that IL-18BP is upregulated in gastric cancer and breast cancer as seen from data obtained from the TCGA and Oncomine databases. Figure 2C shows that PD-1 and IL-18BP expression are highly correlated in a sample of breast and gastric cancer samples (from the TCGA database). R=0.78 and 0.65, respectively.

Figures 3A-3C present results from an exemplary study that demonstrates the construction of human IL-18 independent of IL-18BP using yeast display technology. Figure 3A shows a chemical structure search library for randomization of residues at the surface boundary of IL-18:IL-18BP that was developed and introduced into the yeast display system. Yeast clones that bind to IL-18R and do not bind to IL-18BP were selected using magnetic and fluorescent cell sorting. Figure 3B shows the results of directed development to create IL-18BP-resistant variants of IL-18. Text highlighted in blue indicates positive selection, text highlighted in red indicates negative selection. Figure 3C shows the results of flow cytometric analysis of WT IL-18 (left) or variants after directional development (right) using yeast display technology. Y-axes show IL-18BP binding, X-axes show IL-18Rα binding. After 5 rounds of targeted development, the remaining clones were significantly more IL-18R2 than IL-18BP.

Figure 4 shows results from an exemplary study that characterizes the sequences of human IL-18 decoy receptor-resistant variants ("DR-IL-18", also referred to as "DR-18"). The position of each mutation and the corresponding residue in the mature form of wild-type human IL-18 is indicated at the top of the table. hC4 - hE12 represent sequences obtained after selection by directed development. hCS1-hCS4 are consensus sequences derived from selected sequences. The pink-shaded residues represent the five most conserved mutations observed.

Figures 5A and 5B present results from an example study showing the biophysical characteristics of human DR-IL-18 variants. Figure 5A shows using yeast display technology that the hCS1-hCS4 and A8 variants of human DR-IL-18 are able to bind to hIL-18Rα, with binding isotherms comparable to those of human WT IL-18 (left). In contrast, very weak binding is observed with the same variants and hIL-18BP (right). Figure 5B shows typical surface plasmon resonance sensorgrams between immobilized biotinylated hIL-18BP and DR-IL-18 variants. Recombinant hIL-18 (left) binds IL-18BP with exceptionally high affinity, K _D = 2.0 pM, while hCS1 (right) shows highly attenuated binding with a much higher dissociation rate and K _D = 15.2 nM . These data are shown in tables 6 and 7.

Figures 6A and 6B present results from an exemplary study which demonstrate that human DR-IL-18 variants are not inhibited by IL-18BP. Figure 6A shows using yeast display technology that recombinant IL-18BP inhibits biotinylated IL-18Rα from binding IL-18, but does not affect the DR-IL-18 hCS1-4 and A8 variants (left). In contrast, IL-18BP effectively neutralizes the previously described IL-18 E42A, K89A and E42A/K89A (Kim et al., 2001, Proc. Natl. Acad. Sci., 98 (6): 3304-3309) (right ) [E42 and K89 (Kim et al.) are the E6 and K53 sequences of SEQ ID NO: 30, respectively]. For all samples, a fixed concentration of biotinylated IL-18R2 was maintained at 100 nM. Figure 6B shows that IL-18 WT and hCS1, hCS3 and hCS4 stimulate HEK-Blue IL-18 reporter cells with comparable potency and efficacy (left). In this assay, wild-type IL-18 is highly sensitive to application of recombinant IL-18BP (IC ₅₀ =3 nM), while hCS1 and hCS3 are not inhibited by recombinant IL-18BP even at 1 μM IL-18BP concentrations (right). Maintained a fixed concentration of hIL-18, equal to 5 nm, and a fixed concentration of hCS1 and hCS3, equal to 2.5 nm.

Figures 7A-7C present results from an exemplary study that demonstrates the construction of additional human IL-18 variants independent of IL-18BP (version 2 variants) using yeast display technology. Figure 7A shows data on positions in human IL-18 randomized in library version 2.0. For each position, degenerate codons and a set of encoded amino acids are given. Figure 7B shows the results of directed development to generate IL-18BP-resistant variants of IL-18 version 2.0. Text highlighted in blue indicates positive selection, text highlighted in red indicates negative selection. Figure 7C shows the results of a flow cytometric analysis of the generation of DR-IL-18 version 2.0 variants. Yeasts obtained after rounds 1, 4 and 6 were simultaneously stained with 250 nM streptavidin-PE IL-18BP tetramers or with 100 nM IL-18Rα directly labeled with AlexaFluor647. Y-axes show IL-18BP binding, X-axes show IL-18Rα binding. After 6 rounds of directed development, the remaining clones were significantly more IL-18Rα than IL-18BP.

Figure 8 shows the results from an exemplary study that characterizes the sequences of human IL-18 decoy receptor (DR-IL-18) version 2 variants. top of the table. The rows shaded in different colors indicate the variants of the recurrent sequence obtained both in the 5th round and in the 6th round.

Figure 9 presents results from an exemplary study showing the biophysical characteristics of human version 2.0 variants of DR-IL-18. Figure 9A shows, using yeast display technology, that DR-IL-18 version 2.0 is able to bind to hIL-18Rα, which is characterized by binding isotherms comparable to those of human WT IL-18. Figure 9B shows that, in contrast, very weak binding is observed with the same variants and hIL-18BP. Figure 9C shows the results of evaluating the thermal stability of DR-IL-18 version 2.0 variants by heating yeast display variants over a temperature range for 15 minutes followed by staining with hIL-18Rα. Version 2.0 DR-IL-18 variants were more thermostable than WT IL-18 (Tm=47.6°C) and first generation consensus sequences (Tm=50.9° and 40.2° for hCS1 and hCS2, respectively) . Figure 9D shows the receptor binding characteristics and thermal stability of second generation DR-IL-18 variants. NBD=binding not found. N.D. = value not measured.

Figures 10A-10C present results from an exemplary study that demonstrates the construction of mouse IL-18 variants independent of IL-18BP using yeast display technology. Figure 10A shows the results of directed development to create IL-18BP-resistant variants of mouse IL-18. Text highlighted in blue indicates positive selection, text highlighted in red indicates negative selection. Figure 10B shows the results of a flow cytometric analysis of the resulting yeast display technology of mouse IL-18 variant after 5 rounds of targeted development. Y-axes show IL-18BP binding, X-axes show IL-18Rα binding. Figure 10C presents results that characterize the sequences of mouse IL-18 decoy receptor-resistant variants (DR-IL-18). The position of each mutation and the corresponding residue in the mature form of wild-type mouse IL-18 is indicated at the top of the table. mC1 - mH3 represent sequences obtained after selection by directed development. mCS1 and mCS2 are consensus sequences derived from selected sequences. The pink-shaded residues represent the five most conserved mutations observed.

Figures 11A and 11B present results from an example study showing the biophysical characteristics of mouse DR-IL-18 variants. Figure 11A shows that mA7, mB1, mC1, mE8, mCS1, and mCS2 variants produced by DR-IL-18 yeast display technology are able to bind mIL-18Rα, with binding isotherms comparable to those of mouse WT IL-18c (left ). In contrast, very weak binding is observed with the same variants and mIL-18BP (right). Figure 11B shows typical surface plasmon resonance sensorgrams between immobilized biotinylated mIL-18BP and mouse DR-IL-18 variants. Recombinant mIL-18 (left) binds mIL-18BP with high affinity, K _D =0.8 pM, while mCS2 (right) shows significantly lower binding with a K _D value greater than 10 μM. These data are shown in tables 8 and 9.

Figures 12A-12D show results from an example pharmacodynamic study of DR-IL-18 administered to mice. Figure 12A is a schematic representation of the study design. Mice were injected with placebo (PBS), mIL-18 (1 mg/kg) or DR-IL-18 variant mCS2 (1 mg/kg) once daily for a total of seven doses over seven days (denoted as syringes). Blood samples were taken five hours after injection, two days before the experiment, and on days 0, 3, and 6. Figure 12B shows peripheral blood cell counts for CD4, CD8, NK cells, and monocytes on day 0, day 3, and day 6 Both IL-18 and mCS2 caused an increase in the number of NK cells and monocytes to the same extent by the 3rd day. For each time point (day), the left column is for PBS, the middle column is for IL-18, and the right column is for mCS2. Figure 12C shows data on CD69 expression on peripheral CD4, CD8 and NK cells. mCS2, but not IL-18, stimulated CD69 expression on CD4 and CD8 cells. Both IL-18 and mCS2 increased CD69 on NK cells, but mCS2 treatment resulted in persistent expression of CD69 expressed at day 6 compared to IL-18 which returned CD69 to baseline levels. For each time point (day), the left column is for PBS, the middle column is for IL-18, and the right column is for mCS2. Figure 12D shows the levels of serum cytokines for interferon-γ (IFN-γ), MIP-1b and G-CSF. The introduction of mCS2 gave higher levels of IFN-γ, MIP-1b and G-CSF than the introduction of mIL-18.

Figure 13 shows the results from an exemplary study that demonstrates that administration of DR-IL-18 reduces body fat in mice. Fat content and lean tissue mass were measured in mice administered with 0.01, 0.1 or 1 mg/kg DR-IL-18 mCS2 variant or 1 mg/kg WT mIL-18 every three days. The introduction of mCS2 led to a significant reduction in the content of adipose tissue, expressed as a percentage of the total mass (upper panel). This was expressed as a decrease or stability in body fat content (left panel) calculated as a percentage of total mass (right panel). Placebo-treated mice and mIL-18-treated mice showed an increase in adipose tissue and a stable lean mass over the same treatment period.

Figures 14A-14B present results from an exemplary study that demonstrates that DR-IL-18 is an effective immunotherapeutic agent in a melanoma model. Figure 14A shows tumor growth susceptibility curves in mice with Yummer1.7 melanoma tumors treated with saline (control), WT IL-18 (0.32 mg/kg), DR-IL-18 variant mCS2 (0.32 mg /kg), anti-PD1 antibody (8 mg/kg), IL-18 + anti-PD1 antibody or mCS2 + anti-PD1 antibody twice a week. Figure 14B shows the survival curves in the same groups as in figure 14A. In this model, mCS2 was effective as a monotherapy and synergistic in combination with an anti-PD1 antibody.

Figures 15A and 15B present results from an exemplary study showing that the efficacy of DR-IL-18 in the melanoma model of Figure 14 is dependent on CD4 and CD8 lymphocytes and interferon gamma. Figure 15A shows plots of tumor growth in mice with Yummer1.7 melanoma tumors treated with saline (control) or DR-IL-18 variant mCS2 (0.32 mg/kg) alone or in combination with anti-CD8 depleting antibodies, CD4, interferon gamma or NK1.1. Figure 15B shows the survival curves in the same groups as in figure 15A.

Figure 16 shows results from an example study showing dose dependent activity of DR-IL-18 in the MC38 tumor model. Tumor growth susceptibility curves are shown in mice with MC38 colon cancer tumors treated with PBS (control), 1.0 mg/kg WT IL-18, 1.0 mg/kg mCS2, 0.1 mg/kg mCS2, or 0. 01 mg/kg mCS2 every three days. IL-18 WT showed no activity at 1 mg/kg, while mCS2 had partial activity at 0.1 mg/kg and maximum activity at 1.0 mg/kg.

Figure 17 shows results from an exemplary study showing the activity of DR-IL-18 alone and in combination with immune checkpoint inhibitor anti-PD1 antibody in the MC38 tumor model. Tumor growth susceptibility curves are shown in mice with MC38 colon cancer tumors injected with PBS (control), 0.32 mg/kg WT IL-18, 0.32 mg/kg DR-IL-18 variant mCS2, 5 mg/kg anti-PD1 antibody, combinations of anti-PD1 antibody with WT IL-18, or combinations of anti-PD1 antibody with mCS2. All drugs were administered intraperitoneally twice a week for a total of 6 doses.

Figure 18A and Figure 18B show results from an exemplary study of the mechanism of antitumor activity of DR-IL-18 in mice with MC38 tumors. Figure 18A shows the results of tumor immunophenotyping experiments in mice treated twice weekly with four doses of saline, WT IL-18, or DR-IL-18 mCS2 variant. The introduction of DR-IL-18 resulted in an increase in the number of CD8 and NK cells per milligram of tumor (upper two panels on the left) and increased expression of granzyme B and KLRG1 activation markers on CD8 and NK cells (upper two panels on the right). DR-IL-18 administration did not improve the CD8:Treg ratio compared to saline administration, while WT IL-18 administration made the ratio less favorable. However, administration of DR-IL-18 increased the ratio of CD8 cells to myeloid inhibitory populations, including tumor-associated macrophages (TAMs) and myeloid monocytic and granulocyte suppressor cells (mMDSCs and gMDSCs). Figure 18B shows the results of measurements of the level of cytokines on the device Luminex in serum from the same mice as in figure 18A, taken 24 hours after the 4th dose. DR-IL-18 exhibits a dramatically altered secondary cytokine release profile when treated with WT IL-18, in particular a more than 100-fold increase in interferon-gamma, IL-7 and IL-15 levels.

Figures 19A-19C present results from an exemplary study that demonstrates the ability of DR-IL-18 to effectively therapeutically target tumors that are immune checkpoint inhibitors due to the lack of expression of MHC class I molecules on the surface of tumor cells. Figure 19A shows tumor growth susceptibility curves in B2m-deficient Ymmer1.7 tumor mice treated with saline, anti-PD1 antibody + anti-CTLA4 antibody, DR-IL-18 variant mCS2 or mCS2 with NK cell depletion by anti- NK1.1. DR-IL-18 showed high tumor growth and survival activity (FIG. 19B), allowing 60% of treated mice to be cured in this tumor model, which is completely resistant even to combination therapy with anti-CTLA4 antibody + anti-PD1 antibody. This activity is dependent on NK cells, since the introduction of antibodies against NK1.1 cancels the therapeutic effect of mCS2. Figure 19C shows that NK cells isolated from B2m-deficient tumor Yummer1.7 are dysfunctional and have reduced proliferation (Ki67 stain) and function (interferon-gamma secretion). However, treatment with DR-IL-18 reverses this phenotype, resulting in sustained proliferation and secretion of cytokines.

Figures 20A-20C present results from an exemplary study that demonstrates the construction of human IL-18 variants as IL-18BP (or decoy receptor decoy, D2D) antagonists using yeast display technology. These variants bind IL-18BP but do not signal, thereby antagonizing the effect of IL-18BP on endogenous IL-18. Figure 20A shows positions in human IL-18 randomized in the D2D library. For each position, degenerate codons and a set of encoded amino acids are given. Figure 20B schematically depicts the use of targeted development to generate D2D IL-18 variants that bind and neutralize IL-18BP but do not signal through IL-18R. Text highlighted in blue indicates positive selection, text highlighted in red indicates negative selection. Figure 20C shows the results of a flow cytometric analysis of the generation of D2D hIL-18 variants. Yeast obtained after rounds 1-4 were stained with 1 nM mouse IL-18BP (left panel), with 1 nM human IL-18BP (middle panel), or with 1 μM IL18Rα plus 1 μM IL18Rβ. The selected variants show an increase in IL-18BP binding in all rounds of selection without an increase in IL18Rα or IL18Rβ binding.

Figure 21 shows results from an example study that characterizes the sequences of D2D variants of human IL-18. The position of each mutation and the corresponding residue in the mature form of wild-type human IL-18 is indicated at the top of the table.

Figures 22A-22C present results from an exemplary study showing the biophysical characteristics of decoy receptor decoy (D2D) variants of human IL-28. Figure 22A shows that yeast display D2D variants 5-B02, 5-E08, 5-F10, 5-F02, 5-F01, hD2D-CS1, hD2D-CS2, and hD2D-CS3 for IL-8 are capable of bind hIL-18RBP, which is characterized by binding isotherms comparable to binding isotherms for human WT IL-18. Figure 22b shows that, in contrast, very weak binding is observed with the same variants and hIL-18Rα. Figure 22C shows the receptor binding characteristics of the D2D IL-18 variants. NBD=binding not found.

Figure 23 shows results from an example study that characterizes the sequences of D2D variants of mouse IL-18. The position of each mutation and the corresponding residue in the mature form of wild-type human IL-18 is indicated at the top of the table.

The figure 24 presents the results of measurements of biophysical affinity (sensogram) variants of DR-IL-18 second generation for binding to IL-18Ra and IL-18BP using the method of surface plasmon resonance (SPR). Top row: typical sensograms of indicated IL-18 variants (soluble analytes) for hIL-18Ra (immobilized ligand). Bottom row: representative sensorgrams of the indicated human IL-18 variants (hIL-18BP). The x-axis is time in seconds, and the y-axis is response units (RU). Curves are observed data over time for different concentrations (two-fold dilutions starting at 1 nM) superimposed on curves of best fit calculated based on the 1:1 Langmuir binding model.

Figures 25A and 25B show data demonstrating the activity of DR-IL-18 in the CT26 colorectal tumor model. 250,000 CT26 cells were implanted subcutaneously and treatment started on day 7 after tumors averaged ~60 mm ³ . WT IL-18 and mCS2 were administered at a dose of 0.32 mg/kg twice a week for a total of 5 doses. The anti-PD1 antibody was administered at a dose of 10 mg/kg according to the same schedule. (A) Tumor growth susceptibility curves are shown for animals treated with saline (PBS) as black lines (circles), WT IL-18 as blue lines (squares) and DR-IL-18 (mCS2) as pink lines (triangles). Only administration of DR-IL-18, but not administration of WT IL-18, resulted in tumor growth inhibition and tumor clearance in a subgroup of animals. (B) Survival curves are shown for mice treated with anti-PD-1 antibody, WT IL-18 and DR-IL-18 (mCS2). The number of complete responses is given in brackets. Administration of DR-IL-18, but not administration of WT IL-18, resulted in increased survival and tumor clearance in 40% of mice, an improvement over the anti-PD-1 immune checkpoint inhibitor.

Figures 26A and 26B show data demonstrating the activity of DR-IL-18 in the 4T1 breast cancer model and the B16-F10 melanoma model. (A) Growth curves of 4T1 tumors inoculated in BALB/C mice after saline (PBS; black), WT IL-18 (blue), or DR-IL-18 CS2 variant (pink) are shown. (B) Growth curves of B16-F10 tumors implanted in C57BL/6 mice after saline (PBS; black), WT IL-18 (blue), or DR-IL-18 CS2 variant (pink) are shown. . In both models, only administration of DR-IL-18, but not administration of WT IL-18, resulted in inhibition of tumor growth. The treatment was performed after the average tumor size exceeded 50 mm ³ as indicated in the boxes marked with the letter "t".

Figures 27A and 27B show data that complements the data shown in Figures 19A-19C. The data presented demonstrate the activity of DR-IL-18 in the treatment of additional MHC class I deficient tumor models that are resistant to immune checkpoint inhibitors. (A) B2m-deficient MC38 cells were generated using the CRISPR/Cas9-mediated deletion described for B2m-deficient YUMMER cells. B2m-/-MC38 cells were implanted subcutaneously and treatment started on day 7 after tumors averaged ~65 mm ³ . mCS2 was administered at a dose of 0.32 mg/kg twice a week for a total of 5 doses. Anti-PD1 antibody and anti-CTLA4 antibody were administered at a dose of 8 mg/kg according to the same schedule. (B) RMA/S is a variant of the RMA lymphoma lineage that contains a spontaneous mutation in the TAP-associated glycoprotein. The result is a defect in antigen loading and hence reduced surface expression of class I MHC molecules. It is congenic to the C57BL/6C57BL mouse line and resistant to immune checkpoint inhibitors. Mice were implanted subcutaneously with 1,000,000 RMA/S cells and treatment began on day 7. MKS2 was administered at a dose of 0.32 mg/kg twice a week. The anti-PD1 antibody was administered at a dose of 8 mg/kg according to the same schedule. In both studies, only treatment with the DR-18 mCS2 variant showed an antitumor effect in terms of inhibition of tumor growth (B2m -/- MC38) or tumor clearance (RMA/S).

The figure 28 shows data demonstrating the activity of DR-IL-18 in relation to enhancing antitumor 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 Raja cells were incubated together at an effector:target (E:T) ratio of 1:10 for 25 hours. These samples were treated with the hCS-1 variant of human DR-IL-18 (1 μM), rituximab (10 μg/ml), or a combination of both. Cytotoxicity was determined by flow cytometry and was calculated as the proportion of CFSE cells that became DAPI positive. DR-18 significantly stimulated tumor cell death when used alone, and its effect on cell death was significantly enhanced when combined with the therapeutic antibody rituximab. *p<0.05 by two-way ANOVA with Tukey's correction for multiple comparisons.

Figures 29A and 29B show data demonstrating the antiviral activity of the mCS2 DR-18 variant in the treatment of viral infections (eg, in this case, in the treatment of systemic vaccinia infection). (A) Schematic of the experimental plan. C57BL/6 mice were infected intraperitoneally (IP) with 10 ⁶ pfu of vaccinia virus (VACV) and injected intraperitoneally with 1 mg/kg WT mIL-18 or mCS2. Mice were sacrificed and viral titers in blood and ovaries were determined by reverse transcription polymerase chain reaction (RT-PCR) on the 3rd day after infection. (B) Quantification of viral copies of VACV in the ovaries and blood of treated mice on the 3rd day after infection. Treatment with CS2 showed a significant reduction in viral titers, while treatment with WT IL-18 was ineffective. *p<0.05, **p<0.01, ***p<0.001.

Figure 30A shows data demonstrating the activity of second generation human DR-IL-18 variants. Figure 30A shows that WT IL-18 and h6-12, h6-27, h6-29 and h6-31 stimulate IL-18 HEK-Blue reporter cells. h6-12, h6-27 and h6-29 show increased activity compared to WT hIL-18, while h6-31 has the same activity as WT hIL-18. Thus, the data demonstrate that all second generation human DR-IL-18 variants tested actively signal through IL-18R.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to IL-18 variants that induce or enhance the activation of the IL-18 signaling pathway. In one aspect, the invention relates to IL-18 variants that can bind to the IL-18 receptor (IL-18R) but do not bind to the IL-18 binding protein (IL-18BP), thereby allowing activation of the IL signaling pathway. -18, but which are not inhibited when exposed to IL-18BP. In one aspect, the invention relates to IL-18 variants that bind to IL-18BP, thereby reducing or preventing IL-18BP binding to endogenous IL-18 and inhibition of endogenous IL-18, and thereby allowing activation of the IL-18 signaling pathway. . Therefore, the invention provides compositions and methods for providing activation of the IL-18 signaling pathway even in the presence of IL-18BP.

In various embodiments, the invention is an IL-18 polypeptide variant or fragment thereof that specifically binds to IL-18R and exhibits significantly reduced binding to IL-18BP. In some embodiments, an IL-18 polypeptide variant or fragment thereof that binds to IL-18R but substantially does not bind to IL-18BP is used to provide activation of an IL-18 signaling pathway that is not inhibited by the presence and activity of IL-18R. 18BP. In various embodiments, the invention relates to an IL-18 polypeptide variant, or fragment thereof, that specifically binds to IL-18BP and reduces or prevents the inhibition of endogenous IL-18 by IL-18BP. In some embodiments, an IL-18 polypeptide variant or fragment thereof that binds to IL-18BP is used to inhibit the activity of IL-18BP, thereby inducing, enhancing, or promoting the activity of the IL-18 signaling pathway.

In some embodiments, IL-18 polypeptide variants are used to treat and prevent a disease or disorder. In various embodiments, the disease or disorder is cancer or an infectious disease such as poxviruses that encode an IL-18BP orthologue, a metabolic disease or disorder (including obesity and diabetes), or macular degeneration (e.g., wet macular degeneration, e.g. , wet form of age-related macular degeneration, for example, variant IL-18 can be used as an anti-angiogenic agent, and in some cases, said variant IL-18 polypeptide can, for example, attenuate neovascularization of the choroid). Therefore, in some embodiments, the invention is a composition comprising at least one IL-18 polypeptide variant or fragment thereof. In other embodiments, the invention is a method of administering at least one IL-18 polypeptide variant or fragment thereof for the treatment or prevention of a disease or disorder such as, but not limited to, cancer or an infectious disease, metabolic disease or disorder, or macular degeneration (eg, wet form of macular degeneration, such as wet form of age-related macular degeneration).

In various embodiments, the variant IL-18 polypeptide comprises a mutation relative to the wild form (WT) of the IL-18 polypeptide. In some embodiments, the WT IL-18 polypeptide comprises the amino acid sequence of SEQ ID NO: 30. In other embodiments, the WT IL-18 polypeptide comprises the amino acid sequence of SEQ ID NO: 31.

In some embodiments, variants of an IL-18 polypeptide of the invention exhibit reduced binding affinity for IL-18BP compared to a WT IL-18 polypeptide. In some embodiments, variants of an IL-18 polypeptide of the invention exhibit increased binding affinity for IL-18BP compared to a WT IL-18 polypeptide. In some embodiments, variants of an IL-18 polypeptide of the invention exhibit a binding affinity for IL-18BP that is similar to that of a WT IL-18 polypeptide.

In some embodiments, the variant IL-18 polypeptide exhibits increased binding affinity for IL-18R compared to the WT IL-18 polypeptide. In some embodiments, a variant IL-18 polypeptide exhibits a binding affinity for IL-18R that is similar to that of a WT IL-18 polypeptide. In some embodiments, the variant IL-18 polypeptide exhibits reduced binding affinity for IL-18R compared to the WT IL-18 polypeptide.

In some embodiments, the IL-18 polypeptide variant is a mammalian IL-18 polypeptide variant. In some embodiments, the IL-18 polypeptide variant is a human IL-18 polypeptide variant. In some embodiments, the IL-18 polypeptide variant is a murine human IL-18 polypeptide variant.

In various embodiments, the compositions and methods of the invention include compositions and methods for treating and preventing disease and disorders such as cancer, infectious disease, and metabolic diseases and disorders. In some embodiments, the method includes administering to a subject in need thereof a composition comprising at least one IL-18 polypeptide variant. In some embodiments, the method includes administering to a subject in need thereof a composition comprising at least one IL-18 polypeptide variant and administering to the subject a composition comprising an additional drug.

In one such embodiment, the additional drug comprises an immunotherapeutic agent comprising at least one selected from the group including, but not limited to, altered T cells, chimeric antigen receptor T cells (CAR-T) , armored CAR-T cells, virus, antigen, vaccine, antibody, immune checkpoint inhibitor, small molecule, chemotherapeutic agent and stem cells. In some embodiments, a composition comprising at least one IL-18 polypeptide variant is used in a method to enhance the activity of the immune system before, during, or after infection with a bacteria, virus, or other pathogen. In some embodiments, a composition comprising at least one IL-18 polypeptide variant is used in a method to increase the number and/or activity of immunocytes in vitro, in vivo, or ex vivo, e.g., the number and/or activity of T cells , NK cells and/or myeloid cells.

Definitions

Unless otherwise indicated, all technical and scientific terms used in the invention have the same meanings that are generally accepted by ordinary specialists in the field of technology to which the invention pertains. In describing the present invention and its patent claims, the following terminology will be used.

It should also be borne in mind that the terminology used in the invention serves only to describe specific embodiments and is not a limitation on the invention.

Used in the invention, the singular form denotes in the text one grammatical object or more than one grammatical object (ie, at least one). For example, "element" means one element or more than one element.

As used herein, the term "approximately" in relation to a measurable quantity, such as quantity, time duration, and the like, is intended to include non-limiting deviations of ±40% or ±20% or ±10%, ±5%, ±1%, or ±0.1% of the specified value, if such deviations are acceptable.

As used herein, the term "abnormal" when applied to organisms, tissues, cells, or components thereof, refers to those organisms, tissues, cells, or components thereof that differ in at least one observable or detectable characteristic (e.g., age, treatment, time of day, etc.). e) from those organisms, tissues, cells or their components that exhibit a "normal" (expected) corresponding characteristic. Characteristics that are normal or expected for one type of cell or tissue may be abnormal for another type of cell or tissue.

As used herein, the term "antibody" refers to an immunoglobulin molecule that is capable of specifically binding to a particular epitope on an antigen. The antibodies may be intact immunoglobulins derived from natural sources or from recombinant sources, and may be immunoreactive portions of intact immunoglobulins. The antibodies of the present invention may exist in various forms, including, for example, polyclonal antibodies, monoclonal antibodies, intracellular antibodies ("intraantibodies"), Fv, Fab and F(ab)2, as well as 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 herein, the term "antibody heavy chain" refers to the larger of the two types of polypeptide chains found in all antibody molecules in their natural conformations.

As used herein, the term "antibody light chain" refers to the smaller of the two types of polypeptide chains found in all antibody molecules in their natural conformations. κ and λ light chains refer to the two major isotypes of antibody light chains.

By the term "synthetic antibody" as used herein is meant an antibody which is generated by recombinant DNA, such as, for example, the antibody described in the invention expressed by a bacteriophage. The term should also be construed as meaning an antibody that has been generated by the synthesis of a DNA molecule encoding an antibody, and which DNA molecule expresses an antibody protein, or a signal sequence defining an antibody, where the DNA or amino acid sequence has been obtained using synthetic DNA or amino acid sequencing technology, which is well known and used in the field.

As used herein, the term "immunoassay" refers to any binding assay that uses an antibody capable of specifically binding to a target molecule to detect and quantify the target molecule.

As used herein, the term "specifically binds" in relation to an IL-18 polypeptide variant refers to an IL-18 polypeptide variant that recognizes and binds to a specific receptor, such as IL-18R, or IL-18BP. In some cases, the variant IL-18 polypeptide significantly reduced binding to IL-18BP. For example, a variant of an IL-18 polypeptide that specifically binds to a receptor from one species can also bind to that receptor from one or more species. But such cross-species reactivity does not in itself change the classification of an IL-18 polypeptide variant as specific. In another example, a variant of an IL-18 polypeptide that specifically binds to a receptor can also bind to different allelic forms of the receptor. However, such cross-reactivity does not in itself change the classification of the IL-18 polypeptide variant as specific. In some cases, the terms "specific binding" or "specific binding" may be used in relation to the interaction of an antibody, protein, or peptide with a second chemical species to indicate that the interaction is dependent on the presence of a particular structure (e.g., antigenic determinant or epitope) on the chemical species. ; for example, a variant IL-18 polypeptide recognizes and binds to a specific protein structure rather than to proteins in general.

As used herein, the term "applicator" refers to any device, including, but not limited to, a hypodermic syringe, a pipette, a drug electrophoresis device, a patch, and the like, for administering the compositions of the invention to a subject.

As used herein, the term "cancer" refers to the abnormal growth or division of cells. As a rule, the growth and/or lifespan of cancer cells exceeds and is not consistent with the growth and/or lifespan of normal cells and tissues around them. Cancer can be benign, premalignant, or malignant. Cancer occurs in various cells and tissues, including the oral cavity (eg, mouth, tongue, pharynx, etc.), digestive system (eg, esophagus, stomach, small intestine, colon, rectum, liver, bile duct) , gallbladder, pancreas, and so on), respiratory system (e.g., larynx, lungs, bronchi, and so on), bones, joints, skin (e.g., basal cells, squamous cells, meningioma, and so on), breast, genital organs (such as uterus, ovary, prostate, testis, and so on), urinary system (such as bladder, kidney, ureter, and so on), eye, nervous system (such as brain, and so on), endocrine system (for example, the thyroid gland, and so on) and in the hematopoietic system (for example, lymphoma, myeloma, leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia, and so on).

The term "coding sequence" as used herein refers to a nucleic acid sequence, or its complement, or a portion thereof, which can be transcribed and/or translated to form an mRNA and/or polypeptide or fragment thereof. Coding sequences include exons in genomic DNA or primary transcripts of immature RNA, which combine under the influence of the biochemical mechanisms of cells to form mature mRNA. The antisense strand is the complement of such a nucleic acid and a coding sequence can be derived from it. In contrast, the term "non-coding sequence" as used herein refers to a nucleic acid sequence or its complement, or a portion thereof, that is not translated into an amino acid in vivo, or where the tRNA does not interact to insert or attempt to insert an amino acid. Non-coding sequences include both intron sequences in genomic DNA and primary immature RNA transcripts and gene-associated sequences such as promoters, enhancers, silencers, and the like.

As used herein, the terms "complementary" or "complementarity" are used to refer to polynucleotides (ie, a sequence of nucleotides) linked based on base pairing rules. For example, the sequence "A-G-T" is complementary to the sequence "T-C-A". Complementarity can be "partial", in which only some of the bases of the nucleic acids are fully matched to each other in accordance with the rules of base pairing. Or there may be "total" or "total" complementarity between nucleic acids. The degree of complementarity between nucleic acid strands has a strong influence on the efficiency and intensity of hybridization between nucleic acid strands. This is especially important in amplification reactions, as well as for detection methods that depend on binding between nucleic acids.

"Disease" is a health condition of an animal in which the animal cannot maintain homeostasis and in which, if the disease state is not alleviated, the health of the animal continues to deteriorate. In contrast, a "disorder" in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal's health is less favorable than it would be in the absence of the disorder. Left untreated, the disorder does not necessarily lead to further deterioration in the health of the animal.

Used in the invention, the term "effective amount" means the amount that provides a therapeutic, prophylactic or other desired beneficial effect.

"Encoding" refers to the property inherent in specific nucleotide sequences in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as templates for the synthesis of other polymers and macromolecules in biological processes having either a specific nucleotide sequence (i.e., rRNA, tRNA, and mRNA ), or a particular sequence of amino acids, and the resulting biological properties. Thus, a gene encodes a protein if the transcription and translation of the mRNA corresponding to that gene produces a protein in a cell or other biological system. Both a coding strand whose nucleotide sequence is identical to an mRNA sequence and is commonly listed in sequence listings, and a non-coding strand used as a template for transcription of a gene or cDNA may be referred to as sequences encoding a protein or other product of that gene or cDNA.

Used in the invention, the term "fragment" in relation to the nucleic acid, refers to the sequence of a larger nucleic acid. A nucleic acid "fragment" may be at least about 15 nucleotides in length; for example, at least from 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 from about 1000 nucleotides to about 1500 nucleotides; from about 1500 nucleotides to about 2500 nucleotides; or approximately 2500 nucleotides (and any integer value in between). Used in the invention, the term "fragment" in relation to a protein, polypeptide or peptide, refers to the sequence of a larger protein, polypeptide or peptide. A "fragment" of a protein, polypeptide, or peptide may be at least about 5 amino acids in length; for example, at least about 10 amino acids in length; at least about 20 amino acids in length; at least about 50 amino acids in length; at least about 100 amino acids in length; at least about 200 amino acids in length; or at least approximately 300 amino acids in length (and any integer value in between).

The term "gene" refers to a nucleic acid sequence (eg, DNA) that includes the coding sequences necessary for the production of a polypeptide, precursor, or RNA (eg, mRNA). The polypeptide may be encoded by the full-length coding sequence, or by any portion of the coding sequence, as long as the desired activity or functional property (e.g., enzymatic activity, receptor binding, signal transduction, immunogenicity, and the like) of the full-length sequence or fragment is retained. The term also encompasses the coding region of a structural gene and sequences adjacent to the coding region at both the 5' and 3' ends at a distance of about 2 kb or more at either end such that the gene corresponds to the length of the full length mRNA and 5' regulatory sequences that affect the transcriptional properties of a gene. Sequences located at the 5' position of the coding region and present in mRNA are called 5'-untranslated sequences. 5'-untranslated sequences usually contain regulatory sequences. Sequences located 3' or downstream of the coding region and present in the mRNA are referred to as 3'-untranslated sequences. The term "gene" encompasses both cDNA and genomic forms of a gene. The genomic form or clone of a gene contains a coding region interrupted by non-coding sequences called "introns" or "intermediate regions" or "intermediate sequences". Introns are segments of a gene that are transcribed into nuclear RNA (hnRNA). Introns may contain regulatory elements such as enhancers. Introns are removed or "spliced" from the nuclear or primary transcript, whereby introns are absent from the messenger RNA (mRNA) transcript. mRNA during translation performs the function associated with determining the sequence or order of amino acids in the resulting polypeptide.

The terms "homologous", "identical" or "identity", as used in the invention in relation to two or more sequences of nucleic acids or polypeptides, mean that the sequences have a certain percentage of residues that are the same in the specified area. The percentage can be calculated by optimally aligning two sequences, comparing the two sequences in a specified region, determining the number of positions where an identical residue occurs in both sequences to get 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 give percent sequence identity. In cases where the two sequences are of different lengths or the alignment results in one or more ends of the stepped profile and the specified area of comparison includes only one sequence, the remainders of one sequence are included in the denominator and not in the numerator in the calculation. When comparing DNA and RNA, thymine (T) and uracil (U) can be considered equivalent. Identification can be done manually or with a computer sequencing algorithm such as BLAST or BLAST 2.0.

As used herein, the term "instructive material" includes a publication, record, chart, or any other means of expression that can be used to provide information about the usefulness of a nucleic acid, peptide, polypeptide and/or compound of the invention in a pharmaceutical kit for detecting or facilitating or treatment of various diseases or disorders described in the invention. Optionally, or alternatively, one or more methods for detecting or alleviating diseases or disorders in a cell or tissue of a subject may be described in the guidance material. The instructional material of the pharmaceutical kit, for example, may be attached to a container that contains the nucleic acid, polypeptide and/or compound of the invention, or may be supplied with a container that contains the nucleic acid, polypeptide and/or compound. Alternatively, the guidance material may be sent separately from the container, but on the assumption that the recipient will share the guidance material and the connection.

"Isolated" means modified or released from its natural state. For example, a nucleic acid or polypeptide naturally present in an animal body is not "isolated", but the same nucleic acid or polypeptide partially or completely separated from coexisting materials in its natural state is "isolated". The isolated nucleic acid or protein may exist in substantially pure form, or may exist in a non-native environment such as, for example, a host cell.

"Isolated nucleic acid" refers to a segment or fragment of a nucleic acid that has been separated from the sequences flanking it in its natural state, such as a DNA fragment that has been removed from sequences that are normally adjacent to the fragment, such as from sequences adjacent to the fragment. fragment in the genome in which it occurs naturally. The term is also applied to nucleic acids that have been substantially purified from other components that accompany the nucleic acid in nature, such as RNA or DNA or proteins that accompany it in the cell. Therefore, the term includes, for example, recombinant DNA which is incorporated into a vector, into an autonomously replicating plasmid or virus, or into prokaryotic or eukaryotic genomic DNA, or which exists as a single molecule (for example, as a cDNA or a genomic or cDNA fragment, produced by PCR or restriction endonuclease digestion), independent of other sequences. The term also includes recombinant DNA that is part of a fusion gene encoding an additional polypeptide sequence.

As used herein, the term "label" refers to a detectable compound or composition that is directly or indirectly conjugated to a probe to generate a "labeled" probe. The label may itself be detectable (eg, radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze a chemical change in a detectable substrate compound or composition (eg, avidin-biotin). In some cases, primers may be labeled to detect the PCR product.

By "modulating" as used herein is meant mediating a detectable increase or decrease in the activity and/or level of an mRNA, polypeptide, or response in a subject compared to the activity and/or level of an mRNA, polypeptide, or response in a subject in the absence of treatment or compound, and /or compared with the activity and/or level of mRNA, polypeptide or response in the same, but not treated subject. The term encompasses activation, inhibition, and/or otherwise affecting a native signal or response, thereby mediating a positive therapeutic, prophylactic, or other desired response in a subject, eg, a human.

As used herein, the terms "mutation", "mutant", or "variant" refer to a change in a nucleic acid sequence or a polypeptide sequence relative to a reference sequence (which may be a naturally occurring normal sequence or a "wild-type" sequence), and this change includes translocations, deletions, insertions and substitutions/point mutations. As used herein, the term "mutant" or "variant" refers to a nucleic acid or protein containing a mutation.

The term "nucleic acid" refers to a polynucleotide and includes polyribonucleotides and polydeoxyribonucleotides. The nucleic acids of the present invention may include 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, at 793-800 (Worth Pub. 1982), the entire content of which is incorporated into the present invention by reference). Indeed, the present invention contemplates any deoxyribonucleotide, ribonucleotide, or peptide component of a nucleic acid, and any chemical variants thereof, such as the methylated, hydroxymethylated, or glucosylated forms of these bases, and the like. The polymers or oligomers may be heterogeneous or homogeneous in composition and may be isolated from natural sources or may be produced artificially or synthetically. In addition, nucleic acids may be DNA or RNA, or a mixture thereof, and may exist permanently or transiently in single-stranded or double-stranded form, including homoduplex, heteroduplex, and hybrid states.

"Oligonucleotide" or "polynucleotide" is a nucleic acid with a length in the range of at least 2, preferably at least 8, 15 or 25 nucleotides, but can be up to 50, 100, 1000 or 5000 nucleotides in length, or a compound that specifically hybridizes to a polynucleotide. Polynucleotides include deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) sequences, or mimetics thereof, which can be isolated from natural sources, recombinantly produced, or artificially synthesized. An additional example of a polynucleotide of the present invention may be a peptide nucleic acid (PNA) (see US Pat. No. 6,155,601, the entire content of which is incorporated herein by reference). The invention also includes situations in which unconventional base pairing exists, such as Hoogsteen base pairing, which has been identified in certain tRNA molecules and is believed to exist in the form of a triple helix. The terms "polynucleotide" and "oligonucleotide" are used interchangeably in this invention. It should be understood that when a nucleotide sequence is represented in the invention by a DNA sequence (e.g. A, T, G and C), it also includes the corresponding RNA sequence (e.g. A, U, G, C) in which "U" replaces "T".

The terms "patient", "subject", "individual" and other similar terms are used interchangeably in the invention and refer to any animal or its cells, whether in vivo, in vitro or in situ, to be treated by the methods described in the invention. In specific non-limiting embodiments, the patient, subject, or individual is a human.

Used in the invention, the terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to a compound consisting of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that a protein or peptide sequence may contain. Polypeptides include any peptide or protein containing two or more amino acids connected to each other by peptide bonds. The term used in the invention refers to both short chains, which are also commonly referred to in the art as peptides, oligopeptides and oligomers, and to longer chains, which are commonly referred to in the art as proteins, among which there are many types. "Polypeptides" include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, fusion proteins, and others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, mutant polypeptides, variant polypeptides, or combinations thereof.

As used herein, the term "polynucleotide" includes cDNA, RNA, DNA/RNA hybrid, antisense RNA, ribozyme, genomic DNA, synthetic forms, and mixed polymers, both sense and antisense strands, and a polynucleotide may be chemically or biochemically modified to display to them the properties of non-natural or derivatized synthetic or semi-synthetic nucleotide bases. In addition, the invention contemplates alterations to a wild-type or synthetic gene, including deletion, insertion, substitution of one or more nucleotides, or fusion with other polynucleotide sequences.

As used herein, the term “preventing” a disease or disorder means reducing the severity or frequency of at least one sign or symptom of the disease or disorder that a subject may be experiencing.

Used in the invention, the term "sample" or "biological sample" refers to biological material taken from the subject. The biological sample may contain any biological material suitable for detecting an mRNA, polypeptide, or other marker of a physiological or pathological process in a subject, and may include fluid, tissue, cellular and/or non-cellular material taken from the subject.

Used in the invention, the term "substantially pure" means the practical absence of other components. For example, a substantially purified polypeptide is one that has been separated from other components with which it is normally associated in its natural state.

As used herein, the terms “therapy” or “treatment regimen” refer to those actions taken to prevent, treat, or modify a disease or disorder, such as a course of treatment designed to reduce or eliminate at least one sign or symptom of a disease. or disorders using pharmacological, surgical, dietary and/or other methods. The treatment regimen may include prescribed dosing of one or more compounds or surgery. Therapy will most often have a positive effect and reduce or eliminate at least one sign or symptom of the disorder or disease state, but in some cases, the effect of therapy will be characterized by unwanted or side effects. The effect of therapy will also be influenced by the subject's physiological state, such as age, sex, genetics, body weight, other disease states, and the like.

The term "therapeutically effective amount" refers to the amount of a compound or composition of interest that elicits a biological, physiological, clinical, or medical response in a cell, tissue, organ, system, or subject that the researcher, veterinarian, physician, or other clinician is trying to achieve. The term "therapeutically effective amount" includes that amount of a compound or composition which, when administered, is sufficient to prevent the development of, or to treat to some extent, one or more of the signs or symptoms of the disorder or disease being treated. A therapeutically effective amount will vary depending on the compound or composition, the disease and its severity, as well as the age, body weight and other similar parameters of the subject to be treated.

