NZ755639A - Method for modulating inflammasome activity and inflammation in the lung - Google Patents
Method for modulating inflammasome activity and inflammation in the lungInfo
- Publication number
- NZ755639A NZ755639A NZ755639A NZ75563917A NZ755639A NZ 755639 A NZ755639 A NZ 755639A NZ 755639 A NZ755639 A NZ 755639A NZ 75563917 A NZ75563917 A NZ 75563917A NZ 755639 A NZ755639 A NZ 755639A
- Authority
- NZ
- New Zealand
- Prior art keywords
- antibody
- asc
- lung
- protein
- lungs
- Prior art date
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Abstract
The present invention provides compositions and methods for reducing inflammation in the lungs of a mammal that is afflicted by a condition that leads to inflammation in the lungs. The compositions and methods described herein include agents that inhibit inflammasome signaling in the mammal such as antibodies directed against inflammasome components used alone or in combination with extracellular vesicle uptake inhibitor(s). antibodies directed against inflammasome components used alone or in combination with extracellular vesicle uptake inhibitor(s).
Description
GD FOR NIODULA'I‘ING INFLAMQMASQIWE AC’I‘EVI'E‘Y AND
lNFLAll/EWATEON IN THE LENS
{(130301} This application claims priority from US. Provisional Application Serial No. 62/440,180,
filed er 29, 2016, which is herein incorporated by reference in its entirety for all purposes.
STATENEENT .AS T9 FEEDER/Alli?" SPONSGRED RESEARCH
{8892} This invention was made with US. government support under grant number
4R4ZBSOSSZ'74-02 awarded by the National Institute of Neurological Disorders and Stroke
(NINDS). The US government has certain rights in the invention.
DESCRIPTIQN 017 THE ”HEX"? FILE SUBMITTED ElfiiECTRQNICALLY
{3003} The contents of the text file submitted electronically herewith are incorporated herein
by reference in their entirety: A computer readable format copy of the Sequence Listing ame
UNMlmOl O___Ol VVOMSeqListuST25.txt, date recorded: December 28, 2017, file size 2 kilobytes).
FEELD
lél} The invention relates generally to the fields of immunology and medicine. More
particularly, the invention relates to compositions and methods for modulating ASC (Apoptosis~
associated Speclolilte protein containing a Caspase Activating Recruitment Domain (CARD))
activity and Absent in Melanoma 2. (Alb/1'2) inllammasoine activity in the lungs of a mammal as
treatments for ng inflammation in response to conditions that produce inflammation in the
lungs,
RflUNI}
05} Severe Traumatic Brain lnj ury (TEL) is a major public health concern and is a leading
cause of mortality and morbidity throughout the world (3). in on to direct injury to the brain,
TBl may lead to complications in other organs, such as the lungs. Acute Lung injury (ALI; 2) is a
common pulmonary m after trauma and is associated with a hospital mortality rate of
up from 40% (4). TB} patients, in particular, are susceptible to develop ALL with some studies
reporting an incidence as high as 30% (5): Recent studies have shown that systemic inflammatory
factors may lead to ary dysfunction and lung injury after TB} (6), but the e lar
mechanism underlying TBl—induced lung injury remain poorly defined,
{(3396} A flood of secreted inflammatory l’TlGdialiGI‘S, ing cytolrines, chemolrines, and
damage—associated molecular patterns (DAMPs) released by injured cells contribute to brain
mation and affect distal organs such as the lungs (5} One of the most widely studied s
is the high mobility group box-l (HMO-Bl), which can serve as an early mediator of inflammation
in various pathogenic states> including TBI (7}. A more recent study has shown that HMGBl can
be involved in the mechanism of TBl-induced pulmonary dysfunction (8). l-lMG-Bl release can he
regulated by the inflammasome (9), a multi—protein complex involved in the activation of caspase—
1 and the sing of lL—lfi and IL—1 8 after T31 (10).
{WW} A variety explanations have heen put forth to explaii'i echanisms of pulmonary
complications after TBI, including increased vascular permeability leading to capillary leakage
and infiltration ot‘proteinaceous debris (l l). Extracellular vesicles (EV) are membrane—contained
vesicles that play a role in cell~to—cell communication (l 2) and have been implicated to play a role
in the development ALI in a LPS—induced murine model. Further, it has been shown that EV can
carry hioactive cytokines such as lL—lll and inflainmasome ns (13) (: l4), and may trigger an
immune response and amplify inflammation via its cargo to neighboring and surrounding cells.
l-lowever, it is unknown if EV—rnediated inflainmasome signaling can contribute to the
pathomechanism of induced ALL Further, it is also unknown whether the pathomechanisnis
of TBl—induced ALI are shared by other conditions that produce lung inflammation. in addition,
there is a scarcity of Federal Drug stration (FDA) approved drugs to treat lung
inflammation.
litltltlSl Accordingly, there is an urgent need not only for elucidating the pathornechanisnis of
lung inflammation caused, by 1331 as well as other conditions, but also the development of
eutic compositions and uses thereof for treating and/or preventing lung inflammation.
WARY
{6399] In one , provided herein is a method of ng inflammation in lungs of a patient
in need thereof, the method comprising: administering to the patient a composition sing an
agent that inhibits inllarnniasome signaling, whereby the ii'illanimation in the lungs of the patient
is treated. In some cases, the inflammation in the lungs is caused by a. condition selected from a
central nervous system (CNS) injury, a neurodegenerative disease, an autoimmune disease,
asthma, chronic obstructive ary disease, cystic fibrosis, titial lung disease and acute
respiratory distress syndrome. in some cases, the CNS iniury is selected from the group consisting
of traumatic brain injury (’l‘Bl), stroke and spinal cord injury (SCI). ln some cases, the
egenerative disease is selected from the group consisting of amyotrophic lateral sclerosis
(ALS), multiple sclerosis (MS) and Parkinson’s disease (PD). In some cases, the stration of
the composition results in inhibition of inflammasorne activation in lung cells of the patient. In
some cases, the administration of the composition results in a reduction of caspase—l, nucleotide"
g leucinenrich repeat pyrin domain containing protein l (NLRPl), nucleotide—binding
leucine—rich repeat pyrin domain containing protein 2 (NLRPIZ), nucleotide—binding leucine—rich
repeat pyrin domain containing protein 3 (NLRP3), NLR family CARD doniain~containing protein
4 (NLRCZl), caspase~l l, aninlred, inhibitor of apoptosis protein (XlAP), pannexin—l, Apoptosisn
associated Spec—like protein containing a Caspase Activating Recruitment Domain (ASC),
interleukin~l8 ), high ty group box l (IIllt4lGBl) or absent in melanoma 2 (AIMZ)
levels in lung cells of the patient as compared to a control, wherein the control is an untreated
patient. in some cases, the lung cells are. Type ll ar cells. in some cases, the administration
of the composition results in a reduction in acute lung injury (ALI) as compared to a control,
wherein the control is an untreated patient. In some cases, the reduction in ALI is evidenced by a
reduction in neutrophil infiltration into alveolar and/or interstitial space, d or absent alveolar
septal thickening or a ation f. In some cases, the agent is an extracellular vesicle (EV)
uptake inhibitor, an antibody that binds to an intlarnniasome component or a combination thereof.
In some cases, the EV uptake inhibitor is a compound or an antibody, wherein the antibody is
selected from Table I. In some cases, the agent is an EV uptake inhibitor in combination with an
antibody that binds to an inflanimasome component. In some cases, the EV uptake inhibitor is a
heparin. In some cases, the heparin is Enoxaparini In some cases, the antibody that binds to an
inflamrnasome component is an antibody that specifically binds to a component of a mammalian
AIR/32, NLRPI, NIJRP2, NLRP3 or NIRC4 inflammasoine In some cases, the inflammasome
component is caspase~l ASC or AIMZ In some cases, the inflaniinasoine component is ASC. In
some cases, the antibody binds to an inal PYRIN—PAAD—DAPIN domain (PYD), C—
terminal caspase-recruitnient domain (CARD) domain or an epitope derived from the PYII} or
CARD domain of the ASC protein. In some cases, the antibody binds to a protein having at least
85% sequence identity with an amino acid sequence selected from the group consisting of SEQ ll)
N0: l and SEQ ID NO: 2. In some cases, the antibody inhibits ASC activity in the lungs of the
patient. In some cases, the composition is formulated with a pharinaceutically able carrier
or diluent. In some cases, the composition is administered intracerebroventricularly,
eritoneally, intravenously or by inhalation.
{9916} In another aspect, ed herein is a method of treating ation in lungs of a
patient that has been sub} ected to a central nervous system (CNS) injury, the method comprising:
administering to the patient a composition sing an agent that inhibits inflammasome
signaling, whereby the inflammation in the lungs of the patient is d. In some cases, the CNS
injury is selected from the group consisting of traumatic brain inj ury (TBI), stroke and spinal cord
injury (SCI). In some cases, the administration of the composition results in tion of
inflammasome activation in lung cells of the patient. In some cases, the administration of the
composition results in a reduction of caspase—1, NIRPI, NLRFZ, NIRP3, , caspase—l l,
XIAP pannexin~l, Apoptosis—associated Spec-like protein containing a Caspase Activating
Recruitment Domain (ASC), interleukinmIS (IL—l8), high mobility group box I (HMGBI) or
absent in melanoma 2 (AIME) levels in lung cells of the patient as compared to a control, wherein
the l is an untreated patient. In some cases, the lung cells are Type II alveolar cells. In some
cases, the administration of the composition results in a reduction in acute lung injury (ALI) as
ed to a control, wherein the control is an untreated patient. In some cases, the reduction in
ALI is ced by a reduction in neutrophil infiltration into ar and/or interstitial space,
reduced or absent alveolar septal tl'iichening or a combination thereof. In some cases, the agent is
an extracellular vesicle (EV) uptake inhibitor, an antibody that binds to an inflaniinasome
coniponei'it or a. ation thereof. In some cases, the EV uptake tor is a compound or an
antibody, whereii'i the ai'itibody is selected from Table l. In some cases the agent is an EV uptake
tor in con'ibinatioi'i with an antibody that binds to an inflarnrnasonie component. In some
cases, the EV uptake inhibitor is a heparin. In some cases, the n is Enoxaparin, In some
cases. the antibody that binds to an intl ainrnasorne component is an an tihody that specifically binds
to a component of a rnai'nrnalian Alli/l2, NLRPI, NI_,RP2, NLRPS or NLRCZI inilarnniasonie. In
some cases, the inflainrnasonie component is caspase-1, ASC or AIME. In some cases, the
inasorne component is ASC. In some cases, the antibody binds to the FYI), CARI) domain
or an epitope derived from the I’YD or CARD domain of the ASC protein. In some cases, the
antibody binds to a protein having at least 85% sequence identity with an amino acid sequence
selected from the group consisting of SEQ ID NO: I and SEQ ID N0: 2. In some cases, the
antibody inhibits ASC activity in the lungs of the patient. In some cases, the ition is
formulated with a pharniaceutically acceptable carrier or t. In some cases, the composition
is administered intracerebroventricularly, intraperitoneally, intravenously or by inhalation.
