Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
  • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered

Definitions

• the inflammasome is comprised of caspase-1, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), and a sensor protein such as NOD-like receptorl (NLRP1) or NLRP3.
• ASC caspase recruitment domain
• NLRP1 NOD-like receptorl
• the sensor NLR protein binds to pathogen-associated molecular patterns (PAMPs) or danger-associated molecular patterns (DAM Ps) to activate the inflammasome.
• PAMPs pathogen-associated molecular patterns
• DAM Ps danger-associated molecular patterns
• Inflammasome activation is accompanied by oligomerization of the sensor that then recruits monomeric ASC which oligomerizes with an NOD-like receptor such as NLRP3 via homotypic interactions between the PYRIN domain (PYD) of ASC and the PYD of NLRP3.
• NLRP3 an NOD-like receptor
• PYD PYRIN domain
• pro-caspase-1 is recruited and binds to ASC via homotypic CARD-CARD interactions between the two proteins.
• the activated inflammasome leads to the cleavage of caspase-1 into its active form, leading to the production of mature interleukin (I L)-l(3 and IL-18.
• I L interleukin
• active caspase-1 also cleaves gasdermin-D (GSDM-D).
• GSDM-D gasdermin-D
• the N- terminus is inserted into the cell membrane to form a pore through which IL-1 ⁇ and IL-18 are released, resulting in pyroptosis.
• Pyroptosis leads to inflammasome proteins being released into the extracellular space, including the release of oligomerized ASC in the form of ASC specks that present prion-like properties.
• Extracellular ASC specks remain present for long periods of time, while retaining their ability to cleave pro-IL-1 ⁇ , and thus, perpetuating inflammation. Importantly, this prion-like propagation of inflammation by ASC specks contributes to a progressive inflammatory state that plays a central role in neurodegeneration.
• the administering the monoclonal antibody or the antibody fragment thereof reduces levels of at least inflammatory cytokine.
• the administration of the monoclonal antibody or the antibody fragment thereof results in inhibition of inflammasome activation in the subject.
• the administration of the monoclonal antibody or the antibody fragment thereof results in a reduction in the activity of ASC as compared to a control.
• the control is an untreated subject.
• the administration is intracerebroventricularly, intraperitoneally, intravenously, intranasally or by inhalation.
• the VH region amino acid sequence comprises SEQ ID NO: 18, or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 18; and wherein the VL region amino acid sequence comprises SEQ ID NO: 29 or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 29.
• the VH region amino acid sequence comprises SEQ ID NO: 18, or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 18; and wherein the VL region amino acid sequence comprises SEQ ID NO: 30 or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 30.
• the VH region amino acid sequence comprises SEQ ID NO: 18, or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 18; and wherein the VL region amino acid sequence comprises SEQ ID NO: 31 or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ. ID NO: 31.
• the VH region amino acid sequence comprises SEQ ID NO: 20, or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 20; and wherein the VL region amino acid sequence comprises SEQ ID NO: 28 or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 28.
• the VH region amino acid sequence comprises SEQ ID NO: 20, or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 20; and wherein the VL region amino acid sequence comprises SEQ ID NO: 31 or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 31.
• the VH region amino acid sequence comprises SEQ ID NO: 21, or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ.
• the VL region amino acid sequence comprises SEQ ID NO: 28 or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 28.
• the VH region amino acid sequence comprises SEQ ID NO: 21, or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 21; and wherein the VL region amino acid sequence comprises SEQ ID NO: 29 or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 29.
• the VH region amino acid sequence comprises SEQ ID NO: 22, or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 22; and wherein the VL region amino acid sequence comprises SEQ ID NO: 30 or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 30.
• the monoclonal antibody or the antibody fragment thereof is human, humanized or chimeric.
• an isolated nucleic acid molecule encoding the monoclonal antibody or the antibody fragment thereof.
• an expression vector comprising the nucleic acid molecule.
• the nucleic acid molecule is operatively linked to regulatory sequences suitable for expression of the nucleic acid segment in a host cell.
• a recombinant host cell comprising the expression vector.
• a method for producing an antibody or an antibody fragment that binds specifically to ASC comprising: culturing a recombinant host cell comprising the expression vector under conditions whereby the nucleic acid molecule is expressed, thereby producing the monoclonal antibody or the antibody fragment thereof that binds specifically to ASC.
• a pharmaceutical composition comprising the monoclonal antibody or the antibody fragment thereof, and a pharmaceutically acceptable carrier, diluent or excipient.
• a method of treating inflammation in a subject the method comprises administering to the subject a therapeutically effective amount of the monoclonal antibody or the antibody fragment thereof, thereby treating the inflammation in the subject.
• FIG. 1H shows a representative immunoblot of active caspase-1, ASC, IL-18, IL-0, HMGB1, and AIM2 in lung tissue.
• I, J, K, L, M, N Active caspase-1 (FIG. II), ASC (FIG. 1J), IL-18 (FIG. IK), HMGB1 (FIG. IL), AIM 2 (FIG. IM), and IL-3, (FIG. IN) are significantly elevated in lung tissue 4 and 24 h after TBI.
• Data presented as mean+/ _ SEM. N 4-5 per group, ****p ⁇ 0.001, ***p ⁇ 0.01, **p ⁇ 0.01, *p ⁇ 0.05 compared to sham.
• FIG. 6 illustrates expression of CD81 in serum-derived EV from control and TBI-injured mice. Representative immunoblot of CD81 in serum-derived EV from sham control and TBI-injured mice.
• FIG. 7A-7G illustrates adoptive transfer of EV from TBI animals induce caspase-1 and ASC in the lungs of uninjured mice.
• FIG. 7A illustrates a representative immunoblot showing that caspase-1 (FIG. 7B), ASC (FIG. 7C), IL-18 (FIG. 7D), AIM2 (FIG. 7E), HMGBl (FIG. 7F) are elevated in the lungs of animals that received EV isolated from TBI mice when compared to EV from sham animals.
• FIG. 10A-10F illustrates delivery of serum-derived EV from TBI patients results in increased inflammasome protein expression in pulmonary endothelial cells.
• FIG. 10A shows western blot representation of caspase-1, ASC, AIM2, HMGBl in PMVEC after incubation with TBI-EV and control- EV for 4 hours.
• FIG. 10A-10F illustrates delivery of serum-derived EV from TBI patients results in increased inflammasome protein expression in pulmonary endothelial cells.
• FIG. 10A shows western blot representation of caspase-1, ASC, AIM2, HMGBl in PMVEC after incubation with TBI-EV and control- EV for 4 hours.
• FIG. 10B-10E shows quantification of western blots
• FIG. 18 Illustrates AD neuropathological changes seen with A ⁇ and pTau.
• F Number of neurofibrillary tangles in hippocampal regions of the DG, CAI, CA2, CA3 and subiculum regions and in the entorhinal cortex between intermediate AD cases compared with low AD.
• AD Alzheimer's disease
• a ⁇ beta-Amyloid
• ptau hyperphosphorylated
• DG Dentate Gyrus
• CA3 Cornu Ammonis Field 3
• CA2 Cornu Ammonis Field 2
• CAI Cornu Ammonis Field 1
• Subiculum Sub
• entorhinal cortex EC
• FIG. 21 illustrates differential expression of ASC in neurons and microglia.
• Mouse anti-ASC (A, C and E) cell type binding differs from IC10O's in the hippocampal formation and entorhinal cortex (B, D and F). Region-specific significant changes in cell counts between intermediate cases and low AD cases (E and F). Different morphologies of microglia are present in the CA1-CA2 boundary between low AD (A) and intermediate AD cases (C).
• Stereological analysis of cells stained with mouse anti-ASC was significant higher in intermediate AD cases in the DG, CA2, CAI and Sub regions, whereas IC100 stained mainly neurons in the DG, CA3, CA2 and CAI hippocampal regions of intermediate cases (D) compared with low AD ( B).
• the term "antibody” refers generally and broadly to immunoglobulins (Ig) molecules and immunologically active portions or fragments of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that specifically binds (immunoreacts with) an antigen (e.g., ASC, NLRP1, AIM2, etc.)
• the antibodies provided herein can be polyclonal antibodies, monoclonal antibodies (mAbs), chimeric antibodies, humanized antibodies, anti-idiotypic (anti-ld) antibodies to antibodies that can be labeled in soluble or bound form, as well as active fragments, regions or derivatives thereof.
• the antibodies for use herein may be chimeric, humanized, or human.
• Secreted IgM antibodies are generally composed of five monomers, each monomer composed of two heavy chains and two light chains (as for IgG and IgE antibodies); in this way the IgM molecule has ten antigen binding domains, each again composed of a VH and a VL.
• a cell surface form of IgM also exists and this has two heavy chain/two light chain structure similar to IgG, IgD, and IgE antibodies.
• the fusion protein comprises one or more CDR(s). In another embodiment, the fusion protein comprises CDR H3 (VH CDR3) and/or CDR L3 (VL CDR3).
• a fusion protein contains one or more antibodies and additional amino acid sequence such as for example, a heterologous sequence or a homologous sequence from another region, attached to the N- or C-terminus of the antibody or antibody fragment thereof.
• exemplary heterologous sequences include but are not limited to a "tag" such as a FLAG tag or a 6His tag or an enzyme or a polypeptide which increases the half-life of the antibody in the blood. Tags are well known in the art.
• An antigen binding site can be generally formed by the heavy chain variable region (VH) and the light chain variable region (VL) immunoglobulin domains, with the antigen-binding interface formed by six surface polypeptide loops, termed complementarity determining regions (CDRs). There are three CDRs each in VH (HCDR1, HCDR2, HCDR3) and VL (LCDR1, LCDR2, LCDR3), together with framework regions (FRs).
