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).

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

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
EL-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 die 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)),

chronic obstructive pulmonary disease (COPD), and other conditions involving airway
inflammation, eosinophilia, fibrosis and excess mucus production, e.g., cystic fibrosis and
pulmonary fibrosis; inflammatory 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.

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 ccl or an IL-13 receptor α2) 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-
13 R, 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-

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 EL-13 or IL-4, or IL-13R or IL-4R with high affinity, e.g.,
with an affinity constant of at least about 107 M preferably about 108 M1, and more
preferably, about 109 M"1 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 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 EL-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 EL-4 or an IL-4R. The EL-13 or
the IL-4 antagonist can also be a soluble form of the IL-13R (e.g., soluble IL-13Rα2 or
IL-13Rαl) or the IL-4R (e.g., IL-4Rα), 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

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-13Ral, 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-13Rotl ("IL-13/IL-13αRl"); IL-13 and IL-4Ra
("IL-13/IL-4Ra"); IL-13, IL-13Ral, and IL-4Ra ("IL-13/IL-13Ral/IL-4Rα"); and IL-13
and IL-13Rα2 ("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-13Ral and IL-4Rα. 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-13Ral 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-13Ral and IL-4Ra.
In another embodiment, the IL-13 antagonist, binds to EL-13 and interferes with (e.g.,
inhibits, blocks or otherwise reduces) an interaction, e.g., binding, between IL-13/IL-4Rα
and IL-13Ral. Typically, the IL-13 antagonist interferes with (e.g., inhibits, blocks or
otherwise reduces) an interaction, e.g., binding, of IL-13/IL-13Rαl with IL-4Rα.
Exemplary antibodies inhibit or prevent formation of the ternary complex, EL-13/IL-
13Rαl/IL-4Rα.
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

reduces an interaction (e.g., binding) between IL-4 and an IL-4 receptor, e.g., IL-4Rα
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-4Rα ("IL-4/IL-4Rα"), IL-4 and y common ("IL-4/ycommon"), or IL-4, IL-4Ra,
and y common ("IL-4/IL-4Rα/ 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-4Rα 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-4Rα 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 EL-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 IgGl,
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 IgGl {e.g., human IgGl 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 IgGl constant region

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 IgGl 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-13Rα2 (e.g., the anti-IL13 antibody molecule reduces or inhibits IL-13
interaction with IL-13Ral). The anti-IL13 antibody molecule may prevent formation of
a complex between IL-13 and IL-13Ral, or disrupt or destabilize a complex between
IL-13 and IL-13Ral. In one embodiment, the anti-IL13 antibody molecule inhibits
ternary complex formation, e.g., formation of a complex between IL 13, IL-13Ral 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-IL13 antibody molecule can associate with IL-13 with kinetics in the range of 103 to
108 M"V typically 104 to 107 MV. In one embodiment, the anti-IL13 antibody
molecule binds to human EL-13 with a kon of between 5104 and 8105 NT's1. In yet
another embodiment, the anti-IL13 antibody molecule has dissociation kinetics in the
range of 10"2 to 106 s1, typically 10"2 to 10"5 s1. 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 (BIACORE™).
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

epitope defined by IL-13Rocl binding to human IL-13 or an epitope defined by IL-13Rα2
binding to human IL-13, or an epitope that overlaps with such epitopes. The anti-IL13
antibody molecule may compete with IL-13Rαl 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-13Rαl 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-13Rαl and/or IL-13Rα2. 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 EL-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 EL-13 chosen
from one or more of residues 81-93 and/or 114-132 of human IL-13 (SEQ ED 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 ED NO: 194
[position in mature sequence; SEQ ED 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 ED NO:24 or
SEQ ED NO:178. In one embodiment, the antibody molecule binds to EL-13 irrespective
of the polymorphism present at position 130 in SEQ ED NO:24.
In one embodiment, the antibody molecule includes one, two, three, four, five or
all six CDR's from mAbl3.2, 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.,

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 ED NO:17), mAb 13.2 (SEQ ID NO:196) or C65 (SEQ ID N0.123). 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 ED NO:l 18). The amino acid sequence of the heavy chan variable
domain of MJ2-7 has the amino acid sequence shown as SEQ ED NO: 130. The amino
acid sequence of the light chan variable domain of MJ2-7 has the amino acid sequence
shown as SEQ ED 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 ED NO: 199.
In certain embodiments, the heavy chain variable domain of the antibody
molecule includes one or more of:


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 ED NO:201, SEQ ED NO:202, SEQ
ED NO:203, and SEQ ED 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., mAbl3.2, 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., mAbl3.2, 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., mAbl3.2, MJ2-7, C65, or other antibodies disclosed herein.
In one embodiment, the protein includes all six hypervariable loops from
mAbl3.2, 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 mAbl3.2, 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 mAbl3.2, 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)7. Mol.

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, He, or Leu at position 29; Met, He,
Leu, Val, Thr, Trp, or He 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.,
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 II
subgroup germline sequence or one of the following germline V segment sequences:
A17, Al, 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) EMBO J. 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 lle at position 2; Ser or Pro at position 25; lle 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 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 DPK9 sequence.