BR]EF DESCRIFTEGN OF THE DIM‘WENGS
{9011} FIG. lA—IN illustrateinflainrnasonie activation in C57/BLo niouse cortical and lung
tissue post~'I'BI. FIG. IA shows a representative iniinunoblot of active caspase—1, ASC, IL-l 8, IL—
3, Hts/{GEL and AIME after 'I‘BI. Active caspasenl (FIG. IE), ASC (), IL~I8 ()),
IIIVIGBI (), AIM 2 (), and IL—fi, (), are icantly elevated in cortical
tissue at 4 and 24h post—TEL Data presented as inean+/— SEEM; ****p<0.0t‘il, ***p<0.0l, p<0.05
compared to sham. N=4~5 per group. FIG. Ill-I shows a representative imniunohlot of active
caspase—1, ASC, ILL—l8, IL-B, HMGBI, and AIMZ in lung . I, I, I: L, M, N) Active caspase—
l, (FIG. II), ASC (3),IL-l8 (), HIVIGBI (), AIR/l, 2 (%)., and lL—B,
(FIG. IN) are significantly elevated in lung tissue 4 and 24h after TBI. Data presented as mean+lh
SEM. N: 4-5 per group, **p<:0.0l,., *p<0.05 compared to sham.
{0012} ~2C illustrates Expression. of inflanimasome proteins in Type II alveolar
epithelial cells. shows AIM2, shows active evl and shows ASC
inpnunoreactivity increases in lung tissue after CCI (4, 2-4 h) when compared to mice. Confocal.
images of AIME, caspase~l, and ASC (green) and type ll epithelial cells (surfactant protein C,
red).
{WIS} FIG. SA—SE rates 1781 increases nuclear and asmic I—lMGBl expression in
mice lung. F182}. 3A shows representative immunoblot of nuclear l after TBI. Flt}. 3B
shows nuclear l-lMGB l is significantly elevated in 4 hour injured animals compared to sl’iam. EEG.
3C shows representative irnrnunohlot of cytoplasmic l-lMGBl after TBI. ) shows
cytoplasmic liMGBl is significantly elevated in 4 hour injured s ed to sham. Data
presented as meant/'— SEM; ****p<0.00l) >l‘lwerODl, *p<0.05 compared to sham. N===r4n5 per
group. EEG. 3E shows l-lMGBl iminunoreactivity increased in lung tissue after CCI when
compared to sham mice. Confocal images of l-{MGBl and type ll epithelial cells {surtactant
protein C, red)
{9014} FIG. elk-4C illustrates Pyroptosome formation in mice lungs 4 hours post~TBl
PEG. ill-A shows TBl induces laddering of ASC in lung tissue, indicating formation of
the pyroptosorne, an oligomerization of ASC dimers that leads to activation of caspase—1 and
pyroptosis. shows representative immunohlot and PEG. 4C shows quantification of
gasdermin. Gasdermin—D is significantly elevated in lung tissue post-'I‘Bl. Data presented as
mean+/'~SEM. N: 4—5per group, **p<:0.l_‘ll., *p<0.05 compared to sham.
{9015} FIG. SA—SB illustrates ’l‘Bl induces alveolar morphological changes and acute lung
injury in mice. , shows H8513 ng of lung ns from sham and d animals at
4h and 24 h. Sections show evidence of neutrophil ration (arrow , changes in
morphology of alveolar capillary membranes (asterisk, *), interstitial edema (short arrows),
and ce of thickening ofthe interstitium and the alveolar septum (pound, #3). shows
acute lung injury scoring is significantly increased in injured animals when, compared to sham at
4h and 24 h. Data presented as mean+/—SEM. N: 4—5 per group, **p<0.0l., *p<0.05 compared to
sham.
{lllllfi} illustrates expression of (IDSl in serum-derived EV from control and THE—
injured mice. Representative hlot ol’ C1381 in sernm~derived EV from sliam control and
Till—injured mice.
WO 26009
{$917} -7l4l illustrates adoptive transfer ofEV from TBI animals induce caspase— l and
ASC in the lungs of uninjured mice. EEG. 7A illustrates a representative hlot showing that
caspase- l. (EEG. 7B), ASC (run. 7C}, TEL-18 (Fro. 7n), AIMZ (EEG. 7E), HMGBE (EEG. 73?) are
elevated in the lungs of animals that received EV isolated from TBl mice when compared to EV
from sham animals. Data presented as mean-l1!”— SEM; *p<.0.05 compared to sham. N===3 per group.
EV from T8} mice d alveolar morphological changes (decreased alveolar size) and
infiltration of inflammatory cells as determined by ltl&E staining (EEG. 7G), ALI score is
icantly increased in EV delivered from injured mice compared to uninjured mice (EEG. 7H).
Data presented as meant/'— SEM; *p<,0.05 compared to uninjured group.
{9018} EEG. 8A—8E illustrates treatment with Enoxaparin (3 mg/lrg) and It: lOO (5 nag/kg)
reduces inflainmasome expression in lungs of animals delivered EV from injured mice. EEG. 8A
illustrates a representative hlot showing that caspase—1 (EEG. SB), ASC (EEG. 8C), lL-llfi
(EEG. 3D), Alli/£2 (EEG. 8E1), HMGBl (EEG. 8E) are reduced in the lungs of s that were
d with Enoxaparin and 1C 100 when compared to ted positive coi'itrol animals. Data
presented as meant/— SEM; *p<,0.05 compared to sham. N===4 per group.
{9919] —9E illustrates treatment with Enoxaparin (3 mg/kg) and EC 100 (5 nig/kg)
reduces ALI score in lungs of animals delivered EV from injured mice. EEG. §A~9D illustrates
l-l&E staining of lung sections from saline (}, untreated (EEG. 9E2), Enoxaparin (EEG. 9C)
and K2 l 00 ASC; EEG. 9D) treated mice lungs delivered EV from injured animals. Sections
show evidence of neutroplril infiltration, s in morphology of alveolar capillary membranes,
interstitial edema, and evidence of thickening of the interstitium and the alveolar septum. EEG. 9E
illustrates that acute lung injury g is significantly decreased in animals treated with
Enoxaparin, lC lOO when compared to untreated animals Data presented as meari-t/lSEh/l, NI: 4
per group, **p<10.01,, *p<{),05.
{392G} EEG. 10A~lGE illustrates delivery of serum—derived EV from TBI patients increases
intlarnmasonie protein sion in pulmonary endothelial cells. A shows western blot
entation of caspase~l, ASC, Alli/l2, HMGBl in PMVEC after incubation with TBI-EV and
control—EV for 4 hours, EKG. tflE-EGE) shows quantification of western blots, n====3 filters per
group, n===6 patients, t—test, p~<0.05, FIG. in}? shows immunoassay results ofa significant ii'icrease
in iL—l {3 expression using Ella simple plea assay n====3 filters per group, n--6 ts, t—test, p<0,05.
} FIG. “HA—l it: illustrates delivery of "FBI-EV to pulmonary endothelial cells increases
immunoreaetivity of active caspase—l and cell death. 171G. 11A shows co—loealization of Caspase—
l FLICA and Pl staining and PMVEC incubated with TBI—EV for 4 hours. FE}. ”B shows
caspsae-l FLICA and PI staining in PMVEC incubated with control-EV for 4 hours C
shows scent plate reader analysis C incubated with TBI and control—EV for 4 hours.
n===6, p<i0. GSA
BETAELED DESCRI}?TION
DEFINITIUNL l
{£3622} Unless otherwise defined, all technical terms used herein have the same meaning as
ly understood by one of ordinary skill in the art to WhiCl’l this invention s.
{8923} As used herein, “protein” and “polypeptide" are used synonymously to mean any
peptideslinked chain of amino acids, regardless of length or post—translational modification, e.g
glycosylation or phospliorylation.
{(3024} As used herein, the term ”antibody" refers lly and broadly to globulins,
monoclonal antibodies, and polyclonal antibodies, as well as active fragments thereof. The
fragment may be active in that it binds to the cognate antigen (eg, ASC, NLRPl, AER/l2, etc), or
it may be active in that it is ically functional. The antibodies for use herein may he chimeric,
humanized, or human, using techniques known in the art.
{6925} As used herein, the term "humanized antibody“ refers to an antibody in which minimal
portions of a nonnhuman antibody are introduced into an otherwise human antibody.
{9026} As used herein, the term ”human antibody” refers to an antibody in which substantially
every part of the protein is substantially munogenic in humans, with only minor ce
changes or variations.
{9027} 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 cornpliinentarity determining regions
(CDRs). There are three CDRs each in Vl-l (HCDRl and VL (LCDRl, LCDRZ,
, HCDRZ, HCDRB)
LCDR3), together with framework s (FRs).
{(3328} The term "CDR region" or ”CDR." can be mean the liypemariable regions of the treaty
or light chains of the inirnunoglobulin as defined by Kaba’t et al., l. 991 (Kabat, E A. et al., (1991)
Sequences of Proteins of Immunological Interest, 5th Edition. US Department of Health and
Human Services, Public e, NIH, Washington), and later editions. An antibody typically
contains 3 heavy chain CDRs and 3 light chain CDRs.
{9029} it has been shown that fragments of a whole antibody can also bind antigens. Examples
of binding fragments include: (i) an Fab fragment consisting of VL, Vl-l, CL and (Tl-ll domains
(Ward, E. S. et al., (l 989) Nature 341, 544—546); (ii) an Ed fragment consisting of the Vl-l and CH1
domains ferty et al., @990) Nature, 348, 552—554); (iii) an FV nt consisting of the
VL and VB domains of a single antibody (Holt et al., (2003) Trends in Biotechnology 21, 484"
490); (iv) a dAh nt (Ward, E. S. et al., Nature 341, 544646 (1989), McCafferty et al., (1990)
Nature, 348, 552654, Holt et al., (2003) Trends in Biotechnology 2i, 484490}, which consists of
a VB or a VL domain; (V) isolated CDR regions; (Vi) F(ab')2 fragments, a bivalent fragment
comprising two linked Fab fragments (Vii) single chain EV molecules , wherein aVB domain
and a VL domain are linked by a peptide linker which allows the two domains to ate to form
an antigen g site (Bird et al U988) Science, 242, 423—426, Huston et al., (1988) PNAS
USA, 85, 5879—5883); (viii) hispecific single chain FV dimers {PCT/USQZlG9965) and (ix)
”diahodies”, multiyalent or rnultispecific fragments ucted by gene fusion (Wngl/138(34;
Holliger, P. (£993) et al., Proc. Natl. Acad. Sci. USA 90 6444—6448).
{(3936} EV, scFv or diabody molecules may he stabilized by incorporation of ide bridges
linking the VH and Vi“, domains r, Y. et al., Nature Biotech, l4, l239—1245, l996),
Minihodies comprising a scllv joined to a CH3 domain may also be made (Hu, S. et al. (1996)
Cancer Res, 56, 3055—3061). Other examples ofbinding fragments can he Eab‘, which differs from
Fab fragments by the on of a few residues at the carboxyl terminus of the heavy chain CH l
domain, including one or more cysteines from the antibody hinge region, and Fah'-Sl—l, which is a
Fah' fragment in which the cysteine residue(s) of the constant domains bear a free thiol group.
