U.S. patent application number 14/131406 was filed with the patent office on 2014-05-22 for use of antagonists targeting metallothionein to treat intestinal inflammation.
This patent application is currently assigned to Univeristy of Connecticut. The applicant listed for this patent is Martine De Vos, Lindsey Devisscher, Debby Laukens, Michael A. Lynes. Invention is credited to Martine De Vos, Lindsey Devisscher, Debby Laukens, Michael A. Lynes.
Application Number | 20140141009 14/131406 |
Document ID | / |
Family ID | 44585016 |
Filed Date | 2014-05-22 |
United States Patent
Application |
20140141009 |
Kind Code |
A1 |
De Vos; Martine ; et
al. |
May 22, 2014 |
USE OF ANTAGONISTS TARGETING METALLOTHIONEIN TO TREAT INTESTINAL
INFLAMMATION
Abstract
Metallothioneins are stress inducible proteins with modulating
functions during inflammation. Antagonizing metallothioneins during
gut inflammation reduces markers of inflammations and enhances
recuperation of intestinal inflammation.
Inventors: |
De Vos; Martine; (De Pinte,
BE) ; Laukens; Debby; (Brecht, BE) ;
Devisscher; Lindsey; (Moortsele, BE) ; Lynes; Michael
A.; (Eastford, CT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
De Vos; Martine
Laukens; Debby
Devisscher; Lindsey
Lynes; Michael A. |
De Pinte
Brecht
Moortsele
Eastford |
CT |
BE
BE
BE
US |
|
|
Assignee: |
Univeristy of Connecticut
Farmington
CT
|
Family ID: |
44585016 |
Appl. No.: |
14/131406 |
Filed: |
July 9, 2012 |
PCT Filed: |
July 9, 2012 |
PCT NO: |
PCT/EP2012/063372 |
371 Date: |
January 7, 2014 |
Current U.S.
Class: |
424/145.1 ;
424/158.1 |
Current CPC
Class: |
A01K 2267/0368 20130101;
C07K 16/18 20130101; C07K 2317/76 20130101; A61P 37/06 20180101;
C07K 14/825 20130101; A01K 67/0275 20130101; C07K 2317/24 20130101;
A01K 67/0276 20130101; A01K 2217/075 20130101; A01K 2217/052
20130101; A01K 2227/105 20130101; A61K 2039/505 20130101 |
Class at
Publication: |
424/145.1 ;
424/158.1 |
International
Class: |
C07K 16/18 20060101
C07K016/18 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 8, 2011 |
EP |
11173275.6 |
Claims
1. An antagonist targeting metallothionein for use in treating
intestinal inflammation wherein said antagonist is an
anti-metallothionein antibody or a fragment thereof which
specifically binds to said metallothionein.
2. An antagonist according to claim 1, wherein said intestinal
inflammation is Crohn's disease, ulcerative colitis, collagenous-,
lymphocytic-, ischemic-, diversion- and indeterminate colitis and
Behcet's disease in mammals.
3. An antagonist according to claim 1, wherein said antibody is a
monoclonal anti-metallothionein antibody.
4. An antagonist according to claim 3, wherein said monoclonal
anti-metallothionein antibody is the monoclonal
anti-metallothionein antibody clone UC1MT.
Description
TECHNICAL FIELD
[0001] The present invention is based on the finding that reducing
bioactive metallothionein levels during intestinal inflammation
results in a significant therapeutic effect. Hence, the present
invention relates to antagonists targeting metallothionein, such as
antimetallothionein antibodies, and provides new therapeutics for
the treatment of intestinal inflammation such as inflammatory bowel
diseases.
BACKGROUND ART
[0002] Inflammatory bowel diseases (IBO) comprising Crohn's disease
and ulcerative colitis are inflammatory conditions of the colon
and/or the small intestine. Symptoms may overlap with other forms
of chronic intestinal inflammatory conditions such as collagenous-,
lymphocytic-, ischemic-, diversion- and indeterminate colitis and
Behcet's disease. The exact etiology of these diseases is believed
to be multifactorial but is to date still undefined
(Gastroenterology, Vol 150, No 6 May 2011, 1701-1846). Therefore,
current treatment aims at dampening the ongoing inflammation and is
based on symptoms and presence of lesions. It is clear that new and
efficient therapies for IBD with a minimum of side effects are of
great interest.
