Note: Descriptions are shown in the official language in which they were submitted.
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ANTI-TRKA ANTIBODIES WITH ENHANCED INHIBITORY PROPERTIES AND
DERIVATIVES THEREOF FOR USE IN TREATING BONE ASSOCIATED PAIN
The field of the invention
The present invention relates generally to antibodies directed against TrkA
receptor
and their uses, including humanized anti-TrkA antibodies and methods of
treatment with anti-
TrkA antibodies. In one aspect, the present invention relates to humanized
anti-TrkA
antibodies with enhanced inhibitory properties for use in methods of treating
neuroma and/or
bone associated pain.
In a further aspect, the present invention relates to humanized anti-TrkA
antibodies
with enhanced inhibitory properties comprising a heavy chain variable region,
a light chain
variable region, a human light chain constant region and a variant human IgG4
heavy chain
constant region which exhibit altered exchange properties.
Background of the invention
Neurotrophins are a family of peptide growth factors (Barde YA (1994) J.
Neurobiol.
25(11):1329-33) structurally related to the first member of the family NGF
(Levi-Montalcini
R (1987) EMBO J. 6(5):1145-54). Neurotrophins modulate neuronal
differentiation and
survival, as well as synaptic transmission, both of the peripheral neurons and
of the central
nervous system. Furthermore NGF acts on various non neuronal tissues and
cells, such as
immune cells. NGF acts through two membrane receptors present in the target
cells, the low
affinity p75 receptor, and the 140 kDa high affinity transmembrane
glycoprotein, TrkA
(Kaplan DR et at., (1991) Science 252(5005):554-8; Klein R et at., (1991) Cell
65(1):189-97)
having tyrosine kinase activity. TrkA is expressed in neural-crest neurons, in
sympathetic
neurons as well as in cholinergic neurons of the basal fore-brain and corpus
striatum, where it
represents the crucial mediator of NGF activities (Holtzman DM et at., (1992)
Neuron
9(3):465-78; Verge VM et at., (1992) J. Neurosci. 12(10):4011-22). TrkA is
also expressed in
some non neuronal tissues and cells, including B lymphocytes (Torcia M et at.,
(1996) Cell
85(3):345-56).
Nerve growth factor (NGF) was identified originally as a survival factor for
sensory and
sympathetic neurons in the developing nervous system (Gorin PD & Johnson EM
(1979)
PNAS USA, 76(10):5382-6). In adults, NGF is not required for survival but it
has a crucial
role in the generation of pain and hyperalgesia in several acute and chronic
pain states. The
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expression of NGF is high in injured and inflamed tissues, and activation of
trkA on
nociceptive neurons triggers and potentiates pain signalling by multiple
mechanisms.
Inflammation-related pain can be significantly reduced by neutralizing NGF
bioactivity in
animal models (Woolf CJ et at., (1994) Neuroscience 62(2):327-31; McMahon SB
et at.,
(1995) Nat. Med. 1(8):774-80; Koltzenburg M et at., (1999) Eur. J. Neurosci.
11(5):1698-
704), implying that an enhanced level of this neurotrophin is necessary to
generate the full
hyperalgesic response. Remarkably, the inhibition of other neurotrophins does
not result in
antagonizing the induced hyperalgesia, suggesting that this effect is specific
to NGF
(McMahon SB et at., (1995) Nat. Med. 1(8):774-80); in addition, NGF inhibition
results in
analgesia in different neuropathy-related pain protocols (Koltzenburg M et
at., (1999) Eur. J.
Neurosci. 11(5):1698-704; Ro LS et at., (1999) Pain 79(2-3):265-74; Theodosiou
Met at., (1999)
Pain, 81(3):245-55; Christensen MD & Hulsebosch CE (1997) Exp. Neurol.
147(2):463-75).
There is a large, unmet medical need in the treatment of pain. The dominant
classes of
analgesic drugs, the nonsteroidal anti-inflammatory drugs (NSAIDs) and the
opiates are
limited by their efficacy and tolerability (Hefti FF et at., (2006) Trends
Pharmacol. Sci. 27(2):
85-91). Less than 30% of patients with chronic pain obtain adequate relief
with current
therapies, and there are many adverse effects, particularly with long-term
administration
(Kalso E et at., (2004) Pain 112(3):372-80). The recognition that NGF has a
central role in
pain mechanisms in adults provides an opportunity to develop a completely
novel class of
pain therapeutics.
Targeting TrkA instead of NGF may represent a better therapeutic choice as
this
receptor does not interfere with NGF functions mediated by the p75 receptor,
the latter having
broad function in neuronal development.
The inventors have evaluated the efficacy and safety of using humanized anti-
TrkA antibodies
in methods of treating deep somatic pain and in particular bone associated
pain, such as pain
resulting from damage to bones from an external trauma or as a consequence of
bone
affecting pathology such as cancer which may lead to bone damage,
inappropriate inervation
and/or other effects leading to bone associated pain. The inventors have also
evaluated the
efficacy and safety of using humanized anti-TrkA antibodies in methods of
treating pain
resulting from neuromas that have formed inappropriately as a consequence of a
trauma or
another pathology.
Summary of the invention
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The present disclosure relates generally to humanized anti-TrkA antibodies,
methods for their
preparation and use.
In one aspect, the present disclosure provides a humanized anti-TrkA antibody
or fragment
thereof comprising:
a) a heavy chain variable domain comprising a sequence selected from the group
consisting of
SEQ ID NOs: 1-5, and
b) a light chain variable domain comprising a sequence selected from the group
consisting of
SEQ ID NOs: 6-13,
wherein CDR2 of the heavy chain variable domain comprises at least one amino
acid
substitution and/or wherein the non-CDR region of the heavy chain variable
domain
comprises an amino acid substitution at an amino acid position selected from
the group
consisting of 37, 42 and 89, wherein the amino acid position of each group
member is
indicated utilizing the numbering system set forth in Kabat.
In further aspects, the present disclosure provides an isolated nucleic acid
encoding a
humanized anti-TrkA antibody or fragment thereof, a vector comprising the
isolated nucleic
acid and a host cell comprising the isolated nucleic acid or the vector. Also
provided by the
present disclosure is a method of producing a humanized anti-TrkA antibody or
fragment
thereof
In further aspects, the present disclosure provides a composition comprising a
humanized
anti-TrkA antibody or fragment thereof and an immunoconjugate comprising a
humanized
anti-TrkA antibody or fragment thereof linked to a therapeutic agent.
In particular the present invention relates to a humanized anti-TrkA antibody
or fragment
thereof that bind to human TrkA, wherein said anti-TrkA antibody or fragment
thereof
comprising heavy chain variable domain CDRs 1, 2 and 3 and light chain
variable domain
CDRs 1, 2 and 3, and the heavy chain variable domain comprises a sequence
selected from
the group consisting of SEQ ID NOs: 1-5, and the light chain variable domain
comprises a
sequence selected from the group consisting of SEQ ID NOs: 6-13, wherein the
non-CDR
region of the heavy chain variable domain comprise an amino acid substitution
at an amino
acid position selected from the group consisting of 37, 42, and 89, wherein
the amino acid
position of each group member is indicated utilizing the numbering system set
forth in Kabat
for treating bone associated pain.
In further aspects, the present disclosure provides a humanized anti-TrkA
antibody or
fragment thereof, a composition or an immunoconjugate for use in medicine, for
use in the
treatment of pain, for use in the treatment of chronic pain, for use in the
treatment of acute
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pain, for use in the treatment of pain associated with one or more of the
following:
pancreatitis, kidney stones, endometriosis, IBD, Crohn's disease, post
surgical adhesions, gall
bladder stones, headaches, dysmenorrhea, musculoskeletal pain, sprains,
visceral pain,
ovarian cysts, prostatitis, cystitis, interstitial cystitis, post-operative
pain, migraine, trigeminal
neuralgia, pain from burns and/or wounds, pain associated with trauma,
neuropathic pain,
pain associated with musculoskeletal diseases, rheumatoid arthritis,
osteoarthritis, ankylosing
spondilitis, periarticular pathologies, oncological pain, pain from bone
metastases, HIV
infection, for use in the treatment of cancer, a neuronal disorder,
Alzheimer's disease,
diabetes mellitus, diabetic nephropathy, a viral disorder, an HIV mediated
disorder, leprosy,
or an inflammatory disorder and for use in diagnosis or prognosis.
In a further aspect, the present disclosure relates to methods for treating
deep somatic pain
and in particular bone associated pain comprising at least the step of
administering an
effective amount of a humanized anti-TrkA antibody or fragment thereof
according to the
present invention to an individual in need.
In accordance with the present invention bone associated pain is meant to
refer to any
sensation of pain experienced as a consequence of an injury, deterioration or
any pathology
associated with one or more bones in an individual.
The pain may occur as a feeling of numbness, a tingling sensation, a crawling
sensation, a
sensation of heat or cold, or a feeling of tightness, a dull pain such as be a
feeling of pressure
or tightness, a heavy feeling, or a tingling sensation, a sharp pain such as a
stabbing, shooting,
tearing, or piercing pain, the pain may be constant, throbbing or intermittant
at regular or
irregular intervals of time or occur only when the area is touched.
More specifically the bone associated pain may be a consequence of a bone
fracture, chip,
break or any other injury or inflammation. This may be due to an accident or
as a
consequence of an overuse or repetitive movement injury or result from a
weakened bone due
to a hormone deficiency, infection, bone cancer, a metastatic malignancy,
leukaemia,
myeloma or another cancer affecting blood or bone associated cells, or due to
an interruption
in the blood supply to the bone.
In accordance with the present invention the bone associated pain may be a
consequence of a
bone affecting pathology. A bone affecting pathology refers to any pathology
which directly
or indirectly affects one or more bones in an individual, leading to a
sensation of pain.
Examples of pathologies include cancer, infection and immune system
dysfunction.
In particular the bone associated pain may be associated with or result from
bone cancer and
in particular secondary bone cancer and/or metastatic cancer cells.
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Alternatively the bone associated pain may be a consequence of one or more
neuroma, in
particular resulting from bone cancer or any other pathology.
In particular the method involves the administration of an effective amount of
a humanized
anti-TrkA antibody or fragment thereof according to the present invention, so
as to minimise
5 or reduce abberant nerve sprouting and/or neuroma formation or size.
In accordance with the present invention an effective amount of the humanized
anti-TrkA
antibody or fragment thereof is intended to mean an amount sufficient to lead
to the
alleviation or cessation of one or more pain sensations in an individual in
need thereof, when
administered to the individual by a clinician or other qualified medical
professional.
In a further aspect, the present disclosure relates to a humanized anti-TrkA
antibody or
fragment thereof according to the present invention for use in the treatment
of deep somatic
pain and in particular bone associated pain or neuroma related pain. More
specifically the
bone associated pain may be a consequence of a bone fracture, chip, break or
any other injury
or inflammation. This may be due to an accident or as a consequence of an
overuse or
repetitive movement injury or result from a weakened bone due to a hormone
deficiency,
infection, bone cancer, a metastatic malignancy, leukaemia, myeloma or another
cancer
affecting blood or bone associated cells, or due to an interruption in the
blood supply to the
bone.
In accordance with the present invention the bone associated pain may be a
consequence of a
bone affecting pathology. A bone affecting pathology refers to any pathology
which directly
or indirectly affects one or more bones in an individual, leading to a
sensation of pain.
Examples of pathologies include cancer, infection and immune system
dysfunction.
In particular the bone associated pain may be associated with or result from
bone cancer and
in particular secondary bone cancer and/or metastatic cancer cells.
Alternatively the bone associated pain may be a consequence of one or more
neuroma, in
particular resulting from bone cancer or any other pathology.
In particular the invention relates to a humanized anti-TrkA antibody or
fragment thereof
according to the present invention for use in the treatment to minimise or
reduce aberrant
nerve sprouting and/or neuroma formation or size.
In a further aspect, the present disclosure provides methods for treating
inflammatory pain,
osteoarthritic pain and neuropathic pain. In one aspect, in an in vivo model
of acute
inflammatory hyperalgesia, administration of a humanized anti-TrkA antibody
resulted in a
significant reversal of acute inflammatory hyperalgesia at a dose of
0.01mg/kg. In another
aspect, in an in vivo model of chronic inflammatory hyperalgesia,
administration of a
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humanized anti-TrkA antibody resulted in a significant and sustained reversal
of chronic
inflammatory hyperalgesia at a dose of 0.01mg/kg. In another aspect, in an in
vivo model of
chronic osteoarthritic hyperalgesia, administration of a humanized anti-TrkA
antibody
resulted in a significant and sustained reversal of chronic osteoarthritic
hyperalgesia at a dose
of 0.01mg/kg. In a further aspect, in the in vivo chronic constriction injury
model of
neuropathic pain, administration of a humanized anti-TrkA antibody resulted in
a significant
reversal of neuropathic pain at a dose of 1.0mg/kg.
In a further aspect, the present disclosure provides an article of manufacture
comprising a
humanized anti-TrkA antibody or fragment thereof, a composition or an
immunoconjugate.
In a further aspect, the present disclosure provides a kit comprising a
humanized anti-TrkA
antibody or fragment thereof, a composition or an immunoconjugate.
Particular problems also relate to the generation of stable and efficatious
pharmaceutical
formulations comprising antibodies result from the fact that proteins are
larger and more
complex than traditional organic and inorganic drugs. For a protein to remain
biologically
active, a formulation must preserve intact the conformational integrity of at
least a core
sequence of the protein's amino acids while at the same time protecting the
protein's multple
functional groups from degradation. Degradation pathways for proteins can
involve chemical
instability (i.e. any process which involves modification of the protein by
bond formation or
cleavage resulting in a new chemical entity) or physical instability (i.e.
changes in the higher
order structure of the protein). Chemical instability can result from
deamidation,
racemization, hydrolysis, oxidation, beta elimination or disulfide exchange.
Physical
instability can result from denaturation, aggregation, precipitation or
adsorption
In accordance with a further aspect of the present invention there is provided
a stable aqueous
pharmaceutical formulation comprising a therapeutically effective amount of an
anti-TrkA
antibody, a buffer, a surfactant and a tonicifying agent, wherein the pH of
the formulation is
from about 5 to about 7.
In accordance with this aspect of the present invention the anti-TrkA antibody
comprises:
a) a heavy chain variable domain comprising a sequence selected from the group
consisting of
SEQ ID NOs: 1-5, and
b) a light chain variable domain comprising a sequence selected from the group
consisting of
SEQ ID NOs: 6-13,
wherein CDR2 of the heavy chain variable domain comprises at least one amino
acid
substitution and/or wherein the non-CDR region of the heavy chain variable
domain
comprises an amino acid substitution at an amino acid position selected from
the group
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consisting of 37, 42 and 89, wherein the amino acid position of each group
member is
indicated utilizing the numbering system set forth in Kabat (Kabat EA et at.,
(1991)
Sequences of proteins of immunological interest. 5th Edition - US Department
of Health and
Human Services, NIH publication n 91-3242).
In accordance with this aspect of the present invention the anti-TrkA antibody
is present at a
concentration of at least 0.1 mg/ml.
In accordance with this aspect of the present invention the anti-TrkA antibody
is present at a
concentration of at least 0.2 mg/ml to 175 mg/ml.
In accordance with this aspect of the present invention the anti-TrkA antibody
is present at a
concentration of at least 1 mg/ml.
In accordance with this aspect of the present invention the anti-TrkA antibody
is present at a
concentration of at least 10 mg/ml.
In accordance with this aspect of the present invention the anti-TrkA antibody
is present at a
concentration of at least 100 mg/ml.
In accordance with this aspect of the present invention the anti-TrkA antibody
is present at a
concentration of at least 150 mg/ml.
In accordance with this aspect of the present invention the buffer is selected
from the group
comprising: citrate, acetate, histidine, phosphate, tris
(tris(hydroxymethyl)aminomethane).
In accordance with this aspect of the present invention the
stabilising/tonicifying agents is
selected from the group comprising: sodium acetate, sodium bicarbonate, sodium
carbonate,
sodium chloride, potassium acetate, potassium bicarbonate, potassium
carbonate, potassium
chloride, sucrose, polyols, sugars, amino acids such as histidine, arginine,
and glycine,
methionine, proline, lysine, glutamic acid, amines and trehalose.
In accordance with this aspect of the present invention the surfactant is
selected from the
group comprising: tween 20/40/80, polysorbate 20/80, poloxamer, sodium lauryl
sulphate.
In accordance with this aspect of the present invention the pH of the
formulation is 5.75.
In accordance with this aspect of the present invention the pH of the
formulation is 6Ø
In accordance with this aspect of the present invention, the formulation may
further comprise
one or more excipients and bulking agents.
In accordance with a preferred embodiment of the present invention there is
provided a low
concentration aqueous formulation of an anti-TrkA antibody adjusted to pH 6.0
comprising:
10mg/m1 of an anti-TrkA antibody;
25m1IVI Citrate;
150mM NaCl;
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0.05% Tween 80.
In accordance with a preferred embodiment of the present invention there is
provided a low
concentration aqueous formulation of an anti-TrkA antibody adjusted to pH 6.0
comprising:
at least 10mg/m1 of an anti-TrkA antibody;
50mM histidine;
150mM NaCl;
0.05% Tween 80.
In accordance with a preferred embodiment of the present invention there is
provided a high
concentration aqueous formulation of an anti-TrkA antibody adjusted to pH 5.75
or 6.0,
comprising:
100mg/m1 of an anti-TrkA antibody;
50mM histidine;
150mM NaCl;
0.05% Tween 80.
In accordance with a preferred embodiment of the present invention there is
provided a high
concentration aqueous formulation of an anti-TrkA antibody adjusted to pH 5.75
or 6.0,
comprising:
150mg/m1 of an anti-TrkA antibody;
50mM histidine;
150mM NaCl;
0.05% Tween 80.
The inventors whilst developing these formulations have have overcome issues
associated
with aqeous liquid formulations comprising proteins in general and antibodies
in particular.
Several alternative formulations tried during the course of this research
program lack long
term stability at 5 C, 25 C or 40 C, in particular the antibody was subject to
degradation and
apparent instability in several of the alternative formulations not comprising
the specific
combination of components listed above.
In addition the development of this subcutaneously administered formulation
has increased
the possible dosing, in terms of both the quantity of the anti-TrkA antibody
administered at
each dosing and also the number of dosings possible. In addition preliminary
data also
indicate that the subcutaneously administered formulations according to the
present invention
are a more efficacious way of administering the anti-TrkA antibody by
previously tested
methods and formulations.
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In accordance with a further aspect of the present invention the formulation
is administered to
a person in need at a dose of at least 0.1mg per kg of body weight; in
particular at least
0.3mg/kg, lmg/kg, 3mg/kg, 10mg/kg or 30mg/kg.
In accordance with a further aspect of the present invention the formulation
is administered to
a patient in need once.
In accordance with a further aspect of the present invention the formulation
is administered to
a patient in need at an interval of at least 1 hour between dosing and
preferably 4 hours/6
hours/12 hours/24 hours between dosing.
In accordance with a further aspect of the present invention the formulation
is administered to
a patient in need on demand.
In accordance with a further aspect of the present invention the formulation
is stable at 5 C
for at least 1 year and preferably at least 2 years.
In accordance with a further aspect of the present invention the formulation
is stable at 25 C
for at least 3 months, preferably 6 months and most preferably 1 year.
In accordance with a further aspect of the present invention the formulation
is stable at 40 C
for at least 1 month and preferably 3 months.
Wherein stability is measured with one or more method selected from the group
comprising:
determining changes in the clarity, degree of coloration, degree of
opalescence and particulate
contamination (visible particles) of the formulation, light absorption
measurement of
wavelength 280nm to determine the concentration of protein present in the
formulation; by
SDS-page gel visualisation to determine changes in the weight and or breakdown
of the
antibody; by ELISA to determine any change in the binding properties of the
antibody; by
HPLC-CEX to determine changes in the positive/negative antibody species make
up in the
formulation; by HPLC-IEF so as to determine changes in the IsoElectroFocusing
profile of
the antibody by capillary electrophoresis present in the formulation, by HPLC-
SEC analysis
to determine changes in the antibody in the formulation.
In accordance with a further aspect of the present invention the formulation
comprises an
amount of an anti-TrkA antibody so that the formulation can be administered to
a person in
need at a dose of at least 0.1mg per kg of body weight; in particular at least
0.3mg/kg,
lmg/kg, 3mg/kg, 10mg/kg or 30mg/kg.
In accordance with another aspect of the present invention there is also
provided a method of
treating an individual in pain by administering a formulation according to the
present
invention comprising a therapeutically effective amount of an anti-TrkA
antibody.
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Brief description of the figures
FIG. 1: Surface Plasmon resonance measurements of anti-TrkA antibodies. Data
are
expressed as number of response (abbreviated RU; Y axis) vs. time (X axis).
(FIG. 1A)
MNAC13 antibody. (FIG. 1B) BXhVH5VL1 antibody. (FIG. 1C) GBR VH5(V37A)VL1
5 antibody. (FIG. 1D) GBR VH5(K3Q,V37A)VL1 antibody.
FIG. 2: Thermo stability measurements of anti-TrkA antibodies using
differential scanning
calorimetry. Data are expressed as excess molar heat capacity (abbreviated Cp
[kcal/moU C];
Y axis) vs. temperature (X axis). (FIG. 2A) MNAC13 antibody (FAB fragment Tm
is Tml at
74 C). (FIG. 2B) BXhVH5VL1 antibody (FAB fragment Tm is Tm3 at 76.5 C). (FIG.
2C)
10 GBR VH5(V37A)VL1 antibody (FAB fragment Tm is Tml at 73.6 C). (FIG. 2D)
GBR
VH5(K3Q,V37A)VL1 antibody (FAB fragment Tm is Tml at 73 C). (FIG. 2E) overlay
of
FIG. 2C and FIG. 2D. (FIG. 2F) overlay of FIG. 2B and FIG. 2D. (FIG. 2G)
overlay of FIG.
2A and FIG. 2D.
FIG. 3: Functional bioactivity of anti-TrkA antibodies. Effect of humanized
anti-TrkA
antibodies on the NGF-induced TF-1 cell proliferation; data are expressed as %
of
proliferative response (Y axis) vs. antibody concentration (jig/ml; X axis).
(FIG. 3A) GBR
VH5(V37A)VL1, GBR VH5(K3Q,V37A)VL1 vs. BXhVH5VL1. (FIG. 3B) GBR
VH5(V37A)VL1, GBR VH5(K3Q,V37A)VL1 vs. MNAC13. (FIG. 3C) GBR
VH5(K3Q,V37A)VL1 vs. GBR VH5(K3Q,V37A)VL1 IGHG4 5228P. (FIG. 3D)
BXhVH5VL1, BXhVH5VL3, GBR VH5(V37A)VL1 vs. GBR VH5(V37A)VL3. (FIG. 3E)
BXhVH5VL1, BXhVH3VL1, GBR VH5 (V37A)VL1 vs. GBR VH3 (V37A)VL1 .
FIG. 4: Humanized anti-TrkA antibody reverses acute inflammatory paw pain.
Acute
inflammatory hyperalgesia of the paw was induced by intraplantar injection of
CFA into one
of the hind limb paws of AMB1 mice and measured as the % ratio between weight
bearing on
the ipsilateral (injected) and contralateral (non-injected) paw (%
ipsi/contra, mean s.e.m.).
Weight bearing readings were taken 23hrs post-CFA injection (0 hr) before
treatment
initiation at 24hr post-CFA with a single i.p. injection of 0.0001 (white
bars), 0.001
(horizontally-hatched bars), 0.01 (chequered bars) and 0.1 mg/kg (diagonally
hatched bars)
anti-TrkA antibody or 0.1 mg/kg isotype control antibody (black bars) followed
by weight
bearing measurements at 4, 8, 24, 48, 72, 96 and 120 hrs post-dose. As a
positive control,
mice were treated with 10mg/kg indomethacin p.o. (vertically-hatched bars).
FIG. 5: Humanized anti-TrkA antibody reverses chronic inflammatory joint pain.
Chronic
inflammatory hyperalgesia of the joint was induced by intra-articular
injection of CFA into
one of the hind limb knee joints of AMB1 mice and measured as the % ratio
between weight
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bearing on the ipsilateral (injected) and contralateral (non-injected) limb (%
ipsi/contra, mean
s.e.m.). Weight bearing readings were taken immediately prior to CFA injection
(naive) and
on day 3, 7 and 10 post-CFA before treatment initiation on day 13 post-CFA
with a single i.p.
injection of 0.01 (open square, dashed line), 1 (closed triangle) and 10mg/kg
(open circle)
anti-TrkA antibody or 10 mg/kg isotype control antibody (closed circle)
followed by weight
bearing measurements at 4, 8, 24, 48, 72 and 96 hrs post-dose (in
parentheses). As a positive
control, mice were treated with 60mg/kg celecoxib twice daily (open diamond)
from day 13
onwards and weight bearing was measured at 1 and 8 hrs post-dosing on day 13
post-CFA
and then at 1 hr post-dosing on days 14-17 post-CFA.
FIG. 6: Humanized anti-TrkA antibody reverses chronic osteoarthritic pain.
Chronic
osteoarthritic hyperalgesia was induced by intra-articular injection of MIA
into one of the
hind limb knee joints of AMB1 mice and measured as the % ratio between weight
bearing on
the ipsilateral (injected) and contralateral (non-injected) limb (%
ipsi/contra, mean s.e.m.).
Baseline (BL) weight bearing readings were taken immediately prior to MIA
injection and on
day 3, 7 and 10 days post-MIA before treatment initiation on day 14 post-MIA
with a single
i.p. injection of 1 (open triangle), 10 (open diamond) and 100 g/kg (closed
circle, dashed
line) anti-TrkA antibody or 10 mg/kg isotype control antibody (closed square)
(FIG. 6A). As
a comparator control, animals were treated on day 14 post-MIA with tramadol at
10mg/kg or
pregabalin at 30mg/kg p.o. followed by tramadol at 30mg/kg or pregabalin at
100mg/kg every
other day on days 16-22 post-MIA (FIG. 6B). All animals were assessed using
weight
bearing at 4, 8, and 24 hours post-dosing on day 14 post-MIA followed by every
24 hours for
antibody treated groups and 1 and 24 hours post-dose for the tramadol and
pregabalin treated
groups.
FIG. 7: Humanized anti-TrkA antibody reverses neuropathic pain. Chronic
neuropathic
hyperalgesia and allodynia were induced by chronic constriction injury of the
sciatic nerve of
AMB1 mice and measured as the threshold force required for paw withdrawal (g)
and the
latency time for paw withdrawal from a cold plate (sec), respectively.
Readings were taken
prior to surgery and 7 days after surgery on the day before treatment
initiation (Oh). Animals
were then treated with a single i.p. injection of either 1000 g/kg isotype
control antibody
(closed triangle) or 10 (closed circle), 100 (closed square) and 1000 g/kg
(closed diamond)
anti-TrkA antibody. Pregabalin (closed inverted triangle) at 30 mg/kg/10m1,
p.o. or saline
(open circle) were administered once daily over the seven day post-dose
period. Post-dose
readouts were recorded at 4h, 24h and then every other day until the 7th day
post-dose. Post-
dose readouts were recorded lh after pregabalin or saline dosing on all days.
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FIG. 8: Morphometric analysis of the right SCG (4 mice/treatment =4
ganglia/treatment) of
neonate AMB1 mice treated for 4 weeks from postnatal day 1 with GBR VH5
(K3Q,V37A)
VL1 IGHG4 S228P (label GBR) (triangles), tanezumab (squares) or PBS (circles).
Statistical
analysis was performed as a one-way ANOVA with a Dunnett's post hoc test
comparison to
PBS treatment: *p<0.05, **p<0.01, ***p<0.001.
FIG. 9: Morphometric analysis of the right and left SCG (8-9 mice/treatment
=15-18
ganglia/treatment) of adult AMB1 mice treated for 4 weeks with GBR VH5
(K3Q,V37A)
VL1 IGHG4 5228P (label GBR) (triangles), tanezumab (squares) or PBS (circles).
