Note: Descriptions are shown in the official language in which they were submitted.
DEMANDE OU BREVET VOLUMINEUX
LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVET COMPREND
PLUS D'UN TOME.
CECI EST LE TOME 1 DE 2
CONTENANT LES PAGES 1 A 151
NOTE : Pour les tomes additionels, veuillez contacter le Bureau canadien des
brevets
JUMBO APPLICATIONS/PATENTS
THIS SECTION OF THE APPLICATION/PATENT CONTAINS MORE THAN ONE
VOLUME
THIS IS VOLUME 1 OF 2
CONTAINING PAGES 1 TO 151
NOTE: For additional volumes, please contact the Canadian Patent Office
NOM DU FICHIER / FILE NAME:
NOTE POUR LE TOME / VOLUME NOTE:
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
Methods and Compositions for Treating and Monitoring Treatment of
IL-13-Associated Disorders
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims priority to U.S. Application Serial No. 60/874,333,
filed
on December 11, 2006, and U.S. Application Serial No. 60/925,932, filed on
April 23,
2007, the contents of both of which are hereby incorporated by reference in
their entirety.
SEQUENCE LISTING
A copy of the Sequence Listing in electronic and paper form is being submitted
herewith.
BACKGROUND
Interleukin-13 (IL-13) is a cytokine secreted by T lymphocytes and mast cells
(McKenzie et al. (1993) Proc. Natl. Acad. Sci. USA 90:3735-39; Bost et al.
(1996)
Immunology 87:663-41). IL-13 shares several biological activities with IL-4.
For
example, either IL-4 or IL-13 can cause IgE isotype switching in B cells
(Tomkinson et
al. (2001) .J. Immunol. 166:5792-5800). Additionally, increased levels of cell
surface
CD23 and serum CD23 (sCD23) have been reported in asthmatic patients (Sanchez-
Guererro et al. (1994) Allergy 49:587-92; DiLorenzo et al. (1999) Allergy
Asthma Proc.
20:119-25). In addition, either IL-4 or IL-13 can upregulate the expression of
MHC class
II and the low-affinity IgE receptor (CD23) on B cells and monocytes, which
results in
enhanced antigen presentation and regulated macrophage function (Tomkinson et
al.,
supra). Importantly, either IL-4 or IL-13 can increase the expression of VCAM-
1 on
endothelial cells, which facilitates preferential recruitment of eosinophils
(and T cells) to
the airway tissues (Tomkinson et al., supra). Either IL-4 or IL-13 can also
increase
airway mucus secretion, which can exacerbate airway responsiveness (Tomkinson
et al.,
supra). These observations suggest that although IL- 13 is not necessary for,
or even
capable of, inducing Th2 development, IL-13 may be a key player in the
development of
airway eosinophilia and AHR (Tomkinson et al., supra; Wills-Karp et al. (1998)
Science
282:2258-61).
-1-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
SUMMARY
Methods and compositions for treating and/or monitoring treatment of IL-13-
associated disorders or conditions are disclosed. In one aspect, Applicants
have
discovered that a single administration of an IL-13 antagonist or an IL-4
antagonist to a
subject, prior to the onset of an IL-13 associated disorder or condition,
reduces one or
more symptoms of the disorder or condition, relative to an untreated subject.
Enhanced
reduction of the symptoms of the disorder or condition is detected after co-
administration
of the IL- 13 antagonist with the IL-4 antagonist, relative to the reduction
detected after
administration of the single agent. Thus, methods for reducing or inhibiting,
or
preventing or delaying the onset of, one or more symptoms of an IL- 13-
associated
disorder or condition using an IL-13 antagonist alone or in combination with
an IL-4
antagonist are disclosed. In other embodiments, methods for evaluating the
efficacy of
an IL-13 antagonist in treating or preventing an IL-13-associated disorder or
condition in
a subject, e.g., a human subject, are also disclosed.
Accordingly, in one aspect, the invention features a method of treating or
preventing an IL-13-associated disorder or condition in a subject. The method
includes
administering an IL-13 antagonist and/or an IL-4 antagonist to the subject, in
an amount
effective to reduce one or more symptoms of the disorder or condition (e.g.,
in an amount
effective to reduce one or more of: IgE levels, histamine release, eotaxin
levels, or a
respiratory symptom in the subject). In the case of prophylactic use (e.g., to
prevent,
reduce or delay onset or recurrence of one or more symptoms of the disorder or
condition), the subject may or may not have one or more symptoms of the
disorder or
condition. For example, the IL-13 antagonist and/or IL-4 antagonist can be
administered
prior to any detectable manifestation of the symptoms, or after at least some,
but not all
the symptoms are detected. In the case of therapeutic use, the treatment may
improve,
cure, maintain, or decrease duration of, the disorder or condition in the
subject. In
therapeutic uses, the subject may have a partial or full manifestation of the
symptoms. In
a typical case, treatment improves the disorder or condition of the subject to
an extent
detectable by a physician, or prevents worsening of the disorder or condition.
In one embodiment, the IL-13 antagonist and/or IL-4 antagonist is administered
at
a single treatment interval, e.g., as a single dose, or as a repeated dose of
no more than
-2-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
two or three doses during a single treatment interval, e.g., the repeated dose
is
administered within one week or less from the initial dose. For example, the
IL-13
antagonist and/or the IL-4 antagonist can be administered at a single
treatment interval
prior to the onset or recurrence of one or more symptoms associated with the
IL-13-
disorder or condition, but before a full manifestations of the symptoms
associated with
the disorder or condition. In certain embodiments, the IL-13 antagonist and/or
IL-4
antagonist is administered to the subject prior to exposure to an agent that
triggers or
exacerbates an IL-13-associated disorder or condition, e.g., an allergen, a
pollutant, a
toxic agent, an infection and/or stress. In some embodiments, the IL-13
antagonist and/or
IL-4 antagonist is administered prior to, during, or shortly after exposure to
the agent that
triggers and/or exacerbates the IL- 13 -associated disorder or condition. For
example, the
IL-13 antagonist and/or IL-4 antagonist can be administered 1, 5, 10, 25, or
24 hours; 2,
3, 4, 5, 10, 15, 20, or 30 days; or 4, 5, 6, 7 or 8 weeks, or more before or
after exposure to
the triggering or exacerbating agent. Typically, the IL-13 and/or IL-4
antagonist can be
administered anywhere between 24 hours and 2 days before or after exposure to
the
triggering or exacerbating agent. In those embodiments where administration
occurs
after exposure to the agent, the subject may not be experiencing symptoms or
may be
experiencing a partial manifestation of the symptoms. For example, the subject
may have
symptoms of an early stage of the disorder or condition. Each dose can be
administered
by inhalation or by injection, e.g., subcutaneously, in an amount of about 0.5-
10 mg/kg
(e.g., about 0.7-5 mg/kg, 0.9-4 mg/kg, 1-3 mg/kg, 1.5-2.5 mg/kg, 2 mg/kg).
The IL-13 antagonist and/or IL-4 antagonist can be administered to a subject
having, or at risk of having, an IL-13-associated disorder or condition.
Typically, the
subject is a mammal, e.g., a human (e.g., a child, an adolescent or an adult)
suffering
from or at risk of having an IL-13-associated disorder or condition. Examples
of IL-13-
associated disorders or conditions include, but are not limited to, disorders
chosen from
one or more of: IgE-related disorders, including but not limited to, atopic
disorders, e.g.,
resulting from an increased sensitivity to IL- 13 or IL-4 (e.g., atopic
dermatitis, urticaria,
eczema, and allergic conditions such as allergic rhinitis and allergic
enterogastritis);
respiratory disorders, e.g., asthma (e.g., allergic and nonallergic asthma
(e.g., asthma due
to infection with, e.g., respiratory syncytial virus (RSV), e.g., in younger
children)),
-3-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
chronic obstructive pulmonary disease (COPD), and other conditions involving
airway
inflammation, eosinophilia, fibrosis and excess mucus production, e.g., cystic
fibrosis and
pulmonary fibrosis; inflanunatory and/or autoimmune disorders or conditions,
e.g., skin
inflammatory disorders or conditions (e.g., atopic dermatitis),
gastrointestinal disorders
or conditions (e.g., inflammatory bowel diseases (IBD), ulcerative colitis
and/or Crohn's
disease), liver disorders or conditions (e.g., cirrhosis, hepatocellular
carcinoma), and
scleroderma; tumors or cancers (e.g., soft tissue or solid tumors), such as
leukemia,
glioblastoma, and lymphoma, e.g., Hodgkin's lymphoma; viral infections (e.g.,
from
HTLV-1); fibrosis of other organs, e.g., fibrosis of the liver (e.g., fibrosis
caused by a
hepatitis B and/or C virus); and suppression of expression of protective type
1 immune
responses, (e.g., during vaccination).
For example, the subject can be a human allergic to a seasonal allergen, e.g.,
ragweed, or an asthmatic patient after exposure to a cold or flu virus or
during the cold or
flu season. Prior to the onset of the symptoms (e.g., allergic or asthmatic
symptoms, or
prior to or during an allergy, or cold or flu season), a single dose interval
of the anti-IL-
13 antagonist and/or IL-4 antagonist can be administered to the subject, such
that the
symptoms are reduced and/or the onset of the disorder or condition is delayed.
Similarly,
administration of the IL-13 and/or IL-4 antagonist can be effected prior to
the
manifestation of one or more symptoms (e.g., before a full manifestations of
the
symptoms) associated with the disorder or condition when treating chronic
conditions
that are characterized by recurring flares or episodes of the disorder or
condition. An
exemplary method for treating allergic rhinitis or other allergic disorders
can include
initiating therapy with an IL- 13 and/or IL-4 antagonist prior to exposure to
an allergen,
e.g., prior to seasonal exposure to an allergen, e.g., prior to allergen
blooms. Such
therapy can include a single treatment interval, e.g., a single dose, of the
IL- 13 and/or IL-
4 antagonist. In other embodiments, the single treatment interval of the IL-13
and/or IL-4
antagonist is administered in combination with allergy immunotherapy. For
example the
single treatment interval of the IL- 13 and/or IL-4 antagonist is administered
in
combination with an allergy immunization, e.g., a vaccine containing one or
more
allergens, such as ragweed, dust mite, and ryegrass. The single treatment
interval can be
repeated until a desirable level of immunity is obtained in the subject.
-4-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
In other embodiments, the IL-13 antagonist and/or the IL-4 antagonist is
administered in an amount effective to reduce or inhibit, or prevent or delay
the onset of,
one or more of the symptoms of the IL-13-associated disorder or condition. For
example
the IL-13 and/or IL-4 antagonist can be administered in an amount that
decreases one or
more of: (i) the levels of IL-13 in the subject; (ii) the levels of eotaxin in
the subject; (iii)
the levels of histamine released by basophils (e.g., blood basophils); (iv)
the IgE-titers in
the subject; and/or (v) one or more changes in the respiratory symptoms of the
subject
(e.g., difficulty breathing, wheezing, coughing, shortness of breath and/or
difficulty
performing normal daily activities).
In other embodiments, the IL-13 antagonist and/or the IL-4 antagonist inhibits
or
reduces one or more biological activities of IL-13 or IL-4, or an IL-13
receptor (e.g., an
IL-13 receptor al or an IL-13 receptor a2) or an IL-4 receptor (e.g., an IL-4
receptor
a or a receptor associated subunit thereof, e.g., y-chain). Exemplary
biological activities
that can be reduced using the IL-13 or IL-4 antagonists disclosed herein
include, but is
not limited to, one or more of: induction of CD23 expression; production of
IgE by
human B cells; phosphorylation of a transcription factor, e.g., STAT protein
(e.g., STAT6
protein); antigen-induced eosinophilia in vivo; antigen-induced
bronchoconstriction in
vivo; and/or drug-induced airway hyperreactivity in vivo. Antagonism using an
antagonist of IL-13/IL-13R or IL-4/IL-4R does not necessarily indicate a total
elimination of the biological activity of the IL-13/IL-13R polypeptide and/or
the IL-4/IL-
4R polypeptide.
For purposes of clarity, the term "IL-13 antagonist" or "IL-4 antagonist," as
used
herein, collectively refers to a compound such as a protein (e.g., a multi-
chain
polypeptide, a polypeptide), a peptide, small molecule, or inhibitory nucleic
acid that
reduces, inhibits or otherwise blocks one or more biological activities of IL-
13 and an IL-
13R, or IL-4 and an IL-4R, respectively. In one embodiment, the IL-13
antagonist
interacts with, e.g., binds to, an IL-13 or IL-13R polypeptide (also referred
to herein as an
"antagonistic IL- 13 binding agent." For example, the IL- 13 antagonist can
interact with,
e.g., can bind to, IL-13 or IL-13 receptor, preferably, mammalian, e.g., human
IL-13 or
IL-13R (also individually referred to herein as an "IL-13 antagonist" and "IL-
13R
antagonist," respectively), and reduce or inhibit one or more IL-13- and/or IL-
13R-
-5-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
associated biological activities. In another embodiment, the IL-4 antagonist
interacts
with, e.g., binds to, an IL-4 or an IL-4R polypeptide (e.g., mammalian, e.g.,
human IL-4
or IL-4R (also individually referred to herein as an "IL-4 antagonist" and "IL-
4R
antagonist," respectively)), and reduce or inhibit one or more IL-4 and/or IL-
4R
activities. Antagonists bind to IL-13 or IL-4, or IL-13R or IL-4R with high
affinity, e.g.,
with an affinity constant of at least about 10' M-t, preferably about 108 M-l,
and more
preferably, about 109 M-'to 1010 M-1 or stronger. It is noted that the term
"IL-13
antagonist" or "IL-4 antagonist" includes agents that inhibit or reduce one or
more of the
biological activities disclosed herein, but may not bind to IL- 13 or IL-4
directly.
The terms "anti-IL13 binding agent" and "IL-13 binding agent" are used
interchangeably herein. These terms as used herein refers to any compound,
such as a
protein (e.g., a multi-chain polypeptide, a polypeptide) or a peptide, that
includes an
interface that binds to an IL- 13 protein, e.g., a mammalian IL- 13,
particularly, a human
IL-13. The binding agent generally binds with a Kd of less than 5 x 10-7 M. An
exemplary IL-13 binding agent is a protein that includes an antigen binding
site, e.g., an
antibody molecule. The anti-IL13 binding agent or IL-13 binding agent can be
an IL-13
antagonist that binds to IL13, or can also include IL-13 binding agents that
simply bind to
IL-13, but do not elicit an activity, or may in fact agonize an IL-13
activity. For example,
certain IL-13 binding agents, e.g., anti-IL-13 antibody molecules, that bind
to and inhibit
one or more IL-13 biological activities, e.g., antibodies 13.2, MJ2-7 and C65,
are also
referred to herein as antagonistic IL- 13 binding agents. Examples of IL- 13
antagonists
that are not IL-13 binding agents as defined herein include, e.g., inhibitors
of upstream or
downstream IL-13 signalling (e.g., STAT6 inhibitors).
Additional embodiments may include one or more of the following features:
In some embodiments, the IL- 13 antagonist or the IL4 antagonist can be an
antibody molecule that binds to IL-13 or an IL-13R, or IL-4 or an IL-4R. The
IL-13 or
the IL-4 antagonist can also be a soluble fonn of the IL-13R (e.g., soluble IL-
13R(X2 or
IL-13Ra1) or the IL-4R (e.g., IL-4Ra), alone or fused to another moiety (e.g.,
an
immunoglobulin Fc region) or as a heterodimer of subunits (e.g., a soluble IL-
13R-IL-4R
heterodimer or a soluble IL-4R-y common heterodimer). In other embodiments,
the
antagonist is a cytokine mutein (e.g., an IL-13 or IL-4 mutein that binds to
the
-6-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
corresponding receptor, but does not substantially activate the receptor), or
a cytokine
conjugated to a toxin. In other embodiments, the IL-13 or the IL-4 antagonist
is a small
molecule inhibitor, e.g., a small molecule inhibitor of STAT6, or a peptide
inhibitor. In
yet other embodiments, the IL-13 or IL-4 antagonist is an inhibitor of nucleic
acid
expression. For example, the antagonist is an antisense RNA or siRNA that
blocks or
reduces expression of an IL-13 or IL-13R, or IL-4 or IL-4R gene.
In one embodiment, the IL-13 antagonist or binding agent (e.g., the antibody
molecule, soluble receptor, cytokine mutein, or peptide inhibitor) binds to IL-
13 or an
IL13R and inhibits or reduces an interaction (e.g., binding) between IL-13 and
an IL-13
receptor, e.g., IL-13Ra1, IL-13Ra2, and/or IL-4Ra, thereby reducing or
inhibiting signal
transduction. For example, the IL-13 antagonist can bind to one or more
components of a
complex chosen from, e.g., IL-13 and IL-13Ral ("IL-13/IL-13aRl"); IL-13 and IL-
4Ra
("IL-13/IL-4Ra"); IL-13, IL-13Ra 1, and IL-4Ra ("IL-13/IL-13Ra 1/IL-4R(x");
and IL-13
and IL-13Ra2 ("IL-13/IL13Ra2"). In embodiments, the IL-13 antagonist binds to
IL-13
or an IL-13R and interferes with (e.g., inhibits, blocks or otherwise reduces)
an
interaction, e.g., binding, between IL- 13 and an IL- 13 receptor complex,
e.g., a complex
comprising IL-13Ra1 and IL-4Ra. In other embodiments, the IL-13 antagonist
binds to
IL- 13 and interferes with (e.g., inhibits, blocks or otherwise reduces) an
interaction, e.g.,
binding, between IL-13 and a subunit of the IL-13 receptor complex, e.g., IL-
13Ra1 or
IL-4Ra, individually. In yet another embodiment, the IL-13 antagonist, e.g.,
the anti-IL-
13 antibody or fragment thereof, binds to IL- 13, and interferes with (e.g.,
inhibits, blocks
or otherwise reduces) an interaction, e.g., binding, between IL-13/IL-13Ra1
and IL-4Ra.
In another embodiment, the IL- 13 antagonist, binds to IL- 13 and interferes
with (e.g.,
inhibits, blocks or otherwise reduces) an interaction, e.g., binding, between
IL-13/IL-4Ra
and IL-13Ra1. Typically, the IL- 13 antagonist interferes with (e.g.,
inhibits, blocks or
otherwise reduces) an interaction, e.g., binding, of IL-13/IL-13Ra1 with IL-
4Ra.
Exemplary antibodies inhibit or prevent formation of the ternary complex, IL-
13/IL-
l 3Ra 1 /IL-4Ra.
In another embodiment, the IL-4 antagonist (e.g., the antibody molecule,
soluble
receptor, cytokine mutein, or peptide inhibitor) binds to IL-4 or an IL4R, and
inhibits or
-7-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
reduces an interaction (e.g., binding) between IL-4 and an IL-4 receptor,
e.g., IL-4R(X
and/or y common), thereby reducing or inhibiting signal transduction. For
example, the
IL-4 antagonist can bind to one or more components of a complex chosen from,
e.g., IL-4
and IL-4Ra ("IL-4/IL-4Ra"), IL-4 and y common ("IL-4/ycommon"), or IL-4, IL-
4Ra,
and y common ("IL-4/IL-4R(x/ y common"). In exemplary embodiments, the IL-4
antagonist binds to IL-4 and interferes with (e.g., inhibits, blocks or
otherwise reduces)
an interaction, e.g., binding, between IL-4 and a subunit of the IL-4 receptor
complex,
e.g., IL-4Ra or y common, individually. In yet another embodiment, the IL-4
antagonist,
binds to IL-4, and interferes with (e.g., inhibits, blocks or otherwise
reduces) an
interaction, e.g., binding, between IL-4/IL-4Ra and y common.
In one embodiment, the IL-13/IL-13R or IL-4/IL-4R antagonist or binding agent
is an antibody molecule (e.g., an antibody, or an antigen-binding fragment
thereof) that
binds to IL-13/IL-13R or IL-4/IL-4R. For example, the antibody molecule can be
a full
length monoclonal or single specificity antibody that binds to IL-13 or IL-4,
or an IL-13
receptor or an IL-4 receptor (e.g., an antibody molecule that includes at
least one, and
typically two, complete heavy chains, and at least one, and typically two,
complete light
chains); or an antigen-binding fragment thereof (e.g., a heavy or light chain
variable
domain monomer or dimer (e.g., VH, VHH), an Fab, F(ab')2, Fv, or a single
chain Fv
fragment). Typically, the antibody molecule is a human, camelid, shark,
humanized,
chimeric, or in vitro-generated antibody to human IL- 13 or IL-4, or a human
IL- 13
receptor or IL-4 receptor. In certain embodiments, the antibody molecule
includes a
heavy chain constant region chosen from, e.g., the heavy chain constant
regions of IgGI,
IgG2, IgG3, IgG4, IgM, IgAl, IgA2, IgD, and IgE; particularly, chosen from,
e.g., the
heavy chain constant regions of IgGl, IgG2, IgG3, and IgG4, more particularly,
the
heavy chain constant regions IgGI (e.g., human IgGI or a modified form
thereof). In
another embodiment, the antibody molecule has a light chain constant region
chosen
from, e.g., the light chain constant regions of kappa or lambda, preferably
kappa (e.g.,
human kappa). In one embodiment, the constant region is altered, e.g.,
mutated, to
modify the properties of the antibody molecule (e.g., to increase or decrease
one or more
of: Fc receptor binding, antibody glycosylation, the number of cysteine
residues, effector
cell function, or complement function). For example, the human IgGI constant
region
-8-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
can be mutated at one or more residues, e.g., one or more of residues 234 and
237, as
described in Example 5, to decrease one or more of: Fc receptor binding,
antibody
glycosylation, the number of cysteine residues, effector cell function, or
complement
function. In embodiments, the antibody molecule includes a human IgGI constant
region
mutated at one or more residues of SEQ ID NO:193, e.g., mutated at positions
116 and
119 of SEQ ID NO:193.
In one embodiment, the antibody molecule is a inhibitory or neutralizing
antibody
molecule. For example, the anti-IL13 antibody molecule can have a functional
activity
comparable to IL-13Ra2 (e.g., the anti-IL13 antibody molecule reduces or
inhibits IL-13
interaction with IL-13Ra1). The anti-IL13 antibody molecule may prevent
formation of
a complex between IL- 13 and IL-13Ra1, or disrupt or destabilize a complex
between
IL-13 and IL-13Ra 1. In one embodiment, the anti-IL 13 antibody molecule
inhibits
ternary complex formation, e.g., formation of a complex between IL 13, IL-
13Ra1 and
IL4-R. In one embodiment, the antibody molecule confers a post-injection
protective
effect against exposure to an antigen, e.g., an Ascaris antigen in a sheep
model, at least 6
weeks after injection. In other embodiments, the anti-IL13 antibody molecule
can inhibit
one or more IL-13-associated biological activities with an IC50 of about 50 nM
to 5 pM,
typically about 100 to 250 pM or less, e.g., better inhibition. In one
embodiment, the
anti-IL 13 antibody molecule can associate with IL-13 with kinetics in the
range of 103 to
10g M-ls"1, typically 104 to 107 M"is-I. In one embodiment, the anti-IL13
antibody
molecule binds to human IL-13 with a koõ of between 5 x 104 and 8 x 105 M'1
s"1. In yet
another embodiment, the anti-IL 13 antibody molecule has dissociation kinetics
in the
range of 10-2 to 10-6 s"1, typically 10"2 to 10'5 s'. In one embodiment, the
anti-IL13
antibody molecule binds to IL-13, e.g., human IL-13, with an affinity and/or
kinetics
similar (e.g., within a factor 20, 10, or 5) to monoclonal antibody 13.2, MJ 2-
7 or C65, or
modified forms thereof, e.g., chimeric forms or humanized forms thereof. The
affinity
and binding kinetics of an IL- 13 binding agent can be tested using, e.g.,
biosensor
technology (BIACORETM).
In still another embodiment, the anti-IL13 antibody molecule specifically
binds to
an epitope, e.g., a linear or a conformational epitope, of IL-13, e.g.,
mammalian, e.g.,
human IL-13. For example, the antibody molecule binds to at least one amino
acid in an
-9-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
epitope defined by IL-13Ra1 binding to human IL-13 or an epitope defined by IL-
13Ra2
binding to human IL-13, or an epitope that overlaps with such epitopes. The
anti-IL13
antibody molecule may compete with IL-13Ra1 and/or IL-13Ra2 for binding to IL-
13,
e.g., to human IL-13. The anti-IL13 antibody molecule may competitively
inhibit
binding of IL-13Ra1 and/or IL-13Ra2 to IL-13. The anti-IL13 antibody molecule
may
interact with an epitope on IL-13 which, when bound, sterically prevents
interaction with
IL-13Ra1 and/or IL-13Ra2. In embodiments, the anti-IL13 antibody molecule
binds
specifically to human IL- 13 and competitively inhibits the binding of a
second antibody
to said human IL-13, wherein said second antibody is chosen from 13.2, MJ 2-7
and/or
C65 (or any other anti-IL13 antibody disclosed herein) for binding to IL-13,
e.g., to
human IL-13. The anti-IL13 antibody molecule may competitively inhibit binding
of
13.2, MJ 2-7 and/or C65 to IL-13. The anti-IL13 antibody molecule may
specifically
bind at least one amino acid in an epitope defined by 13.2, MJ 2-7 binding to
human
IL-13 or an epitope defined by C65 binding to human IL-13. In one embodiment,
the
anti-IL13 antibody molecule may bind to an epitope that overlaps with that of
13.2,
MJ 2-7 or C65, e.g., includes at least one, two, three, or four amino acids in
common, or
an epitope that, when bound, sterically prevents interaction with 13.2, MJ 2-7
or C65.
For example, the antibody molecule may contact one or more residues from IL-13
chosen
from one or more of residues 81-93 and/or 114-132 of human IL-13 (SEQ ID NO:
194),
or chosen from one or more of: Glutamate at position 68 [49], Asparagine at
position 72
[53], Glycine at position 88 [69], Proline at position 91 [72], Histidine at
position 92 [73],
Lysine at position 93 [74], and/or Arginine at position 105 [86] of SEQ ID
NO:194
[position in mature sequence; SEQ ID NO:195]. In other embodiments, the
antibody
molecule contacts one or more amino acid residues from IL-13 chosen from one
or more
of residues 116, 117, 118, 122, 123, 124, 125, 126, 127, and/or 128 of SEQ ID
NO:24 or
SEQ ID NO: 178. In one embodiment, the antibody molecule binds to IL- 13
irrespective
of the polymorphism present at position 130 in SEQ ID NO:24.
In one embodiment, the antibody molecule includes one, two, three, four, five
or
all six CDR's from mAb 13.2, MJ2-7, C65, or other antibodies disclosed herein,
or closely
related CDRs, e.g., CDRs which are identical or which have at least one amino
acid
alteration, but not more than two, three or four alterations (e.g.,
substitutions (e.g.,
-10-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
conservative substitutions), deletions, or insertions). Optionally, the
antibody molecule
may include any CDR described herein. In embodiments, the heavy chain
immunoglobulin variable domain comprises a heavy chain CDR3 that differs by
fewer
than 3 amino acid substitutions from a heavy chain CDR3 of monoclonal antibody
MJ2-7
(SEQ ID NO:17), mAb 13.2 (SEQ ID NO:196) or C65 (SEQ ID NO:123). In other
embodiments, the light chain immunoglobulin variable domain comprises a light
chain
CDR1 that differs by fewer than 3 amino acid substitutions from a
corresponding light
chain CDR of monoclonal antibody MJ2-7 (SEQ ID NO: 18), mAb 13.2 (SEQ ID
NO:197) or C65 (SEQ ID NO:118). The amino acid sequence of the heavy chan
variable
domain of MJ2-7 has the amino acid sequence shown as SEQ ID NO:130. The amino
acid sequence of the light chan variable domain of MJ2-7 has the amino acid
sequence
shown as SEQ ID NO:133. The amino acid sequence of the heavy chan variable
domain
of monoclonal antibody 13.2 has the amino acid sequence shown as SEQ ID
NO:198.
The amino acid sequence of the light chan variable domain of monoclonal
antibody 13.2
has the amino acid sequence shown as SEQ ID NO:199.
In certain embodiments, the heavy chain variable domain of the antibody
molecule includes one or more of:
G-(YF)-(NT)-I-K-D-T-Y-(MI)-H (SEQ ID NO:48), in CDR1,
(WR)-I-D-P-(GA)-N-D-N-I-K-Y-(SD)-(PQ-K-F-Q-G (SEQ ID NO:49), in
CDR2, and/or
SEENWYDFFDY (SEQ ID NO:17), in CDR3; or
GFNIKDTYIH (SEQ ID NO:15), in CDR1,
RIDPANDNIKYDPKFQG (SEQ ID NO:16), in CDR2, and/or
SEENWYDFFDY (SEQ ID NO:17) , in CDR3
In other embodiments, the light chain variable domain of the antibody molecule
includes one or more of:
(RK)-S-S-Q-S-(LI)-(KV)-H-S-(ND)-G-N-(TN)-Y-L-(EDNQYAS) (SEQ ID
NO:25), in CDR1,
K-(LVI)-S-(NY)-(RW)-(FD)-S (SEQ ID NO:27), in CDR2, and/or
Q-(GSA)-(ST)-(HEQ)-I-P (SEQ ID NO:28), in CDR3; or
-11-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
RSSQSIVHSNGNTYLE (SEQ ID NO:18), in CDR1
KVSNRFS (SEQ ID NO:19), in CDR2, and
FQGSHIPYT (SEQ ID NO:20), in CDR3.
In other embodiments, the antibody molecule includes one or more CDRs
including an amino acid sequence selected from the group consisting of the
amino acid
sequence of SEQ ID NO:197, SEQ ID NO:200, SEQ ID NO:201, SEQ ID NO:202, SEQ
ID NO:203, and SEQ ID NO:196.
In yet another embodiment, the antibody molecule includes at least one, two,
or
three Chothia hypervariable loops from a heavy chain variable region of an
antibody
chosen from, e.g., mAb 13.2, MJ2-7, C65, or any other antibody disclosed
herein, or at
least particularly the amino acids from those hypervariable loops that contact
IL-13. In
yet another embodiment, the antibody or fragment thereof includes at least
one, two, or
three hypervariable loops from a light chain variable region of an antibody
chosen from,
e.g., mAb 13.2, MJ2-7, C65, or other antibodies disclosed herein, or at least
includes the
amino acids from those hypervariable loops that contact IL-13. In yet another
embodiment, the antibody or fragment thereof includes at least one, two,
three, four, five,
or six hypervariable loops from the heavy and light chain variable regions of
an antibody
chosen from, e.g., mAb13.2, MJ2-7, C65, or other antibodies disclosed herein.
In one embodiment, the protein includes all six hypervariable loops from
mAb13.2, MJ2-7, C65, or other antibodies disclosed herein or closely related
hypervariable loops, e.g., hypervariable loops which are identical or which
have at least
one amino acid alteration, but not more than two, three or four alterations,
from the
sequences disclosed herein. Optionally, the protein may include any
hypervariable loop
described herein.
In still another example, the protein includes at least one, two, or three
hypervariable loops that have the same canonical structures as the
corresponding
hypervariable loop of mAb 13.2, MJ2-7, C65, or other antibodies disclosed
herein, e.g.,
the same canonical structures as at least loop 1 and/or loop 2 of the heavy
and/or light
chain variable domains of mAb13.2, MJ2-7, C65, or other antibodies disclosed
herein.
See, e.g., Chothia et al. (1992) J. Mol. Biol. 227:799-817; Tomlinson et al.
(1992) J. Mol.
-12-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
Biol. 227:776-798 for descriptions of hypervariable loop canonical structures.
These
structures can be determined by inspection of the tables described in these
references.
In one embodiment, the heavy chain framework of the antibody molecule (e.g.,
FR1, FR2, FR3, individually, or a sequence encompassing FR1, FR2, and FR3, but
excluding CDRs) includes an amino acid sequence, which is at least 80%, 85%,
90%,
95%, 97%, 98%, 99% or higher identical to the heavy chain framework of one of
the
following germline V segment sequences: DP-25, DP-1, DP-12, DP-9, DP-7, DP-31,
DP-
32, DP-33, DP-58, or DP-54, or another V gene which is compatible with the
canonical
structure class 1-3 (see, e.g., Chothia et al. (1992) J. Mol. Biol. 227:799-
817; Tomlinson
et al. (1992) J. Mol. Biol. 227:776-798). Other frameworks compatible with the
canonical structure class 1-3 include frameworks with the one or more of the
following
residues according to Kabat numbering: Ala, Gly, Thr, or Val at position 26;
Gly at
position 26; Tyr, Phe, or Gly at position 27; Phe, Val, Ile, or Leu at
position 29; Met, Ile,
Leu, Val, Thr, Trp, or Ile at position 34; Arg, Thr, Ala, Lys at position 94;
Gly, Ser, Asn,
or Asp at position 54; and Arg at position 71.
In one embodiment, the light chain framework of the antibody molecule (e.g.,
FRI, FR2, FR3, individually, or a sequence encompassing FRl, FR2, and FR3, but
excluding CDRs) includes an amino acid sequence, which is at least 80%, 85%,
90%,
95%, 97%, 98%, 99% or higher identical to the light chain framework of a Vx II
subgroup germline sequence or one of the following germline V segment
sequences:
A17, A1, A18, A2, A19/A3, or A23 or another V gene which is compatible with
the
canonical structure class 4-1 (see, e.g., Tomlinson et al. (1995) EMBOJ.
14:4628).
Other frameworks compatible with the canonical structure class 4-1 include
frameworks
with the one or more of the following residues according to Kabat numbering:
Val or Leu
or Ile at position 2; Ser or Pro at position 25; Ile or Leu at position 29;
Gly at position
31d; Phe or Leu at position 33; and Phe at position 71.
In another embodiment, the light chain framework of the antibody molecule
(e.g.,
FR1, FR2, FR3, individually, or a sequence encompassing FRl, FR2, and FR3, but
excluding CDRs) includes an amino acid sequence, which is at least 80%, 85%,
90%,
95%, 97%, 98%, 99% or higher identical to the light chain framework of a VK I
subgroup
germline sequence, e.g., a DPK9 sequence.
-13-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
In another embodiment, the heavy chain framework of the antibody molecule
(e.g., FR1, FR2, FR3, individually, or a sequence encompassing FR1, FR2, and
FR3, but
excluding CDRs) includes an amino acid sequence, which is at least 80%, 85%,
90%,
95%, 97%, 98%, 99% or higher identical to the light chain framework of a VH I
subgroup germline sequence, e.g., a DP-25 sequence or a VH III subgroup
germline
sequence, e.g., a DP-54 sequence.
In certain embodiments, the heavy chain immunoglobulin variable domain of the
antibody molecule includes an amino acid sequence encoded by a nucleotide
sequence
that hybridizes under high stringency conditions to the complement of the
nucleotide
sequence encoding a heavy chain variable domain of V2.1 (SEQ ID NO:71), V2.3
(SEQ
ID NO:73), V2.4 (SEQ ID NO:74), V2.5 (SEQ ID NO:75), V2.6 (SEQ ID NO:76), V2.7
(SEQ ID NO:77), V2.11 (SEQ ID NO:80), ch13.2 (SEQ ID NO:204), h13.2v1 (SEQ ID
NO:205), h13.2v2 (SEQ ID NO:206)or h13.2v3 (SEQ ID NO:207); or includes an
amino
acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or higher
identical
identical to the amino acid sequence of the heavy chain variable domain of
V2.1 (SEQ ID
NO:71), V2.3 (SEQ ID NO:73), V2.4 (SEQ ID NO:74), V2.5 (SEQ ID NO:75), V2.6
(SEQ ID NO:76), V2.7 (SEQ ID NO:77), V2.11 (SEQ ID NO:80); ch13.2 (SEQ ID
NO:208), h13.2v1 (SEQ ID NO:209), h13.2v2 (SEQ ID NO:210) or h13.2v3 (SEQ ID
NO:21 1). In embodiments, the heavy chain immunoglobulin variable domain
includes
the amino acid sequence of SEQ ID NO:80, which may in turn further include a
heavy
chain variable domain framework region 4 (FR4) that includes the amino acid
sequence
ofSEQID NO: 116 or SEQ ID NO:117.
In other embodiments, the light chain immunoglobulin variable domain of the
antibody molecule includes an amino acid sequence encoded by a nucleotide
sequence
that hybridizes under high stringency conditions to the complement of the
nucleotide
sequence encoding a light chain variable domain of V2.11 (SEQ ID NO:36) or
h13.2v2
(SEQ ID NO:212); or includes an amino acid sequence that is at least 80%, 85%,
90%,
95%, 97%, 98%, 99% or higher identical identical to a light chain variable
domain of
V2.11 (SEQ ID NO:36) or h13.2v2 (SEQ ID NO:212). In embodiments, the light
chain
immunoglobulin variable domain includes the amino acid sequence of SEQ ID
NO:36,
-14-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
which may in turn further include a light chain variable domain framework
region 4
(FR4) that includes the amino acid sequence of SEQ ID NO:47.
In yet another embodiment, the antibody molecule includes a framework of the
heavy chain variable domain sequence comprising:
(i) at a position corresponding to 49, Gly;
(ii) at a position corresponding to 72, Ala;
(iii) at positions corresponding to 48, Ile, and to 49, Gly;
(iv) at positions corresponding to 48, Ile, to 49, Gly, and to 72, Ala;
(v) at positions corresponding to 67, Lys, to 68, Ala, and to 72, Ala; and/or
(vi) at positions corresponding to 48, Ile, to 49, Gly, to 72, Ala, to 79,
Ala.
In one embodiment, the anti-IL13 antibody molecule includes at least one light
chain that comprises the amino acid sequence of SEQ ID NO: 177 (or an amino
acid
sequence at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or higher identical
identical to
SEQ ID NO: 177) and at least one heavy chain that comprises the amino acid
sequence of
SEQ ID NO:176 (or an amino acid sequence at least 80%, 85%, 90%, 95%, 97%,
98%,
99% or higher identical identical to SEQ ID NO: 176).
In one embodiment, the anti-IL13 antibody molecule includes two
immunoglobulin chains: a light chain that includes SEQ ID NO:199, 213, 214,
212, or
215 and a heavy chain that includes SEQ ID NO:198, 208, 209, 210, or 211 (or
an amino
acid sequence at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or higher identical
identical
to SEQ ID NO:199, 213, 214, 212, or 215, or SEQ ID NO:198, 208, 209, 210, or
211).
The antibody molecule may further include in the heavy chain the amino acid
sequence
of SEQ ID NO: 193 and in the light chain the amino acid sequence of SEQ ID
NO:216 (or
an amino acid sequence at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or higher
identical identical to SEQ ID NO:193 or SEQ ID NO:216).
Additional examples of anti-IL13 antibody molecules are disclosed in US
07/0128192 or WO 05/007699 and in Blanchard, C. et al. (2005) Clinical and
Experimental Allergy 35(8):1096-1103 disclosing CAT-354; WO 05/062967, WO
05/062972 and Clinical Trials Gov. Identifier: NCT00441818 disclosing TNX-650;
Clinical Trials Gov. Identifier: NCT532233 disclosing QAX-576; US 06/0140948
or WO
06/055638, filed in the name of Abgenix; US 6,468,528 assigned to AMGEN; WO
-15-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
05/091856 naming Centocor, Inc. as the applicant; and in Yang et al. (2004)
Cytokine
28(6):224-32 and Yang et al. (2005) JPharmacol Exp Ther: 313(1):8-15; and anti-
IL13
antibodies as disclosed in WO 07/080174 filed in the name of Glaxo, and as
disclosed in
WO 07/045477 in the name of Novartis.
Additional examples of IL-13 or IL-4 antagonists include, but are not limited
to,
antibody molecules against IL-4 (e.g., pascolizumab and related antibodies
disclosed in
Hart, T.K. et al. (2002) Clin Exp Immunol. 130(1):93-100; Steinke, J.W. (2004)
Immunol. Allergy Clin North Am 24(4):599-614; and in Ramanthan et al. U.S.
6,358,509), IL-4Ra (e.g., AMG-317 and related anti-IL4R antibodies disclosed
in US
05/0118176, US 05/0 1 1 2694 and in Clinical Trials Gov. Identifier:
NCT00436670); IL-
13Ra1 (e.g., anti-13Ra1 antibodies disclosed in WO 03/080675 which names AMRAD
as the applicant); and mono- or bi-specific antibody molecules that bind to
IL4 and/or IL-
13 (disclosed, e.g., in WO 07/085815).
In other embodiments, the IL-13 or IL-4 antagonist is an IL-13 or IL-4 mutein
(e.g., a truncated or variant form of the cytokine that binds to the an IL-13R
or an IL-4
receptor, but does not significantly increase the activity of the receptor),
or a cytokine-
conjugated to a toxin. IL-4 muteins are disclosed by Weinzel et al. (2007)
Lancet
370:1422-31. Additional examples of IL-13/IL-4 inhibiting peptides are
disclosed in
Andrews, A.L. et al. (2006) J. Allergy and Clin Immunol 118:858-865. An
example of a
cytokine-toxin conjugate is disclosed in WO 03/047632, in Kunwar, S. et al.
(2007) J.
Clin Onco125(7):837-44 and in Husain, S. R. et al. (2003) J.
Neuroonco165(1):37-48.
In yet other embodiments, the IL13 antagonist or the IL-4 antagonist is a full
length, or a fragment or modified form of an IL-13 receptor polypeptide (e.g.,
IL-13Ra2
or IL13Ra1) or an IL-4 receptor polypeptide (e.g., IL-4Ra). For example, the
antagonist
can be a soluble form of an IL-13 receptor or an IL-14 receptor (e.g., a
soluble form of
mammalian (e.g., human) IL-13Ra2, IL13Ra1 or IL-4Ra comprising a cytokine-
binding
domain; e.g., a soluble form of an extracellular domain of mammalian (e.g.,
human) IL-
13Ra2, IL13Ra1 or IL-4R(x). Exemplary receptor antagonists include, e.g., IL-
4R-IL-
13R binding fusions as described in WO 05/085284 and Economides, A.N. et al.
(2003)
Nat Med 9(1):47-52, as well as in Borish, L.C. et al. (1999) Am JRespir Crit
Care Med
160(6):1816-23.
-16-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
A soluble form of an IL-13 receptor or IL-4 receptor, or an IL-13 or IL-4
mutein
can be used alone or functionally linked (e.g., by chemical coupling, genetic
or
polypeptide fusion, non-covalent association or otherwise) to a second moiety
to facilitate
expression, steric flexibility, detection and/or isolation or purification,
e.g., an
immunoglobulin Fc domain, serum albumin, pegylation, a GST, Lex-A or an MBP
polypeptide sequence. The fusion proteins may additionally include a linker
sequence
joining the first moiety to the second moiety. For example, a soluble IL-13
receptor or
IL-4 receptor, or an IL- 13 or IL-4 mutein can be fused to a heavy chain
constant region of
the various isotypes, including: IgGI, IgG2, IgG3, IgG4, IgM, IgAl, IgA2, IgD,
and
IgE). Typically, the fusion protein can include the extracellular domain of a
human
soluble IL- 13 receptor or IL-4 receptor, or an IL- 13 or IL-4 mutein (or a
sequence
homologous thereto), and, e.g., fused to, a human immunoglobulin Fc chain,
e.g., human
IgG (e.g., human IgGl or human IgG2, or a mutated form thereof). The Fc
sequence can
be mutated at one or more amino acids to reduce effector cell function, Fc
receptor
binding and/or complement activity.
It will be understood that the antibody molecules and soluble or fusion
proteins
described herein can be functionally linked (e.g., by chemical coupling,
genetic fusion,
non-covalent association or otherwise) to one or more other molecular
entities, such as an
antibody (e.g., a bispecific or a multispecific antibody), toxins,
radioisotopes, cytotoxic
or cytostatic agents.
In another embodiment, the IL- 13 or IL-4 antagonist inhibits the expression
of
nucleic acid encoding an IL- 13 or IL-13R, or an IL-4or IL-4R. Examples of
such
antagonists include nucleic acid molecules, for example, antisense molecules,
ribozymes,
RNAi, siRNA, triple helix molecules that hybridize to a nucleic acid encoding
an IL- 13
or IL-13R, or an IL-4 or IL-4R, or a transcription regulatory region, and
blocks or
reduces mRNA expression of IL-13 or IL-13R, or an IL-4or IL-4R. ISIS-369645
provides an example of an antisense nucleic acid that inhibits expression of
of IL-
4Ra (developed by ISIS Pharmaceuticals and disclosed in, e.g., Karras, J.G. et
al. (2007)
Am JRespir Cell Mol Biol. 36(3):276-86). Exemplary short interference RNAs
(siRNAs)
that interfere with RNA encoding IL-4 or IL-13 are disclosed in WO 07/131274.
-17-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
In yet another embodiment, the IL-13 or IL-4 antagonist is an inhibitor, e.g.,
a
small molecule inhibitor, of upstream or downstream IL-13 signalling (e.g.,
STAT6
inhibitors). Examples of STAT6 inhibitors are disclosed in WO 04/002964, in
Canadian
Patent Application: CA 2490888 and in Nagashima, S. et al. (2007) Bioorg Med
Chem
15(2):1044-55; and in US 6,207,391 and WO 01/083517.
