Compositions and methods for treating diseases or conditions related to inflammasomes

Abstract

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

Description

Technical Field

[0001] Cross - Reference to Related Applications

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

[0002] Description of Research Sponsored by the Federal Government

[0002] The present invention was made under Grant No. 4R42BS086274 - 02 awarded by the National Institute of Neurological Disorders and Stroke (N INDS) and Grant No. 5R42NS086274 - 03 awarded by the National Institute of Health, and was supported by the U.S. government. The U.S. government has certain rights in this invention.

[0003] Description of Electronically Submitted Text File

[0003] The content of the text file electronically submitted with this specification is hereby incorporated by reference in its entirety: a computer-readable format copy of the Sequence Listing (file name: UNMI_010_02WO_SeqList_ST25.txt, recording date: July 3, 2019, file size approximately 19.5 kilobytes).

[0004] Field

[0004] The present invention relates in general 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 mammalian CNS and / or lung, as a treatment to reduce inflammation in response to injury or pathology causing inflammation in the central nervous system (CNS) and / or lung. 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 the leading cause of death 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 brain damage, TBI can lead to complications in other organs (e.g., the lungs). Acute lung injury (ALI;2) is a common post-traumatic cardiopulmonary disorder with an in-hospital mortality rate of over 40% (Rincon F. et al., (2012). Impact of acute lung injury and acute respiratory distress syndrome after traumatic brain injury in the United States. Neurosurgery 71, 795-803). Patients with TBI are particularly susceptible to developing ALI, and some studies have shown that A high incidence rate of 30% has been reported (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 have been shown to contribute to post-TBI lung dysfunction and lung injury (Rincon F. et al., (2012). Impact of acute lung injury and acute respiratory distress syndrome after traumatic brain injury in the United States. Neurosurgery 71, 795-803), but the precise molecular mechanisms underlying TBI-induced lung injury are still not fully defined. It remains the same.

[0006]

[0006] Large amounts of secreted inflammatory mediators (e.g., cytokines, chemokines, and injury-associated molecular patterns (DAMPs)) released from damaged cells contribute to brain inflammation and affect distal organs (e.g., lungs) (Nicolls, MR et al., (2014). Traumatic brain injury: lungs in a RAGE. Sci Transl Med 6, 252fs234). Most widely studied One of the 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). HMG B1 release can be regulated by the inflammasome, a multiprotein complex involved in the activation of caspase-1 after TBI and the processing of IL-1β and IL-18. et al. (2012). Novel role of PKR in inflammasome activation and HMGB1 release. Nature 488, 670-674).

[0007]

[0007] Various explanations have been given to describe the pathological mechanisms of pulmonary complications after TBI (e.g., increased vascular permeability leading to capillary leakage and infiltration of protein debris) (Ware et al., (2000). The Acute Respiratory Distress Syndrome. New England Journal of Medicine 342, 1334-1349). Extracellular vesicles (EVs) are membrane-containing vesicles that play a role in intercellular communication (Yanez-Mo, M. et al., (2015). Biological Properties of extracellular vesicles and their physiological functions. J Extracell Vesicles 4, 27066), and the development of ALI in an LPS-induced mouse model. It is involved in playing a role. Furthermore, EVs can carry biologically active cytokines (e.g., IL-1β and inflammasome proteins) (Qu, Y. et al., (2007). Nonclassical IL-1 beta secretion stimulated by P2X7 receptors is dependent on inflammasome activation and correlated with exosome release in murine macrophages. J Immunol 179, 1913-1925) (de Rivero Vaccari, JP et al., (2015). Exosome-mediated inflammasome signaling after central nervous system injury. J Neurochem), and It has been shown that this can trigger an immune response and amplify inflammation to adjacent and surrounding cells via the cargo. However, it is unclear whether EV-mediated inflammasome signaling can contribute to the pathological mechanism of TBI-induced ALI. Furthermore, it is unclear whether the pathological mechanism of TBI-induced ALI is shared by other conditions that cause pneumonia. In addition, there is a shortage of Federal Drug Administration (FDA) approved drugs to treat pneumonia. . [Overview of the project] [Problems that the invention aims to solve]

[0008]

[0008] Therefore, it is urgent not only to elucidate the pathological mechanisms of pneumonia caused by TBI and other pathological conditions, but also to develop therapeutic compositions and their use for treating and / or preventing pneumonia. [Means for solving the problem]

[0009] overview

[0009] In one embodiment, the Spectral Provision Provides a monoclonal antibody or antibody fragment thereof that binds to a caspase-activating recruitment domain-containing apoptosis-related speck-like protein (ASC), 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 the amino acid sequence of 5 to 10, 10 to 15, or 15 to 20 amino acids of SEQ ID NO: 5. It is a monoclonal antibody or a fragment thereof.

[0010]

[0010] In another embodiment, provided herein is a monoclonal antibody or antibody fragment that specifically binds to ASC, wherein the antibody or antibody fragment comprises 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, comprising variants having at least one amino acid substitution in HCDR1, HCDR2, and / or HCDR3. In some examples, the amino acid sequence of the VH region comprises SEQ ID NOs: 18, 19, 20, 21, and 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 NOs: 18, 19, 20, 21, or 22. In some examples, the ASC is a human ASC protein. In some examples, the antibody fragment is Fab, F(ab')2, Fab', scFv, a single-domain antibody, a bispecific antibody, or a single-chain camel antibody, or a shark antibody. In some examples, the monoclonal antibody or its antibody fragment is human, humanized, or chimeric. In some examples, provided herein is an isolated nucleic acid molecule encoding the monoclonal antibody or its antibody fragment. In some examples, provided herein is an expression vector containing the nucleic acid molecule. In some examples, provided herein is a nucleic acid molecule operably linked to a regulatory sequence suitable for the expression of a nucleic acid segment in a host cell. In some examples, provided herein is a recombinant host cell containing the expression vector. In another embodiment, 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 in which a nucleic acid molecule is expressed, thereby producing a monoclonal antibody or its antibody fragment that specifically binds to ASC. In some examples, what is provided herein is a pharmaceutical composition comprising the monoclonal antibody or an antibody fragment thereof and a pharmaceutically acceptable carrier, diluent, or excipient.In some cases, the methods provided herein are for treating inflammation in a subject, comprising administering a therapeutically effective amount of the monoclonal antibody or a fragment thereof to the subject, thereby treating the inflammation in the subject. In some cases, administration of the monoclonal antibody or a fragment thereof reduces the level of inflammatory cytokines at least. In some cases, the inflammation is inflammasome-related. In some cases, the inflammasome-related inflammation is associated with central nervous system (CNS) injury, autoimmune disease, autoinflammatory disease, or neurodegenerative disease. In some cases, the CNS injury is selected from the group consisting of traumatic brain injury (TBI), stroke, and spinal cord injury (SCI). In some cases, autoimmune or neurodegenerative diseases include 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, inflammation associated with this inflammasome is associated with metabolic diseases or disorders. In some cases, these metabolic diseases include metabolic syndrome, obesity, diabetes mellitus, diabetic nephropathy or diabetic nephropathy (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 some cases, this autoinflammatory disease is cryopyrin-associated periodic syndromes (CAPS). CAPS may encompass familial influenza autoinflammatory syndrome (FCAS), Muckle-Wells syndrome (MWS), and neonatal-onset multiorgan inflammatory disease (NOMID). In some cases, administration of this monoclonal antibody or its antibody fragment inhibits inflammasome activation in the subject. In some cases, administration of this monoclonal antibody or its antibody fragment reduces ASC activity compared to the control. In some cases, the control is an untreated subject. In some cases, the administration is intraventricular, intraperitoneal, intravenous, or by inhalation. In some cases, what is provided herein is for the treatment of multiple sclerosis (MS) in the subject. A method comprising administering a therapeutically effective dose of the monoclonal antibody or a fragment thereof to a subject, thereby treating the subject's MS. In some cases, administration of the monoclonal antibody or a fragment thereof reduces the level of inflammatory cytokines at least. In some cases, administration of the monoclonal antibody or a fragment thereof inhibits inflammasome activation in the subject. In some cases, administration of the monoclonal antibody or a fragment thereof reduces ASC activity compared to a control. In some cases, the control is an untreated subject. In some cases, the administration is intraventricular, intraperitoneal, intravenous, or by inhalation.

[0011]

[0011] In yet another embodiment, provided herein is a monoclonal antibody or antibody fragment that specifically binds to ASC, wherein the antibody or antibody fragment comprises a light chain variable (VL) region and a heavy chain variable (VH) region, the amino acid sequence of the VL region being LCDR1 of SEQ ID NO: 12, LCDR2 of SEQ ID NO: 13, and LCDR3 of SEQ ID NO: 14, or variants thereof, comprising variants 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 NOs: 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 NOs: 28, 29, 30, or 31. In some examples, the ASC is a human ASC protein. In some examples, the antibody fragment is Fab, F(ab')2, Fab', scFv, a single-domain antibody, a bispecific antibody, or a single-chain camel antibody. In some examples, the monoclonal antibody or its antibody fragment is human, humanized, or chimeric. In some examples, provided herein is an isolated nucleic acid molecule encoding the monoclonal antibody or its antibody fragment. In some examples, provided herein is an expression vector containing the nucleic acid molecule. In some examples, provided herein is a nucleic acid molecule operably ligated to a regulatory sequence suitable for the expression of a nucleic acid segment in a host cell. In some examples, provided herein is a recombinant host cell containing the expression vector. In another embodiment, 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 in which a nucleic acid molecule is expressed, thereby producing a monoclonal antibody or its antibody fragment that specifically binds to ASC. In some examples, what is provided herein is a pharmaceutical composition comprising the monoclonal antibody or an antibody fragment thereof and a pharmaceutically acceptable carrier, diluent, or excipient.In some cases, the methods provided herein are for treating inflammation in a subject, comprising administering a therapeutically effective amount of the monoclonal antibody or a fragment thereof to the subject, thereby treating the inflammation in the subject. In some cases, administration of the monoclonal antibody or a fragment thereof reduces the level of inflammatory cytokines at least. In some cases, the inflammation is inflammasome-related. In some cases, the inflammasome-related inflammation is associated with central nervous system (CNS) injury, autoimmune disease, autoinflammatory disease, or neurodegenerative disease. In some cases, the CNS injury is selected from the group consisting of traumatic brain injury (TBI), stroke, and spinal cord injury (SCI). In some cases, autoimmune or neurodegenerative diseases include 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, inflammation associated with this inflammasome is associated with metabolic diseases or disorders. In some cases, these metabolic diseases include metabolic syndrome, obesity, diabetes mellitus, diabetic nephropathy or diabetic nephropathy (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 some cases, this autoinflammatory disease is cryopyrin-associated periodic syndromes (CAPS). CAPS is familial cold autoinflammatory. This may include multiple sclerosis syndrome (FCAS), Muckle-Wells syndrome (MWS), and neonatal onset multiorgan inflammatory disease (NOMID). In some cases, administration of this monoclonal antibody or its antibody fragment inhibits inflammasome activation in the subject. In some cases, administration of this monoclonal antibody or its antibody fragment reduces ASC activity compared to the control. In some cases, the control is an untreated subject. In some cases, the administration is intraventricular, intraperitoneal, intravenous, or by inhalation. In some cases, what is provided herein is a method for treating multiple sclerosis (MS) in a subject, comprising administering a therapeutically effective dose of this monoclonal antibody or its antibody fragment to the subject, thereby treating the subject's MS. In some cases, administration of this monoclonal antibody or its antibody fragment reduces at least the level of inflammatory cytokines. In some cases, administration of this monoclonal antibody or its antibody fragment inhibits inflammasome activation in the subject. In some cases, administration of this monoclonal antibody or its antibody fragment reduces ASC activity compared to the control. In some cases, the control is treatment-naïve. In some cases, the administration is intraventricular, intraperitoneal, intravenous, or by inhalation.

[0012]

[0012] In yet another embodiment, provided herein is a monoclonal antibody or antibody fragment that specifically binds to ASC, wherein the antibody or antibody fragment comprises 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, comprising 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, comprising variants having at least one amino acid substitution in LCDR1, LCDR2, and / or LCDR3. In some cases, the amino acid sequence of this VH region includes SEQ ID NOs. 18, 19, 20, 21, and 22, or includes an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NOs. 18, 19, 20, 21, or 22, and the amino acid sequence of this VL region includes SEQ ID NOs. 28, 29, 30, and 31, or includes an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NOs. 28, 29, 30, or 31. In some cases, the amino acid sequence of this VH region includes SEQ ID NOs. 18, or includes an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NOs. 18, and the amino acid sequence of this VL region includes SEQ ID NOs. 28, or includes an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NOs. 28. In some cases, the amino acid sequence of this VH region includes 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 this VL region includes 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 cases, the amino acid sequence of this VH region includes 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 this VL region includes 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 cases, the amino acid sequence of this VH region includes 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 this VL region includes SEQ ID NO: 31, or an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 31. In some cases, the amino acid sequence of this VH region includes SEQ ID NO: 19, or at least 95% identical to the amino acid sequence of SEQ ID NO: 19. The amino acid sequence in this VL region includes an amino acid sequence that is 96%, 97%, 98%, or 99% identical, and the amino acid sequence in this VL region includes 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 in this VH region includes 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 in this VL region includes 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 cases, the amino acid sequence of this VH region includes 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 this VL region includes 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 cases, the amino acid sequence of this VH region includes 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 this VL region includes 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 cases, the amino acid sequence of this VH region includes 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 this VL region includes 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 cases, the amino acid sequence of this VH region includes 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 this VL region includes 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 cases, the amino acid sequence of this VH region includes 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 this VL region includes SEQ ID NO: 31 or an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 31. In some cases, the amino acid sequence of this VH region includes SEQ ID NO: 21 or an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 21, and the amino acid sequence of this VL region includes 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 cases, the amino acid sequence of this VH region includes SEQ ID NO: 21 or an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 21, and the amino acid sequence of this VL region includes 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 cases, the amino acid sequence of this VH region includes SEQ ID NO: 21, or an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 21, and the amino acid sequence of this VL region includes 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 cases, the amino acid sequence of this VH region includes SEQ ID NO: 21, or an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 21, and the amino acid sequence of this VL region is sequence number. It includes sequence number 31, or an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of sequence number 31. In some cases, the amino acid sequence of this VH region includes sequence number 22, or an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of sequence number 22, and the amino acid sequence of this VL region includes sequence number 28, or an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of sequence number 28. In some cases, the amino acid sequence of this VH region includes sequence number 22, or an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of sequence number 22, and the amino acid sequence of this VL region includes sequence number 29, or an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of sequence number 29. In some cases, the amino acid sequence of this VH region includes 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 this VL region includes 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 cases, the amino acid sequence of this VH region includes 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 this VL region includes SEQ ID NO: 31 or an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 31. In some cases, this ASC is a human ASC protein. In some examples, this antibody fragment is Fab, F(ab')2, Fab', scFv, a single-domain antibody, a bispecific antibody, or a single-chain camel antibody. In some examples, this monoclonal antibody or its antibody fragment is human, humanized, or chimeric. In some examples, what is provided herein is an isolated nucleic acid molecule encoding this monoclonal antibody or its antibody fragment.In some examples, provided herein is an expression vector containing the nucleic acid molecule. In some examples, provided herein is a nucleic acid molecule operably linked to a regulatory sequence suitable for the expression of a nucleic acid segment in a host cell. In some examples, provided herein is a recombinant host cell containing the expression vector. In another embodiment, provided herein is a method for producing an antibody or antibody fragment that specifically binds to ASCs, comprising culturing recombinant host cells containing the expression vector under conditions in which a nucleic acid molecule is expressed, thereby producing a monoclonal antibody or antibody fragment that specifically binds to ASCs. In some examples, provided herein is a pharmaceutical composition comprising the monoclonal antibody or antibody fragment 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 to the subject, thereby treating the inflammation in the subject. In some examples, administration of the monoclonal antibody or antibody fragment reduces the level of at least inflammatory cytokines. In some examples, the inflammation is inflammasome-related inflammation. In some cases, inflammation associated with this inflammasome is associated with central nervous system (CNS) injury, autoimmune disease, autoinflammatory disease, or neurodegenerative disease. In some cases, CNS injury is selected from a group consisting of traumatic brain injury (TBI), stroke, and spinal cord injury (SCI). In some cases, 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, inflammation associated with this inflammasome is associated with metabolic disease or disorder.In some cases, these metabolic disorders include 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 this case, the autoinflammatory disease is cryopyrin-associated periodic syndrome (CAPS). CAPS may include familial influenza autoinflammatory syndrome (FCAS), Muckle-Wells syndrome (MWS), and neonatal onset multiorgan inflammatory disease (NOMID). In some cases, administration of this monoclonal antibody or its antibody fragment inhibits inflammasome activation in the subject. In some cases, administration of this monoclonal antibody or its antibody fragment reduces ASC activity compared to the control. In some cases, the control is an untreated subject. In some cases, the administration is intraventricular, intraperitoneal, intravenous, or by inhalation. In some cases, provided herein is a method for treating multiple sclerosis (MS) in a subject, comprising administering a therapeutically effective dose of this monoclonal antibody or its antibody fragment to the subject, thereby treating the subject's MS. In some cases, administration of this monoclonal antibody or its antibody fragment reduces at least the level of inflammatory cytokines. In some cases, administration of this monoclonal antibody or its antibody fragment inhibits inflammasome activation in the subject. In some cases, administration of this monoclonal antibody or its antibody fragment reduces ASC activity compared to the control. In some cases, the control is an untreated subject. In some cases, the administration is intraventricular, intraperitoneal, intravenous, or by inhalation. [Brief explanation of the drawing]

[0013] Brief explanation of the drawing [Figure 1A]

[0013] We will explain inflammasome activation in the cortical and lung tissue of C57 / BL6 mice after TBI. Figure 1A shows representative immunoblots of active caspase-1, ASC, IL-18, IL-β, HMGB1, and AIM2 after TBI. [Figure 1B]

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

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

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

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

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

[0013] IL-β (Figure 1G) significantly increases at 4 and 24 hours after TBI in cortical tissue. 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]

[0013] Active caspase-1 (Figure 1I) significantly increases at 4 and 24 hours after TBI in lung tissue. 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]

[0013] ASC (Figure 1J) significantly increases at 4 and 24 hours after TBI in lung tissue. 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]

[0013] IL-18 (Figure 1K) significantly increases at 4 and 24 hours after TBI in lung tissue. 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]

[0013] HMGB1 (Figure 1L) significantly increases at 4 and 24 hours after TBI in lung tissue. 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]

[0013] AIM2 (Figure 1M) significantly increases at 4 and 24 hours after TBI in lung tissue. 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]

[0013] IL-β (Figure 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 pseudo. [Figure 2A]

[0014] This explains the expression of inflammasome proteins in type II alveolar epithelial cells. Figure 2A shows AIM2. [Figure 2B]

[0014] Figure 2B shows activated caspase-1. [Figure 2C]

[0014] Figure 2C shows that ASC immunoreactivity increases in lung tissue after CCI (4 and 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] This explains how 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 the sham. Data are presented as mean + / - SEM; *p<0.05 compared to the sham. N=4-5 per group. [Figure 3C]

[0015] Figure 3C shows a typical 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 the sham. Data are presented as mean + / - SEM; *p<0.05 compared to the sham. N=4-5 per group. [Figure 3E]

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

[0016] This describes pyroptosome formation in mouse lungs 4 hours after TBI. Figure 4A shows that TBI induces laddering of ASCs in lung tissue, leading to caspase-1 activation and pyroptosis, which is oligomerization of ASC dimers and the formation of pyroptosomes. [Figure 4B]

[0016] Figure 4B shows a typical immunoblot of gasdermin. [Figure 4C]

[0016] Figure 4C shows the 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 pseudo. [Figure 5A]

[0017] This explains how TBI induces alveolar morphological changes and acute lung injury in mice. Figure 5A shows H and E staining of lung sections from pseudo- and injured animals at 4 and 24 hours. The sections show evidence of neutrophil infiltration (arrowhead), morphological changes in the alveolar capillary membrane (asterisk, *), interstitial edema (short arrow), and thickening of the interstitial and alveolar septa (pound, #). [Figure 5B]

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

[0018] This section describes the expression of CD81 in serum-derived extracellular viable cells (EVs) from control and TBI-injured mice. Representative immunoblots of CD81 in serum-derived EVs from pseudo-control and TBI-injured mice are presented. [Figure 7A]

[0019] This illustrates how adoptive transplantation of extravasation cells (EVs) from TBI animals induces caspase-1 and ASC in the lungs of undamaged mice. Figure 7A illustrates a representative immunoblot 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 pseudo-animals. [Figure 7B]

[0019] Caspase-1 (Figure 7B) is elevated in the lungs of animals receiving EVs isolated from TBI mice compared to EVs from pseudo-animals. Data are presented as +mean / -SEM; *p<0.05 compared to pseudo-animals. N=3 per group. [Figure 7C]

[0019] ASC (Figure 7C) is elevated in the lungs of animals receiving EVs isolated from TBI mice compared to EVs from pseudo-animals. Data are presented as +mean / -SEM; *p<0.05 compared to pseudo-animals. N=3 per group. [Figure 7D]

[0019] This shows that IL-18 (Figure 7D) is elevated in the lungs of animals receiving EVs isolated from TBI mice compared to EVs from pseudo-animals. Data are presented as +mean / -SEM; *p<0.05 compared to pseudo-animals. N=3 per group. [Figure 7E]

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

[0019] HMGB1 (Figure 7F) is elevated in the lungs of animals receiving EVs isolated from TBI mice compared to EVs from pseudo-animals. Data are presented as +mean / -SEM; *p<0.05 compared to pseudo-animals. N=3 per group. [Figure 7G]

[0019] EVs from TBI mice induced alveolar morphological changes (decreased 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 to uninjured mice (Figure 7G). Data are presented as mean + / - SEM; **p<0.01, *p<0.05 compared to the uninjured group. [Figure 8A]

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

[0020] This shows that 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; compared to pseudo, ****p<0.001, ***p<0.01, **p<0.01, *p<0.05. N=4 per group. [Figure 8C]

[0020] ASC (Figure 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 pseudo. N=4 per group. [Figure 8D]

[0020] This shows that IL-1β (Figure 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; compared to pseudo, ****p<0.001, ***p<0.01, **p<0.01, *p<0.05. N=4 per group. [Figure 8E]

[0020] Figure 8E shows that AIM2 is reduced in the lungs of animals treated with enoxaparin and IC100 compared to untreated positive control animals. Data are presented as mean + / - SEM; compared to pseudo, ****p<0.001, ***p<0.01, **p<0.01, *p<0.05. N=4 per group. [Figure 8F]

[0020] This shows that 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; compared to pseudo, ****p<0.001, ***p<0.01, **p<0.01, *p<0.05. N=4 per group. [Figure 9A]

[0021] We explain that treatment with enoxaparin (3 mg / kg) and IC100 (5 mg / kg) reduces the ALI score in the lungs of animals delivered with EVs from injured mice. Figure 9A illustrates H and E staining of lung sections from mouse lungs delivered with EVs from injured mice treated with saline (Figure 9A). The sections show evidence of neutrophil infiltration, morphological changes in the alveolar capillary membranes, interstitial edema, and thickening of the interstitial and alveolar septa. [Figure 9B]

[0021] Figure 9B illustrates H and E staining of lung sections from mouse lungs delivered with EV from an untreated (Figure 9B) injured mouse. [Figure 9C]

[0021] Figure 9C illustrates H and E staining of lung sections from mouse lungs delivered with EV from enoxaparin-treated (Figure 9C) injured mice. [Figure 9D]

[0021] Figure 9D illustrates H and E staining of lung sections from mouse lungs delivered with EV from injured mice treated with IC100 (anti-ASC; Figure 9D). [Figure 9E]

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

[0022] We explain that delivery of serum-derived extracellular viable cells (EVs) from TBI patients leads to increased inflammasome protein expression in lung endothelial cells. Figure 10A shows Western blot representations of caspase-1, ASC, AIM2, and HMGB1 in PMVECs after 4 hours of incubation with TBI-EVs and control EVs. [Figure 10B]

[0022] Figure 10B shows the quantitative analysis of Western blot, with n=3 filters per group, n=6 patients, t-test, ****p<0.001, ***p<0.01, **p<0.01, *p<0.05. [Figure 10C]

[0022] Figure 10C shows the quantitative analysis of Western blot, with n=3 filters per group, n=6 patients, t-test, ****p<0.001, ***p<0.01, **p<0.01, *p<0.05. [Figure 10D]

[0022] Figure 10D shows the quantitative analysis of Western blot, with n=3 filters per group, n=6 patients, t-test, ****p<0.001, ***p<0.01, **p<0.01, *p<0.05. [Figure 10E]

[0022] Figure 10E shows the quantitative analysis of Western blot, with n=3 filters per group, n=6 patients, t-test, ****p<0.001, ***p<0.01, **p<0.01, *p<0.05. [Figure 10F]

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

[0023] We explain that delivery of TBI-EV to lung endothelial cells increases the immunoreactivity and cell death of active caspase-1. Figure 11A shows the co-localization of caspase-1 FLICA and PI staining, as well as PMVEC incubated with TBI-EV for 4 hours. [Figure 11B]

[0023] Figure 11B shows caspase-1FLICA and PI staining in PMVEC incubated with control EV for 4 hours. [Figure 11C]

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

[0024] This paper explains how treatment with a humanized anti-ASC monoclonal antibody (i.e., IC100) improves functional outcomes in EAE. Figure 12A shows the clinical course of MOG35-55 induced EAE in C57BL / 6 mice treated with vehicle or escalating doses of IC100. IC100 administration (10, 30, and 45 mg / kg ip every 4 days) was initiated on day 8, before the mice showed signs of paralysis. Results are expressed as the mean daily clinical score ± SEM for 9-10 mice / group. The curves for 30 and 45 mg / kg are significantly different from the vehicle curve: **p ≤ 0.001 Mann-Whitney U test. [Figure 12B]

[0024] Figure 12B shows a comparison of peak clinical scores (highest disease scores achieved by mice) between groups; *p ≤ 0.05, Student's t-test. [Figure 12C]

[0024] Figure 12C shows a comparison of the cumulative disease index (CDI) between the groups. The CDI is equal to the sum of all scores from the onset date 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 dates between the groups. The onset date is considered to be the day when the mouse first showed EAE symptoms. [Figure 12E]

[0024] Figure 12E shows a comparison of peak disease days between the groups. The peak disease day is the day when the mouse reaches the highest disease score. The onset day is considered to be the day when the mouse first showed EAE symptoms. [Figure 13A]

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

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

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

[0027] This shows the IC100 concentration in tissues. IC100 concentrations (pg / ml) in the brain, spinal cord, liver, and spleen of 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. Each group consisted of 2-10 mice. [Figure 16]

[0028] This study demonstrates that IC100 is incorporated into ASCspeck cells in unstimulated THP-1 cells, and that this incorporation is increased by inflammasome activation. [Figure 17]

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

[0030] This image shows confocal images of spinal nerve cells demonstrating the penetration of the anti-ASC antibody (IC100) into spinal nerve cells. [Figure 19]

[0031] This paper compares the antibody binding of three different antibodies to human ASC against various species. [Figure 20]

[0032] This shows nicotine (500 nM)-induced inflammasome induction in human nucleus pulposus cells, as well as treatment with three different antibodies against human ASCs (H1, H2, H3) and two different antibodies against mouse ASCs (M1, M2). H1 was most effective in preventing IL-1 beta-release / flammasome activation. [Figure 21]

[0033] This shows the biodynamic analysis data of anti-ASC monoclonal antibodies against BLI. [Figure 22]

[0034] This paper compares the dynamics of three different antibodies against human ASC. [Modes for carrying out the invention]

[0014] Detailed explanation definition

[0035] Unless otherwise defined, all technical terms used herein have the same meaning as those generally understood by those skilled in the art to which this invention pertains.