[0003] Metallothioneins are cysteine rich zinc binding proteins
which play a role in different cellular processes such as zinc
homeostasis, oxygen radical scavenging, cell proliferation and
apoptosis. They are considered to be acute phase proteins due to
their rapid induction by different stimuli, including inflammation
(Inoue et al. 2009).
[0004] M. Lynes (US 2003/0007973) indicated that an
anti-metallothionein antibody is particularly useful to stimulate
the immune response in a subject undergoing irradiation or
chemotherapy, autoimmune subjects, subjects exposed to
immunosuppressive agents, neonates and subjects having undergone
transplantation.
[0005] Waeytens et al. (2009) review the role of metallothioneins
in IBD and conclude that there is a deviant metallothionein
expression in IBD but that the role of these proteins during
disease is not clear.
[0006] Tran et al. (2007) and Oz et al. (2005) investigated the
effect of the presence or absence of metallothioneins via
metallothionein knock-out mice but could not observe a significant
and beneficial effect of the absence of metallothioneins.
[0007] Devisscher et al. (2011; abstract) describe that
metallothioneins have been proposed to have a role in the
pathogenesis of IBD and conclude that the low metallothionein
profile in IBD patients may point to a hypoxia-driven adaptive
response in the course of gut inflammation.
[0008] Taken together, it remains unclear whether a reduction of
metallothionein might have a beneficial role during IBD. The
aberrant expression levels in IBD patients and their upregulation
during inflammation (De et al. 1990; Laukens et al. 2009)), even
suggest a protective effect of metallothionein during intestinal
inflammation. The present invention surprisingly demonstrates that
administration of antagonists targeting metallothionein, even
targeting solely secreted metallothioneins by use of antibodies,
results in a significant therapeutic effect of intestinal
inflammation.
SUMMARY
[0009] The inventors have unexpectedly found that a lack of MTs is
beneficial in experimental colitis. Moreover, treatment with an
anti-MT antibody reduces the chemotactic properties of
extracellular MT, resulting in reduced leukocyte infiltration and
dampening of the inflammatory response during colitis and avoids
some or all of the negative side effects of other biologic
treatments currently used for IBD. Hence, anti-MT is an attractive
novel biological in the treatment of IBD patients.
[0010] The invention therefore provides an antagonist targeting
metallothionein for use in treating intestinal inflammation wherein
said antagonist is an anti-metallothionein antibody or a fragment
thereof which specifically binds to said metallothionein.
[0011] In a preferred embodiment, said intestinal inflammation is
selected from the group comprising or consisting of: Crohn's
disease, ulcerative colitis, collagenous-, lymphocytic-, ischemic-,
diversion- and indeterminate colitis and Behcet's disease in
mammals.
[0012] In another preferred embodiment, said antibody is a
monoclonal anti-metallothionein antibody. More preferably, said
monoclonal anti-metallothionein antibody is the monoclonal
anti-metallothionein antibody clone UC1MT.
[0013] The invention further provides a method of treating
intestinal inflammation in a subject, comprising the steps of
administering to said subject a therapeutically-effective amount of
a composition comprising an antibody or a fragment thereof which
specifically binds to metallothioneins.
[0014] In a preferred embodiment of said method, said antagonist is
an anti-metallothionein antibody or a fragment thereof which
specifically binds to said metallothionein.
[0015] In a preferred embodiment of said method, said intestinal
inflammation is selected from the group comprising or consisting
of: Crohn's disease, ulcerative colitis, collagenous-,
lymphocytic-, ischemic-, diversion- and indeterminate colitis and
Behcet's disease in mammals.
[0016] In another preferred embodiment of said method, said
antibody is a monoclonal anti-metallothionein antibody. More
preferably, said monoclonal anti-metallothionein antibody is the
monoclonal anti-metallothionein antibody clone UC1MT.