In another embodiment, one or more IL-13 antagonists are administered in
combination with one or more IL-4 antagonists. The combination therapy can
include the
IL- 13 antagonist formulated with and/or administered with the IL-4
antagonist. The
IL- 13 antagonist and the IL-4 antagonist can be administered simultaneously,
or
sequentially. If administered sequentially, a physician can select an
appropriate sequence
for administering the IL- 13 antagonist in combination with the IL-4
antagonist. The
combination therapy can also include other therapeutic agents chosen from one
or more
of inhaled steroids; beta-agonists, e.g., short-acting or long-acting beta-
agonists;
antagonists of leukotrienes or leukotriene receptors; combination drugs such
as
ADVAIR ; IgE inhibitors, e.g., anti-IgE antibodies (e.g., XOLAIR );
phosphodiesterase
inhibitors (e.g., PDE4 inhibitors); xanthines; anticholinergic drugs; mast
cell-stabilizing
agents such as cromolyn; IL-5 inhibitors; eotaxin/CCR3 inhibitors; and
antihistamines.
Such combinations can be used to treat asthma and other respiratory disorders.
Additional examples of therapeutic agents that can be coadministered and/or
coformulated with an IL-13 binding agent include one or more of: TNF
antagonists (e.g.,
a soluble fragment of a TNF receptor, e.g., p55 or p75 human TNF receptor or
derivatives thereof, e.g., 75 kd TNFR-IgG (75 kD TNF receptor-IgG fusion
protein,
ENBREL"")); TNF enzyme antagonists, e.g., TNFa converting enzyme (TACE)
inhibitors; muscarinic receptor antagonists; TGF-(3 antagonists; interferon
gamma;
perfenidone; chemotherapeutic agents, e.g., methotrexate, leflunomide, or a
sirolimus
(rapamycin) or an analog thereof, e.g., CCI-779; COX2 and cPLA2 inhibitors;
NSAIDs;
immunomodulators; p38 inhibitors, TPL-2, Mk-2 and NFKB inhibitors, among
others.
In another aspect, the application provides a method of evaluating the
efficacy of an IL-
13 antagonistic binding agent, e.g., an anti-IL13 antibody molecule as
described herein,
in treating (e.g., reducing) pulmonary inflammation in a subject, e.g., a
human or non-
human subject.
-18-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
In yet another embodiment, the methods disclosed herein further include the
step(s) of:
evaluating (e.g., detecting) a change in one or more of the following
parameters in
a subject after administration of the IL-13 antagonist and/or IL-4
antagonists: (i)
detecting the levels of IL-13 unbound and/or bound to an IL13 binding agent in
a sample,
e.g., a biological sample (e.g., serum, plasma, blood) as described in the in
vitro detection
methods herein; (ii) measuring eotaxin levels in a sample, e.g., a biological
sample (e.g.,
serum, plasma, blood); (iii) detecting histamine release by basophils; (iv)
detecting IgE-
titers; and/or (v) evaluating changes in the symptoms of the subject (e.g.,
difficulty
breathing, wheezing, coughing, shortness of breath and/or difficulty
performing normal
daily activities). In embodiments, the detection of parameters (i)-(v) can be
carried out
before and/or after administration of the IL-13 antagonistic binding agent
(after single or
multiple administrations) to the subject (e.g., at selected intervals after
initiating therapy).
The detection and/or evaluation of the changes in one or more of (i)-(v) can
be performed
by a clinician or support staff. A change, e.g., a reduction, in one or more
of (i)-(v)
relative to a predetermined level (e.g., comparing before and after treatment)
indicates
that the IL- 13 antagonistic binding agent is effectively reducing lung
inflammation in the
subjects. In embodiments, the subject is a human patient, e.g., an adult or a
child.
In another aspect, the invention provides compositions, e.g., pharmaceutical
compositions, or dose formulations that include a pharmaceutically acceptable
carrier and
at least one IL-13 antagonistic binding agent, e.g., an anti-IL-13 antibody
molecule,
formulated with an IL-4 antagonist. Combinations of the aforesaid antagonists
and
another drug, e.g., a therapeutic agent (e.g., one or more cytokine and growth
factor
inhibitors, immunosuppressants, anti-inflammatory agents (e.g., systemic anti-
inflammatory agents), metabolic inhibitors, enzyme inhibitors, and/or
cytotoxic or
cytostatic agents, as described herein, can also be used.
In yet another aspect, the invention features a kit that includes an IL- 13
antagonist
and/or an IL-4 antagonist for use in the methods disclosed herein with
instructions for
administering the antagonist as a single treatment interval to treat or
prevent an IL- 13
associated disorder or condition (e.g., a disorder or condition as described
herein).
-19-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
In another aspect, the invention features a composition that includes an IL-
13
antagonist and/or an IL-4 antagonist for use in the methods disclosed herein.
In yet another aspect, the invention features the use of a composition that
includes
an IL-13 antagonist and/or an IL-4 antagonist in the manufacture of a
medicament to treat
or prevent an IL- 13 -associated disorder or condition (e.g., a disorder or
condition as
described herein).
In another aspect, this application provides a method for detecting the
presence of
IL-13 in a sample in vitro (e.g., a biological sample, such as serum, plasma,
tissue,
biopsy). The subject method can be used to diagnose a disorder, e.g., an IL-13-
associated
disorder, or to monitor the efficacy of a treatment. The method includes: (i)
contacting
the sample with an IL-13 binding agent, e.g., a first IL-13 binding agent or
anti-IL13
antibody molecule as described herein; and (ii) detecting the formation of a
complex
between the first IL-13 binding agent and IL-13 (e.g., substantially free IL-
13 and/or IL-
13-bound to a second anti-IL-13 binding agent or antibody molecule), in the
sample. A
statistically significant change in the level of IL-13 bound to the first anti-
IL-13 binding
agent or antibody molecule in the sample relative to a reference value or
sample (e.g., a
control sample) is indicative of the presence of the IL-13 in the sample.
In certain embodiments, the first anti-IL- 13 binding agent or antibody
molecule is
immobilized to a support (e.g., a solid support, such as an ELISA plate,
beads).
In other embodiments, the method further includes obtaining a sample from a
subject before and/or after exposure of the subject to a second anti-IL- 13
binding agent or
antibody molecule. The sample can contain substantially free IL-13 and/or IL-
13 bound
to the second anti-IL-13 binding agent or antibody molecule. The sample is
allowed to
contact the immobilized first anti-IL- 13 binding agent or antibody molecule,
under
conditions that allow binding of the IL-13 to the immobilized first anti-IL-13
binding
agent or antibody molecule to occur.
In embodiments, the detection step includes detecting the presence of IL- 13
(e.g.,
substantially free IL-13 and/or IL-13-bound to a second anti-IL-13 binding
agent or
antibody molecule) bound to the immobilized first anti-IL-13 binding agent or
antibody
molecule, e.g., using a labeled third anti-IL-13 binding agent or antibody
molecule, or a
labeled agent that recognizes the complex of IL-13 first or second binding
agent or
-20-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
antibody molecule. The label can be directly or indirectly attached to the
anti-IL- 13
binding agent or antibody molecule, e.g., fluorescence, radioactivity, biotin-
avidin, as
described herein. For example, the anti-IL13 binding agent or antibody
molecule is
directly or indirectly labeled with a detectable substance to facilitate
detection of the
bound or unbound antibody. Suitable detectable substances include various
enzymes,
prosthetic groups, fluorescent materials, luminescent materials and
radioactive materials.
In one embodiment, the first anti-IL- 13 binding agent or antibody molecule
binds
to substantially free IL-13, and does not substantially bind to IL-13 bound to
a second
anti-IL-13 binding agent or antibody molecule. In other embodiments, the first
anti-IL-
13 binding agent or antibody molecule binds to substantially free IL-13 and IL-
13 bound
to a second anti-IL- 13 binding agent or antibody molecule.
In another embodiment, the first, second and/or third anti-IL-13 binding
agents or
antibody molecules bind to different epitopes on IL-13. For example, the first
anti-IL-13
antibody molecule is a mAb 13.2 or a humanized version thereof (disclosed
herein and in
US 06/0063228), or an IL-13 binding agent capable of competing with mAb13.2
for
binding to IL-13; the second anti-IL-13 antibody molecule is an MJ2-7 or a
humanized
version thereof; and/or the third anti-IL-13 antibody molecule is a C65
antibody or a
humanized version thereof (disclosed herein and in US 06/0073148) (or an IL-13
binding
agent capable of competing with mJ2-7 or C65 for binding to IL-13). Any order
of anti-
IL13 antibody molecules can be used in the detection methods.
In embodiment, the complex of IL- 13 bound to the second IL- 13 binding agent,
which is immobilized to the first IL-3 binding agent, is detected by
contacting the
immobilized complex with an Fc binding agent (e.g., an anti-Fc antibody
molecule),
thereby determining the amount of IL-13 bound to the second IL-13 binding
agent in a
sample.
In embodiments, an increase in the level of IL-13 in the sample (e.g., a
biological
sample, such as serum, plasma, tissue, biopsy) of the subject relative to a
predetermined
level is indicative of increased inflammation in the lung.
In yet another aspect, the invention provides a method for detecting the
presence
of IL-13 in vivo (e.g., in vivo imaging in a subject). The subject method can
be used to
diagnose a disorder, e.g., an IL-13-associated disorder, or to measure the
efficacy of a
-21-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
treatment. The method includes: (i) administering a first IL-13 binding agent,
e.g., a first
anti-IL- 13 antibody molecule as described herein, to a subject under
conditions that allow
binding of the first IL- 13 binding agent to IL- 13 to occur; and (ii)
detecting IL- 13 in vivo
(e.g., detecting the formation of a complex between IL-13 and the first IL-13
binding
agent) using a second IL-13 binding agent detectably labeled, wherein a
statistically
significant change in the level of IL-13 in the subject relative to the
control subject is
indicative of the presence of IL-13. In embodiments, an increase in the level
of IL-13 in
the subject relative to a predetermined level is indicative of increased
inflammation in the
lung.
In one embodiment, the IL-13 binding agent and the IL-13 antagonist bind to
substantially free IL-13 and/or IL-13 bound to a second IL-13 binding agent.
In one
embodiment, the IL-13 antagonist and the IL-13 binding agent recognize
different
epitopes on IL-13. For example, the IL-13 antagonist can be a mAb13.2 or a
humanized
version thereof (disclosed herein and in US 06/0063228), or an IL-13
antagonist capable
of competing with mAbl3.2 for binding to IL-13; the IL-13 binding agent is an
MJ2-7 or
a humanized version thereof; or the binding agent is a C65 antibody or a
humanized
version thereof (disclosed herein and in US 06/0073148) (or an IL-13 binding
agent
capable of competing with mJ2-7 or C65 for binding to IL-13). Any order of
anti-IL13
antagonist or binding agents can be used in the detection methods.
In another aspect, the application provides a method of evaluating the
efficacy of
an IL-13 antagonistic binding agent, e.g., an anti-IL13 antibody molecule as
described
herein, in treating (e.g., reducing) pulmonary inflammation in a subject,
e.g., a human or
non-human subject. The method includes:
administering an IL-13 antagonist and/or an IL-4 antagonist to the subject;
2.5 detecting a change in one or more of the following parameters: (i)
detecting the
levels of IL-13 unbound and/or bound to an IL13 binding agent in a sample,
e.g., a
biological sample (e.g., serum, plasma, blood) as described in the in vitro
detection
methods herein, wherein a change in the levels of IL- 13 unbound and/or bound
relative to
a reference value (e.g., a control sample) is indicative of the efficacy of
the agent.
In embodiments, the method further includes: (i) measuring eotaxin levels in a
sample, e.g., a biological sample (e.g., serum, plasma, blood); (ii) detecting
histamine
-22-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
release, e.g., by basophils; (iii) detecting IgE-titers; and/or (iv)
evaluating changes in the
symptoms of the subject (e.g., difficulty breathing, wheezing, coughing,
shortness of
breath and/or difficulty performing normal daily activities). The detection of
parameters
(i)-(v) can be carried out before and/or after administration of the IL- 13
antagonistic
binding agent (after single or multiple administrations) to the subject (e.g.,
at selected
intervals after initiating therapy). The detection and/or evaluation of the
changes in one
or more of (i)-(v) can be performed by a clinician or support staff. A change,
e.g., a
reduction, in one or more of (i)-(v) relative to a predetermined level (e.g.,
comparing
before and after treatment) indicates that the IL- 13 antagonistic binding
agent is
effectively reducing lung inflammation in the subjects. In embodiments, the
subject is a
human patient, e.g., an adult or a child.
In embodiments, the efficacy of an IL-13 binding agent (e.g., an anti-IL13
antibody molecule as described) in neutralizing one or more IL- 13 -associated
activities in
vivo can be evaluated in a subject, e.g., a non-human subject, such as sheep,
rodent, non-
human primate (e.g., a cynomolgus monkey naturally allergic to an antigen,
e.g., Ascaris
suum). For example, the efficacy of IL- 13 binding agents can be evaluated by
measuring
in cynomolgus monkeys naturally allergic to Ascaris suum, before and after
challenge
with the Ascaris antigen in the presence or absence of the IL-13 binding
agent, one or
more of the following: (i) detecting inflammatory cells (e.g., eosinophils,
macrophages,
neutrophils) into the airways; (ii) measuring eotaxin levels; (iii) detecting
in antigen-
specific (e.g., Ascaris-specific) basophil histamine release; and/or (iv)
detecting in
antigen-specific (e.g., Ascaris-specific) IgE titers. A change, e.g., a
reduction, in the
level of one or more of (i)-(iv) relative to a predetermined level (e.g.,
comparison before
and after treatment) indicates that the IL- 13 binding agent is effectively
reducing airway
eosinophilia in the subjects.
Methods of diagnosing an IL- 13 -associated disorder using an IL-13 binding
agent, e.g., an anti-IL13 antibody molecule as described herein are also
disclosed.
As used herein, the articles "a" and "an" refer to one or to more than one
(e.g., to
at least one) of the grammatical object of the article.
The term "or" is used herein to mean, and is used interchangeably with, the
term
"and/or", unless context clearly indicates otherwise.
- 23 -
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
The terms "proteins" and "polypeptides" are used interchangeably herein.
"About" and "approximately" shall generally mean an acceptable degree of error
for the quantity measured given the nature or precision of the measurements.
Exemplary
degrees of error are within 20 percent (%), typically, within 10%, and more
typically,
within 5% of a given value or range of values.
The contents of all publications, pending patent applications, published
patent
applications (inclusive of US 06/0073148 and US 06/0063228), and published
patents
cited throughout this application are hereby incorporated by reference in
their entirety.
Others features, objects and advantages of the invention will be apparent from
the
description and drawings, and from the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 A is an alignment of full-length human and cynomolgus monkey IL-13,
SEQ ID NO: 178 and SEQ ID NO:24, respectively. Amino acid differences are
indicated
by the shaded boxed residues. The location of the R to Q substitution (which
corresponds
to the polymorphism detected in allergic patients) is boxed at position 130.
The location
of the cleavage site is shown by the arrow.
FIG. 1B is a list of exemplary peptides from cynomolgus monkey IL-13, (SEQ ID
NOs: 179-188, respectively).
FIG 2 is a graph depicting the neutralization of NHP IL-13 activity by various
IL- 13 binding agents, as measured by percentage of CD23+ monocytes (y-axis).
Concentration of MJ2-7 (0), C65 (+), and sIL-13Ra2-Fc (*) are indicated on the
x-axis.
FIG 3 is a graph depicting the neutralization of NHP IL- 13 activity by MJ2-7
(murine; =) or humanized MJ2-7 v2.11 (o). NHP IL-13 activity was measured by
phosphorylation of STAT6 (y-axis) as a function of antibody concentration (x-
axis).
FIG 4 is a graph depicting the neutralization of NHP IL-13 activity by MJ2-7
v2.11 (o) or sIL- 1 3Ra2-Fc (A). NHP IL- 13 activity was measured by
phosphorylation
of STAT6 (y-axis) as a function of antagonist concentration (x-axis).
FIG 5 is a graph depicting the neutralization of NHP IL-13 activity by MJ2-7
(0),
C65 (+), or sIL-13Ra2-Fc (o). NHP IL-13 activity was measured by
phosphorylation of
STAT6 (y-axis) as a function of antagonist concentration (x-axis).
-24-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
FIG. 6A is a graph depicting induction of tenascin production (y-axis) by
native
human IL-13 (x-axis).
FIG 6B is a graph depicting the neutralization of NHP IL-13 activity by MJ2-7,
as measured by inhibition of induction of tenascin production (y-axis) as a
function of
antibody concentration (x-axis).
FIG 7 is a graph depicting binding of MJ2-7 or control antibodies to NHP-IL-
13
bound to sIL-13Ra2-Fc coupled to a SPR chip.
FICz 8 is a graph depicting binding of varying concentrations (0.09-600 nM) of
NHP IL-13 to captured hMJ2-7 V2-11 antibody.
FIG 9 is a graph depicting the neutralization of NHP IL-13 activity by mouse
MJ2-7 (9) or humanized Version 1(o), Version 2(*), or Version 3(0) antibodies.
NHP
IL- 13 activity was measured by phosphorylation of STAT6 (y-axis) as a
function of
antibody concentration (x-axis).
FIG 10 is a graph depicting the neutralization of NHP IL-13 activity by
antibodies including mouse MJ2-7 VH and VL (e), mouse VH and humanized Version
2
VL (0), or Version 2 VH and VL (*). NHP IL- 13 activity was measured by
phosphorylation of STAT6 (y-axis) as a function of antibody concentration (x-
axis).
FIGs. 11 A and 11 B are graphs depicting inhibition of binding of IL- 13 to
immobilized IL-13 receptor by MJ2-7 antibody, as measured by ELISA. Binding is
depicted as absorbance at 450 nm (y-axis). Concentration of MJ2-7 antibody is
depicted
on the x-axis. FIG 11A depicts binding to IL-13Ra1. FI('z 11B depicts binding
to
IL-13Ra2.
FIG 12 is an alignment of DPK18 germline amino acid sequence (SEQ ID
NO:126) and humanized MJ2-7 Version 3 VL (SEQ ID NO:190).
FIG. 13A is an amino acid sequence (SEQ ID NO:124) of mature, processed
human IL-13.
FIG. 13B shows an amino acid sequence (SEQ ID NO:125) of human IL-13Ra1.
FIG. 14A-14D shows an increase in the total number of cells/ml and percentage
of inflammatory cells present in BAL fluid post Ascaris challenge compared to
pre-
(baseline) samples.
- 25 -
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
FIGS. 15A-15B show total of BAL cells/ml in BAL fluids in control and
antibody-treated cynomolgus monkeys pre- and post-Ascaris challenge. Control
(circles
(o); MJ2-7-treated samples (open triangles (0)) and mAb 13.2-treated samples
(black
triangles(A)). (Humanized versions of MJ2-7 (MJ2-7v.2) and mAb 13.2 v 2 were
used
in this study).
FIGS. 16A-16B show changes in eotaxin levels in concentrated BAL fluid
collected from antibody-treated cynomolgus monkeys post-Ascaris challenge
relative to
control. FIG. 16A depicts a bar graph showing an increase in eotaxin levels
(pg/ml) post-
Ascaris challenge relative to a baseline, pre-challenge values. FIG. 16B
depicts a
decrease in eotaxin levels in concentrated BAL fluids from cynomolgus monkeys
treated
with mAb 13.2- (grey circles) or MJ2-7-(grey triangles) antibodies compared to
a control.
(Humanized versions of MJ2-7 (MJ2-7v.2) and mAb 13.2 v2 were used in this
study).
FIGS. 17A-17B depict the changes in Ascaris-specific IgE-titers in control and
antibody-treated samples 8-weeks post-challenge. FIG 17A depicts
representative
examples showing no change in Ascaris-specific IgE titer in an individual
monkey treated
with irrelevant Ig (IVIG; animal 20-45; top panel), and decreased titer of
Ascaris-specific
IgE in an individual monkey treated with humanized MJ2-7v.2 (animal 120-434;
bottom
panel). FIG. 17B depicts a decrease in Ascaris-specific IgE-titers in mAb 13.2
or MJ2-7
(black circles) relative to irrelevant Ig-treated cynomolgus monkeys (IVIG
(grey circles))
8-weeks post-Ascaris challenge.
FIGS. 18A-18B show the changes in Ascaris-specific basophil histamine release
in control and antibody-treated samples 24-hours and 8-weeks post-challenge.
FIG. 15A
is a graph depicting the following samples in representative individual
monkeys treated
with saline (left) or humanized mAb 13.2v.2 (right): pre-antibody or Ascaris
challenged
samples (circles); 48-hours post-antibody treatment, 24-hours post-Ascaris
challenged
samples (triangles); and 8 weeks post-Ascaris challenged samples (diamonds).
FIGS.
18B depicts a bar graph showing the changes in normalized histamine levels pre-
and 8-
week post-Ascaris challenge in control (black), humanized mAb 13.2- (white)
and
humanized MJ2-7v.2- (shaded) treated cynomolgus monkeys.
-26-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
FIG. 19 depicts the correlation between Ascaris-specific histamine release and
Ascaris-specific IgE levels in control (open circles) and anti-IL13- or
dexamethasone-
treated samples (black circles).
FIG. 20 is a series of bar graphs depicting the changes in serum IL-13 levels
in
individual cynomolgus monkeys treated with humanized MJ2-7 (hMJ2-7v2). The
label
in each panel (e.g., 120-452) corresponds to the monkey identification number.
The
"pre" sample was collected prior to administration of the antibody. The time
"0" was
collected 24-hours post-antibody administration, but prior to Ascaris
challenge. The
remaining time points were post-Ascaris challenge.
FIG. 21 is a bar graph depicting the STAT6 phosphorylation activity of non-
human primate IL-13 at 0, 1, or 10 ng/ml, either in the absence of serum ("no
serum");
the presence of serum from saline or IVIG-treated animals ("control"); or in
the presence
of serum from anti-IL13 antibody-treated animals, either before antibody
administration
("pre"), or 1-2 weeks post-administration of the indicated antibody. Serum was
tested at
1:4 dilution. (Humanized versions of MJ2-7 (MJ2-7v.2) and niAb 13.2 v2 were
used in
this study).
FIGS. 22A-22C are linear graphs showing that levels of non-human primate IL-13
trapped by humanized MJ2-7 (hMJ2-7v2) in cynomolgus monkey serum correlate
with
the level of inflammation measured in the BAL fluids post-Ascaris challenge.
FIGs. 23A-23B are line graphs showing altered lung function in mice in
response
to human recombinant R110Q IL-13 intratracheal administration; FIG. 23A shows
the
changes in airway resistance (RI) in response to increasing doses of nebulized
metacholine; FIG. 23B shows the changes in dynamic lung compliance (Cdyn) in
response to increasing doses of nebulized metacholine.
FIGs. 24A-24B are bar graphs showing increased lung inflammation and cytokine
production in mice in response to human recombinant R1 lOQ IL-13 intranasal
administration. In FIG. 24A, the percentage of eosinophils and neutrophils in
bronchoalveolar lavage (BAL) were determined by differential cell counts. In
FIG. 24B,
the levels of cytokines, MCP- 1, TNF-a, and IL-6, in BAL were determined by
cytometric
bead array. Data is median s.e.m. of 10 animals per group.
-27-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
FIGs. 25A-25B are dot plots showing humanized MJ2-7-11 (hMJ2-7v.2-1 1)
antibody levels in BAL and serum following intratracheal and intravenous
administration. Animals were treated with human recombinant R110Q IL-13, or an
equivalent volume (20 L) of saline, intratracheally on days 1, 2, and 3.
Humanized
MJ2-7v.2-11 antibody was administered on day 0 and 2 hours before each dose of
human
recombinant R110Q IL-13. FIG. 25A depicts the results when the antibody is
administered intravenously on day 0 and intraperitoneally on days 1, 2, and 3;
or
intranasally on days 0, 1, 2, and 3 (shown in FIG. 25B). Total human IgG
levels in BAL
and serum were assayed by ELISA.
FIGs. 26A-26C show the effect of humanized MJ2-7v.2-11 antibody after
intranasal administration of human recombinant R110Q IL-13-induced altered
lung
function. (A) FIG. 26A shows the changes in lung resistance (RI; cm
Hz0/ml/sec)
expressed as change from baseline. FIG. 26B shows data expressed as
methacholine
dose required to elicit lung resistance (RI) corresponding to a change of 2.5
ml
H20/cm/sec from baseline. Median values are shown for each treatment group. p-
values
were calculated by two-tailed t-test. FIG. 26C shows the median human IgG
levels in
BAL and sera.
FIGs. 27A-27D show the changes in BAL and serum levels of human
recombinant R110Q IL-13 administered alone (FIGs. 27A-27B) or in complex with
humanized MJ2-7v.2-11 antibody (FIGs. 26C-27D) following intratracheal
administration of human recombinant R110Q IL-13 and intranasal administration
of
humanized MJ2-7v.2-11 antibody. Median values are indicated for each group.
n.d. is
not detectable.
FIGs. 28A-28B are dot plots showing eosinophil (FIG. 28A) and neutrophil (FIG.
28B) infiltration into BAL levels following intranasal administration of human
recombinant Rl lOQ IL-13 and intranasal administration of 500, 100, and 20 jig
of
humanized MJ2-7v.2-11 and humanized 13.2v.2, saline, or 500 g of IVIG.
Eosinophil
and neutrophil percentages were determined by differential cell counts. Median
values
for each group are indicated. p-values were determined by two-tailed test and
are
indicated for each antibody-treated group as compared to IVIG.
- 28 -
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
FIGs. 29A-29C are dot plots showing changes in chytokine levels, MCP-1, TNF-
a, and IL-6, respectively, following intranasal administration of human
recombinant
R110Q IL-13 and intranasal administration of 500 g of humanized MJ2-7v.2-1 1,
humanized 13.2v.2, or IVIG, or saline. Dashed line indicates limit of assay
sensitivity.
Data represent median values for each group. p-value was 50.0001, according to
a two-
tailed t-test.
FIGs. 30A-30B are dot plots showing that human recombinant R110Q IL-13
levels are directly related to lung inflammation, as measured by eosinohilia;
and inversely
proportional to humanized MJ2-7v.2-11 BAL levels following intranasal
administration
of human recombinant R110Q IL-13 and intranasal administration of 500, 100, or
20 g
doses of humanized MJ2-7v.2-11 antibody. Humanized MJ2-7v.2-11 antibody BAL
levels were measured by ELISA. Human recombinant R110Q IL-13 BAL levels were
determined by cytometric bead assay. % eosinophil was determined by
differential cell
counting. Associations are shown between levels of; (FIG. 30A) % eosinophilic
inflammation and human recombinant R110Q IL-13, including data from saline
control
animals, mice treated with human recombinant R110Q IL- 13 alone, and mice
treated with
human recombinant RI lOQ IL-13 and 500, 100, and 20 g of humanized MJ2-7v.2-
11
antibody or 500 g IVIG; and (FIG. 30B) humanized MJ2-7v.2-11 and IL-6,
including
data from mice treated with 500, 100, and 20 g of humanized MJ2-7V2-1 1. r2
and p-
values were determined by linear regression analysis.
FIG. 31 shows the schedules for administrating sIL-13Ra2 one day before and
one day after OVA challenge (Schedule 1), and sIL-13Ra2, anti-IL-4 or both one
day
before OVA challenge (Schedule 2).
FIGs. 32A-32C show total serum IgE (FIG. 32A), OVA-specific IgE (FIG. 32B),
and OVA-specific IgGl (FIG. 32C) following treatment with sILRa2.Rc one day
before
and after OVA challenge. The dashed line in FIG. 32B indicates the limit of
assay
sensitivity. n = 20 mice/group
FIGs. 33A-33C depict show total serum IgE (FIG. 33A), OVA-specific IgE (FIG.
33B), and OVA-specific IgGl (FIG. 33C) following single treatment with
sILRa2.Fc one
day before OVA challenge. The dashed line in FIG. 33B indicates the limit of
assay
sensitivity. n = 20 mice/group.
-29-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
FIGs. 34A-34B show total serum IgE (FIG. 34A) and OVA-specific IgE (FIG.
34B) following single treatment of sIL-13Ra2.Fc or anti-IL-4 treatment one day
before
OVA challenge. The dashed line in FIG. 34B indicates the limit of assay
sensitivity. n
20 mice/group.
FIG. 35A-35B show OVA-specific IgGl (FIG. 35A) and OVA-specific IgG3
(FIG. 35B) following single treatment one day prior to OVA challenge with
combined
sIL-13Ra2.Fc and anti-IL-4.
DETAILED DESCRIPTION
Methods and compositions for treating and/or monitoring treatment of IL-13-
associated disorders or conditions are disclosed. In one aspect, Applicants
have
discovered that a single administration of an IL- 13 antagonist or an IL-4
antagonist to a
subject, prior to the onset of an IL- 13 associated disorder or condition,
reduces one or
more symptoms of the disorder or condition, relative to an untreated subject.
Enhanced
reduction of the symptoms of the disorder or condition is detected after co-
administration
of an IL- 13 antagonist with an IL-4 antagonist, relative to the reduction
detected after
administration of the single agent. Thus, methods for reducing or inhibiting,
or
preventing or delaying the onset of, one or more symptoms of an IL-13-
associated
disorder or condition using an IL-13 antagonist, alone or in combination with
an IL-4
antagonist, are disclosed. In other embodiments, methods for evaluating the
efficacy of
an IL-13 antagonist, in a subject, e.g., a human or non-human subject, are
also disclosed.
Definitions
For convenience, certain terms are defined herein. Additional definitions can
be
found throughout the specification.
The term "IL-13" includes the full length unprocessed form of the cytokines
known in the art as IL-13 (irrespective of species origin, and including
mammalian, e.g.,
human and non-human primate IL- 13) as well as mature, processed forms
thereof, as well
as any fragment (of at least 5 amino acids) or variant of such cytokines.
Positions within
the IL-13 sequence can be designated in accordance to the numbering for the
full length,
-unprocessed human IL-13 sequence. For an exemplary full-length monkey IL-13,
see
-30-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
SEQ ID NO:24; for mature, processed monkey IL-13, see SEQ ID NO:14; for full-
length
human IL-13, see SEQ ID NO:178, and for mature, processed human IL-13, see SEQ
ID
NO:124. An exemplary sequence is recited as follows:
MALLLTTVIALTCLGGFASPGPVPPSTALRELIEELVNITQNQKAPLCNGSMV WSI
NLTAGMYCAALESLINVSGCSAIEKTQRMLSGFCPHKVSAGQFSSLHVRDTKIEV
AQFVKDLLLHLKKLFREGRFN (SEQ ID NO:178)
For example, position 130 is a site of a common polymorphism.
Exemplary sequences of IL- 13 receptor proteins and soluble forms thereof
(e.g.,
IL-13Ra1 and IL-13Ra2 or fusions thereof) are described, e.g., in Donaldson et
al.
(1998) .Ilmmunol. 161:2317-24; U.S. 6,214,559; U.S. 6,248,714; and U.S.
6,268,480.
Exemplary sequences and characterization of IL-4, e.g., human IL-4, are
disclosed in Strober et al. (1988) Pediatr. Res. 24:549; and in Ramanthan et
al. U.S.
6,358,509.
Exemplary sequence of IL-4 receptor proteins, soluble forms and fusions
thereof
are described in, e.g., in Stahl et al. U.S. 7,083,949; Seipelt, I. et al.
(1997) Biochem and
Biophys Res Comm 239:534-542; Stahl, N. et al. (1999) FASEB Journal Abstract,
1457;
and Harada, N. et al. (1990) Proc Natl Acad Sci USA 87:857-861. An exemplary
secreted form of human IL-4 receptor is recited as follows:
MGWLCSGLLFPVSCLVLLQVASSGNMKVLQEPTCVSDYMSISTCEWKMNGPTN
CSTELRLLYQLVFLLSEAHTCIPENNGGAGCVCHLLMDDVVSADNYTLDLWAG
QQLLWKGSFKPSEHVKPRAPGNLTVHTNV SDTLLLTW SNPYPPDNYLYNHLTY
AVNIW SENDPADFRIYNV TYLEP S LRIAAS TLKS GIS YRARV RAWAQCYNTTW SE
WSPSTKWHNSNIC (SEQ ID NO:224)
The phrase "a biological activity of' IL-13/IL-13R polypeptide and/or the IL-
4/IL-4R polypeptide refers to one or more of the biological activities of the
corresponding mature IL-13 or IL-4 polypeptide, including, but not limited to,
(1)
interacting with, e.g., binding to, an IL-13R or IL-4R polypeptide (e.g., a
human IL-13R
or IL-4R polypeptide); (2) associating with signal transduction molecules,
e.g., y
common; (3) stimulating phosphorylation and/or activation of stat proteins,
e.g., STAT6;
(4) induction of CD23 expression; (5) production of IgE by human B cells; (6)
induction
of antigen-induced eosinophilia in vivo; (7) induction of antigen-induced
bronchoconstriction in vivo; (8) induction of drug-induced airway
hyperreactivity in vivo;
-31-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
(9) induction of eotoxin levels in vivo; and/or (10) induction histamine
release by
basophils.
An "IL-13 associated disorder or condition" is one in which IL-13 contributes
to a
pathology or symptom of the disorder or condition. Accordingly, an IL-13
binding agent,
e.g., an IL-13 binding agent that is an antagonist of one or more IL-13
associated
activities, can be used to treat or prevent the disorder.
As used herein, a "therapeutically effective amount" of an IL-13/IL-13R
antagonist or an IL-4/IL-4 antagonist refers to an amount of an agent which is
effective,
upon single or multiple dose administration to a subject, e.g., a human
patient, at curing,
reducing the severity of, ameliorating, or preventing one or more symptoms of
a disorder,
or in prolonging the survival of the subject beyond that expected in the
absence of such
treatment.
As used herein, a "prophylactically effective amount" of an IL-13/IL-13R
antagonist or an IL-4/IL-4R antagonist refers to an amount of an IL-13/IL-13R
antagonist
or an IL-4/IL-4R antagonist which is effective, upon single or multiple dose
administration to a subject, e.g., a human patient, in preventing, reducing
the severity, or
delaying the occurrence of the onset or recurrence of an IL-13-associated
disorder or
condition, e.g., a disorder or condition as described herein.
As used herein "a single treatment interval" referes to an amount and/or
frequency
of administration of an IL-13/IL-13R antagonist and/or IL-4/IL-4R antagonist
that when
administered as a single dose, or as a repeated dose of limited frequency
reduces the
severity of, ameliorates, prevents, or delays the occurrence of the onset or
recurrence of,
one or more symptoms of an IL-13-associated disorder or condition, e.g., a
disorder or
condition as described herein. In embodiments, the frequency of administration
is limited
to no more than two or three doses during a single treatment interval, e.g.,
the repeated
dose is administered within one week or less from the initial dose.
The term "isolated" refers to a molecule that is substantially free of its
natural
environment. For instance, an isolated protein is substantially free of
cellular material or
other proteins from the cell or tissue source from which it is derived. The
term refers to
preparations where the isolated protein is sufficiently pure to be
administered as a
therapeutic composition, or at least 70% to 80% (w/w) pure, more preferably,
at least
-32-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
80%-90% (w/w) pure, even more preferably, 90-95% pure; and, most preferably,
at least
95%, 96%, 97%, 98%, 99%, or 100% (w/w) pure. A "separated" compound refers to
a
compound that is removed from at least 90% of at least one component of a
sample from
which the compound was obtained. Any compound described herein can be provided
as
an isolated or separated compound.
As used herein, the term "hybridizes under low stringency, medium stringency,
high stringency, or very high stringency conditions" describes conditions for
hybridization and washing. Guidance for performing hybridization reactions can
be
found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y.
(1989),
6.3.1-6.3.6. Aqueous and nonaqueous methods are described in that reference
and either
can be used. Specific hybridization conditions referred to herein are as
follows: 1) low
stringency hybridization conditions in 6X sodium chloride/sodium citrate (SSC)
at about
45 C, followed by two washes in 0.2X SSC, 0.1% SDS at least at 50 C (the
temperature
of the washes can be increased to 55 C for low stringency conditions); 2)
medium
stringency hybridization conditions in 6X SSC at about 45 C, followed by one
or more
washes in 0.2X SSC, 0.1% SDS at 60 C; 3) high stringency hybridization
conditions in
6X SSC at about 45 C, followed by one or more washes in 0.2X SSC, 0.1% SDS at
65 C; and preferably 4) very high stringency hybridization conditions are 0.5
M sodium
phosphate, 7% SDS at 65 C, followed by one or more washes at 0.2X SSC, 1% SDS
at
65 C. Very high stringency conditions (4) are the preferred conditions and
the ones that
are used unless otherwise specified.
The methods and compositions of the present invention encompass polypeptides
and nucleic acids having the sequences specified, or sequences substantially
identical or
similar thereto, e.g., sequences at least 85%, 90%, 95% identical or higher to
the
sequence specified. In the context of an amino acid sequence, the term
"substantially
identical" is used herein to refer to a first amino acid that contains a
sufficient or
minimum number of amino acid residues that are i) identical to, or ii)
conservative
substitutions of aligned amino acid residues in a second amino acid sequence
such that
the first and second amino acid sequences can have a common structural domain
and/or
common functional activity. For example, amino acid sequences that contain a
common
- 33 -
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
structural domain having at least about 85%, 90%. 91%, 92%, 93%, 94%, 95%,
96%,
97%, 98% or 99% identity to the sequence specified are termed substantially
identical.
In the context of nucleotide sequence, the term "substantially identical" is
used
herein to refer to a first nucleic acid sequence that contains a sufficient or
minimum
number of nucleotides that are identical to aligned nucleotides in a second
nucleic acid
sequence such that the first and second nucleotide sequences encode a
polypeptide having
common functional activity, or encode a common structural polypeptide domain
or a
common functional polypeptide activity. For example, nucleotide sequences
having at
least about 85%, 90%. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity
to
1o the sequence specified are termed substantially identical.
The term "functional variant" refers polypeptides that have a substantially
identical amino acid sequence to the naturally-occurring sequence, or are
encoded by a
substantially identical nucleotide sequence, and are capable of having one or
more
activities of the naturally-occurring sequence.
Calculations of homology or sequence identity between sequences (the terms are
used interchangeably herein) are performed as follows.
To determine the percent identity of two amino acid sequences, or of two
nucleic
acid sequences, the sequences are aligned for optimal comparison purposes
(e.g., gaps
can be introduced in one or both of a first and a second amino acid or nucleic
acid
sequence for optimal alignment and non-homologous sequences can be disregarded
for
comparison purposes). In a preferred embodiment, the length of a reference
sequence
aligned for comparison purposes is at least 30%, preferably at least 40%, more
preferably
at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, 100% of
the length
of the reference sequence. The amino acid residues or nucleotides at
corresponding
amino acid positions or nucleotide positions are then compared. When a
position in the
first sequence is occupied by the same amino acid residue or nucleotide as the
corresponding position in the second sequence, then the molecules are
identical at that
position (as used herein amino acid or nucleic acid "identity" is equivalent
to amino acid
or nucleic acid "homology").
The percent identity between the two sequences is a function of the number of
identical positions shared by the sequences, taking into account the number of
gaps, and
-34-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
the length of each gap, which need to be introduced for optimal alignment of
the two
sequences.
The comparison of sequences and determination of percent identity between two
sequences can be accomplished using a mathematical algorithm. In a preferred
embodiment, the percent identity between two amino acid sequences is
determined using
the Needleman and Wunsch ((1970) J. Mol. Biol. 48:444-453 ) algorithm which
has been
incorporated into the GAP program in the GCG software package (available at
http://www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and
a gap
weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or
6. In yet another
preferred embodiment, the percent identity between two nucleotide sequences is
determined using the GAP program in the GCG software package (available at
http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50,
60,
70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. A particularly preferred
set of
parameters (and the one that should be used unless otherwise specified) are a
Blossum 62
scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a
frameshift gap
penalty of 5.
The percent identity between two amino acid or nucleotide sequences can be
determined using the algorithm of E. Meyers and W. Miller ((1989) CABIOS, 4:11-
17)
which has been incorporated into the ALIGN program (version 2.0), using a
PAM120
weight residue table, a gap length penalty of 12 and a gap penalty of 4.
The nucleic acid and protein sequences described herein can be used as a
"query
sequence" to perform a search against public databases to, for example,
identify other
family members or related sequences. Such searches can be performed using the
NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol.
Biol.
215:403-10. BLAST nucleotide searches can be performed with the NBLAST
program,
score = 100, wordlength = 12 to obtain nucleotide sequences homologous to
nucleic acid
molecules of the invention. BLAST protein searches can be performed with the
XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences
homologous to protein molecules of the invention. To obtain gapped alignments
for
comparison purposes, Gapped BLAST can be utilized as described in Altschul et
al.,
(1997) Nucleic Acids Res. 25:3389-3402. When utilizing BLAST and Gapped BLAST
-35-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
programs, the default parameters of the respective programs (e.g., XBLAST and
NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.
Antibody Molecules
Examples of IL-13 or IL-4 antagonists and/or binding agents include antibody
molecules. As used herein, the term "antibody molecule" refers to a protein
comprising
at least one immunoglobulin variable domain sequence. The term antibody
molecule
includes, for example, full-length, mature antibodies and antigen-binding
fragments of an
antibody. For example, an antibody molecule can include a heavy (H) chain
variable
domain sequence (abbreviated herein as VH), and a light (L) chain variable
domain
sequence (abbreviated herein as VL). In another example, an antibody molecule
includes
one or two heavy (H) chain variable domain sequences and/or one of two light
(L) chain
variable domain sequence. Examples of antigen-binding fragments include: (i) a
Fab
fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains;
(ii) a
F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a
disulfide
bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1
domains;
(iv) a Fv fragment consisting of the VL and VH domains of a single arm of an
antibody,
(v) a VH or VHH domain; (vi) a dAb fragment, which consists of a VH domain;
(vii) a
camelid or camelized variable domain; and (viii) a single chain Fv (scFv).
The VH and VL regions can be further subdivided into regions of
hypervariability, termed "complementarity determining regions" (CDR),
interspersed
with regions that are more conserved, termed "framework regions" (FR). The
extent of
the framework region and CDRs has been precisely defined by a number of
methods (see,
Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest,
Fifth Edition,
U.S. Department of Health and Human Services, NIH Publication No. 91-3242;
Chothia,
C. et al. (1987) J. Mol. Biol. 196:901-917; and the AbM definition used by
Oxford
Molecular's AbM antibody modelling software. See, generally, e.g., Protein
Sequence
and Structure Analysis ofAntibody Variable Domains. In: Antibody Engineering
Lab
Manual (Ed.: Duebel, S. and Kontermann, R., Springer-Verlag, Heidelberg).
Generally,
unless specifically indicated, the following definitions are used: AbM
definition of CDR1
of the heavy chain variable domain and Kabat definitions for the other CDRs.
In
-36-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
addition, embodiments of the invention described with respect to Kabat or AbM
CDRs
may also be implemented using Chothia hypervariable loops. Each VH and VL
typically
includes three CDRs and four FRs, arranged from amino-terminus to carboxy-
terminus in
the following order: FRl, CDR1, FR2, CDR2, FR3, CDR3, FR4.
As used herein, an "immunoglobulin variable domain sequence" refers to an
amino acid sequence which can form the structure of an immunoglobulin variable
domain. For example, the sequence may include all or part of the amino acid
sequence of
a naturally-occurring variable domain. For example, the sequence may or may
not
include one, two, or more N- or C-terminal amino acids, or may include other
alterations
that are compatible with formation of the protein structure.
The term "antigen-binding site" refers to the part of an IL-13 binding agent
that
comprises determinants that form an interface that binds to the IL-13, e.g., a
mammalian
IL-13, e.g., human or non-human primate IL-13, or an epitope thereof. With
respect to
proteins (or protein mimetics), the antigen-binding site typically 'includes
one or more
loops (of at least four amino acids or amino acid mimics) that form an
interface that binds
to IL-13. Typically, the antigen-binding site of an antibody molecule includes
at least
one or two CDRs, or more typically at least three, four, five or six CDRs.
An "epitope" refers to the site on a target compound that is bound by a
binding
agent, e.g., an antibody molecule. An epitope can be a linear or
conformational epitope,
or a combination thereof. In the case where the target compound is a protein,
for
example, an epitope may refer to the amino acids that are bound by the binding
agent.
Overlapping epitopes include at least one common amino acid residue.
The terms "monoclonal antibody" or "monoclonal antibody composition" as used
herein refer to a preparation of antibody molecules of single molecular
composition. A
monoclonal antibody composition displays a single binding specificity and
affinity for a
particular epitope. A monoclonal antibody can be made by hybridoma technology
or by
methods that do not use hybridoma technology (e.g., recombinant methods).