[0015]

[0036] The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described herein. All documents or parts of documents cited herein (e.g., patents, patent applications, articles, books, and professional works) are expressly incorporated herein by reference in their entirety for any purpose. If one or more of the incorporated documents or parts of documents define a term that conflicts with the definitions of terms in this application, the definitions appearing in this application shall prevail. However, any reference to any references, articles, publications, patents, patent application publications, and patent applications cited herein does not acknowledge, suggest, or acknowledge in any way suggest that they constitute valid prior art or form part of the general knowledge in any country of the world.

[0016]

[0037] The terms "a" or "an" refer to one or more entities, i.e., can refer to multiple reference objects. Therefore, the terms "a" or "an," "one or more," and "at least one" are used interchangeably in this specification. In addition, a reference to an "element" with the indefinite article "a" or "an" does not preclude the possibility that the element exists in multiple forms unless the context explicitly requires that the element exists only uniquely.

[0017]

[0038] Unless otherwise required by context, throughout this specification and the claims, the word “comprise” and its variations (e.g., “comprises” and “comprising”) shall be interpreted in an open and inclusive sense of “comprise but not limited to.” It must be understood that the use of alternatives (e.g., "or") means one or both of these alternatives, or any combination thereof. Where used herein, the terms "about" and "essentially from" mean + / - 20% of the indicated range, value, or structure unless otherwise indicated.

[0018]

[0039] Throughout this specification, any reference to “one embodiment” or “an embodiment” refers to specific features, structures, etc., described in relation to this embodiment. This means that the structure or characteristics are included in at least one embodiment of this disclosure. Therefore, the phrase "in one embodiment" appears in various places throughout this specification. The appearance of "in an embodiment" or "in one embodiment" does not necessarily mean that all instances refer to the same embodiment. It is recognized that certain features of the Disclosure described in the context of another embodiment for clarity may also be provided in combination in a single embodiment. Conversely, various features of the Disclosure described in the context of a single embodiment for brevity may also be provided separately or in any suitable partial combination.

[0019]

[0040] Throughout this disclosure, various aspects of the methods and compositions provided herein may be presented in scope form. It should be understood that the scope form is provided solely for convenience and brevity and should not be construed as a rigid limitation to the scope of the invention. A description of a range should be considered to specifically disclose all possible subranges and individual numbers within that range. For example, a description of a range such as 1-6 should be considered to specifically disclose subranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, and individual numbers within that range (e.g., 1, 2, 3, 4, 5, and 6). This applies regardless of the width of the range.

[0020]

[0041] As used herein, “protein” and “polypeptide” are used synonymously to mean any peptide chain of amino acids, regardless of length or post-translational modifications (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 containing antigen-binding sites that specifically bind to (immunely react to) antigens (e.g., ASC, NLRP1, AIM2, etc.). The antibodies provided herein may be polyclonal antibodies, monoclonal antibodies (mAbs), chimeric antibodies, humanized antibodies, anti-idiotype (anti-Id) antibodies against antibodies that can be labeled in soluble or conjugated form, and active fragments, regions, or derivatives thereof. The antibodies for use herein may be chimeric, humanized, or human.

[0022]

[0043] "Specifically binding" or "immunely reacting" means that the antibody reacts with one or more antigenic determinants of a desired antigen and not with other peptides. In certain embodiments, an antibody is said to bind specifically to an antigen if it preferentially recognizes the target antigen of this antibody in a complex mixture of proteins and / or macromolecules. The term "antibody" broadly refers to an immunoglobulin (Ig) molecule (generally comprising four polypeptide chains of two heavy (H) chains and two light (L) chains), or any functional fragment, variant, variant, or derivative of this Ig molecule that retains the essential target-binding properties of the Ig molecule. Such variant antibody forms, variant antibody forms, or derivative antibody forms are known in the art. Such anti-ASC antibodies and anti-NLRP1 antibodies of the present invention can bind to moieties of ASC and NLRP1, respectively, that inhibit caspase-1 activation.

[0023]

[0044] As used herein, the term "humanized antibody" refers to an antibody in which the smallest portion of a non-human antibody has been introduced into another human antibody.

[0024]

[0045] As used herein, the term "human antibody" refers to an antibody in which substantially all of the protein is substantially non-immunogenic in humans, having minor sequence changes or variations.

[0025]

[0046] In full-length antibodies, each heavy chain comprises a heavy chain variable region (hereinafter abbreviated 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 (hereinafter abbreviated 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 more conserved regions (called framework regions (FR)) interspersed with over-denatured regions (called complementarity-determining regions (CDRs)). Each VH and VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), as well as classes (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or subclasses. IgG antibodies, IgD antibodies, and IgE antibodies generally consist of two identical heavy chains and two identical light chains, each comprising a heavy chain variable region (VH) and a light chain variable region (VL). It contains two antigen-binding domains. Generally, IgA antibodies consist of two monomers, each monomer consisting of two heavy chains and two light chains (similar to IgG, IgD, and IgE antibodies), and thus the IgA molecule has four antigen-binding domains, each composed of VH and VL. A particular IgA molecule is a monomer consisting of two heavy chains and two light chains. Secreted IgM antibodies generally consist of five monomers, each monomer consisting of two heavy chains and two light chains (similar to IgG and IgE antibodies), and thus the IgM molecule has ten antigen-binding domains, each composed of VH and VL. A cell surface morphology of IgM also exists, which has a two-heavy-chain / two-light-chain structure similar to IgG, IgD, and IgE antibodies.

[0026]

[0047] The terms “antigen-binding fragment,” “antigen-binding moiety,” “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, part, or site of a binding domain derived from an antibody, that possesses the ability to specifically bind to an antigen (e.g., an ASC protein). Exemplary binding domains include fusion proteins containing a single-chain antibody variable region (e.g., a domain antibody sFv, scFv, scFab), an antibody moiety (e.g., a domain antibody), an extrareceptor domain, and a ligand (e.g., a cytokine, chemokine). 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 the purposes of the present invention, the fusion protein comprises one or more antibodies and an additional amino acid sequence (e.g., a heterologous sequence attached to the N-terminus or C-terminus of the antibody or its antibody fragment, or a homologous sequence from another region). Examples of heterologous sequences include, but are not limited to, “tags” (e.g., FLAG tags or 6His tags), or enzymes or polypeptides that increase the half-life of this antibody in blood. Tags are known in the art. This additional amino acid sequence (which may include amino-terminal fusions and / or carboxyl-terminal fusions) may range in length from one residue to a polypeptide containing 100 or more residues and one or more intrasequence insertions of amino acid residues.

[0027]

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

[0028]

[0049] The term "CDR region" or "CDR" is derived from Kabat et al., 1991 (Kabat, EA et al., (1991) Sequences of Proteins of Immunological Interest, 5th Edition. US Department (of Health and Human Services, Public Service, NIH, Washington), and later editions As defined, it can refer to the hypervariable region of the heavy or light chain of an immunoglobulin. Antibodies typically contain three heavy chain CDRs and three light chain CDRs.

[0029]

[0050] It has been shown that the antigen-binding function of antibodies can be exerted by fragments of full-length antibodies. Embodiments of antibodies and antibody fragments may also be bispecific, triplicate, dual-specific, or multispecific forms, comprising two or more different antigens. It specifically binds to the antigen. Examples of binding fragments included in the antibody term "antigen-binding fragment" are... The following are examples: (i) Fab fragments consisting of a VL domain, VH domain, CL domain, and CH1 domain (Ward, ES et al., (1989) Nature 341, 544-546); (ii) Fd fragments consisting of a VH domain and a CH1 domain (McCafferty et al., (1990) Nature, 348, 552-554); (iii) Fv fragments consisting of a single antibody's VL domain and VH domain (Holt et al., (2003) Trends in Biotechnology 21, 484-490); (iv) dAb fragment consisting of a VH domain or VL domain (Ward, ES et al., Nature 341, 544-546 (1989), McCafferty et al., (1990) Nature, 348, 552-554, Holt et al., (2003) Trends in Biotechnology 21, 484-490); (v) isolated CDR region; (vi) two A single-chain Fv molecule (scFv) (Bird et al., (1988) Science, 242, 423-426; Huston et al., (1988) PNAS USA, 85, 5879-5883) is a bivalent fragment containing a binding Fab fragment, in which the VH and VL domains are linked by a peptide linker that enables these two domains to associate and form an antigen-binding site. The present invention also encompasses the Fab' fragment. Furthermore, although the two domains VL and VH of the Fv fragment are encoded by separate genes, these domains can be linked using recombination by a synthetic linker that enables the VL and VH regions to pair up and form a single protein chain (known as single-chain Fv (scFv)) that forms a monovalent molecule. Such a single-chain antibody is also intended to be encompassed in the antibody term "antigen-binding fragment". In certain embodiments of the present invention, the scFv molecule can be incorporated into a fusion protein. In some embodiments, the present invention includes: (viii) a single-stranded camel antibody; (viii) a bispecific single-stranded Fv dimer (PCT / US No. 92109965); and (ix) a "diabody" which is a polyvalent or multispecific fragment constructed by gene fusion (International Publication No. 94 / 13804; Holliger, P. (1993) et al., Proc. Natl. Acad. Sci. USA 90 6444-6448). The diabody comprises a VH domain and a VL domain. This antibody is expressed on a single polypeptide chain, but uses a linker that is too short to allow pairing between two domains on the same chain, thereby forcing one domain to pair with a complementary domain on another chain to form two antigen-binding sites, resulting in a bivalent, bispecific antibody (e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448). See Poljak, RJ, et al. (1994) Structure 2:1121-1123). Such antibody-binding fragments are known in the art (Kontermann and Dubel eds., Antibody Engineering (2001) Springer-Verlag. New York. 790 pp.). In some embodiments, the present invention This includes single-domain antibodies. Generally, the term “antibody” as used herein encompasses “antibody fragments.” Antibody fragments generally retain the antigen-binding properties of full-length antibodies.

[0030]

[0051] Fv, scFv, or diabody molecules can be stabilized by incorporating disulfide crosslinks that bind the VH and VL domains (Reiter, Y. et al., Nature Biotech, 14, 1239-1245, 1996). Minibodies containing scFv bound to the CH3 domain can also be constructed (Hu, S. et al., (1996) Cancer Res., 56, 3055-3061). Other examples of binding fragments include Fab', which differs from the Fab fragment 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, which is a Fab' fragment in which the cysteine ​​residues of the constant domain support a free thiol group.

[0031]

[0052] "Fv" as used herein may refer to the smallest fragment of an antibody that retains both the antigen recognition site and the antigen binding site. "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] The "Fc region" or "Fc domain" is a part of the source antibody. This refers to polypeptide sequences that correspond to or are derived from the portion involved in binding to the antibody receptor and complement C1q component on the cell surface. Fc is an antibody that readily forms protein crystals. Fc is an abbreviation for "fragment crystal," meaning a fragment. Initially described by protein digestion, separate protein fragments can define the overall general structure of immunoglobulin proteins. According to the earliest definition in the literature, an Fc fragment consists of a disulfide-bonded heavy chain hinge region, a CH2 domain, and a CH3 domain. However, more recently, the term has been applied to single chains consisting of CH3, CH2, and at least a portion of the hinge sufficient to form a disulfide-bonded dimer containing a second such chain. For an overview of the structure and function of immunoglobulins, see Putnam, The Plasma Proteins, Vol. V (Academic Press, Inc., 1987), pp. 49-140; and Padlan, Mol. Immunol. 31:169-217, 1994. As used herein, the term Fc includes naturally occurring sequence variants. In one embodiment, the antibody provided herein or an antibody fragment derived therefrom (e.g., an anti-ASC monoclonal antibody or an antibody fragment thereof) has a modified Fc region or Fc domain. In some cases, this modified Fc region or Fc domain can confer high thermal stability to the resulting antibody or antibody fragment derived therefrom. This high thermal stability can result in a high serum half-life. The Fc region or Fc domain can 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 can be modified to have the deletion of one or more cysteine ​​residues in the hinge region and the substitution of one or more CH3-interface amino acid sulfhydryl-containing residues. In another embodiment, Wozniak-Knopp G, the contents of which are incorporated herein by reference. It's explained in Stadlmann J, Rueker F (2012) Stabilisation of the Fc Fragment of Human IgG1 by Engineered Intradomain Disulfide Bonds. PLoS ONE 7(1): e30083. In addition, the Fc region or Fc domain of the antibody provided herein or an antibody fragment derived therefrom (e.g., an anti-ASC monoclonal antibody or an antibody fragment thereof) can be stabilized by manipulating the Fc region to have an intradomain disulfide bond. In yet another embodiment, the antibody has an Fc region that is modified as described in International Publication No. 99 / 58572, which is incorporated herein by reference. In yet another embodiment, the Fc region or Fc domain can be modified as described in U.S. Patent No. 9574010, whose contents are incorporated herein by reference.

[0033]

[0054] The terms "caspase-activating recruitment domain (CARD)-containing apoptosis-related speck-like protein" and "ASC" refer to the expression product or isoform of the ASC gene, 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. "Functional activity" of a protein is any activity related to the physiological function of the protein. Examples of ASC's functional activity include caspase-1 activation and protein recruitment for the initiation of cell death.

[0034]

[0055] The term “ASC gene” or “ASC nucleic acid” means the natural ASC coding nucleic acid sequence, the genomic sequence on which ASC cDNA can be transcribed, and / or allele variants and homologs thereof. This term encompasses double-stranded DNA, single-stranded DNA, and RNA.

[0035]

[0056] As used herein, the term "inflammasome" refers to a multiprotein (e.g., at least two proteins) complex that activates caspase-1. Furthermore, the term "inflammasome" may refer to a multiprotein complex that activates caspase-1 activity, which then modulates IL-1β, IL-18, and IL-33 processing and activation. These terms are incorporated herein by reference to Arend et al. 2008;L. See i et al. 2008 and Martinon et al. 2002. Term "NLRP1 inflammasome", "NALP1 inflammasome," "NLRP2 inflammasome," "NALP2 inflammasome," "NLRP3 inflammasome," "NALP3 inflammasome," "NLRC4 inflammasome," "IPAF inflammasome," or "AIM2 inflammasome" refers to a protein complex of at least caspase-1 and one adapter protein, e.g., ASC. For example, the terms "NLRP1 inflammasome" and "NALP1 inflammasome" may refer to a multiprotein complex containing NLRP1, ASC, caspase-1, caspase-11 for caspase-1 activation and processing of interleukin-1β, interleukin-18, and interleukin-33, XIAP, and pannexin-1. The terms "NLRP2 inflammasome" and "NALP2 inflammasome" may refer to a multiprotein complex containing NLRP2 (aka NALP2), ASC, and caspase-1, while the terms "NLRP3 inflammasome" and "NALP3 inflammasome" may refer to a multiprotein complex containing NLRP3 (aka NALP3) and ASC, and the terms "NLRC4 inflammasome" and "IPAF inflammasome" may refer to a multiprotein complex containing NLRC4 (aka IPAF), ASC, and caspase-1. Furthermore, the term "AIM2 inflammasome" may refer to a multiprotein complex containing AIM2, ASC, and caspase-1.

[0036]

[0057] As used herein, the term "sequence identity" refers to the proportion of identical subunits at corresponding positions in the two sequences when aligning two sequences (e.g., nucleic acid sequences, amino acid sequences) to maximize subunit matching, i.e., when gaps and insertions are taken into consideration. Sequence identity can be measured using sequence analysis software (e.g., Sequence Analysis Software Package from Accelrys CGC, San Diego, CA). Cut.

[0037]

[0058] The terms “therapeutic effective dose” and “effective dose” mean an amount sufficient to produce a therapeutically (e.g., clinically) desirable outcome; the exact nature of the outcome will vary depending on the nature of the disorder being treated. For example, if the disorder to be treated is SCI, the outcome may be an improvement in motor skills and locomotive function, a reduction in spinal cord lesions, etc. The compositions described herein may be administered once or more daily and once or more weekly. Those skilled in the art will recognize that certain factors, including but not limited to the severity of the disease or disorder, prior treatment, the subject's overall health and / or age, and other pre-existing diseases, may influence the dose and timing required to effectively treat the subject. Furthermore, treatment of a subject with a therapeutic effective dose of the composition of the present invention may consist of a single treatment or a series of treatments.

[0038]

[0059] As used herein, the term “treatment” is defined as the application or administration of a therapeutic agent described herein or identified herein by means of the method herein, to a patient for the purpose of curing, restoring, alleviating, reducing, modifying, rescuing, improving, or influencing a disease, symptoms of a disease, or susceptibility to a disease, or the application or administration of a therapeutic agent to an isolated tissue or cell line from a patient having a disease, symptoms of a disease, or susceptibility to a disease.

[0039]

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

[0040]

[0061] "Absent in melanoma 2" and " "AIM2" may mean 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 numbers NX_014862, NP004824, XP016858337, XP005245673, AAB81613, BAF84731, AAH10940) and exhibits functional activity of AIM2.

[0041]

[0062] As used interchangeably herein, “NACHT, LRR, and PYD domain-containing protein 1,” “NALP1,” and “NLRP1” mean the expression product or isoform of the NALP1 or NLRP1 gene, or 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 numbers AAH51787, NP_001028225, NP_127500, NP_127499, NP_127497, NP055737) and exhibits functional activity of NALP1.

[0042]

[0063] As used interchangeably herein, "NALP2" and "NLRP2" mean 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 numbers 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" mean the expression product or isoform of the NALP3 or NLRP3 gene; or a protein that shares at least 65%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity with NALP3 (e.g., accession numbers 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 functional activity of NALP3.

[0044]

[0065] As used interchangeably herein, "NLRC4" and "IPAF" mean 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 numbers NP_001186067, NP001186068, NP_001289433, or NP_067032) and exhibits functional activity of NLRC4.

[0045]

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

[0046]

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

[0047]

[0068] Methods involving conventional molecular biological techniques are described herein. Such techniques are generally known in the art, and methodological papers, 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 Bi This is detailed in *ology*, ed. Ausubel et al., Greene Publishing and Wiley-Interscience, New York, 1992 (with regular updates). Immunological techniques are generally well-known in this field and are detailed in methodological papers, 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] Similar or equivalent compositions and methods may be used in the practice or testing of the present invention, but preferred compositions and methods are described below. All publications, patent applications, and patents cited herein are incorporated collectively by reference. In case of any conflict, this specification shall prevail, including definitions. The specific embodiments considered below are for illustrative purposes only and are not limiting.

