Compositions and methods for treating inflammasome-associated diseases or disorders

Abstract

The compositions and methods described herein include agents that inhibit inflammasome signaling in a mammal, such as antibodies against inflammasome components, used alone or in combination with one or more extracellular vesicle uptake inhibitors. Also described herein are compositions for treating inflammasome-related diseases or disorders and methods of using the same.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Application Serial No. 16 / 026,482, filed on July 3, 2018, which is incorporated herein by reference in its entirety for all purposes.

[0003] Statement Regarding Federally Sponsored Research

[0004] This invention was made with U.S. Government support under Grant No. 4R42BS086274-02 awarded by the National Institute of Neurological Disorders and Stroke (NINDS) and Grant No. 5R42NS086274-03 awarded by the National Institutes of Health. The U.S. Government has certain rights in this invention.

[0005] Instructions for electronically submitting text files

[0006] The contents of the text file submitted electronically herewith are incorporated herein by reference in its entirety: Computer readable format copy of the Sequence Listing (file name: UNMI_010_02WO_SeqList_ST25.txt, record date: July 3, 2019, file size: approximately 19.5 kilobytes). Technical Field

[0007] The present invention relates generally to the fields of immunology and medicine. More specifically, the present invention relates to compositions and methods for modulating the activity of ASC (apoptosis-associated speck-like protein containing a caspase activation recruitment domain (CARD)) and melanoma deficiency factor 2 (AIM2) inflammasome activity in the central nervous system (CNS) and / or lungs of mammals as a treatment for reducing inflammation in response to an injury or condition that produces inflammation in the CNS and / or lungs. The present invention also relates to monoclonal antibodies or fragments thereof that specifically bind to ASC. Background Art

[0008] Severe traumatic brain injury (TBI) is a major public health problem and is a leading cause of mortality and morbidity worldwide (Summers, CR et al., (2009). Traumatic brain injury in the United States: anepidemiologic overview. Mt Sinai J Med 76, 105-110). In addition to direct damage to the brain, TBI can also lead to complications in other organs (such as the lungs). Acute lung injury (ALI; 2) is a common cardiopulmonary problem after trauma and is associated with an in-hospital mortality rate of up to 40% (Rincon F. et al., (2012). Impact of acute lung injury and acute respiratory distress syndrome after traumatic brain injury in the United States. Neurosurgery 71, 795-803). TBI patients are particularly prone to developing ALI, with some studies reporting an incidence as high as 30% (Nicolls, MR et al., (2014). Traumatic brain injury: lungs in aRAGE. Sci Transl Med 6, 252fs234). Recent studies have shown that systemic inflammatory factors may lead to lung dysfunction and lung injury after TBI (Rincon F. et al., (2012). Impact of acute lung injury and acute respiratory distress syndrome after traumatic brain injury in the United States. Neurosurgery 71, 795-803), but the exact molecular mechanism underlying TBI-induced lung injury is still unclear.

[0009] A large number of secreted inflammatory mediators (including cytokines, chemokines and damage-associated molecular patterns (DAMPs) released by damaged cells) contribute to brain inflammation and affect distal organs such as the lungs (Nicolls, MR et al., (2014). Traumatic brain injury: lungs in a RAGE. Sci Transl Med 6, 252fs234). One of the most widely studied DAMPs is high mobility group box-1 (HMGB1), which can serve as an early inflammatory mediator in a variety of pathogenic states (including TBI) (Andersson U. et al., (2011). Introduction: HMGB1 in inflammation and innate immunity. J Intern Med 270, 296-300). A recent study has shown that HMGB1 may be involved in the mechanism of TBI-induced pulmonary dysfunction (Weber et al., (2014). The HMGB1-RAGE axis mediates traumatic brain injury-induced pulmonary dysfunction in lung transplantation. Sci Transl Med 6, 252ra124). HMGB1 release can be regulated by inflammasomes, which are multiprotein complexes involved in the activation of caspase-1 and the processing of IL-1β and IL-18 after TBI (Lu et al. (2012). Novel role of PKR in inflammasome activation and HMGB1 release. Nature 488, 670-674).

[0010] Various explanations have been proposed to explain the pathological mechanisms of pulmonary complications after TBI, including increased vascular permeability leading to capillary leakage and infiltration of protein debris (Ware et al., (2000). The Acute Respiratory Distress Syndrome. New England Journal of Medicine 342, 1334-1349). Extracellular vesicles (EVs) are membrane-containing vesicles that play a role in intercellular communication (Yanez-Mo, M. et al., (2015). Biological properties of extracellular vesicles and their physiological functions. J Extracell Vesicles 4, 27066) and are thought to play a role in the development of ALI in the LPS-induced murine ALI model. In addition, it has been shown that EVs can carry bioactive cytokines (such as IL-1β and inflammasome proteins) (Qu, Y. et al., (2007). Nonclassical IL-1beta secretion stimulated by P2X7 receptors isdependent on inflammasome activation and correlated with exosome release inmurine macrophages. J Immunol 179, 1913-1925) (de Rivero Vaccari, JP et al., (2015). Exosome-mediated inflammasome signaling after central nervous system injury. J Neurochem), and may trigger immune responses and amplify inflammation via their transport to adjacent and surrounding cells. However, it is not clear whether EV-mediated inflammasome signaling can contribute to the pathological mechanism of TBI-induced ALI. In addition, it is not clear whether the pathological mechanism of TBI-induced ALI is common to other conditions that produce lung inflammation. In addition, there is a lack of drugs approved by the Federal Drug Administration (FDA) for the treatment of lung inflammation.

[0011] Therefore, there is an urgent need not only to elucidate the pathological mechanisms of lung inflammation caused by TBI as well as other conditions, but also to develop therapeutic compositions and their use for treating and / or preventing lung inflammation. Summary of the invention

[0012] On the one hand, the present invention provides a monoclonal antibody or its antibody fragment that binds to apoptosis-associated speck-like protein (ASC) containing a caspase activation recruitment domain, wherein the antibody or the antibody fragment specifically binds to an epitope of ASC, wherein the epitope comprises or consists of SEQ ID NO:5 or an amino acid sequence of 5-10, 10-15 or 15-20 amino acids of SEQ ID NO:5.

[0013] On the other hand, the present invention provides a monoclonal antibody or antibody fragment thereof that specifically binds to ASC, wherein the antibody or the antibody fragment comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region amino acid sequence comprises HCDR1 of SEQ ID NO:6, HCDR2 of SEQ ID NO:7, and HCDR3 of SEQ ID NO:8, or a variant thereof having at least one amino acid substitution in HCDR1, HCDR2, and / or HCDR3. In some cases, the VH region amino acid sequence comprises SEQ ID NO:18, 19, 20, 21, 22, or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence of SEQ ID NO:18, 19, 20, 21 or 22. In some cases, the ASC is a human ASC protein. In some cases, the antibody fragment is Fab, F(ab')2, Fab', scFv, a single domain antibody, a double antibody, or a single chain camel antibody or a shark antibody. In some cases, the monoclonal antibody or its antibody fragment is human, humanized or chimeric. In some cases, a nucleic acid molecule encoding the monoclonal antibody or its antibody fragment is provided herein. In some cases, an expression vector comprising the nucleic acid molecule is provided herein. In some cases, a nucleic acid molecule is provided herein, which is operably connected to a regulatory sequence suitable for expressing a nucleic acid segment in a host cell. In some cases, a recombinant host cell comprising the expression vector is provided herein. On the other hand, a method for producing an antibody or antibody fragment specifically bound to ASC is provided herein, the method comprising: culturing a recombinant host cell comprising the expression vector under conditions of expressing the nucleic acid molecule, thereby producing the monoclonal antibody or its antibody fragment specifically bound to ASC. In some cases, a pharmaceutical composition is provided herein, which comprises the monoclonal antibody or its antibody fragment and a pharmaceutically acceptable carrier, diluent or excipient. In some cases, a method for treating inflammation in a subject is provided herein, the method comprising administering a therapeutically effective amount of the monoclonal antibody or its antibody fragment to the subject, thereby treating inflammation in the subject. In some cases, the administration of the monoclonal antibody or its antibody fragment reduces the level of at least inflammatory cytokines. In some cases, the inflammation is an inflammasome-related inflammation. In some cases, the inflammasome-related inflammation is associated with central nervous system (CNS) damage, autoimmune disease, autoinflammatory disease or neurodegenerative disease. In some cases, the CNS damage is selected from traumatic brain injury (TBI), stroke and spinal cord injury (SCI).In some cases, the autoimmune disease or neurodegenerative disease is amyotrophic lateral sclerosis (ALS), Alzheimer's disease, Parkinson's disease, muscular dystrophy (MD), systemic lupus erythematosus, lupus nephritis, rheumatoid arthritis, inflammatory bowel disease (e.g., Crohn's disease and ulcerative colitis) or multiple sclerosis (MS). In some cases, the inflammasome-associated inflammation is associated with a metabolic disease or disorder. In some cases, the metabolic disease is metabolic syndrome, obesity, diabetes, diabetic nephropathy or diabetic kidney disease (DKD), insulin resistance, atherosclerosis, lipid deposition disorders, glycogen storage disease, medium-chain acyl-CoA dehydrogenase deficiency, nonalcoholic fatty liver disease (e.g., nonalcoholic steatohepatitis (NASH)) and gout. In some cases, the autoinflammatory disease is cryopyrin-associated periodic syndrome (CAPS). CAPS may include familial cold autoinflammatory syndrome (FCAS), Muckle-Wells syndrome (MWS) and neonatal multisystem inflammatory disease (NOMID). In some cases, the administration of the monoclonal antibody or its antibody fragment results in inhibition of inflammasome activation in the subject. In some cases, the administration of the monoclonal antibody or its antibody fragment results in reduced ASC activity compared to the control. In some cases, the control is an untreated subject. In some cases, the administration is via intracerebroventricular, intraperitoneal, intravenous or by inhalation. In some cases, a method for treating multiple sclerosis (MS) in a subject is provided herein, the method comprising administering a therapeutically effective amount of the monoclonal antibody or its antibody fragment to the subject, thereby treating MS in the subject. In some cases, the administration of the monoclonal antibody or its antibody fragment reduces the level of at least inflammatory cytokines. In some cases, the administration of the monoclonal antibody or its antibody fragment results in inhibition of inflammasome activation in the subject. In some cases, the administration of the monoclonal antibody or its antibody fragment results in reduced ASC activity compared to the control. In some cases, the control is an untreated subject. In some cases, the administration is intracerebroventricularly, intraperitoneally, intravenously, or by inhalation.

[0014] In another aspect, the present invention provides a monoclonal antibody or antibody fragment thereof that specifically binds to ASC, wherein the antibody or the antibody fragment comprises a light chain variable (VL) region and a heavy chain variable (VH) region, wherein the VL region amino acid sequence comprises LCDR1 of SEQ ID NO: 12, LCDR2 of SEQ ID NO: 13, and LCDR3 of SEQ ID NO: 14, or a variant thereof having at least one amino acid substitution in LCDR1, LCDR2, and / or LCDR3. In some cases, the VL region amino acid sequence comprises SEQ ID NO: 28, 29, 30, 31, or an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence of SEQ ID NO: 28, 29, 30, or 31. In some cases, the ASC is a human ASC protein. In some cases, the antibody fragment is Fab, F(ab')2, Fab', scFv, a single domain antibody, a double antibody, or a single chain camel antibody. In some cases, the monoclonal antibody or its antibody fragment is human, humanized or chimeric. In some cases, a nucleic acid molecule encoding the monoclonal antibody or its antibody fragment is provided herein. In some cases, an expression vector comprising the nucleic acid molecule is provided herein. In some cases, a nucleic acid molecule is provided herein, which is operably connected to a regulatory sequence suitable for expressing a nucleic acid segment in a host cell. In some cases, a recombinant host cell comprising the expression vector is provided herein. On the other hand, a method for producing an antibody or antibody fragment specifically bound to ASC is provided herein, the method comprising: culturing a recombinant host cell comprising the expression vector under conditions of expressing the nucleic acid molecule, thereby producing the monoclonal antibody or its antibody fragment specifically bound to ASC. In some cases, a pharmaceutical composition is provided herein, which comprises the monoclonal antibody or its antibody fragment and a pharmaceutically acceptable carrier, diluent or excipient. In some cases, a method for treating inflammation in a subject is provided herein, the method comprising administering a therapeutically effective amount of the monoclonal antibody or its antibody fragment to the subject, thereby treating inflammation in the subject. In some cases, the administration of the monoclonal antibody or its antibody fragment reduces the level of at least inflammatory cytokines. In some cases, the inflammation is an inflammasome-related inflammation. In some cases, the inflammasome-related inflammation is associated with central nervous system (CNS) damage, autoimmune disease, autoinflammatory disease or neurodegenerative disease. In some cases, the CNS damage is selected from traumatic brain injury (TBI), stroke and spinal cord injury (SCI).In some cases, the autoimmune disease or neurodegenerative disease is amyotrophic lateral sclerosis (ALS), Alzheimer's disease, Parkinson's disease, muscular dystrophy (MD), systemic lupus erythematosus, lupus nephritis, rheumatoid arthritis, inflammatory bowel disease (e.g., Crohn's disease and ulcerative colitis) or multiple sclerosis (MS). In some cases, the inflammasome-associated inflammation is related to a metabolic disease or disorder. In some cases, the metabolic disease is metabolic syndrome, obesity, diabetes, diabetic nephropathy or diabetic kidney disease (DKD), insulin resistance, atherosclerosis, lipid deposition disorders, glycogen storage disease, medium-chain acyl-CoA dehydrogenase deficiency, nonalcoholic fatty liver disease (e.g., nonalcoholic steatohepatitis (NASH)) and gout. In some cases, the autoinflammatory disease is cryopyrin-associated periodic syndrome (CAPS). CAPS may include familial cold autoinflammatory syndrome (FCAS), Muckle-Wells syndrome (MWS) and neonatal multisystem inflammatory disease (NOMID). In some cases, the administration of the monoclonal antibody or its antibody fragment results in inhibition of inflammasome activation in the subject. In some cases, the administration of the monoclonal antibody or its antibody fragment results in reduced ASC activity compared to the control. In some cases, the control is an untreated subject. In some cases, the administration is via intracerebroventricular, intraperitoneal, intravenous or by inhalation. In some cases, a method for treating multiple sclerosis (MS) in a subject is provided herein, the method comprising administering a therapeutically effective amount of the monoclonal antibody or its antibody fragment to the subject, thereby treating MS in the subject. In some cases, the administration of the monoclonal antibody or its antibody fragment reduces the level of at least inflammatory cytokines. In some cases, the administration of the monoclonal antibody or its antibody fragment results in inhibition of inflammasome activation in the subject. In some cases, the administration of the monoclonal antibody or its antibody fragment results in reduced ASC activity compared to the control. In some cases, the control is an untreated subject. In some cases, the administration is intracerebroventricularly, intraperitoneally, intravenously, or by inhalation.

[0015] In yet another aspect, the present invention provides a monoclonal antibody or an antibody fragment thereof that specifically binds to ASC, wherein the antibody or the antibody fragment comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region amino acid sequence comprises HCDR1 of SEQ ID NO: 6, HCDR2 of SEQ ID NO: 7, and HCDR3 of SEQ ID NO: 8, or a variant thereof having at least one amino acid substitution in HCDR1, HCDR2, and / or HCDR3; and wherein the VL region amino acid sequence comprises LCDR1 of SEQ ID NO: 12, LCDR2 of SEQ ID NO: 13, and LCDR3 of SEQ ID NO: 14, or a variant thereof having at least one amino acid substitution in LCDR1, LCDR2, and / or LCDR3. In some cases, the VH region amino acid sequence comprises SEQ ID NO: 18, 19, 20, 21, 22, or an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence of SEQ ID NO: 18, 19, 20, 21, or 22; and wherein the VL region amino acid sequence comprises SEQ ID NO: 28, 29, 30, 31, or an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence of SEQ ID NO: 28, 29, 30, or 31. In some cases, the VH region amino acid sequence comprises SEQ ID NO: 18, or an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence of SEQ ID NO: 18; and wherein the VL region amino acid sequence comprises SEQ ID NO: 28, or an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence of SEQ ID NO: 28. In some cases, the VH region amino acid sequence comprises SEQ ID NO: 18, or an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence of SEQ ID NO: 18; and wherein the VL region amino acid sequence comprises SEQ ID NO: 29, or an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence of SEQ ID NO: 29. In some cases, the VH region amino acid sequence comprises SEQ ID NO: 18, or an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence of SEQ ID NO: 18; and wherein the VL region amino acid sequence comprises SEQ ID NO: 30, or an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence of SEQ ID NO: 30.In some cases, the VH region amino acid sequence comprises SEQ ID NO: 18, or an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence of SEQ ID NO: 18; and wherein the VL region amino acid sequence comprises SEQ ID NO: 31, or an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence of SEQ ID NO: 31. In some cases, the VH region amino acid sequence comprises SEQ ID NO: 19, or an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence of SEQ ID NO: 19; and wherein the VL region amino acid sequence comprises SEQ ID NO: 28, or an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence of SEQ ID NO: 28. In some cases, the VH region amino acid sequence comprises SEQ ID NO: 19, or an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence of SEQ ID NO: 19; and wherein the VL region amino acid sequence comprises SEQ ID NO: 29, or an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence of SEQ ID NO: 29. In some cases, the VH region amino acid sequence comprises SEQ ID NO: 19, or an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence of SEQ ID NO: 19; and wherein the VL region amino acid sequence comprises SEQ ID NO: 30, or an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence of SEQ ID NO: 30. In some cases, the VH region amino acid sequence comprises SEQ ID NO: 19, or an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence of SEQ ID NO: 19; and wherein the VL region amino acid sequence comprises SEQ ID NO: 31, or an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence of SEQ ID NO: 31. In some cases, the VH region amino acid sequence comprises SEQ ID NO: 20, or an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence of SEQ ID NO: 20; and wherein the VL region amino acid sequence comprises SEQ ID NO: 28, or an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence of SEQ ID NO: 28.In some cases, the VH region amino acid sequence comprises SEQ ID NO: 20, or an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence of SEQ ID NO: 20; and wherein the VL region amino acid sequence comprises SEQ ID NO: 29, or an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence of SEQ ID NO: 29. In some cases, the VH region amino acid sequence comprises SEQ ID NO: 20, or an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence of SEQ ID NO: 20; and wherein the VL region amino acid sequence comprises SEQ ID NO: 30, or an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence of SEQ ID NO: 30. In some cases, the VH region amino acid sequence comprises SEQ ID NO: 20, or an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence of SEQ ID NO: 20; and wherein the VL region amino acid sequence comprises SEQ ID NO: 31, or an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence of SEQ ID NO: 31. In some cases, the VH region amino acid sequence comprises SEQ ID NO: 21, or an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence of SEQ ID NO: 21; and wherein the VL region amino acid sequence comprises SEQ ID NO: 28, or an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence of SEQ ID NO: 28. In some cases, the VH region amino acid sequence comprises SEQ ID NO: 21, or an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence of SEQ ID NO: 21; and wherein the VL region amino acid sequence comprises SEQ ID NO: 29, or an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence of SEQ ID NO: 29. In some cases, the VH region amino acid sequence comprises SEQ ID NO: 21, or an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence of SEQ ID NO: 21; and wherein the VL region amino acid sequence comprises SEQ ID NO: 30, or an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence of SEQ ID NO: 30.In some cases, the VH region amino acid sequence comprises SEQ ID NO: 21, or an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence of SEQ ID NO: 21; and wherein the VL region amino acid sequence comprises SEQ ID NO: 31, or an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence of SEQ ID NO: 31. In some cases, the VH region amino acid sequence comprises SEQ ID NO: 22, or an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence of SEQ ID NO: 22; and wherein the VL region amino acid sequence comprises SEQ ID NO: 28, or an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence of SEQ ID NO: 28. In some cases, the VH region amino acid sequence comprises SEQ ID NO: 22, or an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence of SEQ ID NO: 22; and wherein the VL region amino acid sequence comprises SEQ ID NO: 29, or an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence of SEQ ID NO: 29. In some cases, the VH region amino acid sequence comprises SEQ ID NO: 22, or an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence of SEQ ID NO: 22; and wherein the VL region amino acid sequence comprises SEQ ID NO: 30, or an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence of SEQ ID NO: 30. In some cases, the VH region amino acid sequence comprises SEQ ID NO: 22 or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 22; and wherein the VL region amino acid sequence comprises SEQ ID NO: 31 or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 31. In some cases, the ASC is a human ASC protein. In some cases, the antibody fragment is Fab, F(ab')2, Fab', scFv, a single domain antibody, a double antibody or a single chain camel antibody. In some cases, the monoclonal antibody or its antibody fragment is human, humanized or chimeric. In some cases, an isolated nucleic acid molecule encoding the monoclonal antibody or its antibody fragment is provided herein. In some cases, an expression vector comprising the nucleic acid molecule is provided herein.In some cases, nucleic acid molecules are provided herein, which are operably connected to regulatory sequences suitable for expressing nucleic acid segments in host cells. In some cases, a recombinant host cell comprising the expression vector is provided herein. On the other hand, a method for producing an antibody or antibody fragment specifically bound to ASC is provided herein, the method comprising: culturing a recombinant host cell comprising the expression vector under conditions expressing the nucleic acid molecule, thereby producing the monoclonal antibody or its antibody fragment specifically bound to ASC. In some cases, a pharmaceutical composition is provided herein, which comprises the monoclonal antibody or its antibody fragment and a pharmaceutically acceptable carrier, diluent or excipient. In some cases, a method for treating inflammation in a subject is provided herein, the method comprising administering a therapeutically effective amount of the monoclonal antibody or its antibody fragment to the subject, thereby treating inflammation in the subject. In some cases, the administration of the monoclonal antibody or its antibody fragment reduces the level of at least inflammatory cytokines. In some cases, the inflammation is an inflammasome-related inflammation. In some cases, the inflammasome-related inflammation is associated with central nervous system (CNS) damage, autoimmune disease, autoinflammatory disease or neurodegenerative disease. In some cases, the CNS damage is selected from traumatic brain injury (TBI), stroke and spinal cord injury (SCI). In some cases, the autoimmune disease or neurodegenerative disease are amyotrophic lateral sclerosis (ALS), Alzheimer's disease, Parkinson's disease, muscular dystrophy (MD), systemic lupus erythematosus, lupus nephritis, rheumatoid arthritis, inflammatory bowel disease (e.g., Crohn's disease and ulcerative colitis) or multiple sclerosis (MS). In some cases, the inflammasome-related inflammation is related to metabolic disease or disorder. In some cases, the metabolic disease is metabolic syndrome, obesity, diabetes, diabetic nephropathy or diabetic kidney disease (DKD), insulin resistance, atherosclerosis, lipid deposition disorder, glycogen storage disease, medium chain acyl-CoA dehydrogenase deficiency, non-alcoholic fatty liver disease (e.g., non-alcoholic steatohepatitis (NASH)) and gout. In some cases, the autoinflammatory disease is cryopyrin-associated periodic syndrome (CAPS). CAPS may include familial cold autoinflammatory syndrome (FCAS), Muckle-Wells syndrome (MWS) and neonatal multisystem inflammatory disease (NOMID). In some cases, the administration of the monoclonal antibody or its antibody fragment results in inhibition of inflammasome activation in the subject. In some cases, compared with the control, the administration of the monoclonal antibody or its antibody fragment results in reduced ASC activity. In some cases, the control is an untreated subject.In some cases, the administration is via intraventricular, intraperitoneal, intravenous or by inhalation. In some cases, a method for treating multiple sclerosis (MS) in a subject is provided herein, the method comprising administering a therapeutically effective amount of the monoclonal antibody or its antibody fragment to the subject, thereby treating the MS in the subject. In some cases, the administration of the monoclonal antibody or its antibody fragment reduces the level of at least inflammatory cytokines. In some cases, the administration of the monoclonal antibody or its antibody fragment results in inhibition of inflammasome activation in the subject. In some cases, compared with the control, the administration of the monoclonal antibody or its antibody fragment results in reduced ASC activity. In some cases, the control is an untreated subject. In some cases, the administration is via intraventricular, intraperitoneal, intravenous or by inhalation. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1A-Figure 1N Shown is inflammasome activation in the cortex and lung tissues of C57 / BL6 mice after TBI. Figure 1A Representative immunoblots of active caspase-1, ASC, IL-18, IL-β, HMGB1, and AIM2 after TBI are shown. At 4 and 24 h after TBI, active caspase-1 ( Figure 1B )、ASC( Figure 1C )、IL-18( Figure 1D )、HMGB1( Figure 1E )、AIM 2( Figure 1F ) and IL-β( Figure 1G ) were significantly increased in cortical tissue. Data are expressed as mean + / - SEM; ****p<0.001, ***p<0.01, **p<0.01, *p<0.05 compared with sham operation. N=4-5 / group. Figure 1H Representative immunoblots of active caspase-1, ASC, IL-18, IL-β, HMGB1, and AIM2 in lung tissue are shown. I, J, K, L, M, N) Active caspase-1 (ASC) was expressed in 4 and 24 h after TBI. Fig. 1I )、ASC( Figure 1J )、IL-18( Figure 1K )、HMGB1( Figure 1L )、AIM 2( Figure 1M ) and IL-β( Figure 1N ) were significantly increased in lung tissue. Data are expressed as mean + / - SEM. N = 4-5 / group, ****p<0.001, ***p<0.01, **p<0.01, *p<0.05 compared with sham operation.

[0017] Figure 2A-2CShown is the expression of inflammasome proteins in type II alveolar epithelial cells. Figure 2A AIM2 is shown. Figure 2B showed active caspase-1 and Figure 2C Shown is the increase in ASC immunoreactivity in lung tissues after CCI (4h, 24h) compared to mice. Confocal images of AIM2, caspase-1 and ASC (green) and type II epithelial cells (surfactant protein C, red).

[0018] Figure 3A-3E It was shown that TBI increased nuclear and cytoplasmic HMGB1 expression in mouse lungs. Figure 3A Representative immunoblots of nuclear HMGB1 after TBI are shown. Figure 3B It was shown that nuclear HMGB1 was significantly elevated in 4-hour injured animals compared to sham-operated animals. Figure 3C Representative immunoblots of cytoplasmic HMGB1 after TBI are shown. Figure 3D Cytoplasmic HMGB1 is shown to be significantly elevated in 4-hour injured animals compared to sham. Data are presented as mean + / - SEM; *p<0.05 compared to sham. N=4-5 / group. Figure 3E Shown is an increase in HMGB1 immunoreactivity in lung tissue after CCI when compared to sham-operated mice. Confocal images of HMGB1 and type II epithelial cells (surfactant protein C, red).

[0019] Figure 4A-4C Shown is the formation of pyroptotic bodies in mouse lungs 4 hours after TBI. Figure 4A Shown is a TBI-induced ASC ladder in lung tissue, indicating the formation of pyroptotic bodies (oligomerization of ASC dimers leading to caspase-1 activation and pyroptosis). Figure 4B Representative immunoblots are shown and Figure 4C Quantification of scopolamine is shown. Scopolamine-D was significantly elevated in lung tissue after TBI. Data are presented as mean + / - SEM. N = 4-5 / group, **p < 0.01 compared to sham surgery.

[0020] Figure 5A-Figure 5B Shown are the alveolar morphological changes and acute lung injury in TBI-induced mice. Figure 5A H&E staining of lung sections from sham and injured animals at 4h and 24h is shown. Sections show evidence of neutrophil infiltration (arrows), morphological changes in the alveolar capillary membrane (asterisk, *), interstitial edema (short arrows), and evidence of interstitial and alveolar septal thickening (#). Figure 5BShown are significant increases in acute lung injury scores in injured animals at 4h and 24h when compared to sham surgery. Data are presented as mean + / - SEM. N = 4-5 / group, *p < 0.05 compared to sham surgery.

[0021] Figure 6 Shown is the expression of CD81 in serum-derived EVs from control and TBI-injured mice. Representative immunoblot of CD81 in serum-derived EVs from sham-operated controls and TBI-injured mice.

[0022] Figure 7A-7G Adoptive transfer of EVs from TBI animals was shown to induce caspase-1 and ASC in the lungs of uninjured mice. Fig. 7A Representative immunoblots are shown, showing that caspase-1 ( Figure 7B )、ASC( Figure 7C )、IL-18( Fig.7D )、AIM2( Fig. 7E )、HMGB1( Figure 7F ) were elevated in the lungs of animals that received EVs isolated from TBI mice. Data are presented as mean + / - SEM; *p < 0.05 compared with sham surgery. N = 3 / group. EVs from TBI mice induced alveolar morphological changes (reduced alveolar size) and infiltration of inflammatory cells, as determined by H&E staining ( Figure 7G ). The ALI scores of EVs delivered from injured mice were significantly increased compared to those of uninjured mice ( Figure 7G ). Data are expressed as mean + / - SEM; **p<0.01, *p<0.05 compared with the intact group.

[0023] Figure 8A-8F It was shown that treatment with enoxaparin (3 mg / kg) and IC 100 (5 mg / kg) reduced inflammasome expression in the lungs of animals delivered EVs from injured mice. Fig. 8A Representative immunoblots are shown, showing that caspase-1 ( Figure 8B )、ASC( Figure 8C ), IL-1β( Fig.8D )、AIM2( Fig. 8E )、HMGB1( Fig.8F ) were reduced in the lungs of animals treated with enoxaparin and IC 100. Data are presented as mean + / - SEM; ****p<0.001, ***p<0.01, **p<0.01, *p<0.05 compared to sham. N=4 / group.

[0024] Figure 9A-9EIt was shown that treatment with enoxaparin (3 mg / kg) and IC 100 (5 mg / kg) reduced ALI scores in the lungs of animals delivered EVs from injured mice. Figure 9A-9D Shown are H&E staining of lung sections from: saline ( Fig. 9A ), untreated( Fig. 9B ), Enoxaparin ( Fig. 9C ) and IC 100 (anti-ASC; Fig.9D ) Mouse lungs treated with EVs delivered from injured animals. Sections showed evidence of neutrophil infiltration, changes in alveolar capillary membrane morphology, interstitial edema, and thickening of the interstitium and alveolar septa. Fig.9E It is shown that animals treated with enoxaparin, IC 100, had significantly reduced acute lung injury scores when compared to untreated animals. Data are presented as mean + / - SEM. N=4 / group, ****p<0.01, *p<0.05.

[0025] Figures 10A-10F showed that delivery of serum-derived EVs from TBI patients resulted in increased inflammasome protein expression in lung endothelial cells. Fig. 10A Shown are Western blot representations of caspase-1, ASC, AIM2, HMGB1 in PMVECs after 4 h of incubation with TBI-EVs and control EVs. Figure 10B-10E Quantification of Western blots is shown, n = 3 filters / group, n = 6 patients, t-test, ****p<0.001, ***p<0.01, **p<0.01, *p<0.05. Fig.10F Shown are immunoassay results for significant increase in IL-1β expression using the Ella simple plex assay (n=3 filters / group, n=6 patients, t-test, ****p<0.001, ***p<0.01, **p<0.01, *p<0.05).

[0026] Figure 11A-11C It was shown that delivery of TBI-EVs to lung endothelial cells increased active caspase-1 immunoreactivity and cell death. Fig.11A Caspase-1 FLICA and PI staining and colocalization of PMVECs incubated with TBI-EVs for 4 h are shown. Fig. 11B Shown are caspase-1 FLICA and PI staining in PMVECs incubated with control EVs for 4 h. Fig. 11C Fluorescence plate reader analysis of PMVECs incubated with TBI and control EVs for 4 h is shown. n=6, ***p<0.05.

[0027] Figure 12A-12ETreatment with a humanized anti-ASC monoclonal antibody (ie, IC 100) was shown to improve functional outcomes in EAE. Fig. 12A Shown is the expression of MOG in C57BL / 6 mice treated with vehicle or increasing doses of IC100 35-55 Clinical course of induced EAE. Before mice showed signs of paralysis, IC 100 (10, 30 and 45 mg / Kg intraperitoneally, once every 4 days) was administered starting on day 8. Results are expressed as the average clinical score ± SEM of 9-10 mice / group per day. The 30 and 45 mg / Kg curves were significantly different from the vehicle curve; **p≤0.001, Mann-Whitney test. Fig. 12B Comparison of peak clinical scores (highest disease score reached by mice) between groups is shown; *p≤0.05, Student's t-test. Fig. 12C Comparison of the cumulative disease index (CDI) between groups is shown. CDI is equal to the sum of all scores from the day of onset of disease in each animal and is a measure of the severity of EAE; *p≤0.05, Student's t-test. Fig.12D Comparison of the disease onset day between groups is shown. The disease onset day was considered the day when mice showed the first EAE symptoms. Fig.12E Comparison of peak disease day between groups is shown. Peak disease day is the day when mice reach the highest disease score. Onset day is considered the day when mice show first EAE symptoms.

