Use of cofilin-1 derived peptides to treat inflammatory diseases

Abstract

This disclosure provides compositions comprising isolated polypeptides derived from cofilin- 1, and methods and pharmaceutical compositions for suppressing an immune response in a subject.

Description

MBHB Ref. No.: 24-2385- WOUSE OF COFILIN-1 DERIVED PEPTIDES TO TREAT INFLAMMATORY DISEASESCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 762,445, filed February 24, 2025, which is incorporated by reference herein in its entirety.FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0002] This work was made with government support from the National Human Genome Research Institute Intramural Research Program, Project ZIAHG200373. The government has certain rights in the invention.REFERENCE TO SEQUENCE LISTING

[0003] The instant application contains an electronic Sequence Listing that has been submitted electronically and is hereby incorporated by reference in its entirety. The sequence listing was created on February 16, 2026, is named “24-2385- WO_Sequence-Listing. xml” and is 123,684 bytes in size.BACKGROUND

[0004] This disclosure generally relates to isolated polypeptides, and methods and compositions for suppressing an immune response in a subject.

[0005] The inflammasome is an intracellular multiprotein complex that plays a crucial role in inflammatory responses by promoting the maturation and release of the proinflammatory cytokine interleukin (IL)-10 and IL-18. The NLRP3 inflammasome is the most extensively studied inflammasome and has been demonstrated to be activated by a diverse array of pathogen-associated ordanger-associated molecular patterns (PAMPs, DAMPs). NLRP3 is composed of an N-terminal pyrin domain (PYD), a central nucleotide-binding oligomerization domain (NOD, also called NACHT domain) that mediates ATP hydrolysis, and a C-terminal leucine rich repeat (LRR) domain. Missense mutations in the gene encoding NLRP3 cause a spectrum of dominantly inherited or de novo autoinflammatory diseases known as the cryopyrin-associated periodic syndromes (CAPS) characterized by recurrent episodes of fevers, urticarial skin rash, varying degrees of arthralgia / arthritis, neutrophil-mediated inflammation, and an intense acute-phase response. The activation of the NLRP3 inflammasome also has been implicated in various inflammatory and autoimmune diseases,MBHB Ref. No.: 24-2385- WOsuch as gout, atherosclerosis, type 2 diabetes, Alzheimer's disease, cryopyrin-associated periodic syndromes (CAPS), and inflammatory bowel disease. Understanding the regulation and signaling pathways of NLRP3 inflammasome activation is crucial for developing therapeutic strategies to target inflammatory diseases.

[0006] In the canonical pathway, the activation of the NLRP3 inflammasome involves two distinct signals. The initial signal, also known as the priming signal, induces the expression of components of the NLRP3 inflammasome, including NLRP3 itself, pro-IL-10, and pro-IL-18 in response to inflammatory cytokines or PAMPs, such as lipopolysaccharide (LPS) from bacteria. Under normal conditions, the NLRP3 protein is thought to be maintained in a closed or inactive conformation, resembling a “cage”, preventing spontaneous activation and inappropriate inflammation. However, upon exposure to certain danger signals, such as microbial components, environmental irritants, or endogenous danger signals from damaged cells, the inactive NLRP3 undergoes conformational changes, leading to its activation. These danger signals are believed to disrupt the stability of the inactive conformation and promote the assembly of the NLRP3 inflammasome complex. In addition to the activation signal, interaction of NLRP3 with NEK7 and hydrolysis of ATP by an ATPase activity on the NACHT domain of NLRP3 are required prior to activation of the NLRP3 inflammasome. In response to the activation signals, three intracellular signaling pathways have been proposed for NLRP3 inflammasome activation: depletion of intracellular K+, generation of reactive oxygen species (ROS) from mitochondria, and cathepsin B released from ruptured lysosomes. Despite the diversity of activating signals, ROS has been suggested as a common major factor in NLRP3 inflammasome activation since most extracellular NLRP3 activators induce ROS generation.

[0007] ROS production from macrophages is an important component of the innate immune response against bacterial infections or their components (PAMPs), highlighting the dynamic interplay between host immune cells and pathogens during the infectious process. There is no evidence for post-translational modification of NLRP3 by ROS, as well as for direct interaction of activators with NLRP3. This suggests that ROS might modulate NLRP3 inflammasome activation indirectly through a ROS sensing molecule that binds NLRP3, as seen in the activation mechanism of the pyrin inflammasome, in which there is liberation of inhibitory 14-3-3 proteins from pyrin in response to upstream RhoA GTPase inactivation by certain bacterial toxins.

[0008] Therefore, there remains a need to elucidate the molecular mechanism by which a change in cellular redox state leads to NLRP3 inflammasome activation, as well as the molecular pathogenesis of CAPS.MBHB Ref. No.: 24-2385- WOSUMMARY

[0009] It is against the above background that the present disclosure provides certain advantages over the prior art.

[0010] Although this disclosure as provided herein is not limited to specific advantages or functionalities, the disclosure provides compositions comprising isolated polypeptides derived from cofilin-1, and methods and pharmaceutical compositions for suppressing an immune response in a subject.

[0011] In one aspect, this disclosure provides an isolated polypeptide comprising an amino acid sequence having about 15 amino acids to about 120 amino acids, wherein the amino acid sequence has at least 75% identity to an equal-length portion of residues 35-155 of SEQ ID NO:01.

[0012] In some embodiments, the amino acid sequence of the isolated polypeptide disclosed herein comprises at least one substitution relative to residues 35-155 of SEQ ID NO:01.

[0013] In some embodiments, the isolated polypeptide disclosed herein further comprises at least one modification.

[0014] In some embodiments, the at least one modification improves stability of the polypeptide, extends half-life of the polypeptide, decreases degradation of the polypeptide, and / or promotes delivery of the polypeptide to one or more targets.

[0015] In some embodiments, the isolated polypeptide disclosed herein is a cyclic peptide. In some embodiments, the cyclic peptide comprises head-to-tail cyclic peptides.

[0016] In some embodiments, the amino acid sequence is selected from the group consisting of:95-KKEDLVFIFWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NO:02),94-SKKEDLVFIFWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NQ:03),93-ESKKEDLVFIFWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NQ:04),92-KESKKEDLVFIFWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NQ:05),91-TKESKKEDLVFIFWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NQ:06),90-ETKESKKEDLVFIFWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NQ:07),89-YETKESKKEDLVFIFWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NQ:08),88-TYETKESKKEDLVFIFWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NQ:09), 87-ATYETKESKKEDLVFIFWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NQ:10), 86-DATYETKESKKEDLVFIFWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NO:11), 85-YDATYETKESKKEDLVFIFWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NO: 12), 95-KKEDLVFIFWAPESAPLKSKMIYASSKDAIKK-126 (SEQ ID NO: 13),94-SKKEDLVFIFWAPESAPLKSKMIYASSKDAIK-125 (SEQ ID NO:14),MBHB Ref. No.: 24-2385- WO-ESKKEDLVFIFWAPESAPLKSKMIYASSKDAI-124 (SEQ ID NO: 15), -KESKKEDLVFIFWAPESAPLKSKMIYASSKDA-123 (SEQ ID NO:16), -TKESKKEDLVFIFWAPESAPLKSKMIYASSKD-122 (SEQ ID NO:17), -ETKESKKEDLVFIFWAPESAPLKSKMIYASSK-121 (SEQ ID NO: 18), -YETKESKKEDLVFIFWAPESAPLKSKMIYASS-120 (SEQ ID NO:19), -TYETKESKKEDLVFIFWAPESAPLKSKMIYAS-119 (SEQ ID NQ:20), -ATYETKESKKEDLVFIFWAPESAPLKSKMIYA-118 (SEQ ID NO:21), -DATYETKESKKEDLVFIFWAPESAPLKSKMIY-117 (SEQ ID NO:22), -YDATYETKESKKEDLVFIFWAPESAPLKSKMI-116 (SEQ ID NO:23), -KKEDLVFIYWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NO:24), -SKKEDLVFIYWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NO:25), -ESKKEDLVFIYWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NO:26), -KESKKEDLVFIYWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NO:27), -TKESKKEDLVFIYWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NO:28), -ETKESKKEDLVFIYWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NO:29), -YETKESKKEDLVFIYWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NQ:30), -TYETKESKKEDLVFIYWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NO:31), -ATYETKESKKEDLVFIYWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NO:32), -DATYETKESKKEDLVFIYWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NO:33), -YDATYETKESKKEDLVFIYWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NO:34), -KKEDLVFIYWAPESAPLKSKMIYASSKDAIKK-126 (SEQ ID NO:35), -SKKEDLVFIYWAPESAPLKSKMIYASSKDAIK-125 (SEQ ID NO:36), -ESKKEDLVFIYWAPESAPLKSKMIYASSKDAI-124 (SEQ ID NO:37), -KESKKEDLVFIYWAPESAPLKSKMIYASSKDA-123 (SEQ ID NO:38), -TKESKKEDLVFIYWAPESAPLKSKMIYASSKD-122 (SEQ ID NO:39), -ETKESKKEDLVFIYWAPESAPLKSKMIYASSK-121 (SEQ ID NQ:40), -YETKESKKEDLVFIYWAPESAPLKSKMIYASS-120 (SEQ ID NO:41), -TYETKESKKEDLVFIYWAPESAPLKSKMIYAS-119 (SEQ ID NO:42), -ATYETKESKKEDLVFIYWAPESAPLKSKMIYA-118 (SEQ ID NO:43), -DATYETKESKKEDLVFIYWAPESAPLKSKMIY-117 (SEQ ID NO:44), -YDATYETKESKKEDLVFIYWAPESAPLKSKMI-116 (SEQ ID NO:45), -KKEDLVYIFWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NO:46), -SKKEDLVYIFWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NO:47), -ESKKEDLVYIFWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NO:48), -KESKKEDLVYIFWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NO:49), -TKESKKEDLVYIFWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NQ:50), -ETKESKKEDLVYIFWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NO:51),MBHB Ref. No.: 24-2385- WO-YETKESKKEDLVYIFWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NO:52), -TYETKESKKEDLVYIFWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NO:53), -ATYETKESKKEDLVYIFWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NO:54), -DATYETKESKKEDLVYIFWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NO:55), -YDATYETKESKKEDLVYIFWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NO:56), -KKEDLVYIFWAPESAPLKSKMIYASSKDAIKK-126 (SEQ ID NO:57), -SKKEDLVYIFWAPESAPLKSKMIYASSKDAIK-125 (SEQ ID NO:58), -ESKKEDLVYIFWAPESAPLKSKMIYASSKDAI-124 (SEQ ID NO:59), -KESKKEDLVYIFWAPESAPLKSKMIYASSKDA-123 (SEQ ID NQ:60), -TKESKKEDLVYIFWAPESAPLKSKMIYASSKD-122 (SEQ ID N0:61), -ETKESKKEDLVYIFWAPESAPLKSKMIYASSK-121 (SEQ ID NO:62), -YETKESKKEDLVYIFWAPESAPLKSKMIYASS-120 (SEQ ID NO:63), -TYETKESKKEDLVYIFWAPESAPLKSKMIYAS-119 (SEQ ID NO:64), -ATYETKESKKEDLVYIFWAPESAPLKSKMIYA-118 (SEQ ID NO:65), -DATYETKESKKEDLVYIFWAPESAPLKSKMIY-117 (SEQ ID NO:66), -YDATYETKESKKEDLVYIFWAPESAPLKSKMI-116 (SEQ ID NO:67), -KKEDLVFLFWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NO:68), -SKKEDLVFLFWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NO:69), -ESKKEDLVFLFWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NQ:70), -KESKKEDLVFLFWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID N0:71), -TKESKKEDLVFLFWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NO:72), -ETKESKKEDLVFLFWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NO:73), -YETKESKKEDLVFLFWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NO:74), -TYETKESKKEDLVFLFWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NO:75), -ATYETKESKKEDLVFLFWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NO:76), -DATYETKESKKEDLVFLFWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NO:77), -YDATYETKESKKEDLVFLFWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NO:78), -KKEDLVFLFWAPESAPLKSKMIYASSKDAIKK-126 (SEQ ID NO:79), -SKKEDLVFLFWAPESAPLKSKMIYASSKDAIK-125 (SEQ ID NQ:80), -ESKKEDLVFLFWAPESAPLKSKMIYASSKDAI-124 (SEQ ID N0:81), -KESKKEDLVFLFWAPESAPLKSKMIYASSKDA-123 (SEQ ID NO:82), -TKESKKEDLVFLFWAPESAPLKSKMIYASSKD-122 (SEQ ID NO:83), -ETKESKKEDLVFLFWAPESAPLKSKMIYASSK-121 (SEQ ID NO:84), -YETKESKKEDLVFLFWAPESAPLKSKMIYASS-120 (SEQ ID NO:85), -TYETKESKKEDLVFLFWAPESAPLKSKMIYAS-119 (SEQ ID NO:86), -ATYETKESKKEDLVFLFWAPESAPLKSKMIYA-118 (SEQ ID NO:87), -DATYETKESKKEDLVFLFWAPESAPLKSKMIY-117 (SEQ ID NO:88), orMBHB Ref. No.: 24-2385- WO85-YDATYETKESKKEDLVFLFWAPESAPLKSKMI-116 (SEQ ID NO:89).

[0017] In some embodiments, the amino acid sequence comprises 95- KKEDLVFIFWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NO:02).

[0018] In some embodiments, the amino acid sequence comprises 95- KKEDLVFIYWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NO:24).

[0019] In some embodiments, the amino acid sequence comprises 95- KKEDLVYIFWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NO:46).

[0020] In some embodiments, the amino acid sequence comprises 95- KKEDLVFLFWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NO:68).

[0021] In another aspect, this disclosure provides a nucleic acid composition comprising a nucleic acid sequence encoding the isolated polypeptide disclosed herein.

[0022] In another aspect, this disclosure provides a pharmaceutical composition comprising the isolated polypeptide disclosed herein or the nucleic acid composition disclosed herein.

[0023] In some embodiments, the composition disclosed herein comprises at least one pharmaceutically acceptable carrier or excipient.

[0024] In some embodiments, the pharmaceutical composition disclosed herein further comprises an isotonic agent, a preservative, and / or a buffer.

[0025] In some embodiments, the pharmaceutical composition disclosed herein further comprises an additional active agent.

[0026] In another aspect, this disclosure provides a method of treating an inflammatory condition in a subject, comprising administrating to the subject an effective dose of the pharmaceutical composition disclosed herein.

[0027] In some embodiments, the method of treating an inflammatory condition in a subject comprises repeating the administering at least a second time, at least a third time, at least a fourth time, at least a fifth time, at least a sixth time, or more.

[0028] In some embodiments, the inflammatory condition comprises gout, pericarditis, type 2 diabetes mellitus, atherosclerosis, cryopyrin-associated periodic syndromes (CAPS), or Alzheimer’s disease.

[0029] These and other features and advantages of the present disclosure will be more fully understood from the following detailed description taken together with the accompanying claims. It is noted that the scope of the claims is defined by the recitations therein and not by the specific discussion of features and advantages set forth in the present description.MBHB Ref. No.: 24-2385- WOBRIEF DESCRIPTION OF THE DRAWINGS

[0030] The following detailed description of the embodiments of the present disclosure can be best understood when read in conjunction with the following drawings, where like structure is indicated with like reference numerals and in which:

[0031] FIG. 1A-1E show that NLRP3 interacts with cofilin-1 FIG. 1A PAGE-silver staining analysis of immunoprecipitated (IP) NLRP3 binding proteins in lysates (Lys) of WT or Nlrp3-deficient (Nlrp3-I-) BMDMs treated without or with ATP. FIG. 1B Mass spectrometry analysis of NLRP3 binding proteins in a ~20kDa band, indicated with an arrow in FIG. 1 A, of IP proteins from WT BMDMs treated without or with ATP. FIG. 1C Immunoblot analysis of NLRP3, MYL12b, ARPG4, and cofilin-1 in proteins IP with anti-NLRP3 antibody from Lys of WT or Nlrp3-I- BMDMs treated with or without ATP. FIG. 1D Immunoblot analysis of NLRP3 and cofilin-1 in proteins IP with anti-cofilin- 1 antibody from Lys of WT or Nlrp3- / - BMDMs treated with or without ATP. FIG. 1E, Immunoblot analysis of NLRP3 and cofilin-1 in proteins IP with anti-cofilin-1 , anti-NLRP3 antibodies, or control IgG from mixtures containing 4pg each of purified recombinant human cofilin-1 (95% purity) and human NLRP3 (>80% purity). Data are representative of three independent experiments with similar results (FIG. 1A, 1C-1E).

[0032] FIG. 2A-2F show that cofilin-1 is a negative regulator of the NLRP3 inflammasome FIG. 2A Immunoblot analysis of NLRP3 and cofilin-1 in proteins IP with anti-cofilin-1 antibody from Lys of WT BMDMs treated with indicated NLRP3 inflammasome activators (above lanes).FIG. 2B Immunoblot analysis of IL-10 and caspase-1 in culture supernatants (Sup) and Lys of WT, Asc-, Caspl-, or / V / rp3-deficient BMDMs transiently transfected with negative control siRNA with no substantial sequence similarity to mouse or human gene sequences (N.C.) or siRNA targeting Cfl1, then treated with LPS for 9h). FIG. 2C immunoblot analysis of IL-10 in Sup and Lys of LPS-primed WT BMDMs transiently transfected with negative control siRNA or siRNA targeting Cfl1 , then treated with ATP, nigericin, flagellin or dsDNA (poly(dA:dT)).FIG. 2D Structural model of the predicted cofilin-1 -NLRP3 complex, showing full-length cofilin-1 bound to NLRP3, with NLRP3 domains highlighted by region: PYD, NBD, and LRR. FIG. 2E Immunoblot analysis with antibody to endogenous cofilin-1 in proteins IP with anti-V5 antibody for NLRP3 from Lys of 293T cells transiently expressing full-length NLRP3 or various deletion mutants of NLRP3. Above blots, the schematic structure of full-length NLRP3 and its deletion mutants. PYD, PYRIN domain; NBD, nucleotide-binding domain; LRR, leucine-rich 540 repeats. FIG. 2F Immunoblot analysis with antibody to myc for NLRP3 in proteins IP with antibody to endogenous cofilin-1 from Lys of 293T cells transiently expressing WT or CAPS-MBHB Ref. No.: 24-2385- WOassociated mutant NLRP3. Data are representative of three independent experiments with similar results (FIG. 2A-2C,2E,2F).

[0033] FIG. 3A-3E show that the interaction of cofilin-1 with NLRP3 depends on redox states of cofilin-1. FIG. 3A Schematic diagram of the intramolecular disulfide bond formation between Cys39 and Cys80 after oxidation by ROS. FIG. 3B Immunoblot analysis with monoclonal or polyclonal antibody to cofilin-1 or antibody to phosphorylated cofilin-1 from reduced (with DTT) or non-reduced (without DTT) Lys of LPS-primed BMDMs treated without or with ATP or nigericin. FIG. 3C Immunoblot analyses of Inflammasome activation and oxidation-reduction state of cofilin-1 (top), intracellular ROS measurements (bottom) from LPS-primed BMDMs treated with ATP and various doses of luteolin. n=5 independent replicates from the same BMDM preparation. Data are representative of at least three independent experiments. Data are mean ± s.e.m. P = 0.0317 (*) and P = 0.0079 (**) as determined by unpaired two-tailed Mann-Whitney U test. FIG. 3D Immunoblot analysis of NLRP3 and cofilin-1 under reducing conditions in proteins IP with anti— cofilin- 1 antibody from Lys of LPS-primed WT BMDMs treated with ATP alone or ATP plus luteolin. FIG. 3E Immunoblot analysis of NLRP3 and cofilin-1 under reducing conditions in proteins IP with anti-NLRP3 antibody from mixtures of purified recombinant cofilin-1 and NLRP3 proteins in the presence of various doses of H2O2. Data are representative of three independent experiments with similar results (FIG. 3B-3E).