Statistical
analysis was performed as a one-way ANOVA with a Dunnett's post hoc test
comparison to
PBS treatment: *p<0.05, **p<0.01, ***p<0.001.
FIG. 10: Frequency plot of the diameters of all SCG neuronal cell bodies from
adult AMB1
mice treated with GBR VHS (K3Q,V37A) VL1 IGHG4 5228P (label GBR) (1904
neurons,
circles), PBS (1547 neurons, squares) or tanezumab (1420 neurons, diamonds).
Binning was
performed with Graphpad Prism using standard parameters.
FIG. 11: Representative H&E-stained sections of the SCG from PBS-treated adult
animals,
the smallest of the SCG of tanezumab-treated animals and the largest of the
SCG of GBR
VHS (K3Q,V37A) VL1 IGHG4 5228P (label GBR) - treated animals. The neuronal
cell body
is marked with a black arrow.
FIG. 12: Neuropathic pain was induced by chronic constriction injury of the
sciatic nerve of
AMB1 mice. Mechanical hyperalgesia (A) was measured as the threshold force
required for
paw withdrawal (g) and cold allodynia (B) was measured as the latency time for
paw
withdrawal from a cold plate (sec). Readings were taken prior to surgery (-7d)
and 7 days
after surgery on the day before treatment initiation (Oh). Animals were then
treated with a
single i.p. injection of either saline control (circles) or 0.3 (triangles)
and 1 mg/kg (inverted
triangles) GBR VHS (K3Q,V37A) VL1 IGHG4 5228P (label GBR) or 0.3 (squares) and
1
mg/kg (diamonds) tanezumab. Post-dose readouts were recorded at 4h, 1 day and
then at
every next day until the 9th post-dose day with a final reading at 14 days
post-dose.
FIG. 13: Chronic inflammatory joint pain was induced by multiple intra-
articular injections of
CFA into the knee joint of AMB1 mice. DO: the day of first CFA injection; D3,
D10, D17,
D24: 3, 10, 17 and 24 days after the first CFA injection. Arrows indicate
intra-articular
injections (PBS or CFA) on day 0, 7, 14 and 21. 4h, 8h, 24h, 48h, 76h, 96h and
120h: 4, 8,
24, 48, 76, 96 and 120 hours after dosing on D24. *: P < 0.05, compared to CFA
+ Vehicle
group, one-way ANOVA. n=6 for Sham+vehicle group (diamonds), n=8 for
CFA+vehicle
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group (squares), n=10 for CFA+ GBR VH5 (K3Q,V37A) VL1 IGHG4 S228P (triangles)
and
CFA+Tanezumab (crosses) groups.
FIG. 14: Longitudinal in vivo monitoring of the mice on days 2, 5, 7, 14 and
20 after fracture.
The post-operative values are related to pre-operative values. A) Assessment
of the activity;
B) analysis of the ground reaction force (GRF) of the operated limb. Results
are presented as
the mean SEM; n = 5-7. Asterisk denotes p < 0.05, anti-TrkA vs. PBS.
FIG. 15: Representative histological sections of fractured femurs stained with
safranin-O/fast
green and assessment of the callus composition. A¨C: PBS-treatment, D¨F: anti-
NGF
antibody, G¨I: anti-TrkA antibody. Scale bar = 500 gm. J¨L: Histomorphometric
assessment
of the callus composition on days 7 (J), 14 (K) and 25 (L). Results are
presented as the mean
SEM; n = 5-8. White bars: PBS-treatment, light grey bare: anti-NGF antibody;
dark grey
bars: anti-TrkA antibody.
FIG 16: Flexural rigidity of the frature calli in mice treated with PBS, anti-
NGF antibody or
anti TrkA antibody after a healing period of 25 days. Data is depicted as the
mean SEM; n =
6-8.
FIG 17: Stability data for low concentration (10mg/m1) formulation at 5 C.
FIG 18: Stability data for low concentration (10mg/m1) formulation at 25 C.
FIG 19: Stability data for low concentration (10mg/m1) formulation at 40 C.
FIG 20: Stability data for high concentration (100mg/m1) formulation A (pH
5.75) at 5 C.
FIG 21: Stability data for high concentration (100mg/m1) formulation A (pH
5.75) at 25 C.
FIG 22: Stability data for high concentration (100mg/m1) formulation A (pH
5.75) at 40 C.
FIG 23: Stability data for high concentration (100mg/m1) formulation B (pH 6)
at 5 C.
FIG 24: Stability data for high concentration (100mg/m1) formulation B (pH 6)
at 25 C.
FIG 25: Stability data for high concentration (100mg/m1) formulation B (pH 6)
at 40 C.
FIG 26: Stability data for high concentration (150mg/m1) formulation A (pH
5.75) at 5 C.
FIG 27: Stability data for high concentration (150mg/m1) formulation A (pH
5.75) at 25 C.
FIG 28: Stability data for high concentration (150mg/m1) formulation A (pH
5.75) at 40 C.
FIG 29: Stability data for high concentration (150mg/m1) formulation B (pH 6)
at 5 C.
FIG 30: Stability data for high concentration (150mg/m1) formulation B (pH 6)
at 25 C.
FIG 31: Stability data for high concentration (150mg/m1) formulation B (pH 6)
at 40 C.
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Detailed description of the invention
The present disclosure relates to humanized anti-TrkA antibodies or fragment
thereof,
methods for their preparation and use.
The term "TrkA", "human TrkA", "TrkA receptor" or "human TrkA receptor" are
used herein
equivalently and mean "human TrkA" if not otherwise specifically indicated.
Human TrkA as
used herein include variants, isoforms, and species homologs of human TrkA.
Accordingly,
antibodies of this disclosure may, in certain cases, cross-react with TrkA
from species other
than human. In certain embodiments, the antibodies may be completely specific
for one or
more human TrkA proteins and may not exhibit species or other types of non-
human cross-
reactivity.
TrkA is also known as high affinity nerve growth factor receptor or
neurotrophic tyrosine
kinase receptor type lor TRK1-transforming tyrosine kinase protein or
Tropomyosin-related
kinase A or Tyrosine kinase receptor or Tyrosine kinase receptor A or Trk-A or
gp140trk or
p140-TrkA or MTC or TRK. TrkA is a receptor tyrosine kinase involved in the
development
and the maturation of the central and peripheral nervous systems through
regulation of
proliferation, differentiation and survival of sympathetic and nervous
neurons. TrkA is the
high affinity receptor for NGF which is its primary ligand; it can also bind
and be activated by
NTF3/neurotrophin-3.
The complete amino acid sequence of the four known human TrkA isoforms are
found under
the UniProt/Swiss-Prot accession number P04629 (Consortium TU, (2012) Nucleic
Acids
Res. 40(D1):D71-D5). The four iso forms are produced by alternative splicing:
iso form TrkA-I
is found in most non-neuronal tissues (UniProt/Swiss-Prot accession number
P04629-2),
while iso form TrkA-II is primarily expressed in neuronal cells (UniProt/Swiss-
Prot accession
number P04629-1), and isoform TrkA-III is specifically expressed by
pluripotent neural stem
and neural crest progenitors (UniProt/Swiss-Prot accession number P04629-4). A
fourth
isoform which differs from isoform TrkA-II at residues 1-71 and lacks residues
393 to 398 is
known as isoform 3 (UniProt/Swiss-Prot accession number P04629-3). TrkA-II
isoform is
the major known isoform of TrkA. Isoform TrkA-I has enhanced responsiveness to
NTF3
neurotrophin whereas isoform TrkA-III is constitutively active and does not
bind NGF.
In a preferred embodiment, the TrkA iso form as used herein is the TrkA-II iso
form with SEQ
ID NO: 72 and an extracellular region thereof comprising the sequence of SEQ
ID NO: 25.
The term "anti-TrkA antibody or fragment thereof" or "humanised anti-TrkA
antibody or
fragment thereof" as used herein includes antibodies or a fragment thereof
that bind to human
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TrkA e.g. human TrkA in isolated form, specifically antibodies or fragment
thereof that bind
to the TrkA-II isoform (SEQ ID NO: 72), more specifically antibodies or
fragment thereof
that binds to a monovalent form of the human TrkA extracellular region (SEQ ID
NO: 25) of
the TrkA-II iso form, with an affinity (I(D) of 500 nM or less, preferably 350
nM or less, more
5 preferably 150 nM or less, even more preferably 100 nM or less, most
preferred 50 nM or
less, in particular 30 nM or less. Usually the humanized anti-TrkA antibody or
fragment
thereof is capable of inhibiting the functional activation of TrkA and/or is
capable of blocking
or reducing one or more biological activities that would otherwise be induced
by the binding
of NGF to TrkA.
10 As used herein, an "anti-NGF antibody" refers to an antibody which is
able to bind to NGF,
preferably human NGF. Usually the anti-NGF antibody is capable of inhibiting
the functional
activation of TrkA and/or is capable of blocking or reducing one or more
biological activities
of TrkA. The binding affinity of an anti-NGF antibody to NGF (such as hNGF)
can be 500
nM or less, preferably 100 nM or less. Usually the anti-NGF antibody should
exhibit any one
15 or more of the following characteristics: (a) bind to NGF and inhibit
NGF biological activity
and/or downstream pathways mediated by NGF signaling function; (b) block or
decrease
NGF receptor activation (including TrkA receptor dimerization and/or
autophosphorylation);
(c) increase clearance of NGF; (d) inhibit (reduce) NGF synthesis, production
or release.
Anti-NGF antibodies are known in the art, see, e.g., PCT Publication Nos. WO
01/78698, WO
01/64247, US Patent Nos. 5,844,092, 5,877,016 and 6,153,189; Hongo et at.,
(2000)
Hybridoma, 19:215-227; GenBank Accession Nos. U39608, U39609, L17078 or
L17077.
The term "humanized anti-TrkA antibody or fragment thereof capable of
inhibiting the
functional activation of TrkA" as used herein refers to humanized anti-TrkA
antibodies that
exhibit any one or more of the following characteristics: (a) bind to TrkA and
inhibit TrkA
biological activities and/or downstream pathways mediated by the binding of
NGF or
NTF3/neurotrophin-3 signaling function; (b) prevent, ameliorate, or treat any
aspect of pain;
(c) block or decrease TrkA activation, or dimerization and/or
autophosphorylation; (d)
increase TrkA clearance; (e) inhibit or reduce TrkA synthesis and/or cell
surface expression.
The term "humanized anti-TrkA antibody or fragment thereof capable of blocking
or reducing
one or more biological activities of TrkA" as used herein refers to humanized
anti-TrkA
antibodies which directly or indirectly reduce, inhibit, neutralize, or
abolish TrkA biological
activities.
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The term "biological activities of TrkA" or "Trick biological activities" as
used herein refers
without limitation to any one or more of the following: the ability to bind
NGF or other
neurotrophins; the ability to homo-dimerize, or hetero-dimerize and/or
autophosphorylate;
the ability to activate an NGF induced signalling pathway; the ability to
promote cell
differentiation, proliferation, survival, growth, migration and other changes
in cell physiology,
including (in the case of neurons, including peripheral and central neuron)
change in neuronal
morphology, synaptogenesis, synaptic function, neurotransmitter and/or
neuropeptide release
and regeneration following damage; and the ability to mediate pain and cancer
pain associated
with bone metastasis.
The term "105 0" as used herein describes the half maximal inhibitory
concentration (105 0)
which is a measure of the effectiveness of a compound in inhibiting biological
function, e.g.
inhibition of humanized anti-Trick antibodies on the proliferation of NGF-
induced TF-1 cells.
The term "antibody" as referred to herein includes full-length antibodies and
any antigen
binding fragment or single chains thereof. Antibodies and specifically
naturally occurring
antibodies are glycoproteins which exist as one or more copies of a Y-shaped
unit, composed
of four polypeptide chains. Each "Y" shape contains two identical copies of a
heavy (H)
chain, and two identical copies of a light (L) chain, named as such by their
relative molecular
weights. Each light chain pairs with a heavy chain, and each heavy chain pairs
with another
heavy chain. Covalent interchain disulfide bonds and non covalent interactions
link the chains
together. Antibodies and specifically naturally occurring antibodies contain
variable regions,
which are the two copies of the antigen binding site. Papain, a proteolytic
enzyme splits the
"Y" shape into three separate molecules, two so called "Fab" fragments (Fab =
fragment
antigen binding), and one so called "Fc" fragment or "Fc region" (Fc =
fragment
crystallizable). A Fab fragment consists of the entire light chain and part of
the heavy chain.
The heavy chain contains one variable domain (heavy chain variable domain or
VH) and
either three or four constant domains (CH1, CH2, CH3 and CH4, depending on the
antibody
class or isotype). The region between the CH1 and CH2 domains is called the
hinge region
and permits flexibility between the two Fab arms of the Y-shaped antibody
molecule,
allowing them to open and close to accommodate binding to two antigenic
determinants
separated by a fixed distance. The heavy chains of IgA, IgD and IgG each have
four domains,
i.e. one variable domain (VH) and three constant domains (CH1-3). IgE and IgM
have one
variable and four constant domains (CH1-4) on the heavy chain. The constant
regions of the
antibodies may mediate the binding to host tissues or factors, including
various cells of the
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immune system (e.g., effector cells) and the first component (Clq) of the
complement system
classical pathway. Each light chain is usually linked to a heavy chain by one
covalent
disulfide bond. Each light chain contains one variable domain (light chain
variable domain or
VL) and one light chain constant domain. The light chain constant domain is a
kappa light
chain constant domain designated herein as IGKC or is a lambda light chain
constant domain
designated herein as IGLC. IGKC is used herein equivalently to CI( or CK and
has the same
meaning. IGLC is used herein equivalently to a or CL and has the same meaning.
The term
"an IGLC domain" as used herein refers to all lambda light chain constant
domains e.g. to all
lambda light chain constant domains selected from the group consisting of
IGLC1, IGLC2,
IGLC3, IGLC6 and IGLC7. The VH and VL regions can be further subdivided into
regions of
hypervariability, termed complementarity determining regions (CDR),
interspersed with
regions that are more conserved, termed framework regions (FR or FW or "non-
CDR
regions"). Each VH and VL is composed of three CDRs and four FRs, arranged
from amino-
terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2,
FR3, CDR3,
FR4. The variable regions of the heavy and light chains contain a binding
domain that
interacts with an antigen.
Antibodies are grouped into classes, also referred to as isotypes, as
determined genetically by
the constant region. Human constant light chains are classified as kappa (CK)
and lambda
(a) light chains. Heavy chains are classified as mu (.1), delta (6), gamma
(y), alpha (a), or
epsilon (8), and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE,
respectively.
Thus, "isotype" as used herein is meant any of the classes and/or subclasses
of
immunoglobulins defined by the chemical and antigenic characteristics of their
constant
regions. The known human immunoglobulin isotypes are IgG1 (IGHG1), IgG2
(IGHG2),
IgG3 (IGHG3), IgG4 (IGHG4), IgAl (IGHA1), IgA2 (IGHA2), IgM (IGHM), IgD
(IGHD),
and IgE (IGHE). The so-called human immunoglobulin pseudo-gamma IGHGP gene
represents an additional human immunoglobulin heavy constant region gene which
has been
sequenced but does not encode a protein due to an altered switch region
(Bensmana M et at.,
Nucleic Acids Res. 16(7):3108). In spite of having an altered switch region,
the human
immunoglobulin pseudo-gamma IGHGP gene has open reading frames for all heavy
constant
domains (CH1-CH3) and hinge. All open reading frames for its heavy constant
domains
encode protein domains which align well with all human immunoglobulin constant
domains
with the predicted structural features. This additional pseudo-gamma isotype
is referred herein
as IgGP or IGHGP. Other pseudo immunoglobulin genes have been reported such as
the
human immunoglobulin heavy constant domain epsilon P1 and P2 pseudo-genes
(IGHEP1
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and IGHEP2). The IgG class is the most commonly used for therapeutic purposes.
In humans
this class comprises subclasses IgGl, IgG2, IgG3 and IgG4. In mice this class
comprises
subclasses IgGl, IgG2a, IgG2b, IgG2c and IgG3.
The term "chimeric antibody" or "chimeric anti-TrkA antibody" as used herein
includes
antibodies in which the variable region sequences are derived from one species
and the
constant region sequences are derived from another species, such as an
antibody in which the
variable region sequences are derived from a mouse antibody and the constant
region
sequences are derived from a human antibody.
The term "humanized antibody" or "humanised anti-TrkA antibody" as used herein
includes
antibodies in which CDR sequences derived from the germline of another
mammalian
species, such as a mouse, have been grafted onto human framework sequences.
Additional
framework region modifications may be made within the human framework
sequences as well
as within the CDR sequences derived from the germline of another mammalian
species.
The term "Fab" or "Fab region" as used herein includes the polypeptides that
comprise the
VH, CH1, VL and CL immunoglobulin domains. Fab may refer to this region in
isolation or
this region in the context of a full length antibody or antibody fragment.
The term "Fc" or "Fc region" as used herein includes the polypeptide
comprising the constant
region of an antibody excluding the first constant region immunoglobulin
domain. Thus Fc
refers to the last two constant region immunoglobulin domains of IgA, IgD and
IgG, and the
last three constant region immunoglobulin domains of IgE and IgM, and the
flexible hinge N-
terminal to these domains. For IgA and IgM, Fc may include the J chain. For
IgG, Fc
comprises immunoglobulin domains Cy2 and Cy3 and the hinge between Cyl and
Cy2.
Although the boundaries of the Fc region may vary, the human IgG heavy chain
Fc region is
usually defined to comprise residues C226 or P230 to its carboxyl-terminus,
wherein the
numbering is according to the EU numbering system (Edelman GM et at., (1969)
PNAS USA
63(1): 78-85). For human IgG1 the Fc region is herein defined to comprise
residue P232 to its
carboxyl-terminus, wherein the numbering is according to the EU numbering
system. Fc may
refer to this region in isolation or this region in the context of an Fc
polypeptide, for example
an antibody.
The term "hinge" or "hinge region" or "antibody hinge region" herein includes
the flexible
polypeptide comprising the amino acids between the first and second constant
domains of an
antibody. The "hinge region" as referred to herein is a sequence region of 6-
62 amino acids in
length, only present in IgA, IgD and IgG, which encompasses the cysteine
residues that bridge
the two heavy chains. Structurally, the IgG CH1 domain ends at EU position
220, and the IgG
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CH2 domain begins at residue EU position 237. Thus for IgG the antibody hinge
is herein
defined to include positions 221 (D221 in IgG1) to 231 (A231 in IgG1), wherein
the
numbering is according to the EU numbering system.
The terms "parent antibody", "parent immunoglobulin", "parental antibody" or
"parental
immunoglobulin", which are used equivalently herein include an unmodified
antibody that is
subsequently modified to generate a variant. Said parent antibody may be a
naturally
occurring antibody or a variant or engineered version of a naturally occurring
antibody. Parent
antibody may refer to the antibody itself, compositions that comprise the
parent antibody or
the amino acid sequence that encodes it. By "parental murine antibody" or
"corresponding
parental murine antibody" as used herein is meant an antibody or
immunoglobulin that binds
human TrkA and is modified to generate a variant, specifically the murine
antibody MNAC13
as disclosed in W000/73344.
The term "variant antibody" or "antibody variant" as used herein includes an
antibody
sequence that differs from that of a parent antibody sequence by virtue of at
least one amino
acid modification compared to the parent. The variant antibody sequence herein
will
preferably possess at least about 80%, most preferably at least about 90%,
more preferably at
least about 95% amino acid sequence identity with a parent antibody sequence.
Antibody
variant may refer to the antibody itself, compositions comprising the antibody
variant or the
amino acid sequence that encodes it.
The term "amino acid modification" herein includes an amino acid substitution,
insertion,
and/or deletion in a polypeptide sequence. By "amino acid substitution" or
"substitution"
herein is meant the replacement of an amino acid at a particular position in a
parent
polypeptide sequence with another amino acid. For example, the substitution
R94K refers to a
variant polypeptide, in this case a heavy chain variable framework region
variant, in which
the arginine at position 94 is replaced with a lysine. For the preceding
example, 94K indicates
the substitution of position 94 with a lysine. For the purposes herein,
multiple substitutions
are typically separated by a slash. For example, R94K/L78V refers to a double
variant
comprising the substitutions R94K and L78V. By "amino acid insertion" or
"insertion" as
used herein is meant the addition of an amino acid at a particular position in
a parent
polypeptide sequence. For example, insert -94 designates an insertion at
position 94. By
"amino acid deletion" or "deletion" as used herein is meant the removal of an
amino acid at a
particular position in a parent polypeptide sequence. For example, R94-
designates the
deletion of arginine at position 94.
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As used herein, the term "conservative modifications" or "conservative
sequence
modifications" is intended to refer to amino acid modifications that do not
significantly affect
or alter the binding characteristics of the antibody containing the amino acid
sequence. Such
conservative modifications include amino acid substitutions, insertions and
deletions.
5 Modifications can be introduced into an antibody of the invention by
standard techniques
known in the art, such as site-directed mutagenesis and PCR-mediated
mutagenesis.
Conservative amino acid substitutions are ones in which the amino acid residue
is replaced
with an amino acid residue having a similar side chain. Families of amino acid
residues
having similar side chains have been defined in the art. These families
include amino acids
10 with basic side chains (e.g., lysine, arginine, histidine), acidic side
chains (e.g., aspartic acid,
glutamic acid), uncharged polar side chains (e.g., glycine, asparagine,
glutamine, serine,
threonine, tyrosine, cysteine, tryptophan), non-polar side chains (e.g.,
alanine, valine, leucine,
isoleucine, proline, phenylalanine, methionine), beta-branched side chains
(e.g., threonine,
valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine,
tryptophan,
15 histidine). Thus, one or more amino acid residues within the CDR regions
or within the
framework regions of an antibody of the invention can be replaced with other
amino acid
residues from the same side chain family and the altered antibody (variant
antibody) can be
tested for retained function.
20 For all human immunoglobulin heavy chain constant domains numbering is
according to the
"EU numbering system" (Edelman GM et at., ibid.). For the human kappa
immunoglobulin
light chain constant domain (IGKC), numbering is according to the "EU
numbering system"
(Edelman GM et at., ibid.). For the human lambda immunoglobulin light chain
constant
domains (IGLC1, IGLC2, IGLC3, IGLC6, and IGLC7), numbering is according to the
"Kabat
numbering system" (Kabat EA et at., (1991) Sequences of proteins of
immunological interest.
5th Edition - US Department of Health and Human Services, NIH publication n
91-3242) as
described by Dariavach P et at., (1987) PNAS USA 84(24):9074-8 and Frangione B
et at.,
(1985) PNAS USA 82(10):3415-9.
The term "variable domain" refers to the domains that mediates antigen-binding
and defines
specificity of a particular antibody for a particular antigen. In naturally
occurring antibodies,
the antigen-binding site consists of two variable domains that define
specificity: one located
in the heavy chain, referred herein as heavy chain variable domain (VH) and
the other located
in the light chain, referred herein as light chain variable domain (VL). In
some cases,
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21
specificity may exclusively reside in only one of the two domains as in single-
domain
antibodies from heavy-chain antibodies found in camelids. The V regions are
usually about
110 amino acids long, and consist of relatively invariant stretches of amino
acid sequence
called framework regions (FRs or "non-CDR regions) of 15-30 amino acids
separated by
shorter regions of extreme variability called "hypervariable regions" that are
7-17 amino acids
long. The variable domains of native heavy and light chains comprise four FRs,
largely
adopting a beta-sheet configuration, connected by three hypervariable regions,
which form
loops. The hypervariable regions in each chain are held together in close
proximity by FRs
and, with the hypervariable regions from the other chain, contribute to the
formation of the
antigen binding site of antibodies (see Kabat EA et at., ibid.). The term
"hypervariable
region" as used herein refers to the amino acid residues of an antibody which
are responsible
for antigen binding. The hypervariable region generally comprises amino acid
residues from a
"complementary determining region" or "CDR", the latter being of highest
sequence
variability and/or involved in antigen recognition. For all variable domains
numbering is
according to Kabat (Kabat EA et at., ibid.).
A number of CDR definitions are in use and are encompassed herein. The Kabat
definition is
based on sequence variability and is the most commonly used (Kabat EA et at.,
ibid.). Chothia
refers instead to the location of the structural loops (Chothia & Lesk J.
(1987) Mol. Biol.
196:901-917). The AbM definition is a compromise between the Kabat and the
Chothia
definitions and is used by Oxford Molecular's AbM antibody modelling software
(Martin
ACR et at., (1989) PNAS USA 86:9268-9272; Martin ACR et at., (1991) Methods
Enzymol.
203:121-153; Pedersen JT et at., (1992) Immunomethods 1:126-136; Rees AR et
at., (1996)
In Sternberg M.J.E. (ed.), Protein Structure Prediction. Oxford University
Press, Oxford, 141-
172). The contact definition has been recently introduced (MacCallum RM et
at., (1996) J.
Mol. Biol. 262:732-745) and is based on an analysis of the available complex
structures
available in the Protein Databank. The definition of the CDR by IMGT , the
international
ImMunoGeneTics information system (http://www.imgt.org) is based on the IMGT
numbering for all immunoglobulin and T cell receptor V-REGIONs of all species
(IMGT ,
the international ImMunoGeneTics information system ; Lefranc MP et at.,
(1999) Nucleic
Acids Res. 27(1):209-12; Ruiz M et at., (2000) Nucleic Acids Res. 28(1):219-
21; Lefranc MP
(2001) Nucleic Acids Res. 29(1):207-9; Lefranc MP (2003) Nucleic Acids Res.
31(1):307-10;
Lefranc MP et at., (2005) Dev. Comp. Immunol. 29(3):185-203; Kaas Q et at.,
(2007)
Briefings in Functional Genomics & Proteomics, 6(4):253-64).
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All Complementarity Determining Regions (CDRs) as referred to in the present
invention, are
defined preferably as follows (numbering according to Kabat EA et at., ibid.):
LCDR1: 24-34;
LCDR2: 50-56; LCDR3: 89-98; HCDR1: 26-35; HCDR2: 50-65; HCDR3: 95-102. The
"non-
CDR regions" of the variable domain are known as framework regions (FR). The
"non-CDR
regions" of the VL region as used herein comprise the amino acid sequences: 1-
23 (FR 1), 35-
49 (FR2), 57-88 (FR3), and 99-107 (FR4). The "non-CDR regions" of the VH
region as used
herein comprise the amino acid sequences: 1-25 (FR1), 36-49 (FR2), 66-94
(FR3), and 103-
113 (FR4).
The term "full length antibody" as used herein includes the structure that
constitutes the
natural biological form of an antibody, including variable and constant
regions. For example,
in most mammals, including humans and mice, the full length antibody of the
IgG class is a
tetramer and consists of two identical pairs of two immunoglobulin chains,
each pair having
one light and one heavy chain, each light chain comprising immunoglobulin
domains VL and
CL and each heavy chain comprising immunoglobulin domains VH, CH1 (Cyl), CH2
(Cy2),
and CH3 (Cy3). In some mammals, for example in camels and llamas, IgG
antibodies may
consist of only two heavy chains, each heavy chain comprising a variable
domain attached to
the Fc region.
Antibody fragments as used herein refer to antigen-binding fragments include,
but are not
limited to, (i) the Fab fragment consisting of VL, VH, CL and CH1 domains,
including Fab'
and Fab'-SH, (ii) the Fd fragment consisting of the VH and CH1 domains, (iii)
the Fv
fragment consisting of the VL and VH domains of a single antibody; (iv) the
dAb fragment
(Ward et at. (1989) Nature 341: 544-546) which consists of a single variable
domain, (v)
F(ab')2 fragments, a bivalent fragment comprising two linked Fab fragments
(vi) single chain
Fv molecules (scFv), wherein a VH domain and a VL domain are linked by a
peptide linker
which allows the two domains to associate to form an antigen binding site
(Bird et at. (1988)
Science 242: 423-426; Huston et at. (1988) PNAS USA 85: 5879-5883), (vii)
bispecific
single chain Fv dimers (PCT/1J592/09965), (viii) "diabodies" or "triabodies",
multivalent or
multispecific fragments constructed by gene fusion (Tomlinson I et at., (2000)
Methods
Enzymol. 326:461-479; W094/13804; Holliger et at., (1993) PNAS USA 90:6444-
6448) and
(ix) scFv genetically fused to the same or a different antibody (Coloma &
Morrison (1997)
Nature Biotech. 15:159-163).
