Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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Immunoglobulins
Field of the Invention
The present invention relates to immunoglobulins that specifically bind
Interleukin 13
(IL-13) and in particular human IL-13 (hIL-13). One embodiment of the
invention
relates to antibodies that specifically bind hIL-13. The present invention
also
concerns methods of treating diseases or disorders with said immunoglobulins,
pharmaceutical compositions comprising said immunoglobulins and methods of
manufacture. Other aspects of the present invention will be apparent from the
description below.
Background of the Invention
Interieukin-13 (IL-13)
IL-13 is a 12kDa secreted cytokine originally described as a T cell-derived
cytokine
that inhibits inflammatory cytokine production. Structural studies indicate
that it has a
four-helical bundle arrangement held by two disulphide bonds. Although IL-13
has
four potential glycosylation sites, analysis of native IL-13 from rat lung has
indicated
that it is produced as an unglycosylated molecule. Expression of human IL-13
from
NSO and COS-7 cells confirms this observation (Eisenmesser et al, J. Mol.
Biol.
2001 310(1):231-241; Moy et al, J. Mol. Biol 2001 310(1):219-230; Cannon-
Carlson
et al, Protein Expression and Purification 1998 12(2):239-248).
IL-13 is a pleiotropic cytokine produced by a variety of cell types including
activated
Th2 cells, mast cells, basophils, dendritic cells, keratinocytes and NKT
cells. It can
also be produced by ThO, Th1, CD8 and naive CD45RA+ T cells. IL-13 has
immunoregulatory activities that partially overlap with those of IL4, this
redundancy
may be explained by shared components in the receptors for IL4 and IL-13. IL-
13
signals through the type II IL4 receptor which is a heterodimer composed of
the
IL4Ra and the IL-13Ra1 chains. IL-13Ra1 binds IL-13 with low affinity (Kd = 2-
10
nM), but when paired with IL4Ra it binds with a high affinity (Kd = 400 pM)
and forms
a functional IL-13 receptor (the human receptor is referred to herein as "hIL-
13R")
that signals, resulting in activation of JAK/STAT and IRS-1/IRS-2 pathways. An
additional IL-13 receptor chain has also been characterised (IL-13Ra2) which
binds
IL-13 with high affinity (Kd = 250 pM). IL-13Ra2 is believed to act as a decoy
receptor
modulating IL-13 activity. IL-13 can also signal through the IL13Ra2 chain
(Fichter-
Feig) 2006 Nature Medicine 12:99-106) to induce TGFbetal, and as such may
contribute towards the fibrosis associated with asthma pathology. Functional
receptors for IL-13 are expressed on a wide range of cells including the
airway
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epithelium, smooth muscle, mast cells, eosinophils, basophils, B cells,
fibroblasts,
monocytes and macrophages. T cells do not have functional receptors for IL-13
(Hilton et al, PNAS 1996 93(1):497-501; Caput et al, J. Biol. Chem. 1996
271(28):16921-16926; Hershey GK, J.Allergy Clin. Immunol. 2003 111(4):677-
690).
Both (L-13 and IL-4 act to modify immune and inflammatory responses by
promoting
allergy associated inflammation and suppressing inflammation due to bacteria,
viruses and intracellular pathogens. The principal biological effects of IL-13
include;
induction of B cell proliferation and regulation of isotype switching to IgE;
induction of
MHC II and CD23 expression on B cells and monocytes; up-regulation of VCAM-1
on
endothelial cells; regulation of chemokine production; activation of mast
cell,
eosinophil and neutrophil function as well as inhibition of pro-inflammatory
gene
expression in monocyte and macrophage populations. IL-13 does not have any
proliferative effects on T cells. Thus unlike IL4, IL-13 does not appear to be
important
in the initial differentiation of CD4 T cells into Th2-type cells, but rather
appears to be
important in the effector phase of allergic inflammation (McKenzie et al, PNAS
1993
90(8):3735-3739; Wynn TA, Annu. Rev. Immunol. 2003 21:425-456).
IL-13 and Asthma
Asthma is a chronic lung disease, caused by inflammation of the lower airways
and is
characterised by recurrent breathing problems. Airways of patients are
sensitive and
swollen or inflamed to some degree all the time, even when there are no
symptoms.
Inflammation results in narrowing of the airways and reduces the flow of air
in and out
of the lungs, making breathing difficult and leading to wheezing, chest
tightness and
coughing. Asthma is triggered by super-sensitivity towards allergens (e.g.
dust mites,
pollens, moulds), irritants (e.g. smoke, fumes, strong odours), respiratory
infections,
exercise and dry weather. The triggers irritate the airways and the lining of
the
airways swell to become even more inflamed, mucus then clogs up the airways
and
the muscles around the airways tighten up until breathing becomes difficult
and
stressful and asthma symptoms appear.
There is strong evidence from animal models and patients that asthmatic
inflammation and other pathologies are driven by dysregulated Th2 responses to
aeroallergens and other stimuli (Busse et al, Am. J. Resp. Crit. Care Med.1995
152(1):388-393). ln particular, IL-13 is believed to be the major effector
cytokine
driving a variety of cellular responses in the lung, including airway
hyperreactivity,
eosinophilia, goblet cell metaplasia and mucus hyper-secretion.
Clinical Evidence for the role of IL-13 in asthma
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The gene encoding IL-13 is located on chromosome 5q31. This region also
contains
genes encoding IL-3, IL-4, IL-5, IL-9 and GM-CSF, and has been linked with
asthma.
Genetic variants of IL-13 that are associated with asthma and atopy have been
found
both in the promoter and coding regions (Vercelli D, Curr. Opin. Allergy Clin.
Immunol. 2002 2(5):389-393). Functional study data are available for the
coding
variant, Q130 IL-13 (referred to herein as "Q130 IL-13"). The +2044 G to A
single
nucleotide polymorphism (SNP) found in the fourth exon, results in a
substitution of
an arginine with a glutamine at position 130 (Q130 IL-13). Also note that in
SEQ.ID.NO: 9, this is equivalent to position 110, where the first 'G' amino
acid
residue at the start of the mature human IL-13 amino acid sequence is position
1.
This variant has been found to be associated with asthma, increased IgE levels
and
atopic dermatitis in Japanese and European populations. Q130 IL-13 is believed
to
have enhanced stability compared with wild-type IL-13. It also has slightly
lower
affinity for the IL-13Ra2 decoy receptor and consistent with these
observations,
higher median serum IL-13 levels are found in patients homozygous for the Q130
IL-
13 variant compared with non-homozygous patients. These results indicate that
Q130 IL-13 could influence the local and systemic concentrations of IL-13
(Kazuhiko
et al, J. Allergy Clin. Immunol. 2002 109(6):980-987).
Elevated IL-13 levels have been measured in both atopic and non-atopic
asthmatics.
In one study, average serum IL-13 levels of 50 pg/mi were measured in
asthmatic
patients compared to 8 pg/mi in normal control patients (Lee et al, J. Asthma
2001
38(8):665-671). Increased IL-13 levels have also been measured in plasma,
bronchio-alveoiar lavage fluid, lung biopsy samples and sputum (Berry et al, J
Allergy
Clin. Immunol 2004 114(5):1106-1109; Kroegel et al, Eur Respir. J. 1996
9(5):899-
904; Huang et al, J. Immunol. 1995 155(5):2688-2694; Humbert et al, J. Allergy
Clin.
Immunol. 1997 99(5):657-665).
In vivo evidence for involvement of IL-13 in asthma
A number of studies have defined a critical effector role for IL-13 in driving
pathology
in both acute and chronic mouse models of allergic asthma. The high affinity
IL-13
receptor (IL-13Ra2) or anti-IL-13 polyclonal antibodies have been used to
neutralize
mouse IL-13 bioactivity in these models. Blockade of IL-13 at the time of
allergen
challenge completely inhibited OVA-induced airway hyper-reponsiveness,
eosinophilia and goblet cell metaplasia. In contrast, administration of
antibody to IL-4
after sensitisation and during the allergen challenge phase only partially
reduced the
asthma phenotype. Thus although exogenous IL-4 and IL-13 are both capable of
inducing an asthma-like phenotype, the effector activity for IL-13 appears to
be
superior to that for IL-4. These data suggest a primary role for IL-4 in
immune
induction (particularly for Th2 cell development and recruitment to airways,
and IgE
production), whereas IL-13 is believed to be principally engaged in various
effector
outcomes, including airway hyper-responsiveness, mucus overproduction and
cellular inflammation (Wills-Karp et al, Science 1998 282:2258-2261; Grunig et
al,
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Science 1998 282:2261-2263; Taube et al, J. Immunol. 2002 169:6482-6489;
Blease
at al, J. lmmunol 2001 166(8):5219-5224).
In complementary experiments, lung IL-13 levels have been raised by over-
expression in a transgenic mouse or by instillation of IL-13 protein into the
trachea of
wild-type mice. In both settings, asthma-like characteristics were induced:
non-
specific airway hyper-responsiveness to cholinergic stimulation, pulmonary
eosinophilia, epithelial cell hyperplasia, mucus cell metaplasis, sub-
epithelial fibrosis,
airways obstruction and Charcot-Leyden-like crystals. In addition, IL-13 was
found to
be a potent stimulator of matrix metalloproteinases and cathepsin proteases in
the
lung, resulting in emphysematous changes and mucus metaplasia. Therefore IL-13
may be an important effector molecule both in asthma and COPD disease
phenotypes (Zhu et al, J. Clin. Invest. 1999 103(6):779-788; Zheng et al, J.
Clin.
Invest. 2000 106(9):1081-1093).
These data indicate that IL-13 activity is both necessary and sufficient to
produce
several of the major clinical and pathological features of allergic asthma in
well-
validated animal models.
Chronic Obstructive Pulmonary Disease (COPD)
COPD is a generic term covering several clinical syndromes including emphysema
and chronic bronchitis. Symptoms are similar to asthma and COPD can be treated
with the same drugs. COPD is characterised by a chronic, progressive and
largely
irreversible airflow obstruction. The contribution of the individual to the
course of the
disease is unknown, but smoking cigarettes is thought to cause 90% of the
cases.
Symptoms include coughing, chronic bronchitis, breathlessness and respiratory
infections. Ultimately the disease will lead to severe disability and death.
Chronic
bronchitis is diagnosed in patients with a history of cough or sputum
production on
most days for at least 3 months over 2 years without any other explanation.
Emphysema of the lung is characterised by an abnormal permanent enlargement of
the air spaces and destruction of alveolar walls.
IL-13 may play a role in the development of COPD. Human smokers who develop
COPD have many inflammatory cell types (neutrophils, macrophages, eosinophils)
in
the lung parenchyma. IL-13 is a proinflammatory Th2 cytokine therefore to
model the
progression of emphysema; Zheng et al targeted IL-13 over-expression to the
airway
epithelium in IL-13 transgenic mice. These animals developed airway and lung
parenchymal inflammation and emphysema. They also developed mucus metaplasia
reminiscent of chronic bronchitis (J. Clin. Invest. 2000 106(9):1081-1093).
The IL-13 promoter polymorphism (-1055 C to T) that is associated with
allergic
asthma has also been reported to have an increased frequency in COPD patients
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compared to healthy controls. This implies a functional role for the (L-13
promoter
polymorphism in the enhanced risk to develop COPD (Kraan et al, Genes and
Immunity 2002 3:436-439). In addition, an increased number of IL-13 and IL-4
positive cells were observed in smokers with chronic bronchitis compared to
asymptomatic smokers (Miotto et al, Eur. Resp. J. 2003 22:602-608). However a
recent study to assess the level of IL-13 expression in the lungs of severe
emphysema patients did not find an association between IL-13 levels and
disease
(Boutten et al, Thorax 2004 59:850-854).
Allergic disease including atopic dermatitis and a{lerctic rhinitis
IL-13 has also been implicated in atopic disorders such as atopic rhinitis and
atopic
dermatitis. Allergic rhinitis is the most common atopic disease in the United
States
and is estimated to affect up to 25% of adults and more than 40% of children.
There
is a close relationship between allergic rhinitis and asthma. Both conditions
share
common immunopathology and pathophysiology; they have similar immunologic
processes in which eosinophils and Th2 lymphocytes in nasal and bronchial
tissue
play a role. Excessive production of Th2 cytokines, particularly IL-4 and IL-
5, is
thought to be fundamental in the pathogenesis of allergic disease. IL-13
shares
several characteristics and effector functions with IL-4 and this, combined
with the
functional overlap in IL-4 and IL-13 receptor usage, intracellular signaling
components, and genetic organization provides compelling (albeit indirect)
evidence
for a role of 1L-13 in promoting or maintaining human immediate
hypersensitivity in
vivo. This has been corroborated by Li et al (Li et al. J
Immuno11998;161:7007) who
demonstrated that atopic subjects with seasonal allergic rhinitis exhibited
significantly
stronger IL-13 responses in response to Ag-dependent but not polyclonal
activation.
Atopic dermatitis is a common, chronic, relapsing, highly pruritic
inflammatory skin
disease. The lesional skin of atopic dermatitis patients is histologically
characterized
by an inflammatory T-cell infiltrate, which during acute phases is associated
with a
predominance of IL-4, IL-5 and IL-13 expression (Simon et al, J Allergy Clin
Immunol
2004;114:887; Hamid et al. J Allergy Clin Immunol 1996; 98: 225) In addition,
Tazawa et al have demonstrated that IL-13 mRNA (but not IL-4) is significantly
upregulated in subacute and chronic skin lesions of atopic dermatitis patients
(Tazawa et al, Arch Derm Res 2004;296:459). The frequency of IL-13 expressing
circulating CD4+ and CD8+ T-cetls is also significantly increased in these
patients
(Aleksza et al British J Dermatol 2002;147;1135). This increased IL-13
activity is
thought to result in raised levels of serum IgE, thereby contributing to the
pathogenesis of atopic dermatitis. Furthermore, increased production of IL-13
by
neonatal CD4+ T cells is a useful marker for identifying newborns at high risk
for
subsequent development of allergic diseases, esp. atopic dermatitis (Ohshima
et al.
PediatrRes 2002; 51:195). Additional evidence for the importance of IL-13 in
the
etiology of atopic dermatitis was provided by Simon et al (Simon et al, J
Allergy Clin
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Immuno12004; 114:887); topical treatment with tacrolimus ointment (an
immunosuppressive drug that inhibits intracellular signaling pathways for
cytokine
production) resu(ted in significant clinical and histological improvement of
the atopic
skin lesions accompanied by significant reductions in local expression of Th2
cytokines, including IL-13. Furthermore, IL-13 Ral (a cell surface protein
that
together with IL-4Ra forms a functional receptor for IL-13) has been shown to
be
over-expressed on the suprabasal keratinocytes in the skin of atopic
dermatitis
patients, and IL-13 was able to upregulate IL-13 Ral mRNA in vitro
(Wongpiyabovorn et al., J Dermatol Science 2003;33:31).
These data collectively indicate that IL-13 targeted interventions, including
an IL-13
monoclonal antibody, may provide an effective approach for treatment of human
allergic disease.
Esophagal eosinophilia
The accumulation of eosinophils in the esophagus is a common medical problem
in
patients with diverse diseases, including gastro-esophageal reflux disease,
eosinophiiic esophagitis, eosinophilic gastroenteritis, and parasitic
infections.
Esophageal eosinophilia is associated with allergic responses, and repeated
challenging of mice with aeroallergens established a link between allergic
airway
inflammation and esophagal eosinophilia. Th2 cells are thought to induce
eosinophil-
associated inflammation through the secretion of an array of cytokines
including IL-4
and IL-13 that activate inflammatory and effector pathways both directly and
indirectly. IL-13 appears to be particularly important because it is produced
in high
quantities by Th2-cells and regulates multiple features of allergic disease
(e.g. IgE
production, mucus over-production, eosinophil recruitment and survival, and
airway
hyperreactivity. Eosinophiis can generate functionally active IL-13 after
exposure to
GM-CSF and/or IL-5 under in vitro, ex vivo, and in vivo conditions in
eosinophilic
inflammatory responses. (Schmid-Grendelmeier J Immunology, 2002, 169: 1021-
1027). IL-13 delivered to the lung of wild-type, STAT-6, eotaxin-1 or IL-5
deficient
mice by intratracheal administration, established that pulmonary inflammation,
triggered by IL-13, is associated with the development of esophagal
eosinophilia
(Mishra et al. Gastroentero12003;125:1419). Taken together, these data provide
evidence for a role of IL-13 in esophagal eosinophilia.
Oncology Indications
Another important area of interest is in targeting IL-13 or IL-13 receptors to
inhibit
growth of certain types of tumors. Type 1 T cell-mediated host defenses are
believed
to mediate optimal tumor rejection in vivo, and deviation to a Th2-type
response may
contribute to blocking tumor rejection and/or promotion of tumor recurrence
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(Kobayashi M et al. J. lmmunol. 1998; 160:5869). Several animal studies using
transplantable tumor cell lines support this notion by demonstrating that
Stat6, IL-4,
and IL-13 (produced in part by NKT cells) were capable of inhibiting tumor
rejection
(Terabe et al. Nat. lmmunol. 2000;1:515; Kacha et al. J. Immunol.
2000;165:6024-
28; Ostrand-Rosenberg et al. J. lmmunol. 2000;165:6015). The potent anti-tumor
activity in the absence of Stat-6 was thought to be due to enhancement of
tumor-
specific IFNg production and CTL activity. In addition, a loss of NKT cells
has been
shown to reduce IL-13 production with a concomitant rise in tumor recurrence,
indicating that 1L-13, produced in part by NKT cells is important for
immunosurveillance (Terabe et al. Nat. Immunol. 2000; 1:515). As such, these
findings suggest that IL-13 inhibitors or novel IL-13 antagonists, including
IL-13 mAb,
may be effective as cancer immunotherapeutics by interfering with the negative
regulatory IL-13 plays in downregulating immune responses to tumor cells.
In addition to boosting Th-type-1 -associated anti-tumor defenses, IL-13
inhibitors
may also be able to block tumor cell growth more directly. For example, in B-
cell
chronic lymphocytic leukemia (B-CLL) and Hodgkin's disease, IL-13 either
blocks
apoptosis or promotes tumor cell proliferation (Chaouchi et al. Blood 1996;
87:1022;
Kapp et al. J. Exp Med. 1999; 189:1939). B-CLL is a clinically heterogeneous
disease originating from B lymphocytes that involves apoptotic defect in the
leukemic
cells. IL-13 is not thought to act as a direct growth factor but protects
tumor cells from
in vitro spontaneous apoptosis (Chaouchi et al. Blood 1996; 87:1022; Lai et
al. J.
Immuno1.1999; 162:78) and may contribute to B-CLL by preventing neoplastic
cell
death.
Hodgkin's disease is a type of lymphoma that primarily affects young adults
and
accounts for about 7,500 cases a year in the United States. The cancer is
characterized by the presence of large multi-nucleated Hodgkin/Reed-Sternberg
cells
(H/RS). In a large majority of cases, the malignant cell population arises
from B cells.
Several Hodgkin's disease-derived cell lines, as well as lymph node tissue
taken from
Hodgkin's lymphoma patients, overexpress IL-13 and/or IL-13 receptors. (Kapp
et al.
J. Exp Med. 1999;189:1939, Billard et al. Eur Cytokine Netw 1997;8:19;
Skinnider et
al. Blood 2001; 97:250; Oshima et al, Cell lmmunol 2001 ;211:37). Neutralizing
anti-IL-13 mAbs or IL-13 antagonists have been shown to inhibit H/RS cell
proliferation in a dose-dependent manner (Kapp et al. J. Exp Med. 1999;
189:1939;
Oshima et al, Cell lmmuno12001; 211:37). Similarly, delivery of soluble IL-
13Ra2
decoy receptor to NOD/SCID mice with an implanted Hodgkin's disease-derived
cell
line delayed tumor onset and growth, and enhanced survival, demonstrating that
IL-
13 neutralization can suppress Hodgkin's lymphoma growth in vitro and in vivo
(Trieu
et al. Cancer Research 2004;64:3271). Collectively, these studies indicate
that IL-13
stimulates the proliferation of H/RS cells in an autocrine fashion (Kapp et
al. J. Exp
Med. 1999; 189:1939; Ohshima et al. Histopathology 2001; 38:368).
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Neutralization of IL-13 may therefore represent an attractive and effective
treatment
for Hodgkin's disease and other B cell-associated cancers by inhibiting tumor
cell
growth while at the same time enhancing anti-tumor defenses.
Inflammatory Bowel Diseases
There is a possible role for IL-13 in the pathogenesis of inflammatory bowel
disease
(IBD). Inflammatory bowel disease comprises a number of diseases clinically
classified as ulcerative colitis, Crohn's disease and indeterminate colitis.
Its main
manifestation is chronic intestinal inflammation due to an exaggerated immune
response with an imbalance in the activation of Th1 and Th2 lymphocytes in the
intestinal mucosa. This has been demonstrated in animal models of crohn's
disease
(Bamias et al. Gastroenterol 2005; 128:657) and ulcerative colitis (Heller et
al,
Immunity 2002; 17:629). Neutralization of IL-13 by IL-13Ra2-Fc administration
prevented colitis in a murine Th2 model of human ulcerative colitis (Heller et
al,
Immunity 2002; 17:629). Furthermore, IL-13 production rapidly supersedes that
of IL-
4 in this model, and IL-13 production can be induced by stimulation of NKT
cells,
suggesting that tissue damage may result from toxic activity of IL-13 on the
epithelium cells. There are some human data to support these findings: the
frequency of IL-13 positive rectal biopsy specimens from patients with
ulcerative
colitis was significantly higher than of inflammatory and non-inflammatory
control
subjects, and a higher rate IL-4 and IL-13 expression was observed in acute
than
non-acute ulcerative colitis (Inoue et al. Am J Gastroenterol 1999;94:2441).
In
addition Akido et a/ characterized the immune activity in the muscularis
externa from
intestinal segments of Crohn's disease patients and found that IL-4 and IL-13
mediate hypercontractility of the intestinal smooth muscle cells via a STAT-6
pathway. The authors concluded that this pathway may contribute to the
hypercontractility of intestinal muscles in Crohn's disease (Akiho et al., Am
J Physiol
Gastrointest Liver Physiol 2005; 288:619).
Thus, an IL-13 mAb, possibly in combination with molecules directed at other
cytokines, may provide an approach to stop or slow the progression of IBDs.
Psoriasis and Psoriatic Arthritis
Psoriasis is a chronic skin disease characterized by hyper-proliferation of
keratinocytes and an immunologic cellular infiltrate, including activated T
cells,
producing various cytokines that can influence the phenotype of epidermal
keratinocytes. CDw60 is a carbohydrate-bearing molecule that is upregulated on
the
surface of psoriatic basal and suprabasal keratinocytes of psoriatic skin. IL-
4 and IL-
13 secreted from T cells derived from psoriatic lesions have been shown to
strongly
up-regulate the expression of CDw60 on keratinocytes, (Skov et al., Am J
Pathol
1997;15:675), whereas interferon-gamma blocked IL-4/IL-13 mediated induction
of
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CDw60 on cultured keratinocytes (Huang et al., J Invest Dermatol
2001;116:305).
Thus, CDw60 expression on psoriatic epidermal keratinocytes is thought to be
induced at least in part by IL-13 secreted by activated T cells within the
lesion. In
addition, IL-13 Ral and IL-4Ra, cell surface proteins that together form a
receptor
complex for IL-13, are differently expressed in skin biopsies from patients
with and
without psoriasis (Cancino-Diaz et al., J Invest Dermatol 2002;119:1114;
Wongpiyabovorn et al., J Dermatol Science 2003;33:31), and in vitro
experiments
demonstrated that IL-13 (but not IL-4) could upregulate the expression of IL-
13Ra1
(Wongpiyabovorn et al., J Dermatol Science 2003;33:31). Since IL-13 has an
effect
on a variety of cell types, these studies suggest that the IL-13 receptor may
play a
part in the early inflammatory process of psoriasis.
Psoriatic arthritis is characterized by synovitis which is mediated by both
pro-
inflammatory and anti-inflammatory cytokines. The role of IL-13 in various
forms of
arthritis has been receiving increased interest. Spadaro et al have observed
significantly higher levels of IL-13 in synovial fluid of patients with
psoriatic arthritis
and rheumatoid arthritis than in patients with osteoarthritis. In addition,
synovial fluid
levels of IL-13 were significantly higher than those in serum in patients with
psoriatic
arthritis, and the IL-13 synovial fluid/serum ratio was markedly higher in the
psoriatic
arthritis group than in the rheumatoid arthritis group, suggesting a possible
role for
the locally produced IL-13 in synovial tissues of patients with psoriatic
arthritis
(Spadaro et al., Ann Rheum Dis 2002; 61:174).
Potential Role of IL-13 in other conditions
Acute graft-versus-host disease is a serious cause of morbidity and mortality
following stem cell transplantation and is directly related to the degree of
human
leukocyte antigen (HLA) incompatibility between donor and recipient. Jordan et
al first
identified IL-13 as a typical Th2 cytokine that is abundantly produced during
unrelated, unmatched MLRs (mixed lymphocyte reaction; an in vitro assay for
fine-
tuning donor selection after initial HLA typing) (Jordan et al. J Immunol
Methods;
2002;260:1). The same group subsequently showed that IL-13 production by donor
T-cells is predictive of acute graft-versus-host-disease (aGVHD) following
unrelated
donor stem cell transplantation (Jordan et al. Blood 2004; 103:717). All
patients with
severe, grade III aGVHD following stem cell transplantation had donors who
produced very high pre-transplantation IL-13 responses, demonstrating a
significant
link between IL-13 levels and aGVHD and raising the possibility that IL-13 may
be
directly responsible for some of the aGVHD associated pathology. Consequently,
a
therapy based on specific blocking of IL-13 may be useful for the treatment of
post-
stem cell transplantation aGVHD.
Diabetic nephropathy is one of the major causes of end stage renal disease in
the
Western world. Although the incidence of nephropathy owing to type 1 diabetes
is
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declining, diabetes mellitus type 2 is now the most common single cause of
renal
insufficiency in the USA, Japan and Europe. Furthermore, this group of
patients has
a very poor prognosis on maintenance dialysis owing to extremely high
mortality
caused by cardiovascular events. It is now increasingly clear that
hemodynamic,
metabolic and structural changes are interwoven, and various enzymes,
transcription
factors and growth factors have been identified that play a role in the
pathogenesis of
this disease. Particularly, TGF-R is important in the development of renal
hypertrophy
and accumulation of extracellular matrix components, and is considered the
pivotal
cytokine in mediating collagen formation in the kidney (Cooper. Diabetologia
2001;
44:1957; Wolf. EurJ Clin Invest 2004; 34 (12): 785). In experimental and human
diabetic nephropathy TGF-1 bioactivity is increased and administration of TGF-
P1
antibodies to diabetic mouse led to improvement in renal function and reduced
extra-
cellular matrix accumulation. IL-13 was recently shown in a transgenic mouse
model
of lung fibrosis to mediate its effects at least in part by regulating the
production and
activation of TGF-pi and collagen deposition (Lee et al. J. Exp. Med. 2001;
194:809;
Zhu et al. J. Clin. Invest. 1999; 103:779), thereby establishing a direct
functional link
between IL-13 and TGF-P. Consequently a similar role for IL-13 in regulating
TGF-bl
activity in the diabetic kidney can be envisioned and IL-13 targeted
interventions
could potentially have a role in the management of diabetic nephropathy.
Fibrotic Conditions
Pulmonary fibrosis is a condition of inappropriate and harmful scarring of the
lungs,
leading to disability and often death. The term encompasses a variety of
different
conditions with distinct etiologies, pathologies and responses to treatment.
In some
cases the cause of the fibrosis is identified. Causes include: (1) inhaled
profibrotic
material such as asbestos or silicon, or hard metal dust (2) inhaled organic
material
to which the patient has an idiosyncratic immunological response leading to
fibrosis
(e.g. farmer's lung) (3) drugs, such as nitrofurantoin, amiodarone and
methotrexate
(4) in association with a systemic inflammatory disease, such as Systemic
Sclerosis
or Rheumatoid Arthritis.
However, in many instances no cause or underlying condition is identified.
Many
such patients are diagnosed with Idiopathic Pulmonary Fibrosis (IPF). This is
a
relative rare condition (prevalence 20/100 000). The diagnosis is based on the
absence of an identified cause combined with certain radiological and
pathological
features, particularly honeycombing on the CT or lung biopsy. The disease is
usually
seen in older patients (>50) and often follows a relentless course of
progressive lung
impairment leading to death, with the median survival quoted as 2-5 years.
Moreover,
the patients have the most unpleasant experience of breathlessness progressing
over months or years. This initially restricts physical activity, but in the
terminal phase
- which may last several months - the patient is breathless even at rest and
is
furthermore oxygen dependent.
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At present there is no satisfactory treatment for this disease. Current
treatment
generally takes the form of corticosteroids and immunosuppressives such as
azathioprine. However, corticosteroids may be ineffective in many of patients
and
their side effects may make the situation worse. There are many potential
treatments
under investigation including Interferon gamma, which has shown a trend to
improved survival in a recent large study, and perfenidone.
There is evidence that IL-13 and cytokines associated with the Th2 phenotype
are
involved in the process of fibrosis in tissue repair (Wynn TA, Nat. Rev.
Immunol.
2004 4:583-594; Jakubzick et al, Am. J. Pathol. 2004 164(6):1989-2001;
Jakubzick et
al, Immunol. Res. 2004 30(3):339-349; Jakubzick et al, J. Clin. Pathol. 2004
57:477-
486). IL-13 and IL-4 have been implicated in a variety of fibrotic conditions.
Hepatic
fibrosis induced by Schistosoma appears to be IL-13 dependent and there is
limited
evidence that IL-13 is involved in the pathogenesis of scieroderma (Hasegawa
et al,
J. Rheumatol. 1997 24:328-332; Riccieri et al, Clin. Rheumatol. 2003 22:102-
106)
In terms of pulmonary fibrosis, in vitro studies have shown that IL-13
promotes a
fibrogenic phenotype. Animal studies have shown elevated levels of IL-13
expression
in artificially induced models of fibrosis, and that fibrosis can be reduced
by
elimination of IL-13.
IL-13 promotes a profibrotic phenotype. At a cellular level, there are several
mechanisms by which IL-13 may promote fibrosis. The signal pathways and
importance of these various mechanisms are not well defined.
There is evidence that IL-13 acts on the fibroblast both to promote the
production of
collagen, and to inhibit its breakdown, thus favouring a fibrotic phenotype.
