Compositions and methods for treating inflammasome-associated diseases or conditions

Abstract

The compositions and methods described herein include agents that inhibit inflammasome signaling in mammals, such as antibodies against inflammasome components, used alone or in combination with extracellular vesicle uptake inhibitors. Also described herein are compositions and methods of use for treating inflammasome-related diseases or conditions.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

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

[0002] STATEMENT REGARDING FEDERALLY FUNDED RESEARCH This invention was made with U.S. government support under Grant No. 4R42BS086274-02 awarded by the National Institute of Neurological Disorders and Stroke (NINDS) and Grant No. 5R42NS086274-03 awarded by the National Institutes of Health. The U.S. government has certain rights in this invention.

[0003] Description of electronically submitted text files

[0003] The contents of the text file submitted electronically herewith are incorporated by reference in its entirety into this specification: Computer-readable format copy of the sequence listing (file name: UNMI_010_02WO_SeqList_ST25.txt, date of recording: 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 for reducing inflammation in response to injury or pathology that causes inflammation in the CNS and / or lungs. The present invention also relates to monoclonal antibodies or fragments thereof that specifically bind 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, with some studies reporting an incidence as high as 30% (Nicolls, MR et al., (2014). Traumatic brain injury: lungs in a RAGE. Sci Transl Med 6, 252fs234). Recent studies have 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). However, the precise molecular mechanisms underlying TBI-induced lung injury remain poorly defined.

[0006]

[0006] The release of large amounts of secreted inflammatory mediators (e.g., cytokines, chemokines, and damage-associated molecular patterns (DAMPs)) from damaged cells contributes to brain inflammation and affects distant organs (e.g., lungs) (Nicholls, MR et al., (2014). Traumatic brain injury: lungs in a RAGE. Sci Transl Med 6, 252fs234). One of the most widely studied DAMPs is high mobility group box-1 (HMGB1), which can 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). HMGB1 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 intercellular communication (Yanez-Mo, M. et al., (2015). Biological properties of extracellular vesicles and their physiological functions. J Extracell Vesicles 4, 27066) and have been implicated in playing a role in the development of ALI in an LPS-induced murine model. Furthermore, EVs can carry biologically active cytokines (e.g., IL-1β and inflammasome proteins) (Qu, Y. et al., (2007). Nonclassical IL-1β 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 can trigger immune responses, amplifying inflammation in neighboring and surrounding cells through their cargo. However, it remains unclear whether EV-mediated inflammasome signaling contributes to the pathogenesis of TBI-induced ALI. Furthermore, it remains unclear whether the pathogenesis of TBI-induced ALI is 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

[0009] 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, and the epitope comprises or consists of the amino acid sequence of SEQ ID NO: 5 or an amino acid sequence of 5 to 10, 10 to 15, or 15 to 20 amino acids of SEQ ID NO: 5.

[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, 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, comprising 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 for producing an antibody or antibody fragment that specifically binds to ASC, the method 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 pharmaceutically acceptable carrier, diluent, or excipient.In some examples, provided herein are methods 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 the inflammation in the subject. In some examples, administering the monoclonal antibody or antibody fragment thereof reduces the level of at least 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 cold autoinflammatory syndrome (FCAS), Muckle-Wells syndrome (MWS), and neonatal-onset multisystem inflammatory disease (NOMID). In some cases, administration of the monoclonal antibody or antibody fragment thereof inhibits inflammasome activation in a subject. In some cases, administration of the monoclonal antibody or antibody fragment thereof reduces ASC activity 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.In some examples, provided herein are methods for 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 the subject's MS. In some examples, administering the monoclonal antibody or antibody fragment thereof reduces the level of at least inflammatory cytokines. In some examples, administering the monoclonal antibody or antibody fragment thereof inhibits inflammasome activation in the subject. In some examples, administering 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 intracerebroventricularly, intraperitoneally, intravenously, 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 comprising a light chain variable (VL) region and a heavy chain variable (VH) region, 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, 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 for producing an antibody or antibody fragment that specifically binds to ASC, the method 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 pharmaceutically acceptable carrier, diluent, or excipient.In some examples, provided herein are methods 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 the inflammation in the subject. In some examples, administering the monoclonal antibody or antibody fragment thereof reduces the level of at least 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 cold autoinflammatory syndrome (FCAS), Muckle-Wells syndrome (MWS), and neonatal-onset multisystem inflammatory disease (NOMID). In some cases, administration of the monoclonal antibody or antibody fragment thereof inhibits inflammasome activation in a subject. In some cases, administration of the monoclonal antibody or antibody fragment thereof reduces ASC activity 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.In some examples, provided herein are methods for 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 the subject's MS. In some examples, administering the monoclonal antibody or antibody fragment thereof reduces the level of at least inflammatory cytokines. In some examples, administering the monoclonal antibody or antibody fragment thereof inhibits inflammasome activation in the subject. In some examples, administering 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 intracerebroventricularly, intraperitoneally, intravenously, 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 variants thereof, including variants 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 variants thereof, including variants 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 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 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 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 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 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: 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 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: 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: 20 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: 20, and the amino acid sequence of the VL 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:20 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:20, 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:20 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:20, 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: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 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:21, and the amino acid sequence of the VL 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:21 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:21, 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:21 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:21, 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:21 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:21, 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: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 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: 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: 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 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 comprises SEQ ID NO:31 or an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:31. 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 the 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 for producing an antibody or antibody fragment that specifically binds to ASC, comprising culturing a recombinant host cell containing the expression vector under conditions for expression of the nucleic acid molecule, 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 pharmaceutically 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 the level of at least 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 instances, the CNS injury is selected from the group consisting of traumatic brain injury (TBI), stroke, and spinal cord injury (SCI). In some instances, 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, and inflammatory bowel disease. 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 can include familial cold autoinflammatory syndrome (FCAS), Muckle-Wells syndrome (MWS), and neonatal-onset multisystem inflammatory disease (NOMID). In some cases, 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 intracerebroventricularly, intraperitoneally, intravenously, 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 the level of at least inflammatory cytokines. 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 intracerebroventricularly, intraperitoneally, intravenously, or by inhalation. [Brief explanation 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] Active caspase-1 (Figure 1B) is significantly elevated in cortical tissue at 4 and 24 hours after TBI. Data are 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 (Figure 1C) is significantly elevated in cortical tissue at 4 and 24 hours after TBI. Data are 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 (Figure 1D) is significantly elevated in cortical tissue at 4 and 24 hours after TBI. Data are 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] HMGB1 (Figure 1E) is significantly elevated in cortical tissue at 4 and 24 hours after TBI. Data are 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 (Figure 1F) is significantly elevated in cortical tissue at 4 and 24 hours after TBI. Data are 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 (Figure 1G) is significantly elevated in cortical tissue at 4 and 24 hours after TBI. Data are 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] Active caspase-1 (Figure 1I) is significantly elevated in lung tissue 4 and 24 hours after TBI. Data are 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 (Figure 1J) is significantly elevated in lung tissue 4 and 24 hours after TBI. Data are 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 are 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] HMGB1 (Figure 1L) is significantly elevated in lung tissue 4 and 24 hours after TBI. Data are 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 (Figure 1M) is significantly elevated in lung tissue 4 and 24 hours after TBI. Data are 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 are 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] Figure 2 illustrates 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] We demonstrate 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 are presented as mean + / - SEM; *p<0.05 compared to sham. N=4-5 per group. [Figure 3C]

[0015] Figure 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 are 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 4A illustrates pyroptosome formation in mouse lungs 4 hours after TBI. Figure 4A shows that TBI induces laddering of ASC in lung tissue, resulting in the formation of pyroptosomes, which are oligomerizations of ASC dimers that lead to caspase-1 activation and pyroptosis. [Figure 4B] FIG. 4B shows a representative immunoblot for gasdermin. [Figure 4C]

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

[0017] We demonstrate 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. The sections show evidence of neutrophil infiltration (arrowheads), altered alveolar-capillary membrane morphology (asterisks, *), interstitial edema (short arrows), and thickening of the interstitial and alveolar septa (pounds, #). [Figure 5B] Figure 5B shows that acute lung injury scoring is significantly increased in injured animals compared to sham at 4 and 24 hours. Data are presented as mean + / - SEM. N = 4-5 per group. *p < 0.05 compared to sham. [Figure 6]

[0018] Figure 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 receiving EVs isolated from TBI mice compared to EVs from sham animals. [Figure 7B]

[0019] Caspase-1 (Figure 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]

[0019] ASC (Figure 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<0.05 compared to sham. N=3 per group. [Figure 7D] IL-18 (FIG. 7D) is elevated in the lungs of animals receiving 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 (Figure 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 (Figure 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 (reduced alveolar size) and inflammatory cell infiltration, as determined by H and E staining. ALI scores were significantly increased in EVs delivered from injured mice compared with non-injured mice (Figure 7G). Data are presented as mean + / - SEM; **p<0.01, *p<0.05 compared with the non-injured group. [Figure 8A]

[0020] 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 shows representative immunoblots demonstrating 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 (Figure 8B) is reduced in the lungs of animals treated with enoxaparin and IC100 compared to untreated positive control animals. Data are 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 are 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 are 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]

[0020] AIM2 (Figure 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 (Figure 8F) is reduced in the lungs of animals treated with enoxaparin and IC100 compared to untreated positive control animals. Data are 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] Figure 9A illustrates that treatment with enoxaparin (3 mg / kg) and IC100 (5 mg / kg) reduces ALI scores in the lungs of animals delivered with EVs from injured mice. Figure 9A illustrates H and E staining of lung sections from EV-delivered mouse lungs from saline-treated (Figure 9A) injured mice. The sections show evidence of neutrophil infiltration, altered alveolar-capillary membrane morphology, interstitial edema, and thickening of the interstitial 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] Figure 9E illustrates that acute lung injury scoring was significantly reduced in animals treated with enoxaparin, IC100, compared to untreated animals. Data are 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 increases inflammasome protein expression in pulmonary endothelial cells. Figure 10A shows Western blot analysis of caspase-1, ASC, AIM2, and HMGB1 in PMVECs after 4 hours of 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] Figure 10F shows immunoassay results of a significant increase in IL-1β expression using the Ella Simple Plex 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 demonstrate that delivery of TBI-EVs to lung endothelial cells increases active caspase-1 immunoreactivity and cell death. Figure 11A shows the colocalization of caspase-1 FLICA and PI staining in PMVECs incubated with TBI-EVs for 4 hours. [Figure 11B]

[0023] Figure 11B shows caspase-1 FLICA and PI staining in PMVECs incubated with control EV for 4 hours. [Figure 11C] 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 demonstrate 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 mice showed signs of paralysis. Results are expressed as the mean daily clinical score ± SEM for 9-10 mice per 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 to be the day when the mice showed the first EAE symptoms. [Figure 13A]

[0025] Figure 1 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 demonstrate that IC100 treatment reduces the infiltration of peripheral immune cells into the spinal cord or spleen after EAE. Flow cytometry quantification of spleen-infiltrating leukocyte populations 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 expressed as mean ± SEM of 5 animals / group, *p<0.05, Student's t-test. [Figure 15]

[0027] Figure 1 shows IC100 concentrations in tissues. 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 at 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 by ASC specks in unstimulated THP-1 cells and that uptake is increased by inflammasome activation. [Figure 17]

[0029] Figure 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 across 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 most effective in 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] Figure 1 shows a comparison of the kinetics of three different antibodies against human ASC. DETAILED DESCRIPTION OF THE INVENTION

[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 (e.g., but not limited to, patents, patent applications, articles, 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 that term in this application, the definition that appears in this application shall control. However, reference to any references, articles, publications, patents, patent application publications, and patent applications cited herein is not, and should not be taken as, an acknowledgment or any indication 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., 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 plural, unless the context clearly requires that only one such element be present.

[0017]

[0038] Unless the context requires otherwise, throughout the specification and claims, the word "comprise" and variations thereof (e.g., "comprises" and "comprising") should 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 stated range, value, or structure, unless otherwise indicated.

[0018]

[0039] References throughout this specification to "one embodiment" or "an embodiment" mean that a particular feature, structure, or characteristic described in connection with this embodiment is included in at least one embodiment of the present disclosure. Thus, the appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification 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 an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all 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.)). Antibodies provided herein can be polyclonal antibodies, monoclonal antibodies (mAbs), chimeric antibodies, humanized antibodies, 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 can be chimeric, humanized, or human.

[0022]

[0043] "Specifically binds" or "immunoreacts with" means that an antibody reacts with one or more antigenic determinants of a desired antigen and does not react with other peptides. In certain embodiments, an antibody is said to specifically bind to an antigen when it preferentially recognizes its target antigen in a complex mixture of proteins and / or macromolecules. The term "antibody" broadly refers to an immunoglobulin (Ig) molecule (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] As used herein, the term "humanized antibody" refers to an antibody in which minimal parts of a non-human antibody have been introduced into an otherwise human antibody.

[0024]

[0045] As used herein, the term "human antibody" refers to an antibody in which substantially all portions of the protein are substantially non-immunogenic in humans, with minor sequence changes 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 comprise two identical heavy chains and two identical light chains, and two antigen-binding domains, each composed of a heavy-chain variable region (VH) and a light-chain variable region (VL). IgA antibodies generally comprise two monomers, each composed of two heavy chains and two light chains (as in IgG, IgD, and IgE antibodies); thus, an IgA molecule has four antigen-binding domains, each again composed of a VH and a VL. Certain IgA molecules are monomers composed of two heavy chains and two light chains. Secreted IgM antibodies are generally composed of five monomers, each composed of two heavy and two light chains (as in IgG and IgE antibodies), and thus the IgM molecule has 10 antigen-binding domains, each again composed of a VH and a VL. Cell surface forms of IgM also exist, which have a two heavy chain / two light chain structure similar to IgG, IgD, and IgE antibodies.

[0026]

[0047] The terms "antigen-binding fragment," "antigen-binding portion," "antigen-binding site," "binding domain," or "binding region," as used herein, may refer to a protein, polypeptide, oligopeptide, or peptide, or an antibody, or a domain, region, portion, 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 an additional amino acid sequence (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, a "tag" (e.g., a FLAG tag or a 6His tag), or an enzyme or polypeptide that increases the half-life of the antibody in the 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 containing intrasequence insertions of single or multiple amino acid residues.