[0049] overview

[0070] Provided herein are monoclonal antibodies or antibody fragments thereof that specifically bind to apoptosis-related speck-like proteins (ASCs) containing a caspase-activating recruitment domain. These monoclonal antibodies or antibody fragments may specifically bind to antigenic fragments of ASCs that contain, consist of, or are essentially composed of the amino acid sequence of SEQ ID NO: 5. In addition to these embodiments, the present invention intends to utilize these monoclonal antibodies or antibody fragments in methods for treating a target inflammation. This inflammation may be congenital immunological inflammation. This inflammation may be inflammasome-related inflammation. In one embodiment, the monoclonal antibodies or antibody fragments provided herein may be used in methods for reducing mammalian inflammation, as described in U.S. Patent No. 8,685,400, the contents of which this specification are incorporated herein by reference in their entirety. This inflammation may be present in the lungs and / or central nervous system (CNS). Inflammation in the lungs and / or CNS may result from injury (e.g., traumatic brain injury (TBI) or spinal cord injury (SCI)) or from a disease, condition, or distress of or affecting the CNS. Where provided herein, disease, condition, or distress of or affecting the CNS includes stroke, as well as autoimmune diseases and / or CNS diseases (e.g., amyotrophic lateral sclerosis (ALS) Lou Gehrig's disease, multiple sclerosis (MS), and immunodeficiency muscular CNS breakdown). This may include CNS breakdown, muscular dystrophy (MD), Alzheimer's disease (AD), and Parkinson's disease (PD). The use of this monoclonal antibody or its antibody fragment in a method of treating inflammation may reduce inflammation in the patient's CNS and / or lungs. This reduction may be compared to a control (e.g., an untreated patient and / or a patient before treatment). In one embodiment, MS is treated by administering this monoclonal antibody or an antibody fragment derived from this monoclonal antibody to a patient who has or is suspected of having MS. The monoclonal antibody or its antibody fragment in this embodiment may be present in a composition (e.g., a pharmaceutical composition provided herein). In some cases, this monoclonal antibody or its fragment is used in combination with one or more other agents in a therapeutic method provided herein. Other agents may be any 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 relates to a monoclonal antibody or antibody fragment that specifically binds to ASC, wherein the antibody or antibody fragment comprises 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, H of SEQ ID NO: 7 The invention also includes monoclonal antibodies or antibody fragments thereof, comprising CDR2, and HCDR3 of SEQ ID NO: 8, or variants thereof, having at least one amino acid substitution in HCDR1, HCDR2, and / or HCDR3. In addition to this embodiment, the present invention intends to use this monoclonal antibody or antibody fragment thereof in a method for treating inflammation of interest. This inflammation may be congenital immunological inflammation. This inflammation may be inflammasome-related 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 described in U.S. Patent No. 8,685,400, the contents of this specification being incorporated herein by reference in their entirety. This inflammation may be present in the lungs and / or CNS. Inflammation in the lungs and / or CNS may be a result of injury (e.g., traumatic brain injury (TBI) or spinal cord injury (SCI)) or a result of disease, pathology, or distress of or affecting the CNS. Where provided herein, diseases, conditions, or distress affecting or influencing the CNS may include stroke, as well as autoimmune diseases and / or CNS diseases (e.g., amyotrophic lateral sclerosis (ALS) Lou Gehrig, multiple sclerosis (MS), immunodeficiency muscle CNS breakdown, muscular dystrophy (MD), Alzheimer's disease (AD), and Parkinson's disease (PD)). The use of this monoclonal antibody or its antibody fragment in a method of treating inflammation may reduce inflammation in the patient's CNS and / or lungs. The use of this monoclonal antibody or its antibody fragment in a method of treating inflammation may reduce congenital immune inflammation or inflammasome-related inflammation in the patient. This reduction may be compared to a control (e.g., an untreated patient and / or a patient before treatment). In one embodiment, this monoclonal antibody or an antibody fragment derived from this monoclonal antibody is used to treat central nervous system (CNS) injury, and / or autoimmune diseases, autoinflammatory diseases, metabolic diseases, or neurodegenerative diseases. This CNS injury may be selected from a 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, MS is treated by administering the monoclonal antibody or an antibody fragment derived from the monoclonal antibody to a patient who has or is suspected of having MS. In another embodiment, PD is treated by administering the monoclonal antibody or an antibody fragment derived from the monoclonal antibody to a patient who has or is suspected of having PD. In one embodiment, lupus nephritis is treated by administering the monoclonal antibody or an antibody fragment derived from this monoclonal antibody to a patient who has or is suspected of having lupus nephritis. This metabolic disorder may be selected from metabolic syndrome, obesity, diabetes, diabetic nephropathy or diabetic kidney disease (DKD), insulin resistance, atherosclerosis, lipid storage disorder, glycogen storage disorder, medium-chain acyl coenzyme A dehydrogenase deficiency, non-alcoholic fatty liver disease (e.g., non-alcoholic steatohepatitis (NASH)), and gout. In one embodiment, diabetic nephropathy is treated by administering the monoclonal antibody or an antibody fragment derived from this monoclonal antibody to a patient who has or is suspected of having diabetic nephropathy. In one embodiment, NASH is treated by administering the monoclonal antibody or an antibody fragment derived from the monoclonal antibody to a patient who has NASH or is suspected of having NASH.This autoinflammatory disorder may be cryopyrin-associated periodic syndrome (CAPS). CAPS is also known as familial influenza autoinflammatory syndrome (FCAS) or Mackle-Wells syndrome (MW). S), and neonatal-onset multisystem inflammatory diseases (NOMIDs) may be included. In one embodiment, CAPS is treated by administering the monoclonal antibody or an antibody fragment derived from the monoclonal antibody to a patient who has CAPS or is suspected of having CAPS. The monoclonal antibody or antibody fragment of this embodiment may be present in a composition (e.g., a pharmaceutical composition provided herein). In some cases, the monoclonal antibody or its fragment is used in combination with one or more other agents in the therapeutic methods provided herein. These other agents may be any agent 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 that specifically binds to ASCs, wherein the antibody or antibody fragment comprises a light chain variable (VL) region and a heavy chain variable (VH) region, the amino acid sequence of the VL region being LCDR1 of SEQ ID NO: 12, LCDR2 of SEQ ID NO: 13, and LCDR3 of SEQ ID NO: 14, or variants thereof, comprising variants having at least one amino acid substitution in LCDR1, LCDR2, and / or LCDR3. In addition to these embodiments, the present invention intends to use the monoclonal antibody or antibody fragment in a method for treating inflammation of a target. This inflammation may be congenital immunological inflammation. This inflammation may be inflammasome-related inflammation. In one embodiment, the monoclonal antibody or antibody fragment provided herein may be used in a method for reducing mammalian inflammation as described in U.S. Patent No. 8,685,400, the contents of this specification being incorporated herein by reference in their entirety. This inflammation may be present in the lungs and / or CNS. Inflammation in the lungs and / or CNS may result from injury (e.g., traumatic brain injury (TBI) or spinal cord injury (SCI)) or from a disease, condition, or distress affecting or in the CNS. Where provided herein, diseases, conditions, or distress affecting or in the CNS may include stroke, as well as autoimmune diseases and / or CNS diseases (e.g., amyotrophic lateral sclerosis (ALS) Lou Gehrig, multiple sclerosis (MS), immunodeficiency muscle CNS breakdown, muscular dystrophy (MD), Alzheimer's disease (AD), and Parkinson's disease (PD)). The use of this monoclonal antibody or its antibody fragment in a method of treating inflammation may reduce inflammation in the patient's CNS and / or lungs. The use of this monoclonal antibody or its antibody fragment in a method of treating inflammation may reduce congenital immune inflammation or inflammasome-related inflammation in the patient. This reduction may be compared to a control (e.g., an untreated patient and / or a patient before treatment).In one embodiment, the monoclonal antibody or an antibody fragment derived from this monoclonal antibody is used to treat central nervous system (CNS) injury and / or 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 this monoclonal antibody is used to treat MS in patients who have or are suspected of having MS by administering the monoclonal antibody or an antibody fragment derived from this monoclonal antibody. In one embodiment, PD is treated by administering the monoclonal antibody or an antibody fragment derived from this monoclonal antibody to a patient who has PD or is suspected of having PD. This monoclonal antibody or an antibody fragment derived from this monoclonal antibody is used to treat lupus nephritis in patients who have or are suspected of having lupus nephritis. This metabolic disorder may be selected from metabolic syndrome, obesity, diabetes, diabetic nephropathy or diabetic nephropathy (DKD), insulin resistance, atherosclerosis, lipid storage disorder, glycogen storage disorder, medium-chain acyl coenzyme A dehydrogenase deficiency, non-alcoholic fatty liver disease (e.g., non-alcoholic steatohepatitis (NASH)), and gout. In one embodiment, this monoclonal antibody or an antibody fragment derived from this monoclonal antibody is used to treat diabetic nephropathy in patients who have or are suspected of having diabetic nephropathy by administering this monoclonal antibody or an antibody fragment derived from this monoclonal antibody. In one embodiment, NASH is treated by administering the monoclonal antibody or an antibody fragment derived from this monoclonal antibody to a patient who has or is suspected of having NASH. This autoinflammatory disease may be cryopyrin-associated periodic syndromes (CAPS). CAPS may include familial influenza autoinflammatory syndrome (FCAS), Muckle-Wells syndrome (MWS), and neonatal onset multiorgan inflammatory disease (NOMID). In one embodiment, CAPS is treated by administering the monoclonal antibody or an antibody fragment derived from this monoclonal antibody to a patient who has or is suspected of having CAPS. The monoclonal antibody or antibody fragment of this embodiment may be present in a composition (e.g., a pharmaceutical composition provided herein). In some cases, this monoclonal antibody or a fragment thereof is used in combination with one or more other agents in the therapeutic methods provided herein.Other agents may be any 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 other embodiments, the present invention also provides a monoclonal antibody or antibody fragment that specifically binds to ASCs, wherein the antibody or antibody fragment comprises 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, comprising 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, comprising variants having at least one amino acid substitution in LCDR1, LCDR2, and / or LCDR3. In addition to these embodiments, the present invention intends to use the monoclonal antibody or antibody fragment in a method for treating inflammation of interest. This inflammation may be congenital immunological inflammation. This inflammation may be inflammasome-related inflammation. In one embodiment, a monoclonal antibody or an antibody fragment thereof provided herein may be used in a manner that reduces inflammation in a mammal, as described in U.S. Patent No. 8,685,400, the contents of which this specification are incorporated herein by reference in their entirety. This inflammation may be present in the lungs and / or CNS. Inflammation in the lungs and / or CNS may result from injury (e.g., traumatic brain injury (TBI) or spinal cord injury (SCI)) or from a disease, condition, or distress of or affecting the CNS. Where provided herein, diseases, conditions, or distress of or affecting the CNS may include stroke, as well as autoimmune diseases and / or CNS diseases (e.g., amyotrophic lateral sclerosis (ALS) Lou Gehrig, multiple sclerosis (MS), immunodeficiency muscle CNS breakdown, muscular dystrophy (MD), Alzheimer's disease (AD), and Parkinson's disease (PD)). The use of this monoclonal antibody or its antibody fragment in methods of treating inflammation can reduce inflammation in the patient's CNS and / or lungs. Reduction is possible. The use of this monoclonal antibody or its antibody fragment in methods of treating inflammation may reduce a patient's congenital immune inflammation or inflammation associated with the inflammasome. This reduction may be compared to a control (e.g., an untreated patient and / or a patient before treatment). In one embodiment, this monoclonal antibody or an antibody fragment derived from this monoclonal antibody is used to treat central nervous system (CNS) injury and / or autoimmune disease, autoinflammatory disease, metabolic disease, or neurodegenerative disease. This CNS injury may be selected from the group consisting of traumatic brain injury (TBI), stroke, and spinal cord injury (SCI). This 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, MS is treated by administering the monoclonal antibody or an antibody fragment derived from this monoclonal antibody to a patient who has MS or is suspected of having MS. In another embodiment, PD is treated by administering the monoclonal antibody or an antibody fragment derived from this monoclonal antibody to a patient who has PD or is suspected of having PD. In another embodiment, lupus nephritis is treated by administering the monoclonal antibody or an antibody fragment derived from this monoclonal antibody to a patient who has lupus nephritis or is suspected of having lupus nephritis. These metabolic disorders may be selected from metabolic syndrome, obesity, diabetes mellitus, 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, diabetic nephropathy is treated by administering the monoclonal antibody or an antibody fragment derived from this monoclonal antibody to patients who have or are suspected of having diabetic nephropathy. In another embodiment, NASH is treated by administering the monoclonal antibody or an antibody fragment derived from this monoclonal antibody to patients who have or are suspected of having NASH. This autoinflammatory disease may be cryopyrin-associated periodic syndromes (CAPS). CAPS may include familial influenza autoinflammatory syndrome (FCAS), Muckle-Wells syndrome (MWS), and neonatal onset multiorgan inflammatory disease (NOMID). In one embodiment, CAPS is treated by administering the monoclonal antibody or an antibody fragment derived from the monoclonal antibody to a patient who has CAPS or is suspected of having CAPS. The monoclonal antibody or antibody fragment of this embodiment may be present in a composition (e.g., a pharmaceutical composition provided herein). In some cases, the monoclonal antibody or its fragment is used in combination with one or more other agents in the therapeutic methods provided herein. These other agents may be any 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 congenital immunological inflammation or inflammasome-associated inflammation. In some cases, this inflammasome-associated inflammation is present in the CNS of mammals that have congenital immunological inflammation or inflammasome-associated inflammation, or are exposed to or suffering from conditions that cause this inflammation. In some cases, this inflammasome-associated inflammation is congenital It is present in mammals (e.g., humans) that are associated with, develop, or suffer from conditions that cause immunological inflammation or inflammasome-related inflammation. Conditions that cause or develop congenital immunological inflammation or inflammasome-related inflammation may be CNS damage, autoimmune, autoinflammatory, neurodegenerative, and / or metabolic diseases or disorders. CNS damage may be selected from the group consisting of traumatic brain injury (TBI), stroke, and spinal cord injury (SCI). Autoimmune or neurodegenerative diseases 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). This metabolic disorder may be selected from metabolic syndrome, obesity, diabetes mellitus, 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. This autoinflammatory disorder may be cryopyrin-associated periodic syndromes (CAPS). CAPS may include familial influenza autoinflammatory syndrome (FCAS), Muckle-Wells syndrome (MWS), and neonatal onset multiorgan inflammatory disease (NOMID). The compositions and methods described herein may include antibodies or active fragments thereof provided herein that specifically bind to at least one component (e.g., ASC) of mammalian inflammasomes and / or compounds used as therapeutic agents for mammalian CNS inflammation, modulating (e.g., inhibiting or reducing) extracellular vesicle (EV) uptake and Examples of conditions that may cause inflammation in the central nervous system (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. This composition may be administered in a therapeutically effective dose. This therapeutically effective dose may be the 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 components of the inflammasome, or a combination thereof. The composition may be administered by any suitable route, for example, by inhalation, intravenously, intraperitoneally, or intraventricularly. 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 subjects who have or are suspected of having MS. Methods for treating MS provided herein may involve administering a composition (e.g., a pharmaceutical composition) containing a drug (e.g., IC100) to a subject who has or is suspected of having MS. Multiple sclerosis (MS) is an autoimmune disease affecting the brain and spinal cord. The subject may present with clinical symptoms consistent with MS. The subject may be diagnosed with any type of MS known in the art. MS may be relapsing-remitting MS (RRMS), secondary progressive MS (SPMS), secondary progressive MS (PPMS), or progressive-relapsing MS (PRMS). The MS diagnosis may or may have been determined using any method known in the art. In one embodiment, the subject is diagnosed with MS using the method 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 may be a standard care treatment known in the art for MS, an EV uptake inhibitor (e.g., any EV uptake inhibitor from Table 1), an antibody or antibody fragment thereof provided herein that binds to a component of the inflammasome (e.g., an anti-ASC monoclonal antibody or antibody fragment thereof, e.g., IC100), or any combination thereof. The composition may be administered by any suitable route, for example, by inhalation, intravenously, intraperitoneally, or intraventricularly. The composition may further comprise at least one pharmaceutically acceptable carrier or diluent. Standard care treatment is a treatment for modifying disease outcomes. Treatment may be selected from the following: treatment for managing relapses, treatment for managing symptoms, or any combination thereof. Treatment for modifying disease outcomes 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 subjects who have or are suspected of having PD. Methods for treating PD provided herein may involve administering a composition (e.g., a pharmaceutical composition) containing a drug (e.g., IC100) to a subject who has or is suspected of having PD. Parkinson's disease (PD) is a progressive neurological disorder affecting movement due to the gradual destruction and / or death of nerve cells in the brain of a mammal (e.g., a human) who has PD. PD may develop and / or progress through five stages (i.e., stages 1-5), and the compositions and methods provided herein may be used to treat individuals who have or are suspected of having PD at any of these five stages. A diagnosis of PD may or may have been determined using any method known in the art. In one embodiment, the subject is diagnosed with PD using a method detailed in International Publication No. 2019 / 060516, filed on September 20, 2018 (the contents of which are incorporated herein by reference in their entirety). The agent may be a standard care treatment known in the art for PD, an EV uptake inhibitor (e.g., any EV uptake inhibitor from Table 1), an antibody or antibody fragment thereof provided herein that binds to a component of the inflammasome (e.g., an anti-ASC monoclonal antibody or antibody fragment thereof, e.g., IC100), or any combination thereof. The composition may be administered by any suitable route, for example, by inhalation, intravenously, intraperitoneally, or intraventricularly. The composition may further comprise at least one pharmaceutically acceptable carrier or diluent. Standard care treatment may be selected from carbidopa (Lodosyn), levodopa, carbidopa-levodopa combination, Duopa, dopamine agonists, MAO B inhibitors, catechol O-methyltransferase (COMT) inhibitors, anticholinergics, amantadine, and deep brain stimulation. Dopamine agonists include pramipexole (Mirapex), ropinirole (Requip), and rotigotine (Neupro). MAO B inhibitors include selegiline (Eldepryl, Zelapar) and rasagiline. (Azilect) and safinamide (Xadago) may be selected. COMT inhibitors are used in en Comtan and Tasmar may be selected as the anticholinergic agent. Cogentin or trihexyphenidyl may be selected as the anticholinergic agent.

[0056]

[0077] Provided herein are compositions and methods for treating Alzheimer's disease (AD) in subjects who have or are suspected of having AD. Methods for treating AD provided herein may involve administering a composition (e.g., a pharmaceutical composition) containing a drug (e.g., IC100) to a subject who has or is suspected of having AD. Alzheimer's disease (AD) is a progressive neurological disorder that causes degeneration and death of brain cells in individuals with AD and progresses from mild cognitive impairment (MCI) to complete memory loss, and further to changes in personality and behavior. A diagnosis of AD may or may have been determined using any method known in the art. In one embodiment, the subject has been diagnosed with AD using a method detailed in International Publication No. 2019 / 060516, filed September 20, 2018 (the contents of which are incorporated herein by reference in their entirety). This agent may be a standard care treatment known in the art for AD, an EV uptake inhibitor (e.g., any EV uptake inhibitor from Table 1), an antibody or antibody fragment thereof provided herein that binds to a component of the inflammasome (e.g., an anti-ASC monoclonal antibody or antibody fragment thereof, e.g., IC100), or any combination thereof. This composition may be administered by any suitable route, for example, by inhalation, intravenously, intraperitoneally, or intraventricularly. It can be administered. This composition may further comprise at least one pharmaceutically acceptable carrier or diluent. Standard care treatment involves cholinesterase inhibitors and memantine (Namenda Cholinesterase inhibitors can be selected from: donepezil (Aricept), galantha. The choice may be between Razadyne and rivastigmine (Exelon).

[0057]

[0078] Provided herein are compositions and methods for treating rheumatoid arthritis (RA) in subjects who have or are suspected of having RA. Methods for treating RA provided herein may involve administering a composition (e.g., a pharmaceutical composition) containing a drug (e.g., IC100) to a subject who has or is suspected of having RA. Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disorder that can cause damage to the joints of an individual, as well as to the skin, eyes, lungs, heart, and blood vessels. A diagnosis of RA may or may have been determined using any method known in the art. In one embodiment, the subject has been diagnosed with RA using a method 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 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 conjugates 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 intraventricularly. The composition may further comprise at least one pharmaceutically acceptable carrier or diluent. Standard care treatments may be selected from nonsteroidal anti-inflammatory drugs (NSAIDs), steroids (e.g., prednisone), disease-modifying antirheumatic drugs (DMARDs), and biological agents. Examples of NSAIDs include ibuprofen (Advil, Motrin IB) and naproxen sodium (Aleve). Examples of DMARDs include methotrex. Examples of steroids include SART (Trexall, Otrexup, etc.), leflunomide (Arava), hydroxychloroquine (Plaquenil), and sulfasalazine (Azulfidine). Examples of biological agents include abatacept (Orencia), adalimumab (Humira), anakinra (Kineret), baricitinib (Olumiant), certolizumab (Cimzia), etanercept (Enbrel), golimumab (Simponi), infliximab (Remicade), rituximab (Rituxan), sarilumab (Kevzara), tocilizumab (Actemra), and tofacitinib (Xeljanz). .

[0058]

[0079] Provided herein are compositions and methods for treating lupus nephritis in subjects who have or are suspected of having lupus nephritis. Methods for treating lupus nephritis provided herein may involve administering a composition (e.g., a pharmaceutical composition) containing a drug (e.g., IC100) to subjects who have or are suspected of having lupus nephritis. Lupus nephritis, often simply referred to as lupus, is a type of kidney inflammation that is often a common complication of systemic lupus erythematosus. Lupus nephritis is an autoimmune disease in which lupus autoantibodies affect the structure of the kidneys of an individual, and this autoimmune disease can lead to inflammation of the kidneys and, in some cases, hematuria, proteinuria, hypertension, renal dysfunction, and even renal failure. A diagnosis of lupus nephritis may or may have been determined using any method known in the art. In one embodiment, the subject is diagnosed with lupus nephritis using the method detailed in International Publication No. 2019 / 060516, filed on September 20, 2018 (the contents of which are incorporated herein by reference in their entirety). The agent may be a standard care treatment known in the art for lupus nephritis, an EV uptake inhibitor (e.g., any EV uptake inhibitor from Table 1), an antibody or antibody fragment thereof provided herein that binds to a component of the inflammasome (e.g., an anti-ASC monoclonal antibody or antibody fragment thereof, e.g., IC100), or any combination thereof. It can be administered by an appropriate route of intent, for example, by inhalation, intravenously, intraperitoneally, or intraventricularly. This composition may further comprise at least one pharmaceutically acceptable carrier or diluent. Standard care for lupus nephritis may include medications to control blood pressure and / or special diets low in protein and salt. In addition, standard care for lupus nephritis may include treatment for lupus, which may be, for example, nonsteroidal anti-inflammatory drugs (NSAIDs), antimalarial drugs, corticosteroids (e.g., prednisone; methylprednisolone), immunosuppressants, or biological agents. Examples of NSAIDs include naproxen sodium (Aleve) and ibuprofen (Advil, Motrin IB, etc.). It is possible. An example of an antimalarial drug is hydroxychloroquine (Plaquenil). Examples of inhibitors include azathioprine (Imuran, Azasan), mycophenolate mofetil (CellCept), and methotrexate (Trexall). Examples of biological agents include: Possible options include belimumab (Benlysta) or rituximab (Rituxan).

[0059]

[0080] Provided herein are compositions and methods for treating non-alcoholic steatohepatitis (NASH) in subjects who have or are suspected of having NASH. Methods for treating NASH provided herein may involve administering a composition (e.g., a pharmaceutical composition) containing a drug (e.g., IC100) to subjects who have or are suspected of having NASH. NASH is a type of non-alcoholic fatty liver disease (NAFLD). NAFLD is a broad term encompassing a range of liver conditions affecting people who do not drink alcohol at all or very little. The main features of NAFLD are an excess of fat stored in liver cells and inflammation of the liver, which may progress to scarring and irreversible damage. This damage may be similar to that caused by heavy alcohol consumption. In the most severe cases, non-alcoholic steatohepatitis can progress to cirrhosis and liver failure. A diagnosis of NASH may or may have been determined using any method known in the art. In one embodiment, the subject is diagnosed with NASH using a method detailed in International Publication No. 2019 / 060516, filed on September 20, 2018 (the contents of which are incorporated herein by reference in their entirety). The agent may be a standard care treatment known in the art for NASH, an EV uptake inhibitor (e.g., any EV uptake inhibitor from Table 1), an antibody or antibody fragment thereof provided herein that binds to a component of the inflammasome (e.g., an anti-ASC monoclonal antibody or antibody fragment thereof, e.g., IC100), or any combination thereof. The composition may be administered by any suitable route, for example, by inhalation, intravenously, intraperitoneally, or intraventricularly. The composition may further comprise at least one pharmaceutically acceptable carrier or diluent. Standard care treatments for NASH may include lifestyle changes, such as weight loss, increased exercise, avoidance of drugs that damage the liver, cholesterol reduction, and / or management of diabetes.

[0060]

[0081] Provided herein are compositions and methods for treating diabetic nephropathy in subjects who have or are suspected of having diabetic nephropathy. Methods for treating diabetic nephropathy provided herein may involve administering a composition (e.g., a pharmaceutical composition) containing a drug (e.g., IC100) to a subject who has or is suspected of having diabetic nephropathy. Diabetic nephropathy is a severe kidney-related complication of type 1 and type 2 diabetes, also known as diabetic nephropathy (DKD). A diagnosis of DKD may or may have been determined using any method known in the art. In one embodiment, the subject is diagnosed with DKD using a method detailed in International Publication No. 2019 / 060516, filed September 20, 2018 (the contents of which are incorporated herein by reference in their entirety). This drug is a standard care treatment known in the art for DKD, an EV uptake inhibitor (e.g., any EV uptake inhibitor from Table 1), and a component of the inflammasome. The composition may be an antibody or an antibody fragment thereof provided in the specification (e.g., an anti-ASC monoclonal antibody or an 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 intraventricularly. The composition may further comprise at least one pharmaceutically acceptable carrier or diluent. Standard care treatments for diabetic nephropathy may include lifestyle changes, such as weight loss, increased exercise, cholesterol reduction, control of urinary protein, promotion of bone health, control of hypertension, management of diabetes, and kidney dialysis or transplantation.

[0061]

[0082] Provided herein are compositions and methods for treating inflammatory bowel disease (IBD) in subjects who have or are suspected of having IBD. Methods for treating IBD provided herein may involve administering a composition (e.g., a pharmaceutical composition) containing a drug (e.g., IC100) to subjects who have or are suspected of having IBD. IBD is a broad term used to describe chronic inflammatory disorders of the gastrointestinal tract of an individual. Examples of IBD include ulcerative colitis and Crohn's disease. Ulcerative colitis is a long-term inflammation and erosion (ulceration) of the innermost layer of the large intestine (colon) and rectum, while Crohn's disease is characterized by inflammation of the inner layer of the gastrointestinal tract, which often extends deeply into the affected tissue. IBD diagnosis may or may have been determined using any method known in the art. In one embodiment, the subject is diagnosed with IBD using a method detailed in International Publication No. 2019 / 060516, filed on September 20, 2018 (the contents of which are incorporated herein by reference in their entirety). The agent may be a standard care treatment known in the art for IBD, an EV uptake inhibitor (e.g., any EV uptake inhibitor from Table 1), an antibody or antibody fragment thereof provided herein that binds to a component of the inflammasome (e.g., an anti-ASC monoclonal antibody or antibody fragment thereof, e.g., IC100), or any combination thereof. The composition may be administered by any suitable route, for example, by inhalation, intravenously, intraperitoneally, or intraventricularly. The composition may further comprise at least one pharmaceutically acceptable carrier or diluent. Standard care treatments for IBD may include anti-inflammatory agents, immunosuppressants, antibiotics, antidiarrheals, analgesics, iron supplements, and calcium and vitamin D supplements. Antibiotics that may be used include ciprofloxacin (Cipro) and metronidazole (Flagyl). Examples of immunosuppressants include azathioprine (Azasan, Imuran), mercaptopurine (Purinethol, Purixan), cyclosporine (Gengraf, Neoral, Sandimmune), and methotrexate (Trexall). Other examples of immunosuppressants include tumor necrosis factor (TNF)-associated immunosuppressants. Glutathione inhibitors, or biologics (e.g., infliximab (Remicade), adalimumab (Humira), golimumab (Simponi), natalizumab (Tysabri), vedolizumab (Entyvio)) Possible examples include ), and ustekinumab (Stelara). As an anti-inflammatory agent, corticosteroids Lloyd and aminosalicylates (e.g., mesalamine (Asacol HD, Delzicol), balsa) Possible examples include radid (Colazal) and orsalazine (Dipentum).

[0062]

[0083] Provided herein are compositions and methods for treating Cryopyrin-Associated Periodic Syndromes (CAPS) in subjects who have or are suspected of having CAPS. Methods for treating CAPS provided herein may involve administering a composition (e.g., a pharmaceutical composition) containing a drug (e.g., IC100) to subjects who have or are suspected of having CAPS. Cryopyrin-Associated Periodic Syndromes (CAPS), also known as Cryopyrin-Associated Autoinflammatory Syndromes, consist of the following three autoinflammatory diseases associated with deletions in the same gene (i.e., NLRP3): Neonatal-Onset Multiorgan Inflammatory Disease (NOMID), Muckle-Wells Syndrome (MWS), and Familial Influenza Autoinflammatory Syndrome (FCAS). NOMID is characterized by fever accompanied by inflammation in multiple organs. An early symptom of NOMID is a non-itchy, honeycomb-like rash; Possible symptoms include inflammation of the membranes surrounding the brain, causing headache, blindness, or hearing loss; a swollen appearance of the eyes; and episodes of vomiting. After the age of one, half of children with NOMID may develop arthralgia and swelling. MWS is characterized by symptoms that appear and disappear (e.g., severe headache with skin rash, red eyes, arthralgia, and vomiting). Episodes last 1 to 3 days. Hearing loss, which may be complete, often develops by adolescence. FCAS is characterized by fever, chills, nausea, extreme thirst, headache, and arthralgia. CAPS diagnosis may or may have been determined using any method known in the art. In one embodiment, the subject is diagnosed with CAPS using the method 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 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 conjugates 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 intraventricularly. The composition may further comprise at least one pharmaceutically acceptable carrier or diluent. Standard care treatments for CAPS may include biological agents targeting interleukin-1, as well as physiotherapy, splints for treating joint deformities, and nonsteroidal anti-inflammatory drugs, corticosteroids, or methotrexate for symptom relief.