Skin
fibroblasts possess IL-13 receptors (= the type fi IL-4 receptor) and exposure
of
cultured skin fibroblasts to 1L-131eads to upregulation of collagen generation
(Oriente
et al, J. Pharmacol. Exp. Ther. 2000 292:988-994). IL-4 also has a similar,
but more
transitory effect. A human lung fibroblast cell line (ICIG7) expresses the
type II IL-4
receptor (Jinnin et al, J. Biol. Chem 2004 279:41783-41791). Exposure of these
cells
to IL-13 promotes secretion of a variety of inflammatory and profibrotic
mediators:
GM-CSF, G-CSF, VCAM betal integrin (Doucet et al, Int. Immunol. 1998
10(10):1421-1433).
IL-13 inhibits IL-1 a-induced matrix metalloproteinases 1 and 3 protein
production by
skin fibroblasts which would tend to reduce breakdown of EC matrix (Oriente et
al, J.
Pharmacol. Exp. Ther. 2000 292:988-994). IL-13 acts synergistically with TGF-R
on
human fibroblasts obtained by biopsy of asthma airways to promote expression
of
tissue inhibitor of metalloproteinase 1(TIMP-1). Breakdown of extracellular
matrix is
effected by matrix metalloproteinases, which are inhibited by TIMP-1. This
action of
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12
IL-13 would thus tend to reduce matrix degradation (Zhou et al, Am. J.
Physiol. Cell
Physiol. 2005 288:C435-C442)
Over-expression of IL-13 in transgenic mice leads to subepithelial fibrosis,
epithelial
cell hypertrophy, goblet cell hyperpiasia, crystal deposition (acidic
mammalian
chitinase), airway hyper-responsiveness, interstitial fibrosis, type 2 cell
hypertrophy
and surfactant accumulation (Zhu et al, J. Clin. Invest. 1999 103(6):779-788).
Different strains of mice have different susceptibilities to bleomycin-induced
pulmonary fibrosis. C57B1/6J mice, which are susceptible, exhibit rapid up
regulation
of IL-13, IL-13Ra and IL-4 (as well as TGFR, TNFR(x and IL1 Rs) in response to
bleomycin. BALB/c mice, which are not susceptible, do not show upregulation of
IL-
13.
Belperio et al (Am. J. Respir. Cell Mol. Biol. 2002 27:419-427) studied the
expression
and role of IL-13, IL-4 and the CC chemokine C10 in a mouse bleomycin fibrosis
model. Lung tissue levels of both IL-13 and IL-4 increased in response to
bleomycin.
Prior neutralisation of IL-13 using polyclonal anti IL-13 antibodies
significantly
reduced lung fibrosis in response to bieomycin as assessed by lung
hydroxyproline
levels. Despite the increased expression of IL-4 in the same model,
neutralisation of
IL-4 had no effect on lung fibrosis.
In another model of acute lung fibrosis induced by FITC in the BALB/c mouse,
absence of IL-13 (in knockouts), but not IL-4, protected against lung
fibrosis. There is
no added protection of knockout of IL-4 in IL-13 knockouts (Kolodsick et al,
J.
Immunol. 2004 172:4068-4076). The protective effect of IL-13 absence is not
due to
a difference in cell recruitment into the lung: in all knockouts and BALB/c
total cell
numbers recruited are similar, so the initial inflammatory component seems to
be the
unaffected. Eosinophil recruitment is lower in IL-4 and IL-13 knockouts
compared
with BALB/c, but since IL-4 4- were not protected against fibrosis this cannot
explain
the difference in fibrosis. Perhaps surprisingly, there was no difference in
the levels of
cytokines between IL-13 +/+ and -/-, including for IL10, MCP-1, gamma
interferon,
TGF-i. In addition, the same number of fibroblasts were isolated from lungs of
the
different animals post FITC, but in the IL-13 -/- mice the production of
collagen I is
reduced. This indicates the loss of IL-13 is not simply preventing the
inflammatory
response, but rather is having a more specific anti-fibrotic role. It has been
suggested
that IL-13 might exert its fibrotic effect via TGF-i (Lee et al, J. Exp. Med.
2001
194:809-821). However in this FITC model, expression of TGF-I was not reduced
in
IL-13 knock-out mice.
Interleukin 4 may be expected to exert a similar effect as IL-13 as both act
via the
same receptor. IL-4 is significantly upregulated in the lungs of mice with
bleomycin
induced lung fibrosis (Gharaee-Kermani et al, Cytokine 2001 15:138-147).
However,
comparing bleomycin-induced lung fibrosis in C57BL6/J mice which overexpress
IL-
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13
4, IL-4 knockouts and wild type, lzbicki et al (Am. J. Physiol. Lung Cell Mol.
Physiol
2002 283(5):L1110-L1116) did not find evidence that IL-4 was involved in lung
fibrosis. Fibrosis was not reduced in IL-4 knockouts, and IL-4 over-expressing
mice
had increased levels of fibrosis.
BAL cytokine levels of IL-13 are significantly elevated in patients with a
variety of
forms of pulmonary fibrosis, though with considerable variability. Expression
of IL-13
is significantly upregulated in alveolar macrophages obtained from patients
with lung
fibrosis.
The strongest clinical evidence comes from research at the University of
Michigan.
Jakubzick and colleagues have studied gene expression of IL-13 and IL-4 and
their
receptors in surgical lung biopsies from patients with pulmonary fibrosis. IL-
13 gene
expression is markedly greater in specimens from IPF affected lung than lung
from
normals or other lung fibrotic conditions. Fibroblasts cultured from patients
with
IPF/UIP show heightened expression of the IL-13 and IL-4 receptor, compared
with
tissue and fibroblasts obtained biopsies from patients with normal lungs or
other
forms of lung fibrosis. In particular, the fibroblastic foci, which are
presumably the
epicentre of disease activity, stain particularly strongly for these receptors
(Jakubzick
et al, J. Immunoi 2003 171:2684-2693; Jakubzick et al, Am. J. Pathol. 2003
162:1475-1486; Jakubzick et al, Am. J. Pathol. 2004 164(6):1989-2001;
Jakubzick et
al, Immunol. Res. 2004 30(3):339-349; Jakubzick et al, J. Clin. Pathol. 2004
57:477-
486).
There is good in vitro evidence that Th2 cytokines in general and IL-13 in
particular
promote a profibrotic phenotype. In at least 2 animal models it has been shown
that
chemically-induced fibrosis can be reduced by elimination of IL-13 (either in
gene
knock-out or by anti-IL-13 antibodies). Some evidence indicates that IL-13 is
more
important at promoting pulmonary fibrosis than IL-4. Clinical evidence for the
role of
IL-13 in pulmonary fibrosis suggests that IL-13 and its receptors are
unregulated in
the lungs of patients with IPF.
A growing body of data suggests an important role for IL-13 based therapies
for the
treatment of a variety of fibrotic conditions, including schistosomiasis-
induced hepatic
fibrosis, and various forms of pulmponary fibrosis (e.g. IPF [discussed
elsewhere],
scieroderma).
Experiments in which IL-4 and IL-13 were inhibited independently identified IL-
13 as
the dominant effector cytokine of fibrosis in several models (Chiaramonte et
al J. Clin.
Invest. 1999;104: 777-785; Blease et al. J. Immunol. 2001; 166:5219; Kumar et
al.
Clln. Exp. Allergy 2002; 32:1104). In schistosomiasis, although the egg-
induced
inflammatory response was unaffected by IL-13 blockade, collagen deposition
decreased by more than 85% in chronically infected animals (Chiaramonte et al
J.
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14
Clin. Invest. 1999; 104: 777; Chiaramonte et al Hepatology 2001; 34:273)
despite
continued and undiminished production of IL-4.
The amino acid sequence for h1L-13 is set forth as SEQ.I.D.NO: 9. (This is the
mature protein sequence, that is, no signal sequence is present).
A polynucleotide encoding hIL-13 is set forth in SEQ.I.D.NO:10. (This is the
DNA
sequence for the mature protein sequence, that is, no signal sequence is
present).
All patent and literature references disclosed within the present
specification
(including any patent application to which this application claims priority)
are
expressly and entirely incorporated herein by reference.
Recently vaccines raising immune responses against IL-13 for the treatment of
asthma have been described (WO 02/070711). A role for IL-13 in the
sensitisation of
the skin to environmental allergens has also been recently described (Herrick
et al.,
The Journal of Immunology, 2003, 170:2488-2495).
The present invention provides an antibody that binds hIL-13 and inhibits the
binding
of hIL-13 with both chains of hIL-13R i.e. IL-13Ra1 and IL-13Ra2.
Summary of the Invention
The present invention therefore provides an antibody or antigen binding
fragment
thereof which specifically binds hIL-13 and neutralises the activity of hIL-
13. The
present invention provides an antibody or antigen binding fragment thereof
which
specifically binds hIL-13 and comprises a CDRH3 which is a variant of the
sequence
set forth in SEQ.I.D.NO:3 or a variant in which one or two amino acid residues
within
said CDRH3 of said variant differs from the amino acid residue in the
corresponding
position in SEQ.I.D.NO:3. In one embodiment of the present invention these
differences in amino acid residues are conservative substitutions.
The term "specifically binds" as used throughout the present specification in
relation
to antibodies and antigen binding fragments thereof of the invention means
that the
antibody binds hIL-13 with no or insignificant binding to other human proteins
and in
particular human IL-4. The term however does not exclude the fact that
antibodies of
the invention may also be cross-reactive with cynomolgus IL-13.
The term "neutralises" as used throughout the present specification in
relation to
antibodies and antigen binding fragments thereof of the invention means that
the
biological activity of IL-13 is reduced in the presence of the antibodies and
antigen
binding fragments of the present invention in comparison to the activity of IL-
13 in the
absence of such antibodies and antigen binding fragments thereof. Levels of
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neutralisation can be measured in several ways, for example by use of the
assays as
set out in the examples below, for example in a TF-1 cell proliferation assay
which
may be carried out for example as described in Example 3.3-3.5. The
neutralisation
of IL-13 in this assay is measured by assessing the decreased TF-1 cell
proliferation
in the presence of neutralising antibody.
If an antibody or antigen binding fragment thereof is capable of
neutralisation then
this is indicative of inhibition of the interaction between hIL-13 and its
receptor.
Antibodies which are considered to have neutralising activity against human IL-
13
would have an ND50 of less than 100micrograms/ml, or less than 80micrograms/ml
in
the TF-1 cell proliferation assay as set out in Example 3.3, 3.4 or 3.5.
In an alternative aspect of the present invention there is provided antibodies
or
antigen binding fragments thereof which have equivalent neutralising activity
to the
antibodies exemplified herein, for example antibodies which retain the
neutralising
activity of H2L1 in the TF-1 cell proliferation assay as set out in Example
3.3, 3.4 or
3.5.
As used herein the term "modulates" means inhibition of the binding of IL-13
to its
receptor, and/or blocking of the interaction between IL-13 and its receptor
thereby
decoupling the hIL-13/hIL-13R signalling pathway. This can be inhibition
and/or
blocking of either or both of IL-13Ra1 and IL-13Ra2. The hIL-13 receptor as
used
herein means one or both of these receptors. Inhibition of binding of IL-13 to
its
receptor can be measured in several ways, for example by use of the assays as
set
out in the examples below, for example an ELISA method such as that described
in
Example 6.5 and 6.6.
The antibodies and antigen binding fragments thereof of the present invention
may
be therapeutic antibodies and antigen binding fragments thereof i.e.suitable
for use in
therapy.
In one aspect, there is provided an antibody or antigen binding fragment
thereof
which specifically binds hIL-13, and comprises a CDRH3 comprising the sequence
set forth in SEQ.I.D.NO:3.
In one embodiment the antibody or antigen binding fragment of the present
invention
neutralises human IL-13.
In another embodiment the antibody or antigen binding fragment of the present
invention modulates the binding of human IL-13 to its receptor.
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In another aspect of the present invention there is provided an antibody or
antigen
binding fragment thereof which specifically binds hIL-13 and modulates the
interaction between hIL-13 and hIL-13R.
In certain embodiments, antibodies of the invention at least inhibit the
interaction
between hIL-13 and hIL-13R but may also block the interaction between hIL-13
and
hIL-13R thereby decoupling the hIL-13/hIL-13R signalling pathway.
In another embodiment the present invention provides an antibody or antigen
binding
fragment wherein the CDRH3 comprises the sequence of SEQ ID NO:3. In a further
embodiment the antibody or antigen binding fragment thereof of the present
invention
further comprises one or more of the following sequences CDRH2: SEQ.I.D.NO:2,
CDRH1: SEQ.I.D.NO:1, CDRL1: SEQ.I.D.NO:4, CDRL2: SEQ.I.D.NO:5 and CDRL3:
SEQ.I.D.NO:6. In a further embodiment the present invention further comprises
these
CDR sequences in the context of a human framework, for example as a humanised
antibody or fragment thereof.
In another aspect of the present invention there is provided an antibody or
antigen
binding fragment thereof which specifically binds hIL-13, and comprises the
following
CDRs:
CDRH1: SEQ.I.D.NO:1
CDRH2: SEQ.I.D.NO: 2
CDRH3: SEQ.I.D.NO: 3
CDRL1: SEQ.I.D.NO:4
CDRL2: SEQ.I.D.NO:5
CDRL3: SEQ.I.D.NO:6
In one embodiment of the present invention one or more of the CDRs of the
antibody
or antigen binding fragment may comprise variants of the CDRs set out in the
sequences listed above. Each variant CDR will comprise one or two amino acid
residues which differ from the amino acid residue in the corresponding
position in the
sequence listed above. Such substitutions in amino acid residues may be
conservative substitutions, for example substituting one hydrophobic amino
acid for
an alternative hydrophobic amino acid, for example substituting Leucine with
Valine,
or Isoleucine.
Throughout this specification, amino acid residues in antibody sequences are
numbered according to the Kabat scheme. Similarly, the terms "CDR", "CDRL1",
"CDRL2", "CDRL3", "CDRH1", "CDRH2", "CDRH3" follow the Kabat numbering
system as set forth in Kabat et al; Sequences of proteins of Immunological
Interest
NIH, 1987 with the exception that position 30 of the heavy chain is taken to
be part of
a CDR.
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As used herein the term "comprising" and "comprises" incorporates "consisting
of'
and "consists of'.
In another aspect of the invention there is provided an antibody or antigen
binding
fragment thereof comprising a VH domain comprising the sequence set forth in
SEQ.I.D.NO:7 and a VL domain comprising the sequence set forth in
SEQ.I.D.NO:8.
In another aspect of the invention there is provided an isolated VH domain of
an
antibody comprising the sequence selected from the group consisting of
SEQ.I.D.NO:
7, 11, 12, 13 and 14. In one embodiment the isolated VH domain of an antibody
consists of or consists essentially of an isolated VH domain of an antibody
selected
from the group consisting of SEQ.I.D.NO: 7, 11, 12, 13 and 14.
In another aspect of the invention there is provided an antibody or antigen
binding
fragment thereof comprising a VH domain selected from the group consisting of;
SEQ.I.D.NO:7, 11,12,13 and 14.
In another aspect of the invention there is provided an antibody which
specifically
binds hIL-13 and at least inhibits the interaction between hIL-13 and hIL-13R
which
antibody comprises a heavy chain of SEQ.I.D.NO:18 and a light chain selected
from
the group consisting of; SEQ.I.D.NO:22, 23 and 24.
In another aspect of the invention there is provided an antibody which
specifically
binds hIL-13 and at least inhibits the interaction between hIL-13 and hIL-1 3R
which
antibody comprises a heavy chain of SEQ.I.D.NO:19 and a light chain selected
from
the group consisting of; SEQ.I.D.NO:22, 23 and 24.
In another aspect of the invention there is provided an antibody which
specifically
binds hIL-13 and at least inhibits the interaction between hIL-13 and hIL-13R
which
antibody comprises a heavy chain of SEQ.I.D.NO:20 and a light chain selected
from
the group consisting of; SEQ.I.D.NO:22, 23 and 24.
In another aspect of the invention there is provided an antibody which
specifically
binds hIL-13 and at least inhibits the interaction between hIL-13 and hIL-13R
which
antibody comprises a heavy chain of SEQ.I.D.NO:21 and a light chain selected
from
the group consisting of; SEQ.I.D.NO:22, 23 and 24.
In another aspect of the invention there is provided an antibody or antigen
binding
fragment thereof which inhibits the binding of the antibody comprising a heavy
chain
of SEQ.I.D.NO: 18 and a light chain of SEQ.I.D.NO:22 to hIL-13.
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In accordance with the present invention there is provided a humanised
antibody
which antibody comprises a VH domain of: SEQ.I.D.NO:11 and a VL domain
selected from the group consisting of: SEQ.I.D.NO:15, 16 and 17.
In accordance with the present invention there is provided a humanised
antibody
which antibody comprises a VH domain of: SEQ.I.D.NO:12 and a VL domain
selected from the group consisting of: SEQ.I.D.NO:15, 16 and 17.
In accordance with the present invention there is provided a humanised
antibody
which antibody comprises a VH domain of: SEQ.I.D.NO:13 and a VL domain
selected from the group consisting of: SEQ.I.D.NO:15, 16 and 17.
In accordance with the present invention there is provided a humanised
antibody
which antibody comprises a VH domain of: SEQ.I.D.NO:14 and a VL domain
selected from the group consisting of: SEQ.I.D.NO:15, 16 and 17.
In accordance with the present invention there is provided an antibody or
antigen
binding fragment thereof which binds to the peptides set out in SEQ ID NO:90,
99,
102, 103, 105, 106, 107, 108, 109, 110, 111, 112 and 114 but does not bind the
peptides set out in SEQ ID NO:100, 101, 104 and 113, wherein binding is
defined as
having an equivalent binding activity to the antibodies exemplified herein,
for example
antibodies which retain similar binding activity to 3G4 binding to human IL-13
peptides in the ELISA assay as set out in Example 6.4.
In another aspect of the invention there is provided a method of treating a
human
patient afflicted with a disease or disorder responsive to modulation of the
interaction
between hIL-13 and hIL-13R (such as asthma, COPD, allergic rhinitis, atopic
dermatitis) which method comprises the step of administering to said patient a
therapeutically effective amount of the antibody or antigen binding fragment
thereof
as described herein.
Use of an antibody of the invention in the manufacture of a medicament for the
treatment of a disease or disorder responsive to modulation of the interaction
between hIL-13 and hIL-13R is also provided.
In a further aspect the invention provides a method of selecting an anti-IL13
antibody
suitable for use in therapy, which method comprises i) providing an antibody
which
specifically binds to IL-13R(x1, ii) determining whether the antibody binds
specifically
to IL-13R(x2, and selecting an antibody which binds in step ii) for further
development.
Detailed Description of the Invention
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19
The antibodies of the present invention may be intact antibodies or fragments
thereof; human, chimaeric or humanised antibodies; and mono or bispecific.
1. Antibody Structures
1.1 Intact Antibodies
Intact antibodies include heteromultimeric glycoproteins comprising at least
two
heavy and two light chains. Aside from IgM, intact antibodies are usually
heterotetrameric glycoproteins of approximately 150Kda, composed of two
identical
light (L) chains and two identical heavy (H) chains. Typically, each light
chain is
linked to a heavy chain by one covalent disulfide bond while the number of
disulfide
linkages between the heavy chains of different immunoglobulin isotypes varies.
Each
heavy and light chain also has intrachain disulfide bridges. Each heavy chain
has at
one end a variable domain (VH) followed by a number of constant regions. Each
light chain has a variable domain (VL) and a constant region at its other end;
the
constant region of the light chain is aligned with the first constant region
of the heavy
chain and the light chain variable domain is aligned with the variable domain
of the
heavy chain. The light chains of antibodies from most vertebrate species can
be
assigned to one of two types called Kappa and Lambda based on the amino acid
sequence of the constant region. Depending on the amino acid sequence of the
constant region of their heavy chains, human antibodies can be assigned to
five
different classes, IgA, IgD, IgE, IgG and IgM. IgG and IgA can be further
subdivided
into subclasses, IgG1, IgG2, IgG3 and IgG4; and IgAl and IgA2. Species
variants
exist with mouse and rat having at least IgG2a, IgG2b. The variable domain of
the
antibody confers binding specificity upon the antibody with certain regions
displaying
particular variability called complementarity determining regions (CDRs). The
more
conserved portions of the variable region are called Framework regions (FR).
The
variable domains of intact heavy and light chains each comprise four FR
connected
by three CDRs. The CDRs in each chain are held together in close proximity by
the
FR regions and with the CDRs from the other chain contribute to the formation
of the
antigen binding site of antibodies. The constant regions are not directly
involved in
the binding of the antibody to the antigen but exhibit various effector
functions such
as participation in antibody dependent cell-mediated cytotoxicity (ADCC),
phagocytosis via binding to Fcy receptor, half-life/clearance rate via
neonatal Fc
receptor (FcRn) and complement dependent cytotoxicity via the C1 q component
of
the complement cascade.
In one embodiment therefore we provide an intact antibody that specifically
binds hIL-
13, which antibody modulates the interaction between hIL-13 and hIL-13R, for
example the antibody inhibits the interaction between hIL-13 and its receptor.
The
intact antibody may comprise a constant region of any isotype or subclass
thereof
described supra. In one embodiment, the antibody is of the IgG isotype,
particularly
1
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IgG1. The antibody may be rat, mouse, rabbit, primate or human. In one typical
embodiment, the antibody is primate (such as cynomolgus, Old World monkey or
Great Ape, see e.g. W099/55369, W093/02108) or human.
In another embodiment there is provided an isolated intact antibody comprising
a
CDRH3 of SEQ.I.D.NO: 3. In another embodiment there is provided an intact
antibody comprising a variable region comprising CDRs of SEQ.I.D.NO: 1,
2,3,4,5
and 6.
In another embodiment, there is provided an isolated murine intact antibody or
antigen binding fragment thereof comprising a VH domain comprising the
sequence
of SEQ.I.D.NO: 7 and a VL domain of the sequence of SEQ.I.D.NO: 8.
1.1.2 Human antibodies
Human antibodies may be produced by a number of methods known to those of
skill
in the art. Human antibodies can be made by the hybridoma method using human
myeloma or mouse-human heteromyeloma cells lines see Kozbor J.Immunol 133,
3001, (1984) and Brodeurl Monoclonal Antibody Production Techniques and
Applications, pp51-63 (Marcel Dekker Inc, 1987). Alternative methods include
the
use of phage libraries or transgenic mice both of which utilize human V region
repertories (see Winter G, (1994), Annu.Rev.lmmunol 12,433-455, Green LL
(1999),
J.Immunol.methods 231, 11-23).
Several strains of transgenic mice are now available wherein their mouse
immunoglobulin loci has been replaced with human immunoglobulin gene segments
(see Tomizuka K, (2000) PNAS 97,722-727; Fishwild D.M (1996) Nature
Biotechnol.
14,845-851, Mendez MJ, 1997, Nature Genetics, 15,146-156). Upon antigen
challenge such mice are capable of producing a repertoire of human antibodies
from
which antibodies of interest can be selected. Of particular note is the
TrimeraT""
system (see Eren R et al, (1998) Immunology 93:154-161) where human
lymphocytes are transplanted into irradiated mice, the Selected Lymphocyte
Antibody
System (SLAM, see Babcook et al, PNAS (1996) 93:7843-7848) where human (or
other species) lymphocytes are effectively put through a massive pooled in
vitro
antibody generation procedure followed by deconvulated, limiting dilution and
selection procedure and the Xenomouse lIT~" (Abgenix Inc). An alternative
approach
is available from Morphotek Inc using the MorphodomaTM technology.
Phage display technology can be used to produce human antibodies (and
fragments
thereof), see McCafferty; Nature, 348, 552-553 (1990) and Griffiths AD et al
(1994)
EMBO 13:3245-3260. According to this technique antibody V domain genes are
cloned in frame into either a major or minor coat of protein gene of a
filamentous
bacteriophage such as M13 or fd and displayed (usually with the aid of a
helper
phage) as functional antibody fragments on the surface of the phage particle.
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Selections based on the functional properties of the antibody result in
selection of the
gene encoding the antibody exhibiting those properties. The phage display
technique can be used to select antigen specific antibodies from libraries
made from
human B cells taken from individuals afflicted with a disease or disorder
described
above or alternatively from unimmunized human donors (see Marks; J.Mol.Bio.
222,581-597, 1991). Where an intact human antibody is desired comprising a Fc
domain it is necessary to recione the phage displayed derived fragment into a
mammalian expression vectors comprising the desired constant regions and
establishing stable expressing cell lines.
The technique of affinity maturation (Marks; Bio/technol 10,779-783 (1992))
may be
used to improve binding affinity wherein the affinity of the primary human
antibody is
improved by sequentially replacing the H and L chain V regions with naturally
occurring variants and selecting on the basis of improved binding affinities.
Variants
of this technique such as "epitope imprinting" are now also available see WO
93/06213. See also Waterhouse; Nucl.Acids Res 21, 2265-2266 (1993).
Thus in another embodiment there is provided an isolated human intact antibody
or
antigen binding fragment thereof which specifically binds hIL-13 and modulates
the
interaction between hIL-13 and hIL-13R, for example where it inhibits the
interaction
between hIL-13 and its receptor.
In another aspect there is provided an isolated human intact antibody or
antigen
binding fragment thereof comprising a CDRH3 of SEQ.I.D.NO: 3 which
specifically
binds hIL-13 and modulates the interaction between hIL-13 and hIL-13R, for
example
where it inhibits the interaction between hIL-13 and its receptor. In another
aspect
there is provided an isolated human intact antibody or antigen binding
fragment
thereof comprising a variable region comprising CDRs of SEQ.I.D.NO: 1, 2, 3,
4, 5
and 6 as defined supra.
1.2 Chimaeric and Humanised Antibodies
The use of intact non-human antibodies in the treatment of human diseases or
disorders carries with it the potential for the now well established problems
of
immunogenicity, that is the immune system of the patient may recognise the non-
human intact antibody as non-self and mount a neutralising response. This is
particularly evident upon multiple administration of the non-human antibody to
a
human patient. Various techniques have been developed over the years to
overcome these problems and generally involve reducing the composition of non-
human amino acid sequences in the intact antibody whilst retaining the
relative ease
in obtaining non-human antibodies from an immunised animal e.g. mouse, rat or
rabbit. Broadly two approaches have been used to achieve this. The first are
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22
chimaeric antibodies, which generally comprise a non-human (e.g. rodent such
as
mouse) variable domain fused to a human constant region. Because the antigen-
binding site of an antibody is localised within the variable regions the
chimaeric
antibody retains its binding affinity for the antigen but acquires the
effector functions
of the human constant region and are therefore able to perform effector
functions
such as described supra. Chimaeric antibodies are typically produced using
recombinant DNA methods. DNA encoding the antibodies (e.g. cDNA) is isolated
and sequenced using conventional procedures (e.g. by using oligonucleotide
probes
that are capable of binding specifically to genes encoding the H and L chains
of the
antibody of the invention, e.g. DNA encoding SEQ.I.D.NO 1,2,3,4,5 and 6
described
supra). Hybridoma cells serve as a typical source of such DNA. Once isolated,
the
DNA is placed into expression vectors which are then transfected into host
cells such
as E.Coli, COS cells, CHO cells or myeloma cells that do not otherwise produce
immunoglobulin protein to obtain synthesis of the antibody. The DNA may be
modified by substituting the coding sequence for human L and H chains for the
corresponding non-human (e.g. murine) H and L constant regions see e.g.
Morrison;
PNAS 81, 6851 (1984).
The second approach involves the generation of humanised antibodies wherein
the
non-human content of the antibody is reduced by humanizing the variable
regions.
Two techniques for humanisation have gained popularity. The first is
humanisation
by CDR grafting. CDRs build loops close to the antibody's N-terminus where
they
form a surface mounted in a scaffold provided by the framework regions.
Antigen-
binding specificity of the antibody is mainly defined by the topography and by
the
chemical characteristics of its CDR surface. These features are in turn
determined
by the conformation of the individual CDRs, by the relative disposition of the
CDRs,
and by the nature and disposition of the side chains of the residues
comprising the
CDRs. A large decrease in immunogenicity can be achieved by grafting only the
CDRs of a non-human (e.g. murine) antibodies ("donor" antibodies) onto human
framework ("acceptor framework") and constant regions (see Jones et al (1986)
Nature 321,522-525 and Verhoeyen M etal (1988) Science 239, 1534-1536).
However, CDR grafting per se may not result in the complete retention of
antigen-
binding properties and it is frequently found that some framework residues
(sometimes referred to as "back-mutations") of the donor antibody need to be
preserved in the humanised molecule if significant antigen-binding affinity is
to be
recovered (see Queen C et al (1989) PNAS 86, 10,029-10,033, Co, M et al (1991)
Nature 351, 501-502). In this case, human V regions showing the greatest
sequence
homology to the non-human donor antibody are chosen from a database in order
to
provide the human framework (FR). The selection of human FRs can be made
either
from human consensus or individual human antibodies. Where necessary key
residues from the donor antibody are substituted into the human acceptor
framework
to preserve CDR conformations. Computer modelling of the antibody maybe used
to
help identify such structurally important residues, see W099/48523.
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23
Alternatively, humanisation maybe achieved by a process of "veneering". A
statistical analysis of unique human and murine immunoglobulin heavy and light
chain variable regions revealed that the precise patterns of exposed residues
are
different in human and murine antibodies, and most individual surface
positions have
a strong preference for a small number of different residues (see Padlan E.A.
et al;
(1991) Mol.Immunol.28, 489-498 and Pedersen J.T. et a! (1994) J.Mol.Biol. 235;
959-
973). Therefore it is possible to reduce the immunogenicity of a non-human Fv
by
replacing exposed residues in its framework regions that differ from those
usually
found in human antibodies. Because protein antigenicity may be correlated with
surface accessibility, replacement of the surface residues may be sufficient
to render
the mouse variable region "invisible" to the human immune system (see also
Mark
G.E. et a/ (1994) in Handbook of Experimental Pharmacology vo1.113: The
pharmacology of monoclonal Antibodies, Springer-Verlag, pp105-134). This
procedure of humanisation is referred to as "veneering" because only the
surface of
the antibody is altered, the supporting residues remain undisturbed.
The skilled man will be aware that other methods of antibody humanisation
exist and
are available in the literature.
Thus another embodiment of the invention there is provided a chimaeric
antibody
comprising a non-human (e.g. rodent) variable domain fused to a human constant
region (which maybe of a IgG isotype e.g. IgG1) which specifically binds hIL-
13 and
modulates the interaction between hIL-13 and hIL-13R, for example where it
inhibits
the interaction between hIL-13 and its receptor.
