U.S. patent application number 14/924388 was filed with the patent office on 2016-05-12 for agonistic antibodies to trkc receptors and uses thereof.
The applicant listed for this patent is The Royal Institution for the Advancement of Learning/McGill University, The University of British Columbia. Invention is credited to Neil Cashman, Veronique Guillemard, Horacio Uri Saragovi.
Application Number | 20160130363 14/924388 |
Document ID | / |
Family ID | 45772036 |
Filed Date | 2016-05-12 |
United States Patent
Application |
20160130363 |
Kind Code |
A1 |
Saragovi; Horacio Uri ; et
al. |
May 12, 2016 |
AGONISTIC ANTIBODIES TO TRKC RECEPTORS AND USES THEREOF
Abstract
There are provided herein novel monoclonal antibodies that
selectively bind and/or activate TrkC receptors, pharmaceutical
compositions thereof and the use thereof for treating or preventing
conditions which require activation of TrkC, such as amyotrophic
lateral sclerosis and other neurodegenerative conditions and motor
neuron diseases. The monoclonal antibodies are useful to screen for
agents that share the same binding epitope on the TrkC
receptor.
Inventors: |
Saragovi; Horacio Uri;
(Montreal, CA) ; Guillemard; Veronique; (Bourail,
NC) ; Cashman; Neil; (Vancouver, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The University of British Columbia
The Royal Institution for the Advancement of Learning/McGill
University |
Vancouver
Montreal |
|
CA
CA |
|
|
Family ID: |
45772036 |
Appl. No.: |
14/924388 |
Filed: |
October 27, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13820715 |
Sep 19, 2013 |
9200080 |
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PCT/CA2010/001906 |
Nov 30, 2010 |
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14924388 |
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61379780 |
Sep 3, 2010 |
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Current U.S.
Class: |
424/135.1 ;
424/133.1; 424/136.1; 424/139.1; 530/391.1 |
Current CPC
Class: |
G01N 33/566 20130101;
A61P 25/28 20180101; C07K 16/2863 20130101; C07K 16/40 20130101;
A61K 45/06 20130101; C07K 2317/34 20130101; G01N 2500/02 20130101;
A61K 39/3955 20130101; G01N 2333/475 20130101; C07K 2317/75
20130101; C07K 2317/565 20130101; C07K 2317/14 20130101; A61K
2039/505 20130101; C07K 2317/55 20130101 |
International
Class: |
C07K 16/40 20060101
C07K016/40; A61K 45/06 20060101 A61K045/06; A61K 39/395 20060101
A61K039/395 |
Claims
1-41. (canceled)
42. A dosage form comprising a monoclonal antibody or a fragment,
portion, variant or derivative thereof, wherein said monoclonal
antibody, fragment, portion, variant or derivative thereof,
specifically binds to the juxtamembrane region, a peptide within
the juxtamembrane region, or a peptide having the amino acid
sequence ESTDNFILFDEVSPTPPI (SEQ ID NO: 1), of TrkC; and an
excipient or a carrier.
43. The dosage form of claim 42, wherein said monoclonal antibody
or said fragment, portion, variant or derivative thereof, modulates
TrkC.
44. The dosage form of claim 42, wherein said monoclonal antibody
or said fragment, portion, variant or derivative thereof, does not
bind and/or modulate TrkA, TrkB, p75NTR or a combination
thereof.
45. The dosage form of claim 42, wherein said monoclonal antibody
or said fragment, portion, variant or derivative thereof, binds
TrkC differently from NT-3.
46. The dosage form of claim 42, wherein said monoclonal antibody
or said fragment, portion, variant or derivative thereof, binds an
epitope of human TrkC with a sequence comprising the juxtamembrane
region or a portion thereof of human TrkC.
47. The dosage form of claim 42, wherein said monoclonal antibody
or said fragment, portion, variant or derivative thereof, comprises
all of the six CDRs from a monoclonal antibody produced by a
hybridoma deposited with the International Depositary Authority of
Canada on May 26, 2010 and having accession no. 090310-02.
48. The dosage form of claim 42, wherein said monoclonal antibody
or said fragment, portion, variant or derivative thereof, comprises
a single-chain antibody, a Fab fragment, F(ab').sub.2 fragment, Fd
fragment, dAb fragment, scFv, diabody, or bispecific antibody or
fragment, portion, or variant thereof.
49. The dosage form of claim 42, wherein said monoclonal antibody
or said fragment, portion, variant or derivative thereof, is
humanized, veneered or chimeric.
50. The dosage form of claim 42, wherein said monoclonal antibody
or said fragment, portion, variant or derivative thereof consists
of the monoclonal antibody produced by the hybridoma deposited with
the International Depositary Authority of Canada on May 26, 2010
and having accession no. 090310-02.
51. The dosage form of claim 42, wherein the dosage form is
formulated for parenteral, intravenous, subcutaneous,
intraperitoneal, oral, or intranasal administration.
52. The dosage form of claim 42, wherein the dosage form is
administered in combination with a second therapeutic agent.
53. The dosage form of claim 52, wherein the second therapeutic
agent is selected from the group consisting of a muscle relaxant, a
tranquilizer, an anticonvulsant, a nonsteroidal anti-inflammatory
agent, a benzodiazepine, riluzole and amitriptyline.
54. A method of treating amyotrophic lateral sclerosis (ALS) in a
subject, comprising administering to the subject a dosage form of
said monoclonal antibody or said fragment, portion, variant or
derivative thereof of claim 42.
55. A method of treating or preventing a neurodegenerative
condition or for treating a motor neuron disease in a subject,
comprising administering to the subject a dosage form of said
monoclonal antibody or said fragment, portion, variant or
derivative thereof of claim 42.
56. The method of claim 55, wherein the motor neuron disease is
progressive bulbar palsy, pseudobulbar palsy, primary lateral
sclerosis, progressive muscular atrophy, spinal muscular atrophy,
SMA Type I, SMA type II, SMA type III, Fazio-Londe disease, Kennedy
disease, congenital SMA with arthrogryposis, or post-polio
syndrome.
57. A complex comprising: a monoclonal antibody or, a fragment,
portion, variant or derivative thereof that recognizes the
juxtamembrane region, a peptide within the juxtamembrane region, or
a peptide having the amino acid sequence ESTDNFILFDEVSPTPPI (SEQ ID
NO: 1), of TrkC; and TrkC; wherein said monoclonal antibody or said
fragment, portion, variant or derivative thereof, specifically
binds to the juxtamembrane region, a peptide within the
juxtamembrane region, or a peptide having the amino acid sequence
ESTDNFILFDEVSPTPPI (SEQ ID NO: 1), of TrkC.
58. The complex of claim 57, wherein said monoclonal antibody or
said fragment, portion, variant or derivative thereof, comprises a
single-chain antibody, a Fab fragment, F(ab').sub.2 fragment, Fd
fragment, dAb fragment, scFv, diabody, or bispecific antibody or
fragment, portion, or variant thereof.
59. The complex of claim 57, wherein said monoclonal antibody or
said fragment, portion, variant or derivative thereof, is
humanized, veneered or chimeric.
60. The complex of claim 57, wherein said monoclonal antibody or
said fragment, portion, variant or derivative thereof consists of
the monoclonal antibody produced by the hybridoma deposited with
the International Depositary Authority of Canada on May 26, 2010
and having accession no. 090310-02.
Description
FIELD OF THE INVENTION
[0001] This invention relates to novel monoclonal antibodies that
selectively bind and activate TrkC receptors, pharmaceutical
compositions thereof and use thereof for treating or preventing
conditions which require activation of TrkC, such as amyotrophic
lateral sclerosis, and for inhibiting neurodegeneration.
Functionally active monoclonal antibodies can be used to screen for
TrkC-binding agents that share the epitope on the TrkC receptor, or
that allosterically interfere with the binding of the antibodies to
TrkC receptor.
BACKGROUND OF THE INVENTION
[0002] Trk tyrosine kinase receptors are multi-domain
single-transmembrane receptors that play an important role in a
wide spectrum of neuronal responses including survival,
differentiation, growth and regeneration. They are high affinity
receptors for neurotrophins, a family of protein growth factors
which includes nerve growth factor (NGF), brain derived
neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and
neurotrophins-4/5 (NT-4/5). NT-3, BDNF and NGF are essential growth
factors for the development and maintenance of the nervous system.
The neurotrophins are stable homodimers that bind to either or both
of two types of cell surface receptors termed p75.sup.NTR and
Trk.
[0003] Mature neurotrophins bind a selective Trk receptor with
relatively high affinity (e.g. TrkB-BDNF, TrkA-NGF and TrkC-NT-3).
TrkC is the preferred receptor for NT-3 and mediates the multiple
effects of NT-3, including neuronal death or survival, and cellular
differentiation. The Trk receptor has tyrosine kinase catalytic
activity that is associated with the survival and differentiation
of neurotrophic signals. Neurotrophin-induced Trk activity affords
trophic (growth/survival) responses via MAPK and Aid, whereas
PLC-.gamma. and fibroblast growth factor receptor substrate-2
(FRS-2) activity are involved in differentiation.
[0004] Trk receptors are widely distributed in the central nervous
system and the peripheral nervous system, and play a key role in
neuronal survival, differentiation and maintenance of proper
function. The relevance of Trk receptor function has been
demonstrated in a number of neurodegenerative models, including
stroke, spinal cord injury, optic nerve axotomy, glaucoma and
amyotrophic lateral sclerosis (ALS). For example, motor neurons
express the TrkC receptor, and therefore agents that activate TrkC
may be useful for preventing motor neuron degeneration in disorders
such as ALS. In addition, Trk receptors have also been implicated
in neoplasias, in particular TrkC has been associated with
progression in neuroblastoma, medulloblastoma, prostate cancer, and
breast cancer. In at least some of these diseases of abnormal cell
proliferation, activation of Trk receptors has proven beneficial by
induction of tumor death.
[0005] A Trk receptor ectodomain termed D5 comprises the main
neurotrophin binding site and is required for ligand-dependent
receptor activation. Such receptor sites that define ligand-binding
and functional-activation are termed "hot spots". Previously, it
has been demonstrated that artificial ligands, such as antibodies,
that bind to a receptor hot spot could be functionally active). For
example, an agonistic mAb 5C3 directed to a hot spot of the TrkA D5
domain has been reported (LeSauteur et al., 1996, J. Neurosci. 16:
1308-1316).
[0006] All mature neurotrophins also bind to p75.sup.NTR while the
precursor pro-neurotrophins bind p75.sup.NTR exclusively and do not
bind Trk receptors. It is known that the p75.sup.NTR receptor can
affect Trk-binding or function, although the mechanism is not fully
understood. It has been shown that p75.sup.NTR can unmask a cryptic
"hot spot" of Trk receptors, suggesting the notion of allosteric
regulation.
[0007] ALS is a progressive, fatal, neurodegenerative disease
caused by the degeneration of motor neurons, the nerve cells in the
central nervous system that control voluntary muscle movement. The
disorder causes muscle weakness and atrophy throughout the body as
both the upper and lower motor neurons degenerate, unable to send
messages to the muscles which then degenerate and atrophy due to
their inability to function. ALS is one of the most common
neuromuscular diseases worldwide, with one or two out of 100,000
people developing ALS each year. The disease most commonly strikes
people between 40 and 60 years of age. ALS is fatal, usually within
3 to 5 years of the onset of symptoms.
[0008] Current and prospective treatments for ALS are focused on
neuroprotective agents. One of the few available treatments is the
neuroprotective agent Riluzole, which is believed to reduce damage
to motor neurons by decreasing the release of glutamate, and has
been shown to lengthen patient survival by several months. There is
presently no cure for ALS. There is a need therefore for new and
effective therapies to prevent, inhibit or treat ALS.
SUMMARY OF THE INVENTION
[0009] The present invention relates to novel agonistic anti-TrkC
monoclonal antibodies (mAbs), pharmaceutical compositions thereof,
and use thereof for diagnosing, treating, or preventing conditions
(including symptoms, disorders, or diseases) which require
activation of TrkC, such as diseases involving neurodegeneration
and motor neuron diseases such as amyotrophic lateral sclerosis
(ALS).
[0010] In accordance with one embodiment of the present invention,
there are provided monoclonal antibodies that specifically bind the
juxtamembrane domain of the TrkC receptor, or a peptide sequence
within the juxtamembrane domain of TrkC. Fragments, portions,
variants or derivatives of the monoclonal antibodies which retain
the binding specificity or agonist activity of the full-length
antibodies are also provided herein. In an embodiment, the
antibodies provided herein specifically bind and/or activate
TrkC.
[0011] In another embodiment, the antibodies provided herein can
activate TrkC, i.e. can act as agonists of the TrkC receptor. In
one aspect, the antibodies provided herein are specific for TrkC
and do not bind and/or activate TrkA, TrkB and/or p75NTR receptors.
In a further aspect, the antibodies provided herein bind or
activate TrkC differently from NT-3. The TrkC may be any mammalian
TrkC, including but not limited to human TrkC, murine TrkC and rat
TrkC.