{£3931} "Fv" when used herein can refer to the mil’iil’i’lum fragment of an antibody that s
both antigen-recognition and antigene‘oinding sites. "Fab” when used herein can refer to a fragment
of an antibody that comprises the constant domain of the Eight chain and the CH1 domain of the
heavy chain The term ”niAb" refers to moi'iocional antibody.
{$032} By the terms “Apoptosis—associated Speck-like protein ning a Caspase
ting Recruitment Domain (CARD)” and “ASC” is meant an expression product of an ASC
gene or isoforms thereof, or a protein that shares at Eeast 65% (hut preferabiy 75, 80, 85, 90, 95,
96, 97, 98, or 99%) amino acid sequence identity with ASC (egg NPM037390 (Q9ULZ3ul),
NP___660183 (QQULZZ'i—Z) or QQULZS—S in human, NP___O75747 in mouse or NP___758825
{BACA3754} in rat) and dispiays a functionai activity of ASC. A “functional ty” of a n
is any activity associated with the physiological function of the protein. Functional activities of
ASC include, for example, recruitment of proteins for activation of caspase-1 and initiation of cell
death.
{9033} By the term “ASC gene,” or “ASC nucieic acid” is meant a native ASC—encoding
nucleic acid sequence, genomic Sequences from which ASC cDNA can he transcribed, and/or
alielic variants and, homologues of the foregoing. The terms encompass double—stranded DNA,
single—stranded DNA, and RNA
{9034} As used herein, the term “inflammasome” means a multi—protein (eg at ieast two
n s) x that activates caspased. Further, the term “inflammasome” can refer to a multi-
n compiex that activates e—1 activity, which in turn, reguiates IL—iB, IL—l 8 and Iii—3‘5
processing and activation. See Arend er of. 2008; Li et a]. 2008; and Martinon er al. 2002., each of
which is incorporated by reference in their entireties. The terms “‘16.,RPl inflamrnasome”,“NAIPi
inflammasome”, “NLRPZ asome”, “NAEPZ iasome”, “I‘HRPfi inflammasome”,
“NALPS inflamniasome”, “NLRCZl- inflammasome”, “EPA? inflaniinasome” or “AIME
inflannnasome” mean a protein complex of at least caspased and one adaptor protein, egt, ASC.
For example, the terms “NLRPE intiainrnasorhe” and “NALPE inflainrnasorne” can mean a
multiprotein conipiex containing NLRPl, ASC, caspase~1, caspase—1 l, XIAP, and pannexin-l for
activation of caspase—1 and processing of interleukin—iii, eukin—1% and interleukin—33. The
terms “NLRPZ inflammasonie” and “NALPE infiammasonie” can mean a rotein complex
2017/068713
containing NLRP2 (aka NALP2), ASC and caspase—l,while the terms “NLRP3 intlamrnasorne”
and “NALP3 masome" can mean a multiprotein complex containing NLRPZE (aka NAl_,P3),
ASC and the terms “NLRCll inflanimasorne” and “IFAF intlainmasome” can mean a multiprotein
complex containing } (aha lPAF), ASC and caspase—l Additionally, the term l2
rnasorne” can mean a multiprotein complex comprising AIMZ, ASC and caspase—l.
{(3835} As used herein, the phrase “sequence identity” means the percentage of cal
subunits at corresponding positions in two sequences (e.g nucleic acid sequences, amino acid
sequences} when the two sequences are aligned to maximize subunit matching, i.e., taking into
account gaps and insertions Sequence identity can be measured using sequence analysis software
{e.g., Sequence is Software Package from Accelrys CGC, San Diego, CA).
{9936} By the phrases “therapeutically effective amount” and “effective dosage” is meant an
amount sufficient to produce a therapeutically (eg, clinically) desirable result; the exact nature of
the result will vary depending on the nature of the disorder being treated. For example, Where the
er to he treated is SCI, the result can be an improvement in motor skills and loconiotor
function, a decreased spinal cord lesion, etc. The compositions bed herein can be
administered from one or more times per day to one or more times per week. The d n
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, Moreover, treatment of a
subject with a therapeutically effective amount of the compositions of the invention can e a
single treatment or a series of treatments.
{9037] As used herein, the term “treatment” is defined as the application or administration of
a therapeutic agent described herein, or identified by a method. described herein, to a patient, or
application or administration of the therapeutic agent to an isolated tissue or cell line from a patient,
who has a disease, a symptom of disease or a predisposition toward a disea se, with the purpose to
cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the e, the symptoms
of disease, or the predisposition toward disease.
{$1938} The terms “patient” “subject” and “individual” are used ii'iterchangeahly , and
mean a mammalian t to he treated, with human patients being preferred. In some cases, the
methods of the invention find use in experimental animals, in veterinary applications, and in the
development of animal models for disease, including, but not limited to, rodents including mice,
rats, and hamsters, as well as prin'iates.
{8639} As interchangeably used herein, “Absent in Melanoma 2” and “Alli/I2” can n'iean an
expression product of an AZMZ gene or isct‘crms; or a protein that shares at least 65% (but
preferably '75, 80, 85, 90, 95, 96, 97, 98, or 99%) amino acid sequence ty with Alli/i2 (eg,
accession numbefis) NX___Ol4862, NI’OO4824, XP016858337, XP005245673, AABSI 613,
BAF8473l and displays a functional activity of AlMZ.
, tl‘MO)
{9049} As interchangeably used herein, “NALPl” and “NLRPl’“’ mean an expression product
of an NALPl or NLRPl gene er isofori’ns; or a protein that shares at least 65% (but preferably 75,
80, 85, 9C), 95, 96, 97, 98, or 99%) amino acid sequence identity with NALPl (cg, accession
number(s) 87, 001023225, Issuance, Nifiizrziss, NPW127497, NPOSS737) and
displays a functional activity of l.
{9041} As hangeably used herein, N“ALP2” and “NLRPZ” mean an expression product
of an NALPZ or NL-RPZ gene or isofornis; or a protein that shares at least 65% (but preferably 75,
80, 85, 90, 95, 96, 97, 98, or 99%) amino acid sequence identity with NALPZ (cg, accession
numbers) NP___001167552, Ni>___001167553, Ni>___oei l67554 or Ni>___060322) and displays a
functional activity of NALPZ.
{9942} As interchangeably used herein, “NALPLl” and “NI_RP3” mean an, expressien product
of an NALP3 0r NLRPll gene or iscforins, or a protein that shares at least 65% (but preferably 75,
80, 85, 90, 95, 96, 97, 98, or 99%) amino acid sequence identity with NALPS (eg, accession
numb-eds) NP_00l073289, NP_OOl l20933, NP_GGl l20934, NP_00l230062, NP_004886,
NP_899632, XP_01 l 54,2350, XP_Ol 6855670, XP_01685567l, XP_0l 6855672 or
XP_Gl6855673) and ys a onal activity of NALP3.
[6043} As interchangeably used herein, “NLRCA and “IPAF” mean an expression t of
an NI...RC4 or IPAF gene or lsol’ornis; or a protein that shares at least 65% (but preferably 75, 80,
85, 90, 95, 96, 97, 98, or 99%) amino acid sequence ty with NLRC4 (qu accession
nurnberQs) NP_OOl l 86067, NPOOllBéOéS, NP_00l289433 or NP_O67032) and ys a
functional activity of NLRCAl.
{$9944} By the terms “stroke” and “ischemic stroke” is meant when blood llow is interrupted
to part of the brain or spinal cord,
} By “traumatic injury to the CNS” is meant any insult to the CNS from an external
mechanical force, ly leading to permanent or temporary impairments of CNS function.
{$046} The term “antibody” is meant to include polyclonal antibodies, monoclonal antibodies
(nil/tbs), cliirneric antibodies, humanized antibodies, anti~idiotypic (anti~ld) antibodies to
dies that can be labeled in soluble or bound form, as well as fragments, s or derivatives
thereof, provided by any known technique, such as, but not limited to, enzymatic cleavage, peptide
sis or recombinant techniques. Such anti—ASC and anti—NLRPl antibodies of the present
invention are capable of binding portions of ASC and NLRPl, respectively,that interfere with
caspase—l activation.
{9047} Methods involving conventional lar biology techniques are described herein.
Such techniques are generally known in the art and are described in detail in methodology treatises
such as Molecular Cloning: A Laboratory Manual, 3rd ed, vol. 1—3, ed. Sambrook et al., Cold
Spring Harbor Laboratory Press, Cold Spring , NY, 2001; and Current Protocols in
Molecular Biology, ed. Ausubel et al, Greene Publishing and Wiley-Interscience, New York, £992
(with periodic updates). Immunology techniques are generally known in the art and are described
in detail in methodology treatises such as Advances in Immunology, volume 93, ed. Frederick W.
Alt, Academic Press, gton, MA, 2007; Making and Using Antibodies: A Practical
ok, eds. Gary C, Howard and Matthew R. Kaser, CRC Press, Boca Eaton, FL, 2006;
Medical immunology, 6&1 ed, edited by Gabriel Virella, Informa Healthcare Press, London,
England, 2007; and Harlow and Lane ANTIBODIES: A, laboratory Manual, Cold Spring Harbor
tory l.)ress, Cold. Spring Harbor, NY, l988.
{9948} Although compositions and methods similar or equivalent to those described herein can
be used in the practice or testing of the present invention, le compositions and methods are
described below, All ations, patent applications, and patents mentioned herein are
orated by reference in their entirety. In the case of conflict, the present specification,
including iitions, will control. The particular embodiments discussed below are illustrative
only and not intended to be limiting,
QVERVTE‘W
{6349] Provided herein are compositions and methods for reducing inflamn'iation in the lungs
of a mammal that has been subjected to or is afflicted by a. condition that results in or causes lung
'iniation. The compositions and n'iethods described herein can include antibodies or active
fragments thereof as provided herein that specifically bind to at least one component (e.g ASC)
of a mannnalian intlairiinasome and/or compounds that modulate (cg, inhibit or reduce)
extracellular vesicle (EV) uptake and have use as treatments for lung inflammation in a mammal.
{00350} Described herein are methods for reducing ation in the lungs of a mammal
having a condition that results in and/or causes an inflammatory response in the lungs. in one
embodiment, the method 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 (eg, spinal
cord injury (SCI), traumatic brain injury (TBI) or stroke), a neurodegenerative disease, an
mune disease, asthma, c ctive pulmonary e (COPE), cystic fibrosis,
interstitial lung disease or acute respiratory distress syndrome. The composition can be
administered in a therapeutically effective amount. The eutically 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 f as ed herein that binds to a component of an
masome or a combination thereof, The composition can be administered by any suitable
route, e. g, by inhalation, enously, intraperitoneally, or intracerebroventricularly. The
composition can further include at least one aceutically acceptable carrier or diluent.
{(3951} In one embodiment, administration of the agent can result in a reduction in the activity
an d/or expression level of a component of a mammalian inflammasome in the lungs of the subject
The reduction can be in cells of the lung such as, for example, Type ll 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 sion level of the hillaminasome component(s)
in a subj ect not administered the agent. in one embodiment, administration of the agent results in
the reduction of caspase—l activation in at least the lungs or lung cells of the subject. in one
embodiment, stration of the agent results in the ion of the expression level of one or
more inflammasorne components (eg, ASC, Ali‘s/l2, NALPl NALPS or
, NALPZ, NALPZ,
NLRCél) in at least the lungs or lung cells of the subject.
