Compositions and methods for treating inflammasome related diseases or conditions

Abstract

To provide compositions and methods for treating inflammasome related diseases or conditions.SOLUTION: Compositions and methods described herein include agents that inhibit inflammasome signaling in the mammal such as antibodies directed against inflammasome components used alone or in combination with extracellular vesicle uptake inhibitor(s). Also described herein are compositions and methods of use thereof for treating inflammasome related diseases or conditions.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. patent application Ser. No. 16 / 026,482, filed July 3, 2018, which is incorporated by reference in its entirety for all purposes.

[0002] STATEMENT REGARDING FEDERALLY FUNDED RESEARCH

[0002] The present invention is based on the findings of the National Institute of Neurological Disorders and Stroke (NINS) This invention was made with U.S. Government support under Grant No. 4R42BS086274-02 awarded by the National Institutes of Health (INDS) and Grant No. 5R42NS086274-03 awarded by the National Institutes of Health. The U.S. Government has certain rights in this invention.

[0003] Description of electronically submitted text files

[0003] The contents of the text file submitted electronically herewith are incorporated by reference in their entirety into this specification: Computer-readable format copy of the sequence listing (filename: UNMI_010_02WO_SeqList_ST25.txt, date recorded: July 3, 2019, file size approximately 19.5 kilobytes).

[0004] Field

[0004] The present invention relates generally to the fields of immunology and medicine. More specifically, the present invention relates to compositions and methods for modulating ASC (Apoptosis-associated Speck-like protein containing a Caspase Activating Recruitment Domain (CARD)) activity and Absent in Melanoma 2 (AIM2) inflammasome activity in the central nervous system (CNS) and / or lungs of a mammal as a treatment to reduce inflammation in response to injury or pathology that causes inflammation in the CNS and / or lungs. The present invention also relates to a monoclonal antibody or fragment thereof that specifically binds to ASC. [Background technology]

[0005] background

[0005] Severe traumatic brain injury (TBI) is a major public health problem and a leading cause of mortality and morbidity worldwide (Summers, CR et al., (2009). Traumatic brain injury in the United States: an epidemiologic overview. Mt Sinai J Med 76, 105-110). In addition to direct damage to the brain, TBI can lead to complications in other organs (e.g., the lungs). Acute lung injury (ALI; 2) is a common cardiopulmonary disorder after trauma and is associated with an in-hospital mortality rate of over 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). Patients with TBI are particularly susceptible to developing ALI, and some studies have shown that The incidence rate of RAGE has been reported to be as high as 30% (Nicolls, MR et al., (2014). Traumatic brain injury: lungs in a RAGE. Sci Transl Med 6, 252fs234). It has been shown that systemic inflammatory factors can lead to pulmonary dysfunction and 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 precise molecular mechanisms underlying TBI-induced lung injury remain poorly defined. It remains the same.

[0006]

[0006] The large amount of secreted inflammatory mediators (e.g., cytokines, chemokines, and damage-associated molecular patterns (DAMPs)) released by injured cells contributes to brain inflammation and affects distal organs (e.g., lungs) (Nicolls, MR et al., (2014). Traumatic brain injury: lungs in a RAGE. Sci Transl Med 6, 252fs234). The most widely studied One of the DAMPs that has been identified is high mobility group box-1 (HMGB1), which may function as an early mediator of inflammation in various pathogenic conditions (e.g., TBI) (Andersson U. et al., (2011). Introduction: HMGB1 in inflammation and innate immunity. J Intern Med 270, 296-300). More recent studies have 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). HMG B1 release may be regulated by the inflammasome, a multiprotein complex 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).

[0007]

[0007] Various explanations have been described to explain the pathological mechanisms of pulmonary complications after TBI, such as increased vascular permeability resulting in capillary leakage and infiltration of proteinaceous 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 cell-cell communication (Yanez-Mo, M. et al., (2015). Biological J Extracell Vesicles 4, 27066), and in the development of ALI in an LPS-induced murine model. In addition, EVs can carry biologically active cytokines (e.g., IL-1β and inflammasome proteins) (Qu, Y. et al., (2007). Nonclassical IL-1 beta secretion stimulated by P2X7 receptors is dependent on inflammasome activation and correlated with exosome release in murine 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 It has been shown that EVs can trigger immune responses and amplify inflammation to adjacent and surrounding cells via cargo. However, it is unclear whether EV-mediated inflammasome signaling can contribute to the pathological mechanisms of TBI-induced ALI. Furthermore, it is unclear whether the pathological mechanisms of TBI-induced ALI are shared by other pathologies that cause pulmonary inflammation. In addition, there is a lack of Federal Drug Administration (FDA)-approved drugs to treat pulmonary inflammation. . Summary of the Invention [Problem to be solved by the invention]

[0008]

[0008] Therefore, there is an urgent need not only to elucidate the pathological mechanisms of pulmonary inflammation caused by TBI and other pathological conditions, but also to develop therapeutic compositions and uses thereof for treating and / or preventing pulmonary inflammation. [Means for solving the problem]

[0009] overview In one aspect, provided herein is a monoclonal antibody or antibody fragment thereof that binds to apoptosis-associated speck-like protein (ASC) containing a caspase-activation recruitment domain, wherein the antibody or antibody fragment specifically binds to an epitope of ASC, the epitope comprising or consisting of the amino acid sequence 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. The monoclonal antibody or antibody fragment thereof comprises:

[0010]

[0010] In another aspect, provided herein is a monoclonal antibody or antibody fragment thereof that specifically binds to ASC, the antibody or antibody fragment thereof comprising a heavy chain variable (VH) region and a light chain variable (VL) region, the amino acid sequence of the VH region being HCDR1 of SEQ ID NO:6, HCDR2 of SEQ ID NO:7, and HCDR3 of SEQ ID NO:8, or a variant thereof, comprising a variant having at least one amino acid substitution in HCDR1, HCDR2, and / or HCDR3. In some examples, the amino acid sequence of the VH region 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 some examples, the ASC is a human ASC protein. In some examples, the antibody fragment is a Fab, F(ab')2, Fab', scFv, single domain antibody, bispecific antibody, or single chain camelid antibody, or shark antibody. In some examples, the monoclonal antibody or antibody fragment thereof is human, humanized, or chimeric. In some examples, provided herein is an isolated nucleic acid molecule encoding the monoclonal antibody or antibody fragment thereof. In some examples, provided herein is an expression vector comprising the nucleic acid molecule. In some examples, provided herein is a nucleic acid molecule operably linked to a regulatory sequence suitable for expression of a nucleic acid segment in a host cell. In some examples, provided herein is a recombinant host cell comprising the expression vector. In another aspect, provided herein is a method of producing an antibody or antibody fragment that specifically binds to ASC, comprising culturing a recombinant host cell comprising the expression vector under conditions in which the nucleic acid molecule is expressed, thereby producing a monoclonal antibody or antibody fragment thereof that specifically binds to ASC. In some examples, provided herein is a pharmaceutical composition comprising the monoclonal antibody or antibody fragment thereof and a pharma- ceutically acceptable carrier, diluent, or excipient.In some examples, provided herein is a method for treating inflammation in a subject, comprising administering a therapeutically effective amount of the monoclonal antibody or antibody fragment thereof to the subject, thereby treating inflammation in the subject. In some examples, administering the monoclonal antibody or antibody fragment thereof reduces at least the level of inflammatory cytokines. In some examples, the inflammation is inflammasome-associated inflammation. In some examples, the inflammasome-associated inflammation is associated with central nervous system (CNS) injury, autoimmune disease, autoinflammatory disease, or neurodegenerative disease. In some examples, the CNS injury is selected from the group consisting of 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 storage disorder, glycogen storage disorder, medium-chain acyl-coenzyme A 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 common cold autoinflammatory syndrome (FCAS), Muckle-Wells syndrome (MWS), and neonatal-onset multisystem inflammatory disease (NOMID). In some examples, administration of the monoclonal antibody or antibody fragment thereof inhibits inflammasome activation in a subject. In some examples, administration of the monoclonal antibody or antibody fragment thereof reduces ASC activity compared to a control. In some examples, the control is an untreated subject. In some examples, the administration is intracerebroventricular, intraperitoneal, intravenous, or by inhalation. In some examples, provided herein is a method for treating multiple sclerosis (MS) in a subject. The method includes administering a therapeutically effective amount of the monoclonal antibody or its antibody fragment to the subject, thereby treating the MS of the subject. In some cases, administering the monoclonal antibody or its antibody fragment reduces at least the level of inflammatory cytokines. In some cases, administering the monoclonal antibody or its antibody fragment inhibits inflammasome activation in the subject. In some cases, administering the monoclonal antibody or its antibody fragment reduces the activity of ASC compared to a control. In some cases, the control is an untreated subject. In some cases, the administration is intracerebroventricular, intraperitoneal, intravenous, or by inhalation.

[0011]

[0011] In yet another aspect, provided herein is a monoclonal antibody or antibody fragment thereof that specifically binds to ASC, the antibody or antibody fragment thereof comprising a light chain variable (VL) region and a heavy chain variable (VH) region, the amino acid sequence of the VL region being LCDR1 of SEQ ID NO: 12, LCDR2 of SEQ ID NO: 13, and LCDR3 of SEQ ID NO: 14, or a variant thereof, comprising a variant having at least one amino acid substitution in LCDR1, LCDR2, and / or LCDR3. In some examples, the amino acid sequence of the VL region 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 some examples, the ASC is a human ASC protein. In some examples, the antibody fragment is a Fab, F(ab')2, Fab', scFv, single domain antibody, bispecific antibody, or single chain camelid antibody. In some examples, the monoclonal antibody or antibody fragment thereof is human, humanized, or chimeric. In some examples, provided herein is an isolated nucleic acid molecule encoding the monoclonal antibody or antibody fragment thereof. In some examples, provided herein is an expression vector comprising the nucleic acid molecule. In some examples, provided herein is a nucleic acid molecule operably linked to a regulatory sequence suitable for expression of a nucleic acid segment in a host cell. In some examples, provided herein is a recombinant host cell comprising the expression vector. In another aspect, provided herein is a method of producing an antibody or antibody fragment that specifically binds to ASC, comprising culturing a recombinant host cell comprising the expression vector under conditions in which the nucleic acid molecule is expressed, thereby producing a monoclonal antibody or antibody fragment thereof that specifically binds to ASC. In some examples, provided herein is a pharmaceutical composition comprising the monoclonal antibody or antibody fragment thereof and a pharma- ceutically acceptable carrier, diluent, or excipient.In some examples, provided herein is a method for treating inflammation in a subject, comprising administering a therapeutically effective amount of the monoclonal antibody or antibody fragment thereof to the subject, thereby treating inflammation in the subject. In some examples, administering the monoclonal antibody or antibody fragment thereof reduces at least the level of inflammatory cytokines. In some examples, the inflammation is inflammasome-associated inflammation. In some examples, the inflammasome-associated inflammation is associated with central nervous system (CNS) injury, autoimmune disease, autoinflammatory disease, or neurodegenerative disease. In some examples, the CNS injury is selected from the group consisting of 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 storage disorder, glycogen storage disorder, medium-chain acyl-coenzyme A 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 is familial cold autoinflammatory. Syndrome (FCAS), Muckle-Wells Syndrome (MWS), and Neonatal-onset Multisystem Inflammatory Disease (NOMID). In some examples, administration of the monoclonal antibody or antibody fragment thereof inhibits inflammasome activation in the subject. In some examples, administration of the monoclonal antibody or antibody fragment thereof reduces ASC activity compared to a control. In some examples, the control is an untreated subject. In some examples, the administration is intracerebroventricular, intraperitoneal, intravenous, or by inhalation. In some examples, provided herein is a method of treating multiple sclerosis (MS) in a subject, comprising administering to the subject a therapeutically effective amount of the monoclonal antibody or antibody fragment thereof, thereby treating MS in the subject. In some examples, administration of the monoclonal antibody or antibody fragment thereof reduces at least the level of a proinflammatory cytokine. In some examples, administration of the monoclonal antibody or antibody fragment thereof inhibits inflammasome activation in the subject. In some cases, administration of the monoclonal antibody or antibody fragment thereof reduces the activity of ASC compared to a control. In some cases, the control is an untreated subject. In some cases, the administration is intracerebroventricular, intraperitoneal, intravenous, or by inhalation.

[0012]

[0012] In yet another aspect, provided herein is a monoclonal antibody or antibody fragment thereof that specifically binds to ASC, the antibody or antibody fragment thereof comprising a heavy chain variable (VH) region and a light chain variable (VL) region, the amino acid sequence of the VH region being HCDR1 of SEQ ID NO: 6, HCDR2 of SEQ ID NO: 7, and HCDR3 of SEQ ID NO: 8, or a variant thereof, including a variant having at least one amino acid substitution in HCDR1, HCDR2, and / or HCDR3, and the amino acid sequence of the VL region being LCDR1 of SEQ ID NO: 12, LCDR2 of SEQ ID NO: 13, and LCDR3 of SEQ ID NO: 14, or a variant thereof, including a variant having at least one amino acid substitution in LCDR1, LCDR2, and / or LCDR3. In some examples, the amino acid sequence of the VH region 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, and the amino acid sequence of the VL region 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 some examples, the amino acid sequence of the VH region 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 the amino acid sequence of the VL region 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 some examples, the amino acid sequence of the VH region 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 the amino acid sequence of the VL region 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.In some examples, the amino acid sequence of the VH region comprises SEQ ID NO: 18 or comprises 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 the amino acid sequence of the VL region comprises SEQ ID NO: 30 or comprises 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. In some examples, the amino acid sequence of the VH region comprises SEQ ID NO: 18 or comprises 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 the amino acid sequence of the VL region comprises SEQ ID NO: 31 or comprises 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 examples, the amino acid sequence of the VH region comprises SEQ ID NO: 19 or comprises at least 95% to the amino acid sequence of SEQ ID NO: 19. , 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 28, and the amino acid sequence of the VH region comprises SEQ ID NO: 28 or comprises 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 some examples, the amino acid sequence of the VH region comprises SEQ ID NO: 19 or comprises 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 the amino acid sequence of the VL region comprises SEQ ID NO: 29 or comprises 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. In some examples, the amino acid sequence of the VH region 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 the amino acid sequence of the VL region 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. In some examples, the amino acid sequence of the VH region 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 the amino acid sequence of the VL region 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 examples, the amino acid sequence of the VH region 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 the amino acid sequence of the VL region 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 some examples, the amino acid sequence of the VH region 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 the amino acid sequence of the VL region 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. In some examples, the amino acid sequence of the VH region 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 the amino acid sequence of the VL region 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. In some examples, the amino acid sequence of the VH region 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 the amino acid sequence of the VL region 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 examples, the amino acid sequence of the VH region 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 the amino acid sequence of the VL region 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 some examples, the amino acid sequence of the VH region 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 the amino acid sequence of the VL region 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.In some examples, the amino acid sequence of the VH region 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 the amino acid sequence of the VL region 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. In some examples, the amino acid sequence of the VH region 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 the amino acid sequence of the VL region comprises SEQ ID NO:30. In some examples, the amino acid sequence of the VH region 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 examples, the amino acid sequence of the VH region 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 the amino acid sequence of the VL region 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 some examples, the amino acid sequence of the VH region 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 the amino acid sequence of the VL region 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. In some examples, the amino acid sequence of the VH region comprises SEQ ID NO:22 or comprises 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 the amino acid sequence of the VL region comprises SEQ ID NO:30 or comprises 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. In some examples, the amino acid sequence of the VH region comprises SEQ ID NO:22 or comprises 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 the amino acid sequence of the VL region comprises SEQ ID NO:31 or comprises 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 examples, the ASC is a human ASC protein. In some examples, the antibody fragment is a Fab, F(ab')2, Fab', scFv, single domain antibody, bispecific antibody, or single chain camelid antibody. In some examples, the monoclonal antibody or antibody fragment thereof is human, humanized, or chimeric. In some examples, provided herein is an isolated nucleic acid molecule encoding the monoclonal antibody or antibody fragment thereof.In some examples, provided herein is an expression vector comprising the nucleic acid molecule. In some examples, provided herein is a nucleic acid molecule operably linked to a regulatory sequence suitable for expression of a nucleic acid segment in a host cell. In some examples, provided herein is a recombinant host cell comprising the expression vector. In another aspect, provided herein is a method of producing an antibody or antibody fragment that specifically binds to ASC, comprising culturing a recombinant host cell comprising the expression vector under conditions in which the nucleic acid molecule is expressed, thereby producing a monoclonal antibody or antibody fragment thereof that specifically binds to ASC. In some examples, provided herein is a pharmaceutical composition comprising the monoclonal antibody or antibody fragment thereof and a pharma- ceutically acceptable carrier, diluent, or excipient. In some examples, provided herein is a method of treating inflammation in a subject, comprising administering to the subject a therapeutically effective amount of the monoclonal antibody or antibody fragment thereof, thereby treating inflammation in the subject. In some examples, administering the monoclonal antibody or antibody fragment thereof reduces the level of at least a proinflammatory cytokine. In some examples, the inflammation is an inflammasome-associated inflammation. In some cases, the inflammation associated with this inflammasome is associated with central nervous system (CNS) injury, autoimmune disease, autoinflammatory disease, or neurodegenerative disease. In some cases, the CNS injury is selected from the group consisting of 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 inflammation associated with this inflammasome is associated with metabolic disease or disorder.In some examples, the metabolic disease is metabolic syndrome, obesity, diabetes, diabetic nephropathy or diabetic kidney disease (DKD), insulin resistance, atherosclerosis, lipid storage disorder, glycogen storage disorder, medium-chain acyl-coenzyme A dehydrogenase deficiency, non-alcoholic fatty liver disease (e.g., non-alcoholic steatohepatitis (NASH)), and gout. In the present specification, the autoinflammatory disease is cryopyrin-associated periodic syndrome (CAPS). CAPS can include familial cold autoinflammatory syndrome (FCAS), Muckle-Wells syndrome (MWS), and neonatal-onset multisystem inflammatory disease (NOMID). In some examples, administration of the monoclonal antibody or antibody fragment thereof inhibits inflammasome activation in a subject. In some examples, administration of the monoclonal antibody or antibody fragment thereof reduces ASC activity compared to a control. In some examples, the control is an untreated subject. In some examples, the administration is intracerebroventricular, intraperitoneal, intravenous, or by inhalation. In some examples, provided herein are methods of treating multiple sclerosis (MS) in a subject, comprising administering a therapeutically effective amount of the monoclonal antibody or antibody fragment thereof to the subject, thereby treating MS in the subject. In some examples, administration of the monoclonal antibody or antibody fragment thereof reduces at least the level of a proinflammatory cytokine. In some cases, administering the monoclonal antibody or its antibody fragment inhibits inflammasome activation in the subject.In some cases, administering the monoclonal antibody or its antibody fragment reduces the activity of ASC compared to a control.In some cases, the control is an untreated subject.In some cases, the administration is intracerebroventricular, intraperitoneal, intravenous, or by inhalation. [Brief description of the drawings]

[0013] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1A]

[0013] Illustrating inflammasome activation in C57 / BL6 mouse cortical and lung tissues after TBI, Figure 1A shows representative immunoblots of active caspase-1, ASC, IL-18, IL-β, HMGB1, and AIM2 after TBI. [Figure 1B]

[0013] Active caspase-1 (FIG. 1B) is significantly elevated in cortical tissue at 4 and 24 hours after TBI. Data presented as mean + / - SEM; ****p<0.001, ***p<0.01, **p<0.01, *p<0.05 compared to sham. N=4-5 per group. [Figure 1C] ASC (FIG. 1C) is significantly elevated in cortical tissue at 4 and 24 hours after TBI. Data presented as mean + / - SEM; ****p<0.001, ***p<0.01, **p<0.01, *p<0.05 compared to sham. N=4-5 per group. [Figure 1D] IL-18 (FIG. 1D) is significantly elevated in cortical tissue at 4 and 24 hours after TBI. Data presented as mean + / - SEM; ****p<0.001, ***p<0.01, **p<0.01, *p<0.05 compared to sham. N=4-5 per group. [Figure 1E]

[0013] HMGB1 (FIG. 1E) is significantly elevated in cortical tissue at 4 and 24 hours after TBI. Data presented as mean + / - SEM; ****p<0.001, ***p<0.01, **p<0.01, *p<0.05 compared to sham. N=4-5 per group. [Figure 1F] AIM2 (FIG. 1F) is significantly elevated in cortical tissue at 4 and 24 hours after TBI. Data presented as mean + / - SEM; ****p<0.001, ***p<0.01, **p<0.01, *p<0.05 compared to sham. N=4-5 per group. [Figure 1G]IL-β (FIG. 1G) is significantly elevated in cortical tissue at 4 and 24 hours after TBI. Data presented as mean + / - SEM; ****p<0.001, ***p<0.01, **p<0.01, *p<0.05 compared to sham. N=4-5 per group. [Figure 1H]

[0013] Figure 1H shows representative immunoblots of active caspase-1, ASC, IL-18, IL-β, HMGB1, and AIM2 in lung tissue. [Figure 1I]

[0013] Active caspase-1 (FIG. 1I) is significantly elevated in lung tissue at 4 and 24 hours after TBI. Data presented as mean + / - SEM. N=4-5 per group. ****p<0.001, ***p<0.01, **p<0.01, *p<0.05 compared to sham. [Figure 1J] ASC (FIG. 1J) is significantly elevated in lung tissue at 4 and 24 hours after TBI. Data presented as mean + / - SEM. N=4-5 per group. ****p<0.001, ***p<0.01, **p<0.01, *p<0.05 compared to sham. [Figure 1K] IL-18 (FIG. 1K) is significantly elevated in lung tissue 4 and 24 hours after TBI. Data presented as mean + / - SEM. N=4-5 per group. ****p<0.001, ***p<0.01, **p<0.01, *p<0.05 compared to sham. [Figure 1L]

[0013] HMGB1 (FIG. 1L) is significantly elevated in lung tissue at 4 and 24 hours after TBI. Data presented as mean + / - SEM. N=4-5 per group. ****p<0.001, ***p<0.01, **p<0.01, *p<0.05 compared to sham. [Figure 1M] AIM2 (FIG. 1M) is significantly elevated in lung tissue at 4 and 24 hours after TBI. Data presented as mean + / - SEM. N=4-5 per group. ****p<0.001, ***p<0.01, **p<0.01, *p<0.05 compared to sham. [Figure 1N]IL-β (FIG. 1N) is significantly elevated in lung tissue 4 and 24 hours after TBI. Data presented as mean + / - SEM. N=4-5 per group. ****p<0.001, ***p<0.01, **p<0.01, *p<0.05 compared to sham. [Figure 2A]

[0014] Illustrating the expression of inflammasome proteins in type II alveolar epithelial cells, Figure 2A shows AIM2. [Figure 2B] FIG. 2B shows active caspase-1. [Figure 2C]

[0014] Figure 2C shows that ASC immunoreactivity is increased in lung tissue after CCI (4, 24 hours) compared to mice. Confocal images of AIM2, caspase-1, and ASC (green) and type II epithelial cells (surfactant protein C, red). [Figure 3A]

[0015] Figure 3 illustrates that TBI increases nuclear and cytoplasmic HMGB1 expression in mouse lungs. Figure 3A shows a representative immunoblot of nuclear HMGB1 after TBI. [Figure 3B]

[0015] Figure 3B shows that nuclear HMGB1 is significantly elevated in 4 hour injured animals compared to sham. Data presented as mean + / - SEM; *p<0.05 compared to sham. N=4-5 per group. [Figure 3C]

[0015] FIG. 3C shows a representative immunoblot of cytoplasmic HMGB1 after TBI. [Figure 3D]

[0015] Figure 3D shows that cytoplasmic HMGB1 is significantly elevated in 4 hour injured animals compared to sham. Data presented as mean + / - SEM; *p<0.05 compared to sham. N=4-5 per group. [Figure 3E]

[0015] Figure 3E shows that HMGB1 immunoreactivity was increased in lung tissue after CCI compared to sham mice. Confocal images of HMGB1 and type II epithelial cells (surfactant protein C, red). [Figure 4A]

[0016] Figure 4 illustrates pyroptosome formation in mouse lungs 4 hours after TBI. Figure 4A shows that TBI induces laddering of ASC in lung tissue, showing the formation of pyroptosomes, which are oligomerization of ASC dimers that lead to activation of caspase-1 and pyroptosis. [Figure 4B] FIG. 4B shows a representative immunoblot for gasdermin. [Figure 4C]

[0016] Figure 4C shows quantification of GSDRM. GSDRM-D is significantly elevated in lung tissue after TBI. Data presented as mean + / - SEM. N=4-5 per group, **p<0.01 compared to sham. [Figure 5A]

[0017] We illustrate that TBI induces alveolar morphological changes and acute lung injury in mice. Figure 5A shows H and E staining of lung sections from sham and injured animals at 4 and 24 hours. Sections show evidence of neutrophil infiltration (arrowheads), altered morphology of the alveolar-capillary membrane (asterisk, *), interstitial edema (short arrows), and thickening of the interstitium and alveolar septa (pounds, #). [Figure 5B]

[0017] Figure 5B shows that acute lung injury scoring is significantly increased in injured animals compared to sham at 4 and 24 hours. Data presented as mean + / - SEM. N=4-5 per group, *p<0.05 compared to sham. [Figure 6]

[0018] FIG. 1 illustrates the expression of CD81 in serum-derived EVs from control and TBI injured mice. Representative immunoblots of CD81 in serum-derived EVs from sham control and TBI injured mice. [Figure 7A]

[0019] Adoptive transfer of EVs from TBI animals induces caspase-1 and ASC in the lungs of non-injured mice. Figure 7A illustrates representative immunoblots showing that caspase-1 (Figure 7B), ASC (Figure 7C), IL-18 (Figure 7D), AIM2 (Figure 7E), and HMGB1 (Figure 7F) are elevated in the lungs of animals that received EVs isolated from TBI mice compared to EVs from sham animals. [Figure 7B]

[0019] Caspase-1 (FIG. 7B) is elevated in the lungs of animals that received EVs isolated from TBI mice compared to EVs from sham animals. Data presented as +mean / -SEM; *p<.0.05 compared to sham. N=3 per group. [Figure 7C] ASC (FIG. 7C) is elevated in the lungs of animals that received EVs isolated from TBI mice compared to EVs from sham animals. Data presented as +mean / -SEM; *p<.05 compared to sham. N=3 per group. [Figure 7D] IL-18 (FIG. 7D) is elevated in the lungs of animals that received EVs isolated from TBI mice compared to EVs from sham animals. Data presented as +mean / -SEM; *p<.0.05 compared to sham. N=3 per group. [Figure 7E]

[0019] AIM2 (FIG. 7E) is elevated in the lungs of animals that received EVs isolated from TBI mice compared to EVs from sham animals. Data presented as +mean / -SEM; *p<.0.05 compared to sham. N=3 per group. [Figure 7F]

[0019] HMGB1 (FIG. 7F) is elevated in the lungs of animals that received EVs isolated from TBI mice compared to EVs from sham animals. Data presented as +mean / -SEM; *p<.0.05 compared to sham. N=3 per group. [Figure 7G] EVs from TBI mice induced alveolar morphological changes (reduction in alveolar size) and infiltration of inflammatory cells as determined by H and E staining. ALI scores are significantly increased in EVs delivered from injured mice compared to non-injured mice (Figure 7G). Data presented as mean + / - SEM; **p<0.01, *p<0.05 compared to non-injured group. [Figure 8A]

[0020] Figure 8A illustrates representative immunoblots showing that treatment with enoxaparin (3 mg / kg) and IC100 (5 mg / kg) reduces inflammasome expression in the lungs of animals delivered with EVs from injured mice. Figure 8A illustrates representative immunoblots showing that caspase-1 (Figure 8B), ASC (Figure 8C), IL-1β (Figure 8D), AIM2 (Figure 8E), and HMGB1 (Figure 8F) are reduced in the lungs of animals treated with enoxaparin and IC100 compared to untreated positive control animals. [Figure 8B]

[0020] Caspase-1 (FIG. 8B) is reduced in the lungs of animals treated with enoxaparin and IC100 compared to untreated positive control animals. Data presented as mean + / - SEM; ****p<0.001, ***p<0.01, **p<0.01, *p<0.05 compared to sham. N=4 per group. [Figure 8C] ASC (FIG. 8C) is shown to be reduced in the lungs of animals treated with enoxaparin and IC100 compared to untreated positive control animals. Data presented as mean + / - SEM; ****p<0.001, ***p<0.01, **p<0.01, *p<0.05 compared to sham. N=4 per group. [Figure 8D] IL-1β (FIG. 8D) is reduced in the lungs of animals treated with enoxaparin and IC100 compared to untreated positive control animals. Data presented as mean + / - SEM; ****p<0.001, ***p<0.01, **p<0.01, *p<0.05 compared to sham. N=4 per group. [Figure 8E] AIM2 (FIG. 8E) is reduced in the lungs of animals treated with enoxaparin and IC100 compared to untreated positive control animals. Data presented as mean + / - SEM; ****p<0.001, ***p<0.01, **p<0.01, *p<0.05 compared to sham. N=4 per group. [Figure 8F]

[0020] HMGB1 (FIG. 8F) is reduced in the lungs of animals treated with enoxaparin and IC100 compared to untreated positive control animals. Data presented as mean + / - SEM; ****p<0.001, ***p<0.01, **p<0.01, *p<0.05 compared to sham. N=4 per group. [Figure 9A]

