Note: Descriptions are shown in the official language in which they were submitted.
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TREATMENT OF CHRONIC INFLAMMATORY CONDITIONS
FIELD OF THE INVENTION
This invention relates generally to the treatment of chronic inflammatory
conditions, and in particular to a method for reducing or otherwise
ameliorating the effects
of such conditions in patients. In one particular embodiment, the present
invention relates
to the treatment of arthritis, particularly rheumatoid arthritis which is a
chronic,
inflammatory disease characterised by the presence of numerous inflammatory
mediators
and by the destruction of diarthrodial joints.
BACKGROUND OF THE INVENTION
Interleukin-3 (IL-3) is a cytokine that can improve the body's natural
response to
disease as part of the immune system. IL-3 stimulates the differentiation of
multipotent
hematopoietic stem cells (pluripotent) into myeloid progenitor cells as well
as stimulating
proliferation of all cells in the myeloid lineage (erythrocytes, thrombocytes,
granulocytes,
monocytes, and dendritic cells). It is secreted by activated T cells to
support growth and
differentiation of T cells from the bone marrow in an immune response.
IL-3 exerts its activity through binding to a specific cell surface receptor
known as
the Interleukin-3 receptor (IL-3 R). IL-3 R is a heterodimeric structure
composed of a 70
kDa IL-3 R alpha (CD123) and a 120-140 kDa IL-3 R beta (CD131). The IL-3 R
alpha
chain (IL-3Ra) has a very short intracellular domain while the IL-3 R beta
chain (IL-3RJi)
has a very large cytoplasmic domain. IL-3 R alpha binds IL-3 with relatively
low affinity.
In the presence of IL-3 R beta, however, IL-3 R alpha has a much higher
affinity for IL-3.
It is not clear how signal transduction occurs following IL-3 binding, however
recent
studies suggest signalling requires formation of a higher order complex
comprising a
dodecamer'. The IL-3 R beta chain is also shared by the receptors for IL-5 and
GM-CSF.
Cells known to express IL-3 receptors include hematopoietic progenitors, mast
cells,
basophils and blood monocytes as well as more mature cells of various
hematopoietic
lineages including monocytes, macrophages, neutrophils, basophils, mast cells,
eosinophils, megakaryocytes, erythroid cells, and CD5+ B cell sub-populations
2. Non-
hematopoietic cells have also been shown to express the receptor including
some
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endothelial cells, stromal cells, dendritic cells and Leydig cells 2'3.
IL-3 provides a potentially important connection between the immune and
hemopoietic systems' 4. It may be important for the production and function of
mast cells
and basophils particularly during immune reactions 5. IL-3 can also promote
the growth
and activation of macrophage lineage populations 6-8 and can help to generate
dendritic
cells 9. As noted above, IL-3 signalling is mediated by a common receptor beta-
subunit
(IL-3R13) and a specific ligand-binding alpha-subunit (IL-3Ra), although in
the mouse
there is an additional beta-subunit 10.
Very little is known regarding the role of IL-3 in chronic inflammatory
conditions,
such as rheumatoid arthritis (RA). IL-3 mRNA could not be detected in the
synovium of
RA patients in one study 11 but was found in a later study 12; some but not
all RA patients
have been found to have detectable IL-3 in the circulation13 and there is an
association
between a single-nucleotide polymorphism in the IL-3 gene promoter and RA14.
However,
IL-3 levels decrease during arthritis progression in a rat arthritis model15
and it has been
reported recently that IL-3 administration inhibits murine inflammatory
arthritisl6
In work leading to the present invention, the inventors have found that the
effects
of chronic inflammatory conditions such as RA can be inhibited or reduced by
blocking or
interfering with the ligand/receptor interaction between IL-3 and IL-3R. This
finding is
quite surprising and was unexpected in view of the recent report 16 suggesting
that IL-3
administration has the potential to diminish the inflammatory response and
indirectly arrest
cartilage and bone loss in inflammatory arthritis.
Bibliographic details of the publications referred to in this specification
are
referenced at the end of the description.
The reference in this specification to any prior publication (or information
derived
from it), or to any matter which is known, is not, and should not be taken as
an
acknowledgment or admission or any form of suggestion that that prior
publication (or
information derived from it) or known matter forms part of the common general
knowledge in the field of endeavour to which this specification relates.
Throughout this specification and the claims which follow, unless the context
requires otherwise, the word "comprise", and variations such as "comprises"
and
"comprising", will be understood to imply the inclusion of a stated integer or
step or group
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of integers or steps but not the exclusion of any other integer or step or
group of integers or
steps.
