U.S. patent application number 12/827321 was filed with the patent office on 2011-06-23 for anti-trkb monoclonal antibodies and uses thereof.
This patent application is currently assigned to Wyeth LLC. Invention is credited to Seongeun CHO, Davinder Singh Gill, Ming Diana Qian, Xiang Yang Tan.
Application Number | 20110150893 12/827321 |
Document ID | / |
Family ID | 37400063 |
Filed Date | 2011-06-23 |
United States Patent
Application |
20110150893 |
Kind Code |
A1 |
CHO; Seongeun ; et
al. |
June 23, 2011 |
ANTI-TRKB MONOCLONAL ANTIBODIES AND USES THEREOF
Abstract
The present invention provides monoclonal antibodies for human
TrkB. In certain embodiments the inventive antibodies bind and
activate human TrkB. In certain embodiments the inventive
antibodies are selective for human TrkB in that they do not bind
(or activate) human TrkA or human TrkC. In some embodiments the
inventive monoclonal antibodies cross-react with murine TrkB.
Humanized or veneered versions of the inventive antibodies are also
encompassed. Pharmaceutical compositions that comprise inventive
antibodies are provided as are methods for preparing the inventive
antibodies and methods of using these for treatment, detection or
purification purposes.
Inventors: |
CHO; Seongeun;
(Hillsborough, NJ) ; Gill; Davinder Singh;
(Andover, MA) ; Tan; Xiang Yang; (Reading, MA)
; Qian; Ming Diana; (Needham, MA) |
Assignee: |
Wyeth LLC
Madison
NJ
|
Family ID: |
37400063 |
Appl. No.: |
12/827321 |
Filed: |
June 30, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11446875 |
Jun 5, 2006 |
7750122 |
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12827321 |
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60687705 |
Jun 6, 2005 |
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Current U.S.
Class: |
424/143.1 ;
435/193; 435/334; 436/501; 530/387.9; 530/388.22 |
Current CPC
Class: |
A61P 31/00 20180101;
A61P 17/02 20180101; A61P 3/10 20180101; A61P 21/04 20180101; C07K
2317/34 20130101; C07K 2317/622 20130101; A61P 37/00 20180101; A61P
25/28 20180101; A61K 2039/505 20130101; C07K 2319/30 20130101; A61P
25/00 20180101; C07K 2317/75 20130101; C07K 16/2863 20130101; C07K
2317/92 20130101; A61P 25/16 20180101; A61P 25/14 20180101; A61P
3/00 20180101; A61P 43/00 20180101; A61P 25/18 20180101; A61P 35/00
20180101; A61P 25/24 20180101; C07K 2317/55 20130101; A61P 17/00
20180101; A61P 35/04 20180101 |
Class at
Publication: |
424/143.1 ;
530/388.22; 530/387.9; 435/334; 436/501; 435/193 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C07K 16/28 20060101 C07K016/28; C12N 5/12 20060101
C12N005/12; A61P 25/00 20060101 A61P025/00; A61P 17/02 20060101
A61P017/02; A61P 35/04 20060101 A61P035/04; A61P 3/00 20060101
A61P003/00; A61P 25/28 20060101 A61P025/28; G01N 33/566 20060101
G01N033/566; C12N 9/10 20060101 C12N009/10 |
Claims
1. A monoclonal antibody that binds human TrkB.
2. The monoclonal antibody of claim 1, wherein the binding with
human TrkB has an ED.sub.50 in the range of about 10 .mu.M to about
500 nM.
3-5. (canceled)
6. The monoclonal antibody of claim 1, wherein the monoclonal
antibody activates human TrkB with an EC.sub.50 in the range of
about 10 .mu.M to about 500 nM.
7-9. (canceled)
10. The monoclonal antibody of claim 1, wherein the monoclonal
antibody does not bind and/or does not activate human TrkA.
11. The monoclonal antibody of claim 10, wherein the monoclonal
antibody exhibits no detectable binding with human TrkA and/or does
not cause any detectable activation of human TrkA at antibody
concentrations above about 1 nM.
12-14. (canceled)
15. The monoclonal antibody of claim 1, wherein the monoclonal
antibody does not bind and/or does not activate human TrkC.
16. The monoclonal antibody of claim 15, wherein the monoclonal
antibody exhibits no detectable binding with human TrkC and/or does
not cause any detectable activation of human TrkC at antibody
concentrations above about 1 nM.
17-19. (canceled)
20. The monoclonal antibody of claim 10, wherein the monoclonal
antibody does not bind and/or does not activate human TrkC.
21. The monoclonal antibody of claim 1, wherein the monoclonal
antibody blocks the binding between BDNF and human TrkB.
22. The monoclonal antibody of claim 21, wherein the monoclonal
antibody blocks the binding between BDNF and human TrkB with an
IC.sub.50 in the range of about 100 .mu.M to about 500 nM.
23. (canceled)
24. (canceled)
25. The monoclonal antibody of claim 1, wherein the monoclonal
antibody does not block the binding between BDNF and human
TrkB.
26-37. (canceled)
38. The monoclonal antibody of claim 1, wherein the monoclonal
antibody binds one or both of the epitopes of human TrkB with
sequences KNEYGKD (SEQ ID NO:7) and KGNPKP (SEQ ID NO:8).
39. (canceled)
40. The monoclonal antibody of claim 1, wherein the monoclonal
antibody binds an epitope of human TrkB with the sequence ENLVGED
(SEQ ID NO: 10) and optionally an epitope of human TrkB with the
sequence AGDPVP (SEQ ID NO:11).
41. (canceled)
42. A monoclonal antibody that is produced from the hybridoma
deposited with the ATCC on Aug. 18, 2005 and having ATCC patent
deposit designation PTA-6948 or from a progenitor cell thereof.
43. A monoclonal antibody that binds to the same epitope as a
monoclonal antibody produced from the hybridoma deposited with the
ATCC on Aug. 18, 2005 and having ATCC patent deposit designation
PTA-6949.
44. The monoclonal antibody of claim 1, wherein the monoclonal
antibody binds to the same epitope as the monoclonal antibody of
claim 42.
45. (canceled)
46. A hybridoma that produces a monoclonal antibody according to
claim 1.
47-60. (canceled)
61. A method of activating human TrkB in an individual, comprising:
administering to an individual, a therapeutically effective amount
of a monoclonal antibody of claim 1.
62. The method of claim 61, wherein said individual is suffering
from a neurological condition which requires activation of
TrkB.
63. The method of claim 61, wherein the nervous system of said
individual has been injured by a wound, surgery, ischemia,
infection, a metabolic disease, malnutrition, a malignant tumor or
a toxic drug.
64. The method of claim 61, wherein said individual has suffered a
stroke, a spinal cord injury, or an axotomy.
65. The method of claim 61, wherein said individual is suffering
from a congenital or neurodegenerative condition.
66. The method of claim 65, wherein said condition is selected from
the group consisting of Alzheimer's disease, Parkinson's disease,
Huntington's chorea, and amyotrophic lateral sclerosis (ALS).
67-68. (canceled)
69. A method comprising: combining a monoclonal antibody of claim 1
with a sample that includes an amount of human TrkB under
conditions that allow specific binding between the monoclonal
antibody and human TrkB; and detecting the specific binding,
thereby indicating the presence of human TrkB in the sample.
70. A method comprising: combining a monoclonal antibody of claim 1
with a sample that includes an amount of human TrkB under
conditions that allow specific binding between the monoclonal
antibody and human TrkB, thereby producing an antibody-TrkB
complex; separating the antibody-human TrkB complex from the
remainder of the sample; and separating the antibody from human
TrkB, thereby obtaining purified human TrkB.
Description
PRIORITY INFORMATION
[0001] The present application is a continuation of U.S. Ser. No.
11/446,875, which claims the benefit of U.S. Ser. No. 60/687,705,
filed Jun. 6, 2005, the entire contents of which are hereby
incorporated by reference.
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
neurotrophin-4/5 (NT-4/5). The neurotrophins share highly conserved
structural features, yet their unique amino acid sequences allow
each member to elicit high affinity interactions with the
extracellular domain of specific Trk receptors, namely TrkA, B or
C. Thus, NGF is a preferred ligand for TrkA; BDNF and NT-4/5 are
preferred ligands for TrkB; and NT-3 has been shown to bind TrkC,
although it also appears to bind TrkA and TrkB with lower
affinities.
[0003] Among the Trk receptors, the role of TrkB has been well
characterized in the central nervous system (CNS). TrkB are widely
distributed in the brain, including in the neocortex, hippocampus,
striatum, olfactory formation and brainstem. Using BDNF as a
cognate ligand, the indispensable roles of TrkB in neuronal
survival, differentiation and neuroregeneration have been shown in
a number of neurodegenerative models, including stroke, spinal cord
injury, axotomy and ALS.
[0004] Through various signaling analyses and receptor knockout
systems, the responses of BDNF have been shown to depend on the
binding and activation of TrkB. As noted, Trk receptors are
multi-domain single-transmembrane proteins. They consist of an
extracellular ligand binding domain, a transmembrane region, and an
intracellular tyrosine kinase domain. The extracellular domain is
composed of a leucine-rich motif flanked by two cysteine clusters
and two immunoglobulin(Ig)-like domains. Trk receptors have been
shown to interact with their ligands mainly through the second
Ig-like domain, although contribution of other regions such as a
leucine-rich motif and the first Ig domain in ligand-docking has
been proposed. The crystal structures of the ligand binding domains
of Trk receptors as well as ligand-receptor complexes have been
resolved. The ligand-receptor interface appears to consist of two
patches: one for a conserved binding motif shared among all
neurotrophins and the other specific for each neurotrophin.
[0005] Upon the binding of neurotrophins to Trk receptors, receptor
dimerization and subsequent conformational changes occur, which are
believed to lead to activation of the intracellular tyrosine kinase
domain. There are several conserved tyrosines in the intracellular
domain of Trk receptors. Phosphorylation of the autoregulatory loop
of the kinase domain activates the kinase activity, and
phosphorylation of other residues promotes signaling by creating
docking sites for adaptor proteins that couple these receptors to
intracellular signaling cascades, including Ras/extracellular
signal regulated kinase (ERK) protein kinase pathway, the PI3K/Akt
kinase pathway and phospholipase C-gamma. Although somewhat
overlapping, these individual pathways are involved in discrete
biological activities: Ras/MAPK regulates neuronal differentiation
and proliferation, PI3K/Akt pathways control actin dynamics and
survival and PLC gamma is involved in calcium mobilization.
[0006] To date, there exist no successful examples of reagents that
act as potent and selective in vivo agonists of TrkB. While BDNF,
as a recombinant protein, has been shown to increase neuronal
survival and neuroregeneration in a number of CNS degenerative
models in vitro and in vivo, the outcomes of BDNF protein therapy
in clinics have been negative, most likely because BDNF has a short
in vivo half-life. There is therefore a need in the art for
pharmaceutical reagents that act as potent and selective in vivo
agonists of TrkB.
SUMMARY OF THE INVENTION
[0007] The present invention provides monoclonal antibodies for
human TrkB. In certain embodiments the inventive antibodies bind
and activate human TrkB. In certain embodiments the inventive
antibodies are selective for human TrkB in that they bind
preferentially to TrkB over human TrkA or human TrkC. In some
embodiments the inventive monoclonal antibodies cross-react with
murine TrkB.
[0008] The invention also provides pharmaceutical compositions that
comprise one or more of these antibodies. The invention further
provides methods for preparing the inventive monoclonal antibodies.
Humanized or veneered versions of the inventive antibodies are also
encompassed as are the hybridomas that produce the inventive
antibodies.
[0009] The invention further provides methods of using these
monoclonal antibodies as agonists of TrkB. In certain embodiments
the monoclonal antibodies are used as agonists of human TrkB.
According to such embodiments, the antibodies may be used to treat
conditions which require TrkB activation, including neurological
conditions.
[0010] The invention yet further provides methods for detecting
TrkB in a sample and methods for purifying TrkB from a sample using
the inventive antibodies.
DESCRIPTION OF THE DRAWINGS
[0011] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
[0012] FIG. 1 depicts TrkB-specific antibody activities in immune
sera from five immunized mice (M1-M5). Immune sera binding results
from an ELISA assay with a recombinant protein that includes the
extracellular domain of human TrkB fused to the Fc region of human
IgG1 (rhTrkB-EDC-Fc) are shown in (FIG. 1A). Immune sera binding
results from an ELISA assay with a recombinant protein that
includes the extracellular domain of murine TrkB (rmTrkB-EDC) are
shown in (FIG. 1B). The extent to which immune sera could block the
interaction of rhTrkB-EDC-Fc and BDNF in a competition ELISA assay
are shown in (FIG. 1C). Immune sera binding to surface rhTrkB on
HEK-293 cells are shown in (FIG. 1D).
[0013] FIGS. 2A and 2B depict control results that were obtained
with neurotrophins in a luciferase activity assay that uses HEK-293
cells which express surface rhTrkB. These control results show that
the assay can selectively represent the activation of TrkB.
[0014] FIG. 3 depicts results that were obtained with immune sera
in the same luciferase activity assay as FIG. 2 (FIG. 3A) and by
anti-PY490 immunoblot analysis (FIGS. 3B and 3C). These results
show that incubation of hTrkB cells with immune bleeds
significantly increased the luciferase signals in a dose-dependent
manner.
[0015] FIG. 4 depicts results that were obtained with certain TrkB
antibodies in the same luciferase activity assay as FIG. 2 in graph
(FIG. 4A) and table (FIG. 4B) formats. All of the antibodies caused
dose-dependent increases in the signal with EC.sub.50 in the range
of 10.sup.-10 (M). The maximum signal window was .about.7 fold over
basal for most of the antibodies, which was comparable to the
response induced by BDNF at 200 ng/ml (6.2 fold over basal).
[0016] FIG. 5 depicts the results that were obtained with certain
TrkB antibodies in a neurite outgrowth assay. The assay used human
neuroblastoma SY5Y cells, which are known to express TrkB upon
neuronal differentiation. Addition of BDNF and of most antibodies
promoted neurite outgrowth as demonstrated by increases in the
neurite length (FIG. 5A) and the number of branch points (FIG. 5B).
Representative images of a few treatment groups are shown in (FIG.
5C). The results from full-dose response analyses obtained for a
subset of these antibodies are shown in (FIG. 5D).
[0017] FIG. 6 depicts the results that were obtained with certain
TrkB antibodies in a neuroprotection assay. The assay measures the
survival of differentiated SY5Y cells following serum withdrawal
injury. Results showed that BDNF and several antibodies protected
differentiated SY5Y cells, demonstrating dose-dependent increases
in cell viability.
[0018] FIG. 7 depicts some of the results that were obtained when
three anti-TrkB antibodies, 18C3, 29D7 and 17D11, which were shown
to bind to recombinant murine TrkB (rmTrkB), were tested in rat
cerebellar granule neuron (CGN) cultures for activity against the
endogenous rat TrkB receptor. Results are shown for a neurite
outgrowth assay (FIGS. 7A and 7B) and a neuroprotection assay (29D7
only)(FIG. 7B).