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The term "effector function" as used herein includes a biochemical event that
results from the
interaction of an antibody Fc region with an Fc receptor or ligand. Effector
functions include
FcyR-mediated effector functions such as ADCC (antibody dependent cell-
mediated
cytotoxicity) and ADCP (antibody dependent cell-mediated phagocytosis), and
complement-
mediated effector functions such as CDC (complement dependent cytotoxicity).
An effector
function of an antibody may be altered by altering, i.e. enhancing or
reducing, preferably
enhancing, the affinity of the antibody for an effector molecule such as an Fc
receptor or a
complement component. Binding affinity will generally be varied by modifying
the effector
molecule binding site and in this case it is appropriate to locate the site of
interest and modify
at least part of the site in a suitable way. It is also envisaged that an
alteration in the binding
site on the antibody for the effector molecule need not alter significantly
the overall binding
affinity but may alter the geometry of the interaction rendering the effector
mechanism
ineffective as in non-productive binding. It is further envisaged that an
effector function may
also be altered by modifying a site not directly involved in effector molecule
binding, but
otherwise involved in performance of the effector function. By altering an
effector function of
an antibody it may be possible to control various aspects of the immune
response, eg
enhancing or suppressing various reactions of the immune system, with possible
beneficial
effects in diagnosis and therapy.
As used herein, the term "subject" includes any human or non-human animal. The
term "non-
human animal" includes all vertebrates, e.g., mammals and non-mammals, such as
primates,
sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc.
Preferably the subject is
human.
Antibodies of the invention
In a first aspect the present invention provides a humanized anti-TrkA
antibody or fragment
thereof comprising:
a) a heavy chain variable domain comprising a sequence selected from the group
consisting of
SEQ ID NOs: 1-5, and
b) a light chain variable domain comprising a sequence selected from the group
consisting of
SEQ ID NOs: 6-13,
wherein CDR2 of the heavy chain variable domain comprises at least one amino
acid
substitution and/or wherein the non-CDR region of the heavy chain variable
domain
comprises an amino acid substitution at an amino acid position selected from
the group
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consisting of 37, 42 and 89, wherein the amino acid position of each group
member is
indicated utilizing the numbering system set forth in Kabat.
In some embodiments, the CDR2 of the heavy chain variable domain comprises at
least one
conservative amino acid substitution.
In some embodiments, the non-CDR region of the heavy chain variable domain
comprises a
conservative amino acid substitution at an amino acid position selected from
the group
consisting of 37, 42 and 89.
In some embodiments, the CDR2 of the heavy chain variable domain comprises the
sequence
of SEQ ID NO: 15.
In some embodiments, the heavy chain variable domain comprises a sequence
selected from
the group consisting of SEQ ID NOs: 1, 3 and 5, and the light chain variable
domain
comprises a sequence selected from the group consisting of SEQ ID NOs: 6 and
8.
In some embodiments, the humanized anti-TrkA antibody or fragment thereof
comprises a
combination of a heavy chain variable domain and a light chain variable domain
comprising
the sequences selected from the group consisting of SEQ ID NO: 1 and SEQ ID
NO: 6, SEQ
ID NO: 3 and SEQ ID NO: 6, SEQ ID NO: 3 and SEQ ID NO: 8, SEQ ID NO: 5 and SEQ
ID
NO: 6, and SEQ ID NO: 5 and SEQ ID NO: 8; preferably the sequences of SEQ ID
NO: 5 and
SEQ ID NO: 6 or the sequences of SEQ ID NO: 5 and SEQ ID NO: 8; more
preferably the
sequences of SEQ ID NO: 5 and SEQ ID NO: 6.
In some embodiments, the heavy chain variable domain of the humanized anti-
TrkA antibody
or fragment thereof provided by the present disclosure does not comprise the
sequence of
SEQ ID NO: 71.
In some embodiments, the heavy chain variable domain of the humanized anti-
TrkA antibody
or fragment thereof provided by the present disclosure lacks a serine at
position 87, wherein
the amino acid position of each group member is indicated utilizing the
numbering system set
forth in Kabat.
In some embodiments, the heavy chain variable domain of the humanized anti-
TrkA antibody
or fragment thereof provided by the present disclosure comprises a threonine
at position 87,
wherein the amino acid position of each group member is indicated utilizing
the numbering
system set forth in Kabat.
In some embodiments, the amino acid substitution of the CDR2 of the heavy
chain variable
domain comprises an amino acid substitution at an amino acid position selected
from the
group consisting of 50, 60 and 62, preferably selected from the group
consisting of 60 and 62,
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wherein the amino acid position of each group member is indicated utilizing
the numbering
system set forth in Kabat.
In some embodiments, the amino acid substitution of the CDR2 of the heavy
chain variable
domain does not comprise an amino acid substitution selected from the group
consisting of
5 Y50A, P60A and T62S, wherein the amino acid position of each group member
is indicated
utilizing the numbering system set forth in Kabat.
In some embodiments, if the amino acid substitution of the humanized anti-TrkA
antibody or
fragment in the non-CDR region of the heavy chain variable domain is A49S, the
amino acid
substitution of the humanized anti-TrkA antibody or fragment in the CDR2 of
the heavy chain
10 variable domain is not Y50A.
In some embodiments, the amino acid substitution of the humanized anti-TrkA
antibody or
fragment in the non-CDR region of the heavy chain variable domain is not A49S
and/or the
amino acid substitution of the humanized anti-TrkA antibody or fragment in the
CDR2 of the
heavy chain variable domain is not Y50A.
15 In some embodiments, the amino acid substitution of the non-CDR region
of the heavy chain
variable domain of the antibody or the fragment thereof comprises an amino
acid substitution
selected from the group consisting of V37A, G42E and V89L, preferably V37A,
wherein the
amino acid position is indicated utilizing the numbering system set forth in
Kabat.
In some embodiments, the antibody comprises a heavy chain variable domain
comprising the
20 sequence of SEQ ID NO: 3, wherein the amino acid substitution of the non-
CDR region of the
heavy chain variable domain comprises an amino acid substitution selected from
the group
consisting of V37A, T40A, G42E, R44G, A495 and V89L, preferably selected from
the group
consisting of V37A, T40A, G42E, R44G and V89L wherein the amino acid position
of each
group member is indicated utilizing the numbering system set forth in Kabat.
25 In some embodiments, the antibody comprises a combination of a heavy
chain variable
domain and a light chain variable domain comprising the sequences of SEQ ID
NO: 3 and
SEQ ID NO: 6 or comprising the sequences of SEQ ID NO: 3 and SEQ ID NO: 8,
wherein
the amino acid substitution of the non-CDR region of the heavy chain variable
domain
comprises an amino acid substitution selected from the group consisting of
V37A, T40A,
G42E, R44G, A495 and V89L, preferably selected from the group consisting of
V37A, T40A,
G42E, R44G and V89L, wherein the amino acid position of each group member is
indicated
utilizing the numbering system set forth in Kabat.
In some embodiments, the amino acid substitution of the non-CDR region of the
heavy chain
variable domain comprises an amino acid substitution selected from the group
consisting of
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K3Q, V37A, G42E, A49S, V89L and R94K, preferably selected from the group
consisting of
K3Q, V37A, G42E, V89L and R94K, more preferably comprises the amino acid
substitutions
K3Q and V37A and most preferably comprises the amino acid substitution V37A,
wherein
the amino acid position of each group member is indicated utilizing the
numbering system set
forth in Kabat. Equally most preferred are embodiments wherein the amino acid
substitution
of the non-CDR region of the heavy chain variable domain comprises an amino
acid
substitution selected from the group consisting of V37A and K3Q, V37A and
wherein the
amino acid position of each group member is indicated utilizing the numbering
system set
forth in Kabat.
In some embodiments, the antibody comprises a heavy chain variable domain
comprising the
sequence of SEQ ID NO: 5, wherein the amino acid substitution of the non-CDR
region of the
heavy chain variable domain comprises an amino acid substitution selected from
the group
consisting of K3Q, V37A, G42E, A495, V89L and R94K, preferably selected from
the group
consisting of K3Q, V37A, G42E, V89L and R94K, more preferably comprises the
amino
acid substitutions K3Q and V37A and most preferably comprises the amino acid
substitution
V37A, wherein the amino acid position of each group member is indicated
utilizing the
numbering system set forth in Kabat. Equally most preferred are embodiments
wherein the
antibody comprises a heavy chain variable domain comprising the sequence of
SEQ ID NO:
5, wherein the amino acid substitution of the non-CDR region of the heavy
chain variable
domain comprises an amino acid substitution selected from the group consisting
of V37A and
K3Q, V37A and wherein the amino acid position of each group member is
indicated utilizing
the numbering system set forth in Kabat.
In a further aspect the present invention provides a humanized anti-TrkA
antibody or
fragment thereof comprising:
a) a heavy chain variable domain comprising a sequence selected from the group
consisting of
SEQ ID NOs: 31-49, and
b) a light chain variable domain comprising a sequence selected from the group
consisting of
SEQ ID NOs: 6-13.
In some embodiments the humanized anti-TrkA antibody or fragment thereof
comprises:
a) a heavy chain variable domain comprising a sequence selected from the group
consisting of
SEQ ID NOs: 32, 36, 39, 43, 48 and 49, and
b) a light chain variable domain comprising a sequence selected from the group
consisting of
SEQ ID NOs: 6-13 or selected from the group consisting of SEQ ID NOs: 6 and 8.
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In some embodiments the humanized anti-TrkA antibody or fragment thereof
comprises:
a) a heavy chain variable domain comprising a sequence selected from the group
consisting of
SEQ ID NOs: 32, 36, 48 and 49 and
b) a light chain variable domain comprising a sequence of SEQ ID NO: 6 or SEQ
ID NO: 8.
In some embodiments the humanized anti-TrkA antibody or fragment thereof
comprises
a) a heavy chain variable domain comprising a sequence selected from the group
consisting of
SEQ ID NOs: 32 and 36, and
b) a light chain variable domain comprising the sequence of SEQ ID NO: 6.
In some embodiments the humanized anti-TrkA antibody or fragment thereof
comprises
a) a heavy chain variable domain comprising the sequence of SEQ ID NOs: 36,
and
b) a light chain variable domain comprising the sequence of SEQ ID NO: 6.
In some embodiments, the humanized anti-TrkA antibody comprises a combination
of a
heavy chain variable domain and a light chain variable domain selected from
the group
comprising the sequences of SEQ ID NO: 32 and SEQ ID NO: 6, SEQ ID NO: 32 and
SEQ
ID NO: 8, SEQ ID NO: 36 and SEQ ID NO: 6, SEQ ID NO: 48 and SEQ ID NO: 6, SEQ
ID
NO: 49 and SEQ ID NO: 6, and SEQ ID NO: 49 and SEQ ID NO: 8, preferably
selected from
the group comprising the sequences of SEQ ID NO: 32 and SEQ ID NO: 6, SEQ ID
NO: 32
and SEQ ID NO: 8, SEQ ID NO: 36 and SEQ ID NO: 6, SEQ ID NO: 49 and SEQ ID NO:
6,
and SEQ ID NO: 49 and SEQ ID NO: 8, most preferably selected from the
combination of
sequences of SEQ ID NO: 36 and SEQ ID NO: 6.
In some embodiments the humanized anti-TrkA antibody or fragment thereof
further
comprises heavy and/or light constant regions, preferably heavy and/or light
constant regions
and a hinge region. Preferably the heavy constant regions are of human origin
and are e.g. of
human IgG1 (IGHG1), IgG2 (IGHG2), IgG3 (IGHG3), IgG4 (IGHG4), IgAl (IGHA1),
IgA2
(IGHA2), IgM (IGHM), IgD (IGHD), or IgE (IGHE) isotype. More preferably the
heavy
constant regions are of human IGHG1 isotype or are of human IGHG4 isotype.
Preferably the
light constant regions are of human origin and are human kappa (CK) or human
lambda (Ck)
light constant domains, preferably a human kappa light constant domain.
In some embodiments the humanized anti-TrkA antibody or fragment thereof
further
comprises heavy and/or light constant regions and a hinge region, wherein the
heavy constant
region and the hinge region are of human IGHG1 isotype or are of human IGHG4
isotype.
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In some embodiments the humanized anti-TrkA antibody or fragment thereof
further
comprises heavy and/or light constant regions and a hinge region, wherein the
heavy constant
region and the hinge region are of human IGHG4 isotype and wherein the hinge
region
comprises amino acid substitution S228P, wherein the amino acid position is
indicated
utilizing the EU numbering system.
In a further aspect the present invention provides a humanized anti-TrkA
antibody or
fragment thereof comprising:
a) a heavy chain comprising a sequence selected from the group consisting of
SEQ ID NOs:
50 to 70, and
b) a light chain comprising a sequence selected from the group consisting of
SEQ ID NOs: 29
and 30.
In some embodiments the humanized anti-TrkA antibody or fragment thereof
comprises
a) a heavy chain comprising a sequence selected from the group consisting of
SEQ ID NOs:
51, 52, 56, 57, 60, 64, 69 and 70, and
b) a light chain comprising a sequence selected from the group consisting of
SEQ ID NOs: 29
and 30, preferably SEQ ID NO: 29.
In some embodiments the humanized anti-TrkA antibody or fragment thereof
comprises
a) a heavy chain comprising a sequence selected from the group consisting of
SEQ ID NOs:
51, 52, 56, 57, 69 and 70, and
b) a light chain comprising a sequence selected from the group consisting of
SEQ ID NOs: 29
and 30, preferably SEQ ID NO: 29.
In some embodiments the humanized anti-TrkA antibody or fragment thereof
comprises
a) a heavy chain comprising a sequence selected from the group consisting of
SEQ ID NOs:
51, 52, 56, 57, and 70, and
b) a light chain comprising a sequence selected from the group consisting of
SEQ ID NOs: 29
and 30, preferably SEQ ID NO: 29.
In some embodiments the humanized anti-TrkA antibody or fragment thereof
comprises
a) a heavy chain comprising a sequence selected from the group consisting of
SEQ ID NOs:
51, 52, 56, and 57, and
b) a light chain comprising a sequence selected from the group consisting of
SEQ ID NOs: 29
and 30, preferably SEQ ID NO: 29.
In a preferred embodiment, the humanized anti-TrkA antibody or fragment
thereof
comprises
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a) a heavy chain comprising the sequence of SEQ ID NO: 57, and
b) a light chain comprising the sequence of SEQ ID NO: 29.
In some embodiments the humanized anti-TrkA antibody or fragment thereof is a
full length
antibody.
In some embodiments the humanized anti-TrkA antibody or fragment thereof is an
antibody
fragment selected from the group consisting of Fab, Fab', Fab'-SH, Fd, Fv,
dAb, F(ab')2,
scFv, bispecific single chain Fv dimers, diabodies, triabodies and scFv
genetically fused to the
same or a different antibody; preferably a scFv, or a Fab; more preferably a
scFv dimer or a
diabody or a F(ab)2.
In some embodiments the humanized anti-TrkA antibody or fragment thereof
comprises a
variant Fc region which comprises at least one amino acid modification
relative to the Fc
region of the parent antibody, whereas the antibody comprising the variant Fc
region exhibits
altered effector function compared to the parent antibody.
Amino acid modification within the Fc region typically alter one or more
functional properties
of the antibody, such as serum half-life, complement fixation, effector
function related to Fc
receptor or ligand binding, and/or antigen-dependent cellular cytotoxicity.
Modifications
within the Fc region as outlined below are according to the EU numbering of
residues in the
Fc region. In one embodiment, the hinge region of CH1 is modified such that
the number of
cysteine residues in the hinge region is altered, e.g., increased or
decreased. This approach is
described further in U.S. Patent No. 5,677,425 by Bodmer et at. The number of
cysteine
residues in the hinge region of CH1 is altered to, for example, facilitate
assembly of the light
and heavy chains or to increase or decrease the stability of the antibody. In
another
embodiment, the Fc hinge region of an antibody is mutated to decrease the
biological half life
of the antibody. More specifically, one or more amino acid mutations are
introduced into the
CH2-CH3 domain interface region of the Fc-hinge fragment such that the
antibody has
impaired Staphylococcyl protein A (SpA) binding relative to native Fc-hinge
domain SpA
binding. This approach is described in further detail in U.S. Patent No.
6,165,745 by Ward et
at. In another embodiment, the antibody is modified to increase its biological
half life.
Various approaches are possible. For example, one or more of the following
mutations can be
introduced: T252L, T2545, T256F, as described in U.S. Patent No. 6,277,375 to
Ward.
Alternatively, to increase the biological half life, the antibody can be
altered within the CH1
or CL region to contain a salvage receptor binding epitope taken from two
loops of a CH2
domain of an Fc region of an IgG, as described in U.S. Patent Nos. 5,869,046
and 6,121,022
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by Presta et at. In a further embodiment, an Fe region is altered by replacing
at least one
amino acid residue with a different amino acid residue to alter the effector
function(s) of the
antibody. For example, one or more amino acids selected from amino acid
residues 234, 235,
236, 237, 297, 318, 320 and 322 can be replaced with a different amino acid
residue such that
5 the antibody has an altered affinity for an effector ligand but retains
the antigen- binding
ability of the parent antibody. The effector ligand to which affinity is
altered can be, for
example, an Fe receptor or the Cl component of complement. This approach is
described in
further detail in U.S. Patent Nos. 5,624,821 and 5,648,260, both by Winter et
at. In another
example, one or more amino acids selected from amino acid residues 329, 331
and 322 can be
10 replaced with a different amino acid residue such that the antibody has
altered Cl q binding
and/or reduced or abolished complement dependent cytotoxicity (CDC). This
approach is
described in further detail in U.S. Patent Nos. 6,194,551 by Idusogie et at.
In another
example, one or more amino acid residues within amino acid positions 231 to
238 in the N-
terminal region of the CH2 domain are altered to thereby alter the ability of
the antibody to fix
15 complement. This approach is described further in PCT Publication WO
94/29351 by Bodmer
et at. In yet another example, the Fe region is modified to increase the
ability of the antibody
to mediate antibody dependent cellular cytotoxicity (ADCC) and/or to increase
the affinity of
the antibody for an Fey receptor by modifying one or more amino acids at the
following
positions: 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269,
270, 272, 276,
20 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 301,
303, 305, 307, 309, 312,
315, 320, 322, 324, 326, 327, 329, 330, 331, 333, 334, 335, 337, 338, 340,
360, 373, 376, 378,
382, 388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438 or 439. This
approach is described
further in PCT Publication WO 00/42072 by Presta. Furthermore, an antibody of
the invention
may be chemically modified (e.g., one or more chemical moieties can be
attached to the
25 antibody) or be modified to alter its glycosylation.
Properties of the antibodies of the invention
In some embodiments the humanized anti-TrkA antibody or fragment thereof is
capable of
inhibiting the functional activation of TrkA.
30 In some embodiments the humanized anti-TrkA antibody is capable of
blocking or reducing
one or more biological activities of TrkA.
In some embodiments the humanized anti-TrkA antibody or fragment thereof binds
to human
TrkA with an affinity (KD) of 500 nM or less, preferably 350nM or less, more
preferably 150
nM or less, even more preferably 100 nM or less, most preferably 50 nM or
less, in particular
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30 nM or less e.g. measured by Surface Plasmon Resonance (SPR) using a BIAcore
2000
instrument (GE Healthcare Europe GmbH, Glattbrugg, Switzerland) or equivalent
instrument
known in the art by capturing the antibody on the instrument sensor chip with
a recombinant
monovalent human TrkA extracellular domain (SEQ ID NO: 25) used as analyte.
"Monovalent" as used herein in relation to affinity measurements using TrkA
receptor refers
to a human TrkA receptor domain, like the human TrkA extracellular domain
which is not
artificially dimerized or multimerized as it would be e.g. if the domain would
be amino-
terminally fused to an immunoglobulin Fc portion, or which is not naturally
dimerized as it
would be e.g. if the domain would be associated with its natural ligand NGF.
Standard assays to evaluate the binding ability of the antibodies toward e.g.
human TrkA are
known in the art, including for example, ELISAs, BIAcore, Western blots, RIAs
and flow
cytometry analysis. Suitable assays are described in detail in the Examples.
The binding
kinetics (e.g. binding affinity like KD) of the antibodies also can be
assessed by standard
assays known in the art, such as by Scatchard or Biacore system analysis. The
relative
binding affinity Ki can be assessed by standard competition assays known in
the art.
Engineered anti-TrkA antibodies can be assayed for their ability to inhibit
the functional
activation of TrkA in TF-1 cell proliferation assays. The half maximal
inhibitory
concentration (IC50) which is a measure of the effectiveness of a compound in
inhibiting
biological function can be used to select preferred antibodies.
In some embodiments the humanized anti-TrkA antibody or fragment thereof has
at least an
equivalent or lower IC50 in a TF-1 cell proliferation assay than the
corresponding parental
murine antibody e.g. measured by the ability of the antibody to block cell
surface TrkA/beta-
NGF mediated cell proliferation using the factor dependent human
erythroleukemic cell line
TF-1 (Kitamura T et at., (1989) J. Cellular Physiology 140(2):323-34).
Preferably the
humanized anti-TrkA antibody or fragment thereof has an IC50 in a TF-1 cell
proliferation
assay of 1 jig/ml or less, more preferably of 0.75 jig/ml, even more
preferably of 0.5 jig/ml or
less, most preferably of 0.3 jig/ml or less, in particular of 0.1 jig/ml or
less.
In some embodiments the humanized anti-TrkA antibody or fragment thereof has a
FAB
fragment thermostability temperature greater than 65 C, preferably greater
than 70 C. For
analysis of FAB fragment thermostability differential scanning calorimetry
measurements are
used, whereas a mid-point melting temperature of the FAB fragment in context
of a full-
length IgG is identified. These kind of calorimetric measurements are known to
the skilled
person and can be carried out according to e.g. Garber & Demarest (2007) BBRC
355:751-7.
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Surprisingly, it has been found that the humanized antibody of the present
invention has a
FAB fragment thermostability temperature equivalent to the FAB fragment
thermostability
temperature of the parental murine antibody while having equivalent affinity
as measured by
SPR and improved potency as measured by TF-1 cell proliferation assay. Thus
the present
disclosure also provides a humanized anti-TrkA antibody which has a FAB
fragment
thermostability temperature which is equivalent to the FAB fragment
thermostability
temperature of the parental murine antibody with an equivalent affinity for
human TrkA and
improved inhibitory properties.
"Equivalent to the FAB fragment thermostability temperature of the parental
murine
antibody" as used herein in this context means that the humanized anti-TrkA
antibody or
fragment thereof has a FAB fragment thermostability temperature which is
within a range of
20%, preferably within a range of 15%, more preferably within a range of
10%, even
more preferably within a range of 5% of the FAB fragment thermostability
temperature of
the parental murine antibody. Preferably, the humanized antibody of the
present invention has
a FAB fragment thermostability temperature not more than 15% lower than the
FAB fragment
thermostability temperature of the parental murine antibody.
"Equivalent affinity for human TrkA" as used herein in this context means that
the humanized
anti-TrkA antibody or fragment thereof has an affinity which is within a range
of 20%,
preferably within a range of 15%, more preferably within a range of 10% of
the parental
murine antibody. Preferably, the humanized antibody of the present invention
has a KD which
is at least 5%, preferably at least 10% lower than the KD of the parental
murine antibody.
The present invention also provides a humanized anti-TrkA antibody or fragment
thereof
which can be used to treat pain.
The effect of a humanized anti-TrkA antibody was tested in mice suffering from
acute
inflammatory hyperalgesia induced by intraplantar injection of Complete
Freunds adjuvant
(CFA) into the hind paw (see Example 2). Administration of the humanized anti-
TrkA
antibody at a dose of 0.01mg/kg or above produced a significant reversal of
hyperalgesia,
which was similar to that observed with the NSAID indomethacin.
The effect of a humanized anti-TrkA antibody was tested in mice suffering from
chronic
inflammatory hyperalgesia induced by intra-articular injection of CFA into the
knee joint (see
Example 3). Administration of the humanized anti-TrkA antibody at a single
dose of 0.01
mg/kg or above produced a significant reversal of hyperalgesia, which was
comparable to that
observed with multiple dosing of the COX-2 selective NSAID celecoxib.
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The effect of a humanized anti-TrkA antibody was tested in mice suffering from
chronic
osteoarthritic hyperalgesia induced by intra-articular injection of monosodium
iodoacetate
(MIA) into the knee joint (see Example 4). Administration of the humanized
anti-TrkA
antibody at a single dose of 0.01 mg/kg or above produced a significant
reversal of
hyperalgesia, which was comparable to that observed with multiple dosing of
the opiate
tramadol and pregabalin.
The effect of a humanized anti-TrkA antibody was tested in mice suffering from
neuropathic
pain induced by chronic constriction of the sciatic nerve (CCI model; see
Example 5).
Administration of the humanized anti-TrkA antibody at a single dose of 0.01
mg/kg or above
produced a significant reversal of mechanical hyperalgesia and cold allodynia,
which at the
highest dose tested, 1 mg/kg, was comparable to that observed with multiple
dosing of
pregabalin.
Therefore, a preferred embodiment of the present invention provides a
humanized
anti-TrkA antibody for the treatment of a patient suffering from acute
inflammatory pain,
chronic inflammatory pain, osteoarthritic pain and/or neuropathic pain.
Nucleic acids, Vectors and Host Cells
The present disclosure also provides isolated nucleic acids encoding the anti-
TrkA antibodies
and fragments thereof, vectors, and host cells comprising the nucleic acid or
the vector. The
nucleic acids may be present in whole cells, in a cell lysate, or in a
partially purified or
substantially pure form. A nucleic acid is "isolated" or "rendered
substantially pure" when
purified away from other cellular components or other contaminants, e.g.,
other cellular
nucleic acids or proteins, by standard techniques, including alkaline/SDS
treatment, CsC1
banding, column chromatography, agarose gel electrophoresis and others well
known in the
art, see e.g. Ausubel F et at., ed. (1987) Current Protocols in Molecular
Biology, Greene
Publishing and Wiley Interscience, New York. A nucleic acid of the invention
can be, for
example, DNA or RNA and may or may not contain intronic sequences. In a
preferred
embodiment, the nucleic acid is a cDNA molecule.
Nucleic acids of the invention can be obtained using standard molecular
biology techniques
e.g. cDNAs encoding the light and heavy chains of the antibody or encoding VH
and VL
segments can be obtained by standard PCR amplification or cDNA cloning
techniques. For
antibodies obtained from an immunoglobulin gene library (e.g., using phage
display
techniques), one or more nucleic acids encoding the antibody can be recovered
from the
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library. The methods of introducing exogenous nucleic acid into host cells are
well known in
the art and will vary with the host cell used. Techniques include but are not
limited to dextran-
mediated transfection, calcium phosphate precipitation, calcium chloride
treatment,
polyethylenimine mediated transfection, polybrene mediated transfection,
protoplast fusion,
electroporation, viral or phage infection, encapsulation of the
polynucleotide(s) in liposomes,
and direct microinjection of the DNA into nuclei. In the case of mammalian
cells, transfection
may be either transient or stable.