SUMMARY OF THE INVENTION
In one aspect, the present invention provides a method for the treatment of a
chronic inflammatory condition in a patient, which comprises administration to
the patient
of an agent which blocks or inhibits IL-3 signalling events in the patient.
In another aspect, the invention provides the use of an agent which blocks or
inhibits IL-3 signalling events in, or in the manufacture of a medicament for,
the treatment
of a chronic inflammatory condition in a patient.
In yet another aspect, the invention provides an agent for the treatment of a
chronic
inflammatory condition in a patient, wherein said agent blocks or inhibits IL-
3 signalling
events in the patient.
In other aspects of this invention, the agent may be formulated in a
pharmaceutical
composition together with one or more pharmaceutically acceptable excipients
and/or
diluents, or it may be provided in a kit which optionally includes
instructions to use the
agent in accordance with a method for the treatment of a chronic inflammatory
condition
in a patient.
The chronic inflammatory condition may be, for example, arthritis, more
particularly inflammatory arthritis such as RA.
Preferably, the agent is one which blocks or inhibits IL-3/IL-3R or IL-3/IL-3R
alpha interactions in the patient. Preferably also the patient is a human.
BRIEF DESCRIPTION OF THE DRAWING
Figure 1 shows collagen-induced arthritis (CIA) progression (clinical score)
in
mice receiving anti-IL-3 mAb or PBS (control). Results expressed as mean +
SEM, n=10
mice treated group, n=6 mice control group.
Figure 2 shows effect of IL-3 signalling on the cell viability of monocytes
(CD 14+
derived from PBMC). The monocytes were plated at a density of -1.8 x 106 cells
per 6cm
IWAKI (low adherence) TC dish and cultured for 7 days in RPMI + IO%FCS and: IL-
3
(3ng/ml) alone, IL-3 (3ng/ml) + IL-3-R antibody, or IL-3 (0.3ng/ml) alone, or
IL-3
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(0.3ng/ml) + IL-3-R antibody. IL-3-R antibody was used at 1 g/ml in each case.
At day 4
new IL-3 and IL-3-R antibodies were added. On day 7 cells were removed and
counted.
Figure 3 shows IL-3 induces the basophil activation marker CD203c in a dose-
dependent manner. PBMC from urticaria patients (URT) and a normal donor (NOR)
were
isolated and were stimulated with polyclonal IgE, FMLP, FMLP and IL-3 or an
increasing
concentration of IL-3. The percentage of CD203+ve basophils were calculated
after
staining with an antibody cocktail to identify basophils and with an antibody
against the
basophil activation marker CD203c then analyzing by flow cytometry.
Figure 4 shows anti-IL-3R antibody blocks IL-3 -induced basophil activation.
PBMC from a normal donor were isolated and stimulated with an increasing
concentration
of IL-3 in the presence or absence of a neutralizing anti-IL-3R antibody
(CSL360). The
percentage of CD203+ve basophils were calculated after staining with an
antibody cocktail
to identify basophils and with an antibody against the basophil activation
marker CD203 c
then analyzing by flow cytometry.
Figure 5 shows anti-CD123 monoclonal antibody (CSL362) depletes basophil in a
time-dependent manner. PBMC from a normal donor were isolated and incubated
without
antibody or with a depleting anti-CD123 antibody (CSL362) for various times
(as
indicated). The percentage of basophils remaining were calculated after
staining with an
antibody cocktail to identify basophils and analyzing by flow cytometry.
Figure 6 shows anti-CD123 mAb reproducibly depletes basophils within 24 hr.
PBMC from three normal donors were isolated and incubated without antibody or
with a
depleting anti-CD123 antibody (CSL362) for 24 h. The percentage of basophils
remaining
were calculated after staining with an antibody cocktail to identify basophils
and analyzing
by flow cytometry.
Figure 7 shows anti-CD123 mAb activates NK cells within 24 hr. PBMC from
three normal donors were isolated and incubated without antibody (solid line)
or with a
depleting anti-CD123 antibody (CSL362, dashed line) for 24 h. Activation of NK
cells
(CD56+ve cells) was determined by CD 16 down-regulation. PBMC were stained
with
anti-CD56 antibodies to identify NK cells and with anti-CD 16 antibodies. Loss
of CD 16
staining, as compared with the no antibody control, was determined by flow
cytometry.
Figure 8 shows depletion of murine basophils in peripheral blood by in vivo
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administration of anti-IL-3Ra antibodies. BALB/c mice were intravenously
administered
with an anti-IL-3Ra antibody (1C2), anti-CD200R3 (Ba103) or the isotype
control
antibodies mouse IgG2a (mIgG2a) or rat IgG2b (rIgG2b). All antibodies were
injected at
30 g per mouse except for IC2 which was injected at 18 gg per mouse.
