U.S. patent application number 17/175913 was filed with the patent office on 2021-06-17 for antibodies against mac-1.
The applicant listed for this patent is Albert-Ludwigs-Universitat Freiburg, Baker IDI Heart & Diabetes Institute Holdings Ltd.. Invention is credited to Karlheinz PETER, Dennis WOLF, Andreas ZIRLIK.
Application Number | 20210179717 17/175913 |
Document ID | / |
Family ID | 1000005419679 |
Filed Date | 2021-06-17 |
United States Patent
Application |
20210179717 |
Kind Code |
A1 |
ZIRLIK; Andreas ; et
al. |
June 17, 2021 |
ANTIBODIES AGAINST MAC-1
Abstract
The present invention provides an isolated monoclonal antibody
or an antigen-binding portion thereof which a) binds to Mac-1, b)
specifically inhibits the interaction of CD40L with activated Mac-1
and c) does not induce integrin outside-in signaling.
Inventors: |
ZIRLIK; Andreas; (Freiburg,
DE) ; WOLF; Dennis; (Schopfheim, DE) ; PETER;
Karlheinz; (Hawthorn East, Victoria, AU) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Albert-Ludwigs-Universitat Freiburg
Baker IDI Heart & Diabetes Institute Holdings Ltd. |
Freiburg
Melbourne |
|
DE
AU |
|
|
Family ID: |
1000005419679 |
Appl. No.: |
17/175913 |
Filed: |
February 15, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16312629 |
Dec 21, 2018 |
10919967 |
|
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PCT/EP2017/064339 |
Jun 13, 2017 |
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17175913 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 16/2845 20130101;
C07K 2317/55 20130101; C07K 2317/34 20130101; C07K 2317/76
20130101; C07K 2317/32 20130101; C07K 2317/70 20130101; A61K
2039/505 20130101; C07K 2317/33 20130101 |
International
Class: |
C07K 16/28 20060101
C07K016/28 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 21, 2016 |
EP |
16175382.7 |
Claims
1. An isolated monoclonal antibody or an antigen-binding portion
thereof which a) binds to Mac-1, b) specifically inhibits the
interaction of CD40L with activated Mac-1 and c) does not induce
integrin outside-in signaling, characterized in that the antibody
or antigen-binding portion thereof comprises at least three CDRs
selected from the group consisting of SEQ ID NOs:2-4 and SEQ ID
NOs:6-8.
2. The antibody or antigen-binding portion thereof according to
claim 1 characterized in that the antibody or an antigen-binding
portion thereof does not bind to non-activated Mac-1.
3. The antibody or antigen-binding portion thereof according to
claim 1 characterized in that the antibody or an antigen-binding
portion thereof limits the expression of inflammatory
cytokines.
4. The antibody or antigen-binding portion thereof according to
claim 1 characterized in that the antibody or an antigen-binding
portion thereof blocks leukocyte recruitment in vitro and in vivo
in intravital microscopy.
5. The antibody or antigen-binding portion thereof according to
claim 1 characterized in that the antibody or an antigen-binding
portion thereof does not affect thrombotic and hemostatic functions
of Mac-1.
6. The antibody or antigen-binding portion thereof according to
claim 1 characterized in that the antibody or an antigen-binding
portion thereof binds specifically to a peptide having the sequence
SEQ ID NO: 9
7. The antibody or antigen-binding portion thereof according to
claim 1 characterized in that the antibody or an antigen-binding
portion thereof comprises at least four CDRs selected from the
group consisting of SEQ ID NOs:2-4 and SEQ ID NOs:6-8.
8. The antibody or antigen-binding portion thereof according to
claim 1 characterized in that the antibody or an antigen-binding
portion thereof comprises at least five CDRs selected from the
group consisting of SEQ ID NOs:2-4 and SEQ ID NOs:6-8.
9. The antibody or antigen-binding portion thereof according to
claim 1 characterized in that the antibody or an antigen-binding
portion thereof comprises six CDRs selected from the group
consisting of SEQ ID NOs:2-4 and SEQ ID NOs:6-8.
10. The antibody or antigen-binding portion thereof according to
claim 1 characterized in that the light chain has an identity of at
least 80% to the amino acid sequence of SEQ ID NO:1 and that the
heavy chain has at least 80% identity to the amino acid sequence of
SEQ ID NO:5.
11. The antibody or antigen-binding portion thereof according to
claim 1 characterized in that the light chain has the amino acid
sequence of SEQ ID NO:1.
12. The antibody or antigen-binding portion thereof according to
claim 1 characterized in that the amino acid sequence of the heavy
chain corresponds to SEQ ID NO:5.
13. The antibody or antigen-binding portion thereof according to
claim 1 characterized in that the antibody or an antigen-binding
portion thereof is selected from the group comprising F.sub.ab
fragments, single chain antibodies, diabodies and/or
nanobodies.
14. A pharmaceutical composition characterized in that it comprises
a pharmaceutically active amount of an antibody or antigen-binding
portion thereof according to claim 1.
15. The pharmaceutical composition according to claim 14 for the
treatment of inflammation.
Description
[0001] The present application is a continuation of co-pending U.S.
Ser. No. 16/312,629, filed Dec. 21, 2018, which is a U.S. National
Stage application under 35 U.S.C. .sctn. 371 of International
Application No. PCT/EP2017/064339, filed 13 Jun. 2017, which claims
priority from European Patent Application No. 16175382.7, filed 21
Jun. 2016, which applications are hereby incorporated herein by
reference.
[0002] In the past decades inflammation was identified as driving
force of many pathologies, including atherosclerosis, Type 2
Diabetes, sepsis, myocardial infarction, autoimmune diseases and
neurodegenerative disease. Targeting the inflammatory response has
been proposed as major goal in these pathologies. However, a major
limitation of such strategies remains that the inflammatory
response is critical for regeneration, survival, and host defense.
A safe and reliable anti-inflammatory therapy therefore represents
a major medical need. This is illustrated by glucocorticoids,
potent inhibitors of inflammation that compromise the immune
response, or COX-2 inhibitors, which can suppress inflammation, but
exhibit detrimental effects on the cardiovascular system.
[0003] Inflammation is a process that involves recruitment of
leukocytes to the site of injury mediated by leukocyte integrins,
such as Mac-1 (.alpha..sub.M.beta..sub.2, CD11b/CD18). Mac-1 is a
potent adhesion factor, susceptible to rapid inflammatory
activation by conformational change that exhibits increased
affinity to its ligands resulting in rolling, firm adhesion, and
transmigration of leukocytes into inflamed tissue. Mac-1 is a
powerful target in cardiovascular disease and therapeutic or
genetic inhibition of the integrin and has been shown to be highly
effective in preventing atherosclerosis, neo-intima formation, and
thrombotic glomerulonephritis. Besides its role in inflammation,
Mac-1 was initially named CR (complement receptor) 3 by its ability
to bind complement factors, such as C3bi, reflecting its role in
host defense, wound healing, thrombosis, and various other myeloid
cell effector functions. This broad repertoire of effector
functions is realized by a broad expression on the myeloid lineage,
including on monocytes, macrophages, and neutrophils, but also on
NK cells, and to a smaller extent on activated lymphocytes. Its
functional diversity is furthermore reflected by promiscuous ligand
binding to a large repertoire of proteins and proteoglycans,
including ICAM-1, fibrinogen, fibronectin, heparin, GPIb.alpha.,
RAGE, endothelial protein C-receptor (EPCR), and CD40L. It has been
proposed that integrin antagonism is a promising target in
inflammation. However, its role in host defense and thrombosis may
limit its clinical use.