As used herein, the term "treat" a disease or disorder means reducing the frequency or severity of at least one sign or symptom of the disease or disorder experienced by a subject. The terms "treatment", "treat" and other similar terms are commonly used in the invention to denote the achievement of the desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of completely or partially preventing the disease or symptom(s) thereof and/or may be therapeutic in terms of partially or completely stabilizing or curing the disease and/or adverse effect associated with the disease. The term "treatment" encompasses any treatment of a disease in a mammal, in particular a human, and includes: (a) preventing the onset of the disease and/or symptom(s) in a subject who may be predisposed to the onset of the disease or symptom, but who still has the presence of this disease or symptom has not been diagnosed; (b) inhibition of the disease and/or symptom(s), such as slowing or stopping their development (eg, stopping the growth of tumors, slowing the rate of tumor growth, slowing the rate of proliferation of cancer cells, and so on); or (c) ameliorating the symptom(s) of the disease, ie achieving regression of the disease and/or symptom(s) (eg, achieving reduction in tumor size, reduction in the number of cancer cells present, etc.). Those in need of treatment include patients who are already suffering from a disease (for example, patients suffering from cancer, infection, metabolic disorder, macular degeneration, and so on), as well as patients in need of preventive treatment (for example, patients with an increased susceptibility to cancer, patients with an increased likelihood of infection, patients with suspected cancer, patients with suspected infection, patients with an increased susceptibility to metabolic disease, patients with an increased susceptibility to macular degeneration, and so on).

As used herein, the term "wild type" refers to a gene or gene product isolated from a natural source. A wild-type gene is a gene that is most commonly observed in a population and is therefore arbitrarily referred to as the "normal" or "wild" form of the gene. In contrast, the term "modified", "variant", or "mutant" refers to a gene or gene product that has modifications in sequence and/or functional properties (i.e., altered characteristics) compared to the wild-type gene or gene product. .

Ranges: In this specification, various aspects of the invention may be presented with a range. It should be understood that the range description is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to specifically disclose all possible subranges as well as individual numerical values within that range. For example, a description of a range, such as 1 to 6, should be considered to include the specifically described subranges, such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 up to 6, and so on, as well as individual numbers in that range, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the width of the range.

Description

In some embodiments, compositions and methods of the invention include an activator of IL-18 activity, such as signaling activity through IL-18R. In some embodiments, the activator is a variant IL-18 polypeptide. In some embodiments, the activator is a molecule that is capable of binding to and signaling through IL-18R. In some embodiments, the activator is a molecule that inhibits IL-18BP, thereby promoting activation of the IL-18 signaling pathway.

In some embodiments, the invention provides a variant or fragment of an IL-18 polypeptide that specifically binds to IL-18R and exhibits significantly reduced binding of IL-18BP. In some embodiments, an IL-18 polypeptide variant or fragment thereof that binds to IL-18R but does not substantially bind to IL-18BP is used to confer activity on an IL-18 signaling pathway that is inhibited by the presence and activity of IL-18BP. .

In some embodiments, the variant IL-18 polypeptide is downregulated or resistant to exposure to the IL-18BP polypeptide. In some embodiments, the IL-18BP polypeptide is unable to significantly bind to a variant IL-18 polypeptide. The IL-18 polypeptide variants of the invention exhibit reduced binding affinity for IL-18BP compared to the WT IL-18 polypeptide. In some embodiments, the variant IL-18 polypeptide exhibits increased binding affinity for IL-18R compared to the WT IL-18 polypeptide. In some embodiments, a variant IL-18 polypeptide exhibits similar binding affinity for IL-18R as compared to a WT IL-18 polypeptide. In some embodiments, the variant IL-18 polypeptide exhibits reduced binding affinity for IL-18R compared to the WT IL-18 polypeptide.

In some embodiments, the invention provides a composition comprising an IL-18BP inhibitor, wherein the inhibitor inhibits or reduces the expression, activity of IL-18BP, or both. Examples of IL-18BP inhibitors include, but are not limited to, a chemical compound, a protein, a peptidomimetic, an antibody, a ribozyme, and an antisense nucleic acid molecule. In specific embodiments, the IL-18BP inhibitor comprises an IL-18 variant that binds IL-18BP, thereby reducing or preventing inhibition of IL-18BP by IL-18 exposure and activation of the IL-18 signaling pathway.

In some embodiments, IL-18 polypeptide variants are used to treat or prevent a disease or disorder. In various embodiments, the disease or disorder is cancer or a metabolic disease or disorder, including obesity and diabetes (eg, the proposed method may lead to a decrease in adipose tissue). Thus, in some embodiments, the invention provides a composition comprising at least one IL-18 polypeptide variant or fragment thereof. In other embodiments, the invention provides a method of administering at least one IL-18 polypeptide variant, or fragment thereof, for the treatment or prevention of a disease or disorder, such as, but not limited to, cancer or a metabolic disease or disorder.

In some embodiments, the variant IL-18 polypeptide binds to IL-18R and exhibits significantly reduced binding to IL-18BP. In some embodiments, an IL-18 polypeptide variant that binds to IL-18R and exhibits significantly reduced binding to IL-18BP binds to IL-18BP with a binding affinity that is from about 0.000000000001% to 95% of binding affinity of wild-type IL-18 to IL-18BP.

In some embodiments, a variant IL-18 polypeptide that binds to IL-18R and exhibits significantly reduced binding to IL-18BP binds to IL-18BP with a binding affinity that is approximately 95% of the binding affinity of wild-type IL-18 type with IL-18BP. In some embodiments, an IL-18 polypeptide variant that binds to IL-18R and exhibits significantly reduced binding to IL-18BP binds to IL-18BP with a binding affinity that is approximately 90% of the binding affinity of wild-type IL-18 type with IL-18BP. In some embodiments, a variant IL-18 polypeptide that binds to IL-18R and exhibits significantly reduced binding to IL-18BP binds to IL-18BP with a binding affinity that is approximately 85% of the binding affinity of wild-type IL-18 type with IL-18BP. In some embodiments, an IL-18 polypeptide variant that binds to IL-18R and exhibits significantly reduced binding to IL-18BP binds to IL-18BP with a binding affinity that is approximately 80% of the binding affinity of wild-type IL-18 type with IL-18BP. In some embodiments, a variant IL-18 polypeptide that binds to IL-18R and exhibits significantly reduced binding to IL-18BP binds to IL-18BP with a binding affinity that is approximately 75% of the binding affinity of wild-type IL-18 type with IL-18BP. In some embodiments, a variant IL-18 polypeptide that binds to IL-18R and exhibits significantly reduced binding to IL-18BP binds to IL-18BP with a binding affinity that is approximately 70% of the binding affinity of wild-type IL-18 type with IL-18BP. In some embodiments, a variant IL-18 polypeptide that binds to IL-18R and exhibits significantly reduced binding to IL-18BP binds to IL-18BP with a binding affinity that is approximately 65% of the binding affinity of wild-type IL-18 type with IL-18BP. In some embodiments, an IL-18 polypeptide variant that binds to IL-18R and exhibits significantly reduced binding to IL-18BP binds to IL-18BP with a binding affinity that is approximately 60% of the binding affinity of wild-type IL-18 type with IL-18BP. In some embodiments, an IL-18 polypeptide variant that binds to IL-18R and exhibits significantly reduced binding to IL-18BP binds to IL-18BP with a binding affinity that is approximately 55% of the binding affinity of wild-type IL-18 type with IL-18BP. In some embodiments, a variant IL-18 polypeptide that binds to IL-18R and exhibits significantly reduced binding to IL-18BP binds to IL-18BP with a binding affinity that is approximately 50% of the binding affinity of wild-type IL-18 type with IL-18BP. In some embodiments, a variant IL-18 polypeptide that binds to IL-18R and exhibits significantly reduced binding to IL-18BP binds to IL-18BP with a binding affinity that is approximately 45% of the binding affinity of wild-type IL-18 type with IL-18BP. In some embodiments, a variant IL-18 polypeptide that binds to IL-18R and exhibits significantly reduced binding to IL-18BP binds to IL-18BP with a binding affinity that is approximately 40% of the binding affinity of wild-type IL-18 type with IL-18BP. In some embodiments, a variant IL-18 polypeptide that binds to IL-18R and exhibits significantly reduced binding to IL-18BP binds to IL-18BP with a binding affinity that is approximately 35% of the binding affinity of wild-type IL-18 type with IL-18BP. In some embodiments, a variant IL-18 polypeptide that binds to IL-18R and exhibits significantly reduced binding to IL-18BP binds to IL-18BP with a binding affinity that is approximately 30% of the binding affinity of wild-type IL-18 type with IL-18BP. In some embodiments, a variant IL-18 polypeptide that binds to IL-18R and exhibits significantly reduced binding to IL-18BP binds to IL-18BP with a binding affinity that is approximately 25% of the binding affinity of wild-type IL-18 type with IL-18BP. In some embodiments, a variant IL-18 polypeptide that binds to IL-18R and exhibits significantly reduced binding to IL-18BP binds to IL-18BP with a binding affinity that is approximately 20% of the binding affinity of wild-type IL-18 type with IL-18BP. In some embodiments, an IL-18 polypeptide variant that binds to IL-18R and exhibits significantly reduced binding to IL-18BP binds to IL-18BP with a binding affinity that is approximately 15% of the binding affinity of wild-type IL-18 type with IL-18BP. In some embodiments, a variant IL-18 polypeptide that binds to IL-18R and exhibits significantly reduced binding to IL-18BP binds to IL-18BP with a binding affinity that is approximately 10% of the binding affinity of wild-type IL-18 type with IL-18BP.

In some embodiments, a variant IL-18 polypeptide that binds to IL-18R and exhibits significantly reduced binding to IL-18BP binds to IL-18BP with a binding affinity that is approximately 5% of the binding affinity of wild-type IL-18 type with IL-18BP. In some embodiments, an IL-18 polypeptide variant that binds to IL-18R and exhibits significantly reduced binding to IL-18BP binds to IL-18BP with a binding affinity that is approximately 4% of the binding affinity of wild-type IL-18 type with IL-18BP. In some embodiments, a variant IL-18 polypeptide that binds to IL-18R and exhibits significantly reduced binding to IL-18BP binds to IL-18BP with a binding affinity that is approximately 3% of the binding affinity of wild-type IL-18 type with IL-18BP. In some embodiments, a variant IL-18 polypeptide that binds to IL-18R and exhibits significantly reduced binding to IL-18BP binds to IL-18BP with a binding affinity that is approximately 2% of the binding affinity of wild-type IL-18 type with IL-18BP. In some embodiments, a variant IL-18 polypeptide that binds to IL-18R and exhibits significantly reduced binding to IL-18BP binds to IL-18BP with a binding affinity that is approximately 1% of the binding affinity of wild-type IL-18 type with IL-18BP.

In some embodiments, an IL-18 polypeptide variant that binds to IL-18R and exhibits significantly reduced binding to IL-18BP binds to IL-18BP with a binding affinity that is approximately 0.1% of that of IL-18BP. 18 wt with IL-18BP. In some embodiments, an IL-18 polypeptide variant that binds to IL-18R and exhibits significantly reduced binding to IL-18BP binds to IL-18BP with a binding affinity that is approximately 0.01% of that of IL-18BP. 18 wt with IL-18BP. In some embodiments, an IL-18 polypeptide variant that binds to IL-18R and exhibits significantly reduced binding to IL-18BP binds to IL-18BP with a binding affinity that is approximately 0.001% of the binding affinity of wild-type IL-18 type with IL-18BP. In some embodiments, an IL-18 polypeptide variant that binds to IL-18R and exhibits significantly reduced binding to IL-18BP binds to IL-18BP with a binding affinity that is approximately 0.0001% of that of IL-18BP. 18 wt with IL-18BP. In some embodiments, an IL-18 polypeptide variant that binds to IL-18R and exhibits significantly reduced binding to IL-18BP binds to IL-18BP with a binding affinity that is approximately 0.00001% of that of IL-18BP. 18 wt with IL-18BP. In some embodiments, an IL-18 polypeptide variant that binds to IL-18R and exhibits significantly reduced binding to IL-18BP binds to IL-18BP with a binding affinity that is approximately 0.000001% of that of IL-18BP. 18 wt with IL-18BP. In some embodiments, an IL-18 polypeptide variant that binds to IL-18R and exhibits significantly reduced binding to IL-18BP binds to IL-18BP with a binding affinity that is approximately 0.0000001% of that of IL-18BP. 18 wt with IL-18BP. In some embodiments, an IL-18 polypeptide variant that binds to IL-18R and exhibits significantly reduced binding to IL-18BP binds to IL-18BP with a binding affinity that is approximately 0.00000001% of that of IL-18BP. 18 wt with IL-18BP. In some embodiments, an IL-18 polypeptide variant that binds to IL-18R and exhibits significantly reduced binding to IL-18BP binds to IL-18BP with a binding affinity that is approximately 0.000000001% of that of IL-18BP. 18 wt with IL-18BP. In some embodiments, an IL-18 polypeptide variant that binds to IL-18R and exhibits significantly reduced binding to IL-18BP binds to IL-18BP with a binding affinity that is approximately 0.0000000001% of that of IL-18BP. 18 wt with IL-18BP. In some embodiments, an IL-18 polypeptide variant that binds to IL-18R and exhibits significantly reduced binding to IL-18BP binds to IL-18BP with a binding affinity that is approximately 0.00000000001% of that of IL-18BP. 18 wt with IL-18BP. In some embodiments, an IL-18 polypeptide variant that binds to IL-18R and exhibits significantly reduced binding to IL-18BP binds to IL-18BP with a binding affinity that is approximately 0.000000000001% of that of IL-18BP. 18 wt with IL-18BP.

In some embodiments, a proposed variant IL-18 polypeptide (DR-IL-18) that binds to IL-18R and exhibits significantly reduced binding to IL-18BP has a K _D value for IL-18BP that is 10 nM or more (higher K _D means lower binding affinity). In some embodiments, the proposed DR variant of the IL-18 polypeptide has a K _D value for IL-18BP that is 20 nM or more (e.g., 50 nM or more, 100 nM or more, 500 nM or more, or 1 μM or more).

In some embodiments, the variant IL-18 polypeptide binds to IL-18R and exhibits significantly reduced binding to IL-18BP. In some embodiments, a polypeptide variant that binds to IL-18R and exhibits significantly reduced binding to IL-18BP has an IL-18BP/IL-18R dissociation constant ratio that is at least about 2-fold than the ratio of dissociation constants of IL-18BP/IL-18R wild-type IL-18 (it should be noted that an increased ratio of dissociation constants indicates a relative decrease in IL-18BP binding relative to IL-18R binding). In some embodiments, a polypeptide variant that binds to IL-18R and exhibits significantly reduced binding to IL-18BP has an IL-18BP/IL-18R dissociation constant ratio that is at least 20 times higher, than the ratio of dissociation constants of IL-18BP/IL-18R wild-type IL-18. In some embodiments, a polypeptide variant that binds to IL-18R and exhibits significantly reduced binding to IL-18BP has an IL-18BP/IL-18R dissociation constant ratio that is at least 200-fold higher, than the ratio of dissociation constants of IL-18BP/IL-18R wild-type IL-18. In some embodiments, a polypeptide variant that binds to IL-18R and exhibits significantly reduced binding to IL-18BP has an IL-18BP/IL-18R dissociation constant ratio that is at least 2000-fold higher than than the ratio of dissociation constants of IL-18BP/IL-18R wild-type IL-18. In some embodiments, a polypeptide variant that binds to IL-18R and exhibits significantly reduced binding to IL-18BP has an IL-18BP/IL-18R dissociation constant ratio that is at least about 20,000 times higher, than the ratio of dissociation constants of IL-18BP/IL-18R wild-type IL-18. In some embodiments, a polypeptide variant that binds to IL-18R and exhibits significantly reduced binding to IL-18BP has an IL-18BP/IL-18R dissociation constant ratio that is at least about 200,000 times than the ratio of dissociation constants of IL-18BP/IL-18R wild-type IL-18. In some embodiments, a polypeptide variant that binds to IL-18R and exhibits significantly reduced binding to IL-18BP has an IL-18BP/IL-18R dissociation constant ratio that is at least about 2,000,000 times higher than than the ratio of dissociation constants of IL-18BP/IL-18R wild-type IL-18. In some embodiments, a polypeptide variant that binds to IL-18R and exhibits significantly reduced binding to IL-18BP has an IL-18BP/IL-18R dissociation constant ratio that is at least about 20,000,000 times higher than than the ratio of dissociation constants of IL-18BP/IL-18R wild-type IL-18.

In some embodiments, the variant IL-18 polypeptide binds to IL-18R and exhibits significantly reduced binding to IL-18BP. In some embodiments, a polypeptide variant that binds to IL-18R and exhibits significantly reduced binding to IL-18BP has an IL-18BP/IL-18R dissociation constant ratio that is at least 3-fold higher than the ratio of dissociation constants of IL-18BP/IL-18R wild-type IL-18. In some embodiments, a polypeptide variant that binds to IL-18R and exhibits significantly reduced binding to IL-18BP has an IL-18BP/IL-18R dissociation constant ratio that is at least 30 times higher, than the ratio of dissociation constants of IL-18BP/IL-18R wild-type IL-18. In some embodiments, a polypeptide variant that binds to IL-18R and exhibits significantly reduced binding to IL-18BP has an IL-18BP/IL-18R dissociation constant ratio that is at least 300-fold higher than than the ratio of dissociation constants of IL-18BP/IL-18R wild-type IL-18. In some embodiments, a polypeptide variant that binds to IL-18R and exhibits significantly reduced binding to IL-18BP has an IL-18BP/IL-18R dissociation constant ratio that is at least about 3,000 times than the ratio of dissociation constants of IL-18BP/IL-18R wild-type IL-18. In some embodiments, a polypeptide variant that binds to IL-18R and exhibits significantly reduced binding to IL-18BP has an IL-18BP/IL-18R dissociation constant ratio that is at least about 30,000 times than the ratio of dissociation constants of IL-18BP/IL-18R wild-type IL-18. In some embodiments, a polypeptide variant that binds to IL-18R and exhibits significantly reduced binding to IL-18BP has an IL-18BP/IL-18R dissociation constant ratio that is at least about 300,000 times than the ratio of dissociation constants of IL-18BP/IL-18R wild-type IL-18. In some embodiments, a polypeptide variant that binds to IL-18R and exhibits significantly reduced binding to IL-18BP has an IL-18BP/IL-18R dissociation constant ratio that is at least about 3,000,000 times higher than than the ratio of dissociation constants of IL-18BP/IL-18R wild-type IL-18. In some embodiments, a polypeptide variant that binds to IL-18R and exhibits significantly reduced binding to IL-18BP has an IL-18BP/IL-18R dissociation constant ratio that is at least about 30,000,000 times higher than than the ratio of dissociation constants of IL-18BP/IL-18R wild-type IL-18.

In some embodiments, the variant IL-18 polypeptide binds to IL-18R and exhibits significantly reduced binding to IL-18BP. In some embodiments, a polypeptide variant that binds to IL-18R and exhibits significantly reduced binding to IL-18BP has an IL-18BP/IL-18R dissociation constant ratio that is at least 5-fold higher than the ratio of dissociation constants of IL-18BP/IL-18R wild-type IL-18. In some embodiments, a polypeptide variant that binds to IL-18R and exhibits significantly reduced binding to IL-18BP has an IL-18BP/IL-18R dissociation constant ratio that is at least 50-fold higher than the ratio of dissociation constants of IL-18BP/IL-18R wild-type IL-18. In some embodiments, a polypeptide variant that binds to IL-18R and exhibits significantly reduced binding to IL-18BP has an IL-18BP/IL-18R dissociation constant ratio that is at least 500 times higher than the ratio of dissociation constants of IL-18BP/IL-18R wild-type IL-18. In some embodiments, a polypeptide variant that binds to IL-18R and exhibits significantly reduced binding to IL-18BP has an IL-18BP/IL-18R dissociation constant ratio that is at least 5,000 times higher than than the ratio of dissociation constants of IL-18BP/IL-18R wild-type IL-18. In some embodiments, a polypeptide variant that binds to IL-18R and exhibits significantly reduced binding to IL-18BP has an IL-18BP/IL-18R dissociation constant ratio that is at least about 50,000 times than the ratio of dissociation constants of IL-18BP/IL-18R wild-type IL-18. In some embodiments, a polypeptide variant that binds to IL-18R and exhibits significantly reduced binding to IL-18BP has an IL-18BP/IL-18R dissociation constant ratio that is at least about 500,000 times higher, than the ratio of dissociation constants of IL-18BP/IL-18R wild-type IL-18. In some embodiments, a polypeptide variant that binds to IL-18R and exhibits significantly reduced binding to IL-18BP has an IL-18BP/IL-18R dissociation constant ratio that is at least about 5,000,000 times higher than than the ratio of dissociation constants of IL-18BP/IL-18R wild-type IL-18. In some embodiments, a polypeptide variant that binds to IL-18R and exhibits significantly reduced binding to IL-18BP has an IL-18BP/IL-18R dissociation constant ratio that is at least about 50,000,000 times higher than than the ratio of dissociation constants of IL-18BP/IL-18R wild-type IL-18.

In some embodiments, the variant IL-18 polypeptide binds to IL-18R and exhibits significantly reduced binding to IL-18BP. In some embodiments, a polypeptide variant that binds to IL-18R and exhibits significantly reduced binding to IL-18BP has an IL-18BP/IL-18R dissociation constant ratio that is at least 10-fold higher than the ratio of dissociation constants of IL-18BP/IL-18R wild-type IL-18. In some embodiments, a polypeptide variant that binds to IL-18R and exhibits significantly reduced binding to IL-18BP has an IL-18BP/IL-18R dissociation constant ratio that is at least 100-fold higher, than the ratio of dissociation constants of IL-18BP/IL-18R wild-type IL-18. In some embodiments, a polypeptide variant that binds to IL-18R and exhibits significantly reduced binding to IL-18BP has an IL-18BP/IL-18R dissociation constant ratio that is at least 1000-fold higher than than the ratio of dissociation constants of IL-18BP/IL-18R wild-type IL-18. In some embodiments, a polypeptide variant that binds to IL-18R and exhibits significantly reduced binding to IL-18BP has an IL-18BP/IL-18R dissociation constant ratio that is at least 10,000 times higher than than the ratio of dissociation constants of IL-18BP/IL-18R wild-type IL-18. In some embodiments, a polypeptide variant that binds to IL-18R and exhibits significantly reduced binding to IL-18BP has an IL-18BP/IL-18R dissociation constant ratio that is at least about 100,000 times than the ratio of dissociation constants of IL-18BP/IL-18R wild-type IL-18. In some embodiments, a polypeptide variant that binds to IL-18R and exhibits significantly reduced binding to IL-18BP has an IL-18BP/IL-18R dissociation constant ratio that is at least about 1,000,000 times higher than than the ratio of dissociation constants of IL-18BP/IL-18R wild-type IL-18. In some embodiments, a polypeptide variant that binds to IL-18R and exhibits significantly reduced binding to IL-18BP has an IL-18BP/IL-18R dissociation constant ratio that is at least about 10,000,000 times higher than than the ratio of dissociation constants of IL-18BP/IL-18R wild-type IL-18. In some embodiments, a polypeptide variant that binds to IL-18R and exhibits significantly reduced binding to IL-18BP has an IL-18BP/IL-18R dissociation constant ratio that is at least 100,000,000 times higher than than the ratio of dissociation constants of IL-18BP/IL-18R wild-type IL-18.

In some embodiments, a proposed IL-18 polypeptide variant (DR-IL-18) that binds to IL-18R and exhibits significantly reduced binding to IL-18BP has an inhibition constant (Ki) value for IL-18BP that is greater than 3 nM (eg, 5 nM or more, 10 nM or more, 50 nM or more, 100 nM or more, 500 nM or more, 750 nM or more, or 1 μM or more). In some embodiments, the proposed DR variant of the IL-18 polypeptide has a Ki value for IL-18BP that is 500 nM or more. In some embodiments, the proposed DR variant of the IL-18 polypeptide has a Ki value for IL-18BP that is 1 μM or more.

In some embodiments, a proposed variant IL-18 polypeptide (DR-IL-18) that binds to IL-18R and exhibits significantly reduced binding to IL-18BP has a Ki value for IL-18BP that is greater than 200 nM (eg, 500 nM or more, 750 nM or more, or 1 μM or more). In some embodiments, the proposed DR variant of the IL-18 polypeptide has a Ki value for IL-18BP that is 1 μM or more.

In some embodiments, a proposed IL-18 polypeptide variant (DR-IL-18) that binds to IL-18R and exhibits significantly reduced binding to IL-18BP has an inhibition constant (Ki) value for IL-18BP that is , at least 2-fold higher than the Ki value of wild-type IL-18 for IL-18BP (i.e., the Ki value of the proposed variant IL-18 polypeptide for IL-18BP is at least 2-fold higher than the Ki value of WT IL-18 to IL-18BP). For example, in some instances, a proposed DR variant of an IL-18 polypeptide has a Ki value for IL-18BP that is at least 5-fold higher (e.g., at least 10-fold higher, at least 50 times higher, at least 100 times higher, at least 200 times higher, at least 500 times higher, or at least 1000 times higher) than the Ki value of wild-type IL-18 for IL-18BP.

In some embodiments, a proposed variant IL-18 polypeptide (DR-IL-18) that binds to IL-18R and exhibits significantly reduced binding to IL-18BP has an EC ₅₀ value for IL-18BP that is at least at least 2 times the EC ₅₀ value of wild-type IL-18 for IL-18BP (i.e., the EC ₅₀ value of the proposed variant IL-18 polypeptide for IL-18BP is at least 2 times the EC ₅₀ value of WT IL- 18 for IL-18BP). For example, in some instances, a proposed DR variant of an IL-18 polypeptide has an EC ₅₀ value for IL-18BP that is at least 5-fold higher (e.g., at least 10-fold, at least 50 times, at least 100 times, at least 200 times, at least 500 times, or at least 1000 times higher) IL-18 wild-type EC ₅₀ values for IL-18BP.

In various embodiments, the variant IL-18 polypeptide comprises a mutation relative to the wild-type (WT) IL-18 polypeptide. In some embodiments, the WT IL-18 polypeptide comprises the amino acid sequence of SEQ ID NO: 30. In other embodiments, the WT IL-18 polypeptide comprises the amino acid sequence of SEQ ID NO: 31. Unless otherwise indicated, the symbol "X" is used hereinafter to represent any amino acid.

In various embodiments, the human IL-18 polypeptide variant or fragment comprises at least one mutation (e.g., at least 2, at least 3, at least 4, at least 5, or , at least 6 mutations) selected from the group consisting of Y1X, L5X, K8X, M51X, K53X, S55X, Q56X, P57X, G59X, M60X, E77X, Q103X, S105X, D110X, N111X, M113X, V153X and N155X where X is any amino acid. In various embodiments, the human IL-18 polypeptide variant or fragment thereof comprises at least 4 mutations selected from the group consisting of Y1X, L5X, K8X, M51X, K53X, S55X, Q56X, P57X, G59X, M60X, E77X , Q103X, S105X, D110X, N111X, M113X, V153X and N155X. In various embodiments, the human IL-18 polypeptide variant or fragment thereof comprises at least 6 mutations selected from the group consisting of Y1X, L5X, K8X, M51X, K53X, S55X, Q56X, P57X, G59X, M60X, E77X , Q103X, S105X, D110X, N111X, M113X, V153X and N155X. In various embodiments, the human IL-18 polypeptide variant or fragment comprises at least one mutation (e.g., at least 2, at least 3, at least 4, at least 5, or , at least 6 mutations) selected from the group consisting of Y1X, L5X, K8X, S55X, Q56X, P57X, G59X, E77X, Q103X, S105X, D110X, N111X, M113X, V153X, and N155X a human IL-18 polypeptide, or fragment thereof, has at least one mutation (e.g., at least 2, at least 3, at least 4, at least 5, or at least 6 mutations) selected from the group consisting of Y1H, Y1R, L5H, L5I, L5Y, K8Q, K8R, M51T, M51K, M51D, M51N, M51E, M51R, K53R, K53G, K53S, K53T, S55K, S55R, Q56E, Q56A . D, 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 human IL-18 polypeptide variant comprises at least one IL-18 polypeptide variant or fragment thereof selected from the group consisting of hCS1 (SEQ ID NO: 34), hCS2 (SEQ ID NO: 35) , hCS3 (SEQ ID NO: 36), hCS4 (SEQ ID NO: 37), hC4 (SEQ ID NO: 38), hA8 (SEQ ID NO: 39), hD6 (SEQ ID NO: 40), hH12 (SEQ ID NO: 41), hB11 (SEQ ID NO: 42), hC3 (SEQ ID NO: 43), hC2 (SEQ ID NO: 44), hG10 (SEQ ID NO: 45), hG1 (SEQ ID NO: 46), hF1 (SEQ ID NO: 47), hD2 (SEQ ID NO: 48), hA1 (SEQ ID NO: 49), hB3 (SEQ ID NO: 50), hB4 (SEQ ID NO: 51), hH3 (SEQ ID NO: : 52), hH5 (SEQ ID NO: 53), hH4 (SEQ ID NO: 54), hE1 (SEQ ID NO: 55), hG2 (SEQ ID NO: 56), hB9 (SEQ ID NO: 57), hE12 (SEQ ID NO: 58), hC5 (SEQ ID NO: 59), 5-18 (SEQ ID NO: 73), 5-29 (SEQ ID NO: 74), 5-8 (SEQ ID NO: 75), 5-6 (SEQ ID NO: 76), 5-27 (SEQ ID NO: 77), 5-20 (SEQ ID NO: 78), 5-2 (SEQ ID NO: 79), 5-9 (SEQ ID NO: 80), 5-42 (SEQ ID NO: 81), 5-13 (SEQ ID NO: 82), 5-12 (SEQ ID NO: 83), 5-1 (SEQ ID NO: 84), 5 -33 (SEQ ID NO: 85), 5-21 (SEQ ID NO: 86), 6-31 (SEQ ID NO: 87), 6-20 (SEQ ID NO: 88), 6-12 (SEQ ID NO: : 89), 6-27 (SEQ ID NO: 90), 6-29 (SEQ ID NO: 91), 5-26 (SEQ ID NO: 191), 5-17 (SEQ ID NO: 192), 5- 41 (SEQ ID NO: 193) or a fragment thereof.

In some instances, the proposed DR-IL-18 variant or fragment thereof comprises at least one mutation (e.g., at least 2, at least 3, or at least 4 mutations) selected from the group , consisting of M51X, M60X, S105X, D110X and N111X, relative to SEQ ID NO: 30. In some cases, the proposed DR-IL-18 variant or fragment thereof includes at least 3 mutations selected from the group consisting of M51X , M60X, S105X, D110X, and N111X, relative to SEQ ID NO: 30. , 3, or at least 4 mutations) selected from the group consisting of M51X ₁ , M60X ₂ , S105X ₃ , D110X ₄ and N111X ₅ , relative to SEQ ID NO: 30, where X ₁ is T, K, D, E, R or N; X ₂ is K, Q, L or R; X ₃ represents R, D, K, A or N; X ₄ is H, K, N, Q, E, N, S, or G; and X ₅ is H, D, Y, R, S, or G. at least 3 or at least 4 mutations) selected from the group consisting of M51X ₁ , M60X ₂ , S105X ₃ , D110X ₄ and N111X ₅ relative to SEQ ID NO: 30, where X ₁ is T, K , D, E, R or N; X ₂ is K, Q, L or R; X ₃ represents R, D, K, A or N; X ₄ is H, K, N, Q, E, N, S, or G; and X ₅ is H, D, Y, R, S, or G. at least 3 or at least 4 mutations) selected from the group consisting of M51X ₁ , M60X ₂ , S105X ₃ , D110X ₄ and N111X ₅ relative to SEQ ID NO: 30, where X ₁ is T or K; X ₂ is K or L; X ₃ is D, N or A; X ₄ is K, N, S or G; and X ₅ is H, Y, G, or R.

In some instances, the proposed DR-IL-18 variant, or fragment thereof, includes mutations M51X, M60X, S105X, D110X, and N111X, relative to SEQ ID NO: 30. For example, in some instances, the proposed DR-IL-18 variant, or fragment thereof, includes M51X mutations₁, M60X₂, S105X₃, D110X₄ and N111X₅, relative to SEQ ID NO: 30, where X₁ represents T, K, D, E, R or N; X₂is K, Q, L or R; X₃is R, D, K, A or N; X₄is H, K, N, Q, E, N, S or G; and X₅is H, D, Y, R, S, or G. In some instances, the proposed DR-IL-18 variant or fragment includes M51X mutations₁, M60X₂, S105X₃, D110X₄ and N111X₅, relative to SEQ ID NO: 30, where X₁is T or K; X₂is K or L; X₃is D, N or A; X₄is K, N, S or G; and X₅is H, Y, G, or R. In other words, in some cases, the proposed DR-IL-18 variant or fragment thereof includes {M51T or M51K} mutations; {M60K or M60L}; {S105D, S105N, S105A}; {D110K, D110N, D110S or D110G}; and {N111H, N111Y, N111R or N111G}, relative to SEQ ID NO: 30.

In some instances, the proposed DR-IL-18 variant or fragment thereof comprises at least one mutation (e.g., at least 2, at least 3, or at least 4 mutations) selected from the group, consisting of M51X, K53X, Q56X, S105X and N111X, relative to SEQ ID NO: 30. In some cases, the proposed DR-IL-18 variant or fragment thereof includes at least 3 mutations selected from the group consisting of M51X, K53X, Q56X, S105X and N111X, relative to SEQ ID NO: 30. In some cases, the proposed DR-IL-18 variant or fragment includes at least one mutation (for example, at least 2, at least 3 or at least 4 mutations) selected from the group consisting of M51X ₁ , K53X ₂ , Q56X ₃ , S105X ₄ and N111X ₅ relative to SEQ ID NO: 30, where X ₁ is E, R or K; X ₂ is G, S or T; X ₃ represents E, A, R, V, G, K or L; X ₄ is N, S, K or G; and X ₅ is R, S, G or D. In some instances, the proposed DR-IL-18 variant or fragment thereof includes at least 3 mutations selected from the group consisting of M51X ₁ , K53X ₂ , Q56X ₃ , S105X ₄ and N111X ₅ relative to SEQ ID NO: 30, where X ₁ is E, R or K; X ₂ is G, S or T; X ₃ represents E, A, R, V, G, K or L; X ₄ is N, S, K or G; and X ₅ is R, S, G, or D. In some instances, the proposed DR-IL-18 variant, or fragment thereof, includes at least one mutation (e.g., at least 2, at least 3, or , at least 4 mutations) selected from the group consisting of M51X ₁ , K53X ₂ , Q56X ₃ , S105X ₄ and N111X ₅ relative to SEQ ID NO: 30, where X ₁ is K; X ₂ is G or S; X ₃ is G, R or L; X ₄ is S, N or G; and X ₅ is G or R.

In some instances, the proposed DR-IL-18 variant, or fragment thereof, includes mutations M51X, K53X, Q56X, S105X, and N111X, relative to SEQ ID NO: 30. For example, in some instances, the proposed DR-IL-18 variant, or fragment thereof, includes mutations M51X ₁ , K53X ₂ , Q56X ₃ , S105X ₄ and N111X ₅ , relative to SEQ ID NO: 30, where X ₁ is E, R or K; X ₂ is G, S or T; X ₃ represents E, A, R, V, G, K or L; X ₄ is N, S, K or G; and X ₅ is R, S, G, or D. In some instances, the proposed DR-IL-18 variant or fragment thereof includes mutations M51X ₁ , K53X ₂ , Q56X ₃ , S105X ₄ and N111X ₅ , relative to SEQ ID NO: 30 , where X ₁ represents K; X ₂ is G or S; X ₃ is G, R or L; X ₄ is S, N or G; and X ₅ is G or R. In other words, in some cases, the proposed DR-IL-18 variant or fragment thereof includes {M51K} mutations; {K53G or K53S}; {Q56G, Q56R or Q56L}; {D110S, D110N or D110G}; and {N111R or N111G}, relative to SEQ ID NO: 30.

In some instances, a proposed DR-IL-18 variant or fragment thereof comprises an amino acid sequence having an identity of 85% or more (e.g., 90% or more, 92% or more, 93% or more, 94% or more, 95% or greater than, 96% or greater, 97% or greater, 98% or greater, or 99% or greater) with the wild-type human IL-18 amino acid sequence shown as SEQ ID NO: 30. Accordingly, in some cases, the proposed variant DR -IL-18 or fragment thereof includes an amino acid. A sequence that (i) has an identity of 85% or more (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) with the wild-type human IL-18 amino acid sequence shown as SEQ ID NO: 30; and (ii) includes at least one mutation (e.g., at least 2, at least 3, at least 4, at least 5, or at least 6 mutations) relative to the wild type IL-18 (eg human IL-18).

In some instances, a proposed DR-IL-18 variant or fragment thereof comprises an amino acid sequence having an identity of 85% or more (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) with the amino acid sequence represented by any one of SEQ ID NOs: 34-59, 73-91 and 191-193. Accordingly, in some instances, a proposed DR-IL-18 variant or fragment thereof comprises an amino acid sequence that (i) has an identity of 85% or more (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) with the amino acid sequence represented by any one of SEQ ID NOs: 34-59, 73-91 and 191 -193; and (ii) includes at least one mutation (e.g., at least 2, at least 3, at least 4, at least 5, or at least 6 mutations) relative to the wild type IL-18 (eg human IL-18).

In some instances, a proposed DR-IL-18 variant or fragment thereof includes an amino acid sequence that (i) has an identity of 85% or more (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) with the wild type human IL-18 amino acid sequence shown as SEQ ID NO: 30; and (ii) includes at least one mutation (e.g., at least 2, at least 3, at least 4, at least 5, or at least 6 mutations) selected from group consisting of Y1X, L5X, K8X, M51X, K53X, S55X, Q56X, P57X, G59X, M60X, E77X, Q103X, S105X, D110X, N111X, M113X, V153X and N155X, with respect to SEQ ID NO: 30. In some cases , a proposed DR-IL-18 variant or fragment thereof comprises an amino acid sequence that (i) has an identity of 85% or more (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) with the wild type human IL-18 amino acid sequence shown as SEQ ID NO: 30; and (ii) includes at least 4 mutations selected from the group consisting of Y1X, L5X, K8X, M51X, K53X, S55X, Q56X, P57X, G59X, M60X, E77X, Q103X, S105X, D110X, N111X, M113X , V153X, and N155X, relative to SEQ ID NO: 30. In some instances, a proposed DR-IL-18 variant, or fragment thereof, includes an amino acid sequence that (i) has an identity of 85% or more (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) with the human IL-18 wild-type amino acid sequence represented by as SEQ ID NO: 30; and (ii) includes at least 6 mutations selected from the group consisting of Y1X, L5X, K8X, M51X, K53X, S55X, Q56X, P57X, G59X, M60X, E77X, Q103X, S105X, D110X, N111X, M113X , V153X, and N155X, relative to SEQ ID NO: 30. In some instances, a proposed DR-IL-18 variant, or fragment thereof, includes an amino acid sequence that (i) has an identity of 85% or more (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) with the human IL-18 wild-type amino acid sequence represented by as SEQ ID NO: 30; and (ii) includes at least one mutation (e.g., at least 2, at least 3, at least 4, at least 5, or at least 6 mutations) selected from group consisting of Y1X, L5X, K8X, S55X, Q56X, P57X, G59X, E77X, Q103X, S105X, D110X, N111X, M113X, V153X and N155X, with respect to SEQ ID NO: 30.

In some instances, a proposed DR-IL-18 variant or fragment thereof includes an amino acid sequence that (i) has an identity of 85% or more (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) with the wild type human IL-18 amino acid sequence shown as SEQ ID NO: 30; and (ii) includes at least one mutation (e.g., at least 2, at least 3, or at least 4 mutations) selected from the group consisting of M51X, M60X, S105X, D110X, and N111X, relative to SEQ ID NO: 30. In some instances, a proposed DR-IL-18 variant or fragment thereof includes an amino acid sequence that (i) has an identity of 85% or more (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) with the wild type human IL-18 amino acid sequence shown as SEQ ID NO: 30; and (ii) includes at least 3 mutations selected from the group consisting of M51X, M60X, S105X, D110X and N111X relative to SEQ ID NO: 30. In some cases, the proposed DR-IL-18 variant or fragment thereof includes an amino acid sequence that (i) has an identity of 85% or more (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) with the wild-type amino acid sequence of human IL-18 shown as SEQ ID NO: 30; and (ii) includes at least one mutation (e.g., at least 2, at least 3, or at least 4 mutations) selected from the group consisting of M51X ₁ , M60X ₂ , S105X ₃ , D110X ₄ and N111X ₅ relative to SEQ ID NO: 30, where X ₁ is T, K, D, E, R or N; X ₂ is K, Q, L or R; X ₃ represents R, D, K, A or N; X ₄ is H, K, N, Q, E, N, S, or G; and X ₅ is H, D, Y, R, S, or G. In some cases, the proposed DR-IL-18 variant or fragment includes an amino acid sequence that (i) has an identity of 85% or more (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) with the wild type IL amino acid sequence -18 human shown as SEQ ID NO: 30; and (ii) includes at least 3 mutations selected from the group consisting of M51X ₁ , M60X ₂ , S105X ₃ , D110X ₄ and N111X ₅ relative to SEQ ID NO: 30, where X ₁ is T, K, D, E, R or N; X ₂ is K, Q, L or R; X ₃ represents R, D, K, A or N; X ₄ is H, K, N, Q, E, N, S, or G; and X ₅ is H, D, Y, R, S, or G. In some cases, the proposed DR-IL-18 variant or fragment includes an amino acid sequence that (i) has an identity of 85% or more (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) with the wild type IL amino acid sequence -18 human shown as SEQ ID NO: 30; and (ii) includes at least one mutation (e.g., at least 2, at least 3, or at least 4 mutations) selected from the group consisting of M51X ₁ , M60X ₂ , S105X ₃ , D110X ₄ and N111X ₅ relative to SEQ ID NO: 30, where X ₁ is T or K; X ₂ is K or L; X ₃ is D, N or A; X ₄ is K, N, S or G; and X ₅ is H, Y, G, or R.

In some instances, a proposed DR-IL-18 variant or fragment thereof includes an amino acid sequence that (i) has an identity of 85% or more (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) with the wild type human IL-18 amino acid sequence shown as SEQ ID NO: 30; and (ii) includes mutations M51X, M60X, S105X, D110X, and N111X, relative to SEQ ID NO: 30. In some instances, the proposed DR-IL-18 variant, or fragment thereof, includes an amino acid sequence that (i) has 85% identity, or more than (for example, 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) with the wild-type amino acid sequence of human IL-18 shown as SEQ ID NO: 30; and (ii) includes mutations M51X ₁ , M60X ₂ , S105X ₃ , D110X ₄ and N111X ₅ relative to SEQ ID NO: 30, where X ₁ is T, K, D, E, R, or N; X ₂ is K, Q, L or R; X ₃ represents R, D, K, A or N; X ₄ is H, K, N, Q, E, N, S, or G; and X ₅ is H, D, Y, R, S, or G. In some cases, the proposed DR-IL-18 variant or fragment includes an amino acid sequence that (i) has an identity of 85% or more (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) with the wild type IL amino acid sequence -18 human shown as SEQ ID NO: 30; and (ii) includes mutations M51X ₁ , M60X ₂ , S105X ₃ , D110X ₄ and N111X ₅ relative to SEQ ID NO: 30, where X ₁ is T or K; X ₂ is K or L; X ₃ is D, N or A; X ₄ is K, N, S or G; and X ₅ is H, Y, G, or R.

In some instances, a proposed DR-IL-18 variant or fragment thereof includes an amino acid sequence that (i) has an identity of 85% or more (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) with the wild type human IL-18 amino acid sequence shown as SEQ ID NO: 30; and (ii) includes at least one mutation (e.g., at least 2, at least 3, or at least 4 mutations) selected from the group consisting of M51X, K53X, Q56X, S105X, and N111X, relative to SEQ ID NO: 30. In some instances, a proposed DR-IL-18 variant or fragment thereof includes an amino acid sequence that (i) has an identity of 85% or more (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) with the wild type human IL-18 amino acid sequence shown as SEQ ID NO: 30; and (ii) includes at least 3 mutations selected from the group consisting of M51X, K53X, Q56X, S105X and N111X relative to SEQ ID NO: 30. In some cases, the proposed DR-IL-18 variant or fragment thereof includes an amino acid sequence that (i) has an identity of 85% or more (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) with the wild-type amino acid sequence of human IL-18 shown as SEQ ID NO: 30; and (ii) includes at least one mutation (e.g., at least 2, at least 3, or at least 4 mutations) selected from the group consisting of M51X ₁ , K53X ₂ , Q56X ₃ , S105X ₄ and N111X ₅ relative to SEQ ID NO: 30, where X ₁ is E, R or K; X ₂ is G, S or T; X ₃ represents E, A, R, V, G, K or L; X ₄ is N, S, K or G; and X ₅ is R, S, G, or D. In some cases, the proposed DR-IL-18 variant or fragment includes an amino acid sequence that (i) has an identity of 85% or more (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) with the wild type human IL-18 amino acid sequence, represented as SEQ ID NO: 30; and (ii) includes at least 3 mutations selected from the group consisting of M51X ₁ , K53X ₂ , Q56X ₃ , S105X ₄ and N111X ₅ relative to SEQ ID NO: 30, where X ₁ is E, R or K; X ₂ is G, S or T; X ₃ represents E, A, R, V, G, K or L; X ₄ is N, S, K or G; and X ₅ is R, S, G, or D. In some cases, the proposed DR-IL-18 variant or fragment includes an amino acid sequence that (i) has an identity of 85% or more (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) with the wild type human IL-18 amino acid sequence, represented as SEQ ID NO: 30; and (ii) includes at least one mutation (e.g., at least 2, at least 3, or at least 4 mutations) selected from the group consisting of M51X ₁ , K53X ₂ , Q56X ₃ , S105X ₄ and N111X ₅ relative to SEQ ID NO: 30, where X ₁ is K; X ₂ is G or S; X ₃ is G, R or L; X ₄ is S, N or G; and X ₅ is G or R.