Peripheral blood
cells were isolated 24 h post antibody administration and stained for FccR1a
and CD49b
expression to identify basophils. Representative staining profiles from flow
cytometry
analysis are shown (a). Basophil populations are boxed. The percentage of
basophils per
mouse were calculated from flow cytometry analysis and the mean (+ SEM) of 3
mice per
group are shown (b). * * * : p<0.001, * * : p<0.01.
DETAILED DESCRIPTION OF THE INVENTION
In one aspect, the present invention provides a method for the treatment of a
chronic inflammatory condition in a patient, which comprises administration to
the patient
of an agent which blocks or inhibits IL-3 signalling events in the patient.
Preferably, the agent is one which blocks or inhibits IL-3/IL-3R or IL-3/IL-3R
alpha interactions in the patient.
Chronic inflammatory conditions which may be treated in accordance with the
present invention are well known to persons skilled in this field, and include
in particular
arthritis, more particularly inflammatory arthritis such as adult and juvenile
RA. Other
indications include but are not limited to chronic obstructive pulmonary
disease (COPD);
inflammatory bowel diseases (IBD) such as Crohn's disease and ulcerative
colitis; chronic
inflammatory demyelinating polyneuropathy (CIDP); atherosclerosis;
scleroderma;
systemic lupus erythematosus (SLE); Sjogren's syndrome; gout; osteoarthritis;
polymyalgia rheumatica; seronegative spondyloarthropathies including
ankylosing
spondylitis; Reiter's disease, psoriatic arthritis, mixed connective tissue
disease (MCTD);
chronic Lyme arthritis; Still's disease; chronic urticaria; uveitis associated
with rheumatoid
arthritis and disorders resulting in inflammation of the voluntary muscle and
other muscles,
including dermatomyositis, inclusion body myositis, polymyositis, and
lymphangioleiomyomatosis.
Reference herein to "treatment" is to be considered in its broadest context
and
includes both therapeutic treatment and prophylactic or preventative measures.
Patients in
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need of treatment include those already afflicted with a chronic inflammatory
condition as
well as those in which such a condition is to be prevented. Patients who are
partially or
totally recovered from the condition might also be in need of treatment. The
term
"treatment" does not necessarily imply that a patient is treated until total
recovery.
Accordingly, treatment includes reduction or amelioration of the symptoms of a
particular
chronic inflammatory condition as well as halting or at least retarding the
onset,
development or progress of, reducing the severity of, or eliminating, a
particular chronic
inflammatory condition.
The agent which is administered in accordance with the present invention
blocks or
inhibits the activation of IL-3 signalling events in the patient, preferably
by blocking or
inhibiting IL-3/IL-3R or IL-3/IL-3R alpha interactions. As used herein, a
reference to
"blocks or inhibits IL-3 signalling events in the patient" encompasses any
intervention
which leads to a decreased level of IL-3 initiated signalling. Such
interventions include,
by way of example, the use of agents which specifically block or inhibit the
activation of
IL-3 signalling events (e.g. agents which target IL-3, IL-Ma, IL-3R(3), as
well as the use
of agents designated to selectively target cells capable of IL-3 signalling
and by such
targeting induce cell death (e.g. agents which target IL-3R and carry an anti-
cellular
moiety).
In one embodiment of the invention, the agent may be an antigen binding
molecule
which binds selectively to IL-3, or to IL-3R, the IL-3R alpha or the IL-3R
beta.
As used herein the term "antigen binding molecule" refers to an intact
immunoglobulin, including monoclonal antibodies, such as chimeric, humanized
or human
monoclonal antibodies, or to antigen-binding (including, for example, Fv, Fab,
Fab' and
F(ab')2 fragments) and/or variable-domain-comprising fragments of an
immunoglobulin
that compete with the intact immunoglobulin for specific binding to the
binding partner of
the immunoglobulin, e.g. a host cell protein. Regardless of structure, the
antigen-binding
fragments bind with the same antigen that is recognized by the intact
immunoglobulin.
Antigen-binding fragments may be produced synthetically or by enzymatic or
chemical
cleavage of intact immunoglobulins or they may be genetically engineered by
recombinant
DNA techniques. The methods of production of antigen binding molecules and
fragments
thereof are well known in the art and are described, for example, in
Antibodies, 4
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Laboratory Manual, Edited by E. Harlow and D. Lane (1988), Cold Spring Harbor
Laboratory, Cold Spring Harbor, New York, which is incorporated herein by
reference.
Preferably, the antigen binding molecule is a monoclonal antibody.