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), chl3.2 (SEQ ID NO:204), hl3.2vl (SEQ ID
NO:205), hl3.2v2 (SEQ ED NO:206)or hl3.2v3 (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 ED NO:74), V2.5 (SEQ ID NO:75), V2.6
(SEQ ID NO:76), V2.7 (SEQ ID NO:77), V2.ll (SEQ ID NO:80); chl3.2 (SEQ ED
NO:208), hl3.2vl (SEQ ID NO:209), hl3.2v2 (SEQ ID NO:210) or hl3.2v3 (SEQ ID
NO:211). 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
of SEQ ID NO:l 16 or SEQ ID NO:l 17.
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 hl3.2v2
(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 hl3.2v2 (SEQ ID NO:212). In embodiments, the light chain
immunoglobulin variable domain includes the amino acid sequence of SEQ ED NO:36,

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, lle, and to 49, Gly;
(iv) at positions corresponding to 48, lle, 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, lle, 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 YD 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

05/091856 naming Centocor, Inc. as the applicant; and in Yang et al. (2004) Cytokine
28(6):224-32 and Yang et al. (2005) J Pharmacol 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-4Rα (e.g., AMG-317 and related anti-IL4R antibodies disclosed in US
05/0118176, US 05/0112694 and in Clinical Trials Gov. Identifier: NCT00436670); IL-
13Ral (e.g., anti-13Ral antibodies disclosed in WO 03/080675 which names AMRAD
as the applicant); and mono- or bi-specific antibody molecules that bind to EL4 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 Oncol 25(7):837-44 and in Husain, S. R. et al. (2003) J. Neurooncol 65(l):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 IL13Ral) or an IL-4 receptor polypeptide (e.g., EL-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-13Rct2, IL13Rαl or IL-4Ra comprising a cytokine-binding
domain; e.g., a soluble form of an extracellular domain of mammalian (e.g., human) IL-
13Rα2, IL13Rαl or IL-4Rα). 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(l):47-52, as well as in Borish, L.C. et al. (1999) Am JRespir Crit Care Med
160(6):1816-23.

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: IgGl, IgG2, IgG3, IgG4, IgM, IgAl, IgA2, IgD, and
IgE). Typically, the fusion protein can include the extracellular domain of a human
soluble EL-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-
4Rα (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.

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) BioorgMed 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; EL-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-P 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.

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).

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

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 EL-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 mAbl3.2 or a humanized version thereof (disclosed herein and in
US 06/0063228), or an IL-13 binding agent capable of competing with mAbl3.2 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 EL-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

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 mAbl3.2 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 C6S 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;
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

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
suurti). 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., Ascara-specific) basophil histamine release; and/or (iv) detecting in
antigen-specific (e.g., cam-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.

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. 1A 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. IB 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 (A), 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-13Ra2-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 (A),
C65 (♦), or sIL-13Ra2-Fc (•). NHP IL-13 activity was measured by phosphorylation of
STAT6 (y-axis) as a function of antagonist concentration (x-axis).

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.
FIG 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 (•) or humanized Version 1 (o), Version 2 (♦), or Version 3 (A) 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 (•), mouse VH and humanized Version 2
VL (A), 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. 11A and 1 IB are graphs depicting inhibition of binding of IL-13 to
immobilized EL-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
onthex-axis. FIG 11A depicts binding to IL-13 Reel. FIG 1 IB depicts binding to
IL-13Ra2.
FIG 12 is an alignment of DPK18 germline amino acid sequence (SEQ ED
NO: 126) and humanized MJ2-7 Version 3 VL (SEQ ED NO: 190).
FIG. 13A is an amino acid sequence (SEQ ED NO: 124) of mature, processed
human IL-13.
FIG. 13B shows an amino acid sequence (SEQ ID NO:125) of human IL-13Ral.
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.

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 (A)) 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 Ascara-specific IgE-titers in control and
antibody-treated samples 8-weeks post-challenge. FIG 17A depicts representative
examples showing no change in hearts-specific IgE titer in an individual monkey treated
with irrelevant Ig (WIG; animal 20-45; top panel), and decreased titer of Ascaris-specifxc
IgE in an individual monkey treated with humanized MJ2-7v.2 (animal 120-434; bottom
panel). FIG. 17B depicts a decrease in Ascara-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 cam-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 mAbl3.2v.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.

FIG. 19 depicts the correlation between Ascaris-specific histamine release and
cam-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 mAb 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 Rl 10Q 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 Rl 10Q 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.

FIGs. 25A-25B are dot plots showing humanized MJ2-7-11 (hMJ2-7v.2-ll)
antibody levels in BAL and serum following intratracheal and intravenous
administration. Animals were treated with human recombinant Rl 10Q IL-13, or an
equivalent volume (20 uL) of saline, intratracheally on days 1, 2, and 3. Humanized
MJ2-7v.2-l 1 antibody was administered on day 0 and 2 hours before each dose of human
recombinant Rl 10Q 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-l 1 antibody after
intranasal administration of human recombinant Rl 10Q IL-13-induced altered lung
function. (A) FIG. 26A shows the changes in lung resistance (Rl; cm H20/ml/sec)
expressed as change from baseline. FIG. 26B shows data expressed as methacholine
dose required to elicit lung resistance (Rl) 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 Rl 10Q IL-13 administered alone (FIGs. 27A-27B) or in complex with
humanized MJ2-7v.2-l 1 antibody (FIGs. 26C-27D) following intratracheal
administration of human recombinant Rl 10Q IL-13 and intranasal administration of
humanized MJ2-7v.2-l 1 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 10Q IL-13 and intranasal administration of 500, 100, and 20 ug of
humanized MJ2-7v.2-l 1 and humanized 13.2v.2, saline, or 500 ug of WIG. 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.

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
Rl 10Q IL-13 and intranasal administration of 500 pig of humanized MJ2-7v.2-l 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 <0.0001, according to a two-
tailed t-test.
FIGs. 30A-30B are dot plots showing that human recombinant Rl 10Q IL-13
levels are directly related to lung inflammation, as measured by eosinohilia; and inversely
proportional to humanized MJ2-7v.2-l 1 BAL levels following intranasal administration
of human recombinant Rl 10Q IL-13 and intranasal administration of 500, 100, or 20 ug
doses of humanized MJ2-7v.2-11 antibody. Humanized MJ2-7v.2-11 antibody BAL
levels were measured by ELISA. Human recombinant Rl 10Q 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 Rl 10Q IL-13, including data from saline control
animals, mice treated with human recombinant Rl 10Q IL-13 alone, and mice treated with
human recombinant Rl 10Q IL-13 and 500, 100, and 20 ug of humanized MJ2-7v.2-l 1
antibody or 500 pig IVIG; and (FIG. 30B) humanized MJ2-7v.2-l 1 and IL-6, including
data from mice treated with 500, 100, and 20 ug of humanized MJ2-7V2-11. 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 sELRa2.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.