In some embodiments the isolated nucleic acids encoding the anti-TrkA
antibodies and
fragments thereof comprise a nucleic acid sequence selected from the group
consisting of
SEQ ID NOs 73-116, usually nucleic acids molecules encoding the light chain
variable region
or the light chain selected from the group consisting of SEQ ID NOs: 113, 114,
115 and 116
and/or the heavy chain variable region or the heavy chain selected from the
group consisting
of SEQ ID NOs: 73-112.
Preferred nucleic acids molecules of the invention are those encoding the
light chain variable
region selected from the group consisting of SEQ ID NOs: 113 and 114 and/or
the heavy
chain variable region selected from the group consisting of SEQ ID NOs: 74,
78, 81, 85, 90
and 91. More preferred are nucleic acids molecules encoding the heavy chain
variable region
selected from the group consisting of SEQ ID NOs: 74 and 78 and/or encoding
the light chain
variable region selected from the group consisting of SEQ ID NOs: 113 and 114.
Most
preferred are nucleic acids molecules encoding the heavy chain variable region
comprising
the nucleic acid sequence of SEQ ID NO: 74 or 78 and/or encoding the light
chain variable
region comprising the nucleic acid sequence of SEQ ID NO: 113.
Further preferred nucleic acids molecules of the invention are those encoding
the light chain
selected from the group consisting of SEQ ID NO: 115 and 116 and/or the heavy
chain
selected from the group consisting of SEQ ID NO: 93, 94, 98, 99, 102, 106, 111
and 112.
More preferred are nucleic acids molecules encoding the heavy chain comprising
the nucleic
acid sequence of SEQ ID NO: 93, 94, 98, and 99 and/or encoding the light chain
selected
from the group consisting of of SEQ ID NO: 115 and 116. Most preferred are
nucleic acids
molecules encoding the heavy chain comprising the nucleic acid sequence of SEQ
ID NO: 93,
94, 98, and 99 and/or encoding the light chain comprising the nucleic acid
sequence of SEQ
ID NO: 115.
Once DNA fragments encoding VH and VL segments are obtained, these DNA
fragments can
be further manipulated by standard recombinant DNA techniques, for example to
convert the
variable region genes to full-length antibody chain genes, or to fragment
genes corresponding
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to the fragments described above like e.g. Fab fragment genes or to a scFv
gene. In these
manipulations, a VL- or VH-encoding DNA fragment is operatively linked to
another DNA
fragment encoding another protein, such as an antibody constant region or a
flexible linker.
The term "operatively linked", as used in this context, is intended to mean
that the two DNA
5 fragments are joined such that the amino acid sequences encoded by the
two DNA fragments
remain in-frame. The isolated DNA encoding the VH region can be converted to a
full-length
heavy chain gene by operatively linking the VH-encoding DNA to another DNA
molecule
encoding heavy chain constant regions (CH1, CH2 and CH3). The sequences of
human heavy
chain constant region genes are known in the art (see e.g., Kabat, EA et at.,
ibid.) and DNA
10 fragments encompassing these regions can be obtained by standard PCR
amplification. The
heavy chain constant region can be an IgGl, IgG2, IgG3, IgG4, IgA, IgE, IgM or
IgD
constant region, but most preferably is an IgG4 constant region, preferably a
human IGHG4
constant region wherein the hinge region comprises amino acid substitution
S228P. For a Fab
fragment heavy chain gene, the VH encoding DNA can be operatively linked to
another DNA
15 molecule encoding only the heavy chain CH1 constant region. The isolated
DNA encoding
the VL region can be converted to a full-length light chain gene (as well as a
Fab light chain
gene) by operatively linking the VL-encoding DNA to another DNA molecule
encoding the
light chain constant region, CL. The sequences of human light chain constant
region genes are
known in the art (see e.g., Kabat EA et at., ibid.) and DNA fragments
encompassing these
20 regions can be obtained by standard PCR amplification. In preferred
embodiments, the light
chain constant region can be a kappa or lambda constant region, preferably a
kappa constant
region. To create a scFv gene, the VH- and VL-encoding DNA fragments are
operatively
linked to another fragment encoding a flexible linker, e.g., encoding the
amino acid sequence
(G1y4 -Ser)3, such that the VH and VL sequences can be expressed as a
contiguous single-
25 chain protein, with the VL and VH regions joined by the flexible linker
(see e.g., Bird et at.,
ibid.; Huston et at., ibid.; McCafferty et at., (1990) Nature 348: 552-554).
Various techniques
have been developed for the production of antibody fragments of antibodies.
Traditionally,
these fragments were derived via proteolytic digestion of intact antibodies
(see e.g. Morimoto
et at., (1992) J. Biochem. Biophysical Methods, 24: 107-117 and Brennan et
at., (1985)
30 Science, 229:81). However, these fragments can now be produced directly
by recombinant
host cells. For example, the antibody fragments can be isolated from the
antibody phage
libraries discussed above. Alternatively, Fab'-SH fragments can be directly
recovered from E.
coli and chemically coupled to form F(ab')2 fragments (Carter et at., (1992)
Bio/Technology,
10:163-167). According to another approach, F(ab')2 fragments can be isolated
directly from
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recombinant host cell culture. Other techniques for the production of antibody
fragments will
be apparent to the skilled practitioner. In other embodiments, the antibody of
choice is a
single-chain Fv fragment (scFv), see e.g. W093/16185; US Patent Nos. 5,571,894
and
5,587,458. The antibody fragment may also be a "linear antibody", e.g., as
described in US
Patent No. 5,641,870, for example.
The nucleic acids that encode the antibodies of the present invention may be
incorporated into
a vector, preferably an expression vector in order to express the protein. A
variety of
expression vectors may be utilized for protein expression. Expression vectors
may comprise
self-replicating extra- chromosomal vectors or vectors which integrate into a
host genome.
Expression vectors are constructed to be compatible with the host cell type.
Thus vectors,
preferably expression vectors, which find use in the present invention include
but are not
limited to those which enable protein expression in mammalian cells, bacteria,
insect cells,
yeast, and in in vitro systems. As is known in the art, a variety of
expression vectors are
available, commercially or otherwise, that may find use in the present
invention for
expressing antibodies.
Expression vectors typically comprise a protein operably linked with control
or regulatory
sequences, selectable markers, any fusion partners, and/or additional
elements. By "operably
linked" herein is meant that the nucleic acid is placed into a functional
relationship with
another nucleic acid sequence. The term "regulatory sequence" is intended to
include
promoters, enhancers and other expression control elements (e.g.,
polyadenylation signals)
that control the transcription or translation of the antibody chain genes.
Such regulatory
sequences are described, for example, in Goeddel (Gene Expression Technology,
Methods in
Enzymology 185, Academic Press, San Diego, CA (1990)). Generally, these
expression
vectors include transcriptional and translational regulatory nucleic acid
operably linked to the
nucleic acid encoding the antibody, and are typically appropriate to the host
cell used to
express the protein. In general, the transcriptional and translational
regulatory sequences may
include promoter sequences, ribosomal binding sites, transcriptional start and
stop sequences,
translational start and stop sequences, and enhancer or activator sequences.
As is also known
in the art, expression vectors typically contain a selection gene or marker to
allow the
selection of transformed host cells containing the expression vector.
Selection genes are well
known in the art and will vary with the host cell used. For example, typically
the selectable
marker gene confers resistance to drugs, such as G418, hygromycin or
methotrexate, on a host
cell into which the vector has been introduced. Preferred selectable marker
genes include the
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dihydrofo late reductase (DHFR) gene (for use in dhfr- host cells with
methotrexate
selection/amplification) and the neo gene (for G418 selection).
Suitable host cells for cloning or expressing the DNA in the vectors herein
are prokaryote,
yeast, or higher eukaryote cells. Suitable prokaryotes for this purpose
include eubacteria,
including gram-negative or gram-positive organisms, for example,
Enterobacteriaceae such
as Escherichia, e.g., E. coli, Enterobacter, 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, Pseudomonas such as P. aeruginosa and
Streptomyces. Suitable
E. coli cloning hosts include E. coli 294 (ATCC 31,446), E. coli B, E. coli
X1776 (ATCC
31,537), and E. coli W3110 (ATCC 27,325).
In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or
yeast are suitable
cloning or expression hosts. Saccharomyces cerevisiae or common baker's yeast
is the most
commonly used among lower eukaryotic host microorganisms. Host cells for
expressing the
recombinant antibodies of the invention are preferably mammalian host cells
which include
Chinese Hamster Ovary (CHO cells) (including dhfr- CHO cells, described in
Urlaub &
Chasin (1980) PNAS USA 77:4216-4220, used with a DHFR selectable marker, e.g.,
as
described in Kaufman & Sharp (1982) J. Mol. Biol. 159:601-621), NSO myeloma
cells, COS
cells , 5P2 cells and HEK293-EBNA1 cells (ATCC catalogue number: CRL-10852).
In
particular, for use with NSO myeloma cells, another preferred expression
system is the GS
gene expression system disclosed in W087/04462, W089/01036 and EP0338841.
Construction and Production of Antibodies
Antibodies generated against the TrkA polypeptide may be obtained by
immunisation of an
animal i.e. by administering the polypeptides to an animal, preferably a non-
human animal,
using well-known and routine protocols, see for example Handbook of
Experimental
Immunology, Weir DM (ed.), Vol 4, Blackwell Scientific Publishers, Oxford,
England,
(1986). Many warm-blooded animals, such as rabbits, mice, rats, sheep, cows,
camels or pigs
may be immunized. However, mice, rabbits, pigs and rats in particular mice are
generally
most suitable. Antibodies can be produced as well by recombinant DNA
techniques known to
the skilled person. In additional antibodies can be produced by enzymatic or
chemical
cleavage of naturally occurring antibodies. Humanized antibodies of the
present invention
may be constructed by transferring one or more CDRs or portions thereof from
VH and/or VL
regions from a non-human animal (e.g., mouse) to one or more framework regions
from
human VH and/or VL regions. Preferably the humanized antibodies of the present
invention
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are constructed by transferring one or more CDRs or portions thereof from VH
and/or VL
regions from murine MNAC13 antibody as disclosed in W000/73344 to one or more
framework regions from human VH and/or VL regions. Optionally, human framework
residues thus present in the VH and/or VL regions may be replaced by
corresponding non-
human (e.g., mouse) residues when needed or desired for decreasing
immunogenicity of the
antibody and/or maintaining binding affinity. Optionally, non-human amino acid
residues
present in the CDRs may be replaced with human residues. Chimeric or humanized
antibodies
of the present invention can be prepared based on the sequence of a non-human
monoclonal
antibody prepared as described above. DNA encoding the heavy and light chain
immunoglobulins can be obtained from the non- human hybridoma of interest and
engineered
to contain non-murine (e.g., human) immunoglobulin sequences using standard
molecular
biology techniques. For example, to create a chimeric antibody, murine
variable regions can
be linked to human constant regions using methods known in the art (see e.g.,
US Patent No.
4,816,567 to Cabilly et al). To create a humanized antibody, murine CDR
regions can be
inserted into a human framework using methods known in the art (see e.g., US
Patent No.
5,225,539 to Winter and US Patent Nos. 5,530,101; 5,585,089; 5,693,762 and
6,180,370 to
Queen et al).
Humanized antibodies of the present invention may be constructed wherein the
human
acceptor molecule for the heavy chain variable region is selected based on
homology
considerations between potential acceptor molecule variable regions and the
heavy chain
variable region of the murine antibody. Germline candidate human acceptor
molecules are
preferred to reduce potential immunogenicity. Germline databases are made up
of antibody
sequences that read through the end of the heavy chain FW3 region and
partially into the
CDR3 sequence. For selection of a FW4 region, databases of mature antibody
sequences
which have been derived from the selected germline molecule can be searched or
antibody
sequences which have been derived from the selected germline molecule from a
human donor
can be used. Human acceptor molecules are preferably selected from the same
heavy chain
class as the murine donor molecule, and of the same canonical structural class
of the variable
region of the murine donor molecule. Secondary considerations for selection of
the human
acceptor molecule for the heavy chain variable region include homology in CDR
length
between the murine donor molecule and the human acceptor molecule. Human
acceptor
antibody molecules are preferably selected by homology search to the V-BASE
database,
although other databases such as the Kabat and the public NCBI databases may
be used as
well.
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Humanized antibodies of the present invention may be constructed wherein the
human
acceptor molecule for the light chain variable region is selected based on
homology
considerations between potential acceptor molecule variable regions and with
the light chain
variable region of the murine antibody. Germline candidate human acceptor
molecules are
preferred to reduce potential immunogenicity. Germline databases are made up
of antibody
sequences that read through the end of the heavy chain FW3 region and
partially into the
CDR3 sequence. For selection of a FW4 region, databases of mature antibody
sequences
which have been derived from the selected germline molecule can be searched or
antibody
sequences which have been derived from the selected germline molecule from a
human donor
can be used. Human acceptor molecules are preferably selected from the same
light chain
class as the murine donor molecule and of the same canonical structural class
of the variable
region of the murine donor molecule. Secondary considerations for selection of
the human
acceptor molecule for the light chain variable region include homology in CDR
length
between the murine donor molecule and the human acceptor molecule. Human
acceptor
antibody molecules are preferably selected by homology searches to the V-BASE
database,
and other databases such as the Kabat and the public NCBI databases may be
used as well.
When the antibody is produced as recombinant antibody by e.g. introducing
genes into
mammalian host cells, the antibodies are produced by culturing the host cells
for a period of
time sufficient to allow for expression of the antibody in the host cells or,
more preferably, for
secretion of the antibody into the culture medium in which the host cells are
grown. Host cells
useful for producing antibodies that bind to human TrkA may be cultured in a
variety of
media. Commercially available media such as Ham's F10 (Sigma-Aldrich Chemie
GmbH,
Buchs, Switzerland), Minimal Essential Medium (MEM; Sigma-Aldrich Chemie
GmbH),
RPMI-1640 (Sigma-Aldrich Chemie GmbH, Basel, Switzerland), EX-CELL 293, HEK293-
serum-free medium (Sigma, Buchs, Switzerland) and Dulbecco's Modified Eagle's
Medium
((DMEM; Sigma-Aldrich Chemie GmbH) are suitable for culturing the host cells.
Antibodies
can be recovered from the culture medium using standard protein purification
methods.
Antibodies may be operably linked to a fusion partner to enable targeting of
the expressed
protein, purification, screening, display and the like. Fusion partners may be
linked to the
antibody sequence via a linker sequences. The linker sequence will generally
comprise a
small number of amino acids, typically less than ten, although longer linkers
may also be
used. Typically, linker sequences are selected to be flexible and resistant to
degradation. As
will be appreciated by those skilled in the art, any of a wide variety of
sequences may be used
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as linkers. For example, a common linker sequence comprises the amino acid
sequence
GGGGS. A fusion partner may be a targeting or signal sequence that directs
antibody and any
associated fusion partners to a desired cellular location or to the
extracellular media. As is
known in the art, certain signalling sequences may target a protein to be
either secreted into
5 the growth media, or into the periplasmic space, located between the
inner and outer
membrane of the cell. A fusion partner may also be a sequence that encodes a
peptide or
protein that enables purification and/or screening. Such fusion partners
include but are not
limited to polyhistidine tags (His-tags) (for example H6 and H10 or other tags
for use with
Immobilized Metal Affinity Chromatography (IMAC) systems (e.g. Ni2'affinity
columns)),
10 GST fusions, MBP fusions, Strep-tag, the BSP biotinylation target
sequence of the bacterial
enzyme BirA and epitope tags which are targeted by antibodies (for example c-
myc tags, flag-
tags and the like). As will be appreciated by those skilled in the art, such
tags may be useful
for purification, for screening or for both.
Characterization and Purification of Anti-TrkA antibodies
15 Screening for antibodies can be performed using assays to measure
binding to human TrkA
and/or assays to measure the ability to block the binding of TrkA to its
ligand NGF. An
example of a binding assay is an ELISA. In addition, Surface Plasmon Resonance
(SPR)
analysis as e.g. used in the examples can be applied to measure the
association and
dissociation rate constants for the binding kinetics of the antibodies. An
example of a
20 blocking assay is a flow cytometry based assay measuring the blocking of
NGF binding to
TrkA. As an assay for evaluating the functional activity of anti-TrkA
antibodies e.g. a TF-1
cell proliferation assay can be used, wherein the ability of antibodies to
block cell surface
TrkA/beta-NGF mediated cell proliferation is assayed using the factor
dependent human
erythroleukemic cell line TF-1 (Kitamura T et at., (1989) J. Cellular
Physiology 140(2):323-
25 34).
Antibodies of the present invention may be isolated or purified in a variety
of ways known to
those skilled in the art. Standard purification methods include
chromatographic techniques,
including ion exchange, hydrophobic interaction, affinity, sizing or gel
filtration and reversed-
phase, carried out at atmospheric pressure or at high pressure using systems
such as FPLC and
30 HPLC. Purification methods also include electrophoretic, immunological,
precipitation,
dialysis and chromatofocusing techniques. Ultrafiltration and diafiltration
techniques, in
conjunction with protein concentration, are also useful. To purify TrkA
antibodies, selected
host cells can be grown in e.g. spinner-flasks for monoclonal antibody
purification.
Supernatants can be filtered and concentrated before affinity chromatography
with protein A-
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sepharose (Pharmacia, Piscataway, NJ). Eluted antibodies can be checked by gel
electrophoresis and high performance liquid chromatography to ensure purity. A
preferred
antibody of the present invention is thus an isolated and/or purified antibody
that binds to
human TrkA.
Immunoconjugates
In another aspect, the present invention provides anTrkA antibody or a
fragment thereof that
binds to human TrkA, linked to a therapeutic agent, such as a cytotoxin, a
drug (e.g., an
immunosuppressant) or a radiotoxin. Such conjugates are referred to herein as
"immunoconjugates". Immunoconjugates that include one or more cytotoxins are
referred to
as "immunotoxins." A cytotoxin or cytotoxic agent includes any agent that is
detrimental to
(e.g., kills) cells. Examples include taxol, cytochalasin B, gramicidin D,
ethidium bromide,
emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine,
colchicin, doxorubicin,
daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin,
actinomycin D, 1-
dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine,
propranolol, and
puromycin and analogs or homologs thereof Therapeutic agents also include, for
example,
antimetabolites (e.g., methotrexate, 6- mercaptopurine, 6-thioguanine,
cytarabine, 5-
fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa
chlorambucil,
melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan,
dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum
(II) (DDP)
cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and
doxorubicin),
antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin,
mithramycin, and
anthramycin (AMC)) and anti-mitotic agents (e.g., vincristine and
vinblastine). Other
examples of therapeutic cytotoxins that can be linked to an antibody of the
invention include
duocarmycins, calicheamicins, maytansines and auristatins and derivatives
thereof An
example of a calicheamicin antibody conjugate is commercially available
(Mylotarg(R);
American Home Products). Cytotoxins can be linked to antibodies of the
invention using
linker technology available in the art. Examples of linker types that have
been used to
conjugate a cytotoxin to an antibody include, but are not limited to,
hydrazones, thioethers,
esters, disulfides and peptide-containing linkers. A linker can be chosen that
is, for example,
susceptible to cleavage by low pH within the lysosomal compartment or
susceptible to
cleavage by proteases, such as proteases preferentially expressed in tumor
tissue such as
cathepsins (e.g., cathepsins B, C, D). For further discussion of types of
cytotoxins, linkers and
methods for conjugating therapeutic agents to antibodies, see also Saito G et
at., (2003) Adv.
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Drug Deliv. Rev. 55:199-215; Trail PA et at., (2003) Cancer Immunol.
Immunother. 52:328-
337; Payne G (2003) Cancer Cell 3:207-212; Allen TM (2002) Nat. Rev. Cancer
2:750-763;
Pastan I & Kreitman RJ (2002) Cum Opin. Investig. Drugs 3:1089-1091; Senter PD
&
Springer CJ (2001) Adv. Drug Deliv. Rev. 53:247-264. Antibodies of the present
invention
also can be linked to a radioactive isotope to generate cytotoxic
radiopharmaceuticals, also
referred to as radioimmunoconjugates. Examples of radioactive isotopes that
can be
conjugated to antibodies for use diagnostically or therapeutically include,
but are not limited
to, iodine-131, indium-111, yttrium-90 and lutetium-177. Methods for preparing
radioimmunconjugates are established in the art. Examples of
radioimmunoconjugates are
commercially available, including Zevalin (EDEC Pharmaceuticals) and Bexxar
(Corixa
Pharmaceuticals) and similar methods can be used to prepare
radioimmunoconjugates using
the antibodies of the invention. The antibody immunoconjugates of the
invention can be used
to modify a given biological response, and the drug moiety is not to be
construed as limited to
classical chemical therapeutic agents. For example, the drug moiety may be a
protein or
polypeptide possessing a desired biological activity. Such proteins may
include, for example,
an enzymatically active toxin, or active fragment thereof, such as abrin,
ricin A, pseudomonas
exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor or
interferon-y; or
biological response modifiers such as, for example, lymphokines, interleukin-1
(IL-1),
interleukin-2 (IL-2), interleukin-6 (IL-6), granulocyte macrophage colony
stimulating factor
(GM-CSF), granulocyte colony stimulating factor (G-CSF), or other growth
factors.
Techniques for linking such therapeutic agents to antibodies are well known,
see, e.g., Amon
et at., "Monoclonal Antibodies for Immunotargeting of Drugs in Cancer
Therapy", in
Monoclonal Antibodies and Cancer Therapy, Reisfeld et at., (eds.), pp. 243- 56
(Alan R. Liss,
Inc. 1985); Hellstrom et at., "Antibodies For Drug Delivery", in Controlled
Drug Delivery
(2nd Ed.), Robinson et at. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987);
Thorpe, "Antibody
Carriers Of Cytotoxic Agents In Cancer Therapy: A Review", in Monoclonal
Antibodies '84:
Biological And Clinical Applications, Pinchera et at. (eds.), pp. 475-506
(1985); "Analysis,
Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled
Antibody In
Cancer Therapy", in Monoclonal Antibodies For Cancer Detection And Therapy,
Baldwin et
at. (eds.), pp. 303-16 (Academic Press 1985) and Thorpe et at., Immunol. Rev.
62:119-58
(1982).
In another aspect, the present invention provides an anti-TrkA antibody or a
fragment thereof
that binds to human TrkA, administered together with a therapeutic agent, such
as a cytotoxin,
a drug (e.g., an immunosuppressant) or a radiotoxin.
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Pharmaceutical Compositions
In another aspect, the present invention provides a composition, e.g., a
pharmaceutical
composition, comprising the antibody or fragment thereof of the present
invention and a
pharmaceutically acceptable carrier. Such compositions may include one or a
combination of
(e.g., two or more different) antibodies, and/or immunoconjugates of the
invention and/or a
therapeutic agent, such as a cytotoxin, a drug (e.g., an immunosuppressant) or
a radiotoxin as
described supra. For example, a pharmaceutical composition of the invention
can comprise a
combination of antibodies (or immunoconjugates) that bind to different
epitopes on the target
antigen or that have complementary activities. Pharmaceutical compositions of
the invention
also can be administered in combination therapy, i.e., combined with other
agents as outlined
further below.
As used herein, "pharmaceutically acceptable carrier" includes any and all
solvents,
dispersion media, coatings, antibacterial and antifungal agents, isotonic and
absorption
delaying agents and the like that are physiologically compatible. Preferably,
the carrier is
suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or
epidermal
administration (e.g. by injection or infusion). Depending on the route of
administration, the
active compound, i.e. antibody or immunoconjugate may be coated in a material
to protect the
compound from the action of acids and other natural conditions that may
inactivate the
compound. Pharmaceutically acceptable carriers include sterile aqueous
solutions or
dispersions and sterile powders for the extemporaneous preparation of sterile
injectable
solutions or dispersion. The use of such media and agents for pharmaceutically
active
substances is known in the art. Except insofar as any conventional media or
agent is
incompatible with the active compound, use thereof in the pharmaceutical
compositions of the
invention is contemplated. Supplementary active compounds can also be
incorporated into the
compositions.
In another aspect, the present invention provides a composition comprising the
humanized
anti-TrkA antibody or fragment thereof of the present invention and another
pharmaceutically
active agent. Preferably the pharmaceutically active agent is one or more of:
a) an analgesic agent, b) another anti-TrkA antibody, c) NGF, d) an anti-
cancer agent,
e) an anti-NGF antibody as outlined further below.
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In another aspect, the present invention provides a composition comprising an
immunoconjugate comprising the antibody or fragment thereof that binds to
human TrkA
linked to a therapeutic agent and a pharmaceutically acceptable carrier.
Immunoconjugates
and therapeutic agents which can be used are as described supra.
A pharmaceutical composition of the invention may also include a
pharmaceutically
acceptable antioxidant. Examples of pharmaceutically acceptable antioxidants
include: (1)
water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride,
sodium bisulfate,
sodium metabisulfite, sodium sulfite and the like; (2) oil- soluble
antioxidants, such as
ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene
(BHT),
lecithin, propyl gallate, alpha-tocopherol and the like; and (3) metal
chelating agents, such as
citric acid, ethylenediamine tetraacetic-acid (EDTA), sorbitol, tartaric acid,
phosphoric acid
and the like. Examples of suitable aqueous and non-aqueous carriers that may
be employed in
the pharmaceutical compositions of the invention include water, ethanol,
polyols (such as
glycerol, propylene glycol, polyethylene glycol and the like) and suitable
mixtures thereof,
vegetable oils, such as olive oil and injectable organic esters, such as ethyl
oleate. Proper
fluidity can be maintained, for example, by the use of coating materials, such
as lecithin, by
the maintenance of the required particle size in the case of dispersions and
by the use of
surfactants. These compositions may also contain adjuvants such as
preservatives, wetting
agents, emulsifying agents and dispersing agents. Prevention of presence of
microorganisms
may be ensured both by sterilization procedures, supra and by the inclusion of
various
antibacterial and antifungal agents, for example, paraben, chlorobutanol,
phenol sorbic acid
and the like. It may also be desirable to include isotonic agents, such as
sugars, sodium
chloride and the like into the compositions. In addition, prolonged absorption
of the injectable
pharmaceutical form may be brought about by the inclusion of agents which
delay absorption
such as aluminum monostearate and gelatin.
Therapeutic and other uses
Antibodies of the present invention can be used in medicine to treat various
disorders/
conditions, as set out in various categories below.
The invention thus provides a method of treatment of the below mentioned
conditions which
comprises administering to a subject, suitably a mammalian subject, especially
a human
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subject in need thereof, a therapeutically effective amount of an antibody or
derivative as
described herein such that the condition is thereby treated.
The invention also provides use of an antibody or derivative as described
herein in the
manufacture of a medicament for the treatment of the below mentioned
conditions.
5 Here the term "treatment" includes therapeutic treatment of an existing
disorder/condition. It
also includes prophylactic treatment. It further includes the amelioration of
one or more
adverse symptoms, even if a patient is not cured of a given
disorder/condition. For example,
pain may be alleviated or reduced.
10 A preferred medical use is in the treatment of pain. According to
International Association for
the Study of Pain ("IASP") pain is generally defined as "An unpleasant sensory
and emotional
experience associated with actual or potential tissue damage, or described in
terms of such
damage or both". The essential element in all forms of pain is the activation
of specialized
high-threshold receptors and nerve fibers to warn the organism of potential
tissue damage.
15 The involvement of inflammatory cells and processes is a common element
in many pain
states. The term "acute pain" means immediate, generally high threshold, pain
brought about
by injury such as a cut, crush, burn, or by chemical stimulation. The term
"chronic pain," as
used herein, means pain other than acute pain, both of inflammatory and
neuropathic origin. It
is understood that chronic pain often is of relatively long duration, for
example, months or
20 years and can be continuous or intermittent. Antibodies of the present
invention can be used
to treat chronic pain or acute pain. The treatment of chronic pain is
preferred.