[0021] FIG. 9A illustrates H and E staining of lung sections from EV-delivered mouse lungs from saline (FIG. 9A)-treated injured mice. Sections show evidence of neutrophil infiltration, altered alveolar-capillary membrane morphology, interstitial edema, and thickening of the interstitium and alveolar septa. [Figure 9B] FIG. 9B illustrates H and E staining of lung sections from EV-delivered mouse lungs from injured mice without treatment (FIG. 9B). [Figure 9C] FIG. 9C illustrates H and E staining of lung sections from EV-delivered mouse lungs from injured mice treated with enoxaparin (FIG. 9C). [Figure 9D] FIG. 9D illustrates H and E staining of lung sections from EV-delivered mouse lungs from IC100 (anti-ASC; FIG. 9D)-treated injured mice. [Figure 9E]

[0021] Figure 9E illustrates that acute lung injury scoring is significantly reduced in animals treated with enoxaparin, IC100 compared to untreated animals. Data presented as mean + / - SEM. N=4 per group, ****p<0.01. *p<0.05. [Figure 10A]

[0022] We demonstrate that delivery of serum-derived EVs from TBI patients leads to increased inflammasome protein expression in pulmonary endothelial cells. Figure 10A shows Western blot display of caspase-1, ASC, AIM2, and HMGB1 in PMVECs after 4 h incubation with TBI-EVs and control EVs. [Figure 10B] FIG. 10B shows quantification of Western blots, n=3 filters per group, n=6 patients, t-test, ****p<0.001, ***p<0.01, **p<0.01, *p<0.05. [Figure 10C] FIG. 10C shows quantification of Western blots, n=3 filters per group, n=6 patients, t-test, ****p<0.001, ***p<0.01, **p<0.01, *p<0.05. [Figure 10D] FIG. 10D shows quantification of Western blots, n=3 filters per group, n=6 patients, t-test, ****p<0.001, ***p<0.01, **p<0.01, *p<0.05. [Figure 10E] FIG. 10E shows quantification of Western blots, n=3 filters per group, n=6 patients, t-test, ****p<0.001, ***p<0.01, **p<0.01, *p<0.05. [Figure 10F]

[0022] Figure 10F shows immunoassay results of significant increase in IL-1β expression using the Ella Simpleplex assay, n=3 filters per group, n=6 patients, t-test, ****p<0.001, ***p<0.01, **p<0.01, *p<0.05. [Figure 11A]

[0023] We illustrate that delivery of TBI-EVs to pulmonary endothelial cells increases active caspase-1 immunoreactivity and cell death. Figure 11A shows co-localization of caspase-1 FLICA and PI staining and PMVECs incubated with TBI-EVs for 4 hours. [Figure 11B] FIG. 11B shows caspase-1 FLICA and PI staining in PMVECs incubated with control EV for 4 hours. [Figure 11C]

[0023] Figure 11C shows fluorescence plate reader analysis of PMVECs incubated with TBI and control EVs for 4 hours, n=6, ***p<0.05. [Figure 12A]

[0024] We illustrate that treatment with a humanized anti-ASC monoclonal antibody (i.e., IC100) improves functional outcome in EAE. Figure 12A shows the clinical course of MOG35-55-induced EAE in C57BL / 6 mice treated with vehicle or increasing doses of IC100. Administration of IC100 (10, 30, and 45 mg / kg ip every 4 days) began on day 8, before the mice showed signs of paralysis. Results are expressed as the mean daily clinical score ± SEM for 9-10 mice / group. The 30 and 45 mg / kg curves are significantly different from the vehicle curve: **p ≤ 0.001 Mann-Whitney test. [Figure 12B] FIG. 12B shows a comparison of peak clinical scores (highest disease score reached by a mouse) between groups; *p≦0.05, Student's t test. [Figure 12C]

[0024] Figure 12C shows a comparison of the cumulative disease index (CDI) between groups. CDI is equal to the sum of all scores from the day of onset for each animal and is a measure of EAE severity; *p≦0.05, Student's t-test. [Figure 12D]

[0024] Figure 12D shows a comparison of the onset date between groups. The onset date is considered as the day when the mice showed the first EAE symptoms. [Figure 12E]

[0024] Figure 12E shows a comparison of the peak disease day between groups. The peak disease day is the day when the mice reached the highest disease score. The onset day is considered as the day when the mice showed the first EAE symptoms. [Figure 13A]

[0025] Figure 2 illustrates that IC100 treatment reduces infiltration of peripheral immune cells into the spinal cord or spleen after EAE. Flow cytometry quantification of leukocyte populations infiltrating the spinal cord 35 dpi after EAE. Results are expressed as mean ± SEM of 5 mice / group, *p<0.05, **p<0.001, Student's t-test. [Figure 13B]

[0025] We illustrate that IC100 treatment reduces infiltration of peripheral immune cells into the spinal cord or spleen after EAE. Flow cytometry quantification of leukocyte populations infiltrating the spleen 35 dpi after EAE. Results are expressed as mean ± SEM of 5 mice / group, *p<0.05, **p<0.001, Student's t-test. [Figure 14]

[0026] Figure 1 shows that IC100 treatment reduces microglia in the spinal cord after EAE. Flow cytometry quantification of total and MHCII+ activated microglia in the spinal cord at 35 dpi after EAE. Results are presented as mean ± SEM of 5 mice / group, *p<0.05, Student's t-test. [Figure 15]

[0027] Tissue IC100 concentrations are shown. Concentrations of IC100 (pg / ml) in brain, spinal cord, liver, and spleen in control mice and mice treated with IC100 at 10, 30, and 45 mg / kg 35 dpi after EAE. Data are shown as mean ± SEM. N=2-10 mice per group. [Figure 16]

[0028] We show that IC100 is taken up into ASC specks in unstimulated THP-1 cells and that uptake is increased by inflammasome activation. [Figure 17]

[0029] 1 shows that IC100 prevented IL1-beta release from THP-1 cells. [Figure 18]

[0030] 1 shows confocal images of spinal cord neurons, demonstrating that anti-ASC antibody (IC100) penetrates into spinal cord neurons. [Figure 19]

[0031] 1 shows a comparison of antibody binding of three different antibodies to human ASC to various species. [Figure 20]

[0032] Figure 1 shows inflammasome induction by nicotine (500 nM) in human nucleus pulposus cells and treated with three different antibodies against human ASC (H1, H2, H3) and two different antibodies against mouse ASC (M1, M2). H1 was the most effective at preventing IL-1 beta release / inflammasome activation. [Figure 21]

[0033] 1 shows live BLI kinetic analysis data of anti-ASC monoclonal antibody. [Figure 22]

[0034] 1 shows a comparison of the kinetics of three different antibodies against human ASC. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] Detailed Description definition

[0035] 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.

[0015]

[0036] The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described. All documents or portions of documents cited herein (such as, but not limited to, patents, patent applications, papers, books, and treatises) are expressly incorporated herein by reference in their entirety for all purposes. In the event that one or more of the incorporated documents or portions of documents defines a term that contradicts the definition of the term in this application, the definition that appears in this application shall control. However, reference to any references, papers, publications, patents, patent application publications, and patent applications cited herein is not an admission or any suggestion, and should not be taken as an admission or in any way suggestion, that they constitute valid prior art or form part of the general knowledge in any country throughout the world.

[0016]

[0037] The term "a" or "an" refers to one or more entities, i.e., it may refer to more than one referent. Thus, the terms "a" or "an", "one or more", and "at least one" are used interchangeably herein. In addition, reference to an "element" by the indefinite article "a" or "an" does not exclude the possibility of the element being present in a plurality, unless the context clearly requires that the element be present singly.

[0017]

[0038] Unless the context requires otherwise, throughout this specification and claims, the word "comprise" and variations thereof (e.g., "comprises" and "comprising") shall be interpreted in its open, inclusive sense of "including but not limited to." The use of the alternatives (e.g., "or") should be understood to mean either one, both, or any combination of those alternatives. As used herein, the terms "about" and "consisting essentially of" mean + / - 20% of the indicated range, value, or structure, unless otherwise indicated.

[0018]

[0039] References throughout this specification to "one embodiment" or "an embodiment" refer to particular features, configurations, and features that are described in connection with this embodiment. It means that the structure, feature, or characteristic is included in at least one embodiment of the present disclosure. Thus, the phrase "in one embodiment" in various places throughout this specification. The appearances of "in one embodiment" or "in an embodiment" may not necessarily all refer to the same embodiment. It is recognized that certain features of the present disclosure, which are for clarity described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the present disclosure, which are for brevity described in the context of a single embodiment, may also be provided separately or in any suitable subcombination.

[0019]

[0040] Throughout this disclosure, various aspects of the methods and compositions provided herein may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as a rigid limitation on the scope of the invention. Thus, ranges The description of a range should be considered to have specifically disclosed all the possible subranges and individual numerical values ​​within that range. For example, the description of a range such as 1 to 6 should be considered to have specifically disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numerical values ​​within that range (e.g., 1, 2, 3, 4, 5, and 6). This is true regardless of the breadth of the range.

[0020]

[0041] As used herein, "protein" and "polypeptide" are used interchangeably to mean any peptide-linked chain of amino acids, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation).

[0021]

[0042] 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 that contain an antigen-binding site that specifically binds (immunoreacts with) an antigen (e.g., ASC, NLRP1, AIM2, etc.). The antibodies provided herein may be polyclonal, monoclonal (mAb), chimeric, humanized, anti-idiotypic (anti-Id) antibodies to antibodies that may be labeled in soluble or bound form, and active fragments, regions, or derivatives thereof. Antibodies for use herein may be chimeric, humanized, or human.

[0022]

[0043] "Specifically bind" or "immunoreact with" means that the antibody reacts with one or more antigenic determinants of the desired antigen and does not react with other peptides. In certain embodiments, an antibody is said to specifically bind 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 (generally comprising four polypeptide chains, two heavy (H) chains and two light (L) chains), or any functional fragment, mutant, variant, or derivative of this Ig molecule that retains the essential target binding properties of the Ig molecule. Such mutant, variant, or derivative antibody forms are known in the art. Such anti-ASC and anti-NLRP1 antibodies of the present invention may bind to portions of ASC and NLRP1, respectively, that prevent caspase-1 activation.

[0023]

[0044] The term "humanized antibody" as used herein refers to an antibody in which minimal parts of a non-human antibody have been introduced into an otherwise human antibody.

[0024]

[0045] The term "human antibody," as used herein, refers to an antibody in which substantially all portions of the protein are substantially non-immunogenic in humans, with minor sequence alterations or variations.

[0025]

[0046] In full-length antibodies, 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 and VL regions can be further subdivided into regions of hypervariability (called complementarity determining regions (CDRs)) interspersed with more conserved regions (called framework regions (FRs)). Each VH and VL is composed of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY) and class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or subclass. IgG, IgD, and IgE antibodies generally consist of two identical heavy chains and two identical light chains, each with a heavy chain variable region (VH) and a light chain variable region (VL). IgA antibodies are generally composed of two monomers, each of which is composed of two heavy and two light chains (as in IgG, IgD, and IgE antibodies), and thus the IgA molecule has four antigen-binding domains, each of which is again composed of a VH and a VL. Certain IgA molecules are monomers, each of which is composed of two heavy and two light chains. Secreted IgM antibodies are generally composed of five monomers, each of which is composed of two heavy and two light chains (as in IgG and IgE antibodies), and thus the IgM molecule has ten antigen-binding domains, each of which is again composed of a VH and a VL. Cell surface forms of IgM also exist, which have a similar two heavy / two light chain structure as IgG, IgD, and IgE antibodies.

[0026]

[0047] The term "antigen-binding fragment", or "antigen-binding portion", or "antigen-binding site", or "binding domain", or "binding region", as used herein, may refer to a protein, polypeptide, oligopeptide, or peptide, or an antibody, or a domain, region, part, or portion of a binding domain derived from an antibody, that retains the ability to specifically bind to an antigen (e.g., an 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 ectodomains, 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 (VH CDR3) and / or CDR L3 (VL CDR3). For purposes of the present invention, a fusion protein comprises one or more antibodies and additional amino acid sequences (e.g., a heterologous sequence or a homologous sequence from another region attached to the N-terminus or C-terminus of the antibody or antibody fragment thereof). Exemplary heterologous sequences include, but are not limited to, "tags" (e.g., FLAG tags or 6His tags), or enzymes or polypeptides that increase the half-life of the antibody in blood. Tags are known in the art. This additional amino acid sequence (which may include amino- and / or carboxyl-terminal fusions) can range in length from one residue to polypeptides of 100 or more residues and including intrasequence insertions of single or multiple amino acid residues.

[0027]

[0048] An antigen-binding site may generally be formed by a heavy chain variable region (VH) immunoglobulin domain and a light chain variable region (VL) immunoglobulin domain, with the antigen-binding interface being formed by six surface polypeptide loops called complementarity determining regions (CDRs). Three CDRs are present in the VH (HCDR1, HCDR2, HCDR3) and VL (LCDR1, LCDR2, LCDR3), respectively, together with framework regions (FRs). In certain embodiments, the binding domain comprises or consists of an antigen-binding site (e.g., variable heavy and variable light chain sequences from an antibody arranged in alternative framework regions (FRs) (e.g., human FRs, optionally containing one or more amino acid substitutions) or three light chain complementarity determining regions (CDRs) and three heavy chain CDRs).

[0028]

[0049] The term "CDR region" or "CDR" refers to the CDR region of a polypeptide as described in Kabat et al., 1991 (Kabat, EA et al., (1991) Sequences of Proteins of Immunological Interest, 5th Edition. US Department of of Health and Human Services, Public Service, NIH, Washington, and later editions. As defined herein, it may refer to the hypervariable region of an immunoglobulin heavy or light chain. An antibody typically contains three heavy chain CDRs and three light chain CDRs.

[0029]

[0050] 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 form, comprising two or more different antigen-binding domains. Examples of binding fragments encompassed by the term "antigen-binding fragment" of an antibody are: Examples of such fragments include: (i) a Fab fragment consisting of a VL domain, a VH domain, a CL domain, and a CH1 domain (Ward, ES et al., (1989) Nature 341, 544-546); (ii) an Fd fragment consisting of a VH domain and a CH1 domain (McCafferty et al., (1990) Nature, 348, 552-554); (iii) an Fv fragment consisting of a VL domain and a VH domain of a single antibody (Holt et al., (2003) Trends in Biotechnology 21, 484-490); (iv) dAb fragments consisting of the VH or VL domains (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]; (v) isolated CDR regions; (vi) two F(ab')2 fragments, which are bivalent fragments that contain a binding Fab fragment; (vii) single chain Fv molecules (scFv) in which the VH and VL domains are linked by a peptide linker that allows the two domains to associate and 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 encompasses Fab' fragments. Furthermore, although the two domains VL and VH of the Fv fragment are encoded by separate genes, these domains can be linked using recombinant methods by a synthetic linker that allows the domains to be formed as a single protein chain (known as single chain Fv (scFv)) in which the VL and VH regions pair to form a monovalent molecule. Such single chain antibodies are also intended to be encompassed by the term "antigen-binding fragment" of an antibody. In certain embodiments of the present 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 / US92109965), and (ix) "diabodies," which are multivalent or multispecific fragments constructed by genetic fusion (WO 94 / 13804; Holliger, P. (1993) et al., Proc. Natl. Acad. Sci. USA 90 6444-6448). Diabodies are comprised of a VH domain and a VL domain. are expressed on a single polypeptide chain, but use a linker that is too short to allow pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains on another chain to form two antigen-binding sites (see, e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448). (See Poljak, RJ, et al. (1994) Structure 2:1121-1123). Such antibody binding fragments are known in the art (Kontermann and Dubel eds., Antibody Engineering (2001) Springer-Verlag. New York. 790 pp.). In some embodiments, the present invention provides includes single domain antibodies. In general, the term "antibody" as used herein encompasses "antibody fragments." Antibody fragments generally retain the antigen-binding properties of the full-length antibody.

[0030]

[0051] Fv, scFv or diabody molecules can be stabilized by the incorporation of disulfide bridges linking the VH and VL domains (Reiter, Y. et al., Nature Biotech, 14, 1239-1245, 1996). Minibodies can also be made that contain scFvs linked to the CH3 domain (Hu, S. et al., (1996) Cancer Res., 56, 3055-3061). Other examples of binding fragments can be Fab', which differs from Fab fragments by the addition of a few residues at the carboxyl terminus of the heavy chain CH1 domain, e.g., one or more cysteines from the antibody hinge region, and Fab'-SH, which is a Fab' fragment in which the cysteine ​​residues of the constant domains bear free thiol groups.

[0031]

[0052] "Fv" as used herein may refer to the minimum fragment of an antibody that retains both the antigen recognition and binding sites. "Fab" as used herein 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.

[0032]

[0053] An "Fc region" or "Fc domain" refers to the cellular component of a source antibody. Fc refers to the polypeptide sequence corresponding to or derived from the portion of an antibody that is involved in binding to the antibody receptor on cells and the C1q component of complement. Fc is a polypeptide sequence that readily forms protein crystals. Fc is an abbreviation for "fragment crystal", which is a fragment of immunoglobulin. Separate protein fragments, initially described by proteolytic digestion, may define the overall general structure of immunoglobulin proteins. According to the original definition in the literature, the Fc fragment consists of the disulfide-bonded heavy chain hinge region, CH2 domain, and CH3 domain. However, more recently, the term has been applied to a single chain consisting of at least a portion of the hinge sufficient to form a disulfide-bonded dimer containing CH3, CH2, and 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 naturally occurring sequence variants. In one embodiment, the antibody or antibody fragment derived therefrom (e.g., anti-ASC monoclonal antibody or antibody fragment thereof) provided herein has a modified Fc region or Fc domain. In some cases, the modified Fc region or Fc domain may confer high thermal stability to the resulting antibody or antibody fragment derived therefrom. This high thermal stability may result in a high serum half-life. The Fc region or Fc domain may be modified as described in US Patent Publication No. 20160193295, the contents of which are incorporated herein by reference. As described in US Patent Publication No. 20160193295, the Fc region or Fc domain may be modified to have one or more cysteine ​​residues in the hinge region deleted and one or more CH3-interface amino acids replaced with sulfhydryl-containing residues. In another embodiment, the modified Fc region or Fc domain may be modified as described in Wozniak-Knopp G, the contents of which are incorporated herein by reference. As described in Stadlmann J, Rueker F (2012) Stabilisation of the Fc Fragment of Human IgG1 by Engineered Intradomain Disulfide Bonds. PLoS ONE 7(1): e30083 Thus, the Fc region or Fc domain of the antibodies provided herein or antibody fragments derived therefrom (e.g., anti-ASC monoclonal antibodies or antibody fragments thereof) may be stabilized by engineering the Fc region to have intradomain disulfide bonds. In yet another embodiment, the antibody has an Fc region that has been modified as described in WO 99 / 58572, the contents of which are incorporated herein by reference. In yet other embodiments, the Fc region or Fc domain may be modified as described in U.S. Patent No. 9,574,010, the contents of which are incorporated herein by reference.

[0033]

[0054] The terms "caspase-activating recruitment domain (CARD)-containing apoptosis-associated speck-like protein" and "ASC" refer to the expression product of the ASC gene or an isoform thereof, or a protein that shares 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 exhibits the functional activity of ASC. A "functional activity" of a protein is any activity related to the physiological function of the protein. Functional activities of ASC include, for example, activation of caspase-1 and recruitment of the protein to initiate cell death.

[0034]

[0055] The term "ASC gene" or "ASC nucleic acid" refers to a naturally occurring ASC-encoding nucleic acid sequence, a genomic sequence from which ASC cDNA can be transcribed, and / or allelic variants and homologs of the above. The term encompasses double-stranded DNA, single-stranded DNA, and RNA.

[0035]

[0056] As used herein, the term "inflammasome" refers to a multiprotein (e.g., at least two proteins) complex that activates caspase-1. Additionally, the term "inflammasome" can refer to a multiprotein complex that activates caspase-1 activity, which in turn regulates IL-1β, IL-18, and IL-33 processing and activation. Arend et al. 2008; L, each of which is incorporated herein by reference in its entirety. See Martinon et al. 2002; and Martinon et al. 2008. "NALP1 inflammasome", "NLRP2 inflammasome", "NALP2 inflammasome", "NLRP3 inflammasome", "NALP3 inflammasome", "NLRC4 inflammasome", "IPAF inflammasome" or "AIM2 inflammasome" refers to a protein complex of at least caspase-1 and one adaptor protein, such as ASC. For example, the terms "NLRP1 inflammasome" and "NALP1 inflammasome" can refer to a multiprotein complex containing NLRP1, ASC, caspase-1, caspase-11 for activation of caspase-1 and processing of interleukin-1β, interleukin-18 and interleukin-33, XIAP, and pannexin-1. The terms "NLRP2 inflammasome" and "NALP2 inflammasome" may refer to a multiprotein complex containing NLRP2 (aka NALP2), ASC and caspase-1, while the terms "NLRP3 inflammasome" and "NALP3 inflammasome" may refer to a multiprotein complex containing NLRP3 (aka NALP3), ASC, and the terms "NLRC4 inflammasome" and "IPAF inflammasome" may refer to a multiprotein complex containing NLRC4 (aka IPAF), ASC and caspase-1. Additionally, the term "AIM2 inflammasome" may refer to a multiprotein complex containing AIM2, ASC and caspase-1.

[0036]

[0057] As used herein, the phrase "sequence identity" refers to the alignment of two sequences (e.g., nucleic acid sequences, amino acid sequences) to maximize subunit matching, i.e., the percentage of identical subunits at corresponding positions in the two sequences, taking into account gaps and insertions. Sequence identity can be measured using sequence analysis software (e.g., Sequence Analysis Software Package from Accelrys CGC, San Diego, Calif.). Cut.

[0037]

[0058] The phrases "therapeutically effective amount" and "effective dosage" refer to an amount sufficient to produce a therapeutically (e.g., clinically) desired result; the exact nature of the result will vary depending on the nature of the disorder being treated. For example, if the disorder being treated is SCI, the result may be improved motor skills and locomotor function, reduced spinal cord pathology, and the like. The compositions described herein may be administered one or more times daily to one or more times weekly. One of skill in the art will recognize that certain factors, including but not limited to the severity of the disease or disorder, prior treatments, the general health and / or age of the subject, and other diseases present, may affect the dosage and timing required to effectively treat a subject. Furthermore, treatment of a subject with a therapeutically effective amount of a composition of the invention may include a single treatment or a series of treatments.

[0038]

[0059] The term "treatment" as used herein is defined as the application or administration of a therapeutic agent described herein or identified by the methods described herein to a patient, or to an isolated tissue or cell line from a patient having a disease, a symptom of a disease, or a predisposition to a disease, for the purpose of curing, ameliorating, alleviating, altering, rescuing, ameliorating, or affecting the disease, a symptom of a disease, or a predisposition to a disease.

[0039]

[0060] The terms "patient," "subject," and "individual" are used interchangeably herein to refer to a mammalian subject to be treated. In one embodiment, the mammalian subject is a human. In some instances, the methods of the present invention are used in laboratory animals, in veterinary applications, and in developing animal models for disease, including, but not limited to, rodents, such as mice, rats, and hamsters, and primates.

[0040]

[0061] As used herein interchangeably, "absentee melanoma 2" and " "AIM2" can mean an expression product or isoform of the AIM2 gene; or a protein sharing at least 65%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity with AIM2 (e.g., Accession Nos. NX_014862, NP004824, XP016858337, XP005245673, AAB81613, BAF84731, AAH10940) and exhibiting a functional activity of AIM2.

[0041]

[0062] As used interchangeably herein, "NACHT, LRR and PYD domain-containing protein 1," "NALP1," and "NLRP1" refer to the expression product or isoform of the NALP1 or NLRP1 gene, or to a protein that shares at least 65%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity with NALP1 (e.g., Accession Nos. AAH51787, NP_001028225, NP_127500, NP_127499, NP_127497, NP055737) and exhibits the functional activity of NALP1.

[0042]

[0063] "NALP2" and "NLRP2," as used interchangeably herein, refer to the expression product or isoform of the NALP2 or NLRP2 gene; or a protein that shares at least 65%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity with NALP2 (e.g., Accession Nos. NP_001167552, NP_001167553, NP_001167554, or NP_060322) and exhibits the functional activity of NALP2.

[0043]

[0064] "NALP3" and "NLRP3," as used interchangeably herein, refer to the expression product or isoform of the NALP3 or NLRP3 gene; or a protein that shares at least 65%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity with 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 exhibits a functional activity of NALP3.

[0044]

[0065] "NLRC4" and "IPAF," as used interchangeably herein, refer to the expression product or isoform of the NLRC4 or IPAF gene; 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 the functional activity of NLRC4.

[0045]

[0066] The terms "stroke" and "ischemic stroke" refer to a part of the brain or spinal cord where blood flow is interrupted.

[0046]

[0067] "CNS traumatic injury" means any injury to the CNS from an external mechanical force that may result in permanent or temporary impairment of CNS function.

[0047]

[0068] Methods involving conventional molecular biology techniques are described herein. Such techniques are generally known in the art and can be found in methodological treatises, such as Molecular Cloning: A Laboratory Manual, 3rd ed., vol. 1-3, ed. Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001; and Current Protocols in Molecular Biology. Immunology, ed. 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 methodological treatises such as Advances in Immunology, volume 93, ed. Frederick W. Alt, Academic Press, Burlington, MA, 2007; Making and Using Antibodies: A Practical Handbook, eds. Gary C. Howard and Matthew R. Kaser, CRC Press, Boca Raton, FL, 2006; Medical Immunology, 6th ed., edited by Gabriel Virella, Informa Healthcare Press, London, England, 2007; and Harlow and Lane ANTIBODIES: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1988.

[0048]

[0069] Although compositions and methods similar or equivalent to those 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 case of conflict, the present specification, including definitions, will control. The specific embodiments discussed below are for illustrative purposes only and are not limiting.

[0049] overview

[0070] Provided herein is a monoclonal antibody or antibody fragment thereof that specifically binds to apoptosis-associated speck-like protein (ASC) containing a caspase-activating recruitment domain. The monoclonal antibody or fragment thereof can specifically bind to an antigen fragment of ASC that comprises, consists of, or consists essentially of the amino acid sequence of SEQ ID NO:5. In addition to this embodiment, the present invention contemplates the use of the monoclonal antibody or antibody fragment thereof 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 antibody fragment thereof provided herein can be used in a method for reducing inflammation in a mammal as described in U.S. Pat. No. 8,685,400, the contents of which are incorporated herein by reference in their entirety. The inflammation can be in the lungs and / or the central nervous system (CNS). Inflammation in the lungs and / or CNS can be the result of injury (e.g., traumatic brain injury (TBI) or spinal cord injury (SCI)) or can be the result of a disease, condition, or affliction of or affecting the CNS. As provided herein, diseases, conditions, or afflictions of or affecting the CNS include stroke, as well as autoimmune and / or CNS diseases (e.g., amyotrophic lateral sclerosis (ALS) Lou Gehrig's, multiple sclerosis (MS), immune dysfunction muscular dyscrasia (IMD), and CNS disorders (e.g., pulmonary edema ... The monoclonal antibody or antibody fragment thereof may be a marker of a disease, such as a pulmonary fibrosis (CNS breakdown), muscular dystrophy (MD), Alzheimer's disease (AD), or Parkinson's disease (PD). The use of the monoclonal antibody or antibody fragment thereof in a method for treating inflammation may reduce inflammation in the CNS and / or lungs of a patient. The reduction may be in comparison to a control (e.g., an untreated patient and / or a patient prior to treatment). In one embodiment, the monoclonal antibody or antibody fragment derived from the monoclonal antibody is used to treat MS by administering the monoclonal antibody or antibody fragment derived from the monoclonal antibody to a patient suffering from or suspected of suffering from MS. The monoclonal antibody or antibody fragment thereof of this embodiment may be present in a composition (e.g., a pharmaceutical composition provided herein). In some cases, the monoclonal antibody or antibody fragment thereof is used in combination with one or more other agents in the treatment methods provided herein. The other agent can be any agent provided herein (e.g., an EV uptake inhibitor) and / or an antibody or antibody fragment against other inflammasome components (e.g., IL-18, caspase-1, NALP1, AIM2, etc.).