• VH heavy chain variable region
• VL light chain variable region
• CDRs complementarity determining regions
• the binding domain comprises or consists of an antigen binding site (e.g., comprising a variable heavy chain sequence and variable light chain sequence or three light chain complementary determining regions (CDRs) and three heavy chain CDRs from an antibody placed into alternative framework regions (FRs) (e.g., human FRs optionally comprising one or more amino acid substitutions).
• an antigen binding site e.g., comprising a variable heavy chain sequence and variable light chain sequence or three light chain complementary determining regions (CDRs) and three heavy chain CDRs from an antibody placed into alternative framework regions (FRs) (e.g., human FRs optionally comprising one or more amino acid substitutions).
• FRs alternative framework regions
• the invention also encompasses an Fab' fragment.
• VL and VH are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv).
• single chain Fv Single chain Fv
• scFv molecules may be incorporated into a fusion protein.
• the invention includes a single chain camelid antibody; (viii) bispecific single chain Fv dimers (PCT/U.S. Pat. No.
• Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creatingtwo antigen binding sites (see e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci.
• Fv, scFv or diabody molecules may be stabilized by incorporation of disulfide bridges linking the VH and VL domains (Reiter, Y. et aL, Nature Biotech, 14, 1239-1245, 1996).
• Minibodies comprising a scFv joined to a CH3 domain may also be made (Hu, S. et al., (1996) Cancer Res., 56, 3055-3061).
• the modified Fc region or domain can confer increased thermal stability to the resultant antibody or antibody fragment derived therefrom.
• the increased thermal stability can result in increased serum half-life.
• the Fc region or domain can be modified as described in US20160193295, the contents of which are herein incorporated by reference. As described in US20160193295, the Fc region or domain can be modified to possess a deletion of one or more cysteine residues in the hinge region and substitution with a sulfhydryl-containing residue of one or more CH3-interface amino acids.
• ASC gene or "ASC nucleic acid” is meant a native ASC-encoding nucleic acid sequence, genomic sequences from which ASC cDNA can be transcribed, and/or allelic variants and homologues of the foregoing.
• the terms encompass double-stranded DNA, single-stranded DNA, and
• NLRP1 inflammasome and "NALP1 inflammasome” can mean a multiprotein complex containing NLRP1, ASC, caspase-1, caspase- 11, XIAP, and pannexin-1 for activation of caspase-1 and processing of interleukin-1 ⁇ , interleukin-18 and interleukin-33.
• compositions described herein can be administered from one or more times per day to one or more times per week. The skilled artisan will appreciate that certain factors can influence the dosage and timing required to effectively treat a subject, including but not limited to, the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present.
• “Absent in Melanoma 2” and “AIM2” can mean an expression product of an AIM2 gene or isoforms; 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 number(s) NX 014862, NP004824, XP016858337, XP005245673, AAB81613, BAF84731, AAH10940) and displays a functional activity of AIM2.
• AIM2 accession number(s) NX 014862, NP004824, XP016858337, XP005245673, AAB81613, BAF84731, AAH10940
• NALP2 and NLRP2 mean an expression product of an NALP2 or NLRP2 gene or isoforms; 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 number(s) NP_001167552, NP_001167553, NP_001167554 or NP_060322) and displays a functional activity of NALP2.
• accession number(s) NP_001167552, NP_001167553, NP_001167554 or NP_060322 e.g., accession number(s) NP_001167552, NP_001167553, NP_001167554 or NP_060322
• NLRC4 and I PAF mean an expression product of an NLRC4 or IPAF gene or isoforms; 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 number(s) NP_001186067, NP001186068, NP_001289433 or NP_067032) and displays a functional activity of NLRC4.
• stroke and "ischemic stroke” is meant when blood flow is interrupted to part of the brain or spinal cord.
• traumatic injury to the CNS is meant any insult to the CNS from an external mechanical force, possibly leading to permanent or temporary impairments of CNS function.
• ASC Caspase Activating Recruitment Domain
• the monoclonal antibodies or fragments thereof can bind specifically to an antigenic fragment of ASC that comprises, consists of or consists essentially of an amino acid sequence of SEQ. ID NO. 5.
• the invention contemplates use of the monoclonal antibodies or antibody fragments thereof in a method for treating inflammation in a subject.
• the inflammation can be an innate immune inflammation.
• the inflammation can be an inflammasome-related inflammation.
• the viral infection can be caused by a coronavirus, such as SARS-CoV-2, or an influenza virus, such as influenza A H5N1 (avian influenza) and influenza A H1N1 (swine flu).
• a coronavirus such as SARS-CoV-2
• influenza virus such as influenza A H5N1 (avian influenza) and influenza A H1N1 (swine flu).
• the disease, condition or affliction of the CNS or affecting the CNS can be stroke as well as autoimmune diseases and/or CNS diseases including amyotrophic lateral sclerosis (ALS) Lou Gehrig's, multiple sclerosis (MS), immune dysfunction muscular CNS breakdown, muscular dystrophy (MD), Alzheimer's disease (AD), Parkinson's disease (PD).
• ALS amyotrophic lateral sclerosis
• MS multiple sclerosis
• MD muscular dystrophy
• AD Alzheimer's disease
• PD Parkinson's disease
• the monoclonal antibodies and fragments thereof described herein may be used prophy
• the patients to be treated by the methods of the invention may be experiencing a hyperinflammatory response also known as cytokine storm syndrome.
• Cytokine storm syndrome is a secondary haemophagocytic lymphohistocytosis (sHLH) characterized by fulminant hypercytokemia that results in multiorgan failure (Mehta, McAuley et al. 2020).
• the antibodies, antibody fragments and methods of the invention may be used to treat or prevent hyperinflammation or cytokine storm syndrome associated with a viral infection, including hyperinflammation and cytokine storms associated with SARS-CoV-2 infections.
• the invention also encompasses monoclonal antibodies or antibody fragments thereof that binds specifically to ASC, wherein the antibody or the antibody fragment comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region amino acid sequence comprises HCDR1 of SEQ. ID NO: 6, HCDR2 of SEQ ID NO: 7 and HCDR3 of SEQ ID NO: 8, or a variant thereof having at least one amino acid substitution in HCDR1, HCDR2, and/or HCDR3.
• the invention contemplates use of the monoclonal antibody or antibody fragment thereof in a method for treating inflammation in a subject.
• the inflammation can be an innate immune inflammation.
• the inflammation can be an inflammasome-related inflammation.
• the disease, condition or affliction of the CNS or affecting the CNS can be stroke as well as autoimmune diseases and/or CNS diseases including amyotrophic lateral sclerosis (ALS) Lou Gehrig's, multiple sclerosis (MS), immune dysfunction muscular CNS breakdown, muscular dystrophy (MD), Alzheimer's disease (AD), Parkinson's disease (PD).
• ALS amyotrophic lateral sclerosis
• MS multiple sclerosis
• MD muscular dystrophy
• AD Alzheimer's disease
• PD Parkinson's disease
• Use of the monoclonal antibody or antibody fragment thereof in a method fortreating inflammation can reduce inflammation in the CNS and/or lungs of the patient.
• Use of the monoclonal antibody or antibody fragment thereof in a method for treating inflammation can reduce innate immune or inflammasome-related inflammation in the patient.
• the invention contemplates use of the monoclonal antibody or antibody fragment thereof in a method for treating inflammation in a subject.
• the inflammation can be an innate immune inflammation.
• the inflammation can be an inflammasome-related inflammation.
• the monoclonal antibodies or antibody fragments thereof provided herein can be used in a method for reducing inflammation in a mammal as described in U.S. Pat. No. 8,685,400, the contents of which are herein incorporated by reference in their entirety.
• the inflammation can be in the lungs and/or the CNS.
• the monoclonal antibody or antibody fragment derived therefrom is used to treat MS by administering the monoclonal antibody or antibody fragment derived therefrom to a patient suffering from or suspected of suffering from MS.
• the monoclonal antibody or antibody fragment thereof of this embodiment can be present in a composition such as, for example, a pharmaceutical composition as provided herein.
• the monoclonal antibody or fragment thereof is used in combination with one or more other agents in the methods of treatment provided herein.
• the other agents can be any agent provided herein (e.g., EV uptake inhibitors) and/or antibodies or antibody fragments directed against other inflammasome components (e.g., IL-18, caspase-1, NALP1, AIM2, etc.)
• Examples of conditions that can lead to inflammation in the CNS include a viral infection, a CNS injury (e.g., spinal cord injury (SCI), traumatic brain injury (TBI) or stroke), a neurodegenerative disease, an autoimmune disease (e.g., MS), asthma, chronic obstructive pulmonary disease (COPD), cystic fibrosis, interstitial lung disease or acute respiratory distress syndrome.
• the composition can be administered in a therapeutically effective amount.
• the therapeutically effective amount can be a dose as provided herein.
• the agent can be an extracellular vesicle (EV) uptake inhibitor and/or an antibody or an active fragment thereof as provided herein that binds to a component of an inflammasome or a combination thereof.
• the composition can be administered by any suitable route, e.g., by inhalation, intravenously, intraperitoneally, intranasally or intracerebroventricularly.
• the composition can further include at least one pharmaceutically acceptable carrier or diluent.
• compositions and methods for treating a viral infection or lung inflammation in a subject that is suffering from or is suspected of suffering from a viral infection including ARDS.
• the methods for treating viral-associated lung inflammation provided herein can entail administering a composition (e.g., a pharmaceutical composition) comprising an agent to the subject suffering from or suspected of suffering from lung inflammation.
• a composition e.g., a pharmaceutical composition
• the subject can present with clinical symptoms consistent with viral infection.
• the subject can be diagnosed with any type of virus known in the art.