In some instances, a proposed DR-IL-18 variant or fragment thereof includes an amino acid sequence that (i) has an identity of 85% or more (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) with the wild type human IL-18 amino acid sequence shown as SEQ ID NO: 30; and (ii) includes mutations M51X, K53X, Q56X, S105X and N111X, relative to SEQ ID NO: 30. In some cases, the proposed DR-IL-18 variant or fragment thereof includes an amino acid sequence that (i) has 85% identity or more than (for example, 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) with the wild-type amino acid sequence of human IL-18 shown as SEQ ID NO: 30; and (ii) includes mutations M51X ₁ , K53X ₂ , Q56X ₃ , S105X ₄ and N111X ₅ relative to SEQ ID NO: 30, where X ₁ is E, R or K; X ₂ is G, S or T; X ₃ represents E, A, R, V, G, K or L; X ₄ is N, S, K or G; and X ₅ is R, S, G, or D. In some cases, the proposed DR-IL-18 variant or fragment includes an amino acid sequence that (i) has an identity of 85% or more (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) with the wild type human IL-18 amino acid sequence, represented as SEQ ID NO: 30; and (ii) includes mutations M51X ₁ , K53X ₂ , Q56X ₃ , S105X ₄ and N111X ₅ relative to SEQ ID NO: 30, where X ₁ is K; X ₂ is G or S; X ₃ is G, R or L; X ₄ is S, N or G; and X ₅ is G or R.

In some embodiments, the mouse IL-18 polypeptide variant includes at least one mutation (e.g., at least 2, at least 3, at least 4, at least 5, or at least , 6 mutations) selected from the group consisting of N1X, M50X, Y51X, K52X, S54X, E55X, V56X, R57X, G58X, L59X, R104X, N109X and L151X, where X is any amino acid. In some embodiments, the murine IL-18 polypeptide variant, or fragment thereof, comprises at least one mutation (e.g., at least 2, at least 3, at least 4, at least 5, or, at least 6 mutations) selected from the group consisting of N1H, N1Y, M50A, M50S, M50V, M50G, M50T, Y51R, K52V, K52S, K52T, K52G, K52A, S54R, S54K, S54G, S54N, E55R, E55H, E55N, E55D, E55G, V56L, V56M, V56R, V56A, V56S, V56Q, R57G, R57K, G58A, L59K, L59R, L59V, R104K, R104L, R104Q, R104S, N109D and L151V. In some embodiments, the murine IL-18 polypeptide variant includes at least one variant selected from the group consisting of 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), or a fragment thereof.

In some embodiments, the invention provides a nucleic acid (eg, DNA, cDNA, mRNA, and others) that encodes at least one IL-18 polypeptide variant.

Therapeutic compositions and methods for inhibition

In various embodiments, the present invention includes compositions and methods for inhibiting IL-18BP to treat or prevent a disease or disorder in which it is desirable to reduce the activity or level of IL-18BP. Indications for use for such a drug include the above indications developed for the DR-IL-18 variant. Non-limiting examples of diseases or disorders in which it is desirable to reduce the activity or level of IL-18BP and which can be treated or prevented by the compositions and methods of the invention include cancer, infectious disease, metabolic diseases and disorders, and macular degeneration. In various embodiments, compositions and methods for inhibiting IL-18BP to treat or prevent a disease or disorder of the invention reduce the amount of an IL-18BP polypeptide, the amount of IL-18BP mRNA, the amount of IL-18BP enzymatic activity, the amount of IL-18BP substrate binding activity, or their combinations.

As a result of reading the description of the present invention, it will become apparent to one of ordinary skill in the art that a decrease in the level of IL-18BP implies a decrease in the expression of IL-18BP, including transcription, translation, or both. Once familiar with the teachings of the present invention, it will also be apparent to one of ordinary skill in the art that a decrease in IL-18BP levels implies a decrease in IL-18BP activity (eg, enzymatic activity, substrate binding activity, etc.). Thus, a decrease in the level or activity of IL-18BP includes, but is not limited to, a decrease in transcription, translation, or both, of a nucleic acid encoding IL-18BP; and this also includes the reduction of any activity of the IL-18BP polypeptide. Compositions and methods for inhibiting IL-18BP of the invention may selectively inhibit IL-18BP or may inhibit both IL-18BP and another molecule.

Inhibition of IL-18BP can be assessed using a wide variety of methods, including methods that are described in the present invention, as well as methods known in the art or methods that will be developed in the future. Thus, after reading the description of the present invention, it should be obvious to a person of ordinary skill in the art that a decrease in the level or activity of IL-18BP can be easily assessed using methods that allow assessment of the level of nucleic acid encoding IL-18BP (for example, mRNA ), the level of the IL-18BP polypeptide present in the biological sample, the level of IL-18BP activity (eg, enzymatic activity, substrate binding activity, etc.), or combinations thereof.

Upon reading the description of the present invention, it should be apparent to one of ordinary skill in the art that the invention can be used to treat or prevent a disease or disorder in a subject in need thereof, whether or not the subject is being treated with another drug or therapy. . In addition, after becoming familiar with the teachings of the present invention, it should be apparent to one of ordinary skill in the art that a disease or disorder that can be treated with the compositions and methods described herein includes any disease or disorder in which a certain IL-18BP plays a role and in which a decrease in the level or decrease in the activity of IL-18BP contributes to a positive therapeutic result.

Compositions and methods for inhibiting IL-18BP of the invention that reduce the level or activity (e.g., enzymatic activity, ligand binding activity, etc.) of IL-18BP include, but are not limited to, chemical compound, polypeptide, peptide, peptidomimetic , an antibody, a ribozyme, a small molecular weight chemical compound, an antisense nucleic acid molecule (eg, siRNA, microRNA, etc.), or combinations thereof. Upon reading the description of the present invention, it should be apparent to one of ordinary skill in the art that a composition for inhibiting IL-18BP includes a chemical compound that reduces the level or activity of IL-18BP. In addition, the composition for inhibiting IL-18BP includes chemically modified compounds and derivatives, which are well known to any specialist in the field of chemistry.

Compositions and methods for inhibiting IL-18BP of the invention that reduce the level or activity (eg, enzymatic activity, ligand binding activity, etc.) of IL-18BP include antibodies. Antibodies of the invention include a wide variety of antibody forms, including, for example, polyclonal antibodies, monoclonal antibodies, intracellular antibodies ("intraantibodies"), Fv, Fab and F(ab)2, single chain antibodies (scFv), heavy chain antibodies ( such as camel antibodies), synthetic antibodies, chimeric antibodies and humanized antibodies. In some embodiments, the antibody of the invention is an antibody that specifically binds to IL-18BP.

In some embodiments, the IL-18BP inhibitor comprises an engineered variant of IL-18 that is designed to bind to IL-18BP but does not substantially bind or interact with IL-18R (eg, IL-18Rα and IL-18Rβ). This variant, "receptor decoy with receptor decoy" (D2D), serves to bind to IL-18BP, thereby preventing the ability of IL-18BP to inhibit endogenously produced or therapeutically administered IL-18. A decoy receptor with decoy receptor variant could be constructed using much the same selection strategy using yeast display technology as described elsewhere in the invention, but with positive selection for IL-18BP binding and negative selection against significant binding to IL- 18Ra.

In various embodiments, a variant IL-18 polypeptide that binds and inhibits IL-18BP comprises a mutation relative to the wild-type (WT) IL-18 polypeptide. In some embodiments, the WT IL-18 polypeptide comprises the amino acid sequence of SEQ ID NO: 30. In other embodiments, the WT IL-18 polypeptide comprises the amino acid sequence of SEQ ID NO: 31.

In various embodiments, the IL-18 polypeptide variant that binds and inhibits IL-18BP comprises a human IL-18 polypeptide variant or fragment thereof comprising at least one mutation (e.g., at least 2, at least , 3, at least 4, at least 5 or at least 6 mutations) selected from the group consisting of Y1X, L5X, D17X, E31X, T34X, D35X, S36X, D37X, D40X, N41X, M51X, Q56X, M60X, Q103X, H109X, M113X and R131X, where X is any amino acid. In some embodiments, the IL-18 polypeptide variant that binds and inhibits IL-18BP comprises a human IL-18 polypeptide variant or fragment thereof comprising at least one mutation (e.g., at least 2, at least , 3, at least 4, at least 5 or at least 6 mutations) selected from the group consisting of Y1D, Y1F, Y1H, Y1L, L5F, L5H, D17A, D17G, D17R, D17H, E31A, E31T, E31G, E31K, E31R, T34A, T34K T34E, D35S, D35A, D35Y, S36N, S36K, S36R, D37P, D37A, D37R, D37H, D37L, D37V, D40Y D40S, D40A, N41K, N41S , N41R, M51F, M51L, M51I, Q56H, M60L, M60F, M60I, Q103L, Q103I, H109A, H109P, H109D, M113L, M113I, M113F and R131S. In some embodiments, the IL-18 polypeptide variant that binds and inhibits IL-18BP comprises a human IL-18 polypeptide variant or fragment thereof selected from the group consisting of hD2D-5F12 (SEQ ID NO: 92), hD2D-5F11 (SEQ ID NO: 93), hD2D-5F10 (SEQ ID NO: 94), hD2D-5F08 (SEQ ID NO: 95), hD2D-5F06 (SEQ ID NO: 96), hD2D-5F04 (SEQ ID NO: 97 ), hD2D-5F02 (SEQ ID NO: 98), hD2D-5F01 (SEQ ID NO: 99), hD2D-5E10 (SEQ ID NO: 100), hD2D-5E08 (SEQ ID NO: 101), hD2D-5E03 ( SEQ ID NO: 102), hD2D-5E02 (SEQ ID NO: 103), hD2D-5D10 (SEQ ID NO: 104), hD2D-5D08 (SEQ ID NO: 105), hD2D-5D06 (SEQ ID NO: 106) , hD2D-5D05 (SEQ ID NO: 107), hD2D-5D03 (SEQ ID NO: 108), hD2D-5D02 (SEQ ID NO: 109), hD2D-5C10 (SEQ ID NO: 110), hD2D-5C09 (SEQ ID NO: 111), hD2D-5C08 (SEQ ID NO: 112), hD2D-5C05 (SEQ ID NO: 113), hD2D-5C04 (SEQ ID NO: 114), hD2D-5C03 (SEQ ID NO: 115), hD2D-5B11 (SEQ ID NO: 116), hD2D-5B10 (SEQ ID NO: 117), hD2D-5B06 (SEQ ID NO: 118), hD2D-5B05 (SEQ ID NO: 119), hD2D-5B02 (SEQ ID NO: 120), hD2D-5A09 (SEQ ID NO: 121), hD2D-5A02 (SEQ ID NO: 122), hD2D-CS1 (SEQ ID NO: 123), hD2D-CS2 (SEQ ID NO: 124), hD2D -CS3 (SEQ ID NO: 125), or a fragment thereof.

In some instances, the proposed D2D-IL-18 variant or fragment thereof comprises at least one mutation (e.g., at least 2, at least 3, or at least 4 mutations) selected from the group consisting of D17X, E30X and Q103X, relative to SEQ ID NO: 30. In some cases, the proposed D2D-IL-18 variant or fragment includes at least one mutation (for example, at least 2, at least 3 or at least 4 mutations) selected from the group consisting of D17X ₁ , E30X ₂ and Q103X ₃ relative to SEQ ID NO: 30, where X ₁ is G, H, R or A; X ₂ is A, T, G, K or R; and X ₃ is I or L. In some instances, the proposed D2D-IL-18 variant, or fragment thereof, includes at least one mutation (e.g., at least 2, at least 3, or at least , 4 mutations) selected from the group consisting of D17G, E30A and (Q103L or Q103I).

In some instances, the proposed D2D-IL-18 variant or fragment thereof includes D17X, E30X, and Q103X mutations, relative to SEQ ID NO: 30. For example, in some instances, the proposed D2D-IL-18 variant or fragment thereof includes D17X ₁ mutations. E30X ₂ and Q103X ₃ relative to SEQ ID NO: 30, where X ₁ is G, H, R or A; X ₂ is A, T, G, K or R; and X ₃ is I or L. In some instances, the proposed D2D-IL-18 variant or fragment thereof includes mutations D17G, E30A and (Q103L or Q103I).

In some instances, the proposed D2D-IL-18 variant or fragment thereof comprises at least one mutation (e.g., at least 2, at least 3, or at least 4 mutations) selected from the group consisting of D17X, E30X, D35X, M51X and Q103X, relative to SEQ ID NO: 30. In some cases, the proposed D2D-IL-18 variant or fragment thereof includes at least one mutation (for example, at least at least 3 or at least 4 mutations) selected from the group consisting of D17X ₁ , E30X ₂ , D35X ₃ , M51X ₄ and Q103X ₃ relative to SEQ ID NO: 30, where X ₁ is G, H, R or A; X ₂ is A, T, G, K or R; X ₃ is S, A or Y; X ₄ is F, I or L; and X ₅ is I or L. In some instances, the proposed D2D-IL-18 variant, or fragment thereof, includes at least one mutation (e.g., at least 2, at least 3, or at least , 4 mutations) selected from the group consisting of D17G, E30A, D35S, M51F and (Q103L or Q103I) relative to SEQ ID NO: 30.

In some instances, the proposed D2D-IL-18 variant, or fragment thereof, includes mutations D17X, E30X, D35X, M51X, and Q103X, relative to SEQ ID NO: 30. For example, in some instances, the proposed D2D-IL-18 variant, or fragment thereof, includes mutations D17X ₁ , E30X ₂ , D35X ₃ , M51X ₄ and Q103X ₃ relative to SEQ ID NO: 30, where X ₁ is G, H, R or A; X ₂ is A, T, G, K or R; X ₃ is S, A or Y; X ₄ is F, I or L; and X ₅ is I or L. In some instances, the proposed D2D-IL-18 variant or fragment thereof includes mutations D17G, E30A, D35S, M51F and (Q103L or Q103I) relative to SEQ ID NO: 30.

In some instances, the proposed D2D-IL-18 variant or fragment thereof comprises an amino acid sequence having an identity of 85% or more (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) with the amino acid sequence of the wild-type human IL-18 shown as SEQ ID NO: 30. Accordingly, in some cases, the proposed variant D2D -IL-18, or a fragment thereof, comprises an amino acid sequence that (i) has an identity of 85% or more (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) with the wild type human IL-18 amino acid sequence shown as SEQ ID NO: 30; and (ii) includes at least one mutation (e.g., at least 2, at least 3, at least 4, at least 5, or at least 6 mutations) relative to the wild type IL-18 (eg human IL-18).

In some instances, the proposed D2D-IL-18 variant or fragment thereof comprises an amino acid sequence having an identity of 85% or more (e.g., 90% or more, 92% or more, 93% or more, 94% or more, 95% or more than, 96% or more, 97% or more, 98% or more, or 99% or more) with the amino acid sequence represented by any one of SEQ ID NOs: 126-190. Accordingly, in some instances, a proposed D2D-IL-18 variant or fragment thereof comprises an amino acid sequence that (i) has an identity of 85% or more (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) with the amino acid sequence represented by any one of SEQ ID NOs: 126-190; and (ii) includes at least one mutation (e.g., at least 2, at least 3, at least 4, at least 5, or at least 6 mutations) relative to the wild type IL-18 (eg human IL-18).

In some instances, a proposed DR-IL-18 variant or fragment thereof includes an amino acid sequence that (i) has an identity of 85% or more (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) with the wild type human IL-18 amino acid sequence shown as SEQ ID NO: 30; and (ii) includes at least one mutation (e.g., at least 2, at least 3, at least 4, at least 5, or at least 6 mutations) selected from group consisting of Y1X, L5X, D17X, E31X, T34X, D35X, S36X, D37X, D40X, N41X, M51X, Q56X, M60X, Q103X, H109X, M113X and R131X.

In some instances, a proposed DR-IL-18 variant or fragment thereof includes an amino acid sequence that (i) has an identity of 85% or more (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) with the wild type human IL-18 amino acid sequence shown as SEQ ID NO: 30; and (ii) includes at least one mutation (e.g., at least 2, at least 3, at least 4, at least 5, or at least 6 mutations) selected from group consisting of D17X, E30X and Q103X, relative to SEQ ID NO: 30. In some cases, the proposed DR-IL-18 variant or fragment includes an amino acid sequence that (i) has an identity of 85% or more (for example, 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) with the wild type IL amino acid sequence -18 human shown as SEQ ID NO: 30; and (ii) includes at least one mutation (e.g., at least 2, at least 3, at least 4, at least 5, or at least 6 mutations) selected from the group consisting of D17X ₁ , E30X ₂ and Q103X ₃ relative to SEQ ID NO: 30, where X ₁ is G, H, R or A; X ₂ is A, T. In some instances, the proposed DR-IL-18 variant, or fragment thereof, includes an amino acid sequence that (i) has an identity of 85% or more (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) with the wild type human IL-18 amino acid sequence shown as SEQ ID NO : thirty; and (ii) includes at least one mutation (e.g., at least 2, at least 3, at least 4, at least 5, or at least 6 mutations) selected from group consisting of D17G, E30A and (Q103L or Q103I).

In some instances, a proposed DR-IL-18 variant or fragment thereof includes an amino acid sequence that (i) has an identity of 85% or more (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) with the wild type human IL-18 amino acid sequence shown as SEQ ID NO: 30; and (ii) includes mutations D17X, E30X, and Q103X, relative to SEQ ID NO: 30. In some instances, a proposed DR-IL-18 variant, or fragment thereof, includes an amino acid sequence that (i) has 85% or more 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) with the wild-type amino acid sequence human IL-18 type, shown as SEQ ID NO: 30; and (ii) includes mutations D17X ₁ , E30X ₂ and Q103X ₃ relative to SEQ ID NO: 30, where X ₁ is G, H, R or A; X ₂ is A, T. In some instances, the proposed DR-IL-18 variant, or fragment thereof, includes an amino acid sequence that (i) has an identity of 85% or more (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) with the wild type human IL-18 amino acid sequence shown as SEQ ID NO : thirty; and (ii) includes mutations D17G, E30A and (Q103L or Q103I).

In some instances, a proposed DR-IL-18 variant or fragment thereof includes an amino acid sequence that (i) has an identity of 85% or more (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) with the wild type human IL-18 amino acid sequence shown as SEQ ID NO: 30; and (ii) includes at least one mutation (e.g., at least 2, at least 3, at least 4, at least 5, or at least 6 mutations) selected from a group consisting of D17X, E30X, D35X, M51X and Q103X, relative to SEQ ID NO: 30. In some cases, the proposed DR-IL-18 variant or fragment thereof includes an amino acid sequence that (i) has an identity of 85% or more ( 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) with an amino acid sequence of wild-type human IL-18, presented as SEQ ID NO: 30; and (ii) includes at least one mutation (e.g., at least 2, at least 3, at least 4, at least 5, or at least 6 mutations) selected from a group consisting of D17X ₁ , E30X ₂ , D35X ₃ , M51X ₄ and Q103X ₃ relative to SEQ ID NO: 30, where X ₁ is G, H, R or A; X ₂ is A, T, G, K or R; X ₃ is S, A or Y; X ₄ is F, I or L; and X ₅ is I or L. In some instances, the proposed DR-IL-18 variant, or fragment thereof, comprises an amino acid sequence that (i) has an identity of 85% or more (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) with the wild type human IL-18 amino acid sequence shown as SEQ ID NO: 30; and (ii) includes at least one mutation (e.g., at least 2, at least 3, at least 4, at least 5, or at least 6 mutations) selected from the group consisting of D17G, E30A, D35S, M51F and (Q103L or Q103I) with respect to SEQ ID NO: 30.

In some instances, a proposed DR-IL-18 variant or fragment thereof includes an amino acid sequence that (i) has an identity of 85% or more (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) with the wild type human IL-18 amino acid sequence shown as SEQ ID NO: 30; and (ii) includes mutations D17X, E30X, D35X, M51X, and Q103X, relative to SEQ ID NO: 30. In some instances, the proposed DR-IL-18 variant, or fragment thereof, includes an amino acid sequence that (i) has 85% identity, or more than (for example, 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) with the wild-type amino acid sequence of human IL-18 shown as SEQ ID NO: 30; and (ii) includes mutations D17X ₁ , E30X ₂ , D35X ₃ , M51X ₄ and Q103X ₃ relative to SEQ ID NO: 30, where X ₁ is G, H, R or A; X ₂ is A, T, G, K or R; X ₃ is S, A or Y; X ₄ is F, I or L; and X ₅ is I or L. In some instances, the proposed DR-IL-18 variant, or fragment thereof, comprises an amino acid sequence that (i) has an identity of 85% or more (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) with the wild type human IL-18 amino acid sequence shown as SEQ ID NO: 30; and (ii) includes mutations D17G, E30A, D35S, M51F and (Q103L or Q103I), relative to SEQ ID NO: 30.

In some embodiments, a variant IL-18 polypeptide that binds and inhibits IL-18BP comprises a murine variant of an IL-18 polypeptide comprising at least one mutation (e.g., at least 2, at least 3, at least 4, at least 5 or at least 6 mutations) selected from the group consisting of N1X, L5X, D17X, E30X, T33X, D34X, I35X, D36X, M50X, Q102X, R104, H108X, N109X, M111X, D129X and D130X, where X is any amino acid. In some embodiments, the IL-18 polypeptide variant that binds and inhibits IL-18BP comprises a murine IL-18 polypeptide variant or fragment thereof comprising at least one mutation (e.g., at least 2, at least , 3, at least 4, at least 5 or at least 6 mutations) selected from the group consisting of N1Y, N1D, N1H, N1L, N1F, N1V, N1I, L5Y, L5H, D17Q, D17G, D17A, D17E, D17S, D17N, E30A, E30R, E30K, E30T, E30G, T33G, T33A, T33E, T33R, T33K, D34Y, D34S, D34A, I35T, I35K, I35R, D36V, D36A, D36G, D3 6H, D36P, D36R, D36L, M50F, M50L, Q102L, Q102I, R104E, R104A, R104P, R104G, R104Q, R104H, H108D, H108A, N109R, N109S, N109T, N109I, M111L, M111I, D 129A, D129F, D129V, D129Y, D129S, D130E, D130T, D130G, D130N, D130R, D130S, D130Q and D130H. In some embodiments, the IL-18 polypeptide variant that binds and inhibits IL-18BP comprises a murine IL-18 polypeptide variant or fragment thereof selected from the group consisting of mD2D-A5 (SEQ ID NO: 126), mD2D-A6 (SEQ ID NO: 127), mD2D-A7 (SEQ ID NO: 128), mD2D-A8 (SEQ ID NO: 129), mD2D-A9 (SEQ ID NO: 130), mD2D-A11 (SEQ ID NO: 131 ), mD2D-A12 (SEQ ID NO: 132), mD2D-B4 (SEQ ID NO: 133), mD2D-B7 (SEQ ID NO: 134), mD2D-B11 (SEQ ID NO: 135), mD2D-B12 ( SEQ ID NO: 136), mD2D-C1 (SEQ ID NO: 137), mD2D-C3 (SEQ ID NO: 138), mD2D-C5 (SEQ ID NO: 139), mD2D-C6 (SEQ ID NO: 140) , mD2D-C9 (SEQ ID NO: 141), mD2D-C10 (SEQ ID NO: 142), mD2D-C11 (SEQ ID NO: 143), mD2D-D1 (SEQ ID NO: 144), mD2D-D9 (SEQ ID NO: 145), mD2D-D12 (SEQ ID NO: 146), mD2D-E3 (SEQ ID NO: 147), mD2D-E4 (SEQ ID NO: 148), mD2D-E5 (SEQ ID NO: 149), mD2D-E7 (SEQ ID NO: 150), mD2D-E8 (SEQ ID NO: 151), mD2D-E9 (SEQ ID NO: 152), mD2D-E10 (SEQ ID NO: 153), mD2D-E11 (SEQ ID NO: 154), mD2D-E12 (SEQ ID NO: 155), mD2D-F3 (SEQ ID NO: 156), mD2D-F4 (SEQ ID NO: 157), mD2D-F5 (SEQ ID NO: 158), mD2D -F7 (SEQ ID NO: 159), mD2D-F8 (SEQ ID NO: 160), mD2D-F9 (SEQ ID NO: 161), mD2D-G1 (SEQ ID NO: 162), mD2D-G7 (SEQ ID NO: : 163), mD2D-G9 (SEQ ID NO: 164), mD2D-H7 (SEQ ID NO: 165), mD2D-E1 (SEQ ID NO: 166), mD2D-G8 (SEQ ID NO: 167), mD2D- H3 (SEQ ID NO: 168), mD2D-A10 (SEQ ID NO: 169), mD2D-H1 (SEQ ID NO: 170), mD2D-F12 (SEQ ID NO: 171), mD2D-G10 (SEQ ID NO: 172), mD2D-G12 (SEQ ID NO: 173), mD2D-E2 (SEQ ID NO: 174), mD2D-G11 (SEQ ID NO: 175), mD2D-C4 (SEQ ID NO: 176), mD2D-F11 (SEQ ID NO: 177), mD2D-C2 (SEQ ID NO: 178), mD2D-F10 (SEQ ID NO: 179), mD2D-A2 (SEQ ID NO: 180), mD2D-F6 (SEQ ID NO: 181 ), mD2D-A1 (SEQ ID NO: 182), mD2D-E6 (SEQ ID NO: 183), mD2D-D4 (SEQ ID NO: 184), mD2D-D6 (SEQ ID NO: 185), mD2D-A3 ( SEQ ID NO: 186), mD2D-A4 (SEQ ID NO: 187), mD2D-B10 (SEQ ID NO: 188), mD2D-B8 (SEQ ID NO: 189), mD2D-B9 (SEQ ID NO: 190) , or a fragment of it.

In some embodiments, the invention provides a nucleic acid (eg, DNA, cDNA, mRNA, etc.) that encodes at least one IL-18 polypeptide variant.

In some embodiments, the IL-18BP inhibitor is an IL-18 polypeptide variant that binds to IL-18BP and exhibits significantly reduced binding to IL-18R. In some embodiments, an IL-18BP inhibitor that binds to IL-18BP and exhibits significantly reduced binding to IL-18R binds to IL-18R with a binding affinity that is from about 0.000000000001% to about 95% of binding affinity of wild-type IL-18 to IL-18R.

In some embodiments, an IL-18 polypeptide variant that binds to IL-18BP and exhibits significantly reduced binding to IL-18R binds to IL-18R with a binding affinity that is approximately 95% of the binding affinity of wild-type IL-18 type with IL-18R. In some embodiments, a variant IL-18 polypeptide that binds to IL-18BP and exhibits significantly reduced binding to IL-18R binds to IL-18R with a binding affinity that is approximately 90% of the binding affinity of wild-type IL-18 type with IL-18R. In some embodiments, a variant IL-18 polypeptide that binds to IL-18BP and exhibits significantly reduced binding to IL-18R binds to IL-18R with a binding affinity that is approximately 85% of the binding affinity of wild-type IL-18 type with IL-18R. In some embodiments, a variant IL-18 polypeptide that binds to IL-18BP and exhibits significantly reduced binding to IL-18R binds to IL-18R with a binding affinity that is approximately 80% of the binding affinity of wild-type IL-18 type with IL-18R. In some embodiments, a variant IL-18 polypeptide that binds to IL-18BP and exhibits significantly reduced binding to IL-18R binds to IL-18R with a binding affinity that is approximately 75% of the binding affinity of wild-type IL-18 type with IL-18R. In some embodiments, a variant IL-18 polypeptide that binds to IL-18BP and exhibits significantly reduced binding to IL-18R binds to IL-18R with a binding affinity that is approximately 70% of the binding affinity of wild-type IL-18 type with IL-18R. In some embodiments, an IL-18 polypeptide variant that binds to IL-18BP and exhibits significantly reduced binding to IL-18R binds to IL-18R with a binding affinity that is approximately 65% of the binding affinity of wild-type IL-18 type with IL-18R. In some embodiments, an IL-18 polypeptide variant that binds to IL-18BP and exhibits significantly reduced binding to IL-18R binds to IL-18R with a binding affinity that is approximately 60% of the binding affinity of wild-type IL-18 type with IL-18R. In some embodiments, an IL-18 polypeptide variant that binds to IL-18BP and exhibits significantly reduced binding to IL-18R binds to IL-18R with a binding affinity that is approximately 55% of the binding affinity of wild-type IL-18 type with IL-18R. In some embodiments, a variant IL-18 polypeptide that binds to IL-18BP and exhibits significantly reduced binding to IL-18R binds to IL-18R with a binding affinity that is approximately 50% of the binding affinity of wild-type IL-18 type with IL-18R. In some embodiments, an IL-18 polypeptide variant that binds to IL-18BP and exhibits significantly reduced binding to IL-18R binds to IL-18R with a binding affinity that is approximately 45% of the binding affinity of wild-type IL-18 type with IL-18R. In some embodiments, a variant IL-18 polypeptide that binds to IL-18BP and exhibits significantly reduced binding to IL-18R binds to IL-18R with a binding affinity that is approximately 40% of the binding affinity of wild-type IL-18 type with IL-18R. In some embodiments, a variant IL-18 polypeptide that binds to IL-18BP and exhibits significantly reduced binding to IL-18R binds to IL-18R with a binding affinity that is approximately 35% of the binding affinity of wild-type IL-18 type with IL-18R. In some embodiments, an IL-18 polypeptide variant that binds to IL-18BP and exhibits significantly reduced binding to IL-18R binds to IL-18R with a binding affinity that is approximately 30% of the binding affinity of wild-type IL-18 type with IL-18R. In some embodiments, an IL-18 polypeptide variant that binds to IL-18BP and exhibits significantly reduced binding to IL-18R binds to IL-18R with a binding affinity that is approximately 25% of the binding affinity of wild-type IL-18 type with IL-18R. In some embodiments, a variant IL-18 polypeptide that binds to IL-18BP and exhibits significantly reduced binding to IL-18R binds to IL-18R with a binding affinity that is approximately 20% of the binding affinity of wild-type IL-18 type with IL-18R. In some embodiments, a variant IL-18 polypeptide that binds to IL-18BP and exhibits significantly reduced binding to IL-18R binds to IL-18R with a binding affinity that is approximately 15% of the binding affinity of wild-type IL-18 type with IL-18R. In some embodiments, a variant IL-18 polypeptide that binds to IL-18BP and exhibits significantly reduced binding to IL-18R binds to IL-18R with a binding affinity that is approximately 10% of the binding affinity of wild-type IL-18 type with IL-18R.

In some embodiments, a variant IL-18 polypeptide that binds to IL-18BP and exhibits significantly reduced binding to IL-18R binds to IL-18R with a binding affinity that is approximately 5% of the binding affinity of wild-type IL-18 type with IL-18R. In some embodiments, a variant IL-18 polypeptide that binds to IL-18BP and exhibits significantly reduced binding to IL-18R binds to IL-18R with a binding affinity that is approximately 4% of the binding affinity of wild-type IL-18 type with IL-18R. In some embodiments, a variant IL-18 polypeptide that binds to IL-18BP and exhibits significantly reduced binding to IL-18R binds to IL-18R with a binding affinity that is approximately 3% of the binding affinity of wild-type IL-18 type with IL-18R. In some embodiments, an IL-18 polypeptide variant that binds to IL-18BP and exhibits significantly reduced binding to IL-18R binds to IL-18R with a binding affinity that is approximately 2% of the binding affinity of wild-type IL-18 type with IL-18R. In some embodiments, an IL-18 polypeptide variant that binds to IL-18BP and exhibits significantly reduced binding to IL-18R binds to IL-18R with a binding affinity that is approximately 1% of the binding affinity of wild-type IL-18 type with IL-18R.

In some embodiments, an IL-18 polypeptide variant that binds to IL-18BP and exhibits significantly reduced binding to IL-18R binds to IL-18R with a binding affinity that is approximately 0.1% of that of IL-18R. 18 wt with IL-18R. In some embodiments, an IL-18 polypeptide variant that binds to IL-18BP and exhibits significantly reduced binding to IL-18R binds to IL-18R with a binding affinity that is approximately 0.01% of the binding affinity of IL-18R. 18 wt with IL-18R. In some embodiments, a variant IL-18 polypeptide that binds to IL-18BP and exhibits significantly reduced binding to IL-18R binds to IL-18R with a binding affinity that is approximately 0.001% of the binding affinity of wild-type IL-18 type with IL-18R. In some embodiments, an IL-18 polypeptide variant that binds to IL-18BP and exhibits significantly reduced binding to IL-18R binds to IL-18R with a binding affinity that is approximately 0.0001% of the binding affinity of IL-18R. 18 wt with IL-18R. In some embodiments, an IL-18 polypeptide variant that binds to IL-18BP and exhibits significantly reduced binding to IL-18R binds to IL-18R with a binding affinity that is approximately 0.00001% of that of IL-18R. 18 wt with IL-18R. In some embodiments, an IL-18 polypeptide variant that binds to IL-18BP and exhibits significantly reduced binding to IL-18R binds to IL-18R with a binding affinity that is approximately 0.000001% of that of IL-18R. 18 wt with IL-18R. In some embodiments, an IL-18 polypeptide variant that binds to IL-18BP and exhibits significantly reduced binding to IL-18R binds to IL-18R with a binding affinity that is approximately 0.0000001% of that of IL-18R. 18 wt with IL-18R. In some embodiments, an IL-18 polypeptide variant that binds to IL-18BP and exhibits significantly reduced binding to IL-18R binds to IL-18R with a binding affinity that is approximately 0.00000001% of that of IL-18R. 18 wt with IL-18R. In some embodiments, an IL-18 polypeptide variant that binds to IL-18BP and exhibits significantly reduced binding to IL-18R binds to IL-18R with a binding affinity that is approximately 0.000000001% of that of IL-18R. 18 wt with IL-18R. In some embodiments, a variant IL-18 polypeptide that binds to IL-18BP and exhibits significantly reduced binding to IL-18R binds to IL-18R with a binding affinity that is approximately 0.0000000001% of the binding affinity of wild-type IL-18 type with IL-18R. In some embodiments, an IL-18 polypeptide variant that binds to IL-18BP and exhibits significantly reduced binding to IL-18R binds to IL-18R with a binding affinity that is approximately 0.00000000001% of that of IL-18R. 18 wt with IL-18R. In some embodiments, an IL-18 polypeptide variant that binds to IL-18BP and exhibits significantly reduced binding to IL-18R binds to IL-18R with a binding affinity that is approximately 0.000000000001% of that of IL-18R. 18 wt with IL-18R.

In addition, for a person skilled in the art, after familiarization with this invention and the examples of methods given in the invention, it should be obvious that the composition for inhibitors of IL-18BP includes such inhibitors that will be created in the future based on criteria well known in pharmacology, such as physiological results of inhibition of IL-18BP, as detailed in the invention and/or known in the art. Therefore, the present invention is in no way limited to any particular composition for inhibiting IL-18BP exemplified or described herein; moreover, the invention covers even those inhibitor compositions which are known to the ordinary person skilled in the art and which will be created in the future.

In addition, methods of identifying and preparing compositions of IL-18BP inhibitors are well known to those of ordinary skill in the art, including, but not limited to, obtaining the inhibitor from a natural source (e.g., Streptomyces sp., Pseudomonas sp., Stylotella aurantium, etc.) . Alternatively, the IL-18BP inhibitor can be chemically synthesized. In addition, based on the teachings of the invention, it should be apparent to those skilled in the art that the IL-18BP inhibitor composition may be derived from a recombinant organism. Compositions and methods for the chemical synthesis of IL-18BP inhibitors and their preparation from natural sources are well known in the art and described in the scientific literature.

It is apparent to one of ordinary skill in the art that the inhibitor may be administered in the form of a small molecule chemical, protein, antibody, nucleic acid construct encoding a protein, antisense nucleic acid, nucleic acid construct encoding an antisense nucleic acid, or combinations thereof. Numerous vectors and other compositions and methods are well known for introducing a protein or nucleic acid construct encoding a protein into cells or tissues. Therefore, the invention includes a method of introducing a protein or nucleic acid encoding a protein that is an inhibitor of IL-18BP. (Sambrook et al., 2012, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York; Ausubel et al., 1997, Current Protocols in Molecular Biology, John Wiley & Sons, New York).

It should be apparent to any person skilled in the art that reducing the amount or activity of a molecule that itself increases the amount or activity of IL-18BP can be used in the compositions and methods of the present invention to reduce the amount or activity of IL-18BP.

Antisense oligonucleotides are DNA or RNA molecules that are complementary to some portion of the RNA molecule. Once in cells, antisense oligonucleotides hybridize to an existing RNA molecule and inhibit translation in the gene product. Inhibition of gene expression using an antisense oligonucleotide is well known (Marcus-Sekura, 1988, Anal. Biochem. 172:289), as are methods of expressing an antisense oligonucleotide in a cell (Inoue, U.S. Pat. No. 5,190,931). The methods of the invention include the use of an antisense oligonucleotide to decrease the amount of IL-18BP, or to decrease the amount of molecules that cause an increase in the amount or activity of IL-18BP, resulting in a decrease in the amount or activity of IL-18BP.

The present invention provides antisense oligonucleotides that are synthesized and delivered into the cell using methods well known to those of ordinary skill in the art. For example, an antisense oligonucleotide can be synthesized having a length in the range of about 10 to about 100 nucleotides, more typically about 15 to about 50 nucleotides. The synthesis of nucleic acid molecules is well known for the synthesis of modified antisense oligonucleotides in order to increase biological activity compared to unmodified antisense oligonucleotides (Tullis, 1991, U.S. Pat. No. 5023243).

Similarly, gene expression can be inhibited by hybridization of the antisense molecule to a promoter or other regulatory element of the gene, due to the effect on gene transcription. Methods for identifying a promoter or other regulatory element that interacts with the corresponding gene are well known and include methods such as the yeast two hybrid system (Bartel and Fields, eds., In: The Yeast Two Hybrid System, Oxford University Press, Cary, N.C. ).

Alternatively, inhibition of a gene expressing IL-18BP or a gene expressing a protein that increases the level or activity of IL-18BP can be accomplished by using a ribozyme. The use of ribozymes to inhibit gene expression is well known to those skilled in the art (see, for example, Cech et al., 1992, J. Biol. Chem. 267:17479; Hampel et al., 1989, Biochemistry 28:4929; Altman et al. , U.S. Pat. No. 5,168,053). Ribozymes are catalytic RNA molecules that have the ability to cleave other single-stranded RNA molecules. Ribozymes are known to have sequence selectivity and therefore can be modified to recognize a specific nucleotide sequence (Cech, 1988, J. Amer. Med. Assn. 260:3030), allowing selective cleavage of specific mRNA molecules. Given the nucleotide sequence of the molecule, any person skilled in the art can synthesize an antisense oligonucleotide or ribozyme without undue experimentation, using the description of the present invention and the references included in the invention.

It will be appreciated by one of ordinary skill in the art that IL-18BP inhibitors may be administered short term (eg, for a short period of time such as a day, week, or month) or continuously (eg, over an extended period of time, such as several months or year or more). For any person skilled in the art it is obvious that inhibitors of IL-18BP can be administered alone or in any combination with other drugs. In addition, the IL-18BP inhibitors may be administered singly or in any combination as long as they may be administered simultaneously and/or before and/or after each other. It will be apparent to any person skilled in the art from the foregoing description of the invention that IL-18BP inhibitor compositions may be used to treat or prevent a disease or disorder in a subject in need thereof, and that the inhibitor composition may be used alone or in any combination with another drug in order to achieve a therapeutic result.

In various embodiments, any of the IL-18BP inhibitors of the invention described herein may be administered alone or in combination with other inhibitors of other molecules associated with the disease or disorder described in the invention or known in the art.

Upon reading the description of the present invention, including the methods described in detail, it should be apparent to any person skilled in the art that the invention is not limited to the treatment of a disease or disorder that has already been identified. In particular, the disease or disorder need not already be present to the detriment of the health of the subject; on the contrary, the disease or disorder need not be identified in the subject prior to treatment. That is, the disease or disorder must not be reliably identified before the application of the present invention can have a beneficial effect. Therefore, the present invention includes a method for preventing a disease or disorder in a subject, wherein the IL-18BP inhibitor composition described herein can be administered to the subject before the onset of the disease or disorder, thereby preventing the development of the disease or disorder. The prophylactic methods described herein also include treating a subject in remission to prevent recurrence of the disease or disorder.

Upon reading the description of the present invention, it should be apparent to any person skilled in the art that preventing a disease or disorder includes administering an IL-18BP inhibitor composition to a subject as a prophylactic measure against the onset of the disease or disorder. As already described in more detail in the invention, methods for reducing the level or activity of IL-18BP include a wide variety of methods for reducing not only the activity of IL-18BP, but also methods for reducing the expression of a nucleic acid encoding IL-18BP, including either reducing transcription, or reduction in translation, or both.

In addition, after becoming familiar with the ideas of the present invention, it should be obvious to any person skilled in the art that the present invention includes a method for the prevention of a number of diseases, disorders and pathologies in which a decrease in the expression and/or activity of IL-18BP mediates, has a therapeutic effect or prevents the occurrence of a disease, disorder or pathology. Methods for assessing the association of disease with IL-18BP levels or activity are well known. In addition, the invention includes the treatment or prevention of such diseases that may occur in the future.

The invention includes administering an IL-18BP inhibitor to carry out the methods of the invention; after reading the description of the present invention, it is obvious to any person skilled in the art how to prepare and administer an appropriate IL-18BP inhibitor to a subject. However, the present invention is not limited to any particular route of administration or regimen.

Cytokine inhibitors

In some embodiments, the composition of the present invention includes an inhibitor of one or more cytokines. In some embodiments, an inhibitor of one or more cytokines includes a chemical compound, protein, peptide, peptidomimetic, antibody, ribozyme, small molecule chemical compound, or antisense nucleic acid molecule (e.g., siRNA, microRNA, etc.) that inhibits expression, activity, or both, 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 enhances the activity of the administered IL-18 polypeptide variant or IL-18BP inhibitor.

Compositions and methods for treating and preventing disease

In various embodiments, the present invention includes compositions comprising an activator of IL-18 activity, such as activating a signaling pathway through at least one IL-18R, and methods for increasing IL-18 activity, such as activating a signaling pathway, at least , via one IL-18R, in a cell, organ tissue, system, or subject in need thereof. In various embodiments, the IL-18 activity activator in the compositions and treatments of the invention increases the amount of IL-18R signaling pathway activation, the amount of immunocyte activity, or both. In various embodiments, diseases or disorders in which increased activation of the IL-18R signaling pathway may improve therapeutic effects include, but are not limited to, cancer, infectious diseases, macular degeneration, and metabolic diseases and disorders.