[0019] FIG. 8 depicts the results that were obtained when certain
anti-TrkB antibodies were evaluated in Western analysis for
induction of TrkB autophosphorylation. All antibodies led to robust
TrkB phosphorylation, and these effects were antagonized by
treatment with the kinase inhibitor K252a, indicating that these
TrkB-binding antibodies caused activation of TrkB.
[0020] FIG. 9 depicts the results from a FACS TrkA binding assay
(FIG. 9A) and a TrkA luciferase activity assay (FIG. 9B) that were
obtained with certain anti-TrkB antibodies. These results show that
inventive antibodies do not bind or activate human TrkA cells.
[0021] FIG. 10 depicts the results that were obtained when certain
anti-TrkB antibodies were evaluated in a TrkC luciferase activity
assay. These results show that inventive antibodies do not activate
human TrkC cells.
[0022] FIG. 11 depicts the rodent model of neonatal
hypoxia-ischemia (HI) that is based on the Levine procedure.
[0023] FIG. 12 shows the phage binding ELISA data from a
representative set of TrkB positive scFv clones (BMV library
panning selection).
[0024] FIGS. 13A-13D show the binding specificity of TrkB selected
scFv antibodies tested using the FACS assay. The data shows that
TrkB selected scFv antibodies react with membrane associated TrkB
in a specific manner (filled histograms) with no cross-reactivity
to control cells (open histograms).
[0025] FIG. 14 show ELISA binding results for 29D7 IgG antibody and
its Fab fragment with human (FIG. 14A) and mouse (FIG. 14B) TrkB.
Both total IgG and Fab of 29D7 showed dose-dependent binding
activities to human and mouse TrkB. However, the ED.sub.50 value of
the Fab was about 100 fold lower than that of intact 29D7. Neither
an isotype control IgG1 nor its Fab fragment bound to TrkB.
[0026] FIG. 15 shows luciferase activity in HEK-293 cells measured
16 hours post-treatment with 29D7 IgG or 29D7 Fab. Both total IgG
and Fab of 29D7 induced dose-dependent luciferase activities,
indicating activation of TrkB. However, the EC.sub.50 value of 29D7
Fab was about 27 fold higher than that of 29D7 IgG (0.083 nM and
2.28 nM for 29D7 IgG and 29D7 Fab, respectively). Neither an
isotype control IgG1 nor its Fab fragment had effects on TrkB
activation.
[0027] FIG. 16 shows results from the epitope mapping analysis of
anti-TrkB monoclonal antibodies 17D11, 29D7, 7F5, 11E1 and 19E12.
The 17D11 monoclonal antibody recognizes loop-3 of the IgG-2
segment of human TrkB (KNEYGKD, SEQ ID NO:7, amino acids 364 to 370
of SEQ ID NO:1); and loop-1 of the IgG-2 segment of human TrkB
(KGNPKP, SEQ ID NO:8, amino acids 308 to 313 of SEQ ID NO:1). The
29D7, 7F5, 11E1 and 19E12 monoclonal antibodies all recognize
loop-3 of the IgG-1 segment of human TrkB (ENLVGED, SEQ ID NO:10,
amino acids 269 to 275 of SEQ ID NO:1); and may also recognize
loop-1 of the IgG-1 segment of human TrkB (AGDPVP, SEQ ID NO:11,
amino acids 221 to 226 of SEQ ID NO:1). The IgG-1 and IgG-2
segments of TrkB shown in FIG. 16 correspond to SEQ ID NOs: 12 and
13, respectively.
[0028] FIG. 17 compares the stains that were obtained from the
striatum and hippocampus for different treatments after
hypoxic-ischemic (HI) injury.
[0029] FIG. 18 compares the total brain tissue loss (A) and
sub-regional tissue loss (B) for different treatments after HI
injury. The data are shown as mean and standard error of the mean
(S.E.M.). BDNF and anti-TrkB monoclonal antibody 29D7 showed
significant dose-dependent protection of the brain from HI injury.
The protection was not localized to any specific sub-region of the
brain but greatest protection was generally observed in the
cortex.
[0030] FIG. 19 shows the results of a DEVD-AMC cleavage assay that
was used to determine caspase-3 activities for different treatments
after HI injury. The results for different regions of the brain are
shown as mean.+-.S.E.M. BDNF and anti-TrkB monoclonal antibody 29D7
blocked caspase-3 activation caused by HI injury.
[0031] FIG. 20 shows immunoblots of protein samples taken from mice
given different treatments after HI injury. The samples were
separated by SDS-PAGE and subjected to immunoblotting with
antibodies against certain caspase-3 substrates (PARP and
.alpha.-spectrin). Antibodies against .beta.-actin were used as
controls to verify equal loading of proteins. The results show that
cleavage of these caspase-3 substrates was inhibited by BDNF and
anti-TrkB monoclonal antibody 29D7 (C=contra and I=ipsi sides of
carotid ligation).
[0032] FIG. 21 shows immunoblots of protein samples taken from
normal mice after an intracerebroventricular injection of anti-TrkB
antibody 29D7 (or vehicle as control). Samples were taken 1, 2, 6,
12 and 24 hours after injection. Samples were separated by SDS-PAGE
and subjected to immunoblotting with antibodies specific to
proteins that are phosphorylated as a result of TrkB activation
(phospho-ERK1/2 and phospho-AKT). Antibodies against .beta.-actin
were used as controls to verify equal loading of proteins. The
results show that anti-TrkB monoclonal antibody 29D7 induced
time-dependent phosphorylation of ERK1/2 and AKT (data from
hippocampal and cortical samples are shown and are representative
of results obtained from four other samples).
[0033] FIG. 22 is a densitometric quantification of the ERK
phosphorylation shown in FIG. 21.
[0034] FIG. 23 is a densitometric quantification of the AKT
phosphorylation shown in FIG. 21.
[0035] FIG. 24 shows vehicle and 29D7 treated brain tissues that
have been fixed and immunoflourescently labeled with an antibody
for the neuron-specific marker NeuN (left) and an
anti-phospho-ERK1/2 antibody (center). The right hand figures show
these two merged. ERK1/2 phosphorylation is significantly stronger
in the 29D7 treated samples.
[0036] FIG. 25 shows the time-course of ERK1/2 activation in the
cortical and hippocampal tissues following intracerebroventricular
injection of anti-TrkB monoclonal antibody 29D7.
[0037] FIG. 26 shows the dose-dependent inhibition of neurite
outgrowth of primary cerebellar granule neurons caused by (A) MAG
and (B) myelin.
[0038] FIG. 27 shows the reversal of (A) MAG- and (B)
myelin-mediated neurite inhibition that is caused by anti-TrkB
monoclonal antibody 29D7.
DESCRIPTION OF CERTAIN EMBODIMENTS
[0039] The present invention stemmed in part from the realization
that monoclonal antibodies that specifically bind to the
extracellular domains of the TrkB receptor might dimerize TrkB and
be sufficient to induce the activation of the receptor and
biological responses similar to those mediated by BDNF.
[0040] Monoclonal antibodies in general represent a unique class of
proteins that have diverse utilities in research, medical
diagnosis, and the clinical treatment of disease. Advantageously,
monoclonal antibodies are thought to have greater pharmacokinetic
stability than recombinant proteins such as BDNF thereby allowing
for sustainable pharmacological effects that have a significant
benefit for clinical applications. For example, the current
clinically approved uses of monoclonal antibody medications include
prevention of organ transplant rejection, treatment of cancers
(e.g., breast, colon, non-Hodgkin's lymphoma, leukemia), rheumatoid
arthritis, prophylaxis against respiratory syncytial virus disease,
Crohn's disease, percutaneous coronary intervention, and asthma.
Other medical applications for treating a variety of human diseases
with monoclonal antibodies are also currently in clinical trials.
The biological effects of these monoclonal antibodies are generally
conveyed by blocking or neutralizing molecular events exerted by
endogenous ligands, thus primarily acting as specific high-affinity
antagonists. In the present invention, monoclonal antibodies are
used as agonists that mimic the biological effects of
receptor-ligand interactions.
1. Monoclonal Antibodies and Hybridomas
[0041] In one aspect, the present invention provides monoclonal
antibodies that bind human TrkB (SEQ ID NO:1). In certain
embodiments, these antibodies bind human TrkB (SEQ ID NO:1) with an
ED.sub.50 in the range of about 10 pM to about 500 nM, for example
in the range of about 10 pM to about 1 nM, about 10 pM to about 100
pM, or about 10 pm to about 50 pM. As used herein the term "about"
is defined to encompass variations of .+-.15%.
[0042] In one embodiment the inventive antibodies are also agonists
of human TrkB. In certain embodiments, these antibodies activate
human TrkB (SEQ ID NO:1) with an EC.sub.50 in the range of about 10
pM to about 500 nM, for example in the range of about 10 pM to
about 1 nM, about 10 pM to about 100 pM, or about 10 pM to about 50
pM.
[0043] In one embodiment, inventive antibodies are selective for
human TrkB (SEQ ID NO:1) in that they bond preferentially to TrkB
(SEQ ID NO:1) over human TrkA (SEQ ID NO:5) or human TrkC (SEQ ID
NO:6). In some embodiments, inventive antibodies do not bind to
human TrkA or human TrkC. As defined herein, an inventive antibody
"does not bind" human TrkA (SEQ ID NO:5) or human TrkC (SEQ ID
NO:6) if it exhibits no detectable binding with these receptors at
concentrations above about 1 nM, for example above about 10 nM,
above about 100 nM or above about 1 .mu.M.
[0044] In one embodiment, inventive antibodies are selective for
human TrkB in that they do not activate human TrkA (SEQ ID NO:5) or
human TrkC (SEQ ID NO:6). As defined herein, an inventive antibody
"does not activate" human TrkA (SEQ ID NO:5) or human TrkC (SEQ ID
NO:6) if it causes no detectable activation with these receptors at
concentrations above about 1 nM, for example above about 10 nM,
above about 100 nM or above about 1 .mu.M.
[0045] In one embodiment, inventive antibodies block the binding
between BDNF and human TrkB (SEQ ID NO:1) with an IC.sub.50 in the
range of about 100 .mu.M to about 500 nM, for example in the range
of about 100 pM to about 1 nM, or about 100 pM to about 500 pM. In
other embodiments these antibodies do not block the binding between
BDNF and human TrkB (SEQ ID NO:1). As defined herein, an inventive
antibody "does not block" the binding between BDNF and human TrkB
(SEQ ID NO:1) if it exhibits no detectable blocking activity at
concentrations above about 1 nM, for example above about 10 nM,
above about 100 nM or above about 1 .mu.M.
[0046] In certain embodiments the antibodies of the invention
belong to an IgG isotype, e.g., the IgG1, IgG2a or IgG2b
isotype.
[0047] In another aspect, the present invention provides monoclonal
antibodies with any of the properties from above that further bind
and/or activate mouse TrkB (SEQ ID NO:2). In certain embodiments,
these antibodies bind and/or activate mouse TrkB (SEQ ID NO:2) with
an ED.sub.50 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.
[0048] In another aspect, the present invention provides monoclonal
antibodies with any of the properties from above that further bind
one or more specific epitopes of human TrkB (SEQ ID NO:1) and
optionally one or more specific epitopes of mouse TrkB (SEQ ID
NO:2). In certain embodiments, these antibodies bind one or both
epitopes of human TrkB with the sequences KNEYGKD (SEQ ID NO:7,
amino acids 364 to 370 of SEQ ID NO:1) and KGNPKP (SEQ ID NO:8,
amino acids 308 to 313 of SEQ ID NO:1). In one embodiment, these
antibodies also bind one or both epitopes of mouse TrkB with the
sequences KNEYGKD (SEQ ID NO:7, amino acids 364 to 370 of SEQ ID
NO:2) and RGNPKP (SEQ ID NO:9, amino acids 308 to 313 of SEQ ID
NO:2). In other embodiments, the present invention provides
antibodies that bind an epitope of human TrkB with the sequence
ENLVGED (SEQ ID NO:10, amino acids 269 to 275 of SEQ ID NO:1) and
optionally an epitope of human TrkB with the sequence AGDPVP (SEQ
ID NO:11, amino acids 221 to 226 of SEQ ID NO:1). In one
embodiment, these antibodies also bind an epitope of mouse TrkB
with the sequence ENLVGED (SEQ ID NO:10, amino acids 269 to 275 of
SEQ ID NO:2).
[0049] In yet another aspect, the present invention provides
hybridomas that produce any of these monoclonal antibodies. For
example, hybridomas that were deposited with the ATCC on Aug. 18,
2005 and have been given ATCC patent deposit designations PTA-6948
(17D11) and PTA-6949 (29D7) are provided.
[0050] In still another aspect, the present invention provides
monoclonal antibodies that are produced by the hybridomas that were
deposited with the ATCC on Aug. 18, 2005 and have been given ATCC
patent deposit designations PTA-6948 (17D11) and PTA-6949 (29D7),
respectively. The present invention also provides antibodies that
block the binding of these antibodies and therefore share the same
binding epitope on human TrkB (SEQ ID NO:1).
2. Preparation of Monoclonal Antibodies and Hybridomas
[0051] 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 inventive antibodies can be prepared by initially
immunizing an animal with human TrkB or a derivative thereof (e.g.,
a recombinant protein that includes the extracellular domain of
human TrkB) and then preparing monoclonals from suitably prepared
hybridomas.
[0052] In one aspect, the monoclonal antibodies of the present
invention are prepared by standard hybridoma technology using at
least two protein immunogens that are derived from TrkB proteins of
different species. For example, the immunogens may include a first
immunogen that was derived from human TrkB and a second immunogen
that was derived from a non-human TrkB. Preferably the immunogens
each include the extracellular domain of TrkB. In certain
embodiments the at least two immunogens are combined as a mixture
for immunization purposes.
[0053] In one embodiment, the first of these immunogens is a
recombinant protein that includes the extracellular domain (ECD) of
human TrkB. The ECD of human TrkB is comprised of amino acid
residues C32-H430 from the full length protein (which is set forth
as SEQ ID NO:1, GenBank Accession No. NP.sub.--006171). Note that
all protein and nucleic acid sequences that are present as of the
filing date in the GenBank, SwissProt, EMBL databases or any other
publicly available database are incorporated herein by reference
(including without limitation the sequences of the neurotrophins,
e.g., NGF, BDNF, NT-3, NT-4/5, etc.). The second immunogen is a
recombinant protein that includes the extracellular domain (ECD) of
murine TrkB. The ECD of murine TrkB is comprised of amino acid
residues C32-H429 from the full length protein (which is set forth
as SEQ ID NO:2, GenBank Accession No. P15209). Those skilled in the
art will appreciate that suitable immunogens that include these
domains 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).
[0054] In certain embodiments, the first and second immunogens are
administered in a ratio (by weight) that is greater than about 1.
For example, the ratio may be about 2, 3, 4, 5, 6, 7, 8, 9, 10 or
more. In one embodiment the ratio is greater than about 5. In one
embodiment the ratio is about 10. In one embodiment the first and
second immunogens are administered simultaneously as a mixture.
[0055] In one embodiment, one or both immunogens include versions
of their respective extracellular domains that differ slightly from
the naturally-occurring domains. For example, amino acids at the N-
or C-terminus of the extracellular domain may be missing.