In this embodiment of the invention, the antigen binding molecule may comprise
a
modified Fc region, more particularly a Fc region which has been modified to
provide
enhanced effector functions, such as enhanced binding affinity to Fc
receptors, antibody-
dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated
phagocytosis (ADCP) and complement-dependent cytotoxicity (CDC). For the IgG
class
of antibodies, these effector functions are governed by engagement of the Fc
region with a
family of receptors referred to as the Fcy receptors (FcyRs) which are
expressed on a
variety of immune cells. Formation of the Fc/ FcyR complex recruits these
cells to sites of
bound antigen, typically resulting in signalling and subsequent immune
responses.
Methods for optimizing the binding affinity of the FcyRs to the antibody Fe
region in order
to enhance the effector functions, in particular to alter the ADCC and/or CDC
activity
relative to the "parent" Fc region, are well known to persons skilled in the
art, and are
described, for example, in International Patent Publication No. WO
2009/070844. These
methods can include modification of the Fc region of the antibody to enhance
its
interaction with relevant Fe receptors and increase its potential to
facilitate ADCC and
ADCP. Enhancements in ADCC activity have also been described following the
modification of the oligosaccharide covalently attached to IgGI antibodies at
the
conserved Asn297 in the Fc region.
The term "binds selectively", as used herein, in reference to the interaction
of an
antigen binding molecule, e.g. an antibody or antibody fragment, and its
binding partner,
e.g. an antigen, means that the interaction is dependent upon the presence of
a particular
structure, e.g. an antigenic determinant or epitope, on the binding partner.
In other words,
the antibody or antibody fragment preferentially binds or recognizes the
binding partner
even when the binding partner is present in a mixture of other molecules or
organisms.
Without wishing to be bound by any particular theory, it is believed that in
this
embodiment of the invention, by binding selectively to IL-3, or to IL-3R, IL-
3R alpha or
IL-3R beta, an antigen binding molecule such as an antibody or antibody
fragment blocks
or inhibits the ligand/receptor interaction and thereby interferes with IL-3
signal activation.
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in one embodiment, the antigen binding molecule may be a monoclonal antibody
which binds selectively to IL-3R alpha (CD123). Thus, the antigen binding
molecule may
be monoclonal antibody (MAb) 7G3, raised against CD 123, which has previously
been
shown to inhibit IL-3 mediated proliferation and activation of both leukaemic
cell lines and
primary cells (see US Patent No. 6,177,678 to Lopez). Alternatively, the agent
may be the
monoclonal antibody CSL360, a chimeric antibody obtained by grafting the light
variable
and heavy variable regions of the mouse monoclonal antibody 7G3 onto a human
IgG1
constant region (see International Patent Publication No. WO 2009/070844).
Like 7G3,
CSL360 binds to CD123 (human IL-3Ra.) with high affinity, competes with IL-3
for
binding to the receptor and blocks its biological activities. CSL360 also has
the advantage
of potential utility as a human therapeutic agent by virtue of its human IgG1
Fc region
which would be able to initiate effector activity in a human setting.
Moreover, it is likely
that in humans it would show reduced clearance relative to the mouse 7G3
equivalent and
be less likely to be immunogenic. Further examples of this antigen binding
molecule
include humanised antibody variants of 7G3 or CSL360, fully human anti-CD123
antibodies and anti-CD123 antibodies with enhanced effector function (such as
ADCC
activity) as described, for example in Example 4 of International Patent
Publication No.
W02009/070844.
In another embodiment of the present invention, the agent may be an IL-3
mutein
which binds to IL-3R but either does not lead to or at least results in
reduced IL-3 signal
activation. Generally, these `IL-3 muteins' include natural or artificial
mutants differing
by the addition, deletion and/or substitution of one or more contiguous or non-
contiguous
amino acid residues. An example of an IL-3 mutein which binds to IL-3R but
exhibits
reduced IL-3 signal activation is a 16/84 C-->A mutant17. IL-3 muteins may
also include
modified polypeptides in which one or more residues are modified to, for
example,
increase their in vivo half life. This could be achieved by attaching other
elements such as
a PEG group. Methods for the PEGylation of polypeptides are well known in the
art.
In another embodiment of the present invention, the agent is a soluble
receptor
which is capable of binding to IL-3. Examples of such soluble receptors
include the
extracellular portion of IL-3R alpha or a fusion protein comprising the
extracellular portion
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of IL-3R alpha fused to the extracellular portion of IL-3R beta.
In yet another embodiment, the agent may comprise an anti-cellular moiety that
can
be targeted to cells capable of IL-3 signalling to induce cell death. In some
embodiments,
the agent may comprise the anti-cellular moiety conjugated to an antigen
binding molecule
which binds selectively to IL-3, or to IL-3R, IL-3R alpha or IL-3R beta. In
other
embodiments the agent may comprise an anticellular moiety conjugated to a IL-3
mutein.