[0063]

[0084] Provided herein are compositions and methods for reducing inflammation in the lungs of mammals that are affected by, or suffering from, conditions that cause pneumonia. The compositions and methods described herein may include antibodies or active fragments thereof (e.g., IC100) provided herein that modulate (e.g., inhibit or reduce) extracellular vesicle (EV) uptake and specifically bind to at least one component (e.g., ASC) of a mammalian inflammasome and / or compound used as a treatment for mammalian pneumonia.

[0064]

[0085] This specification describes methods for reducing inflammation in the lungs of mammals having conditions that cause and / or induce an inflammatory response in the lungs. In one embodiment, a 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., IC100). The mammal may be a patient or subject provided herein. Examples of conditions that may cause inflammation in the lungs include central nervous system (CNS) injuries (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 dose. The therapeutically effective dose may be the dose provided herein. The agent may be an extracellular vesicle (EV) uptake inhibitor, an antibody or active fragment thereof provided herein that binds to components of the inflammasome, or a combination thereof. The composition can be administered by any preferred route, for example, by inhalation, intravenously, intraperitoneally, or intraventricularly. The composition may further comprise at least one pharmaceutically acceptable carrier or diluent.

[0065]

[0086] Provided herein are compositions and methods for reducing kidney inflammation in mammals that are affected by, or suffering from, conditions that cause kidney inflammation. The compositions and methods described herein involve antibodies or active fragments thereof provided herein that regulate (e.g., inhibit or reduce) the uptake of extracellular vesicles (EVs) and specifically bind to at least one component (e.g., ASC) of the mammalian inflammasome and / or compound used as a treatment for mammalian kidney inflammation. It may include IC100).

[0066]

[0087] Described herein are methods for reducing renal inflammation in mammals having conditions that cause and / or induce an immune response in the kidney. In one embodiment, a 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 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. This therapeutically effective amount may be the dose provided herein. The agent may be an extracellular vesicle (EV) uptake inhibitor, an antibody or active fragment thereof provided herein that binds to components of the inflammasome, or a combination thereof. The composition may be administered by any suitable route, for example, by inhalation, intravenously, intraperitoneally, or intraventricularly. The composition may further comprise at least one pharmaceutically acceptable carrier or diluent.

[0067]

[0088] In one embodiment, administration of a drug (e.g., an antibody or antibody fragment derived from such antibody (e.g., IC100)) in a manner provided herein may result in a decrease in the activity and / or expression levels of mammalian inflammasome components in the CNS, kidney, or lung of a subject. This decrease may occur in lung cells, such as type II alveolar cells. This decrease may be in comparison to a control, which may be a subject before administration of the drug. This control may be the activity and / or expression levels of inflammasome components in a subject not administered the drug. In one embodiment, administration of the drug results in a reduction of caspase-1 activation in at least the CNS or CNS cells of the subject. In one embodiment, administration of the drug results in a reduction of caspase-1 activation in at least the lung or lung cells of the subject. In one embodiment, administration of this drug 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 this drug 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 a drug (e.g., an antibody or antibody fragment derived from such antibody, either alone or in combination with an EV uptake inhibitor) may result in a reduction or elimination of acute lung injury (ALI). In one embodiment, the reduction of ALI is demonstrated 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. This reduction may be in comparison to a control. This control may be ALI in a subject before administration of the drug. This control may be ALI in a subject suffering from ALI but not administered the drug.

[0069]

[0090] In yet another embodiment, administration of a drug (e.g., an antibody or antibody fragment derived from this antibody (e.g., IC100), either alone or in combination with an EV uptake inhibitor) may result in a reduction or elimination of pyroptosis in the subject's CNS or lung. Pyroptosis is a pro-inflammatory form of cell death accompanied by caspase-1 activation. Pyroptosis can be induced by caspase-1-mediated cleavage of gasdermin D (GSDMD). In one embodiment, a reduction in pyroptosis is demonstrated by a decrease or absence of GSDMD cleavage in the subject's lung or lung cells (e.g., type II alveolar cells). The reduction or elimination of pyroptosis may be compared to a control. The decrease or absence of GSDMD cleavage may be compared to a control. This control is the level of pyroptosis in the subject before administration of the drug. This could be the case. This control could be the level of pyroptosis in subjects suffering from pyroptosis who have not been administered this drug.

[0070]

[0091] The success of or response to the therapeutic methods provided herein (e.g., treatment of CNS injury, autoimmune diseases, autoinflammatory diseases, neurodegenerative diseases, or metabolic diseases (e.g., MS, PD, lupus nephritis, NASH, DKD, CAPS, inflammatory bowel disease (IBD); AD, rheumatoid arthritis), congenital immune inflammation or inflammation associated with the inflammasome, CNS inflammation, and / or pneumonia) can be monitored by measuring the level of at least one inflammasome protein. Accordingly, in some embodiments, the therapeutic methods provided herein further include measuring the level of at least one inflammasome protein in a biological sample obtained from a subject after treatment, creating a therapeutic inflammasome protein signature associated with a positive response to the treatment, wherein the therapeutic protein signature includes a decrease in the level of at least one inflammasome protein, and identifying a subject that exhibits 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 this subject. The one or more inflammasome proteins measured in samples obtained after treatment may be the same as or different from those measured in samples obtained before treatment. The level of inflammasome proteins may be used to adjust the dosage or frequency of treatment. The level of these inflammasome proteins can be confirmed using methods and techniques as 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 by the therapeutic method provided herein is an EV reuptake inhibitor. The EV reuptake inhibitor may be a compound, antisense RNA, siRNA, peptide, an antibody or its active fragment provided herein, or a combination thereof. The compound or peptide may be one or more compounds selected from heparin, α-difluoromethylornithine (DFMO), enoxaparin, asialofetine, human receptor-related protein (RAP), RGD (Arg-Gly-Asp) peptide, cytochalasin D, cytochalasin B, ethylenediaminetetraacetic acid (EDTA), latruncrine A, latruncrine B, NSC23766, dinosaur, chlorpromazine, 5-(N-ethyl-N-isopropyl)amylolide (EIPA), amylolide, bafilomycin A, monensin and chloroquine, annexin-V, wartmannin, LY294002, methyl-β-cyclodextrin (MβCD), philipin, simvastatin, fumonisin B1 and N-butyldeoxynojirimycin hydrochloride, U0126, or proton pump inhibitors. EV uptake inhibitors, antibodies or active fragments thereof provided herein may be one or more antibodies or active fragments thereof directed against the protein targets listed in Table 1. Compositions for treating and / or reducing inflammation in the CNS or lungs of mammals using EV uptake inhibitors 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 provided herein or an active fragment thereof, directed against a component of a mammalian inflammasome or an antigen or epitope derived therefrom. In another embodiment, the agent to be administered is an antisense RNA or siRNA directed against a component of a mammalian inflammasome. The inflammasome component may be any inflammasome known in the art, such as the NAPL1, NALP2, NALP3, NLRC4, or AIM2 inflammasome. In a typical embodiment, the antibody specifically binds to the ASC or an antigen or epitope derived therefrom. However, antibodies against any other component of a mammalian inflammasome (e.g., NALP1, NALP2, NALP3, NLRC4, or AIM2 inflammasome) can be used.

[0078]

[0095] The antibodies described herein may be monoclonal or polyclonal antibodies or their active fragments. The antibodies or active fragments may be chimeric, human, or humanized as described herein.

[0079]

[0096] Any suitable antibody or active fragment thereof provided herein that specifically binds to ASC, for example, target CNS (e.g., CNS cells) or lung cells (e.g., type II alveoli). Antibodies that inhibit ASC activity in cells can be used. In one embodiment, the antibody specifically binds to an amino acid sequence having at least 85% sequence identity with amino acid sequence SEQ ID NO: 1 or SEQ ID NO: 2. In another embodiment, the antibody or a fragment thereof binds to an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with 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 yet another embodiment, 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 epitopes of the ASC to which the antibody or antibody fragment binds (e.g., epitopes having amino acid SEQ ID NO: 5) are continuous. In some embodiments, the epitopes of the ASC to which the antibody or antibody fragment binds (e.g., epitopes having amino acid SEQ ID NO: 5) are discontinuous. In some examples, the monoclonal antibodies or antibody fragments provided herein inhibit or reduce the activity of the ASC.

[0080]

[0097] As used herein, the term “epitope” includes any protein determinant capable of specifically binding to an immunoglobulin or immunoglobulin fragment. Epitope determinants typically consist of chemically active surface groups of molecules, such as amino acids or sugar side chains, and usually possess specific three-dimensional structural and charge properties. The term “epitope” also refers to a structural unit conventionally bound by a pair of heavy-chain variable (VH) and light-chain variable (VL) regions of an immunoglobulin. Epitopes can define the minimum binding site of an antibody and thus represent the target of the antibody's specificity.

[0081]

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

[0082]

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

[0083]

[0100] In one embodiment, this method involves mammalian inflammasomes (e.g., AI) The present invention provides a therapeutically effective amount of a composition comprising an antibody provided herein or an active fragment thereof that specifically binds to at least one component (e.g., ASC) of a mammalian inflammasome (e.g., AIM2 inflammasome); and administers the composition to a mammal suffering from CNS or pneumonia or MS, wherein the administration of the composition to the mammal results in a reduction of caspase-1 activation in the mammalian CNS or lung. In another embodiment, the present invention provides 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 administers the composition to a mammal suffering from CNS or pneumonia or MS, wherein the administration of the composition to the mammal results in a reduction of caspase-1 activation in the mammalian CNS or lung. This results in a reduction in the levels of one or more inflammasome components (e.g., ASCs). In yet another embodiment, the method comprises 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 pneumonia or MS, wherein the administration of the composition to the mammal results in a reduction of ALI. CNS or pneumonia may be the result of CNS damage by inflammatory components (e.g., SCI or TBI), asthma, chronic obstructive pulmonary disease (COPD), neurodegenerative disease, or autoimmune disease. In one embodiment, pneumonia is caused by CNS damage, e.g., TBI or SCI.

[0084]

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

[0085] Antibodies that specifically bind to at least one component of mammalian inflammasomes.

[0102] The following methods reduce inflammation in the CNS and / or lungs of mammals as described herein. The method comprises a composition comprising an antibody or an active fragment thereof provided herein that specifically binds to at least one component (e.g., ASC, AIM2) of a mammalian inflammasome (e.g., AIM2 inflammasome). Compositions for treating and / or reducing inflammation in the mammalian CNS and / or lung may further comprise at least one pharmaceutically acceptable carrier or diluent. Exemplary antibodies directed against components of mammalian inflammasomes 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. Exemplary monoclonal antibodies or antibody fragments (e.g., monoclonal antibodies or antibody fragments comprising a VH region, the amino acid sequence of which is such that it comprises HCDR1 of SEQ ID NO: 6, HCDR2 of SEQ ID NO: 7, and HCDR3 of SEQ ID NO: 8, and a VL region, the amino acid sequence of which is such that it comprises LCDR1 of SEQ ID NO: 12, LCDR2 of SEQ ID NO: 13, and LCDR3 of SEQ ID NO: 14) are also provided herein.

[0086]

[0103] In one embodiment, inflammation in the CNS or lungs of mammals is treated, and The composition for / or reduction comprises an antibody or active fragment thereof provided herein that specifically binds to a domain or part thereof of a mammalian ASC protein, such as human, mouse, or rat ASC protein. Any suitable anti-ASC antibody may be used, some of which are commercially available. An example of an anti-ASC antibody for use in the method herein may be found in U.S. Patent No. 8,685,400, the contents of which are incorporated herein by reference in their entirety. An example of a commercially available anti-ASC antibody for use in the method provided herein is, but is not limited to, 04-147 anti-ASC, MilliporeSig Clone 2EI-7 mouse monoclonal antibody from ma, AB3 from Millipore Sigma Examples include 607-anti-ASC antibody, orb194021 anti-ASC from Biobyt, LS-C331318-50 anti-ASC from LifeSpan Biosciences, AF3805 anti-ASC from R&D Systems, NBP1-78977 anti-ASC from Novus Biologicals, 600-401-Y67 anti-ASC from Rockland Immunochemicals, D086-3 anti-ASC from MBL International, AL177 anti-ASC from Adipogen, monoclonal anti-ASC (clone o93E9) antibody, anti-ASC antibody (F-9) from Santa Cruz Biotechnology, anti-ASC antibody (B-3) from Santa Cruz Biotechnology, ASC polyclonal antibody-ADI-905-173 from Enzo Life Sciences, or A161 anti-human ASC-Leinco Technologies. Human ASC proteins may have accession numbers NP_037390.2 (Q9ULZ3-1), NP_660183 (Q9ULZ3-2), or Q9ULZ3-3. Rat ASC proteins may have accession number NP_758825 (BAC43754). Mouse ASC proteins may have accession number NP_075747.3. In one embodiment, the antibody binds to the PYRIN-PAAD-DAPIN domain (PYD) or a part or fragment thereof of a mammalian ASC protein (e.g., human, mouse, or rat ASC). 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 or fragment thereof of a human, mouse, or rat ASC. In one embodiment, the antibody binds to the C-terminal caspase recruitment domain (CARD) or a portion or fragment thereof of a mammalian ASC protein (e.g., human, mouse, or rat ASC). In this embodiment, the antibody 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 or fragment thereof of a human, mouse, or rat ASC. 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) localized between the PYD and CARD domains. In another embodiment, a composition for treating and / or reducing inflammation in the CNS and / or lung of a mammal includes an antibody that specifically binds to a region of rat ASC, for example, the amino acid sequence ALRQTQPYLVTDLEQS (SEQ ID NO: 1) (i.e., residues 178-193 of rat ASC, accession number 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 with the rat ASC amino acid sequence ALRQTQPYLVTDLEQS (SEQ ID NO: 1).In another embodiment, a composition for treating and / or reducing inflammation in the mammalian CNS and / or lung comprises an antibody that specifically binds to a region of the human ASC, for example, the amino acid sequence RESQSYLVEDLERS (SEQ ID NO: 2). In yet another embodiment, a composition for treating and / or reducing inflammation in the mammalian CNS and / or lung comprises an antibody that specifically binds to a region of the human ASC (for example, the amino acid sequence KKFKLKLLSVPLREGYGRIPR (SEQ ID NO: 5; i.e., residues 21-41 of the 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 the ASC domain or a fragment thereof, as described herein, inhibits ASC activity in lung cells (e.g., mammalian type II alveolar cells). In another embodiment, an antibody that binds to the ASC domain or a fragment thereof, as described herein (e.g., a monoclonal anti-ASC antibody or an antibody fragment thereof provided herein), inhibits ASC activity in the CNS of mammals that are or are suspected of being affected by CNS damage or impairment. Examples of CNS damage or impairment include TBI, SCI, stroke, amyotrophic lateral sclerosis (ALS) Lou Gehrig's disease, multiple sclerosis (MS), immunodeficiency muscle CNS degradation, muscular dystrophy (MD), Alzheimer's disease (AD), and Parkinson's disease (PD).

[0087]

[0104] In a particular embodiment, the present invention specifically binds to ASC and is shown in Table 2. The present invention provides antibodies and antibody fragments comprising one or more amino acid sequences. Similarly, the present invention provides isolated nucleic acid molecules encoding monoclonal antibodies or antibody fragments thereof comprising the nucleic acid sequences shown in Table 2. In some examples, expression vectors comprising the nucleic acid molecules in Table 2 are provided. These expression vectors may comprise a heavy chain constant region or a light chain constant region. An example of a light chain 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 chain or light chain nucleic acid molecules may be operably linked to a regulatory sequence suitable for the 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, the molecule provided herein specifically binds to ASC. A noclonal antibody or an antibody fragment thereof, wherein the antibody or antibody fragment is heavy chain variable ( A monoclonal antibody or antibody fragment thereof comprising a VH) region and a light or kappa chain variable (VL) region, wherein the amino acid sequence of the VH region comprises SEQ ID NOs. 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 NOs. 18, 19, 20, 21, or 22. In addition to this embodiment, provided herein is the use of this monoclonal antibody or antibody fragment thereof in a method for treating inflammation of interest. This inflammation may be congenital immunological inflammation. This inflammation may be inflammasome-related inflammation. This inflammation may be present in the lungs and / or CNS. Inflammation in the lungs and / or CNS may be the result of injury (e.g., traumatic brain injury (TBI) or spinal cord injury (SCI)) or the result of a disease, condition, or distress of or affecting the CNS. Where provided herein, diseases, conditions, or distress affecting or influencing the CNS may include stroke, as well as autoimmune diseases and / or CNS diseases (e.g., amyotrophic lateral sclerosis (ALS) Lou Gehrig's disease, multiple sclerosis (MS), immunodeficiency muscle CNS breakdown, muscular dystrophy (MD), Alzheimer's disease (AD), and Parkinson's disease (PD)). The use of this monoclonal antibody or its antibody fragment in a method of treating inflammation may reduce inflammation in the patient's CNS and / or lungs. This reduction may be compared to a control (e.g., an untreated patient and / or a patient before treatment). In one embodiment, MS is treated by administering this monoclonal antibody or an antibody fragment derived from this monoclonal antibody to a patient who has or is suspected of having MS. In some examples, the monoclonal antibody or its antibody fragment of this embodiment is present in a composition. This composition may be a pharmaceutical composition provided herein.

[0094]

[0107] In one embodiment, the molecule provided herein specifically binds to ASC. A monoclonal antibody or antibody fragment thereof, wherein the antibody or antibody fragment comprises a heavy chain variable (VH) region and a light or kappa chain variable (VL) region, the amino acid sequence of the VL region comprising SEQ ID NOs. 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 NOs. 28, 29, 30, or 31. In addition to this embodiment, provided herein is the use of the monoclonal antibody or antibody fragment thereof in a method for treating inflammation of interest. This inflammation may be congenital immunological inflammation. This inflammation may be inflammasome-related inflammation. This inflammation may be present in the lungs and / or CNS. Inflammation in the lungs and / or CNS may be the result of injury (e.g., traumatic brain injury (TBI) or spinal cord injury (SCI)) or the result of a disease, condition, or distress of or affecting the CNS. Where provided herein, diseases, conditions, or distress affecting or influencing the CNS may include stroke, as well as autoimmune diseases and / or CNS diseases (e.g., amyotrophic lateral sclerosis (ALS) Lou Gehrig's disease, multiple sclerosis (MS), immunodeficiency muscle CNS breakdown, muscular dystrophy (MD), Alzheimer's disease (AD), and Parkinson's disease (PD)). The use of this monoclonal antibody or its antibody fragment in a method of treating inflammation may reduce inflammation in the patient's CNS and / or lungs. This reduction may be compared to a control (e.g., an untreated patient and / or a patient before treatment). In one embodiment, MS is treated by administering this monoclonal antibody or an antibody fragment derived from this monoclonal antibody to a patient who has or is suspected of having MS. In some examples, the monoclonal antibody or its antibody fragment of this embodiment is present in a composition. This composition may be a pharmaceutical composition provided herein.

[0095]

[0108] In one embodiment, the molecule provided herein specifically binds to ASC. A noclonal antibody or an antibody fragment thereof, wherein the antibody or antibody fragment is heavy chain variable ( A monoclonal antibody or antibody fragment thereof comprising a VH) region and a light or kappa chain variable (VL) region, wherein the amino acid sequence of the VH region comprises SEQ ID NOs. 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 NOs. 18, 19, 20, 21, or 22, and the amino acid sequence of the VL region comprises SEQ ID NOs. 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 NOs. 31. In addition to this embodiment, provided herein is the use of this monoclonal antibody or antibody fragment thereof in a method for treating inflammation of interest. This inflammation may be congenital immunological inflammation. This inflammation may be inflammasome-related inflammation. This inflammation may be present in the lungs and / or CNS. Inflammation in the lungs and / or CNS may result from injury (e.g., traumatic brain injury (TBI) or spinal cord injury (SCI)) or from a disease, condition, or distress affecting or in the CNS. Where provided herein, diseases, conditions, or distress affecting or in the CNS may include stroke, as well as autoimmune diseases and / or CNS diseases (e.g., amyotrophic lateral sclerosis (ALS) Lou Gehrig's disease, multiple sclerosis (MS), immunodeficiency muscle CNS breakdown, muscular dystrophy (MD), Alzheimer's disease (AD), and Parkinson's disease (PD)). The use of this monoclonal antibody or its antibody fragment in a method of treating inflammation may reduce inflammation in the patient's CNS and / or lungs. This reduction may be compared to a control (e.g., an untreated patient and / or a patient before treatment). In one embodiment, MS is treated by administering the monoclonal antibody or an antibody fragment derived from the monoclonal antibody to a patient who has or is suspected of having MS. In some examples, the monoclonal antibody or the antibody fragment of the monoclonal antibody of this embodiment is present in a composition. This composition may be a pharmaceutical composition provided herein.

[0096]

[0109] In one embodiment, the molecule provided herein specifically binds to ASC. A monoclonal antibody or antibody fragment thereof, wherein the antibody or antibody fragment comprises a heavy chain variable (VH) region and a light or kappa chain variable (VL) region, the amino acid sequence of the VH region comprising SEQ ID NO: 18 or 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 comprising SEQ ID NO: 28 or an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 28. In addition to this embodiment, provided herein is the use of the monoclonal antibody or antibody fragment thereof in a method for treating inflammation of interest. This inflammation may be congenital immunological inflammation. This inflammation may be inflammasome-related inflammation. This inflammation may be present in the lungs and / or CNS. Inflammation in the lungs and / or CNS may result from injury (e.g., traumatic brain injury (TBI) or spinal cord injury (SCI)) or from a disease, condition, or distress affecting or in the CNS. Where provided herein, diseases, conditions, or distress affecting or in the CNS may include stroke, as well as autoimmune diseases and / or CNS diseases (e.g., amyotrophic lateral sclerosis (ALS) Lou Gehrig's disease, multiple sclerosis (MS), immunodeficiency muscle CNS breakdown, muscular dystrophy (MD), Alzheimer's disease (AD), and Parkinson's disease (PD)). The use of this monoclonal antibody or its antibody fragment in a method of treating inflammation may reduce inflammation in the patient's CNS and / or lungs. This reduction may be compared to a control (e.g., an untreated patient and / or a patient before treatment). In one embodiment, MS is treated by administering the monoclonal antibody or an antibody fragment derived from the monoclonal antibody to a patient who has or is suspected of having MS. In some examples, the monoclonal antibody or the antibody fragment of the monoclonal antibody of this embodiment is in the composition. It exists in [location]. This composition may be a pharmaceutical composition provided herein.

[0097]

[0110] In one embodiment, the molecule provided herein specifically binds to ASC. A monoclonal antibody or antibody fragment thereof, wherein the antibody or antibody fragment comprises a heavy chain variable (VH) region and a light or kappa chain variable (VL) region, the amino acid sequence of the VH region comprising SEQ ID NO: 18 or 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 comprising 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 addition to this embodiment, provided herein is the use of the monoclonal antibody or antibody fragment thereof in a method for treating inflammation of interest. This inflammation may be congenital immunological inflammation. This inflammation may be inflammasome-related inflammation. This inflammation may be present in the lungs and / or CNS. Inflammation in the lungs and / or CNS may result from injury (e.g., traumatic brain injury (TBI) or spinal cord injury (SCI)) or from a disease, condition, or distress affecting or in the CNS. Where provided herein, diseases, conditions, or distress affecting or in the CNS may include stroke, as well as autoimmune diseases and / or CNS diseases (e.g., amyotrophic lateral sclerosis (ALS) Lou Gehrig's disease, multiple sclerosis (MS), immunodeficiency muscle CNS breakdown, muscular dystrophy (MD), Alzheimer's disease (AD), and Parkinson's disease (PD)). The use of this monoclonal antibody or its antibody fragment in a method of treating inflammation may reduce inflammation in the patient's CNS and / or lungs. This reduction may be compared to a control (e.g., an untreated patient and / or a patient before treatment). In one embodiment, MS is treated by administering the monoclonal antibody or an antibody fragment derived from the monoclonal antibody to a patient who has or is suspected of having MS. In some examples, the monoclonal antibody or the antibody fragment of the monoclonal antibody of this embodiment is present in a composition. This composition may be a pharmaceutical composition provided herein.

[0098]

[0111] In one embodiment, the molecule provided herein specifically binds to ASC. A monoclonal antibody or antibody fragment thereof, wherein the antibody or antibody fragment comprises a heavy chain variable (VH) region and a light or kappa chain variable (VL) region, the amino acid sequence of the VH region comprising SEQ ID NO: 18 or 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 comprising SEQ ID NO: 30 or an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 30. In addition to this embodiment, provided herein is the use of the monoclonal antibody or antibody fragment thereof in a method for treating inflammation of interest. This inflammation may be congenital immunological inflammation. This inflammation may be inflammasome-related inflammation. This inflammation may be present in the lungs and / or CNS. Inflammation in the lungs and / or CNS may result from injury (e.g., traumatic brain injury (TBI) or spinal cord injury (SCI)) or from a disease, condition, or distress affecting or in the CNS. Where provided herein, diseases, conditions, or distress affecting or in the CNS may include stroke, as well as autoimmune diseases and / or CNS diseases (e.g., amyotrophic lateral sclerosis (ALS) Lou Gehrig's disease, multiple sclerosis (MS), immunodeficiency muscle CNS breakdown, muscular dystrophy (MD), Alzheimer's disease (AD), and Parkinson's disease (PD)). The use of this monoclonal antibody or its antibody fragment in a method of treating inflammation may reduce inflammation in the patient's CNS and / or lungs. This reduction may be compared to a control (e.g., an untreated patient and / or a patient before treatment). In one embodiment, this monoclonal antibody or an antibody fragment derived from this monoclonal antibody is used to treat patients who have or are suspected of having MS. MS is treated by administering an antibody or an antibody fragment derived from this monoclonal antibody. In some cases, the monoclonal antibody or its antibody fragment of this embodiment is present in the composition. This composition may be a pharmaceutical composition provided herein.