[0028] Figure 13A-13B IC100 treatment reduces the infiltration of peripheral immune cells into the spinal cord or spleen after EAE. Fig.13A ) or in the spleen ( Fig. 13B ) were quantified by flow cytometry. The results are shown as mean ± SEM of 5 mice / group, *p<0.05, **p<0.001, Student's t test.

[0029] Fig.14 IC100 treatment reduces microglia in the spinal cord after EAE. Total microglia and MHCII in the spinal cord at 35 dpi after EAE + Activated microglia were quantified by flow cytometry. The results are presented as mean ± SEM of 5 mice / group, *p < 0.05, Student's t-test.

[0030] Fig.15 IC100 concentrations in tissues are shown. Concentrations of IC100 in brain, spinal cord, liver and spleen in control mice and mice treated with 10, 30 and 45 mg / kg of IC100 at 35 dpi after EAE (in pg / ml). Data are shown as mean ± SEM. N = 2-10 mice / group.

[0031] Fig.16 IC100 was shown to be taken up into ASC specks in unstimulated THP-1 cells and uptake was increased by inflammasome activation.

[0032] Fig.17 The IC100 for blocking IL1-β release from THP-1 cells is shown.

[0033] Fig.18 Confocal images of spinal cord neurons are shown, illustrating the penetration of anti-ASC antibody (IC 100) into spinal cord neurons.

[0034] Fig.19 A comparison of the binding of three different antibodies against human ASC to different classes of antibodies is shown.

[0035] Fig. 20 Shown is induction of inflammasomes by nicotine (500 nM) in human nucleus pulposus cells treated with three different anti-human ASC antibodies (H1, H2, H3) and two anti-mouse ASC antibodies (M1, M2). H1 was most effective in preventing IL-1β release / inflammasome activation.

[0036] Fig.21 Raw BLI kinetic analysis data for anti-ASC monoclonal antibodies are shown.

[0037] Fig. 22 Shown is a comparison of the kinetics of three different anti-human ASC antibodies. DETAILED DESCRIPTION

[0038] definition

[0039] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0040] The section headings used herein are only for organizational purposes and should not be interpreted as limiting the subject matter described. All documents or portions of documents cited herein (including but not limited to patents, patent applications, articles, books and papers) are hereby expressly incorporated by reference in their entirety for any purpose. If one or more incorporated documents or portions of documents define a term that contradicts the definition of the term in this application, the definition that appears in this application shall prevail. However, any reference, article, publication, patent, patent publication and patent application cited herein are not and should not be regarded as an admission or any form of suggestion that they constitute effective prior art or form part of the common knowledge of any country in the world.

[0041] The term "a" or "an" refers to one or more of the entity, i.e., may refer to a plural referent. Thus, the terms "a" or "a kind", "one or more", and "at least one" are used interchangeably herein. Furthermore, unless the context clearly requires one and only one element, reference to "an element" by the indefinite article "a" or "an" does not exclude the possibility that more than one element is present.

[0042] Unless the context requires otherwise, throughout this specification and claims, the word "comprise" and variations such as "comprises" and "comprising" are to be interpreted in an open, inclusive sense, i.e., "including, but not limited to." The use of alternatives (e.g., "or") should be understood to mean one, both, or any combination of the alternatives. As used herein, unless otherwise indicated, the terms "about" and "consisting essentially of" mean + / - 20% of the indicated range, value, or structure.

[0043] References throughout this specification to "one embodiment" or "an embodiment" mean that a particular feature, structure, or characteristic described in conjunction with the embodiment may be included in at least one embodiment of the present disclosure. Thus, the phrases "in one embodiment" or "in an embodiment" appearing in various places throughout this specification may not necessarily all refer to the same embodiment. It should be understood that certain features of the present disclosure that are described in the context of separate embodiments for clarity may also be provided in combination in a single embodiment. Conversely, various features of the present disclosure that are described in the context of a single embodiment for brevity may also be provided separately or in any suitable subcombination.

[0044] Throughout this disclosure, the various aspects of the methods and compositions provided herein can be presented in a range format. It should be understood that the description of the range format is only for convenience and brevity, and should not be construed as a rigid limitation on the scope of the present invention. Therefore, it should be considered that the description of the range specifically discloses all possible sub-ranges and each numerical value in the range. For example, the description of the range (such as from 1 to 6) should be considered to have particularly disclosed a sub-range (such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc.) and a single number in the range, such as 1, 2, 3, 4, 5 and 6. Regardless of the width of the scope, this applies.

[0045] As used herein, "protein" and "polypeptide" are used synonymously to refer to any peptide-linked chain of amino acids, regardless of length or post-translational modifications (eg, glycosylation or phosphorylation).

[0046] As used herein, the term "antibody" generally and broadly refers to immunoglobulin (Ig) molecules and immunologically active portions or fragments of immunoglobulin molecules, i.e., molecules containing an antigen binding site that specifically binds to (immunoreacts with) an antigen (e.g., ASC, NLRP1, AIM2, etc.). The antibodies provided herein may be polyclonal antibodies, monoclonal antibodies (mAbs), chimeric antibodies, humanized antibodies, anti-idiotypic (anti-Id) antibodies directed against antibodies that can be labeled in soluble or bound form, and active fragments, regions, or derivatives thereof. The antibodies used for the purposes herein may be chimeric, humanized, or human.

[0047] "Specific binding" or "immunoreactive with" means that the antibody reacts with one or more antigenic determinants of the desired antigen and does not react with other polypeptides. In certain embodiments, an antibody is said to specifically bind to an antigen when it preferentially recognizes its target antigen in a complex mixture of proteins and / or macromolecules. The term "antibody" broadly refers to an immunoglobulin (Ig) molecule, or any functional fragment, mutant, variant or derivative thereof that retains the basic target binding characteristics of an Ig molecule, wherein the immunoglobulin molecule typically comprises four polypeptide chains, namely two heavy (H) chains and two light (L) chains. Such mutant, variant or derivative antibody forms are known in the art. Such anti-ASC and anti-NLRP1 antibodies of the present invention are capable of binding to portions of ASC and NLRP1 that interfere with caspase-1 activation, respectively.

[0048] As used herein, the term "humanized antibody" refers to an antibody in which a minimal portion of a non-human antibody is introduced into an original human antibody.

[0049] As used herein, the term "human antibody" refers to an antibody in which substantially every portion of the protein is substantially non-immunogenic in humans, with only minor sequence alterations or variations.

[0050] In a full-length antibody, each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region comprises three domains, CH1, CH2 and CH3. Each light chain comprises a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region comprises one domain CL. The VH region and the VL region can be further subdivided into regions with high variability, called complementary determining regions (CDRs), interspersed with more conservative regions, called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Immunoglobulin molecules can belong to any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY) and category (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass. IgG, IgD and IgE antibodies generally contain two identical heavy chains and two identical light chains and two antigen binding domains, each of which is composed of a heavy chain variable region (VH) and a light chain variable region (VL). Generally speaking, IgA antibodies are composed of two monomers, each of which is composed of two heavy chains and two light chains (such as for IgG, IgD and IgE antibodies); in this way, the IgA molecule has four antigen binding domains, each of which is also composed of VH and VL. Some IgA antibodies are monomeric because they are composed of two heavy chains and two light chains. Secretory IgM antibodies are generally composed of five monomers, each of which is composed of two heavy chains and two light chains (such as for IgG and IgE antibodies); in this way, the IgM molecule has ten antigen binding domains, each of which is also composed of VH and VL. There is also a cell surface form of IgM, and it has a two heavy chain / two light chain structure similar to IgG, IgD and IgE antibodies.

[0051] As used herein, the term "antigen binding fragment" or "antigen binding portion" or "antigen binding site" or "binding domain" or "binding region" may refer to a domain, region, part or site of a protein, polypeptide, oligopeptide or peptide, or an antibody or a binding domain derived from an antibody that retains the ability to specifically bind to an antigen (e.g., ASC protein). Exemplary binding domains include single-chain antibody variable regions (e.g., domain antibodies, sFv, scFv, scFab), fusion proteins comprising antibody portions (e.g., domain antibodies), receptor extracellular domains, and ligands (e.g., cytokines, chemokines). In one embodiment, the fusion protein comprises one or more CDRs. In another embodiment, the fusion protein comprises CDR H3 (VHCDR3) and / or CDR L3 (VL CDR3). For the purposes of the present invention, the fusion protein contains one or more antibodies and additional amino acid sequences, such as a heterologous sequence or homologous sequence from another region attached to the N- or C-terminus of the antibody or its antibody fragment. Exemplary heterologous sequences include, but are not limited to, "tags" (such as FLAG tags or 6His tags) or enzymes or polypeptides that increase the half-life of the antibody in the blood. Tags are well known in the art. The additional amino acid sequences that may include amino- and / or carboxyl-terminal fusions may range in length from one residue to polypeptides containing one hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues.

[0052] The antigen binding site can generally be formed by heavy chain variable region (VH) and light chain variable region (VL) immunoglobulin domains, wherein the antigen binding interface is formed by six surface polypeptide loops called complementary determining regions (CDRs). There are three CDRs in each of VH (HCDR1, HCDR2, HCDR3) and VL (LCDR1, LCDR2, LCDR3), and a framework region (FR). In certain embodiments, the binding domain comprises or consists of an antigen binding site (e.g., comprising variable heavy chain sequences and variable light chain sequences or three light chain complementary determining regions (CDRs) and three heavy chain CDRs from an antibody placed in alternating framework regions (FRs) (e.g., human FRs optionally comprising one or more amino acid substitutions).

[0053] The term "CDR region" or "CDR" may refer to a hypervariable region of an immunoglobulin heavy or light chain as defined by Kabat et al., 1991 (Kabat, EA et al., (1991) Sequences of Proteins of Immunological Interest, 5th Edition US Department of Health and Human Services, Public Service, NIH, Washington) and later versions. An antibody typically contains three heavy chain CDRs and three light chain CDRs.

[0054] It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Antibody and antibody fragment embodiments can also be in bispecific, trispecific, dual-specific or multispecific formats; specifically binding to two or more different antigens. Examples of binding fragments encompassed by the term "antigen-binding fragment" of an antibody include: (i) a Fab fragment consisting of the VL, VH, CL and CH1 domains (Ward, ES et al., (1989) Nature 341, 544-546); (ii) a Fd fragment consisting of the VH and CH1 domains (McCafferty et al., (1990) Nature, 348, 552-554); (iii) a Fv fragment consisting of the VL and VH domains of a single antibody (Holt et al., (2003) Trends in Biotechnology 21, 484-490); (iv) a dAb fragment (Ward, ES et al., Nature 341, 544-546 (1989), McCafferty et al., (1990) Nature, 348, 552-554, Holt et al., (2003) Trends in Biotechnology 21,484-490), which consists of a VH or VL domain; (v) an isolated CDR region; (vi) a F(ab')2 fragment, i.e., a bivalent fragment comprising two connected Fab fragments; (vii) a single-chain Fv molecule (scFv), in which the VH domain and the VL domain are connected by a peptide linker that allows the two domains to associate to form an antigen binding site (Bird et al., (1988) Science, 242,423-426, Huston et al., (1988) PNAS USA, 85,5879-5883). The present invention also includes Fab' fragments. In addition, although the two domains VL and VH of the Fv fragment are encoded by separate genes, they can be joined using recombinant methods through synthetic linkers to make them a single protein chain, in which the VL region and the VH region are paired to form a monovalent molecule (called a single-chain Fv (scFv)). Such single-chain antibodies are also intended to be included in the term "antigen-binding fragment" of an antibody. In certain embodiments of the invention, scFv molecules can be incorporated into fusion proteins. In some embodiments, the invention includes single-chain camelid antibodies; (viii) bispecific single-chain Fv dimers (PCT / US 92109965) and (ix) "diabodies", multivalent or multispecific fragments constructed by gene fusion (WO 94 / 13804; Holliger, P. (1993) et al., Proc. Natl. Acad. Sci. USA 90 6444-6448).Diabodies are bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but a linker that is too short to allow pairing between the two domains on the same chain is used, thereby forcing the domains to pair with the complementary domains of another chain and produce two antigen binding sites (see, e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6444-6448; Poljak, RJ, et al. (1994) Structure 2: 1121-1123). Such antibody binding fragments are known in the art (Kontermann and Dubel, ed., Antibody Engineering (2001) Springer-Verlag. New York p. 790). In some aspects, the present invention includes single domain antibodies. In general, when used herein, the term "antibody" includes "antibody fragments". Antibody fragments generally retain the antigen binding properties of full-length antibodies.

[0055] Fv, scFv or diabody molecules can be stabilized by incorporating disulfide bridges connecting the VH and VL domains (Reiter, Y. et al., Nature Biotech, 14, 1239-1245, 1996). Minibodies comprising scFv connected to the CH3 domain can also be prepared (Hu, S. et al., (1996) Cancer Res., 56, 3055-3061). Other examples of binding fragments may be Fab', which differs from the Fab fragment by adding a small number of residues at the carboxyl terminus of the heavy chain CH1 domain, the small number of residues including one or more cysteines from the antibody hinge region; and may be Fab'-SH, which is a Fab' fragment in which one or more cysteine ​​residues of the constant domain carry a free thiol group.

[0056] As used herein, "Fv" may refer to the smallest fragment of an antibody that retains both antigen recognition and antigen binding sites. As used herein, "Fab" may refer to a fragment of an antibody that contains the constant domain of the light chain and the CH1 domain of the heavy chain. The term "mAb" refers to a monoclonal antibody.

[0057] "Fc region" or "Fc domain" refers to a polypeptide sequence corresponding to or derived from a portion of a source antibody that is responsible for binding to antibody receptors on cells and the C1q component of complement. Fc stands for "fragment crystallization," i.e., antibody fragments that readily form protein crystals. The different protein fragments, originally described by proteolytic digestion, can define the overall general structure of immunoglobulin proteins. As originally defined in the literature, the Fc fragment consists of a heavy chain hinge region, CH2, and CH3 domains linked by disulfides. However, the term has recently been applied to a single chain consisting of CH3, CH2, and at least a portion of a hinge that is sufficient to form a disulfide-linked dimer with a second such chain. For a review of immunoglobulin structure and function, see Putnam, The Plasma Proteins, Vol. V (Academic Press, Inc., 1987), pp. 49-140; and Padlan, Mol. Immunol. 31: 169-217, 1994. As used herein, the term Fc includes variants of naturally occurring sequences. In one embodiment, the antibody provided herein or the antibody fragment derived therefrom (e.g., anti-ASC monoclonal antibody or its antibody fragment) has a modified Fc region or domain. In some cases, the modified Fc region or domain can confer increased thermal stability to the resulting antibody or the antibody fragment derived therefrom. Increased thermal stability can lead to increased serum half-life. The Fc region or domain can be modified as described in US20160193295, and its contents are incorporated herein by reference. As described in US20160193295, the Fc region or domain can be modified to have a deletion of one or more cysteine ​​residues in the hinge region and a substitution of a sulfhydryl-containing residue of one or more CH3 interface amino acids. In another embodiment, the Fc region or domain of the antibody or antibody fragment derived therefrom provided herein (e.g., an anti-ASC monoclonal antibody or an antibody fragment thereof) can be stabilized by engineering the Fc region to have intradomain disulfide bonds as described in Wozniak-Knopp G, Stadlmann J, Rüker F (2012) Stabilisation of the Fc Fragment of Human IgG1 by Engineered Intradomain Disulfide Bonds. PLoS ONE 7 (1): e30083, the contents of which are incorporated herein by reference. In yet another embodiment, the antibody has an Fc region modified as described in WO 99 / 58572, the contents of which are incorporated herein by reference. In still other embodiments, the Fc region or domain can be modified as described in US9574010, the contents of which are incorporated herein by reference.

[0058] The terms "apoptosis-associated speck-like protein containing a caspase activation recruitment domain (CARD)" and "ASC" mean the expression product of the ASC gene or its isoform, or a protein having at least 65%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity with ASC (e.g., NP_037390 (Q9ULZ3-1), NP_660183 (Q9ULZ3-2) or Q9ULZ3-3 in humans, NP_075747 in mice or NP_758825 (BAC43754) in rats) and showing the functional activity of ASC. The "functional activity" of a protein is any activity associated with the physiological function of the protein. The functional activity of ASC includes, for example, recruiting proteins to activate caspase-1 and triggering cell death.

[0059] The term "ASC gene" or "ASC nucleic acid" means a natural nucleic acid sequence encoding ASC, a genomic sequence from which ASC cDNA can be transcribed, and / or allelic variants and homologs of the foregoing. The term includes double-stranded DNA, single-stranded DNA and RNA.

[0060] As used herein, the term "inflammasome" means a multi-protein (e.g., at least two proteins) complex that activates caspase-1. In addition, the term "inflammasome" may refer to a multi-protein complex that activates caspase-1 activity and regulates the processing and activation of IL-1β, IL-18, and IL-33. See Arend et al. 2008; Li et al. 2008; and Martinon et al. 2002, each of which is incorporated by reference in its entirety. The term "NLRP1 inflammasome", "NALP1 inflammasome", "NLRP2 inflammasome", "NALP2 inflammasome", "NLRP3 inflammasome", "NALP3 inflammasome", "NLRC4 inflammasome", "IPAF inflammasome" or "AIM2 inflammasome" means a protein complex of at least caspase-1 and an adaptor protein (e.g., ASC). For example, the terms "NLRP1 inflammasome" and "NALP1 inflammasome" may refer to a multiprotein complex containing NLRP1, ASC, caspase-1, caspase-11, XIAP, and pan-linker protein-1 for activating caspase-1 and processing interleukin-1β, interleukin-18, and interleukin-33. The terms "NLRP2 inflammasome" and "NALP2 inflammasome" may refer to a multiprotein complex containing NLRP2 (also known as NALP2), ASC, and caspase-1, while the terms "NLRP3 inflammasome" and "NALP3 inflammasome" may refer to a multiprotein complex containing NLRP3 (also known as NALP3), ASC, and the terms "NLRC4 inflammasome" and "IPAF inflammasome" may refer to a multiprotein complex containing NLRC4 (also known as IPAF), ASC, and caspase-1. In addition, the term "AIM2 inflammasome" may mean a multiprotein complex including AIM2, ASC, and caspase-1.

[0061] As used herein, the phrase "sequence identity" means the percentage of identical subunits at corresponding positions in two sequences (e.g., nucleic acid sequences, amino acid sequences) when the two sequences are aligned to maximize subunit matching (i.e., taking into account gaps and insertions). Sequence identity can be measured using sequence analysis software (e.g., sequence analysis software packages from Accelrys CGC, San Diego, CA).

[0062] The phrases "therapeutically effective amount" and "effective dose" mean an amount sufficient to produce a desired result for treatment (e.g., clinical); the exact nature of the result will vary depending on the nature of the disorder being treated. For example, in the case where the disorder to be treated is SCI, the result may be improvements in motor skills and motor function, reduced spinal cord lesions, etc. The compositions described herein may be administered once or more per day to once or more per week. Those skilled in the art will appreciate that certain factors may affect the dosage and timing required for effective treatment of the subject, including, but not limited to, the severity of the disease or disorder, previous treatment, the general health and / or age of the subject, and other diseases present. In addition, treatment of a subject with a therapeutically effective amount of the composition of the present invention may include a single treatment or a series of treatments.

[0063] As used herein, the term "treatment" is defined as the application or administration of a therapeutic agent described herein or identified by the methods described herein to a patient having a disease, disease symptom, or disease predisposition, or to an isolated tissue or cell line from a patient, with the purpose of treating, curing, alleviating, relieving, altering, remedying, ameliorating, alleviating, or affecting the disease, disease symptom, or disease predisposition.

[0064] The terms "patient," "subject," and "individual" are used interchangeably herein and refer to a mammalian subject to be treated. In one embodiment, the mammalian patient is a human. In some cases, the methods of the present invention can be used in experimental animals, veterinary applications, and the development of animal models of disease, including but not limited to rodents (including mice, rats, and hamsters) and primates.

[0065] As used interchangeably herein, "melanoma deficiency factor 2" and "AIM2" may refer to the expression product of the AIM2 gene or isoform; or a protein having at least 65%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to AIM2 (e.g., Accession Nos. NX_014862, NP004824, XP016858337, XP005245673, AAB81613, BAF84731, AAH10940) and exhibiting the functional activity of AIM2.

[0066] As used interchangeably herein, "NACHT, LRR and PYD domain-containing protein 1", "NALP1" and "NLRP1" mean the expression product of the NALP1 or NLRP1 gene or isoform; or a protein having at least 65%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to NALP1 (e.g., Accession Nos. AAH51787, NP_001028225, NP_127500, NP_127499, NP_127497, NP055737) and exhibiting NALP1 functional activity.

[0067] As used interchangeably herein, "NALP2" and "NLRP2" mean the expression product of a NALP2 or NLRP2 gene or isoform; or a protein having at least 65%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to NALP2 (e.g., Accession Nos. NP_001167552, NP_001167553, NP_001167554 or NP_060322) and exhibiting NALP2 functional activity.

[0068] As used interchangeably herein, "NALP3" and "NLRP3" mean the expression product of a NALP3 or NLRP3 gene or isoform; or a protein having at least 65%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to NALP3 (e.g., Accession Nos. NP_001073289, NP_001120933, NP_001120934, NP_001230062, NP_004886, NP_899632, XP_011542350, XP_016855670, XP_016855671, XP_016855672 or XP_016855673) and exhibiting NALP3 functional activity.

[0069] As used interchangeably herein, "NLRC4" and "IPAF" mean the expression product of a NLRC4 or IPAF gene or isoform; or a protein that shares at least 65%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity with NLRC4 (e.g., Accession Nos. NP_001186067, NP001186068, NP_001289433 or NP_067032) and exhibits NLRC4 functional activity.

[0070] The terms "stroke" and "ischemic stroke" refer to a condition when blood flow to a part of the brain or spinal cord is interrupted.

[0071] "Traumatic injury to the CNS" refers to any injury to the CNS by external mechanical forces that may result in permanent or temporary impairment of CNS function.

[0072] Methods involving conventional molecular biology techniques are described herein. Such techniques are generally known in the art and are described in detail in methodological papers, such as the following documents: Molecular Cloning: A Laboratory Manual, 3rd Edition, Volumes 1-3, Editors Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001; and Current Protocols in Molecular Biology, Editors Ausubel et al., Greene Publishing and Wiley-Interscience, New York, 1992 (regularly updated). Immunological techniques are generally known in the art and are described in detail in the following methodological treatises: e.g., Advances in Immunology, Vol. 93, Frederick W. Alt, ed., Academic Press, Burlington, MA, 2007; Making and Using Antibodies: A Practical Handbook, Gary C. Howard and Matthew R. Kaser, eds., CRC Press, Boca Raton, FL, 2006; Medical Immunology, 6th ed., Gabriel Virella, ed., Informa Healthcare Press, London, UK, 2007; and Harlow and Lane ANTIBODIES: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1988.

[0073] Although compositions and methods similar or equivalent to those compositions and methods described herein can be used in the practice or testing of the present invention, suitable compositions and methods are described below. All publications, patent applications and patents mentioned herein are incorporated by reference in their entirety. In the event of a conflict, the present specification including definitions shall prevail. The specific embodiments discussed below are illustrative only and are not intended to be restrictive.

[0074] Overview

[0075] Provided herein are monoclonal antibodies or antibody fragments thereof that specifically bind to apoptosis-associated speck-like proteins (ASC) containing caspase activation recruitment domains. The monoclonal antibody or its fragment can specifically bind to an antigenic fragment of ASC, and the antigenic fragment of ASC is composed of or substantially composed of the amino acid sequence of SEQ ID NO.5. With respect to this embodiment, the present invention contemplates the use of the monoclonal antibody or its antibody fragment in a method for treating inflammation in a subject. The inflammation can be innate immune inflammation. The inflammation can be inflammasome-associated inflammation. In one embodiment, the monoclonal antibody or its antibody fragment provided herein can be used in a method for reducing mammalian inflammation as described in US 8,685,400, the contents of which are incorporated herein by reference in their entirety. The inflammation may be in the lungs and / or the central nervous system (CNS). Inflammation in the lungs and / or CNS may be the result of damage (e.g., traumatic brain injury (TBI) or spinal cord injury (SCI)) or a disease, disorder or suffering of the CNS or a disease, disorder or suffering that affects the CNS. As provided herein, the disease, illness or suffering of CNS or the disease, illness or suffering affecting CNS can be stroke and autoimmune disease and / or CNS disease, including amyotrophic lateral sclerosis (ALS) (Lou Gehrig's disease), multiple sclerosis (MS), immune dysfunction muscle CNS collapse, muscular dystrophy (MD), Alzheimer's disease (AD), Parkinson's disease (PD). In the method for treating inflammation, the monoclonal antibody or its antibody fragment can be used to reduce the inflammation in the CNS and / or lung of the patient. The reduction can be compared with a control (e.g., an untreated patient and / or a patient before treatment). In one embodiment, the monoclonal antibody or the antibody fragment derived therefrom is applied to a patient suffering from or suspected of suffering from MS, and the monoclonal antibody or the antibody fragment derived therefrom is used to treat MS. The monoclonal antibody or its antibody fragment of this embodiment can be present in a composition (e.g., as a pharmaceutical composition provided herein). In some cases, in the method for treating inflammation provided herein, the monoclonal antibody or its fragment is used in combination with one or more other agents. The additional agent can be any agent provided herein (e.g., an EV uptake inhibitor) and / or an antibody or antibody fragment directed against other inflammasome components (e.g., IL-18, caspase-1, NALP1, AIM2, etc.).

[0076] The present invention also includes a monoclonal antibody or an antibody fragment thereof that specifically binds to ASC, wherein the antibody or the antibody fragment comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region amino acid sequence comprises HCDR1 of SEQ ID NO:6, HCDR2 of SEQ ID NO:7, and HCDR3 of SEQ ID NO:8, or a variant thereof having at least one amino acid substitution in HCDR1, HCDR2, and / or HCDR3. With respect to this embodiment, the present invention contemplates the use of the monoclonal antibody or its antibody fragment in a method for treating inflammation in a subject. The inflammation may be an innate immune inflammation. The inflammation may be an inflammasome-associated inflammation. In one embodiment, the monoclonal antibody or its antibody fragment provided herein can be used in a method for reducing mammalian inflammation as described in US 8,685,400, the contents of which are incorporated herein by reference in their entirety. The inflammation may be in the lungs and / or CNS. Inflammation in the lungs and / or CNS may be the result of an injury (e.g., traumatic brain injury (TBI) or spinal cord injury (SCI)) or a disease, disorder, or pain of the CNS or a disease, disorder, or pain that affects the CNS. As provided herein, the disease, illness or suffering of CNS or the disease, illness or suffering affecting CNS can be stroke and autoimmune disease and / or CNS disease, including amyotrophic lateral sclerosis (ALS) (Lou Gehrig's disease), multiple sclerosis (MS), immune dysfunction muscle CNS collapse, muscular dystrophy (MD), Alzheimer's disease (AD), Parkinson's disease (PD). The use of the monoclonal antibody or its antibody fragment in the method for treating inflammation can reduce the inflammation in the CNS and / or lung of the patient. The use of the monoclonal antibody or its antibody fragment in the method for treating inflammation can reduce the innate immune inflammation or inflammasome-related inflammation of the patient. The reduction can be compared with a control (e.g., an untreated patient and / or a patient before treatment). In one embodiment, the monoclonal antibody or the antibody fragment derived therefrom is used to treat central nervous system (CNS) damage and / or autoimmune disease, autoinflammatory disease, metabolic disease or neurodegenerative disease. The CNS damage can be selected from traumatic brain injury (TBI), stroke and spinal cord injury (SCI). The autoimmune disease or neurodegenerative disease can be selected from amyotrophic lateral sclerosis (ALS), Alzheimer's disease, Parkinson's disease (PD), muscular dystrophy (MD), systemic lupus erythematosus, lupus nephritis, rheumatoid arthritis, inflammatory bowel disease (e.g. (PD), Crohn's disease and ulcerative colitis) and multiple sclerosis (MS). In one embodiment, the monoclonal antibody or the antibody fragment derived therefrom is used to treat MS by administering the monoclonal antibody or the antibody fragment derived therefrom to a patient suffering from or suspected of suffering from MS.In one embodiment, by administering the monoclonal antibody or an antibody fragment derived therefrom to a patient suffering from or suspected of having PD, the monoclonal antibody or an antibody fragment derived therefrom is used to treat PD. In one embodiment, by administering the monoclonal antibody or an antibody fragment derived therefrom to a patient suffering from or suspected of having lupus nephritis, the monoclonal antibody or an antibody fragment derived therefrom is used to treat lupus nephritis. The metabolic disease may be selected from metabolic syndrome, obesity, diabetes, diabetic nephropathy or diabetic kidney disease (DKD), insulin resistance, atherosclerosis, lipid deposition disorders, glycogen storage disease, medium chain acyl-CoA dehydrogenase deficiency, non-alcoholic fatty liver disease (e.g., non-alcoholic steatohepatitis (NASH)) and gout. In one embodiment, by administering the monoclonal antibody or an antibody fragment derived therefrom to a patient suffering from or suspected of having diabetic nephropathy, the monoclonal antibody or an antibody fragment derived therefrom is used to treat diabetic nephropathy. In one embodiment, the monoclonal antibody or the antibody fragment derived therefrom is applied to a patient suffering from or suspected of having NASH, and the monoclonal antibody or the antibody fragment derived therefrom is used to treat NASH. The autoinflammatory disease may be cold pyrine-associated periodic syndrome (CAPS). CAPS may include familial cold autoinflammatory syndrome (FCAS), Muckle-Wells syndrome (MWS) and neonatal onset multisystem inflammatory disease (NOMID). In one embodiment, the monoclonal antibody or the antibody fragment derived therefrom is applied to a patient suffering from or suspected of having CAPS, and the monoclonal antibody or the antibody fragment derived therefrom is used to treat CAPS. The monoclonal antibody or its antibody fragment of this embodiment may be present in a composition (e.g., a pharmaceutical composition as provided herein). In some cases, in the methods of treatment provided herein, the monoclonal antibody or its fragment is used in combination with one or more other agents. The additional agent can be any agent provided herein (e.g., an EV uptake inhibitor) and / or an antibody or antibody fragment directed against other inflammasome components (e.g., IL-18, caspase-1, NALP1, AIM2, etc.).