The following are non-limiting examples of types of cancer that can be treated or prevented using the methods and compositions of the invention: acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, appendix cancer, basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, tumors brain and spinal cord, brainstem glioma, brain tumor, breast cancer, bronchial tumors, Burkitt's lymphoma, carcinoid tumor, atypical teratoid/rhabdoid tumor of the central nervous system, embryonic tumors of the central nervous system, lymphoma of the central nervous system, cerebellar astrocytoma, cerebral astrocytoma/malignant glioma, cervical cancer, pediatric optic pathway tumor, chordoma, chronic lymphocytic leukemia, chronic myeloid leukemia, chronic myeloproliferative disorders, colon cancer, colorectal cancer, craniopharyngioma, skin cancer, cutaneous T-cell lymphoma, endometrial cancer, ependymoblastoma, ependymoma, esophageal cancer, Ewing tumor family, extracranial cancer, extragonadal germ cell tumor, extrahepatic biliary tract cancer, extrahepatic cancer, eye cancer, mycosis fungoides, gallbladder cancer, gastric cancer, gastrointestinal tract cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, germ cell tumor, gestational cancer, gestational trophoblastic tumor, glioblastoma, glioma, hairy cell leukemia, head and neck cancer, hepatocellular carcinoma (liver cancer), histiocytosis, Hodgkin's lymphoma, hypopharyngeal cancer, hypothalamic and optic pathway glioma, hypothalamic tumor , intraocular cancer (cancer of the eye), intraocular melanoma, insulinomas, Kaposi's sarcoma, kidney cancer (renal cell carcinoma), Langerhans cell carcinoma, Langerhans cell histiocytosis, cancer of the larynx, leukemia, cancer of the lips and mouth, liver cancer, lung cancer, lymphoma, macroglobulinemia, malignant fibrous bone histiocytoma and osteosarcoma, medulloblastoma, medulloepithelioma, melanoma, Merkel cell carcinoma, mesothelioma, metastatic squamous cell carcinoma of the neck of unknown origin, oral cancer, multiple endocrine neoplasia syndrome, multiple myeloma, mycosis, myelodysplastic syndromes, myelodysplastic/myeloproles ferative diseases , myelogenous leukemia, myeloid leukemia, myeloma, myeloproliferative disorders, cancer of the nasal cavity and paranasal sinuses, nasopharyngeal cancer, neuroblastoma, non-Hodgkin's lymphoma, non-small cell lung cancer, oral cancer, oropharyngeal cancer, osteosarcoma and malignant fibrous histiocytoma, osteosarcoma and malignant fibrous histiocytoma cytoma bone, ovarian cancer, epithelial ovarian cancer, ovarian germ cell tumor, borderline ovarian tumor, pancreatic cancer, papillomatosis, paraganglioma, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, parenchymal pineal tumors of intermediate differentiation, pineoblastoma and supratentorial primitive neuroectodermal tumors, pituitary tumor, plasma cell neoplasia, plasma cell neoplasia/multiple myeloma, pleuropulmonary blastoma, primary central nervous system cancer, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell carcinoma (kidney cancer), kidney cancer pelvis and ureter, respiratory tract carcinoma containing the NUT gene on chromosome 15, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, Cesari syndrome, skin cancer (melanoma), non-melanocytic skin cancer, skin carcinoma, small cell lung cancer, cancer of the small intestine, cancer soft tissue, soft tissue sarcoma, squamous cell carcinoma, squamous cell neck cancer, gastric cancer, supratentorial primitive neuroectodermal tumors, supratentorial primitive neuroectodermal tumors and pineoblastoma, T-cell lymphoma, testicular cancer, throat cancer, thymoma and carcinoma of the thymus, thyroid cancer, transitional cell carcinoma, transitional cell carcinoma of the renal pelvis and ureter, trophoblastic tumor, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, optic pathway and hypothalamic glioma, vaginal cancer, Waldenström macroglobulinemia, and Wilms' tumor.

Thus, non-limiting examples of types of cancer that can be treated or prevented by the methods and compositions of the invention include solid cancers, liquid cancers, blood cancers, teratomas, sarcomas, and carcinomas.

In some embodiments, the method of the present invention is used to treat or prevent a tumor or cancer that is resistant to immune checkpoint inhibitors (ICIs). Examples of immune checkpoint inhibitors include, but are not limited to, PD1 antibody (eg, nivolumab), CTLA4 antibody (eg, ipilimumab), TIM3 antibody, TIGIT antibody, LAG3 antibody, B7H3 antibody, B7H4 antibody, anti-VISTA antibody, anti-ICOS antibody, anti-GITR antibody, anti-41BB antibody, anti-OX40 antibody, and anti-CD40 antibody. Examples of immune checkpoint inhibitor targets include, but are not limited to: PD-L1, PD1, CTLA4, TIM3, TIGIT, LAG3, B7H3, B7H4, VISTA, ICOS, GITR, 41BB, OX40, and CD40. Thus, examples of immune checkpoint inhibitors include drugs that inhibit proteins such as PD-L1, PD1, CTLA4, TIM3, TIGIT, LAG3, B7H3, B7H4, VISTA, ICOS, GITR, 41BB, OX40, or CD40. In some instances, a proposed IL-18 polypeptide variant (e.g., DR-IL-18 variant, D2D-IL-18 variant) is co-administered with an immune checkpoint inhibitor (e.g., an agent that inhibits PD-L1, PD1, CTLA4, TIM3, TIGIT, LAG3, B7H3, B7H4, VISTA, ICOS, GITR, 41BB, OX40, or CD40, or any combination of these).

Mergers/conjugations

In some embodiments, a variant IL-18 polypeptide of the present invention is fused to another protein, i.e., a variant IL-18 polypeptide or fragment thereof can be fused in frame to a second polypeptide (with a fusion partner). In some embodiments, the second polypeptide (fusion partner) is capable of increasing the overall size of the fusion protein, such that the fusion protein is not rapidly cleared from the circulation, for example. In some cases, a variant IL-18 polypeptide or fragment thereof is not fused to a second polypeptide.

In some embodiments, the second polypeptide (fusion partner with a variant IL-18 polypeptide or fragment thereof) is part or all of an immunoglobulin Fc region (ie, an antibody Fc sequence). In other embodiments, the second polypeptide is any suitable polypeptide that is substantially similar to Fc, such as allowing for size increase, domain multimerization, and/or additional binding or interaction with Ig molecules. In some embodiments, the second polypeptide is part of or is all 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 agent that binds or interacts with HSA. These fusion proteins may facilitate purification, multimerization and have an increased in vivo half-life. Fusion proteins having multimeric structures attached by disulfide bonds can also be, in some cases, more effective at binding and neutralizing other molecules.

When fused to a heterologous polypeptide, the portion corresponding to the variant IL-18 polypeptide or fragment thereof may be referred to as the "variant IL-18 polypeptide portion" of the proposed variant IL-18 polypeptide. In some instances, the "IL-18 polypeptide variant part" may be 100 amino acids or more in length (e.g., 110 amino acids or more, 125 amino acids or more, 150 amino acids or more, 90 amino acids or more, 95 amino acids or more, 100 amino acids or more, 105 amino acids or more, 110 amino acids or more, 115 amino acids or more, 120 amino acids or more, 125 amino acids or more, 130 amino acids or more, 140 amino acids or more, or 150 amino acids or more), up to full-length IL- 18 and may be fused to a heterologous polypeptide.

In some instances, the IL-18 polypeptide variant portion of the proposed IL-18 polypeptide variant has a length in the range of 100 amino acids to 157 amino acids (e.g., 100 amino acids to 150 amino acids, 100 amino acids to 140 amino acids, 140 amino acids to 157 amino acids, 140 amino acids to 150 amino acids, 145 amino acids to 157 amino acids, or 150 amino acids to 157 amino acids).

In some instances, the second polypeptide is a marker sequence (eg, an affinity tag) such as a peptide that facilitates purification of the fused polypeptide. For example, the marker amino acid sequence may be, inter alia, a histidine hexapeptide such as the tag introduced into the pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), many of which are commercially available. For example, as described in Gentz et al., Proc. Natl. Acad. sci. USA 86: 821-824, 1989, histidine hexapeptide allows for easy purification of the fusion protein. Another peptide tag used for purification, the "HA" tag, corresponds to an epitope derived from the hemagglutinin protein of the influenza virus (Wilson et al., Cell 37: 767, 1984). The addition of peptide fragments to facilitate manipulation of polypeptides has long been known and accepted.

The proposed IL-18 polypeptide variant may be modified, eg combined/conjugated with a variety of other oligopeptides, proteins and/or non-protein fragments, for various purposes. For example, a proposed IL-18 polypeptide variant may be post-translationally modified, for example, by prenylation, acetylation, amidation, carboxylation, glycosylation, pegylation (by covalent attachment of polyethylene glycol (PEG) polymer chains), and so on. Such modifications may also include glycosylation modifications, for example, modifications made by modifying the glycosylation profiles of the polypeptide during its synthesis and processing, or at additional processing steps; for example, by exposing the polypeptide to enzymes that adversely affect glycosylation, such as mammalian glycosylation or deglycosylation enzymes. In some embodiments, the proposed IL-18 polypeptide variant has one or more phosphorylated amino acid residues, for example, phosphotyrosine, phosphoserine, or phosphothreonine.

In some other embodiments, the IL-18 polypeptide variants of the invention comprise reagents further modified to improve their resistance to proteolytic disintegration or to optimize solubility or to make them more suitable for use as a therapeutic agent. For example, embodiments of the present invention further include analogs containing residues that are not natural L-amino acid residues, such as D-amino acid residues or unnatural synthetic amino acids. Some or all of the amino acid residues may be replaced by D-amino acids.

Co-administration and multispecific variants of the IL-18 polypeptide

As already noted in this invention, in some cases, the proposed version of the IL-18 polypeptide is administered with an additional drug. The terms "co-administration", "co-administration" and "in combination with" include the administration of two or more therapeutic agents (for example, a proposed variant of IL-18, such as DR-IL-18 or D2D, in combination with an additional drug) or simultaneously, in parallel, or sequentially without a specific indication of time points. In some embodiments, the drugs are present in the cells or in the body of the subject at the same time, or exert their biological or therapeutic effects at the same time. In some embodiments, the therapeutic agents are in the same single dose composition or dosage form. In other embodiments, the therapeutic agents are in separate compositions or single dose dosage forms. In specific embodiments, the first drug may be administered over (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 before), at the same time, 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 after ) administration of the second therapeutic agent.

In some cases, the proposed IL-18 variant (eg, DR-IL-18 variant or D2D variant) (eg, formulated in the form of a pharmaceutical composition) is co-administered with an anti-cancer therapeutic, an infection therapeutic, or an anti-cancer-targeted antibody. Such administration may include concurrent (ie at the same time), prior or subsequent administration of the drug/antibody relative to the administration of the drug or drugs of the invention. It will not be difficult for a person of ordinary skill in the art to determine the appropriate times, sequence, and doses for the administration of particular drugs and compositions of the present invention.

In some embodiments, treatment is by administering a combination (co-administration) of the proposed IL-18 variant (e.g., DR-IL-18 variant or D2D variant) with another drug (e.g., an immunostimulant, a chronic infection agent, cytotoxic agent, anticancer agent, and the like). One example of a class of cytotoxic agents that can be used is a chemotherapeutic agent. Examples of chemotherapeutic agents include, but are not limited to, aldesleukin, altretamine, amifostine, asparaginase, bleomycin, capecitabine, carboplatin, carmustine, cladribine, cisapride, cisplatin, cyclophosphamide, cytarabine, dacarbazine (DTIC), dactinomycin, docetaxel, doxorubicin, dronabinol, duocar mycin , etoposide, filgrastim, fludarabine, fluorouracil, gemcitabine, granisetron, hydroxyurea, idarubicin, ifosfamide, interferon alfa, irinotecan, lansoprazole, levamisole, leucovorin, megestrol, mesnu, methotrexate, metoclopramide, mitomycin, mitotane, mitoxantrone, omepra zol, ondansetron, paclitaxel ( taxol ^TM ), pilocarpine, prochlorperazine, rituximab, saproin, tamoxifen, taxol, topotecan hydrochloride, trastuzumab, vinblastine, vincristine, and vinorelbine tartrate.

A proposed IL-18 variant (e.g., DR-IL-18 variant or D2D variant) optionally, but optionally, may be prepared with one or more medicines, which increase the activity or which increase the therapeutic effect. In some embodiments, treatment is by administering a combination (co-administration) of a proposed IL-18 variant (eg, DR-IL-18 variant) and an agent that opsonizes the target cell. Therefore, the invention also contemplates compositions (and methods in which the compositions are used) that include: (a) an inventive variant of IL-18 (eg, variant DR-IL-18); and (b) an agent that opsonizes the target cell. In some cases, the agent that opsonizes the target cell is rituximab. In some cases, this agent that opsonizes the target cell is cetuximab.

An "agent that opsonizes a target cell" ("opsonizing agent") is any agent that can bind to a target cell (e.g., a cancer cell, a cell containing an intracellular pathogen, and the like) and opsonize the cell- target (eg, label the target cell for phagocytosis and/or antibody-dependent cell-mediated cytotoxicity (ADCC)). For example, any antibody that can bind to a target cell (e.g., a cancer cell such as a tumor cell) and that has an FC region is considered to be an agent that opsonizes the target cell and is an antibody that binds to the target cell. a target (eg, an anti-tumor antibody, an anti-cancer antibody, an anti-infective antibody, and the like).

For example, antibodies selective for tumor cell markers, radiation, surgical and/or hormonal deprivation are described in Kwon et al., Proc. Natl. Acad. Sci U.S.A., 96: 15074-9, 1999. Angiogenesis inhibitors may also be used in combination with the methods of the invention. A number of antibodies are currently used in clinical practice for the treatment of cancer, and other antibodies are in various stages of clinical trials. For example, a number of antigens and corresponding monoclonal antibodies are known for the treatment of B-cell malignancies. One target antigen is CD20. Rituximab is a chimeric, unconjugated monoclonal antibody that targets the CD20 antigen. CD20 plays an important functional role in the activation, proliferation and differentiation of B cells. The CD52 antigen is targeted by the monoclonal antibody alemtuzumab, which is prescribed for the treatment of chronic lymphocytic leukemia. A number of antibodies target CD22 and have recently been shown to be active in combination with a toxin in chemotherapy-resistant hairy cell leukemia. Two new CD20-targeting monoclonal antibodies, tositumomab and ibritumomab, have been submitted for registration with the US Food and Drug Administration (FDA). These antibodies are conjugated to radioisotopes. Alemtuzumab (Campat) is used in the treatment of chronic lymphocytic leukemia; gemtuzumab (mylotarg) is used in the treatment of acute myeloid leukemia; ibritumomab (zevalin) is used in the treatment of non-Hodgkin's lymphoma; Panitumumab (Vectibix) is being used in the treatment of colon cancer.

Monoclonal antibodies useful in the methods of the invention that have found use in the treatment of solid tumors include, without limitation, edrecolomab and trastuzumab (Herceptin). Edrecolomab targets the 17-1A antigen found in colon and rectal cancers and has been approved for use in Europe for these indications. Trastuzumab targets the HER-2/neu antigen. Cetuximab (Erbitux) is also of interest for use in the methods of the invention. This antibody binds to the EGF receptor (EGFR) and has been used in the treatment of solid tumors including colon cancer and small cell carcinoma of the head and neck (SCCHN).

The proposed IL-18 polypeptide variant (eg, DR-IL-18 variant, D2D-IL-18 variant) can be combined with any of the agents mentioned above (eg, agents such as antibodies that opsonize target cells). Therefore, in some cases, the proposed IL-18 polypeptide variant (e.g., DR-IL-18 variant, D2D-IL-18 variant) is used in combination therapy (co-administered) with one or more cancer cell selective opsonizing agents, such as tumor cells. In some cases, a proposed IL-18 polypeptide variant (eg, DR-IL-18 variant, D2D-IL-18 variant) is used in combination therapy (co-administered) with one or more of the following: cetuximab (binds EGFR), panitumumab (binds EGFR), rituximab (binds CD20), trastuzumab (binds HER2), pertuzumab (binds HER2), alemtuzumab (binds CD52), brentuximab (binds CD30), tositumomab, ibritumomab, gemtuzumab, ibritumomab and edrecolomab (binds 17-1A) or their combinations.

In some instances, a proposed IL-18 polypeptide variant (eg, DR-IL-18 variant, D2D-IL-18 variant) is co-administered with a cancer cell opsonizing agent (eg, an agent that includes an antigen-binding region that targets 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 ganglioside, MUC1, CanAg mucin, mesothelin, endoglin, Lewis Y antigen, CEA, CEACAM1, CEACAM5, CA-125, PSMA, BAFF, FGFR2, TAG-72, gelatinase B, glypican 3, nectin-4, BCMA, CSF1R, SLAMF7, integrin α _v β ₃ , TYRP1, GPNMB, CLDN18.2, FOLR1, CCR4, CXCR4, MICA, C242 antigen, DLL3, DLL4, EGFL7, vimentin, fibronectin extra domain-B, TROP-2, LRRC15, FAP, SLITRK6, NOTCH2, NOTCH3, tanascin-3, STEAP1, or NRP1, or any combination thereof).

In some instances, a proposed IL-18 polypeptide variant (e.g., DR-IL-18 variant, D2D-IL-18 variant) is co-administered with an agent that targets one or more antigens selected from 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 instances, a proposed IL-18 polypeptide variant (e.g., DR-IL-18 variant, D2D-IL-18 variant) is used in combination therapy (co-administered) with any suitable immunomodulatory agent (e.g., anti-CTLA4 antibody, anti- PD-1, anti-PD-L1, anti-TIGIT, anti-TIM3, anti-LAG3, anti-VISTA, anti-B7H3, anti-B7H4, CD40 agonist, 4-1BB modulator (e.g. 41BB agonist), with an OX-40 modulator (for example, an OX-40 agonist), with a GITR modulator (for example, a GITR agonist), with a CD47 binding agent, such as an anti-CD47 antibody or a high affinity CD47 binding agent, with a SIRPA binding agent, such as an anti-SIRPA antibody or a high affinity SIRPA binding agent with a TGFbeta antagonist such as an anti-TGF antibody with a cytokine or cytokine variant including IL-1, IL-2, IL-10, IL-12, IL-15 , IL-18, IL-21, IL-33, with interferon alpha, interferon beta, interferon gamma, TNF, TRAIL, lymphotoxin, LIGHT/TNSF14, or a Toll-like receptor agonist including TLR2, TLR4, TLR5, TLR7, TLR9 , an inflammasome agonist, a STING/cGAS pathway agonist or a RIG-I pathway agonist, an A2aR/A2bR adenosine receptor antagonist, an arylhydrocarboxylic receptor antagonist, an IDO and/or TDO antagonist, or an oncolytic virus.

In some instances, a contemplated IL-18 polypeptide variant (eg, DR-IL-18 variant, D2D-IL-18 variant) is used in combination therapy (co-administered) with a BTLA and/or CD160 inhibitor. In some instances, a proposed IL-18 polypeptide variant (e.g., DR-IL-18 variant, D2D-IL-18 variant) is used in combination therapy (co-administered) with an anti-CD47/SIRPA antibody (e.g., anti-CD47 antibody, anti- SIRPA, high affinity CD47 binding agent, high affinity SIRPA binding agent, and the like). In some instances, a contemplated IL-18 polypeptide variant (eg, DR-IL-18 variant, D2D-IL-18 variant) is used in combination therapy (co-administered) with a TIM3 and/or CEACAM1 inhibitor.

As mentioned above, in some cases, the proposed version of the IL-18 polypeptide is fused with another protein (ie, "fusion partner", "second polypeptide"). In some embodiments, the second polypeptide (fusion partner for the proposed variant IL-18 polypeptide) specifically binds to a target molecule that is not a target molecule linked through a portion of the variant IL-18 polypeptide of the fusion protein (e.g., that is not target molecule for IL-18R variants that bind IL-18R; or which is not a target molecule for IL-18BP variants that bind IL-18BP).

Therefore, in some embodiments, the proposed IL-18 polypeptide variant is multispecific (eg, bispecific). The terms "multispecific" or "bispecific" are commonly used when referring to drugs (eg, ligands or antibodies) that recognize two or more different antigens because they possess at least one region (eg. , ligand or Fab of the first antibody) that is specific for the first target, and at least a second region (eg, ligand or Fab of the second antibody) that is specific for the second target. A bispecific agent binds specifically to two targets and is one type of multispecific agent.

In some embodiments, the proposed variant IL-18 polypeptide is multispecific (e.g., bispecific) due to the fact that the first region of the polypeptide includes the sequence of the proposed variant IL-18 polypeptide (i.e., the first region includes the variant IL-18 polypeptide) and the second region, that specifically binds to another target molecule (for example, an antigen). For example, in some cases, an IL-18 polypeptide variant is fused to a second polypeptide that specifically binds to a target molecule that is not the target molecule bound by the IL-18 polypeptide variant.

Any one of the agents discussed above in relation to co-administration may be conjugated to the proposed IL-18 polypeptide variant. As used herein, the term "co-administration" is intended to include such conjugated compounds. For example, in the case where agent 1 is co-administered with agent 2, the term is meant to include embodiments in which agent 1 and agent 2 are not conjugated to each other, but the term is also meant to include embodiments in which where agent 1 and agent 2 are conjugated to each other (for example, when agent 1 and agent 2 are both proteins and agent 1 is fused to agent 2).

In some instances, the second region of the multispecific IL-18 polypeptide variant is an immune checkpoint inhibitor. In some cases, the second region of the multispecific IL-18 polypeptide variant inhibits one or more proteins selected from PD-L1, PD1, CTLA4, TIM3, TIGIT, LAG3, B7H3, B7H4, VISTA, ICOS, GITR, 41BB, OX40, and CD40.

In some instances, the second region of the multispecific IL-18 polypeptide variant is a cancer cell opsonizing agent. In some instances, the second region of the multispecific IL-18 polypeptide variant targets one or more proteins selected from 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 instances, the second region of a multispecific IL-18 polypeptide variant is an opsonizing agent that targets one or more proteins selected from 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 instances, the second region of a multispecific IL-18 polypeptide variant includes an ectodomain, e.g., an ectodomain from PD-1, PD-L1, CD47 (e.g., a high affinity CD47/polypeptide variant), or SIRPA (e.g., a high affinity SIRPA variant). affinity/polypeptide). In some cases, the second region of the multispecific IL-18 polypeptide variant specifically binds an antigen selected from CTLA-4, Lag-3, BTLA, Tim-3, CD244, CD40, CD40L, CD47, SIRPA, PD-1, and PD-L1.

In some embodiments, the proposed IL-18 polypeptide variant includes a linker (eg, a polypeptide linker). For example, in some embodiments, the proposed IL-18 polypeptide variant and the fusion partner are separated by a linker (eg, a polypeptide linker). The polypeptide linker may have any of a wide variety of amino acid sequences. Proteins may be connected via a polypeptide linker, which may be flexible in nature (eg, via a flexible polypeptide linker), although other chemical linkages are not excluded. Suitable linkers include polypeptides ranging in length from about 6 amino acids to about 40 amino acids, or from about 6 amino acids to about 25 amino acids. These linkers can be made by using synthetic linker-encoding oligonucleotides to bind to proteins. Peptide linkers with a certain degree of flexibility can be used. Linker peptides may have essentially any amino acid sequence, whereas in some cases, linkers may have a sequence that results in a flexible peptide. The use of small amino acids such as glycine and alanine finds use in the creation of a flexible peptide. The creation of such sequences is well known to those skilled in the art. A number of different linkers are commercially available and can be used.

In some embodiments, the IL-18 polypeptide variant is co-administered with an engineered immunocyte, such as a CAR-T or CAR-NK cell, or a T or NK cell transfected with the engineered T cell receptor. In other embodiments, a variant IL-18 polypeptide is co-administered with an oncolytic virus.

In some embodiments, a nucleic acid encoding a variant IL-18 polypeptide is introduced into an engineered ("modified") immunocyte, such as a CAR-T or CAR-NK cell, or a T or NK cell transfected with the engineered receptor T cells. In this case, the engineered cell (eg, altered T cell, altered NK cell) will secrete a variant IL-18 polypeptide. The ability to secrete a variant IL-18 peptide can be regulated in a contextual manner (eg, by regulation in the tumor microenvironment), for example, by the synthetic receptor NOTCHα.

In some embodiments, a nucleic acid encoding a variant IL-18 polypeptide is introduced into an oncolytic virus. In this case, cells infected with an oncolytic virus will secrete a variant IL-18 polypeptide.

In some embodiments, the method of the present invention is used to treat or prevent a tumor or cancers that lack the expression of MHC class I molecules on the surface of tumor cells; such as a tumor that lacks the B2m, MHC locus, or that has mutations in other members of the antigen presentation and/or antigen loading complex such as TAP-associated glycoprotein.

Metabolic diseases and disorders include various metabolic and endocrine related diseases and disorders. The following are non-limiting examples of metabolic and endocrine related diseases that can be treated or prevented by the methods and compositions of the invention: obesity, diabetes, prediabetes, type II diabetes, juvenile adult type 1 diabetes (MODY), hyperglycemia , metabolic syndrome, dyslipidemia, hypertriglyceridemia and hypercholesterolemia.

Non-limiting examples of other diseases and disorders that can be treated or prevented by the compositions and methods of the invention include viral infections, bacterial infections, parasitic infections, and low immune activity. In some embodiments, the viral infection is at least one of poxvirus, variola virus, molluscum contagiosum, human papillomavirus, and wart virus infections. In some embodiments, the infection is a systemic infection. In some embodiments, the viral infection is a 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, the low immune activity is neutropenia, such as that resulting from chemotherapy.

Non-limiting examples of other diseases and disorders that can be treated or prevented by the compositions and methods of the invention include macular degeneration. For example, in some cases, the disease or disorder is a wet form of macular degeneration, and in some cases, the disease or disorder is a wet form of age-related macular degeneration. In some such cases, the IL-18 variant may be used as an anti-angiogenic agent. For example, the proposed IL-18 polypeptide variant may, in some cases, reduce choroidal neovascularization.

Therefore, the present invention relates to the prevention and treatment of a disease or disorder by administering a therapeutically effective amount of an IL-18 polypeptide variant, a recombinant IL-18 polypeptide variant, an active fragment of an IL-18 polypeptide variant (e.g., an IL-18 peptide variant, and the like), an IL-18 variant expression or activity activator, or a nucleic acid (e.g., DNA, cDNA, mRNA, etc.) that encodes at least one IL-18 polypeptide variant, into a cell, organ tissue, or subject in need of this, for the purpose of treating or preventing a disease or disorder or associated signs, symptoms or pathologies.

In some embodiments, a composition of the invention is administered to a cell, organ tissue, system, or subject to treat or prevent a disease or disorder. In some embodiments, a variant human IL-18 polypeptide is introduced into a cell, organ tissue, system, or subject. In some embodiments, a nucleic acid (eg, DNA, cDNA, mRNA, etc.) encoding at least one variant human IL-18 polypeptide is introduced into a cell, organ tissue, system, or subject.

In various embodiments, the human IL-18 polypeptide variant or fragment comprises at least one mutation (e.g., at least 2, at least 3, at least 4, at least 5, or, at least 6 mutations) selected from the group consisting of Y1X, L5X, K8X, M51X, K53X, S55X, Q56X, P57X, G59X, M60X, E77X, Q103X, S105X, D110X, N111X, M113X, V153X and N155X, where X is any amino acid. In various embodiments, the human IL-18 polypeptide variant or fragment thereof comprises at least 4 mutations selected from the group consisting of Y1X, L5X, K8X, M51X, K53X, S55X, Q56X, P57X, G59X, M60X, E77X , Q103X, S105X, D110X, N111X, M113X, V153X and N155X, where X is any amino acid. In various embodiments, the human IL-18 polypeptide variant or fragment thereof comprises at least 6 mutations selected from the group consisting of Y1X, L5X, K8X, M51X, K53X, S55X, Q56X, P57X, G59X, M60X, E77X , Q103X, S105X, D110X, N111X, M113X, V153X and N155X. In various embodiments, the human IL-18 polypeptide variant or fragment comprises at least one mutation (e.g., at least 2, at least 3, at least 4, at least 5, or, at least 6 mutations) selected from the group consisting of Y1X, L5X, K8X, S55X, Q56X, P57X, G59X, E77X, Q103X, S105X, D110X, N111X, M113X, V153X and N155X.

In some embodiments, the human IL-18 polypeptide variant or fragment comprises at least one mutation (e.g., at least 2, at least 3, at least 4, at least 5, or, at least 6 mutations) selected from the group consisting of 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, G59A, M60K, M60Q, M60R, M60L, E77D, Q103E, Q103K, Q103P, Q103A, Q10 3R, S105R, S105D, S105K, S105N, S105A, D110H, D110K, D110N, D110Q, D110E, D110S, D110G, N111H, N111Y, N111D, N111R, N111S, N111G, M113V, M113R, M113T, M1 13K, V153I, V153T, V153A, N155K and N155H. In some embodiments, the human IL-18 polypeptide variant comprises at least one IL-18 polypeptide variant or fragment thereof selected from the group consisting of hCS1 (SEQ ID NO: 34), hCS2 (SEQ ID NO: 35) , hCS3 (SEQ ID NO: 36), hCS4 (SEQ ID NO: 37), hC4 (SEQ ID NO: 38), hA8 (SEQ ID NO: 39), hD6 (SEQ ID NO: 40), hH12 (SEQ ID NO: 41), hB11 (SEQ ID NO: 42), hC3 (SEQ ID NO: 43), hC2 (SEQ ID NO: 44), hG10 (SEQ ID NO: 45), hG1 (SEQ ID NO: 46), hF1 (SEQ ID NO: 47), hD2 (SEQ ID NO: 48), hA1 (SEQ ID NO: 49), hB3 (SEQ ID NO: 50), hB4 (SEQ ID NO: 51), hH3 (SEQ ID NO: : 52), hH5 (SEQ ID NO: 53), hH4 (SEQ ID NO: 54), hE1 (SEQ ID NO: 55), hG2 (SEQ ID NO: 56), hB9 (SEQ ID NO: 57), hE12 (SEQ ID NO: 58), hC5 (SEQ ID NO: 59), 5-18 (SEQ ID NO: 73), 5-29 (SEQ ID NO: 74), 5-8 (SEQ ID NO: 75), 5-6 (SEQ ID NO: 76), 5-27 (SEQ ID NO: 77), 5-20 (SEQ ID NO: 78), 5-2 (SEQ ID NO: 79), 5-9 (SEQ ID NO: 80), 5-42 (SEQ ID NO: 81), 5-13 (SEQ ID NO: 82), 5-12 (SEQ ID NO: 83), 5-1 (SEQ ID NO: 84), 5 -33 (SEQ ID NO: 85), 5-21 (SEQ ID NO: 86), 6-31 (SEQ ID NO: 87), 6-20 (SEQ ID NO: 88), 6-12 (SEQ ID NO: : 89), 6-27 (SEQ ID NO: 90), 6-29 (SEQ ID NO: 91), 5-26 (SEQ ID NO: 191), 5-17 (SEQ ID NO: 192), 5- 41 (SEQ ID NO: 193), or a fragment thereof.

In some instances, the proposed DR-IL-18 variant or fragment thereof comprises at least one mutation (e.g., at least 2, at least 3, or at least 4 mutations) selected from the group, consisting of M51X, M60X, S105X, D110X and N111X, relative to SEQ ID NO: 30. In some cases, the proposed DR-IL-18 variant or fragment thereof includes at least 3 mutations selected from the group consisting of M51X, M60X, S105X, D110X and N111X, relative to SEQ ID NO: 30. In some cases, the proposed DR-IL-18 variant or fragment includes at least one mutation (for example, at least 2 3 or at least 4 mutations) selected from the group consisting of M51X ₁ , M60X ₂ , S105X ₃ , D110X ₄ and N111X ₅ relative to SEQ ID NO: 30, where X ₁ is T, K, D, E, R or N; X ₂ is K, Q, L or R; X ₃ represents R, D, K, A or N; X ₄ is H, K, N, Q, E, N, S, or G; and X ₅ is H, D, Y, R, S, or G. In some instances, the proposed DR-IL-18 variant or fragment thereof includes at least 3 mutations selected from the group consisting of M51X ₁ , M60X ₂ , S105X ₃ , D110X ₄ and N111X ₅ relative to SEQ ID NO: 30, where X ₁ is T, K, D, E, R, or N; X ₂ is K, Q, L or R; X ₃ represents R, D, K, A or N; X ₄ is H, K, N, Q, E, N, S, or G; and X ₅ is H, D, Y, R, S, or G. at least 3 or at least 4 mutations) selected from the group consisting of M51X ₁ , M60X ₂ , S105X ₃ , D110X ₄ and N111X ₅ relative to SEQ ID NO: 30, where X ₁ is T or K; X ₂ is K or L; X ₃ is D, N or A; X ₄ is K, N, S or G; and X ₅ is H, Y, G, or R.

In some instances, the proposed DR-IL-18 variant or fragment thereof includes mutations M51X, M60X, S105X, D110X, and N111X relative to SEQ ID NO: 30. For example, in some instances, the proposed DR-IL-18 variant or fragment thereof includes mutations M51X ₁ , M60X ₂ , S105X ₃ , D110X ₄ and N111X ₅ relative to SEQ ID NO: 30, where X ₁ is T, K, D, E, R or N; X ₂ is K, Q, L or R; X ₃ represents R, D, K, A or N; X ₄ is H, K, N, Q, E, N, S, or G; and X ₅ is H, D, Y, R, S or G. In some instances, the proposed DR-IL-18 variant or fragment thereof includes mutations M51X ₁ , M60X ₂ , S105X ₃ , D110X ₄ and N111X ₅ relative to SEQ ID NO: 30, where X ₁ represents T or K; X ₂ is K or L; X ₃ is D, N or A; X ₄ is K, N, S or G; and X ₅ is H, Y, G, or R. In other words, in some instances, the proposed DR-IL-18 variant or fragment thereof includes {M51T or M51K} mutations; {M60K or M60L}; {S105D, S105N, S105A}; {D110K, D110N, D110S or D110G}; and {N111H, N111Y, N111R or N111G} relative to SEQ ID NO: 30.

In some instances, the proposed DR-IL-18 variant or fragment thereof comprises at least one mutation (e.g., at least 2, at least 3, or at least 4 mutations) selected from the group, consisting of M51X, K53X, Q56X, S105X and N111X, relative to SEQ ID NO: 30. In some cases, the proposed DR-IL-18 variant or fragment thereof includes at least 3 mutations selected from the group consisting of M51X, K53X, Q56X, S105X and N111X, relative to SEQ ID NO: 30. In some cases, the proposed DR-IL-18 variant or fragment includes at least one mutation (for example, at least 2, at least 3 or at least 4 mutations) selected from the group consisting of M51X ₁ , K53X ₂ , Q56X ₃ , S105X ₄ and N111X ₅ relative to SEQ ID NO: 30, where X ₁ is E, R or K; X ₂ is G, S or T; X ₃ represents E, A, R, V, G, K or L; X ₄ is N, S, K or G; and X ₅ is R, S, G or D. In some instances, the proposed DR-IL-18 variant or fragment thereof includes at least 3 mutations selected from the group consisting of M51X ₁ , K53X ₂ , Q56X ₃ , S105X ₄ and N111X ₅ relative to SEQ ID NO: 30, where X ₁ is E, R or K; X ₂ is G, S or T; X ₃ represents E, A, R, V, G, K or L; X ₄ is N, S, K or G; and X ₅ is R, S, G, or D. In some instances, the proposed DR-IL-18 variant, or fragment thereof, includes at least one mutation (e.g., at least 2, at least 3, or , at least 4 mutations) selected from the group consisting of M51X ₁ , K53X ₂ , Q56X ₃ , S105X ₄ and N111X ₅ relative to SEQ ID NO: 30, where X ₁ is K; X ₂ is G or S; X ₃ is G, R or L; X ₄ is S, N or G; and X ₅ is G or R.

In some instances, the proposed DR-IL-18 variant or fragment thereof includes mutations M51X, K53X, Q56X, S105X, and N111X relative to SEQ ID NO: 30. For example, in some instances, the proposed DR-IL-18 variant or fragment thereof includes mutations M51X ₁ , K53X ₂ , Q56X ₃ , S105X ₄ and N111X ₅ relative to SEQ ID NO: 30, where X ₁ is E, R or K; X ₂ is G, S or T; X ₃ represents E, A, R, V, G, K or L; X ₄ is N, S, K or G; and X ₅ is R, S, G, or D. In some instances, the proposed DR-IL-18 variant or fragment thereof includes mutations M51X ₁ , K53X ₂ , Q56X ₃ , S105X ₄ and N111X ₅ relative to SEQ ID NO: 30, where X ₁ represents K; X ₂ is G or S; X ₃ is G, R or L; X ₄ is S, N or G; and X ₅ is G or R. In other words, in some instances, the proposed DR-IL-18 variant or fragment thereof includes {M51K} mutations; {K53G or K53S}; {Q56G, Q56R or Q56L}; {D110S, D110N or D110G}; and {N111R or N111G} relative to SEQ ID NO: 30.

In some instances, a proposed DR-IL-18 variant or fragment thereof comprises an amino acid sequence having an identity of 85% or more (e.g., 90% or more, 92% or more, 93% or more, 94% or more, 95% or greater than, 96% or greater, 97% or greater, 98% or greater, or 99% or greater) with the human IL-18 wild-type amino acid sequence shown as SEQ ID NO: 30. Therefore, in some cases, the proposed a DR-IL-18 variant, or fragment thereof, comprises an amino acid sequence that (i) has an identity of 85% or more (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) with the wild type human IL-18 amino acid sequence shown as SEQ ID NO: 30; and (ii) includes at least one mutation (e.g., at least 2, at least 3, at least 4, at least 5, or at least 6 mutations) relative to the wild type IL-18 (eg human IL-18).

In some instances, a proposed DR-IL-18 variant or fragment thereof includes an amino acid sequence that (i) has an identity of 85% or more (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) with the wild type human IL-18 amino acid sequence shown as SEQ ID NO: 30; and (ii) includes at least one mutation (e.g., at least 2, at least 3, at least 4, at least 5, or at least 6 mutations) selected from group consisting of Y1X, L5X, K8X, M51X, K53X, S55X, Q56X, P57X, G59X, M60X, E77X, Q103X, S105X, D110X, N111X, M113X, V153X and N155X, with respect to SEQ ID NO: 30. In some cases , a proposed DR-IL-18 variant or fragment thereof comprises an amino acid sequence that (i) has an identity of 85% or more (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) with the wild type human IL-18 amino acid sequence shown as SEQ ID NO: 30; and (ii) includes at least 4 mutations selected from the group consisting of Y1X, L5X, K8X, M51X, K53X, S55X, Q56X, P57X, G59X, M60X, E77X, Q103X, S105X, D110X, N111X, M113X , V153X, and N155X, relative to SEQ ID NO: 30. In some instances, a proposed DR-IL-18 variant, or fragment thereof, includes an amino acid sequence that (i) has an identity of 85% or more (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) with the human IL-18 wild-type amino acid sequence represented by as SEQ ID NO: 30; and (ii) includes at least 6 mutations selected from the group consisting of Y1X, L5X, K8X, M51X, K53X, S55X, Q56X, P57X, G59X, M60X, E77X, Q103X, S105X, D110X, N111X, M113X , V153X, and N155X, relative to SEQ ID NO: 30. In some instances, a proposed DR-IL-18 variant, or fragment thereof, includes an amino acid sequence that (i) has an identity of 85% or more (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) with the human IL-18 wild-type amino acid sequence represented by as SEQ ID NO: 30; and (ii) includes at least one mutation (e.g., at least 2, at least 3, at least 4, at least 5, or at least 6 mutations) selected from group consisting of Y1X, L5X, K8X, S55X, Q56X, P57X, G59X, E77X, Q103X, S105X, D110X, N111X, M113X, V153X and N155X, with respect to SEQ ID NO: 30.

In some instances, a proposed DR-IL-18 variant or fragment thereof includes an amino acid sequence that (i) has an identity of 85% or more (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) with the wild type human IL-18 amino acid sequence shown as SEQ ID NO: 30; and (ii) includes at least one mutation (e.g., at least 2, at least 3, or at least 4 mutations) selected from the group consisting of M51X, M60X, S105X, D110X, and N111X, relative to SEQ ID NO: 30. In some instances, a proposed DR-IL-18 variant or fragment thereof includes an amino acid sequence that (i) has an identity of 85% or more (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) with the wild type human IL-18 amino acid sequence shown as SEQ ID NO: 30; and (ii) includes at least 3 mutations (e.g., at least 2, at least 3, or at least 4 mutations) selected from the group consisting of M51X, M60X, S105X, D110X, and N111X , relative to SEQ ID NO: 30. In some instances, a proposed variant of DR-IL-18 or a fragment thereof includes an amino acid sequence that (i) has an identity of 85% or more (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) with the wild type human IL-18 amino acid sequence shown as SEQ ID NO : thirty; and (ii) includes at least one mutation (e.g., at least 2, at least 3, or at least 4 mutations) selected from the group consisting of M51X ₁ , M60X ₂ , S105X ₃ , D110X ₄ and N111X ₅ relative to SEQ ID NO: 30, where X ₁ is T, K, D, E, R or N; X ₂ is K, Q, L or R; X ₃ represents R, D, K, A or N; X ₄ is H, K, N, Q, E, N, S, or G; and X ₅ is H, D, Y, R, S, or G. In some cases, the proposed DR-IL-18 variant or fragment includes an amino acid sequence that (i) has an identity of 85% or more (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) with the wild type IL amino acid sequence -18 human shown as SEQ ID NO: 30; and (ii) includes at least 3 mutations selected from the group consisting of M51X ₁ , M60X ₂ , S105X ₃ , D110X ₄ and N111X ₅ relative to SEQ ID NO: 30, where X ₁ is T, K, D, E, R or N; X ₂ is K, Q, L or R; X ₃ represents R, D, K, A or N; X ₄ is H, K, N, Q, E, N, S, or G; and X ₅ is H, D, Y, R, S, or G. In some cases, the proposed DR-IL-18 variant or fragment includes an amino acid sequence that (i) has an identity of 85% or more (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) with the wild type IL amino acid sequence -18 human shown as SEQ ID NO: 30; and (ii) includes at least one mutation (e.g., at least 2, at least 3, or at least 4 mutations) selected from the group consisting of M51X ₁ , M60X ₂ , S105X ₃ , D110X ₄ and N111X ₅ relative to SEQ ID NO: 30, where X ₁ is T or K; X ₂ is K or L; X ₃ is D, N or A; X ₄ is K, N, S or G; and X ₅ is H, Y, G, or R.

In some instances, a proposed DR-IL-18 variant or fragment thereof includes an amino acid sequence that (i) has an identity of 85% or more (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) with the wild type human IL-18 amino acid sequence shown as SEQ ID NO: 30; and (ii) includes mutations M51X, M60X, S105X, D110X, and N111X, relative to SEQ ID NO: 30. In some instances, the proposed DR-IL-18 variant, or fragment thereof, includes an amino acid sequence that (i) has 85% identity, or more than (for example, 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) with the wild-type amino acid sequence of human IL-18 shown as SEQ ID NO: 30; and (ii) includes mutations M51X ₁ , M60X ₂ , S105X ₃ , D110X ₄ and N111X ₅ relative to SEQ ID NO: 30, where X ₁ is T, K, D, E, R, or N; X ₂ is K, Q, L or R; X ₃ represents R, D, K, A or N; X ₄ is H, K, N, Q, E, N, S, or G; and X ₅ is H, D, Y, R, S, or G. In some cases, the proposed DR-IL-18 variant or fragment includes an amino acid sequence that (i) has an identity of 85% or more (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) with the wild type IL amino acid sequence -18 human shown as SEQ ID NO: 30; and (ii) includes mutations M51X ₁ , M60X ₂ , S105X ₃ , D110X ₄ and N111X ₅ relative to SEQ ID NO: 30, where X ₁ is T or K; X ₂ is K or L; X ₃ is D, N or A; X ₄ is K, N, S or G; and X ₅ is H, Y, G, or R.

In some instances, a proposed DR-IL-18 variant or fragment thereof includes an amino acid sequence that (i) has an identity of 85% or more (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) with the wild type human IL-18 amino acid sequence shown as SEQ ID NO: 30; and (ii) includes at least one mutation (e.g., at least 2, at least 3, or at least 4 mutations) selected from the group consisting of M51X, K53X, Q56X, S105X, and N111X, relative to SEQ ID NO: 30. In some instances, a proposed DR-IL-18 variant or fragment thereof includes an amino acid sequence that (i) has an identity of 85% or more (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) with the wild type human IL-18 amino acid sequence shown as SEQ ID NO: 30; and (ii) includes at least 3 mutations selected from the group consisting of M51X, K53X, Q56X, S105X and N111X relative to SEQ ID NO: 30. In some cases, the proposed DR-IL-18 variant or fragment thereof includes an amino acid sequence that (i) has an identity of 85% or more (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) with the wild-type amino acid sequence of human IL-18 shown as SEQ ID NO: 30; and (ii) includes at least one mutation (e.g., at least 2, at least 3, or at least 4 mutations) selected from the group consisting of M51X ₁ , K53X ₂ , Q56X ₃ , S105X ₄ and N111X ₅ relative to SEQ ID NO: 30, where X ₁ is E, R or K; X ₂ is G, S or T; X ₃ represents E, A, R, V, G, K or L; X ₄ is N, S, K or G; and X ₅ is R, S, G, or D. In some cases, the proposed DR-IL-18 variant or fragment includes an amino acid sequence that (i) has an identity of 85% or more (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) with the wild type human IL-18 amino acid sequence, represented as SEQ ID NO: 30; and (ii) includes at least 3 mutations selected from the group consisting of M51X ₁ , K53X ₂ , Q56X ₃ , S105X ₄ and N111X ₅ relative to SEQ ID NO: 30, where X ₁ is E, R or K; X ₂ is G, S or T; X ₃ represents E, A, R, V, G, K or L; X ₄ is N, S, K or G; and X ₅ is R, S, G, or D. In some cases, the proposed DR-IL-18 variant or fragment includes an amino acid sequence that (i) has an identity of 85% or more (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) with the wild type human IL-18 amino acid sequence, represented as SEQ ID NO: 30; and (ii) includes at least one mutation (e.g., at least 2, at least 3, or at least 4 mutations) selected from the group consisting of M51X ₁ , K53X ₂ , Q56X ₃ , S105X ₄ and N111X ₅ relative to SEQ ID NO: 30, where X ₁ is K; X ₂ is G or S; X ₃ is G, R or L; X ₄ is S, N or G; and X ₅ is G or R.