[0034] FIG. 4A-4F show that Extracellular potassium and MCC950 inhibit dissociation of cofilin-1 from NLRP3. FIG.4A Immunoblot analyses of IL-10 release (top), NLRP3 and cofilin-1 in proteins IP with anti-cofilin-1 antibody (middle), and oxidation-reduction state of cofilin-1 (bottom) from Sup or Lys of LPS-primed WT BMDMs treated with ATP in the presence of 100nM MCC950 or 20mM KOI. FIG. 4B Immunoblot analysis of NLRP3 and cofilin-1 in proteins IP with antibody to NLRP3 from mixtures of purified recombinant cofilin-1 and NLRP3 in the presence of various doses of MCC950 (above lanes). FIG. 4C and FIG.4D Visualization of mitochondrial ROS production in LPS-primed BMDMs treated with ATP and increasing doses of MCC950 (FIG. 4C) or KCI (FIG. 4D). Cells were stained with MitoSOX Green to detect mitochondrial superoxide and counterstained with DAPI to visualize nuclei. All images are shown at the same magnification. Scale bar, 50 pm. FIG. 4E and FIG. 4F Immunoblot analysis of IL-10 release (top) and measurements of intracellular ROS (bottom) from LPS-primed BMDMs treated with ATP and various doses of MCC950 (FIG. 4E) or KCI (FIG. 4F). n=5 independent replicates from the same BMDM preparation. Data are representative of at least three independent experiments. Data are mean + s.e.m, P= 0.0159 (*) and P = 0.0079 (**) as determined by unpaired two-tailed Mann-Whitney U test. FIG. 4G and FIG. 4H Immunoblot analysis of IL-10 release from LPS-primed PBMCs of CAPS patients (1 and 2)MBHB Ref. No.: 24-2385- WOwith various mutations (above lanes) in NLRP3 and treated with various doses of KCI (FIG.4G) or luteolin (FIG. 4H). Data are representative of three independent experiments with similar results (FIG. 4A- FIG. 4F).

[0035] FIG. 5A-5D show that Oxidation-resistant C39A / C80A mutant cofilin- 1 suppresses NLRP3 inflammasome activation. FIG. 5A Immunoblot analysis with antibody to V5 or myc for cofilin-1 in proteins IP with antibody to V5 from Lys of A / m2-deficient BMDMs transiently expressing both V5-tagged and myc-tagged WT cofilin-1 or cofilin-1 with various oxidation resistant cysteine to alanine substitutions (above lanes) and treated with ATP. FIG. 5B Structural comparison of reduced and oxidized cofilin-1. Distance between Cys39 and Cys80 is indicated with dotted line. FIG. 5C Immunoblot analysis of IL-10 release from retroviral-transduced J774A.1 cells, expressing WT or indicated mutant cofilin-1 proteins (above lanes) and treated with ATP, Poly(dA:dT), and flagellin. FIG. 5D GST-pulldown assay of Lys of LPS-primed A / m2-deficient BMDMs transiently expressing GST-tagged WT or indicated mutant cofilin-1 and treated with or without ATP. Data are representative of three independent experiments with similar results (FIG. 5A, 5C and 5D).

[0036] FIG. 6A-6E show that the four amino acid residues from 101 to 104 of cofilin-1 are essential for interaction with NLRP3. FIG. 6A GST-pulldown assay of Lys of LPS-primed U937 cells, expressing endogenous NLRP3, with recombinant GST-tagged full-length or indicated fragments of cofilin-1 proteins (above lanes). Above blots, the schematic structure of full-length cofilin-1 with lines corresponding to N-terminal (aa 1-84), middle (aa 41-127), and C-terminal (aa 85-166) halves of cofilin-1. NLS, nuclear localization signal; ADF-H, actindepolymerizing factor homology domain; PI(4,5)P2; phosphatidylinositol 4,5-bisphosphate binding site. FIG. 6B GST-pulldown assay of Lys of LPS-primed U937 cells, expressing endogenous NLRP3, with recombinant GST-tagged WT (SEQ ID NO:02) or mutant (above lanes) aa 95-127 cofilin-1 fragments. FIG. 6C Predicted changes in binding affinity (AAG_binding) between NLRP3 and cofilin-1 variants with single-residue substitutions at each position of the binding motif (Phe101-lle102-Phe103-Trp104), relative to WT cofilin-1. FIG. 6D Magnified views of interaction interfaces between NLRP3 and cofilin-1, with WT (F103) or alanine (A103) or tyrosine (Y103) substitution at residue 103 (F103). Putative interactions are indicated by dotted lines. FIG. 6E GST-pulldown assay of Lys of LPS-primed U937 cells, expressing endogenous NLRP3, with recombinant GST-tagged WT or F103A or F103Y mutant aa 95-127 fragments of cofilin-1. Data are representative of three independent experiments with similar results (FIG. 6A, 6B and 6E).

[0037] FIG. 7A-7E shows that peptides containing the NLRP3-binding motif of cofilin-1 suppress NLRP3 inflammasome activation. FIG. 7A Measurements of ATPase activity from purified recombinant human NLRP3 (expressed in HEK293 cells) with purified GST (control),MBHB Ref. No.: 24-2385- WOGST-tagged full-length (FL) cofilin-1 , or GST-tagged WT or F103A mutant aa 95-127 cofilin-1 fragment. n=7 independent in vitro assays. Data are mean + s.e.m. P = 0.0006 (***) as determined by unpaired two-tailed Mann-Whitney ( / test. FIG. 7B GST-pulldown assay of Lys of LPS-primed U937 cells, expressing endogenous NLRP3, with recombinant GST-tagged full-length cofilin-1 or aa 95-127 cofilin-1 fragment in the presence of various doses of H2O2 (above 624 lanes). FIG. 7C Immunoblot analysis of IL-1 and caspase-1 (Casp-1) from Sup or Lys of LPS-primed A / m2-deficient BMDMs transiently transfected with synthesized peptides corresponding to aa 95-127 or aa 134-166 of cofilin-1 and treated with 1.25 pM nigericin, 5 mM ATP, or flagell in . FIG.7D and FIG. 7E Immunoblot analysis of IL-10 and Casp-1 from Sup or Lys of LPS-primed A / m2-deficient BMDMs (FIG. 7D) or PBMCs of CAPS patients (3, 4 and 5) with various NLRP3 mutations (above blots) (FIG. 7E), transiently transfected with synthesized WT or F103A or F103Y mutant peptides corresponding to aa 95-127 of cofilin-1 and treated with 1.25 pM nigericin, 5 mM ATP, or flagellin (FIG. 7D) or LPS-priming alone (FIG. 7E). Data are representative of three independent experiments with similar results (FIG.7A-7E).

[0038] FIG. 8 shows the proposed molecular mechanism for ROS-induced NLRP3 inflammasome activation. In the primed resting state, NLRP3 exists as both monomers and in a self-inhibitory decameric cage. Monomeric NLRP3 is restrained by cofilin-1 binding, while the cage prevents activation through autoinhibition, collectively blocking NEK7 association. Upon stimulation with NLRP3 activators, ROS oxidizes cofilin-1, leading to its dissociation from monomeric NLRP3. Freed NLRP3 then oligomerizes and engages NEK7, driving assembly of the active inflammasome complex. The closed cage, the NLRP3-NEK7 complex, and the active NLRP3 inflammasome disk are derived from PDB entries 7PZC, 6NPY, and 8EJ4, respectively.

[0039] FIG.9A-9C show that cofilin-1 is a negative regulator of the NLRP3 inflammasome.FIG. 9A immunoblot analysis of NLRP3 and cofilin-1 in proteins IP with anti-cofilin- 1 antibody or control IgG (1stlane) from Lys of WT BMDMs treated with indicated NLRP3 inflammasome activators (above lanes). FIG. 9B, Immunoblot analysis with antibody to cofilin-1, caspase-1, 11-1 b, GSDMD, ASC, and NLRP3 in cell lysates or crosslinked pellets of WT BMDMs transiently transfected with negative control siRNA (N.C.) or siRNA targeting CfH, then treated with or without LPS for 9h. FIG. 9C, IL-10 measurements of culture supernatants from WT, Nlrc4-, Mefv-, and A / m2-deficient BMDMs transiently transfected with negative control siRNA (N.C.) or siRNA targeting Cfl1, then treated with LPS for 9h. Data are mean + s.e.m. n=3 independent replicates from the same BMDM preparation. Data are representative of at least three independent experiments. Statistical significance was determined by two-tailed Student’s f-test.MBHB Ref. No.: 24-2385- WO

[0040] FIG. 10A-10D show Cofilin-1 and NLRP3 interaction is reduced by CAPS-associated NLRP3 missense mutations. FIG. 10A Immunoblot analysis with antibody to cofilin-1 in proteins IP with antibody to myc from Lys of 293T cells transiently expressing WT, the V198M variant of uncertain significance, or CAPS-associated D303G mutant NLRP3 proteins. FIG. 10B, Structural model of the cofilin-1-NLRP3 complex highlighting the positions of eight CAPS-associated NLRP3 mutations. FIG. 10C, Predicted changes in binding affinity (AAG_binding) between cofilin-1 and NLRP3 variants harboring CAPS-associated mutations, relative to WT NLRP3. FIG. 10D, GST-pulldown assay of Lys of 293T cells transiently expressing WT or mutant NLRP3 carrying substitutions in conserved polybasic residues within the PYD-NACHT linker with recombinant GST-tagged cofilin-1. Data are representative of three independent experiments with similar results (FIG. 10A and 10D).

[0041] FIG. 11A-11C show epitope mapping of a monoclonal cofilin-1 antibody. FIG. 11A and FIG. 11B, Immunoblot analysis of recombinant GST-tagged full-length or indicated fragments of cofilin-1 proteins (above lanes) with antibody to GST (FIG. 11A) or with monoclonal antibody to cofilin-1 (FIG. 11AB). FIG. 11C, The schematic structure of full-length cofilin-1 with lines corresponding to N-terminal (aa 1-84), middle (aa 41-127), and C-terminal (aa 85-166) halves of cofilin-1. NLS, nuclear localization signal; ADF-H, actin-depolymerizing factor homology domain; PI(4,5)P2; phosphatidylinositol 4,5-bisphosphate binding site.

[0042] FIG. 12A-12E show IL-1|3 releases are not suppressed in BMDMs of KI mice harboring single cysteine to alanine mutation. FIG. 12A-12E show IL-1 measurements of culture supernatants of LPS-primed BMDMs from WT (Cof1+ / +), Cof1+I- (FIG. 12A), Cof7C39A / C39A (FIG. 12B), C0HC8OA / + (FIG. 12C), Cof1C139A / C139A (FIG. 12D), and Cof1 C147A / C147A (FIG. 12E) mice treated with 5 mM ATP. Data are mean ± s.e.m. n=4-6 independent mice.

[0043] FIG. 13A-13B show that cofilin-1 forms intermolecular disulfide bonds. FIG. 13A, Immunoblot analysis with antibody to V5 or myc for cofilin-1 in proteins IP with anti-V5 antibody from Lys of A / m2-deficient BMDMs transiently expressing both V5-tagged and myc-tagged WT cofilin-1 or cofilin-1 with various oxidation resistant cysteine to alanine substitutions (above lanes) and treated with or without ATP. Data are representative of three independent experiments with similar results. FIG. 13B, Proposed model for formation of intramolecular disulfide bond in WT cofilin-1 and intermolecular disulfide bond between two C39A or C80A mutant cofilin-1 molecules when oxidized by ROS.

[0044] FIG. 14A - 14E show that cofilin-1 interferes with NLRP3 oligomerization and interaction with NEK7. FIG. 14A, Immunoblot analysis with antibody to V5 or myc for NLRP3 in proteins IP with anti-V5 antibody from Lys of PT67 cells transiently expressing both V5- andMBHB Ref. No.: 24-2385- WOmyc-tagged WT NLRP3 and treated with or without nigericin. FIG. 14B, Immunoblot analysis with antibody to V5 or myc for NLRP3, or GST for cofilin- 1 in proteins IP with anti-V5 antibody from Lys of PT67 cells transiently expressing V5- and myc-tagged WT NLRP3 with GST-tagged WT or C39A / C80A mutant cofilin-1 and treated with or without nigericin. FIG. 14C, Structural overlay of cofilin-1 (orange) onto the human NLRP3-NLRP3 dimer (blue) extracted from the cryo-EM cage structure (PDB: 7PZC). The NLRP3 structure from the cofilin-1-NLRP3 model was aligned to the lower NLRP3 subunit in this dimer. Red dashed lines highlight atomic clashes between cofilin-1 and the opposing NLRP3 subunit at the dimer interface. FIG. 14D, Structural overlay of cryo-EM NLRP3-NEK7 complex (PDB: 6NPY) and modeled NLRP3-cofilin-1 complex (blue), showing that NEK7 and cofilin-1 occupy overlapping interaction interfaces on NLRP3. The pyrin domain is not present in the cryo-EM structure. FIG. 14E, Immunoblot analysis with antibody to V5 and NEK7 in proteins IP with anti-NEK7 antibody from Lys of PT67 cells transiently expressing V5-tagged WT NLRP3 with GST-tagged WT or C39A / C80A mutant cofilin-1 and treated with or without nigericin. Data are representative of three independent experiments with similar results (FIG. 14A, 14B, and 14E).

[0045] FIG. 15 shows inhibition of the NLRP3 inflammasome by cofilin-1 -derived peptides containing substitutions within the NLRP3 binding motif of cofilin-1. Released IL-1|3 were measured from culture supernatants of LPS-primed BMDMs transiently transfected with synthesized WT or F101A, F101Y, I102A, or I102L mutant peptides corresponding to aa 95-127 of cofilin-1 and treated with 5 mM ATP.

[0046] FIG. 16 shows inhibition of the NLRP3 inflammasome by cofilin-1 -derived head-to-tail cyclized peptides. Released IL-1 p were measured from culture supernatants of LPS-primed BMDMs transiently transfected with synthesized WT or F101A linear or cyclic WT or F103A, F103Y mutant peptides corresponding to aa 95-127 of cofilin-1 and treated with 5 mM ATP.

[0047] Skilled artisans will appreciate that elements in the Figures are illustrated for simplicity and clarity and have not necessarily been drawn to scale. For example, the dimensions of some of the elements in the Figures can be exaggerated relative to other elements to help improve understanding of the embodiment(s) of the present disclosure.DETAILED DESCRIPTION OF THE DISCLOSURE

[0048] All publications, patents and patent applications cited herein are hereby expressly incorporated by reference for all purposes.

[0049] Before describing the present disclosure in detail, a number of terms will be defined. Unless otherwise required by context, singular terms shall include pluralities andMBHB Ref. No.: 24-2385- WOplural terms shall include the singular. For example, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.

[0050] It is noted that terms like “preferably,” “commonly,” and “typically” are not utilized herein to limit the scope of the claimed subject matter or to imply that certain features are critical, essential, or even important to the structure or function of the claimed subject matter. Rather, these terms are merely intended to highlight alternative or additional features that can or cannot be utilized in a particular embodiment of the present disclosure.

[0051] For the purposes of describing and defining the present disclosure, it is noted that the term “substantially” is utilized herein to represent the inherent degree of uncertainty that can be attributed to any quantitative comparison, value, measurement, or other representation. The term “substantially” is also utilized herein to represent the degree by which a quantitative representation can vary from a stated reference without resulting in a change in the basic function of the subject matter at issue.

[0052] As utilized in accordance with the present disclosure, unless otherwise indicated, all technical and scientific terms shall be understood to have the same meaning as commonly understood by one of ordinary skill in the art.

[0053] In the present disclosure, any concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated.

[0054] As used herein, the term “about” means ±10% of the indicated range, value, sequence, or structure, unless otherwise indicated.

[0055] It is noted that terms like “preferably,” “commonly,” and “typically” are not utilized herein to limit the scope of the claimed subject matter or to imply that certain features are critical, essential, or even important to the structure or function of the claimed subject matter. Rather, these terms are merely intended to highlight alternative or additional features that can or cannot be utilized in a particular embodiment of the present disclosure.

[0056] Unless expressly specified otherwise, the term “comprising” is used in the context of the present disclosure to indicate that further members may optionally be present in addition to the members of the list introduced by “comprising”. It is, however, contemplated as a specific embodiment of the present disclosure that the term “comprising” encompasses the possibility of no further members being present, i.e., for the purpose of this embodiment “comprising” is to be understood as having the meaning of “consisting of’.MBHB Ref. No.: 24-2385- WO

[0057] Methods well known to those skilled in the art can be used to construct genetic expression constructs and recombinant cells according to this disclosure. These methods include in vitro recombinant DNA techniques, synthetic techniques, in vivo recombination techniques, and polymerase chain reaction (PCR) techniques. See, for example, techniques as described in Green & Sambrook, 2012, MOLECULAR CLONING: A LABORATORY MANUAL, Fourth Edition, Cold Spring Harbor Laboratory, New York; Ausubel et al., 1989, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Greene Publishing Associates and Wiley Interscience, New York, and PCR Protocols: A Guide to Methods and Applications (Innis et al., 1990, Academic Press, San Diego, CA).

[0058] NLRP3 has a pivotal role in nucleating the inflammasome, a cytoplasmic multiprotein complex that mediates the maturation of the proinflammatory cytokines interleukin-1 p (IL-1P) and IL-18 by activating caspase-1. Mutations in the gene encoding human NLRP3 cause a spectrum of autoinflammatory disease, the cryopyrin-associated periodic syndromes (CAPS), and also has been reported to be involved in the pathogenesis of other inflammatory conditions, including gout, type 2 diabetes mellitus, atherosclerosis, cryopyrin-associated periodic syndromes (CAPS), and Alzheimer’s disease.

[0059] Cryopyrin-associated periodic syndromes (CAPS) are a group of conditions that have overlapping signs and symptoms. CAPS prevalence is estimated to be 2.7 to 5.5 per 1 million and might be higher, as CAPS is not widely known, and difficult to diagnose. The cryopyrin-associated periodic syndromes are usually caused by heterozygous gain-of-function variants in the NLRP3 gene. The CAPS spectrum includes mild, moderate, and severe phenotypes. The mild phenotype is called familial cold autoinflammatory syndrome (FCAS), the moderate phenotype is also known as Muckle-Wells syndrome (MWS), and the neonatal-onset multisystem inflammatory disease (NOMID)Zchronic infantile neurologic cutaneous articular syndrome (CINCA) describes the severe phenotype. CAPS is the result in excessive inflammasome activation with subsequent overproduction of interleukin (IL)-ip, and one possible treatment is a targeted anti-IL-1 therapy.

[0060] This disclosure demonstrates that cofilin-1 is a key component in regulating the NLRP3 inflammasome, and that polypeptides containing the NLRP3-binding motif of cofilin-1 could be developed into compositions and treatments, not only for cryopyrin-associated periodic syndromes (CAPS), but also several acquired inflammatory diseases, in which the NLRP3 inflammasome has been implicated.

[0061] NLRP3 has a pivotal role in nucleating the inflammasome, a cytoplasmic multiprotein complex that mediates the maturation of the proinflammatory cytokines interleukin-1 p (IL-1 ) and IL-18 by activating caspase-1. Mutations in the gene encodingMBHB Ref. No.: 24-2385- WOhuman NLRP3 cause a spectrum of autoinflammatory disease, the cryopyrin-associated periodic syndromes (CAPS). The NLRP3 inflammasome also has been reported to be involved in the pathogenesis of other inflammatory conditions, including gout (1), type 2 diabetes mellitus (2), atherosclerosis (3), and Alzheimer’s disease (4). Reactive oxygen species (ROS) are a major factor for NLRP3 inflammasome activation induced by many extracellular activators. However, the molecular mechanism by which a change in cellular redox state leads to NLRP3 inflammasome activation, as well as the molecular pathogenesis of CAPS, has not been elucidated. Here it is shown that cofilin- 1 , an actin severing protein, is a negative regulator of the NLRP3 inflammasome that is released by ROS.

[0062] In the absence of NLRP3 inflammasome activators, cofilin-1 directly bound to the nucleotide-binding domain (NBD) of NLRP3 protein in LPS-primed mouse bone marrow derived macrophages (BMDMs). When the cells were stimulated with NLRP3 inflammasome activators, cofilin-1 was oxidized and dissociated from NLRP3. On the other hand, oxidationresistant C39A / C80A mutant cofilin-1 was not dissociated from NLRP3 and suppressed inflammasome activation by NLRP3 inflammasome activators. The dissociation of cofilin-1 from NLRP3 in inflammasome-activated cells was inhibited by extracellular potassium and MCC950, known inhibitors of the NLRP3 inflammasome. Knockdown of Cfl1 induced spontaneous IL-10 release, which was dependent on the NLRP3 inflammasome but not NLRC4, AIM2, or pyrin inflammasomes. The binding of cofilin-1 to CAPS-associated mutant human NLRP3 was substantially decreased relative to binding to wild-type NLRP3. Four amino acid residues from 101 to 104 of cofilin-1, Phe-lle-Phe-Trp (SEQ ID NO:90) were essential for the interaction with NLRP3. A polypeptide synthesized with 33 amino acids from residues 95 to 127 of cofilin-1 containing the NLRP3-binding motif was surprisingly shown to suppress IL-1 p release induced by CAPS-associated NLRP3 mutations as well as NLRP3 inflammasome activators.

[0063] Taken together, these results demonstrate that cofilin-1 is a key component in regulating the NLRP3 inflammasome in response to ROS, and that polypeptides containing the NLRP3-binding motif of cofilin-1 could be developed into compositions and treatments, not only for CAPS, but also several acquired inflammatory diseases, in which the NLRP3 inflammasome has been implicated.