[0012] As used herein, the term "differently from" in relation to
binding or activation refers to binding to a different binding site
or binding differently to a site (e.g. binding more strongly),
and/or having a different effect once bound (e.g. different
activation properties, causing different biological consequences,
and so on). For example, the 2B7 antibody binds to a different
binding site on TrkC than the natural ligand NT-3 (e.g.
juxtamembrane domain rather than D5 domain), the 2B7 antibody
causes activation of some but not all of the TrkC signaling
pathways that NT-3 can activate (e.g. phospho-AKT is efficiently
activated but phospho-MAPK is only poorly activated), and 2B7
causes some but not all of the biological consequences that NT-3
can cause (e.g. neuronal survival but not neuronal
differentiation). The expression of p75NTR also regulates 2B7
binding and activity on TrkC differently from that of NT-3, as 2B7
binding and efficacy is reduced by p75NTR, whereas NT-3 binding is
enhanced by p75NTR. Thus binding or activating "differently" refers
to any difference in binding and/or activation properties or any
combination of these properties.
[0013] In yet another embodiment, the antibodies provided herein
specifically bind an epitope of TrkC with a sequence comprising the
juxtamembrane domain of TrkC. In an embodiment, the antibodies
provided herein specifically bind an epitope of TrkC located
between the transmembrane domain and the D5 domain. In another
embodiment, the antibodies provided herein bind selectively to
native TrkC on the cell surface, near the juxtamembrane region, and
do not bind to p75, TrkB, or TrkA. The TrkC may be any mammalian
TrkC, including but not limited to human TrkC, murine TrkC and rat
TrkC.
[0014] There is also provided herein a monoclonal antibody that is
produced from the hybridoma deposited with the International
Depositary Authority of Canada on May 26, 2010 and having accession
no. 090310-02 or from a progenitor cell thereof. In another aspect,
antibodies (or fragments, portions, variants or derivatives
thereof) binding to the same epitope as the monoclonal antibody
produced from the hybridoma deposited with the International
Depositary Authority of Canada on May 26, 2010 and having accession
no. 090310-02 are provided. The antibodies of the invention may be
humanized or modified in any way which provides benefit without
altering the binding properties or the biological activity of the
antibodies. Non-limiting examples of fragments, portions, variants
or derivatives of the antibodies include single chain antibodies
and Fab fragments thereof. A hybridoma that produces a monoclonal
antibody according to the invention is also encompassed herein.
[0015] There is further provided herein an antibody which comprises
complementarity-determining regions (CDRs) and/or hypervariable
domains of an antibody produced by a hybridoma having ATCC patent
deposit designation 090310-02.
[0016] In a further embodiment, pharmaceutical compositions
comprising the antibodies of the invention or the fragments,
portions, variants or derivatives thereof, and a pharmaceutically
acceptable carrier, are provided.
[0017] In accordance with another embodiment of the invention,
there is provided a method of activating TrkC in a subject,
comprising administering a therapeutically effective amount of a
monoclonal antibody of the invention or a fragment, portion,
variant or derivative thereof to the subject, such that TrkC is
activated in the subject. In an aspect, the subject is human and
the TrkC is human TrkC. In another aspect, the subject suffers from
a neurological or neurodegenerative condition which requires
activation of TrkC. For example, the subject may have been injured
by a wound, surgery, ischemia, infection, a metabolic disease,
malnutrition, a malignant tumor or a toxic drug, or may have
suffered a stroke, spinal cord injury or an axotomy. In one aspect,
the subject suffers from a neurodegenerative disease which is
amyotrophic lateral sclerosis (ALS). In an aspect, the subject
suffers from a motor neuron disease.
[0018] In an aspect, the antibodies of the invention may be
administered to a subject parenterally, intravenously,
subcutaneously or interperitoneally. In another aspect, the
antibodies of the invention may be administered in combination with
a second therapeutic agent, such as an agent for treating ALS.
[0019] In other embodiments, methods for treating ALS or for
treating or preventing a neurodegenerative condition or a motor
neuron disease in a subject, comprising administering a
therapeutically effective amount of a monoclonal antibody of the
invention, or a fragment, portion, variant or derivative thereof,
are provided.
[0020] In further embodiments, fragments, portions, variants or
derivatives of the monoclonal antibody produced by the hybridoma
having ATCC patent deposit designation 090310-02, said fragments,
portions, variants or derivatives binding specifically to the same
epitope as the monoclonal antibody, are provided herein. The
monoclonal antibody produced by the hybridoma having ATCC patent
deposit designation 090310-02 or antigen-binding fragments,
portions, variants or derivatives thereof may also be humanized,
veneered, or chimeric.
[0021] In some embodiments, the monoclonal antibody produced by the
hybridoma having ATCC patent deposit designation 090310-02 or
antigen-binding fragments, portions, variants or derivatives
thereof specifically bind TrkC receptor, or may specifically bind
TrkC receptor juxtamembrane domain, or the region between the
transmembrane domain and the D5 domain. In additional embodiments,
the monoclonal antibody produced by the hybridoma having ATCC
patent deposit designation 090310-02 or antigen-binding fragments,
portions, variants or derivatives thereof activate TrkC
receptor.
[0022] There are also provided herein methods of in vitro screening
for an agent which binds to TrkC receptor and can thereby affect
TrkC receptor biological activity, comprising combining the
antibodies or the fragments, portions, variants or derivatives of
the invention with TrkC receptor, in the presence or absence of a
candidate agent, and determining whether binding of the antibodies
to TrkC receptor or to a fragment or peptide thereof is reduced in
the presence of the candidate agent, wherein a reduction in
antibody binding in the presence of the candidate agent indicates
that said candidate agent binds directly to, or allosterically
alters TrkC, and can thereby modulate TrkC receptor biological
activity.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] Particular embodiments of the present invention will now be
explained by way of example and with reference to the accompanying
drawings, in which:
[0024] FIG. 1 shows that mAb 2B7 binds selectively to TrkC receptor
expressing cells. All data are representative of at least three
independent assays. (A-F) show FACScan binding assays with mAb 2B7.
The indicated cells were incubated with a saturating concentration
of mAb 2B7 (.about.65 nM or higher). (G, H) show dose-dependent
competition of mAb 2B7 binding by NT-3. NIH-TrkC or
NIH-TrkC.sup.+p75 cells were studied. Note reduced mAb 2B7
immunofluorescence in NIH-TrkC.sup.+p75 cells although they express
the same levels of TrkC (see panel K). These data suggest
modulation of the 2B7 TrkC-binding site, which is likely
allosteric. (I) shows comparable FACScan saturability profiles in
NIH TrkC cells for mAb 2B7 and 2B7 Fabs. (J) shows that 2B7
recognizes TrkC in Western blots, under non-reducing conditions.
(K) shows that total levels of TrkC receptor are similar in
NIH-TrkC and NIH-TrkC.sup.+p75 cells. NIH-TrkC.sup.+p75 cells
express high levels of p75. It is noted that the doublet seen for
TrkC may be differentially glycosylated receptor.
[0025] FIG. 2 shows that TrkC, Akt and MAPK activation are induced
by mAb 2B7 or mAb 2B7 Fabs. NIH TrkC cells were treated with the
indicated ligands for 12 minutes and cell lysates were analyzed by
Western blot. (A) shows anti-P-Tyr, anti-P-Akt, anti-P-MAPK, and
total Trk (anti-sera 203, from Dr. David Kaplan). A representative
experiment is shown. (B) shows a summary of data quantification by
densitometry standardized to total Trk and presented as % relative
to 10 nM NT-3 (Anti-P-Tyr, n=3; anti-p-Akt, n=3; anti-p-MAPK n=2).
(C) shows anti-p-Akt blots as in (A), wherein NIH TrkC cells were
treated with the indicated ligands for 12 or 30 minutes.
[0026] FIG. 3 shows that co-expression of p75 with TrkC hinders the
trophic protection of mAb2B7. The survival of NIH-TrkC or
NIH-TrkC.sup.+p75 cells was tested in MTT assays after culture in
SFM supplemented with the indicated ligands or controls (5%
serum=100%, untreated=0%, n=4 for each assay). Data are
representative from 3 independent experiments. * indicates
statistical significance, and ns indicates not statistically
significant.
[0027] FIG. 4 shows that mAb 2B7 does not induce differentiation of
nnr5 TrkC cells. (A) shows representative pictures of the
differentiation of nnr5-TrkC cells in response to treatment with
the indicated ligand for 48-72 hours. After treatment with the
ligand, cells were fixed and immunostained with MAP-2 antibody
(Chemicon) followed by goat anti-rabbit Cy3 (Jackson
Immunochemicals) and analyzed as described (Ivanisevic et al.,
2003, Oncogene 22 5677-5685). (B) shows a quantitative summary
(.+-.SD) of 3 independent experiments. Cells were plated with the
indicated treatments or controls, and differentiation was scored as
% of cells with neurites (>2 cell body long). * indicates
statistical significance relative to 100 nM mIgG, p.ltoreq.0.05.
The figure shows that 50 pM NT-3 affords significantly lower
cellular differentiation than 10 nM NT-3, while a combination of 50
pM NT-3+10 nM NGF (as a p75 ligand) affords significantly higher
cellular differentiation than each ligand alone, and achieves
levels comparable to 10 nM NT-3.
[0028] FIG. 5 shows that mAb 2B7 does not induce differentiation of
primary neuronal cultures. Hippocampal neurons were plated with
indicated treatments, and differentiation was scored as % of
control cells. (A) shows a Sholl analysis of dendritic
intersections as a % of untreated control cells. NT-3 and BDNF have
a significant effect on the morphology of the dendritic arbor, with
a higher number of primary dendrites and branching. NT-3 has longer
dendrites compared to BDNF, whereas mAb 2B7 did not have any effect
on the development of the dendritic arborization. In (B) the
average of the total length of neurites and average of branch
length per branch order was further assessed using NeuronJ. BDNF
and NT-3 significantly increased the total length of neurites
compared to control-untreated cells, but NT-3 treatment was more
effective. The mAb 2B7 did not have any effect on the growth of
neurites compared to control. All comparisons are statistically
significant (p<0.05) except control versus 2B7 treatment. (C)
shows analysis of the branch length per branch order. MAb 2B7 did
not cause any change in the elongation of neurites. NT-3 increases
the length of both secondary (p<0.001) and tertiary order
branches (p<0.05), whereas BDNF increases the length of
secondary order branches (p<0.01). Asterisk indicates
p<0.05.
[0029] FIG. 6 shows MAb 2B7 immunostaining of primary neuronal
cultures. Representative pictures of E18 primary hippoccampal
neurons immunostained with mAb 2B7 are shown, wherein A, B, C show
confocal images and D shows epifluoresence. Control immunostaining
with no 2B7 primary antibody results in no detectable fluorescent
signal (inset square, panel A). The image in panel B is a
magnification of the area in yellow ellipse from panel A.
[0030] FIG. 7 shows survival distribution function in G93A mutant
SOD1 transgenic mice. Survival plot showing % of mice surviving at
the indicated age (days). The indicated treatment was started at
age .about.day 98 (when early symptoms are evident, e.g. poor hind
leg reflex in the cohort). Route: intraperitoneal injections, three
times a week, 0.5 mg/kg each dose (.about.10 micrograms per mouse
per injection). Duration of treatment 12 weeks. Control=saline
vehicle. TX1=treatment with 2B7 Fab monovalent. TX7=treatment with
2B7 IgG.
[0031] FIG. 8 shows average rotarod scores, indicative of motor
function, in G93A mutant SOD1 transgenic mice. Average rotarod
performance (in seconds) at the indicated age (weeks).
Control=saline vehicle. TX1=treatment with 2B7 Fab monovalent.
TX7=treatment with 2B7 IgG.
DETAILED DESCRIPTION OF THE INVENTION
[0032] The present invention provides monoclonal antibodies (mAbs)
that selectively target TrkC. Agonist monoclonal antibodies that
activate TrkC, pharmaceutical compositions thereof, and use thereof
for treating diseases involving neurodegeneration and/or for
providing neuroprotection, including motor neuron diseases such as
amyotrophic lateral sclerosis (ALS), are provided herein.
[0033] The present invention is based, at least in part, on the
principle that monoclonal antibodies that specifically bind to the
TrkC receptor are sufficient to induce the activation of the
receptor, and therefore induce biological responses similar to
those mediated by, for example, NT-3. Monoclonal antibodies such as
those provided herein can act as agonists that mimic the biological
effects of receptor-ligand interactions.