[0050]

[0071] The present invention also relates to a monoclonal antibody or an antibody fragment thereof that specifically binds to ASC, the antibody or the antibody fragment thereof comprising a heavy chain variable (VH) region and a light chain variable (VL) region, the amino acid sequence of the VH region being represented by HCDR1 of SEQ ID NO:6, HDR2 of SEQ ID NO:7, Also encompassed is a monoclonal antibody or antibody fragment thereof comprising CDR2 and HCDR3 of SEQ ID NO: 8, or a variant thereof, with at least one amino acid substitution in HCDR1, HCDR2, and / or HCDR3. In addition to this embodiment, the present invention contemplates the use of this monoclonal antibody or antibody fragment thereof 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 antibody fragment thereof provided herein can be used in a method for reducing inflammation in a mammal as described in U.S. Pat. No. 8,685,400, the contents of which are incorporated herein by reference in their entirety. The inflammation can be present in the lungs and / or CNS. Inflammation in the lungs and / or CNS can be the result of injury (e.g., traumatic brain injury (TBI) or spinal cord injury (SCI)) or can be the result of a disease, condition, or affliction in or affecting the CNS. As provided herein, the disease, condition, or affliction of or affecting the CNS may be stroke, as well as autoimmune and / or CNS diseases (e.g., amyotrophic lateral sclerosis (ALS) Lou Gehrig's, multiple sclerosis (MS), immune dysfunction muscle CNS degeneration, muscular dystrophy (MD), Alzheimer's disease (AD), Parkinson's disease (PD)). Use of the monoclonal antibody or antibody fragment thereof in a method for treating inflammation may reduce inflammation in the CNS and / or lungs of a patient. Use of the monoclonal antibody or antibody fragment thereof in a method for treating inflammation may reduce innate immune inflammation or inflammasome-associated inflammation in a patient. The reduction may be relative to a control (e.g., an untreated patient and / or a patient prior to treatment). In one embodiment, the monoclonal antibody or antibody fragment derived from the monoclonal antibody is used to treat central nervous system (CNS) injury and / or an autoimmune, autoinflammatory, metabolic, or neurodegenerative disease. The CNS injury may be selected from the group consisting of traumatic brain injury (TBI), stroke, and spinal cord injury (SCI).The autoimmune or neurodegenerative disease may 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., Crohn's disease and ulcerative colitis), and multiple sclerosis (MS). In one embodiment, the monoclonal antibody or an antibody fragment derived from the monoclonal antibody is used to treat MS by administering the monoclonal antibody or an antibody fragment derived from the monoclonal antibody to a patient suffering from or suspected of suffering from MS. In one embodiment, the monoclonal antibody or an antibody fragment derived from the monoclonal antibody is used to treat PD by administering the monoclonal antibody or an antibody fragment derived from the monoclonal antibody to a patient suffering from or suspected of suffering from PD. In one embodiment, the monoclonal antibody or an antibody fragment derived from the monoclonal antibody is used to treat lupus nephritis by administering the monoclonal antibody or an antibody fragment derived from the monoclonal antibody to a patient suffering from or suspected of suffering from lupus nephritis. The metabolic disease may be selected from metabolic syndrome, obesity, diabetes, diabetic nephropathy or diabetic kidney disease (DKD), insulin resistance, atherosclerosis, lipid storage disorder, glycogen storage disorder, medium-chain acyl-coenzyme A dehydrogenase deficiency, nonalcoholic fatty liver disease (e.g., nonalcoholic steatohepatitis (NASH)), and gout. In one embodiment, the monoclonal antibody or an antibody fragment derived from the monoclonal antibody is used to treat diabetic nephropathy by administering the monoclonal antibody or an antibody fragment derived from the monoclonal antibody to a patient suffering from or suspected of suffering from diabetic nephropathy. In one embodiment, the monoclonal antibody, or an antibody fragment derived from the monoclonal antibody, is used to treat NASH by administering the monoclonal antibody, or an antibody fragment derived from the monoclonal antibody, to a patient suffering from or suspected of suffering from NASH.This autoinflammatory disease may be cryopyrin-associated periodic syndrome (CAPS), which is also known as familial common cold autoinflammatory syndrome (FCAS), Muckle-Wells syndrome (MW). In one embodiment, the monoclonal antibody or an antibody fragment derived from the monoclonal antibody is used to treat CAPS by administering the monoclonal antibody or an antibody fragment derived from the monoclonal antibody to a patient suffering from or suspected of suffering from CAPS. The monoclonal antibody or antibody fragment of the monoclonal antibody of this embodiment may be present in a composition (e.g., a pharmaceutical composition provided herein). In some cases, the monoclonal antibody or fragment thereof is used in combination with one or more other agents in the therapeutic methods provided herein. The other agents may be any of the agents provided herein (e.g., EV uptake inhibitors) and / or antibodies or antibody fragments against other inflammasome components (e.g., IL-18, caspase-1, NALP1, AIM2, etc.).

[0051]

[0072] In some embodiments, the present invention provides a monoclonal antibody or antibody fragment thereof that specifically binds to ASC, the antibody or antibody fragment thereof comprising a light chain variable (VL) region and a heavy chain variable (VH) region, the amino acid sequence of the VL region being LCDR1 of SEQ ID NO: 12, LCDR2 of SEQ ID NO: 13, and LCDR3 of SEQ ID NO: 14, or a variant thereof, comprising a variant having at least one amino acid substitution in LCDR1, LCDR2, and / or LCDR3. In addition to this embodiment, the present invention contemplates 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. In one embodiment, the monoclonal antibody or antibody fragment thereof provided herein may be used in a method for reducing inflammation in a mammal as described in U.S. Pat. No. 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 can be the result of injury (e.g., traumatic brain injury (TBI) or spinal cord injury (SCI)) or can be the result of a disease, condition, or affliction of or affecting the CNS. As provided herein, the disease, condition, or affliction of or affecting the CNS can be stroke, as well as autoimmune and / or CNS diseases (e.g., amyotrophic lateral sclerosis (ALS) Lou Gehrig's, multiple sclerosis (MS), immune dysfunction muscle CNS degeneration, muscular dystrophy (MD), Alzheimer's disease (AD), Parkinson's disease (PD)). Use of the monoclonal antibody or antibody fragment thereof in a method of treating inflammation can reduce inflammation in the CNS and / or lungs of a patient. Use of the monoclonal antibody or antibody fragment thereof in a method of treating inflammation can reduce innate immune inflammation or inflammasome-associated inflammation in a patient. The reduction can be in comparison to a control (e.g., an untreated patient and / or a patient prior to treatment).In one embodiment, the monoclonal antibody or an antibody fragment derived from the monoclonal antibody is used to treat central nervous system (CNS) injury and / or autoimmune, autoinflammatory, metabolic, or neurodegenerative disease. The CNS injury may be selected from the group consisting of traumatic brain injury (TBI), stroke, and spinal cord injury (SCI). The autoimmune or neurodegenerative disease may 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., Crohn's disease and ulcerative colitis), and multiple sclerosis (MS). In one embodiment, the monoclonal antibody or an antibody fragment derived from the monoclonal antibody is used to treat MS by administering the monoclonal antibody or an antibody fragment derived from the monoclonal antibody to a patient suffering from or suspected of suffering from MS. In one embodiment, the monoclonal antibody or an antibody fragment derived from the monoclonal antibody is used to treat PD by administering the monoclonal antibody or an antibody fragment derived from the monoclonal antibody to a patient suffering from or suspected of suffering from PD. The monoclonal antibody or an antibody fragment derived from the monoclonal antibody is used to treat lupus nephritis by administering the monoclonal antibody or an antibody fragment derived from the monoclonal antibody to a patient suffering from or suspected of suffering from lupus nephritis. The metabolic disease may be selected from metabolic syndrome, obesity, diabetes, diabetic nephropathy or diabetic kidney disease (DKD), insulin resistance, atherosclerosis, lipid storage disorder, glycogen storage disorder, medium-chain acyl-coenzyme A dehydrogenase deficiency, nonalcoholic fatty liver disease (e.g., nonalcoholic steatohepatitis (NASH)), and gout. In one embodiment, the monoclonal antibody or an antibody fragment derived from the monoclonal antibody is used to treat diabetic nephropathy by administering the monoclonal antibody or an antibody fragment derived from the monoclonal antibody to a patient suffering from or suspected of suffering from diabetic nephropathy. In one embodiment, the monoclonal antibody or an antibody fragment derived from the monoclonal antibody is used to treat NASH by administering the monoclonal antibody or an antibody fragment derived from the monoclonal antibody to a patient suffering from or suspected of suffering from NASH. The autoinflammatory disease may be cryopyrin-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 an antibody fragment derived from the monoclonal antibody is used to treat CAPS by administering the monoclonal antibody or an antibody fragment derived from the monoclonal antibody to a patient suffering from or suspected of suffering from CAPS. The monoclonal antibody or antibody fragment of this embodiment may be present in a composition (e.g., a pharmaceutical composition provided herein). In some cases, the monoclonal antibodies or fragments thereof are used in combination with one or more other agents in the therapeutic methods provided herein.The other agent can be any agent provided herein (e.g., an EV uptake inhibitor) and / or an antibody or antibody fragment against other inflammasome components (e.g., IL-18, caspase-1, NALP1, AIM2, etc.).

[0052]

[0073] In another embodiment, the present invention also provides a monoclonal antibody or antibody fragment thereof that specifically binds to ASC, the antibody or antibody fragment thereof comprising a heavy chain variable (VH) region and a light chain variable (VL) region, the amino acid sequence of the VH region being HCDR1 of SEQ ID NO: 6, HCDR2 of SEQ ID NO: 7, and HCDR3 of SEQ ID NO: 8, or a variant thereof, with at least one amino acid substitution in HCDR1, HCDR2, and / or HCDR3, and the amino acid sequence of the VL region being LCDR1 of SEQ ID NO: 12, LCDR2 of SEQ ID NO: 13, and LCDR3 of SEQ ID NO: 14, or a variant thereof, with at least one amino acid substitution in LCDR1, LCDR2, and / or LCDR3. In addition to this embodiment, the present invention contemplates 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. In one embodiment, the monoclonal antibodies or antibody fragments thereof provided herein may be used in a method of reducing inflammation in a mammal as described in U.S. Patent No. 8,685,400, the contents of which are incorporated herein by reference in their entirety. The inflammation may be present in the lungs and / or CNS. The 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 may be the result of a disease, condition, or affliction of or affecting the CNS. As provided herein, the disease, condition, or affliction of or affecting the CNS may be stroke, as well as autoimmune and / or CNS diseases (e.g., amyotrophic lateral sclerosis (ALS) Lou Gehrig's, multiple sclerosis (MS), immune dysfunction muscle CNS degeneration, muscular dystrophy (MD), Alzheimer's disease (AD), Parkinson's disease (PD)). The use of this monoclonal antibody or antibody fragment thereof in a method for treating inflammation reduces inflammation in the CNS and / or lungs of a patient. The use of the monoclonal antibody or antibody fragment thereof in a method for treating inflammation may reduce innate immune inflammation or inflammasome-associated inflammation in a patient. The reduction may be in comparison to a control (e.g., an untreated patient and / or a patient prior to treatment). In one embodiment, the monoclonal antibody or an antibody fragment derived from the monoclonal antibody is used to treat central nervous system (CNS) injury and / or an autoimmune disease, an autoinflammatory disease, a metabolic disease, or a neurodegenerative disease. The CNS injury may be selected from the group consisting of traumatic brain injury (TBI), stroke, and spinal cord injury (SCI). The autoimmune disease or neurodegenerative disease may 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., Crohn's disease and ulcerative colitis), and multiple sclerosis (MS). In one embodiment, the monoclonal antibody or an antibody fragment derived from the monoclonal antibody is used to treat MS by administering the monoclonal antibody or an antibody fragment derived from the monoclonal antibody to a patient suffering from or suspected of suffering from MS. In one embodiment, the monoclonal antibody or an antibody fragment derived from the monoclonal antibody is used to treat PD by administering the monoclonal antibody or an antibody fragment derived from the monoclonal antibody to a patient suffering from or suspected of suffering from PD. In one embodiment, the monoclonal antibody or an antibody fragment derived from the monoclonal antibody is used to treat lupus nephritis by administering the monoclonal antibody or an antibody fragment derived from the monoclonal antibody to a patient suffering from or suspected of suffering from lupus nephritis. The metabolic disease may be selected from metabolic syndrome, obesity, diabetes, diabetic nephropathy or diabetic kidney disease (DKD), insulin resistance, atherosclerosis, lipid storage disorder, glycogen storage disorder, medium-chain acyl-coenzyme A dehydrogenase deficiency, non-alcoholic fatty liver disease (e.g., non-alcoholic steatohepatitis (NASH)), and gout.In one embodiment, the monoclonal antibody or an antibody fragment derived from the monoclonal antibody is used to treat diabetic nephropathy by administering the monoclonal antibody or an antibody fragment derived from the monoclonal antibody to a patient suffering from or suspected of suffering from diabetic nephropathy. In one embodiment, the monoclonal antibody or an antibody fragment derived from the monoclonal antibody is used to treat NASH by administering the monoclonal antibody or an antibody fragment derived from the monoclonal antibody to a patient suffering from or suspected of suffering from NASH. The autoinflammatory disease may be cryopyrin-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 an antibody fragment derived from the monoclonal antibody is used to treat CAPS by administering the monoclonal antibody or an antibody fragment derived from the monoclonal antibody to a patient suffering from or suspected of suffering from CAPS. The monoclonal antibody or antibody fragment of this embodiment may be present in a composition (e.g., a pharmaceutical composition provided herein). In some cases, the monoclonal antibody or fragment thereof is used in combination with one or more other agents in the therapeutic methods provided herein. The other agents may be any of the agents provided herein (e.g., EV uptake inhibitors) and / or antibodies or antibody fragments against other inflammasome components (e.g., IL-18, caspase-1, NALP1, AIM2, etc.).

[0053]

[0074] Provided herein are compositions and methods for reducing innate immune inflammation or inflammasome-associated inflammation. In some instances, the inflammasome-associated inflammation is present in the CNS of a mammalian subject to or afflicted by a condition that results in or causes innate immune inflammation or inflammasome-associated inflammation. In some instances, the inflammasome-associated inflammation is present in the CNS of a mammalian subject that is exposed to or afflicted by a condition that results in or causes innate immune inflammation or inflammasome-associated inflammation. The present invention relates to a mammal (e.g., a human) suffering from or suspected to suffer from a condition associated with, resulting in, or causing immune inflammation or inflammasome-associated inflammation. The condition resulting in or causing congenital immune inflammation or inflammasome-associated inflammation may be a CNS injury, an autoimmune, autoinflammatory, neurodegenerative, and / or metabolic disease or disorder. The CNS injury may be selected from the group consisting of traumatic brain injury (TBI), stroke, and spinal cord injury (SCI). The autoimmune or neurodegenerative disease may 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., Crohn's disease and ulcerative colitis), and multiple sclerosis (MS). The metabolic disease may be selected from metabolic syndrome, obesity, diabetes, diabetic nephropathy or diabetic kidney disease (DKD), insulin resistance, atherosclerosis, lipid storage disorder, glycogen storage disorder, medium-chain acyl-coenzyme A dehydrogenase deficiency, nonalcoholic fatty liver disease (e.g., nonalcoholic steatohepatitis (NASH)), and gout. The autoinflammatory disease may be cryopyrin-associated periodic syndromes (CAPS). CAPS may include familial cold autoinflammatory syndrome (FCAS), Muckle-Wells syndrome (MWS), and neonatal-onset multisystem inflammatory disease (NOMID). The compositions and methods described herein may include an antibody or active fragment thereof provided herein that specifically binds to at least one component (e.g., ASC) of a mammalian inflammasome and / or a compound that modulates (e.g., inhibits or reduces) uptake of extracellular vesicles (EVs) and is used as a treatment for CNS inflammation in a mammal. Examples of conditions that may cause inflammation in the CNS include CNS trauma (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. The therapeutically effective amount may be a dose provided herein.The agent may be an extracellular vesicle (EV) uptake inhibitor and / or an antibody or an active fragment thereof provided herein that binds to a component of inflammasome, or a combination thereof. The composition may be administered by any suitable route, for example, by inhalation, intravenously, intraperitoneally, or intracerebroventricularly. The composition may further comprise at least one pharma- ceutical acceptable carrier or diluent.

[0054]

[0075] Provided herein are compositions and methods for treating multiple sclerosis (MS) in a subject suffering from or suspected of suffering from MS. The methods of treating MS provided herein can involve administering a composition (e.g., a pharmaceutical composition) containing an agent (e.g., IC100) to a subject suffering from or suspected of suffering from MS. Multiple sclerosis (MS) is an autoimmune disease that affects the brain and spinal cord. The subject can exhibit clinical symptoms consistent with MS. The subject can be diagnosed with any type of MS known in the art. The MS can be relapsing-remitting MS (RRMS), secondary progressive MS (SPMS), secondary progressive MS (PPMS), or progressive relapsing MS (PRMS). The MS diagnosis can be or has been determined using any method known in the art. In one embodiment, the subject has been diagnosed with MS using the methods detailed in US Patent Application No. 62 / 560,963, filed September 20, 2017, the contents of which are incorporated herein by reference in their entirety. The agent may be a standard of care treatment known in the art for MS, an EV uptake inhibitor (e.g., any EV uptake inhibitor from Table 1), an antibody or antibody fragment thereof provided herein that binds to a component of the inflammasome (e.g., an anti-ASC monoclonal antibody or antibody fragment thereof, e.g., IC100), or any combination thereof. The composition may be administered by any suitable route, for example, by inhalation, intravenously, intraperitoneally, or intracerebroventricularly. The composition may further comprise at least one pharma- ceutical acceptable carrier or diluent. Standard of care treatments include treatments for modifying disease outcomes, The treatment may be selected from a treatment for managing recurrence, a treatment for managing symptoms, or any combination thereof. The treatment for modifying disease outcome may be selected from beta-interferon, glatiramer acetate, fingolimod, teriflunomide, dimethyl fumarate, mitoxantrone, ocrelizumab, alemtuzumab, daclizumab, and natalizumab.

[0055]

[0076] Provided herein are compositions and methods for treating Parkinson's disease (PD) in a subject suffering from or suspected of suffering from PD. The methods of treating PD provided herein can involve administering a composition (e.g., a pharmaceutical composition) containing an agent (e.g., IC100) to a subject suffering from or suspected of suffering from PD. Parkinson's disease (PD) is a progressive nervous system disorder that affects movement due to the gradual destruction and / or death of nerve cells in the brain of a mammal (e.g., a human) suffering from PD. PD can develop and / or progress through five stages (i.e., stages 1-5), and the compositions and methods provided herein can be used to treat individuals suffering from or suspected of suffering from PD at any of the five stages. PD diagnosis can be determined or may have been determined using any method known in the art. In one embodiment, the subject has been diagnosed with PD using the methods detailed in International Publication WO 2019 / 060516, filed September 20, 2018, the contents of which are incorporated herein by reference in their entirety. The agent may be a standard of care treatment known in the art for PD, an EV uptake inhibitor (e.g., any EV uptake inhibitor from Table 1), an antibody or antibody fragment thereof provided herein that binds to a component of the inflammasome (e.g., an anti-ASC monoclonal antibody or antibody fragment thereof, e.g., IC100), or any combination thereof. The composition may be administered by any suitable route, for example, by inhalation, intravenously, intraperitoneally, or intracerebroventricularly. The composition may further comprise at least one pharma- ceutically acceptable carrier or diluent. Standard care treatments may be selected from carbidopa (Lodosyn), levodopa carbidopa-levodopa combinations, Duopa, dopamine agonists, MAO B inhibitors, catechol O-methyltransferase (COMT) inhibitors, anticholinergics, amantadine, and deep brain stimulation. Dopamine agonists may be selected from pramipexole (Mirapex), ropinirole (Requip), and rotigotine (Neupro). The MAO B inhibitors may be selected from selegiline (Eldepryl, Zelapar), rasagiline (Azilect), and safinamide (Xadago). The anticholinergic agent may be selected from takapone (Comtan) and tolcapone (Tasmar). The anticholinergic agent may be selected from benztropine (Cogentin) or trihexyphenidyl.

[0056]

[0077] Provided herein are compositions and methods for treating Alzheimer's disease (AD) in a subject suffering from AD or suspected of suffering from PD. The methods of treating AD provided herein can involve administering a composition (e.g., a pharmaceutical composition) containing an agent (e.g., IC100) to a subject suffering from AD or suspected of suffering from AD. Alzheimer's disease (AD) is a progressive nervous system disorder that causes brain cell degeneration and death in individuals suffering from AD, and progresses from mild cognitive impairment (MCI) to complete memory loss and even personality and behavior changes. An AD diagnosis can be or may have been determined using any method known in the art. In one embodiment, the subject has been diagnosed with AD using the methods detailed in International Publication WO 2019 / 060516, filed September 20, 2018, the contents of which are incorporated herein by reference in their entirety. The agent may be a standard of care treatment known in the art for AD, an EV uptake inhibitor (e.g., any EV uptake inhibitor from Table 1), an antibody or antibody fragment thereof provided herein that binds to a component of the inflammasome (e.g., an anti-ASC monoclonal antibody or antibody fragment thereof, e.g., IC100), or any combination thereof. The composition may be administered by any suitable route, for example, by inhalation, intravenously, intraperitoneally, or intracerebroventricularly. The composition may be administered. The composition may further comprise at least one pharma- ceutically acceptable carrier or diluent. Standard care treatments include cholinesterase inhibitors and memantine (Memantine). The cholinesterase inhibitors may be selected from donepezil (Aricept), galantana The agent may be selected from among ribastigmine (Razadyne), and rivastigmine (Exelon).

[0057]

[0078] Provided herein are compositions and methods for treating rheumatoid arthritis (RA) in a subject suffering from or suspected of suffering from RA. The methods of treating RA provided herein can involve administering a composition (e.g., a pharmaceutical composition) containing an agent (e.g., IC100) to a subject suffering from or suspected of suffering from RA. Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disorder that can cause damage to an individual's joints, as well as the skin, eyes, lungs, heart, and blood vessels. RA diagnosis can be or has been determined using any method known in the art. In one embodiment, the subject has been diagnosed with RA using the method detailed in International Publication WO 2019 / 060516, filed September 20, 2018, the contents of which are incorporated herein by reference in their entirety. The agent may be a standard of care treatment known in the art for RA, an EV uptake inhibitor (e.g., any EV uptake inhibitor from Table 1), an antibody or antibody fragment thereof provided herein that binds to a component of the inflammasome (e.g., an anti-ASC monoclonal antibody or antibody fragment thereof, e.g., IC100), or any combination thereof. The composition may be administered by any suitable route, for example, by inhalation, intravenously, intraperitoneally, or intracerebroventricularly. The composition may further comprise at least one pharma- ceutical acceptable carrier or diluent. The standard of care treatment may be selected from nonsteroidal anti-inflammatory drugs (NSAIDs), steroids (e.g., prednisone), disease-modifying antirheumatic drugs (DMARDs), and biologics. NSAIDs may include ibuprofen (Advil, Motrin IB), and naproxen sodium (Aleve). DMARDs may include methotrexate, ... Medications may include sartan (Trexall, Otrexup, others), leflunomide (Arava), hydroxychloroquine (Plaquenil), and sulfasalazine (Azulfidine). Biologic agents may include abatacept (Orencia), adalimumab (Humira), anakinra (Kineret), baricitinib (Olumiant), certolizumab (Cimzia), etanercept (Enbrel), golimumab (Simponi), infliximab (Remicade), rituximab (Rituxan), sarilumab (Kevzara), tocilizumab (Actemra), and tofacitinib (Xeljanz). .

[0058]

[0079] Provided herein are compositions and methods for treating lupus nephritis in subjects suffering from or suspected of suffering from lupus nephritis. The methods for treating lupus nephritis provided herein can involve administering a composition (e.g., pharmaceutical composition) containing an agent (e.g., IC100) to a subject suffering from or suspected of suffering from lupus nephritis. Lupus nephritis is a type of kidney inflammation that is often a common complication of systemic lupus erythematosus, often simply referred to as lupus. Lupus nephritis is an autoimmune disease in which lupus autoantibodies affect the structure of an individual's kidney, which can lead to kidney inflammation and hematuria, proteinuria, high blood pressure, kidney dysfunction, and even kidney failure. Lupus nephritis diagnosis can be or has been determined using any method known in the art. In one embodiment, the subject has been diagnosed with lupus nephritis using the methods detailed in International Publication WO 2019 / 060516, filed September 20, 2018, the contents of which are incorporated herein by reference in their entirety. The agent can be a standard of care treatment known in the art for lupus nephritis, an EV uptake inhibitor (e.g., any EV uptake inhibitor from Table 1), an antibody or antibody fragment thereof provided herein that binds to a component of the inflammasome (e.g., an anti-ASC monoclonal antibody or antibody fragment thereof, e.g., IC100), or any combination thereof. The composition is administered to any The composition may be administered by any suitable route, for example, by inhalation, intravenously, intraperitoneally, or intracerebroventricularly. The composition may further comprise at least one pharma- ceutically acceptable carrier or diluent. Standard care treatments for lupus nephritis may include medications to control blood pressure, and / or special diets that are low in protein and 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 include naproxen sodium (Aleve), and ibuprofen (Advil, Motrin IB, etc.). An example of an antimalarial drug may be hydroxychloroquine (Plaquenil). Examples of inhibitors may include azathioprine (Imuran, Azasan), mycophenolate mofetil (CellCept), and methotrexate (Trexall). Belimumab (Benlysta), or Rituximab (Rituxan) may be mentioned.

[0059]

[0080] Provided herein are compositions and methods for treating non-alcoholic steatohepatitis (NASH) in subjects suffering from or suspected of suffering from NASH. Provided herein are methods for treating NASH that can involve administering a composition (e.g., pharmaceutical composition) containing an agent (e.g., IC100) to a subject suffering from or suspected of suffering from NASH. NASH is a type of non-alcoholic fatty liver disease (NAFLD). NAFLD is an umbrella term for a variety of liver conditions that affect people who do not drink alcohol or drink very little alcohol. The main feature of NAFLD is an excessive amount of fat stored in liver cells and is characterized by inflammation of the liver, which can progress to scarring and irreversible damage. This damage can be similar to the damage caused by heavy alcohol consumption. At its most severe, non-alcoholic steatohepatitis can progress to cirrhosis and liver failure. NASH diagnosis can be or has been determined using any method known in the art. In one embodiment, the subject has been diagnosed with NASH using the methods detailed in International Publication WO 2019 / 060516, filed September 20, 2018, the contents of which are incorporated herein by reference in their entirety. The agent may be a standard of care treatment known in the art for NASH, an EV uptake inhibitor (e.g., any EV uptake inhibitor from Table 1), an antibody or antibody fragment thereof provided herein that binds to a component of the inflammasome (e.g., an anti-ASC monoclonal antibody or antibody fragment thereof, e.g., IC100), or any combination thereof. The composition may be administered by any suitable route, for example, by inhalation, intravenously, intraperitoneally, or intracerebroventricularly. The composition may further comprise at least one pharma- ceutically acceptable carrier or diluent. Standard of care treatment for NASH may include lifestyle changes, such as weight loss, increased exercise, avoidance of liver-damaging drugs, cholesterol reduction, and / or diabetes management.

[0060]

[0081] Provided herein are compositions and methods for treating diabetic nephropathy in a subject suffering from or suspected of suffering from diabetic nephropathy. The methods of treating diabetic nephropathy provided herein may involve administering a composition (e.g., a pharmaceutical composition) comprising an agent (e.g., IC100) to a subject suffering from or suspected of suffering from diabetic nephropathy. Diabetic nephropathy is a severe kidney-related complication of type 1 and type 2 diabetes, which may also be referred to as diabetic nephropathy (DKD). DKD diagnosis may be or may have been determined using any method known in the art. In one embodiment, the subject has been diagnosed with DKD using the methods detailed in International Publication WO 2019 / 060516, filed September 20, 2018, the contents of which are incorporated herein by reference in their entirety. The agents include standard of care treatments known in the art for DKD, EV uptake inhibitors (e.g., any EV uptake inhibitor from Table 1), and agents that bind to components of the inflammasome. The composition may be an antibody or antibody fragment thereof provided herein (e.g., an anti-ASC monoclonal antibody or antibody fragment thereof, e.g., IC100), or any combination thereof. The composition may be administered by any suitable route, for example, by inhalation, intravenously, intraperitoneally, or intracerebroventricularly. The composition may further comprise at least one pharma- ceutical 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, promoting bone health, controlling high blood pressure, managing diabetes, kidney dialysis, or transplantation.

[0061]

[0082] Provided herein are compositions and methods for treating inflammatory bowel disease (IBD) in subjects suffering from or suspected of suffering from IBD. The methods of treating IBD provided herein can involve administering a composition (e.g., pharmaceutical composition) containing an agent (e.g., IC100) to a subject suffering from or suspected of suffering from IBD. IBD is a comprehensive term used to describe disorders involving chronic inflammation of an individual's digestive tract. IBD can include ulcerative colitis and Crohn's disease. Ulcerative colitis is a long-term inflammation and sores (ulcers) of the innermost layer of the large intestine (colon) and rectum, while Crohn's disease is characterized by inflammation of the digestive tract lining, which often extends deep into the affected tissue. IBD diagnosis can be determined or may have been determined using any method known in the art. In one embodiment, the subject has been diagnosed with IBD using the methods detailed in International Publication WO 2019 / 060516, filed September 20, 2018, the contents of which are incorporated herein by reference in their entirety. The agent may be a standard of care treatment known in the art for IBD, an EV uptake inhibitor (e.g., any EV uptake inhibitor from Table 1), an antibody or antibody fragment thereof provided herein that binds to a component of the inflammasome (e.g., an anti-ASC monoclonal antibody or antibody fragment thereof, e.g., IC100), or any combination thereof. The composition may be administered by any suitable route, for example, by inhalation, intravenously, intraperitoneally, or intracerebroventricularly. The composition may further comprise at least one pharma- ceutically acceptable carrier or diluent. Standard of care treatment for IBD may include anti-inflammatory agents, immune system suppressants, antibiotics, antidiarrheals, painkillers, iron supplements, and calcium and vitamin D supplements. Antibiotics may include ciprofloxacin (Cipro) and metronidazole (Flagyl). Examples of suppressants may include azathioprine (Azasan, Imuran), mercaptopurine (Purinethol, Purixan), cyclosporine (Gengraf, Neoral, Sandimmune), and methotrexate (Trexall). Other examples of immunosuppressants include tumor necrosis factor (TNF)-associated leukemia inhibitors, or biologics (e.g., infliximab (Remicade), adalimumab (Humira), golimumab (Simponi), natalizumab (Tysabri), vedolizumab (Entyvio) Anti-inflammatory agents may include corticosteroids. steroids and aminosalicylates (e.g., mesalamine (Asacol HD, Delzicol), valproate, azide (Colazal), and olsalazine (Dipentum).