{(3352} In another embodiment, administration of the agent can result in a reduction in or
elimination of acute lung injury (ALE). In one embodiment, the reduction in ALI is evidenced by
a reduction in neutropl'iil infiltration into alveolar and/or interstitial space, reduced or absent
ar septal thickening or a nation f. The reduction can be in comparison to a
control. The control can be AM in the subject prior to administration of the agent. The control can
be ALI in a t suffering from ALI not administered the agent.
{(13053} ln still another ment, administration of the agent can result in a reduction in or
elimination of pyroptosis in the lungs of the suhj ect. osis is a proinflaniinatoi'jy' form of cell
death that involves tion of caspase—1. Pyroptosis can be triggered hy the caspase—1 mediated
cleavage of gasderniin l) (GSDMD). in one embodiment, the reduction in pyroptosis is evidenced
by a reduction in or lack of cleavage of GSDl‘le in the lungs or lung cells (eg, Type II alveolar
cells) of the suhj ect. The reduction or elimination of pyroptosis can he in comparison to a control.
The reduction in or lack of cleavage of GSDMD can be in comparison to a control. The control
can be the level of pyroptosis in the subject prior to administration of the agent. The control can
he the level of pyroptosis in a subject suffering from pyroptosis not administered the agent.
{(3954} in one embodiment, the agent to be administered is an EV uptake inhibitor. The EV
uptake inhibitor can be a compound, antisense RNA, siRNA, e, 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, d-difluoromethylornithine (DEMO), arin,
Asialofetoin, Human receptor—associated protein (RAP), RGD (Arg-Gly-Asp) peptide
Cytochalasin D, Cytochalasin B, Ethylenediaininetetra acetic acid (EDTA), l_.atrnnculin A,
latrunculin B, NSC23766, Dynasore, Chlorpromazine, 5—(N~Ethyl.~N—i.sopropyl)amiloride
(EPA), Amiloride? Bafilornycin A Mortensin and Chloroquine, Annexin—V, Wortmannin,
1-,Y294002, Methyl~B~cyclodextrin (MBCD), Filipin, Simvastatin, Fumonisin El and N~
hiityldeoxynojirirnycin hydrochloride, UOl 26 or a proton pump inhibitor. The EV uptake inhibitor
antibody or an active nt thereof as provided herein can he one or more antibodies or active
fragments thereof ed against n targets listed in Tabie l. A composition for treating
and/or reducing inflannnation in the lungs oi‘a mammal using an EV uptake in‘nibitor can further
ineiude at least one pi'iartnaoeutiealiy acceptable carrier or diluent.
{(3355} Tobie t. Exernpiary targets. and ponding antibodies for use in blocking EV
uptake.
Gene Symbol Gene Name Exempiary Antibodies
iCAl‘t/ini interoeitular Adhesion Meteorite 1 ogen iCAIt/i—i antibody (Life
Technologies, 07~5403); CD54
(ICAl‘i/I— 1 ) Monoclonal Antibody
(R65), eBiogcienc-e'm
LFA—l Lymphocyte function—associated Abbiotec LFAnl antibody (Abbioteo,
antigen '1 ); pmental Studies
oma Bank LFAPI antibody
(Developmental Studies Hybridorna
Bank, MHMZZzt)
T~ceii membrane protein 4 BioLegend TiMD4 antibody
(Bioiegend, 354004); I_.ifeSpan
Biosoienoes TIMD4 antibody
(lifespan Biosoien ces, 1.3—3 1 4 13)
‘MFG—ES Milk Fat Giobuie-EGF Factor 8 MEL internationai Vii:G138 antibody
n (MEL, 13199—3); Santa Cruz E
Biotechnology b’iFGE/S antibody
(Santa Cruz, sc—SOZQ); MEL E
International MFGES antibody (its/3131.17;
1 8,426.10) i
Gene Syniboi Gene Name Exempiary dies
D I—SIGN tio Cali—Specific eiiuiar lnvitrogen DC SELGN antibody
adhesion moiecule~3~Grabbing Now (eBioscience, eB—h209, 112099-48;
integrin BD Biosciences DC SIGN antibody
(Bo, DCN46, 551 186)
................................................................................................................................................................................................................................
DECZOS cluster of differentiation 205 EMU Millipore LY75 antibody
(Millipore, H1330); BioLegend a
LY75 dy (BioLegend, 342203)
BioLegend HZ—Kl antibody
“Evil-1C Class I (H—ZKd) (BioLegend, 28—86, 114603); a
BioLegend I-{2_K1 antibody
(BioLegend, 28—14-8, 146999-85) :
TSpanS 'I‘eti'aspanin-S R and D SyS’tems 'I‘SPANS antibody
(R3213 Sy Stems, MAB4734) ‘
'I‘SpanZQ '1‘ettaspanin—29 Santa Cruz Biotechnology CD9
antibody (Santa Cruz, 5059140);
Envitrogen CD9 antibody
{eBioscienoe, eBioSN4; BI)
Bioscienoes CDQ antibody (BD
Pharmingen, 555370)
ITGAL Integtin subunit aipba L ’1‘81/22.1.1.13.3; REM/4.4.11.9
___________________________________________________________________________________________________________________________________________________________________________________________________________________________________.€
I'I‘GAM Integtin t aipba M CD111) Monoclonal Antibody
(VB/HEX CD1 1B00); BB Biosciencesi
WO 26009
Gene Symbni Gene Name Exempiai'y Antibodies
CD1 Eb antibody (BD ngen,
ICRF44; 555385) E
I'I‘GAX Integrin subunit aipha X Anti—integrin dX Antibody, clone :
N418 (MABISQQZ); BD Biosciences
CD} 10 antibody (8D Bioscience, B~
136; 560369) E
Cluster of differentiation 44 iny'iti‘ngen CD44 antibody a
(eBioscience, VFF—"i; MAl—8239‘Z);
invittngen CD44 antibody :
(eBinsoience, 1M7; MAI -1 0225);
Invitmgeii CD44 antibody i
(eBins-cience, SFIZ; MASJ 2.394); BD:
Biosciences CD44 antibody (BD
Biosciences, 515; 550990 OR 550988);
ITGAB in subunit alpha 3 £5th Miiiipote integrin aipha3
antibody (Miliipoie, P135;
MAB195ZZ 0R MAB 1 9521’}
ETGA4 Integtin subunit alpha 4 Bio X Cell 4 antibody {BioXceiLé
PS/Z) (BEOO'HnSl‘t/IG); Bi)
Biosoiences I'I'GA4 antibody (BB
ences, 561892); 1810 encesi
I'I'GA4 antibody (@331); 340976); EMLD
Miiiipore ITGA4 antibody (Miiiiporefi
PdCZ; MABiQSS) i
Gene Symbol Genie Name Exemplary Antibodies
I'I‘GAV lntegrin subunit alpha V Ahcam integrin alpha V antibody
(Abcam, 31377996); Abcam integrin
alpha v antibody (Aboam, ab78289);
Abcam in alpha V dy
{Abcaim abl6821}; Invitrogen integrinE
alpha y antibody (Thermo Fisher
Scientific, 27247136, MA} 81669); RE
& D Systems integrin alpha 3; antibodyE
(R&D Systems, MAB/”3528)
......................................................................................................................................................................................................................................
Integrin subunit beta :3 Abcam in beta}. antibody
(Absam, ab78289); Abnova intogrin
beta3 antibody (Abnova, MHFZI, E
EILABT’OQS)
im L BioLegend CD62L antibody
(Biologend, 304804); BioLegend l
CD621... antibody gend, 304810)
C081 molecule ED Biosoimoos CD?“ antibody (BI)
Phaimingen, 555675); R and I) E
Systems CD81 antibody (R3513
Systems, MAB-4Q 5)
LDL receptor related protein 1 ogen LRPl antibody (Life
Technologiesfi 0); Invilrogen
LRPl antibody (Thermo Fisher, MAl—E
27198) E
2017/068713
Gene Symbol Gene Name Exempiar‘y Antibodies
VCAMl vascular cell adhesion molecule 1 invitrogen VCAM-l antibody (Caltagj
1511.131; MAS—16429);
lmrnnnoteeh anti—VCAl‘t/iul
antibody
......................................................................................................................................................................................................................................
CD151 C0151 molecule (Raph biood group) BD Biosciences CD151 antibody
(Becton Dickinson, );
Epitomics CD151 antibody
(Epitomics, 5901 — 1)
{9956] In one embodiment, the agent to be stered is an antibody or an active fragment
thereof as provided herein directed against a component of a mammalian inilarnmasorne or an
antigen or epitope derived therefrom. In another embodiment, the agent to be administered is an
antisense RNA or siRNA directed against a component of a mammalian inflammasome. The
ini‘larnmasome component can be a component of any asorne known in the art, such as,
for exarnpie, the NAPLl NLRCd or AIR/12 inilarnrnasonie. in a,
, NALPZ, NALP3, typical
embodiment, the antibody specifically binds to ASC or an antigen or epitope derived tl'ierel'iom.
yer, an antibody against any other component of a niai'nrnaiian asome (eg, the
NALPL NAI_,P2, NALPS, NLRC4 or Alb/i2 inflammasome) may be used.
[(1857] An antibody as described herein can be a. inonocional or onal antibody or active
fragments thereof. Said antibodies or active fragments can be chimeric, human or humanized as
described herein.
{(1958} Any suitable antibody or an active fragment thereof as provided herein that specifically
binds ASE? can he used, e.a an antibody that inhibits ASC activity in lung cells (eg ’JType 11
alveolar cells) of the subject. in a typical embodiment, the antibody icaily binds to an amino
acid sequence having at least 85% sequence identity with amino acid sequence SEQ 1D N011 or
SEQ 11) N02. rly, in another embodiment, the inflannnasome is the NALPl
2t).
inflammasome, and the at least one component is NALPl tie, NLRPl). in this embodiment, the
antibody or an active fragment thereof as provided herein specifically 1oinds to an amino acid
sequence having at least 85% sequence identity with amino acid sequence SEQ ID NO: 3 or SEQ
1D NO: 4.
{$059} in yet another embodiment, the agent is one or more EV uptake inhibitors in
coinbina‘ti on with one or more antibodies or active fragments thereof as provided herein that hind
a ent of an inflammasoine. The EV uptake inhibitor can be any EV uptake inhibitor as
provided herein. The antibody that binds a ent of an inflammasome can any antibody that
binds any imasome component as provided herein in one embodiment, the agent
administered to a suhj ect suffering from lung inflammation comprises a n (e.g, Enoxaparin)
in combination with an antibody that binds a component of the Alli/l2 inflammasome (eg, ASC).