[0027]

[0048] An antigen-binding site can 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 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, along with framework regions (FRs). In certain embodiments, a binding domain comprises or consists of an antigen-binding site (e.g., antibody-derived variable heavy and variable light chain sequences or three light chain complementarity-determining regions (CDRs) and three heavy chain CDRs arranged in alternative framework regions (FRs) (e.g., human FRs, optionally containing one or more amino acid substitutions)).

[0028]

[0049] The term "CDR region" or "CDR" can refer to the hypervariable region of an immunoglobulin heavy or light chain as defined by Kabat et al., 1991 (Kabat, E. A. et al., (1991) Sequences of Proteins of Immunological Interest, 5th Edition. U.S. Department of Health and Human Services, Public Service, NIH, Washington), and later editions. Antibodies typically contain 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 may also be bispecific, trispecific, dual specific, or multispecific, specifically binding to two or more different antigens. Examples of binding fragments encompassed by the term "antigen-binding fragment" of an antibody include: (i) a Fab fragment consisting of the VL domain, VH domain, CL domain, and CH1 domain (Ward, ES et al., (1989) Nature 341, 544-546); (ii) a Fd fragment consisting of the VH domain and CH1 domain (McCafferty et al., (1990) Nature, 348, 552-554); (iii) a Fv fragment consisting of the VL domain and VH domain of a single antibody (Holt et al., (2003) Trends in Biotechnology 21, 484-490); and (iv) a dAb fragment consisting of the VH domain or the VL domain (Ward, ES et al., Nature 341, 544-546 (1989), McCafferty et al., (1990) Nature, 348, 552-554, Holt et al. al., (2003) Trends in Biotechnology 21, 484-490]; (v) isolated CDR regions; (vi) F(ab')2 fragments, which are bivalent fragments containing two binding Fab fragments; and (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 of an Fv fragment, VL and VH, are encoded by separate genes, they can be linked using recombinant methods by a synthetic linker that allows them to form a single protein chain (known as a 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 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 gene fusion (WO 94 / 13804; Holliger, P. (1993) et al., Proc. Natl. Acad. Sci. USA 90 6444-6448). Diabodies are bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but with 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; 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 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 containing scFvs linked to the CH3 domain can also be produced (Hu, S. et al., (1996) Cancer Res., 56, 3055-3061). Other examples of binding fragments are Fab', which differs from Fab fragments only 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, 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 antigen-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] "Fc region" or "Fc domain" refers to the polypeptide sequence corresponding to or derived from the portion of a source antibody involved in binding to the antibody receptor on cells and the C1q component of complement. Fc is an abbreviation for "fragment crystallographic," a fragment of an antibody that readily forms protein crystals. Separate protein fragments, initially elucidated by proteolytic digestion, can define the overall general structure of immunoglobulin proteins. According to the original definition in the literature, an 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 the CH3, CH2, and at least a portion of the hinge sufficient to form a disulfide-bonded dimer containing 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 antibodies provided herein or antibody fragments derived therefrom (e.g., anti-ASC monoclonal antibodies or antibody fragments thereof) have a modified Fc region or Fc domain. In some cases, this modified Fc region or Fc domain may confer increased thermal stability to the resulting antibody or antibody fragment derived therefrom. This increased thermal stability may result in an increased serum half-life. The Fc region or Fc domain may be modified as described in U.S. Patent Application Publication No. 20160193295, the contents of which are incorporated herein by reference. As described in U.S. Patent Application 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 Fc region or Fc domain of an antibody provided herein or an antibody fragment derived therefrom (e.g., an anti-ASC monoclonal antibody or antibody fragment thereof) can be stabilized by engineering the Fc region to contain intradomain disulfide bonds, as described in Wozniak-Knopp G, Stadlmann J, Rueker F (2012) Stabilization of the Fc Fragment of Human IgG1 by Engineered Intradomain Disulfide Bonds. PLoS ONE 7(1): e30083, the contents of which are incorporated herein by reference. In yet another embodiment, the antibody has an Fc region that has been modified as described in WO 99 / 58572, the contents of which are incorporated herein by reference. In yet another embodiment, the Fc region or Fc domain can be modified as described in U.S. Pat. No. 9,574,010, the contents of which are incorporated herein by reference.

[0033]

[0054] The terms "caspase-activation 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 proteins 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 an 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 protein) 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. See Arend et al. 2008; Li et al. 2008; and Martinon et al. 2002, each of which is incorporated herein by reference in its entirety. The terms "NLRP1 inflammasome," "NALP1 inflammasome," "NLRP2 inflammasome," "NALP2 inflammasome," "NLRP3 inflammasome," "NALP3 inflammasome," "NLRC4 inflammasome," "IPAF inflammasome," or "AIM2 inflammasome" refer to a protein complex of at least caspase-1 and one adaptor protein, e.g., ASC. For example, the terms "NLRP1 inflammasome" and "NALP1 inflammasome" can refer to a multiprotein complex containing NLRP1, ASC, caspase-1, caspase-11 for activating caspase-1 and processing interleukin-1β, interleukin-18, and interleukin-33, XIAP, and pannexin-1. The terms "NLRP2 inflammasome" and "NALP2 inflammasome" can refer to a multiprotein complex containing NLRP2 (aka NALP2), ASC, and caspase-1, while the terms "NLRP3 inflammasome" and "NALP3 inflammasome" can refer to a multiprotein complex containing NLRP3 (aka NALP3), ASC, and the terms "NLRC4 inflammasome" and "IPAF inflammasome" can refer to a multiprotein complex containing NLRC4 (aka IPAF), ASC, and caspase-1. Furthermore, the term "AIM2 inflammasome" may refer to a multiprotein complex comprising 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, CA).

[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, a reduction in spinal cord pathology, and the like. The compositions described herein can 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, previous treatments, the subject's general health and / or age, and other diseases present, can influence 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 can 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, mitigating, altering, rescuing, ameliorating, improving, or affecting the disease, the symptoms of a disease, or the 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] "Absent in melanoma 2" and "AIM2," as used interchangeably herein, can refer to the expression product or isoform of the AIM2 gene; or a protein that shares 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 exhibits the 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," 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] As used interchangeably herein, "NALP3" and "NLRP3" refer to an 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 the functional activity of NALP3.

[0044]

[0065] "NLRC4" and "IPAF," 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 in which blood flow is interrupted.

[0046]

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

[0047]

[0068] Methods including conventional molecular biology techniques are described herein.Such techniques are generally known in the art and are described in detail 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, 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 detail 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 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-activation recruitment domain. The monoclonal antibody or fragment thereof can specifically bind to an antigenic fragment of ASC comprising, consisting of, or consisting 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 may be innate immune-mediated inflammation. The inflammation may 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, such as those 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 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, the disease, condition, or affliction of or affecting the CNS can be stroke, as well as autoimmune and / or CNS disorders (e.g., amyotrophic lateral sclerosis (ALS), Lou Gehrig's disease, multiple sclerosis (MS), immune dysfunction muscular CNS breakdown, muscular dystrophy (MD), Alzheimer's disease (AD), and Parkinson's disease (PD)). Use of this monoclonal antibody or antibody fragment thereof in methods 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. The monoclonal antibody or antibody fragment thereof of this embodiment can be present in a composition (e.g., a pharmaceutical composition provided herein).In some cases, the monoclonal antibody or fragment thereof is used in the therapeutic methods provided herein in combination with one or more other agents, which can 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.).

[0050]

[0071] The present invention also encompasses a monoclonal antibody or antibody fragment thereof that specifically binds to ASC, the antibody or antibody fragment 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, comprising at least one amino acid substitution in HCDR1, HCDR2, and / or HCDR3. 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 provided herein may be used in a method for reducing inflammation in a mammal, such as those 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 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 disease, multiple sclerosis (MS), immune dysfunction muscle CNS degeneration, muscular dystrophy (MD), Alzheimer's disease (AD), and Parkinson's disease (PD)). Use of this monoclonal antibody or antibody fragment thereof in methods of treating inflammation can reduce inflammation in the CNS and / or lungs of a patient. Use of this monoclonal antibody or antibody fragment thereof in methods of treating inflammation can reduce innate immune inflammation or inflammasome-associated inflammation in 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 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 disorders, glycogen storage disorders, medium-chain acyl-coenzyme A dehydrogenase deficiency, non-alcoholic fatty liver disease (e.g., non-alcoholic steatohepatitis (NASH)), and gout. In one embodiment, this monoclonal antibody or an antibody fragment derived from this monoclonal antibody is used to treat diabetic nephropathy by administering the monoclonal antibody or an antibody fragment derived from this monoclonal antibody to a patient suffering from or suspected of suffering from diabetic nephropathy.In one embodiment, this monoclonal antibody or an antibody fragment derived from this monoclonal antibody is used to treat NASH by administering the monoclonal antibody or an antibody fragment derived from this monoclonal antibody to a patient suffering from or suspected of suffering from NASH. 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). In one embodiment, this monoclonal antibody or an antibody fragment derived from this monoclonal antibody is used to treat CAPS by administering the monoclonal antibody or an antibody fragment derived from this monoclonal antibody to a patient suffering from or suspected of suffering from CAPS. 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 fragment thereof is used in the therapeutic methods provided herein in combination with one or more other agents, which can 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 comprising a light chain variable (VL) region and a heavy chain variable (VH) region, 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. In addition to this embodiment, the present invention contemplates 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, such as those 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 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 disease, multiple sclerosis (MS), immune dysfunction muscle CNS degeneration, muscular dystrophy (MD), Alzheimer's disease (AD), and Parkinson's disease (PD)). Use of this monoclonal antibody or antibody fragment thereof in methods of treating inflammation can reduce inflammation in the CNS and / or lungs of a patient. Use of this monoclonal antibody or antibody fragment thereof in methods of treating inflammation can reduce innate immune inflammation or inflammasome-associated inflammation in 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 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 disorders, glycogen storage disorders, medium-chain acyl-coenzyme A dehydrogenase deficiency, non-alcoholic fatty liver disease (e.g., non-alcoholic steatohepatitis (NASH)), and gout. In one embodiment, this monoclonal antibody or an antibody fragment derived from this monoclonal antibody is used to treat diabetic nephropathy by administering the monoclonal antibody or an antibody fragment derived from this monoclonal antibody to a patient suffering from or suspected of suffering from diabetic nephropathy.In one embodiment, this monoclonal antibody or an antibody fragment derived from this monoclonal antibody is used to treat NASH by administering the monoclonal antibody or an antibody fragment derived from this monoclonal antibody to a patient suffering from or suspected of suffering from NASH. 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). In one embodiment, this monoclonal antibody or an antibody fragment derived from this monoclonal antibody is used to treat CAPS by administering the monoclonal antibody or an antibody fragment derived from this monoclonal antibody to a patient suffering from or suspected of suffering from CAPS. 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 fragment thereof is used in the therapeutic methods provided herein in combination with one or more other agents, which can 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.).