[0062]

[0083] Provided herein are compositions and methods for treating cryopyrin-associated periodic syndromes (CAPS) in a subject suffering from or suspected of suffering from CAPS. The methods for treating CAPS provided herein can involve administering a composition (e.g., a pharmaceutical composition) containing an agent (e.g., IC100) to a subject suffering from or suspected of suffering from CAPS. Cryopyrin-associated periodic syndromes (CAPS), also referred to as cryopyrin-associated autoinflammatory syndromes, consist of three autoinflammatory diseases associated with deletions in the same gene (i.e., NLRP3): neonatal-onset multisystem inflammatory disease (NOMID), Muckle-Wells syndrome (MWS), and familial common cold autoinflammatory syndrome (FCAS). NOMID is characterized by fever accompanied by inflammation in multiple organs. Initial symptoms of NOMID include a non-itchy honeycomb rash; Symptoms may include inflammation of the membrane surrounding the brain, causing headaches, blindness, or hearing loss; swollen appearance of the eyes; and episodes of vomiting. After age 1, half of children with NOMID may develop joint pain and swelling. MWS is characterized by symptoms that come and go (e.g., severe headaches accompanied by skin rashes, red eyes, joint pain, and vomiting). Episodes last 1-3 days. Hearing loss, which may be complete, often develops by the teenage years. FCAS is characterized by fever, chills, nausea, extreme thirst, headache, and joint pain. CAPS diagnosis may be or has been determined using any method known in the art. In one embodiment, the subject has been diagnosed with CAPS using the methods detailed in International Publication WO 2019 / 060516, filed September 20, 2018, the contents of which are incorporated herein by reference in their entirety. The agent may be a standard of care treatment known in the art for CAPS, an EV uptake inhibitor (e.g., any EV uptake inhibitor from Table 1), an antibody or antibody fragment thereof provided herein that binds to a component of the inflammasome (e.g., an anti-ASC monoclonal antibody or antibody fragment thereof, e.g., IC100), or any combination thereof. The composition may be administered by any suitable route, for example, by inhalation, intravenously, intraperitoneally, or intracerebroventricularly. The composition may further comprise at least one pharma- ceutical acceptable carrier or diluent. Standard of care treatment for CAPS may include biologic agents targeting interleukin-1, as well as physical therapy, splints to treat joint deformities, and nonsteroidal anti-inflammatory drugs, corticosteroids, or methotrexate to relieve symptoms.

[0063]

[0084] Provided herein are compositions and methods for reducing inflammation in the lungs of a mammal exposed to or afflicted by a condition that results in or causes pulmonary inflammation. The compositions and methods described herein may include an antibody or active fragment thereof (e.g., IC100) provided herein that specifically binds to at least one component (e.g., ASC) of a mammalian inflammasome and / or a compound that modulates (e.g., inhibits or reduces) the uptake of extracellular vesicles (EVs) and is used as a treatment for pulmonary inflammation in a mammal.

[0064]

[0085] Described herein is a method of reducing inflammation in the lungs of a mammal having a condition that results in and / or causes an inflammatory response in the lungs. In one embodiment, a method of treating inflammation in the lungs of a mammal comprises administering to the mammal a composition comprising an agent that inhibits inflammasome signaling (e.g., IC 100). The mammal may be a patient or subject as provided herein. Examples of conditions that may result in inflammation in the lungs include central nervous system (CNS) injury (e.g., spinal cord injury (SCI), traumatic brain injury (TBI) or stroke), neurodegenerative disease, autoimmune disease (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. The therapeutically effective amount may be a dose as provided herein. The agent may be an extracellular vesicle (EV) uptake inhibitor, an antibody or active fragment thereof as provided herein that binds to a component of the inflammasome, or a combination thereof. The composition may be administered by any suitable route, for example, by inhalation, intravenously, intraperitoneally, or intracerebroventricularly. The composition may further comprise at least one pharma- ceutically acceptable carrier or diluent.

[0065]

[0086] Provided herein are compositions and methods for reducing renal inflammation in a mammal exposed to or afflicted with a condition that results in or causes renal inflammation. The compositions and methods described herein include ... an antibody or active fragment thereof (e.g., an antibody or an active fragment thereof) provided herein that specifically binds to at least one component (e.g., ASC) of a mammalian inflammasome and / or a compound used as a treatment for renal inflammation in a mammal. IC100).

[0066]

[0087] Described herein is a method of reducing renal inflammation in a mammal having a condition that results in and / or causes an immune response in the kidney. In one embodiment, a method of treating renal inflammation in a mammal includes administering to the mammal a composition comprising an agent (e.g., IC100) that inhibits inflammasome signaling. The mammal may be a patient or subject as provided herein. An example of a condition that may cause inflammation in the kidney includes lupus nephritis. The composition may be administered in a therapeutically effective amount. The therapeutically effective amount may be a dose as provided herein. The agent may be an extracellular vesicle (EV) uptake inhibitor, an antibody or active fragment thereof provided herein that binds to a component of the inflammasome, or a combination thereof. The composition may be administered by any suitable route, for example, by inhalation, intravenously, intraperitoneally, or intracerebroventricularly. The composition may further comprise at least one pharma-ceutically acceptable carrier or diluent.

[0067]

[0088] In one embodiment, administration of an agent in the methods provided herein (e.g., an antibody or an antibody fragment derived from the antibody (e.g., IC100), alone or in combination with, e.g., an EV uptake inhibitor) can result in a decrease in activity and / or expression levels of a mammalian inflammasome component in the CNS, kidney, or lung of a subject. The decrease can be in cells of the lung, such as, e.g., type II pneumocytes. The decrease can be compared to a control. The control can be a subject prior to administration of the agent. The control can be the activity and / or expression levels of an inflammasome component in a subject to which the agent is not administered. In one embodiment, administration of the agent results in a decrease in caspase-1 activation in at least the CNS or CNS cells of the subject. In one embodiment, administration of the agent results in a decrease in caspase-1 activation in at least the lung or lung cells of the 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 the 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.

[0068]

[0089] In another embodiment, administration of an agent (e.g., an antibody or an antibody fragment derived from the antibody, alone or in combination with, e.g., an EV uptake inhibitor) may result in a reduction or elimination of acute lung injury (ALI). In one embodiment, a reduction in ALI is evidenced by a reduction in neutrophil infiltration into the alveolar and / or interstitial spaces, a reduction or absence of alveolar septal thickening, or a combination thereof. The reduction may be compared to a control. The control may be an ALI in a subject prior to administration of the agent. The control may be an ALI in a subject suffering from ALI to which the agent is not administered.

[0069]

[0090] In yet another embodiment, administration of an agent (e.g., an antibody or an antibody fragment derived from this antibody (e.g., IC100), alone or in combination with, e.g., an EV uptake inhibitor) may result in the reduction or elimination of pyroptosis in the CNS or lungs of a subject. Pyroptosis is a pro-inflammatory form of cell death that involves activation of caspase-1. Pyroptosis may be caused by caspase-1 mediated cleavage of gasdermin D (GSDMD). In one embodiment, the reduction in pyroptosis is evidenced by a reduction or lack of cleavage of GSDMD in the lungs or lung cells (e.g., type II alveolar cells) of the subject. The reduction or elimination of pyroptosis may be in comparison to a control. The reduction or lack of cleavage of GSDMD may be in comparison to a control. The control may be the level of pyroptosis in the subject prior to administration of the agent. The control can be the level of pyroptosis in a subject suffering from pyroptosis to which the agent has not been administered.

[0070]

[0091] The success or response to the therapeutic 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-associated inflammation, CNS inflammation, and / or pulmonary inflammation) may be monitored by measuring the level of at least one inflammasome protein. Thus, in some embodiments, the therapeutic methods provided herein further include measuring the level of at least one inflammasome protein in a biological sample obtained from the subject after treatment, creating a therapeutic inflammasome protein signature associated with a positive response to the treatment, the therapeutic protein signature comprising a decreased level of at least one inflammasome protein, and identifying subjects exhibiting the presence of the therapeutic protein signature as a positive response to the treatment. A decrease in the level, abundance, or concentration of one or more inflammasome proteins (e.g., ASC, IL-18, or caspase-1) indicates the effectiveness of the treatment in the subject. The one or more inflammasome proteins measured in a sample obtained after treatment may be the same as or different from the inflammasome proteins measured in a sample obtained before treatment. The level of the inflammasome protein may be used to adjust the dosage or frequency of treatment. The level of the inflammasome protein may be confirmed using methods and techniques such as those described herein or found in U.S. Patent Application No. 62 / 560,963, filed September 20, 2017.

[0071]

[0092] In one embodiment, the agent administered in the therapeutic methods provided herein is an EV uptake inhibitor. The EV uptake inhibitor can be a compound, an antisense RNA, an siRNA, a peptide, an antibody or an active fragment thereof provided herein, or a combination thereof. The compound or peptide can be one or more compounds selected from 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, dynasoar, 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, antibody or active fragment thereof provided herein can 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 can further comprise at least one pharma- ceutically acceptable carrier or diluent.

[0072]

[0093] [Table 1]

[0073] [Table 2]

[0074] [Table 3]

[0075] [Table 4]

[0076] [Table 5]

[0077]

[0094] In one embodiment, the agent to be administered is an antibody or an active fragment thereof provided herein directed against a component of a mammalian inflammasome or an antigen or epitope derived therefrom. In another embodiment, the agent to be administered is an antisense RNA or siRNA directed against a component of a mammalian inflammasome. The inflammasome component can be any component of an inflammasome known in the art, such as 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 against any other component of a mammalian inflammasome (e.g., NALP1, NALP2, NALP3, NLRC4 or AIM2 inflammasome) can be used.

[0078]

[0095] The antibodies 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.

[0079]

[0096] Any suitable antibody or active fragment thereof provided herein that specifically binds to ASC, such as an antibody that specifically binds to the CNS (e.g., CNS cells) or lung cells (e.g., type II alveolar cells) of a subject. An antibody that inhibits ASC activity in a mammalian cell (such as a mammalian cell) can be used. In one embodiment, the antibody specifically binds to an amino acid sequence having at least 85% sequence identity with the amino acid sequence SEQ ID NO: 1 or SEQ ID NO: 2. In another embodiment, the antibody or a fragment thereof binds to an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence KKFKLKLLSVPLREGYGRIPR (SEQ ID NO: 5). In yet another embodiment, the antibody or fragment thereof binds to the amino acid sequence KKFKLKLLSVPLREGYGRIPR (SEQ ID NO: 5), or binds to 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 further embodiments, the antibody or fragment thereof binds to 2-5, 5-10, 10-15, or 15-20 amino acids of SEQ ID NO: 5. In some embodiments, the epitope of ASC to which the antibody or antibody fragment binds (e.g., the epitope having the amino acid sequence SEQ ID NO: 5) is contiguous. In some embodiments, the epitope of ASC to which the antibody or antibody fragment binds (e.g., the epitope having the amino acid sequence SEQ ID NO: 5) is discontinuous. In some examples, the monoclonal antibodies or antibody fragments provided herein inhibit or reduce the activity of ASC.

[0080]

[0097] As used herein, the term "epitope" includes any protein determinant capable of specific binding to an immunoglobulin or immunoglobulin fragment. Epitope determinants usually consist of chemically active surface groupings 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 unit of structure conventionally bound by a pair of variable heavy (VH) and variable light (VL) chains of an immunoglobulin. An epitope may define the minimal binding site of an antibody and thus represent the target of the specificity of the antibody.

[0081]

[0098] Similarly, in another embodiment, the inflammasome is the NALP1 inflammasome, and at least one component is NALP1 (i.e., NLRP1). In this embodiment, the antibody or active fragment thereof provided herein specifically binds to an amino acid sequence having at least 85% sequence identity with the amino acid sequence SEQ ID NO:3 or SEQ ID NO:4.

[0082]

[0099] In yet another embodiment, the agent is one or more EV uptake inhibitors in combination with one or more antibodies or active fragments thereof provided herein that bind to an inflammasome component. The EV uptake inhibitor can be any EV uptake inhibitor provided herein. The antibody that binds to an inflammasome component can be any antibody that binds to any inflammasome component provided herein. In one embodiment, the agent administered to a subject suffering from CNS or pulmonary inflammation comprises heparin (e.g., enoxaparin) in combination with an antibody that binds to an AIM2 inflammasome component (e.g., ASC).

[0083]

[0100] In one embodiment, the method comprises detecting a mammalian inflammasome (e.g., an AI and administering the composition to a mammal suffering from CNS or pulmonary inflammation or MS, wherein administering the composition to the mammal results in reduced 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); ... 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 pulmonary inflammation or MS, where administration of the composition to the mammal results in a reduction in ALI. The CNS or pulmonary inflammation can be the result of CNS injury (e.g., SCI or TBI), asthma, chronic obstructive pulmonary disorder (COPD), neurodegenerative disease, or autoimmune disease due to inflammatory components. In one embodiment, the pulmonary inflammation is caused by CNS injury, e.g., TBI or SCI.

[0084]

[0101] In one embodiment, the methods provided herein involve treating CNS or pulmonary inflammation or The method further includes detecting the level or activity of one or more components of a mammalian inflammasome in a sample from a subject suspected of having MS. The method of detecting the level or activity includes measuring the level of at least one inflammasome protein (e.g., ASC or AIM2) in a sample obtained from the subject; determining the presence or absence of an increase in the 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 can be elevated 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 the protein signature can be elevated relative to a predetermined reference value or range of reference values. The at least one inflammasome protein can be nucleotide-binding leucine-rich repeat pyrin domain-containing protein 1 (NLRP1), NLRP2, NLRP3, NLRC4, AIM2, caspase recruitment domain-containing apoptosis-associated speck-like protein (ASC), caspase-1, or a combination thereof. The sample may be cerebrospinal fluid (CSF), saliva, blood, serum, plasma, urine or lung aspirate.

[0085] Antibodies that specifically bind to at least one component of a mammalian inflammasome

[0102] The method of claim 1, wherein the method comprises administering to the mammal a method for treating inflammation in the CNS and / or lungs of the mammal. The method includes a composition comprising an antibody or an active fragment thereof 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 lungs of a mammal may further comprise at least one pharma- ceutically acceptable carrier or diluent. Exemplary antibodies directed against a component of a mammalian inflammasome for use in the method herein may be those found in U.S. Pat. No. 8,685,400, the contents of which are incorporated herein by reference in their entirety. Exemplary monoclonal antibodies or antibody fragments (e.g., a monoclonal antibody or antibody fragment comprising a VH region, the amino acid sequence of which comprises HCDR1 of SEQ ID NO:6, HCDR2 of SEQ ID NO:7, and HCDR3 of SEQ ID NO:8, and a VL region, the amino acid sequence of which comprises LCDR1 of SEQ ID NO:12, LCDR2 of SEQ ID NO:13, and LCDR3 of SEQ ID NO:14) are also provided herein.

[0086]

[0103] In one embodiment, treating inflammation in the CNS or lungs of a mammal, and The compositions for reducing / reducing include the antibodies or active fragments thereof provided herein that specifically bind to a domain or portion thereof of a mammalian ASC protein, such as human, mouse or rat ASC protein. Any suitable anti-ASC antibody can be used, and some are commercially available. Examples of anti-ASC antibodies for use in the methods provided herein can be those found in U.S. Patent No. 8,685,400, 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, MilliporeSig. Clone 2EI-7 mouse monoclonal antibody from ma, AB3 from Millipore Sigma 607-anti-ASC antibody, orb194021 anti-ASC from Bioorbyt, 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 Santa Cruz Biotechnology, ASC polyclonal antibody-ADI-905-173 from Enzo Life Sciences, or A161 anti-human ASC-Leinco Technologies. The human ASC protein may have accession number NP_037390.2 (Q9ULZ3-1), NP_660183 (Q9ULZ3-2) or Q9ULZ3-3. The rat ASC protein may have accession number NP_758825 (BAC43754). The mouse ASC protein may have 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 described herein specifically binds to an amino acid sequence having at least 65% (e.g., 65, 70, 75, 80, 85%) sequence identity to the PYD domain of human, mouse or rat ASC or a fragment thereof. In one embodiment, the antibody binds to the C-terminal caspase recruitment domain (CARD) of a mammalian ASC protein (e.g., human, mouse, or rat ASC), or a portion or fragment thereof. In this embodiment, the antibody described herein specifically binds to an amino acid sequence having at least 65% (e.g., 65, 70, 75, 80, 85%) sequence identity to the CARD domain of human, mouse, or rat ASC, or a fragment thereof. In yet another embodiment, the antibody binds to a portion or fragment thereof of a mammalian ASC protein sequence (e.g., human, mouse, or rat ASC) located between the PYD and CARD domains. 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., the amino acid sequence ALRQTQPYLVTDLEQS (SEQ ID NO: 1) (i.e., residues 178-193 of rat ASC, Accession No. BAC43754). In this embodiment, the antibodies described herein specifically bind to an amino acid sequence having at least 65% (eg, 65, 70, 75, 80, 85%) sequence identity to the amino acid sequence ALRQTQPYLVTDLEQS (SEQ ID NO:1) of 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 human ASC, e.g., the amino acid sequence RESQSYLVEDLERS (SEQ ID NO: 2). In yet 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 human ASC (e.g., the amino acid sequence KKFKLKLLSVPLREGYGRIPR (SEQ ID NO: 5; i.e., residues 21-41 of human ASC), or 5-10, 10-15, or 15-20 amino acids of SEQ ID NO: 5). In one embodiment, an antibody that binds to an ASC domain or fragment thereof as described herein inhibits ASC activity in lung cells (e.g., mammalian type II alveolar cells). In another embodiment, an antibody that binds to an ASC domain or fragment thereof as described herein (e.g., a monoclonal anti-ASC antibody or antibody fragment thereof provided herein) inhibits ASC activity in the CNS of a mammal suffering from or suspected of suffering from a CNS injury or disorder. Examples of CNS injury or disorder may include TBI, SCI, stroke, amyotrophic lateral sclerosis (ALS) Lou Gehrig's, multiple sclerosis (MS), immune dysfunction muscle CNS degeneration, muscular dystrophy (MD), Alzheimer's disease (AD), Parkinson's disease (PD).

[0087]

[0104] In certain embodiments, the present invention provides a method for the preparation of a polypeptide that specifically binds to ASC and is shown in Table 2. Also provided herein are isolated nucleic acid molecules encoding monoclonal antibodies or antibody fragments thereof comprising one or more amino acid sequences as set forth in Table 2. In some examples, an expression vector comprising a nucleic acid molecule of Table 2. The expression vector may comprise a heavy chain constant region or a light chain constant region. An example of a light and heavy chain expression vector system for use in the compositions and methods provided herein is the Antitope pANT expression vector system for IgG4(S241P) heavy chain and kappa light chain. The heavy or light chain nucleic acid molecule may be operably linked to a regulatory sequence suitable for expression of the nucleic acid segment in a host cell.

[0088]

[0105] [Table 6]

[0089] [Table 7]

[0090] [Table 8]

[0091] [Table 9]

[0092] [Table 10]

[0093]

[0106] In one embodiment, provided herein is a molecule that specifically binds to ASC. A monoclonal antibody or an antibody fragment thereof, which antibody or an antibody fragment thereof comprises a heavy chain variable ( A monoclonal antibody or antibody fragment thereof, comprising a variable light or kappa chain (VH) region and a variable light or kappa chain (VL) region, wherein the amino acid sequence of the VH region 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 addition to this embodiment, provided herein is the use of the monoclonal antibody or antibody fragment thereof in a method of treating inflammation in a subject. The inflammation can be innate immune inflammation. The inflammation can be inflammasome-associated inflammation. The inflammation can be present in the lungs and / or CNS. The inflammation in the lungs and / or CNS can be the result of injury (e.g., traumatic brain injury (TBI) or spinal cord injury (SCI)) or can be the result of a disease, condition, or affliction in or affecting the CNS. As provided herein, the disease, condition, or affliction of or affecting the CNS may be stroke, as well as autoimmune and / or CNS diseases (e.g., amyotrophic lateral sclerosis (ALS) Lou Gehrig's, multiple sclerosis (MS), immune dysfunction muscle CNS degeneration, muscular dystrophy (MD), Alzheimer's disease (AD), Parkinson's disease (PD)). Use of the monoclonal antibody or antibody fragment thereof in a method of treating inflammation may reduce inflammation in the CNS and / or lungs of a patient. The reduction may be compared to a control (e.g., an untreated patient and / or a patient prior to treatment). In one embodiment, the monoclonal antibody or antibody fragment derived from the monoclonal antibody is used to treat MS by administering the monoclonal antibody or antibody fragment derived from the monoclonal antibody to a patient suffering from or suspected of suffering from MS. In some examples, the monoclonal antibody or antibody fragment of the embodiment is present in a composition. The composition may be a pharmaceutical composition provided herein.

[0094]

[0107] In one embodiment, provided herein is a molecule that specifically binds to ASC. A monoclonal antibody or antibody fragment thereof, the antibody or antibody fragment thereof comprising a heavy chain variable (VH) region and a light or kappa chain variable (VL) region, the amino acid sequence of the VL region comprising SEQ ID NO: 28, 29, 30, 31, or comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 28, 29, 30, or 31. In addition 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 a congenital immune inflammation. The inflammation may be an inflammasome-associated inflammation. The inflammation may be present in the lungs and / or CNS. The 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 may be the result of a disease, condition, or affliction in or affecting the CNS. As provided herein, the disease, condition, or affliction of or affecting the CNS may be stroke, as well as autoimmune and / or CNS diseases (e.g., amyotrophic lateral sclerosis (ALS) Lou Gehrig's, multiple sclerosis (MS), immune dysfunction muscle CNS degeneration, muscular dystrophy (MD), Alzheimer's disease (AD), Parkinson's disease (PD)). Use of the monoclonal antibody or antibody fragment thereof in a method of treating inflammation may reduce inflammation in the CNS and / or lungs of a patient. The reduction may be compared to a control (e.g., an untreated patient and / or a patient prior to treatment). In one embodiment, the monoclonal antibody or antibody fragment derived from the monoclonal antibody is used to treat MS by administering the monoclonal antibody or antibody fragment derived from the monoclonal antibody to a patient suffering from or suspected of suffering from MS. In some examples, the monoclonal antibody or antibody fragment of the embodiment is present in a composition. The composition may be a pharmaceutical composition provided herein.

[0095]

[0108] In one embodiment, provided herein is a molecule that specifically binds to ASC. A monoclonal antibody or an antibody fragment thereof, which antibody or an antibody fragment thereof comprises a heavy chain variable ( A monoclonal antibody or antibody fragment thereof comprising a light or kappa chain variable (VH) region and a light or kappa chain variable (VL) region, wherein the amino acid sequence of the VH region comprises SEQ ID NO: 18, 19, 20, 21, 22 or comprises 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, and the amino acid sequence of the VL region comprises SEQ ID NO: 28, 29, 30, 31 or comprises 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 addition to this embodiment, provided herein is the use of the monoclonal antibody or antibody fragment thereof in a method of treating inflammation in a subject. The inflammation can be innate immune inflammation. The inflammation can be inflammasome-associated inflammation. The inflammation can be present in the lung and / or CNS. Inflammation in the lungs and / or CNS can be the result of injury (e.g., traumatic brain injury (TBI) or spinal cord injury (SCI)) or can be the result of a disease, condition, or affliction of or affecting the CNS. As provided herein, the disease, condition, or affliction of or affecting the CNS can be stroke, as well as autoimmune and / or CNS diseases (e.g., amyotrophic lateral sclerosis (ALS) Lou Gehrig's, multiple sclerosis (MS), immune dysfunction muscle CNS degeneration, muscular dystrophy (MD), Alzheimer's disease (AD), Parkinson's disease (PD)). Use of this monoclonal antibody or antibody fragment thereof in a method of treating inflammation can reduce inflammation in the CNS and / or lungs of a patient. This reduction can be in comparison to a control (e.g., an untreated patient and / or a patient prior to treatment). In one embodiment, the monoclonal antibody or an antibody fragment derived from the monoclonal antibody is used to treat MS by administering the monoclonal antibody or an antibody fragment derived from the monoclonal antibody to a patient suffering from or suspected of suffering from MS. In some examples, the monoclonal antibody or antibody fragment thereof of this embodiment is present in a composition. The composition may be a pharmaceutical composition as provided herein.

[0096]

[0109] In one embodiment, provided herein is a molecule that specifically binds to ASC. A monoclonal antibody or antibody fragment thereof, the antibody or antibody fragment thereof comprising a heavy chain variable (VH) region and a light or kappa chain variable (VL) region, the amino acid sequence of the VH region comprising SEQ ID NO: 18 or comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 18, and the amino acid sequence of the VL region comprising SEQ ID NO: 28 or comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 28. In addition 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 can be innate immune inflammation. The inflammation can be inflammasome-associated inflammation. The inflammation can be present in the lung and / or CNS. Inflammation in the lungs and / or CNS can be the result of injury (e.g., traumatic brain injury (TBI) or spinal cord injury (SCI)) or can be the result of a disease, condition, or affliction of or affecting the CNS. As provided herein, the disease, condition, or affliction of or affecting the CNS can be stroke, as well as autoimmune and / or CNS diseases (e.g., amyotrophic lateral sclerosis (ALS) Lou Gehrig's, multiple sclerosis (MS), immune dysfunction muscle CNS degeneration, muscular dystrophy (MD), Alzheimer's disease (AD), Parkinson's disease (PD)). Use of this monoclonal antibody or antibody fragment thereof in a method of treating inflammation can reduce inflammation in the CNS and / or lungs of a patient. This reduction can be in comparison to a control (e.g., an untreated patient and / or a patient prior to treatment). In one embodiment, the monoclonal antibody or an antibody fragment derived from the monoclonal antibody is used to treat MS by administering the monoclonal antibody or an antibody fragment derived from the monoclonal antibody to a patient suffering from or suspected of suffering from MS. In some examples, the monoclonal antibody or antibody fragment thereof of this embodiment is present in a composition. The composition can be a pharmaceutical composition provided herein.

[0097]

[0110] In one embodiment, provided herein is a molecule that specifically binds to ASC. A monoclonal antibody or antibody fragment thereof, the antibody or antibody fragment thereof comprising a heavy chain variable (VH) region and a light or kappa chain variable (VL) region, the amino acid sequence of the VH region comprising SEQ ID NO: 18 or comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 18, and the amino acid sequence of the VL region comprising SEQ ID NO: 29 or comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 29. In addition 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 can be innate immune inflammation. The inflammation can be inflammasome-associated inflammation. The inflammation can be present in the lung and / or CNS. Inflammation in the lungs and / or CNS can be the result of injury (e.g., traumatic brain injury (TBI) or spinal cord injury (SCI)) or can be the result of a disease, condition, or affliction of or affecting the CNS. As provided herein, the disease, condition, or affliction of or affecting the CNS can be stroke, as well as autoimmune and / or CNS diseases (e.g., amyotrophic lateral sclerosis (ALS) Lou Gehrig's, multiple sclerosis (MS), immune dysfunction muscle CNS degeneration, muscular dystrophy (MD), Alzheimer's disease (AD), Parkinson's disease (PD)). Use of this monoclonal antibody or antibody fragment thereof in a method of treating inflammation can reduce inflammation in the CNS and / or lungs of a patient. This reduction can be in comparison to a control (e.g., an untreated patient and / or a patient prior to treatment). In one embodiment, the monoclonal antibody or an antibody fragment derived from the monoclonal antibody is used to treat MS by administering the monoclonal antibody or an antibody fragment derived from the monoclonal antibody to a patient suffering from or suspected of suffering from MS. In some examples, the monoclonal antibody or antibody fragment thereof of this embodiment is present in a composition. The composition may be a pharmaceutical composition as provided herein.

[0098]

[0111] In one embodiment, provided herein is a molecule that specifically binds to ASC. A monoclonal antibody or antibody fragment thereof, the antibody or antibody fragment thereof comprising a heavy chain variable (VH) region and a light or kappa chain variable (VL) region, the amino acid sequence of the VH region comprising SEQ ID NO: 18 or comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 18, and the amino acid sequence of the VL region comprising SEQ ID NO: 30 or comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 30. In addition 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 can be innate immune inflammation. The inflammation can be inflammasome-associated inflammation. The inflammation can be present in the lung and / or CNS. Inflammation in the lungs and / or CNS can be the result of injury (e.g., traumatic brain injury (TBI) or spinal cord injury (SCI)) or can be the result of a disease, condition, or affliction of or affecting the CNS. As provided herein, the disease, condition, or affliction of or affecting the CNS can be stroke, as well as autoimmune and / or CNS diseases (e.g., amyotrophic lateral sclerosis (ALS) Lou Gehrig's, multiple sclerosis (MS), immune dysfunction muscle CNS degeneration, muscular dystrophy (MD), Alzheimer's disease (AD), Parkinson's disease (PD)). Use of this monoclonal antibody or antibody fragment thereof in a method of treating inflammation can reduce inflammation in the CNS and / or lungs of a patient. This reduction can be in comparison to a control (e.g., an untreated patient and / or a patient prior to treatment). In one embodiment, the monoclonal antibody or an antibody fragment derived from the monoclonal antibody is used to administer the monoclonal antibody to a patient suffering from or suspected of suffering from MS. The antibody or antibody fragment derived from this monoclonal antibody is administered to treat MS. In some examples, the monoclonal antibody or antibody fragment thereof of this embodiment is present in a composition. The composition may be a pharmaceutical composition as provided herein.