[0034] There is provided herein a monoclonal antibody (mAb) 2B7
targeting the juxtamembrane domain of TrkC receptors. MAb 2B7 binds
to murine and human TrkC receptors and is a functional agonist that
affords activation of TrkC, AKT and MAPK. These signals result in
cell survival but not in cellular differentiation. Monomeric 2B7
Fabs also afford cell survival. Binding of 2B7 mAb and 2B7 Fabs to
TrkC are blocked by NT-3 in a dose-dependent manner, but not by
pro-NT-3. Expression of p75.sup.NTR co-receptors on the cell
surface block the binding and function of mAb 2B7, whereas NT-3
binding and function are enhanced. MAb 2B7 defines a previously
unknown neurotrophin receptor functional hot spot. The antibody
exclusively generates survival signals and can be activated by
non-dimeric ligands.
[0035] The hot spot defined by MAb 2B7, and other "hot spots" on
Trk receptors, should allow different modes of activation for the
receptors. Agents that bind at such hot spots and modulate receptor
activity might be useful for treating disorders such as
neurodegeneration or cancer. Agents that bind at such hot spots and
modulate receptor activity might also be useful as a tool to screen
for agents that also bind to the same hot spots.
[0036] In one aspect, the present invention provides monoclonal
antibodies that bind specifically to TrkC. In certain embodiments
the antibodies bind to human TrkC. In another embodiment the
antibodies bind to rat and/or mouse TrkC. In certain embodiments,
the antibodies bind preferentially to human TrkC, and do not bind
to TrkA, TrkB and/or p75NTR receptors.
[0037] In one embodiment the antibodies provided herein are also
agonists of TrkC.
[0038] In another embodiment the antibodies provided herein bind to
the ESTDNFILFDEVSPTPPI peptide, which is near the D5 domain. In an
embodiment, the receptor site for antibody binding is located
between the transmembrane domain and the D5 domain of TrkC. In
another embodiment, the receptor site for antibody binding is
located in the extracellular domain of TrkC. In one embodiment, the
antibodies of the invention bind selectively to native TrkC on the
cell surface, near the juxtamembrane region, and do not bind to
p75, TrkB, or TrkA. In another aspect, the present invention
provides any monoclonal antibodies with the properties described
herein that bind and/or activate human, rat or mouse TrkC.
[0039] In certain embodiments, these antibodies bind and/or
activate TrkC with an ED50 in the range of about 10 pM to about 500
nM, for example in the range of about 10 pM to about 1 nM,
including in the range of about 10 pM to about 500 pM and the range
of about 10 pM to about 100 pM.
[0040] In another aspect, the present invention provides monoclonal
antibodies with any of the properties described herein that bind
one or more specific epitopes near the juxtamembrane region of
human TrkC. In yet another aspect, the present invention provides
monoclonal antibodies with any of the properties described herein
that bind specifically to the region between the transmembrane
domain and the D5 domain of human, rat or mouse TrkC.
[0041] In yet another aspect, the present invention provides
hybridomas that produce any of the monoclonal antibodies of the
invention. For example, the hybridoma that was deposited with the
International Depositary Authority of Canada on May 26, 2010 and
given accession no. 090310-02 is provided. In still another aspect,
the present invention provides the monoclonal antibody produced by
the hybridoma that was deposited with the IDAC on May 26, 2010 and
given accession no. 090310-02, or antigen-binding fragments
thereof. The present invention also provides antibodies that block
the binding of this antibody and therefore share the same binding
epitope on human, rat or mouse TrkC.
[0042] It is to be understood that the monoclonal antibodies of the
invention can be prepared by any known method. For example, they
can be prepared using synthetic, recombinant or hybridoma
technology (e.g., as described in Antibodies: A Laboratory Manual,
Ed. by E. Harlow and D. Lane, Cold Spring Harbor Laboratory Press,
1988 or Monoclonal Antibodies: Principles and Practice by J. W.
Goding, Academic Press, 1996). In particular it will be appreciated
that the antibodies provided herein can be prepared by initially
immunizing an animal with human TrkC or a derivative thereof (e.g.,
a recombinant protein that includes the desired domain of human
TrkC, such as recombinant human TrkC juxtamembrane region) and then
preparing monoclonals from suitably prepared hybridomas. Those
skilled in the art will appreciate that suitable immunogens can be
prepared using standard recombinant technology (e.g., see Protocols
in Molecular Biology Ed. by Ausubel et al., John Wiley & Sons,
New York, N.Y., 1989 and Molecular Cloning: A Laboratory Manual Ed.
by Sambrook et al., Cold Spring Harbor Press, Plainview, N. Y.,
1989, the contents of which are incorporated herein by
reference).
[0043] In certain embodiments, the immunogen used does not include
any of the amino acids that are found in the intracellular domain
of TrkC or in the D5 domain of TrkC. Once suitable immunogens have
been prepared, the immunogens are injected into any of a wide
variety of animals (e.g., mice, rats, rabbits, etc.) and antibodies
are prepared using standard, art-recognized techniques.
[0044] When using the antibodies provided herein for therapeutic
purposes it may prove advantageous to use a humanized or veneered
version of the antibody of interest to reduce any potential
immunogenic reaction. In general, humanized or veneered antibodies
minimize unwanted immunological responses that limit the duration
and effectiveness of therapeutic applications of non-human
antibodies in human recipients.
[0045] A number of methods for preparing humanized antibodies
comprising an antigen binding portion derived from a non-human
antibody have been described in the art. In particular, antibodies
with rodent variable regions and their associated
complementarity-determining regions (CDRs) fused to human constant
domains have been described, as have rodent CDRs grafted into a
human supporting framework region (FR) prior to fusion with an
appropriate human antibody constant domain. Completely human
antibodies are particularly desirable for therapeutic treatment of
human patients. Such antibodies can be produced using transgenic
mice that are incapable of expressing endogenous immunoglobulin
heavy and light chain genes, but which can express human heavy and
light chain genes. It should be understood that partially or
completely humanized versions of the antibodies provided herein are
encompassed by the present invention.
[0046] Veneered versions of the antibodies provided herein may also
be used in the methods of the present invention. The process of
veneering involves selectively replacing FR residues from, e.g., a
murine heavy or light chain variable region, with human FR residues
in order to provide an antibody that comprises an antigen binding
portion which retains substantially all of the native FR protein
folding structure. Veneering techniques are based on the
understanding that the antigen binding characteristics of an
antigen binding portion are determined primarily by the structure
and relative disposition of the heavy and light chain CDR sets
within the antigen-association surface (e.g., see Davies et al.,
Ann Rev. Biochem. 59:439, 1990). Thus, antigen association
specificity can be preserved in a humanized antibody only wherein
the CDR structures, their interaction with each other and their
interaction with the rest of the variable region domains are
carefully maintained. By using veneering techniques, exterior
(e.g., solvent-accessible) FR residues which are readily
encountered by the immune system are selectively replaced with
human residues to provide a hybrid molecule that comprises either a
weakly immunogenic, or substantially non-immunogenic veneered
surface. It should be understood that veneered versions of the
antibodies provided herein are encompassed by the present
invention.
[0047] The term "antibody", as used herein, broadly refers to any
immunoglobulin (Ig) molecule comprised of four polypeptide chains,
two heavy (H) chains and two light (L) chains, or any functional
fragment, mutant, variant, or derivative thereof, which retains the
essential epitope binding features of an Ig molecule. Such mutant,
variant, or derivative antibody formats are known in the art. A
"monoclonal antibody" as used herein is intended to refer to a
preparation of antibody molecules, which share a common heavy chain
and common light chain amino acid sequence, in contrast with
"polyclonal" antibody preparations that contain a mixture of
different antibodies. Monoclonal antibodies can be generated by
several novel technologies like phage, bacteria, yeast or ribosomal
display, as well as classical methods exemplified by
hybridoma-derived antibodies (e.g., an antibody secreted by a
hybridoma prepared by hybridoma technology, such as the standard
Kohler and Milstein hybridoma methodology ((1975) Nature
256:495-497).
[0048] In a full-length antibody, each heavy chain is comprised of
a heavy chain variable region (abbreviated herein as HCVR or VH)
and a heavy chain constant region. The heavy chain constant region
is comprised of three domains, CH1, CH2 and CH3. Each light chain
is comprised of a light chain variable region (abbreviated herein
as LCVR or VL) and a light chain constant region. The light chain
constant region is comprised of one domain, CL. 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).
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. Immunoglobulin molecules can
be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class
(e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass.
[0049] The term "antigen-binding portion" or "antigen-binding
fragment" of an antibody (or simply "antibody portion" or "antibody
fragment"), as used herein, refers to one or more fragments of an
antibody that retain the ability to specifically bind to an antigen
(e.g., juxtamembrane region domain of TrkC). It has been shown that
the antigen-binding function of an antibody can be performed by
fragments of a full-length antibody. Such antibody embodiments may
also be bispecific, dual specific, or multi-specific formats;
specifically binding to two or more different antigens. Examples of
binding fragments encompassed within the term "antigen-binding
portion" of an antibody include (i) a Fab fragment, a monovalent
fragment consisting of the VL, VH, CL and CH1 domains; (ii) a
F(ab').sub.2 fragment, a bivalent fragment comprising two Fab
fragments linked by a disulfide bridge at the hinge region; (iii) a
Fd fragment consisting of the VH and CH1 domains; (iv) a Fv
fragment consisting of the VL and VH domains of a single arm of an
antibody, (v) a dAb fragment (Ward et al., (1989) Nature
341:544-546, Winter et al., PCT publication WO 90/05144 A1, herein
incorporated by reference), which comprises a single variable
domain; and (vi) an isolated complementarity determining region
(CDR). Furthermore, although the two domains of the Fv fragment, VL
and VH, are coded for by separate genes, they can be joined, using
recombinant methods, by a synthetic linker that enables them to be
made as a single protein chain in which the VL and VH regions pair
to form monovalent molecules (known as single chain Fv (scFv); see
e.g., Bird et al. (1988) Science 242:423-426; and Huston et al.
(1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chain
antibodies are also intended to be encompassed within the present
invention. Other forms of single chain antibodies, such as
diabodies are also encompassed. Diabodies are bivalent, bispecific
antibodies in which VH and VL domains are expressed on a single
polypeptide chain, but using a linker that is too short to allow
for pairing between the two domains on the same chain, thereby
forcing the domains to pair with complementary domains of another
chain and creating two antigen binding sites (see e.g., Holliger,
P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak,
R. J., et al. (1994) Structure 2:1121-1123). Such antibody binding
portions are known in the art (Kontermann and Dubel eds., Antibody
Engineering (2001) Springer-Verlag. New York. 790 pp. (ISBN
3-540-41354-5).
[0050] It should be understood that the antibodies of the invention
include fragments, portions, variants or derivatives thereof, such
as single-chain antibodies or Fab fragments, that retain the same
binding properties (e.g. specificity or affinity) of the
full-length antibodies.
[0051] The antibodies of the invention also include functional
equivalents that include polypeptides with amino acid sequences
substantially the same as the amino acid sequence of the variable
or hypervariable regions of the antibodies of the present
invention. "Substantially the same" amino acid sequence includes an
amino acid sequence with at least 70%, preferably at least 80%, and
more preferably at least 90% identity to another amino acid
sequence when the amino acids of the two sequences are optimally
aligned and compared to determine exact matches of amino acids
between the two sequences. "Substantially the same" amino acid
sequence also includes an amino acid sequence with at least 70%,
preferably at least 80%, and more preferably at least 90% homology
to another amino acid sequence, as determined by the FASTA search
method in accordance with Pearson and Lipman, Proc. Natl. Acad.
Sci. USA 85, 2444-8 (1988).
[0052] In addition, proteins and non-protein agents may be
conjugated to the antibodies by methods that are known in the art.
Conjugation methods include direct linkage, linkage via covalently
attached linkers, and specific binding pair members (e.g.,
avidin-biotin). For example, an antibody of the invention may
include modifications that retain specificity for juxtamembrane
region of TrkC. Such modifications include, but are not limited to,
conjugation to an effector molecule such as another therapeutic
agent or conjugation to detectable reporter moieties. For example
conjugation of a therapeutic agent to an antibody of the invention
may be used to deliver the therapeutic agent, e.g. a drug or
pro-drug, to a cell via the TrkC receptor. Many anti-cancer
therapeutics are known in the art, and it is contemplated that such
therapeutics, for example, may be conjugated to an antibody of the
invention for TrkC-mediated delivery in a subject. Modifications
that extend antibody half-life (e.g., pegylation) are also
included.
[0053] In certain embodiments, the antibodies presented herein are
characterized for their binding activities to human TrkC protein
(e.g., using ELISA, FACS, Surface Plasmon Resonance, and/or other
methods known in the art). In certain embodiments binding to human
TrkC proteins that are expressed on a cell surface may also be
assessed (e.g., using cell lines, as known in the art), as well as
determination of functional properties using said cell lines.
Antibodies may also be tested for their cross-species binding
activity; this allows monoclonal antibodies that bind TrkC from
more than one species to be identified. In an embodiment, the mAbs
bind to both human TrkC and rat TrkC. These antibodies are of
interest since they can be tested in animal models with the
knowledge that they can also be applied in human clinical
trials.