• the virus can be a coronavirus, such as SARS-CoV-2, MERS coronavirus, Herpes viruses, such as herpes simplex virus (HSV) and cytomegalovirus (CMV), or an influenza virus, such as influenza A H5N1 (avian influenza) and influenza A H1N1 (swine flu).
• a coronavirus such as SARS-CoV-2, MERS coronavirus
• Herpes viruses such as herpes simplex virus (HSV) and cytomegalovirus (CMV)
• an influenza virus such as influenza A H5N1 (avian influenza) and influenza A H1N1 (swine flu).
• the MS diagnosis can be or can have been determined using any method known in the art.
• the subject has been diagnosed as having MS using the methods detail in U.S. 62/560,963, filed Sep. 20, 2017, the contents of which are herein incorporated by reference in their entirety.
• the agent can be a standard of care treatment known in the art for MS or viral infections, an EV uptake inhibitor (e.g., any EV uptake inhibitor from Table 1), an antibody or antibody fragment thereof as provided herein that binds to a component of an inflammasome (e.g., an anti-ASC monoclonal antibody or antibody fragment thereof) or any combination thereof.
• the composition can be administered by any suitable route, e.g., by inhalation, intravenously, intraperitoneally, intranasally or intracerebroventricularly.
• the composition can further include at least one pharmaceutically acceptable carrier or diluent.
• the standard of care treatment can be selected from therapies directed towards modifying disease outcome, managing relapses, managing symptoms or any combination thereof.
• the therapies directed toward modifying disease outcome can be selected from betainterferons, glatiramer acetate, fingolimod, teriflunomide, dimethyl fumarate, mitoxantrone, ocrelizumab, alemtuzumab, daclizumab and natalizumab.
• the method of treating inflammation in the lungs of a mammal comprises administering to the mammal a composition comprising an agent that inhibits inflammasome signaling.
• the mammal can be a patient or subject as provided herein.
• Examples of conditions that can lead to inflammation in the lungs include a central nervous system (CNS) injury (e.g., spinal cord injury (SCI), traumatic brain injury (TBI) or stroke), a neurodegenerative disease, an autoimmune disease (e.g., MS), asthma, chronic obstructive pulmonary disease (COPD), cystic fibrosis, interstitial lung disease or acute respiratory distress syndrome.
• the composition can be administered in a therapeutically effective amount.
• the therapeutically effective amount can be a dose as provided herein.
• the agent can be an extracellular vesicle (EV) uptake inhibitor, an antibody or an active fragment thereof as provided herein that binds to a component of an inflammasome or a combination thereof.
• the composition can be administered by any suitable route, e.g., by inhalation, intravenously, intraperitoneally, intranasally, or intracerebroventricularly.
• the composition can further include at least one pharmaceutically acceptable carrier or diluent.
• administration of an agent in the methods provided herein can result in a reduction in the activity and/or expression level of a component of a mammalian inflammasome in the CNS or lungs of the subject.
• the reduction can be in cells of the lung such as, for example, Type II alveolar cells.
• the reduction can be in comparison to a control.
• the control can be the subject prior to administration of the agent.
• the control can be the activity and/or expression level of the inflammasome component(s) in a subject not administered the agent.
• administration of the agent can result in a reduction in or elimination of acute lung injury (ALI).
• ALI acute lung injury
• the reduction in ALI is evidenced by a reduction in neutrophil infiltration into alveolar and/or interstitial space, reduced or absent alveolar septal thickening or a combination thereof.
• the reduction can be in comparison to a control.
• the control can be ALI in the subject prior to administration of the agent.
• the control can be ALI in a subject suffering from ALI not administered the agent.
• a reduction in the level, abundance, or concentration of one or more inflammasome proteins is indicative of the efficacy of the treatment in the subject.
• the one or more inflammasome proteins measured in the sample obtained following treatment may be the same as or differentthan the inflammasome proteins measured in a sample obtained prior to treatment.
• the inflammasome protein levels may also be used to adjust dosage or frequency of a treatment.
• the inflammasome protein levels can be ascertained using the methods and techniques provided herein or as found in US WO 2019/060516, filed Sep. 20, 2018.
• the agent to be administered in the method of treatments provided herein is an EV uptake inhibitor.
• the EV uptake inhibitor can be a compound, antisense RNA, siRNA, peptide, antibody or an active fragment thereof as provided herein or a combination thereof.
• the compound or peptide can be one or more compounds selected from heparin, a- difluoromethylornithine (DFMO), Enoxaparin, Asialofetuin, Human receptor-associated protein (RAP), RGD (Arg-Gly-Asp) peptide, Cytochalasin D, Cytochalasin B, Ethylenediamine tetraacetic acid (EDTA), Latrunculin A, Latrunculin B, NSC23766, Dynasore, Chlorpromazine, 5-(N-Ethyl-N-isopropyl)amiloride (EIPA), Amiloride, Bafilomycin A Monensin and Chloroquine, Annexin-V, Wortmannin, LY294002, Methyl-
• the agent to be administered is an antibody or an active fragment thereof as provided herein directed against a component of a mammalian inflammasome or an antigen or epitope derived therefrom.
• the agent to be administered is an antisense RNA or siRNA directed against a component of a mammalian inflammasome.
• the inflammasome component can be a component of any inflammasome known in the art, such as, for example, the NAPL1, NALP2, NALP3, NLRC4 or AIM2 inflammasome.
• the antibody specifically binds to ASC or an antigen or epitope derived therefrom.
• an antibody against any other component of a mammalian inflammasome may be used.
• the agent is one or more EV uptake inhibitors in combination with one or more antibodies or active fragments thereof as provided herein that bind a component of an inflammasome.
• the EV uptake inhibitor can be any EV uptake inhibitor as provided herein.
• the antibody that binds a component of an inflammasome can be any antibody that binds any inflammasome component as provided herein.
• the agent administered to a subject suffering from CNS or lung inflammation comprises a heparin (e.g., Enoxaparin) in combination with an antibody that binds a component of the AIM2 inflammasome (e.g., ASC).
• the methods provided herein further entail detecting a level or activity of one or more components of a mammalian inflammasome in a sample from a subject suspected of suffering from CNS or lung inflammation or MS.
• the method of detecting the level or activity entails measuring the level of at least one inflammasome protein (e.g., ASC or AIM2) in the sample obtained from the subject; determining the presence or absence of an elevated level or activity of said at least one inflammasome protein (e.g., ASC or AIM2).
• the level or activity of said at least one inflammasome protein can be enhanced relative to the level of said at least one inflammasome protein in a control sample.
• Antibodies that Bind Specifically to at Least One Component of a Mammalian Inflammasome.
• compositions including an antibody or an active fragment thereof as provided herein that specifically binds to at least one component (e.g., ASC, AIM2) of a mammalian inflammasome (e.g., the AIM2 inflammasome).
• a composition for treating and/or reducing inflammation in the CNS and/or lungs of a mammal can further include at least one pharmaceutically acceptable carrier or diluent.
• Exemplary antibodies directed against components of a mammalian inflammasome for use in the methods herein can be those found in U.S. Pat. No. 8,685,400, the contents of which are herein incorporated by reference in its entirety.
• Exemplary monoclonal antibodies or antibody fragments are also provided herein, such as, for example, the monoclonal antibody or antibody fragment comprising a VH region such that the VH region amino acid sequence comprises HCDR1 of SEQ ID NO: 6, HCDR2 of SEQ ID NO: 7 and HCDR3 of SEQ ID NO: 8, and a VL region such that the VL region amino acid sequence comprises LCDR1 of SEQ ID NO: 12, LCDR2 of SEQ ID NO: 13 and LCDR3 of SEQ ID NO: 14.
• a composition for treating and/or reducing inflammation in the CNS or lungs of a mammal includes an antibody or an active fragment thereof as provided herein that specifically binds to a domain or portion thereof of a mammalian ASC protein such as, for example, a human, mouse or rat ASC protein.
• a mammalian ASC protein such as, for example, a human, mouse or rat ASC protein.
• Any suitable anti-ASC antibody can be used, and several are commercially available. Examples of anti-ASC antibodies for use in the methods herein can be those found in U.S. Pat. No. 8,685,400, the contents of which are herein incorporated by reference in its entirety.
• anti-ASC antibodies for use in the methods provided herein include, but are not limited to 04-147 Anti-ASC, clone 2EI-7 mouse monoclonal antibody from MilliporeSigma, AB3607— Anti-ASC Antibody from Millipore Sigma, orbl94021 Anti-ASC from Biorbyt, LS-C331318-50 Anti-ASC from LifeSpan Biosciences, AF3805 Anti-ASC from R & D Systems, NBP1-78977 Anti-ASC from Novus Biologicals, 600-401-Y67 Anti-ASC from Rockland Immunochemicals, D086-3 Anti-ASC from MBL International, AL177 anti-ASC from Adipogen, monoclonal anti-ASC (clone o93E9) antibody, anti-ASC antibody ( F-9) from Santa Cruz Biotechnology, anti-ASC antibody ( B-3) from Santa Cruz Biotechnology, ASC polyclonal antibody— ADI-905-173 from Enzo Life Sciences, or A161 AntiHu
• the antibody binds to a portion or fragment thereof of a mammalian ASC protein sequence (e.g., human, mouse or rat ASC) located between the PYD and CARD domains.
• a composition for treating and/or reducing inflammation in the CNS and/or lungs of a mammal includes an antibody that specifically binds to a region of rat ASC, e.g., amino acid sequence ALRQTQPYLVTDLEQS (SEQ ID NO:1) (i.e., residues 178-193 of rat ASC, accession number BAC43754).
• an antibody that binds to an ASC domain or fragment thereof as described herein inhibits ASC activity in the CNS of a mammal suffering or suspected of suffering from a CNS injury or disorder.