{9066} in one embodiment, the method comprises: providing a eutically ive
amount of a composition including an antibody or an active fragment thereof as provided herein
that specifically binds to at least one component (e. g, ASC) of a mammalian inflammasome (eg,
Alli/12 inflammasome); and administering the composition to the mammal suffering from lung
inflammation wherein administering the composition to the mammal results in a reduction of
caspase—1 activation in the lungs of the mammal. in another embodiment, the method comprises:
providing a therapeutically effective amount of a composition including an dy that
specifically binds to at least one component (eg ASC) of a mammalian inflammasonie (eg,
AIMZ masome); and administering the composition to the mammal suffering from lung
inflammation, wherein administering the composition to the mammal results in a reduction in the
levels of one or more inflammasome components (eg, ASC). in yet another embodiment, the
method. comprises: providing a eutically ive amount of a composition including an
antibody that specifically binds to at least one component (eg ASC) of a mammalian
inflantmasome (eig, AIME inflammasome); and. administering the composition. to the mammal
suffering from lung ii'illa,nirria.titii'i, wherein administering the ition to the mammal results
in a reduction ALI. The lung inflammation can he the result of a CNS injury (egg SCI or TEE),
asthma, chronic obstructive pulmonary disorder (COPE), a neurodegenerative disease, or an
autoimmune disease with an intlarnrnatow ent in one embodimen t, the lung inflammation
is caused by a CNS injury such as TBI or SCl.
{fillet} in one embodiment, the methods provided herein further entail detecting a level or
activity of one or more components of a ian iritlanii'na,sr3rne in a sample from a suhj ect
suspected ofsuffering from lung inflammation. The method of detecting the level or activity entails
measuring the level of at least one intlamrnasome protein (eg ASC or AIMZ) in the sample
obtained from the subject; determining the presence or absence of an elevated level or activity of
said at least one inflammasome n (eg ASC or AIMZ). The level or activity of said at least
one inflainmasonie protein can be enhanced relative to the level of said at least one inflammasome
n in a control sample. The level or ty of said at least one inflarnmasonie protein in the
protein ure can be enhanced relative to a predetermined reference value or range of
reference values. The at least one inflammasome n can be nucleotidenbinding leucineurich
repeat pyrin domain containing protein 1 (NLRPl), NLRPZ, NLRP3 NLRC4, AIMZ, apoptosisn
associated speck—like protein containing a caspase recruitment domain (ASC), e—l, or
combinations thereof. The sample can be cerebrospinal fluid (CSF), saliva, blood, serum, plasma,
urine or a hang aspirate.
Antibodies Thin Bind Specificalij.’ to At Least Gite Component of A
iii/IflMH’iafiflfi Inflomnmsome
{95362} The s described herein for reducing mation in the hangs of a mammal
include compositions including an antibody or an active fragment thereof as provided herein that
specifically binds to at least one component (egg ASC, AIMZ) of a mammalian inflammasome
(egg, the AIME inflammasome). A composition for treating and/or reducing inflammation in the
lungs of a mammal can r 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 US$685400, the contents of which are herein incorporated
by reference in its entirety.
{(3363} In one embodiment, a composition for treating and/or reducing inflammation in the
lungs of a mammal includes an antibody or an active fragment thereof as provided herein that
specifically binds to a domain or n f of a mammalian ASC protein such as, for
example, a human, mouse or rat ASC protein. Any suitable SC dy can be used, and
several are commercially ble, Examples of anti-ASC antibodies for use in the methods
herein can be those found in 1188685400, the contents of which are herein incorporated by
reference in its entirety, Examples of rcially available anti—ASC antibodies for use in the
methods provided herein include, but are not limited to Od—l47 Anti—ASC, clone 23—7 mouse
monoclonal antibody from MilliporeSigma, [US$607 ~ Anti-ASC Antibody from Millipore Sigma,
orb19402l Anti-ASC from Biorbyt, LS~C331318~50 Anti-ASC from LifeSpan ences,
[$3805 Anti—ASK: from R 8; D Systems, NBPlu78977 Anti-ASC from Novas Biologicals, 600~
401-Y67 Anti—ASE". from Rockland lmmunochemicals, D0863 AntiuASC from MEL
international, AL177 anti—ASC from Adipogen, monoclonal anti—ASC (clone 093 139) antibody,
anti—ASC antibody (Ii—9:) from Santa Cruz Biotechnology, anti—ASC antibody (B3) from Santa
Cruz Biotechnology, ASC onal antibody n ADLQGSJ’ZE} from Enzo Life Sciences, or Alol
Anti—Human ASC — Leinco 'l‘echnologies. The human ASC n can be accession number
NP___037390.2 {Q9ULZ3nl}, NP___660183 (Q9ULZS—2} or Qatirzso. The rat ASC protein can be
accession number NP____758825 (BAC43754). The mouse ASC protein can be ion number
NP___O75747.3. In one ment, the antibody binds to a PY’RlN-PAAD—DAPIN domain (PYD)
or a portion or fragment thereof of a mammalian ASC protein (egg. human, mouse or rat ASC). in
this embodiment, an antibody as described herein specifically binds to an amino acid. sequence
having at least 65% (eg, 65, 70, 75, 80, 85%) sequence identity with a FYI) domain or fragment
thereof of human, mouse or rat ASC. In one embodiment, the antibody binds to a C~terininal
evrecruitment domain (CARD) or a portion or fragment thereof of a mammalian ASC
protein {eg human, mouse or rat ASC). In this embodiment, an antibody as described herein
specifically binds to an amino acid sequence having at least 65% (eg, 65, 7", 75, 80, 85%)
sequence identity with a CARD domain or tiragment f of human, mouse or rat ASC. In still
r embodiment, the dy binds to a portion or fragment thereof of a mammalian ASC
protein sequence leg. human, mouse or rat ASC) located between the PYD and CARD domains,
In another embodiment, a composition for treating and/or reducing inflammation in the lungs of a
mammal includes an dy that specifically binds to a region of rat ASC, e. g, amino acid
sequence ALRQTQPYLVTDI_,EQS (SEQ ID NO:1) (ie, residues l78-l 93 of rat ASC, accession
number BAC43754) ln this embodiment, an antibody as described herein specifically binds to an
amino acid sequence having at least 65% (eg, 65, 70, 75, 80, 85%) sequence identity with amino
acid sequence ALRQTQPYLVTDLEQS (SEQ ll) N0:l) of rat A80 in another embodiment, a
composition for treating and/or reducing inflammation in the CNS of a mammal includes an
antibody that specifically binds to a region of human ASC, e.g., amino acid ce
RESQSWJVEDLERS (SEQ ID N02). in one embodiment, an antibody that binds to an ASC
domain or fragment thereof as described herein inhibits ASC activity in lung cells, eg Type ll
alveolar cells of a mammal.
{9064} ln another embodiment, a composition for reducing ation in the lungs of a
mammal includes an antibody or an active fragment thereof as provided herein that specifically
binds to NLRPl (eg, antinNLRPl chicken antibody) or a domain thereof. Any le anti“
NLRPl dy can be used. and several are commercially ble. Examples of antinNLRPl
antibodies for use in the methods herein can be those found in US$685400, the contents of which
are herein orated by reference in its entirety. Examples of commercially available anti—
NLRPl antibodies for use in the methods ed herein include, but are not limited to human
NLRPl polyclonal antibody [£36788 from R&D Systems, EIVID ore rabbit polyclonal anti—
NLRPl ABFZZ, Novus Biologicals rabbit polyclonal anthNLRPl NBlOOééld—S, SigmamAldrich
mouse polyclonal anti—NLRPl SM3140715L Abcam rabbit polyclonal anti—NLRPl g
Biorbyt rabbit polyolonal anti—NLRB, orb325922 mybiosource rabbit onal anti~NLRPl
l‘vaS700'l225, Rail?) systems sheep polyclonal AF6788, Aviva Systems mouse onal anti—
NLRPl oaed00344, Aviva s rabbit polyelonal anti~Nl_..RPl AR,054478_P05Q Origene
rabbit polyclonal anti—NLRB 75PU-N, Antibodies online rabbit polyelonal anti—NlRPl
ABIN’768983, Prosci rabbit polyclonal anti—NLRPl 3037, Proteinteeh rabbit polyclonal anti—
NlRPl l2256—l—AP, Enzo mouse monoclonal anti,~Nl_.-RPl ALK—SOd-SOS—Clfltt lnvitrogen
mouse monoclonal. a.n.ti-Nl...RPl_ MAl.~258/-’l2, GeneTex mouse monoclonal anti—NLRH.
GTXl 60%? Rockland rabbit polyclonal anti—NLRPl 200—401(in or Cell ing Technology
rabbit polyclonal antiuNLRPl 4990. The human NlRPl protein can he accession number
AAl—l5l787, NP_001028225, NP_055737, NP_l27497, NP_l 27499, or NP_l27500. in one
embodiment, the antibody binds to a Pyrin, NACHT, LRRl ~6, FllND or CARD domain or a
portion or fragment thereof of a, mamn'ialian NLRPl protein (eg, human NLRPl). ln this
embodiment, an antibody as described herein specifically binds to an amino acid sequence having
at least 65% (eg, 65, 70, 75, 80, 85%) sequence identity with a specific domain (eg, Pyrin,
NACHT, LRRl ~67 FHND or CARD) or fragment thereof ot‘hun'ian NLRPl. In one ment,
a. n anti—NI..RPl polyclonal that was custom—designed and produced by Ayes Laboratories
is used for reducing lung inflammation. This antibody can be directed t the following amino
acid sequence in human NLRP]: CEYYTEIREREREKSEKGR (SEQ ll) N023). In one
embodiment, an antibody that binds to a NLRPl domain or fragment thereof as described herein
ts NLRl’l activity in lung cells, cg, Type ll alveolar cells of a mammal.
{0065} In yet another ment, a eomposition for reducing inflammation in the lungs of a
mammal includes an antibody or an active fragment f as provided herein that specifically
binds to Alb/£2 or a domain f. Any suitable anti~AlM2 antibody can be used, and several are
commercially available. Examples of commercially available antinAle/l antibodies for use in the
methods provided herein include, but are not limited to a rabbit polyclonal antinAlMZ cat. Number
ZOSQO—lmAP from Proteinteclin Abcam anti-AlMS antibody (abl 19791), rabbit polyclonal anti—
AlM'j’, (bl—terminal region) Cat. Number AP3851 from ECM ences, rabbit polyclonal anti—
ASC Cat. Number E-ABn30449 from Elabsciences,, AntiwAlMZ mouse monoclonal antibody
called Alli/£2 Antibody (3C4Gl l) with catalog number 50293174 from Santa Cruz Biotechnology,
mouse monoclonal Alb/{Z antibody with catalog number TA} 24972 from efi AlMZ
monoclonal antibody ( ) from fisher Scientifid AIMZ rabbit polyclonal antibody
ABIN928372 or ARIN/”60766 from Antibodies—online, Biomatix coat anti,~AlMZ polyclonal
antibody with cat, Number CAE02l53. Anti-AIMZ polyclaonl antibody (OABFOloTEZ) from
Aviva s Biology, rabbit polyclonal anti- Alb/l2 antibody LS—C3S4l27 from LSBio-
C354127, rabbit monoclonal anti—AIMZ antibody from Cell Signaling Technology; with cat
number b/lAS-liSZSQi Rabbit polyclonal anti-Albiz monoclonal. antibody from Fab Gennix
international incorporated, Cat. Number Alb/l2 201M), MyBiosouree rabbit polyclonal anti—
AIMZ cat number MBSSSSTQO, Signalway rabbit polyclonal anti Alb/l2 g number 36253,
Noyes Biological rabbit polyclonal anti—MMZ catalog number 02, GeneTex rabbit
polclonal anti-A342 GTXS49lO, Prosci, rabbit polyclonal anti—AIMZ 2.6-540, Biorby't mouse
monoclonal anti~AlM2 orh333902, Abcain rabbit polyclonal anti-All‘dZ ab93015), Abcam rabbit
polyclonal anti~/-‘\.lMZ ab76423, Signma Aldrich mouse onal lh/lfiz SABl 406827, or
Biolegend anti—AlMT/l. 3Bl0. The human AIMZ protein can be accession number NX_0l4862,
W004824, X13016853337, XP005245673, AABRléB, BAF34’73l or AAl-il0940. in one
enibodii’nent, the antibody binds to a Pyrin or l-llN—ZOO domain or a portion or fragment thereof of
a mammalian Alli/l2 protein (eg, human Alli/l2). In this embodiment, an antibody as bed
herein specifically binds to an amino acid sequence having at least 65% (eg 65, 70, 75, 80, 85%)
sequence ty with a specific domain (eg, Pyrin or l-llN—ZOO) or fragment thereof of human
AIMZ. in one embodiment, an antibody that binds to a AIMZ domain or fragment thereof as
described herein inhibits AIMZ activity in lung cells, eg, Type II alveolar cells of a mammal.