Alternatively a few amino acids within the naturally-occurring
sequence may be mutated. Preferably these mutations are
conservative substitutions. It is to be understood that these
non-naturally occurring versions should include an amino acid
sequence that is at least 95%, preferably at least 96%, more
preferably at least 97%, yet more preferably at least 98% and even
more preferably at least 99% identical to the extracellular domains
that are found in SEQ ID NO:1 or SEQ ID NO:2.
[0056] In certain embodiments, the first and/or second immunogens
do not include any of the amino acids that are found outside of the
extracellular domain of TrkB (e.g., they do not include the amino
acids that are found in transmembrane and/or intracellular domains
of TrkB). In certain embodiments, the immunogens may include one or
more terminal amino acids that are absent from the naturally
occurring TrkB proteins. In particular, terminal amino acids may be
added to increase expression of the recombinant protein, as a
consequence of the vector used for expression, etc. In addition,
amino acid segments that are absent from the protein allergen may
be added to the amino and/or carboxyl terminus of the recombinant
protein, e.g., tags for purification, labels for detection, tags
that increase the solubility of the recombinant allergen, tags that
increase the stability of the immunogens, fusion with an unrelated
protein or adjuvant carrier protein, etc. A proteolytic cleavage
site may be introduced at the junction of the added amino acid
segment and the recombinant protein terminus to enable removal of
the added segment after the recombinant protein has been purified,
absorbed, etc. Common terminal modifications used in recombinant
technology are described in Current 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.
[0057] In one embodiment, the first and second immunogens have the
same composition as the two immunogens that are described in the
Examples and that were obtained from R&D systems, Inc. (Cat.
No. 397-TR/CF and 1494-TB/CF, respectively).
[0058] In certain embodiments, the first immunogen is as described
above but the second immunogen includes the extracellular domain
(ECD) from a non-human TrkB species other than murine (e.g., rat,
chicken, rabbit, etc.). The ECD of rat TrkB is comprised of amino
acid residues C32-H429 from the full length protein (which is set
forth as SEQ ID NO:3, GenBank Accession No. NP 036863). The ECD of
chicken TrkB is comprised of amino acid residues C32-T428 from the
full length protein (which is set forth as SEQ ID NO:4, GenBank
Accession No. CAA54468).
[0059] Once suitable immunogens have been prepared, the immunogens
are injected into any of a wide variety of animals (e.g., mice,
rats, rabbits, etc.). In one embodiment the immunogens are injected
into mice as described in the Examples. For example, the immunogens
are injected subcutaneously and intraperitoneally with complete
Freund's adjuvant. In certain embodiments, each animal is injected
subcutaneously at multiple different sites. In this step, the
recombinant proteins may serve as immunogens without further
modification. Alternatively, a superior immune response may be
elicited if the recombinant proteins are joined to an adjuvant
carrier protein, such as bovine serum albumin or keyhole limpet
hemocyanin (KLH). The immunogens are injected into the animal host,
preferably according to a predetermined schedule incorporating one
or more booster immunizations and the animals are bled
periodically. For example, in certain embodiments, one or more
booster immunizations are administered intravenously. Binding
between immune sera and the first (and optionally the second)
immunogen is then optionally assessed to confirm that a suitable
titer of antibodies has been raised.
[0060] Monoclonal antibodies that are specific for one or both of
the immunogens may be prepared by any standard method. For example,
the technique of Kohler and Milstein, Eur. J. Immunol. 6:511, 1976
and improvements thereto may be used. Briefly, these methods
generally involve the preparation of immortal cell lines capable of
producing antibodies having the desired specificity. Such cell
lines may be produced, for example, from spleen cells obtained from
one or more animals immunized as described above. The spleen cells
are then immortalized by, for example, fusion with a myeloma cell
fusion partner, preferably one that is syngeneic with the immunized
animal. In certain embodiments animals with suitable sera are
boosted one or more times with the first and/or second immunogen
before the spleen cells are removed. A variety of fusion techniques
may be employed. For example, the spleen cells and myeloma cells
may be combined with a nonionic detergent for a few minutes and
then plated at low density on a selective medium that supports the
growth of hybrid cells, but not myeloma cells. A certain selection
technique uses HAT (hypoxanthine, aminopterin, thymidine)
selection. After a sufficient time, usually about 1 to 2 weeks,
colonies of hybrids are observed. Single colonies are selected and
their culture supernatants tested for binding activity against the
first (and optionally the second) immunogen as described below.
[0061] Monoclonal antibodies may be isolated from the supernatants
of growing hybridoma colonies. In addition, various techniques may
be employed to enhance the yield, such as injection of the
hybridoma cell line into the peritoneal cavity of a suitable
vertebrate host, such as a murine. Monoclonal antibodies may then
be harvested from the ascites fluid or the blood. Contaminants may
be removed from the antibodies by conventional techniques, such as
chromatography, gel filtration, precipitation and extraction. The
antibody isotypes can be determined using standard methods. As
discussed in the Examples and shown in Table 1, we have prepared
specific murine antibodies belonging to isotypes IgG1, IgG2a and
IgG2b using these methods.
3. Characterization of Antibody Binding In certain embodiments,
inventive antibodies are characterized for their binding activities
to human TrkB (e.g., using ELISA and/or FACS as described in the
Examples). In certain embodiments binding to human TrkB proteins
that are expressed on a cell surface may also be assessed (e.g.,
using HEK293 cells as described in the Examples). Preferably,
inventive antibodies are also tested for their cross-species
binding activity (e.g., with the second immunogen). This allows
monoclonal antibodies that bind TrkB from both species to be
identified. 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.
[0062] 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
TrkB and BDNF. One may also assess whether the antibodies bind
non-human TrkB and/or human TrkA or TrkC.
[0063] Mapping of the relative antibody binding epitopes on TrkB
(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 TrkB. 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 TrkB.
4. Characterization of Antibody Function
[0064] In certain embodiments, inventive antibodies are
characterized for their functional ability to activate human TrkB.
Any agonist assay may be used. The Examples describe an exemplary
luciferase assay that has been shown to selectively represent the
activation of TrkB. TrkB autophosphorylation (e.g., as measured by
Western blot) can also be used as a measure of TrkB activation.
[0065] Alternatively or additionally, the human TrkB agonist
activity of the inventive antibodies can be assessed in an assay
that involves endogenous TrkB (e.g., in human neuroblastoma SY5Y
cells). As described in the Examples, such assays test for the
ability of each antibody to promote neurite growth or to increase
survival of differentiated cells following injury (e.g., serum
withdrawal injury). In certain embodiments the dose-dependent
effects of the inventive antibodies on neurite growth and/or cell
viability are measured.
[0066] In certain embodiments the purified monoclonal antibodies
are also characterized for their functional ability to activate
non-human TrkB (e.g., murine, rat, chicken, rabbit, etc.). The
Examples describe an assay in which the antibodies were tested in
rat cerebellar granule neuron (CGN) cultures for activity against
the endogenous rat TrkB receptor. As with the human cells a neurite
outgrowth assay and a neuroprotection assay can be performed. Other
useful assays are known in the art and will be recognized by those
skilled in the art.
[0067] In yet other embodiments and as described in the Examples,
the purified monoclonal antibodies are further characterized for
their functional ability to activate human TrkA and/or TrkC.
5. Humanized and Veneered Antibodies
[0068] When using an inventive antibody 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.
[0069] 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 (e.g., see Winter et al., Nature
349:293, 1991; Lobuglio et al., Proc. Nat. Acad. Sci. USA 86:4220,
1989; Shaw et al., J. Immunol. 138:4534, 1987; and Brown et al.,
Cancer Res. 47:3577, 1987). Rodent CDRs grafted into a human
supporting framework region (FR) prior to fusion with an
appropriate human antibody constant domain (e.g., see Riechmann et
al., Nature 332:323, 1988; Verhoeyen et al., Science 239:1534,
1988; and Jones et al. Nature 321:522, 1986) and rodent CDRs
supported by recombinantly veneered rodent FRs have also been
described (e.g., see EPO Patent Pub. No. 519,596).
[0070] 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 chains genes, but which
can express human heavy and light chain genes (e.g., see Lonberg
and Huszar Int. Rev. Immunol. 13:65-93, 1995 and U.S. Pat. Nos.
5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016).
[0071] Veneered versions of the inventive antibodies 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.
6. Single-Chain Antibodies
[0072] Single chain antibodies can also be prepared based on the
inventive antibodies. For example, a single-chain antibody (scFv)
can be engineered as described in, for example, Colcher et al.,
Ann. N Y Acad. Sci. 880:263-80, 1999; and Reiter, Clin. Cancer Res.
2:245-52, 1996. Specific methods are described in the Examples. The
single-chain antibody can be dimerized or multimerized to generate
multivalent antibodies having specificities for different epitopes
of human TrkB.
7. Pharmaceutical Compositions
[0073] Monoclonal antibodies of the invention may be administered
neat in order to activate TrkB in accordance with the present
invention. More commonly, however, they 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.
[0074] 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,
i.e., treatment, prevention or amelioration of a condition which
requires TrkB 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.
[0075] 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). 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.
[0076] 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.
[0077] 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.
[0078] 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.
[0079] 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.
[0080] 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.
[0081] Thus, the present invention also provides a pharmaceutical
composition in unit dosage form for activating TrkB, 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.
[0082] 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 TrkB
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.
8. Other Pharmaceutical Agents
[0083] According to the present invention, monoclonal antibodies of
the invention may be administered alone to modulate TrkB 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 TrkB activity.
[0084] For example, other pharmaceutical agents that can modulate
TrkB activity may be used in combination with the monoclonal
antibodies of the invention, including other activators of TrkB.
U.S. Pat. Nos. 5,770,577; 6,077,829; 6,723,701 and 6,800,607 (each
incorporated herein by reference in their entirety) describe BDNF
derivatives and compositions that may be useful in accordance with
the practice of the present invention.
[0085] 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. 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.). 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.
9. Therapeutic Uses
[0086] In one aspect, inventive antibodies are useful for treating
conditions (including symptoms, disorders, or diseases) which
require activation of TrkB. Such methods involve administering a
therapeutically effective amount of one or more inventive
antibodies to the individual. In certain embodiments, the invention
provides methods for treating neurological conditions. For example,
and without limitation, inventive antibodies 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 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. The benefits of TrkB activation in treating non-neurological
diseases such as cancer and diabetes has also been described and
the inventive antibodies may therefore find utility in such
contexts (e.g., U.S. Pat. Nos. 5,877,016 and 6,800,607 describe the
benefits of TrkB activation for treating cancer and diabetes,
respectively).
[0087] The methods of this invention are useful for treating the
conditions described herein in adults and children. They may also
be utilized for veterinary applications, particularly including
canine and feline applications. If desired, the methods herein may
also be used with farm animals, such as ovine, bovine, porcine and
equine breeds.
[0088] Inventive methods involve delivery of inventive monoclonal
antibodies via 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.
[0089] 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
by infusion or needleless injection techniques. For such parenteral
administration, the antibodies of the invention may be prepared and
maintained in conventional lyophylized 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.
[0090] 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.
[0091] 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.
[0092] 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.
[0093] For inventive methods utilizing oral delivery, such delivery
may be accomplished using solid or liquid formulations, for example
in the form of tablets, capsules, multi-particulates, 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.
[0094] 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-enhacing 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.
10. Diagnostic Uses
[0095] In another aspect, inventive antibodies may be used for
detecting TrkB in a sample (e.g., in order to diagnose a disorder
characterized by over or under expression of TrkB). According to
such methods an inventive antibody is combined with a sample under
conditions to allow specific binding. The specific binding is then
detected thereby indicating the presence of TrkB in the sample.
[0096] The sample can be derived from a body fluid (e.g., from
cerebrospinal fluid, blood, serum, urine, etc.) or an extract of
cells or tissue (e.g., a biopsy sample). Detection of binding can
be facilitated by coupling (i.e., physically linking) the antibody
to a detectable substance (i.e., antibody labeling). Examples of
detectable substances include various enzymes, prosthetic groups,
fluorescent materials, luminescent materials, bioluminescent
materials, and radioactive materials. Examples of suitable enzymes
include horseradish peroxidase, alkaline phosphatase,
.beta.-galactosidase, or acetylcholinesterase; examples of suitable
prosthetic group complexes include streptavidin/biotin and
avidin/biotin; examples of suitable fluorescent materials include
umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride or
phycoerythrin; an example of a luminescent material includes
luminol; examples of bioluminescent materials include luciferase,
luciferin, and aequorin; and examples of suitable radioactive
material include .sup.125I, .sup.131I, .sup.35S or .sup.3H.
[0097] A variety of protocols for measuring antibody binding,
including ELISA and FACS, are known in the art and provide a basis
for diagnosing altered or abnormal levels of TrkB expression. ELISA
and FACS are further described in the Examples. Normal or standard
values for TrkB expression are established by combining samples
taken from normal individuals with an inventive antibody under
conditions suitable for complex formation. The amount of standard
complex formation may be quantified by various methods depending on
the nature of the detectable substance. Preferably the amount of
standard complex formation is quantified by photometric means.
Levels of TrkB expression in samples from diseased individuals are
then compared with the standard values. Deviation between standard
and diseased values establishes the parameters for diagnosing
disease.
[0098] In certain embodiments, the inventive methods may be used to
diagnose neurological conditions that are characterized by over or
under expression of TrkB. For example, and without limitation,
inventive methods may be used to identify nervous systems that have
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 methods may
also be used to diagnose disorders such as attention-deficit
hyperactivity disorder (ADHD), depression and age-associated mental
impairment (i.e., by providing cognitive enhancement). The
inventive methods may also be used to diagnose congenital or
neurodegenerative conditions including Alzheimer's disease,
Parkinson's disease, Huntington's chorea, amyotrophic lateral
sclerosis (ALS) and conditions related to these.
11. Purification Uses
[0099] The invention also provides a method of using an antibody to
purify TrkB from a sample comprising combining an inventive
antibody with a sample under conditions to allow specific binding,
thereby producing an antibody-TrkB receptor complex, separating the
antibody-TrkB receptor complex from the remainder of the sample and
then separating the antibody from the TrkB receptor, thereby
obtaining a purified TrkB receptor. It will be appreciated that the
inventive antibodies can be used to isolate TrkB by any standard
technique, such as affinity chromatography or
immunoprecipitation.
EXAMPLES
[0100] The present invention is further illustrated and supported
by the following examples. However, these examples should in no way
be considered to further limit the scope of the invention. To the
contrary, one having ordinary skill in the art would readily
understand that there are other embodiments, modifications, and
equivalents of the present invention without departing from the
spirit of the present invention and/or the scope of the appended
claims.
Example 1
[0101] This example describes the preparation and in vitro
characterization and testing of a plurality of TrkB antibodies.
Materials and Methods
Immunogens
[0102] Murine anti-TrkB antibodies were prepared using a mixture of
two protein immunogens: a first recombinant protein that includes
the extracellular domain (ECD) of human TrkB (rhTrkB-ECD) (R&D
systems, Inc., Cat. No. 397-TR/CF) and a second recombinant protein
that includes the extracellular domain of murine TrkB (rmTrkB-ECD)
(R&D system, Inc., Cat. No. 1494-TB/CF).