• CNS injuries or disorders can include TBI, SCI, stroke, amyotrophic lateral sclerosis (ALS) Lou Gehrig's, multiple sclerosis (MS), immune dysfunction muscular CNS breakdown, muscular dystrophy (MD), Alzheimer's disease (AD), Parkinson's disease (PD).
• the invention provides antibodies and antibody fragments that bind specifically to ASC and that comprise one or more amino acid sequences shown Table 2.
• isolated nucleic acid molecules encoding the monoclonal antibodies or the antibody fragments thereof that comprise nucleic acid sequences shown in Table 2.
• expression vectors comprising the nucleic acid molecules of Table 2.
• the expression vector can comprise heavy chain or light chain constant regions.
• 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 lgG4 (S241P) heavy and kappa light chain.
• the nucleic acid molecule for the heavy or light chain can be operatively linked to regulatory sequences suitable for expression of the nucleic acid segments in a host cell.
• a monoclonal antibody or an antibody fragment thereof that binds specifically ASC wherein the antibody or the antibody fragment thereof comprises a heavy chain variable (VH) region and a light or kappa chain variable (VL) region, wherein the VH region amino acid sequence comprises SEQ ID NO: 18, 19, 20, 21, 22, or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 18, 19, 20, 21, or 22.
• VH region amino acid sequence comprises SEQ ID NO: 18, 19, 20, 21, 22, or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 18, 19, 20, 21, or 22.
• the monoclonal antibody or antibody fragment thereof in a method for treating inflammation in a subject.
• the inflammation can be an innate immune inflammation.
• the inflammation can be an inflammasome-related inflammation.
• the inflammation can be in the lungs and/or the CNS.
• the inflammation in the lungs and/or the CNS can be the result of a viral infection, an injury (e.g., traumatic brain injury (TBI) or spinal cord injury (SCI)) or disease, condition or affliction of the CNS or affecting the CNS.
• TBI traumatic brain injury
• SCI spinal cord injury
• the disease, condition or affliction of the CNS or affecting the CNS can be stroke as well as autoimmune diseases and/or CNS diseases including amyotrophic lateral sclerosis (ALS) Lou Gehrig's, multiple sclerosis (MS), immune dysfunction muscular CNS breakdown, muscular dystrophy (MD), Alzheimer's disease (AD), Parkinson's disease (PD).
• ALS amyotrophic lateral sclerosis
• MS multiple sclerosis
• MD muscular dystrophy
• AD Alzheimer's disease
• PD Parkinson's disease
• Use of the monoclonal antibody or antibody fragment thereof in a method for treating inflammation can reduce inflammation in the CNS and/or lungs of the patient. The reduction can be as compared to a control (e.g., untreated patient and/or patient prior to treatment).
• the monoclonal antibody or antibody fragment derived therefrom is used to treat MS by administering the monoclonal antibody or antibody fragment derived therefrom to a patient suffering from or suspected of suffering from MS.
• the monoclonal antibody or the antibody fragment thereof of this embodiment is present in a composition.
• the composition can be a pharmaceutical composition as provided herein.
• a monoclonal antibody or an antibody fragment thereof that binds specifically ASC wherein the antibody or the antibody fragment thereof comprises a heavy chain variable (VH) region and a light or kappa chain variable (VL) region, wherein the VL region amino acid sequence comprises SEQ ID NO: 28, 29, 30, 31, or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ. ID NO: 28, 29, 30 or 31.
• VH heavy chain variable
• VL light or kappa chain variable
• the VL region amino acid sequence comprises SEQ ID NO: 28, 29, 30, 31, or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ. ID NO: 28, 29, 30 or 31.
• the monoclonal antibody or antibody fragment thereof in a method for treating inflammation in a subject.
• the inflammation can be an innate immune inflammation.
• the inflammation can be an inflammasome-related inflammation.
• the inflammation can be an innate immune inflammation.
• the inflammation can be an inflammasome- related inflammation.
• the inflammation can be in the lungs and/or the CNS.
• a monoclonal antibody or an antibody fragment thereof that binds specifically ASC wherein the antibody or the antibody fragment thereof comprises a heavy chain variable (VH) region and a light or kappa chain variable (VL) region, wherein the VH region amino acid sequence comprises SEQ ID NO: 18, or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 18; and wherein the VL region amino acid sequence comprises SEQ ID NO: 28 or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 28.
• VH heavy chain variable
• VL light or kappa chain variable
• the inflammation can be an innate immune inflammation.
• the inflammation can be an inflammasome-related inflammation.
• the inflammation can be in the lungs and/or the CNS.
• the inflammation in the lungs and/or the CNS can be the result of a viral infection, an injury (e.g., traumatic brain injury (TBI) or spinal cord injury (SCI)) or disease, condition or affliction of the CNS or affecting the CNS.
• TBI traumatic brain injury
• SCI spinal cord injury
• the disease, condition or affliction of the CNS or affecting the CNS can be stroke as well as autoimmune diseases and/or CNS diseases including amyotrophic lateral sclerosis (ALS) Lou Gehrig's, multiple sclerosis (MS), immune dysfunction muscular CNS breakdown, muscular dystrophy (MD), Alzheimer's disease (AD), Parkinson's disease (PD).
• ALS amyotrophic lateral sclerosis
• MS multiple sclerosis
• MD muscular dystrophy
• AD Alzheimer's disease
• PD Parkinson's disease
• Use of the monoclonal antibody or antibody fragment thereof in a method for treating inflammation can reduce inflammation in the CNS and/or lungs of the patient. The reduction can be as compared to a control (e.g., untreated patient and/or patient prior to treatment).
• the monoclonal antibody or antibody fragment derived therefrom is used to treat MS by administering the monoclonal antibody or antibody fragment derived therefrom to a patient suffering from or suspected of suffering from MS.
• the monoclonal antibody or the antibody fragment thereof of this embodiment is present in a composition.
• the composition can be a pharmaceutical composition as provided herein.
• a monoclonal antibody or an antibody fragment thereof that binds specifically ASC wherein the antibody or the antibody fragment thereof comprises a heavy chain variable (VH) region and a light or kappa chain variable (VL) region, wherein the VH region amino acid sequence comprises SEQ ID NO: 18, or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 18; and wherein the VL region amino acid sequence comprises SEQ ID NO: 30 or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 30.
• VH heavy chain variable
• VL light or kappa chain variable
• the monoclonal antibody or antibody fragment derived therefrom is used to treat MS by administering the monoclonal antibody or antibody fragment derived therefrom to a patient suffering from or suspected of suffering from MS.
• the monoclonal antibody or the antibody fragment thereof of this embodiment is present in a composition.
• the composition can be a pharmaceutical composition as provided herein.
• the monoclonal antibody or antibody fragment derived therefrom is used to treat MS by administering the monoclonal antibody or antibody fragment derived therefrom to a patient suffering from or suspected of suffering from MS.
• the monoclonal antibody or the antibody fragment thereof of this embodiment is present in a composition.
• the composition can be a pharmaceutical composition as provided herein.
• Use of the monoclonal antibody or antibody fragment thereof in a method for treating inflammation can reduce inflammation in the CNS and/or lungs of the patient. The reduction can be as compared to a control (e.g., untreated patient and/or patient prior to treatment).
• the monoclonal antibody or antibody fragment derived therefrom is used to treat MS by administering the monoclonal antibody or antibody fragment derived therefrom to a patient suffering from or suspected of suffering from MS.
• the monoclonal antibody or the antibody fragment thereof of this embodiment is present in a composition.
• the composition can be a pharmaceutical composition as provided herein.
• a monoclonal antibody or an antibody fragment thereof that binds specifically ASC wherein the antibody or the antibody fragment thereof comprises a heavy chain variable (VH) region and a light or kappa chain variable (VL) region, wherein the VH region amino acid sequence comprises SEQ ID NO: 20, or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 20; and wherein the VL region amino acid sequence comprises SEQ ID NO: 28 or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 28.
• VH region amino acid sequence comprises SEQ ID NO: 20
• VL region amino acid sequence comprises SEQ ID NO: 28 or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 28.
• the monoclonal antibody or antibody fragment derived therefrom is used to treat MS by administering the monoclonal antibody or antibody fragment derived therefrom to a patient suffering from or suspected of suffering from MS.
• the monoclonal antibody or the antibody fragment thereof of this embodiment is present in a composition.
• the composition can be a pharmaceutical composition as provided herein.
• a monoclonal antibody or an antibody fragment thereof that binds specifically ASC wherein the antibody or the antibody fragment thereof comprises a heavy chain variable (VH) region and a light or kappa chain variable (VL) region, wherein the VH region amino acid sequence comprises SEQ ID NO: 20, or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ. ID NO: 20; and wherein the VL region amino acid sequence comprises SEQ ID NO: 29 or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 29.
• VH heavy chain variable
• VL light or kappa chain variable
• the inflammation can be an innate immune inflammation.
• the inflammation can be an inflammasome-related inflammation.
• the inflammation can be in the lungs and/or the CNS.
• the inflammation in the lungs and/or the CNS can be the result of a viral infection, an injury (e.g., traumatic brain injury (TBI) or spinal cord injury (SCI)) or disease, condition or affliction of the CNS or affecting the CNS.
• TBI traumatic brain injury
• SCI spinal cord injury
• the monoclonal antibody or antibody fragment derived therefrom is used to treat MS by administering the monoclonal antibody or antibody fragment derived therefrom to a patient suffering from or suspected of suffering from MS.
• the monoclonal antibody or the antibody fragment thereof of this embodiment is present in a composition.
• the composition can be a pharmaceutical composition as provided herein.