[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 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, comprising at least one amino acid substitution in HCDR1, HCDR2, and / or HCDR3, and 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, comprising at least one amino acid substitution in LCDR1, LCDR2, and / or LCDR3. In addition to this embodiment, the present invention contemplates 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 can be used in a method for reducing inflammation in a mammal, such as those described in U.S. Patent No. 8,685,400, the contents of which are incorporated herein by reference in their entirety. This 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, 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 disease, multiple sclerosis (MS), immune dysfunction, muscle CNS degeneration, muscular dystrophy (MD), Alzheimer's disease (AD), and Parkinson's disease (PD)). Use of this monoclonal antibody or antibody fragment thereof in methods for treating inflammation may reduce inflammation in the CNS and / or lungs of a patient. Use of this monoclonal antibody or antibody fragment thereof in methods 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 an antibody fragment derived from the monoclonal antibody is used to treat central nervous system (CNS) injury and / or an autoimmune disease, autoinflammatory disease, metabolic disease, 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 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 having or suspected of having 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 having or suspected of having 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 having or suspected of having lupus nephritis. The metabolic disorder may be selected from metabolic syndrome, obesity, diabetes, diabetic nephropathy or diabetic kidney disease (DKD), insulin resistance, atherosclerosis, lipid storage disorders, glycogen storage disorders, medium-chain acyl-coenzyme A dehydrogenase deficiency, non-alcoholic fatty liver disease (e.g., non-alcoholic steatohepatitis (NASH)), and gout.In one embodiment, this monoclonal antibody or an antibody fragment derived from this monoclonal antibody is used to treat diabetic nephropathy by administering the monoclonal antibody or an antibody fragment derived from this monoclonal antibody to a patient suffering from or suspected of suffering from diabetic nephropathy. In one embodiment, this monoclonal antibody or an antibody fragment derived from this monoclonal antibody is used to treat NASH by administering the monoclonal antibody or an antibody fragment derived from this monoclonal antibody to a patient suffering from or suspected of suffering from NASH. 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). In one embodiment, this monoclonal antibody or an antibody fragment derived from this monoclonal antibody is used to treat CAPS by administering the monoclonal antibody or an antibody fragment derived from this monoclonal antibody to a patient suffering from or suspected of suffering from CAPS. 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, this monoclonal antibody or fragment thereof is used in combination with one or more other agents in the treatment 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 examples, the inflammasome-associated inflammation is present in the CNS of a mammal exposed to or afflicted with a condition that results in or causes innate immune inflammation or inflammasome-associated inflammation. In some cases, the inflammasome-associated inflammation is present in a mammal (e.g., a human) that is afflicted with or suspected to be afflicted with a condition that results in or causes innate immune inflammation or inflammasome-associated inflammation. The condition that results in or causes innate immune inflammation or inflammasome-associated inflammation can be a CNS injury, an autoimmune, autoinflammatory, neurodegenerative, and / or metabolic disease or disorder. The CNS injury can 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 disorders, glycogen storage disorders, 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 can include familial cold autoinflammatory syndrome (FCAS), Muckle-Wells syndrome (MWS), and neonatal-onset multisystem inflammatory disease (NOMID). The compositions and methods described herein can 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) extracellular vesicle (EV) uptake and is used as a treatment for CNS inflammation in a mammal.Examples of conditions that can 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 active fragment thereof provided herein that binds to a component of an inflammasome, or a combination thereof. The composition may be administered by any suitable route, for example, by inhalation, intravenously, intraperitoneally, or intracerebroventricularly. The composition may further comprise at least one pharmaceutically 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 U.S. Patent Application No. 62 / 560,963, filed September 20, 2017, 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 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 an inflammasome component (e.g., an anti-ASC monoclonal antibody or antibody fragment thereof, e.g., IC100), or any combination thereof. The composition can be administered by any appropriate route, for example, by inhalation, intravenously, intraperitoneally, or intracerebroventricularly. The composition can further comprise at least one pharmaceutically acceptable carrier or diluent. The standard of care treatment can be selected from a treatment for modifying disease outcome, a treatment for managing relapse, a treatment for managing symptoms, or any combination thereof. Therapies 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 these 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 No. 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 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 an inflammasome component (e.g., an anti-ASC monoclonal antibody or antibody fragment thereof, e.g., IC100), or any combination thereof. The composition can be administered by any appropriate route, for example, by inhalation, intravenously, intraperitoneally, or intracerebroventricularly. The composition can further comprise at least one pharmaceutically 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 inhibitor may be selected from selegiline (Eldepryl, Zelapar), rasagiline (Azilect), and safinamide (Xadago). The COMT inhibitor may be selected from entacapone (Comtan) and tolcapone (Tasmar). The anticholinergic 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 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 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 with AD and progresses from mild cognitive impairment (MCI) to complete memory loss and personality and behavior changes. An AD diagnosis can be determined or may have been determined using any method known in the art. In one embodiment, the subject is diagnosed with AD using the methods detailed in International Publication No. 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 an inflammasome component (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 appropriate route, for example, by inhalation, intravenously, intraperitoneally, or intracerebroventricularly. The composition may further comprise at least one pharmaceutically acceptable carrier or diluent. The standard of care treatment may be selected from a cholinesterase inhibitor and memantine (Namenda). The cholinesterase inhibitor may be selected from donepezil (Aricept), galantamine (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. An RA 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 RA using the methods detailed in International Publication No. 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 appropriate route, for example, by inhalation, intravenously, intraperitoneally, or intracerebroventricularly. The composition may further comprise at least one pharmaceutically acceptable carrier or diluent. The standard of care treatment may be selected from a nonsteroidal anti-inflammatory drug (NSAID), a steroid (e.g., prednisone), a disease-modifying antirheumatic drug (DMARD), and a biologic agent. NSAIDs may include ibuprofen (Advil, Motrin IB), and naproxen sodium (Aleve). DMARDs can include methotrexate (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 a subject suffering from or suspected of suffering from lupus nephritis. The methods for treating lupus nephritis provided herein may 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 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 kidneys, potentially resulting in kidney inflammation and hematuria, proteinuria, hypertension, impaired kidney function, and even kidney failure. A lupus nephritis diagnosis may 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 No. 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 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 an inflammasome component (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 appropriate route, for example, by inhalation, intravenously, intraperitoneally, or intracerebroventricularly. The composition may further comprise at least one pharmaceutically acceptable carrier or diluent. Standard of care treatment for lupus nephritis may include medications to control blood pressure and / or a special diet low in protein and salt. Additionally, the standard of care treatment for lupus nephritis may be a treatment for lupus, such as a nonsteroidal anti-inflammatory drug (NSAID), an antimalarial drug, a corticosteroid (e.g., prednisone; methylprednisolone), an immunosuppressant, or a biologic agent.Examples of NSAIDs may include naproxen sodium (Aleve) and ibuprofen (Advil, Motrin IB, etc.). An example of an antimalarial drug may be hydroxychloroquine (Plaquenil). Examples of immunosuppressants may include azathioprine (Imuran, Azasan), mycophenolate mofetil (CellCept), and methotrexate (Trexall). Examples of biologics may include belimumab (Benlysta) or rituximab (Rituxan).

[0059]

[0080] Provided herein are compositions and methods for treating nonalcoholic steatohepatitis (NASH) in a subject suffering from or suspected of suffering from NASH. The methods for treating NASH 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 NASH. NASH is a type of nonalcoholic fatty liver disease (NAFLD). NAFLD is an umbrella term for various liver conditions that affect people who rarely or never drink alcohol. NAFLD is primarily characterized by excessive fat storage in liver cells and liver inflammation, which can progress to scarring and irreversible damage. This damage can be similar to that caused by heavy alcohol consumption. In its most severe form, nonalcoholic steatohepatitis can progress to cirrhosis and liver failure. A diagnosis of NASH 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 No. 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 an inflammasome component (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 appropriate route, for example, by inhalation, intravenously, intraperitoneally, or intracerebroventricularly. The composition may further comprise at least one pharmaceutically 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 for treating diabetic nephropathy 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 diabetic nephropathy. Diabetic nephropathy is a severe kidney-related complication of type 1 and type 2 diabetes, which can also be referred to as diabetic nephropathy (DKD). A DKD diagnosis can be determined or may have been determined using any method known in the art. In one embodiment, the subject is 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 agent may be a standard of care treatment known in the art for DKD, 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 appropriate route, for example, by inhalation, intravenously, intraperitoneally, or intracerebroventricularly. The composition may further comprise at least one pharmaceutically acceptable carrier or diluent. Standard of care treatment for diabetic nephropathy may include lifestyle changes, such as weight loss, increased exercise, lowering cholesterol, controlling urinary protein, promoting bone health, controlling high blood pressure, managing diabetes, and kidney dialysis or transplantation.

[0061]

[0082] Provided herein are compositions and methods for treating inflammatory bowel disease (IBD) in a subject suffering from or suspected of suffering from IBD. The methods of treating IBD 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 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) in the innermost lining 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. An 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 No. 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 an inflammasome component (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 appropriate route, for example, by inhalation, intravenously, intraperitoneally, or intracerebroventricularly. The composition may further comprise at least one pharmaceutically 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 immunosuppressants may include azathioprine (Azasan, Imuran), mercaptopurine (Purinethol, Purixan), cyclosporine (Gengraf, Neoral, Sandimmune), and methotrexate (Trexall). Other examples of immunosuppressants may include tumor necrosis factor (TNF)-alpha inhibitors or biologics (e.g., infliximab (Remicade), adalimumab (Humira), golimumab (Simponi), natalizumab (Tysabri), vedolizumab (Entyvio), and ustekinumab (Stelara)). Anti-inflammatory agents may include corticosteroids and aminosalicylates (e.g., mesalamine (Asacol HD, Delzicol), balsalazide (Colazal), and olsalazine (Dipentum)).

[0062]

[0083] Provided herein are compositions and methods for treating cryopyrin-associated periodic syndromes (CAPS) in subjects 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 known as cryopyrin-associated autoinflammatory syndromes, consist of three autoinflammatory diseases associated with defects 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. Early symptoms of NOMID may include a non-itchy, honeycomb-like rash; inflammation of the membranes surrounding the brain, causing headaches, blindness, or hearing loss; swollen 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., skin rash, red eyes, joint pain, and severe headache accompanied by 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. A 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 No. 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 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 pharmaceutically acceptable carrier or diluent. Standard care treatments 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 with 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 are methods for 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, the method for 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 can be administered in a therapeutically effective amount. The therapeutically effective amount can be a dose provided herein. The agent can 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 can be administered by any suitable route, for example, by inhalation, intravenously, intraperitoneally, or intracerebroventricularly. The composition can further comprise at least one pharmaceutically 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 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 renal inflammation in a mammal.

[0066]

[0087] Described herein are methods for reducing renal inflammation in a mammal with a condition that results in and / or causes an immune response in the kidney. In one embodiment, the method for treating renal inflammation in a mammal comprises 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 is lupus nephritis. 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, an antibody or active fragment thereof provided herein that binds to an inflammasome component, 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 pharmaceutically 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 this antibody (e.g., IC100), alone or in combination with, e.g., an EV uptake inhibitor) can result in a decrease in the activity and / or expression level of a mammalian inflammasome component in the CNS, kidney, or lung of a subject. The decrease can be in lung cells, 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 level of an inflammasome component in a subject to which the agent has not been 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 antibody fragment derived from this antibody, alone or in combination with, for example, an EV uptake inhibitor) can result in the reduction or elimination of acute lung injury (ALI). In one embodiment, the reduction in ALI is evidenced by reduced neutrophil infiltration into the alveolar and / or interstitial spaces, reduced or absent alveolar septal thickening, or a combination thereof. The reduction can be compared to a control. The control can be ALI in a subject prior to administration of the agent. The control can be ALI in a subject with ALI who has not been administered the agent.

[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, for example, an EV uptake inhibitor) can 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 can be caused by caspase-1-mediated cleavage of gasdermin D (GSDMD). In one embodiment, the reduction in pyroptosis is evidenced by reduced or absent cleavage of GSDMD in the lungs or lung cells (e.g., type II pneumocytes) of the subject. The reduction or elimination of pyroptosis can be compared to a control. The reduced or absent cleavage of GSDMD can be compared to a control. The control can be the level of pyroptosis in a subject before administration of the agent. The control can be the level of pyroptosis in a subject suffering from pyroptosis to which the agent is not 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) can be monitored by measuring the level of at least one inflammasome protein. Thus, in some embodiments, the therapeutic methods provided herein further comprise 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 decrease in the 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 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 inflammasome proteins may be used to adjust the dosage or frequency of treatment. The level of inflammasome proteins 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 treatment methods provided herein is an EV uptake inhibitor, which can be a compound, antisense RNA, siRNA, a peptide, an antibody or 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, Dynasoa, 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 may further comprise at least one pharmaceutically 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 mammalian inflammasome component or an antigen or epitope derived therefrom. In another embodiment, the agent to be administered is an antisense RNA or siRNA directed against a mammalian inflammasome component. The inflammasome component can be any component 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 can be monoclonal or polyclonal antibodies or active fragments thereof. The antibodies or active fragments can be chimeric, human, or humanized as described herein.

[0079]

[0096] Any suitable antibody or active fragment thereof provided herein that specifically binds to ASC can be used, for example, an antibody that inhibits ASC activity in the CNS (e.g., CNS cells) or lung cells (e.g., type II alveolar cells) of a subject. In one embodiment, the antibody specifically binds to an amino acid sequence having at least 85% sequence identity to the amino acid sequence SEQ ID NO: 1 or SEQ ID NO: 2. In another embodiment, the antibody or 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 to 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 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 amino acid 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 amino acid SEQ ID NO: 5) is discontinuous. In some examples, the monoclonal antibodies or antibody fragments thereof 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 customarily bound by a pair of immunoglobulin heavy-chain (VH) and light-chain (VL) domains. An epitope may define the minimal binding site of an antibody and thus represent the target of antibody specificity.

[0081]

[0098] Similarly, in another embodiment, the inflammasome is an 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 of 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 includes providing a therapeutically effective amount of a composition comprising an antibody or active fragment thereof provided herein that specifically binds to at least one component (e.g., ASC) of a mammalian inflammasome (e.g., AIM2 inflammasome); and administering the composition to a mammal suffering from CNS or 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); 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 levels of one or more inflammasome components (e.g., ASC). In yet another embodiment, the method includes providing a therapeutically effective amount of a composition comprising an antibody that specifically binds to at least one component (e.g., ASC) of a mammalian inflammasome (e.g., AIM2 inflammasome); and administering the composition to a mammal suffering from CNS or pulmonary inflammation or MS, wherein administering the composition to the mammal results in a reduction of 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 method provided herein further comprises detecting the level or activity of one or more components of a mammalian inflammasome in a sample from a subject suspected of having CNS or pulmonary inflammation or MS. The method for detecting the level or activity comprises measuring the level of at least one inflammasome protein (e.g., ASC or AIM2) in a sample obtained from the subject; and determining the presence or absence of an elevated level or activity of the at least one inflammasome protein (e.g., ASC or AIM2). The level or activity of the at least one inflammasome protein may be 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 may be elevated relative to a predetermined reference value or range of reference values. The at least one inflammasome protein may 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 can 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 methods described herein for reducing inflammation in the CNS and / or lungs of a mammal include compositions comprising the antibodies provided herein or active fragments thereof that specifically bind to at least one component (e.g., ASC, AIM2) of a mammalian inflammasome (e.g., AIM2 inflammasome). Compositions for treating and / or reducing inflammation in the CNS and / or lungs of a mammal may further comprise at least one pharmaceutically acceptable carrier or diluent. Exemplary antibodies directed against components of a mammalian inflammasome for use in the methods herein may be those found in U.S. Patent No. 8,685,400, the contents of which are incorporated herein by reference in their entirety. Also provided herein are exemplary monoclonal antibodies or antibody fragments (e.g., monoclonal antibodies or antibody fragments comprising a VH region, the amino acid sequence of which comprises an HCDR1 of SEQ ID NO: 6, an HCDR2 of SEQ ID NO: 7, and an HCDR3 of SEQ ID NO: 8, and a VL region, the amino acid sequence of which comprises an LCDR1 of SEQ ID NO: 12, an LCDR2 of SEQ ID NO: 13, and an LCDR3 of SEQ ID NO: 14).

[0086]

[0103] In one embodiment, a composition for treating and / or reducing inflammation in the CNS or lungs of a mammal comprises an antibody or active fragment thereof provided herein that specifically binds to a domain or portion thereof of a mammalian ASC protein, such as a 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 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, clone 2EI-7 mouse monoclonal antibody from MilliporeSigma, AB3607-anti-ASC antibody from MilliporeSigma, orb194021 anti-ASC from Biorbyt, LS-C331318-50 anti-ASC from LifeSpan Biosciences, AF3805 anti-ASC from R&D Systems, NBP1-78977 anti-ASC from Novus Biologicals, 600-401-Y67 anti-ASC from Rockland Immunochemicals, D086-3 anti-ASC from MBL International, AL177 anti-ASC from Adipogen, monoclonal anti-ASC (clone o93E9) antibody, anti-ASC antibody (F-9) from Santa Cruz Biotechnology, anti-ASC antibody (B-3) from Santa Cruz Biotechnology, Enzo Life Examples of antibodies include ASC polyclonal antibody ADI-905-173 from Biosciences, or A161 anti-human ASC from Leinco Technologies. Human ASC proteins can be under accession numbers NP_037390.2 (Q9ULZ3-1), NP_660183 (Q9ULZ3-2), or Q9ULZ3-3. Rat ASC proteins can be under accession numbers NP_758825 (BAC43754). Mouse ASC proteins can be under accession numbers 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 with the PYD domain of human, mouse, or rat ASC, or a fragment thereof. In one embodiment, the antibody binds to the C-terminal caspase 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 with 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 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 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 pneumocytes). 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 damage or disorders can include TBI, SCI, stroke, amyotrophic lateral sclerosis (ALS) Lou Gehrig's disease, multiple sclerosis (MS), immune dysfunction, muscle CNS degradation, muscular dystrophy (MD), Alzheimer's disease (AD), and Parkinson's disease (PD).