[0099]

[0112] In one embodiment, provided herein is a molecule that specifically binds to ASC. A monoclonal antibody or antibody fragment thereof, the antibody or antibody fragment thereof comprising a heavy chain variable (VH) region and a light or kappa chain variable (VL) region, the amino acid sequence of the VH region comprising SEQ ID NO: 18 or comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 18, and the amino acid sequence of the VL region comprising SEQ ID NO: 31 or comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 31. In addition 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 can be innate immune inflammation. The inflammation can be inflammasome-associated inflammation. The inflammation can be present in the lung and / or CNS. Inflammation in the lungs and / or CNS can be the result of injury (e.g., traumatic brain injury (TBI) or spinal cord injury (SCI)) or can be the result of a disease, condition, or affliction of or affecting the CNS. As provided herein, the disease, condition, or affliction of or affecting the CNS can be stroke, as well as autoimmune and / or CNS diseases (e.g., amyotrophic lateral sclerosis (ALS) Lou Gehrig's, multiple sclerosis (MS), immune dysfunction muscle CNS degeneration, muscular dystrophy (MD), Alzheimer's disease (AD), Parkinson's disease (PD)). Use of this monoclonal antibody or antibody fragment thereof in a method of treating inflammation can reduce inflammation in the CNS and / or lungs of a patient. This reduction can be in comparison to a control (e.g., an untreated patient and / or a patient prior to treatment). In one embodiment, the monoclonal antibody or an antibody fragment derived from the monoclonal antibody is used to treat MS by administering the monoclonal antibody or an antibody fragment derived from the monoclonal antibody to a patient suffering from or suspected of suffering from MS. In some examples, the monoclonal antibody or antibody fragment thereof of this embodiment is present in a composition. The composition may be a pharmaceutical composition as provided herein.

[0100]

[0113] In one embodiment, provided herein is a molecule that specifically binds to ASC. A monoclonal antibody or antibody fragment thereof, the antibody or antibody fragment thereof comprising a heavy chain variable (VH) region and a light or kappa chain variable (VL) region, the amino acid sequence of the VH region comprising SEQ ID NO: 19 or comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 19, and the amino acid sequence of the VL region comprising SEQ ID NO: 28 or comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 28. In addition 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 can be innate immune inflammation. The inflammation can be inflammasome-associated inflammation. The inflammation can be present in the lung and / or CNS. Inflammation in the lungs and / or CNS can be the result of injury (e.g., traumatic brain injury (TBI) or spinal cord injury (SCI)) or can be the result of a disease, condition, or affliction of or affecting the CNS. As provided herein, the disease, condition, or affliction of or affecting the CNS can be stroke, as well as autoimmune and / or CNS diseases (e.g., amyotrophic lateral sclerosis (ALS) Lou Gehrig's, multiple sclerosis (MS), immune dysfunction muscle CNS degeneration, muscular dystrophy (MD), Alzheimer's disease (AD), Parkinson's disease (PD)). Use of this monoclonal antibody or antibody fragment thereof in a method of treating inflammation can reduce inflammation in the CNS and / or lungs of a patient. This reduction can be compared to a control (e.g., an untreated patient and / or a patient prior to treatment). In one embodiment, The monoclonal antibody or an antibody fragment derived from the monoclonal antibody is used to treat MS by administering the monoclonal antibody or an antibody fragment derived from the monoclonal antibody to a patient suffering from or suspected of suffering from MS. In some examples, the monoclonal antibody or antibody fragment thereof of this embodiment is present in a composition. The composition may be a pharmaceutical composition as provided herein.

[0101]

[0114] In one embodiment, provided herein is a molecule that specifically binds to ASC. A monoclonal antibody or antibody fragment thereof, the antibody or antibody fragment thereof comprising a heavy chain variable (VH) region and a light or kappa chain variable (VL) region, the amino acid sequence of the VH region comprising SEQ ID NO: 19 or comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 19, and the amino acid sequence of the VL region comprising SEQ ID NO: 29 or comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 29. In addition 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 can be innate immune inflammation. The inflammation can be inflammasome-associated inflammation. The inflammation can be present in the lung and / or CNS. Inflammation in the lungs and / or CNS can be the result of injury (e.g., traumatic brain injury (TBI) or spinal cord injury (SCI)) or can be the result of a disease, condition, or affliction of or affecting the CNS. As provided herein, the disease, condition, or affliction of or affecting the CNS can be stroke, as well as autoimmune and / or CNS diseases (e.g., amyotrophic lateral sclerosis (ALS) Lou Gehrig's, multiple sclerosis (MS), immune dysfunction muscle CNS degeneration, muscular dystrophy (MD), Alzheimer's disease (AD), Parkinson's disease (PD)). Use of this monoclonal antibody or antibody fragment thereof in a method of treating inflammation can reduce inflammation in the CNS and / or lungs of a patient. This reduction can be in comparison to a control (e.g., an untreated patient and / or a patient prior to treatment). In one embodiment, the monoclonal antibody or an antibody fragment derived from the monoclonal antibody is used to treat MS by administering the monoclonal antibody or an antibody fragment derived from the monoclonal antibody to a patient suffering from or suspected of suffering from MS. In some examples, the monoclonal antibody or antibody fragment thereof of this embodiment is present in a composition. The composition may be a pharmaceutical composition as provided herein.

[0102]

[0115] In one embodiment, provided herein is a molecule that specifically binds to ASC. A monoclonal antibody or antibody fragment thereof, the antibody or antibody fragment thereof comprising a heavy chain variable (VH) region and a light or kappa chain variable (VL) region, the amino acid sequence of the VH region comprising SEQ ID NO: 19 or comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 19, and the amino acid sequence of the VL region comprising SEQ ID NO: 30 or comprising 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 examples, a monoclonal antibody or an antibody fragment derived from the monoclonal antibody comprising an amino acid sequence of the VH region comprising SEQ ID NO: 19 and an amino acid sequence of the VL region comprising SEQ ID NO: 30 may be referred to as IC100. In addition 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 can be inflammasome-associated inflammation. The inflammation can be present in the lungs and / or CNS. Inflammation in the lungs and / or CNS can be the result of injury (e.g., traumatic brain injury (TBI) or spinal cord injury (SCI)) or can be the result of a disease, condition, or affliction of or affecting the CNS. As provided herein, diseases, conditions, or afflictions of or affecting the CNS include stroke, and autoimmune and / or CNS diseases (e.g., amyotrophic lateral sclerosis (ALS), Lou Gehrig's syndrome, The monoclonal antibody or antibody fragment thereof may be used to treat inflammation in the patient's CNS and / or lungs. The use of the monoclonal antibody or antibody fragment thereof in a method of treating inflammation may reduce inflammation in the patient's CNS and / or lungs. The reduction may be compared to a control (e.g., an untreated patient and / or a patient prior to treatment). In one embodiment, the monoclonal antibody or antibody fragment thereof is used to treat MS by administering the monoclonal antibody or antibody fragment thereof to a patient suffering from or suspected of suffering from MS. In some examples, the monoclonal antibody or antibody fragment thereof of this embodiment is present in a composition. The composition may be a pharmaceutical composition provided herein.

[0103]

[0116] In one embodiment, provided herein is a molecule that specifically binds to ASC. A monoclonal antibody or antibody fragment thereof, the antibody or antibody fragment thereof comprising a heavy chain variable (VH) region and a light or kappa chain variable (VL) region, the amino acid sequence of the VH region comprising SEQ ID NO: 19 or comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 19, and the amino acid sequence of the VL region comprising SEQ ID NO: 31 or comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 31. In addition 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 can be innate immune inflammation. The inflammation can be inflammasome-associated inflammation. The inflammation can be present in the lung and / or CNS. Inflammation in the lungs and / or CNS can be the result of injury (e.g., traumatic brain injury (TBI) or spinal cord injury (SCI)) or can be the result of a disease, condition, or affliction of or affecting the CNS. As provided herein, the disease, condition, or affliction of or affecting the CNS can be stroke, as well as autoimmune and / or CNS diseases (e.g., amyotrophic lateral sclerosis (ALS) Lou Gehrig's, multiple sclerosis (MS), immune dysfunction muscle CNS degeneration, muscular dystrophy (MD), Alzheimer's disease (AD), Parkinson's disease (PD)). Use of this monoclonal antibody or antibody fragment thereof in a method of treating inflammation can reduce inflammation in the CNS and / or lungs of a patient. This reduction can be in comparison to a control (e.g., an untreated patient and / or a patient prior to treatment). In one embodiment, the monoclonal antibody or an antibody fragment derived from the monoclonal antibody is used to treat MS by administering the monoclonal antibody or an antibody fragment derived from the monoclonal antibody to a patient suffering from or suspected of suffering from MS. In some examples, the monoclonal antibody or antibody fragment thereof of this embodiment is present in a composition. The composition may be a pharmaceutical composition as provided herein.

[0104]

[0117] In one embodiment, provided herein is a molecule that specifically binds to ASC. A monoclonal antibody or antibody fragment thereof, the antibody or antibody fragment thereof comprising a heavy chain variable (VH) region and a light or kappa chain variable (VL) region, the amino acid sequence of the VH region comprising SEQ ID NO:20 or comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:20, and the amino acid sequence of the VL region comprising SEQ ID NO:28 or comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:28. In addition 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 can be innate immune inflammation. The inflammation can be inflammasome-associated inflammation. The inflammation can be present in the lung and / or CNS. Inflammation in the lungs and / or CNS can be the result of injury (e.g., traumatic brain injury (TBI) or spinal cord injury (SCI)) or can be associated with a disease, condition, or affliction of or affecting the CNS. The disease, condition, or affliction of or affecting the CNS may be a result of stroke, as well as autoimmune and / or CNS diseases (e.g., amyotrophic lateral sclerosis (ALS) Lou Gehrig's, multiple sclerosis (MS), immune dysfunction muscle CNS degeneration, muscular dystrophy (MD), Alzheimer's disease (AD), Parkinson's disease (PD)). The use of the monoclonal antibody or antibody fragment thereof in a method of treating inflammation may reduce inflammation in the CNS and / or lungs of a patient. The reduction may be compared to a control (e.g., an untreated patient and / or a patient prior to treatment). In one embodiment, the monoclonal antibody or antibody fragment derived from the monoclonal antibody is used to treat MS by administering the monoclonal antibody or antibody fragment derived from the monoclonal antibody to a patient suffering from or suspected of suffering from MS. In some examples, the monoclonal antibody or antibody fragment of the embodiment is present in a composition. The composition can be a pharmaceutical composition provided herein.

[0105]

[0118] In one embodiment, provided herein is a molecule that specifically binds to ASC. A monoclonal antibody or antibody fragment thereof, the antibody or antibody fragment thereof comprising a heavy chain variable (VH) region and a light or kappa chain variable (VL) region, the amino acid sequence of the VH region comprising SEQ ID NO:20 or comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:20, and the amino acid sequence of the VL region comprising SEQ ID NO:29 or comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:29. In addition 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 can be innate immune inflammation. The inflammation can be inflammasome-associated inflammation. The inflammation can be present in the lung and / or CNS. Inflammation in the lungs and / or CNS can be the result of injury (e.g., traumatic brain injury (TBI) or spinal cord injury (SCI)) or can be the result of a disease, condition, or affliction of or affecting the CNS. As provided herein, the disease, condition, or affliction of or affecting the CNS can be stroke, as well as autoimmune and / or CNS diseases (e.g., amyotrophic lateral sclerosis (ALS) Lou Gehrig's, multiple sclerosis (MS), immune dysfunction muscle CNS degeneration, muscular dystrophy (MD), Alzheimer's disease (AD), Parkinson's disease (PD)). Use of this monoclonal antibody or antibody fragment thereof in a method of treating inflammation can reduce inflammation in the CNS and / or lungs of a patient. This reduction can be in comparison to a control (e.g., an untreated patient and / or a patient prior to treatment). In one embodiment, the monoclonal antibody or an antibody fragment derived from the monoclonal antibody is used to treat MS by administering the monoclonal antibody or an antibody fragment derived from the monoclonal antibody to a patient suffering from or suspected of suffering from MS. In some examples, the monoclonal antibody or antibody fragment thereof of this embodiment is present in a composition. The composition may be a pharmaceutical composition as provided herein.

[0106]

[0119] In one embodiment, provided herein is a molecule that specifically binds to ASC. A monoclonal antibody or antibody fragment thereof, the antibody or antibody fragment thereof comprising a heavy chain variable (VH) region and a light or kappa chain variable (VL) region, the amino acid sequence of the VH region comprising SEQ ID NO:20 or comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:20, and the amino acid sequence of the VL region comprising SEQ ID NO:30 or comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:30. In addition to this embodiment, provided herein is the use of the monoclonal antibody or antibody fragment thereof in a method of treating inflammation in a subject. The inflammation can be innate immune inflammation. The inflammation can be inflammasome-associated inflammation. The inflammation can be present in the lungs and / or CNS. The inflammation can be present in the lungs and / or CNS. Inflammation in the S may be the result of injury (e.g., traumatic brain injury (TBI) or spinal cord injury (SCI)) or may be the result of a disease, condition, or affliction of or affecting the CNS. As provided herein, the disease, condition, or affliction of or affecting the CNS may be stroke, as well as autoimmune and / or CNS diseases (e.g., amyotrophic lateral sclerosis (ALS) Lou Gehrig's, multiple sclerosis (MS), immune dysfunction muscle CNS degeneration, muscular dystrophy (MD), Alzheimer's disease (AD), Parkinson's disease (PD)). Use of this monoclonal antibody or antibody fragment thereof in a method of treating inflammation may reduce inflammation in the CNS and / or lungs of a patient. This reduction may be in comparison to a control (e.g., an untreated patient and / or a patient prior to treatment). In one embodiment, the monoclonal antibody or an antibody fragment derived from the monoclonal antibody is used to treat MS by administering the monoclonal antibody or an antibody fragment derived from the monoclonal antibody to a patient suffering from or suspected of suffering from MS. In some examples, the monoclonal antibody or antibody fragment thereof of this embodiment is present in a composition. The composition may be a pharmaceutical composition as provided herein.

[0107]

[0120] In one embodiment, provided herein is a molecule that specifically binds to ASC. A monoclonal antibody or antibody fragment thereof, the antibody or antibody fragment thereof comprising a heavy chain variable (VH) region and a light or kappa chain variable (VL) region, the amino acid sequence of the VH region comprising SEQ ID NO:20 or comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:20, and the amino acid sequence of the VL region comprising SEQ ID NO:31 or comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:31. In addition 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 can be innate immune inflammation. The inflammation can be inflammasome-associated inflammation. The inflammation can be present in the lung and / or CNS. Inflammation in the lungs and / or CNS can be the result of injury (e.g., traumatic brain injury (TBI) or spinal cord injury (SCI)) or can be the result of a disease, condition, or affliction of or affecting the CNS. As provided herein, the disease, condition, or affliction of or affecting the CNS can be stroke, as well as autoimmune and / or CNS diseases (e.g., amyotrophic lateral sclerosis (ALS) Lou Gehrig's, multiple sclerosis (MS), immune dysfunction muscle CNS degeneration, muscular dystrophy (MD), Alzheimer's disease (AD), Parkinson's disease (PD)). Use of this monoclonal antibody or antibody fragment thereof in a method of treating inflammation can reduce inflammation in the CNS and / or lungs of a patient. This reduction can be in comparison to a control (e.g., an untreated patient and / or a patient prior to treatment). In one embodiment, the monoclonal antibody or an antibody fragment derived from the monoclonal antibody is used to treat MS by administering the monoclonal antibody or an antibody fragment derived from the monoclonal antibody to a patient suffering from or suspected of suffering from MS. In some examples, the monoclonal antibody or antibody fragment thereof of this embodiment is present in a composition. The composition may be a pharmaceutical composition as provided herein.

[0108]

[0121] In one embodiment, provided herein is a molecule that specifically binds to ASC. and a monoclonal antibody or antibody fragment thereof, the antibody or antibody fragment thereof comprising a heavy chain variable (VH) region and a light or kappa chain variable (VL) region, the amino acid sequence of the VH region comprising SEQ ID NO:21 or comprising 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 the amino acid sequence of the VL region comprising SEQ ID NO:28 or comprising 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 addition to this embodiment, provided herein is the use of the monoclonal antibody or antibody fragment thereof in a method of treating inflammation in a subject. The inflammation may be innate immune inflammation. The inflammation may be inflammasome-associated inflammation. The inflammation may be present in the lungs and / or CNS. The 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 may be the result of a disease, condition, or affliction of or affecting the CNS. As provided herein, the disease, condition, or affliction of or affecting the CNS may be stroke, as well as autoimmune and / or CNS diseases (e.g., amyotrophic lateral sclerosis (ALS) Lou Gehrig's, multiple sclerosis (MS), immune dysfunction muscle CNS degeneration, muscular dystrophy (MD), Alzheimer's disease (AD), Parkinson's disease (PD)). Use of the monoclonal antibody or antibody fragment thereof in a method of treating inflammation may reduce inflammation in the CNS and / or lungs of a patient. The reduction may be compared to a control (e.g., an untreated patient and / or a patient prior to treatment). In one embodiment, the monoclonal antibody or an antibody fragment derived from the monoclonal antibody is used to treat MS by administering the monoclonal antibody or an antibody fragment derived from the monoclonal antibody to a patient suffering from or suspected of suffering from MS. In some examples, the monoclonal antibody or antibody fragment thereof of this embodiment is present in a composition. The composition may be a pharmaceutical composition as provided herein.

[0109]

[0122] In one embodiment, provided herein is a molecule that specifically binds to ASC. A monoclonal antibody or antibody fragment thereof, the antibody or antibody fragment thereof comprising a heavy chain variable (VH) region and a light or kappa chain variable (VL) region, the amino acid sequence of the VH region comprising SEQ ID NO:21 or comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:21, and the amino acid sequence of the VL region comprising SEQ ID NO:29 or comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:29. In addition 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 can be innate immune inflammation. The inflammation can be inflammasome-associated inflammation. The inflammation can be present in the lung and / or CNS. Inflammation in the lungs and / or CNS can be the result of injury (e.g., traumatic brain injury (TBI) or spinal cord injury (SCI)) or can be the result of a disease, condition, or affliction of or affecting the CNS. As provided herein, the disease, condition, or affliction of or affecting the CNS can be stroke, as well as autoimmune and / or CNS diseases (e.g., amyotrophic lateral sclerosis (ALS) Lou Gehrig's, multiple sclerosis (MS), immune dysfunction muscle CNS degeneration, muscular dystrophy (MD), Alzheimer's disease (AD), Parkinson's disease (PD)). Use of this monoclonal antibody or antibody fragment thereof in a method of treating inflammation can reduce inflammation in the CNS and / or lungs of a patient. This reduction can be in comparison to a control (e.g., an untreated patient and / or a patient prior to treatment). In one embodiment, the monoclonal antibody or an antibody fragment derived from the monoclonal antibody is used to treat MS by administering the monoclonal antibody or an antibody fragment derived from the monoclonal antibody to a patient suffering from or suspected of suffering from MS. In some examples, the monoclonal antibody or antibody fragment thereof of this embodiment is present in a composition. The composition may be a pharmaceutical composition as provided herein.

[0110]

[0123] In one embodiment, provided herein is a molecule that specifically binds to ASC. a monoclonal antibody or antibody fragment thereof, the antibody or antibody fragment thereof comprising a heavy chain variable (VH) region and a light or kappa chain variable (VL) region, the amino acid sequence of the VH region comprising SEQ ID NO:21 or comprising 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 the amino acid sequence of the VL region comprising SEQ ID NO:30 or comprising 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; In addition to this embodiment, provided herein is the use of this monoclonal antibody or antibody fragment thereof in a method of treating inflammation in a subject. The inflammation can be innate immune inflammation. The inflammation can be inflammasome-associated inflammation. The inflammation can be present in the lungs and / or CNS. The inflammation in the lungs and / or CNS can be the result of injury (e.g., traumatic brain injury (TBI) or spinal cord injury (SCI)) or can be the result of a disease, condition, or affliction of or affecting the CNS. As provided herein, the disease, condition, or affliction of or affecting the CNS can be stroke, as well as autoimmune diseases and / or CNS diseases (e.g., amyotrophic lateral sclerosis (ALS) Lou Gehrig's, multiple sclerosis (MS), immune dysfunction muscle CNS degeneration, muscular dystrophy (MD), Alzheimer's disease (AD), Parkinson's disease (PD)). The use of this monoclonal antibody or its antibody fragment in a method for treating inflammation may reduce inflammation in the CNS and / or lungs of a patient. This reduction may be in comparison to a control (e.g., an untreated patient and / or a patient prior to treatment). In one embodiment, this monoclonal antibody or an antibody fragment derived from this monoclonal antibody is used to treat MS by administering this monoclonal antibody or an antibody fragment derived from this monoclonal antibody to a patient suffering from or suspected of suffering from MS. In some examples, the monoclonal antibody or its antibody fragment of this embodiment is present in a composition. This composition may be a pharmaceutical composition as provided herein.

[0111]

[0124] In one embodiment, provided herein is a molecule that specifically binds to ASC. A monoclonal antibody or antibody fragment thereof, the antibody or antibody fragment thereof comprising a heavy chain variable (VH) region and a light or kappa chain variable (VL) region, the amino acid sequence of the VH region comprising SEQ ID NO:21 or comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:21, and the amino acid sequence of the VL region comprising SEQ ID NO:31 or comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:31. In addition 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 can be innate immune inflammation. The inflammation can be inflammasome-associated inflammation. The inflammation can be present in the lung and / or CNS. Inflammation in the lungs and / or CNS can be the result of injury (e.g., traumatic brain injury (TBI) or spinal cord injury (SCI)) or can be the result of a disease, condition, or affliction of or affecting the CNS. As provided herein, the disease, condition, or affliction of or affecting the CNS can be stroke, as well as autoimmune and / or CNS diseases (e.g., amyotrophic lateral sclerosis (ALS) Lou Gehrig's, multiple sclerosis (MS), immune dysfunction muscle CNS degeneration, muscular dystrophy (MD), Alzheimer's disease (AD), Parkinson's disease (PD)). Use of this monoclonal antibody or antibody fragment thereof in a method of treating inflammation can reduce inflammation in the CNS and / or lungs of a patient. This reduction can be in comparison to a control (e.g., an untreated patient and / or a patient prior to treatment). In one embodiment, the monoclonal antibody or an antibody fragment derived from the monoclonal antibody is used to treat MS by administering the monoclonal antibody or an antibody fragment derived from the monoclonal antibody to a patient suffering from or suspected of suffering from MS. In some examples, the monoclonal antibody or antibody fragment thereof of this embodiment is present in a composition. The composition may be a pharmaceutical composition as provided herein.

[0112]

[0125] In one embodiment, provided herein is a molecule that specifically binds to ASC. and a monoclonal antibody or antibody fragment thereof, the antibody or antibody fragment thereof comprising a heavy chain variable (VH) region and a light or kappa chain variable (VL) region, the amino acid sequence of the VH region comprising 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 the amino acid sequence of the VL region comprising SEQ ID NO:22. The amino acid sequence is a monoclonal antibody or antibody fragment thereof comprising 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 addition to this embodiment, provided herein is the use of this monoclonal antibody or antibody fragment thereof in a method for treating inflammation in a subject. The inflammation can be innate immune inflammation. The inflammation can be inflammasome-associated inflammation. The inflammation can be present in the lungs and / or CNS. The inflammation in the lungs and / or CNS can be the result of injury (e.g., traumatic brain injury (TBI) or spinal cord injury (SCI)) or can be the result of a disease, condition, or affliction in or affecting the CNS. As provided herein, the disease, condition, or affliction of or affecting the CNS may be stroke, as well as autoimmune and / or CNS diseases (e.g., amyotrophic lateral sclerosis (ALS) Lou Gehrig's, multiple sclerosis (MS), immune dysfunction muscle CNS degeneration, muscular dystrophy (MD), Alzheimer's disease (AD), Parkinson's disease (PD)). Use of the monoclonal antibody or antibody fragment thereof in a method of treating inflammation may reduce inflammation in the CNS and / or lungs of a patient. The reduction may be compared to a control (e.g., an untreated patient and / or a patient prior to treatment). In one embodiment, the monoclonal antibody or antibody fragment derived from the monoclonal antibody is used to treat MS by administering the monoclonal antibody or antibody fragment derived from the monoclonal antibody to a patient suffering from or suspected of suffering from MS. In some examples, the monoclonal antibody or antibody fragment of the embodiment is present in a composition. The composition may be a pharmaceutical composition provided herein.

[0113]

[0126] In one embodiment, provided herein is a molecule that specifically binds to ASC. A monoclonal antibody or antibody fragment thereof, the antibody or antibody fragment thereof comprising a heavy chain variable (VH) region and a light or kappa chain variable (VL) region, the amino acid sequence of the VH region comprising SEQ ID NO:22 or comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:22, and the amino acid sequence of the VL region comprising SEQ ID NO:29 or comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:29. In addition 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 can be innate immune inflammation. The inflammation can be inflammasome-associated inflammation. The inflammation can be present in the lung and / or CNS. Inflammation in the lungs and / or CNS can be the result of injury (e.g., traumatic brain injury (TBI) or spinal cord injury (SCI)) or can be the result of a disease, condition, or affliction of or affecting the CNS. As provided herein, the disease, condition, or affliction of or affecting the CNS can be stroke, as well as autoimmune and / or CNS diseases (e.g., amyotrophic lateral sclerosis (ALS) Lou Gehrig's, multiple sclerosis (MS), immune dysfunction muscle CNS degeneration, muscular dystrophy (MD), Alzheimer's disease (AD), Parkinson's disease (PD)). Use of this monoclonal antibody or antibody fragment thereof in a method of treating inflammation can reduce inflammation in the CNS and / or lungs of a patient. This reduction can be in comparison to a control (e.g., an untreated patient and / or a patient prior to treatment). In one embodiment, the monoclonal antibody or an antibody fragment derived from the monoclonal antibody is used to treat MS by administering the monoclonal antibody or an antibody fragment derived from the monoclonal antibody to a patient suffering from or suspected of suffering from MS. In some examples, the monoclonal antibody or antibody fragment thereof of this embodiment is present in a composition. The composition may be a pharmaceutical composition as provided herein.

[0114]

[0127] In one embodiment, provided herein is a molecule that specifically binds to ASC. A monoclonal antibody or antibody fragment thereof, the antibody or antibody fragment thereof comprising a heavy chain variable (VH) region and a light or kappa chain variable (VL) region, the amino acid sequence of the VH region being , comprising 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 the amino acid sequence of the VL region 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. In addition to this embodiment, provided herein is the use of this monoclonal antibody or antibody fragment thereof in a method of treating inflammation in a subject. The inflammation can be innate immune inflammation. The inflammation can be inflammasome-associated inflammation. The inflammation can be present in the lungs and / or CNS. The inflammation in the lungs and / or CNS can be the result of injury (e.g., traumatic brain injury (TBI) or spinal cord injury (SCI)) or can be the result of a disease, condition, or affliction in or affecting the CNS. As provided herein, the disease, condition, or affliction of or affecting the CNS may be stroke, as well as autoimmune and / or CNS diseases (e.g., amyotrophic lateral sclerosis (ALS) Lou Gehrig's, multiple sclerosis (MS), immune dysfunction muscle CNS degeneration, muscular dystrophy (MD), Alzheimer's disease (AD), Parkinson's disease (PD)). Use of the monoclonal antibody or antibody fragment thereof in a method of treating inflammation may reduce inflammation in the CNS and / or lungs of a patient. The reduction may be compared to a control (e.g., an untreated patient and / or a patient prior to treatment). In one embodiment, the monoclonal antibody or antibody fragment derived from the monoclonal antibody is used to treat MS by administering the monoclonal antibody or antibody fragment derived from the monoclonal antibody to a patient suffering from or suspected of suffering from MS. In some examples, the monoclonal antibody or antibody fragment of the embodiment is present in a composition. The composition may be a pharmaceutical composition provided herein.

[0115]

[0128] In one embodiment, provided herein is a molecule that specifically binds to ASC. A monoclonal antibody or antibody fragment thereof, the antibody or antibody fragment thereof comprising a heavy chain variable (VH) region and a light or kappa chain variable (VL) region, the amino acid sequence of the VH region comprising SEQ ID NO:22 or comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:22, and the amino acid sequence of the VL region comprising SEQ ID NO:31 or comprising an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:31. In addition 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 can be innate immune inflammation. The inflammation can be inflammasome-associated inflammation. In one embodiment, the monoclonal antibodies or antibody fragments thereof provided herein may be used in a method of reducing inflammation in a mammal as described in U.S. Pat. No. 8,685,400, the contents of which are incorporated herein by reference in their entirety. The inflammation may be present in the lungs and / or CNS. The 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 may be the result of a disease, condition, or affliction of or affecting the CNS. As provided herein, the disease, condition, or affliction of or affecting the CNS may be stroke, as well as autoimmune and / or CNS diseases (e.g., amyotrophic lateral sclerosis (ALS) Lou Gehrig's, multiple sclerosis (MS), immune dysfunction muscle CNS degeneration, muscular dystrophy (MD), Alzheimer's disease (AD), Parkinson's disease (PD)). Use of this monoclonal antibody or antibody fragment thereof in a method for treating inflammation may reduce inflammation in the CNS and / or lungs of a patient. This reduction may be in comparison to a control (e.g., an untreated patient and / or a patient prior to treatment). In one embodiment, this monoclonal antibody or an antibody fragment derived from this monoclonal antibody is used to treat MS by administering the monoclonal antibody or an antibody fragment derived from this monoclonal antibody to a patient suffering from or suspected of suffering from MS.In some examples, the monoclonal antibody or antibody fragment thereof of this embodiment is present in a composition. The composition can be a pharmaceutical composition provided herein.