BRIEF DESCRIPTION OF FIGURES
[0017] FIG. 1: Kaplan-Meier survival curve (A and C, censored data)
and body weight evolution (B and D) for DSS-treated wild type (WT),
MT knockout (MT.sup.-/-) and transgenic (MT.sup.+/+) mice and for
DSS-treated UC1MT antibody and IgG1IgG11 control treated mice
respectively. Body weight evolution (E) and colon length (F) for
TNBS-treated control IgG1IgG11 and UC1MT treated mice compared to
mice not receiving TNBS (control). A higher survival rate and less
weight loss was observed for MT.sup.-/- mice and UC1MT antibody
treated mice compared to control mice. Mice treated with the UC1MT
antibody tended to lose less weight with significantly less colon
shortening after TNBS treatment compared to control treated mice.
Data are presented as mean.+-.SEM.*p<0.05.
[0018] FIG. 2: Histological inflammation score (A) and
myeloperoxidase activity (neutrophil infiltration) (B) for
DSS-treated wild type (WT), MT knockout (MT.sup.-/-) and MT
transgenic (MT.sup.+1+) mice at day 10. Representative pictures for
macrophage infiltration (F4/80 staining) in IgG1IgG11 control and
UC1MT treated mice after DSS (C) and TNBS (D) treatment. MT.sup.-/-
mice scored significantly better for histological inflammation and
neutrophil infiltration. UC1MT treated mice had significantly less
macrophage infiltrate compared to IgG1IgG11 control treated mice
after DSS- and TNBS-colitis induction (p<0.05). Data are
presented as mean.+-.SEM.
[0019] FIG. 3: Metallothioneins are secreted from intestinal
epithelial cells and function as chemokines in vitro.
Ficoll-isolated blood leukocyte migration towards supernatant (SN)
of zinc treated (to induce MT synthesis) HT29 colonic epithelial
cells in Boyden transwell chambers is higher compared to SN of
untreated cells. Chemokine function is inhibited by the addition of
anti-MT antibodies as shown by reduced leukocyte migration towards
SN containing anti-MT antibodies.
DETAILED DESCRIPTION
[0020] The term `an antagonist targeting metallothionein` means an
antibody which specifically or selectively binds to a
metallothionein. It is further clear that the antibodies of the
present invention can be prepared by any method known in the art.
Examples of the latter methods are e.g. described in WO 2010/037864
and US 2003/007973.
[0021] `Metallothionein` means any of the 18 isoforms of
metallothionein that have been identified in humans and are grouped
as MT1 to MT4. Where MT3 and MT4 are constitutively expressed, MT1
and MT2 are highly inducible and can be secreted (Lynes et al.
2006; Laukens et al. 2009). The antibodies of the present invention
specifically target or bind to the secreted metallothioneins.
[0022] `Inflammatory bowel disease,` comprising Crohn's disease and
ulcerative colitis, means a group of inflammatory conditions of the
colon and/or the small intestine (i.e. inflammation of the
intestine). The term `intestinal inflammation` more specifically
refers to Crohn's disease, ulcerative colitis, collagenous-,
lymphocytic-, ischemic-, diversion- and indeterminate colitis and
Behcet's disease in mammals.
[0023] The term `antibody or a fragment thereof` relates to an
antibody characterized as being specifically directed against
metallothionein or any derivative thereof specifically binding to
said metallothionein or an antigen-binding fragment thereof
specifically binding to said metallothionein, particularly of the
F(ab')2, F(ab) or single chain Fv (scFv) type, or any type of
recombinant antibody thereof such as a nanobody specifically
binding to said metallothionein. According to particular
embodiments, the anti-metallothionein antibodies are monoclonal
antibodies that selectively and preferentially bind to
metallothioneins. More specifically, the present invention relates
to the antibody denominated as clone `UC1MT` as described in US
2003/0007973 and as commercially available from Abcam Inc,
Cambridge, Mass. Clone UC1MT has also been described by Lynes et
al. (Toxicology 1993 (85): 161-177): The monoclonal antibodies of
the present invention can be further humanized or may be human
monoclonal antibodies. The antibodies of the present invention can
be further modified for various uses as is known in the art.