An "effectively human" protein is a protein that does not evoke a neutralizing
antibody response, e.g., the human anti-murine antibody (HAMA) response. HAMA
can
be problematic in a number of circumstances, e.g., if the antibody molecule is
administered repeatedly, e.g., in treatment of a chronic or recurrent disease
condition. A
-37-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
HAMA response can make repeated antibody administration potentially
ineffective
because of an increased antibody clearance from the serum (see, e.g., Saleh et
al., Cancer
Immunol. Immunother., 32:180-190 (1990)) and also because of potential
allergic
reactions (see, e.g., LoBuglio et al., Hybridoma, 5:5117-5123 (1986)).Numerous
methods
are available for obtaining antibody molecules.
One exemplary method of generating antibody molecules includes screening
protein expression libraries, e.g., phage or ribosome display libraries. Phage
display is
described, for example, in Ladner et al., U.S. Patent No. 5,223,409; Smith
(1985) Science
228:1315-1317; WO 92/18619; WO 91/17271; WO 92/20791; WO 92/15679; WO
1o 93/01288; WO 92/01047; WO 92/09690; and WO 90/02809. In addition to the use
of
display libraries, other methods can be used to obtain an anti-IL-13 antibody
molecule.
For example, an IL- 13 protein or a peptide thereof can be used as an antigen
in a non-
human animal, e.g., a rodent, e.g., a mouse, hamster, or rat.
In one embodiment, the non-human animal includes at least a part of a human
immunoglobulin gene. For example, it is possible to engineer mouse strains
deficient in
mouse antibody production with large fragments of the human Ig loci. Using the
hybridoma technology, antigen-specific monoclonal antibodies derived from the
genes
with the desired specificity may be produced and selected. See, e.g.,
XENOMOUSETM,
Green et al. (1994) Nature Genetics 7:13-21, US 2003-0070185, WO 96/34096,
published Oct. 31, 1996, and PCT Application No. PCT/US96/05928, filed Apr.
29,
1996.
In another embodiment, a monoclonal antibody is obtained from the non-human
animal, and then modified, e.g., humanized or deimmunized. Winter describes an
exemplary CDR-grafting method that may be used to prepare the humanized
antibodies
described herein (U.S. Patent No. 5,225,539). All of the CDRs of a particular
human
antibody may be replaced with at least a portion of a non-human CDR, or only
some of
the CDRs may be replaced with non-human CDRs. It is only necessary to replace
the
number of CDRs required for binding of the humanized antibody to a
predetermined
antigen.
Humanized antibodies can be generated by replacing sequences of the Fv
variable
domain that are not directly involved in antigen binding with equivalent
sequences from
-38-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
human Fv variable domains. Exemplary methods for generating humanized antibody
molecules are provided by Morrison (1985) Science 229:1202-1207; by Oi et al.
(1986)
BioTechniques 4:214; and by US 5,585,089; US 5,693,761; US 5,693,762; US
5,859,205;
and US 6,407,213. Those methods include isolating, manipulating, and
expressing the
nucleic acid sequences that encode all or part of immunoglobulin Fv variable
domains
from at least one of a heavy or light chain. Such nucleic acids may be
obtained from a
hybridoma producing an antibody against a predetermined target, as described
above, as
well as from other sources. The recombinant DNA encoding the humanized
antibody
molecule can then be cloned into an appropriate expression vector.
An antibody molecule may also be modified by specific deletion of human T cell
epitopes or "deimmunization" by the methods disclosed in WO 98/52976 and WO
00/34317. Briefly, the heavy and light chain variable domains of an antibody
can be
analyzed for peptides that bind to MHC Class II; these peptides represent
potential T-cell
epitopes (as defined in WO 98/52976 and WO 00/34317). For detection of
potential T-
cell epitopes, a computer modeling approach termed "peptide threading" can be
applied,
and in addition a database of human MHC class II binding peptides can be
searched for
motifs present in the VH and VL sequences, as described in WO 98/52976 and WO
00/34317. These motifs bind to any of the 18 major MHC class II DR allotypes,
and thus
constitute potential T cell epitopes. Potential T-cell epitopes detected can
be eliminated
by substituting small numbers of amino acid residues in the variable domains,
or
preferably, by single amino acid substitutions. Typically, conservative
substitutions are
made. Often, but not exclusively, an amino acid common to a position in human
germline antibody sequences may be used.
Human germline sequences, e.g., are disclosed in Tomlinson, et al. (1992) J.
Mol.
Biol. 227:776-798; Cook, G. P. et al. (1995) Immunol. Today Vol. 16 (5): 237-
242;
Chothia, D. et al. (1992) J. Mol. Biol. 227:799-817; and Tomlinson et al.
(1995) EMBO
J 14:4628-4638. The V BASE directory provides a comprehensive directory of
human
immunoglobulin variable region sequences (compiled by Tomlinson, I.A. et al.
MRC
Centre for Protein Engineering, Cambridge, UK). These sequences can be used as
a
source of human sequence, e.g., for framework regions and CDRs. Consensus
human
framework regions can also be used, e.g., as described in US 6,300,064.
-39-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
Additionally, chimeric, humanized, and single-chain antibody molecules (e.g.,
proteins that include both human and nonhuman portions), may be produced using
standard recombinant DNA techniques. Humanized antibodies may also be
produced, for
example, using transgenic mice that express human heavy and light chain genes,
but are
incapable of expressing the endogenous mouse immunoglobulin heavy and light
chain
genes.
Additionally, the antibody molecules described herein also include those that
bind
to IL-13, interfere with the formation of a functional IL-13 signaling
complex, and have
mutations in the constant regions of the heavy chain. It is sometimes
desirable to mutate
and inactivate certain fragments of the constant region. For example,
mutations in the
heavy constant region can be made to produce antibodies with reduced binding
to the Fc
receptor (FcR) and/or complement; such mutations are well known in the art. An
example of such a mutation to the amino sequence of the constant region of the
heavy
chain of IgG is provided in SEQ ID NO:128. Certain active fragments of the CL
and CH
subunits (e.g., CH1) are covalently link to each other. A further aspect
provides a
method for obtaining an antigen-binding site that is specific for a surface of
IL- 13 that
participates in forming a functional IL-13 signaling complex.
Exemplary antibody molecules can include sequences of VL chains as set forth
in
SEQ ID NOs:30-46, and/or of VH chains as set forth in and SEQ ID NOs:50-115,
but
also can include variants of these sequences that retain IL- 13 binding
ability. Such
variants may be derived from the provided sequences using techniques well
known in the
art. Amino acid substitutions, deletions, or additions, can be made in either
the FRs or in
the CDRs. Whereas changes in the framework regions are usually designed to
improve
stability and reduce immunogenicity of the antibody molecule, changes in the
CDRs are
usually designed to increase affinity of the antibody molecule for its target.
Such
affinity-increasing changes are typically determined empirically by altering
the CDR
region and testing the antibody molecule. Such alterations can be made
according to the
methods described in Antibody Engineering, 2nd. ed. (1995), ed. Borrebaeck,
Oxford
University Press.
An exemplary method for obtaining a heavy chain variable domain sequence that
is a variant of a heavy chain variable domain sequence described herein,
includes adding,
-40-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
deleting, substituting, or inserting one or more amino acids in a heavy chain
variable
domain sequence described herein, optionally combiirning the heavy chain
variable
domain sequence with one or more light chain variable domain sequences, and
testing a
protein that includes the modified heavy chain variable domain sequence for
specific
binding to IL-13, and (preferably) testing the ability of such antigen-binding
domain to
modulate one or more IL-13-associated activities. An analogous method may be
employed using one or more sequence variants of a light chain variable domain
sequence
described herein.
Variants of antibody molecules can be prepared by creating libraries with one
or
more varied CDRs and screening the libraries to find members that bind to IL-
13, e.g.,
with improved affinity. For example, Marks et al. (Bio/Technology (1992)
10:779-83)
describe methods of producing repertoires of antibody variable domains in
which
consensus primers directed at or adjacent to the 5' end of the variable domain
area are
used in conjunction with consensus primers to the third framework region of
human VH
genes to provide a repertoire of VH variable domains lacking a CDR3. The
repertoire
may be combined with a CDR3 of a particular antibody. Further, the CDR3-
derived
sequences may be shuffled with repertoires of VH or VL domains lacking a CDR3,
and
the shuffled complete VH or VL domains combined with a cognate VL or VH domain
to
provide specific antigen-binding fragments. The repertoire may then be
displayed in a
suitable host system such as the phage display system of WO 92/01047, so that
suitable
antigen-binding fragments can be selected. Analogous shuffling or
combinatorial
techniques are also disclosed by Stemmer (Nature (1994) 370:389-91). A further
alternative is to generate altered VH or VL regions using random mutagenesis
of one or
more selected VH and/or VL genes to generate mutations within the entire
variable
domain. See, e.g., Gram et al. Proc. Nat. Acad. Sci. USA (1992) 89:3576-80.
Another method that may be used is to direct mutagenesis to CDR regions of VH
or VL genes. Such techniques are disclosed by, e.g., Barbas et al. (Proc. Nat.
Acad. Sci.
USA (1994) 91:3809-13) and Schier et al. (J. Mol. Biol. (1996) 263:551-67).
Similarly,
one or more, or all three CDRs may be grafted into a repertoire of VH or VL
domains, or
even some other scaffold (such as a fibronectin domain). The resulting protein
is
evaluated for ability to bind to IL-13.
-41-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
In one embodiment, a binding agent that binds to a target is modified, e.g.,
by
mutagenesis, to provide a pool of modified binding agents. The modified
binding agents
are then evaluated to identify one or more altered binding agents which have
altered
functional properties (e.g., improved binding, improved stability, lengthened
stability in
vivo). In one implementation, display library technology is used to select or
screen the
pool of modified binding agents. Higher affinity binding agents are then
identified from
the second library, e.g., by using higher stringency or more competitive
binding and
washing conditions. Other screening techniques can also be used.
In some embodiments, the mutagenesis is targeted to regions known or likely to
be at the binding interface. If, for example, the identified binding agents
are antibody
molecules, then mutagenesis can be directed to the CDR regions of the heavy or
light
chains as described herein. Further, mutagenesis can be directed to framework
regions
near or adjacent to the CDRs, e.g., framework regions, particular within 10,
5, or 3 amino
acids of a CDR junction. In the case of antibodies, mutagenesis can also be
limited to
one or a few of the CDRs, e.g., to make step-wise improvements.
In one embodiment, mutagenesis is used to make an antibody more similar to one
or more gennline sequences. One exemplary germlining method can include:
identifying
one or more gennline sequences that are similar (e.g., most similar in a
particular
database) to the sequence of the isolated antibody. Then mutations (at the
amino acid
level) can be made in the isolated antibody, either incrementally, in
combination, or both.
For example, a nucleic acid library that includes sequences encoding some or
all possible
germline mutations is made. The mutated antibodies are then evaluated, e.g.,
to identify
an antibody that has one or more additional germline residues relative to the
isolated
antibody and that is still useful (e.g., has a functional activity). In one
embodiment, as
many germline residues are introduced into an isolated antibody as possible.
In one embodiment, mutagenesis is used to substitute or insert one or more
germline residues into a CDR region. For example, the germline CDR residue can
be
from a germline sequence that is similar (e.g., most similar) to the variable
domain being
modified. After mutagenesis, activity (e.g., binding or other functional
activity) of the
antibody can be evaluated to detennine if the germline residue or residues are
tolerated.
Similar mutagenesis can be performed in the framework regions.
-42-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
Selecting a germline sequence can be performed in different ways. For example,
a germline sequence can be selected if it meets a predetermined criteria for
selectivity or
similarity, e.g., at least a certain percentage identity, e.g., at least 75,
80, 85, 90, 91, 92,
93, 94, 95, 96, 97, 98, 99, or 99.5% identity. The selection can be performed
using at
least 2, 3, 5, or 10 germline sequences. In the case of CDR1 and CDR2,
identifying a
similar germline sequence can include selecting one such sequence. In the case
of CDR3,
identifying a similar germline sequence can include selecting one such
sequence, but may
including using two germline sequences that separately contribute to the amino-
terminal
portion and the carboxy-terminal portion. In other implementations more than
one or two
germline sequences are used, e.g., to form a consensus sequence.
In other embodiments, the antibody may be modified to have an altered
glycosylation pattern (i.e., altered from the original or native glycosylation
pattern). As
used in this context, "altered" means having one or more carbohydrate moieties
deleted,
and/or having one or more glycosylation sites added to the original antibody.
Addition of
glycosylation sites to the presently disclosed antibodies may be accomplished
by altering
the amino acid sequence to contain glycosylation site consensus sequences;
such
techniques are well known in the art. Another means of increasing the number
of
carbohydrate moieties on the antibodies is by chemical or enzymatic coupling
of
glycosides to the amino acid residues of the antibody. These methods are
described in,
e.g., WO 87/05330, and Aplin and Wriston (1981) CRC Crit. Rev. Biochem. 22:259-
306.
Removal of any carbohydrate moieties present on the antibodies may be
accomplished
chemically or enzymatically as described in the art (Hakimuddin et al. (1987)
Arch.
Biochem. Biophys. 259:52; Edge et al. (1981) Anal. Biochem. 118:131; and
Thotakura et
al. (1987) Meth. Enzymol. 138:350). See, e.g., U.S. 5,869,046 for a
modification that
increases in vivo half life by providing a salvage receptor binding epitope.
In one embodiment, the anti-IL-13 antibody molecule includes at least one, two
and preferably three CDRs from the light or heavy chain variable domain of an
antibody
disclosed herein, e.g., MJ 2-7. For example, the protein includes one or more
of the
following sequences within a CDR region:
GFNIKDTYIH (SEQ ID NO:15),
RIDPANDNIKYDPKFQG (SEQ ID NO:16),
- 43 -
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
SEENWYDFFDY (SEQ ID NO:17),
RSSQSIVHSNGNTYLE (SEQ ID NO:18),
KVSNRFS (SEQ ID NO:19), and
FQGSHIPYT (SEQ ID NO:20), or a CDR having an amino acid sequence that
differs by no more than 4, 3, 2.5, 2, 1.5, 1, or 0.5 alterations (e.g.,
substitutions, insertions
or deletions) for every 10 amino acids (e.g., the number of differences being
proportional
to the CDR length) relative to a sequence listed above, e.g., at least one
alteration but not
more than two, three, or four per CDR.
For example, the anti-IL- 13 antibody molecule can include, in the light chain
variable domain sequence, at least one, two, or three of the following
sequences within a
CDR region:
RSSQSIVHSNGNTYLE (SEQ ID NO:18),
KVSNRFS (SEQ ID NO:19), and
FQGSHIPYT (SEQ ID NO:20), or an amino acid sequence that differs by no
more than 4, 3, 2.5, 2, 1.5, 1, or 0.5 substitutions, insertions or deletions
for every 10
amino acids relative to a sequence listed above.
The anti-IL- 13 antibody molecule can include, in the heavy chain variable
domain
sequence, at least one, two, or three of the following sequences within a CDR
region:
GFNIKDTYIH (SEQ ID NO:15),
RIDPANDNIKYDPKFQG (SEQ ID NO:16), and
SEENWYDFFDY (SEQ ID NO: 17), or an amino acid sequence that differs by no
more than 4, 3, 2.5, 2, 1.5, 1, or 0.5 substitutions, insertions or deletions
for every 10
amino acids relative to a sequence listed above. The heavy chain CDR3 region
can be
less than 13 or less than 12 amino acids in length, e.g., 11 amino acids in
length (either
using Chothia or Kabat definitions).
In another example, the anti-IL-13 antibody molecule can include, in the light
chain variable domain sequence, at least one, two, or three of the following
sequences
within a CDR region (amino acids in parentheses represent alternatives for a
particular
position):
-44-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
(i) (RK)-S-S-Q-S-(LI)-(KV)-H-S-(ND)-G-N-(TN)-Y-L-(EDNQYAS) (SEQ ID
NO:25) or (RK)-S-S-Q-S-(LI)-(KV)-H-S-(ND)-G-N-(TN)-Y-L-E (SEQ ID NO:26), or
(RK)-S-S-Q-S-(LI)-(KV)-H-S-N-G-N-T-Y-L-(EDNQYAS) (SEQ ID NO:21),
(ii) K-(LVI)-S-(NY)-(RW)-(FD)-S (SEQ ID NO:27), or K-(LV)-S-(NY)-R-F-S
(SEQ ID NO:22), and
(iii) Q-(GSA)-(ST)-(HEQ)-I-P (SEQ ID NO:28), F-Q-(GSA)-(SIT)-(HEQ)-(IL)-P
(SEQ ID NO:23), or Q-(GSA)-(ST)-(HEQ)-I-P-Y-T (SEQ ID NO:194), or F-Q-(GSA)-
(SIT)-(HEQ)-(IL)-P-Y-T (SEQ ID NO:29).
In one preferred embodiment, the anti-IL-13 antibody molecule includes all six
CDR's from MJ 2-7 or closely related CDRs, e.g., CDRs which are identical or
which
have at least one amino acid alteration, but not more than two, three or four
alterations
(e.g., substitutions, deletions, or insertions). The IL-13 binding agent can
include at least
two, three, four, five, six, or seven IL-13 contacting amino acid residues of
MJ 2-7
In still another example, the anti-IL-13 antibody molecule includes at least
one,
two, or three CDR regions that have the same canonical structures and the
corresponding
CDR regions of MJ 2-7, e.g., at least CDR1 and CDR2 of the heavy and/or light
chain
variable domains of MJ 2-7.
In another example, the anti-IL-13 antibody molecule can include, in the heavy
chain variable domain sequence, at least one, two, or three of the following
sequences
within a CDR region (amino acids in parentheses represent alternatives for a
particular
position):
(i) G-(YF)-(NT)-I-K-D-T-Y-(MI)-H (SEQ ID NO:48),
(ii) (WR)-I-D-P-(GA)-N-D-N-I-K-Y-(SD)-(PQ-K-F-Q-G (SEQ ID NO:49), and
(iii) SEENWYDFFDY (SEQ ID NO:17).
In one embodiment, the anti-IL-13 antibody molecule includes at least one, two
and preferably three CDR's from the light or heavy chain variable domain of an
antibody
disclosed herein, e.g., C65. For example, the anti-IL-13 antibody molecule
includes one
or more of the following sequences within a CDR region:
QASQGTSINLN (SEQ ID NO: 118),
GASNLED (SEQ ID NO: 119), and
LQHSYLPWT (SEQ ID NO:120)
-45-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
GFSLTGYGVN (SEQ ID NO:121),
IIWGDGSTDYNSAL (SEQ ID NO:122), and
DKTFYYDGFYRGRMDY (SEQ ID NO:123), or a CDR having an amino acid
sequence that differs by no more than 4, 3, 2.5, 2, 1.5, 1, or 0.5
substitutions, insertions or
deletions for every 10 amino acids (e.g., the number of differences being
proportional to
the CDR length) relative to a sequence listed above, e.g., at least one
alteration but not
more than two, three, or four per CDR. For example, the protein can include,
in the light
chain variable domain sequence, at least one, two, or three of the following
sequences
within a CDR region:
QASQGTSINLN (SEQ ID NO: 118),
GASNLED (SEQ ID NO:119), and
LQHSYLPWT (SEQ ID NO: 120), or an amino acid sequence that differs by no
more than 4, 3, 2.5, 2, 1.5, 1, or 0.5 substitutions, insertions or deletions
for every 10
amino acids relative to a sequence listed above.
The anti-IL-13 antibody molecule can include, in the heavy chain variable
domain
sequence, at least one, two, or three of the following sequences within a CDR
region:
GFSLTGYGVN (SEQ ID NO:121),
IIWGDGSTDYNSAL (SEQ ID NO:122), and
DKTFYYDGFYRGRMDY (SEQ ID NO:123), or an amino acid sequence that
differs by no more than 4, 3, 2.5, 2, 1.5, 1, or 0.5 substitutions, insertions
or deletions for
every 10 amino acids relative to a sequence listed above.
In embodiments, the IL- 13 antibody molecule can include one of the following
sequences:
^ DIVMTQTPLSLPVTPGEPASISCRSSQSIVHSNGNTYLEWYLQKPGQSP
QLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC
FQGSHIPYT (SEQ ID NO:30)
= DVVMTQSPLSLPVTLGQPASISCRSSQSIVHSNGNTYLEWFQQRPGQSP
RRLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC
FQGSHIPYT (SEQ ID NO:31)
-46-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
= DIVMTQTPLSLSVTPGQPASISCRSSQSIVHSNGNTYLEWYLQKPGQSP
QLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC
FQGSHIPYT (SEQ ID NO:32)
^ DIVMTQTPLSLSVTPGQPASISCRSSQSIVHSNGNTYLEWYLQKPGQPP
QLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC
FQGSHIPYT (SEQ ID NO:33)
^ DIVMTQSPLSLPVTPGEPASISCRSSQSIVHSNGNTYLEWYLQKPGQSP
QLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC
FQGSHIPYT (SEQ ID NO:34)
^ DIVMTQTPLSSPVTLGQPASISCRSSQSIVHSNGNTYLEWLQQRPGQPP
RLLIYKVSNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYC
FQGSHIPYT (SEQ ID NO:35)
^ DIQMTQSPSSLSASVGDRVTITCRSSQSIVHSNGNTYLEWYQQKPGKAP
KLLIYKVSNRFSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC
FQGSHIPYT (SEQ ID NO:36)
^ DVVMTQSPLSLPVTLGQPASISCRSSQSLVYSDGNTYLNWFQQRPGQSP
RRLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC
FQGSHIPYT (SEQ ID NO:37)
= DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSP
KLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYC
FQGSHIPYT (SEQ ID NO:38)
or a sequence that has fewer than eight, seven, six, five, four, three, or two
alterations
(e.g., substitutions, insertions or deletions, e.g., conservative
substitutions or a
substitution for an amino acid residue at a corresponding position in MJ 2-7).
Exemplary
substitutions are at one of the following Kabat positions: 2, 4, 6, 35, 36,
38, 44, 47, 49,
62, 64-69, 85, 87, 98, 99, 101, and 102. The substitutions can, for example,
substitute an
amino acid at a corresponding position from MJ 2-7 into a human framework
region.
The IL-13 antibody molecule may also include one of the following sequences:
^ DIVMTQTPLSLPVTPGEPASISC-(RK)-S-S-Q-S-(LI)-(KV)-H-
S- (ND) -G-N- (TN) -Y-L- (EDNQYAS) WYLQKPGQSPQLLIYK-
(LVI) -S- (NY) - (RW) - (FD) -
-47-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
SGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC F-Q-(GSA)-
(SIT) - (HEQ) - (IL) -P (SEQ ID NO:39)
^ DVVMTQSPLSLPVTLGQPASISC-(RK)-S-S-Q-S-(LI)-(KV)-H-
S- (ND) -G-N- (TN) -Y-L- (EDNQYAS) WFQQRPGQSPRRLIYK-
(LVI) -S- (NY) - (RW) - (FD) -
SGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCF-Q-(GSA)-(SIT)-
(HEQ) - (IL) -P (SEQ ID NO:40)
= DIVMTQTPLSLSVTPGQPASISC-(RK)-S-S-Q-S-(LI)-(KV)-H-
S-(ND)-G-N-(TN)-Y-L-(EDNQYAS)WYLQKPGQSPQLLIYK-
(LVI) -S- (NY) - (RW) - (FD) -
SGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCF-Q-(GSA)-(SIT)-
(HEQ) - (IL) -P (SEQ ID NO:41)
= DIVMTQTPLSLSVTPGQPASISC(RK)-S-S-Q-S-(LI)-(KV)-H-
S-(ND)-G-N-(TN)-Y-L-(EDNQYAS)WYLQKPGQPPQLLIYK-
(LVI) -S- (NY) - (RW) - (FD) -
SGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCF-Q-(GSA)-(SIT)-
(HEQ) - (IL) -P (SEQ ID NO:42)
^ DIVMTQSPLSLPVTPGEPASISC(RK)-S-S-Q-S-(LI)-(KV)-H-
S- (ND) -G-N- (TN) -Y-L- (EDNQYAS) WYLQKPGQSPQLLIYK-
(LVI) -S- (NY) - (RW) - (FD) -
SGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCF-Q-(GSA)-(SIT)-
(HEQ) - (IL) -P (SEQ ID NO:43)
= DIVMTQTPLSSPVTLGQPASISC(RK)-S-S-Q-S-(LI)-(KV)-H-
S-(ND)-G-N-(TN)-Y-L-(EDNQYAS)WLQQRPGQPPRLLIYK-
(LVI) -S- (NY) - (RW) - (FD) -
SGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCF-Q-(GSA)-(SIT)-
(HEQ)-(IL)-P (SEQ ID NO:44)
^ DIQMTQSPSSLSASVGDRVTITC(RK) -S-S-Q-S- (LI) - (KV) -H-
S-(ND)-G-N-(TN)-Y-L-(EDNQYAS)WYQQKPGKAPKLLIYK-
(LVI) -S- (NY) - (RW) - (FD) -
- 48 -
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
SGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCF-Q-(GSA)-(SIT)-
(HEQ) - (IL) -P (SEQ ID NO:45)
^ DVLMTQTPLSLPVSLGDQASISC(RK)-S-S-Q-S-(LI)-(KV)-H-
S-(ND)-G-N-(TN)-Y-L-(EDNQYAS)WYLQKPGQSPKLLIYK-
(LVI) -S- (NY) - (RW) - (FD) -
SGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCF-Q-(GSA)-(SIT)-
(HEQ)-(IL)-P (SEQ ID NO:46)
or a sequence that has fewer than eight, seven, six, five, four, three, or two
alterations
(e.g., substitutions, insertions or deletions, e.g., conservative
substitutions or a
substitution for an amino acid residue at a corresponding position in MJ 2-7)
in the
framework region. Exemplary substitutions are at one or more of the following
Kabat
positions: 2, 4, 6, 35, 36, 38, 44, 47, 49, 62, 64-69, 85, 87, 98, 99, 101,
and 102. The
substitutions can, for example, substitute an amino acid at a corresponding
position from
MJ 2-7 into a human framework region. The sequences may also be followed by
the
dipeptide Tyr-Thr. The FR4 region can include, e.g., the sequence FGGGTKVEIKR
(SEQ ID NO:47).
In other embodiments, the IL- 13 antibody molecule can include one of the
following sequences:
^ QVQLVQSGAEVKKPGASVKVSCKASGFNIKDTYIHWVRQAPGQGLEWMG
RIDPANDNIKYDPKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCAR
SEENWYDFFDY (SEQ ID NO:50)
^ QVQLVQSGAEVKKPGASVKVSCKASGFNIKDTYIHWVRQAPGQRLEWMG
RIDPANDNIKYDPKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCAR
SEENWYDFFDY (SEQ ID NO:51)
^ QVQLVQSGAEVKKPGASVKVSCKASGFNIKDTYIHWVRQATGQGLEWMG
RIDPANDNIKYDPKFQGRVTMTRNTSISTAYMELSSLRSEDTAVYYCAR
SEENWYDFFDY (SEQ ID NO:52)
^ QVQLVQSGAEVKKPGASVKVSCKASGFNIKDTYIHWVRQAPGQGLEWMG
RIDPANDNIKYDPKFQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCAR
SEENWYDFFDY (SEQ ID NO:53)
-49-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
^ QVQLVQSGAEVKKPGASVKVSCKVSGFNIKDTYIHWVRQAPGKGLEWMG
RIDPANDNIKYDPKFQGRVTMTEDTSTDTAYMELSSLRSEDTAVYYCAT
SEENWYDFFDY (SEQ ID NO:54)
^ QMQLVQSGAEVKKTGSSVKVSCKASGFNIKDTYIHWVRQAPGQALEWMG
RIDPANDNIKYDPKFQGRVTITRDRSMSTAYMELSSLRSEDTAMYYCAR
SEENWYDFFDY (SEQ ID NO:55)
^ QVQLVQSGAEVKKPGASVKVSCKASGFNIKDTYIHWVRQAPGQGLEWMG
RIDPANDNIKYDPKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR
SEENWYDFFDY (SEQ ID NO:56)
^ QMQLVQSGPEVKKPGTSVKVSCKASGFNIKDTYIHWVRQARGQRLEWIG
RIDPANDNIKYDPKFQGRVTITRDMSTSTAYMELSSLRSEDTAVYYCAA
SEENWYDFFDY (SEQ ID NO:57)
^ EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVA
RIDPANDNIKYDPKFQGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR
SEENWYDFFDY (SEQ ID NO:58)
^ EVQLVESGGGLVQPGRSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVS
RIDPANDNIKYDPKFQGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAK
DSEENWYDFFDY (SEQ ID NO:59)
^ QVQLVESGGGLVKPGGSLRLSCAASGFNIKDTYIHWIRQAPGKGLEWVS
RIDPANDNIKYDPKFQGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR
SEENWYDFFDY (SEQ ID NO:60)
^ EVQLVESGGGLVKPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVG
RIDPANDNIKYDPKFQGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTT
SEENWYDFFDY (SEQ ID NO:61)
^ EVQLVESGGGVVRPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVS
RIDPANDNIKYDPKFQGRFTISRDNAKNSLYLQMNSLRAEDTALYHCAR
SEENWYDFFDY (SEQ ID NO:62)
^ EVQLVESGGGLVKPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVS
RIDPANDNIKYDPKFQGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR
SEENWYDFFDY (SEQ ID NO:63)
-50-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
= EVQLLESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVS
RIDPANDNIKYDPKFQGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK
SEENWYDFFDY (SEQ ID NO:64)
^ QVQLVESGGGWQPGRSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVA
RIDPANDNIKYDPKFQGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK
SEENWYDFFDY (SEQ ID NO:65)
= QVQLVESGGGWQPGRSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVA
RIDPANDNIKYDPKFQGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR
SEENWYDFFDY (SEQ ID NO:66)
^ EVQLVESGGVVVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVS
RIDPANDNIKYDPKFQGRFTISRDNSKNSLYLQMNSLRTEDTALYYCAK
DSEENWYDFFDY (SEQ ID NO:67)
' EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVS
RIDPANDNIKYDPKFQGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCAR
SEENWYDFFDY (SEQ ID NO:68)
= EVQLVESGGGLVQPGRSLRLSCTASGFNIKDTYIHWFRQAPGKGLEWVG
RIDPANDNIKYDPKFQGRFTISRDGSKSIAYLQMNSLKTEDTAVYYCTR
SEENWYDFFDY (SEQ ID NO:69)
^ EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEYVS
RIDPANDNIKYDPKFQGRFTISRDNSKNTLYLQMGSLRAEDMAVYYCAR
SEENWYDFFDY (SEQ ID NO:70)
^ EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWIG
RIDPANDNIKYDPKFQGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR
SEENWYDFFDY (SEQ ID NO:71)
^ EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVA
RIDPANDNIKYDPKFQGKATISRDNAKNSLYLQMNSLRAEDTAVYYCAR
SEENWYDFFDY (SEQ ID NO:72)
^ EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVA
RIDPANDNIKYDPKFQGRFTISADNAKNSLYLQMNSLRAEDTAVYYCAR
SEENWYDFFDY (SEQ ID NO:73)
-51-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
= EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVG
RIDPANDNIKYDPKFQGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR
SEENWYDFFDY (SEQ ID NO:74)
^ EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVA
RIDPANDNIKYDPKFQGKATISADNAKNSLYLQMNSLRAEDTAVYYCAR
SEENWYDFFDY (SEQ ID NO:75)
^ EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWIG
RIDPANDNIKYDPKFQGRFTISADNAKNSLYLQMNSLRAEDTAVYYCAR
SEENWYDFFDY (SEQ ID NO:76)
= EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVG
RIDPANDNIKYDPKFQGRFTISADNAKNSLYLQNNSLRAEDTAVYYCAR
SEENWYDFFDY (SEQ ID NO:77)
^ EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVA
RIDPANDNIKYDPKFQGRFTISRDNAKNSAYLQMNSLRAEDTAVYYCAR
SEENWYDFFDY (SEQ ID NO:78)
^ EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVG
RIDPANDNIKYDPKFQGRFTISADNAKNSAYLQMNSLRAEDTAVYYCAR
SEENWYDFFDY (SEQ ID NO:79)
= EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWIG
RIDPANDNIKYDPKFQGRFTISADNAKNSAYLQMNSLRAEDTAVYYCAR
SEENWYDFFDY (SEQ ID NO:80)
^ EVQLVESGGGLVQPGGSLRLSCTGSGFNIKDTYIHWVRQAPGKGLEWIG
RIDPANDNIKYDPKFQGRFTISADNAKNSLYLQMNSLRAEDTAVYYCAR
SEENWYDFFDY (SEQ ID NO:81)
= EVQLQQSGAELVKPGASVKLSCTGSGFNIKDTYIHWVKQRPEQGLEWIG
RIDPANDNIKYDPKFQGKATITADTSSNTAYLQLNSLTSEDTAVYYCAR
SEENWYDFFDY (SEQ ID NO:82)
or a sequence that has fewer than eight, seven, six, five, four, three, or two
alterations
(e.g., substitutions, insertions or deletions, e.g., conservative
substitutions or a
substitution for an amino acid residue at a corresponding position in MJ 2-7).
Exemplary
substitutions are at one or more of the following Kabat positions: 2, 4, 6,
25, 36, 37, 39,
-52-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
47, 48, 93, 94, 103, 104, 106, and 107. Exemplary substitutions can also be at
one or
more of the following positions (accordingly to sequential numbering): 48, 49,
67, 68, 72,
and 79. The substitutions can, for example, substitute an amino acid at a
corresponding
position from MJ 2-7 into a human framework region. In one embodiment, the
sequence
includes (accordingly to sequential numbering) one or more of the following:
Ile at 48,
Gly at 49, Lys at 67, Ala at 68, Ala at 72, and Ala at 79; preferably, e.g.,
Ile at 48, Gly at
49, Ala at 72, and Ala at 79.
Further, the frameworks of the heavy chain variable domain sequence can
include: (i) at a position corresponding to 49, Gly; (ii) at a position
corresponding to 72,
Ala; (iii) at positions corresponding to 48, Ile, and to 49, Gly; (iv) at
positions
corresponding to 48, Ile, to 49, Gly, and to 72, Ala; (v) at positions
corresponding to 67,
Lys, to 68, Ala, and to 72, Ala; and/or (vi) at positions corresponding to 48,
Ile, to 49,
Gly, to 72, Ala, to 79, Ala.
The IL-13 antibody molecule may also include one of the following sequences:
^ QVQLVQSGAEVKKPGASVKVSCKASG-(YF)-(NT)-I-K-D-T-Y-
(MI)-H,WVRQAPGQGLEWMG(WR)-I-D-P-(GA)-N-D-N-I-K-Y-
(SD)-(PQ)-K-F-Q-GRVTMTRDTSISTAYMELSRLRSDDTAVYYCAR
SEENWYDFFDY (SEQ ID NO:83)
^ QVQLVQSGAEVKKPGASVKVSCKASG-(YF)-(NT)-I-K-D-T-Y-
(MI) -H, WVRQAPGQRLEWMG (WR) -I-D-P- (GA) -N-D-N-I-K-Y-
(SD)-(PQ)-K-F-Q-GRVTITRDTSASTAYMELSSLRSEDTAVYYCAR
SEENWYDFFDY (SEQ ID NO:84)
^ QVQLVQSGAEVKKPGASVKVSCKASG-(YF)-(NT)-I-K-D-T-Y-
(MI)-H,WVRQATGQGLEWMG(WR)-I-D-P-(GA)-N-D-N-I-K-Y-
(SD)-(PQ)-K-F-Q-GRVTMTRNTSISTAYMELSSLRSEDTAVYYCAR
SEENWYDFFDY (SEQ ID NO:85)
^ QVQLVQSGAEVKKPGASVKVSCKASG-(YF)-(NT)-I-K-D-T-Y-
(MI)-H,WVRQAPGQGLEWMG(WR)-I-D-P-(GA)-N-D-N-I-K-Y-
(SD)-(PQ)-K-F-Q-GRVTMTTDTSTSTAYMELRSLRSDDTAVYYCAR
SEENWYDFFDY (SEQ ID NO:86)
-53-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
^ QVQLVQSGAEVKKPGASVKVSCKVSG-(YF)-(NT)-I-K-D-T-Y-
(MI)-H,WVRQAPGKGLEWMG(WR)-I-D-P-(GA)-N-D-N-I-K-Y-
(SD)-(PQ)-K-F-Q-GRVTMTEDTSTDTAYMELSSLRSEDTAVYYCAT
SEENWYDFFDY (SEQ ID NO:87)
= QMQLVQSGAEVKKTGSSVKVSCKASG-(YF)-(NT)-I-K-D-T-Y-
(MI)-H,WVRQAPGQALEWMG(WR)-I-D-P-(GA)-N-D-N-I-K-Y-
(SD)-(PQ)-K-F-Q-GRVTITRDRSMSTAYMELSSLRSEDTAMYYCAR
SEENWYDFFDY (SEQ ID NO:88)
^ QVQLVQSGAEVKKPGASVKVSCKASG-(YF)-(NT)-I-K-D-T-Y-
(MI)-H,WVRQAPGQGLEWMG(WR)-I-D-P-(GA)-N-D-N-I-K-Y-
(SD)-(PQ)-K-F-Q-GRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR
SEENWYDFFDY (SEQ ID NO:89)
^ QMQLVQSGPEVKKPGTSVKVSCKASG-(YF)-(NT)-I-K-D-T-Y-
(MI)-H,WVRQARGQRLEWIG(WR)-I-D-P-(GA)-N-D-N-I-K-Y-
(SD)-(PQ)-K-F-Q-GRVTITRDMSTSTAYMELSSLRSEDTAVYYCAA
SEENWYDFFDY (SEQ ID NO:90)
^ EVQLVESGGGLVQPGGSLRLSCAASG-(YF)-(NT)-I-K-D-T-Y-
(MI)-H,WVRQAPGKGLEWVA(WR)-I-D-P-(GA)-N-D-N-I-K-Y-
(SD)-(PQ)-K-F-Q-GRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR
SEENWYDFFDY (SEQ ID NO:91)
^ EVQLVESGGGLVQPGRSLRLSCAASG-(YF)-(NT)-I-K-D-T-Y-
(MI)-H,WVRQAPGKGLEWVS(WR)-I-D-P-(GA)-N-D-N-I-K-Y-
(SD)-(PQ)-K-F-Q-GRFTISRDNAKNSLYLQMNSLRAEDTALYYCAK
DSEENWYDFFDY (SEQ ID NO:92)
^ QVQLVESGGGLVKPGGSLRLSCAASG-(YF)-(NT)-I-K-D-T-Y-
(MI)-H,WIRQAPGKGLEWVS(WR)-I-D-P-(GA)-N-D-N-I-K-Y-
(SD)-(PQ)-K-F-Q-GRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR
SEENWYDFFDY (SEQ ID NO:93)
^ EVQLVESGGGLVKPGGSLRLSCAASG-(YF)-(NT)-I-K-D-T-Y-
(MI) -H, WVRQAPGKGLEWVG (WR) -I-D-P- (GA) -N-D-N-I-K-Y-
-54-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
(SD)-(PQ)-K-F-Q-GRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTT
SEENWYDFFDY (SEQ ID NO:94)
^ EVQLVESGGGWRPGGSLRLSCAASG-(YF)-(NT)-I-K-D-T-Y-
(MI)-H,WVRQAPGKGLEWVS(WR)-I-D-P-(GA)-N-D-N-I-K-Y-
(SD)-(PQ)-K-F-Q-GRFTISRDNAKNSLYLQMNSLRAEDTALYHCAR
SEENWYDFFDY (SEQ ID NO:95)
^ EVQLVESGGGLVKPGGSLRLSCAASG-(YF)-(NT)-I-K-D-T-Y-
(MI)-H,WVRQAPGKGLEWVS(WR)-I-D-P-(GA)-N-D-N-I-K-Y-
(SD)-(PQ)-K-F-Q-GRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR
SEENWYDFFDY (SEQ ID NO:96)
^ EVQLLESGGGLVQPGGSLRLSCAASG-(YF)-(NT)-I-K-D-T-Y-
(MI) -H, WVRQAPGKGLEWVS (WR) -I-D-P- (GA) -N-D-N-I-K-Y-
(SD)-(PQ)-K-F-Q-GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK
SEENWYDFFDY (SEQ ID NO:97)
^ QVQLVESGGGVVQPGRSLRLSCAASG-(YF)-(NT)-I-K-D-T-Y-
(MI)-H,WVRQAPGKGLEWVA(WR)-I-D-P-(GA)-N-D-N-I-K-Y-
(SD)-(PQ)-K-F-Q-GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK
SEENWYDFFDY (SEQ ID NO:98)
^ QVQLVESGGGWQPGRSLRLSCAASG-(YF)-(NT)-I-K-D-T-Y-
(MI) -H,WVRQAPGKGLEWVA(WR) -I-D-P- (GA) -N-D-N-I-K-Y-
(SD)-(PQ)-K-F-Q-GRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR
SEENWYDFFDY (SEQ ID NO:99)
^ EVQLVESGGVWQPGGSLRLSCAASG- (YF) - (NT) -I-K-D-T-Y-
(MI)-H,WVRQAPGKGLEWVS(WR)-I-D-P-(GA)-N-D-N-I-K-Y-
(SD)-(PQ)-K-F-Q-GRFTISRDNSKNSLYLQMNSLRTEDTALYYCAK
DSEENWYDFFDY (SEQ ID NO:100)
= EVQLVESGGGLVQPGGSLRLSCAASG-(YF)-(NT)-I-K-D-T-Y-
(MI) -H, WVRQAPGKGLEWVS (WR) -I-D-P- (GA) -N-D-N-I-K-Y-
(SD)-(PQ)-K-F-Q-GRFTISRDNAKNSLYLQMNSLRDEDTAVYYCAR
SEENWYDFFDY (SEQ ID NO:101)
-55-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
^ EVQLVESGGGLVQPGRSLRLSCTASG-(YF)-(NT)-I-K-D-T-Y-
(MI)-H,WFRQAPGKGLEWVG(WR)-I-D-P-(GA)-N-D-N-I-K-Y-
(SD)-(PQ)-K-F-Q-GRFTISRDGSKSIAYLQMNSLKTEDTAVYYCTR
SEENWYDFFDY (SEQ ID NO:102)
^ EVQLVESGGGLVQPGGSLRLSCAASG-(YF)-(NT)-I-K-D-T-Y-
(MI ) -H, WVRQAPGKGLEYVS (WR) - I -D- P- (GA) -N-D-N- I -K-Y-
(SD)-(PQ)-K-F-Q-GRFTISRDNSKNTLYLQMGSLRAEDMAVYYCAR
SEENWYDFFDY (SEQ ID NO:103)
^ EVQLVESGGGLVQPGGSLRLSCAASG-(YF)-(NT)-I-K-D-T-Y-
(MI ) -H, WVRQAPGKGLEWIG (WR) -I-D-P- (GA) -N-D-N-I-K-Y-
(SD)-(PQ)-K-F-Q-GRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR
SEENWYDFFDY (SEQ ID NO:104)
^ EVQLVESGGGLVQPGGSLRLSCAASG-(YF)-(NT)-I-K-D-T-Y-
(MI)-H,WVRQAPGKGLEWVA(WR)-I-D-P-(GA)-N-D-N-I-K-Y-
(SD)-(PQ)-K-F-Q-GKATISRDNAKNSLYLQMNSLRAEDTAVYYCAR
SEENWYDFFDY (SEQ ID NO:105)
^ EVQLVESGGGLVQPGGSLRLSCAASG-(YF)-(NT)-I-K-D-T-Y-
(MI)-H,WVRQAPGKGLEWVA(WR)-I-D-P-(GA)-N-D-N-I-K-Y-
(SD)-(PQ)-K-F-Q-GRFTISADNAKNSLYLQMNSLRAEDTAVYYCAR
SEENWYDFFDY (SEQ ID NO:106)
^ EVQLVESGGGLVQPGGSLRLSCAASG-(YF)-(NT)-I-K-D-T-Y-
(MI)-H,WVRQAPGKGLEWVG(WR)-I-D-P-(GA)-N-D-N-I-K-Y-
(SD)-(PQ)-K-F-Q-GRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR
SEENWYDFFDY (SEQ ID NO:107)
^ EVQLVESGGGLVQPGGSLRLSCAASG-(YF)-(NT)-I-K-D-T-Y-
(MI)-H,WVRQAPGKGLEWVA(WR)-I-D-P-(GA)-N-D-N-I-K-Y-
(SD)-(PQ)-K-F-Q-GKATISADNAKNSLYLQMNSLRAEDTAVYYCAR
SEENWYDFFDY (SEQ ID NO:108)
^ EVQLVESGGGLVQPGGSLRLSCAASG-(YF)-(NT)-I-K-D-T-Y-
(MI) -H,WVRQAPGKGLEWIG(WR) -I-D-P- (GA) -N-D-N-I-K-Y-
-56-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
(SD)-(PQ)-K-F-Q-GRFTISADNAKNSLYLQMNSLRAEDTAVYYCAR
SEENWYDFFDY (SEQ ID NO:109)
^ EVQLVESGGGLVQPGGSLRLSCAASG-(YF)-(NT)-I-K-D-T-Y-
(MI)-H,WVRQAPGKGLEWVG(WR)-I-D-P-(GA)-N-D-N-I-K-Y-
(SD)-(PQ)-K-F-Q-GRFTISADNAKNSLYLQMNSLRAEDTAVYYCAR
SEENWYDFFDY (SEQ ID NO:110)
^ EVQLVESGGGLVQPGGSLRLSCAASG-(YF)-(NT)-I-K-D-T-Y-
(MI)-H,WVRQAPGKGLEWVA(WR)-I-D-P-(GA)-N-D-N-I-K-Y-
(SD)-(PQ)-K-F-Q-GRFTISRDNAKNSAYLQMNSLRAEDTAVYYCAR
SEENWYDFFDY (SEQ ID NO:111)
^ EVQLVESGGGLVQPGGSLRLSCAASG-(YF)-(NT)-I-K-D-T-Y-
(MI)-H,WVRQAPGKGLEWVG(WR)-I-D-P-(GA)-N-D-N-I-K-Y-
(SD)-(PQ)-K-F-Q-GRFTISADNAKNSAYLQMNSLRAEDTAVYYCAR
SEENWYDFFDY (SEQ ID NO:112)
^ EVQLVESGGGLVQPGGSLRLSCAASG-(YF)-(NT)-I-K-D-T-Y-
(MI)-H,WVRQAPGKGLEWIG(WR)-I-D-P-(GA)-N-D-N-I-K-Y-
(SD)-(PQ)-K-F-Q-GRFTISADNAKNSAYLQMNSLRAEDTAVYYCAR
SEENWYDFFDY (SEQ ID NO:113)
^ EVQLVESGGGLVQPGGSLRLSCTGSG-(YF)-(NT)-I-K-D-T-Y-
(MI)-H,WVRQAPGKGLEWIG(WR)-I-D-P-(GA)-N-D-N-I-K-Y-
(SD)-(PQ)-K-F-Q-GRFTISADNAKNSLYLQMNSLRAEDTAVYYCAR
SEENWYDFFDY (SEQ ID NO:114)
^ EVQLQQSGAELVKPGASVKLSCTGSG-(YF)-(NT)-I-K-D-T-Y-
(MI)-H,WVKQRPEQGLEWIG(WR)-I-D-P-(GA)-N-D-N-I-K-Y-
(SD)-(PQ)-K-F-Q-GKATITADTSSNTAYLQLNSLTSEDTAVYYCAR
SEENWYDFFDY (SEQ ID NO:115)
or a sequence that has fewer than eight, seven, six, five, four, three, or two
alterations
(e.g., substitutions, insertions or deletions, e.g., conservative
substitutions or a
substitution for an amino acid residue at a corresponding position in MJ 2-7)
in the
framework region. Exemplary substitutions are at one or more of the following
Kabat
positions: 2, 4, 6, 25, 36, 37, 39, 47, 48, 93, 94, 103, 104, 106, and 107.