[0103] The extracellular domain of human TrkB is comprised of amino
acid residues C32-H430 of the full length protein (which is set
forth as SEQ ID NO:1, GenBank Accession No. NP.sub.--006171).
rhTrkB-ECD was expressed in murine myeloma cell line NSO. The
calculated molecular mass of monomeric rhTrkB-ECD is 44 kDa;
however, when glycosylated it migrates as a broad band of 80-100
kDa in SDS-PAGE under reducing conditions.
[0104] The extracellular domain of murine TrkB is comprised of
amino acid residues C32-H429 of the full length protein (which is
set forth as SEQ ID NO:2, GenBank Accession No. P15209). In the
mhTrkB-ECD used for this Example, this sequence is flanked by an
N-terminal human CD33 signal peptide which is cleaved during
expression and a C-terminal H is Tag. rhTrkB-ECD was also expressed
in murine myeloma cell line NSO. The calculated molecular mass of
the monomeric mhTrkB-ECD is 45.9 kDa; however, when glycosylated it
migrates as a broad band of 75-100 kDa in SDS-PAGE under reducing
conditions.
Immunization Schedules
[0105] Five 8-week old female BALB/c mice were immunized with 10
.mu.g of rhTrkB-ECD that was pre-mixed with complete Freund's
adjuvant (CFA). The mixture was separated into portions that were
injected subcutaneously and intraperitoneally 4 times biweekly
(i.e., at weeks 0, 2, 4, and 6). The mice were also immunized at
week 7 by subcutaneous and intraperitoneal injection with 1 .mu.g
of rmTrkB-ECD that was pre-mixed with complete Freund's adjuvant
(CFA). Mice bleeds were collected at weeks 5 and 7 and antibody
responses in the sera were evaluated.
Generation of Murine Anti-TrkB Monoclonal Antibodies (mABs)
[0106] Three of the five mice were additionally boosted
intravenously with 10 .mu.g rhTrkB and 1 .mu.g rmTrkB 3 days prior
to the cell fusion (which occurred at week 12). Splenocytes from
these three mice were fused with murine myeloma cells P3X63Ag8.653
(ATCC, Cat. No. CRL-1580) at 4:1 ratio using 50% polyethylene
glycol (MW 1500) (Roche Diagnostics Corp., Cat. No. 783641). After
fusion, cells were seeded and cultured in 96-well plates at
1.times.10.sup.5 cells/well in selection medium (RPMI1640
containing 20% FBS and 5% Origen) (IGEN International, Inc., Cat.
No. 210001), 2 mM L-glutamine, 100 U/ml penicillin, 100 .mu.g/ml
streptomycin, 1.times.HEPES and 1.times.HAT
(hypoxanthine-aminopterin-thymidine) (Sigma, Cat. No. H0262).
Hybridoma supernatants were screened for binding with rhTrkB by
ELISA and staining on rhTrkB-expressing HEK293 stable cells by FACS
analysis (see below). The hybridoma supernatants selected to be
positive were subsequently tested for agonist activities on rhTrkB
using a luciferase assay (see below). Selected hybridomas were
subcloned four times by serial dilutions and once by FACS sorting
(see below). Conditional medium was harvested from the stable
hybridoma culture. Prosep-A (Montage Antibody Purification Spin
columns, Millipore, Cat. No. P36486) was used to purify IgG from
the hybridoma conditional medium. The Ig class of each mAb was
determined with a murine mAb isotyping kit (IsoStrip, Boehringer
Mannheim Corp., Cat. No. 1493027).
ELISAs
[0107] To measure the presence of TrkB-specific antibodies, 96-well
plates (Maxisorp, Nunc) were coated with 1 .mu.g/ml rhTrkB-EDC-Fc
(R&D system, Cat. No. 688-TK) or rmTrkB-Fc (R&D system) and
incubated overnight at 4.degree. C. After washing the plates and
blocking the wells with PBS (10 mM sodium phosphate, 150 mM NaCl,
pH 7.2) containing 1% BSA and 0.05% Tween-20, 100 .mu.l of diluted
immune serum or hybridoma supernatants were added and incubated for
1 hr at room temperature. The plates were washed, and the bound
anti-TrkB antibodies were detected using peroxidase conjugated goat
anti-murine IgG (H+ L) (PIERCE, Cat. No. 31434) followed by
incubation with the substrate TMB (BioFX Laboratories, Cat. No.
TMBW.sub.--1000-01). The absorbance values were determined at 450
nm in a spectrophotometer.
[0108] To determine the mAb concentration in the hybridoma
supernatant, 96-well plates were coated with 1 .mu.g/ml goat
anti-murine IgG (Fc.gamma.) (PIERCE, Cat. No. 31123) in PBS and
incubated overnight at 4.degree. C. After washing and blocking the
wells with PBS containing 1% BSA and 0.05% Tween-20, 100 .mu.l of
diluted hybridoma supernatants were added for 1 hr at room
temperature. Purified isotype-matching murine IgG was used as a
standard for the quantification of anti-TrkB IgG concentrations.
The plates were washed, and HRP labeled goat anti-murine IgG-Fc
were added and incubated for 1 hr at room temperature. After
washing the wells, the substrate TMB was added. Absorbance was
determined at 450 nm.
Characterization of Relative Antibody Binding Epitopes Using
Competition Binding
[0109] To determine how the binding of anti-TrkB IgG to TrkB
protein affects the BDNF interaction with TrkB, a 96-well plate was
coated with 0.3 .mu.g/ml of BDNF (R&D system, Cat. No.
248-BD/CF) in PBS and incubated overnight at 4.degree. C. After
washing and blocking the wells with PBS containing 1% BSA and 0.05%
Tween-20, 100 .mu.l of pre-incubated hybridoma supernatant (or
diluted immune serum) mixture with rhTrkB-EDC-Fc was added to the
plate and incubated for 1 hr at room temperature. After washing the
plate, peroxidase conjugated goat anti-human IgG, (Fc.gamma.)
(PIERCE, Cat. No. 31416) was added and incubated for 1 hr at room
temperature. The wells were washed and the substrate TMB was added.
Absorbance was determined at 450 nm.
[0110] To map the relative antibody binding epitopes on rhTrkB, a
96-well plate was coated with 1 mg/ml of each individual
TrkB-specific mAb in PBS and incubated overnight at 4.degree. C.
After washing and blocking the wells with PBS containing 1% BSA and
0.05% Tween-20, 100 .mu.l of mixture of each individual
pre-incubated TrkB mAb (20 .mu.g/ml) and rhTrkB-EDC-Fc (0.1
.mu.g/ml) was added to the wells and incubated for 1 hr at room
temperature. The plate was washed and incubated for 1 hr with
peroxidase conjugated goat ant-human IgG, (Fc.gamma.). After
washing the plate, the substrate TMB was added. Absorbance was
determined at 450 nm.
Luciferase Assay
[0111] A stable line of HEK-293 cells expressing rhTrkB was
generated by transfecting HEK-293 cells with pcDNA-hTrkB (full
length, see GenBank Accession No. NM.sub.--006180). Transfected
cells were selected in the presence of hygromycin for 2 wks with
limited dilutions. Following initial evaluation, a single clone of
cells was chosen for the study. For the luciferase assay, cells
were plated at 1.5.times.10.sup.4 cells/well in 100 .mu.l growth
medium in 96-well plates. The next day, cells were treated with 10
.mu.l of 10.times. final concentration of BDNF or test antibodies.
Luciferase activities were measured 16 hr after the treatments
using the Promega Steady-Glo assay kit according to the
manufacturer's protocol. In brief, media was replaced with 100
.mu.l of PBS and 100 .mu.l of Steady-Glo reagent was added. After
sealing the plates with TopSeal, the plates were shaken at Titer
Plate Shaker at speed .about.5 for 5 minutes and then luminescence
was measured using a TopCount NXT v2.13 instrument (Packard).
FACS Analysis
[0112] HEK-293 cells expressing rhTrkB were detached from the
plates with PBS containing 5 mM EDTA and transferred in to 5 ml
Falcon tubes (Becton Dickinson, Cat. No. 352063) with
2.times.10.sup.5 cells per tube. Cells were washed once with PBS by
centrifuging at 800 rpm at 4.degree. C. for 3 min, and incubated
for 30 min at 4.degree. C. with 100 .mu.l of hybridoma culture
supernatant, purified antibodies or immune serum diluted in PBS
with 1% FBS. The cells were washed 3 times with 1 ml PBS containing
1% FBS and incubated for 30 min at 4.degree. C. in the dark with PE
labeled goat anti-murine IgG, F(ab').sub.2 fragment (DAKO
Corporation, Cat. No. R0480) in PBS containing 1% FBS. Cells were
washed three times again and re-suspended in 250 .mu.l PBS
containing 1% FBS. Popidium iodide was used for detection of dead
cells, which were excluded from analysis. The fluorescence of 5000
cells/tube was counted by a FACScan flow cytofluorometer (Becton
Dickinson).
TrkB Autophosphorylation Assay
[0113] HEK-293 cells expressing rhTrkB were plated in 24 well
plates at 2.times.10.sup.5 cells/well in a DMEM growth media. The
next day, cells were incubated in serum-free DMEM for 90 min, then
stimulated with BDNF or testing antibodies at different
concentrations for 30 min at 37.degree. C. After washing with PBS
once, cells were lysed in Laemmli Sample Buffer (Bio-Rad) preheated
at 95.degree. C. Lysates were run through QIAshredder column
(Qiagen) and 20 .mu.l of samples were resolved on 4-12% Bis-Tris
gel (Invitrogen). Following transfer to nitrocellulose membranes,
phosphorylated TrkB bands were detected using PY490
phospho-Trk-specific antibody (1:100, Cell Signaling, Cat. No.
9141) followed by incubation with HRP-conjugated anti-rabbit
secondary antibody (Molecular Probes). The signals were developed
using ECL plus kit (Amersham).
Neurite Outgrowth Assay
[0114] Human neuroblastoma SH-SY5Y cells were grown in
DMEM:F12(1:1) supplemented with 2 mM L-glutamine, 15% FBS and
pen/strep. For the neurite outgrowth assay, cells were plated in 96
well tissue culture plates at the density of 4.times.10.sup.3
cells/well and incubated with 10 .mu.M all-trans-retinoic acid (RA)
to induce neuronal differentiation. On the third day, the media was
replaced with fresh growth media with or without BDNF or testing
antibodies, as indicated in the results. After three additional
days in culture, cells were fixed by IC-Fix for 30 mM at room
temperature and further processed for immunostaining of
beta-Tubulin III. First, cells were permeabilized by brief
incubation in 0.2% Triton in phosphate-buffered solution (TPBS).
Then samples were incubated in 1.5% normal goat serum (NGS) in TPBS
for 30 min to block non-specific binding, followed by incubation
with anti-beta-Tubulin III mAb (Tuj1, 1:1000, Covance) in 1.5%
NGS/TPBS. Tuj1 signals were detected using Alexa 488 murine
anti-goat antibody (1:500, Molecular Probes) and neurite outgrowth
was analyzed using Cellomics arrayscan.
[0115] For the measurement of neurite promoting effects in the
primary neurons, rat or murine cerebellar granular neuron (CGN)
cultures were prepared. Briefly, the cerebellum was dissected from
animals postnatal day 7 and cut into small pieces. The tissue was
treated with papain (Worthington Biochemical Corp.) for 30 min at
37.degree. C. and dispersed by gentle trituration. Following
centrifugation at 300 g for 5 min, dissociated cells were
reconstituted in Neurobasal medium containing B27 supplement, 0.5
mM 1-glutamine, and 25 mM potassium chloride and plated in 96 well
Biocoat plates precoated with poly-d-lysin (BD bioscience) at a
density of 1.2.times.10.sup.4 cells/well. Cells were treated with
BDNF or testing antibodies for 24 hrs, fixed with IC-Fix for 30 min
at room temperature and processed for Tuj1 immunostaining as
described above.
Cell Survival Assay
[0116] SH-SY5Y cells were plated in 96 well plates at
1.times.10.sup.4 cells/well and incubated with RA (10 .mu.M) to
induce neuronal differentiation. After 3 days, the culture media
was switched to growth media without serum (serum-free media) and
cells were treated with BDNF, testing antibodies or vehicle.
Following two additional days in culture, cell viability was
measured by MTT assay using the CellTiter 96 Non-Radioactive Cell
Proliferation Assay Kit (Promega) according to the manufacturer's
protocol.
Neuroprotection Assay
[0117] Rat or murine CGN cultures were prepared from 7 day-old pups
as described above and plated in 96 well Biocoat plates precoated
with poly-d-lysin (BD bioscience) at a density of
7.3.times.10.sup.4 cells/well. 24 hrs after plating, cells were
subjected to potassium serum deprivation (KSD) injury, which is
known to result in significant death of cerebellar granular
neurons. Sister cultures were co-treated with BDNF or testing
antibodies. Following 24 hr incubation, cell viability was measured
using a CellTiter 96 Non-Radioactive Cell Proliferation Assay Kit
(Promega).
Results
[0118] Evaluation of Antibody Responses from TrkB Immunized
Mice
[0119] To evaluate the specific immune responses to TrkB, the five
immunized mice (M1-M5) were bled one week following the third and
fourth immunizations. High anti-TrkB antibody titers in the serum
were determined by ELISA and FACS analysis in both the third and
the fourth bleeds. FIG. 1 represents results from the bleeds after
the fourth immunization. All five mice generated high titers
recognizing both rhTrkB-ECD and rmTrkB-ECD in ELISA (FIGS. 1A and
1B).
[0120] The location of antibody binding epitopes on hTrkB relative
to the BDNF binding site was also evaluated. Results from
competition ELISA showed that the immune bleeds can block
rhTrkB-EDC-Fc and BDNF interactions in an order of
M2=M5>M3>M4=M1 (FIG. 1C). Interestingly, the efficacies of
antibodies in blocking the rhTrkB-BDNF interaction correlated with
the antibody binding titers (compare FIGS. 1A and 1C). All immune
bleeds were also shown to bind to cell surface expressed rhTrkB on
HEK-293 cells as demonstrated by FACS analysis (FIG. 1D).
[0121] Following the observation of high titer specific binding of
immune sera to hTrkB, these immune bleeds were evaluated in a
luciferase reporter assay to test their agonistic activities. The
luciferase activity in HEK-293 cells expressing rhTrkB has been
shown to selectively represent the activation of TrkB (FIG. 2).
Incubation of these cells with immune bleeds significantly
increased the luciferase signals in a dose-dependent manner (FIG.
3). Again, the efficacy of agonistic activities was in an order of
M2=M5>M3>M4=M1. The three mice with the highest antibody
titers and robust agonist activities (M2, M3 and M5) were chosen
for subsequent hybridoma generation.
Production of Monoclonal Antibodies
[0122] As described previously, the three selected mice (M2, M3 and
M5) were boosted intravenously with 10 .mu.g of rhTrkB-ECD and 1
.mu.g of rmTrkB-ECD three days prior to the fusion. From the first
round of hybridoma screening, ninety-four clones were picked up
which bound strongly to rhTrkB-EDC-Fc in an ELISA assay. Twenty of
these clones cross-reacted with rmTrkB-ECD in an ELISA assay. FACS
analysis confirmed that fifty-four clones bound to rhTrkB expressed
on the surface of HEK-293 cells.