[0087]

[0104] In certain embodiments, the present invention provides antibodies and antibody fragments that specifically bind to ASC and comprise one or more amino acid sequences set forth in Table 2. Also provided herein are isolated nucleic acid molecules encoding monoclonal antibodies or antibody fragments thereof, comprising a nucleic acid sequence set forth in Table 2. In some examples, an expression vector comprises 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 the heavy chain and kappa light chain of IgG4(S241P). The heavy or light chain nucleic acid molecule may be operably linked to suitable regulatory sequences 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 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 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 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 use of the monoclonal antibody or antibody fragment thereof in a method for treating inflammation in a subject. The inflammation may be innate immune inflammation. The inflammation may be inflammasome-associated inflammation. The inflammation may be 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 disease, multiple sclerosis (MS), immune dysfunction, muscle CNS degeneration, muscular dystrophy (MD), Alzheimer's disease (AD), and Parkinson's disease (PD)). Use of this monoclonal antibody or antibody fragment thereof in methods 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 provided herein.

[0094]

[0107] In one embodiment, 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 or kappa chain variable (VL) region, wherein 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 addition to this embodiment, provided herein is use of the monoclonal antibody or antibody fragment thereof in a method for treating inflammation in a subject. The inflammation may be innate immune inflammation. The inflammation may be inflammasome-associated inflammation. The inflammation may be 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 disease, multiple sclerosis (MS), immune dysfunction, muscle CNS degeneration, muscular dystrophy (MD), Alzheimer's disease (AD), and Parkinson's disease (PD)). Use of this monoclonal antibody or antibody fragment thereof in methods 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 provided herein.

[0095]

[0108] In one embodiment, 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 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 an amino acid sequence 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 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 use of the monoclonal antibody or antibody fragment thereof in a method for treating inflammation in a subject. The inflammation may be innate immune-mediated 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 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 disease, multiple sclerosis (MS), immune dysfunction, muscle CNS degeneration, muscular dystrophy (MD), Alzheimer's disease (AD), and Parkinson's disease (PD)). Use of this monoclonal antibody or antibody fragment thereof in a method for 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, which may be a pharmaceutical composition as provided herein.

[0096]

[0109] In one embodiment, 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 or kappa chain variable (VL) region, wherein the amino acid sequence of the VH region comprises SEQ ID NO: 18 or 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 comprises SEQ ID NO: 28 or 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 use of the monoclonal antibody or antibody fragment thereof in a method for treating inflammation in a subject. The inflammation may be innate immune inflammation. The inflammation may be inflammasome-associated inflammation. The inflammation may be 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 disease, multiple sclerosis (MS), immune dysfunction, muscle CNS degeneration, muscular dystrophy (MD), Alzheimer's disease (AD), and Parkinson's disease (PD)). Use of this monoclonal antibody or antibody fragment thereof in methods 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 provided herein.

[0097]

[0110] In one embodiment, 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 or kappa chain variable (VL) region, wherein the amino acid sequence of the VH region comprises SEQ ID NO: 18 or 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 comprises SEQ ID NO: 29 or 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 use of the monoclonal antibody or antibody fragment thereof in a method for treating inflammation in a subject. The inflammation may be innate immune inflammation. The inflammation may be inflammasome-associated inflammation. The inflammation may be 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 disease, multiple sclerosis (MS), immune dysfunction, muscle CNS degeneration, muscular dystrophy (MD), Alzheimer's disease (AD), and Parkinson's disease (PD)). Use of this monoclonal antibody or antibody fragment thereof in methods 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 provided herein.

[0098]

[0111] In one embodiment, 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 or kappa chain variable (VL) region, wherein the amino acid sequence of the VH region comprises SEQ ID NO: 18 or 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 comprises SEQ ID NO: 30 or an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 30. In addition to this embodiment, provided herein is use of the monoclonal antibody or antibody fragment thereof in a method for treating inflammation in a subject. The inflammation may be innate immune inflammation. The inflammation may be inflammasome-associated inflammation. The inflammation may be 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 disease, multiple sclerosis (MS), immune dysfunction, muscle CNS degeneration, muscular dystrophy (MD), Alzheimer's disease (AD), and Parkinson's disease (PD)). Use of this monoclonal antibody or antibody fragment thereof in methods 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 provided herein.

[0099]

[0112] In one embodiment, 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 or kappa chain variable (VL) region, wherein the amino acid sequence of the VH region comprises SEQ ID NO: 18 or 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 comprises SEQ ID NO: 31 or an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 31. In addition to this embodiment, provided herein is use of the monoclonal antibody or antibody fragment thereof in a method for treating inflammation in a subject. The inflammation may be innate immune inflammation. The inflammation may be inflammasome-associated inflammation. The inflammation may be 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 disease, multiple sclerosis (MS), immune dysfunction, muscle CNS degeneration, muscular dystrophy (MD), Alzheimer's disease (AD), and Parkinson's disease (PD)). Use of this monoclonal antibody or antibody fragment thereof in methods 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 provided herein.

[0100]

[0113] In one embodiment, 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 or kappa chain variable (VL) region, wherein the amino acid sequence of the VH region comprises SEQ ID NO: 19 or comprises 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 comprises SEQ ID NO: 28 or comprises 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 use of the monoclonal antibody or antibody fragment thereof in a method for treating inflammation in a subject. The inflammation may be innate immune inflammation. The inflammation may be inflammasome-associated inflammation. The inflammation may be 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 disease, multiple sclerosis (MS), immune dysfunction, muscle CNS degeneration, muscular dystrophy (MD), Alzheimer's disease (AD), and Parkinson's disease (PD)). Use of this monoclonal antibody or antibody fragment thereof in methods 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 provided herein.

[0101]

[0114] In one embodiment, 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 or kappa chain variable (VL) region, wherein the amino acid sequence of the VH region comprises SEQ ID NO: 19 or comprises 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 comprises SEQ ID NO: 29 or comprises 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 use of the monoclonal antibody or antibody fragment thereof in a method for treating inflammation in a subject. The inflammation may be innate immune inflammation. The inflammation may be inflammasome-associated inflammation. The inflammation may be 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 disease, multiple sclerosis (MS), immune dysfunction, muscle CNS degeneration, muscular dystrophy (MD), Alzheimer's disease (AD), and Parkinson's disease (PD)). Use of this monoclonal antibody or antibody fragment thereof in methods 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 provided herein.

[0102]

[0115] In one embodiment, 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 or kappa chain variable (VL) region, wherein the amino acid sequence of the VH region comprises SEQ ID NO: 19 or an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 19, and the amino acid sequence of the VL region comprises SEQ ID NO: 30 or an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 30. In some examples, a monoclonal antibody or antibody fragment derived from this monoclonal antibody comprising an amino acid sequence of a VH region comprising SEQ ID NO: 19 and an amino acid sequence of a VL region comprising SEQ ID NO: 30 may be referred to as IC100. 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 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 disease, multiple sclerosis (MS), immune dysfunction muscle CNS degeneration, muscular dystrophy (MD), Alzheimer's disease (AD), and Parkinson's disease (PD)). Use of this monoclonal antibody or antibody fragment thereof in a method for treating inflammation can reduce inflammation in the CNS and / or lungs of a patient. The reduction can be relative to a control (eg, an untreated patient and / or a patient before 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 provided herein.

[0103]

[0116] In one embodiment, 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 or kappa chain variable (VL) region, wherein the amino acid sequence of the VH region comprises SEQ ID NO: 19 or comprises 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 comprises SEQ ID NO: 31 or comprises 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 use of the monoclonal antibody or antibody fragment thereof in a method for treating inflammation in a subject. The inflammation may be innate immune inflammation. The inflammation may be inflammasome-associated inflammation. The inflammation may be 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 disease, multiple sclerosis (MS), immune dysfunction, muscle CNS degeneration, muscular dystrophy (MD), Alzheimer's disease (AD), and Parkinson's disease (PD)). Use of this monoclonal antibody or antibody fragment thereof in methods 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 provided herein.

[0104]

[0117] In one embodiment, 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 or kappa chain variable (VL) region, wherein the amino acid sequence of the VH region comprises SEQ ID NO:20 or 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 comprises SEQ ID NO:28 or 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 use of the monoclonal antibody or antibody fragment thereof in a method for treating inflammation in a subject. The inflammation may be innate immune inflammation. The inflammation may be inflammasome-associated inflammation. The inflammation may be 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 disease, multiple sclerosis (MS), immune dysfunction, muscle CNS degeneration, muscular dystrophy (MD), Alzheimer's disease (AD), and Parkinson's disease (PD)). Use of this monoclonal antibody or antibody fragment thereof in methods 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 provided herein.

[0105]

[0118] In one embodiment, 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 or kappa chain variable (VL) region, wherein the amino acid sequence of the VH region comprises SEQ ID NO:20 or 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 comprises SEQ ID NO:29 or 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 use of the monoclonal antibody or antibody fragment thereof in a method for treating inflammation in a subject. The inflammation may be innate immune inflammation. The inflammation may be inflammasome-associated inflammation. The inflammation may be 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 disease, multiple sclerosis (MS), immune dysfunction, muscle CNS degeneration, muscular dystrophy (MD), Alzheimer's disease (AD), and Parkinson's disease (PD)). Use of this monoclonal antibody or antibody fragment thereof in methods 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 provided herein.

[0106]

[0119] In one embodiment, 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 or kappa chain variable (VL) region, wherein the amino acid sequence of the VH region comprises SEQ ID NO:20 or comprises 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 comprises SEQ ID NO:30 or comprises 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 use of the monoclonal antibody or antibody fragment thereof in a method for treating inflammation in a subject. The inflammation may be innate immune inflammation. The inflammation may be inflammasome-associated inflammation. The inflammation may be 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 disease, multiple sclerosis (MS), immune dysfunction, muscle CNS degeneration, muscular dystrophy (MD), Alzheimer's disease (AD), and Parkinson's disease (PD)). Use of this monoclonal antibody or antibody fragment thereof in methods 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 provided herein.

[0107]

[0120] In one embodiment, 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 or kappa chain variable (VL) region, wherein the amino acid sequence of the VH region comprises SEQ ID NO:20 or 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 comprises SEQ ID NO:31 or an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:31. In addition to this embodiment, provided herein is use of the monoclonal antibody or antibody fragment thereof in a method for treating inflammation in a subject. The inflammation may be innate immune inflammation. The inflammation may be inflammasome-associated inflammation. The inflammation may be 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 disease, multiple sclerosis (MS), immune dysfunction, muscle CNS degeneration, muscular dystrophy (MD), Alzheimer's disease (AD), and Parkinson's disease (PD)). Use of this monoclonal antibody or antibody fragment thereof in methods 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 provided herein.

[0108]

[0121] In one embodiment, 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 or kappa chain variable (VL) region, wherein the amino acid sequence of the VH region comprises SEQ ID NO:21 or an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:21, and the amino acid sequence of the VL region comprises SEQ ID NO:28 or 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 use of the monoclonal antibody or antibody fragment thereof in a method for treating inflammation in a subject. The inflammation may be innate immune inflammation. The inflammation may be inflammasome-associated inflammation. The inflammation may be 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 disease, multiple sclerosis (MS), immune dysfunction, muscle CNS degeneration, muscular dystrophy (MD), Alzheimer's disease (AD), and Parkinson's disease (PD)). Use of this monoclonal antibody or antibody fragment thereof in methods 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 provided herein.

[0109]

[0122] In one embodiment, 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 or kappa chain variable (VL) region, wherein the amino acid sequence of the VH region comprises SEQ ID NO:21 or an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:21, and the amino acid sequence of the VL region comprises SEQ ID NO:29 or 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 use of the monoclonal antibody or antibody fragment thereof in a method for treating inflammation in a subject. The inflammation may be innate immune inflammation. The inflammation may be inflammasome-associated inflammation. The inflammation may be 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 disease, multiple sclerosis (MS), immune dysfunction, muscle CNS degeneration, muscular dystrophy (MD), Alzheimer's disease (AD), and Parkinson's disease (PD)). Use of this monoclonal antibody or antibody fragment thereof in methods 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 provided herein.

[0110]

[0123] In one embodiment, 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 or kappa chain variable (VL) region, wherein the amino acid sequence of the VH region comprises SEQ ID NO:21 or an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:21, and the amino acid sequence of the VL region comprises SEQ ID NO:30 or an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:30. In addition to this embodiment, provided herein is use of the monoclonal antibody or antibody fragment thereof in a method for treating inflammation in a subject. The inflammation may be innate immune inflammation. The inflammation may be inflammasome-associated inflammation. The inflammation may be 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 disease, multiple sclerosis (MS), immune dysfunction, muscle CNS degeneration, muscular dystrophy (MD), Alzheimer's disease (AD), and Parkinson's disease (PD)). Use of this monoclonal antibody or antibody fragment thereof in methods 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 provided herein.

[0111]

[0124] In one embodiment, 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 or kappa chain variable (VL) region, wherein the amino acid sequence of the VH region comprises SEQ ID NO:21 or comprises 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 comprises SEQ ID NO:31 or comprises 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 use of the monoclonal antibody or antibody fragment thereof in a method for treating inflammation in a subject. The inflammation may be innate immune inflammation. The inflammation may be inflammasome-associated inflammation. The inflammation may be 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 disease, multiple sclerosis (MS), immune dysfunction, muscle CNS degeneration, muscular dystrophy (MD), Alzheimer's disease (AD), and Parkinson's disease (PD)). Use of this monoclonal antibody or antibody fragment thereof in methods 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 provided herein.