[0099]

[0112] In one embodiment, the molecule provided herein specifically binds to ASC. A monoclonal antibody or antibody fragment thereof, wherein the antibody or antibody fragment comprises a heavy chain variable (VH) region and a light or kappa chain variable (VL) region, the amino acid sequence of the VH region comprising SEQ ID NO: 18 or 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 comprising 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 addition to this embodiment, provided herein is the use of the monoclonal antibody or antibody fragment thereof in a method for treating inflammation of interest. This inflammation may be congenital immunological inflammation. This inflammation may be inflammasome-related inflammation. This inflammation may be present in the lungs and / or CNS. Inflammation in the lungs and / or CNS may result from injury (e.g., traumatic brain injury (TBI) or spinal cord injury (SCI)) or from a disease, condition, or distress affecting or in the CNS. Where provided herein, diseases, conditions, or distress affecting or in the CNS may include stroke, as well as autoimmune diseases and / or CNS diseases (e.g., amyotrophic lateral sclerosis (ALS) Lou Gehrig's disease, multiple sclerosis (MS), immunodeficiency muscle CNS breakdown, muscular dystrophy (MD), Alzheimer's disease (AD), and Parkinson's disease (PD)). The use of this monoclonal antibody or its antibody fragment in a method of treating inflammation may reduce inflammation in the patient's CNS and / or lungs. This reduction may be compared to a control (e.g., an untreated patient and / or a patient before treatment). In one embodiment, MS is treated by administering the monoclonal antibody or an antibody fragment derived from the monoclonal antibody to a patient who has or is suspected of having MS. In some examples, the monoclonal antibody or the antibody fragment of the monoclonal antibody of this embodiment is present in a composition. This composition may be a pharmaceutical composition provided herein.

[0100]

[0113] In one embodiment, the molecule provided herein specifically binds to ASC. A monoclonal antibody or antibody fragment thereof, wherein the antibody or antibody fragment comprises a heavy chain variable (VH) region and a light or kappa chain variable (VL) region, the amino acid sequence of the VH region comprising SEQ ID NO: 19 or 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 comprising SEQ ID NO: 28 or an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 28. In addition to this embodiment, provided herein is the use of the monoclonal antibody or antibody fragment thereof in a method for treating inflammation of interest. This inflammation may be congenital immunological inflammation. This inflammation may be inflammasome-related inflammation. This inflammation may be present in the lungs and / or CNS. Inflammation in the lungs and / or CNS may result from injury (e.g., traumatic brain injury (TBI) or spinal cord injury (SCI)) or from a disease, condition, or distress affecting or in the CNS. Where provided herein, diseases, conditions, or distress affecting or in the CNS may include stroke, as well as autoimmune diseases and / or CNS diseases (e.g., amyotrophic lateral sclerosis (ALS) Lou Gehrig, multiple sclerosis (MS), immunodeficiency muscle CNS breakdown, muscular dystrophy (MD), Alzheimer's disease (AD), and Parkinson's disease (PD)). The use of this monoclonal antibody or its antibody fragment in a method of treating inflammation may reduce inflammation in the patient's CNS and / or lungs. This reduction may be compared to a control (e.g., an untreated patient and / or a patient before treatment). In one embodiment, This monoclonal antibody or an antibody fragment derived from this monoclonal antibody is used to treat MS in patients who have or are suspected of having MS by administering this monoclonal antibody or an antibody fragment derived from this monoclonal antibody. In some examples, the monoclonal antibody or the antibody fragment of this embodiment is present in a composition. This composition may be a pharmaceutical composition provided herein.

[0101]

[0114] In one embodiment, the molecule provided herein specifically binds to ASC. A monoclonal antibody or antibody fragment thereof, wherein the antibody or antibody fragment comprises a heavy chain variable (VH) region and a light or kappa chain variable (VL) region, the amino acid sequence of the VH region comprising SEQ ID NO: 19 or 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 comprising 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 addition to this embodiment, provided herein is the use of the monoclonal antibody or antibody fragment thereof in a method for treating inflammation of interest. This inflammation may be congenital immunological inflammation. This inflammation may be inflammasome-related inflammation. This inflammation may be present in the lungs and / or CNS. Inflammation in the lungs and / or CNS may result from injury (e.g., traumatic brain injury (TBI) or spinal cord injury (SCI)) or from a disease, condition, or distress affecting or in the CNS. Where provided herein, diseases, conditions, or distress affecting or in the CNS may include stroke, as well as autoimmune diseases and / or CNS diseases (e.g., amyotrophic lateral sclerosis (ALS) Lou Gehrig's disease, multiple sclerosis (MS), immunodeficiency muscle CNS breakdown, muscular dystrophy (MD), Alzheimer's disease (AD), and Parkinson's disease (PD)). The use of this monoclonal antibody or its antibody fragment in a method of treating inflammation may reduce inflammation in the patient's CNS and / or lungs. This reduction may be compared to a control (e.g., an untreated patient and / or a patient before treatment). In one embodiment, MS is treated by administering the monoclonal antibody or an antibody fragment derived from the monoclonal antibody to a patient who has or is suspected of having MS. In some examples, the monoclonal antibody or the antibody fragment of the monoclonal antibody of this embodiment is present in a composition. This composition may be a pharmaceutical composition provided herein.

[0102]

[0115] In one embodiment, the molecule provided herein specifically binds to ASC. A monoclonal antibody or antibody fragment thereof, wherein the antibody or antibody fragment comprises a heavy chain variable (VH) region and a light or kappa chain variable (VL) region, the amino acid sequence of the VH region comprising SEQ ID NO: 19 or 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 comprising 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, a monoclonal antibody or antibody fragment derived from this monoclonal antibody comprising the amino acid sequence of the VH region comprising SEQ ID NO: 19 and the amino acid sequence of the VL region comprising SEQ ID NO: 30 may be referred to as IC100. In addition to this embodiment, provided herein is the use of this monoclonal antibody or antibody fragment thereof in a method for treating an inflammation of interest, which may be a congenital immunological inflammation. This inflammation may be inflammasome-related inflammation. This inflammation may be present in the lungs and / or CNS. Inflammation in the lungs and / or CNS may be a result of injury (e.g., traumatic brain injury (TBI) or spinal cord injury (SCI)) or a result of a disease, condition, or distress of or affecting the CNS. Where provided herein, diseases, conditions, or distress of or affecting the CNS include stroke, as well as autoimmune diseases and / or CNS diseases (e.g., amyotrophic lateral sclerosis (ALS) Lou Gehrig's disease). This may include multiple sclerosis (MS), immunodeficiency muscle CNS breakdown, muscular dystrophy (MD), Alzheimer's disease (AD), and Parkinson's disease (PD). The use of this monoclonal antibody or its antibody fragment in a method of treating inflammation may reduce inflammation in the patient's CNS and / or lungs. This reduction may be compared to a control (e.g., an untreated patient and / or a patient before treatment). In one embodiment, MS is treated by administering this monoclonal antibody or an antibody fragment derived from this monoclonal antibody to a patient who has or is suspected of having MS. In some examples, the monoclonal antibody or its antibody fragment of this embodiment is present in a composition. This composition may be a pharmaceutical composition provided herein.

[0103]

[0116] In one embodiment, the molecule provided herein specifically binds to ASC. A monoclonal antibody or antibody fragment thereof, wherein the antibody or antibody fragment comprises a heavy chain variable (VH) region and a light or kappa chain variable (VL) region, the amino acid sequence of the VH region comprising SEQ ID NO: 19 or 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 comprising 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 addition to this embodiment, provided herein is the use of this monoclonal antibody or antibody fragment thereof in a method for treating inflammation of interest. This inflammation may be congenital immunological inflammation. This inflammation may be inflammasome-related inflammation. This inflammation may be present in the lungs and / or CNS. Inflammation in the lungs and / or CNS may result from injury (e.g., traumatic brain injury (TBI) or spinal cord injury (SCI)) or from a disease, condition, or distress affecting or in the CNS. Where provided herein, diseases, conditions, or distress affecting or in the CNS may include stroke, as well as autoimmune diseases and / or CNS diseases (e.g., amyotrophic lateral sclerosis (ALS) Lou Gehrig's disease, multiple sclerosis (MS), immunodeficiency muscle CNS breakdown, muscular dystrophy (MD), Alzheimer's disease (AD), and Parkinson's disease (PD)). The use of this monoclonal antibody or its antibody fragment in a method of treating inflammation may reduce inflammation in the patient's CNS and / or lungs. This reduction may be compared to a control (e.g., an untreated patient and / or a patient before treatment). In one embodiment, MS is treated by administering the monoclonal antibody or an antibody fragment derived from the monoclonal antibody to a patient who has or is suspected of having MS. In some examples, the monoclonal antibody or the antibody fragment of the monoclonal antibody of this embodiment is present in a composition. This composition may be a pharmaceutical composition provided herein.

[0104]

[0117] In one embodiment, the molecule provided herein specifically binds to ASC. A monoclonal antibody or antibody fragment thereof, wherein the antibody or antibody fragment comprises a heavy chain variable (VH) region and a light or kappa chain variable (VL) region, the amino acid sequence of the VH region comprising SEQ ID NO: 20 or 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 comprising SEQ ID NO: 28 or an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 28. In addition to this embodiment, provided herein is the use of the monoclonal antibody or antibody fragment thereof in a method for treating inflammation of interest. This inflammation may be congenital immunological inflammation. This inflammation may be inflammasome-related inflammation. This inflammation may be present in the lungs and / or CNS. Inflammation in the lungs and / or CNS may be a result of injury (e.g., traumatic brain injury (TBI) or spinal cord injury (SCI)) or a disease, condition, or distress affecting or influencing the CNS. Pain may result from this. Where provided herein, diseases, conditions, or pain of or affecting the CNS may include stroke, as well as autoimmune diseases and / or CNS diseases (e.g., amyotrophic lateral sclerosis (ALS) Lou Gehrig, multiple sclerosis (MS), immunodeficiency muscle CNS breakdown, muscular dystrophy (MD), Alzheimer's disease (AD), and Parkinson's disease (PD)). The use of this monoclonal antibody or its antibody fragment in a method of treating inflammation may reduce inflammation in the patient's CNS and / or lungs. This reduction may be compared to a control (e.g., an untreated patient and / or a patient before treatment). In one embodiment, MS is treated by administering this monoclonal antibody or an antibody fragment derived from this monoclonal antibody to a patient who has or is suspected of having MS. In some examples, the monoclonal antibody or its antibody fragment of this embodiment is present in a composition. This composition may be a pharmaceutical composition provided herein.

[0105]

[0118] In one embodiment, the molecule provided herein specifically binds to ASC. A monoclonal antibody or antibody fragment thereof, wherein the antibody or antibody fragment comprises a heavy chain variable (VH) region and a light or kappa chain variable (VL) region, the amino acid sequence of the VH region comprising SEQ ID NO: 20 or 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 comprising 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 addition to this embodiment, provided herein is the use of the monoclonal antibody or antibody fragment thereof in a method for treating inflammation of interest. This inflammation may be congenital immunological inflammation. This inflammation may be inflammasome-related inflammation. This inflammation may be present in the lungs and / or CNS. Inflammation in the lungs and / or CNS may result from injury (e.g., traumatic brain injury (TBI) or spinal cord injury (SCI)) or from a disease, condition, or distress affecting or in the CNS. Where provided herein, diseases, conditions, or distress affecting or in the CNS may include stroke, as well as autoimmune diseases and / or CNS diseases (e.g., amyotrophic lateral sclerosis (ALS) Lou Gehrig's disease, multiple sclerosis (MS), immunodeficiency muscle CNS breakdown, muscular dystrophy (MD), Alzheimer's disease (AD), and Parkinson's disease (PD)). The use of this monoclonal antibody or its antibody fragment in a method of treating inflammation may reduce inflammation in the patient's CNS and / or lungs. This reduction may be compared to a control (e.g., an untreated patient and / or a patient before treatment). In one embodiment, MS is treated by administering the monoclonal antibody or an antibody fragment derived from the monoclonal antibody to a patient who has or is suspected of having MS. In some examples, the monoclonal antibody or the antibody fragment of the monoclonal antibody of this embodiment is present in a composition. This composition may be a pharmaceutical composition provided herein.

[0106]

[0119] In one embodiment, the molecule provided herein specifically binds to ASC. A monoclonal antibody or antibody fragment thereof, wherein the antibody or antibody fragment comprises a heavy chain variable (VH) region and a light or kappa chain variable (VL) region, the amino acid sequence of the VH region comprising SEQ ID NO: 20 or 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 comprising SEQ ID NO: 30 or an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 30. In addition to this embodiment, provided herein is the use of this monoclonal antibody or antibody fragment thereof in a method for treating inflammation of interest. This inflammation may be congenital immunological inflammation. This inflammation may be inflammasome-related inflammation. This inflammation may be present in the lungs and / or CNS. Inflammation in the pulmonary system (S) may result from injury (e.g., traumatic brain injury (TBI) or spinal cord injury (SCI)) or from a disease, condition, or distress of or affecting the CNS. Where provided herein, diseases, conditions, or distress of or affecting the CNS may include stroke, as well as autoimmune diseases and / or CNS diseases (e.g., amyotrophic lateral sclerosis (ALS) Lou Gehrig, multiple sclerosis (MS), immunodeficiency muscle CNS breakdown, muscular dystrophy (MD), Alzheimer's disease (AD), and Parkinson's disease (PD)). The use of this monoclonal antibody or its antibody fragment in a method of treating inflammation may reduce inflammation in the patient's CNS and / or lungs. This reduction may be compared to a control (e.g., an untreated patient and / or a patient before treatment). In one embodiment, MS is treated by administering the monoclonal antibody or an antibody fragment derived from the monoclonal antibody to a patient who has or is suspected of having MS. In some examples, the monoclonal antibody or the antibody fragment of the monoclonal antibody of this embodiment is present in a composition. This composition may be a pharmaceutical composition provided herein.

[0107]

[0120] In one embodiment, the molecule provided herein specifically binds to ASC. A monoclonal antibody or antibody fragment thereof, wherein the antibody or antibody fragment comprises a heavy chain variable (VH) region and a light or kappa chain variable (VL) region, the amino acid sequence of the VH region comprising SEQ ID NO: 20 or 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 comprising 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 addition to this embodiment, provided herein is the use of the monoclonal antibody or antibody fragment thereof in a method for treating inflammation of interest. This inflammation may be congenital immunological inflammation. This inflammation may be inflammasome-related inflammation. This inflammation may be present in the lungs and / or CNS. Inflammation in the lungs and / or CNS may result from injury (e.g., traumatic brain injury (TBI) or spinal cord injury (SCI)) or from a disease, condition, or distress affecting or in the CNS. Where provided herein, diseases, conditions, or distress affecting or in the CNS may include stroke, as well as autoimmune diseases and / or CNS diseases (e.g., amyotrophic lateral sclerosis (ALS) Lou Gehrig's disease, multiple sclerosis (MS), immunodeficiency muscle CNS breakdown, muscular dystrophy (MD), Alzheimer's disease (AD), and Parkinson's disease (PD)). The use of this monoclonal antibody or its antibody fragment in a method of treating inflammation may reduce inflammation in the patient's CNS and / or lungs. This reduction may be compared to a control (e.g., an untreated patient and / or a patient before treatment). In one embodiment, MS is treated by administering the monoclonal antibody or an antibody fragment derived from the monoclonal antibody to a patient who has or is suspected of having MS. In some examples, the monoclonal antibody or the antibody fragment of the monoclonal antibody of this embodiment is present in a composition. This composition may be a pharmaceutical composition provided herein.

[0108]

[0121] In one embodiment, the molecule provided herein specifically binds to ASC. A monoclonal antibody or antibody fragment thereof, wherein the antibody or antibody fragment comprises a heavy chain variable (VH) region and a light or kappa chain variable (VL) region, the amino acid sequence of the VH region comprising SEQ ID NO: 21 or an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 21, and the amino acid sequence of the VL region comprising SEQ ID NO: 28 or an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 28. In addition to this embodiment, the use of the monoclonal antibody or antibody fragment thereof in a method for treating the inflammation of the subject is provided herein. This inflammation may be congenital immune inflammation. This inflammation may be inflammasome-related inflammation. This inflammation may be present in the lungs and / or CNS. Inflammation in the lungs and / or CNS may result from injury (e.g., traumatic brain injury (TBI) or spinal cord injury (SCI)) or from a disease, condition, or distress of or affecting the CNS. Where provided herein, diseases, conditions, or distress of or affecting the CNS may include stroke, as well as autoimmune diseases and / or CNS diseases (e.g., amyotrophic lateral sclerosis (ALS) Lou Gehrig, multiple sclerosis (MS), immunodeficiency muscle CNS breakdown, muscular dystrophy (MD), Alzheimer's disease (AD), and Parkinson's disease (PD)). The use of this monoclonal antibody or its antibody fragment in a method of treating inflammation may reduce inflammation in the patient's CNS and / or lungs. This reduction may be compared to a control (e.g., an untreated patient and / or a patient before treatment). In one embodiment, MS is treated by administering the monoclonal antibody or an antibody fragment derived from the monoclonal antibody to a patient who has or is suspected of having MS. In some examples, the monoclonal antibody or the antibody fragment thereof of this embodiment is present in a composition. This composition may be a pharmaceutical composition provided herein.

[0109]

[0122] In one embodiment, the molecule provided herein specifically binds to ASC. A monoclonal antibody or antibody fragment thereof, wherein the antibody or antibody fragment comprises a heavy chain variable (VH) region and a light or kappa chain variable (VL) region, the amino acid sequence of the VH region comprising SEQ ID NO: 21 or an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 21, and the amino acid sequence of the VL region comprising SEQ ID NO: 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 addition to this embodiment, provided herein is the use of the monoclonal antibody or antibody fragment thereof in a method for treating inflammation of interest. This inflammation may be congenital immunological inflammation. This inflammation may be inflammasome-related inflammation. This inflammation may be present in the lungs and / or CNS. Inflammation in the lungs and / or CNS may result from injury (e.g., traumatic brain injury (TBI) or spinal cord injury (SCI)) or from a disease, condition, or distress affecting or in the CNS. Where provided herein, diseases, conditions, or distress affecting or in the CNS may include stroke, as well as autoimmune diseases and / or CNS diseases (e.g., amyotrophic lateral sclerosis (ALS) Lou Gehrig's disease, multiple sclerosis (MS), immunodeficiency muscle CNS breakdown, muscular dystrophy (MD), Alzheimer's disease (AD), and Parkinson's disease (PD)). The use of this monoclonal antibody or its antibody fragment in a method of treating inflammation may reduce inflammation in the patient's CNS and / or lungs. This reduction may be compared to a control (e.g., an untreated patient and / or a patient before treatment). In one embodiment, MS is treated by administering the monoclonal antibody or an antibody fragment derived from the monoclonal antibody to a patient who has or is suspected of having MS. In some examples, the monoclonal antibody or the antibody fragment of the monoclonal antibody of this embodiment is present in a composition. This composition may be a pharmaceutical composition provided herein.

[0110]

[0123] In one embodiment, the molecule provided herein specifically binds to ASC. A noclonal antibody or antibody fragment thereof, wherein the antibody or antibody fragment comprises a heavy chain variable (VH) region and a light or kappa chain variable (VL) region, wherein the amino acid sequence of the VH region includes SEQ ID NO: 21 or an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 21, and the amino acid sequence of the VL region includes 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, monoclonal This is a monoclonal antibody or a fragment thereof. In addition to this embodiment, provided herein is the use of this monoclonal antibody or a fragment thereof in a method for treating inflammation of interest. This inflammation may be a congenital immune inflammation. This inflammation may be an inflammasome-related inflammation. This inflammation may be present in the lungs and / or CNS. Inflammation in the lungs and / or CNS may be the result of injury (e.g., traumatic brain injury (TBI) or spinal cord injury (SCI)) or the result of a disease, condition, or distress of or affecting the CNS. Where provided herein, diseases, conditions, or distress of or affecting the CNS may be stroke, as well as autoimmune diseases and / or CNS diseases (e.g., amyotrophic lateral sclerosis (ALS) Lou Gehrig, multiple sclerosis (MS), immunodeficiency muscle CNS breakdown, muscular dystrophy (MD), Alzheimer's disease (AD), Parkinson's disease (PD)). The use of this monoclonal antibody or its antibody fragment in a method of treating inflammation may reduce inflammation in the patient's CNS and / or lungs. This reduction may be compared to a control (e.g., an untreated patient and / or a patient before treatment). In one embodiment, MS is treated by administering this monoclonal antibody or an antibody fragment derived from this monoclonal antibody to a patient who has or is suspected of having MS. In some examples, the monoclonal antibody or its antibody fragment of this embodiment is present in a composition. This composition may be a pharmaceutical composition provided herein.

[0111]

[0124] In one embodiment, the molecule provided herein specifically binds to ASC. A monoclonal antibody or antibody fragment thereof, wherein the antibody or antibody fragment comprises a heavy chain variable (VH) region and a light or kappa chain variable (VL) region, the amino acid sequence of the VH region comprising SEQ ID NO: 21 or an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 21, and the amino acid sequence of the VL region comprising SEQ ID NO: 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 addition to this embodiment, provided herein is the use of the monoclonal antibody or antibody fragment thereof in a method for treating inflammation of interest. This inflammation may be congenital immunological inflammation. This inflammation may be inflammasome-related inflammation. This inflammation may be present in the lungs and / or CNS. Inflammation in the lungs and / or CNS may result from injury (e.g., traumatic brain injury (TBI) or spinal cord injury (SCI)) or from a disease, condition, or distress affecting or in the CNS. Where provided herein, diseases, conditions, or distress affecting or in the CNS may include stroke, as well as autoimmune diseases and / or CNS diseases (e.g., amyotrophic lateral sclerosis (ALS) Lou Gehrig's disease, multiple sclerosis (MS), immunodeficiency muscle CNS breakdown, muscular dystrophy (MD), Alzheimer's disease (AD), and Parkinson's disease (PD)). The use of this monoclonal antibody or its antibody fragment in a method of treating inflammation may reduce inflammation in the patient's CNS and / or lungs. This reduction may be compared to a control (e.g., an untreated patient and / or a patient before treatment). In one embodiment, MS is treated by administering the monoclonal antibody or an antibody fragment derived from the monoclonal antibody to a patient who has or is suspected of having MS. In some examples, the monoclonal antibody or the antibody fragment of the monoclonal antibody of this embodiment is present in a composition. This composition may be a pharmaceutical composition provided herein.

[0112]

[0125] In one embodiment, the molecule provided herein specifically binds to ASC. A noclonal antibody or antibody fragment thereof, wherein the antibody or antibody fragment comprises a heavy chain variable (VH) region and a light or kappa chain variable (VL) region, 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 The no-acid sequence is a monoclonal antibody or an antibody fragment thereof comprising an amino acid sequence containing SEQ ID NO: 28, or an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 28. In addition to this embodiment, provided herein is the use of this monoclonal antibody or an antibody fragment thereof in a method for treating inflammation of interest. This inflammation may be a congenital immunological inflammation. This inflammation may be an inflammasome-related inflammation. This inflammation may be present in the lungs and / or CNS. Inflammation in the lungs and / or CNS may be a result of injury (e.g., traumatic brain injury (TBI) or spinal cord injury (SCI)), or a result of a disease, condition, or distress of or affecting the CNS. Where provided herein, diseases, conditions, or distress affecting or influencing the CNS may include stroke, as well as autoimmune diseases and / or CNS diseases (e.g., amyotrophic lateral sclerosis (ALS) Lou Gehrig's disease, multiple sclerosis (MS), immunodeficiency muscle CNS breakdown, muscular dystrophy (MD), Alzheimer's disease (AD), and Parkinson's disease (PD)). The use of this monoclonal antibody or its antibody fragment in a method of treating inflammation may reduce inflammation in the patient's CNS and / or lungs. This reduction may be compared to a control (e.g., an untreated patient and / or a patient before treatment). In one embodiment, MS is treated by administering this monoclonal antibody or an antibody fragment derived from this monoclonal antibody to a patient who has or is suspected of having MS. In some examples, the monoclonal antibody or its antibody fragment of this embodiment is present in a composition. This composition may be a pharmaceutical composition provided herein.

[0113]

[0126] In one embodiment, the molecule provided herein specifically binds to ASC. A monoclonal antibody or antibody fragment thereof, wherein the antibody or antibody fragment comprises a heavy chain variable (VH) region and a light or kappa chain variable (VL) region, the amino acid sequence of the VH region comprising SEQ ID NO: 22 or an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 22, and the amino acid sequence of the VL region comprising SEQ ID NO: 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 addition to this embodiment, provided herein is the use of the monoclonal antibody or antibody fragment thereof in a method for treating inflammation of interest. This inflammation may be congenital immunological inflammation. This inflammation may be inflammasome-related inflammation. This inflammation may be present in the lungs and / or CNS. Inflammation in the lungs and / or CNS may result from injury (e.g., traumatic brain injury (TBI) or spinal cord injury (SCI)) or from a disease, condition, or distress affecting or in the CNS. Where provided herein, diseases, conditions, or distress affecting or in the CNS may include stroke, as well as autoimmune diseases and / or CNS diseases (e.g., amyotrophic lateral sclerosis (ALS) Lou Gehrig's disease, multiple sclerosis (MS), immunodeficiency muscle CNS breakdown, muscular dystrophy (MD), Alzheimer's disease (AD), and Parkinson's disease (PD)). The use of this monoclonal antibody or its antibody fragment in a method of treating inflammation may reduce inflammation in the patient's CNS and / or lungs. This reduction may be compared to a control (e.g., an untreated patient and / or a patient before treatment). In one embodiment, MS is treated by administering the monoclonal antibody or an antibody fragment derived from the monoclonal antibody to a patient who has or is suspected of having MS. In some examples, the monoclonal antibody or the antibody fragment of the monoclonal antibody of this embodiment is present in a composition. This composition may be a pharmaceutical composition provided herein.