Examples of suitable anti-cellular moieties include chemotherapeutics,
radioisotopes or
cytotoxins. Chemotherapeutics include a hormone such as a steroid; an anti-
metabolite
such as cytosine arabinoside, fluorouracil, methotrexate or aminopterin; an
anthracycline ;
mitomycin C; a vinca alkaloid; demecolcine; etoposide; mithramycin;
calicheamycin, CC-
1065 and derivatives thereof, or an alkylating agent such as chlorambucil or
melphalan, a
coagulant, a cytokine, growth factor, bacterial endotoxin or the lipid A
moiety of bacterial
endotoxin. Radioisotopes include a-emitters such as, for example, 21
lAstatine,
212Bismuth and 213Bismuth, as well as (3-emitters such as, for example,
131Iodine,
90Yttrium, 177Lutetium, 153Samarium and 109Palladium, and Auger emitters such
as, for
example, 111 Indium. Cytotoxins include generally a plant-, fungus-or bacteria-
derived
toxin, such as an A chain toxin, a ribosome inactivating protein, a-sarcin,
aspergillin,
restirictocin, a ribonuclease, diphtheria toxin or pseudomonas exotoxin, to
mention just a
few examples, as well as cytotoxins derived from marine organisms such as
sponges, such
as Kahalalide F, Ecteinascidin (YondelisTM), or Variolin B, for example.
The agent is administered in an effective amount. An "effective amount" means
an
amount necessary at least partly to attain the desired response or to delay or
inhibit
progression or halt altogether, the progression of the particular condition
being treated.
The amount varies depending upon the health and physical condition of the
individual to
be treated, the racial background of the individual to be treated, the degree
of protection
desired, the formulation of the composition, the assessment of the medical
situation, and
other relevant factors. It is expected that the amount will fall in a
relatively broad range
that can be determined through routine trials. If necessary, the
administration of the agent
may be repeated one or several times. The actual amount administered will be
determined
both by the nature of the condition which is being treated and by the rate at
which the
agent is being administered.
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Preferably, the patient is a human, however the present invention extends to
treatment and/or prophylaxis of other mammalian patients including primates,
livestock
animals (e.g. sheep, pigs, cattle, horses, donkeys), laboratory test animals
(e.g. mice,
rabbits, rats, guinea pigs), companion animals (e.g. dogs, cats) and captive
wild animals.
In accordance with the present invention, the agent is preferably administered
to a
patient by a parenteral route of administration. Parenteral administration
includes any
route of administration that is not through the alimentary canal (that is, not
enteral),
including administration by injection, infusion and the like. Administration
by injection
includes, by way of example, into a vein (intravenous), an artery
(intraarterial), a muscle
(intramuscular) and under the skin (subcutaneous). The agent may also be
administered in
a depot or slow release formulation, for example, subcutaneously,
intradermally or
intramuscularly, in a dosage which is sufficient to obtain the desired
pharmacological
effect.
In another aspect, the present invention provides the use of an agent which
blocks
or inhibits IL-3 signalling events in, or in the manufacture of a medicament
for, the
treatment of a chronic inflammatory condition in a patient.
In yet another aspect, the present invention provides an agent for the
treatment of a
chronic inflammatory condition in a patient, wherein said agent blocks or
inhibits IL-3
signalling events in the patient.
In this aspect of the invention, the agent as described above may be
formulated in a
pharmaceutical composition together with one or more pharmaceutically
acceptable
excipients and/or diluents.
Compositions suitable for parenteral administration conveniently comprise a
sterile
aqueous preparation of the active component which is preferably isotonic with
the blood of
the recipient. This aqueous preparation may be formulated according to known
methods
using suitable dispersing or wetting agents and suspending agents. The sterile
injectable
preparation may also be a sterile injectable solution or suspension in a non-
toxic
parenterally-acceptable diluent or solvent, for example as a solution in
polyethylene glycol
and lactic acid. Among the acceptable vehicles and solvents that may be
employed are
water, Ringer's solution, suitable carbohydrates (e.g. sucrose, maltose,
trehalose, glucose)
and isotonic sodium chloride solution. In addition, sterile, fixed oils are
conveniently
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employed as a solvent or suspending medium. For this purpose, any bland fixed
oil may
be employed including synthetic mono- or di-glycerides. In addition, fatty
acids such as
oleic acid find use in the preparation of injectables.