[0024] The terms `specifically or selectively binding` to
metallothionein refer to a binding reaction that is determinative
of the presence of a metallothionein in a heterogeneous population
of proteins and other biologics. Thus, under designated immunoassay
conditions, the specified antibodies of the present invention bind
to a metallothionein at least two times the background and do not
substantially bind in a significant amount to other proteins than
metallothioneins present in the sample. Specific binding to an
antibody under such conditions may thus require an antibody that is
selected for its specificity to a metallothionein.
[0025] By the term `treatment` is meant the medical management of a
patient with the intent to cure, ameliorate, stabilize, or prevent
the onset of intestinal inflammation in mammals such as human
patients via administering an effective amount of the
antagonist/antibody of the present invention. It is further
understood that appropriate doses of said compounds (which can also
be denominated as drugs or pharmaceutical compositions) depends
upon a number of factors within the knowledge of the ordinary
skilled physician. The dose of these compounds will vary, for
example, depending upon the identity, size, and condition of the
patient being treated, upon the route of administration of said
compounds (i.e. parenteral (intravenous, intradermal,
subcutaneous), oral, transdermal, transmucosal or rectal) and upon
the effect which the skilled physician desires the compound to
have. A pharmaceutical composition is formulated to be compatible
with its intended route of administration. Suitable diluents,
solvents, antioxidants, chelating agents, buffers, carriers,
isotonic agents, binding agents, adjuvants, flavoring agents,
propellants, detergents and the like are described in detail in,
for example, WO 03/004989.
[0026] The present invention thus relates to a method of treating
or inhibiting inflammatory bowel disease or other inflammatory
diseases of a patient in need thereof, comprising: administering to
said patient a therapeutically-effective amount of a composition
comprising an antagonist targeting metallothionein of the present
invention.
[0027] The following examples are provided to better illustrate
particular embodiments, and they should not be considered limiting
the invention.
EXAMPLES
Animals.
[0028] MT knockout (MT.sup.-/-), transgenic (MT.sup.+1+) and wild
type (WT) mice (all C57BL/6 background) were kindly provided by
Prof. Michael Lynes, University of Connecticut. C57BL/6J-Mttm1Bri
Mt2tm1Bri mice were bred from C57BL/6J backcrossed to the Mt1,2
knockout construct obtained from Jackson Laboratory Stock Number
002211, strain 129S7/SvEvBrd-Mt1tm1Bri Mt2tm1Bri/J as described in
Crowthers et al. (2000), whereas the C57BL/6J-Tg(Mt1) mice were
bred from C57BL/6J backcrossed to the Mt1 transgene obtained from
Jackson Laboratory Stock Number 002209, strain Tg(Mt1)174Bri/J.
Animals were housed and reared in the laboratory animal facility at
the University Hospital Ghent according to the institutional animal
healthcare guidelines. This study was approved by the Institutional
Review Board of the Faculty of Medicine and Health Science of Ghent
University (ECD 10/11).
Dextran Suphate Sodium (DSS)-Induced Colitis in WT, MT.sup.-/- and
MT.sup.+/+ Mice.
[0029] Thirty 8 to 10 week old animals of each group received 4%
DSS in their drinking water for 7 days followed by 7 days of normal
drinking water. Mice were matched for initial body weight prior to
start of the experiment and body weight was further measured daily.
Five animals of each group were anaesthetized with isoflurane in
oxygen for blood sampling and sacrificed by cervical dislocation at
day 0 (D0), 3 (D3), 7 (D7), 10 (D10) and 15 (D15). Serum samples
were stored at -80.degree. C. Colonic epithelial cells were
isolated using the Cell Recovery Solution (BD Bioscience, Belford,
Mass.) and lysed for RNA and protein extraction. Tissue samples for
myeloperoxidase assay (MPO) and protein lysates were flash frozen,
colon tissue for immunohistochemistry was fixed in formalin.
Treatment With the UC1MT Anti-MT Antibody or IgG1 Control During
Colitis.
[0030] DSS-induced colitis model: Twenty-four 8 week old C57BL/6
mice received 4% DSS in their drinking water for 7 days and were
followed up for weight loss and survival until D10. Mice were
randomized at D4 and treated intraperitoneally with 4 mg/kg anti-MT
antibody or an equivalent volume IgG1 (control) on D4, D6 and D8.