In another embodiment there is provided a chimaeric antibody comprising a non-
human (e.g. rodent) variable region and a human constant region (which maybe
of an
IgG isotype e.g. IgG1) which specifically binds hIL-13, which antibody further
comprises a CDRH3 of SEQ.I.D.N03. Such antibodies may further comprise a
human constant region of the IgG isotype, e.g. IgG1
In another embodiment there is chimaeric antibody comprising a non-human (e.g.
rodent) variable region and a human constant region (which maybe of a IgG
isotype,
for example IgG1) which specifically binds hIL-13 comprising the CDRs of
SEQ.I.D.NO: 1, 2,3,4,5 and 6.
In another embodiment there is provided a chimaeric antibody comprising a VH
domain of SEQ.I.D.NO:7 and a VL domain of SEQ.I.D.NO:8 and a human constant
region of an IgG isotype, e.g. IgG1 which specifically binds hIL-13 and
modulates the
interaction between hIL-13 and hIL-13R, for example where it inhibits the
interaction
between h1L-13 and its receptor.
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24
In another embodiment there is provided a humanised antibody or antigen
binding
fragment thereof which specifically binds hIL-13 and modulates the interaction
between hIL-1 3 and hIL-1 3R, for example where it inhibits the interaction
between
hIL-13 and its receptor.
In another embodiment there is provided a humanised antibody or antigen
binding
fragment thereof which specifically binds hIL-13 and comprises a CDRH3 of
SEQ.I.D.NO: 3. Such antibodies may comprise a human constant region of the IgG
isotype, e.g. IgG1.
In another embodiment there is provided a humanised antibody or antigen
binding
fragment thereof which specifically binds hIL-13 and comprises CDRs of
SEQ.I.D.NO1, 2,3,4,5 and 6. Such antibodies may comprise a human constant
region of the IgG isotype, e.g. IgG1.
In accordance with the present invention there is provided a humanised
antibody
which antibody comprises a VH domain selected from the group of:
SEQ.I.D.NO:11,
and a VL domain selected from the group of: SEQ.I.D.NO:15,16,17. Such
antibodies
may comprise a human constant region of the IgG isotype e.g. IgG1.
In accordance with the present invention there is provided a humanised
antibody
which antibody comprises a VH domain selected from the group of:
SEQ.I.D.NO:12,
and a VL domain selected from the group of: SEQ.I.D.NO:15,16,17. Such
antibodies
may comprise a human constant region of the IgG isotype e.g. IgG1.
In accordance with the present invention there is provided a humanised
antibody
which antibody comprises a VH domain selected from the group of:
SEQ.I.D.NO:13,
and a VL domain selected from the group of: SEQ.I.D.NO:15,16,17. Such
antibodies
may comprise a human constant region of the IgG isotype e.g. IgG1.
In accordance with the present invention there is provided a humanised
antibody
which antibody comprises a VH domain selected from the group of:
SEQ.I.D.NO:14,
and a VL domain selected from the group of: SEQ.I.D.NO:15,16,17. Such
antibodies
may comprise a human constant region of the IgG isotype e.g. IgG1
In another embodiment there is provided a humanised antibody which antibody
comprises a VH domain of SEQ.I.D.NO: 11 and a VL domain of SEQ.I.D.NO:15.
In another embodiment there is provided a humanised antibody which antibody
comprises a VH domain of SEQ.I.D.NO: 12 and a VL domain of SEQ.I.D.NO:15.
In another embodiment there is provided a humanised antibody which antibody
comprises a VH domain of SEQ.I.D.NO: 13 and a VL domain of SEQ.I.D.NO:15.
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In another embodiment there is provided a humanised antibody which antibody
comprises a VH domain of SEQ.l.D.NO: 14 and a VL domain of SEQ.I.D.N0:15.
In another embodiment there is provided a humanised antibody which antibody
comprises a VH domain of SEQ.I.D.NO: 11 and a VL domain of SEQ.I.D.NO:16.
In another embodiment there is provided a humanised antibody which antibody
comprises a VH domain of SEQ.I.D.NO: 12 and a VL domain of SEQ.I.D.NO:16.
In another embodiment there is provided a humanised antibody which antibody
comprises a VH domain of SEQ.I.D.NO: 13 and a VL domain of SEQ.I.D.NO:16.
In another embodiment there is provided a humanised antibody which antibody
comprises a VH domain of SEQ.I.D.NO: 14 and a VL domain of SEQ.I.D.NO:16.
In another embodiment there is provided a humanised antibody which antibody
comprises a VH domain of SEQ.I.D.NO: 11 and a VL domain of SEQ.I.D.NO:17.
In another embodiment there is provided a humanised antibody which antibody
comprises a VH domain of SEQ.I.D.NO: 12 and a VL domain of SEQ.I.D.NO:17.
In another embodiment there is provided a humanised antibody which antibody
comprises a VH domain of SEQ.I.D.NO: 13 and a VL domain of SEQ.I.D.NO:17.
In another embodiment there is provided a humanised antibody which antibody
comprises a VH domain of SEQ.I.D.NO: 14 and a VL domain of SEQ.I.D.NO:17.
In another embodiment, there is provided a humanised antibody or antigen
binding
fragment thereof which specifically binds hIL-13 wherein said antibody or
fragment
thereof comprises CDRH3 of SEQ.I.D.NO:3 optionally further comprising one or
more
of CDRs of SEQ.I.D.NO:1, 2, 4, 5, and 6 wherein one or more of the residue(s)
selected from the group consisting of position 10, 30, 67, 69, 71, 73 and 93
of the
human acceptor heavy chain framework and one or both residue(s) at position 76
and 98 of the human acceptor light chain framework are substituted by the
corresponding residues found in the donor antibody framework from which CDRH3
is
derived (which is set out in SEQ.I.D.NO:7).
In another embodiment there is provided a humanised antibody or antigen
binding
fragment thereof which specifically binds hIL-13 wherein said antibody or
fragment
thereof comprises CDRH3 of SEQ.I.D.NO:3, optionally further comprising one or
more CDRs of SEQ.I.D.NO:1,2,4,5 and 6 wherein the human heavy chain framework
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26
comprises one or more (e.g. all) of the following residues (or conservative
substitution thereof);
Position Residue
D
30 1
67 A
69 L
71 A
73 K
93 T
and a light chain framework comprising either or both of the following
residues (or
conservative substitute thereof);
76 N
98 L
It will be apparent to those skilled in the art that the term "derived" is
intended to
define not only the source in the sense of it being the physical origin for
the material
but also to define material which is structurally identical (in terms of
primary amino
acid sequence) to the material but which does not originate from the reference
source. Thus "residues found in the donor antibody from which CDRH3 is
derived"
need not necessarily have been purified from the donor antibody.
It is well recognised in the art that certain amino acid substitutions are
regarded as
being "conservative". Amino acids are divided into groups based on common side-
chain properties and substitutions within groups that maintain all or
substantially all of
the binding affinity of the antibody of the invention or antigen binding
fragment thereof
are regarded as conservative substitutions, see table 1:
Table 1
Side chain Members
H dro hobic met, ala,val,Ieu,ile
neutral h dro hilic c s, ser, thr
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Acidic as , glu
Basic asn, In, his, I s, ar
residues that influence chain gly, pro
orientation
Aromatic trp, tyr, phe
In accordance with the present invention there is provided a humanised
antibody
comprising a heavy chain selected from the group consisting of: SEQ.I.D.NO:
18,19,20,21 and a light chain selected from the group consisting of;
SEQ.I.D.NO:22,
23, 24.
In one embodiment of the invention there is provided a humanised antibody
which
specifically binds hIL-13 comprising a heavy chain of SEQ.I.D.NO:18 and a
light
chain of SEQ.I.D.NO:22.
In one embodiment of the invention there is provided a humanised antibody
which
specifically binds hIL-13 comprising a heavy chain of SEQ.I.D.NO:19 and a
light
chain of SEQ.I.D.NO:22.
In one embodiment of the invention there is provided a humanised antibody
which
specifically binds hIL-13 comprising a heavy chain of SEQ.I.D.NO:20 and a
light
chain of SEQ.I.D.NO:22.
In one embodiment of the invention there is provided a humanised antibody
which
specifically binds hIL-13 comprising a heavy chain of SEQ.I.D.NO:21 and a
light
chain of SEQ.I.D.NO:22.
In one embodiment of the invention there is provided a humanised antibody
which
specifically binds hIL-13 comprising a heavy chain of SEQ.I.D.NO:18 and a
light
chain of SEQ.I.D.NO:23.
In one embodiment of the invention there is provided a humanised antibody
which
specifically binds hIL-13 comprising a heavy chain of SEQ.I.D.NO:19 and a
light
chain of SEQ.I.D.NO:23.
In one embodiment of the invention there is provided a humanised antibody
which
specifically binds hIL-13 comprising a heavy chain of SEQ.I.D.NO:20 and a
light
chain of SEQ.I.D.NO:23.
In one embodiment of the invention there is provided a humanised antibody
which
specifically binds hIL-13 comprising a heavy chain of SEQ.I.D.NO:21 and a
light
chain of SEQ.I.D.NO:23.
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28
In one embodiment of the invention there is provided a humanised antibody
which
specifically binds hIL-13 comprising a heavy chain of SEQ.I.D.NO:18 and a
light
chain of SEQ.I.D.NO:24.
In one embodiment of the invention there is provided a humanised antibody
which
specifically binds hIL-13 comprising a heavy chain of SEQ.I.D.NO:19 and a
light
chain of SEQ.I.D.NO:24.
In one embodiment of the invention there is provided a humanised antibody
which
specifically binds hIL-13 comprising a heavy chain of SEQ.I.D.NO:20 and a
light
chain of SEQ.I.D.NO:24.
In one embodiment of the invention there is provided a humanised antibody
which
specifically binds hIL-13 comprising a heavy chain of SEQ.I.D.NO:21 and a
light
chain of SEQ.I.D.NO:24.
In one embodiment of the present invention there is provided a human or
humanised
heavy chain variable region comprising each of the CDRs listed in SEQ ID NO 1-
3. In
another embodiment of the present invention there is provided a humanised
heavy
chain variable region comprising the CDRs listed in SEQ ID NO 1-3 within the
larger
sequence of a human heavy chain variable region. In yet another embodiment the
humanised heavy chain variable region comprises the CDRs listed in SEQ ID NO 1-
3
within an acceptor antibody framework having greater than 40% identity in the
framework regions, or greater than 50%, or greater than 60%, or greater than
65%
identity to the murine 3G4 donor antibody heavy chain variable region (SEQ ID
NO.
7).
In one aspect of the present invention the antibodies comprise a heavy chain
variable
region comprising the amino acid sequence of SEQ ID NO. 44 further comprising
a
number of substitutions at one or more of positions 10, 30, 67, 69, 71, 73, 93
(Kabat
numbering system); wherein each substituted amino acid residue is replaced
with the
amino acid residue at the equivalent position in SEQ ID NO. 7 (the heavy chain
variable region of the donor antibody 3G4) and the number of substitutions is
between 0 and 7. In other embodiments the number of substitutions is 0, or 1,
or 2, or
3, or 4, or 5, or 6, or 7.
In one embodiment of the present invention there is provided a human or
humanised
light chain variable region comprising each of the CDRs listed in SEQ ID NO 4-
6. In
another embodiment of the present invention there is provided a humanised
light
chain variable region comprising the CDRs listed in SEQ ID NO 4-6 within the
larger
sequence of a human light chain variable region. In yet another embodiment the
humanised light chain variable region comprises the CDRs listed in SEQ ID NO 4-
6
within an acceptor antibody framework having greater than 40% identity in the
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29
framework regions, or greater than 50%, or greater than 60%, or greater than
65%
identity to the murine 3G4 donor antibody heavy chain variable region (SEQ ID
NO.
8).
in one aspect of the present invention the antibodies comprise a light chain
variable
region comprising the amino acid sequence of SEQ ID NO. 45 further comprising
a
number of substitutions at one or more of positions 76, 98 (Kabat numbering
system);
wherein each substituted amino acid residue is replaced with the amino acid
residue
at the equivalent position in SEQ ID NO. 8 (the light chain variable region of
the
donor antibody 3G4) and the number of substitutions is between 0 and 2. In
other
embodiments the number of substitutions is 0 or 1 or 2.
1.3 Bispecific antibodies
A bispecific antibody is an antibody having binding specificities for at least
two
different epitopes. Methods of making such antibodies are known in the art.
Traditionally, the recombinant production of bispecific antibodies is based on
the
coexpression of two immunoglobulin H chain-L chain pairs, where the two H
chains
have different binding specificities see Millstein et al, Nature 305 537-539
(1983),
WO93/08829 and Traunecker et al EMBO, 10, 1991, 3655-3659. Because of the
random assortment of H and L chains, a potential mixture of ten different
antibody
structures are produced of which only one has the desired binding specificity.
An
alternative approach involves fusing the variable domains with the desired
binding
specificities to heavy chain constant region comprising at least part of the
hinge
region, CH2 and CH3 regions. It is preferred to have the CH1 region containing
the
site necessary for light chain binding present in at least one of the fusions.
DNA
encoding these fusions, and if desired the L chain are inserted into separate
expression vectors and are then cotransfected into a suitable host organism.
It is
possible though to insert the coding sequences for two or all three chains
into one
expression vector. In one preferred approach, the bispecific antibody is
composed
of a H chain with a first binding specificity in one arm and a H-L chain pair,
providing
a second binding specificity in the other arm, see W094104690. Also see Suresh
et
a/ Methods in Enzymology 121, 210, 1986.
In one embodiment of the invention there is provided a bispecific antibody
wherein at
least one binding specificity of said antibody is for hIL-13, wherein said
antibody
modulates the interaction between hiL-13 and IL-13R, for example where it
inhibits
the interaction between hIL-13 and its receptor. Such antibodies may further
comprise a human constant region of the IgG isotype, e.g. IgG1. In some
embodiments, the bispecific therapeutic antibody has a first binding
specificity for hIL-
13 and modulates the interaction between hIL-13 and hIL-13R, for example where
it
inhibits the interaction between hIL-13 and its receptor and a second binding
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specificity for hIL-4 and modulates the interaction between hIL-4 and a
receptor for
hIL-4, for example where it inhibits the interaction between hIL-4 and its
receptor.
In one embodiment of the invention there is provided a bispecific antibody
wherein at
least one binding specificity of said antibody is for hlL-13, wherein said
antibody
comprises a CDRH3 of SEQ.I.D.NO: 3. Such antibodies may further comprise a
human constant region of the IgG isotype, e.g. IgG1.
In one embodiment of the invention there is provided a bispecific antibody
wherein at
least one binding specificity of said antibody is for hIL-13, wherein said
antibody
comprises at least CDRs of SEQ.I.D.NO: 1, 2,3,4,5 and 6. Such antibodies may
further comprise a human constant region of the IgG isotype, e.g. IgG1.
1.4 Antibody Fragments
In certain embodiments of the invention there is provided antibody fragments
which
modulate the interaction between hIL-13 and hIL-13R, for example where the
fragments inhibit the interaction between hIL-13 and its receptor. Such
fragments
may be functional antigen binding fragments of intact and/or humanised and/or
chimaeric antibodies such as Fab, Fab', F(ab')2, Fv, ScFv fragments of the
antibodies
described supra. Traditionally such fragments are produced by the proteolytic
digestion of intact antibodies by e.g. papain digestion (see for example, WO
94/29348) but may be produced directly from recombinantly transformed host
cells.
For the production of ScFv, see Bird et al ;(1988) Science, 242, 423-426. In
addition,
antibody fragments may be produced using a variety of engineering techniques
as
described below.
Fv fragments appear to have lower interaction energy of their two chains than
Fab
fragments. To stablise the association of the VH and VL domains, they have
been
Iinked with peptides (Bird et al, (1988) Science 242, 423-426, Huston et al,
PNAS,
85, 5879-5883), disulphide bridges (Glockshuber et al, (1990) Biochemistry,
29,
1362-1367) and "knob in hole" mutations (Zhu et al (1997), Protein Sci., 6,
781-788).
ScFv fragments can be produced by methods well known to those skilled in the
art
see Whitlow et al (1991) Methods companion Methods Enzymol, 2, 97-105 and
Huston et al (1993) Int.Rev.lmmunol 10, 195-217. ScFv may be produced in
bacterial cells such as E.Coli but are more preferably produced in eukaryotic
cells.
One disadvantage of ScFv is the monovalency of the product, which precludes an
increased avidity due to polyvalent binding, and their short half-life.
Attempts to
overcome these problems include bivalent (ScFv')2 produced from ScFV
containing
an additional C terminal cysteine by chemical coupling (Adams et a/ (1993)
Can.Res
53, 4026-4034 and McCartney et a/ (1995) Protein Eng. 8, 301-314) or by
spontaneous site-specific dimerization of ScFv containing an unpaired C
terminal
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31
cysteine residue (see Kipriyanov et al (1995) Cell. Biophys 26, 187-204).
Alternatively, ScFv can be forced to form multimers by shortening the peptide
linker
to 3 to 12 residues to form "diabodies", see Holliger et al PNAS (1993), 90,
6444-
6448. Reducing the linker still further can result in ScFV trimers
("triabodies", see
Kortt et al (1997) Protein Eng, 10, 423-433) and tetramers ("tetrabodies", see
Le Gall
et a/ (1999) FEBS Lett, 453, 164-168). Construction of bivalent ScFV molecules
can
also be achieved by genetic fusion with protein dimerizing motifs to form
"miniantibodies" (see Pack et al (1992) Biochemistry 31, 1579-1584) and
"minibodies" (see Hu et a/ (1996), Cancer Res. 56, 3055-3061). ScFv-Sc-Fv
tandems ((ScFV)2) may also be produced by linking two ScFv units by a third
peptide
linker, see Kurucz et al (1995) J.Immol.154, 4576-4582. Bispecific diabodies
can be
produced through the noncovalent association of two single chain fusion
products
consisting of VH domain from one antibody connected by a short linker to the
VL
domain of another antibody, see Kipriyanov et al (1998), Int.J.Can 77,763-772.
The
stability of such bispecific diabodies can be enhanced by the introduction of
disuiphide bridges or "knob in hole" mutations as described supra or by the
formation
of single chain diabodies (ScDb) wherein two hybrid ScFv fragments are
connected
through a peptide linker see Kontermann et al (1999) J.Immunol.Methods 226 179-
188. Tetravalent bispecific molecules are available by e.g. fusing a ScFv
fragment to
the CH3 domain of an IgG molecule or to a Fab fragment through the hinge
region
see Coloma et a/ (1997) Nature Biotechnol. 15, 159-163. Alternatively,
tetravalent
bispecific molecules have been created by the fusion of bispecific single
chain
diabodies (see Alt et al, (1999) FEBS Lett 454, 90-94. Smaller tetravalent
bispecific
molecules can also be formed by the dimerization of either ScFv-ScFv tandems
with
a linker containing a helix-loop-helix motif (DiBi miniantibodies, see Muller
et al
(1998) FEBS Lett 432, 45-49) or a single chain molecule comprising four
antibody
variable domains (VH and VL) in an orientation preventing intramolecular
pairing
(tandem diabody, see Kipriyanov et al, (1999) J.Mol.Biol. 293, 41-56).
Bispecific
F(ab')2 fragments can be created by chemical coupling of Fab' fragments or by
heterodimerization through leucine zippers (see Shalaby et al, (1992)
J.Exp.Med.
175, 217-225 and Kostelny et a/ (1992), J.Immunol. 148, 1547-1553). Also
available
are isolated VH and VL domains (Domantis plc), see US 6, 248,516; US
6,291,158;
US 6, 172,197.
In one embodiment there is provided an antibody fragment (e.g. ScFv, Fab,
Fab',
F(ab')2) or an engineered antibody fragment as described supra that
specifically
binds hIL-13 and modulates the interaction between hIL-13 and hIL-13R, for
example
where such fragments inhibit the interaction between hIL-13 and its receptor.
The
antibody fragment may comprise a CDRH3 comprising the sequence of SEQ.I.D.NO:
3 optionally together with further CDRs comprising one or more of the
sequences as
set out in SEQ.I.D.NO: 1, 2, 4, 5 and 6.
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32
A ScFv may be made comprising the VH and VL regions of the antibodies of the
present invention. For example a ScFv may comprise SEQ.I.D.NO: 12 and 15, or
for
example may comprise SEQ.I.D.NO: 13 and 15. This could be made by the
polynucleotides according to the invention, for example the sequences set out
in
SEQ.I.D.NO: 93 and 94, or for example by the sequences set out in SEQ.I.D.NO:
28
and 31. In one embodiment of the invention is provided a ScFv comprising a
protein
encoded by the sequences as set out in SEQ.I.D.NO: 93 and 94.
1.5 Heteroconiuqate antibodies
Heteroconjugate antibodies also form an embodiment of the present invention.
Heteroconjugate antibodies are composed of two covalently joined antibodies
formed
using any convenient cross-linking methods. See, for example, US 4,676,980.
1.6 Other Modifications.
The interaction between the Fc region of an antibody and various Fc receptors
(FcyR) is believed to mediate the effector functions of the antibody which
include
antibody-dependent cellular cytotoxicity (ADCC), fixation of complement,
phagocytosis and half-life/clearance of the antibody. Various modifications to
the Fc
region of antibodies of the invention may be carried out depending on the
desired
property. For example, specific mutations in the Fc region to render an
otherwise
lytic antibody, non-lytic is detailed in EP 0629 240B1 and EP 0307 434B2 or
one may
incorporate a salvage receptor binding epitope into the antibody to increase
serum
half life see US 5,739,277. There are five currently recognised human Fcy
receptors, FcyR (I), FcyRlla, FcyRllb, FcyRIIIa and neonatal FcRn. Shields et
al,
(2001) J.Biol.Chem 276, 6591-6604 demonstrated that a common set of IgG1
residues is involved in binding all Fc7Rs, while FcyRll and FcyRIIi utilize
distinct sites
outside of this common set. One group of IgG1 residues reduced binding to all
FcyRs when altered to alanine: Pro-238, Asp-265, Asp-270, Asn-297 and Pro-239.
All are in the IgG CH2 domain and clustered near the hinge joining CH1 and
CH2.
While FcyRI utilizes only the common set of IgG1 residues for binding, FcyRII
and
FcyRIIi interact with distinct residues in addition to the common set.
Alteration of
some residues reduced binding only to FcyRll (e.g. Arg-292) or FcyRIIl (e.g.
Glu-
293). Some variants showed improved binding to FcyRll or FcyRIII but did not
affect
binding to the other receptor (e.g. Ser-267AIa improved binding to FcyRil but
binding
to FcyRili was unaffected). Other variants exhibited improved binding to
FcyRII or
FcyRlll with reduction in binding to the other receptor (e.g. Ser-298AIa
improved
binding to FcyRIIl and reduced binding to FcyR11). For FcyRIIIa, the best
binding
IgG1 variants had combined alanine substitutions at Ser-298, Glu-333 and Lys-
334.
The neonatal FcRn receptor is believed to be involved in both antibody
clearance and
the transcytosis across tissues (see Junghans R.P (1997) Immunol.Res 16. 29-57
and Ghetie et al (2000) Annu.Rev.lmmunol. 18, 739-766). Human IgGI residues
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33
determined to interact directly with human FcRn includes I1e253, Ser254,
Lys288,
Thr307, GIn311, Asn434 and His435. Switches at any of these positions
described in
this Example may enable increased serum half-life and/or altered effector
properties
of antibodies of the invention.
Other modifications include glycosylation variants of the antibodies of the
invention.
Glycosylation of antibodies at conserved positions in their constant regions
is known
to have a profound effect on antibody function, particularly effector
functioning such
as those described above, see for example, Boyd et al (1996), Mol.lmmunol. 32,
1311-1318. Glycosylation variants of the antibodies or antigen binding
fragments
thereof of the present invention wherein one or more carbohydrate moiety is
added,
substituted, deleted or modified are contemplated. Introduction of an
asparagine-X-
serine or asparagine-X-threonine motif creates a potential site for enzymatic
attachment of carbonhydrate moieties and may therefore be used to manipulate
the
glycosylation of an antibody. In Raju et at (2001) Biochemistry 40, 8868-8876
the
terminal sialyation of a TNFR-IgG immunoadhesin was increased through a
process
of regalactosylation and/or resialylation using beta-1, 4-
galactosyltransferace and/or
alpha, 2,3 sialyltransferase. Increasing the terminal sialylation is believed
to increase
the half-life of the immunoglobulin. Antibodies, in common with most
glycoproteins,
are typically produced as a mixture of glycoforms. This mixture is
particularly
apparent when antibodies are produced in eukaryotic, particularly mammalian
cells.
A variety of methods have been developed to manufacture defined glycoforms,
see
Zhang et al Science (2004), 303, 371, Sears et al, Science, (2001) 291, 2344,
Wacker et al (2002) Science, 298 1790, Davis et a/ (2002) Chem.Rev. 102, 579,
Hang et al (2001) Acc.Chem.Res 34, 727. Thus the invention contemplates a
plurality of (monoclonal) antibodies (which maybe of the IgG isotype, e.g.
IgG1) as
herein described comprising a defined number (e.g. 7 or less, for example 5 or
less
such as two or a single) glycoform(s) of said antibodies or antigen binding
fragments
thereof.
Further embodiments of the invention include antibodies of the invention or
antigen
binding fragments thereof coupled to a non-proteinaeous polymer such as
polyethylene glycol (PEG), polypropylene glycol or polyoxyalkylene.
Conjugation of
proteins to PEG is an established technique for increasing half-life of
proteins, as well
as reducing antigenicity and immunogenicity of proteins. The use of PEGylation
with
different molecular weights and styles (linear or branched) has been
investigated with
intact antibodies as well as Fab' fragments, see Koumenis I.L. et a/ (2000)
I nt. J. P h a rm a ceut. 198 : 83-95 .
2. Production Methods
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Antibodies of the invention maybe produced as a polyclonal population but are
more
preferably produced as a monoclonal population (that is as a substantially
homogenous population of identical antibodies directed against a specific
antigenic
binding site). It will of course be apparent to those skilled in the art that
a population
implies more than one antibody entity. Antibodies of the present invention may
be
produced in transgenic organisms such as goats (see Pollock et al (1999),
J.Immunol.Methods 231:147-157), chickens (see Morrow KJJ (2000)
Genet.Eng.News 20:1-55, mice (see Pollock et al) or plants (see Doran PM,
(2000)
Curr.Opinion Biotechnol. 11, 199-204, Ma JK-C (1998), Nat.Med. 4; 601-606,
Baez J
et al, BioPharm (2000) 13: 50-54, Stoger E et al; (2000) Plant Mol.Biol.
42:583-590).
Antibodies may also be produced by chemical synthesis. However, antibodies of
the
invention are typically produced using recombinant cell culturing technology
well
known to those skilled in the art. A polynucleotide encoding the antibody is
isolated
and inserted into a replicable vector such as a plasmid for further cloning
(amplification) or expression. One useful expression system is a glutamate
synthetase system (such as sold by Lonza Biologics), particularly where the
host cell
is CHO or NSO (see below). Polynucleotide encoding the antibody is readily
isolated
and sequenced using conventional procedures (e.g. oligonucleotide probes).
Vectors
that may be used include plasmid, virus, phage, transposons, minichromsomes of
which plasmids are a typical embodiment. Generally such vectors further
include a
signal sequence, origin of replication, one or more marker genes, an enhancer
element, a promoter and transcription termination sequences operably linked to
the
light and/or heavy chain polynucleotide so as to facilitate expression.
Polynucleotide
encoding the light and heavy chains may be inserted into separate vectors and
transfected into the same host cell or, if desired both the heavy chain and
light chain
can be inserted into the same vector for transfection into the host cell. Thus
according to one aspect of the present invention there is provided a process
of
constructing a vector encoding the light and/or heavy chains of an antibody or
antigen binding fragment thereof of the invention, which method comprises
inserting
into a vector, a polynucleotide encoding either a light chain and/or heavy
chain of an
antibody of the invention.
In other aspect of the invention there is provided a polynucleotide encoding a
murine
VH domain comprising the sequence set forth as SEQ.I.D.NO:25.
In another aspect of the invention there is provided polynucleotide encoding a
murine
VL domain comprising the sequence set forth as SEQ.I.D.NO: 26.
In another embodiment there is provided a polynucletotide encoding a VH domain
comprising the sequence selected from the group consisting of SEQ.I.D.NO:27,
28,
29,30
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In another embodiment there is provided a polynucletotide encoding a VL domain
comprising the sequence selected from the group consisting of; SEQ.I.D.NO: 31,
32,
33
In accordance with the present invention there is provided a polynucleotide
encoding
a heavy chain of the invention which polynucleotide is selected from the group
consisting of; SEQ.I.D.NO:34, 35, 36, 37.
In accordance with the present invention there is provided a polynucleotide
encoding
a light chain of the invention which polynucleotide is selected from the group
consisting of; SEQ.I.D.NO:38, 39, 40.
It will be immediately apparent to those skilled in the art that due to the
redundancy of
the genetic code, alternative polynucleotides to those disclosed herein
(particularly
those codon optimised for expression in a given host cell) are also available
that will
encode the polypeptides of the invention.
3.1 Signal sequences
Antibodies of the present invention maybe produced as a fusion protein with a
heterologous signal sequence having a specific cleavage site at the N terminus
of the
mature protein. The signal sequence should be recognised and processed by the
host cell. For prokaryotic host cells, the signal sequence may be an alkaline
phosphatase, penicillinase, or heat stable enterotoxin II leaders. For yeast
secretion
the signal sequences may be a yeast invertase leader, a factor leader or acid
phosphatase leaders see e.g. W090/13646. In mammalian cell systems, viral
secretory leaders such as herpes simplex gD signal and a native immunoglobulin
signal sequence are available. Typically the signal sequence is ligated in
reading
frame to DNA encoding the antibody of the invention.
3.2 Origin of replication
Origin of replications are well known in the art with pBR322 suitable for most
gram-
negative bacteria, 2 plasmid for most yeast and various viral origins such as
SV40,
polyoma, adenovirus, VSV or BPV for most mammalian cells. Generally the origin
of
replication component is not needed for mammalian expression vectors but the
SV40
may be used since it contains the early promoter.
3.3 Selection marker
Typical selection genes encode proteins that (a) confer resistance to
antibiotics or
other toxins e.g. ampicillin, neomycin, methotrexate or tetracycline or (b)
complement
auxiotrophic deficiencies or supply nutrients not available in the complex
media. The
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36
selection scheme may involve arresting growth of the host cell. Cells, which
have
been successfully transformed with the genes encoding the antibody of the
present
invention, survive due to e.g. drug resistance conferred by the selection
marker.
Another example is the so-called DHFR selection marker wherein transformants
are
cultured in the presence of methotrexate. In typical embodiments, cells are
cultured
in the presence of increasing amounts of methotrexate to amplify the copy
number of
the exogenous gene of interest. CHO cells are a particularly useful cell line
for the
DHFR selection. A further example is the glutamate synthetase expression
system
(Lonza Biologics). A suitable selection gene for use in yeast is the trpl
gene, see
Stinchcomb et al Nature 282, 38, 1979.
3.4 Promoters
Suitable promoters for expressing antibodies of the invention are operably
linked to
DNA/polynucleotide encoding the antibody. Promoters for prokaryotic hosts
include
phoA promoter, Beta-lactamase and lactose promoter systems, alkaline
phosphatase, tryptophan and hybrid promoters such as Tac. Promoters suitable
for
expression in yeast cells include 3-phosphoglycerate kinase or other
glycolytic
enzymes e.g. enolase, glyceralderhyde 3 phosphate dehydrogenase, hexokinase,
pyruvate decarboxylase, phosphofructokinase, glucose 6 phosphate isomerase, 3-
phosphoglycerate mutase and glucokinase. Inducible yeast promoters include
alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, metallothionein
and
enzymes responsible for nitrogen metabolism or maltose/galactose utilization.
Promoters for expression in mammalian cell systems include viral promoters
such as
polyoma, fowlpox and adenoviruses (e.g. adenovirus 2), bovine papilloma virus,
avian sarcoma virus, cytomegalovirus (in particular the immediate early gene
promoter), retrovirus, hepatitis B virus, actin, rous sarcoma virus (RSV)
promoter and
the early or late Simian virus 40. Of course the choice of promoter is based
upon
suitable compatibility with the host cell used for expression. In one
embodiment
therefore there is provided a first plasmid comprising a RSV and/or SV40
and/or
CMV promoter, DNA encoding light chain V region (VL) of the invention, KC
region
together with neomycin and ampicillin resistance selection markers and a
second
plasmid comprising a RSV or SV40 promoter, DNA encoding the heavy chain V
region (VH) of the invention, DNA encoding the 71 constant region, DHFR and
ampicillin resistance markers
3.5 Enhancer element
Where appropriate, e.g. for expression in higher eukaroytics, an enhancer
element
operably linked to the promoter element in a vector may be used. Suitable
mammalian enhancer sequences include enhancer elements from globin, elastase,
albumin, fetoprotein and insulin. Alternatively, one may use an enhancer
element
from a eukaroytic cell virus such as SV40 enhancer (at bp100-270),
cytomegalovirus
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37
early promoter enhancer, polyma enhancer, baculoviral enhancer or murine IgG2a
locus (see W004/009823). The enhancer is preferably located on the vector at a
site
upstream to the promoter.
3.5.5 - Polyadenyiation signals
In eukaryotic systems, polyadenylation signals are operably linked to
DNA/polynucleotide encoding the antibody of this invention. Such signals are
typically placed 3' of the open reading frame. In mammalian systems, non-
limiting
example include signals derived from growth hormones, elongation factor-1
alpha
and viral (eg SV40) genes or retroviral long terminal repeats. In yeast
systems non-
limiting examples of polydenylation/termination signals include those derived
from the
phosphoglycerate kinase (PGK) and the alcohol dehydrogenase 1(ADH) genes. In
prokaryotic system polyadenylation signals are typically not required and it
is instead
usual to employ shorter and more defined terminator sequences. Of course the
choice of polyadenylation/termination sequences is based upon suitable
compatibility
with the host cell used for expression.
3.6 Host cells
Suitable host cells for cloning or expressing vectors encoding antibodies of
the
invention are prokaroytic, yeast or higher eukaryotic cells. Suitable
prokaryotic cells
include eubacteria e.g. enterobacteriaceae such as Escherichia e.g. E.Coli
(for
example ATCC 31,446; 31,537; 27,325), Enterobacter, Erwinia, Klebsiella
Proteus,
Salmonella e.g. Salmonella typhimurium, Serratia e.g. Serratia marcescans and
Shigella as well as Bacilli such as B.subtilis and B.licheniformis (see DD 266
710),
Pseudomonas such as P.aeruginosa and Streptomyces. Of the yeast host cells,
Saccharomyces cerevisiae, schizosaccharomyces pombe, Kluyveromyces (e.g.
ATCC 16,045; 12,424; 24178; 56,500), yarrowia (EP402, 226), Pichia Pastoris
(EP183, 070, see also Peng et al J.Biotechnol. 108 (2004) 185-192), Candida,
Trichoderma reesia (EP244, 234), Penicillin, Tolypocladium and Aspergillus
hosts
such as A.nidulans and A.niger are also contemplated.
Although Prokaryotic and yeast host cells are specifically contemplated by the
invention, preferably however, host cells of the present invention are higher
eukaryotic cells. Suitable higher eukaryotic host cells include mammalian
cells such
as COS-1 (ATCC No.CRL 1650) COS-7 (ATCC CRL 1651), human embryonic
kidney line 293, baby hamster kidney cells (BHK) (ATCC CRL.1632), BHK570 (ATCC
NO: CRL 10314), 293 (ATCC NO.CRL 1573), Chinese hamster ovary cells CHO (e.g.
CHO-K1, ATCC NO: CCL 61, DHFR-CHO cell line such as DG44 (see Urlaub et al,
(1986) Somatic Cell Mol.Genet.12, 555-556)), particularly those CHO cell lines
adapted for suspension culture, mouse sertoli cells, monkey kidney cells,
African
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38
green monkey kidney cells (ATCC CRL-1 587), HELA cells, canine kidney cells
(ATCC CCL 34), human lung cells (ATCC CCL 75), Hep G2 and myeloma or
lymphoma cells e.g. NSO (see US 5,807,715), Sp2/0, Y0.
Thus in one embodiment of the invention there is provided a stably transformed
host
cell comprising a vector encoding a heavy chain and/or light chain of the
antibody or
antigen binding fragment thereof as herein described. Preferably such host
cells
comprise a first vector encoding the light chain and a second vector encoding
said
heavy chain.
Bacterial fermentation
Bacterial systems are particularly suited for the expression of antibody
fragments.
Such fragments are localised intracellularly or within the periplasma.
Insoluble
periplasmic proteins can be extracted and refolded to form active proteins
according
to methods known to those skilled in the art, see Sanchez et a/ (1999)
J.Biotechnol.
72, 13-20 and Cupit PM et al (1999) Lett Appl Microbiol, 29, 273-277.
3.7 Cell Culturing Methods.
Host cells transformed with vectors encoding the antibodies of the invention
or
antigen binding fragments thereof may be cultured by any method known to those
skilled in the art. Host cells may be cultured in spinner flasks, roller
bottles or hollow
fibre systems but it is preferred for large scale production that stirred tank
reactors
are used particularly for suspension cultures. Preferably the stirred tankers
are
adapted for aeration using e.g. spargers, baffles or low shear impellers. For
bubble
columns and airlift reactors direct aeration with air or oxygen bubbles maybe
used.
Where the host cells are cultured in a serum free culture media it is
preferred that the
media is supplemented with a cell protective agent such as pluronic F-68 to
help
prevent cell damage as a result of the aeration process. Depending on the host
cell
characteristics, either microcarriers maybe used as growth substrates for
anchorage
dependent cell lines or the cells maybe adapted to suspension culture (which
is
typical). The culturing of host cells, particularly invertebrate host cells
may utilise a
variety of operational modes such as fed-batch, repeated batch processing (see
Drapeau et a/ (1994) cytotechnology 15: 103-109), extended batch process or
perfusion culture. Although recombinantly transformed mammalian host cells may
be
cultured in serum-containing media such as fetal calf serum (FCS), it is
preferred that
such host cells are cultured in synthetic serum -free media such as disclosed
in
Keen et al (1995) Cytotechnology 17:153-163, or commercially available media
such
as ProCHO-CDM or UltraCHOT"" (Cambrex NJ, USA), supplemented where
necessary with an energy source such as glucose and synthetic growth factors
such
as recombinant insulin. The serum-free culturing of host cells may require
that those
cells are adapted to grow in serum free conditions. One adaptation approach is
to
culture such host cells in serum containing media and repeatedly exchange 80%
of
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39
the culture medium for the serum-free media so that the host cells learn to
adapt in
serum free conditions (see e.g. Scharfenberg K et al (1995) in Animal Cell
technology: Developments towards the 21st century (Beuvery E.C. et al eds),
pp619-
623, Kluwer Academic publishers).
Antibodies of the invention secreted into the media may be recovered and
purified
using a variety of techniques to provide a degree of purification suitable for
the
intended use. For example the use of antibodies of the invention for the
treatment of
human patients typically mandates at least 95% purity, more typically 98% or
99% or
greater purity (compared to the crude culture medium). In the first instance,
cell
debris from the culture media is typically removed using centrifugation
followed by a
clarification step of the supernatant using e.g. microfiltration,
ultrafiltration and/or
depth filtration. A variety of other techniques such as dialysis and gel
electrophoresis
and chromatographic techniques such as hydroxyapatite (HA), affinity
chromatography (optionally involving an affinity tagging system such as
polyhistidine)
and/or hydrophobic interaction chromatography (HIC, see US 5, 429,746) are
available. In one embodiment, the antibodies of the invention, following
various
clarification steps, are captured using Protein A or G affinity chromatography
followed
by further chromatography steps such as ion exchange and/or HA chromatography,
anion or cation exchange, size exclusion chromatography and ammonium sulphate
precipitation. Typically, various virus removal steps are also employed (e.g.
nanofiltration using e.g. a DV-20 filter). Following these various steps, a
purified
(preferably monoclonal) preparation comprising at least 75mg/ml or greater
e.g.
100mg/mI or greater of the antibody of the invention or antigen binding
fragment
thereof is provided and therefore forms an embodiment of the invention.
Suitably
such preparations are substantially free of aggregated forms of antibodies of
the
invention.
4. Pharmaceutical Compositions
Purified preparations of antibodies of the invention (particularly monoclonal
preparations) as described supra, may be incorporated into pharmaceutical
compositions for use in the treatment of human diseases and disorders such as
atopic diseases e.g. asthma, allergic rhinitis, COPD. Typically such
compositions
comprise a pharmaceutically acceptable carrier as known and called for by
acceptable pharmaceutical practice, see e.g. Remingtons Pharmaceutical
Sciences,
16th edition, (1980), Mack Publishing Co. Examples of such carriers include
sterilised carrier such as saline, Ringers solution or dextrose solution,
buffered with
suitable buffers to a pH within a range of 5 to 8. Pharmaceutical compositions
for
injection (e.g. by intravenous, intraperitoneal, intradermal, subcutaneous,
intramuscular or intraportal) or continuous infusion are suitably free of
visible
particulate matter and may comprise between 0.1 ng to 100mg of antibody,
preferably
between 5mg and 25mg of antibody. Methods for the preparation of such
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pharmaceutical compositions are well known to those skilled in the art. In one
embodiment, pharmaceutical compositions comprise between 0.1 ng to 100mg of
antibodies of the invention in unit dosage form, optionally together with
instructions
for use. Pharmaceutical compositions of the invention may be lyophilised
(freeze
dried) for reconstitution prior to administration according to methods well
known or
apparent to those skilled in the art. Where embodiments of the invention
comprise
antibodies of the invention with an IgG1 isotype, a chelator of copper such as
citrate
(e.g. sodium citrate) or EDTA or histidine may be added to pharmaceutical
composition to reduce the degree of copper-mediated degradation of antibodies
of
this isotype, see EP0612251. Anti-hlL-13 treatment maybe given orally, by
inhalation, topically (for example, intraocular, intransnasal, rectal into
wounds on the
skin).
Effective doses and treatment regimes for administering the antibody of the
invention
are generally determined empirically and are dependent on factors such as the
age,
weight and health status of the patient and disease or disorder to be treated.
Such
factors are within the purview of the attending physician. Guidance in
selecting
appropriate doses may be found in e.g. Smith et al (1977) Antibodies in human
diagnosis and therapy, Raven Press, New York but will in general be between 1
mg
and 1000mg.
Depending on the disease or disorder to be treated (but particularly asthma),
pharmaceutical compositions comprising a therapeutically effective amount of
the
antibody of the invention may be used simultaneously, separately or
sequentially with
an effective amount of another medicament such as anti-inflammatory agents
(e.g.
corticosteroid or an NSAID), anticholinergic agents (particularly M1/M2/M3
receptor
antagonists), (32 adrenoreceptor agonists, antiinfective agents (e.g.
antibiotics,
antivirals), antihistamines, PDE4 inhibitor. Examples of [32 adrenoreceptor
agonists
include salmeterol, salbutamol, formoterol, salmefamol, fenoterol,
terbutaline.
Preferred long acting (32 adrenoreceptor agonists include those described in
W002/66422A, W002/270490, W002/076933, W003/024439 and W003/072539.
Suitable corticosteroids include methyl prednisolone, prednisolone,
dexamethasone,
fluticasone propionate, 6a,9a-difluoro-17a-[(2-furanylcarbonyl)oxy]-11 [3-
hydroxy-16a-
methyl-3-oxo-androsta-1,4-diene-1 7R-carbothioic acid S-fluoromethyl ester,
6a,9a-
difluoro-11 R-hydroxy-16a-methyl-3-oxo-17a-propionyloxy- androsta-1,4-diene-
17[3-
carbothioic acid S-(2-oxo-tetrahydro-furan-3S-yl) ester, beclomethasone esters
(eg.
the 17-propionate ester or the 17,21-dipropionate ester), budesonide,
flunisolide,
mometasone esters (eg. the furoate ester), triamcinolone acetonide,
rofieponide,
ciclesonide (1 6a, 1 7-[[(R)-cyclohexylmethylene]bis(oxy)]-1 1 R,21-dihydroxy-
pregna-
1,4-diene-3,20-dione), butixocort propionate, RPR-106541, and ST-126.
Preferred
corticosteroids include fluticasone propionate, 6a,9a-difluoro-11(3-hydroxy-
16a-
methyl-17a-[(4-methyl-1,3-thiazole-5-carbonyl)oxy]-3-oxo-androsta-1,4-diene-
17R-
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41
carbothioic acid S-fluoromethyl ester and 6a,9a-difluoro-17a-[(2-
furanylcarbonyl)oxy]-11 [3-hydroxy-16a-methyl-3-oxo-androsta-1,4-diene-17R-
carbothioic acid S-fluoromethyl ester, more preferably 6a,9a-difluoro-17a-[(2-
furanylcarbonyl)oxy]-11 R-hydroxy-16a-methyl-3-oxo-androsta-1,4-diene-17[i-
carbothioic acid S-fluoromethyl ester.
Non-steroidal compounds having glucocorticoid agonism that may posess
selectivity
for transrepression over transactivation and that may be useful in combination
therapy include those covered in the following patents: W003/082827,
WO01/10143,
W098/54159, W004/005229, W004/009016, W004/009017, W004/018429,
W003/104195, W003/082787, W003/082280, W003/059899, W003/101932,
W002/02565, W001/16128, W000/66590, W003/086294, W004/026248,
W003/061651, W003/08277.
Suitable anti-inflammatory agents include non-steroidal anti-inflammatory
drugs
(NSAID's).
Suitable NSAID's include sodium cromoglycate, nedocromil sodium,
phosphodiesterase (PDE) inhibitors (e.g. theophylline, PDE4 inhibitors or
mixed
PDE3/PDE4 inhibitors), leukotriene antagonists, inhibitors of leukotriene
synthesis
(eg. montelukast), iNOS inhibitors, tryptase and elastase inhibitors, beta-2
integrin
antagonists and adenosine receptor agonists or antagonists (e.g. adenosine 2a
agonists), cytokine antagonists (e.g. chemokine antagonists, such as a CCR3
antagonist) or inhibitors of cytokine synthesis, or 5-lipoxygenase inhibitors.
Suitable
other R2-adrenoreceptor agonists include salmeterol (e.g. as the xinafoate),
salbutamol (e.g. as the sulphate or the free base), formoterol (e.g. as the
fumarate),
fenoterol or terbutaline and salts thereof. An iNOS (inducible nitric oxide
synthase
inhibitor) is preferably for oral administration. Suitable iNOS inhibitors
include those
disclosed in W093/13055, W098/30537, W002/50021, W095/34534 and
W099/62875. Suitable CCR3 inhibitors include those disclosed in W002/26722.
Of particular interest is use of the antibodies of the invention in
combination with a
phosphodiesterase 4 (PDE4) inhibitor. The PDE4-specific inhibitor useful in
this
aspect of the invention may be any compound that is known to inhibit the PDE4
enzyme or which is discovered to act as a PDE4 inhibitor, and which are only
PDE4
inhibitors, not compounds which inhibit other members of the PDE family, such
as
PDE3 and PDE5, as well as PDE4.
Compounds of interest include cis-4-cyano-4-(3-cyclopentytoxy-4-
methoxyphenyl)cyclohexan-1-carboxylic acid, 2-carbomethoxy-4-cyano-4-(3-
cyclopropylmethoxy-4-difluoromethoxyphenyl)cyclohexan-1-one and cis-[4-cyano-4-
(3-cyclopropylmethoxy-4-difluoromethoxyphenyl)cyclohexan-l-ol]. Also, cis-4-
cyano-
4-[3-(cyclopentyloxy)-4-methoxyphenyl]cyclohexane-1-carboxylic acid (also
known as
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42
cilomilast) and its salts, esters, pro-drugs or physical forms, which is
described in
U.S. patent 5,552,438 issued 03 September, 1996; this patent and the compounds
it
discloses are incorporated herein in full by reference.
AWD-12-281 from Elbion (Hofgen, N. et al. 15th EFMC Int Symp Med Chem (Sept 6-
10, Edinburgh) 1998, Abst P.98; CAS reference No. 247584020-9); a 9-
benzyladenine derivative nominated NCS-613 (INSERM); D-4418 from Chiroscience
and Schering-Plough; a benzodiazepine PDE4 inhibitor identified as CI-1018 (PD-
168787) and attributed to Pfizer; a benzodioxole derivative disclosed by Kyowa
Hakko in W099/16766; K-34 from Kyowa Hakko; V-11294A from Napp (Landells,
L.J. et al. Eur Resp J [Annu Cong Eur Resp Soc (Sept 19-23, Geneva) 1998]
1998,
12 (Suppl. 28): Abst P2393); roflumilast (CAS reference No 162401-32-3) and a
pthalazinone (W099/47505, the disclosure of which is hereby incorporated by
reference) from Byk-Gulden; Pumafentrine, (-)-p-[(4aR*,10bS*)-9-ethoxy-
1,2,3,4,4a,10b-hexahydro-8-methoxy-2-methylbenzo[c][1,6]naphthyridin-6-yl]-N,N-
diisopropylbenzamide which is a mixed PDE3/PDE4 inhibitor which has been
prepared and published on by Byk-Gulden, now Altana; arofylline under
development
by Almirall-Prodesfarma; VM554/UM565 from Vernalis; or T-440 (Tanabe Seiyaku;
Fuji, K. et al. J Pharmacol Exp Ther,1998, 284(1): 162), and T2585.
Further compounds of interest are disclosed in the published international
patent
application W004/024728 (Glaxo Group Ltd), PCT/EP2003/014867 (Glaxo Group
Ltd) and PCT/EP2004/005494 (Glaxo Group Ltd).
Suitable anticholinergic agents are those compounds that act as antagonists at
the
muscarinic receptors, in particular those compounds which are antagonists of
the M,
or M3 receptors, dual antagonists of the M1/M3 or M2/M3, receptors or pan-
antagonists of the M1/M2/M3 receptors. Exemplary compounds for administration
via
inhalation include ipratropium (e.g. as the bromide, CAS 22254-24-6, sold
under the
name Atrovent), oxitropium (e.g. as the bromide, CAS 30286-75-0) and
tiotropium
(e.g. as the bromide, CAS 136310-93-5, sold under the name Spiriva). Also of
interest are revatropate (e.g. as the hydrobromide, CAS 262586-79-8) and LAS-
34273 which is disclosed in WO01/04118. Exemplary compounds for oral
administration include pirenzepine (CAS 28797-61-7), darifenacin (CAS 133099-
04-
4, or CAS 133099-07-7 for the hydrobromide sold under the name Enablex),
oxybutynin (CAS 5633-20-5, sold under the name Ditropan), terodiline (CAS
15793-
40-5), tolterodine (CAS 124937-51-5, or CAS 124937-52-6 for the tartrate, sold
under
the name Detrol), otilonium (e.g. as the bromide, CAS 26095-59-0, sold under
the
name Spasmomen), trospium chloride (CAS 10405-02-4) and solifenacin (CAS
242478-37-1, or CAS 242478-38-2 for the succinate also known as YM-905 and
sold
under the name Vesicare).
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43
Other suitable anticholinergic agents include compounds of formula (XXI),
which are
disclosed in US patent application 60/487981:
N X
H (XXI)
R 31
32
in which the preferred orientation of the alkyl chain attached to the tropane
ring is
endo;
R 31 and R32 are, independently, selected from the group consisting of
straight or
branched chain lower alkyl groups having preferably from 1 to 6 carbon atoms,
cycloalkyl groups having from 5 to 6 carbon atoms, cycloalkyl-alkyl having 6
to 10
carbon atoms, 2-thienyl, 2-pyridyl, phenyl, phenyl substituted with an alkyl
group
having not in excess of 4 carbon atoms and phenyl substituted with an alkoxy
group
having not in excess of 4 carbon atoms;
X- represents an anion associated with the positive charge of the N atom. X-
may be
but is not limited to chloride, bromide, iodide, sulfate, benzene sulfonate,
and toluene
sulfonate,
including, for example:
(3-endo)-3-(2,2-di-2-thienylethenyl)-8,8-dimethyl-8-azoniabicyclo[3.2.1
]octane bromide;
(3-endo)-3-(2,2-diphenylethenyl)-8,8-dimethyl-8-azoniabicyclo[3.2.1]octane
bromide;
(3-endo)-3-(2,2-diphenylethenyl)-8,8-dimethyl-8-azoniabicyclo[3.2.1]octane 4-
methylbenzenesulfonate;
(3-endo)-8,8-d i methyl-3-[2-phe nyl-2-(2-th ie nyl )eth enyl]-8-azo n ia
bicyclo[3.2.1 ]octane
bromide; and/or
(3-endo)-8,8-dimethyl-3-[2-phenyl-2-(2-pyridinyl)ethenyl]-8-
azoniabicyclo[3.2.1 ]octane bromide.
Further suitable anticholinergic agents include compounds of formula (XXII) or
(XXIII), which are disclosed in US patent application 60/511009:
~
N+ R41- ----N
H (XXII) H (XXIII)
R43 43
R44 R42 R44 R42
wherein:
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44
the H atom indicated is in the exo position;
R41- represents an anion associated with the positive charge of the N atom. R1-
may
be but is not limited to chloride, bromide, iodide, sulfate, benzene sulfonate
and
toluene sulfonate;
R42 and R43 are independently selected from the group consisting of straight
or
branched chain lower alkyl groups (having preferably from 1 to 6 carbon
atoms),
cycloalkyl groups (having from 5 to 6 carbon atoms), cycloalkyl-alkyl (having
6 to 10
carbon atoms), heterocycloalkyl (having 5 to 6 carbon atoms) and N or 0 as the
heteroatom, heterocycloalkyl-alkyl (having 6 to10 carbon atoms) and N or 0 as
the
heteroatom, aryl, optionally substituted aryl, heteroaryl, and optionally
substituted
heteroaryl;
R44 is slected from the group consisting of P-C6)alkyl, (C3-C12)cycloalkyl,
(C3-
C7)heterocycloalkyl, (C,-C6)alkyl(C3-C12)cycloalkyl, (C1-C6)alkyl(C3-
C7)heterocycloalkyl, aryl, heteroaryl, (Cl-C6)alkyl-aryl, (Cl-C6)alkyl-
heteroaryl, -OR45,
-CH20R45, -CH2OH, -CN, -CF3, -CH2O(CO)R46, -C02R47, -CH2NH2, -
CH2N(R47)S02R45, -SO2N(R47 )(R4$), -CON(R47 )(R4s) -CH2N(R48)CO(R46), -
CH2N(R48)S02(R46), -CH2N(R48)CO2(R45), -CH2N(R48)CONH(R47);
R45 is selected from the group consisting of (C,-C6)alkyl, (C1-C6)alkyl(C3-
C12)cycloalkyl, (C,-
C6)alkyl(C3-C,)heterocycloalkyl, (C,-C6)alkyl-aryl, (C,-C6)alkyl-heteroaryl;
R46 is selected from the group consisting of (C,-C6)alkyl, (C3-C12)cycloalkyl,
(C3-
C7)heterocycloalkyl, (C,-C6)alkyl(C3-C12)cycloalkyl, (C,-C6)alkyl(C3-
C7)heterocycloalkyl, aryl,
heteroaryl, (C,-C6)alkyl-aryl, (C,-C6)alkyl-heteroaryl;
R47 and R48 are, independently, selected from the group consisting of H, (Cl-
C6)alkyl,
(C3-C12)cycloalkyl, (C3-C7)heterocycloalkyl, (Cj-C6)alkyl(C3-C12)cycloalkyl,
(Cl-
C6)alkyl(C3-C7)heterocycloalkyl, (Cl-C6)alkyl-aryl, and (C,-C6)alkyl-
heteroaryl,
including, for example:
(Endo)-3-(2-methoxy-2,2-d i-th iophen-2-yl-ethyl )-8,8-d imethyl-8-azon ia-
bicyclo[3.2.1 ]octane iodide;
3-((Endo)-8-methyl-8-aza-bicyclo[3.2.1 ]oct-3-yl )-2,2-d iphenyl-
propionitrile;
(Endo)-8-methyl-3-(2,2,2-triphenyl-ethyl )-8-aza-bicyclo[3.2.1 ]octane;
3-((Endo)-8-methyl-8-aza-bicyclo[3.2. 1 ]oct-3-yl)-2,2-diphenyl-propionamide;
3-((Endo)-8-methyl-8-aza-bicyclo[3.2.1 ]oct-3-yl)-2,2-diphenyl-propionic acid;
(Endo)-3-(2-cyano-2,2-diphenyl-ethyl)-8,8-dimethyl-8-azonia-bicyclo[3.2.1
]octane
iodide;
(Endo)-3-(2-cyano-2,2-diphenyl-ethyl)-8,8-d imethyl-8-azonia-bicyclo[3.2.1
]octane
bromide;
3-((Endo)-8-methyl-8-aza-bicyclo[3.2.1 ]oct-3-yl)-2,2-diphenyl-propan-1 -ol;
N-Benzyl-3-((endo)-8-methyl-8-aza-bicyclo[3.2.1 ]oct-3-yl)-2,2-diphenyl-
propionamide;
(Endo)-3-(2-carbamoyl-2,2-diphenyl-ethyl )-8,8-dimethyl-8-azonia-bicyclo[3.2.1
]octane
iodide;
1-Benzyl-3-[3-((endo)-8-methyl-8-aza-bicyclo[3.2.1 ]oct-3-yl)-2,2-diphenyl-
propyl]-
urea;
1 -Ethyl-3-[3-((endo)-8-methyl-8-aza-bicyclo[3.2. 1 ]oct-3-yl)-2,2-diphenyl-
propyl]-urea;
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N-[3-((Endo)-8-methyl-8-aza-bicyclo[3.2.1 ]oct-3-yl )-2,2-d iphenyl-propyl]-
acetamide;
N-[3-((Endo)-8-methyl-8-aza-bicyclo[3.2. 1 ]oct-3-yl)-2,2-diphenyl-propyl]-
benzamide;
3-((Endo)-8-methyl-8-aza-bicyclo[3.2. 1 ]oct-3-yl)-2,2-di-thiophen-2-yl-
propionitrile;
(Endo)-3-(2-cyano-2,2-di-thiophen-2-yl-ethyl)-8,8-dimethyl-8-azonia-
bicyclo[3.2.1 ]octane iodide;
N-[3-((Endo)-8-methyl-8-aza-bicyclo[3.2.1 ]oct-3-yl )-2,2-d iphenyl-propyl]-
benzenesulfonamide;
[3-((Endo)-8-methyl-8-aza-bicyclo[3.2.1 ]oct-3-yl)-2,2-d iphenyl-propyl]-urea;
N-[3-((Endo)-8-methyl-8-aza-bicyclo[3.2.1 ]oct-3-yl)-2,2-d iphenyl-propyl]-
methanesulfonamide; and/or
(Endo)-3-{2,2-diphenyl-3-[(1-phenyl-methanoyl)-amino]-propyl}-8,8-dimethyl-8-
azonia-bicyclo[3.2.1 ]octane bromide.
More preferred compounds useful in the present invention include:
(Endo)-3-(2-methoxy-2,2-di-thiophen-2-yl-ethyl)-8,8-dimethyl-8-azonia-
bicyclo[3.2.1 ]octane iodide;
(Endo)-3-(2-cyano-2,2-d iphenyl-ethyl )-8,8-d imethyl-8-azonia-bicyclo[3.2.1
]octane
iodide;
(Endo)-3-(2-cyano-2,2-diphenyl-ethyl)-8,8-d imethyl-8-azonia-bicyclo[3.2.1
]octane
bromide;
(Endo)-3-(2-carbamoyl-2,2-diphenyl-ethyl)-8,8-dimethyl-8-azonia-bicyclo[3.2.1
]octane
iodide;
(Endo)-3-(2-cyano-2,2-di-thiophen-2-yl-ethyl)-8,8-dimethyl-8-azonia-
bicyclo[3.2.1]octane iodide; and/or
(Endo)-3-{2,2-diphenyl-3-[(1-phenyl-methanoyl)-amino]-propyl}-8,8-dimethyl-8-
azonia-bicyclo[3.2.1 ]octane bromide.
Suitable antihistamines (also referred to as H1-receptor antagonists) include
any one
or more of the numerous antagonists known which inhibit H 1-receptors, and are
safe
for human use. First generation antagonists, include derivatives of
ethanolamines,
ethylenediamines, and alkylamines, e.g diphenylhydramine, pyrilamine,
clemastine,
chlropheniramine. Second generation antagonists, which are non-sedating,
include
loratidine, desloratidine,terfenadine,astemizole,acrivastine, azelastine,
levocetirizine
fexofenadine and cetirizine.
Examples of preferred anti-histamines include loratidine, desloratidine,
fexofenadine
and cetirizine.
Other contemplated combinations include the use of antibodies of the invention
in
combination with an anti-IL-4 agent (e.g. anti-IL-4 antibody such as
pascolizumab)
and/or anti-IL-5 agent (e.g. anti-IL-5 antibody such as mepolizumab) and/or
anti-IgE
agent (e.g. anti-IgE antibody such as omalizumab (XolairTM) or talizumab).