{9966} iIfinti—inflarnmasonie (cg, AntinASC, antimNLRPl or anti—AlMZ) dies as
bed herein include polyclonal and monoclonal rodent antibodies, polyclonal and monoclonal
human antibodies, or any portions thereof, having at least one n binding region of an
ininiunoglobulin variable region, which antibody specifically binds to a component of a
mammalian inflammasome (cg, Alli/£2 inflammasorne) such as, for e, ASC or AIME in
some cases, the antibody is specific for ASC such that an antibody is specific for ASC if it is
ed against an epitope of the polypeptide and binds to at least part of the natural or
recombinant protein.
{9967} Methods for ining monoclonal antibody specificity and affinity by competitive
inhibition can be found in Harlow, et al, Antibodies: A Laboratory Manual, Cold Spring Harbor
tory Press, Cold Spring Harbor, NY, l988, Colligan et al eds, Current Protocols in
immunology, Greene Publishing Assoc. and ‘Wiley lnterscience, NY, (l99‘2, 1993), and Muller,
Moth. Enzymol. 91589601, l. 983, which references are entirely incorporated herein. by reference.
{9968} Anti—inflainmasome (eg, Anti—ASC and anti—AIME) antibodies of the present
ion can be routinely made according to methods such as, but not d to inoculation of an
appropriate animal with the polypeptide or an antigenic fragment, in Wire stimulation of
lymphocyte populations, synthetic methods, omas, and/or recombinant cells sing
nucleic acid encoding such anti~ASC or anti-NLRl antibodies. immunization of an animal using
purified recombinant ASC or peptide fragments thereof, e. g, residues l78— l 93 {SEQ ID NO: l) of
rat ASC (cg, ion number BAC43754) or SEQ ll) N8] of human ASC, is an example of a
method of preparing anti—ASC antibodies, Similarly, immunization of an animal using purified
recombinant NLRPl or peptide fragments thereof, eg, residues MEE SQS KEE SNT EG—cys
(SEQ ll) N04) of rat NALPl or SEQ ll) NOE of human NALPl, is an e ol’a method of
preparing an ti —NLRPl antibodies.
} Monoclonal dies that specifically bind ASC or NlRPl may be obtained by
methods known to those skilled in the art. See, for example Kohler and Milstem, Nature 2563:4951"
497, 1975; US. Pat. No. 4,376,l l0; l et al, eds, Current Protocols in Molecular Biology,
Greene Publishing Assoc. and Wiley lnterscience, NY, (1987, l992); ow and Lane
AN'l’lBODlES: A Laboratory Manual Cold Spring Harbor Laboratory Press, Cold Spring ,
NY, l988; Colligan et al, eds, Current Protocols in immunology, Greene Publishing Assoc. and
Wiley lnterscience, NY, (1992, 1993), the contents of which are incorporated entirely herein by
reference. Such antibodies may be of any immunoglobulin class including lgG, lgM, lgE, lgA,
Gill) and any subclass thereof A hybridoma producing a monoclonal antibody of the present
invention may be ated in virro, in sin: or in Villa.
Administration of Compositions
{8976} The compositions of the invention may be administered to mammals (cg, rodents,
) in any suitable ation. For example, anti—ASC antibodies may be formulated in
pharmaceutically acceptable carriers or diluents such as physiological saline or a buffered salt
solution. Suitable carriers and diluents can be selected on the basis of mode and, route of
administration and standard. pharmaceutical ce. A description of exemplary
pharmaceutically acceptable carriers and diluents, as well as pharmaceutical formulations, can be
found in Remington’s Pharmaceutical Sciences, a standard text in this field, and in USPNF Other
substances may be added to the compositions to stabilize and/or preserve the compositions.
{3071} The compositions of the invention may be administered to mammals by any
conventional technique, Typically, such administration. will be by inhalation. or parenteral (cg,
intravenous, subcutaneous, intratumoral, intramuscular, intraperitoneal, or intrathecal
introduction). The compositions may also be administered directly to a target site by, for example,
surgical delivery to an internal or external target, site, or by catheter to a site accessible by a blood
vessel. The compositions may be administered in a single bolus, multiple injections, or by
uous infusion (e,g intravenously, by peritonealr is, pump on), For parenteral
administration, the compositions are preferably formulated in a sterilized pyrogenfree form.
Effective Doses
{lltl’72} The compositions described above are preferably administered to a mammal (eg, a
rat, human) in an effective amount, that is, an amount capable of producing a desirable result in a
treated mammal (eg, reducing inflammation in the CNS of a mammal ted to a traumatic
injury to the CNS or stroke or having an autoimmune or CNS disease). Such a therapeutically
ellective amount can be determined as bed below.
{9073} ty 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 like (the dose lethal to 50% of the population), The
dose ratio between toxic and therapeutic effects is the therapeutic index and it can be sed as
the ratio LDso/I'ED-so. Those compositions that t large therapeutic indices are red.
While those that exhibit toxic side effects may be used, care should he taken to design a delivery
system that minimizes the potential damage of such side effects. The dosage of preferred
compositions lies preferably within a range that includes an E1350 with little or no toxicity. The
dosage may vary within this range depending upon the dosage form employed and the route of
administration utilized.
{9074} As is well known in the medical and veterinary arts, 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.
{9875} The present ion is further illustrated by the following specific examples. The
examples are provided. for illustration only and should not be construed as limiting the scope of
the invention in any way.
Example 1: Role ofEV mediated inl‘larnmasome signaling in ALI following T’Bl and effects
of its neutralization
{sins} Pulmonary dysfunction often presents as a complication of Severe Traumatic Brain Injury
(l), Approximately 20—25 percent of "fill subjects develop acute lung iniuiy (All) (2), but the
mechanisms mediating the pathology of duced AU remain poorly defined. Previous
literature has supported the idea that pulmonary dysfunction after TBl is due to the sympathetic
response to increased intracranial pressure leading to cardiopulmonary dysfunction (42). More
recent studies, however, have shown that a systemic inflammatory response also plays a key role
in TBl—induced lung injury (43). ically, the l-lMGBl IRAGE ligand receptor pathway serves
as central transduction ism for pulmonary dysfunction after ’l’Bl {8). in addition, HMGBl
induces AIMZ inflanunasome activation (:37). Furthermore previous literature reveals that
pathogens secrete EV that carry DAMl’s, such as ELMGBl, and trigger mation (Buzas et al,
2014). Various s have shown that the blood brain barrier (BBB) is permeable after 181 as
early as 3—6 hours after injury resulting in damage to the protective barrier between the brain and
the intravascular compartment and leads to leakage of proteins and fluid (44). Disruption of the
BBB after injury results in the secretion of inflammatory mediators such as , which can
further hrain inflammation and damage distal organs (5). Several inflammatory mediators can act
as clear markers for brain injury, however their validity is not widely accepted (45). Furthermore,
there is currently no clinically ed ent or hioinarlcer for TBLinduced All Recently,
EV have become an area of st in ker research for a several different types of diseases,
including lung injury (46) and T81 (47), it has been previously shown that in EV isolated from the
cerebrospinal fluid of patient with TBl, there is an increase of inflainmasonie proteins when
compared to control samples (14), In this Example, the contribution of EV mediated
inflammasome signaling in the etiology of TBl—induced All was examined
Materials and Methods
Animals and tic Brain. injury
{9977} All animal procedures were approved by the lnstitutional Animal Care and Use
Committee of the University of Miami Miller School of Medicine (Animal e Assurance
A3224-Ol) and were done ing to the NEH Guide for the Care and Use of Laboratory
Animals. The ARRIVE ines were followed when conducting this study. All CSWBLc mice
were S—l 2 weeks and 24 to 32 grams. Mice were prospectively randomized to experimental groups
(sham, 4h, 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
procedure but were not injured. For adoptive transfer treatment studies, the sham—saline group
underwent surgical procedures and received saline as vehicle treatment. Naive animals underwent
no surgical procedures. A sample size of 5 to 6 was used for each group based on power analysis
(using (3* power analysis, with an effect size F2085, a set a 0.05) and historical data 49’ sol All
mice were housed in the Viral antigen free (VAF) animal ty at the Lois Pope Life Center at
the sity 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. s were observed post—op, where they were lrept on a g pad and body
ature was controlled with a rectal probe where it was maintained at 370C, in our operation
room and then transferred to the animal quarters.
{0078] Prior to surgery s were anesthetized with ne and xylazine (intraperitoneal,
i.p.). The anesthetized animals were then placed on a heating pad to ensure a hotly ature of
37°C. TEE was performed using a Controlled Cortical Impact (CCI) model. A 5 mm craniotomy
was made on the right cortex {---2. 5 mm posterior, 2.0 mm lateral from Bregina). lnj ury was induced
using the ECCI~63 device (Custom Design & Fabrication, Richmond, VA, USA) with a 3 mm
impounder at 6 rn/s velocity, 0.8 mm depth, and 150 ms impact duration (l 5). Following these
procedures animals were returned to their cages and given food and water. Animals were sacrificed
at 4 hours and 24 hours after TBl as described. Sham animals were anesthetized and subjected to
the same pie—surgical incision as injured animals but did not undergo a craniotomy or contusion.
’l‘issue collection
{9979} All animals were etized with ketamine and xylazine, prior to perfusion. Animals then
underwent al perfusion. Lungs were infused with 4% paraformaldehyde (PFA) using a
tracheal catheter at 20 cm H20 and then fixed in 4% PFA overnight at 40C. Fixed lung tissues
were n embedded and 5 pm sections were processed (16): Right lung tissue was collected
for protein isolation and molecular analyses. Animals then underwent decapitation and right
al tissue was collected for protein isolation and molecular analyses.