[0054] In certain embodiments it may prove advantageous to further
characterize the binding properties of any given monoclonal
antibody. In particular, one may use a competition assay (e.g., an
ELISA) to determine whether the antibodies block the interaction of
TrkC and NT-3. One may also assess whether the antibodies bind
non-human TrkC and/or human TrkA, TrkB or p75NTR. Mapping of the
relative antibody binding epitopes on TrkC (human or other) may
also be conducted, e.g., by examining the activity of each
individual antibody in blocking the binding of other antibodies to
TrkC. For example, the observation that two antibodies block each
other's binding suggests these antibodies may bind to the same
epitope or overlapping epitopes on TrkC. Methods for mapping
epitopes are well-known in the art.
[0055] In certain embodiments, the antibodies presented herein are
characterized for their functional ability to activate TrkC which
may be human or non-human TrkC (e.g. murine, rat, chicken, etc).
Any agonist assay may be used. For example, MTT-based
survival/proliferation assays in cell lines may be used. These
assays and other useful assays are known in the art and will be
recognized by those skilled in the art.
Pharmaceutical Compositions
[0056] In one aspect, the monoclonal antibodies provided herein are
administered to a subject in order to activate TrkC, in accordance
with the present invention. In another aspect, the monoclonal
antibodies provided herein are administered in the context of a
pharmaceutical composition, that contains a therapeutically
effective amount of one or more antibodies together with one or
more other ingredients known to those skilled in the art for
formulating pharmaceutical compositions. As used herein, the terms
"pharmaceutically effective amount" or "therapeutically effective
amount" mean the total amount of each active ingredient of the
pharmaceutical composition or method that is sufficient to show a
meaningful patient benefit, e.g., treatment, prevention or
amelioration of a condition which requires TrkC activation. When
applied to an individual active ingredient that is administered
alone, the term refers to that ingredient alone. When applied to a
combination of active ingredients, the term refers to combined
amounts of the active ingredients that result in the therapeutic
effect, whether administered in combination, serially or
simultaneously.
[0057] In certain embodiments of the invention, inventive
antibodies are administered with a weekly dose in the range of
about 0.1 to about 1000 mg/kg body weight, or about 1 to about 500
mg/kg body weight, in certain embodiments about 10 to about 300
mg/kg body weight. Doses may be administered as a single regimen or
as a continuous regimen divided by two or more doses over the
course of a day or week. Delivery may be as a bolus or in certain
embodiments as a gradual infusion (e.g., by injection over 30 mins)
or as a continuous infusion (e.g. days) using minipumps, or using
slow release delivery particles or cells that have been engineered
to secrete the inventive antibodies. In certain embodiments one or
more higher doses (e.g., 2, 3 or 4 fold higher) may be administered
initially followed by one or more, lower maintenance doses. The
higher dose(s) may be administered at the onset of treatment only
or at the beginning of each treatment cycle. These dosage levels
and other dosage levels herein are for intravenous or
intraperitoneal administration. The skilled person will readily be
able to determine the dosage levels required for a different route
of administration. It will be appreciated that, in general, the
precise dose used will be as determined by the prescribing
physician and will depend not only on the weight of the subject and
the route of administration, but also on the age of the subject and
the severity of the symptoms.
[0058] Additional ingredients useful in preparing pharmaceutical
compositions in accordance with the present invention include, for
example, carriers (e.g., in liquid or solid form), flavoring
agents, lubricants, solubilizers, suspending agents, fillers,
glidants, compression aids, binders, tablet-disintegrating agents,
encapsulating materials, emulsifiers, buffers, preservatives,
sweeteners, thickening agents, coloring agents, viscosity
regulators, stabilizers or osmo-regulators, or combinations
thereof. Liquid pharmaceutical compositions preferably contain one
or more monoclonal antibodies of the invention and one or more
liquid carriers to form solutions, suspensions, emulsions, syrups,
elixirs, or pressurized compositions. Pharmaceutically acceptable
liquid carriers include, for example water, organic solvents,
pharmaceutically acceptable oils or fat, or combinations thereof.
The liquid carrier can contain other suitable pharmaceutical
additives such as solubilizers, emulsifiers, buffers,
preservatives, sweeteners, flavoring agents, suspending agents,
thickening agents, colors, viscosity regulators, stabilizers or
osmo-regulators, or combinations thereof. If the liquid formulation
is intended for pediatric use, it is generally desirable to avoid
inclusion of alcohol.
[0059] Examples of liquid carriers suitable for oral or parenteral
administration include water (preferably containing additives such
as cellulose derivatives such as sodium carboxymethyl cellulose),
alcohols or their derivatives (including monohydric alcohols or
polyhydric alcohols such as glycols) or oils (e.g., fractionated
coconut oil and arachis oil). For parenteral administration the
carrier can also be an oily ester such as ethyl oleate and
isopropyl myristate. The liquid carrier for pressurized
compositions can be halogenated hydrocarbons or other
pharmaceutically acceptable propellant.
[0060] Solid pharmaceutical compositions preferably contain one or
more solid carriers, and optionally one or more other additives
such as flavoring agents, lubricants, solubilizers, suspending
agents, fillers, glidants, compression aids, binders or
tablet-disintegrating agents or an encapsulating material. Suitable
solid carriers include, for example, calcium phosphate, magnesium
stearate, talc, sugars, lactose, dextrin, starch, gelatin,
cellulose, methyl cellulose, sodium carboxymethyl cellulose,
polyvinylpyrrolidine, low melting waxes or ion exchange resins, or
combinations thereof. In powder pharmaceutical compositions, the
carrier is preferably a finely divided solid which is in admixture
with the finely divided active ingredient. In tablets, the active
ingredient(s) are generally mixed with a carrier having the
necessary compression properties in suitable proportions, and
optionally, other additives, and compacted into the desired shape
and size.
[0061] In some embodiments of the invention, pharmaceutical
compositions are provided in unit dosage form, such as tablets or
capsules. In such form, the composition is sub-divided in unit dose
containing appropriate quantities of the active ingredient(s). The
unit dosage forms can be packaged compositions, for example
packeted powders, vials, ampoules, pre-filled syringes or sachets
containing liquids. The unit dosage form can be, for example, a
capsule or tablet itself, or it can be an appropriate number of any
such compositions in package form. Thus, the present invention also
provides a pharmaceutical composition in unit dosage form for
activating TrkC, where the composition contains a therapeutically
effective unit dosage of at least one monoclonal antibody of the
invention. As one skilled in the art will recognize, the certain
therapeutically effective unit dosage will depend on the method of
administration. The present invention also provides a therapeutic
package for dispensing the monoclonal antibodies of the invention
to an individual being treated for a condition which requires TrkC
activation. In some embodiments, the therapeutic package contains
one or more unit dosages of at least one inventive monoclonal
antibody, a container containing the one or more unit dosages, and
labeling directing the use of the package for treatment. In certain
embodiments, the unit dose is in tablet or capsule form. In some
cases, each unit dosage is a therapeutically effective amount.
[0062] According to the present invention, monoclonal antibodies of
the invention may be administered alone to modulate TrkC activity.
Alternatively the antibodies may be administered in combination
with (whether simultaneously or sequentially) one or more other
pharmaceutical agents useful in the treatment, prevention or
amelioration of one or more other conditions (including symptoms,
disorders, or diseases) which require TrkC activity. For example,
other pharmaceutical agents that can modulate TrkC activity may be
used in combination with the monoclonal antibodies of the
invention, including other activators of TrkC, including but not
limited to NT-3 derivatives and compositions.
[0063] Additionally or alternatively, the monoclonal antibodies may
be used in conjunction with other pharmaceutical agents that are
useful in the treatment, prevention or amelioration of neurological
disorders and diseases or of motor neuron disorders and diseases.
In certain embodiments, the monoclonal antibodies are combined with
agents that are useful in the treatment, prevention or amelioration
of disorders and diseases caused by injuries to the nervous system
(e.g., by wound, surgery, ischemia, infection, metabolic diseases,
malnutrition, malignant tumor, toxic drugs, etc.), particularly to
the motor neurons. It is to be understood that any suitable agent
known in the art may be used, including those listed in the
Physicians' Desk Reference, 55.sup.th Edition, 2001, published by
Medical Economics Company, Inc. at Monvale, N.J., the relevant
portions of which are incorporated herein by reference.
[0064] Currently the only prescribed drug approved by the U.S. Food
and Drug Administration to treat ALS is the drug riluzole
(Rilutek.RTM.), which prolongs life by 2-3 months but does not
relieve symptoms. The drug reduces the body's natural production of
the neurotransmitter glutamate, which carries signals to the motor
neurons. Scientists believe that too much glutamate can harm motor
neurons and inhibit nerve signaling.
[0065] Other treatments are symptomatic. Muscle relaxants such as
baclofen, tizanidine, and the benzodiazepines may reduce
spasticity. Glycopyrrolate and atropine may reduce the flow of
saliva. Quinine or phenytoin may decrease cramps. Anticonvulsants
and nonsteroidal anti-inflammatory drugs may help relieve pain, and
other drugs can be prescribed to treat depression. Tranquilizers
often help with sleeping problems. Some individuals with PPS
develop sleep apnea (a potentially life-threatening condition
characterized by interruptions of breathing during sleep), which
can be treated with decongestant therapy, assisted breathing at
night, or surgery to remove any blockage to the airway. Panic
attacks over fears of choking to death can be treated with
benzodiazepines. Botulinum toxin may be used to treat jaw spasms or
drooling. Amitriptyline and other drugs can help control excess
drooling. Some individuals may eventually require stronger
medicines such as morphine to cope with musculoskeletal
abnormalities or pain, and opiates are used to provide comfort care
in terminal stages of the disease.
[0066] In one embodiment, the monoclonal antibodies may be used in
conjunction with riluzole and/or with symptomatic treatments for
motor neuron diseases, such as for example muscle relaxants,
tranquilizers, anticonvulsants, nonsteroidal anti-inflammatory
drugs, benzodiazepines and amitriptyline.
[0067] In the therapeutic methods provided herein, the monoclonal
antibodies may be delivered to a subject using any appropriate
route of administration including, for example, parenteral,
intravenous, topical, nasal, oral (including buccal or sublingual),
rectal or other modes. In general, the antibodies may be formulated
for immediate, delayed, modified, sustained, pulsed, or
controlled-release delivery.
[0068] In certain embodiments, the antibodies are formulated for
delivery by injection. In such embodiments, administration may be,
for example, intracavernous, intravenous, intra-arterial,
intraperitoneal, intrathecal, intraventricular, intraurethral,
intrasternal, intracranial, intramuscular or subcutaneous, or via
infusion or needle-less injection techniques. For such parenteral
administration, the antibodies of the invention may be prepared and
maintained in conventional lyophilized formulations and
reconstituted prior to administration with a pharmaceutically
acceptable saline solution, such as a 0.9% saline solution. The pH
of the injectable formulation can be adjusted, as is known in the
art, with a pharmaceutically acceptable acid, such as
methanesulfonic acid. Other acceptable vehicles and solvents that
may be employed include Ringer's solution and U.S.P. In addition,
sterile, fixed oils are conventionally employed as a solvent or
suspending medium. For this purpose any bland fixed oil can be
employed including synthetic mono- or diglycerides. In addition,
fatty acids such as oleic acid are used in the preparation of
injectables. The injectable formulations can be sterilized, for
example, by filtration through a bacterial-retaining filter, or by
incorporating sterilizing agents in the form of sterile solid
compositions which can be dissolved or dispersed in sterile water
or other sterile injectable medium prior to use.
[0069] In order to prolong the effect of the inventive antibody, it
may be desirable to slow its absorption from an intramuscular or
subcutaneous injection. Delayed absorption of such an administered
antibody may be accomplished by dissolving or suspending the agent
in an oil vehicle. Injectable depot forms are made by forming
microencapsule matrices of the antibody in biodegradable polymers
such as polylactide-polyglycolide. Depending upon the ratio of
antibody to polymer and the nature of the particular polymer
employed, the rate of antibody release can be controlled. Examples
of other biodegradable polymers include poly(orthoesters) and
poly(anhydrides). Depot injectable formulations are also prepared
by entrapping the antibodies in liposomes or microemulsions which
are compatible with body tissues.
[0070] For application topically to the skin, the antibodies can be
formulated as a suitable ointment containing the active ingredient
suspended or dissolved in, for example, a mixture with one or more
of the following mineral oil, liquid petrolatum, white petrolatum,
propylene glycol, polyoxyethylene polyoxypropylene compound,
emulsifying wax and water. Alternatively, they can be formulated as
a suitable lotion or cream, suspended or dissolved in, for example,
a mixture of one or more of the following: mineral oil, sorbitan
monostearate, a polyethylene glycol, liquid paraffin, polysorbate
60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl
alcohol and water. The inventive antibodies can also be
administered intranasally or by inhalation and are conveniently
delivered in the form of a dry powder inhaler or an aerosol spray
presentation from a pressurized container, pump, spray, atomiser or
nebuliser, with or without the use of a suitable propellant, e.g.
dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane, a hydrofluoroalkane, carbon dioxide or
other suitable gas. In the case of a pressurized aerosol, the
dosage unit may be determined by providing a valve to deliver a
metered amount. The pressurized container, pump, spray, atomiser or
nebuliser may contain a solution or suspension of the antibody,
e.g., using a mixture of ethanol and the propellant as the solvent,
which may additionally contain a lubricant, e.g., sorbitan
trioleate. Capsules and cartridges (made, for example, from
gelatin) for use in an inhaler or insufflator may be formulated to
contain a powder mix of the antibodies of the invention and a
suitable powder base such as lactose or starch.