[0123] The TrkB agonist activity of each clone was tested using a
luciferase assay and seventeen hybridoma clones with highest
activities were selected for further characterization. Following
stabilization of these clones, three rounds of subcloning with
serial dilution, and one round of subcloning with FACS sorting, the
monoclonal antibodies from each culture conditional medium were
collected and purified by using ProSep-A (Montage Antibody
Purification Kits). The IgG isotypes of each antibody were
determined by Murine Isotyping test kit (Table 1). The antibody
concentrations were determined by Murine IgG quantification ELISA
and all seventeen clones produced good levels of Ig in the culture
supernatants.
TABLE-US-00001 TABLE 1 mAb Murine IgG Isotype Ig concentration
(.mu.g/ml) 2E8 IgG2b, k 14 4C7 IgG2b, k 8 5D8 IgG2b, k 28 5E11
IgG2b, k 55 6D5 IgG2b, k 26 6E2 IgG2b, k 18 6E6 IgG2b, k 14 7E1
IgG2b, k 26 7F5 IgG2b, k 16 11E1 IgG1, k 20 16E11 IgG2b, k 5 17D11
IgG1, k 22 18C3 IgG1, k 18 19E12 IgG2a, k 10 29D7 IgG1, k 52
Characterization of Monoclonal Antibodies
[0124] The purified TrkB-specific monoclonal antibodies were
characterized for their binding activities to hTrkB by ELISA. Most
of the antibodies bound to rhTrkB with high binding affinities
(ED.sub.50=10.sup.-11(M)), except for a clone 29D7, for which the
ED.sub.50 dropped from 10.sup.-11(M) to 10.sup.-10 (M) after
purification (data not shown). Two clones 12F4 and 18C8 lost their
binding activities after purification and were therefore deselected
from the priority list (and thus were not included in Table 1).
Using FACS analyses, all of the fifteen remaining TrkB binding mAbs
were also shown to specifically bind to hTrkB expressed on the
surface of HEK293 cells. Antibodies were also tested for their
cross-species binding activities to rmTrkB by ELISA. While most of
the antibodies were found to bind rmTrkB weakly, clones 17D11, 18C3
and 29D7 were found to bind rmTrkB with
ED.sub.50=10.sup.-10.about.11 (M) (Table 2).
[0125] A competition ELISA assay was used to determine whether the
antibodies block the interaction of rhTrkB and BDNF. Clone 29D7
showed no blocking activity, while clones 17D11, 18C3 and 19E12
partially blocked rhTrkB-BDNF interaction. All of the other clones
blocked rhTrkB-BDNF interaction with IC.sub.50=3-5.times.10.sup.-10
(M) (Table 2).
TABLE-US-00002 TABLE 2 Binding Activity Blocking activity rhTrkB
rmTrkB 293-hTrkB hTrkB-BDNF mAb [ED.sub.50 (nM)] [ED.sub.50 (nM)]
[Mean value] [IC.sub.50 (nM)] 2E8 0.020 W* 251.88 0.30 4C7 0.017 W*
241.26 0.30 5D8 0.019 W* 247.43 0.30 5E11 0.016 W* 263.27 0.40 6D5
0.021 W* 258.74 0.58 6E2 0.016 W* 235.47 0.28 6E6 0.019 W* 246.15
0.40 7E1 0.016 W* 237.89 0.27 7F5 0.016 W* 240.03 0.30 11E1 0.020
NB** 117.83 0.30 16E11 0.020 W* 224.55 0.28 17D11 0.020 0.300
134.52 Partial block 18C3 0.020 0.300 132.03 Partial block 19E12
0.031 W* 194.41 Partial block 29D7 0.120 0.013 95.59 No block W*:
Weak binding NB**: No binding
[0126] Mapping of the relative antibody binding epitopes on rhTrkB
was conducted by examining the activity of each individual antibody
in blocking the binding of other antibodies to rhTrkB. For example,
the observation that two antibodies blocked each other's binding
with rhTrkB suggests these antibodies may bind to the same epitope
or overlapping epitopes on rhTrkB (Table 3). In Table 3, the
pre-bound antibodies are presented in each row while the competing
(i.e., coating) antibodies are presented in each column. The
results showed that clones 11E1, 19E12 and 29D7 may recognize
unique epitopes. Clones 17D11 and 18C3 appear to compete for the
same binding site. All the remaining clones competed with each
other and may share the same binding epitope.
TABLE-US-00003 TABLE 3 2E8 4C7 5D8 5E11 6D5 6E2 6E6 7E1 7F5 11E1
16E11 17D11 18C3 19E12 29D7 c mAb* 2E8 + + + + + + + + + +/- + - -
- - - 4C7 + + + + + + + + + +/- + - - - - - 5D8 + + + + + + + + +
+/- + - - - - - 5E11 + + + + + + + + + +/- + - - - - - 6D5 + + + +
+ + + + + +/- + - - - - - 6E2 + + + + + + + + + +/- + - - - - - 6E6
+ + + + + + + + + +/- + - - - - - 7E1 + + + + + + + + + +/- + - - -
- - 7F5 + + + + + + + + + +/- + - - - - - 11E1 + + + + + + + + + +
+ - - - +/- - 16E11 + + + + + + + + + +/- + - - - - - 17D11 - - - -
- - - - - - - + + +/- - - 18C3 - - - - - - - - - - - + + +/- - -
19E12 - - - - - - - - - - - + + + - - 29D7 - - - - - - - - - - - -
- - + - c mAb* - - - - - - - - - - - - - - - - +: Complete block.
+/-: Partial block. -: No block. c mAb*: A mixture of murine IgG1,
IgG2a and IgG2b.
Luciferase Activities
[0127] The purified TrkB-specific antibodies were examined using a
luciferase assay to demonstrate agonist activities. All of the
antibodies caused dose-dependent increases in the signal with
EC.sub.50 in the range of 10.sup.-10 (M) (FIG. 4). The maximum
signal window was .about.7 fold over basal for most of the
antibodies, which was comparable to the response induced by BDNF at
200 ng/ml (6.2 fold over basal).
Functional Activities of mAbs
[0128] To test if the TrkB-binding antibodies have functional
agonist activities mediated by endogenous TrkB receptor activation,
neurite promoting effects were evaluated following treatments with
these antibodies. Based on the species specific binding
characteristic of the antibodies, we first utilized human
neuroblastoma SY5Y cells, which are known to express TrkB upon
neuronal differentiation. Consistent with previous reports, we
observed that the addition of BDNF promoted neurite outgrowth as
demonstrated by increases in the neurite length and the number of
branch points (FIGS. 5A and 5B). Importantly, most antibodies also
significantly increased neurite outgrowth with efficacies
comparable or even superior to BDNF (FIGS. 5A and 5B). Examples of
representative images of a few treatment groups are shown in FIG.
5C.
[0129] Seven TrkB antibodies with highest activities (6E2, 7F5,
11E1, 16E11, 17D11, 19E12 and 29D7) were selected for full
dose-response analysis. They induced dose-dependent increases in
the neurite growth with EC.sub.50 of around 10.sup.-10 (M) (FIG.
5D, data was not obtained for 6E2). These antibodies were also
tested for their ability to increase survival of differentiated
SY5Y cells following serum withdrawal injury. Results showed that
BDNF and several antibodies protected differentiated SY5Y cells,
demonstrating dose-dependent increases in cell viability (FIG.
6).
[0130] Two TrkB antibodies, 17D11, 18C3 and 29D7, which were shown
to bind to rmTrkB, were also tested in rat cerebellar granule
neuron (CGN) cultures for activity against the endogenous rat TrkB
receptor. In both the neurite outgrowth assay and the
neuroprotection assay, only 29D7 showed activities comparable to
those of BDNF, while 17D11 and 18C3 were inactive (FIG. 7 and data
not shown). It may suggest that 17D11 and 18C3 recognize an epitope
on murine TrkB that contains different amino acids in rat TrkB.
TrkB Phosphorylation Analysis
[0131] Seven TrkB antibodies (6E2, 7F5, 11E1, 16E11, 17D11, 19E12
and 29D7) selected based on all activities described above were
evaluated in Western analysis for induction of TrkB
autophosphorylation. All antibodies led to robust TrkB
phosphorylation, and these effects were antagonized by treatment
with the kinase inhibitor K252a, indicating that these TrkB-binding
antibodies caused activation of TrkB (FIG. 8).
[0132] Two TrkB antibodies, 17D11, 18C3 and 29D7, which were shown
to bind to rmTrkB, were also tested in rat cerebellar granule
neuron (CGN) cultures for activity against the endogenous rat TrkB
receptor. In both the neurite outgrowth assay and the
neuroprotection assay, only 29D7 showed activities comparable to
those of BDNF, while 17D11 and 18C3 were inactive (FIG. 7 and data
not shown). It may suggest that 17D11 and 18C3 recognize an epitope
on murine TrkB that contains different amino acids in rat TrkB.
TrkB Phosphorylation Analysis
[0133] Seven TrkB antibodies (6E2, 7F5, 11E1, 16E11, 17D11, 19E12
and 29D7) selected based on all activities described above were
evaluated in Western analysis for induction of TrkB
autophosphorylation. All antibodies led to robust TrkB
phosphorylation, and these effects were antagonized by treatment
with the kinase inhibitor K252a, indicating that these TrkB-binding
antibodies caused activation of TrkB (FIG. 8).
ATCC Deposits
[0134] The hybridomas that produced the 17D11 and 29D7 TrkB
antibodies were deposited with the ATCC on Aug. 18, 2005 and have
been given ATCC patent deposit designations PTA-6948 and PTA-6949,
respectively.
Example 2
[0135] This example describes further in vitro characterization and
testing of a plurality of TrkB antibodies. In particular, this
example describes experiments that were performed to assess the
TrkB vs. TrkA and TrkC specificity of some of the antibodies of
Example 1.
Materials and Methods
FACS Analysis
[0136] HEK-293 cells expressing human TrkA were detached from the
plates with PBS containing 5 mM EDTA and transferred in to 5 ml
Falcon tubes (Becton Dickinson, Cat. No. 352063) with
2.times.10.sup.5 cells per tube. Cells were washed once with PBS by
centrifuging at 800 rpm at 4.degree. C. for 3 min, and incubated
for 30 min at 4.degree. C. with 100 .mu.l of hybridoma culture
supernatant or immune serum diluted in PBS with 1% FBS. The cells
were washed 3 times with 1 ml PBS containing 1% FBS and incubated
for 30 min at 4.degree. C. in the dark with PE labeled goat
anti-murine IgG, F(ab').sub.2 fragment (DAKO Corporation, Cat. No.
R0480) in PBS containing 1% FBS. Cells were washed three times
again and re-suspended in 250 .mu.l PBS containing 1% FBS. Popidium
iodide was used for detection of dead cells, which were excluded
from analysis. The fluorescence of 5000 cells/tube was counted by a
FACScan flow cytofluorometer (Becton Dickinson).
Luciferase Assay
[0137] Stable lines of HEK-293 cells expressing human TrkA (or
human TrkC) were prepared. Transfected cells were selected in the
presence of hygromycin for 2 wks with limited dilutions. Following
initial evaluation, a single clone of cells was chosen for the
study. For the luciferase assay, cells were plated at
1.5.times.10.sup.4 cells/well in 100 .mu.l growth medium in 96-well
plates. The next day, cells were treated with 10 .mu.l of 10.times.
final concentration of NGF (or NT-3) or test antibodies. Luciferase
activities were measured 16 hr after the treatments using the
Promega Steady-Glo assay kit according to the manufacturer's
protocol. In brief, media was replaced with 100 .mu.l of PBS and
100 .mu.l of Steady-Glo reagent was added. After sealing the plates
with TopSeal, the plates were shaken at Titer Plate Shaker at speed
.about.5 for 5 minutes and then luminescence was measured using a
TopCount NXT v2.13 instrument (Packard).
Results
FACS Analysis of Monoclonal Antibodies
[0138] The neat conditioned media for each of the TrkB-specific
monoclonal antibodies of Table 1 were characterized for their
binding activities to human TrkA by FACS. All of the tested
antibodies failed to bind to human TrkA. Specifically, the
antibodies were tested at concentrations ranging from about 5 to
about 60 .mu.g/ml (see specific concentrations for each antibody in
Table 1, these correspond to concentrations in the range of about
30 to about 500 nM) and each antibody failed to show any detectable
binding. The FACS data is shown in FIG. 9A.
Luciferase Activities
[0139] A subset of the purified TrkB-specific antibodies of Table 2
were also examined using a TrkA or TrkC luciferase assay to assess
agonist activities. None of the tested antibodies activated TrkA or
TrkC. Specifically, the antibodies were tested at concentrations of
up to about 3 .mu.g/ml (about 20 nM) and in each case the
antibodies failed to cause detectable increase above basal (see
FIG. 9B for TrkA and FIG. 10 for TrkC). In contrast, NGF induced a
.about.6 fold increase over basal for TrkA at 300 ng/ml (FIG. 9A)
and NT-3 induced a .about.6 fold signal increase over basal for
TrkC at 300 ng/ml (FIG. 10).
Example 3
[0140] This example describes the in vivo testing of a plurality of
TrkB antibodies in a rodent model of neonatal hypoxia-ischemia
(HI).
Materials and Methods
Animals and Surgical Procedures
[0141] The rodent model of neonatal hypoxia-ischemia (HI) was based
on the Levine procedure that is set forth in FIG. 11 (e.g., see
Levine, Am. J. Pathol. 36:1-17, 1960; Rice et al., Ann. Neurol.,
9:131-141, 1981 and Gidday et al., Neurosci. Lett. 168:221-224,
1994 each of which is incorporated herein by reference). Briefly,
pups at P7 were anesthetized with 2.5% halothane and the left
common carotid artery was permanently ligated. After the incisions
were sutured, pups were returned to the home cage for recovery and
feeding. Two hours later, pups were placed in individual
containers, through which humidified 8% oxygen was flowed. After
2.5 hrs of a hypoxic ischemic period, the pups were returned to
their home cages. For treatment, animals received a 5 .mu.l
intracerebroventricular injection of 0.1 or 0.3 nmole of either
BDNF, anti-TrkB monoclonal antibody 29D7, control IgG1 or vehicle
just prior to the hypoxic insult.
Assessment of Brain Tissue Loss
[0142] At P14, brain sections were prepared from some of the mice
to determine damage caused by the HI injury. The coronal sections
of the striatum, cortex and hippocampus were stained with cresyl
violet and the percent area loss in the lesioned hemisphere was
compared with the intact area. FIG. 17 compares the stains that
were obtained from the striatum and hippocampus for different
treatments. FIGS. 18A and 18B compare the total brain tissue loss
and sub-regional tissue loss, respectively, for different
treatments. The data are shown as mean and standard error of the
mean (S.E.M.). BDNF and anti-TrkB monoclonal antibody 29D7 showed
significant dose-dependent protection of the brain from HI damage.
The protection was not localized to any specific sub-region of the
brain but greatest protection was generally observed in the
cortex.
Biochemical Analysis
[0143] 24 hours after the HI injury, brain sections were prepared
from a subset of the mice for biochemical analysis. Hippocampal and
cortical brain tissues were dissected, lysed and subjected to
several biochemical assays.