[0004] CD40 ligand (CD40L) is a transmembrane molecule of crucial
interest in cell signaling in innate and adaptive immunity. It is
expressed by a variety of cells, but mainly by activated
T-lymphocytes and platelets. CD40L may be cleaved into a soluble
form (sCD40L) that has a cytokine-like activity. Both forms bind to
several receptors, including CD40. This interaction is necessary
for the antigen specific immune response. CD40L binds also to
different receptors whereby Mac-1 (.alpha.M.beta.2) is one receptor
whereby said interaction plays a role in arterial neo-intima
formation, leukocyte recruitment and atherosclerosis, pathogenesis
of atherothrombosis, monocyte adhesion and neutrophil infiltration
and release of pro-inflammatory cytokines (IL-8, IL-6).
[0005] Mac-1 is a classical adhesion factor involved in a variety
of inflammatory pathologies. Despite its promoting effect on
leukocyte recruitment in atherosclerosis and peritoneal
inflammation, Mac-1 targeted therapy is limited by various side
effects, such as impaired wound healing and host defense. This is
further reflected by the human Leukocyte Adhesion Deficiency (LAD),
which is characterized by a defect of the integrin Mac-1, LFA-1,
and CD11c in the .beta.-subunit that impairs host defense.
Unspecific attempts to therapeutically inhibit Mac-1 seem therefore
not favorable. To circumvent these limitations novel monoclonal
antibodies are provided that specifically target the binding of
CD40L to Mac-1's major ligand binding I-domain within the
am-subunit of the integrin. CD40L represents a biased agonist for
Mac-1, mediating its pro-inflammatory function by serving as
endothelial adhesion factor for CD40L, but not by activation of
outside-in signaling pathways. CD40L/Mac-1 binding does not
interfere with CD40L-CD40 or Mac-1-GP1 balpha and Mac-1-ICAM-1
binding, suggesting unique binding epitopes on each of the protein
surfaces.
[0006] Integrins are major adhesion receptors that transmit signals
bidirectionally across the plasma membrane, playing significant
roles in diverse biological processes including immune response.
Integrins contain two non-covalently associated type 1
transmembrane glycoprotein .alpha. and .beta. subunits; each
subunit contains large extracellular domains, a single-spanning
transmembrane domain and short cytoplasmic domain. The ability of
the integrin's extracellular domain to bind ligands depends on an
open-extended confirmation of the .alpha.M subunit ("activation")
and regulates cell adhesion and signal transduction, both
outside-in and inside-out signaling. The present invention relates
to a specific modification of the interaction between CD40L and the
integrin .alpha..sub.M.beta.2 (Mac-1).
[0007] It has been found that inactivation of distinct integrin
functions involved in inflammatory, but not in regenerative or
immune pathways could be achieved by selectively blocking Mac-1's
interaction to specific ligands, while not affecting others.
[0008] Monoclonal antibodies, specifically targeting the EQLKKSKTL
(SEQ ID NO:9) binding motif in Mac-1, which we have demonstrated to
be required for binding to its adhesive, pro-inflammatory ligand
CD40L have been constructed.
[0009] The present invention provides therefore isolated monoclonal
antibodies or antigen-binding portions thereof, which inhibit the
recruitment of leukocytes without undesired side effects. Such
antibodies or antigen-binding portions thereof
a) bind to Mac-1, b) specifically inhibit the interaction of CD40L
with activated Mac-1, and c) do not induce integrin outside-in
signaling.
[0010] In the course of the present invention monoclonal antibodies
have been constructed whereby the most preferred embodiment is the
antibody in the following designated as anti-M7. The sequence of
the antibody has been determined and the CDRs were identified. With
this information and computational and conventional binding studies
it is possible to provide suitable other antibodies or
antigen-binding fragments thereof, which are derived from this
antibody. Since antibody technology has gained much interest in the
therapeutic area there are several engineered antibody fragments
available which can be used in practice. The term "monoclonal
antibody or antigen-binding fragment thereof" is understood in a
broad sense and includes therefore not only the F.sub.ab fragments
but also single-chained Fv fragments (scFv), diabodies which may be
bispecific, bispecific single chain fragments, triabodies,
tetrabodies or minibodies. The sequence information provided herein
can also be used to produce nanobodies which are derived from
camelite immunoglobulins. Many of those structures are summarized
in the review article of Holliger et al. (Nature Biotechnology,
vol. 23, no. 9 (2005), pp 1126-1136).
[0011] It is a preferred property of the isolated monoclonal
antibodies or antigen-binding portions thereof that they bind to
Mac-1, whereby, however, the binding to the activated Mac-1 is
preferred whereas the antibody structures of the present invention
should not bind to non-activated Mac-1. A distinction between
activated and non-activated Mac-1 can be performed by quantifying
the binding kinetics as for example described by Li et al. (Journal
of Immunology (2013), pp 4371-4381).
[0012] Another preferred embodiment of the isolated monoclonal
antibodies or antigen-binding portions thereof of the present
invention is that they limit the expression of inflammatory
cytokines.
[0013] Another preferred property of the isolated monoclonal
antibodies or antigen-binding portions thereof is that they block
leukocyte recruitment in vitro and preferably in vivo. Such
blockage can be observed and measured in intravital microscopy as
shown in the examples of the present application.
[0014] A further preferred embodiment of the monoclonal antibodies
or antigen-binding portions thereof is that the thrombotic and
hemostatic functions of Mac-1 are not effected. This can be
measured by using suitable in vivo experiments.
[0015] The preferred embodiment disclosed herein designated as
anti-M7 was produced as a monoclonal antibody in the mouse system.
It is well-known to the skilled person that monoclonal murine
antibodies cannot be used in the therapy of humans since after
repeated administration of such murine antibodies anti-mouse
antibodies are generated in the patient. Therefore, the monoclonal
antibodies or antigen-binding portions thereof are preferably
humanized. Humanization means that the mouse framework of the
antibody is replaced by a human framework structure of an antibody
which has high similarity to the mouse antibody. By using suitable
computational models further adaptations of the amino acid
structure can be made in order to reduce the mouse character of the
antibody. It has, however, to be checked whether the proposed
changes in the amino acid sequence reduce the binding strength of
the humanized antibody or antigen-binding construct. Only such
amino acid substitutions are performed which do not negatively
affect the binding properties and in particular the
specificity.
[0016] It is assumed that such modified antibodies or
antigen-binding portions thereof should comprise at least three
CDRs. The CDRs are disclosed and have SEQ ID NOs:2-4 and 6-8,
respectively. In a more preferred embodiment the monoclonal
antibodies or antigen-binding portions thereof according to the
present invention comprise at least four, more preferred five and
in particular preferred six CDRs having the sequences of SEQ ID
NOs:2-4 and 6-8, respectively.
[0017] The light chain of the anti-M7 antibody has the amino acid
sequence as provided in SEQ ID NO:1 and the heavy chain corresponds
to SEQ ID NO:5. As already explained above, in the course of
humanization amino acid sequence changes are introduced into the
amino acid sequence. In preferred embodiments the isolated
monoclonal antibodies or antigen-binding portions of the present
invention have a light chain which has an amino acid identity of at
least 80%, preferred of at least 85%, more preferred of at least
90% and in particular preferred of at least 95% identity to SEQ ID
NO:1.