[0064] In order to study the mechanism of NLRP3 inflammasome activation by ROS and molecular pathogenesis of CAPS, NLRP3 interacting proteins were screened for polypeptides that inhibit inflammasome activation, and cofilin-1 was identified. Cofilin-1 is one of the major regulators of actin dynamics by mediating filament severing and polymerization and its activity is regulated by phosphorylation and oxidation. It was surprisingly discovered that cofilin-1MBHB Ref. No.: 24-2385- WOregulates NLRP3 inflammasome activation by sensing alterations in the redox milieu independently of its actin-regulating activity.

[0065] In one aspect, this disclosure provides an isolated polypeptide comprising an amino acid sequence having about 15 amino acids to about 120 amino acids, wherein the amino acid sequence has at least 75% identity to an equal-length portion of residues 35-155 of SEQ ID NQ:01.

[0066] SEQ ID NO:1 Cofilin-1 :MASGVAVSDG VIKVFNDMKV RKSSTPEEVK KRKKAVLFCL SEDKKNIILE EGKEILVGDV GQTVDDPYAT FVKMLPDKDC RYALYDATYE TKESKKEDLV FIFWAPESAP LKSKMIYASS KDAIKKKLTG IKHELQANCY EEVKDRCTLA EKLGGSAVIS LEGKPL (SEQ ID NO:1)

[0067] As disclosed herein, the binding of cofilin-1 to CAPS-associated mutant human NLRP3 was substantially decreased relative to binding to wild-type NLRP3, and four amino acid residues from residues 101 to 104 of cofilin-1 , namely Phe-lle-Phe-Trp (SEQ ID NQ:90) were essential for the interaction with NLRP3. Thus, the isolated polypeptides as disclosed herein comprise at least residues 101 to 104 of SEQ ID NQ:01 , and comprise about 15 amino acids to about 120 amino acids between residues 35-155 of SEQ ID NQ:01.

[0068] In certain embodiments, the amino acid sequence of the isolated polypeptide comprises at least one substitution relative to residues 35-155 of SEQ ID NQ:01. In some embodiments of the isolated polypeptide, the amino acid sequence comprises about 15 amino acids to about 120 amino acids between residues 35-155 of SEQ ID NQ:01. For example, in some embodiments of the recombinant protein, the isolated polypeptide comprises at least one substitution relative to residues 35-155 of SEQ ID NQ:01 and shares at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to all or an equal length portion of the amino acid sequence of SEQ ID NQ:01.

[0069] In some embodiments of the isolated polypeptide, the amino acid sequence comprises 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111 , 112, 113, 114, 115, 116, 117, 118, 119, or 120 consecutive amino acids from between residues 35-155 of SEQ ID NQ:01, and shares at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to all or an equal length portion of the amino acid sequence of SEQ ID NQ:01. For example, in some embodiments, the isolated polypeptide comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequenceMBHB Ref. No.: 24-2385- WOidentity to any one of SEQ ID NO:02-89. For example, in some embodiments, the isolated polypeptide consists of an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to any one of SEQ ID NO:02-89.

[0070] In some embodiments of the isolated polypeptide, the amino acid sequence comprises 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111 , 112, 113, 114, 115, 116, 117, 118, 119, or 120 consecutive amino acids from between residues 35-155 of SEQ ID NQ:01. For example, in some embodiments, the isolated polypeptide comprises the amino acid sequence of any one of SEQ ID NQ:02-89. For example, in some embodiments, the isolated polypeptide consists of the amino acid sequence of any one of SEQ ID NQ:02-89.

[0071] In some embodiments, the isolated polypeptide comprises at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs:1-89. In some embodiments, the isolated polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:1-89. In some embodiments, the isolated polypeptide consists of an amino acid sequence selected from the group consisting of SEQ ID NOs:1-89.

[0072] In certain embodiments, the amino acid sequence of the isolated polypeptide comprises at least one substitution relative to residues 35-155 of SEQ ID NQ:01. For example, oxidation sites, such as cysteine 39, cysteine 80, cysteine 139, and cysteine 147, can be substituted for another amino acid, such as, for example, alanine. Cofilin can form intramolecular disulfide bond (e.g., C39-C80) and / or (e.g., C139-C147) by oxidative stress. These intermolecular disulfide bridges lead to cofilin dimers or oligomers that can disrupt interaction with NLRP3, and thus, in certain embodiments, the isolated polypeptides as disclosed herein, do not comprise one or more of cysteine 39, cysteine 80, cysteine 139, and cysteine 147, and / or comprise a substitution or cysteine 39, cysteine 80, cysteine 139, and cysteine 147.

[0073] In certain embodiments, the amino acid sequence of the isolated polypeptide comprises at least one substitution relative to residues 35-155 of SEQ ID NQ:01. For example, the amino acid sequence of the isolated polypeptide can comprise one or more conservative amino acid substitutions.MBHB Ref. No.: 24-2385- WO

[0074] As used herein the term, “conservative amino acid substitution” refers to amino acid substitutions that take place within a family of amino acids that are related in their side chains. For example, it is reasonable to expect that an isolated substitution of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid will not have a major effect on the binding or properties of the resulting molecule, especially if the substitution does not involve an amino acid within a framework site. Whether an amino acid change results in a functional peptide can readily be determined by assaying the specific activity of the polypeptide derivative. Examples of such variants may be derived from the sequences and conserved amino acid substitutions thereof listed below by a skilled artisan using techniques well known in the art.

[0075] As used herein, the terms “sequence identity” or “percent identity,” in the context of two or polypeptide sequences (or nucleotide sequences), refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues (or nucleotides) that are the same (e.g., about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region (a polypeptide sequence comprising conserved elements), when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manualMBHB Ref. No.: 24-2385- WOalignment and visual inspection (see e.g., NCBI web site or the like). Such sequences are then said to be “substantially identical.” This definition also refers to, or can be applied to, the compliment of a test sequence. The definition also includes sequences that have deletions and / or additions, as well as those that have substitutions. As described below, the preferred algorithms can account for gaps and the like. Preferably, identity exists over a region that is at least about 15, 20, 25, 50, 75, 100, 150, 200 amino acids (or nucleotides in length), and oftentimes over a region that is 225, 250, 300, 350, 400, 450, 500 amino acids (or nucleotides) in length or over the full-length of an amino acid or nucleic acid sequences.

[0076] For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Preferably, default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

[0077] A preferred example of an algorithm that is suitable for determining percent sequence identity and sequence similarity are the BLAST algorithms, which are described in Altschul eta!., Nuc. Acids Res. 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410 (1990), respectively. BLAST software is publicly available through the National Center for Biotechnology Information. Both default parameters or other non-default parameters can be used. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11 , an expectation (E) of 10, M=5, N=-4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)) alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands.

[0078] As used herein, the terms “isolated” and / or “purified” refer to a polypeptide ( / .e., the isolated polypeptide, the synthesized polypeptide, or the recombinant polypeptide as disclosed herein; ora nucleotide sequence), that is present in the substantial absence of other biological macromolecules of the same type. The term “purified” as used herein in particular means at least 75%, 85%, 95%, or 98% by weight, of biological macromolecules of the same type are present. An “isolated” polypeptide (or an isolated nucleic acid molecule that encodes a particular polypeptide) refers to a polypeptide that is substantially free of other polypeptides; however, the molecule may include some additional residues or moieties, which do not deleteriously affect the basic characteristics of the composition. The terms “substantially pure”MBHB Ref. No.: 24-2385- WOor “substantially purified,” as used herein, refer to a compound or species that is the predominant species present ( / .e., on a molar basis it is more abundant than any other individual species in the composition). In some embodiments, a substantially purified fraction is a composition wherein the species comprises at least about 50% (on a molar basis) of all macromolecular species present. In other embodiments, a substantially pure composition will comprise more than about 80%, 85%, 90%, 95%, or 99% of all macromolar species present in the composition. In still other embodiments, the species is purified to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single macromolecular species.

[0079] In certain embodiments, the isolated polypeptides as disclosed herein further comprise at least one modification that improves stability of the polypeptide, extends half-life of the polypeptide, decreases degradation of the polypeptide, and / or promotes delivery of the polypeptide to one or more targets.

[0080] The isolated polypeptides as disclosed herein can be modified in order to extend or increase half-life of the polypeptide, decrease degradation of the polypeptide, and / or promote delivery of the polypeptide to one or more targets cells and / or tissues. In an embodiment, the isolated polypeptide as disclosed herein further comprises at least one amino acid sequence, protein, polypeptide, or other entity to increase the half-life as compared to the unmodified isolated polypeptide. Such modifications and / or combinations can be prepared and used according to art-recognized methods. Generally, such isolated polypeptides preferably have a half-life that is at least 1.5 times, or at least 2 times, or at least 5 times, or at least 10 times, or at least 20 times greater, or more than 100 times greater than the half-life of the corresponding unmodified isolated polypeptide. The term “half-life,” as used herein, refers to the time taken for the serum concentration of the isolated polypeptides of the disclosure to be reduced by 50%, in vivo, as a result, for example, of the degradation of the molecule and / or clearance or sequestration of the isolated polypeptide by physiological mechanisms. Methods for pharmacokinetic analysis and determination of half-life are familiar to those skilled in the art. For example, additions or subtraction of chemical groups, peptides, or proteins to the amino acid sequence of the isolated polypeptide sequences as disclosed herein that result in variant isolated polypeptides with increased half-life, decreased degradation of the polypeptide, enhanced delivery of the isolated polypeptides, or increased solubility in biological matrices to one or more target cells and / or tissues (such as in blood, brain, cerebrospinal fluid, or various human tissues, such as pancreas) than that of unmodified isolated polypeptides.

[0081] Examples of modifications can include, but are not limited to, fusion of the isolated polypeptides as disclosed herein to: Fc domain(s) of an antibody (e.g., lgG1 , lgG2, lgG3, orMBHB Ref. No.: 24-2385- WOI gG4) , human serum albumin (albumin), transferrin, or a proprietary recombinant polypeptide, such as XTEN®. In some embodiments, the isolated polypeptides as disclosed herein can be PEGylated, polysialylated, or lipidated. For example, the isolated polypeptides as disclosed herein can be covalently attached to one or more high molecular weight chains of polyethylene glycol (PEG). In certain embodiments, nanoparticle (NP) mediated delivery can be used to create the desired increased half-life, decreased degradation of the polypeptide, enhanced delivery of the isolated polypeptides, or increased solubility in biological matrices to one or more target cells and / or tissues.

[0082] In certain embodiments, protein cyclisation can be used to improve stability of the isolated polypeptide, extend the half-life of the isolated polypeptide, or decrease degradation of the isolated polypeptide as disclosed herein. Cyclisation can be categorized into four general classes: side chain-to-side chain, head-to-tail (also known as backbone cyclisation), head-to-side chain, and side chain-to-tail. In head-to-tail cyclization, the amino terminus (N) is linked to the carboxyl terminus (C) to form a cyclic peptide bond. In side-chain-to-side-chain cyclization, links are formed between functional groups, such as the thiol groups of cysteine residues (disulfide bridge) or between lysine and aspartic / glutamic acid. In head-to-side-chain I side-chain-to-tail cyclization, one end of the peptide is connected to an internal amino acid side chain (e.g., e-NH2 of Lys or y-COOH of Glu). Peptide cyclisation can lead to improved biological activity by enabling enhanced binding towards the target molecule. Head-to-tail cyclic peptides have increased resistance to hydrolysis by exopeptidases due to the absence of the free termini. Membrane permeability and cytoplasmic delivery of the molecule is enhanced. In certain embodiments, the isolated polypeptides as disclosed herein comprise cyclic peptides. In certain embodiments, the isolated polypeptides as disclosed herein comprise head-to-tail cyclic peptides.

[0083] Methods for producing cyclic peptides have been described. In some embodiments, the cyclic peptides can be generated by a lactam cyclization strategy, including head to-tail (head-tail) lactam cyclization (between the terminal residues of the acyclic peptide) and / or lactam linkage between other residues. In some embodiments, the cyclic peptides can be generated by “click” chemistry and / or olefin metathesis. In some embodiments, the head-to-tail cyclic peptides were synthesized by intramolecular amide bond formation between the terminal amine and carboxyl groups of the peptide chain.

[0084] In certain embodiments, either the N- or C-terminus can be modified in order to improve stability of the isolated polypeptide, extend the half-life of the isolated polypeptide, or decrease degradation of the isolated polypeptide as disclosed herein. For example, adding or having a cysteine on the N-terminus can increase stabilization of the isolated polypeptides as disclosed herein.MBHB Ref. No.: 24-2385- WO

[0085] As used herein, the term “N-terminus” (also known as the amino-terminus, NH2-terminus, N-terminal end or amine-terminus) refers to the start of a protein or polypeptide, referring to the free amine group (-NH2) located at the end of a polypeptide. Within a peptide, the amine group is bonded to the carboxylic group of another amino acid, making it a chain. That leaves a free carboxylic group at one end of the peptide, called the C-terminus, and a free amine group on the other end called the N-terminus. By convention, peptide sequences are written N-terminus to C-terminus, left to right. This correlates the translation direction to the text direction, because when a protein is translated from messenger RNA, it is created from the N-terminus to the C-terminus, as amino acids are added to the carboxyl end of the protein. As used herein, the “C-terminus” (also known as the carboxyl-terminus, carboxyterminus, C-terminal tail, C-terminal end, or COOH-terminus) refers to the end of an amino acid chain (protein or polypeptide), terminated by a free carboxyl group (-COOH). The convention for writing peptide sequences is to put the C-terminal end on the right and write the sequence from N- to C-terminus.

[0086] In another aspect, this disclosure provides for nucleic acid compositions comprising a nucleic acid sequence encoding the isolated polypeptides as disclosed herein.

[0087] As used herein, the terms “polynucleotide,” “nucleotide,” and “nucleic acid” can be used interchangeably to refer to nucleic acid comprising DNA, RNA, derivatives thereof, or combinations thereof, in either single-stranded or double-stranded embodiments depending on context as understood by the skilled worker. In the present disclosure, a “nucleic acid” molecule can include, DNA, cDNA and genomic DNA sequences, RNA, messenger RNA, and synthetic nucleic acid sequences. In some embodiments, the nucleic acid molecules are codon-optimized for expression. Thus, “nucleic acid” also encompasses embodiments in which analogs of DNA and RNA are employed. In some embodiments, the nucleic acid component may comprise one or more RNA molecules, such as viral RNA molecules or mRNA molecules that encode the isolated polypeptide of interest as disclosed herein. For example, vector constructs comprising a nucleotide sequence encoding an amino acid sequence having about 15 amino acids to about 120 amino acids, wherein the amino acid sequence has at least 75% identity to an equal-length portion of residues 35-155 of SEQ ID NO:01 , as disclosed herein.

[0088] The terms "recombinant" and "engineered," as used herein and applied to a particular molecule, such as a polypeptide, refers to a molecule that has been modified or manipulated, such as by mutation, truncation, deletion, substitution, addition, conjugation, or by otherwise changing the primary sequence, chemical or three-dimensional structure, chemical signature, folding behavior, glycosylation state, or any other attribute of the molecule, such that the molecule differs from its naturally occurring counterpart.MBHB Ref. No.: 24-2385- WO

[0089] The term “vector,” as used herein, refers to any molecule (e.g., nucleic acid, plasmid, or virus) that is used to transfer coding information to a host cell. One type of vector is a “plasmid,” which refers to a circular double-stranded DNA molecule into which additional DNA segments may be inserted. Another type of vector is a viral vector, wherein additional DNA segments may be inserted into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell and thereby are replicated along with the host genome. In addition, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0090] Vectors normally contain components known in the art and generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker or selection genes, sequences facilitating and / or enhancing translation, an enhancer element and so on. Thus, expression vectors include a nucleotide sequence operably linked to such suitable transcriptional or translational regulatory nucleotide sequences such as those derived from mammalian, microbial, viral, or insect genes. Examples of additional regulatory sequences include operators, mRNA ribosomal binding sites, and / or other appropriate sequences which control transcription and translation, such as initiation and termination thereof. Nucleotide sequences are “operably linked” when the regulatory sequence functionally relates to the nucleotide sequence for the appropriate polypeptide. Thus, a promoter nucleotide sequence is operably linked to, e.g., the antibody heavy chain sequence if the promoter nucleotide sequence controls the transcription of that nucleotide sequence.

[0091] The vector may be a plasmid, a single-stranded or double-stranded viral vector, a single-stranded or double-stranded RNA or DNA phage vector, a phagemid, a cosmid or any other carrier of a transgene of interest. Such vectors may be introduced into cells as polynucleotides by well-known techniques for introducing DNA and RNA into cells. The vectors, in the case of phage and viral vectors also may be introduced into cells as packaged or encapsulated virus by well-known techniques for infection and transduction. Viral vectors may be replication competent or replication defective.

[0092] The term “operably linked,” as used herein, refers to an arrangement of flanking sequences wherein the flanking sequences so described are configured or assembled so as to perform their usual function. Thus, a flanking sequence operably linked to a coding sequence may be capable of effecting the replication, transcription, and / or translation of the coding sequence. For example, a coding sequence is operably linked to a promoter when theMBHB Ref. No.: 24-2385- WOpromoter is capable of directing transcription of that coding sequence. A flanking sequence need not be contiguous with the coding sequence, so long as it functions correctly.

[0093] In certain embodiments, the vector of this disclosure can be formulated for pharmaceutical administration. While any suitable carrier known to those of ordinary skill in the art may be employed in the pharmaceutical compositions of this disclosure, the type of carrier will vary depending on the mode of administration. For parenteral administration, including intranasal, intradermal, subcutaneous or intramuscular injection or electroporation, the carrier preferably comprises water, saline, and optionally an alcohol, a fat, a polymer, a wax, one or more stabilizing amino acids or a buffer. General formulation technologies are known to those of skill in the art (see, for example, Remington: The Science and Practice of Pharmacy (20th edition), Gennaro, ed., 2000, Lippincott Williams & Wilkins; Injectable Dispersed Systems: Formulation, Processing And Performance, Burgess, ed., 2005, CRC Press; and Pharmaceutical Formulation Development of Peptides and Proteins, Frkjr et al., eds., 2000, Taylor & Francis).

[0094] DNA vectors can be administered in solution (e.g., a phosphate-buffered saline solution) by injection, usually by an intra-arterial, intravenous, subcutaneous or intramuscular route. Compositions comprising a DNA vector can be administered once or multiple times. For vaccination with a vector, administration can be performed more than once, for example, 2, 3, 4, 5, 6, 7, 8, 10, 15, 20 or more times as needed to induce the desired response (e.g., specific antigenic response or proliferation of immune cells). Multiple administrations can be administered, for example, bi-weekly, weekly, bi-monthly, monthly, or more or less often, as needed, for a time period sufficient to achieve the desired response.

[0095] The vectors of this disclosure can be administered to a mammalian host. The mammalian host usually is a human or a primate.

[0096] The vectors encoding the recombinant proteins as disclosed herein can be formulated in accordance with standard techniques well known to those skilled in the pharmaceutical art. Such compositions can be administered in dosages and by techniques well known to those skilled in the medical arts taking into consideration such factors as the age, sex, weight, and condition of the particular patient, and the route of administration.

[0097] In some embodiments, the vector comprises an mRNA sequence encoding the recombinant protein of interest (e.g., the isolated polypeptides as disclosed herein). In an embodiment, the mRNA sequence is a natural and non-modified mRNA. Within the context of the present disclosure, natural and non-modified mRNA encompasses mRNA generated in vitro, without chemical modifications or changes in the sequence. In certain embodiments, the mRNA can be an artificial mRNA. In the context of the present disclosure, artificial mRNAMBHB Ref. No.: 24-2385- WOencompasses mRNA with chemical modifications, sequence modifications or non-natural sequences. The vector comprising an RNA construct of this disclosure can be administered to a mammalian host. The mammalian host usually is a human or a primate.

[0098] In another aspect, this disclosure provides for pharmaceutical compositions comprising the isolated polypeptide as disclosed herein or the nucleic acid composition as disclosed herein. In some embodiments, the pharmaceutical composition comprises at least one pharmaceutically acceptable carrier or excipient. In certain embodiments, the pharmaceutical composition can further comprise an isotonic agent, a preservative, and / or a buffer.

[0099] The term “pharmaceutical composition” as used herein refers to a composition comprising the isolated polypeptides as disclosed herein capable of inducing a desired therapeutic effect when properly administered to a patient. In some embodiments, the disclosure provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of the isolated polypeptides of the disclosure. The terms “pharmaceutically acceptable carrier” or “physiologically acceptable carrier” as used herein refer to one or more formulation materials suitable for accomplishing or enhancing the delivery of the isolated polypeptides of the disclosure.

[0100] In some embodiments, the isolated polypeptides as disclosed herein may be formulated with a pharmaceutically acceptable carrier, excipient, or stabilizer, as pharmaceutical compositions. In certain embodiments, such pharmaceutical compositions are suitable for administration to a human or non-human animal via any one or more routes of administration using methods known in the art. The term “pharmaceutically acceptable carrier” means one or more non-toxic materials that do not interfere with the effectiveness of the biological activity of the active ingredients. Such preparations may routinely contain salts, buffering agents, preservatives, compatible carriers, and optionally other therapeutic agents. Such pharmaceutically acceptable preparations may also contain compatible solid or liquid fillers, diluents or encapsulating substances which are suitable for administration into a human. Other contemplated carriers, excipients, and / or additives, which may be utilized in the formulations described herein include, for example, flavoring agents, antimicrobial agents, sweeteners, antioxidants, antistatic agents, lipids, protein excipients such as serum albumin, gelatin, casein, salt-forming counter-ions such as sodium, and the like. These and additional known pharmaceutical carriers, excipients, and / or additives suitable for use in the formulations described herein are known in the art, for example, as listed in “Remington: The Science & Practice of Pharmacy,” 2lst ed., Lippincott Williams & Wilkins, (2005), and in the "Physician's Desk Reference," 60th ed., Medical Economics, Montvale, N.J. (2005). PharmaceuticallyMBHB Ref. No.: 24-2385- WOacceptable carriers can be selected that are suitable for the mode of administration, solubility, and / or stability desired or required.

[0101] In certain embodiments, the isolated polypeptides as disclosed herein can be formulated for liposomal delivery. The liposomes may be unilamellar or multilamellar vesicles formed from one or more pharmaceutically acceptable lipids. The lipid components may include, without limitation, phospholipids (e.g., phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol), sphingolipids, cholesterol or cholesterol derivatives, and polyethylene glycol (PEG)-modified lipids. In some embodiments, the liposomes comprise neutral, anionic, or cationic lipids, or combinations thereof. The isolated polypeptides as disclosed herein may be encapsulated within the aqueous core of the liposome, associated with the lipid bilayer, or conjugated to the liposome surface. Encapsulation may be achieved using methods known in the art, including but not limited tothin-film hydration, ethanol injection, reverse-phase evaporation, microfluidic mixing, active loading, or combinations thereof. In certain embodiments, the liposomes may further comprise surface modifications to enhance stability, circulation time, or cell-specific targeting. Such modifications may include PEGylation, incorporation of targeting ligands (e.g., antibodies, peptides, carbohydrates, or receptor-binding moieties), or inclusion of charged lipids to modulate cellular uptake. In some embodiments, the liposomes may have an average particle diameter ranging from about 50 nm to about 500 nm, and may be formulated in a pharmaceutically acceptable carrier suitable for parenteral administration, including intravenous, intraperitoneal, subcutaneous, or intratumoral injection.

[0102] In some embodiments, the pharmaceutical composition comprising the isolated polypeptides as disclosed herein can further comprise an additional active agent or an additional anti-inflammatory agent. In certain embodiments, an additional therapeutic can be administered to the subject in need of treatment. In an embodiment, the additional therapeutic is an anti-inflammatory therapeutic. The term “active agent” or “additional therapeutic”, as used herein, refers to any therapeutic that is used in addition to treatment with the isolated polypeptides as disclosed herein. For example, when the pharmaceutical composition comprising the isolated polypeptides as disclosed herein is used in the treatment of a subject, the method can comprise the use of an additional therapeutic, wherein the additional active agent is an anti-inflammatory therapeutic in addition to the isolated polypeptides as disclosed herein. The additional active agent may be administered at the same time or at a different time and / or via the same mode of administration or via a different mode of administration, as that of the isolated polypeptides as disclosed herein. In some embodiments, the additional active agent will be given at a time and in a way that will provide a benefit to the subject during the effective treatment window of the isolated polypeptides as disclosed herein. When twoMBHB Ref. No.: 24-2385- WOcompositions are administered with a specific time period, generally the time period is measured from the start of the first composition to the start of the second composition. As used herein, when two compositions are given within an hour, for example, the time before the start of the administration of the first composition is about an hour before the start of the administration of the second composition.

[0103] In some embodiments, the additional active agent includes, but is not limited to, an anti-IL-1 treatment. For example, the additional active agent comprises an anti-IL-1 treatment selected from anakinra (recombinant human IL-1Ra), rilonacept (a soluble decoy receptor ‘trap’, binding both IL-1a and IL-1P), and canakinumab (human monoclonal anti-IL-ip antibody). Anakinra can be administered daily subcutaneously, and typical dosing regimen varies from 1 to 2 mg / kg / day up to 10 mg / kg / day. Rilonacept can be administered weekly in adults up to about 160 mg / week and varies from 2.2 to 4.4 mg / kg / week in children. Canakinumab can be administered subcutaneously every four to eight weeks, at up to about 150 mg of canakinumab (if the body weight is >40 kg) or it can be dosed with 2 mg / kg for patients from >15 to < 40 kg, every four to eight weeks. Additional active agents can also include non-steroidal anti-inflammatory drugs or a corticosteroid. In some embodiments, an additional active agent can include the diarylsulfonylurea compound MCC950 (a small molecule inhibitor of NLRP3), or p-hydroxybutyrate (BHB).

[0104] In some embodiments, the additional active agent includes treatments effective in the treatment of one or more of gout, diabetes, Alzheimer’s disease, cryopyrin-associated periodic syndromes (CAPS), and / or pericarditis. In an embodiment, the additional active agent includes an anti-gout flare treatment colchicine or an NSAID. In an embodiment, the additional active agent includes one or more treatments for diabetes. For example, a biguanide (e.g., metformin), a Dpp-4 inhibitor (e.g., sitagliptin saxagliptin, and linaglipti), an inhibitor of Sodium-Glucose cotransporter-2 (SGLT2; e.g., canagliflozin, dapagliflozin, and empagliflozin), an alpha-glucosidase inhibitor, a GLP-1 receptor agonist (e.g., exenatide, dulaglutide, lixisenatide, liraglutide and semaglutide), insulin, insulin-releasing medicines (e.g., sulfonylureas, such as glimepiride, glipizide, or glyburide; or meglitinides, such as repaglinide or nateglinide), or thiazolidinedione (e.g., pioglitazone or rosiglitazone). In an embodiment, the additional active agent includes one or more treatments for Alzheimer’s disease. For example, brexpiprazole, donepezil, galantamine, benzgalantamine, memantine, a combination of memantine and donepezil, rivastigmine, lecanemab, or donanemab. In an embodiment, the additional active agent includes one or more treatments for pericarditis. For example, pain relievers (e.g., aspirin and ibuprofen), colchicine (e.g., colcrys, or mitigare), corticosteroids, immunosuppressants or immunomodulators (e.g., an interleukin 1 blocker).MBHB Ref. No.: 24-2385- WO

[0105] In some embodiments, the one or more further active agents are selected from: antibodies (e.g., abciximab, adalimumab, alemtuzumab, atlizumab, basiliximab, belimumab, bevacizumab, bretuximab vedotin, canakinumab, cetuximab, ceertolizumab pegol, daclizumab, denosumab, eculizumab, efalizumab, gemtuzumab, golimumab, ibritumomab tiuxetan, infliximab, ipilimumab, muromonab-CD3, natalizumab, ofatumumab, omalizumab, palivizumab, panitumuab, ranibizumab, rituximab, tocilizumab, tositumomab, and / or trastuzumab); alkylating agents (e.g., cisplatin, carboplatin, mechlorethamine, cyclophosphamide, chlorambucil, ifosfamide and / or oxaliplatin); anti-angiogenic agents (e.g., endostatin, angiogenin inhibitors, angiostatin, angioarrestin, angiostatin, basementmembrane collagen-derived anti-angiogenic factors (tumstatin, canstatin, or arrestin)); anti-angiogenic antithrombin III, and / or cartilage-derived inhibitor (GDI); alkaloids and / or terpenoids (e.g., a vinca alkaloid, a podophyllotoxin and / or a taxane, vincristine, vinblastine, vinorelbine and / or vindesine, taxol, paclitaxel, docetaxel and / or ortataxel, etoposide, and / or teniposide); mTOR inhibitors (e.g., rapamycin, everolimus, temsirolimus and / or deforolimus); stilbenoids (e.g., resveratrol, piceatannol, pinosylvin, pterostilbene, alpha-viniferin, ampelopsin A, ampelopsin E, diptoindonesin C, diptoindonesin F, epsilon-vinferin, flexuosol A, gnetin H, hemsleyanol D, hopeaphenol, trans-diptoindonesin B, astringin, piceid and / or diptoindonesin A); immunomodulatory agents (e.g., modulator of CTLA-4, PD-1, PD-L1, PD-L2, T cell immunoglobulin and mucin 3 (TIM3 or HAVCR2), galectin 9, phosphatidylserine, lymphocyte activation gene 3 protein (LAG3), MHC class I, MHC class II, 4-1 BB, 4-1 BBL, 0X40, OX40L, GITR, GITRL, CD27, CD70, TNFRSF25, TL1A, CD40, CD40L, HVEM, LIGHT, BTLA, CD160, CD80, CD244, CD48, ICOS, ICOSL, B7-H3, B7-H4, VISTA, TMIGD2, HHLA2, TMIGD2, a butyrophilin (including BTNL2), a Siglec family member, TIGIT, PVR, a killer-cell immunoglobulin-like receptor, an ILT, a leukocyte immunoglobulin-like receptor, NKG2D, NKG2A, MICA, MICB, CD28, CD86, SIRPA, CD47, VEGF, neuropilin, CD30, CD39, CD73, CXCR4, and / or CXCL12); antibiotics (e.g., amikacin, gentamicin, kanamycin, neomycin, netilmicin, tobramycin, paromomycin, streptomycin, spectinomycin, geldanamycin, herbimycin, rifaximin, loracarbef, ertapenem, doripenem, imipenem, cilastatin, meropenem, cefadroxil, cefazolin, cefalotin, cefalothin, cefalexin, cefaclor, cefamandole, cefoxitin, cefprozil, cefuroxime, cefixime, cefdinir, cefditoren, cefoperazone, cefotaxime, cefpodoxime, ceftazidime, ceftibuten, ceftizoxime, ceftriaxone, cefepime, ceftaroline fosamil, ceftobiprole, teicoplanin, vancomycin, telavancin, dalbavancin, oritavancin, clindamycin, lincomycin, daptomycin, azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin, troleandomycin, telithromycin, spiramycin, aztreonam, furazolidone, nitrofurantoin, linezolid, posizolid, radezolid, torezolid, amoxicillin, ampicillin, azlocillin, carbenicillin, cloxacillin, dicloxacillin, flucloxacillin, mezlocillin, methicillin, nafcillin, oxacillin, penicillin G, penicillin V, piperacillin, temocillin, ticarcillin, calvulanate, ampicillin, subbactam, tazobactam, ticarcillin,MBHB Ref. No.: 24-2385- WOclavulanate, bacitracin, colistin, polymyxin B, ciprofloxacin, enoxacin, gatifloxacin, gemifloxacin, levofloxacin, lomefloxacin, moxifloxacin, nalidixic acid, norfloxacin, ofloxacin, trovafloxacin, grepafloxacin, sparfloxacin, temafloxacin, mafenide, sulfacetamide, sulfadiazine, silver sulfadiazine, sulfadimethoxine, sulfamethoxazole, sulfanamide, sulfasalazine, sulfisoxazole, trimethoprim-sulfamethoxazole, sulfonamideochrysoidine, demeclocycline, minocycline, oytetracycline, tetracycline, clofazimine, dapsone, dapreomycin, cycloserine, ethambutol, ethionamide, isoniazid, pyrazinamide, rifampicin, rifabutin, rifapentine, streptomycin, arsphenamine, chloramphenicol, fosfomycin, fusidic acid, metronidazole, mupirocin, platensimycin, quinupristin, dalopristin, thiamphenicol, tigecycyline, tinidazole, trimethoprim, and / or teixobactin); or anti-fungal agents (e.g., bifonazole, butoconazole, clotrimazole, econazole, ketoconazole, luliconazole, miconazole, omoconazole, oxiconazole, sertaconazole, sulconazole, tioconazole, albaconazole, efinaconazole, epoziconazole, fluconazole, isavuconazole, itraconazole, posaconazole, propiconazole, ravusconazole, terconazole, voriconazole, abafungin, amorolfin, butenafine, naftifine, terbinafine, anidulafungin, caspofungin, micafungin, benzoic acid, ciclopirox, flucytosine, 5-fluorocytosine, griseofulvin, haloprogin, tolnaflate, undecylenic acid, and / or balsam of Peru).

[0106] Any of the additional active agents disclosed herein can be administered by oral, parenteral (including intravenous, subcutaneous, intramuscular, intradermal, intratracheal, intraperitoneal, intraarticular, intracranial and epidural), airway (aerosol), rectal, vaginal, ocular or topical (including transdermal, buccal, mucosal, sublingual and topical ocular) administration.

[0107] In some aspects, this disclosure provides for a method of treating an inflammatory condition in a subject, comprising administrating to the subject an effective dose of the pharmaceutical composition comprising the isolated polypeptides as disclosed herein. In some embodiments, the method further comprises repeating the administering at least a second time, at least a third time, at least a fourth time, at least a fifth time, at least a sixth time, or more. In some embodiments, the inflammatory condition is selected from the group consisting of: gout, pericarditis, type 2 diabetes mellitus, atherosclerosis, cryopyrin-associated periodic syndromes (CAPS), and Alzheimer’s disease. In some embodiments, this disclosure provides for methods of treating gout, pericarditis, type 2 diabetes mellitus, atherosclerosis, cryopyrin-associated periodic syndromes (CAPS), and Alzheimer’s disease by administration of the isolated polypeptides as disclosed herein.

[0108] As used herein, the terms “treatment,” “treat,” or “treating” refer to a method of reducing the effects of a disease or condition or symptom of the disease or condition. Thus, in the methods disclosed herein, treatment can refer to a 5%, 10%, 20%, 30%, 40%, 50%,MBHB Ref. No.: 24-2385- WO60%, 70%, 80%, 90%, or 100% reduction in the severity of an established disease or condition or symptom of the disease or condition that is an inflammatory condition selected from gout, pericarditis, type 2 diabetes mellitus, atherosclerosis, cryopyrin-associated periodic syndromes (CAPS), and Alzheimer’s disease. For example, a method of treating a disease is considered to be a treatment if there is a 5% reduction in one or more symptoms of the disease in a subject as compared to a control. Thus, the reduction can be a 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or any percent reduction between 5% and 100% as compared to native or control levels. It is understood that treatment does not necessarily refer to a cure or complete ablation of the disease, condition, or symptoms of the disease or condition.

[0109] The term “patient” is intended to include human and non-human animals, particularly mammals. In some embodiments, mammals can include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the patient is a human.

[0110] The terms “administration” or “administering” as used herein refer to providing, contacting, and / or delivering the isolated polypeptides disclosed herein by any appropriate route to achieve the desired effect. Administration may include, but is not limited to, oral, sublingual, parenteral (e.g., intravenous, subcutaneous, intracutaneous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional, or intracranial injection), transdermal, topical, buccal, rectal, vaginal, nasal, ophthalmic, via inhalation, and implants.

[0111] An “effective amount” of the isolated polypeptides disclosed herein (or a pharmaceutical formulation), refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result. In some embodiments, a therapeutically effective amount of the isolated polypeptides administered to a patient will be in the range of about 0.01 to about 50 mg / kg of patient body weight, whether by one or more administrations. In some instances, the amount of the isolated polypeptide used is about 0.01 mg / kg to about 45 mg / kg, about 0.01 mg / kg to about 40 mg / kg, about 0.01 mg / kg to about 35 mg / kg, about 0.01 mg / kg to about 30 mg / kg, about 0.01 mg / kg to about 25 mg / kg, about 0.01 mg / kg to about 20 mg / kg, about 0.01 mg / kg to about 15 mg / kg, about 0.01 mg / kg to about 10 mg / kg, about 0.01 mg / kg to about 5 mg / kg, about 0.2 mg / kg to about 0.8 mg / kg, or about 0.01 mg / kg to about 1 mg / kg administered daily, weekly, every two weeks, every three weeks, or monthly, for example. In some instances, the isolated polypeptide is administered at about 0.2 mg / kg to about 0.8 mg / kg. However, other dosage regimens may be useful. In one instance, the isolated polypeptide described herein is administered to a human at a dose ofMBHB Ref. No.: 24-2385- WOabout 10 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 60 mg, about 70 mg, about 75 mg, about 80 mg, about 90 mg, about 100 mg, about 125 mg, about 150 mg, about 175 mg, about 200 mg, about 225 mg, about 250 mg, about 275 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg, about 550 mg, about 600 mg, about 650 mg, about 700 mg, about 750 mg, about 800 mg, about 850 mg, about 900 mg, about 950 mg, or about 1000 mg daily, weekly, every two weeks, every three weeks, or monthly. In some instances, the isolated polypeptide is administered at about 45 mg intravenously daily, every two days, every three days, every four days, every five days, every six days, weekly, every two weeks, every three weeks, or monthly. The dose may be administered as a single dose or as multiple doses (e.g., 2 or 3 doses), such as infusions. A dosing schedule of, for example, once / week, twice / week, three times / week, four times / week, five times / week, six times / week, seven times / week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, and the like, can be used. The dosing schedules encompass dosing for a total period of time of, for example, one week, two weeks, three weeks, four weeks, five weeks, six weeks, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, and up to twelve months or more.

[0112] In certain embodiments, the dose of the isolated polypeptide administered in a combination treatment may be reduced as compared to a single treatment. The progress of this therapy is easily monitored by conventional techniques.

[0113] In some embodiments, the methods further involve administering to the patient an effective amount of an additional active agent or an additional therapeutic agent. In some instances, the additional active agent is selected from an anti-IL-1 treatment (anakinra, rilonacept, or canakinumab), a non-steroidal anti-inflammatory drugs, a corticosteroid, the diarylsulfonylurea compound MCC950, p-hydroxybutyrate (BHB), an anti-gout flare treatment, one or more treatments for diabetes, one or more treatments for cryopyrin-associated periodic syndromes (CAPS), one or more treatments for Alzheimer’s disease, one or more treatments for pericarditis, an alkylating agent, an anti-angiogenic agent, an alkaloid, a terpenoid, a mTOR inhibitor, a stilbenoid, an immunomodulatory agent, an antibiotic, or an anti-fungal agent.

[0114] A skilled artisan can determine the optimal pharmaceutical composition comprising the isolated polypeptides disclosed herein depending upon, for example, the intended route of administration, delivery format, and desired dosage.

[0115] In some embodiments, the pharmaceutical compositions of the disclosure can also be selected for parenteral delivery. Alternatively, the pharmaceutical compositions can be selected for inhalation or for delivery through the digestive tract, such as orally. TheMBHB Ref. No.: 24-2385- WOpreparation of such pharmaceutical compositions is within the knowledge of one of skill in the art. Additional pharmaceutical compositions will be evident to those of skill in the art, including formulations involving sustained- or controlled-delivery formulations.

[0116] A number of autoinflammatory and autoimmune diseases are known to involve NLRP3, and therefore which may be treated or prevented by the isolated polypeptides as disclosed herein, include multiple sclerosis, type-1 diabetes (T1D), psoriasis, rheumatoid arthritis (RA), Behcet's disease, Schnitzler syndrome, macrophage activation syndrome, systemic lupus erythematosus, systemic sclerosis, lung diseases including chronic obstructive pulmonary disorder (COPD), asthma (including steroid-resistant asthma), central nervous system conditions, including Parkinson's disease (PD), cryopyrin-associated periodic syndromes (CAPS), Alzheimer's disease (AD), dementia, Huntington's disease, cerebral malaria, brain injury from pneumococcal meningitis, intracranial aneurysms, and traumatic brain injury; metabolic diseases including type 2 diabetes (T2D), atherosclerosis, obesity, gout, pseudo-gout, metabolic syndrome, and non-alcoholic steatohepatitis; atherosclerosis, myocardial infarction, heart failure, aortic aneurysm and dissection, and other cardiovascular events; ocular diseases such as both wet and dry age-related macular degeneration, diabetic retinopathy and optic nerve damage; liver diseases including non-alcoholic steatohepatitis (NASH); inflammatory reactions in the lung and skin including contact hypersensitivity (such as bullous pemphigoid, atopic dermatitis, Hidradenitis suppurativa, acne vulgaris, and sarcoidosis); inflammatory reactions in the joints; amyotrophic lateral sclerosis; cystic fibrosis; stroke; chronic kidney disease; and inflammatory bowel diseases including ulcerative colitis and Crohn's disease. Additionally, examples of diseases, disorders or conditions which may be treated or prevented by the isolated polypeptides as disclosed herein include any disease where an individual has been determined to carry a germline or somatic non-silent mutation in NLRP3.

[0117] In some embodiments, the disease, disorder or condition which may be treated or prevented by the isolated polypeptides as disclosed herein include acne; atopic dermatitis; Alzheimer's disease; amyotrophic lateral sclerosis; age-related macular degeneration (AMD); anaplastic thyroid cancer; chronic kidney disease; congestive heart failure; contact dermatitis; Crohn's disease; cryopyrin-associated periodic syndromes (CAPS); cystic fibrosis; diabetes; familial cold autoinflammatory syndrome (FCAS); gout; Huntington's disease; heart failure; ischemic reperfusion injury; juvenile idiopathic arthritis; myocardial infarction; macrophage activation syndrome; myelodysplastic syndrome; multiple myeloma; motor neuron disease; multiple sclerosis; Muckle-Wells syndrome (MWS); non-alcoholic steatohepatitis (NASH); neonatal-onset multisystem inflammatory disease (NOMID); Parkinson's disease; pericarditis;MBHB Ref. No.: 24-2385- WOsystemic juvenile idiopathic arthritis; systemic lupus erythematosus; traumatic brain injury; transient ischemic attack; and ulcerative colitis.

[0118] In one aspect, the isolated polypeptide disclosed herein comprises an amino acid sequence having about 15 amino acids to about 120 amino acids, wherein the amino acid sequence has at least 75% identity to an equal-length portion of residues 35-155 of SEQ ID NO:01. In some embodiments, the amino acid sequence of the isolated polypeptide disclosed herein comprises at least one substitution relative to residues 35-155 of SEQ ID NO:01. In some embodiments, the isolated polypeptide disclosed herein further comprises at least one modification. In some embodiments, the at least one modification improves stability of the polypeptide, extends half-life of the polypeptide, decreases degradation of the polypeptide, and / or promotes delivery of the polypeptide to one or more targets. In some embodiments, the amino acid sequence is selected from the group consisting of SEQ ID NQ:02 through SEQ ID NO:89. In some embodiments, the amino acid sequence comprises 95- KKEDLVF IFWAPESAPLKSKM I YASSKDAI KKK- 127 (SEQ ID NQ:02). In some embodiments, the amino acid sequence comprises 95- KKEDLVFIYWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NO:24). In some embodiments, the amino acid sequence comprises 95- KKEDLVYIFWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NO:46). In some embodiments, the amino acid sequence comprises 95- KKEDLVFLFWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NO:68).

[0119] In another aspect, the nucleic acid composition disclosed herein, comprises a nucleic acid sequence encoding the isolated polypeptide comprising the amino acid sequence having about 15 amino acids to about 120 amino acids, wherein the amino acid sequence has at least 75% identity to an equal-length portion of residues 35-155 of SEQ ID NQ:01. In some embodiments, the amino acid sequence of the isolated polypeptide disclosed herein comprises at least one substitution relative to residues 35-155 of SEQ ID NQ:01. In some embodiments, the isolated polypeptide disclosed herein further comprises at least one modification. In some embodiments, the at least one modification improves stability of the polypeptide, extends half-life of the polypeptide, decreases degradation of the polypeptide, and / or promotes delivery of the polypeptide to one or more targets. In some embodiments, the amino acid sequence is selected from the group consisting of SEQ ID NO:02 through SEQ ID NO:89. In some embodiments, the amino acid sequence comprises 95- KKEDLVF IFWAPESAPLKSKM I YASSKDAI KKK- 127 (SEQ ID NO:02). In some embodiments, the amino acid sequence comprises 95- KKEDLVFIYWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NO:24). In some embodiments, the amino acid sequence comprises 95-MBHB Ref. No.: 24-2385- WOKKEDLVYIFWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NO:46). In some embodiments, the amino acid sequence comprises 95-KKEDLVFLFWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NO:68).

[0120] In another aspect, the pharmaceutical composition disclosed herein, comprises the isolated polypeptide comprising amino acid sequence having about 15 amino acids to about 120 amino acids, wherein the amino acid sequence has at least 75% identity to an equallength portion of residues 35-155 of SEQ ID NQ:01. In some embodiments, the amino acid sequence of the isolated polypeptide disclosed herein comprises at least one substitution relative to residues 35-155 of SEQ ID NQ:01. In some embodiments, the isolated polypeptide disclosed herein further comprises at least one modification. In some embodiments, the at least one modification improves stability of the polypeptide, extends half-life of the polypeptide, decreases degradation of the polypeptide, and / or promotes delivery of the polypeptide to one or more targets. In some embodiments, the amino acid sequence is selected from the group consisting of SEQ ID NQ:02 through SEQ ID NO:89. In some embodiments, the amino acid sequence comprises 95-KKEDLVFIFWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NQ:02). In some embodiments, the amino acid sequence comprises 95- KKEDLVFIYWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NO:24). In some embodiments, the amino acid sequence comprises 95- KKEDLVYIFWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NO:46). In some embodiments, the amino acid sequence comprises 95- KKEDLVFLFWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NO:68)

[0121] In another aspect, the pharmaceutical composition disclosed herein, comprises the nucleic acid composition, comprising the nucleic acid sequence encoding the isolated polypeptide comprising the amino acid sequence having about 15 amino acids to about 120 amino acids, wherein the amino acid sequence has at least 75% identity to an equal-length portion of residues 35-155 of SEQ ID NQ:01. In some embodiments, the amino acid sequence of the isolated polypeptide disclosed herein comprises at least one substitution relative to residues 35-155 of SEQ ID NQ:01. In some embodiments, the isolated polypeptide disclosed herein further comprises at least one modification. In some embodiments, the at least one modification improves stability of the polypeptide, extends half-life of the polypeptide, decreases degradation of the polypeptide, and / or promotes delivery of the polypeptide to one or more targets. In some embodiments, the amino acid sequence is selected from the group consisting of SEQ ID NQ:02 through SEQ ID NO:89. In some embodiments, the amino acid sequence comprises 95-KKEDLVFIFWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NQ:02). In some embodiments, the amino acid sequence comprises 95-KKEDLVFIYWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NO:24). In someMBHB Ref. No.: 24-2385- WOembodiments, the amino acid sequence comprises 95- KKEDLVYIFWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NO:46). In some embodiments, the amino acid sequence comprises 95- KKEDLVFLFWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NO:68). In some embodiments, the composition disclosed herein comprises at least one pharmaceutically acceptable carrier or excipient. In some embodiments, the pharmaceutical composition disclosed herein further comprises an isotonic agent, a preservative, and / or a buffer. In some embodiments, the pharmaceutical composition disclosed herein further comprises an additional active agent.

[0122] In another aspect, this disclosure provides a method of treating an inflammatory condition (e.g., gout, pericarditis, type 2 diabetes mellitus, atherosclerosis, cryopyrin-associated periodic syndromes (CAPS), or Alzheimer’s disease ) in a subject, comprising administrating to the subject an effective dose of the pharmaceutical composition, comprising the isolated polypeptide, comprising amino acid sequence having about 15 amino acids to about 120 amino acids, wherein the amino acid sequence has at least 75% identity to an equal-length portion of residues 35-155 of SEQ ID NQ:01. In some embodiments, the amino acid sequence of the isolated polypeptide disclosed herein comprises at least one substitution relative to residues 35-155 of SEQ ID NQ:01. In some embodiments, the isolated polypeptide disclosed herein further comprises at least one modification. In some embodiments, the at least one modification improves stability of the polypeptide, extends half-life of the polypeptide, decreases degradation of the polypeptide, and / or promotes delivery of the polypeptide to one or more targets. In some embodiments, the amino acid sequence is selected from the group consisting of SEQ ID NQ:02 through SEQ ID NO:89. In some embodiments, the amino acid sequence comprises 95-KKEDLVFIFWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NO:02). In some embodiments, the amino acid sequence comprises 95- KKEDLVFIYWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NO:24). I In some embodiments, the amino acid sequence comprises 95- KKEDLVYIFWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NO:46). In some embodiments, the amino acid sequence comprises 95- KKEDLVFLFWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NO:68).

[0123] In another aspect, this disclosure provides a method of treating an inflammatory condition (e.g., gout, pericarditis, type 2 diabetes mellitus, atherosclerosis, cryopyrin-associated periodic syndromes (CAPS), or Alzheimer’s disease ) in a subject, comprising administrating to the subject an effective dose of the pharmaceutical composition, comprising the nucleic acid composition, comprising the nucleic acid sequence encoding the isolated polypeptide comprising the amino acid sequence having about 15 amino acids to about 120MBHB Ref. No.: 24-2385- WOamino acids, wherein the amino acid sequence has at least 75% identity to an equal-length portion of residues 35-155 of SEQ ID NO:01. In some embodiments, the amino acid sequence of the isolated polypeptide disclosed herein comprises at least one substitution relative to residues 35-155 of SEQ ID NQ:01. In some embodiments, the isolated polypeptide disclosed herein further comprises at least one modification. In some embodiments, the at least one modification improves stability of the polypeptide, extends half-life of the polypeptide, decreases degradation of the polypeptide, and / or promotes delivery of the polypeptide to one or more targets. In some embodiments, the amino acid sequence is selected from the group consisting of SEQ ID NQ:02 through SEQ ID NO:89. In some embodiments, the amino acid sequence comprises 95-KKEDLVFIFWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NQ:02). In some embodiments, the amino acid sequence comprises 95- KKEDLVFIYWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NO:24). I In some embodiments, the amino acid sequence comprises 95- KKEDLVYIFWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NO:46). In some embodiments, the amino acid sequence comprises 95- KKEDLVFLFWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NO:68). In some embodiments, the composition disclosed herein comprises at least one pharmaceutically acceptable carrier or excipient. In some embodiments, the pharmaceutical composition disclosed herein further comprises an isotonic agent, a preservative, and / or a buffer. In some embodiments, the pharmaceutical composition disclosed herein further comprises an additional active agent. In some embodiments, the method of treating an inflammatory condition in a subject comprises repeating the administering at least a second time, at least a third time, at least a fourth time, at least a fifth time, at least a sixth time, or more. In some embodiments, the inflammatory condition comprises gout, pericarditis, type 2 diabetes mellitus, atherosclerosis, cryopyrin-associated periodic syndromes (CAPS), or Alzheimer’s disease.

[0124] The disclosure will be further described in the following examples, which do not limit the scope of the disclosure described in the claims.EXAMPLES

[0125] The Examples that follow are illustrative of specific embodiments of the disclosure, and various uses thereof. They are set forth for explanatory purposes only, and should not be construed as limiting the scope of the disclosure in any way.METHODSReagentsMBHB Ref. No.: 24-2385- WO

[0126] Ultra-pure flagellin (tlrl-pstfla), ATP (tlrl-atpl), poly (dA:dT) (tlrl-patn), nigericin (tlrl-nig), MSU (tlrl-msu), and ultra-pure LPS (tlrl-3pelps) were obtained from InvivoGen. Luteolin (2874), MCC950 (5479), and 845 KH7 (3834) were from Tocris Bioscience. Recombinant human NLRP3 protein (TP320952) was from Origene and recombinant human Cofilin- 1 protein (CF01) was from Cytoskeleton.Mice

[0127] 8-week to 16-week-old male and female mice (Mus musculus) were used on a C57BL / 6J background in the experiments. Wild-type (WT) C57BL / 6J mice were obtained from the Jackson Laboratory. In each experiment, experimental and control mice were age and sex matched, with both male and female mice used depending on availability. All animal studies were performed in accordance with US National Institutes of Health guidelines and were approved by the Institutional Animal Care and Use Committee of the National Human Genome Research Institute.

[0128] Cofilin- 1 -Deficient Mice were generated using a targeting construct by inserting a 4.3 kb genomic fragment encompassing upstream and exon 1 (5’ arm), 3.0-kb genomic fragment encompassing exon 2-4 flanking two loxP with neomycin resistant cassette (flanked with two FRT), and 4.0-kb genomic fragment of downstream of cof1. (3’ arm) into pPNT-double loxP. Linearized constructs were introduced into embryonic stem (ES) cells of C57BL / 6 background. G418-resistant clones were screened for homologous recombination by PCR. Heterozygotes generated from founders were crossed with Flip C transgenic mice to remove the neo cassette and crossed with Rosa Tamoxifen Cre or Ella-Cre transgenic mice (Jackson Laboratory) to remove exon 2-4.

[0129] Four knockin (KI) mice bearing oxidation-resistant cysteine to alanine mutations (C39A, C80A, C139A, and C147A) for each of the four cysteine residues in cofilin-1 were generated by homology-directed repair (HDR) using CRISPR-Cas9 genome editing. Single guide RNAs (sgRNAs) were:5’-GGTGCTCTTTTGCCTGAGTG-3’ for C39A (SEQ ID NO:91),5’-TTGCATCATAGAGTGCATAG-3’ for C80A(SEQ ID NO:92),5’-ATTACAAGCTAACTGCTACG-3’ for C139A(SEQ ID NO:93), and5’-GGTCAAGGACCGCTGCACCC-3’ for C147A(SEQ ID NO:94).HDR donor oligonucleotides wereMBHB Ref. No.: 24-2385- WO5’CAAAAGTGGTGTAGGGGTCGTCCACAGTCTGCCCCACATCTCCTACCAGGATCTCCT TGCCCTCCTCCAGGATGATGTTCTTCTTGTCTTCACTCAGTGCAAAGAGCACCGCCTTC TTGCGTTTCTTCACTTCTTC-3’ for C39A (SEQ ID NO:95), 5’CATCCTGGAGGAGGGCAAGGAGATCCTGGTAGGAGATGTGGGGCAGACTGTGGACG ACCCCTACACCACTTTTGTCAAGATGCTGCCAGACAAGGACGCGCGCTATGCACTCTAT GATGCAACCTATGAGACCAAG-3’ for C80A (SEQ ID NO:96), 5’GAGGTGGCTCACAAAGGCTTGCCCTCCAGGGAAATGACGGCGCTGCCACCTAGTTTC TCTGCCAGGGTGCAGCGGTCCTTGACCTCTTCGTACGCGTTAGCTTGTAATTCATGCTT GATTCCTACAGGGTG-3’ for C139A (SEQ ID NO:97), and 5’AAATGACGGCGCTGCCACCTAGTTTCTCTGCAAGGGTGGCGCGGTCCTTGACCTCCT CGTAGCAGTTAGCTTGTAATTCATGCTTGATTCCTACAGGGTGAAAAGAGAGCAGTGAG CAGGAAGCACTGGGG-3’ for C147A (SEQ ID NO:98).Cas9, sgRNA, and HDR donor oligonucleotides were transfected to mouse embryos at 0.5 day post coitum by electroporation. Electroporated zygotes were transferred to the oviducts of pseudopregnant females on the day of the vaginal plug.Patients

[0130] Blood specimens from 5 CAPS patients were drawn after obtaining informed consent under a protocol approved by the National Institute of Arthritis and Musculoskeletal and Skin Diseases / National Institute of Diabetes and Digestive and Kidney Diseases Institutional Review Board.Cell preparation, inflammasome activation and immunoblot

[0131] Bone marrow progenitors were isolated from the tibia and femur of 8-16-week-old male orfemale mice. The isolated progenitors were differentiated into macrophages (BMDMs) by culturing in Iscove's Modified Dulbecco’s Media (IMDM; Gibco) supplemented with 20 ng / ml M-CSF (PeproTech), 10% heat-inactivated fetal bovine serum (FBS; Invitrogen), 1 mM sodium pyruvate (Invitrogen), 100 U / ml penicillin (Invitrogen), and 100 g / ml streptomycin (Invitrogen). The differentiation process was carried out for 7 days in a humidified incubator at 37°C with 5% CO2. BMDMs were replated in 12-well plates (1.0 x 106cells per well) in DMEM (Invitrogen) containing 10% FBS and antibiotics.1 day prior to experiments. Human peripheral blood mononuclear cells (PBMCs) were isolated by LSM-905 Lymphocyte Separation Medium (50494, MP Biomedicals) from freshly drawn peripheral venous blood from patients, and plated in 12-well plates(2.0 x 106cells per well) in RPMI 1640 (Invitrogen) containing 10% FBS and antibiotics. cDNAs encoding WT or various mutant cofilin-1 was cloned into pLNCX2 vector (631503,Takara) and then transfected into RetroPack PT67 cells (631510, Takara).MBHB Ref. No.: 24-2385- WOThe cell culture medium of the transfected PT67 cells, which contain viral particles, was collected by filtration with a low protein-binding 0.22-pm syringe filter. The viral particlecontaining medium was transferred to J774 cells for viral infection in the presence of polybrene (8 pg ml-1). Cells were centrifuged at 1,200g for 90 min at 32 °C and incubated for 8 hours. Transduced J774 cells were selected with G418 (800 pg ml-1) for 3 weeks and replated in 12-well plates (1.0 x 106cells per well) in DMEM containing 10% FBS and antibiotics.1 day prior to experiments. BMDMs, PBMCs, or J774 cells were primed with 1 pg ml-1 LPS for 3 hours. For AIM2 or NLRC4 inflammasome activation, 1 pg ml-1 of dsDNA with 2.5 pl ml-1 of Lipofectamine 2000 (Invitrogen) or 0.5 pg ml-1 flagellin with 25 pl ml-1 DOTAP (Sigma), respectively, were mixed in Opti-MEM and incubated for 10 minutes before treating the cells. For NLRP3 inflammasome activation, cells were treated with ATP (5 mM), nigericin (10 pM), MSU (200 pg ml-1), KH7 (50 pM), CaCI2 (1 mM), and Imiquimod (100 pM). None of the reagents in these experiments induced cytotoxicity as confirmed by LDH assay (K311-400, BioVision). Fornoncanonical inflammasome activation, BMDMs were treated with Pam3CSK4 (100 ng ml-1) for 3h and transfect LPS (6 pg) with Lipofectamine 2000 for 6 hours.

[0132] Cell culture supernatants were collected, and cells were lysed with M-PER mammalian protein extraction reagent (78501 , Thermo Fisher Scientific). Lysates and supernatants were analyzed by immunoblot with antibodies to human IL-1 p (AF-201-NA, R&D Systems); mouse IL-1 (AF-401-NA, R&D Systems); cofilin-1 (sc-33779, rabbit polyclonal; sc-53934, mouse monoclonal, specific for reduced cofilin-1; sc-376476, mouse monoclonal, Santa Cruz Biotechnology); MYL12B (sc-130331 Santa Cruz Biotechnology); ARPC4 (ab217056, abeam); human Caspase-1 (p20) (AG-20B-0048, Adipogen Life Sciences); mouse Caspase-1 (p20) (AG-20B-0042, Adipogen Life Sciences); actin (SC-1615HRP; Santa Cruz Biotechnology); myc (SC-40HRP, Santa Cruz Biotechnology); NLRP3 (AG-20B-0014-C100, Adipogen; 15101 , Cell Signaling); GSDMD (NBP2-33422, R&D Systems) or V5 (R961-25, Thermo Fisher scientific). For the ASC pyroptosome, pellets from whole-cell 936 lysates were crosslinked with disuccinimidyl suberate (DSS) and analysed by immunoblotting with antibody to ASC (sc-271054, Santa Cruz Biotechnology). Released IL-ip was also measured in supernatants by ELISA (MLB00C, R&D Systems).Mass-Compatible Silver Staining and Mass Spectrometry

[0133] Immunoprecipitated protein samples with NLRP3 specific antibody were separated on a 4-20 % SDS-polyacrylamide gel. The gel was fixed in 50% methanol and 12% acetic acid for 30 min, followed by washing with 50% ethanol for 20 min and then with 10% ethanol for 10 min. The gel was sensitized with 0.02% sodium thiosulfate for 1 min and washed with distilled water (D.W) three times. Staining was performed with 0.1% silver nitrate for 30 minutes in theMBHB Ref. No.: 24-2385- WOdark, followed by three quick washes with D.W. Development was carried out in a solution containing 2% sodium carbonate and 0.04% formaldehyde until protein bands became visible. The reaction was halted by incubating the gel in 5% acetic acid for 10 minutes, followed by thorough rinsing with distilled water. The stained bands were then excised from the gel using a clean scalpel, digested in-gel with sequencing grade modified trypsin, and analyzed on a NanoLC-ESI-MS / MS system. The mass spectrometric data was used to search against the most recent non-redundant protein database (NR database) from NCBI with ProtTech’s ProtQuest software suite.Site-directed mutagenesis and gene knockdown assay

[0134] Mutated human cofilin constructs were generated by site-directed mutagenesis using QuickChange Lightning Kit (210519-5, Agilent Technologies) according to the manufacturer's instructions. siRNAs targeting mouse cofilin- 1 and negative control siRNA (4390847) were purchased from Invitrogen. The siRNA for mouse Cof1 knockdown was 5'-AGGAGAUCCUGGUAGGAGATT -3' (S121365; SEQ ID NO:99). For siRNA gene knockdown experiments, 50 - 250 pmol siRNA was transfected into BMDMs (1x106cells per well) by electroporation and replated in 12-well plates. After 40 hours, the siRNA-transfected cells were primed with LPS for 7h and the culture medium was replaced with serum free medium. Cell culture supernatants and lysates were collected after 2 hours and analyzed by immunoblot or ELISA.Immunoprecipitation and Pull-down assay

[0135] LPS-primed BMDMs treated with activators or inhibitors, and 293T or PT67 cells transiently transfected with expression constructs for the WT, CAPS-associated mutant, cagebreaking polybasic mutant, or various deleted forms of NLRP3 or WT or mutant cofilin- 1 were lysed in an IP lysis buffer (30 mM Tris-CI pH 7.4, 120 mM NaCI, 2 mM KCI, 2 mM EDTA, 10% Glycerol, 0.2% NP-40) or GST lysis buffer (20 mM Tris-CI pH 8.0, 300 mM NaCI, 1 mM EDTA, 0.5% NP-40). The cell lysates were incubated overnight with antibodies to cofilin-1 (sc-376476, Santa Cruz Biotechnology); NLRP3 (AG-20B-0014-C100, Adipogen); V5 (R961-25, Thermo Fisher scientific); myc (2278, Cell Signaling Technology); or NEK7 (ab133514, abeam) followed by incubation with protein A / G beads for 1 hours at 4°C. After washing with the lysis buffer or PBS, bound proteins were eluted by 2x SDS sample buffer from the beads and analyzed by immunoblot with antibodies to cofilin-1 (sc-53934, Santa Cruz Biotechnology); NLRP3 (15101 , Cell Signaling or AG-20B-0014-C100, Adipogen); V5 (R961-25, Thermo Fisher Scientific); or myc (sc-40HRP, Santa Cruz Biotechnology). For in vitro GST pull-down assay, purified GST-tagged full-length and various deleted cofilin-1 were bound to glutathione magnetic agarose beads (78602, Thermo Fisher Scientific) and incubated withMBHB Ref. No.: 24-2385- WOphorbol 12-myristate 13-acetate (PMA) treated U937 cell lysates in a GST lysis buffer for 30 minutes and washed with the GST lysis buffer and PBS. Bound proteins were eluted by 2* SDS sample buffer from the beads and analyzed by immunoblot with antibodies to NLRP3 or GST (sc-138 HRP, Santa Cruz Biotechnology). Recombinant cofilin-1 and NLRP3 proteins were incubated in the presence or absence of H2O2for 30 minutes at 37°C, immunoprecipitated with antibodies to NLRP3, cofilin-1, or normal mouse IgG, and analyzed by immunoblot.Measurements of Intracellular ROS and potassium

[0136] BMDMs were plated 6.25 x 103cells / well in 96 well cell culture plate or 0.3 x 1O6cells / well in 4 chamber cell culture slide one day before treatment. The cells were primed with LPS (100 ng ml-1) for 3 hours, then treated with 5 or 10 pM MitoSoxTM Green or Red mitochondrial superoxide indicators (M36009, Thermo Fisher Scientific) or Asante potassium green-2 AM y(ab142806, abeam) for 10 minutes. Following washing with PBS, cells were treated with 5 mM ATP for 5 min co-treatment with luteolin, KCI, or MCC950. Fluorescent signals were measured 995 by a plate reader (Victor3TM, Perkin Elmer) or captured by confocal microscopy using Confocal Laser Scanning microscope (LSM 710; Carl Zeiss) and analyzed to assess the levels of intracellular ROS.Measurement of the ATPase activity of NLRP3

[0137] Human recombinant 0.15 ug NLRP3 protein (100190; BPS Bioscience) was incubated with purified GST, GST-tagged full-length cofilin-1, or GST-tagged WT or F103A mutant aa 95-127 cofilin-1 fragment in a reaction buffer (100 mM Tris pH 7.8, 2.8 mM EDTA, 100 mM MgCI2, 15 mM KCI, 665 mM NaCI) at RT for 1 hour followed by the addition of ATP (1 mM) for 40 min at 37 °C. The hydrolysis of ATP by NLRP3 was measured using ADP-Glo max assay (V7001, Promega, Madison, USA) according to the manufacturer’s instruction. Transfection of Peptides

[0138] Peptides were chemically synthesized with >95% purity by custom peptide synthesis service from Biomatik Corporation, Ontario, Canada. BMDMs from AIM2-deficient mice were plated 0.5 x6cells / well in 12 well cell culture plate one day before transfection and PBMCs from CAPS patients were plated 2 x 106cells / well in 12 well cell culture plate on the day of transfection. Cells were primed with LPS (1 pg ml-1 for BMDMs, 0.1 pg ml-1 for PBMCs) for 3 hours and changed the culture media with 350 ul of FBS-free DMEM (BMDMs) or RPMI (PBMCs). Peptides (20 uM, final concentration) were transfected into BMDMs using the Xfect™ protein transfection reagent (631324, Takara Bio Inc.), according to the manufacturer’s instruction. After 15 minutes, BMDMs were treated with ATP (5 mM), nigericinMBHB Ref. No.: 24-2385- WO(1.25 uM), or flagellin (0.5 g ml-1 with 25 pl ml-1 DOTAP) for 30 minutes, and PBMCs were incubated for 1 hour.Protein structure acquisition and complex prediction.

[0139] Initial monomeric structures for human cofilin-1 and NLRP3 were obtained from the AlphaFold Protein Structure Database (AF-P23528-F1 and AF-Q96P20-F1). Canonical protein sequences retrieved from UniProt (CFL1: sp|P23528|COF1 JHUMAN; NLRP3:sp |Q96P20| NALP3_HUMAN) were submitted to the AlphaFold3 server using the complex prediction mode to generate de novo models of protein-protein interaction24. Two configurations were modeled: full-length cofilin-1 with full-length NLRP3 (FL-FL), and a C-terminal peptide of cofilin-1024 1 (residues 95-127) with full-length NLRP3 (P-FL). Chain A was designated as cofilin-1 (or peptide-cofilin-1) and chain B as NLRP3 in all models. A spin movie of the cofilin-1-NLRP3 complex was created using UCSF ChimeraX (version 1.9) (34).Mutagenesis and structural analysis.

[0140] Point mutations were introduced into each model manually using PyMOL v3.058, and mutant structures were evaluated without further energy minimization. PyMOL was also used to visualize side-chain level residue contacts and evaluate structural interaction types, including van der Waals forces, hydrophobic packing, hydrogen bonding, aromatic stacking, and ionic contacts. Both the FL-FL and P-FL complexes were analyzed independently. In addition, interatomic contacts within 5 A were calculated for all wild-type and mutant models to quantify the number of angstrom-level interactions at the protein-protein interface.Stability, flexibility, and binding prediction.

[0141] The effect of each mutation on protein stability, conformational flexibility, and protein-protein binding affinity was assessed using multiple complementary in silico prediction tools. Free energy changes (AAG) were calculated as the difference between wild-type and mutant states (AAG = AG_WT - AG_Mutant), where negative values reflect destabilization or reduced binding affinity [4-10], Mutational effects on AAG were predicted using mCSM59, DUET60, DynaMut61, DynaMut262, DDMut63, SDM64, and ENCoM65. ENCoM was further used to compute changes in vibrational entropy (AAS_vib). These predictors estimate how missense variants affect either monomeric protein stability or complex integrity using graphbased approaches and statistical models trained on experimental AAG measurements. Changes in protein-protein binding affinity (AAG_binding) were estimated using DDMut-PPI66 and mCSM-PPI267. All predictions were calculated using both full-length and peptide-bound models of the Cofilin-1-NLRP3 complex. A consensus approach between tools was used to evaluate overall mutation effect.MBHB Ref. No.: 24-2385- WOVariant impact predictions.

[0142] Variants were annotated using the Ensembl Variant Effect Predictor (VEP). Pathogenicity scores were obtained from AlphaMissense38, CADD v1.739, and REVEL40. Allele frequencies and population-level constraint metrics were derived from the gnomAD database68 to assess variant rarity and potential functional impact.Domain-level interaction prediction.

[0143] To assess which domains of NLRP3 contribute most to cofilin-1 binding, we generated models of cofilin-1 with truncated NLRP3 constructs (PYD, PYD+NACHT, LRR, NACHT+LRR). Binding affinities (AG) and dissociation constants (Kd) were calculated using the PRODIGY (PROtein binDIng enerGY prediction) web server69. Interface contact types and non-interacting surfaces were also extracted for interaction profiling.Structural modeling of oxidized cofilin-1.

[0144] Using Robetta70, which utilizes the Rosetta software suite (33), we submitted the monomeric structure for human cofilin-1 (AF-P23528-F1) for structural refinement to reduce the distance between Cys39 and Cys80, which were initially 11.6 A apart. The PDB output of Rosetta was examined using ChimeraX for the appropriate bond geometry, ensuring that the sulfur atoms of the cysteine residues were within a typical bond distance (approximately 2.0-2.5 A). We verified that the final model of cofilin-1 after Robetta optimization placed Cys39 and Cys802.1 A apart, facilitating the formation of a disulfide bond. We then used ChimeraX to manually introduce a disulfide bond between the Cys39 and Cys80 residues, representing the hypothetical oxidized form of cofilin-1. The input and output cofilin-1 structures were aligned, compared, and a spin movie animation was created to illustrate the dynamic structural differences between the two conformations. Solvent accessible surface area (SASA) values for reduced and oxidized cofilin-1 models were calculated using ChimeraX. The ‘measure sasa’ command was applied to each model using the default probe radius of 1.4 A and vertex density of 2.0. Total SASA values were recorded and compared to assess conformational changes resulting from disulfide bond formation. We then used the HADDOCK2.4 web server35 to generate a model for the oxidized cofilin-1-NLRP3 complex. The Rosetta / ChimeraX output oxidized cofilin-1 PDB and NLRP3 PDB obtained from the AlphaFold Protein Structure Database (AF-Q96P20-F1) were used as input files. Data obtained from the Alphafold3 model of cofilin-1 and NLRP3 were used to define the probable interface residues. Among the HADDOCK output data for the oxidized cofilin-1-NLRP3 complex, two clusters displayed top ranking scores (Clusters 1 and 3).

[0145] Although Cluster 3 had a slightly lower average HADDOCK score, we selected Cluster 1 for subsequent analysis, as the most reliable docking solution. This decision wasMBHB Ref. No.: 24-2385- WObased on its substantially larger cluster size (49 vs. 14), indicating better sampling convergence and increased confidence in the modeled interaction. Additionally, Cluster 1 showed advantages in terms of interface quality, with stronger electrostatic energy and greater buried surface area — both suggestive of a stronger and more stable interface. To enable direct comparison, we also evaluated the AlphaFold3-predicted cofilin-1-NLRP3 complex using the HADDOCK scoring protocol, by submitting the pre-assembled complex structure.Statistics and reproducibility

[0146] Statistical analyses were performed using the nonparametric Mann-Whitney U test in Prism software (GraphPad) or unpaired two-tailed Student’s t-test in Microsoft Excel, as indicated in the figure legends. A P value of < 0.05 was considered statistically significant. The number of reproduced experimental repeats is described in the relevant figure legends. The investigators were not blinded to allocation during experiments and outcome assessment, except as noted above.Example 1. NLRP3 interacts with cofilin-1 and the interaction is substantially reduced by ATP.

[0147] To identify negative regulators of the NLRP3 inflammasome, proteins were screened to identify proteins that interact with NLRP3 and dissociate after NLRP3 inflammasome activation. NLRP3 protein complexes were pulled down with an NLRP3-specific antibody from lysates of bone-marrow-derived macrophages (BMDMs) treated with LPS alone or with ATP after LPS priming. Immunoprecipitated complexes were separated on PAGE, and a band around 20 kDa was identified that was present only in lysates of LPS-primed WT BMDMs but not in ATP-treated WT BMDMs nor NLRP3-deficient BMDMs (Fig.1a). Mass spectrometry analysis identified five proteins in the ~20 kDa band, of which three proteins, including myosin regulatory light chain 12B (MYL12b), actin-related protein 2 / 3 complex subunit 4 (ARPC4), and cofilin-1 disappeared when the inflammasome was activated by ATP (Fig. 1b). Subsequent immunoblot analysis 104 revealed that cofilin-1 is an interacting partner of NLRP3 in LPS-primed BMDMs while MYL12b and ARPC4 did not associate with NLRP3 (Fig. 1c). The interaction of cofilin-1 with NLRP3 was confirmed in a reciprocal coimmunoprecipitation assay with a cofilin-1 specific antibody (Fig. 1d). Cofilin-1 is a 19 kDa actin-severing enzyme that binds to actin, and NLRP3 is also known to bind to actin21. Thus, to examine whether the interaction of cofilin-1 and NLRP3 is due to direct binding or indirect binding mediated by actin, an in vitro pull down assay was performed using purified recombinant cofilin-1 and NLRP3. Recombinant cofilin-1 was observed to co-immunoprecipitate with recombinant NLRP3, and vice versa, which indicates a direct interaction of cofilin-1 with NLRP3 (Fig. 1e).MBHB Ref. No.: 24-2385- WOExample 2. Cofilin-1 is a negative regulator of the NLRP3 inflammasome.

[0148] The NLRP3 inflammasome is activated by a wide range of PAMPs or DAMPs. The effect of various NLRP3 inflammasome activators was examined on the NLRP3-cofilin-1 interaction. As seen in cells treated with ATP, the NLRP3-cofilin-1 interaction was also substantially decreased by nigericin, monosodium urate (MSU) crystals, KH7 (an adenylate cyclase inhibitor that lowers intracellular cyclic AMP, which activates NLRP3)22, extracellular calcium22, cytosolic LPS (which triggers non-canonical NLRP3 activation), or Imiquimod (a potassium-efflux-independent NLRP3 activator) (23) (Fig. 2a and Fig, 9a). Given that cofilin-1 is dissociated from NLRP3 upon its activation signals, it was hypothesized that cofilin-1 may suppress the NLRP3 inflammasome.

[0149] To examine the role of cofilin-1 in NLRP3 inflammasome activation further, the cofilin-1 gene (Cfl 1) was knocked down in mouse BMDMs by short interfering RNA (siRNA). Knockdown of Cfl1 in LPS-primed WT BMDMs induced spontaneous inflammasome activation, which was dependent on the NLRP3 inflammasome, but independent on the NLRC4 (IPAF, ICE protease-activating factor), pyrin, or AIM2 (absent in melanoma 2) inflammasomes (Fig. 2b and Fig. 9b, c). Moreover, it was observed that IL-1 release from the BMDMs with Cfl1 knockdown was substantially increased in response to NLRP3 inflammasome activators, ATP or nigericin, but not by NLRC4 or AIM2 inflammasome activators, flagellin or double-stranded DNA (dsDNA), respectively (Fig 2c). Taken together, these results indicate that cofilin-1 specifically suppresses NLRP3 inflammasome activation. Notably, although caspase-1 and GSDMD cleavage as well as IL-10 release were not detected, Cfl 1 -knockdown cells exhibited increased ASC oligomerization even without LPS priming, at levels comparable to primed cells (Fig. 9b). This indicates that relief of cofilin-1— mediated NLRP3 suppression does not require priming.

[0150] To investigate the structural basis ofthe NLRP3-cofilin-1 interaction, the full-length complex was modeled using AlphaFold324. The predicted structure revealed a well-defined binding interface between cofilin-1 and both the NBD and LRR domain of NLRP3, comprising 46 atomic contacts within 5 A (Fig. 2d). To dissect domain-specific interactions, additional models were generated with cofilin-1 bound to individual NLRP3 domains. The highest predicted binding affinity was observed with the combined NBD-LRR region, yielding a binding free energy (AG) of -20.6 kcal / mol and a dissociation constant (Kd) of 3.0 x 10’15 M. Consistent with this prediction, pull-down assays using NLRP3 constructs lackingl individual domains demonstrated that the interaction with cofilin-1 is mediated through the NBD and LRR domains of NLRP3 (Fig. 2e).MBHB Ref. No.: 24-2385- WO

[0151] Because the NBD of NLRP3 is the most frequent site of CAPS-associated mutations, how disease-associated variants affect its interaction with cofilin-1 was examined. The interaction between cofilin-1 and NLRP3 harboring any of the eight missense mutations — validated as pathogenic in the international INFEVERS registry and distributed across the NBD and LRR domains — was markedly diminished compared with WT NLRP3 or a variant of uncertain significance V198M (rs121908147) (Fig. 2f and Fig. 10a). Structural modeling of these eight mutations in complex with WT cofilin-1 predicted reduced binding affinity for all substitutions (AAG_binding < 0) (Fig. 10b,c). Taken together, these results suggest that the spontaneous inflammasome activation observed in myeloid cells from CAPS patients may result from diminished inhibition of NLRP3 activation by cofilin-1. In addition, increased binding of cofilin-1 to an NLRP3 mutant carrying substitutions in conserved polybasic residues within the PYD-NACHT linker was observed and that were previously shown to have reduced cage formationl l , compared with WT NLRP3 (Fig, 10d), suggesting that cofilin-1 preferentially binds to inactive NLRP3 monomers.Example 3. The interaction of cofilin-1 with NLRP3 depends on redox states of cofilin- 1.

[0152] Despite the diverse range of activators for the NLRP3 inflammasome, mitochondrial ROS generation is a common cellular response that plays a critical role in inflammasome activation16. Thiol groups of cysteines are among the most prominent targets for ROS-mediated oxidation in proteins25. The human or mouse cofilin-1 molecules contain four cysteine residues - Cys39, Cys80, Cys139, and Cys147 - that are potential targets for ROS. It has been reported that oxidation of these cysteines leads to the formation of intramolecular disulfide bridges, which cause conformational changes of cofilin-1 and prevent its phosphorylation at Ser326, 27, 28. T aken together, these observations suggest that NLRP3 inflammasome activators may induce cofilin-1 oxidation, leading to the formation of intramolecular disulfide bonds (Fig 3a).

[0153] To determine the redox status of cofilin-1 in response to NLRP3 inflammasome activators, BMDMs were analyzed by immunoblot using a monoclonal antibody that recognizes an epitope between Cys39 and Cys80 of cofilin-1 when cofilin-1 is in its reduced state (Fig. 11 a-11 c). It was found that cofilin-1 in ATP- or nigericin-treated BMDMs was not readily detectable using the monoclonal antibody under non reducing conditions, whereas it was detectable under reducing conditions (Fig. 3b). A loss of Ser3 phosphorylation of cofilin-1 upon treatment of BMDMs with ATP or nigericin was also observed (Fig. 3b), which is also evidence for cofilin-1 oxidation by NLRP3 activators26. To further investigate the role of ROS on cofilin-1 oxidation and NLRP3 inflammasome activation, ROS in ATP-treated BMDMs was blocked with an antioxidant, luteolin. In LPS-primed BMDMs, ATP-induced caspase-1MBHB Ref. No.: 24-2385- WOactivation and IL-ip release were substantially diminished at the doses of luteolin that decreased oxidized cofilin-1 (Fig. 3c). It is noteworthy that LPS-induced priming, the expression of NLRP3 and pro-IL-1 were not attenuated by luteolin (Fig. 3c). Furthermore, in the luteolin-treated BMDMs, cofilin-1 was not dissociated from NLRP3 by ATP (Fig. 3d). These data suggest that the reduced form of cofilin-1 binds to NLRP3 to suppress its activation, and that cofilin-1 is dissociated from NLRP3 when cofilin-1 is oxidized by ROS generated by NLRP3 activators. Indeed, in an in vitro pull-down assay with recombinant cofilin-1 and NLRP3, the NLRP3-cofilin-1 interaction was decreased in a dose-dependent manner by H2O2 that is known to oxidize cofilin-1 in vitro26 (Fig. 3e).Example 4. Potassium and MCC950 inhibit dissociation of cofilin-1 from NLRP3.

[0154] Hhow the interaction of cofilin-1 with NLRP3 is affected by the NLRP3 inflammasome inhibitors, MCC95029 and high concentrations of extracellular potassium15 was studied. In LPS-primed BMDMs, ATP-driven IL-10 release was significantly inhibited by 100 nM of MCC950 or 25 mM of KCI. Interestingly, with both inhibitors, cofilin-1 remained bound to NLRP3 without being dissociated in ATP-treated BMDMs (Fig. 4a). Moreover, in the inhibitor-treated cells, cofilin-1 remained in the reduced form without being oxidized by ATP-induced ROS (Fig. 4a).

[0155] MCC950 stabilizes the inactive state of NLRP3 by binding to a cleft spanned by the NBD, HD1, WHD, HD2, and transition LRR subdomains, precluding the conformational changes required for ATP-binding, and suppressing the ATPase activity required for NLRP3 activation 30. In an in vitro pull-down assay, it was observed that MCC950 had no appreciable effect on the binding of recombinant NLRP3 to recombinant cofilin-1 (Fig. 4b). The in vitro pulldown assay also demonstrated that potassium is not necessary for the NLRP3-cofilin-1 direct interaction, as the assay was conducted in a potassium-free buffer (Fig. 4b). To understand how MCC950 and extracellular potassium prevent dissociation of cofilin-1 from NLRP3 in ATP-treated BMDMs, changes were assessed in ROS levels within the BMDMs. Both MCC950 and extracellular potassium significantly suppressed ATP-induced ROS production in LPS-primed BMDMs (Fig. 4c-f), suggesting an indirect mechanism by which MCC950 and high concentrations of extracellular potassium prevent the dissociation of cofilin-1 from NLRP3.

[0156] To gain further insight into the role of potassium and ROS in NLRP3 inflammasome activation, the effects of high concentrations of extracellular potassium or antioxidant on inflammasome activation were examined in mutation-positive CAPS patients’ PBMCs, in which NLRP3 is constitutively activated22. Contrary to a previous study with BMDMs from Kl-mice harboring CAPS-associated mutations31 , extracellular potassium substantially blocked IL-10 release from LPS-primed CAPS PBMCs in a dose dependent manner (Fig. 4g).MBHB Ref. No.: 24-2385- WOMoreover, it was also observed dose-dependent inhibition of IL-1 release by the antioxidant luteolin in LPS-primed CAPS PBMCs (Fig. 4h). Taken together, these results are consistent with the hypothesis that cofilin-1 regulates the NLRP3 inflammasome by sensing ROS.Example 5. Oxidation-resistant cofilin-1 suppresses NLRP3 inflammasome activation.

[0157] To investigate cysteine residues of cofilin-1 that regulate the interaction with NLRP3 in response to ROS in vivo, cofilin-1 knockout (KO) and four knock-in (KI) mice bearing oxidation-resistant cysteine to alanine mutations (C39A, C80A, C139A, and C147A) were generated for each of the four cysteine residues. Cofilin-1 KO mice were generated using a Cre-inducible gene deletion strategy (Cof1fl / fl) and bred either with mice expressing Cre recombinase during early embryonic development to obtain constitutive KO mice (Cof1 “), or with tamoxifen-inducible Cre mice to generate inducible KO mice (Cof1fl / 242 fl;Cre / +). Homozygous constitutive KO mice (Cof17“) were embryonic lethal (Fig. 12a), and BMDMs from Cof1fl / fl;Cre / + mice did not survive following tamoxifen treatment. Likewise, homozygous C80A KI mice (Cof1 C80A / C80A) were embryonic lethal. In contrast, homozygous Cof1 C39A / C39A, Cof1C139A / C139A, and Cof1C147A / C147A mice were produced and developed normally (Fig. 12b-12e). Considering that oxidation of cysteines of cofilin-1 results in the formation of an intramolecular disulfide bond between the two cysteines, we hypothesized that alanine substitution of a cysteine involved in disulfide bond formation may inhibit the dissociation of cofilin-1 from NLRP3 by ROS and subsequently suppress inflammasome activation. However, no suppression of NLRP3 inflammasome activation by ATP was observed in LPS-primed BMDMs of all mutant KI mice, Cof 1 C39A / C39A, Cof1C139A / C139A, Cof1C147A / C147A, and Cof1C80A / + (Fig. 12b-12e). These results suggest that when cofilin-1 is oxidized by NLRP3 activators, more than one disulfide bond may form, leading to the dissociation of cofilin-1 from NLRP3.

[0158] In addition to the Cys39-Cys80 intramolecular bonds, cofilin-1 has been shown to form dimers in vitro through intermolecular disulfide bonding when oxidized32. To examine whether cofilin-1 can be dimerized by intermolecular disulfide bonds in vivo, we expressed two kinds of cofilin-1 proteins each labeled with two different tags, myc or V5, in BMDMs. No intermolecular interaction was observed between WT cofilin-1 proteins, whereas cofilin-1 mutants with alanine substitutions at Cys39 (C39A) or Cys80 (C80A) interacted each other and the interaction was increased when the cells were treated with ATP (Fig. 5a and Fig. 13a). However, cofilin-1 failed to interact with itself when both Cys39 and Cys80 were substituted with alanine (Fig. 5a). These data indicate that Cys39 and Cys80 of WT cofilin-1 preferentiallyMBHB Ref. No.: 24-2385- WOform an intramolecular disulfide bond, but when one of the cysteine residues is mutated, the remaining cysteine forms a disulfide bond with that of another cofilin- 1 (Fig. 13b).

[0159] Based on these data, a structural model of oxidized cofilin-1 was generated with Rosetta33, a bioinformatictool, and manually introduced a disulfide bond between Cys39 and Cys80 using ChimeraX (version 1.9)34 (Fig. 5b). Structural alignment of the reduced and oxidized cofilin-1 revealed conformational differences (Fig. 5b). Quantitative analysis showed a 558 A2reduction in total solvent-accessible surface area in the oxidized form, indicative of a more compact tertiary structure following disulfide bond formation between Cys39 and Cys80. To assess the impact of cofilin-1 oxidation on its interaction with NLRP3, docking simulations were conducted using the HADDOCK2.4 web server35 and selected the top-ranked cluster based on scoring parameters. The oxidized cofilin-1-NLRP3 complex displayed a significantly higher HADDOCK score (-52.5 ± 2.5) compared to the reduced form (-89.3 ± 1.9), along with increased van der Waals energy and a reduced buried surface area, indicating a weakened interaction following oxidation. These structural and computational analyses support a regulatory role for the Cys39-Cys80 disulfide bond in modulating cofilin-1-NLRP3 binding.

[0160] Therefore, mouse macrophage J774A.1 cells ectopically expressing all combinations of mutant cofilin-1 proteins in which each of the two cysteines was substituted with alanine by retroviral transduction were generated. Consistent with the BMDMs of the Kl-mice, no inhibition of 288 ATP-induced IL-1 release was observed in retroviral-transduced J774A.1 cells expressing mutant cofilin-1 with a single cysteine to alanine substitution (Fig.5c). However, the level of released IL-ip by ATP was significantly reduced in the cells expressing cofilin-1 with C39A / C80A double mutations among the J774A.1 cells expressing cofilin-1 with two cysteine-to-alanine mutations (Fig. 5c). In contrast, AIM2 inflammasome activation induced by dsDNA and NLRC4 inflammasome activation induced by flagellin were not suppressed by C39A / C80A mutant cofilin-1 (Fig. 5c). These data indicate that oxidation of both Cys39 and Cys80 in cofilin-1 is required for NLRP3 inflammasome activation and C39A / C80A mutant cofilin-1 suppresses NLRP3. Consistent with this hypothesis, in a pulldown assay with BMDMs ectopically expressing WT and mutant cofilin-1 proteins, we observed that the C39A / C80A mutant cofilin-1 was not dissociated from NLRP3 by ATP, while cofilin-1 with a single mutation, C39A or C80A, was dissociated from NLRP3 by ATP, as was WT cofilin-1 (Fig. 5d).Example 6. Cofilin-1 interferes with NLRP3 oligomerization and NEK7 interaction.

[0161] To investigate the structural mechanism by which cofilin-1 regulates NLRP3 inflammasome activation, we examined its effects on NLRP3 oligomerization and interactionMBHB Ref. No.: 24-2385- WOwith NEK7, hypothesizing that cofilin-1 binding interferes with these processes and is lost upon NLRP3 activation. Myc- and V5-tagged NLRP3 were co-expressed in PT67 cells and assessed oligomerization by pull-down assays. Upon nigericin treatment, NLRP3 oligomerization increased in a time-dependent manner (Fig. 14a). However, co-expression of the oxidation-resistant C39A / C80A mutant cofilin-1 markedly attenuated nigericin-induced NLRP3 oligomerization compared with WT cofilin-1 (Fig. 14b). Consistent with this, the C39A / C80A mutant remained bound to NLRP3 following nigericin stimulation, whereas WT cofilin-1 was released (Fig. 14b), indicating that NLRP3 oligomerization is inhibited by the binding of cofilin-1. Because NLRP3 can also exist in a self-inhibitory cage-like oligomer under steady state after priming36, we next asked whether cofilin-1 is able to associate with this closed architecture. As direct visualization of the cage in living cells is currently not feasible, we performed in silico structural analysis to assess the steric compatibility of cofilin-1 binding within the cage. Alignment of the cofilin-1 -bound NLRP3 model (Fig. 2d) with an NLRP3-NLRP3 dimer extracted from the human cryo-EM structure of the closed decameric cage (PDB: 7PZC)30 revealed 1,276 atomic clashes, indicating that cofilin-1 cannot access its binding site once NLRP3 is incorporated into the closed oligomeric state (Fig. 14c).

[0162] It was examined whether cofilin-1 binding interferes with NEK7 recruitment to NLRP3. Structurally alignment of our cofilin-1-bound NLRP3 model with the cryo-EM NEK7-NLRP3 complex (PDB: 6NPY)37 revealed 1 ,225 atomic clashes between NEK7 and cofilin-1 , strongly suggesting that two proteins cannot bind simultaneously to NLRP3 (Fig. 14d). This supports a model in which cofilin-1 physically blocks NEK7 recruitment and thereby prevents NLRP3 activation. Consistent with this prediction, co-expression of the oxidation-resistant C39A / C80A mutant cofilin-1, which remains more tightly bound to NLRP3 than WT cofilin-1 , markedly reduced the interaction between NLRP3 and NEK7 (Fig. 14e).Example 7. Four residues in cofilin-1 are essential for interaction with NLRP3

[0163] Cofilin-1 is a 19,335 kDa protein consisting of 166 amino acids, most of which comprise an actin-depolymerizing factor homology (ADF-H) domain containing a nuclear localization signal and a phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] binding site (Fig.6a). To determine the motif through which cofilin-1 binds to NLRP3, we pulled down recombinant GST fusion proteins containing the N-terminal (aa 2-84), middle (aa 41-127), or C-terminal (aa 85-166) half, or the full length cofilin-1, from lysates of LPS primed differentiated U937 cells expressing endogenous NLRP3. Immunoblot analysis for NLRP3 revealed that the middle and C-terminal halves of cofilin-1 bound to NLRP3 (Fig. 6a, left panel). Since there is an overlapping region between the middle and C terminal halves, it was hypothesized thatMBHB Ref. No.: 24-2385- WOresidues 85-127 harbor an NLRP3-binding motif. Indeed, no binding defect with NLRP3 was observed when the C-terminal end of cofilin-1 was deleted up to aa 135 (Fig. 6a, left panel), but in contrast, a binding defect with NLRP3 was observed when the C-terminal half was deleted from its N-terminus up to aa 105 (Fig. 6a, right panel). Taken together with the observation that the C-terminal portion of cofilin-1 with aa 100-166 still binds to NLRP3 (Fig.6a, right panel), the binding motif of cofilin-1 for NLRP3 could be localized between aa 100 and aa 105.

[0164] It was also found that a cofilin-1 fragment (aa 95-127) consisting of 33 amino acid residues from 95 to 127, which includes the putative binding motif, strongly binds to NLRP3 (Fig. 6a). To determine the amino acid sequence of the binding motif, each amino acid residue from 99 to 106 of the aa 95-127 fragment was substituted with alanine or threonine. The cofilin-1 fragment spanning amino acids 95-127 failed to interact with NLRP3 when individual residues at positions 101 to 104 were substituted, indicating that 357 the core binding motif comprises Phe101-lle102-Phe103-Trp104 (Fig. 6b; SEQ ID NO:90).

[0165] To evaluate the structural and energetic contributions of this motif to NLRP3 binding, in silico modeling of full-length cofilin-1 variants containing alanine substitutions at each position was performed. All four alanine substitutions resulted in marked reductions in both the intrinsic stability of cofilin-1 and the thermodynamic stability of the cofilin-1-NLRP3 complex. Additionally, binding affinity, as estimated by AAG binding values, was consistently decreased across all variants, supporting the critical role of this aromatic and hydrophobic motif in mediating high-affinity interactions with NLRP3 (Fig. 6c, d). In contrast, substitution of Phe103 with tyrosine (F103Y) preserved the hydrophobic character and resulted in minimal changes in complex stability (AAG binding = -0.09 kcal / mol) (Fig. 6c, d). Further, F103Y was predicted to have milder impact by AlphaMissense38, CADD39, and REVEL40, suggesting that an aromatic amino acid at this site is tolerated. This prediction was experimentally validated by a pull-down assay, which showed that the F103Y mutation within the 95-127 fragment did not compromise NLRP3 binding (Fig. 6e).Example 8. Cofilin-1 blocks ATPase activity of NLRP3

[0166] It was investigated how the NLRP3 inflammasome is inhibited by the cofilin-1 binding. Given that cofilin-1 binds to the NBD, which has ATPase activity, and that ATP hydrolysis is essential for NLRP3 inflammasome assembly41, 42 and activation, we hypothesized that cofilin-1 may inhibit NLRP3 inflammasome activation by suppressing the ATPase activity of the NLRP3 NBD. In an assay measuring ATP hydrolysis, we observed that full-length cofilin-1 significantly suppressed the ATPase activity of NLRP3 (Fig. 7a). Moreover,MBHB Ref. No.: 24-2385- WOthe ATPase activity of NLRP3 was significantly suppressed by the WT aa 95-127 fragment, but not by a mutant fragment with a substitution of Phe103 to alanine (aa 95-127 F103A), which did not bind to NLRP3 (Fig. 6b and Fig. 7a). These results demonstrate that cofilin-1 binds to the NBD of NLRP3 and thereby blocks the ATPase activity of NLRP3.Example 9. Peptides from cofilin-1 inhibit NLRP3 inflammasome activation.

[0167] The cofilin-1 fragment comprising aa 95-127 contains the NLRP3-binding motif but lacks the cysteines responsible for regulating cofilin-1 dissociation from NLRP3 through oxidation-mediated disulfide bond formation. It was observed that the binding of this fragment to NLRP3 remained unaffected by H2O2, while the binding of full length cofilin-1 was decreased by H2O2 (Fig. 7b), suggesting that fragment aa 95-127 could be utilized as an inhibitor for the NLRP3 inflammasome. To investigate the impact of the aa 95-127 fragment on the NLRP3 inflammasome, we initially synthesized this fragment in peptide form along with another peptide corresponding to aa 134-166 of cofilin-1, excluding the NLRP3-binding motif. To avoid unintended activation of the AIM2 inflammasome during transfection, these peptides were delivered into BMDMs from AIM2 deficient mice. ATP- or nigericin-induced IL-10 release and caspase- 1 activation were markedly inhibited by aa 95-127 but not by aa 134-166, whereas NLRC4 inflammasome activation induced by flagellin was not suppressed by aa 95-127 (Fig. 7c).

[0168] As demonstrated previously, substitution of phenylalanine with alanine at position 103 (F103A) within the cofilin-1 fragment (aa 95-127) abrogated binding to NLRP3, whereas replacement with tyrosine (F103Y) preserved the interaction (Fig. 6b, e). Consistent with these binding data, the F103A-substituted peptide (aa 95-127) failed to inhibit NLRP3 inflammasome activation. In contrast, the F103Y-substituted peptide, which preserved NLRP3 binding, effectively suppressed NLRP3 inflammasome activity while exhibiting no inhibitory effect on NLRC4 inflammasome activation (Fig. 7d). Additionally, we observed that the constitutive inflammasome activation of CAPS PBMCs was also substantially suppressed by peptides aa 95-127 or aa 95-127 F103Y, but not by peptide aa 95-127 F103A (Fig. 7e). Taken together, these data strongly support the proposal of a role for cofilin-1 in suppressing the NLRP3 inflammasome and suggest that fragment aa 95-127 of cofilin-1 potentially serves as an inhibitor for the NLRP3 inflammasome.

[0169] Moreover, this finding holds significant promise for the development of therapeutic interventions targeting NLRP3-mediated inflammatory diseases, offering a novel approach to managing several more common acquired inflammatory diseases in which the NLRP3 inflammasome has been implicated.MBHB Ref. No.: 24-2385- WO

[0170] Through an unbiased proteomic approach complemented with structural modeling, this paper presents strong evidence that cofilin- 1 binds to NLRP3 and blocks the activation of the NLRP3 inflammasome, that cofilin-1 oxidation provides a mechanism for sensing intracellular ROS and releasing tonic NLRP3 inhibition, and that specific oxidationindependent cofilin-1-derived peptides inhibit NLRP3 inflammasome activation. The data add substantially to the emerging concept of homeostatic regulation of innate immunity43, and may also extend the abilities to functionally validate NLRP3 variants in a systematic fashion44, 45.

[0171] The NLRP3 inflammasome is remarkable in its ability to respond to a broad spectrum of stimuli, including pathogens, cellular damage, metabolic stress, and environmental stressors. Rather than directly binding to these activators, NLRP3 detects subsequent intracellular physiological changes, such as potassium efflux and ROS generation. The biochemical and structural modeling data presented herein demonstrate that in resting macrophages, cofilin-1 binds directly to the NBD and LRR domains of NLRP3 to prevent oligomerization and NEK7 engagement. This interaction may help explain the distribution of most common disease-associated NLRP3 variants within these domains. ROS-induced oxidative modification of cofilin-1 leads to its dissociation from NLRP3, releasing NLRP3 monomers for dimerization, NEK7 binding, and inflammasome assembly (Fig. 8). Blocking cofilin-1 oxidation, either by cysteine mutations or antioxidant treatment, sustained NLRP3-cofilin-1 binding and suppressed inflammasome activation, underscoring the critical role of redox regulation in this process. Consistent with prior reports that NEK7 cannot bind to the closed decameric cage structure of NLRP330, 46, our structural clash analyses further indicate that cofilin-1 is also incompatible with this conformation and instead associates with monomeric NLRP3. Taken together, these findings suggest that cofilin-1 serves as a redox sensitive checkpoint that restrains NLRP3 monomers under resting conditions, thereby contributing a critical layer of regulation to the NLRP3 activation pathway.

[0172] This study also provides new mechanistic insights into CAPS-associated mutant NLRP3 activation. We observed that mutant NLRP3 proteins causing CAPS exhibit significantly reduced binding to cofilin-1 compared to WT NLRP3, suggesting that impaired cofilin-1 -mediated regulation contributes to the constitutive activation of these mutants. This finding further strengthens the concept that loss of negative regulatory mechanisms, rather than merely increased sensitivity to activators, plays a key role in the pathogenesis of CAPS. These findings suggest that NLRP3 inflammasome activation follows a guard-type mechanism conserved in plant defense, in which host sensors detect common physiological disturbances rather than directly recognizing PAMPs, allowing the immune system to respond broadly to a wide range of infections and cellular stressors47.MBHB Ref. No.: 24-2385- WO

[0173] Thioredoxin-interacting protein (TXNIP) has been proposed to be required for NLRP3 activation in response to ROS through direct interaction in islet cells (48). However, the role of TXNIP as a positive regulator mediating ROS-induced NLRP3 activation remains controversial. TXNIP activation primarily enhances IL-10 mRNA and intracellular pro-IL-10 levels ratherthan directly promoting IL- 10 secretion (49), and in BMDMsfrom TXNIP KO mice, no significant difference in IL-10 release was observed in response to inflammasome activators (50). In contrast, our BMDM data demonstrate that cofilin-1 knockdown induces NLRP3 inflammasome activation without exogenous stimulation. Cofilin-1 oxidation may also create an autocrine positive feedback loop because oxidized cofilin-1 translocates to mitochondria, leading to mitochondrial dysfunction and increased ROS generation (51, 52).

[0174] Importantly, this study identified a conserved motif (Phe-lle-Phe-Trp; SEQ ID NO:90) in cofilin-1 that is essential for NLRP3 binding. An oxidation-independent synthetic peptide containing this NLRP3-binding motif effectively suppressed IL-10 release induced by both CAPS associated NLRP3 mutations and canonical inflammasome activators. This finding opens new avenues for therapeutic development, as peptides mimicking this binding interface could serve as potential inhibitors of excessive NLRP3 activation, with a potentially broader impact than biologies that target IL-10, IL-18, or Gasdermin D individually. Given the broad involvement of NLRP3 in various inflammatory diseases, including gout4, type 2 diabetes (6, 4) atherosclerosis (5), Alzheimer’s disease (7, 8), and pericarditis (9, 10), targeting the NLRP3-cofilin-1 interaction may represent an important opportunity for treating a spectrum of autoinflammatory and acquired inflammatory conditions.