[0114]

[0127] In one embodiment, the molecule provided herein specifically binds to ASC. A noclonal antibody or an antibody fragment thereof, wherein the antibody or antibody fragment comprises a heavy chain variable (VH) region and a light or kappa chain variable (VL) region, and the amino acid sequence of the VH region is A monoclonal antibody or antibody fragment thereof comprising an amino acid sequence containing 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, wherein the amino acid sequence of the VL region contains SEQ ID NO: 30, or an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 30. In addition to this embodiment, provided herein is the use of this monoclonal antibody or antibody fragment thereof in a method for treating inflammation of interest. This inflammation may be congenital immunological inflammation. This inflammation may be inflammasome-related inflammation. This inflammation may be present in the lungs and / or CNS. Inflammation in the lungs and / or CNS may be the result of injury (e.g., traumatic brain injury (TBI) or spinal cord injury (SCI)), or the result of a disease, condition, or distress of or affecting the CNS. Where provided herein, diseases, conditions, or distress affecting or influencing the CNS may include stroke, as well as autoimmune diseases and / or CNS diseases (e.g., amyotrophic lateral sclerosis (ALS) Lou Gehrig's disease, multiple sclerosis (MS), immunodeficiency muscle CNS breakdown, muscular dystrophy (MD), Alzheimer's disease (AD), and Parkinson's disease (PD)). The use of this monoclonal antibody or its antibody fragment in a method of treating inflammation may reduce inflammation in the patient's CNS and / or lungs. This reduction may be compared to a control (e.g., an untreated patient and / or a patient before treatment). In one embodiment, MS is treated by administering this monoclonal antibody or an antibody fragment derived from this monoclonal antibody to a patient who has or is suspected of having MS. In some examples, the monoclonal antibody or its antibody fragment of this embodiment is present in a composition. This composition may be a pharmaceutical composition provided herein.

[0115]

[0128] In one embodiment, the molecule provided herein specifically binds to ASC. A monoclonal antibody or antibody fragment thereof, wherein the antibody or antibody fragment comprises a heavy chain variable (VH) region and a light or kappa chain variable (VL) region, the amino acid sequence of the VH region comprising SEQ ID NO: 22 or an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 22, and the amino acid sequence of the VL region comprising SEQ ID NO: 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 addition to this embodiment, provided herein is the use of the monoclonal antibody or antibody fragment thereof in a method for treating inflammation of interest. This inflammation may be congenital immunological inflammation. This inflammation may be inflammasome-related inflammation. In one embodiment, a monoclonal antibody or an antibody fragment thereof provided herein may be used in a manner that reduces inflammation in a mammal, as described in U.S. Patent No. 8,685,400 (the contents of which are incorporated herein by reference in their entirety). This inflammation may be present in the lungs and / or CNS. Inflammation in the lungs and / or CNS may result from injury (e.g., traumatic brain injury (TBI) or spinal cord injury (SCI)) or from a disease, condition, or distress of or affecting the CNS. Where provided herein, the disease, condition, or distress of or affecting the CNS may be stroke, as well as autoimmune diseases and / or CNS diseases (e.g., amyotrophic lateral sclerosis (ALS) Lou Gehrig, multiple sclerosis (MS), immunodeficiency muscle CNS breakdown, muscular dystrophy (MD), Alzheimer's disease (AD), Parkinson's disease (PD)). The use of this monoclonal antibody or its antibody fragment in a method of treating inflammation may reduce inflammation in the patient's CNS and / or lungs. This reduction may be compared to a control (e.g., an untreated patient and / or a patient before treatment). In one embodiment, MS is treated by administering this monoclonal antibody or an antibody fragment derived from this monoclonal antibody to a patient who has or is suspected of having MS.In some cases, the monoclonal antibody or antibody fragment of this embodiment is present in the composition. This composition may be a pharmaceutical composition provided herein.

[0116]

[0129] In another embodiment, reducing inflammation in the CNS or lungs of mammals. The composition for use comprises an antibody provided herein (e.g., an anti-NLRP1 chicken antibody) or an active fragment thereof that specifically binds to NLRP1 or its domain. Any suitable anti-NLRP1 antibody can be used, some of which are commercially available. Examples of anti-NLRP1 antibodies for use in the methods provided herein may be 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, and Novus Biologicals rabbit polyclonal antibody Liclonal anti-NLRP1 NB100-56148, Sigma-Aldrich mouse polyclonal Abcam Rabbit Polyclonal Anti-NLRP1 SAB1407151, Abcam Rabbit Polyclonal Anti-NLRP1 ab3683, Biobyt Rabbit Polyclonal Anti-NLRP1 orb325922 mybiosource Rabbit Polyclonal Anti-NLRP1 MBS7001225, R&D Systems Sheep Polyclonal AF6788, Aviva Systems Mouse Monoclonal Anti-NLRP1 OA ed00344, Aviva Systems Rabbit Polyclonal Anti-NLRP1 ARO54478 _P050, Origene Rabbit Polyclonal Anti-NLRP1 APO7775PU-N, Antibodies online Rabbit Polyclonal Anti-NLRP1 ABIN768983, Prosci Rabbit Polyclonal Anti-NLRP1 3037, Proteintech Rabbit Polyclonal Anti-NLR P1 12256-1-AP, Enzo Mouse Monoclonal Anti-NLRP1 ALX-804-803-C100, Invitrogen Mouse Monoclonal Anti-NLRP1 MA1-25842, GeneTex Mouse Monoclonal Anti-NLRP1 GTX16091, Rockland Rabbit Liclonal anti-NLRP1 200-401-CX5, or Cell Signaling Technology Rabbit polyclonal anti-NLRP1 4990 is an example. The human NLRP1 protein may 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 or part or fragment thereof of the mammalian NLRP1 protein (e.g., human NLRP1). 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 specified domain (e.g., Pyrin, NACHT, LRR1-6, FIIND, or CARD) or fragment thereof of human NLRP1. In one embodiment, chicken anti-NLRP1 polyclonal antibody, custom designed and produced by Ayes Laboratories, binds to the Pyrin, NACHT, LRR1-6, FIIND, or CARD domain or part or fragment thereof. This antibody is used to reduce pneumonia. This antibody may be directed to the following amino acid sequence CEYYTEIREREREKSEKGR (SEQ ID NO: 3) in human NLRP1. In one embodiment, an antibody that binds to the NLRP1 domain or a fragment thereof as described herein inhibits NLRP1 activity in mammalian lung cells, e.g., type II alveolar cells.

[0117]

[0130] In yet another embodiment, inflammation in the CNS or lungs of mammals is reduced. The composition for this purpose comprises an antibody provided herein or an active fragment thereof that specifically binds to AIM2 or its domain. Any suitable anti-AIM2 antibody can be used, several of which are commercially available. Examples of commercially available anti-AIM2 antibodies for use in the method provided herein include, but are not limited to, rabbit polyc from Proteintech. Ronal anti-AIM2 catalog number 20590-1-AP, Abcam anti-AIMS antibody (ab1 19791), Rabbit polyclonal anti-AIM2 (N-terminal region) from ECM biosciences Catalog number AP3851, Rabbit polyclonal anti-ASC from Elabsciences; Catalog number E-AB-30449, Anti-AIM2 mouse monoclonal antibody called AIM2 antibody (3C4G11) from Santa Cruz Biotechnology; Catalog number sc-293174, Mouse monoclonal AIM2 antibody from Origene; Catalog number TA324972, T AIM2 monoclonal antibody (10M2B3) from Hermofisher Scientific, AIM2 rabbit polyclonal antibody ABIN928372 or ABIN from Antibodies-online 760766, Biomatix-coated anti-AIM2 polyclonal antibody, catalog number CAE02153, anti-AIM2 polyclonal antibody from Aviva Systems Biology (OABF016 32) Rabbit polyclonal anti-AIM2 antibody LS-C354127 7. Rabbit monoclonal anti-AIM2 antibody from Cell Signaling Technology, catalog Rabbit Polyc, number MA5-16259, from Fab Gennix International Incorporated Ronal anti-AIM2 monoclonal antibody, catalog number AIM2 201AP; MyBiosource rabbit polyclonal anti-AIM2 catalog number MBS855320; Signalway rabbit polyclonal anti-AIM2 catalog number 36253; Novus Biological rabbit polyclonal anti-AIM2 catalog number 43900002; GeneTex rabbit polyclonal anti-AIM2 M2 GTX54910, Prosci, Rabbit Polyclonal Anti-AIM2 26-540, Biorbyt Mouse Monoclonal Anti-AIM2 orb333902, Abcam Rabbit Polyclonal Anti-AIM2 ab93015, Abcam Rabbit Polyclonal Anti-AIM2 ab764 23. Examples include Signma Aldrich mouse polyclonal anti-AIM2 SAB1406827, or Biolegend anti-AIM2 3B10. Human AIM2 protein is accession The antibody may be code NX_014862, NP004824, XP016858337, XP005245673, AAB81613, BAF84731, or AAH10940. In one embodiment, the antibody binds to the Pyrin or HIN-200 domain or part or fragment thereof of the mammalian AIM2 protein (e.g., human AIM2). 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 specified domain (e.g., Pyrin or HIN-200) or fragment thereof of human AIM2. In one embodiment, the antibody that binds to the AIM2 domain or fragment thereof described herein inhibits AIM2 activity in mammalian lung cells, e.g., type II alveolar cells.

[0118]

[0131] Anti-inflammasomes described herein (e.g., anti-ASC, anti-NLRP1 or Antibodies against AIM2 include polyclonal and monoclonal rodent antibodies, polyclonal and monoclonal human antibodies, or any part thereof, having at least one antigen-binding region of an immunoglobulin variable region that specifically binds to a component of the mammalian inflammasome (e.g., the AIM2 inflammasome), such as ASC or AIM2. In some examples, the antibody is specific to ASC if the antibody is produced against a polypeptide epitope and binds to at least a part of a natural or recombinant protein.

[0119]

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

[0120]

[0133] In certain embodiments, conservative amino acid substitutions are disclosed herein. This is performed at one or more locations in the amino acid sequence of the antibody or antibody fragment. "Conservative amino acid substitution" is performed by replacing an amino acid residue with an amino acid residue that has a similar side chain. In certain embodiments, conservative amino acid substitutions are performed only in the FR sequence and not in the CDR sequence 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 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). Therefore, for example, an amino acid residue in the polypeptide of an anti-ASC monoclonal antibody or ASC-binding antibody fragment may be replaced with another amino acid residue from the same side chain family. In certain embodiments, the amino acid string may be replaced with a structurally similar string having a different order and / or composition of side-chain family members. Those skilled in the art may evaluate whether an anti-ASC monoclonal antibody or ASC-binding antibody fragment containing a polypeptide having one or more amino acid substitutions, deletions, or insertions compared to a polypeptide having one or more amino acid sequences from SEQ ID NOs: 6-8, 12-14, 18-22, or 28-31 by utilizing routine methods known in the art (e.g., ELISA, Western blotting, phage display, etc.).

[0121]

[0134] Sequence homology or sequence identity between sequences (these terms are interchangeable in this specification) The calculation of (used in) can be carried out as follows.

[0122]

[0135] To determine the percentage identity of two amino acid sequences or two nucleic acid sequences These sequences are then aligned for optimal comparison purposes (for example, gaps may be introduced in one or both of the first and second amino acid sequences or nucleic acid sequences for optimal alignment, and non-homologous sequences may be ignored for comparison purposes). In exemplary embodiments, the length of the reference sequence aligned for comparison purposes is at least 30%, 40%, 50%, 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the length of the reference sequence. Then, amino acid residues or nucleotides are compared at the corresponding amino acid or nucleotide positions. If a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then these molecules are identical at that position (as used herein, "identity" of an amino acid or nucleic acid is equivalent to "homology" of an amino acid or nucleic acid). Percent identity between two sequences is a function of the number of identical positions shared by these sequences, taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap.

[0123]

[0136] The comparison of sequences and the determination of percentage identity between two sequences can be performed using a mathematical algorithm. This can be achieved using BLOSUM. In one embodiment, percentage identity between two amino acid sequences is determined using either a BLOSUM 62 matrix or a PAM250 matrix, as well as 16, 14, 1 Using gap weights of 2, 10, 8, 6, or 4 and length weights of 1, 2, 3, 4, 5, or 6, the GAP program in the GCG software package (available at www.gcg.com) The algorithm of Needleman et al. ((1970) J. Mol. Biol. 48:444-453) is incorporated and used to determine the percentage identity between two nucleotide sequences using the NWSgapdna CMP matrix and a gap of 40, 50, 60, 70, or 80. Using the P weight and length weights of 1, 2, 3, 4, 5, or 6, the GCG software package Determine using the GAP program in the page (available at www.gcg.com). Parameters (and whether the molecule is within the limits of sequence identity or sequence homology of the present invention) One set of parameters (which may be used when experts are unsure which parameters to apply) is the BLOSUM 62 scoring matrix, which includes 12 gap penalties, 4 gap extension penalties, and 5 frame shift gap penalties.

[0124]

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

[0125]

[0138] In a particular embodiment, the antibody is a monoclonal antibody. In another embodiment, the antibody The body is composed of polyclonal antibodies. The term "monoclonal antibody" refers to a group of antibody molecules that contain only one antigen-binding site capable of immunely responding to a specific epitope of an antigen. Therefore, a monoclonal antibody composition typically exhibits a single binding affinity to the specific protein to which this monoclonal antibody composition immunely responds.

[0126]

[0139] In some embodiments, the antibody of the present invention (anti-ASC monoclonal antibody or AS The C-binding antibody fragment is either humanized, chimeric, or human.

[0127]

[0140] In some embodiments, the antibody of the present invention is a humanized antibody.

[0128]

[0141] When this term is used herein, "humanized antibody" refers to a heavy chain and / or This refers to an antibody that has been engineered to include one or more human framework regions in its variable region, along with a non-human (e.g., mouse, rat, or hamster) complementarity-determining region (CDR) of the light chain. In certain embodiments, the humanized antibody contains a sequence that is entirely human except for this CDR region. In some cases, the Fv framework region (FR) residues of human immunoglobulin are replaced with corresponding non-human residues. Furthermore, the humanized antibody may contain residues that are not found in the humanized antibody or the transferred CDR or framework sequence, but are included to further refine and optimize the antibody's performance. Generally, the humanized antibody contains substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR region corresponds to that of a non-human immunoglobulin, and all or substantially all of the FR region is from the human immunoglobulin consensus sequence. This RF region can be modified in any way known in the art and / or provided herein. This modification may confer desired properties (e.g., increased half-life, and / or improved expression in host cells). In one embodiment, this FR region 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). Humanized antibodies also optimally include at least a portion of the immunoglobulin constant region or domain (Fc), typically at least a portion of human immunoglobulin.

[0129]

[0142] Humanized antibodies typically exhibit lower immunogenicity against humans compared to non-humanized antibodies. The risk is low, and therefore offers therapeutic benefits in certain situations. For example, the constant region of the antibody can be manipulated to be immunologically inactive (e.g., not cause 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 techniques suitable for the production of such humanized antibodies. See, for example, Hwang, WYK, et al., Methods 36:35, 2005; Queen et al., Proc. Natl. Acad. Sci. USA, 86:10029-10033, 1989; Jones et al., Nature, 321:522-25, 1986. See also Riechmann et al., Nature, 332:323-27, 1988; Verhoeyen et al., Science, 239:1534-36, 1988; Orlandi et al., Proc. Natl. Acad. Sci. USA, 86:3833-37, 1989; U.S. Patent Nos. 5,225,539, 5,530,101, 5,585,089, 5,693,761, 5,693,762, and 6,180,370; and Selick et al., International Publication No. 90 / 07861 (each of these is incorporated herein by reference in its entirety). Another method for humanizing antibodies that can be used similarly is disclosed by Daugherty et al., Nucl. Acids Res. 19:2471-2476, 1991. The present invention is disclosed in U.S. Patents No. 6,180,377; No. 6,054,297; No. 5,997,867; No. 5,866,692; No. 6,210,671; and No. 6,350,861, and International Publication No. 01 / 27160, each of which is incorporated herein by reference in whole. For example, the anti-ASC antibody or anti-ASC antigen binding fragment of the present invention may include an amino acid sequence of the 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 the 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 is ASC It binds specifically. In some cases, this anti-ASC chimeric antibody reduces the activity of ASC. When this term is used herein, “chimeric antibody” refers to an antibody that has been engineered to include at least one human constant region. For example, one or all of the variable regions of the light chain and / or one or all of the variable regions of the heavy chain of a mouse antibody (e.g., a mouse monoclonal antibody) may be linked to a human constant region (e.g., but not limited to the IgG1 human constant region). Chimeric antibodies typically have lower immunogenicity to humans compared to non-chimeric antibodies and therefore offer therapeutic advantages in certain situations. Those skilled in the art will recognize chimeric antibodies and also recognize techniques suitable for the production of such chimeric antibodies. For example, Cabilly et al., U.S. Patent No. 4,816,567; Shoemaker et al., U.S. Patent No. 4,978,775; Beavers et al., U.S. Patent No. 4,975,369; and Boss et al., U.S. Patent No. 4,816, See Patent No. 397 (each of which is incorporated herein by reference in whole). For example, the antibody or antigen-binding fragment of the present invention may include a VH region containing SEQ ID NO: 22; a VL region containing SEQ ID NO: 31; and a human constant region.

[0131]

[0144] As used herein, the terms “immunological binding” and “immunological binding properties” are used herein. "Immunological binding" refers to a type of non-covalent interaction that occurs between an immunoglobulin molecule (e.g., an antibody) and an antigen to which this 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, where a smaller Kd indicates a higher affinity. The immunological binding properties of a selected polypeptide can be quantified using methods known in the art. One such method involves measuring the rate of formation and dissociation of the antigen-binding site / antigen complex, which depends on geometric parameters that equally affect the concentration of the complex partner, the affinity of the interaction, and the rate 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. (Nature) See 361:186-87 (1993). The Koff / Kon ratio allows for the dissociation of all parameters not related to affinity and is equal to the dissociation constant Kd. (Generally, Davies See et al. (1990) Annual Rev Biochem 59:439-473). The antibodies of the present invention are said to specifically bind to epitopes (e.g., ASC fragments having the amino acids of SEQ ID NO: 5) when measured by assays such as radioligand binding assays or similar assays known to those skilled in the art, the equilibrium binding constant (Kd) is ≤10 μM, ≤10 nM, ≤10 nM, and ≤100 pM to about 1 pM.

[0132]

[0145] In certain embodiments, the antibody of the present invention is monovalent or bivalent, and single-stranded or Includes double-stranded antibodies. Functionally, the binding affinity of an antibody can be in the range of 10⁻⁵M to 10⁻¹²M. For example, the binding affinity of an antibody can be 10⁻⁶M to 10⁻¹²M, 10⁻⁷M to 10⁻¹²M, 10⁻⁸M to 10⁻¹²M, 10⁻⁵M to 10⁻¹¹M, 10⁶M to 10⁻¹¹M, 10⁷M to 10⁻¹¹M, 10⁹M to 10⁻¹¹M, 10⁵M to 10⁻¹⁰M, 10⁶M to 10⁻¹¹M. 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] A method for determining monoclonal antibody specificity and affinity by competitive inhibition is described below. The findings 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 are incorporated herein by reference. It is possible.

[0134]

[0147] The anti-inflammasome (e.g., anti-ASC and anti-AIM2) antibodies of the present invention are Antibodies can be systematically prepared by methods such as inoculation of appropriate animals with polypeptides or antigenic fragments, in vitro stimulation of lymphocyte populations, synthesis, hybridomas, and / or recombinant cells expressing nucleic acids encoding such anti-ASC or anti-NLR1 antibodies. Immunization of animals using purified recombinant ASC or its peptide fragments (e.g., residues 178-193 of rat ASC (e.g., accession number BAC43754) (SEQ ID 1), EQ ID NO:2 of human ASC, or residues 21-41 of human ASC (e.g., accession number NP_037390.2) (SEQ ID 5)) is one example of a method for preparing anti-ASC antibodies. Similarly, immunization of animals using purified recombinant NLRP1 or its peptide fragments, e.g., residues MEE SQS KEE SNT EG-cys of rat NALP1 (SEQ ID 4), or SEQ ID 3 of human NALP1, is one example of a method for preparing anti-NLRP1 antibodies.

[0135]

[0148] Monoclonal antibodies that specifically bind to ASC or NLRP1 are available to those skilled in the art. It can be obtained by known methods. For example, the contents of which are incorporated herein in whole by reference are Kohler and Milstein, Nature 256:495-497, 1975; U.S. 4,376, 1 No. 10; Ausubel et al., eds., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley Interscience, NY, (1987, 1992); Harlow and Lane ANTIBODIES:A See Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1988; Colligan et al., eds., Current Protocols in Immunology, Greene Publishing Assoc. and Wiley Interscience, NY, (1992, 1993). Such antibodies are available in any immunoglobulin. The phospholipids may be, for example, IgG, IgM, IgE, IgA, GILD and any subclass thereof. The hybridoma producing the monoclonal antibody of the present invention can be cultured in vitro, in situ, or in vivo. In one embodiment, the hybridoma producing the anti-ASC monoclonal antibody of the present disclosure is an ICCN1.OH hybridoma. In another embodiment, the hybridoma producing the anti-ASC monoclonal antibody of the present disclosure is 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 variants thereof, and produces a monoclonal antibody comprising variants having at least one amino acid substitution in HCDR1, HCDR2, and / or HCDR3. In another embodiment, the hybridoma producing the anti-ASC monoclonal antibody of the present disclosure comprises a heavy chain variable (VH) region and a light chain variable A monoclonal antibody containing a variant (VL) region, wherein the amino acid sequence of this 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, and the antibody produces a monoclonal antibody containing a variant having at least one amino acid substitution in LCDR1, LCDR2, and / or LCDR3. In yet another embodiment, the hybridoma producing the anti-ASC monoclonal antibody of the present disclosure is 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 variants thereof, and includes variants 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 variants thereof, and includes variants having at least one amino acid substitution in LCDR1, LCDR2, and / or LCDR3, thereby producing a monoclonal antibody.

[0136] Administration of composition

[0149] The composition of the present invention can be used in any suitable formulation for mammals (e.g., rodents, rhinoceros It can be administered to (t). For example, the anti-ASC antibody can be formulated in a pharmaceutically acceptable carrier or diluent, such as physiological saline or buffer salt solution. Suitable carriers and diluents can be selected based on the method 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 the standard textbooks in the art, Remington's Pharmaceutical Sciences and USP / NF. Other substances can be added to the composition to stabilize and / or preserve it.

[0137]

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

[0138] Effective dose

[0151] The above-mentioned composition, in an effective amount, i.e., in the treated mammal, yields the desired result. It can be administered to mammals (e.g., rats, humans) in amounts that may produce (for example, reduce inflammation in the CNS of mammals that have suffered traumatic injury or stroke to the CNS, or have autoimmune diseases, autoinflammatory diseases, metabolic diseases, neurodegenerative diseases, or CNS diseases). Such therapeutically effective doses may be determined as described below. The therapeutically effective dose of a composition comprising the agent provided herein (e.g., a monoclonal antibody provided herein, such as IC100, or an antibody fragment derived from this monoclonal antibody) may 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. The therapeutically effective dose of a composition containing the agent provided herein (e.g., a monoclonal antibody provided herein, such as IC100, or an antibody fragment derived from this monoclonal antibody) is generally about 0.001 to about 200 mg / kg of patient body weight. The therapeutically effective dose of a composition containing the agent provided herein (e.g., a monoclonal antibody provided herein, such as IC100, or an antibody fragment derived from this monoclonal antibody) is generally about 0.001 mg / kg to about 0.01 mg / kg, or about 0.01 mg / kg. The dose may be approximately 0.1 mg / kg to 1 mg / kg, approximately 1 mg / kg to 10 mg / kg, approximately 10 mg / kg to 25 mg / kg, approximately 25 mg / kg to 50 mg / kg, approximately 50 mg / kg to 75 mg / kg, approximately 75 mg / kg to 100 mg / kg, approximately 100 mg / kg to 125 mg / kg, approximately 125 mg / kg to 150 mg / kg, approximately 150 mg / kg to 175 mg / kg, or approximately 175 mg / kg to 200 mg / kg of the subject's body weight. Compositions containing the agents provided herein (e.g., monoclonal antibodies provided herein, such as IC100, or antibody fragments derived from such monoclonal antibodies) may be administered in single or multiple doses.

[0139]

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

[0140]

[0153] As is well known in the fields of medicine and veterinary medicine, The dosage depends on many factors, such as the size of the subject, body surface area, age, the specific composition to be administered, the time and route of administration, the subject's overall health, and any other drugs administered concurrently. [Examples]

[0141] Examples

[0154] The present invention will be further explained by the following specific examples. The examples are for illustrative purposes only. This merely provides the information and should not be interpreted as limiting the scope of the present invention.