The substitutions
-57-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
can, for example, substitute an amino acid at a corresponding position from MJ
2-7 into a
human framework region. The FR4 region can include, e.g., the sequence
WGQGTTLTVSS (SEQ ID NO:116) or WGQGTLVTVSS (SEQ ID NO:117).
Additional examples of IL- 13 antibodies, that interfere with IL- 13 binding
to IL-
13R (e.g., an IL-13 receptor complex), or a subunit thereof, include "mAbl3.2"
and
modified, e.g., chimeric or humanized forms thereof. The amino acid and
nucleotide
sequences for the heavy chain variable region of mAb13.2 are set forth herein
as SEQ ID
NO:198 and SEQ ID NO:217, respectively. The amino acid and nucleotide
sequences for
the light chain variable region of mAb 13.2 are set forth herein as SEQ ID
NO:199 and
SEQ ID NO:218, respectively. An exemplary chimeric form (e.g., a form
comprising the
heavy and light chain variable region of mAb13.2) is referred to herein as
"chl3.2." The
amino acid and nucleotide sequences for the heavy chain variable region of
ch13.2 are set
forth herein as SEQ ID NO:208 and SEQ ID NO:204, respectively. The amino acid
and
nucleotide sequences for the light chain variable region of chl3.2 are set
forth herein as
SEQ ID NO:213 and SEQ ID NO:219, respectively. A humanized form of mAbl3.2,
which is referred to herein as "h13.2v1," has amino acid and nucleotide
sequences for the
heavy chain variable region set forth herein as SEQ ID NO:209 and SEQ ID
NO:205,
respectively. The amino acid and nucleotide sequences for the light chain
variable region
of h13.2v1 are set forth herein as SEQ ID NO:214 and SEQ ID NO:220,
respectively.
Another humanized form of mAbl3.2, which is referred to herein as "h13.2v2,"
has
amino acid and nucleotide sequences for the heavy chain variable region set
forth herein
as SEQ ID NO:210 and SEQ ID NO:206, respectively. The amino acid and
nucleotide
sequences for the light chain variable region of h13.2v2 are set forth herein
as SEQ ID
NO:212 and SEQ ID NO:221, respectively. Another humanized form of mAb13.2,
which
is referred to herein as "h13.2v3," has amino acid and nucleotide sequences
for the heavy
chain variable region set forth herein as SEQ ID NO:211 and SEQ ID NO:207,
respectively. The amino acid and nucleotide sequences for the light chain
variable region
of h13.2v3 are set forth herein as SEQ ID NO:35 and SEQ ID NO:223,
respectively.
In another embodiment, the anti-IL- 13 antibody molecule comprises at least
one,
two, three, or four antigen-binding regions, e.g., variable regions, having an
amino acid
sequence as set forth in SEQ ID NOs:198, 208, 209, 210, or 211 for VH, and/or
SEQ ID
-58-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
NOs:199, 213, 214, 212, or 215 for VL), or a sequence substantially identical
thereto
(e.g., a sequence at least about 85%, 90%, 95%, 99% or more identical thereto,
or which
differs by no more than 1, 2, 5, 10, or 15 amino acid residues from SEQ ID
NOs:199,
213, 214, 212, 198, 208, 209, 210, 215, or 211). In another embodiment, the
antibody
includes a VH and/or VL domain encoded by a nucleic acid having a nucleotide
sequence
as set forth in SEQ ID NOs222, 204, 205, 208, or 207 for VH, and/or SEQ ID
NOs:218,
219, 220, 221, or 223 for VL), or a sequence substantially identical thereto
(e.g., a
sequence at least about 85%, 90%, 95%, 99% or more identical thereto, or which
differs
by no more than 3, 6, 15, 30, or 45 nucleotides from SEQ ID NOs:218, 219, 220,
221,
lo 222, 204, 205, 206, 223, or 207). In yet another embodiment, the antibody
or fragment
thereof comprises at least one, two, or three CDRs from a heavy chain variable
region
having an amino acid sequence as set forth in SEQ ID NOs:202, 203, or 196 for
VH
CDRs 1-3, respectively, or a sequence substantially homologous thereto (e.g.,
a sequence
at least about 85%, 90%, 95%, 99% or more identical thereto, and/or having one
or more
substitutions, e.g., conserved substitutions). In yet another embodiment, the
antibody or
fragment thereof comprises at least one, two, or three CDRs from a light chain
variable
region having an amino acid sequence as set forth in SEQ ID NOs:197, 200, or
201 for
VL CDRs 1-3, respectively, or a sequence substantially homologous thereto
(e.g., a
sequence at least about 85%, 90%, 95%, 99% or more identical thereto, and/or
having
one or more substitutions, e.g., conserved substitutions). In yet another
embodiment, the
antibody or fragment thereof comprises at least one, two, three, four, five or
six CDRs
from heavy and light chain variable regions having an amino acid sequence as
set forth in
SEQ ID NOs:202, 203, 196 for VH CDRs 1-3, respectively; and SEQ ID NO:197,
200, or
201 for VL CDRs 1-3, respectively, or a sequence substantially homologous
thereto (e.g.,
a sequence at least about 85%, 90%, 95%, 99% or more identical thereto, and/or
having
one or more substitutions, e.g., conserved substitutions).
In one embodiment, the anti-IL-13 antibody molecule includes all six CDRs from
C65 or closely related CDRs, e.g., CDRs which are identical or which have at
least one
amino acid alteration, but not more than two, three or four alterations (e.g.,
substitutions,
deletions, or insertions).
-59-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
In still another embodiment, the IL-13 binding agent includes at least one,
two or
three CDR regions that have the same canonical structures and the
corresponding CDR
regions of C65, e.g., at least CDR1 and CDR2 of the heavy and/or light chain
variable
domains of C65.
In one embodiment, the heavy chain framework (e.g., FRl, FR2, FR3,
individually, or a sequence encompassing FRI, FR2, and FR3, but excluding
CDRs)
includes an amino acid sequence, which is at least 80%, 85%, 90%, 95%, 97%,
98%,
99% or higher identical to the heavy chain framework of one of the following
germline V
segment sequences: DP-71 or DP-67 or another V gene which is compatible with
the
canonical structure class of C65 (see, e.g., Chothia et al. (1992) J. Mol.
Biol. 227:799-
817; Tomlinson et al. (1992) J. Mol. Biol. 227:776-798).
In one embodiment, the light chain framework (e.g., FR1, FR2, FR3,
individually,
or a sequence encompassing FR1, FR2, and FR3, but excluding CDRs) includes an
amino
acid sequence, which is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or higher
identical to the light chain framework of DPK-1 or DPK-9 germline sequence or
another
V gene which is compatible with the canonical structure class of C65 (see,
e.g.,
Tomlinson et al. (1995) EMBO J. 14:4628).
In another embodiment, the light chain framework (e.g., FR1, FR2, FR3,
individually, or a sequence encompassing FR1, FR2, and FR3, but excluding
CDRs)
includes an amino acid sequence, which is at least 80%, 85%, 90%, 95%, 97%,
98%,
99% or higher identical to the light chain framework of a VK I subgroup
germline
sequence, e.g., a DPK-9 or DPK-1 sequence.
In another embodiment, the heavy chain framework (e.g., FR1, FR2, FR3,
individually, or a sequence encompassing FR1, FR2, and FR3, but excluding
CDRs)
includes an amino acid sequence, which is at least 80%, 85%, 90%, 95%, 97%,
98%,
99% or higher identical to the light chain framework of a VH IV subgroup
germline
sequence, e.g., a DP-71 or DP-67 sequence.
In one embodiment, the light or the heavy chain variable framework (e.g., the
region encompassing at least FRI, FR2, FR3, and optionally FR4) can be chosen
from:
(a) a light or heavy chain variable framework including at least 80%, 85%,
90%, 95%, or
100% of the amino acid residues from a human light or heavy chain variable
framework,
-60-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
e.g., a light or heavy chain variable framework residue from a human mature
antibody, a
human germline sequence, a human consensus sequence, or a human antibody
described
herein; (b) a light or heavy chain variable framework including from 20% to
80%, 40% to
60%, 60% to 90%, or 70% to 95% of the amino acid residues from a human light
or
heavy chain variable framework, e.g., a light or heavy chain variable
framework residue
from a human mature antibody, a human germline sequence, a human consensus
sequence; (c) a non-human framework (e.g., a rodent framework); or (d) a non-
human
framework that has been modified, e.g., to remove antigenic or cytotoxic
determinants,
e.g., deimmunized, or partially humanized. In one embodiment, the heavy chain
variable
domain sequence includes human residues or human consensus sequence residues
at one
or more of the following positions (preferably at least five, ten, twelve, or
all): (in the FR
of the variable domain of the light chain) 4L, 35L, 36L, 38L, 43L, 44L, 58L,
46L, 62L,
63L, 64L, 65L, 66L, 67L, 68L, 69L, 70L, 71L, 73L, 85L, 87L, 98L, and/or (in
the FR of
the variable domain of the heavy chain) 2H, 4H, 24H, 36H, 37H, 39H, 43H, 45H,
49H,
58H, 60H, 67H, 68H, 69H, 70H, 73H, 74H, 75H, 78H, 91H, 92H, 93H, and/or 103H
(according to the Kabat numbering).
In one embodiment, the anti-IL13 antibody molecules includes at least one non-
human CDR, e.g., a murine CDR, e.g., a CDR from e.g., mAb 13.2, MJ2-7, C65,
and/or
modified forms thereof (e.g., humanized or chimeric variansts thereof), and at
least one
framework which differs from a framework of e.g., mAb 13.2, MJ2-7, C65, and/or
modified forms thereof (e.g., humanized or chimeric variansts thereof) by at
least one
amino acid, e.g., at least 5, 8, 10, 12, 15, or 18 amino acids. For example,
the proteins
include one, two, three, four, five, or six such non-human CDRs and includes
at least one
amino acid difference in at least three of HC FR1, HC FR2, HC FR3, LC FR1, LC
FR2,
and LC FR3.
In one embodiment, the heavy or light chain variable domain sequence of the
anti-
IL-13 antibody molecule includes an amino acid sequence, which is at least
80%, 85%,
90%, 95%, 97%, 98%, 99% or higher identical to a variable domain sequence of
an
antibody described herein, e.g., mAb13.2, MJ2-7, C65, and/or modified forms
thereof
(e.g., humanized or chimeric variansts thereof); or which differs at at least
1 or 5
residues, but less than 40, 30, 20, or 10 residues, from a variable domain
sequence of an
-61-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
antibody described herein, e.g., mAb13.2, MJ2-7, C65, and/or modified forms
thereof
(e.g., humanized or chimeric variansts thereof). In one embodiment, the heavy
or light
chain variable domain sequence of the protein includes an amino acid sequence
encoded
by a nucleic acid sequence described herein or a nucleic acid that hybridizes
to a nucleic
acid sequence described herein or its complement, e.g., under low stringency,
medium
stringency, high stringency, or very high stringency conditions.
In one embodiment, one or both of the variable domain sequences include amino
acid positions in the framework region that are variously derived from both a
non-human
antibody (e.g., a murine antibody such as mAb 13.2) and a human antibody or
germline
sequence. For example, a variable domain sequence can include a number of
positions at
which the amino acid residue is identical to both the non-human antibody and
the human
antibody (or human germline sequence) because the two are identical at that
position. Of
the remaining framework positions where the non-human and human differ, at
least 50,
60, 70, 80, or 90% of the positions of the variable domain are preferably
identical to the
human antibody (or human germline sequence) rather than the non-human. For
example,
none, or at least one, two, three, or four of such remaining framework
position may be
identical to the non-human antibody rather than to the human. For example, in
HC FR1,
one or two such positions can be non-human; in HC FR2, one or two such
positions can
be non-human; in FR3, one, two, three, or four such positions can be non-
human; in LC
FR1, one, two, three, or four such positions can be non-human; in LC FR2, one
or two
such positions can be non-human; in LC FR3, one or two such positions can be
non-
human. The frameworks can include additional non-human positions.
In one embodiment, an antibody molecule has CDR sequences that differ only
insubstantially from those of MJ 2-7, C65, or 13.2. Insubstantial differences
include
minor amino acid changes, such as substitutions of 1 or 2 out of any of
typically 5-7
amino acids in the sequence of a CDR, e.g., a Chothia or Kabat CDR. Typically,
an
amino acid is substituted by a related amino acid having similar charge,
hydrophobic, or
stereochemical characteristics. Such substitutions are within the ordinary
skills of an
artisan. Unlike in CDRs, more substantial changes in structure framework
regions (FRs)
can be made without adversely affecting the binding properties of an antibody.
Changes
to FRs include, but are not limited to, humanizing a nonhuman-derived
framework or
-62-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
engineering certain framework residues that are important for antigen contact
or for
stabilizing the binding site, e.g., changing the class or subclass of the
constant region,
changing specific amino acid residues which might alter an effector function
such as Fc
receptor binding (Lund et al. (1991) J. Immunol. 147:2657-62; Morgan et al.
(1995)
Immunology 86:319-24), or changing the species from which the constant region
is
derived. Antibodies may have mutations in the CH2 region of the heavy chain
that
reduce or alter effector function, e.g., Fc receptor binding and complement
activation.
For example, antibodies may have mutations such as those described in U.S.
Patent Nos.
5,624,821 and 5,648,260. In the IgGI or IgG2 heavy chain, for example, such
mutations
may be made to resemble the amino acid sequence set forth in SEQ ID NO: 17.
Antibodies may also have mutations that stabilize the disulfide bond between
the two
heavy chains of an immunoglobulin, such as mutations in the hinge region of
IgG4, as
disclosed in the art (e.g., Angal et al. (1993) Mol. Immunol. 30:105-08).
The anti-IL-13 antibody molecule can be in the form of intact antibodies,
antigen-
binding fragments of antibodies, e.g., Fab, F(ab')2, Fd, dAb, and scFv
fragments, and
intact antibodies and fragments that have been mutated either in their
constant and/or
variable domain (e.g., mutations to produce chimeric, partially humanized, or
fully
humanized antibodies, as well as to produce antibodies with a desired trait,
e.g., enhanced
IL-13 binding and/or reduced FcR binding).
The anti-IL-13 antibody molecule can be derivatized or linked to another
functional molecule, e.g., another peptide or protein (e.g., an Fab fragment).
For
example, the binding agent can be functionally linked (e.g., by chemical
coupling,
genetic fusion, noncovalent association or otherwise) to one or more other
molecular
entities, such as another antibody molecule (e.g., to form a bispecific or a
multispecific
antibody molecule), toxins, radioisotopes, cytotoxic or cytostatic agents,
among others.
Additional IL-13/IL-13R or IL-4/IL-4R Binding Agents
Also provided are other binding agents, other than antibody molecules, that
bind
to IL-13 or IL-4 polypeptide or nucleic acid, or an IL-13R or IL-4R
polypeptide or
nucleic acid. In embodiments, the other binding agents described herein are
antagonists
and thus reduce, inhibit or otherwise diminish one or more biological
activities of IL- 13
- 63 -
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
and/or IL-4 (e.g., one or more biological activities of IL-13 and/or IL-4 as
described
herein).
Binding agents can be identified by a number of means, including modifying a
variable domain described herein or grafting one or more CDRs of a variable
domain
described herein onto another scaffold domain. Binding agents can also be
identified
from diverse libraries, e.g., by screening. One method for screening protein
libraries uses
phage display. Particular regions of a protein are varied and proteins that
interact with
IL-13 or IL-4, or its receptors, are identified, e.g., by retention on a solid
support or by
other physical association. For example, to identify particular binding agents
that bind to
the same epitope or an overlapping epitope as MJ2-7, C65 or mAb 13.2 on IL-13,
binding
agents can be eluted by adding MJ2-7, C65 or mAb 13.2 (or related antibody),
or binding
agents can be evaluated in competition experiments with MJ2-7, C65 or mAb 13.2
(or
related antibody). It is also possible to deplete the library of agents that
bind to other
epitopes by contacting the library to a complex that contains IL- 13 and MJ2-
7, C65 or
mAb 13.2 (or related antibody). The depleted library can then be contacted to
IL-13 to
obtain a binding agent that binds to IL- 13 but not to IL-13 when it is bound
by MJ 2-7,
C65 or mAbl3.2. It is also possible to use peptides from IL-13 that contain
the MJ 2-7,
C65 epitope, or mAb13.2 as a target.
Phage display is described, for example, in U.S. Patent No. 5,223,409; Smith
(1985) Science 228:1315-1317; WO 92/18619; WO 91/17271; WO 92/20791; WO
92/15679; WO 93/01288; WO 92/01047; WO 92/09690; WO 90/02809; WO 94/05781;
Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay et al. (1992) Hum Antibod
Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281; Griffiths et al.
(1993)
EMBO J 12:725-734; Hawkins et al. (1992) JMoI Bio1226:889-896; Clackson et al.
(1991) Nature 352:624-628; Gram et al. (1992) PNAS 89:3576-3580; Garrard et
al.
(1991) Bio/Technology 9:1373-1377; Rebar et al. (1996) Methods Enzymol.
267:129-49;
and Barbas et al. (1991) PNAS 88:7978-7982. Yeast surface display is
described, e.g., in
Boder and Wittrup (1997) Nat. Biotechnol. 15:553-557. Another form of display
is
ribosome display. See, e.g., Mattheakis et al. (1994) Proc. Natl. Acad. Sci.
USA 91:9022
and Hanes et al. (2000) Nat Biotechnol. 18:1287-92; Hanes et al. (2000)
Methods
Enzymol. 328:404-30. and Schaffitzel et al. (1999) Jlmmunol Methods. 231(1-
2):119-35.
-64-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
Binding agents that bind to IL-13 or IL-4, or its receptors, can have
structural
features of one scaffold proteins, e.g., a folded domain. An exemplary
scaffold domain,
based on an antibody, is a "minibody" scaffold has been designed by deleting
three beta
strands from a heavy chain variable domain of a monoclonal antibody
(Tramontano et al.,
1994, J. Mol. Recognit. 7:9; and Martin et al., 1994, EMBO J. 13:5303-5309).
This
domain includes 61 residues and can be used to present two hypervariable
loops, e.g., one
or more hypervariable loops of a variable domain described herein or a variant
described
herein. In another approach, the binding agent includes a scaffold domain that
is a V-like
domain (Coia et al. WO 99/45110). V-like domains refer to a domain that has
similar
structural features to the variable heavy (VH) or variable light (VL) domains
of
antibodies. Another scaffold domain is derived from tendamistatin, a 74
residue, six-
strand beta sheet sandwich held together by two disulfide bonds (McConnell and
Hoess,
1995, J Mol. Biol. 250:460). This parent protein includes three loops. The
loops can be
modified (e.g., using CDRs or hypervariable loops described herein) or varied,
e.g., to
select domains that bind to IL-13 or IL-4, or its receptors. WO 00/60070
describes a(3-
sandwich structure derived from the naturally occurring extracellular domain
of CTLA-4
that can be used as a scaffold domain.
Still another scaffold domain for an IL-13/13R or IL-4/IL-4R binding agent is
a
domain based on the fibronectin type III domain or related fibronectin-like
proteins. The
overall fold of the fibronectin type III (Fn3) domain is closely related to
that of the
smallest functional antibody fragment, the variable domain of the antibody
heavy chain.
Fn3 is a(3-sandwich similar to that of the antibody VH domain, except that Fn3
has seven
(3-strands instead of nine. There are three loops at the end of Fn3; the
positions of BC,
DE and FG loops approximately correspond to those of CDR1, 2 and 3 of the VH
domain
of an antibody. Fn3 is advantageous because it does not have disulfide bonds.
Therefore,
Fn3 is stable under reducing conditions, unlike antibodies and their fragments
(see WO
98/56915; WO 01/64942; WO 00/34784). An Fn3 domain can be modified (e.g.,
using
CDRs or hypervariable loops described herein) or varied, e.g., to select
domains that bind
to IL-13 or IL-4, or its receptors.
Still other exemplary scaffold domains include: T-cell receptors; MHC
proteins;
extracellular domains (e.g., fibronectin Type III repeats, EGF repeats);
protease inhibitors
- 65 -
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
(e.g., Kunitz domains, ecotin, BPTI, and so forth); TPR repeats; trifoil
structures; zinc
finger domains; DNA-binding proteins; particularly monomeric DNA binding
proteins;
RNA binding proteins; enzymes, e.g., proteases (particularly inactivated
proteases),
RNase; chaperones, e.g., thioredoxin, and heat shock proteins; and
intracellular signaling
domains (such as SH2 and SH3 domains). US 20040009530 describes examples of
some
alternative scaffolds.
Examples of small scaffold domains include: Kunitz domains (58 amino acids, 3
disulfide bonds), Cucurbida maxima trypsin inhibitor domains (31 amino acids,
3
disulfide bonds), domains related to guanylin (14 amino acids, 2 disulfide
bonds),
domains related to heat-stable enterotoxin IA from gram negative bacteria (18
amino
acids, 3 disulfide bonds), EGF domains (50 amino acids, 3 disulfide bonds),
kringle
domains (60 amino acids, 3 disulfide bonds), fungal carbohydrate-binding
domains (35
amino acids, 2 disulfide bonds), endothelin domains (18 amino acids, 2
disulfide bonds),
and Streptococcal G IgG-binding domain (35 amino acids, no disulfide bonds).
Examples of small intracellular scaffold domains include SH2, SH3, and EVH
domains.
Generally, any modular domain, intracellular or extracellular, can be used.
Exemplary criteria for evaluating a scaffold domain can include: (1) amino
acid
sequence, (2) sequences of several homologous domains, (3) 3-dimensional
structure,
and/or (4) stability data over a range of pH, temperature, salinity, organic
solvent, oxidant
concentration. In one embodiment, the scaffold domain is a small, stable
protein
domains, e.g., a protein of less than 100, 70, 50, 40 or 30 amino acids. The
domain may
include one or more disulfide bonds or may chelate a metal, e.g., zinc.
Still other binding agents are based on peptides, e.g., proteins with an amino
acid
sequence that are less than 30, 25, 24, 20, 18, 15, or 12 amino acids.
Peptides can be
incorporated in a larger protein, but typically a region that can
independently bind to
IL-13, e.g., to an epitope described herein. Peptides can be identified by
phage display.
See, e.g., US 20040071705.
A binding agent may include non-peptide linkages and other chemical
modification. For example, part or all of the binding agent may be synthesized
as a
peptidomimetic, e.g., a peptoid (see, e.g., Simon et al. (1992) Proc. Natl.
Acad. Sci. USA
89:9367-71 and Horwell (1995) Trends Biotechnol. 13:132-4). A binding agent
may
-66-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
include one or more (e.g., all) non-hydrolyzable bonds. Many non-hydrolyzable
peptide
bonds are known in the art, along with procedures for synthesis of peptides
containing
such bonds. Exemplary non-hydrolyzable bonds include --[CH2NH]-- reduced amide
peptide bonds, --[COCH2]-- ketomethylene peptide bonds, --[CH(CN)NH]--
(cyanomethylene)amino peptide bonds, --[CH2CH(OH)]-- hydroxyethylene peptide
bonds, --[CH2O]--peptide bonds, and --[CH2S]-- thiomethylene peptide bonds
(see e.g.,
U.S. Pat. No. 6,172,043).
In another embodiment, the IL-13 or IL-4 antagonist is derived from a
lipocalin,
e.g., a human lipocalin scaffold.
Soluble Receptors
A soluble form of an IL-13 or an IL-4 receptor or a modified antagonistic
cytokine can be used alone or functionally linked (e.g., by chemical coupling,
genetic or
polypeptide fusion, non-covalent association or otherwise) to a second moiety,
e.g., an
immunoglobulin Fc domain, serum albumin, pegylation, a GST, Lex-A or an MBP
polypeptide sequence. As used herein, a "fusion protein" refers to a protein
containing
two or more operably associated, e.g., linked, moieties, e.g., protein
moieties. Typically,
the moieties are covalently associated. The moieties can be directly
associate, or
connected via a spacer or linker.
The fusion proteins may additionally include a linker sequence joining the
first
moiety to the second moiety. For example, the fusion protein can include a
peptide
linker, e.g., a peptide linker of about 4 to 20, more preferably, 5 to 10,
amino acids in
length; the peptide linker is 8 amino acids in length. Each of the amino acids
in the
peptide linker is selected from the group consisting of Gly, Ser, Asn, Thr and
Ala; the
peptide linker includes a Gly-Ser element. In other embodiments, the fusion
protein
includes a peptide linker and the peptide linker includes a sequence having
the formula
(Ser-Gly-Gly-Gly-Gly)y wherein y is 1, 2, 3, 4, 5, 6, 7, or 8.
In other embodiments, additional amino acid sequences can be added to the N-
or
C-terminus of the fusion protein to facilitate expression, detection and/or
isolation or
purification. For example, the receptor fusion protein may be linked to one or
more
additional moieties, e.g., GST, His6 tag, FLAG tag. For example, the fusion
protein may
-67-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
additionally be linked to a GST fusion protein in which the fusion protein
sequences are
fused to the C-terminus of the GST (i.e., glutathione S-transferase)
sequences. Such
fusion proteins can facilitate the purification of the receptor fusion
protein.
In another embodiment, the fusion protein is includes a heterologous signal
sequence
(i.e., a polypeptide sequence that is not present in a polypeptide encoded by
a receptor
nucleic acid) at its N-terminus. For example, the native receptor signal
sequence can be
removed and replaced with a signal sequence from another protein. In certain
host cells
(e.g., mammalian host cells), expression and/or secretion of receptor can be
increased
through use of a heterologous signal sequence.
A chimeric or fusion protein of the invention can be produced by standard
recombinant DNA techniques. For example, DNA fragments coding for the
different
polypeptide sequences are ligated together in-frame in accordance with
conventional
techniques, e.g., by employing blunt-ended or stagger-ended termini for
ligation,
restriction enzyme digestion to provide for appropriate termini, filling-in of
cohesive ends
as appropriate, alkaline phosphatase treatment to avoid undesirable joining,
and
enzymatic ligation. In another embodiment, the fusion gene can be synthesized
by
conventional techniques including automated DNA synthesizers. Alternatively,
PCR
amplification of gene fragments can be carried out using anchor primers that
give rise to
complementary overhangs between two consecutive gene fragments that can
subsequently be annealed and reamplified to generate a chimeric gene sequence
(see, for
example, Ausubel et al. (eds.) Current Protocols in Molecular Biology, John
Wiley &
Sons, 1992). Moreover, many expression vectors are commercially available that
encode
a fusion moiety (e.g., an Fc region of an immunoglobulin heavy chain). A
receptor
encoding nucleic acid can be cloned into such an expression vector such that
the fusion
moiety is linked in-frame to the immunoglobulin protein.
In some embodiments, fusion polypeptides exist as oligomers, such as dimers or
trimers.
In other embodiments, the receptor polypeptide moiety is provided as a variant
receptor polypeptide having a mutation in the naturally-occurring receptor
sequence (wild
type) that results in higher affinity (relative to the non-mutated sequence)
binding of the
receptor polypeptide to cytokine.
-68-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
In other embodiments, additional amino acid sequences can be added to the N-
or
C-terminus of the fusion protein to facilitate expression, steric flexibility,
detection
and/or isolation or purification. The second polypeptide is preferably
soluble. In some
embodiments, the second polypeptide enhances the half-life, (e.g., the serum
half-life) of
the linked polypeptide. In some embodiments, the second polypeptide includes a
sequence that facilitates association of the fusion polypeptide with a second
BMP-10
receptor polypeptide. In embodiments, the second polypeptide includes at least
a region
of an immunoglobulin polypeptide. Immunoglobulin fusion polypeptide are known
in the
art and are described in e.g., U.S. Pat. Nos. 5,516,964; 5,225,538; 5,428,130;
5,514,582;
5,714,147; and 5,455,165. For example, a soluble form of a BMP-10 receptor or
a BMP-
10 antagonistic propeptide can be fused to a heavy chain constant region of
the various
isotypes, including: IgG1, IgG2, IgG3, IgG4, IgM, IgAl, IgA2, IgD, and IgE).
Typically,
the fusion protein can include the extracellular domain of a human BMP- 10
receptor, or a
BMP-10 propeptide (or a sequence homologous thereto), and, e.g., fused to, a
human
immunoglobulin Fc chain, e.g., human IgG (e.g., human IgGl or human IgG2, or a
mutated form thereof).
The Fc sequence can be mutated at one or more amino acids to reduce effector
cell function, Fc receptor binding and/or complement activity. Methods for
altering an
antibody constant region are known in the art. Antibodies with altered
function, e.g.
altered affinity for an effector ligand, such as FcR on a cell, or the C 1
component of
complement can be produced by replacing at least one amino acid residue in the
constant
portion of the antibody with a different residue (see e.g., EP 388,151 Al,
U.S. Pat. No.
5,624,821 and U.S. Pat. No. 5,648,260). Similar type of alterations could be
described
which if applied to the murine, or other species immunoglobulin would reduce
or
eliminate these functions. For example, it is possible to alter the affinity
of an Fc region
of an antibody (e.g., an IgG, such as a human IgG) for an FcR (e.g., Fc gamma
R1), or
for C 1 q binding by replacing the specified residue(s) with a residue(s)
having an
appropriate functionality on its side chain, or by introducing a charged
functional group,
such as glutamate or aspartate, or perhaps an aromatic non-polar residue such
as
phenylalanine, tyrosine, tryptophan or alanine (see e.g., U.S. Pat. No.
5,624,821).
In embodiments, the second polypeptide has less effector function that the
-69-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
effector function of a Fc region of a wild-type immunoglobulin heavy chain. Fc
effector
function includes for example, Fc receptor binding, complement fixation and T
cell
depleting activity (see for example, U.S. Pat. No. 6,136,310). Methods for
assaying T
cell depleting activity, Fc effector function, and antibody stability are
known in the art.
In one embodiment, the second polypeptide has low or no detectable affinity
for the Fc
receptor. In an alternative embodiment, the second polypeptide has low or no
detectable
affinity for complement protein C 1 q.
It will be understood that the antibody molecules and soluble receptor or
fusion
proteins described herein can be functionally linked (e.g., by chemical
coupling, genetic
fusion, non-covalent association or otherwise) to one or more other molecular
entities,
such as an antibody (e.g., a bispecific or a multispecific antibody), toxins,
radioisotopes,
cytotoxic or cytostatic agents, among others.
Nucleic Acid Antagonists
In yet another embodiment, the antagonist inhibits the expression of nucleic
acid
encoding an IL-13 or IL-13R, or an IL-4 or IL-4R. Examples of such antagonists
include
nucleic acid molecules, for example, antisense molecules, ribozymes, RNAi,
triple helix
molecules that hybridize to a nucleic acid encoding an IL-13 or IL-13R, or an
IL-4 or IL-
4R, or a transcription regulatory region, and blocks or reduces mRNA
expression of an
IL-13 or IL-13R, or an IL-4 or IL-4R.
In embodiments, nucleic acid antagonists are used to decrease expression of an
endogenous gene encoding an IL-13 or IL-13R, or an IL-4 or IL-4R. In one
embodiment,
the nucleic acid antagonist is an siRNA that targets mRNA encoding an IL-13 or
IL-13R,
or an IL-4 or IL-4R. Other types of antagonistic nucleic acids can also be
used, e.g., a
dsRNA, a ribozyme, a triple-helix former, or an antisense nucleic acid.
Accordingly,
isolated nucleic acid molecules that are nucleic acid inhibitors, e.g.,
antisense, RNAi, to a
an IL-13 or IL-13R, or an IL-4 or IL-4R-encoding nucleic acid molecule are
provided.
An "antisense" nucleic acid can include a nucleotide sequence which is
complementary to a "sense" nucleic acid encoding a protein, e.g.,
complementary to the
coding strand of a double-stranded cDNA molecule or complementary to an mRNA
sequence. The antisense nucleic acid can be complementary to an entire an IL-
13 or IL-
-70-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
13R, or an IL-4 or IL-4R coding strand, or to only a portion thereof. In
another
embodiment, the antisense nucleic acid molecule is antisense to a "noncoding
region" of
the coding strand of a nucleotide sequence encoding an IL-13 or IL-13R, or an
IL-4 or
IL-4R (e.g., the 5' and 3' untranslated regions). Anti-sense agents can
include, for
example, from about 8 to about 80 nucleobases (i.e. from about 8 to about 80
nucleotides), e.g., about 8 to about 50 nucleobases, or about 12 to about 30
nucleobases.
Anti-sense compounds include ribozymes, external guide sequence (EGS)
oligonucleotides (oligozymes), and other short catalytic RNAs or catalytic
oligonucleotides which hybridize to the target nucleic acid and modulate its
expression.
Anti-sense compounds can include a stretch of at least eight consecutive
nucleobases that
are complementary to a sequence in the target gene. An oligonucleotide need
not be
100% complementary to its target nucleic acid sequence to be specifically
hybridizable.
An oligonucleotide is specifically hybridizable when binding of the
oligonucleotide to the
target interferes with the normal function of the target molecule to cause a
loss of utility,
and there is a sufficient degree of complementarity to avoid non-specific
binding of the
oligonucleotide to non-target sequences under conditions in which specific
binding is
desired, i.e., under physiological conditions in the case of in vivo assays or
therapeutic
treatment or, in the case of in vitro assays, under conditions in which the
assays are
conducted.
Hybridization of antisense oligonucleotides with mRNA can interfere with one
or
more of the normal functions of mRNA. The functions of mRNA to be interfered
with
include all key functions such as, for example, translocation of the RNA to
the site of
protein translation, translation of protein from the RNA, splicing of the RNA
to yield one
or more mRNA species, and catalytic activity which may be engaged in by the
RNA.
Binding of specific protein(s) to the RNA may also be interfered with by
antisense
oligonucleotide hybridization to the RNA.
Exemplary antisense compounds include DNA or RNA sequences that
specifically hybridize to the target nucleic acid, e.g., the mRNA encoding BMP-
10/BMP-
10 receptor. The complementary region can extend for between about 8 to about
80
nucleobases. The compounds can include one or more modified nucleobases.
Modified
nucleobases may include, e.g., 5-substituted pyrimidines such as 5-iodouracil,
5-
-71-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
iodocytosine, and C5-propynyl pyrimidines such as C5-propynylcytosine and C5-
propynyluracil. Other suitable modified nucleobases include N4 --(C1 -C12)
alkylaminocytosines and N4,N4 --(CI -C12) dialkylaminocytosines. Modified
nucleobases
may also include 7-substituted-8-aza-7-deazapurines and 7-substituted-7-
deazapurines
such as, for example, 7-iodo-7-deazapurines, 7-cyano-7-deazapurines, 7-
aminocarbonyl-
7-deazapurines. Examples of these include 6-amino-7-iodo-7-deazapurines, 6-
amino-7-
cyano-7-deazapurines, 6-amino-7-aminocarbonyl-7-deazapurines, 2-amino-6-
hydroxy-7-
iodo-7-deazapurines, 2-amino-6-hydroxy-7-cyano-7-deazapurines, and 2-amino-6-
hydroxy-7-aminocarbonyl-7-deazapurines. Furthermore, N6 --(C1 -C12)
alkylaminopurines and N6,N6 --(CI -C12) dialkylaminopurines, including N6 -
methylaminoadenine and N6,N6 -dimethylaminoadenine, are also suitable modified
nucleobases. Similarly, other 6-substituted purines including, for example, 6-
thioguanine
may constitute appropriate modified nucleobases. Other suitable nucleobases
include 2-
thiouracil, 8-bromoadenine, 8-bromoguanine, 2-fluoroadenine, and 2-
fluoroguanine.
Derivatives of any of the aforementioned modified nucleobases are also
appropriate.
Substituents of any of the preceding compounds may include C, -C30 alkyl, C2 -
C30
alkenyl, C2 -C30 alkynyl, aryl, aralkyl, heteroaryl, halo, amino, amido,
nitro, thio,
sulfonyl, carboxyl, alkoxy, alkylcarbonyl, alkoxycarbonyl, and the like.
Descriptions of
other types of nucleic acid agents are also available. See, e.g., U.S. Patent
Nos.
4,987,071;. 5,116,742; and 5,093,246; Woolf et al. (1992) Proc Natl Acad Sci
USA;
Antisense RNA and DNA, D.A. Melton, Ed., Cold Spring Harbor Laboratory, Cold
Spring
Harbor, N.Y. (1988); 89:7305-9; Haselhoff and Gerlach (1988) Nature 334:585-
59;
Helene, C. (1991) Anticancer Drug Des. 6:569-84; Helene (1992) Ann. N. Y.
Acad. Sci.
660:27-36; and Maher (1992) Bioassays 14:807-15.
The antisense nucleic acid molecules of the invention are typically
administered to
a subject (e.g., by direct injection at a tissue site), or generated in situ
such that they
hybridize with or bind to cellular mRNA and/or genomic DNA encoding a BMP-
10BMP-10 receptor protein to thereby inhibit expression of the protein, e.g.,
by
inhibiting transcription and/or translation. Alternatively, antisense nucleic
acid
molecules can be modified to target selected cells and then administered
systemically.
For systemic administration, antisense molecules can be modified such that
they
-72-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
specifically bind to receptors or antigens expressed on a selected cell
surface, e.g., by
linking the antisense nucleic acid molecules to peptides or antibodies which
bind to cell
surface receptors or antigens. The antisense nucleic acid molecules can also
be delivered
to cells using the vectors described herein. To achieve sufficient
intracellular
concentrations of the antisense molecules, vector constructs in which the
antisense
nucleic acid molecule is placed under the control of a strong pol II or pol
III promoter are
preferred.
In yet another embodiment, the antisense nucleic acid molecule of the
invention is
an a-anomeric nucleic acid molecule. An a-anomeric nucleic acid molecule forms
specific double-stranded hybrids with complementary RNA in which, contrary to
the
usual (3-units, the strands run parallel to each other (Gaultier et al. (1987)
Nucleic Acids.
Res. 15:6625-6641). The antisense nucleic acid molecule can also comprise a 2'-
o-
methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res. 15:6131-6148) or
a
chimeric RNA-DNA analogue (Inoue et al. (1987) FEBSLett. 215:327-330).
siRNAs are small double stranded RNAs (dsRNAs) that optionally include
overhangs. For example, the duplex region of an siRNA is about 18 to 25
nucleotides in
length, e.g., about 19, 20, 21, 22, 23, or 24 nucleotides in length.
Typically, the siRNA
sequences are exactly complementary to the target mRNA. dsRNAs and siRNAs in
particular can be used to silence gene expression in mammalian cells (e.g.,
human cells).
siRNAs also include short hairpin RNAs (shRNAs) with 29-base-pair stems and 2-
nucleotide 3' overhangs. See, e.g., Clemens et al. (2000) Proc. Natl. Acad.
Sci. USA
97:6499-6503; Billy et al. (2001) Proc. Natl. Sci. USA 98:14428-14433;
Elbashir et al.
(2001) Nature. 411:494-8; Yang et al. (2002) Proc. Natl. Acad. Sci. USA
99:9942-9947;
Siolas et al. (2005), Nat. Biotechnol. 23(2):227-31; 20040086884; U.S.
20030166282;
20030143204; 20040038278; and 20030224432.
In still another embodiment, an antisense nucleic acid of the invention is a
ribozyme. A ribozyme having specificity for an IL-13 or IL-13R, or an IL-4 or
IL-4R-
encoding nucleic acid can include one or more sequences complementary to the
nucleotide sequence of an IL-13 or IL-13R, or an IL-4 or IL-4R cDNA disclosed
herein,
and a sequence having known catalytic sequence responsible for mRNA cleavage
(see
U.S. Pat. No. 5,093,246 or Haselhoff and Gerlach (1988) Nature 334:585-591).
For
-73-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
example, a derivative of a Tetrahymena L- 19 IVS RNA can be constructed in
which the
nucleotide sequence of the active site is complementary to the nucleotide
sequence to be
cleaved in a BMP-10BMP-10 receptor-encoding mRNA. See, e.g., Cech et al. U.S.
Patent No. 4,987,071; and Cech et al. U.S. Patent No. 5,116,742.
Alternatively, BMP-
10BMP-10 receptor mRNA can be used to select a catalytic RNA having a specific
ribonuclease activity from a pool of RNA molecules. See, e.g., Bartel, D. and
Szostak,
J.W. (1993) Science 261:1411-1418.
an IL-13 or IL-13R, or an IL-4 or IL-4R gene expression can be inhibited by
targeting nucleotide sequences complementary to the regulatory region of the
an IL-13 or
IL-13R, or an IL-4 or IL-4R (e.g., the an IL-13 or IL-13R, or an IL-4 or IL-4R
promoter
and/or enhancers) to form triple helical structures that prevent transcription
of an IL- 13 or
IL-13R, or an IL-4 or IL-4R gene in target cells. See generally, Helene, C.
(1991)
Anticancer Drug Des. 6:569-84; Helene, C. i (1992) Ann. N. Y. Acad. Sci.
660:27-36; and
Maher, L.J. (1992) Bioassays 14:807-15. The potential sequences that can be
targeted for
triple helix formation can be increased by creating a so-called "switchback"
nucleic acid
molecule. Switchback molecules are synthesized in an alternating 5'-3', 3'-5'
manner,
such that they base pair with first one strand of a duplex and then the other,
eliminating
the necessity for a sizeable stretch of either purines or pyrimidines to be
present on one
strand of a duplex.
The invention also provides detectably labeled oligonucleotide primer and
probe
molecules. Typically, such labels are chemiluminescent, fluorescent,
radioactive, or
colorimetric.
An IL- 13 or IL-13R, or an IL-4 or IL-4R nucleic acid molecule can be modified
at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the
stability,
hybridization, or solubility of the molecule. For non-limiting examples of
synthetic
oligonucleotides with modifications see Toulme (2001) Nature Biotech. 19:17
and Faria
et al. (2001) Nature Biotech, 19:40-44. Such phosphoramidite oligonucleotides
can be
effective antisense agents.