The formulation of such therapeutic compositions is well known to persons
skilled
in this field. Suitable pharmaceutically acceptable carriers and/or diluents
include any and
all conventional solvents, dispersion media, fillers, solid carriers, aqueous
solutions,
coatings, antibacterial and antifungal agents, isotonic and absorption
delaying agents, and
the like. The use of such media and agents for pharmaceutically active
substances is well
known in the art, and it is described, by way of example, in Remington's
Pharmaceutical
Sciences, 18th Edition, Mack Publishing Company, Pennsylvania, USA. Except
insofar as
any conventional media or agent is incompatible with the active ingredient,
use thereof in
the pharmaceutical compositions of the present invention is contemplated.
Supplementary
active ingredients can also be incorporated into the compositions.
In a further aspect of the invention, there is provided a kit comprising (i)
an agent
as described above, and optionally (ii) instructions to use the agent in
accordance with a
method for the treatment of a chronic inflammatory condition in a patient.
The present invention is further illustrated by the following non-limiting
Examples.
EXAMPLE I
The effect of a neutralizing monoclonal antibody (mAb) to murine IL-3 on
disease
progression was tested in the collagen-induced arthritis (CIA) model, the most
widely used
murine model for rheumatoid arthritis.
Male DBA/1 mice (8-12 weeks old, 10 mice per group) were immunized
intradermally with type II collagen in adjuvant on days 0 and 21 18
Mice were assessed for redness and swelling of limbs and a clinical score was
allocated for each limb using an established scoring system as follows: 0 -
normal; 1 -
slight swelling and/or erythema; 2 - extensive swelling and/or erythema; 3 -
severe
swelling; 4 - severe swelling and/or rigidity. Severity of arthritis is
expressed in terms of
the mean clinical score totalled for all four limbs (range 0-16 per mouse).
Mice were treated with 250 g anti-IL-3 mAb (Southern Biotech)/mouse or PBS on
day 21, 23, 25, 28 and 30. As can be seen in Figure 1, there was a suppression
of disease
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severity in the anti-IL-3-treated group.
This experiment is particularly encouraging as in this particular experiment
there
was a very rapid induction of a very severe disease with the plateau in the
control mice
being reached quickly. This plateau is normally reached about one week later.
The literature data for the relatively acute murine CIA indicates that
neutralizing
anti-IL-3 Ab is required soon after disease induction and is ineffective if
delayed post
disease onset 19. This does not necessarily mean that IL-3 would not be a
target in
inflammation. We do not yet understand the background inflammatory/autoimmune
events responsible for driving diseases such as rheumatoid arthritis (RA) and
how closely
these events are mirrored in animal models. Even for a chronic condition such
as RA there
are patients with "acute onset" disease. Also, anti-IL-3 therapy might still
be beneficial in
RA, for example, since the disease often relapses providing opportunities to
suppress its
exacerbations. It should also be borne in mind that the commonly used anti-
inflammatory
glucocorticoids are widely believed to act by down-regulating inflammatory
mediator gene
expression at the transcriptional level - they do not work in vitro in
surrogate inflammation
assays if added subsequent to the inciting stimulus.
EXAMPLE 2
The role of IL-3 signalling as a pro-inflammatory cytokine in CD 14+ monocytes
was investigated. Peripheral Blood Mononuclear Cells (PBMC) were isolated from
a Red
Cross donor buffy pack. CD 14+ monocytes were then purified by negative
selection
(MACS separation) such that approximately 80% of the cells were CD 14+ as
assessed by
flow cytometry. Monocytes were plated at a density of -1.8 x 106 cells per 6cm
IWAKI
(low adherence) TC dish and cultured for 7 days in RPMI + 10%FCS and: IL-3
(3ng/ml)
alone, IL-3 (3ng/ml) + IL-3-R antibody, IL-3 (0.3ng/ml) alone, or IL-3
(0.3ng/ml) + IL-3-
R antibody. IL-3-R antibody was used at lug/ml in each case. At day 4 new IL-3
and IL-
3-R antibodies were added. On day 7 cells were removed and counted before
being lysed
in RNA lysis buffer. The results indicate that IL-3 provides a pro-survival
stimulus for the
monocytes in a dose dependent manner and this effect was overcome by anti-IL-
3R
antibody (Figure 2). Thus blocking or inhibiting IL-3 signalling in CD 14+
monocytes
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prevents their survival and accumulation at sites of inflammation and as such
provides a
means to control inflammation.