[0077] In some embodiments, the present invention provides a monoclonal antibody or an antibody fragment thereof that specifically binds to ASC, wherein the antibody or the antibody fragment comprises a light chain variable (VL) region and a heavy chain variable (VH) region, wherein the VL region amino acid sequence comprises LCDR1 of SEQ ID NO: 12, LCDR2 of SEQ ID NO: 13, and LCDR3 of SEQ ID NO: 14, or a variant thereof having at least one amino acid substitution in LCDR1, LCDR2, and / or LCDR3. With respect to this embodiment, the present invention contemplates the use of the monoclonal antibody or its antibody fragment in a method for treating inflammation in a subject. The inflammation may be an innate immune inflammation. The inflammation may be an inflammasome-associated inflammation. In one embodiment, the monoclonal antibody or its antibody fragment provided herein can be used in a method for reducing mammalian inflammation as described in US8,685,400, the contents of which are incorporated herein by reference in their entirety. The inflammation may be in the lungs and / or CNS. Inflammation in lung and / or CNS may be the result of damage (e.g., traumatic brain injury (TBI) or spinal cord injury (SCI)) or disease, illness or suffering of CNS or disease, illness or suffering affecting CNS. As provided herein, disease, illness or suffering of CNS or disease, illness or suffering affecting CNS may be stroke and autoimmune disease and / or CNS disease, including amyotrophic lateral sclerosis (ALS) (Lou Gehrig's disease), multiple sclerosis (MS), immune dysfunction muscle CNS collapse, muscular dystrophy (MD), Alzheimer's disease (AD), Parkinson's disease (PD). In the method for treating inflammation, the monoclonal antibody or its antibody fragment can be used to reduce the inflammation in the CNS and / or lung of the patient. The use of the monoclonal antibody or its antibody fragment in the method for treating inflammation can reduce the innate immune inflammation of the patient or the inflammation-related inflammation of the inflammasome. The reduction can be compared with a control (e.g., an untreated patient and / or a patient before treatment). In one embodiment, the monoclonal antibody or the antibody fragment derived therefrom is used to treat central nervous system (CNS) damage and / or autoimmune disease, autoinflammatory disease, metabolic disease or neurodegenerative disease. The CNS damage can be selected from traumatic brain injury (TBI), stroke and spinal cord injury (SCI). The autoimmune disease or neurodegenerative disease can be selected from amyotrophic lateral sclerosis (ALS), Alzheimer's disease, Parkinson's disease (PD), muscular dystrophy (MD), systemic lupus erythematosus, lupus nephritis, rheumatoid arthritis, inflammatory bowel disease (e.g. (PD), Crohn's disease and ulcerative colitis) and multiple sclerosis (MS). In one embodiment, by applying the monoclonal antibody or the antibody fragment derived therefrom to a patient suffering from or suspected of suffering from MS, the monoclonal antibody or the antibody fragment derived therefrom is used to treat MS.In one embodiment, by administering the monoclonal antibody or an antibody fragment derived therefrom to a patient suffering from or suspected of having PD, the monoclonal antibody or an antibody fragment derived therefrom is used to treat PD. In one embodiment, by administering the monoclonal antibody or an antibody fragment derived therefrom to a patient suffering from or suspected of having lupus nephritis, the monoclonal antibody or an antibody fragment derived therefrom is used to treat lupus nephritis. The metabolic disease may be selected from metabolic syndrome, obesity, diabetes, diabetic nephropathy or diabetic kidney disease (DKD), insulin resistance, atherosclerosis, lipid deposition disorders, glycogen storage disease, medium chain acyl-CoA dehydrogenase deficiency, non-alcoholic fatty liver disease (e.g., non-alcoholic steatohepatitis (NASH)) and gout. In one embodiment, by administering the monoclonal antibody or an antibody fragment derived therefrom to a patient suffering from or suspected of having diabetic nephropathy, the monoclonal antibody or an antibody fragment derived therefrom is used to treat diabetic nephropathy. In one embodiment, the monoclonal antibody or the antibody fragment derived therefrom is applied to a patient suffering from or suspected of having NASH, and the monoclonal antibody or the antibody fragment derived therefrom is used to treat NASH. The autoinflammatory disease may be cold pyrine-associated periodic syndrome (CAPS). CAPS may include familial cold autoinflammatory syndrome (FCAS), Muckle-Wells syndrome (MWS) and neonatal onset multisystem inflammatory disease (NOMID). In one embodiment, the monoclonal antibody or the antibody fragment derived therefrom is applied to a patient suffering from or suspected of having CAPS, and the monoclonal antibody or the antibody fragment derived therefrom is used to treat CAPS. The monoclonal antibody or its antibody fragment of this embodiment may be present in a composition (e.g., a pharmaceutical composition as provided herein). In some cases, in the methods of treatment provided herein, the monoclonal antibody or its fragment is used in combination with one or more other agents. The additional agent can be any agent provided herein (e.g., an EV uptake inhibitor) and / or an antibody or antibody fragment directed against other inflammasome components (e.g., IL-18, caspase-1, NALP1, AIM2, etc.).

[0078] In other embodiments, the present invention also provides a monoclonal antibody or an antibody fragment thereof that specifically binds to ASC, wherein the antibody or the antibody fragment comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region amino acid sequence comprises HCDR1 of SEQ ID NO: 6, HCDR2 of SEQ ID NO: 7, and HCDR3 of SEQ ID NO: 8, or a variant thereof having at least one amino acid substitution in HCDR1, HCDR2, and / or HCDR3; and wherein the VL region amino acid sequence comprises LCDR1 of SEQ ID NO: 12, LCDR2 of SEQ ID NO: 13, and LCDR3 of SEQ ID NO: 14, or a variant thereof having at least one amino acid substitution in LCDR1, LCDR2, and / or LCDR3. With respect to this embodiment, the present invention contemplates the use of the monoclonal antibody or its antibody fragment in a method for treating inflammation in a subject. The inflammation may be innate immune inflammation. The inflammation may be inflammasome-associated inflammation. In one embodiment, the monoclonal antibody or its antibody fragment provided herein can be used for the method for reducing mammalian inflammation as described in US 8,685,400, and its content is incorporated herein by reference in its entirety. The inflammation may be in the lung and / or CNS. The inflammation in the lung and / or CNS may be the result of damage (e.g., traumatic brain injury (TBI) or spinal cord injury (SCI)) or the disease, illness or suffering of the CNS or the disease, illness or suffering affecting the CNS. As provided herein, the disease, illness or suffering of the CNS or the disease, illness or suffering affecting the CNS can be apoplexy and autoimmune diseases and / or CNS diseases, including amyotrophic lateral sclerosis (ALS) (Lou Gehrig's disease), multiple sclerosis (MS), immune dysfunction muscle CNS collapse, muscular dystrophy (MD), Alzheimer's disease (AD), Parkinson's disease (PD). In the method for treating inflammation, the monoclonal antibody or its antibody fragment can be used to reduce the inflammation in the CNS and / or lung of the patient. The use of the monoclonal antibody or its antibody fragment in the method for treating inflammation can reduce the innate immune inflammation or inflammasome-related inflammation of the patient. The reduction can be compared to a control (e.g., an untreated patient and / or a patient before treatment). In one embodiment, the monoclonal antibody or antibody fragment derived therefrom is used to treat central nervous system (CNS) damage and / or autoimmune disease, autoinflammatory disease, metabolic disease or neurodegenerative disease. The CNS damage can be selected from traumatic brain injury (TBI), stroke and spinal cord injury (SCI).The autoimmune disease or neurodegenerative disease can be selected from amyotrophic lateral sclerosis (ALS), Alzheimer's disease, Parkinson's disease (PD), muscular dystrophy (MD), systemic lupus erythematosus, lupus nephritis, rheumatoid arthritis, inflammatory bowel disease (e.g., (PD), Crohn's disease and ulcerative colitis) and multiple sclerosis (MS). In one embodiment, the monoclonal antibody or the antibody fragment derived therefrom is used to treat MS by administering the monoclonal antibody or the antibody fragment derived therefrom to a patient suffering from or suspected of suffering from MS. In one embodiment, the monoclonal antibody or the antibody fragment derived therefrom is used to treat PD by administering the monoclonal antibody or the antibody fragment derived therefrom to a patient suffering from or suspected of suffering from PD. In one embodiment, the monoclonal antibody or the antibody fragment derived therefrom is used to treat lupus nephritis by administering the monoclonal antibody or the antibody fragment derived therefrom to a patient suffering from or suspected of suffering from lupus nephritis. The metabolic disease can be selected from metabolic syndrome, obesity, diabetes, diabetic nephropathy or diabetic kidney disease (DKD), insulin resistance, atherosclerosis, lipid deposition disorders, glycogen storage disease, medium chain acyl-CoA dehydrogenase deficiency, non-alcoholic fatty liver disease (e.g., non-alcoholic steatohepatitis (NASH)) and gout. In one embodiment, the monoclonal antibody or the antibody fragment derived therefrom is administered to a patient suffering from or suspected of having diabetic nephropathy, and the monoclonal antibody or the antibody fragment derived therefrom is used to treat diabetic nephropathy. In one embodiment, the monoclonal antibody or the antibody fragment derived therefrom is administered to a patient suffering from or suspected of having NASH, and the monoclonal antibody or the antibody fragment derived therefrom is used to treat NASH. The autoinflammatory disease can be cold pyrine-associated periodic syndrome (CAPS). CAPS can include familial cold autoinflammatory syndrome (FCAS), Muckle-Wells syndrome (MWS) and neonatal multisystem inflammatory disease (NOMID). In one embodiment, the monoclonal antibody or antibody fragment derived therefrom is used to treat CAPS by administering the monoclonal antibody or antibody fragment derived therefrom to a patient suffering from or suspected of having CAPS. The monoclonal antibody or antibody fragment thereof of this embodiment may be present in a composition (e.g., a pharmaceutical composition such as provided herein). In some cases, in the methods of treatment provided herein, the monoclonal antibody or fragment thereof is used in combination with one or more other agents. The other agent may be any agent provided herein (e.g., an EV uptake inhibitor) and / or an antibody or antibody fragment directed against other inflammasome components (e.g., IL-18, caspase-1, NALP1, AIM2, etc.).

[0079] Provided herein are compositions and methods for reducing innate immune inflammation or inflammasome-related inflammation.In some cases, the inflammasome-related inflammation is in the CNS of a mammal that has been subjected to or suffered from the following illness, and the illness causes or causes innate immune inflammation or inflammasome-related inflammation.In some cases, the inflammasome-related inflammation is in a mammal (e.g., people) that suffers or suspects suffering from the following illness, and the illness is related to innate immune inflammation or inflammasome-related inflammation, causes or causes innate immune inflammation or inflammasome-related inflammation.Causing or causing the illness of innate immune inflammation or inflammasome-related inflammation can be CNS damage, autoimmune disease or disorder, autoinflammatory disease or disorder, neurodegenerative disease or disorder and / or metabolic disease or disorder.The CNS damage can be selected from traumatic brain injury (TBI), stroke and spinal cord injury (SCI). The autoimmune disease or neurodegenerative disease can be selected from amyotrophic lateral sclerosis (ALS), Alzheimer's disease, Parkinson's disease (PD), muscular dystrophy (MD), systemic lupus erythematosus, lupus nephritis, rheumatoid arthritis, inflammatory bowel disease (e.g. (PD), Crohn's disease and ulcerative colitis) and multiple sclerosis (MS). The metabolic disease can be selected from metabolic syndrome, obesity, diabetes, diabetic nephropathy or diabetic kidney disease (DKD), insulin resistance, atherosclerosis, lipid deposition disorder, glycogen storage disease, medium chain acyl-CoA dehydrogenase deficiency, non-alcoholic fatty liver disease (e.g., non-alcoholic steatohepatitis (NASH)) and gout. The autoinflammatory disease can be cold pyrine associated periodic syndrome (CAPS). CAPS can include familial cold autoinflammatory syndrome (FCAS), Muckle-Wells syndrome (Muckle-Wells syndrome, MWS) and neonatal onset multisystem inflammatory disease (NOMID). The compositions and methods described herein may include antibodies or active fragments thereof as provided herein that specifically bind to at least one component (e.g., ASC) of a mammalian inflammasome, and / or compounds that regulate (e.g., inhibit or reduce) extracellular vesicle (EV) uptake; and used as treatments for CNS inflammation in mammals. Examples of conditions that may cause inflammation in the CNS include CNS injury (e.g., spinal cord injury (SCI), traumatic brain injury (TBI) or stroke), neurodegenerative diseases, autoimmune diseases (e.g., MS), asthma, chronic obstructive pulmonary disease (COPD), cystic fibrosis, interstitial lung disease, or acute respiratory distress syndrome. The composition may be administered in a therapeutically effective amount. A therapeutically effective amount may be a dose as provided herein. The agent may be an extracellular vesicle (EV) uptake inhibitor, and / or an antibody or active fragment thereof as provided herein that binds to a component of an inflammasome, or a combination thereof. The composition may be administered by any suitable route, for example, by inhalation, intravenous, intraperitoneal, or intracerebroventricular administration.The composition may further include at least one pharmaceutically acceptable carrier or diluent.

[0080] Provided herein are compositions and methods for treating MS in a subject suffering from or suspected of having multiple sclerosis (MS). The method provided herein for treating MS may include administering a composition (e.g., pharmaceutical composition) comprising a medicament (e.g., IC 100) to a subject suffering from or suspected of having MS. Multiple sclerosis (MS) is an autoimmune disease involving the brain and spinal cord. The subject may present clinical symptoms consistent with MS. The subject may be diagnosed with any type of MS known in the art. MS may be relapsing-remitting MS (RRMS), secondary progressive MS (SPMS), primary progressive MS (PPMS), or progressive relapsing MS (PRMS). Any method known in the art may be used or may have been used to determine MS diagnosis. In one embodiment, the subject has been diagnosed as suffering from MS using the method detailed in US 62 / 560,963 filed September 20, 2017, and the contents of the document are incorporated herein by reference in their entirety. The agent can be a standard care treatment for MS known in the art, an EV uptake inhibitor (e.g., any EV uptake inhibitor from Table 1), an antibody or antibody fragment thereof as provided herein that binds to a component of the inflammasome (e.g., an anti-ASC monoclonal antibody or an antibody fragment thereof, such as IC 100), or any combination thereof. The composition can be administered by any suitable route, for example, by inhalation, intravenous, intraperitoneal, or intracerebroventricular administration. The composition may further include at least one pharmaceutically acceptable carrier or diluent. The standard care treatment can be selected from therapies for improving disease outcomes, managing relapses, managing symptoms, or any combination thereof. The therapy for improving disease outcomes can be selected from beta-interferon, glatiramer acetate, fingolimod, teriflunomide, dimethyl fumarate, mitoxantrone, ocrelizumab, alemtuzumab, daclizumab, and natalizumab.

[0081] Provided herein are compositions and methods for treating PD in a subject suffering from or suspected of having Parkinson's disease (PD). The method provided herein for treating PD may include administering a composition (e.g., pharmaceutical composition) comprising a medicament (e.g., IC 100) to a subject suffering from or suspected of having PD. Parkinson's disease (PD) is a progressive nervous system disorder that affects movement due to the gradual decomposition and / or death of nerve cells in the brain of a mammal (e.g., a person) suffering from PD. The appearance and / or progression of PD can be through five phases (i.e., phases 1-5), and provided herein are compositions and methods that can be used to treat an individual suffering from or suspected of having PD in any one of the five phases. Any method known in the art can be used or can be used to determine PD diagnosis. In one embodiment, the subject has been diagnosed as suffering from PD using the method detailed in WO 2019 / 060516 filed on September 20, 2018, and the contents of the document are incorporated herein by reference in their entirety. The agent can be a standard care treatment for PD known in the art, an EV uptake inhibitor (e.g., any EV uptake inhibitor from Table 1), an antibody or antibody fragment thereof as provided herein that binds to a component of an inflammasome (e.g., an anti-ASC monoclonal antibody or an antibody fragment thereof, such as IC 100), or any combination thereof. The composition can be administered by any suitable route, for example, by inhalation, intravenous, intraperitoneal, or intracerebroventricular administration. The composition may further include at least one pharmaceutically acceptable carrier or diluent. Standard care treatment can be selected from carbidopa (Lodosyn), levodopa carbidopa-levodopa combination, Duopa, dopamine agonists, MAO B inhibitors, catechol O-methyltransferase (COMT) inhibitors, anticholinergic drugs, amantadine, and deep brain stimulation. The dopamine agonist can be selected from pramipexole (Lebok), ropinirole (Libiping) and rotigotine (Nupro). The MAO B inhibitor can be selected from selegiline (Zelapar), rasagiline (Rasagiline Tablets) and safinamide (Xadago). The COMT inhibitor can be selected from entacapone (Norcontin) and tolcapone (Tosmetofen). The anticholinergic drug can be selected from benztropine (Cogentin) or trihexyphenidyl.

[0082] Provided herein are compositions and methods for treating AD in a subject suffering from or suspected of having Alzheimer's disease (AD). The method provided herein for treating AD may include administering a composition (e.g., pharmaceutical composition) comprising a medicament (e.g., IC 100) to a subject suffering from or suspected of having AD. Alzheimer's disease (AD) is a progressive nervous system disorder that causes brain cell degeneration and death in individuals with AD, and progresses from mild cognitive impairment (MCI) to complete memory loss, and even personality and behavioral changes. Any method known in the art can be used or may have been used to determine AD diagnosis. In one embodiment, the subject has been diagnosed as suffering from AD using the method detailed in WO 2019 / 060516 submitted on September 20, 2018, and the contents of the document are incorporated herein by reference in their entirety. The agent can be a standard care treatment for AD known in the art, an EV uptake inhibitor (e.g., any EV uptake inhibitor from Table 1), an antibody or antibody fragment thereof as provided herein that binds to a component of the inflammasome (e.g., an anti-ASC monoclonal antibody or an antibody fragment thereof, such as, for example, IC 100), or any combination thereof. The composition can be administered by any suitable route, for example, by inhalation, intravenous, intraperitoneal, or intracerebroventricular administration. The composition may further include at least one pharmaceutically acceptable carrier or diluent. Standard care treatments can be selected from cholinesterase inhibitors and memantine (Namenda). The cholinesterase inhibitor can be selected from donepezil (Aricept), galantamine (Razadyne), and rivastigmine (Aceton).

[0083] Provided herein are compositions and methods for treating RA in subjects suffering from or suspected of having rheumatoid arthritis (RA). The method provided herein for treating RA may include administering a composition (e.g., pharmaceutical composition) comprising a medicament (e.g., IC 100) to a subject suffering from or suspected of having RA. Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disorder that causes damage to the joints, skin, eyes, lungs, heart, and blood vessels of an individual. Any method known in the art may be used or may have been used to determine RA diagnosis. In one embodiment, the subject has been diagnosed as suffering from RA using the method detailed in WO 2019 / 060516 filed on September 20, 2018, and the contents of the document are incorporated herein by reference in their entirety. The medicament may be a standard of care treatment for RA known in the art, an EV uptake inhibitor (e.g., any EV uptake inhibitor from Table 1), an antibody or its antibody fragment as provided herein (e.g., an anti-ASC monoclonal antibody or its antibody fragment, such as IC 100) or any combination thereof that is combined with a component of an inflammasome. The composition can be administered by any suitable route, for example, by inhalation, intravenous, intraperitoneal or intraventricular administration. The composition may further include at least one pharmaceutically acceptable carrier or diluent. The standard of care treatment may be selected from nonsteroidal anti-inflammatory drugs (NSAIDs), steroids (e.g., prednisone), anti-rheumatic drugs (DMARDs) and biological agents that improve the condition. NSAIDs may include ibuprofen (Ideville, Motrin IB) and naproxen sodium (naproxen). DMARDs may include methotrexate (Trexall, Otrexup, etc.), leflunomide (Ai Ruo Hua), hydroxychloroquine (Sai ​​Neng) and sulfasalazine (Azulfidine). Biologics can include abatacept (Original), adalimumab (Humira), anakinra (Kineret), baricitinib (Olumiant), certolizumab (Cimilon), etanercept (Enbrel), golimumab (Simpson), infliximab (Remicade), rituximab (MabThera), sarrelumab (Kevzara), tocilizumab (Antinib), and tofacitinib (Xeljanz).

[0084] Provided herein are compositions and methods for treating lupus nephritis in a subject suffering from or suspected of having lupus nephritis. The method provided herein for treating lupus nephritis may include administering a composition (e.g., pharmaceutical composition) comprising a medicament (e.g., IC 100) to a subject suffering from or suspected of having lupus nephritis. Lupus nephritis is a type of renal inflammation, which is usually a common complication of systemic lupus erythematosus, usually referred to as lupus. Lupus nephritis is an autoimmune disease, in which lupus autoantibodies affect the structure in the kidney of an individual, which may lead to renal inflammation and hematuria, proteinuria, hypertension, impaired renal function and even renal failure. Lupus nephritis diagnosis can be determined using or may have been determined using any method known in the art. In one embodiment, the subject has been diagnosed as suffering from lupus nephritis using the method detailed in WO 2019 / 060516 submitted on September 20, 2018, and the content of the document is incorporated herein by reference in its entirety. The agent may be a standard care treatment for lupus nephritis known in the art, an EV uptake inhibitor (e.g., any EV uptake inhibitor from Table 1), an antibody or antibody fragment thereof as provided herein that binds to a component of an inflammasome (e.g., an anti-ASC monoclonal antibody or an antibody fragment thereof, such as IC 100), or any combination thereof. The composition may be administered by any suitable route, for example, by inhalation, intravenous, intraperitoneal, or intracerebroventricular administration. The composition may further include at least one pharmaceutically acceptable carrier or diluent. Standard care treatments for lupus nephritis may include drugs to control blood pressure and / or a specific diet with low protein and low salt. In addition, standard care treatments for lupus nephritis may be treatments for lupus, such as nonsteroidal anti-inflammatory drugs (NSAIDs), antimalarials, corticosteroids (e.g., prednisone; methylprednisolone), immunosuppressants, or biologics. Examples of NSAIDs may include naproxen sodium (naproxen) and ibuprofen (Adville, Motrin IB, etc.). An example of an antimalarial drug may be hydroxychloroquine (Sene). Examples of immunosuppressants may include azathioprine (Azasan), mycophenolate mofetil (CellCept), and methotrexate (Trexall). Examples of biologics may include belimumab (Benlista) or rituximab (Rituxan).

[0085] Provided herein are compositions and methods for treating NASH in a subject suffering from or suspected of having nonalcoholic steatohepatitis (NASH). The method provided herein for treating NASH may include administering a composition (e.g., a pharmaceutical composition) comprising a medicament (e.g., IC 100) to a subject suffering from or suspected of having NASH. NASH is a type of nonalcoholic fatty liver disease (NAFLD). NAFLD is an umbrella term for a series of liver disorders that affect people who rarely drink or do not drink. The main feature of NAFLD is that too much fat is stored in hepatocytes and is marked by liver inflammation, which may progress to scarring and irreversible damage. This damage may be similar to the damage caused by drinking a lot of alcohol. In the most serious cases, nonalcoholic steatohepatitis may progress to cirrhosis and liver failure. Any method known in the art may be used or may have been used to determine NASH diagnosis. In one embodiment, the subject has been diagnosed as having NASH using the method detailed in WO 2019 / 060516 filed on September 20, 2018, and the contents of the document are incorporated herein by reference in their entirety. The agent can be a standard care treatment for NASH known in the art, an EV uptake inhibitor (e.g., any EV uptake inhibitor from Table 1), an antibody or antibody fragment thereof as provided herein that binds to a component of the inflammasome (e.g., an anti-ASC monoclonal antibody or an antibody fragment thereof, such as IC 100), or any combination thereof. The composition can be administered by any suitable route, for example, by inhalation, intravenous, intraperitoneal, or intracerebroventricular administration. The composition may further include at least one pharmaceutically acceptable carrier or diluent. Standard care treatments for NASH can include lifestyle changes (e.g., weight loss, increased exercise), avoiding drugs that damage the liver, lowering cholesterol, and / or managing diabetes.

[0086] Provided herein are compositions and methods for treating diabetic nephropathy in subjects with or suspected of having diabetic nephropathy. The method provided herein for treating diabetic nephropathy may include administering a composition (e.g., pharmaceutical composition) comprising a medicament (e.g., IC 100) to a subject with or suspected of having diabetic nephropathy. Diabetic nephropathy is a serious kidney-related complication of type 1 diabetes and type 2 diabetes, which may also be referred to as diabetic kidney disease (DKD). Any method known in the art may be used or may have been used to determine DKD diagnosis. In one embodiment, the subject has been diagnosed as having DKD using the method detailed in WO 2019 / 060516 filed on September 20, 2018, and the contents of the document are incorporated herein by reference in their entirety. The medicament may be a DKD standard of care treatment known in the art, an EV uptake inhibitor (e.g., any EV uptake inhibitor from Table 1), an antibody or its antibody fragment as provided herein (e.g., an anti-ASC monoclonal antibody or its antibody fragment, such as IC 100) or any combination thereof that is combined with a component of an inflammasome. The composition can be administered by any suitable route, for example, by inhalation, intravenous, intraperitoneal or intraventricular administration. The composition may further include at least one pharmaceutically acceptable carrier or diluent. Standard care treatments for diabetic nephropathy may include lifestyle changes (such as weight loss, increased exercise), lowering cholesterol, controlling protein in urine, cultivating bone health, controlling hypertension, managing diabetes, renal dialysis or transplantation.

[0087] Provided herein are compositions and methods for treating IBD in a subject suffering from or suspected of having inflammatory bowel disease (IBD).The method for treating IBD provided herein may include administering a composition (e.g., pharmaceutical composition) comprising a medicament (e.g., IC 100) to a subject suffering from or suspected of having IBD. IBD is an inclusive term for describing the obstruction of chronic inflammation of the digestive tract including an individual. IBD may include ulcerative colitis and Crohn's disease. Ulcerative colitis is a long-term inflammation and sores (ulcers) in the innermost layer of your large intestine (colon) and rectum, and Crohn's disease is characterized by inflammation of the inner layer of the digestive tract, which often spreads to the depths of the involved tissues. Any method known in the art may be used or may have been used to determine IBD diagnosis. In one embodiment, the subject has been diagnosed as suffering from IBD using the method detailed in WO 2019 / 060516 submitted on September 20, 2018, and the contents of the document are incorporated herein by reference in their entirety. The agent can be a standard care treatment for IBD known in the art, an EV uptake inhibitor (e.g., any EV uptake inhibitor from Table 1), an antibody or antibody fragment thereof as provided herein that binds to a component of an inflammasome (e.g., an anti-ASC monoclonal antibody or an antibody fragment thereof, such as IC 100), or any combination thereof. The composition can be administered by any suitable route, for example, by inhalation, intravenous, intraperitoneal, or intracerebroventricular administration. The composition may further include at least one pharmaceutically acceptable carrier or diluent. Standard care treatments for IBD may include anti-inflammatory drugs, immune system suppressants, antibiotics, antidiarrheal drugs, analgesics, iron supplements, and calcium and vitamin D supplements. Antibiotics may include ciprofloxacin (Cipro) and metronidazole (Flagyl). Examples of immunosuppressive drugs may include thiopurine (Azasan, Mikropurin), mercaptopurine (mercaptopurine, Purixan), cyclosporine (Gengraf, Neoral, Shandiming) and methotrexate (Trexall). Other examples of immunosuppressants can include tumor necrosis factor (TNF)-α inhibitors or biologics, such as infliximab (Remicade), adalimumab (Humira), golimumab (Simpson), natalizumab (Tysabri), vedolizumab (Anjiu), and ustekinumab (Sedano). Anti-inflammatory drugs can include corticosteroids and aminosalicylates, such as mesalamine (Asacol HD, Mesalazine), balsalazide (Colazal), and olsalazine (Dipentum).

[0088] Provided herein are compositions and methods for treating CAPS in a subject suffering from or suspected of suffering from cold pyrine-associated periodic syndrome (CAPS). The methods provided herein for treating CAPS may include administering a composition (e.g., a pharmaceutical composition) comprising a medicament (e.g., IC 100) to a subject suffering from or suspected of suffering from CAPS. Cold pyrine-associated periodic syndrome (CAPS), also known as cold pyrine-associated autoinflammatory syndrome, is composed of the following three autoinflammatory diseases associated with defects in the same gene (i.e., NLRP3): neonatal onset multisystem inflammatory disease (NOMID), Mu-Wei syndrome (MWS), and familial cold autoinflammatory syndrome (FCAS). NOMID is characterized by fever and inflammation in multiple organs. Early symptoms of NOMID may include a non-itchy honeycomb rash; inflammation of the membranes surrounding the brain, which causes headaches, blindness, or hearing loss; bulging eyes; and episodes of vomiting. After the age of 1, half of children with NOMID may develop joint pain and swelling. MWS is characterized by recurring symptoms, including rash, red eyes, joint pain, and severe headaches and vomiting. The attack lasts between one and three days. Hearing loss (which may be complete) usually occurs in adolescence. FCAS is characterized by fever, chills, nausea, extreme thirst, headache and joint pain. Any method known in the art can be used or may have been used to determine the CAPS diagnosis. In one embodiment, the subject has been diagnosed with CAPS using the method detailed in WO 2019 / 060516 filed on September 20, 2018, the contents of which are incorporated herein by reference in their entirety. The agent can be a standard of care treatment for CAPS known in the art, an EV uptake inhibitor (e.g., any EV uptake inhibitor from Table 1), an antibody or an antibody fragment thereof as provided herein (e.g., an anti-ASC monoclonal antibody or an antibody fragment thereof, such as IC 100), or any combination thereof that binds to a component of an inflammasome. The composition can be administered by any suitable route, for example, by inhalation, intravenous, intraperitoneal or intracerebroventricular administration. The composition may further include at least one pharmaceutically acceptable carrier or diluent. Standard-of-care treatment for CAPS can include biologics that target interleukin-1 as well as physical therapy, splints to treat joint deformities, and nonsteroidal anti-inflammatory drugs, corticosteroids, or methotrexate to reduce symptoms.

[0089] Provided herein are compositions and methods for reducing lung inflammation in mammals that have been subjected to a disorder that causes or causes lung inflammation or are afflicted with it. The compositions and methods described herein may include antibodies or active fragments thereof (e.g., IC 100) specifically bound to at least one component (e.g., ASC) of a mammalian inflammasome, and / or a compound that regulates (e.g., inhibits or reduces) extracellular vesicle (EV) uptake; and can be used as a treatment for lung inflammation in mammals.

[0090] Described herein is a method for reducing lung inflammation in a mammal suffering from a condition that causes and / or causes an inflammatory response in the lung. In one embodiment, a method for treating inflammation in a mammalian lung includes administering to a mammal a composition comprising an agent (e.g., IC 100) that inhibits inflammasome signaling. The mammal may be a patient or subject as provided herein. Examples of conditions that may cause inflammation in the lung include central nervous system (CNS) damage (e.g., spinal cord injury (SCI), traumatic brain injury (TBI) or stroke), neurodegenerative diseases, autoimmune diseases (e.g., MS), asthma, chronic obstructive pulmonary disease (COPD), cystic fibrosis, interstitial lung disease, or acute respiratory distress syndrome. The composition may be administered in a therapeutically effective amount. A therapeutically effective amount may be a dosage as provided herein. The agent may be an extracellular vesicle (EV) uptake inhibitor, an antibody or its active fragment as provided herein that is combined with a component of an inflammasome, or a combination thereof. The composition may be administered by any suitable route, for example, by inhalation, intravenous, intraperitoneal, or intraventricular administration. The composition may further include at least one pharmaceutically acceptable carrier or diluent.

[0091] Provided herein are compositions and methods for reducing renal inflammation in mammals that have been subjected to or suffered from the following conditions, which conditions cause or cause renal inflammation. Compositions and methods described herein may include antibodies or their active fragments (e.g., IC100) specifically bound to at least one component (e.g., ASC) of a mammalian inflammasome, and / or a compound that regulates (e.g., inhibits or reduces) extracellular vesicles (EV) uptake; and used as a treatment for renal inflammation in mammals.

[0092] Described herein is a method for reducing renal inflammation in a mammal suffering from a condition that causes and / or causes a renal inflammatory response. In one embodiment, a method for treating inflammation in the kidney of a mammal includes administering to the mammal a composition comprising an agent (e.g., IC 100) that inhibits inflammasome signaling. The mammal may be a patient or subject as provided herein. Examples of conditions that may cause inflammation in the kidney include lupus nephritis. The composition may be administered in a therapeutically effective amount. A therapeutically effective amount may be a dosage as provided herein. The agent may be an extracellular vesicle (EV) uptake inhibitor, an antibody or its active fragment as provided herein that is combined with a component of an inflammasome, or a combination thereof. The composition may be administered by any suitable route, for example, by inhalation, intravenous, intraperitoneal or intracerebroventricular administration. The composition may further include at least one pharmaceutically acceptable carrier or diluent.

[0093] In one embodiment, administration of an agent in the method provided herein (e.g., an antibody alone or in combination with, for example, an EV uptake inhibitor or an antibody fragment derived therefrom (e.g., IC 100)) can result in a decrease in the activity and / or expression level of a component of a mammalian inflammasome in the CNS, kidney, or lung of a subject. The decrease can be in lung cells (e.g., such as type II alveolar cells). The decrease can be compared to a control. The control can be a subject before the administration of the agent. The control can be the activity and / or expression level of one or more inflammasome components in a subject to which the agent is not administered. In one embodiment, administration of the agent results in a decrease in the activation of caspase-1 in at least the CNS or CNS cells of a subject. In one embodiment, administration of the agent results in a decrease in the activation of caspase-1 in at least the lungs or lung cells of a subject. In one embodiment, administration of the agent results in a decrease in the expression level of one or more inflammasome components (e.g., ASC, AIM2, NALP1, NALP2, NALP2, NALP3, or NLRC4) in at least the CNS or CNS cells of a subject. In one embodiment, administration of the agent results in a decrease in the expression level of one or more inflammasome components (e.g., ASC, AIM2, NALP1, NALP2, NALP2, NALP3, or NLRC4) in at least the lung or lung cells of the subject.