The pain may for example be or may be associated with any of the following:
inflammatory
pain, post-surgical pain, post-operative pain (including dental pain),
neuropathic pain,
peripheral neuropathy, diabetic neuropathy, diabetic nephropathy, fracture
pain, gout joint
25 pain, post-herpetic neuralgia, cancer pain, osteoarthritis or rheumatoid
arthritis pain, sciatica,
pains associated with sickle cell crises, headaches (e.g., migraines, tension
headache, cluster
headache), dysmenorrhea, endometriosis, uterine fibroids, musculoskeletal
pain, chronic low
back pain, fibromyalgia, sprains, visceral pain, ovarian cysts, prostatitis,
chronic pelvic pain
syndrome, cystitis, interstitial cystitis, painful bladder syndrome and/or
bladder pain
30 syndrome, pain associated with chronic abacterial prostatitis,
incisional pain, migraine,
trigeminal neuralgia, pain from burns and/or wounds, pain associated with
trauma, pain
associated with musculoskeletal diseases, ankylosing spondilitis,
periarticular pathologies,
pain from bone metastases, pain from HIV, erythromelalgia or pain caused by
pancreatitis or
kidney stones, malignant melanoma, Sjogren's syndrome, asthma, (e.g.,
uncontrolled asthma
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with severe airway hyper-responsiveness), intractable cough, demyelinating
diseases, chronic
alcoholism, stroke, thalamic pain syndrome, pain from toxins, pain from
chemotherapy,
inflammatory bowel disorders, irritable bowel syndrome, inflammatory eye
disorders,
inflammatory or unstable bladder disorders, psoriasis, skin complaints with
inflammatory
components, sunburn, carditis, dermatitis, myositis, neuritis, collagen
vascular diseases,
chronic inflammatory conditions, inflammatory pain and associated hyperalgesia
and
allodynia, neuropathic pain and associated hyperalgesia or allodynia, diabetic
neuropathy
pain, causalgia, sympathetically maintained pain, deafferentation syndromes,
epithelial tissue
damage or dysfunction, disturbances of visceral motility at respiratory,
genitourinary,
gastrointestinal or vascular regions, allergic skin reactions, pruritis,
vitiligo, general
gastrointestinal disorders, colitis, gastric ulceration, duodenal ulcers,
vasomotor or allergic
rhinitis, bronchial disorders, dyspepsia, gastroesophageal reflux,
pancreatitis, visceralgia and
fibrous dysplasia of bone (FD).
The pain may for example be or may be associated with any of the following:
pancreatitis,
kidney stones, endometriosis, IBD, Crohn's disease, post surgical adhesions,
gall bladder
stones, headaches, dysmenorrhea, musculoskeletal pain, sprains, visceral pain,
ovarian cysts,
prostatitis, cystitis, interstitial cystitis, post-operative pain, migraine,
trigeminal neuralgia,
pain from burns and/or wounds, pain associated with trauma, neuropathic pain,
pain
associated with musculo skeletal diseases, rheumatoid arthritis,
osteoarthritis, ankylosing
spondilitis, periarticular pathologies, oncological pain, pain from bone
metastases, HIV
infection.
Various models are known for assessing pain and can be used in screening
antibodies/derivatives thereof For example, the nociception hot plate test can
be used, as
disclosed in WO 00/73344, for example. The experiment can be carried out
according to
McMahon et at., 1995 (Nature Med. 1:774-780), using the antibody/derivative as
immunoadhesin. The antibody/derivative is infused subcutaneously into hind paw
of an adult
rat for a period of three weeks or by an osmotic mini-pump. The nociception
sensitivity is
evaluated at intervals using a hot plate test (Eddy & Leimbach (1953) J. Phar.
Exp. Ther. 107:
385-393), which mimics hyperalgesia situations following inflammation or
partial damage to
the nerve. The nociceptive stimulus induces in such a case a response (paw
licking and/or
jumping) which presumes an integrated coordination higher than simple reflex.
According to
the test, the animal is put in a pen having a plate heated to the desired
temperature as base,
usually 56 C. The latency of any of two responses (paw licking and jumping) is
measured in
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control animals (treated with non relevant antibody) and in those treated with
the anti-TrkA
antibody/derivative.
As an alternative to the hot plate test, the nociceptive response to formalin
can be assessed.
This test is disclosed by Porro & Cavazzuti, 1993 (Prog. Neurobiol, 41:565-
607) and was
used in W006/137106. It involves assessing the reduction in pain response by
analyzing any
subsequent reduction paw licking when a given candidate is administered prior
to testing.
Saline is typically used as a negative control.
An assessment of hyperalgesia can be determined using a weight bearing method.
Mice
normally distribute their body weight equally between their two limbs.
Following a painful
stimulus to the limb, for example by the local injection of Complete Freunds
adjuvant (CFA)
or monosodium iodoacetate (MIA) to the hind paw (intraplantar) or knee joint
(intra-
articular), the weight is redistributed to reduce that placed on the injected
limb and increase
that placed on the non-injected limb. Weight bearing is measured using an
incapacitance
tester with the hind paws placed on separate sensors and the average force
exerted by both
hind limbs recorded. Weight bearing can be used to assess acute inflammatory
hyperalgesia
resulting from intraplantar injection of CFA, chronic inflammatory
hyperalgesia resulting
from intra-articular injection of CFA or chronic osteoarthritic hyperalgesia
resulting from
intra-articular injection of MIA, as detailed in Examples 2, 3 and 4,
respectively.
An assessment of mechanical hyperalgesia can be performed by a number of
methods, for
example, Von Frey filaments or a Randall-Selitto analgesiometer. Paw withdraw
thresholds
are measured in response to increasing pressure stimuli applied to the plantar
hind paw
surface by von Frey filaments or to the dorsal hind paw surface by a wedge-
shaped probe of a
Randall-Selitto analgesiometer. The latency of hind paw withdraw is measured
in control
animals and in those treated with the anti-TrkA antibody/derivative. These
methods can be
used to assess mechanical hyperalgesia resulting from the Chronic Constriction
Injury (CCI)
animal model as detailed in Example 5.
The CCI model is also a well known animal model. It involves chronic
constriction of the
sciatic nerve and is used to induce chronic pain of a neuropathic nature in
rodents, such as
mice or rats. This model is described by Bennett & Xie, 1998 in Pain 33:87-
107. It was used
in WO 06/131592, for example. A major feature of many neuropathic pain states
is that
normally innocuous cool stimuli begin to produce pain. An increased response
to a non-
painful stimulus is termed allodynia. The extent of the neuropathic pain state
induced can
therefore be measured using a cold plate test for allodynia, as detailed in
Example 5. The
animal is put in a pen having a plate cooled to the desired temperature, which
can be in the
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range of -5 C to 15 C. The latency of hind paw withdraw is measured in control
animals and
in those treated with the anti-TrkA antibody/derivative. Typically the latency
for withdrawal
becomes different from the baseline at surface temperatures of 5 C and below.
The antibodies can be used in the treatment of cancer, a neuronal disorder,
Alzheimer's
disease, diabetes mellitus, diabetic nephropathy, a viral disorder, an HIV
mediated disorder,
leprosy or an inflammatory disorder. In addition, the antibodies are also
useful in treating
other diseases that may be associated with increased levels of NGF including,
for example,
lupus erythematosus, shingles, postherpetic neuralgia, and hyperalgesia.
Various cancers express TrkA. The interaction of TrkA with NGF may be involved
in tumour
development (e.g. of prostate and pancreatic cancers). Indeed in certain forms
of cancer, an
excess of NGF can facilitate the growth and infiltration of nerve fibres. By
blocking the action
of NGF it is possible to significantly reduce the formation of neuromas.
Furthermore, as an
alternative to simply providing a blocking effect, the antibodies can be
coupled to a cytotoxic
agent and can be used to target cancer cells expressing TrkA. It is not
however necessary to
couple the antibodies to toxins. ADCC (antibody-dependent cell-mediated
cytotoxicity) arises
due to an immune response in which antibodies , by coating target cells, can
make them
vulnerable to attack by the system (e.g. by T cells, by complement activation,
etc.) Preferred
cancers to be treated are prostate cancer, thyroid cancer, lung cancer,
prolactinoma, melanoma
or bone cancer pain including cancer pain associated with bone metastasis. A
preferred cancer
to be treated is bone cancer pain including cancer pain associated with bone
metastasis.
The antibodies can also be used in the treatment of various neuronal disorders
which comprise
neurodegenerative disorders e.g. the antibodies can be used to reduce the
formation of
neuromas. They can also be used in the treatment of Alzheimer's disease or in
neuroregenerative therapies. Antibodies of the present invention may be useful
in such
treatments to reduce undesired agonist effects of NGF (see also the
"Combination therapy"
section below). Furthermore, the antibodies can be used to treat neuropathic
pain, as discussed
above. This may be associated with a lesion or a dysfunction of the nervous
system. NGF has
potential use in the treatment of diabetes and leprosy but has undesired
agonist properties
including an increase in pain sensitivity which could be avoided by using the
antibodies of the
present invention.
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A still further application is in the treatment of inflammatory disorders. NGF
is released by
mast cells, fibroblasts and other cell types in the peripheral sites where
inflammatory
processes occur. In particular, mast cells appear to play a fundamental role.
They produce
NGF and at the same time express functional TrkA receptors at their surface.
The NGF/TrkA
system appears to mediate mastocyte activation through an autocrine positive
feedback
mechanism which allows local amplification of the algogenic inflammatory
signal. Examples
of inflammatory disorders that may be treated include inflammatory forms of
the urinary tract
and of the pelvic region, osteoarthritis, multiple sclerosis, colitis,
inflammatory bowel disease,
bladder cystitis, eczema, contact dermititis, arthritis, including chronic
arthritis and
rheumatoid arthritis, Crohn's disease, psoriasis and asthma.
Antibodies of the present invention may be useful in such treatments as
mentioned above to
reduce undesired agonist effects of NGF.
Antibodies or derivatives thereof of the present invention may be used
together with one or
more other active agents, e.g. pharmaceutically active agents in combination
therapy. They
may be used for simultaneous, sequential or concerted administration in
medicine.
For example, the antibody or derivative may be combined with an analgesic
agent such as an
analgesic opioid or a non-opioid analgesic. It is disclosed in W006/137106
that small
amounts of molecules able to block TrkA biological activity can potentiate the
analgesic
effects of opioids. Such analgesic opioids include one or more compounds
selected from the
following: morphine, codeine, dihydrocodeine diacetylmorphine, hydrocodone,
hydromorphone, levorphanol, oxymorphone, alfentanil, buprenorphine,
butorphanol, fentanyl,
sufentanyl, meperidine, methadone, nabumetone , propoxyphene, pentazocine; and
their
pharmaceutically acceptable derivatives thereof (e.g. pharmaceutically
acceptable salts
thereof). Suitable non-opioid analgesics include non-steroidal inflammatory
drugs (NSAIDs)
as well as other analgesics such as acetaminophen. Commonly available NSAIDs
to treat
acute inflammatory pain include asprin, ibuprofen, indomethacin, naproxen and
ketoprofen
and to treat chronic inflammatory pain include celecoxib and meloxicam.
A further combination is that of one or more antibodies of the present
invention together with
one or more other antibodies. A preferred combination is with one or more
other anti-TrkA
and/or an anti-NGF antibody. Such combinations may provide increased efficacy
in treating
one or more of the disorders discussed herein, relative to treatment with a
single antibody. For
example combinations of two or more antibodies found to be amongst the most
effective in
assay procedures used herein may be used.
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A further combination is that of the antibody of the present invention with
NGF. As discussed
above, the use of NGF in the treatment of various disorders, including
Alzheimer's disease,
diabetes mellitus, leprosy, etc., had been proposed, but an increase in pain
sensitivity had been
noted arising from agonist properties towards peripheral targets. Again, by
using an antibody
5 or derivative of the present invention, pain sensitivity can be reduced,
thereby making NGF-
based therapies more attractive.
A further combination is that of the antibody of the present invention with an
anti-cancer
agent such as e.g. an alkylating agent, an antimetabolite, a topoisomerase II
inhibitor, a
topoisomerase I inhibitor, an antimitotic drug or a platinum derivative.
The antibodies of the present invention can be administered by any appropriate
route.
This includes (but is not limited to) intraperitoneal, intramuscular,
intravenous, subcutaneous,
intratracheal, oral, enteral, parenteral, intranasal or dermal administration.
The antibodies can typically be administered for local application by
injection (intraperitoneal
or intracranial-typically in a cerebral ventricle-or intrapericardiac or
intrabursal) of liquid
formulations or by ingestion of solid formulations (in the form of pills,
tablets, capsules) or of
liquid formulations (in the form of emulsions and solutions).
Compositions for parenteral administration commonly comprise a solution of
immunoglobulin dissolved in a compatible, preferably aqueous solution. The
concentration of
the antibody/derivative in these formulations can vary from less than 0.005%
to 15-20% w/v.
It is selected mainly according to the volumes of the liquid, viscosity, etc,
and according to
the particular administration mode desired. Alternatively, the antibodies can
be prepared for
administration in solid form. The antibodies can be combined with different
inert or excipient
substances, which can include ligands such as microcrystalline cellulose,
gelatin or Arabic
rubber; recipients such lactose or starch; agents such as alginic acid,
Primogel or corn starch;
lubricants such as magnesium stearate, colloidal silicon dioxide; sweeteners
such as
saccharose or saccharin; or flavours, such as mint and methyl salicylate.
Other pharmaceutical
administration systems include hydrogel, hydroxymethylcellulose, liposomes,
microcapsules,
microemulsions, microspheres, etc.
Local injections directly at a site affected by a disorder /close thereto are
a preferred mode of
administration if a disorder is localised. The anti-TrkA antibodies are
suitably administered
systemically. Systemic administration can be performed by injection, e.g.
continuous
intravenous infusion, bolus intravenous infusion, subcutaneous or
intramuscular injection.
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Alternatively, other forms of administration (e.g. oral, mucosal, via
inhalation, sublingually,
etc.) may also be used. If desired, however, delivery of the antibody/
derivative can be
performed by local administration (e.g. intra-articular injection or
subcutaneous,
intramuscular injection) in the vicinity of affected tissues.
The anti-TrkA antibody/derivative will suitably be formulated in a
pharmaceutical
composition appropriate for the intended route of administration. Solutions
for injection will
suitably contain the antibody/derivative dissolved or dispersed in an aqueous
medium (e.g.
water for injection) as appropriate containing appropriate buffers and
molarity modifiers (e.g.
phosphate, salt and/or dextrose).
The treatment regime (i.e. dose, timing and repetition), can be represented by
single or
repeated administrations (e.g. injections) of the product by the chosen
administration route.
The interval of dose administration can be subject to modifications depending
on the extent
and duration of the clinical response, as well as the particular individual
and the individual
clinical history.
Suitably the anti-TrkA antibody/derivative has a long duration of action. In
particular the
clinical effect of the antibody extends following administration may be as
long as 21 days as
determined from animal studies. Furthermore, anti-TrkA antibodies may manifest
clinical
benefit for a longer period than that in which its presence can be detected in
a relevant
biological matrix such as serum or plasma following its administration.
In light of the intended long duration of action (i.e. an effect suitably
lasting at least one week,
or preferably at least two weeks e.g. at least three weeks or at least four
weeks), suitably the
antibody/derivative may be administered to subjects at a frequency of not more
than once per
week e.g. not more than once per two weeks or once per three weeks or once per
four weeks.
A suitable daily dose of the anti-TrkA antibody/derivative will typically
range from 0.1 mg/kg
to 10 mg/kg body weight. A suitable dose of the anti-TrkA antibody for the
treatment of acute
and chronic inflammatory pain is at least 0.01mg/kg (see Examples 2 and 3). A
suitable dose
of the anti-TrkA antibody for the treatment of osteoarthritic pain is at least
0.01mg/kg (see
Example 4). A suitable dose of the anti-TrkA antibody for the treatment of
neuropathic pain is
at least 0.01 mg/kg, preferably 0.1 mg/kg and most preferably 1 mg/kg (see
Example 5).
These doses relate to an in vivo condition in mice only. The present invention
relates the use
of a humanized anti-TrkA antibody in the treatment of acute inflammatory pain,
wherein
hyperalgesia is effectively reversed to the same extent as that observed with
administration of
a NSAID. The present invention also relates to the use of a humanized anti-
TrkA antibody in
the treatment of chronic inflammatory pain, wherein hyperalgesia is
effectively reversed to
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the same extent as that observed with administration of a NSAID. The present
invention also
relates to the use of a humanized anti-TrkA antibody in the treatment of
osteoarthritic pain,
wherein hyperalgesia is effectively reversed to the same extent as that
observed with
administration of pregabalin and an opiate. The present invention also relates
to the use of a
humanized anti-TrkA antibody in the treatment of neuropathic pain, wherein
hyperalgesia is
effectively reversed to the same extent as that observed with administration
of pregabalin.
Turning now to administration specifically in respect of tumours,
administration may be
through direct and localized injection into a tumour or a tissue near the
tumour site. For
systemic administration, doses vary from 0.05 mg/kg per day to 500 mg/kg per
day, although
dosages in the lower region of the range are preferred because they are easier
to administer.
Dosages can be calibrated for example to guarantee a particular level in the
plasma of the
antibody/derivative (in the range of about 5-30 mg/ml, preferably between 10-
15 mg/ml) and
maintain this level for a given period of time until the clinical results are
achieved.
Effective methods for measuring or assessing the stage of pancreatic or
prostatic tumours are
based on the measurement of the prostate specific antigen (PSA) in blood, on
the
measurement of the survival time for pancreas tumours, on the measurement of
the slowing or
inhibition of diffusion for metastases in the case of both tumour types.
For direct injection at the level of a tumour site, dosage depends on
different factors including
the type, stage and volume of the tumour, along with many other variables.
Depending on tumour volume, typical therapeutic doses may vary from 0.01 mg/ml
and 10
mg/ml injections which can be administered with the necessary frequency.
Whatever the nature of the therapy, humanised antibodies may be eliminated
much more
slowly and require lower dosages to maintain an effective level in the plasma
than non-
humanised antibodies. Moreover, with high affinity antibodies, administration
may be less
frequent and less sizable than with antibodies having lower affinity.
The therapeutically effective dosage of each antibody/ derivative can be
determined during
the treatment by a skilled medical practitioner. If necessary, dosages can be
reduced (e.g. to
reduce side effects) or increased (to increase therapeutic effects).
Prior to administration, preparations of antibodies of the invention can be
stored by being
frozen or lyophilized. They may then be reconstituted immediately before use
in a suitable
buffer. Given that lyophilisation and reconstitution can result in a loss in
activity, antibody
administration levels can be calibrated to compensate for this fact. (For
conventional
immunoglobulins, IgM antibodies tend to have a greater loss of activity than
IgG antibodies.)
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A shelf life may also be assigned so that antibodies are not used after a
certain period
of storage.
An antibody or derivative thereof of the present invention can be used in the
diagnosis or
prognosis of any of the diseases/ conditions discussed above in relation to
medical uses.
For example it may be used to facilitate detection of TrkA positive tumour
markers, as a
precocious marker of the insurgence of Alzheimer's disease, etc.
It may also be used in the diagnosis of CIPA ("congenital insensitivity to
pain with
anhydrosis"). This is a hereditary, recessive, autosomal syndrome
characterised by recurrent
episodic fever, anhydrosis, the absence of reaction to nociceptive stimuli,
mental retardation
and a tendency to self-mutilation. It results from mutations in the TrkA gene.
Indeed an
antibody or derivative of the present invention may be used in the diagnosis
or prognosis of a
wide range of conditions involving aberrant expression of TrkA (compared to
expression of
TrkA in a healthy individual or a healthy tissue sample) or an aberrant
activity involving
TrkA. The present invention therefore includes within its scope a method
comprising
obtaining a biological sample obtained from a patient and contacting the
sample with an
antibody or derivative of the present invention. If desired, the
antibody/derivative may be
immobilised. The method may then include assaying the binding of the antibody!
derivative
to said sample in a quantitative or qualitative manner. If desired, this may
be done with
reference and/or to a positive control (indicating a healthy state) or a
negative control
(indicating the presence/likelihood of a disorder). For diagnostic purposes,
the antibodies can
be both marked with a detectable marker or can be unmarked. (The term "marker"
is used
herein to include labels or any other detectable moiety/moiety that can
trigger a detectable
change.)
Article of manufacture and kit
In another embodiment of the disclosure, an article of manufacture comprising
the antibody or
fragment thereof, the composition or the immunoconjugate of the invention for
the treatment
of one or more of the above mentioned diseases/conditions. The article of
manufacture may
comprise a container and a label or package insert on or associated with the
container.
Suitable containers include, for example, bottles, vials or syringes. The
containers may be
formed from a variety of materials such as glass or plastic. The container
holds a composition
that may be effective for treating the condition and may have a sterile access
port (e.g., the
container may be an intravenous solution bag or a vial having a stopper
pierceable by a
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hypodermic injection needle). At least one active agent in the composition may
be the
antibody described herein. The label or package insert may indicate that the
composition may
be used for treating the condition of choice.
Moreover, the article of manufacture may comprise (a) a first container with a
composition
contained therein, wherein the composition comprises the antibody herein, and
(b) a second
container with a composition contained therein, wherein the composition
comprises a
therapeutic agent other than the antibody. The article of manufacture in this
embodiment of
the disclosure may further comprise a package insert indicating that the first
and second
compositions can be used in combination. Such therapeutic agent may be any of
the adjunct
therapies described in the preceding section (e.g., a thrombolytic agent, an
anti-platelet agent,
a chemotherapeutic agent, an anti-angiogenic agent, an anti-hormonal compound,
a
cardioprotectant, and/or a regulator of immune function in a mammal, including
a cytokine).
Alternatively, or additionally, the article of manufacture may further
comprise a second (or
third) container comprising a pharmaceutically acceptable buffer, such as
bacteriostatic water
for injection (BWFI), phosphate-buffered saline, Ringer's solution and
dextrose solution. It
may further include other materials desirable from a commercial and user
standpoint,
including other buffers, diluents, filters, needles, and syringes.
Also within the scope of the present invention are kits comprising the
antibody, the
compositions or the immunoconjugates of the invention and instructions for
use. The kit can
further contain one or more additional reagents, such as an immunosuppressive
reagent, a
cytotoxic agent or a radiotoxic agent, or one or more additional antibodies of
the invention
(e.g., an antibody having a complementary activity which binds to an epitope
in the TrkA
antigen distinct from the first antibody).
Without further description, it is believed that one of ordinary skill in the
art may, using the
preceding description and the following illustrative examples, make and
utilize the agents of
the present disclosure and practice the claimed methods. The following working
examples are
provided to facilitate the practice of the present disclosure and are not to
be construed as
limiting in any way the remainder of the disclosure.
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Examples
The humanized anti-TrkA antibodies described herein represent a novel subgroup
of
anti-TrkA antibodies, which are highly effective at inhibiting the functional
activation of
5 TrkA. The anti-TrkA mouse monoclonal antibody known as MNAC13 disclosed
in
W000/73344, and its humanized variants disclosed in W009/098238 do not achieve
the same
level of inhibition, which is desired under certain circumstances, e.g. for
the treatment of pain
and associated cancer pain, where a high level of inhibition can lead to
increased efficacy of
treatment. Thus, it would be desirable to develop humanized anti-TrkA
antibodies that would
10 exhibit inhibition of TrkA to a high degree.
Methods to assess antibody mediated inhibition of the functional activation of
TrkA
are well known in the art and include TF-1 cell proliferation assays, wherein
the ability of
antibodies to block cell surface TrkA/beta-NGF mediated cell proliferation is
assayed using
15 the factor dependent human erythroleukemic cell line TF-1 (Kitamura T et
at,. (1989) J.
Cellular Physiology 140(2):323-34). Humanized antibodies disclosed in
W009/098238 have
demonstrated inhibitory activity in TF-1 proliferation assays with the
BXhVH5VL1 candidate
having the highest degree of inhibition amongst humanized variants of the
MNAC13 mouse
antibody. In addition humanized antibodies disclosed in W009/098238 have
demonstrated
20 direct binding to the extracellular region of TrkA using Surface Plasmon
Resonance (SPR)
technique. Hence, to design new humanized variants of the mouse MNAC13
antibody with
improved binding affinities to human TrkA and more importantly improved
inhibition
potencies in TF-1 cell proliferation assays over the BXhVH5VL1 humanized
variant, a
selected subset of the humanized antibodies disclosed in W009/098238 was used
as a starting
25 point for engineering.
The letters following "BXh" are VH or VL or both. VH or VL indicates a heavy
chain
having IGHG1 isotype with a specific heavy chain variable domain denoted by a
specific
number e.g. BXhVH5, or a light chain having kappa isotype with a specific
light chain
30 variable domain denoted by a specific number e.g. BXhVL1, respectively.
When both a VH
with a specific number as well as a VL with a specific number are presented
after "BXh" e.g.
BXhVH5VL1, they indicate a specific antibody molecule consisting of a specific
combination
of heavy and light chains e.g. BXhVH5VL1 antibody refers to an antibody having
BXhVH5
heavy chain and BXhVL1 light chain. Newly engineered humanized variants based
on the
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selected subset of VH and VL variable domains described in W009/098238 were
termed
"GBR" antibodies. Conversely, the same nomenclature system is in force with
regard to the
GBR antibodies. Specific substitutions made within variable heavy chain
domains as used
herein are further indicated in brackets. All GBR antibodies were of IGHG1
heavy chain
isotype with kappa isotype light chains except as otherwise stated.
Example 1: Antibody Engineering
Out of the forty antibodies disclosed in W009/098238, five humanized
antibodies
were selected as inputs for engineering: BXhVH1VL1 having heavy chain variable
domain
VH1 (SEQ ID NO: 1) and light chain variable domain VL1 ( SEQ ID NO: 6),
BXhVH3VL1
having heavy chain variable domain VH3 (SEQ ID NO: 3) and light chain variable
domain
VL1, BXhVH3VL3 having heavy chain variable domain VH3 and light chain variable
domain VL3 (SEQ ID NO: 8), BXhVH5VL1 having heavy chain variable domain VHS
(SEQ
ID NO: 5) and light chain variable domain VL1, and BXhVH5VL3 having heavy
chain
variable domain VHS and light chain variable domain VL1. MNAC13 mouse antibody
as
used herein has heavy chain variable domain MNAC13 VH with SEQ ID NO: 20,
heavy
chain with SEQ ID NO 21, and light chain with SEQ ID NO: 22.
Surprisingly, all engineered antibodies benefited from a simple change of
valine to
alanine (mouse back-mutation) in the heavy chain variable domain at position
37 (Kabat
numbering (Kabat EA et at, ibid.). This substitution had the unique property
of broadly
enhancing the affinity for human TrkA amongst all engineered antibodies as
measured by
SPR. More importantly, the same substitution led to a potency increase in TF-1
cell
proliferation assays for most variants, which unexpectedly when introduced in
the VHS
domain paired with the VL1 domain (GBR VHS (V37A)VL1 antibody) was a ten-fold
increase in comparison to the BXhVH5VL1 humanized antibody. Notably, the same
GBR
VH5(V37A)VL1 antibody also exhibited a three-fold potency increase in TF-1
cell
proliferation assays when compared to the MNAC13 mouse antibody.
Since an increase in the number of mouse-derived residues in humanized
antibodies
can increase their risk of immunogenicity (Harding FA et at., (2010) MAbs
2(3):256-65),
further investigation was made to reduce the number of mouse residues in the
engineered
antibodies. The change of mouse residue lysine at position 3 for a glutamine
in the GBR
VH5(V37A)VL1 antibody resulted in the GBR VH5(K3Q,V37A)VL1 antibody having the
same number of mouse residues compared to the BXhVH5VL1 humanized antibody,
but with
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increased binding affinity, equivalent FAB thermo-stability to the parental
mouse MNAC13
antibody, and increased inhibitory potency.
Finally, since antibody-mediated effector functions can be detrimental to
therapies
wherein only TrkA blockade is required, it is desirable to develop anti-TrkA
antibodies that
would inhibit TrkA without mediating the killing of TrkA expressing cells by
immune cells.