• a monoclonal antibody or an antibody fragment thereof that binds specifically ASC wherein the antibody or the antibody fragment thereof comprises a heavy chain variable (VH) region and a light or kappa chain variable (VL) region, wherein the VH region amino acid sequence comprises SEQ ID NO: 20, or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ. ID NO: 20; and wherein the VL region amino acid sequence comprises SEQ ID NO: 30 or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 30.
• VH heavy chain variable
• VL light or kappa chain variable
• the inflammation can be an innate immune inflammation.
• the inflammation can be an inflammasome-related inflammation.
• the inflammation can be in the lungs and/or the CNS.
• the inflammation in the lungs and/or the CNS can be the result of a viral infection, an injury (e.g., traumatic brain injury (TBI) or spinal cord injury (SCI)) or disease, condition or affliction of the CNS or affecting the CNS.
• TBI traumatic brain injury
• SCI spinal cord injury
• the disease, condition or affliction of the CNS or affecting the CNS can be stroke as well as autoimmune diseases and/or CNS diseases including amyotrophic lateral sclerosis (ALS) Lou Gehrig's, multiple sclerosis (MS), immune dysfunction muscular CNS breakdown, muscular dystrophy (MD), Alzheimer's disease (AD), Parkinson's disease (PD).
• ALS amyotrophic lateral sclerosis
• MS multiple sclerosis
• MD muscular dystrophy
• AD Alzheimer's disease
• PD Parkinson's disease
• Use of the monoclonal antibody or antibody fragment thereof in a method for treating inflammation can reduce inflammation in the CNS and/or lungs of the patient. The reduction can be as compared to a control (e.g., untreated patient and/or patient prior to treatment).
• a monoclonal antibody or an antibody fragment thereof that binds specifically ASC wherein the antibody or the antibody fragment thereof comprises a heavy chain variable (VH) region and a light or kappa chain variable (VL) region, wherein the VH region amino acid sequence comprises SEQ ID NO: 20, or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 20; and wherein the VL region amino acid sequence comprises SEQ ID NO: 31 or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 31.
• VH heavy chain variable
• VL light or kappa chain variable
• the inflammation can be an innate immune inflammation.
• the inflammation can be an inflammasome-related inflammation.
• the inflammation can be in the lungs and/or the CNS.
• the inflammation in the lungs and/or the CNS can be the result of a viral infection, an injury (e.g., traumatic brain injury (TBI) or spinal cord injury (SCI)) or disease, condition or affliction of the CNS or affecting the CNS.
• TBI traumatic brain injury
• SCI spinal cord injury
• the disease, condition or affliction of the CNS or affecting the CNS can be stroke as well as autoimmune diseases and/or CNS diseases including amyotrophic lateral sclerosis (ALS) Lou Gehrig's, multiple sclerosis (MS), immune dysfunction muscular CNS breakdown, muscular dystrophy (MD), Alzheimer's disease (AD), Parkinson's disease (PD).
• ALS amyotrophic lateral sclerosis
• MS multiple sclerosis
• MD muscular dystrophy
• AD Alzheimer's disease
• PD Parkinson's disease
• Use of the monoclonal antibody or antibody fragment thereof in a method for treating inflammation can reduce inflammation in the CNS and/or lungs of the patient. The reduction can be as compared to a control (e.g., untreated patient and/or patient prior to treatment).
• the monoclonal antibody or antibody fragment derived therefrom is used to treat MS by administering the monoclonal antibody or antibody fragment derived therefrom to a patient suffering from or suspected of suffering from MS.
• the monoclonal antibody or the antibody fragment thereof of this embodiment is present in a composition.
• the composition can be a pharmaceutical composition as provided herein.
• the disease, condition or affliction of the CNS or affecting the CNS can be stroke as well as autoimmune diseases and/or CNS diseases including amyotrophic lateral sclerosis (ALS) Lou Gehrig's, multiple sclerosis (MS), immune dysfunction muscular CNS breakdown, muscular dystrophy (MD), Alzheimer's disease (AD), Parkinson's disease (PD).
• ALS amyotrophic lateral sclerosis
• MS multiple sclerosis
• MD muscular dystrophy
• AD Alzheimer's disease
• PD Parkinson's disease
• Use of the monoclonal antibody or antibody fragment thereof in a method for treating inflammation can reduce inflammation in the CNS and/or lungs of the patient. The reduction can be as compared to a control (e.g., untreated patient and/or patient prior to treatment).
• the monoclonal antibody or antibody fragment derived therefrom is used to treat MS by administering the monoclonal antibody or antibody fragment derived therefrom to a patient suffering from or suspected of suffering from MS.
• the monoclonal antibody or the antibody fragment thereof of this embodiment is present in a composition.
• the composition can be a pharmaceutical composition as provided herein.
• the disease, condition or affliction of the CNS or affecting the CNS can be stroke as well as autoimmune diseases and/or CNS diseases including amyotrophic lateral sclerosis (ALS) Lou Gehrig's, multiple sclerosis (MS), immune dysfunction muscular CNS breakdown, muscular dystrophy (MD), Alzheimer's disease (AD), Parkinson's disease (PD).
• ALS amyotrophic lateral sclerosis
• MS multiple sclerosis
• MD muscular dystrophy
• AD Alzheimer's disease
• PD Parkinson's disease
• Use of the monoclonal antibody or antibody fragment thereof in a method for treating inflammation can reduce inflammation in the CNS and/or lungs of the patient. The reduction can be as compared to a control (e.g., untreated patient and/or patient prior to treatment).
• the disease, condition or affliction of the CNS or affecting the CNS can be stroke as well as autoimmune diseases and/or CNS diseases including amyotrophic lateral sclerosis (ALS) Lou Gehrig's, multiple sclerosis (MS), immune dysfunction muscular CNS breakdown, muscular dystrophy (MD), Alzheimer's disease (AD), Parkinson's disease (PD).
• ALS amyotrophic lateral sclerosis
• MS multiple sclerosis
• MD muscular dystrophy
• AD Alzheimer's disease
• PD Parkinson's disease
• Use of the monoclonal antibody or antibody fragment thereof in a method for treating inflammation can reduce inflammation in the CNS and/or lungs of the patient. The reduction can be as compared to a control (e.g., untreated patient and/or patient prior to treatment).
• a monoclonal antibody or an antibody fragment thereof that binds specifically ASC wherein the antibody or the antibody fragment thereof comprises a heavy chain variable (VH) region and a light or kappa chain variable (VL) region, wherein the VH region amino acid sequence comprises SEQ ID NO: 22, or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 22; and wherein the VL region amino acid sequence comprises SEQ ID NO: 28 or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 28.
• VH heavy chain variable
• VL light or kappa chain variable
• the inflammation can be an innate immune inflammation.
• the inflammation can be an inflammasome-related inflammation.
• the inflammation can be in the lungs and/or the CNS.
• the inflammation in the lungs and/or the CNS can be the result of a viral infection, an injury (e.g., traumatic brain injury (TBI) or spinal cord injury (SCI)) or disease, condition or affliction of the CNS or affecting the CNS.
• TBI traumatic brain injury
• SCI spinal cord injury
• the disease, condition or affliction of the CNS or affecting the CNS can be stroke as well as autoimmune diseases and/or CNS diseases including amyotrophic lateral sclerosis (ALS) Lou Gehrig's, multiple sclerosis (MS), immune dysfunction muscular CNS breakdown, muscular dystrophy (MD), Alzheimer's disease (AD), Parkinson's disease (PD).
• ALS amyotrophic lateral sclerosis
• MS multiple sclerosis
• MD muscular dystrophy
• AD Alzheimer's disease
• PD Parkinson's disease
• Use of the monoclonal antibody or antibody fragment thereof in a method for treating inflammation can reduce inflammation in the CNS and/or lungs of the patient. The reduction can be as compared to a control (e.g., untreated patient and/or patient prior to treatment).
• the monoclonal antibody or antibody fragment derived therefrom is used to treat MS by administering the monoclonal antibody or antibody fragment derived therefrom to a patient suffering from or suspected of suffering from MS.
• the monoclonal antibody or the antibody fragment thereof of this embodiment is present in a composition.
• the composition can be a pharmaceutical composition as provided herein.
• a monoclonal antibody or an antibody fragment thereof that binds specifically ASC wherein the antibody or the antibody fragment thereof comprises a heavy chain variable (VH) region and a light or kappa chain variable (VL) region, wherein the VH region amino acid sequence comprises SEQ ID NO: 22, or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ. ID NO: 22; and wherein the VL region amino acid sequence comprises SEQ ID NO: 29 or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 29.
• VH heavy chain variable
• VL light or kappa chain variable
• the inflammation can be an innate immune inflammation.
• the inflammation can be an inflammasome-related inflammation.
• the inflammation can be in the lungs and/or the CNS.
• the inflammation in the lungs and/or the CNS can be the result of a viral infection, an injury (e.g., traumatic brain injury (TBI) or spinal cord injury (SCI)) or disease, condition or affliction of the CNS or affecting the CNS.
• TBI traumatic brain injury
• SCI spinal cord injury
• the disease, condition or affliction of the CNS or affecting the CNS can be stroke as well as autoimmune diseases and/or CNS diseases including amyotrophic lateral sclerosis (ALS) Lou Gehrig's, multiple sclerosis (MS), immune dysfunction muscular CNS breakdown, muscular dystrophy (MD), Alzheimer's disease (AD), Parkinson's disease (PD).
• ALS amyotrophic lateral sclerosis
• MS multiple sclerosis
• MD muscular dystrophy
• AD Alzheimer's disease
• PD Parkinson's disease
• Use of the monoclonal antibody or antibody fragment thereof in a method for treating inflammation can reduce inflammation in the CNS and/or lungs of the patient. The reduction can be as compared to a control (e.g., untreated patient and/or patient prior to treatment).