[0094] In another embodiment, administration of the agent (e.g., an antibody or antibody fragment derived therefrom, alone or in combination with, for example, an EV uptake inhibitor) can result in a reduction or elimination of acute lung injury (ALI). In one embodiment, the reduction in ALI is demonstrated by a reduction in the infiltration of neutrophils into the alveoli and / or interstitial spaces, a reduction or absence of alveolar septal thickening, or a combination thereof. The reduction can be compared to a control. The control can be the ALI of a subject before administration of the agent. The control can be the ALI of a subject with ALI who has not been administered the agent.

[0095] In still another embodiment, administration of the agent (e.g., an antibody or antibody fragment derived therefrom (e.g., IC 100) alone or in combination with, for example, an EV uptake inhibitor) can result in a reduction or elimination of pyroptosis in the CNS or lungs of a subject. Pyroptosis is a proinflammatory form of cell death that involves activation of caspase-1. Pyroptosis can be triggered by caspase-1-mediated cleavage of glucosamine D (GSDMD). In one embodiment, a reduction in pyroptosis is demonstrated by a reduction or lack of GSDMD cleavage in the lungs or lung cells (e.g., type II alveolar cells) of a subject. The reduction or elimination of pyroptosis can be compared to a control. The reduction or lack of GSDMD cleavage can be compared to a control. The control can be the level of pyroptosis in a subject before administration of the agent. The control can be the level of pyroptosis in a subject with pyroptosis who has not been administered the agent.

[0096] The success of the treatment methods provided herein (e.g., treatment of CNS injury, autoimmune disease, autoinflammatory disease, neurodegenerative disease or metabolic disease (e.g., MS, PD, lupus nephritis, NASH, DKD, CAPS, inflammatory bowel disease (IBD); AD, rheumatoid arthritis), innate immune inflammation or inflammasome-related inflammation, CNS inflammation and / or lung inflammation) or the response thereto can be monitored by measuring the level of at least one inflammasome protein. Therefore, in some embodiments, the treatment methods provided herein further include: measuring the level of at least one inflammasome protein in a biological sample obtained from a subject after treatment; preparing a therapeutic inflammasome protein signature associated with a positive response to the treatment, wherein the therapeutic protein signature comprises a reduced level of at least one inflammasome protein; and identifying a subject exhibiting the presence of the therapeutic protein signature as having a positive response to the treatment. A reduction in the level, abundance or concentration of one or more inflammasome proteins (e.g., ASC, IL-18 or caspase-1) indicates the efficacy of the treatment in the subject. One or more inflammasome proteins measured in a sample obtained after treatment may be the same or different from the inflammasome proteins measured in a sample obtained before treatment. Inflammasome protein levels may also be used to adjust the dosage or frequency of treatment. Inflammasome protein levels may be determined using methods and techniques provided herein or found in US 62 / 560,963 filed September 20, 2017.

[0097] In one embodiment, the agent administered in the treatment methods provided herein is an EV uptake inhibitor. The EV uptake inhibitor can be a compound, antisense RNA, siRNA, peptide, antibody or active fragment thereof, or a combination thereof as provided herein. The compound or peptide may be one or more compounds selected from the group consisting of heparin, α-difluoromethylornithine (DFMO), enoxaparin, asialofetuin, human receptor-associated protein (RAP), RGD (Arg-Gly-Asp) peptide, cytochalasin D, cytochalasin B, ethylenediaminetetraacetic acid (EDTA), Latrunculin A, Latrunculin B, NSC23766, Dynasore, chlorpromazine, 5-(N-ethyl-N-isopropyl) amiloride (EIPA), amiloride, bafilomycin A monensin and chloroquine, annexin-V, wortmannin, LY294002, methyl-β-cyclodextrin (MβCD), filipin, simvastatin, fumonisin B1 and N-butyldeoxynojirimycin hydrochloride, U0126 or a proton pump inhibitor. The EV uptake inhibitor antibodies or active fragments thereof as provided herein may be one or more antibodies or active fragments thereof directed against the protein targets listed in Table 1. The composition for treating and / or reducing inflammation in the CNS or lungs of a mammal using an EV uptake inhibitor may further comprise at least one pharmaceutically acceptable carrier or diluent.

[0098] Table 1. Exemplary targets and corresponding antibodies for blocking EV uptake.

[0099]

[0100]

[0101]

[0102] In one embodiment, the medicament to be administered is an antibody or its active fragment as provided herein, which is directed to a mammalian inflammasome component or an antigen or epitope derived therefrom. In another embodiment, the medicament to be administered is an antisense RNA or siRNA directed to a mammalian inflammasome component. The inflammasome component can be a component of any inflammasome known in the art (e.g., NAPL1, NALP2, NALP3, NLRC4 or AIM2 inflammasome). In a typical embodiment, the antibody specifically binds to ASC or an antigen or epitope derived therefrom. However, antibodies to any other components of a mammalian inflammasome (e.g., NALP1, NALP2, NALP3, NLRC4 or AIM2 inflammasome) can be used.

[0103] The antibodies as described herein may be monoclonal or polyclonal antibodies or active fragments thereof. The antibodies or active fragments may be chimeric, human or humanized as described herein.

[0104] Any suitable antibody or its active fragment as provided herein that specifically binds to ASC can be used, for example, an antibody that inhibits the activity of ASC in the CNS (e.g., CNS cells) or lung cells (e.g., type II alveolar cells) of a subject. In one embodiment, the antibody specifically binds to the following amino acid sequence, and the amino acid sequence has at least 85% sequence identity to the amino acid sequence SEQ ID NO: 1 or SEQ ID NO: 2. In another embodiment, the antibody or its fragment is bound to the following amino acid sequence, and the amino acid sequence has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence KKFKLKLLSVPLREGYGRIPR (SEQ ID NO: 5). In yet another embodiment, the antibody or its fragment binds to the amino acid sequence KKFKLKLLSVPLREGYGRIPR (SEQ ID NO: 5) or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids of SEQ ID NO: 5. In still another embodiment, the antibody or its fragment binds to 2-5, 5-10, 10-15 or 15-20 amino acids of SEQ ID NO: 5. In some embodiments, the ASC epitope (e.g., an epitope with amino acid SEQ ID NO: 5) bound by the antibody or antibody fragment is continuous. In some embodiments, the ASC epitope (e.g., an epitope with amino acid SEQ ID NO: 5) bound by the antibody or antibody fragment is discontinuous. In some cases, the monoclonal antibodies or their antibody fragments provided herein inhibit or reduce the activity of ASC.

[0105] As used herein, the term "epitope" includes any protein determinant that can specifically bind to an immunoglobulin or immunoglobulin fragment. Epitope determinants are usually composed of chemically active surface groups of molecules (such as amino acids or sugar side chains) and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics. The term "epitope" also refers to the structural unit that is conventionally bound by the variable (VH) region of the immunoglobulin heavy chain and the variable (VL) region of the light chain. An epitope can define the minimum binding site of an antibody, thereby representing the specific target of an antibody.

[0106] Similarly, in another embodiment, the inflammasome is a NALP1 inflammasome and the at least one component is NALP1 (ie, NLRP1). In this embodiment, the antibody or active fragment thereof as provided herein specifically binds to an amino acid sequence having at least 85% sequence identity to the amino acid sequence SEQ ID NO: 3 or SEQ ID NO: 4.

[0107] In yet another embodiment, the agent is a combination of one or more EV uptake inhibitors and one or more antibodies or active fragments thereof as provided herein that bind to inflammasome components. The EV uptake inhibitor can be any EV uptake inhibitor as provided herein. The antibody that binds to the inflammasome component can be any antibody that binds to any inflammasome component as provided herein. In one embodiment, the agent administered to a subject with CNS or lung inflammation comprises a combination of heparin (e.g., enoxaparin) and an antibody that binds to an AIM2 inflammasome component (e.g., ASC).

[0108] In one embodiment, the method includes: providing a therapeutically effective amount of a composition comprising an antibody or an active fragment thereof as provided herein that specifically binds to at least one component (e.g., ASC) of a mammalian inflammasome (e.g., AIM2 inflammasome); and administering the composition to a mammal suffering from CNS or lung inflammation or MS, wherein administration of the composition to the mammal results in a reduction in caspase-1 activation in the CNS or lungs of the mammal. In another embodiment, the method includes: providing a therapeutically effective amount of a composition comprising an antibody that specifically binds to at least one component (e.g., ASC) of a mammalian inflammasome (e.g., AIM2 inflammasome); and administering the composition to a mammal suffering from CNS or lung inflammation or MS, wherein administration of the composition to the mammal results in a reduction in the level of one or more inflammasome components (e.g., ASC). In yet another embodiment, the method includes: providing a therapeutically effective amount of a composition comprising an antibody that specifically binds to at least one component (e.g., ASC) of a mammalian inflammasome (e.g., AIM2 inflammasome); and administering the composition to a mammal suffering from CNS or lung inflammation or MS, wherein administering the composition to the mammal results in a decrease in ALI. The CNS or lung inflammation can be the result of CNS injury (e.g., SCI or TBI), asthma, chronic obstructive pulmonary disease (COPD), neurodegenerative disease, or an autoimmune disease with an inflammatory component. In one embodiment, the lung inflammation is caused by CNS injury (e.g., TBI or SCI).

[0109] In one embodiment, the methods provided herein also require the detection of the level or activity of one or more components of the mammalian inflammasome in a sample from a subject suspected of having CNS or lung inflammation or MS. The method for detecting the level or activity requires measuring the level of at least one inflammasome protein (e.g., ASC or AIM2) in a sample obtained from a subject; determining the presence or absence of an elevated level or activity of the at least one inflammasome protein (e.g., ASC or AIM2). The level or activity of the at least one inflammasome protein may be enhanced relative to the level of the at least one inflammasome protein in a control sample. The level or activity of the at least one inflammasome protein in a protein feature may be enhanced relative to a predetermined reference value or reference value range. The at least one inflammasome protein may be a protein 1 (NLRP1) containing a nucleotide binding leucine-rich repeat hot protein domain, NLRP2, NLRP3, NLRC4, AIM2, apoptosis-associated speck-like protein (ASC) containing a caspase activation recruitment domain, caspase-1, or a combination thereof. The sample may be cerebrospinal fluid (CSF), saliva, blood, serum, plasma, urine, or lung aspirate.

[0110] Antibodies that specifically bind to at least one component of a mammalian inflammasome

[0111] The method described herein for reducing inflammation in the CNS and / or lung of a mammal includes a composition comprising an antibody or its active fragment as provided herein that specifically binds to at least one component (e.g., ASC, AIM2) of a mammalian inflammasome (e.g., AIM2 inflammasome). The composition for treating and / or reducing inflammation in the CNS and / or lung of a mammal may further comprise at least one pharmaceutically acceptable carrier or diluent. Exemplary antibodies for mammalian inflammasome components for use in the methods herein may be those found in US 8685400, the contents of which are incorporated herein by reference in their entirety. Also provided herein are exemplary monoclonal antibodies or antibody fragments, such as, for example, monoclonal antibodies or antibody fragments comprising a VH region such that the VH region amino acid sequence comprises HCDR1 of SEQ ID NO:6, HCDR2 of SEQ ID NO:7, and HCDR3 of SEQ ID NO:8, and a VL region such that the VL region amino acid sequence comprises LCDR1 of SEQ ID NO:12, LCDR2 of SEQ ID NO:13, and LCDR3 of SEQ ID NO:14.

[0112] In one embodiment, the composition for treating and / or reducing inflammation in the CNS or lungs of a mammal comprises an antibody or its active fragment as provided herein, which specifically binds to a domain or portion thereof of a mammalian ASC protein (e.g., such as a human mouse or rat ASC protein). Any suitable anti-ASC antibody can be used, and several are commercially available. Examples of anti-ASC antibodies for use in the methods herein may be those found in US 8685400, the contents of which are incorporated herein by reference in their entirety. Examples of commercially available anti-ASC antibodies for use in the methods provided herein include, but are not limited to, 04-147 anti-ASC, clone 2EI-7 mouse monoclonal antibody from MilliporeSigma, AB3607-anti-ASC antibody from MilliporeSigma, orb194021 anti-ASC from Biorbyt, LS-C331318-50 anti-ASC from LifeSpan Biosciences, AF3805 anti-ASC from R&D Systems, NBP1-78977 anti-ASC from Novus Biologicals, 600-401-Y67 anti-ASC from Rockland Immunochemicals, D086-3 anti-ASC from MBL International, AL177 anti-ASC from Adipogen, monoclonal anti-ASC (clone o93E9) antibody, anti-ASC antibody (F-9) from Santa Cruz Biotechnology, anti-ASC antibody (B-3) from Enzo Life Sciences' ASC polyclonal antibody - ADI-905-173 or A161 anti-human ASC - Leinco Technologies. The human ASC protein can be accession number NP_037390.2 (Q9ULZ3-1), NP_660183 (Q9ULZ3-2) or Q9ULZ3-3. The rat ASC protein can be accession number NP_758825 (BAC43754). The mouse ASC protein can be accession number NP_075747.3. In one embodiment, the antibody binds to the PYRIN-PAAD-DAPIN domain (PYD) of a mammalian ASC protein (e.g., human, mouse or rat ASC) or a portion or fragment thereof. In this embodiment, the antibody as described herein specifically binds to the following amino acid sequence, which has an amino acid sequence with at least 65% (e.g., 65%, 70%, 75%, 80%, 85%) sequence identity with the PYD domain of human, mouse or rat ASC or a fragment thereof.In one embodiment, the antibody binds to the C-terminal caspase-recruiting domain (CARD) or a portion or fragment thereof of a mammalian ASC protein (e.g., human, mouse or rat ASC). In this embodiment, the antibody as described herein specifically binds to the following amino acid sequence, which has at least 65% (e.g., 65%, 70%, 75%, 80%, 85%) sequence identity with the CARD domain or fragment thereof of human, mouse or rat ASC. In yet another embodiment, the antibody binds to a portion or fragment thereof located between the PYD and CARD domains of a mammalian ASC protein sequence (e.g., human, mouse or rat ASC). In another embodiment, a composition for treating and / or reducing inflammation in the CNS and / or lungs of a mammal comprises an antibody that specifically binds to a region of rat ASC (e.g., amino acid sequence ALRQTQPYLVTDLEQS (SEQ ID NO: 1)) (i.e., residues 178-193 of rat ASC, accession number BAC43754). In this embodiment, the antibody as described herein specifically binds to the following amino acid sequence, which has at least 65% (e.g., 65%, 70%, 75%, 80%, 85%) sequence identity with the amino acid sequence ALRQTQPYLVTDLEQS (SEQ ID NO: 1) of rat ASC. In another embodiment, the composition for treating and / or reducing inflammation in the CNS and / or lungs of a mammal comprises an antibody that specifically binds to a region of human ASC (e.g., the amino acid sequence RESQSYLVEDLERS (SEQ ID NO: 2)). In still another embodiment, the composition for treating and / or reducing inflammation in the CNS and / or lungs of a mammal comprises an antibody that specifically binds to a region of human ASC, such as the amino acid sequence KKFKLKLLSVPLREGYGRIPR (SEQ ID NO: 5; i.e., residues 21-41 of human ASC) or SEQ ID NO: 5. 5-10, 10-15, or 15-20 amino acids. In one embodiment, an antibody as described herein that is bound to an ASC domain or a fragment thereof inhibits ASC activity in mammalian lung cells (e.g., type II alveolar cells). In another embodiment, an antibody as described herein that is bound to an ASC domain or a fragment thereof (e.g., a monoclonal anti-ASC antibody or an antibody fragment thereof provided herein) inhibits ASC activity in the CNS of a mammal suffering from or suspected of suffering from CNS damage or disorder. Examples of CNS damage or disorder may include TBI, SCI, stroke, amyotrophic lateral sclerosis (ALS) (Lou Gehrig's disease), multiple sclerosis (MS), immune dysfunction muscle CNS collapse, muscular dystrophy (MD), Alzheimer's disease (AD), Parkinson's disease (PD).

[0113] In certain embodiments, the present invention provides antibodies and antibody fragments that specifically bind to ASC and comprise one or more amino acid sequences shown in Table 2. Also provided herein are isolated nucleic acid molecules encoding the monoclonal antibodies or antibody fragments thereof, the isolated nucleic acid molecules comprising the nucleic acid sequences shown in Table 2. In some cases, the expression vector comprises the nucleic acid molecule of Table 2. The expression vector may comprise a heavy chain or light chain constant region. An example of a light chain and heavy chain expression vector system used in the compositions and methods provided herein is the Antitope pANT expression vector system for IgG4 (S241P) heavy chain and κ light chain. Nucleic acid molecules for heavy or light chains can be operably linked to regulatory sequences suitable for expressing nucleic acid segments in host cells.

[0114] Table 2. Variable heavy chain and variable light (κ) chain sequences of the anti-ASC antibodies or antibody fragments thereof of the present invention.

[0115]

[0116]

[0117]

[0118]

[0119]

[0120]

[0121] In one embodiment, provided herein is a monoclonal antibody or antibody fragment thereof that specifically binds to ASC, wherein the antibody or antibody fragment thereof comprises a heavy chain variable (VH) region and a light chain or kappa chain variable (VL) region, wherein the VH region amino acid sequence comprises SEQ ID NO: 18, 19, 20, 21, 22 or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 18, 19, 20, 21 or 22. In terms of this embodiment, provided herein is the use of the monoclonal antibody or antibody fragment thereof in a method for treating inflammation in a subject. The inflammation may be innate immune inflammation. The inflammation may be inflammasome-associated inflammation. The inflammation may be in the lungs and / or CNS. Inflammation in the lungs and / or CNS may be the result of an injury (e.g., traumatic brain injury (TBI) or spinal cord injury (SCI)) or a disease, disorder or pain of the CNS or a disease, disorder or pain that affects the CNS. As provided herein, the disease, illness or suffering of CNS or the disease, illness or suffering affecting CNS can be stroke and autoimmune disease and / or CNS disease, including amyotrophic lateral sclerosis (ALS) (Lou Gehrig's disease), multiple sclerosis (MS), immune dysfunction muscle CNS collapse, muscular dystrophy (MD), Alzheimer's disease (AD), Parkinson's disease (PD). In the method for treating inflammation, the monoclonal antibody or its antibody fragment can be used to reduce the inflammation in the CNS and / or lung of the patient. The reduction can be compared with a control (e.g., an untreated patient and / or a patient before treatment). In one embodiment, the monoclonal antibody or the antibody fragment derived therefrom is applied to a patient suffering from or suspected of suffering from MS, and the monoclonal antibody or the antibody fragment derived therefrom is used to treat MS. In some cases, the monoclonal antibody or its antibody fragment of this embodiment is present in a composition. The composition can be a pharmaceutical composition as provided herein.

[0122] In one embodiment, provided herein is a monoclonal antibody or antibody fragment thereof that specifically binds to ASC, wherein the antibody or antibody fragment thereof comprises a heavy chain variable (VH) region and a light chain or kappa chain variable (VL) region, wherein the VL region amino acid sequence comprises SEQ ID NO: 28, 29, 30, 31 or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 28, 29, 30 or 31. In terms of this embodiment, provided herein is the use of the monoclonal antibody or antibody fragment thereof in a method for treating inflammation in a subject. The inflammation may be innate immune inflammation. The inflammation may be inflammasome-associated inflammation. The inflammation may be in the lungs and / or CNS. Inflammation in the lungs and / or CNS may be the result of an injury (e.g., traumatic brain injury (TBI) or spinal cord injury (SCI)) or a disease, disorder or pain of the CNS or a disease, disorder or pain that affects the CNS. As provided herein, the disease, illness or suffering of CNS or the disease, illness or suffering affecting CNS can be stroke and autoimmune disease and / or CNS disease, including amyotrophic lateral sclerosis (ALS) (Lou Gehrig's disease), multiple sclerosis (MS), immune dysfunction muscle CNS collapse, muscular dystrophy (MD), Alzheimer's disease (AD), Parkinson's disease (PD). In the method for treating inflammation, the monoclonal antibody or its antibody fragment can be used to reduce the inflammation in the CNS and / or lung of the patient. The reduction can be compared with a control (e.g., an untreated patient and / or a patient before treatment). In one embodiment, the monoclonal antibody or the antibody fragment derived therefrom is applied to a patient suffering from or suspected of suffering from MS, and the monoclonal antibody or the antibody fragment derived therefrom is used to treat MS. In some cases, the monoclonal antibody or its antibody fragment of this embodiment is present in a composition. The composition can be a pharmaceutical composition as provided herein.

[0123] In one embodiment, provided herein is a monoclonal antibody or antibody fragment thereof that specifically binds to ASC, wherein the antibody or antibody fragment thereof comprises a heavy chain variable (VH) region and a light chain or kappa chain variable (VL) region, wherein the VH region amino acid sequence comprises SEQ ID NO: 18, 19, 20, 21, 22, or an amino acid sequence at least 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence of SEQ ID NO: 18, 19, 20, 21 or 22; and wherein the VL region amino acid sequence comprises SEQ ID NO: 28, 29, 30, 31, or an amino acid sequence at least 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence of SEQ ID NO: 28, 29, 30 or 31. In terms of this embodiment, provided herein is the use of the monoclonal antibody or antibody fragment thereof in a method for treating inflammation in a subject. The inflammation may be an innate immune inflammation. The inflammation may be an inflammasome-associated inflammation. The inflammation may be in the lungs and / or CNS. Inflammation in the lung and / or CNS may be the result of damage (e.g., traumatic brain injury (TBI) or spinal cord injury (SCI)) or a disease, disorder or pain of the CNS or a disease, disorder or pain that affects the CNS. As provided herein, a disease, disorder or pain of the CNS or a disease, disorder or pain that affects the CNS may be a stroke and an autoimmune disease and / or a CNS disease, including amyotrophic lateral sclerosis (ALS) (Lou Gehrig's disease), multiple sclerosis (MS), immune dysfunction muscle CNS collapse, muscular dystrophy (MD), Alzheimer's disease (AD), Parkinson's disease (PD). In the method for treating inflammation, the monoclonal antibody or its antibody fragment can be used to reduce the inflammation in the CNS and / or lung of the patient. The reduction can be compared with a control (e.g., an untreated patient and / or a patient before treatment). In one embodiment, the monoclonal antibody or an antibody fragment derived therefrom is applied to a patient suffering from or suspected of suffering from MS, and the monoclonal antibody or an antibody fragment derived therefrom is used to treat MS. In some cases, the monoclonal antibody or its antibody fragment of this embodiment is present in the composition. The composition may be a pharmaceutical composition as provided herein.

[0124] In one embodiment, provided herein is a monoclonal antibody or antibody fragment thereof that specifically binds to ASC, wherein the antibody or antibody fragment thereof comprises a heavy chain variable (VH) region and a light chain or kappa chain variable (VL) region, wherein the VH region amino acid sequence comprises SEQ ID NO: 18 or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 18; and wherein the VL region amino acid sequence comprises SEQ ID NO: 28 or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 28. In terms of this embodiment, provided herein is the use of the monoclonal antibody or antibody fragment thereof in a method for treating inflammation in a subject. The inflammation may be innate immune inflammation. The inflammation may be inflammasome-associated inflammation. The inflammation may be in the lungs and / or CNS. Inflammation in the lungs and / or CNS may be the result of an injury (e.g., traumatic brain injury (TBI) or spinal cord injury (SCI)) or a disease, disorder or affliction of the CNS or a disease, disorder or affliction that affects the CNS. As provided herein, the disease, illness or suffering of CNS or the disease, illness or suffering affecting CNS can be stroke and autoimmune disease and / or CNS disease, including amyotrophic lateral sclerosis (ALS) (Lou Gehrig's disease), multiple sclerosis (MS), immune dysfunction muscle CNS collapse, muscular dystrophy (MD), Alzheimer's disease (AD), Parkinson's disease (PD). In the method for treating inflammation, the monoclonal antibody or its antibody fragment can be used to reduce the inflammation in the CNS and / or lung of the patient. The reduction can be compared with a control (e.g., an untreated patient and / or a patient before treatment). In one embodiment, the monoclonal antibody or the antibody fragment derived therefrom is applied to a patient suffering from or suspected of suffering from MS, and the monoclonal antibody or the antibody fragment derived therefrom is used to treat MS. In some cases, the monoclonal antibody or its antibody fragment of this embodiment is present in a composition. The composition can be a pharmaceutical composition as provided herein.

[0125] In one embodiment, provided herein is a monoclonal antibody or antibody fragment thereof that specifically binds to ASC, wherein the antibody or antibody fragment thereof comprises a heavy chain variable (VH) region and a light chain or kappa chain variable (VL) region, wherein the VH region amino acid sequence comprises SEQ ID NO: 18 or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 18; and wherein the VL region amino acid sequence comprises SEQ ID NO: 29 or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 29. With respect to this embodiment, provided herein is the use of the monoclonal antibody or antibody fragment thereof in a method for treating inflammation in a subject. The inflammation may be innate immune inflammation. The inflammation may be inflammasome-associated inflammation. The inflammation may be in the lungs and / or CNS. Inflammation in the lungs and / or CNS may be the result of an injury (e.g., traumatic brain injury (TBI) or spinal cord injury (SCI)) or a disease, disorder or affliction of the CNS or a disease, disorder or affliction that affects the CNS. As provided herein, the disease, illness or suffering of CNS or the disease, illness or suffering affecting CNS can be stroke and autoimmune disease and / or CNS disease, including amyotrophic lateral sclerosis (ALS) (Lou Gehrig's disease), multiple sclerosis (MS), immune dysfunction muscle CNS collapse, muscular dystrophy (MD), Alzheimer's disease (AD), Parkinson's disease (PD). In the method for treating inflammation, the monoclonal antibody or its antibody fragment can be used to reduce the inflammation in the CNS and / or lung of the patient. The reduction can be compared with a control (e.g., an untreated patient and / or a patient before treatment). In one embodiment, the monoclonal antibody or the antibody fragment derived therefrom is applied to a patient suffering from or suspected of suffering from MS, and the monoclonal antibody or the antibody fragment derived therefrom is used to treat MS. In some cases, the monoclonal antibody or its antibody fragment of this embodiment is present in a composition. The composition can be a pharmaceutical composition as provided herein.

[0126] In one embodiment, provided herein is a monoclonal antibody or antibody fragment thereof that specifically binds to ASC, wherein the antibody or antibody fragment thereof comprises a heavy chain variable (VH) region and a light chain or kappa chain variable (VL) region, wherein the VH region amino acid sequence comprises SEQ ID NO: 18 or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 18; and wherein the VL region amino acid sequence comprises SEQ ID NO: 30 or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 30. With respect to this embodiment, provided herein is the use of the monoclonal antibody or antibody fragment thereof in a method for treating inflammation in a subject. The inflammation may be innate immune inflammation. The inflammation may be inflammasome-associated inflammation. The inflammation may be in the lungs and / or CNS. Inflammation in the lungs and / or CNS may be the result of an injury (e.g., traumatic brain injury (TBI) or spinal cord injury (SCI)) or a disease, disorder or affliction of the CNS or a disease, disorder or affliction that affects the CNS. As provided herein, the disease, illness or suffering of CNS or the disease, illness or suffering affecting CNS can be stroke and autoimmune disease and / or CNS disease, including amyotrophic lateral sclerosis (ALS) (Lou Gehrig's disease), multiple sclerosis (MS), immune dysfunction muscle CNS collapse, muscular dystrophy (MD), Alzheimer's disease (AD), Parkinson's disease (PD). In the method for treating inflammation, the monoclonal antibody or its antibody fragment can be used to reduce the inflammation in the CNS and / or lung of the patient. The reduction can be compared with a control (e.g., an untreated patient and / or a patient before treatment). In one embodiment, the monoclonal antibody or the antibody fragment derived therefrom is applied to a patient suffering from or suspected of suffering from MS, and the monoclonal antibody or the antibody fragment derived therefrom is used to treat MS. In some cases, the monoclonal antibody or its antibody fragment of this embodiment is present in a composition. The composition can be a pharmaceutical composition as provided herein.

[0127] In one embodiment, provided herein is a monoclonal antibody or antibody fragment thereof that specifically binds to ASC, wherein the antibody or antibody fragment thereof comprises a heavy chain variable (VH) region and a light chain or kappa chain variable (VL) region, wherein the VH region amino acid sequence comprises SEQ ID NO: 18 or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 18; and wherein the VL region amino acid sequence comprises SEQ ID NO: 31 or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 31. With respect to this embodiment, provided herein is the use of the monoclonal antibody or antibody fragment thereof in a method for treating inflammation in a subject. The inflammation may be innate immune inflammation. The inflammation may be inflammasome-associated inflammation. The inflammation may be in the lungs and / or CNS. Inflammation in the lungs and / or CNS may be the result of an injury (e.g., traumatic brain injury (TBI) or spinal cord injury (SCI)) or a disease, disorder or affliction of the CNS or a disease, disorder or affliction that affects the CNS. As provided herein, the disease, illness or suffering of CNS or the disease, illness or suffering affecting CNS can be stroke and autoimmune disease and / or CNS disease, including amyotrophic lateral sclerosis (ALS) (Lou Gehrig's disease), multiple sclerosis (MS), immune dysfunction muscle CNS collapse, muscular dystrophy (MD), Alzheimer's disease (AD), Parkinson's disease (PD). In the method for treating inflammation, the monoclonal antibody or its antibody fragment can be used to reduce the inflammation in the CNS and / or lung of the patient. The reduction can be compared with a control (e.g., an untreated patient and / or a patient before treatment). In one embodiment, the monoclonal antibody or the antibody fragment derived therefrom is applied to a patient suffering from or suspected of suffering from MS, and the monoclonal antibody or the antibody fragment derived therefrom is used to treat MS. In some cases, the monoclonal antibody or its antibody fragment of this embodiment is present in a composition. The composition can be a pharmaceutical composition as provided herein.

[0128] In one embodiment, provided herein is a monoclonal antibody or antibody fragment thereof that specifically binds to ASC, wherein the antibody or antibody fragment thereof comprises a heavy chain variable (VH) region and a light chain or kappa chain variable (VL) region, wherein the VH region amino acid sequence comprises SEQ ID NO: 19 or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 19; and wherein the VL region amino acid sequence comprises SEQ ID NO: 28 or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 28. In terms of this embodiment, provided herein is the use of the monoclonal antibody or antibody fragment thereof in a method for treating inflammation in a subject. The inflammation may be innate immune inflammation. The inflammation may be inflammasome-associated inflammation. The inflammation may be in the lungs and / or CNS. Inflammation in the lungs and / or CNS may be the result of an injury (e.g., traumatic brain injury (TBI) or spinal cord injury (SCI)) or a disease, disorder or affliction of the CNS or a disease, disorder or affliction that affects the CNS. As provided herein, the disease, illness or suffering of CNS or the disease, illness or suffering affecting CNS can be stroke and autoimmune disease and / or CNS disease, including amyotrophic lateral sclerosis (ALS) (Lou Gehrig's disease), multiple sclerosis (MS), immune dysfunction muscle CNS collapse, muscular dystrophy (MD), Alzheimer's disease (AD), Parkinson's disease (PD). In the method for treating inflammation, the monoclonal antibody or its antibody fragment can be used to reduce the inflammation in the CNS and / or lung of the patient. The reduction can be compared with a control (e.g., an untreated patient and / or a patient before treatment). In one embodiment, the monoclonal antibody or the antibody fragment derived therefrom is applied to a patient suffering from or suspected of suffering from MS, and the monoclonal antibody or the antibody fragment derived therefrom is used to treat MS. In some cases, the monoclonal antibody or its antibody fragment of this embodiment is present in a composition. The composition can be a pharmaceutical composition as provided herein.