[0018] In preferred embodiments the isolated monoclonal antibodies
or antigen-binding portions of the present invention have a heavy
chain which has an amino acid identity of at least 80%, preferred
of at least 85%, more preferred of at least 90% and in particular
preferred of at least 95% identity to SEQ ID NO:5.
[0019] The term "identity" means that the sequence of the original
murine sequence and the sequence of the humanized construct are
compared to each other. An identity of for example 90% means that
90% of the amino acids are at the corresponding amino positions
identical in the original mouse sequence and in the humanized
sequence.
[0020] The isolated monoclonal antibodies or antigen-binding
portions thereof of the present invention can preferably be used in
pharmaceutical compositions which comprise a pharmaceutically
active amount of the antibody or antigen-binding portion thereof
together with additives suitable for the application to a patient
whereby intraperitoneal application is especially preferred. The
pharmaceutical compositions of the present invention can preferably
be used for the inhibition of inflammation.
[0021] It turned out that the monoclonal antibodies or
antigen-binding portions thereof according to the present invention
can preferably be used in the treatment of inflammatory
complications following myocardial infarction. In such
complications, which occur frequently after myocardial infarction
inflammatory leukocytes attracted to the area which is affected by
the myocardial infarction cause and contribute to an inflammatory
response that aggravates wound healing and may inhibit the recovery
after myocardial infarction. In such embodiments the antibodies and
antigen-binding portions thereof according to the present invention
are preferably used.
[0022] The inhibition of inflammation by anti-M7 or the derivatives
derived therefrom provides several advantages over a conventional
anti-Mac-1 therapy. It has been observed that mice treated with
formerly known anti-Mac-1 antibodies showed increased mortality
compared to control mice. These data confirm previous studies in
which Mac-1 deficient mice were not protected from bacterial
sepsis, an effect most likely caused by the inability to bind
complement factors and promote clearance of bacterial particles
e.g. by C3bi-mediated phagocytosis.
[0023] Previous epitope mapping studies have revealed and located
the binding of C3bi to the residues P.sup.147-R.sup.152,
P.sup.201-K.sup.217, and K.sup.245-R.sup.261 within the
.alpha..sub.m I-domain, demonstrating a binding epitope that is
distinct from the binding sequence required for CD40L
(E.sup.162-L.sup.170). It has been shown that mice treated with
anti-M7 show increased survival compared to anti-Mac-1 and
IgG-control treated mice, indicating that anti-M7 does not only
lack detrimental properties, but induces protective effects.
[0024] It is assumed that suppression of pro-inflammatory leukocyte
adhesion in the peritoneum helps to slow-down the overwhelming
pro-inflammatory response accompanying the initial attempt to
remove and fight the bacterial invasion. It is recognized that the
balance between protective and disease aggravating pathways is
disturbed in many conditions and might potentially been shifted to
the protective side by limiting leukocyte recruitment. This
hypothesis is further supported by the fact that anti-M7 protected
from pro-inflammatory cytokine levels in plasma compared with
control animals, while anti-Mac-1 raised cytokine levels. Thus, the
reduction in cytokine levels may be secondary to diminished
leukocyte activation and activation in target tissues. Indeed,
intimal mononuclear cells produce pro-inflammatory cytokines, such
as TNF.alpha., IL-1, IFN.gamma. as well as anti-inflammatory
mediators IL-10. In plasma, mice challenged with TNF.alpha. and
treated with anti-M7 antibody showed a reduction of the
pro-inflammatory cytokines IL-6, TNF.alpha. and MCP-1, while
anti-Mac-1 induced enhanced cytokine expression.
[0025] However, treatment with other anti-Mac-1 antibodies, such as
the clone M1/70 (which is used as control), might not entirely
reflect the genetic knock-out. It is noteworthy to mention that
M1/70 induces a strong pro-inflammatory response in Mac-1
expressing cells, in particular in macrophages, and elevates
cytokine expression. The latter is also confirmed by our results,
demonstrating that a single injection of anti-Mac-1 results in
strongly up-regulated cytokine plasma levels, likely affecting
wound healing. It has been suggested that over-stimulation as
provided by M1/70 could represent a feasible strategy to resolve
inflammation by activation of apoptotic pathways. Indeed, it has
previously been shown that apoptosis of cells resident in the
peritoneal cavity was enhanced after a single injection of
anti-Mac-1 clone M1/70. This could potently support anti-Mac-1's
effect in decreasing peritoneal cell accumulation. However, an
apoptosis inducing therapy, accompanied by a cytokine-storm is
likely unfavorable in the clinical practice.
[0026] Mac-1 supports interaction to multiple other molecules and
more are likely of not been discovered so far. More than 40
different protein interactions have been described, but molecular
binding properties of only some of these is known. Therefore it is
not to exclude that the binding site of CD40L is shared by other
ligands as wells. However, the data presented herein unveil and
confirm previous suggestions that CD40L binding to Mac-1 does not
share many features with binding properties to other conventional
ligands: [0027] (1) While binding epitopes identified for
fibrinogen and other ligands show overlapping regions, the
EQLKKSKTL (SEQ ID NO:9) motif within Mac-1's I-domain is not
involved in binding of alternative ligands, [0028] (2) neither
CD40L itself, nor anti-M7 did induce integrin outside-in signaling,
while this feature of integrin physiology has been considered as
paradigm in integrin ligand binding so far, [0029] (3) CD40L's
interaction with Mac-1 does not expand on immune or haemostatic
function, while most of Mac-1 ligands, such as Fibrinogen, are
involved in multiple of those pathologies.
[0030] The data presented herein propose that the interaction of
CD40L with Mac-1 is primarily required for firm adhesion of
inflammatory leukocytes, presumably of granulocytes in a variety of
inflammatory pathologies. The results do not rule out, but
emphasize that immune function, haemostatic parameters and
regenerative response do not involve binding of CD40L to Mac-1.
[0031] It has been shown previously that treatment with the
specific inhibitor of the CD40L/Mac-1 interaction, cM7, attenuates
inflammatory leukocyte recruitment in a model of intravital
microscopy in inflamed cremaster venules, and in a model of sterile
peritonitis. It is demonstrated that treatment with either the full
IgG antibody anti-M7 or F.sub.ab fragments thereof, directed
against the CD40L binding site on Mac-1, significantly reduces
leukocyte adhesion. Interestingly, the inhibitory efficiency of
anti-M7 is comparable to that of anti-Mac-1 treatment, suggesting
the CD40L/Mac-1 interaction as instrumental for leukocyte
recruitment. This does not falsify previous reports, but does
extend the repertoire of Mac-1's ligands expressed on the
endothelium, ICAM-1 and RAGE, by CD40L. In this regard, it is
plausible that patterns of counter-receptor binding depend on
pathologies and the inflammatory burden. Thereby, it is either
possible that the interaction of CD40L and Mac-1 is disease
specific and regulated by either expression of endothelial CD40L,
by conformational change of Mac-1 or that some pathologies are more
dependent on leukocyte invasion than others. For example,
atherosclerosis--a disease in which myeloid cell recruitment is
needed at least in early stages of disease--was strongly
susceptible to blockade of the CD40L/Mac-1 interaction, while
neo-intima formation after a wire injury was not inhibited by
blocking CD40L/Mac-1, but by anti-Mac-1 or in Mac-1 knock-out
mice.
[0032] The data obtained in the course of the present invention
show that anti-M7 was most effective in blocking the interaction to
activated Mac-1, but not to non-activated Mac-1. This proposes that
the interaction may play a more important role in pathologies
associated with a higher inflammatory burden, rather than under
baseline conditions.