• the monoclonal antibody or antibody fragment derived therefrom is used to treat MS by administering the monoclonal antibody or antibody fragment derived therefrom to a patient suffering from or suspected of suffering from MS.
• the monoclonal antibody or the antibody fragment thereof of this embodiment is present in a composition.
• the composition can be a pharmaceutical composition as provided herein.
• a monoclonal antibody or an antibody fragment thereof that binds specifically ASC wherein the antibody or the antibody fragment thereof comprises a heavy chain variable (VH) region and a light or kappa chain variable (VL) region, wherein the VH region amino acid sequence comprises SEQ ID NO: 22, or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ. ID NO: 22; and wherein the VL region amino acid sequence comprises SEQ ID NO: 30 or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 30.
• VH heavy chain variable
• VL light or kappa chain variable
• an antibody provided herein comprises a polypeptide having one or more amino acid substitutions, deletions or insertions.
• an anti-ASC monoclonal antibody or an ASC binding antibody fragment comprises a polypeptide having one or more amino acid substitutions, deletions or insertions as compared to a polypeptide having an amino acid sequence of one or more of SEQ. ID NOs: 6-8, 12-14, 18-22 or 28-31.
• An antibody provided herein may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid substitutions, deletions or insertions.
• an anti-ASC monoclonal antibody or an ASC binding antibody fragment comprising a polypeptide having one or more amino acid substitutions, deletions or insertions as compared to a polypeptide having an amino acid sequence of one or more of SEQ ID NOs: 6-8, 12-14, 18-22 or 28-31 binds ASC protein by utilizing routine, art-recognized methods including, but not limited to, ELISAs, Western blots, phage display, etc.
• the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes).
• the length of a 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.
• amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared.
• a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid "identity” is equivalent to amino acid or nucleic acid "homology”).
• the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
• the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6.
• One set of parameters (and the one that can be used if the practitioner is uncertain about what parameters should be applied to determine if a molecule is within a sequence identity or homology limitation of the invention) is a BLOSUM 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.
• an antibody is a monoclonal antibody.
• an antibody is a polyclonal antibody.
• the term "monoclonal antibody” refers to a population of antibody molecules that contain only one species of an antigen binding site capable of immunoreacting with a particular epitope of an antigen. A monoclonal antibody composition thus typically displays a single binding affinity for a particular protein with which it immunoreacts.
• an antibody of the invention is humanized, chimeric or human.
• an antibody of the invention is a humanized antibody.
• Humanized antibody refers to an antibody that has been engineered to comprise one or more human framework regions in the variable region together with non-human (e.g., mouse, rat, or hamster) complementarity-determining regions (CDRs) of the heavy and/or light chain.
• CDRs complementarity-determining regions
• a humanized antibody comprises sequences that are entirely human except for the CDR regions.
• Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues.
• the humanized antibody may comprise residues that are found neither in the human form of the antibody nor in the imported CDR or framework sequences but are included to further refine and optimize antibody performance.
• the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence.
• the FR region can be modified in any manner known in the art and/or provided herein. The modifications can confer desirable properties such as increased half-life and/or improved expression in host cells. In one embodiment, the FR region(s) can be modified or mutated as described in US20150232557, which is herein incorporated by reference.
• humanized antibodies can have one or more CDRs (CDR LI, CDR L2, CDR L3, CDR Hl, CDR H2, or CDR H3) which are altered with respect to the original antibody, which are also termed one or more CDRs "derived from” one or more CDRs from the original antibody.
• the humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin.
• Humanized antibodies are typically less immunogenic to humans, relative to non-humanized antibodies, and thus offer therapeutic benefits in certain situations.
• the antibody constant region can be engineered such that it is immunologically inert (e.g., does not trigger complement lysis).
• PCT Publication No. PCT/GB99/01441; UK Patent Application No. 9809951.8 each of which is incorporated herein by reference in its entirety.
• Those skilled in the art will be aware of humanized antibodies and will also be aware of suitable techniques for their generation. See for example, Hwang, W. Y. K., et al., Methods 36:35, 2005; Queen et al., Proc. Natl. Acad. Sci.
• an anti-ASC antibody or anti-ASC antigen-binding fragment of the invention may comprise a VH region amino acid sequence that comprises HCDR1 of SEQ ID NO: 6, HCDR2 of SEQ ID NO: 7 and HCDR3 of SEQ ID NO: 8; and a VL region amino acid sequence that comprises 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.
• an antibody of the invention is a chimeric antibody and binds, specifically ASC.
• the anti-ASC chimeric antibody reduces the activity of ASC.
• "Chimeric antibody” as the term is used herein refers to an antibody that has been engineered to comprise at least one human constant region.
• one or all the variable regions of the light chain(s) and/or one or all the variable regions of the heavy chain(s) of a mouse antibody may each be joined to a human constant region, such as, without limitation an IgGl human constant region.
• Chimeric antibodies are typically less immunogenic to humans, relative to non-chimeric antibodies, and thus offer therapeutic benefits in certain situations.
• an antibody or antigen-binding fragment of the invention may comprise a VH region comprising SEQ ID NO: 22; a VL region comprising SEQ ID NO: 31, and a human constant region.
• immunological binding refers to the non-covalent interactions of the type which occur between an immunoglobulin molecule (e.g., antibody) and an antigen for which the immunoglobulin is specific.
• the strength, or affinity of immunological binding interactions can be expressed in terms of the dissociation constant (Kd) of the interaction, wherein a smaller Kd represents a greater affinity.
• Immunological binding properties of selected polypeptides can be quantified using methods well known in the art.
• One such method entails measuring the rates of antigen-binding site/antigen complex formation and dissociation, wherein those rates depend on the concentrations of the complex partners, the affinity of the interaction, and geometric parameters that equally influence the rate in both directions.
• both the "on rate constant” (K on ) and the “off rate constant” (K off ) can be determined by calculation of the concentrations and the actual rates of association and dissociation.
• K on the "on rate constant”
• K off K off
• the ratio of K off / K on enables the cancellation of all parameters not related to affinity and is equal to the dissociation constant Kd. (See, generally, Davies et al. (1990) Annual Rev Biochem 59:439-473).
• Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, GILD and any subclass thereof.
• a hybridoma producing a monoclonal antibody of the present invention may be cultivated in vitro, in situ or in vivo.
• a hybridoma producing an anti-ASC monoclonal antibody of the present disclosure is the ICCN1.OH hybridoma.
• Toxicity and therapeutic efficacy of the compositions utilized in methods of the invention can be determined by standard pharmaceutical procedures, using either cells in culture or experimental animals to determine the LD 50 (the dose lethal to 50% of the population).
• the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD 50 /ED 50
• the compositions provided herein exhibit large therapeutic indices. While those that exhibit toxic side effects may be used, care should be taken to design a delivery system that minimizes the potential damage of such side effects.
• the dosage of compositions provided herein lies within a range that includes an ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
• dosage for any one subject depends on many factors, including the subject's size, body surface area, age, the particular composition to be administered, time and route of administration, general health, and other drugs being administered concurrently.
• HMGB1-RAGE ligand receptor pathway serves as central transduction mechanism for pulmonary dysfunction after TBI.
• HMGB1 induces AIM2 inflammasome activation.
• mice were 8-12 weeks and 24 to 32 grams. Mice were prospectively randomized to experimental groups (sham, 4 h, 24) for TBI, experimental groups (naive, sham-saline, untreated, enoxaparin, anti-ASC) for adoptive transfer and treatment. For TBI experiment-groups, sham animals underwent surgical procedures but were not injured.
• mice were housed in the viral antigen free (VAF) animal facility at the Lois Pope Life Center at the University of Miami on 12-hour light/dark cycles and food and water were supplied ad libitum. The facility conducts husbandry procedures twice a week and checks on the conditions of the animals daily. Animals were observed post-op, where they were kept on a heating pad and body temperature was controlled with a rectal probe where it was maintained at 37° C., in our operation room and then transferred to the animal quarters.
• VAF viral antigen free
• mice lung tissue lysates were filtered through a 5 ⁇ m low-binding polyvinylidene difluoride (PVDF) membrane (Millipore). After filtration, the supernatant was centrifuged at 2,700xg for 8 minutes. The pellet was resuspended in 40 pl of 3[(3-cholamidopropyl) dimethylammonio]- propanesulfonic acid (CHAPS) buffer (20 mmol/L HEPES-KOH, pH 7.5, 5 mmol/L MgCI2, 0.5 mmol/L EGTA, 0.1 mmol/L phenylmethylsulfonyl fluoride, protease inhibitor cocktail, and 0.1% CHAPS).
• CHAPS 3[(3-cholamidopropyl) dimethylammonio]- propanesulfonic acid
• the pyroptosome was pelleted by centrifugation at 2,700xg for 8 minutes. The pellet was then resuspended and incubated in 27.8 pl of CHAPS buffer with 2.2 ⁇ l of disuccinimidyl substrate for 30 minutes at room temperature to cross-link ASC dimers. Lastly, an equal amount of 2x Laemmli buffer was added, and proteins were analyzed by immunoblotting using commercially available antibodies to ASC and Gasdermin D (GSD).
• GSD Gasdermin D
• Nuclear and Cytoplasmic fractions were extracted using the NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific) according to manufacturer instructions. Briefly, mice lung tissue samples were cut into 20-100 mg pieces and centrifuged at 500xg for 5 minutes. Tissue pieces were then homogenized with the Cytoplasmic Extraction Reagent and centrifuged at 16,000xg for 5 minutes. Then the supernatant (cellular extract) was removed and the pellet was centrifuged with Nuclear Extraction Reagent (Thermo Scientific) at 16,000xg for 10 minutes. This supernatant corresponded to the nuclear fraction, which was removed and stored at -80° C.
• Lung and brain tissue samples were snap frozen in liquid nitrogen and stored in -80° C.
• 2-mm sections of right lower lung and right cortical tissue were homogenized in extraction buffer containing protease and phosphatase inhibitor cocktail (Sigma, St Louis, Mo., USA) and resolved in 4-20% Tris-TGX Criterion precast gels (Bio-Rad, Hercules, Calif., USA) as described in de Rivero Vaccari et al. 2015 using antibodies to caspase-1 (Novus Biologicals), ASC (Santa Cruz), IL-1 (Cell Signaling), IL-18 (Abeam) AIM2 (Santa Cruz) and HMGB1 (Millipore). Quantification of band density was performed using Image Lab and all data were normalized to 0-actin.
• Tissue sections were deparaffinized in xylene and then rehydrated using ethanol and Tris buffer saline. Immunohistochemical procedures were then carried out for double staining as previously described. Sections were incubated overnight at 4° C. with antibodies against Caspase-1 and ASC (Millipore), AIM2 (Santa Cruz), HMGB1 (Millipore) and SPC (Millipore). Immunostained lung sections of sham, 4-hour, and 24-hour mice were examined with a Zeiss laser scanning confocal microscope (Zeiss, Inc., Thornwood, N.Y., USA). Lung sections were analyzed by individuals who were blinded to the groups.
• EV were isolated from serum from TBI-injured mice and injury mice using the Total Exosome Isolation solution according to manufacturer's instructions (Invitrogen). Briefly, 100 pl of each sample were centrifuged at 2000xg for 30 minutes. The supernatant was then incubated with 20 pl of Total Exosome Isolation (TEI) reagent for 30 minutes at 4° C. followed by centrifugation at 10,000xg for 10 minutes at room temperature. Supernatants were discarded and the pellet was resuspended in 100 pl of PBS. EV were characterized by the expression of CD81 and by Nanosight tracking analysis (FIG. 6).
• TEI Total Exosome Isolation
• the following groups were used: 1) the naive group received no treatment, 2) the sham saline group was used as a negative control and underwent jugular vein injection of only saline, 3) the untreated group received EV from TBI mice without any treatment and was used as a positive control, 4) the ENOX group received EV from TBI mice and Enoxaparin, and 5) the Anti-ASC group received EV from TBI mice and Anti-ASC. The order of treatment was randomized. Lung and brain tissues were collected 24 hours after injection for analysis. It should be noted that the anti-ASC antibody used in the treatment experiments was a humanized monoclonal antibody against ASC and recognizes murine, human and swine ASC.
• Lung tissue sections were stained by a standard hematoxylin and eosin method for histology, morphometry and ALI scoring. Lung sections were scored by a blinded pathologist using the Lung Injury Scoring System from the American Thoracic Society Workshop Report. Twenty random high-power fields were chosen for scoring. Criteria for ALI scoring was based on number of neutrophils in the alveolar space, interstitial space, hyaline membranes, proteinaceous debris filling the airspaces and alveolar septal thickening. Based on these criteria a score between 0 (no injury) and 1 (severe injury) was given.
• FIGS. 1H, IL shows that HMGB1 expression increased at 4 and 24 hours after TBI, indicating that the Al M2 inflammasome and H MGB1 play a role in the inflammatory response in the lungs post-TBI.
• TBI Increases Immunoreactivity of Inflammasome Proteins in Type II Alveolar Epithelial Cells
• TBI may lead to capillary leak, resulting in increased vascular permeability and damage to specialized alveolar epithelial cells, called type II pneumocytes.
• immunohistochemical analysis was performed in lung sections of sham, 4 hour, and 24 hour injured animals.
• Type II alveolar epithelial cells are known to be the main type of lung cells injured in ALL Lung sections were stained with antibodies against AIM2, caspase-1, and ASC (green) and co-stained with Pro-surfactant protein C (Pro-SPC, red), a marker of type II epithelial cells, and DAPI nuclear staining (blue).
• Pro-surfactant protein C Pro-surfactant protein C
• FIG. 2A-2C active caspase-1 (FIG. 2A), ASC (FIG. 2B), as well as AIM2 (FIG. 2C) are present in SPC-positive cells (arrow). Immunoreactivity of these inflammasome proteins increased after TBI.
• ALI can be characterized by inflammatory processes, which lead to alveolar and interstitial edema as well as infiltration of inflammatory cells into the alveolar space.
• Histopathological analysis of lung tissue (FIG. 5A) indicate that severe TBI causes substantial changes in the lung architecture and morphology at 4 and 24 hours after injury. Sham animals showed a normal alveolar morphology, whereas injured animals showed acute changes in alveolar edema but decreased slightly by 24 hours after injury (long arrows).
• neutrophil infiltration arrow heads
• changes in morphology of alveolar capillary membranes *
• Injured animals showed signs of interstitial edema, which was more pronounced at 4 hours post-injury, but was still evident at 24 hours post injury (short arrows). Lastly, injured animals also showed evidence of thickening of the interstitial area and the alveolar septum (pound, #).
• exosome uptake blockade was attempted by treatment with either Enoxaparin or a monoclonal antibody against ASC after adoptive transfer of EV from injured to naive mice. Negative control animals received saline and positive control animals received no treatment.
• FIG. 8A-8F Caspase-1 (FIG. 8A, 8B), ASC (FIG. 8A, 8C), TL-1 ⁇ (FIG. 8A, 8D), AIM2 (FIG. 8A, 8E), and HMGB1 (FIG.
• this Example showed that AIM2 inflammasome signaling plays a central role in the pathomechanism of lung injury after TBI and demonstrates a mechanism of TBI-induced ALI involving EV-mediated inflammasome signaling.
• EV-mediated inflammasome signaling can play a central role involving a Neuronal-Respiratory-Inflammatory Axis. Therefore, targeting this axis with antibodies against inflammasome proteins or drugs that block EV uptake may provide a therapeutic approach in Neurotrauma-induced ALI in all areas of critical care medicine.
• the disclosed therapeutic strategies may be useful for the treatment of inflammatory diseases of the lung in general.
• HBSS w/o cold Hanks' Balanced Salt Solution without Mg 2+ and Ca 2+
• Samples were manually dissociated into single cell suspensions through a 70-um strainer and washed in HBSS w/o.
• the spleen samples were spun at 1200 r ⁇ m for 10 min at 4° C., supernatants were removed, and red blood cells (RBCs) lysed in 2 ml RBC lysis buffer (eBioscience) according to the manufacturer's instructions. Spleen cells were then resuspended in PBS.
• RBCs red blood cells
• IC-100 significantly improved functional recovery when used at the doses of 30 and 45 mg/Kg, with a robust reduction of the clinical disease scores throughout the duration of the experiment (FIG. 12A).
• Treatment reduced the average peak clinical scores (FIG. 12B) as well as the overall severity of EAE measured as a reduction in the cumulative disease index (CDI) (FIG. 12C).
• Mice treated with 30 and 45 mg/Kg IC-100 also showed a tendency to a delayed disease onset (FIG. 12D). No differences in the day the mice reached their peak disease score were observed (FIG. 12E).
• the murine coronavirus, mouse hepatitis virus strain A59 (MHV-A59) model is used to study the pathogenesis and the response of the host to coronavirus infections.
• This model virus has been shown to infect the lungs, liver, spleen, brain and spinal cord, among others.
• this model also shows demyelination consistent with a multiple sclerosis-like phenotype (Weiss and Leibowitz 2011).
• IC100 bind to ASC to inhibit inflammasome activation as a result of viral infection, resulting in increased survival and decreased production of pro-inflammatory cytokines, including IL- 1P, TNF and IL-6.
• Paraffin-embedded tissue sections were then taken from the hippocampus formation, which included the entorhinal cortex, the Cornu Ammonis (CA) regions CA1-CA3, the subiculum and the dentate gyrus (DG) and were used for immunohistochemical and histochemical staining as described below.
• Paraffin-embedded tissueblocks were sectioned at a 20- ⁇ m thickness with 20 retrieved serial sections using a Leica RM2245 microtome (Leica Microsystems Ltd.). Three sections based on a systematic sampling principle and a section-sampling fraction of 1/5 were selected from each block for further investigations.
• Sections were then blocked in 5% goat serum (Vector Laboratories, Burlingame, Calif., USA) for 20 min before being incubated overnight at 4°C in a solution of mouse anti-Phospho-Tau (Ser202, Thr205) Antibody (AT-8; 0.4 ⁇ g/mL; ThermoFisher Chemicals, Richmond, VA., USA), mouse anti- ⁇ -Amyloid (6E10; 1.0 ⁇ g/mL; Biolegend, San Diego, CA., USA), rabbit anti-NLRP3 (3.0 ⁇ g/mL; MilliporeSigma, St.
• H&E staining was used to evaluate the general morphology of the brain tissue and orientation of the brain regions as described previously.
• standard tissue paraffin block was sectioned at 20-ptn thickness and the slides were allowed to dry and heated at 60’ C for 30 minutes. Prior to staining, sections were deparaffinized in three changes of xylene and rehydrated through graded concentrations of ethanol. Sampling areas and the strategies for identifying regions of interest were shown using standard H&E-stained sections (Fig. 17A).
• ROIs were determined by the cellular architecture as described previously and sampled from the postmortem brains: CAI was sampled from regions anterior to the DG, CA2 was sampled from the region posterior to the DG and CA3 was sampled from regions that are adjacent to the opening of the DG. For each region, the CA scans encompassed all layers of the strata.
• the Braak score in the low AD group ranged from 0-11; none to low AD neuropathological change.
• the neuropathological score for the distribution of neurofibrillary tangles ranged from B0-B1 with a mode of Bl.
• causes of death were due to complications of cardiovascular disease (14 donors), renal failure (1 donors), cancer (1 donor) and liver failure (1 donor).
• Donors with intermediate AD had Braak Scores that ranged from lll-VI; intermediate neuropathological change.
• any changes in cellular density in a CA region could result from shrinkage in the strata due to neuropil changes.
• a decrease in strata volume may be related to a difference in layer thickness.
• the hippocampal strata was measured between the gray/white matter interface and the alveus surface. There was no significant difference in hippocampal thickness between intermediate AD cases and low AD (Fig. 17B) when the hippocampal thickness was measured in the CAI, CA2 and CA3 regions (p>0.05), indicating that changes in cellular populations is not related to changes in hippocampal volume.
• the number of A ⁇ clusters do not differ between intermediate AD and low AD in the hippocampus
• the intermediate AD cases had an average Thai score of "A2” and an average CERAD (e.g., the extent of neuritic amyloid plaques or C score) score of "C2", whereas in the low AD group, the average Thai score was "Al” and the average CERAD was "CO".
• the increase of A ⁇ plaques in seen in the subiculum and the entorhinal cortex demonstrates that there are numerous regions that are prone to have a significant change in protein accumulation as the neural degeneration progresses.
• NLRP3 may contribute to the chronic rieuroinflammation vfo the production of IL-1 ⁇ , resulting in reduced clearance of A ⁇ plaques.
• NLRP3 expression was seen in microglia adjacent to neurons or was present as clusters. It is possible that the cluster distribution of NLRP3 configuration may be mediated by TANK-binding kinase 1 (TBK1) that interacts with tau proteins.
• NLRP1 is expressed in the cytoplasm of hippocampal neurons, and is upregulated in cases with intermediate AD. This observation is consistent with our previous studies that show NLRP- 1 is present in motor neurons in the ventral horn of the human spinal cord and is upregulated after spinal cord injury, traumatic brain injury, stroke and the aging brain.
• Saresella et al. and Yap et al. reported that NLRP1 is primarily expressed by pyramidal neurons of the hippocampus and is activated by aggregated A ⁇ .
• the hippocampal formation cortical band did not differ in thickness between low AD and intermediate AD, indicating that neuronal death and atrophy were not evident in these cases.
• Two other studies examined hippocampal thickness and neuronal numbers in AD cases staged at different Braak stages V-VI and report hippocampal neuronal loss at Braak stages V-Vi, thus supporting our findings.
• the authors evaluated neuropathologicai changes between the low AD and the intermediate groups in all of the hippocampal CA regions and found that the number of A[3 plaques clusters did not significantly differ between the low and intermediate groups. However, here we found that the morphology of the plaques were more dense than those seen in low AD than in the intermediate group. In contrast, in the intermediate group, we saw a significant increase in the number of neurons labeled with pTau in the subiculum, CAI, CA2 and DG regions, consistent with previous studies on tau pathology in post-mortem AD cases.
• composition is administered intracerebroventricularly, intraperitoneally, intravenously, intranasally or by inhalation.
• a method of treating inflammation in lungs of a patient that has been subjected to a viral infection, a central nervous system (CNS) injury comprising: administering to the patient a composition comprising an agent that inhibits inflammasome signaling, whereby the inflammation in the lungs of the patient is treated.
• CNS central nervous system
• CNS injury is selected from the group consisting of traumatic brain injury (TBI), stroke and spinal cord injury (SCI).
• TBI traumatic brain injury
• SCI spinal cord injury
• [0261] 42 The method of any one of embodiments 23-41, wherein the composition is administered intrace rebroventricularly, intraperitoneally, intravenously, intranasally or by inhalation.
• 43 A monoclonal antibody or an antibody fragment thereof that binds to Apoptosis- associated Speck-like protein containing a Caspase Activating Recruitment Domain (ASC), wherein the antibody or the antibody fragment binds specifically to an epitope of ASC, wherein the epitope comprises or consists of the amino acid sequence of SEQ ID NO: 5 or 5-10, 10-15 or 15-20 amino acids of SEQ ID NO: 5.
• ASC Caspase Activating Recruitment Domain
• a monoclonal antibody or an antibody fragment thereof that binds specifically to ASC wherein the antibody or the antibody fragment comprises a light chain variable (VL) region and a heavy chain variable (VH) region, wherein the VL region amino acid sequence comprises LCDR1 of SEQ ID NO: 12, LCDR2 of SEQ ID NO: 13 and LCDR3 of SEQ ID NO: 14, or a variant thereof having at least one amino acid substitution in LCDR1, LCDR2, and/or LCDR3.
• VL light chain variable
• VH heavy chain variable
• a monoclonal antibody or an antibody fragment thereof that binds specifically to ASC wherein the antibody or the antibody fragment comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH region amino acid sequence comprises HCDR1 of SEQ ID NO: 6, HCDR2 of SEQ ID NO: 7 and HCDR3 of SEQ ID NO: 8, or a variant thereof having at least one amino acid substitution in HCDR1, HCDR2, and/or HCDR3; and wherein the VL region amino acid sequence comprises LCDR1 of SEQ ID NO: 12, LCDR2 of SEQ ID NO: 13 and LCDR3 of SEQ ID NO: 14, or a variant thereof having at least one amino acid substitution in LCDR1, LCDR2, and/or LCDR3.
• VH heavy chain variable
• VL light chain variable
• VH region amino acid sequence comprises SEQ ID NO: 18, 19, 20, 21, 22, or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 18, 19, 20, 21, or 22.
• VL region amino acid sequence comprises SEQ ID NO: 28, 29, 30, 31, or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 28, 29,
• VH region amino acid sequence comprises SEQ ID NO: 18, 19, 20, 21, 22, or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ. ID NO: 18, 19, 20, 21 or 22; and wherein the VL region amino acid sequence comprises SEQ ID NO: 28, 29, 30, 31, or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 28, 29, 30 or 31.
• VH region amino acid sequence comprises SEQ ID NO: 18, or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 18; and wherein the VL region amino acid sequence comprises SEQ ID NO: 28 or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 28.
• VH region amino acid sequence comprises SEQ ID NO: 18, or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 18; and wherein the VL region amino acid sequence comprises SEQ ID NO: 29 or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 29.
• VH region amino acid sequence comprises SEQ ID NO: 18, or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 18; and wherein the VL region amino acid sequence comprises SEQ ID NO: 30 or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 30.
• VH region amino acid sequence comprises SEQ ID NO: 18, or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 18; and wherein the VL region amino acid sequence comprises SEQ ID NO: 31 or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 31.
• VH region amino acid sequence comprises SEQ ID NO: 19, or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 19; and wherein the VL region amino acid sequence comprises SEQ ID NO: 28 or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ. ID NO: 28.
• VH region amino acid sequence comprises SEQ ID NO: 19, or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 19; and wherein the VL region amino acid sequence comprises SEQ ID NO: 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.
• VH region amino acid sequence comprises SEQ ID NO: 19, or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 19; and wherein the VL region amino acid sequence comprises SEQ ID NO: 31 or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 31.
• VH region amino acid sequence comprises SEQ ID NO: 20, or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 20; and wherein the VL region amino acid sequence comprises SEQ ID NO: 28 or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 28.
• VH region amino acid sequence comprises SEQ ID NO: 20, or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 20; and wherein the VL region amino acid sequence comprises SEQ ID NO: 29 or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 29.
• VH region amino acid sequence comprises SEQ ID NO: 20, or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 20; and wherein the VL region amino acid sequence comprises SEQ ID NO: 30 or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 30.
• VH region amino acid sequence comprises SEQ ID NO: 20, or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 20; and wherein the VL region amino acid sequence comprises SEQ ID NO: 31 or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 31.
• VH region amino acid sequence comprises SEQ ID NO: 21, or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 21; and wherein the VL region amino acid sequence comprises SEQ ID NO: 28 or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 28.
• VH region amino acid sequence comprises SEQ ID NO: 21, or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 21; and wherein the VL region amino acid sequence comprises SEQ ID NO: 29 or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 29.
• VH region amino acid sequence comprises SEQ ID NO: 21, or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 21; and wherein the VL region amino acid sequence comprises SEQ ID NO: 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.
• VH region amino acid sequence comprises SEQ ID NO: 21, or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 21; and wherein the VL region amino acid sequence comprises SEQ ID NO: 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.
• VH region amino acid sequence comprises SEQ ID NO: 22, or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 22; and wherein the VL region amino acid sequence comprises SEQ ID NO: 28 or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 28.
• VH region amino acid sequence comprises SEQ ID NO: 22, or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 22; and wherein the VL region amino acid sequence comprises SEQ ID NO: 29 or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 29.
• VH region amino acid sequence comprises SEQ ID NO: 22, or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 22; and wherein the VL region amino acid sequence comprises SEQ ID NO: 30 or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 30.
• VH region amino acid sequence comprises SEQ ID NO: 22, or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 22; and wherein the VL region amino acid sequence comprises SEQ ID NO: 31 or an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 31.
• a method for producing an antibody or an antibody fragment that binds specifically to ASC comprising: culturing a recombinant host cell comprising the expression vector of embodiment 74 or 75 under conditions whereby the nucleic acid molecule is expressed, thereby producing the monoclonal antibody or the antibody fragment thereof that binds specifically to ASC.
• a pharmaceutical composition comprising the monoclonal antibody or the antibody fragment thereof of any one of embodiments 44-72, and a pharmaceutically acceptable carrier, diluent or excipient.
• CNS injury selected from the group consisting of traumatic brain injury (TBI), stroke and spinal cord injury (SCI).
• a method of treating multiple sclerosis (MS) in a subject comprises administering to the subject a therapeutically effective amount of the monoclonal antibody or the antibody fragment thereof of any one of embodiments 44-72, thereby treating MS in the subject.

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