[0129] In one embodiment, provided herein is a monoclonal antibody or antibody fragment thereof that specifically binds to ASC, wherein the antibody or antibody fragment thereof comprises a heavy chain variable (VH) region and a light chain or kappa chain variable (VL) region, wherein the VH region amino acid sequence comprises SEQ ID NO: 19 or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 19; and wherein the VL region amino acid sequence comprises SEQ ID NO: 29 or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 29. With respect to this embodiment, provided herein is the use of the monoclonal antibody or antibody fragment thereof in a method for treating inflammation in a subject. The inflammation may be innate immune inflammation. The inflammation may be inflammasome-associated inflammation. The inflammation may be in the lungs and / or CNS. Inflammation in the lungs and / or CNS may be the result of an injury (e.g., traumatic brain injury (TBI) or spinal cord injury (SCI)) or a disease, disorder or affliction of the CNS or a disease, disorder or affliction that affects the CNS. As provided herein, the disease, illness or suffering of CNS or the disease, illness or suffering affecting CNS can be stroke and autoimmune disease and / or CNS disease, including amyotrophic lateral sclerosis (ALS) (Lou Gehrig's disease), multiple sclerosis (MS), immune dysfunction muscle CNS collapse, muscular dystrophy (MD), Alzheimer's disease (AD), Parkinson's disease (PD). In the method for treating inflammation, the monoclonal antibody or its antibody fragment can be used to reduce the inflammation in the CNS and / or lung of the patient. The reduction can be compared with a control (e.g., an untreated patient and / or a patient before treatment). In one embodiment, the monoclonal antibody or the antibody fragment derived therefrom is applied to a patient suffering from or suspected of suffering from MS, and the monoclonal antibody or the antibody fragment derived therefrom is used to treat MS. In some cases, the monoclonal antibody or its antibody fragment of this embodiment is present in a composition. The composition can be a pharmaceutical composition as provided herein.

[0130] In one embodiment, provided herein is a monoclonal antibody or antibody fragment thereof that specifically binds to ASC, wherein the antibody or antibody fragment thereof comprises a heavy chain variable (VH) region and a light chain or kappa chain variable (VL) region, wherein the VH region amino acid sequence comprises SEQ ID NO: 19 or an amino acid sequence at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 19; and wherein the VL region amino acid sequence comprises SEQ ID NO: 30 or an amino acid sequence at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 30. In some cases, a monoclonal antibody or an antibody fragment derived therefrom comprising a VH region amino acid sequence of SEQ ID NO: 19 and a VL region amino acid sequence of SEQ ID NO: 30 may be referred to as IC 100. With respect to this embodiment, provided herein is the use of the monoclonal antibody or antibody fragment thereof in a method for treating inflammation in a subject. The inflammation may be innate immune inflammation. The inflammation may be inflammation associated with an inflammasome. The inflammation may be in the lungs and / or CNS. Inflammation in the lung and / or CNS may be the result of damage (e.g., traumatic brain injury (TBI) or spinal cord injury (SCI)) or a disease, disorder or pain of the CNS or a disease, disorder or pain that affects the CNS. As provided herein, a disease, disorder or pain of the CNS or a disease, disorder or pain that affects the CNS may be a stroke and an autoimmune disease and / or a CNS disease, including amyotrophic lateral sclerosis (ALS) (Lou Gehrig's disease), multiple sclerosis (MS), immune dysfunction muscle CNS collapse, muscular dystrophy (MD), Alzheimer's disease (AD), Parkinson's disease (PD). In the method for treating inflammation, the monoclonal antibody or its antibody fragment can be used to reduce the inflammation in the CNS and / or lung of the patient. The reduction can be compared with a control (e.g., an untreated patient and / or a patient before treatment). In one embodiment, the monoclonal antibody or an antibody fragment derived therefrom is applied to a patient suffering from or suspected of suffering from MS, and the monoclonal antibody or an antibody fragment derived therefrom is used to treat MS. In some cases, the monoclonal antibody or its antibody fragment of this embodiment is present in the composition. The composition may be a pharmaceutical composition as provided herein.

[0131] In one embodiment, provided herein is a monoclonal antibody or antibody fragment thereof that specifically binds to ASC, wherein the antibody or antibody fragment thereof comprises a heavy chain variable (VH) region and a light chain or kappa chain variable (VL) region, wherein the VH region amino acid sequence comprises SEQ ID NO: 19 or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 19; and wherein the VL region amino acid sequence comprises SEQ ID NO: 31 or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 31. With respect to this embodiment, provided herein is the use of the monoclonal antibody or antibody fragment thereof in a method for treating inflammation in a subject. The inflammation may be innate immune inflammation. The inflammation may be inflammasome-associated inflammation. The inflammation may be in the lungs and / or CNS. Inflammation in the lungs and / or CNS may be the result of an injury (e.g., traumatic brain injury (TBI) or spinal cord injury (SCI)) or a disease, disorder or affliction of the CNS or a disease, disorder or affliction that affects the CNS. As provided herein, the disease, illness or suffering of CNS or the disease, illness or suffering affecting CNS can be stroke and autoimmune disease and / or CNS disease, including amyotrophic lateral sclerosis (ALS) (Lou Gehrig's disease), multiple sclerosis (MS), immune dysfunction muscle CNS collapse, muscular dystrophy (MD), Alzheimer's disease (AD), Parkinson's disease (PD). In the method for treating inflammation, the monoclonal antibody or its antibody fragment can be used to reduce the inflammation in the CNS and / or lung of the patient. The reduction can be compared with a control (e.g., an untreated patient and / or a patient before treatment). In one embodiment, the monoclonal antibody or the antibody fragment derived therefrom is applied to a patient suffering from or suspected of suffering from MS, and the monoclonal antibody or the antibody fragment derived therefrom is used to treat MS. In some cases, the monoclonal antibody or its antibody fragment of this embodiment is present in a composition. The composition can be a pharmaceutical composition as provided herein.

[0132] In one embodiment, provided herein is a monoclonal antibody or antibody fragment thereof that specifically binds to ASC, wherein the antibody or antibody fragment thereof comprises a heavy chain variable (VH) region and a light chain or kappa chain variable (VL) region, wherein the VH region amino acid sequence comprises SEQ ID NO: 20 or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 20; and wherein the VL region amino acid sequence comprises SEQ ID NO: 28 or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 28. In terms of this embodiment, provided herein is the use of the monoclonal antibody or antibody fragment thereof in a method for treating inflammation in a subject. The inflammation may be innate immune inflammation. The inflammation may be inflammasome-associated inflammation. The inflammation may be in the lungs and / or CNS. Inflammation in the lungs and / or CNS may be the result of an injury (e.g., traumatic brain injury (TBI) or spinal cord injury (SCI)) or a disease, disorder or affliction of the CNS or a disease, disorder or affliction that affects the CNS. As provided herein, the disease, illness or suffering of CNS or the disease, illness or suffering affecting CNS can be stroke and autoimmune disease and / or CNS disease, including amyotrophic lateral sclerosis (ALS) (Lou Gehrig's disease), multiple sclerosis (MS), immune dysfunction muscle CNS collapse, muscular dystrophy (MD), Alzheimer's disease (AD), Parkinson's disease (PD). In the method for treating inflammation, the monoclonal antibody or its antibody fragment can be used to reduce the inflammation in the CNS and / or lung of the patient. The reduction can be compared with a control (e.g., an untreated patient and / or a patient before treatment). In one embodiment, the monoclonal antibody or the antibody fragment derived therefrom is applied to a patient suffering from or suspected of suffering from MS, and the monoclonal antibody or the antibody fragment derived therefrom is used to treat MS. In some cases, the monoclonal antibody or its antibody fragment of this embodiment is present in a composition. The composition can be a pharmaceutical composition as provided herein.

[0133] In one embodiment, provided herein is a monoclonal antibody or antibody fragment thereof that specifically binds to ASC, wherein the antibody or antibody fragment thereof comprises a heavy chain variable (VH) region and a light chain or kappa chain variable (VL) region, wherein the VH region amino acid sequence comprises SEQ ID NO: 20 or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 20; and wherein the VL region amino acid sequence comprises SEQ ID NO: 29 or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 29. With respect to this embodiment, provided herein is the use of the monoclonal antibody or antibody fragment thereof in a method for treating inflammation in a subject. The inflammation may be innate immune inflammation. The inflammation may be inflammasome-associated inflammation. The inflammation may be in the lungs and / or CNS. Inflammation in the lungs and / or CNS may be the result of an injury (e.g., traumatic brain injury (TBI) or spinal cord injury (SCI)) or a disease, disorder or affliction of the CNS or a disease, disorder or affliction that affects the CNS. As provided herein, the disease, illness or suffering of CNS or the disease, illness or suffering affecting CNS can be stroke and autoimmune disease and / or CNS disease, including amyotrophic lateral sclerosis (ALS) (Lou Gehrig's disease), multiple sclerosis (MS), immune dysfunction muscle CNS collapse, muscular dystrophy (MD), Alzheimer's disease (AD), Parkinson's disease (PD). In the method for treating inflammation, the monoclonal antibody or its antibody fragment can be used to reduce the inflammation in the CNS and / or lung of the patient. The reduction can be compared with a control (e.g., an untreated patient and / or a patient before treatment). In one embodiment, the monoclonal antibody or the antibody fragment derived therefrom is applied to a patient suffering from or suspected of suffering from MS, and the monoclonal antibody or the antibody fragment derived therefrom is used to treat MS. In some cases, the monoclonal antibody or its antibody fragment of this embodiment is present in a composition. The composition can be a pharmaceutical composition as provided herein.

[0134] In one embodiment, provided herein is a monoclonal antibody or antibody fragment thereof that specifically binds to ASC, wherein the antibody or antibody fragment thereof comprises a heavy chain variable (VH) region and a light chain or kappa chain variable (VL) region, wherein the VH region amino acid sequence comprises SEQ ID NO: 20 or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 20; and wherein the VL region amino acid sequence comprises SEQ ID NO: 30 or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 30. With respect to this embodiment, provided herein is the use of the monoclonal antibody or antibody fragment thereof in a method for treating inflammation in a subject. The inflammation may be innate immune inflammation. The inflammation may be inflammasome-associated inflammation. The inflammation may be in the lungs and / or CNS. Inflammation in the lungs and / or CNS may be the result of an injury (e.g., traumatic brain injury (TBI) or spinal cord injury (SCI)) or a disease, disorder or affliction of the CNS or a disease, disorder or affliction that affects the CNS. As provided herein, the disease, illness or suffering of CNS or the disease, illness or suffering affecting CNS can be stroke and autoimmune disease and / or CNS disease, including amyotrophic lateral sclerosis (ALS) (Lou Gehrig's disease), multiple sclerosis (MS), immune dysfunction muscle CNS collapse, muscular dystrophy (MD), Alzheimer's disease (AD), Parkinson's disease (PD). In the method for treating inflammation, the monoclonal antibody or its antibody fragment can be used to reduce the inflammation in the CNS and / or lung of the patient. The reduction can be compared with a control (e.g., an untreated patient and / or a patient before treatment). In one embodiment, the monoclonal antibody or the antibody fragment derived therefrom is applied to a patient suffering from or suspected of suffering from MS, and the monoclonal antibody or the antibody fragment derived therefrom is used to treat MS. In some cases, the monoclonal antibody or its antibody fragment of this embodiment is present in a composition. The composition can be a pharmaceutical composition as provided herein.

[0135] In one embodiment, provided herein is a monoclonal antibody or antibody fragment thereof that specifically binds to ASC, wherein the antibody or antibody fragment thereof comprises a heavy chain variable (VH) region and a light chain or kappa chain variable (VL) region, wherein the VH region amino acid sequence comprises SEQ ID NO: 20 or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 20; and wherein the VL region amino acid sequence comprises SEQ ID NO: 31 or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 31. With respect to this embodiment, provided herein is the use of the monoclonal antibody or antibody fragment thereof in a method for treating inflammation in a subject. The inflammation may be innate immune inflammation. The inflammation may be inflammasome-associated inflammation. The inflammation may be in the lungs and / or CNS. Inflammation in the lungs and / or CNS may be the result of an injury (e.g., traumatic brain injury (TBI) or spinal cord injury (SCI)) or a disease, disorder or pain of the CNS or a disease, disorder or pain that affects the CNS. As provided herein, the disease, illness or suffering of CNS or the disease, illness or suffering affecting CNS can be stroke and autoimmune disease and / or CNS disease, including amyotrophic lateral sclerosis (ALS) (Lou Gehrig's disease), multiple sclerosis (MS), immune dysfunction muscle CNS collapse, muscular dystrophy (MD), Alzheimer's disease (AD), Parkinson's disease (PD). In the method for treating inflammation, the monoclonal antibody or its antibody fragment can be used to reduce the inflammation in the CNS and / or lung of the patient. The reduction can be compared with a control (e.g., an untreated patient and / or a patient before treatment). In one embodiment, the monoclonal antibody or the antibody fragment derived therefrom is applied to a patient suffering from or suspected of suffering from MS, and the monoclonal antibody or the antibody fragment derived therefrom is used to treat MS. In some cases, the monoclonal antibody or its antibody fragment of this embodiment is present in a composition. The composition can be a pharmaceutical composition as provided herein.

[0136] In one embodiment, provided herein is a monoclonal antibody or antibody fragment thereof that specifically binds to ASC, wherein the antibody or antibody fragment thereof comprises a heavy chain variable (VH) region and a light chain or kappa chain variable (VL) region, wherein the VH region amino acid sequence comprises SEQ ID NO: 21 or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 21; and wherein the VL region amino acid sequence comprises SEQ ID NO: 28 or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 28. In terms of this embodiment, provided herein is the use of the monoclonal antibody or antibody fragment thereof in a method for treating inflammation in a subject. The inflammation may be innate immune inflammation. The inflammation may be inflammasome-associated inflammation. The inflammation may be in the lungs and / or CNS. Inflammation in the lungs and / or CNS may be the result of an injury (e.g., traumatic brain injury (TBI) or spinal cord injury (SCI)) or a disease, disorder or affliction of the CNS or a disease, disorder or affliction that affects the CNS. As provided herein, the disease, illness or suffering of CNS or the disease, illness or suffering affecting CNS can be stroke and autoimmune disease and / or CNS disease, including amyotrophic lateral sclerosis (ALS) (Lou Gehrig's disease), multiple sclerosis (MS), immune dysfunction muscle CNS collapse, muscular dystrophy (MD), Alzheimer's disease (AD), Parkinson's disease (PD). In the method for treating inflammation, the monoclonal antibody or its antibody fragment can be used to reduce the inflammation in the CNS and / or lung of the patient. The reduction can be compared with a control (e.g., an untreated patient and / or a patient before treatment). In one embodiment, the monoclonal antibody or the antibody fragment derived therefrom is applied to a patient suffering from or suspected of suffering from MS, and the monoclonal antibody or the antibody fragment derived therefrom is used to treat MS. In some cases, the monoclonal antibody or its antibody fragment of this embodiment is present in a composition. The composition can be a pharmaceutical composition as provided herein.

[0137] In one embodiment, provided herein is a monoclonal antibody or antibody fragment thereof that specifically binds to ASC, wherein the antibody or antibody fragment thereof comprises a heavy chain variable (VH) region and a light chain or kappa chain variable (VL) region, wherein the VH region amino acid sequence comprises SEQ ID NO: 21 or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 21; and wherein the VL region amino acid sequence comprises SEQ ID NO: 29 or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 29. With respect to this embodiment, provided herein is the use of the monoclonal antibody or antibody fragment thereof in a method for treating inflammation in a subject. The inflammation may be an innate immune inflammation. The inflammation may be an inflammasome-associated inflammation. The inflammation may be in the lungs and / or CNS. Inflammation in the lungs and / or CNS may be the result of an injury (e.g., traumatic brain injury (TBI) or spinal cord injury (SCI)) or a disease, disorder or affliction of the CNS or a disease, disorder or affliction that affects the CNS. As provided herein, the disease, illness or suffering of CNS or the disease, illness or suffering affecting CNS can be stroke and autoimmune disease and / or CNS disease, including amyotrophic lateral sclerosis (ALS) (Lou Gehrig's disease), multiple sclerosis (MS), immune dysfunction muscle CNS collapse, muscular dystrophy (MD), Alzheimer's disease (AD), Parkinson's disease (PD). In the method for treating inflammation, the monoclonal antibody or its antibody fragment can be used to reduce the inflammation in the CNS and / or lung of the patient. The reduction can be compared with a control (e.g., an untreated patient and / or a patient before treatment). In one embodiment, the monoclonal antibody or the antibody fragment derived therefrom is applied to a patient suffering from or suspected of suffering from MS, and the monoclonal antibody or the antibody fragment derived therefrom is used to treat MS. In some cases, the monoclonal antibody or its antibody fragment of this embodiment is present in a composition. The composition can be a pharmaceutical composition as provided herein.

[0138] In one embodiment, provided herein is a monoclonal antibody or antibody fragment thereof that specifically binds to ASC, wherein the antibody or antibody fragment thereof comprises a heavy chain variable (VH) region and a light chain or kappa chain variable (VL) region, wherein the VH region amino acid sequence comprises SEQ ID NO: 21 or an amino acid sequence at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 21; and wherein the VL region amino acid sequence comprises SEQ ID NO: 30 or an amino acid sequence at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 30. With respect to this embodiment, provided herein is the use of the monoclonal antibody or antibody fragment thereof in a method for treating inflammation in a subject. The inflammation may be innate immune inflammation. The inflammation may be inflammasome-associated inflammation. The inflammation may be in the lungs and / or CNS. Inflammation in the lungs and / or CNS may be the result of an injury (e.g., traumatic brain injury (TBI) or spinal cord injury (SCI)) or a disease, disorder or affliction of the CNS or a disease, disorder or affliction affecting the CNS. As provided herein, the disease, illness or suffering of CNS or the disease, illness or suffering affecting CNS can be stroke and autoimmune disease and / or CNS disease, including amyotrophic lateral sclerosis (ALS) (Lou Gehrig's disease), multiple sclerosis (MS), immune dysfunction muscle CNS collapse, muscular dystrophy (MD), Alzheimer's disease (AD), Parkinson's disease (PD). In the method for treating inflammation, the monoclonal antibody or its antibody fragment can be used to reduce the inflammation in the CNS and / or lung of the patient. The reduction can be compared with a control (e.g., an untreated patient and / or a patient before treatment). In one embodiment, the monoclonal antibody or the antibody fragment derived therefrom is applied to a patient suffering from or suspected of suffering from MS, and the monoclonal antibody or the antibody fragment derived therefrom is used to treat MS. In some cases, the monoclonal antibody or its antibody fragment of this embodiment is present in a composition. The composition can be a pharmaceutical composition as provided herein.

[0139] In one embodiment, provided herein is a monoclonal antibody or antibody fragment thereof that specifically binds to ASC, wherein the antibody or antibody fragment thereof comprises a heavy chain variable (VH) region and a light chain or kappa chain variable (VL) region, wherein the VH region amino acid sequence comprises SEQ ID NO: 21 or an amino acid sequence at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 21; and wherein the VL region amino acid sequence comprises SEQ ID NO: 31 or an amino acid sequence at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 31. With respect to this embodiment, provided herein is the use of the monoclonal antibody or antibody fragment thereof in a method for treating inflammation in a subject. The inflammation may be innate immune inflammation. The inflammation may be inflammasome-associated inflammation. The inflammation may be in the lungs and / or CNS. Inflammation in the lungs and / or CNS may be the result of an injury (e.g., traumatic brain injury (TBI) or spinal cord injury (SCI)) or a disease, disorder or affliction of the CNS or a disease, disorder or affliction affecting the CNS. As provided herein, the disease, illness or suffering of CNS or the disease, illness or suffering affecting CNS can be stroke and autoimmune disease and / or CNS disease, including amyotrophic lateral sclerosis (ALS) (Lou Gehrig's disease), multiple sclerosis (MS), immune dysfunction muscle CNS collapse, muscular dystrophy (MD), Alzheimer's disease (AD), Parkinson's disease (PD). In the method for treating inflammation, the monoclonal antibody or its antibody fragment can be used to reduce the inflammation in the CNS and / or lung of the patient. The reduction can be compared with a control (e.g., an untreated patient and / or a patient before treatment). In one embodiment, the monoclonal antibody or the antibody fragment derived therefrom is applied to a patient suffering from or suspected of suffering from MS, and the monoclonal antibody or the antibody fragment derived therefrom is used to treat MS. In some cases, the monoclonal antibody or its antibody fragment of this embodiment is present in a composition. The composition can be a pharmaceutical composition as provided herein.

[0140] In one embodiment, provided herein is a monoclonal antibody or antibody fragment thereof that specifically binds to ASC, wherein the antibody or antibody fragment thereof comprises a heavy chain variable (VH) region and a light chain or kappa chain variable (VL) region, wherein the VH region amino acid sequence comprises SEQ ID NO: 22 or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 22; and wherein the VL region amino acid sequence comprises SEQ ID NO: 28 or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 28. In terms of this embodiment, provided herein is the use of the monoclonal antibody or antibody fragment thereof in a method for treating inflammation in a subject. The inflammation may be innate immune inflammation. The inflammation may be inflammasome-associated inflammation. The inflammation may be in the lungs and / or CNS. Inflammation in the lungs and / or CNS may be the result of an injury (e.g., traumatic brain injury (TBI) or spinal cord injury (SCI)) or a disease, disorder or affliction of the CNS or a disease, disorder or affliction that affects the CNS. As provided herein, the disease, illness or suffering of CNS or the disease, illness or suffering affecting CNS can be stroke and autoimmune disease and / or CNS disease, including amyotrophic lateral sclerosis (ALS) (Lou Gehrig's disease), multiple sclerosis (MS), immune dysfunction muscle CNS collapse, muscular dystrophy (MD), Alzheimer's disease (AD), Parkinson's disease (PD). In the method for treating inflammation, the monoclonal antibody or its antibody fragment can be used to reduce the inflammation in the CNS and / or lung of the patient. The reduction can be compared with a control (e.g., an untreated patient and / or a patient before treatment). In one embodiment, the monoclonal antibody or the antibody fragment derived therefrom is applied to a patient suffering from or suspected of suffering from MS, and the monoclonal antibody or the antibody fragment derived therefrom is used to treat MS. In some cases, the monoclonal antibody or its antibody fragment of this embodiment is present in a composition. The composition can be a pharmaceutical composition as provided herein.

[0141] In one embodiment, provided herein is a monoclonal antibody or antibody fragment thereof that specifically binds to ASC, wherein the antibody or antibody fragment thereof comprises a heavy chain variable (VH) region and a light chain or kappa chain variable (VL) region, wherein the VH region amino acid sequence comprises SEQ ID NO: 22 or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 22; and wherein the VL region amino acid sequence comprises SEQ ID NO: 29 or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 29. With respect to this embodiment, provided herein is the use of the monoclonal antibody or antibody fragment thereof in a method for treating inflammation in a subject. The inflammation may be an innate immune inflammation. The inflammation may be an inflammasome-associated inflammation. The inflammation may be in the lungs and / or CNS. Inflammation in the lungs and / or CNS may be the result of an injury (e.g., traumatic brain injury (TBI) or spinal cord injury (SCI)) or a disease, disorder or affliction of the CNS or a disease, disorder or affliction that affects the CNS. As provided herein, the disease, illness or suffering of CNS or the disease, illness or suffering affecting CNS can be stroke and autoimmune disease and / or CNS disease, including amyotrophic lateral sclerosis (ALS) (Lou Gehrig's disease), multiple sclerosis (MS), immune dysfunction muscle CNS collapse, muscular dystrophy (MD), Alzheimer's disease (AD), Parkinson's disease (PD). In the method for treating inflammation, the monoclonal antibody or its antibody fragment can be used to reduce the inflammation in the CNS and / or lung of the patient. The reduction can be compared with a control (e.g., an untreated patient and / or a patient before treatment). In one embodiment, the monoclonal antibody or the antibody fragment derived therefrom is applied to a patient suffering from or suspected of suffering from MS, and the monoclonal antibody or the antibody fragment derived therefrom is used to treat MS. In some cases, the monoclonal antibody or its antibody fragment of this embodiment is present in a composition. The composition can be a pharmaceutical composition as provided herein.

[0142] In one embodiment, provided herein is a monoclonal antibody or antibody fragment thereof that specifically binds to ASC, wherein the antibody or antibody fragment thereof comprises a heavy chain variable (VH) region and a light chain or kappa chain variable (VL) region, wherein the VH region amino acid sequence comprises SEQ ID NO: 22 or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 22; and wherein the VL region amino acid sequence comprises SEQ ID NO: 30 or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 30. With respect to this embodiment, provided herein is the use of the monoclonal antibody or antibody fragment thereof in a method for treating inflammation in a subject. The inflammation may be innate immune inflammation. The inflammation may be inflammasome-associated inflammation. The inflammation may be in the lungs and / or CNS. Inflammation in the lungs and / or CNS may be the result of an injury (e.g., traumatic brain injury (TBI) or spinal cord injury (SCI)) or a disease, disorder or affliction of the CNS or a disease, disorder or affliction that affects the CNS. As provided herein, the disease, illness or suffering of CNS or the disease, illness or suffering affecting CNS can be stroke and autoimmune disease and / or CNS disease, including amyotrophic lateral sclerosis (ALS) (Lou Gehrig's disease), multiple sclerosis (MS), immune dysfunction muscle CNS collapse, muscular dystrophy (MD), Alzheimer's disease (AD), Parkinson's disease (PD). In the method for treating inflammation, the monoclonal antibody or its antibody fragment can be used to reduce the inflammation in the CNS and / or lung of the patient. The reduction can be compared with a control (e.g., an untreated patient and / or a patient before treatment). In one embodiment, the monoclonal antibody or the antibody fragment derived therefrom is applied to a patient suffering from or suspected of suffering from MS, and the monoclonal antibody or the antibody fragment derived therefrom is used to treat MS. In some cases, the monoclonal antibody or its antibody fragment of this embodiment is present in a composition. The composition can be a pharmaceutical composition as provided herein.

[0143] In one embodiment, provided herein is a monoclonal antibody or antibody fragment thereof that specifically binds to ASC, wherein the antibody or antibody fragment thereof comprises a heavy chain variable (VH) region and a light chain or kappa chain variable (VL) region, wherein the VH region amino acid sequence comprises SEQ ID NO: 22 or an amino acid sequence at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 22; and wherein the VL region amino acid sequence comprises SEQ ID NO: 31 or an amino acid sequence at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 31. With respect to this embodiment, provided herein is the use of the monoclonal antibody or antibody fragment thereof in a method for treating inflammation in a subject. The inflammation may be an innate immune inflammation. The inflammation may be an inflammasome-associated inflammation. In one embodiment, the monoclonal antibody or antibody fragment thereof provided herein can be used in a method for reducing inflammation in a mammal as described in US 8,685,400, the contents of which are incorporated herein by reference in their entirety. The inflammation may be in the lungs and / or CNS. Inflammation in the lung and / or CNS may be the result of damage (e.g., traumatic brain injury (TBI) or spinal cord injury (SCI)) or a disease, disorder or pain of the CNS or a disease, disorder or pain that affects the CNS. As provided herein, a disease, disorder or pain of the CNS or a disease, disorder or pain that affects the CNS may be a stroke and an autoimmune disease and / or a CNS disease, including amyotrophic lateral sclerosis (ALS) (Lou Gehrig's disease), multiple sclerosis (MS), immune dysfunction muscle CNS collapse, muscular dystrophy (MD), Alzheimer's disease (AD), Parkinson's disease (PD). In the method for treating inflammation, the monoclonal antibody or its antibody fragment can be used to reduce the inflammation in the CNS and / or lung of the patient. The reduction can be compared with a control (e.g., an untreated patient and / or a patient before treatment). In one embodiment, the monoclonal antibody or an antibody fragment derived therefrom is applied to a patient suffering from or suspected of suffering from MS, and the monoclonal antibody or an antibody fragment derived therefrom is used to treat MS. In some cases, the monoclonal antibody or its antibody fragment of this embodiment is present in the composition. The composition may be a pharmaceutical composition as provided herein.

[0144] In another embodiment, a composition for reducing inflammation in the CNS or lung of a mammal comprises an antibody or an active fragment thereof as provided herein that specifically binds to NLRP1 (e.g., an anti-NLRP1 chicken antibody) or a domain thereof. Any suitable anti-NLRP1 antibody may be used, and several are commercially available. Examples of anti-NLRP1 antibodies for use in the methods herein may be those found in US 8685400, the contents of which are incorporated herein by reference in their entirety. Examples of commercially available anti-NLRP1 antibodies for use in the methods provided herein include, but are not limited to, human NLRP1 polyclonal antibody AF6788 from R&D Systems, EMD Millipore rabbit polyclonal anti-NLRP1 ABF22, Novus Biologicals rabbit polyclonal anti-NLRP1 NB100-56148, Sigma-Aldrich mouse polyclonal anti-NLRP1 SAB1407151, Abcam rabbit polyclonal anti-NLRP1 ab3683, Biorbyt rabbit polyclonal anti-NLRP1 orb325922 mybiosource rabbit polyclonal anti-NLRP1 MBS7001225, R&D systems sheep polyclonal AF6788, Aviva Systems mouse monoclonal anti-NLRP1 oaed00344, Aviva Systems rabbit polyclonal anti-NLRP1 ARO54478_P050, Origene rabbit polyclonal anti-NLRP1 APO7775PU-N, Antibodies online rabbit polyclonal anti-NLRP1 ABIN768983, Prosci rabbit polyclonal anti-NLRP1 3037, Proteintech rabbit polyclonal anti-NLRP1 12256-1-AP, Enzo mouse monoclonal anti-NLRP1 ALX-804-803-C100, Invitrogen mouse monoclonal anti-NLRP1 MA1-25842, GeneTex mouse monoclonal anti-NLRP1 GTX16091, Rockland rabbit polyclonal anti-NLRP1 200-401-CX5, or Cell Signaling Technology rabbit polyclonal anti-NLRP1 4990. Human NLRP1 protein can be accession number AAH51787, NP_001028225, NP_055737, NP_127497, NP_127499, or NP_127500. In one embodiment, the antibody binds to the Pyrin, NACHT, LRR1-6, FIIND or CARD domain of a mammalian NLRP1 protein (eg, human NLRP1), or a portion or fragment thereof.In this embodiment, the antibodies as described herein specifically bind to an amino acid sequence having at least 65% (e.g., 65%, 70%, 75%, 80%, 85%) sequence identity to a specific domain of human NLRP1 (e.g., Pyrin, NACHT, LRR1-6, FIIND, or CARD) or a fragment thereof. In one embodiment, a chicken anti-NLRP1 polyclonal custom designed and produced by Ayes Laboratories is used to reduce lung inflammation. The antibody may be directed against the following amino acid sequence in human NLRP1: CEYYTEIREREREKSEKGR (SEQ ID NO: 3). In one embodiment, an antibody as described herein that binds to a NLRP1 domain or a fragment thereof inhibits NLRP1 activity in mammalian lung cells (e.g., type II alveolar cells).

[0145] In yet another embodiment, the composition for reducing inflammation in the CNS or lung of a mammal comprises an antibody or an active fragment thereof as provided herein, which specifically binds to AIM2 or its domain. Any suitable anti-AIM2 antibody can be used, and several are commercially available. Examples of commercially available anti-AIM2 antibodies for use in the methods provided herein include, but are not limited to, rabbit polyclonal anti-AIM2 catalog number 20590-1-AP from Proteintech, Abcam anti-AIMS antibody (ab119791), rabbit polyclonal anti-AIM2 (N-terminal region) catalog number AP3851 from ECM biosciences, rabbit polyclonal anti-ASC catalog number E-AB-30449 from Elabsciences, and commercially available anti-AIM2 antibodies from Santa Clara, California. Cruz Biotechnology's anti-AIM2 mouse monoclonal antibody called AIM2 antibody (3C4G11) with catalog number sc-293174, Origene's mouse monoclonal AIM2 antibody with catalog number TA324972, Thermofisher Scientific's AIM2 monoclonal antibody (10M2B3), Antibodies-online's AIM2 rabbit polyclonal antibody ABIN928372 or ABIN760766, Biomatix coat's anti-AIM2 polyclonal antibody with catalog number CAE02153, Viva Systems Biology's anti-AIM2 polyclonal antibody (OABF01632), LSBio-C354127's rabbit polyclonal anti-AIM2 antibody LS-C354127, and Cell Signaling Technology's rabbit monoclonal anti-AIM2 antibody with catalog number MA5-16259. Rabbit polyclonal anti-AIM2 monoclonal antibody from Fab Gennix International Incorporated catalog number AIM2 201AP, MyBiosource rabbit polyclonal anti-AIM2 catalog number MBS855320, Signalway rabbit polyclonal anti-AIM2 catalog number 36253, Novus Biological rabbit polyclonal anti-AIM2 catalog number 43900002, GeneTex rabbit polyclonal anti-AIM2 GTX54910, Prosci rabbit polyclonal anti-AIM2 26-540, Bioorbyt mouse monoclonal anti-AIM2 orb333902, Abcam rabbit polyclonal anti-AIM2 ab93015), Abcam rabbit polyclonal anti-AIM2 ab76423, SignmaAldrich mouse polyclonal anti-AIM2 SAB1406827, or Biolegend anti-AIM2 3B10.The human AIM2 protein can be accession number NX_014862, NP004824, XP016858337, XP005245673, AAB81613, BAF84731 or AAH10940. In one embodiment, the antibody binds to the Pyrin or HIN-200 domain of a mammalian AIM2 protein (e.g., human AIM2), or a portion or fragment thereof. In this embodiment, the antibody as described herein specifically binds to the following amino acid sequence antibody, the amino acid sequence having at least 65% (e.g., 65%, 70%, 75%, 80%, 85%) sequence identity with a specific domain of human AIM2 (e.g., Pyrin or HIN-200) or a fragment thereof. In one embodiment, the antibody as described herein that binds to the AIM2 domain or a fragment thereof inhibits AIM2 activity in mammalian lung cells (e.g., type II alveolar cells).