[0071] For oral delivery, such delivery may be accomplished using
solid or liquid formulations, for example in the form of tablets,
capsules, multiparticulates, gels, films, ovules, elixirs,
solutions or suspensions. In certain embodiments, the monoclonal
antibodies are administered as oral tablets or capsules. Such
preparations may be mixed chewable or liquid formulations or food
materials or liquids if desirable, for example to facilitate
administration to children, to individuals whose ability to swallow
tablets is compromised, or to animals. Compositions for rectal
administration are preferably suppositories which can be prepared
by mixing the inventive antibodies with suitable non-irritating
excipients or carriers such as cocoa butter, polyethylene glycol or
a suppository wax which are solid at ambient temperature but liquid
at body temperature and therefore melt in the rectal vault and
release the antibodies. Retention enemas and rectal catheters can
also be used as is known in the art. Viscosity-enhancing carriers
such as hydroxypropyl cellulose are also certain carriers of the
invention for rectal administration since they facilitate retention
of the pharmaceutical composition within the rectum. Generally, the
volume of carrier that is added to the pharmaceutical composition
is selected in order to maximize retention of the composition. In
particular, the volume should not be so large as to jeopardize
retention of the administered composition in the rectal vault.
Therapeutic Uses
[0072] In one aspect, inventive antibodies and compositions are
useful for treating or preventing conditions (including symptoms,
disorders, or diseases) which require activation of TrkC. Such
methods involve administering a therapeutically effective amount of
one or more of the antibodies provided herein to a subject. In
certain embodiments, the invention provides methods for treating
neurological conditions, neurodegenerative diseases and/or for
providing neuroprotection, and/or for treating motor neuron
diseases, and/or for diagnosing a condition such as cancer.
[0073] For example, and without limitation, the antibodies provided
herein may be used to treat individuals with a nervous system that
has been injured by wound, surgery, ischemia, infection, metabolic
diseases, malnutrition, malignant tumor, toxic drug, etc. Specific
examples include stroke, spinal cord injury, traumatic brain
injury, retinal degeneration and axotomy. The inventive antibodies
may also be used to treat disorders such as attention-deficit
hyperactivity disorder (ADHD), depression and age-associated mental
impairment (i.e., by providing cognitive enhancement). The
inventive antibodies and compositions may also be used to treat
congenital or neurodegenerative conditions including Alzheimer's
disease, Parkinson's disease, Huntington's chorea, amyotrophic
lateral sclerosis (ALS) and conditions related to these.
[0074] In one embodiment, inventive antibodies and compositions are
useful for treating or preventing motor neuron diseases. Motor
neurons express the TrkC receptor and are dependent on NT-3. Motor
neuron diseases are a group of progressive neurological disorders
that destroy motor neurons, the cells that control essential
voluntary muscle activity such as speaking, walking, breathing, and
swallowing. Normally, messages from nerve cells in the brain
(called upper motor neurons) are transmitted to nerve cells in the
brain stem and spinal cord (called lower motor neurons) and from
them to particular muscles. Upper motor neurons direct the lower
motor neurons to produce movements such as walking or chewing.
Lower motor neurons control movement in the arms, legs, chest,
face, throat, and tongue. When there are disruptions in these
signals, the muscles do not work properly; the result can be
gradual weakening, wasting away, and uncontrollable twitching
(called fasciculations). When upper motor neurons are affected, the
manifestations include spasticity or stiffness of limb muscles and
over-activity of tendon reflexes such as knee and ankle jerks.
Eventually, the ability to control voluntary movement can be lost.
Motor neuron diseases may be inherited or sporadic.
[0075] Many motor neuron diseases are known. Common motor neuron
diseases include amyotrophic lateral sclerosis (ALS), which affects
both upper and lower motor neurons. Progressive bulbar palsy
affects the lower motor neurons of the brain stem, causing slurred
speech and difficulty chewing and swallowing. Primary lateral
sclerosis is a disease of the upper motor neurons, while
progressive muscular atrophy affects only lower motor neurons in
the spinal cord.
[0076] Amyotrophic lateral sclerosis (ALS), also called Lou
Gehrig's disease or classical motor neuron disease, is a
progressive, ultimately fatal disorder that eventually disrupts
signals to all voluntary muscles. In the United States, doctors use
the terms motor neuron disease and ALS interchangeably. Both upper
and lower motor neurons are affected. Approximately 75 percent of
people with classic ALS will also develop weakness and wasting of
the bulbar muscles (muscles that control speech, swallowing, and
chewing). Other symptoms include spasticity, exaggerated reflexes,
muscle cramps, fasciculations, and increased problems with
swallowing and speaking. When muscles of the diaphragm and chest
wall fail to function properly, individuals lose the ability to
breathe without mechanical support. Although the disease does not
usually impair a person's mind or personality, several recent
studies suggest that some people with ALS may have alterations in
cognitive functions such as problems with decision-making and
memory. ALS most commonly strikes people between 40 and 60 years of
age, but younger and older people also can develop the disease. Men
are affected more often than women. Most cases of ALS occur
sporadically, and family members of those individuals are not
considered to be at increased risk for developing the disease,
although there is a familial form of ALS in adults, which often
results from mutation of the superoxide dismutase gene, or SOD1,
located on chromosome 21. A rare juvenile-onset form of ALS is
genetic. Most individuals with ALS die from respiratory failure,
usually within 3 to 5 years from the onset of symptoms. However,
about 10 percent of affected individuals survive for 10 or more
years.
[0077] Progressive bulbar palsy, also called progressive bulbar
atrophy, involves the bulb-shaped brain stem--the region that
controls lower motor neurons needed for swallowing, speaking,
chewing, and other functions. Symptoms include pharyngeal muscle
weakness (involved with swallowing), weak jaw and facial muscles,
progressive loss of speech, and tongue muscle atrophy. Limb
weakness with both lower and upper motor neuron signs is almost
always evident but less prominent. Affected persons have outbursts
of laughing or crying (called emotional lability). Individuals
eventually become unable to eat or speak and are at increased risk
of choking and aspiration pneumonia, which is caused by the passage
of liquids and food through the vocal folds and into the lower
airways and lungs.
[0078] Pseudobulbar palsy, which shares many symptoms of
progressive bulbar palsy, is characterized by upper motor neuron
degeneration and progressive loss of the ability to speak, chew,
and swallow. Progressive weakness in facial muscles leads to an
expressionless face. Individuals may develop a gravelly voice and
an increased gag reflex. The tongue may become immobile and unable
to protrude from the mouth. Individuals may also experience
emotional lability.
[0079] Primary lateral sclerosis (PLS) affects only upper motor
neurons and is nearly twice as common in men as in women. Onset
generally occurs after age 50. PLS occurs when specific nerve cells
in the cerebral cortex (the thin layer of cells covering the brain
which is responsible for most higher level mental functions) that
control voluntary movement gradually degenerate, causing the
muscles under their control to weaken. The syndrome--which
scientists believe is only rarely hereditary--progresses gradually
over years or decades, leading to stiffness and clumsiness of the
affected muscles. The disorder usually affects the legs first,
followed by the body trunk, arms and hands, and, finally, the
bulbar muscles. Symptoms may include difficulty with balance,
weakness and stiffness in the legs, clumsiness, spasticity in the
legs which produces slowness and stiffness of movement, dragging of
the feet (leading to an inability to walk), and facial involvement
resulting in dysarthria (poorly articulated speech). Major
differences between ALS and PLS (considered a variant of ALS) are
the motor neurons involved and the rate of disease progression. PLS
may be mistaken for spastic paraplegia, a hereditary disorder of
the upper motor neurons that causes spasticity in the legs and
usually starts in adolescence. PLS often develops into ALS.
[0080] Progressive muscular atrophy is marked by slow but
progressive degeneration of only the lower motor neurons. Weakness
is typically seen first in the hands and then spreads into the
lower body, where it can be severe. Other symptoms may include
muscle wasting, clumsy hand movements, fasciculations, and muscle
cramps. The trunk muscles and respiration may become affected.
Exposure to cold can worsen symptoms. The disease develops into ALS
in many instances.
[0081] Spinal muscular atrophy (SMA) is a hereditary disease
affecting the lower motor neurons. Weakness and wasting of the
skeletal muscles is caused by progressive degeneration of the
anterior horn cells of the spinal cord. This weakness is often more
severe in the legs than in the arms. SMA has various forms, with
different ages of onset, patterns of inheritance, and severity and
progression of symptoms. Some of the more common SMAs are described
below.
[0082] SMA type I, also called Werdnig-Hoffmann disease, is evident
by the time a child is 6 months old. Symptoms may include hypotonia
(severely reduced muscle tone), diminished limb movements, lack of
tendon reflexes, fasciculations, tremors, swallowing and feeding
difficulties, and impaired breathing. Some children also develop
scoliosis (curvature of the spine) or other skeletal abnormalities.
Affected children never sit or stand and the vast majority usually
die of respiratory failure before the age of 2.
[0083] Symptoms of SMA type II usually begin after the child is 6
months of age. Features may include inability to stand or walk,
respiratory problems, hypotonia, decreased or absent tendon
reflexes, and fasciculations. These children may learn to sit but
do not stand. Life expectancy varies, and some individuals live
into adolescence or later.
[0084] Symptoms of SMA type III (Kugelberg-Welander disease) appear
between 2 and 17 years of age and include abnormal gait; difficulty
running, climbing steps, or rising from a chair; and a fine tremor
of the fingers. The lower extremities are most often affected.
Complications include scoliosis and joint contractures--chronic
shortening of muscles or tendons around joints, caused by abnormal
muscle tone and weakness, which prevents the joints from moving
freely.
[0085] Symptoms of Fazio-Londe disease appear between 1 and 12
years of age and may include facial weakness, dysphagia (difficulty
swallowing), stridor (a high-pitched respiratory sound often
associated with acute blockage of the larynx), difficulty speaking
(dysarthria), and paralysis of the eye muscles. Most individuals
with SMA type III die from breathing complications.
[0086] Kennedy disease, also known as progressive spinobulbar
muscular atrophy, is an X-linked recessive disease. Daughters of
individuals with Kennedy disease are carriers and have a 50 percent
chance of having a son affected with the disease. Onset occurs
between 15 and 60 years of age. Symptoms include weakness of the
facial and tongue muscles, hand tremor, muscle cramps, dysphagia,
dysarthria, and excessive development of male breasts and mammary
glands. Weakness usually begins in the pelvis before spreading to
the limbs. Some individuals develop noninsulin-dependent diabetes
mellitus. The course of the disorder varies but is generally slowly
progressive. Individuals tend to remain ambulatory until late in
the disease. The life expectancy for individuals with Kennedy
disease is usually normal.
[0087] Congenital SMA with arthrogryposis (persistent contracture
of joints with fixed abnormal posture of the limb) is a rare
disorder. Manifestations include severe contractures, scoliosis,
chest deformity, respiratory problems, unusually small jaws, and
drooping of the upper eyelids.
[0088] Post-polio syndrome (PPS) is a condition that can strike
polio survivors decades after their recovery from poliomyelitis.
PPS is believed to occur when injury, illness (such as degenerative
joint disease), weight gain, or the aging process damages or kills
spinal cord motor neurons that remained functional after the
initial polio attack. Many scientists believe PPS is latent
weakness among muscles previously affected by poliomyelitis and not
a new motor neuron disease. Symptoms include fatigue, slowly
progressive muscle weakness, muscle atrophy, fasciculations, cold
intolerance, and muscle and joint pain. These symptoms appear most
often among muscle groups affected by the initial disease. Other
symptoms include skeletal deformities such as scoliosis and
difficulty breathing, swallowing, or sleeping. Symptoms are more
frequent among older people and those individuals most severely
affected by the earlier disease. Some individuals experience only
minor symptoms, while others develop SMA and, rarely, what appears
to be, but is not, a form of ALS.
[0089] In certain embodiments, the antibodies and compositions
provided herein may be used to prevent or treat motor neuron
diseases, such as those described herein, and in particular ALS. In
an embodiment, the antibodies provided herein are used to prevent
or treat ALS in a subject in need thereof. In another embodiment,
the antibodies and compositions provided herein are used to prevent
or treat a motor neuron disease selected from the group consisting
of ALS, progressive bulbar palsy, pseudobulbar palsy, primary
lateral sclerosis, progressive muscular atrophy, spinal muscular
atrophy, SMA Type I, SMA type II, SMA type III, Fazio-Londe
disease, Kennedy disease, congenital SMA with arthrogryposis, and
post-polio syndrome.