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 ofIL-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

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 EL-13, see SEQ ID
NO: 124. An exemplary sequence is recited as follows:
MALLLTTVIALTCLGGFASPGPVPPSTALRELIEELVNITQNQKAPLCNGSMVWSI
NLTAGMYCAALESLINVSGCSAIEKTQPxMLSGFCPHKVSAGQFSSLHVRDTKIEV
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-13Rαl and IL-13Rα2 or fusions thereof) are described, e.g., in Donaldson et al.
(1998) JImmunol. 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
CSTELRLLYQLVFLLSEAHTCffENNGGAGCVCHLLMDDWSADNYTLDLWAG
QQLLWKGSFKPSEHVKPRAPGNLTVHTNVSDTLLLTWSNPYPPDNYLYNHLTY
AVNrWSENDPADFRIYNVTYLEPSLRIAASTLKSGISYRARVRAWAQCYNTTWSE
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;

(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

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

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
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

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

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 CHI 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 CHI 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 of Antibody 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

addition, embodiments of the invention described with respect to Kabat or AbM CDRs
may also be implemented using Chothia hvpervariable loops. Each VH and VL typically
includes three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in
the following order: FR1, 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. KAMA 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

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
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., XENOMOUSE™,
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

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 IIDR 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, LA. 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.

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 ED NO: 128. Certain active fragments of the CL and CH
subunits (e.g., CHI) 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-l 15, 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,

deleting, substituting, or inserting one or more amino acids in a heavy chain variable
domain sequence described herein, optionally combining 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 EL-13.

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 germline sequences. One exemplary germlining method can include: identifying
one or more germline 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 determine if the germline residue or residues are tolerated.
Similar mutagenesis can be performed in the framework regions.

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 etal. (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),

SEENWYDFFDY (SEQ ED NO: 17),
RSSQSIVHSNGNTYLE (SEQ ED NO: 18),
KVSNRFS(SEQIDNO:19), and
FQGSHEPYT (SEQ ED 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 ED NO: 18),
KVSNRFS (SEQ ED NO: 19), and
FQGSHEPYT (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:
GFNEKDTYEH (SEQ ED NO: 15),
REDPANDNEKYDPKFQG (SEQ ID NO: 16), and
SEENWYDFFDY (SEQ ED 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):

(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 ED 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:l 18),
GASNLED (SEQ ID NO: 119), and
LQHSYLPWT (SEQ ID NO: 120)

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:l 19), 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),
IIWGDGSTDYNS AL (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
PQGSHIPYT (SEQ ID NO:30)
DWMTQSPLSLPVTLGQPASISCRSSQSIVHSNGNTYLEWFQQRPGQSP
RRLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC
PQGSHIPYT (SEQ ID NO:31)

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

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.

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

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:l 16) or WGQGTLVTVSS (SEQ ID NO:l 17).
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 mAbl3.2 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 mAbl3.2 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 mAbl3.2) is referred to herein as "chl3.2." The
amino acid and nucleotide sequences for the heavy chain variable region of chl3.2 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 "hl3.2vl," 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 hl3.2vl 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 "hl3.2v2," 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 hl3.2v2 are set forth herein as SEQ ID
NO:212 and SEQ ID NO:221, respectively. Another humanized form of mAbl3.2, which
is referred to herein as "hl3.2v3," 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 hl3.2v3 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

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,
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).

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., 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-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) EMBOJ. 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 FR1, 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,

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., mAbl3.2, 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., mAbl3.2, 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., mAbl3.2, 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

antibody described herein, e.g., mAbl3.2, 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 mAbl3.2) 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

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 IgGl 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

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 mAbl3.2 (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
mAbl3.2 (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 mAb 13.2 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) JMol Biol 226:889-896; 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 etal (1999) JImmunol Methods. 231(1-2):! 19-35.

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, EMBOJ. 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 P-
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 EL-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 p-sandwich similar to that of the antibody VH domain, except that Fn3 has seven
p-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

(e.g., Kunitz domains, ecotin, BPTI, and so forth); TPR repeats; trifoil structures; zinc
finger domains; DNA-binding proteins; particularly monomelic 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

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, -[CH20]--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

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.

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: IgGl, 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 CI 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 Rl), or
for Clq 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

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 C1 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-

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-

iodocytosine, and C5-propynyl pyrimidines such as C5-propynylcytosine and C5-
propynyluracil. Other suitable modified nucleobases include N4 ~(Ci -C12)
alkylaminocytosines and N N4 —(C1 -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 N6N6 (C1 -C12) dialkylaminopurines, including N6 -
methylaminoadenine and N6N6 -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 C1 -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 andDNA, 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-
10/BMP-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

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 ot-anomeric nucleic acid molecule forms
specific double-stranded hybrids with complementary RNA in which, contrary to the
usual p-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

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-10/BMP-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-
10/BMP-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., Battel, 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
etal. (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

"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., SI 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 a/. (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

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) J Immunol 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., NS0 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 dhff 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

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 Fey 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: BIACORE™ 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

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 EL-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
BIAcore 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.

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)7. 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, EL-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

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.

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 PROTONLX® 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 Symptoms 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.

Symptoms of allergic rhinitis (hay fever) include itchy, runny, sneezing, or stuffy
nose, and itchy eyes. An EL-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 dermatitis 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

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)7. 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

progression ofliver 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 normal 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.

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,

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 rag/m2 or about 5 to 20 mg/m2.

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 propel lant that forces aerosolized

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., dichlorodifiuoromethane, 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

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
(urn) 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
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., phosphatidylcholines, L-alpha-phosphatidylcholine dipalmitoyl

(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
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.

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.