[0146] Anti-inflammasome (e.g., anti-ASC, anti-NLRP1 or anti-AIM2) antibodies as described herein include polyclonal and monoclonal rodent antibodies, polyclonal and monoclonal human antibodies, or any portion thereof, having at least one antigen binding region of an immunoglobulin variable region, which specifically bind to a component of a mammalian inflammasome (e.g., an AIM2 inflammasome), such as, for example, ASC or AIM2. In some cases, the antibody is specific for ASC, such that if the antibody is generated against an epitope of a polypeptide and binds to at least a portion of a native or recombinant protein, the antibody is specific for ASC.

[0147] In certain embodiments, the antibodies provided herein include polypeptides having one or more amino acid substitutions, deletions or insertions. For example, an anti-ASC monoclonal antibody or an ASC binding antibody fragment comprises a polypeptide having one or more amino acid substitutions, deletions or insertions compared to a polypeptide having an amino acid sequence of one or more of SEQ ID NO: 6-8, 12-14, 18-22 or 28-31. The antibodies provided herein may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions, deletions or insertions. For example, an anti-ASC monoclonal antibody or an ASC binding antibody fragment may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions, deletions or insertions. Substitutions, deletions or insertions may be introduced by standard techniques, such as site-directed mutagenesis or PCR-mediated mutagenesis of a nucleic acid molecule encoding a polypeptide of an anti-ASC antibody or an ASC binding antibody fragment.

[0148] In certain embodiments, conservative amino acid substitutions are performed at one or more positions in the amino acid sequence of the antibody or antibody fragment disclosed herein. "Conservative amino acid substitution" is the substitution of amino acid residues with amino acid residues having similar side chains. In certain embodiments, conservative amino acid substitutions are performed only in the FR sequence of the antibody or antibody fragment and not in the CDR sequence. Families of amino acid residues with similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, for example, the amino acid residues in the polypeptide of the anti-ASC monoclonal antibody or ASC binding antibody fragment can be replaced by another amino acid residue from the same side chain family. In certain embodiments, the amino acid string may be replaced by a structurally similar string but with a different order and / or composition of side chain family members. One skilled in the art will be able to evaluate whether an anti-ASC monoclonal antibody or ASC binding antibody fragment comprising a polypeptide having one or more amino acid substitutions, deletions or insertions compared to a polypeptide having an amino acid sequence of one or more of SEQ ID NOs: 6-8, 12-14, 18-22 or 28-31 binds to an ASC protein by utilizing conventional, art-recognized methods (including but not limited to ELISA, Western blot, phage display, etc.).

[0149] Calculations of sequence homology or identity (the terms used interchangeably herein) between sequences can be performed as follows.

[0150] To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of the first and second amino acid or nucleic acid sequences to achieve optimal alignment, and non-homologous sequences can be ignored for comparison purposes). In an exemplary embodiment, the length of the reference sequence aligned for comparison purposes is at least 30%, 40%, 50%, 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein, amino acid or nucleic acid "identity" is equivalent to amino acid or nucleic acid "homology"). The percent identity between the two sequences is a function of the number of identical positions the sequences have, taking into account the number of gaps that need to be introduced to achieve optimal alignment of the two sequences, and the length of each gap.

[0151] Mathematical algorithms can be used to complete the determination of the percent identity between sequence alignment and two sequences. In one embodiment, the algorithm of Needleman et al. ((1970) J.Mol.Biol.48:444-453) in the GAP program incorporated into the GCG software package (available at www.gcg.com) is used, using the BLOSUM 62 matrix or PAM250 matrix and gap weights 16, 14, 12, 10, 8, 6 or 4 and length weights 1, 2, 3, 4, 5 or 6 to determine the percent identity between two amino acid sequences. In another embodiment, the GAP program in the GCG software package (available at www.gcg.com) is used, using the NWSgapdna CMP matrix and gap weights 40, 50, 60, 70 or 80 and length weights 1, 2, 3, 4, 5 or 6 to determine the percent identity between two nucleotide sequences. One set of parameters (and parameters that can be used if the practitioner is unsure what parameters should be applied to determine whether a molecule is within the sequence identity or homology limits of the invention) is the BLOSUM 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

[0152] The percent identity between two amino acid or nucleotide sequences can be determined using the algorithm of Meyers et al. ((1989) CABIOS 4:11-17) incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4.

[0153] In some aspects, the antibody is a monoclonal antibody. In other aspects, the antibody is a polyclonal antibody. The term "monoclonal antibody" refers to a population of antibody molecules that contain only one antigen binding site that can immunoreact with a specific antigenic epitope. Therefore, a monoclonal antibody composition typically exhibits a single binding affinity for a specific protein that immunoreacts with it.

[0154] In some aspects, the antibodies (anti-ASC monoclonal antibodies or ASC binding antibody fragments) of the invention are humanized, chimeric or human.

[0155] In some embodiments, the antibodies of the invention are humanized antibodies.

[0156] The term "humanized antibody" used herein refers to an antibody that has been engineered to include one or more human framework regions and non-human (e.g., mouse, rat, or hamster) complementary determining regions (CDRs) of heavy and / or light chains in the variable region. In certain embodiments, a humanized antibody includes a sequence in which, except for the CDR region, the sequence is completely human. In some cases, the Fv framework region (FR) residues of human immunoglobulin are replaced by corresponding non-human residues. In addition, the humanized antibody may include residues that are neither found in the human form of the antibody nor in the CDR or framework sequence of the import, but are included to further improve and optimize antibody performance. Typically, the humanized antibody will substantially include all of at least one (and typically two) variable domains, wherein all or substantially all CDR regions correspond to those of non-human immunoglobulins, and all or substantially all FR regions are those of human immunoglobulin consensus sequences. The FR region may be modified in any manner known in the art and / or provided herein. The modification may confer desired characteristics, such as increased half-life and / or improved expression in a host cell. In one embodiment, one or more FR regions may be modified or mutated as described in US 20150232557, which is incorporated herein by reference. Other forms of humanized antibodies may have one or more CDRs (CDR L1, CDR L2, CDR L3, CDR H1, CDR H2, or CDR H3) that are altered with respect to the original antibody, which are also referred to as one or more CDRs "derived from" one or more CDRs of the original antibody. The humanized antibody also optimally comprises at least a portion of an immunoglobulin constant region or constant domain (Fc), typically at least a portion of a constant region or constant domain of a human immunoglobulin.

[0157] Relative to non-humanized antibodies, humanized antibodies are generally less immunogenic to humans, and therefore provide therapeutic benefits in some cases. For example, the antibody constant region can be engineered to make it immunologically inert (e.g., not triggering complement lysis). See, for example, PCT Publication No. PCT / GB99 / 01441; UK Patent Application No. 9809951.8, each of which is incorporated herein by reference in its entirety. Those skilled in the art will be aware of humanized antibodies, and will also be aware of suitable techniques for producing them. See, for example, Hwang, WYK, et al., Methods 36:35, 2005; Queen et al., Proc. Natl. Acad. Sci. USA, 86:10029-10033, 1989; Jones et al., Nature, 321:522-25, 1986; Riechmann et al., Nature, 332:323-27, 1988; Verhoeyen et al., Science, 239:1534-36, 1988; Orlandi et al., Proc. Natl. Acad. Sci. USA, 86:3833-37, 1989; U.S. Patent Nos. 5,225,539, 5,530,101, 5,585,089, 5,693,761, 5,693,762, 6,180,370; and Selick et al., WO 90 / 07861, each of which is incorporated herein by reference in its entirety. Other methods of humanizing antibodies that can also be used are disclosed in the following literature: Daugherty et al., Nucl. Acids Res. 19:2471-2476, 1991; and U.S. Patent Nos. 6,180,377, 6,054,297, 5,997,867, 5,866,692, 6,210,671 and 6,350,861; and PCT Publication No. WO 01 / 27160, each of which is incorporated herein by reference in its entirety. For example, the anti-ASC antibody or anti-ASC antigen-binding fragment of the present invention may comprise a VH region amino acid sequence comprising HCDR1 of SEQ ID NO:6, HCDR2 of SEQ ID NO:7, and HCDR3 of SEQ ID NO:8; and a VL region amino acid sequence comprising LCDR1 of SEQ ID NO:12, LCDR2 of SEQ ID NO:13, and LCDR3 of SEQ ID NO:14; and one or more human framework region sequences.

[0158] In some embodiments, the antibody of the present invention is a chimeric antibody and specifically binds to ASC. In some cases, the anti-ASC chimeric antibody reduces the activity of ASC. As used herein, the term "chimeric antibody" refers to an antibody that has been engineered to include at least one human constant region. For example, one or all variable regions of one or two light chains of a mouse antibody (e.g., a mouse monoclonal antibody) and / or one or all variable regions of one or two heavy chains can each be connected to a human constant region (such as, but not limited to, an IgG1 human constant region). Relative to non-chimeric antibodies, chimeric antibodies are generally less immunogenic to humans, and therefore provide therapeutic benefits in some cases. Those skilled in the art will be aware of chimeric antibodies, and will also be aware of suitable techniques for producing them. See, for example, Cabilly et al., U.S. Patent No. 4,816,567; Shoemaker et al., U.S. Patent No. 4,978,775; Beavers et al., U.S. Patent No. 4,975,369; and Boss et al., U.S. Patent No. 4,816,397, each of which is incorporated herein by reference in its entirety. For example, an antibody or antigen-binding fragment of the invention may comprise a VH region comprising SEQ ID NO: 22, a VL region comprising SEQ ID NO: 31, and a human constant region.

[0159] As used herein, the terms "immunobinding" and "immunobinding properties" refer to the type of non-covalent interaction that occurs between an immunoglobulin molecule (e.g., an antibody) and an antigen to which the immunoglobulin is specific. The strength or affinity of an immunobinding interaction can be expressed as the dissociation constant (K) of the interaction. d ), where the smaller K d =K indicates greater affinity. The immunological binding properties of a selected polypeptide can be quantified using methods well known in the art. One such method requires measuring the rates of formation and dissociation of the antigen binding site / antigen complex, where those rates depend on the concentration of the complex partners, the affinity of the interaction, and geometric parameters that also affect the rates in both directions. Thus, the "association rate constant" (K on ) and the “dissociation rate constant” (K off ) Both can be determined by calculating the concentration and the actual association and dissociation rates. (See Nature 361: 186-87 (1993)). off / K on The ratio of allows cancellation of all affinity-independent parameters and is equal to the dissociation constant K d (See generally Davies et al. (1990) Annual Rev Biochem 59:439-473). When the equilibrium binding constant (K d) is ≤10 μM, ≤10 nM, ≤10 nM and ≤100 pM to about 1 pM, as measured by an assay (such as a radioligand binding assay or similar assays known to those skilled in the art), the antibodies of the present invention are said to specifically bind to an epitope (e.g., an ASC fragment having amino acid SEQ ID NO: 5).

[0160] In certain aspects, the antibodies of the invention are monovalent or divalent and comprise single or double chains. Functionally, the binding affinity of the antibodies can be between 10 -5 M to 10 -12 M. For example, the binding affinity of an antibody ranges from 10 -6 M to 10 -12 M. From 10 -7 M to 10 -12 M. From 10 -8 M to 10 -12 M. From 10 -9 M to 10 -12 M. From 10 -5 M to 10 -11 M. From 10 -6 M to 10 -11 M. From 10 - 7 M to 10 -11 M. From 10 -8 M to 10 -11 M. From 10 -9 M to 10 -11 M. From 10 -10 M to 10 -11 M. From 10 -5 M to 10 -10 M. From 10 -6 M to 10 -10 M. From 10 -7 M to 10 -10 M. From 10 -8 M to 10 -10 M. From 10 -9 M to 10 -10 M. From 10 -5 M to 10 -9 M. From 10 -6 M to 10 -9 M. From 10 -7 M to 10 -9 M. From 10 -8 M to 10 -9 M. From 10 -5 M to 10 -8 M. From 10 -6 M to 10 -8 M. From 10 -7 M to 10-8 M. From 10 -5 M to 10 -7 M. From 10 -6 M to 10 -7 M or from 10 -5 M to 10 -6 M.

[0161] Methods for determining monoclonal antibody specificity and affinity by competitive inhibition can be found in Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1988; Colligan et al., eds., Current Protocols in Immunology, Greene Publishing Assoc. and Wiley Interscience, New York, (1992, 1993); and Muller, Meth. Enzymol. 92:589-601, 1983, the entireties of which are incorporated herein by reference.

[0162] Anti-inflammasome (e.g., anti-ASC and anti-AIM2) antibodies of the invention can be routinely prepared according to methods such as, but not limited to, inoculation of appropriate animals with polypeptides or antigenic fragments, in vitro stimulation of lymphocyte populations, synthetic methods, hybridomas, and / or recombinant cells expressing nucleic acids encoding such anti-ASC or anti-NLR1 antibodies. Immunization of animals with purified recombinant ASC or peptide fragments thereof (e.g., residues 178-193 (SEQ ID NO: 1) of rat ASC (e.g., Accession No. BAC43754), EQID NO: 2 of human ASC, or residues 21-41 (SEQ ID NO: 5) of human ASC (e.g., Accession No. NP_037390.2) is an example of a method for preparing anti-ASC antibodies. Similarly, immunization of animals with purified recombinant NLRP1 or peptide fragments thereof (e.g., residues MEE SQS KEE SNT EG-cys (SEQ ID NO: 4) of rat NALP1 or SEQ ID NO: 3 of human NALP1 is an example of a method for preparing anti-NLRP1 antibodies.

[0163] Monoclonal antibodies that specifically bind to ASC or NLRP1 can be obtained by methods known to those skilled in the art. See, for example, Kohler and Milstein, Nature 256: 495-497, 1975; U.S. Patent No. 4,376,110; Ausubel et al., ed., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley Interscience, New York, (1987, 1992); Harlow and Lane ANTIBODIES: A Laboratory Manual Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1988; Colligan et al., ed., Current Protocols in Immunology, Greene Publishing Assoc. and Wiley Interscience, New York, (1992, 1993), the contents of which are incorporated herein by reference in their entirety. Such antibodies may belong to any immunoglobulin class, including IgG, IgM, IgE, IgA, GILD, and any subclass thereof. Hybridomas producing the monoclonal antibodies of the present invention can be cultured in vitro, in situ, or in vivo. In one embodiment, the hybridoma producing the anti-ASC monoclonal antibodies of the present disclosure is an ICCN1.OH hybridoma. In another embodiment, the hybridoma producing the anti-ASC monoclonal antibodies of the present disclosure produces a monoclonal antibody comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region amino acid sequence comprises HCDR1 of SEQ ID NO: 6, HCDR2 of SEQ ID NO: 7, and HCDR3 of SEQ ID NO: 8, or a variant thereof having at least one amino acid substitution in HCDR1, HCDR2, and / or HCDR3. In another embodiment, the hybridoma producing the anti-ASC monoclonal antibodies of the present disclosure produces a monoclonal antibody comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VL region amino acid sequence comprises LCDR1 of SEQ ID NO: 12, LCDR2 of SEQ ID NO: 13, and LCDR3 of SEQ ID NO: 14, or a variant thereof having at least one amino acid substitution in LCDR1, LCDR2, and / or LCDR3.In yet another embodiment, the hybridoma producing the anti-ASC monoclonal antibody of the present disclosure produces a monoclonal antibody comprising a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region amino acid sequence comprises HCDR1 of SEQ ID NO:6, HCDR2 of SEQ ID NO:7, and HCDR3 of SEQ ID NO:8, or a variant thereof having at least one amino acid substitution in HCDR1, HCDR2, and / or HCDR3; and wherein the VL region amino acid sequence comprises LCDR1 of SEQ ID NO:12, LCDR2 of SEQ ID NO:13, and LCDR3 of SEQ ID NO:14, or a variant thereof having at least one amino acid substitution in LCDR1, LCDR2, and / or LCDR3.

[0164] Administration of the composition

[0165] The compositions of the invention can be administered to mammals (e.g., rodents, humans) in any suitable formulation. For example, the anti-ASC antibody can be formulated in a pharmaceutically acceptable carrier or diluent (such as saline or buffered saline). Suitable carriers and diluents can be selected based on the mode and route of administration and standard pharmaceutical practice. Descriptions of exemplary pharmaceutically acceptable carriers and diluents and pharmaceutical formulations can be found in Remington's Pharmaceutical Sciences (a standard text in this field) and USP / NF. Other substances may be added to the composition to stabilize and / or preserve the composition.

[0166] The composition of the present invention can be administered to mammals by any conventional technique. Typically, this administration will be by suction or parenteral (e.g., intravenous, subcutaneous, intratumoral, intramuscular, intraperitoneal or intrathecal introduction). The composition can also be directly administered to the target site by, for example, surgical delivery to an internal or external target site or by delivery to a vascular accessible site by a catheter. The composition can be administered by a single bolus, multiple injections or by continuous infusion (e.g., intravenous, by peritoneal dialysis, pump infusion). For parenteral administration, the composition can be prepared in a sterile, pyrogen-free form.

[0167] Effective dose

[0168] The above-described compositions can be administered to a mammal (e.g., rat, human) in an effective amount (i.e., an amount capable of producing a desired result in the treated mammal (e.g., reducing inflammation in the CNS of a mammal that has undergone traumatic injury to the CNS or a stroke or suffers from an autoimmune disease, an autoinflammatory disease, a metabolic disease, a neurodegenerative disease, or a CNS disease). Such a therapeutically effective amount can be determined as described below. The therapeutically effective amount of a composition comprising an agent as provided herein (e.g., a monoclonal antibody as provided herein or an antibody fragment derived therefrom, such as, for example, IC 100) can typically be about 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 2, 4, 6, 8, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175 or 200 mg / kg patient body weight. The therapeutically effective amount of a composition comprising an agent as provided herein (e.g., a monoclonal antibody as provided herein or an antibody fragment derived therefrom, such as IC 100) can typically be about 0.001 to about 200 mg / kg patient body weight. A therapeutically effective amount of a composition comprising an agent as provided herein (e.g., a monoclonal antibody as provided herein or an antibody fragment derived therefrom, such as, for example, IC100) can generally be from about 0.001 mg / kg to about 0.01 mg / kg, from about 0.01 mg / kg to about 0.1 mg / kg, from about 0.1 mg / kg to about 1 mg / kg, from about 1 mg / kg to about 10 mg / kg, from about 10 mg / kg to about 25 mg / kg, from about 25 mg / kg to about 50 mg / kg, from about 50 mg / kg to about 75 mg / kg, from about 75 mg / kg to about 100 mg / kg, from about 100 mg / kg to about 125 mg / kg, from about 125 mg / kg to about 150 mg / kg, from about 150 mg / kg to about 175 mg / kg, or from about 175 mg / kg to about 200 mg / kg of the subject's body weight. A composition comprising an agent as provided herein (eg, a monoclonal antibody as provided herein or an antibody fragment derived therefrom, such as, for example, IC100) can be administered in a single dose or in multiple doses.

[0169] Toxicity and therapeutic efficacy of the compositions used in the methods of the invention can be determined by standard pharmaceutical procedures, such as by determining the LD using cells in culture or experimental animals. 50 (the dose lethal to 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as LD 50 / ED 50In some cases, the compositions provided herein exhibit a large therapeutic index. Although compositions that exhibit toxic side effects may be used, it should be noted that a delivery system can be designed that minimizes the potential damage of such side effects. In some cases, the dosage of the compositions provided herein falls within the range of ED 50 The dosage may vary within this range depending on the dosage form employed and the route of administration utilized.

[0170] As is well known in the medical and veterinary arts, the dosage for any one subject depends on many factors, including the subject's size, body surface area, age, the specific composition to be administered, the time and route of administration, general health, and other drugs administered concurrently.

[0171] Example

[0172] The present invention is further illustrated by the following specific examples. The examples are provided for illustration only and should not be construed as limiting the scope of the present invention in any way.

[0173] Example 1: Role of EV-mediated inflammasome signaling in ALI after TBI and the impact of its neutralization

[0174] Pulmonary dysfunction frequently manifests as a complication of severe traumatic brain injury (1). Approximately 20%-25% of TBI subjects develop acute lung injury (ALI) (2), but the mechanisms mediating TBI-induced ALI pathology remain poorly defined. Previous literature supports the view that lung dysfunction after TBI is due to sympathetic nervous system responses to increased intracranial pressure, leading to cardiopulmonary dysfunction (42). However, recent studies have shown that systemic inflammatory responses also play a key role in TBI-induced lung injury (43). Specifically, the HMGB1-RAGE ligand receptor pathway serves as a central transduction mechanism of lung dysfunction after TBI (8). In addition, HMGB1 induces AIM2 inflammasome activation (37). In addition, previous literature revealed that pathogens secrete EVs carrying DAMPs (such as HMGB1) and trigger inflammation (Buzas et al., 2014). Various studies have shown that the blood-brain barrier (BBB) ​​is permeable after TBI as early as 3-6 hours after injury, resulting in a compromised protective barrier between the brain and the intravascular compartment and leading to protein and fluid leakage (44). Disruption of the BBB after injury leads to the secretion of inflammatory mediators (such as DAMPs), which may further lead to brain inflammation and damage to distal organs (5). Several inflammatory mediators can serve as clear markers of brain injury, however their effectiveness has not been widely accepted (45). In addition, there are currently no clinically approved treatments or biomarkers for TBI-induced ALI. Recently, EVs have become an area of ​​interest in biomarker research for several different types of diseases, including lung injury (46) and TBI (47). It has been previously shown that inflammasome proteins are increased in EVs isolated from the cerebrospinal fluid of patients with TBI when compared to control samples (14). In this example, the contribution of EV-mediated inflammasome signaling in the etiology of TBI-induced ALI was examined.

[0175] Materials and methods

[0176] Animals and Traumatic Brain Injury

[0177] All animal procedures were approved by the Institutional Animal Care and Use Committee of the University of Miami Miller School of Medicine (Animal Welfare Assurance A3224-01) and were performed in accordance with the NIH Guidelines for the Care and Use of Laboratory Animals. The ARRIVE guidelines were followed in conducting this study. All C57 / BL6 mice were 8-12 weeks and 24 to 32 grams. The mice were prospectively randomized to experimental groups for TBI (sham, 4h, 24h), experimental groups for adoptive transfer and treatment (naive, sham-saline, untreated, enoxaparin, anti-ASC). For the TBI experimental group, sham animals underwent surgery but were not injured. For adoptive transfer treatment studies, the sham-saline group underwent surgery and received saline as a vehicle treatment. Naive animals did not undergo surgery. Based on power analysis (using G* power analysis, where the effect size F=0.85, α was set to 0.05) and historical data 49,50 , using a sample size of 5 to 6 per group. All mice were housed in a viral antigen-free (VAF) animal facility at the Lois Pope Life Center at the University of Miami, with a 12-hour light / dark cycle, and food and water were provided ad libitum. The facility performed a feeding program twice a week, and the condition of the animals was checked daily. Animals were observed postoperatively in our operating room, kept on a heating pad, and body temperature was controlled with a rectal probe to maintain it at 37°C, and then transferred to an animal dormitory.

[0178] Prior to surgery, animals were anesthetized with ketamine and xylazine (intraperitoneal, ip). The anesthetized animals were then placed on a heating pad to ensure that the body temperature was 37°C. TBI was performed using a controlled cortical impact (CCI) model. A 5mm craniotomy was performed on the right cortex (posterior -2.5mm, lateral to the bregma 2.0mm). Injury was induced using an ECCI-6.3 device (Custom Design & Fabrication, Richmond, VA, USA), in which a 3mm impactor was subjected to a speed of 6m / s, a depth of 0.8mm, and an impact duration of 150ms (15). After these procedures, the animals were returned to the cage and given food and water. As described above, animals were killed 4 hours and 24 hours after TBI. Sham-operated animals were anesthetized and subjected to the same preoperative incision as injured animals but without craniotomy or contusion.

[0179] Tissue collection

[0180] Prior to perfusion, all animals were anesthetized with ketamine and xylazine. Animals were then perfused intratracheally. Lungs were infused with 4% paraformaldehyde (PFA) at 20 cm H2O using a tracheal tube and then fixed overnight in 4% PFA at 4°C. Fixed lung tissue was paraffin-embedded and processed for 5 μm sections (16). Right lung tissue was collected for protein isolation and molecular analysis. Animals were then sacrificed by decapitation and right cortical tissue was collected for protein isolation and molecular analysis.

[0181] Pyroptotic body isolation assay

[0182] Mouse lung tissue lysate was filtered through a 5 μm low-binding polyvinylidene fluoride (PVDF) membrane (Millipore). After filtration, the supernatant was centrifuged at 2,700 x g for 8 minutes. The pellet was resuspended in 40 μl 3[(3-cholamidopropyl)dimethylamino]-propanesulfonic acid (CHAPS) buffer (20 mmol / L HEPES-KOH, pH 7.5, 5 mmol / L MgCl2, 0.5 mmol / LEGTA, 0.1 mmol / L phenylmethylsulfonyl fluoride, protease inhibitor cocktail and 0.1% CHAPS). Pyroptotic bodies were precipitated by centrifugation at 2,700 x g for 8 minutes. The pellet was then resuspended and incubated in 27.8 μl CHAPS buffer with 2.2 μl disuccinimide substrate (9) at room temperature for 30 minutes to crosslink ASC dimers. Finally, an equal amount of 2x Laemmli buffer was added and proteins were analyzed by immunoblotting using commercially available antibodies against ASC and GSD.

[0183] Nuclear and cytoplasmic extraction

[0184] According to the manufacturer's instructions, NE-PER nuclear and cytoplasmic extraction reagent (Thermo Scientific) was used to extract the nuclear and cytoplasmic fractions. In short, mouse lung tissue samples were cut into 20-100 mg fragments and centrifuged at 500 x g for 5 minutes. The tissue fragments were homogenized with cytoplasmic extraction reagent and centrifuged at 16,000 x g for 5 minutes. The supernatant (cell extract) was then removed and the precipitate was centrifuged at 16,000 x g for 10 minutes with nuclear extraction reagent (Thermo Scientific). This supernatant corresponds to the nuclear fraction, which was removed and stored at -80 ° C.

[0185] Immunoblotting

[0186] Lung and brain tissue samples were snap frozen in liquid nitrogen and stored at -80°C. 2 mm sections of right lower lung and right cortical tissue were homogenized in extraction buffer containing a cocktail of protease and phosphatase inhibitors (Sigma, St Louis, MO, USA) and resolved in 4%-20% Tris-TGX Criterion precast gels (Bio-Rad, Hercules, CA, USA) using antibodies against caspase-1 (Novus Biologicals), ASC (Santa Cruz), IL-1β (Cell Signaling), IL-18 (Abcam), AIM2 (Santa Cruz), and HMGB1 (Millipore) as described by de Rivero Vaccari et al. 2015 (13). Band density was quantified using Image Lab, and all data were normalized to β-actin.

[0187] Immunohistochemistry

[0188] The tissue sections were deparaffinized in xylene and then rehydrated with ethanol and Tris-buffered saline. Immunohistochemical procedures were then performed as previously described for double staining (16). The sections were incubated overnight at 4 °C with antibodies against caspase-1 and ASC (Millipore), AIM2 (Santa Cruz), HMGB1 (Millipore), and SPC (Millipore). Immunostained lung sections of sham-operated, 4-hour, and 24-hour mice were examined with a Zeiss laser scanning confocal microscope (Zeiss, Inc., Thornwood, NY, USA). Lung sections were analyzed by individuals who were unaware of the grouping.

[0189] EV isolation

[0190] EVs were isolated from the serum of TBI-injured mice and injured mice using total exosome isolation solution according to the manufacturer's instructions (Invitrogen). Briefly, 100 μl of each sample was centrifuged at 2000 x g for 30 minutes. The supernatant was then incubated with 20 μl of total exosome isolation (TEI) reagent at 4 °C for 30 minutes, followed by centrifugation at 10,000 x g for 10 minutes at room temperature. The supernatant was discarded and the pellet was resuspended in 100 μl PBS. EVs were characterized by expression of CD81 and Nanosight tracking analysis ( Figure 6 ).

[0191] Adoptive transfer of EVs

[0192] Serum-derived EVs from C57BL-6 TBI and sham-operated mice were injected via the jugular vein at 1.0 × 1010 Particles / g / body weight 48 The dose of was injected into naive C57BL-6 mice. Particle counts were measured by Nanosight tracking analysis and samples were diluted accordingly. Prior to surgery, animals were anesthetized with ketamine and xylene. A 1-2 cm incision was made between the jaw and clavicle. The jugular vein was elevated and tied, followed by a catheter. Serum-derived EVs were transferred and lung and brain tissues were collected for analysis 24 hours after injection (n=5).

[0193] Enoxaparin and anti-ASC therapy

[0194] EVs derived from serum of TBI mice were injected into naive C57-BL6 mice by jugular vein injection. One hour later, enoxaparin (3 mg / kg) (n=4) and anti-ASC (IC100; 5 mg / kg) (n=4) were administered to the recipient animals. The following groupings were used: 1) the naive group received no treatment, 2) the sham saline group was used as a negative control and received only jugular vein injection of saline, 3) the untreated group received EVs from TBI mice without any treatment and was used as a positive control, 4) the ENOX group received EVs and enoxaparin from TBI mice, and 5) the anti-ASC group received EVs and anti-ASC from TBI mice. The order of treatment was randomized. Lung and brain tissues were collected 24 hours after injection for analysis. It should be noted that the anti-ASC antibody used in the treatment experiments is a humanized monoclonal antibody against ASC and recognizes mouse, human and porcine ASC.

[0195] Histology and lung injury scores

[0196] Lung tissue sections were stained by standard hematoxylin and eosin method for histology, morphometry and ALI scoring. Lung sections were scored by blinded pathologists using the lung injury scoring system reported from the American Thoracic Society workshop (17). Twenty random high-power fields were selected for scoring. The criteria for ALI scoring are based on the number of neutrophils in the alveolar cavity, interstitial space, hyaline membrane, protein debris filling the air space, and thickening of the alveolar septa. Based on these criteria, scores between 0 (no damage) and 1 (severe damage) were given.

[0197] Statistical analysis

[0198] Data were analyzed using Student's T test and one-way ANOVA for two groups and Tukey's multiple comparison test for two or more groups (GraphPad Prism version 7.0). The D'Agostino-Pearson test was used to test normality. Data are expressed as mean + / - SEM. The significance P value used was *p<0.05.

[0199] result

[0200] Severe TBI increases AIM2 inflammasome protein and HMGB1 expression in mouse brain

[0201] Excessive levels of the proinflammatory cytokines IL-1β and IL-18 and inflammasome proteins have been associated with secondary injury following fluid-induced brain injury (18). To determine whether severe CCI induces changes in the processing of proinflammatory cytokines and inflammasome protein levels, cortical lysates were analyzed; however, studies of inflammasome activation in severe TBI are limited. In this example, following severe CCI, cortical lysates were examined for caspase-1 ( Figure 1A , Figure 1B )(p<0.001), ASC( Figure 1A , Figure 1C )(p=0.003), IL-18( Figure 1A , Figure 1D )(p=0.0042), AIM2( Figure 1A , Figure 1F )(p=0.0197) and IL-1β( Figure 1A , Figure 1G )(p=0.0141) levels. Levels of caspase-1, ASC, AIM2, and IL-Iβ peaked at 4 h after CCI and decreased at 24 h. The time course of inflammatory cytokine maturation was slightly different, peaking at 24 h after TBI. As other proteins have shown a role for the inflammasome DAMP HMGB1 in activating the AIM2 inflammasome, the levels of these proteins were also determined in cortical lysates. Figure 1A , Figure 1E As shown, CCI induced a significant increase in HMGB1 levels at 4 and 24 hours after injury ( Figure 1A , Figure 1E )(p=0.0121). These data indicate that after severe CCI in mice, the level of AIM2 inflammasome protein is significantly elevated in the post-injury cortex.