The human IGHG4 isotype does not carry effector functions such as ADCC or CDC,
and as
such is a particularly well suited antibody format when effector functions are
not required or
detrimental to therapy. However, one drawback of the naturally occurring human
IGHG4
isotype is known as the "FAB exchange" phenomenon by which naturally occurring
human
IGHG4 antibodies are known to exchange heavy chains in vivo (Labrijn AF et
at., (2009) Nat.
Biotechnol. 27(8):767-71). Methods to block heavy chain exchange between
recombinant and
endogenous human IGHG4s are known in the art and the exchange is known to be
efficiently
blocked by substitution of the serine residue at position 228 (EU numbering,
Edelman GM et
at., ibid.) located in the hinge region for a proline residue, thereby
mimicking the
conformational hinge structure found in human IGHG1 isotype (Lewis KB et at.,
(2009) Mol.
Immunol. 46(16):3488-94). GBR VH5(V37A)VL1 and GBR VH5(K3Q,V37A)VL1 were
both formatted as IGHG4 S228P immunoglobulins for the aforementioned
therapeutic
purpose and showed equivalent potency to their IGHG1 isotype counterpart,
thereby making
both GBR VH5(V37A)VL1 IGHG4 S228P and GBR VH5(K3Q,V37A)VL1 IGHG4 S228P
antibodies suitable for human therapies wherein only TrkA blockade is
required.
Methods
Design of engineered variants
A 3D model for the VH5-VL1 pair of variable domains was calculated using the
structure homology-modelling server SWISS-MODEL (Arnold K et at., (2006)
Bioinformatics 22(2) : 195-201; http ://swis smo del. exp asy. org) set in
automated mode. The
retrieved model template was the experimentally solved MNAC13 FAB 3D structure
(PDB
code 1SEQ, www.pdb.org, Berman HM et at., (2000) Nucleic Acids Res. 28(1):235-
42,
Covaceuszach S et at., (2005) Proteins 58(3):717-27). Sequence alignment of
MNAC13 VH,
VH1, VH3, VHS and germline VH3-023*01 (SEQ ID NO: 23) domains showed different
amino acid content at positions: 3, 5, 19, 37, 40, 42, 44, 49, 50, 52A, 53,
60, 62, 74, 82A, 83,
84, 89, and 94 (Kabat numbering). Model analysis allowed the selection of a
subset of
positions based on their putative influence on CDR regions and/or heavy chain-
light chain
variable domain packing. This subset of positions consisted of: 3, 37, 40, 42,
44, 49, 50, 60,
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62, 89, and 94 (Kabat numbering). Hence engineered antibodies had heavy chain
variable
domains encompassing substitutions at single or multiple positions in the
aforementioned
variable domain sequences (VH1, VH3, and VH5 with SEQ ID NO: 1, 3, 5,
respectively)
selected from the subset of positions described above. More specifically,
engineered
antibodies consisted of the substituted heavy chain variable domains as
described above
paired with one of the two previously mentioned light chain variable domains
VL1 or VL3
(SEQ ID NO 6 and 8).
Molecular Biology
Engineered heavy chain variable domain coding DNA sequences (cDNAs) were
created by standard mutagenesis techniques using the vector DNAs for BXhVH1,
BXhVH3,
and BXhVH5 described in WO 09/098238 as PCR templates. Similarly, light chain
variable
domain cDNAs were directly amplified from the vector DNAs for BXhVL1 and
BXhVL3
described in WO 09/098238. Variable domain cDNAs were further assembled
upstream of
their respective constant domain cDNA sequences using PCR assembly techniques.
Finally,
the complete heavy and light chain cDNAs were ligated in independent vectors
that are based
on a modified pcDNA3.1 vector (Invitrogen, CA, USA) carrying the CMV promoter
and a
Bovine Growth Hormone poly-adenylation signal. The light chain specific vector
allowed
expression of kappa isotype light chains by ligation of the light chain
variable domain cDNA
of interest in front of the kappa light chain constant domain cDNA using BamHI
and BsiWI
restriction enzyme sites; while the heavy chain specific vector was engineered
to allow
ligation of the heavy chain variable domain cDNA of interest in front of the
cDNA sequence
encoding the IGHG1 CH1, IGHG1 hinge region, IGHG1 CH2, and IGHG1 CH3 constant
domains using BamHI and Sall restriction enzyme sites. In both heavy and light
chain
expression vectors, secretion was driven by the murine VJ2C leader peptide
containing the
BamHI site. The BsiWI restriction enzyme site is located in the kappa constant
domain;
whereas the Sall restriction enzyme site is found in the IGHG1 CH1 domain.
IGHG4 immunoglobulin formatting having substitution 5228P was achieved by
replacing the cDNA sequence encoding the IGHG1 CH1, IGHG1 hinge region, IGHG1
CH2,
and IGHG1 CH3 constant domains for a cDNA sequence encoding the IGHG4 CH1,
IGHG4
hinge region having 5228P substitution, IGHG4 CH2, and IGHG4 CH3 constant
domains in
the heavy chain specific vector described above. Substitution 5228P was
introduced in a
human IGHG4 heavy chain cDNA template by standard PCR mutagenesis techniques.
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Antibody production
For transient expression of antibodies, equal quantities of heavy and light
chains
vectors were co-transfected into suspension-adapted HEK293-EBNA1 cells (ATCCO
catalogue number: CRL-10852) using polyethylenimine (jetPEI , Polyplus
transfection,
Illkirch Cedex, France). Typically, 100 ml of cells in suspension at a density
of 0.8-1.2
million cells per ml is transfected with a DNA-jetPEI mixture containing 50
iug of
expression vector encoding the heavy chain and 50 iug of expression vector
encoding the light
chain. When recombinant expression vectors encoding antibody genes are
introduced into the
host cells, antibodies are produced by further culturing the cells for a
period of 4 to 5 days to
allow for secretion into the culture medium (EX-CELL 293, HEK293-serum-free
medium;
Sigma, Buchs, Switzerland), supplemented with 0.1% pluronic acid, 4 mM
glutamine, and
0.25 ug/m1 geneticin).
Antibodies were purified from cell-free supernatant using recombinant protein-
A
streamline media (GE Healthcare Europe GmbH, Glattbrugg, Switzerland), and
buffered
exchanged into phosphate buffer saline prior to assays.
Stability testing using differential scanning calorimetry
Calorimetric measurements were carried out on a VP-DSC differential scanning
microcalorimeter (GE Healthcare Europe GmbH, Glattbrugg, Switzerland). The
cell volume
was 0.128 ml, the heating rate was 200 C/h, and the excess pressure was kept
at 65 p.s.i. All
antibodies were used at a concentration of 1 mg/ml in PBS (pH 7.4). The molar
heat capacity
of each protein was estimated by comparison with duplicate samples containing
identical
buffer from which the protein had been omitted. The partial molar heat
capacities and melting
curves were analyzed using standard procedures. Thermograms were baseline-
corrected and
concentration-normalized before being further analyzed using a Non-Two State
model in the
Origin software (v7.0, GE Healthcare Europe GmbH, Glattbrugg, Switzerland).
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Affinity measurements by SPR
SPR analysis was used to measure the association and dissociation rate
constants for
the binding kinetics of the different antibodies (mouse and humanized
antibodies). The
binding kinetics of the mouse antibody and humanized variants were measured on
a
5 BIACORE 2000 instrument (GE Healthcare Europe GmbH, Glattbrugg,
Switzerland) at room
temperature, and analyzed with the BiaEvaluation software (v4.1, GE Healthcare
Europe
GmbH, Glattbrugg, Switzerland).
Since antibodies are bivalent molecules, it is best to immobilize antibodies
onto the
sensor chip. If antibodies are used as analytes, SPR measurements will bear a
valency
10 component in addition to affinity. Although the BIAcore evaluation
software can model
bivalency and extract affinity constants, it is preferable to circumvent any
valency bias by
working with a monovalent analyte whenever possible. To this aim, a monovalent
form of the
human TrkA extracellular region was produced by digestion of a recombinant
human TrkA-
Fc fusion protein (SEQ ID NO: 24) made in HEK293-EBNA1 cells. The fusion
protein
15 consisted of the human TrkA extracellular region (SEQ ID NO: 25) fused
to an IGHG1 Fc
region wherein a protease-specific cleavage sequence was included between the
two regions
(TEV cleavage protease amino acid sequence: ENLYFQS). Following protease
cleavage, the
monovalent form of the human TrkA extracellular region was further purified to
homogeneity
using standard chromatographic techniques which included a protein-A step to
remove Fc
20 fragments. Finally, the monovalent human TrkA extracellular region
protein was buffer-
exchanged into PBS before being diluted in Biacore running buffer for affinity
measurements.
Sample purity, homogeneity and molecular weight were confirmed by SDS-PAGE and
size-
exclusion analysis.
25 Antibodies were immobilized via capture of their Fc portion to allow
for correct
orientation on the sensor chip surface. A monoclonal mouse anti-human IgG (Fc)
antibody
sensor chip was used to capture all humanized antibodies regardless of their
isotypes (Human
Antibody Capture Kit, catalogue number BR-1008-39, GE Healthcare Europe GmbH),
and a
polyclonal rabbit anti-mouse immunoglobulin sensor chip was used to capture
the MNAC13
30 mouse antibody (Mouse Antibody Capture Kit, catalogue number BR-1008-38,
GE
Healthcare Europe GmbH).
Data (sensorgram: fc2-fcl) were fitted with a 1:1 Langmuir model with mass
transfer
in spite of having little mass transfer limitation in mass transfer tests. To
account for the
experimental variations in captured antibody at the beginning of each
measurement, the Rmax
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value was set to local in all fits. Dissociation times were of at least 350
seconds.
Measurements were performed in triplicate, and included zero-concentration
samples for
referencing. Both Chi2 and residual values were used to evaluate the quality
of a fit between
the experimental data and individual binding models.
Proliferation assay with TF-1 cells
Suspension adapted TF-1 cells (ATCC number: CRL-2003) were grown in complete
RPMI medium containing 10% FCS, and 5 ng/ml of rhul3NGF (recombinant human
beta-
NGF, R&D Systems Europe Ltd, Abingdon, UK). For the assay, TF-1 cells were
incubated
for 5h in complete RPMI without human beta-NGF. Following this starvation
step, cells were
centrifuged and seeded in flat-bottomed 96 well plates with various
concentrations of each
antibody and a fixed concentration of recombinant beta-NGF. TF-1 cells were
seeded at a
density of 7000 cells/well and the total antibody-cell mixture volume was 200
ul with 5 ng/ml
of human beta-NGF (final concentration). This mixture was incubated for 4 days
at 37 C with
CO2 supplementation. At day 3, the colorimetric dye Alamar blue (AbD Serotec,
Morphosys
AbD GmbH, Dusseldorf, Germany) was added to each well without any change to
the
incubation conditions. At day 4, fluorescence was read on a Bio-Tek SynergyTM
2
spectrophotometer/microplate reader (BioTek Instruments GmbH, Luzern,
Switzerland) with
a 530-to-560 nm excitation wavelength and a 590 nm emission wavelength.
Experiments were
performed at least twice and measurements for each antibody concentration were
done in
triplicates.
Results
Engineering new humanized anti-TrkA antibodies based on BXhVH5VL1
Based on a 3D model for the VH5-VL1 pair of variable domains, a subset of
common
3D positions within the different heavy chain variable domains (VH1, VH3, and
VHS) were
selected for mouse-to-human as well as human-to-mouse mutations; this group
consisted of
positions: 3, 37, 40, 42, 44, 49, 50, 60, 62, 89, and 94 (Kabat numbering).
The engineering
strategy with regard to the combination of substitutions as used herein was
based on the
complementarity of the different substitutions in terms of on their putative
influence on CDR
regions and/or variable domain packing and/or immunogenicity.
In a first approach mouse-to-human and human-to-mouse mutations were
engineered
in the BXhVH5VL1 candidate in the context of an IGHG1 isotype format.
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The mouse-to-human mutations undertaken included the following substitutions:
K3Q, T40A,
R44G, A49S, Y50A, P60A, T62S, and R94K. Production yields and affinities of
some of the
engineered anti-TrkA antibodies based on these single or combination of
substitutions are
shown in Table 1 and 2, respectively. Combining most or all mouse-to-human
mutations led
to poor production yields and complete loss of binding to TrkA. For example,
mouse-to-
human substitution A49S combined with Y50A induced a complete abrogation of
binding,
which was not rescued by the other mouse-to-human substitutions and mouse-to-
human
substitutions K3Q, T40A, R44G, and R94K did not lead to any improvement in
affinity either
alone or in combination with other mouse-to-human substitutions.
The human-to-mouse mutations undertaken included the following substitutions:
V37A,
G42E, and V89L. Human-to-mouse substitution V37A had the most positive impact
and led
to at least a two-fold increase in affinity (KD is decreased by at least two-
fold) (Table 2 and
FIG. 1); a 2.5 fold increase in affinity was recorded for the GBR VH5(V37A)VL1
IGHG1
antibody . This increase in affinity was abrogated when combining human-to-
mouse
substitution V37A with mouse-to-human substitutions P60A and T62S, but
maintained when
making combinations with mouse-to-human substitutions K3Q and/or R44G.
Taken together these results identified the importance of positions 49 and 50
to allow binding
and the unique property of the human-to-mouse substitution V37A in enhancing
the affinity
of BXhVH5VL1 by at least two-fold. Since antibody-mediated effector functions
can be
detrimental to therapies wherein only TrkA blockade is required, the GBR
VH5(V37A)VL1
and the GBR VH5(K3Q,V37A)VL1 were both reformatted in the aforementioned IGHG4
isotype having the 5228P substitution. Both engineered antibodies showed
similar affinities to
their IGHG1 isotype counterparts, indicating that the affinity improvement
brought by the
V37A substitution was not affected by the isotype switch.
Engineering new humanized anti-TrkA antibodies based on BXhVH1VL1, BXhVH3VL1
BXhVH3VL1 and BXhVH5VL3
In a second approach, the V37A substitution was introduced in the other
selected
candidates originating from WO 09/098238, and the affinities of the engineered
anti-TrkA
antibodies benchmarked against BXhVH5VL1 and MNAC13 antibodies by SPR. Table 3
and
4 show, respectively, the production yields and affinities of the V37A
substituted antibodies.
Although engineered antibodies had differences in their production yields, all
engineered
antibodies exhibited an enhancement in affinity upon introduction of the V37A
substitution.
Affinities were improved by 8, 20, 30, and 55%, for BXhVH3VL1, BXhVH1VL1,
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BXhVH3VL3, and BXhVH5VL3, respectively. Thus, the human-to-mouse substitution
V37A
did not only lead to an increase in affinity when introduced in BXhVH5VL1 but,
and most
unexpectedly also led to an increase in affinity amongst all humanized
variants. Notably,
GBR VH5(V37A)VL1 or GBR VH5(K3Q,V37A)VL1 both as IGHG1 or IGHG4 S228P
isotype had affinities matching the affinity measured for the MNAC13 mouse
antibody
(Tables 2 and 4); both being increased by at least two-fold when compared to
the
BXhVH5VL1 antibody (KD is decrease by 2.5 to 2.7 fold, respectively).
Table 3 and FIG.2 includes the melting temperatures of the FAB portions
measured
within the engineered antibodies. Monoclonal antibodies melting profiles are
characteristic of
their isotypes but the mid-point melting temperature (Tm) of the FAB fragment
can be easily
identified even in the context of a full-length immunoglobulin (Garber E and
Demarest SJ
(2007) Biochem. Biophys. Res. Commun. 355(3):751-7). Such mid-point melting of
the FAB
portion was used to monitor the stability of the engineered candidates. GBR
VH5(V37A)VL1
and GBR VH5(K3Q,V37A)VL1 FAB fragments each displayed a single transition at
73.6 and
73 C respectively, both having equivalent thermo-stability with a shape and an
amplitude of
the FAB transition consistent with a cooperative unfolding which is generally
observed for a
compactly folded FAB fragment indicating that the engineering process was
successful at
retaining FAB stability. This is further exemplified, when comparing the FAB
Tm of GBR
VH5(V37A)VL1 or GBR VH5(K3Q,V37A)VL1 with the MNAC13 FAB Tm (74 C), the
differences being within 1 C or less.
Functional testing of engineered anti-TrkA antibodies
Engineered anti-TrkA antibodies were assayed for their ability to inhibit the
functional
activation of TrkA in TF-1 cell proliferation assays (FIG. 3). The half
maximal inhibitory
concentration (IC50) which is a measure of the effectiveness of a compound in
inhibiting
biological function was calculated for each curve and the results are given in
Table 5. Note
that GBR VH1(V37A)VL1 was not assayed due to its lower affinity by SRP.
It was found that GBR VH5(V37A)VL1 was the best performer followed by GBR
VH5(K3Q, V37A)VL1, GBR VH5(K3Q,V37A)VL1 IGHG4 5228P, and GBR
VH3(V37A)VL1 which had identical IC50s. GBR VH3(V37A)VL3 and GBR
VH5(V37A)VL3 antibodies had slightly higher IC5Os but all tested engineered
antibodies
performed better than BXhVH5VL1, GBR VH5(V37A)VL1 being ten-fold better.
Notably,
the GBR VH5(V37A)VL1 antibody also exhibited a three-fold potency increase in
TF-1 cell
proliferation assays when compared to the MNAC13 mouse antibody. All V37A
variants had
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equivalent or better IC50 when compared to the mouse parental antibody.
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Table 1: production yields from a selection of engineered anti-TrkA antibodies
based on
BXhVH5VL1; all antibodies are of the IGHG1 isotype except as otherwise stated.
(*) IGHG4
isotype with S228P substitution. ' Includes mouse-to-human and human-to-mouse
mutations
as described in Example 1.
Humanized anti-TrkA antibodies SEQ ID Number of
Transient
NOs mutations +
expression
(mg/1)
BXhVH5VL1 28,29 8 10
GBR VH5(K3Q)VL1 50,29 7 16
GBR VH5(V37A)VL1 51, 29 9 19
GBR VH5(V37A)VL1 (*) 52, 29 9 11
GBR VH5(G42E)VL1 53, 29 9 19
GBR VH5(V89L)VL1 54,29 9 17
GBR VH5(R94K)VL1 55, 29 7 15
GBR VH5(K3Q,V37A)VL1 56, 29 8 52
GBR VH5(K3Q,V37A)VL1 (*) 57, 29 8 11
GBR VH5(K3Q,T40A)VL1 58, 29 6 3
GBR VH5(P60A,T62S)VL1 59, 29 6 7
GBR VH5(K3Q,V37A,R44G)VL1 60, 29 7 24
GBR VH5(K3Q,A49S,Y50A)VL1 61,29 5 15
GBR VH5(K3Q,P60A,T62S)VL1 62, 29 5 7
GBR VH5(K3Q,T40A,P60A,T62S)VL1 63, 29 4 8
GBR VH5(K3Q,V37A,T40A,P60A,T62S)VL1 64, 29 5 12
GBR VH5(K3Q,T40A,R44G,A49S,Y50A)VL1 65, 29 3 22
GBR VH5(K3Q,A49S,Y50A,P60A,T62S)VL1 66, 29 3 16
GBR VH5(K3Q,T40A,R44G,A49S,Y50A,P60A,T62S)VL1 67, 29 1 3
GBR VH5(K3Q,T40A,R44G,A49S,Y50A,P60A,T62S,R94K)VL1 68, 29 0 3
5
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Table 2: affinities from a selection of engineered anti-TrkA antibodies based
on
BXhVH5VL1; all antibodies are of the IGHG1 isotype except as otherwise stated.
(*) IGHG4 isotype with S228P substitution. N.B. indicates no binding.
Humanized anti-TrkA antibodies KD
(nM)
BXhVH5VL1 40.9
GBR VH5(V37A)VL1 16
GBR VH5(V37A)VL1 (*) 15
GBR VH5(R94K)VL1 52
GBR VH5(K3Q,V37A)VL1 15.4
GBR VH5(K3Q,V37A)VL1 (*) 15
GBR VH5(K3Q,V37A,R44G)VL1 20
GBR VH5(K3Q,A49S,Y50A)VL1 N.B.
GBR VH5(K3Q,V37A,T40A,P60A,T62S)VL1 48
GBR VH5(K3Q,T40A,R44G,A49S,Y50A)VL1 N.B.
GBR VH5(K3Q,A49S,Y50A,P60A,T62S)VL1 N.B.
GBR VH5(K3Q,T40A,R44G,A49S,Y50A,P60A,T62S)VL1 N.B.
GBR VH5(K3Q,T40A,R44G,A49S,Y50A,P60A,T62S,R94K)VL1 N.B.
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Table 3: production yields and FAB thermo-stability data from a selection of
engineered anti-
TrkA antibodies; all antibodies are of the IGHG1 isotype.
Anti-TrkA antibodies SEQ ID NOs Expression Tm
Fab
(mg/1) ( C)
MNAC13 21,22 5 74
BXhVH1VL1 26,29 10 64.7
GBR VH1(V37A)VL1 69,29 16 65.3
BXhVH3VL1 27, 29 9 71.3
GBR VH3(V37A)VL1 70, 29 1 70.2
BXhVH3VL3 27, 30 6 71
GBR VH3(V37A)VL3 70,30 9 69.3
BXhVH5VL1 28, 29 16 76.5
GBR VH5(V37A)VL1 51,29 19 73.6
GBR VH5(K3Q,V37A)VL1 56,29 52 73
BXhVH5VL3 28, 30 16 76.4
GBR VH5(V37A)VL3 51,30 19 73.3
Table 4: affinities of V37A engineered anti-TrkA antibodies; all antibodies
are of the IGHG1
isotype.
Anti-TrkA antibodies kon (1/Ms) koff (Vs) KD (nM)
MNAC13 6.02x104 1.09x10-3 18
BXhVH1VL1 3.13x104 1.33x10-2 426
GBR VH1(V37A)VL1 1.96x104 6.63x10-3 338
BXhVH3VL1 4.65x104 6.19x10-3 133
GBR VH3(V37A)VL1 3.82x104 4.71x10-3 123
BXhVH3VL3 3.14x104 4.59x10-3 134
GBR VH3(V37A)VL3 3.1x104 2.95x10-3 95
BXhVH5VL1 1.04x105 4.26x10-3 40.9
GBR VH5(V37A)VL1 1.4x105 2.25x10-3 16
GBR VH5(K3Q,V37A)VL1 1.7x105 2.62x10-3 15.4
GBR VH5VL3 6.23x104 3.95x10-3 63
GBR VH5(V37A)VL3 7.45x104 2.05x10-3 28
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Table 5: TF-1 cell proliferation assay IC5Os for V37A engineered anti-TrkA
antibodies; all
antibodies are of the IGHG1 isotype except as otherwise stated. (*) IGHG4
isotype with
S228P substitution. N.D. indicates not determined.
Anti-TrkA antibodies 1050 tug/mil
MNAC13 0.23
BXhVH5VL1 0.73
GBR VH1(V37A)VL1 N.D.
GBR VH3 (V37A)VL1 0.15
GBR VH3 (V37A)VL3 0.29
GBR VH5 (V37A)VL1 0.075
GBR VH5 (K3Q ,V37A)VL1 0.15
GBR VH5 (K3 Q,V37A)VL1(*) 0.15
GBR VH5 (V37A)VL3 0.22
Example 2: Humanized anti-TrkA antibody reverses acute inflammatory
hyperalgesia
of the paw induced by intraplantar injection of CFA.
Intraplantar injection of Complete Freunds adjuvant (CFA) into one of the hind
paws of mice
causes acute inflammatory hyperalgesia that can be assessed using the weight
bearing method.
Naive mice normally distribute their body weight equally between their two
hind paws.
However, when the CFA injected hind paw is inflamed and painful, the weight is
re-
distributed to lessen that placed on the injected paw (ipsilateral) and
increase that on the non-
injected (contralateral) hind paw.
Methods
Weight bearing through each hind limb was measured using an incapacitance
tester (Linton
Instruments, UK). Mice were placed in the incapacitance tester with the hind
paws on
separate sensors and the average force exerted by both hind limbs was recorded
over 2
seconds. Naïve AMB1 mice were acclimatized to the procedure room in their home
cages,
with food and water available ad libitum. The AMB1 mice were generated by
replacement of
exon 1 of mouse TrkA with that of its human counterpart and as such they
exclusively express
the human TrkA protein. Habituation to the incapacitance tester was performed
over several
days. Baseline weight bearing recordings were taken prior to induction of
insult.
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Inflammatory hyperalgesia was induced by intraplantar injection of CFA (20u1
of 1.5mg/m1
solution) into the left hind paw. A pre-treatment weight bearing reading was
taken to assess
hyperalgesia 23 hours post-CFA. Animals were then ranked and randomised
according to
CFA window in a Latin square design. 24 hours post-CFA, animals were treated
with a single
i.p. injection of 0.1mg/kg isotype control i.p. or 0.0001, 0.001, 0.01 and
0.1mg/kg of the
antibody GBR VH5(K3Q,V37A)VL1 IGHG4 S228P i.p. (10m1/kg dose volume). The non-
selective NSAID indomethacin was used as a positive control at 10mg/kg p.o.
(5m1/kg dose
volume). Weight bearing readings were taken at 4, 8, 24, 48, 72, 96 and 120
hours post-
antibody/drug treatment.
Behavioural assessments were performed blind. Weight bearing (g) readings were
taken for
both ipsilateral and contralateral hind paws and expressed as % weight bearing
difference (%
ipsi/contra). Data were analysed by comparing treatment groups to isotype
control group at
each time point. Statistical analysis was performed as a repeated measures
ANOVA followed
by Planned comparison test using InVivoStat, (p<0.05 considered significant).
Results
Intraplantar injection of CFA induced marked hyperalgesia as detected 23 hrs
post-CFA by a
shift in weight bearing from the ipsilateral to the contralateral hind paw
resulting in a drop in
the % ipsi/contra ratio (Fig. 4). The isotype control treatment initiated at
24 hrs post-CFA
showed no effect on the hyperalgesia. A significant reversal of the
hyperalgesia was observed
with 0.01 and 0.1mg/kg of the antibody GBR VH5(K3Q,V37A)VL1 IGHG4 S228P from 4-
48 hours post-dose when compared to the isotype control. Doses of 0.001mg/kg
of the
antibody GBR VH5(K3Q,V37A)VL1 IGHG4 S228P or below were ineffective at
reversing
the hyperalgesia. Indomethacin (10mg/kg) showed a significant reversal of
hyperalgesia at all
time points tested. In conclusion, the antibody GBR VH5(K3Q,V37A)VL1 IGHG4
S228P
gave a significant reversal of acute inflammatory hyperalgesia of the paw
similar to
indomethacin. Furthermore, the effective doses of GBR VH5(K3Q,V37A)VL1 IGHG4
S228P
administered (0.01 and 0.1 mg/kg) are many fold less than the effective dose
of indomethacin
(10mg/kg), demonstrating that the anti-TrkA antibody can be administered at a
much lower
dose than the standard treatment for this condition.
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Example 3: Humanized anti-TrkA antibody reverses chronic inflammatory
hyperalgesia
of the knee joint induced by intra-articular injection of CFA
Intra-articular injection of CFA into one of the knee joints of the hind limbs
of mice induces
chronic inflammatory hyperalgesia that can be assessed using the weight
bearing method.
5 When the CFA injected joint is inflamed and painful, the weight is re-
distributed to lessen that
placed on the limb with the injected (ipsilateral) joint and increase that on
the limb with the
non-injected (contralateral) joint. The development of these signs in this
animal model are
believed to be clinically relevant as they reflect symptoms displayed by
patients presenting
chronic inflammatory joint pain associated with underlying conditions such as
osteoarthritis
10 and rheumatoid arthritis.
Methods
Naïve AMB1 mice were acclimatised to the procedure room in their home cages,
with food
and water available ad libitum. The AMB1 mice were generated by replacement of
exon 1 of
15 mouse TrkA with that of its human counterpart and as such they
exclusively express the
human TrkA protein. Habituation to the incapacitance tester was performed.