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Conveniently, a pharmaceutical composition comprising a kit of parts of the
antibody
of the invention or antigen binding fragment thereof together with such
another
medicaments optionally together with instructions for use is also contemplated
by the
present invention.
The invention furthermore contemplates a pharmaceutical composition comprising
a
therapeutically effective amount of monoclonal therapeutic antibody or antigen
binding fragment thereof as herein described for use in the treatment of
diseases
responsive to modulation of the interaction between hIL-13 and hIL-13R.
In accordance with the present invention there is provided a pharmaceutical
composition comprising a therapeutically effective amount of a monoclonal
humanised therapeutic antibody which antibody comprises a VH domain selected
from the group consisting of: SEQ.I.D.NO:11,12,13,14 and a VL domain selected
from the group consisting of: SEQ.I.D.NO:15,16, 17.
In accordance with the present invention there is provided a pharmaceutical
composition comprising a monoclonal antibody comprising a heavy chain selected
from the group consisting of: SEQ.I.D.NO: 18,19,20,21 and a light chain
selected
from the group consisting of; SEQ.I.D.NO:22, 23,24
In accordance with the present invention there is provided a pharmaceutical
composition comprising a monoclonal antibody comprising a heavy chain of
SEQ.I.D.NO:18 and a light chain of SEQ.I.D.NO:22 and a pharmaceutically
acceptable carrier.
In accordance with the present invention there is provided a pharmaceutical
composition comprising a monoclonal antibody comprising (or consisting
essentially
of) a heavy chain of SEQ.I.D.NO:18 and a light chain of SEQ.I.D.NO:22 and a
pharmaceutically acceptable carrier.
In accordance with the present invention there is provided a pharmaceutical
composition comprising a pharmaceutically acceptable carrier and a
therapeutically
effective amount of a monoclonal population of antibody which antibody
comprises a
heavy chain of SEQ.I.D.NO:18 and a light chain of SEQ.I.D.NO:22.
In accordance with the present invention there is provided a pharmaceutical
composition comprising a pharmaceutically acceptable carrier and a
therapeutically
effective amount of a monoclonal population of antibody which antibody
comprises a
heavy chain of SEQ.I.D.NO:18 and a light chain of SEQ.I.D.NO:23.
In accordance with the present invention there is provided a pharmaceutical
composition comprising a pharmaceutically acceptable carrier and a
therapeutically
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47
effective amount of a monoclonal population of antibody which antibody
comprises a
heavy chain of SEQ.I.D.NO:18 and a light chain of SEQ.I.D.NO:24.
In accordance with the present invention there is provided a pharmaceutical
composition comprising a pharmaceutically acceptable carrier and a
therapeutically
effective amount of a monoclonal population of antibody which antibody
comprises a
heavy chain of SEQ.I.D.NO:19 and a light chain of SEQ.I.D.NO:22.
In accordance with the present invention there is provided a pharmaceutical
composition comprising a pharmaceutically acceptable carrier and a
therapeutically
effective amount of a monoclonal population of antibody which antibody
comprises a
heavy chain of SEQ.I.D.NO:19 and a light chain of SEQ.I.D.NO:23.
In accordance with the present invention there is provided a pharmaceutical
composition comprising a pharmaceutically acceptable carrier and a
therapeutically
effective amount of a monoclonal population of antibody which antibody
comprises a
heavy chain of SEQ.I.D.NO:19 and a light chain of SEQ.I.D.NO:24.
In accordance with the present invention there is provided a pharmaceutical
composition comprising a pharmaceutically acceptable carrier and a
therapeutically
effective amount of a monoclonal population of antibody which antibody
comprises a
heavy chain of SEQ.I.D.NO:20 and a light chain of SEQ.I.D.NO:22.
In accordance with the present invention there is provided a pharmaceutical
composition comprising a pharmaceutically acceptable carrier and a
therapeutically
effective amount of a monocional population of antibody which antibody
comprises a
heavy chain of SEQ.I.D.NO:20 and a light chain of SEQ.I.D.NO:23
In accordance with the present invention there is provided a pharmaceutical
composition comprising a pharmaceutically acceptable carrier and a
therapeutically
effective amount of a monoclonal population of antibody which antibody
comprises a
heavy chain of SEQ.I.D.NO:20 and a light chain of SEQ.I.D.NO:24.
In accordance with the present invention there is provided a pharmaceutical
composition comprising a pharmaceutically acceptable carrier and a
therapeutically
effective amount of a monoclonal population of antibody which antibody
comprises a
heavy chain of SEQ.I.D.NO:21 and a light chain of SEQ.I.D.NO:22.
In accordance with the present invention there is provided a pharmaceutical
composition comprising a pharmaceutically acceptable carrier and a
therapeutically
effective amount of a monoclonal population of antibody which antibody
comprises a
heavy chain of SEQ.I.D.NO:21 and a light chain of SEQ.I.D.NO:23.
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In accordance with the present invention there is provided a pharmaceutical
composition comprising a pharmaceutically acceptable carrier and a
therapeutically
effective amount of a monoclonal population of antibody which antibody
comprises a
heavy chain of SEQ.I.D.NO:21 and a light chain of SEQ.I.D.NO:24.
5. Clinical uses.
Antibodies of the invention may be used in the treatment of atopic diseases/
disorders and chronic inflammatory diseases/disorders. Of particular interest
is their
use in the treatment of asthma, such as allergic asthma, particularly severe
asthma
(that is asthma that is unresponsive to current treatment, including
systemically
administered corticosteroids; see Busse WW et al, J Allergy Clin. Immunol
2000, 106:
1033-1042), "difficult" asthma (defined as the asthmatic phenotype
characterised by
failure to achieve control despite maximally recommended doses of prescribed
inhaled steroids, see Barnes PJ (1998), Eur Respir J 12:1208-1218), "brittle"
asthma
(defines a subgroup of patients with severe, unstable asthma who maintain a
wide
peak expiratory flow (PEF) variability despite high doses of inhaled steroids,
see
Ayres JG et al (1998) Thorax 58:315-321), nocturnal asthma, premenstrual
asthma,
steroid resistant asthma (see Woodcock AJ (1993) Eur Respir J 6:743-747),
steroid
dependent asthma (defined as asthma that can be controlled only with high
doses of
oral steroids), aspirin induced asthma, adult-onset asthma, paediatric asthma
.
Antibodies of the invention maybe used to prevent, reduce the frequency of, or
mitigate the effects of acute, asthmatic episodes (status asthmaticus).
Antibodies of
the invention may also be used to reduce the dosing required (either in terms
of
amount administered or frequency of dosing) of other medicaments used in the
treatment of asthma. For example, antibodies of the invention may be used to
reduce the dosing required for steroid treatment of asthma such as
corticosteroid
treatment ("steroid sparing"). Other diseases or disorders that may be treated
with
antibodies of the invention include atopic dermatitis, allergic rhinitis,
Crohn's disease,
chronic obstructive pulmonary disease (COPD), eosinophilic esophagitis,
fibrotic
diseases or disorders such as idiopathic pulmonary fibrosis, progressive
systemic
sclerosis (scleroderma), hepatic fibrosis, hepatic granulomas,
schistosomiasis,
leishmaniasis, and diseases of cell cycle regulation, e.g. Hodgkins disease, B
cell
chronic lymphocytic leukaemia. Further diseases or disorders that may be
treated
with antibodies of the invention are detailed in the Background of the
invention
Example above.
In one embodiment of the invention there is provided a method of treating a
human
patient afflicted with an asthmatic condition which is refractory to treatment
with
corticosteroids which method comprises the step of administering to said
patient a
therapeutically effective amount of an antibody of the invention.
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In another embodiment there is provided a method of preventing an acute
asthmatic
attack in a human patient which method comprises the step of administering to
said
patient a therapeutically effective amount of an antibody of the invention.
In another embodiment there is provided a method of reducing the frequency of
and/or mitigating the effects of an acute asthmatic attack in a human patient
which
method comprises the step of administering to said patient a therapeutically
effective
amount of an antibody of the invention.
In another embodiment of the invention there is provided a method of biasing T
helper cell response towards a Th1 type response following an inflammatory
and/or
allergic insult in a human patient which method comprises administering to
said
patient a therapeutically effective amount of an antibody or antigen binding
fragment
thereof of the invention.
In another embodiment of the invention there is provided a method of treating
a
human patient having the Q130hIL-13 variant which patient is afflicted with
asthma,
such as severe asthma, said method comprising the step of administering to
said
patient a therapeutically effective amount of an antibody or antigen binding
fragment
thereof of the invention.
Although the present invention has been described principally in relation to
the
treatment of human diseases or disorders, the present invention may also have
applications in the treatment of similar diseases or disorders in non-human
mammals.
The present invention is now described by way of example only.
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Brief Description of the Drawings
Figure 1
Sandwich ELISA illustrating the binding of mouse monoclonal antibody 3G4 to
recombinant E.coli-expressed human IL-13 at increasing concentrations.
Figure 2a
Neutralisation assay illustrating the ability of mouse monoclonal antibody 3G4
at
increasing concentrations to inhibit the bioactivity of recombinant E.coli-
expressed
human IL-13 in a TF-1 cell proliferation assay.
Figure 2b
Neutralisation assay illustrating the ability of chimaeric 3G4 at increasing
concentrations to inhibit the bioactivity of recombinant E.coli-expressed
human IL-13
in a TF-1 cell proliferation assay.
Figure 3
Neutralisation assay illustrating the ability of mouse monoclonal antibody 3G4
(and
chimaeric 3G4) at increasing concentrations to inhibit the bioactivity of
recombinant
E.coli-expressed cynomolgus IL-13 in a TF-1 cell proliferation assay. The
curve
labelled 'Campath' is that obtained using anti-CD52 humanised antibody
alemtuzumab, acting as an irrelevant antibody control in this experiment. The
line
labelled 'anti-hIL13 poly' is that obtained using a neutralising polyclonal
anti-IL13
preparation (R&D Systems, catalogue number AF-213-NA) as a positive control.
Figure 4
Neutralisation assay illustrating the ability of mouse monoclonal antibody 3G4
(and
chimaeric 3G4) at increasing concentrations to inhibit the bioactivity of
mammalian-
expressed (CHO cell) human IL-13 in a TF-1 cell proliferation assay. Campath
and
anti-hIL13 are control reagents as described supra.
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51
Figure 5
Neutralisation assay illustrating the ability of mouse monoclonal antibody 3G4
and
(chimaeric 3G4) at increasing concentrations to inhibit the bioactivity of
recombinant
E.coli-expressed Q130 human IL-13 in a TF-1 cell proliferation assay. Campath
and
anti-hIL13 are control reagents as described supra.
Figure 6
An epitope mapping ELISA to determine the binding epitope for mouse monoclonal
antibody 3G4 on human and cynomoigus IL-13.
Figure 7a
An epitope mapping ELISA to identify the fine binding specificity of mouse
monoclonal antibody 3G4 on human IL-13
Figure 7b
An epitope mapping ELISA to identify the fine binding specificity of mouse
monoclonal antibody 3G4 on cynomolgus IL-13
Figure 8
An epitope mapping ELISA to determine the key amino acid residues required for
binding of mouse monoclonal antibody 3G4 to human IL-13.
Figure 9
An epitope mapping ELISA to determine the key amino acid residues required for
binding of mouse monoclonal antibody 3G4 to human IL-13
Figure 10
Sandwich ELISA illustrating the binding of mouse monoclonal antibody 3G4, 3G4
chimaera and humanised mAbs H2L1, H3L1 to recombinant E.coli-expressed human
IL-13 at increasing concentrations.
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Figure 11
Neutralisation assay illustrating the ability of mouse monoclonal antibody
3G4, 3G4
chimera and humanised mAbs H2L1, H3L1 at increasing concentrations to inhibit
the
bioactivity of recombinant E.coli-expressed human IL-13 in a TF-1 cell
proliferation
assay.
Figure 12
Neutralisation assay illustrating the ability of mouse monoclonal antibody
3G4, 3G4
chimera and humanised mAbs H2L1, H3L1 at increasing concentrations to inhibit
the
bioactivity of recombinant E.coli-expressed cynomolgus IL-13 in a TF-1 cell
proliferation assay.
Figure 13
Neutralisation assay illustrating the ability of mouse monoclonal antibody
3G4, 3G4
chimera and humanised mAbs H2L1, H3L1 at increasing concentrations to inhibit
the
bioactivity of mammalian-expressed (CHO cell) human IL-13 in a TF-1 cell
proliferation assay.
Figure 14
Neutralisation assay illustrating the ability of mouse monoclonal antibody
3G4, 3G4
chimera and humanised mAbs H2L1, H3L1 at increasing concentrations to inhibit
the
bioactivity of recombinant E.coli-expressed Q130 human IL-13 in a TF-1 cell
proliferation assay.
Figure 15
Sandwich ELISA demonstrating that mouse monoclonal antibody 3G4, 3G4 chimera
and humanised mAbs H2L1, H3L1 do not bind recombinant E.coli-expressed human
IL-4.
Figure 16
Sandwich ELISA demonstrating that mouse monoclonal antibody 3G4, 3G4 chimera
and humanised mAbs H2L1, H3L1 do not bind recombinant E.coli-expressed human
IL-5.
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Figure 17
Direct binding ELISA demonstrating that mouse monoclonal antibody 3G4, 3G4
chimera and humanised mAbs H2L1, H3L1 do not bind human GM-CSF.
Figure 18
Sandwich ELISA illustrating the binding of mouse monoclonal antibody 3G4, 3G4
chimera and humanised mAbs H2L1, H3L1 to native human IL-13 at increasing
concentrations.
Figure 19
ELISA illustrating the ability of mouse monoclonal antibody 3G4, 3G4 chimera
and
humanised mAbs H2L1, H3L1 at increasing concentrations to inhibit recombinant
E.coli-expressed human IL-13 binding to the human IL-13 receptor a 1 chain.
Figure 20
ELISA illustrating the ability of mouse monoclonal antibody 3G4, 3G4 chimera
and
humanised mAbs H2L1, H3L1 at increasing concentrations to inhibit recombinant
E.coli-expressed human IL-13 binding to the human IL-13 receptor a 2 chain.
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Examples
1. GENERATION OF MONOCLONAL ANTIBODIES AND CHARACTERISATION
OF MOUSE MONOCLONAL ANTIBODY 3G4
Five SJL mice were immunised by intraperitoneal injection each with 2pg
recombinant human IL-13 derived from E.Coli (Cambridge Bioscience, Cat. No. CH-
013). Spleen cells from the mice were removed and B lymphocytes fused with
mouse myeloma cells derived from P3X cells using PEG1500 (Boehringer) to
generate hybridomas. Individual hybridoma cell lines were cloned by limiting
dilution
(E Harlow and D Lane). Wells containing single colonies were identified
microscopically and supernatants tested for activity. Cells from the most
active clones
were expanded for cryopreservation, antibody production etc.
Initially, hybridoma supernatants were screened for binding activity against
an E.coli-
expressed recombinant det-1 tagged human IL-13 protein (made in-house) in a
sandwich assay format. A secondary screen of these positives was completed
using
a BlAcoreTM method to detect for binding to the det-1 tagged human IL-13
protein.
Samples from these hybridomas were then tested for ability to neutralise the
bioactivity of E.coli-expressed recombinant human IL-13 (Cambridge Bioscience,
cat.
no CH-013) in a TF-1 cell bioassay.
Positives identified from the human IL-13 neutralising bioassay were subcloned
by
limiting dilution to generate stable monoclonal cell lines. Immunoglobulins
from these
hybridomas, grown in cell factories under serum free conditions, were purified
using
immobilised Protein A columns. These purified mAbs were then re-screened in
the
same three assay systems.
Monoclonal antibody 3G4 was identified as a potent antibody that neutralised
human
IL-13 bioactivity.
The 3G4 antibody has the VH region amino acid sequence as set out in
SEQ.I.D.NO:7
The 3G4 antibody has the VL region amino acid sequence as set out in
SEQ.I.D.NO:8
A chimaeric antibody was constructed by taking variable regions from the 3G4
murine monoclonal antibody and grafting these on to human IgGl/k wild type C
regions. A human signal sequence (as shown in SEQ.I.D.NO: 43) was used in the
construction of these constructs. This chimearic antibody was termed 3G4C.
1.1 Binding to E.Coli-expressed recombinant human IL-13
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3G4 bound E.Coli-expressed recombinant human IL-13 in a sandwich ELISA, the
method was carried out as described in Example 6.1 (See Figure 1).
1.2 Neutralisation of E.CoIi-expressed recombinant human and cynomoigus IL-
13 in a TF-1 cell proliferation bioassay
TF-1 cells proliferate in response to human IL-13 and cynomolgus IL-13. A
bioassay
was developed to assess the neutralisation capacity of an anti-IL-13 mAb on
human
and cynomoigus IL-13-induced TF-1 cell proliferation. The method was carried
out as
described in Example 6.2.
The amino acid sequence and a cDNA sequence for cynomolgus IL-13 (not
including
signal sequence) is set forth as SEQ.I.D.NO:41 and SEQ.I.D.NO:42 respectively.
3G4 neutralised the bioactivity of recombinant human IL-13 in a TF-1 cell
bioassay
(see figure 2a).
3G4 neutralised cynomolgus IL-13 bioactivity less potently than human IL-13
(see
figure 3).
An average ND50 value of 0.13pg/ml was calculated for the neutralisation of
approximately 10ng/mI E.coli-expressed recombinant human IL-13 bioactivity in
a TF-
1 cell bioassay by monoclonal antibody 3G4.
An ND50 value of 29pg/ml was calculated for the neutralisation of
approximately
10ng/mI E.coli-expressed recombinant cynomoigus IL-13 bioactivity in a TF-1
cell
bioassay by monoclonal antibody 3G4. [The ND50 (neutralisation dose) value is
the
concentration of monoclonal antibody required to reduce TF-1 cell
proliferation by
50%, in response to a set concentration of IL-13].
1.3 Neutralisation of mammalian-expressed (CHO cell) human IL-13 in a TF-1
cell proliferation bioassay
The neutralisation capacity of monoclonal antibody 3G4 for human IL-13
expressed
from CHO cells was assessed in a TF-1 cell proliferation assay. The method was
carried out as described in Example 6.2. 3G4 neutralised mammalian-expressed
human IL-13 more potently than a commercially available anti-human IL-13
polyclonal reagent. An ND50 value of 0.31 pg/mI was calculated for the
neutralisation
of - 20ng/mI mammalian-expressed human IL-13 in a TF-1 cell bioassay by
monoclonal antibody 3G4. See Figure 4.
1.4 Neutralisation of recombinant Q130 human IL-13 variant in a TF-1 cell
proliferation bioassay
The neutralisation capacity of monoclonal antibody 3G4 for E.coli-expressed
recombinant Q130 human IL-13 (Peprotech, Cat. No. 200-13A) was assessed in a
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TF-1 cell proliferation assay. The method was carried out as described in
Example
6.2. 3G4 neutralised Q130 human IL-13 more potently than a commercially
available
anti-human IL-13 polyclonal reagent. An ND50 value of 0.025pg/ml was
calculated for
the neutralisation of - 60ng/ml Q130 human IL-13 bioactivity in a TF-1 cell
bioassay
by monoclonal antibody 3G4. See Figure 5.
1.5 BlAcoreTM analysis
The affinity of 3G4 mouse mAb for recombinant human and cynomolgus IL-13 was
assessed by BlAcoreTM (surface plasmon resonance) analysis. See Table 2.
BlAcoreTM analyses were carried out using anti-mouse IgG capture. An anti-
mouse
IgG antibody was coupled onto a CM5 chip by primary amine coupling in
accordance
with the manufacturers recommendation. 3G4 parental mouse mAb was then
captured onto this surface and human or cynomolgus IL-13 passed over at
defined
concentrations. The surface was regenerated back to the anti-mouse IgG surface
using mild acid elution conditions, this did not significantly affect the
ability to capture
antibody for a subsequent IL-13 binding event. This work was carried out on
the
Biacore 3000 and analysed using the evaluation software inherent in the
machine
and the data analysed using the 1:1 model of binding. The data for human IL-13
binding was generated using recombinant E.coli-expressed Det-1 tagged human IL-
13 as well as a commercial recombinant E.coli-expressed untagged human IL-13
reagent (supplied by Peprotech). Recombinant E.coli-expressed cynomolgus IL-13
was generated at GSK. All Biacore runs were carried out at 25 degrees C.
Table 2. Biacore 3000 data for parental 3G4 mouse mAb binding to human and
cynomoigus IL-13
3G4 mouse mAb ka Kd KD (nM)
binding to
Det-1 tagged human 5.61e6 1.68e-4 0.031
IL-13 (1.49e6) (7.07e-7) (0.009)
Human IL-13 2.25e6 1.37e-4 0.061
Pe rotech
Cynomolgus IL-13 4.11e5 1.3e-3 3.15
For Det-1 tagged human IL-13, the data were produced from 2 independent
experiments with both runs carried out in duplicate. The data are presented as
the
mean and standard deviation (in brackets) of these results.
For the human IL-13 (supplied by Peprotech) and cynomolgus IL-13 (made at
GSK),
the data are the result of one experiment, carried out in duplicate.
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For the data described above, duplicates were analysed together to give one
value
for the run.
These data indicate that 3G4 mouse mAb has a very high binding affinity for
human
IL-13, the binding affinity of 3G4 mouse mAb for cynomolgus IL-13 is less
potent in
comparison.
2. HUMANISATION OF CLONE 3G4
2.1 Sequence analysis
A comparison was made between the sequences of the 3G4 variable regions and
other murine and human immunoglobulin sequences. This was done using the
FASTA and BLAST programs and by inspection.
The following framework residues in 3G4 were identified as being potentially
important in the design of a CDR-grafted (humanised) version of the antibody:
Position 3G4 VH
D
30 1
93 T
Position is according to the Kabat et a/ numbering system.
Position 3G4 VL
98 L
A suitable human acceptor framework for the 3G4 VH was identified:
SEQ.I.D.NO:44
A suitable human acceptor framework for the 3G4 VL was identified:
SEQ.l.D.NO:45
In SEQ.I.D.NO:44 CRDH1 and H2 are present and CDRH3 is represented by
XXXXXXXXXX. In SEQ.I.D.NO:45 CRDL1 and L2 are present and CDRL3 is
represented by XXXXXXXXX.
In CDR grafting, it is typical to require one or more framework residues from
the
donor antibody to be included in place of their orthologues in the acceptor
frameworks in order to obtain satisfactory binding. In addition to those
listed above,
the following murine framework residues were also considered for possible
retention
in a humanised antibody design:
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Position (Kabat#) 3G4 VH Human VH
D E
30 I T
67 A V
69 L M
71 A R
73 K T
93 T A
Position (Kabat#) mouse 3G4 VL Human VL
76 N S
98 L F
4 humanised VH constructs with different back-mutations were designed to
obtain a
humanised antibody with satisfactory activity. These are numbered H1 to H4.
H1 consists of a CDR graft of the 3G4 VH CDRs into the specified acceptor
sequence. Additionally, H1 contains the murine residue at position 30
(isoleucine).
This is outside the Kabat definition of CDR, but within the CDR H1 definition
of
Chothia. In this case, this residue is considered to be part of the CDR rather
than a
true framework back-mutation.
H2 is identical to H1, but with a back-mutation where the amino acid at
position 93 is
threonine instead of alanine.
H3 is identical to H2, but with an additional back-mutation where the amino
acid at
position 10 is aspartic acid instead of glutamic acid.
H4 is identical to H3, but contains four additional back-mutations at
positions 67
(alanine in place of valine), 69 (leucine in place of methionine), 71 (alanine
in place of
arginine) and 73 (lysine in place of threonine).
3 humanised VL constructs with different back-mutations were designed to
obtain a
humanised antibody with satisfactory activity. These are numbered L1 to L3.
L1 consists of a CDR graft of the 3G4 VL CDRs into the specified acceptor
sequence,
using the Kabat definition of CDRs.
L2 is identical to L1, but with a back-mutation where the amino acid at
position 98 is
leucine in place of phenylalanine.
L3 is identical to L2, but with an additional back-mutation where the amino
acid at
position 76 is asparagine in place of serine.
Humanised VH construct H1:
SEQ.I.D.NO:11
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Humanised VH construct H2:
SEQ.I.D.N0:12
Humanised VH construct H3:
SEQ.I.D.N0:13
Humanised VH construct H4:
SEQ.I.D.N0:14
Humanised VL construct LI:
SEQ.I.D.N0:15
Humanised Vs construct L2:
SEQ.I.D.N0:16
Humanised VL construct L3:
SEQ.I.D.N0:17
2.2 Humanisation of 3G4
Humanised VH and VL constructs were prepared by de novo build up of
overlapping
oligonucleotides including restriction sites for cloning into RId and Rln
mammalian
expression vectors as well as a human signal sequence. Hind III and Spe I
restriction
sites were introduced to frame the VH domain containing the human signal
sequence
(SEQ.I.D.NO: 43) for cloning into RId containing the human yl wild type
constant
region. Hind III and BsiW I restriction sites were introduced to frame the VL
domain
containing the human signal sequence for cloning into RIn containing the human
kappa constant region. This is essentially as described in WO 2004/014953.
3. EXPRESSION AND CHARACTERISATION OF HUMANISED ANTIBODIES
Humanised VH constructs (H1, H2, H3 and H4) and two humanised VL constructs
(L1, and L2) were prepared in Rid hCy1wt and Rln hCK mammalian expression
vectors. Plasmid heavy chain-light chain combinations (H1 L1, H1L2, H2L1,
H2L2,,
H3L1, H3L2, H4L1, H4L2) were transiently co-transfected into CHO cells and
expressed at small scale to give eight different humanised antibodies.
The antibodies produced in the CHO cell supernatant and subsequent batches of
purified antibodies were analysed for activity in the human IL-13 binding
ELISA, in
the human IL-13 neutralisation bioassay, and binding to human IL-13 by
BlAcoreTM
All eight humanised mAbs showed binding and/or neutralisation of human IL-13
in
each of these assays. H2L1 and H3L1 were selected for further analysis due to
better
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performance in the human IL-13 neutralisation bioassay, and binding to human
IL-13
by BlAcoreTM, whilst offering a limited number of back-mutations.
3.1 Binding to E-coli expressed recombinant human IL-13
3G4C, H2LI and H3L1 bound E.coli expressed recombinant human IL13 in a
sandwich ELISA with similar profiles. The method was carried out as described
in
Example 6.1A. Table 3 shows average ED50 values (see also figure 10).
[The ED50 (effective dose) is the concentration of antibody required for half
maximal
binding to IL-13 in this ELISA]
Table 3
mAb ED50 Standard Error
(pg/mi)
chimaeric 3G4 0.04 0.021
H2L1 0.04 0.001
H3L1 0.05 0.007
The 3G4, 3G4C, H2L1, and H3L1 have also been shown to inhibit human IL-13
binding to human IL-13 receptor chains (IL13R(x1 and IL13Ra2) by ELISA. This
method was carried out as described in Example 6.5 and 6.6.
Figure 19 shows the data demonstrating inhibition for binding to IL13Ra1.
Table 4 shows the average IC50 values for the inhibition for binding to
IL13Ra2.
[The IC50 is the concentration of mAb required to inhibit binding of a fixed
amount of
human IL-13 to IL13Ra2 by 50%].
Table 4
IL13Ra2
mAb IC50 Standard error
(pg/ml)
Parental 3G4 mouse 0.025 0.0048
mAb
chimaeric 3G4 0.063 0.0060
H2L1 0.067 0.0097
H3L1 0.068 0.0121
Figure 19 illustrates that 3G4, 3G4, H2L1 and H3L1 inhibit human IL-13 binding
to
IL13Ra1 with similar potency.
Figure 20 illustrates that 3G4, 3G4C, H2L1 and H3L1 inhibit human IL-13
binding to
IL13Ra2 with similar potency.
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3.2 Binding to native human IL-13
The HDLM-2 cell line (a Reed-Steinberg-like cell line, originally derived from
bone
marrow) makes human IL-13 and uses it in an autocrine fashion for growth. This
native human IL-13 is secreted into HDLM-2 cell supernatant. This was used to
assess the binding of 3G4, 3G4C, H2L1 and H3L1 to native human IL-13 using a
method as described in Example 6.1 B. By ELISA, all four antibodies bound
native
human IL-13 in the HDLM2 cell supernatant with very similar performance to
that of
the parental 3G4 mAb. See figure 18.
3.3 Neutralisation of E-coli expressed recombinant human and cynomologus
IL-13 in a TF-1 cell proliferation bioassay.
3G4, 3G4C, H2L1 and H3L1 neutralised the bioactivity of E-coli expressed
recombinant human and cynomomogus IL-13. The method was carried out as
described in Example 6.2A.
3G4, 3G4C, H2L1 and H3L1 neutralised the bioactivity of E-coli expressed
recombinant cynomomogus IL-13 less potently than human IL-
[ND50 (neutralising dose) is the concentration of mAb required to reduce TF-1
cell
proliferation by 50%, in response to a set concentration of IL-13].
See table 5 below and figures 11 and 12.
Table 5
mAb Mean ND50 for Standard Error
IL-13 variant minimum of 2 assays
(p /ml
E.coli-expressed chimaeric 3G4 0.089 0.029
human IL-13 H2L1 0.210 0.085
H3L1 0.217 0.061
3G4 parental mAb 0.049 0.018
E.coli-expressed chimaeric 3G4 23.95 7.25
cynomolgus IL- H2L1 36.59 4.61
13 H3L1 35.44 0.36
3G4 arental mAb 34.45 3.95
The average ND50 value for 3G4 was 0.049pg/ml as calculated for the
neutralisation
of approximately lOng/mi E.coli-expressed recombinant human IL-13 bioactivity
in
the TF-1 assay. The results in table 5 are the average of four separate
repeats. The
value obtained is comparable although approximately two fold lower than the
ND50
value previously obtained (see Example 1.2).
The level of neutralisation of E.coli expressed human IL13 achieved for
parental 3G4
mouse mAb is comparable to that achieved by chimaeric 3G4. The potency of H2L1
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and H3L1 were reduced in comparison to both parental 3G4 mouse mAb and
chimaeric 3G4.
The average ND50 value for 3G4 parental is 34pg/ml. This was calculated for
the
neutralisation of approximately 10ng/ml E.coli-expressed recombinant cyno IL-
13
bioactivity in the TF-1 assay. This value was similar to the value previously
reported
for parental 3G4 (see Example 1.2). H2L1 and H3L1 also showed similar potency
against cyno IL13 as 3G4.
3.4 Neutralisation of mammalian expressed (CHO cell) human IL-13 in a TF-1
cell proliferation bioassay.
The neutralisation capacity of monoclonal antibody 3G4,3G4C, H2L1, and H3L1
for
human IL-13 expressed from CHO cells was assessed in a TF-1 cell proliferation
assay according to the method as set out in Example 6.2A.
3G4, 3G4C H2L1 and H3L1 neutralised the bioactivity of recombinant CHO-
expressed human IL13 (see table 6 and figure 13).
[ND50 (neutralising dose) is the concentration of mAb required to reduce TF-1
cell
proliferation by 50%, in response to a set concentration of IL-13]
Table 6
mAb Mean ND50 of 4 Standard Error
IL-13 variant assays
(pg/mI)
Mammalian chimaeric 3G4 0.12 0.05
(CHO-cell) H2L1 0.227 0.028
human IL-13 H3L1 0.215 0.003
3G4 parental 0.0515 0.0015
mAb
The average ND50 value for 3G4 parental was 0.05iag/ml. This was calculated
for the
neutralisation of - 10ng/ml mammalian-expressed human IL-13 in a TF-1 cell
bioassay. This value differs to that previously obtained (see Example 1.3).
However
the amount of human IL13 used to stimulate proliferation of the TF-1 cells in
these
experiments was lower than previously used (10ng/mI).
The level of neutralisation of CHO expressed human IL13 achieved by the
parental
3G4 mAb was slightly better than the level for 3G4C. The potencies of H2L1 and
H3L1 were reduced in comparison with both parental 3G4 mAb and chimaeric 3G4.
3.5 Neutralisation of recombinant Q130 human IL-13 variant in a TF-1 cell
proliferation bioassay.
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The neutralisation capacity of 3G4, 3G4C, H2L1 and H3L1 for E.coli-expressed
recombinant Q130 human IL-13 was assessed in a TF-1 cell proliferation assay.
The
method was carried out as described in Example 6.2A.
An ND50 value of 0.274pg/ml was obtained. This differs from the ND50 value
previously obtained (see Example 1.4). This assay was repeated several times
to
confirm these ND50 values . The quality of the commercially-sourced Q130 human
IL-
13 preparation used in both sets of experiments (carried out at different
times) may
explain the differences observed for these 2 data sets.
Table 7 sets out the potencies of 3G4C, H2L1 and H3L1, which were all similar.
See
also Figure 14.
Table 7
mAb Mean ND50 for a Standard Error
IL-13 variant minimum of 2
assays
(lig/mi)
E.coli-expressed chimaeric 3G4 0.37925 0.151153
Q130 human IL- H2L1 0.385 0.160282
13 H3L1 0.416 0.102
3G4 parental 0.274 0.089982
mAb
3.6 BlAcoreTM analysis
The affinity of 3G4C, H2L1 and H3L1 for recombinant human and cynomolgus IL-13
was assessed by BlAcoreTM analysis. See tables 8, 9 and 10.
Analyses were carried out using Protein A capture. Briefly, Protein A was
coupled
onto a CM5 chip by primary amine coupiing in accordance with the manufactures
recommendations. Chimeric antibody or humanised antibodies were then captured
onto this surface and human or cynomolgus IL-13 passed over at defined
concentrations. The surface was regenerated back to the Protein A surface
using
mild acid elution conditions, this did not significantly affect the ability to
capture
antibody for a subsequent IL-13 binding event. The work was carried out on the
Biacore 3000 and the T100 Biacore machines, the data were analysed using the
evaluation software inherent in the machines and fitted to the 1:1 model of
binding.
The data differed slightly between the two machines, though the differences
seen
between the kinetics for the chimera and humanised antibodies binding to human
IL-
13 was similar for both machines. The binding data for human IL-13 fitted well
to the
1:1 model for all constructs, however the fit for binding to cynomolgus IL-13
was
worse, raising the possibility that the actual values may be slightly worse
(e.g. a 2- 3
fold difference) than reported due to this poorer fit and the difficulty in
the analyses.
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The data were generated using untagged recombinant human or cynomolgus IL-13
(made at GSK). All Biacore runs were carried out at 25 degrees C.
Table 8: Biacore 3000 data for chimeric and humanised antibodies binding to
human
IL-13
Antibody ka kd KD (nM)
Chimeric 3G4 3.29e6 1.92e-4 0.060
(6.31 e5) (3.68e-5) (0.00)
H2LI 2.77e6 3.30e-4 0.120
(1.43e5) (9.19e-5) (0.03)
H3L1 2.84e6 3.77e-4 0.130
(9.11 e4) (1.94e-5) (0.01)
For chimaeric 3G4, the data were produced from 4 independent experiments (two
of
which were carried out in duplicate, with each duplicate being analysed
separately).
For H2L1 and H3L1 humanised mAbs, the data were produced from two independent
experiments carried out in duplicate (with each duplicate being analysed
separately).
The data are presented as the mean and standard deviation (in brackets) of
these
results.
Table 9: T100 data for chimeric and humanised antibodies bindin to human IL-13
Antibody ka kd KD (nM)
Chimeric 3G4 1.12e7 2.5e-4 0.023
(7.07e4) (2.4e-5) (0.002)
H2L1 1.07e7 5.68e-4 0.066
(7.76e6) (1.38e-4) (0.035)
H3L1 7.21e6 5.31e-4 0.077
(2.09e6) (3.25e-5) (0.018)
The data were produced from 2 independent experiments and are presented as the
mean and standard deviation (in brackets) of these results.
The 3G4 chimaeric antibody and the humanised mAbs H2L1 and H3L1 bind with high
affinity to human IL-13, and these data are comparable to the bind affinity of
the
parental 3G4 mouse mAb for human IL-13.
Table 10: T100 data for chimeric and humanised antibody binding to cynomolgus
IL-
13
Antibody ka kd KD (nM)
Chimeric 3G4 1.97e6 1.77e-3 0.899
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H2L1 3.18e5 1.12e-3 3.5
H3L1 3.65e5 1.04e-3 2.9
The data were produced from a single experiment.
The 3G4 chimaeric antibody and the humanised mAbs H2L1 and H3L1 bind to
cynomolgus IL-13 with a lower affinity in comparison to their binding affinity
for
human IL-13.
3,7 Specificity of 3G4C, H2L1 and H3L1 for binding to human IL-13.
The specificities of 3G4C, H2L1 and H3L1_for human IL-13 were assessed by
analysis of the cross-reactivity potential against human IL-4, human IL-5 and
human
GM-CSF in binding ELISAs. These methods were carried as described in section
6.7,
6.8 and 6.9 respectively. See figures 15, 16 and 17. These mAbs were found to
be
specific for binding to IL-13, with no cross-reactivity for human IL-4 human
IL-5 or
human GM-CSF at mAb concentrations up to 30pg/ml. 3G4 chimera appeared to
show some binding to human IL-5 at 301ag/mi, this is probably due to a
pipetting error
as no such observation was made for humanised mAbs H2L1 and H3L1 at a similar
concentration.
4. EPITOPE MAPPING OF 3G4 USING BIOTINYLATED PEPTIDES
A series of epitope mapping experiments were performed to determine which
amino
acid residues in IL-13 were required for binding of the 3G4 mouse mAb.
4.1 Crude mapping of the 3G4 mouse mAb binding epitope on human and
cynomoigus IL-13
Biotinylated 16 mer peptides offset by 4 were synthesised to map the location
of the
binding epitope recognised by mouse mAb 3G4 on human and cynomolgus IL-13. An
ELISA method as described in Example 6.3 was used to detect binding of
immobilised biotinylated peptide to the parental mouse mAb 3G4.
Details of 16 mer custom designed Peptides: 88 x 16 mers, offset by 4
(supplied by
Mimotopes, Australia).
Format: Peptides 25 & 44 = Biotin-SGSG-PEPTIDE-acid
Peptides 2-24 & 27-43 = Biotin-SGSG-PEPTIDE-amide
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# Hydro Mo1Wt N-term Sequence C-term
2 0.42 2,311.66 Biotin- SEQ.I.D.NO:46 -NH2
3 0.27 2,453.82 Biotin- SEQ.I.D.NO:47 -NH2
4 0.38 2,326.70 Biotin- SEQ.I.D.NO:48 -NH2
0.31 2,231.58 Biotin- SEQ.I.D.NO:49 -NH2
6 0.43 2,289.66 Biotin- SEQ.I.D.NO:50 -NH2
7 0.59 2,190.57 Biotin- SEQ.I.D.NO:51 -NH2
8 0.57 2,260.64 Biotin- SEQ.I.D.NO:52 -NH2
9 0.62* 2,255.64 Biotin- SEQ.I.D.NO:53 -NH2
0.51 2,197.56 Biotin- SEQ.I.D.NO:54 -NH2
11 0.56 2,144.52 Biotin- SEQ.I.D.NO:55 -NH2
12 0.46 2,090.38 Biotin- SEQ.I.D.NO:56 -NH2
13 0.29 2,219.54 Biotin- SEQ.I.D.NO:57 -NH2
14 0.29 2,180.53 Biotin- SEQ.I.D.NO:58 -NH2
0.36 2,318.70 Biotin- SEQ.I.D.NO:59 -NH2
16 0.32 2,303.73 Biotin- SEQ.I.D.NO:60 -NH2
17 0.47 2,209.57 Biotin- SEQ.I.D.NO:61 -NH2
18 0.48 2,257.60 Biotin- SEQ.I.D.NO:62 -NH2
19 0.17 2,273.57 Biotin- SEQ.I.D.NO:63 -NH2
0.27 2,300.60 Biotin- SEQ.I.D.NO:64 -NH2
21 0.29 2,383.77 Biotin- SEQ.I.D.NO:65 -NH2
22 0.35 2,401.83 Biotin- SEQ.I.D.NO:66 -NH2
23 0.45 2,407.92 Biotin- SEQ.I.D.NO:67 -NH2
24 0.42 2,541.08 Biotin- SEQ.I.D.NO:68 -NH2
0.33 2,513.97 Biotin- SEQ.I.D.NO:69 -OH
27 0.42 2,283.64 Biotin- SEQ.I.D.NO:70 -NH2
28 0.27 2,425.81 Biotin- SEQ.I.D.NO:71 -NH2
29 0.57 2,228.57 Biotin- SEQ.I.D.NO:72 -NH2
0.62* 2,223.57 Biotin- SEQ.I.D.NO:73 -NH2
31 0.51 2,165.49 Biotin- SEQ.I.D.NO:74 -NH2
32 0.56 2,112.45 Biotin- SEQ.I.D.NO:75 -NH2
33 0.27 2,207.56 Biotin- SEQ.I.D.NO:76 -NH2
34 0.33 2,345.73 Biotin- SEQ.I.D.NO:77 -NH2
0.29 2,330.76 Biotin- SEQ.I.D.NO:78 -NH2
36 0.45 2,236.60 Biotin- SEQ.I.D.NO:79 -NH2
37 0.43 2,276.64 Biotin- SEQ.I.D.NO:80 -NH2
38 0.12 2,292.62 Biotin- SEQ.I.D.NO:81 -NH2
39 0.22 2,319.64 Biotin- SEQ.I.D.NO:82 -NH2
0.24 2,402.82 Biotin- SEQ.I.D.NO:83 -NH2
41 0.33 2,387.80 Biotin- SEQ.I.D.NO:84 -NH2
42 0.43 2,393.90 Biotin- SEQ.I.D.NO:85 -NH2
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43 0.39 2,527.05 Biotin- SEQ.I.D.NO:86 -NH2
44 0.35 2,471.88 Biotin- SEQ.I.D.NO:87 -OH
(* indicates a high hydrophobicity value)
The results indicated that 3G4 mouse mAb bound to the two peptides shown below
(also see figure 6).
Peptide 25: DLLLHLKKLFREGRFN (SEQ.I.D.NO:88)
Peptide 44: DLLVHLKKLFREGQFN (SEQ.I.D.NO:89)
Peptide 25 is derived from human IL-13.
Peptide 44 is derived from cynomolgus IL-13.
NB: BOLD indicates residue differences between human IL-13 and the cynomoigus
IL-13 orthologue.
4.2 Fine mapping of the 3G4 mouse mAb binding epitope on human and
cynomoigus IL-13 using biotinyiated peptides
The minimal binding epitope for mouse mAb 3G4 on human IL-13 was determined
using a peptide set based around the peptide sequence,
QFVKDLLLHLKKLFREGRFN (SEQ.I.D.NO:90). Peptides were obtained with 1 amino
acid sequentially removed from either the N or C-terminus of this parental
peptide
sequence in order to define the precise linear binding epitope for mouse mAb
3G4. A
similar approach was taken to map binding to cynomolgus IL-13.
An ELISA method (carried out as described in Example 6.4) was used to detect
binding of immobilised biotinylated peptide to the parental mouse mAb 3G4
(figures
7a and 7b).
The results indicate that parental mouse mAb 3G4 binds to the minimal amino
acid
epitope LLHLKKLFREG (SEQ.I.D.NO:91) at the C-terminal region of human IL-13.
However, the 2 amino acids (D and L) located adjacent to the N-terminal end of
the
above peptide sequence (in the human IL-13 sequence) may also be important for
binding, as the binding signal is enhanced when these residues are present.
Similarly, the 3 amino acids (R, F and N) located adjacent to the C-terminal
end of
the above peptide sequence (in the human IL-13 sequence) may also be important
for binding, as the binding signal is lost when the R and F residues are
present, but
the binding signal is restored when the N residue is present.
Similar results were obtained for binding of mouse mAb 3G4 to the cynomoigus
IL-13
peptide set. The results indicate that parental mouse mAb 3G4 binds to the
minimal
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amino acid epitope LLVHLKKLFREG (SEQ.I.D.NO:98) in the C-terminal region of
cynomolgus IL-13. However, the 1 amino acid (D) located adjacent to the N-
terminal
end of the above peptide sequence (in the cynomolgus IL-13 sequence) may also
be
important for binding, as the binding signal is enhanced when this residue is
present.
Similarly, the 3 amino acids (Q, F and N) located adjacent to the C-terminal
end of
the above peptide sequence (in the cynomoigus IL-13 sequence) may also be
important for binding, as the binding signal is lost when the Q and F residues
are
present, but the binding signal is restored when the N residue is present.
4.3 Alanine scanning of the mouse mAb 3G4 binding epitope using biotinyiated
peptides
In order to identify the key residues involved in the interaction of human IL-
13 with
mouse mAb 3G4, an alanine scanning approach was adopted using parental peptide
sequence QFVKDLLLHLKKLFREGRFN (SEQ.I.D.NO:90). For this analysis, peptides
as set out in Table 11 were obtained (AnaSpec Inc) where one amino acid was
sequentially substituted for an alanine residue at each amino acid position in
the
LKKLFRE (SEQ.I.D.NO:92) portion of the parental QFVKDLLLHLKKLFREGRFN
(SEQ.I.D.NO:90) epitope.
Table 11
SEQ.I.D.NO N-terminus Peptide sequence
SEQ.I.D.NO:90 Biotin-SGSG QFVKDLLLHLKKLFREGRFN
SEQ.I.D.NO:99 Biotin-SGSG QFVKDLLLHAKKLFREGRFN
SEQ.I.D.NO:100 Biotin-SGSG QFVKDLLLHLAKLFREGRFN
SEQ.I.D.NO:101 Biotin-SGSG QFVKDLLLHLKALFREGRFN
SEQ.I.D.NO:102 Biotin-SGSG QFVKDLLLHLKKAFREGRFN
SEQ.I.D.NO:103 Biotin-SGSG QFVKDLLLHLKKLAREGRFN
SEQ.I.D.NO:104 Biotin-SGSG QFVKDLLLHLKKLFAEGRFN
SEQ.I.D.NO:105 Biotin-SGSG QFVKDLLLHLKKLFRAGRFN
An ELISA method was used (similar to that set out in Example 6.4) to detect
binding
of immobilised biotinylated peptide to the parental mouse mAb 3G4 (see Figure
8)
These data confirm that the key amino acid residues involved in the
interaction of
parental mouse mAb 3G4 with human IL-13 are at least, arginine (R) at position
107,
lysine (K) at position 103 and lysine (K) at position 104. The numbering of
these is as
previously described in W02006/003407.
As the above analysis had only scanned the LKKLFRE (SEQ ID NO:92) portion of
the minimal binding epitope, a further set of peptides as set out in Table 12
were
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obtained (Mimotopes) to expand the alanine scanning study to the other amino
acid
residues in the minimal binding epitope.
Table 12:
SEQ.I.D.NO N-terminus Peptide sequence
SEQ.I.D.NO:90 Biotin-SGSG QFVKDLLLHLKKLFREGRFN
SEQ.I.D.NO:104 Biotin-SGSG QFVKDLLLHLKKLFAEGRFN
SEQ.I.D.NO:106 Biotin-SGSG QFVKDLLLALKKLFREGRFN
SEQ.I.D.NO:107 Biotin-SGSG QFVKDLLAHLKKLFREGRFN
SEQ.I.D.NO:108 Biotin-SGSG QFVKDLALHLKKLFREGRFN
SEQ.I.D.NO:109 Biotin-SGSG QFVKDALLHLKKLFREGRFN
SEQ.I.D.NO:110 Biotin-SGSG QFVKALLLHLKKLFREGRFN
SEQ.I.D.NO:111 Biotin-SGSG QFVKDLLLHLKKLFREARFN
SEQ.I.D.NO:112 Biotin-SGSG QFVKDLLLHLKKLFREGAFN
SEQ.I.D.NO:113 Biotin-SGSG QFVKDLLLHLKKLFREGRAN
SEQ.I.D.NO:114 Biotin-SGSG QFVKDLLLHLKKLFREGRFA
Again, an ELISA method (similar to that used in Example 6.4) was used to
detect
binding of immobilised biotinylated peptide to the parental mouse mAb 3G4. See
Figure 9 (in this experiment peptide QFVKDLLLHLKKLFREGRFN (SEQ.I.D.NO:90)
is the positive control demonstrating maximal binding, and peptide
QFVKDLLLHLKKLFAEGRFN (SEQ ID No 104) is the negative control demonstrating
minimal binding).
These data suggest that the phenylalanine (F) residue at position 111 is also
important for the interaction of parental mouse mAb 3G4 with human IL-13. The
numbering of this residue is as previously described in WO2006/003407.
5. Efficacy of a humanised anti-IL-13 mAb in cynomolgus asthma model.
This Example is prophetic.
The model of Ascaris suum-induced (A.suum) pulmonary bronchoconstriction in
cynomolgus monkeys (Macaca fascicularis) is widely recognised as the non-
clinical
model most similar and relevant to asthma in humans (Patterson R, et al Trans.
Assoc. Am. Physicians 1980 93:317-325; Patterson R, et al J. Lab. Clin. Med.
1983
101:864-872).
In this model, animals having an innate pulmonary sensitivity to A.suum are
exposed
to nebulised A.suum to induce an asthmatic response. This asthmatic response
can
be characterised by measuring airways hyper-responsiveness (AHR), cellular
infiltration as measured in broncho alveolar lavage (BAL) fluid and serum IgE
levels.
Experimental methods are similar to those previously described by Mauser P, et
al in
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Am. J. Resp. Crit. Care Med. 1995 204:467-472 and by Evanoff H, et a/ in
Immunologic Investigation 1992 21:39.
This study uses 30 animals, pre-selected for entry having demonstrated a
positive
bronchoconstrictor response to a specific dose of A.suum antigen.
A.suum is administered at the optimal response dose (ORD) for each animal. It
is a
pre-determined dose of A.suum that produces an increase in RL (lung
resistance) of
at least 40% and a decrease in CDYN (dynamic compliance) of at least 35%, by
aerosol inhalation (for a single dose given over 15 breaths using a
neublizer).
The study takes place in 2 phases. During phase 1, AHR is assessed in response
to
intravenous (i/v) histamine challenge (that is a dose of histamine sufficient
to induce
an increase in RL of at least 30% above baseline (PC30)) both before (the
baseline
pulmonary function assessment on day 1) and after (on day 11) administering
A.suum antigen (on days 9 and 10, when A.suum is administered at an optimal
pre-
determined dose for each animal by aerosol inhalation).
Phase 2 is identical to phase 1 except that animals receive treatment with
antibody
(see below), each antibody is given as 3 doses of approximately 30mg/kg
administered by i/v infusion on days 1, 5 and 9.
Group 1(n=12): A humanised anti-IL-13 mAb (30mg/kg)
Group 2 (n=12): A humanised anti-IL-13 mAb (30mg/kg) and Pascolizumab
(humanised anti-IL4 mAb, 30mg/kg)
Group 3 (n=6): vehicle alone negative control treatment
The AHR readouts from phases 1 and 2 are calculated by taking pressure and
airflow
readings - lung resistance (RL) and dynamic compliance (CDYN) - in response to
histamine, using the Buxco pulmonary mechanics system. The maximum percentage
change from the baseline compared to post A.suum antigen challenge [for lung
resistance (RL) and dynamic compliance (CpYN)] is compared for phases 1 and 2
i.e.
with or without antibody treatment, and these data are used to assess the AHR
phenotype.
In addition BAL samples are taken at days 1 and 11 in phases 1 and 2, to
measure
cellular infiltration and in particular eosinophilia. Serum samples are also
taken to
monitor IgE levels.
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6.1. Human or cynomolgus IL-13 binding ELISA
This assay describes an ELISA that detects binding of an antibody to human or
cynomolgus IL-13. It is a sandwich ELISA format.
6.1.1 Materials
1. Nunc Immunoplate 1 F96 Maxisorp (Life Technologies, 4-39454A)
2. Human IL-13 (E.coli expressed from Cambridge Biosciences, cat. no. CH1-
013)
3. Cynomolgus IL-13 (made by GlaxoSmithkline)
4. Goat anti-human IL-13 polyclonal antibody (R+D Systems, cat. no. AF-213-
NA)
5. Anti-human IgG-HRP (Sigma, Cat No. A-6029)
6. Anti-mouse IgG-HRP (Sigma, Cat No. A-9309)
7. Carbonate/bicarbonate buffer (Sigma; cat. no. C-3041)
8. TBST [Tris buffered saline (6.06g Tris + 8.06g NaCI + 0.2g KCI + H20 to 1
L) +
0.05% Tween 20]
9. BSA (Sigma A-7030)
10. OPD (Sigma, Cat. No. P-9187)
11. Sulphuric acid
6.1.2 Method
1. Blocking solution is 3% BSA+TBST
2. Washing solution is TBST
3. Coat 'Nunc Maxisorp' ELISA plates with 50u1 of 5ug/ml goat anti-human IL-13
polyclonal antibody (R+D Systems, cat. no. AF-213-NA. Made up at a stock
concentration of 500ug/ml according to maufacturers instructions, and stored
in
aliquots at -20C) in carbonate/bicarbonate buffer (Sigma; cat. no. C-3041,
made up as per maufacturers instructions), cover with a plate sealer and
incubate overnight at 4 C.
4. Block with 100ul of 3% BSA/TBST incubate at rtp for 1 hr.
5. Wash X3 in TBST (at least 200u1 wash solution per well per wash).
6. Add 20ng per well (in a 50ul volume) human IL-13 (Cambridge Bioscience,
cat.
no. CH1-013. Made up at a stock concentration of 100ng/uI according to
maufacturers instructions, and stored in aliquots at -20C) or 20ng per well
cynomoigus IL-13, in block solution and incubate at room temperature for 1 hr.
7. Wash X3 in TBST.
8. Add 50u1 antibody sample (titrate out to obtain end-point titre data, if
required) in
block solution, incubate at rtp for 1 hr.
9. Wash X3 in TBST.
10. For 3G4 chimaeric antibody or humanised antibody, detect binding using
50u1
per well anti-human IgG-HRP (Sigma, Cat No. A-6029) at a 1/2000 dilution in
block solution for 1 hr at rtp. For 3G4 mouse monoclonal antibody, detect
binding
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using 50ul per well anti-mouse IgG-HRP (Sigma, Cat No. A-9309) at a 1/1000
dilution in block solution for 1 hr at rtp.
11. Wash X3 in TBST.
12. Develop with 100ul OPD (Sigma, Cat. No. P-9187. Made up as per
maufacturers instructions), stop with 50u1 3M H2SO4, read at an absorbance of
490nm. Development time is - 12 minutes.
6.1A Human IL-13 binding ELISA
This assay describes an ELISA that detects binding of an antibody to human IL-
13. It
is a sandwich ELISA format and differs only slightly to that described in
Example 6.1.
6.1A.1 Materials
1. Costar 96 well EIA plate (Corning Costar cat. no 3369)
2. Human IL-13 (Peprotech cat. no. 200-13)
3. Goat anti-human IL-13 polyclonal antibody (R+D Systems, cat. no. AF-213-
NA)
4. Anti-human kappa light chain-HRP (Sigma, Cat No. A7164)
5. Carbonate/bicarbonate buffer (Sigma; cat. no. C-3041)
6. TBST [Tris buffered saline (6.06g Tris + 8.06g NaCI + 0.2g KCI + H20 to 1
L) +
0.05% Tween 20]
7. BSA (Sigma A-7030)
8. OPD (Sigma, Cat. No. P-9187)
9. Sulphuric acid
6.1A.2 Method
1. Blocking solution is 3% BSA+TBST
2. Washing solution is TBST
3. Coat 'Costar E1A/RIA' ELISA plates with 50u1 of 5ug/mI goat anti-human IL-
13 polyclonal antibody (R+D Systems, cat. no. AF-213-NA. Made up at a
stock concentration of 500ug/ml according to maufacturers instructions, and
stored in aliquots at -20C) in carbonate/bicarbonate buffer (Sigma; cat. no.
C-3041, made up as per maufacturers instructions), cover with a plate sealer
and incubate overnight at 4 C.
4. Block with 100ul of 3% BSA/TBST incubate at rtp for 1 hr or minimum of
overnight 4 C.
5. Wash X2 in TBST (at least 200ul wash solution per well per wash).
6. Add 20ng per well (in a 50u1 volume) human IL-13 (Peprotech cat. no. 200-
13). Made up at a stock concentration of 100ng/ul according to maufacturers
instructions, and stored in aliquots at -20C), dilute in block solution and
incubate at room temperature for 1 hr.
7. Wash X2 in TBST.
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8. Add 50ul antibody sample (titrate out to obtain end-point titre data, if
required) in block solution, incubate at rtp for 1 hr.
9. Wash X2 in TBST.
10. For 3G4 chimaeric antibody or humanised antibody, detect binding using
50u1 per well anti-human kappa light chain-HRP (Sigma, Cat No. A7164) at a
1/2000 dilution in block solution for 1 hr at rtp.
11. Wash X2 in TBST.
12. Develop with 100ul OPD (Sigma, Cat. No. P-9187. Made up as per
maufacturers instructions), stop with 50ul 3M H2SO4, read at an absorbance
of 490nm.
6.1113 Native human IL-13 binding ELISA
This assay describes an ELISA that detects binding of an antibody to native
human
IL-13. It is a sandwich ELISA format.
6.1 B.1 Materials
1. Nunc Immunoplate 1 F96 Maxisorp (Life Technologies, 4-39454A)
2. Native human IL-13 (HDLM-2 cell supernatant)
3. Anti-human IL13 antibody (Pharmingen, Cat. No. 554570)
4. Biotinylated Anti-human IL13 antibody (Pharmingen, Cat. No. 555054)
5. Streptavidin-HRP (Sigma cat no. E2886)
6. Carbonate/bicarbonate buffer (Sigma; cat. no. C-3041)
7. RPMI +20%FBS+2mMglutamine
8. PBST (PBS + 0.05% Tween 20)
9. BSA (Sigma A-7030)
10.OPD (Sigma, Cat. No. P-9187)
11. Sulphuric acid
6.1 B.2 Method
1. Blocking solution is 1 % BSA in PBST
2. Washing solution is PBST
3. Coat 'Nunc Maxisorp' ELISA plates with 50u1 of 5ug/ml of 3G4 chimeric or
humanised antibody or 2ug/ml anti human IL13 (Pharmingen cat no. 554570)
dilution in Carbonate/bicarbonate buffer, cover with a plate sealer and
incubate
overnight at 4 C.
4. Block with 200ul of 1% BSA/PBST incubate at room temperature for 1 hr.
5. Wash X3 in PBST.
6. Add 50u1 native IL13 present in HDLM2 supernatent sample (titrate out)
dilute in
RPMI +20%FBS+2mMglutamine solution, incubate at rtp for 1 hr.
7. Wash X3 in PBST.
8. Add 50u1 per well biotinylated anti-human IL13 1 ug/mI (Pharmingen, Cat.
No.
555054) dilute in PBST + 1 /oBSA _incubate at rtp for 1 hr.
9. Wash X3 in PBST.
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10. Add 50ul per well streptavidin-HRP conjugate at 1/1000 dilution dilute in
PBST +
1%BSA. Incubate for 1 hour at room temperature.
11. Wash x3 in TBST
12.Develop with 100ul per well OPD (Sigma, Cat. No. P-9187. Made up as per
maufacturers instructions), stop with 50u1 per well 3M H2SO4, read at an
absorbance of 490nm.
6.2. IL-13 neutralisation bioassay (TF-1 cell proliferation assay)
This is an IL-13 bioassay that can be used to determine the neutralisation
capacity of
an anti-IL-13 antibody. The method described below uses recombinant human or
cynomolgus IL-13. Mammalian-expressed human IL-13 or the Q130 human IL-13
variant can also be used in this assay too.
6.2.1 Materials
1. TF-1 cell line (TF-1 cell line obtained in-house, NB, ATCC version
available)
2. 96 well tissue culture plates (Invitrogen)
3. Human IL-13 (Cambridge Bioscience, cat. no. CH1-013)
4. CeIlTiter 96 non-radioactive cell proliferation assay (Promega, Cat. No.
G4000)
6.2.2 Method
1. Method to measure the ability of an anti-human IL-13 mAb to neutralise the
bioactivity of recombinant human or cynomolgus IL-13 in a TF-1 cell bioassay.
2. This assay is performed in sterile 96 well tissue culture plates
(Invitrogen), under
sterile conditions. All tests are performed in triplicate.
3. Pre-incubate 10ng/ml human IL-13 (Cambridge Bioscience, cat. no. CH1-013.
Make up at a stock concentration of 100ng/ul according to maufacturers
instructions using sterile technique in a class 2 tissue culture hood, store
in small
aliquots at -20C) or 10ng/ml cynomolgus IL-13 (generated at GSK) with various
dilutions of the anti-human IL-13 mAb (diluted from 6ug/ml in 3 fold dilutions
down
to 0.025ug/ml) in a total volume of 50u1 for 1 hour at 37C. Also included will
be
positive control wells, having IL-13 present but no anti-human IL-13 mAb. In
addition, negative control wells will have no IL-13 and no anti-human IL-13
mAb
present. Use a sterile, low protein binding, round bottom 96 well plate for
this pre-
incubation. (Note that the concentration of IL-13 and anti-human IL-13 mAb
will be
halved at a later stage when cells are added).
4. Plate out 50ul of TF-1 cells at 2x105 per ml in a sterile 96 well tissue
culture plate.
After the 1 hour pre-incubation, add the IL-13 and anti-human IL-13 mAb sample
to the cells. The final 100ul assay volume, containing various anti-human IL-
13
mAb dilutions, recombinant IL-13 and TF-1 cells, is incubated at 37 C for -70
hours in a humidified CO2 incubator.
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5. At - 66hrs, scan the wells to confirm that they are sterile and that no
bacterial
contamination has occurred.
6. Add 15ul of filter sterilised MTT substrate per well (Cat. No. G4000,
Promega.
Made up as per maufacturers instructions) for the final 4 hours of incubation.
7. Stop the reaction with 100ul of stop solution (provided in the MTT kit) to
solubilise
the metabolised blue formazan product. Leave for at least 2 hours, then
pipette up
and down to help dissolve the crystals. Alternatively, cover with a plate
sealer and
leave at 4C overnight, then pipette up and down the next day (this is easier
in
terms of pipetting)
8. Read the absorbance of the solution in each well in a 96-well plate reader
at
570nm wavelength.
9. The capacity of the anti-human IL-13 mAb to neutralise human or cynomoigus
IL-
13 bioactivity is expressed as, that concentration of anti-human IL-13 mAb
required to neutralise the bioactivity of a defined amount of human or
cynomolgus
IL-13 (5ng/ml) by 50% (= ND50). The lower the concentration required, the more
potent the neutralisation capacity.
6.2.A IL-13 neutralisation bioassay (TF-1 cell proliferation assay)
This is an IL-13 bioassay that can be used to determine the neutralisation
capacity of
an anti-IL-13 antibody. The method described below uses recombinant human and
cynomolgus IL-13. Mammalian-expressed human IL-13 or the Q130 human IL-13
variant can also be used in this assay too. Note that this method differs only
slightly
to that described in Example 6.2
6.2.A.1 Materials
1. TF-1 cell line (TF-1 cell line obtained in-house, NB, ATCC version
available)
2. 96 well tissue culture plates (Corning costar, cat no. 3596)
3. Human IL-13 (Peprotech, cat. no. 200-13)
4. Human IL-13 (CHO cell expressed) generated at GSK.
5. Human IL-13 Q130 variant (Peprotech, cat. no. 200-13A)
6. Cynomolgus IL-13 (generated at GSK).
7. Polyclonal anti-human IL13 (R&D systems AF-213-NA)
8. 96 well tissue culture plates (Corning costar, cat no.3790)
9. CeIlTiter 96 non-radioactive cell proliferation assay (Promega, Cat. No.
G4000)
6.2.A.2 Method
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1. Method to measure the ability of an anti-human IL-13 mAb to neutralise the
bioactivity of recombinant human and cynomolgus IL-13 in a TF-1 cell
bioassay.
2. This assay is performed in sterile 96 well tissue culture plates (Corning
costar,
cat no. 3596), under sterile conditions. All tests are performed in
triplicate.
3. Pre-incubate 10ng/ml human IL-13 (Peprotech, cat. no. 200-13), or 10ng/ml
CHO expressed human IL13 (generated at GSK) or 60ng/mI human IL13
Q130 variant (Peprotech, cat. no. 200-13A), or 10ng/ml cynomoigus IL-13
(generated at GSK) make up commercial Ab at a stock concentration
according to maufacturers instructions using sterile technique in a class 2
tissue culture hood, store in small aliquots at -20C. With various dilutions
of
the anti-human IL-13 mAb (diluted from 6ug/mi or 2ug/ml or 180 ug/mI in 3 fold
dilutions down to 0.025ug/ml or 0.008 ug/ml or 0.74 ug/mI respectively) in a
total volume of 50u1 for 1 hour at 37C. Also included will be positive control
wells, having IL-13 present but no anti-human IL-13 mAb. In addition, negative
control wells will have no IL-13 and no anti-human IL-13 mAb present. Use a
sterile, low protein binding, round bottom 96 well plate for this pre-
incubation
(Corning costar, cat no.3790). (Note that the concentration of IL-13 and anti-
human IL-13 mAb will be halved at a later stage when cells are added).
4. Plate out 50u1 of TF-1 cells at 2x105 per ml in a sterile 96 well tissue
culture
plate (Corning costar, cat no. 3596). After the 1 hour pre-incubation, add the
IL-13 and anti-human IL-13 mAb sample to the cells. The final 100u1 assay
volume, containing various anti-human IL-13 mAb dilutions, recombinant IL-13
and TF-1 cells, is incubated at 37 C for 3 days in a humidified CO2 incubator.
5. Scan the wells to confirm that they are sterile and that no bacterial
contamination has occurred.
6. Add 15u1 of MTT substrate per well (Cat. No. G4000, Promega) for the final
4
hours of incubation.
7. Stop the reaction with 100ul of stop solution (provided in the MTT kit) to
solubilise the metabolised blue formazan product. Leave for at least 2 hours
at
RT, then shake plates on plate shaker for -30min. Alternatively, cover with a
plate sealer and leave at 4C overnight, then shake plates on plate shaker for
-30min.Read the absorbance of the solution in each well in a 96-well plate
reader at 570nm wavelength.
8. The capacity of the anti-human IL-13 mAb to neutralise human and
cynomolgus IL-13 bioactivity is expressed as, that concentration of anti-human
IL-13 mAb required to neutralise the bioactivity of a defined amount of human
or cynomolgus IL-13 by 50% (= ND50). The lower the concentration required,
the more potent the neutralisation capacity.
6.3. Epitope crude mapping ELISA
This assay describes an ELISA that detects binding of mouse mAb 3G4 to human
or
cynomolgus IL-13 peptides.
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6.3.1 Materials
1. Nunc Immunoplate 1 F96 Maxisorp (Life Technologies, 4-39454A)
2. ImmunoPure Streptavidin (Pierce, cat. no. 21125)
3. PBST (Phosphate buffered saline + 0.05% Tween 20)
4. BSA (Sigma A-7030)
5. Human and cynomolgus IL-13 16 mer peptides, offset = 4 (Mimotopes custom
order)
6. Positive and negative control 20 mer peptides (Supplied with Mimotopes
custom order)
7. 3G4 mouse mAb
8. Control Ab (Supplied with Mimotopes custom order)
9. Rabbit anti-mouse Ig HRP conjugated (DAKO, code no. P0260)
10.OPD (Sigma, Cat. No. P-9187)
11. 3M Sulphuric acid
6.3.2 Method
1. Blocking solution is 3% BSA+PBST.
2. Washing solution is PBST.
3. Coat 'Nunc Maxisorp' ELISA plates with 100 1 of 5 g/ml ImmunoPure
Streptavidin (Pierce, cat. no. 21125 made up at a stock concentration of
1 mg/mI according to manufacturers instructions, and stored in aliquots at
+4 C) using PBST as a dilutent. Incubate overnight at 37 C to allow solution
to
dry.
4. Block with 200 1 of 3% BSA/PBST. Add plate sealer and incubate at rtp for
1 hr.
5. Wash X3 in PBST (at least 200 1 wash solution per well per wash).
6. In duplicate and using PBST as a dilutent, add 100 1 per well (except
control
wells) of 1,000-fold dilutions of each peptide (dissolved as per manufacturers
instructions in 200 1 40% Acetonitrile 60% Water, then aliquoted in 10-fold
dilutions in the same solvent and stored at -20 C).
7. In the control wells, in duplicate and using PBST as a dilutent add 100 1
per
well of 10-fold dilutions of control peptides (dissolved as per manufacturers
instructions in 1 ml 40% Acetonitrile 60% Water and stored at -20 C). Add
plate sealer and incubate at rtp for 1 hr on a shaking table.
8. Wash X3 in PBST (at least 200 1 wash solution per well per wash).
9. Add 100 1 per well (except control wells) of 1.506 g/mI mouse mAb in PBST.
10.Add 100 1 per well to control wells only, 4, 16 and 32-fold dilutions of
control
antibody (used as supplied by the manufacturer and stored at -20 C) using
PBST as a dilutent. Add plate sealer and incubate at rtp (room temperature
and pressure) for 1 hr on a shaking table.
11. Wash X3 in PBST (at least 200 1 wash solution per well per wash).
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12.Add 100 1 per well of 2,000-fold dilution of rabbit anti-mouse Ig HRP-
conjugated (DAKO, code no. P0260 used as supplied, stored at +4 C) using
PBST as a dilutent. Add plate sealer and incubate at rtp for 1 hr on a shaking
table.
13. Wash X3 in PBST (at least 200 1 wash solution per well per wash).
14. Develop with 100 l OPD (Sigma, Cat. No. P-9187. Made up as per
manufacturers instructions), stop with 50 l 3M H2SO4, read at an absorbency
of 490nm. Development time is - 10 minutes.
6.4. Epitope fine mapping ELISA
This assay describes an ELISA that detects binding of mAb 3G4 to human or
cynomolgus IL-13 peptides.
6.4.1 Materials
1. Nunc Immunoplate 1 F96 Maxisorp (Life Technologies, 4-39454A)
2. ImmunoPure Streptavidin (Pierce, cat. no. 21125)
3. PBST (Phosphate buffered saline + 0.05% Tween 20)
4. BSA (Sigma A-7030)
5. Human and cynomolgus IL-13 partial window net peptides (14-mer truncated
by one amino acid at a time from both the N- and C-terminal ends; Mimotopes
custom order)
6. Positive control 16 mer peptide (Supplied with previous Mimotopes custom
order)
7. 3G4 mAb (made in-house)
8. Goat anti-mouse IgG (Fc specific) HRP conjugated antibody (Sigma A-9309)
9. OPD (Sigma, Cat. No. P-9187)
10.3M Sulphuric acid
6.4.2 Method
1. Blocking solution is 3% BSA+PBST.
2. Washing solution is PBST.
3. Coat 'Nunc Maxisorp' ELISA plates with 100 l of 5 g/ml ImmunoPure
Streptavidin in ultra pure water (Pierce, cat. no. 21125 made up at a stock
concentration of 1 mg/mi according to manufacturer's instructions, and stored
at +4 C). Incubate overnight at +37 C.
4. Block with 200 1 of 3% BSA in PBST. Add plate sealer and incubate overnight
at +4 C.
5. Wash X3 in PBST (at least 200 1 wash solution per well per wash).
6. In duplicate and using 3% BSA in PBST as a dilutent, add 100 1 per well of
1,000-fold dilutions of each peptide (dissolved as per manufacturers
instructions in 200 1 of 40% Acetonitrile 60% Water and stored at -20 C). Add
plate sealer and incubate at room temperature for 1 hour on a shaking table.
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7. Wash X3 in PBST (at least 200 l wash solution per well per wash).
8. Add 100 l per well of 3 g/ml 3G4 diluted in 3% BSA in PBST. Add plate
sealer and incubate at room temperature for 1 hour on a shaking table.
9. Wash X3 in PBST (at least 200 1 wash solution per well per wash).
10.Add 100 l per well of 1,000-fold dilution of goat anti-mouse IgG HRP-
conjugated antibody (Sigma A-9309 used as supplied, stored at +4 C) using
3% BSA in PBST as a dilutent. Add plate sealer and incubate at room
temperature for 1 hour on a shaking table.
11. Wash X3 in PBST (at least 200 I wash solution per well per wash).
12. Develop with 100 1 OPD (Sigma, Cat. No. P-9187. Made up as per
manufacturers instructions), stop with 50 1 3M H2SO4, read at an absorbency
of 490nm. Development time is - 10 minutes.
6.5 Human IL-13 binding to the human IL-13Ra1 chain ELISA
This ELISA determines whether an antibody can inhibit human IL-13 binding to
the
human IL13Ra1 chain.
6.5.1 Materials
1. Nunc Immunoplate 1 F96 Maxisorp (Life Technologies, 4-39454A)
2. Human IL13Ra1-Fc (R&D Systems, cat.no. 146-IR)
3. human IL-13 (made in-house)
4. Biotinylated anti-human IL-13 (R&D Systems, cat. no. BAF213)
5. Streptavidin-HRP (Sigma cat no. E2886)
6. Carbonate/bicarbonate buffer (Sigma; cat. no. C-3041)
7. TBST [Tris buffered saline (6.06g Tris + 8.06g NaCI + 0.2g KCI + H20 to 1L)
+
0.05% Tween 20]
8. BSA (Sigma A-7030)
9. OPD (Sigma, Cat. No. P-9187)
10. Sulphuric acid
6.5.2 Method
1. Blocking solution is 3% BSA+TBST
2. Washing solution is TBST
3. Coat'Nunc Maxisorp' ELISA plates with 50ul of 5ng/ul human IL-13Ra1-Fc in
carbonate/bicarbonate buffer. Cover with a plate sealer and incubate overnight
at
4 C.
4. Block with 100ul of 3% BSA/TBST incubate at rtp for 1 hr.
5. Wash X3 TBST (at least 200ul wash solution per well per wash).
6. In a total volume of 50ul, pre-incubate 0.4ng/ul human IL-13 with antibody
sample
(titrated) in block solution for 30 minutes. Add the pre-incubated sample to
the
receptor-coated ELISA plate and incubate at room temperature for 1 hr.
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7. Wash x3 in TBST
8. Detect any bound human IL-13 using 50u1 per well biotinylated anti-human IL-
13
diluted at 1 ug/ml. Incubate for 1 hour at room temperature
9. Wash x3 in TBST
10.Add 50u1 per well streptavidin-HRP conjugate at 1/1000 dilution. Incubate
for
1 hour at room temperature.
11. Wash x3 in TBST
12. Develop with 100ul per well OPD (Sigma, Cat. No. P-9187. Made up as per
maufacturers instructions), stop with 50u1 per well 3M H2SO4, read at an
absorbance of 490nm.
6.6 Human IL-13 binding to the human IL13Ra2 chain ELISA
This ELISA determines whether an antibody can inhibit human IL-13 binding to
the
human IL13Ra2 chain.
6.6.1 Materials
1. Nunc Immunoplate 1 F96 Maxisorp (Life Technologies, 4-39454A)
2. Human IL13Ra2-Fc (R&D Systems, cat.no. 614-IR)
3. human IL-13 (generated at GSK)
4. Biotinylated anti-human IL-13 (R&D Systems, cat. no. BAF213)
5. Streptavidin-HRP
6. Carbonate/bicarbonate buffer (Sigma; cat. no. C-3041)
7. TBST [Tris buffered saline (6.06g Tris + 8.06g NaCI + 0.2g KCI + H20 to 1
L) +
0.05% Tween 20]
8. BSA (Sigma A-7030)
9. OPD (Sigma, Cat. No. P-9187)
10. Sulphuric acid
6.6.2 Method
1. Blocking solution is 3% BSA+TBST
2. Washing solution is TBST
3. Coat 'Nunc Maxisorp' ELISA plates with 50u1 of 5ng/ul human IL-13Ra2-Fc in
carbonate/bicarbonate buffer. Cover with a plate sealer and incubate overnight
at
4 C.
4. Block with 100ul of 3% BSA/TBST incubate at rtp for 1 hr.
5. Wash X3 TBST (at least 200u1 wash solution per well per wash).
6. In a total volume of 50u1, pre-incubate 0.01 ng/ul human IL-13 with
antibody
sample (titrated) in block solution for 60 minutes. Add the pre-incubated
sample to
the receptor-coated ELISA plate and incubate at room temperature for 1 hr.
7. Wash x3 in TBST
8. Detect any bound human IL-13 using 50uI per well biotinylated anti-human IL-
13
diluted at 0.5ug/ml. Incubate for 1 hour at room temperature.
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9. Wash x3 in TBST
10.Add 50u1 per well streptavidin-HRP conjugate at 1/1000 dilution. Incubate
for
1 hour at room temperature.
11. Wash x3 in TBST
12.Develop with 100ul per well OPD (Sigma, Cat. No. P-9187. Made up as per
maufacturers instructions), stop with 50u1 per well 3M H2SO4, read at an
absorbance of 490nm. Development time is - 2 minutes.
6.7 Human IL-4 binding ELISA
This assay describes an ELISA that detects binding of an antibody to human IL-
4. It
is a sandwich ELISA format.
6.7.1 Materials
12. Nunc Immunoplate 1 F96 Maxisorp (Life Technologies, 4-39454A)
13. Human IL-4 (R+D Systems, cat. no. 2041L)
14. Goat anti-human IL-4 polyclonal antibody (R+D Systems, Cat. No. AF-204-NA)
15.Biotinylated rat anti-human IL-4 antibody (R & D systems, Cat. No BAF204.)
16.Anti-mouse IgG-HRP (Dako, Cat No. P0260)
17.Anti-human kappa light chain-HRP (Sigma A7164)
18.Streptavidin-HRP (Sigma cat no. E2886)
19. Carbonate/bicarbonate buffer (Sigma; cat. no. C-3041)
20.TBST [Tris buffered saline (6.06g Tris + 8.06g NaCI + 0.2g KCI + H20 to 1L)
+
0.05% Tween 20]
21. BSA (Sigma A-7030)
22.OPD (Sigma, Cat. No. P-9187)
23.Sulphuric acid
6.7.2 Method
13. Blocking solution is 3% BSA in TBST
14. Washing solution is TBST
15. Coat 'Nunc Maxisorp' ELISA plates with 50ul of 5ug/ml goat anti-human IL-4
polyclonal antibody (R+D Systems, cat. no. AF-204-NA. Made up at a stock
concentration of 500ug/ml according to maufacturers instructions, and stored
in
aliquots at -20C) in carbonate/bicarbonate buffer (Sigma; cat. no. C-3041,
made
up as per manufacturers instructions), cover with a plate sealer and incubate
overnight at 4 C.
16. Block with 100ul of 3% BSA/TBST incubate at room temperature pressure
(rtp) for
1 hr.
17. Wash X3 in TBST (at least 200ul wash solution per well per wash).
18.Add 1ng/ml (in a 50u1 volume) human IL-4 in block solution and incubate at
room
temperature for 1 hr.
19. Wash X3 in TBST.
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20.Add 50u1 antibody sample (titrate out to obtain end-point titre data, if
required) in
block solution, incubate at rtp for 1 hr. As a positive control for binding to
human
IL-4, use a biotinylated anti-human IL-4 monoclonal antibody (titrated out).
21.Wash X3 in TBST.
22. For 3G4 mouse monoclonal antibody, detect binding using 50u1 per well anti-
mouse IgG-HRP (Dako, Cat No. P0260) at a 1/2000 dilution in block solution for
1 hr at rtp. For 3G4 chimaeric antibody or humanised antibody, detect binding
using 50u1 per well anti-human kappa light chain-HRP (Sigma, Cat No. Sigma
A7164) at a 1/2000 dilution in block solution for 1 hr at rtp. For the
positive control
biotinylated rat anti-human IL-4 monoclonal antibody, detect using a
streptavidin-
HRP conjugated antibody (Sigma cat no. E2886) at 1/1000 dilution in block
solution for 1 hr at rtp.
23. Wash X3 in TBST.
24. Develop with 100ul OPD (Sigma, Cat. No. P-9187. Made up as per
maufacturers
instructions), stop with 50u1 3M H2SO4, read at an absorbance of 490nm.
6.8 Human IL-5 binding ELISA
This assay describes an ELISA that detects binding of an antibody to human IL-
5. It
is a sandwich ELISA format.
6.8.1 Materials
24. Nunc Immunoplate 1 F96 Maxisorp (Life Technologies, 4-39454A)
25. Human IL-5 (R+D Systems, cat. no. 2051L)
26. anti-human IL-5 polyclonal antibody (R+D Systems, Cat. No. AF-205-NA)
27.anti-human IL-5 Mepolizumab (in house clinical grade)
28.Anti-mouse IgG-HRP (Dako, Cat No. P0260 )
29.Anti-human kappa light chain-HRP (Sigma A7164)
30. Carbonate/bicarbonate buffer (Sigma; cat. no. C-3041)
31.TBST [Tris buffered saline (6.06g Tris + 8.06g NaCI + 0.2g KCI + H20 to 1L)
+
0.05% Tween 20]
32. BSA (Sigma A-7030)
33.OPD (Sigma, Cat. No. P-9187)
34.Sulphuric acid
6.8.2 Method
25. Blocking solution is 3% BSA in TBST
26. Washing solution is TPBST
27.Coat 'Nunc Maxisorp' ELISA plates with 50ul of 5ug/ml goat anti-human IL-5
polyclonal antibody (R+D Systems, cat. no. AF-205-NA. Made up at a stock
concentration of 500ug/ml according to maufacturers instructions, and stored
in
aliquots at -20C) in carbonate/bicarbonate buffer (Sigma; cat. no. C-3041,
made
up as per manufacturers instructions), cover with a plate sealer and incubate
overnight at 4 C.
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28. Block with 100u1 of 3% BSA/TBST incubate at room temperature pressure
(rtp) for
1 hr.
29. Wash X3 in TBST (at least 200ul wash solution per well per wash).
30.Add 100ng/ml (in a 50u1 volume) human IL-5 (R+D Systems, cat. no. 2051L)in
block solution and incubate at room temperature for 1 hr.
31. Wash X3 in TBST.
32.Add 50u1 antibody sample (titrate out to obtain end-point titre data, if
required) in
block solution, incubate at rtp for 1 hr. As a positive control for binding to
human
IL-5, use a anti-human IL-5 Mepolizumab antibody (titrated out).
33. Wash X3 in TBST.
34. For 3G4 mouse monoclonal antibody, detect binding using 50u1 per well anti-
mouse IgG-HRP (Dako, Cat No. P0260) at a 1/2000 dilution in block solution for
1 hr at rtp. For 3G4 chimaeric antibody or humanised antibody and anti IL5
Mepolizumab, detect binding using 50u1 per well anti-human kappa light chain-
HRP (Sigma, Cat No. Sigma A7164) at a 1/2000 dilution in block solution for
1hr
at rtp.
35. Wash X3 in TBST.
36. Develop with 100ul OPD (Sigma, Cat. No. P-9187. Made up as per
maufacturers
instructions), stop with 50ul 3M H2SO4, read at an absorbance of 490nm.
6.9 Human GM-CSF binding ELISA
This assay describes an ELISA that detects binding of an antibody to human GM-
CSF. It is a direct binding ELISA format.
6.9.1 Materials
35. Nunc Immunoplate 1 F96 Maxisorp (Life Technologies, 4-39454A)
36. Human GM-CSF (clinical grade in house)
37.Anti-human GM-CSF monoclonal antibody (R+D Systems, Cat. No. MAB215)
38.Anti-mouse lgG-HRP (Dako, Cat No. P0260 )
39.Anti-human kappa light chain-HRP (Sigma A7164)
40. Carbonate/bicarbonate buffer (Sigma; cat. no. C-3041)
41. PBST (PBS + 0.05% Tween 20)
42. BSA (Sigma A-7030)
43.OPD (Sigma, Cat. No. P-9187)
44. Sulphuric acid
6.9.2 Method
37. Blocking solution is 3% BSA in PBST
38. Washing solution is PBST
39. Coat 'Nunc Maxisorp' ELISA plates with 50ul of 2ug/ml human GM-CSF
dilution in
PBS, cover with a plate sealer and incubate overnight at 4 C.
40. Block with 200u1 of 3% BSA/PBST incubate at room temperature pressure
(rtp)
for 1 hr.
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41. Wash X3 in PBST.
42.Add 50u1 antibody sample (titrate out to obtain end-point titre data) in
block
solution, incubate at rtp for 1 hr. As a positive control for binding to human
IL-GM-
CSF, use anti-human GM-CSF (R&D systems cat no.MAB215 antibody (titrated
out).
43. Wash X3 in PBST.
44. For anti GM CSF mouse monoclonal antibody, detect binding using 50u1 per
well
anti-mouse IgG-HRP (Dako, Cat No. P0260) at a 1/2000 dilution in block
solution
for 1 hr at rtp. For 3G4 chimaeric antibody or humanised antibody, detect
binding
using 50u1 per well anti-human kappa light chain-HRP (Sigma, Cat No. Sigma
A7164) at a 1/2000 dilution in block solution for 1 hr at rtp.
45. Wash X3 in PBST.
46. Develop with 100u1 OPD (Sigma, Cat. No. P-9187. Made up as per
maufacturers
instructions), stop with 50u1 3M H2SO4, read at an absorbance of 490nm.
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Table 13:
Protein or polynucleotide description Sequence identifier
SEQ.I.D.NO:
3G4, CDRH1 1
3G4,CDRH2 2
3G4,CDRH3 3
3G4, CDRL1 4
3G4, CDRL2 5
3G4, CDRL3 6
3G4, VH murine 7
3G4, VL (murine) 8
hIL-13 9
Polynucleotide encoding hIL-13 10
3G4, VH, humanised construct H1 11
3G4, VH, humanised construct H2 12
3G4, VH, humanised construct H3 13
3G4, VH, humanised construct H4 14
3G4, VL, humanised construct L1 15
3G4, VL, humanised construct L2 16
3G4, VL, humanised construct L3 17
3G4, heavy chain, humanised construct H1 18
3G4, heav chain humanised construct H2 19
3G4, heavy chain humanised construct H3 20
3G4, heavy chain humanised construct H4 21
3G4, light chain humanised construct L1 22
3G4, light chain humanised construct L2 23
3G4, light chain humanised construct L3 24
Polynucleotide encoding 3G4 VH (murine) (SEQ.I.D.NO:7 25
Pol nucleotide encoding 3G4 VL (murine) (SEQ.I.D.N0:8) 26
Polynucleotide encoding 3G4 VH humanised construct HI 27
SEQ.I.D.NO:11)
Polynucleotide encoding 3G4 VH humanised construct H2 28
SEQ.I.D.NO:12)
Polynucleotide encoding 3G4 VH humanised construct H3 29
SEQ.I.D.N0:13)
Polynucleotide encoding 3G4 VH humanised construct H4 30
SEQ.I.D.NO:14
Polynucleotide encoding 3G4 VL humanised construct L1 31
SEQ.I.D.NO:15
Polynucleotide encoding 3G4 VL humanised construct 32
L2 SEQ.I.D.NO:16)
Polynucleotide encoding 3G4 VL humanised construct L3 33
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SEQ.I.D.NO:17
Polynucleotide encoding 3G4 heavy chain, humanised construct 34
H1 SEQ.I.D.NO:18)
Palynucleotide encoding 3G4 heavy chain humanised construct 35
H2 SEQ.I.D.NO:19)
Polynucleotide encoding 3G4 heavy chain humanised construct 36
H3 SEQ.I.D.NO:20)
Polynucleotide encoding 3G4 heavy chain humanised construct 37
H4 SEQ.I.D.NO:21)
Polynucleotide encoding 3G4 light chain humanised construct 38
L1 SEQ.I.D.NO:22
Polynucleotide encoding 3G4 light chain humanised construct 39
L2 (SEQ.I.D.NO:23)
Polynucleotide encoding 3G4 light chain humanised construct 40
L3 SEQ.I.D.NO:24
C nomol us IL-13 41
C nomol us IL-13 ( ol nucleotide 42
Human signal sequence 43
Human acceptor framework sequence for 3G4 VH 44
Human acceptor framework sequence for 3G4 VL 45
Alternative polynucleotide encoding 3G4 VH humanised 93
construct H2 (SEQ.I.D.NO:12
Alternative polynucleotide encoding 3G4 VL humanised 94
construct L1 (SEQ.I.D.NO:15
Alternative polynucleotide encoding heavy chain humanised 95
construct H2 (SEQ.I.D.NO:19
Alternative polynucleotide encoding heavy chain humanised 96
construct H3 (SEQ.I.D.NO:20)
Alternative polynucleotide encoding light chain humanised 97
construct L1 (SEQ.I.D.NO:22)
Note: Protein or DNA polynucleotide sequence in SEQ.ID numbers from 11 to 24
and 27 to 40 (inclusive) also include the signal sequence.