[0202] Severe TBI increases AIM2 inflammasome protein and HMGB1 expression in mouse lungs

[0203] To determine whether CCI induces inflammasome activation in the lung, lung lysates were subjected to caspase-1 ( Figure 1H , Fig. 1I )(p=0.0026), ASC( Figure 1H , Figure 1J )(p=0.0427), IL-18( Figure 1H , Figure 1K )(p=0.0025), IL-1β( Figure 1H , Figure 1N )(p=0.0012) and AIM2( Figure 1H , Figure 1M Immunoblotting analysis of NLRP3 (p < 0.001) and NLRP3 (p = 0.0047) (Supplementary Figure 1). Compared with sham-operated controls, increased levels of caspase-1, ASC, IL-18, and AIM2 were significantly higher at 4 and 24 hours after injury. However, the time course of increased protein expression was slightly different from that observed in the brain, where they peaked at 24 hours after CCI. Since the HMGB1-RAGE axis plays a role in the mechanism of TBI-induced lung dysfunction (8), lung lysates were analyzed for HMGB1 protein expression levels. Figure 1H , Figure 1L (p=0.0158) showed that HMGB1 expression was increased at 4 and 24 hours after TBI, suggesting that AIM2 inflammasome and HMGB1 play a role in the inflammatory response in the lung after TBI.

[0204] TBI induces pyroptosis in the lungs of mice

[0205] As previously shown, activation of the AIM2 inflammasome in cortical neurons leads to pyroptotic cell death (19). To investigate whether TBI induces pyroptosis in mouse lung tissue, pyroptotic bodies were isolated from lung tissue after TBI. TBI animals sacrificed 4 h after injury showed evidence of ASC oligomerization compared to sham-operated animals ( Figure 4A ). ASC dimers and trimers (50 and 75 kDA, respectively) were observed in TBI animals. These results suggest the formation of pyroptotic bodies, which can be characterized by supramolecular assembly of ASC oligomers. In addition, sclerotin D (GSDMD), which is cleaved upon activation of caspase-1 and triggers pyroptosis and IL-1β release (20), was significantly increased in the lungs of TBI animals compared with sham surgery ( Figure 4B and Figure 4C )(p=0.0001). These findings suggest that pyroptosis contributes to cell death in lung tissue after TBI.

[0206] TBI increases the immunoreactivity of inflammasome proteins in type II alveolar epithelial cells

[0207] TBI may cause capillary leakage, resulting in increased vascular permeability and damage to specialized alveolar epithelial cells, called type II pneumocytes (5). To examine the cellular effects of TBI on inflammasome expression in the lung following injury, immunohistochemistry was performed in lung sections from sham-operated, 4-hour, and 24-hour injured animals. Type II alveolar epithelial cells are known to be the major lung cell type damaged in ALI (17). Lung sections were stained with antibodies against AIM2, caspase-1, and ASC (green) and co-stained with Pro-surfactant protein C (Pro-SPC, red), a marker for type II epithelial cells, and DAPI nuclear stain (blue). Figure 2A-2C As shown, active caspase-1 ( Figure 2A )、ASC( Figure 2B ) and AIM2( Figure 2C ) are present in SPC-positive cells (arrows). The immunoreactivity of these inflammasome proteins is increased after TBI. These findings suggest that inflammasome proteins are expressed in type II alveolar epithelial cells and that TBI leads to increased immunoreactivity in these cells.

[0208] TBI increases nuclear and cytoplasmic HMGB1 expression

[0209] To determine the cellular distribution of HMGB1 in lung cells after TBI, nuclear and cytoplasmic fractions from lung homogenates were separated ( Figure 3A , Figure 3C )(p=0.0337). Immunoblotting showed that HMGB1 expression in both fractions increased significantly 4 hours after TBI ( Figure 3B , Figure 3D )(p=0.0345). Immunohistochemical analysis of HMGB1 was also performed to determine changes in immunoreactivity in lung sections after TBI. Sections were co-stained for HMGB1 (green) and SPC (red) as well as DAPI nuclear staining (blue). When compared to sham surgery, HMGB1 immunoreactivity increased at 4 and 24 hours. Weak immunoreactivity of HMGB1 was observed in SPC-positive cells (arrows) ( Figure 3E ); therefore, it is suggested that the changes of HMGB1 in injured lung tissue may be cytoplasmic.

[0210] TBI induces changes in lung morphology and induces ALI

[0211] ALI can be characterized by an inflammatory process that results in alveolar and interstitial edema and infiltration of inflammatory cells into the alveolar spaces (23). Figure 5A) shows that severe TBI causes significant changes in lung structure and morphology 4 and 24 hours after injury. Sham-operated animals show normal alveolar morphology, while injured animals show dramatic changes in alveolar edema, but slightly decreased 24 hours after injury (long arrows). In addition, there is evidence of neutrophil infiltration (arrows) and morphological changes in alveolar capillary membranes (*) at two time points. Injured animals show signs of interstitial edema, which is more obvious 4 hours after injury, but still obvious 24 hours after injury (short arrows). Finally, injured animals also show evidence of thickening of interstitial areas and alveolar septa (pounds, #).

[0212] To confirm that severe injury induced ALI, tissue sections were analyzed using the ALI scoring system defined by the American Thoracic Society (17). This system is based on evidence of neutrophil infiltration into the alveoli and interstitial spaces, hyaline membrane formation, proteinaceous debris filling the air spaces, and thickening of the alveolar septa (17). These features were significantly elevated in injured animals compared with sham surgery, and ALI scores were generally higher in TBI animals ( Figure 5B )(p=0.0017).

[0213] Enoxaparin and anti-ASC antibody treatment significantly reduced inflammasome expression and ALI after adoptive transfer of EVs from TBI mice

[0214] To provide evidence that EVs and their transporters released into the circulation after TBI can induce inflammasome activation in the lung, classical adoptive transfer experiments were performed using serum-derived EVs from mice with severe CCI. EV preparations were validated using Western blotting for the EV marker CD81 ( Figure 6 ). Controls received EVs isolated from sham-operated or naive animals. Figure 7A-7F As shown, active caspase-1 ( Fig. 7A , Figure 7B )、ASC( Fig. 7A , Figure 7C )、IL-18( Fig. 7A , Fig.7D )、AIM2( Fig. 7A , Fig. 7E ) and HMGB1( Fig. 7A , Figure 7F ) was significantly increased. In addition, in the lungs treated with EVs from TBI mice, infiltration of inflammatory cells (arrows) was evident ( Figure 7G Finally, ALI scores were also significantly higher in animals that received EVs from injured mice ( Figure 7G). These studies provide evidence for a neuro-respiratory-inflammasome axis, in which EV release into the circulation after TBI activation of inflammasomes in lung target cells contributes to the pathogenesis of ALI.

[0215] Next, exosome uptake blockade was attempted by treatment with enoxaparin or a monoclonal antibody against ASC (IC 100) following adoptive transfer of EVs from injured mice to naive mice. Negative control animals received saline, and positive control animals received no treatment. Figure 8A-8F As shown, after treatment with enoxaparin or a humanized monoclonal anti-ASC antibody (e.g., IC100 antibody), caspase-1 ( Fig. 8A , Figure 8B )、ASC( Fig. 8A , Figure 8C ), IL-1β( Fig. 8A , Fig.8D )、AIM2( Fig. 8A , Fig. 8E ) and HMGB1( Fig. 8A , Fig.8F ) was significantly reduced compared to the untreated (positive control) group (p = <0.0001). In addition, H&E-stained lung sections showed a significant reduction in neutrophil infiltration into the alveolar and interstitial spaces, and no signs of septal thickening ( Figure 9A-9D Animals treated with enoxaparin and anti-ASC antibody (IC 100) had significantly lower ALI scores compared to the untreated group ( Fig.9E )(p=<0.0001). Thus, EV release into the circulation after TBI plays a role in inflammasome activation in lung cells leading to ALI.

[0216] in conclusion:

[0217] TBI may be associated with a higher incidence of certain medical complications, especially pulmonary and central nervous system dysfunction. In this example, severe TBI is shown to increase HMGB1 and inflammasome expression in cortical and lung tissues (e.g., AIM2, caspase-1, and ASC expression) and induce lung morphological changes consistent with ALI (e.g., neutrophil infiltration into alveolar and interstitial spaces, alveolar septal thickening, alveolar edema and hemorrhage), and introduce the concept of the Neural Respiratory Inflammatory Axis. Importantly, TBI causes cell pyroptosis in lung tissue (e.g., the presence of GSDMD cleavage) and increased expression of inflammasome proteins in type II alveolar epithelial cells. In addition, adoptive transfer of EVs from TBI mice activates inflammasomes and induces ALI, indicating that brain injury induces the release of EVs containing a bunch of inflammasome proteins, which then proceed to cause ALI. Furthermore, the results showed that by inhibiting EV uptake (enoxaparin) and inflammasome activation (anti-ASC antibody (IC 100) treatment), inflammasome protein expression and the development of ALI were reduced.

[0218] In summary, this example demonstrates that AIM2 inflammasome signaling plays a central role in the pathomechanism of lung injury after TBI and demonstrates a mechanism in which TBI-induced ALI involves EV-mediated inflammasome signaling. These data provide evidence that EV-mediated inflammasome signaling can play a central role involving the neuronal-respiratory-inflammatory axis. Therefore, targeting this axis with antibodies to inflammasome proteins or drugs that block EV uptake may provide a therapeutic approach for neurotrauma-induced ALI in all areas of critical care medicine. In view of these results, the disclosed therapeutic strategy may be generally useful for treating inflammatory diseases of the lung.

[0219] Incorporated by Reference

[0220] The following references are incorporated by reference in their entirety for all purposes.

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[0271] Example 2: Role of EV-mediated inflammasome signaling in ALI after TBI in human patients

[0272] As a follow-up to the experiments on mice in Example 1, the effects of EVs isolated from human TBI patients on inflammasome signaling in human lung endothelial cells were examined.

[0273] In the first experiment, serum-derived EVs were isolated from TBI and control patients using a total exosome isolation kit (Thermofisher). Pulmonary human microvascular endothelial cells (HMVEC-Lonza) were cultured and plated on 12-well plates. After reaching confluence, isolated EVs (1.94 x 108 particles / ml) from TBI and control patients were delivered to the cells for a 4-hour incubation period. After incubation, cells were harvested with 200ul lysis buffer and cell lysates were used for Western blot analysis.

[0274] In the second experiment, serum-derived EVs were isolated from TBI and control patients using a total exosome isolation kit (Thermofisher). Pulmonary human microvascular endothelial cells (HMVE C-Lonza) were cultured and plated on 96-well plates. After reaching confluence, isolated EVs (1.94x 108 particles / ml) from TBI and control patients were delivered to the cells for 3 hours of incubation, and then incubated for 1 hour with caspase-1FAM FLICA (Immunohistochemistry Technologies) at a 1:30 volume ratio. After incubation, the culture medium was removed and the cells were washed 3 times with apoptosis wash buffer (Immunohistochemistry Technologies). The cells were then co-stained, with Hoechst used for nuclear staining, and propidium iodide used for cell death staining. Images were taken using an E VOS microscope, and the cells were then read under a fluorescent plate reader with an excitation wavelength of 492nm and an emission wavelength of 520nm.

[0275] result

[0276] like Figures 10A-10F As shown, delivery of serum-derived EVs from TBI patients increased inflammasome protein expression in lung endothelial cells. Figure 10A-10E The results showed that caspase-1, ASC, AIM2, and HMGB1 were elevated in PMVECs incubated with TBI-EVs for 4 h compared to PMVECs incubated with control-EVs for 4 h. Immunoassay results using the Ella simple plex assay showed a significant increase in IL-1β expression ( Fig.10F ).

[0277] like Figure 11A-11C As shown, delivery of TBI-EVs to lung endothelial cells increased caspase-1 immunoreactivity and cell death.

[0278] in conclusion

[0279] These studies provide additional evidence for a neuro-respiratory-inflammasome axis, in which EV release into the circulation following TBI activation of inflammasomes in lung target cells contributes to the pathogenesis of ALI.

[0280] Example 3: Effect of using humanized anti-ASC antibodies in multiple sclerosis animal models

[0281] To determine the utility of humanized anti-ASC monoclonal antibodies in treating MS, the antibodies were administered to experimental allergic encephalomyelitis (EAE) mice. EAE is an animal (i.e., rodent) model of MS, as described in the following literature: R, Leisser M, Bauer J, Lassmann H (December 2015). "Autoimmuneencephalitis in humans: how closely does it reflect multiple sclerosis?". ActaNeuropathol Commun. 3(1):80 and Lassman Hans (February 2010). "Acute disseminatedencephalomyelitis and multiple sclerosis. Brain. 133:317–319 and L. Gómez Vicente et al. Relapse in a paucisymptomatic form of multiple sclerosis in a patient treated with nivolumab, Neuro Oncol (2016) 18(Suppl 4):iv25.

[0282] method

[0283] Induction of EAE and treatment with IC 100

[0284] Myelin oligodendrocyte glycoprotein 35-55 peptide (MOG 35-55 Active EAE was induced in 2-month-old C57BL / 6 female mice by ELISA (BioSynthesis). Briefly, mice received an intraperitoneal (ip) injection of pertussis toxin (dissolved in PBS (350 ng / mouse; day 0), followed by subcutaneous administration of MOG emulsified in complete Freund's adjuvant. 35-55 (300 ng / mouse; day 1), and a second intraperitoneal injection of pertussis toxin (350 ng / mouse; day 2). Starting from day 8 after EAE induction, mice were administered vehicle (0.9% saline) or three different doses (10, 30 and 45 mg / kg) of IC100 via intraperitoneal injection once every 4 days. Clinical symptoms of EAE were assessed daily on a scale of 0 to 6 as follows: 0, no clinical signs; 1, loss of tail tone; 2, flaccid tail; 3, complete hind limb paralysis; 4, complete forelimb paralysis; 5, moribund; 6, death.

[0285] Cell separation for flow cytometry

[0286] After transcardial perfusion with PBS, the spinal cord was harvested and placed in a Mg-free 2+ and Ca2+ The samples were manually separated into single cell suspensions through a 70um filter and washed in HBSS w / o. The spleen samples were rotated at 1200rpm for 10min at 4°C, the supernatant was removed, and red blood cells (RBC) were lysed in 2ml RBC lysis buffer (eBioscience) according to the manufacturer's instructions. Splenocytes were resuspended in PBS. The cells separated from the spinal cord were resuspended in flow cytometry buffer (FCB, eBioscience) and incubated with myelin removal beads II (Miltenyi). Myelin was depleted using LS magnetic columns (Miltenyi) as described in the manufacturer's protocol. Similar to spleen cells, spinal cord cells were resuspended in PBS and stained as described below.

[0287] Immunolabeling and flow cytometric analysis

[0288] For experiments evaluating caspase-1, FAM FLICA was used according to the manufacturer's instructions. TM Caspase 1 kit (BioRad). Cells were incubated in FLICA solution (BioRad) for 30 min at 4°C, washed with apoptosis wash buffer (BioRad) and resuspended in 1 ml PBS. Samples were then incubated with fixable live / dead cell stain (TonboBiosciences) for 30 min at 4°C, spun at 1200 rpm for 10 min at 4°C, and the supernatant removed. Cells were resuspended in 100 ul FACS buffer, blocked with anti-CD16 / 32 (FcR block, eBioscience) for 5 min at room temperature, immunostained for 30 min at 4°C, and fixed with 1% PFA. Samples were analyzed using a CytoFLEX S flow cytometer (Beckman Coulter) equipped with CytExpert 2.1 software. Spinal cord leukocyte counts were determined using 123count eBeads (eBioscience). Splenic leukocyte counts were determined by flow cytometry combined with trypan blue exclusion counting using a TC20TM automated cell counter (Bio-Rad). Table 3 below provides a list of flow cytometry antibodies.

[0289] Table 3. Flow cytometry antibodies used in the methods provided herein.

[0290] antigen color Dilution Provider Catalog Number CD45 FITC 1:1000 eBioscience 11-0451-82 CD45 PE 1:1000 eBioscience 12-0451-82 CD4 PE / Cy7 1:200 eBioscience 25-0042-81 CD8 Percp-Cy 5.5 1:200 Biolegend 100734 B220 PE 1:200 Biolegend 103208 B220 APC eFluor780 1:200 eBioscience 47-0452-80 CD11b APC eFluor780 1:200 eBioscience 47-0112-82 CD11b PE / Cy7 1:200 Biolegend 101215 MHCII APC 1:200 eBioscience 17-5321-81 Ly6-G Percp-Cy5.5 1:200 Biolegend 127616 NK1.1 APC 1:200 Tonbo Biosciences 20-5941-U025 Live / Dead Violet 450 1:1000 Tonbo Biosciences 13-0863-T500

[0291] Luxol fast blue staining and quantification of demyelinated white matter volume

[0292] The spinal cord segments fixed with paraformaldehyde (PFA) were embedded in paraffin and cut into 10 mm thick cross sections with a Leica RM 2135 ultrathin slicer and stained with luxol fast blue (LFB). The volume of demyelinated white matter was estimated with ten 50 μm interval serial sections. The outline of the demyelinated area was displayed with an Olympus BX51 microscope, and the volume of demyelinated white matter was quantified with Stereoinvestigator software (MicroBrightfield). The demyelinated spinal cord was reconstructed in 3D on the same serial sections with Neurolucida software (MBF Bioscience).

[0293] Quantification of IC100 in tissues

[0294] IC100 in brain, spinal cord, liver and spleen at 35 days post-EAE induction (dpi) was quantified using an assay developed by InflamaCORE, LLC using Meso Scale Technology. The assay was read using a QuickPlex SQ 120 instrument (Meso Scale Diagnostics, Maryland).

[0295] Similar experiments were performed in a rat model of contusive cervical spinal cord injury to determine whether anti-ASC infiltrated spinal cord neurons.

[0296] To determine whether IC100 is taken up by cells, fluorescein-labeled IC100 was added to tissue culture medium containing THP-1 cells (a human monocytic cell line).

[0297] result

[0298] Treatment with the anti-ASC antibody IC 100 improves functional outcomes in experimental autoimmune encephalomyelitis (EAE)

[0299] To evaluate the therapeutic potential of IC100, two-month-old female C57BL / 6 mice were treated with MOG 35-55 Peptide induced EAE (Brambilla et al., 2014), and IC 100 or vehicle alone was administered starting at 8 days after induction of the disease (dpi). Administration was repeated every four days until sacrifice, which was set at 35 dpi. Three doses were tested: 10, 30 and 45 mg / Kg.

[0300] When used at doses of 30 and 45 mg / Kg, IC100 significantly improved functional recovery, with clinical disease scores significantly reduced throughout the experimental period ( Fig. 12A Treatment reduced mean peak clinical scores ( Fig. 12B) and the overall severity of EAE measured by a reduction in the cumulative disease index (CDI) ( Fig. 12C Mice treated with 30 and 45 mg / Kg IC 100 also showed a trend of delayed onset ( Fig.12D No differences were observed in the date that mice reached their peak disease scores ( Fig.12E ).

[0301] Treatment with the anti-ASC antibody IC 100 reduces the infiltration of peripheral immune cells into the spinal cord after EAE

[0302] The onset, persistence and severity of clinical symptoms of EAE are directly related to the infiltration of immune cells into the spinal cord. To evaluate whether IC 100 affects this process, immune cell populations isolated from the spinal cord at 35 dpi were analyzed by flow cytometry. Treatment with 30 mg / kg IC 100 significantly reduced the number of encephalitogenic CD4 + T cells and CD8 + The total number of T cells (i.e., the most critical immune cell population in driving EAE pathology) Fig.13A All other immune cell populations showed a clear trend toward decreases. No differences in cell numbers were observed in the spleen at any IC100 dose, suggesting that treatment did not interfere with the ability of mice to mount an adequate immune response to EAE challenge ( Fig. 13B ).

[0303] Treatment with anti-ASC antibody IC 100 reduces the number and activation state of microglia after EAE

[0304] Microglia are involved in the immune inflammatory response to CNS diseases. As their activation state increases, they proliferate and upregulate surface expression of MHCII. To assess whether IC100 affects this response, MHCII in total microglia and spinal cord was quantified by flow cytometry. + Both populations were significantly reduced by treatment with 30 mg / Kg IC 100, indicating that at this dose IC 100 is effective in inhibiting microglial activation and microglial-mediated neuroinflammation (see Fig.14 ).

[0305] IC 100 penetrates into the brain and spinal cord

[0306] An important parameter when designing a drug for the treatment of MS is to determine whether the drug penetrates into the CNS at therapeutic levels. This is an important feature, especially when treating progressive forms of MS, as the blood-brain barrier appears relatively intact at this stage of the disease (Lassman et al. 2012). Therefore, we harvested brain, spinal cord, liver, and spleen to determine the IC100 levels in these tissues. Fig.15 As shown in Figure 2, IC100 penetrated all of these tissues, including brain and spinal cord, at all three doses. Interestingly, the levels of IC100 in the spinal cord were higher at the 30 mg / kg dose, consistent with a greater therapeutic effect at this dose.

[0307] Fluorescein-labeled IC100 was taken up by THP-1 cells (a human monocytic cell line) and incorporated into ASC specks when added to tissue culture medium. In addition, IC100 labeled with inflammasome induction stimuli of these cells was taken up into ASC specks together with rhodamine-labeled dextran, indicating that IC100 uptake is mediated by endocytosis (see Fig.16 ).

[0308] Likewise, IC100 blocked the release of IL-1β from THP-1 cells ( Fig.17 ).

[0309] In a rat model of contusive cervical spinal cord injury, anti-ASC antibodies (eg, IC100) also infiltrated spinal cord neurons.

[0310] IC 100 can act both inside and outside the cell. Inside the cell, it can act by binding to the ASC protein and inhibiting it, thereby preventing the assembly of the multiprotein inflammasome and the initiation of the inflammatory response. It can also bind to ASC in ASC specks, preventing the propagation of large filamentous signaling platforms, thereby inhibiting the extracellular activation of pro-ILβ, which is responsible for perpetuating inflammation in chronic inflammatory diseases.

[0311] Incorporated by Reference

[0312] The following references are incorporated by reference in their entirety for all purposes.

[0313] Brambilla R, Morton PD, Ashbaugh JJ, Karmally S, Lambertsen K, and Bethea JR (2014) Astrocytes play a key role in EAE pathophysiology by orchestrating in the CNS the inflammatory response of resident and peripheral immune cells and by suppressing remyelination. GLIA, 62:452-457.

[0314] Lassmann, H., van Horssen, J., and Mahad, D. Progressive multiple sclerosis: pathology and pathogenesis. Nat Rev Neurol 8, 647-656, doi:10.1038 / nrneurol.2012.168 (2012).

[0315] Example 4: Kinetic Analysis of Candidate Anti-ASC Monoclonal Antibodies

[0316] The candidate anti-ASC monoclonal antibodies were subjected to kinetic analysis using bio-layer interferometry (BLI). In BLI, association or dissociation with a surface results in a change in the wavelength of reflected light and measuring this change over time enables determination of binding kinetics.

[0317] The BLI assay consists of the following:

[0318] Sensor check (30 s) --> Load Ab / supernatant (700 s) --> Baseline (300 s) --> Ab association (600 s) --> Dissociation (600 s) --> Repeat

[0319] Candidate mouse IgG antibody supernatants were tested for binding to human ASC peptide (SEQ ID NO: 5) at 7 different concentrations (i.e., 540 nM, 180 nM, 60 nM, 20 nM, 6.67 nM, 2.22 nM, 0.741 nM). The antibodies tested were ICCN 1.0H (i.e., IC 100); ICCN 2.0H; and ICCN 3.0H. AMC (anti-mouse IgG Fc) biosensor was loaded with mouse IgG from undiluted supernatant. Raw antibody kinetic data for the 3 candidate antibodies are shown in Fig.21 In Fig. 22 Displays the global KD value.

[0320] Example 5: Absorption, distribution, metabolism and excretion studies of IC 100

[0321] Absorption, distribution, metabolism and excretion (ADME) experiments will be performed to characterize the disposition of IC100 in CD-1 male rats.

[0322] In the first experiment, 30 six-week-old CD-1 male rats will be obtained from Charles River. The experiment will be conducted at Bolder BioPath (BBP). The mice will be acclimated for at least seven days after arrival at BBP. The mice will be housed four animals per cage.

[0323] Animals will be randomized into treatment groups (nine per group) based on body weight and doses of IC 100 will be administered intravenously (IV). Treatment groups will receive 5 mg / kg, 15 mg / kg or 30 mg / kg IC 100. Plasma will be collected at different times after a single IV dose for pharmacokinetic (PK) monitoring. An exemplary schedule for plasma collection is shown in Table 4 below. Acute toxicity will be monitored by clinical observation. Plasma will be shipped to Antibody Solutions for analysis. The experiment will last for ten weeks.

[0324] Body weight measurements will be taken on days 0, 7, 14, 21, 28, and 35.

[0325] Retrobulbar bleeding will be used to collect sufficient blood volumes; mice will be anesthetized with isoflurane prior to sample collection. Animals 1, 2, and 3 in each group will be bled on days 1 and 10; animals 4, 5, and 6 will be bled on days 2 and 15; and animals 7, 8, and 9 will be bled on days 5 and 20.

[0326] At the final time point, animals were anesthetized with isoflurane and bled to exsanguination, followed by bilateral pneumothorax. Animals 1, 2, and 3 will be sacrificed on day 25. Animals 4, 5, and 6 will be sacrificed on day 30. Animals 7, 8, and 9 will be sacrificed on day 35.

[0327] Table 4. Sample collection list

[0328] Group Animal No. Time 1,2,3 1-3 Day 1 (24 hours) 1,2,3 4-6 Day 2 (48 hours) 1,2,3 7-9 Day 5 1,2,3 1-3 Day 10 1,2,3 4-6 Day 15 1,2,3 7-9 Day 20 1,2,3 1-3 Day 25 (End) 1,2,3 4-6 Day 30 (end) 1,2,3 7-9 Day 35 (end)

[0329] *Necropsy was performed at the end point blood collection

[0330] Example 6. IC100 biodistribution and fluorescence imaging using female B6 albino mice

[0331] In the second experiment, IC 100 and control mouse IgG will be labeled with VivoTag 680XL fluorescent labeling dye according to the established VivoTag protocol and the binding affinity will be determined. Briefly, according to the VivoTag protocol, the labeling protocol will include:

[0332] 1. Prepare antibody (>7kDa) solution to 1-10mg / mL in PBS. The antibody should not contain ammonium ions or primary amines to reduce competition with the active dye.

[0333] 2. Dissolve 0.25 mg of VivoTag 680XL in 10 μL of dry DMSO. After reconstitution, VivoTag 680XL is stable for up to 7 days when stored at 2°C-8°C and protected from light.

[0334] 3. Add 0.5 mL protein (0.5-5 mg), 50 μL sodium bicarbonate, 2 μL VivoTag 680XL (for each mg protein) to an eppendorf tube. Incubate the mixture in the dark at room temperature with shaking for 2 hours.

[0335] 4. Separate the protein conjugate from the free dye. Unscrew the bottom closure of the column and loosen the cap. Place the column on a 15 mL conical collection tube and centrifuge at 1,000 x g for 2 min. Add 2 mL of PBS to the column and centrifuge at 1,000 x g for 2 min. Repeat the wash two more times.

[0336] 5. Place the column on a new 15 mL conical collection tube. Load all protein samples (200-700 μL) onto the column and centrifuge at 1,000 xg for 2 min. Collect the protein sample that flows through.

[0337] 6. The collected labeled antibody samples can be analyzed for degree of labeling (DOL). The absorbance of the purified conjugate is measured at 280 nm and 668 nm.

[0338] 7. Adjust the absorbance of the purified protein at 280 nm by subtracting the absorbance of the VivoTag 680XL at 280 nm, which is 16% of the absorbance at 668 nm.

[0339] 8. Absorbance analysis can be performed with a UV spectrophotometer or a Nanodrop spectrophotometer. To use the latter, the sample needs to be diluted to the 0.5-2 mg / mL range and then measured. Since the optical path is 1 mm, the reading should be normalized using a factor of 10.

[0340] In the third experiment, the biodistribution of IC 100 will be determined. Fifteen 8-12 week old female B6 albino (C57BL6) mice will be used in the study. Animals will be randomized into groups based on body weight on the first day.

[0341] Female B6 albino mice will receive no treatment (negative control), a single dose of IC 100 labeled with VivoTag 680XL, or a single dose of mouse IgG labeled with VivoTag 680XL (negative control). Treatments will be administered intravenously (volume = 200 μL) at a dose of 100 μg / animal.

[0342] In vivo fluorescence imaging will be performed at 2hr, 8hr, 24hr, 48hr, 72hr and 96hr after treatment, and in vivo whole body images of the back and abdomen will be captured using fluorescence imaging at various time intervals until 96 hours after treatment. Ex vivo imaging will be performed on the brain, eyes with optic nerves, heart, left and right kidneys, large intestine (including terminal colon), liver, lungs, ovaries, pancreas, small intestine, spine, stomach, thyroid and bladder of all animals.

[0343] Whole blood will be collected and the immune infiltrate will be analyzed for CD4+T cells (CD4+CD11b-CD3+CD8-), CD8+T cells (CD8+CD11b-CD3+CD4-), B cells (CD3-CD111b-CD45R+), monocytes (CD3-CD11b+CD115+) and NK cells (CD3-CD 49b+CD335+). The expression of Ab-VivoTag 680XL will be quantified to determine the level of the delivered labeled antibody. LIVE / DEAD dye will be included in the flow cytometry detection panel to quantify the number of viable cells. The entire antibody detection panel will include antibodies for CD3, CD4, CD8, CD11b, CD115, CD45R, CD49b, CD335 and LIVE / DEAD dye.

[0344] Example 7: Effects of IC 100 Administration on Inflammasome Signaling

[0345] Blood from patients with nonalcoholic steatohepatitis (NASH), diabetic nephropathy, and lupus nephritis will be obtained by BioReclamation IVT for biomarker analysis of inflammasome proteins. Tissues from patients with NASH, diabetic nephropathy, and lupus nephritis will be obtained by BolderBioPath, and protein lysates will be obtained from those tissues and analyzed for expression of inflammasome signaling proteins, including caspase-1 and ASC, by immunoblotting and other biochemical techniques.

[0346] In addition, human cancer cell lines will be used to examine ASC-dependent inflammasome activation in real time. The study will consist of two objectives and will be initiated over a 6-9 week time period.

[0347] Specific target 1: Antibody labeling

[0348] 2 mg IC 100 was added with IgG-680XL-IFC ( 680XL, PerkinElmer #-NEV11120) fluorescent marker dye labeling, and the stoichiometry of labeling will be determined according to the manufacturer's instructions. Control mouse IgG (provided by Charles River Laboratories (CRL)) was similarly labeled and analyzed.

[0349] Specific objective 2: Binding affinity determination

[0350] The human cancer cell line THP-1 was cultured in logarithmic phase and plated at 20,000 cells per well in a 100 μL volume of culture medium in white polystyrene 6-well microplates ( 96-well flat bottom plates, catalog number 3917). Labeled antibodies were then added to the wells in duplicate (10 point dose response, 1:3 dilution, highest concentration 200 nM). All wells were tested for bound antibody according to the Charles River protocol. Bound antibody (mean fluorescence intensity) was plotted as a function of antibody concentration, and binding affinity (Kd) was estimated by fitting the data to the following equation: Y = Bmax*X / (Kd+X).

[0351] Example 8: In vivo study of IC100 in NASH rat model

[0352] Male Wistar Han rats with liver fibrosis fed a choline-deficient high-fat diet (CDHFD) are used as an animal model for NASH. 55 mice will be used in this study. 8-9 week old Wistar Han rats will be obtained from Envigo or Charles River. Rats will be acclimated for 3-7 days after arrival at Bolder BioPath. Rats will be housed 2-3 animals per cage. Animals are fed a standard diet.

[0353] On day 0, animals were randomly divided into five groups based on body weight. Group 1 was fed Teklad Global Diets-Rodent 2014, a standard diet. Groups 2, 3, 4, and 5 were fed a CDHFD diet.