[0144] A DEVD-AMC cleavage assay was used to determine caspase-3
activities (e.g., see Nagase et al., Immunol Lett. 84:23, 2002
incorporated herein by reference). The results for different
regions of the brain are shown in FIG. 19 for mice that were given
0.1 nmole of different test reagents (mean.+-.S.E.M.). BDNF and
anti-TrkB monoclonal antibody 29D7 blocked caspase-3 activation
caused by HI injury.
[0145] Protein samples (30 .mu.g/lane) from mice treated with 0.3
nmole of different test reagents were separated by SDS-PAGE and
subjected to immunoblotting with antibodies against certain
caspase-3 substrates (PARP and .alpha.-spectrin). Antibodies
against .beta.-actin were used as controls to verify equal loading
of proteins. The results shown in FIG. 20 demonstrate inhibition of
cleavage of these caspase-3 substrates by BDNF and anti-TrkB
monoclonal antibody 29D7 (C=contra and I=ipsi sides of carotid
ligation).
Follow Up Experiments
[0146] The following are prophetic follow up experiments that could
be performed on the mice of this study. The performance of the
remaining mice in spatial learning and memory tests could be
assessed (e.g., without limitation the tests described in Almli et
al., Exp. Neurology 166:99-114, 2000 incorporated herein by
reference). For example, the mice could be tested between P20-P30.
Optionally, the performance could be assessed over several days
during that period or even at later time points.
[0147] Treatment protocols that produce a positive outcome (as
measured by a reduction in brain tissue loss, an improvement in
spatial learning and memory or otherwise) could be repeated over an
even greater range of dosages. Alternatively or additionally,
treatment protocols could be repeated with different modes of
administration (e.g., intraperitoneal administration, intravenous
administration, etc.); with different start points (e.g., before
ligation, immediately after hypoxia, with a variable delay after
hypoxia, etc.); and/or with different duration or frequency (e.g.,
daily treatment for 2 wks post injury, etc.).
Example 4
[0148] This example describes the preparation and testing of human
single-chain Fv (scFv) antibodies against human TrkB. Human
antibody phage display libraries (CS, BMV and DP-47; Cambridge
Antibody Technology) containing single-chain Fv fragments were
selected for TrkB binding using the following methods.
Materials and Methods
Selection of Antibodies by Panning
[0149] 10 .mu.g of hTrkB-EDC-Fc was coated on Nunc Maxisorp plate
in 100 .mu.l and left overnight at 4.degree. C. A phage library was
pre-blocked (50 .mu.l phage aliquot added to 50 .mu.l of 6% skimmed
milk in 2.times.PBS) and negatively selected against PSGL-Fc
(Wyeth, Lot. No. 00H25M004) for 1 hr at room temperature to deplete
phage reactive against Fc. Deselected phage were transferred to the
target protein-coated plate (hTrkB-EDC-Fc) and incubated at room
temperature for 2 hrs. Wells were washed 10 times with PBS/0.1%
Tween 20 and 5 times with PBS. Bound phage were eluted with 50
.mu.l/well of freshly made 100 mM TEA (140 .mu.l of TEA in 10 ml of
ultra pure water). Eluted phage were neutralized using 25 .mu.l of
sterile 1 M Tris-HCl pH 7.5. Phage were infected into 10 ml of a
mid-log (O.D at 600 nm=0.5) of E. coli TG1 cells. Transformed cells
were spread onto a 2.times.TYAG agar Bioassay plate and incubated
overnight at 30.degree. C.
Soluble Phase Selection (Biotin Selection)
[0150] Phage antibodies were selected using biotinylated
hTrkB-EDC-Fc according to the protocol described above with the
following exceptions. Pre-blocked phage were deselected first
against 100 nM biotinylated PSGL-Fc followed by positive selection
on 100 nM biotinylated hTrkB-EDC-Fc. Human TrkB specific phage were
captured using pre-blocked magnetic streptavidin beads (Dynabeads
M-280 streptavidin, Cat. No. 112.06). Beads were washed 10 times
with PBS/0.1% Tween 20 and 3 times with PBS. Bound phage were
eluted with 200 .mu.l of freshly made 100 mM TEA (140 .mu.l of TEA
in 10 ml of ultra pure water) and neutralized using 100 .mu.l of
sterile 1 M Tris-HCl pH 7.5 to the eluted phage to neutralize the
TEA. Phage were infected into E. coli and propagated as described
in the previous step.
Phage Rescue
[0151] E. coli infected with phage were scrapped off the Bioassay
agar plates and mixed with 10 ml 2.times.TYAG per Bioassay plate
(2.times.TY with 100 .mu.g/ml Amp and 2% glucose). 20 ml
2.times.TYAG was inoculated with 100 .mu.l of cell suspension and
grown at 37.degree. C. (300 rpm) to OD at 600 nm=0.3-0.5. E. coli
were superinfected with 3.3 .mu.l of MK13KO7 helper phage and
incubated at 37.degree. C. (150 rpm) for 1 hr. Superinfected cells
were re-suspended in 20 ml 2.times.TYAK medium (2.times.TY/100
.mu.g/ml Amp/50 .mu.g/ml Kanamycin) and grown overnight at
25.degree. C. at 280 rpm. E. coli were spun at 3500 rpm for 15 min
and the supernatant containing the phage were used for the next
round of selection.
Phage Antibody Preparation for ELISA
[0152] Single colonies of infected E. coli were picked into
microtitre wells (Costat Cellwells) containing 150 ml of
2.times.TYAG media (2% glucose) per well. Clones were allowed to
grow at 37.degree. C. (100-120 rpm) for 5-6 hrs (OD at 600 nm=0.5).
M13K07 helper phage stock (10.sup.13 pfu/ml) was diluted 1:1000
with 2.times.TYAG medium and 20 .mu.l was added to each well. The
wells were incubated at 37.degree. C. (100 rpm) for 1 hr. Plates
were centrifuged at 3200 rpm for 10 min, the supernatant was
removed and the cells were resuspended in 150 .mu.l of 2.times.TYAK
medium. Cultures were grown overnight at 25.degree. C. (120 rpm).
The next day, plates were centrifuged at 3200 rpm for 15 min and
the supernatant was transferred to a fresh plate for the phage
ELISA assay.
Phage ELISA
[0153] ELISA plates were coated with 50 .mu.l per well of 1
.mu.g/ml of hTrkB-EDC-Fc (BSA as a control) in PBS at 4.degree. C.
overnight. Wells were rinsed 3 times with PBS and blocked with 300
.mu.l per well of PBS/3% skimmed milk at room temperature for 1 hr.
Phage were blocked with equal volume of PBS/6% skimmed milk and
incubated at room temperature for 1 hr. 50 .mu.l of blocked phage
were added to ELISA wells and incubated at room temperature for 1
hr. Plates were washed 3 times with PBS/0.1% Tween followed by 3
times with PBS. 50 .mu.l of HRP-Mouse anti-M13 antibody in PBS/3%
skimmed milk (1:5000, Amersham Pharmacia biotech, Cat. No.
27-9421-01) was added to each well and incubated at room
temperature for 1 hr. Plates were washed as before and 50 .mu.l TMB
substrate was added to each well and developed for 2-5 min. The
reaction was stopped by adding 50 .mu.l of 0.5 M sulphuric acid and
the absorbance was read at 450 nm.
Single-Chain Fv Preparation for FACS
[0154] Single colonies were picked and grown in deepwell microtiter
plates containing 0.9 ml 2.times.TYAG media at 37.degree. C. for
5-6 hrs. scFv expression was induced by adding IPTG to a final
concentration of 0.02 mM in 2.times.TY medium and growth at
30.degree. C. overnight. E. coli were harvested after overnight
growth and osmotically shocked with 150 .mu.l TES buffer diluted
1:5 in water and incubated on ice for 30 min. Cells were
centrifuged and the supernatant transferred to a fresh plate for
the FACS assay. 293 cells stably transfected with TrkB were stained
with scFv prepared as described. Cells were incubated on ice for 30
min and then washed with PBS buffer. Single-chain Fv was detected
using a solution containing 9E10 anti-myc antibody diluted 1:1000
followed by addition of PE conjugated anti mouse IgG-Fc antibody
diluted 1:500. Stained cells were analysed on a Bectin-Dickinson
Flow Cytometer.
Results
Phage ELISA
[0155] Table 4 shows the results from phage ELISA assays from the
different phage library selections (panning and soluble
selections). FIG. 12 shows the phage binding ELISA data from a
representative set of TrkB positive clones (BMV library panning
selection). The majority of TrkB selected clones showed strong and
specific reactivity against TrkB-EDC-Fc and not to the control
protein PSGL-Fc.
TABLE-US-00004 TABLE 4 phage ELISA Sequence Antigen (selection)
Library (% positives) (% diversity) TrkB-EDC-Fc CS 78 53 (panning)
BMV 58 23 DP-47 45 39 Biotinylated CS 3 100 TrkB-EDC-Fc (soluble)
BMV 6 50 DP-47 10 50
[0156] After two rounds of panning selection, 78% of randomly
picked clones from the CS library were positive for TrkB-EDC-Fc
binding but not for PSGL-Fc binding. Likewise, 3% of clones were
positive after two rounds of the soluble selections. The numbers
for the BMV and DP-47 libraries were 58% and 45% positive clones
from panning selection and 6% and 10% from soluble selection. Using
DNA sequencing, we confirmed that 53% of the CS panning clones were
unique whereas 100% of the CS soluble selection clones were unique.
The numbers for the BMV and DP-47 libraries were 23% and 39% unique
from panning selections and 50% and 50% unique from soluble
selections.
FACS Analysis
[0157] FIG. 13 shows the binding specificity of TrkB selected scFv
antibodies tested using the FACS assay. The data shows that TrkB
selected scFv antibodies react with membrane associated TrkB in a
specific manner (filled blue histograms) with no cross-reactivity
to control cells (open green histograms).
Example 5
[0158] This example compares the binding activities of the 29D7
TrkB antibody and a 29D7 Fab fragment against human and mouse TrkB.
Mouse (rhTrkB-EDC-Fc) and human (rmTrkB-EDC) TrkB were coated on
ELISA plates as described above in Example 1.
[0159] FIGS. 14A and 14B show the ELISA binding results for human
and mouse TrkB, respectively. Both the 29D7 IgG antibody and its
Fab fragment have dose-dependent binding activities to human and
mouse TrkB. However, the ED.sub.50 value of the Fab was about 100
fold lower than that of intact 29D7. Neither an isotype control
IgG1 nor its Fab fragment bound to TrkB.
Example 6
[0160] This example compares the agonistic activities of the 29D7
TrkB antibody and the Fab fragment of Example 5 against human TrkB.
Agonistic activities were examined using a luciferase assay and
HEK-293 cells which express surface rhTrkB as described in Example
1.
[0161] HEK-293 cells were treated with 29D7 IgG or 29D7 Fab at
indicated concentrations and accumulating luciferase activities
were measured 16 hours post-treatment. As shown in FIG. 15, both
total IgG and Fab of 29D7 induced dose-dependent luciferase
activities, indicating activation of TrkB. However, the EC.sub.50
value of 29D7 Fab was about 27 fold higher than that of 29D7 IgG
(0.083 nM and 2.28 nM for 29D7 IgG and 29D7 Fab, respectively).
Neither an isotype control IgG1 nor its Fab fragment had effects on
TrkB activation.
Example 7
[0162] This example describes the epitope mapping analysis of
certain inventive TrkB monoclonal antibodies. The mapping was
performed against linear, single looped and double looped peptides
that were deduced from sequences within the human TrkB
extracellular domain.
[0163] As shown in FIG. 16, the 17D11 monoclonal antibody
recognizes loop-3 of the IgG-2 segment of human TrkB (KNEYGKD, SEQ
ID NO:7, amino acids 364 to 370 of SEQ ID NO:1); and loop-1 of the
IgG-2 segment of human TrkB (KGNPKP, SEQ ID NO:8, amino acids 308
to 313 of SEQ ID NO:1). The mouse sequence for the former epitope
(KNEYGKD, SEQ ID NO:7, amino acids 364 to 370 of SEQ ID NO:2) is
identical to the human sequence. The mouse sequence for the latter
epitope (RGNPKP, SEQ ID NO:9, amino acids 308 to 313 of SEQ ID
NO:2) differs by one amino acid.
[0164] As shown in FIG. 16, the 29D7, 7F5, 11E1 and 19E12
monoclonal antibodies all recognize loop-3 of the IgG-1 segment of
human TrkB (ENLVGED, SEQ ID NO:10, amino acids 269 to 275 of SEQ ID
NO:1); and may also recognize loop-1 of the IgG-1 segment of human
TrkB (AGDPVP, SEQ ID NO:11, amino acids 221 to 226 of SEQ ID NO:1).
The mouse sequence for the former epitope (ENLVGED, SEQ ID NO:10,
amino acids 269 to 275 of SEQ ID NO:2) is identical to the human
sequence. The mouse sequence for the latter epitope (GGDPLP, SEQ ID
NO:12, amino acids 221 to 226 of SEQ ID NO:2) differs by two amino
acids.
Example 8
[0165] This example describes in vivo TrkB activation experiments
that were performed with the anti-TrkB antibody 29D7. Briefly, pups
at P7 received a 5 .mu.l intracerebroventricular injection of 0.3
nmole of either anti-TrkB monoclonal antibody 29D7 or vehicle.
Brain tissues were then dissected 1, 2, 6, 12 and 24 hours after
injection. Tissues were lysed and equal amounts of protein samples
(30 .mu.g/lane) were separated by SDS-PAGE and subjected to
immunoblotting with antibodies specific to proteins that are
phosphorylated as a result of TrkB activation (phospho-ERK1/2 and
phospho-AKT). Antibodies against .beta.-actin were used as controls
to verify equal loading of proteins. The results of FIG. 21 show
that anti-TrkB monoclonal antibody 29D7 induced time-dependent
phosphorylation of ERK1/2 and AKT (data from hippocampal and
cortical samples are shown and are representative of results
obtained from four other samples).
[0166] FIG. 22 is a densitometric quantification of the ERK
phosphorylation shown in FIG. 21. FIG. 23 is a densitometric
quantification of the AKT phosphorylation shown in FIG. 21. FIG. 24
shows vehicle and 29D7 treated brain tissues that have been fixed
and immunoflourescently labeled with an antibody for the
neuron-specific marker NeuN (left) and an anti-phospho-ERK1/2
antibody (center). The right hand figures show these two merged.
ERK1/2 phosphorylation is significantly stronger in the 29D7
treated samples. FIG. 25 shows the time-course of ERK1/2 activation
in the cortical and hippocampal tissues following
intracerebroventricular injection of anti-TrkB monoclonal antibody
29D7.
Example 9
[0167] This example describes a MAG/myelin-induced neurite
inhibition assay that was performed with the anti-TrkB antibody
29D7. Briefly, aliquots of 50 .mu.l recombinant rat MAG(1-5) or
purified rat myelin were added to 96 well flat bottom tissue
culture plates and air-dried overnight at room temperature. The
total amounts of MAG(1-5) or myelin were 0.25-0.5 .mu.g per well,
unless indicated differently. Next day, the MAG- or myelin-coated
plates were treated with 50 .mu.l of poly-D-lysine (17 .mu.g/ml)
for 1.5 hours, followed by incubation with a media containing 10%
FBS for 1 hour at 37.degree. C. After aspiration of the media, rat
CGN cells were plated at a density of 8,000 cells/well in
B27-supplemented Neurobasal growth media containing 200 mM
L-Glutamine, 2 M KCl, and 100 U/ml penicillin and streptomycin.