For example, the deoxyribose phosphate backbone of the nucleic acid molecules
can be modified to generate peptide nucleic acids (see Hyrup B. et al. (1996)
Bioorganic
& Medicinal Chemistry 4: 5-23). As used herein, the terms "peptide nucleic
acid" or
-74-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
"PNA" refers to a nucleic acid mimic, e.g., a DNA mimic, in which the
deoxyribose
phosphate backbone is replaced by a pseudopeptide backbone and only the four
natural
nucleobases are retained. The neutral backbone of a PNA can allow for specific
hybridization to DNA and RNA under conditions of low ionic strength. The
synthesis of
PNA oligomers can be performed using standard solid phase peptide synthesis
protocols
as described in Hyrup B. et al. (1996) supra and Perry-O'Keefe et al. Proc.
Natl. Acad.
Sci. 93: 14670-675.
PNAs can be used in therapeutic and diagnostic applications. For example, PNAs
can be used as antisense or antigene agents for sequence-specific modulation
of gene
expression by, for example, inducing transcription or translation arrest or
inhibiting
replication. PNAs of nucleic acid molecules can also be used in the analysis
of single
base pair mutations in a gene, (e.g., by PNA-directed PCR clamping); as
'artificial
restriction enzymes' when used in combination with other enzymes, (e.g., S 1
nucleases
(Hyrup B. et al. (1996) supra)); or as probes or primers for DNA sequencing or
hybridization (Hyrup B. et al. (1996) supra; Perry-O'Keefe supra).
In other embodiments, the oligonucleotide may include other appended groups
such as peptides (e.g., for targeting host cell receptors in vivo), or agents
facilitating
transport across the cell membrane (see, e.g., Letsinger et al. (1989) Proc.
Natl. Acad.
Sci. USA 86:6553-6556; Lemaitre et al. (1987) Proc. Natl. Acad. Sci. USA
84:648-652;
W088/09810) or the blood-brain barrier (see, e.g., WO 89/10134). In addition,
oligonucleotides can be modified with hybridization-triggered cleavage agents
(see, e.g.,
Krol et al. (1988) Bio-Techniques 6:958-976) or intercalating agents (See,
e.g., Zon
(1988) Pharm. Res. 5:539-549). To this end, the oligonucleotide may be
conjugated to
another molecule, (e.g., a peptide, hybridization triggered cross-linking
agent, transport
agent, or hybridization-triggered cleavage agent).
Binding Agent Production
Some antibody molecules, e.g., Fabs, or binding agents can be produced in
bacterial cells, e.g., E. coli cells. For example, if the Fab is encoded by
sequences in a
phage display vector that includes a suppressible stop codon between the
display entity
and a bacteriophage protein (or fragment thereof), the vector nucleic acid can
be
-75-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
transferred into a bacterial cell that cannot suppress a stop codon. In this
case, the Fab is
not fused to the gene III protein and is secreted into the periplasm and/or
media.
Antibody molecules can also be produced in eukaryotic cells. In one
embodiment, the antibodies (e.g., scFv's) are expressed in a yeast cell such
as Pichia
(see, e.g., Powers et al. (2001) Jlmmunol Methods. 251:123-35), Hanseula, or
Saccharomyces.
In one embodiment, antibody molecules are produced in mammalian cells.
Typical mammalian host cells for expressing the clone antibodies or antigen-
binding
fragments thereof include Chinese Hamster Ovary (CHO cells) (including dhfr
CHO
cells, described in Urlaub and Chasin (1980) Proc. Natl. Acad. Sci. USA
77:4216-4220,
used with a DHFR selectable marker, e.g., as described in Kaufman and Sharp
(1982)
Mol. Biol. 159:601-621), lymphocytic cell lines, e.g., NSO myeloma cells and
SP2 cells,
COS cells, and a cell from a transgenic animal, e.g., a transgenic mammal. For
example,
the cell is a mammary epithelial cell.
In addition to the nucleic acid sequences encoding the antibody molecule, the
recombinant expression vectors may carry additional sequences, such as
sequences that
regulate replication of the vector in host cells (e.g., origins of
replication) and selectable
marker genes. The selectable marker gene facilitates selection of host cells
into which
the vector has been introduced (see e.g., U.S. Patents Nos. 4,399,216,
4,634,665 and
5,179,017). For example, typically the selectable marker gene confers
resistance to
drugs, such as G418, hygromycin, or methotrexate, on a host cell into which
the vector
has been introduced.
In an exemplary system for recombinant expression of an antibody molecule, a
recombinant expression vector encoding both the antibody heavy chain and the
antibody
light chain is introduced into dhfr CHO cells by calcium phosphate-mediated
transfection. Within the recombinant expression vector, the antibody heavy and
light
chain genes are each operatively linked to enhancer/promoter regulatory
elements (e.g.,
derived from SV40, CMV, adenovirus and the like, such as a CMV enhancer/AdMLP
promoter regulatory element or an SV40 enhancer/AdMLP promoter regulatory
element)
to drive high levels of transcription of the genes. The recombinant expression
vector also
carries a DHFR gene, which allows for selection of CHO cells that have been
transfected
-76-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
with the vector using methotrexate selection/amplification. The selected
transformant
host cells can be cultured to allow for expression of the antibody heavy and
light chains
and intact antibody is recovered from the culture medium. Standard molecular
biology
techniques can be used to prepare the recombinant expression vector, transfect
the host
cells, select for transformants, culture the host cells and recover the
antibody molecule
from the culture medium. For example, some antibody molecules can be isolated
by
affinity chromatography with a Protein A or Protein G coupled matrix.
For antibody molecules that include an Fc domain, the antibody production
system preferably synthesizes antibodies in which the Fc region is
glycosylated. For
example, the Fc domain of IgG molecules is glycosylated at asparagine 297 in
the CH2
domain. This asparagine is the site for modification with biantennary-type
oligosaccharides. It has been demonstrated that this glycosylation is required
for effector
functions mediated by Fcy receptors and complement Clq (Burton and Woof (1992)
Adv.
Immunol. 51:1-84; Jefferis et al. (1998) Immunol. Rev. 163:59-76). In one
embodiment,
the Fc domain is produced in a mammalian expression system that appropriately
glycosylates the residue corresponding to asparagine 297. The Fc domain can
also
include other eukaryotic post-translational modifications.
Antibody molecules can also be produced by a transgenic animal. For example,
U.S. Patent No. 5,849,992 describes a method of expressing an antibody in the
mammary
gland of a transgenic mammal. A transgene is constructed that includes a milk-
specific
promoter and nucleic acids encoding the antibody molecule and a signal
sequence for
secretion. The milk produced by females of such transgenic mammals includes,
secreted-
therein, the antibody of interest. The antibody molecule can be purified from
the milk, or
for some applications, used directly.
Characterization of Binding Agents
The binding properties of a binding agent may be measured by any method, e.g.,
one of the following methods: BIACORETM analysis, Enzyme Linked Immunosorbent
Assay (ELISA), x-ray crystallography, sequence analysis and scanning
mutagenesis. The
ability of a protein to neutralize and/or inhibit one or more IL-13-associated
activities
may be measured by the following methods: assays for measuring the
proliferation of an
-77-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
IL-13 dependent cell line, e.g. TFI; assays for measuring the expression of IL-
13-
mediated polypeptides, e.g., flow cytometric analysis of the expression of
CD23; assays
evaluating the activity of downstream signaling molecules, e.g., STAT6; assays
evaluating production of tenascin; assays testing the efficiency of an
antibody described
herein to prevent asthma in a relevant animal model, e.g., the cynomolgus
monkey, and
other assays. An IL-13 binding agent, particularly an IL-13 antibody molecule,
can have
a statistically significant effect in one or more of these assays. Exemplary
assays for
binding properties include the following.
The binding interaction of a IL-13 or IL-4 binding agent and a target (e.g.,
IL-13
or IL-4) can be analyzed using surface plasmon resonance (SPR). SPR or
Biomolecular
Interaction Analysis (BIA) detects biospecific interactions in real time,
without labeling
any of the interactants. Changes in the mass at the binding surface
(indicative of a
binding event) of the BIA chip result in alterations of the refractive index
of light near the
surface. The changes in the refractivity generate a detectable signal, which
are measured
as an indication of real-time reactions between biological molecules. Methods
for using
SPR are described, for example, in U.S. Patent No. 5,641,640; Raether (1988)
Surface
Plasmons Springer Verlag; Sjolander and Urbaniczky (1991) Anal. Chem. 63:2338-
2345;
Szabo et al. (1995) Curr. Opin. Struct. Biol. 5:699-705 and on-line resources
provide by
BlAcore International AB (Uppsala, Sweden).
Information from SPR can be used to provide an accurate and quantitative
measure of the equilibrium dissociation constant (Kd), and kinetic parameters,
including
Kon and Koff, for the binding of a molecule to a target. Such data can be used
to compare
different molecules. Information from SPR can also be used to develop
structure-activity
relationships (SAR). For example, the kinetic and equilibrium binding
parameters of
different antibody molecule can be evaluated. Variant amino acids at given
positions can
be identified that correlate with particular binding parameters, e.g., high
affinity and slow
Koff. This information can be combined with structural modeling (e.g., using
homology
modeling, energy minimization, or structure determination by x-ray
crystallography or
NMR). As a result, an understanding of the physical interaction between the
protein and
its target can be formulated and used to guide other design processes.
-78-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
Respiratory Disorders
An IL- 13 and/or IL-4 antagonist can be used to treat or prevent respiratory
disorders including, but are not limited to asthma (e.g., allergic and
nonallergic asthma
(e.g., due to infection, e.g., with respiratory syncytial virus (RSV), e.g.,
in younger
children)); bronchitis (e.g., chronic bronchitis); chronic obstructive
pulmonary disease
(COPD) (e.g., emphysema (e.g., cigarette-induced emphysema); conditions
involving
airway inflammation, eosinophilia, fibrosis and excess mucus production, e.g.,
cystic
fibrosis, pulmonary fibrosis, and allergic rhinitis. For example, an IL-13
binding agent
(e.g., an anti-IL-13 antibody molecule) can be administered in an amount
effective to
treat or prevent the disorder or to ameliorate at least one symptom of the
disorder.
Asthma can be triggered by myriad conditions, e.g., inhalation of an allergen,
presence of an upper-respiratory or ear infection, etc. (Opperwall (2003)
Nurs. Clin.
North Am. 38:697-711). Allergic asthma is characterized by airway
hyperresponsiveness
(AHR) to a variety of specific and nonspecific stimuli, elevated serum
immunoglobulin E
(IgE), excessive airway mucus production, edema, and bronchial epithelial
injury (Wills-
Karp, supra). Allergic asthma begins when the allergen provokes an immediate
early
airway response, which is frequently followed several hours later by a delayed
late-phase
airway response (LAR) (Henderson et al. (2000) J. Immunol. 164:1086-95).
During
LAR, there is an influx of eosinophils, lymphocytes, and macrophages
throughout the
airway wall and the bronchial fluid. (Henderson et al., supra). Lung
eosinophilia is a
hallmark of allergic asthma and is responsible for much of the damage to the
respiratory
epithelium (Li et al. (1999) J. Immunol. 162:2477-87).
CD4+ T helper (Th) cells are important for the chronic inflammation associated
with asthma (Henderson et al., supra). Several studies have shown that
commitment of
CD4+ cells to type 2 T helper (Th2) cells and the subsequent production of
type 2
cytokines (e.g., IL-4, IL-5, IL-10, and IL-13) are important in the allergic
inflammatory
response leading to AHR (Tomkinson et al. (2001) J. Immunol. 166:5792-5800,
and
references cited therein). First, CD4+ T cells have been shown to be necessary
for
allergy-induced asthma in murine models. Second, CD4+ T cells producing type 2
cytokines undergo expansion not only in these animal models but also in
patients with
allergic asthma. Third, type 2 cytokine levels are increased in the airway
tissues of
-79-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
animal models and asthmatics. Fourth, Th2 cytokines have been implicated as
playing a
central role in eosinophil recruitment in murine models of allergic asthma,
and adoptively
transferred Th2 cells have been correlated with increased levels of eotaxin (a
potent
eosinophil chemoattractant) in the lung as well as lung eosinophilia (Wills-
Karp et al.,
supra; Li et al., supra).
The methods for treating or preventing asthma described herein include those
for
extrinsic asthma (also known as allergic asthma or atopic asthma), intrinsic
asthma (also
known as non-allergic asthma or non-atopic asthma) or combinations of both,
which has
been referred to as mixed asthma. Extrinsic or allergic asthma includes
incidents caused
by, or associated with, e.g., allergens, such as pollens, spores, grasses or
weeds, pet
danders, dust, mites, etc. As allergens and other irritants present themselves
at varying
points over the year, these types of incidents are also referred to as
seasonal asthma. Also
included in the group of extrinsic asthma is bronchial asthma and allergic
bronchopulmonary aspergillosis.
Disorders that can be treated or alleviated by the agents described herein
include
those respiratory disorders and asthma caused by infectious agents, such as
viruses (e.g.,
cold and flu viruses, respiratory syncytial virus (RSV), paramyxovirus,
rhinovirus and
influenza viruses. RSV, rhinovirus and influenza virus infections are common
in
children, and are one leading cause of respiratory tract illnesses in infants
and young
children. Children with viral bronchiolitis can develop chronic wheezing and
asthma,
which can be treated using the methods described herein. Also included are the
asthma
conditions which may be brought about in some asthmatics by exercise and/or
cold air.
The methods are useful for asthmas associated with smoke exposure (e.g.,
cigarette-
induced and industrial smoke), as well as industrial and occupational
exposures, such as
smoke, ozone, noxious gases, sulfur dioxide, nitrous oxide, fumes, including
isocyanates,
from paint, plastics, polyurethanes, varnishes, etc., wood, plant or other
organic dusts,
etc. The methods are also useful for asthmatic incidents associated with food
additives,
preservatives or pharmacological agents. Also included are methods for
treating,
inhibiting or alleviating the types of asthma referred to as silent asthma or
cough variant
asthma.
-80-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
The methods disclosed herein are also useful for treatment and alleviation of
asthma associated with gastroesophageal reflux (GERD), which can stimulate
bronchoconstriction. GERD, along with retained bodily secretions, suppressed
cough,
and exposure to allergens and irritants in the bedroom can contribute to
asthmatic
conditions and have been collectively referred to as nighttime asthma or
nocturnal
asthma. In methods of treatment, inhibition or alleviation of asthma
associated with
GERD, a pharmaceutically effective amount of the IL-13 and/or IL-4 antagonist
can be
used as described herein in combination with a pharmaceutically effective
amount of an
agent for treating GERD. These agents include, but are not limited to, proton
pump
inhibiting agents like PROTONIX brand of delayed-release pantoprazole sodium
tablets, PRILOSEC brand omeprazole delayed release capsules, ACIPHEX brand
rebeprazole sodium delayed release tablets or PREVACID brand delayed release
lansoprazole capsules.
Atopic Disorders and S rnptoms Thereof
It has been observed that cells from atopic patients have enhanced sensitivity
to
IL-13. Accordingly, an IL-13 and/or IL-4 antagonist can be administered in an
amount
effective to treat or prevent an atopic disorder. ."Atopic" refers to a group
of diseases in
which there is often an inherited tendency to develop an allergic reaction.
Examples of atopic disorders include allergy, allergic rhinitis, atopic
dermatitis,
asthma and hay fever. Asthma is a phenotypically heterogeneous disorder
associated
with intermittent respiratory symptoms such as, e.g., bronchial
hyperresponsiveness and
reversible airflow obstruction. Immunohistopathologic features of asthma
include, e.g.,
denudation of airway epithelium, collagen deposition beneath the basement
membrane;
edema; mast cell activation; and inflammatory cell infiltration (e.g., by
neutrophils,
eosinophils, and lymphocytes). Airway inflammation can further contribute to
airway
hyperresponsiveness, airflow limitation, acute bronchoconstriction, mucus plug
formation, airway wall remodeling, and other respiratory symptoms. An IL-13
binding
agent (e.g., an IL-13 binding agent such as an antibody molecule described
herein) can be
administered in an amount effective to ameliorate one or more of these
symptoms.
-81-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
Symptoms of allergic rhinitis (hay fever) include itchy, runny, sneezing, or
stuffy
nose, and itchy eyes. An IL- 13 and/or IL-4 antagonist can be administered to
ameliorate
one or more of these symptoms. Atopic dermatitis is a chronic (long-lasting)
disease that
affects the skin. Information about atopic denmatitis is available, e.g., from
NIH
Publication No. 03-4272. In atopic dermatitis, the skin can become extremely
itchy,
leading to redness, swelling, cracking, weeping clear fluid, and finally,
crusting and
scaling. In many cases, there are periods of time when the disease is worse
(called
exacerbations or flares) followed by periods when the skin improves or clears
up entirely
(called remissions). Atopic dermatitis is often referred to as "eczema," which
is a general
term for the several types of inflammation of the skin. Atopic dermatitis is
the most
common of the many types of eczema. Examples of atopic dermatitis include:
allergic
contact eczema (dermatitis: a red, itchy, weepy reaction where the skin has
come into
contact with a substance that the immune system recognizes as foreign, such as
poison
ivy or certain preservatives in creams and lotions); contact eczema (a
localized reaction
that includes redness, itching, and burning where the skin has come into
contact with an
allergen (an allergy-causing substance) or with an irritant such as an acid, a
cleaning
agent, or other chemical); dyshidrotic eczema (irritation of the skin on the
palms of hands
and soles of the feet characterized by clear, deep blisters that itch and
burn);
neurodermatitis (scaly patches of the skin on the head, lower legs, wrists, or
forearms
caused by a localized itch (such as an insect bite) that become intensely
irritated when
scratched); nummular eczema (coin-shaped patches of irritated skin-most common
on the
arms, back, buttocks, and lower legs-that may be crusted, scaling, and
extremely itchy);
seborrheic eczema (yellowish, oily, scaly patches of skin on the scalp, face,
and
occasionally other parts of the body). Additional particular symptoms include
stasis
dermatitis, atopic pleat (Dennie-Morgan fold), cheilitis, hyperlinear palms,
hyperpigmented eyelids (eyelids that have become darker in color from
inflammation or
hay fever), ichthyosis, keratosis pilaris, lichenification, papules, and
urticaria. An IL-13
or IL-4 antagonist can be administered to ameliorate one or more of these
symptoms.
An exemplary method for treating allergic rhinitis or other allergic disorder
can
include initiating therapy with an IL- 13 and/or IL-4 antagonist prior to
exposure to an
-82-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
allergen, e.g., prior to seasonal exposure to an allergen, e.g., prior to
allergen blooms.
Such therapy can include one or more doses, e.g., doses at regular intervals.
Cancer
IL-13 and its receptors may be involved in the development of at least some
types
of cancer, e.g., a cancer derived from hematopoietic cells or a cancer derived
from brain
or neuronal cells (e.g., a glioblastoma). For example, blockade of the IL-13
signaling
pathway, e.g., via use of a soluble IL- 13 receptor or a STAT6 -/- deficient
mouse, leads
to delayed tumor onset and/or growth of Hodgkins lymphoma cell lines or a
metastatic
mammary carcinoma, respectively (Trieu et al. (2004) Cancer Res. 64: 3271-75;
Ostrand-
Rosenberg et al. (2000) J. Immunol. 165: 6015-6019). Cancers that express IL-
13R(2
(Husain and Puri (2003) J. Neurooncol. 65:37-48; Mintz et al. (2003) J.
Neurooncol.
64:117-23) can be specifically targeted by anti-IL-13 antibodies described
herein. IL-13
antagonists can be useful to inhibit cancer cell proliferation or other cancer
cell activity.
A cancer refers to one or more cells that has a loss of responsiveness to
normal growth
controls, and typically proliferates with reduced regulation relative to a
corresponding
normal cell.
Examples of cancers against which IL-13 antagonists (e.g., an IL-13 binding
agent such as an antibody or antigen binding fragment described herein) can be
used for
treatment include leukemias, e.g., B-cell chronic lymphocytic leukemia, acute
myelogenous leukemia, and human T-cell leukemia virus type 1(HTLV-1)
transformed
T cells; lymphomas, e.g. T cell lymphoma, Hodgkin's lymphoma; glioblastomas;
pancreatic cancers; renal cell carcinoma; ovarian carcinoma; AIDS-Kaposi's
sarcoma,
and breast cancer (as described in Aspord, C. et al. (2007) JEM 204:1037-
1047). For
example, an IL-13 binding agent (e.g., an anti-IL-13 antibody molecule) can be
administered in an amount effective to treat or prevent the disorder, e.g., to
reduce cell
proliferation, or to ameliorate at least one symptom of the disorder.
Fibrosis
IL- 13 and/or IL-4 antagonists can also be useful in treating inflammation and
fibrosis, e.g., fibrosis of the liver. IL-13 production has been correlated
with the
-83-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
progression of liver inflammation (e.g., viral hepatitis) toward cirrhosis,
and possibly,
hepatocellular carcinoma (de Lalla et al. (2004) J. Immunol. 173:1417-1425).
Fibrosis
occurs, e.g., when nonnal tissue is replaced by scar tissue, often following
inflammation.
Hepatitis B and hepatitis C viruses both cause a fibrotic reaction in the
liver, which can
progress to cirrhosis. Cirrhosis, in turn, can evolve into severe
complications such as
liver failure or hepatocellular carcinoma. Blocking IL- 13 activity using the
IL- 13 and/or
IL-4 antagonists described herein can reduce inflammation and fibrosis, e.g.,
the
inflammation, fibrosis, and cirrhosis associated with liver diseases,
especially hepatitis B
and C. For example, the antagonists(s) can be administered in an amount
effective to
treat or prevent the disorder or to ameliorate at least one symptom of the
inflammatory
and/or fibrotic disorder.
Inflammatory Bowel Disease
Inflammatory bowel disease (IBD) is the general name for diseases that cause
inflammation of the intestines. Two examples of inflammatory bowel disease are
Crohn's
disease and ulcerative colitis. IL-13/STAT6 signaling has been found to be
involved in
inflammation-induced hypercontractivity of mouse smooth muscle, a model of
inflammatory bowel disease (Akiho et al. (2002) Am. J. Physiol. Gastrointest.
Liver
Physiol. 282:G226-232). For example, an IL-13 and/or IL-4 antagonist can be
administered in an amount effective to treat or prevent the disorder or to
ameliorate at
least one symptom of the inflammatory bowel disorder.
Pharmaceutical Compositions
The IL-13 and/or IL-4 antagonists (such as those described herein) can be used
in
vitro, ex vivo, or in vivo. They can be incorporated into a pharmaceutical
composition,
e.g., by combining the IL-13 binding agent with a pharmaceutically acceptable
carrier.
Such a composition may contain, in addition to the IL-13 binding agent and
carrier,
various diluents, fillers, salts, buffers, stabilizers, solubilizers, and
other materials well
known in the art. Pharmaceutically acceptable materials is generally a
nontoxic material
that does not interfere with the effectiveness of the biological activity of
an IL- 13 binding
agent. The characteristics of the carrier can depend on the route of
administration.
-84-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
The pharmaceutical composition described herein may also contain other
factors,
such as, but not limited to, other anti-cytokine antibody molecules or other
anti-
inflammatory agents as described in more detail below. Such additional factors
and/or
agents may be included in the pharmaceutical composition to produce a
synergistic effect
with an IL-13 and/or IL-4 antagonist described herein. For example, in the
treatment of
allergic asthma, a pharmaceutical composition described herein may include
anti-IL-4
antibody molecules or drugs known to reduce an allergic response.
The pharmaceutical composition described herein may be in the form of a
liposome in which an IL-13 and/or IL-4 antagonist, such as one described
herein is
combined, in addition to other pharmaceutically acceptable carriers, with
amphipathic
agents such as lipids that exist in aggregated form as micelles, insoluble
monolayers,
liquid crystals, or lamellar layers while in aqueous solution. Suitable lipids
for liposomal
formulation include, without limitation, monoglycerides, diglycerides,
sulfatides,
lysolecithin, phospholipids, saponin, bile acids, and the like. Exemplary
methods for
preparing such liposomal formulations include methods described in U.S. Patent
Nos.
4,235,871; 4,501,728; 4,837,028; and 4,737,323.
As used herein, the term "therapeutically effective amount" means the total
amount of each active component of the pharmaceutical composition or method
that is
sufficient to show a meaningful patient benefit, e.g., amelioration of
symptoms of,
healing of, or increase in rate of healing of such conditions. When applied to
an
individual active ingredient, administered alone, the term refers to that
ingredient alone.
When applied to a combination, the term refers to combined amounts of the
active
ingredients that result in the therapeutic effect, whether administered in
combination,
serially or simultaneously.
Administration of an an IL- 13 and/or IL-4 antagonist used in the
pharmaceutical
composition can be carried out in a variety of conventional ways, such as oral
ingestion,
inhalation, or cutaneous, subcutaneous, or intravenous injection. When a
therapeutically
effective amount of an IL- 13 and/or IL-4 antagonist is administered by
intravenous,
cutaneous or subcutaneous injection, the binding agent can be prepared as a
pyrogen-free,
parenterally acceptable aqueous solution. The composition of such parenterally
acceptable protein solutions can be adapted in view factors such as pH,
isotonicity,
-85-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
stability, and the like, e.g., to optimize the composition for physiological
conditions,
binding agent stability, and so forth. A pharmaceutical composition for
intravenous,
cutaneous, or subcutaneous injection can contain, e.g., an isotonic vehicle
such as
Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose
and Sodium
Chloride Injection, Lactated Ringer's Injection, or other vehicle as known in
the art. The
pharmaceutical composition may also contain stabilizers, preservatives,
buffers,
antioxidants, or other additive.
The amount of an IL- 13 and/or IL-4 antagonist in the pharmaceutical
composition
can depend upon the nature and severity of the condition being treated, and on
the nature
of prior treatments that the patient has undergone. The pharmaceutical
composition can
be administered to normal patients or patients who do not show symptoms, e.g.,
in a
prophylactic mode. An attending physician may decide the amount of IL-13
and/or IL-4
antagonist with which to treat each individual patient. For example, an
attending
physician can administer low doses of antagonist and observe the patient's
response.
Larger doses of antagonist may be administered until the optimal therapeutic
effect is
obtained for the patient, and at that point the dosage is not generally
increased further.
For example, a pharmaceutical may contain between about 0.1 mg to 50 mg
antibody per
kg body weight, e.g., between about 0.1 mg and 5 mg or between about 8 mg and
50 mg
antibody per kg body weight. In one embodiment in which the antibody is
delivered
subcutaneously at a frequency of no more than twice per month, e.g., every
other week or
monthly, the composition includes an amount of about 0.7-3.3, e.g., 1.0-3.0
mg/kg, e.g.,
about 0.8-1.2, 1.2-2.8, or 2.8-3.3 mg/kg.
The duration of therapy using the pharmaceutical composition may vary,
depending on the severity of the disease being treated and the condition and
potential
idiosyncratic response of each individual patient. In one embodiment, the IL-
13 and/or
IL-4 antagonist can also be administered via the subcutaneous route, e.g., in
the range of
once a week, once every 24, 48, 96 hours, or not more frequently than such
intervals.
Exemplary dosages can be in the range of 0.1-20 mg/kg, more preferably 1-10
mg/kg.
The agent can be administered, e.g., by intravenous infusion at a rate of less
than 20, 10,
5, or 1 mg/min to reach a dose of about 1 to 50 mg/mz or about 5 to 20 mg/m2.
-86-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
In one embodiment, an administration of a an IL-13 and/or IL-4 antagonist to
the
patient includes varying the dosage of the protein, e.g., to reduce or
minimize side
effects. For example, the subject can be administered a first dosage, e.g., a
dosage less
than a therapeutically effective amount. In a subsequent interval, e.g., at
least 6, 12, 24,
or 48 hours later, the patient can be administered a second dosage, e.g., a
dosage that is at
least 25, 50, 75, or 100% greater than the first dosage. For example, the
second and/or a
comparable third, fourth and fifth dosage can be at least about 70, 80, 90, or
100% of a
therapeutically effective amount.
Inhalation
A composition that includes an an IL-13 and/or IL-4 antagonist can be
formulated
for inhalation or other mode of pulmonary delivery. The term "pulmonary
tissue" as used
herein refers to any tissue of the respiratory tract and includes both the
upper and lower
respiratory tract, except where otherwise indicated. An an IL-13 and/or IL-4
antagonist
can be administered in combination with one or more of the existing modalities
for
treating pulmonary diseases.
In one example the an IL-13 and/or IL-4 antagonist is formulated for a
nebulizer.
In one embodiment, the an IL- 13 and/or IL-4 antagonist can be stored in a
lyophilized
form (e.g., at room temperature) and reconstituted in solution prior to
inhalation. It is
also possible to formulate the an IL- 13 and/or IL-4 antagonist for inhalation
using a
medical device, e.g., an inhaler. See, e.g., U.S. 6,102,035 (a powder inhaler)
and
6,012,454 (a dry powder inhaler). The inhaler can include separate
compartments for the
the IL-13 and/or IL-4 antagonist at a pH suitable for storage and another
compartment for
a neutralizing buffer and a mechanism for combining the IL-13 and/or IL-4
antagonist
with a neutralizing buffer immediately prior to atomization. In one
embodiment, the
inhaler is a metered dose inhaler.
The three common systems used to deliver drugs locally to the pulmonary air
passages include dry powder inhalers (DPIs), metered dose inhalers (MDIs) and
nebulizers. MDIs, the most popular method of inhalation administration, may be
used to
deliver medicaments in a solubilized form or as a dispersion. Typically MDIs
comprise a
Freon or other relatively high vapor pressure propellant that forces
aerosolized
-87-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
medication into the respiratory tract upon activation of the device. Unlike
MDIs, DPIs
generally rely entirely on the inspiratory efforts of the patient to introduce
a medicament
in a dry powder form to the lungs. Nebulizers form a medicament aerosol to be
inhaled
by imparting energy to a liquid solution. Direct pulmonary delivery of drugs
during
liquid ventilation or pulmonary lavage using a fluorochemical medium has also
been
explored. These and other methods can be used to deliver an an IL-13 and/or IL-
4
antagonist. In one embodiment, the an IL-13 and/or IL-4 antagonist is
associated with a
polymer, e.g., a polymer that stabilizes or increases half-life of the
compound.
For example, for administration by inhalation, an IL- 13 and/or IL-4
antagonist is
delivered in the form of an aerosol spray from pressured container or
dispenser which
contains a suitable propellant or a nebulizer. The IL-13 and/or IL-4
antagonist may be in
the form of a dry particle or as a liquid. Particles that include the IL-13
and/or IL-4
antagonist can be prepared, e.g., by spray drying, by drying an aqueous
solution of the
IL- 13 and/or IL-4 antagonist with a charge neutralizing agent and then
creating particles
from the dried powder or by drying an aqueous solution in an organic modifier
and then
creating particles from the dried powder.
The IL- 13 and/or IL-4 antagonist may be conveniently delivered in the form of
an
aerosol spray presentation from pressurized packs or a nebulizer, with the use
of a
suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case
of a
pressurized aerosol, the dosage unit may be determined by providing a valve to
deliver a
metered amount. Capsules and cartridges for use in an inhaler or insufflator
may be
formulated containing a powder mix of an an IL-13 and/or IL-4 antagonist and a
suitable
powder base such as lactose or starch, if the particle is a formulated
particle. In addition
to the formulated or unformulated compound, other materials such as 100% DPPC
or
other surfactants can be mixed with the an IL- 13 and/or IL-4 antagonist to
promote the
delivery and dispersion of formulated or unformulated compound. Methods of
preparing
dry particles are described, for example, in WO 02/32406.
An IL-13 and/or IL-4 antagonist can be formulated for aerosol delivery, e.g.,
as
dry aerosol particles, such that when administered it can be rapidly absorbed
and can
produce a rapid local or systemic therapeutic result. Administration can be
tailored to
-88-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
provide detectable activity within 2 minutes, 5 minutes, 1 hour, or 3 hours of
administration. In some embodiments, the peak activity can be achieved even
more
quickly, e.g., within one half hour or even within ten minutes. An IL-13
and/or IL-4
antagonist can be formulated for longer biological half-life (e.g., by
association with a
polymer such as PEG) for use as an alternative to other modes of
administration, e.g.,
such that the IL- 13 and/or IL-4 antagonist enters circulation from the lung
and is
distributed to other organs or to a particular target organ.
In one embodiment, the IL-13 and/or IL-4 antagonist is delivered in an amount
such that at least 5% of the mass of the polypeptide is delivered to the lower
respiratory
tract or the deep lung. Deep lung has an extremely rich capillary network. The
respiratory membrane separating capillary lumen from the alveolar air space is
very thin
(:56 m) and extremely permeable. In addition, the liquid layer lining the
alveolar surface
is rich in lung surfactants. In other embodiments, at least 2%, 3%, 5%, 10%,
20%, 30%,
40%, 50%, 60%, 70%, or 80% of the composition of an IL-13 and/or IL-4
antagonist is
delivered to the lower respiratory tract or to the deep lung. Delivery to
either or both of
these tissues results in efficient absorption of the IL-13 and/or IL-4
antagonist and high
bioavailability. In one embodiment, the IL- 13 and/or IL-4 antagonist is
provided in a
metered dose using, e.g., an inhaler or nebulizer. For example, the IL-13
binding agent is
delivered in a dosage unit form of at least about 0.02, 0.1, 0.5, 1, 1.5, 2,
5, 10, 20, 40, or
50 mg/puff or more. The percent bioavailability can be calculated as follows:
the percent
bioavailability =(AUCnon-invasive/AUCi.v. or s.c.) X(dOsei,v, or s.c./dOSenon-
invasive) X 100.
Although not necessary, delivery enhancers such as surfactants can be used to
further enhance pulmonary delivery. A "surfactant" as used herein refers to a
IL IL- 13
and/or IL-4 antagonist having a hydrophilic and lipophilic moiety, which
promotes
absorption of a drug by interacting with an interface between two immiscible
phases.
Surfactants are useful in the dry particles for several reasons, e.g.,
reduction of particle
agglomeration, reduction of macrophage phagocytosis, etc. When coupled with
lung
surfactant, a more efficient absorption of the IL- 13 and/or IL-4 antagonist
can be
achieved because surfactants, such as DPPC, will greatly facilitate diffusion
of the
compound. Surfactants are well known in the art and include but are not
limited to
phosphoglycerides, e.g., phosphatidyicholines, L-alpha-phosphatidylcholine
dipalmitoyl
-89-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
(DPPC) and diphosphatidyl glycerol (DPPG); hexadecanol; fatty acids;
polyethylene
glycol (PEG); polyoxyethylene-9-; auryl ether; palmitic acid; oleic acid;
sorbitan trioleate
(Span 85); glycocholate; surfactin; poloxomer; sorbitan fatty acid ester;
sorbitan trioleate;
tyloxapol; and phospholipids.
Stabilization
In one embodiment, an IL-13 and/or IL-4 antagonist is physically associated
with
a moiety that improves its stabilization and/or retention in circulation,
e.g., in blood,
serum, lymph, bronchopulmonary lavage, or other tissues, e.g., by at least
1.5, 2, 5, 10, or
lo 50 fold.
For example, an IL-13 and/or IL-4 antagonist can be associated with a polymer,
e.g., a substantially non-antigenic polymers, such as polyalkylene oxides or
polyethylene
oxides. Suitable polymers will vary substantially by weight. Polymers having
molecular
number average weights ranging from about 200 to about 35,000 (or about 1,000
to about
15,000, and 2,000 to about 12,500) can be used.
For example, an IL-13 and/or IL-4 antagonist can be conjugated to a water
soluble
polymer, e.g., hydrophilic polyvinyl polymers, e.g. polyvinylalcohol and
polyvinylpyrrolidone. A non-limiting list of such polymers includes
polyalkylene oxide
homopolymers such as polyethylene glycol (PEG) or polypropylene glycols,
polyoxyethylenated polyols, copolymers thereof and block copolymers thereof,
provided
that the water solubility of the block copolymers is maintained. Additional
useful
polymers include polyoxyalkylenes such as polyoxyethylene, polyoxypropylene,
and
block copolymers of polyoxyethylene and polyoxypropylene (Pluronics);
polymethacrylates; carbomers; branched or unbranched polysaccharides which
comprise
the saccharide monomers D-mannose, D- and L-galactose, fucose, fructose, D-
xylose, L-
arabinose, D-glucuronic acid, sialic acid, D-galacturonic acid, D-mannuronic
acid (e.g.
polymannuronic acid, or alginic acid), D-glucosamine, D-galactosamine, D-
glucose and
neuraminic acid including homopolysaccharides and heteropolysaccharides such
as
lactose, amylopectin, starch, hydroxyethyl starch, amylose, dextran sulfate,
dextran,
dextrins, glycogen, or the polysaccharide subunit of acid mucopolysaccharides,
e.g.
-90-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
hyaluronic acid; polymers of sugar alcohols such as polysorbitol and
polymannitol;
heparin or heparan.
The conjugates of an IL-13 and/or IL-4 antagonist and a polymer can be
separated
from the unreacted starting materials, e.g., by gel filtration or ion exchange
chromatography, e.g., HPLC. Heterologous species of the conjugates are
purified from
one another in the same fashion. Resolution of different species (e.g.
containing one or
two PEG residues) is also possible due to the difference in the ionic
properties of the
unreacted amino acids. See, e.g., WO 96/34015.
Other Uses of IL- 13 and/or IL-4 Antagonists
In yet another aspect, the invention features a method for modulating (e.g.,
decreasing, neutralizing and/or inhibiting) one or more associated activities
of IL- 13 in
vivo by administering an IL-13 and/or IL-4 antagonist described herein in an
amount
sufficient to inhibit its activity. An IL-13 and/or IL-4 antagonist can also
be administered
to subjects for whom inhibition of an IL- 13 -mediated inflammatory response
is required.
These conditions include, e.g., airway inflammation, asthma, fibrosis,
eosinophilia and
increased mucus production.
The efficacy of an IL-13 and/or IL-4 antagonist described herein can be
evaluated, e.g., by evaluating ability of the antagonist to modulate airway
inflammation
in cynomolgus monkeys exposed to an Ascaris suum allergen. An IL- 13 and/or IL-
4
antagonist can be used to neutralize or inhibit one or more IL- 13 -associated
activities,
e.g., to reduce IL-13 mediated inflammation in vivo, e.g., for treating or
preventing
IL-13-associated pathologies, including asthma and/or its associated symptoms.
In one embodiment, an IL- 13 and/or IL-4 antagonist, or a pharmaceutical
compositions thereof, is administered in combination therapy, i.e., combined
with other
agents, e.g., therapeutic agents, that are useful for treating pathological
conditions or
disorders, such as allergic and inflammatory disorders. The term "in
combination" in this
context means that the agents are given substantially contemporaneously,
either
simultaneously or sequentially. If given sequentially, at the onset of
administration of the
second compound, the first of the two compounds is preferably still detectable
at
effective concentrations at the site of treatment.
-91-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
For example, the combination therapy can include one or more IL- 13 binding
agents (e.g., the IL-13 antagonist alone or in combination with the IL-4
antagonist) that
bind to IL- 13 and interfere with the formation of a functional IL- 13
signaling complex,
coformulated with, and/or coadministered with, one or more additional
therapeutic
agents, e.g., one or more cytokine and growth factor inhibitors,
immunosuppressants,
anti-inflanunatory agents, metabolic inhibitors, enzyme inhibitors, and/or
cytotoxic or
cytostatic agents, as described in more detail below. Furthermore, one or more
IL-13
binding agents (e.g., the IL-13 antagonist alone or in combination with the IL-
4
antagonist) may be used in combination with two or more of the therapeutic
agents
described herein. Such combination therapies may advantageously utilize lower
dosages
of the administered therapeutic agents, thus avoiding possible toxicities or
complications
associated with the various monotherapies. Moreover, the therapeutic agents
disclosed
herein act on pathways that differ from the IL-13 / IL-13-receptor pathway,
and thus are
expected to enhance and/or synergize with the effects of the IL- 13 binding
agents.
Therapeutic agents that interfere with different triggers of asthma or airway
inflammation, e.g., therapeutic agents used in the treatment of allergy, upper
respiratory
infections, or ear infections, may be used in combination with an IL- 13
binding agent
(e.g., the IL-13 antagonist alone or in combination with the IL-4 antagonist).
In one
embodiment, one or more IL-13 binding agents (e.g., the IL-13 antagonist alone
or in
combination with the IL-4 antagonist) may be coformulated with, and/or
coadministered
with, one or more additional agents, such as other cytokine or growth factor
antagonists
(e.g., soluble receptors, peptide inhibitors, small molecules, adhesins),
antibody
molecules that bind to other targets (e.g., antibodies that bind to other
cytokines or
growth factors, their receptors, or other cell surface molecules), and anti-
inflammatory
cytokines or agonists thereof. Non-limiting examples of the agents that can be
used in
combination with IL-13 binding agents (e.g., the IL-13 antagonist alone or in
combination with the IL-4 antagonist) include, but are not limited to, inhaled
steroids;
beta-agonists, e.g., short-acting or long-acting beta-agonists; antagonists of
leukotrienes
or leukotriene receptors; combination drugs such as ADVAIR ; IgE inhibitors,
e.g., anti-
IgE antibodies (e.g., XOLAIR~); phosphodiesterase inhibitors (e.g., PDE4
inhibitors);
-92-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
xanthines; anticholinergic drugs; mast cell-stabilizing agents such as
cromolyn; IL-5
inhibitors; eotaxin/CCR3 inhibitors; and antihistamines.
In other embodiments, one or more IL- 13 antagonists alone or in combination
with one or more IL-4 antagonists can be co-formulated with, and/or
coadministered
with, one or more anti-inflammatory drugs, immunosuppressants, or metabolic or
enzymatic inhibitors. Examples of the drugs or inhibitors that can be used in
combination with the IL- 13 binding agents include, but are not limited to,
one or more of:
TNF antagonists (e.g., a soluble fragment of a TNF receptor, e.g., p55 or p75
human TNF
receptor or derivatives thereof, e.g., 75 kd TNFR-IgG (75 kD TNF receptor-IgG
fusion
protein, ENBREL'T')); TNF enzyme antagonists, e.g., TNFa converting enzyme
(TACE)
inhibitors; muscarinic receptor antagonists; TGF-(3 antagonists; interferon
gamma;
perfenidone; chemotherapeutic agents, e.g., methotrexate, leflunomide, or a
sirolimus
(rapamycin) or an analog thereof, e.g., CCI-779; COX2 and cPLA2 inhibitors;
NSAIDs;
immunomodulators; p38 inhibitors, TPL-2, Mk-2 and NFKB inhibitors.
Vaccine Formulations
In another aspect, the invention features a method of modifying an immune
response associated with immunization. An IL-13 antagonist, alone or in
combination
with an IL-4 antagonist, can be used to increase the efficacy of immunization
by
inhibiting IL-13 activity. Antagonists can be administered before, during, or
after
delivery of an immunogen, e.g., administration of a vaccine. In one
embodiment, the
immunity raised by the vaccination is a cellular immunity, e.g., an immunity
against
cancer cells or virus infected, e.g., retrovirus infected, e.g., HIV infected,
cells. In one
embodiment, the vaccine formulation contains one or more antagonists and an
antigen,
e.g., an immunogen. In one embodiment, the IL-13 and/or IL-4 antagonists are
administered in combination with immunotherapy (e.g., in combination with an
allergy
immunization with one or more immunogens chosen from ragweed, ryegrass, dust
mite
and the like. In another embodiment, the antagonist and the immunogen are
administered
separately, e.g., within one hour, three hours, one day, or two days of each
other.
-93-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
Inhibition of IL-13 can improve the efficacy of, e.g., cellular vaccines,
e.g.,
vaccines against diseases such as cancer and viral infection, e.g., retroviral
infection, e.g.,
HIV infection. Induction of CD8+ cytotoxic T lymphocytes (CTL) by vaccines is
down
modulated by CD4+ T cells, likely through the cytokine IL-13. Inhibition of IL-
13 has
been shown to enhance vaccine induction of CTL response (Ahlers et al. (2002)
Proc.
Natl. Acad. Sci. USA 99:13020-10325). An IL-13 antagonist can be used in
conjunction
with a vaccine to increase vaccine efficacy. Cancer and viral infection (such
as retroviral
(e.g., HIV) infection) are exemplary disorders against which a cellular
vaccine response
can be effective. Vaccine efficacy is enhanced by blocking IL- 13 signaling at
the time of
vaccination (Ahlers et al. (2002) Proc. Nat. Acad. Sci. USA 99:13020-25). A
vaccine
formulation may be administered to a subject in the form of a pharmaceutical
or
therapeutic composition.
Methods for Diagnosing, Prognosing, and Monitoring Disorders
IL-13 binding agents can be used in vitro and in vivo as diagnostic agents.
One
exemplary method includes: (i) administering the IL-13 binding agent (e.g., an
IL-13
antibody molecule) to a subject; and (ii) detecting the IL-13 binding agent in
the subject.
The detecting can include determining location of the IL-13 binding agent in
the subject.
Another exemplary method includes contacting an IL-13 binding agent to a
sample, e.g.,
a sample from a subject. The presence or absence of IL-13 or the level of IL-
13 (either
qualitative or quantitative) in the sample can be determined.