[0116]

[0129] In another embodiment, the method reduces inflammation in the CNS or lungs of a mammal. The compositions for use in the methods provided herein include an antibody (e.g., an anti-NLRP1 chicken antibody) or an active fragment thereof that specifically binds to NLRP1 or a domain thereof, as provided herein. Any suitable anti-NLRP1 antibody can be used, and some are commercially available. Examples of anti-NLRP1 antibodies for use in the methods provided herein can be those found in U.S. Pat. No. 8,685,400, 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, rabbit polyclonal anti-NLRP1 ABF22 from EMD Millipore, rabbit polyclonal anti-NLRP1 ABF23 from Novus Biologicals, and rabbit polyclonal anti-NLRP1 ABF24 from Novus Biologicals. Reclonal anti-NLRP1 NB100-56148, Sigma-Aldrich mouse polyclonal antibody 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 oa ed00344, 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-NLR P1 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 porcine Reclonal anti-NLRP1 200-401-CX5, or Cell Signaling Technology Examples of antibodies include rabbit polyclonal anti-NLRP1 4990. The human NLRP1 protein can be accession numbers 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 (e.g., human NLRP1), or a portion or fragment thereof. In this embodiment, the antibody described herein specifically binds to an amino acid sequence having at least 65% (e.g., 65, 70, 75, 80, 85%) sequence identity with a defined domain (e.g., Pyrin, NACHT, LRR1-6, FIIND, or CARD) of human NLRP1, or a fragment thereof. In one embodiment, a chicken anti-NLRP1 polyclonal antibody custom designed and produced by Ayes Laboratories is used. The antibody may be directed against the following amino acid sequence in human NLRP1: CEYYTEIREREREKSEKGR (SEQ ID NO: 3). In one embodiment, the antibody that binds to the NLRP1 domain or a fragment thereof described herein inhibits NLRP1 activity in mammalian lung cells, e.g., type II alveolar cells.

[0117]

[0130] In yet another embodiment, the method comprises the steps of: reducing inflammation in the CNS or lungs of a mammal; The composition for performing the method includes an antibody or an active fragment thereof provided herein that specifically binds to AIM2 or a domain thereof. Any suitable anti-AIM2 antibody can be used, and some 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 antibody from Proteintech. Local anti-AIM2 Catalog No. 20590-1-AP, Abcam anti-AIMS antibody (ab1 19791), rabbit polyclonal anti-AIM2 (N-terminal region) from ECM biosciences Catalog number AP3851, rabbit polyclonal anti-ASC catalog number E-AB-30449 from Elabsciences, anti-AIM2 mouse monoclonal antibody called AIM2 antibody (3C4G11) from Santa Cruz Biotechnology, catalog number sc-293174, mouse monoclonal AIM2 antibody from Origene, catalog number TA324972, T AIM2 monoclonal antibody (10M2B3) from Hermofisher Scientific, AIM2 rabbit polyclonal antibody ABIN928372 or ABIN from Antibodies-online 760766, Biomatix coated anti-AIM2 polyclonal antibody, catalog number CAE02153, anti-AIM2 polyclonal antibody from Aviva Systems Biology (OABF016 32), rabbit polyclonal anti-AIM2 antibody LS-C35412 from LSBio-C354127 7. Rabbit monoclonal anti-AIM2 antibody from Cell Signaling Technology, catalog No. MA5-16259, rabbit polyclonal antibody from Fab Gennix International Incorporated monoclonal anti-AIM2 monoclonal antibody, Catalog No. AIM2 201AP, MyBiosource rabbit polyclonal anti-AIM2 Catalog No. MBS855320, Signalway rabbit polyclonal anti-AIM2 Catalog No. 36253, Novus Biological rabbit polyclonal anti-AIM2 Catalog No. 43900002, GeneTex rabbit polyclonal anti-AI M2 GTX54910, Prosci, rabbit polyclonal anti-AIM2 26-540, Biorbyt mouse monoclonal anti-AIM2 orb333902, Abcam rabbit polyclonal anti-AIM2 ab93015), Abcam rabbit polyclonal anti-AIM2 ab764 23, Sigma Aldrich mouse polyclonal anti-AIM2 SAB1406827, or Biolegend anti-AIM2 3B10. Human AIM2 protein was obtained from the accession number The antibody may be of the following gene numbers: 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 described herein specifically binds to an amino acid sequence having at least 65% (e.g., 65, 70, 75, 80, 85%) sequence identity with a defined domain of human AIM2 (e.g., Pyrin or HIN-200) or a fragment thereof. In one embodiment, the antibody that binds to the AIM2 domain or a fragment thereof described herein inhibits AIM2 activity in mammalian lung cells, e.g., type II alveolar cells.

[0118]

[0131] Anti-inflammasome antibodies described herein (e.g., anti-ASC, anti-NLRP1 or (anti-AIM2) antibodies 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 that specifically binds to a component of a mammalian inflammasome (e.g., the AIM2 inflammasome), such as ASC or AIM2. In some instances, the antibody is specific for ASC, such that the antibody is specific for ASC when it is raised against an epitope of a polypeptide and binds to at least a portion of a natural or recombinant protein.

[0119]

[0132] In certain embodiments, the antibodies provided herein comprise one or more For example, the anti-ASC monoclonal antibody or ASC-binding antibody fragment includes a polypeptide having one or more amino acid substitutions, deletions, or insertions compared to a polypeptide having one or more amino acid sequences of SEQ ID NOs: 6-8, 12-14, 18-22, or 28-31. The antibodies provided herein may have one, two, three, four, five, six, seven, eight, nine, ten, or more amino acid substitutions, deletions, or insertions. For example, the anti-ASC monoclonal antibody or ASC-binding antibody fragment may have one, two, three, four, five, six, seven, eight, nine, ten, or more amino acid substitutions, deletions, or insertions. The 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 the anti-ASC antibody or ASC-binding antibody fragment polypeptide.

[0120]

[0133] In certain embodiments, conservative amino acid substitutions are those disclosed herein. A "conservative amino acid substitution" is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. In certain embodiments, conservative amino acid substitutions are made only in the FR sequences and not in the CDR sequences of the antibody or antibody fragment. Families of amino acid residues having 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), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan; histidine). Thus, for example, an amino acid residue in an anti-ASC monoclonal antibody or ASC-binding antibody fragment polypeptide can be replaced with another amino acid residue from the same side chain family. In certain embodiments, the string of amino acids may be replaced with a structurally similar string that differs in order and / or in the composition of side chain family members. One skilled in the art could assess 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 one or more of the amino acid sequences of SEQ ID NOs: 6-8, 12-14, 18-22, or 28-31 binds to an ASC protein by utilizing routine art-recognized methods (e.g., but not limited to, ELISA, Western blot, phage display, etc.).

[0121]

[0134] Sequence homology or sequence identity between sequences (the terms are used interchangeably herein) Calculation of (used in) may be performed as follows:

[0122]

[0135] To determine the percent identity of two amino acid sequences or two nucleic acid sequences These sequences are then aligned for optimal comparison purposes (e.g., gaps may be introduced into one or both of the first and second amino acid or nucleic acid sequences for optimal alignment, and non-homologous sequences may be ignored for comparison purposes). In exemplary embodiments, 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 two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap that need to be introduced for optimal alignment of the two sequences.

[0123]

[0136] The comparison of sequences and determination of percent identity between two sequences is carried out using a mathematical algorithm. In one embodiment, the percent identity between two amino acid sequences can be determined using either the BLOSUM 62 matrix or the PAM250 matrix, as well as the 16, 14, 1 Gap weights of 2, 10, 8, 6, or 4 and length weights of 1, 2, 3, 4, 5, or 6 were used with the GAP program in the GCG software package (available at www.gcg.com). In yet another embodiment, the percent identity between two nucleotide sequences is determined using the algorithm of Needleman et al. ((1970) J. Mol. Biol. 48:444-453) incorporated herein. In yet another embodiment, the percent identity between two nucleotide sequences is determined using the NWSgapdna CMP matrix, as well as gaps of 40, 50, 60, 70, or 80. The GCG software package uses a group weight and a length weight of 1, 2, 3, 4, 5, or 6. The parameters (and how they are used to determine whether a molecule is within the sequence identity or sequence homology limits of the invention) are determined using the GAP program in the GCG page (available at www.gcg.com). One set of scoring matrices (which may be used when the expert is unsure which parameters to apply) is the BLOSUM 62 scoring matrix, which includes a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

[0124]

[0137] The percent identity between two amino acid or nucleotide sequences was calculated using the PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4, incorporated into the ALIGN program (version 2.0). This can be determined using the algorithm of Meyers et al. ((1989) CABIOS 4:11-17), which is incorporated herein by reference.

[0125]

[0138] In certain embodiments, the antibody is a monoclonal antibody. The monoclonal antibody is a polyclonal antibody. The term "monoclonal antibody" refers to a population of antibody molecules that contain only one species of antigen-binding site capable of immunoreacting with a particular epitope of an antigen. Thus, a monoclonal antibody composition typically exhibits a single binding affinity for a particular protein with which it immunoreacts.

[0126]

[0139] In some embodiments, the antibody of the present invention (anti-ASC monoclonal antibody or ASC C-binding antibody fragments) may be humanized, chimeric or human.

[0127]

[0140] In some embodiments, the antibodies of the invention are humanized antibodies.

[0128]

[0141] A "humanized antibody," as that term is used herein, refers to an antibody having a heavy chain and / or It refers to an antibody that has been engineered to contain one or more human framework regions in the variable regions with non-human (e.g., mouse, rat, or hamster) complementarity determining regions (CDRs) of the light chain. In certain embodiments, a humanized antibody comprises sequences that are completely human except for the CDR regions. In some cases, Fv framework region (FR) residues of the human immunoglobulin are replaced with corresponding non-human residues. Furthermore, a humanized antibody may contain residues that are found neither in the human antibody nor in the imported CDR or framework sequences, but are included to further refine and optimize antibody performance. In general, a humanized antibody comprises substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are of human immunoglobulin consensus sequences. The RF regions may be modified in any manner known in the art and / or provided herein. This modification may confer desirable properties (e.g., increased half-life and / or improved expression in a host cell). In one embodiment, the FR region may be modified or mutated as described in US Patent Publication No. 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 modified with respect to the original antibody (also referred to as one or more CDRs "derived" from one or more CDRs from the original antibody). The humanized antibody optimally also includes at least a portion of an immunoglobulin constant region or domain (Fc), typically at least a portion of a human immunoglobulin.

[0129]

[0142] Humanized antibodies typically have reduced immunogenicity in humans compared to non-human antibodies. The constant region of an antibody has a low affinity for a given antibody, thereby providing a therapeutic advantage in certain circumstances. For example, the constant region of an antibody can be engineered to be immunologically inert (e.g., not trigger complement lysis). See, e.g., 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 recognize humanized antibodies and techniques suitable for generating the same. See, e.g., 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. Pat. 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 by reference in its entirety). Other methods for humanizing antibodies that can also be used are disclosed by 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 WO 01 / 27160, each of which is incorporated herein by reference in its entirety. For example, an anti-ASC antibody or antigen-binding fragment of the invention may comprise an amino acid sequence of a VH region comprising an HCDR1 of SEQ ID NO:6, an HCDR2 of SEQ ID NO:7, and an HCDR3 of SEQ ID NO:8, an amino acid sequence of a VL region comprising an LCDR1 of SEQ ID NO:12, an LCDR2 of SEQ ID NO:13, and an LCDR3 of SEQ ID NO:14, and one or more human framework region sequences.

[0130]

[0143] In some embodiments, the antibodies of the invention are chimeric antibodies and bind to ASC. Specifically binds to ASC. In some examples, the anti-ASC chimeric antibody reduces the activity of ASC. "Chimeric antibody", as the term is used herein, refers to an antibody that has been engineered to include at least one human constant region. For example, one or all of the variable regions of the light chain and / or one or all of the variable regions of the heavy chain of a mouse antibody (e.g., a mouse monoclonal antibody) can each be linked to a human constant region (e.g., but not limited to, an IgG1 human constant region). Chimeric antibodies are typically less immunogenic to humans compared to non-chimeric antibodies, and therefore provide therapeutic advantages in certain circumstances. Those skilled in the art will recognize chimeric antibodies and also recognize techniques suitable for generating the same. See, for example, Cabilly et al., U.S. Pat. No. 4,816,567; Shoemaker et al., U.S. Pat. No. 4,978,775; Beavers et al., U.S. Pat. No. 4,975,369; and Boss et al., U.S. Pat. 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 can comprise a VH region comprising SEQ ID NO:22; a VL region comprising SEQ ID NO:31; and a human constant region.

[0131]

[0144] As used herein, the terms "immunological binding" and "immunological binding properties" refer to " refers to the type of non-covalent interaction that occurs between an immunoglobulin molecule (e.g., an antibody) and the antigen for which the immunoglobulin is specific. The strength or affinity of an immunological binding interaction can be expressed in terms of the dissociation constant (Kd) of the interaction, with a smaller Kd representing a higher affinity. The immunological binding properties of a selected polypeptide can be quantified using methods known in the art. One such method involves measuring the rates of formation and dissociation of the antigen-binding site / antigen complex, which rates depend on the concentrations of the complex partners, the affinity of the interaction, and geometric parameters that affect the rates equally in both directions. Thus, both the "on-rate constant" (Kon) and the "off-rate constant" (Koff) can be determined by calculation of the concentrations and the actual association and dissociation rates. (Nature 361:186-87 (1993). The ratio Koff / Kon allows for the cancellation of all parameters not related to affinity and is equal to the dissociation constant Kd. (See generally Davies (See, e.g., Hoffmann et al. (1990) Annual Rev Biochem 59:439-473). An antibody of the invention is said to specifically bind an epitope (e.g., an ASC fragment having the amino acid sequence of SEQ ID NO:5) if it has an equilibrium binding constant (Kd) of ≦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 assay known to one of skill in the art.

[0132]

[0145] In certain aspects, the antibodies of the invention are monovalent or bivalent and single chain or Functionally, the binding affinity of the antibody can be in the range of 10-5M to 10-12M. For example, the binding affinity of the antibody can be in the range of 10-6M to 10-12M, 10-7M to 10-12M, 10-8M to 10-12M, 10-9M to 10-12M, 10-5M to 10-11M, 10-6M to 10-11M, 10-7M to 10-11M, 10-8M to 10-11M, 10-9M to 10-11M, 10-10M to 10-11M, 10-5M to 10-10M, 10-6M to 10- 10M, 10-7M~10-10M, 10-8M~10-10M, 10-9M~10-10M, 10-5M~10-9M, 10-6M~10-9M, 10-7M~10-9M, 10-8M ~10-9M, 10-5M~10-8M, 10-6M~10-8M, 10-7M~10-8M, 10-5M~10-7M, 10-6M~10-7M, or 10-5M~10-6M.

[0133]

[0146] Methods for determining monoclonal antibody specificity and affinity by competitive inhibition are described in Harlow, et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1988; Colligan et al., eds., Current Protocols in Immunology, Greene Publishing Assoc. and Wiley Interscience, NY, (1992, 1993); and Muller, Meth. Enzymol. 92:589-601, 1983, which are incorporated herein by reference in their entireties. can be done.

[0134]

[0147] The anti-inflammasome (e.g., anti-ASC and anti-AIM2) antibodies of the present invention are Anti-ASC or anti-NLR1 antibodies can be routinely produced by methods such as, but not limited to, inoculation of suitable animals with a polypeptide or antigenic fragment, 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 an animal with purified recombinant ASC or a peptide fragment thereof (e.g., residues 178-193 (SEQ ID NO: 1) of rat ASC (e.g., accession number BAC43754), EQ ID NO: 2 of human ASC, or residues 21-41 (SEQ ID NO: 5) of human ASC (e.g., accession number NP_037390.2)) is an example of a method for preparing anti-ASC antibodies. Similarly, immunization of an animal with purified recombinant NLRP1 or a peptide fragment thereof, such as 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.

[0135]

[0148] Monoclonal antibodies that specifically bind to ASC or NLRP1 are available to those skilled in the art. These can be obtained by known methods, see, for example, Kohler and Milstein, Nature 256:495-497, 1975; U.S. Pat. No. 4,376,113, the contents of which are incorporated herein by reference in their entirety. No. 10; Ausubel et al., eds., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley Interscience, NY, (1987, 1992); Harlow and Lane ANTIBODIES:A See Laboratory Manual Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1988; Colligan et al., eds., Current Protocols in Immunology, Greene Publishing Assoc. and Wiley Interscience, NY, (1992, 1993). Such antibodies can be produced by any immunoglobulin. The monoclonal antibody of the present disclosure may be of any phosphoclass, for example, IgG, IgM, IgE, IgA, GILD, and any subclass thereof. The hybridoma producing the monoclonal antibody of the present disclosure may be cultured in vitro, in situ, or in vivo. In one embodiment, the hybridoma producing the anti-ASC monoclonal antibody of the present disclosure is an ICCN1.OH hybridoma. In 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, the amino acid sequence of the VH region being HCDR1 of SEQ ID NO:6, HCDR2 of SEQ ID NO:7, and HCDR3 of SEQ ID NO:8, or a variant thereof, comprising a variant having at least one amino acid substitution in HCDR1, HCDR2, and / or HCDR3. In 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, the amino acid sequence of the VH region being HCDR1 of SEQ ID NO:6, HCDR2 of SEQ ID NO:7, and HCDR3 of SEQ ID NO:8, or a variant thereof, comprising at least one amino acid substitution in HCDR1, HCDR2, and / or HCDR3. The present invention relates to a method for producing a monoclonal antibody comprising: producing a variant (VL) region, the amino acid sequence of which is an LCDR1 of SEQ ID NO: 12, an LCDR2 of SEQ ID NO: 13, and an LCDR3 of SEQ ID NO: 14, or a variant thereof, the variant 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 amino acid sequence of the VH region is HCDR1 of SEQ ID NO: 6, HCDR2 of SEQ ID NO: 7, and HCDR3 of SEQ ID NO: 8, or a variant thereof, including a variant having at least one amino acid substitution in HCDR1, HCDR2, and / or HCDR3, and wherein the amino acid sequence of the VL region is LCDR1 of SEQ ID NO: 12, LCDR2 of SEQ ID NO: 13, and LCDR3 of SEQ ID NO: 14, or a variant thereof, including a variant having at least one amino acid substitution in LCDR1, LCDR2, and / or LCDR3.

[0136] Administration of the Composition

[0149] The compositions of the present 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 pharma- ceutically acceptable carrier or diluent, such as physiological saline or a buffered salt solution. Suitable carriers and diluents can be selected based on the mode and route of administration and standard pharmaceutical practice. Descriptions of exemplary pharma-ceutically acceptable carriers and diluents, as well as pharmaceutical formulations, can be found in Remington's Pharmaceutical Sciences, and USP / NF, standard textbooks in the field. Other substances can be added to the composition to stabilize and / or preserve the composition.

[0137]

[0150] The compositions of the present invention can be administered to a mammal by any conventional technique. Typically, such administration may be by inhalation or parenterally (e.g., intravenous, subcutaneous, intratumoral, intramuscular, intraperitoneal, or intrathecal introduction). The compositions may also be administered directly to the target site, for example, by surgical delivery to an internal or external target site, or by a catheter to a site accessible by blood vessels. The compositions may be administered in a single bolus, multiple injections, or by continuous infusion (e.g., intravenously, by peritoneal dialysis, pump infusion). For parenteral administration, the compositions may be formulated in a sterile, pyrogen-free form.

[0138] Effective Dose

[0151] The compositions described above are administered in an amount effective to produce the desired result in the treated mammal. The therapeutically effective amount may be administered to a mammal (e.g., rat, human) in an amount that can produce a therapeutic effect (e.g., reduce inflammation in the CNS of a mammal that has suffered a traumatic injury to the CNS or a stroke, or has an autoimmune disease, an autoinflammatory disease, a metabolic disease, a neurodegenerative disease, or a CNS disease). Such a therapeutically effective amount may be determined as described below. A therapeutically effective amount of a composition comprising an agent provided herein (e.g., a monoclonal antibody provided herein or an antibody fragment derived from the monoclonal antibody, such as IC100) can generally 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 of patient body weight. A therapeutically effective amount of a composition comprising an agent provided herein (e.g., a monoclonal antibody provided herein, such as IC100, or an antibody fragment derived from this monoclonal antibody) can generally be about 0.001 to about 200 mg / kg of patient body weight. A therapeutically effective amount of a composition comprising an agent provided herein (e.g., a monoclonal antibody provided herein, such as IC100, or an antibody fragment derived from this monoclonal antibody) can generally be about 0.001 mg / kg to about 0.01 mg / kg, about 0.01 mg / kg. The dosage may be from about 0.1 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 provided herein (e.g., a monoclonal antibody provided herein, such as IC100, or an antibody fragment derived from this monoclonal antibody) may be administered in a single or multiple doses.

[0139]

[0152] The toxicity and therapeutic efficacy of the compositions utilized in the methods of the present invention are determined by the LD50 (the dose lethal to 50% of the population) can be determined by standard pharmaceutical procedures using either cells or experimental animals. The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as the ratio LD50 / ED50. In some instances, the compositions herein exhibit a large therapeutic index. While those that exhibit toxic side effects can be used, care should be taken to design a delivery system that minimizes the potential damage of such side effects. In some instances, the dosage of the compositions herein is within a range that includes the ED50 with little or no toxicity. Dosage can vary within this range depending on the dosage form used and the route of administration utilized.

[0140]

[0153] As is well known in the medical and veterinary fields, the efficacy and safety of any one subject is Dosage will depend on many factors, including the subject's size, body surface area, age, the particular composition being administered, the time and route of administration, general health, and other drugs being co-administered. EXAMPLES

[0141] Working Example

[0154] The present invention is further described by the following specific examples, which are provided for illustrative purposes only. It is provided merely for purposes of illustration and should not be construed as limiting the scope of the invention in any way.

[0142] Example 1: The role of EV-mediated inflammasome signaling in ALI after TBI and the effect of its neutralization

[0155] Pulmonary dysfunction is a frequent complication of severe traumatic brain injury (1). Approximately 20-25 percent of TBI subjects develop acute lung injury (ALI) (2), but the mechanisms mediating the pathology of TBI-induced ALI remain poorly defined. Conventional literature supports the idea that pulmonary dysfunction after TBI results from a sympathetic response to increased intracranial pressure resulting in cardiopulmonary dysfunction (42). However, more recent studies have shown that the systemic inflammatory response also plays a key role in TBI-induced lung injury (43). Specifically, the HMGB1-RAGE ligand receptor pathway serves as a central transmitter mechanism for pulmonary dysfunction after TBI (8). Furthermore, HMGB1 induces AIM2 inflammasome activation (37). Further conventional literature reveals that pathogens secrete DAMPs, e.g., EVs carrying HMGB1, triggering inflammation (Buzas et al., 2014). Various studies have shown that the blood-brain barrier (BBB) ​​is penetrated after TBI as early as 3-6 hours after injury. It has been shown that EVs become toxic and cause damage to the protective barrier between the brain and intravascular compartments, resulting in the leakage of proteins and fluids (44). Disruption of the BBB after injury leads to the secretion of inflammatory mediators, e.g., DAMPs, which can promote brain inflammation and damage distal organs (5). Some inflammatory mediators can act as markers of clear brain injury, but their validity is not widely accepted (45). Furthermore, there are currently no clinically approved treatments or biomarkers for TBI-induced ALI. In recent years, EVs have become an area of ​​interest in biomarker research for several different types of diseases, e.g., lung injury (46) and TBI (47). In EVs isolated from the cerebrospinal fluid of patients with TBI, inflammasome proteins were significantly increased compared to control samples. It has been previously shown that EV-mediated inflammasome signaling is increased in TBI-induced ALI. (14) In this example, we examined the contribution of EV-mediated inflammasome signaling in the pathogenesis of TBI-induced ALI.

[0143] Materials and Methods Animals and traumatic brain injury

[0156] 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 Guide for the Care and Use of Laboratory Animals. The ARRIVE guidelines were followed when conducting this study. All C57 / BL6 mice were 8-12 weeks old and 24-32 grams. Mice were prospectively randomized into experimental groups for TBI (sham, 4 hr, 24), adoptive transfer and treatment (naive, sham saline, untreated, enoxaparin, anti-ASC). For the TBI experimental groups, sham animals underwent surgery but were not injured. For the adoptive transfer treatment study, the sham saline group underwent surgery and received saline as the vehicle treatment. Naive animals did not undergo surgery. A sample size of 5–6 was used for each group based on power analysis (G* power analysis, effect size F = 0.85, alpha setting 0.05) and historical data49,50. 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-h light-dark cycle and provided food and water ad libitum. The facility performed husbandry procedures twice weekly and checked the animals' condition daily. The animals were observed postoperatively, keeping them on a heating pad, body temperature controlled by a rectal probe, and body temperature maintained at 37°C in the operating room, then transferred to the animal housing room.

[0144]

[0157] Before surgery, animals were anesthetized with ketamine and xylazine (intraperitoneal, i.p.). The anesthetized animals were then placed on a heating pad to ensure a body temperature of 37°C. TBI was performed using the Controlled Cortical Impact (CCI) model. A 5 mm craniotomy was performed over the right cortex (-2.5 mm posterior and 2.0 mm lateral from bregma). An ECCI-6.3 device (Custom Design & Fabrication, Richmond, VA, USA) was inserted into the craniotomy chamber at a velocity of 6 m / s. Injury was induced using a 3 mm impounder at 100 rpm, 0.8 mm depth, and 150 ms impact duration (15). After these procedures, animals were placed in their They were returned to their cages and provided with food and water. Animals were sacrificed 4 and 24 hours after TBI as described. Sham animals were anesthetized and subjected to the same pre-operative incisions as the injured animals, but did not receive a craniotomy or contusion.

[0145] Tissue collection

[0158] All animals were anesthetized with ketamine and xylazine prior to perfusion. Animals underwent tracheal perfusion. The lungs were perfused with 4% paraformaldehyde (PFA) using a tracheal catheter in 20 cm of HO and then fixed in 4% PFA overnight at 4°C. Fixed lung tissue was paraffin embedded and 5 μm sections were processed (16). Right lung tissue was collected for protein isolation and molecular analysis. Animals then underwent decapitation and right cortical tissue was collected for protein isolation and molecular analysis.

[0146] Pyroptosome isolation assay

[0159] Mouse lung tissue lysates were stained with 5 μm low-binding polyvinylidene difluoride (PV After filtration, the supernatant was centrifuged at 2,700 × g for 8 min. The pellet was resuspended in 40 μl of 3[(3-cholamidopropyl)dimethylammonio]-propanesulfonic acid (CHAPS) buffer (20 mmol / L HEPES-KOH, pH 7.5, 5 mmol / L MgCl2, 0.5 mmol / L EGTA, 0.1 mmol / L phenylmethylsulfonyl fluoride, protease inhibitor cocktail, and 0.1% CHAPS). Pyroptosomes were diluted with 2,700× The cells were pelleted by centrifugation at 4°C for 8 min. The pellet was then resuspended in 27.8 μl of CHAPS buffer with 2.2 μl of disuccinimidyl substrate (9) and incubated at room temperature for 30 min to crosslink ASC dimers. Finally, an equal volume of 2× Laemmli buffer was added and commercial antibodies against ASC and gasdermin-D (GSD) were added. Proteins were analyzed by immunoblotting using

[0147] Nuclear and cytoplasmic extraction

[0160] Nuclear and cytoplasmic fractions were extracted using NE-PER Nuclear Cytoplasmic Extraction Reagent (Thermo Scientific) according to the manufacturer's instructions. Briefly, mouse lung tissue samples were cut into small pieces of 20-100 mg and centrifuged at 500 × g for 5 min. The tissue pieces were homogenized with cytoplasmic extraction reagent and centrifuged at 16,000 × g for 5 min. The supernatant (cell extract) was then removed and the pellet was centrifuged with nuclear extraction reagent (Thermo Scientific) for 16,000 × g. The supernatant corresponds to the nuclear fraction and was removed and stored at -80°C.

[0148] Immunoblotting

[0161] Lung and brain tissue samples were snap frozen in liquid nitrogen and stored at -80°C. Protease and phosphatase inhibitor cocktail (Sigma, St Louis, MO, USA) was included. Two-mm sections of right lower lung and right cortical tissue were homogenized in extraction buffer containing 100% ethanol and analyzed in 4–20% Tris-TGX Criterion precast gels (Bio-Rad, Hercules, CA, USA) for caspase-1 (Novus Biologicals), 100% ethanol, and 100% ethanol as described by de Rivero Vaccari et al. 2015 (13). ASC (Santa Cruz), IL-1β (Cell Signaling), IL-18 (Abcam) AIM2 (Santa Cruz) and HMGB1 (Millipore) antibodies. Quantitation of band density was performed using Image Lab and all data was normalized to β-actin.

[0149] Immunohistochemistry

[0162] Tissue sections were deparaffinized in xylene, then buffered with ethanol and Tris. The sections were rehydrated using buffered saline. Immunohistochemistry procedures were then performed as previously described (16) for double staining. Sections were stained for caspase-1 and ASC (Millipore). The mice were incubated overnight at 4° C. with antibodies against AIM2 (Santa Cruz), HMGB1 (Millipore), and SPC (Millipore). Immunostained lung sections from sham, 4-h, and 24-h mice were examined with a Zeiss laser scanning confocal microscope (Zeiss, Inc., Thornwood, NY, USA). Lung sections were analyzed by a blinded study.