[0090] As used herein the term "subject" may include animals, such
as mammals, such as dogs, cats, cows, pigs, sheep and horses, and
human. In a particular embodiment, the subject is a human. In yet
another embodiment, the subject is an adult human.
EXAMPLES
[0091] The present invention will be more readily understood by
referring to the following examples, which are provided to
illustrate the invention and are not to be construed as limiting
the scope thereof in any manner.
Experimental Procedures
Cell Lines
[0092] Mouse SP2/0 myelomas; nnr5 cells (derived from rat PC12
pheochromocytoma) and which express p75 but not Trk receptors,
nnrr5 cells stably transfected with human TrkC cDNA (nnr5-TrkC),
NIH-3T3 transfected with human trkC cDNA (NIH-TrkC cells) or human
trkA cDNA (NIH-TrkA cells), and wild type NIH-3T3 cells were used.
All cells were cultured in RPMI media supplemented with 5% fetal
bovine serum (FBS) and antibiotics (Gibco). Stable transfectants
were added the appropriate drug selection, and protein expression
was routinely verified.
Co-Expression of Full Length p75 Receptors
[0093] NIH-TrkC cells were stably transfected with full length rat
p75 receptor with pcDNA3.1/Zeo(+) p75 construct. Stable
transfectants were selected by treatment with Zeocin (200
.mu.g/ml).
Antibodies
[0094] Rat anti-mouse IgG (.alpha.mIgG; Sigma, St. Louis, Mo.),
anti-phosphotyrosine mAb 4G10 (Upstate Biotechnology, Lake Placid,
N.Y.), anti-phospho-AKT (ser473) antibody (Cell Signaling),
anti-phospho-MAPK (p42/44, thr202/tyr204) antibody (Cell
Signaling), and fluoresceinated [fluorescin isothiocyanate (FITC)]
goat anti-mouse IgG (FITC-G.alpha.mIgG) (Sigma, St. Louis, Mo.) and
goat anti mouse Fab (GamFab) antibodies were purchased
commercially. MAb 5C3 was developed and grown in our laboratory
(LeSauteur et al., 1996, J. Neurosci. 16: 1308-1316) and is an
agonistic anti-TrkA mAb directed to the TrkA-D5/juxtamembrane
domain. Rabbit antisera 203 that binds to all Trks was a gift of
David Kaplan (Univ. of Toronto) and rabbit antisera to TrkC
ectodomain protein was a gift of Lino Tessarrollo (National Cancer
Institute).
Peptide Immunogen
[0095] A peptide (ESTDNFILFDEVSPTPPI) spanning a sequence near the
D5 domain of human TrkC was synthesized and was conjugated to KLH
as carrier. The 18 amino acid ectodomain sequence is located at the
linker region and ends 10 residues before the predicted
transmembrane domain. The sequence matches perfectly and with no
gaps most primates (e.g. chimpanzee), and has high homology with
mouse and rat sequences (ESTDFFDFESDASPTPPI). The alignment for
human/mouse/rat is ESTD-F--FD---+-SPTPPI.
MAb 2B7 Generation and Purification
[0096] All animal protocols were approved by McGill Animal Care
Committee. Female Balb/c mice (8 weeks old) were immunized three
times. Splenocytes were fused to SP2/0 myelomas, and hybridomas
were screened by differential binding in an Enzyme-Linked
Immunosorbent Assay (ELISA) using the original peptide immunogen
conjugated to BSA. Specific binding data to native cell surface
receptors were obtained using a Fluorescent Activated Cell Scanner
(FACScan) (Becton Dickinson, San Jose, Calif.) (see below). MAb 2B7
[IgG1 (.kappa.)] was identified by IsoStrip (Roche) and subcloned
three times. MAb 2B7 was purified onto a Protein G-Sepharose column
(Sigma). The binding and biochemical properties of purified mAb 2B7
were characterized by ELISA, FACScan, and SDS-PAGE.
Monomeric mAb 2B7 Fabs
[0097] MAb 2B7 was purified (8 mg/ml) as above and digested with
0.02 mg/ml papain (Gibco, Toronto, Ontario, Canada) for 6 hours
(LeSauteur et al., 1996, J. Neurosci. 16: 1308-1316). Fabs were
re-purified on Protein A-Sepharose and dialyzed against PBS.
Products were characterized by SDS-PAGE under non-reducing
conditions.
FACScan
[0098] Cells (2.5.times.10.sup.5) in 0.1 ml of binding buffer
[Hanks' Balanced Salt Solution (HBSS), 0.1% bovine serum albumin
(BSA), and 0.1% NaN.sub.3] were incubated with the indicated
concentration of mAbs or Fabs for 20 min at 4.degree. C., washed in
binding buffer to remove excess primary antibody, and immunostained
with FITC-G.alpha.mIgG secondary antibody for 20 min at 4.degree.
C. Cells were acquired and analyzed on a FACScan-BD Sciences using
the Cell Quest program. As negative controls no primary (background
fluorescence), or irrelevant mouse IgG (Sigma) were used followed
by secondary antibody. Specificity was gauged using various cells
expressing different receptors.
Western Blot Analysis
[0099] Assays were performed as previously described (Maliartchouk
and Saragovi, 1997, J. Neurosci. 17: 6031-6037). The activation of
each protein (Trk, Akt and MAPK) was studied after treatment of
live cells with different concentrations of ligands mAb 2B7, mAb
2B7 Fabs, NT-3 for 12 minutes at 37.degree. C., cells were
solubilized and protein concentrations were determined with Bio-Rad
Detergent Compatible Protein Assay (Bio-Rad). Western blot analysis
was performed with the indicated reagents. Blots were visualized
using the enhanced chemiluminiscence system (PerkinElmer Life
Sciences). Re-blotting the membranes with anti-serum directed to
total Trk (203 serum from Dr. David Kaplan) or anti-actin antibody
(Sigma) confirmed equal protein loading. Quantification of Western
blots was done by densitometric analysis relative to total protein
levels. Quantification data are presented as percent relative to
optimal (10 nM NT-3) as 100%. Statistical analysis were performed
by two-tailed t-tests; statistical significance (p.ltoreq.0.05) is
indicated by an asterisk (*).
Binding Inhibition Assays
[0100] MAb 2B7 and mAb 5C3 were labeled with biotin (Pierce).
Competition of mAb 2B7 binding to NIH-TrkC or NIH-TrkC+p75 cells
was tested with NT-3 or NGF as irrelevant control. The binding
assays were first optimized to quantify saturation. Cells were
first incubated with various concentrations of the test inhibitor
(20 minutes at 4.degree. C.) followed by saturating (.about.67 nM)
of mAb 2B7-biotin, mAb 5C3-biotin as irrelevant primary, or
negative control mouse IgG for another 20 minutes at 4.degree. C.
Then, FITC-goatamIgG or FITC-avidin was added as secondary reagent.
After washing, cells were analyzed by FACScan as previously
described. The conditions used (4.degree. C. and Na azide in the
buffer) did not allow internalization.
Proliferation/Survival Assays
[0101] NIH-TrkC or NIH-TrkC.sup.+p75 cells (7,500 cells/well) in
serum-free media (PFHM-II; Gibco) supplemented with 0.2% BSA were
added to 96-well plates (Falcon, Lincoln Park, N.J.) containing
NT-3, mAb 2B7, mAb 2B7 Fabs, negative control mouse IgG, or serum
(final 5% FBS, normal culture conditions). Where indicated, mAb 2B7
Fabs were cross-linked with goat anti-mouse Fab (Gam Fab). Wells
containing all culture conditions but no cells were used as blanks.
The growth/survival profile of the cells was quantified using the
tetrazolium salt reagent
4-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT;
Sigma) 48-72 hours after plating. Optical density readings of MTT
were done in a Benchmark Plus microplate Spectrophotometer (BioRad)
at 595 nm with blanks subtracted. For the cells used in this paper
we have validated the MTT method for measuring cell viability.
Almost all the cells die by apoptosis within 72 hours. In this
assay "partial survival" or "slower death" correlates with
TrkC-pTyr activation.
Differentiation Assays in Cell Lines
[0102] nnr5 cells stably transfected with TrkC cDNA (nnr5-TrkC)
were plated on cover-slips with full media in 24-well plates. 24
hours after plating the indicated treatments were added for an
additional 48-72 hours of culture. Cellular differentiation was
gauged by immunocytochemistry after cover-slips were fixed and
stained with MAP-2 antibody (Chemicon) followed by goat anti-rabbit
Cy3 (Jackson Immunochemicals) and analyzed as described earlier
(Ivanisevic et al, 2003, Oncogene 22: 5677-5685).
Dendritic Development Assays in Hippocampal Neurons
[0103] Primary cultures of hippocampal neurons were prepared from
rats (E18) as previously described (Kaech and Banker, 2006, Nat.
Protoc. 1: 2406-2415). Neurons were plated on poly-L-lysine coated
coverslips at low density (.about.6000 neurons/cm.sup.2) in minimum
essential medium (MEM) containing 10% horse serum, 0.6% glucose, 2
mM glutamine and antibiotics. After 5-8 hours, medium was replaced
for Neurobasal media (GIBCO, Invitrogen, USA) containing 1 mM
glutamine, B-27 supplement and antibiotics. These neuronal cultures
do not express detectable p75 (data not shown, see (Bronfman et
al., 2007, Dev. Neurobiol. 67: 1183-1203)). This is consistent with
this stage for neurons in vivo, where p75 is undetectable by
immunohistochemistry in the hippocampal formation of the adult rat
brain. One day after plating, neurons were treated with saturating
concentrations of neurotrophins (NT-3, BDNF, 6.7 nM) or 2B7 (100
nM). After 7 days of treatment, neurons were fixed 15 minutes with
3% paraformaldehyde, 4% sucrose in PBS. For immunostaining, fixed
neurons were incubated with glycine 0.15 M, pH 7.4, for 10 minutes
and then washed. Neurons are then permeabilized with 0.2% saponin,
non-specific binding was blocked with 3% BSA, and immunostaining
was done with mAb MAP2 (Chemicon, Millipore), followed by
incubation with anti-Mouse Alexa 555 secondary antibody (Molecular
Probes).
Image Analysis and Quantification
[0104] Z-series of individual immunostained neurons was acquired
with a Zeiss LSM Pascal 5 (Carl Zeiss, USA) connected to an
inverted microscope (Axiovert 2000) with a 63.times. objective.
Z-series of each neuron were integrated in a single image for
morphometrical analysis of the complete dendritic arbor.
Quantitative analysis of dendritic arborization was performed using
ImageJ software (NIH, USA) as previously described by Sholl (Sholl,
1953, J. Anat. 67: 387-406). For each neuron, concentric circles
spaced 10 .mu.m apart starting from the center of cell body was
traced. The number of dendrites that intersect each circle was
counted and plotted as a function of distance from the soma. The
total length of neurites and branch length per branch order was
analyzed using the "ImageJ" plugin called "NeuronJ". Neurites were
traced manually and labeled as primary (originated directly from
the soma), secondary (branching from a primary) and tertiary
(branching from a secondary). Since the MAP2 antibody labels all
neurites, it is not possible to accurately assign axons and
dendrite nomenclature. Instead, the program uses an order of
primary-secondary-tertiary branches to perform the calculations.
Once branch order nomenclatures were assigned, the neurite tracings
appeared color-coded by type and a text file containing neurite
total length and branch length per branch order measurement data
was generated. The average of the total length of neurites and
average of branch length per branch order were calculated and
t-test was applied for statistical analysis (p0.05 considered
significant).
ALS Therapeutic Paradigms
Transgenic Murine Models of ALS
[0105] Transgenic mice are available that express mutant human SOD1
protein and develop a motor neuron syndrome clinically and
neuropathologically similar to human ALS. B6.Cg-Tg(SOD1*G93A)1Gur/J
(JAX Labs, stock #004435) mice over express the human SOD1 gene
with the G93A mutation in CBL/black 6 background; these mice have
an average 50% survival at 157.1+9.3 days.
Mouse Behavior
[0106] Mice were weighed and assessed for motor function and
behavior at least twice prior to the treatment.
Hindlimb Extension Reflex
[0107] Reduction in hindlimb extension when animals are lifted by
the tail is an early deficit observed in mutant SOD1 transgenic
mice Animals were lifted by the base of the tail and hindlimb
extension was scored. Score 4 indicates full extension of both
hindlimbs. Score 3 indicates normal extension of one hindlimb, but
poor or inconsistent extension of the other. Score 2 indicates
normal extension of one hindlimb, and no extension of the other.
Score 1 indicates poor or inconsistent extension of one hindlimb,
and no extension of the other. Score 0 indicates no hindlimb
movement. This test was done weekly until death or euthanasia.