In some instances, a proposed DR-IL-18 variant or fragment thereof includes an amino acid sequence that (i) has an identity of 85% or more (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) with the wild type human IL-18 amino acid sequence shown as SEQ ID NO: 30; and (ii) includes mutations M51X, K53X, Q56X, S105X, and N111X relative to SEQ ID NO: 30. In some instances, a proposed DR-IL-18 variant or fragment thereof includes an amino acid sequence that (i) has an identity of 85% or more (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) with the wild-type amino acid sequence of human IL-18 shown as SEQ ID NO: 30; and (ii) includes mutations M51X ₁ , K53X ₂ , Q56X ₃ , S105X ₄ and N111X ₅ relative to SEQ ID NO: 30, where X ₁ is E, R or K; X ₂ is G, S or T; X ₃ represents E, A, R, V, G, K or L; X ₄ is N, S, K or G; and X ₅ is R, S, G, or D. In some cases, the proposed DR-IL-18 variant or fragment includes an amino acid sequence that (i) has an identity of 85% or more (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) with the wild type human IL-18 amino acid sequence, represented as SEQ ID NO: 30; and (ii) includes mutations M51X ₁ , K53X ₂ , Q56X ₃ , S105X ₄ and N111X ₅ relative to SEQ ID NO: 30, where X ₁ is K; X ₂ is G or S; X ₃ is G, R or L; X ₄ is S, N or G; and X ₅ is G or R.

In various embodiments, the IL-18 polypeptide variant that binds and inhibits IL-18BP comprises a human IL-18 polypeptide variant or fragment thereof comprising at least one mutation (e.g., at least 2, at least , 3, at least 4, at least 5 or at least 6 mutations) selected from the group consisting of Y1X, L5X, D17X, E31X, T34X, D35X, S36X, D37X, D40X, N41X, M51X, Q56X, M60X, Q103X, H109X, M113X and R131X, where X is any amino acid. In some embodiments, the IL-18 polypeptide variant that binds and inhibits IL-18BP comprises a human IL-18 polypeptide variant or fragment thereof comprising at least one mutation (e.g., at least 2, at least , 3, at least 4, at least 5 or at least 6 mutations) selected from the group consisting of Y1D, Y1F, Y1H, Y1L, L5F, L5H, D17A, D17G, D17R, D17H, E31A, E31T, E31G, E31K, E31R, T34A, T34K T34E, D35S, D35A, D35Y, S36N, S36K, S36R, D37P, D37A, D37R, D37H, D37L, D37V, D40Y D40S, D40A, N41K, N41S , N41R, M51F, M51L, M51I, Q56H, M60L, M60F, M60I, Q103L, Q103I, H109A, H109P, H109D, M113L, M113I, M113F and R131S. In some embodiments, the IL-18 polypeptide variant that binds and inhibits IL-18BP comprises a human IL-18 polypeptide variant or fragment thereof selected from the group consisting of hD2D-5F12 (SEQ ID NO: 92), hD2D-5F11 (SEQ ID NO: 93), hD2D-5F10 (SEQ ID NO: 94), hD2D-5F08 (SEQ ID NO: 95), hD2D-5F06 (SEQ ID NO: 96), hD2D-5F04 (SEQ ID NO: 97 ), hD2D-5F02 (SEQ ID NO: 98), hD2D-5F01 (SEQ ID NO: 99), hD2D-5E10 (SEQ ID NO: 100), hD2D-5E08 (SEQ ID NO: 101), hD2D-5E03 ( SEQ ID NO: 102), hD2D-5E02 (SEQ ID NO: 103), hD2D-5D10 (SEQ ID NO: 104), hD2D-5D08 (SEQ ID NO: 105), hD2D-5D06 (SEQ ID NO: 106) , hD2D-5D05 (SEQ ID NO: 107), hD2D-5D03 (SEQ ID NO: 108), hD2D-5D02 (SEQ ID NO: 109), hD2D-5C10 (SEQ ID NO: 110), hD2D-5C09 (SEQ ID NO: 111), hD2D-5C08 (SEQ ID NO: 112), hD2D-5C05 (SEQ ID NO: 113), hD2D-5C04 (SEQ ID NO: 114), hD2D-5C03 (SEQ ID NO: 115), hD2D-5B11 (SEQ ID NO: 116), hD2D-5B10 (SEQ ID NO: 117), hD2D-5B06 (SEQ ID NO: 118), hD2D-5B05 (SEQ ID NO: 119), hD2D-5B02 (SEQ ID NO: 120), hD2D-5A09 (SEQ ID NO: 121), hD2D-5A02 (SEQ ID NO: 122), hD2D-CS1 (SEQ ID NO: 123), hD2D-CS2 (SEQ ID NO: 124), hD2D -CS3 (SEQ ID NO: 125), or a fragment thereof.

In some instances, the proposed D2D-IL-18 variant or fragment thereof comprises at least one mutation (e.g., at least 2, at least 3, or at least 4 mutations) selected from the group consisting of D17X, E30X and Q103X, relative to SEQ ID NO: 30. In some cases, the proposed D2D-IL-18 variant or fragment includes at least one mutation (for example, at least 2, at least 3 or at least 4 mutations) selected from the group consisting of D17X ₁ , E30X ₂ and Q103X ₃ relative to SEQ ID NO: 30, where X ₁ is G, H, R or A; X ₂ is A, T, G, K or R; and X ₃ is I or L. In some instances, the proposed D2D-IL-18 variant, or fragment thereof, includes at least one mutation (e.g., at least 2, at least 3, or at least , 4 mutations) selected from the group consisting of D17G, E30A and (Q103L or Q103I).

In some instances, the proposed D2D-IL-18 variant or fragment thereof includes mutations D17X, E30X and Q103X relative to SEQ ID NO: 30. For example, in some instances, the proposed D2D-IL-18 variant or fragment thereof includes mutations D17X ₁ , E30X ₂ and Q103X ₃ with respect to SEQ ID NO: 30, where X ₁ is G, H, R or A; X ₂ is A, T, G, K or R; and X ₃ is I or L. In some instances, the proposed D2D-IL-18 variant or fragment thereof includes mutations D17G, E30A and (Q103L or Q103I).

In some instances, the proposed D2D-IL-18 variant or fragment thereof comprises at least one mutation (e.g., at least 2, at least 3, or at least 4 mutations) selected from the group consisting of D17X, E30X, D35X, M51X and Q103X, relative to SEQ ID NO: 30. In some cases, the proposed D2D-IL-18 variant or fragment includes at least one mutation (for example, at least 2, at least 3 or at least 4 mutations) selected from the group consisting of D17X ₁ , E30X ₂ , D35X ₃ , M51X ₄ and Q103X ₃ relative to SEQ ID NO: 30, where X ₁ is G, H, R or A; X ₂ is A, T, G, K or R; X ₃ is S, A or Y; X ₄ is F, I or L; and X ₅ is I or L. In some instances, the proposed D2D-IL-18 variant, or fragment thereof, includes at least one mutation (e.g., at least 2, at least 3, or at least , 4 mutations) selected from the group consisting of D17G, E30A, D35S, M51F and (Q103L or Q103I) relative to SEQ ID NO: 30.

In some instances, the proposed D2D-IL-18 variant or fragment thereof includes mutations D17X, E30X, D35X, M51X, and Q103X relative to SEQ ID NO: 30. For example, in some instances, the proposed D2D-IL-18 variant or fragment thereof includes mutations D17X ₁ , E30X ₂ , D35X ₃ , M51X ₄ and Q103X ₃ relative to SEQ ID NO: 30, where X ₁ is G, H, R or A; X ₂ is A, T, G, K or R; X ₃ is S, A or Y; X ₄ is F, I or L; and X ₅ is I or L. In some instances, the proposed D2D-IL-18 variant or fragment thereof includes mutations D17G, E30A, D35S, M51F and (Q103L or Q103I) relative to SEQ ID NO: 30.

In some instances, a proposed D2D-IL-18 variant or fragment thereof comprises an amino acid sequence having an identity of 85% or more (e.g., 90% or more, 92% or more, 93% or more, 94% or more, 95% or greater than, 96% or greater, 97% or greater, 98% or greater, or 99% or greater) with the wild-type human IL-18 amino acid sequence shown as SEQ ID NO: 30. Accordingly, in some cases, the proposed D2D variant -IL-18, or a fragment thereof, comprises an amino acid sequence that (i) has an identity of 85% or more (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) with the wild type human IL-18 amino acid sequence shown as SEQ ID NO: 30; and (ii) includes at least one mutation (e.g., at least 2, at least 3, at least 4, at least 5, or at least 6 mutations) relative to the wild type IL-18 (eg human IL-18).

In some instances, a proposed DR-IL-18 variant or fragment thereof includes an amino acid sequence that (i) has an identity of 85% or more (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) with the wild type human IL-18 amino acid sequence shown as SEQ ID NO: 30; and (ii) includes at least one mutation (e.g., at least 2, at least 3, at least 4, at least 5, or at least 6 mutations) selected from group consisting of Y1X, L5X, D17X, E31X, T34X, D35X, S36X, D37X, D40X, N41X, M51X, Q56X, M60X, Q103X, H109X, M113X and R131X.

In some instances, a proposed DR-IL-18 variant or fragment thereof includes an amino acid sequence that (i) has an identity of 85% or more (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) with the wild type human IL-18 amino acid sequence shown as SEQ ID NO: 30; and (ii) includes at least one mutation (e.g., at least 2, at least 3, at least 4, at least 5, or at least 6 mutations) selected from group consisting of D17X, E30X and Q103X, relative to SEQ ID NO: 30. In some cases, the proposed DR-IL-18 variant or fragment includes an amino acid sequence that (i) has an identity of 85% or more (for example, 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) with the wild type IL amino acid sequence -18 human shown as SEQ ID NO: 30; and (ii) includes at least one mutation (e.g., at least 2, at least 3, at least 4, at least 5, or at least 6 mutations) selected from the group consisting of D17X ₁ , E30X ₂ and Q103X ₃ relative to SEQ ID NO: 30, where X ₁ is G, H, R or A; X ₂ is A, T. In some instances, the proposed DR-IL-18 variant, or fragment thereof, includes an amino acid sequence that (i) has an identity of 85% or more (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) with the wild type human IL-18 amino acid sequence shown as SEQ ID NO : thirty; and (ii) includes at least one mutation (e.g., at least 2, at least 3, at least 4, at least 5, or at least 6 mutations) selected from group consisting of D17G, E30A and (Q103L or Q103I).

In some instances, a proposed DR-IL-18 variant or fragment thereof includes an amino acid sequence that (i) has an identity of 85% or more (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) with the wild type human IL-18 amino acid sequence shown as SEQ ID NO: 30; and (ii) includes mutations D17X, E30X, and Q103X relative to SEQ ID NO: 30. In some instances, the proposed DR-IL-18 variant, or fragment thereof, includes an amino acid sequence that (i) has an identity of 85% or more (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) with wild-type amino acid sequence Human IL-18 shown as SEQ ID NO: 30; and (ii) includes mutations D17X ₁ , E30X ₂ and Q103X ₃ relative to SEQ ID NO: 30, where X ₁ is G, H, R or A; X ₂ is A, T. In some instances, the proposed DR-IL-18 variant, or fragment thereof, includes an amino acid sequence that (i) has an identity of 85% or more (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) with the wild type human IL-18 amino acid sequence shown as SEQ ID NO : thirty; and (ii) includes mutations D17G, E30A and (Q103L or Q103I).

In some instances, a proposed DR-IL-18 variant or fragment thereof includes an amino acid sequence that (i) has an identity of 85% or more (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) with the wild type human IL-18 amino acid sequence shown as SEQ ID NO: 30; and (ii) includes at least one mutation (e.g., at least 2, at least 3, at least 4, at least 5, or at least 6 mutations) selected from a group consisting of D17X, E30X, D35X, M51X and Q103X, relative to SEQ ID NO: 30. In some cases, the proposed DR-IL-18 variant or fragment thereof includes an amino acid sequence that (i) has an identity of 85% or more ( 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) with an amino acid sequence of wild-type human IL-18, presented as SEQ ID NO: 30; and (ii) includes at least one mutation (e.g., at least 2, at least 3, at least 4, at least 5, or at least 6 mutations) selected from a group consisting of D17X ₁ , E30X ₂ , D35X ₃ , M51X ₄ and Q103X ₃ relative to SEQ ID NO: 30, where X ₁ is G, H, R or A; X ₂ is A, T, G, K or R; X ₃ is S, A or Y; X ₄ is F, I or L; and X ₅ is I or L. In some instances, the proposed DR-IL-18 variant, or fragment thereof, comprises an amino acid sequence that (i) has an identity of 85% or more (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) with the wild type human IL-18 amino acid sequence shown as SEQ ID NO: 30; and (ii) includes at least one mutation (e.g., at least 2, at least 3, at least 4, at least 5, or at least 6 mutations) selected from the group consisting of D17G, E30A, D35S, M51F and (Q103L or Q103I) with respect to SEQ ID NO: 30.

In some instances, a proposed DR-IL-18 variant or fragment thereof includes an amino acid sequence that (i) has an identity of 85% or more (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) with the wild type human IL-18 amino acid sequence shown as SEQ ID NO: 30; and (ii) includes mutations D17X, E30X, D35X, M51X, and Q103X relative to SEQ ID NO: 30. In some cases, a proposed DR-IL-18 variant or fragment thereof includes an amino acid sequence that (i) has an identity of 85% or more (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) with the wild-type amino acid sequence of human IL-18 shown as SEQ ID NO: 30; and (ii) includes mutations D17X ₁ , E30X ₂ , D35X ₃ , M51X ₄ and Q103X ₃ relative to SEQ ID NO: 30, where X ₁ is G, H, R or A; X ₂ is A, T, G, K or R; X ₃ is S, A or Y; X ₄ is F, I or L; and X ₅ is I or L. In some instances, the proposed DR-IL-18 variant, or fragment thereof, comprises an amino acid sequence that (i) has an identity of 85% or more (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) with the wild type human IL-18 amino acid sequence shown as SEQ ID NO: 30; and (ii) includes mutations D17G, E30A, D35S, M51F and (Q103L or Q103I) relative to SEQ ID NO: 30.

In some embodiments, a composition of the invention is administered to a mouse cell, organ tissue, system, or subject to treat or prevent a disease or disorder. In some embodiments, a murine IL-18 polypeptide variant or fragment thereof is introduced into a cell, organ tissue, system, or subject (eg, a human cell, organ tissue, system, or subject). In some embodiments, a nucleic acid (eg, DNA, cDNA, mRNA, etc.) encoding at least one murine IL-18 polypeptide variant is introduced into a cell, organ tissue, system, or subject.

In some embodiments, the murine IL-18 polypeptide variant, or fragment thereof, comprises at least one mutation (e.g., at least 2, at least 3, at least 4, at least 5, or, at least 6 mutations) selected from the group consisting of N1X, M50X, Y51X, K52X, S54X, E55X, V56X, R57X, G58X, L59X, R104X, N109X and L151X, where X is any amino acid. In some embodiments, the murine IL-18 polypeptide variant, or fragment thereof, comprises at least one mutation (e.g., at least 2, at least 3, at least 4, at least 5, or, at least 6 mutations) selected from the group consisting of N1H, N1Y, M50A, M50S, M50V, M50G, M50T, Y51R, K52V, K52S, K52T, K52G, K52A, S54R, S54K, S54G, S54N, E55R, E55H, E55N, E55D, E55G, V56L, V56M, V56R, V56A, V56S, V56Q, R57G, R57K, G58A, L59K, L59R, L59V, R104K, R104L, R104Q, R104S, N109D and L151V. In some embodiments, the murine IL-18 polypeptide variant includes at least one variant selected from the group consisting of 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), or a fragment thereof.

In some embodiments, a variant IL-18 polypeptide that binds and inhibits IL-18BP comprises a murine variant of an IL-18 polypeptide comprising at least one mutation (e.g., at least 2, at least 3, at least 4, at least 5 or at least 6 mutations) selected from the group consisting of N1X, L5X, D17X, E30X, T33X, D34X, I35X, D36X, M50X, Q102X, R104, H108X, N109X, M111X, D129X and D130X, where X is any amino acid. In some embodiments, the IL-18 polypeptide variant that binds and inhibits IL-18BP comprises a murine IL-18 polypeptide variant or fragment thereof comprising at least one mutation (e.g., at least 2, at least , 3, at least 4, at least 5 or at least 6 mutations) selected from the group consisting of N1Y, N1D, N1H, N1L, N1F, N1V, N1I, L5Y, L5H, D17Q, D17G D17A D17E D17S D17N E30A E30R E30K E30T E30G T33G T33A T33E T33R T33K D34Y D34S D34A I35T I35K I35R D36V D36A D36G D3 6h, D36P, D36R, D36L, M50F, M50L, Q102L, Q102I, R104E, R104A, R104P, R104G, R104Q, R104H, H108D, H108A, N109R, N109S, N109T, N109I, M111L, M111I, D 129A, D129F, D129V, D129Y, D129S, D130E, D130T, D130G, D130N, D130R, D130S, D130Q and D130H. An IL-18 polypeptide variant that binds and inhibits IL-18BP includes a murine IL-18 polypeptide variant or fragment thereof selected from the group consisting of mD2D-A5 (SEQ ID NO: 126), mD2D-A6 (SEQ ID NO: 127), mD2D-A7 (SEQ ID NO: 128), mD2D-A8 (SEQ ID NO: 129), mD2D-A9 (SEQ ID NO: 130), mD2D-A11 (SEQ ID NO: 131), mD2D-A12 (SEQ ID NO: 132), mD2D-B4 (SEQ ID NO: 133), mD2D-B7 (SEQ ID NO: 134), mD2D-B11 (SEQ ID NO: 135), mD2D-B12 (SEQ ID NO: 136 ), mD2D-C1 (SEQ ID NO: 137), mD2D-C3 (SEQ ID NO: 138), mD2D-C5 (SEQ ID NO: 139), mD2D-C6 (SEQ ID NO: 140), mD2D-C9 ( SEQ ID NO: 141), mD2D-C10 (SEQ ID NO: 142), mD2D-C11 (SEQ ID NO: 143), mD2D-D1 (SEQ ID NO: 144), mD2D-D9 (SEQ ID NO: 145) , mD2D-D12 (SEQ ID NO: 146), mD2D-E3 (SEQ ID NO: 147), mD2D-E4 (SEQ ID NO: 148), mD2D-E5 (SEQ ID NO: 149), mD2D-E7 (SEQ ID NO: 150), mD2D-E8 (SEQ ID NO: 151), mD2D-E9 (SEQ ID NO: 152), mD2D-E10 (SEQ ID NO: 153), mD2D-E11 (SEQ ID NO: 154), mD2D-E12 (SEQ ID NO: 155), mD2D-F3 (SEQ ID NO: 156), mD2D-F4 (SEQ ID NO: 157), mD2D-F5 (SEQ ID NO: 158), mD2D-F7 (SEQ ID NO: 159), mD2D-F8 (SEQ ID NO: 160), mD2D-F9 (SEQ ID NO: 161), mD2D-G1 (SEQ ID NO: 162), mD2D-G7 (SEQ ID NO: 163), mD2D -G9 (SEQ ID NO: 164), mD2D-H7 (SEQ ID NO: 165), mD2D-E1 (SEQ ID NO: 166), mD2D-G8 (SEQ ID NO: 167), mD2D-H3 (SEQ ID NO: : 168), mD2D-A10 (SEQ ID NO: 169), mD2D-H1 (SEQ ID NO: 170), mD2D-F12 (SEQ ID NO: 171), mD2D-G10 (SEQ ID NO: 172), mD2D- G12 (SEQ ID NO: 173), mD2D-E2 (SEQ ID NO: 174), mD2D-G11 (SEQ ID NO: 175), mD2D-C4 (SEQ ID NO: 176), mD2D-F11 (SEQ ID NO: 177), mD2D-C2 (SEQ ID NO: 178), mD2D-F10 (SEQ ID NO: 179), mD2D-A2 (SEQ ID NO: 180), mD2D-F6 (SEQ ID NO: 181), mD2D-A1 (SEQ ID NO: 182), mD2D-E6 (SEQ ID NO: 183), mD2D-D4 (SEQ ID NO: 184), mD2D-D6 (SEQ ID NO: 185), mD2D-A3 (SEQ ID NO: 186 ), mD2D-A4 (SEQ ID NO: 187), mD2D-B10 (SEQ ID NO: 188), mD2D-B8 (SEQ ID NO: 189), mD2D-B9 (SEQ ID NO: 190), or a fragment thereof.

In some embodiments, the method includes introducing an isolated nucleic acid molecule encoding one or more of the IL-18 peptide variants described above to a subject, into a cell or tissue.

It should be apparent to any person skilled in the art that increasing the level of activation of the IL-18 signaling pathway through IL-18R includes increasing the amount of IL-18 or an IL-18 polypeptide variant available for IL-18R binding and activation. This can be done by increasing the level or activation of IL-18, which includes, but is not limited to, direct or indirect administration of IL-18, direct or indirect administration of an IL-18 polypeptide variant, and direct or indirect administration of an IL-18BP inhibitor, as well as an increase in transcription, translation, or both, of a nucleic acid encoding IL-18 or a variant IL-18 polypeptide; and this also includes an increase in any IL-18 activity as well as an IL-18 polypeptide variant.

Increasing the level of activation of the IL-18 signaling pathway, including by using a variant IL-18 polypeptide, can be assessed by a variety of methods, including methods described in the invention, as well as methods that are known in the art or will be developed in the future. That is, after reading the description of the invention, it should be obvious to a person of ordinary skill in the art that the increase in the level and activity of the IL-18 signaling pathway can be easily assessed using methods that allow the assessment of the level of nucleic acid (for example, mRNA) encoding IL-18 or IL-18 polypeptide variant or fragment, level of IL-18 or IL-18 polypeptide variant or polypeptide fragment and/or activity level of IL-18 or IL-18 polypeptide variant or fragment in a biological sample taken from a subject.

After reading the description of the present invention, it should be obvious to any person skilled in the art that the invention can be applied to subjects who, in whole (for example, systemically) or in part (for example, locally, at the level of cells, tissue of an organ), are exposed to currently or will be undergoing treatment for a disease or disorder in which an increase in the activity of the IL-18 signaling pathway may have a beneficial effect. Based on the teachings of the invention, it should be apparent to one of ordinary skill in the art that the diseases and disorders treated with the compositions and methods of the invention include any disease or disorder in which an increase in the activity of the IL-18 signaling pathway would enhance a positive biological, physiological, clinical or therapeutic outcome.

It should be apparent to any person skilled in the art that in addition to directly increasing the activity of the IL-18 signaling pathway, reducing the amount or activity of a molecule that itself reduces the amount or activity of the IL-18 signaling pathway can be used to increase the amount of IL-18 or IL-18 signaling activity. Therefore, an IL-18 activity activator may include, but is not limited to, a chemical compound, a protein, a peptidomimetic, an antibody, a ribozyme, and an antisense nucleic acid molecule. Based on reading the description of the invention, it should be obvious to any person skilled in the art that an activator of IL-18 activity includes a compound that increases the level of activation of the IL-18 signaling pathway. In addition, an IL-18 activity activator includes a compound that inhibits the level or activity of a molecule that itself reduces the amount of IL-18 or the activation of an IL-18 signaling pathway (ie, IL-18BP). The present invention provides IL-18BP antagonists that include, but are not limited to, monoclonal antibodies, small molecule therapeutics that neutralize IL-18BP, and an engineered variant of IL-18 that binds to IL-18BP but that does not substantially bind or does not interact with IL-18R, and as a result of inhibition of IL-18BP in this way, the activity of endogenously produced IL-18 is increased due to disinhibition (disinhibition).

It should also be apparent to a person skilled in the art upon review of the ideas set forth in the specification that an increase in the level of activation of the IL-18 signaling pathway includes an increase in the level of IL-18 or activation of IL-18 (e.g., receptor binding activity, signaling activity). receptor pathway, and so on). Therefore, increasing the level or activity of the IL-18 signaling pathway includes, but is not limited to, increasing the amount of available IL-18 polypeptide or variant IL-18 polypeptide, increasing transcription, translation, or both, of the nucleic acid encoding the IL-18 polypeptide. 18 or a variant IL-18 polypeptide; and it also includes increasing any activity of an IL-18 polypeptide or also a variant IL-18 polypeptide. The IL-18 activity activator in the compositions and methods of the invention may selectively activate the IL-18 signaling pathway, or may activate both the IL-18 signaling pathway and the signaling pathway of another molecule. Therefore, the present invention relates to the administration of an IL-18 activity activator, a recombinant IL-18 polypeptide activity activator, an L-18 polypeptide fragment activity activator, or an IL-18 signaling pathway component expression or activity activator.

In addition, after reviewing the description of this invention and the examples given, it should be obvious to any person skilled in the art that an IL-18 activity activator includes activators that will be created in the future based on well-known criteria in the field of pharmacology, such as physiological results of activation of the IL-18 signaling pathway as described in detail in the invention and/or known in the art. Therefore, the present invention is in no way limited to any particular IL-18 activity activator exemplified or described in the invention; and moreover, the invention includes activators known in the art or created in the future, which in the opinion of experts in this field can be used.

In addition, methods of identifying and obtaining an IL-18 activity activator are well known to those of ordinary skill in the art, including, but not limited to, obtaining the activator from a natural source (e.g., Streptomyces sp., Pseudomonas sp., Stylotella aurantium, etc.) . Alternatively, the IL-18 activity activator can be chemically synthesized. In addition, after becoming familiar with the teachings of the present invention, it should be apparent to a person of ordinary skill in the art that an IL-18 activity activator can be obtained from a recombinant organism. Compositions and methods for chemically synthesizing the IL-18 activity activator and for obtaining them from natural sources are well known and described in the art.

For any person skilled in the art it is obvious that the activator can be introduced in the form of a small chemical compound, a polypeptide, a nucleic acid construct encoding a polypeptide, or combinations thereof. Numerous vectors and other compositions and methods are well known for introducing a polypeptide or nucleic acid encoding a polypeptide into cells or tissues. Therefore, the invention includes a method of introducing a polypeptide or nucleic acid encoding a polypeptide that is an activator of the IL-18 signaling pathway (Sambrook et al., 2012, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York; Ausubel et al., 1997 , Current Protocols in Molecular Biology, John Wiley & Sons, New York).

It should be apparent to any person skilled in the art that reducing the amount or activity of a molecule that itself reduces the amount or activity of the IL-18 signaling pathway can be used to increase the amount or activity of the IL-18 signaling pathway (e.g., IL-18BP ). Antisense oligonucleotides are DNA or RNA molecules that are complementary to some portion of the mRNA molecule. When present in cells, antisense oligonucleotides hybridize to an existing mRNA molecule and inhibit translation into the gene product. It is well known in the art to inhibit gene expression using an antisense oligonucleotide (Marcus-Sekura, 1988, Anal. Biochem. 172:289) as methods for expressing an antisense oligonucleotide in a cell (Inoue, U.S. Pat. No. 5190931). The methods of the invention include the use of an antisense oligonucleotide to reduce the amount of a molecule that reduces the amount or activity of the IL-18 signaling pathway, thereby increasing the amount or activity of the IL-18 signaling pathway. The present invention provides antisense oligonucleotides that are synthesized and delivered into the cell using methods that are well known to those of ordinary skill in the art. By way of example, an antisense oligonucleotide can be synthesized with a length ranging from about 10 to about 100, more preferably from about 15 to about 50 nucleotides. The synthesis of nucleic acid molecules is well known as the synthesis of modified antisense oligonucleotides to enhance their biological activity compared to unmodified antisense oligonucleotides (Tullis, 1991, U.S. Pat. No. 5023243).

Similarly, gene expression can be inhibited by hybridization of the antisense molecule to a promoter or other regulatory element of the gene, thereby affecting the transcription of the gene. Methods for identifying a promoter or other regulatory element that interacts with a gene of interest are well known in the art and include methods such as the yeast two hybrid system (Bartel and Fields, eds., In: The Yeast Two Hybrid System, Oxford University Press, Cary , N.C.).

Alternatively, inhibition of gene expression by proteins that reduces the level or activity of the IL-18 signaling pathway can be accomplished by using a ribozyme. The use of ribozymes to inhibit gene expression is well known to those skilled in the art (see, for example, Cech et al., 1992, J. Biol. Chem. 267:17479; Hampel et al., 1989, Biochemistry 28:4929; Altman et al. , U.S. Pat. No. 5168053). Ribozymes are catalytic RNA molecules that have the ability to cleave other single-stranded RNA molecules. Ribozymes are known to be sequence-specific and therefore can be modified to recognize a specific nucleotide sequence (Cech, 1988, J. Amer. Med. Assn. 260:3030), allowing selective cleavage of specific mRNA molecules. Given the nucleotide sequence of the molecule, one of ordinary skill in the art can synthesize an antisense oligonucleotide or ribozyme without undue experimentation, guided by the present invention and the references included therein.

It will be apparent to any person skilled in the art that an IL-18 activity activator, such as an IL-18 polypeptide variant or fragment, or a nucleic acid (e.g., DNA, cDNA, mRNA, etc.) encoding an IL-18 polypeptide variant or a fragment thereof, may be administered singly or in any combination thereof.

It will also be apparent to any person skilled in the art that administration may be short-term (eg, for a short period of time, such as a day, week, or month) or continuous (eg, over a long period of time, such as several months or a year or more). In addition, an IL-18 activity activator, such as an IL-18 polypeptide variant or fragment thereof, or a nucleic acid (e.g., DNA, cDNA, mRNA, etc.) encoding an IL-18 polypeptide variant or fragment thereof, can be administered via alone or in any combination with other drugs, as long as they are administered at the same time, before and/or after each other. It will be apparent to any person skilled in the art from the foregoing description that an IL-18 activity activator, an IL-18 polypeptide activity activator, a recombinant IL-18 polypeptide activity activator, or an IL-18 polypeptide fragment activity activator may be used alone or in combination. any combination with another IL-18 activity activator, IL-18 polypeptide activity activator, recombinant IL-18 polypeptide activity activator, or IL-18 polypeptide fragment activity activator to achieve a therapeutic result.

Upon reading the description of the present invention, including the methods described in detail, it should be apparent to any person skilled in the art that the invention is not limited to the treatment of a disease or disorder that has already been identified. In particular, the symptoms of a disease or disorder need not be manifested to the detriment of the subject's health; on the contrary, the disease or disorder need not be identified in the subject prior to treatment. That is, significant pathology as a result of the disease or disorder must not occur before the application of the present invention can have a beneficial effect. Therefore, described in great detail, the present invention includes a method for preventing a disease or disorder in a subject, in which an activator of the activity of an IL-18 molecule (for example, a polypeptide, a peptide, etc.) or an activator of the activity of IL-18 described in the invention can be administered to the subject, before the onset of the disease or disorder, thereby preventing the development of the disease or disorder.

The invention includes administering an IL-18 activity activator, an IL-18 polypeptide activity activator, a recombinant IL-18 polypeptide signaling pathway, or an IL-18 polypeptide fragment signaling pathway activation to carry out the methods of the invention; after reading the description of the present invention, it should be apparent to one skilled in the art how to prepare and administer an appropriate activator of IL-18 activity, an activator of IL-18 polypeptide activity, a recombinant IL-18 polypeptide signaling pathway, or an IL-18 fragment signaling pathway activity to a subject. However, the present invention is not limited to any particular route of administration or regimen. This is especially true in those cases where it is apparent to any person skilled in the art, as a result of reading the description of the invention, including the practice of using a disease model recognized in the art, that methods of administering an activator of IL-18 activity, an IL polypeptide signaling pathway - 18, the signaling pathway of a recombinant IL-18 polypeptide or the signaling pathway of a fragment of an IL-18 polypeptide can be determined by any person skilled in the art of pharmacology.

In some embodiments, the method includes administering to a subject in need thereof a composition comprising at least one IL-18 polypeptide variant and administering to the subject a composition comprising an additional drug. In one such embodiment, the additional drug comprises an immunotherapeutic agent, including at least an immunotherapeutic agent selected from the group including, but not limited to, altered T cells, chimeric antigen receptor T cells (CAR-T) , armored CAR-T cells, virus, antigen, vaccine, antibody, immune checkpoint inhibitor, small molecule, chemotherapeutic agent and stem cells. In some embodiments, a composition comprising at least one IL-18 polypeptide variant is used in a method of enhancing immune system activity before, during, or after a bacterial infection, viral infection, or infection with another pathogen. In some embodiments, a composition comprising at least one IL-18 polypeptide variant is used in a method for increasing the number and/or activity of immunocytes in vitro, in vivo, or ex vivo, such as the number and/or activity of T cells, NK cells and/or myeloid cells.

In some embodiments, the additional drug comprises an inhibitor of one or more cytokines. In some embodiments, an inhibitor of one or more cytokines includes a chemical compound, protein, peptide, peptidomimetic, antibody, ribozyme, small molecule chemical compound, or antisense nucleic acid molecule (e.g., siRNA, microRNA, etc.) that inhibits expression, activity, or both, 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 a cytokine inhibitor reduces toxicity. In some embodiments, administration of a cytokine inhibitor increases the activity of the administered IL-18 polypeptide variant or IL-18BP inhibitor.

As used herein, the term "pharmaceutically acceptable carrier" means a chemical composition with which its respective IL-18 signaling pathway activation modulator can be combined and which, once combined, can be used as a vehicle for administering its respective IL-18 signaling pathway activation modulator. 18 subject.

Sets

The present invention also relates to kits used in the methods of the invention. Such kits include various combinations of components used in any of the methods described herein, including, for example, an activator of IL-18 activity, such as an IL-18 polypeptide variant and/or an IL-18BP inhibitor, and/or IL-18 polypeptide variant assay materials. -18 or variant IL-18 nucleic acid, and/or materials for evaluating the activity of a variant IL-18 polypeptide or variant IL-18 nucleic acid, and/or instructional material. For example, in some embodiments, the kit includes components used to quantify IL-18 variant nucleic acid in a biological sample. In another embodiment, the kit includes components used to quantify an IL-18 polypeptide variant in a biological sample. In yet another embodiment, the kit includes components used to evaluate the activity (eg, enzymatic activity, ligand binding activity, and so on) of an IL-18 polypeptide variant in a biological sample.

In yet another embodiment, the kit includes components for monitoring the effectiveness of treatment of a subject in need thereof, containing instructional material and components for determining whether the level of activation of the IL-18 signaling pathway in a biological sample taken from the subject is modulated during or after treatment. In various embodiments, to determine whether the level of activation of the IL-18 signaling pathway is modulated in a biological sample taken from a subject, the level of activation of the IL-18 signaling pathway is compared with the level of at least one comparative control contained in the kit, such as a positive control, negative control, historical control, historical norm, or the level of another reference molecule in a biological sample. In specific embodiments, as an aid in monitoring treatment, the ratio of IL-18 signaling pathway activation to a reference molecule is determined.

Pharmaceutical Compositions and Their Administration

Compositions comprising a polypeptide, a fragment of a polypeptide, an IL-18 signaling pathway level or activity activator, or an IL-18BP level or activity inhibitor can be prepared and administered to a subject as described herein. As non-limiting examples, a composition identified as an activator of IL-18 activity, including IL-18 polypeptide variants, recombinant IL-18 polypeptide variants, and active fragments of IL-18 polypeptide variants, for the treatment and/or prevention of a disease or disorder, can be prepared and administered to the subject as described in the invention. As additional non-limiting examples, a composition identified as an IL-18BP inhibitor to be used, including a chemical compound, a protein, a peptide, a peptidomimetic, an antibody, a ribozyme, a small molecule chemical compound, an antisense nucleic acid molecule (e.g., siRNA, miRNA, etc.) , for the treatment and/or prevention of a disease or disorder, may be prepared and administered to a subject, as described in the invention.

The invention includes the preparation and use of pharmaceutical compositions comprising a composition used to treat or prevent the disease or disorder described in the invention as an active ingredient. Such a pharmaceutical composition may consist of only one active ingredient in a form suitable for administration to a subject, or the pharmaceutical composition may include the active ingredient and one or more pharmaceutically acceptable carriers, one or more additional ingredients, or some combination of both. The active ingredient may be present in the pharmaceutical composition in the form of a physiologically acceptable ester or salt, for example in combination with a physiologically acceptable cation or anion, as is known in the art. In various embodiments, the active ingredient is a polypeptide described herein, a fragment of a polypeptide, an IL-18 signaling pathway or activity level activator, an IL-18BP level or activity inhibitor, or a combination thereof.

As used herein, the term "pharmaceutically acceptable carrier" means a chemical composition with which its respective IL-18 signaling pathway activation modulator can be combined and which, once combined, can be used as a vehicle for administering its respective modulator (e.g., activator, inhibitor, and so on) to the subject.

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

Pharmaceutical compositions used to practice the invention may be administered to deliver doses ranging from about 0.1 ng/kg/day to 100 mg/kg/day or more.

In various embodiments, the pharmaceutical compositions used in the methods of the invention may be administered, for example, systemically, parenterally, or topically, for example, in oral dosage forms, inhalation dosage forms, including solid or aerosol forms, and in topical dosage forms. applications or in other similar forms. In addition to the appropriate therapeutic composition, such pharmaceutical compositions may contain pharmaceutically acceptable carriers and other known ingredients for more effective or more convenient administration of the drug. Other possible forms such as nanoparticles, liposomes, other preparations containing the active ingredient, and immunological systems can also be used to administer the appropriate modulator in accordance with the methods of the invention.

The carrier may carry the agent of interest (eg, a variant IL-18 polypeptide) in a variety of ways, including covalent bonding, either directly or through a linker group, and non-covalent associations. Suitable carriers that form a covalent bond include proteins such as albumins, peptides, and polysaccharides such as aminodextran, each of which has many fragment attachment sites. The carrier may also carry a variant IL-18 polypeptide by non-covalent associations, such as non-covalent binding, or by encapsulation. For the purposes of the present invention, the solvent may be soluble or insoluble.

Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the doses and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechin; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 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 dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (eg Zn-protein complexes); and/or nonionic surfactants such as TWEEN ^TM , PLURONICS ^TM or polyethylene glycol (PEG). Dosage forms intended for in vivo administration must be sterile. This is easily achieved by filtration through sterile filter membranes.

The active ingredients may also be encapsulated in a microcapsule prepared, for example, by coacervation or interfacial polymerization methods, e.g., hydroxymethyl cellulose or gelatin microcapsule and polymethyl methacrylate microcapsule, respectively, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions). , nanoparticles and nanocapsules) or in macroemulsion. These methods are described in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

The compositions may be in the form of injectables, either in the form of liquid solutions or suspensions; they may also be prepared in solid form for use in dissolution or suspension in liquid media prior to injection. The drug may also be emulsified or encapsulated in liposomes or microparticles such as a polyglycolide or copolymer to enhance the adjuvant effect described above (Langer, Science 249: 1527, 1990 and Hanes, Advanced Drug Delivery Reviews 28: 97-119, 1997) . The medicaments of this invention may be administered in the form of a depot injection or an implantable preparation which may be formulated in such a way as to allow sustained or pulsatile release of the active ingredient. Pharmaceutical compositions are typically prepared sterile, substantially isotonic, and in strict adherence to all US Food and Drug Administration Good Manufacturing Practice (GMP) guidelines.

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 of the other ingredients of a pharmaceutical composition and that does not adversely affect the subject to whom the composition is administered.

Dosage forms of the pharmaceutical compositions described in the invention can be prepared by any method known in pharmacology or a method that will be developed in the future. Typically, such preparations include the step of contacting the active ingredient with the carrier or one or more other accessory ingredients and then, if necessary or desired, shaping or packing the product into the desired single dose or multi-dose dosage form.

While the descriptions of pharmaceutical compositions provided herein are primarily directed to pharmaceutical compositions that are used by a physician for administration to humans, it should nevertheless be apparent to one of ordinary skill in the art that such compositions can be widely used for administration to a variety of animals. Modification of pharmaceutical compositions for administration to humans to provide compositions for administration to various animals is well known and one of ordinary skill in the art of veterinary pharmacology can consider and implement such modification, if necessary, by simple experimentation.

Pharmaceutical compositions that are used in the methods of the invention may be formulated, packaged or marketed in dosage forms for oral, rectal, vaginal, parenteral, topical, pulmonary, intranasal, buccal, intravenous, transdermal, intralesional, subcutaneous , intramuscular, ophthalmic, intrathecal and other known routes of administration. Other proposed dosage forms include promising nanoparticles, liposomal formulations, other formulations containing the active ingredient, and immunologically based formulations.

The pharmaceutical composition of the invention may be formulated, packaged, or marketed in bulk, single dose, or multiple single dose formulations. Used in the invention, the term "single dose" means a discrete amount of a pharmaceutical composition comprising a given amount of the active ingredient. The amount of active ingredient is usually equal to the dose of the active ingredient to be administered to the subject, or equal to a convenient portion of such dose, for example, half or one third of such dose.

The relative amounts of active ingredient, pharmaceutically acceptable carrier, and any additional ingredients in a pharmaceutical composition of the invention will vary depending on the individual, height and weight, and general health of the subject being treated, and furthermore, depending on the route of administration of the composition. As an example, the composition may contain from 0.1 to 100% (by weight) of the active ingredient.

In addition to the active ingredient, the pharmaceutical composition of the invention may further comprise one or more additional pharmaceutically active agents.

Controlled or sustained release dosage forms of the pharmaceutical composition of the invention can be prepared using conventional technology.

The dosage form of the pharmaceutical composition of the invention for oral administration may be formulated, packaged, or marketed as a discrete solid unit dose including, but not limited to, a tablet, hard or soft capsule, cachet, lozenge, or lozenge. containing a predetermined amount of the active ingredient. Other dosage forms useful for oral administration include, but are not limited to, powder or granular form, aqueous or oily suspension, aqueous or oily solution or emulsion.

Pharmaceutically acceptable excipients used in the manufacture of pharmaceutical compositions include, but are not limited to, inert diluents, granulating and disintegrating agents, binders, and lubricants. Known dispersants include, but are not limited to, potato starch and sodium starch glycolate. 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 granulating and disintegrating agents include, but are not limited to, cornstarch and alginic acid. Known binders include, but are not limited to, gelatin, gum arabic, pregelatinized cornstarch, polyvinylpyrrolidone, and hydroxypropyl methylcellulose. Known lubricants include, but are not limited to, magnesium stearate, stearic acid, silica, and talc.

Liquid dosage forms of the pharmaceutical composition of the invention may be prepared, packaged or marketed in either liquid form or as a dry product intended to be reconstituted with water or other suitable vehicle prior to use.

Liquid suspensions may be prepared using conventional methods for suspension of the active ingredient in an aqueous or oily medium. Aqueous media include, for example, water and isotonic saline. Oil media include, for example, almond oil, oil esters, ethyl alcohol, vegetable oils such as peanut, olive, sesame or coconut oil, fractionated vegetable oils, and mineral oils such as liquid paraffin. Liquid suspensions may additionally contain one or more additional ingredients, including, but not limited to, suspending agents, dispersing or wetting agents, emulsifiers, emollients, preservatives, buffers, salts, flavors, colors, and sweeteners. Oil suspensions may additionally include a thickener.

Known suspending agents include, but are not limited to, sorbitol syrup, hydrogenated edible fats, sodium alginate, polyvinylpyrrolidone, gum tragacanth, gum arabic, and cellulose derivatives such as sodium carboxymethylcellulose, methylcellulose, and hydroxypropylmethylcellulose. Known dispersing or wetting agents include, but are not limited to, natural phosphatides such as lecithin, alkylene oxide condensates with a fatty acid, with a long chain aliphatic alcohol, with a partial ester derived from a fatty acid and hexitol, or with a partial ester derived from from fatty acid and hexitan anhydride (for example, polyoxyethylene stearate, heptadecaethyleneoxycetanol, polyoxyethylene sorbitol monooleate and polyoxyethylene 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 para-hydroxybenzoates, ascorbic acid and sorbic acid. Known sweeteners include, for example, glycerin, propylene glycol, sorbitol, sucrose and saccharin. Known thickeners for oil suspensions include, for example, beeswax, paraffin wax and cetyl alcohol.

Liquid solutions of the active ingredient in aqueous or oily vehicles can be prepared in much the same manner as liquid suspensions, with the main difference being that the active ingredient is dissolved rather than suspended in a solvent. Liquid solutions of the pharmaceutical composition of the invention may include each of the components described in connection with liquid suspensions, it should be borne in mind that suspending agents will not necessarily help dissolve the active ingredients in the solvent. Aqueous solvents include, for example, water and isotonic saline. Oil solvents include, for example, almond oil, oil esters, ethyl alcohol, vegetable oils such as peanut, olive, sesame or coconut oil, fractionated vegetable oils and mineral oils such as liquid paraffin.