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-inflammatory 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);

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™)); TNF enzyme antagonists, e.g., TNFa converting enzyme (TACE)
inhibitors; muscarinic receptor antagonists; TGF-p 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., Hrv 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.

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.

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

(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

include the fluoresceins and rhodamines. Another group of fluorescent compounds are
the naphthylamines. Once labeled with a fiuorophore 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, e3,1-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.

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 as 131I,
1In, 123I, 99mTc, 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.

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 I4C, 3H, 35S, ,25I, 99mTc, 32P,
33P, and 131I) 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 Tl 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
Tl) 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+, Gd3+). Other agents can be
in the form of particles, e.g., less than 10 \im 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

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

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
inventions but are not intended to, and should not be construed to, limit its scope in any
way.
EXAMPLES
Example 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

human IL-13 is shown in FIG. 1 A. 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 IgGl Fc tail
(sIL13Roc2-Fc). By binding to IL-13 and sequestering the cytokine from the cell surface
IL13Rccl-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 monocytes. 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 phosphorylation 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

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. IB. 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

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-13Rctl-Fc by ELISA. Wells were coated with soluble IL-13Rctl-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 sIL13Ral-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
sIL13Rccl-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 ALEXA™ 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.

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/ml 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 sIL13Ral-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.

Example 2: MJ 2-7 Antibody
Total RNA was prepared from MJ 2-7 hybridoma cells using the QIAGEN
RNEASY™ Mini Kit (Qiagen). RNA was reverse transcribed to cDNA using the
SMART™ PCR Synthesis Kit (BD Biosciences Clontech). The variable region of
MJ 2-7 heavy chain was extrapolated by PCR using SMART™ oligonucleotide as a
forward primer and mlgGl primer annealing to DNA encoding the N-terminal part of
CHI domain of mouse IgGl constant region as a reverse primer. The DNA fragment
encoding MJ 2-7 light chain variable region was generated using SMART™ and mouse
kappa specific primers. The PCR reaction was performed using DEEP VENT™ 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 EL-13 and which has characteristics which suggest that
it may interact with human IL-13 are as follows:



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

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:

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-13Rα2-/-, 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.

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 BLUE™ 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-13Rcc2. Mice deficient in
IL-13 had virtually undetectable levels of serum IgE. In contrast, mice lacking the
inhibitory receptor IL-13Rct2 displayed elevated levels of serum IgE. These results
demonstrate that blocking IL-13 can be useful for treating or preventing atopic disorders.
Example 6: EL-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.
Example 7: Nucleotide and amino acid sequences of mouse MJ 2-7 antibody
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

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.
STAT6phosphorylation 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 ALEXA™ Fluor 488-labeled antibody to STAT6 (BD Biosciences).

Fluorescence was analyzed with a FACSCAN™ and CELLQUEST™ 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/ml streptomycin, 2 mM L-glutamine, and
plated in a 48-well tissue culture plate (Costar/Corning). 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% CO2 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 CD1 lb (BD Biosciences). Monocytes were gated
based on high forward and side light scatter, and expression of CD1 lb. CD23 expression
on monocytes was determined by flow cytometry using a FACSCAN™ (BD
Biosciences), and the percentage of CD23+ cells was analyzed with CELLQUEST™
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/Corning), 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 uCi / well 3H-
thymidine (Perkin Elmer / New England Nuclear). They were incubated an additional

4.5 hours, then cells were harvested onto filter mats using a TOMTEK™ 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
supematants 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
supematants 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 ran.
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 ALEXA™ 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-


Example 11: Binding Interaction Site Between IL-13 and IL-13Rctl
A complex of IL-13, the extracellular domain of IL-13Ral (residues 27-342 of
SEQ ED 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 EL-13Ral
and IL-13 results in the formation of an extended beta sheet spanning the two molecules.
Residues Thr88 [Thrl07], Lys89 [Lysl08], Ile90 [Ilel09], and Glu91 [Glul 10] of EL-13
(SEQ ID NO: 124, mature sequence [full-length sequence (SEQ ED NO: 178)]) form a beta
strand that interacts with residues Lys76, Lys77, Ile78 and Ala79 of the receptor (SEQ ED
NO: 125). Additionally, the side chain of Met33 [Met52] of EL-13 (SEQ ED NO: 124
[SEQ ID NO:l 78]) 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 (Phel07 [Phel26]) of IL-13 (SEQ ED NO:124 [SEQ ED NO:178])
into a hydrophobic pocket in Ig domain 3 of the receptor IL-13Ral. The hydrophobic
pocket of IL-13Ral is formed by the side chains of residues Leu319, Cys257, Arg256,
and Cys320 (SEQ ED NO:125). The interaction with Phel07 [Phel26] of IL-13 (SEQ ID

NO: 124 [SEQ ID NO: 178]) results in an extensive set of van der Waals interactions
between amino acid residues Ile254, Ser255, Arg256, Lys318, Cys320, and Tyr321 of
IL-13Ral (SEQ ID NO: 125) and amino acid residues Argl 1 [Arg30], Glul2 [Glu31],
Leul3 [Leu32], Ilel4 [Ile33], Glul5 [Ile34], Lysl04 [Lysl23], Lysl05 [Lysl24], Leul06
[Leul25], Phel07 [Phel26], and Argl08 [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-13Ral 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 TRANSITIT™-LT1
Transfection reagent (Minis) 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 IgG 1/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 VI 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 VI 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

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 ug/ml) in the transient COS transfection experiments.

The CDR grafted MJ 2-7 VI 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 VI (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 VI and V2. The CDR grafted MJ 2-7 V2 had a 3-fold higher expression level
then CDR grafted MJ 2-7 VI in the same assay conditions.



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 (V48I 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.


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 (VI, 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 l^g/ml of anti-FLAG
monoclonal antibody M2 (Sigma). The FLAG NHP IL-13 protein at concentration of
10 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-
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. BIACORE™ analysis also confirmed that NHP IL-13 had rapid
binding to and slow dissociation to immobilized hluMJ 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), 1N8Z (co-crystal structure of
human Her2 in complex with Herceptin Fab) and F13.2 (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, 1N8Z and F13.2
(available from 16163-029001) were determined based on the Ccc 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-

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
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 1QBL and
1QBM, 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:I48
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
V48I.

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,
L115TandR87T.
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 CYCHROME™-
labeled antibody to the monocyte-specific marker, CD1 lb, 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 ALEXA™ 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
(V2.11) were 0.48 nM and 0.52 nM respectively. The potencies of MJ2-7 and
sEL-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

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 MJ2-7antibodv by SPR
sIL-13Ra2-Fc was directly coated onto a CM5 chip by standard amine coupling.
NHP-IL-13 at 100 nM concentration was injected, and its binding to the immobilized IL-
13Ra2-Fc was detected by BIACORE™. 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-EL-13 when it was in a complex with hu IL-13Rα2, whereas a positive
control anti-IL-13 antibody did (FIG. 7). These results indicate that hu EL-13Ra2 and
MJ2-7 bind to the same or overlapping epitopes of NHP IL-13.
Example 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 l-ethyI-3-(3-
dimethylaminopropyl) carbodiimide (EDC) and 0.05 M N-Hydroxysuccinimide (NHS).
The capturing antibody was injected at a concentration of 10 ug/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 ul of a 1 ug/ml 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

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 ul 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 NaCl, 3 mM EDTA and
0.005% (v/v) Surfactant P20) for 5 minutes at the same flow rate followed by two 5 ul
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 BIAEVALUATION™ software 3.0.2
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'V1 and 8.89x10"4 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 bioassavs
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 VI, 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 EL-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 VI 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 VI only
partially retained the neutralization activity the version with humanized VL V2 was as

active as parental mouse antibody. Therefore, a series of back-mutations were introduced
into the VI 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-13Ral 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-13Ral 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)7. 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 El 10,
El28, and LI22 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-13Ral (Eisenmesser
et al. (2001)7. Mol. Biol. 310:231-241; Moy et al. (2001)7. 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-13Ral 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 1 IB). This
provides a basis for the neutralization of IL-13 bioactivity by MJ2-7.

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 NHPIL-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 VI 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 CDR1 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.



Example 21: Neutralizing Activities of Anti-IL13 Antibodies in Cvnomolgus 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 scans-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

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-l 1 and humanized mAbl3.2v.2) were
administered to cynomolgus monkeys 24 hours prior to challenge with Ascaris suum
antigen. (mAb 13.2 and its humanized form hmAbl3.2v2 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-l 1, hmAbl3.2v2, 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,
hmAbl3.2v2- and hMJ2-7v2-l 1- treated samples are shown as dark and light triangles,
respectively. hMJ2-7v2-l 1 and hmAbl3.2v2 showed comparable efficacy in this model.
FIG. 15B shows a linear graph depicting the concentration of either hMJ2-7v2-l 1 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
(FIG 16A). Both hMJ2-7v2-l I and hmAbl3.2v2 reduced eotaxin levels detected in BAL
fluids from cynomolgus monkeys 24 hours after to challenge with Ascaris suum antigen,
compared to saline treated controls.

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-l 1 and hmAbl3.2v2 in IgE- and
basophil levels, cynomolgus monkeys dosed with humanized hMJ2-7v.2, hmAbl3.2v2,
irrelevant Ig (IVIG), or saline, as described above, were bled 8 weeks post-Ascaris
challenge, and levels of total and ;4scam-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-l 1. 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-l 1 relative to the pre-
challenge values in all dilutions assayed FIG 17A depicts representative examples
showing no change in Ascam-specific IgE titer in an individual monkey treated with
irrelevant Ig (IVIG; animal 20-45; top panel), and decreased titer of Ascaris-speciRc IgE
in an individual monkey treated with hMJ2-7v2-l 1 (animal 120-434; bottom panel).
Animals treated with either humanized hMJ2-7v.2-l 1 or hmAbl3.2v2 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-l 1. 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-l 1 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

FIGS. 18A-18B, the control animals demonstrated increased levels ofAscara-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-l 1 or hmAbl3.2v2 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-l 1 or
hmAbl3.2v2 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 scara-specific histamine release and
5cam-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 mAbl3.2v.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 mAbl3.2 reduced airway inflammation induced by Ascaris suum
antigen at comparable levels as detected by cytokine levels in BAL samples, serum levels
of scam-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-l 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-l 1 or hmAb3.2v2

antibodies. More specifically, ELISA plates (MaxiSorp; Nunc, Rochester, NY), were
coated overnight at 4°C with 0.5 ug/ml mAbl3.2 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 MJ1-64 (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 mAbl3.2, 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-IL13 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-l 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-l 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.

Example 22: Effects of Humanized Anti-IL-13 Antibodies on Airway Inflammation.
Lung Resistance, and Dynamic Lung Compliance Induced by Administration of Human
IL-13toMice
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-l 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-l 1
includes a GLN at position 110. In humans, however, position 110 is a polymorphic
variant, typically with ARG replacing GLN (e.g., Rl 10). The Rl 10Q polymorphic
variant is widely associated with increased prevalence of atopic disease.
In this example, recombinant human Rl 10Q 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 hl3.2v.2). Antibody MJ2-7 (IgGl, 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-l 1 (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 lOmM
L-histidine, pH 6, containing 5% sucrose.
To analyze the mouse lung response to the presence of recombinant human
Rl 10Q IL-13, BABL/c female mice were treated with 5 ug of recombinant human
Rl 10Q IL-13 (e.g., approximately 250 pg/kg), or an equivalent volume of saline (20 [iL),
administered intratracheally on days 1, 2, and 3. On day 4, animals were tested for signs
of airway resistance (Rl) 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

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 of recombinant human
Rl 10Q IL-13, or an equivalent volume (50 uL) 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 fim 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 Rl 10Q
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 Rl 10Q 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-l 1, antibody
levels in BAL and serum were analyzed following intraperitoneal and intravenous, or
intranasal administration following treatment with recombinant human Rl 10Q IL-13
administered intranasally or intratracheally. Briefly, BALB/c female mice were

administered 5 ug of recombinant human Rl 10Q 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 ug humanized MJ2-7v.2administered intravenously
on day 0, and by IP on days 1, 2, and 3 (FIG. 25 A). Alternatively, 500 ug of humanized
MJ2-7v.2-l 1 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 µl/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-l 1 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. 25 A shows elevated levels of human IgG in serum compared to BAL
following intraperitoneal and intravenous administraton of the humanized MJ2-7v.2-l 1
antibody. As shown in FIG. 25B, total IgG levels in ug/ml were significantly higher in
BAL than serum levels following intranasal administration of humanized MJ2-7v.2-l 1
antibody.
To determine if the humanized MJ2-7v.2-l 1 antibody was capable of modulating
the above observed lung function and inflammatory response, airway
hyperresponsiveness was induced by intratracheal administration of 5 ug recombinant
human Rl 10Q IL-13 or an equivalent volume (20 uL) of saline on days 1, 2, and 3. On
day 0, and 2 hours before administering each dose of recombinant human Rl 10Q IL-13,
animals were treated with 500 fig of humanized MJ2-7v.2-l 1, 500 ug dose of IVIG, or
an equivalent volume of saline, administered intranasally. Animals were tested on day 4
for airway resistance (Rl) and compliance (Cdyn) in response to increasing doses of
nebulized methacholine, as described above. Humanized MJ2-7v.2 and IVIG levels in

BAL and serum were analyzed by ELISA, as described above. As shown in FIGs. 26A-
26B, humanized MJ2-7v.2-l 1 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-l 1 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 µl well mouse anti-IL-13 antibody, mAbl3.2, 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 /xg/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 0.01M 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-l 1 complex formation, an ELISA was developed to specifically
detect IL-13 and humanized MJ2-7v.2-l 1 in complex. Briefly, ELISA plates (Nunc

Maxi-Sorp) were coated overnight with 30 µl well mouse anti-IL-13 antibody,
mAbl3.2, 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 0.01M
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-l 1 complexes were
recovered from BAL and serum of mice in this model. This observation indicates that
humanized MJ2-7v.2-l 1 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-l 1 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, C57BL/6 female mice (10/group) were
treated with 5 µg of recombinant human Rl 10Q IL-13 (e.g., approximately 250 fig/kg),
or an equivalent volume (50 µl) 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 µg, 100 µg, or 20 µg of humanized MJ2-7v.2-l 1 or humanized
13.2v.2. Control groups received 500 µg 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
Rl 10Q IL-13 treatment evoked an increase in eosinophil and neutrophil infiltration

levels. Interestingly, humanized MJ2-7v.2-l 1 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-l 1 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 µg of recombinant human
Rl 10Q IL-13 (e.g., approximately 250 µg/kg) or an equivalent volume (50 µl) 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 µg
of humanized MJ2-7v.2-l 1. On day 4, animals were sacrificed and BAL collected.
Humanized MJ2-7v.2-l 1 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-l 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 µg 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 µg I 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-l 1 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-l 1 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,

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-l 1 in vivo.
Example 23: Effects of IL-13 and / or IL-4 Neutralization at the Time of Allergen
Challenge on Allergen-Specific IgE 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.

Groups of mice were immunized by intraperitoneal injections with 200 µl solution
containing 20 µg OVA (grade V, Sigma-Aldrich, St Louis, MO) emulsified with 4 mg
aluminum hydroxide/magnesium hydroxide (ImjectAlum; Pierce, Rockford, EL) in PBS
on days 0 and 13 (FIG. 31). Sensitized mice were administered 200 µg/dose soluble
murine IL-13Ra2.IgG fusion protein (sIL-13Ra2.Fc; Wyeth Research) or 200 µg/dose
rat anti-mouse IL-4 monoclonal antibody (clone 30340; rat IgGl 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 Lmpac6 system (VetEquip, Pleasanton, CA) and
challenged intranasally with 20 µg OVA/mouse in 50 µl 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 IgGl was quantitated
with biotinylated rat anti-mouse IgGl 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

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 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, EL-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-13Rc2.Fc (200 µg)
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-13Rot2.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 IgGl (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-
13Rα2.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-13Rα2.Fc (p <0.01;
FIG. 33B). There was no change in titer of OVA-specific IgGl.
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
µg 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 u,g each). Neutralization of either IL-

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-13Rα2.Fc together with anti-EL-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 IgGl (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. etal. (1997) J Asthma 34:195-201; Yang, G. etal. (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-13Rct2.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 (-

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 IgGl, 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-13Roc2.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 IgGl were
not affected. Total serum IgE concentrations were also reduced in most experiments.
Interestingly, delivery of two doses of sIL-13Rα2.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 IgGl nor OVA-specific IgG3 titers were affected by the combined
treatment with sIL-13Rα2.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.

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.

WHAT IS CLAIMED IS:
1. A method of treating or preventing an IL-13-associated disorder or condition in
a subject, comprising administering to the subject, as a single treatment interval, an IL-13
antagonist and/or an IL-4 antagonist in an amount effective to reduce or delay the onset
or recurrence of one or more symptoms of the disorder or condition.
2. The method of claim 1, wherein the single treatment interval is a single dose of
the IL-13 antagonist and/or the IL-4 antagonist.
3. The method of claim 1, wherein single treatment interval consists essentially
of two or three doses of the IL-13 antagonist and/or the IL-4 antagonist within one week
or less from the initial dose.
4. The method of any of claims 1-3, wherein the administration of the IL-13
antagonist and/or an IL-4 antagonist occurs prior to any detectable manifestation of the
symptoms of the disorder or condition.
5. The method of any of claims 1-3, wherein the administration of the IL-13
antagonist and/or an IL-4 antagonist occurs after a partial manifestation of the symptoms
of the disorder or condition.
6. The method of any of claims 1-5, wherein the IL-13 antagonist and/or IL-4
antagonist is administered to the subject prior to exposure to an agent that triggers or
exacerbates the IL-13-associated disorder or condition.
7. The method of claim 6, wherein the IL-13 antagonist and/or IL-4 antagonist is
administered prior to seasonal exposure to an allergen.
8. The method of claim 4 or 5, wherein the IL-13 antagonist and/or IL-4
antagonist is administered prior to the recurrence of a flare or episode of the IL13-
associated disorder or condition.