[0354] On the 38th day of the study, blood was collected from the animals for clinical chemistry and placed in the treatment group according to the alanine aminotransferase (ALT) concentration. On the 42nd day of the study, treatment was started. The following dosage was used to test the efficacy of IC 100, which was determined based on the pharmacokinetic data obtained from Example 5. One group of patients received treatment with vehicle to serve as a negative control. The group receiving a standard diet will also receive treatment with vehicle. Body weight, food intake and cage-side clinical observations were measured weekly. On the 38th and 63rd days, whole blood was obtained via tail vein collection. Autopsy will be performed on the 84th day. Animals will be killed under isoflurane anesthesia, blood was collected to exsanguination, and then bilateral pneumothorax.

[0355] Animals will be weighed on study days -1, 0, 2, 4, 6, 7, 14, 21, 28, 35, 42, 45, 49, 52, 56, 59, 63, 66, 70, 73, 77, 80, and 83.

[0356] Weekly updates of food intake (g / day / rat) will be recorded on Days 0, 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77 and 84 of the study.

[0357] Clinical cage-side observations will be performed on days 0-7, 14, 21, 28, 35, 42, 45, 49, 52, 56, 59, 63, 66, 70, 73, 77, 80, 83. Animals will be observed and weighed daily if they begin to show clinical signs of toxicity or illness.

[0358] At autopsy, the weight of liver, brown adipose tissue and right inguinal adipose tissue will be measured.4x 7mm biopsy of the left lateral lobe of the liver will be obtained, frozen in liquid nitrogen, and stored at -80°C.3mm transverse sections of the inner lobe, left lateral lobe and right lateral lobe of the liver will be obtained and fixed in 10% formalin for 36-48 hours, then stored in 70% ethanol at ambient temperature for histopathology.Three 100mg pieces of adipose tissue were quickly frozen in liquid nitrogen and stored in Eppendorf safety buckle tubes at -80°C.Three equal-sized pieces of brown adipose tissue were quickly frozen in liquid nitrogen and stored in Eppendorf safety buckle tubes at -80°C.

[0359] Inguinal subcutaneous adipose tissue, i.e., white adipose tissue depot (WAT), will be collected according to the following scheme. The inguinal triangle SQ depot will be exposed by tearing off the lower half of the mouse. One hand holds the upper appendages and chest, and the other hand pulls the skin down toward the feet. The mouse is positioned in a supine position, taking care not to contaminate the exposed depot with hair. Clean the surgical instruments and change gloves. Subsequently, the triangle of subcutaneous fat will be dissected, being careful not to contaminate the sample with muscle, adjacent fat, mammary gland, or blood. If the boundary cannot be clearly determined, a dissecting microscope is used. The fat depot is removed and transferred to 10% neutral buffered formalin with a fixative and a tissue volume of 50:1, and fixed at room temperature for 36-48 hours. If RNA or protein is to be extracted, the tissue is frozen by immersion in liquid nitrogen and stored at -80°C to prevent degradation. Efforts are made to avoid cross contamination between fat depots by frequently changing gloves.

[0360] Histological processing will be performed by Histotox Laboratories. LLL, MLL and RLL histological processing will be performed on three liver transverse sections / animal. Samples will be stained with Sirius Red and Hematoxylin and Eosin (H&E).

[0361] Example 9: In vivo study of the effect of IC100 on diabetic nephropathy in the BTBR Ob / Ob mouse model

[0362] The mouse strain BTBR with the ob / ob leptin deficiency mutation serves as a mouse model for diabetic nephropathy. Male BTBR Ob / Ob mice will be used to evaluate the effect of IC 100 in reversing diabetic nephropathy. Five male wild-type (WT) BTBR mice will be used as negative controls and will not receive any treatment. 50 BTBR Ob / Ob mice will be used and divided into five groups. The groups will receive vehicle, control, or IC 100 at a dose determined based on Example 5. The experiment will be conducted for six weeks.

[0363] Histology of mouse kidneys will be performed by the Histotox laboratory. Appropriate parameters will be determined by a pathologist using current BolderBioPATH methods.

[0364] Blood glucose measurements will be made by snipping the tail and applying a drop of blood (approximately 5 μL) to a test strip compatible with a True Metrix blood glucose meter. Blood glucose measurements will be made twice weekly until completion of the study.

[0365] Proteinuria will be scored by inverting the mouse and applying pressure to the abdomen to express urine from the mouse. The amount of protein in the urine will be determined using Albustix reagent strips.

[0366] If an animal is found dead, no samples will be taken. If an animal requires euthanasia, for whatever reason, samples will be taken as at necropsy after study day 7.

[0367] Example 10: In vivo study of the effect of IC100 on lupus nephritis in a mouse model

[0368] The lupus nephritis model will be developed using 12 week old female MRL / MpJ-Tnfrsf6lpr / J mice. Mice will be obtained from Bolder BioPath. Mice will be randomized into treatment groups based on body weight. Animals will be observed daily for vital clinical signs, moribundity, and death. The onset of lupus nephritis will occur at approximately 12-14 weeks of age.

[0369] After the onset of lupus nephritis, mice were divided into five groups and treated with different doses of IC100 (according to the results of Example 5), vehicle or control IgG.

[0370] Body weight, urine proteinuria, lymphadenopathy scores, and skin lesion scores will be collected. Necropsy will be performed at week 20, and tissue and whole blood will be collected for analysis. The experiment is expected to last 15 weeks.

[0371] Example 11: Intravenous bolus toxicity study in albino rats with acute and 21-day IC100 ranges

[0372] Albino rats were obtained from Charles River. The IC100 dose level will be determined for a definitive 28-day multiple dose toxicity and pharmacokinetic study in rats. Rats will be divided into four groups, each containing three rats / sex / dose. An up and down study will be performed.

[0373] Repeated dose studies will be conducted for three weeks. Rats will be divided into groups containing five rats, either all male or all female. Rats will be dosed weekly at a dose level of IC 100. Vital parameters, including death, clinical signs, body weight, and clinical tests with toxicokinetics will be performed.

[0374] Example 12: Maximum Tolerated Dose and 22-Day Dose Range Finding Study of IC100 in Cynomolgus Monkeys

[0375] IC100 dose levels and pharmacokinetics are determined in cynomolgus monkeys. The experiment will be conducted in Charles River. The cynomolgus monkeys will be divided into four groups, each containing one cynomolgus monkey / sex / dose. The up and down study will be conducted for 28 days.

[0376] Repeated dose studies will be conducted for three weeks. Cynomolgus monkeys will be divided into groups containing two cynomolgus monkeys, all male or all female. Cynomolgus monkeys will be dosed weekly at a dose level of IC100. Vital parameters, including death, clinical signs, body weight, and clinical tests with toxicokinetics will be performed.

[0377] The experiment is expected to last 15 weeks.

[0378] Example 13: IC100 Toxicity Study in Rats with Subsequent 4-Week Recovery Period

[0379] Toxicity and toxicokinetics will be determined in rats obtained from Charles River following intravenous administration of IC100 followed by a recovery period.

[0380] Rats will be divided into 10-15 rats / sex / group at three dose levels plus controls. Additional groups will include a high dose group and a control group containing 5 rats / sex with four weeks of recovery.

[0381] Death, body weight, food intake, clinical observations, clinical pathology (hematology and clinical chemistry), necropsy findings, organ histopathology, and toxicokinetics will be measured. After recovery, the same parameters will be measured, but toxicokinetics will not be included.

[0382] The experiment is expected to last six months.

[0383] Example 14: IC100 and toxicity study in cynomolgus monkeys with subsequent 4-week recovery period

[0384] Toxicity and toxicokinetics will be determined in nonhuman primates at Charles River following intravenous administration of IC100 followed by a recovery period.

[0385] The cynomolgus monkeys will be divided into 3 cynomolgus monkeys / sex / group at three dose levels plus control. Additional groups will include a high dose group and a control group, which contains 2 cynomolgus monkeys / sex, with four weeks of recovery.

[0386] Death, body weight, food intake, clinical observations, clinical pathology (hematology and clinical chemistry), necropsy findings, organ histopathology, and toxicokinetics will be measured. After recovery, the same parameters will be measured, but toxicokinetics will not be included.

[0387] The experiment is expected to last six months.

[0388] Example 15: In vitro cardiovascular studies using the hERG assay

[0389] The potential for cardiovascular toxicity (QT prolongation) will be evaluated in an in vitro assay with CHO or HEK293 cells.

[0390] This experiment will determine the IC50 for an IC100 to block the HERG channel.

[0391] The experiment is expected to last two months.

[0392] Example 16: In vitro blood hemolysis study

[0393] The potential of the IC 100 intravenous formulation to cause hemolysis of human erythrocytes in vitro will be evaluated. Various concentrations of IC 100 (determined based on Example 5) will be mixed with erythrocytes in an in vitro system. The extent of hemolysis will be determined. The experiment is expected to last two months.

[0394] Example 17: Examination of the in vivo efficacy of IC100 in the treatment of Parkinson's disease (PD)

[0395] The efficacy of IC 100 in treating PD was evaluated by administering IC 100 in several animal (ie rodent) models of PD. 5 mg / kg, 15 mg / kg, 30 mg / kg.

[0396] The 6-OHDA rat model of PD is a chemically induced unidirectional model of PD (lesions within the striatum or the median forebrain bundle), in which mice exhibit behavioral deficits including rotational asymmetry and motor deficits. Histologically, the 6-OHDA model exhibits reduced dopamine, DOPAC, and HVA content in the striatum, and shows a decrease in TH-positive cells in the substantia nigra.

[0397] In one set of experiments using the 6-OHDA model of PD, a total of 45 male rats were divided into 3 experimental groups (n=15 rats / group) and were treated as follows:

[0398] 1. Rats induced by sham surgery will be treated with vehicle;

[0399] 2. Treated 6-OHDA-induced rats will be treated with vehicle; and

[0400] 3. Treated 6-OHDA induced rats will be treated with IC100 dose 1 (selected based on PK / half-life studies).

[0401] A single 6-OHDA / sham infusion was performed on study day 0;

[0402] Daily dose formulation and administration (QD, oral) will occur on Study Days 15-28.

[0403] Body weight tracking and behavioral testing will be performed on Days 14 (baseline), 28, and 42, and behavioral testing will include amphetamine-induced rotations.

[0404] Endpoint blood, CSF, and brain sampling was performed on study day 42, followed by HPLC for DA, DOPAC, and HVA in the striatum, and IHC for TH+ cells in the SNpc.

[0405] In the second set of experiments using the 6-OHDA model of PD, a total of 45 male rats were divided into 3 experimental groups (n=15 rats / group) and were treated as follows:

[0406] 1. Rats induced by sham surgery will be treated with vehicle;

[0407] 2. Treated 6-OHDA-induced rats will be treated with vehicle; and

[0408] 3. Treated 6-OHDA induced rats will be treated with IC100 dose 1 (selected based on PK / half-life studies).

[0409] The experiment will be conducted over a 6-week period with a single 6-OHDA / sham infusion on study day 0;

[0410] Daily dosing and administration (QD, oral) will begin on Study Day 1 and will continue until 6 weeks following 6-OHDA infusion.

[0411] Behavioral testing will be performed on Days 14 (baseline), 28, and 42 and will include amphetamine-induced rotations and cylinder tests.

[0412] Endpoint brain sampling was performed on study day 42, followed by HPLC for DA, DOPAC, and HVA in the striatum, and IHC for TH and Iba-1 (bidirectional) in the SNc.

[0413] In the third set of experiments using the 6-OHDA model of PD, a total of 90 male rats were divided into 6 experimental groups (n=18 rats / group at the beginning until day 14 baseline, n=15 rats / group as target), which will be treated as follows:

[0414] 1. Treat sham-surgery-induced rats with vehicle

[0415] 2. 6-OHDA-induced rats were treated with vehicle

[0416] 3. 6-OHDA-induced rats were treated with IC 100 dose 1

[0417] 4. 6-OHDA-induced rats were treated with IC 100 dose 2

[0418] 5. 6-OHDA-induced rats were treated with IC 100 dose 3

[0419] 6. Treat 6-OHDA-induced rats with IC 100 dose 4

[0420] The experiment will be conducted over a 6-week period with a single 6-OHDA / sham infusion on study day 0;

[0421] Daily dose formulation and administration (QD, oral) will occur on Study Days 15-42.

[0422] Behavioral testing will be performed on Days 14 (baseline), 28, and 42 and will include amphetamine-induced rotations and cylinder tests.

[0423] Endpoint brain sampling was performed on study day 42, followed by HPLC for DA, DOPAC, and HVA in the striatum, and IHC for TH and Iba-1 (bidirectional) in the SNc.

[0424] Example 18: Examination of the in vitro efficacy of IC100 in the treatment of Parkinson's disease (PD)

[0425] TOM20 assay:

[0426] PD pathogenesis has been linked to mitochondrial dysfunction by several studies, starting with the discovery that the mitochondrial complex I inhibitor rotenone induces parkinsonism. In addition, mutations in genes encoding proteins involved in the selective clearance of dysfunctional and superfluous mitochondria (mitophagy), such as PARK2 and PINK1, are present in the majority of autosomal recessive PD cases. Similarly, increasing evidence suggests that mitochondrial dysfunction is associated with other neurodegenerative disorders such as AD, ALS, and Huntington's disease (HD). Therefore, phenotypic readouts for measuring mitochondrial (dys) function in a disease-relevant cellular context are considered powerful predictive tools for probing neurodegenerative pathobiology and identifying potential therapeutics that can enhance mitophagy.

[0427] TOM20 is a subunit of the mitochondrial outer membrane translocase (TOM) complex and represents a biomarker of mitochondrial abundance. By analyzing therapeutic candidates in the absence (monotherapy) and presence (co-treatment) of established mitophagy-inducing triggers, candidate molecules (e.g., IC 100) can be selected that enhance trigger-induced mitochondrial clearance without directly damaging mitochondria.

[0428] The TOM20 loss assay is a scalable and rapid in vitro assay that is used to screen compounds for their ability to enhance mitophagy in a neuronal context.

[0429] The TOM20 assay will utilize immortalized human midbrain progenitor cells (ReNcell VM) seeded at 50,000 cells / well in laminin-coated 96-well plates, which will be differentiated by the following method: withdrawal of growth factors (bFGF, EGF) and addition of differentiation-promoting factors (cAMP, GDNF) for seven days starting from day 0 (D0), and replenishment of feed (refreshment) on days 1 (D1) and 4 (D4). Cells will then be treated with IC 100 for 18 hours on day 7 in the absence and presence of 1 micromolar oligomycin / antimycin (O / A; a commonly used mitotic trigger), followed by fixed immunocytochemical staining of TOM20 with anti-TOM20 antibody and DAPI staining (i.e., on day 8). Finally, the assay will contain a 1uM O / A positive control and a 0.1% DMSO negative control.

[0430] Combining mitophagy enhancing compounds with 1uM O / A treatment will result in a significant reduction in TOM20 levels that exceeds the reduction induced by O / A treatment alone. The TOM20 immunostaining intensity will be quantified using a high content analysis (HCA) algorithm developed by Charles River Laboratories. Nuclear counts will be quantified to identify potential compound (e.g., IC 100) induced cytotoxicity.

[0431] α-Synuclein Aggregation Assay:

[0432] Aggregates of the presynaptic protein α-synuclein are considered the major biomarker for PD, and there is evidence that α-synuclein aggregates directly mediate neuronal death. Therefore, strategies aimed at reducing α-synuclein aggregation and toxicity may have therapeutic potential.

[0433] In this assay, immortalized human midbrain progenitor cells (ReNcell VM) seeded at 10,000 cells / well in laminin-coated 96-well plates will be differentiated by the following method: growth factors (bFGF, EGF) are withdrawn from day 0 (D0) and differentiation factors (cAMP, GDNF) are added for seven days. Also at D0, cells are transduced with adenovirus encoding wild-type human α-synuclein. After 24 hours, cells are then treated with IC 100, and α-synuclein expression and aggregation are detected 6 days later by the following method: immunocytochemistry using antibodies detecting α / β-synuclein (Syn205; Cell Signaling Technology) and aggregated α-synuclein (MJFR14; Abcam), followed by HCA quantification as described for the TOM20 assay. Finally, the assay will also contain a 10 uM KU 0063794 positive control and a 0.1% DMSO negative control added at D1 and D4.

[0434] Numbered embodiments of the present disclosure

[0435] Other subject matter contemplated by the present disclosure is set forth in the following numbered embodiments:

[0436] 1. A method for treating inflammation in the lungs of a patient in need thereof, the method comprising: administering to the patient a composition comprising an agent that inhibits inflammasome signaling, thereby treating inflammation in the lungs of the patient.

[0437] 2. The method of embodiment 1, wherein the inflammation in the lung is caused by a condition selected from the group consisting of central nervous system (CNS) damage, neurodegenerative diseases, autoimmune diseases, asthma, chronic obstructive pulmonary disease, cystic fibrosis, interstitial lung disease, and acute respiratory distress syndrome.

[0438] 3. The method of embodiment 2, wherein the CNS injury is selected from traumatic brain injury (TBI), stroke, and spinal cord injury (SCI).

[0439] 4. The method according to embodiment 2, wherein the neurodegenerative disease is selected from amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS) and Parkinson's disease (PD).

[0440] 5. A method according to any one of the above embodiments, wherein administration of the composition results in inhibition of inflammasome activation in lung cells of the patient.

[0441] 6. A method according to any one of embodiments 1-4, wherein administration of the composition results in a decrease in the level of caspase-1, nucleotide-binding leucine-rich repeat-pyrin domain-containing protein 1 (NLRP1), nucleotide-binding leucine-rich repeat-pyrin domain-containing protein 2 (NLRP2), nucleotide-binding leucine-rich repeat-pyrin domain-containing protein 3 (NLRP3), NLR family CARD domain-containing protein 4 (NLRC4), caspase-11, X-linked inhibitor of apoptosis protein (XIAP), pan-connexin-1, apoptosis-associated speck-like protein containing a caspase activation recruitment domain (ASC), interleukin-18 (IL-18), high-mobility group protein B1 (HMGB1), or melanoma deficiency factor 2 (AIM2) in the patient's lung cells compared to a control, wherein the control is an untreated patient.

[0442] 7. A method according to embodiment 5 or 6, wherein the lung cells are type II alveolar cells.

[0443] 8. The method of any one of embodiments 1-5, wherein administration of the composition results in a reduction in acute lung injury (ALI) compared to a control, wherein the control is an untreated patient.

[0444] 9. The method of embodiment 8, wherein the reduction in ALI is demonstrated by reduced infiltration of neutrophils into the alveolar and / or interstitial spaces, reduced or absent alveolar septal thickening, or a combination thereof.

[0445] 10. A method according to any one of the above embodiments, wherein the agent is an extracellular vesicle (EV) uptake inhibitor, an antibody that binds to an inflammasome component, or a combination thereof.

[0446] 11. The method of embodiment 10, wherein the EV uptake inhibitor is a compound or an antibody, wherein the antibody is selected from Table 1.

[0447] 12. A method according to any one of embodiments 10-11, wherein the agent is a combination of an EV uptake inhibitor and an antibody that binds to an inflammasome component.

[0448] 13. A method according to embodiment 12, wherein the EV uptake inhibitor is heparin.

[0449] 14. The method of embodiment 13, wherein the heparin is enoxaparin.

[0450] 15. A method according to any one of embodiments 10-14, wherein the antibody that binds to the inflammasome component is an antibody that specifically binds to a component of the mammalian AIM2, NLRP1, NLRP2, NLRP3 or NLRC4 inflammasome.

[0451] 16. The method of embodiment 10 or 15, wherein the inflammasome component is caspase-1, ASC or AIM2.

[0452] 17. A method according to embodiment 16, wherein the inflammasome component is ASC.

[0453] 18. A method according to embodiment 17, wherein the antibody binds to an N-terminal PYRIN-PAAD-DAPIN domain (PYD), a C-terminal caspase recruitment domain (CARD) domain, or an epitope derived from the PYD or CARD domain of the ASC protein.

[0454] 19. The method of embodiment 17, wherein the antibody binds to an amino acid having at least 85% sequence identity with an amino acid sequence selected from SEQ ID NO: 1 and SEQ ID NO: 2.

[0455] 20. The method of any one of embodiments 17-19, wherein the antibody inhibits ASC activity in the lungs of the patient.

[0456] 21. The method according to any one of the preceding claims, wherein the composition is formulated with a pharmaceutically acceptable carrier or diluent.

[0457] 22. The method according to any one of the above embodiments, wherein the composition is administered intracerebroventricularly, intraperitoneally, intravenously, or by inhalation.

[0458] 23. A method of treating inflammation in the lungs of a patient who has sustained central nervous system (CNS) injury, the method comprising: administering to the patient a composition comprising an agent that inhibits inflammasome signaling, thereby treating inflammation in the lungs of the patient.

[0459] 24. The method of embodiment 23, wherein the CNS injury is selected from traumatic brain injury (TBI), stroke, and spinal cord injury (SCI).

[0460] 25. A method according to any one of embodiments 23-24, wherein administration of the composition results in inhibition of inflammasome activation in lung cells of the patient.

[0461] 26. A method according to any one of embodiments 23-24, wherein administration of the composition results in a decrease in the level of caspase-1, NLRP1, NLRP2, NLRP3, NLRC4, caspase-11, XIAP, pan-connexin-1, apoptosis-associated speck-like protein containing a caspase activation recruitment domain (ASC), interleukin-18 (IL-18), high mobility group protein B1 (HMGB1), or melanoma deficiency factor 2 (AIM2) in the patient's lung cells compared to a control, wherein the control is an untreated patient.

[0462] 27. A method according to embodiment 25 or 26, wherein the lung cells are type II alveolar cells.

[0463] 28. The method of any one of embodiments 23-27, wherein administration of the composition results in a reduction in acute lung injury (ALI) compared to a control, wherein the control is an untreated patient.

[0464] 29. The method of embodiment 28, wherein the reduction in ALI is evidenced by reduced infiltration of neutrophils into the alveolar and / or interstitial spaces, reduced or absent alveolar septal thickening, or a combination thereof.

[0465] 30. A method according to any one of embodiments 23-29, wherein the agent is an extracellular vesicle (EV) uptake inhibitor, an antibody that binds to an inflammasome component, or a combination thereof.

[0466] 31. A method according to embodiment 30, wherein the EV uptake inhibitor is a compound or an antibody, wherein the antibody is selected from Table 1.

[0467] 32. A method according to any one of embodiments 30-31, wherein the agent is a combination of an EV uptake inhibitor and an antibody that binds to an inflammasome component.

[0468] 33. A method according to embodiment 32, wherein the EV uptake inhibitor is heparin.

[0469] 34. The method of embodiment 33, wherein the heparin is enoxaparin.

[0470] 35. A method according to any one of embodiments 30-34, wherein the antibody that binds to an inflammasome component is an antibody that specifically binds to a component of a mammalian AIM2, NLRP1, NLRP2, NLRP3 or NLRC4 inflammasome.

[0471] 36. A method according to embodiment 30 or 35, wherein the inflammasome component is caspase-1, ASC or AIM2.

[0472] 37. A method according to embodiment 36, wherein the inflammasome component is ASC.

[0473] 38. The method of embodiment 37, wherein the antibody binds to the PYD, CARD domain, or an epitope derived from the PYD or CARD domain of the ASC protein.

[0474] 39. A method according to embodiment 37, wherein the antibody binds to the following amino acids, which have at least 85% sequence identity with an amino acid sequence selected from SEQ ID NO:1 and SEQ ID NO:2.

[0475] 40. A method according to any one of embodi...

Claims

1. A monoclonal antibody or an antibody fragment thereof that specifically binds to an apoptosis-associated speck-like protein containing a caspase activation recruitment domain, wherein the antibody or the antibody fragment comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region amino acid sequence comprises HCDR1 of SEQ ID NO: 6, HCDR2 of SEQ ID NO: 7, and HCDR3 of SEQ ID NO: 8; and wherein the VL region amino acid sequence comprises LCDR1 of SEQ ID NO: 12, LCDR2 of SEQ ID NO: 13, and LCDR3 of SEQ ID NO:

14.

2. The monoclonal antibody or antibody fragment thereof according to claim 1, wherein the VH region amino acid sequence comprises SEQ ID NO: 18, 19, 20, 21, 22.

3. The monoclonal antibody or antibody fragment thereof according to claim 1, wherein the VH region amino acid sequence comprises SEQ ID NO: 18, 19, 20, 21, 22; and Wherein the VL region amino acid sequence comprises SEQ ID NO: 28, 29, 30, 31.

4. The monoclonal antibody or antibody fragment thereof according to claim 1, wherein the VH region amino acid sequence comprises SEQ ID NO: 18; and Wherein the VL region amino acid sequence comprises SEQ ID NO:

28.

5. The monoclonal antibody or antibody fragment thereof according to claim 1, wherein the VH region amino acid sequence comprises SEQ ID NO: 18; and Wherein the VL region amino acid sequence comprises SEQ ID NO:

29.

6. The monoclonal antibody or antibody fragment thereof according to claim 1, wherein the VH region amino acid sequence comprises SEQ ID NO: 18; and Wherein the VL region amino acid sequence comprises SEQ ID NO:

30.

7. The monoclonal antibody or antibody fragment thereof according to claim 1, wherein the VH region amino acid sequence comprises SEQ ID NO: 18; and Wherein the VL region amino acid sequence comprises SEQ ID NO:

31.

8. The monoclonal antibody or antibody fragment thereof according to claim 1, wherein the VH region amino acid sequence comprises SEQ ID NO: 19; and Wherein the VL region amino acid sequence comprises SEQ ID NO:

28.

9. The monoclonal antibody or antibody fragment thereof according to claim 1, wherein the VH region amino acid sequence comprises SEQ ID NO: 19; and Wherein the VL region amino acid sequence comprises SEQ ID NO:

29.

10. The monoclonal antibody or antibody fragment thereof according to claim 1, wherein the VH region amino acid sequence comprises SEQ ID NO: 19; and Wherein the VL region amino acid sequence comprises SEQ ID NO:

30.

11. The monoclonal antibody or antibody fragment thereof according to claim 1, wherein the VH region amino acid sequence comprises SEQ ID NO: 19; and Wherein the VL region amino acid sequence comprises SEQ ID NO:

31.

12. The monoclonal antibody or antibody fragment thereof according to claim 1, wherein the VH region amino acid sequence comprises SEQ ID NO: 20; and Wherein the VL region amino acid sequence comprises SEQ ID NO:

28.

13. The monoclonal antibody or antibody fragment thereof according to claim 1, wherein the VH region amino acid sequence comprises SEQ ID NO: 20; and Wherein the VL region amino acid sequence comprises SEQ ID NO:

29.

14. The monoclonal antibody or antibody fragment thereof according to claim 1, wherein the VH region amino acid sequence comprises SEQ ID NO: 20; and Wherein the VL region amino acid sequence comprises SEQ ID NO:

30.

15. The monoclonal antibody or antibody fragment thereof according to claim 1, wherein the VH region amino acid sequence comprises SEQ ID NO: 20; and Wherein the VL region amino acid sequence comprises SEQ ID NO:

31.

16. The monoclonal antibody or antibody fragment thereof according to claim 1, wherein the VH region amino acid sequence comprises SEQ ID NO: 21; and Wherein the VL region amino acid sequence comprises SEQ ID NO:

28.

17. The monoclonal antibody or antibody fragment thereof according to claim 1, wherein the VH region amino acid sequence comprises SEQ ID NO: 21; and Wherein the VL region amino acid sequence comprises SEQ ID NO:

29.

18. The monoclonal antibody or antibody fragment thereof according to claim 1, wherein the VH region amino acid sequence comprises SEQ ID NO: 21; and Wherein the VL region amino acid sequence comprises SEQ ID NO:

30.

19. The monoclonal antibody or antibody fragment thereof according to claim 1, wherein the VH region amino acid sequence comprises SEQ ID NO: 21; and Wherein the VL region amino acid sequence comprises SEQ ID NO:

31.

20. The monoclonal antibody or antibody fragment thereof according to claim 1, wherein the VH region amino acid sequence comprises SEQ ID NO: 22; and Wherein the VL region amino acid sequence comprises SEQ ID NO:

28.

21. The monoclonal antibody or antibody fragment thereof according to claim 1, wherein the VH region amino acid sequence comprises SEQ ID NO: 22; and Wherein the VL region amino acid sequence comprises SEQ ID NO:

29.

22. The monoclonal antibody or antibody fragment thereof according to claim 1, wherein the VH region amino acid sequence comprises SEQ ID NO: 22; and Wherein the VL region amino acid sequence comprises SEQ ID NO:

30.

23. The monoclonal antibody or antibody fragment thereof according to claim 1, wherein the VH region amino acid sequence comprises SEQ ID NO: 22; and Wherein the VL region amino acid sequence comprises SEQ ID NO:

31. 24 . The monoclonal antibody or antibody fragment thereof according to any one of claims 1 to 23 , wherein the apoptosis-associated speck-like protein containing a caspase activation recruitment domain is human apoptosis-associated speck-like protein containing a caspase activation recruitment domain.

25. The monoclonal antibody or antibody fragment thereof according to any one of claims 1 to 23, wherein the antibody fragment is Fab, F(ab')2, Fab', scFv, or diabody.

26. The monoclonal antibody or antibody fragment thereof according to any one of claims 1-23, wherein the monoclonal antibody or antibody fragment thereof is human, humanized or chimeric.

27. An isolated nucleic acid molecule encoding the monoclonal antibody or antibody fragment thereof according to any one of claims 1-23.

28. An expression vector comprising the nucleic acid molecule according to claim 27.

29. The expression vector of claim 28, wherein the nucleic acid molecule is operably linked to regulatory sequences suitable for expressing the nucleic acid segment in a host cell.

30. A recombinant host cell comprising the expression vector according to claim 28.

31. A method for producing an antibody or antibody fragment that specifically binds to an apoptosis-associated speck-like protein containing a caspase activation recruitment domain, the method comprising: The recombinant host cell comprising the expression vector according to claim 28 is cultured under conditions for expressing the nucleic acid molecule, thereby producing the monoclonal antibody or antibody fragment thereof that specifically binds to apoptosis-associated speck-like protein containing a caspase activation recruitment domain.

32. A pharmaceutical composition comprising the monoclonal antibody or antibody fragment thereof according to any one of claims 1 to 23 and a pharmaceutically acceptable carrier, diluent or excipient.

33. Use of the monoclonal antibody or antibody fragment thereof according to any one of claims 1 to 23 in the preparation of a medicament for treating traumatic brain injury or acute lung injury in a subject.

34. The use according to claim 33, wherein administration of the monoclonal antibody or antibody fragment thereof reduces the level of at least an inflammatory cytokine.

35. The use according to claim 33, wherein administration of the monoclonal antibody or antibody fragment thereof results in inhibition of inflammasome activation in the subject.

36. The use according to claim 33, wherein administration of the monoclonal antibody or antibody fragment thereof results in a decrease in apoptosis-associated speck-like protein containing a caspase activation recruitment domain activity compared to a control.

37. Use according to claim 36, wherein the control is an untreated subject.

38. The use according to claim 33, wherein the drug is administered intracerebroventricularly, intraperitoneally, intravenously or by inhalation.

39. Use of the monoclonal antibody or antibody fragment thereof according to any one of claims 1 to 23 in the preparation of a medicament for treating experimental autoimmune encephalomyelitis in a subject.

40. The use according to claim 39, wherein administration of the monoclonal antibody or antibody fragment thereof reduces the level of at least an inflammatory cytokine.

41. The use according to claim 39, wherein administration of the monoclonal antibody or antibody fragment thereof results in inhibition of inflammasome activation in the subject.

42. The use according to claim 39, wherein administration of the monoclonal antibody or antibody fragment thereof results in a decrease in apoptosis-associated speck-like protein containing a caspase activation recruitment domain activity compared to a control.

43. The use according to claim 42, wherein the control is an untreated subject.

44. The use according to claim 39, wherein the drug is administered intracerebroventricularly, intraperitoneally, intravenously or by inhalation.

45. A monoclonal antibody or antibody fragment thereof according to any one of claims 1 to 26 that binds to apoptosis-associated speck-like protein containing a caspase activation recruitment domain, wherein the antibody or the antibody fragment specifically binds to an epitope of apoptosis-associated speck-like protein containing a caspase activation recruitment domain, wherein the epitope comprises or consists of SEQ ID NO: 5 or an amino acid sequence of 5-10, 10-15 or 15-20 amino acids of SEQ ID NO: 5.

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