In another aspect, the present invention provides a diagnostic method for
detecting the presence of a IL-13, in vitro (e.g., a biological sample, such
as tissue,
biopsy) or in vivo (e.g., in vivo imaging in a subject). The method includes:
(i) contacting
a sample with IL- 13 binding agent; and (ii) detecting formation of a complex
between the
IL-13 binding agent and the sample. The method can also include contacting a
reference
sample (e.g., a control sample) with the binding agent, and determining the
extent of
formation of the complex between the binding agent an the sample relative to
the same
for the reference sample. A change, e.g., a statistically significant change,
in the
formation of the complex in the sample or subject relative to the control
sample or
subject can be indicative of the presence of IL-13 in the sample.
-94-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
Another method includes: (i) administering the IL-13 binding agent to a
subject;
and (ii) detecting formation of a complex between the IL-13 binding agent and
the
subject. The detecting can include determining location or time of formation
of the
complex.
The IL- 13 binding agent can be directly or indirectly labeled with a
detectable
substance to facilitate detection of the bound or unbound protein. Suitable
detectable
substances include various enzymes, prosthetic groups, fluorescent materials,
luminescent materials and radioactive materials.
Complex formation between the IL- 13 binding agent and IL- 13 can be detected
by measuring or visualizing either the binding agent bound to the IL- 13 or
unbound
binding agent. Conventional detection assays can be used, e.g., an enzyme-
linked
immunosorbent assays (ELISA), a radioimmunoassay (RIA) or tissue
immunohistochemistry. Further to labeling the IL-13 binding agent, the
presence of
IL-13 can be assayed in a sample by a competition immunoassay utilizing
standards
labeled with a detectable substance and an unlabeled IL- 13 binding agent. In
one
example of this assay, the biological sample, the labeled standards and the IL-
13 binding
agent are combined and the amount of labeled standard bound to the unlabeled
binding
agent is determined. The amount of IL-13 in the sample is inversely
proportional to the
amount of labeled standard bound to the IL- 13 binding agent.
Methods for Diagnosing, Prognosing, and/or Monitoring Asthma
The binding agents described herein can be used, e.g., in methods for
diagnosing,
prognosing, and monitoring the progress of asthma by measuring the level of IL-
13 in a
biological sample. In addition, this discovery enables the identification of
new inhibitors
of IL-13 signaling, which will also be useful in the treatment of asthma. Such
methods
for diagnosing allergic and nonallergic asthma can include detecting an
alteration (e.g., a
decrease or increase) of IL-13 in a biological sample, e.g., serum, plasma,
bronchoalveolar lavage fluid, sputum, etc. "Diagnostic" or "diagnosing" means
identifying the presence or absence of a pathologic condition. Diagnostic
methods
involve detecting the presence of IL-13 by determining a test amount of IL-13
polypeptide in a biological sample, e.g., in bronchoalveolar lavage fluid,
from a subject
-95-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
(human or nonhuman mammal), and comparing the test amount with a normal amount
or
range (i.e., an amount or range from an individual(s) known not to suffer from
asthma)
for the IL- 13 polypeptide. While a particular diagnostic method may not
provide a
definitive diagnosis of asthma, it suffices if the method provides a positive
indication that
aids in diagnosis.
Methods for prognosing asthma and/or atopic disorders can include detecting
upregulation of IL-13, at the mRNA or protein level. "Prognostic" or
"prognosing"
means predicting the probable development and/or severity of a pathologic
condition.
Prognostic methods involve determining the test amount of IL- 13 in a
biological sample
from a subject, and comparing the test amount to a prognostic amount or range
(i.e., an
amount or range from individuals with varying severities of asthma) for IL-13.
Various
amounts of the IL- 13 in a test sample are consistent with certain prognoses
for asthma.
The detection of an amount of IL- 13 at a particular prognostic level provides
a prognosis
for the subject.
The present application also provides methods for monitoring the course of
asthma by detecting the upregulation of IL-13. Monitoring methods involve
determining
the test amounts of IL-13 in biological samples taken from a subject at a
first and second
time, and comparing the amounts. A change in amount of IL-13 between the first
and
second time can indicate a change in the course of asthma and/or atopic
disorder, with a
decrease in amount indicating remission of asthma, and an increase in amount
indicating
progression of asthma and/or atopic disorder. Such monitoring assays are also
useful for
evaluating the efficacy of a particular therapeutic intervention (e.g.,
disease attenuation
and/or reversal) in patients being treated for an IL- 13 associated disorder.
Fluorophore- and chromophore-labeled binding agents can be prepared. The
fluorescent moieties can be selected to have substantial absorption at
wavelengths above
310 nm, and preferably above 400 nm. A variety of suitable fluorescers and
chromophores are described by Stryer (1968) Science, 162:526 and Brand, L. et
al.
(1972) Annual Review of Biochemistry, 41:843-868. The binding agents can be
labeled
with fluorescent chromophore groups by conventional procedures such as those
disclosed
in U.S. Patent Nos. 3,940,475, 4,289,747, and 4,376,110. One group of
fluorescers
having a number of the desirable properties described above is the xanthene
dyes, which
-96-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
include the fluoresceins and rhodamines. Another group of fluorescent
compounds are
the naphthylamines. Once labeled with a fluorophore or chromophore, the
binding agent
can be used to detect the presence or localization of the IL-13 in a sample,
e.g., using
fluorescent microscopy (such as confocal or deconvolution microscopy).
Histological Analysis. Immunohistochemistry can be performed using the
binding agents described herein. For example, in the case of an antibody, the
antibody
can synthesized with a label (such as a purification or epitope tag), or can
be detectably
labeled, e.g., by conjugating a label or label-binding group. For example, a
chelator can
be attached to the antibody. The antibody is then contacted to a histological
preparation,
e.g., a fixed section of tissue that is on a microscope slide. After an
incubation for
binding, the preparation is washed to remove unbound antibody. The preparation
is then
analyzed, e.g., using microscopy, to identify if the antibody bound to the
preparation.
The antibody (or other polypeptide or peptide) can be unlabeled at the time of
binding.
After binding and washing, the antibody is labeled in order to render it
detectable.
Protein Arrays. An IL-13 binding agent (e.g., a protein that is an IL-13
binding
agent) can also be immobilized on a protein array. The protein array can be
used as a
diagnostic tool, e.g., to screen medical samples (such as isolated cells,
blood, sera,
biopsies, and the like). The protein array can also include other binding
agents, e.g., ones
that bind to IL-13 or to other target molecules.
Methods of producing protein arrays are described, e.g., in De Wildt et al.
(2000)
Nat. Biotechnol. 18:989-994; Lueking et al. (1999) Anal. Biochem. 270:103-111;
Ge
(2000) Nucleic Acids Res. 28, e3, I-VII; MacBeath and Schreiber (2000) Science
289:1760-1763; WO 01/40803 and WO 99/51773A1. Polypeptides for the array can
be
spotted at high speed, e.g., using commercially available robotic apparati,
e.g., from
Genetic MicroSystems or BioRobotics. The array substrate can be, for example,
nitrocellulose, plastic, glass, e.g., surface-modified glass. The array can
also include a
porous matrix, e.g., acrylamide, agarose, or another polymer. For example, the
array can
be an array of antibodies, e.g., as described in De Wildt, supra. Cells that
produce the
protein can be grown on a filter in an arrayed format. proteins production is
induced, and
the expressed protein are immobilized to the filter at the location of the
cell.
-97-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
A protein array can be contacted with a sample to determine the extent of IL-
13 in
the sample. If the sample is unlabeled, a sandwich method can be used, e.g.,
using a
labeled probe, to detect binding of the IL-13. Information about the extent of
binding at
each address of the array can be stored as a profile, e.g., in a computer
database. The
protein array can be produced in replicates and used to compare binding
profiles, e.g., of
different samples.
Flow Cytometry. The IL-13 binding agent can be used to label cells, e.g.,
cells in
a sample (e.g., a patient sample). The binding agent can be attached (or
attachable) to a
fluorescent compound. The cells can then be analyzed by flow cytometry and/or
sorted
using fluorescent activated cell sorted (e.g., using a sorter available from
Becton
Dickinson Immunocytometry Systems, San Jose CA; see also U.S. Patent No.
5,627,037;
5,030,002; and 5,137,809). As cells pass through the sorter, a laser beam
excites the
fluorescent compound while a detector counts cells that pass through and
determines
whether a fluorescent compound is attached to the cell by detecting
fluorescence. The
amount of label bound to each cell can be quantified and analyzed to
characterize the
sample. The sorter can also deflect the cell and separate cells bound by the
binding agent
from those cells not bound by the binding agent. The separated cells can be
cultured
and/or characterized.
In vivo Imaging. In still another embodiment, the invention provides a method
for
detecting the presence of a IL-13 within a subject in vivo. The method
includes (i)
administering to a subject (e.g., a patient having an IL-13 associated
disorder) an anti-
IL-13 antibody molecule, conjugated to a detectable marker; (ii) exposing the
subject to a
means for detecting the detectable marker. For example, the subject is imaged,
e.g., by
NMR or other tomographic means.
Examples of labels useful for diagnostic imaging include radiolabels such
as13'I,
>> iin, 123I, 99m.I.c, 32P, 33P, 125I, 3H, 14C, and 188Rh, fluorescent labels
such as fluorescein
and rhodamine, nuclear magnetic resonance active labels, positron emitting
isotopes
detectable by a positron emission tomography ("PET") scanner, chemiluminescers
such
as luciferin, and enzymatic markers such as peroxidase or phosphatase. Short-
range
radiation emitters, such as isotopes detectable by short-range detector probes
can also be
employed. The binding agent can be labeled with such reagents using known
techniques.
-98-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
For example, see Wensel and Meares (1983) Radioimmunoimaging and
Radioimmunotherapy, Elsevier, New York for techniques relating to the
radiolabeling of
antibodies and Colcher et al. (1986) Meth. Enzymol. 121: 802-816. A
radiolabeled
binding agent can also be used for in vitro diagnostic tests. The specific
activity of a
isotopically-labeled binding agent depends upon the half-life, the isotopic
purity of the
radioactive label, and how the label is incorporated into the antibody.
Procedures for
labeling polypeptides with the radioactive isotopes (such as 14C, 3H, 35S,
125I, 99mTc, 32P,
33P, and 1311) are generally known. See, e.g., U.S. 4,302,438; Goding, J.W.
(Monoclonal
antibodies : principles and practice : production and application of
monoclonal
antibodies in cell biology, biochemistry, and immunology 2nd ed. London;
Orlando:
Academic Press, 1986. pp 124-126) and the references cited therein; and A.R.
Bradwell
et al., "Developments in Antibody Imaging", Monoclonal Antibodies for Cancer
Detection and Therapy, R.W. Baldwin et al., (eds.), pp 65-85 (Academic Press
1985).
IL-13 binding agents described herein can be conjugated to Magnetic Resonance
Imaging (MRI) contrast agents. Some MRI techniques are summarized in EP-A-0
502
814. Generally, the differences in relaxation time constants T1 and T2 of
water protons
in different environments is used to generate an image. However, these
differences can
be insufficient to provide sharp high resolution images. The differences in
these
relaxation time constants can be enhanced by contrast agents. Examples of such
contrast
agents include a number of magnetic agents paramagnetic agents (which
primarily alter
T1) and ferromagnetic or superparamagnetic (which primarily alter T2
response).
Chelates (e.g., EDTA, DTPA and NTA chelates) can be used to attach (and reduce
toxicity) of some paramagnetic substances (e.g., Fe3+, Mn2+, Gd 3). Other
agents can be
in the form of particles, e.g., less than 10 m to about 10 nm in diameter)
and having
ferromagnetic, antiferromagnetic, or superparamagnetic properties. The IL-13
binding
agents can also be labeled with an indicating group containing the NMR active
19F atom,
as described by Pykett (1982) Scientific American, 246:78-88 to locate and
image IL-13
distribution.
Also within the scope described herein are kits comprising an IL- 13 binding
agent
and instructions for diagnostic use, e.g., the use of the IL-13 binding agent
(e.g., an
antibody molecule or other polypeptide or peptide) to detect IL-13, in vitro,
e.g., in a
-99-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
sample, e.g., a biopsy or cells from a patient having an IL-13 associated
disorder, or in
vivo, e.g., by imaging a subject. The kit can further contain a least one
additional reagent,
such as a label or additional diagnostic agent. For in vivo use the binding
agent can be
formulated as a pharmaceutical composition.
Kits
An IL-13 binding agent, e.g., an anti-IL-13 antibody molecule, and/or the IL-4
antagonist can be provided in a kit, e.g., as a component of a kit. For
example, the kit
includes (a) an IL-13 binding agent, e.g., an anti-IL-13 antibody molecule,
and/or the IL-
4 antagonist and, optionally (b) informational material. The informational
material can
be descriptive, instructional, marketing or other material that relates to a
method, e.g., a
method described herein. The informational material of the kits is not limited
in its form.
In one embodiment, the informational material can include information about
production
of the compound, molecular weight of the compound, concentration, date of
expiration,
batch or production site information, and so forth. In one embodiment, the
informational
material relates to using the IL- 13 binding agent to treat, prevent,
diagnose, prognose, or
monitor a disorder described herein. In one embodiment the informational
material
includes instructions for administration of the IL-13 binding as a single
treatment
interval.
In one embodiment, the informational material can include instructions to
administer an IL-13 binding agent, e.g., an anti-IL-13 antibody molecule, in a
suitable
manner to perform the methods described herein, e.g., in a suitable dose,
dosage form, or
mode of administration (e.g., a dose, dosage form, or mode of administration
described
herein). In another embodiment, the informational material can include
instructions to
administer an IL-13 binding agent, e.g., an anti-IL-13 antibody molecule, to a
suitable
subject, e.g., a human, e.g., a human having, or at risk for, allergic asthma,
non-allergic
asthma, or an IL-13 mediated disorder, e.g., an allergic and/or inflammatory
disorder, or
HTLV-1 infection. IL-13 production has been correlated with HTLV-1 infection
(Chung
et al., (2003) Blood 102: 4130-36).
For example, the material can include instructions to administer an IL- 13
binding
agent, e.g., an anti-IL-13 antibody molecule, to a patient, a patient with or
at risk for
-100-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
allergic asthma, non-allergic asthma, or an IL- 13 mediated disorder, e.g., an
allergic
and/or inflammatory disorder, or HTLV-1 infection.
The kit can include one or more containers for the composition containing an
IL-13 binding agent, e.g., an anti-IL-13 antibody molecule. In some
embodiments, the
kit contains separate containers, dividers or compartments for the composition
and
informational material. For example, the composition can be contained in a
bottle, vial,
or syringe, and the informational material can be contained in a plastic
sleeve or packet.
In other embodiments, the separate elements of the kit are contained within a
single,
undivided container. For example, the composition is contained in a bottle,
vial or
syringe that has attached thereto the informational material in the form of a
label. In
some embodiments, the kit includes a plurality (e.g., a pack) of individual
containers,
each containing one or more unit dosage forms (e.g., a dosage form described
herein) of
an IL-13 binding agent, e.g., anti-IL-13 antibody molecule. For example, the
kit includes
a plurality of syringes, ampules, foil packets, atomizers or inhalation
devices, each
containing a single unit dose of an IL-13 binding agent, e.g., an anti-IL-13
antibody
molecule, or multiple unit doses.
The kit optionally includes a device suitable for administration of the
composition, e.g., a syringe, inhalant, pipette, forceps, measured spoon,
dropper (e.g., eye
dropper), swab (e.g., a cotton swab or wooden swab), or any such delivery
device. In a
preferred embodiment, the device is an implantable device that dispenses
metered doses
of the binding agent.
The Examples that follow are set forth to aid in the understanding of the
iriventions but are not intended to, and should not be construed to, limit its
scope in any
way.
EXAMPLES
Examnle 1
(a) Cloning of NHP-IL-13 and homology to human IL-13
The cynomolgus monkey IL- 13 (NHP IL- 13) was cloned using hybridization
probes. A comparison of the cynomolgus monkey IL- 13 amino acid sequence to
that of
- 101 -
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
human IL-13 is shown in FIG. lA. There is 94% amino acid identity between the
two
sequences, due to 8 amino acid differences. One of these differences, R130Q,
represents
a common human polymorphism preferentially expressed in asthmatic subjects
(Heinzmann et al. (2000) Hum. Mol. Genet. 9:549-559).
(b) Binding of NHP-IL-13 to human IL13Ra2
Human IL- 13 binds with high affinity to the alpha2 form of IL- 13 receptor
(IL13Ra2). A soluble form of this receptor was expressed with a human IgGI Fc
tail
(sIL13R(x2-Fc). By binding to IL-13 and sequestering the cytokine from the
cell surface
IL13Ral-IL4R signaling complex, sIL13Ra2-Fc can act as a potent inhibitor of
human
IL-13 bioactivity. sIL13Ra2-Fc was shown to bind to NHP-IL-13 produced by CHO
cells or E. coli.
(c) Bioactivity of NHP-IL-13 on human monocytes
(i) CD23 expression on human monoc, es. cDNA encoding cynomolgus
monkey IL- 13 was expressed in E. coli and refolded to maintain bioactivity.
Reactivity
of human cells to cynomolgus IL-13 was demonstrated using a bioassay in which
normal
peripheral blood mononuclear cells from healthy donors were treated with IL-13
overnight at 37 C. This induced up-regulation of CD23 expression on the
surface of
monocytes. Results showed that cynomolgus IL-13 had bioactivity on primary
human
monocytes.
(ii) STAT6 phosphorvlation on HT-29 cells. The human HT-29 epithelial cell
line responds to IL- 13 by undergoing STAT6 phosphorylation, a consequence of
signal
transduction through the IL-13 receptor. To assay the ability of recombinant
NHP-IL-13
to induce STAT6 phosphorylation, HT-29 cells were challenged with the NHP-IL-
13 for
30 minutes at 37 C, then fixed, permeabilized, and stained with fluorescent
antibody to
phospho-STAT6. Results showed that cynomolgus IL-13 efficiently induced STAT6
phosphorylation in this human cell line.
(d) Generation of antibodies that bind to NHP-IL-13
- 102 -
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
Mice or other appropriate animals may be immunized and boosted with
cynomolgus IL-13, e.g., using one or more of the following methods. One method
for
immunization may be combined with either the same or different method for
boosting:
(i) Immunization with cynomolgus IL-13 protein expressed in E. coli, purified
from inclusion bodies, and refolded to preserve biological activity. For
immunization,
the protein is emulsified with complete Freund's adjuvant (CFA), and mice are
immunized according to standard protocols. For boosting, the same protein is
emulsified
with incomplete Freund's adjuvant (IFA).
(ii) Immunization with peptides spanning the entire sequence of mature
cynomolgus IL-13. Each peptide contains at least one amino acid that is unique
to
cynomolgus IL-13 and not present in the human protein. See FIG. 1B. Where the
peptide has a C-terminal residue other than cysteine, a cysteine is added for
conjugation
to a carrier protein. The peptides are conjugated to an immunogenic carrier
protein such
as KLH, and used to immunize mice according to standard protocols. For
immunization,
the protein is emulsified with complete Freund's adjuvant (CFA), and mice are
immunized according to standard protocols. For boosting, the same protein is
emulsified
with incomplete Freund's adjuvant (IFA).
(iii) Immunization with NHP-IL-13 - encoding cDNA expressed. The cDNA
encoding NHP-IL-13, including leader sequence, is cloned into an appropriate
vector.
This DNA is coated onto gold beads which are injected intradermally by gene
gun.
(iv) The protein or peptides can be used as a target for screening a protein
library,
e.g., a phage or ribosome display library. For example, the library can
display varied
immunoglobulin molecules, e.g., Fab's, scFv's, or Fd's.
(e) Selection of antibody clones cross-reactive with NHP and optionally a
human
IL-13, e.g., a native human IL-13.
Primary screen
-103-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
The primary screen for antibodies was selection for binding to recombinant NHP-
IL-13 by ELISA. In this ELISA, wells are coated with recombinant NHP IL-13.
The
immune serum was added in serial dilutions and incubated for one hour at room
temperature. Wells were washed with PBS containing 0.05% TWEEN-20 (PBS-
Tween). Bound antibody was detected using horseradish peroxidase (HRP)-labeled
anti-
mouse IgG and tetramethylbenzidene (TMB) substrate. Absorbance was read at 450
nm.
Typically, all immunized mice generated high titers of antibody to NHP-IL-13.
Secondary screen
The secondary screen was selection for inhibition of binding of recombinant
NHP-IL-13 to sIL-13Ra1-Fc by ELISA. Wells were coated with soluble IL-13Ral-
Fc,
to which FLAG-tagged NHP-IL- 13 could bind. This binding was detected with
anti-
FLAG antibody conjugated to HRP. Hydrolysis of TMB substrate was read as
absorbance at 450 nm. In the assay, the FLAG-tagged NHP-IL-13 was added
together
with increasing concentrations of immune serum. If the immune serum contained
antibody that bound to NHP-IL-13 and prevented its binding to the sIL13Ra1-Fc
coating
the wells, the ELISA signal was decreased. All immunized mice produced
antibody that
competed with sIL13Ral-Fc binding to NHP-IL-13, but the titers varied from
mouse to
mouse. Spleens were selected for fusion from animals whose serum showed
inhibited
sIL13Ra1-Fc binding to NHP-IL-13 at the highest dilution.
Tertiary screen
The tertiary screen tested for inhibition of NHP-IL-13 bioactivity. Several
bioassays were available to be used, including the TF-1 proliferation assay,
the monocyte
CD23 expression assay, and the HT-29 cell STAT6 phosphorylation assay. Immune
sera
were tested for inhibition of NHP-IL-13 - mediated STAT6 phosphorylation. The
HT-29
human epithelial cell line was challenged for 30 minutes at 37 C with
recombinant
NHP-IL-13 in the presence or absence of the indicated concentration of mouse
immune
serum. Cells were then fixed, permeabilized, and stained with ALEXAT"' Fluor
488-
conjugated mAb to phospho-STAT6 (Pharmingen). The percentage of cells
responding
to IL- 13 by undergoing STAT6 phosphorylation was determined by flow
cytometry.
- 104 -
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
Spleens of mice with the most potent neutralization activity, determined as
the strongest
inhibition of NHP-IL- 13 bioactivity at a high serum dilution, were selected
for generation
of hybridomas.
Quaternary Screen
A crude preparation containing human IL- 13 was generated from human
umbilical cord blood mononuclear cells (BioWhittaker/Cambrex). The cells were
cultured in a 37 C incubator at 5% C02, in RPMI media containing 10% heat-
inactivated
FCS, 50 U/ml penicillin, 50 mg/mi streptomycin, and 2 mM L-glutamine. Cells
were
stimulated for 3 days with the mitogen PHA-P (Sigma), and skewed toward Th2
with
recombinant human IL-4 (R&D Systems) and anti-human IL-12. The Th2 cells were
expanded for one week with IL-2, then activated to produce cytokine by
treatment with
phorbol 12-myristate 13-acetate (PMA) and ionomycin for three days. The
supernatant
was collected and dialyzed to remove PMA and ionomycin. To deplete GM-CSF and
IL-
4, which could interfere with bioassays for IL-13, the supernatant was treated
with
biotinylated antibodies to GM-CSF and IL-4 (R&D Systems, Inc), then incubated
with
streptavidin-coated magnetic beads (Dynal). The final concentration of IL-13
was
determined by ELISA (Biosource), and for total protein by Bradford assay (Bio-
Rad).
The typical preparation contains < 0.0005% IL-13 by weight.
Selection of hybridoma clones
Using established methods, hybridomas were generated from spleens of mice
selected as above, fused to the P3X63 AG8.653 myeloma cell line (ATCC). Cells
were
plated at limiting dilution and clones were selected according to the
screening criteria
described above. Data was collected for the selection of clones based on
ability to
compete for NHP-IL-13 binding to sIL13Ra1-Fc by ELISA. Clones were further
tested
for ability to neutralize the bioactivity of NHP-IL-13. Supernatants of the
hybridomas
were tested for competition of STAT-6 phosphorylation induced by NHP-IL- 13 in
the
HT-29 human epithelial cell line.
-105-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
Example 2: MJ 2-7 Antibody
Total RNA was prepared from MJ 2-7 hybridoma cells using the QIAGEN
RNEASYT'" Mini Kit (Qiagen). RNA was reverse transcribed to cDNA using the
SMARTT"" PCR Synthesis Kit (BD Biosciences Clontech). The variable region of
MJ 2-7 heavy chain was extrapolated by PCR using SMARTT'" oligonucleotide as a
forward primer and mIgGl primer annealing to DNA encoding the N-terminal part
of
CH1 domain of mouse IgGI constant region as a reverse primer. The DNA fragment
encoding MJ 2-7 light chain variable region was generated using SMARTT"" and
mouse
kappa specific primers. The PCR reaction was performed using DEEP VENTT"' DNA
polymerase (New England Biolabs) and 25 nM of dNTPs for 24 cycles (94 C for 1
minute, 60 C for 1 minute, 72 C for 1 minute). The PCR products were
subcloned into
the pED6 vector, and the sequence of the inserts was identified by DNA
sequencing. N-
terminal protein sequencing of the purified mouse MJ 2-7 antibody was used to
confirm
that the translated sequences corresponded to the observed protein sequence.
Exemplary nucleotide and amino acid sequences of mouse monoclonal antibody
MJ 2-7 which interacts with NHP IL-13 and which has characteristics which
suggest that
it may interact with human IL-13 are as follows:
An exemplary nucleotide sequence encoding the heavy chain variable domain
includes:
GAG GTTCAGCTGC AGCAGTCTGG GGCAGAGCTT GTGAAGCCAG
GGGCCTCAGT CAAGTTGTCC TGCACAGGTT CTGGCTTCAA CATTAAAGAC
ACCTATATAC ACTGGGTGAA GCAGAGGCCT GAACAGGGCC TGGAGTGGAT
TGGAAGGATT GATCCTGCGA ATGATAATAT TAAATATGAC CCGAAGTTCC
AGGGCAAGGC CACTATAACA GCAGACACAT CCTCCAACAC AGCCTACCTA
CAGCTCAACA GCCTGACATC TGAGGACACT GCCGTCTATT ACTGTGCTAG
ATCTGAGGAA AATTGGTACG ACTTTTTTGA CTACTGGGGC CAAGGCACCA
CTCTCACAGT CTCCTCA (SEQ ID NO:129)
An exemplary amino acid sequence for the heavy chain variable domain includes:
-106-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
EVQLQQS GAELVKPGAS VKLSCTGSGFNIKDTYIHW VKQRPEQGLEWIGRIDPA
NDNIKYDPKFQGKATITADTS SNTAYLQLNSLTSEDTAVYYCARSEENWYDFF
DYWGQGTTLTVSS (SEQ ID NO:130)
CDRs are underlined. The variable domain optionally is preceded by a leader
sequence. e.g., MKCSWVIFFLMAVVTGVNS (SEQ ID NO:131). An exemplary
nucleotide sequence encoding the light chain variable domain includes:
GAT GTTTTGATGA CCCAAACTCC ACTCTCCCTG CCTGTCAGTC
TTGGAGATCA AGCCTCCATC TCTTGCAGGT CTAGTCAGAG CATTGTACAT
AGTAATGGAA ACACCTATTT AGAATGGTAC CTGCAGAAAC CAGGCCAGTC
TCCAAAGCTC CTGATCTACA AAGTTTCCAA CCGATTTTCT GGGGTCCCAG
ACAGGTTCAG TGGCAGTGGA TCAGGGACAG ATTTCACACT CAAGATTAGC
AGAGTGGAGG CTGAGGATCT GGGAGTTTAT TACTGCTTTC AAGGTTCACA
TATTCCGTAC ACGTTCGGAG GGGGGACCAA GCTGGAAATA AAA (SEQ ID NO:132)
An exemplary amino acid sequence for the light chain variable domain includes:
DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSP
KLLIYKVSNRFSGVPDRFS GSGSGTDFTLKISRVEAEDLGVYYCFQGSHIPYTFG
GGTKLEIK (SEQ ID NO:133)
CDRs are underlined. The amino acid sequence optionally is preceded by a
leader sequence, e.g., MKLPVRLLVLMFWIPASSS (SEQ ID NO: 134). The term
"MJ 2-7" is used interchangeably with the term "mAb7.1.1," herein.
Example 3: C65 Antibody
Exemplary nucleotide and amino acid sequences of mouse monoclonal antibody
C65, which interacts with NHP IL- 13 and which has characteristics that
suggest that it
may interact with human IL- 13 are as follows:
An exemplary nucleic acid sequence for the heavy chain variable domain
includes:
1 ATGGCTGTCC TGGCATTACT CTTCTGCCTG GTAACATTCC CAAGCTGTAT
51 CCTTTCCCAG GTGCAGCTGA AGGAGTCAGG ACCTGGCCTG GTGGCGCCCT
- 107 -
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
101 CACAGAGCCT GTCCATCACA TGCACCGTCT CAGGGTTCTC ATTAACCGGC
151 TATGGTGTAA ACTGGGTTCG CCAGCCTCCA GGAAAGGGTC TGGAGTGGCT
201 GGGAATAATT TGGGGTGATG GAAGCACAGA CTATAATTCA GCTCTCAAAT
251 CCAGACTGAT CATCAACAAG GACAACTCCA AGAGCCAAGT TTTCTTAAAA
301 ATGAACAGTC TGCAAACTGA TGACACAGCC AGGTACTTCT GTGCCAGAGA
351 TAAGACTTTT TACTACGATG GTTTCTACAG GGGCAGGATG GACTACTGGG
401 GTCAAGGAAC CTCAGTCACC GTCTCCTCA (SEQ ID N0:135)
An exemplary amino acid sequence for the heavy chain variable domain includes:
QVQLKESGPGL VAPSQSLSIT CTVSGFSLTG YGVNWVRQPP GKGLEWLGII
WGDGSTDYNS ALKSRLIINK DNSKSQVFLK MNSLQTDDTA RYFCARDKTF
YYDGFYRGRM DYWGQGTSVT VSS (SEQ ID NO:136)
CDRs are underlined. The amino acid sequence optionally is preceded by a
leader
sequence, e.g., MAVLALLFCL VTFPSCILS (SEQ ID NO:137).
An exemplary nucleotide sequence encoding the light chain variable domain
includes:
1 ATGAACACGA GGGCCCCTGC TGAGTTCCTT GGGTTCCTGT TGCTCTGGTT
51 TTTAGGTGCC AGATGTGATG TCCAGATGAT TCAGTCTCCA TCCTCCCTGT
101 CTGCATCTTT GGGAGACATT GTCACCATGA CTTGCCAGGC AAGTCAGGGC
151 ACTAGCATTA ATTTAAACTG GTTTCAGCAA AAACCAGGGA AAGCTCCTAA
201 GCTCCTGATC TTTGGTGCAA GCAACTTGGA AGATGGGGTC CCATCAAGGT
251 TCAGTGGCAG TAGATATGGG ACAAATTTCA CTCTCACCAT CAGCAGCCTG
301 GAGGATGAAG ATATGGCAAC TTATTTCTGT CTACAGCATA GTTATCTCCC
351 GTGGACGTTC GGTGGCGGCA CCAAACTGGA AATCAAA (SEQ ID NO:138)
An exemplary amino acid sequence for the light chain variable domain includes:
DVQMIQSP SSLSASLGDI VTMTCQASQG TSINLNWFQQ KPGKAPKLLI
FGASNLEDGV PSRFSGSRYG TNFTLTISSL EDEDMATYFC LQHSYLPWTF
GGGTKLEIK (SEQ ID NO:139)
CDRs are underlined. The amino acid sequence optionally is preceded by a
leader
sequence, e.g., MNTRAPAEFLGFLLLWFLGARC (SEQ ID NO:140).
- 108 -
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
Example 4: Fc sequences
The Ser at position #1 of SEQ ID NO: 128 represents amino acid residue #119 in
a
first exemplary full length antibody numbering scheme in which the Ser is
preceded by
residue #118 of a heavy chain variable domain. In the first exemplary full
length
antibody numbering scheme, mutated amino acids are at numbered 234 and 237,
and
correspond to positions 116 and 119 of SEQ ID NO:128. Thus, the following
sequence
represents an Fc domain with two mutations: L234A and G237A, according to the
first
exemplary full length antibody numbering scheme.
Mus musculus (SEQ ID NO:128)
The following is another exemplary human Fc domain sequence:
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV
LQS S GLYS LS S V V TVP S S S LGTQTYICNVNHKP SNTKVDKKV EPKS CDKTHTCPP
CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE
VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS
KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY
KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSP
GK (SEQ ID NO:141)
Other exemplary alterations that can be used to decrease effector function
include
L234A;L235A), ( L235A;G237A ), and N297A.
Example 5: IL-13 and IgE in Mice
IL- 13 is involved in the production of IgE, an important mediator of atopic
disease. Mice deficient in IL- 13 had partial reductions in serum IgE and mast
cell IgE
responses, whereas mice lacking the natural IL-13 binding agent, IL-13Ra2 /-,
had
enhanced levels of IgE and IgE effector function.
BALB/c female mice were obtained from Jackson Laboratories (Bar Harbor,
ME). IL-13Ra2-/- mice are described, e.g., in Wood et al. (2003) J. Exp. Med.
197:703-
9. Mice deficient in IL-13 are described, e.g., in McKenzie et al. (1998)
Immunity 9:423-
32. All mutant strains were on the BALB/c background.
- 109 -
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
Serum IgE levels were measured by ELISA. ELISA plates (MaxiSorp; Nunc,
Rochester, NY) were coated overnight at 4 C with rat anti-mouse IgE (BD
Biosciences,
San Diego, CA). Plates were blocked for 1 hour at room temperature with 0.5%
gelatin
in PBS, washed in PBS containing 0.05% TWEEN-20 (PBS-Tween), and incubated for
six hours at room temperature with purified mouse IgE (BD Biosciences) as
standards or
with serum dilutions. Binding was detected with biotinylated anti-mouse IgE
(BD
Biosciences) using mouse IgG (Sigma-Aldrich, St. Louis, MO) as a blocker.
Binding
was detected with peroxidase-linked streptavidin (Southern Biotechnology
Associates,
Inc., Birmingham, AL) and SURE BLUET" substrate (KPL Inc., Gaithersburg, MD).
In order to investigate the requirement for IL- 13 to support resting IgE
levels in
naive mice, serum was examined in the absence of specific immunization from
wild-type
mice and from mice genetically deficient in IL-13 and IL-13Ra2. Mice deficient
in
IL- 13 had virtually undetectable levels of serum IgE. In contrast, mice
lacking the
inhibitory receptor IL-13Ra2 displayed elevated levels of serum IgE. These
results
demonstrate that blocking IL- 13 can be useful for treating or preventing
atopic disorders.
Example 6: IL-13 and Atopic Disorders
The ability of MJ2-7 to inhibit the bioactivity of native human IL-13 (at 1
ng/ml)
was evaluated in an assay for STAT6 phosphorylation. MJ2-7 inhibited the
activity of
native human IL-13 with an IC50 of about 0.293 nM in this assay. An antibody
with the
murine heavy chain of MJ2-7 and a humanized light chain inhibited the activity
of native
human IL-13 with an IC50 of about 0.554 nM in this assay.
The ability of MJ2-7 to inhibit non-human primate IL- 13 (at 1 ng/ml) was
evaluated in an assay for CD23 expression. The MJ2-7 inhibited the activity of
non-
human primate IL-13 with an IC50 of about 0.242 nM in this assay. An antibody
with
the murine heavy chain of MJ2-7 and a humanized light chain inhibited the
activity of
non-human primate IL-13 with an IC50 of about 0.308 nM in this assay.
Examnle 7: Nucleotide and amino acid sequences of mouse MJ 2-7 antibod
The nucleotide sequence encoding the heavy chain variable region (with an
optional leader) is as follows:
1 ATGAAATGCA GCTGGGTTAT CTTCTTCCTG ATGGCAGTGG TTACAGGGGT
51 CAATTCAGAG GTTCAGCTGC AGCAGTCTGG GGCAGAGCTT GTGAAGCCAG
- 110 -
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
101 GGGCCTCAGT CAAGTTGTCC TGCACAGGTT CTGGCTTCAA CATTAAAGAC
151 ACCTATATAC ACTGGGTGAA GCAGAGGCCT GAACAGGGCC TGGAGTGGAT
201 TGGAAGGATT GATCCTGCGA ATGATAATAT TAAATATGAC CCGAAGTTCC
251 AGGGCAAGGC CACTATAACA GCAGACACAT CCTCCAACAC AGCCTACCTA
301 CAGCTCAACA GCCTGACATC TGAGGACACT GCCGTCTATT ACTGTGCTAG
351 ATCTGAGGAA AATTGGTACG ACTTTTTTGA CTACTGGGGC CAAGGCACCA
401 CTCTCACAGT CTCCTCA (SEQ ID NO:142)
The amino acid sequence of the heavy chain variable region with an optional
leader (underscored) is as follows:
1 MKCSWVIFFL MAVVTGVNSE VQLQQSGAEL VKPGASVKLS CTGSGFNIKD
51 TYIHWVKQRP EQGLEWIGRI DPANDNIKYD PKFQGKATIT ADTSSNTAYL
101 QLNSLTSEDT AVYYCARSEE NWYDFFDYWG QGTTLTVSS
(SEQ ID NO:143)
The nucleotide sequence encoding the light chain variable region is as
follows:
1 ATGAAGTTGC CTGTTAGGCT GTTGGTGCTG ATGTTCTGGA TTCCTGCTTC
51 CAGCAGTGAT GTTTTGATGA CCCAAACTCC ACTCTCCCTG CCTGTCAGTC
101 TTGGAGATCA AGCCTCCATC TCTTGCAGGT CTAGTCAGAG CATTGTACAT
151 AGTAATGGAA ACACCTATTT AGAATGGTAC CTGCAGAAAC CAGGCCAGTC
201 TCCAAAGCTC CTGATCTACA AAGTTTCCAA CCGATTTTCT GGGGTCCCAG
251 ACAGGTTCAG TGGCAGTGGA TCAGGGACAG ATTTCACACT CAAGATTAGC
301 AGAGTGGAGG CTGAGGATCT GGGAGTTTAT TACTGCTTTC AAGGTTCACA
351TATTCCGTAC ACGTTCGGAG GGGGGACCAA GCTGGAAATA AAA (SEQ ID NO:144)
The amino acid sequence of the light chain variable region with an optional
leader
(underscored) is as follows:
1 MKLPVRLLVL MFWIPASSSD VLMTQTPLSL PVSLGDQASI SCRSSQSIVH
51 SNGNTYLEWY LQKPGQSPKL LIYKVSNRFS GVPDRFSGSG SGTDFTLKIS
101 RVEAEDLGVY YCFQGSHIPY TFGGGTKLEI K(SEQ IDNO:145)
- 111 -
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
Example 8: Nucleotide and amino acid sequences of exemplarv first humanized
variants
of the MJ 2-7 antibody
Humanized antibody Version 1(V1) is based on the closest human germline
clones. The nucleotide sequence of hMJ 2-7 V 1 heavy chain variable region
(hMJ 2-7
VH V1) (with a sequence encoding an optional leader sequence) is as follows:
1 ATGGATTGGA CCTGGCGCAT CCTGTTCCTG GTGGCCGCTG CCACCGGCGC
51 TCACTCTCAG GTGCAGCTGG TGCAGTCTGG CGCCGAGGTG AAGAAGCCTG
101 GCGCTTCCGT GAAGGTGTCC TGTAAGGCCT CCGGCTTCAA CATCAAGGAC
151 ACCTACATCC ACTGGGTGCG GCAGGCTCCC GGCCAGCGGC TGGAGTGGAT
201 GGGCCGGATC GATCCTGCCA ACGACAACAT CAAGTACGAC CCCAAGTTTC
251 AGGGCCGCGT GACCATCACC CGCGATACCT CCGCTTCTAC CGCCTACATG
301 GAGCTGTCTA GCCTGCGGAG CGAGGATACC GCCGTGTACT ACTGCGCCCG
351 CTCCGAGGAG AACTGGTACG ACTTCTTCGA CTACTGGGGC CAGGGCACCC
401 TGGTGACCGT GTCCTCT (SEQ ID NO:146)
The amino acid sequence of the heavy chain variable region (hMJ 2-7 V 1) is
based on a CDR grafted to DP- 25, VH-I, 1-03. The amino acid sequence with an
optional leader (first underscored region; CDRs based on AbM definition shown
in
subsequent underscored regions) is as follows:
1 MDWTWRILFL VAAATGAHS - Q VQLVQSGAEV KKPGASVKVS CKASGFNIKD
51 TYIHWVRQAP GQRLEWMGRI DPANDNIKYD PKFOGRVTIT RDTSASTAYM
101 ELSSLRSEDT AVYYCARSEE NWYDFFDYWG QGTLVTVSSG ESCR (SEQ IDNO:147)
The nucleotide sequence of the hMJ 2-7 V I light chain variable region (hMJ 2-
7
VL V 1) (with a sequence encoding an optional leader sequence) is as follows:
1 ATGCGGCTGC CCGCTCAGCT GCTGGGCCTG CTGATGCTGT GGGTGCCCGG
51 CTCTTCCGGC GACGTGGTGA TGACCCAGTC CCCTCTGTCT CTGCCCGTGA
101 CCCTGGGCCA GCCCGCTTCT ATCTCTTGCC GGTCCTCCCA GTCCATCGTG
151 CACTCCAACG GCAACACCTA CCTGGAGTGG TTTCAGCAGA GACCCGGCCA
201 GTCTCCTCGG CGGCTGATCT ACAAGGTGTC CAACCGCTTT TCCGGCGTGC
251 CCGATCGGTT CTCCGGCAGC GGCTCCGGCA CCGATTTCAC CCTGAAGATC
- 112 -
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
301 AGCCGCGTGG AGGCCGAGGA TGTGGGCGTG TACTACTGCT TCCAGGGCTC
351 CCACATCCCT TACACCTTTG GCGGCGGAAC CAAGGTGGAG ATCAAG
(SEQ ID N0:148)
This version is based on a CDR graft to DPK18, V kappaII. The amino acid
sequence of hMJ 2-7 V 1 light chain variable region (hMJ 2-7 VL V 1) (with
optional
leader as first underscored region; CDRs based on AbM definition in subsequent
underscored regions) is as follows:
1 MRLPAQLLGL LMLWVPGSSG -DVVMTQSPLS LPVTLGQPAS ISCRSSOSIV
51 HSNGNTYLEW FQQRPGQSPR RLIYKVSNRF SGVPDRFSGS GSGTDFTLKI
101 SRVEAEDVGV YYCFOGSHIP YTFGGGTKVE IK (SEQ ID NO:149)
Example 9: Nucleotide and amino acid sequences of exemplary second humanized
variants of the MJ 2-7 antibody
The following heavy chain variable region is based on a CDR graft to DP-54,
VH-3, 3-07. The nucleotide sequence of hMJ 2-7 Version 2 (V2) heavy chain
variable
region (hMJ 2-7 VH V2) (with a sequence encoding an optional leader sequence)
is as
follows:
1 ATGGAGCTGG GCCTGTCTTG GGTGTTCCTG GTGGCTATCC TGGAGGGCGT
51 GCAGTGCGAG GTGCAGCTGG TGGAGTCTGG CGGCGGACTG GTGCAGCCTG
101 GCGGCTCTCT GCGGCTGTCT TGCGCCGCTT CCGGCTTCAA CATCAAGGAC
151 ACCTACATCC ACTGGGTGCG GCAGGCTCCC GGCAAGGGCC TGGAGTGGGT
201 GGCCCGGATC GATCCTGCCA ACGACAACAT CAAGTACGAC CCCAAGTTCC
251 AGGGCCGGTT CACCATCTCT CGCGACAACG CCAAGAACTC CCTGTACCTC
301 CAGATGAACT CTCTGCGCGC CGAGGATACC GCCGTGTACT ACTGCGCCCG
351 GAGCGAGGAG AACTGGTACG ACTTCTTCGA CTACTGGGGC CAGGGCACCC
401 TGGTGACCGT GTCCTCT (SEQ ID NO:150)
The amino acid sequence of hMJ 2-7 V2 heavy chain variable region (hMJ 2-7
VH V2) with an optional leader (first underscored region; CDRs based on AbM
definition shown in subsequent underscored regions) is as follows:
1 MELGLSWVFL VAILEGVOC- E VQLVESGGGL VQPGGSLRLS CAASGFNIKD
- 113 -
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
51 TYIHWVRQAP GKGLEWVARI DPANDNIKYD PKFOGRFTIS RDNAKNSLYL
101 QMNSLRAEDT AVYYCARSEE NWYDFFDYWG QGTLVTVSS (SEQ ID NO:151)
The hMJ 2-7 V2 light chain variable region was based on a CDR graft to DPK9,
V kappal, 02. The nucleotide sequence of hMJ 2-7 V2 light chain variable
region (hMJ
2-7 VL V2) (with a sequence encoding an optional leader sequence) is as
follows:
1 ATGGATATGC GCGTGCCCGC TCAGCTGCTG GGCCTGCTGC TGCTGTGGCT
51 GCGCGGAGCC CGCTGCGATA TCCAGATGAC CCAGTCCCCT TCTTCTCTGT
101 CCGCCTCTGT GGGCGATCGC GTGACCATCA CCTGTCGGTC CTCCCAGTCC
151 ATCGTGCACT CCAACGGCAA CACCTACCTG GAGTGGTATC AGCAGAAGCC
201 CGGCAAGGCC CCTAAGCTGC TGATCTACAA GGTGTCCAAC CGCTTTTCCG
251 GCGTGCCTTC TCGGTTCTCC GGCTCCGGCT CCGGCACCGA TTTCACCCTG
301 ACCATCTCCT CCCTCCAGCC CGAGGATTTC GCCACCTACT ACTGCTTCCA
351 GGGCTCCCAC ATCCCTTACA CCTTTGGCGG CGGAACCAAG GTGGAGATCA
401 AGCGT (SEQ ID NO:152)
The amino acid sequence of the light chain variable region of hMJ 2-7 V2 light
chain variable region (hMJ 2-7 VL V2) (with optional leader peptide
underscored and
CDRs based on AbM definition shown in subsequent underscored regions) is as
follows:
1 MDMRVPAQLL GLLLLWLRGA RC -DIQMTQSP SSLSASVGDR VTITCRSSOS
51 IVHSNGNTYL EWYQQKPGKA PKLLIYKVSN RFSGVPSRFS GSGSGTDFTL
101 TISSLQPEDF ATYYCFOGSH IPYTFGGGTK VEIKR (SEQ ID NO:153)
Additional humanized versions of MJ 2-7 V2 heavy chain variable region were
made. These versions included backmutations that have murine amino acids at
selected
framework positions.