[0112]

[0125] In one embodiment, 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 or kappa chain variable (VL) region, wherein the amino acid sequence of the VH region comprises SEQ ID NO: 22 or an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 22, and the amino acid sequence of the VL region comprises SEQ ID NO: 28 or 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 use of the monoclonal antibody or antibody fragment thereof in a method for treating inflammation in a subject. The inflammation may be innate immune inflammation. The inflammation may be inflammasome-associated inflammation. The inflammation may be 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 disease, multiple sclerosis (MS), immune dysfunction, muscle CNS degeneration, muscular dystrophy (MD), Alzheimer's disease (AD), and Parkinson's disease (PD)). Use of this monoclonal antibody or antibody fragment thereof in methods 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 provided herein.

[0113]

[0126] In one embodiment, 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 or kappa chain variable (VL) region, wherein the amino acid sequence of the VH region comprises SEQ ID NO:22 or an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:22, and the amino acid sequence of the VL region comprises SEQ ID NO:29 or 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 use of the monoclonal antibody or antibody fragment thereof in a method for treating inflammation in a subject. The inflammation may be innate immune inflammation. The inflammation may be inflammasome-associated inflammation. The inflammation may be 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 disease, multiple sclerosis (MS), immune dysfunction, muscle CNS degeneration, muscular dystrophy (MD), Alzheimer's disease (AD), and Parkinson's disease (PD)). Use of this monoclonal antibody or antibody fragment thereof in methods 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 provided herein.

[0114]

[0127] In one embodiment, 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 or kappa chain variable (VL) region, wherein the amino acid sequence of the VH region comprises SEQ ID NO:22 or an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:22, and the amino acid sequence of the VL region comprises SEQ ID NO:30 or an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:30. In addition to this embodiment, provided herein is use of the monoclonal antibody or antibody fragment thereof in a method for treating inflammation in a subject. The inflammation may be innate immune inflammation. The inflammation may be inflammasome-associated inflammation. The inflammation may be 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 disease, multiple sclerosis (MS), immune dysfunction, muscle CNS degeneration, muscular dystrophy (MD), Alzheimer's disease (AD), and Parkinson's disease (PD)). Use of this monoclonal antibody or antibody fragment thereof in methods 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 provided herein.

[0115]

[0128] In one embodiment, 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 or kappa chain variable (VL) region, wherein the amino acid sequence of the VH region comprises SEQ ID NO:22 or an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:22, and the amino acid sequence of the VL region comprises SEQ ID NO:31 or an amino acid sequence at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:31. In addition to this embodiment, provided herein is 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 can be used in a method for reducing inflammation in a mammal, such as those described in U.S. Patent No. 8,685,400, the contents of which are incorporated herein by reference in their entirety. This 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, the disease, condition, or affliction of or affecting the CNS can be stroke, as well as autoimmune and / or CNS disorders (e.g., amyotrophic lateral sclerosis (ALS), Lou Gehrig's disease, multiple sclerosis (MS), immune dysfunction, muscle CNS degeneration, muscular dystrophy (MD), Alzheimer's disease (AD), and 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, which 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 provided herein.

[0116]

[0129] In another embodiment, the composition for reducing inflammation in the CNS or lungs of a mammal comprises an antibody (e.g., an anti-NLRP1 chicken antibody) or an active fragment thereof provided herein that specifically binds to NLRP1 or a domain thereof. Any suitable anti-NLRP1 antibody can be used, and some are commercially available. Examples of anti-NLRP1 antibodies for use in the methods described 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-NLRP1 antibodies for use in the methods provided herein include, but are not limited to, human NLRP1 polyclonal antibody AF6788 from R&D Systems, EMD Millipore rabbit polyclonal anti-NLRP1 ABF22, Novus Biologicals rabbit polyclonal anti-NLRP1 NB100-56148, Sigma-Aldrich mouse polyclonal anti-NLRP1 SAB1407151, Abcam rabbit polyclonal anti-NLRP1 ab3683, Biorbyt rabbit polyclonal anti-NLRP1 orb325922, mybiosource rabbit polyclonal anti-NLRP1 MBS7001225, R&D systems sheep polyclonal AF6788, Aviva Systems mouse monoclonal anti-NLRP1 oaed00344, Aviva Systems rabbit polyclonal anti-NLRP1 ARO54478_P050, Origene rabbit polyclonal anti-NLRP1 APO7775PU-N, Antibodies online rabbit polyclonal anti-NLRP1 ABIN768983, Prosci rabbit polyclonal anti-NLRP1 3037, Proteintech rabbit polyclonal anti-NLRP1 12256-1-AP, Enzo mouse monoclonal anti-NLRP1 ALX-804-803-C100, Invitrogen mouse monoclonal anti-NLRP1 MA1-25842, GeneTex mouse monoclonal anti-NLRP1 GTX16091, Rockland rabbit polyclonal anti-NLRP1 200-401-CX5, or Cell Signaling Technology rabbit polyclonal anti-NLRP1 4990. The human NLRP1 protein can be accession number AAH51787, NP_001028225, NP_055737, NP_127497, NP_127499, or NP_127500. In one embodiment, the antibody binds to the Pyrin, NACHT, LRR1-6, FIIND, or CARD domain of a mammalian NLRP1 protein (e.g., human NLRP1), or a portion or fragment thereof.In this embodiment, the antibodies described herein specifically bind to an amino acid sequence having at least 65% (e.g., 65, 70, 75, 80, 85%) sequence identity with a defined domain of human NLRP1 (e.g., Pyrin, NACHT, LRR1-6, FIIND, or CARD) or a fragment thereof. In one embodiment, a chicken anti-NLRP1 polyclonal antibody custom designed and produced by Ayes Laboratories is used to reduce pulmonary inflammation. This antibody may be directed against the following amino acid sequence in human NLRP1: CEYYTEIREREREKSEKGR (SEQ ID NO: 3). In one embodiment, an antibody that binds to the NLRP1 domain or a fragment thereof described herein inhibits NLRP1 activity in mammalian lung cells, such as type II pneumocytes.

[0117]

[0130] In yet another embodiment, a composition for reducing inflammation in the CNS or lungs of a mammal comprises an antibody or 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 anti-AIM2 catalog number 20590-1-AP from Proteintech, Abcam anti-AIMS antibody (ab119791), rabbit polyclonal anti-AIM2 (N-terminal region) catalog number AP3851 from ECM biosciences, rabbit polyclonal anti-ASC catalog number E-AB-30449 from Elabsciences, anti-AIM2 mouse monoclonal antibody designated AIM2 antibody (3C4G11) from Santa Cruz Biotechnology, catalog number sc-293174, mouse monoclonal AIM2 antibody from Origene, catalog number TA324972, and Thermofisher. AIM2 monoclonal antibody (10M2B3) from Scientific, AIM2 rabbit polyclonal antibody ABIN928372 or ABIN760766 from Antibodies-online, Biomatix Coat anti-AIM2 polyclonal antibody, catalog number CAE02153, anti-AIM2 polyclonal antibody (OABF01632) from Aviva Systems Biology, rabbit polyclonal anti-AIM2 antibody LS-C354127 from LSBio-C354127, rabbit monoclonal anti-AIM2 antibody from Cell Signaling Technology, catalog number MA5-16259, rabbit polyclonal anti-AIM2 monoclonal antibody from Fab Gennix International Incorporated, catalog number AIM2 201AP, MyBiosource rabbit polyclonal anti-AIM2 catalog number MBS855320, Signalway rabbit polyclonal anti-AIM2 catalog number 36253, NovusExamples include Biological rabbit polyclonal anti-AIM2 catalog number 43900002, GeneTex rabbit polyclonal anti-AIM2 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 ab76423, Sigma Aldrich mouse polyclonal anti-AIM2 SAB1406827, or Biolegend anti-AIM2 3B10. Human AIM2 protein can be under the accession 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 part 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, such as type II pneumocytes.

[0118]

[0131] Anti-inflammasome (e.g., anti-ASC, anti-NLRP1, or anti-AIM2) antibodies described herein include polyclonal and monoclonal rodent antibodies, polyclonal and monoclonal human antibodies, or any portion thereof, that have 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 raised against an epitope of a polypeptide and is specific for ASC when it binds to at least a portion of a natural or recombinant protein.

[0119]

[0132] In certain embodiments, the antibodies provided herein include polypeptides having one or more amino acid substitutions, deletions, or insertions. For example, anti-ASC monoclonal antibodies or ASC-binding antibody fragments include polypeptides having one or more amino acid substitutions, deletions, or insertions compared to a polypeptide having one or more amino acid sequences selected from 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, anti-ASC monoclonal antibodies or ASC-binding antibody fragments may have one, two, three, four, five, six, seven, eight, nine, ten, or more amino acid substitutions, deletions, or insertions. Substitutions, deletions or insertions may be introduced by standard techniques, such as site-directed mutagenesis or PCR-mediated mutagenesis of a nucleic acid molecule encoding an anti-ASC antibody or ASC-binding antibody fragment polypeptide.

[0120]

[0133] In certain embodiments, conservative amino acid substitutions are made at one or more positions in the amino acid sequence of an antibody or antibody fragment 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, a string of amino acids can be replaced with a structurally similar string that differs in the order and / or composition of side chain family members. A skilled artisan would be able to 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 amino acid sequences of SEQ ID NOs: 6-8, 12-14, 18-22, or 28-31 binds to an ASC protein by using routine art-recognized methods (e.g., but not limited to, ELISA, Western blot, phage display, etc.).

[0121]

[0134] Calculations of sequence homology or sequence identity between sequences (the terms are used interchangeably herein) may be performed as follows.

[0122]

[0135] To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., for optimal alignment, gaps can be introduced into one or both of the first and second amino acid or nucleic acid sequences, and non-homologous sequences can 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, 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] Comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In one embodiment, percent identity between two amino acid sequences is determined using the algorithm of Needleman et al. ((1970) J. Mol. Biol. 48:444-453) as incorporated into the GAP program in the GCG software package (available at www.gcg.com) using either a BLOSUM 62 matrix or a PAM250 matrix, and gap weights of 16, 14, 12, 10, 8, 6, or 4 and length weights of 1, 2, 3, 4, 5, or 6. In yet another embodiment, percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at www.gcg.com) using a NWSgapdna CMP matrix, and gap weights of 40, 50, 60, 70, or 80 and length weights of 1, 2, 3, 4, 5, or 6. One set of parameters (and which may be used when the expert is unsure which parameters to apply to determine whether a molecule is within the sequence identity or sequence homology limits of the invention) 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 may be determined using the algorithm of Meyers et al. ((1989) CABIOS 4:11-17) as incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4.

[0125]

[0138] In certain embodiments, the antibody is a monoclonal antibody. In other embodiments, the 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 antibodies (anti-ASC monoclonal antibodies or ASC binding antibody fragments) of the present invention are humanized, chimeric or human.

[0127]

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

[0128]

[0141] "Humanized antibody," as the term is used herein, refers to an antibody that has been engineered to contain one or more human framework regions in the variable regions along with non-human (e.g., mouse, rat, or hamster) complementarity-determining regions (CDRs) of the heavy and / or light chains. In certain embodiments, a humanized antibody comprises sequences that are entirely 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, humanized antibodies may comprise 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 will comprise 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 those of a human immunoglobulin consensus sequence. The RF regions may be modified by any method known in the art and / or provided herein. The modifications may confer desirable properties (e.g., increased half-life and / or improved expression in host cells). In one embodiment, the FR regions may be modified or mutated as described in U.S. Patent Application 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 comprises at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin.

[0129]

[0142] Humanized antibodies are typically less immunogenic to humans than non-humanized antibodies, and therefore offer therapeutic advantages in certain situations. For example, the constant region of an antibody can be engineered to be immunologically inert (e.g., not induce complement lysis). See, for example, PCT Publication No. PCT / GB99 / 01441; UK Patent Application No. 9809951.8 (each of which is incorporated herein by reference in its entirety). Those skilled in the art will recognize humanized antibodies and will also recognize techniques suitable for producing such humanized antibodies. For example, Hwang, WYK, et al., Methods 36:35, 2005;Queen et al., Proc. Natl. Acad. Sci. 1988;Verhoeyen et al., Science, 239:1534-36, 1988;Orlandi et al., Proc. Natl. Acad. Sci. USA, 86:3833-37, 1989; U.S. Patent Nos. 5,225,539; 5,530,101; 5,585,089; 5,693,761; 5,693,762; 6,180,370; and Selick et al., WO 90 / 07861 (each of which is incorporated by reference in its entirety). Other methods for humanizing antibodies that may also be utilized are disclosed by Daugherty et al., Nucl. Acids Res. 19:2471-2476, 1991, and in U.S. Pat. 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 anti-ASC antigen-binding fragment of the present invention may comprise an amino acid sequence of a VH region comprising HCDR1 of SEQ ID NO: 6, HCDR2 of SEQ ID NO: 7, and HCDR3 of SEQ ID NO: 8, an amino acid sequence of a VL region comprising LCDR1 of SEQ ID NO: 12, LCDR2 of SEQ ID NO: 13, and LCDR3 of SEQ ID NO: 14, and one or more human framework region sequences.

[0130]

[0143] In some embodiments, the antibody of the present invention is a chimeric antibody and specifically binds to ASC. In some examples, this anti-ASC chimeric antibody reduces the activity of ASC. As used herein, a "chimeric antibody" refers to an antibody that has been engineered to contain 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., an IgG1 human constant region). Chimeric antibodies are typically less immunogenic in humans than non-chimeric antibodies, and therefore offer therapeutic advantages in certain situations. Those skilled in the art will recognize chimeric antibodies and techniques suitable for generating such chimeric antibodies. See, e.g., Cabilly et al., U.S. Patent No. 4,816,567; Shoemaker et al., U.S. Patent No. 4,978,775; Beavers et al., U.S. Patent No. 4,975,369; and Boss et al., U.S. Patent No. 4,816,397 (each of which is incorporated by reference herein 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 characteristics" refer to the type of noncovalent interactions that occur between an immunoglobulin molecule (e.g., an antibody) and an 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 characteristics 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 depend on the concentrations of the complex partners, the affinity of the interaction, and geometric parameters that affect the rate equally in both directions. Thus, both the "on-rate constant" (Kon) and the "off-rate constant" (Koff) can be determined by calculating the concentration and the actual association and dissociation rates. (See Nature 361:186-87 (1993)). The ratio Koff / Kon allows for the elimination of all parameters unrelated to affinity and is equal to the dissociation constant Kd. (See generally Davies et al. (1990) Annual Rev Biochem 59:439-473.) Antibodies of the invention are said to specifically bind to an epitope (e.g., an ASC fragment having the amino acid sequence of SEQ ID NO: 5) if they have 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 those of skill in the art.

[0132]

[0145] In certain embodiments, the antibodies of the present invention are monovalent or bivalent and comprise single or double chains. 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 can be found 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.

[0134]

[0147] Anti-inflammasome (e.g., anti-ASC and anti-AIM2) antibodies of the invention can be routinely produced by methods such as, but not limited to, inoculation of appropriate animals with polypeptides or antigenic fragments, in vitro stimulation of lymphocyte populations, synthetic methods, hybridomas, and / or recombinant cells expressing nucleic acids encoding such anti-ASC or anti-NLR1 antibodies. Immunization of animals with purified recombinant ASC or a peptide fragment thereof (e.g., residues 178-193 (SEQ ID NO: 1) of rat ASC (e.g., Accession No. BAC43754), EQ ID NO: 2 of human ASC, or residues 21-41 (SEQ ID NO: 5) of human ASC (e.g., Accession No. NP_037390.2)) is an example of a method for preparing anti-ASC antibodies. Similarly, immunization of animals with purified recombinant NLRP1 or 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 can be obtained by methods known to those skilled in the art. See, for example, Kohler and Milstein, Nature 256:495-497, 1975; U.S. Patent No. 4,376,110; Ausubel et al., eds., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley Interscience, NY, (1987, 1992); Harlow and Lane 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), the contents of which are incorporated herein by reference in their entirety. Such antibodies may belong to any immunoglobulin class, such as IgG, IgM, IgE, IgA, GILD, and any subclass thereof. Hybridomas producing the monoclonal antibodies of the present disclosure can be cultured in vitro, in situ, or in vivo. In one embodiment, a hybridoma producing an anti-ASC monoclonal antibody of the present disclosure is an ICCN1.OH hybridoma. In another embodiment, a hybridoma producing an 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 sequences of the VH region are HCDR1 of SEQ ID NO: 6, HCDR2 of SEQ ID NO: 7, and HCDR3 of SEQ ID NO: 8, or variants thereof, with 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, wherein the amino acid sequences of the VL region are LCDR1 of SEQ ID NO: 12, LCDR2 of SEQ ID NO: 13, and LCDR3 of SEQ ID NO: 14, or variants thereof, including variants 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 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, anti-ASC antibodies can be formulated in a pharmaceutically 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 pharmaceutically acceptable carriers and diluents, as well as pharmaceutical formulations, can be found in standard textbooks in this field, such as Remington's Pharmaceutical Sciences and USP / NF. 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 can be by inhalation or parenterally (e.g., intravenous, subcutaneous, intratumoral, intramuscular, intraperitoneal, or intrathecal introduction). The compositions can 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 vascularly accessible site. The compositions can 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 can be administered to a mammal (e.g., a rat, a human) in an effective amount, i.e., an amount capable of producing a desired result in the treated mammal (e.g., reducing inflammation in the CNS of a mammal that has 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 can be determined as described below. 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, 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 to about 0.1 mg / kg, about 0.1 mg / kg to about 1 mg / kg, about 1 mg / kg to about 10 mg / kg, about 10 mg / kg to about 25 mg / kg, about 25 mg / kg to about 50 mg / kg, about 50 mg / kg to about 75 mg / kg, about 75 mg / kg to about 100 mg / kg, about 100 mg / kg to about 125 mg / kg, about 125 mg / kg to about 150 mg / kg, about 150 mg / kg to about 175 mg / kg, or 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) can be administered in single or multiple doses.

[0139]

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

[0140]

[0153] As is well known in the medical and veterinary fields, the dosage for any one subject 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. [Example]

[0141] Example

[0154] The present invention is further described by the following specific examples, which are provided for illustrative purposes only 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 effects of its neutralization

[0155] Pulmonary dysfunction is a common 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. Previous literature supports the idea that post-TBI pulmonary dysfunction results from a sympathetic response to increased intracranial pressure, which leads to cardiopulmonary dysfunction (42). However, more recent studies have shown that the systemic inflammatory response also plays an important role in TBI-induced lung injury (43). Specifically, the HMGB1-RAGE ligand receptor pathway serves as a central transmitter of post-TBI pulmonary dysfunction (8). Furthermore, HMGB1 induces AIM2 inflammasome activation (37). Further previous literature reveals that pathogens secrete DAMPs, such as HMGB1-carrying EVs, which trigger inflammation (Buzas et al., 2014). Various studies have shown that the blood-brain barrier (BBB) ​​becomes permeable after TBI as early as 3–6 h after injury, resulting in damage to the protective barrier between the brain and intravascular compartments and resulting in the leakage of proteins and fluids (44). Post-injury BBB disruption leads to the secretion of inflammatory mediators, such as DAMPs, which can promote brain inflammation and damage distal organs (5). While some inflammatory mediators can act as distinct markers of brain injury, their validity is not widely recognized (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, such as lung injury (46) and TBI (47). Increased inflammasome proteins have already been shown in EVs isolated from the cerebrospinal fluid of patients with TBI compared with control samples (14). In this study, 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 University of Miami Miller School of Medicine's Institutional Animal Care and Use Committee (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 weighed 24-32 grams. Mice were prospectively randomized into experimental groups for TBI (sham, 4 h, 24 h) and experimental groups for adoptive transfer and treatment (naive, sham saline, untreated, enoxaparin, anti-ASC). For the TBI experimental group, sham animals underwent surgery but were not injured. For the adoptive transfer treatment study, the sham saline group underwent surgery and received saline as 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 data. All mice were housed in a viral antigen-free (VAF) animal facility at the Lois Pope Life Center at the University of Miami under a 12-hour light-dark cycle and provided food and water ad libitum. The facility performed animal husbandry procedures twice weekly and checked the animals daily. Animals were observed postoperatively; they were kept on a heating pad, and their body temperature was controlled with a rectal probe. Body temperature was maintained at 37°C in the operating room before being transferred to the animal care room.

[0144]

[0157] Before surgery, animals were anesthetized with ketamine and xylazine (intraperitoneally, i.p.). The anesthetized animals were then placed on a heating pad to maintain 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). Injury was induced using an ECCI-6.3 device (Custom Design & Fabrication, Richmond, VA, USA) with a 3 mm impounder at a velocity of 6 m / s, a depth of 0.8 mm, and an impact duration of 150 ms (15). After these procedures, animals 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 preoperative 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 before perfusion. Then, 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 embedded in paraffin and processed into 5 μm sections (16). Right lung tissue was collected for protein isolation and molecular analysis. The 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 filtered through a 5 μm low-binding polyvinylidene difluoride (PVDF) membrane (Millipore). After filtration, the supernatant was centrifuged at 2,700 × g for 8 minutes. 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 pelleted by centrifugation at 2,700 × g for 8 minutes. 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 minutes to crosslink ASC dimers. Finally, an equal volume of 2x Laemmli buffer was added and proteins were analyzed by immunoblotting using commercially available antibodies against ASC and gasdermin-D (GSD).

[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 20-100 mg pieces and centrifuged at 500 × g for 5 minutes. The tissue pieces were homogenized with cytoplasmic extraction reagent and centrifuged at 16,000 × g for 5 minutes. The supernatant (cell extract) was then removed, and the pellet was centrifuged at 16,000 × g for 10 minutes with nuclear extraction reagent (Thermo Scientific). This supernatant, which corresponds to the nuclear fraction, 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. Two-mm sections of right lower lung and right cortical tissue were homogenized in extraction buffer containing a protease and phosphatase inhibitor cocktail (Sigma, St. Louis, MO, USA) and resolved in 4-20% Tris-TGX Criterion precast gels (Bio-Rad, Hercules, CA, USA) using antibodies against caspase-1 (Novus Biologicals), ASC (Santa Cruz), IL-1β (Cell Signaling), IL-18 (Abcam), AIM2 (Santa Cruz), and HMGB1 (Millipore) as described by de Rivero Vaccari et al. (2015) (13). Quantitation of band density was performed using Image Lab, and all data were normalized to β-actin.

[0149] Immunohistochemistry

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

[0150] EV isolation

[0163] EVs were isolated from serum from TBI-injured and injured mice using total exosome extraction solution according to the manufacturer's instructions (Invitrogen). Briefly, 100 μl of each sample was centrifuged at 2000 × g for 30 minutes. The supernatant was then incubated with 20 μl of total exosome extraction (TEI) reagent for 30 minutes at 4°C and then centrifuged at 10,000 × g for 10 minutes 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 (Figure 6).

[0151] Adoptive transfer of EVs

[0164] Serum-derived EVs from C57BL-6 TBI and sham mice were injected into naive C57BL-6 mice via the jugular vein at a dose of 1.0 × 10 particles per gram of body weight. The particle count was measured using Nanosight tracking analysis, and samples were diluted accordingly. Prior to surgery, animals were anesthetized with ketamine and xylene. A 1-2 cm incision was made between the chin and clavicle. The jugular vein was elevated and restrained, and a catheter was then placed. Serum-derived EVs were transplanted, and lung and brain tissues were collected 24 hours after injection for analysis (n = 5).

[0152] Enoxaparin and anti-ASC treatment

[0165] Serum-derived EVs from TBI mice were injected into naive C57-BL6 mice via jugular vein injection. One hour later, enoxaparin (3 mg / kg) (n = 4) and anti-ASC (IC100, 5 mg / kg) (n = 4) were administered to recipient animals. The following groups were used: 1) naive group, which received no treatment; 2) sham saline group, which served as a negative control and received a jugular vein injection of saline alone; 3) untreated group, which received EVs from TBI mice without any treatment and served as a positive control; 4) ENOX group, which received EVs from TBI mice and enoxaparin; and 5) anti-ASC group, which 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. Note that the anti-ASC antibody used in the treatment experiments is a humanized monoclonal antibody against ASC, which recognizes murine, human, and porcine ASC.

[0153] Histological examination and lung injury scoring

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

[0154] statistical analysis

[0167] Data were analyzed using Student's t-test for two groups and one-way ANOVA followed by Tukey's multiple comparison test for more than two groups (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 the mouse brain

[0168] Expression levels of the pro-inflammatory cytokines IL-1β and IL-18, as well as 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 pro-inflammatory cytokine processing and inflammasome protein levels, but research into inflammasome activation in severe TBI has been limited. In this example, after severe CCI, cortical lysates were examined for levels of caspase-1 (Figure 1A,B) (p<0.001), ASC (Figure 1A,C) (p=0.003), IL-18 (Figure 1A,D) (p=0.0042), AIM2 (Figure 1A,F) (p=0.0197), and IL-1β (Figure 1A,G) (p=0.0141) at 4 and 24 h postinjury. Levels of caspase-1, ASC, AIM2, and IL-Iβ peaked at 4 h after CCI and decreased by 24 h. The time course of inflammatory cytokine maturation was slightly different, but peaked by 24 h after TBI. Because others have demonstrated a role for the inflammasome DAMP HMGB1 in activating the AIM2 inflammasome, the levels of these proteins were also determined in cortical lysates. As shown in Figures 1A and 1E, CCI induced a significant increase in the levels of HMGB1 (Figures 1A and 1E) (p = 0.0121) at 4 and 24 h 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, immunoblot analysis of lung lysates was performed for caspase-1 (Figure 1H,I) (p = .0026), ASC (Figure 1H,J) (p = .0427), IL-18 (Figure 1H,K) (p = .0025), IL-1β (Figure 1H,N) (p = .0012), and AIM2 (Figure 1H,M) (p < .001), and NLRP3 (p = .0047) (Supplementary Figure 1). Increased levels of caspase-1, ASC, IL-18, and AIM2 were significantly increased at 4 and 24 hours after injury compared with sham controls. However, the time course of increased protein expression differed slightly 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 lung dysfunction (8), lung lysates were analyzed for levels of HMGB1 protein expression. Figures 1H and 1L (p = .0158) show that HMGB1 expression 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 the lungs of mice

[0170] As previously shown, activation of the AIM2 inflammasome in cortical neurons leads to pyroptotic cell death (19). To investigate whether TBI leads to pyroptosis in mouse lung tissue, pyroptosomes were isolated from lung tissue 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 observed 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 IL-1β release (20), was significantly increased in the lungs of TBI animals compared to sham animals (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 can result in capillary leakage, increased vascular permeability, and damage to specialized alveolar epithelial cells called type II pneumocytes (5). To examine the cellular effects of TBI on inflammasome expression in the lung after injury, immunohistochemical analysis was performed on lung sections from sham, 4-hour, and 24-hour injured animals. Type II alveolar epithelial cells are known to be the predominant type of injured lung cell in ALI (17). Lung sections were stained with antibodies against AIM2, caspase-1, and ASC (green), and co-stained with pro-surfactant protein C (Pro-SPC, red), a marker for type II epithelial cells, and DAPI nuclear stain (blue). 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 increased after TBI. These findings indicate that inflammasome proteins are expressed in type II alveolar epithelial cells and that TBI leads to increased immune reactivity in these cells.

[0159] TBI increases nuclear and cytoplasmic HMGB1 expression

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

[0160] TBI induces changes in lung morphology and ALI

[0173] ALI can be characterized by an inflammatory process resulting in alveolar and interstitial edema and infiltration of inflammatory cells into the alveolar space (23). Histopathological analysis of lung tissue (Figure 5A) shows that severe TBI induces considerable changes in lung structure and morphology at 4 and 24 h after injury. Sham animals exhibited normal alveolar morphology, while injured animals showed a rapid increase in alveolar edema, which slightly decreased by 24 h after injury (long arrows). Furthermore, there was evidence of neutrophil infiltration (arrowheads) and altered alveolar-capillary membrane morphology (*) at both time points. Injured animals showed signs of interstitial edema, which was more pronounced at 4 h after injury but was still evident at 24 h after injury (short arrows). Finally, injured animals also showed evidence of thickening of the interstitial areas and alveolar septa (lb, #).

[0161]

[0174] To confirm 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 animals (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] To provide evidence that EVs and their cargoes, which may be released into the circulation after TBI, can induce inflammasome activation in the lung, we performed a classical adoptive transfer experiment using serum-derived EVs from mice with severe CCI. EV preparations were validated using Western blots for the EV marker CD81 (Figure 6). Controls received EVs isolated from sham and naive 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 with the lungs of uninjured or naive mice or animals receiving EVs from naive mice. Furthermore, inflammatory cell infiltration (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, contributing to the pathology of ALI.

[0163]

[0176] Next, we attempted to block exosome uptake by treatment with either enoxaparin or a monoclonal antibody against ASC (IC100) after adoptive transfer of EVs from injured mice to naive mice. Negative control animals received saline, and positive control animals received no treatment. As shown in Figures 8A–8F, caspase-1 (Figures 8A, 8B), ASC (Figures 8A, 8C), IL-1β (Figures 8A, 8D), AIM2 (Figures 8A, 8E), and HMGB1 (Figures 8A, 8F) were significantly reduced after treatment with enoxaparin or a 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 (Figures 9A–D). The ALI scores for animals treated with enoxaparin and anti-ASC antibody (IC100) were significantly lower than those for the untreated group (Figure 9E) (p=<.0001). Thus, EVs released into the circulation after TBI play a role in inflammasome activation in lung cells, leading to ALI.

[0164] conclusion

[0177] TBI may be associated with a higher rate of certain medical complications, particularly pulmonary and central nervous system dysfunction. In this example, we show that severe TBI increases HMGB1 and inflammasome expression (e.g., AIM2, caspase-1, and ASC expression) in cortical and lung tissue and induces 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 concept of a neurorespiratory-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 inflammasome protein cargo, 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 plays a central role in the pathogenesis of lung injury after TBI and demonstrates a mechanism for TBI-induced ALI involving EV-mediated inflammasome signaling. These data provide evidence that EV-mediated inflammasome signaling may play a central role in the neuronal-respiratory-inflammatory axis. Therefore, targeting this axis with antibodies against inflammasome proteins or drugs that block EV uptake may provide a therapeutic approach for neurotrauma-induced ALI in all areas of critical care medicine. In light of these results, the disclosed therapeutic strategies 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.

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[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, we examined the role of EVs isolated from human TBI patients on inflammasome signaling in human pulmonary endothelial cells.

[0168]

[0231] In the first experiment, serum-derived EVs were isolated from TBI and control patients using a whole exosome extraction kit (Thermofisher). Human pulmonary microvascular endothelial cells (HMVEC-Lonza) were cultured and plated on 12-well plates. After reaching confluency, isolated EVs from TBI and control patients were delivered to the cells (1.94 × 10 particles / ml) for a 4-hour incubation period. After incubation, cells were harvested with 200 μl of lysis buffer, and cell lysates were used for Western blot analysis.

[0169]

[0232] In the second experiment, serum-derived EVs were isolated from TBI and control patients using a whole exosome extraction kit (Thermofisher). Human pulmonary microvascular endothelial cells (HMVEC-Lonza) were cultured and plated on 96-well plates. After reaching confluency, isolated EVs from TBI and control patients were delivered to the cells (1.94 × 10 particles / ml) for a 3-hour incubation period and then incubated with caspase-1 FAM FLICA (Immunohistochemistry Technologies) at a 1:30 volume / volume ratio for an additional 1 hour. After incubation, the medium was removed, and the cells were washed three times with apoptosis wash buffer (Immunohistochemistry Technologies). Cells were then co-stained with Hoechst for nuclear staining and propidium iodide for cell death. Images were taken using an EVOS microscope, and the cells were then read under a fluorescence 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 increased inflammasome protein expression in lung endothelial cells. Figures 10A-10E showed that caspase-1, ASC, AIM2, and HMGB1 were elevated in PMVECs incubated with TBI-EVs for 4 hours compared to PMVECs incubated with control EVs for 4 hours. Immunoassay results showed a significant increase in IL-1 beta expression using the Ella Simpleplex assay (Figure 10F).

[0171]

[0234] As shown in Figures 11A-11C, delivery of TBI-EVs to lung endothelial cells increased caspase-1 immunoreactivity and cell death.

[0172] conclusion

[0235] These studies provided further evidence for a neuronal-respiratory-inflammasome axis in which EVs released into the circulation after TBI activate inflammasomes in lung target cells that contribute to the pathology of ALI.

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

[0236] To determine the utility of a humanized anti-ASC monoclonal antibody in treating MS, the antibody was administered to mice with experimental allergic encephalomyelitis (EAE). EAE is an animal (i.e., rodent) model of MS, as described 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 Lassmann 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.

[0174] method Induction of EAE and treatment with IC100

[0237] Active EAE was induced in 2-month-old C57BL / 6 female mice with myelin oligodendrocyte glycoprotein 35-55 peptide (MOG35-55, BioSynthesis) as previously described (Brambilla et al., 2014). Briefly, mice were intraperitoneally (i.p.) injected 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 another i.p. injection of pertussis toxin (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 i.p. injection every 4 days, starting on day 8 after EAE induction. 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, spinal cords were harvested and placed in cold Mg2+- and Ca2+-free Hank's balanced salt solution (HBSS w / o). Samples were manually dissociated into single-cell suspensions by passing them through a 70-μm strainer and washed with HBSS w / o. Spleen samples were spun at 1200 rpm for 10 min at 4°C, the supernatant removed, and red blood cells (RBCs) were lysed in 2 ml of RBC lysis buffer (eBioscience) according to the manufacturer's instructions. Spleen cells were then resuspended in PBS. Cells isolated from spinal cords 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 splenocytes, spinal cord cells were resuspended in PBS and stained as described below.

[0176] Immunolabeling and flow cytometry analysis

[0239] In experiments evaluating caspase-1, the FAM FLICA™ Caspase 1 kit was used (BioRad) according to the manufacturer's instructions. Cells were incubated in FLICA solution (BioRad) for 30 minutes at 4°C, washed with Apoptosis Wash Buffer (BioRad), and resuspended in 1 ml of PBS. Samples were then incubated with a fixative live / dead stain (Tonbo Biosciences) for 30 minutes at 4°C, spun at 1200 rpm for 10 minutes at 4°C, and the supernatant was removed. Cells were resuspended in 100 μl of FACS buffer, blocked with anti-CD16 / 32 (FcR block, eBioscience) for 5 minutes at room temperature, immunostained for 30 minutes at 4°C, and fixed with 1% PFA. Samples were analyzed using a CytoFLEX S flow cytometer (Beckman Coulter) equipped with CytExpert 2.1 software. The number of leukocytes in the spinal cord was determined using 123count eBeads (eBioscience). The number of leukocytes in the spleen was determined by flow cytometry combined with trypan blue exclusion counting using a TC20™ Automated Cell Counter (Bio-Rad). A list of antibodies for flow cytometry is listed in Table 3 below.

[0177]

[0240] [Table 11]

[0178]

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

[0242] Paraformaldehyde (PFA)-fixed segments of spinal cord were embedded in paraffin, sectioned into 10-mm-thick sections using a Leica RM 2135 microtome, and stained with Luxol Fast Blue (LFB). Ten serial sections spaced 50 μm apart were used to estimate the volume of demyelinated white matter. The demyelinated area was outlined using an Olympus BX51 microscope, and the volume of demyelinated white matter was quantified using Stereoinvestigator software (MicroBrightfield). 3D reconstruction of the demyelinated spinal cord was performed on the same serial sections using Neurolucida software (MBF Bioscience).

[0179] Quantification of IC100 in tissues

[0243] IC100 was quantified in brain, spinal cord, liver, and spleen at 35 days post-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, similar experiments were performed in a rat model of crush cervical spinal cord injury.

[0181]

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

[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 induced with EAE using the MOG35-55 peptide (Brambilla et al., 2014) and administered IC100 or vehicle alone starting 8 days post-induction of the disease (dpi). This 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] When used at doses of 30 and 45 mg / Kg, IC100 significantly improved functional recovery, accompanied by a robust reduction in clinical disease scores throughout the duration of the experiment (Figure 12A). Treatment reduced the mean peak clinical score (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 day the mice reached their peak disease score (Figure 12E).

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

[0248] The onset, persistence, and severity of EAE clinical symptoms directly correlate with immune cell infiltration into the spinal cord. To assess whether IC100 affected this process, immune cell populations isolated from the spinal cord at 35 dpi were characterized by flow cytometry. Treatment with 30 mg / kg IC100 significantly reduced the total numbers 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 toward reduction. In the spleen, no significant differences in cell numbers were observed at either dose of IC100, suggesting that treatment did not interfere with 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. As their activation state increases, microglia proliferate and upregulate MHCII surface expression. To assess whether IC100 affected 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 30 mg / kg IC100, indicating that at this dose, IC100 is effective in suppressing microglial activation and microglial-mediated neuroinflammation (see Figure 14).

[0186] IC100 penetrates the brain and spinal cord

[0250] A key parameter in designing a drug to reverse MS is determining whether the drug penetrates the CNS at therapeutic levels. This is an important feature, especially in the treatment of progressive MS, because the blood-brain barrier appears to be relatively intact at this stage of the disease (Lassman et al. 2012). Therefore, we harvested the brain, spinal cord, liver, and spleen and determined IC100 levels in these tissues. As shown in Figure 15, IC100 penetrated all of these tissues, including the brain and spinal cord, at all three doses. Interestingly, IC100 levels in the spinal cord were highest at the 30 mg / kg dose, consistent with the greater therapeutic effect at this dose.

[0187]

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

[0188]

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

[0189]

[0253] Anti-ASC antibodies (e.g., IC100) also penetrated spinal cord neurons in a rat model of crush cervical spinal cord injury.

[0190]

[0254] IC100 can function both intracellularly and extracellularly. Intracellularly, IC100 can 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 can 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-IL-β, which is involved in perpetuating inflammation in chronic inflammatory diseases.

[0191] Incorporation by Reference

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

[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] Kinetic analysis of candidate anti-ASC monoclonal antibodies was performed using biolayer interferometry (BLI), in which association and dissociation from a surface causes a shift in the wavelength of reflected light, which, when measured over time, allows the determination of binding kinetics.

[0195]

[0259] The BLI assay consisted of:

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

[0196]

[0261] Candidate mouse IgG antibody supernatants were tested for binding to human ASC peptide (SEQ ID NO: 5) at seven different concentrations (i.e., 540 nM; 180 nM; 60 nM; 20 nM; 6.67 nM; 2.22 nM; 0.741 nM). The antibodies tested were ICCN 1.0H (i.e., IC100); ICCN 2.0H; and ICCN 3.0H. An AMC (anti-mouse IgG Fc) biosensor was loaded with mouse IgG from undiluted supernatants. Raw antibody kinetic data for these three candidate antibodies is shown in Figure 21, and global KD values ​​are shown in Figure 22.

[0197] Example 5: Absorption, Distribution, Metabolism, and Excretion Study of IC100 Pharmacokinetic Study

[0262] Absorption, distribution, metabolism, and excretion (ADME) studies are performed to describe the pharmacokinetics of IC100 in CD-1 male rats.

[0198]

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

[0199]

[0264] Animals are randomly divided into treatment groups (9 mice per group) based on body weight and administered a dose of IC100 intravenously (IV). Treatment groups receive 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 observation. 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 mice were anesthetized with isoflurane prior to sample collection. Animals 1, 2, and 3 in each group were bled on days 1 and 10, animals 4, 5, and 6 on days 2 and 15, and animals 7, 8, and 9 on days 5 and 20.

[0202]

[0267] At the end of the day, animals are anesthetized with isoflurane and bled to exsanguination and subsequent 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 using female B6 Albino mice and fluorescence imaging

[0268] In a second experiment, IC100 and control mouse IgG were 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:

[0205]

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

[0206]

[0270] 2. Dissolve 0.25 mg of VivoTag 680XL in 10 μL of dry DMSO. Once reconstituted, VivoTag 680XL is stable for up to 7 days when stored at 2-8° C. and protected from light.

[0207]

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

[0208]

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

[0209]

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

[0210]

[0274] 6. Collected labeled antibody samples can be analyzed for degree of labeling (DOL). Determine the absorbance of the purified conjugate at 280 nm and 668 nm.

[0211]

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

[0212]

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

[0213]

[0277] In the third experiment, the biodistribution of IC100 will be determined. This study will utilize 15 8-12 week old female B6 Albino (C57BL6) mice. Animals will be randomly divided into groups based on their body weight on day 1.

[0214]

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

[0215]

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

[0216]

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

[0217] Example 7: Effect of IC100 administration on inflammasome signaling

[0281] Blood samples from human patients with nonalcoholic steatohepatitis (NASH), diabetic nephropathy, and lupus nephritis are 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 the expression of inflammasome signaling proteins, such as caspase-1 and ASC.

[0218]

[0282] Additionally, we will use human cancer cell lines to examine ASC-dependent inflammasome activation in real time. This study will be two-fold 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 dye, and the labeling stoichiometry is determined according to the manufacturer's instructions. Control mouse IgG (provided by Charles River Laboratories (CRL)) is similarly labeled and analyzed.

[0220]

[0285] Specific Aim 2: Determining Binding Affinity

[0286] Human cancer cell line THP-1 is cultured in logarithmic phase and plated in a white polystyrene 6-well microculture plate (Corning® Costar® 96-well flat-bottom plate, catalog number 3917) at 20,000 cells per well in a volume of 100 μL of medium. Labeled antibody is then added to the wells in duplicate (10-point dose response, 1:3 dilution, highest concentration 200 nM). Bound antibody is detected in all wells according to the Charles River protocol. Bound antibody (mean fluorescence intensity) is plotted as a function of antibody concentration, and 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 a NASH rat model

[0287] Male Wistar Han rats with liver fibrosis fed a choline-deficient, high-fat diet (CDHFD) serve as an animal model of NASH. 55 mice are utilized for this study. 8-9 week old Wistar Han rats are obtained from Envigo or Charles River. Rats are allowed to acclimate for 3-7 days after 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 are randomly divided into 5 groups based on body weight: Group 1 is fed a standard diet, Teklad Global Diets - Rodent 2014. Groups 2, 3, 4, and 5 are fed a CDHFD diet.

[0223]

[0289] On study day 38, animals are bled for clinical chemistry and classified into treatment groups based on alanine aminotransferase (ALT) levels. Treatment begins on study day 42. The efficacy of IC100 is tested using a dose determined based on the pharmacokinetic data obtained 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 intake, and cage-side clinical observations are measured weekly. Whole blood is obtained by tail vein collection on days 38 and 63. Necropsy is performed on day 84. Animals are sacrificed with isoflurane anesthesia and bled to exsanguination and subsequent bilateral pneumothorax.