[0150] EV isolation

[0163] Total exosome extraction solution was used according to the manufacturer's instructions (Invitrogen). EVs were isolated from serum from TBI-injured and injured mice using a ELISA kit. Briefly, 100 μl of each sample was centrifuged at 2000×g for 30 min. The supernatant was then incubated with 20 μl of total exosome extraction (TEI) reagent for 30 min at 4° C. and then centrifuged at 10,000×g for 10 min at room temperature. The supernatant was discarded and the pellet was resuspended in 100 μl of PBS. EVs were characterized by CD81 expression and by Nanosight tracking analysis (FIG. 6).

[0151] Adoptive transfer of EVs

[0164] Serum-derived EVs from C57BL-6TBI and sham mice were cultured in naïve C5 The IgG was injected into 7BL-6 mice via the jugular vein at a dose of 1.0 × 10 particles per gram of body weight. 48 Particle counts were measured using Nanosight tracking analysis. Samples were diluted according to the following formula: Preoperatively, 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 restrained, and a catheter was then placed. Serum-derived EVs were implanted, and lung and brain tissues were harvested 24 hours after injection for analysis (n=5).

[0152] Enoxaparin and anti-ASC treatment

[0165] Serum-derived EVs from TBI mice were transfused cervically into naïve C57-BL6 mice. EVs from TBI mice were injected via intravenous 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 groups were used: 1) naive group received no treatment, 2) sham saline group was used as a negative control and received a jugular vein injection of saline only, 3) no treatment group received EVs from TBI mice without any treatment and was used as a positive control, 4) ENOX group received EVs from TBI mice and enoxaparin, and 5) anti-ASC group received EVs from TBI mice and anti-ASC. The treatment order 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 murine, human and porcine ASC.

[0153] Histological examination and lung injury scoring

[0166] Standard hematocrit was used for histological examination, morphometry, and ALI scoring. Lung tissue sections were stained by the silanol and eosin method. Lung injury was scored by a blinded pathologist using the lung injury scoring system from the American Thoracic Society Workshop Report (17). Sections were scored. Twenty random high-power fields were selected for scoring. Criteria for ALI scoring were based on the number of neutrophils in the alveolar space, interstitial space, hyaline membranes, proteinaceous debris filling the voids, and alveolar septal thickening. Based on these criteria, a score between 0 (no damage) and 1 (severe damage) was assigned.

[0154] statistical analysis

[0167] Student's T-test for two groups and one-way test for more than two groups Data were analyzed using ANOVA followed by Tukey's multiple comparison test (GraphPad Prism version 7.0). Normality was tested using the D'Agostino-Parson test. Data are expressed as mean + / - SEM. The P value for significance used was *p<0.05.

[0155] result Severe TBI increases AIM2 inflammasome protein and HMGB1 expression in mouse brain

[0168] Proinflammatory cytokines IL-1β and IL-18, as well as inflammasomes Expression levels of inflammasome proteins are associated with secondary injury after fluid percussion brain injury (18). Cortical lysates were analyzed to determine whether severe CCI induced alterations in the processing of proinflammatory cytokines and inflammasome protein levels, but studies on inflammasome activation in severe TBI have been limited. In this example, after severe CCI, cortical lysates were examined for levels of caspase-1 (Figure 1A,B) (p<.001), ASC (Figure 1A,C) (p=.003), IL-18 (Figure 1A,D) (p=.0042), AIM2 (Figure 1A,F) (p=0.0197), and IL-1β (Figure 1A,G) (p=0.0141) at 4 and 24 hours after injury. Caspase-1, ASC, AIM2, and IL-1β levels peaked at 4 hours after CCI and decreased by 24 hours. The time course of maturation of inflammatory cytokines differed slightly, but peaked by 24 hours after TBI. Because others have shown a role for the inflammasome DAMP HMGB1 in activating the AIM2 inflammasome, the levels of those proteins were also determined in cortical lysates. As shown in Figure 1A, 1E, CCI significantly increased the levels of HMGB1 (Figure 1A, 1E) (p=0.0121) at 4 and 24 hours after injury. These data indicate that after severe CCI in mice, the levels of AIM2 inflammasome protein were significantly elevated in the post-injury cortex.

[0156] Severe TBI increases AIM2 inflammasome protein and HMGB1 expression in mouse lungs

[0169] To determine whether CCI induced inflammasome activation in the lung, To investigate the role of HMGB1 in the induction of pulmonary dysfunction, immunoblot analysis of lung lysates was performed for caspase-1 (Fig. 1H,I) (p=.0026), ASC (Fig. 1H,J) (p=.0427), IL-18 (Fig. 1H,K) (p=.0025), IL-1β (Fig. 1H,N) (p=.0012), and AIM2 (Fig. 1H,M) (p<.001), and NLRP3 (p=.0047) (Supplementary Fig. 1). Increased levels of caspase-1, ASC, IL-18, and AIM2 were significantly increased at 4 and 24 hours after injury compared to sham controls. 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. Because the HMGB1-RAGE axis plays a role in the mechanism by which TBI induces pulmonary dysfunction (8), lung lysates were analyzed for the level of HMGB1 protein expression. Figures 1H, 1L (p=0.0158) show that HMGB1 expression was increased at 4 and 24 hours after TBI, indicating that the AIM2 inflammasome and HMGB1 play a role in the inflammatory response in the lung after TBI.

[0157] TBI induces pyroptosis in mouse lungs

[0170] As previously shown, the AIM2 inflammasome in cortical neurons Activation of ASC leads to pyroptotic cell death (19). To investigate whether TBI leads to pyroptosis in mouse lung tissue, pyroptosomes in lung tissue were isolated after TBI. TBI animals sacrificed 4 hours after injury showed evidence of ASC oligomerization compared to sham animals (Figure 4A). ASC dimers and trimers were found in TBI animals (50 and 75 kDA, respectively). These results indicate pyroptosome formation, which can be characterized by supramolecular assembly of ASC oligomers. Furthermore, gasdermin-D (GSDMD), which is cleaved upon caspase-1 activation and triggers pyroptosis and release of IL-1β (20), was significantly increased in the lungs of TBI animals compared to sham (Figures 4B and 4C) (p = 0.0001). These findings indicated that pyroptosis contributes to cell death in lung tissue after TBI.

[0158] TBI increases inflammasome protein immunoreactivity in type II alveolar epithelial cells

[0171] TBI leads to capillary leakage, increased vascular permeability and the proliferation of type II pneumocytes. Inflammasome expression in the lung after injury can result in injury to specialized alveolar epithelial cells called inflammasome-associated pulmonary epithelial cells (5). To test the cellular effects of TBI on inflammasome expression in the lung after injury, immunohistochemical analysis was performed on lung sections from sham, 4 h, and 24 h injured animals. Type II alveolar epithelial cells are known to be the main type of injured lung cells 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 of type II epithelial cells, and DAPI nuclear stain (blue). As shown in Figures 2A-2C, 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 was increased after TBI. These findings indicate that inflammasome proteins are expressed in type II alveolar epithelial cells and that TBI results in increased immunoreactivity in these cells.

[0159] TBI increases nuclear and cytoplasmic HMGB1 expression

[0172] To determine the cellular distribution of HMGB1 in lung cells after TBI, lung homogenates were Nuclear and cytoplasmic fractions from sham were isolated (Figure 3A, 3C) (p=.0337). Immunoblotting showed that both fractions had a significant increase in HMGB1 expression 4 hours after TBI (Figure 3B, 3D) (p=.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) and DAPI nuclear stain (blue). HMGB1 immunoreactivity was increased at 4 hours and 24 hours compared to sham. Weak immunoreactivity of HMGB1 was observed in SPC positive cells (arrows) (Figure 3E); thus, suggesting that HMGB1 changes in injured lung tissue may be cytoplasmic.

[0160] TBI induces changes in lung morphology and induces ALI

[0173] ALI results in alveolar and interstitial edema and infiltration of inflammatory cells into the alveolar space. The lesions can be characterized by an inflammatory process that is characterized by a pulmonary edema ...

[0161]

[0174] To support that severe injury induces ALI, histological 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 alveolar and interstitial spaces, hyaline membrane formation, proteinaceous debris filling the airspace, and alveolar septal thickening (17). These features were significantly elevated in injured animals, and ALI scores were overall higher in TBI animals compared to sham (Figure 5B) (p=0.0017).

[0162] Enoxaparin and anti-ASC antibody treatment significantly reduces inflammasome expression and ALI after adoptive transfer of EVs from TBI mice

[0175] EVs and their cargo that can be released into the circulation after TBI are in the lungs. To provide evidence that flammasome activation can be induced, classical adoptive transfer experiments were performed using serum-derived EVs from severe CCI mice. EV preparations were validated using Western blots for the EV marker CD81 (Figure 6). Controls received EVs isolated from sham and naïve animals. As shown in Figures 7A-7F, active caspase-1 (Figures 7A, 7B), ASC (Figures 7A, 7C), IL-18 (Figures 7A, 7D), AIM2 (Figures 7A, 7E) and HMGB1 (Figures 7A, 7F) were significantly elevated in the lungs of animals receiving EVs from TBI injured animals compared to the lungs of non-injured or naïve mice or animals receiving EVs from naïve mice. Furthermore, infiltration of inflammatory cells (arrows) was evident in lungs treated with EVs from TBI mice (Figure 7G). Finally, ALI scores were also significantly higher in animals receiving EVs from injured mice (Figure 7G). These studies provided evidence for a neural-respiratory-inflammasome axis in which EVs released into the circulation after TBI activate inflammasomes in pulmonary target cells that contribute to the pathology of ALI.

[0163]

[0176] Next, enoxaparin after adoptive transfer of EVs from injured mice to naïve mice We attempted to block exosome uptake by treatment with either IgG4 or a monoclonal antibody against ASC (IC100). Negative control animals received saline, and positive control animals received no treatment. As shown in Figures 8A-8F, there were no significant increases in the expression of caspase-1 (Figures 8A, 8B), ASC (Figures 8A, 8C), IL-1β (Figures 8A, 8D), AIM2 (Figures 8A, 8E), and IL-1β (Figures 8A, 8F). 8E) and HMGB1 (Fig. 8A, 8F) were significantly reduced after treatment with enoxaparin or humanized monoclonal anti-ASC antibody (e.g., IC100 antibody) compared to the untreated (positive control) group (p=<.0001). Furthermore, H- and E-stained lung sections showed significantly less neutrophil infiltration into the alveolar and interstitial spaces and no signs of septal thickening (Fig. 9A-D). The ALI scores for animals treated with enoxaparin and anti-ASC antibody (IC100) were significantly lower compared to the untreated group (Fig. 9E) (p=<.0001). Thus, EVs released into the circulation after TBI play a role in inflammasome activation in lung cells resulting in ALI.

[0164] conclusion

[0177] TBI is associated with higher rates of medical complications, particularly pulmonary and central nervous system dysfunction. In this example, severe TBI was shown to increase HMGB1 and inflammasome expression (e.g., AIM2, caspase-1, and ASC expression) in cortical and lung tissues and induce changes in lung morphology consistent with ALI (e.g., neutrophil infiltration into alveolar and interstitial spaces, alveolar septal thickening, and alveolar edema and hemorrhage), introducing the idea of ​​a Neural Respiratory Inflammatory Axis. Importantly, TBI led to pyroptosis in lung tissue (e.g., in the presence of GSDMD cleavage) and increased the expression of inflammasome proteins in type II alveolar epithelial cells. Furthermore, adoptive transfer of EVs from TBI mice activated inflammasomes and induced ALI, indicating that brain injury induces the release of EVs containing the cargo of inflammasome proteins, which then leads to ALI. Furthermore, inhibition of both EV uptake (enoxaparin) and inflammasome activation (anti-ASC antibody (IC100) treatment) was shown to reduce inflammasome protein expression and the occurrence of ALI.

[0165]

[0178] In summary, this example demonstrates that AIM2 inflammasome signaling mediates TBI These data demonstrate that EV-mediated inflammasome signaling plays a central role in the pathological mechanisms of subsequent lung injury and demonstrate the mechanism of TBI-induced ALI involving EV-mediated inflammasome signaling. These data provided evidence that EV-mediated inflammasome signaling may play a central role in the neuronal-respiratory-inflammatory axis. Thus, targeting this axis with antibodies against inflammasome proteins or drugs that block EV uptake may provide a therapeutic approach in neurotrauma-induced ALI in all areas of critical care medicine. In light of these results, the disclosed therapeutic strategy may be useful for the treatment of pulmonary inflammatory diseases in general.

[0166] Incorporation by Reference

[0179] The following references are incorporated by reference in their entirety for all purposes: る。

[0180] 1. Pfeifer R, et al. (2015) Development of a standardized trauma-related lung injury model. J Surg Res 196(2):388-394.

[0181] 2. Summers CR, Ivins B, & Schwab KA (2009) Traumatic brain injury in the United States: an epidemiologic overview. The Mount Sinai journal of medicine, New York 76(2):105-110.

[0182] 3. Erickson SE, et al. (2009) Recent trends in acute lung injury mortality: 1996-2005. Crit Care Med 37(5):1574-1579.

[0183] 4. Nicolls MR & Laubach VE (2014) Traumatic brain injury: lungs in a RAGE. Sci Transl Med 6(252):252fs234.

[0184] 5. 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(4):795-803.

[0185] 6. Andersson U & Rauvala H (2011) Introduction: HMGB1 in inflammation and innate immunity. J Intern Med 270(4):296-300.

[0186] 7. Weber DJ, et al. (2014) The HMGB1-RAGE axis mediates traumatic brain injury-induced pulmonary dysfunction in lung transplantation. Sci Transl Med 6(252):252ra124.

[0187] 8. Lu B, et al. (2012) Novel role of PKR in inflammasome activation and HMGB1 release. Nature 488(7413):670-674.

[0188] 9. de Rivero Vaccari JP, Dietrich WD, & Keane RW (2014) Activation and regulation of cellular inflammasomes: gaps in our knowledge for central nervous system injury. Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism 34(3):369-375.

[0189] 10. Ware LB & Matthay MA (2000) The Acute Respiratory Distress Syndrome. New England Journal of Medicine 342(18):1334-1349.

[0190] 11. Yanez-Mo M, et al. (2015) Biological properties of extracellular vesicles and their physiological functions. J Extracell Vesicles 4:27066.

[0191] 12. Qu Y, Franchi L, Nunez G, & Dubyak GR (2007) Nonclassical IL-1 beta secretion stimulated by P2X7 receptors is dependent on inflammasome activation and correlated with exosome release in murine macrophages. J Immunol 179(3):1913-1925.

[0192] 13. de Rivero Vaccari JP, et al. (2015) Exosome-mediated inflammasome signaling after central nervous system injury. J Neurochem. Jan;136 Suppl 1:39-48. doi: 10.1111 / jnc.13036.

[0193] 14. Atkins CM, Cepero ML, Kang Y, Liebl DJ, & Dietrich WD (2013) Effects of early rolipram treatment on histopathological outcome after controlled cortical impact injury in mice. Neurosci Lett 532:1-6.

[0194] 15. Wu S, et al. (2010) Conditional overexpression of connective tissue growth factor disrupts postnatal lung development. American journal of respiratory cell and molecular biology 42(5):552-563.

[0195] 16. Matute-Bello G, et al. (2011) An official American Thoracic Society workshop report: features and measurements of experimental acute lung injury in animals. American journal of respiratory cell and molecular biology 44(5):725-738.

[0196] 17. de Rivero Vaccari JP, et al. (2009) Therapeutic neutralization of the NLRP1 inflammasome reduces the innate immune response and improves histopathology after traumatic brain injury. Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism 29(7):1251-1261.

[0197] 18. Adamczak SE, et al. (2014) Pyroptotic neuronal cell death mediated by the AIM2 inflammasome. Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism 34(4):621-629.

[0198] 19. Liu X, et al. (2016) Inflammasome-activated gasdermin D causes pyroptosis by forming membrane pores. Nature 535(7610):153-158.

[0199] 20. Dolinay T, et al. (2012) Inflammasome-regulated cytokines are critical mediators of acute lung injury. Am J Respir Crit Care Med 185(11):1225-1234.

[0200] 21. Muller MC, et al. (2014) Contribution of damage-associated molecular patterns to transfusion-related acute lung injury in cardiac surgery. Blood transfusion = Trasfusione del sangue 12(3):368-375.

[0201] 22. Ragaller M & Richter T (2010) Acute lung injury and acute respira tory distress syndrome. Journal of emergencies, trauma, and shock 3(1):43-51.

[0202] 23. Lee K & Rincon F (2012) Pulmonary complications in patients with severe brain injury. Critical care research and practice 2012:207247.

[0203] 24. Yasui H, Donahue DL, Walsh M, Castellino FJ, & Ploplis VA (2016) Early coagulation events induce acute lung injury in a rat model of blunt traumatic brain injury. American journal of physiology. Lung cellular and molecular physiology 311(1):L74-86.

[0204] 25. Hendrickson CM, et al. (2016) The acute respiratory distress syndrome following isolated severe traumatic brain injury. J Trauma Acute Care Surg.

[0205] 26. Cross LJ & Matthay MA (2011) Biomarkers in acute lung injury: insights into the pathogenesis of acute lung injury. Crit Care Clin 27(2):355-377.

[0206] 27. Butt Y, Kurdowska A, & Allen TC (2016) Acute Lung Injury: A Clinical and Molecular Review. Archives of pathology & laboratory medicine 140(4):345-350.

[0207] 28. Luh SP & Chiang CH (2007) Acute lung injury / acute respiratory distress syndrome (ALI / ARDS): the mechanism, present strategies and future perspectives of therapies. Journal of Zhejiang University. Science. B 8(1):60-69.

[0208] 29. Matute-Bello G & Martin TR (2003) Science review: apoptosis in acute lung injury. Critical care 7(5):355-358.

[0209] 30. Miao EA, Rajan JV, & Aderem A (2011) Caspase-1-induced pyroptotic cell death. Immunological reviews 243(1):206-214.

[0210] 31. Hornung V, et al. (2009) AIM2 recognizes cytosolic dsDNA and forms a caspase-1-activating inflammasome with ASC. Nature 458(7237):514-518.

[0211] 32. Lam NY, Rainer TH, Chan LY, Joynt GM, & Lo YM (2003) Time course of early and late changes in plasma DNA in trauma patients. Clinical chemistry 49(8):1286-1291.

[0212] 33. Fernandes-Alnemri T & Alnemri ES (2008) Assembly, purification, and assay of the activity of the ASC pyroptosome. Methods Enzymol 442:251-270.

[0213] 34. Man SM & Kanneganti TD (2016) Converging roles of caspases in inflammasome activation, cell death and innate immunity. Nature reviews. Immunology 16(1):7-21.

[0214] 35. Liu L, et al. (2014) HMGB1-DNA complex-induced autophagy limits AIM2 inflammasome activation through RAGE. Biochem Biophys Res Commun 450(1):851-856.

[0215] 36. Hoesch RE, et al. (2012) Acute lung injury in critical neurological illness. Critical care medicine 40(2):587-593.

[0216] 37. Kalsotra A, Zhao J, Anakk S, Dash PK, & Strobel HW (2007) Brain trauma leads to enhanced lung inflammation and injury: evidence for role of P4504Fs in resolution. Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism 27(5):963-974.

[0217] 38. Hay (2015) Blood-Brain Barrier Disruption Is an Early Event That May Persist for Many Years After Traumatic Brain Injury in Humans. J Neuropathol Exp Neurol 74(12):1147-1157.

[0218] 39. Zygun DA, Kortbeek JB, Fick GH, Laupland KB, & Doig CJ (2005) Non-neurologic organ dysfunction in severe traumatic brain injury. Critical care medicine 33(3):654-660.

[0219] 40. Peltz ED ME, Eckels PC, Damle SS, Tsuruta Y, Johnson JL, Sauaia A, Silliman CC, Banerjee A, Abraham E. (209) HMGB1 is markedly elevated within 6 hours of mechanical trauma in humans. Shock 32(1):17-22.

[0220] 41. Chi W, et al. (2015) HMGB1 promotes the activation of NLRP3 and caspase-8 inflammasomes via NF-kappaB pathway in acute glaucoma. Journal of neuroinflammation 12:137.

[0221] 42. Woodcock T & Morganti-Kossmann MC (2013) The role of markers of inflammation in traumatic brain injury. Frontiers in neurology 4:18.

[0222] 43. Monsel A, Zhu YG, Gudapati V, Lim H, & Lee JW (2016) Mesenchymal stem cell derived secretome and extracellular vesicles for acute lung injury and other inflammatory lung diseases. Expert opinion on biological therapy 16(7):859-871.

[0223] 44. Taylor DD & Gercel-Taylor C (2014) Exosome platform for diagnosis and monitoring of traumatic brain injury. Philosophical transactions of the Royal Society of London. Series B, Biological sciences 369(1652).

[0224] 45. Guo H, Callaway JB, & Ting JP (2015) Inflammasomes: mechanism of action, role in disease, and therapeutics. Nature medicine 21(7):677-687.

[0225] 46. Silverman WR, et al. (2009) The pannexin 1 channel activates the inflammasome in neurons and astrocytes. The Journal of biological chemistry 284(27):18143-18151.

[0226] 47. Tomura S, de Rivero Vaccari JP, Keane RW, Bramlett HM, & Dietrich WD (2012) Effects of therapeutic hypothermia on inflammasome signaling after traumatic brain injury. Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism 32(10):1939-1947.

[0227] 48. Wiklander, O.P., Nordin, J.Z., O’Loughlin, A., Gustafsson, Y., Corso, G., Mager, I., Vader, P., Lee, Y., Sork, H., Seow, Y., Heldring, N., Alvarez-Erviti, L., Smith, C.I., Le Blanc, K., Macchiarini, P., Jungebluth, P., Wood, MJ and Andaloussi, SE (2015). Extracellular vesicle in vivo biodistribution is determined by cell source, route of administration and targeting. J Extracell Vesicles 4, 26316.

[0228] 49. de Rivero Vaccari, JP, Lotocki, G., Marcillo, AE, Dietrich, WD and Keane, RW (2008). A molecular platform in neurons regulates inflammation after spinal cord injury. J Neurosci 28, 3404-3414.

[0229] 50. Assis-Nascimento, P., Umland, O., Cepero, ML and Liebl, DJ (2016). A flow cytometric approach to analyzing mature and progenitor endothelial cells following traumatic brain injury. J Neurosci Methods 263, 57-67.

[0167] Example 2: Role of EV-mediated inflammasome signaling in ALI after TBI in human patients

[0230] As a follow-up to the mouse experiments in Example 1, human lung We examined the role of EVs isolated from human TBI patients on inflammasome signaling in epithelial cells.

[0168]

[0231] In the first experiment, a total exosome extraction kit (Thermofisher) was used. Serum-derived EVs were isolated from TBI and control patients using the method described above. Lung human microvascular endothelial cells (HMVEC-Lonza) were cultured and plated on 12-well plates at confluency. After reaching a total cell viability of 1.94×108 particles / ml, isolated EVs from TBI and control patients were delivered to cells for a 4-h incubation period. After incubation, cells were harvested with 200 ul of lysis buffer and cell lysates were used for Western blot analysis.

[0169]

[0232] In the second experiment, a total exosome extraction kit (Thermofisher) was used. We isolated serum-derived EVs from TBI and control patients using human pulmonary microvascular endothelial cells (HMVEs). C-Lonza) and plated on a 96-well plate. After reaching a pH of 10.0, isolated EVs from TBI and control patients were delivered to cells (1.94 × 108 particles / ml) for a 3-h incubation period and then incubated with Caspase-1 FAM FLICA (Immunohistochemistry Technologies) at a 1:30 volume / volume ratio for an additional 1 h. After incubation, the medium was removed and cells were washed three times with Apoptosis Wash Buffer (Immunohistochemistry Technologies). Cells were then analyzed for nuclear staining. The cells were co-stained with Hoechst for cytotoxicity and propidium iodide for cell death. Images were taken using an EVOS microscope, and the cells were then read under a fluorescent plate reader at an excitation wavelength of 492 nm and an emission wavelength of 520 nm.

[0170] result

[0233] As shown in Figures 10A-10F, delivery of serum-derived EVs from TBI patients TBI-EVs increased inflammasome protein expression in pulmonary endothelial cells. Figures 10A-10E 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 showed a significant increase in IL-1beta expression using the Ella simpleplex assay (Figure 10F).

[0171]

[0234] As shown in Figures 11A to 11C, TBI-EVs were translocated into pulmonary endothelial cells. Delivery increased caspase-1 immunoreactivity and cell death.

[0172] conclusion

[0235] These studies suggest that EVs released into the circulation after TBI contribute to the pathology of ALI. We provide further evidence for a neuronal-respiratory-inflammasome axis that activates inflammasomes in pulmonary target cells that mediate inflammation.

[0173] Example 3: Effect of the use of humanized anti-ASC antibodies in an animal model of multiple sclerosis

[0236] To determine the utility of humanized anti-ASC monoclonal antibodies in the treatment of MS The antibody was then administered to mice with experimental allergic encephalomyelitis (EAE). EAE is a condition that has been reported in Hoeftberger R, Leisser M, Bauer J, Lassmann H (Dec 2015). “Autoimmune encephalitis in humans: how closely does it reflect multiple sclerosis?”. Acta Neuropathol Commun. 3 (1): 80, and Lassman Hans (Feb 2010). “Acute disseminated encephalomyelitis and multiple sclerosis”. Brain. 133: 317-319, and L. Gomez Vicente et al. Relapse in a paucisymptomatic form of multiple sclerosis in a patient treated with nivolumab, Neuro Oncol (2016) 18 (suppl 4): iv25, in an animal (i.e., rodent) model of MS.

[0174] method Induction of EAE and treatment with IC100

[0237] Active EAE was determined as previously described ( Brambilla et al., 2014 ). EAE was induced in 2-month-old C57BL / 6 female mice with myelin oligodendrocyte glycoprotein 35-55 peptide (MOG35-55, BioSynthesis). Briefly, mice were injected intraperitoneally (ip) with pertussis toxin dissolved in PBS (350 ng / mouse; day 0), followed by subcutaneous administration of MOG35-55 emulsified in complete Freund's adjuvant (300 ng / mouse; day 1), and pertussis toxin was injected ip again (350 ng / mouse; day 2). Mice were administered vehicle (0.9% saline) or IC100 at three different doses (10, 30, and 45 mg / kg) by ip injection every 4 days, starting on day 8 after induction of EAE. 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 paralysis of hind limbs; 4, complete paralysis of forelimbs; 5, moribund; and 6, death.

[0175] Cell isolation for flow cytometry

[0238] After transcardial perfusion with PBS, the spinal cord was harvested and purified with Mg2+ and Ca2+. The samples were manually dissociated into a single cell suspension by passing through a 70um strainer and washed with HBSS w / o. Spleen samples were spun at 1200 rpm for 10 minutes at 4°C, the supernatant removed and red blood cells (RBCs) lysed in 2 ml of RBC lysis buffer (eBioscience) according to the manufacturer's instructions. The spleen cells were then resuspended in PBS. Cells isolated from the spinal cord were resuspended in flow cytometry buffer (FCB, eBioscience) and incubated with Myelin Removal Beads II (Miltenyi). Myelin was depleted using an LS magnetic column as described in the manufacturer's protocol (Miltenyi). Similar to the splenocytes, the spinal cord cells were resuspended in PBS and stained as described below.

[0176] Immunolabeling and flow cytometric analysis

[0239] In experiments evaluating caspase-1, the FAM FLICA™ Caspase 1 kit was used according to the manufacturer's instructions (BioRad). Cells were incubated in FLICA solution (BioRad) for 30 minutes at 4° C., washed with Apoptosis Wash Buffer (BioRad), and then incubated for 30 minutes at 4° C. for 30 minutes. The samples were then incubated with a fixative live / dead stain (Tonbo Biosciences) for 30 min at 4° C. and incubated with PBS for 10 min at 4° C. Spin at 1200 rpm and remove supernatant. Cells were resuspended in 100 ul of FACS buffer and blocked with anti-CD16 / 32 (FcR block, eBioscience) for 5 minutes at room temperature. The cells were locked, immunostained for 30 min at 4°C, and fixed with 1% PFA. Samples were analyzed on a CytoFLEX S flow cytometer (Beckman Coulter) equipped with CytExpert 2.1 software. Spinal cord leukocyte counts were determined with 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). A list of antibodies for flow cytometry is provided in Table 3 below.

[0177]

[0240] [Table 11]

[0178]

[0241] Luxol fast blue staining and quantification of demyelinated white matter volume

[0242] Embedding paraformaldehyde (PFA)-fixed segments of spinal cord in paraffin The tissue was then cut into 10 mm-thick sections using a Leica RM 2135 microtome and then transfected with a Luxol Fast Batch Microtome. The sections were stained with Leu (LFB). Ten serial sections at 50 um intervals were used to estimate the demyelinated white matter volume. The demyelinated areas were outlined with an Olympus BX51 microscope, and the demyelinated white matter volume was quantified with Stereoinvestigator software (MicroBrightfield). 3D reconstruction of the demyelinated spinal cord was performed on the same serial sections with Neurolucida software (MBF Bioscience).

[0179] Quantification of IC100 in tissues

[0243] IC100 was quantified in brain, spinal cord, liver, and spleen at 35 days after induction of EAE (dpi) 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).

[0180]

[0244] To determine whether anti-ASC penetrates spinal cord neurons, we performed crush cervical spinal cord injury. Similar experiments were performed in a rat model of spinal cord injury.

[0181]

[0245] To determine whether IC100 was taken up by cells, fluorescein was used. Polyamine-labeled IC100 was added to tissue culture medium containing THP-1 cells (a human monocytic cell line).