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

[0155] Pulmonary insufficiency is often found as a complication of severe traumatic brain injury (1) Approximately 20–25 percent of TBI patients develop acute lung injury (ALI) (2), but the mechanisms mediating TBI-induced ALI remain poorly defined. Previous literature supports the idea that post-TBI lung dysfunction is due to a sympathetic response to increased intracranial pressure leading to cardiopulmonary dysfunction (42). However, more recent studies have shown that systemic inflammatory responses also play a significant role in TBI-induced lung injury (43). Specifically, the HMGB1-RAGE ligand receptor pathway functions as a central transmission mechanism for post-TBI lung dysfunction (8). Furthermore, HMGB1 induces AIM2 inflammasome activation (37). Further previous literature has revealed that pathogens secrete DAMPs, such as HMGB1-carrying EVs, triggering inflammation (Buzas et al., 2014). Various studies suggest that the blood-brain barrier (BBB) ​​penetrates post-TBI approximately 3–6 hours earlier than injury. This can lead to damage to the protective barriers between the brain and intravascular compartments, resulting in leakage of proteins and fluids (44). Post-injury disruption of the BBB can lead to the secretion of inflammatory mediators, such as DAMP, which can promote brain inflammation and damage distal organs (5). While some inflammatory mediators may act as clear markers of brain injury, their validity is not widely accepted (45). Furthermore, there are currently no clinically approved treatments or biomarkers for TBI-induced ALI. In recent years, EV has become an area of ​​interest for biomarker research for several different types of diseases, e.g., lung injury (46) and TBI (47). In EV isolated from cerebrospinal fluid of patients with TBI, inflammasome proteins have been shown to be higher compared to control samples. It has already been shown that increases (14). In this embodiment, we investigated the contribution of EV-mediated inflammasome signaling to the pathogenesis of TBI-induced ALI.

[0143] Materials and methods Animals and traumatic brain injury

[0156] All animal procedures were approved by the Institutional Animal Care and Use Committee (Animal Welfare Assurance A3224-01) of the University of Miami Miller School of Medicine and performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals. When conducting this study, the ARRIVE guidelines were followed. All C57 / BL6 mice were 8–12 weeks old and weighed 24–32 grams. Mice were prospectively randomized to either a TBI experimental group (simulated, 4 hours, 24) or an adoptive transplant and treatment group (naive, simulated saline, untreated, enoxaparin, anti-ASC). In the TBI experimental group, simulated animals underwent surgery but were not injured. In the adoptive transplant and treatment trial, the simulated saline group underwent surgery and received saline as the vehicle treatment. Naive animals did not undergo surgery. Based on power analysis (G* power analysis, effect size F=0.85, α setting 0.05) and historical data, 5-6 sample sizes were used for each group (49, 50). All mice were housed in a virus-free (VAF) animal facility at the Lois Pope Life Center at the University of Miami, under a 12-hour light-dark cycle, with feed and water provided ad libitum. The facility performed livestock procedures twice a week, and the animals' condition was checked daily. Postoperatively, the animals were observed, held on heating pads, their body temperature controlled by a rectal probe, and their body temperature maintained at 37°C in the operating room before being moved to the animal housing room.

[0144]

[0157] Preoperatively, the animals were anesthetized with ketamine and xylazine (intraperitoneal, IP). Next, the anesthetized animals were placed on a heating pad to maintain a body temperature of 37°C. TBI was performed using a Controlled Cortical Impact (CCI) model. A 5mm craniotomy was performed on the right cortex (2.5mm posterior and 2.0mm lateral from the bregma). An ECCI-6.3 device (Custom Design & Fabrication, Richmond, VA, USA) was used at 6 m / s. Injuries were induced using a 3 mm impactor at a speed, depth of 0.8 mm, and impact duration of 150 ms (15). After these procedures, the animals were subjected to their The animals were returned to their cages and given feed and water. The animals were slaughtered 4 and 24 hours after TBI as described. The dummy animals were anesthetized and subjected to the same preoperative incision as the injured animals, but did not undergo craniotomy or contusions.

[0145] Organizational Recovery

[0158] All animals were anesthetized with ketamine and xylazine before perfusion. Then, the animals The animals underwent tracheal perfusion. A tracheal catheter was used to perfuse the lungs with 4% paraformaldehyde (PFA) in 20 cm of H2O, and the animals were then fixed overnight in 4% PFA at 4°C. The fixed lung tissue was embedded in paraffin, and 5 μm sections were prepared (16). Right lung tissue was collected for protein isolation and molecular analysis. The animals then underwent femoral head resection, and right cortical tissue was collected for protein isolation and molecular analysis.

[0146] Pyroptosome isolation assay

[0159] Mouse lung tissue lysates were treated with 5 μm low-binding polyvinylidene difluoride (PV). The solution was filtered through a DF (dihydrodynamic) membrane (Millipore). After filtration, the supernatant was incubated at 2,700 × g for 8 minutes. The pellet was centrifuged. The pellet was resuspended in 40 μl of 3[(3-collamidopropyl)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). The piroptosomes were subjected to 2,700 × The pellet was pelletized by centrifugation at 1g for 8 minutes. The pellet was then resuspended in 27.8 μl of CHAPS buffer containing 2.2 μl of disuccinimidyl substrate (9), and incubated at room temperature for 30 minutes to crosslink the ASC dimers. Finally, an equal volume of 2×Laemmli buffer was added, and a commercially available antibody against ASC and gasdermin-D (GSD) was added. Proteins were analyzed using immunoblotting.

[0147] Nucleus and cytoplasmic extraction

[0160] Nuclear and cytoplasmic fractions were extracted using NE-PER nucleocytoplasmic 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 the cytoplasmic extraction reagent and centrifuged at 16,000 × g for 5 minutes. Then, the supernatant (cell extract) was removed, and the pellet was homogenized with the nuclear extraction reagent (Thermo Scientific) at 16, The mixture was centrifuged at 000×g for 10 minutes. The supernatant, corresponding 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. It contains a cocktail of protease and phosphatase inhibitors (Sigma, St. Louis, MO, USA). Two mm sections of right lower lung and right cortical tissue were homogenized in the extraction buffer, and caspase-1 (Novus Biologicals) was extracted in 4-20% Tris-TGX Criterion precast gel (Bio-Rad, Hercules, CA, USA) as described in de Rivero Vaccari et al. 2015(13). ASC (Santa Cruz), IL-1β (Cell Signaling), IL-18 (Abcam) AIM2 The enzyme was degraded using antibodies against (Santa Cruz) and HMGB1 (Millipore). Band density was quantified using ImageLab, and all data were normalized to β-actin.

[0149] Immunohistochemical examination

[0162] Tissue sections were deparaffinized in xylene, then treated with ethanol and Tris solution. The tissue was rehydrated with saline solution. Then, the immunohistochemical procedure was carried out for double staining as previously described (16). Sections were stained with caspase-1 and ASC(Millipore) The cells were incubated overnight at 4°C with antibodies against AIM2 (Santa Cruz), HMGB1 (Millipore), and SPC (Millipore). Immunostained lung sections from simulated, 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 blinded participants.

[0150] EV isolation

[0163] Use the whole exosome extract solution according to the manufacturer's instructions (Invitrogen). Extracellular viable cells (EVs) were isolated from TBI-injured mice and serum from injured mice. 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 at 4°C for 30 minutes, followed by centrifugation at 10,000 × g at room temperature for 10 minutes. 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] EV adoption transplant

[0164] Serum-derived EVs from C57BL-6TBI and pseudo-mice were used in naive C5 7BL-6 mice were injected via the jugular vein at a dose of 1.0 × 10¹⁰ particles per gram of body weight.48 The number of particles was measured by Nanosight tracking analysis, and based on this... The sample was diluted. Preoperatively, the animals were anesthetized with ketamine and xylene. A 1-2 cm incision was made between the jaw and clavicle. The jugular vein was elevated and restrained, and then the catheter was inserted. Serum-derived extracellular organisms (EVs) were transplanted, and lung and brain tissue were collected 24 hours after injection for analysis (n=5).

[0152] Enoxaparin and anti-ASC treatment

[0165] Serum-derived EVs from TBI mice were introduced into naive C57-BL6 mice. Injections were made via intravenous 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) The naive group received no treatment. 2) The sham saline group was used as a negative control and received an intravenous injection of only saline. 3) The untreated group received EVs from TBI mice without any treatment and was used as a positive control. 4) The ENOX group received EVs from TBI mice and enoxaparin. And 5) The anti-ASC group received EVs from TBI mice and anti-ASC. The treatment order was randomized. For analysis, lung and brain tissues were collected 24 hours after injection. It should be noted that the anti-ASC antibody used in the treatment experiment is a humanized monoclonal antibody against ASC and recognizes murine, human, and porcine ASC.

[0153] Histological examination and lung injury scoring

[0166] For histological examination, morphometry, and ALI scoring, lung tissue sections were stained by standard hematoxylin and eosin method. 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. The criteria for ALI scoring were based on the number of neutrophils in the alveolar lumen and interstitial space, hyaline membranes, proteinaceous debris filling the airspaces, and alveolar septal thickening. Based on these criteria, scores between 0 (no injury) and 1 (severe injury) were assigned. and eosin method to stain lung tissue sections. Lung sections were scored by a blinded pathologist using the lung injury scoring system from the American Thoracic Society Workshop Report (17). For scoring, 20 random high-power fields were selected. The criteria for ALI scoring were based on the number of neutrophils in the alveolar lumen, interstitial space, hyaline membranes, proteinaceous debris filling the airspaces, and alveolar septal thickening. Based on these criteria, scores between 0 (no injury) and 1 (severe injury) were assigned.

[0154] Statistical analysis [[ID=,17]]

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

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

[0168] Pro-inflammatory cytokines IL-1β and IL-18, and inflammas Inflammasome protein expression levels are associated with secondary injury following fluid percussion brain injury (18). Cortical lysates were analyzed to determine whether severe CCI induced pro-inflammatory cytokine processing and alteration of inflammasome protein levels, but studies on inflammasome activation in severe TBI have been limited. In this embodiment, after severe CCI, cortical lysates were tested for the levels of caspase-1 (Figure 1A, B) (p<.001), ASC (Figure 1A, C) (p=.003), IL-18 (Figure 1A, D) (p=.0042), AIM2 (Figure 1A, F) (p=0.0197), and IL-1β (Figure 1A, G) (p=0.0141) at 4 and 24 hours after injury. The levels of caspase-1, ASC, AIM2, and IL-1β peaked at 4 hours after CCI and decreased by 24 hours. The time course of inflammatory cytokine maturation differed slightly, but peaked by 24 hours after TBI. Since others have shown the role of 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 was elevated at 4 and 24 hours after injury by HMGB1 (Figures 1A and 1E) (p=0.0121). This induced a significant increase in the level of [protein name]. These data indicate that after severe CCI in mice, the level of AIM2 inflammasome protein was significantly elevated in the post-injury cortex.

[0156] Severe TBI increases AIM2 inflammasome protein and HMGB1 expression in the lungs of mice.

[0169] To determine whether CCI induced inflammasome activation in the lungs Immunoblotting of pulmonary 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), AIM2 (Figure 1H, M) (p<.001), and NLRP3 (p=.0047) (Supplementary Figure 1). Increases in the levels of caspase-1, ASC, IL-18, and AIM2 were significantly higher at 4 and 24 hours after injury compared to the quasi-control. However, the time course of the increase in protein expression differed slightly from that observed in the brain, where they peaked at 24 hours after CCI. Since the HMGB1-RAGE axis plays a role in the mechanism by which TBI induces pulmonary dysfunction (8), pulmonary lysates were analyzed for the level of HMGB1 protein expression. Figures 1H and 1L (p=.0158) show that HMGB1 expression increased 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 already shown, the AIM2 inflammasome in cortical neurons Activation leads to pyroptosis and 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 pseudo-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) (20), which is cleaved upon caspase-1 activation and triggers pyroptosis and IL-1β release, was significantly increased in the lungs of TBI animals compared to pseudo-animals (Figures 4B and 4C) (p=0.0001). These findings indicate that pyroptosis contributes to cell death in lung tissue after TBI.

[0158] TBI increases the immunoreactivity of inflammasome proteins in type II alveolar epithelial cells.

[0171] TBI leads to capillary leakage, increased vascular permeability, and type II lung cells This can result in damage to specialized alveolar epithelial cells called inflammasomes (5). To test the cellular effects of TBI on inflammasome expression in the lung after injury, immunohistochemical analysis was performed on lung sections of quasi-, 4-hour, and 24-hour injured animals. Type II alveolar epithelial cells are known to be the injured lung cells in the main type of 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) and DAPI nuclear staining (blue), which are markers of type II epithelial cells. 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 results in increased immunoreactivity in these cells.

[0159] TBI increases nuclear and cytoplasmic HMGB1 expression.

[0172] To determine the cellular distribution of HMGB1 in lung cells after TBI, lung homogeneity Nuclear and cytoplasmic fractions were isolated from the nate (Figure 3A, 3C) (p=.0337). Immunoblotting showed that both fractions had a significant increase in HMGB1 expression 4 hours after TBI (Figure 3B, 3D) (p=.0345). Immunohistochemical analysis of HMGB1 was also performed to determine changes in immunoreactivity in lung sections after TBI. Sections were co-stained for HMGB1 (green), SPC (red), and DAPI nuclear staining (blue). Immunoreactivity of HMGB1 increased at 4 and 24 hours compared to the pseudo-sequence. Weak immunoreactivity of HMGB1 was observed in SPC-positive cells (arrow) (Figure 3E); therefore, this suggests that the HMGB1 changes in injured lung tissue may be cytoplasmic.

[0160] TBI induces changes in lung morphology and leads to ALI.

[0173] ALI results in alveolar and interstitial edema, as well as infiltration of inflammatory cells into the alveolar space. It can be characterized by an inflammatory process (23). Histopathological analysis of lung tissue (Figure 5A) shows that severe TBI causes significant changes in lung structure and morphology at 4 and 24 hours after injury. Pseudoanimals showed normal alveolar morphology, while injured animals showed a rapid change in alveolar edema, which had decreased slightly by 24 hours after injury (long arrow). Furthermore, evidence of neutrophil infiltration (arrow head) and morphological changes in the alveolar capillary membrane (*) was present at both time points. Injured animals showed signs of interstitial edema, which was more pronounced at 4 hours after injury but was still evident at 24 hours after injury (short arrow). Finally, injured animals also showed evidence of thickening of the interstitial segments and alveolar septa (pound, #).

[0161]

[0174] To support the induction of ALI by severe injury, 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 alveoli and interstitial spaces, hyaline membrane formation, proteinous debris filling the spaces, and alveolar septal thickening (17). These features were significantly elevated in injured animals, and ALI scores were generally higher in TBI animals compared to pseudo-TBI animals (Figure 5B) (p=0.0017).

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

[0175] EVs and their loads that may be released in circulation after TBI enter the lungs To provide evidence that flammosome activation can be induced, classical adoptive transplantation experiments were performed using serum-derived EVs from severely CCI mice. EV preparations were validated using Western blotting for the EV marker CD81 (Figure 6). Controls received EVs isolated from pseudo- 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 to the lungs of uninjured or naive mice or animals receiving EVs from naive mice. Furthermore, inflammatory cell infiltration (arrows) was evident in the 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 neuro-respiratory-inflammasome axis in which extravasation vesicles (EVs) released into circulation after TBI activate inflammasomes in lung target cells that contribute to ALI lesions.

[0163]

[0176] Next, enoxapari after adoptive transplantation of EV from injured mice to naive mice. Exosome uptake blockade was attempted by treatment with either a monoclonal antibody (IC100) against caspase-1 or ASC. Negative control animals accepted physiological saline, while positive control animals did not accept the treatment. As shown in Figures 8A-8F, caspase-1 (Figures 8A, 8B), ASC (Figures 8A, 8C), IL-1β (Figures 8A, 8D), AIM2 (Figure 8A, 8E) and HMGB1 (Figures 8A, 8F) were significantly reduced after treatment with enoxaparin or humanized monoclonal anti-ASC antibody (e.g., IC100 antibody) compared to the untreated (positive control) group (p=<.0001). Furthermore, H and E stained lung sections showed significantly less neutrophil infiltration into the alveoli and interstitial space and no signs of septal thickening (Figures 9A-D). ALI scores in animals treated with enoxaparin and anti-ASC antibody (IC100) were significantly lower compared to the untreated group (Figure 9E) (p=<.0001). Therefore, EVs released into circulation after TBI play a role in inflammasome activation in lung cells that lead to ALI.

[0164] conclusion

[0177] TBI has a higher rate of certain medical complications, particularly pulmonary and central nervous system dysfunction. This may be relevant to all. In this embodiment, it was shown that severe TBI increases HMGB1 and inflammasome expression (e.g., AIM2, caspase-1, and ASC expression) in cortical and lung tissue and induces lung morphological changes consistent with ALI (e.g., neutrophil infiltration into alveoli and interstitial spaces, alveolar septal thickening, and alveolar edema and hemorrhage), introducing the concept of a neural respiratory-inflammatory axis. Importantly, TBI resulted in pyroptosis in lung tissue (e.g., in the presence of GSDMD cleavage) and increased inflammasome protein expression in type II alveolar epithelial cells. Furthermore, adoptive transplantation of EVs from TBI mice activated the inflammasome and induced ALI, indicating that brain injury induces the release of EVs containing a cargo of inflammasome proteins, which then leads to ALI. Furthermore, it was shown that inhibition of both EV uptake (enoxaparin) and inflammasome activation (treatment with anti-ASC antibody (IC100)) reduced inflammasome protein expression and ALI development.

[0165]

[0178] In summary, this embodiment demonstrates that AIM2 inflammasome signaling is used in TBI This study demonstrates the central role of EV-mediated inflammasome signaling in the pathological mechanisms of subsequent lung injury, and elucidates the mechanisms of TBI-induced ALI, including EV-mediated inflammasome signaling. These data provide evidence that EV-mediated inflammasome signaling may play a central role, including in the Neuronal-Respiratory-Inflammatory Axis. Therefore, targeting this axis with antibodies against inflammasome proteins or drugs that block EV uptake may offer therapeutic approaches for neurotrauma-induced ALI in all areas of life-saving medicine. In light of these results, the disclosed therapeutic strategies may be useful in the treatment of inflammatory diseases of the lung in general.

[0166] Built-in by reference

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

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

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

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

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

[0184] 5. Rincon F, et al. (2012) Impact of acute lung injury and acute respiratory distress syndrome after traumatic brain injury in the United States. Neurosurgery 71(4):795-803.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0230] As a follow-up to the experiment on mice in Example 1, in human lungs We investigated the role of EVs isolated from human TBI patients in inflammasome signaling in cortical cells.

[0168]

[0231] In the first experiment, a whole exosome extraction kit (Thermofisher) was used. Serum-derived extracellular endothelial cells (EVs) were isolated from TBI and control patients. Human pulmonary microvascular endothelial cells (HMVEC-Lonza) were cultured and plated on 12-well plates. Confluence After reaching a certain temperature, isolated extracellular molecules (EVs) from TBI and control patients were delivered to the cells for a 4-hour incubation period (1.94 × 10⁸ particles / ml). Following incubation, the cells were harvested with 200 μl of lysis buffer, and the cell lysates were used for Western blot analysis.

[0169]

[0232] In the second experiment, a whole exosome extraction kit (Thermofisher) was used. Serum-derived EVs were isolated from TBI and control patients. Human pulmonary microvascular endothelial cells (HMVE) C-Lonza) was cultured and plated on a 96-well plate. Confluence After reaching the target level, isolated extracellular fluid (EVs) from TBI and control patients were delivered to the cells for a 3-hour incubation period (1.94 × 10⁸ particles / ml), followed by a further 1-hour incubation with caspase-1 FAM FLICA (Immunohistochemistry Technologies) in a 1:30 volume / volume ratio. After incubation, the medium was removed and the cells were washed three times with apoptosis wash buffer (Immunohistochemistry Technologies). Subsequently, the cells were subjected to nuclear staining. The cells were co-stained with propidium iodide for Hoechst and 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 extracellular molecules from TBI patients. This increased inflammasome protein expression in lung endothelial cells. Figures 10A–10E show that caspase-1, ASC, AIM2, and HMGB1 were elevated in PMVEC incubated with TBI-EV for 4 hours compared to PMVEC incubated with control EV for 4 hours. Immunosay 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, TBI-EV to pulmonary endothelial cells Delivery increased caspase-1 immunoreactivity and cell death.

[0172] conclusion

[0235] These studies suggest that extravascular ventricular activity (EV) released into circulation after TBI contributes to ALI lesions. This provided further evidence for a neuronal-respiratory-inflammasome axis that activates inflammasomes in lung target cells.

[0173] Example 3: Effects of using humanized anti-ASC antibodies in an animal model of multiple sclerosis.

[0236] To determine the usefulness of humanized anti-ASC monoclonal antibodies in the treatment of MS The aforementioned antibody was administered to mice with experimental allergic encephalomyelitis (EAE). EAE is 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 Lassman Hans (Feb 2010). “Acute disseminated encephalomyelitis and multiple sclerosis”. Brain. 133: 317-319, and L. Gomez Vicente et al. Relapse in a paucisymptomatic form of multiple sclerosis in a patient treated As described in *with nivolumab, Neuro Oncol (2016) 18 (suppl 4): iv25*, this is an animal (i.e., rodent) model of MS.

[0174] method EAE induction and treatment with IC100

[0237] Active EAEs, as already explained (Brambilla et al., 2014) EAE induction was performed in 2-month-old C57BL / 6 female mice using myelin oligodendrocyte glycoprotein 35-55 peptide (MOG35-55, BioSynthesis). Briefly, mice were intraperitoneally (ip) injected with pertussis toxin dissolved in PBS (350 ng / mouse; day 0), followed by subcutaneous administration of MOG35-55 emulsified with complete Freund's adjuvant (300 ng / mouse; day 1), and then again ip-injected pertussis toxin (350 ng / mouse; day 2). Starting 8 days after EAE induction, mice were administered three different doses (10, 30, and 45 mg / kg) of vehicle (0.9% saline) or IC100 by ip injection every 4 days. Clinical symptoms of EAE were assessed daily on a scale of 0 to 6 as follows: 0, no clinical signs; 1, loss of tail tone; 2, tail flaccidity; 3, complete paralysis of the hind limbs; 4, complete paralysis of the forelimbs; 5, mortally ill; 6, death.

[0175] Cell isolation for flow cytometry

[0238] After transcardiac perfusion with PBS, the spinal cord was retrieved and contained Mg2+ and Ca2+. The samples were placed in cold Hanks equilibrium salt solution (HBSS w / o). The samples were manually dissociated into single-cell suspensions by passing them through a 70 μm strainer and washed with HBSS w / o. The spleen samples were rotated at 1200 rpm for 10 minutes at 4°C, the supernatant was removed, and the red blood cells (RBCs) were dissolved in 2 ml of RBC lysis buffer (eBioscience) according to the manufacturer's instructions. Next, the spleen cells were resuspended in PBS. Cells isolated from the spinal cord were resuspended in flow cytometry buffer (FCB, eBioscience) and incubated with Myelin Removal Beads II (Miltenyi). Myelin was depleted using an LS magnetic column as described in the manufacturer's protocol (Miltenyi). Similar to the spleen cells, the 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 according to the manufacturer's instructions (BioRad). Cells were incubated in FLICA solution (BioRad) at 4°C for 30 minutes and washed with Apoptosis Wash Buffer (BioRad). The sample was resuspended in 1 ml of PBS. Then, the sample was incubated with a fixative viable / dead stain (Tonbo Biosciences) at 4°C for 30 minutes, and then incubated at 4°C for 10 minutes. The cells were rotated at 1200 rpm and the supernatant was removed. The cells were resuspended in 100 μl of FACS buffer and bled for 5 minutes at room temperature with anti-CD16 / 32 (FcR block, eBioscience). The samples were locked, immunostained at 4°C for 30 minutes, and fixed with 1% PFA. The samples were analyzed using a CytoFLEX S flow cytometry system (Beckman Coulter) with CytExpert 2.1 software. Spinal leukocyte counts were determined using 123-count eBeads (eBioscience). Spleen leukocyte counts were determined by flow cytometry combined with trypan blue exclusion counting using a TC20™ Automated Cell Counter (Bio-Rad). A list of flow cytometry antibodies is provided in Table 3 below.

[0177]

[0240] [Table 11]

[0178]

[0241] Luxor Fast Blue staining and quantification of demyelinated white mass

[0242] Paraformaldehyde (PFA) fixed segments of the spinal cord are embedded in paraffin. The sample was then sectioned into 10mm thick sections using a Leica RM 2135 microtome, and Luxor Fastob was used. The tissue was stained with LFB (Lou). Demyelinated white mass was estimated using 10 serial sections spaced 50 μm apart. The contour of the demyelinated region was drawn using an Olympus BX51 microscope, and the demyelinated white mass 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] Quantitative determination of IC100 in tissues

[0243] IC100 was quantified in the brain, spinal cord, liver, and spleen 35 days (dpi) after EAE induction using an assay developed by InflamaCORE, LLC using Meso Scale Technology. This assay was read using a QuickPlex SQ 120 instrument (Meso Scale Diagnostics, Maryland).

[0180]

[0244] To determine whether anti-ASC penetrates spinal nerve cells, compress the cervical spine. Similar experiments were conducted using a rat model of spinal cord injury.

[0181]

[0245] To determine whether IC100 is taken up by cells, fluorescein IC100 labeled with 2C was added to tissue culture medium containing THP-1 cells (human monocyte cell lineage).

[0182] result Treatment with the anti-ASC antibody IC100 leads to remission of functional outcomes in experimental autoimmune encephalomyelitis (EAE).

[0246] To evaluate the therapeutic potential of IC100, a 2-month-old female C57BL / 6 ma EAA was induced in muskets with MOG35-55 peptide (Brambilla et al., 2014). Starting on day 8 (dpi) after disease induction, either IC100 or vehicle alone was administered. This administration was repeated every 4 days until slaughter, set at 35 dpi. Three doses—10, 30, and 45 mg / kg—were tested.