[0224]

[0290] Animals will be weighed on days -1, 0, 2, 4, 6, 7, 14, 21, 28, 35, 42, 45, 49, 52, 56, 59, 63, 66, 70, 73, 77, 80, and 83 of the study.

[0225]

[0291] Weekly updates of food intake (grams / day / rat) are recorded on days 0, 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, and 84 of the study.

[0226]

[0292] Cageside clinical observations are performed on days 0-7, 14, 21, 28, 35, 42, 45, 49, 52, 56, 59, 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 liver, brown adipose tissue, and right inguinal adipose tissue were weighed. A 4 x 7 mm biopsy of the left lateral lobe of the liver was obtained, frozen in liquid nitrogen, and stored at -80°C. Three 3 mm transverse sections of the median, left, and right lateral lobes of the liver were obtained and fixed in 10% formalin for 36-48 hours before being stored in 70% ethanol at room temperature for histopathology. Three 100 mg fragments of adipose tissue were snap-frozen in liquid nitrogen and stored in Eppendorf safe-lock tubes at -80°C. Three equal-sized fragments of brown adipose tissue were snap-frozen in liquid nitrogen and stored in Eppendorf safe-lock tubes at -80°C.

[0228]

[0294] Inguinal subcutaneous adipose tissue, a white adipose tissue depot (WAT), was harvested according to the following protocol. Degloving the mouse's lower body revealed the inguinal triangle SQ depot. The upper limbs and chest were held with one hand, while the skin was pulled down toward the feet with the other. The mouse was placed in a supine position, taking care not to contaminate the exposed depot with hair. After cleaning the surgical instruments and changing gloves, the subcutaneous fat triangle was then dissected, taking care not to contaminate the sample with muscle, adjacent fat, mammary gland, or blood. If the borders were not clearly defined, a dissecting microscope was used. The adipose depot was removed and transferred to 10% neutral-buffered formal at a 50:1 fixative-to-tissue volume ratio and fixed at room temperature for 36–48 hours. For RNA or protein extraction, the tissue was 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 changing gloves frequently.

[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 carries the ob / ob leptin deficiency mutation, serves as a mouse model of diabetic nephropathy. 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 receive no treatment. 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 conducted over a six-week period.

[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 compatible with the True Metrix blood glucose meter. Blood glucose levels are measured twice weekly until the completion of the study.

[0233]

[0299] Proteinuria scoring is performed by expressing urine from mice by holding them upside down and applying pressure to the abdomen, and the amount of protein in the urine is determined with the aid of Albustix reagent paper.

[0234]

[0300] If an animal is found dead, no samples will be taken. If an animal needs to be euthanized, regardless of the reason, samples will be taken at necropsy on study day 7.

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

[0301] A model of lupus nephritis is developed using 12-week-old female MRL / MpJ-Tnfrsf6lpr / J mice. Mice are obtained from Bolder BioPath. Mice are randomly assigned to treatment groups based on body weight. Animals are observed daily for significant 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 are divided into five groups and treated with either various doses of IC100 according to the results of Example 5, vehicle, or control IgG.

[0237]

[0303] Body weight, urine protein, lymphadenopathy score, and skin lesion score will be collected. Necropsy will be performed at 20 weeks 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. An IC100 dose level is 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] Repeat dose studies are conducted over a 3-week period. Rats are divided into all-male or all-female groups containing 5 rats. Rats are administered a single dose level of IC100 weekly. Clinical examinations are performed for survival parameters such as mortality, clinical signs, body weight, and toxicokinetics.

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

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

[0241]

[0307] The repeat dose study will be conducted over a 3 week period. The monkeys will be divided into all male or all female groups containing 2 monkeys. The monkeys will be administered a single dose level of IC100 weekly. Clinical examinations will be performed with survival parameters such as 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 are established following intravenous administration of IC100 in rats obtained from Charles River and a subsequent recovery period.

[0244]

[0310] Rats are sorted into groups of 10-15 rats / sex containing three dose levels and a control. Additional groups include a high dose group and a control group containing 5 rats / sex for 4 weeks of recovery.

[0245]

[0311] Mortality, body weight, food consumption, clinical observations, clinical pathology (hematology and clinical chemistry), necropsy findings, 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 and toxicokinetics following intravenous administration of IC100 in non-human primates and a subsequent recovery period will be established at Charles River.

[0248]

[0314] The monkeys are divided into groups of 3 monkeys / sex / group containing 3 dose levels and a control. 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 findings, 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] Potential cardiovascular toxicity (QT prolongation) is assessed in an in vitro assay with CHO or HEK293 cells.

[0252]

[0318] This experiment establishes the IC50 for HERG channel blockade of IC100.

[0253]

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

[0254] Example 16: In vitro blood hemolysis studies

[0320] The potential of an intravenous formulation of IC100 to cause hemolysis of human red blood cells in vitro 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 two months.

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

[0321] The efficacy of IC100 in treating PD is evaluated in several animal (i.e., rodent) models of PD by administering IC100 at 5 mg / kg, 15 mg / kg, and 30 mg / kg.

[0256]

[0322] The 6-OHDA rat model of PD is a chemically induced unilateral model of PD (intrastriatal or medial forebrain bundle lesion) in which mice exhibit behavioral deficits, including rotational asymmetry and motor deficits. The 6-OHDA model shows reduced dopamine, DOPAC, and HVA content in the striatum, and histologically 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 male 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 with vehicle;

[0326] 3. Treat 6-OHDA-induced rats with IC100 dose 1 (selected based on PK / half-life studies);

[0327] Unilateral 6-OHDA / sham infusion was performed on study day 0;

[0328] Daily dose formulation and administration (QD, po) will occur on study days 15-28;

[0329] Body weight is followed and behavioral tests are performed on days -14 (baseline), 28, and 42 and include amphetamine-induced rotation.

[0258]

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

[0259]

[0331] In a second set of experiments using the 6-OHDA model of PD, a total of 45 male rats will be 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 with vehicle;

[0334] 3. Treat 6-OHDA-induced rats with IC100 dose 1 (selected based on PK / half-life studies);

[0335] The experiment was conducted over a 6-week period, with unilateral 6-OHDA / sham infusions administered on study day 0;

[0336] Daily dose formulation and administration (QD, po) occurs on study day 1 and continues for up to 6 weeks after 6-OHDA injection.

[0260]

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

[0261]

[0338] Terminal brain sampling will be 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 a third set of experiments using the 6-OHDA model of PD, a total of 90 male rats will be divided into six experimental groups (n=18 rats / group from start to day 14 baseline, with a target 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 of 4;

[0346] The experiment was conducted over a 6-week period, with unilateral 6-OHDA / sham infusions administered on study day 0;

[0347] Daily dose formulation and administration (QD, po) will occur on study days 15-42.

[0263]

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

[0264]

[0349] Terminal brain sampling will be 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 the treatment of Parkinson's disease (PD)

[0350] TOM20 assay:

[0351] The pathogenesis of PD has been linked to mitochondrial dysfunction through several lines of research, including the discovery that the mitochondrial complex I inhibitor rotenone induces parkinsonism. Additionally, most autosomal recessive cases of PD harbor mutations in genes encoding proteins involved in the selective clearance of dysfunctional and redundant mitochondria (mitophagy), such as PARK2 and PINK1. Similarly, mounting evidence implicates mitochondrial dysfunction in other neurodegenerative disorders (e.g., AD, ALS, and Huntington's disease (HD)). Therefore, phenotypic readouts measuring mitochondrial (dys)function in disease-relevant cellular backgrounds may represent powerful predictive tools for investigating neurodegenerative pathology and identifying potential therapies that can enhance mitophagy.

[0266]

[0352] TOM20 is a subunit of the mitochondrial translocase of the outer membrane (TOM) complex and represents a biomarker for mitochondrial abundance. Profiling therapeutic candidates in the absence (single treatment) and presence (combination treatment) of established mitophagy-inducing triggers allows for the selection of candidate molecules (e.g., IC100) that enhance trigger-induced mitochondrial clearance without directly damaging mitochondria.

[0267]

[0353] The TOM20 loss assay is a scalable and rapid in vitro assay for screening compounds for their ability to enhance mitophagy in a neuronal background.

[0268]

[0354] The TOM20 assay utilizes immortalized human midbrain progenitor cells (ReNcell VM) seeded at 50,000 cells / well in laminin-coated 96-well plates. They are differentiated by withdrawal of growth factors (bEGF, EGF) and addition of differentiation factors (cAMP, GDNF) over a 7-day period, beginning on day 0 (D0) and followed by refreshment on days 1 (D1) and 4 (D4). Cells are then treated with IC100 on day 7 in the absence and presence of 1 micromolar oligomycin / antimycin (O / A; a commonly used mitophagy trigger) for 18 hours, followed by fixed immunocytochemical staining of TOM20 with an anti-TOM20 antibody and DAPI staining (i.e., on day 8). Finally, the assay includes a 1 μM O / A positive control and a 0.1% DMSO negative control.

[0269]

[0355] Co-treatment of mitophagy-enhancing compounds with 1 uM O / A results in a dramatic reduction in TOM20 levels, in addition to the reduction induced by O / A treatment alone. The intensity of TOM20 immunostaining is quantified using a high content analysis-based (HCA) algorithm developed by Charles River Labs. The number of nuclei is quantified to confirm cytotoxicity induced by potential compounds (e.g., IC100).

[0270]

[0356] Alpha-synuclein aggregation assay:

[0357] Aggregates of the presynaptic protein alpha-synuclein are considered a primary biomarker of PD, and evidence suggests that alpha-synuclein aggregates directly mediate neuronal cell death. Therefore, strategies aimed at reducing alpha-synuclein aggregation and toxicity may have therapeutic potential.

[0271]

[0358] In this assay, immortalized human mesencephalic progenitor cells (ReNcell VM) seeded at 10,000 cells / well in laminin-coated 96-well plates are differentiated for 7 days, beginning on day 0 (D0), by withdrawal of growth factors (bEGF, EGF) and addition of differentiation factors (cAMP, GDNF). Also on D0, cells are transduced with adenovirus encoding wild-type human alpha-synuclein. After 24 hours, cells are then treated with IC100, and alpha-synuclein expression and aggregation are detected 6 days later by immunocytochemistry using antibodies detecting alpha / beta-synuclein (Syn205; Cell Signaling Technology) and aggregated alpha-synuclein (MJFR14; Abcam), followed by quantitation by HSA as described for the TOM20 assay. Finally, the assay also includes a 10 uM KU 0063794 positive control and a 0.1% DMSO negative control added at D1 and D4.

[0272] Numbered Embodiments of the Present Disclosure

[0359] Other subject matter contemplated by this disclosure is described in the numbered embodiments below.

[0273]

[0360] 1. A method for treating pulmonary inflammation in a patient in need thereof, comprising administering to the patient a composition comprising an agent that inhibits inflammasome signaling, thereby treating the pulmonary inflammation in the patient.

[0274]

[0361] 2. The method of embodiment 1, wherein the pulmonary inflammation is caused by a condition selected from central nervous system (CNS) injury, a neurodegenerative disease, an autoimmune disease, asthma, chronic obstructive pulmonary disease, cystic fibrosis, interstitial lung disease, and acute respiratory distress syndrome.

[0275]

[0362] 3. The method of embodiment 2, wherein the CNS injury is selected from the group consisting of traumatic brain injury (TBI), stroke, and spinal cord injury (SCI).

[0276]

[0363] 4. The method according to 2, wherein the neurodegenerative disease is selected from the group consisting of amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), and Parkinson's disease (PD).

[0277]

[0364] 5. The method of any one of the above embodiments, wherein administration of the composition results in inhibition of inflammasome activation in lung cells of the patient.

[0278]

[0365] 6. The method of any one of embodiments 1-4, wherein administration of the composition results in a decrease in the level of caspase-1, nucleotide-binding leucine-rich repeat pyrin domain-containing protein 1 (NLRP1), nucleotide-bind...

Claims

1. A monoclonal antibody or antibody fragment thereof that specifically binds to apoptosis-associated speck-like protein (ASC) containing a caspase-activation recruitment domain, wherein the antibody or the antibody fragment comprises a heavy chain variable (VH) region and a light chain variable (VL) region; 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. A monoclonal antibody or an antibody fragment thereof.

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 monoclonal antibody or antibody fragment thereof according to claim 1.

3. The monoclonal antibody or antibody fragment thereof according to claim 1 or 2, wherein the ASC is a human ASC protein.

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

5. The monoclonal antibody or antibody fragment thereof according to any one of claims 1 to 4, wherein the monoclonal antibody or antibody fragment thereof is humanized or chimeric.

6. A pharmaceutical composition comprising the monoclonal antibody or antibody fragment thereof according to any one of claims 1 to 5 and a pharmaceutically acceptable carrier, diluent, or excipient.

7. 7. The pharmaceutical composition of claim 6 for use in treating inflammasome-associated inflammation associated with acute lung injury (ALI) in the lungs of a patient suffering from acute lung injury (ALI).

8. The pharmaceutical composition of claim 7, wherein the ALI is acute respiratory distress syndrome.

9. 9. The pharmaceutical composition of claim 8, wherein the ALI is caused by an injury to the central nervous system (CNS), and the CNS injury is selected from the group consisting of traumatic brain injury (TBI), stroke, and spinal cord injury (SCI).

10. 10. The pharmaceutical composition of any one of claims 7 to 9, wherein use of the pharmaceutical composition in treating inflammasome-associated inflammation associated with ALI inhibits inflammasome activation in lung cells of the patient, and the lung cells are type II pneumocytes.

11. The pharmaceutical composition according to any one of claims 7 to 10, wherein use of the pharmaceutical composition in treating inflammasome-associated inflammation associated with ALI reduces ALI compared to a control, wherein the control is an untreated patient.

12. 12. The pharmaceutical composition of claim 11, wherein the 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.

13. 13. The pharmaceutical composition of any one of claims 7 to 12, wherein use of said pharmaceutical composition in treating inflammasome-associated inflammation associated with ALI reduces pro-inflammatory cytokines in the lungs of said patient.

14. 14. The pharmaceutical composition of claim 13, wherein the pro-inflammatory cytokine is selected from the group consisting of IL-1β and IL-18.

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