[0182] result Treatment with anti-ASC antibody IC100 ameliorates functional outcomes in experimental autoimmune encephalomyelitis (EAE)

[0246] To evaluate the therapeutic potential of IC100, 2-month-old female C57BL / 6 mice were Mice were induced with EAE with MOG35-55 peptide (Brambilla et al., 2014) and administered IC100 or vehicle alone starting 8 days after induction of the disease (dpi). The treatment was repeated every 4 days until sacrifice, which was set at 35 dpi. Three doses were tested: 10, 30, and 45 mg / kg.

[0183]

[0247] The IC100 was 4.28 mg / kg when used at doses of 30 and 45 mg / kg. Treatment significantly improved functional recovery with a robust reduction in clinical disease scores throughout the duration of the study (Figure 12A). Treatment reduced the mean peak clinical scores (Figure 12B) and the overall severity of EAE measured as a reduction in the cumulative disease index (CDI) (Figure 12C). Mice treated with 30 and 45 mg / Kg IC100 also showed a trend toward delayed disease onset (Figure 12D). No significant differences were observed in the days when the mice reached their peak disease scores (Figure 12E).

[0184] Treatment with anti-ASC antibody IC100 reduces infiltration of peripheral immune cells into the spinal cord after EAE

[0248] The onset, duration, and severity of clinical symptoms of EAE are determined by immune cell infiltration into the spinal cord. directly correlated. To assess whether IC100 influenced this process, immune cell populations isolated from spinal cord at 35 dpi were characterized by flow cytometry. Treatment with 30 mg / Kg IC100 significantly reduced the total number of encephalitogenic CD4+ and CD8+ T cells, the immune cell populations most important in driving EAE pathology (Figure 13A). All other immune cell populations showed a clear trend towards reduction. In the spleen, no significant differences in cell numbers were observed at either dose of IC100, suggesting that treatment did not impede the ability of mice to mount a sufficient immune response to EAE challenge (Figure 13B).

[0185] Treatment with anti-ASC antibody IC100 reduces the number and activation state of microglia after EAE

[0249] Microglia are involved in the immune-inflammatory response to CNS diseases. Upon increasing activation state, microglia proliferate and upregulate surface expression of MHCII. To assess whether IC100 influenced this response, the number of total microglia and MHCII+ activated microglia in the spinal cord was quantified by flow cytometry. Both populations were significantly reduced by treatment with 30mg / Kg IC100, indicating that at this dose, IC100 is effective in suppressing microglial activation and microglial-mediated neuroinflammation (see FIG. 14).

[0186] IC100 penetrates the brain and spinal cord

[0250] A key parameter in the design of a drug to reverse MS is whether the drug is curative or not. The aim of the present invention is to determine whether the therapeutic level of IL-16 can penetrate the CNS, which is an important feature in the treatment of progressive MS, in particular, because the blood-brain barrier appears to be relatively intact at this stage of the disease (Lassman et al. 2012). They harvested the brain, spinal cord, liver, and spleen and determined the levels of IC100 in these tissues. As shown in Figure 15, IC100 penetrated all these tissues, including the brain and spinal cord, at all three doses. Interestingly, the level of IC100 in the spinal cord was highest at the 30 mg / kg dose, consistent with a greater therapeutic effect at this dose.

[0187]

[0251] When fluorescein-labeled IC100 was added to tissue culture medium, T It is taken up by HP-1 cells (a human monocytic cell line) and incorporated into ASC specks. In addition, inflammasome induction in these cells stimulates the uptake of labeled IC100 into ASC specks together with rhodamine-labeled dextran, suggesting that uptake of IC100 is mediated by endocytosis (see FIG. 16).

[0188]

[0252] Similarly, IC100 prevented IL-1β release from THP-1 cells (Figure (See 17.)

[0189]

[0253] Anti-ASC antibodies (e.g., IC100) were shown to be effective in rat models of crush cervical spinal cord injury. also penetrated spinal cord neurons.

[0190]

[0254] IC100 can function both intracellularly and extracellularly. IC100 may function by binding to and inhibiting the ASC protein, thus preventing the assembly of the multiprotein inflammasome and the initiation of the inflammatory response. IC100 may also bind to the ASC of the ASC speck, preventing the propagation of the large filamentous signaling platform, thereby inhibiting the extracellular activation of pro-ILβ, which is involved in the perpetuation of inflammation in chronic inflammatory diseases.

[0191] Incorporation by Reference

[0255] The following references are incorporated by reference in their entirety for all purposes: do.

[0192]

[0256] 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.

[0193]

[0257] Lassmann, H., van Horssen, J. & Mahad, D. Progressive multiple sclerosis: pathology and pathogenesis. Nat Rev Neurol 8, 647-656, doi:10.1038 / nrneurol.2012.168 (2012).

[0194] Example 4: Kinetic analysis of candidate anti-ASC monoclonal antibodies

[0258] Biolayer Interferometry (BLI) was used to characterize the dynamics of candidate anti-ASC monoclonal antibodies. In BLI, association and dissociation from a surface causes a shift in the wavelength of reflected light, and by measuring this shift over time, binding kinetics can be determined.

[0195]

[0259] The BLI assay consisted of:

[0260] Sensor check (30 seconds) → Load Ab / Supnt. (700 seconds) → Base Baseline (300 sec) → Ab Assoc. (600 sec) → Dissoc. (600 sec) → Repeat.

[0196]

[0261] Candidate mouse IgG antibody supernatants were incubated at seven different concentrations (i.e., 540 nM; The antibodies were tested for binding to human ASC peptide (SEQ ID NO:5) at ICCN 1.0H (i.e. IC100); ICCN 2.0H; and ICCN 3.0H. An AMC (anti-mouse IgG Fc) biosensor was loaded with mouse IgG from undiluted supernatant. The raw antibody kinetic data for these three candidate antibodies is shown in Figure 21 and the global KD values ​​are shown in Figure 22.

[0197] Example 5: Absorption, distribution, metabolism, and excretion study of IC100 Pharmacokinetic study

[0262] To explain the pharmacokinetics of IC100 in male CD-1 rats, absorption, distribution , metabolism, and excretion (ADME) experiments are carried out.

[0198]

[0263] In the first experiment, 30 6-week-old CD-1 male rats will be obtained from Charles River. Experiments will be performed at Bolder BioPath (BBP). Mice will be kept at least 1 year after arrival at BBP. Allow 7 days for acclimation. Mice are housed 4 animals per cage.

[0199]

[0264] Animals were randomized into treatment groups (9 mice per group) based on body weight. The subjects are divided into groups and given a dose of IC100 intravenously (IV). Treatment groups are given either 5 mg / kg, 15 mg / kg, or 30 mg / kg of IC100. Plasma is collected at various times after the single IV dose for pharmacokinetic (PK) monitoring. An exemplary timetable for plasma collection is shown in Table 4 below. Acute toxicity is monitored by clinical observations. Plasma is sent to Antibody Solutions for analysis. The study will continue for 10 weeks.

[0200]

[0265] Body weight measurements are taken on days 0, 7, 14, 21, 28, and 35.

[0201]

[0266] Retro-orbital bleeding was employed to collect sufficient blood, and the mice were then Animals 1, 2, and 3 of each group are bled on days 1 and 10, animals 4, 5, and 6 are bled on days 2 and 15, and animals 7, 8, and 9 are bled on days 5 and 20.

[0202]

[0267] At the final time point, animals were anesthetized with isoflurane and exsanguinated, followed by bilateral pneumothorax. Animals 1, 2, and 3 are sacrificed on day 25. Animals 4, 5, and 6 are sacrificed on day 30. Animals 7, 8, and 9 are sacrificed on day 35.

[0203] [Table 12]

[0204] Example 6. Biodistribution of IC100 in Female B6 Albino Mice and Fluorescence Imaging

[0268] In a second experiment, IC100 and control mouse IgG are labeled with VivoTag 680XL fluorescent labeling dye according to the established VivoTag protocol to determine binding affinity. Briefly, according to the VivoTag protocol, the labeling protocol involves the following:

[0205]

[0269] 1. Prepare antibody (>7 kDa) solution at 1-10 mg / mL in PBS The antibodies do not contain ammonium ions or primary amines to reduce competition for reaction with reactive dyes.

[0206]

[0270] 2. Dissolve 0.25 mg of VivoTag 680XL in 10 μL of dry DMSO. Upon reconstitution, VivoTag 680XL will retain up to It is stable for 7 days.

[0207]

[0271] 3. 0.5 mL (0.5-5 mg) protein, 50 μl sodium bicarbonate Add 2 μL of VivoTag 680XL per mg of protein into an Eppendorf tube. The mixture is incubated in the dark with shaking at room temperature for 2 hours.

[0208]

[0272] 4. Separating the protein conjugate from the free dye. Twist the strands and loosen the cap. Place the column over a 15 mL conical collection tube and centrifuge at 1,000 x g for 2 minutes. Add 2 mL of PBS to the column and centrifuge at 1,000 x g for 2 minutes. Repeat this wash two more times.

[0209]

[0273] 5. Place the column onto a new 15mL conical collection tube. Load the entire protein sample (200-700 µL) onto this column and centrifuge at 1,000 x g for 2 min. Collect the flow-through protein sample.

[0210]

[0274] 6. Collected labeled antibody samples can be analyzed for degree of labeling (DOL). The absorbance of the purified conjugate is determined at 80 nm and 668 nm.

[0211]

[0275] 7. Adjust the absorbance at 280 nm of the purified protein by subtracting the absorbance at 280 nm of the VivoTag 680XL, which is 16% of the absorbance at 668 nm.

[0212]

[0276] 8. Absorbance analysis was performed using either a UV spectrophotometer or a Nanodrop spectrophotometer. This can be done by either: To use the latter, samples should be diluted to the 0.5-2 mg / mL range before measurement; the light path is 1 mm, so readings should be normalized by a factor of 10.

[0213]

[0277] In the third experiment, the biodistribution of IC100 was determined. This study included 15 Utilize 8-12 week old female B6 Albino (C57BL6) mice. Randomly divide the animals into groups based on their body weight on day 1.

[0214]

[0278] Female B6 Albino mice were either untreated (negative control), administered a single dose of IC100 labeled with VivoTag 680XL, or administered a single dose of IC100 labeled with VivoTag 680XL. A single dose of IgG is administered (negative control). Treatments are administered intravenously (volume = 200 μL) at a dose of 100 μg / animal.

[0215]

[0279] In vivo fluorescence imaging was performed for 2, 8, 24, 48, 72, and Treatment is performed at 10-96 hours and 96 hours, with dorsal and ventral in vivo whole-body images captured using fluorescence imaging at various time intervals up to 96 hours after treatment. Ex vivo imaging is performed of the brain, eye including optic nerve, heart, left and right kidneys, large intestine (including terminal colon), liver, lungs, ovaries, pancreas, small intestine, spine, stomach, thyroid, and bladder from all animals.

[0216]

[0280] Whole blood was collected and the immune infiltrate was analyzed using CD4+ T cells (CD4+CD11b-CD Ab-VivoTag 680 is analyzed for CD8+ T cells (CD8+CD11b-CD3+CD4-), B cells (CD3-CD111b-CD45R+), monocytes (CD3-CD11b+CD115+), and NK cells (CD3-CD49b+CD335+). Expression of XL is quantified to determine the level of labeled antibody delivered. The number of viable cells is quantified. The flow cytometry panel includes a live / dead dye to detect CD3, CD4, CD8, CD11b, CD115, CD45R, CD49b, CD335, and a live / dead dye.

[0217] Example 7: Effect of IC100 Administration on Inflammasome Signaling

[0281] Nonalcoholic steatohepatitis (NASH), diabetic nephropathy, and lupus nephritis of human patient blood is obtained from BioReclamation IVT for biomarker analysis of inflammasome proteins. Tissues from patients with NASH, diabetic nephropathy, and lupus nephritis are obtained from Bolder BioPath, and protein lysates are obtained from these tissues and analyzed by immunoblotting and other biochemical techniques for expression of inflammasome signaling proteins such as caspase-1 and ASC.

[0218]

[0282] In addition, human cancer cell lines were used to investigate ASC-dependent inflammasome activity. The study will examine the effects of serotonin on cellular metabolism in real time. The study has two objectives and will begin over a 6-9 week period.

[0219]

[0283] Specific purpose 1: Antibody labeling

[0284] 2 mg of IC100 is labeled with IgG-680 XL-IFC (VivoTag® 680 XL, PerkinElmer #-NEV11120) fluorescent labeling dye and the stoichiometry of labeling is determined according to the manufacturer's instructions. Control Mouse IgG (provided by Charles River Laboratories (CRL)) are similarly labeled and analyzed.

[0220]

[0285] Specific objective 2: Determination of binding affinity

[0286] The human cancer cell line THP-1 was cultured in logarithmic phase in white polystyrene 6-well plates. Microculture plate (Corning® Costar® 96-well flat-bottom plate) Cells are plated at 20,000 cells per well in a volume of 100 μL of medium (Cat. No. 3917). Labeled antibody is then added to the wells in duplicate (10-point dose response, 1:3 dilution, top concentration 200 nM). Bound antibody is analyzed using a Charles River protocol. The bound antibody (mean fluorescence intensity) is plotted as a function of antibody concentration, and the binding affinity (Kd) is estimated by fitting the following equation to the data: Y=Bmax*X / (Kd+X).

[0221] Example 8: In vivo study of IC100 in NASH rat model

[0287] Those with liver fibrosis who are fed a choline-deficient high-fat diet (CDHFD) Male Wistar Han rats served as an animal model of NASH. Fifty-five mice were utilized in this study. Eight to nine week old Wistar Han rats were cultured in Envigo or Charles River The rats are obtained from Bolder Biopath, Inc. Rats are allowed to acclimate for 3-7 days upon arrival at Bolder Biopath. Rats are housed 2-3 animals per cage. Animals are fed a standard chow diet.

[0222]

[0288] On day 0, animals were randomly divided into 5 groups based on body weight: Group 1 contained: They were fed the standard diet, Teklad Global Diets - Rodent 2014. Groups 2, 3, 4 and and 5 are fed the CDHFD diet.

[0223]

[0289] On study day 38, animals were bled for clinical chemistry and alanine aminotransferase was administered. Mice are divided into treatment groups based on ALT levels. Treatment begins on day 42 of the study. Efficacy of IC100 is tested using doses determined based on pharmacokinetic data from Example 5. One group is treated with vehicle to serve as a negative control. A group receiving standard chow is also treated with vehicle. Body weight, food consumption, and cage side clinical observations are measured weekly. Whole blood is obtained by tail vein sampling on days 38 and 63. Necropsy is performed on day 84. Animals are sacrificed by isoflurane anesthesia and bled to exsanguination and subsequent bilateral pneumothorax.

[0224]

[0290] In this study, -1, 0, 2, 4, 6, 7, 14, 21, 28, 35, 42, 4 On days 5, 49, 52, 56, 59, 63, 66, 70, 73, 77, 80, and 83, the animals are weighed.

[0225]

[0291] 0, 7, 14, 21, 28, 35, 42, 49, 56, 63, 7 of this study Weekly updates of food intake (grams / day / rat) are recorded on days 0, 77, and 84.

[0226]

[0292] 0~7, 14, 21, 28, 35, 42, 45, 49, 52, 56, 59, Cageside clinical observations are performed on days 63, 66, 70, 73, 77, 80, and 83. Animals are observed and weighed daily if they begin to show clinical signs of toxicity or disease.

[0227]

[0293] At necropsy, the weights of the liver, brown adipose tissue, and right inguinal adipose tissue were measured. A 4 x 7 mm biopsy of the left lateral lobe of the liver is obtained, frozen in liquid nitrogen, and stored at -80°C. 3 mm transverse sections of the median, left, and right lateral lobes of the liver are obtained and fixed in 10% formalin for 36-48 hours and then stored in 70% ethanol at room temperature for histopathology. Three 100 mg pieces of adipose tissue are snap frozen in liquid nitrogen and stored in Eppendorf safe-lock tubes at -80°C. Three equal sized pieces of brown adipose tissue are snap frozen in liquid nitrogen and stored in Eppendorf safe-lock tubes at -80°C.

[0228]

[0294] White adipose tissue depot (WAT) was prepared from inguinal subcutaneous adipose tissue using the following protocol. The mice were degloved in the lower body and the inguinal The triangular SQ depot becomes apparent. Hold the upper limbs and chest with one hand and pull the skin down towards the feet with the other. Turn the mouse into a supine position, taking care not to contaminate the exposed depot with hair. Clean surgical instruments and change gloves. Then dissect out a triangle of subcutaneous fat, taking care not to contaminate the sample with muscle, adjacent fat, mammary gland, or blood. If the borders are not clearly defined, utilize a dissecting microscope. Remove the fat depot and transfer it to 10% neutral buffered formal at 50:1 fixative to tissue volume and fix for 36-48 hours at room temperature. 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.

[0229]

[0295] Histological processing is performed by Histotox Labs. Histological processing of LLL, MLL, and RLL is performed on 3 liver sections / animal. Samples are stained with Sirius Red and Hematoxylin and Eosin (H&E).

[0230] Example 9: In vivo study of the effects of IC100 on diabetic nephropathy in the BTBR Ob / Ob mouse model

[0296] The mouse strain BTBR, which has an ob / ob leptin deficiency mutation, is a model for diabetic nephropathy. Serving as a mouse model, male BTBR Ob / Ob mice are used to evaluate the effect of IC100 in reversing the effects of diabetic nephropathy. Five male wild type (WT) BTBR mice are used as negative controls and are not treated. Fifty BTBR Ob / Ob mice are used and divided into five groups. These groups are administered either vehicle, control, or IC100 at doses determined according to Example 5. The experiment is carried out over a period of six weeks.

[0231]

[0297] Histological examination is performed on mouse kidneys by Histotox Labs. Appropriate parameters are determined by a pathologist using current Bolder BioPATH methods.

[0232]

[0298] Blood glucose levels are measured by snipping the tail and depositing blood (approximately 5 μL) onto a test strip that fits the True Metrix blood glucose meter. Blood glucose levels are measured twice weekly until the completion of the study.

[0233]

[0299] Proteinuria was scored by holding the mouse upside down and applying abdominal pressure. This is performed by expressing urine from the mice by means of Albustix reagent paper and the amount of protein in the urine is determined.

[0234]

[0300] If an animal is found dead, do not take a sample. Euthanize the animal. If necessary, for any reason, samples will be taken at necropsy on study day 7.

[0235] Example 10: In vivo study of the effect of IC100 on lupus nephritis in a mouse model

[0301] Using 12-week-old female MRL / MpJ-Tnfrsf6lpr / J mice, To develop a model of lupus nephritis, mice are obtained from Bolder BioPath. Mice are randomly divided into treatment groups based on body weight. Animals are observed daily for noticeable clinical signs, moribundity, and mortality. The onset of lupus nephritis occurs at approximately 12-14 weeks of age.

[0236]

[0302] After the onset of lupus nephritis, the mice were divided into five groups according to the results of Example 5. Treatment will be with either various doses of IC100, vehicle, or control IgG.

[0237]

[0303] Body weight, urine protein, lymphadenopathy score, and skin lesion score are collected. Necropsies will be performed at week 20 to collect tissues and whole blood for analysis. The study is expected to last 15 weeks.

[0238] Example 11: Acute and 21-day range-finding IC100 intravenous bolus injection toxicity studies in Albino rats

[0304] Albino rats are obtained from Charles River. IC100 dose levels are established for a defined 28-day multiple-dose toxicity and pharmacokinetic study in rats. Rats are divided into four groups containing three rats / sex / dose. An up and down study is performed.

[0239]

[0305] The repeated dose study was conducted over a period of 3 weeks. All rats were divided into 5 groups. Rats are divided into either male or all-female groups. Rats are dosed weekly with a single dose level of IC100. Clinical examinations are performed for survival parameters such as mortality, clinical signs, body weight, and toxicokinetics.

[0240] Example 12: Maximum Tolerated Dose of IC100 and 22-Day Dose-Range Finding Study in Cynomolgus Monkeys

[0306] Dose levels and pharmacokinetics of IC100 are established in cynomolgus monkeys. Experiments are performed at Charles River. Monkeys are divided into 4 groups, including 1 monkey / sex / dose. An up-and-down study is performed over 28 days.

[0241]

[0307] A repeated dose study was conducted over a 3-week period. The monkeys were all males, including two monkeys. Monkeys are dosed weekly with a single dose level of IC100. Clinical examinations are performed with survival parameters including mortality, clinical signs, body weight, and toxicokinetics.

[0242]

[0308] The experiment is expected to last 15 weeks.

[0243] Example 13: Toxicity study of IC100 in rats followed by a 4-week recovery period

[0309] Toxicity and toxicokinetics following intravenous administration of IC100 in rats obtained from Charles River and a subsequent recovery period are established.

[0244]

[0310] Rats were grouped into 10-15 rats / sex / group, including three dose levels and controls. Additional groups include a high dose group and a control group containing 5 rats / sex for 4 weeks recovery.

[0245]

[0311] Mortality, body weight, food consumption, clinical observations, clinical pathology (hematology and clinical chemistry), necropsy Observations, organ histopathology, and toxicokinetics are measured. After recovery, the same parameters are measured except for toxicokinetics.

[0246]

[0312] The experiment is expected to last six months.

[0247] Example 14: Toxicity Study of IC100 in Cynomolgus Monkeys Followed by a 4-Week Recovery Period

[0313] Toxicity after intravenous administration of IC100 and a subsequent recovery period in non-human primates. and toxicokinetics will be established at Charles River.

[0248]

[0314] The monkeys are divided into groups of 3 monkeys / sex / group containing 3 dose levels and controls. Additional groups include a high dose group and a control group containing 2 monkeys / sex for 4 weeks of recovery.

[0249]

[0315] Mortality, body weight, food consumption, clinical observations, clinical pathology (hematology and clinical chemistry), necropsy Observations, organ histopathology, and toxicokinetics are measured. After recovery, the same parameters are measured except for toxicokinetics.

[0250]

[0316] The experiment is expected to last six months.

[0251] Example 15: In vitro cardiovascular studies using the hERG assay

[0317] The possibility of cardiovascular toxicity (QT prolongation) was examined using CHO cells or HEK293 cells. The effect is evaluated in an in vitro assay.

[0252]

[0318] This experiment established the IC50 for HERG channel blockade of IC100. It will be established.

[0253]

[0319] The experiment is expected to last two months.

[0254] Example 16: In vitro blood hemolysis studies

[0320] The potential of an intravenous preparation of IC100 to induce hemolysis of human red blood cells in vitro The activity of the antibody is evaluated. The concentration of IC100 (determined according to Example 5) is mixed with red blood cells in an in vitro system. The degree of hemolysis is established. This experiment is expected to last for 2 months.

[0255] Example 17: Investigating the in vivo efficacy of IC100 in the treatment of Parkinson's disease (PD)

[0321] The efficacy of IC100 in treating PD was evaluated in several animal models of PD (i.e. rodents). ) model by administering IC100: 5mg / kg, 15mg / kg, 30mg / kg.

[0256]

[0322] The 6-OHDA rat model of PD shows that mice exhibit rotational asymmetry and motor deficits. It is a chemically induced unilateral model of PD (lesion of the striatum or medial forebrain bundle) that exhibits behavioral deficits including depression. The 6-OHDA model shows reduced dopamine, DOPAC, and HVA content in the striatum, and histological examination shows a reduction in TH-positive cells in the substantia nigra.

[0257]

[0323] In one set of experiments using the 6-OHDA model of PD, a total of 45 males Rats are divided into three experimental groups (n=15 rats / group) and treated as follows:

[0324] 1. Sham-induced rats were treated with vehicle;

[0325] 2. Treated 6-OHDA-induced rats were treated with vehicle;

[0326] 3. Treated 6-OHDA-induced rats were treated with IC100 dose 1 (PK / half-life Treatment with 100 mg / kg / day of chemotherapy (selected based on early stage studies);

[0327] Unilateral 6-OHDA / sham injections were performed on study day 0;

[0328] Daily doses were formulated and administered (QD, po) on study days 15-28. stomach;

[0329] Body weight was monitored and behavioral tests were performed on days -14 (baseline), 28 (days), and 14 (days). The experiments were performed on days 1, 2, and 42 and included amphetamine-induced rotations.

[0258]

[0330] Terminal blood, CSF, and brain sampling was performed on study day 42, followed by HPLC will be performed to examine DA, DOPAC, and HVA in the striatum, and IHC will be performed to examine TH+ cells in the SNpc.

[0259]

[0331] In the second set of experiments using the 6-OHDA model of PD, a total of 45 mice were Male rats were divided into three experimental groups (n=15 rats / group) and treated as follows:

[0332] 1. Sham-induced rats were treated with vehicle;

[0333] 2. Treated 6-OHDA-induced rats were treated with vehicle;

[0334] 3. Treated 6-OHDA-induced rats were treated with IC100 dose 1 (PK / half-life Treatment with 100 mg / kg / day of chemotherapy (selected based on early stage studies);

[0335] The study was conducted over a 6-week period and included a unilateral 6-OHDA / sham injection study. Done on day 0;

[0336] The daily dose was formulated and administered (QD, po) on Study Day 1, and The subjects were then treated with 6-OHDA for up to 6 weeks after injection.

[0260]

[0337] Behavioral testing was performed on days -14 (baseline), 28, and 42. and includes the amphetamine-induced rotation and cylinder tests.

[0261]

[0338] Terminal brain sampling was performed on study day 42, followed by HPLC. to examine DA, DOPAC, and HVA in the striatum, and IHC to examine TH and Iba-1 in the SNpc (bilaterally).

[0262]

[0339] In the third set of experiments using the 6-OHDA model of PD, a total of 90 mice were Male rats are divided into 6 experimental groups (n=18 rats / group from baseline to day 14 baseline, with a goal of n=15 rats / group) and treated as follows:

[0340] 1. Sham-induced rats were treated with vehicle;

[0341] 2. 6-OHDA-induced rats were treated with vehicle;

[0342] 3. 6-OHDA-induced rats were treated with IC100 dose 1;

[0343] 4. 6-OHDA-induced rats were treated with IC100 dose 2;

[0344] 5. 6-OHDA-induced rats were treated with IC100 dose 3;

[0345] 6. 6-OHDA-induced rats were treated with IC100 dose 4;

[0346] The study was conducted over a 6-week period and included a unilateral 6-OHDA / sham injection study. Done on day 0;

[0347] Daily doses were formulated and administered (QD, po) on study days 15-42. Now.

[0263]

[0348] Behavioral testing was performed on days -14 (baseline), 28, and 42. and includes the amphetamine-induced rotation and cylinder tests.

[0264]

[0349] Terminal brain sampling was performed on study day 42, followed by HPLC. to examine DA, DOPAC, and HVA in the striatum, and IHC to examine TH and Iba-1 in the SNpc (bilaterally).

[0265] Example 18: In vitro efficacy study of IC100 in treating Parkinson's disease (PD)

[0350] TOM20 Assay:

[0351] The onset of PD is thought to be due to the mitochondrial complex I inhibitor rotenone inducing Parkinsonism. Several lines of research have implicated mitochondrial dysfunction, including the discovery that PARK2 induces PD in most autosomal recessive cases. There are mutations in genes encoding proteins involved in the selective clearance of dysfunctional and redundant mitochondria (mitophagy), such as PINK1 and PINK2. Similarly, mounting evidence implicates mitochondrial dysfunction in other neurodegenerative disorders (e.g., AD, ALS, and Huntington's disease (HD)). Thus, phenotypic readouts to measure mitochondrial (dys)function in disease-relevant cellular backgrounds may represent powerful predictive tools for investigating neurodegenerative pathologies and for identifying potential therapeutics that may enhance mitophagy.

[0266]

[0352] TOM20 is a subunit of the mitochondrial translocase of the outer membrane (TOM) complex and represents a biomarker for mitochondrial abun...

Claims

1. A nucleic acid encoding a monoclonal antibody or an antibody fragment thereof that specifically binds to apoptosis-associated speck-like protein (ASC) containing a caspase-activation recruitment domain, the antibody or the antibody fragment comprising a heavy chain variable (VH) region and a light chain variable (VL) region; The amino acid sequence of the VH region comprises an HCDR1 of SEQ ID NO:6, an HCDR2 of SEQ ID NO:7, and an HCDR3 of SEQ ID NO:8; The amino acid sequence of the VL region comprises an LCDR1 of SEQ ID NO: 12, an LCDR2 of SEQ ID NO: 13, and an LCDR3 of SEQ ID NO: 14; Nucleic acid.

2. The amino acid sequence of the VH region comprises SEQ ID NO: 18, 19, 20, 21 or 22, The amino acid sequence of the VL region comprises SEQ ID NO: 28, 29, 30 or 31; The nucleic acid of claim 1.

3. The nucleic acid described in claim 1 or 2, wherein the ASC is a human ASC protein.

4. The nucleic acid of any one of claims 1 to 3, wherein the antibody fragment is a Fab, F(ab')2, Fab', scFv, a single domain antibody, or a bispecific antibody.

5. A nucleic acid described in any one of claims 1 to 4, wherein the monoclonal antibody or antibody fragment thereof is humanized or chimeric.

6. An expression vector comprising a nucleic acid molecule described in any one of claims 1 to 5.

7. The expression vector of claim 6, wherein the nucleic acid molecule is operably linked to a regulatory sequence suitable for expression of the nucleic acid segment in a host cell.

8. A recombinant host cell comprising an expression vector described in claim 6 or 7.

9. A method for producing an antibody or antibody fragment that specifically binds to apoptosis-associated speck-like protein (ASC) containing a caspase-activating recruitment domain, comprising culturing a recombinant host cell containing the expression vector described in claim 6 or 7 under conditions in which the nucleic acid molecule is expressed, thereby producing a monoclonal antibody or antibody fragment thereof that specifically binds to ASC.

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