[0183]

[0247] When IC100 is used at doses of 30 and 45 mg / kg, this practical The treatment significantly improved functional recovery, accompanied by a robust reduction in clinical disease scores throughout the entire duration of the trial (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 tendency toward delayed disease onset (Figure 12D). No significant difference was observed on the day the mice reached their peak disease score (Figure 12E).

[0184] Treatment with the 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 are related to the infiltration of immune cells into the spinal cord. A direct correlation was observed. To assess whether IC100 influenced this process, the characteristics of immune cell populations isolated from the spinal cord at 35 dpi were revealed by flow cytometry. Treatment with 30 mg / kg IC100 significantly reduced the total number of encephalitis-inducing CD4+ T cells and CD8+ T cells, which are the most important immune cell populations driving EAE pathology (Figure 13A). All other immune cell populations showed a clear trend toward reduction. In the spleen, no significant difference in cell number was observed at any dose of IC100, suggesting that the treatment did not impede the mice's ability to initiate a sufficient immune response to the EAE challenge (Figure 13B).

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

[0249] Microglia are involved in the immune-inflammatory response to CNS diseases. As the activation state increases, microglia proliferate and upregulate the surface expression of MHCII. 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 activating microglia and suppressing microglia-mediated neuroinflammation (see Figure 14).

[0186] IC100 penetrates the brain and spinal cord.

[0250] A key parameter in designing drugs to reduce MS is whether the drug can cure the condition. The objective is to determine whether the drug penetrates the CNS at a therapeutic level. This is a particularly important characteristic in the treatment of progressive MS, because the blood-brain barrier appears relatively intact in this stage of the disease (Lassman et al. 2012). Therefore, the present invention The researchers collected samples from the brain, spinal cord, liver, and spleen and determined the levels of IC100 in these tissues. As shown in Figure 15, IC100 penetrated all of these tissues, including the brain and spinal cord, at all three dosages. Interestingly, the level of IC100 in the spinal cord was highest at a dose of 30 mg / kg, which is consistent with the greater therapeutic effect at this dose.

[0187]

[0251] When fluorescein-labeled IC100 is added to tissue culture medium, T It is taken up by HP-1 cells (human monocyte cell lineage) and incorporated into ASCspek. In addition, inflammasome induction in these cells, along with rhodamine-labeled dextran, stimulates the uptake of labeled IC100 into ASCspek, suggesting that IC100 uptake is mediated by endocytosis (see Figure 16).

[0188]

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

[0189]

[0253] Anti-ASC antibodies (e.g., IC100) were found in a rat model of crush cervical spinal cord injury. It also infiltrated the spinal nerve cells.

[0190]

[0254] IC100 can function both intracellularly and extracellularly. Intracellularly, IC1 IC100 can function by binding to and inhibiting ASC proteins, thereby preventing the construction of multiprotein inflammasomes and the initiation of inflammatory responses. IC100 can also bind to ASCs of the ASC spec, preventing the propagation of the large filamentous signaling platform, and thereby inhibiting the extracellular activation of pro-ILβ, which is involved in the persistence of inflammation in chronic inflammatory diseases.

[0191] Built-in by reference

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

[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: Dynamic analysis of candidate anti-ASC monoclonal antibodies

[0258] Using biolayer interference (BLI), the dynamics of candidate anti-ASC monoclonal antibodies were analyzed. A dynamic analysis was performed. In BLI, association and dissociation with the surface cause a shift in the wavelength of reflected light, and by measuring this shift over time, it becomes possible to determine the coupling dynamics.

[0195]

[0259] This BLI assay consisted of the following:

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

[0196]

[0261] Candidate mouse IgG antibody supernatant was tested at seven different concentrations (i.e., 540 nM; 18 The antibodies were tested for binding to human ASC peptide (SEQ ID NO: 5) at concentrations of 0nM, 60nM, 20nM, 6.67nM, 2.22nM, and 0.741nM. The tested antibodies were ICCN 1.0H (i.e., IC100), ICCN 2.0H, and ICCN 3.0H. Mouse IgG was loaded onto an AMC (anti-mouse IgG Fc) biosensor from the undiluted supernatant. The biokinetic data for these three candidate antibodies are shown in Figure 21, and the global KD values ​​are shown in Figure 22.

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

[0262] To describe the pharmacokinetics of IC100 in male CD-1 rats, absorption and distribution are discussed. Metabolism and excretion (ADME) experiments will be conducted.

[0198]

[0263] In the first experiment, 30 six-week-old male CD-1 rats will be obtained from Charles River. The experiment will be conducted at Bolder BioPath (BBP). The mice will be kept at least after arrival at BBP. The mice will be allowed to adapt for 7 days. They will be housed in cages of four animals each.

[0199]

[0264] The animals were randomly assigned to treatment groups (9 mice per group) based on their weight. The treatment group will be divided and administered IC100 intravenously (IV). Each treatment group will receive either 5 mg / kg, 15 mg / kg, or 30 mg / kg of IC100. Plasma will be collected at various times after a single IV dose for pharmacokinetic (PK) monitoring. An exemplary timetable for plasma collection is shown in Table 4 below. Acute toxicity will be monitored by clinical observation. Plasma will be sent to Antibody Solutions for analysis. This experiment will continue for 10 weeks.

[0200]

[0265] Weight measurements will be taken on days 0, 7, 14, 21, 28, and 35.

[0201]

[0266] A sufficient amount of blood was collected using posterior orbital hemorrhage, and the mice were prepared before sample collection. The animals are anesthetized with isoflurane. Animals 1, 2, and 3 in each group are induced to bleed on day 1 and day 10, animals 4, 5, and 6 are induced to bleed on day 2 and day 15, and animals 7, 8, and 9 are induced to bleed on day 5 and day 20.

[0202]

[0267] At the final stage, the animals were anesthetized with isoflurane to treat blood loss and subsequent bilateral pneumothorax. Let them bleed. Slaughter animals 1, 2, and 3 on day 25. Slaughter animals 4, 5, and 6 on day 30. Slaughter animals 7, 8, and 9 on day 35.

[0203] [Table 12]

[0204] Example 6. In vivo distribution of IC100 using female B6 Albino mice and fluorescence imaging.

[0268] In the second experiment, IC100 and control mouse IgG were labeled with VivoTag 680XL fluorescent dye according to the established VivoTag protocol, and their binding affinity was determined. To put it simply, according to the VivoTag protocol, this labeling protocol involves the following:

[0205]

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

[0206]

[0270] 2. Dissolve 0.25 mg of VivoTag 680XL in 10 μL of dry DMSO. When reconstituted, VivoTag 680XL will have a maximum capacity when stored at 2-8°C and protected from light. It has remained stable for 7 days.

[0207]

[0271] 3. Protein 0.5 mL (0.5-5 mg), sodium bicarbonate 50 μg Place 2 μL of VivoTag 680XL per 1 mg of protein into an Eppendorf tube. Incubate this mixture in the dark at room temperature for 2 hours while shaking it.

[0208]

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

[0209]

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

[0210]

[0274] 6. The collected labeled antibody samples can be analyzed in terms of their degree of labeling (DOL). The absorbance of the purified conjugate was determined at 80 nm and 668 nm.

[0211]

[0275] The absorbance of the purified protein at 280 nm is adjusted by subtracting the 280 nm absorbance of VivoTag 680XL, which is 16% of the absorbance at 7.668 nm.

[0212]

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

[0213]

[0277] In the third experiment, the in vivo distribution of IC100 will be determined. This study involves 15 We used 8-12 week old female B6 Albino (C57BL6) mice. The animals were randomly divided into groups based on their body weight on day 1.

[0214]

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

[0215]

[0279] In vivo fluorescence imaging was performed at 2 hours, 8 hours, 24 hours, 48 ​​hours, 72 hours, and The procedure is performed over 96 hours, and fluorescence imaging is used at various time intervals up to 96 hours post-treatment to acquire dorsal and ventral in vivo whole-body images. Ex vivo imaging is performed on all animals of the brain, eyes (including optic nerve), heart, left and right kidneys, large intestine (including terminal colon), liver, lungs, ovaries, pancreas, small intestine, spine, stomach, thyroid, and bladder.

[0216]

[0280] Whole blood is collected, and the immune infiltrates are identified as CD4+ T cells (CD4+CD11b-CD4). We will analyze the following cells: 3+CD8-, CD8+ T cells (CD8+CD11b-CD3+CD4-), B cells (CD3-CD111b-CD45R+), monocytes (CD3-CD11b+CD115+), and NK cells (CD3-CD49b+CD335+). Ab-VivoTag 680 Quantify XL expression to determine the level of delivered labeled antibody. Quantify the number of viable cells. To this end, the flow cytometry panel includes viable / dead dyes. The total antibody panel includes antibodies against CD3, CD4, CD8, CD11b, CD115, CD45R, CD49b, and CD335, along with viable / dead dyes.

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

[0281] Non-alcoholic steatohepatitis (NASH), diabetic nephropathy, and lupus nephritis Blood samples from human patients will be obtained from BioReclamation IVT for biomarker analysis of inflammasome proteins. Tissues from patients with NASH, diabetic nephropathy, and lupus nephritis will be obtained from Bolder BioPath, and protein lysates will be obtained from these tissues and analyzed for the expression of inflammasome signaling proteins such as caspase-1 and ASC using immunoblotting and other biochemical techniques.

[0218]

[0282] In addition, human cancer cell lines were used to study the activity of ASC-dependent inflammasomes. The study will investigate the changes in real time. This research has two objectives and will begin over a period of 6 to 9 weeks.

[0219]

[0283] Specific objective 1: Labeling of antibodies

[0284] IC100 2 mg was labeled with IgG-680 XL-IFC (VivoTag® 680 XL, PerkinElmer #-NEV11120) fluorescent dye, and the stoichiometry of the label was determined according to the manufacturer's instructions. Control mouse IgG (provided by Charles River Laboratories (CRL)) These are then labeled and analyzed in the same manner.

[0220]

[0285] Specific Objective 2: Determining Binding Affinity

[0286] Human cancer cell lineage THP-1 was cultured in logarithmic phase and then transferred to a white polystyrene 6-well tray. Microculture Plate (Corning® Costar® 96-well flat-bottom plate) Cells are seeded in 100 μL of medium (catalog number 3917) at a rate of 20,000 cells per well. Then, the labeled antibody is added in double dose to this well (10-point dose-response, 1:3 dilution, maximum concentration 200 nM). The bound antibody is then processed using Charles River's Prototype. Detection is performed in all wells according to the col. The bound antibody (average fluorescence intensity) is plotted as a function of antibody concentration, and the binding affinity (Kd) is estimated by fitting the following formula to this data: Y = Bmax * X / (Kd + X).

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

[0287] A patient with hepatic fibrosis is being fed a choline-deficient, high-fat diet (CDHFD). Male Wistar Han rats serve as an animal model for NASH. This study utilizes 55 mice. 8-9 week old Wistar Han rats were fed to Envigo or Charles River. Obtain from [source]. Allow the rats to acclimate for 3–7 days after arrival at Bolder Biopath. House the rats in cages of 2–3 animals each. Feed the animals standard solid feed.

[0222]

[0288] On day 0, the animals are randomly divided into 5 groups based on their body weight. Group 1 includes: Feed the standard diet, Teklad Global Diets - Rodent 2014. Groups 2, 3, 4, and For the fifth stage, feed them CDHFD feed.

[0223]

[0289] On day 38 of the study, the animals were induced to bleed for clinical chemistry, and alanine amino acid tranquilizers were administered. Participants will be classified into treatment groups based on their encephalase (ALT) concentration. Treatment will be initiated on day 42 of the study. The efficacy of IC100 will be tested using a dose determined based on pharmacokinetic data obtained from Example 5. One group will be treated with a vehicle to serve as a negative control. The group receiving standard feed will also be treated with a vehicle. Body weight, food intake, and clinical observations from the cage sides will be measured weekly. Whole blood will be obtained by tail vein collection on days 38 and 63. Necropsy will be performed on day 84. Animals will be sacrificed under isoflurane anesthesia and allowed to bleed until blood loss and subsequent bilateral pneumothorax occur.

[0224]

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

[0225]

[0291] In this study, 0, 7, 14, 21, 28, 35, 42, 49, 56, 63, 7 On days 0, 77, and 84, record weekly updates on food intake (grams / day / rat).

[0226]

[0292] 0-7, 14, 21, 28, 35, 42, 45, 49, 52, 56, 59, Clinical observations from the side of the cage will be performed on days 63, 66, 70, 73, 77, 80, and 83. If the animals begin to show clinical signs of toxicity or disease, they will be observed and weighed daily.

[0227]

[0293] During autopsy, the weights of the liver, brown adipose tissue, and right inguinal adipose tissue are measured. Obtain a 4×7mm biopsy of the left lobe of the liver, freeze it in liquid nitrogen, and store it at -80°C. Obtain 3mm transverse sections of the middle, left, and right lobes of the liver, fix them in 10% formalin for 36-48 hours, and then store them in 70% ethanol at room temperature for histopathology. Snap-freeze three 100mg fragments of adipose tissue in liquid nitrogen and store them at -80°C in Eppendorf SafeLock tubes. Snap-freeze three equal-sized fragments of brown adipose tissue in liquid nitrogen and store them at -80°C in Eppendorf SafeLock tubes.

[0228]

[0294] The white adipose tissue depot (WAT), which is the subcutaneous adipose tissue of the inguinal region, is used in the following protocol. Collect according to the instructions. Degloving of the lower body of the mouse will collect samples from the inguinal region. The triangular square depot is revealed. Hold the upper limbs and chest with one hand and pull the skin down towards the feet with the other hand. Position the mouse supine, taking care not to contaminate the exposed depot with hair. Clean the surgical instruments and change gloves. Then, dissect the subcutaneous fat triangle, taking care not to contaminate the sample with muscle, adjacent fat, mammary gland, or blood. Use a dissecting microscope if the boundary is not clearly defined. Remove the fat depot and transfer it to 10% neutral buffered formal at a fixative-to-tissue volume ratio of 50:1 and fix at room temperature for 36-48 hours. If RNA or protein is to be extracted, freeze the tissue by immersion in liquid nitrogen and store at -80°C to prevent degradation. Take care to prevent cross-contamination between fat depots by frequently changing gloves.

[0229]

[0295] Histological processing will be performed by Histotox Labs. Histological processing for LLL, MLL, and RLL will be performed on three liver sections / animals. Samples will be stained with Sirius Red, hematoxylin, and eosin (H & E).

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

[0296] The BTBR mouse strain, which has the ob / ob leptin deficiency mutation, is associated with diabetic nephropathy. This study will serve as a mouse model. Using male BTBR Ob / Ob mice, we will evaluate the effect of IC100 in reversing the effects of diabetic nephropathy. Five male wild-type (WT) BTBR mice will be used as negative controls and will not receive any treatment. Fifty BTBR Ob / Ob mice will be divided into five groups. These groups will be administered either the vehicle, control, or IC100 at the dose determined based on Example 5. This experiment will be conducted over six weeks.

[0231]

[0297] Histological examination will be performed on mouse kidneys by Histotox Labs. Appropriate parameters will be determined by a pathologist using the current Bolder BioPATH method.

[0232]

[0298] Blood glucose levels will be measured by cutting the tail and dropping approximately 5 μL of blood onto a test piece compatible with the True Metrix blood glucose meter. Blood glucose levels will be measured twice a week until the completion of the study.

[0233]

[0299] Proteinuria scoring involves holding the mouse upside down and compressing its abdomen. This procedure is performed by extracting urine from mice. The amount of protein in this urine is determined using Albustix reagent paper.

[0234]

[0300] If an animal is found dead, no sample will be taken. If the animal is euthanized... If necessary, regardless of the reason, samples will be taken at the autopsy on the 7th day after the study.

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

[0301] Using 12-week-old female MRL / MpJ-Tnfrsf6lpr / J mice To develop a model of lupus nephritis, mice will be obtained from Bolder BioPath. Mice will be randomly assigned to treatment groups based on body weight. Animals will be observed daily for significant clinical signs, morbidity, and mortality. Lupus nephritis typically develops at approximately 12–14 weeks of age.

[0236]

[0302] After the onset of lupus nephritis, mice were classified into five groups, following the results of Example 5. Treatment is administered with various doses of IC100, a vehicle, or a control IgG.

[0237]

[0303] Collect body weight, urine protein, lymph node swelling score, and skin lesion score. An autopsy will be performed at 20 weeks, and tissue and whole blood will be collected for analysis. This experiment is expected to last 15 weeks.

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

[0304] Albino rats will be obtained from Charles River. IC100 dose levels will be established for a confirmed 28-day multi-dose toxicity and pharmacokinetic study in rats. The rats will be classified into four groups, each containing three rat types / sex / dose. An up-and-down study will be conducted.

[0239]

[0305] A repeated dose study will be conducted over three weeks. All rats, including five rats, will be used. The rats are classified into male or all-female groups. They are administered weekly at a single dose level of IC100. Survival parameters, such as mortality, clinical signs, body weight, and toxicokinetics are monitored.

[0240] Example 12: Study to determine the maximum tolerated IC100 and 22-day dose range in cynomolgus monkeys

[0306] The dose levels and pharmacokinetics of IC100 will be established in cynomolgus monkeys. The experiment will be conducted at Charles River. The monkeys will be classified into four groups, each consisting of one monkey species, sex, and dose. An up-and-down study will be conducted over 28 days.

[0241]

[0307] A repeated dose study will be conducted over three weeks. The monkeys will be all male, including two male monkeys. Alternatively, classify the monkeys into groups consisting entirely of females. Administer the monkeys weekly at a single dose level of IC100. Perform clinical examinations using survival parameters such as mortality, clinical signs, body weight, and toxicological data.

[0242]

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

[0243] Example 13: Toxicity study of IC100 in rats and subsequent 4-week recovery period.

[0309] To establish the toxicity and toxicological effects of intravenous administration of IC100 in rats obtained from Charles River, and the subsequent recovery period.

[0244]

[0310] Rats were divided into groups of 10-15 rats / sex / including 3 dose levels and controls. The rats will be classified into groups. Additional groups will include a high-dose group and a control group, each containing 5 rats / sex for 4 weeks of recovery.

[0245]

[0311] Mortality rate, body weight, food intake, clinical observation, clinicopathology (hematology and clinical chemistry), autopsy Findings, organ histopathology, and toxicology are measured. After recovery, the same parameters are measured, except for toxicology.

[0246]

[0312] This experiment is expected to last for six months.

[0247] Example 14: Toxicity study of IC100 in cynomolgus monkeys and subsequent 4-week recovery period.

[0313] Toxicity of IC100 after intravenous administration and subsequent recovery period in non-human primates Furthermore, the toxicology will be established in the Charles River.

[0248]

[0314] The monkeys are classified into three groups / sex / groups, each containing three different dose levels and a control group. The additional groups included a high-dose group and a control group, each containing two monkeys / sex for four weeks of recovery.

[0249]

[0315] Mortality rate, body weight, food intake, clinical observation, clinicopathology (hematology and clinical chemistry), autopsy Findings, organ histopathology, and toxicology are measured. After recovery, the same parameters are measured, except for toxicology.

[0250]

[0316] This experiment is expected to last for six months.

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

[0317] The possibility of cardiovascular toxicity (QT prolongation) can be assessed using CHO cells or HEK293 cells. It will be evaluated using an in vitro assay.

[0252]

[0318] This experiment confirmed that IC50 is related to HERG channel occlusion by IC100. It is erected.

[0253]

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

[0254] Example 16: In vitro study of blood hemolysis

[0320] Intravenous formulations of IC100 may cause hemolysis of human red blood cells in vitro. Evaluate the properties. Mix the IC100 concentration (determined based on Example 5) with red blood cells in vitro. Establish the degree of hemolysis. This experiment is expected to last for two months.

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

[0321] The effectiveness of IC100 in the treatment of PD was investigated in several animals with PD (i.e., rodents). The model is evaluated by administering IC100 at doses of 5 mg / kg, 15 mg / kg, and 30 mg / kg.

[0256]

[0322] The 6-OHDA rat model of PD is used in mice to describe rotational asymmetry and lack of movement. This is a chemically induced unilateral model of Parkinson's disease (PD) presenting with behavioral defects including depression (lesion of the striatum or medial forebrain bundle). The 6-OHDA model shows decreased dopamine, DOPAC, and HVA content in the striatum, and histological examination shows a decrease in TH-positive cells in the substantia nigra.

[0257]

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

[0324] 1. Treat the sham-induced rats with the vehicle;

[0325] 2. Treat the 6-OHDA-induced rats with the vehicle;

[0326] 3. Treat 6-OHDA-induced rats with an IC100 dose of 1 (PK / half dose). Treatment is selected based on the study period;

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

[0328] The daily dose formulation and administration (QD, po) were carried out on days 15-28 of the study. stomach;

[0329] Weight tracking was conducted, and behavioral tests were performed on day 14 (baseline) and day 28. This procedure is performed on the 42nd day and includes amphetamine-induced rotation.

[0258]

[0330] End-stage blood, CSF, and brain samples were taken on day 42 of the study, followed by... Then, HPLC is performed to examine DA, DOPAC, and HVA in the striatum, and IHC is performed to examine TH+ cells in SNpc.

[0259]

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

[0332] 1. Treat the sham-induced rats with the vehicle;

[0333] 2. Treat the 6-OHDA-induced rats with the vehicle;

[0334] 3. Treat 6-OHDA-induced rats with an IC100 dose of 1 (PK / half dose). Treatment is selected based on the study period;

[0335] This experiment was conducted over a period of six weeks to study unilateral 6-OHDA / sham injection. Performed on day 0;

[0336] The daily dose formulation and administration (QD, po) were carried out on the first day of the study, and Continue for up to 6 weeks after 6-OHDA injection.

[0260]

[0337] Behavioral tests were conducted on days -14 (baseline), 28, and 42. and also include amphetamine-induced rotation and cylinder tests.

[0261]

[0338] Brain sampling was performed on day 42 of the study, followed by HPLC. Then, DA, DOPAC, and HVA in the striatum are examined, and IHC is performed to examine TH and Iba-1 in the SNpc (bilateral).

[0262]

[0339] In the third set of experiments using the PD 6-OHDA model, a total of 90 individuals were used. The male rats were divided into six experimental groups (n=18 rats / group from the start to baseline at day 14, with a target of n=15 rats / group), and treated as follows:

[0340] 1. Treat the sham-induced rats with the vehicle;

[0341] 2.6-OHDA-induced rats were treated with a vehicle;

[0342] 3.6-OHDA-induced rats were treated with an IC100 dose of 1;

[0343] 4.6-OHDA-induced rats were treated with an IC100 dose of 2;

[0344] 5.6-OHDA-induced rats were treated with an IC100 dose of 3;

[0345] 6.6-OHDA-induced rats were treated with an IC100 dose of 4;

[0346] This experiment was conducted over a period of six weeks to study unilateral 6-OHDA / sham injection. Performed on day 0;

[0347] The daily dose formulation and administration (QD, po) were carried out from day 15 to day 42 of the study. Now.

[0263]

[0348] Behavioral tests were conducted on days -14 (baseline), 28, and 42. and also include amphetamine-induced rotation and cylinder tests.

[0264]

[0349] Brain sampling was performed on day 42 of the study, followed by HPLC. Then, DA, DOPAC, and HVA in the striatum are examined, and IHC is performed to examine TH and Iba-1 in the SNpc (bilateral).

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

[0350] TOM20 assay:

[0351] The onset of Parkinson's disease is caused by the mitochondrial complex I inhibitor rotenone. Several lines of research, including the discovery that it induces mitochondrial dysfunction, have linked it to PARK2. In addition, most autosomal recessive cases of PD have PARK2 Furthermore, dysfunctions such as PINK1 and mutations in genes encoding proteins involved in the selective clearance of redundant mitochondria (mitophagy) are present. Similarly, there is growing evidence of mitochondrial dysfunction in other neurodegenerative disorders (e.g., AD, ALS, and Huntington's disease (HD)). Therefore, phenotypic reading to measure mitochondrial (dys) function in disease-associated cellular backgrounds is considered a powerful predictive tool for investigating neurodegenerative pathologies and identifying potential therapies that may enhance mitophagy.

[0266]

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

[0267]

[0353] TOM20 loss assay performs mitophagy in a neuronal background. This is a scalable and rapid in vitro assay for screening compounds based on their ability to enhance other substances.

[0268]

[0354] The TOM20 assay begins on day 0 (D0), followed by day 1 (D1) and day 4. Immortalized human midbrain progenitor cells (ReNcell VM), seeded at 50,000 cells / well in laminin-coated 96-well plates, are di...

Claims

1. A nucleic acid encoding a monoclonal antibody or antibody fragment thereof that specifically binds to apoptosis-related speck-like protein (ASC) containing a caspase-activating recruitment domain, wherein the antibody or antibody fragment comprises a heavy chain variable (VH) region and a light chain variable (VL) region. The amino acid sequence of the VH region includes HCDR1 of SEQ ID NO: 6, HCDR2 of SEQ ID NO: 7, and HCDR3 of SEQ ID NO:

8. The amino acid sequence of the VL region includes LCDR1 of SEQ ID NO: 12, LCDR2 of SEQ ID NO: 13, and LCDR3 of SEQ ID NO:

14. Nucleic acid.

2. The amino acid sequence of the VH region includes SEQ ID NOs: 18, 19, 20, 21, or 22. The amino acid sequence of the VL region includes sequence numbers 28, 29, 30, or 31. The nucleic acid according to claim 1.

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

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

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

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

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

8. Recombinant host cells comprising the expression vector according to claim 6 or 7.

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

Images