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SEQ.I.D.NO 1
DYEIH
SEQ.I.D.NO 2
AIDPETGGTAYNQKFKG
SEQ.I.D.NO 3
ILLYYYPMDY
SEQ.I.D.NO 4
RASQNISDYLH
SEQ.I.D.NO 5
YASQSIS
SEQ.I.D.NO 6
QNGHSFPLT
SEQ.I.D.NO 7
QVQLQQSGADLVRPGASVTLSCKASGYTFIDYEIHWMKQTPVHGLEWIGAIDPETGGTAYNQ
KFKGKAILTADKSSSTAYMELRSLTSEDSAVYYCTRILLYYYPMDYWGQGTSVTVSS
SEQ.I.D.NO 8
DIVMTQSPATLSVTPGDRVSLSCRASQNISDYLHWYQQKSHESPRLLIKYASQSISGIPSRF
SGSGSGSDFTLSINSVEPEDVGVYYCQNGHSFPLTLGAGTKLELK
SEQ.I.D.NO:9
GPVPPSTALRELIEELVNITQNQKAPLCNGSMVWSINLTAGMYCAALESLINVSGCSAIEKT
QRMLSGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDLLLHLKKLFREGRFN
SEQ.I.D.NO:10
CA 02635972 2008-07-10
WO 2007/080174 PCT/EP2007/050219
88
GGCCCTGTGCCTCCCTCTACAGCCCTCAGGGAGCTCATTGAGGAGCTGGTCAACATCACCCA
GAACCAGAAGGCTCCGCTCTGCAATGGCAGCATGGTATGGAGCATCAACCTGACAGCTGGCA
TGTACTGTGCAGCCCTGGAATCCCTGATCAACGTGTCAGGCTGCAGTGCCATCGAGAAGACC
CAGAGGATGCTGAGCGGATTCTGCCCGCACAAGGTCTCAGCTGGGCAGTTTTCCAGCTTGCA
TGTCCGAGACACCAAAATCGAGGTGGCCCAGTTTGTAAAGGACCTGCTCTTACATTTAAAGA
AACTTTTTCGCGAGGGACGGTTCAACTGA
SEQ.I.D.NO ll
MGWSCIILFLVATATGVHSQVQLVQSGAEVKKPGASVKVSCKASGYTFIDYEIHWVRQAPGQ
GLEWMGAIDPETGGTAYNQKFKGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARILLYYYP
MDYWGQGTLVTVSS
SEQ.I.D.NO 12
MGWSCIILFLVATATGVHSQVQLVQSGAEVKKPGASVKVSCKASGYTFIDYEIHWVRQAPGQ
GLEWMGAIDPETGGTAYNQKFKGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCTRILLYYYP
MDYWGQGTLVTVSS
SEQ.I.D.NO 13
MGWSCIILFLVATATGVHSQVQLVQSGADVKKPGASVKVSCKASGYTFIDYEIHWVRQAPGQ
GLEWMGAIDPETGGTAYNQKFKGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCTRILLYYYP
MDYWGQGTLVTVSS
SEQ.I.D.NO 14
MGWSCIILFLVATATGVHSQVQLVQSGADVKKPGASVKVSCKASGYTFIDYEIHWVRQAPGQ
GLEWMGAIDPETGGTAYNQKFKGRATLTADKSTSTAYMELRSLRSDDTAVYYCTRILLYYYP
MDYWGQGTLVTVSS
SEQ.I.D.NO 15
MGWSCIILFLVATATGVHSEIVLTQSPATLSLSPGERATLSCRASQNISDYLHWYQQKPGQA
PRLLIYYASQSISGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQNGHSFPLTFGGGTKVE
IK
SEQ.I.D.NO 16
MGWSCIILFLVATATGVHSEIVLTQSPATLSLSPGERATLSCRASQNISDYLHWYQQKPGQA
PRLLIYYASQSISGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQNGHSFPLTLGGGTKVE
IK
CA 02635972 2008-07-10
WO 2007/080174 PCT/EP2007/050219
89
SEQ.I.D.NO 17
MGWSCIILFLVATATGVHSEIVLTQSPATLSLSPGERATLSCRASQNISDYLHWYQQKPGQA
PRLLIYYASQSISGIPARFSGSGSGTDFTLTINSLEPEDFAVYYCQNGHSFPLTLGGGTKVE
IK
SEQ.I.D.NO 18
MGWSCIILFLVATATGVHSQVQLVQSGAEVKKPGASVKVSCKASGYTFIDYEIHWVRQAPGQ
GLEWMGAIDPETGGTAYNQKFKGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARILLYYYP
MDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS
GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP
PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT
KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL
PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD
KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ.I.D.NO 19
MGWSCIILFLVATATGVHSQVQLVQSGAEVKKPGASVKVSCKASGYTFIDYEIHWVRQAPGQ
GLEWMGAIDPETGGTAYNQKFKGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCTRILLYYYP
MDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS
GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP
PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT
KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL
PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD
KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ.I.D.NO 20
MGWSCIILFLVATATGVHSQVQLVQSGADVKKPGASVKVSCKASGYTFIDYEIHWVRQAPGQ
GLEWMGAIDPETGGTAYNQKFKGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCTRILLYYYP
MDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS
GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP
PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT
KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL
PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD
KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ.I.D.NO 21
MGWSCIILFLVATATGVHSQVQLVQSGADVKKPGASVKVSCKASGYTFIDYEIHWVRQAPGQ
GLEWMGAIDPETGGTAYNQKFKGRATLTADKSTSTAYMELRSLRSDDTAVYYCTRILLYYYP
CA 02635972 2008-07-10
WO 2007/080174 PCT/EP2007/050219
MDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS
GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP
PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT
KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL
PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD
KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ.I.D.NO 22
MGWSCIILFLVATATGVHSEIVLTQSPATLSLSPGERATLSCRASQNISDYLHWYQQKPGQA
PRLLIYYASQSISGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQNGHSFPLTFGGGTKVE
IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD
SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
SEQ.I.D.NO 23
MGWSCIILFLVATATGVHSEIVLTQSPATLSLSPGERATLSCRASQNISDYLHWYQQKPGQA
PRLLIYYASQSISGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQNGHSFPLTLGGGTKVE
IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD
SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
SEQ.I.D.NO 24
MGWSCIILFLVATATGVHSEIVLTQSPATLSLSPGERATLSCRASQNISDYLHWYQQKPGQA
PRLLIYYASQSISGIPARFSGSGSGTDFTLTINSLEPEDFAVYYCQNGHSFPLTLGGGTKVE
IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD
SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
SEQ.I.D.NO 25
CAGGTTCAACTGCAGCAGTCTGGGGCTGACCTGGTGAGGCCTGGGGCTTCAGTGACGCTGTC
CTGCAAGGCTTCGGGCTACACATTTATTGACTATGAAATACACTGGATGAAGCAGACACCTG
TGCATGGCCTGGAATGGATTGGAGCTATTGATCCTGAAACTGGTGGTACAGCCTATAATCAG
AAGTTCAAGGGCAAGGCCATTCTGACTGCAGACAAATCCTCCAGTACAGCCTACATGGAGCT
CCGCAGCCTGACATCTGAGGACTCTGCCGTCTATTACTGTACAAGAATTCTCTTATATTACT
ATCCTATGGACTACTGGGGTCAAGGGACCTCAGTCACAGTCTCCTCA
SEQ.I.D.NO 26
GACATTGTGATGACTCAGTCTCCAGCCACCCTGTCTGTGACTCCAGGAGATAGAGTCTCTCT
TTCCTGCAGGGCCAGCCAGAATATTAGCGACTACTTACACTGGTATCAACAAAAATCACATG
AGTCTCCAAGGCTTCTCATCAAATATGCTTCCCAATCCATCTCTGGGATCCCCTCCAGGTTC
AGTGGCAGTGGATCAGGGTCAGATTTCACTCTCAGTATCAACAGTGTGGAACCTGAAGATGT
CA 02635972 2008-07-10
WO 2007/080174 PCT/EP2007/050219
91
TGGAGTGTATTACTGTCAAAATGGTCACAGCTTTCCGCTCACGCTCGGTGCTGGGACCAAGC
TGGAGCTGAAA
SEQ.I.D.NO 27
ATGGGCTGGAGCTGCATCATCCTGTTCCTGGTGGCCACCGCCACCGGCGTGCACAGCCAGGT
GCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCGCCAGCGTGAAGGTGAGCTGCA
AGGCCAGCGGCTACACCTTCATCGACTACGAGATCCACTGGGTGCGCCAGGCCCCCGGCCAG
GGCCTGGAGTGGATGGGCGCCATCGACCCCGAGACCGGCGGCACCGCCTACAACCAGAAGTT
CAAGGGCCGCGTGACCATGACCACCGACACCAGCACCAGCACCGCCTACATGGAGCTGCGCA
GCCTGCGCAGCGACGACACCGCCGTGTACTACTGCGCCCGCATCCTGCTGTACTACTACCCC
ATGGACTACTGGGGCCAGGGCACACTAGTCACAGTCTCCTCA
SEQ.I.D.NO 28
ATGGGCTGGAGCTGCATCATCCTGTTCCTGGTGGCCACCGCCACCGGCGTGCACAGCCAGGT
GCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCGCCAGCGTGAAGGTGAGCTGCA
AGGCCAGCGGCTACACCTTCATCGACTACGAGATCCACTGGGTGCGCCAGGCCCCCGGCCAG
GGCCTGGAGTGGATGGGCGCCATCGACCCCGAGACCGGCGGCACCGCCTACAACCAGAAGTT
CAAGGGCCGCGTGACCATGACCACCGACACCAGCACCAGCACCGCCTACATGGAGCTGCGCA
GCCTGCGCAGCGACGACACCGCCGTGTACTACTGCACCCGCATCCTGCTGTACTACTACCCC
ATGGACTACTGGGGCCAGGGCACACTAGTCACAGTCTCCTCA
SEQ.I.D.NO 29
ATGGGCTGGAGCTGCATCATCCTGTTCCTGGTGGCCACCGCCACCGGCGTGCACAGCCAGGT
GCAGCTGGTGCAGAGCGGCGCCGACGTGAAGAAGCCCGGCGCCAGCGTGAAGGTGAGCTGCA
AGGCCAGCGGCTACACCTTCATCGACTACGAGATCCACTGGGTGCGCCAGGCCCCCGGCCAG
GGCCTGGAGTGGATGGGCGCCATCGACCCCGAGACCGGCGGCACCGCCTACAACCAGAAGTT
CAAGGGCCGCGTGACCATGACCACCGACACCAGCACCAGCACCGCCTACATGGAGCTGCGCA
GCCTGCGCAGCGACGACACCGCCGTGTACTACTGCACCCGCATCCTGCTGTACTACTACCCC
ATGGACTACTGGGGCCAGGGCACACTAGTCACAGTCTCCTCA
SEQ.I.D.NO 30
ATGGGCTGGAGCTGCATCATCCTGTTCCTGGTGGCCACCGCCACCGGCGTGCACAGCCAGGT
GCAGCTGGTGCAGAGCGGCGCCGACGTGAAGAAGCCCGGCGCCAGCGTGAAGGTGAGCTGCA
AGGCCAGCGGCTACACCTTCATCGACTACGAGATCCACTGGGTGCGCCAGGCCCCCGGCCAG
GGCCTGGAGTGGATGGGCGCCATCGACCCCGAGACCGGCGGCACCGCCTACAACCAGAAGTT
CAAGGGCCGCGCCACCCTGACCGCCGACAAGAGCACCAGCACCGCCTACATGGAGCTGCGCA
GCCTGCGCAGCGACGACACCGCCGTGTACTACTGCACCCGCATCCTGCTGTACTACTACCCC
ATGGACTACTGGGGCCAGGGCACACTAGTCACAGTCTCCTCA
CA 02635972 2008-07-10
WO 2007/080174 PCT/EP2007/050219
92
SEQ.I.D.NO 31
ATGGGCTGGAGCTGCATCATCCTGTTCCTGGTGGCCACCGCCACCGGCGTGCACAGCGAGAT
CGTGCTGACCCAGAGCCCCGCCACCCTGAGCCTGAGCCCCGGCGAGCGCGCCACCCTGAGCT
GCCGCGCCAGCCAGAACATCAGCGACTACCTGCACTGGTACCAGCAGAAGCCCGGCCAGGCC
CCCCGCCTGCTGATCTACTACGCCAGCCAGAGCATCAGCGGCATCCCCGCCCGCTTCAGCGG
CAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGGAGCCCGAGGACTTCGCCG
TGTACTACTGCCAGAACGGCCACAGCTTCCCCCTGACCTTCGGCGGCGGCACCAAGGTGGAG
ATCAAG
SEQ.I.D.NO 32
ATGGGCTGGAGCTGCATCATCCTGTTCCTGGTGGCCACCGCCACCGGCGTGCACAGCGAGAT
CGTGCTGACCCAGAGCCCCGCCACCCTGAGCCTGAGCCCCGGCGAGCGCGCCACCCTGAGCT
GCCGCGCCAGCCAGAACATCAGCGACTACCTGCACTGGTACCAGCAGAAGCCCGGCCAGGCC
CCCCGCCTGCTGATCTACTACGCCAGCCAGAGCATCAGCGGCATCCCCGCCCGCTTCAGCGG
CAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGGAGCCCGAGGACTTCGCCG
TGTACTACTGCCAGAACGGCCACAGCTTCCCCCTGACCCTGGGCGGCGGCACCAAGGTGGAG
ATCAAG
SEQ.I.D.NO 33
ATGGGCTGGAGCTGCATCATCCTGTTCCTGGTGGCCACCGCCACCGGCGTGCACAGCGAGAT
CGTGCTGACCCAGAGCCCCGCCACCCTGAGCCTGAGCCCCGGCGAGCGCGCCACCCTGAGCT
GCCGCGCCAGCCAGAACATCAGCGACTACCTGCACTGGTACCAGCAGAAGCCCGGCCAGGCC
CCCCGCCTGCTGATCTACTACGCCAGCCAGAGCATCAGCGGCATCCCCGCCCGCTTCAGCGG
CAGCGGCAGCGGCACCGACTTCACCCTGACCATCAACAGCCTGGAGCCCGAGGACTTCGCCG
TGTACTACTGCCAGAACGGCCACAGCTTCCCCCTGACCCTGGGCGGCGGCACCAAGGTGGAG
ATCAAG
SEQ.I.D.NO 34
ATGGGCTGGAGCTGCATCATCCTGTTCCTGGTGGCCACCGCCACCGGCGTGCACAGCCAGGT
GCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCGCCAGCGTGAAGGTGAGCTGCA
AGGCCAGCGGCTACACCTTCATCGACTACGAGATCCACTGGGTGCGCCAGGCCCCCGGCCAG
GGCCTGGAGTGGATGGGCGCCATCGACCCCGAGACCGGCGGCACCGCCTACAACCAGAAGTT
CAAGGGCCGCGTGACCATGACCACCGACACCAGCACCAGCACCGCCTACATGGAGCTGCGCA
GCCTGCGCAGCGACGACACCGCCGTGTACTACTGCGCCCGCATCCTGCTGTACTACTACCCC
ATGGACTACTGGGGCCAGGGCACACTAGTCACAGTCTCCTCAGCCTCCACCAAGGGCCCATC
GGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCC
TGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGC
GGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGT
CA 02635972 2008-07-10
WO 2007/080174 PCT/EP2007/050219
93
GACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCA
GCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCA
CCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAA
GGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACG
AAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACA
AAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCA
CCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCC
CCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTG
CCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTT
CTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGA
CCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGAC
AAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAA
CCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA
SEQ.I.D.NO 35
ATGGGCTGGAGCTGCATCATCCTGTTCCTGGTGGCCACCGCCACCGGCGTGCACAGCCAGGT
GCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCGCCAGCGTGAAGGTGAGCTGCA
AGGCCAGCGGCTACACCTTCATCGACTACGAGATCCACTGGGTGCGCCAGGCCCCCGGCCAG
GGCCTGGAGTGGATGGGCGCCATCGACCCCGAGACCGGCGGCACCGCCTACAACCAGAAGTT
CAAGGGCCGCGTGACCATGACCACCGACACCAGCACCAGCACCGCCTACATGGAGCTGCGCA
GCCTGCGCAGCGACGACACCGCCGTGTACTACTGCACCCGCATCCTGCTGTACTACTACCCC
ATGGACTACTGGGGCCAGGGCACACTAGTCACAGTCTCCTCAGCCTCCACCAAGGGCCCATC
GGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCC
TGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGC
GGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGT
GACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCA
GCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCA
CCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAA
GGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACG
AAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACA
AAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCA
CCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCC
CCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTG
CCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTT
CTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGA
CCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGAC
AAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAA
CCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA
SEQ.I.D.NO 36
CA 02635972 2008-07-10
WO 2007/080174 PCT/EP2007/050219
94
ATGGGCTGGAGCTGCATCATCCTGTTCCTGGTGGCCACCGCCACCGGCGTGCACAGCCAGGT
GCAGCTGGTGCAGAGCGGCGCCGACGTGAAGAAGCCCGGCGCCAGCGTGAAGGTGAGCTGCA
AGGCCAGCGGCTACACCTTCATCGACTACGAGATCCACTGGGTGCGCCAGGCCCCCGGCCAG
GGCCTGGAGTGGATGGGCGCCATCGACCCCGAGACCGGCGGCACCGCCTACAACCAGAAGTT
CAAGGGCCGCGTGACCATGACCACCGACACCAGCACCAGCACCGCCTACATGGAGCTGCGCA
GCCTGCGCAGCGACGACACCGCCGTGTACTACTGCACCCGCATCCTGCTGTACTACTACCCC
ATGGACTACTGGGGCCAGGGCACACTAGTCACAGTCTCCTCAGCCTCCACCAAGGGCCCATC
GGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCC
TGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGC
GGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGT
GACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCA
GCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCA
CCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAA
GGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACG
AAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACA
AAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCA
CCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCC
CCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTG
CCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTT
CTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGA
CCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGAC
AAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAA
CCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA
SEQ.I.D.NO 37
ATGGGCTGGAGCTGCATCATCCTGTTCCTGGTGGCCACCGCCACCGGCGTGCACAGCCAGGT
GCAGCTGGTGCAGAGCGGCGCCGACGTGAAGAAGCCCGGCGCCAGCGTGAAGGTGAGCTGCA
AGGCCAGCGGCTACACCTTCATCGACTACGAGATCCACTGGGTGCGCCAGGCCCCCGGCCAG
GGCCTGGAGTGGATGGGCGCCATCGACCCCGAGACCGGCGGCACCGCCTACAACCAGAAGTT
CAAGGGCCGCGCCACCCTGACCGCCGACAAGAGCACCAGCACCGCCTACATGGAGCTGCGCA
GCCTGCGCAGCGACGACACCGCCGTGTACTACTGCACCCGCATCCTGCTGTACTACTACCCC
ATGGACTACTGGGGCCAGGGCACACTAGTCACAGTCTCCTCAGCCTCCACCAAGGGCCCATC
GGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCC
TGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGC
GGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGT
GACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCA
GCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCA
CCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAA
GGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACG
AAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACA
AAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCA
CCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCC
CA 02635972 2008-07-10
WO 2007/080174 PCT/EP2007/050219
CCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTG
CCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTT
CTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGA
CCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGAC
AAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAA
CCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA
SEQ.I.D.NO 38
ATGGGCTGGAGCTGCATCATCCTGTTCCTGGTGGCCACCGCCACCGGCGTGCACAGCGAGAT
CGTGCTGACCCAGAGCCCCGCCACCCTGAGCCTGAGCCCCGGCGAGCGCGCCACCCTGAGCT
GCCGCGCCAGCCAGAACATCAGCGACTACCTGCACTGGTACCAGCAGAAGCCCGGCCAGGCC
CCCCGCCTGCTGATCTACTACGCCAGCCAGAGCATCAGCGGCATCCCCGCCCGCTTCAGCGG
CAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGGAGCCCGAGGACTTCGCCG
TGTACTACTGCCAGAACGGCCACAGCTTCCCCCTGACCTTCGGCGGCGGCACCAAGGTGGAG
ATCAAGCGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAA
ATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTAC
AGTGGAAGGTGGACAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGAC
AGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAA
ACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCT
TCAACAGGGGAGAGTGTTAG
SEQ.I.D.NO 39
ATGGGCTGGAGCTGCATCATCCTGTTCCTGGTGGCCACCGCCACCGGCGTGCACAGCGAGAT
CGTGCTGACCCAGAGCCCCGCCACCCTGAGCCTGAGCCCCGGCGAGCGCGCCACCCTGAGCT
GCCGCGCCAGCCAGAACATCAGCGACTACCTGCACTGGTACCAGCAGAAGCCCGGCCAGGCC
CCCCGCCTGCTGATCTACTACGCCAGCCAGAGCATCAGCGGCATCCCCGCCCGCTTCAGCGG
CAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGGAGCCCGAGGACTTCGCCG
TGTACTACTGCCAGAACGGCCACAGCTTCCCCCTGACCCTGGGCGGCGGCACCAAGGTGGAG
ATCAAGCGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAA
ATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTAC
AGTGGAAGGTGGACAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGAC
AGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAA
ACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCT
TCAACAGGGGAGAGTGTTAG
SEQ.I.D.NO 40
ATGGGCTGGAGCTGCATCATCCTGTTCCTGGTGGCCACCGCCACCGGCGTGCACAGCGAGAT
CGTGCTGACCCAGAGCCCCGCCACCCTGAGCCTGAGCCCCGGCGAGCGCGCCACCCTGAGCT
GCCGCGCCAGCCAGAACATCAGCGACTACCTGCACTGGTACCAGCAGAAGCCCGGCCAGGCC
CA 02635972 2008-07-10
WO 2007/080174 PCT/EP2007/050219
96
CCCCGCCTGCTGATCTACTACGCCAGCCAGAGCATCAGCGGCATCCCCGCCCGCTTCAGCGG
CAGCGGCAGCGGCACCGACTTCACCCTGACCATCAACAGCCTGGAGCCCGAGGACTTCGCCG
TGTACTACTGCCAGAACGGCCACAGCTTCCCCCTGACCCTGGGCGGCGGCACCAAGGTGGAG
ATCAAGCGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAA
ATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTAC
AGTGGAAGGTGGACAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGAC
AGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAA
ACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCT
TCAACAGGGGAGAGTGTTAG
SEQ.I.D.NO:41
SPVPPSTALKELIEELVNITQNQKAPLCNGSMVWSINLTAGVYCAALESLINVSGCSAIEKT
QRMLNGFCPHKVSAGQFSSLRVRDTKIEVAQFVKDLLVHLKKLFREGQFN
SEQ.I.D.NO:42
AGCCCTGTGCCTCCCTCTACAGCCCTCAAGGAGCTCATTGAGGAGCTGGTCAACATCACCCA
GAACCAGAAGGCCCCGCTCTGCAATGGCAGCATGGTGTGGAGCATCAACCTGACAGCTGGCG
TGTACTGTGCAGCCCTGGAATCCCTGATCAACGTGTCAGGCTGCAGTGCCATCGAGAAGACC
CAGAGGATGCTGAACGGATTCTGCCCGCACAAGGTCTCAGCTGGGCAGTTTTCCAGCTTGCG
TGTCCGAGACACCAAAATCGAGGTGGCCCAGTTTGTAAAGGACCTGCTCGTACATTTAAAGA
AACTTTTTCGCGAGGGACAGTTCAACTGA
SEQ.I.D.NO:43
MGWSCIILFLVATATGVHS
SEQ.I.D.NO:44
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYNGNTNYAQ
KLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARXXXXXXXXXXWGQGTLVTVSS
SEQ.I.D.NO:45
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARF
SGSGSGTDFTLTISSLEPEDFAVYYCXXXXXXXXXFGGGTKVEIK
SEQ.I.D.NO:46
SGSGPSTALRELIEELVNIT
CA 02635972 2008-07-10
WO 2007/080174 PCT/EP2007/050219
97
SEQ.I.D.NO:47
SGSGLRELIEELVNITQNQK
SEQ.I.D.NO:48
SGSGIEELVNITQNQKAPLC
SEQ.I.D.NO:49
SGSGVNITQNQKAPLCNGSM
SEQ.I.D.NO:50
SGSGQNQKAPLCNGSMVWSI
SEQ.I.D.NO:51
SGSGAPLCNGSMVWSINLTA
SEQ.I.D.NO:52
SGSGNGSMVWSINLTAGMYC
SEQ.I.D.NO:53
SGSGVWSINLTAGMYCAALE
SEQ.I.D.NO:54
SGSGNLTAGMYCAALESLIN
SEQ.I.D.NO:55
SGSGGMYCAALESLINVSGC
SEQ.I.D.NO:56
SGSGAALESLINVSGCSAIE
SEQ.I.D.NO:57
SGSGSLINVSGCSAIEKTQR
CA 02635972 2008-07-10
WO 2007/080174 PCT/EP2007/050219
98
SEQ.I.D.NO:58
SGSGVSGCSAIEKTQRMLSG
SEQ.I.D.NO:59
SGSGSAIEKTQRMLSGFCPH
SEQ.I.D.NO:60
SGSGKTQRMLSGFCPHKVSA
SEQ.I.D.NO:61
SGSGMLSGFCPHKVSAGQFS
SEQ.I.D.NO:62
SGSGFCPHKVSAGQFSSLHV
SEQ.I.D.NO:63
SGSGKVSAGQFSSLHVRDTK
SEQ.I.D.NO:64
SGSGGQFSSLHVRDTKIEVA
SEQ.I.D.NO:65
SGSGSLHVRDTKIEVAQFVK
SEQ.I.D.NO:66
SGSGRDTKIEVAQFVKDLLL
SEQ.I.D.NO:67
SGSGIEVAQFVKDLLLHLKK
SEQ.I.D.NO:68
CA 02635972 2008-07-10
WO 2007/080174 PCT/EP2007/050219
99
SGSGQFVKDLLLHLKKLFRE
SEQ.I.D.NO:69
SGSGDLLLHLKKLFREGRFN
SEQ.I.D.NO:70
SGSGPSTALKELIEELVNIT
SEQ.I.D.NO:71
SGSGLKELIEELVNITQNQK
SEQ.I.D.NO:72
SGSGNGSMVWSINLTAGVYC
SEQ.I.D.NO:73
SGSGVWSINLTAGVYCAALE
SEQ.I.D.NO:74
SGSGNLTAGVYCAALESLIN
SEQ.I.D.NO:75
SGSGGVYCAALESLINVSGC
SEQ.I.D.NO:76
SGSGVSGCSAIEKTQRMLNG
SEQ.I.D.NO:77
SGSGSAIEKTQRMLNGFCPH
SEQ.I.D.NO:78
SGSGKTQRMLNGFCPHKVSA
CA 02635972 2008-07-10
WO 2007/080174 PCT/EP2007/050219
100
SEQ.I.D.NO:79
SGSGMLNGFCPHKVSAGQFS
SEQ.I.D.NO:80
SGSGFCPHKVSAGQFSSLRV
SEQ.I.D.NO:81
SGSGKVSAGQFSSLRVRDTK
SEQ.I.D.NO:82
SGSGGQFSSLRVRDTKIEVA
SEQ.I.D.NO:83
SGSGSLRVRDTKIEVAQFVK
SEQ.I.D.NO:84
SGSGRDTKIEVAQFVKDLLV
SEQ.I.D.NO:85
SGSGIEVAQFVKDLLVHLKK
SEQ.I.D.NO:86
SGSGQFVKDLLVHLKKLFRE
SEQ.I.D.NO:87
SGSGDLLVHLKKLFREGQFN
SEQ.I.D.NO:88
DLLLHLKKLFREGRFN
SEQ.I.D.NO:89
CA 02635972 2008-07-10
WO 2007/080174 PCT/EP2007/050219
101
DLLVHLKKLFREGQFN
SEQ.I.D.NO:90
QFVKDLLLHLKKLFREGRFN
SEQ.I.D.NO:91
LLHLKKLFREG
SEQ.I.D.NO:92
LKKLFRE
SEQ ID No. 93
CAGGTGCAGCTGGTGCAGAGCGGAGCCGAGGTGAAGAAGCCTGGCGCCAGCGTCAAGGTG
TCCTGCAAGGCCAGCGGCTACACCTTCATCGACTACGAGATCCACTGGGTGCGGCAGGCT
CCTGGACAGGGCCTGGAATGGATGGGCGCCATCGACCCCGAGACAGGCGGCACCGCCTAC
AACCAGAAGTTCAAGGGCCGGGTCACCATGACCACCGACACCAGCACCAGCACCGCCTAT
ATGGAACTGCGGAGCCTGAGAAGCGACGACACCGCCGTGTACTACTGCACCCGGATCCTG
CTGTACTACTACCCCATGGACTACTGGGGCCAGGGCACACTAGTCACCGTGAGCAGC
SEQ ID No. 94
GAGATCGTGCTGACCCAGAGCCCCGCCACCCTGAGCCTGAGCCCTGGCGAGCGGGCCACC
CTGTCCTGCCGGGCCAGCCAGAACATCAGCGACTACCTGCACTGGTATCAGCAGAAGCCC
GGCCAGGCCCCCAGGCTGCTGATCTACTACGCCAGCCAGTCCATCTCCGGCATCCCCGCC
AGGTTCAGCGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCTCTCTGGAACCC
GAGGACTTCGCCGTGTATTATTGCCAGAACGGCCACAGCTTCCCCCTGACCTTTGGCGGC
GGAACAAAGGTGGAGATCAAG
SEQ ID No. 95
ATGGGATGGAGCTGCATCATCCTCTTCCTGGTGGCCACGGCTACCGGCGTGCATAGCCAGGT
GCAGCTCGTCCAGTCTGGGGCCGAGGTGAAGAAGCCCGGAGCTTCTGTGAAGGTGTCCTGCA
AGGCCAGCGGCTATACCTTCATCGACTACGAGATCCATTGGGTGAGGCAGGCTCCCGGGCAG
GGCCTGGAGTGGATGGGCGCCATCGACCCAGAGACCGGAGGCACGGCGTACAACCAGAAGTT
CAAGGGACGGGTCACCATGACAACCGATACCAGCACCTCCACCGCTTACATGGAGCTGCGCA
GCCTGAGAAGCGACGACACCGCGGTGTACTACTGTACGCGCATCCTGCTCTACTACTACCCC
ATGGATTACTGGGGCCAGGGCACACTAGTCACAGTCTCCTCAGCCTCCACCAAGGGCCCATC
CA 02635972 2008-07-10
WO 2007/080174 PCT/EP2007/050219
102
GGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCC
TGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGC
GGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGT
GACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCA
GCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCA
CCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAA
GGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACG
AAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACA
AAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCA
CCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCC
CCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTG
CCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTT
CTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGA
CCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGAC
AAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAA
CCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA
SEQ ID No. 96
ATGGGATGGAGCTGCATCATCCTCTTCCTGGTGGCCACGGCTACCGGCGTGCATAGCCAGGT
GCAGCTCGTCCAGTCTGGGGCCGACGTGAAGAAGCCCGGAGCTTCTGTGAAGGTGTCCTGCA
AGGCCAGCGGCTATACCTTCATCGACTACGAGATCCATTGGGTGAGGCAGGCTCCCGGGCAG
GGCCTGGAGTGGATGGGCGCCATCGACCCAGAGACCGGAGGCACGGCGTACAACCAGAAGTT
CAAGGGACGGGTCACCATGACAACCGATACCAGCACCTCCACCGCTTACATGGAGCTGCGCA
GCCTGAGAAGCGACGACACCGCGGTGTACTACTGTACGCGCATCCTGCTCTACTACTACCCC
ATGGATTACTGGGGCCAGGGCACACTAGTCACAGTCTCCTCAGCCTCCACCAAGGGCCCATC
GGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCC
TGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGC
GGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGT
GACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCA
GCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCA
CCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAA
GGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACG
AAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACA
AAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCA
CCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCC
CCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTG
CCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTT
CTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGA
CCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGAC
AAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAA
CCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA
CA 02635972 2008-07-10
WO 2007/080174 PCT/EP2007/050219
103
SEQ ID No. 97
ATGGGATGGTCTTGTATCATCCTGTTCCTGGTGGCGACCGCCACCGGCGTGCACTCCGAGAT
CGTGCTGACCCAGAGTCCAGCCACCCTCAGCCTGAGCCCTGGGGAACGCGCCACCCTGTCCT
GCCGGGCGAGTCAGAACATCTCCGACTACCTGCATTGGTACCAGCAGAAGCCCGGCCAGGCC
CCTCGCCTGCTGATCTACTACGCCTCCCAGAGCATCAGCGGAATCCCCGCCCGGTTCTCCGG
AAGTGGGTCCGGAACCGACTTTACCCTGACCATCAGCTCTCTCGAGCCAGAGGACTTCGCGG
TGTACTACTGCCAGAACGGGCATAGTTTCCCACTGACCTTCGGAGGGGGCACAAAGGTGGAG
ATCAAGCGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAA
ATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTAC
AGTGGAAGGTGGACAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGAC
AGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAA
ACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCT
TCAACAGGGGAGAGTGTTAG
SEQ ID NO:98
LLVHLKKLFREG