When indicated, treatment reagents were added at the time of cell
plating. Neurons were grown in a 37.degree. C. incubator
equilibrated with 5% CO.sub.2. Approximately 20 hours post plating,
cells were fixed with 4% paraformaldehyde and proceeded for Tuj1
staining. Cells were permeabilized with 0.2% Triton X/PBS (TPBS)
for 5 minutes at room temperature followed by incubation with 1.5%
normal goat serum in TPBS (S-TPBS) for 30 minutes to block
non-specific binding. An aliquot of anti-Neuronal Class III
b-Tubulin monoclonal antibody (Tuj1, 1:1000; Covance # MMS-435P)
was added to the cells. After 1 hour incubation at room
temperature, unbound antibodies were washed with PBS three times
and an aliquot of Alexa Fluor 488 mouse anti-goat IgG antibody
(1:500, Molecular Probe # A-11001) was added to visualize the
signals. Hoechst 33342 (Molecular Probe # H-3570, 2 .mu.g/ml) was
included to label the nucleus. After washing, plates were sealed
and analyzed for neurite growth using a Cellomics array scan.
Typically around 300 cells from 9 fields were analyzed per well and
each treatment was conducted in quadruplets.
[0168] MAG and myelin inhibit neurite outgrowth of primary
cerebellar granule neurons in cultures. Increasing concentrations
of recombinant rat MAG(1-5) or purified rat myelin were coated on
96 well tissue culture plates and the neurite extension of primary
neurons were measured at 20 hours as described above. As shown in
FIG. 26, (A) MAG and (B) myelin resulted in dose-dependent
inhibition of neurite outgrowth.
[0169] CGN neurons were then plated on either MAG or myelin-coated
plates with control or with a range of concentrations of the TrkB
antibody 29D7. Neurite extension was measured 20 hours
post-treatment. As shown in FIG. 27, 29D7 led to reversal of (A)
MAG- and (B) myelin-mediated neurite inhibition.
Other Embodiments
[0170] Other embodiments of the invention will be apparent to those
skilled in the art from a consideration of the specification or
practice of the invention disclosed herein. It is intended that the
specification and examples be considered as exemplary only, with
the true scope of the invention being indicated by the following
claims.
Sequence CWU 1
1
131838PRTArtificialHuman - (homo sapiens) TrkB 1Met Ser Ser Trp Ile
Arg Trp His Gly Pro Ala Met Ala Arg Leu Trp1 5 10 15Gly Phe Cys Trp
Leu Val Val Gly Phe Trp Arg Ala Ala Phe Ala Cys 20 25 30Pro Thr Ser
Cys Lys Cys Ser Ala Ser Arg Ile Trp Cys Ser Asp Pro 35 40 45Ser Pro
Gly Ile Val Ala Phe Pro Arg Leu Glu Pro Asn Ser Val Asp 50 55 60Pro
Glu Asn Ile Thr Glu Ile Phe Ile Ala Asn Gln Lys Arg Leu Glu65 70 75
80Ile Ile Asn Glu Asp Asp Val Glu Ala Tyr Val Gly Leu Arg Asn Leu
85 90 95Thr Ile Val Asp Ser Gly Leu Lys Phe Val Ala His Lys Ala Phe
Leu 100 105 110Lys Asn Ser Asn Leu Gln His Ile Asn Phe Thr Arg Asn
Lys Leu Thr 115 120 125Ser Leu Ser Arg Lys His Phe Arg His Leu Asp
Leu Ser Glu Leu Ile 130 135 140Leu Val Gly Asn Pro Phe Thr Cys Ser
Cys Asp Ile Met Trp Ile Lys145 150 155 160Thr Leu Gln Glu Ala Lys
Ser Ser Pro Asp Thr Gln Asp Leu Tyr Cys 165 170 175Leu Asn Glu Ser
Ser Lys Asn Ile Pro Leu Ala Asn Leu Gln Ile Pro 180 185 190Asn Cys
Gly Leu Pro Ser Ala Asn Leu Ala Ala Pro Asn Leu Thr Val 195 200
205Glu Glu Gly Lys Ser Ile Thr Leu Ser Cys Ser Val Ala Gly Asp Pro
210 215 220Val Pro Asn Met Tyr Trp Asp Val Gly Asn Leu Val Ser Lys
His Met225 230 235 240Asn Glu Thr Ser His Thr Gln Gly Ser Leu Arg
Ile Thr Asn Ile Ser 245 250 255Ser Asp Asp Ser Gly Lys Gln Ile Ser
Cys Val Ala Glu Asn Leu Val 260 265 270Gly Glu Asp Gln Asp Ser Val
Asn Leu Thr Val His Phe Ala Pro Thr 275 280 285Ile Thr Phe Leu Glu
Ser Pro Thr Ser Asp His His Trp Cys Ile Pro 290 295 300Phe Thr Val
Lys Gly Asn Pro Lys Pro Ala Leu Gln Trp Phe Tyr Asn305 310 315
320Gly Ala Ile Leu Asn Glu Ser Lys Tyr Ile Cys Thr Lys Ile His Val
325 330 335Thr Asn His Thr Glu Tyr His Gly Cys Leu Gln Leu Asp Asn
Pro Thr 340 345 350His Met Asn Asn Gly Asp Tyr Thr Leu Ile Ala Lys
Asn Glu Tyr Gly 355 360 365Lys Asp Glu Lys Gln Ile Ser Ala His Phe
Met Gly Trp Pro Gly Ile 370 375 380Asp Asp Gly Ala Asn Pro Asn Tyr
Pro Asp Val Ile Tyr Glu Asp Tyr385 390 395 400Gly Thr Ala Ala Asn
Asp Ile Gly Asp Thr Thr Asn Arg Ser Asn Glu 405 410 415Ile Pro Ser
Thr Asp Val Thr Asp Lys Thr Gly Arg Glu His Leu Ser 420 425 430Val
Tyr Ala Val Val Val Ile Ala Ser Val Val Gly Phe Cys Leu Leu 435 440
445Val Met Leu Phe Leu Leu Lys Leu Ala Arg His Ser Lys Phe Gly Met
450 455 460Lys Asp Phe Ser Trp Phe Gly Phe Gly Lys Val Lys Ser Arg
Gln Gly465 470 475 480Val Gly Pro Ala Ser Val Ile Ser Asn Asp Asp
Asp Ser Ala Ser Pro 485 490 495Leu His His Ile Ser Asn Gly Ser Asn
Thr Pro Ser Ser Ser Glu Gly 500 505 510Gly Pro Asp Ala Val Ile Ile
Gly Met Thr Lys Ile Pro Val Ile Glu 515 520 525Asn Pro Gln Tyr Phe
Gly Ile Thr Asn Ser Gln Leu Lys Pro Asp Thr 530 535 540Phe Val Gln
His Ile Lys Arg His Asn Ile Val Leu Lys Arg Glu Leu545 550 555
560Gly Glu Gly Ala Phe Gly Lys Val Phe Leu Ala Glu Cys Tyr Asn Leu
565 570 575Cys Pro Glu Gln Asp Lys Ile Leu Val Ala Val Lys Thr Leu
Lys Asp 580 585 590Ala Ser Asp Asn Ala Arg Lys Asp Phe His Arg Glu
Ala Glu Leu Leu 595 600 605Thr Asn Leu Gln His Glu His Ile Val Lys
Phe Tyr Gly Val Cys Val 610 615 620Glu Gly Asp Pro Leu Ile Met Val
Phe Glu Tyr Met Lys His Gly Asp625 630 635 640Leu Asn Lys Phe Leu
Arg Ala His Gly Pro Asp Ala Val Leu Met Ala 645 650 655Glu Gly Asn
Pro Pro Thr Glu Leu Thr Gln Ser Gln Met Leu His Ile 660 665 670Ala
Gln Gln Ile Ala Ala Gly Met Val Tyr Leu Ala Ser Gln His Phe 675 680
685Val His Arg Asp Leu Ala Thr Arg Asn Cys Leu Val Gly Glu Asn Leu
690 695 700Leu Val Lys Ile Gly Asp Phe Gly Met Ser Arg Asp Val Tyr
Ser Thr705 710 715 720Asp Tyr Tyr Arg Val Gly Gly His Thr Met Leu
Pro Ile Arg Trp Met 725 730 735Pro Pro Glu Ser Ile Met Tyr Arg Lys
Phe Thr Thr Glu Ser Asp Val 740 745 750Trp Ser Leu Gly Val Val Leu
Trp Glu Ile Phe Thr Tyr Gly Lys Gln 755 760 765Pro Trp Tyr Gln Leu
Ser Asn Asn Glu Val Ile Glu Cys Ile Thr Gln 770 775 780Gly Arg Val
Leu Gln Arg Pro Arg Thr Cys Pro Gln Glu Val Tyr Glu785 790 795
800Leu Met Leu Gly Cys Trp Gln Arg Glu Pro His Met Arg Lys Asn Ile
805 810 815Lys Gly Ile His Thr Leu Leu Gln Asn Leu Ala Lys Ala Ser
Pro Val 820 825 830Tyr Leu Asp Ile Leu Gly
8352821PRTArtificialmurine (Mus musculus) TrkB 2Met Ser Pro Trp Leu
Lys Trp His Gly Pro Ala Met Ala Arg Leu Trp1 5 10 15Gly Leu Cys Leu
Leu Val Leu Gly Phe Trp Arg Ala Ser Leu Ala Cys 20 25 30Pro Thr Ser
Cys Lys Cys Ser Ser Ala Arg Ile Trp Cys Thr Glu Pro 35 40 45Ser Pro
Gly Ile Val Ala Phe Pro Arg Leu Glu Pro Asn Ser Val Asp 50 55 60Pro
Glu Asn Ile Thr Glu Ile Leu Ile Ala Asn Gln Lys Arg Leu Glu65 70 75
80Ile Ile Asn Glu Asp Asp Val Glu Ala Tyr Val Gly Leu Arg Asn Leu
85 90 95Thr Ile Val Asp Ser Gly Leu Lys Phe Val Ala Tyr Lys Ala Phe
Leu 100 105 110Lys Asn Ser Asn Leu Arg His Ile Asn Phe Thr Arg Asn
Lys Leu Thr 115 120 125Ser Leu Ser Arg Arg His Phe Arg His Leu Asp
Leu Ser Asp Leu Ile 130 135 140Leu Thr Gly Asn Pro Phe Thr Cys Ser
Cys Asp Ile Met Trp Leu Lys145 150 155 160Thr Leu Gln Glu Thr Lys
Ser Ser Pro Asp Thr Gln Asp Leu Tyr Cys 165 170 175Leu Asn Glu Ser
Ser Lys Asn Met Pro Leu Ala Asn Leu Gln Ile Pro 180 185 190Asn Cys
Gly Leu Pro Ser Ala Arg Leu Ala Ala Pro Asn Leu Thr Val 195 200
205Glu Glu Gly Lys Ser Val Thr Leu Ser Cys Ser Val Gly Gly Asp Pro
210 215 220Leu Pro Thr Leu Tyr Trp Asp Val Gly Asn Leu Val Ser Lys
His Met225 230 235 240Asn Glu Thr Ser His Thr Gln Gly Ser Leu Arg
Ile Thr Asn Ile Ser 245 250 255Ser Asp Asp Ser Gly Lys Gln Ile Ser
Cys Val Ala Glu Asn Leu Val 260 265 270Gly Glu Asp Gln Asp Ser Val
Asn Leu Thr Val His Phe Ala Pro Thr 275 280 285Ile Thr Phe Leu Glu
Ser Pro Thr Ser Asp His His Trp Cys Ile Pro 290 295 300Phe Thr Val
Arg Gly Asn Pro Lys Pro Ala Leu Gln Trp Phe Tyr Asn305 310 315
320Gly Ala Ile Leu Asn Glu Ser Lys Tyr Ile Cys Thr Lys Ile His Val
325 330 335Thr Asn His Thr Glu Tyr His Gly Cys Leu Gln Leu Asp Asn
Pro Thr 340 345 350His Met Asn Asn Gly Asp Tyr Thr Leu Met Ala Lys
Asn Glu Tyr Gly 355 360 365Lys Asp Glu Arg Gln Ile Ser Ala His Phe
Met Gly Arg Pro Gly Val 370 375 380Asp Tyr Glu Thr Asn Pro Asn Tyr
Pro Glu Val Leu Tyr Glu Asp Trp385 390 395 400Thr Thr Pro Thr Asp
Ile Gly Asp Thr Thr Asn Lys Ser Asn Glu Ile 405 410 415Pro Ser Thr
Asp Val Ala Asp Gln Ser Asn Arg Glu His Leu Ser Val 420 425 430Tyr
Ala Val Val Val Ile Ala Ser Val Val Gly Phe Cys Leu Leu Val 435 440
445Met Leu Leu Leu Leu Lys Leu Ala Arg His Ser Lys Phe Gly Met Lys
450 455 460Gly Pro Ala Ser Val Ile Ser Asn Asp Asp Asp Ser Ala Ser
Pro Leu465 470 475 480His His Ile Ser Asn Gly Ser Asn Thr Pro Ser
Ser Ser Glu Gly Gly 485 490 495Pro Asp Ala Val Ile Ile Gly Met Thr
Lys Ile Pro Val Ile Glu Asn 500 505 510Pro Gln Tyr Phe Gly Ile Thr
Asn Ser Gln Leu Lys Pro Asp Thr Phe 515 520 525Val Gln His Ile Lys
Arg His Asn Ile Val Leu Lys Arg Glu Leu Gly 530 535 540Glu Gly Ala
Phe Gly Lys Val Phe Leu Ala Glu Cys Tyr Asn Leu Cys545 550 555
560Pro Glu Gln Asp Lys Ile Leu Val Ala Val Lys Thr Leu Lys Asp Ala
565 570 575Ser Asp Asn Ala Arg Lys Asp Phe His Arg Glu Ala Glu Leu
Leu Thr 580 585 590Asn Leu Gln His Glu His Ile Val Lys Phe Tyr Gly
Val Cys Val Glu 595 600 605Gly Asp Pro Leu Ile Met Val Phe Glu Tyr
Met Lys His Gly Asp Leu 610 615 620Asn Lys Phe Leu Arg Ala His Gly