The UC1MT monoclonal anti-MT antibody was kindly provided by Prof.
Michael Lynes, University of Connecticut. Samples were prelevated
in the same manner as described for the former experiment.
[0031] 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis
model: Fifteen 8 week old WT mice were treated intrarectally with
100 .mu.l of a 1:1 solution of absolute ethanol and 5% TNBS on DO,
as described earlier. Mice were treated every day with 4 mg/kg
UC1MT antibody or an equivalent volume IgG1 (control). Four mice
received 50% ethanol alone as control for the TNBS. Samples were
taken at D3, as described in the DSS-induced colitis.
MT.sup.-/- Mice Develop a Less Severe Form of DSS Colitis.
[0032] The role of MTs during intestinal inflammation was first
explored using mice lacking or over-expressing MTs in DSS-induced
colitis. Wild type, MT.sup.-/- and MT.sup.+/+ mice were treated for
7 days with DSS, followed by 7 days of normal drinking water to
evaluate both the induction and recovery from acute colitis. Data
were obtained at different time points.
[0033] Most strikingly, 48% of the MT.sup.+/+ mice died during the
course of colitis, whereas this was only the case for 10% of the
MT.sup.-/- mice (p<0.05, FIG. 1A). This higher survival rate was
accompanied with significant less body weight loss for MT.sup.-/-
mice at D6 and D7 compared to MT.sup.+/+ and at D9 and D10 compared
to WT mice (p<0.05, FIG. 1B). Moreover, MT.sup.-/- mice showed
reduced histological inflammation and neutrophil infiltration,
assessed by MPO assay (p<0.05 at D10, for both parameters, FIG.
2A and B). No differences could be observed for colonic cytokine
expression, mucosal macrophage infiltrate, mucosal hypoxia,
vascularization or proliferation between groups.
Monoclonal Anti-MT Treatment Reduces Signs of Inflammation in
Experimental Colitis.
[0034] To further explore the observed protection in case of MT
absence, the effect of targeting MTs using the monoclonal UC1MT
anti-MT antibody was tested in two experimental models for acute
colitis. Mice received DSS in their drinking water from D0 to D7.
At D4, mice were randomized in 2 groups receiving 4 mg/kg UC1MT
antibody or an isotype control at D4, 6 and 8. Mice were sacrificed
for sampling at D10. Confirming the protective effect in the
MT.sup.-/- mice, mice receiving anti-MT antibody tended to score
better for weight loss and survival (FIG. 1C and D). Significantly
less inflammatory cell infiltrate was present at D10 for UC1MT
treated mice and the observed infiltrate represented macrophages,
as demonstrated by immunohistochemical staining for F4/80
(p<0.05, FIG. 2C). Finally, we evaluated the potency of the
UC1MT antibody in TNBS-induced acute colitis, characterized by a
transmural inflammation 4 days after intra-rectal instillation of
TNBS. Anti-MT treated mice tended to lose less weight with
significantly less colon shortening compared to control treated
mice (FIG. 1E and F). Complementary to the results of the
DSS-experiment, UC1MT treated mice showed significant less mucosal
macrophage infiltrate compared to control treated mice (FIG.
2D).
Metallothionein Acts a Potent Chemokine.
[0035] Supernatant of zinc treated HT29 cells containing high MT
levels attracts more blood leukocytes in a Boyden transwell assay
than untreated cells (FIG. 3).
The UC1MT Antibody Abolishes the Chemotactic Capacity of MTs.
[0036] In line with previous findings published by Yin et al.,
significantly less leukocyte migration could be detected using
supernatant containing the UC1MT antibody compared to control
supernatant (FIG. 3).
[0037] In summary, the inventors found that a lack of MTs is
beneficial in experimental colitis. Moreover, treatment with an
anti-MT antibody reduced the chemotactic properties of
extracellular MT, resulting in reduced leukocyte infiltration and
dampening of the inflammatory response during colitis that intends
to avoid some or all of the negative side effects of other biologic
treatments currently used for IBD. Hence, anti-MT is an attractive
novel biological in the treatment of IBD patients.
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