T’yroptosnme ion assay
{$989} Mice lung tissue s were filtered through a 5 pm low—binding polyvinylidene
difluoride (PVTW) membrane (Millipore). After filtration; the supernatant was centrifuged at 2,700
xg for 8 minutes. The pellet was resuspended in 40 pl of 3{(3~cholainidopropyl)
dimethylammonio]~propanesulfonic acid. (CHAPS) buffer (ZGmmOl/L HEPES—KOH, pH 7.5,
mmol/L MgClZ, O.51n,mol/L EGTAfi (ll nnnol/L phenylinethylsulfonyl fluoride, protease
inhibitor cocktail, and O.l% CHAPS). The pyroptosoine was pelleted by centrifugation at 2,700
xg for 8 minutes. The pellet was then resuspended and incubated in 27.8 pl of CHAPS buffer with
2.2 u} of disuecinimidyl substrate (9) for 30 minutes at room temperature to cross—link ASC dimers
Lastly, an equal amount of 2x Laernmli buffer was added and ns were analyzed by
inununohlottinq usin rciallv available dies to ASC and Clasderrnin D 6813
C‘ ..
Nuclear and Cytoplasmic Extraction
{9981] Nuclear and Cytoplasmic fractions were extracted using the NE—PER Nuclear and
Cytoplasmic Extraction Reagents (Therino Scientific) according to manufacturer instructions,
Briefly, rniee lung tissue samples were cut into 20-10% rng pieces and centrifuged at 500 x g for 5
s Tissue pieces were the homogenized with the Cytoplasmic Extraction t and
centrifuged at l6,000 x g for 5 n'iinutes, Then the supernatant (cellular extract) was removed and
the pellet was centrifuged with Nuclear tion Reagent (Therrno Scientific) at l6,000 x g for
minutes. This supernatant corresponded to the miclear fraction, which was removed and stored
at ~80“ C
lmmnnohlotting
{$982} Lung and brain tissue san'iples were snap frozen in liquid nitrogen and stored in ~8OCC 2-
mm sections of right lower lung and right cortical tissue were nized in extraction butter
containing protease and. phosphatase inhibitor cocktail (Sigma, St Louis, MO, USA) and resolved
in 4—Zt % Tris—TGX Criterion precasted gels (Bio—Rad, l-lercules, CA, USA) as bed in de
Rivera Vaccari et all 20l 5 (l3) using antibodies to caspase-l (Novas Biologicals), ASC (Santa
Cruz), lL—l ll (Cell Signaling), lL—lS (Aboarn) Alli/l2 (Santa Cruz) and l-lMGBl pore).
Quantification of band density was performed using Image Lab and all data. were normalized to B~
actin.
lmmunohisteclremistry
{0083] Tissue ns were deparall‘inized in xylene and then reliydrated using ethanol and Tris
buffer saline. lnununohistochenrical procedures were then carried out for double staining as
previously described (l6). Sections were incubated overnight at 40C with antibodies against
Caspase—l and. ASC pore), Alli/l2 (Santa Cruz) HWIGBI (Millipore) and SPC pore).
ostained lung sections of sham, 4 hour, and 24 hour mice were examined with a Zeiss laser
seaming confocal microscope (Zeiss, Inc, Thornwood, NY, USA). lung sections were analyzed
by individuals who were blinded to the groups.
EV Isolation
{0084} EV were isolated from serum from Till-injured mice and injury mice using the Total
Exoson'ie lsolation solution ing to manufacturer’s instructions (lnvitrogen). Briefly? l00 ul
of each sample were centrifuged at 2000 x g for 30 minutes. The supernatant was then incubated
with. 20 ill of Total Exosome isolation (TED reagent for '30 minutes at 40C ed by
centrifugation at l0,000 X g for 10 minutes at room temperature. Supernatants were discarded and
the pellet was resuspertded in 100 pl of PBS. EV were characterized by the expression of CD81
and by Nanosigbt ng analysis (.
Adoptive Transfer of EV
{0085} Serum-derived EV from 6 T31 and sham mice were injected into naive C578L—6
mice through the jugular vein at a dose of l0 X l010 particles per grain/body weight 48. Particle
count was measured by Nanosigltt Tracking analysis and samples were diluted accordingly. Prior
to surgery animals were anesthetized with ketamine and xylene. .A l. -2 cm incision. was made
between the jaw and the clavicle The jugular vein was elevated and tied, followed by catheter
ent. Serum-derived EV were erred and lung and brain tissues were collected 24 hours
after injection for analysis (n==5).
Ennxaparin and Ami—ASC treatment
{@886} Serum-derived EV from TBl mice were injected into naive (157—8146 mice through a
jugular vein inj ection. One hour later, Enoxaparm (3 rng/lig) (r124) and Anti-ASC (Sing/kg) (n===4)
were adn'iinistered to recipient animals The following groups were used: 1) the naive group
received no treatment, 2) the sham saline group was used as a negative control and underwent
jugular vein injection of only saline, 3) the untreated group received EV from TB] mice without
any treatment and was used as a positive control, 4) the ENOX group received EV from TBI mice
and Ei’ioxaparin, and S) the Anti~ASC group received EV from TB], mice and /-‘\.nti~ASC The order
of treatment was randomized. Lung and brain tissues were collected 24 hours after injection for
analysislt should he noted that the anti-ASC antibody used in the treatment ments was a
humanized monoclonal antibody against ASC and recognizes murine, human and swine ASC.
lilistology and Lung injury Scoring
{@087} Lung tissue sections were stained by a standard xylin and eosin method for
ogy, morphometry and ALI scoring. Lung sections were scored by a d ogist
using the Lung injury Scoring System from the American ic Society Workshop Report (l 7).
'l‘wenty random high power fields were chosen for scoring. ia for ALI scoring was based on
number of neutrophils in the alveolar space, interstitial space, hyaline membranes, proteinaceous
debris filling the aii‘spaces and alveolar septal thickening. Based on these criteria a score between
0 (no ) and 1 (severe injury) was given.
Statistical Analysis
{9088} Data were analyzed using a student’s ”lFtest for two groups and a oneuway ANOVA
followed by Tukey’s le comparison tests, (GraphPad Prism version 7.0) for two or more
groups. D’iltgostinod’earson test was used to test for normality. Data are expressed as mean .+./_
SEM. P values ofsignificance used were * p<0.05.
Results
Severe TB} increases Alli/12 infiaininason‘ie proteins and thGBl expression in the brain of
mice
{8889} Excessive levels of the proini‘larnmatory cytoltine lL—l f5 and llml 8, and intiamrnasorne
proteins are associated with secondary damage after fluid—percussion brain injury (l8) To
determine whether severe CCl induced processing of prointlarrnnatoiy nes and alterations
in levels of inflarnmasonie proteins, al lysates were analyzed, however there is limited
research on inflammasonie activation in severe TEL In this Example, following severe CCL
cortical lysates were examined for the levels of the caspase-l (Fig EA, B) (p<.00l), ASC (Fig EA,
C) (pm .003}, IL—is (Fig 1, A, n) (p===z0042), AIMZ (Fig. 1A, F) s?) and iL-is (Fig 1 A,
G) (p===0.0l 41) at 4 and 24 hrs after injury” levels of caspase—1, ASC, Ally/£2, and lL-lfi peaked
at 4 hours after CG and decreased by 24 hrs. The time course for maturation of inflammatory
cytokines ed slightly but peaked by 24 hours after TEL Since others have shown a role for
the masome fl’ ELMGBl activating the Alli/12 inflammasome, the levels of these
proteins were also ined in cortical lysates. As shown in FIGS. 1A, 1E, CCi induced a
significant increase in the levels ofHMIGBl (.) 1E) (13:0.0l 2i) at 4 and 24 hrs after injury,
These data indicate that following severe CCI in mice the levels of the Ali‘s/l2 inflammasome
proteins were significantly elevated in the cortex following inj ury.
Severe TBI increases Aihfl inflammasome protein and HhiGBl expression on the lungs of
mice
{9999} To determine whether CCI d inflammasome activation in the lungs, an inoblot
analysis of lung lysates was performed for caspase~l (Fig l H, I) (p=.0026), ASC (Fig 1 H.) J)
(p=.0427)3 HAS (Fig 111.) K) (p=.0025), IL-lfs (Fig 1 HM) (p=_0012) and AIMZ (Fig l HJVE)
(p<.001), and NLRP3 (p=.0047) (Supplemental Figure 1). increased levels of caspase—1, ASC, IL—
18 and AIME were significantly increased at 4 hrs and 24 hrs after injury as compared to the sham
control. However the time course of the increase in protein sion differed slightly from that
observed in brain in which they peaked at 24 hr after CCl. Since, the thGBl "RAGE axis plays
a role in the mechanism by which TBI induces lung dysfunction (8), long lysates were analyzed
for levels ei‘HMGBl pretein expression. Fle. EH, 1L (pZDlSS) shows lMGBl expression
increased at 4 and 24 hours after TBI, indicating that the AIMZ ini‘lai'nrnaserne and HMGBl play
a role in the inflammatory response in the lungs FBI.
TEE induces pyreptesis in the lungs of mice
{Milli} As shown previously, activation of the 2 intlarnrnasorne in cortical neurons leads to
pyroptotic cell death (19)?) investigate whether TBI results in pyrcptnsis in mice lung tissue, the
pyroptosoi'ne in lung tissue was isolated after T81. TBl animals, sacrificed at 4 hours post-injury
showed ce ofASC oligoinerization compared to sham animals (). ASC dirneis and
triniers were seen in TBl animals (50, 75 kDA respectively) These results were indicative of
pyroptoscme formation, which can he characterized by the supramolecular assembly of ASC
ners. in addition, gasderniin D(GSDl\/1D), which is cleaved upon activation ofcaspase—l and
triggers pyroptosis and the release of lLulfi (20), was icantly sed in the lungs of TBI
animals canipared to sham {Flt}, 4B: and 4C) (p===0.000l). These findings indicated that pyroptosis
contributes to cell death in lung tissue after ”l‘Bl.
TB} increases lmmnnureactivity of asome pruteins in type H alvenlar epithelial cells
{M1592} TB} may lead to capillary leak, resulting in increased vascular permeability and damage
to specialized alveolar epithelial cells, called type ll pneumocytes (5). To examine the cellular
effects of TBI on inflamniasonie expression in the lungs after injury, immunohistcchemical
is was perfernied in lung sections of shani, 4 hear, and 24 hour injured s. Type ll
alveolar lial cells are known to he the main type of lung cells injured in All (17), Lung
sections were stained with antibodies against AlMI-Z, caspase-l, and ASC (green) and, constained
with Pro—surfactant protein C (PromSPC, red), a marker of type ll lial cells, and SAP}; nuclear
staining (blue). As Shawn in FIG. ZA—ZC‘, active caspasenl (), ASC (FIG. Ell), as well as
AIMZZ () are present in Sl’C—positive cells (arrow). lmniunoreactivity of these
inflaniniasoine proteins sed after 'l‘Bl. These findings indicate that inflainniasorne proteins
are expressed in type ll alveolar epithelial cells and that TBI results in increased innnuncreactivity
in these cells.