EXAMPLE 3
Urticaria, commonly known as hives, is an inflammatory condition that
manifests
as recurrent wheals that range in size from several centimeters down to just a
few
millimeters. Generally the wheals are pink in color with a pale center and can
be
associated with a burning or prickly sensation. Urticaria can present at any
age and 1% -
5% of the population will present with urticaria at some point in their
lifetime. Chronic
urticaria greatly impacts on quality of life, similar to patients with severe
atopic dermatitis,
psoriasis or acne. Most cases of chronic urticaria are idiopathic in nature;
however, it is
becoming increasingly clear that in many cases (35 % - 50%) auto-antibodies to
the high
affinity IgE receptor (FccRl) or IgE itself are present, suggesting that
chronic urticaria
may be an autoimmune disease. Mast cells and basophils are the main cell types
that
express FcsRl and respond to auto-antibodies in urticaria patients. When
activated, mast
cells and basophils release large amounts of histamine, which is the main
effector molecule
driving urticarial wheal formation. Both mast cells and basophils express the
IL-3 receptor
and IL-3 can prime these cells to produce increased levels of effector
molecules, such as
histamine, when exposed to triggers such as IgE and C5a.
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PBMC from seven urticaria patient samples and four normal donor samples have
been analyzed. IL-3 can activate human basophils ex vivo (Figure 3) and a
neutralizing
anti-IL-Ma antibody (CSL360- see International Patent Publication No.
W02009/070844)
can inhibit IL-3 induced human basophil activation (Figure 4). An ADCC
optimized anti-
IL-3Ra antibody (CSL362-an afucosylated variant of humanized and affinity
matured anti-
CD123 mAb 168-26 as described in International Patent Publication No. WO
2009/070844) can deplete human basophils from PBMC in a time dependent manner
(Figure 5). Complete or near-complete basophil depletion was observed in three
independent donors within 24 hours of CSL362 addition (Figure 6). NK cell
activation
was observed within 24 hours of addition of CSL362 suggesting that the
depletion of
basophils is via NK-cell mediated ADCC (Figure 7).
EXAMPLE 4
This example demonstrates that anti-IL-3R antibodies can also affect the
number
and level of activation of basophils in vivo. Female BALB/c mice (10-12 weeks
old) were
treated with a tail vein intravenous injection of anti-IL3Ra antibody 1C2 (see
Example 5)
or the control mouse IgG2a. A commercial antibody Bal 03 that targets against
CD200R3
and has been shown to specifically deplete mouse basophils20, and its control
antibody rat
IgG2b were tested alongside as the controls. All antibodies were injected at
30 g in 200 l
of PBS with the exception of 1C2 which was administered at 18 g. A day later,
peripheral
blood and peritoneal cells were isolated and single suspension of cells
prepared. Cells were
pre-incubated with anti-FcyRII-III to prevent nonspecific binding. Cells were
stained with
FITC-conjugated anti-FcsRIa monoclonal antibody and PE-conjugated anti-CD49b
monoclonal antibody to identify basophils (FcsRIa+ CD49b). FITC-hamster IgG
and PE-
rat IgM were used as the isotype controls for FccRIa and CD49b antibodies,
respectively.
Debris was gated out using a forward scatter (FSC) versus side scatter (SSC)
and dead
cells were discriminated with 7-amino-actinomycin D (7-AAD) staining. Stained
cells
were then analysed with FACSCantoTM (BD Biosciences) and data analysed using
FlowJo
software.
A single intravenous injection of 18 g anti-IL3Ra antibody 1C2 induced a
drastic
reduction in basophil frequency in peripheral blood, to approximately 23% of
the level in
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the isotype control (mouse IgG2a)-treated mice (Figure 8). This depletion
efficacy was
comparable to that from the administration of 30 g Ba103. Unlike the effect on
basophils,
no significant reduction in the frequency of peritoneal mast cells was noted
in the mice
treated with anti-IL3Ra or Ba103 antibodies (data not shown). The observation
with
Ba103 antibody on mast cells is consistent with the studies by Obata and
colleagues 20
EXAMPLE 5
1) Generation of murine IL3 receptor specific monoclonal antibodies
Antibody sequences that specifically recognised murine IL-3R alpha (mIL3Ra)
were
isolated from a library of human antibody sequences expressed as Fab fragments
fused to
the gIII protein on the surface of the filamentous bacteriophage M13 (Dyax
Corp.). Anti-
mIL3Ra phage-displayed antibody fragments were isolated by incubation of the
phage
library with a commercially-available purified recombinant fusion protein
consisting of
amino acids 17-331 of mIL3Ra fused to residues 100-330 of human IgG1 (Fc
fragment)
with a short polypeptide linker (sequence: IEGRID) supplied by R&D systems
Inc.
Specifically-bound phage were enriched and isolated as individual clones using
standard
methods. Individual clones were tested for specific binding to both the
original target
(mIL3Ra - Fc fusion) and to the extracellular domain of mIL3Ra expressed as
residues 1-
331 with a C-terminal hexa-histidine tag (msIL-3R-6His). Phage clones that
bound to these
targets and not to control proteins were selected for further analysis. Unique
clones were
identified by DNA sequencing of both polypeptide chains of the encoded
antibody and the
binding affinity of these clones for mIL-3R-6His was quantified by competitive
ELISA.