The nucleotide sequence encoding the heavy chain variable region "Version 2.1
"
or V2.1 with the back mutations V481,A29G is as follows:
1 GAGGTGCAGC TGGTGGAGTC TGGCGGCGGA CTGGTGCAGC CTGGCGGCTC
51 TCTGCGGCTG TCTTGCGCCG CTTCCGGCTT CAACATCAAG GACACCTACA
101 TCCACTGGGT GCGGCAGGCT CCCGGCAAGG GCCTGGAGTG GATCGGCCGG
-114-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
151 ATCGATCCTG CCAACGACAA CATCAAGTAC GACCCCAAGT TCCAGGGCCG
201 GTTCACCATC TCTCGCGACA ACGCCAAGAA CTCCCTGTAC CTCCAGATGA
251 ACTCTCTGCG CGCCGAGGAT ACCGCCGTGT ACTACTGCGC CCGGAGCGAG
301 GAGAACTGGT ACGACTTCTT CGACTACTGG GGCCAGGGCA CCCTGGTGAC
351 CGTGTCCTCT (SEQ ID NO:154)
The amino acid sequence of the heavy chain variable region of V2.1 (CDRs based
on AbM definition shown in subsequent underscored regions) is as follows:
1 EVQLVESGGG LVQPGGSLRL SCAASGFNIK DTYIHWVRQA PGKGLEWIGR
51 IDPANDNIKY DPKFOGRFTI SRDNAKNSLY LQMNSLRAED TAVYYCARSE
101 ENWYDFFDYW GQGTLVTVSS (SEQ ID NO:155)
The nucleotide sequence encoding the heavy chain variable region V2.2 with the
back mutations (R67K,=F68A) is as follows:
1 GAGGTGCAGC TGGTGGAGTC TGGCGGCGGA CTGGTGCAGC CTGGCGGCTC
51 TCTGCGGCTG TCTTGCGCCG CTTCCGGCTT CAACATCAAG GACACCTACA
101 TCCACTGGGT GCGGCAGGCT CCCGGCAAGG GCCTGGAGTG GGTGGCCCGG
151 ATCGATCCTG CCAACGACAA CATCAAGTAC GACCCCAAGT TCCAGGGCAA
201 GGCCACCATC TCTCGCGACA ACGCCAAGAA CTCCCTGTAC CTCCAGATGA
251 ACTCTCTGCG CGCCGAGGAT ACCGCCGTGT ACTACTGCGC CCGGAGCGAG
301 GAGAACTGGT ACGACTTCTT CGACTACTGG GGCCAGGGCA CCCTGGTGAC
351 CGTGTCCTCT (SEQ ID NO:156)
The amino acid sequence of the heavy chain variable region of V2.2 (CDRs based
on AbM definition shown in subsequent underscored regions) is as follows:
1 EVQLVESGGG LVQPGGSLRL SCAASGFNIK DTYIHWVRQA PGKGLEWVAR
51 IDPANDNIKY DPKFOGKATI SRDNAKNSLY LQMNSLRAED TAVYYCARSE
102 ENWYDFFDYW GQGTLVTVSS (SEQ ID NO:157)
The nucleotide sequence encoding the heavy chain variable region V2.3 with the
back mutations (R72A):
- 115 -
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
1 GAGGTGCAGC TGGTGGAGTC TGGCGGCGGA CTGGTGCAGC CTGGCGGCTC
51 TCTGCGGCTG TCTTGCGCCG CTTCCGGCTT CAACATCAAG GACACCTACA
101 TCCACTGGGT GCGGCAGGCT CCCGGCAAGG GCCTGGAGTG GGTGGCCCGG
151 ATCGATCCTG CCAACGACAA CATCAAGTAC GACCCCAAGT TCCAGGGCCG
201 GTTCACCATC TCTGCCGACA ACGCCAAGAA CTCCCTGTAC CTCCAGATGA
251 ACTCTCTGCG CGCCGAGGAT ACCGCCGTGT ACTACTGCGC CCGGAGCGAG
301 GAGAACTGGT ACGACTTCTT CGACTACTGG GGCCAGGGCA CCCTGGTGAC
351 CGTGTCCTCT (SEQ ID NO:158)
The amino acid sequence of the heavy chain variable region of V2.3 (CDRs based
on AbM definition shown in subsequent underscored regions) is as follows:
1 EVQLVESGGG LVQPGGSLRL SCAASGFNIK DTYIHWVRQA PGKGLEWVAR
51 IDPANDNIKY DPKFOGRFTI SADNAKNSLY LQMNSLRAED TAVYYCARSE
103 ENWYDFFDYW GQGTLVTVSS (SEQ ID NO:159)
The nucleotide sequence encoding the heavy chain variable region V2.4 with the
back mutations (A49G) is as follows:
1 GAGGTGCAGC TGGTGGAGTC TGGCGGCGGA CTGGTGCAGC CTGGCGGCTC
51 TCTGCGGCTG TCTTGCGCCG CTTCCGGCTT CAACATCAAG GACACCTACA
101 TCCACTGGGT GCGGCAGGCT CCCGGCAAGG GCCTGGAGTG GGTGGGCCGG
151 ATCGATCCTG CCAACGACAA CATCAAGTAC GACCCCAAGT TCCAGGGCCG
201 GTTCACCATC TCTCGCGACA ACGCCAAGAA CTCCCTGTAC CTCCAGATGA
251 ACTCTCTGCG CGCCGAGGAT ACCGCCGTGT ACTACTGCGC CCGGAGCGAG
301 GAGAACTGGT ACGACTTCTT CGACTACTGG GGCCAGGGCA CCCTGGTGAC
351 CGTGTCCTCT (SEQ ID NO:160)
The amino acid sequence of the heavy chain variable region of V2.4 (CDRs based
on AbM definition shown in subsequent underscored regions) is as follows:
I EVQLVESGGG LVQPGGSLRL SCAASGFNIK DTYIHWVRQA PGKGLEWVGR
51 IDPANDNIKY DPKFOGRFTI SRDNAKNSLY LQMNSLRAED TAVYYCARSE
104 ENWYDFFDYW GQGTLVTVSS (SEQ ID NO:161)
- 116 -
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
The nucleotide sequence encoding the heavy chain variable region V2.5 with the
back mutations (R67K;F68A;R72A) is as follows:
1 GAGGTGCAGC TGGTGGAGTC TGGCGGCGGA CTGGTGCAGC CTGGCGGCTC
51 TCTGCGGCTG TCTTGCGCCG CTTCCGGCTT CAACATCAAG GACACCTACA
101 TCCACTGGGT GCGGCAGGCT CCCGGCAAGG GCCTGGAGTG GGTGGCCCGG
151 ATCGATCCTG CCAACGACAA CATCAAGTAC GACCCCAAGT TCCAGGGCAA
201 GGCCACCATC TCTGCCGACA ACGCCAAGAA CTCCCTGTAC CTCCAGATGA
251 ACTCTCTGCG CGCCGAGGAT ACCGCCGTGT ACTACTGCGC CCGGAGCGAG
301 GAGAACTGGT ACGACTTCTT CGACTACTGG GGCCAGGGCA CCCTGGTGAC
352 CGTGTCCTCT (SEQ ID NO:162)
The amino acid sequence of the heavy chain variable region of V2.5 (CDRs based
on AbM definition shown in subsequent underscored regions) is as follows:
I EVQLVESGGG LVQPGGSLRL SCAASGFNIK DTYIHWVRQA PGKGLEWVAR
51 IDPANDNIKY DPKFOGKATI SADNAKNSLY LQMNSLRAED TAVYYCARSE
105 ENWYDFFDYW GQGTLVTVSS (SEQ ID NO:163)
The nucleotide sequence encoding the heavy chain variable region V2.6 with the
back mutations (V481;A49G;R72A) is as follows:
1 GAGGTGCAGC TGGTGGAGTC TGGCGGCGGA CTGGTGCAGC CTGGCGGCTC
51 TCTGCGGCTG TCTTGCGCCG CTTCCGGCTT CAACATCAAG GACACCTACA
101 TCCACTGGGT GCGGCAGGCT CCCGGCAAGG GCCTGGAGTG GATCGGCCGG
151 ATCGATCCTG CCAACGACAA CATCAAGTAC GACCCCAAGT TCCAGGGCCG
201 GTTCACCATC TCTGCCGACA ACGCCAAGAA CTCCCTGTAC CTCCAGATGA
251 ACTCTCTGCG CGCCGAGGAT ACCGCCGTGT ACTACTGCGC CCGGAGCGAG
301 GAGAACTGGT ACGACTTCTT CGACTACTGG GGCCAGGGCA CCCTGGTGAC
351 CGTGTCCTCT (SEQ ID NO: 164)
The amino acid sequence of the heavy chain variable region of V2.6 (CDRs based
on AbM definition shown in subsequent underscored regions) is as follows:
1 EVQLVESGGG LVQPGGSLRL SCAASGFNIK DTYIHWVRQA PGKGLEWIGR
- 117 -
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
51 IDPANDNIKY DPKFOGRFTI SADNAKNSLY LQMNSLRAED TAVYYCARSE
106 ENWYDFFDYW GQGTLVTVSS (SEQ ID NO: 165)
The nucleotide sequence encoding the heavy chain variable region V2.7 with the
back mutations (A49G;R72A) is as follows:
1 GAGGTGCAGC TGGTGGAGTC TGGCGGCGGA CTGGTGCAGC CTGGCGGCTC
51 TCTGCGGCTG TCTTGCGCCG CTTCCGGCTT CAACATCAAG GACACCTACA
101 TCCACTGGGT GCGGCAGGCT CCCGGCAAGG GCCTGGAGTG GGTGGGCCGG
151 ATCGATCCTG CCAACGACAA CATCAAGTAC GACCCCAAGT TCCAGGGCCG
201 GTTCACCATC TCTGCCGACA ACGCCAAGAA CTCCCTGTAC CTCCAGATGA
251 ACTCTCTGCG CGCCGAGGAT ACCGCCGTGT ACTACTGCGC CCGGAGCGAG
301 GAGAACTGGT ACGACTTCTT CGACTACTGG GGCCAGGGCA CCCTGGTGAC
351 CGTGTCCTCT (SEQ ID NO:166)
The amino acid sequence of the heavy chain variable region of V2.7 (CDRs based
on AbM definition shown in subsequent underscored regions) is as follows:
1 EVQLVESGGG LVQPGGSLRL SCAASGFNIK DTYIHWVRQA PGKGLEWVGR
51 IDPANDNIKY DPKFOGRFTI SADNAKNSLY LQMNSLRAED TAVYYCARSE
107 ENWYDFFDYW GQGTLVTVSS (SEQ ID NO:167)
The nucleotide sequence encoding the heavy chain variable region V2.8 with the
back mutations (L79A) is as follows:
1 GAGGTGCAGC TGGTGGAGTC TGGCGGCGGA CTGGTGCAGC CTGGCGGCTC
51 TCTGCGGCTG TCTTGCGCCG CTTCCGGCTT CAACATCAAG GACACCTACA
101 TCCACTGGGT GCGGCAGGCT CCCGGCAAGG GCCTGGAGTG GGTGGCCCGG
151 ATCGATCCTG CCAACGACAA CATCAAGTAC GACCCCAAGT TCCAGGGCCG
201 GTTCACCATC TCTCGCGACA ACGCCAAGAA CTCCGCCTAC CTCCAGATGA
251 ACTCTCTGCG CGCCGAGGAT ACCGCCGTGT ACTACTGCGC CCGGAGCGAG
301 GAGAACTGGT ACGACTTCTT CGACTACTGG GGCCAGGGCA CCCTGGTGAC
351 CGTGTCCTCT (SEQ ID NO:168)
- 118 -
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
The amino acid sequence of the heavy chain variable region of V2.8 (CDRs based
on AbM definition shown in subsequent underscored regions) is as follows:
1 EVQLVESGGG LVQPGGSLRL SCAASGFNIK DTYIHWVRQA PGKGLEWVAR
51 IDPANDNIKY DPKFOGRFTI SRDNAKNSAY LQMNSLRAED TAVYYCARSE
108 ENWYDFFDYW GQGTLVTVSS (SEQ ID NO:169)
The nucleotide sequence encoding the heavy chain variable region V2. 10 with
the
back mutations (A49G;R72A;L79A) is as follows:
1 GAGGTGCAGC TGGTGGAGTC TGGCGGCGGA CTGGTGCAGC CTGGCGGCTC
51 TCTGCGGCTG TCTTGCGCCG CTTCCGGCTT CAACATCAAG GACACCTACA
101 TCCACTGGGT GCGGCAGGCT CCCGGCAAGG GCCTGGAGTG GGTGGGCCGG
151 ATCGATCCTG CCAACGACAA CATCAAGTAC GACCCCAAGT TCCAGGGCCG
201 GTTCACCATC TCTGCCGACA ACGCCAAGAA CTCCGCCTAC CTCCAGATGA
251 ACTCTCTGCG CGCCGAGGAT ACCGCCGTGT ACTACTGCGC CCGGAGCGAG
301 GAGAACTGGT ACGACTTCTT CGACTACTGG GGCCAGGGCA CCCTGGTGAC
351 CGTGTCCTCT (SEQ ID NO:170)
The amino acid sequence of the heavy chain variable region of V2.10 (CDRs
based on AbM definition shown in subsequent underscored regions) is as
follows:
1 EVQLVESGGG LVQPGGSLRL SCAASGFNIK DTYIHWVRQA PGKGLEWVGR
51 IDPANDNIKY DPKFOGRFTI SADNAKNSAY LQMNSLRAED TAVYYCARSE
109 ENWYDFFDYW GQGTLVTVSS (SEQ ID NO:171)
The nucleotide sequence encoding the heavy chain variable region V2.11 with
the
back mutations (V481;A49G;R72A;L79A) is as follows:
1 GAGGTGCAGC TGGTGGAGTC TGGCGGCGGA CTGGTGCAGC CTGGCGGCTC
51 TCTGCGGCTG TCTTGCGCCG CTTCCGGCTT CAACATCAAG GACACCTACA
101 TCCACTGGGT GCGGCAGGCT CCCGGCAAGG GCCTGGAGTG GATCGGCCGG
151 ATCGATCCTG CCAACGACAA CATCAAGTAC GACCCCAAGT TCCAGGGCCG
201 GTTCACCATC TCTGCCGACA ACGCCAAGAA CTCCGCCTAC CTCCAGATGA
251 ACTCTCTGCG CGCCGAGGAT ACCGCCGTGT ACTACTGCGC CCGGAGCGAG
-119-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
301 GAGAACTGGT ACGACTTCTT CGACTACTGG GGCCAGGGCA CCCTGGTGAC
351 CGTGTCCTCT (SEQ ID NO:172)
The amino acid sequence of the heavy chain variable region of V2.11 (CDRs
based on AbM definition shown in subsequent underscored regions) is as
follows:
1 EVQLVESGGG LVQPGGSLRL SCAASGFNIK DTYIHWVRQA PGKGLEWIGR
51 IDPANDNIKY DPKFOGRFTI SADNAKNSAY LQMNSLRAED TAVYYCARSE
110 ENWYDFFDYW GQGTLVTVSS (SEQ ID NO: 173)
The nucleotide sequence encoding the heavy chain variable region V2.16 with
the
back mutations (V481;A49G;R72A) is as follows:
1 GAGGTGCAGC TGGTGGAGTC TGGCGGCGGA CTGGTGCAGC CTGGCGGCTC
51 TCTGCGGCTG TCTFGCACCG GCTCCGGCTT CAACATCAAG GACACCTACA
101 TCCACTGGGT GCGGCAGGCT CCCGGCAAGG GCCTGGAGTG GATCGGCCGG
151 ATCGATCCTG CCAACGACAA CATCAAGTAC GACCCCAAGT TCCAGGGCCG
201 GTTCACCATC TCTGCCGACA ACGCCAAGAA CTCCCTGTAC CTCCAGATGA
251 ACTCTCTGCG CGCCGAGGAT ACCGCCGTGT ACTACTGCGC CCGGAGCGAG
301 GAGAACTGGT ACGACTTCTr CGACTACTGG GGCCAGGGCA CCCTGGTGAC
351 CGTGTCCTCT (SEQ ID NO:174)
The amino acid sequence of the heavy chain variable region of V2.16 (CDRs
based on AbM definition shown in subsequent underscored regions) is as
follows:
1 EVQLVESGGG LVQPGGSLRL SCTGSGFNIK DTYIHWVRQA PGKGLEWIGR
51 IDPANDNIKY DPKFOGRFTI SADNAKNSLY LQMNSLRAED TAVYYCARSE
111 ENWYDFFDYW GQGTLVTVSS (SEQ ID NO: 175)
The following is the amino acid sequence of a humanized MH 2-7 V2.11 IgGI
with a mutated CH2 domain:
EVQLVES GGGLV QPGGSLRLSCAASGFNIKDTYIH W VRQAPGKGLE WIGRIDPA
NDNIKYDPKFQGRFTISADNAKNSAYLQMNSLRAEDTAVYYCARSEENWYDFF
DYWGQGTLVTV S SASTKGPS VFPLAPS SKSTSGGTAALGCLVKDYFPEPVTV S W
NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK
KVEPKSCDKTHTCPPCPAPEALGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE
-120-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
DPEVKFNWYVD GV E V HNAKTKPREEQYNS TYRV V S V LT V LHQD WLNGKEYKC
KV SNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQV SLTCLVKGFYPSDI
AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE
ALHNHYTQKSLSLSPGK (SEQ ID NO:176)
The variable domain is at amino acids 1-120; CH1 at 121-218; hinge at 219-233;
CH2 at 234-343; and CH3 at 344-450. The light chain includes the following
sequence
with variable domain at 1-133.
DIQMTQSPS SLSAS VGDRVTITCRS SQSIVHSNGNTYLEWYQQKPGKAPKLLIYK
VSNRFSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCFQGSHIPYTFGGGTKVEIK
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQE
SVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
(SEQ ID NO:177)
Example 10: Functional Assays of Exemplary Variants of MJ2-7
The ability of the MJ2-7 antibody and humanized variants was evaluated to
inhibit human IL- 13 in assays for IL- 13 activity.
STAT6 phosphorylation assay.
HT-29 human colonic epithelial cells (ATCC) were grown as an adherent
monolayer in McCoy's 5A medium containing 10% FBS, Pen-Strep, glutamine, and
sodium bicarbonate. For assay, the cells were dislodged from the flask using
trypsin,
washed into fresh medium, and distributed into 12x75 mm polystyrene tubes.
Recombinant human IL-13 (R&D Systems, Inc.) was added at concentrations
ranging
from 100 - 0.01 ng/ml. For assays testing the ability of antibody to inhibit
the IL-13
response, 1 ng/ml recombinant human IL-13 was added along with dilutions of
antibody
ranging from 500 - 0.4 ng/ml. Cells were incubated in a 37 C water bath for 30-
60
minutes, then washed into ice-cold PBS containing 1% BSA. Cells were fixed by
incubating in 1% paraformaldehyde in PBS for 15 minutes at 37 C, then washed
into
PBS containing 1% BSA. To permeabilize the nucleus, cells were incubated
overnight at
-20 C in absolute methanol. They were washed into PBS containing 1% BSA, then
stained with ALEXAT"" Fluor 488-labeled antibody to STAT6 (BD Biosciences).
- 121 -
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
Fluorescence was analyzed with a FACSCANT"' and CELLQUESTTM' software (BD
Biosciences).
CD23 induction on human monocytes
Mononuclear cells were isolated from human peripheral blood by layering over
HISTOPAQUE (Sigma). Cells were washed into RPMI containing 10% heat-
inactivated FCS, 50 U/ml penicillin, 50 mg/mi streptomycin, 2 mM L-glutamine,
and
plated in a 48-well tissue culture plate (Costar/Coming). Recombinant human IL-
13
(R&D Systems, Inc.) was added at dilutions ranging from 100 - 0.01 ng/ml. For
assays
testing the ability of antibody to inhibit the IL-13 response, 1 ng/ml
recombinant human
IL-13 was added along with dilutions of antibody ranging from 500 - 0.4 ng/ml.
Cells
were incubated overnight at 37 C in a 5% COz incubator. The next day, cells
were
harvested from wells using non-enzymatic Cell Dissociation Solution (Sigma),
then
washed into ice-cold PBS containing 1% BSA. Cells were incubated with
phycoerythrin
(PE)-labeled antibody to human CD23 (BD Biosciences, San Diego, CA), and Cy-
Chrome-labeled antibody to human CD11b (BD Biosciences). Monocytes were gated
based on high forward and side light scatter, and expression of CDl lb. CD23
expression
on monocytes was determined by flow cytometry using a FACSCANTM (BD
Biosciences), and the percentage of CD23+ cells was analyzed with CELLQUESTT""
software (BD Biosciences).
TF-1 cell proliferation
TF-1 cells are a factor-dependent human hemopoietic cell line requiring
interleukin 3 (IL-3) or granulocyte/macrophage colony-stimulating factor (GM-
CSF) for
their long-term growth. TF-1 cells also respond to a variety of other
cytokines, including
interleukin 13 (IL-13). TF-1 cells (ATCC) were maintained in RPMI medium
containing
10% heat-inactivated FCS, 50 U/ml penicillin, 50 mg/ml streptomycin, 2 mM
L-glutamine, and 5 ng/ml recombinant human GM-CSF (R&D Systems). Prior to
assay,
cells were starved of GM-CSF overnight. For assay, TF-1 cells were plated in
duplicate
at 5000 cells / well in 96-well flat-bottom microtiter plates (Costar/Coming),
and
challenged with human IL-13 (R&D Systems), ranging from 100 - 0.01 ng/ml.
After 72
hours in a 37 C incubator with 5% CO2, the cells were pulsed with 1 Ci /
we113H-
thymidine (Perkin Elmer / New England Nuclear). They were incubated an
additional
-122-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
4.5 hours, then cells were harvested onto filter mats using a TOMTEKTM
harvester. 3H-
thymidine incorporation was assessed by liquid scintillation counting.
Tenascin production assay
BEAS-2B human bronchial epithelial cells (ATCC) were maintained BEGM
media with supplements (Clonetics). Cells were plated at 20,000 per well in a
96-well
flat-bottom culture plate overnight. Fresh media is added containing IL-13 in
the
presence or absence of the indicated antibody. After overnight incubation, the
supernatants are harvested, and assayed for the presence of the extracellular
matrix
component, tenascin C, by ELISA. ELISA plates are coated overnight with 1
ug/ml of
murine monoclonal antibody to human tenascin (IgGl, k; Chemicon International)
in
PBS. Plates are washed with PBS containing 0.05% TWEEN -20 (PBS-Tween), and
blocked with PBS containing 1% BSA. Fresh blocking solution was added every 6
minutes for a total of three changes. Plates were washed 3X with PBS-Tween.
Cell
supernatants or human tenascin standard (Chemicon International) were added
and
incubated for 60 minutes at 37 C. Plates were washed 3X with PBS-Tween.
Tenascin
was detected with murine monoclonal antibody to tenascin (IgG2a, k; Biohit).
Binding
was detected with HRP-labeled antibody to mouse IgG2a, followed by TMB
substrate.
The reaction was stopped with 0.01 N sulfuric acid. Absorbance was read at 450
nm.
The HT 29 human epithelial cell line can be used to assay STAT6
phosphorylation. HT 29 cells are incubated with 1 ng/ml native human IL- 13
crude
preparation in the presence of increasing concentrations of the test antibody
for 30
minutes at 37 C. Western blot analysis of cell lysates with an antibody to
phosphorylated STAT6 can be used to detect dose-dependent IL 13-mediated
phosphorylation of STAT6. Similarly, flow cytometric analysis can detect
phosphorylated STAT6 in HT 29 cells that were treated with a saturating
concentration of
IL-13 for 30 minutes at 37 C, fixed, permeabilized, and stained with an
ALEXATM Fluor
488-labeled mAb to phospho-STAT6. An exemplary set of results is set forth in
the
Table 1. The inhibitory activity of V2.11 was comparable to that of sIL-13Ra2-
Fc.
-123-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
Table 1
Construct Backmutations Expression Native hIL-13
VH g/ml / STAT6 assay
H VL COS; 48h IC 50, nM
2.0 2 one, CDR grafted 8-10 >100
CDR graft
2.1 2 V481; A49G 9-14 2.8
2.2 2 67K; F68A 5-6 >100
2.3 2 72A 8-9 1.67 - 2.6
2.4 2 49G 10 17.5
2.5 2 67K; F68A; R72A 4-5 1.75
2.6 2 481; A49G: R72A 11-12 1.074 - 3.37
2.7 2 49G; R72A 10-11 1.7
2.11 2 481; A49G: 24 0.25 - 0.55
72A:L79A
Example 11: Binding Interaction Site Between IL-13 and IL-13Ra1
A complex of IL-13, the extracellular domain of IL-13Ral (residues 27-342 of
SEQ ID NO: 125), and an antibody that binds human IL-13 was studied by x-ray
crystallography. See, e.g., US 07/0048785. Two points of substantial
interaction were
found between IL- 13 and IL-13Ral. The interaction between Ig domain 1 of IL-
13Ra1
and IL-13 results in the formation of an extended beta sheet spanning the two
molecules.
Residues Thr88 [Thr107], Lys89 [Lys108], I1e90 [I1e109], and G1u91 [G1u110] of
IL-13
(SEQ ID NO:124, mature sequence [full-length sequence (SEQ ID NO:178)]) form a
beta
strand that interacts with residues Lys76, Lys77, I1e78 and A1a79 of the
receptor (SEQ ID
NO:125). Additionally, the side chain of Met33 [Met52] of IL-13 (SEQ ID NO:124
[SEQ ID NO: 178]) extends into a hydrophobic pocket that is created by the
side chains of
these adjoining strands.
The predominant feature of the interaction with Ig domain 3 is the insertion
of a
hydrophobic residue (Phe107 [Phe126]) of IL-13 (SEQ ID NO:124 [SEQ ID NO:178])
into a hydrophobic pocket in Ig domain 3 of the receptor IL-13Ral. The
hydrophobic
pocket of IL-13Ra1 is formed by the side chains of residues Leu319, Cys257,
Arg256,
and Cys320 (SEQ IDNO:125). The interaction with Phe107 [Phe126] of IL-13 (SEQ
ID
-124-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
NO: 124 [SEQ ID NO: 178]) results in an extensive set of van der Waals
interactions
between amino acid residues I1e254, Ser255, Arg256, Lys318, Cys320, and Tyr321
of
IL-13Ra1 (SEQ ID NO:125) and amino acid residues Argl 1[Arg30], G1u12 [G1u31],
Leu13 [Leu32], I1e14 [Ile33], Glul5 [I1e34], Lys104 [Lys123], Lys105 [Lys124],
Leu106
[Leu125], Phe107 [Phe126], and Arg108 [Arg 127] of IL-13 (SEQ ID NO:124 [SEQ
ID
NO: 178]). These results demonstrate that an IL- 13 binding agent that binds
to the
regions of IL-13 involved in interaction with IL-13Ra1 can be used to inhibit
IL-13
signaling.
Example 12: Expression of humanized MJ 2-7 antibody in COS cells
To evaluate the production of chimeric anti-NHP IL13 antibodies in the
mammalian recombinant system, the variable regions of mouse MJ 2-7 antibody
were
subcloned into a pED6 expression vector containing human kappa and IgGlmut
constant
regions. Monkey kidney COS-1 cells were grown in DME media (Gibco) containing
10% heat-inactivated fetal bovine serum, 1 mM glutamine and 0.1 mg/ml
Penicillin/
Streptomycin. Transfection of COS cells was performed using TRANSITITT"'-LT1
Transfection reagent (Mirus) according to the protocol suggested by the
reagent supplier.
Transfected COS cells were incubated for 24 hours at 37 C in the presence of
10% CO2,
washed with sterile PBS, and then grown in serum-free media R1CD1 (Gibco) for
48
hours to allow antibody secretion and accumulation in the conditioned media.
The
expression of chMJ 2-7 antibody was quantified by total human IgG ELISA using
purified human IgGI/kappa antibody as a standard.
The production of chimeric MJ 2-7 antibody in COS cells was significantly
lower
then the control chimeric antibody (Table 2). Therefore, optimization of Ab
expression
was included in the MJ 2-7 humanization process. The humanized MJ 2-7 V 1 was
constructed by CDR grafting of mouse MJ 2-7 heavy chain CDRs onto the most
homologous human germline clone, DP 25, which is well expressed and
represented in
typical human antibody response. The CDRs of light chain were subcloned onto
human
germline clone DPK 18 in order to generate huMJ 2-7 V 1 VL. The humanized MJ 2-
7
V2 was made by CDR grafting of CDRs MJ 2-7 heavy chain variable region onto
DP54
human germline gene framework and CDRs of MJ 2-7 light chain variable region
onto
- 125 -
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
DPK9 human germline gene framework. The DP 54 clone belongs to human VH III
germline subgroup and DPK9 is from the V kappa I subgroup of human germline
genes.
Antibody molecules that include VH III and V kappa I frameworks have high
expression
level in E. coli system and possess high stability and solubility in aqueous
solutions (see,
e.g., Stefan Ewert et al., J. Mol. Biol. (2003), 325; 531-553, Adrian Auf et
al., Methods
(2004) 34:215-224). We have used the combination of DP54/DPK9 human frameworks
in the production of several recombinant antibodies and have achieved a high
expression
of antibody (> 20 g/ml) in the transient COS transfection experiments.
Table 2
mAb Expression, g/ml
3D6 10.166
Ch MJ 2-7 pED6 (1) 2.44
Ch MJ 2-7pED6 (2) 2.035
h12A11 V2 1.639
The CDR grafted MJ 2-7 V 1 and V2 VH and VL genes were subcloned into two
mammalian expression vector systems (pED6kappa/pED6 IgGlmut and pSMEN2kappa/
pSMED2IgGlmut), and the production of humanized MJ 2-7 antibodies was
evaluated in
transient COS transfection experiments as described above. In the first set of
the
experiments the effect of various combinations of huMJ 2-7 VL and VH on the
antibody
expression was evaluated (Table 3). Changing of MJ 2-7 VL framework regions to
DKP9 increased the antibody production 8-10 fold, whereas VL V1 (CDR grafted
onto
DPK 18) showed only a moderate increase in antibody production. This effect
was
observed when humanized VL was combined with chimeric MJ 2-7 VH and humanized
MJ 2-7 V 1 and V2. The CDR grafted MJ 2-7 V2 had a 3-fold higher expression
level
then CDR grafted MJ 2-7 V 1 in the same assay conditions.
Table 3
mAb Expression, g/ml
- 126 -
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
ChMJ 2-7 1.83
hVH V1/ mVL 3.04
hVH V 1/ hVL V 1 6.34
hVH Vl/ hVL V2 15.4
hVH-V2 / mVL 0.2
mVH / hVL-V2 18.41
hVH-V2 / hVL-V1 5.13
hVH-V2 / hVL-V2 10.79
Similar experiments were performed with huMJ 2-7 V2 containing back
mutations in the heavy chain variable regions (Table 4). The highest
expression level
was detected for huMJ 2-7 V2.11 that retained the antigen binding and
neutralization
properties of mouse MJ 2-7 antibody. Introduction of back mutations at the
positions 48
and 49 (V481 and A49G) increased the production of huMJ 2-7 V2 antibody in COS
cells, whereas the back mutations of amino acids at the positions 23, 24, 67
and 68
(A23T; A24G; R67K and F68A) had a negative impact on antibody expression.
Table 4
mAb Expression, g/ml
V2 8.27
V2.1 12.1
V2.2 5.29
V2.3 9.60
V2.4 8.20
V2.5 6.05
V2.6 11.3
V2.10 9.84
V2.11 14.85
V2.16 1.765
- 127 -
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
Example 13: Evaluation of antigen binding properties of humanized MJ 2-7
antibodies
by NHP IL- 13 FLAG ELISA
The ability of fully humanized MJ 2-7 mAb (V 1, V2 v2) to compete with
biotinylated mouse MJ 2-7 Ab for binding to NHP IL-13-FLAG was evaluated by
ELISA. The microtiter plates (Costar) were coated with 1 g/ml of anti-FLAG
monoclonal antibody M2 (Sigma). The FLAG NHP IL-13 protein at concentration of
ng/ml was mixed with 10 ng/ml of biotin labeled mouse MJ 2-7 antibody and
various
concentrations of unlabeled mouse and humanized MJ 2-7 antibody. The mixture
was
incubated for 2 hours at room temperature and then added to the anti-FLAG
antibody-
10 coated plate. Binding of FLAG NHP-IL-13/ bioMJ2-7 Ab complexes was detected
with
streptavidin-HRP and 3,3',5,5'-tetramethylbenzidine (TMB). The humanized MJ 2-
7 V2
significantly lost activity whereas huMJ 2-7 V2.11 completely restored the
antigen
binding activity and was capable of competing with biotinylated MJ 2-7 mAb for
binding
to FLAG-NHP IL-13. BIACORETM analysis also confirmed that NHP IL-13 had rapid
binding to and slow dissociation to immobilized h1uMJ 2-7 v2.11.
Example 14: Molecular modeling of humanized MJ2-7 V2VH
Structure templates for modeling humanized MJ2-7 heavy chain version 2 (MJ2-7
V2VH) were selected based on BLAST homology searches against Protein Data Bank
(PDB). Besides the two structures selected from the BLAST search output, an
additional
template was selected from an in-house database of protein structures. Model
of MJ2-7
V2VH was built using the three template structures 1JPS (co-crystal structure
of human
tissue factor in complex with humanized Fab D3h44), IN8Z (co-crystal structure
of
human Her2 in complex with Herceptin Fab) and F 13.2 (IL-13 in complex with
mouse
antibody Fab fragment) as templates and the Homology module of Insightll
(Accelrys,
San Diego). The structurally conserved regions (SCRs) of 1JPS, IN8Z and F 13.2
(available from 16163-029001) were determined based on the Ca distance matrix
for
each molecule and the template structures were superimposed based on minimum
RMS
deviation of corresponding atoms in SCRs. The sequence of the target protein
MJ2-7
V2VH was aligned to the sequences of the superimposed templates proteins and
coordinates of the SCRs were assigned to the corresponding residues of the
target protein.
Based on the degree of sequence similarity between the target and the
templates in each
- 128 -
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
of the SCRs, coordinates from different templates were used for different
SCRs.
Coordinates for loops and variable regions not included in the SCRs were
generated by
Search Loop or Generate Loop methods as implemented in Homology module.
Briefly,
Search Loop method scans protein structures that would fit properly between
two SCRs
by comparing the Ca distance matrix of flanking SCR residues with a pre-
calculated
matrix derived from protein structures that have the same number of flanking
residues
and an intervening peptide segment of a given length. Generate Loop method
that
generate atom coordinates de novo was used in those cases where Search Loops
did not
produce desired results. Conformation of amino acid side chains was kept the
same as
1o that in the template if the amino acid residue was identical in the
template and the target.
However, a conformational search of rotamers was done and the energetically
most
favorable conformation was retained for those residues that are not identical
in the
template and target. This was followed by Splice Repair that sets up a
molecular
mechanics simulation to derive proper bond lengths and bond angles at
junctions between
two SCRs or between SCR and a variable region. Finally the model was subjected
to
energy minimization using Steepest Descents algorithm until a maximum
derivative of
5 kcal/(mol A) or 500 cycles and Conjugate Gradients algorithm until a maximum
derivative of 5 kcal/(mol A) or 2000 cycles. Quality of the model was
evaluated using
ProStat/Struct Check command.
Molecular model of mouse MJ2-7 VH was built by following the procedure
described for humanized MJ2-7 V2VH except the templates used were 1 QBL and
1 QBM, crystal structures for horse anti-cytochrome c antibody FabE8.
Potential differences in CDR-Framework H-bonds predicted by the models
hMJ2-7 V2VH:G26 - hMJ2-7 V2VH:A24
hMJ2-7 V2VH:Y109 - hMJ2-7 V2VH:S25
mMJ2-7 VH:D61 - mMJ2-7 VH:148
mMJ2-7 VH:K63 - mMJ2-7 VH:E46
mMJ2-7 VH:Y109 - mMJ2-7 VH:R98
These differences suggested the following optional back mutations: A23T, A24G
and
V481.
-129-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
Other optional back mutations suggested based on significant RMS deviation of
individual amino acids and differences in amino acid residues adjacent to
these are: G9A,
L115T and R87T.
Example 15: IL-13 neutralization activity of MJ2-7 and C65
The IL-13 neutralization capacities of MJ2-7 and C65 were tested in a series
of
bioassays. First, the ability of these antibodies to neutralize the
bioactivity of NHP IL-13
was tested in a monocyte CD23 expression assay. Freshly isolated human PBMC
were
incubated overnight with 3 ng/ml NHP IL- 13 in the presence of increasing
concentrations
of MJ2-7, C65, or sIL-13Ra2-Fc. Cells were harvested, stained with CYCHROMET"'-
labeled antibody to the monocyte-specific marker, CD11b, and with PE-labeled
antibody
to CD23. In response to IL- 13 treatment, CD23 expression is up-regulated on
the surface
of monocytes, which were gated based on expression of CD1 lb. MJ2-7, C65, and
sIL13Ra2-Fc all were able to neutralize the acitivity of NHP IL-13 in this
assay. The
potencies of MJ2-7 and sIL-13Ra2-Fc were equivalent. C65 was approximately 20-
fold
less active (FIG. 2).
In a second bioassay, the neutralization capacities of MJ2-7 and C65 for
native
human IL- 13 were tested in a STAT6 phosphorylation assay. The HT-29
epithelial cell
line was incubated with 0.3 ng/ml native human IL-13 in the presence of
increasing
concentrations of MJ2-7, C65, or sIL-13Ra2-Fc, for 30 minutes at 37 C. Cells
were
fixed, permeabilized, and stained with ALEXATM Fluor 488-labeled antibody to
phosphorylated STAT6. IL-13 treatment stimulated STAT6 phosphorylation. MJ2-7,
C65, and sIL13Ra2-Fc all were able to neutralize the acitivity of native human
IL-13 in
this assay (FIG. 3). The IC50's for the murine MJ-27 antibody and the
humanized form
(V2.1 1) were 0.48 nM and 0.52 nM respectively. The potencies of MJ2-7 and
sIL-13Ra2-Fc were approximately equivalent. The IC50 for sIL-13Ra2-Fc was 0.33
nM
(FIG. 4). C65 was approximately 20-fold less active (FIG. 5).
In a third bioassay, the ability of MJ2-7 to neutralize native human IL-13 was
tested in a tenascin production assay. The human BEAS-2B lung epithelial cell
line was
incubated overnight with 3 ng/ml native human IL- 13 in the presence of
increasing
concentrations of MJ2-7. Supernatants were harvested and tested for production
of the
- 130 -
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
extracellular matrix protein, tenascin C, by ELISA (FIG. 6A). MJ2-7 inhibited
this
response with IC50 of approximately 0.1 nM (FIG. 6B).
These results demonstrate that MJ2-7 is an effective neutralizer of both NHP
IL-
13 and native human IL-13. The IL-13 neutralization capacity of MJ2-7 is
equivalent to
that of sIL-13Ra2-Fc. MJ1-65 also has IL-13 neutralization activity, but is
approximately 20-fold less potent than MJ2-7.
Example 16: Epitope mapping of MJ2-7antibody by SPR
sIL-13Ra2-Fc was directly coated onto a CM5 chip by standard amine coupling.
lo NHP-IL-13 at 100 nM concentration was injected, and its binding to the
immobilized IL-
13Ra2-Fc was detected by BIACORET"'. An additional injection of 100 nM of anti
IL-
13 antibodies was added, and changes in binding were monitored. MJ2-7 antibody
did
not bind to NHP-IL-13 when it was in a complex with hu IL-13Ra2, whereas a
positive
control anti-IL-13 antibody did (FIG. 7). These results indicate that hu IL-
13Ra2 and
MJ2-7 bind to the same or overlapping epitopes of NHP IL-13.
Examnle 17: Measurement of kinetic rate constants for the interaction between
NHP-
IL- 13 and humanized MJ2-7 V2-11 antibody
To prepare the biosensor surface, goat anti-human IgG Fc specific antibody was
immobilized onto a research-grade carboxy methyl dextran chip (CM5) using
amine
coupling. The surface was activated with a mixture of 0.1 M 1-ethyl-3-(3-
dimethylaminopropyl) carbodiimide (EDC) and 0.05 M N-Hydroxysuccinimide (NHS).
The capturing antibody was injected at a concentration of 10 g/ml in sodium
acetate
buffer (pH 5.5). Remaining activated groups were blocked with 1.0 M
ethanolamine (pH
8.0). As a control, the first flow cell was used as a reference surface to
correct for bulk
refractive index, matrix effect,s and non-specific binding, the second, third
and fourth
flow cells were coated with the capturing molecule.
For kinetic analysis, the monoclonal antibody hMJ2-7 V2-11 was captured onto
the anti IgG antibody surface by injecting 40 l of a 1 g/mi solution. The
net difference
between the baseline and the point approximately 30 seconds after completion
of
injection was taken to represent the amount of target bound. Solutions of NHP-
IL-13 at
- 131 -
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
600, 200, 66.6, 22.2, 7.4, 2.5, 0.8, 0.27, 0.09 and 0 nM concentrations were
injected in
triplicate at a flow rate of 100 l per min for 2 minutes, and the amount of
bound material
as a function of time was recorded (FIG. 8). The dissociation phase was
monitored in
HBS/EP buffer (10 mM HEPES, pH 7.4, containing 150 mM NaCI, 3 mM EDTA and
0.005% (v/v) Surfactant P20) for 5 minutes at the same flow rate followed by
two 5 111
injections of glycine, pH 1.5, to regenerate a fully active capturing surface.
All kinetic
experiments were done at 22.5 C in HBS/EP buffer. Blank and buffer effects
were
subtracted for each sensorgram using double referencing.
The kinetic data were analyzed using BIAEVALUATIONT"' software 3Ø2
applied to a 1:1 model. The apparent dissociation (kd) and association (ka)
rate constants
were calculated from the appropriate regions of the sensorgrams using a global
analysis.
The affinity constant of the interaction between antibody and NHP IL- 13 was
calculated
from the kinetic rate constants by the following formula: Kd = kd / ka. These
results
indicate that huMJ2-7 V2-11 has on and off-rates of 2.05x107 M-'s"1 and
8.89x10' 1/s,
respectively, resulting in an antibody with 43 pM affinity for NHP-IL- 13.
Example 18: Inhibitory activity of MJ2-7 humanization intermediates in
bioassays
The inhibitory activity of various intermediates in the humanization process
was
tested by STAT6 phosphorylation and tenascin production bioassays. A sub-
maximal
level of NHP IL-13 or native human IL-13 crude preparation was used to elicit
the
biological response, and the concentration of the humanized version of MJ2-7
required
for half-maximal inhibition of the response was determined. Analysis hMJ2-7 V
1, hMJ2-
7 V2 and hMJ2-7 V3, expressed with the human IgGl, and kappa constant regions,
showed that Version 2 retained neutralization activity against native human IL-
13. This
concentration of the Version 2 humanized antibody required for half-maximal
inhibition
of native human IL-13 bioactivity was approximately 110-fold greater than that
of murine
MJ2-7 (FIG. 9). Analysis of a semi-humanized form, in which the V 1 or V2 VL
was
combined with murine MJ2-7 VH, demonstrated that the reduction of native human
IL-
13 neutralization activity was not due to to the humanized VL, but rather to
the VH
sequence (FIG. 10). Whereas the semi-humanized MJ2-7 antibody with VL V 1 only
partially retained the neutralization activity the version with humanized VL
V2 was as
- 132 -
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
active as parental mouse antibody. Therefore, a series of back-mutations were
introduced
into the V1 VH sequence to improve the native human IL-13 neutralization
activity of
murine MJ2-7.