Powder and granular dosage forms of the pharmaceutical preparation according to the invention can be prepared using known methods. Such dosage forms may be administered directly to the subject, but may be used, for example, to form tablets, to fill capsules, or to prepare an aqueous or oily suspension or solution by adding an aqueous or oily medium thereto. Each of these dosage forms may further include one or more dispersing or wetting agents, suspending agents and preservatives. Additional excipients such as fillers and sweeteners, flavors or colors may also be included in these dosage forms.

The pharmaceutical composition of the invention may also be formulated, packaged or marketed as an oil-in-water emulsion or a water-in-oil emulsion. The oil phase may be a vegetable oil such as olive or peanut oil, a mineral oil such as liquid paraffin, or a combination thereof. Such compositions may additionally contain one or more emulsifiers such as natural gums such as gum arabic or gum tragacanth, natural phosphatides such as soy or lecithin phosphatide, esters or partial esters derived from combinations of fatty acids and hexitol anhydrides, such as sorbitan monooleate, and the condensation products of such partial esters with ethylene oxide, such as polyoxyethylene sorbitan monooleate. These emulsions may also contain additional ingredients including, for example, sweeteners or flavors.

Techniques for impregnating with a chemical composition or coating a material with a chemical composition are well known in the art and include, but are not limited to, methods for applying or bonding a chemical composition to a surface, methods for introducing a chemical composition into a material structure during material synthesis ( that is, such a material that decomposes as a result of physiological action) and methods for absorbing an aqueous or oily solution or suspension into the absorbent material, with or without subsequent drying.

As used herein, the term "parenteral administration" of a pharmaceutical composition includes any route of administration characterized by the formation of a puncture or incision in the tissue of the subject by mechanical action and the introduction of the pharmaceutical composition through the puncture or incision in the tissue. Therefore, parenteral administration includes, but is not limited to, administering the pharmaceutical composition by injecting the composition, by applying the composition through a surgical incision, by applying the composition through a tissue-penetrating non-surgical wound, and the like. In particular, parenteral administration is intended to include, but is not limited to, dermal, subcutaneous, intraperitoneal, intravenous, intramuscular, intracisternal injection, and renal dialysis infusion techniques.

Dosage forms of the pharmaceutical composition used for parenteral administration contain the active ingredient in combination with a pharmaceutically acceptable carrier such as sterile water or sterile isotonic saline. Such dosage forms may be formulated, packaged, or marketed as drugs for bolus administration or for continuous administration. Injectable dosage forms may be prepared, packaged or marketed in single dose dosage form, for example, in ampoules or in multi-dose containers containing a preservative. Dosage forms for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained release or biodegradable dosage forms. Such dosage forms may further contain one or more additional ingredients including, but not limited to, suspending, stabilizing or dispersing agents. In some parenteral formulation embodiments, the active ingredient is provided in dry (i.e., powder or granular) form for dissolution in a suitable medium (e.g., sterile, pyrogen-free water) prior to parenteral administration of the composition thus prepared.

Pharmaceutical compositions may be formulated, packaged or marketed as a sterile injectable aqueous or oily suspension or solution. This suspension or solution may be prepared in accordance with the prior art and may include, in addition to the active ingredients, additional ingredients such as the dispersing agents, wetting agents or suspending agents described in the invention. Such sterile injectable dosage forms may be prepared using a non-toxic parenterally acceptable diluent or solvent such as, for example, water or 1,3-butanediol. Other suitable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or diglycerides. Other parenterally useful dosage forms include dosage forms that contain the active ingredient in microcrystalline form, in a liposomal formulation, or as a component of biodegradable polymer systems. Compositions for sustained release or for implantation may contain pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt.

Dosage forms suitable for topical administration include, but are not limited to, liquid or semi-liquid preparations such as liniments, lotions, oil-in-water or water-in-oil emulsions such as creams, ointments or pastes, and solutions. or suspensions. Dosage forms for topical administration may contain, for example, from about 1% to about 10% (by weight) of the active ingredient, although the concentration of the active ingredient may reach the solubility limit of the active ingredient in the solvent. In addition, topical dosage forms may include one or more additional ingredients described in the invention.

The pharmaceutical composition of the invention may be formulated, packaged or marketed in a dosage form for pulmonary administration through the oral cavity. Such a dosage form may include dry particles that contain the active ingredient and that have a diameter in the range from about 0.5 to about 7 nanometers, and preferably from about 1 to about 6 nanometers. Conveniently, such compositions are in the form of dry powders for administration using a device containing a dry powder reservoir into which a propellant stream can be directed to disperse the powder, or using a self-propelled solvent dispensing powder container, such as a device comprising an active ingredient. , dissolved or suspended in a low boiling propellant in a sealed container. Preferably, such powders include particles in which at least 98% of the particles, by mass, have a diameter greater than 0.5 nanometers, and at least 95% of the particles, by number, have a diameter of less than 7 nanometers. More preferably, at least 95% of the particles, by mass, have a diameter greater than 1 nanometer, and at least 90% of the particles, by number, have a diameter of less than 6 nanometers. Preferably, the dry powder compositions comprise a solid, finely divided powder diluent such as sugar, and are conveniently formulated in a single dose dosage form.

Low boiling propellants typically include liquid propellants having a boiling point below 18° C. at atmospheric pressure. Typically, the propellant may comprise from 50 to 99.9% (by weight) of the total weight of the composition, and the active ingredients may comprise from 0.1 to 20% (by weight) of the total weight of the composition. In addition, the propellant may include additional ingredients such as a liquid nonionic or solid anionic surfactant or a solid diluent (preferably having a particle size of the same order as the particles comprising the active ingredient).

Pharmaceutical compositions of the invention formulated for pulmonary delivery may also provide for administration of the active ingredient in the form of droplets of a solution or suspension. Such dosage forms may be prepared, dispensed or marketed as aqueous or diluted alcoholic solutions or suspensions, optionally sterile, containing the active ingredients, and they may be conveniently administered using any nebulization or pulverization device. In addition, such dosage forms may include one or more additional ingredients, including, but not limited to, a flavoring agent such as sodium saccharinate, a volatile oil, a buffering agent, a surfactant, or a preservative such as methyl hydroxybenzoate. Preferably, the droplets delivered by this route of administration have an average diameter in the range of about 0.1 to about 200 nanometers. The dosage forms described herein for pulmonary delivery may also be used for intranasal delivery of the pharmaceutical composition of the invention. Another dosage form used for intranasal administration is a large particle size powder comprising the active ingredient and having an average particle size of from about 0.2 to 500 micrometers.

Such a dosage form is administered by inhaling through the nose, that is, by rapidly inhaling the powder from a container held close to the nostrils through the nasal passage. Dosage forms used for nasal administration may, for example, include as little as about 0.1% (w/w) up to 100% (w/w) of the active ingredient, and may further include one or more of the inventive additional ingredients.

The pharmaceutical composition of the invention may be formulated, packaged or marketed in a dosage form suitable for buccal administration. Such dosage forms may be, for example, tablets or lozenges prepared in a conventional manner and contain, for example, from 0.1 to 20% (by weight) of the active ingredient, with the remainder comprising an orally dissolving or disintegrating composition and optionally one or more of the additional ingredients described in the invention. Alternatively, buccal dosage forms may include a powder or a spray or spray solution or suspension containing the active ingredients. Preferably, such powder, spray, or spray dosage forms, when dispersed, have an average particle or droplet size in the range of from about 0.1 to about 200 nanometers and, in addition, may contain one or more of the additional ingredients described in the invention.

The pharmaceutical composition of the invention may be formulated, packaged or marketed in a dosage form for ophthalmic administration. Such dosage forms may be, for example, eye drops comprising, for example, 0.1-1.0% (by weight) of a solution or suspension of the active ingredients in an aqueous or oily liquid carrier. Such drops may additionally include buffer substances, salts, or one or more other additional ingredients described in the invention. Other useful ophthalmically administered dosage forms include those that contain the active ingredients in microcrystalline form or in a liposomal formulation.

Used in the invention "additional ingredients" include, but are not limited to, one or more of the following ingredients: excipients; surfactants; dispersing agents, inert diluents, granulating and disintegrating agents, binders, lubricants, sweetening agents, flavoring agents, coloring agents, preservatives, physiologically degradable compositions such as gelatin, aqueous media and solvents, oily media and solvents, suspending agents, dispersing or wetting agents, emulsifiers, emollients, buffers, salts, thickeners, fillers, emulsifiers, antioxidants, antibiotics, antifungals, stabilizers, and pharmaceutically acceptable polymeric or hydrophobic materials. Other "additional ingredients" that may be included in the pharmaceutical compositions of the invention are known in the art and are described, for example, in Genaro, ed., 1985, Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA., full content which is included in the present invention by reference to it.

Typical dosages of a compound of the invention that may be administered to an animal, preferably a human, are in the range of about 0.001 to about 1000 mg per kilogram of animal body weight. The exact amount of dose administered will vary depending on a number of factors including, but not limited to, the type of animal and the type of disease or disorder being treated, the age of the animal, and the route of administration. Preferably, the dose of the compound will range from about 0.1 to about 10 mg per kilogram of animal body weight. The compound may be administered to the animal as frequently as several times a day, or it may be administered less frequently, 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 even more rarely. Dosing frequency can be readily determined by one of skill in the art and will depend on a number of factors such as, but not limited to, the type and severity of the disease or disorder being treated, the type and age of the animal, and other such factors.

EXAMPLES OF EXPERIMENTAL STUDIES

Further, the invention is described in detail using the following examples of experimental studies. These examples are provided for illustrative purposes only and should not be construed as limitations unless otherwise noted. Therefore, the invention should in no way be considered to be limited to the following examples, on the contrary, it should be considered that the invention includes all possible variations that become apparent as a result of familiarization with the ideas described in the invention.

It can be assumed that, even without further description of the invention, any person skilled in the art, using the above description and the following illustrative examples, can recreate and use the present invention and practice the methods claimed in the patent claims. Therefore, the following working examples should not be considered in any way as limiting the rest of the invention.

Example 1 IL-18 polypeptide variants

IL-18 is a pro-inflammatory cytokine that can stimulate T cells, NK cells, and myeloid cells. It has been proposed as an immunotherapeutic agent in the treatment of cancer due to its ability to stimulate antitumor immunocytes. As shown in the invention, the therapeutic efficacy of treatment with recombinant IL-18 is largely limited as a result of overregulation of its natural endogenous soluble inhibitor IL-18BP. The present invention is based, inter alia, on the development of human and murine IL-18 variants that are substantially independent of IL-18BP. These cytokine variants are characterized by a change in relative preference for receptors (IL-18Rα and IL-18BP) ranging from hundreds of thousands to more than a million times. These variants demonstrate potent antitumor activity in preclinical tumor models, both as monotherapy and in combination with immune checkpoint inhibitors such as anti-PD-1 antibody. With regard to additional application, it is well known that IL-18 also has a positive therapeutic effect in the treatment of obesity, and the invention shows that the introduction of these variants significantly reduces the content of adipose tissue compared with treatment using WT IL-18. Therefore, in addition to antitumor immunotherapy, these new options can be used in the treatment of endocrinological, metabolic disorders and obesity.

In addition, the invention describes a further set of IL-18 variants that act as IL-18BP antagonists by exclusively binding to IL-18BP, but with no or significantly reduced binding of IL-18Rα. It is assumed that these proteins can be used to enhance the activity of endogenous IL-18 by neutralizing IL-18BP.

The following describes the materials and methods used in these experiments.

Protein expression and purification

Human IL-18, mouse IL-18 (amino acids 1-157) and variants thereof were assembled as blocks of gBlocks gene fragments (Integrated DNA Technologies, IDT) and cloned into the pET28a-smt vector to express N-terminal proteins labeled with SUMO , and C-terminal proteins labeled with hexahistidine in E. coli BL21 (DE3) Rosetta. Protein expression was induced with 0.5 mM IPTG (isopropylthiogalactoside) at 16°C for 20 hours. Fusion proteins were first purified using nickel loaded chelating resins and then SUMO tags were cleaved with SUMO protease. After that, the proteins were separated from the aggregates by successive precipitation of fractions with ammonium sulfate, and the aggregates were precipitated with 20% ammonium sulfate solution, and the target proteins were precipitated with 70% ammonium sulfate solution. Protein pellets were resuspended and reloaded onto nickel loaded chelating resins to remove SUMO marks and washed with 0.1% Triton X-114 to remove endotoxin. Finally, the eluted protein was buffer exchanged with PBS using a PD-10 column (GE Healthcare). The protein sample was tested for monodispersity by gel permeation chromatography using an FPLC column (Bio-Rad) and SEC650 (Bio-Rad).

Human IL-18Rα ectodomain (amino acids 19-329), human IL-18Rβ ectodomain (amino acids 15-356) and IL-18BP (amino acids 31-194) were isolated and purified using a baculovirus expression system. Briefly, all construct sequences were cloned into the pAcBN-BH3 vector (BD Biosciences) with the gp67 N-terminal signal peptide and the AviTag™ C-terminal tag and hexahistidine tag. Insect Spodoptera frugiperda (Sf9) cells cultured at 27° C. in serum-free SF900 II medium (Invitrogen) were transfected with plasmid constructs to generate a high titer recombinant virus, which was then amplified. Insect Trichopulsia ni (High-Five) (Invitrogen) cells grown in Insect Xpress medium (Lonza) at 27°C were infected with viruses to express the recombinant protein. Three days post-infection, proteins were recovered by Ni-NTA affinity chromatography (QIAGEN), concentrated and purified to >98% homogeneity using SEC650 biosizing columns (Bio-Rad) equilibrated in 10 mM HEPES ( pH 7.5) and 150 mM NaCl.

The ectodomain of mouse IL-18Rα (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 pEZT_D_Lux vector of the BacMam expression system with an N-terminal H7 signal peptide and an AviTag™ C-terminal tag and a hexahistidine tag. Expi293 cells cultured at 37°C in Expi293 expression medium (Thermo Fisher) were transfected with plasmids using the ExpiFectamine 293 transfection kit (Thermo Fisher) according to the manufacturer's instructions. Cells were harvested 3-5 days after transfection. Protein purification methods were the same as for human proteins.

For protein biotinylation, the C-terminal biotin acceptor peptide (AviTag)-GLNDIFEAQKIEWHE was fused to all IL-18 receptor constructs. Protein biotinylation was performed with BirA biotin ligase enzyme dissolved in 0.1 mM bicine (pH 8.3), 10 mM ATP, 10 mM magnesium acetate and 0.5 mM biotin (Sigma). Proteins were purified using SEC650 biomolecular size separation columns as described above.

Yeast Display IL-18

The human and mouse IL-18 (IDT) gene block was synthesized and cloned into the pYAL vector and replicated on the Saccharomyces cerevisiae EBY100 strain. Grow individual colonies of yeast IL-18 overnight at 30°C in SDCAA liquid medium, and induced in SGCAA liquid medium for 1 day at 20°C. Yeast IL-18 display levels were confirmed by flow cytometry using an anti-cMyc tag antibody (anti-myc-PE; Cell Signaling Technologies). Receptor staining was performed with biotinylated IL-18Rα (in the presence or absence of IL-18Rβ) or with biotinylated IL-18BP in PBS supplemented with 0.5% BSA and 2 mM EDTA (PBE) on ice. All analysis was performed on a Sony SA3800 flow cytometer.

Construction of a human IL-18 library and selection

For the first library of human IL-18 resistant to the decoy receptor, fourteen contact residues of hIL-18Rα and hIL-18BP were identified at homologous positions in hIL-18 (Table 1) by aligning the structure of the hIL-18/hIL-18Rα/hIL- 18Rβ (Protein Data Bank (PDB ID) code 3OW4) with structure IL-18/IL-18BP (PDB ID 3F62). A library was designed to randomize these residues using assembly PCR with the degenerate primers shown in Table 2. The theoretical diversity of the library was ~1.96 x 10 ¹¹ unique protein sequences. The PCR products were then amplified with primers having homology to the pYAL vector and co-electroporated along with linearized pYAL into EBY100 yeast. The resulting library contained 2.5×10 ⁸ transformants.

For the second decoy receptor resistant human IL-18 library in hIL-18, eleven contact residues of hIL-18Rα and hIL-18BP were selected for randomization with a theoretical diversity of 3.44 x 10 ⁹ variants (shown in Figure 7A). A library randomizing these residues was constructed using assembly PCR with degenerate primers and co-electroporated with pYAL into yeast EBY100. The resulting library had a diversity of 6×10 ⁸ transformants.

Table 1 Design of the first human IL-18 library

Remainder codon Potential Remains 1Y YNT Y, F, S, C, L, P, H, R L5 NWT L, F, I, Y, H, N, V, D 8K MRA K, R, R, Q 51M RNS M, I, T, N, K, S, R, V, A, D, E, G 53K ARA K, R 55S RRW S, R, G, G, N, K, D, E 59G RNA G, E, A, V, I, T, K, R 60M VDG M, K, R, L, Q, R, V, E, G 103Q VAW Q, K, E, D, N, H 105S RRW S, K, R, N, D, E, G, G 110D VAW D, E, K, N, Q, H 111N NAT N, D, H, Y 153V RHT V, A, D, I, T, N 155N VAW N, K, D, E, Q, H

Table 2 Assembly primers of the first human IL-18 library

primer Sequence (from 5' to 3') hIL18Lib1 CATTTTCATTAAGATGCAGTTACTTCGCTGTTTTTCAATATTTTCTGTTATTGCTAGC (SEQ ID NO: 1) hIL18Lib2 AATTACGGATGACCGAAAGTYKGGATTCAWNCTTGCCGAAANRTGCTAAAACGCTAGCAATAACAGAAAATATTGAAAAA (SEQ ID NO: 2) hIL18Lib3 ACTTTCGGTCATCCGTAATTTGAACGACCAAGTCCTTTTTATTTGACCAGGG (SEQ ID NO: 3) hIL18Lib4 ACTATCCGTCATATCCTCGAATAAGGGACGATTGCCCTGGTCAATAAAAAGGACT (SEQ ID NO: 4) hIL18Lib5 CTTATTCGAGGATATGACGGATAGTGATTGCCGTGACAACGCCC (SEQ ID NO: 5) hIL18Lib6 ACTGAGATTGTTACGCCHBTNYACGGGGTTGWYYATCTYTATASNYAGAGATGATGAAAATTGTACGAGGGGCGTTGTCACGG (SEQ ID NO: 6) hIL18Lib7 GGCGGTAACAATCTCAGTTAAGTGCGAAAAAATCTCGACACTTTCTTGTGAA (SEQ ID NO: 7) hIL18Lib8 GGTTCATTTCCTTGAACGAAATGATCTTGTTTTCACAAGAAAGTGTCGAGATT (SEQ ID NO: 8) hIL18Lib9 CATTTCGTTCAAGGAAATGAACCCGCCGGATAATATCAAGGATACAAAATCAGATATTATTT (SEQ ID NO: 9) hIL18Lib10 TGATGAGCTCTCGAATTGCATCTTATNWTBGTGTCCAGGCACWYYACGWTBGAAGAAAATAATATCTGATTTTGTATCCTTGATATTA (SEQ ID NO: 10) hIL18Lib11 ATAAGATGCAATTCGAGAGCTCATCATACGAAGGTTACTTTTTAGCCTGCG (SEQ ID NO: 11) hIL18Lib12 AATTAACTTAAACAGGTCGCGCTCCTTCTCGCAGGCTAAAAAGTAACCTT (SEQ ID NO: 12) hIL18Lib13 GCGACCTGTTTAAGTTAATTCTTAAGAAAGAAGATGAGTTGGGGGATCG (SEQ ID NO: 13) hIL18Lib14 CCAGAACCACCGTCCTCWTBCTGADYGGTAAACATGATGCTACGATCCCCCCAACTCATCTT (SEQ ID NO: 14) hIL18Lib15 GAGGACGGTGGTTCTGGATCCGAACAAAAGCTTATCTCCGAAGAAGACTTGG (SEQ ID NO: 15) hIL18Lib16 CCACCAGATCCACCACCACCCAAGTCTTCTTCGGAGATAAG (SEQ ID NO: 16)

For both libraries, transformed yeasts were recovered and propagated in liquid synthetic dextrose with casamino acids (SDCAA) medium at 30°C and induced by a 1:10 dilution in liquid synthetic galactose with casamino acids (SGCAA) medium and cultured at 20°C for 24 hours. An appropriate amount of induced yeast was used in each round to ensure at least 10-fold coverage of the expected library diversity at each stage and no less than 10 ⁸ cells. All selection steps were performed at 4° C. using PBE buffer (PBS with 0.5% BSA and 2 mM EDTA). For the first generation library, the screening reagents for each round are shown in Table 3. In the first round, yeast was negatively screened with anti-Cy5/AlexaFluor 647 microspheres (Miltenyi) and an LS MACS column (Miltenyi) to remove non-specific binding beads. Positive selection was performed by labeling the yeast with 1 μM biotinylated hIL-18Rα for 1 hour at 4°C followed by magnetic selection using anti-Cy5/AlexaFluor 647 microspheres and an LS MACS column. In the second round, negative selection was performed with 1 μM biotinylated IL-18BP and positive selection was performed in the same way as in the first round. For rounds 3-5, selection was performed by incubating the yeast with 100 nM (rounds 3-4) or 10 nM (round 5) biotinylated IL-18Rα and 250 nM preformed hIL-18BP/SA-PE tetramers coated with biotin. After competitive binding, the yeast was washed and labeled with SA AlexaFluor 647 to detect IL-18Rα. Display levels were determined by staining with AlexaFluor 488 conjugated anti-cMyc antibody (Cell Signaling Technologies) and the top 1% of display-normalized binding IL-18Rα (from non-binding IL-18BP) was isolated by FACS using a Sony cell sorter SA3800. After each selection round, the recovered yeasts were propagated in SDCAA medium at 30° C. overnight and then induced at 20° C. by a 1:10 dilution in SGCAA medium for 24 hours.

Library V-2.0 human DR-IL-18 was selected in a similar way by carrying out specific stages of selection, presented in detail in figure 7B.

Mouse IL-18 library construction and selection

The design and selection procedure is the same as for human IL-18, but with the following changes. Library construction was based on information obtained from computer simulations of the structure of the mouse IL-18/receptor complex (predicted using the Phyer2.0 program). Thirteen positions were selected for randomization (Table 3) using the primers described in Table 4. As a result of co-electroporation with pYAL, a library of 4×10 ⁸ transformants was obtained. Screening reagents used for each round are shown in Table 5.

Table 3 Mouse IL-18 Library Design

Remainder codon Potential Remains 1N NWT F, Y, L, H, I, N, V, D 50M RNS M, I, T, N, K, S, R, V, A, D, E, G 51Y NRN Y, K, R, D, E 52K VNS L, P, H, Q, R, I, M, T, N, K, S, V, A, D, E, G 54S RRW S, R, G, G, N, K, D, E 55E VRN E, K, N, R, S, R, H 56V VNV V, S, P, T, A, K, R 57R RVW R, D, E, S, T 58G RNA G, E, A, V, I, T, K, R 59L VDR L, K, R, Q, R, V, E, G 104R NDH R, D, E, N, Y, F, I, L, V 109N NAT N, D, H, Y 151L VHY L, V, A, D, I, T, N

Table 4 Assembly Primers of the Mouse IL-18 Library

primer Sequence (from 5' to 3') mIL18lib1 CATTTTCATTAAGATGCAGTTACTTCGCTGTTTTTCAATATTTTCTGTTATTGCTAGCGTTT (SEQ ID NO: 17) mIL18lib2 TTGTACAGTGAAGTCGGCCAAAAWNTGCTAAAACGCTAGCAATAACAGAAAATAT (SEQ ID NO: 18) mIL18lib3 GCCGACTTCACTGTACAACGCAGTAATACGGAATATAAATGACCAAGTTCTCTTCGTT (SEQ ID NO: 19) mIL18lib4 TTGATCAATATCAGTCATATCCTCGAACACAGGCTGTCTTTTGTCAACGAAGAGAACTTGGTCATTT (SEQ ID NO: 20) mIL18lib5 GTGTTCGAGGATATGACTGATATTGATCAAAGTGCCAGTGAACCCCAGACCAGA (SEQ ID NO: 21) mIL18lib6 TCACAGAGAGGGTCACAGCYHBTNYWBYBNBNYBWYYGTCSNBNYNSNYGTATATTATCAGTCTGGTCTGGGGTTCAC (SEQ ID NO: 22) mIL18lib7 GCTGTGACCCTCTCTGTGAAGGATAGTAAAATGTCTACCCTCTCCTGTAAGAACAAGA (SEQ ID NO: 23) mIL18lib8 GTATATCATCAATATTTTCAGGTGGATCCATTTCCTCAAAGGAAATGATCTTGTTCTTACAGGAGAGGG (SEQ ID NO: 24) mIL18lib9 AATGGATCCACTGAAAATATTGATGATATACAAAGTGATCTCATATTCTTTCAGAAANDHGTTCCAGGACACNATAAGATGGAGTTTGAATCTTCACT (SEQ ID NO: 25) mIL18lib10 CCTTTTGGCAAGCAAGAAAGTGTCCTTCATACAGTGAAGATTCAAACTCCATCTTAT (SEQ ID NO: 26) mIL18lib11 CTTTCTTGCTTGCCAAAAGGAAGATGATGCTTCAAACTCATTCTGAAAAAAAAGGATGA (SEQ ID NO: 27) mIL18lib12 CCACCACTTTGATGTAAGTTAGTRDBAGTGAACATTACAGATTTATCCCCATTTTCATCCTTTTTTTTCAGAATGAG (SEQ ID NO: 28) mIL18lib13 ACTAACTTACATCAAAGTGGTGGTTCTGGATCCGAACAAAAGCTTATCTCCGAAGAAGA (SEQ ID NO: 29)

Table 5. Brief description of reagents for library screening

Human IL-18 library selection Mouse IL-18 library selection negative selection positive selection negative selection positive selection Round 1 Only SA spheres 1 μM hIL-18Rα-SA spheres ------ 1000 nM IL-18Ra-SA spheres Round 2 1 μM IL-18BP 1 μM IL-18Rα-SA spheres ------ 1 μM IL-18Rα Round 3 1 μM IL-18BP 100 nM hIL-18Ra 1 µM IL-18BP 1 μM IL-18Rα Round 4 1 µM IL-18BP 10 nM hIL-18Rα 1 µM IL-18BP 100 nM IL-18Ra Round 5 250 nM IL-18BP tetramer 10 nM hIL-18Rα 1 µM IL-18BP 10 nM IL-18Ra Round 6 ------ ------ 250 nM IL-18BP tetramer 200 nM IL-18Ra

Surface plasmon resonance

The experiments were carried out using the Biacore T100 system at 25°C. Biotinylated IL-18Rα or IL-18BP was immobilized on a Biacore biotin capture chip (Series S CAP sensor chip, GE Healthcare) to give Rmax ~50 RU (IL-18Rα) or ~10 RU (IL-18BP). Measurements were made for serially diluted IL-18 variants in HEPES buffered Saline-P+ (10 mM HEPES, pH 7.4, 150 mM NaCl, 0.005% P20 surfactant). The surface was regenerated with three 60 second injections of regeneration buffer (3/4 (v/v) 8 M guanidine hydrochloride with 1/4 (v/v) 1 M sodium hydroxide). The experiments were carried out simultaneously on several channels to increase the number of observations. All data were analyzed with Biacore T100 scoring software version 2.0 with a 1:1 Langmuir binding model.

cell lines

HEK-Blue IL-18 sensory 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 blasticidin, 180 µg/ml zeocin and 200 µg/ml hygromycin. YUMMER1.7 melanoma cells were cultured and prepared as described in Wang et al., 2017, Pigment Cell Melanoma Res., 30 (4): 428-435.

Investigation of cytokine activity using HEK-Blue cells

To measure cytokine activity, 50,000 HEK-Blue IL-18 sensor cells per well of a 96-well flat bottom plate were incubated with recombinant human IL-18 at successively decreasing concentrations in a total volume of 200 µl of complete medium. After 20-24 hours incubation at 37°C and 5% CO ₂ , 30 µl of cell culture supernatant was mixed with 170 µl of QUANTI-Blue assay medium (InvivoGen) and incubated at 37°C and 5% CO ₂ until until an observable color change from pink to blue occurs (0.5-4 hours). Alkaline phosphatase levels were quantified using a spectrophotometer at a wavelength of 655 nm. Cytokine activity was determined by calculating the relative absorbance value (as a percentage of the maximum absorbance measured at 655 nm) for each cytokine under study.

In IL-18BP blockade experiments, a fixed concentration of recombinant human IL-18 was preincubated with recombinant human IL-18BP at successively decreasing concentrations for 1 hour at 4°C. After that, protein mixtures were added to HEK-Blue IL-18 sensor cells and analyzed as described.

Mice

For in vivo experiments in mice, wild-type C57BL/6 mice (aged 6-9 weeks) from the Jackson laboratory were used. The experimental groups were comparable in terms of body weight, sex and age. All animal experiments were performed in accordance with the approval of the Yale University Institutional Animal Care and Use Committee.

Pharmacodynamic and pharmacokinetic studies in vivo

Mice (n=9 per group) were injected daily intraperitoneally (i.p.) with 1 mg/kg of recombinant IL-18 (WT or mCS2 variant) or PBS as control placebo. On day 1, day 4 and day 7 of the experiment, 3 mice per group were sacrificed 5 hours after injection for blood sampling by cardiac puncture and subsequent analysis of blood plasma or leukocytes (see ELISA assay method for mouse IL-18BP, a multiplex immunoassay method based on Luminex technology for the analysis of mouse cytokines, as well as an immunophenotyping method using flow cytometry). During the 7 days of the experiment, body temperature was monitored daily using a Rodent BIO-TK8851 thermometer (Bioseb) and a RET 3 rectal probe for mice (Braintree Scientific Inc.). Body weight was monitored daily.

Preparation of plasma from whole blood

Plasma was prepared from whole blood using EDTA-coated Microtainer (BD) plasma separator tubes according to the manufacturer's instructions. Plasma samples were frozen once at -20°C before being used for analysis.

Analysis of IFN-γ and IL-18BP by ELISA

Human IFN-γ ELISA MAX Deluxe Set (BioLegend) with a sensitivity of 4 pg/mL and a detection range of 7.8-500 pg/mL was used to measure human IFN-γ levels in the cell culture supernatant according to the manufacturer's instructions. Quantikine Human IL-18BP Immunoassay (R&D Systems) was used to quantify human IL-18BP in cell culture supernatant with a sensitivity of 7.52 pg/mL and a detection range of 26.6-1700 pg/mL. Plasma levels of mouse IL-18BP were 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 analyzes, including sample preparation, were performed in accordance with the manufacturer's instructions.

Multiplex immunoassay method based on Luminex technology for the analysis of mouse cytokines

To quantify the levels of various cytokines in the blood plasma of mice, including IFN-γ and IL-12, a multiplex immunoassay based on Luminex technology (Bio-Rad) was performed using the Bio-Plex 200 system (Bio-Rad). Cytokines of interest were analyzed using Bio-Plex Pro Mouse Cytokine Standard 23-Plex (Group I) reconstituted in DMEM according to the manufacturer's instructions.

Immunophenotyping by flow cytometry

For leukocyte analysis, 100 µl of whole blood was collected into an EDTA-coated Microtainer (BD) plasma separator tube additionally containing 50 µl of heparin solution, and mixed by tipping the tube upside down several times. RBCs were lysed by adding ACK Lysing Buffer (VWR) and incubating for 3-5 minutes at room temperature. After addition of 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 specific fluorescently labeled antibodies for staining for subsequent analysis by flow cytometry. Staining was performed for 30 minutes at 4°C using the following antibodies: αCD4-AF700 (BioLegend), αCD8-APC (BioLegend), B220-APC-Cy7 (BioLegend), CD11b-PB (BioLegend), NK1.1-PE (BioLegend), NKp46-PE (BioLegend) and CD69-FITC (BioLegend). The leukocytes were then washed twice with MACS buffer as described above. Finally, cells were resuspended in 100 μl of MACS buffer and samples were taken for analysis on a flow cytometer (Sony SA3800). A 10 μl aliquot was taken for cell counting on an Invitrogen Countess II Automated Cell Counter (Thermo Fisher Scientific). FlowJo v10.3 software was used for data analysis, and leukocytes and single events were gated using forward and side scatter.

Conducting Tumor Treatment Experiments

0.5×10 ⁶ YUMMER1.7 cells were implanted subcutaneously in C57BL/6J mice. 7 days after implantation, when the mass of tumors reached approximately 50 mg, treatment was started. Mice were divided into treatment groups which included: 1) placebo (saline), 2) anti-PD1 antibody (rat clone RMP1-14, Bio X Cell, West Lebanon, New Hampshire, US), 3) wild-type IL-18 type, 4) mCS2, 5) wild-type IL-18+anti-PD-1 antibody, and 6) CS2 IL-18+anti-PD1 antibody. Anti-PD-1, wild-type IL-18 and mCS2 IL-18 were administered by intraperitoneal injection twice a week at a dose of 8 mg/kg, 0.32 mg/kg and 0.32 mg/kg, respectively. Mice were monitored for signs of clinical toxicity and tumor growth was determined twice a week with calipers. Mice were sacrificed when the tumor diameter at the site of greatest measurement reached or exceeded 1.5 cm; this was considered the end point of the survival study of the survival analysis.

Similarly, with minor changes, studies of B2m-deficient YUMMER1.7 cells were performed. 1.0 x 10 ⁶ cells were transplanted because the tumors grew more slowly than the parent strain. Treatment consisted of saline, anti-PD1 plus anti-CTLA4 and mCS2 administered in the same schedule and at the same doses as in the above studies.

The results of the experiments are described below.

IL-18 Axis as a Target in Cancer Immunotherapy

In order to identify potential signaling pathway nodes for immunotherapeutic intervention, we analyzed single cell RNA sequencing data from antitumor effector lymphocytes for the expression of cytokine signaling pathway components (Singer et al., 2016, Cell, 166: 1500-1511, e1509). As seen in Figure 1A, the receptor subunits for IL-18-IL-18Rα (i.e., IL-18R1) and IL-18Rβ (i.e., IL-18RAP), as well as IL-18 itself, were upregulated as in the programs activated lymphocytes, and in programs of dysfunctional lymphocytes. Further analysis of the immunological genome database (ImmGen) showed that the expression of both IL-18 receptor subunits correlated with the expression of T-cell depletion markers in CD4 and CD8 cells, including PD-1, Tim3, Lag3, and TIGIT, after long-term antigen exposure. (figure 1B). These expression features suggest that the IL-18 pathway can be used to selectively stimulate activated and dysfunctional/depleted T cells in tumors as a theoretically sound immunotherapy approach.

IL-18 is a Th1 cytokine, originally named "interferon gamma inducing factor" (IGIF) because of its ability to reliably stimulate T cells and NK cells to release interferon gamma (IFN-γ). IL-18 feedback inhibition achieved by IFN-γ-triggered induction of IL-18BP, a secreted high-affinity decoy receptor for IL-18 that sterically interferes with the ability of IL-18 to bind and activate its receptor (Figure 2A). Without advocating any particular theory, however, it is possible that this mechanism resembles the induction of PD-L1 by IFN-γ, indicating that IL-18BP may act as a "soluble immune checkpoint". Based on this hypothesis, it was found using the Cancer Gene Anthology and Genome Atlas (TCGA) database and the ONCOMINE database that IL-18BP is overregulated in several types of cancer, particularly breast cancer, gastric cancer, and brain cancer (Figure 2B). In addition, IL-18BP expression strongly correlates with expression of the key immune checkpoint PD-1 in tumors (r=0.65 and 0.78 in gastric and breast cancer, respectively, Figure 2C), suggesting that IL -18BP may also contribute to tumor escape from immunological surveillance and lymphocyte depletion.

Recombinant IL-18 has been administered to cancer patients in numerous clinical trials. It was found to be well tolerated even at high doses of 2 mg/kg with stable pharmacodynamic results including an increase in the number of activated CD69 ⁺ natural killer (NK) cells and a sharp increase in serum IFN-γ levels. However, a phase II clinical trial in patients with melanoma was terminated due to lack of treatment efficacy. An analysis of the presented pharmacodynamic results from these clinical trials shows that the efficacy of rIL-18 decreases with repeated dosing, while tachyphylaxis is observed in relation to the activation / expansion of peripheral NK cells and the release of cytokines (including IFN-γ and GM- CSF). The decrease in rIL-18 efficacy coincides with a marked increase in serum IL-18BP levels, more than doubling pre-treatment levels and often exceeding 100 ng/mL. Without wishing to be bound by any particular theory, however, it can be hypothesized that IL-18BP limits the efficacy of rIL-18 therapy and that IL-18 variants that do not respond to the inhibitory effect of IL-18BP may serve as effective antitumor immunotherapies. In addition, inhibitors of IL-18BP are likely to be effective in tumor immunotherapy.

Construction of IL-18 variants that are resistant to the inhibitory effect of IL-18BP (human DR-IL-18 variants)

To obtain variants of IL-18 that can signal through IL-18Rα/IL-18Rβ, but are not subject to the inhibitory effect of IL-18BP, directed development using yeast surface display technology was used. First, the structure of the ternary human IL-18:IL-18Rα:IL-18Rβ signaling pathway complex (PDB=3OW4) was analyzed and IL-18 residues that share a common interface with the signaling pathway complex and IL-18BP were identified (Figure 3A). Since the structure of hIL-18:hIL-18BP was not determined, a cognate complex between IL-18 and the viral (murine pox virus) orthologue IL-18BP (PDB=3F62) was used. A combinatorial library was created randomizing this set of residues to a defined set of alternatives (see Table 1) using degenerate oligonucleotide primers and assembly PCR. This library was electroporated into yeast along with the pYAL N-terminal yeast display vector to obtain a library with 2.5 x 10 ⁸ transformants. Using this library, directed development was carried out by performing sequential rounds of selection using magnetic and fluorescent cell sorting (FACS) with recombinant hIL-18Rα and negative selection with hIL-18BP, as shown in figure 3B. After five rounds of selection, a clear majority of the library clones reversed their relative preferences for hIL-18BP and hIL-18Rα compared to WT hIL-18 (Figure 3C). These clones were referred to as "DR-hIL-18" variants, where "DR" stands for decoy receptor resistant.

Sequencing of 96 clones from the pool after the fifth round revealed 21 unique sequences, which were analyzed for the possibility of creating four "consensus sequences", hCS1-4 (figure 4). To assess the binding affinity of these variants for hIL-18Rα and hIL-18BP, binding isotherms for binding hIL-18Rα and IL-18BP were generated using cytokine variants obtained by yeast display technology and flow cytometry. As seen in Figure 5A, DR-hIL-18 variants bound hIL-18Rα with comparable affinity to IL-18 WT, but exhibited significantly weaker binding to hIL-18BP, with apparent EC values₅₀for binding significantly exceeded 1 μM. To further characterize the receptor binding activity of DR-IL-18 variants, cytokines were recombinantly expressed and IL-18Rα and IL-18BP assayed by surface plasmon resonance (see representative sensorgrams in Figure 5B). These results are presented in Tables 6 and 7 and show that the DR-hIL-18 variants have a significantly lower, by several orders of magnitude, preference for IL-18BP compared to preference for IL-18Rα.

Table 6 Binding affinities of human IL-18 variants to IL-18Rα and IL-18BP based on binding isotherms obtained by yeast display technology.

Variant IL-18 K _D IL-18Rα (M) K _D IL-18BP (M) Ratio K _D :
IL-18BP/IL-18Ra Dissociation constant ratio normalized to WT IL-18 hIL-18WT 2.40E-08 7.08E-09 2.95E-01 1 hA8 5.77E-08 NBD > 3.47E+02 > 1.17E+03 hH3 8.38E-08 NBD > 2.39E+02 > 8.09E+02 hB9 1.27E-07 NBD > 1.57E+02 > 5.34E+02 hCS1 6.44E-08 1.93E-05 3.00E+02 1.02E+03 hCS2 9.15E-08 NBD > 2.19E+02 > 7.41E+02 hCS3 1.13E-07 1.16E-05 1.03E+02 3.48E+02 hCS4 1.60E-07 NBD > 1.25E+02 > 4.24E+02 6-31 4.1E-08 NBD 4.9E+02 > 7.2E+03 6-20 N.D. 3.4E-07 ------ ------ 6-12 1.7E-08 NBD 1.2E+03 >1.7E+04 6-27 4.2E-08 NBD 4.8E+02 >7.0E+03 6-29 3.7E-08 NBD 5.4E+02 >8.0E+03

NBD, no binding detected (20 μM was used for ratio calculations);

------, the value was not determined.

Table 7 Binding affinities of human IL-18 variants to IL-18Rα and IL-18BP based on Surface Plasmon Resonance (SPR) studies

Variant IL-18 K _D IL-18Rα (M) K _D IL-18BP (M) Ratio K _D :
IL-18BP/IL-18Ra Dissociation constant ratio normalized to WT IL-18 hIL-18WT 2.93E-09 1.90E-12 6.48E-04 1 hA8 ------ ------ ------ ------ hH3 ------ ------ ------ ------ hB9 ------ ------ ------ ------ hCS1 8.05E-09 1.94E-08 2.41E+00 3.72E+03 hCS2 1.31E-08 ------ ------ ------ hCS3 8.18E-09 1.86E-08 2.27E+00 3.50E+03 hCS4 4.38E-09 1.83E-07 4.18E+01 6.45E+04

------, the value was not determined.

Functional characterization of human DR-IL-18 variants

The aforementioned publication Kim et al., 2001, Proc Natl Acad Sci USA, 98 (6): 3304-9) describes 3 variants of hIL-18 with increased activity and an expected decrease in the inhibitory effect of IL-18BP on them, namely: E42A , K89A and E42A/K89A. These cytokine variants were generated by yeast display technology and the inhibitory effect of IL-18BP on IL-18Rα binding was assessed by flow cytometry. As seen in Figure 6A, while the DR-hIL-18 variants were refractory to inhibition of hIL-18Rα binding when exposed to hIL-18BP, the variants described in Kim et al. showed approximately equivalent neutralization of hIL-18BP compared to WT hIL-18. These results indicate that the DR-hIL-18 variants are independent of IL-18BP, while the variants described in Kim et al. are highly sensitive to the inhibitory effect of IL-18BP, similar to WT hIL-18.

To confirm that DR-hIL-18 can provide efficient signaling through the IL-18 receptor in cells, concentration-response experiments were performed using the HEK-blue IL-18 reporter cell line. In this system, IL-18R signaling is read by expression of secreted alkaline phosphatase (SEAP) downstream of NFκb/AP1 promoter molecules. In the absence of IL-18BP, DR-hIL-18 variants produced signaling EC ₅₀ values commensurate with WT hIL-18. However, DR-hIL-18 variants showed little to no inhibition when exposed to hIL-18BP, with no inhibition detected at 1 μM IL-18BP (Figure 6B). Taken together, the results of these studies indicate that DR-hIL-18 variants are biologically active and immune to the neutralizing action of IL-18BP in cell signaling.

Construction and characterization of second-generation variants of human IL-18 resistant to the inhibitory effects of IL-18BP (human DR-IL-18 variants v2.0)

To generate additional potentially more active human DR-IL-18 variants, a second human IL-18 library randomized at 11 positions was constructed (Figure 7A) and yeast was transformed as described above. The resulting library containing 6 x 10 ⁸ transformants was selected as shown in Figure 7B, resulting in a consistent preference for IL-18Rα over IL-18BP through successive selection steps (Figure 7C). Extracted 17 unique sequences after 5-6 rounds of selection (figure 8). Figure 9A shows that, compared to WT IL-18, clones 6-12, 6-27, 6-29, and 6-31 had equivalent or stronger binding to IL18Rα as measured from biotinylated IL18Rα binding isotherms using yeast display technology. However, Figure 9B shows that these clones do not show significant binding to IL-18BP. Figure 9C shows a thermostability measurement of yeast-display clones over a temperature range, which indicates that the clones are more thermostable than WT IL-18 over a temperature range of 7-13°C. These results are presented in Figure 9D.

Construction of IL-18 variants that are resistant to the inhibitory effect of IL-18BP (DR-IL-18 mouse variants)

Since the cross-species cross-reactivity of human and mouse IL-18 relative to IL-18Rα is low, mouse equivalents of DR-IL-18 variants were created that could be used for research in mice. Similar to the approach used above for hIL-18, a combinatorial library of mIL-18 variants was created randomizing a similar set of mIL-18Rα/mIL-18BP contact residues (Table 3), resulting in a library of 4 x 10 ⁸ transformants. This library was targeted development in a manner similar to that of the human IL-18 library; the selection strategy is shown in Figure 10A. After completion of six rounds of selection, the remaining clones had an almost complete preference for mIL-18Rα over mIL-18BP (Figure 10B). Analysis of 96 clones revealed 11 unique sequences from which two consensus sequences mCS1 and mCS2 were generated (Figure 10C). Yeast display binding isotherms and surface plasmon resonance experiments confirmed that these DR-IL-18 clones have even greater IL-18BP independence than the human IL-18 variants described here, with the mIL-18BP binding KD of well above 1 μm, and the amount of binding mIL-18Rα remains approximately equivalent to the amount of binding for WT mIL-18 (figure 11A, figure 11B, tables 8 and 9).