Clones with acceptable affinity were selected for re-engineering and
expression as
chimeric antibodies (human variable regions and murine IgG2a / kappa constant
regions)
for further analysis.
2) Mammalian expression vector construction for transient expression
Heavy and light chain variable regions for the mIL3Ra-specific antibodies were
PCR
amplified from the phagemid vectors using standard molecular biology
techniques. The
heavy chain variable region was then cloned into the mammalian expression
vector
pcDNA3. 1 (+)-mlgG2a, which is based on the pcDNA3.1(+) expression vector
(Invitrogen)
modified to include the murine IgG2a constant region and a terminal stop
codon. The light
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chain variable region was cloned into the expression vector pcDNA3. I (+)-mx,
which is
based on the pcDNA3.1(+) expression vector modified to include the murine
kappa
constant region. The expression vectors also contained a Kozak translation
initiation
sequence, an ATG start codon and appropriate signal peptides.
3) Cell Culture
Serum-free suspension adapted 293-T cells were obtained from Genechoice Inc.
Cells
were cultured in FreeStyleTM Expression Medium (Invitrogen) supplemented with
penicillin/streptomycin/fungizone reagent (Invitrogen). Prior to transfection
the cells were
maintained at 37 C in humidified incubators with an atmosphere of 8% C02.
4) Transient Tansfection
Transient transfection of the anti-mIL3Ra expression plasmids using 293-T
cells was
performed using 293fectin transfection reagent (Invitrogen) according to the
manufacturer's instructions. The light and heavy chain expression vectors were
combined
and co-transfected with the 293-T cells. Cells (1000 ml) were transfected at a
final
concentration of 1 x 106 viable cells/ml and incubated in a Cellbag 2L (Wave
Biotech/GE
Healthcare) for 5 days at 37 C with an atmosphere of 8% C02 on a 2/10 Wave
Bioreactor
system 2/10 or 20/50 (Wave Biotech/GE Healthcare). The culture conditions were
35
rocks per minute with an angle of 8 . Pluronic F-68 (Invitrogen), to a final
concentration
of 0.1% v/v, was added 4 hours post-transfection. 24 hours post-transfection
the cell
cultures were supplemented with Tryptone Ni (Organotechnie, France) to a final
concentration of 0.5 % v/v. The cell culture supernatants were harvested by
centrifugation
at 2500 rpm and were then passed through a 0.45 M filter (Nalgene) prior to
purification.
5) Analysis of Protein Expression
After 5 days 20 l of culture supernatant was electrophoresed on a 4-20% Tris-
Glycine
SDS polyacrylamide gel and the antibody was visualised by staining with
Coomassie Blue
reagent.
6) Antibody Purification
Anti-mIL3Ra antibodies were purified using protein A affinity chromatography
at 4 C,
where MabSelect resin (5 ml, GE Healthcare, UK) was packed into a 30 ml Poly-
Prep
empty column (Bio-Rad, CA). The resin was first washed with 10 column volumes
of
pyrogen free GIBCO Distilled Water (Invitrogen, CA) to remove storage ethanol
and then
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equilibrated with 5 column volumes of pyrogen free phosphate buffered saline
(PBS)
(GIBCO PBS, Invitrogen, CA). The filtered conditioned cell culture media (1L)
was
loaded onto the resin by gravity feed. The resin was then washed with 5 column
volumes
of pyrogen free PBS to remove non-specific proteins. The bound antibody was
eluted with
2 column volumes of 0.1 M glycine pH 2.8 (Sigma, MO) into a fraction
containing 0.2
column volumes of 2M Tris-HC1 pH 8.0 (Sigma, MO) to neutralise the low pH. The
eluted antibody was dialysed for 18 hrs at 4 C in a 12m1 Slide-A-Lyzer
cassette MW
cutoff 3.5kD (Pierce, IL) against 5L PBS. The antibody concentration was
determined by
measuring the absorbance at 280 nm using an Ultraspec 3000 (GE Healthcare, UK)
spectrophotometer. The purity of the antibody was analysed by SDS-PAGE, were 2
tig
protein in reducing Sample Buffer (Invitrogen, CA) was loaded onto a Novex 10-
20% Tris
Glycine Gel (Invitrogen, CA) and a constant voltage of 150V was applied for 90
minutes
in an XCell SureLock Mini-Cell (Invitrogen, CA) with Tris Glycine SDS running
buffer
before being visualised using Coomassie Stain, as per the manufacturer's
instructions.
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