Baseline weight
bearing readings were taken immediately prior to CFA injection. Animals were
anaesthetised
using isoflurane and oxygen mixed 3:1 in sterile conditions. The knee area was
shaved and
cleaned with a dilute hibiscrub solution. The left knee was injected with 1 OW
of 10mg/m1
20 CFA. Animals were allowed to recover in a warmed environment before
returning to their
home cage. Animals were assessed for development of inflammatory hyperalgesia
using the
weight bearing method at days 4, 7 and 10 post-CFA. Based on the day 10 post-
CFA
measurement, animals were ranked and randomised to treatment groups according
to their
CFA window. At 13 days post-CFA when the hyperalgesia was well-established,
animals
25 were treated with a single injection of either isotype control antibody
at 10mg/kg i.p. or the
antibody GBR VH5(K3Q,V37A)VL1 IGHG4 S228P at 0.01, 1 and 10mg/kg i.p. The COX-
2
selective NSAID celecoxib was used as a positive control at 60mg/kg p.o. twice
daily. For
the antibody treated groups, weight bearing was measured at 4, 8, 24 and 96
hrs post-dosing.
For the celecoxib treated group, weight bearing was measured at 1 and 8 hrs
post-dosing on
30 day 13 post-CFA and then at 1 hr post-dosing on days 14-17 post-CFA.
Behavioural
assessments were performed blind. Weight bearing (g) readings were taken for
both ipsilateral
and contralateral hind limbs and expressed as % weight bearing difference (%
ipsi/contra).
Data were analysed by comparing treatment groups to isotype control group at
each time
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point. Statistical analysis was performed as a repeated measures ANOVA
followed by
Planned comparison test using InVivoStat, (p<0.05 considered significant).
Results
Injection of CFA into the knee joint caused marked and long-lasting
hyperalgesia from day 3
onwards as detected by a shift in weight bearing from the ipsilateral to the
contralateral hind
limb resulting in a drop in the % ipsi/contra ratio (Fig. 5). The isotype
control antibody
treatment had no effect on the hyperalgesia. A significant reversal of
hyperalgesia was seen
with celecoxib (60mg/kg) 1 hour post dose during the course of the treatment.
A significant
reversal of the hyperalgesia was observed with a single injection of 0.01, 1
and 10mg/kg of
the antibody GBR VH5(K3Q,V37A)VL1 IGHG4 5228P from 4-72 hours post-dose, with
only the highest two doses being significant at 96 hours when compared to the
isotype
control. In conclusion, a single dose of 0.01 mg/kg the antibody GBR
VH5(K3Q,V37A)VL1
IGHG4 5228P or above gave a significant and sustained reversal of chronic
inflammatory
hyperalgesia of the knee joint similar to multiple dosings of celecoxib.
Furthermore, only a
single dose of 0.01 mg/kg of the antibody GBR VH5(K3Q,V37A)VL1 IGHG4 5228P was
required to achieve an effect compared with multiple doses of celecoxib at 60
mg/kg
indicating that the antibody GBR VH5(K3Q,V37A)VL1 IGHG4 5228P can be given at
a
much lower dose and less frequently than the NSAID celecoxib, which is the
current standard
of care.
Example 4: Humanized anti-TrkA antibody reverses chronic osteoarthritic
hyperalgesia
of the knee joint induced by intra-articular injection of MIA
Intra-articular injection of monosodium iodoacetate (MIA) into the knee joint
of mice leads to
the development of chronic hyperalgesia associated with local joint
inflammation coupled
with cartilage degradation. As such, the hyperalgesia measured in the MIA
model closely
resembles osteoarthritic-associated pain. Furthermore, the cartilage
degradation leads to
chronic neuronal damage with neuropathic-like characteristics. Since
pregabalin, an
anticonvuldant drug is the recommended first-line drug in the treatment of
neuropathic pain it
is used here as a comparator compound. Tramadol, a weak u-opioid receptor
agonist, is
increasingly being used for the treatment of OA because, in contrast to
NSAIDs, tramadol
does not produce gastrointestinal bleeding or renal problems nor does it
affect articular
cartilage. For this reason tramadol was used alongside pregabalin as a
comparator to the
antibody GBR VH5(K3Q,V37A)VL1 IGHG4 5228P.
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Methods
Naïve AMB1 mice were acclimatised to the procedure room in their home cages,
with food
and water available ad libitum. The AMB1 mice were generated by replacement of
exon 1 of
mouse TrkA with that of its human counterpart and as such they exclusively
express the
human TrkA protein. Habituation to the incapacitance tester was performed over
several days.
Base line weight bearing readings measurements were taken. Animals were
anaesthetised
using isoflurane and oxygen mixed 3:1 in sterile conditions. The knee area was
shaved and
cleaned with a dilute hibiscrub solution. MIA, Sul of 100mg/m1 (500 g) or
saline (sham) was
injected into the knee joint of the left hind leg. Animals were allowed to
recover in a warmed
environment, before returning to their home cage. On days 3 ¨ 23 post-MIA,
animals were
assessed at regular intervals using weight bearing for development of knee
joint hyperalgesia.
On day 14 post-MIA, weight bearing measurements were taken and animals were
ranked and
randomised to treatment groups according to their MIA response window followed
by
treatment with a single i.p. injection of 1, 10, 100 g/kg antibody GBR
VH5(K3Q,V37A)VL1
IGHG4 S228P or 100 g/kg isotype control antibody (5m1/kg dose volume). As a
comparator
control, animals were treated on day 14 post-MIA with tramadol at 10mg/kg or
pregabalin at
30mg/kg p.o. (5m1/kg dose volume) followed by tramadol at 30mg/kg or
pregabalin at
100mg/kg every other day on days 16-22 post-MIA (5m1/kg dose volume). All
animals were
assessed using weight bearing at 4, 8, and 24 hours post-dosing on day 14 post-
MIA followed
by every 24 hours for antibody treated groups and 1 and 24 hours post-dose for
tramadol and
pregabalin treated groups. Behavioural assessments were performed blind.
Weight bearing (g)
readings were taken for both ipsilateral and contralateral hind limbs and
expressed as %
weight bearing difference (% ipsi/contra). Data were analysed by comparing
treatment
groups to sham group at each time point (day 3-14 post-MIA), for effects of
MIA on
hyperalgesia. Post-dosing data were analysed by comparing treatment groups to
the isotype
control group at each time point. Statistical analysis was performed as a
repeated measures
ANOVA followed by Planned comparison test using InVivoStat, (p<0.05 considered
significant).
Results
Injection of MIA at 500 g into the knee joint caused a marked hyperalgesia
from day 3
onwards as detected by a shift in weight bearing from the ipsilateral to the
contralateral hind
limb resulting in a drop in the % ipsi/contra ratio. A significant reversal of
hyperalgesia was
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seen with a single injection of 10 and 100ug/kg of the antibody GBR
VH5(K3Q,V37A)VL1
IGHG4 S228P from 4hrs ¨ 7 days post-dose when compared to isotype control at
each time
point (Fig. 6A). The antibody GBR VH5 (K3Q,V37A) VL1 IGHG4 S228P at lug/kg had
no
effect on the hyperalgesia. Tramadol (10mg/kg) and pregabalin (30mg/kg) had no
effect at 1
hour post-dosing on day 14 post-MIA, but showed significant reversal of
hyperalgesia by 4
hours post-dose (Fig. 6B). Due to lack of effect at 1 hour post-dosing on day
14 post-MIA,
tramadol was increased to 30mg/kg and pregabalin was increased to 100mg/kg,
which both
showed significant reversal of hyperalgesia at 1 hour post-dose on day 16, 18
and 20 post-
MIA. In conclusion, a single dose of 10 g/kg of the antibody GBR VH5
(K3Q,V37A)VL1
IGHG4 S228P or above gave a significant and sustained reversal of chronic
osteoarthritic
hyperalgesia of the knee joint similar to multiple dosings of tramadol and
pregabalin.
Furthermore, only a single dose of the antibody GBR VHS (K3Q,V37A)VL1 IGHG4
S228P
at 10 g/kg was required to achieve an effect compared with multiple doses of
two drugs,
tramadol and pregabalin, used at far higher doses (30mg/kg and 100mg/kg,
respectively).
Therefore the anti-TrkA antibody can be administered at a much lower dose and
with a lower
dosing frequency than pregabalin and tramadol.
Example 5: Humanized anti-TrkA antibody reverses peripheral neuropathic
hyperalgesia and allodynia induced by chronic constriction injury of the
sciatic nerve
Peripheral neuropathic pain is a chronic form of pain arising from injury,
dysfunction
or disease of the peripheral nervous system. It typically comprises
hyperalgesia (heightened
response to painful stimuli) as well as allodynia (painful response to non-
painful stimuli).
Neuropathic pain can be induced in animals by experimental injury of
peripheral nerves and
this is typically achieved by chronic constriction injury (CCI) of the sciatic
nerve via loose
ligation (Bennett & Xie (1998) Pain, 33: 87- 107). Since pregabalin is the
recommended first-
line drug in the treatment of neuropathic pain it is used here as a comparator
compound.
Methods
CCI surgery was performed in male and female AMB1 mice under ketamine xylazine
(100
and 10 mg/kg/10m1, i.p.) anaesthesia. AMB1 mice were generated by replacement
of exon 1
of mouse TrkA with that of its human counterpart and as such they exclusively
express the
human TrkA protein. Following hair clipping, the left sciatic nerve was
exposed at mid-thigh
level, aseptically. Three loose ligature (using 4-0 black silk) were place
around the sciatic
nerve at 1-2 mm before the branching of nerve. The surgical site was treated
with betadine
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solution following skin suture. Animals were allowed to recover for the next
seven days. One
day before the experiment, animals were acclimatized to the measuring device
(cold plate)
and the pre-dose paw withdrawal thresholds were recorded (Ohr). Animals were
then
regrouped into six groups. The following day, animals were treated with a
single i.p. injection
of isotype control antibody (1000 ug/kg/10m1) or different doses of the
antibody GBR
VH5(K3Q,V37A)VL1 IGHG4 S228P (10, 100 & 1000 ug/kg/10m1). Pregabalin (30
mg/kg/10m1, p.o.) or saline were administered once daily over a seven day post-
dose period.
Post-dose readouts were recorded at 4h, 24h and then every other day until the
7th day post-
dose. Post-dose readouts were recorded lh after pregabalin or saline dosing on
all days. Post-
dose readouts were recorded first for mechanical hyperalgesia and 5 min later
for cold
allodynia. Mechanical hyperalgesia was measured as the threshold force (g)
required to elicit
paw withdrawal using a dynamic plantar aesthesiometer (Plantar Von Frey
instrument; Ugo
Basile Srl, Italy). Cold allodynia was measured as time taken for paw
withdrawal from a cold
plate in seconds (IITC Life Science Inc., USA).
Results
Seven days after CCI surgery, mice exhibited marked mechanical hyperalgesia
(Fig. 7A) and
cold allodynia (Fig. 7B) as seen by a sharp reduction in their paw withdrawal
threshold and
paw withdrawal latency time, respectively. All doses of the antibody GBR VHS
(K3Q,V37A)VL1 IGHG4 5228P gave a reversal of mechanical hyperalgesia, as seen
by an
increase in the paw withdrawal threshold (Fig. 7A) and cold allodynia (Fig.
7B), as seen by an
increase in the paw withdrawal latency. The extent and duration of this
reversal was clearly
dose dependent and was comparable to pregabalin at the highest dose of 1 mg/kg
(1000
1..tg/kg) for both readouts. This dose of the antibody GBR VHS (K3Q,V37A)VL1
IGHG4
5228P of 1 mg/kg was effective when administered as a single dose, compared to
multiple
doses of pregabalin of 30 mg/kg, again showing that the anti-TrkA antibody can
be
administered at a lower dose and lower dosing frequency compared to the
standard treatment
of pregabalin.
Example 6: Effects of an anti-TrkA antibody on sensory and sympathetic neurons
during neonatal development
NGF/TrkA signalling is critically required for the survival of sensory and
sympathetic
neurons during development (Bibel and Barde, 2000 Genes Dev 14(23):2919-37).
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Corresponding, blockade of NGF by either active or passive immunization during
the
embryonic or neonatal periods results in substantial loss of these neurons
along with
pronounced atrophy of the neuronal cell body and associated neuronal
structures such as the
sympathetic ganglia (Cattaneo, (2013) Proc Natl Acad Sci U S A. 2013 Mar
26;110(13):4877-
5 85). Conversely, treatment of neonatal animals with NGF results in
hypertrophy and
hyperplasia of sympathetic neurons leading to enlarged sympathetic ganglia
(Levi-Montalcini
and Booker, 1960a Proc Natl Acad Sci U S A. Mar;46(3):373-84.; Levi-Montalcini
and
Cohen, 1960 Ann N Y Acad Sci. 1960 Mar 29;85:324-41; Banks et al., 1975 J
Physiol.May;247(2):289-98).
10 To determine the impact of anti-TrkA antibody treatment during the
neonatal period on the
survival and morphology of sympathetic neurons compared to an anti-NGF
antibody, a
detailed quantitative and morphometric analysis of sympathetic neurons located
in the
superior cervical ganglia (SCG) was performed following a 4 week treatment of
human TrkA
knock-in (AMB1) mice. Human TrkA knock-in mice were required as GBR VHS
15 (K3Q,V37A) VL1 IGHG4 S228P has weak cross-reactivity to rodent TrkA.
Neonatal mice
were treated intraperitoneally (i.p.) once per week for 4 weeks from postnatal
day 1 with
100mg/kg GBR VHS (K3Q,V37A) VL1 IGHG4 S228P, an anti-NGF based on the sequence
of tanezumab which is fully cross-reactive to mouse NGF (Cattaneo, 2010 Curr
Opin Mol
Ther, 12, 94-106) or PBS.
20 At the end of the treatment period, mice were anesthetized with a
mixture of Ketalar [(Parke-
Davis) 75 mg/kg of body weight] and Xylazine [(Streuli) 10 mg/kg of body
weight] and then
perfused with 0.9% NaC1, followed by freshly prepared 4% paraformaldehyde in
0.1M
phosphate buffer, pH 7.3.
The right superior cervical ganglion (SCG) in the neck was dissected,
protected with foam in
25 plastic embedding cassettes and post-fixed in the same fixative for 24
hours at 4 C. The SCG
was dissected with the surrounding arteries to allow for a better
identification during the
histological procedure. They were then embedded in paraffin (TCA 44-720 Medite
automate)
and cut with a Leica-microtome (3 gm thin sections; Leica RM2135). Bands of
consecutive
sections were prepared and each 10th (adult) or 20th (neonate) section was
mounted on
30 "Superfrost plus" (Menzel) glass slides and stained with hematoxylin and
eosin (H&E). The
slices were scanned with a Hamamatsu (Nanozoomer) slide scan system and were
magnified
with a 40x objective. The pictures (.ndpi) were magnified in the NDP-Viewer2
software with
either a 10x or 20x objective and then exported in jpeg format for the volume
determination.
The volume of a ganglion was determined according to the Cavalieri method
(Estimating
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Volume in Biological Structures; Cold Spring Harb Protoc 2012 Nov 1;
2012(11):1129-39;
doi: 10.1101/pdb.top071787). The ImageJ software with appropriate plugins
("grid" and "cell
counter") was used for the volume determination. Grid size was chosen to get
approximately
100 points or more per ganglion.
The volume was calculated as follows: Volume of Ganglion = number of points on
ganglion
tissue x grid area x thickness x distance between slices. The mean volume of a
cell was
determined according to the nucleator principle (The nucleator, J Microsc.
1988 Jul; 15 l(Pt
1):3-21). The area (A) and the radius (R) of an object correlate with its
volume (V), also for
irregular objects like cells. Therefore, by measuring the area of a large
number of cells, a very
good approximation of the mean cell volume in a ganglion can be obtained. The
area of 100
or more randomly chosen cells per ganglion was measured. For each area (A),
the radius (r =
Ai (A/ 7r)) and then the volume (volume = (4/3) er3) were calculated. The
average of all
volumes was taken as mean cell volume for the ganglion. The cell number in one
ganglion
was calculated according to the formula: Total cell volume of ganglion/mean
cell volume in
ganglion. The cell density was calculated according to the formula: Number of
Cells in
Ganglion/Volume of Ganglion.
Treatment of neonatal mice with tanezumab resulted in a significant reduction
in the SCG
neuronal cell diameter, an atrophy of the overall SCG and a significant
reduction in the
number of neuronal cells per ganglion in comparison to PBS treatment (Fig. 8).
In contrast,
GBR VHS (K3Q,V37A) VL1 IGHG4 5228P treatment resulted in a significant
increase in the
SCG neuronal cell diameter, a hypertrophy of the overall SCG and a significant
increase in
the number of neuronal cells per ganglion in comparison to PBS treatment.
Example 7: Effects of an anti-TrkA antibody on sympathetic neuron survival
during
adulthood
Following the neonatal period, sensory neuronal survival becomes NGF-
independent. For
sympathetic neurons, however, NGF continues in adulthood to modulate their
morphology. In
adult rodents, NGF blockade by either active or passive immunization leads to
sympathetic
neuronal cell body atrophy that overall contributes to an atrophy of the
sympathetic ganglia
(Levi-Montalcini and Booker, 1960b Proc Natl Acad Sci U S A. Mar;46(3):384-91;
Angeletti
et al., 1971a . Brain Res. Apr 2;27(2):343-55; Bjerre et al., 1975 Brain Res.
Jul 11;92(2):257-
78; Goedert et al, 1978 Brain Res. Jun 9;148(1):264-8; Otten et al., 1979
Brain Res. Oct
26;176(1):79-90.; Gorin and Johnson, 1980 Brain Res. 1980 Sep 29;198(1):27-
42.; Johnson et
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al., 1982; Ruit et al., 1990 J Neurosci. Jul;10(7):2412-9.). On the other
hand, treatment of
adult rodents with NGF leads to hypertrophy of sympathetic neurons but, unlike
treatment in
the neonate, does not lead to hyperplasia (Levi-Montalcini and Booker, 1960b
Proc Natl Acad
Sci U S A. Mar;46(3):384-91; Angeletti et al., 1971b J Ultrastruct Res. 1971b
Jul;36(1):24-
36.; Bueker and Schenkein, 1964 Ann N Y Acad Sci. Oct 9;118:183-205.).
To determine the impact of GBR VH5 (K3Q,V37A) VL1 IGHG4 S228P treatment during
the
adult period compared to an anti-NGF on the survival and morphology of
sympathetic
neurons, adult (2.5 months old) mice were treated i.p. once per week for 4
weeks with
100mg/kg GBR VH5 (K3Q,V37A) VL1 IGHG4 S228P, tanezumab or PBS and both the
left
and right SCG were analysed as described in example 6.
Treatment of adult mice with tanezumab resulted in a significant reduction in
the SCG
neuronal cell diameter, an atrophy of the overall SCG but, unlike in the
neonate, did not affect
the number of neuronal cells per ganglion (Fig. 9). GBR VHS (K3Q,V37A) VL1
IGHG4
5228P treatment resulted in a significant increase in the SCG neuronal cell
diameter, a
hypertrophy of the overall SCG but again the neuronal cell number per ganglion
was
unaffected. The atrophy effects of tanezumab were to a similar degree in both
neonates and
adults, whereas the hypertrophy effects of GBR VHS (K3Q,V37A) VL1 IGHG4 5228P
were
far more pronounced in neonates than adults (compare Figs 8 and 9).
Although the effect of GBR VHS (K3Q,V37A) VL1 IGHG4 5228P on SCG neuronal cell
diameter was significant in the adult mice, the range of cell diameters of GBR
VHS
(K3Q,V37A) VL1 IGHG4 5228P treated mice entirely overlapped with that of PBS-
treated
animals, whereas a significant proportion of SCG neuronal cell diameters of
tanezumab-
treated animals lay outside and below the range for PBS-treated animals (Fig.
10).
Visually, the SCG neuronal cell bodies of GBR VHS (K3Q,V37A) VL1 IGHG4 5228P
treated animals appeared similar to those of PBS-treated animals, whereas
those of
tanezumab-treated animals appeared shrunken with a condensed cytoplasm (Fig.
11).
The results obtained with tanezumab are consistent with previous studies
demonstrating that
NGF blockade leads to sympathetic neuronal cell loss in neonates and atrophy
of the
sympathetic neuronal cell body and ganglia in both neonates and adults. The
hypertrophy and,
in neonates, hyperplasia effects of GBR VHS (K3Q,V37A) VL1 IGHG4 5228P on the
SCG
are akin to that reported for NGF treatment. Thus, GBR VHS (K3Q,V37A) VL1
IGHG4
5228P and tanezumab clearly exert opposing effects on sympathetic neurons in
both neonate
and adult animals, although the effects of GBR VHS (K3Q,V37A) VL1 IGHG4 5228P
are
diminished in the latter.
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Example 8: Effects of an anti-TrkA antibody on Peripheral neuropathic pain
Peripheral neuropathic pain is a chronic form of pain arising from
injury/dysfunction/disease
of the peripheral nervous system (Bridges et al., (2001) Br J Anaesth.
u1;87(1):12-26.). It
typically comprises hyperalgesia (heightened response to painful stimuli) as
well as allodynia
(painful response to non-painful stimuli). Neuropathic pain can be induced in
animals by
experimental injury of peripheral nerves and this is typically achieved by
chronic constriction
injury (CCI) of the sciatic nerve via loose ligation.
In male and female AMB1 mice, mechanical hyperalgesia was measured as the
threshold
force (g) required to elicit paw withdrawal and cold allodynia was measured as
time taken for
paw withdrawal from a cold plate (seconds). CCI surgery was then performed
under ketamine
xylazine (100 and 10 mg/kg/10m1, i.p.) anesthesia. Following hair clipping,
the left sciatic
nerve was exposed at mid-thigh level aseptically. Three loose ligature (using
4-0 black silk)
were place around the sciatic nerve at 1-2 mm before the branching of nerve.
The surgical site
was treated with betadine solution following skin suture. Animals were allowed
to recover
for the next seven days. Animals were acclimatized to the measuring device
(cold plate) and
the pre-dose paw withdrawal threholds were recorded (Ohr). Animals were then
treated with a
single i.p. injection of 0.3 and lmg/kg GBR VHS (K3Q,V37A) VL1 IGHG4 S228P,
tanezumab or saline. Post-dose readouts were recorded at 4h, 1 day and then at
every next day
until the 9th post-dose day with a final reading at 14d post-dose.
Post-dose readouts were recorded first for mechanical hyperalgesia and 5 min
later for cold
allodynia. 7 days after CCI surgery (Oh), mice exhibited marked mechanical
allodynia (Fig.
12A) and cold allodynia (Fig. 12B) as seen by a sharp reduction in their paw
withdrawal
threshold and paw withdrawal latency time, respectively. GBR VHS (K3Q,V37A)
VL1
IGHG4 S228P at both doses gave a long-lasting reversal of both mechanical
hyperalgesia as
seen by an increase in the paw withdrawal threshold, and cold allodynia as
seen by an
increase in the paw withdrawal latency. At equivalent doses, GBR VHS
(K3Q,V37A) VL1
IGHG4 S228P clearly gave a higher reversal of mechanical hyperalgesia and cold
allodynia
than the anti-NGF antibody based on tanezumab.
Example 9: Effects of anti-TrkA antibody on abberant sensory and sympathetic
nerve
sprouting
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Persistent knee joint inflammation induced by multiple intra-articular
injections of Complete
Freunds adjuvant (CFA) over several weeks in mice leads to ectopic sensory and
sympathetic
neuronal sprouting within the joint along with nociceptive behaviours
(Ghilardi et al., 2012
Arthritis Rheum. Jul;64(7):2223-32). Similar ectopic sprouting is also
observed in mouse
models of bone cancer pain and has been proposed to be involved in the
maintenance of pain
(Jimenez-Andrade et al., 2011 Pain. Nov;152(11):2564-74). In both models, anti-
NGF
treatment reduces ectopic neuronal sprouting and nociceptive behaviours,
indicative of a role
for NGF in this process (Jimenez-Andrade et al., 2011 Pain. Nov;152(11):2564-
74; Ghilardi
et al., 2012 Arthritis Rheum. Jul;64(7):2223-32).
To assess the effect of GBR VH5 (K3Q,V37A) VL1 IGHG4 5228P in comparison to an
anti-
NGF antibody based on tanezumab in the multi-CFA knee joint pain model, adult
female
AMB1 mice were lightly anaesthetized with 3% isoflurane mixed with 95% oxygen.
The left
knee joint was injected with 10 1 CFA (H37 RA; Difco Laboratories, USA, 2.5
mg/ml in
mineral oil, Sigma). After CFA injection, the animals were returned to their
home cages.
Regular observations were carried out to monitor the condition of the animals
after injection.
The CFA injection was repeated on days 7, 14 and 21.
One group of animals (n=6) was intra-articularly injected with 10 1 of
phosphate buffer
solution (PBS) to serve as a sham control. The mice were acclimatised to the
environment for
experiments for three days by training them to stand on the pads of an
Incapacitance Meter
(weight-bearing apparatus, Linton) for 2-3 min. The average values of weight
load exerted by
the left (ipsilateral) and right (contralateral) hind-paws over a period of 5
sec were noted.
Three measurements were taken and the mean calculated for each time point. The
baseline
values of weight placed on the pad were examined on the day before the first
injection of CFA
(Day 0) and re-assessed 3 days after each follow-on injections on days 3, 10,
17 and 24, as
well as 4, 8, 24, 48, 72, 96 and 120 hours following compound administration
on day 24.
Data resulting from behavioural experiments were calculated as the mean
S.E.M of the
weight bearing ratio (WBR=weight bearing on ipsilateral side/weight bearing on
contralateral
side x100). One group of 6 mice were randomly selected as the sham control and
received 1
PBS injection into the left knee joint every 7 days for 21 days yielding a
total of 4 injections
(Sham CFA group), and received vehicle i.p. on day 24 following the first knee
injection.
Other mice received a total of 4 injections of CFA at days 0, 7, 14 and 21. On
day 24, mice
with a weight-bearing ratio lower than 70% were randomly divided into vehicle
control
group, 1 mg/kg GBR VHS (K3Q,V37A) VL1 IGHG4 5228P group and 1 mg/kg tanezumab
group. All vehicle/compounds were dosed i.p. at a single dose of 5 ml/kg.
Multiple intra-
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articular injections of CFA induced sustained hyperalgesia as detected by a
shift in weight
bearing from the ipsilateral to the contralateral hind paw resulting in a drop
in the
%ipsi/contra ratio to around 50% (Fig. 13). After a single i.p. injection on
day 24 when
hyperalgesia was well established, GBR VH5 (K3Q,V37A) VL1 IGHG4 S228P at
lmg/kg
5 gave a significant reversal of hyperalgesia when compared to the vehicle
control, whereas the
anti-NGF antibody based on tanezumab had no significant effect at the same
dose.
Example 10: Effects of an anti-TrkA antibody on bone healing
10 Introduction
Adequate fracture-associated pain treatment is important for patient welfare.
However,
currently available analgesics may exert considerable side effects. Opioids
used to treat
moderate to severe pain often cause nausea, sedation and drowsiness as well as
respiratory
depression. Furthermore, opioid addiction may occur and it is unclear whether
this substance
15 class can influence fracture healing. In contrast, non-steroidal anti-
inflammatory drugs
(NSAID) that are often prescribed for the treatment of mild to moderate
musculoskeletal pain
have been shown to exert considerable gastrointestinal side effects (Dib et
al.(2014), Scand J
Gastroenterol, 49, 785-9). The anti-inflammatory action of NSAID is based on
the selective
or non-selective inhibition of cyclooxygenase (COX)-2, thus blocking the
synthesis of
20 prostaglandins (PGs) from arachidonic acid. However, PGE-2 has
pleiotropic effects on bone,
and COX-2 function is essential for bone healing (Blackwell et al.(2010)
Trends Endocrinol
Metab, 21, 294-301; Simon et al., (2002) J Bone Miner Res, 17, 963-76). In
rodents, there is
strong evidence for disturbed fracture healing when NSAID are applied during
the healing
period (Spiro et al., (2010) J Orthop Res, 28, 785-791; Krischak et al.,
(2007a) Arch Orthop
25 Trauma Surg, 127, 453-8: Krischak et al.,( 2007b) Arch Orthop Trauma
Surg, 127, 3-9). In
addition, NSAID in humans are also assumed to have negative effects on bone
healing.