Table 8 Binding affinities of IL-18Rα and IL-18BP variants of mouse IL-18 based on binding isotherms obtained by yeast display technology

Variant IL-18 K _D IL-18Rα (M) K _D IL-18BP (M) Ratio K _D :
IL-18BP/IL-18Ra Dissociation constant ratio normalized to WT IL-18 mIL-18 WT 1.13E-08 2.13E-09 1.88E-01 1 mA7 1.35E-08 NBD > 7.41E+02 > 3.93E+03 mB1 1.79E-08 NBD > 5.59E+02 > 2.96E+03 mE8 4.20E-08 NBD > 2.38E+02 > 1.26E+03 mC1 4.30E-08 NBD > 2.33E+02 > 1.23E+03 mCS1 1.07E-08 NBD > 9.35E+02 > 4.96E+03 mCS2 1.13E-08 NBD > 8.85E+02 > 4.69E+03

NBD, no binding was found (10 μM was used for ratio calculations).

Table 9 Binding affinities of mouse IL-18 variants to IL-18Rα and IL-18BP based on Surface Plasmon Resonance (SPR) studies

Variant IL-18 K _D IL-18Rα (M) K _D IL-18BP (M) Ratio K _D :
IL-18BP/IL-18Ra Dissociation constant ratio normalized to WT IL-18 mIL-18 WT 6.00E-10 1.10E-12 1.83E-03 1 mA7 2.20E-10 1.39E-05 6.32E+04 3.45E+07 mB1 7.00E-10 1.47E-05 2.10E+04 1.15E+07 mE8 1.69E-09 NBD > 1.78E+04 > 9.68E+06 mC1 1.09E-09 2.87E-05 2.63E+04 1.44E+07 mCS1 5.40E-10 3.80E-06 7.04E+03 3.84E+06 mCS2 7.90E-11 1.05E-05 1.33E+05 7.25E+07

NBD, no binding was found (30 μM was used for ratio calculations).

In vivo pharmacodynamic studies of DR-IL-18 variants

In order to evaluate the biological effects resulting from the administration of DR-IL-18 variants in vivo, pharmacodynamic studies were performed in mice comparing WT mIL-18 with mCS2. In the first study, mice were treated with placebo (PBS), mIL-18 (1 mg/kg/day) or mCS2 (1 mg/kg/day) for a total of seven injections (Figure 12A). Analysis of peripheral blood phenotypes by flow cytometry showed that administration of both WT mIL-18 and mCS2 led to an increase in the number of peripheral blood NK cells by more than ten times and the number of peripheral blood monocytes by more than five times compared with placebo administration. without significantly affecting the total number of CD4 and CD8 cells (figure 12B). A study of cellular activation status by CD69 induction showed that mCS2 treatment dramatically increased CD69 levels in CD4 and CD8 cells compared with mIL-18 or placebo, achieving 30% to over 50% positive results for the CD4 and CD8 subpopulations. respectively (figure 12C). While mIL-18 and mCS2 stimulated CD69 expression on peripheral blood NK cells to levels greater than 20% of positivity by day 3, CD69 levels decreased to negligible levels with mIL-18 administration on day 6 but remained significantly increased with the introduction of mCS2 (figure 12C). Peripheral blood cytokine levels were also measured by multiplex immunoassay based on Luminex technology. As seen in Figure 12D, mIL-18 and mCS2 administration increased serum levels of IFN-g, MIP1b, and G-CSF compared to placebo administration, but mCS2 achieved much higher levels than mIL-18 at 6th day in the case of each of these cytokines, since mIL-18 was characterized by the manifestation of tachyphylaxis with stabilization or decrease in the level of induced cytokines upon subsequent administration.

The effect of mCS2 on the content of adipose tissue

In order to evaluate the effect of DR-IL-18 variants on the content of adipose tissue, C57BL/6 mice were injected intraperitoneally every day for three days with WT IL-18 at a dose of 1 mg/kg, or 0.01, 0.1 or 1 mg/kg mCS2. The content of adipose tissue and non-adipose tissue was monitored by the method based on EchoMRI™ technology. All tested doses of mCS2 (1 mg/kg, 0.1 mg/kg and 0.01 mg/kg) resulted in a dramatic decrease in the total percentage of adipose tissue on the 12th day, while in the case of mice injected with placebo and mIL-18, there was no significant change in total adipose tissue (Figure 13, top). In particular, mice treated with mCS2 had either reduced or stable levels of total fat mass during the experiment (Figure 13, bottom left), but they had a significant increase in total lean tissue (Figure 13, bottom right). These results indicate that mCS2 and other variants disclosed in the invention can be used for therapeutic reduction of adipose tissue (eg, in the treatment of obesity, diabetes and/or metabolic syndrome).

Antitumor activity of DR-IL-18 variants

The antitumor activity of DR-IL-18 (mCS2) was evaluated in the transplantable syngeneic tumor model of malignant melanoma YUMMER1.7. on mice. Mice bearing YUMMER1.7WT tumors were treated biweekly with mIL-18 and mCS2 at 0.32 mg/kg with or without co-administration of anti-PD1 antibodies (8 mg/kg/q3d). In accordance with previously published data on the use of WT IL-18 in mice and humans, WT IL-18 did not affect tumor growth or survival compared to placebo (saline) and only slightly increased the activity of anti-PD1 antibodies when administered in combination. However, the use of mCS2 in the form of monotherapy allowed to achieve recovery in 27% of treated mice and partial remission in another 27% of mice, that is, to achieve an effect that is commensurate with treatment with anti-PD1 antibody. The combination of mCS2 with anti-PD1 antibody allowed recovery in 80% of treated mice (Figure 14A and Figure 14B).

To elucidate the mechanism of action of DR-IL-18 on YUMMER1.7 tumors, cell depletion studies were performed using antibodies against CD8, CD4, NK1.1 and interferon-gamma. As seen in Figure 15A and Figure 15B, depletion of CD8 cells or neutralization of interferon-gamma completely abolishes DR-IL-18 activity. Depletion of CD4 cells did not affect the initial activity of DR-IL-18 in terms of tumor growth, however, in CD4 treated mice, therapeutic responses are erratic, suggesting a role for CD4 cells in maintaining antitumor immunity. Depletion of NK cells did not affect tumor growth or survival of YUMMER1.7 cells.

In addition, the activity of DR-IL-18 was further evaluated in the MC38 immunogenic colorectal tumor model. A dose titration study was first conducted with saline, IL-18 WT (1 mg/kg twice weekly) or DR-IL-18 in the dose range of 0.01 mg/kg, 0.1 mg/kg, or 1 mg/kg twice a week. As seen in Figure 16, WT IL-18 had no effect on tumor growth, while DR-IL-18 (mCS2) exhibited dose-dependent activity, slowing down tumor growth at a dose of 0.1 mg/kg and causing tumor regression at a dose of 1 mg /kg. The groups were then expanded and the manifestation of a possible synergistic effect in combination with an immune checkpoint inhibitor was evaluated. And in this case, WT IL-18 had no effect when used in the form of monotherapy and did not affect the increase in activity of antibodies against PD1. In contrast, DR-IL-18 exhibited high monotherapy activity commensurate with or superior to that of anti-PD1 antibody, and combination therapy with anti-PD1 antibody had an extremely high synergistic effect, leading to complete tumor regression in all treated mice (figure 17 ).

To further elucidate the mechanism of action of DR-IL-18, flow cytometric studies were performed on the immune infiltrate of MC38 tumors in mice injected with saline, WT IL-18, or DR-IL-18 (mCS2). Compared to saline or WT IL-18, administration of DR-IL-18 increased CD8 and NK cell infiltration per mg of tumor and furthermore resulted in upregulation of effector cell activation markers such as granzyme B and KLRG1 (Figure 18A , top row). Unlike other cytokine therapies such as IL-2 or IL-15, DR-IL-18 administration does not increase the CD8:Treg ratio in tumors compared to saline administration. However, administration of DR-IL-18 results in a more favorable tumor immune microenvironment by increasing the ratio of CD8 cells to tumor macrophages (TAMs) and monocytic and granulocytic myeloid-derived suppressor cells (MDSCs). In addition, the release profile of secondary cytokines in the serum of the same mice was also measured using the Luminex assay. As seen in Figure 18B, DR-IL-18 administration increased systemic levels of interferon-gamma, IL-7, and IL-15 by more than 100-fold compared to WT IL-18 administration. Taken together, these results indicate that DR-IL-18 has antitumor activity due to a unique mechanism of action different from that of IL-2, IL-15, or WT IL-18.

One would speculate that some of the secondary cytokines induced by DR-IL-18 therapy could potentially contribute to toxicity and/or decreased activity. For example, IL-17, which is more than 100-fold overregulated by DR-IL-18, promotes colitis and psoriasis and furthermore stimulates granulocytes, which can become immunosuppressive suppressor cells derived from myeloid cells. IL-5 and IL-13 are type 2 cytokines that are also upregulated by DR-IL-18 and may contribute to allergy, asthma exacerbation, or anaphylaxis. Type 2 T cells (Th2) do not promote immunotherapeutic responses and may promote the development of immunosuppressive Treg cells. As such, in some cases, the activity and safety of DR-IL-18 can be increased by selectively inhibiting unwanted secondary cytokines such as IL-17, IL-5 and IL-13, for example, with a neutralizing antibody.

Many tumors are resistant to immune checkpoint inhibition, either at initial presentation (primary resistance) or after an initial response to treatment (secondary resistance). The most common cause of resistance to treatment by checkpoint inhibition is loss of antigen presentation through MHC class I molecules. Loss of surface MHC class I molecules is classically associated with NK cell-mediated cytolysis, however NK cells can be depleted in MHC I-deficient tumors. Since NK cells express IL-18R, and previous results in MC38 showed that NK cells proliferate and become activated when exposed to DR-IL-18, therefore, studies were conducted on the possibility of using DR-IL-18 to stimulate the attack of NK cells. against MHC I-deficient tumors. CRISPR/cas9 was used to knock out B2m in the Yummer1.7 cell line and implanted B2m-deficient YUMMER1.7 tumors were found to be refractory even to combination therapy with both anti-CTLA4 antibody and anti-PD1 antibody (Figure 19A and Figure 19B), to a combination that typically cures almost 100% of parental Yummer1.7 tumors. However, monotherapy with DR-IL-18 (mCS2) cured 60% of B2m-deficient Yummer1.7 tumors in an NK cell dependent manner, as anti-NK1.1 depletion abolished this effect (Figure 19A and Figure 19B) . Experiments were performed to elucidate the effect of DR-IL-18 on intratumoral NK cells in MHC I-deficient tumors. Immunophenotyping studies were performed by flow cytometry on B2m-deficient Yummer 17 tumors in mice treated with saline or DR-IL-18. 24 hours after the 3rd dose, the mice were sacrificed, the tumors dissociated, and the cell suspension was treated with PMA/ionomycin for four hours. Then the proliferative index and functional capacity of NK cells were determined by intracellular flow cytometry using Ki67 and interferon-gamma. As seen in Figure 19C, NK cells from B2m-deficient tumors in Yummer1.7 mice injected with saline had low levels of interferon-gamma and Ki67 production, indicating a depleted phenotype. In contrast, NK cells from tumors of mice injected with DR-IL-18 had high levels of interferon-gamma and Ki67 production, with the majority of NK cells positive for both markers. Thus, these results suggest that DR-IL-18 is active in the treatment of MHC class I deficient tumors that are resistant to immune checkpoint blockade in an NK cell dependent manner.

These results suggest that DR-IL-18 is a very promising immunotherapeutic anticancer agent and provides strong evidence that IL-18BP significantly limits the efficacy of IL-18 therapy given the significantly higher activity of the mCS2 variant of DR-IL-18. Based on these results, it can be hypothesized that other strategies, such as blocking IL-18BP with an antibody, small protein, and/or small molecule, may enhance the efficacy of IL-18 therapy and other immunotherapy regimens.

Efforts have been made to construct an IL-18BP antagonist by creating decoy receptor decoy (D2D) or IL-18 variants that specifically bind IL-18BP but do not bind IL-18Rα and therefore do not signal. The potential advantage of such a drug is that it can serve to neutralize IL-18BP and increase the activity of endogenous IL-18, as opposed to triggering systemic IL-18R signaling. Therefore, IL-18 was randomized at contact positions to IL-18Rα (Figure 20A), and a human and mouse DR-IL-18 library was prepared using the yeast display technology described above. The resulting library of 3.9 x 10 ⁸ transformants was selected for 3 rounds as shown in Figure 20B, selecting for residual binding to IL-18BP while negatively selecting against IL-18Rα. As seen in Figure 20C, each round of selection resulted in an enrichment in binding to IL-18BP (human and mouse) but no acquisition of IL-18Rα binding. 96 clones were sequenced to obtain 31 unique sequences from which three consensus sequences hD2D-CS1, hD2D-CS2 and hD2D-CS3 were generated (Figure 21). Biophysical examination of the resulting clones indicated that they had similar IL-18BP binding isotherms to WT IL-18 (FIG. 22A), but greatly reduced/absent binding to IL-18Rα (FIG. 22B). These data are presented in Figure 22C. A similar selection process was carried out for murine IL-18 with the creation of a library of 2.0 x 10 ⁸ transformants, resulting in a selection with 51 unique sequences (results are shown in figure 23).

Example 2 Measurement of Binding Affinity of Second Generation Variants

Surface plasmon resonance (SPR) was used to biophysically measure the affinity of second generation DR-IL-18 variants (binding to IL-18R versus binding to IL-18BP). The figure 24 presents the obtained sensograms. The results of the kinetic studies are shown in Table 10, the results of the affinity measurements are shown in Table 11, and Table 12 summarizes the results, including the dissociation constant ratios of the second generation DR-IL-18 variants.

Table 10 SPR Kinetic Data for Second Generation hDR-IL-18 Variants

Surface ligand analyte ka (1/ms) kd
(1/sec) KD(M)
experiment 2 KD(M)
experiment 1 Rmax% hIL-18Ra hIL-18 5.55E+05 2.97E-03 5.36E-09 5.35E-09 32 hIL-18Ra 6-12 4.95E+05 9.10E-04 1.84E-09 2.24E-09 35 hIL-18Ra 6-27 6.31E+05 2.43E-03 3.85E-09 3.48E-09 35 hIL-18Ra 6-29 5.75E+05 1.19E-03 2.07E-09 2.65E-09 36 hIL-18Ra 6-31 2.18E+05 3.32E-03 1.52E-08 1.94E-08 19 hIL-18BP hIL-18 5.18E+05 2.23E-07 4.30E-13 6.94E-13 48 hIL-18BP 6-12 Signal too weak to measure -1 hIL-18BP 6-27 Signal too weak to measure 2 hIL-18BP 6-29 Signal too weak to measure 0 hIL-18BP 6-31 Signal too weak to measure -1

Table 11 SPR affinity data for second generation hDR-IL-18 variants

Sample KD _apparent hIL-18Ra (nM) KD _apparent hIL-18BP (nM) hIL-18 5.4, 5.4 < 0.1 6-12 1.8, 2.2 too weak 6-27 3.9, 3.5 too weak 6-29 2.1, 2.7 too weak 6-31 15.2, 19.4 too weak

Table 12. Results of affinity measurements by the SPR method. Results of measurements of the affinity of second generation hDR-IL-18 variants for IL-18Ra and IL-18BP by the SPR method. The IL-18 BP:Ra dissociation constant ratio is the ratio of KD for IL-18BP to KD for IL-18Ra normalized to the same ratio for WT IL-18. The higher value of this ratio indicates that the IL-18 variant has an increased preference for IL-18Ra binding over IL-18BP binding compared to WT IL-18. *Average of 2 measurements. k is a multiple of 1000, m is a multiple of 1000000.

Protein SPR method:
K _D Ra (nM) SPR method:
K _D BP (nM) The ratio of dissociation constants of IL-18 BP:Ra WT hIL-18 4.1* 0.002 1 hCS1 8.0* 11.8* 3.024 hCS3 9.1* 19.3* 4.348 hCS4 7.7* 121* 32.215 6-12 2.2 > 10000 > 9318275 6-27 3.5 > 10000 > 5857201 6-29 2.7 > 10000 > 7592669 6-31 19.4 > 10000 > 1056712 WT mIL-18 0.60 0.0011 1 mCS2 0.08 11000 > 75000136 A7, B1, C1, E8 0.22-1.7 14k - 29k 9.3m - 35m

Example 3 Efficacy in the Treatment of Cancer

The efficacy of DR-IL-18 variants was studied using various cancer models, including models of colorectal tumors, breast cancer, melanoma, and MHC class I deficient tumors that are resistant to immune checkpoint inhibitors. The results show that DR-IL-18 variants predominantly binding IL-18R but not IL-18BP can be used to treat a wide range of cancers (and not just the types of cancer that have been investigated).

Figures 25A and 25B present data demonstrating the efficacy of DR-IL-18 in the CT26 colorectal tumor model. Mice were implanted subcutaneously with 250,000 CT26 cells and treatment started on day 7 after tumors averaged ~60 mm ³ . WT IL-18 and mCS2 were administered at a dose of 0.32 mg/kg twice a week for a total of 5 doses. An antibody against PD1 was administered at a dose of 10 mg/kg according to the same scheme. (A) Tumor growth susceptibility curves are shown for animals treated with saline (PBS) as black lines (circles), WT IL-18 as blue lines (squares) and DR-IL-18 (mCS2) as pink lines (triangles). Only administration of DR-IL-18, but not WT IL-18, resulted in tumor growth inhibition and tumor clearance in a subgroup of animals. (B) Survival curves are shown for mice treated with anti-PD-1 antibody, WT IL-18 and DR-IL-18 (mCS2). The number of complete responses is given in brackets. Administration of DR-IL-18, but not WT IL-18, resulted in increased survival and tumor clearance in 40% of mice, an improvement over the anti-PD-1 immune checkpoint inhibitor.

Figures 26A and 26B show data demonstrating the activity of DR-IL-18 in the 4T1 breast cancer model and the B16-F10 melanoma model. (A) Growth curves of 4T1 tumors inoculated in BALB/C mice after saline (PBS; black), WT IL-18 (blue), or DR-IL-18 CS2 variant (pink) are shown. (B) Growth curves of B16-F10 tumors implanted in C57BL/6 mice after saline (PBS; black), WT IL-18 (blue), or DR-IL-18 CS2 variant (pink) are shown. . In both models, only administration of DR-IL-18, but not WT IL-18, resulted in inhibition of tumor growth. The introduction was carried out after the average tumor size exceeded 50 mm ³ as indicated in the boxes marked with the letter "t".

Figures 27A and 27B show data that complements the data shown in Figures 19A-19C. The data presented demonstrate the activity of DR-IL-18 in the treatment of additional MHC class I deficient tumor models that are resistant to immune checkpoint inhibitors. (A) B2m-deficient MC38 cells were generated using the CRISPR/Cas9-mediated deletion described for B2m-deficient YUMMER cells. Implanted subcutaneously cells B2m-/-MC38, and began treatment on the 7th day after the size of the tumors averaged ~65 mm ³ . mCS2 was administered at a dose of 0.32 mg/kg twice a week for a total of 5 doses. Anti-PD1 antibody and anti-CTLA4 antibody were administered at a dose of 8 mg/kg according to the same schedule. (B) RMA/S is a variant of the RMA lymphoma lineage that contains a spontaneous mutation in the TAP-associated glycoprotein. The result is a defect in antigen loading and hence reduced surface expression of MHC class I molecules. It is congenic to the C57BL/6C57BL mouse line and resistant to immune checkpoint inhibitors. Mice were implanted subcutaneously with 1,000,000 RMA/S cells and treatment began on day 7. MKS2 was administered at a dose of 0.32 mg/kg twice a week. The anti-PD1 antibody was administered at a dose of 8 mg/kg according to the same schedule.

Example 4 Combination Therapy

Figure 28 shows data demonstrating the activity of DR-IL-18 in enhancing antitumor antibody-dependent cell-mediated cytotoxicity (ADCC) (maintenance combination therapy with opsonizing agents such as tumor-targeting antibodies). Ex vivo cytotoxicity studies used CFSE-labeled Raji (B-cell lymphoma) cells and isolated human peripheral blood mononuclear cells (PBMCs). PBMCs and labeled Raja cells were incubated together at an effector:target (E:T) ratio of 1:10 for 25 hours. These samples were treated with the hCS-1 variant of human DR-IL-18 (1 μM), rituximab (10 μg/ml), or a combination of both. Cytotoxicity was determined by flow cytometry and was calculated as the proportion of CFSE cells that became DAPI positive. *p<0.05 by two-way ANOVA with Tukey's correction for multiple comparisons.

Example 5 Efficacy Against Viral Infection

Figures 29A and 29B show data demonstrating the antiviral activity of DR-18 variants in the treatment of viral infections (e.g., viral infection, such as in this illustrative example, the mCS2 variant was used in the treatment of systemic vaccinia infection). (A) Schematic of the experimental design. C57BL/6 mice were infected intraperitoneally (IP) with 10 ⁶ PFU of vaccinia virus (VACV) and injected intraperitoneally with 1 mg/kg WT mIL-18 or mCS2. Mice were sacrificed and viral titers in blood and ovaries were determined by reverse transcription polymerase chain reaction (RT-PCR) on the 3rd day after infection. (B) Quantification of viral copies of VACV in the ovaries and blood of treated mice on the 3rd day after infection. Treatment with CS2 showed a significant reduction in viral titers, while treatment with WT IL-18 was ineffective. *p<0.05, **p<0.01, ***p<0.001.

Example 6 Second Generation Human DR-IL-18 Variants

Figure 30A shows data demonstrating the activity of second generation human DR-IL-18 variants. Figure 30A shows that WT IL-18 and h6-12, h6-27, h6-29 and h6-31 stimulate IL-18 HEK-Blue reporter cells. h6-12, h6-27 and h6-29 show increased activity compared to WT hIL-18, while h6-31 has the same activity as WT hIL-18. Thus, the data demonstrate that all second generation human DR-IL-18 variants tested actively signal through IL-18R.

Amino acid sequences of wild-type IL-18

Human interleukin-18 (mature form)

YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 30)

Mouse interleukin-18 (mature form)

NFGRLHCTTAVIRNINDQVLFVDKRQPVFEDMTDIDQSASEPQTRLIIYMYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFQKRVPGHNKMEFESSLYEGHFLACQKEDDAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 31)

Q14116|IL18_human interleukin-18 (uncleaved precursor)

MAAEPVEDNCINFVAMKFIDNTLYFIAEDDENLESDYFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 32)

P70380|IL18_mouse interleukin-18 (uncleaved precursor)

SEQ ID NO: 33)

Amino acid sequences of human interleukin-18 generation 1 variants resistant to decoy receptor

hCS1 YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISTYKDSQPRGKAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRDVPGHKHKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 34) hCS2 YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISTYKDKQPRAKAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRDVPGHKHKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTIQNED (SEQ ID NO: 35) hCS3 RFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAAPRTIFIISTYKDSQPRGKAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRDVPGHKHKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 36) hCS4 RFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAAPRTIFIISTYRDSQPRGKAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRNVPGHKYKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 37) hC4 YFGKLESQLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAAPRTIFIISTYKDKQPRTKAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRRVPGHHNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQKED (SEQ ID NO: 38) hA8 YFGKLESRLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISKYKDKQPRAQAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRDVPGHKHKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTIQNED (SEQ ID NO: 39) hd6 YFGKLESRLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISDYKDKQPRAXAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRDVPGHKHKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTIQNED (SEQ ID NO: 40) hH12 YFGKHESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAAPRTIFIISTYRDSQPRGKAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRDVPGHNNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTTQNED (SEQ ID NO: 41) hB11 (SEQ ID NO: 42) hC3 YFGKIESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISTYKDRQPRGKAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFERDVPGHHHKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTIQNED (SEQ ID NO: 43) hC2 (SEQ ID NO: 44) hG10 YFGKIESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISTYKDKQPRAKAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRRVPGHHHKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTIQKED (SEQ ID NO: 45) hG1 YFGKIESRLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAAPRTIFIISTYKDKQPRGKAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRDVPGHDYKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTIQKED (SEQ ID NO: 46) hF1 YFGKYESRLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAAPRTIFIISTYRDSQPRGKAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRDVPGHEHKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQKED (SEQ ID NO: 47) hD2 (SEQ ID NO: 48) hA1 RFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISTYRDSQPRAKAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRDVPGHQHKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTAQKED (SEQ ID NO: 49) hB3 RFGKLESRLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAAPRTIFIISDYRDSQPRGRAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFKRNVPGHKYKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQHED (SEQ ID NO: 50) hB4 RFGKLESRLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAAPRTIFIISNYRDSQPRGQAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFKRRVPGHNHKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQKED (SEQ ID NO: 51) hH3 RFGKLESRLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISTYKDSQPRGKAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRDVPGHKHKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 52) hH5 RFGKHESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAAPRTIFIISTYRDSQPRGKAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFERNVPGHKYKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 53) hH4 RFGKLESRLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISTYRDSQPRAKAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFERDVPGHQHKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTIQXED (SEQ ID NO: 54) hE1 RFGKLESRLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAAPRTIFIISTYRDSQPRTKAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRNVPGHHDKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQHED (SEQ ID NO: 55) hG2 RFGKLESRLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISTYKDSQPRAKAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFERDVPGHQHKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTIQKED (SEQ ID NO: 56) hB9 RFGKHESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAAPRTIFIISTYRDSQPRGKAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFERNVPGHKYKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 57) hE12 RFGKYESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPTIFIISTYKDSQPRTKAVTISVKCEKISTLSCDNKIISFKEMNPPDNIKDTKSDIIFFQRDVPGHKHKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 58) hC5 RFGKLESRLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAAPRTIFIISTYRDSQPRTKAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRKVPGHNHKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQKED (SEQ ID NO: 59)

Amino acid sequences of human interleukin-18 generation 2 variants resistant to decoy receptor

5-18 YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISEYKDSELRGRAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFPRAVPGHNRKVQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 73) 5-29 YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISKYKDSAGRGLAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFERDVPGHSNKVQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 74) 5-8 YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISKYGDSAARGLAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 75) 5-6 YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISKYGDSRGRGLAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFERDVPGHNSKRQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 76) 5-27 YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISKYGDSVPRGLAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFARAVPGHSRKTQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 77) 5-20 YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISKYSDSGARGLAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFARAVPGHGRKTQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 78) 5-2 YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISKYSDSKARGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFARDVPGHSSKRQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 79) 5-9 YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISKYSDSLARGLAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRDVPGHSRKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 80) 5-42 YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISKYSDSRARGLAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRNVPGHGRKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 81) 5-13 YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISKYSDSRARGLAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFARSVPGHGRKTQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 82) 5-12 YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISKYSDSRARGLAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFARDVPGHSGKRQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 83) 5-1 YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISKYTDSRPRGLAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFERDVPGHSSKKQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 84) 5-33 YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISKYTDSRARGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFERDVPGHNDKRQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 85) 5-21 YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISRYKDSGKRGLAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFRRSVPGHSRKVQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 86) 6-31 YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISKYGDSGARGLAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFERDVPGHSGKVQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 87) 6-20 YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAAPRTIFIISKYGDSRPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRAVPGHNRKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 88) 6-12 YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISKYSDSLARGLAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRDVPGHSRKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 89) 6-27 YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISKYSDSRARGLAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFARSVPGHGRKTQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 90) 6-29 YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISKYSDSRARGLAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRNVPGHGRKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 91) 5-26 YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISKYGDSVPRGLAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFARAVPGHSRKTQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 191) 5-17 YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISKYSDSRARGLAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFARSVPGHGRKTQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 192) 5-41 YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISKYSDSRARGLAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFARDVPGHSGKRQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 193)

Amino acid sequences of murine interleukin-18 variants resistant to decoy receptor

mCS1 (SEQ ID NO: 60) mCS2 HFGRLHCTTAVIRNINDQVLFVDKRQPVFEDMTDIDQSASEPQTRLIIYAYGDSRARGKAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFQKRVPGHNKMEFESSLYEGHFLACQKEDDAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 61) mC1 NFGRLHCTTAVIRNINDQVLFVDKRQPVFEDMTDIDQSASEPQTRLIIYAYVDRRLRGKAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFQKKVPGHNKMEFESSLYEGHFLACQKEDDAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 62) mA12 NFGRLHCTTAVIRNINDQVLFVDKRQPVFEDMTDIDQSASEPQTRLIIYSYSDKHMRGKAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFQKLVPGHNKMEFESSLYEGHFLACQKEDDAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 63) mE8 NFGRLHCTTAVIRNINDQVLFVDKRQPVFEDMTDIDQSASEPQTRLIIYVYTDGRRRGKAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFQKKVPGHDKMEFESSLYEGHFLACQKEDDAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 64) mC10 HFGRLHCTTAVIRNINDQVLFVDKRQPVFEDMTDIDQSASEPQTRLIIYAYGDSHMRGKAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFQKQVPGHNKMEFESSLYEGHFLACQKEDDAFKLILKKKDENGDKSVMFTVTNLHQS (SEQ ID NO: 65) mB7 HFGRLHCTTAVIRNINDQVLFVDKRQPVFEDMTDIDQSASEPQTRLIIYAYGDSNAGGRAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFQKKVPGHNKMEFESSLYEGHFLACQKEDDAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 66) mB1 (SEQ ID NO: 67) mD1 HFGRLHCTTAVIRNINDQVLFVDKRQPVFEDMTDIDQSASEPQTRLIIYGYSDRGSKGKAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFQKQVPGHNKMEFESSLYEGHFLACQKEDDAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 68) mH7 YFGRLHCTTAVIRNINDQVLFVDKRQPVFEDMTDIDQSASEPQTRLIIYMYADRRARGKAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFQKKVPGHDKMEFESSLYEGHFLACQKEDDAFKLILKKKDENGDKSVMFTVTNLHQS (SEQ ID NO: 69) mA7 YFGRLHCTTAVIRNINDQVLFVDKRQPVFEDMTDIDQSASEPQTRLIIYAYGDNRVRGKAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFQKRVPGHNKMEFESSLYEGHFLACQKEDDAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 70) mE1 YFGRLHCTTAVIRNINDQVLFVDKRQPVFEDMTDIDQSASEPQTRLIIYGYGDSERGGRAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFQKRVPGHDKMEFESSLYEGHFLACQKEDDAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 71) mH3 YFGRLHCTTAVIRNINDQVLFVDKRQPVFEDMTDIDQSASEPQTRLIIYTRTDGGQKGVAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFQKRVPGHDKMEFESSLYEGHFLACQKEDDAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 72)

Amino acid sequences of human decoy receptor decoy (D2D) variants

hD2D-5F12 (SEQ ID NO: 92) hD2D-5F11 (SEQ ID NO: 93) hD2D-5F10 (SEQ ID NO: 94) hD2D-5F08 (SEQ ID NO: 95) hD2D-5F06 (SEQ ID NO: 96) hD2D-5F04 YFGKLESKLSVIRNLNRQVLFIDQGNRPLFTDMTYKDCRDNAPRTIFIISFYKDSQPRGFAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFIRSVPGADNKIQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 97) hD2D-5F02 (SEQ ID NO: 98) hD2D-5F01 (SEQ ID NO: 99) hD2D-5E10 (SEQ ID NO: 100) hD2D-5E08 YFGKLESKLSVIRNLNGQVLFIDQGNRPLFTDMTASPCRDNAPRTIFIISFYKDSQPRGLAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFLRSVPGHDNKIQFESSSYEGYFLACEKERSLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 101) hD2D-5E03 (SEQ ID NO: 102) hD2D-5E02 (SEQ ID NO: 103) hD2D-5D10 (SEQ ID NO: 104) hD2D-5D08 YFGKLESKLSVIRNLNDQVLFIDQGNRPLFADMKAKACRSNAPTIFIISFYKDSQPRGFAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFLRSVPGADNKIQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 105) hD2D-5D06 (SEQ ID NO: 106) hD2D-5D05 YFGKLESKLSVIRNLNGQVLFIDQGNRPLFTDMKSNLCRSNAPRTIFIISFYKDSQPRGIAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFIRSVPGDDNKIQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 107) hD2D-5D03 (SEQ ID NO: 108) hD2D-5D02 YFGKLESKLSVIRNLNDQVLFIDQGNRPLFADMASNPCRYKAPRTIFIISMYKDSQPRGLAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFLRSVPGADNKLQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 109) hD2D-5C10 (SEQ ID NO: 110) hD2D-5C09 (SEQ ID NO: 111) hD2D-5C08 YFGKLESKLSVIRNLNGQVLFIDQGNRPLFTDMTASHCRSSAPRTIFIISLYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFLRSVPGHDNKFQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 112) hD2D-5C05 YFGKLESKLSVIRNLNGQVLFIDQGNRPLFTDMEYRLCRANAPRTIFIISFYKDSHPRGLAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFLRSVPGDDNKLQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 113) hD2D-5C04 (SEQ ID NO: 114) hD2D-5C03 YFGKLESKLSVIRNLNGQVLFIDQGNRPLFKDMEANDCRSSAPRTIFIISIYKDSQPRGLAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFIRSVPGADNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 115) hD2D-5B11 (SEQ ID NO: 116) hD2D-5B10 (SEQ ID NO: 117) hD2D-5B06 (SEQ ID NO: 118) hD2D-5B05 (SEQ ID NO: 119) hD2D-5B02 (SEQ ID NO: 120) hD2D-5A09 (SEQ ID NO: 121) hD2D-5A02 YFGKLESKLSVIRNLNAQVLFIDQGNRPLFTDMTASPCRSSAPRTIFIISLYKDSQPRGLAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFLRSVPGPDNKIQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 122) hD2D-CS1 (SEQ ID NO: 123) hD2D-CS2 YFGKLESKLSVIRNLNGQVLFIDQGNRPLFADMTSSDCRDNAPRTIFIISFYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFLRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 124) hD2D-CS3 YFGKLESKLSVIRNLNGQVLFIDQGNRPLFADMESSDCRDNAPRTIFIISFYKDSQPRGLAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFLRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (SEQ ID NO: 125)

Amino acid sequences of mouse receptor decoy receptor (D2D) variants

mD2D-A5 YFGRYHCTTAVIRNINQQVLFVDKRQPVFADMGYTVQSASEPQTRLIIYMYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFLKEVPGHRKLEFESSLYEGHFLACQKEDEAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 126) mD2D-A6 DFGRLHCTTAVIRNINDQVLFVDKRQPVFADMGSIAQSASEPQTRLIIYFYKDSEVRGLAVTLSVKDSKMYTLSCKNKIISFEEMDPPENIDDIQSDLIFFLKAVPGDNKIEFESSLYEGHFLACQKEATAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 127) mD2D-A7 YFGRLHCTTAVIRNINGQVLFVDKRQPVFRDMADTVQSASEPQTRLIIYFYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFIKPVPGASKMEFESSLYEGHFLACQKEAGAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 128) mD2D-A8 HFGRLHCTTAVIRNINDQVLFVDKRQPVFKDMEYTVQSASEPQTRLIIYMYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFIKAVPGDRKIEFESSLYEGHFLACQKEDNAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 129) mD2D-A9 YFGRLHCTTAVIRNINAQVLFVDKRQPVFADMADKGQSASEPQTRLIIYMYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFLKPVPGDTKMEFESSLYEGHFLACQKEFGAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 130) mD2D-A11 YFGRLHCTTAVIRNINEQVLFVDKRQPVFADMGDRHQSASEPQTRLIIYMYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFIKPVPGASKLEFESSLYEGHFLACQKEDDAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 131) mD2D-A12 HFGRLHCTTAVIRNINDQVLFVDKRQPVFRDMGAIGQSASEPQTRLIIYFYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFIKPVPGDSKLEFESSLYEGHFLACQKEVDAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 132) mD2D-B4 HFGRLHCTTAVIRNINSQVLFVDKRQPVFTDMGSIVQSASEPQTRLIIYMYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFIKGVPGDNKIEFESSLYEGHFLACQKEDRAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 133) mD2D-B7 YFGRLHCTTAVIRNINSQVLFVDKRQPVFRDMEDTPQSASEPQTRLIIYFYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFIKRVPGDSKLEFESSLYEGHFLACQKEFEAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 134) mD2D-B11 HFGRLHCTTAVIRNINAQVLFVDKRQPVFGDMTATVQSASEPQTRLIIYFYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFIKPVPGDSKLEFESSLYEGHFLACQKEDNAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 135) mD2D-B12 NFGRLHCTTAVIRNINNQVLFVDKRQPVFKDMEYTLQSASEPQTRLIIYFYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFIKPVPGDNKLEFESSLYEGHFLACQKEYEAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 136) mD2D-C1 YFGRLHCTTAVIRNINGQVLFVDKRQPVFADMEATRQSASEPQTRLIIYFYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFIKGVPGANKMEFESSLYEGHFLACQKEDGAFKLILKKKDENGDNSVMFTLTNLHQS (SEQ ID NO: 137) mD2D-C3 (SEQ ID NO: 138) mD2D-C5 YFGRLHCTTAVIRNINAQVLFVDKRQPVFADMEATAQSASEPQTRLIIYFYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFIKGVPGASKMEFESSLYEGHFLACQKEDGAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 139) mD2D-C6 LFGRLHCTTAVIRNINGQVLFVDKRQPVFADMGATLQSASEPQTRLIIYMYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFLKPVPGDTKMEFESSLYEGHFLACQKEASAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 140) mD2D-C9 NFGRLHCTTAVIRNINGQVLFVDKRQPVFEDMAYTVQSASEPQTRLIIYFYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFIKGVPGDSKMEFESSLYEGHFLACQKEYDAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 141) mD2D-C10 DFGRLHCTTAVIRNINDQVLFVDKRQPVFKDMESKPQSASEPQTRLIIYFYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFLKAVPGASKLEFESSLYEGHFLACQKEANAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 142) mD2D-C11 (SEQ ID NO: 143) mD2D-D1 YFGRHHCTTAVIRNINQQVLFVDKRQPVFRDMAATRQSASEPQTRLIIYMYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFLKGVPGDNKMEFESSLYEGHFLACQKEDDAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 144) mD2D-D9 NFGRLHCTTAVIRNINQQVLFVDKRQPVFTDMESIGQSASEPQTRLIIYFYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFLKAVPGANKLEFESSLYEGHFLACQKEDSAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 145) mD2D-D12 (SEQ ID NO: 146) mD2D-E3 VFGRHHCTTAVIRNINGQVLFVDKRQPVFKDMTYIDQSASEPQTRLIIYMYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFLKAVPGDTKMEFESSLYEGHFLACQKEAQAFKLILKKKDEIGDKSVMFTLTNLHQS (SEQ ID NO: 147) mD2D-E4 NFGRLHCTTAVIRNINGQVLFVDKRQPVFADMTATRQSASEPQTRLIIYMYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFIKQVPGANKIEFESSLYEGHFLACQKEFRAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 148) mD2D-E5 DFGRLHCTTAVIRNINGQVLFVDKRQPVFGDMAYIGQSASEPQTRLIIYFYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFIKAVPGHSKIEFESSLYEGHFLACQKESGAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 149) mD2D-E7 YFGRLHCTTAVIRNINDQVLFVDKRQPVFRDMGSIAQSASEPQTRLIIYMYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFIKPVPGATKLEFESSLYEGHFLACQKEDGAFKLILKKKDENGDNSVMFTLTNLHQS (SEQ ID NO: 150) mD2D-E8 YFGRLHCTTAVIRNINEQVLFVDKRQPVFTDMEAIGQSASEPQTRLIIYFYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFIKGVPGDRKMEFESSLYEGHFLACQKEDGAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 151) mD2D-E9 (SEQ ID NO: 152) mD2D-E10 NFGRLHCTTAVIRNINNQVLFVDKRQPVFADMEDRLQSASEPQTRLIIYMYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFLKGVPGDNKMEFESSLYEGHFLACQKEDHAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 153) mD2D-E11 YFGRLHCTTAVIRNINAQVLFVDKRQPVFRDMGYILQSASEPQTRLIIYLYKDSEVRGLAVTLSVKESKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFLKPVPGDTKIEFESSLYEGHFLACQKEDNAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 154) mD2D-E12 YFGRLHCTTAVIRNINDQVLFVDKRQPVFGDMADTAQSASEPQTRLIIYFYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFIKPVPGDSKMEFESSLYEGHFLACQKEADAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 155) mD2D-F3 DFGRLHCTTAVIRNINGQVLFVDKRQPVFEDMAYIAQSASEPQTRLIIYFYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFIKPVPGDSKIEFESSLYEGHFLACQKEADAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 156) mD2D-F4 NFGRLHCTTAVIRNINEQVLSVDKRQPVFRDMKYILQSASEPQTRLIIYFYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFLKGVPGDNKMEFESSLYEGHFLACQKEYGAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 157) mD2D-F5 DFGRLHCTTAVIRNINEQVLFVDKRQPVFTDMAYILQSASEPQTRLIIYFYKDSEVRGLAVTLSVKESKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFIKAVPGDSKLEFESSLYEGHFLACQKEDTAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 158) mD2D-F7 DFGRLHCTTAVIRNINNQVLFVDKRQPVFKDMESTAQSASEPQTRLIIYMYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFLKGVPGASKLEFESSLYEGHFLACQKEAGAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 159) mD2D-F8 (SEQ ID NO: 160) mD2D-F9 (SEQ ID NO: 161) mD2D-G1 YFGRLHCTTAVIRNINAQVLFVDKRQPVFTDMGYTLQSASEPQTRLIIYLYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFIKPVPGHNKIEFESSLYEGHFLACQKEDRAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 162) mD2D-G7 NFGRLHCTTAVIRNINNQVLFVDKRQPVFRDMASTAQSASEPQTRLIIYMYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFIKGVPGANKIEFESSLYEGHFLACQKEDDAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 163) mD2D-G9 (SEQ ID NO: 164) mD2D-H7 NFGRLHCTTAVIRNINEQVLFVDKRQPVFADMTDIAQSASEPQTRLIIYMYKDSEVRGLAVTLSVKESKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFLKPVPGDIKMEFESSLYEGHFLACQKEYGAFKLILKKKDENGDNSVMFTLTNLHQS (SEQ ID NO: 165) mD2D-E1 YFGRLHCTTAVIRNINEQVLFVDKRQPVFADMTDTLQSASEPQTRLIIYFYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFLKGVPGDNKMEFESSLYEGHFLACQKEDTAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 166) mD2D-G8 YFGRLHCTTAVIRNINEQVLFVDKRQPVFADMTDTLQSASEPQTRLIIYFYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFLKGVPGDNKMEFESSLYEGHFLACQKEDTAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 167) mD2D-H3 YFGRLHCTTAVIRNINEQVLFVDKRQPVFADMTDTLQSASEPQTRLIIYFYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFLKGVPGDNKMEFESSLYEGHFLACQKEDTAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 168) mD2D-A10 (SEQ ID NO: 169) mD2D-H1 (SEQ ID NO: 170) mD2D-F12 YFGRLHCTTAVIRNINGQVLFVDKRQPVFEDMKAKAQSASEPQTRLIIYFYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFIKPVPGASKMEFESSLYEGHFLACQKEDGAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 171) mD2D-G10 YFGRLHCTTAVIRNINGQVLFVDKRQPVFEDMKAKAQSASEPQTRLIIYFYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFIKPVPGASKMEFESSLYEGHFLACQKEDGAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 172) mD2D-G12 YFGRLHCTTAVIRNINGQVLFVDKRQPVFEDMKAKAQSASEPQTRLIIYFYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFIKPVPGASKMEFESSLYEGHFLACQKEDGAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 173) mD2D-E2 (SEQ ID NO: 174) mD2D-G11 (SEQ ID NO: 175) mD2D-C4 YFGRLHCTTAVIRNINSQVLFVDKRQPVFTDMAYTVQSASEPQTRLIIYFYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFIKAVPGDSKLEFESSLYEGHFLACQKEDNAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 176) mD2D-F11 YFGRLHCTTAVIRNINSQVLFVDKRQPVFTDMAYTVQSASEPQTRLIIYFYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFIKAVPGDSKLEFESSLYEGHFLACQKEDNAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 177) mD2D-C2 YFGRLHCTTAVIRNINGQVLFVDKRQPVFTDMGARVQSASEPQTRLIIYFYKDSEVRGLAVTLSVKDSKMYTLSCKNKIISFEEMDPPENIDDIQSDLIFFLKPVPGDNKLEFESSLYEGHFLACQKESGAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 178) mD2D-F10 YFGRLHCTTAVIRNINGQVLFVDKRQPVFTDMGARVQSASEPQTRLIIYFYKDSEVRGLAVTLSVKDSKMYTLSCKNKIISFEEMDPPENIDDIQSDLIFFLKPVPGDNKLEFESSLYEGHFLACQKESGAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 179) mD2D-A2 (SEQ ID NO: 180) mD2D-F6 (SEQ ID NO: 181) mD2D-A1 DFGRLHCTTAVIRNINSQVLFVDKRQPVFRDMGSIHQSASEPQTRLIIYFYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFLKAVPGANKLEFESSLYEGHFLACQKEDGAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 182) mD2D-E6 DFGRLHCTTAVIRNINSQVLFVDKRQPVFRDMGSIHQSASEPQTRLIIYFYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFLKAVPGANKLEFESSLYEGHFLACQKEDGAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 183) mD2D-D4 YFGRLHCTTAVIRNINEQVLFVDKRQPVFKDMKDKLQSASEPQTRLIIYFYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFLKGVPGDNKLEFESSLYEGHFLACQKEFGAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 184) mD2D-D6 YFGRLHCTTAVIRNINEQVLFVDKRQPVFKDMKDKLQSASEPQTRLIIYFYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFLKGVPGDNKLEFESSLYEGHFLACQKEFGAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 185) mD2D-A3 YFGRLHCTTAVIRNINGQVLFVDKRQPVFADMASTHQSASEPQTRLIIYFYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFLKGVPGANKIEFESSLYEGHFLACQKEDDAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 186) mD2D-A4 YFGRLHCTTAVIRNINGQVLFVDKRQPVFADMASTHQSASEPQTRLIIYFYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFLKGVPGANKIEFESSLYEGHFLACQKEDDAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 187) mD2D-B10 YFGRLHCTTAVIRNINGQVLFVDKRQPVFADMASTHQSASEPQTRLIIYFYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFLKGVPGANKIEFESSLYEGHFLACQKEDDAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 188) mD2D-B8 YFGRLHCTTAVIRNINSQVLFVDKRQPVFGDMKYIVQSASEPQTRLIIYFYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFLKGVPGDTKMEFESSLYEGHFLACQKESGAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 189) mD2D-B9 YFGRLHCTTAVIRNINSQVLFVDKRQPVFGDMKYIVQSASEPQTRLIIYFYKDSEVRGLAVTLSVKDSKMSTLSCKNKIISFEEMDPPENIDDIQSDLIFFLKGVPGDTKMEFESSLYEGHFLACQKESGAFKLILKKKDENGDKSVMFTLTNLHQS (SEQ ID NO: 190)

Examples of non-limiting aspects of the invention

The aspects described above, including embodiments, of the subject matter of the present invention allow a beneficial effect to be achieved alone or in combination with one or more other aspects or embodiments. Without limiting the foregoing description of the invention, the following are specific non-limiting aspects of the invention in ASPECTS A and ASPECTS B. Upon reading the description of the invention, it should be apparent to one of ordinary skill in the art that each of the numbered aspects may be used alone or in combination. with any of the preceding or following individually numbered aspects. This is intended to support all such aspect combinations and is not limited to the aspect combinations expressly provided below. It should be apparent to one of ordinary skill in the art that various changes and modifications can be made without departing from the spirit or scope of the invention.