2017/068713
"Elli ses r and cytoplasmic Hill/{Gill expression
{6393] In order to determine the ar distribution of HMGBl in lung cells after Tidal,
r and cytoplasmic fractions from lung homogenates were isolated (, 3C) (p:=.0337).
lmmunohlotting indicated that both fractions had significant increases in lthGBl expression at 4
hrs post—T31 ( 3D) (p:,0345), lmmunchistocheniical analysis of HMGBl was also
performed in order to determine the changes in imniunoreactivity in lung sections after TBl.
Sections were co—stained for HMG-Bl ) and SPC (red) and DAPI. nuclear staining (blue).
lmmunoreactivity of l-lMGBl was increased at 4 hrs and 24 hrs when compared to sham. Weak
immunoreactivity of HMGBl was observed in SPCupositive cells (arrow) (PEG. 3511‘); therefore,
suggesting that l—lMGBl changes in the injured lung tissue may he asmic.
Till induces changes in lung morphology and induces AL}
{0694} AM can he characterized by inflammatory processes, which lead to ar and interstitial
edema as well as infiltration of inflammatory cells into the alveolar space (23). l-listopathological
analysis of lung tissue (Fifi. 5A) indicate that severe TBl 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
sed ly by 24 hours after injury (long arrows). In addition, there was evidence of
neutrophil infiltration (arrow heads) and changes in morphology of alveolar capillary membranes
(*7) at both time points. Injured animals showed signs of titial 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, d).
{(3095} To confirm that severe injury induces All, histological sections were analyzed using the
All g system defined by the American Thoracic Society (l7), This system is based on
ce of neutrophil ration into the alveolar and interstitial spaces, hyaline membrane
formation, proteinaceous debris filling the airspaces, and alveolar septal thickening.(l7). These
characteristics were significantly elevated in injured animals and All scores were higher overall
in. T31 animals compared to sham (FIG. SB) (p=0.0017).
Enoxaparin and antiwASC antibody treatment significantly s inflammasome
expression and ALI after adoptive transfer at EV from TBI mice
{91396} in order to provide evidence that EV and their cargo that can he released into the
circulation after fill may induce inflammasonie activation in the lung, a classic adoptive transfer
experiment was performed using serum—derived EV from severe CCl mice. EV ations were
validated using Western Blot for EV marker CD8l (. Controls received EV isolated from
sham or naive animals. As shown in -7F, active caspase—1 (, 7B), .ASC ( ,
’7C), lLulS (Fifi. 7A, ’71)), AIMEE (, 7E) and lilleBl (Fifi. 7A, 7 7) were significantly
elevated in the lungs of animals that received the EV from TBl injured animals when ed to
the lungs of animals that receive EV from red or naive mice or naive mice. Furthermore,
infiltration of inflammatory cells (arrows) was apparent in lungs treated with EV from TB} mice
(PEG. 7G). Lastly, ALl score was also significantly higher in animals that received EV from
injured, mice (PEG. 7H). These studies provided evidence for a neuralnrespiratoryninflammasome
axis in which EV released into the circulation after ’I'Bl activate the inflammasome in lung target
cells contributing to the pathogenesis of ALl
{0097] Next, 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 s ed saline and positive control s received no treatment. As
shown in, ~8E e—l (, SB), ASC (FIG. SA, 8C), lL~ll3 (, 81)),
AIMZ (Fifi. 8A, 832), and l-IMGBl {Flt}. 8A, 8F) were icantly reduced (:p====<.000l) as
compared to untreated (positive control} group after treatment with Enoxaparin or a humanized
monoclonal anti—ASC antibody l: e. g. lC lfil} antibody). in addition, H&E stained lung ns
showed significantly less neutrophil infiltration into alveolar and, interstitial space, as well as no
signs of septal thickening (FIG. Stilt—D). ALI scores for animals treated, with Enoxaparin and anti—
.ASC dy (1C 100) were significantly lower compared to untreated group ()
(p=<.GGOl). Thus, EV released into the ation after TBI play a role in inflammasome
activation in lung cells leading to ALL
Conclusions
2017/068713
{$998} TBl can he associated with higher rates of certain rnedical complications, especially
pulmonary and central s system dysfunction. In this Example, severe TBl was shown to
increase HMGBl and intlainrnasorne expression (e.g, talk/l2, caspase- l. and ASC expression) in
cortical and lung tissue and induce cl'ianges in lung morphology consistent with All (eg,
infiltration of neutrophils into the alveolar and interstitial space, alveolar septal thickening, and
alveolar edema and hemorrhage) and introduces the idea of a Neural atory inflammatory
Axis. Iniportantly, TBI resulted in pyroptosis in lung tissue (eg presence of @813th cleavage)
and increased expression of inflammasorne proteins in Type ll alveolar epithelial cells.
Additionally, ve transfer of EV from 'I‘Bl mice activated the inflaminasoine and induced
ALI, indicating that brain injury induces the release of EV containing a cargo of inllainmasorne
proteins that are then carried to the ing in AU. Moreover, it was shown that by both inhibiting
EV uptake (Enoxaparin) and inflammasome activation (anti—ASC antihody (1C lOO) treatment),
there is a ion in inflamniasonie protein expression and in the pment of ALI.
{9099} in summary, this Example showed that AlMZ inflaininasorne signaling plays a central role
in the pathoineclianism of lung injury after ’l'Bl and demonstrates a mechanism of Till—induced
ALI involving EV-inediated inflammasorne signaling. These data provided ce that EV-
mediated, inflamniasonie signaling can play a central role involving a al—Respiratory—
lnflaniinatoiy Axis. Therefore. targeting this axis with antibodies t inflamniasoine ns
or drugs that block. EV uptake may provide a therapeutic approach in Neurotraurna—induced All
in all areas of critical care medicine. In light of these results, the disclosed therapeutic strategies
may be useful for the treatment of inflammatory diseases of the lung, in general.
Incorporation by reference
lillllllllll The following references are incorporated by reference in their entireties for all
purposes
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Example 2: Role of EV mediated inflammasome signaling in ALE following TBI in human
{@151} As a follow up to the ments on mice in Example 1, the role of EV isolated from
human ’I'Bl ts on inflamniasome signaling in human pulmonary endothelial cells was
examined.
{89152} in a first experiment, serum—derived EV were isolated from 'l‘Bl and control patients
using Total Exosome Isolation lrit ('l‘lierrnofisher). Pulmonary Human Microvascular Endothelial
Cells- (HMVECnLonza) were cultured and plated on a 12—well plate. After confluency was reached,
isolated EV from TB} and control patients were delivered (l .94 X 108 particles/ml) to cells for an
incubation period of 4 hours. After incubation cells were harvested, with 200 ul of lysis buffer and
cell lysates were used for Western Blot analysis.
{$91.53} In a second experiment, nderived EV were isolated from 'l'Bl and, control patients
using Total Exosome Isolation kit (’l‘herrnofisher). Pulmonary Human Microvascular Endothelial
Cells (Elli/WED Lonza) were cultured and plated on a l plate. After confluency was
reached, isolated EV from T31 and control patients were delivered {1.94 x lOS paiticles/ml) to
2017/068713
cells for an incubation period of 3 hours and then 1 additional hour with caspase—l FAR/lFl_,lC1-li
(lnnnunohistochernistry Technologies) with a, l:30 volume to volume ratio After incubation,
media, was removed and cells were washed 3 times with sis wash buffer
(lnnnunohistochernistry Technologies) Cells were then ined with Hceclist for r
staining and Propidiuin Iodide for cell death. Images were taken using an EVOS microscope and
then cells were read under a fluorescent plate reader at an excitation wavelength of 492 nrn and an
emission ngth of 5’20 nrn.
Results
{4919154} As shown in FIG. ltlAulflF, delivery ofserurn—derived EV from TB} patients increased
inl’laniinasorne protein sion in pulmonary elial cells. FIG. MBA—10E showed that
caspase—1 , ASC, AlMZ, and HMGBl were elevated in Ph/WEC incubated with EV for 4 hours
as compared to PMVEC incubated with controluE’V for 4 hours. Immunoassay results showed a
significant increase in lL—lheta expression using Ella simple plex assay {Flt}. NF),
{$0155} As shown in EEG. llA-llC, delivery of TELEV to pulmonary endothelial cells
increased immunoreactivity of caspase-l and cell death.
Cnneluslon
{90156} These studies provided further evidence for a neural—respiratory»inflannnasorne axis in
which EV released into the circulation after 'l'Bl. activate the inflammasome in lung target cells
contributing to the pathogenesis of AM.
{89157} The various embodiments described above can he combined to provide further
embodiments All ofthe U. S patents, US patent application publications, US patent application,
foreign patents, foreign patent application and non—patent publications referred to in this
specification are orated herein by reference, in their entirety. Aspects of the embodiments
can be modified, if necessary to employ concepts of the various patents, application and
publications to provide yet further embodiments.
{4919158} These and other changes can be made to the embodiments in light of the above—
detailed description In general, in the following claims, the terms used should not be construed
to limit the claims to the specific eh'tbodimehts disclcsed in the ication and the claims, but
should be construed to include all possible embodiments along with the full scope of equivalents
to which such claims are entitled. Accordingly, the claims are not limited by the disclos die.
Claims (1)
- What is claimed is: 1. l. A method of treating inflannnation in lungs of a patient in need thereof, the method sing: administering to the patient a ition comprising an agent that inhibits intlarnniasonie signaling, whereby the inflammation in the lungs of the t is d. .N The method of claim 1, wherein the ation in the lungs is caused by a condition selected from a l nervous system (CNS) inj my, a neurodegenerative disease, an autoimmune disease, asthma, chronic obstructive pulmonary disease, cystic fibrosis, interstitial lung disease and acute respiratory distress syndrome. U} The method of claim 2, wherein the CNS injury is selected from the group consisting of traumatic brain iniury , stroke and spinal cord iniury (SCI). The method of 2, n the neurodegeneratiye disease is selected from the group consisting of aniyotrophic lateral sclerosis (ALE), multiple sclerosis (MS) and Parkinson’s disease (PD). The method of any one of the above claims, wherein the administration of the composition results in inhibition of nmasorne activation in lung cells of the patient. 6. The method of any one of claims 1—4, wherein the administration of the composition results in a reduction of caspasenl leucine~rich repeat pyrin domain containing , nucleotide—binding protein 1 (NLRPl), nucleotide—binding lencine~rich repeat pyrin domain containing n 2 (NTRP2), tide~hinding lencine—rich repeat pyrin domain containing protein 3 (NI_RP3), NLR family CARD domain—containing protein 4 (NLRCZl), caspase-ll, X— linked inhibitor of apoptosis protein (XIAP), pannexin~l, Apoptosis~associated Spec—like protein containing a Caspase Activating Recruitment Domain (ASC), interleukin—l 8 (Hr l8), high. mobility group hex .l (HMGBl) or absent in melanoma 2 (AIME) levels in lung cells of the patient as compared to a control, wherein the l is an untreated patient. The method of claim 5 or 6, wherein. the lung cells are Type ll alveolar cells. The method of any one of claims l—S, wherein the administration of the composition results in a reduction in acute lung injury (ALI) as compared to a, control, wherein the control is an untreated patient.
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