Rotarod
[0108] Mice were placed on a 11/4 inch diameter drum that is
rotated at a pre-determined speed, and the time taken until they
drop was automatically recorded by a sensor in the landing
platform. This test was performed weekly.
Endpoints and Analysis
[0109] Once the mice have a hindlimb reflex score of less than 2
they are considered to be in late stage disease and are monitored
and weighed on a daily basis. At this stage of disease progression,
mice were tested daily for a righting reflex. The animal is placed
on its back or side and the time it takes for the animal to stand
up on its paws is measured. The animal is considered to have
reached its study endpoints if it cannot right itself within 30
seconds of being placed on its side or back or if it loses more
than 20% of its body weight. If either of these endpoints is
reached, the animal must be euthanized. Survival is scored until
death ensues or euthanasia is mandated.
Example 1
Generation and Initial Screening of mAb 2B7 Binding
[0110] Linear peptide NH.sub.2-ESTDNFILFDEVSPTPPI-COOH was
conjugated through the N-terminus to KLH, and was used to immunize
mice. After fusion of splenocytes with SP2 myeloma cells, culture
supernatant from hybridomas was screened by ELISA using either the
immunizing peptide conjugated to BSA, or free peptide immobilized
on the ELISA plate. Several independent wells with hybridoma cells
producing antibodies with selectivity to the immunizing peptide
were identified, and they were subcloned three times by limiting
dilution. MAb 2B7 was chosen for further work.
Example 2
Characterization of mAb 2B7 Binding
[0111] To assess mAb 2B7 specificity for cell surface TrkC, cells
expressing or lacking TrkC were screened for differential binding
by FACScan.
[0112] MAb 2B7 binds strongly to NIH-TrkC transfectants (FIG. 1C)
and nnr5-TrkC transfectants (FIG. 1F). In controls, it does not
bind to wild type NIH-3T3 cells (FIG. 1A), NIH-TrkA transfectants
(FIG. 1B), or wild type nnr5 cells (FIG. 1E) above mIgG background
control. In additional assays mAb 2B7 does not bind to SYSY cells
transfected with human TrkB cDNA (data not shown). Binding of mAb
2B7 to non-permeabilized cells indicates that it recognizes the
extracellular domain of TrkC. The specific epitope on TrkC is
located between the transmembrane and the D5 domain. Therefore, mAb
2B7 binds selectively to native TrkC on the cell surface; near the
juxtamembrane region; and it does not bind to p75, TrkB, or
TrkA.
[0113] The concentration of mAb 2B7 and mAb 2B7 Fab required for
saturation of TrkC (FIG. 1I) is .about.65 nM mAb 2B7 and .about.75
nM 2B7 Fab. The slight difference in fluorescent intensity at
saturation with intact mAb versus Fabs is due to the use of
different fluorescinated secondary reagents.
[0114] Western blot analysis with mAb 2B7 reveals a band at M.sub.r
145 kDa (p145) for lysates from NIH-TrkC, but no bands for lysates
of control wild type NIH-3T3 cells (FIG. 1J), or for NIH-TrkA cells
(data not shown). MAb 2B7 is effective in western blot analysis
only when samples were prepared under non-reducing conditions,
which suggests the influence of a disulfide bond.
Example 3
Ligand Competition Studies
[0115] FACScan analysis demonstrated that NT-3 blocks, in a
dose-dependent manner, mAb 2B7 binding sites. NT-3 at 1 nM blocks
.about.40% of the mAb 2B7 binding sites in NIH-TrkC (FIG. 1G) and
in NIH-TrkC+p75 cells (FIG. 1H; see Table 1 for a summary). Thus,
mAb 2B7 binds to a receptor hot spot topologically related to the
NT-3 binding site, but which is outside the known NT-3 binding site
demonstrated to be exclusively at the D5 domain of TrkC. The block
of NT-3 upon 2B7 binding could be due to steric inhibition or
allosteric inhibition. These results suggest that assays to screen
for agents that inhibit mAb 2B7 binding to TrkC can be used to
identify TrkC ligands.
[0116] Table 1 shows a summary of FACScan data from FIGS. 1G and
1H. Raw mean channel fluorescence (MCF) for a single experiment is
shown, and mAb 2B7 inhibition of binding by NT-3 are presented as %
inhibition.+-.sem, n=3 independent experiments. Mouse IgG is used
as background control (no binding). MAb 2B7 is used at saturating
concentrations.
TABLE-US-00001 TABLE 1 NT-3 competes 2B7 binding Mean Channel %
inhibition of Added Fluorescence 2B7 binding Fluorescent NT-3 TrkC
TrkC + p75 TrkC TrkC + p75 Antibody Competitor cells cells cells
cells 2B7 0 nM 95 56 2B7 0.1 nM 87 42 14 .+-. 4 30 .+-. 2 2B7 1.0
nM 63 33 43 .+-. 8 49 .+-. 5 mIgG -- 11 10 control
Example 4
Co-Expression of p75NTR Reduces the 2B7 mAb Binding Sites on TrkC,
an Effect that Requires the Extracellular Domain of p75NTR
[0117] Co-expression of full length p75.sup.NTR in NIH-TrkC cells
reduces the cell surface 2B7 binding sites by .about.50-60% in
quantitative FACScan assays. This reduction was observed in all
twelve NIH-TrkC.sup.+p75 clones that were independently isolated
(FIG. 1D, also see Table 1). Higher concentrations of mAb 2B7 do
not overcome the reduction in binding sites elicited by expression
of p75.
[0118] Quantitative Western blot analyses of three
NIH-TrkC.sup.+p75 clones, using mAb 2B7, demonstrated levels of
total TrkC comparable to those in parental NIH-TrkC cells (FIG.
1K), and similar data were obtained using anti-TrkC rabbit serum
(data not shown). These data indicate that expression of p75 does
not reduce expression of TrkC. Rather, expression of p75 may induce
conformational changes or steric hindrance at or near the mAb 2B7
epitope on TrkC. This would be suggestive of physical or allosteric
p75.cndot.TrkC interactions that prevent mAb from binding to TrkC.
This suggestion is further supported by the observed inverse
correlation between p75 levels and the level of blocking of mAb 2B7
binding to TrkC. In different clones, low expression of p75 reduces
2B7 binding weakly, while high expression of p75 reduces 2B7
binding strongly (data not shown).
[0119] To determine which domain of p75 was relevant to block the
2B7 mAb binding sites on TrkC, we transfected a p75 construct
expressing the transmembrane domain (TM) and intracellular domain
(ICD) but which had the ectodomain (ECD) deleted. High expression
of the deletion p75 mutant was verified through an engineered tag.
The expressed p75-TM-ICD is not sufficient to block mAb 2B7 binding
to TrkC (data not shown). Thus, we conclude that the p75 ECD is
required for blocking mAb 2B7 binding to TrkC.
Example 5
p75NTR Blocking of 2B7 mAb is not Regulated by p75 Ligands
[0120] Because the p75 blocking of 2B7 mAb binding requires the
p75-ECD, we tested whether selective ligands of p75 could release
the hindrance to 2B7.cndot.TrkC interactions. In NIH-TrkC.sup.+p75
cells, NGF, BDNF, and pro-NT-3 (Alomone Labs) were added first to
engage p75, because in this paradigm they act as p75-selective
ligands. Then, cells were analyzed in quantitative FACScan assays
with mAb 2B7. None of the p75 ligands afford an increase or a
decrease in 2B7 binding (data not shown). The data suggest that
whether or not it is liganded, p75 can reduce 2B7.cndot.TrkC
binding.
[0121] As an interesting side point, pro-NT-3 does not reduce the
mAb 2B7 binding sites on NIH-TrkC cells either (data not shown),
whereas mature NT-3 does (FIG. 1G). These data indicate that
pro-NT-3 does not bind to this particular region of TrkC
receptors.
Example 6
Agonism by mAb 2B7 and Monomeric mAb 2B7 Fabs
[0122] Biochemical assays (FIG. 2), survival assays (FIG. 3), and
differentiation assays (FIG. 4) were undertaken to determine if mAb
2B7 has NT-3-like agonistic activity.
[0123] Phosphorylation of TrkC, AKT and MAPK were studied in
lysates from cells that had been exposed for 12 minutes to ligands
or controls (FIG. 2). NT-3, mAb 2B7 and 2B7 Fabs (with or without
cross-linking using GamFab antibodies) afford significant tyrosine
phosphorylation of TrkC (p-TrkC) over basal levels in untreated
cells (FIG. 2A).
[0124] NT-3, mAb 2B7 and 2B7 Fabs also activate downstream
signaling proteins MAPK (--2-fold over baseline) and AKT
(.about.5-fold over baseline) (FIG. 2A). The quantification of
phosphorylated proteins after 12 minutes of activation, relative to
total Trk protein loaded, are presented as % of optimal NT-3 (10
nM, 100% efficacy) (FIG. 2B). p-TrkC is induced by 0.1 nM NT-3 or
by 10 nM 2B7 with .about.30% efficacy, and by 2B7 Fabs (or 2B7 Fabs
cross-linked with GamFabs) with .about.15% efficacy. p-Akt and
p-MAPK are induced by 0.1 nM NT-3 and 10 nM 2B7 with .about.60%
efficacy, and by 2B7 Fabs (or 2B7 Fabs cross-linked with GamFabs)
with .about.30% efficacy. In cellular controls studying NIH-TrkA
cells, there is no increase in p-TrkA, p-AKT, or p-MAPK after
treatment with 2B7 mAb, or 2B7 Fabs (data not shown).
[0125] A longer time-course study of p-AKT (12 minutes, and 30
minutes of ligand treatment) showed that activation by NT-3 was
sustained, while activation by 2B7 was less efficient long term. At
the 30 min point p-AKT by NT-3 remains high .about.70% relative to
that seen at 12 min, whereas p-AKT by 2B7 is significantly reduced
and is <20% relative to that seen at 12 min (FIG. 2C).
Therefore, activation by mAb 2B7 is transient compared to that of
NT-3.
[0126] These ligands were then tested for their ability to protect
cells from death induced by culture in serum-free media (SFM) (FIG.
3). This cellular death is known to be apoptotic. In MTT assays
NT-3, mAb 2B7, and monovalent 2B7 Fabs significantly delay the
death of NIH-TrkC cells in a dose-dependent manner. Compared versus
optimal NT-3, mAb 2B7 has a maximal efficacy of .about.45%.
Monomeric 2B7 Fabs have a maximal efficacy of .about.35% (data not
shown). These data correlate with induction of pAKT, which is known
to be involved in mediation of trophic support.
[0127] Similar MTT assays compared the effects of TrkC agonists on
NIH-TrkC cells versus NIH-TrkC.sup.+p75 cells (FIG. 3). The purpose
was to test the effect of p75 expression on 2B7 agonistic activity.
Because these two cell lines have different survival profiles in
response to NT-3, the data here is standardized to normal serum
growth conditions (100%). The death of NIH-TrkC and
NIH-TrkC.sup.+p75 cells are both reduced by 10 nM NT-3 to a
comparable degree, .about.65-70%. As expected, expression of
full-length rat p75 in NIH-TrkC cells significantly enhances the
efficacy of 0.1 nM NT-3 (35% survival in NIH-TrkC.sup.+p75 versus
20% in NIH-TrkC). In contrast, p75 expression significantly reduces
the efficacy of mAb 2B7 (20% survival in NIH-TrkC.sup.+p75 cells
versus 45% in NIH-TrkC).
[0128] Thus, expression of p75 improves the binding and the
function of NT-3 but reduces the binding and the function of mAb
2B7. These data correlate with a reduction of mAb 2B7 binding when
p75 is co-expressed.
Example 7
Effect of p75 Ligands on 2B7 Agonistic Activity
[0129] We have previously shown that p75 negatively regulates the
efficacy of selective TrkA agonists such as mAb 5C3 (LeSauteur et
al., 1996, J. Neurosci. 16: 1308-1316). In this scenario, p75
ligands such as anti-p75 mAb MC192 neutralize the negative
regulation of p75 and thus allow full TrkA activation (Maliartchouk
and Saragovi, 1997, J. Neurosci. 17: 6031-6037). We therefore
performed survival assays with mAb 2B7.+-.engagement of p75 with
NGF, BDNF, or anti-p75 mAb MC192.
[0130] The ligands are p75-selective in NIH-TrkC.sup.+p75 cells,
and do not enhance or reduce the survival-promoting signals of mAb
2B7 (data not shown). Thus, the negative regulation of p75 upon mAb
2B7 survival function is not affected by p75-ligands. These data
are consistent with our earlier data showing that p75-ligands do
not reverse the block to mAb 2B7 binding; and with a report that
p75.cndot.TrkC functional interactions differ from p75.cndot.TrkA
functional interactions (Ivanisevic et al., 2003, Oncogene 22
5677-5685).