Example 19: MJ2-7 blocks IL-13 interaction with IL-13Ra1 and IL-13Ra2
MJ2-7 is specific for the C-terminal 19-mer of NHP IL-13, corresponding to
amino acid residues 114 - 132 of the immature protein (SEQ ID NO:24), and
residues 95
- 113 of the mature protein (SEQ ID NO:14). For human IL-13, this region,
which forms
part of the D alpha-helix of the protein, has been reported to contain
residues important
for binding to both IL-13Ra1 and IL-13Ra2. Analysis of human IL-13 mutants
identified the A, C, and D-helices as containing important contacts site for
the IL-13Ral /
IL-4Ra signaling complex (Thompson and Debinski (1999) J. Biol. Chem. 274:
29944-
50). Alanine scanning mutagenesis of the D-helix identified residues K123,
K124, and
R127 (SEQ ID NO:24) as responsible for interaction with IL-13Ra2, and residues
E110,
E128, and L122 as important contacts for IL-13Ral (Madhankmuar et al. (2002)
J. Biol.
Chem. 277: 43194-205). High resolution solution structures of human IL-13
determined
by NMR have predicted the IL-13 binding interactions based on similarities to
related
ligand-receptor pairs of known structure. These NMR studies have supported a
key role
for the IL-13 A and D-helices in making important contacts with IL-13Ra1
(Eisenmesser
et al. (2001) J Mol. Biol. 310:231-241; Moy et al. (2001) J. Mol. Biol.
310:219-230).
Binding of MJ2-7 to this epitope located in the C-terminal, D-helix of IL-13
was
predicted to disrupt interaction of IL-13 with IL-13Ral and IL-13Ra2.
The ability of MJ2-7 to inhibit binding of NHP IL-13 to IL-13Ra l and IL-13Ra2
was tested by ELISA. Recombinant soluble forms of human IL-13Ral-Fc and IL-
13Ra2-Fc were coated onto ELISA plates. FLAG-tagged NHP IL-13 was added in the
presence of increasing concentrations of MJ2-7. Results showed that MJ2-7
competed
with both soluble receptor forms for binding to NHP IL-13 (FIGs. 11A and 11B).
This
provides a basis for the neutralization of IL-13 bioactivity by MJ2-7.
- 133 -
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
Example 20: The MJ 2-7 light chain CDRs contribute to antigen binding
To evaluate if all three light chain CDR regions are required for the binding
of MJ
2-7 antibody to NHP IL-13, two additional humanized versions of MJ 2-7 VL were
constructed by CDR grafting. The VL version 3 was designed based on human
germline
clone DPK18, contained CDR1 and CDR2 of the human germline clone and CDR3 from
mouse MJ2-7 antibody (FIG 12). In the second construct (hMJ 2-7 V4), only CDR1
and
CDR2 of MJ 2-7 antibody were grafted onto DPK 18 framework, and CDR3 was
derived
from irrelevant mouse monoclonal antibody.
The humanized MJ 2-7 V3 and V4 were produced in COS cells by combining
hMJ 2-7 VH V 1 with hMJ 2-7 VL V3 and V4. The antigen binding properties of
the
antibodies were examined by direct NHP IL-13 binding ELISA. The hMJ 2-7 V4 in
which MJ 2-7 light chain CDR3 was absent retained the ability to bind NHP IL-
13,
whereas V3 that contained human germline CDRI and CDR2 in the light chain did
not
bind to immobilized NHP IL-13. These results demonstrate that CDR1 and CDR2 of
MJ
2-7 antibody light chain are most likely responsible for the antigen binding
properties of
this antibody.
Nucleotide sequence of hMJ 2-7 VL V3
1 ATGCGGCTGC CCGCTCAGCT GCTGGGCCTG CTGATGCTGT GGGTGCCCGG
51 CTCTTCCGGC GACGTGGTGA TGACCCAGTC CCCTCTGTCT CTGCCCGTGA
101 CCCTGGGCCA GCCCGCTTCT ATCTCTTGCC GGTCCTCCCA GTCCCTGGTG
151 TACTCCGACG GCAACACCTA CCTGAACTGG TTCCAGCAGA GACCCGGCCA
201 GTCTCCTCGG CGGCTGATCT ACAAGGTGTC CAACCGCTTT TCCGGCGTGC
251 CCGATCGGTT CTCCGGCTCC GGCAGCGGCA CCGATTTCAC CCTGAAGATC
301 AGCCGCGTGG AGGCCGAGGA TGTGGGCGTG TACTACTGCT TCCAGGGCTC
351 CCACATCCCT TACACCTTTG GCGGCGGAAC CAAGGTGGAG ATCAAG
(SEQ ID NO:189)
Amino acid sequence of hMJ 2-7 VL V3
MRLPAQLLGLLMLWVPGSSG- DVVMTQSPLSLPVTLGQPASISCRSSOSLVYSDGNTYLNW
FQQRPGQSPRRLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCF GSHIP
- 134 -
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
YTFGGGTKVEIK (SEQ ID NO:190)
Nucleotide sequence of hMJ 2-7 VL V4
GATGTTGTGATGACCCAATCTCCACTCTCCCTGCCTGTCACTCCTGGAGAGCCAGCCTCC
ATCTCTTGCAGATCTAGTCAGAGCATTGTGCATAGTAATGGAAACACCTACCTGGAATGG
TACCTGCAGAAACCAGGCCAGTCTCCACAGCTCCTGATCTACAAAGTTTCCAACCGATTT
TCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATC
AGCAGAGTGGAGGCTGAGGATGTGGGAGTTTATTACTGCTTTCAAAGTTCACATGTTCCT
CTCACCTTCGGTCAGGGGACCAAGCTGGAGATCAAA (SEQ ID NO:191)
Amino acid sequence of hMJ 2-7 VL V4
DVVMTQSPLS LPVTPGEPAS ISCRSSOSIV HSNGNTYLEW YLQKPGQSPQ LLIYKVSNRF
SGVPDRFSGS GSGTDFTLKISRVEAEDVGV YYCFOSSHVP LTFGQGTKLE IK (SEQ ID NO:192)
Example 21: Neutralizing Activities of Anti-IL13 Antibodies in Cynomolgus
Monkey
Model
The efficacy of an IL-13 binding agent (e.g., an anti-IL13 antibody) in
neutralizing one or more IL-13-associated activities in vivo can be tested
using a model of
antigen-induced airway inflammation in cynomolgus monkeys naturally allergic
to
Ascaris suum. These assays can be used to confirm that the binding agent
effectively
reduces airway eosinophilia in allergic animals challenged with an allergen.
In this
model, challenge of an allergic monkey with Ascaris suum antigen results in
one or more
of the following: (i) an influx of inflammatory cells, e.g., eosinophils into
the airways;
(ii) increased eotaxin levels; (iii) increase in Ascaris-specific basophil
histamine release;
and/or (iv) increase in Ascaris-specific IgE titers.
To test the ability of an anti-IL- 13 antibody to prevent the influx of
inflammatory
cells, the antibody can be administered 24 hours prior to challenge with
Ascaris suum
antigen. On the day of challenge, a baseline bronchoalveolar lavage (BAL)
sample can
be obtained from the left lung. Ascaris suum antigen can be instilled
intratracheally into
the right lung. Twenty-four hours later, the right lung is lavaged, and the
BAL fluid from
animals treated intravenously with the antibody were compared to BAL fluid
from
untreated animals. If the antibody reduces airway inflammation, an increase in
percent
-135-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
BAL eosinophils may be observed among the untreated group, but not for the
antibody-
treated group.
FIGS. 14A-14D depict an increase in the total number of cells and percentage
of
inflammatory cells, for example, eosinophils (FIG. 14B), neutrophils (FIG.
14C) and
macrophages (FIG. 14D) 24-hours following airway challenge with Ascaris. A
statistically significant increase in the percentage of inflammatory cells was
observed 24
hours after challenge compared to the baseline values.
Anti-IL13 antibodies (humanized MJ2-7v.2-11 and humanized mAb13.2v.2) were
administered to cynomolgus monkeys 24 hours prior to challenge with Ascaris
suum
antigen. (mAb 13.2 and its humanized form hmAb13.2v2 were described in
commonly
owned PCT application WO 05/123126, the contents of which are incorporated
herein by
reference in their entirety). Control monkeys were treated with saline. 10
mg/kg of
hMJ2-7v2-11, hmAb13.2v2, or irrelevant human Ig (IVIG) were administered
intravenously. The following day, prechallenged BAL samples from control and
treated
monkeys (referred to in FIG. 15A as "control pre" and "Ab pre") were collected
from the
left lung of the monkeys. The monkeys were treated with 0.75 micrograms of
Ascaris
suum antigen intratracheally into the right lung. Twenty-four hours post-
challenge, BAL
samples were collected from the right lung of control and treated monkeys, and
assayed
for cellular infiltrate (referred to in FIG. 15B as "control post" and "Ab
post,"
respectively). BAL samples collected from antibody-treated monkeys showed a
statistically significant reduction in the total number of cell infiltrate
compared to control
animals (FIG. 15A). Control samples are represented in FIG. 15A as circles,
hmAb13.2v2- and hMJ2-7v2-1 1- treated samples are shown as dark and light
triangles,
respectively. hMJ2-7v2-11 and hmAb 13.2v2 showed comparable efficacy in this
model.
FIG. 15B shows a linear graph depicting the concentration of either hMJ2-7v2-
11 or
hmAbl3.2v2 with respect to days post-Ascaris infusion. A comparable decrease
kinetics
is detected for both antibodies.
Eotaxin levels were significantly increased 24 hours following Ascaris
challenge
(FICx 16A). Both hMJ2-7v2-11 and hmAb13.2v2 reduced eotaxin levels detected in
BAL
fluids from cynomolgus monkeys 24 hours after to challenge with Ascaris suum
antigen,
compared to saline treated controls.
- 136 -
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
Cynomolgus monkeys sensitized to Ascaris suum develop IgE to Ascaris antigen.
The IgE binds to FcsRI on circulating basophils, such that in vitro challenge
of peripheral
blood basophils with Ascaris antigen induces degranulation and release of
histamine.
Repeated antigen exposure boosts basophil sensitization, resulting in enhanced
histamine
release responses. To test the effects of hMJ2-7v2-11 and hmAbl3.2v2 in IgE-
and
basophil levels, cynomolgus monkeys dosed with humanized hMJ2-7v.2,
hmAb13.2v2,
irrelevant Ig (IVIG), or saline, as described above, were bled 8 weeks post-
Ascaris
challenge, and levels of total and Ascaris-specific IgE in plasma were
determined by
ELISA. FIG. 17A shows a linear graph of the changes in absorbance with respect
to
dilution of samples obtained pre- and 8-weeks post-challenge from animals
treated with
IVIG or hMJ2-7v2-11. Open-circles represent pre-bleed measurements; filled
circles
represent post-treatment measurements. A significant reduction in absorbance
was
detected in post-challenged samples treated with hMJ2-7v2-11 relative to the
pre-
challenge values in all dilutions assayed FIG 17A depicts representative
examples
showing no change in Ascaris-specific IgE titer in an individual monkey
treated with
irrelevant Ig (IVIG; animal 20-45; top panel), and decreased titer of Ascaris-
specific IgE
in an individual monkey treated with hMJ2-7v2-11 (animal 120-434; bottom
panel).
Animals treated with either humanized hMJ2-7v.2-11 or hmAb13.2v2 showed a
significant reduction in levels of circulating IgE-specific for Ascaris in
cynomolgus
monkey sera (FIG. 17B). There was no significant change in total IgE titer for
any of the
treatment groups. FIG. 17A shows a linear graph of the changes in absorbance
with
respect to dilution of samples obtained pre- and 8-weeks post-challenge from
animals
treated with IVIG or hMJ2-7v2-11. Open-circles represent pre-bleed
measurements;
filled circles represent post-treatment measurements. A significant reduction
in
absorbance was detected in post-challenged samples treated with hMJ2-7v2-11
relative to
the pre-challenge values in all dilutions assayed. The designations "20-45"
and "120-
434" refer to the cynomolgus monkey identification number.
To evaluate the effects of anti-IL13 antibodies on basophil histamine release,
the
animals were bled at 24 hours and 8 weeks post-Ascaris challenge. Whole blood
was
challenged with Ascaris antigen for 30 minutes at 37 C, and histamine released
into the
supernatant was quantitated by ELISA (Beckman Coulter, Fullerton, CA). As
shown in
- 137 -
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
FIGS. 18A-18B, the control animals demonstrated increased levels of Ascaris-
induced
basophil histamine release particularly 8 weeks following antigen challenge
(represented
by the diamonds in FIG. 18A and left-hand bar in FIG. 18B). In contrast, the
animals
treated with either humanized hMJ2-7v.2-11 or hmAb 13.2v2 did not show this
increase
in basophil sensitization in response to Ascaris 8 weeks after challenge
(FIGS. 18A-18B).
The majority of individual animals treated with humanized hMJ2-7v.2-11 or
hmAb 13.2v2 showed either a decrease (example in FIG 18A) or no change in
basophil
histamine release 8 weeks post-challenge compared to pre- or 24 hour post-
challenge.
Thus, a single administration of the humanized anti-IL13 antibody had a
lasting effect in
modifying histamine release in this model.
FIG. 19 depicts the correlation between Ascaris-specific histamine release and
Ascaris-specific IgE levels. Higher values were detected in control samples
(saline- or
IVIG-treated samples) (light blue circles) compared to anti-IL13 antibody- or
dexamethasone (dex)-treated (dark red circles). Humanized anti-IL13 antibody
(humanized mAb13.2v.2) administered i.v. 24 hours prior to Ascaris challenge,
or
dexamethasone administered intramuscular in two injections each one at a
concentration
of 1 mg/kg 24 hours and 30 mins. prior to Ascaris challenge. Twenty four hours
post-
challenge, BAL lavage was collected from the right lung and assayed for
histamine
release and IgE levels.
The results shown herein demonstrated that pretreatment of cynomolgus monkeys
with either MJ2-7 or mAb 13.2 reduced airway inflammation induced by Ascaris
suum
antigen at comparable levels as detected by cytokine levels in BAL samples,
serum levels
of Ascaris-specific IgE's and basophil histamine release in response to
Ascaris challenge
in vitro.
FIG. 20 is a series of bar graphs depicting the increases in serum IL-13
levels in
individual cynomolgus monkeys treated with humanized MJ2-7 (hMJ2-7v2-1 1). The
label in each panel (e.g., 120-452) corresponds to the monkey identification
number. The
"pre" sample was collected prior to administration of the antibody. The time
"0" was
collected 24-hours post-antibody administration, but prior to Ascaris
challenge. The
remaining time points were post-Ascaris challenge. The assays used to detect
IL-13
levels are able to detect IL-13 in the presence of hMJ2-7v2-11 or hmAbl3.2v2
- 138 -
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
antibodies. More specifically, ELISA plates (MaxiSorp; Nunc, Rochester, NY),
were
coated overnight at 4 C with 0.5 ug/ml mAb13.2 in PBS. Plates were washed in
PBS
containing 0.05% Tween-20 (PBS-Tween). NHP IL-13 standards, or serum dilutions
from cynomolgus monkeys, were added and incubated for 2 hours at room
temperature.
Plates were washed, and 0.3 ug/ml biotinylated MJl-64 (referred to herein as
C65
antibody) was added in PBS-Tween. Plates were incubated 2 hours, room
temperature,
washed, and binding detected using HRP-streptavidin (Southern Biotechnology
Associates) and Sure Blue substrate (Kirkegaard and Perry Labs). For detection
of IL-13
in the presence of mAb 13.2, the same protocol was followed, excepts that
ELISA plates
were coated with 0.5 ug/ml MJ2-7.
FIG. 21 shows data demonstrating that sera from cynomolgus monkeys treated
with anti-IL 13 antibodies have residual IL- 13 neutralization capacity at the
concentrations of non-human primate IL-13 tested. FIG. 21 is a bar graph
depicting the
STAT6 phosphorylation activity of non-human primate IL-13 at 0, 1, or 10
ng/ml, either
in the absence of serum ("no serum"); the presence of serum from saline or
IVIG-treated
animals ("control"); or in the presence of serum from anti-IL13 antibody-
treated animals,
either before antibody administration ("pre"), or 1-2 weeks post-
administration of the
indicated antibody. Serum was tested at 1:4 dilution. A humanized version of
MJ2-7
(MJ2-7v.2-1 1) was used in this study. Assays for measuring STAT6
phosphorylation are
disclosed herein.
FIG. 22 are linear graphs showing that levels of non-human primate IL- 13
trapped
by humanized MJ2-7 (hMJ2-7v2-1 1) at a 1-week time point in cynomolgus monkey
serum correlate with the level of inflammation measured in the BAL fluids post-
Ascaris
challenge. Such correlation supports that detection of serum IL- 13 (either
unbound or
bound to an anti-IL13 antibody) as a biomarker for detecting subjects having
inflammation. Subjects having more severe inflammation showed higher levels of
serum
IL- 13. Although levels of unbound IL- 13 are typically difficult to
quantitate, the assays
disclosed herein above in FIG. 20 provides a reliable assay for measuring IL-
13 bound to
an anti-IL-13 antibody.
-139-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
Example 22: Effects of Humanized Anti-IL-13 Antibodies on Airway Inflammation,
Lung Resistance, and Dynamic Luniz Compliance Induced by Administration of
Human
IL-13 to Mice
Murine models of asthma have proved invaluable tools for understanding the
role
of IL-13 in this disease. The use of this model to evaluate in vivo efficacies
of the IMA
antibody series (humanized 13.2v.2 and humanized MJ2-7v.2-1 1) was initially
hampered
by the inability of these antibodies to cross react with rodent IL- 13. This
limitation was
circumvented herein by administering human recombinant IL-13 to mice. Human IL-
13
is capable of binding to the murine IL-13 receptor, and when administered
exogenously
induces airway inflammation, hyperresponsiveness, and other correlates of
asthma.
In non-human primates, the IL-13 epitope recognized by humanized MJ2-7v.2-11
includes a GLN at position 110. In humans, however, position 110 is a
polymorphic
variant, typically with ARG replacing GLN (e.g., R110). The R110Q polymorphic
variant is widely associated with increased prevalence of atopic disease.
In this example, recombinant human R110Q IL-13 was expressed in E. coli and
refolded. Antibody 13.2 (IgGl, k) was cloned from BALB/c mice immunized with
human IL-13, and the humanized version of this antibody is designated
humanized
13.2v.2 (or h13.2v.2). Antibody MJ2-7 (IgGI, k) was cloned from BALB/c mice
immunized with the N-terminal 19 amino acids of nonhuman primate IL-13, and
the
humanized version of this antibody is designated humanized MJ2-7v.2-11 (or
hMJ2-7v.2-
11). Both antibodies were formulated in 10 mM L-histidine, pH 6, containing 5%
sucrose. Carimune NH immune globulin intravenous (human IVIG) (ZLB Bioplasma
Inc., Switzerland) was purified by Protein A chromatography and formulated in
10mM
L-histidine, pH 6, containing 5% sucrose.
To analyze the mouse lung response to the presence of recombinant human
R110Q IL-13, BABL/c female mice were treated with 5 g of recombinant human
Rl 10Q IL-13 (e.g., approximately 250 g/kg), or an equivalent volume of
saline (20 L),
administered intratracheally on days 1, 2, and 3. On day 4, animals were
tested for signs
of airway resistance (RI) and compliance (Cdyn) in response to increasing
doses of
nebulized methacholine. Briefly, anesthetized and tracheostomized mice were
placed
into whole body plethysmographs, each with a manifold built into the head
plate of the
-140-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
chamber, with ports to connect to the trachea, to the inspiration and
expiration ports of a
ventilator, and to a pressure transducer, monitoring the tracheal pressure. A
pneumotachograph in the wall of each plethysmograph monitored the airflow into
and
out of the chamber, due to the thoracic movement of the ventilated animal.
Animals were
ventilated at a rate of 150 breaths/min and a tidal volume of 150 ml.
Resistance
computations were derived from the tracheal pressure and airflow signals,
using an
algorithm of covariance.
As shown in FIGs. 23A-23B, intratracheal administration of recombinant human
Rl 10Q IL-13 elicited increased lung resistance and decreased dynamic
compliance in
response to methacholine challenge. These observations were not, however,
accompanied by strong lung inflammation.
To enhance the lung inflammatory response in mice, 5 g of recombinant human
R110Q IL-13, or an equivalent volume (50 L) of saline, was administered to
C57BL/6
mice intranasally on days 1, 2, and 3. Animals were sacrificed on day 4 and
bronchoalveolar lavage (BAL) fluid collected. Pre-analysis, BAL was filtered
through a
70 m cell strainer and centrifuged at 2,000 rpm for 15 minutes to pellet
cells. Cell
fractions were analyzed for total leukocyte count, spun onto microscope slides
(Cytospin;
Pittsburgh, PA), and stained with Diff-Quick (Dade Behring, Inc. Newark DE)
for
differential analysis. IL-6, TNFa, and MCP-1 levels were determined by
cytometric bead
array (CBA; BD Pharmingen, San Diego, CA). The limits of assay sensitivity
were 1
pg/ml for IL-6, and 5 pg/ml for TNFa and MCP- 1.
As shown in FIG. 24A, intranasal administration of recombinant human R110Q
IL- 13 induced a strong airway inflammatory response, as indicated by elevated
eosinophil and neutrophil infiltration into BAL. Cell infiltrates consisted
primarily of
eosinophils (e.g., approximately 40%). As shown in FIG. 24B, intranasal
administration
of recombinant human R110Q IL-13 also significantly increased the levels of
several
cytokines in BAL including, for example, MCP- 1, TNF-a, and IL-6.
To determine the best delivery method for humanized MJ2-7v.2-11, antibody
levels in BAL and serum were analyzed following intraperitoneal and
intravenous, or
intranasal administration following treatment with recombinant human R110Q IL-
13
administered intranasally or intratracheally. Briefly, BALB/c female mice were
-141-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
administered 5 g of recombinant human R1 l OQ IL-13 or an equivalent volume
of saline
intratracheally on days 1, 2, and 3. On day 0, and 2 hours prior to
administering each IL-
13 dose, mice were treated with 500 g humanized MJ2-7v.2administered
intravenously
on day 0, and by IP on days 1, 2, and 3 (FIG. 25A). Alternatively, 500 g of
humanized
MJ2-7v.2-11 were administered intranasally on days 0, 1, 2, and 3. Total human
IgG was
measured by ELISA, as follows: ELISA plates (MaxiSorp; Nunc, Rochester, NY)
were
coated overnight at 4 C with 1:1500 dilution of goat anti-human Ig (M+G+A) Fc
(ICN-
Cappel, Costa Mesa, CA) at 50 l/well in 25 mM carbonate - bicarbonate buffer,
pH 9.6.
Plates were blocked for 1 hour at room temperature with 0.5% gelatin in PBS,
washed in
PBS containing 0.05% Tween-20 (PBS-Tween). Humanized MJ2-7v.2-11 standard or 6
x 1:2 dilutions of sheep serum starting at 1:500 - 1:50,000 were added and
incubated for
2 hours at room temperature. Plates were washed with PBS-Tween, and a 1:5000
dilution of biotinylated mouse anti-human IgG (Southern Biotechnology
Associates) was
incubated for 2 hours at room temperature. Plates were washed with PBS-Tween,
and
binding was detected with peroxidase-linked streptavidin (Southern
Biotechnology
Associates) and Sure Blue substrate (KPL Inc.). Assay sensitive was 0.5 ng/ml
human
IgG.
FIG. 25A shows elevated levels of human IgG in serum compared to BAL
following intraperitoneal and intravenous administraton of the humanized MJ2-
7v.2-11
antibody. As shown in FIG. 25B, total IgG levels in g/ml were significantly
higher in
BAL than serum levels following intranasal administration of humanized MJ2-
7v.2-11
antibody.
To determine if the humanized MJ2-7v.2-11 antibody was capable of modulating
the above observed lung function and inflammatory response, airway
hyperresponsiveness was induced by intratracheal administration of 5 g
recombinant
human R110Q IL-13 or an equivalent volume (20 L) of saline on days 1, 2, and
3. On
day 0, and 2 hours before administering each dose of recombinant human Ri lOQ
IL-13,
animals were treated with 500 g of humanized MJ2-7v.2-11, 500 g dose of
IVIG, or
an equivalent volume of saline, administered intranasally. Animals were tested
on day 4
for airway resistance (RI) and compliance (Cdyn) in response to increasing
doses of
nebulized methacholine, as described above. Humanized MJ2-7v.2 and IVIG levels
in
-142-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
BAL and serum were analyzed by ELISA, as described above. As shown in FIGs.
26A-
26B, humanized MJ2-7v.2-11 effectively reduced the asthmatic response,
resulting in a
significant reduction in the dose of methacholine required to achieve half-
maximal
degree of lung resistance. In contrast, an equivalent dose of IVIG had no
effect.
Changes in dynamic lung compliance were not apparent under these conditions.
As
shown in FIG. 26C, BAL IgG antibody levels were approximately 10 - 20 times
higher
than serum levels.
To determine if humanized MJ2-7v.2-11 anti-IL-13 antibody administration
promoted an increase in the circulating levels of IL-13, BAL and sera were
assayed for
IL-13 levels by ELISA, as follows: Briefly, BALB/c female mice were treated as
described for FIG. 26A-26B. ELISA plates (Nunc Maxi-Sorp) were coated
overnight
with 50 l / well mouse anti-IL-13 antibody, mAb13.2, diluted to 0.5 mg/ml in
PBS.
Plates were washed 3 times with PBS containing 0.05% Tween-20 (PBS-Tween) and
blocked for 2 hours at room temperature with 0.5% gelatin in PBS. Plates were
then
washed and human IL-13 standard (Wyeth, Cambridge, MA), or dilutions of mouse
serum (serial 3X dilutions starting at 1:4) were added, in PBS-Tween
containing 2% fetal
calf serum (FCS). Plates were incubated for a further 4 hours at room
temperature, and
washed. Biotinylated mouse anti-human IL-13 antibody, C65, was added at 0.3
g/ml in
PBS-Tween. Plates were incubated for 1 - 2 hours at room temperature, washed,
then
incubated with HRP-streptavidin (Southern Biotechnology Associates,
Birmingham, AL)
for 1 hour at room temperature. Color was developed using Sure Blue peroxidase
substrate (KPL, Gaithersburg, MD), and the reaction stopped with 0.O1M
sulfuric acid.
Absorbance was read at 450 nm in read in a SpectraMax plate reader (Molecular
Devices
Corp., Sunnyvale, CA). Serum IL-13 levels were determined by reference to a
human
IL- 13 standard curve, which was independently established for each plate.
As shown in FIGs. 27A-27B, consistent with FIG. 26C, IL-13 levels were
elevated in BAL of antibody-treated mice, but not serum. In addition, we
observed that
IL- 13 isolated from these samples had no detectable biological activity (data
not shown).
To determine if this observed lack of IL-13 biological activity was due to IL-
13 and
humanized MJ2-7v.2-11 complex formation, an ELISA was developed to
specifically
detect IL-13 and humanized MJ2-7v.2-11 in complex. Briefly, ELISA plates (Nunc
-143-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
Maxi-Sorp) were coated overnight with 50 l / well inouse anti-IL-13 antibody,
mAb13.2, diluted to 0.5 mg/ml in PBS. Plates were washed 3 times with PBS
containing
0.05% Tween-20 (PBS-Tween) and blocked for 2 hours at room temperature with
0.5%
gelatin in PBS. Plates were then rewashed, and human IL-13 standard (Wyeth,
Cambridge, MA), or dilutions of mouse serum (serial 3X dilutions starting at
1:4) were
added, in PBS-Tween containing 2% fetal calf serum (FCS). Plates were
subsequently
incubated for 4 hours at room temperature. Biotinylated anti-human IgG (Fc
specific)
(Southern Biotechnology Associates, Birmingham, AL) diluted 1:5000 in PBS-
Tween
was then added. Plates were incubated for 1- 2 hours at room temperature,
washed, and
finally incubated with HRP-streptavidin (Southern Biotechnology Associates,
Birmingham, AL) for 1 hour at room temperature. Color was developed using Sure
Blue
peroxidase substrate (KPL, Gaithersburg, MD), and the reaction stopped with
0.01 M
sulfuric acid. Absorbance was read at 450 nm in read in a SpectraMax plate
reader
(Molecular Devices Corp., Sunnyvale, CA).
As shown in FIGs. 27D-27E, IL-13 and humanized MJ2-7v.2-11 complexes were
recovered from BAL and serum of mice in this model. This observation indicates
that
humanized MJ2-7v.2-11 is capable of binding IL-13 in vivo, and that this
interaction may
negate IL-13 biological activity.
The effects of humanized MJ2-7v.2-11 on human IL-13-mediated lung
inflammation and cytokine production were tested in mice, and compared with a
second
antibody, humanized 13.2v.2, as follows. Briefly, C57BU6 female mice
(10/group) were
treated with 5 g of recombinant human R110Q IL-13 (e.g., approximately 250
g/kg),
or an equivalent volume (50 l) of saline, on days 1, 2, and 3, administered
intranasally.
On day 0, and 2 hours before administering each dose of IL-13, mice were given
intranasal doses of 500 g, 100 g, or 20 g of humanized MJ2-7v.2-11 or
humanized
13.2v.2. Control groups received 500 g IVIG, or an equivalent volume of
saline.
Animals were sacrificed on day 4, and BAL collected. Eosinophil and neutrophil
infiltration into BAL were determined by differential cell count and expressed
as a
percentage.
As shown in FIGs. 28A-28B, consistent with FIG. 24A, recombinant human
R110Q IL-13 treatment evoked an increase in eosinophil and neutrophil
infiltration
- 144 -
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
levels. Interestingly, humanized MJ2-7v.2-11 and humanized 13.2v.2
significantly
reduced eosinophil (FIG. 28A) and neutrophil (FIG. 28B) infiltration compared
to
controls (e.g., saline, IL-13, IVIG). As shown in FIG. 29A-29C, HMJ2-7V2-11
and
humanized MJ2-7v.2-11 also abrogated increases in MCP-1, TNF-a, and IL-6
cytokine
levels.
To confirmation that BAL cytokine levels accurately represent the degree of
inflammation C57BL/6 female mice were treated with 5 g of recombinant human
R110Q IL-13 (e.g., approximately 250 g/kg) or an equivalent volume (50 l) of
saline
on days 1, 2, and 3, administered intranasally. On day 0, and 2 hours before
administering each dose of IL-13, mice were given intranasal doses of 500,
100, or 20 g
of humanized MJ2-7v.2-1 1. On day 4, animals were sacrificed and BAL
collected.
Humanized MJ2-7v.2-11 antibody levels in BAL were determined by ELISA, as
described above. BAL IL-6 levels were determined by cytometric bead array.
Eosinophil percentages were determined by differential cell counting.
As shown in FIGs. 30A-30B, IL-6 BAL cytokine levels were related to the degree
of inflammation. Furthermore, higher levels of humanized MJ2-7v.2-i 1 in BAL
fluid
inversely correlated with cytokine concentration, strongly implying a
treatment effect.
The levels of antibody required to reduce IL-13 bioactivity in vivo in this
model
were high. The best efficacy was seen at a dose of 500 g antibody,
corresponding to
approximately 25 mg/kg in the mouse. This high dose requirement for antibody
is most
likely a consequence of the high levels of IL- 13 (5 g / dose x 3 doses) used
to elicit lung
responses. Interestingly, good neutralization of in vivo IL-13 bioactivity was
seen only
when humanized MJ2-7v.2-11 was administered intranasally, and not when the
antibody
was administered via intravenous or intraperitoneal. Distribution studies
showed that
following intravenous and intraperitoneal dosing, high levels of antibody were
recovered
in serum at the time of sacrifice, but very low levels were found in BAL. In
contrast,
following intranasal dosing, comparable levels of antibody were found in serum
and in
BAL. Thus, levels of humanized MJ2-7v.2-11 in BAL fluid were approximately 100-
fold higher following intranasal dosing than intravenous and intraperitoneal
dosing. The
observation that intranasal dosing was efficacious but intravenous and
intraperitoneal
dosing was not indicates that in this model, the site of antibody action was
the lung. This
site of action is expected based on the intratracheal or intranasal delivery
route of IL-13,
-145-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
and was confirmed by the observation that antibody trapped IL-13 in the BAL
fluid, but
very little antibody / IL-13 complex was seen in the serum.
In conclusion, these findings further support the IL-13 neutralization
activity of
humanized MJ2-7v.2-11 in vivo.
Example 23: Effects of IL-13 and / or IL-4 Neutralization at the Time of
Allergen
Challenge on Allergen-Specific I Eg Titer
IL- 13 and IL-4 drive the production of IgE, an important mediator of allergic
disease (Oettgen, H.C. (2000) Curr Opin Immunol 12:618-623; Wynn, T.A. (2003)
Anuu
Rev. Immunol. 21:425-456). The effects of a single administration of IL-4 or
IL-13
antagonist, delivered 24 hours prior to challenge, on allergen-specific IgE
levels were
examined. These questions were addressed using a standard murine OVA
sensitization
and challenge model.
Female Balb/c mice between 6 and 8 weeks of age were purchased from Jackson
Laboratory. Mice were housed in environmentally controlled, pathogen-free
conditions
for 2 weeks before the study and for the duration of the experiments. All
procedures
were reviewed and approved by the Institutional Animal Care and Use Committee
at
Wyeth Research.
-146-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
Groups of mice were immunized by intraperitoneal injections with 200 l
solution
containing 20 g OVA (grade V, Sigma-Aldrich, St Louis, MO) emulsified with 4
mg
aluminum hydroxide/magnesium hydroxide(ImjectAlum; Pierce, Rockford, IL) in
PBS
on days 0 and 13 (FIG. 31). Sensitized mice were administered 200 g/dose
soluble
murine IL-13Ra2.IgG fusion protein (sIL-13Ra2.Fc; Wyeth Research) or 200
g/dose
rat anti-mouse IL-4 monoclonal antibody (clone 30340; rat IgGI anti-mouse IL-
4; R&D
Systems, Minneapolis, MN), by intraperitoneal injection one day before
challenge.
Control animals received mouse IgG2a (Wyeth Research) or purified rat IgGl
(Wyeth
Reserach). Some groups were treated with sIL-13Ra2.Fc or control one day
before and
one day after challenge. On day 21, the mice were anesthetized with isoflurane
solution
(Henry Schein, Melville, NY) using an Impac6 system (VetEquip, Pleasanton, CA)
and
challenged intranasally with 20 g OVA/mouse in 50 l PBS.
Mice were sacrificed on day 28 and blood collected by cardiac puncture. Serum
was obtained by use of gel barrier with clotting activator tubes (CapiJect;
Terumo
Medical, Somerset, NJ).
To assay IgE titers, ELISA plates (MaxiSorp; Nunc) were coated with rat anti-
mouse IgE (BD Biosciences, San Jose, CA). Plates were blocked with 0.5%
gelatin in
PBS for 1 hour; washed in PBS containing 0.05% Tween-20 (PBS-Tween); incubated
6
hours at room temperature with purified mouse IgE (BD Biosciences) as
standard, or
dilutions of serum, in the presence of mouse IgG (Sigma-Aldrich, St. Louis,
MO) as
blocker. The assay was developed using peroxidase-linked streptavidin
(Southern
Biotechnology Associates, Birmingham, AL) and TMB-substrate solution
(SureBlue;
Kirkegaard & Perry Laboratories, Gaithersberg, MD). For determination of OVA-
specfic
IgE or IgG subtypes, plates were coated overnight with OVA (Sigma-Aldrich).
Bound
IgE was quantitated with biotinylated rat anti-mouse IgE (BD Biosciences) in
the
presence of mouse IgG blocking agent (Sigma-Aldrich). Bound IgGI was
quantitated
with biotinylated rat anti-mouse IgGi or rat anti-mouse IgG3 (BD Biosciences).
Total
IgE concentrations were determined by reference to a standard curve of
purified mouse
IgE (BD Biosciences). The limit of detection was 2 ng/ml. OVA-specific Ig
titer was
quantitated as the serum dilution required to reach a given absorbance value,
relative to a
- 147 -
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
reference standard. The limit of detection was a relative titer of 0.5. Serial
dilutions of
serum were run in each assay, with each sample run in at least three separate
assays.
For each test, average values for replicate determinations from each animal
were
included. Groups of 20 animals were run in each assay. Data were analyzed
using
GraphPad Prism software. All reported p values were determined by unpaired
Student's t
test.
To address the requirement for IL- 13 in driving IgE production in response to
allergen challenge, IL- 13 antagonist (sIL-13Ra2.Fc) was administered to OVA-
immunized mice 24 hours before and 24 hours after intranasal challenge with
the antigen.
As outlined in FIG. 31, mice were immunized i.p. with OVA/alum on day 0,
boosted with
OVA/alum on day 13, and challenged intranasally on day 21. sIL-13Ra2.Fc (200
g)
was administered i.p. on both days 20 and 22. Animals were sacrificed on day
28, and
blood collected into serum separator tubes. Total serum IgE was quantitated by
ELISA.
There was no difference in total IgE titer in animals treated with sIL-
13Ra2.Fc as
compared to those given control mouse IgG2a (FIG. 32A). Animals treated both
before
and after challenge with the IL-13 antagonist had reduced OVA-specific IgE
titer as
compared to animals treated with the isotype control, but this difference
failed to reach
statitstical significance because of the presence of several animals in the
control group
with no detectable titer of OVA-specific IgE (FIG. 32B). There was no
significant
difference in titers of OVA-specific IgGI (FIG. 32C).
Because there was a trend toward reduced titers of OVA-specific IgE in animals
treated with sIL-13Ra2.Fc both before and after challenge, we evaluated the
effectiveness of a single administration of sIL-13Ra2.Fc, given 24 hours
before
challenge. Total serum IgE concentration was reduced in the mice treated with
sIL-
13Ra2.Fc as compared to those given IgG2a control (p < 0.05; FIG. 33A). OVA-
specific
IgE titer was also reduced following a single administration of sIL-13Ra2.Fc
(p <0.01;
FIG. 33B). There was no change in titer of OVA-specific IgGI.
To evaluate whether IL-4 neutralization could affect the IgE response to OVA
challenge in a similar way to IL- 13 neutralization, mice were given a single
dose of 200
g anti-IL-4 i.p., 24 hours pre-challenge. An additional group of mice was
treated with a
combination of sIL-13Ra2.Fc and anti-IL-4 (200 g each). Neutralization of
either IL-
-148-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
13 (p < 0.05) or IL-4 (p < 0.02) produced a significant reduction in total
serum IgE titer
(FIG. 34A). OVA-specific IgE titers were also significantly reduced following
treatment
with either anti-IL-4 (p < 0.02) or sIL-13Ra2.Fc (p < 0.02) (FIG. 34B). OVA-
specific
IgGl titers were unaffected by either treatment (FIG. 35A). OVA-specific IgG3
titers
were also measured in this study and showed a significant reduction with IL-
13
antagonist (p < 0.001), but not with anti-IL-4 treatment (FIG. 35B).
Administration of sIL-13Ra2.Fc together with anti-IL-4 produced a greater
reduction in total serum IgE titer than that produced by either agent alone (p
< 0.001)
(FIG. 34A). Similarly, OVA-specific IgE titers were reduced to a greater
extent
following treatment with sIL-13Ra2.Fc and anti-IL-4 than was seen by blocking
either
cytokine alone (p < 0.001) (FIG. 34B). Mice treated with the combination of
sIL-
13Ra2.Fc and anti-IL-4 did not differ in titers of OVA-specific IgG1 (FIG.
35A) or
OVA-sepcific IgG3 (FIG. 35B) compared to control animals.
Several studies have examined the utility of IL-4 or IL- 13 neutralization,
delivered throughout the course of OVA immunization and/or challenge, in
modulating
IgE responses (Zhou, C.Y. et al. (1997) JAsthma 34:195-201; Yang, G. et al.
(2004)
Cytokine 28:224-232). Although this treatment paradigm is effective, studies
in the NHP
model, discussed herein, indicate that effective IL- 13 neutralization could
have a lasting
impact on IgE responses. Therefore, the requirement for multiple
administrations of an
IL-4 or IL-13 neutralizing agent was addressed in a mouse model. We determined
whether, under optimal conditions of sensitization and challenge, a single
treatment with
IL-4 or IL- 13 neutralizing agent could effectively modulate IgE responses to
antigen.
sIL-13Ra2.Fc is a potent IL-13 antagonist, that has been shown to block lung
inflammation, AHR, and mucus production in animal models of asthma (Wills-
Karp, M.
et al. (1998) Science 282:2258-2261). In previous studies addressing its
effects on IgE
production, mice were given two rounds of lung challenge with OVA either 10
days
(Wills-Karp, M. et al. (1998) supra) or 6 weeks (Taube, C. et al. (2002) J.
Immunol.
169:6482-6489) following the initial challenge. sIL-13Ra2.Fc delivered only at
the time
of secondary allergen challenge did not alter the serum titer of OVA-specific
IgE (Wills-
Karp, M. et al. (1998) supra, Taube, C. et al. (2002) supra). The lack of
effect on IgE
titer was not surprising given the robust IgE response seen with a secondary
challenge (-
-149-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
Karp, M. et al. (1998) supra). Consistent with this, delivery of several doses
of IL- 13
antagonist, beginning at the initial challenge, has been more effective. Serum
levels of
allergen-specific IgE, but not IgG1, were reduced when antibody to IL-13 was
administered prior to each of 5 weekly intranasal challenges with OVA in a
chronic
asthma model (Zhou, C.Y. et al. (1997) supra).
To address whether a single dosing paradigm with IL- 13 neutralizing agent
would
affect specific IgE production in mice, sIL-13Ra2.Fc was administered before
intranasal
challenge with OVA. Mice were sensitized with OVA/alum on days 0 and 13, then
given
a single intranasal challenge with OVA on day 21. Results showed that a single
administration of sIL-13Ra2.Fc, delivered 24 hours before challenge, reduced
titers of
OVA-specific IgE at the time of sacrifice, on day 28. Titers of OVA-specific
IgGI were
not affected. Total serum IgE concentrations were also reduced in most
experiments.
Interestingly, delivery of two doses of sIL-13Ra2.Fc, at 24 hours before and
24 hours
after challenge, did not improve the efficacy of this treatment.
To compare the efficacy of IL-13 and IL-4 neutralization, groups of mice were
sensitized and challenged with OVA as described above, and treated 24 hours
before
challenge either with sIL-13Ra2.Fc, antibody to IL-4, or both sIL-13Ra2.Fc and
anti-IL-
4. Treatment with either sIL-13Ra2.Fc or anti-IL-4 significantly reduced
titers of OVA-
specific IgE. Total serum IgE concentration was also significantly, reduced.
Administration of both sIL-13Ra2.Fc and anti-IL-4 produced a greater magnitude
of
change in OVA-specific titer and in total serum IgE concentration than was
seen with
either treatment alone. These effects appeared specific for IgE, however, as
neither
OVA-specific IgGI nor OVA-specific IgG3 titers were affected by the combined
treatment with sIL-13Ra2.Fc and anti-IL-4.
These findings support the observations from NHP studies, that delivery of an
IL-
13 neutralizing agent in single administration prior to allergen challenge can
reduce the
IgE response to allergen. An IL-4 neutralizing agent can have similar
activity.
Neutralization of both IL-4 and IL- 13 had a more potent effect on reduction
of IgE
responses than neutralization of either cytokine alone. These findings
emphasize the
critical requirement for IL-4 and IL-13 at the time of allergen challenge.
1 -150-
CA 02672215 2009-06-10
WO 2008/073463 PCT/US2007/025418
Those skilled in the art will recognize, or be able to ascertain using no more
than
routine experimentation, many equivalents of the specific embodiments
described herein
described herein. Other embodiments are within the following claims.
-151-
DEMANDE OU BREVET VOLUMINEUX
LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVET COMPREND
PLUS D'UN TOME.
CECI EST LE TOME 1 DE 2
CONTENANT LES PAGES 1 A 151
NOTE : Pour les tomes additionels, veuillez contacter le Bureau canadien des
brevets
JUMBO APPLICATIONS/PATENTS
THIS SECTION OF THE APPLICATION/PATENT CONTAINS MORE THAN ONE
VOLUME
THIS IS VOLUME 1 OF 2
CONTAINING PAGES 1 TO 151
NOTE: For additional volumes, please contact the Canadian Patent Office
NOM DU FICHIER / FILE NAME:
NOTE POUR LE TOME / VOLUME NOTE:
