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. Serial No. 60/926,078 and U.S. Serial No.
60/925,932, both of which were filed on April 23,2007. The contents of the
aforementioned applications are hereby incorporated by reference in their entirety.
SEQUENCE LISTING
An electronic copy of the Sequence Listing in both pdf and txt formats 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 etal. (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 embodiment, Applicants have
discovered that administration of an IL-13 antagonist, e.g., an IL-13 antibody molecule,
reduces at least one symptom of an allergen-induced early and/or a late asthmatic
response in a subject, e.g., a human subject, relative to an untreated subject. The
reduction in one or more asthmatic symptoms is detected within minutes following
exposure of the subject to the allergen, and during an early asthmatic response (e.g., up to
about 3 hours after exposure to the allergen). The reduction in symptoms is maintained
during a late asthmatic response (e.g., for a period of about 3 to 24 hours after allergen
exposure). In other embodiments, methods of evaluating an anti-IL13 antibody molecule
and/or treatment modalities associated with said antibody molecule are disclosed. The
evaluation methods include detecting at least one pharmacokinetic/pharmacodynamic
(PK/PD) parameter of the anti-IL13 antibody molecule in the subject. Thus, uses of IL-
13 binding agents or antagonists for reducing or inhibiting, and/or preventing or delaying
the onset of, in a subject, one or more symptoms associated with an early and/or a late
phase of an IL-13-associated disorder or condition are disclosed. In other embodiments,
methods for evaluating the kinetics and/or efficacy of an IL-13 binding agent or
antagonist in treating or preventing the IL-13-associated disorder or condition in a subject
are also disclosed..
Accordingly, in one aspect, the invention features a method of treating or
preventing an early and/or a late phase of an IL-13-associated disorder or condition in a
subject. The method includes administering an IL-13 binding agent or an 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: a respiratory symptom
(e.g., bronchoconstriction), IgE levels, release or levels of histamine or leukotriene, or
eotaxin levels 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 binding agent or antagonist can be administered prior to exposure to
an insult, or prior to the onset of 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 binding agent or antagonist inhibits or reduces one
or more symptoms associated with an early phase of the IL-13 associated disorder, e.g.,
an "early asthmatic response" or "EAR". For example, the IL-13 binding agent or
antagonist reduces one or more symptoms associated with an EAR, e.g., about 0.25,
about 0.5, about 1, about 1.5, about 2, about 2.5, or about 3 hours after an insult (e.g.,
allergen exposure) until about 3 hours after insult (e.g., allergen exposure). The IL-13
binding agent or antagonist can decrease or prevent one or more symptoms of the EAR
including, but not limited to, one or more of: a release of at least one allergic mediator
such as a leukotriene (e.g., LTA4, LTB4, LTC4, LTD4, LTE4, and/or LTF4) and/or
histamine, e.g., from airway mast or basophil cells; an increase in the levels of at least
one allergic mediator such as a leukotriene and/or histamine; bronchoconstriction; and/or
airway edema. The IL-13 binding agent or antagonist can cause a decrease in one or
more of these EAR symptoms in the subject, e.g., as compared to the level or degree of
the symptom in the subject in the absence of the IL-13 binding agent or antagonist.
Alternatively, the IL-13 binding agent or antagonist can prevent as large of an increase in
the symptom, e.g., as compared to the level or degree of the symptom in the subject in the
absence of the the IL-13 binding agent or antagonist).
In other embodiments, the IL-13 binding agent or antagonist inhibits or reduces
one or more symptoms associated with a late phase of an IL-13 associated disorder, e.g.,
a "late asthmatic response" or "LAR". For example, the IL-13 binding agent or
antagonist reduces one or more symptoms associated with an LAR, e.g., at least about 3,
about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 8,
about 9, about 10, about 11, about 12, or about 13 hours after an insult (e.g., allergen
exposure) up to about 24 hours after an insult (e.g., allergen exposure). For example, the
IL-13 binding agent or antagonist can decrease or prevent one or more symptoms of the
LAR, e.g., one or more of: airway reactivity and/or an influx and/or activation of
inflammatory cells, such as lymphocytes, eosinophils and/or macrophages, e.g., in the
airways and/or bronchial mucosa. The IL-13 binding agent or antagonist can cause a
decrease in one or more of these symptoms of an LAR in a subject, e.g., as compared to
the level or degree of the symptom in the subject in the absence of the the IL-13 binding
agent or antagonist. Alternatively, the IL-13 binding agent or antagonist can prevent as
large of an increase in the symptom, e.g., as compared to the level or degree of the
symptom in the subject in the absence of the the IL-13 binding agent or antagonist).
The IL-13 binding agent or antagonist can be administered prior to the onset or
recurrence of one or more symptoms associated with the IL-13-disorder or condition, but
before a full manifestation of the symptoms associated with the disorder or condition. In
certain embodiments, the IL-13 binding agent or 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 binding agent or antagonist is administered prior to, during, or
shortly after exposure to the agent that triggers and/or exacerbates the IL-13-associated
disorder or condition. For example, the IL-13 binding agent or 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 binding agent or antagonist can be administered anywhere between
24 hours and 2 days before or after exposure to the triggering or exacerbating agent. In
those embodiments where administration occurs after exposure to the agent, the subject
may not be experiencing symptoms or may be experiencing a partial manifestation of the
symptoms. For example, the subject may have symptoms of an early stage of the
disorder or condition. Each dose can be administered by inhalation or by injection, e.g.,
subcutaneously, in an amount of about 0.5-10 mg/kg (e.g., about 0.7-5 mg/kg, about 0.9-
4 mg/kg, about 1-3 mg/kg, about 1.5-2.5 mg/kg, or about 2 mg/kg). In one embodiment,
the single treatment interval includes two subcutaneous doses of about 1-3 mg/kg, about
1.5-2.5 mg/kg, or about 2 mg/kg of an anti-IL13 antibody molecule at least 4, 7, 9 or 14
days apart. For example, the single treatment interval can include two subcutaneous
doses of about 2 mg/kg of an anti-IL13 antibody molecule 7 days apart. In some
embodiments, a flat dose of an anti-IL13 antibody molecule is administered to the
subject, e.g., a flat dose of between about 50 mg and 500 mg, about 60mg and 490 mg,
about 70 mg to 480 mg, about 75 mg to 460 mg, about 80 mg to 450, about 100 mg and
about 450 mg, about 150 mg to about 400 mg, about 200 mg to about 300 mg, about 200
mg to about 250 mg; or about 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 90 mg, 100 mg, 125
mg, 150 mg, 175 mg, 200 mg, 225 mg, or 250 mg. The flat dose (e.g., about 75 mg, 100
mg, 200 mg or 225 of the anti-IL13 antibody molecule) (or any combination of the flat
dose) can be administered as a schedule of about once a week, every two weeks, every
three weeks, four weeks, or month, or any combination thereof, or as determined by a
clinician. An exemplary schedule of a flat dose of the anti-IL13 antibody is as follows:
initial dose at day 1, followed by doses at about days 8, 28, 42, 56, 70 and 84.
In one embodiment, the IL-13 binding agent or 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.
The IL-13 antagonist or binding agent 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 (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).
In certain embodiments, the subject is a human having mild, moderate or severe
asthma, e.g., atopic asthma The therapeutic and prophylactic methods disclosed herein
can be practiced prior to, during or aften allergen exposure. 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 binding agent or 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 binding agent or
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 binding agent or 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 binding agent or antagonist. In other embodiments, the IL-13 binding
agent or antagonist is administered in combination with allergy immunotherapy. For
example the IL-13 binding agent or 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 administration of the 11-13 binding agent or antagonist can
be repeated until a predetermined level of immunity is obtained in the subject.
In other embodiments, the IL-13 binding agent or 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 binding
agent or antagonist can be administered in an amount that decreases one or more of: (i)
the levels of IL-13 (e.g., free IL-13) in the subject; (ii) the levels of eotaxin in the subject;
(iii) the levels of histamine or leukotrienes in the subject; (iv) the amount of histamine or
leukotrienes released by mast cells or basophils (e.g., blood basophils); (v) the IgE-titers
in the subject; and/or (vi) one or more changes in the respiratory symptoms of the subject
(e.g., bronchoconstriction, e.g., difficulty breathing, wheezing, coughing, shortness of
breath and/or difficulty performing normal daily activities).
In other embodiments, the IL-13 binding agent or antagonist inhibits or reduces
one or more biological activities of IL-13 or an IL-13 receptor (e.g., an IL-13 receptor al
or an IL-13 receptor a2). Exemplary biological activities that can be reduced using the
IL-13 binding agent or antagonist 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 does not necessarily indicate a total elimination of the biological activity of
the IL-13/IL-13R polypeptide.
In one embodiment, the anti-IL-13 antibody molecule used in the therapeutic and
prophylactic methods is described herein. In other embodiments, the anti-IL13 antibody
molecule used in the methods is described in WO 05/123126, published on December 29,
2005 or its U.S. equivalent U.S. 06/0063228 (the entire contents of both applications are
incorporated herein by reference). For example, the antibody molecule is an antibody
that interferes with (e.g., inhibits, blocks or otherwise reduces) binding of IL-13 to an
epitope in either IL-13Rod or IL-13Ra2. In other embodiments, the antibody molecule
binds to a complex that includes IL-13 and IL-13Ral. In embodiments, the antibody
molecule binds to IL-13 and interferes with (e.g., inhibits blocks or otherwise reduces)
binding between a complex of IL-13 and IL-13Ral with IL-4Ra. In other embodiments,
the antibody molecule can, e.g., confer a post-injection protective effect against exposure
to Ascaris antigen in a sheep model at least 6 weeks after injection.
In one embodiment, the IL-13 binding agent or antagonist is administered in
combination with another therapeutic agent. The combination therapy can include an
IL-13 binding agent, e.g., an anti-IL-13 antibody molecule, co-formulated with and/or co-
administered with one or more additional therapeutic agents, 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 in more herein. The IL-13 binding agent and
the other therapeutic can also be administered separately.
Examples of preferred additional therapeutic agents that can be coadministered
and/or coformulated with an IL-13 binding agent include: 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-4 inhibitors (e.g.,
an IL-4 inhibitor antibody, IL-4 receptor fusion or an IL-4 mutein); IL-5 inhibitors;
eotaxin/CCR3 inhibitors; and antihistamines. Such combinations can be used to treat
asthma and other respiratory disorders. Additional examples of therapeutic agents that
can be co-administered and/or co-formulated 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-& 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 NFPB
inhibitors, among others.
In another aspect, this application provides compositions, e.g., pharmaceutical
compositions, that include a pharmaceutically acceptable carrier and at least one IL-13
binding agent, e.g., an anti-IL-13 antibody molecule. In one embodiment, the
compositions, e.g., pharmaceutical compositions, comprise a combination of two or more
IL-13 binding agents, e.g., two or more anti-IL-13 antibody molecules. A combinations
of the IL-13 binding agent, e.g., the anti-IL-13 antibody molecule, and a drug, e.g., a
therapeutic agent (e.g., one or more of an anti-histamine, an anti-leukotriene, a cytokine
or a growth factor inhibitor, an immunosuppressant, an anti-inflammatory agent (e.g.,
systemic anti-inflammatory agent), a metabolic inhibitor, an enzyme inhibitor, and/or a
cytotoxic or cytostatic agent, as described herein, can also be used.
In yet another embodiment, the methods disclosed herein further include:
evaluating the efficacy of an IL-13 binding agent (e.g., an anti-IL13 antibody molecule as
described herein or in WO 05/123126), in a subject, e.g., a human or non-human subject.
The method of evaluating the efficacy of the IL-13 binding agent can be practiced alone,
or in addition to the therapeutic and/or diagnostic methods described herein. In
embodiments, the efficacy of the IL-13 binding agent in reducing pulmonary symptoms
(e.g., eosinophilia, mucus production, bronchoconstriction, bronchospasm) is evaluated
by assessing one or more of the following parameters: (i) detecting the levels of IL-13 in
a sample (e.g., detecting the levels of IL-13 unbound and/or bound to an anti-IL13
antibody as described herein); (ii) measuring eotaxin levels in a sample; (iii) detecting the
levels or release of histamine and/or leukotrienes; (iv) detecting IgE-titers (total and/or
allergen-specific IgE); (v) detecting any changes to cysteinyl leukotriene receptor 1 or 2
protein or mRNA levels; (vi) evaluating changes in the symptoms of the subject (e.g.,
bronchoconstriction, e.g., difficulty breathing, wheezing, coughing, shortness of breath
and/or difficulty performing normal daily activities); (vii) evaluating lung function in a
subject (e.g., forced expiratory volume in 1 second (FEV1); (viii) evaluating a change in
the level of one or more cytokines (e.g., MCP-1, TNFa and/or interleukin-6 (IL-6); (ix)
evaluating a change in an inflammatory cell and/or marker in a sample from a subject;
and/or (x) evaluating at least one pharmacokinetic/ pharmacodynamic (PK/PD) parameter
of the IL-13 binding agent, e.g., a PK/PD parameter as described herein. The evaluation
of parameters (i)-(x) can be carried out before and/or after administration of the IL-13
binding agent (after single or multiple administrations) to the subject (e.g., at selected
intervals after initiating therapy). The evaluation can be performed by a clinician or
support staff. The sample can be a biological sample, such as serum, plasma, blood, or
sputum or tissue sample. A change, e.g., a reduction, in one or more of (i)-(x) relative to
a predetermined level (e.g., comparison before and after treatment) indicates that the IL-
13 binding agent is effectively reducing lung inflammation in the subject. In
embodiments, the subject is a human patient, e.g., an adult or a child.
In embodiments, the efficacy value, or an indication of whether the preselected
efficacy standard is met, is recorded or memorialized, e.g., in a computer readable
medium. Such values or indications of meeting pre-selected efficacy standard can be
listed on the product insert, a compendium (e.g., the U.S. Pharmacopeia), or any other
materials, e.g., labeling that may be distributed, e.g., for commercial use, or for
submission to a U.S. or foreign regulatory agency.
In another aspect, the invention features a method of evaluating or selecting an
IL-13 binding agent or antagonist, e.g., an anti-IL13 antibody molecule (e.g., an IL-13
antibody as described herein or in WO 05/123126). The method includes:
providing a. test value, e.g., a mean test value, for at least one pharmacokinetic/
pharmacodynamic (PK/PD) parameter of the IL-13 binding agent in a subject, e.g., a
human or animal subject; and
comparing the test value, e.g., mean test value, provided with at least one
reference value, to thereby evaluate or select the IL-13 binding agent.
The PK/PD parameter can be estimated using non-compartmental methods,
compartmental methods (e.g., two-compartmental model methods), and/or a PK-PD
model. The PK/PD parameter can be chosen from one or more of: an in vivo
concentration of the anti-IL13 antibody molecule (e.g., a concentration in blood, serum,
plasma and/or tissue); clearance of the anti-IL-13 antibody molecule (CL); steady-volume
distribution of the anti-IL-13 antibody molecule (Vdss); half-life of the anti-IL-13
antibody molecule (Un); bioavailability of the anti-IL-13 antibody molecule; dose
normalized maximum blood, serum or plasma concentration of the anti-IL-13 antibody
molecule; dose normalized exposure of the anti-IL-13 antibody molecule; or tissue-to-
serum ratio of the anti-IL-13 antibody molecule.
In a related embodiment, the PK/PD parameter can be estimated from the two-
compartmental or the PK-PD model. The PK/PD parameter can be chosen from one or
more of: clearance from the central compartment (CLab,); a distribution clearance
between the central compartment and the peripheral compartment (CLd,Ab); an association
rate constant (Kon); a dissociation rate constant (Koff); a serum clearance of the Ab-IL-13
complex (CLcomplex); an endogenous rate constant for IL-13 production divided by a
serum clearance of IL-13 (Ksyn/CLIL-13); an in vivo concentration of anti-IL-13 antibody
-IL-13 complex (CAb-IL-13and C(Ab-in-13)2) in blood, serum, plasma, or tissue; or an in
vivo concentration of free IL-13 (QIL-13) in blood, serum, plasma, or tissue.
The comparison can include determining if the test value has a pre-selected
relationship with the reference value, e.g., determining if it falls within the range of the
reference value (either inclusive or exclusive of the endpoints of the range); is equal to or
greater than the reference value. In embodiments, if the test value meets a preselected
relationship, e.g., falls within the reference value, the IL-13 binding agent is selected.
In embodiments where the IL-13 binding agent includes a full-length antibody,
the reference value, e.g., the mean reference value, includes one or more of: a clearance
(CL) mean value in the range of about 0.05 to 0.9, 0.06 to 0.5, 0.065 to 0.3, or 0.067 to
0.2 mL/hr/kg after intravenous administration of the IL-13 binding agent to the subject
(e.g., a mean CL value is in the range of about 0.05 to 0.5, 0.06 to 0.1, or 0.065 to 0.15
mL/hr/kg after intravenous administration to a human); a mean steady state volume of
distribution (Vdss) value of less than about 150, 130,120, 110, 100,90, 80, or 70 mL/kg
after intravenous administration to the subject (e.g., a control or diseased subject); a mean
half-life (tl/2) of about 50-800, 70-750, 100 to 600, 400-800, 500-700, 550 to 750, 552 to
696, 576 to 720,600 to 800,650 to 750,670 to 725, or 670 to 710 hours after
administration, e.g., intravenous, subcutaneous, intraperitoneal administration, to the
subject (e.g., a mean t1/2 of about 400-800,480-780, or 500-700 after intravenous or
subcutaneous administration to a human); a mean bioavailability of about 50 to 100, 60 to
90, or 70 to 85% after administration, e.g., subcutaneous or intraperitoneal
administration, to the subject; a dose normalized (a parameter value divided by the
dosage) mean maximum serum or plasma concentration of about 2 to 40,4 to 25, 5 to 22,
10 to 20,20 to 40, or 11 to 15 µg/ml after intravenous administration to the subject, or
about 0.1 to 30, 0.5 to 15, 0.75 to 12, 1 to 10, or 3 to 8 µg/ml after subcutaneous
administration to the subject; a mean Tmax of about or 6-200, 6-40,20-50, or 40-120
hours after subcutaneous administration to the subject; a mean dose normalized exposure
(i.e., mean value for area under the concentration-time profile curve from time zero to
infinity divided by the dosage) of about 800 to 18,000, 600 to 15,000, 500 to 12,000, 300
to 10,000, 150 to 5,000 (u.ghr/mL)/(mg/kg) after intravenous administration to the
subject, or 400 to 18000, 500 to 15,000, 600 to 12,000, 800 to 10,000, 1,000 to 5,000
(ughx/mL)/(mg/kg) after subcutaneous administration to the subject; a mean tissue-to-
serum ratio of less than about 0.8,0.6. 0.5. 0.4; or a mean preferential exposure of
antibody molecule in a tissue selected from the group consisting of lung, kidney, liver,
heart and spleen (e.g., an exposure or tissue concentration at a given time-point of greater
than 50%, 60%, 70% or greater than other organs).
In embodiments where the IL-13 binding agent includes an antigen-binding site of
the antibody molecule (e.g., a single chan antibody, a Fab fragment, a (Fab)'2, a Vh, a
Vhh). an Fv, a single chain Fv fragment, or a fusion protein containing an antigen-binding
site of the antibody molecule), the reference value, e.g., the mean reference value,
includes one or more of: a mean half-life (t1/2) of about 0.1 to 100, 0.2 to 75, 0.3 to 50,
0.4 to 45, 0.5 to 30, 0.5 to 15, 0.5 to 10, or 0.5 to 5 hours after administration, e.g.,
subcutaneous, intravenous, intraperitoneal administration, to the subject.
In embodiments where the IL-13 binding agent is complexed to IL-13, the
reference value, e.g., the mean reference value, includes a mean clearance of less 0.02
mL/hr/kg, 0.009 ml/hr/kg, 0.004 mL/hr/kg, 0.003 mL/hr/kg, or 0.002 mL/hr/kg after
administration e.g., subcutaneous, intravenous, intraperitoneal administration, to the non-
human primate or human subject. In other embodiments, the IL-13 binding agent is
evaluated using a two-compartmental integrated PK-PD model (e.g., "sequential
binding") as described herein. The model includes a central compartment (CAb, V) and a
peripheral compartment (C2,Ab,V2)- In those embodiments, one or more of the following
PK/PD parameters are evaluated: an in vivo concentration of the anti-IL13 antibody
molecule (e.g., a concentration in serum, plasma, blood, and/or tissue) (CAb); a clearance
from the central compartment (CLAb); a distribution clearance between the central
compartment and the peripheral compartment (CLd,Ab); an association rate constant (Kon);
a dissociation rate constant (Koff); a clearance of the Ab-IL-13 complex (CLcomplex); or an
endogenous rate constant for IL-13 production divided by a clearance (e.g., serum
clearance) of IL-13 (Ksyn/CLIL-13).
Exemplary reference values, e.g., mean reference values, of IL-13 binding agents
evaluated using a two-compartmental model where the IL-13 binding agent is a full-
length antibody includes one or more of: a clearance from the central compartment
(CLAb) mean value in the range of about 0.05 to 0.9, 0.06 to 0.5, 0.065 to 0.3, or 0.67 to
0.2 mL/hr/kg after intravenous administration of the IL-13 binding agent to the subject
(e.g., a mean CLAb value is in the range of about 0.05 to 0.5 , 0.06 to 0.1, or 0.065 to 0.15
mL/hr/kg after intravenous administration to a human); a volume of distribution in the
central compartment of less than about 150,130, 120,110, 90, 80, or 70 mL/kg after
intravenous administration to the subject (e.g., less than about 120, 90, 80, or 70 mL/kg
after intravenous administration to a human); a distribution clearance between the central
compartment and the peripheral compartment (CL,Ab) mean value in the range of about
0.0001-6.0, 0.0005 to 5.0,0.00067 to 4.5, 0.001 to 4.0 mL/hr/kg after intravenous
administration to the subject (e.g., 0.0002 to 5.7, or 0.0005 to 4.6 mL/hr/kg after
intravenous administration to a human); a volume distribution of the peripheral
compartment (V2) mean value of less than 150,130, 120, 110,90, 80, or 70 mL/kg after
intravenous administration to the subject (e.g., less than about 120, 90, 80, or 70 mL/kg
after intravenous administration to a human); an association rate constant (Kon) mean
value in the range of about 0.9 to 0.001, 0.5 to 0.01,0.3 to 0.02, or 0.026 to 0.06
nM-1day-1, a dissociation rate constant (Koff) mean value in the range of about 0.4 to
0.00001, 0.3 to 0.0001, 0.2 to 0.001, or 0.19 to 0.01; a serum clearance of the Ab-IL-13
complex (CLcomplex) mean value of about 0.40 to 0.00083, 0.25 to 0.0042, 0.17 to 0.0083,
0.15 to 0.0125 mL/hr/kg, or an endogenous rate constant for IL-13 production divided by
a serum clearance of IL-13 (Ksyn/CLIL-13) mean value of about 0.09 to 0.0001, 0.06 to
0.001, 0.05 to 0.003, 0.045 to 0.005 nM.
In embodiments, the test value, or an indication of whether the preselected
relationship is met, is recorded or memorialized, e.g., in a computer readable medium.
Such test values or indications of meeting pre-selected relationship can be listed on the
product insert, a compendium (e.g., the U.S. Pharmacopeia), or any other materials, e.g.,
labeling that may be distributed, e.g., for commercial use, or for submission to a U.S. or
foreign regulatory agency.
In embodiments, the step of providing a test value includes obtaining a sample of
the antibody molecule, e.g., a sample batch of an antibody culture, and testing for at least
one of the pharmacokinetic parameters described herein. Methods disclosed herein can
be useful from a process standpoint, e.g., to monitor or ensure batch-to-batch consistency
or quality.
In embodiments, a decision or step is taken depending on whether the test value
meets the pre-selected relationship (e.g., falls within the range provided for the reference
value). For example, the IL-13 binding agent, e.g., the anti-IL13 antibody molecule, can
be classified, selected, accepted, released (e.g., released into commerce) or withheld,
processed into a drag product, shipped, moved to a new location, formulated, labeled,
packaged, sold, or offered for sale.
In other embodiments, the test value provided is obtained after single or multiple
administrations of the antibody molecule at a dose of about 1 to 100 mg/kg, 1 to 10
mg/kg, or 1 to 2 mg/kg.
In other embodiments, the subject is a human or non-human animal, e.g., a rodent
or a primate. For example, the subject can be chosen from one or more of, e.g., rodent
(e.g., a mouse, rat), a primate (e.g., a monkey or a human, e.g., a patient). The human
can have a body weight of about 45-130 kg, or about 50-80 kg, typically 60 kg.
In another aspect, the invention provides a method of determining a treatment
modality (e.g., a dosage, timing, or mode of administration) of an IL-13 binding agent
(e.g., an anti-IL13 antibody molecule (e.g., an IL-13 antibody as described herein or in
WO 05/123126) for an IL-13-mediated disorder, in a subject. The method includes:
providing a test value, e.g., a mean test value, for at least one pharmacokinetic/
pharmacodynamic (PK/PD) parameter of the IL-13 binding agent in a subject, e.g., a
human or animal subject;
comparing the test value, e.g., mean test value, provided with at least one
reference value, e.g., mean reference value; and
selecting one or more of dosage, timing, or mode of administration based on the
comparison of at least one test value to the reference value.
The PK/PD parameter can be estimated using non-compartmental methods,
compartmental metihods (e.g., two-compartmental model methods), and/or a PK-PD
model. The PK/PD parameter can be chosen from one or more of: an in vivo
concentration of the anti-IL13 antibody molecule (e.g., a concentration in blood, serum,
plasma and/or tissue); clearance of the anti-IL-13 antibody molecule (CL); steady-volume
distribution of the anti-IL-13 antibody molecule (Vdss); half-life of the anti-IL-13
antibody molecule (t1/2); bioavailability of the anti-IL-13 antibody molecule; dose
normalized maximum blood, serum or plasma concentration of the anti-IL-13 antibody
molecule; dose normalized exposure of the anti-IL-13 antibody molecule; or tissue-to-
serum ratio of the anti-IL-13 antibody molecule.
In a related embodiment, the PK/PD parameter can be estimated from the two-
compartmental or the PK-PD model. The PK/PD parameter can be chosen from one or
more of: clearance from the central compartment (CLAb); a distribution clearance
between the central compartment and the peripheral compartment (CLd,Ab); an association
rate constant (Kon); a dissociation rate constant (Koff); a serum clearance of the Ab-IL-13
complex (CLcomplex); an endogenous rate constant for IL-13 production divided by a
serum clearance of IL-13 (Ksyn/CLIL-13); an in vivo concentration of anti-IL-13 antibody
-IL-13 complex (Cahl-h and C(Ab-il-13)2) in blood, serum, plasma, or tissue; or an in
vivo concentration of free IL-13 (CIL-13) in blood, serum, plasma, or tissue.
The comparison can include determining if the test value has a pre-selected
relationship with the reference value, e.g., determining if it falls within the range of the
reference value (either inclusive or exclusive of the endpoints of the range); is equal to or
greater than the reference value. In embodiments, if the test value meets a preselected
relationship, e.g., falls within the reference value, the IL-13 binding agent is selected.
In embodiments where the IL-13 binding agent includes a full-length antibody,
the reference value, e.g., the mean reference value, includes one or more of: a clearance
(CL) mean value in the range of about 0.05 to 0.9, 0.06 to 0.5, 0.065 to 0.3, or 0.067 to
0.2 mL/hr/kg after intravenous administration of the IL-13 binding agent to the subject
(e.g., a mean CL value is in the range of about 0.05 to 0.5, 0.06 to 0.1, or 0.065 to 0.15
mL/hr/kg after intravenous administration to a human); a mean steady state volume of
distribution (Vdss) value of less than about 150,130, 120, 110,100, 90, 80, or 70 mL/kg
after intravenous administration to the subject (e.g., a control or diseased subject); a mean
half-life (t1/2) of about 50-800, 70-750, 100 to 600,400-800, 500-700, 550 to 750, 552 to
696, 576 to 720,600 to 800, 650 to 750, 670 to 725, or 670 to 710 hours after
administration, e.g., intravenous, subcutaneous, intraperitoneal administration, to the
subject (e.g., a mean t1/2 of about 400-800,480-780, or 500-700 after intravenous or
subcutaneous administration to a human); a mean bioavailability of about 50 to 100, 60 to
90, or 70 to 85% after administration, e.g., subcutaneous or intraperitoneal
administration, to the subject; a dose normalized (a parameter value divided by the
dosage) mean maximum serum or plasma concentration of about 2 to 40, 4 to 25, 5 to 22,
10 to 20,20 to 40, or 11 to 15 (µg/ml after intravenous administration to the subject, or
about 0.1 to 30, 0.5 to 15,0.75 to 12, 1 to 10, or 3 to 8 µg/ml after subcutaneous
administration to the subject; a mean Tmax of about or 6-200,6-40,20-50, or 40-120
hours after subcutaneous administration to the subject; a mean dose normalized exposure
(i.e., mean value for area under the concentration-time profile curve from time zero to
infinity divided by the dosage) of about 800 to 18,000, 600 to 15,000, 500 to 12,000, 300
to 10,000, 150 to 5,000 (µghr/mL)/(mg/kg) after intravenous administration to the
subject, or 400 to 18000, 500 to 15,000, 600 to 12,000, 800 to 10,000,1,000 to 5,000
(µghr/mL)/(mg/kg) after subcutaneous administration to the subject; a mean tissue-to-
serum ratio of less than about 0.8, 0.6. 0.5. 0.4; or a mean preferential exposure of
antibody molecule in a tissue selected from the group consisting of lung, kidney, liver,
heart and spleen (e.g., an exposure or tissue concentration at a given time-point of greater
than 50%, 60%, 70% or greater than other organs).
In embodiments where the IL-13 binding agent includes an antigen-binding site of
the antibody molecule (e.g., a single chan antibody, a Fab fragment, a (Fab)'2, a VH, a
Vhh), an Fv, a single chain Fv fragment, or a fusion protein containing an antigen-binding
site of the antibody molecule), the reference value, e.g., the mean reference value,
includes one or more of: a mean half-life (t^) of about 0.1 to 100, 0.2 to 75, 0.3 to 50,
0.4 to 45, 0.5 to 30, 0.5 to 15, 0.5 to 10, or 0.5 to 5 hours after administration, e.g.,
subcutaneous, intravenous, intraperitoneal administration, to the subject.
In embodiments where the IL-13 binding agent is complexed to IL-13, the
reference value, e.g., the mean reference value, includes a mean clearance of less 0.02
mL/hr/kg, 0.009 ml/hr/kg, 0.004 mL/hr/kg, 0.003 mL/hr/kg, or 0.002 mL/hr/kg after
administration e.g., subcutaneous, intravenous, intraperitoneal administration, to the non-
human primate or human subject. In other embodiments, the IL-13 binding agent is
evaluated using a two-compartmental integrated PK-PD model (e.g., "sequential
binding") as described herein. The model includes a central compartment (CAb, V) and a
peripheral compartment (C2,Ab,V2). In those embodiments, one or more of the following
PK/PD parameters are evaluated: an in vivo concentration of the anti-IL13 antibody
molecule (e.g., a concentration in serum, plasma, blood, and/or tissue) (CAb); a clearance
from the central compartment (CLAb); a distribution clearance between the central
compartment and the peripheral compartment (CLd,Ab); an association rate constant (Kon);
a dissociation rate constant (Koff); a clearance of the Ab-IL-13 complex (CLcomplex); or an
endogenous rate constant for IL-13 production divided by a clearance (e.g., serum
clearance) of IL-13 (Ksyn/CLIL-13).
Exemplary reference values, e.g., mean reference values, of IL-13 binding agents
evaluated using a two-compartmental model where the IL-13 binding agent is a full-
length antibody includes one or more of: a clearance from the central compartment
(CLAb) mean value in the range of about 0.05 to 0.9, 0.06 to 0.5, 0.065 to 0.3, or 0.67 to
0.2 mL/hr/kg after intravenous administration of the IL-13 binding agent to the subject
(e.g., a mean CLAb value is in the range of about 0.05 to 0.5 , 0.06 to 0.1, or 0.065 to 0.15
mL/hr/kg after intravenous administration to a human); a volume of distribution in the
central compartment of less than about 150, 130, 120,110, 90, 80, or 70 mL/kg after
intravenous administration to the subject (e.g., less than about 120, 90, 80, or 70 mL/kg
after intravenous administration to a human); a distribution clearance between the central
compartment and the peripheral compartment (CLd,Ab) mean value in the range of about
0.0001-6.0, 0.0005 to 5.0, 0.00067 to 4.5, 0.001 to 4.0 mL/hr/kg after intravenous
administration to the subject (e.g., 0.0002 to 5.7, or 0.0005 to 4.6 mL/hr/kg after
intravenous administration to a human); a volume distribution of the peripheral
compartment (V2) mean value of less than 150,130, 120, 110, 90, 80, or 70 mL/kg after
intravenous administration to the subject (e.g., less than about 120, 90, 80, or 70 mL/kg
after intravenous administration to a human); an association rate constant (Kon) mean
value in the range of about 0.9 to 0.001, 0.5 to 0.01, 0.3 to 0.02, or 0.026 to 0.06
nM-1day-1, a dissociation rate constant (Koff) mean value in the range of about 0.4 to
0.00001, 0.3 to 0.0001, 0.2 to 0.001, or 0.19 to 0.01; a serum clearance of the Ab-IL-13
complex (CLcomplex) mean value of about 0.40 to 0.00083, 0.25 to 0.0042, 0.17 to 0.0083,
0.15 to 0.0125 mL/hr/kg, or an endogenous rate constant for IL-13 production divided by
a serum clearance of IL-13 (Ksyn/CLIL-13) mean value of about 0.09 to 0.0001, 0.06 to
0.001, 0.05 to 0.003, 0.045 to 0.005 nM.
The selection of treatment modality (e.g., a dosage, timing, or mode of
administration) can be based, in part, on the comparison of the test value and the
reference value. The comparison can include determining if the test value has a pre-
selected relationship with the reference value, e.g., determining if it falls within the range
of the reference value (either inclusive or exclusive of the endpoints of the range); is
equal to or greater than the reference value. For example, if the half-life of the binding
agent falls within the range specified in the reference value, a practitioner may determine
that the frequency of administration can be reduced to, e.g., once or twice per month. In
combination or independently, a low dose of the binding agent can be administered, e.g.,
less than one of 5,4, 3, 2,1 mg/kg. Treatment modalities chosen based on the
comparison can vary depending on the IL-13-associated disorder at issue. For respiratory
disorders, e.g., asthma, the IL-13 binding agent can be delivered by inhalation,
subcutaneously or intravenously.
In embodiments, the subject is a human or non-human animal, e.g., a rodent or a
primate. For example, the subject can be chosen from one or more of, e.g., rodent (e.g., a
mouse, rat), a primate (e.g., a monkey or a human, e.g., a patient). The human can have a
body weight of about 45-130 kg, or about 50-80 kg, typically 60 kg. The human may be
a control or diseased subject.
In another aspect, the invention features a method of treating an IL-13-associated
disorder (e.g., an IL-13 disorder as described herein) in a subject, e.g., a subject as
described herein, that includes administering, to a subject having, or being at risk of
having, the IL-13-associated disorder, an effective amount of the IL-13 binding agent,
e.g., the anti-IL-13 antibody molecule evaluated or selected using one or more of the
PK/PD parameters described herein.
In another aspect, the invention features a method of instructing, or transferring
information to, a recipient (e.g., a patient, a pharmacist, a caregiver, a clinician, a member
of a medical staff, a manufacturer, or a distributor) on the use of an IL-13 binding agent,
e.g., an anti-IL13 antibody molecule, to treat an IL-13-associated disorder. The method
includes instructing, or sending information to, the recipient that said IL-13 binding agent
has at least one test value, e.g., mean test value, for a PK/PD parameter selected from the
group consisting of:
a clearance (CL) mean value in the range of about 0.05 to 0.9, 0.06 to 0.5, 0.065
to 0.3, or 0.067 to 0.2 mL/hr/kg after intravenous administration of the IL-13 binding
agent to a subject (e.g., a mean CL value is in the range of about 0.05 to 0.5, 0.06 to 0.1,
or 0.065 to 0.15 mL/hr/kg after intravenous administration to a human), wherein the IL-
13 binding agent includes a full-length antibody; a mean steady state volume of
distribution (Vdss) value of less than about 150,130, 120, 110, 100, 90, 80, or 70 mL/kg
after intravenous administration to the subject (e.g., a control or diseased subject),
wherein the IL-13 binding agent includes a full-length antibody; a mean half-life (tm) of
about 50-800, 70-750, 100 to 600,400-800, 500-700, 550 to 750, 552 to 696, 576 to 720,
600 to 800, 650 to 750, 670 to 725, or 670 to 710 hours after administration, e.g.,
intravenous, subcutaneous, intraperitoneal administration, to the subject (e.g., a mean ti/2
of about 400-800,480-780, or 500-700 after intravenous or subcutaneous administration
to a human); a mean bioavailability of about 50 to 100,60 to 90, or 70 to 85% after
administration, e.g., subcutaneous or intraperitoneal administration, to the subject; a dose
normalized (a parameter value divided by the dosage) mean maximum serum or plasma
concentration of about 2 to 40, 4 to 25, 5 to 22, 10 to 20,20 to 40, or 11 to 15 µg/ml after
intravenous administration to the subject, or about 0.1 to 30, 0.5 to 15, 0.75 to 12, 1 to 10,
or 3 to 8 µg/ml after subcutaneous administration to the subject; a mean Tmax of about or
6-200, 6-40, 20-50, or 40-120 hours after subcutaneous administration to the subject; a
mean dose normalized exposure (i.e., mean value for area under the concentration-time
profile curve from time zero to infinity divided by the dosage) of about 800 to 18,000,
600 to 15,000, 500 to 12,000, 300 to 10,000, 150 to 5,000 (µghr/mL)/(mg/kg) after
intravenous administration to the subject, or 400 to 18000, 500 to 15,000,600 to 12,000,
800 to 10,000,1,000 to 5,000 (µghr/mL)/(mg/kg) after subcutaneous administration to
the subject; a mean tissue-to-serum ratio of less than about 0.8, 0.6. 0.5. 0.4; or a mean
preferential exposure of antibody molecule in a tissue selected from the group consisting
of lung, kidney, liver, heart and spleen (e.g., an exposure or tissue concentration at a
given time-point of greater than 50%, 60%, 70% or greater than other organs), wherein
the IL-13 binding agent includes a full-length antibody; a mean half-life (t1/2) of about 0.1
to 100,0.2 to 75, 0.3 to 50, 0.4 to 45, 0.5 to 30, 0.5 to 15, 0.5 to 10, 0.5 to 5 hours after
administration, e.g., subcutaneous, intravenous, intraperitoneal administration, to the
subject, wherein the IL-13 binding agent includes an antigen-binding site of the antibody
molecule (e.g., a single chan antibody, a Fab fragment, a (Fab)"2, a VH, a Vhh) an Fv, a
single chain Fv fragment, or a fusion protein containing an antigen-binding site of the
antibody molecule); and a mean clearance rate of less than 0.004 mL/hr/kg, 0.003
mL/hr/kg, or 0.002 mL/hr/kg after administration to the subject, wherein the IL-13
binding agent is complexed to IL-13.
In other embodiments, the PK/PD parameter of the IL-13 binding agent is
evaluated using a two-compartmental (e.g., "sequential binding") model as described
herein. The two-compartmental model includes a central compartment (CAb, V) and a
peripheral compartment (C2,Ab,V2). In those embodiments, one or more of the following
PK/PD parameters are evaluated: an in vivo concentration of the anti-IL13 antibody
molecule (e.g., a concentration in serum, plasma, and/or tissue) (CLAb), a distribution
clearance between the central compartment and the peripheral compartment (CLd,Ab), an
association rate constant (Kon), a dissociation rate constant (Koff), a serum clearance of
the Ab-IL-13 complex (CLcomplex), or an endogenous rate constant for IL-13 production
divided by a serum clearance of IL-13 (Ksyn/CLIL-13).
Exemplary reference values, e.g., mean reference values, of IL-13 binding agents
evaluated using a two-compartmental model where the IL-13 binding agent is a full-
length antibody includes one or more of: a clearance from the central compartment
(CLAb) mean value in the range of about 0.05 to 0.9,0.06 to 0.5,0.065 to 0.3, or 0.67 to
0.2 mL/hr/kg after intravenous administration of the IL-13 binding agent to the subject
(e.g., a mean CLAb value is in the range of about 0.05 to 0.5 , 0.06 to 0.1, or 0.065 to 0.15
mL/hr/kg after intravenous administration to a human); a volume of distribution in the
central compartment of less than about 150, 130, 120,110, 90, 80, or 70 mL/kg after
intravenous administration to the subject (e.g., less than about 120, 90, 80, or 70 mL/kg
after intravenous administration to a human); a distribution clearance between the central
compartment and the peripheral compartment (CLd,Ab) mean value in the range of about
0.0001-6.0, 0.0005 to 5.0, 0.00067 to 4.5, 0.001 to 4.0 mL/hr/kg after intravenous
administration to the subject (e.g., 0.0002 to 5.7, or 0.0005 to 4.6 mL/hr/kg after
intravenous administration to a human); a volume distribution of the peripheral
compartment (V2) mean value of less than 150,130, 120, 110, 90, 80, or 70 mL/kg after
intravenous administration to the subject (e.g., less than about 120,90, 80, or 70 mL/kg
after intravenous administration to a human); an association rate constant (Kon) mean
value in the range of about 0.9 to 0.001, 0.5 to 0.01, 0.3 to 0.02, or 0.026 to 0.06 nM-1day-
1, a dissociation rate constant (Koff) mean value in the range of about 0.4 to 0.00001, 0.3
to 0.0001, 0.2 to 0.001, or 0.19 to 0.01; a serum clearance of the Ab-1L-13 complex
(CLcomplex) mean value of about 0.40 to 0.00083, 0.25 to 0.0042, 0.17 to 0.0083, 0.15 to
0.0125 mL/hr/kg, or an endogenous rate constant for IL-13 production divided by a
serum clearance of IL-13 (Ksyn/CLIL-13) mean value of about 0.09 to 0.0001, 0.06 to
0.001, 0.05 to 0.003, 0.045 to 0.005 nM.
In embodiments, the method includes recording or memorializing, e.g., in a
computer readable medium, one of more of the test values. Such test values can be listed
on the product insert, a compendium (e.g., the U.S. Pharmacopeia), or any other
materials, e.g., labeling that may be distributed, e.g., for commercial use, or for
submission to a U.S. or foreign regulatory agency.
In embodiments, the method further includes administering the IL-13 binding
agent to the patient. In embodiments, one or more of dosage, timing, or mode of
administration of the binding agent, e.g., antibody molecule, is based, at least in part, on
the comparison of the test value at least one PK/PD parameter of the antibody molecule
with a reference value, e.g., a reference value as described herein.
In another aspect, the invention features method of treating an IL-13-associated
disorder in a subject having, or being at risk of having, the IL-13-associated disorder.
The method includes:
instructing a caregiver or a patient that an IL-13 binding agent, e.g., an anti-IL13
antibody has at least one test value, e.g., mean test value, for a PK/PD parameter selected
from the group consisting of:
a clearance (CL) mean value in the range of about 0.05 to 0.9, 0.06 to 0.5, 0.065
to 0.3, or 0.067 to 0.2 mL/hr/kg after intravenous administration of the IL-13 binding
agent to a subject (e.g., a mean CL value is in the range of about 0.05 to 0.5, 0.06 to 0.1,
or 0.065 to 0.15 mL/hr/kg after intravenous administration to a human), wherein the IL-
13 binding agent includes a full-length antibody; a mean steady state volume of
distribution (Vdss) value of less than about 150, 130,120,110, 100, 90, 80, or 70 mL/kg
after intravenous administration to the subject (e.g., a control or diseased subject),
wherein the IL-13 binding agent includes a full-length antibody; a mean half-life (t1/2) of
about 50-800, 70-750, 100 to 600,400-800, 500-700, 550 to 750, 552 to 696, 576 to 720,
600 to 800, 650 to 750, 670 to 725, or 670 to 710 hours after administration, e.g.,
intravenous, subcutaneous, intraperitoneal administration, to the subject (e.g., a mean t1/2
of about 400-800, 480-780, or 500-700 after intravenous or subcutaneous administration
to a human); a mean bioavailability of about 50 to 100, 60 to 90, or 70 to 85% after
administration, e.g., subcutaneous or intraperitoneal administration, to the subject; a dose
normalized (a parameter value divided by the dosage) mean maximum serum or plasma
concentration of about 2 to 40,4 to 25, 5 to 22, 10 to 20,20 to 40, or 11 to 15 µg/ml after
intravenous administration to the subject, or about 0.1 to 30, 0.5 to 15, 0.75 to 12, 1 to 10,
or 3 to 8 µg/ml after subcutaneous administration to the subject; a mean Tmax of about or
6-200, 6-40, 20-50, or 40-120 hours after subcutaneous administration to the subject; a
mean dose normalized exposure (i.e., mean value for area under the concentration-time
profile curve from time zero to infinity divided by the dosage) of about 800 to 18,000,
600 to 15,000, 500 to 12,000, 300 to 10,000, 150 to 5,000 (µghr/mL)/(mg/kg) after
intravenous administration to the subject, or 400 to 18000, 500 to 15,000, 600 to 12,000,
800 to 10,000, 1,000 to 5,000 (µghr/mL)/(mg/kg) after subcutaneous administration to
the subject; a mean tissue-to-serum ratio of less than about 0.8, 0.6. 0.5. 0.4; or a mean
preferential exposure of antibody molecule in a tissue selected from the group consisting
of lung, kidney, liver, heart and spleen (e.g., an exposure or tissue concentration at a
given time-point of greater than 50%, 60%, 70% or greater than other organs), wherein
the IL-13 binding agent includes a full-length antibody; a mean half-life (tm) of about 0.1
to 100, 0.2 to 75, 0.3 to 50, 0.4 to 45, 0.5 to 30,0.5 to 15, 0.5 to 10, 0.5 to 5 hours after
administration, e.g., subcutaneous, intravenous, intraperitoneal administration, to the
subject, wherein the IL-13 binding agent includes an antigen-binding site of the antibody
molecule (e.g., a single chan antibody, a Fab fragment, a (Fab)'2, a VH, a VHh), an Fv, a
single chain Fv fragment, or a fusion protein containing an antigen-binding site of the
antibody molecule); and a mean clearance rate of less than 0.004 mL/hr/kg, 0.003
mL/hr/kg, or 0.002 mL/hr/kg after administration to the subject, wherein the IL-13
binding agent is complexed to IL-13; and
administering the IL-13 binding agent, e.g., the anti-IL13 antibody molecule, to
the patient. The administration step can be performed by the patient directly, e.g., self-
administration, or by another party, e.g., a caregiver.
In other embodiments, the PK/PD parameter of the IL-13 binding agent is
evaluated using a two-compartmental model as described herein. The two-
compartmental model includes a central compartment (CAb, V) and a peripheral
compartment (C2,Ab,V2). In those embodiments, one or more of the following PK/PD
parameters are evaluated: an in vivo concentration of the anti-EL13 antibody molecule
(e.g., a concentration in serum, plasma, and/or tissue) (CLAb), a distribution clearance
between the central compartment and the peripheral compartment (CLd,Ab), an association
rate constant (Kon), a dissociation rate constant (Koff), a serum clearance of the Ab-IL-13
complex (CLCompkex), or an endogenous rate constant for IL-13 production divided by a
serum clearance of IL-13 (Ksyn/CLIL-13).
Exemplary reference values, e.g., mean reference values, of IL-13 binding agents
evaluated using a two-compartmental model where the IL-13 binding agent is a full-
length antibody include one or more of: a clearance from the central compartment
(CLAb) mean value in the range of about 0.05 to 0.9,0.06 to 0.5, 0.065 to 0.3, or 0.67 to
0.2 mL/hr/kg after intravenous administration of the IL-13 binding agent to the subject
(e.g., a mean CLAb, value is in the range of about 0.05 to 0.5 , 0.06 to 0.1, or 0.065 to 0.15
mL/hr/kg after intravenous administration to a human); a volume of distribution in the
central compartment of less than about 150, 130, 120,110, 90, 80, or 70 mL/kg after
intravenous administration to the subject (e.g., less than about 120, 90, 80, or 70 mL/kg
after intravenous administration to a human); a distribution clearance between the central
compartment and the peripheral compartment (CLd,Ab) mean value in the range of about
0.0001-6.0, 0.0005 to 5.0, 0.00067 to 4.5, 0.001 to 4.0 mL/hr/kg after intravenous
administration to the subject (e.g., 0.0002 to 5.7, or 0.0005 to 4.6 mL/hr/kg after
intravenous administration to a human); a volume distribution of the peripheral
compartment (V2) mean value of less than 150, 130, 120, 110, 90, 80, or 70 mL/kg after
intravenous administration to the subject (e.g., less than about 120, 90, 80, or 70 mL/kg
after intravenous administration to a human); an association rate constant (Kon) mean
value in the range of about 0.9 to 0.001, 0.5 to 0.01, 0.3 to 0.02, or 0.026 to 0.06
nM-1day-1, a dissociation rate constant (Koff) mean value in the range of about 0.4 to
0.00001, 0.3 to 0.0001, 0.2 to 0.001, or 0.19 to 0.01; a serum clearance of the Ab-IL-13
complex (CLcomplex) mean value of about 0.40 to 0.00083, 0.25 to 0.0042, 0.17 to 0.0083,
0.15 to 0.0125 mL/hr/kg, or an endogenous rate constant for IL-13 production divided by
a serum clearance of IL-13 (Ksyn/CLIL-13) mean value of about 0.09 to 0.0001,0.06 to
0.001, 0.05 to 0.003, 0.045 to 0.005 nM.
In embodiments, one or more of dosage, timing, or mode of administration of the
binding agent, e.g., antibody molecule, is based, at least in part, on a comparison of the
test value at least one PK/PD parameter of the antibody molecule with a reference value,
e.g., a reference value as described herein.
In another aspect, the invention features a kit or package that includes an IL-13
binding agent, e.g., an anti-IL13 antibody molecule as described herein or in WO
05/123126), or a pharmaceutical composition thereof, and instructions for use. In
embodiments, the IL-13 binding agent included in the kit is or has been evaluated or
selected based, at least in part, on a comparison of a test value with a reference value, as
described herein. In other embodiments, the IL-13 binding agent has at least one test
value for a PK/PD parameter as described herein. In embodiments, the kit includes an
IL-13 antibody molecule packaged to be administered as a flat dose, e.g., a flat dose as
described herein, and instruction for administration as a flat dose.
In yet another aspect, the invention features an IL-13 binding agent, e.g., an anti-
IL13 molecule, selected or evaluated by comparing a test value for a pharmacokinetic
parameter with a reference value, as described herein. In embodiments, the binding agent
is other than 13.2, MJ2-7 and C65 (or humanized versions thereof).
In another aspect, the invention features a method of evaluating the amount of a
drug-ligand complex in a subject using a two-compartmental PK-PD model that includes
a central compartment (CAb, V) and a peripheral compartment (C2,Ab,V2). The method
includes:
providing at least one pharmacokinetic or pharmacodynamic parameter value of
the drug-ligand concentration in the subject at a predetermined time interval, said value
chosen from one oir more of the following PK/PD parameters: an in vivo concentration of
the drug, e.g., anti-ILI3 antibody molecule (e.g., a concentration in blood, serum, plasma,
and/or tissue) (CLAb); an in vivo concentration of unbound 11-13, anti-IL-13-bound IL-13
or total IL-13 ((e.g., a concentration in blood, serum, plasma, and/or tissue) ) a
distribution clearance between the central compartment and the peripheral compartment
(CLd,Ab); an association rate constant (Kon); a dissociation rate constant (Koff); a serum
clearance of the drug-ligand (e.g., Ab-IL-13) complex (CLcomplex); or an endogenous rate
constant for ligand, e.g., IL-13, production divided by a serum clearance of the ligand,
e.g., IL-13, (Ksyn/CLIL-13);
evaluating the at least one pharmacokinetic parameter in the subject using the
two-comparmental PK-PD model as represented in FIG. 33.
In embodiments, the two-compartmental model is represented as follows:

For 2vbolus close:
In(t) = Dose (6)
Forsc dose:
Ir{f) = Ka*F*Dose (7)
wherein,
CAb is a concentration of antibody (binding agent);
In(t) is a dose administered (for a bolus dose), and In(t) is Ka*F*Dose for
a subcutaneous does, wherein Ka is a first order rate constant and F is an estimate
of bioavailability;
CLd,Ab is a distribution clearance between the central compartment and the
peripheral compartment;
C2,Ab i is a concentration of the ligand binding agent in the peripheral
compartment;
V is a volume distribution in a central component;
Kon is a second order rate constant;
C ligand (or CIL-13) is a concentration of ligand;
CAb(ligand) (or CAb-(IL-13))is a concentration of ligand binding agent/ligand
complex;
Koff is a first order disassociation rate constant, V2 is a volume of
distribution in a peripheral compartment;
CLcomplex is the serum clearance of the ligand binding agent/ligand
complex; and
Ksyn is a zero order rate constant for endogenous ligand.
In certain embodiments, the method evaluates the amount of a drug-ligand
complex selected from the group consisting of a ligand-antibody complex and a ligand-
soluble receptor complex. For example, the ligand can be a cytokine, e.g., IL-5, IL-6, IL-
12, IL-13, IL-21, IL-22; or a growth factor, e.g., VEGF, TNFa; and the drug can be an
antibody against the ligand, or a soluble receptor.
In certain embodiments, the method evaluates the amount of IL-13-antibody
complex in the subject. For example, the two compartmental model used in the methods
includes pharmacokinetic/pharmacodynamic values for one the following:
the Ligand is IL-13 and the ligand binding agent (Ab) is a drug (e.g., is an
antibody molecule (e.g., hMJ2-7v.2-11HMJ2-7v.2-l 1));
Complex is a drug-ligand complex (e.g., hMJ2-7v.2-l 1 HMJ2-7v.2-l l/IL-13
complex);
CLd,Ab and CLAbare distribution clearance and serum clearance of the drug (e.g.,
an antibody molecule (e.g., hMJ2-7v.2-l lHMJ2-7v.2-l 1)), respectively;
CLcomplex and C LLigand (or CLIL-13) are serum clearance of the complex and the
ligand, e.g., IL-13, respectively;
Ksyn is a zero-order ligand, e.g., IL-13, synthesis rate constant;
Kon is a second-order association rate constant;
Koff is a first-order dissociation rate constant; and V and V2 are volumes of
distribution of the drug (e.g., hMJ2-7v.2-l 1 HMJ2-7v.2-l 1) in the serum (central) and the
second compartment, respectively.
In some aspects, the invention features a method of treating an IL-13-associated
disorder in a subject, e.g., using a flat dose of anti-IL-13 antibody. The method includes
administering, to a subject having, or being at risk of having, the IL-13-associated
disorder, a flat dose of an anti-IL-13 antibody molecule (e.g., hMJ2-7v.2-l 1 HMJ2-7v.2-
11 or 13.2v2).
In some embodiments, the flat dose is a dose of between about 50 mg and 500
mg, about 60mg and 490 mg, about 70 mg to 480 mg, about 75 mg to 460 mg, about 80
mg to 450, about 100 mg and about 450 mg, about 150 mg to about 400 mg, about 200
mg to about 300 mg, about 200 mg to about 250 mg; or about 60 mg, 65 mg, 70 mg, 75
mg, 80 mg, 90 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, or 250 mg of an
anti-IL-13 antibody molecule (e.g., hMJ2-7v.2-l 1 HMJ2-7v.2-l 1 or 13.2v2).
In some embodiments, the flat dose is about 75,200 or 225 mg of an anti-IL-13
antibody molecule (e.g., hMJ2-7v.2-l 1 HMJ2-7v.2-l 1 or 13.2v2).
In some embodiments, the flat dose is administered to the subject approximately
every week, approximately every 2 weeks, approximately every 3 weeks, or
approximately every 4 weeks.
For purposes of clarity, the term "IL-13 antagonist" as used herein collectively
refers to a compound such as a protein (e.g., a multi-chain polypeptide, a polypeptide), a
peptide, small molecule, or inhibitory nucleic acid that reduces, inhibits or otherwise
blocks one or more biological activities of IL-13 and an IL-13R. 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 EL-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. Antagonists bind to IL-13 or IL-
13R with high affinity, e.g., with an affinity constant of at least about 107 NT-1, preferably
about 108 M-1, and more preferably, about 109 M-1 to 1010 M-1 or stronger. It is noted that
the term "IL-13 antagonist" includes agents that inhibit or reduce one or more of the
biological activities disclosed herein, but may not bind to IL-13 directly.
The terms "anti-IL13 binding agent" and "IL-13 binding agent" are used
interchangeably herein. These terms as used herein refers to any compound, such as a
protein (e.g., a multi-chain polypeptide, a polypeptide) or a peptide, that includes an
interface that binds to an IL-13 protein, e.g., a mammalian IL-13, particularly, a human
IL-13. The binding agent generally binds with a Kd of less than 5 x 10-7 M. An
exemplary IL-13 binding agent is a protein that includes an antigen binding site, e.g., an
antibody molecule. The anti-IL13 binding agent or IL-13 binding agent can be an IL-13
antagonist that binds to IL13, or can also include IL-13 binding agents that simply bind to
IL-13, but do not elicit an activity, or may in fact agonize an IL-13 activity. For example,
certain IL-13 binding agents, e.g., anti-IL-13 antibody molecules, that bind to and inhibit
one or more IL-13 biological activities, e.g., antibodies 13.2, MJ2-7 and C65, are also
referred to herein as antagonistic IL-13 binding agents. Examples of IL-13 antagonists
that are not IL-13 binding agents as defined herein include, e.g., inhibitors of upstream or
downstream IL-13 signalling (e.g., STAT6 inhibitors).
Additional embodiments of the methods disclosed herein may include one or
more of the following features:
In some embodiments, the IL-13 antagonist can be an antibody molecule that
binds to IL-13 or an IL-13R. The IL-13 can also be a soluble form of the IL-13R (e.g.,
soluble IL-13Ra2 or IL-13Ral), 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). In other embodiments, the antagonist is a cytokine mutein (e.g., an IL-13 mutein
that binds to the corresponding receptor, but does not substantially activate the receptor).
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-4RI, thereby reducing or inhibiting signal
transduction. For example, the IL-13 antagonist can bind to one or more components of a
complex chosen from, e.g., IL-13 and IL-13Ral ("IL-13/IL-13aRl"); IL-13 and IL-4Ra
("IL-13/IL-4Ra"); IL-13, IL-13Ral, and IL-4Ra ("IL-13/IL-13Ral/IL-4Ra"); and IL-13
and IL-13Ra2 ("IL-13/IL13Ra2"). In embodiments, the DL-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-4Ra. In other embodiments, the IL-13 antagonist binds to
IL-13 and interferes with (e.g., inhibits, blocks or otherwise reduces) an interaction, e.g.,
binding, between IL-13 and a subunit of the IL-13 receptor complex, e.g., IL-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 IL-13 and interferes with (e.g.,
inhibits, blocks or otherwise reduces) an interaction, e.g., binding, between IL-13/IL-4Ra
and IL-13Rocl. Typically, the IL-13 antagonist interferes with (e.g., inhibits, blocks or
otherwise reduces) an interaction, e.g., binding, of IL-13/IL-13Ral with IL-4Ra.
Exemplary antibodies inhibit or prevent formation of the ternary complex, IL-13/IL-
13Ral/IL-4Ra.
In one embodiment, the IL-13/IL-13R 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. For example, the antibody molecule can be a full length monoclonal or single
specificity antibody that binds to IL-13 or an IL-13 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 a
human IL-13 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 aintibody 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 IgG 1 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 an inhibitory or neutralizing
antibody molecule. For example, the anti-IL13 antibody molecule can have a functional
activity comparable to IL-13Ra2 (e.g., the anti-IL13 antibody molecule reduces or
inhibits IL-13 interaction with IL-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-1s-1, typically 104 to 107 M-1s-1. In one
embodiment, the anti-IL13 antibody molecule binds to human IL-13 with a kon of
between 5x104 and 8*105 M-1s-1. In yet another embodiment, the anti-IL13 antibody
molecule has dissociation kinetics in the range of 10-2 to 10-6 s-1, typically 10-2 to 10-5 s-1.
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-13Rcd binding to human IL-13 or an epitope defined by IL-13Ra2
binding to human IL-13, or an epitope that overlaps with such epitopes. The anti-IL13
antibody molecule may compete with IL-13Ral 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-13Ral 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-13Ral and/or IL-13Ra2. In embodiments, the anti-IL13 antibody molecule binds
specifically to human IL-13 and competitively inhibits the binding of a second antibody
to said human IL-13, wherein said second antibody is chosen from 13.2, MJ 2-7 and/or
C65 (or any other anti-IL13 antibody disclosed herein) for binding to IL-13, e.g., to
human IL-13. The anti-IL13 antibody molecule may competitively inhibit binding of
13.2, MJ 2-7 and/or C65 to IL-13. The anti-IL13 antibody molecule may specifically
bind at least one amino acid in an epitope defined by 13.2, MJ 2-7 binding to human
IL-13 or an epitope defined by C65 binding to human IL-13. In one embodiment, the
anti-IL13 antibody molecule may bind to an epitope that overlaps with that of 13.2,
MJ 2-7 or C65, e.g., includes at least one, two, three, or four amino acids in common, or
an epitope that, when bound, sterically prevents interaction with 13.2, MJ 2-7 or C65.
For example, the antibody molecule may contact one or more residues from IL-13 chosen
from one or more of residues 81-93 and/or 114-132 of human IL-13 (SEQ ID NO: 194),
or chosen from one or more of: Glutamate at position 68 [49], Asparagine at position 72
[53], Glycine at position 88 [69], Proline at position 91 [72], Histidine at position 92 [73],
Lysine at position 93 [74], and/or Arginine at position 105 [86] of SEQ ID NO: 194
[position in mature sequence; SEQ ID NO: 195]. In other embodiments, the antibody
molecule contacts: one or more amino acid residues from IL-13 chosen from one or more
of residues 116, 117, 118, 122, 123,124,125, 126,127, and/or 128 of SEQ ID NO:24 or
SEQ ID NO: 178. In one embodiment, the antibody molecule binds to IL-13 irrespective
of the polymorphism present at position 130 in SEQ ID NO:24.
In one embodiment, the antibody molecule includes one, two, three, four, five or
all six CDR's from mAM3.2, 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 ID NO:17), mAb 13.2 (SEQ ID NO:196) or C65 (SEQ ID NO:123). In other
embodiments, the light chain immunoglobulin variable domain comprises a light chain
CDR1 that differs by fewer than 3 amino acid substitutions from a corresponding light
chain CDR of monoclonal antibody MJ2-7 (SEQ ID NO: 18), mAb 13.2 (SEQ ID
NO: 197) or C65 (SEQ ID NO:l 18). The amino acid sequence of the heavy chan variable
domain of MJ2-7 has the amino acid sequence shown as SEQ ID NO: 130. The amino
acid sequence of the light chan variable domain of MJ2-7 has the amino acid sequence
shown as SEQ ID NO: 133. The amino acid sequence of the heavy chan variable domain
of monoclonal antibody 13.2 has the amino acid sequence shown as SEQ ID NO:198.
The amino acid sequence of the light chan variable domain of monoclonal antibody 13.2
has the amino acid sequence shown as SEQ ID NO: 199.
In certain embodiments, the heavy chain variable domain of the antibody
molecule includes one or more of:
G-(YF)-(NT)-I-K-D-T-Y-(MI)-H (SEQ ID NO:48), in CDR1,
(WR)-I-D-P-(GA)-N-D-N-I-K-Y-(SD)-(PQ)-K-F-Q-G (SEQ ID NO:49), in
CDR2, and/or
SEENWYDFFDY (SEQ ID NO:17), in CDR3; or
GFNIKDTYIH (SEQ ID NO: 15), in CDR1,
RIDPANDNIKYDPKFQG (SEQ ID NO: 16), in CDR2, and/or
SEENWYDFFDY (SEQ ID NO: 17), in CDR3
In other embodiments, the light chain variable domain of the antibody molecule
includes one or more of:
(RK)-S-S-Q-S-(LI)-(KV)-H-S-(ND)-G-N-(TN)-Y-L-(EDNQYAS) (SEQ ID
NO:25), in CDR1,
K-(LVI)-S-(NY)-(RW)-(FD)-S (SEQ ID NO:27), in CDR2, and/or
Q-(GSA)-(ST)-(HEQ)-I-P (SEQ ID NO:28), in CDR3; or
RSSQSIVHSNGNTYLE (SEQ ID NO: 18), in CDR1
KVSNRFS (SEQ ID NO: 19), in CDR2, and
FQGSHIPYT (SEQ ID NO:20), in CDR3.
In other embodiments, the antibody molecule includes one or more CDRs
including an amino acid sequence selected from the group consisting of the amino acid
sequence of SEQ ID NO: 197, SEQ ID NO:200, SEQ ID NO:201, SEQ ID NO:202, SEQ
IDNO:203, and SEQ ID NO: 196.
In yet another embodiment, the antibody molecule includes at least one, two, or
three Chothia hypeirvariable loops from a heavy chain variable region of an antibody
chosen from, e.g., rnAbl3.2, 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., mAb13.2, 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 mAb13.2, MJ2-7, C65, or other antibodies disclosed herein, e.g.,
the same canonical structures as at least loop 1 and/or loop 2 of the heavy and/or light
chain variable domains of mAb13.2, MJ2-7, C65, or other antibodies disclosed herein.
See, e.g., Chothia et al. (1992)7. Mol. Biol. 227:799-817; Tomlinson etal. (1992) J. 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 He at position 2; Ser or Pro at position 25; He or Leu at position 29; Gly at position
3 Id; 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 VHI
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), h!3.2vl (SEQ ID
NO:205), hl3.2v2 (SEQ IDNO: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 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). 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: 11 (5 or SEQ ID NO: 117.
In other embodiments, the light chain immunoglobulin variable domain of the
antibody molecule includes an amino acid sequence encoded by a nucleotide sequence
that hybridizes under high stringency conditions to the complement of the nucleotide
sequence encoding a light chain variable domain of V2.11 (SEQ ID NO:36) or 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 ID 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, He, and to 49, Gly;
(iv) at positions corresponding to 48, He, 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, He, to 49, Gly, to 72, Ala, to 79, Ala.
In one embodiment, the anti-IL13 antibody molecule includes at least one light
chain that comprises the amino acid sequence of SEQ ID NO: 177 (or an amino acid
sequence at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or higher identical identical to
SEQ ID NO: 177) and at least one heavy chain that comprises the amino acid sequence of
SEQ ID NO: 176 (or an amino acid sequence at least 80%, 85%, 90%, 95%, 97%, 98%,
99% or higher identical identical to SEQ ID NO: 176).
In one embodiment, the anti-IL13 antibody molecule includes two
immunoglobulin chains: a light chain that includes SEQ ID NO:199, 213, 214, 212, or
215 and a heavy chain that includes SEQ ID NO: 198, 208,209,210, or 211 (or an amino
acid sequence at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or higher identical identical
to SEQ ID NO:199, 213, 214,212, or 215, or SEQ ID NO:198, 208,209, 210, or 211).
The antibody molecule may further include in the heavy chain the amino acid sequence
of SEQ ID NO:193 and in the light chain the amino acid sequence of SEQ ID NO:216 (or
an amino acid sequence at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or higher
identical identical to SEQ ID NO: 193 or SEQ ID NO:216).
In another embodiment, the IL-13 binding agent, e.g., anti-IL-13 antibody
molecule, interferes with the interaction of IL-13 with the receptor IL-13RI1. In one
embodiment, the IL-13 binding agent can interfere with the interaction of Phe107 of
IL-13 (SEQ ID NO:124; FIG. 13A) with a hydrophobic pocket of IL-13Ral formed by
the side chains of residues Leu319, Cys257, Arg256, and Cys320 (SEQ ID NO: 125; FIG.
13B), e.g., by direct binding to these residues or steric hindrance. In another
embodiment, the IL-13 binding agent can interfere with 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, Glul2, Leul3, Ilel4,
Glul5, Lys104, Lysl05, Leu106, Phe107, and Arg108 of IL-13 (SEQ ID NO: 124), e.g.,
by direct binding to these residues or steric hindrance.
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 mis 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. 1B is a list of exemplary peptides from cynomolgus monkey IL-13 (SEQ ID
NO: 179-188, respectively) that can be used for generating anti-IL13 antibodies.
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-13RI2-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 v.211 (o) (referred to interchangeably herein as "hMJ2-
7v.2-11 or "MJ2-7v.2-11"). 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
v.211 (o) or sIL-13RI2-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-13RI2-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-13RI2-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 v.2-11 antibody.
FIG 9 is a graph depicting the neutralization of NHPIL-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 IL-13 receptor by MJ2-7 antibody, as measured by ELISA. Binding is
depicted as absorbance at 450 nm (y-axis). Concentration of MJ2-7 antibody is depicted
on the x-axis. FIG 11A depicts binding to IL-13Ral. FIG 1 IB depicts binding to
IL-13Ra2.
FIG 12 is an alignment of DPK18 germline amino acid sequence (SEQ ID
NO:126) and humanized MJ2-7 Version 3 VL (SEQ ID NO:190).
FIG. 13A is an amino acid sequence (SEQ ID NO: 124) of mature, processed
human IL-13.
FIG. 13B shows an amino acid sequence (SEQ ID NO: 125) of human IL-13Ral.
FIGs. 14A-14D show 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 (light
circles (o); MJ2-7v.2-l 1-treated samples (light triangles (lght triangles)) and mAb
13.2v2-treated samples (dark triangles( ?)). (Humanized versions of MJ2-7 (MJ2-7v.2-
11) and mAb 13.2v2 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- (gray circles) or MJ2-7-(gray triangles) antibodies compared to a control
(dark circles). (Humanized versions of MJ2-7 (MJ2-7v.2-l 1) and mAb 13.2 v2 were
used in this study).
FIGs. 17A-17B depict the changes in Ascaris-specific IgE-titers in control and
antibody-treated samples 8-weeks post-challenge. FIG 17A depicts representative
examples showing no change in y4scam-specific IgE titer in an individual monkey treated
with irrelevant Ig (IVIG; animal 20-45; top panel), and decreased titer of ^.scam-specific
IgE in an individual monkey treated with humanized MJ2-7v.2-l 1 (animal 120-434;
bottom panel). FIG. 17B depicts a decrease in ^cam-specific IgE-titers in mAb 13.2 or
hMJ2-7-l 1 (dark circles) relative to irrelevant Ig-treated cynomolgus monkeys (IVIG
(gray circles)) 8-weeks post-Ascaris challenge.
FIGs. 18A-18B show the changes in A scam-specific basophil histamine release
in control and antibody-treated samples 24-hours and 8-weeks post-challenge. FIG. 18A
is a graph depicting the following samples in representative individual monkeys treated
with saline (left) or humanized mAb13.2v.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). FIG. 18B
depicts a bar graph showing the changes in normalized histamine levels pre- and 8-week
post-Ascaris challenge in control (solid black bar), humanized mAb 13.2- (white bar) and
humanized MJ2-7v.2-l 1- (hatched bar) treated cynomolgus monkeys.
FIG. 19 depicts the correlation between ^scam-specifie histamine release and
v4scam-specific IgE levels in control (light circles) and anti-IL13- or dexamethasone-
treated samples (dark 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 (hMJ2-7v.2-11). 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-l 1) 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 humani2:ed MJ2-7 (hMJ2-7v.2-l 1) 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 R110Q 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-I, 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-l 1)
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 µL) 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 R110Q IL-13. FIG. 25A depicts the results when the antibody is
administered intravenously on day 0 and intraperitoneally on days 1, 2, and 3; or
intranasally on days 0, 1,2, and 3 (shown in FIG. 25B). Total human IgG levels in BAL
and serum were assayed by ELISA.
FIGs. 26A-26C show the effect of humanized MJ2-7v.2-11 antibody after
intranasal administration of human recombinant Rl 10Q IL-13-induced altered lung
function. (A) FIG. 26A shows the changes in lung resistance (RI; cm HzO/ml/sec)
expressed as change from baseline. FIG. 26B shows data expressed as methacholine
dose required to elicit lung resistance (RI) corresponding to a change of 2.5 ml
HbO/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 R110QIL-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 R110Q 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 R110Q IL-13 and intranasal administration of 500,100, and 20 µg of
humanized MJ2-7v.2-l 1 and humanized 13.2v.2, saline, or 500 µg of IVIG. Eosinophil
and neutrophil percentages were determined by differential cell counts. Median values
for each group are indicated, p-values were determined by two-tailed test and are
indicated for each antibody-treated group as compared to IVIG.
FIGs. 29A-29C are dot plots showing changes in cytokine levels, MCP-1, TNF-I,
and IL-6, respectively, following intranasal administration of human recombinant
R110Q IL-13 and intranasal administration of 500 Tg 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 R110Q 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 R110Q IL-13 and intranasal administration of 500, 100, or 20 u.g
doses of humanized MJ2-7v.2-l 1 antibody. Humanized MJ2-7v.2-l 1 antibody BAL
levels were measured by ELISA. Human recombinant R110Q IL-13 BAL levels were
determined by cytometric bead assay. % eosinophil was determined by differential cell
counting. Associations are shown between levels of: (FIG. 30A) % eosinophilic
inflammation and human recombinant R110Q IL-13, including data from saline control
animals, mice treated with human recombinant R110Q IL-13 alone, and mice treated with
human recombinant R110Q IL-13 and 500, 100, and 20 Tg of humanized MJ2-7v.2-l 1
antibody or 500 u.g IVIG; and (FIG. 30B) humanized MJ2-7v.2-l 1 and IL-6, including
data from mice treated with 500,100, and 20 u.g of humanized MJ2-7v.2-l 1. r2 and p-
values were determined by linear regression analysis.
FIGs. 31A-31B are line graphs showing concentrations of [125I]-labeled
humanized 13.2v.2 anti-IL-13 antibody and [125I]-labeled humanized MJ2-7v.2-l 1
antibody in various mouse and rat tissue, respectively. Following IV administration of
anti-IL-13 antibodies, tissue samples were collected at 1,24,168, and 336 hours (FIG.
31A) or 1, 48, 168, 336, and 840 hours (FIG. 31B).
FIGs. 32A-32B are line graphs showing observed and predicted IL-13 and anti-
IL-13 antibody levels overtime. In FIG. 32A, 1 mg/kg of humanized MJ2-7v.2-l 1
antibody was administered to naive cynomolgus monkeys. Total IL-13 and humanized
MJ2-7v.2-l 1 serum levels were quantified over a period of 0 - 45 days using a specific
ELISA. Predicted IL-13 and humanized MJ2-7v.2-l 1 antibody levels based on model
shown in FIG. 40 are shown for comparison. In FIG. 32B, humanized 13.2v.2 and
humanized MJ2-7v.2-l 1 antibodies were administered to cynomolgus monkeys at day 0
and Ascaris challenge was performed at day 1. Total IL-13 serum levels were quantified
over a period of up to 120 days using a specific ELISA.
FIG. 33 is a schematic representation of PK-PD model of humanized MJ2-7v.2-
11. Ab is hMJ2-7v.2-l 1. Complex is hMJ2-7v.2-l l/IL-13 complex. CLd,Ab and CLAb are
distribution clearance and serum clearance of hMJ2-7v.2-ll, respectively. CLcomplex and
CLIL-13 are serum; clearance of the complex and IL-13, respectively. Ksyn is a zero-order
IL-13 synthesis rate constant, Konis a second-order association rate constant, and Koff is a
first-order dissociation rate constant. V and V2 are volumes of distribution of hMJ2-7v.2-
11 in the serum (central) and the second compartment, respectively.
FIGs. 34A-34C show mean hMJ2-7v.2-l 1 and total IL-13 concentration time-
profiles in cynomolgus monkeys. A single 1 mg/kg IV or 2 mg/kg SC dosage of hMJ2-
7v.2-l 1 was administered to naive cynomolgus monkey and a single 10 mg/kg IV dosage
of hMJ2-7v.2-11 was given to Ascaris-challenged cynomolgus monkeys. The challenge
was performed that with 0.75 µg of Ascaris suum antigen 24 hours post administration of
the hMJ2-7v.2-11. hMJ2-7v.2-11 (A, B) and total IL-13 (C) concentrations were
determined using quantitative ELISAs. Data point show individual animal values (A) or
mean values (B and C). For the mean values, N=3 for 1 mg/kg-IV group, N=2 for 2
mg/kg- SC group, and N=8 for 10 mg/kg-IV group, with Monkey #5 in the SC group
being excluded from calculations of the mean values. Error bars indicated standard
deviation from the mean values. M=monkey.
FIGs. 35A-35D are a series of goodness-of-fit plots showing hMJ2-7v.2-11
(closed circle) and total IL-13 (open circle) concentrations following a single dosage of
hMJ2-7v.2-11 fitted using the integrated PK-PD model depicted in FIG. 33. Individual
observed versus individual predicted concentrations (A) and individual weighted
residuals versus individual predicted concentrations (B) following a single dosage of
hMJ2-7v.2-11 are shown for five naive (N=3,1 mg/kg IV and N=2,2 mg/kg SC) and
eight /4.scara-challenged cynomolgus monkeys (10 mg/kg, IV). One animal in the SC
group was excluded from these analyses due to a sharp decline in hMJ2-7v.2-11 and total
IL-13 levels in the terminal phase, compared to other naive monkeys in the study.
Representative individual fits after IV administration of hMJ2-7v.2-11 are shown for a
naive (C) and an ./tscara-challenged monkey (D), with predicted hMJ2-7v.2-11 and total
IL-13 levels shown by solid line and dotted lines, respectively.
FIGs. 36A and 36B are graphs depicting simulated free IL-13 and total IL-13
concentration-time profiles after a single IV administration of hMJ2-7v.2-11 to
cynomolgus monkeys. For naive monkeys (FIG. 36A), a 1 mg/kg dosage was assumed as
in Study 1, while for Ascaris-challenged monkeys (FIG. 36B), a 10 mg/kg dosage and
Ascaris challenge 24-hour post- hMJ2-7v.2-11 administration (Day 1) was assumed as in
Study 2. Free IL-13 is shown by solid lines, while total IL-13 is shown by dotted lines.
FIGs. 37A and 37 B are graphs showing simulated free IL-13 concentration-time
profiles after different dosing regimens of hMJ2-7v.2-11 to cynomolgus monkeys. A
single 1,5, 10, 20, or 50 mg/kg IV bolus dosage of hMJ2-7v.2-11 (as indicated) was
assumed for both naive (FIG. 37A) and Ascaris -challenged (FIG. 37B) monkeys. Ascaris
challenge was assumed at pre-dose (Day 0) to mimic the "established airway
inflammation" situation.
FIG. 38 is a line graph plotted from PK data showing concentration-time profiles
of humanized MJ2-7v.2-11 in normal versus v4.scara-challenged cynomolgus monkeys.
FIG. 39 is a line graph plotted from PK data showing concentration-time profiles
of humanized 13.2v.2 in normal versus Ascaris-challenged cynomolgus monkeys.
FIG. 40 is a stoichiometric PK-PD model of IL-13 and anti-IL-13 antibody
disposition in cynomolgus monkeys, wherein; Ab is anti-IL-13 antibody; Complex is an
Ab and IL-13 complex; Comp is compartment; CLdAb and CLAb are distribution
clearance and serum clearance of Ab, respectively; CLcomplex is serum clearance of the
complex; Ksyn is the zero-order IL-13 synthesis rate constant; Kdeg is the first-order IL-
13 degradation constant; Kon is the third-order association rate constant; Koff is the first-
order dissociation rate constant; VAb and V2Ab are apparent volumes of distribution in the
serum and the second compartment, respectively; and the model is based on the
assumptions that Kon is 3rd order; anti-IL-13 and IL-13 have a 1:2 molar binding ratio;
and Vanti-IL-13VComplex=VIL-13:=V.
FIG. 41 is a line graph showing predicted serum concentrations of free and
humanized MJ2-7v.2-11-bound IL-13 following 1 mg/kg IV administration of humanized
MJ2-7v.2-11 to naive cynomolgus monkeys. Data were predicted using the
concentration-time profiles from studies described in Table 8 and depicted in FIG. 34,
and the model presented in FIG. 40, and is represented for a period of up to 50 days.
FIG. 42 is a line graph showing predicted serum concentrations of free and
humanized MJ2-7V.2-11-bound IL-13 following 1 mg/kg IV administration of humanized
MJ2-7v.2-11 to Ascaris-challenged cynomolgus monkeys. Data were predicted using the
concentration-time profile from studies described in Table 8 and depicted in FIG. 34, and
the model presented in FIG. 40, and is represented for a period of up to 150 days.
FIG. 43 is a series of line graphs showing allometric scaling of humanized MJ2-
7v.2-11 for three PK parameters, CL, Vdss and t1/2- Solid line represents the fitted curve
based on a linear regression using data from mice, rats and monkeys. The dotted lines
represent the 95% confidence intervals.
FIG. 44 is a line graph showing the percent change in FEV1 (% Change in FEV1)
at various time points after allergen challenge (Time after allergen challenge (h)) for
human subjects that will be treated with anti-IL-13 antibody treated (open circles) or
placebo treated (closed circles). The results shown are for allergen challenge on the
screening day two weeks prior to the initial administration of anti-IL-13 antibody or
placebo, (h): hours; EAR: early asthmatic response; LAR: late asthmatic response.
FIG. 45 is a line graph showing the percent change in FEV1 (% Change in FEV1)
at various time points after allergen challenge (Time after allergen challenge (h)) for anti-
IL-13 antibody treated (open circles) or placebo treated (closed circles) human subjects.
The results shown are for allergen challenge on day 14 after initial administration of anti-
IL-13 antibody or placebo, (h): hours; EAR: early asthmatic response; LAR: late
asthmatic response.
FIG. 46 is a line graph showing the percent change in FEV1 (% Change in FEV1)
at various time points after allergen challenge (Time after allergen challenge (h)) for anti-
IL-13 antibody treated (open circles) or placebo treated (closed circles) human subjects.
The results shown are for allergen challenge on day 35 after initial administration of anti-
IL-13 antibody or placebo, (h): hours; EAR: early asthmatic response; LAR: late
asthmatic response.
FIG. 47 is a graph showing serum concentration (ng/mL) of antibody at Day 14
and Day 35.
FIG. 48 is a table showing the maximum percent drop (max % drop) and area
under the curve percent drop (AUC % drop) during the EAR (early phase) and LAR (late
phase) on Day 14 and Day 35 after initial antibody (or placebo) administration. P values
(P-val) are also indicated.
FIG. 49 is a line graph showing the 13.2v2 antibody serum concentration (ng/ml)
in human subjects over time (days) after administration. The thin lines depict the PK
profiles for 13.2v2 antibody administered in a single ascending dose of 4 mg/kg. The
thicker lines depict the PK profiles for 13.2v2 antibody administered as two doses of 2
mg/kg. Administration of the two doses was separated by a week.
FIG. 50 is a graph showing individual AUC normalized by mg/kg dose against
respective body weight in 81 subjects from both study A and study B.
FIG. 51 is a graph showing individual AUC normalized by total dose (body
weight * mg/kg dose) against respective body weight in 81 subjects from both study A
and study B.
FIG. 52 is a graph showing 13.2v2 AUC exposure normalized by actual dose
(body weight*mg/kg dose).
DETAILED DESCRWTION
Methods and compositions for treating and/or monitoring treatment of IL-13-
associated disorders or conditions are disclosed. In one embodiment, Applicants have
discovered that administration of an IL-13 antagonist, e.g., an IL-13 antibody molecule,
reduces at least one symptom of an allergen-induced early and/or a late asthmatic
response in a subject, e.g., a human subject, relative to an untreated subject. The
reduction in one or more asthmatic symptoms is detected within minutes following
exposure of the subject to an insult, e.g., an allergen, and during an early asthmatic
response (e.g., up to about 3 hours after exposure to the insult). The reduction in
symptoms is maintained during a late asthmatic response (e.g., for a period of about 3 to
24 hours after insult exposure). In other embodiments, methods of evaluating an anti-
IL13 antibody molecule and/or treatment modalities associated with said antibody
molecule are disclosed. The evaluation methods include detecting at least one
pharmacokinetic/pharmacodynamic (PK/PD) parameter of the anti-IL13 antibody
molecule in the subject. Thus, uses of IL-13 binding agents or antagonists for reducing
or inhibiting, andfor preventing or delaying the onset of, one or more symptoms
associated with an early and/or a late phase of an IL-13-associated disorder or condition
in a subject are disclosed. In other embodiments, methods for evaluating the kinetics
and/or efficacy of an IL-13 binding agent or antagonist in treating or preventing the IL-
13-associated disorder or condition in a 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 IL-13, see SEQ ID
NO:124 (FIG. 1). An exemplary sequence is recited as follows:
MALLLTTVIALTCLGGFASPGPVPPSTALRELIEELVNITQNQKAPLCNGSMVWSI
NLTAGMYCAALESLINVSGCSAIEKTQRMLSGFCPHKVSAGQFSSLHVRDTKIEV
AQFVKDLLLHLKKLFREGRFN (SEQ ID NO: 178)
There is about 94% amino acid sequence identity between the human and cyno
monkey IL-13 sequences, due to 8 amino acid sequences. One of these differences,
R130Q, represents a common human polymorphism typically expressed in asthmatic
subjects (Heinzmann et al. (2000) Human Mol Genet. 9:549-559).
Exemplary sequences of IL-13 receptor proteins and soluble forms thereof (e.g.,
IL-13Ra1 and IL-13Ra2 or fusions thereof) are described, e.g., in Donaldson et al.
(1998) JImmunol 161:2317-24; U.S. 6,214,559; U.S. 6,248,714; and U.S. 6,268,480.
The phrase "a biological activity of IL-13/IL-13R polypeptide refers to one or
more of the biological activities of the corresponding mature IL-13 polypeptide,
including, but not limited to, (1) interacting with, e.g., binding to, an IL-13R polypeptide
(e.g., a human IL-13R polypeptide); (2) associating with signal transduction molecules,
e.g., ? 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 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 refers to an amount of an IL-13/IL-13R 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 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.
The term "early asthmatic response" or "EAR" refers to the initial period of
response after a subject's exposure to an allergen. For example, the response occurring in
the first 3 hours (e.g., about 2.5, about 2.75, about 2.9, about 3, about 3.25, about 3.5
hours) following exposure to an allergen is considered to be the EAR. For example, the
maximum airway construction can occur within about 15-30 minutes after exposure.
Events that occur during the EAR can include the release of mediators such as
leukotrienes (e.g., LTA4, LTB4, LTC4, LTD4, LTE4, and/or LTF4) and/or histamine from
airway mast cells, e.g., leading to bronchoconstriction and/or airway edema, and/or
increase in the levels of leukotrienes and/or histamine (e.g., an increase relative to the
level of leukotrienes and/or histamine in the subject prior to exposure to allergen).
Treatments for EAR include administration of an anti-IL-13 antibody (e.g., an antibody
described herein), an anti-histamine (e.g., loratidine (e.g., CLARITIN®), cetirizine (e.g.,
ZYRTEC®), diphenhydramine), an anti-leukotriene (e.g., zafirlukast, montelukast (e.g.,
SINGULAIR®)), an IL-4 variant (e.g., pintrakinra), or a combination of two or more of
these agents.
The term "late asthmatic response" or "LAR" refers to the period of response
after a subject's exposure to an allergen that occurs after the EAR, or the response that
begins about 3 hours after a subject's exposure to an allergen. As a further example, the
LAR commences after about 3-5 hours, is maximal at about 6-12 hours, and can persist
for up to about 24 hours. In contrast to the EAR, the LAR involves inflammatory cells
and/or an increase in mucus. For example, the LAR can be associated with increases in
airway reactivity and/or with an influx and activation of inflammatory cells, such as
lymphocytes, eosinophils, and macrophages, e.g., in the airways and/or bronchial mucosa
(e.g., an increase relative to the level of inflammatory cells, such as lymphocytes,
eosinophils, and macrophages, e.g., in the airways and/or bronchial mucosa in the subject
prior to exposure to allergen). Treatments for LAR include administration of an anti-IL-
13 antibody (e.g., am antibody described herein), a steroid (e.g., inhaled steroid), a beta-
agonist (e.g., albuterol (e.g., VENTOLIN®; PROVENTIL®, SALBUTAMOL®),
metaproteronol (e.g., ALUPENT®, METAPREL®), terbutaline (e.g., BRETHINE®,
BRICANYL®, or BRETHAIRE®) or a combination of two or more of these agents.
A "flat" dose of a therapeutic agent (e.g., anti-IL-13 antibody) refers to a dose that
is administered to a subject without regard for the weight or body surface area of the
subject. The flat dose is not provided as a mg/kg dose, but rather as an absolute amount
of the therapeutic agent.
Antibody Molecules
Examples of IL-13 antagonists and/or binding agents include antibody molecules.
As used herein, the term "antibody molecule" refers to a protein comprising at least one
immunoglobulin variable domain sequence. The term antibody molecule includes, for
example, full-length, mature antibodies and antigen-binding fragments of an antibody.
For example, an antibody molecule can include a heavy (H) chain variable domain
sequence (abbreviated herein as VH), and a light (L) chain variable domain sequence
(abbreviated herein as VL). In another example, an antibody molecule includes one or
two heavy (H) chain variable domain sequences and/or one of two light (L) chain
variable domain sequence. Examples of antigen-binding fragments include: (i) a Fab
fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a
F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide
bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains;
(iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody,
(v) a VH or VHH domain; (vi) a dAb fragment, which consists of a VH domain; (vii) a
camelid or camelized variable domain; and (viii) a single chain Fv (scFv).
The VH and VL regions can be further subdivided into regions of
hypervariability, termed "complementarity determining regions" (CDR), interspersed
with regions that are more conserved, termed "framework regions" (FR). The extent of
the framework region and CDRs has been precisely defined by a number of methods (see,
Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition,
U.S. Department of Health and Human Services, NIH Publication No. 91-3242; Chothia,
C. et al. (1987) J. Mol. Biol. 196:901-917; and the AbM definition used by Oxford
Molecular's AbM antibody modelling software. See, generally, e.g., Protein Sequence
and Structure Analysis 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 hypervariable 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. HAMA can
be problematic in a number of circumstances, e.g., if the antibody molecule is
administered repeatedly, e.g., in treatment of a chronic or recurrent disease condition. A
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. etal. (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 ID 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 IL-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 tine 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 Thotakuraef
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 ID NO: 17),
RSSQSIVHSNGNTYLE (SEQ ID NO: 18),
KVSNRFS (SEQ ID N0:19), and
FQGSHIPYT (SEQ ID NO:20), or a CDR having an amino acid sequence that
differs by no more than 4,3,2.5,2, 1.5,1, or 0.5 alterations (e.g., substitutions, insertions
or deletions) for every 10 amino acids (e.g., the number of differences being proportional
to the CDR length) relative to a sequence listed above, e.g., at least one alteration but not
more than two, three, or four per CDR.
For example, the anti-IL-13 antibody molecule can include, in the light chain
variable domain sequence, at least one, two, or three of the following sequences within a
CDR region:
RSSQSIVHSNGNTYLE (SEQ ID NO: 18),
KVSNRFS (SEQ ID NO: 19), and
FQGSHIPYT (SEQ ID NO:20), or an amino acid sequence that differs by no
more than 4,3, 2.5, 2,1.5, 1, or 0.5 substitutions, insertions or deletions for every 10
amino acids relative to a sequence listed above.
The anti-IL-13 antibody molecule can include, in the heavy chain variable domain
sequence, at least one, two, or three of the following sequences within a CDR region:
GFNIKDTYIH (SEQ ID NO: 15),
RIDPANDNIKYDPKFQG (SEQ ID NO: 16), and
SEENWYDFFDY (SEQ ID NO: 17), or an amino acid sequence that differs by no
more than 4, 3, 2.5, 2,1.5, 1, or 0.5 substitutions, insertions or deletions for every 10
amino acids relative to a sequence listed above. The heavy chain CDR3 region can be
less than 13 or less than 12 amino acids in length, e.g., 11 amino acids in length (either
using Chothia or Kabat definitions).
In another example, the anti-IL-13 antibody molecule can include, in the light
chain variable domain sequence, at least one, two, or three of the following sequences
within a CDR region (amino acids in parentheses represent alternatives for a particular
position):
(i) (RK)-S-S-Q-S-(LI)-(KV)-H-S-(ND)-G-N-(TN)-Y-L-(EDNQYAS) (SEQ ID
NO:25) or (RK)-S-S-Q-S-(LI)-(KV)-H-S-(ND)-G-N-(TN)-Y-L-E (SEQ ID NO:26), or
(RK)-S-S-Q-S-(LI)-(KV)-H-S-N-G-N-T-Y-L-(EDNQYAS) (SEQ ID NO:21),
(ii) K-(LVI)-S-(NY)-(RW)-(FD)-S (SEQ ID NO:27), or K-(LV)-S-(NY)-R-F-S
(SEQIDNO: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)-aL)-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 IDNO: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),
IIWGDGSTDYNSAL (SEQ ID NO: 122), and
DKTFYYDGFYRGRMDY (SEQ ID NO: 123), or an amino acid sequence that
differs by no more than 4, 3,2.5,2,1.5,1, or 0.5 substitutions, insertions or deletions for
every 10 amino acids relative to a sequence listed above.
In embodiments, the IL-13 antibody molecule can include one of the following
sequences:

or a sequence that has fewer than eight, seven, six, five, four, three, or two alterations
(e.g., substitutions, insertions or deletions, e.g., conservative substitutions or a
substitution for an amino acid residue at a corresponding position in MJ 2-7). Exemplary
substitutions are at one of the following Kabat positions: 2,4, 6, 35, 36, 38,44,47,49,
62, 64-69, 85, 87, 98, 99, 101, and 102. The substitutions can, for example, substitute an
amino acid at a corresponding position from MJ 2-7 into a human framework region.
The IL-13 antibody molecule may also include one of the following sequences:

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
(SEQIDNO:47).
In other embodiments, the IL-13 antibody molecule can include one of the
following sequences:

or a sequence thai has fewer than eight, seven, six, five, four, three, or two alterations
(e.g., substitutions, insertions or deletions, e.g., conservative substitutions or a
substitution for an amino acid residue at a corresponding position in MJ 2-7). Exemplary
substitutions are at one or more of the following Kabat positions: 2, 4, 6, 25, 36, 37, 39,
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: He at 48,
Gly at 49, Lys at (57, Ala at 68, Ala at 72, and Ala at 79; preferably, e.g., He 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, IIe, and to 49, Gly; (iv) at positions
corresponding to 48, He, 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, He, to 49,
Gly, to 72, Ala, to 79, Ala.
The IL-13 antibody molecule may also include one of the following sequences:

or a sequence thai: 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 IDNO: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., mAb13.2, 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., mAb13.2, MJ2-7, C65, and/or
modified forms thereof (e.g., humanized or chimeric variansts thereof) by at least one
amino acid, e.g., at least 5, 8, 10, 12, 15, or 18 amino acids. For example, the proteins
include one, two, three, four, five, or six such non-human CDRs and includes at least one
amino acid difference in at least three of HC FR1, HC FR2, HC FR3, LC FR1, LC FR2,
and LC FR3.
In one embodiment, the heavy or light chain variable domain sequence of the anti-
IL-13 antibody molecule includes an amino acid sequence, which is at least 80%, 85%,
90%, 95%, 97%, 98%, 99% or higher identical to a variable domain sequence of an
antibody described herein, e.g., mAb13.2, MJ2-7, C65, and/or modified forms thereof
(e.g., humanized or chimeric variansts thereof); or which differs at at least 1 or 5
residues, but less than 40, 30, 20, or 10 residues, from a variable domain sequence of an
antibody described herein, e.g., mAb13.2, MJ2-7, C65, and/or modified forms thereof
(e.g., humanized or chimeric variansts thereof). In one embodiment, the heavy or light
chain variable domain sequence of the protein includes an amino acid sequence encoded
by a nucleic acid sequence described herein or a nucleic acid that hybridizes to a nucleic
acid sequence described herein or its complement, e.g., under low stringency, medium
stringency, high stringency, or very high stringency conditions.
In one embodiment, one or both of the variable domain sequences include amino
acid positions in the framework region that are variously derived from both a non-human
antibody (e.g., a murine antibody such as mAb13.2) 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 eta!. (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).
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) JPharmacol Exp Ther: 313(1):8-15; and anti-IL13
antibodies as disclosed in WO 07/080174 filed in the name of Glaxo, and as disclosed in
WO 07/045477 in the name of Novartis.
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 Binding Agents
Also provided are other binding agents, other than antibody molecules, that bind
to IL-13 polypeptide or nucleic acid, or an IL-13R 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 (e.g., one or more
biological activities of IL-13 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 anodier 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 its receptors, are identified, e.g., by retention on a solid support or by other
physical association. For example, to identify particular binding agents that bind to the
same epitope or an overlapping epitope as MJ2-7, C65 or mAb 13.2 on IL-13, binding
agents can be eluted by adding MJ2-7, C65 or mAb 13.2 (or related antibody), or binding
agents can be evaluated in competition experiments with MJ2-7, C65 or mAb 13.2 (or
related antibody). It is also possible to deplete the library of agents that bind to other
epitopes by contacting the library to a complex that contains IL-13 and MJ2-7, C65 or
mAb 13.2 (or related antibody). The depleted library can then be contacted to IL-13 to
obtain a binding agent that binds to IL-13 but not to IL-13 when it is bound by MJ 2-7,
C65 or mAb 13.2. 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)MethodsEnzymol. 267:129-49;
and Barbas et al. (1991) PNAS 88:7978-7982. Yeast surface display is described, e.g., in
Boder and Wittrup (1997) Nat. Biotechnol. 15:553-557. Another form of display is
ribosome display. See, e.g., Mattheakis et al. (1994) Proc. Natl. Acad. Sci. USA 91:9022
and Hanes et al. (2000) Nat Biotechnol. 18:1287-92; Hanes et al. (2000) Methods
Enzymol. 328:404-30. and Schaffitzel et al (1999) J Immunol Methods. 231(1-2):119-35.
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, EMBO J. 13:5303-5309). This
domain includes 61 residues and can be used to present two hypervariable loops, e.g., one
or more hypervariable loops of a variable domain described herein or a variant described
herein. In another approach, the binding agent includes a scaffold domain that is a V-like
domain (Coia et al. WO 99/45110). V-like domains refer to a domain that has similar
structural features to the variable heavy (VH) or variable light (VL) domains of
antibodies. Another scaffold domain is derived from tendamistatin, a 74 residue, six-
strand beta sheet sandwich held together by two disulfide bonds (McConnell and Hoess,
1995, J. Mol. Biol. 250:460). This parent protein includes three loops. The loops can be
modified (e.g., using CDRs or hypervariable loops described herein) or varied, e.g., to
select domains that bind to IL-13 or IL-4, or its receptors. WO 00/60070 describes a |3-
sandwich structure derived from the naturally occurring extracellular domain of CTLA-4
that can be used as a scaffold domain.
Still another scaffold domain for an IL-13/13R binding agent is a domain based
on the fibronectin type III domain or related fibronectin-like proteins. The overall fold of
the fibronectin type III (Fn3) domain is closely related to that of the smallest functional
antibody fragment, the variable domain of the antibody heavy chain. Fn3 is a (3-sandwich
similar to that of the antibody VH domain, except that Fn3 has seven 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, 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, ~[CH2O]-peptide bonds, and -
[CH2S]-- thiomethylene peptide bonds (see e.g., U.S. Pat. No. 6,172,043).
In another embodiment, the IL-13 antagonist is derived from a lipocalin, e.g., a
human lipocalin scaffold.
Variant IL-13 Binding Molecules
In yet another embodiment, the IL-13 binding agent, antagonist is a variant
molecule or a small molecule. An example of a variant molecule typically includes a
binding domain polypeptide that is fused or otherwise connected to a hinge or hinge-
acting region polypeptide, which in turn is fused or otherwise connected to a region
comprising one or more native or engineered constant regions from a heavy chain, other
than CH1, for example, the CH2 and CH3 regions of IgG and IgA, or the CH3 and CH4
regions of IgE (see e.g., U.S. 05/0136049 by Ledbetter, J. et al. for a more complete
description). The binding domain-fusion protein can further include a region that
includes a native or engineered heavy chain CH2 constant region polypeptide (or CH3 in
the case of a construct derived in whole or in part from IgE) that is fused or otherwise
connected to the hinge region polypeptide and a native or engineered heavy chain CH3
constant region polypeptide (or CH4 in the case of a construct derived in whole or in part
from IgE) that is; fused or otherwise connected to the CH2 constant region polypeptide (or
CH3 in the case of a construct derived in whole or in part from IgE). Typically, such
binding domain-fusion proteins are capable of at least one activity selected from the
group consisting of fusion protein- dependent cell-mediated cytotoxicity, complement
fixation, and/or binding to a target, for example, a IL-13.
Another example of an IL-13 binding variant is a soluble form of an IL-13
receptor or a fusion thereof. For example, a modified soluble receptor form 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 associated, 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, 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 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 receptor or a ligand binding
fusion can be fused to a heavy chain constant region of the various isotypes, including:
IgGl, IgG2, IgG3, IgG4, IgM, IgAl, IgA2, IgD, and IgE).
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 C1 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 fo 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 CIq 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 C1q.
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. 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 a transcription regulatory
region, and blocks; or reduces mRNA expression of an IL-13 or IL-13R.
In embodiments, nucleic acid antagonists are used to decrease expression of an
endogenous gene encoding an IL-13 or IL-13R. In one embodiment, the nucleic acid
antagonist is an siRNA that targets mRNA encoding an IL-13 or IL-13R. 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 an IL-13 or IL-13R-encoding nucleic
acid molecule are provided.
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 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 a-anomeric nucleic acid molecule forms
specific double-stranded hybrids with complementary RNA in which, contrary to the
usual ß-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 etal. (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 etal. (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 rVS RNA can be constructed in which the
nucleotide sequence of the active site is complementary to the nucleotide sequence to be
cleaved in a 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, mRNA can be used to select a
catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules.
See, e.g., Bartel, D. and Szostak, J.W. (1993) Science 261:1411-1418.
IL-13 or IL-13R gene expression can be inhibited by targeting nucleotide
sequences complementary to the regulatory region of the IL-13 or IL-13R (e.g., the an
IL-13 or IL-13R promoter and/or enhancers) to form triple helical structures that prevent
transcription of an IL-13 or IL-13R gene in target cells. See generally, Helene, C. (1991)
Anticancer Drug Des. 6:569-84; Helene, C. (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 nucleic acid molecule can be modified at the base moiety,
sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or
solubility of the molecule. For non-limiting examples of synthetic oligonucleotides with
modifications see Toulme (2001) Nature Biotech. 19:17 and Faria et al. (2001) Nature
Biotech. 19:40-44. Such phosphoramidite oligonucleotides can be effective antisense
agents. For example, the deoxyribose phosphate backbone of the nucleic acid molecules
can be modified to generate peptide nucleic acids (see Hyrup B. et al. (1996) Bioorganic
& Medicinal Chemistry 4: 5-23). As used herein, the terms "peptide nucleic acid" or
"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.
In other embodiments, the oligonucleotide may include other appended groups
such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating
transport across the cell membrane (see, e.g., Letsinger et al. (1989) Proc. Natl. Acad.
Sci. USA 86:6553-6556; Lemaitre et al. (1987) Proc. Natl. Acad. Sci. USA 84:648-652;
W088/09810) or the blood-brain barrier (see, e.g., W0 89/10134). In addition,
oligonucleotides can be modified with hybridization-triggered cleavage agents (see, e.g.,
Krol etal. (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 dhfr- CHO cells by calcium phosphate-mediated
transfection. Within the recombinant expression vector, the antibody heavy and light
chain genes are each operatively linked to enhancer/promoter regulatory elements (e.g.,
derived from SV40, CMV, adenovirus and the like, such as a CMV enhancer/AdMLP
promoter regulatory element or an SV40 enhancer/AdMLP promoter regulatory element)
to drive high levels of transcription of the genes. The recombinant expression vector also
carries a DHFR gene, which allows for selection of CHO cells that have been transfected
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 IL-13 or IL-4 binding agent and a target (e.g., IL-13)
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 (Ka), 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 binding agent or antagonist can be used to treat or prevent respiratory
disorders including, but are not limited to asthma (e.g., allergic and nonallergic asthma
(e.g., due to infection, e.g., with respiratory syncytial virus (RSV), e.g., in younger
children)); bronchitis (e.g., chronic bronchitis); chronic obstructive pulmonary disease
(COPD) (e.g., emphysema (e.g., cigarette-induced emphysema); conditions involving
airway inflammation, eosinophilia, fibrosis and excess mucus production, e.g., cystic
fibrosis, pulmonary fibrosis, and allergic rhinitis. For example, an IL-13 binding agent
(e.g., an anti-IL-13 antibody molecule) can be administered in an amount effective to
treat or prevent the disorder or to ameliorate at least one symptom of the disorder.
Asthma can be triggered by myriad conditions, e.g., inhalation of an allergen,
presence of an upper-respiratory or ear infection, etc. (Opperwall (2003) Nurs. Clin.
North Am. 38:697-711). Allergic asthma is characterized by airway hyperresponsiveness
(AHR) to a variety of specific and nonspecific stimuli, elevated serum immunoglobulin E
(IgE), excessive airway mucus production, edema, and bronchial epithelial injury (Wills-
Karp, supra). Allergic asthma begins when the allergen provokes an immediate early
airway response, which is frequently followed several hours later by a delayed late-phase
airway response (LAR) (Henderson et al. (2000) J. Immunol. 164:1086-95). During
LAR, there is an influx of eosinophils, lymphocytes, and macrophages throughout the
airway wall and the bronchial fluid. (Henderson et al., supra). Lung eosinophilia is a
hallmark of allergic asthma and is responsible for much of the damage to the respiratory
epithelium (Li et al. (1999) J. Immunol. 162:2477-87).
CD4+ T helper (Th) cells are important for the chronic inflammation associated
with asthma (Henderson et al., supra). Several studies have shown that commitment of
CD4+ cells to type 2 T helper (Th2) cells and the subsequent production of type 2
cytokines (e.g., IL-4, IL-5, IL-10, and IL-13) are important in the allergic inflammatory
response leading to AHR (Tomkinson et al. (2001) J. Immunol. 166:5792-5800, and
references cited therein). First, CD4+ T cells have been shown to be necessary for
allergy-induced asthma in murine models. Second, CD4+ T cells producing type 2
cytokines undergo expansion not only in these animal models but also in patients with
allergic asthma. Third, type 2 cytokine levels are increased in the airway tissues of
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 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 PROTGNIX® 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 IL-13 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 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 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) J. Neurooncol.
64:117-23) can be specifically targeted by anti-IL-13 antibodies described herein. IL-13
antagonists can be useful to inhibit cancer cell proliferation or other cancer cell activity.
A cancer refers to one or more cells that has a loss of responsiveness to normal growth
controls, and typically proliferates with reduced regulation relative to a corresponding
normal cell.
Examples of cancers against which IL-13 antagonists (e.g., an IL-13 binding
agent such as an antibody or antigen binding fragment described herein) can be used for
treatment include leukemias, e.g., B-cell chronic lymphocytic leukemia, acute
myelogenous leukemia, and human T-cell leukemia virus type 1 (HTLV-1) transformed
T cells; lymphomas, e.g. T cell lymphoma, Hodgkin's lymphoma; glioblastomas;
pancreatic cancers; renal cell carcinoma; ovarian carcinoma; AIDS-Kaposi's sarcoma,
and breast cancer (as described in Aspord, C. et al. (2007) JEM 204:1037-1047). For
example, an IL-13 binding agent (e.g., an anti-IL-13 antibody molecule) can be
administered in an amount effective to treat or prevent the disorder, e.g., to reduce cell
proliferation, or to ameliorate at least one symptom of the disorder.
Fibrosis
IL-13 and/or IL-4 antagonists can also be useful in treating inflammation and
fibrosis, e.g., fibrosis of the liver. IL-13 production has been correlated with the
progression of liver inflammation (e.g., viral hepatitis) toward cirrhosis, and possibly,
hepatocellular carcinoma (de Lalla et al. (2004) J. Immunol. 173:1417-1425). Fibrosis
occurs, e.g., when 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 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 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-EL-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 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 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 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 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. In other embodiments, 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).
In one embodiment, the single treatment interval includes two subcutaneous doses of
about 1-3 mg/kg, 1.5-2.5 mg/kg, 2 mg/kg of an anti-IL13 antibody molecule at least 4, 7,
9 or 14 days apart. For example, the single treatment interval can include two
subcutaneous doses of about 2 mg/kg of an anti-IL13 antibody molecule 7 days apart.
The duration of therapy using the pharmaceutical composition may vary,
depending on the severity of the disease being treated and the condition and potential
idiosyncratic response of each individual patient. In one embodiment, the IL-13 and/or
IL-4 antagonist can also be administered via the subcutaneous route, e.g., in the range of
once a week, once every 24, 48, 96 hours, or not more frequently than such intervals.
Exemplary dosages can be in the range of 0.1-20 mg/kg, more preferably 1-10 mg/kg.
The agent can be administered, e.g., by intravenous infusion at a rate of less than 20, 10,
5, or 1 mg/min to reach a dose of about 1 to 50 mg/m2 or about 5 to 20 mg/m2.
In one embodiment, an administration of an IL-13 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 IL-13 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 IL-13 antagonist is formulated for a nebulizer. In one
embodiment, the IL-13 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 IL-13 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 IL-13 antagonist at a pH suitable for
storage and another compartment for a neutralizing buffer and a mechanism for
combining the IL-13 antagonist with a neutralizing buffer immediately prior to
atomization. In one embodiment, the inhaler is a metered dose inhaler.
The three common systems used to deliver drugs locally to the pulmonary air
passages include dry powder inhalers (DPIs), metered dose inhalers (MDIs) and
nebulizers. MDIs, the most popular method of inhalation administration, may be used to
deliver medicaments in a solubilized form or as a dispersion. Typically MDIs comprise a
Freon or other relatively high vapor pressure propellant that forces aerosolized
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 IL-13 antagonist. In one
embodiment, the IL-13 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 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 antagonist may be in the form of a dry
particle or as a liquid. Particles that include the IL-13 antagonist can be prepared, e.g., by
spray drying, by drying an aqueous solution of the IL-13 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 antagonist may be conveniently delivered in the form of an aerosol
spray presentation from pressurized packs or a nebulizer, with the use of a suitable
propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a
pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a
metered amount. Capsules and cartridges for use in an inhaler or insufflator may be
formulated containing a powder mix of an IL-13 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 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 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 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 antagonist enters
circulation from the lung and is distributed to other organs or to a particular target organ.
In one embodiment, the IL-13 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 (<6 Tm)
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 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 antagonist and high bioavailability. In one
embodiment, the IL-13 antagonist is provided in a metered dose using, e.g., an inhaler or
nebulizer. For example, the IL-13 binding agent is delivered in a dosage unit form of at
least about 0.02, 0.1,0.5, 1, 1.5,2, 5,10, 20,40, or 50 mg/puff or more. The percent
bioavailability can be calculated as follows: the percent bioavailability =

Although not necessary, delivery enhancers such as surfactants can be used to
further enhance pulmonary delivery. A "surfactant" as used herein refers to an IL-13
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 peirticles 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 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 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 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 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 EL-13 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 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 antagonist described herein in an amount sufficient to
inhibit its activity. An IL-13 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 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 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 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 firsi: 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 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. 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 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.t 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, the IL-13 binding agents can be administered in
combination with an IL-4 antagonist. Examples of IL-4 antagonists include, but are not
limited to, antibody molecules against IL-4 (e.g., pascolizumab and related antibodies
disclosed in Hart, T.K. et al. (2002) Clin Exp Immunol. 130(1):93-100; Steinke, J.W.
(2004) Immunol. Allergy Clin North Am 24(4):599-614; and in Ramanthan et al. U.S.
6,358,509), IL-4Ra (e.g., AMG-317 and related anti-IL4R antibodies disclosed in US
05/0118176, US 05/0112694 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 IL4 and/or IL-
13 (disclosed, e.g., in WO 07/085815).
In other embodiments, the IL-13 or IL-4 antagonist is an IL-13 or IL-4 mutein
(e.g., a truncated or variant form of the cytokine that binds to the 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-13Rot2
or IL13Rotl) or an IL-4 receptor polypeptide (e.g., IL-4Ra). For example, the antagonist
can be a soluble form of an IL-13 receptor or an IL-14 receptor (e.g., a soluble form of
mammalian (e.g., human) IL-13Ra2, IL13Ral or IL-4Ra comprising a cytokine-binding
domain; e.g., a soluble form of an extracellular domain of mammalian (e.g., human) IL-
13Ra2, IL13Ral or IL-4Ra). 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(\):A1-52, 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 IL-13 receptor or IL-4 receptor, or an IL-13 or IL-4 mutein (or a sequence
homologous thereto), and, e.g., fused to, a human immunoglobulin Fc chain, e.g., human
IgG (e.g., human IgG1 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-4R
. developed by ISIS Pharmaceuticals and disclosed in, e.g., Karras, J.G. et al. (2007) Am J
Respir 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) Bioorg Med Chem
15(2): 1044-55; and in US 6,207,391 and WO 01/083517.
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-S 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 NFPB inhibitors.
Vaccine Formulations
In another aspect, the invention features a method of modifying an immune
response associated with immunization. An DL-13 antagonist, alone or in combination
with an IL-4 antagonist, can be used to increase the efficacy of immunization by
inhibiting IL-13 activity. Antagonists can be administered before, during, or after
delivery of an immunogen, e.g., administration of a vaccine. In one embodiment, the
immunity raised by the vaccination is a cellular immunity, e.g., an immunity against
cancer cells or virus infected, e.g., retrovirus infected, e.g., HIV infected, cells. In one
embodiment, the vaccine formulation contains one or more antagonists and an antigen,
e.g., an immunogen. In one embodiment, the IL-13 and/or IL-4 antagonists are
administered in combination with immunotherapy (e.g., in combination with an allergy
immunization with one or more immunogens chosen from ragweed, ryegrass, dust mite
and the like. In another embodiment, the antagonist and the immunogen are administered
separately, e.g., within one hour, three hours, one day, or two days of each other.
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/or Monitoring IL-13-Associated Disorders
The binding agents described herein can be used, e.g., in methods for diagnosing,
prognosing, and monitoring the progress of IL-13- associated disorders, e.g., 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 IL-13- associated disorders, e.g., 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 fluorophore or chromophore, the binding agent
can be used to detect the presence or localization of the IL-13 in a sample, e.g., using
fluorescent microscopy (such as confocal or deconvolution microscopy).
Histological Analysis. Immunohistochemistry can be performed using the
binding agents described herein. For example, in the case of an antibody, the antibody
can synthesized with a label (such as a purification or epitope tag), or can be detectably
labeled, e.g., by conjugating a label or label-binding group. For example, a chelator can
be attached to the antibody. The antibody is then contacted to a histological preparation,
e.g., a fixed section of tissue that is on a microscope slide. After an incubation for
binding, the preparation is washed to remove unbound antibody. The preparation is then
analyzed, e.g., using microscopy, to identify if the antibody bound to the preparation.
The antibody (or other polypeptide or peptide) can be unlabeled at the time of binding.
After binding and washing, the antibody is labeled in order to render it detectable.
Protein Arrays. An IL-13 binding agent (e.g., a protein that is an IL-13 binding
agent) can also be immobilized on a protein array. The protein array can be used as a
diagnostic tool, e.g., to screen medical samples (such as isolated cells, blood, sera,
biopsies, and the like). The protein array can also include other binding agents, e.g., ones
that bind to IL-13 or to other target molecules.
Methods of producing protein arrays are described, e.g., in De Wildt et al. (2000)
Nat. Biotechnol. 18:989-994; Lueking et al. (1999) Anal. Biochem. 270:103-111; Ge
(2000) Nucleic Acids Res. 28, e3,1-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,
111In, 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 14C, 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 µm 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, mode of administration,
pharmacokinetic/phamacodynamic properties 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:: MJ 2-7 Antibody
Total RNA was prepared from MJ 2-7 hybridoma cells using the QIAGEN
RNEASY3 Mini Kit (Qiagen). RNA was reverse transcribed to cDNA using the
SMART3 PCR Synthesis Kit (BD Biosciences Clontech). The variable region of MJ 2-7
heavy chain was extrapolated by PCR using SMART3 oligonucleotide as a forward
primer and mlgGl primer annealing to DNA encoding the N-terminal part of CH1
domain of mouse IgG1 constant region as a reverse primer. The DNA fragment encoding
MJ 2-7 light chain variable region was generated using SMART3 and mouse kappa
specific primers. The PCR reaction was performed using DEEP VENT3 DNA
polymerase (New England Biolabs) and 25 nM of dNTPs for 24 cycles (94 °C for 1
minute, 60 °C for I 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 NHPIL-13 and which has characteristics which suggest that
it may interact with human IL-13 are as follows:
An exemplary nucleotide sequence encoding the heavy chain variable domain
includes:

An exemplary amino acid sequence for the heavy chain variable domain includes:

CDRs are underlined. The variable domain optionally is preceded by a leader
sequence, e.g., MKCSWVIFFLMAVVTGVNS (SEQ ID NO:131). An exemplary
nucleotide sequence encoding the light chain variable domain includes:

An exemplary amino acid sequence for the light chain variable domain includes:

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 2: 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:

An exemplary amino acid sequence for the heavy chain variable domain includes:

CDRs are underlined. The amino acid sequence optionally is preceded by a leader
sequence, e.g., MAVLALLFCL VTFPSCILS (SEQ ID NO: 137).
An exemplary nucleotide sequence encoding the light chain variable domain
includes:
)
An exemplary amino acid sequence for the light chain variable domain includes:

CDRs are underlined. The amino acid sequence optionally is preceded by a leader
sequence, e.g., MNTRAPAEFLGFLLLWFLGARC (SEQ ID NO: 140).
Example 3: 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:

Other exemplary alterations that can be used to decrease effector function include
L234A;L235A), (L235A;G237A), and N297A.
Example 4: IL-13 and Atopic Disorders
The ability of MJ2-7 to inhibit the bioactivity of native human IL-13 (at 1 ng/ml)
was evaluated in an assay for STAT6 phosphorylation. MJ2-7 inhibited the activity of
native human IL-13 with an IC50 of about 0.293 nM in this assay. An antibody with the
murine heavy chain of MJ2-7 and a humanized light chain inhibited the activity of native
human IL-13 with an IC50 of about 0.554 nM in this assay.
The ability of MJ2-7 to inhibit non-human primate IL-13 (at 1 ng/ml) was
evaluated in an assay for CD23 expression. The MJ2-7 inhibited the activity of non-
human primate IL-13 with an IC50 of about 0.242 nM in this assay. An antibody with
the murine heavy chain of MJ2-7 and a humanized light chain inhibited the activity of
non-human primate IL-13 with an IC50 of about 0.308 nM in this assay.
Example 5: 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:

The amino acid sequence of the heavy chain variable region with an optional
leader (underscored) is as follows:

The nucleotide sequence encoding the light chain variable region is as follows:

The amino acid sequence of the light chain variable region with an optional leader
(underscored) is as follows:

Example 6: Nucleotide and amino acid sequences of exemplary first humanized
variants of the MJ 2-7 antibody
Humanized antibody Version 1 (VI) is based on the closest human germline
clones. The nucleotide sequence of hMJ 2-7 VI heavy chain variable region (hMJ 2-7
VH VI) (with a sequence encoding an optional leader sequence) is as follows:

The amino acid sequence of the heavy chain variable region (hMJ 2-7 VI) is
based on a CDR grafted to DP- 25, VH-1,1-03. The amino acid sequence with an
optional leader (first underscored region; CDRs based on AbM definition shown in
subsequent underscored regions) is as follows:

The nucleotide sequence of the hMJ 2-7 VI light chain variable region (hMJ 2-7
VL VI) (with a sequence encoding an optional leader sequence) is as follows:

This version is based on a CDR graft to DPK18, V kappall. The amino acid
sequence of hMJ 2-7 VI light chain variable region (hMJ 2-7 VL VI) (with optional
leader as first underscored region; CDRs based on AbM definition in subsequent
underscored regions) is as follows:

The following heavy chain variable region is based on a CDR graft to DP-54,
VH-3,3-07. The nucleotide sequence of hMJ 2-7 Version 2 (V2) heavy chain variable
region (hMJ 2-7 VH V2) (with a sequence encoding an optional leader sequence) is as
follows:

The amino acid sequence of hMJ 2-7 V2 heavy chain variable region (hMJ 2-7
VH V2) with an optional leader (first underscored region; CDRs based on AbM
definition shown in subsequent underscored regions) is as follows:

The hMJ 2-7 V2 light chain variable region was based on a CDR graft to DPK9,
V kappal, 02. The nucleotide sequence of hMJ 2-7 V2 light chain variable region (hMJ
2-7 VL V2) (with a sequence encoding an optional leader sequence) is as follows:

The amino acid sequence of the light chain variable region of hMJ 2-7 V2 light
chain variable region (hMJ 2-7 VL V2) (with optional leader peptide underscored and
CDRs based on AbM definition shown in subsequent underscored regions) is as follows:

Additional humanized versions of MJ 2-7 V2 heavy chain variable region were
made. These versions included backmutations that have murine amino acids at selected
framework positions.
The nucleotide sequence encoding the heavy chain variable region "Version 2.1"
or V2.1 with the back mutations V48I,A29G is as follows:

The amino acid sequence of the heavy chain variable region of V2.1 (CDRs based
on AbM definition shown in subsequent underscored regions) is as follows:
1 EVQLVESGGG LVQPGGSLRL SCAASGFNIK PTYIHWVRQA PGKGLEWIGR

The nucleotide sequence encoding the heavy chain variable region V2.2 with the
back mutations (R67K;F68A) is as follows:

The amino acid sequence of the heavy chain variable region of V2.2 (CDRs based
on AbM definition shown in subsequent underscored regions) is as follows:

The amino acid sequence of the heavy chain variable region of V2.3 (CDRs based
on AbM definition shown in subsequent underscored regions) is as follows:

The nucleotide sequence encoding the heavy chain variable region V2.4 with the
back mutations (A49G) is as follows:

The amino acid sequence of the heavy chain variable region of V2.4 (CDRs based
on AbM definition shown in subsequent underscored regions) is as follows:

The nucleotide sequence encoding the heavy chain variable region V2.5 with the
back mutations (R67K;F68A;R72A) is as follows:

The amino acid sequence of the heavy chain variable region of V2.5 (CDRs based
on AbM definition shown in subsequent underscored regions) is as follows:

The nucleotide sequence encoding the heavy chain variable region V2.6 with the
back mutations (V48I;A49G;R72A) is as follows:

The amino acid sequence of the heavy chain variable region of V2.6 (CDRs based
on AbM definition shown in subsequent underscored regions) is as follows:

The nucleotide sequence encoding the heavy chain variable region V2.7 with the
back mutations (A49G;R72A) is as follows:

The amino acid sequence of the heavy chain variable region of V2.7 (CDRs based
on AbM definition shown in subsequent underscored regions) is as follows:

The nucleotide sequence encoding the heavy chain variable region V2.8 with the
back mutations (L79A) is as follows:

The amino acid sequence of the heavy chain variable region of V2.8 (CDRs based
on AbM definition shown in subsequent underscored regions) is as follows:

The nucleotide sequence encoding the heavy chain variable region V2.10 with the
back mutations (A49G;R72A;L79A) is as follows:

The amino acid sequence of the heavy chain variable region of V2.10 (CDRs
based on AbM definition shown in subsequent underscored regions) is as follows:

The nucleotide sequence encoding the heavy chain variable region V2.ll with the
back mutations (V48I;A49G;R72A;L79A) is as follows:

The amino acid sequence of the heavy chain variable region of V2.11 (CDRs
based on AbM definition shown in subsequent underscored regions) is as follows:

The nucleotide sequence encoding the heavy chain variable region V2.16 with the
back mutations (V48I;A49G;R72A) is as follows:

The amino acid sequence of the heavy chain variable region of V2.16 (CDRs
based on AbM definition shown in subsequent underscored regions) is as follows:

The following is the amino acid sequence of a humanized MH 2-7 V2.11 IgGl
with a mutated CH2 domain:

The variable domain is at amino acids 1-120; CH1 at 121-218; hinge at 219-233;
CH2 at 234-343; and CH3 at 344-450. The light chain includes the following sequence
with variable domain at 1-133.

Example 8: Functional Assays of Exemplary Variants of MJ2-7
We evaluated the ability of the MJ2-7 antibody and humanized variants 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 ALEXA3 Fluor 488-labeled antibody to STAT6 (BD Biosciences).
Fluorescence was analyzed with a FACSCAN3 and CELLQUEST3 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 raM 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 CD11b (BD Biosciences). Monocytes were gated
based on high forward and side light scatter, and expression of CD11b. CD23 expression
on monocytes was determined by flow cytometry using a FACSCAN3 (BD Biosciences),
and the percentage of CD23+ cells was analyzed with CELLQUEST3 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 TCi / 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 TOMTEK3 harvester. 3H-
thymidine incorporation was assessed by liquid scintillation counting.
Tenascin production assay
BEAS-2B human bronchial epithelial cells (ATCC) were maintained BEGM
media with supplements (Clonetics). Cells were plated at 20,000 per well in a 96-well
flat-bottom culture plate overnight. Fresh media is added containing IL-13 in the
presence or absence of the indicated antibody. After overnight incubation, the
supernatants are harvested, and assayed for the presence of the extracellular matrix
component, tenascin C, by ELISA. ELISA plates are coated overnight with 1 ug/ml of
murine monoclonal antibody to human tenascin (IgGl, k; Chemicon International) in
PBS. Plates are washed with PBS containing 0.05% TWEEN®-20 (PBS-Tween), and
blocked with PBS containing 1% BSA. Fresh blocking solution was added every 6
minutes for a total of three changes. Plates were washed 3X with PBS-Tween. Cell
supernatants or human tenascin standard (Chemicon International) were added and
incubated for 60 minutes at 37 °C. Plates were washed 3X with PBS-Tween. Tenascin
was detected with murine monoclonal antibody to tenascin (IgG2a, k; Biohit). Binding
was detected with HRP-labeled antibody to mouse IgG2a, followed by TMB substrate.
The reaction was stopped with 0.01 N sulfuric acid. Absorbance was read at 450 nm.
The HT 29 human epithelial cell line can be used to assay STAT6
phosphorylation. HT 29 cells are incubated with 1 ng/ml native human IL-13 crude
preparation in the presence of increasing concentrations of the test antibody for 30
minutes at 37 °C. Western blot analysis of cell lysates with an antibody to
phosphorylated STAT6 can be used to detect dose-dependent IL 13-mediated
phosphorylation of STAT6. Similarly, flow cytometric analysis can detect
phosphorylated STAT6 in HT 29 cells that were treated with a saturating concentration of
IL-13 for 30 minutes at 37 °C, fixed, permeabilized, and stained with an 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.

Example 9: Binding Interaction Site Between IL-13 and IL-13RI1
A complex of IL-13, the extracellular domain of IL-13RI1 (residues 27-342 of
SEQ ID NO:125), and an antibody that binds human IL-13 was studied by x-ray
crystallography. See, e.g., 16163-029001. Two points of substantial interaction were
found between IL-13 and IL-13Ra1. The interaction between Ig domain 1 of IL-13Ral
and IL-13 results in the formation of an extended beta sheet spanning the two molecules.
Residues Thr88 [Thr107], Lys89 [Lys108], Ile90 [Ile109], and Glu91 [Glul 10] of IL-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 ID
NO:125). Additionally, the side chain of Met33 [Met52] of IL-13 (SEQ ID NO:124
[SEQ ID NO: 178]) extends into a hydrophobic pocket that is created by the side chains of
these adjoining strands.
The predominant feature of the interaction with Ig domain 3 is the insertion of a
hydrophobic residue (Phel07 [Phe126]) of IL-13 (SEQ ID NO:124 [SEQ ID NO:178])
into a hydrophobic pocket in Ig domain 3 of the receptor IL-13Ral. The hydrophobic
pocket of IL-13Ral is formed by the side chains of residues Leu319, Cys257, Arg256,
and Cys320 (SEQ ID 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 IIe254, Ser255, Arg256, Lys318, Cys320, and Tyr321 of
IL-13Ral (SEQ ID NO:125) and amino acid residues Argl 1 [Arg30], GIu12 [Glu31],
Leul3 [Leu32], Ile14 [Ue33], Glul5 [Ile34], Lys104 [Lys123], Lys105 [Lys124], Leu106
[Leu125], Phe107 [Phe126], and Arg108 [Arg 127] of IL-13 (SEQ ID N0.124 [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-13RI1 can be used to inhibit IL-13
signaling.
Example 10: 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 raM glutamine and 0.1 mg/ml Penicillin/
Streptomycin. Transfection of COS cells was performed using TRANSITIT3-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 Tg/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 V1 and V2. The CDR grafted MJ 2-7 V2 had a 3-fold higher expression level
then CDR grafted MJ 2-7 V1 in the same assay conditions.
Table 3

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 11: Molecular modeling of humanized MJ2-7 v.2VH
Structure templates for modeling humanized MJ2-7 heavy chain version 2 (MJ2-7
v.2VH) 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
v.2VH 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 UPS, 1N8Z and F13.2
(available from WO05/121177) were determined based on the Ca distance matrix for
each molecule and the template structures were superimposed based on minimum RMS
deviation of corresponding atoms in SCRs. The sequence of the target protein MJ2-7
v.2VH 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
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 v.2VH 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 v.2VH:G26 - hMJ2-7 v.2VH:A24
hMJ2-7 v.2VH:Y109 - hMJ2-7 v.2VH: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 12: 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-13RI2-Fc. Cells were harvested, stained with CYCHROME3-
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 CD11b. MJ2-7, C65, and
sIL13RI2-Fc all were able to neutralize the acitivity of NHP IL-13 in this assay. The
potencies of MJ2-7 and sIL-13RI2-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-13RI2-Fc, for 30 minutes at 37 °C. Cells were
fixed, permeabilized, and stained with ALEXA3 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
sIL-13RI2-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 NHPIL-
13 and native human IL-13. The IL-13 neutralization capacity of MJ2-7 is equivalent to
that of sIL-13RI2-Fc. MJ1-65 also has IL-13 neutralization activity, but is approximately
20-fold less potent than MJ2-7.
Example 13: Epitope mapping of MJ2-7antibody by SPR
sIL-13RI2-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-
13RI2-Fc was detected by BIACORE3. An additional injection of 100 nM of anti IL-13
antibodies was added, and changes in binding were monitored. MJ2-7 antibody did not
bind to NHP-IL-13 when it was in a complex with hu IL-13RI2, whereas a positive
control anti-IL-13 antibody did (FIG. 7). These results indicate mat hu IL-13RI2 and
MJ2-7 bind to the same or overlapping epitopes of NHP IL-13.
Example 14: Measurement of kinetic rate constants for the interaction between
NHP-IL-13 and humanized MJ2-7 v.2-11 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-ethyl-3-(3-
dimethylaminopropyl) carbodiimide (EDC) and 0.05 M N-Hydroxysuccinimide (NHS).
The capturing antibody was injected at a concentration of 10 Tg/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 v.2-11 was captured onto
the anti IgG antibody surface by injecting 40 Tl of a 1 Tg/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 Tl 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 Tl
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 BIAEVALUATION3 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 v.2-11 has on and off-rates of 2.05xl07 N-1s-1 and 8.89X10-4 1/s,
respectively, resulting in an antibody with 43 pM affinity for NHP-IL-13.
Example 15: 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 IL-13. This
concentration of the Version 2 humanized antibody required for half-maximal inhibition
of native human IL-13 bioactivity was approximately 110-fold greater than that of murine
MJ2-7 (FIG. 9). Analysis of a semi-humanized form, in which the V1 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 V1 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 V1 VH sequence to improve the native human IL-13 neutralization activity of
murine MJ2-7.
Example 16; MJ2-7 blocks IL-13 interaction with IL-13RI1 and IL-13RI2
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-13RI1 and IL-13RI2. Analysis of human IL-13 mutants identified
the A, C, and D-helices as containing important contacts site for the IL-13RI1 / IL-4RI
signaling complex (Thompson and Debinski (1999) J. Biol. Chem. 21 A: 29944-50).
Alanine scanning mutagenesis of the D-helix identified residues K123, K124, and R127
(SEQ ID NO:24) sis responsible for interaction with IL-13RI2, and residues El 10, E128,
and L122 as important contacts for IL-13RI1 (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-13RI1 (Eisenmesser
et al. (2001) J. Mol. Biol. 310:231-241; Moy et al. (2001) J. Mol. Biol. 310:219-230).
Binding of MJ2-7 to this epitope located in the C-terminal, D-helix of IL-13 was
predicted to disrupt interaction of IL-13 with IL-13RI1 and IL-13RI2.
The ability of MJ2-7 to inhibit binding of NHP IL-13 to IL-13RI1 and IL-13RI2
was tested by ELISA. Recombinant soluble forms of human IL-13RI1-Fc and IL-13RI2-
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 17: 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 EL-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.
Nucleotide sequence of hMJ 2-7 VL V3

Example 18: Neutralizing Activities of Anti-IL-13 Antibodies in Cynomolgus
Monkey Model
The efficacy of an IL-13 binding agent (e.g., an anti-IL-13 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 j4.scara-specific basophil histamine release;
and/or (iv) increase in ^(scam-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.2-11 and humanized mAb13.2v.2) were
administered to cynomolgus monkeys 24 hours prior to challenge with Ascaris suum
antigen. (mAb 13.2 and its humanized form hmAb13.2v2 were described in commonly
owned PCT application WO 05/123126, the contents of which are incorporated herein by
reference in their entirety). Control monkeys were treated with saline. lOmg/kgof
hMJ2-7v.2-11, hmAb13.2v2, or irrelevant human Ig (IVIG) were administered
intravenously. The following day, prechallenged BAL samples from control and treated
monkeys (referred to in FIG. 15A as "control pre" and "Ab pre") were collected from the
left lung of the monkeys. The monkeys were treated with 0.75 micrograms of Ascaris
suum antigen intratracheally into the right lung. Twenty-four hours post-challenge, BAL
samples were collected from the right lung of control and treated monkeys, and assayed
for cellular infiltrate (referred to in FIG. 15B as "control post" and "Ab post,"
respectively). BAL samples collected from antibody-treated monkeys showed a
statistically significant reduction in the total number of cell infiltrate compared to control
animals (FIG. 15A). Control samples are represented in FIG. 15A as circles,
hmAb13.2v2- and hMJ2-7v.2-11- treated samples are shown as dark and light triangles,
respectively. hMJ2-7v.2-11 and hmAb13.2v2 showed comparable efficacy in this model.
FIG. 15B shows a linear graph depicting the concentration of either hMJ2-7v.2-11 or
hmAb13.2v2 with respect to days post-Ascaris infusion. A comparable decrease in
kinetics is detected for both antibodies.
Eotaxin levels were significantly increased 24 hours following Ascaris challenge
(FIG. 16A). Both hMJ2-7v.2-11 and hmAb13.2v2 reduced eotaxin levels detected in
BAL fluids from cynomolgus monkeys 24 hours after to challenge with Ascaris suum
antigen, compared to saline treated controls.
Cynomolgus monkeys sensitized to Ascaris suum develop IgE to Ascaris antigen.
The IgE binds to FceRI 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 hMJ2-7v.2-11 and hmAb13.2v2 in IgE- and
basophil levels, cynomolgus monkeys dosed with humanized hMJ2-7v.2, hmAb13.2v2,
irrelevant Ig (TVIG), or saline, as described above, were bled 8 weeks post- Ascaric
challenge, and levels of total and ylyajra-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 hMJ2-7v.2-11. Open-circles represent pre-bleed measurements; filled circles
represent post-treatment measurements. A significant reduction in absorbance was
detected in post-challenged samples treated with hMJ2-7v.2-11 relative to the pre-
challenge values in all dilutions assayed FIG 17A depicts representative examples
showing no change in Ascaric-specific IgE titer in an individual monkey treated with
irrelevant Ig (TVIG; animal 20-45; top panel), and decreased titer of Ascaric-specific IgE
in an individual monkey treated with hMJ2-7v.2-11 (animal 120-434; bottom panel).
Animals treated with either humanized hMJ2-7v.2-11 or hmAb13.2v2 showed a
significant reduction in levels of circulating IgE-specific for Ascaris in cynomolgus
monkey sera (FIG. 17B). There was no significant change in total IgE titer for any of the
treatment groups. FIG. 17A shows a linear graph of the changes in absorbance with
respect to dilution of samples obtained pre- and 8-weeks post-challenge from animals
treated with IVIG or hMJ2-7v.2-11. Open-circles represent pre-bleed measurements;
filled circles represent post-treatment measurements. A significant reduction in
absorbance was detected in post-challenged samples treated with hMJ2-7v.2-11 relative
to the pre-challenge values in all dilutions assayed. The designations "20-45" and "120-
434" refer to the cynomolgus monkey identification number.
To evaluate the effects of anti-IL13 antibodies on basophil histamine release, the
animals were bled at 24 hours and 8 weeks post-Ascaris challenge. Whole blood was
challenged with Ascaris antigen for 30 minutes at 37°C, and histamine released into the
supernatant was quantitated by ELISA (Beckman Coulter, Fullerton, CA). As shown in
FIGS. 18A-18B, trie control animals demonstrated increased levels of ^cam-induced
basophil histamine release particularly 8 weeks following antigen challenge (represented
by the diamonds in FIG. 18A and left-hand bar in FIG. 18B). In contrast, the animals
treated with either humanized hMJ2-7v.2-11 or hmAb13.2v2 did not show this increase
in basophil sensitization in response to Ascaris 8 weeks after challenge (FIGS. 18A-18B).
The majority of individual animals treated with humanized hMJ2-7v.2-11 or
hmAb13.2v2 showed either a decrease (example in FIG 18A) or no change in basophil
histamine release 8 weeks post-challenge compared to pre- or 24 hour post-challenge.
Thus, a single administration of the humanized anti-IL13 antibody had a lasting effect in
modifying histamine release in this model.
FIG. 19 depicts the correlation between Ascaris-specific histamine release and
Ascaris-specific IgE levels. Higher values were detected in control samples (saline- or
IVIG-treated samples) (light blue circles) compared to anti-IL13 antibody- or
dexamethasone (dex)-treated (dark red circles). Humanized anti-IL13 antibody
(humanized mAb13.2v.2) administered i.v. 24 hours prior to Ascaris challenge, or
dexamethasone administered intramuscular in two injections each one at a concentration
of 1 mg/kg 24 hours and 30 mins. prior to Ascaris challenge. Twenty four hours post-
challenge, BAL lavage was collected from the right lung and assayed for histamine
release and IgE levels.
The results shown herein demonstrated that pretreatment of cynomolgus monkeys
with either MJ2-7 or mAb13.2 reduced airway inflammation induced by Ascaris suum
antigen at comparable levels as detected by cytokine levels in BAL samples, serum levels
of Ascaris-specific IgE's and basophil histamine release in response to Ascaris challenge
in vitro.
FIG. 20 is a series of bar graphs depicting the increases in serum IL-13 levels in
individual cynomolgus monkeys treated with humanized MJ2-7 (hMJ2-7v.2-11). 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 hMJ2-7v.2-11 or hmAb13.2v2
antibodies. More specifically, ELISA plates (MaxiSorp; Nunc, Rochester, NY), were
coated overnight at 4°C with 0.5 ug/ml mAM3.2 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 mAb13.2, the same protocol was followed, except 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.2-11) 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 (hMJ2-7v.2-11) 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 19: Effects of Humanized Anti-IL-13 Antibodies on Airway
Inflammation. Lung Resistance, and Dynamic Lung Compliance Induced by
Administration of Human IL-13 to Mice
Murine models of asthma have proved invaluable tools for understanding the role
of IL-13 in this disease. The use of this model to evaluate in vivo efficacies of the
antibody series (humanized 13.2v.2 and humanized MJ2-7v.2-11) was initially hampered
by the inability of these antibodies to cross react with rodent IL-13. This limitation was
circumvented herein by administering human recombinant IL-13 to mice. Human IL-13
is capable of binding to the murine IL-13 receptor, and when administered exogenously
induces airway inflammation, hyperresponsiveness, and other correlates of asthma.
In non-human primates, the IL-13 epitope recognized by humanized MJ2-7v.2-11
includes a GLN at position 110. In humans, however, position 110 is a polymorphic
variant, typically with ARG replacing GLN (e.g., R110). The R110Q polymorphic
variant is widely associated with increased prevalence of atopic disease.
In this example, recombinant human R110Q IL-13 was expressed in E. coli and
refolded. Antibody 13.2 (IgGl, k) was cloned from BALB/c mice immunized with
human IL-13, and the humanized version of this antibody is designated humanized
13.2v.2 (or 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.2-11 (or hMJ2-7v.2-
11). Both antibodies were formulated in 10 mM L-histidine, pH 6, containing 5%
sucrose. Carimune NH immune globulin intravenous (human IVIG) (ZLB Bioplasma
Inc., Switzerland) was purified by Protein A chromatography and formulated in 10mM
L-histidine, pH 6, containing 5% sucrose.
To analyze the mouse lung response to the presence of recombinant human
R110Q IL-13, BABL/c female mice were treated with 5 µg of recombinant human
R110Q IL-13 (e.g., approximately 250 Tg/kg), or an equivalent volume of saline (20 µL),
administered intratracheally on days 1,2, and 3. On day 4, animals were tested for signs
of airway resistance (RI) and compliance (Cdyn) in response to increasing doses of
nebulized methacholine. Briefly, anesthetized and tracheostomized mice were placed
into whole body plethysmographs, each with a manifold built into the head plate of the
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
R110QIL-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 ug of recombinant human
R110Q IL-13, or an equivalent volume (50 µL) 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 urn cell strainer and centrifuged at 2,000 rpm for 15 minutes to pellet cells. Cell
fractions were analyzed for total leukocyte count, spun onto microscope slides (Cytospin;
Pittsburgh, PA), and stained with Diff-Quick (Dade Behring, Inc. Newark DE) for
differential analysis. IL-6, TNFa, and MCP-1 levels were determined by cytometric bead
array (CBA; BD Pharmingen, San Diego, CA). The limits of assay sensitivity were 1
pg/ml for IL-6, and 5 pg/ml for TNFa and MCP-1.
As shown in FIG. 24A, intranasal administration of recombinant human R110Q
IL-13 induced a strong airway inflammatory response, as indicated by elevated
eosinophil and neutrophil infiltration into BAL. Cell infiltrates consisted primarily of
eosinophils (e.g., approximately 40%). As shown in FIG. 24B, intranasal administration
of recombinant human R110Q IL-13 also significantly increased the levels of several
cytokines in BAL including, for example, MCP-1, TNF-I, and IL-6.
To determine the best delivery method for humanized MJ2-7v.2-11, antibody
levels in BAL and serum were analyzed following intraperitoneal and intravenous, or
intranasal administration following treatment with recombinant human R110Q IL-13
administered intranasally or intratracheally. Briefly, BALB/c female mice were
administered 5 µg of recombinant human R110Q 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 jig humanized MJ2-7v.2 administered intravenously
on day 0, and by IP on days 1, 2, and 3 (FIG. 25A). Alternatively, 500 µg of humanized
MJ2-7v.2-11 were administered intranasally on days 0, 1,2, and 3. Total human IgG was
measured by ELISA, as follows: ELISA plates (MaxiSorp; Nunc, Rochester, NY) were
coated overnight at 4°C with 1:1500 dilution of goat anti-human Ig (M+G+A) Fc (ICN-
Cappel, Costa Mesa, CA) at 50 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.2-11 standard or 6
x 1:2 dilutions of sheep serum starting at 1:500 - 1:50,000 were added and incubated for
2 hours at room temperature. Plates were washed with PBS-Tween, and a 1:5000
dilution of biotinylated mouse anti-human IgG (Southern Biotechnology Associates) was
incubated for 2 hours at room temperature. Plates were washed with PBS-Tween, and
binding was detected with peroxidase-linked streptavidin (Southern Biotechnology
Associates) and Sure Blue substrate (KPL Inc.). Assay sensitive was 0.5 ng/ml human
IgG.
FIG. 25A shows elevated levels of human IgG in serum compared to BAL
following intraperitoneal and intravenous administrator! of the humanized MJ2-7v.2-11
antibody. As shown in FIG. 25B, total IgG levels in ng/ml were significantly higher in
BAL than serum levels following intranasal administration of humanized MJ2-7v.2-11
antibody.
To determine if the humanized MJ2-7v.2-11 antibody was capable of modulating
the above observed lung function and inflammatory response, airway
hyperresponsiveness was induced by intratracheal administration of 5 µg recombinant
human R110Q IL--13 or an equivalent volume (20 µL) of saline on days 1, 2, and 3. On
day 0, and 2 hours; before administering each dose of recombinant human R110Q IL-13,
animals were treated with 500 |xg of humanized MJ2-7v.2-11, 500 u.g dose of IVIG, or an
equivalent volume of saline, administered intranasally. Animals were tested on day 4 for
airway resistance (RI) and compliance (Cdyn) in response to increasing doses of
nebulized methacholine, as described above. Humanized MJ2-7v.2 and IVIG levels in
BAL and serum were analyzed by ELISA, as described above. As shown in FIGs. 26A-
26B, humanized MJ2-7v.2-11 effectively reduced the asthmatic response, resulting in a
significant reduction in the dose of methacholine required to achieve half-maximal
degree of lung resistance. In contrast, an equivalent dose of IVIG had no effect.
Changes in dynamic lung compliance were not apparent under these conditions. As
shown in FIG. 26C, BAL IgG antibody levels were approximately 10-20 times higher
than serum levels.
To determine if humanized MJ2-7v.2-11 anti-IL-13 antibody administration
promoted an increase in the circulating levels of IL-13, BAL and sera were assayed for
IL-13 levels by ELISA, as follows: Briefly, BALB/c female mice were treated as
described for FIG. 26A-B. ELISA plates (Nunc Maxi-Sorp) were coated overnight with
50 µl / well mouse anti-IL-13 antibody, mAb13.2, diluted to 0.5 mg/ml in PBS. Plates
were washed 3 times with PBS containing 0.05% Tween-20 (PBS-Tween) and blocked
for 2 hours at room temperature with 0.5% gelatin in PBS. Plates were then washed and
human IL-13 standard (Wyeth, Cambridge, MA), or dilutions of mouse serum (serial 3X
dilutions starting at 1:4) were added, in PBS-Tween containing 2% fetal calf serum
(FCS). Plates were incubated for a further 4 hours at room temperature, and washed.
Biotinylated mouse anti-human IL-13 antibody, MJ1-64, was added at 0.3 µg/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 cui-ve, which was independently established for each plate.
As shown in FIGs. 27A-27B, consistent with FIG. 26C, IL-13 levels were
elevated in BAL of antibody-treated mice, but not serum. In addition, we observed that
IL-13 isolated from these samples had no detectable biological activity (data not shown).
To determine if this observed lack of IL-13 biological activity was due to IL-13 and
humanized MJ2-7v.2-11 complex formation, an ELISA was developed to specifically
detect IL-13 and humanized MJ2-7v.2-11 in complex. Briefly, ELISA plates (Nunc
Maxi-Sorp) were coated overnight with 50 µl / well mouse anti-IL-13 antibody,
mAb13.2, diluted to 0.5 mg/ml in PBS. Plates were washed 3 times with PBS containing
0.05% Tween-20 (PBS-Tween) and blocked for 2 hours at room temperature with 0.5%
gelatin in PBS. Plates were then 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. 27C-27D, IL-13 and humanized MJ2-7v.2-11 complexes were
recovered from BAL and serum of mice in this model. This observation indicates that
humanized MJ2-7v.2-11 is capable of binding IL-13 in vivo, and that this interaction may
negate IL-13 biological activity.
The effects of humanized MJ2-7v.2-11 on human IL-13-mediated lung
inflammation and cytokine production were tested in mice, and compared with a second
antibody, humanized 13.2v.2, as follows. Briefly, C57BL/6 female mice (10/group) were
treated with 5 jig of recombinant human R110Q 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 µg, 100 ug, or 20 µg of humanized MJ2-7v.2-11 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
R110Q IL-13 treatment evoked an increase in eosinophil and neutrophil infiltration
levels. Interestingly, humanized MJ2-7v.2-11 and humanized 13.2v.2 significantly
reduced eosinophil (FIG. 28A) and neutrophil (FIG. 28B) infiltration compared to
controls (e.g., saline, IL-13, IVIG). As shown in FIG. 29A-29C, HMJ2-7V2-11 and
humanized MJ2-7v.2-11 also abrogated increases in MCP-1, TNF-I, 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 ug of recombinant human
R110QIL-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.2-11. On day 4, animals were sacrificed and BAL collected.
Humanized MJ2-7v.2-11 antibody levels in BAL were determined by ELISA, as
described above. BAL IL-6 levels were determined by cytometric bead array.
Eosinophil percentages were determined by differential cell counting.
As shown in FIGs. 30A-30B, IL-6 BAL cytokine levels were related to the degree
of inflammation. Furthermore, higher levels of humanized MJ2-7v.2-11 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 ug 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 / dose x 3 doses) used to elicit lung
responses. Interestingly, good neutralization of in vivo IL-13 bioactivity was seen only
when humanized MJ2-7v.2-11 was administered intranasally, and not when the antibody
was administered via intravenous or intraperitoneal. Distribution studies showed that
following intravenous and intraperitoneal dosing, high levels of antibody were recovered
in serum at the time of sacrifice, but very low levels were found in BAL. In contrast,
following intranasal dosing, comparable levels of antibody were found in serum and in
BAL. Thus, levels of humanized MJ2-7v.2-11 in BAL fluid were approximately 100-
fold higher following intranasal dosing than intravenous and intraperitoneal dosing. The
observation that intranasal dosing was efficacious but intravenous and intraperitoneal
dosing was not indicates that in this model, the site of antibody action was the lung. This
site of action is expected based on the intratracheal or intranasal delivery route of IL-13,
and was confirmed by the observation that antibody trapped IL-13 in the BAL fluid, but
very little antibody /IL-13 complex was seen in the serum.
In conclusion, these findings further support the IL-13 neutralization activity of
humanized MJ2-7v.2-11 in vivo.
Example 20: Pharmacokinetics, Pharmacodynamics, and Interspecies Scaling of
Humanized Anti-IL-13 Antibodies
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."
Antibody MJ2-7 (IgGl, k) was cloned from BALB/c mice immunized with the N-
terminal 19 amino acids of non-human primate IL-13, and the humanized version of this
antibody is designated herein as "humanized MJ2-7v.2-11" or "hMJ2-7v.2-11." Both
antibodies were formulated in 10 mM L-histidine, pH 6, containing 5% sucrose. Both
anti-IL-13 antibodies are cross reactive with monkey IL-13, and humanized 13.2v.2 is
cross reactive with sheep IL-13. However, humanized 13.2v.2 and humanized MJ2-7v.2-
11 antibodies do not cross react with rodent (e.g., mouse and rat) IL-13.
To support pre-clinical testing of anti-IL-13 antibodies, single dose
pharmacokinetic (PK) and pharmacodynamic (PD) studies were performed in mice, rats,
sheep, and cynomolgus monkeys after IV and SC administration. In addition, PK studies
were conducted using the Ascaris-challenged monkey model, described in Example 21a,
and an ^jcara-challenged sheep model, described below. PK parameters were
calculated using non-compartmental models and WinNonLin software (Model 201 and
200). Finally, PK animal profiles have been extrapolated using PK-PD modeling to
predict the disposition of anti-IL-13 in humans.
Single dose PK studies were performed in mice (e.g., male A/J for humanized
13.2v.2 and female BALB/c for humanized MJ2-7v.2-11), male Spraugue-Dawley rats,
naive male cynomolgus monkeys, and the Ascaris-chaMmged cynomolgus monkey
model, described in Example 21a. IV doses were administered, according to the most
recent scheduled body weights, as a single bolus injection into the tail vein, jugular vein
via catheter, or saphenous vein for mice, rats, and monkeys, respectively.
For the Ascora-challenged cynomolgus monkey model, animals selected
according to the protocol described in Example 21a, were treated with humanized MJ2-
7v.2 administered via a short (e.g., approximately 10 minutes) IV infusion as described
supra. 24 hours post IV infusion, animals were challenged with 0.75 u.g Ascaris suum
antigen reconstituted with PBS (Greer Diagnostics, Lenoir, NC) and administered by
aerosol delivery.
For the Aycam-challenged sheep model, female sheep, pre-screened for airway
hypersensitivity to Ascaris suum antigen, were treated with an IV bolus injection of
humanized 13.2v.2 (2 mg/kg) or IVIG (2 mg/kg). yiscara-challenge was then
administered 24 hours later using aerosol delivery.
Following the appropriate treatment, described above, blood samples were
collected at pre-determined time points into serum separator tubes and allowed to clot at
room temperature for 15 minutes, before processing by serum centrifiigation (e.g.,
approximately 11,000 rpm for 10 minutes). Pre-determined time points were; pre-test
and 5 minutes to 42 days in the humanized 13.2v.2 A/J mouse studies; 5 minutes to 21
days in the humanized MJ2-7v.2-11 BALB/c mouse studies, with 3-4 animals analyzed
per time point; pre-test and 5 minutes to 35 days in both humanized 13.2v.2 and
humanized MJ2-7v.2-11 rat studies; pre-test and 5 minutes to 42 days in the 1 mg/kg and
5 minutes to 55 days in the 100 mg/kg humanized 13.2v.2 and humanized MJ2-7v.2-11
naive cynomolgus monkey studies; 5 minutes to 42 days in the humanized 13.2v.2
Ascaris-challenged sheep studies; and 24 hours to 113 days in the^lscam-challenged
cynomolgus monkey studies.
The concentrations of anti-IL-13 antibodies in mouse, rat, and cynomolgus
monkey serum samples were determined using quantitative enzyme-linked
immunosorbant assays (ELISA). In this assay, an IL-13 ligand, which contains a FLAG
octapeptide fusion tag (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys), was captured onto a
microtiter plate by an anti-FLAG monoclonal antibody. After blocking and washing,
serum samples containing anti-IL-13 antibodies or anti-IL-13 standards were incubated
on the plate to allow for binding to the FLAG tagged IL-13. Bound anti-IL-13 antibodies
or anti-IL-13 standards were detected using a mouse anti-human IgG (Fc) antibody fused
to horse radish peroxidase (HRP). Finally, bound antibodies were quantified using the
HRP subtrate 2,2' azino-di (3-ethyl-benzthiazoline-6-sulfonate (ABTS) and optical
densities were measured at 405 nm.
The ELISA to quantify humanized 13.2v.2 in sheep was performed as follows.
Briefly, biotinylated humanized 13.2v.2 was pre-incubated with recombinant human IL-
13-FLAG in the presence of either unlabeled humanized 13.2v.2 standards or humanized
13.2v.2-containing sheep serum. This mixture was transferred to a pre-washed and
blocked anti-FLAG coated ELISA plate. Following a second incubation, the plate was
washed and biotinylated humanized 13.2v.2 was detected with peroxidase-linked
streptavidin. ELISA sample concentrations were determined by interpolation from a
calibration curve fit using a 4-parameter equation (Softmax Pro).
Mouse PK parameters were based on mean concentrations for 3-4 animals at each
time point, whereas rat and monkey PK parameters were determined for individual
animals, as follows. All data was generated using a non-compartmental analysis module
of the pharmacokinetic software package, WinNonlin (Pharsight). The area under the
serum concentration versus time curve (AUC) was calculated using the linear trapezoidal
model. The slope of the apparent terminal phase was estimated by log-linear regression
using at least 3 date points and the terminal rate constant (£) was derived from the slope.
AUC0-co was estimated as the sum of the AUQu (t = time of last measurable
concentration) and C1/S. The apparent terminal half-life (t1/2) was calculated as 0.693/E.
Human PK parameters were predicted for a subject with a body weight of 60 Kg
using an allometric scaling approach, as follows. PK parameters calculated from each
species were plotted on log-coordinates, and the allometric coefficient (a) and allometric
exponent (b) were estimated from the linear regression: log Y = log (a) + log (w) * b
(where log (a) = y intercept; b = slope of fit). PK parameters were then scaled using the
equation: Y = a • Wb (where; Y = PK parameter of interest; W = body weight of species;
a = allometric coefficient; b = allometric exponent), as shown in Table 7. PK data is
presented in Tables 5A-5C.
Table 5A. Interspecies Comparison of Mean (+SD)Pharmacokinetic Parameters for
Humanized 13.2v.2 and Humanized MJ2-7v.2-ll Following Single IV
Administration
Table 5B. Interspecies Comparison of Mean Pharmacokinetic Parameters for
Humanized 13.2v.2 and Humanized MJ2-7v.2-ll Following Single IV
Administration
Table 5C. Dose-Normalized Exposure of Humanized 13.2v.2 and Humanized MJ2-
7v.2-11 Following Single IV Administration
PK profiles were determined for humanized 13.2v.2 and humanized MJ2-7v.2-11
in mice, rats, sheep, and monkeys as described above. As shown in Table 5A-5B, in
general, PK parameters were comparable for all species analyzed. More specifically, PK
data clearly demonstrates the elimination of both anti-IL-13 antibodies was slow, with
serum clearances (CL) ranging from 0.13 mL/hr/kg in monkeys and sheep to 0.81
mL/hr/kg in mice. Steady state volume of distribution (Vdss) was also low for all species
(< 120 mL/kg), indicating that the anti-IL-13 antibodies were present mainly in the
vascular circulation. Interestingly, the apparent terminal half life (T1/2) was 3-6 days in
mice (a non-binding species) compared to 14-17 days in monkeys and sheep (IL-13
binding species). In monkeys, PK parameters were determined at both 1 mg/kg and 100
mg/kg dosage levels. PK parameters for humanized 13.2v.2 and humanized MJ2-7v.2-11
antibodies were approximately dose-proportional in the 1-100 mg/kg range, as CL, ti/2,
Vdss, and dose-normalized exposure (AUC/dose) were not significantly different between
the 1 and 100 mg/kg dosage levels. In general, PK parameters in naive and Ascaris-
challenged monkeys were not significantly different, suggesting that there is no apparent
target-mediated clearance of the anti-IL-13 antibodies at the therapeutic dose level.
However, the Vdss of humanized MJ2-7v.2-11 was lower in Ascaris-challenged monkeys,
particularly when compared to 1 mg/kg of humanized MJ2-7v.2-11 -treated nai've
monkeys, possibly due to IL-13 redistribution caused by vascular re-modeling.
Allometric scaling was applied to predict PK of humanized 13.2v.2 and
humanized MJ2-7v.2-11 antibodies in humans after IV administration. As shown in
Tables 5A-5B and FIG. 43, both anti-IL-13 antibodies were predicted to have a highly
favorable PK profile in humans with a low CL (e.g., approximately 0.07 - 0.1 mL/hr/kg),
a low Vdss (e.g., approximately 68 - 90 mL/kg), and a long t1/2 (e.g., approximately 27 -
29 days).
Dose-normalized exposure data (AUC0-ocVDose) obtained from the above
described IV studies were used to calculate bioavailability following 2 mg/kg
subcutaneous (SC) administration of humanized 13.2v.2 and humanized MJ2-7v.2-11
antibodies.
As shown in Table 6, the bioavailability of anti-IL-13 antibodies was 60 - 100%
in all species tested. The maximum serum concentration (Cmax) observed at 1-3 days post
dosing ranged from 7.25 µg/mL in mice to 22.6 µg/kg in monkeys for humanized
13.2v.2, and 24.2 µg/mL in mice to 22.5 µg/mL in monkeys for humanized MJ2-7v.2-11.
Absorption from the injection site of both anti-IL-13 antibodies was slow; however,
slightly faster for humanized MJ2-7v.2-11. Based on the high levels of SC
bioavailability in preclinical species, both anti-BL-13 antibodies were predicted to have >
50% bioavailability in humans.
As described above, human PK parameters were predicted for a subject with a
body weight of 60 kg using an allometric scaling approach. Briefly, PK parameters
presented in Table 5 for mice, rats, and monkeys, were regressed against body weights
(e.g., PK parameter = a • Weightb) to obtain R2. PK parameters for each species were then
plotted on log coordinates and the allometric coefficient (a) and the allometric exponent
(b) were estimated from the linear regression, as shown in Table 7.
Table 7. Allometric Scaling of Anti-IL-13 Antibody PK Parameters
Table 7 shows the allometric coefficients (a), allometric exponents (b), and R2
values obtained from regression of PK parameters against body weight and the CL, Un,
and Vdss for both anti-IL-13 antibodies.
Humanized 13.2v.2 and humanized MJ2-7v.2-11 antibody biodistribution assays
were performed in A/J mice and Sprague-Dawley rats, respectively, using radio labeled
anti-IL-13 antibodies. Briefly, humanized 13.2v.2 was labeled using the Iodo-gen
reagent (1,3,4,6-tetrachloro-3,6-diphenylgIycoluril, supplied by Pierce). A 20 µL aliquot
of Iodo-gen solution was combined with 1 mCi [125I] dissolved in 100 TL PBS and 10 µL
of humanized 13.2v.2 antibody and incubated for 15 minutes at room temperature. [125I]-
labeled humanized 13.2v.2 was purified using a NAP 5 column (Pharmacia, Uppsala,
Sweden). Similarly, humanized MJ2-7v.2 was iodinated using the IODO-BEADS
method (Pierce, Rockford, IL) in which 300 µg of humanized MJ2-7v.2-11 antibody was
incubated for 25 minutes with 3 mCi of [125I], IODO BEADS, and PBS. Unincorporated
[125I] was separated from the IODO BEADS by filtration (Centricon, 10 kD cut-off), and
the resulting [125I]-labeled humanized MJ2-7v.2-11 antibody was mixed with unlabeled
HMJ2-7V2-11. The specific activities of [125I]-labeled humanized 13.2v.2 and [125I]-
labeled HMJ2-7v.2-11 anti-IL-13 antibodies were 2.79 x 108 cpm/mg (unincorporated
iodine = 5%) and 2.56 x 107 cpm/mg (unincorporated iodine = 1.1%), respectively.
[125I]-labeled humanized 13.2v.2 was then administered IV at a dose of 1 mg/kg and
[l25I]-labeled humanized MJ2-7v.2-11 was administered at a dose of 2 mg/kg. Tissue
samples were subsequently collected at 1, 24, 168, and 336 hours for the [125I] labeled
humanized 13.2v.2 mouse study and at 1,48, 168, 336 and 840 hours for the [125I]-
labeled humanized MJ2-7v.2-11 rat study. Tissues including, for example, spleen, lung,
heart, liver, kidney, skeletal muscle, stomach, small intestine, large intestine, lymph node,
skin, and fat were collected immediately after blood sampling and whole body perfusion
with heparinized PBS at 25 U/mL.
Anti-IL-13 antibody levels, defined as radioactive equivalent concentrations, in
serum (µg eq./mL) and tissue (µg eq./g) were estimated by gamma-counting
trichloroacetic acid (TCA)- precipitable or total radioactivity, respectively, and the
following formulas: For serum; [average TCA precipitable cpm/EXP(0.693/60.2 x (ts -
tD))]/[specific activity x sample volume]: For tissue; [average TCA precipitable
cpm/EXP(0.693/60.2 x (ts - tD))]/[specific activity x sample weight], where ts is dates of
sample and to is dosing solution
measurement after correction for the half-life of [125I].
As shown in FIGs. 31A-31B, following IV administration of [125I] labeled
humanized 13.2v.2 and [125I]-labeled humanized MJ2-7v.2-11 antibodies, the highest
levels of both antibodies were detected in the serum, confirming that both anti-IL-13
antibodies are present predominantly in the vasculature. Other tissues with high levels of
both anti-IL-13 antibodies include highly perfused tissues, for example, lung, kidney,
liver, heart, and spleen. Of all the tissue compartments analyzed, humanized 13.2v.2 and
humanized MJ2-7v.2-11 antibody levels were highest at the 1 hour time point in the lung,
indicating that both anti-IL-13 antibodies are rapidly delivered to this tissue, which is also
the desired target organ for future therapeutic application. Finally, both humanized
13.2v.2 and humanized MJ2-7v.2-11 antibody levels declined over the duration of this
study, and only trace amounts were detected at the final time points.
Humanized 13.2v.2 and humanized MJ2-7v.2-11 antibody pharmacodynamics
(PD) were also analyzed using the ELISA described above. As shown in FIG. 32A-B,
total IL-13 levels transiently increased following IV administration of both humanized
13.2v.2 and humanized MJ2-7v.2-11 antibodies in naive and Ascaric-challenged
cynomolgus monkeys. Importantly, however, IL-13 in the serum of these animals had no
biological activity when tested in a cell-based potency assay (data not shown). IL-13 was
not detectable at all time points in sera from IVIG or saline-treated animals (data not
shown).
Further analysis of IL-13 levels following administration of anti-IL-13 antibodies
was conducted using allometric scaling, as described above. Briefly, as shown in FIGS.
38 and 39, concentration-time profiles were calculated for humanized MJ2-7v.2-11 and
humanized 13.2v.2, respectcively, in naive versus normal cynomolgus monkeys. This
data was combined with PK data presented in Table 5 and applied to the model depicted
in FIG. 40 and the equation described above. The resulting allometric scaling data for
humanized MJ2-7v.2-11 in naive cynomolgus monkeys is presented in FIG. 36 and Table
8. The resulting allometric data for humanized MJ2-7v.2-11 in Ascaric-challenged
monkeys is presented in FIG. 42 and Table 10.
Example 21a: Pharmacokinetic and Pharmacodynamic Modeling of a Humanized
Anti-IL-13 Antibody in Naive and Ascaric-Challenged Cynomolgus Monkeys
("Sequential Model")
This example discusses an integrated model that describes pharmacokinetics and
pharmacodynamics of an anti-IL-13 antibody in both naive animals and in the animal
pharmacology study. The model is used to characterize the kinetics of IL-13
neutralization by an anti-IL-13 antibody in both naTve and pharmacology-study settings.
The model exemplified herein with IL-13 can be extended to evaluate other drug-ligand
interaction, particularly where free cytokine levels are difficult to assay directly.
Cytokine neutralization by monoclonal antibodies or cytokine receptor/Fc fusion
proteins is being explored as a therapeutic approach for a variety of cytokine-mediated
disorders, including autoimmune diseases, such as rheumatoid arthritis (RA), asthma, and
systemic lupus erythematosus (SLE) (Ichinose et al., Curr Drug Targets Inflamm Allergy
2004;3(3):263-9; Economides et al., Nat Med 2003;9(1):47-52; Toussirot et al., Expert
Opin Pharmacother 2007;8(13):2089-107; and Anolik et al., Best Pract Res Clin
Rheumatol 2005;19(5):859-78). A common problem in the development of therapeutic
proteins is that cytokine neutralization cannot be directly monitored in the presence of a
drug, due to unavailability of an assay method of sufficient sensitivity to measure free
cytokine levels. Instead, total (free plus drug-bound) cytokine levels are often used as a
surrogate pharmacodynamic (PD) marker of drug activity. There are several examples of
anti-cytokine proteins acting as "cytokine traps", resulting in increased total circulating
cytokine levels following drug administration, presumably due to slower elimination of a
drug-bound circulating cytokine, compared to that of a free circulating cytokine
(Margolin et al., J Clin Oncol 2001;19(3):851-6; Charles et al., J Immunol
1999;163(3):1521-8; Ito et al., Gastroenterology 2004;126(4):989-96; discussion 947).
When free cytokine levels (in the presence and often in the absence of an anti-
cytokine protein) are difficult to assay directly, PK-PD modeling can be a useful tool for
delineating a relationship between the kinetics of ligand neutralization and the
concentration-time! profile of an anti-cytokine therapeutic, using total cytokine levels as a
PD marker. These models can be especially useful when data from both healthy and
disease subjects (animals or humans) subjects are available, so that the free cytokine
levels can be estimated before and after therapy in both settings. Establishing a
relationship between the kinetics of ligand neutralization and the concentration-time
profile of potential therapeutic, combined with efficacy data, can be useful for design of
an optimal dosing regimen in animal pharmacology or in clinical studies.
Neutralization of interleukin-13 (IL-13) is an attractive approach for therapeutic
intervention in asthma, as this Th2 cytokine plays an important role in asthma
pathogenesis in animal models of asthma (Andrews et al, J Biol Chem
2002;277(48):46073-8; Corry et al., Am J Respir Med 2002; 1 (3): 185-93; Wills-Karp et
al., Curr Allergy Asthma Rep 2004;4(2): 123-31; Grunig et al., Science
1998;282(5397):2261-3; Padilla et al., J Immunol 2005;174(12):8097-105; Taube et al., J
Immunol 2002;169(11):6482-9). In addition, there are consistent correlations between
polymorphism in the IL-13 gene and asthma susceptibility in humans (Vercelli, Curr
Opin Allergy Clin Immunol 2002;2(5):389-93). Neutralization of IL-13 with anti-IL-13
antibodies or with IL-13 receptor a2/Fc fusion protein (IL-13Ra2-Fc) prevents airway
hyperresponsiveness and other asthmatic changes in mice (Taube et al.; Grunig et al;
Kumar, Am J Respir Crit Care Med 2004; 170(10): 1043-8; Wills-Karp et al., Science
1998;282(5397):2258-61; Yang et al., J Pharmacol Exp Ther 2005;313(1):8-15), sheep
(Kasaian et al., Am J Respir Cell Mol Biol 2007;36(3):368-76), and cynomolgus monkeys
(Bree et al., J Allergy Clin Immunol 2007;119(5):1251-7).
IL-13 signals via a receptor complex consisting of IL-13 receptor al (IL13aRl)
and interleukin-4 receptor alpha (IL-4Ra) subunits (Andrews et al., J Biol Chem
2002;277(48):46073-8; Corry et &l,Am J Respir Med 2002; 1(3): 185-93). IL-13 first
undergoes a low affinity interaction with IL-13Ral, which recruits IL-4Ra to form an
active signaling complex with high affinity for IL-13, leading to phosphorylation of
STAT6 and downstream cellular activation events.
hMJ2-7v.2-11, discussed herein is a humanized anti-IL-13 antibody that blocks
binding of IL13Ral to human and non-human primate IL-13. hMJ2-7v.2-11 does not
substantially cross-react with either rodent or sheep IL-13; thus non-human primates
were used as pharmacological species. As discussed herein, hMJ2-7v.2-11 has been
shown to be efficacious (at 10 mg/kg IV dose) in the model of acute airway inflammation
induced by Ascaris challenge in cynomolgus monkeys. In this example, the PK and total
IL-13 (PD) data following hMJ2-7v.2-11 administration to naive and yi^cam-challenged
monkeys were used to establish an integrated PK-PD model and characterize the kinetics
of IL-13 neutralization.
The study design is summarized in Table 8. Single dose pharmacokinetic studies
in protein-free adult fed cynomolgus monkeys were conducted at Wyeth Research (Pearl
River, NY and Andover, MA for Study land Study 2, respectively), as previously
described. hMJ2-7v.2-11 was administered by IV injection into saphenous vein or by SC
route. The dose was based on the most recent scheduled body weights, prior to dosing.
Blood samples were collected into serum separator tubes at the designated time-points
(Table 8), allowed to clot at room temperature for approximately 15 minutes, and
processed for serum by centrifugation (approximately 11,000 rpm for 10 minutes).
Table 8. Study Design
Ascaris-challenge study protocol was described previously (Bree et al., J Allergy
Clin Immunol 2007; 119(5): 1251 -7). In brief, several months prior to the study untreated
monkeys were given an initial screening challenge with Ascaris suum antigen. Monkeys
that responded with at least a 2-fold increase in bronchoalveolar lavage (BAL) eosinophil
content 24 hours post-challenge were selected for the study. Animals were administered
either hMJ2-7v.2-11(10 mg/kg) or a negative control (IVIG, 10 mg/kg) by IV route and
were challenged with 0.75 µg Ascaris suum antigen (obtained from Greer Diagnostics,
Lenoir, NC and reconstituted with PBS) 24 hours post administration of hMJ2-7v.2-11 or
a negative control.
The concentrations of hMJ2-7v.2-11 in serum samples were determined using
quantitative enzyme-linked immunosorbent assays (ELISA). In this assay, the
recombinant human IL-13 ligand, which contains a FLAG ocatapeptide fusion tag (Asp-
Tyr-Lys-Asp-Asp-Asp-Asp-Lys) was captured onto a microtiter plate by an anti-FLAG
monoclonal antibody. After blocking and washing, the serum samples containing hMJ2-
7v.2-11 or the hMJ2-7v.2-11 standards were incubated on the plate to allow for binding
to the IL-13. Bound hMJ2-7v.2-11 were detected with a mouse anti-human IgG (Fc)
antibody conjugated to horseradish peroxidase (HRP). The enzyme substrate 2,2' azino-
di (3-ethyl-benzothiazoline-6-sulfonate (ABTS) was added and optical densities were
measured at 405 nm. The low limit of quantitation of the assay was approximately 10.5
ng/mL.
The concentrations of total IL-13 in serum samples obtained from hMJ2-7v.2-11-
treated monkeys were determined using quantitative ELISA. In this assay, an anti-IL-13
antibody (humanized 13.2 antibody, 13.2v.2, Wyeth Research) that was able to bind IL-
13 in the presence of hMJ2-7v.2-11 HMJ2-7v.2-11 was used as a capture. After blocking
and washing, the serum samples containing IL-13 from in vivo studies or the non-human
primate IL-13 standards were incubated on the plate to allow for binding to the anti-IL-13
capture antibody. Total IL-13 was detected with a biotinylated Jin2, an anti-IL-13
antibody that binds to an IL-13 epitope that is distinct from those of humanized 13.2 and
hMJ2-7v.2-11. Streptavidin conjugated to HRP and the enzyme substrate 3,3',5,5'-
tetramethylbenzidine (TMB) peroxidase were added and optical densities were measured
at 450 nm. The low limit of quantitation of the assay was approximately 0.15 ng/mL.
An integrated pharmacokinetic and pharmacodynamic model that described the
relationship between observed serum concentrations of hMJ2-7v.2-11 and total IL-13,
was developed using WinNonlin software V 5.1.1 (Pharsight, Mountain View, CA) (FIG.
33). The pharmacokinetics of hMJ2-7v.2-11 was evaluated with a two-compartmental
model including a central compartment (CAb, V) and a peripheral compartment (C2, Ab
V2). CLd,Ab represented the distribution clearance between these two compartments.
Clearance (CLAb) of hMJ2-7v.2-11 was assumed only through the central compartment.
The pharmacodynamics of hMJ2-7v.2-11 was characterized with the neutralization of
endogenous IL-13. Based on the bivalent feature of IgG, the model assumed that each
hMJ2-7v.2-11 molecule had two independent binding sites for IL-13 with identical
association (Kon) and disassociation (Koff) rate constants. Kon was a 2nd order rate constant
governing the formation of hMJ2-7v.2-11 /IL-13 (Ab-IL-13) complex and Koff was a 1st
order rate constant governing the disassociation of Ab-IL-13 complex. CLcomplex
represented the serum clearance of Ab-IL-13 complex. The homeostasis of IL-13 was
assumed to be regulated by IL-13 production (zero order, Ksyn) and degradation (CLIL-13).
Differential equations derived from the model scheme in FIG. 33 are as follows:
Since preliminary modeling indicated that hMJ2-7v.2-11, IL-13 and Ab-IL-13
complex had similar estimates of volume of distribution in a central compartment
(approximately 0.1 to -.3 L), a single volume variable (V) was used in the final modeling
for model parsimony. A 1st order absorption rate constant (Ka) was used to describe the
absorption process for a subcutaneous dose.
Except for estimate of bioavailability (F), PK/PD parameter estimates were
obtained by simultaneously fitting the model to both serum hMJ2-7v.2-11 HMJ2-7v.2-
1 land total LL-13 concentration-time profiles from either individual naive or Ascaris-
challenged monkeys. The integrated PK/PD model was fitted first to data from naive
monkeys with IV (n=3) and SC (n=3) doses. Bioavailability (F) of the anti-IL-13
antibody after the SC dose was estimated with non-compartmental analysis as shown in
Example 21b. One naive monkey (Monkey #5) in the SC arm of the study, had a sharp
decline in hMJ2-7v.2-11 HMJ2-7v.2-11 levels in the terminal phase (and a faster drop of
total IL-13 levels), compared to other naive monkeys in both the IV and SC arms (FIG.
34A), likely due to formation of antibodies against hMJ2-7v.2-11. Therefore Monkey #5
was excluded from the calculation of the mean model parameters in the naive-model
settings. It was assumed that Kon and Koff were not altered by Ascaris challenge.
Therefore, mean Kon and Koff estimates obtained from naive monkeys were used in the
model fitting for Ascaris-challenged monkeys. The onset of inflammation in Ascaris-
challenged monkeys was assumed to occur instantaneously after the challenge at 24 hours
post dose. Thus, naive condition was assumed for Ascaris-challenged monkeys in the pre-
challenge period (0-24 hr) by fitting the data with mean parameters obtained from naive
monkeys. All data were reported as mean ± SD (n= 5 for naive and n= 8 for Ascaris-
challenged monkeys). Statistical significance (p<0.05) was assessed with unpaired
Student t-test.
Simulations for concentrations of hMJ2-7v.2-11, total IL-13, and free (unbound)
IL-13 in naive or ,4scara-challenge settings after different dose regimens of hMJ2-7v.2-
11, were conducted with the corresponding mean parameters obtained from PK/PD
modeling. When Ascaris challenge was assumed at Day 1 (as used in the experiment
design of Study 2), simulations for the 0-24 hours period were performed with mean
parameter estimates from naive settings, while simulations for Day 1 onward were
performed with mean parameter estimates from the Ascaris-challenge settings. When
Ascaris challenge was assumed at Day 0 (for a hypothetic "established inflammation"
situation), simulations for all time-points were performed with mean parameter estimates
from the /iscaris-challenge settings.
Mean concentration-time profiles of hMJ2-7v.2-11 (1 mg/kg, IV and 2 mg/kg SC,
Study 1) in naive cynomolgus monkeys were reported in Example 21b. Individual
concentration-time profiles of hMJ2-7v.2-11 in Study 1 are shown in FIG. 34A. A sharp
decline of hMJ2-7v.2-11 serum levels after approximately 14 days post-dose was
observed in one animal (Monkey #5) in the SC arm of the study, relative to other five
animals (three in Ihe IV arm and two in the SC arm) in Study 1. Mean concentration-time
profiles of hMJ2-7v.2-11 in Ascaris-challenged monkeys (10 mg/kg, IV, with Ascaris
challenge 24 hours post-dose, Study 2) together with those in naive monkeys are
summarized in FIG. 34B.
Quantitative ELISA were developed to measure total IL-13 levels in the absence
or presence of hM J2-7v.2-11. Serum IL-13 levels were undetectable by these assays in
pre-dose samples or in all samples from control animals treated with IVIG (data not
shown). After hMJ2-7v.2-11 administration, total IL-13 levels were transiently increased
in both Study 1 (naive monkeys; 1 mg/kg IV or 2 mg/kg SC) and in Study 2 (10 mg/kg
IV, with Ascaris challenge 24 hours post-dose) (FIG. 34C). There was high inter-animal
variability in the concentration-time profiles of total IL-13. However, Monkey #5 in the
SC arm of Study 1 had an apparent sharp decline in the total IL-13 levels, compared to
other five naive monkeys on Study 1 that were treated with hMJ2-7v.2-11, likely due to
formation of anti- hMJ2-7v.2-11 antibodies in this animal. The onset of decline in total
IL-13 in Monkey #5 coincided with that in hMJ2-7v.2-11 levels in this monkey (data not
shown).
Results of the previously reported cell-based assay performed with sera from
^5,cara-challenge(i animals indicated that samples with detectable levels of total IL-13
had no IL-13 -mediated biological activity (Kasaian et al., submitted), suggesting that the
transient increase In total IL-13 levels in naive and ^^cam-challenged monkeys was due
to the increase in hMJ2-7v.2-11-bound IL-13. However, the concentration-time profile
of free, (biologically active) IL-13 following hMJ2-7v.2-11 administration to naive or
Ascaris-challenged animals remained to be characterized.
An integrated drug-ligand binding PK-PD model depicted in FIG. 33 was
developed to describe the relationship between the observed total serum concentrations of
IL-13 and hMJ2-7v.2-ll in naive andAycaro-challenged monkeys. In this model, the
pharmacokinetics of hMJ2-7v.2-11 was described with a two-compartmental model and
the pharmacodynamics of hMJ2-7v.2-11 was characterized with the neutralization of
endogenous IL-13. Based on the bivalent feature of IgG, the models were developed
under the assumption that hMJ2-7v.2-11 can bind either one or two IL-13 molecules, in a
sequential manner. The homeostasis of IL-13 was assumed to be regulated by the zero-
order synthesis (Ksyn) and degradation (CLil-13) of IL-13.
For PK-PD modeling, raw concentration data (measured in ng/mL or |ig/mL) was
converted to nM units, using molecular weights of 150 kDa and 10 kDa for hMJ2-7v.2-
11 and IL-13 respectively.
Table 9. Summary of hMJ2-7v.2-ll Pharmacokinetic and Pharmacodynamic
Parameters from Individual Fittings of Data for Naive and Ascaris-Challenged
Cynomolgus Monkeys
a. For estimation of mean parameters in naive animals, three animals in the 1 mg/kg, IV group and 2
animals in the 2 mg/kg, SC group were used. One animal in the SC group was excluded from calculations
of mean parameters due to a sharp decline in hMJ2-7v.2-11 levels (and total IL-13 levels) in the terminal
phase, compared to other naive monkeys in the study.
Stars (* or ***) indicate that a mean parameter in the Ascaris-challenged animals was significantly
different (p=0.05 or =0.001, respectively) from a corresponding value in naive monkeys, based on unpaired
Student t test.
In general, this model adequately characterized the animal data (FIG. 35A and
Table 9). The residuals were evenly distributed, without noticeable systematic bias (FIG.
35B). The representative fits for naive (Study 1) and Ascaris-challenged (Study 2)
monkeys are shown in FIG. 35C and 35D, respectively. However, the sharp decline of
both hMJ2-7v.2-11 and total IL-13 serum levels in Monkey #5 from the SC arm of Study
1 could not be described by this integrated PK/PD model. Therefore, the PK parameters
from Monkey #5 were excluded from the calculation of the mean model parameters in the
naive-animal settings.
PK and PE) parameters generated from the model fitting for both naive and
/4scam-challenged monkeys are summarized in Table 9. The clearance of unbound
hMJ2-7v.2-11 (CLAb) from the central compartment was low (approximately 0.013-0.015
L/day) and was similar between the naive and ylscam-challenged monkeys. In naive
animals, the clearance of hMJ2-7v.2-11/IL-13 complex from the central compartment
(CLcomplex) was approximately 5-6 fold lower, compared to CLAb- In Ascaris-challenged
animals, CLcomplex was similar to CLAb- Thus, CLcomplex was approximately 5-fold higher
in Ascaris-challenged animals, when compared to that in naive monkeys. The volume of
hMJ2-7v.2-11 in the central compartment (V) was found to be similar to the average
plasma volume in cynomolgus monkeys for both naive and ^scam-challenged animals.
However, V and the distribution clearance of hMJ2-7v.2-11 (CLd,Ab) were significantly
lower in the Ascaris-challenged monkeys, when compared to that in naive monkeys. This
result is in accord with the lower estimate for the volume of distribution in Ascaris-
challenged monkeys obtained with earlier non-compartment analysis (Vugmeyster et al.,
submitted).
The neutralization of IL-13 was governed by Kon and Koff, the rate constants of
the coupling/uncoupling of hMJ2-7v.2-11 and free IL-13. The mean Kon and Koff
estimates were 0.0896 nM-1day-1 and 0.1630 day-1, respectively. Baseline IL-13 levels
were defined by the ratio of endogenous IL-13 synthesis rate (Ksyn) and the clearance of
IL-13 from the central compartment (CLil-13) (Benincosa et al., J Pharmacol Exp Ther
2000;292(2):810-6; Ng et al., Pharm Res 2006;23(1):95-103; Mager et al., J
Pharmacokinet Pharmacodyn 2001;28(6):507-32). The estimated baseline IL-13 level
was approximately 0.0115 nM in naive monkeys and it was approximately 3-fold higher
(approximately 0.0346 nM) in Ascaris-challenged monkeys (p<0.001).
Model simulation with mean parameter estimates of the integrated PK-PD model
were used to predict the levels of free and hMJ2-7v.2-11-bound IL-13 post hMJ2-7v.2-11
administration. These simulations predicted that the transient increase in total IL-13
levels in both Study 1 (naive) and Study 2 (Ascaris-challenged at Day 1) was due to the
increase in hMJ2-7v.2-11-bound IL-13, while free IL-13 was decreased after the IV
administration of hMJ2-7v.2-11 (FIGs 36A and 36B). The decrease in free IL-13
appeared more dramatic in Ascaris-challenged monkeys, because of the higher hMJ2-
7v.2-11 dose (10 img/kg) used, relative to naive monkeys (1 mg/kg). In the Ascaris-
challenge monkeys (Study 2), free IL-13 levels were predicted to remain at or below the
estimate of free IL-13 levels in naive monkeys (i.e. below 0.0115 nM) for approximately
35 days post 10 mg/kg single IV administration of hMJ2-7v.2-11. Free IL-13 levels in
^scaris-challenged monkeys were predicted to rise above the naive baseline average
when hMJ2-7v.2-11 concentration was approximately 160 nM. Along with the
elimination of hMJ2-7v.2-11, free IL-13 levels in naive and ylscara-challenged monkeys
gradually rose to the corresponding baseline levels (defined by Ksyn/CLIL-13)-
The kinetics of IL-13 neutralization was also simulated with the different IV
doses of hMJ2-7v.2-11 (1-50 mg/kg) in monkeys with a hypothetic established airway
inflammation, i.e. assuming Ascaris challenge at Day 0. Predicted free IL-13 levels in
naive monkeys and in monkeys with established airway inflammation after a single IV
administration of hMJ2-7v.2-1 ] are shown in FIGs. 37A and 37B. In both naive monkeys
and in monkeys with established airway inflammation, the time at which free IL-13 levels
were below baseline IL-13 levels increased with hMJ2-7v.2-11 dosage used for the
simulations. However, the extent and duration of IL-13 neutralization by hMJ2-7v.2-11
appeared to differ between the naive monkeys and the monkeys with established airway
inflammation. For example, after 10-mg/kg IV dosage of hMJ2-7v.2-11 to naive
monkeys, most of IL-13 appeared to be hMJ2-7v.2-11-bound as late as Day 40 post-dose,
with free IL-13 levels of O.001 nM (or <7% of baseline). In contrast, after 10-mg/kg IV
dosage of hMJ2-7v.2-11 to monkeys with established airway inflammation, there was an
initial drop in free IL-13 to nearly-zero levels followed by a steady rise to approximately
0.008 nM or 21% of baseline at Day 40.
In this example, an integrated antibody-ligand binding PK-PD model was
developed that described the relationship between the total serum concentrations of IL-13
and hMJ2-7v.2-11, an anti-IL-13 humanized IgG1 antibody, in naive cynomolgus
monkeys and in the disease model of acute airway inflammation induced by Ascaris
challenge to cynomolgus monkeys. Due to lack of a bioanalytical method of sufficient
sensitivity, free IL-13 levels could not be directly measured in either the presence or the
absence of hMJ2-7v.2-11. Therefore, total IL-13 levels were used as a PD marker, as
total IL-13 levels were transiently increased in both naive and ^^cam-challenged
monkeys. The model presented in this report was developed under the assumption that
hMJ2-7v.2-11 can bind either one or two IL-13 molecules, in a sequential manner. This
assumption is based on the physiological mechanism of anti-IL-13/IL-13 interaction and
is different from those used in the previously published integrated antibody-ligand
binding PK-PD models for therapeutic antibodies, in which either 1:1 or 1:2
stoichiometry was assumed (Benincosa et al, J Pharmacol Exp Ther 2000;292(2):810-6;
Mager et al., J Pharmacokinet Pharmacodyn 2001 ;28(6):507-32; Ng et al., Pharm Res
2006;23(1):95-103; Hayashi et al., Br J Clin Pharmacol 2007;63(5):548-61; Chow et al.,
Clin Pharmacol Ther 2002;71(4):235-45).
The novel PK-PD model presented in this example described the data in both
naive and Ascaris -challenge settings reasonable well and this model was used for analysis
of the kinetics of neutralization of IL-13 by hMJ2-7v.2-11.
The hMJ2-7v.2-11 PK parameters estimated by the integrated PK/PD modeling
were consistent with those estimated by non-compartmental analysis in Example 21b.
hMJ2-7v.2-11 had a low clearance and a small volume of distribution in monkeys, typical
of those seen for other humanized IgGl therapeutic proteins (Adams et al., Cancer
Immunol Immunother 2006;55(6):717-27; Lin et al., J Pharmacol Exp Ther
1999;288(1):371-S; Zia-Amirhosseini et al., J Pharmacol Exp Ther 1999;291(3): 1060-7).
The integrated PK/PD modeling further confirmed that hMJ2-7v.2-11 volume of
distribution was smaller in Ascaris-challenged monkeys, when compared to that in naive
monkeys, in line with the results of non-compartmental analysis. Volume of distribution
of hMJ2-7v.2-11 in the central (V) and, to some degree, the peripheral (V2)
compartments, as well as the distribution clearance (CLd,Ab) of hMJ2-7v.2-11 between
these two compartments were decreased in v4scam-challenged monkeys when compared
to those in naive monkeys. The difference of hMJ2-7v.2-11 volume of distribution
between naive and Ascaris-challenged monkeys was unlikely due to the difference in
hMJ2-7v.2-11 dosage used (1 or 2 mg/kg in naive monkey and 10 mg/kg in Ascaris-
challenged monkeys), since the steady-state volume of distribution (Vdss) of hMJ2-7v.2-
11 was similar among naive monkeys over a wide dose range (l-100mg/kg) (Example
21b).
For both naive and Ascaris-challenged monkeys, the model also demonstrated
that the transient increase in total IL-13 levels in Ascaris-challenged and naive animals
was due to the increase in hMJ2-7v.2-11-bound IL-13, while free IL-13 was decreased.
The neutralization of IL-13, leading to decrease in free IL-13 levels, is the intended
biological effect of hMJ2-7v.2-11 and is consistent the observed efficacy of hMJ2-7v.2-
11 in reducing airway inflammation in the yiscam-challenged animals (Study 2), as well
as with the lack of IL-13 -mediated biological activity in the sera obtained from these
animals.
Results of the PK-PD modeling and simulations indicated a number of differences
in IL-13 neutralization between the naive and Ascaris-challenge settings. In the Ascaris-
challenged animals, baseline IL-13 levels were estimated be approximately 3-fold higher,
when compared to those in naive monkeys. This estimation was consistent with the
notion that acute airway inflammation induced by Ascaris challenge in cynomolgus
monkeys was mediated by IL-13. In human subjects, including normal human volunteers
and subjects with a variety of disorders, there is a wide range of reported baseline IL-13
levels (from < 10 pg/mL to > 150 pg/mL), in part dependent on assay methodology
employed for the measurements (Fiumara et al., Blood 2001 ;98(9):2877-8; Wang et al., J
Clin Virol 2006;37(1):47-52). In general, baseline IL-13 levels in estimated for naive
monkeys (approximately 100 pg/mL or approximately 0.01 nM) appeared to be higher,
compared to those reported for healthy humans.
In Ascaris-challenged animals (Study 2), free circulating IL-13 levels were
maintained below the average free IL-13 levels in naive monkeys for approximately one
month after a 10 mg/kg IV administration of hMJ2-7v.2-11. Modeling indicated that for
a given dose level of hMJ2-7v.2-11, extent and duration of hMJ2-7v.2-11 -mediated IL-13
neutralization in the naive- and Ascaris-challenged monkeys were different. Thus,
caution should be used when applying PK-PD data from normal human volunteers to the
design of clinical studies in subjects with airway inflammation.
It should be noted that the levels of free IL-13 in the target tissue (lung) may be a
more direct indicator of effectiveness of IL-13 neutralization by a therapeutic protein.
However, the level at which tissue (and circulating) IL-13 needs to be maintained to
suppress ^.ream-induced airway inflammation in monkeys (and in asthmatic patients), as
well as the required duration of the neutralization is not known. Total IL-13 levels were
below the limit of detection in BAL (bronchoalveolar lavage) fluid of animals in Study 2
(data not shown), so that it was not possible to obtain a PD readout in the tissue
compartment.
In summary, a novel PK-PD model was developed that described the relationship
between the total serum concentrations of IL-13 and hMJ2-7v.2-11, in naive and Ascaris-
challenged monkeys. The model prediction on IL-13 neutralization were the following:
(1) The estimated circulating IL-13 levels were increased approximately 3-fold after the
Ascaris-challenge, consistent with the notion that Ascaris-induced acute airway
inflammation was IL-13-mediated; (2) the transient increase in total IL-13 levels
observed in both naive and .4.scara-challenged monkeys, was due to the increase in
hMJ2-7V.2-l 1-bound IL-13, while free IL-13 was decreased after IV administration of
hMJ2-7V.2-l 1; and (3) when identical hMJ2-7v.2-11 dose regimens were used for
simulations, the extent and duration of IL-13 neutralization in the circulation were
different in naive and airway inflammation settings. However, this prediction needs to be
interpreted with caution, as the model does not describe neutralization of IL-13 in the
lung, the target organ. The PK-PD model presented in this Example can be applied to
study drug-ligand interactions for other therapeutics proteins, in cases when free ligand
(such as a cytokine or growth factor) cannot be readily assayed directly but total ligand
levels change with drug administration. The differences in the ligand neutralization by a
therapeutic protein between the healthy and pharmacology-model settings described in
this report, illustrates the importance of conducting preclinical PK-PD studies in both
settings, if practically feasible.
Example 21b. Pharmacokinetic and Pharmacodynamic Modeling of a Humanized
Anti-IL-13 Antibody in Naive and Ascaris-Challenged Cvnomolgus Monkeys
(''Stoichiometric Model")
Prior to conducting PK-PD modeling using the "sequential" integrated PK-PD
model described in Example 21a, hMJ2-7v.2-11 PK-PD profile after 1 mg/kg IV
administration of hMJ2-7v.2-11 to unchallenged monkeys (Table 8, Study 1), was
analyzed using a "stoichiometrc" PK-PD model. The hMJ2-7v.2-11 PK concentration
and total IL-13 concentration data-sets used for modeling was from Study 1, described in
Table 8 and obtained using bioanalytical methods described in Example 21a.
The "stoichiometric" PK-PD model assumes two-to-one stoichiometry for the IL-
13- hMJ2-7v.2-11 complex, i.e., one antibody molecule is bound to the two IL-13
molecules bound. The stoichiometric model is similar to previously published models in
which either 1:1 or 2:1 stoichiometry was assumed. (Benincosa et al., J Pharmacol Exp
Ther 2000;292(2)::810-6; Mager et al., J Pharmacokinet Pharmacodyn 2001 ;28(6):507-
32; Ng et al., Pharm Res 2006;23(1):95-103; Hayashi et al., Br J Clin Pharmacol
2007;63(5):548-6I; Chow et al., Clin Pharmacol Ther 2002;71(4):235-45).)
Specifically, an integrated "stoichiometric" pharmacokinetic and
pharmacodynamic model that described the relationship between observed serum
concentrations of hMJ2-7v.2-11 and total IL-13, was developed using WinNonlin
software V 5.1.1 (Pharsight, Mountain View, CA) (FIG. 41). The pharmacokinetics of
hMJ2-7v.2-11 was evaluated with a two-compartmental model including a central
compartment (CAb., V) and a peripheral compartment (C2, Ab, V2). CLd,Ab represented the
distribution clearance between these two compartments. Clearance (CLAb) of hMJ2-7v.2-
11 was assumed only through the central compartment. The pharmacodynamics of
hMJ2-7v.2-11 was characterized with the neutralization of endogenous IL-13. Based on
the bivalent femnature of IgG, the model assumed that each hMJ2-7v.2-11 molecule
binds two IL-13 molecules simulatenousely with association (K^) and disassociation
(Koff) rate constants. Kon was a 3d order rate constant governing the formation of hMJ2-
7v.2-11 /(IL-13)2 (Ab-IL-13) complex and Koff was a 1st order rate constant governing the
disassociation of Ab-IL-13 complex. CLcomplex represented the serum clearance of Ab-IL-
13 complex. The homeostasis of IL-13 was assumed to be regulated by IL-13 production
(zero order, Ksyn) and degradation (CLil-13). The following assumptions were also used
(similar to that in Example 21a): Vanti-IL-13=Vcomplex=VIL-13=V for model parsimony. The
integrated PK/PD model was fitted to individual PK-PD data from 3 naive animals. The
representative fit is shown in FIG. 32A.
The PK-PD parameters of hMJ2-7v.2-11 after 1 mg/kg IV administration to naive
(unchallenged) cynomolgus monkeys, as derived from the "stoichiometric" PK-PD model
are shown in Table 10.
Table 10. Mean Parameter Estimates from a Stoichiometric PK-PD Model of
Humanized MJ2-7v.2-ll and IL13 Disposition in Unchallenged Cynomolgus
Monkeys
The mean model parameters described in Table 10 were used to simulate levels of
free IL-13 and anti-IL-13-bound IL-13 using the WinNonlin software V 5.1.1
(Pharsight, Mountain View, CA).
In general, the results of the simulations of free and ant-IL-13 bound IL-13, after a
single 1 mg/kg IV dosage to naive monkeys, were similar for the "stoichiometric" (this
example) and "sequential" models (Example 21a). As shown in FIG. 41, the
"stoichiometric" model predicted a transient increase in total IL-13 following IV
administration of 1 mg/kg of humanized MJ2-7v.2-11 to naive monkeys. Following
administration of anti-IL-13 antibody, the majority of IL-13 is in complex with
humanized MJ2-7v.2-11 antibody. Thus, the results of stoichiometric model are
consistent with those of sequential model (Example 21a) and suggest that the transient
increase observed for total IL-13 due to increased levels of IL-13/anti-IL-13 antibody
complex, while free IL-13 levels are decreased.
The stoichiometric model was also used to fit PK-PD from Ascaris-challenged
monkeys (Study 2: in Table 8), obtain a set of PK-PD parameters and then simulate free,
anti-IL- 13-bound, and total IL-13 levels after 1 mg/kg IV dosage to Ascaris-challenged
monkeys. The results of these simulations are shown in FIG. 41. Similar to simulations
results for naive monkeys, the 'stoichiometirc" PK-PD model predicted that the transient
increase observed for total IL-13 due to increased levels of IL-13/anti-IL-13 antibody
complex, while free IL-13 levels are decreased (FIG. 42).
Example 22: Humanized 13.2v2 Antibody Effective in Allergen Challenge Study
in Human Subjects
Study Design: Subjects with mild allergic asthma and dual airway responses to
allergen challenge (AC) were randomized to receive two subcutaneous 2 mg/kg doses of
a humanized anti-IL-13 antibody, 13.2v2, (n=14) or placebo (n=13) one week apart, in a
multi-centre, double-blind, placebo controlled parallel-group study. AC was performed 2
weeks (Day 14) and 5 weeks (Day 35) after the first dose. Allergen-induced early (EAR)
and late (LAR) asthmatic responses and airway hyperresponsiveness to methacholine
were measured at each AC. Safety, tolerability and pharmacokinetics (PK) were
evaluated throughout the study.
Results and Discussion: Humanized anti-IL-13 antibody, 13.2v2, was well
tolerated, and was not associated with any serious adverse events, changes in blood
hematology, chemistry, or vital signs. The frequency of adverse events was similar in the
antibody 13.2v2 and placebo groups.
Human subjects with mild atopic asthma were selected for the study. Fourteen of
the subjects were selected to receive anti-IL-13 antibody, and 13 subjects to receive
placebo. The percent change in FEV1 for each subject was measured over 7 hours at
various time points after allergen challenge. FEV1 (Forced Expiratory Volume in the
first second) is the volume of air that can be forced out in one second after taking a deep
breath, an important measure of lung function. A negative change in FEV1 indicates a
decrease of lung function.
The subjects were challenged with allergen (Ag) on the screening visit (two
weeks before the first administration of antibody). The allergen challenge was
administered and the percent change in FEV1 was measured for each subject over 7 hours
at various time points after allergen challenge. The results are shown in FIG. 44 as the
mean (including standard error (STERR)) in FEV1 over time. Both groups of subjects
responded similarly to the allergen challenge during the screening period.
Two weeks later, the subjects were administered 2 mg/kg of antibody (or a
placebo control) subcutaneously. One week later, the subjects received another dose of
2 mg/kg of antibody (or a placebo control) subcutaneously.
Peak plasma concentrations were reached on ~Day 14 of the study (two weeks
after the initial dose of antibody was administered).
On Day 14, an allergen challenge was administered and the percent change in
FEV1 was measured for each subject over 7 hours at various time points after allergen
challenge. The results of the study are shown in FIG. 45 as the mean (including standard
error (STERR)) in FEV1 over time. As indicated in the figure, subjects that received the
13.2v2 antibody had less of a percent change in FEV1 at all time points tested as
compared to the placebo-treated control subjects. The differences in percent change in
FEV1 were statistically significant for the early asthmatic response (EAR; 0-3 hours after
challenge, p= 0.042) and nearly reached significance for the late asthmatic response
(LAR; 3-7 hours after challenge) time points (p= 0.095). Also on Day 14, area under the
curve (AUC) measurements were taken, and the area of the EAR and LAR were both
significantly inhibited by the 13.2v2 antibody compared to placebo (EAR AUC0-3h: 46.3
% inhibition versus placebo, p=0.030; LAR AUC3-7h: 49.0 % inhibition versus placebo,
p=0.039).
The percent change in FEV1 was also measured over 7 hours at various time
points after allergen challenge on Day 35 (relative to the day of the first administration of
antibody). The results of the study are shown in FIG. 46 as the mean (including standard
error (STERR)) in FEV1 over time. As indicated in the figure, subjects that received the
antibody had less of a percent change in FEV1 at all time points tested as compared to the
placebo-treated control subjects. The differences in percent change in FEV1 were seen at
both the early asthmatic response (EAR; 0-3 hours after challenge) and late asthmatic
response (LAR; 3-7 hours after challenge) time points, and continued the trend seen on
Day 14. Also on Day 35, area under the curve (AUC) measurements were taken. There
was a similar trend for inhibition of the area of the EAR and LAR at week 5 (Day 35),
however this did not reach statistical significance (p=0.13 for both).
The serum concentration (ng/mL) of the 13.2v2 antibody on Day 14 and Day 35
are shown in FIG. 47.
The results of repeated measures and statistical analysis for late phase (LAR) and
early phase (EAR) maximum percent drop in FEV1 and AUC percent drop at Day 14 and
Day 35 of this study are shown in FIG. 48. The differences (Diff) are shown as the value
measured for the 13.2v2 antibody (AB) group minus the value measured for the placebo
(PBO) group (AB-PBO). P values (P-Val) are also provided. Statistical significance is
indicated by an asterisk (*). The statistical 95% confidence interval (CI) is also provided.
The ability of the antibody to affect allergen-induced hyperresponsiveness to
methacholine was also measured at Days 14 and 35. No effect was seen on this
parameter on either day.
Conclusions: Allergen-induced EAR and LAR at Day 14 were significantly
inhibited by antibody 13.2v2, which also corresponded with peak plasma PK levels.
These data demonstrate that IL-13 has a significant role in the early and late allergen-
induced bronchoconstriction in humans.
Example 23: PK Profiles for Antibody 13.2v2 in Human Subjects
The PK profiles of 13.2v2 in human subjects were determined. Serum antibody
concentration (ng/ml) was measured over a time course (days). The antibody was
administered subcutaneously as a single ascending dose (SAD) of 4 mg/kg, or as two 2
mg/kg doses that were administered a week apart for the allergen challenge (AC) study.
The results are shown in FIG. 49.
The half life of the antibody is approximately 23-29 days.
Example 23: Antibody 13.2v2 Pharmacokinetics and Product Metabolism in
Humans
Pharmacokinetic data were obtained for non-Asian patients with mild asthma in
SAD study A; and for healthy Japanese and non-Asian volunteers in SAD study B.
Except for an additional IV cohort of 3 mg/kg dose in study A, both SAD studies were of
similar design with 4 SC cohorts of 13.2v2 doses of 0.3, 1, 2, and 4 mg/kg. The mean
(SD) serum concentration-time profiles of 13.2v2 in mild asthmatic non-Asian patients in
study A and non-Asian volunteers in study B were determined. The pharmacokinetic
profiles of 13.2v2 were consistent and parallel from 0.3 mg/kg to 4 mg/kg in both studies.
Non-compartment analysis of serum 13.2v2 data in Japanese and foreign subjects:
The serum 13.2v2 concentration time data in both study A and study B were analyzed
using model independent noncompartment methods. The summary statistics on
noncompartmental pharmacokinetic parameters of 13.2v2 are presented in Table 11 for
study A and Table 12 for study B.
Since body weight normalized 13.2v2 dosing was employed for both studies,
subject with larger body weight received a larger dose of 13.2v2. The effect of body
weight on 13.2v2 exposures was graphically assessed in FIGS. 50 and 51.
In FIG. 50, AUC exposure normalized by respective mg/kg dose in all 81 subjects
in both studies appeared to be positively correlated to body weight, suggesting the
difference in exposure is related to body weight difference.
In FIG. 51, exposure normalized by actual doses appeared to be consistent across
all doses in all 81 subjects, suggesting that body weight is not a significant factor
affecting 13.2v2 exposure. Furthermore, when exposure normalized by actual doses were
compared in mild asthmatic US subjects and healthy Japanese and US subjects in FIG.
52, the 13.2v2 AUC per unit of 13.2v2 dose were independent of mg/kg dose and
consistent between study A and B. This suggests that 13.2v2 exposure increases
approximately with the dose increment, and neither ethnicity nor presence of mild asthma
remarkably affects 13.2v2 exposure. In addition, the 13.2v2 AUC per unit of 13.2v2 SC
dose is close to the 13.2v2 AUC per unit of IV dose suggest that close to complete
systemic absorption of 13.2v2 following SC administration.
Population pharmacokinetic analyses of 13.2v2 exposure data in Japanese and foreign
subjects:
In addition to the non-compartmental analysis, serum 13.2v2 concentration data in
both study A and B were combined and analyzed using population pharmacokinetic
methodology based on nonlinear mixed effect pharmacostatistical model implemented in
NONMEM software package. While the PK exposure and parameters derived from
distinct dose levels by non-compartmental analysis are based on a small number of
subjects (5-8), the point estimate of the mean and variability is expected to vary from
dose to dose and prone to chance findings. In comparison, the population analyses took
advantage of the mixed effect model methodology, provides a systemic framework to
examine 13.2v2 exposure and potential important covariate across all dose in both
Japanese and Non-Asian populations. The population method is more sensitive than non-
compartment method to detect significant covariate.
The population PK analyses employed NONMEM PREDD library routine
ADVAN3 with TRANS3 in NONMEM version VI. The first order conditional
estimation method with ?-e was used throughout the model building and covariate
analysis process. The analysis identified an optimal base population PK model consisting
of a two-compartmental structure PK model component and combined proportional and
additive error model components. Covariate analyses were performed based on the base
population PK model. Body weight, body surface area, ethnicity and presence of mild
asthma/health status were evaluated as potential covariates, and none of these factors was
found to affect 13.2v2 serum exposure in a statistically significant manner. The base and
optimal population PK model parameters are listed in Table 13.
Note: The population PK model was developed based on 13.2v2 exposure data
from both study A and study B.
The final model adequately describes the serum 13.2v2 observations in both
studies, as measured by Postier predictive checks of the base and optimal population PK
model of 13.2v2. Furthermore, the PK parameters derived from the population analysis
are consistent with those derived from the non-compartmental analyses.
Based on the optimal population PK model, a series of simulations were
performed based on the optimal population PK model to compare 13.2v2 exposure and
associated variability of 3 mg/kg dosing versus flat dosing of 225 mg (3 mg/kg in a 75 kg
subject) in typical subjects with body weight of 50 kg, 75 kg and 130 kg, respectively.
The 90% confidence interval of expected 13.2v2 exposure in these typical subjects was
determined. Flat dosing produced consistent 13.2v2 exposure in these subjects of
different body weight, while mg/kg dose resulted in higher 13.2v2 exposure in subjects
with larger body weights, lower 13.2v2 exposure in subjects with lower body weights.
When these subjects were pulled together, as expected in any clinical study enrolling
subjects of various body weights, the mg/kg dosing resulted in larger variability than flat
dosing.
Summary of pharmacokinetic findings in study A and study B:
• 13.2v2 exposure increases with dose increment from 0.3 mg to 4 mg/kg in both
asthmatic US subjects and healthy Japanese and US subjects;
• Ethnicity close not affect 13.2v2 pharmacokinetics, 13.2v2 exposure in Japanese
subjects was similar to that in non-Asian subjects receiving identical doses;
• Body weight does not affect 13.2v2 pharmacokinetics, as a result, flat dosing is
better than mg/kg dosing and results in less exposure variability;
• Being healthy or having mild asthma does not affect 13.2v2 pharmacokinetics, the
13.2v2 exposure in healthy Japanese and non-Asian are similar to that in asthmatic US
patients.
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 evaluating an anti-IL13 antibody molecule, comprising:
providing a mean test value for at least one pharmacokinetic/pharmacodynamic
(PK/PD) parameter of the anti-IL13 antibody molecule in a subject; and
comparing the mean test value provided with at least one mean reference value,
thereby evaluating the anti-IL13 antibody molecule,
wherein the mean reference value is selected from the group consisting of:
a mean CL value in the range of about 0.05 to 0.9 mL/hr/kg after intravenous
administration of the anti-IL13 antibody molecule to the subject; a mean Vdss value of
less than about 150 mL/kg after intravenous administration to the subject; a mean half-
life (t1/2) of about 500 to 800 hours after intravenous administration in a human; a dose
normalized mean maximum serum or plasma concentration of about 2 to 40 µg/ml after
intravenous administration to the subject, or about 0.1 to 30 µg/ml after subcutaneous
administration to the subject; a mean dose normalized exposure of about 800 to 18,000
(µghr/mL)/(mg/kg) after intravenous administration to the subject, or 400 to 18000
(µghr/mL)/(mg/kg) after subcutaneous administration to the subject; a bioavailability of
about 60 to 90% after subcutaneous administration to the subject; and a tissue-to-serum
ratio of less than about 0.5, wherein the anti-IL13 antibody molecule comprises a a full-
length antibody;
a mean half-life (t1/2) of about 0.5 to 30 hours after subcutaneous or intravenous
administration, to the subject, wherein the anti-IL-13 antibody molecule comprises an
antigen-binding site of the antibody molecule; and
a mean clearance rate of less than 0.004 mL/hr/kg after administration to the
subject, wherein the anti-IL-13 antibody molecule is complexed to IL-13.
2. A method of determining a treatment modality of an anti-IL13 antibody
molecule for an IL-13-mediated disorder, in a subject, comprising:
providing a mean test value for at least one PK/PD parameter of the anti-IL13
antibody molecule in a subject;
comparing the mean test value provided with at least one mean reference value;
and
selecting one or more of dosage, timing, or mode of administration based on the
comparison of at least one mean test value to the mean reference value,
wherein the mean reference value is selected from the group consisting of:
a mean CL value in the range of about 0.05 to 0.9 mL/hr/kg after intravenous
administration of the anti-IL13 antibody molecule to the subject; a mean Vdss value of
less than about 150 mL/kg after intravenous administration to the subject; a mean half-
life (t1/2) of about 500 to 800 hours after intravenous administration in a human; a dose
normalized mean maximum serum or plasma concentration of about 2 to 40 µg/ml after
intravenous administration to the subject, or about 0.1 to 30 µg/ml after subcutaneous
administration to the subject; a mean dose normalized exposure of about 800 to 18,000
(µghr/mL)/(mg/kg) after intravenous administration to the subject, or 400 to 18000
(µghr/mL)/(mg/kg) after subcutaneous administration to the subject; a bioavailability of
about 60 to 90% after subcutaneous administration to the subject; and a tissue-to-serum
ratio of less than about 0.5, wherein the anti-IL13 antibody molecule comprises a a full-
length antibody;
a mean half-life (t1/2) of about 0.5 to 30 hours after subcutaneous or intravenous
administration, to the subject, wherein the anti-IL-13 antibody molecule comprises an
antigen-binding site of the antibody molecule; and
a mean clearance rate of less than 0.004 mL/hr/kg after administration to the
subject, wherein the anti-IL-13 antibody molecule is complexed to IL-13.
3. The method of claim 1 or 2, wherein the mean reference value comprises a
mean serum clearance (CL) value in the range of about 0.065 to 0.3 mL/hr/kg after
intravenous administration to the subject.
4. The method of claim 1 or 2, wherein the mean reference value comprises a
mean steady state volume of distribution (Vdss) value of less than about 110 mL/kg after
intravenous administration to the subject.
5. The method of claim 1 or 2, wherein the mean reference value comprises a
mean half-life (t1/2) of about 670 to 725.
6. The method of claim 1 or 2, wherein the mean test value, or an indication of
whether the preselected relationship is met, is memorialized.
7. The method of claim 1, wherein the step of providing a mean test value
comprises obtaining a sample of the antibody molecule and testing at least one of said
PK/PD parameters;.
8. The method of claim 1 or 2, wherein the subject is a rodent or a primate.
9. The method of claim 1 or 2, wherein the subject is a human.
10. The method of claim 9, wherein the human has a body weight of about 50-80
kg-
11. A method of treating an IL-13-associated disorder in a subject, comprising:
administering, to a subject having, or being at risk of having, the IL-13-associated
disorder, an effective amount of an anti-IL-13 antibody molecule evaluated by the
method of claim 1,
12. A method of treating an IL-13-associated disorder in a subject, comprising:
administering, to a subject having, or being at risk of having, the IL-13-associated
disorder, an anti-IL-13 antibody molecule at a dosage, timing or mode of administration
determined by the method of claim 2.
13. The method of claim 12 or 13, wherein the IL-13 associated disorder is
selected from the group consisting of: asthmatic disorders, atopic disorders, chronic
obstructive pulmonary disease (COPD), conditions involving airway inflammation,
eosinophilia, fibrosis and excess mucus production, inflammatory conditions,
autoimmune conditions, tumors or cancers, viral infection, and suppression of expression
of protective type 1 immune responses.
14. A method of instructing a recipient on the use of an anti-IL13 antibody
molecule to treat an IL-13-associated disorder, comprising:
instructing the recipient that the anti-IL13 antibody molecule has at least one
mean test value for a PK/PD parameter selected from the group consisting of:
wherein the mean reference value is selected from the group consisting of:
a mean CL value in the range of about 0.05 to 0.9 mL/hr/kg after intravenous
administration of the anti-IL13 antibody molecule to the subject; a mean VdSS value of
less than about 150 mL/kg after intravenous administration to the subject; a mean half-
life (tia) of about 500 to 800 hours after intravenous administration in a human; a dose
normalized mean maximum serum or plasma concentration of about 2 to 40 |a.g/ml after
intravenous administration to the subject, or about 0.1 to 30 u,g/ml after subcutaneous
administration to the subject; a mean dose normalized exposure of about 800 to 18,000
(µghr/mL)/(mg/kg) after intravenous administration to the subject, or 400 to 18000
(µghr/mL)/(mg/kg) after subcutaneous administration to the subject; a bioavailability of
about 60 to 90% after subcutaneous administration to the subject; and a tissue-to-serum
ratio of less than about 0.5, wherein the anti-IL13 antibody molecule comprises a a full-
length antibody;
a mean half-life (t1/2) of about 0.5 to 30 hours after subcutaneous or intravenous
administration, to the subject, wherein the anti-IL-13 antibody molecule comprises an
antigen-binding site of the antibody molecule; and
a mean clearance rate of less than 0.004 mL/hr/kg after administration to the
subject, wherein the anti-IL-13 antibody molecule is complexed to IL-13.
15. The method of claim 14, wherein the recipient is a patient, a pharmacist, a
caregiver, a clinician, a member of a medical staff, a manufacturer, or a distributor.
16. The method of claim 14, wherein the method further comprises recording or
memorializing one of more of the test values of the antibody molecule.
17. A method of treating an IL-13-associated disorder in a subject having, or
being at risk of having, the IL-13-associated disorder, comprising:
instructing a caregiver or a patient that an anti-IL13 antibody has at least one
mean test value for a PK/PD parameter selected from the group consisting of:
wherein the mean reference value is selected from the group consisting of:
a mean CL value in the range of about 0.05 to 0.9 mL/hr/kg after intravenous
administration of the ahti-EL13 antibody molecule to the subject; a mean Vdss value of
less than about 150 mL/kg after intravenous administration to the subject; a mean half-
life (t1/2) of about 500 to 800 hours after intravenous administration in a human; a dose
normalized mean maximum serum or plasma concentration of about 2 to 40 µg/ml after
intravenous administration to the subject, or about 0.1 to 30 µg/ml after subcutaneous
administration to the subject; a mean dose normalized exposure of about 800 to 18,000
(µghr/mL)/(mg/kg) after intravenous administration to the subject, or 400 to 18000
(µghr/mL)/(mg/kg) after subcutaneous administration to the subject; a bioavailability of
about 60 to 90% after subcutaneous administration to the subject; and a tissue-to-serum
ratio of less than about 0.5, wherein the anti-IL13 antibody molecule comprises a a full-
length antibody;
a mean half-life (t1/2) of about 0.5 to 30 hours after subcutaneous or intravenous
administration, to the subject, wherein the anti-IL-13 antibody molecule comprises an
antigen-binding site of the antibody molecule; and
a mean clearance rate of less than 0.004 mL/hr/kg after administration to the
subject, wherein the anti-IL-13 antibody molecule is complexed to IL-13.
18. The method of any of claims 1, 2, 11, 12, 14 or 17, wherein the anti-IL-13
antibody molecule comprises a heavy chain immunoglobulin variable domain sequence
and a light chain immunoglobulin variable domain sequence that form an antigen binding
site that binds to IL-13 with a KD of less than 10-7 M, wherein the antibody molecule has
one or more of the following properties:
(a) the heavy chain immunoglobulin variable domain sequence comprises a heavy
chain CDR3 that differs by fewer than 3 amino acid substitutions from a heavy chain
CDR3 of mAb MJ2-7;
(b) the light chain immunoglobulin variable domain sequence comprises a light
chain CDR that differs by fewer than 3 amino acid substitutions from a corresponding
light chain CDR of mAb MJ2-7;
(c) the heavy chain immunoglobulin variable domain sequence comprises a
sequence encoded by a nucleic acid that hybridizes under high stringency conditions to
the complement of a nucleic acid encoding a heavy chain variable domain of V2.1, V2.3,
V2.4, V2.5, V2.6, V2.7, or V2.11;
(d) the light chain immunoglobulin variable domain sequence comprises a
sequence encoded by a nucleic acid that hybridizes under high stringency conditions to
the complement of a nucleic acid encoding a light chain variable domain of V2.11;
(e) the heavy chain immunoglobulin variable domain sequence is at least 90%
identical a heavy chain variable domain of V2.1, V2.3, V2.4, V2.5, V2.6, V2.7, or V2.11;
(f) the light chain immunoglobulin variable domain sequence is at least 90%
identical a light chain variable domain of V2.11;
(g) the antibody molecule competes with mAb MJ2-7 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 heavy chain variable domain sequence has the same canonical structure as
mAb MJ2"7 in hypervariable loops 1,2 and/or 3;
(j) the light chain variable domain sequence has the same canonical structure as
mAb MJ2-7 in hypervariable loops 1, 2 and/or 3; and
(k) 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.
19. The method of claim 18, wherein the anti-lL-13 antibody molecule is a full
length antibody or a fragment thereof.
20. The method of claim 18, wherein the anti-IL-13 antibody molecule reduces
the ability of IL-13 to bind to IL-13RI1 or IL-13RI2.
21. The method of claim 18, wherein the anti-IL-13 antibody molecule comprises
a heavy chain variable domain sequence having a sequence:
(i) G-(YF)-(NT)-I-K-D-T-Y-(Mr)-H (SEQ ID NO:48), in CDR1,
(ii) (WR)-I-E)-P-(GA)-N-D-N-I-K-Y-(SD)-(PQ)-K-F-Q-G (SEQ ID NO:49), in
CDR2, and
(iii) SEENWYDFFDY (SEQ ID NO: 17), in CDR3; and
a light chain variable domain sequence having the sequence:
(i) (RK)-S-S-Q-S-(LI)-(KV)-H-S-(ND)-G-N-(TN)-Y-L-(EDNQYAS) (SEQ ID
NO:25), in CDR1,
(ii) K-(LVI)-S-(NY)-(RW)-(FD)-S (SEQ ID NO:27), in CDR2, and
(iii) Q-(GSA)-(ST)-(HEQ)-I-P (SEQ IDNO:28), in CDR3.
22. The method of claim 18, wherein the anti-IL-13 antibody molecule comprises
a heavy chain variable domain sequence having a sequence:
GFNIKDTYIH (SEQ ID NO: 15), in CDR1,
RIDPANDNIKYDPKFQG (SEQ ID NO: 16), in CDR2, and
SEENWYDFFDY (SEQ ID NO:17) , in CDR3; and
a light chain variable domain sequence having the sequence:
RSSQSIVHSNGNTYLE (SEQ ID NO: 18), in CDR1
KVSNRFS (SEQ ID NO: 19), in CDR2, and
FQGSHIPYT (SEQ ID NO:20), in CDR3.
23. A method of evaluating the amount of a drug-ligand complex in a subject
using a two-compartmental model that includes a central compartment (CAb, V) and a
peripheral compartment (C2,Ab,V2), said method comprising:
providing at least one pharmacokinetic parameter value of the drug-ligand
concentration in the subject at a predetermined time interval, said value chosen from one
or more of: a clearance of the drug from the central compartment (CLAb); a distribution
clearance between the central compartment and the peripheral compartment (CLd,Ab); an
association rate constant (Kon); a dissociation rate constant (Koff); a serum clearance of
the drug-ligand complex (CLcomplex); or an endogenous rate constant for ligand production
divided by a serum clearance of the ligand (Ksyn/CLIL-13);
evaluating the at least one pharmacokinetic parameter in the subject using the
two-compartmental model as represented in Figure 39.
24. The method of claim 23, wherein the two-compartmental model is
represented as follows:
wherein,
CAb is a concentration of antibody (binding agent);
In(t) is a dose administered (for a bolus dose), and In(t) is Ka*F*Dose for
a subcutaneous does, wherein Ka is a first order rate constant and F is an estimate
of bioavailability;
CLd,Ab is a distribution clearance between the central compartment and the
peripheral compartment;
C2,Ab is a concentration of the ligand binding agent in the peripheral
compartment;
V is a volume distribution in a central component;
Kon is a second order rate constant;
Cligand (or CIL-13) is a concentration of ligand;
CAb-(ligand) (or CAb-(IL-13)) is a concentration of ligand binding agent/ligand
complex;
Koff is a first order disassociation rate constant, V2 is a volume of
distribution in a peripheral compartment;
CLcomplex is the serum clearance of the ligand binding agent/ligand
complex; and
Ksyn is a zero order rate constant for endogenous ligand.
25. The method of claim 23 or 24, wherein drug-ligand complex is a ligand-
antibody complex or a ligand-soluble receptor complex.
26. A method of treating or preventing an early asthmatic response (EAR) in a
subject, the method comprising administering, to a subject having, or being at risk of
having, an EAR, an anti-IL-13 antibody molecule.
27. The method of claim 26, wherein the anti-IL-13 antibody molecule decreases
or prevents one or more one or more of: a release of at least one allergic mediator such as
a leukotriene and/or histamine; an increase in the levels of at least one allergic mediator
such as a leukotriene and/or histamine; bronchoconstriction; and/or airway edema.
28. A method of treating or preventing an early asthmatic response (EAR) in a
subject, the method comprising:
administering, to a subject having, or being at risk of having, an EAR, an anti-IL-
13 antibody molecule at a dosage, timing or mode of administration determined by the
method of claim 2.
29. A method of treating or preventing a late asthmatic response (LAR) in a
subject, the method comprising administering, to a subject having, or being at risk of
having, an LAR, an anti-IL-13 antibody molecule.
30. A method of treating or preventing a late asthmatic response (LAR) in a
subject, the method comprising:
administering, to a subject having, or being at risk of having, an LAR, an anti-IL-
13 antibody molecule at a dosage, timing or mode of administration determined by the
method of claim 2.
31. The method of any of claims 26 to 30, wherein the anti-IL-13 antibody
molecule comprises a heavy chain immunoglobulin variable domain sequence and a light
chain immunoglobulin variable domain sequence that form an antigen binding site that
binds to EL-13 with a Kd of less than 10-7 M, wherein the antibody molecule has one or
more of the following properties:
(a) the heavy chain immunoglobulin variable domain sequence comprises a heavy
chain CDR3 that differs by fewer than 3 amino acid substitutions from a heavy chain
CDR3 of mAb MJ2-7;
(b) the light chain immunoglobulin variable domain sequence comprises a light
chain CDR that differs by fewer than 3 amino acid substitutions from a corresponding
light chain CDR of mAb MJ2-7;
(c) the heavy chain immunoglobulin variable domain sequence comprises a
sequence encoded by a nucleic acid that hybridizes under high stringency conditions to
the complement of a nucleic acid encoding a heavy chain variable domain of V2.1, V2.3,
V2.4, V2.5, V2.6, V2.7, or V2.11;
(d) the light chain immunoglobulin variable domain sequence comprises a
sequence encoded by a nucleic acid that hybridizes under high stringency conditions to
the complement of a nucleic acid encoding a light chain variable domain of V2.11;
(e) the heavy chain immunoglobulin variable domain sequence is at least 90%
identical a heavy chain variable domain of V2.1, V2.3, V2.4, V2.5, V2.6, V2.7, or V2.11;
(f) the light chain immunoglobulin variable domain sequence is at least 90%
identical a light chain variable domain of V2.11;
(g) the antibody molecule competes with mAb MJ2-7 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 heavy chain variable domain sequence has the same canonical structure as
mAb MJ2"7 in hypeirvariable loops 1,2 and/or 3;
(j) the light chain variable domain sequence has the same canonical structure as
mAb MJ2-7 in hypervariable loops 1, 2 and/or 3; and
(k) 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.
32. A method of treating an IL-13-associated disorder in a subject, the method
comprising:
administering, to a subject having, or being at risk of having, the IL-13-associated
disorder, one or more flat doses of an anti-IL-13 antibody molecule.
33. The method of claim 32, wherein the flat dose is between about 75 mg and
about 500 mg.
34. The method of claim 33, wherein the flat dose is about 75 mg, 100 mg, 200
mg or 225 mg.
35. The method of any of claims 32-34, wherein the flat dose is administered to
the subject approximately every week, approximately every 2 weeks, approximately
every 3 weeks, approximately every 4 weeks, or approximately every month.

Methods and compositions for reducing or inhibiting, or preventing or delaying the onset of, one or more symptoms
associated with an early and/or a late phase of an IL- 13-associated disorder or condition using IL-13 binding agents are disclosed.
Methods for evaluating the kinetics and/or efficacy of an IL-13 binding agent in treating or preventing an IL-13 -associated disorder
or condition in a subject, e.g., a human subject, are also disclosed.