Example 8
Effect of mAb 2B7 on the Differentiation of Cell Lines
[0131] We next tested neurite outgrowth in response to mAb 2B7
(FIG. 4A, data summarized in FIG. 4B). Treatment with mAb 2B7 does
not induce the differentiation of nnr5-TrkC cells. This was
puzzling because mAb 2B7 binds to the cell surface of nnr5-TrkC
cells (FIG. 1F). In positive control assays, nnr5-TrkC cells
differentiate in response to NT-3, in a dose-dependent manner. NT-3
increases the percent of cells bearing>2 axons with axonal
length>2 cell bodies. In negative control assays, treatment with
mIgG or 10 nM NGF do not differentiate nnr5-TrkC cells.
[0132] Because p75 can regulate TrkC-mediated signals, including
cellular differentiation (Ivanisevic et al., 2003, Oncogene 22
5677-5685), we tested whether mAb 2B7 in combination with selective
p75 ligands may afford cellular differentiation. mAb 2B7 combined
with the p75-selective ligands NGF (FIG. 4) or BDNF (data not
shown) do not stimulate differentiation. In contrast, in positive
controls, a suboptimal concentration of 50 pM NT-3 in combination
with NGF as a p75-selective ligand increases cellular
differentiation. This control combination achieves levels
comparable to optimal 10 nM NT-3, as reported previously
(Ivanisevic et al., 2003, Oncogene 22 5677-5685).
[0133] Thus, mAb 2B7 activates TrkC but it does not have intrinsic
neuritogenic activity in the nnr5-TrkC cell line, and the use of
p75 ligands does not potentiate neurogenesis either.
Example 9
Effect of BDNF, NT-3, and mAb 2B7 on Dendritic Arborization in
Primary Neuronal Cultures
[0134] Primary cultures of E18 rat hippocampal neurons were
prepared (Kaech and Banker, 2006, Nat. Protocol 1: 2406-2415) and
plated at low density (.about.6000 neurons/cm.sup.2) as described
in Neurobasal/B27. Under these conditions expression of p75 is
barely detectable by western blotting and is undetectable by
immunohistochemistry (Bronfman et al., 2007, Dev. Neurobiol. 67:
1183-1203). This is consistent with this stage of development for
neurons in vivo, where p75 is undetectable by immunohistochemistry
in the hippocampal formation of the adult rat brain. Neuronal
cultures were treated with saturating concentrations of NT-3, BDNF
(6.7 nM) or 2B7 (100 nM), and the morphology of the dendritic arbor
was analyzed after 7 days treatment as described in the
Methods.
[0135] The data of the morphology of the dendritic arbor are
illustrated in FIG. 5. The histogram on FIG. 5A represents the
Sholl analysis of dendritic intersections as % of untreated control
cells. Both NT-3 and BDNF have a significant but differential
effect on the morphology of the dendritic arbor. Both NT-3 and BDNF
treatment result in a higher number of primary dendrites
(projections from the cell body of the neuron) and an increase in
dendritic branching. However, NT-3 resulted in longer dendrites
compared to BDNF. Compared to NT-3 or BDNF the mAb 2B7 did not have
any effect on the development of the dendritic arborization of
hippocampal neurons, suggesting that the mAb is not able to trigger
morphological differentiation as NT-3.
[0136] The average of the total length of neurites and average of
branch length per branch order was further assessed using NeuronJ
(FIG. 5B). Both BDNF and NT-3 significantly increased the total
length of neurites compared to control-untreated cells (p<0.05,
p<0.01, respectively). Interestingly, NT-3 treatment resulted in
longer neurites compared to BDNF (p<0.05). The mAb 2B7 did not
have any effect on the growth of neurites compared to control.
[0137] A further detailed analysis of the branch length per branch
order (FIG. 5C) reveals that, compared to control, BDNF increases
the length of secondary order neurites (p<0.01) whereas NT-3
increases the length of both, secondary (p<0.001) and tertiary
order neurites (p<0.05). NT-3 had a bigger effect on the length
of secondary and tertiary branches compared to BDNF (p<0.05),
indicating that NT-3 results in longer neurites, as we have
indicated by Sholl analysis.
[0138] Representative confocal immunofluorescence and
epifluorescent pictures of neuronal cultures immunostained with mAb
2B7 are shown (FIG. 6). The neuronal cultures look healthy, even if
they are not supplemented with growth factor or antibody. Low
magnification shows widespread and intense immunostaining of soma
and dendrites (FIGS. 6A and 6B). High magnification shows punctuate
immunostaining in the axons, that appear to be vesicular (FIGS. 6C
and 6D).
[0139] Thus, the mAb 2B7 did not induce any change in the
elongation of neurites in hippocampal neurons. These data are
consistent with the results using cell lines, and suggest that 2B7
can support cell survival but does not have any effect on the
differentiation process.
Example 10
Effect of mAb 2B7 and 2B7 Monovalent Fabs In Vivo, in a Mouse Model
of ALS (G93A SOD1 Mutant Transgenic c57black/6 Mouse)
[0140] Transgenic mice that express mutant human SOD1 protein and
develop a motor neuron syndrome clinically and neuropathologically
similar to human ALS have an average 50% survival at 157.1+9.3
days. These mice were treated with mAb 2B7 IgG, with 2B7 monovalent
Fabs, or with saline vehicle. Treatment was initiated when the
cohort exhibited signs of disease, based on poor hindlimb extension
reflex tests (approximately at age 100 days). Treatment was done by
intraperitoneal injections of 0.5 mg/kg of test reagent, in 100
microliters of saline, three times a week, for 10-12 weeks.
[0141] FIG. 7 shows that G93A mutant SOD1 transgenic mice survive
longer when treated with a TrkC agonist mAb 2B7 or with 2B7 Fabs.
FIG. 8 shows that the motor performance of the same mice in rotarod
tests, indicative of motor function, is improved when treated with
a TrkC agonist mAb 2B7 or 2B7 Fabs.
[0142] The above Examples show that the mAb 2B7 reported herein
specifically binds to the TrkC ectodomain, at the juxtamembrane
linker region. The results indicate that the mAb 2B7 reported
herein is useful for FACScan, immunofluorescence analysis,
immunoprecipitation, Western blot analysis, and immunocytochemistry
of TrkC.
[0143] The epitope of mAb 2B7 defines a previously unknown "hot
spot" of TrkC, between the second immunoglobulin domain (D5) and
the transmembrane domain. Competition studies between NT-3 and mAb
2B7, shown herein, indicate topological closeness at their binding
sites such that at least steric inhibition or allosteric inhibition
can occur. Moreover, because mAb 2B7 in western blots can bind to
TrkC only under non-reducing conditions, the results suggest that
mAb 2B7 recognizes a TrkC conformation stabilized or influenced by
a disulfide bond. This suggests a conformationally sensitive
docking site because the epitope contains no cysteines and there
are no reported disulfide bonds in this region of TrkC.
[0144] Biological studies uncovered a unique signal transduction
mechanism. MAb 2B7 and its monovalent Fabs mimic NT-3 binding and
function. Functional mimicry by mAb 2B7 is indicated by
phosphorylation and activation of TrkC, and its downstream
signaling partners, and promotion of trophic cell survival. By
these criteria, mAb 2B7 is a TrkC partial agonist.
[0145] The partial agonistic signals induced by mAb 2B7 include
MAPK activation (.about.2-fold over baseline) and AKT activation
(.about.5-fold over baseline), and both of these levels of
activation are comparable to that resulting form treatment with 100
pM NT-3.
[0146] However, there are important biological differences between
2B7 and NT-3. First, mAb 2B7 only affords trophic survival but does
not induce neuritogenic differentiation in cell lines or in primary
neuronal cultures expressing TrkC, and therefore it can be defined
as a biased partial agonist. Second, mAb 2B7 does not bind to p75
and is therefore a more selective ligand than NT-3. Third, 2B7 is
less potent than NT-3.
[0147] The results also suggest that 2B7 Fabs may cause
conformational changes in TrkC that induce or stabilize
receptor-receptor interactions.
[0148] The above examples also show that expression of p75 leads to
a reduction of mAb 2B7 binding sites, without a concomitant
reduction in TrkC expression, suggesting that that the mAb 2B7 hot
spot is either involved in or close to sites of interactions for
TrkC.cndot.p75.
[0149] The above examples further demonstrate that for blocking mAb
2B7 binding to TrkC, the p75-TM-ICD are not sufficient, and the
p75-ECD is required. Because the p75-ECD is the domain where
ligands can bind p75, we predicted that p75 ligands might alter the
"p75-mediated block" of mAb 2B7.cndot.TrkC interactions. However,
the "p75-mediated block" of mAb 2B7.cndot.TrkC interactions appears
to be independent of p75 ligands (e.g. they are not required) and
furthermore the block is insensitive to the presence of p75 ligands
(in this report we used NGF, BDNF, or pro-NT-3, which did not alter
the block).
[0150] Using mAb 2B7 as a selective TrkC agonist, we show herein
that expression of p75 causes a reduction of 2B7.cndot.TrkC signals
both in terms of potency (e.g. potency requires higher ligand
concentrations) and in terms of efficacy (e.g. the overall strength
of the response is lower). This is a striking contrast to NT-3,
because expression of p75 can enhance the potency of
NT-3.cndot.TrkC signals without affecting the overall efficacy.
[0151] Lower mAb 2B7 potency obviously stems from the fact that p75
causes a reduction in mAb 2B7 binding sites; meaning that fewer
TrkC receptors are activated. However, lower efficacy of mAb 2B7
can only be the consequence of the suppression of TrkC signals by
p75.
[0152] This interpretation would be consistent with a reported
reciprocal interplay between TrkA and p75 receptors that regulates
signal cascades and ligand binding. Notably, however, there is an
important difference between TrkA-p75 and TrkC-p75. Suppression of
TrkA signals by p75 is responsive to p75 ligands. In contrast,
suppression of TrkC signals by p75 is not responsive to p75 ligands
(Ivanisevic et al., 2003, Oncogene 22 5677-5685).
[0153] The examples presented herein also show that mAb 2B7 can
afford trophic survival but completely lacks neuritogenic
differentiation. This result indicates that it is possible to
uncouple these signals at the level of the ligand acting through a
wild type receptor. It is quite unusual for an agonistic ligand
binding at the ectodomain to uncouple signals that are mediated by
intracellular adaptor proteins. In this case we detected efficient
pAKT but poor pMAPK signals. This would require a limited
conformational change in the receptor activation state such that
only some (but not all) adaptor proteins can be activated. mAb 2B7
may be a biased agonist because the ankyrin-rich membrane
spanning)/Kidins220 protein (ARMS) protein interacts with Trks
through their transmembrane domains, leading to prolonged MAPK
signaling and differentiation; TrkC receptors within a putative
TrkC-ARMS-p75 complex may not be recognized by mAb 2B7, causing the
consequent poor MAPK activation. Alternatively, distinct kinetics
of ligand-induced receptor internalization can affect the
functional outcome towards neuritogenic differentiation or trophic
survival, and mAb 2B7 appears to have slower activation kinetics
than NT-3.
[0154] We have shown herein that MAb 2B7 fulfills the criteria of a
receptor ligand: selective binding, high affinity, and
saturability. Functional assays demonstrate biased agonistic
activity. Biased agonists are of great biological interest, and
very few have been documented for receptor tyrosine kinases. In
particular, it is intriguing that mAb 2B7 is a biased agonistic
ligand because it binds to a "hot spot" partially overlapping with
NT-3. These data suggest that engaging a TrkC receptor "hot spot"
in the juxtamembrane-linker domain can induce survival signals
only. Moreover, this "hot spot" can potentially be regulated by
expression of p75, either through direct steric hindrance, or
through direct or indirect changes to the TrkC conformation.
[0155] Aberrant expression of trkC mRNA have been correlated with
neurodegenerative diseases such as Alzheimer's disease (AD), motor
neuron diseases such as amyotropic lateral sclerosis (ALS) and some
types of cancers, as well as with photoreceptor disorders and
glaucoma. Thus, mAb 2B7 or its derivatives are of particular
interest for diagnostic, therapeutic, or prophylactic use for these
diseases. Also, mAb 2B7 may be useful in disorders where
TrkC-mediated trophic support is desired without inducing neuritic
growth, differentiation, or new connections.
[0156] The contents of all documents and references cited herein
are hereby incorporated by reference in their entirety.
[0157] Unless defined otherwise or the context clearly dictates
otherwise, all technical and scientific terms used herein have the
same meaning as commonly understood by one of ordinary skill in the
art to which this invention belongs. It should be understood that
any methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the invention.
[0158] While the invention has been described in connection with
specific embodiments thereof, it will be understood that it is
capable of further modifications and this application is intended
to cover any variations, uses, or adaptations of the invention
following, in general, the principles of the invention and
including such departures from the present disclosure as come
within known or customary practice within the art to which the
invention pertains and as may be applied to the essential features
hereinbefore set forth, and as follows in the scope of the appended
claims.
Sequence CWU 1
1
1118PRTMus musculus 1Glu Ser Thr Asp Asn Phe Ile Leu Phe Asp Glu
Val Ser Pro Thr Pro 1 5 10 15 Pro Ile
* * * * *