SET OF ASPECTS A

1. A composition comprising an IL-18 polypeptide variant, wherein the IL-18 polypeptide variant specifically binds to the IL-18 receptor (IL-18R) and wherein the IL-18 polypeptide variant exhibits significantly reduced binding to the IL-18 binding protein ( IL-18BP).

2. The composition of claim 1, wherein the variant IL-18 polypeptide comprises at least one mutation relative to wild-type IL-18 (WT IL-18).

3. The composition of claim 2, wherein WT IL-18 is human IL-18 comprising the amino acid sequence of SEQ ID NO: 30.

4. The composition of claim 2, wherein WT IL-18 is murine IL-18 comprising the amino acid sequence of SEQ ID NO: 31.

5. The composition of claim 3, wherein the variant IL-18 polypeptide comprises at least one mutation selected from the group consisting of Y1X, L5X, K8X, M51X, K53X, S55X, Q56X, P57X, G59X, M60X, E77X , Q103X, S105X, D110X, N111X, M113X, V153X and N155X, relative to SEQ ID NO: 30.

6. The composition of claim 3, wherein the IL-18 polypeptide variant comprises at least one mutation selected from the group consisting of 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, G59A, M60K, M60Q, M60R, M60L , E77D, Q103E, Q103K, Q103P, Q103A, Q103R, S105R, S105D, S105K, S105N, S105A, D110H, D110K, D110N, D110Q, D110E, D110S, D110G, N111H, N111Y, N 111D, N111R, N111S, N111G, M113V , M113R, M113T, M113K, V153I, V153T, V153A, N155K and N155H, relative to SEQ ID NO: 30.

7. The composition of claim 2, wherein the IL-18 polypeptide variant comprises at least one IL-18 polypeptide variant selected from the group consisting of SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36 , SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO : 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 73, SEQ ID NO: 74 , SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO : 91, or a fragment of it.

8. The composition of claim 4, wherein the IL-18 polypeptide variant comprises at least one mutation selected from the group consisting of N1X, M50X, Y51X, K52X, S54X, E55X, V56X, R57X, G58X, L59X, R104X , N109X and L151X, relative to SEQ ID NO: 31.

9. The composition of claim 4, wherein the IL-18 polypeptide variant comprises at least one mutation selected from the group consisting of N1H, N1Y, M50A, M50S, M50V, M50G, M50T, Y51R, K52V, K52S, K52T , K52G, K52A, S54R, S54K, S54G, S54N, E55R, E55H, E55N, E55D, E55G, V56L, V56M, V56R, V56A, V56S, V56Q, R57G, R57K, G58A, L59K, L59R, L59V, R104K , R104L , R104Q, R104S, N109D and L151V, relative to SEQ ID NO: 31.

10. The composition of claim 2, wherein the IL-18 polypeptide variant comprises at least one IL-18 polypeptide variant selected from the group consisting of SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62 , SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, or a fragment thereof.

11. A composition comprising a nucleic acid encoding at least one of the variants of the IL-18 polypeptide according to paragraphs. 1-10.

12. A method of treating or preventing a disease or disorder in a subject in need thereof, comprising administering to the subject at least one composition according to paragraphs. 1-11.

13. The method of claim 12 wherein the disease or disorder is cancer.

14. The method of claim 13 wherein the cancer is a cancer that is resistant to immune checkpoint inhibitors (ICIs).

15. The method of claim 13, wherein the cancer is associated with a tumor that lacks the expression of class I MHC molecules.

16. The method of claim 12 wherein the disease or disorder is a metabolic disease or disorder.

17. The method of claim 12, wherein the disease or disorder is an infectious disease.

18. The method of claim 12, further comprising administering at least one other agent to the subject.

19. The method of claim 18 wherein at least one other agent comprises an inhibitor of one or more secondary cytokines.

20. The method of claim 19 wherein the one or more secondary cytokines is at least one cytokine selected from the group consisting of IL-17, IL-5 and IL-13.

21. The method of claim 19, wherein the inhibitor of one or more secondary cytokines comprises at least one inhibitor selected from the group consisting of a chemical compound, a polypeptide, a peptide, a peptidomimetic, an antibody, a ribozyme, a small molecule chemical compound, and an antisense nucleic acid molecule. acids.

22. A composition comprising an IL-18BP inhibitor or IL-18BP antagonist, wherein the inhibitor or antagonist inhibits the ability of IL18BP to neutralize endogenous IL-18.

23. The composition of claim 22, wherein the inhibitor or antagonist comprises at least one inhibitor or antagonist selected from the group consisting of a chemical compound, a polypeptide, a peptide, a peptidomimetic, an antibody, a ribozyme, a small molecule chemical compound, and an antisense nucleic acid molecule .

24. The composition of claim 22, comprising an IL-18 polypeptide variant, wherein the IL-18 polypeptide variant specifically binds to an IL-18 binding protein (IL-18BP) and wherein the IL-18 polypeptide variant exhibits significantly reduced receptor binding IL-18 (IL-18R).

25. The composition of claim 24, wherein the variant IL-18 polypeptide comprises at least one mutation relative to wild-type IL-18 (WT IL-18).

26. The composition of claim 24, wherein WT IL-18 is human IL-18 comprising the amino acid sequence of SEQ ID NO: 30.

27. The composition of claim 24, wherein WT IL-18 is murine IL-18 comprising the amino acid sequence of SEQ ID NO: 31.

28. The composition of claim 26, wherein the IL-18 polypeptide variant comprises at least one mutation selected from the group consisting of Y1X, L5X, D17X, E31X, T34X, D35X, S36X, D37X, D40X, N41X, M51X , Q56X, M60X, Q103X, H109X, M113X and R131X, relative to SEQ ID NO: 30.

29. The composition of claim 26, wherein the variant IL-18 polypeptide comprises at least one mutation selected from the group consisting of Y1D, Y1F, Y1H, Y1L, L5F, L5H, D17A, D17G, D17R, D17H, E31A . 1R, M51F , M51L, M51I, Q56H, M60L, M60F, M60I, Q103L, Q103I, H109A, H109P, H109D, M113L, M113I, M113F and R131S, with respect to SEQ ID NO: 30.

30. The composition of claim 24, wherein the IL-18 polypeptide variant comprises at least one IL-18 polypeptide variant selected from the group consisting of SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94 , SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO : 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119 , SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, or a fragment thereof.

31. The composition of claim 27, wherein the IL-18 polypeptide variant comprises at least one mutation selected from the group consisting of N1X, L5X, D17X, E30X, T33X, D34X, I35X, D36X, M50X, Q102X, R104 , H108X, N109X, M111X, D129X and D130X, relative to SEQ ID NO: 31.

32. The composition of claim 4, wherein the variant IL-18 polypeptide comprises at least one mutation selected from the group consisting of N1Y, N1D, N1H, N1L, N1F, N1V, N1I, L5Y, L5H, D17Q, D17G , D17A, D17E, D17S, D17N, E30A, E30R, E30K, E30T, E30G, T33G, T33A, T33E, T33R, T33K, D34Y, D34S, D34A, I35T, I35K, I35R, D36V, D36A, D36G, D36H, D36P . A, D129F, D129V, D129Y, D129S , D130E, D130T, D130G, D130N, D130R, D130S, D130Q and D130H, relative to SEQ ID NO: 31.

33. The composition of claim 2, wherein the IL-18 polypeptide variant comprises at least one IL-18 polypeptide variant selected from the group consisting of SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128 , SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO : 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153 , SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO : 170, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178 , SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, or a fragment thereof.

34. A composition comprising a nucleic acid encoding at least one of the variants of the IL-18 polypeptide according to paragraphs. 24-33.

35. A method of treating or preventing a disease or disorder in a subject in need thereof, comprising administering to the subject at least one composition according to paragraphs. 22-34.

36. The method of claim 35 wherein the disease or disorder is cancer.

37. The method of claim 36 wherein the cancer is a cancer that is resistant to immune checkpoint inhibitors (ICIs).

38. The method of claim 36 wherein the cancer is associated with a tumor lacking the expression of class I MHC molecules.

39. The method of claim 35, wherein the disease or disorder is a metabolic disease or disorder.

40. The method of claim 35, wherein the disease or disorder is an infectious disease.

41. The method of claim 35, further comprising administering at least one other agent to the subject.

42. The method of claim 41 wherein at least one other agent comprises an inhibitor of one or more secondary cytokines.

43. The method of claim 42, wherein the one or more secondary cytokines is at least one cytokine selected from the group consisting of IL-17, IL-5, and IL-13.

44. The method of claim 42, wherein the inhibitor of one or more secondary cytokines comprises at least one inhibitor selected from the group consisting of a chemical compound, a polypeptide, a peptide, a peptidomimetic, an antibody, a ribozyme, a small molecule chemical compound, and an antisense nucleic acid molecule. acids.

SET OF ASPECTS B

1. A composition comprising an IL-18 polypeptide variant, wherein the IL-18 polypeptide variant specifically binds to the IL-18 receptor (IL-18R) and wherein the IL-18 polypeptide variant exhibits significantly reduced binding to the IL-18 binding protein ( IL-18BP).

2. The composition of claim 1, wherein the variant IL-18 polypeptide comprises at least one mutation relative to wild-type IL-18 (WT IL-18).

3. The composition of claim 2, wherein WT IL-18 is human IL-18 comprising the amino acid sequence of SEQ ID NO: 30.

4. The composition of claim 2, wherein WT IL-18 is murine IL-18 comprising the amino acid sequence of SEQ ID NO: 31.

5. The composition of claim 3, wherein the variant IL-18 polypeptide comprises at least one mutation selected from the group consisting of Y1X, L5X, K8X, M51X, K53X, S55X, Q56X, P57X, G59X, M60X, E77X , Q103X, S105X, D110X, N111X, M113X, V153X and N155X, relative to SEQ ID NO: 30.

6. The composition of claim 3, wherein the IL-18 polypeptide variant comprises at least one mutation selected from the group consisting of 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, G59A, M60K, M60Q, M60R, M60L , E77D, Q103E, Q103K, Q103P, Q103A, Q103R, S105R, S105D, S105K, S105N, S105A, D110H, D110K, D110N, D110Q, D110E, D110S, D110G, N111H, N111Y, N 111D, N111R, N111S, N111G, M113V , M113R, M113T, M113K, V153I, V153T, V153A, N155K and N155H, relative to SEQ ID NO: 30.

7. The composition of claim 3, wherein the variant IL-18 polypeptide comprises mutations M51X, M60X, S105X, D110X and N111X relative to SEQ ID NO: 30.

8. The composition of claim 3, wherein the IL-18 polypeptide variant comprises the M51X, K53X, Q56X, S105X, and N111X mutations relative to SEQ ID NO: 30.

9. The composition of claim 2, wherein the variant IL-18 polypeptide comprises the amino acid sequence represented by any one of SEQ ID NOs.: 34-59, 73-91, 191-193, or a fragment thereof.

10. The composition of claim 4, wherein the variant IL-18 polypeptide comprises at least one mutation selected from the group consisting of N1X, M50X, Y51X, K52X, S54X, E55X, V56X, R57X, G58X, L59X, R104X , N109X and L151X, relative to SEQ ID NO: 31.

11. The composition of claim 4, wherein the variant IL-18 polypeptide comprises at least one mutation selected from the group consisting of N1H, N1Y, M50A, M50S, M50V, M50G, M50T, Y51R, K52V, K52S, K52T , K52G, K52A, S54R, S54K, S54G, S54N, E55R, E55H, E55N, E55D, E55G, V56L, V56M, V56R, V56A, V56S, V56Q, R57G, R57K, G58A, L59K, L59R, L59V, R104K , R104L , R104Q, R104S, N109D and L151V, relative to SEQ ID NO: 31.

12. The composition of claim 2, wherein the variant IL-18 polypeptide comprises the amino acid sequence represented by any one of SEQ ID NOs.: 60-72, or a fragment thereof.

13. A composition comprising a nucleic acid encoding a variant IL-18 polypeptide of the composition according to any one of paragraphs. 1-12.

14. Composition according to any one of paragraphs. 1-13, further comprising one or more selected from (i) an immune checkpoint inhibitor; (ii) an agent that inhibits one or more proteins selected from PD-L1, PD1, CTLA4, TIM3, TIGIT, LAG3, B7H3, B7H4, VISTA, ICOS, GITR, 41BB, OX40 and CD40; (iii) a cancer cell opsonizing agent; and (iv) an agent that targets one or more antigens selected from 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 ganglioside, MUC1 , CanAg mucin, mesothelin, endoglin, Lewis Y antigen, CEA, CEACAM1, CEACAM5, CA-125, PSMA, BAFF, FGFR2, TAG-72, gelatinase B, glypican 3, nectin-4, BCMA, CSF1R, SLAMF7, integrin α _v β ₃ , TYRP1, GPNMB, CLDN18.2, FOLR1, CCR4, CXCR4, MICA, C242 antigen, DLL3, DLL4, EGFL7, vimentin, fibronectin extra domain-B, TROP-2, LRRC15, FAP, SLITRK6, NOTCH2, NOTCH3 , tanascin-3, STEAP1 and NRP1.

15. A method of treating or preventing a disease or disorder in a subject in need thereof, comprising administering to the subject a composition according to any one of paragraphs. 1-14.

16. The method of claim 15 wherein the disease or disorder is cancer.

17. The method of claim 16 wherein the cancer is a cancer that is immune checkpoint inhibitor (ICI) resistant.

18. The method of claim 16 wherein the cancer is associated with a tumor lacking the expression of class I MHC molecules.

19. The method of claim 15 wherein the disease or disorder is a metabolic disease or disorder.

20. The method of claim 15, wherein the disease or disorder is an infectious disease.

21. The method according to any one of paragraphs. 15-20, wherein the method comprises administering to the subject an IL-18 polypeptide variant and at least one other agent.

22. The method of claim 21 wherein at least one other agent comprises an immune checkpoint inhibitor.

23. The method of claim 22, wherein the immune checkpoint inhibitor is an agent that inhibits PD-L1, PD1, CTLA4, TIM3, TIGIT, LAG3, B7H3, B7H4, VISTA, ICOS, GITR, 41BB, OX40, or CD40, or any their combination.

24. The method of claim 21, wherein the at least one other agent comprises an agent that opsonizes cancer cells.

25. The method of claim 24, wherein at least one other agent targets one or more antigens selected from 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), ganglioside GD2, MUC1, CanAg mucin, mesothelin, endoglin, Lewis Y antigen, CEA, CEACAM1, CEACAM5, CA-125, PSMA, BAFF, FGFR2, TAG-72, gelatinase B, glypican 3, nectin- 4, BCMA, CSF1R, SLAMF7, integrin α _v β ₃ , TYRP1, GPNMB, CLDN18.2, FOLR1, CCR4, CXCR4, MICA, C242 antigen, DLL3, DLL4, EGFL7, vimentin, fibronectin extra domain-B, TROP-2 , LRRC15, FAP, SLITRK6, NOTCH2, NOTCH3, tanascin-3, STEAP1 and NRP1.

26. The method according to any one of paragraphs. 21-25, wherein at least one other agent is conjugated to a variant IL-18 polypeptide.

27. The method of claim 21 wherein at least one other agent is altered T cells or NK cells.

28. The method of claim 21 wherein at least one other agent is an oncolytic virus.

29. A composition comprising an IL-18BP inhibitor or IL-18BP antagonist, wherein the inhibitor or antagonist inhibits the ability of IL18BP to neutralize endogenous IL-18.

30. The composition of claim 29, wherein the inhibitor or antagonist comprises at least one inhibitor or antagonist selected from the group consisting of a chemical compound, a polypeptide, a peptide, a peptidomimetic, an antibody, a ribozyme, a small molecule chemical compound, and an antisense nucleic acid molecule .

31. The composition of claim 30, comprising an IL-18 polypeptide variant, wherein the IL-18 polypeptide variant specifically binds to an IL-18 binding protein (IL-18BP) and wherein the IL-18 polypeptide variant exhibits significantly reduced receptor binding IL-18 (IL-18R).

32. The composition of claim 31, wherein the variant IL-18 polypeptide comprises at least one mutation relative to wild-type IL-18 (WT IL-18).

33. The composition of claim 32, wherein WT IL-18 is human IL-18 comprising the amino acid sequence of SEQ ID NO: 30.

34. The composition of claim 32, wherein WT IL-18 is murine IL-18 comprising the amino acid sequence of SEQ ID NO: 31.

35. The composition of claim 33, wherein the IL-18 polypeptide variant comprises at least one mutation selected from the group consisting of Y1X, L5X, D17X, E31X, T34X, D35X, S36X, D37X, D40X, N41X, M51X , Q56X, M60X, Q103X, H109X, M113X and R131X, relative to SEQ ID NO: 30.

36. The composition of claim 33, wherein the variant IL-18 polypeptide comprises at least one mutation selected from the group consisting of Y1D, Y1F, Y1H, Y1L, L5F, L5H, D17A, D17G, D17R, D17H, E31A . 1R, M51F , M51L, M51I, Q56H, M60L, M60F, M60I, Q103L, Q103I, H109A, H109P, H109D, M113L, M113I, M113F and R131S, with respect to SEQ ID NO: 30.

37. The composition of claim 33, wherein the variant IL-18 polypeptide comprises the amino acid sequence represented by any one of SEQ ID NOs.: 92-125, or a fragment thereof.

38. The composition of claim 33, wherein the IL-18 polypeptide variant includes D17X, E30X and Q103X mutations relative to SEQ ID NO: 30.

39. The composition of claim 33, wherein the variant IL-18 polypeptide comprises mutations D17X, E30X, D35X, M51X, and Q103X relative to SEQ ID NO: 30.

40. The composition of claim 34, wherein the variant IL-18 polypeptide comprises at least one mutation selected from the group consisting of N1X, L5X, D17X, E30X, T33X, D34X, I35X, D36X, M50X, Q102X, R104 , H108X, N109X, M111X, D129X and D130X, relative to SEQ ID NO: 31.

41. The composition of claim 34, wherein the IL-18 polypeptide variant comprises at least one mutation selected from the group consisting of N1Y, N1D, N1H, N1L, N1F, N1V, N1I, L5Y, L5H, D17Q, D17G , D17A, D17E, D17S, D17N, E30A, E30R, E30K, E30T, E30G, T33G, T33A, T33E, T33R, T33K, D34Y, D34S, D34A, I35T, I35K, I35R, D36V, D36A, D36G, D36H, D36P . A, D129F, D129V, D129Y, D129S , D130E, D130T, D130G, D130N, D130R, D130S, D130Q and D130H, relative to SEQ ID NO: 31.

42. The composition of claim 34, wherein the variant IL-18 polypeptide comprises the amino acid sequence represented by any one of SEQ ID NOs.: 126-190, or a fragment thereof.

43. A composition comprising a nucleic acid encoding a variant IL-18 polypeptide of the composition according to any one of paragraphs. 31-42.

44. Composition according to any one of paragraphs. 19-43, further comprising one or more selected from (i) an immune checkpoint inhibitor; (ii) an agent that inhibits one or more proteins selected from PD-L1, PD1, CTLA4, TIM3, TIGIT, LAG3, B7H3, B7H4, VISTA, ICOS, GITR, 41BB, OX40 and CD40; (iii) a cancer cell opsonizing agent; and (iv) an agent that targets one or more antigens selected from 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 ganglioside, MUC1 , CanAg mucin, mesothelin, endoglin, Lewis Y antigen, CEA, CEACAM1, CEACAM5, CA-125, PSMA, BAFF, FGFR2, TAG-72, gelatinase B, glypican 3, nectin-4, BCMA, CSF1R, SLAMF7, integrin α _v β ₃ , TYRP1, GPNMB, CLDN18.2, FOLR1, CCR4, CXCR4, MICA, C242 antigen, DLL3, DLL4, EGFL7, vimentin, fibronectin extra domain-B, TROP-2, LRRC15, FAP, SLITRK6, NOTCH2, NOTCH3 , tanascin-3, STEAP1 and NRP1.

45. A method of treating or preventing a disease or disorder in a subject in need thereof, comprising administering to the subject a composition according to any one of paragraphs. 29-44.

46. The method of claim 45 wherein the disease or disorder is cancer.

47. The method of claim 46, wherein the cancer is a cancer that is resistant to immune checkpoint inhibitors (ICIs).

48. The method of claim 46, wherein the cancer is associated with a tumor that lacks the expression of class I MHC molecules.

49. The method of claim 45 wherein the disease or disorder is a metabolic disease or disorder.

50. The method of claim 45, wherein the disease or disorder is an infectious disease.

51. The method according to any one of paragraphs. 45-50, wherein the method comprises administering to the subject at least one agent other than an IL-18BP inhibitor or IL-18BP antagonist.

52. The method of claim 51 wherein at least one other agent comprises an immune checkpoint inhibitor.

53. The method of claim 52, wherein the immune checkpoint inhibitor is an agent that inhibits PD-L1, PD1, CTLA4, TIM3, TIGIT, LAG3, B7H3, B7H4, VISTA, ICOS, GITR, 41BB, OX40, or CD40, or any their combination.

54. The method of claim 51, wherein the at least one other agent comprises an agent that opsonizes cancer cells.

55. The method according to p. 51, where at least one other agent specifically affects one or more antigens selected from 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), ganglioside GD2, MUC1, CanAg mucin, mesothelin, endoglin, Lewis Y antigen, CEA, CEACAM1, CEACAM5, CA-125, PSMA, BAFF, FGFR2, TAG-72, gelatinase B, glypican 3, nectin- 4, BCMA, CSF1R, SLAMF7, integrin α _v β ₃ , TYRP1, GPNMB, CLDN18.2, FOLR1, CCR4, CXCR4, MICA, C242 antigen, DLL3, DLL4, EGFL7, vimentin, fibronectin extra domain-B, TROP-2 , LRRC15, FAP, SLITRK6, NOTCH2, NOTCH3, tanascin-3, STEAP1 and NRP1.

56. The method according to any one of paragraphs. 51-55, wherein the inhibitor or antagonist is an IL-18 polypeptide variant and at least one other agent conjugated to an IL-18 polypeptide variant.

57. The method of claim 51 wherein at least one other agent is altered T cells or NK cells.

58. The method of claim 51 wherein at least one other agent is an oncolytic virus.

The entire content of each patent, patent application and publication cited in the invention is incorporated into the present invention by reference to them. Although this invention has been disclosed through the description of specific embodiments, however, it is obvious that other embodiments and variations of this invention can be developed by other specialists in this field without deviating from the essence and scope of the invention. The appended claims are intended to include all such embodiments and equivalents.

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SEQUENCE LIST

<110> Ring, Aaron

Zhou, Ting

Fisher, Susan

<120> INTERLEUKIN-18 OPTIONS AND THEIR APPLICATIONS

<130> 047162-5249-00-WO.607779

<150> US 62/554,605

<151> 2017-09-06

<150> US 62/652,279

<151> 2018-04-03

<160> 193

<170>PatentIn version 3.5

<210> 1

<211> 58

<212> DNA

<213> artificial sequence

<220>

<223> Synthetic sequence

<400> 1

cattttcatt aagatgcagt tacttcgctg tttttcaata ttttctgtta ttgctagc 58

<210> 2

<211> 80

<212> DNA

<213> artificial sequence

<220>

<223> Synthetic sequence

<220>

<221> other_features

<222> (31)..(31)

<223> n is a, c, g or t

<220>

<221> other_features

<222> (42)..(42)

<223> n is a, c, g or t

<400> 2

aattacggat gaccgaaagt ykggattcaw ncttgccgaa anrtgctaaa acgctagcaa 60

taacagaaaa tattgaaaaa 80

<210> 3

<211> 51

<212> DNA

<213> artificial sequence

<220>

<223> Synthetic sequence

<400> 3

actttcggtc atccgtaatt tgaacgacca agtccttttt attgaccagg g 51

<210> 4

<211> 55

<212> DNA

<213> artificial sequence

<220>

<223> Synthetic sequence

<400> 4

actatccgtc atatcctcga ataagggacg attgccctgg tcaataaaaa ggact 55

<210> 5

<211> 44

<212> DNA

<213> artificial sequence

<220>

<223> Synthetic sequence

<400> 5

cttattcgag gatatgacgg atagtgattg ccgtgacaac gccc 44

<210> 6

<211> 84

<212> DNA

<213> artificial sequence

<220>

<223> Synthetic sequence

<220>

<221> other_features

<222> (22)..(22)

<223> n is a, c, g or t

<220>

<221> other_features

<222> (46)..(46)

<223> n is a, c, g or t

<400> 6

actgagattg ttaccgcchb tnyacggggt tgwyyatcty tatasnyaga gatgatgaaa 60

attgtacgag gggcgttgtc acgg 84

<210> 7

<211> 52

<212> DNA

<213> artificial sequence

<220>

<223> Synthetic sequence

<400> 7

ggcggtaaca atctcagtta agtgcgaaaa aatctcgaca ctttcttgtg aa 52

<210> 8

<211> 53

<212> DNA

<213> artificial sequence

<220>

<223> Synthetic sequence

<400> 8

ggttcatttc cttgaacgaa atgatcttgt tttcacaaga aagtgtcgag att 53

<210> 9

<211> 62

<212> DNA

<213> artificial sequence

<220>

<223> Synthetic sequence

<400> 9

catttcgttc aaggaaatga acccgccggga taatatcaag gatacaaaat cagatattat 60

tt62

<210> 10

<211> 88

<212> DNA

<213> artificial sequence

<220>

<223> Synthetic sequence

<220>

<221> other_features

<222> (27)..(27)

<223> n is a, c, g or t

<400> 10

tgatgagctc tcgaattgca tcttatnwtb gtgtccaggc acwyyacgwt bgaagaaaat 60

aatatctgat tttgtatcct tgatatta 88

<210> 11

<211> 51

<212> DNA

<213> artificial sequence

<220>

<223> Synthetic sequence

<400> 11

ataagatgca attcgagagc tcatcatacg aaggttactt tttagcctgc g 51

<210> 12

<211> 50

<212> DNA

<213> artificial sequence

<220>

<223> Synthetic sequence

<400> 12

aattaactta aacaggtcgc gctccttctc gcaggctaaa aagtaacctt 50

<210> 13

<211> 49

<212> DNA

<213> artificial sequence

<220>

<223> Synthetic sequence

<400> 13

gcgacctgtt taagttaatt cttaagaaag aagatgagtt gggggatcg 49

<210> 14

<211> 61

<212> DNA

<213> artificial sequence

<220>

<223> Synthetic sequence

<400> 14

ccagaaccac cgtcctcwtb ctgadyggta aacatgatgc tacgatcccc caactcatct 60

t61

<210> 15

<211> 52

<212> DNA

<213> artificial sequence

<220>

<223> Synthetic sequence

<400> 15

gaggacggtg gttctggatc cgaacaaaag cttatctccg aagaagactt gg 52

<210> 16

<211> 41

<212> DNA

<213> artificial sequence

<220>

<223> Synthetic sequence

<400> 16

ccaccagatc caccaccacc caagtcttct tcggagataa g 41

<210> 17

<211> 62

<212> DNA

<213> artificial sequence

<220>

<223> Synthetic sequence

<400> 17

cattttcatt aagatgcagt tacttcgctg tttttcaata ttttctgtta ttgctagcgt 60

tt62

<210> 18

<211> 55

<212> DNA

<213> artificial sequence

<220>

<223> Synthetic sequence

<220>

<221> other_features

<222> (25)..(25)

<223> n is a, c, g or t

<400> 18

ttgtacagtg aagtcggcca aaawntgcta aaacgctagc aataacagaa aatat 55

<210> 19

<211> 59

<212> DNA

<213> artificial sequence

<220>

<223> Synthetic sequence

<400> 19

gccgacttca ctgtacaacc gcagtaatac ggaatataaa tgaccaagtt ctcttcgtt 59

<210> 20

<211> 67

<212> DNA

<213> artificial sequence

<220>

<223> Synthetic sequence

<400> 20

ttgatcaata tcagtcatat cctcgaacac aggctgtctt ttgtcaacga agagaacttg 60

gtcattt 67

<210> 21

<211> 54

<212> DNA

<213> artificial sequence

<220>

<223> Synthetic sequence

<400> 21

gtgttcgagg atatgactga tattgatcaa agtgccagtg aaccccagac caga 54

<210> 22

<211> 78

<212> DNA

<213> artificial sequence

<220>

<223> Synthetic sequence

<220>

<221> other_features

<222> (24)..(24)

<223> n is a, c, g or t

<220>

<221> other_features

<222> (30)..(30)

<223> n is a, c, g or t

<220>

<221> other_features

<222> (32)..(32)

<223> n is a, c, g or t

<220>

<221> other_features

<222> (42)..(42)

<223> n is a, c, g or t

<220>

<221> other_features

<222> (44)..(44)

<223> n is a, c, g or t

<220>

<221> other_features

<222> (46)..(46)

<223> n is a, c, g or t

<220>

<221> other_features

<222> (48)..(48)

<223> n is a, c, g or t

<400> 22

tcacagagag ggtcacagcy hbtnywbybn bnybwyygtc snbnynsnyg tatattatca 60

gtctggtctg gggttcac 78

<210> 23

<211> 58

<212> DNA

<213> artificial sequence

<220>

<223> Synthetic sequence

<400> 23

gctgtgaccc tctctgtgaa ggatagtaaa atgtctaccc tctcctgtaa gaacaaga 58

<210> 24

<211> 69

<212> DNA

<213> artificial sequence

<220>

<223> Synthetic sequence

<400> 24

gtatatcatc aatattttca ggtggatcca ttttcctcaaa ggaaatgatc ttgttcttac 60

aggagagg 69

<210> 25

<211> 99

<212> DNA

<213> artificial sequence

<220>

<223> Synthetic sequence

<220>

<221> other_features

<222> (59)..(59)

<223> n is a, c, g or t

<220>

<221> other_features

<222> (74)..(74)

<223> n is a, c, g or t

<400> 25

aatggatcca cctgaaaata ttgatgatat acaaagtgat ctcatattct ttcagaaand 60

hgttccagga cacnataaga tggagtttga atcttcact 99

<210> 26

<211> 57

<212> DNA

<213> artificial sequence

<220>

<223> Synthetic sequence

<400> 26

ccttttggca agcaagaaag tgtccttcat acagtgaaga ttcaaactcc atcttat 57

<210> 27

<211> 60

<212> DNA

<213> artificial sequence

<220>

<223> Synthetic sequence

<400> 27

ctttcttgct tgccaaaagg aagatgatgc tttcaaactc attctgaaaa aaaaggatga 60

<210> 28

<211> 77

<212> DNA

<213> artificial sequence

<220>

<223> Synthetic sequence

<400> 28

ccaccacttt gatgtaagtt agtrdbagtg aacattacag atttatcccc attttcatcc 60

ttttttttca gaatgag 77

<210> 29

<211> 59

<212> DNA

<213> artificial sequence

<220>

<223> Synthetic sequence

<400> 29

actaacttac atcaaagtgg tggttctgga tccgaacaaa agcttatctc cgaagaaga 59

<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 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> homo sapiens

<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> house mouse

<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> Synthetic 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 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> Synthetic 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 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> Synthetic 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 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> Synthetic 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 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> Synthetic 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 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> Synthetic 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 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> Synthetic sequence

<220>

<221> other_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 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> Synthetic 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 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> Synthetic 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 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> Synthetic 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 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> Synthetic 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 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> Synthetic 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 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> Synthetic 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 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> Synthetic 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 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> Synthetic 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 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> Synthetic 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 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> Synthetic 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 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> Synthetic 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 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> Synthetic 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 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> Synthetic 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 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> Synthetic sequence

<220>

<221> other_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 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> Synthetic 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 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> Synthetic 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 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> Synthetic 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 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> Synthetic 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 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> Synthetic 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 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> Synthetic 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> Synthetic 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> Synthetic 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> Synthetic 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> Synthetic 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> Synthetic 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> Synthetic 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> Synthetic 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> Synthetic 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> Synthetic 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> Synthetic 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> Synthetic 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> Synthetic 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> Synthetic 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 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> Synthetic 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 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> Synthetic 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 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> Synthetic 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> Synthetic 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 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> Synthetic 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 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> Synthetic 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> Synthetic 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 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> Synthetic 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 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> Synthetic 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 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> Synthetic 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> Synthetic 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> Synthetic 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> Synthetic 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 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> Synthetic 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 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> Synthetic 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 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> Synthetic 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 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> Synthetic 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 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> Synthetic 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 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> Synthetic 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> Synthetic 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 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> Synthetic 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 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> Synthetic 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> Synthetic 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> Synthetic 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> Synthetic 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> Synthetic 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> Synthetic 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> Synthetic 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> Synthetic 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> Synthetic 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> Synthetic 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> Synthetic 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> Synthetic 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 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> Synthetic 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> Synthetic 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> Synthetic 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> Synthetic 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> Synthetic 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> Synthetic 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> Synthetic 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> Synthetic 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> Synthetic 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 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> Synthetic 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> Synthetic 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> Synthetic 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> Synthetic 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 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> Synthetic 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> Synthetic 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 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> Synthetic 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> Synthetic 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 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> Synthetic 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 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> Synthetic 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 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> Synthetic 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 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> Synthetic 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> Synthetic 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 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> Synthetic 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 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> Synthetic 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> Synthetic 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 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> Synthetic 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> Synthetic 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> Synthetic 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 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> Synthetic 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 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> Synthetic 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 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> Synthetic 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 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> Synthetic 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> Synthetic 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 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> Synthetic 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 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> Synthetic 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 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> Synthetic 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 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> Synthetic 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 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> Synthetic 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 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> Synthetic 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> Synthetic 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 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> Synthetic 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> Synthetic 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 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> Synthetic 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> Synthetic 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> Synthetic 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> Synthetic 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 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> Synthetic 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 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> Synthetic 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> Synthetic 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> Synthetic 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> Synthetic 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> Synthetic 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> Synthetic 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 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> Synthetic 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> Synthetic 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> Synthetic 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 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> Synthetic 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 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> Synthetic 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 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> Synthetic 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> Synthetic 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 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> Synthetic 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 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> Synthetic 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 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> Synthetic 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> Synthetic 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> Synthetic 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> Synthetic 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> Synthetic 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> Synthetic 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> Synthetic 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> Synthetic 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 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> Synthetic 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 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> Synthetic 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 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> Synthetic 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 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> Synthetic 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 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> Synthetic 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 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> Synthetic 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> Synthetic 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> Synthetic 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> Synthetic 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> Synthetic 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 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> Synthetic 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 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> Synthetic 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 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> Synthetic 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> Synthetic 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> Synthetic 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 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> Synthetic 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 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> Synthetic 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

<---

Claims (35)

1. Composition to enhance the activity of IL-18, including: (a) a therapeutically effective amount of an interleukin-18 (IL-18) polypeptide variant, wherein said IL-18 polypeptide variant contains five or more mutations relative to wild-type (WT) IL-18 shown in SEQ ID NO: 30, wherein at least at least five of said five or more mutations are at positions selected from M51, K53, Q56, P57, M60, S105, D110 and N111; And (b) a pharmaceutically acceptable excipient. 2. The composition of claim 1, wherein said variant IL-18 polypeptide binds to IL-18BP with a binding affinity that is approximately 10% or less of that of wild-type IL-18 to IL-18BP. 3. The composition of claim 1, wherein said IL-18 polypeptide variant has a K _D value for IL-18BP that is 10 nM or more. 4. Composition for use in the treatment of cancer, comprising: (a) a pharmaceutically acceptable excipient; (b) a therapeutically effective amount of an interleukin-18 (IL-18) polypeptide variant, wherein said IL-18 polypeptide variant specifically binds to the IL-18 receptor (IL-18R) and contains five or more mutations relative to wild-type (WT) IL-18 ) shown in SEQ ID NO: 30, wherein at least five of said five or more mutations are at positions selected from M51, K53, Q56, P57, M60, S105, D110, and N111 relative to WT IL-18, and where compared to WT IL-18 shown in SEQ ID NO: 30, this IL-18 polypeptide variant exhibits reduced binding to IL-18 binding protein (IL-18BP). 5. The composition of claim 4, wherein the variant IL-18 polypeptide comprises at least one mutation selected from the group consisting of Y1X, L5X, K8X, M51X, K53X, S55X, Q56X, P57X, G59X, M60X, E77X , Q103X, S105X, D110X, N111X, M113X, V153X and N155X, relative to SEQ ID NO: 30, where X is any amino acid. 6. The composition of claim 5, wherein the variant IL-18 polypeptide comprises at least one mutation selected from the group consisting of 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, G59A, M60K, M60Q, M60R, M60L , E77D, Q103E, Q103K, Q103P, Q103A, Q103R, S105R, S105D, S105K, S105N, S105A, D110H, D110K, D110N, D110Q, D110E, D110S, D110G, N111H, N111Y, N 111D, N111R, N111S, N111G, M113V , M113R, M113T, M113K, V153I, V153T, V153A, N155K and N155H, relative to SEQ ID NO: 30. 7. The composition of claim 1, wherein the variant IL-18 polypeptide comprises at least one mutation selected from the group consisting of M51X, M60X, S105X, D110X, and N111X relative to SEQ ID NO: 30, where X is any amino acid. 8. The composition according to claim 7, where the IL-18 polypeptide variant contains mutations M51X, M60X, S105X, D110X and N111X, where M at position 51 is mutated to T, K, D, E, R or N; M at position 60 is mutated to K, Q, L, or R; S at position 105 is mutated to R, D, K, A, or N; D at position 110 is mutated to H, K, N, Q, E, N, S, or G; and N at position 111 is mutated to H, D, Y, R, S, or G. 9. The composition according to claim 7, where the IL-18 polypeptide variant contains mutations M51X, M60X, S105X, D110X and N111X, where M at position 51 is mutated to T or K; M at position 60 is mutated to K or L; S at position 105 is mutated to D, N, or A; D at position 110 is mutated to K, N, S, or G; and N at position 111 is mutated to H, Y, G, or R. 10. The composition of claim 1, wherein the variant IL-18 polypeptide comprises at least one mutation selected from the group consisting of M51X, K53X, Q56X, D110X, and N111X relative to SEQ ID NO: 30, where X is any amino acid. 11. The composition according to claim 10, where the IL-18 polypeptide variant contains mutations M51X, K53X, Q56X, D110X and N111X, where M at position 51 is mutated to E, R or K; K at position 53 is mutated to G, S, or T; Q at position 56 is mutated to E, A, R, V, G, K, or L; D at position 110 is mutated to N, S, K, or G; and N at position 111 is mutated to R, S, G, or D. 12. The composition according to claim 10, where the IL-18 polypeptide variant contains mutations M51X, K53X, Q56X, D110X and N111X, where M at position 51 is mutated to K; K at position 53 is mutated to G or S; the Q at position 56 is mutated to G, R, or L; D at position 110 is mutated to S, N, or G; and N at position 111 is mutated to G or R. 13. The composition of claim 1, wherein the variant IL-18 polypeptide comprises an amino acid sequence having an identity of 85% or more with the amino acid sequence represented by any one of SEQ ID NOs: 34-59, 73-91, 191-193. 14. The composition according to any one of paragraphs. 8, 9 and 11, 12, wherein the variant IL-18 polypeptide comprises an amino acid sequence having 85% or more identity with the amino acid sequence shown in SEQ ID NO: 30. 15. The composition according to any one of paragraphs. 1-14 for use in the treatment of cancer, wherein a therapeutically effective amount of an IL-18 polypeptide variant is administered as a nucleic acid. 16. A method of treating or preventing a tumor in a subject in need thereof, comprising administering to the subject a composition according to any one of paragraphs. 1-15. 17. The method of claim 16 wherein the tumor is cancer. 18. The method of claim 17 wherein the cancer is a cancer that is resistant to immune checkpoint inhibitors (ICIs). 19. The method of claim 17 wherein the cancer is associated with a tumor lacking the expression of class I MHC molecules. 20. The method according to any one of paragraphs. 16-19, wherein the method comprises administering to the subject an IL-18 polypeptide variant and at least one other agent selected from an immune checkpoint inhibitor. 21. The method of claim 20 wherein the immune checkpoint inhibitor is (a) an agent that inhibits PD-L1, PD1, CTLA4, TIM3, TIGIT, LAG3, B7H3, B7H4, or VISTA; (b) a CD40 agonist, 41BB agonist, OX40 agonist, or GITR agonist; (c) a cancer cell opsonizing agent; (d) an agent that targets one or more antigens selected from 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 ganglioside, MUC1, mucin CanAg, mesothelin, endoglin, Lewis antigen Y, CEA, CEACAM1, CEACAM5, CA-125, PSMA, BAFF, FGFR2, TAG-72, gelatinase B, glypican 3, nectin-4, BCMA, CSF1R, SLAMF7, integrin α _v β ₃ , TYRP1, GPNMB, CLDN18.2, FOLR1, CCR4, CXCR4, MICA, C242 antigen, DLL3, DLL4, EGFL7, vimentin, fibronectin extra domain-B, TROP-2, LRRC15, FAP, SLITRK6, NOTCH2, NOTCH3, tanascin-3, STEAP1 and NRP1; (e) an altered T cell or NK cell; (f) oncolytic virus; (g) a cytokine optionally selected from IL-1, IL-2, IL-10, IL-12, IL-15, IL-18, IL-21, IL-33, interferon-alpha, interferon-beta, interferon- gamma and TNF; (h) a Toll-like receptor agonist, optionally selected from TLR2, TLR4, TLR5, TLR7 and TLR9; (i) an inflammasome agonist; (j) an agonist of the STING/cGAS pathway; And (k) RIG-I pathway agonist.

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