9. The method of any of claims 1-5, wherein the IL-13 antagonist and/or IL-4
antagonist is administered anywhere between 1 to 5 days before or after exposure to the
triggering or exacerbating agent.
10. The method of claim 6 or 9, wherein the agent that triggers or exacerbates the
IL-13-associated disorder is selected from the group consisting of an allergen, a pollutant,
a toxic agent, an infection and stress.
11. The method of claim 1-10, wherein the symptoms of the IL-13 associated
disorder or condition comprise one or more of: increased IgE levels, increase histamine
release, increase eotaxin levels, or a respiratory symptom.
12. The method of claim 11, wherein the respiratory symptom comprises one or
more of: difficulty breathing, wheezing, coughing, shortness of breath and/or difficulty
performing normal daily activities.
13. The method of any of claim 1-12, wherein the subject is a human adult, an
adolescent, or a child having, or at risk of having, the IL-13 associated disorder or
condition.
14. The method of any of claim 1-13, wherein the IL-13-associated disorder or
condition is an inflammatory, a respiratory, an allergic, or an autoimmune disorder or
condition.
15. The method of any of claim 1-14, wherein the EL-13-associated disorder or
condition is chosen from one or more of: IgE-related disorders, atopic disorders, atopic
dermatitis, urticaria, eczema, allergic rhinitis allergic enterogastritis, asthma, chronic
obstructive pulmonary disease (COPD), and/or conditions involving airway
inflammation, eosinophilia, fibrosis and excess mucus production.

16. The method of any of claims 1-15, wherein the IL-13 associated disorder or
condition is chosen from one or more of: autoimmune conditions of the skin, atopic
dermatitis, inflammatory bowel disease (BBD), ulcerative colitis, Crohn's disease,
cirrhosis, hepatocellular carcinoma, scleroderma, tumors, cancers, leukemia,
glioblastoma, lymphoma, viral infections, and/or fibrosis of the liver.
17. The method of any of claims 1-16, wherein the single treatment interval
comprises a dose of the IL-13 antagonist in an amount of about 1-3 mg/kg.
18. The method of claim 17, wherein the IL-13 antagonist is administered by
inhalation, by injection or orally.
19. The method of any of claims 1-18, wherein 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 or an IL-4 receptor.
20. The method of claim 19, wherein the biological activities is chosen from one
or more of: induction of CD23 expression, production of IgE by human B cells,
phosphorylation of a transcription factor, activation of STAT6 protein, antigen-induced
eosinophilia in vivo; antigen-induced bronchoconstriction in vivo, and/or drug-induced
airway hyperreactivity in vivo.
21. The method of any of claims 1-21, wherein the IL-13 antagonist and/or the
IL4 antagonist is an antibody molecule that binds to IL-13, IL-13R, IL-4 or IL-4Roc; a
soluble form of the IL-13R or the IL-4Ra; an IL-13 or IL-4 mutein that binds to the
corresponding receptor, but does not substantially activate the receptor; a small molecule
inhibitor of STAT6; a peptide inhibitor; or an inhibitor of nucleic acid expression.
22. The method of claim 21, wherein the IL-13R is an IL-13Ra2 or an IL-
l3Ral.

23. The method of claim 21, wherein the IL-13 antagonist reduces formation of a
complex chosen from IL-13/IL-13aRl, IL-13/IL-4Ra, IL-13, IL-13/IL-13Ral/IL-4Ra;
orIL-13/IL13Ra2.
24. The method of claim 21, wherein the IL-4 antagonist reduces formation of a
complex chosen from IL-4/IL-4Rα, IL-4/ycommon, or IL-4/IL-4Rα/  common.
25. The method of claim 21, wherein the antibody molecule is an antibody, or an
antigen-binding fragment thereof that binds to IL-13 or IL-13R, or IL-4 or IL-4R.
26. The method of claim 21, wherein the antibody molecule binds to IL-13 with a
KD of less than 10"7 M, and has one or more of the following properties:

(a) 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 ED NO: 196) or C65 (SEQ
ED NO: 123);
(b) 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 ED NO: 18), mAb 13.2 (SEQ ED
NO: 197) or C65 (SEQ ID NO: 118);
(c) the heavy chain immunoglobulin variable domain comprises a 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 ED NO:73), V2.4 (SEQ ED NO:74), V2.5
(SEQ ED NO:75), V2.6 (SEQ ED NO:76), V2.7 (SEQ ED NO:77), V2.11 (SEQ ID
NO:80), chl3.2 (SEQ ED NO:204), hl3.2vl (SEQ ED NO:205), hl3.2v2 (SEQ ED
NO:206)or hl3.2v3 (SEQ ED NO:207);
(d) the light chain immunoglobulin variable domain comprises 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 ED NO:36) or hl3.2v2 (SEQ ED NO:212);