[0033] Also, it remains to be answered whether an antibody such as
anti-M7 can actively modulate or conserve different conformations
of the integrin as previously proposed. This could explain that
only the permanently-activated integrin, but not the integrin in
native condition was targeted as the data show. However, for the
determination of the exact binding properties a more detailed
structural analyses may be helpful.
[0034] Finally, it cannot be excluded that CD40L/Mac-1 interaction
may be responsible for the egress and mobilization of monocyte from
the bone marrow or the spleen as previously suggested. As observed
herein, inflammatory monocytosis during sepsis could be completely
reversed by anti-M7 treatment. Whether this is caused by impaired
monocyte reservoirs, e.g. by impaired migration to the spleen,
shall be determined in further experiments.
[0035] The antibodies of the present invention follow a strategy to
selectively target the EQLKKSKTL (SEQ ID NO:9) binding motif,
representing CD40L's binding site within the Mac-1 I-domain, by a
monoclonal antibody anti-M7. This antibody is highly selective for
the targeted binding site, does not interfere with alternative
binding partners, and--in contrast to conventional anti-Mac-1
antibodies--does not affect haemostasis, host defense and wound
healing. In preferred embodiments the antibodies of the present
invention do not interfere with alternate binding partners and are
therefore highly selective for the targeted binding site. The
proposed ligand-targeted anti-integrin therapy is superior to an
unselective approach and represents an advantage to refine and
adjust anti-integrin therapy against inflammatory disease.
[0036] The results, experiments and advantages obtainable by the
present invention are summarized in the Figures and the Examples.
Figures and Examples show preferred embodiments of the present
invention, in particular the most preferred antibody anti-M7, but
it should be understood that Figures and Examples should not be
considered as limiting the present invention.
BRIEF DESCRIPTION TO DRAWINGS
[0037] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
[0038] The preferred embodiments of the invention are shown in the
Figures and in the Examples:
[0039] FIG. 1 shows that A mouse monoclonal antibody raised against
the CD40L binding site within human Mac-1, anti-M7, is effective in
targeting the human integrin. The peptide sequence M7 within the
Mac-1, required for binding of CD40L, is a highly conserved binding
motif between the human (SEQ ID NO:9) and murine (SEQ ID NO:10)
integrin (FIG. 1A).
[0040] Furthermore, FIG. 1A shows the peptide M1 of human origin
(SEQ ID NO:14) and the corresponding peptide M1 derived from Mus
musculus having SEQ ID NO:15. The human peptide having the
designation M8 corresponds to SEQ ID NO:13 and the peptide M8
derived from Mus musculus has SEQ ID NO:16.
[0041] Antibody anti-M7 generated by immunization of mice with the
binding peptide VMEQLKKAKTLMQ (SEQ ID NO:11) coupled to diphtheria
toxoid bound to a CHO cell line over-expressing native (WT) and
permanently activated Mac-1 (del), but not to control CHO cells in
western blot (FIG. 1B).
[0042] Specific binding of the antibody anti-M7 to the immobilized
peptides M7 (EQLKKSKTL) (SEQ ID NO:9), sM7 (KLSLEKQTK) (SEQ ID
NO:12), and M8 (EEFRIHFT) (SEQ ID NO:13) was tested in a solid
phase binding with immobilized peptides (FIG. 1C).
[0043] Binding was quantified by binding of biotinylated anti-mouse
IgG and color reaction after incubation with HRP-coupled
streptavidin. Specific binding was calculated by subtraction of
binding of mouse IgG to the peptides. Anti-M7 was coupled with the
fluorochrome Alexa647 and binding to human leukocyte subsets was
quantified in FACS. Alexa647 Isotype antibody served as control
(FIG. 1D).
[0044] FIG. 2 shows that Anti-M7 selectively blocks the interaction
of permanently activated Mac-1 with CD40L, but not of the native
integrin or to alternative Mac-1 ligands. CHO-cells over-expressing
the permanently activated Mac-1 mutant (Mac-1-del) adhered to
immobilized CD40L in a static adhesion assay (FIG. 2A, 2B).
[0045] Cells were incubated with anti-M7 or the human pan-I-Domain
blocking reference clone 2LPM19c 15 min prior to adhesion.
Alternatively, adhesion of the native, non-activated Mac-1 integrin
was tested (FIG. 2C). To exclude unspecific Fc-mediated
interaction, F.sub.ab-fragment preparation of anti-M7 or anti-Mac-1
were used as inhibitor (FIG. 2D).
[0046] To test whether anti-M7 is specific for CD40L, a panel of
classical Mac-1 ligands were separately immobilized and adhesion of
permanently activated Mac-1 CHO cells was quantified in the
presence of anti-M7 or pan I-Domain blocking anti-Mac-1 (FIG.
2E).
[0047] FIG. 3 shows that Anti-M7 does not induce integrin
outside-in signaling, while conventional anti-Mac-1 antibodies
induce activation of MAP-kinases and inflammatory cytokine
expression in vitro and in vivo.
[0048] Murine macrophages were isolated by injection of 4%
thioglycollate in the peritoneum of C57Bl/6 mice and incubation for
72 hours. Peritoneal cells were collected by peritoneal lavage,
FACS analysis confirmed purity of >90 percent F4/80.sup.+
macrophages. Macrophages were cultured in 5% FCS RPMI overnight and
stimulated with 10 .mu.g/ml of mouse IgG, anti-human Mac-1 (clone
2LPM19c), anti-mouse Mac-1 (clone M1/70) or anti-M7 for 30 min.
Cells were lysed and phosphorylated ERK1/2, Nf.kappa.B and p38 were
visualized by western blot (FIG. 3A), and the ratio of
phosphorylated fractions was calculated (FIG. 3B). Values were
calculated as relative arbitrary units (AU) normalized to signal of
cells stimulated with saline alone. Mac-1 antibody clones were
injected i.p. in mice and serum concentration of IL-6, TNF.alpha.,
and MCP-1 was measured by cytometric bead array 4 hours after
injection (FIG. 3C). Anti-Mac-1 clone 1/70 was used as control.
[0049] FIG. 4 shows that treatment with anti-M7 prevents
inflammatory leukocyte recruitment in vitro and in vivo and
decreases inflammatory cytokine expression. Murine RAW-cells were
allowed to adhere on isolated and TNF.alpha.-primed murine
endothelial cells in vitro in a flow chamber assay. Number of
adhering cells was quantified in the presence of an anti-mouse IgG
or anti-M7 antibody (FIG. 4A). C57Bl/6 mice were injected with 200
ng TNF.alpha. i.p. to induce peritoneal and mesenteric
inflammation. Simultaneously, either IgG isotype control or
anti-mouse anti-Mac-1 (clone M1/70) F.sub.ab-fragment preparations
were injected. Leukocyte recruitment to inflamed mesenteric venules
was monitored by intravital microscopy 4 hours after injection
(FIG. 4B). Number of adhering and rolling leukocytes were
quantified, as well as leukocyte rolling velocity, displayed as
cumulative frequency (FIG. 4C-E). Mice expressing GFP in monocytes
(CX3CR1-GFP) were subjected to intravital microscopy in the
presence of IgG or anti-M7 F.sub.ab preparations (FIG. 4F).
Migrated monocytes (white arrows) were quantified in the
para-vascular space in the viewing field (FIG. 4G). Plasma cytokine
levels in mice subjected to intravital microscopy after IgG or
anti-M7 F.sub.ab treatment were assessed by CBA bead array (FIG.
4H).
[0050] FIG. 5 shows that Anti-M7 does not affect venous thrombosis
and platelet effector function in vivo. Venous thrombosis was
induced in mesenteric venules of C57Bl/6 mice by ferric chloride.
Thrombus formation was visualized by in vivo rhodamine staining in
intravital microscopy (FIG. 5A). Time to thrombus-occlusion of the
vessel and rate of emboli (/min) was monitored and quantified (FIG.
5B, 5C). Mice were treated with either F.sub.ab-preparation of
mouse IgG, anti-M7 or anti-Mac-1 (50 .mu.g) by intraperitoneal
injection 15 min prior to thrombus induction. Formation of
platelet-monocyte aggregates was quantified by detection of
CD41.sup.+ monocytes in flow cytometry after treatment with
anti-Mac-1 antibody clones (FIG. 5D).
[0051] FIG. 6 shows that specific inhibition of Mac-1's interaction
to CD40L, but not to other ligands, improves skin wound healing.
Aseptic skin wounds were induced by a 4-mm biopsy punch after
injection of anti-Mac-1 or anti-M7 F.sub.ab preparations. After 6
days skin wounds were photographed (FIG. 6A) and wound area was
calculated (FIG. 6B).
[0052] FIG. 7 shows that Anti-M7 improves host defense, bacterial
clearance, and inflammation during bacterial sepsis, while
unspecific blockade of Mac-1 potentiates bacteremia in mice. To
test whether blockade of Mac-1 or specifically of the CD40L binding
site affects host defense and inflammation during bacterial sepsis,
coecal-ligation and puncture sepsis (CLP) was induced. 20 hours
after CLP procedure inflammatory and patrolling monocytes
circulating in blood were quantified by flow cytometry (FIG. 7A).
Granulocytes (F4/80.sup.-Gr-1.sup.+) invading into the peritoneal
cavity were identified by flow cytometry (FIG. 7B) and total
numbers were calculated (FIG. 7C). Levels of the acute phase
protein SAA (FIG. 7D) and of bacterial LPS titers (FIG. 7E) were
quantified in plasma. Accumulation of granulocytes in kidney
parenchyma was determined by staining against DAP and Ly6G (FIG.
7F) and quantified as ratio of granulocytes/total cell nuclei (FIG.
7G).
[0053] FIG. 8 shows that Anti-M7 improves, while anti-Mac-1
decreases, survival during CLP-sepsis. Coecal-ligation and puncture
sepsis (CLP) was induced. To assess if treatment with Mac-1
antibody clones affects survival, mice were treated by
intraperitoneal injection with either anti-Mac-1 or anti-M7
F.sub.ab preparations at 0, 48, and 96 hours after induction of CLP
sepsis. Relative survival was calculated and displayed as
Kaplan-Maier survival cure.
[0054] FIG. 9 shows that treatment with Anti-M7 blocks inflammatory
leukocyte infiltration in the injured myocardium following
myocardial infarction. Myocardial infarction was induced by a
surgical ligation of the left anterior descending coronary artery
(LAD). Leukocytes infiltrating the infarcted myocardium were
quantified by flow cytometry in digested hearts after myocardial
infarction. Anti-M7 decreased the infiltration with monocytes and
neutrophils and attenuated heart failure as assessed by
echocardiography.
[0055] The results summarized in the Figure were obtained in the
following examples:
EXAMPLE 1
[0056] Male mice on a C57BL/6N background received a standard chow
diet. All mice were maintained under standardized conditions
(12-hour light, 12-hour dark cycle) and had access to food and
water ad libidum. At the age of 8 weeks, mice were subjected to
intravital microscopy, wound healing or CLP sepsis as indicated.
Treatment with antibodies was performed by intraperitoneal
injection in the indicated concentration at a volume of 100 uL per
injection. In some intravital experiments, GFP-transgene animals
under the control of CXCR3-promoter (CXCR3-GFP) were used to track
leukocytes. All experimental protocols were approved by the animal
ethics committee of the Alfred Medical Research and Education
Precinct (AMREP), Melbourne, Australia and the local animal ethics
committee at the University of Freiburg. All procedures were
carried out in accordance with institutional guidelines.
[0057] An antibody specific for a peptide corresponding to Mac-1
I-domain sequence V160-S172 was obtained by immunizing mice with
the peptide C-VMEQLKKSKTLFS-NH2 (SEQ ID NO:17) coupled to
diphtheria toxoid (Monash Antibody Technologies Facility, Monash
University, Melbourne, Australia). Solid phase binding assays was
employed to screen binding of sera to the immobilized peptide M7.
Among different clones binding with high affinity to M7, the
preferred clone RC3 (termed anti-M7) was further characterized.
EXAMPLE 2
[0058] A mouse monoclonal antibody raised against the CD40L binding
site within human Mac-1, anti-M7, is effective in targeting the
human integrin.
[0059] It has previously been shown that CD40L selectively binds to
the EQLKKSKTL (SEQ ID NO:9) motif within the major Mac-1
ligand-binding domain. To obtain a specific inhibitor of the human
binding site, mice were immunized with the human peptide V160-S172
containing the binding peptide M7. Interestingly, the M7 sequence
was highly conserved between the human and murine protein sequence
(FIG. 1A). Among several hybridoma clones with high-affinity
binding of the according supernatant to the immobilized peptide M7
in a solid-phase binding assay, clone RC3 (mouse IgG2b.kappa.)
showed specific inhibition of Mac-1-CD40L binding, but not of the
interaction to other ligands. This antibody clone, subsequently
termed anti-M7, bound to a CHO cell line over-expressing native
(WT) and permanently activated Mac-1 (del), but not to control CHO
cells in western blot (FIG. 1B), confirming successful binding to
the target protein.
[0060] Moreover, anti-M7 bound to the immobilized peptides M7
(EQLKKSKTL) (SEQ ID NO:9), but not to the control peptides
scrambled sM7 (KLSLEKQTK) (SEQ ID NO:12) or the peptide M8
(EEFRIHFT) (SEQ ID NO:13) in a solid phase binding (FIG. 1C),
indicating that anti-M7 specifically binds to the immunized
peptide. To test binding of anti-M7 to Mac-1 expressing human
cells, we coupled the antibody with the fluorochrome Alexa647 and
quantified binding to human leukocyte subsets in flow cytometry.
Interestingly anti-M7 showed concentration-dependent binding to
human leukocytes expressing Mac-1, such as monocytes and
neutrophils, but not to lymphocytes as expected (FIG. 1D). Binding
of anti-Mac-1 clone M1/70 served as control and showed the same
binding properties with highest binding to myeloid cells. These
findings demonstrate that the binding sequence M7 within the human
Mac-1 I-domain is accessible to binding with the monoclonal
antibody anti-M7. Further DNA sequencing revealed CDRs and exact
protein sequence of anti-M7 variable regions of heavy and light
chain. This is shown in Table 1:
TABLE-US-00001 TABLE 1 Protein sequence of anti-M7 variable regions
Light chain DIQMTQSPSSLSASLGERVSLTCRASQEISGYLSWHQQKPDGTIKRLLYS
TSTLDSGVPKRFSGSRSGSDYSLTISSLESEDFADYYCLQYAISPPTFGG GTKLEIK (SEQ ID
NO: 1) Heavy chain
QVTLKESGPGILQTSQTLSLTCSFSGFSLSTSGMGVSWIRQPSGKGLEWL
AHIYWDDDKRYNPSLKSRLTISKDTSRNQVFLKITSVDTTDTATYYCALN
YYNSTYNFDFWGQGTTLTVSS (SEQ ID NO: 5) Position of CDR 1, 2, 3 is
underlined
EXAMPLE 3
[0061] Specific binding of the antibody anti-M7 to the immobilized
peptides M7 (EQLKKSKTL) (SEQ ID NO:9), sM7 (KLSLEKQTK) (SEQ ID
NO:12), and M8 (EEFRIHFT) (SEQ ID NO:13) was tested in a solid
phase binding with immobilized peptides in 96-well ELISA plates
(Nunc). Binding of anti-M7 was detected by addition of biotinylated
anti-mouse IgG and subsequent color reaction after incubation with
HRP-coupled streptavidin and TMB-substrate. Specific binding was
calculated by subtraction of binding of mouse IgG to the peptides.
To test binding of the antibody anti-M7 to human leukocytes,
anti-M7 was labeled with Alexa Fluor 647 according to the
manufacturers protocols (Monoclonal Antibody Labeling Kit, Life
Technologies). Human leukocytes were isolated from healthy donors
by centrifugation and Red Blood Cell lysis-Leukocytes were
stimulated with PMA (200 ng/ml), incubated with anti-M7-Alexa 647
(1 .mu.g and 5 .mu.g) and antibody binding was quantified by flow
cytometry.
[0062] It was found that anti-M7 is a ligand- and activation
specific inhibitor of Mac-1's interaction with CD40L.
[0063] To test whether anti-M7 is able to functionally block the
interaction of Mac-1 and CD40L, the adhesion of CHO-cells
over-expressing a permanently activated Mac-1 mutant (Mac-1-del) to
immobilized CD40L was tested in a static adhesion assay.
Interestingly, anti-M7 blocked the cell adhesion by 65.6.+-.7.2%,
an effect nearly as strong as the anti-human pan-I-Domain blocking
reference clone 2LPM19c (inhibition by 92.7.+-.2.0%, FIG. 2A, B).
In the experiment a concentration of 10 .mu.g/ml was used. It can
be concluded therefrom that in general concentrations of the
antibody ranging from 1 to 50 .mu.g/ml and preferably from 5 to 20
.mu.g/ml are used. Most interestingly, in contrast to the reference
anti-Mac-1 antibody, anti-M7 did not block adhesion of CHO cells
expressing the native, non-activated Mac-1 integrin (FIG. 2C),
indicating that blockade by anti-M7 was specific to high-affinity
conformation of the integrin. Moreover, inhibition by anti-M7 was
not restricted to human proteins, since interaction of murine
macrophages and murine CD40L was significantly blocked by anti-M7.
Furthermore, blocking by anti-M7 was not unspecifically caused by
the F.sub.e-fragments of the antibody, since F.sub.ab-fragment
preparations of anti-M7 or anti-Mac-1 were as effective as the
whole antibody preparation (FIG. 2D). Different ligands can bind to
separate or overlapping binding regions within the Mac-1 I-domain.
To test whether anti-M7 is specific for the CD40L binding epitope,
a panel of classical Mac-1 ligands, such as Fibrinogen, ICAM-1,
NIF, heparin, and RAGE was separately immobilized and binding of
Mac-1-del cells was tested in the presence of anti-M7 and
anti-Mac-1 (FIG. 2E). Notably, anti-Mac-1 blocked each of the
interactions, while blocking capacity of anti-M7 was restricted to
CD40L. These data unveil that anti-M7 is an effective and specific
inhibitor of the CD40L/Mac-1 interaction.
EXAMPLE 4
[0064] Murine peritoneal macrophages were obtained as described
above. Flow cytometry revealed that the majority (>90%) of PECs
were positive for the macrophage marker F4/80. After overnight
starvation macrophages were stimulated with the indicated
antibodies against Mac-1 in a concentration of 10 .mu.g/ml for 30
minutes. After the indicated time points, cells were lysed,
proteins were separated by SDS-PAGE and blotted to polyvinylidene
difluoride membranes. Total protein and the phosphorylated fraction
of NF.kappa.B, ERK1/2 and p38 were detected by specific antibody
binding in western blot (Cell Signaling). The ratio of
phosphorylated fractions was calculated and expressed as relative
arbitrary unit (AU) normalized to signal of cells stimulated with
saline alone.
[0065] The test results show that anti-M7 does not induce integrin
outside-in signaling, while conventional anti-Mac-1 antibodies
induce activation of MAP-kinases and inflammatory cytokine
expression in vitro and in vivo.
[0066] Conventional anti-Mac-1 antibodies induce activation of the
integrin, termed outside-in-signaling mediated by downstream
activation of MAP-kinases, such as ERK and p38 upon ligand and
antibody binding. It has previously been shown that CD40L is a
biased agonist not inducing outside-in signaling events upon
binding. To test whether anti-M7 would induce cell activation,
thioglycollate-elicited peritoneal macrophages from male, 8 week
old C57Bl/6 mice were collected. After overnight starvation in 5%
FCS containing RPMI, the macrophages were stimulated with 10
.mu.g/ml of either mouse IgG, anti-human Mac-1 (clone 2LPM19c),
anti-mouse Mac-1 (clone M1/70) or anti-M7 for 30 min. Anti-Mac-1
treatment induced phosphorylation of ERK and p38 as quantified by
an elevated ratio of the phosphorylated epitopes in western blot
(FIG. 3A), while anti-M7 had no effects, indicating that the
binding epitope targeted by anti-M7 is not involved in outside-in
signaling (FIG. 3B). To assess whether this effect is relevant for
an in vivo treatment, Mac-1 antibody clones were injected i.p. in
mice and serum concentration of IL-6, TNF.alpha., and MCP-1 were
quantified 4 hours after injection. Surprisingly, the Mac-1
reference clone M1/70 (control) strongly elevated cytokine levels,
while anti-M7 did not (FIG. 3C). In accordance, levels of
pro-inflammatory cytokines increased in in vitro culture of
macrophages after antibody stimulation. These findings indicate
that anti-M7 is targeting an epitope not causing unwanted
outside-in signaling during integrin blockade.
EXAMPLE 5
[0067] Before enzymatic digestion, the antibody was dialyzed in a
SnakeSkin Dialysis Tubing 10k MWCO against PBS overnight at
4.degree. C. Immobilized papain was used to prepare F.sub.ab
fragments from anti-M7, anti-Mac-1 (clone M1/70) and an IgG isotype
control as indicated according to the manufacturer's instructions
(Pierce F.sub.ab Preparation Kit, Thermo Scientific). Briefly,
F.sub.ab-fragments were generated in the presence of 25 mM cysteine
for 3 h at 37.degree. C., followed by purification on NAb Protein A
Spin Columns. Purity of F.sub.ab-fragments was evaluated on
SDS-PAGE.
[0068] 96-well plates (Nunc) were coated with sCD40L (10 .mu.g/ml)
and incubated with CHO-cells expressing constitutively activated
Mac-1. Cells were pre-incubated with blocking antibodies (10
.mu.g/mL) as indicated and allowed to adhere for 50 minutes.
Adhering cells were counted after repeated washing with PBS. For
dynamic adhesion assays, human umbilical endothelial cells (HUVECs)
were grown to confluency in 35 mm cell culture dishes, stimulated
with TNF.alpha. overnight and placed in a parallel flow chamber
system (Glycotech). Number of adhering cells was quantified at the
indicated shear rate in the presence of the indicated antibodies
(10 .mu.g/mL).
[0069] For intravital microscopy mice received an intraperitoneal
injection of 100 .mu.g of antibodies or 50 .mu.g of
F.sub.ab-fragments i.p. After 15 minutes mice were injected i.p.
with 200 ng murine TNF.alpha. (R&D Systems). Surgery started 4
hours after TNF.alpha. administration. Briefly, mice were
anesthetized by intraperitoneal injection of ketamine hydrochloride
(Essex) and xylazin (Bayer, Leverkusen, Germany). The mesentery was
exteriorized and placed under an upright intravital microscope
(AxioVision, Carl Zeiss). Videos of rolling and adhering in
mesenteric venules were taken after retro-orbital injection of
rhodamine. Rolling leukocyte flux was defined as the number of
leukocytes moving at a velocity less than erythrocytes. Adherent
leukocytes were defined as cells that remained stationary for at
least 30 seconds.
[0070] Flow cytometry: Peritoneal exudate cells (PECs) and blood
leukocytes were obtained as described below. Remaining red blood
cells were removed by incubation with a red blood cell lysing
buffer (155 mM NH4Cl, 5.7 mM K2HPO4, 0.1 mM EDTA, pH7.3). Cells
were washed in PBS, and Fc-Receptors were blocked by anti-CD16/CD32
(eBioscience) for 10 minutes on ice. Cells were then labeled with
the indicated antibodies before quantification with a flow
cytometer (FACS Calibur, BD Biosciences). All antibodies were
obtained from eBioscience. Distinct leukocyte populations were
identified upon cell surface expression of the indicated antigens:
granulocytes (Gr-1.sup.+F4/80.sup.-CD11b.sup.+CD115.sup.-),
macrophages (F4/80.sup.+CD11b.sup.+CD115.sup.-), inflammatory
monocytes (CD11b.sup.+CD115.sup.+Gr-1+F4/80.sup.-),
non-inflammatory monocytes
(CD11b.sup.+CD115.sup.+Gr-1.sup.-F4/80.sup.-).
[0071] Isolation and cultivation of murine peritoneal macrophages:
Antibodies were injected i.p. 30 min before WT mice received an
injection of 2 mL of 4% thioglycollate broth (Sigma). A peritoneal
lavage was performed after 72 hours. Peritoneal exudate cells
(PECs) were quantified and characterized by FACS as described
above. In CLP experiments a peritoneal lavage was performed 20
hours after surgery.
[0072] It could be shown that treatment with anti-M7 prevents
inflammatory leukocyte recruitment in vitro and in vivo and
decreases inflammatory cytokine expression.
[0073] Mac-1 is a powerful adhesion factor, likely mediating its
adhesive function through interaction with different ligands
expressed at the endothelium, including ICAM-1, RAGE, and CD40L. To
test if anti-M7 blocks cellular adhesion, murine monocyte-like
RAW-cells were allowed to adhere on isolated and TNF.alpha.-primed
murine endothelial cells in vitro in a flow chamber assay. Number
of adhering cells decreased after incubation with anti-M7,
indicating that CD40L/Mac-1 interaction is required for leukocyte
arrest (FIG. 3A). To test for relevance of these findings in vivo,
F.sub.ab-fragment preparation of anti-M7 and an according isotype
were injected i.p. prior to intravital microscopy (FIG. 4B).
Leukocyte recruitment to inflamed mesenteric venules was monitored
after simultaneous stimulation with TNF.alpha. for 4 hours to
induce inflammatory leukocyte recruitment. Consistently with our in
vitro results, we observed that the number of adhering (FIG. 4C),
but not of rolling leukocytes (FIG. 4D) was reduced after anti-M7
injection. In accord, leukocyte rolling velocity, displayed as
cumulative frequency, was not changed (FIG. 4E), indicating that
firm adhesion, but not rolling properties of leukocyte is blocked
by anti-M7. To exclude that anti-M7 induces leukocyte depletion we
injected anti-M7 or an according isotype control i.p., and
quantified leukocyte populations. Of note, no changes were observed
in both groups. To test if impaired monocyte arrest would affect
down-stream effects, such as transmigration, mice expressing GFP in
monocytes (CX3CR1-GFP) were subjected to intravital microscopy in
the presence of IgG or anti-M7 F.sub.ab preparations after a
TNF.alpha. challenge for 4 hours (FIG. 4F). In accordance, we
observed that anti-M7 treated animals showed lower numbers of
monocytes migrated to the perivascular space (FIG. 4G). Finally, we
observed that plasma levels of the pro-inflammatory cytokines
TNF.alpha., IL-6, and MCP-1 were significantly reduced in mice
subjected to intravital microscopy after anti-M7 F.sub.ab treatment
compared with IgG F.sub.ab treated control animals (FIG. 4H). These
results clearly indicate that leukocyte adhesion proceeds by the
interaction of CD40L and Mac-1 and that this interaction can be
functionally blocked by anti-M7 antibody.
EXAMPLE 6
[0074] It has also been shown that anti-M7 does not affect venous
thrombosis and platelet effector function in vivo.
[0075] Mac-1 participates in haemostasis and thrombus formation,
presumably by its interaction to the platelet glycoprotein
GP1b.alpha.. Also, CD40L stabilizes thrombi and its therapeutic
inhibition raises thromboembolic complications. To exclude that an
antibody according to the invention would induce unwanted thrombus
destabilization, venous thrombosis was induced in mesenteric
venules of C57Bl/6 mice by ferric chloride. Thrombus formation was
visualized by in vivo rhodamine staining in intravital microscopy
(FIG. 5A). As described previously, inhibition of Mac-1 by an i.p.
injected F.sub.ab-fragment prolonged vessel occlusion time and
increased the release of thrombotic emboli (FIG. 5B, C), confirming
that Mac-1 is needed to stabilize thrombi. However, inhibition by
anti-M7 did not cause significant changes in vessel occlusion time
or release of thrombotic emboli, proposing that participating
pathways were not affected. Accordingly, formation of
leukocyte-platelet aggregates was diminished by unspecific blockade
of Mac-1, but not by specific inhibition of the CD40L/Mac-1
interaction (FIG. 5D). These data propose that anti-M7 is likely
not inducing unwanted effects on the haemostatic system.
EXAMPLE 7
[0076] Interaction of Mac-1 to CD40L, but not to other ligands,
improves skin wound healing. Leukocyte engagement is a critically
step in wound healing and delayed wound healing has been reported
in Mac-1 null mice. To test whether these effects are mediated by
Mac-1's interaction to CD40L, we treated C57Bl/6 mice with
i.p.-injections of F.sub.ab-fragments of either anti-M7, anti-Mac-1
or an according isotype control directly after induction of 4 mm
dorsal skin wounds. Interestingly, during the time course of the
experiment delayed wound healing in anti-Mac-1 treated mice was not
detected. However, skin wounds tent to close faster in Kaplan-Maier
wound closure analysis in anti-M7 treated mice and demonstrated a
smaller wound surface 6 days after wound induction (FIG. 6A,B).
This indicates that specific inhibition of the CD40L/Mac-1
interaction does not affect, but instead seems to exhibit
protective effects on skin wound healing.
EXAMPLE 8
[0077] Unselective inhibition of Mac-1 aggravates, while specific
blockade of its interaction to CD40L improves bacterial clearance,
inflammation, and survival during bacterial sepsis.
[0078] It has recently been shown that mice with a genetic
deficiency of Mac-1 demonstrated decreased survival during
bacterial sepsis, highlighting the potential role of the leukocyte
integrin in host defense and clearance of bacteria. To elucidate
whether ligand-specific blockade of Mac-1 and CD40L is rather
beneficial during bacterial sepsis, a model of coecal-ligation and
puncture sepsis (CLP) was performed. 20 hours after CLP procedure
inflammatory and patrolling monocytes circulating in blood and
basic inflammatory parameters were quantified. Interestingly, CLP
induced a strong mobilization of inflammatory Gr-1.sup.+ monocytes
to the circulation, reaching a percentage of the inflammatory
subset of about 82.4.+-.4.6% of all monocytes in IgG
F.sub.ab-fragment treated mice. This response was not affected by
F.sub.ab anti-Mac-1 treatment (77.4.+-.6.0%), but nearly reversed
by F.sub.ab anti-M7 treatment (56.8.+-.3.7%, FIG. 7A). During CLP,
myeloid cells populate the peritoneal cavity. Granulocytes
(F4/80.sup.-Gr-1.sup.+) invading the peritoneal cavity were
identified by flow cytometry (FIG. 7B). Both, anti-Mac-1 and
anti-M7, strongly reduced granulocyte accumulation by 59.9.+-.12.2%
and 73.8.+-.7.1% for anti-Mac-1 and anti-M7, respectively (FIG.
7C). The anti-inflammatory effect of anti-M7 treatment was further
reflected by a strong decrease of the acute-phase protein SAA by
63.4.+-.19.7% (FIG. 7D). Notably, anti-M7 improved bacterial
clearance in the plasma, while anti-Mac-1 worsened bacterial load
in both, plasma and the peritoneal cavity (FIG. 7E). During CLP,
accumulation of neutrophils is observed in the periphery, such as
the kidney and lung. To quantify granulocyte trafficking to the
spleen, ICH was performed against the granulocyte marker Ly6G in
kidney sections (FIG. 7F). Notably, both anti-integrin therapies
prevented neutrophil accumulation with a stronger effect in
anti-Mac-1 treated animals (FIG. 7F). Finally, it was assessed if
the new ligand-specific approach according to the invention is
beneficial in surviving sepsis. Therefore, CLP was induced and
animals were subsequently treated with F.sub.ab-preparations of
IgG, anti-Mac-1 and anti-M7 at 0, 48, and 96 hours after induction
of CLP operation. Survival rate was calculated employing
Kaplan-Maier analysis and log-rank testing. Animals treated with
anti-Mac-1 showed significantly decreased mean survival compared to
IgG-control treated animals (0% vs. 6.7% after 169 hours after
CLP-induction for anti-Mac-1 and IgG, respectively). Notably,
anti-M7 treated showed a survival rate of 40.0% at the end of the
study (FIG. 8), demonstrating that ligand-directed therapy is
superior to unspecific inhibition.
EXAMPLE 9
[0079] Treatment with anti-M7 improves the infiltration with
inflammatory leukocytes in the injured myocardium following
myocardial infarction. Accumulation of inflammatory leukocyte
occurs after myocardial infarction within days. Inflammatory
leukocyte recruited to the infarcted heart cause an inflammatory
response that aggravates wound healing and drives heart failure
after myocardial infarction. Inhibition of leukocyte infiltration
has been proposed to represent a therapeutic strategy, but not such
strategy is available. After induction of myocardial infarction in
mice by a surgical ligation of the left anterior descending
coronary artery (LAD) and treatment with anti-M7 less infiltrating
monocytes and neutrophils, a subclass of inflammatory leukocytes
that express Mac-1, were found in the injured myocardium. As a
result, anti-M7 attenuated heart failure.
Sequence CWU 1
1
171107PRTartificial sequencelight chain 1Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly1 5 10 15Glu Arg Val Ser Leu
Thr Cys Arg Ala Ser Gln Glu Ile Ser Gly Tyr 20 25 30Leu Ser Trp His
Gln Gln Lys Pro Asp Gly Thr Ile Lys Arg Leu Leu 35 40 45Tyr Ser Thr
Ser Thr Leu Asp Ser Gly Val Pro Lys Arg Phe Ser Gly 50 55 60Ser Arg
Ser Gly Ser Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Ser65 70 75
80Glu Asp Phe Ala Asp Tyr Tyr Cys Leu Gln Tyr Ala Ile Ser Pro Pro
85 90 95Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100
10526PRTartificial sequenceCDR 1 2Gln Glu Ile Ser Gly Tyr1
533PRTartificial sequenceCDR 2 3Ser Thr Ser149PRTartificial
sequenceCDR 3 4Leu Gln Tyr Ala Ile Ser Pro Pro Thr1
55121PRTartificial sequenceheavy chain 5Gln Val Thr Leu Lys Glu Ser
Gly Pro Gly Ile Leu Gln Thr Ser Gln1 5 10 15Thr Leu Ser Leu Thr Cys
Ser Phe Ser Gly Phe Ser Leu Ser Thr Ser 20 25 30Gly Met Gly Val Ser
Trp Ile Arg Gln Pro Ser Gly Lys Gly Leu Glu 35 40 45Trp Leu Ala His
Ile Tyr Trp Asp Asp Asp Lys Arg Tyr Asn Pro Ser 50 55 60Leu Lys Ser
Arg Leu Thr Ile Ser Lys Asp Thr Ser Arg Asn Gln Val65 70 75 80Phe
Leu Lys Ile Thr Ser Val Asp Thr Thr Asp Thr Ala Thr Tyr Tyr 85 90
95Cys Ala Leu Asn Tyr Tyr Asn Ser Thr Tyr Asn Phe Asp Phe Trp Gly
100 105 110Gln Gly Thr Thr Leu Thr Val Ser Ser 115
120610PRTartificial sequenceCDR1 6Gly Phe Ser Leu Ser Thr Ser Gly
Met Gly1 5 1077PRTartificial sequenceCDR 2 7Ile Tyr Trp Asp Asp Asp
Lys1 5813PRTartificial sequenceCDR 3 8Ala Leu Asn Tyr Tyr Asn Ser
Thr Tyr Asn Phe Asp Phe1 5 1099PRTartificial sequencepeptide 9Glu
Gln Leu Lys Lys Ser Lys Thr Leu1 5109PRTartificial sequencepeptide
M7 10Glu Gln Phe Lys Lys Ser Lys Thr Leu1 51113PRTartificial
sequencebinding peptide 11Val Met Glu Gln Leu Lys Lys Ala Lys Thr
Leu Met Gln1 5 10129PRTartificial sequencepeptide 12Lys Leu Ser Leu
Glu Lys Gln Thr Lys1 5138PRTartificial sequencepeptide 13Glu Glu
Phe Arg Ile His Phe Thr1 51413PRTartificial sequencepeptide M1
14Pro His Asp Phe Arg Arg Met Lys Glu Phe Val Ser Thr1 5
101513PRTartificial sequencepeptide M1 15Asn Ile Asp Phe Gln Lys
Met Lys Glu Phe Val Ser Thr1 5 10168PRTartificial sequencepeptide
M8 16Asp Glu Phe Arg Ile His Phe Thr1 51713PRTartificial
sequencepeptide 17Val Met Glu Gln Leu Lys Lys Ser Lys Thr Leu Phe
Ser1 5 10
* * * * *