An alternative approach to treat fracture-associated pain may be the blockade
of nerve growth
factor (NGF)/neurothrophic tyrosine kinase receptor type 1 (TrkA) signalling.
Blockade of
this signalling pathway using an anti-NGF monoclonal antibody was shown to be
effective for
30 the treatment of chronic low back pain and arthrosis (Katz et al.,(2011)
Pain, 152, 2248-
2258:, McKelvey et al., (2013) J Neurochem, 124, 276-89). The binding of NGF
to TrkA
leads to receptor dimerization and activation by receptor auto-
phosphorylation. Ligand-
receptor interaction can be abolished by the application of specific
antibodies that selectively
bind and neutralize NGF or by the application of small molecular weight
inhibitors of the
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kinase activity of Trk receptors (Kumar and Mahal, (2012) J Pain Res, 5, 279-
87: Watson et
al., (2008) . Biodrugs, 22, 349-359).
The influence of NGF/TrkA signalling on fracture repair has not been fully
elucidated.
Recently, two studies reported a significant reduction in pain-related
behaviour after blocking
NGF/TrkA signalling by the application of a neutralizing anti-NGF monoclonal
antibody or
small molecular weight Trk kinase inhibitors (Koewler et al., (2007) J Bone
Miner Res, 22,
1732-42.:, Ghilardi et al., (2011) Bone, 48, 389-98). However, both studies
reported an
increase in callus size and a slight reduction of the biomechanical properties
of the healed
bone was also indicated in one report (Koewler et al., (2007) J Bone Miner
Res, 22, 1732-42),
possibly indicating interference of the fracture-healing process. Because the
effects observed
in both studies were relatively minor, further clarification is warranted.
Therefore, we
addressed this issue by applying neutralizing monoclonal antibodies that
target NGF and
TrkA, respectively, in a mechanically defined diaphyseal fracture-healing
mouse model.
Methods
Antibodies
The neutralizing anti-NGF monoclonal antibody is a human IgG2 based on the
sequence of
tanezumab and is fully cross-reactive to mouse NGF (Cattaneo, (2010) Curr Opin
Mol Ther,
12, 94-106). The neutralizing anti-TrkA antibody is GBR VHS (K3Q,V37A) VL1
IGHG4
S228P.
Animal Model and Husbandry
The animal experiment was performed according to the national and
international guidelines
for the care and use of laboratory animals and was approved by the local
ethics committee
(Regierungsprasidium Tubingen, No. 1144). Male AMB1 mice have a knock-in for
human
TrkA at the mouse TrkA locus and were obtained from Charles River (Charles
River Italia,
Calco, Italy). The mice were housed in groups of up to four animals with a 14-
h light, 10-h
dark cycle at 23 C and 55 10% humidity. Standard rodent chow and water were
available
ad libitum. Surgical procedures were conducted under sterile conditions.
Study Design
To investigate the influence of NGF-TrkA signal blockade on fracture healing,
a standardized
osteotomy in the right femur of 13-week-old mice as described previously in
detail (Röntgen
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et al., (2010) J Orthop Res, 28, 1456-62) was created. Briefly, under general
anaesthesia (2
vol-% isoflurane, Forene0, Abbott, Wiesbaden, Germany), an osteotomy gap was
created in
the mid-shaft of the right femur using a Gigli wire-saw (RISystem, Davos,
Switzerland). The
osteotomy was stabilized using an external fixator (stiffness 3 Nxmm 1;
RISystem, Davos,
Switzerland). Prior to surgery, the mice received a single dose of antibiotic
(clindamycin-2-
dihydrogenphosphate, 45 mgxkg-1; Clindamycin, Ratiopharm, Ulm, Germany). For
analgesia,
tramadol-hydrochloride (25 mgxL-1, Griinenthal, Aachen, Germany) was provided
via the
drinking water 1 day before and after surgery.
Prior to surgery, the mice were randomly allocated to three groups for the
application of
phosphate-buffered saline (PBS, PAA Laboratories, Linz, Austria) (control, n =
24), anti-NGF
antibody (n = 25) or anti-TrkA antibody (n = 24) (both antibodies were
provided by Glenmark
Pharmaceuticals Ltd, Switzerland). The antibodies were administered
intraperitoneally at a
concentration of 10 mgxkg-1 bodyweight. The substances were applied on days 1,
6 and 11
after surgery.
The mice were euthanized 7, 14 or 25 days after surgery, and the operated and
contra-lateral
femurs were explanted.
Measurement of Activity and Ground Reaction Force
To determine the analgesic effects of the administered antibodies, the
activity of the mice
after surgery and the vertical ground reaction force (GRF) of the operated
limb was assessed,
because pain leads to reduced activity and presumably reduced loading of the
osteotomised
limb. The analyses were performed on days 2, 5, 7, 14 and 20 after surgery. To
assess the
vertical GRF, the mice were allowed to move freely through an acrylic glass
tunnel containing
a force plate in the floor (HE6x6, AMTI, Watertown, MA, USA). The peak
vertical GRF
during the stance phase of the operated limb was recorded by a blinded
observer and averaged
from a minimum of four measurements of each mouse per time point. The activity
of the mice
was recorded overnight using an infrared beam system fitted to specialized
cages (ActiMot,
TSE Systems GmbH, Bad Homburg, Germany). The postoperative values were related
to the
preoperative measurement.
Biomechanical testing
To investigate the mechanical properties, intact and osteotomised femurs
explanted on day 25
were subjected to a non-destructive three-point bending test as described
previously (Röntgen
et al., (2010) J Orthop Res, 28, 1456-62; Wehrle et al., (2014) J Orthop Res,
32, 1006-13).
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Briefly, the proximal end of the femur was fixed to an aluminium cylinder,
which in turn was
fixed to a hinge joint of the 3-point-bending setup (Z10, Zwick Roell, Ulm,
Germany). The
femur condyles rested unfixed on a bending support. An axial load was applied
to the mid-
shaft of the femur in the sagittal plane. Flexural rigidity was calculated
from the slope (k) of
the linear region of the force-deflection curve. The distance between the load
vector and the
proximal (a) and distal (b) supports was considered when the fracture callus
was not located
exactly at the middle between the supports (//2). Flexural rigidity (El) was
calculated
according to the formula for asymmetrical bending: EI=k((a2b2)x3r1).
Micro-computed tomography (uCT)
Femurs harvested on days 14 and 25 were scanned using a CT device (Skyscan
1172,
Skyscan, Kontich, Belgium) at a resolution of 8 m per pixel at a voltage of
50 kV and 200
A. Within each scan, two phantoms with a defined density of hydroxyapatite
(HA) (250 and
750 mgxcm 3) were scanned to determine the bone mineral density (BMD).
On day 14, the whole callus was analysed for total volume (TV), bone volume
(BV), bone
volume fraction (BV/TV) and BMD. On day 25, two regions of interest, the whole
callus and
the former osteotomy gap, were analysed for the above-indicated parameters. To
distinguish
between mineralized and non-mineralized tissue, a global threshold
corresponding to
641.9 mg HAxcm 3 was applied (Morgan et al., (2009) Bone, 44, 335-44).
Histomorphometry
Femurs were fixed in 4% formalin for 24 h, decalcified in 20%
ethylenediaminetetraacetic
acid (pH 7.2-7.4) for 10-12 days and embedded in paraffin (Paraplast, Leica
Biosystems,
Wetzlar, Germany). Longitudinal sections of 6- m thickness were cut and
stained using
Safranin-O and fast green. Evaluation of the tissue composition was performed
using light
microscopy (Leica DMI6000 B; Software MetaMorph , Leica Microsystems,
Mannheim,
Germany) under 50-fold magnification. At all time points, the whole callus,
consisting of the
periosteal callus and the osteotomy gap, was analysed for the relative amounts
of fibrous
tissue, cartilage and bone.
Statistical analysis
The results are presented as the means standard error of the mean (SEM).
Data were tested
for normal distribution using the Shapiro-Wilk test. For data analysis, SPSS
statistics software
(Version 21, IBM Corp, Chicago, IL, USA) was used. Groups were compared using
ANOVA.
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Post-hoc analysis was performed using the Fisher's LSD test. Significance was
assumed at p
< 0.05.
Results
Assessment of activity and GRF
The activity and vertical GRF of the mice were assessed 1 to 2 days before and
2, 5, 7, 14 and
20 days following surgery. As expected, the activity declined following
surgery in all groups.
On day 2, the anti-TrkA-antibody-treated mice displayed significantly higher
activity
compared to PBS-treated animals (Fig. 14A), indicating that the antibody-
treated mice
suffered less pain. There were no significant differences between PBS and anti-
NGF-antibody
or anti-NGF-antibody and anti-TrkA-antibody treatment. At later time points,
no significant
inter-group differences were detected (Figure 14A).
Analysis of the vertical GRF demonstrated a decrease to 83-90% of the pre-
operative values
in all treatment groups on day 2 (Fig. 14B). The vertical GRF decreased
further by day 5, then
slowly increased until day 20, when the GRF attained 90-100% of the pre-
operative values.
There were no significant differences between the groups at any given time
point (Fig. 14B).
Influence of NGF-TrkA signalling blockade on fracture healing
Histomorphometric assessment of the callus composition was performed on days
7, 14 and
25. Representative sections are depicted in Fig. 15 A¨I. We did not detect any
statistically
significant differences in the callus composition between the treatment groups
at any time
point (Fig. 15 J¨L). Three-dimensional assessment of the callus on day 14
using CT showed
no significant differences in callus size, BV, BV/TV or BMD (Table 6). The
same was found
on day 25 on analysing the osteotomy or the whole callus (Table 7). The
flexural rigidity of
the calli, determined using a non-destructive three-point bending test, was
not significantly
influenced by anti-NGF antibody or anti-TrkA antibody compared to PBS
administration
(Figure 16).
Table 6: CT-analysis of the fracture callus on day 14 after osteotomy.
PBS Anti-NGF Anti-TrkA
TV (mm3) 6.48 0.67 7.06 0.52 7.75
0.73
BV (mm3) 0.77 0.09 0.66 0.05 0.73
0.10
BV/TV (%) 12.03 0.98 9.89 1.16 9.59
1.14
BMD 288 14 269 17 264 17
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TV, total callus volume; BV, bone volume; BV/TV, bone volume per callus
volume; BMD,
bone mineral density. Data is presented as the mean SEM; n = 7-8.
Table 7: CT-analyses of the osteotomy and whole fracture calli of mice
treated with PBS,
anti-NGF antibody or anti-TrkA antibody after a healing period of 25 days.
Osteotomy, day 25 Whole callus, day 25
PBS Anti-NGF Anti-TrkA PBS Anti-NGF Anti-TrkA
TV (mm3)
0.60 0.11 0.64 0.11 0.65 0.09 6.49 0.49 6.23 0.61 6.50 0.62
BV (mm3)
0.15 0.04 0.20 0.04 0.18 0.04 2.60 0.24 2.71 0.19 2.93 0.27
BV/TV (%) 25.07 2.60 30.56 3.11 27.95 4,88 40.11 2.33 46.36 3.52 45.65 2.87
BMD 404 28 466 33 428 47 554 30 620 38
612 30
TV, total callus volume; BV, bone volume; BV/TV, bone volume per callus
volume; BMD,
bone mineral density. Data is presented as the mean SEM; n = 7-8.
5 Taken together, the data indicated that blocking NGF/TrkA signalling did
not negatively
influence fracture healing.
Discussion and Conclusion
Abatement of fracture-related pain is an important issue in patient treatment.
However,
10 common treatment using NSAID appears disadvantageous for fracture
healing.
Here, it was investigated whether blockade of NGF/TrkA signalling for
analgesia influences
fracture healing. Our results indicate that bone regeneration is unaffected by
NGF/TrkA
signalling blockade using selective antibodies as callus formation and
maturation occurred
normally in animals treated with neutralizing anti-NGF or anti-TrkA
antibodies.
15 No significant differences in the callus composition assessed
histologically on days 7, 14 and
25 or by CT analyses performed on days 14 and 25 in animals treated with PBS,
anti-NGF
or anti-TrkA antibodies were observed. Furthermore, flexural rigidity was
unaffected
indicating that analgesia via NGF/TrkA blockade may be safe in relation to
bone healing.
There is evidence that NGF/TrkA signalling may regulate bone formation and
healing,
20 because both NGF and its receptor TrkA are expressed by bone cells
(Asaumi et al., (2000)
Bone, 26, 625-33.). In vitro data suggests an anti-apoptotic action of
NGF/TrkA in pre-
osteoblastic MC3T3-E1 cells (Mogi et al., (2000) Life Sci, 67, 1197-206) and
induction of
alkaline phosphatase in these cells after NGF-treatment, thereby promoting
osteoblast
differentiation (Yada et al., (1994) Biochem Biophys Res Commun, 205, 1187-
93). Studies in
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mice demonstrated TrkA expression during fracture healing in proliferating,
mature and
hypertrophic chondrocytes as well as in osteoblasts (Asaumi et al., (2000)
Bone, 26, 625-33.).
In addition, NGF expression was demonstrated in proliferating, mature and
hypertrophic
chondrocytes and in osteoblasts near the ossification front. Although these
findings imply a
function for NGF/TrkA signalling during fracture healing, the exact role has
not been
elucidated. It was demonstrated that topical NGF application improved fracture
healing in a
rib fracture model in rats (Grills et al., (1997) J Orthop Res, 15, 235-42).
Although the
underlying mechanism has not been extensively investigated, the authors
speculated that
increased sympathetic innervation stimulated the differentiation of
osteoprogenitor cells
towards chondrocytes (Grills et al., (1997) J Orthop Res, 15, 235-42). After
NGF-signalling
blockade by the application of a neutralizing anti-NGF monoclonal antibody or
small
molecular weight Trk kinase inhibitors during fracture healing, the formation
of a
significantly larger callus was reported (Koewler et al., (2007) J Bone Miner
Res, 22, 1732-
42: Ghilardi et al., (2011) Bone, 48, 389-98). These findings imply a slight
delay of the
healing process. However, our findings together with those of others (Koewler
et al., (2007) J
Bone Miner Res, 22, 1732-42: Ghilardi et al., (2011) Bone, 48, 389-98) imply a
subordinate
role, if any, of this signalling pathway in bone cells and their precursors
during fracture
healing.
To assess the analgesic effects of the antibody treatment, we determined the
mice's activity
and the GRF of the operated limb longitudinally. Significantly higher activity
was found for
the anti-TrkA antibody-treated mice compared to the PBS-treated animals on day
2 following
surgery. There was also a tendency for greater activity in the mice treated
with anti-NGF
antibody but this did not reach statistical significance. In contrast, we
found no statistically
significant differences when comparing both antibodies, thus indicating at
least an equivalent
analgesic effects. These activity findings are corroborated by the observation
of Koewler et at
J Bone Miner Res, 22, 1732-42., who reported a significant reduction of pain-
related
behaviour after the application of a neutralizing anti-NGF antibody (Koewler
et al., 2007 J
Bone Miner Res, 22, 1732-42). Generally, a trend for greater activity in the
mice treated with
anti-TrkA antibody in comparison with PBS treatment over the healing period
was found.
This may indicate that the mice receiving PBS were affected more by the
surgery and post-
surgical pain than animals that received one of the analgesic antibodies,
indicating a positive
effect of adequate early pain control on the mouse's welfare over the entire
healing process.
Loading of the operated limb represented by the vertical GRF did not show
significant inter-
group differences at any time point. This is also in agreement with the report
of others
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(Koewler et al., (2007) J Bone Miner Res, 22, 1732-42). We suggest that the
slight reduction
in the GRF may be independent of fracture-related pain. We assume that the
dissection of the
muscles during surgery alters function, thus causing the reduced loading.
In conclusion, the results indicate no negative effect of a blockade of
NGF/TrkA signalling on
fracture healing in rodents using specific antibodies because biomechanical
properties and the
callus composition were unaltered by antibody treatment.
Example 11 ¨ Stability data for low concentration aqueous formulation
The inventors have generated and tested a first low concentration liquid
formulation of an
anti-TrkA antibody adjusted to pH 6.0, comprising:
10mg/m1 of an anti-TrkA antibody comprising a light chain variable sequence
comprising:
SEQ ID NO: 5 and a heavy chain variable region comprising SEQ ID NO: 7;
25m1IVI Citrate;
150mM NaCl;
0.05% Tween 80.
The inventors have characterised the stability of the anti-TrkA antibody using
a number of
criteria at 5 C, 25 C and 40 C at time points (in months) TO, Ti, T2, T3, T6,
T9, T12, T18,
T24, T30.
In particular the antibody present in the formulation was characterised at the
relevant time
point, by determining the clarity, degree of coloration, degree of opalescence
and particulate
contamination (visible particles) of the formulation or a portion thereof;
light absorption
measurement of wavelength 280nm to determine the concentration of protein
present in the
formulation; by SDS-page gel visualisation to determine changes in the weight
and or
breakdown of the antibody; by ELISA to determine any change in the binding
properties of
the antibody; by HPLC-CEX to determine changes in the positive/negative
antibody species
make up in the formulation; by HPLC-IEF so as to determine changes in the
IsoElectroFocusing profile of the antibody by capillary electrophoresis
present in the
formulation, by HPLC-SEC analysis so as to determine changes in the the
antibody in the
formulation.
Each of the assessments of the properties of the formulation and the antibody
therein were
made using standard techniques.
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In each case comparison at least being made between one or more time points
and the TO
value and/or a previously established standard for the antibody used as a
standard material for
quality assurance and quality control purposes.
Stability data is provided for 5 C in figure 17, 25 C in figure 18 and 40 C in
figure 19.
Example 12 ¨ Stability data for high concentration aqueous formulation
The inventors have generated and tested a liquid formulation of an anti-TrkA
antibody
adjusted to pH 5.75 or 6.0, comprising:
100mg/m1 of an anti-TrkA antibody comprising a light chain variable sequence
comprising:
SEQ ID NO: 5 and a heavy chain variable region comprising SEQ ID NO: 7;
50mM Histidine;
150mM NaCl;
0.05% Tween 80.
The inventors have characterised the stability of the anti-TrkA antibody using
a number of
criteria at 5 C, 25 C and 40 C at time points (in months) TO, Ti, T2, T3, T6,
T9, T12, T18,
T24, T30.
In particular the antibody present in the formulation was characterised at one
or more time
points by determining changes in the clarity, degree of coloration, degree of
opalescence and
particulate contamination (visible particles) of the formulation, light
absorption measurement
of wavelength 280nm to determine the concentration of protein present in the
formulation; by
SDS-page gel visualisation to determine changes in the weight and or breakdown
of the
antibody; by ELISA to determine any change in the binding properties of the
antibody; by
HPLC-CEX to determine changes in the positive/negative antibody species make
up in the
formulation; by HPLC-IEF so as to determine changes in the IsoElectroFocusing
profile of
the antibody by capillary electrophoresis present in the formulation, by HPLC-
SEC analysis
to determine changes in the antibody in the formulation.. In each case
comparison between
made between each time point of the TO value and a previously established
standard for the
antibody used as a standard material for quality assurance and quality control
purposes.
Stability data is provided for the formulation adjusted to pH 5.75, at 5 C in
figure 20, 25 C in
figure 21 and 40 C in figure 22. Stability data is provided for the
formulation adjusted to pH
6, at 5 C in figure 23, 25 C in figure 24 and 40 C in figure 25.
Example 13 ¨ Stability data for high concentration aqueous formulation
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The inventors have generated and tested a first low concentration liquid
formulation of an
anti-TrkA antibody comprising:
150mg/m1 of an anti-TrkA antibody comprising a light chain variable sequence
comprising:
SEQ ID NO: 5 and a heavy chain variable region comprising SEQ ID NO: 7;
50mM Histidine;
150mM NaCl;
0.05% Tween 80.
The inventors have characterised the stability of the anti-TrkA antibody using
a number of
criteria at 5 C, 25 C and 40 C at time points (in months) TO, Ti, T2, T3, T6,
T9, T12, T18,
T24, T30.
In particular the antibody present in the formulation was characterised at one
or more time
points by determining changes in the clarity, degree of coloration, degree of
opalescence and
particulate contamination (visible particles) of the formulation, light
absorption measurement
of wavelength 280nm to determine the concentration of protein present in the
formulation; by
SDS-page gel visualisation to determine changes in the weight and or breakdown
of the
antibody; by ELISA to determine any change in the binding properties of the
antibody; by
HPLC-CEX to determine changes in the positive/negative antibody species make
up in the
formulation; by HPLC-IEF so as to determine changes in the IsoElectroFocusing
profile of
the antibody by capillary electrophoresis present in the formulation, by HPLC-
SEC analysis
to determine changes in the antibody in the formulation.
In each case comparison was made between each time point of the TO value and a
previously
established standard for the antibody used as a standard material for quality
assurance and
quality control purposes.
Stability data is provided for the formulation adjusted to pH 5.75, at 5 C in
figure 26, 25 C in
figure 27 and 40 C in figure 28. Stability data is provided for the
formulation adjusted to pH
6, at 5 C in figure 29, 25 C in figure 30 and 40 C in figure 31.
Example 14 ¨ Sub-visible particle, viscocity and syringeability of 100mg/m1
and
150mg/m1 formulations
In order to further characterise the properties of the high concentration
liquid formulations of
the present invention a further series of experiments were performed to
determine the
presence of sub-visible particles, as well as the viscosity and syringeability
of the
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formulations. Experiments were performed on samples stored at 5 C for between
9 and 12
months.
- Sub-visible particles
5
One means by which the efficacy of therapeutic proteins can be compromised is
by inducing
an unwanted immune response, resulting in antibody-mediated neutralization of
the protein's
activity or alterations in bioavailability. It is well established that
protein aggregates in
therapeutic protein products can enhance immunogenicity and such an effect is
therefore an
10 important risk factor to consider when assessing product quality. Such
aggregates appear as
sub-visible particles in the formulation.
Proteins usually aggregate from partially unfolded molecules, which can be
part of the native
state ensemble of molecules. Even though product formulations are developed to
maximize
15 and maintain the fraction of the protein molecules present in the native
state, significant
amounts of aggregates can form, especially over pharmaceutically-relevant time
scales and
under stress conditions. The levels and sizes of protein particles present in
a given product
can be changed by many factors relevant to commercial production of
therapeutic proteins.
20 It is known that large protein assemblies with repetitive arrays of
antigens, in which the
protein molecules have native conformation, are usually the most potent at
inducing immune
responses. Furthermore, efforts to develop more effective vaccines have shown
that adsorbing
antigenic proteins to nano- or microparticles comprised of other materials
(e.g., colloidal
aluminum salts or polystyrene) can greatly increase immunogenicity. Applying
these lessons
25 to therapeutic protein products, it has been argued that large
aggregates containing protein
molecules with native-like conformation pose the greatest risk of causing
adverse immune
responses in patients.
The standard test for sub-visible particulate analysis, specifies that
particulates >10 [tm in size
30 are controlled at or below 6000 particles per ml and particles >25 [im
are limited to at or
below 600 particles per ml.
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Sub visible particle analysis was performed using HIAC system (Beckman Coulter
Lifesciences) using the protocol provided by the manufacturer. Measurement is
based on light
obscuration and involves measurement of particles based on the geometry of the
particles.
Formulation Concentration Number of particles Number of
particles
>10um per ml >25um per ml
50mM Histidine, 150mM 100 mg/ml 262 20
NaC1, Tween 80 ¨ 0.05 %,
pH 5.75
50mM Histidine, 150mM 100 mg/ml 358 30
NaC1, Tween 80 ¨ 0.05 %,
pH 6
50mM Histidine, 150mM 150 mg/ml 1672 30
NaC1, Tween 80 ¨ 0.05 %,
pH 5.75
50mM Histidine, 150mM 150 mg/ml 288 24
NaC1, Tween 80 ¨ 0.05 %,
pH 6
Table 6 HIAC sub-visible analysis
All formulations are well under acceptance levels of sub-visible particles,
the observed lack of
particles provides a good indication of a high level of protein stability of
the formulated
antibody.
- Viscocity
The use of monoclonal antibodies as therapeutic agents generally requires the
delivery of a
large dose of the protein. Such concentrated solutions are associated with a
number of
challenges, including solution viscosity. Although no industry standards exist
for acceptable
viscosity of an injected solution, a value of between 25-30 mPA.s is generally
considered a
soft upper limit, with a viscosity of greater than 50 mPA.s generally
requiring non-standard
administration techniques/equipment to allow the injection to be performed.
Viscoscity analysis was performed using a Haake Reostress 1 (Thermo
Scientific) using the
protocol provided by the manufacturer.
Formulation Concentration Viscosity (mPa.$)
at 100
s-1
50mM Histidine, 150mM NaC1, Tween 80 ¨ 100 mg/ml 2.51
0.05 %, pH 5.75
50mM Histidine, 150mM NaC1, Tween 80 ¨ 100 mg/ml 3.03
0.05 %, pH 6
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50mM Histidine, 150mM NaC1, Tween 80 ¨ 150 mg/ml 4.94
0.05 %, pH 5.75
50mM Histidine, 150mM NaC1, Tween 80 ¨ 150 mg/ml 12.50
0.05 %, pH 6
Table 7 Viscosity
Surprisingly the 150mg/m1 formulation A (50mM Histidine, 150mM NaC1, Tween 80
¨ 0.05
%, pH 5.75, 150 mg/ml) showed exceptional properties having a viscosity close
to that of the
tested 100mg/m1 formulations. The second tested 150mg/m1 formulation (50mM
Histidine,
150mM NaC1, Tween 80 ¨ 0.05 %, pH 6, 150 mg/ml) also showed good properties
and better
that comparable marketed 150mg/m1 therapeutic antibody formulations.
- Syringeability
Syringeability is a key-product performance parameter of any parenteral dosage
form. This
refers to the ability of an injectable therapeutic to pass easily through a
hypodermic needle on
transfer from a vial prior to an injection. Syringeability includes such
factors as ease of
withdrawal, clogging and foaming tendencies, and accuracy of dose
measurements.
Syringeability can be affected by the needle geometry, i.e. inner diameter,
length, shape of the
opening, as well as the surface finish of the syringe (4). This is of
particular significance for
self-injection devices, such as pens and auto-injectors, which are equipped
with very thin
needles. Indeed, patients can use pen injectors which employ 29-31-G needles.
As far as pre-
filled syringes are concerned, common needle configurations for subcutaneous
dosing are
27 G and 25 G (4,5). While reducing the pain of injection, fine needles
require an increased
force to inject the drug. At the present time, no compendial testing
procedures are specified in
Pharmacopoeias, but in general a force less than 5-8 N is considered
acceptable for a human
injection.
Syringeability analysis was performed using a TA-XT Plus (Stable Micro
Systems) using the
protocol provided by the manufacturer.
Formulation Concentration Needle
Fmax(N)
oUaoe
50mM Histidine, 150mM NaC1, Tween 80 ¨ 0.05 150 mg/ml 27 0.86
%, pH 5.75
50mM Histidine, 150mM NaC1, Tween 80 ¨ 0.05 150 mg/ml 27 1.3
%, pH 6
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50mM Histidine, 150mM NaC1, Tween 80 ¨ 0.05 150 mg/ml 30 1.5
%, pH 5.75
50mM Histidine, 150mM NaC1, Tween 80 ¨ 0.05 150 mg/ml 30 3.3
%, pH 6
Table 8 Syringeability
The syringeability for the 27G needle data was good, while exceptional for 30G
needle.