(e) the heavy chain immunoglobulin variable domain comprises an amino acid
sequence that is at least 90% 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); chl3.2 (SEQ ID NO:208), hl3.2vl (SEQ ID NO:209), hl3.2v2 (SEQ
ID NO:210) or hl3.2v3 (SEQ ID NO:211);
(f) the light chain immunoglobulin variable domain sequence is at least 90%
identical a light chain variable domain of V2.11 (SEQ ID NO:36)or hl3.2v2 (SEQ ID
NO:212);
(g) the antibody molecule competes with mAb MJ2-7, mAbl3.2 or C65 for
binding to human IL-13;
(h) the antibody molecule contacts one or more amino acid residues from IL-13
selected from the group consisting of residues 116, 117, 118, 122, 123, 124, 125, 126,
127, and 128 of SEQ ID NO:24 or SEQ ID NO: 178,
(i) the antibody molecule contacts one or more residues from IL-13 selected from
the group consisting of residues 81-93 and 114-132 of human IL-13 (SEQ ID NO: 194),
or selected from the group consisting 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 Arginine at position 105 [86] of SEQ ID
NO: 194 [position in mature sequence; SEQ ID NO: 195];
(j) the heavy chain variable domain sequence has the same canonical structure as
mAb MJ2"7, mAb 13.2 or C65 in hypervariable loops 1, 2 and/or 3;
(k) the light chain variable domain sequence has the same canonical structure as
mAb MJ2-7, mAb 13.2 or C65 in in hypervariable loops 1, 2 and/or 3; and
(1) the heavy chain variable domain sequence and/or the light chain variable
domain sequence has FR1, FR2, and FR3 framework regions from VH segments encoded
by germline genes DP-54 and DPK-9 respectively or a sequence at least 95% identical to
VH segments encoded by germline genes DP-54 and DPK-9; and
(m) confers a post-injection protective effect against exposure to Ascaris antigen
in a sheep model at least 6 weeks after injection.

27. The method of any of claims 1-26, wherein the IL-13 antagonist and the IL-4
antagonist are administered in combination simultaneously or sequentially.
28. The method of claim 27, wherein the IL-13 antagonist and the IL-4 antagonist
are co-formulated.
29. The method of claim 27, wherein the IL-13 antagonist and the IL-4 antagonist
are administered in combination with other therapeutic agents chosen from one or more
of: inhaled steroids, beta-agonists, antagonists of leukotrienes or leukotriene receptors,
IgE inhibitors, PDE4 inhibitors, xanthines, anticholinergic drugs, IL-5 inhibitors,
eotaxin/CCR3 inhibitors or anti-histamines.
30. A composition or a dose-formulation comprising an IL-13 antagonist and an
IL-4 antagonist, wherein the IL4 antagonist is selected from the group consisting of an
antibody molecule that binds to IL-4 or IL-4Ra; a soluble form of IL-4Rα; an IL-4
mutein; a small molecule inhibitor of STAT6; a peptide inhibitor; or an inhibitor of
nucleic acid expression, and the IL-13 antagonist is an antibody molecule competes with
mAb MJ2-7, mAbl3.2 or C65 for binding to human IL-13, or a soluble fragment of an
IL-13Rcx2.
31. A method for detecting the presence of IL-13 in a sample in vitro, comprising
providing a first anti-IL-13 antibody molecule immobilized to a support;
providing a sample obtained from a subject after exposure of the subject to a
second anti-IL-13 antibody molecule;
contacting the sample with the first anti-IL-13 antibody, under conditions that
allow binding of the IL-13 to the immobilized first anti-IL-13 antibody molecule to
occur; and
detecting IL-13 in the sample relative to a reference value,
wherein the first and second anti-IL13 antibodies bind to different epitopes on IL-13.

32. The method of claim 31, wherein the first anti-IL-13 antibody molecule binds
to substantially free IL-13, and does not substantially bind to IL-13 bound to the second
anti-IL-13 antibody molecule.
33. The method of claim 31, wherein the first anti-IL-13 antibody molecule binds
to substantially free IL-13 and IL-13 bound to a second anti-IL-13 antibody molecule.
34. The method of claim 31, wherein the detecting of the presence of IL-13
bound to the immobilized first anti-IL-13 antibody molecule is carried out using a labeled
third anti-IL-13 antibody molecule, or a labeled agent that recognizes the complex of IL-
13 first or second antibody molecule.
35. The method of claim 31, wherein a change in the level of IL-13 bound to the
first anti-IL-13 antibody molecule in the sample relative to a control sample is indicative
of the presence of the IL-13 in the sample
36. The method of claim 35, wherein the change is an increase in the level of IL-
13 in the sample relative to a predetermined level, wherein said increase is indicative of
increased inflammation in the lung.
37. The method of claim 31, wherein the sample is a biological sample selected
form the group consisting of a serum sample, a plasma sample, a tissue sample and a
biopsy.
38. A method for evaluating the efficacy of an IL-13 antagonistic binding agent,
in reducing pulmonary inflammation in a subject, comprising:
detecting the levels of IL-13 unbound and/or bound to an IL-13 antagonistic
binding agent in a sample according to the method of any of claims 31-37,
wherein a change in the levels of IL-13 unbound and/or bound relative to a reference
sample is indicative of the efficacy of the IL-13 antagonistic binding agent.

39. The method of claim 38, further comprising evaluating a change in one or
more of eotaxin levels in a sample, histamine release by basophils, IgE-titers, or
evaluating changes in the symptoms of the subject.
40. The method of claim 38 or 39, wherein a reduction in the levels of IL-13
unbound and/or bound to an IL-13 antagonistic binding agent, or an increase in the level
of IL-13 bound to the antagonistic binding agent is indicative that the IL-13 antagonistic
binding agent is effectively reducing lung inflammation in the subject.

41. A kit comprising an IL-13 antagonist and/or an IL-4 antagonist for use in any
of claims 1-29 with instructions for use as at a single treatment interval in treating or
preventing an IL-13 associated disorder or condition.
42. A composition for use in a method according to any of claims 1-29.
43. Use of a composition comprising an IL-13 antagonist and/or an IL-4
antagonist in the manufacture of a medicament for treating or preventing as a single
treatment interval an IL-13 associated disorder or condition.

Methods and compositions for treating and/or monitoring treatment of IL-13-associated disorders or conditions are disclosed.