[0175] Having described the disclosure in detail and by reference to specific embodiments thereof, it will be apparent that modifications and variations are possible without departing from the scope of the disclosure defined in the appended claims. More specifically, although some aspects of the present disclosure are identified herein as particularly advantageous, it is contemplated that the present disclosure is not necessarily limited to these particular aspects of the disclosure.References1. Fu, J., Schroder, K. & Wu, H. Mechanistic insights from inflammasome structures. Nat. Rev. Immunol. 24, 518-535 (2024).2. Hoffman, H.M., Mueller, J.L., Broide, D.H., Wanderer, A.A. & Kolodner, R.D. Mutation of a new gene encoding a putative pyrin-like protein causes familial cold autoinflammatory syndrome and Muckle- Wells syndrome. Nat. Genet. 29, 301-305 (2001).3. Aksentijevich, I. et al. De novo CIAS1 mutations, cytokine activation, and evidence for genetic heterogeneity in patients with neonatal-onset multisystem inflammatory diseaseMBHB Ref. No.: 24-2385- WO(NOMID): a new member of the expanding family of pyrin-associated autoinflammatory diseases. Arthritis Rheum. 46, 3340-3348 (2002).4. Martinon, F., Petrilli, V., Mayor, A., Tardivel, A. & Tschopp, J. Gout-associated uric acid crystals activate the NALP3 inflammasome. Nature 440, 237-241 (2006).5. Duewell, P. et al. NLRP3 inflammasomes are required for atherogenesis and activated by cholesterol crystals. Nature 464, 1357-1361 (2010).6. Vandanmagsar, B. et al. The NLRP3 inflammasome instigates obesity-induced inflammation and insulin resistance. Nature medicine 17, 179-188 (2011).7. Halle, A. et al. The NALP3 inflammasome is involved in the innate immune response to amyloid-beta. Nat. Immunol. 9, 857-865 (2008).8. Heneka, M.T. et al. NLRP3 is activated in Alzheimer's disease and contributes to pathology in APP / PS1 mice. Nature 493, 674-678 (2013).9. Mauro, A.G. et al. The Role of NLRP3 Inflammasome in Pericarditis: Potential for Therapeutic Approaches. JACC Basic Transl Sci 6, 137-150 (2021).10. Vecchie, A. et al. Interleukin-1 and the NLRP3 Inflammasome in Pericardial Disease. Curr Cardiol Rep 23, 157 (2021).11. Andreeva, L. et al. NLRP3 cages revealed by full-length mouse NLRP3 structure control pathway activation. Cell 184, 6299-6312 e6222 (2021).12. He, Y., Zeng, M.Y., Yang, D., Motro, B. & Nunez, G. NEK7 is an essential mediator of NLRP3 activation downstream of potassium efflux. Nature 530, 354-357 (2016).13. Shi, H. et al. NLRP3 activation and mitosis are mutually exclusive events coordinated by NEK7, a new inflammasome component. Nat. Immunol. 17, 250-258 (2016).14. Schmacke, N.A. et al. IKKbeta primes inflammasome formation by recruiting NLRP3 to the trans-Golgi network. Immunity 55, 2271-2284 e2277 (2022).15. Petrilli, V. et al. Activation of the NALP3 inflammasome is triggered by low intracellular potassium concentration. Cell Death Differ. 14, 1583-1589 (2007).16. Zhou, R., Yazdi, A.S., Menu, P. & Tschopp, J. A role for mitochondria in NLRP3 inflammasome activation. Nature 469, 221-225 (2011).17. Bruchard, M. et al. Chemotherapy-triggered cathepsin B release in myeloid derived suppressor cells activates the Nlrp3 inflammasome and promotes tumor growth. Nature medicine 19, 57-64 (2013).18. Tassi, S. et al. Altered redox state of monocytes from cryopyrin-associated periodic syndromes causes accelerated IL-1 beta secretion. Proc. Natl. Acad. Sci. USA. 107, 9789-9794 (2010).19. Balow, J.E., Jr. et al. Microarray-based gene expression profiling in patients with cryopyrin-associated periodic syndromes defines a disease-related signature and IL-1 -responsive transcripts. Annals of the rheumatic diseases 72, 1064-1070 (2013).MBHB Ref. No.: 24-2385- WO20. Park, Y.H., Wood, G., Kastner, D.L. & Chae, J. J. Pyrin inflammasome activation and RhoA signaling in the autoinflammatory diseases FMF and HIDS. Nat. Immunol. 17, 914-921 (2016).21. Burger, D., Fickentscher, C., de Moerloose, P. & Brandt, K.J. F-actin dampens NLRP3 inflammasome activity via Flightless-I and LRRFIP2. Sci Rep 6, 29834 (2016).22. Lee, G.S. et al. The calcium-sensing receptor regulates the NLRP3 inflammasome through Ca2+ and cAMP. Nature 492, 123-127 (2012).23. Gross, C.J. et al. K(+) Efflux-Independent NLRP3 Inflammasome Activation by Small Molecules Targeting Mitochondria. Immunity 45, 761-773 (2016).24. Abramson, J. et al. Accurate structure prediction of biomolecular interactions with AlphaFold 3. Nature 630, 493-500 (2024).25. Finkel, T. Signal transduction by reactive oxygen species. J. Cell Biol. 194, 7-15 (2011).26. Klemke, M. et al. Oxidation of cofilin mediates T cell hyporesponsiveness under oxidative stress conditions. Immunity 29, 404-413 (2008).27. Klejnot, M. et al. Analysis of the human cofilin 1 structure reveals conformational changes required for actin binding. Acta Crystallogr D Biol Crystallogr 69, 1780-1788 (2013).28. Cameron, J.M. et al. Polarized cell motility induces hydrogen peroxide to inhibit cofilin via cysteine oxidation. Curr. Biol. 25, 1520-1525 (2015).29. Coll, R.C. et al. A small-molecule inhibitor of the NLRP3 inflammasome for the treatment of inflammatory diseases. Nature medicine 21, 248-255 (2015).30. Hochheiser, I.V. et al. Structure of the NLRP3 decamer bound to the cytokine release inhibitor CRID3. Nature 604, 184-189 (2022).31. Munoz-Planillo, R. et al. K(+) efflux is the common trigger of NLRP3 inflammasome activation by bacterial toxins and particulate matter. Immunity 38, 1142-1153 (2013).32. Pfannstiel, J. 755 et al. Human cofilin forms oligomers exhibiting actin bundling activity. J. Biol. Chem. 276, 49476-49484 (2001).33. Leaver-Fay, A. et al. ROSETTA3: an object-oriented software suite for the simulation and design of macromolecules. Methods Enzymol 487, 545-574 (2011).34. Goddard, T.D. et al. UCSF ChimeraX: Meeting modern challenges in visualization and analysis. Protein Sci 27, 14-25 (2018).35. Honorato, R.V. et al. The HADDOCK2.4 web server for integrative modeling of biomolecular complexes. Nature protocols 19, 3219-3241 (2024).36. Xiao, L., Magupalli, V.G. & Wu, H. Cryo-EM structures of the active NLRP3 inflammasome disc. Nature 613, 595-600 (2023).37. Sharif, H. et al. Structural mechanism for NEK7-licensed activation of NLRP3 inflammasome. Nature 570, 338-343 (2019).38. Cheng, J. et al. Accurate proteome-wide missense variant effect prediction with AlphaMissense. Science 381 , eadg7492 (2023).MBHB Ref. No.: 24-2385- WO39. Schubach, M., Maass, T., Nazaretyan, L., Roner, S. & Kircher, M. CADD v1.7: using protein language models, regulatory CNNs and other nucleotide-level scores to improve genome-wide variant predictions. Nucleic acids research 52, D1143-D1154 (2024).40. loannidis, N.M. et al. REVEL: An Ensemble Method for Predicting the Pathogenicity of Rare Missense Variants. American journal of human genetics 99, 877-885 (2016).41. Duncan, J.A. et al. Cryopyrin / NALP3 binds ATP / dATP, is an ATPase, and requires ATP binding to mediate inflammatory signaling. Proc. Natl. Acad. Sci. USA. 104, 8041-8046 (2007).42. Brinkschulte, R. et al. ATP-binding and hydrolysis of human NLRP3. Commun Biol 5, 1176 (2022).43. Liston, A. & Masters, S.L. Homeostasis-altering molecular processes as mechanisms of inflammasome activation. Nat. Rev. Immunol. 17, 208-214 (2017).44. Feng, S. et al. Mechanisms of NLRP3 activation and inhibition elucidated by functional analysis of disease-associated variants. Nat. Immunol. 26, 511-523 (2025).45. Cosson, C. et al. Functional diversity of NLRP3 gain-of-function mutants associated with CAPS autoinflammation. J. Exp. Med. 221 (2024).46. Yu, X. et al. Structural basis for the oligomerization-facilitated NLRP3 activation. Nat Commun 15, 1164 (2024).47. Jones, J.D. & Dangl, J.L. The plant immune system. Nature 444, 323-329 (2006).48. Zhou, R., Tardivel, A., Thorens, B., Choi, I. & Tschopp, J. Thioredoxin-interacting protein links oxidative stress to inflammasome activation. Nat. Immunol. 11 , 136-140 (2010).49. Koenen, T.B. et al. Hyperglycemia activates caspase-1 and TXNIP-mediated IL-1 beta transcription in human adipose tissue. Diabetes 60, 517-524 (2011).50. Masters, S.L. et al. Activation of the NLRP3 inflammasome by islet amyloid polypeptide provides a mechanism for enhanced IL-1 beta in type 2 diabetes. Nat. Immunol. 11 , 897-904 (2010).51. Klamt, F. et al. Oxidant-induced apoptosis is mediated by oxidation of the actin regulatory protein cofilin. Nat Cell Biol 11, 1241-1246 (2009).52. Hoffmann, L. et al. Cofilin 1 oxidation links oxidative distress to mitochondrial demise and neuronal cell death. Cell Death Dis 12, 953 (2021).53. Paunovska, K., Loughrey, D. & Dahlman, J.E. Drug delivery systems for RNA therapeutics. Nat Rev Genet 23, 265-280 (2022).54. Taya, T., Kami, D., Teruyama, F., Matoba, S. & Gojo, S. Peptide-encoding gene transfer to modulate intracellular protein-protein interactions. Mol Ther Methods Clin Dev 32, 101226 (2024).55. Li, H. et al. Therapeutic potential of MCC950, a specific inhibitor of NLRP3 inflammasome. Eur J Pharmacol 928, 175091 (2022).MBHB Ref. No.: 24-2385- WO56. Tang, F. et al. First-in-human phase 1 trial evaluating safety, pharmacokinetics, and pharmacodynamics of NLRP3 inflammasome inhibitor, GDC-2394, in healthy volunteers. Clin Transl Sci 16, 1653-1666 (2023).

Claims

MBHB Ref. No.: 24-2385- WOWHAT IS CLAIMED IS:

1. An isolated polypeptide comprising an amino acid sequence having about 15 amino acids to about 120 amino acids, wherein the amino acid sequence has at least 75% identity to an equal-length portion of residues 35-155 of SEQ ID NO:01.

2. The isolated polypeptide of claim 1, wherein the amino acid sequence of the isolated polypeptide comprises at least one substitution relative to residues 35-155 of SEQ ID NQ:01.

3. The isolated polypeptide of either claim 1 or claim 2, wherein the isolated polypeptide further comprises at least one modification.

4. The isolated polypeptide of any one of claims 1 -3, wherein the at least one modification improves stability of the polypeptide, extends half-life of the polypeptide, decreases degradation of the polypeptide, and / or promotes delivery of the polypeptide to one or more targets.

5. The isolated polypeptide of any one of claims 1-4, wherein the isolated polypeptide is a cyclic peptide.

6. The isolated polypeptide of claim 5, wherein the cyclic peptide comprises head-to-tail cyclic peptides.

7. The isolated polypeptide of any one of claims 1-6, wherein the amino acid sequence is selected from the group consisting of:95-KKEDLVFIFWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NQ:02),94-SKKEDLVFIFWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NQ:03),93-ESKKEDLVFIFWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NQ:04), 92-KESKKEDLVFIFWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NQ:05), 91-TKESKKEDLVFIFWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NQ:06), 90-ETKESKKEDLVFIFWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NQ:07), 89-YETKESKKEDLVFIFWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NQ:08), 88-TYETKESKKEDLVFIFWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NQ:09), 87-ATYETKESKKEDLVFIFWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NQ:10),MBHB Ref. No.: 24-2385- WO86-DATYETKESKKEDLVFIFWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NO:11),85-YDATYETKESKKEDLVFIFWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NO:12),95-KKEDLVFIFWAPESAPLKSKMIYASSKDAIKK-126 (SEQ ID N0:13),94-SKKEDLVFIFWAPESAPLKSKMIYASSKDAIK-125 (SEQ ID N0:14),93-ESKKEDLVFIFWAPESAPLKSKMIYASSKDAI-124 (SEQ ID N0:15),92-KESKKEDLVFIFWAPESAPLKSKMIYASSKDA-123 (SEQ ID N0:16),91-TKESKKEDLVFIFWAPESAPLKSKMIYASSKD-122 (SEQ ID N0:17),90-ETKESKKEDLVFIFWAPESAPLKSKMIYASSK-121 (SEQ ID N0:18),89-YETKESKKEDLVFIFWAPESAPLKSKMIYASS-120 (SEQ ID N0:19),88-TYETKESKKEDLVFIFWAPESAPLKSKMIYAS-119 (SEQ ID NQ:20),87-ATYETKESKKEDLVFIFWAPESAPLKSKMIYA-118 (SEQ ID N0:21),86-DATYETKESKKEDLVFIFWAPESAPLKSKMIY-117 (SEQ ID NO:22),85-YDATYETKESKKEDLVFIFWAPESAPLKSKMI-116 (SEQ ID NO:23),95-KKEDLVFIYWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NO:24),94-SKKEDLVFIYWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NO:25),93-ESKKEDLVFIYWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NO:26), 92-KESKKEDLVFIYWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NO:27), 91-TKESKKEDLVFIYWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NO:28), 90-ETKESKKEDLVFIYWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NO:29), 89-YETKESKKEDLVFIYWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NQ:30), 88-TYETKESKKEDLVFIYWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID N0:31), 87-ATYETKESKKEDLVFIYWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NO:32), 86-DATYETKESKKEDLVFIYWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NO:33),85-YDATYETKESKKEDLVFIYWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NO: 34),95-KKEDLVFIYWAPESAPLKSKMIYASSKDAIKK-126 (SEQ ID NO:35),94-SKKEDLVFIYWAPESAPLKSKMIYASSKDAIK-125 (SEQ ID NO:36),93-ESKKEDLVFIYWAPESAPLKSKMIYASSKDAI-124 (SEQ ID NO:37),92-KESKKEDLVFIYWAPESAPLKSKMIYASSKDA-123 (SEQ ID NO:38),91-TKESKKEDLVFIYWAPESAPLKSKMIYASSKD-122 (SEQ ID NO:39),90-ETKESKKEDLVFIYWAPESAPLKSKMIYASSK-121 (SEQ ID NQ:40),89-YETKESKKEDLVFIYWAPESAPLKSKMIYASS-120 (SEQ ID NO:41),88-TYETKESKKEDLVFIYWAPESAPLKSKMIYAS-119 (SEQ ID NO:42),87-ATYETKESKKEDLVFIYWAPESAPLKSKMIYA-118 (SEQ ID NO:43),MBHB Ref. No.: 24-2385- WO86-DATYETKESKKEDLVFIYWAPESAPLKSKMIY-117 (SEQ ID NO:44),85-YDATYETKESKKEDLVFIYWAPESAPLKSKMI-116 (SEQ ID NO:45),95-KKEDLVYIFWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NO:46),94-SKKEDLVYIFWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NO:47),93-ESKKEDLVYIFWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NO:48), 92-KESKKEDLVYIFWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NO:49), 91-TKESKKEDLVYIFWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NQ:50), 90-ETKESKKEDLVYIFWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NO:51), 89-YETKESKKEDLVYIFWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NO:52), 88-TYETKESKKEDLVYIFWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NO:53), 87-ATYETKESKKEDLVYIFWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NO:54), 86-DATYETKESKKEDLVYIFWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NO:55),85-YDATYETKESKKEDLVYIFWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NO: 56),95-KKEDLVYIFWAPESAPLKSKMIYASSKDAIKK-126 (SEQ ID NO:57),94-SKKEDLVYIFWAPESAPLKSKMIYASSKDAIK-125 (SEQ ID NO:58),93-ESKKEDLVYIFWAPESAPLKSKMIYASSKDAI-124 (SEQ ID NO:59),92-KESKKEDLVYIFWAPESAPLKSKMIYASSKDA-123 (SEQ ID NQ:60),91-TKESKKEDLVYIFWAPESAPLKSKMIYASSKD-122 (SEQ ID NO:61),90-ETKESKKEDLVYIFWAPESAPLKSKMIYASSK-121 (SEQ ID NO:62),89-YETKESKKEDLVYIFWAPESAPLKSKMIYASS-120 (SEQ ID NO:63),88-TYETKESKKEDLVYIFWAPESAPLKSKMIYAS-119 (SEQ ID NO:64),87-ATYETKESKKEDLVYIFWAPESAPLKSKMIYA-118 (SEQ ID NO:65),86-DATYETKESKKEDLVYIFWAPESAPLKSKMIY-117 (SEQ ID NO:66),85-YDATYETKESKKEDLVYIFWAPESAPLKSKMI-116 (SEQ ID NO:67),95-KKEDLVFLFWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NO:68),94-SKKEDLVFLFWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NO:69),93-ESKKEDLVFLFWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NQ:70), 92-KESKKEDLVFLFWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NO:71), 91-TKESKKEDLVFLFWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NO:72), 90-ETKESKKEDLVFLFWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NO:73), 89-YETKESKKEDLVFLFWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NO:74), 88-TYETKESKKEDLVFLFWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NO:75), 87-ATYETKESKKEDLVFLFWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NO:76), 86-DATYETKESKKEDLVFLFWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NO:77),MBHB Ref. No.: 24-2385- WO85-YDATYETKESKKEDLVFLFWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NO:78),95-KKEDLVFLFWAPESAPLKSKMIYASSKDAIKK-126 (SEQ ID NO:79),94-SKKEDLVFLFWAPESAPLKSKMIYASSKDAIK-125 (SEQ ID NQ:80),93-ESKKEDLVFLFWAPESAPLKSKMIYASSKDAI-124 (SEQ ID N0:81),92-KESKKEDLVFLFWAPESAPLKSKMIYASSKDA-123 (SEQ ID NO:82),91-TKESKKEDLVFLFWAPESAPLKSKMIYASSKD-122 (SEQ ID NO:83),90-ETKESKKEDLVFLFWAPESAPLKSKMIYASSK-121 (SEQ ID NO:84),89-YETKESKKEDLVFLFWAPESAPLKSKMIYASS-120 (SEQ ID NO:85),88-TYETKESKKEDLVFLFWAPESAPLKSKMIYAS-119 (SEQ ID NO:86),87-ATYETKESKKEDLVFLFWAPESAPLKSKMIYA-118 (SEQ ID NO:87),86-DATYETKESKKEDLVFLFWAPESAPLKSKMIY-117 (SEQ ID NO:88), or85-YDATYETKESKKEDLVFLFWAPESAPLKSKMI-116 (SEQ ID NO:89).

8. The isolated polypeptide of any one of claims 1-7, wherein the amino acid sequence comprises 95-KKEDLVFIFWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NQ:02).

9. The isolated polypeptide of any one of claims 1-7, wherein the amino acid sequence comprises 95-KKEDLVFIYWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NO:24).

10. The isolated polypeptide of any one of claims 1-7, wherein the amino acid sequence comprises 95-KKEDLVYIFWAPESAPLKSKMIYASSKDAIKKK-127 (SEQ ID NO:46).

11. A nucleic acid composition, comprising a nucleic acid sequence encoding the isolated polypeptide of any one of claims 1-10.

12. A pharmaceutical composition, comprising the isolated polypeptide of any one of claims 1 -9 or the nucleic acid composition of claim 11.

13. The pharmaceutical composition of claim 12, wherein the composition comprises at least one pharmaceutically acceptable carrier or excipient.

14. The pharmaceutical composition of either claim 12 or claim 13, further comprising an isotonic agent, a preservative, and / or a buffer.

15. The pharmaceutical composition of any one of claims 12-14, further comprising an additional active agent.MBHB Ref. No.: 24-2385- WO16. A method of treating an inflammatory condition in a subject, comprising administrating to the subject an effective dose of the pharmaceutical composition of any one of claims 12-15.

17. The method of claim 16, further comprising repeating the administering at least a second time, at least a third time, at least a fourth time, at least a fifth time, at least a sixth time, or more.

18. The method of either claim 16 or claim 17, wherein the inflammatory condition comprises gout, pericarditis, type 2 diabetes mellitus, atherosclerosis, cryopyrin- associated periodic syndromes (CAPS), and Alzheimer’s disease.

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