Pro Asp Ala Val Leu Met Ala Glu625 630 635 640Gly Asn Pro Pro Thr
Glu Leu Thr Gln Ser Gln Met Leu His Ile Ala 645 650 655Gln Gln Ile
Ala Ala Gly Met Val Tyr Leu Ala Ser Gln His Phe Val 660 665 670His
Arg Asp Leu Ala Thr Arg Asn Cys Leu Val Gly Glu Asn Leu Leu 675 680
685Val Lys Ile Gly Asp Phe Gly Met Ser Arg Asp Val Tyr Ser Thr Asp
690 695 700Tyr Tyr Arg Val Gly Gly His Thr Met Leu Pro Ile Arg Trp
Met Pro705 710 715 720Pro Glu Ser Ile Met Tyr Arg Lys Phe Thr Thr
Glu Ser Asp Val Trp 725 730 735Ser Leu Gly Val Val Leu Trp Glu Ile
Phe Thr Tyr Gly Lys Gln Pro 740 745 750Trp Tyr Gln Leu Ser Asn Asn
Glu Val Ile Glu Cys Ile Thr Gln Gly 755 760 765Arg Val Leu Gln Arg
Pro Arg Thr Cys Pro Gln Glu Val Tyr Glu Leu 770 775 780Met Leu Gly
Cys Trp Gln Arg Glu Pro His Thr Arg Lys Asn Ile Lys785 790 795
800Ser Ile His Thr Leu Leu Gln Asn Leu Ala Lys Ala Ser Pro Val Tyr
805 810 815Leu Asp Ile Leu Gly 8203821PRTArtificialrat - (Rattus
norvegicus) TrkB 3Met Ser Pro Trp Pro Arg Trp His Gly Pro Ala Met
Ala Arg Leu Trp1 5 10 15Gly Leu Cys Leu Leu Val Leu Gly Phe Trp Arg
Ala Ser Leu Ala Cys 20 25 30Pro Met Ser Cys Lys Cys Ser Thr Thr Arg
Ile Trp Cys Thr Glu Pro 35 40 45Ser Pro Gly Ile Val Ala Phe Pro Arg
Leu Glu Pro Asn Ser Ile Asp 50 55 60Pro Glu Asn Ile Thr Glu Ile Leu
Ile Ala Asn Gln Lys Arg Leu Glu65 70 75 80Ile Ile Asn Glu Asp Asp
Val Glu Ala Tyr Val Gly Leu Lys Asn Leu 85 90 95Thr Ile Val Asp Ser
Gly Leu Lys Phe Val Ala Tyr Lys Ala Phe Leu 100 105 110Lys Asn Gly
Asn Leu Arg His Ile Asn Phe Thr Arg Asn Lys Leu Thr 115 120 125Ser
Leu Ser Arg Arg His Phe Arg His Leu Asp Leu Ser Asp Leu Ile 130 135
140Leu Thr Gly Asn Pro Phe Thr Cys Ser Cys Asp Ile Met Trp Leu
Lys145 150 155 160Thr Leu Gln Glu Thr Lys Ser Ser Pro Asp Thr Gln
Asp Leu Tyr Cys 165 170 175Leu Asn Glu Ser Ser Lys Asn Thr Pro Leu
Ala Asn Leu Gln Ile Pro 180 185 190Asn Cys Gly Leu Pro Ser Ala Arg
Leu Ala Ala Pro Asn Leu Thr Val 195 200 205Glu Glu Gly Lys Ser Val
Thr Ile Ser Cys Ser Val Gly Gly Asp Pro 210 215 220Leu Pro Thr Leu
Tyr Trp Asp Val Gly Asn Leu Val Ser Lys His Met225 230 235 240Asn
Glu Thr Ser His Thr Gln Gly Ser Leu Arg Ile Thr Asn Ile Ser 245 250
255Ser Asp Asp Ser Gly Lys Gln Ile Ser Cys Val Ala Glu Asn Leu Val
260 265 270Gly Glu Asp Gln Asp Ser Val Asn Leu Thr Val His Phe Ala
Pro Thr 275 280 285Ile Thr Phe Leu Glu Ser Pro Thr Ser Asp His His
Trp Cys Ile Pro 290 295 300Phe Thr Val Arg Gly Asn Pro Lys Pro Ala
Leu Gln Trp Phe Tyr Asn305 310 315 320Gly Ala Ile Leu Asn Glu Ser
Lys Tyr Ile Cys Thr Lys Ile His Val 325 330 335Thr Asn His Thr Glu
Tyr His Gly Cys Leu Gln Leu Asp Asn Pro Thr 340 345 350His Met Asn
Asn Gly Asp Tyr Thr Leu Met Ala Lys Asn Glu Tyr Gly 355 360 365Lys
Asp Glu Arg Gln Ile Ser Ala His Phe Met Gly Arg Pro Gly Val 370 375
380Asp Tyr Glu Thr Asn Pro Asn Tyr Pro Glu Val Leu Tyr Glu Asp
Trp385 390 395 400Thr Thr Pro Thr Asp Ile Gly Asp Thr Thr Asn Lys
Ser Asn Glu Ile 405 410 415Pro Ser Thr Asp Val Ala Asp Gln Thr Asn
Arg Glu His Leu Ser Val 420 425 430Tyr Ala Val Val Val Ile Ala Ser
Val Val Gly Phe Cys Leu Leu Val 435 440 445Met Leu Leu Leu Leu Lys
Leu Ala Arg His Ser Lys Phe Gly Met Lys 450 455 460Gly Pro Ala Ser
Val Ile Ser Asn Asp Asp Asp Ser Ala Ser Pro Leu465 470 475 480His
His Ile Ser Asn Gly Ser Asn Thr Pro Ser Ser Ser Glu Gly Gly 485 490
495Pro Asp Ala Val Ile Ile Gly Met Thr Lys Ile Pro Val Ile Glu Asn
500 505 510Pro Gln Tyr Phe Gly Ile Thr Asn Ser Gln Leu Lys Pro Asp
Thr Phe 515 520 525Val Gln His Ile Lys Arg His Asn Ile Val Leu Lys
Arg Glu Leu Gly 530 535 540Glu Gly Ala Phe Gly Lys Val Phe Leu Ala
Glu Cys Tyr Asn Leu Cys545 550 555 560Pro Glu Gln Asp Lys Ile Leu
Val Ala Val Lys Thr Leu Lys Asp Ala 565 570 575Ser Asp Asn Ala Arg
Lys Asp Phe His Arg Glu Ala Glu Leu Leu Thr 580 585 590Asn Leu Gln
His Glu His Ile Val Lys Phe Tyr Gly Val Cys Val Glu 595 600 605Gly
Asp Pro Leu Ile Met Val Phe Glu Tyr Met Lys His Gly Asp Leu 610 615
620Asn Lys Phe Leu Arg Ala His Gly Pro Asp Ala Val Leu Met Ala
Glu625 630 635 640Gly Asn Pro Pro Thr Glu Leu Thr Gln Ser Gln Met
Leu His Ile Ala 645 650 655Gln Gln Ile Ala Ala Gly Met Val Tyr Leu
Ala Ser Gln His Phe Val 660 665 670His Arg Asp Leu Ala Thr Arg Asn
Cys Leu Val Gly Glu Asn Leu Leu 675 680 685Val Lys Ile Gly Asp Phe
Gly Met Ser Arg Asp Val Tyr Ser Thr Asp 690 695 700Tyr Tyr Arg Val
Gly Gly His Thr Met Leu Pro Ile Arg Trp Met Pro705 710 715 720Pro
Glu Ser Ile Met Tyr Arg Lys Phe Thr Thr Glu Ser Asp Val Trp 725 730
735Ser Leu Gly Val Val Leu Trp Glu Ile Phe Thr Tyr Gly Lys Gln Pro
740 745 750Trp Tyr Gln Leu Ser Asn Asn Glu Val Ile Glu Cys Ile Thr
Gln Gly 755 760 765Arg Val Leu Gln Arg Pro Arg Thr Cys Pro Gln Glu
Val Tyr Glu Leu 770 775 780Met Leu Gly Cys Trp Gln Arg Glu Pro His
Thr Arg Lys Asn Ile Lys785 790
795 800Asn Ile His Thr Leu Leu Gln Asn Leu Ala Lys Ala Ser Pro Val
Tyr 805 810 815Leu Asp Ile Leu Gly 8204818PRTArtificialchicken -
(Gallus gallus) TrkB 4Met Val Ser Trp Arg Arg Arg Pro Gly Pro Gly
Leu Ala Arg Leu Trp1 5 10 15Gly Leu Cys Cys Leu Val Leu Gly Cys Trp
Arg Gly Ala Leu Gly Cys 20 25 30Pro Ala Ser Cys Arg Cys Ser Ser Trp
Arg Ile Trp Cys Ser Glu Pro 35 40 45Val Pro Gly Ile Thr Ser Phe Pro
Val Pro Gln Arg Ser Thr Glu Asp 50 55 60Asp Asn Val Thr Glu Ile Tyr
Ile Ala Asn Gln Arg Lys Leu Glu Ser65 70 75 80Ile Asn Asp Asn Glu
Val Gly Phe Tyr Val Gly Leu Lys Asn Leu Thr 85 90 95Val Val Asp Ser
Gly Leu Arg Phe Val Ser Arg Gln Ala Phe Val Lys 100 105 110Asn Ile
Asn Leu Gln Tyr Ile Asn Leu Ser Arg Asn Lys Leu Ser Ser 115 120
125Leu Ser Lys Lys Pro Phe Arg His Leu Gly Leu Ser Asp Leu Ile Leu
130 135 140Val Asp Asn Pro Phe Lys Cys Ser Cys Glu Ile Met Trp Ile
Lys Lys145 150 155 160Phe Gln Glu Thr Lys Phe Tyr Thr Glu Ala Gln
Asp Ile Tyr Cys Val 165 170 175Asp Asp Asn Asn Lys Arg Ile Ala Leu
Met Asp Met Lys Val Pro Asn 180 185 190Cys Asp Leu Pro Ser Ala Asn
Leu Ser Asn Tyr Asn Ile Thr Val Val 195 200 205Glu Gly Lys Ser Ile
Thr Leu Tyr Cys Asp Thr Thr Gly Gly Pro Pro 210 215 220Pro Asn Val
Ser Trp Val Leu Thr Asn Leu Val Ser Asn His Glu Ser225 230 235
240Asp Thr Ser Lys Asn Pro Ala Ser Leu Thr Ile Lys Asn Val Ser Ser
245 250 255Met Asp Ser Gly Leu Trp Ile Ser Cys Val Ala Glu Asn Ile
Val Gly 260 265 270Glu Val Gln Thr Ser Ala Glu Leu Thr Val Phe Phe
Ala Pro Asn Ile 275 280 285Thr Phe Ile Glu Ser Pro Thr Pro Asp His
His Trp Cys Ile Pro Phe 290 295 300Thr Val Lys Gly Asn Pro Lys Pro
Thr Leu Gln Trp Phe Tyr Glu Gly305 310 315 320Ala Ile Leu Asn Glu
Ser Glu Tyr Ile Cys Thr Lys Ile His Val Ile 325 330 335Asn Gln Ser
Glu Tyr His Gly Cys Leu Gln Leu Asp Asn Pro Thr His 340 345 350Leu
Asn Asn Gly Ala Tyr Thr Leu Leu Ala Lys Asn Glu Tyr Gly Glu 355 360
365Asp Glu Lys Arg Val Asp Ala His Phe Met Ser Val Pro Gly Asp Gly
370 375 380Ser Gly Pro Ile Val Asp Pro Asp Val Tyr Glu Tyr Glu Thr
Thr Pro385 390 395 400Asn Asp Leu Gly Asp Thr Thr Asn Asn Ser Asn
Gln Ile Thr Ser Pro 405 410 415Asp Val Ser Asn Lys Glu Asn Glu Asp
Ser Ile Thr Val Tyr Val Val 420 425 430Val Gly Ile Ala Ala Leu Val
Cys Thr Gly Leu Val Ile Met Leu Ile 435 440 445Ile Leu Lys Phe Gly
Arg His Ser Lys Phe Gly Met Lys Gly Pro Ser 450 455 460Ser Val Ile
Ser Asn Asp Asp Asp Ser Ala Ser Pro Leu His His Ile465 470 475
480Ser Asn Gly Ser Asn Thr Pro Ser Ser Ser Glu Gly Gly Pro Asp Ala
485 490 495Val Ile Ile Gly Met Thr Lys Ile Pro Val Ile Glu Asn Pro
Gln Tyr 500 505 510Phe Gly Ile Thr Asn Ser Gln Leu Lys Pro Asp Thr
Phe Val Gln His 515 520 525Ile Lys Arg His Asn Ile Val Leu Lys Arg
Glu Leu Gly Glu Gly Ala 530 535 540Phe Gly Lys Val Phe Leu Ala Glu
Cys Tyr Asn Leu Cys Pro Glu Gln545 550 555 560Asp Lys Ile Leu Val
Ala Val Lys Thr Leu Lys Asp Ala Ser Asp Asn 565 570 575Ala Arg Lys
Asp Phe His Arg Glu Ala Glu Leu Leu Thr Asn Leu Gln 580 585 590His
Glu His Ile Val Lys Phe Tyr Gly Val Cys Val Glu Gly Asp Pro 595 600
605Leu Ile Met Val Phe Glu Tyr Met Lys His Gly Asp Leu Asn Lys Phe
610 615 620Leu Arg Ala His Gly Pro Asp Ala Val Leu Met Ala Glu Gly
Asn Arg625 630 635 640Pro Ala Glu Leu Thr Gln Ser Gln Met Leu His
Ile Ala Gln Gln Ile 645 650 655Ala Ala Gly Met Val Tyr Leu Ala Ser
Gln His Phe Val His Arg Asp 660 665 670Leu Ala Thr Arg Asn Cys Leu
Val Gly Glu Asn Leu Leu Val Lys Ile 675 680 685Gly Asp Phe Gly Met
Ser Arg Asp Val Tyr Ser Thr Asp Tyr Tyr Arg 690 695 700Val Gly Gly
His Thr Met Leu Pro Ile Arg Trp Met Pro Pro Glu Ser705 710 715
720Ile Met Tyr Arg Lys Phe Thr Thr Glu Ser Asp Val Trp Ser Leu Gly
725 730 735Val Val Leu Trp Glu Ile Phe Thr Tyr Gly Lys Gln Pro Trp
Tyr Gln 740 745 750Leu Ser Asn Asn Glu Val Ile Glu Cys Ile Thr Gln
Gly Arg Val Leu 755 760 765Gln Arg Pro Arg Thr Cys Pro Lys Glu Val
Tyr Asp Leu Met Leu Gly 770 775 780Cys Trp Gln Arg Glu Pro His Met
Arg Leu Asn Ile Lys Glu Ile His785 790 795 800Ser Leu Leu Gln Asn
Leu Ala Lys Ala Ser Pro Val Tyr Leu Asp Ile 805 810 815Leu
Gly5796PRTArtificialhuman - (Homo sapiens) TrkA 5Met Leu Arg Gly
Gly Arg Arg Gly Gln Leu Gly Trp His Ser Trp Ala1 5 10 15Ala Gly Pro
Gly Ser Leu Leu Ala Trp Leu Ile Leu Ala Ser Ala Gly 20 25 30Ala Ala
Pro Cys Pro Asp Ala Cys Cys Pro His Gly Ser Ser Gly Leu 35 40 45Arg
Cys Thr Arg Asp Gly Ala Leu Asp Ser Leu His His Leu Pro Gly 50 55
60Ala Glu Asn Leu Thr Glu Leu Tyr Ile Glu Asn Gln Gln His Leu Gln65
70 75 80His Leu Glu Leu Arg Asp Leu Arg Gly Leu Gly Glu Leu Arg Asn
Leu 85 90 95Thr Ile Val Lys Ser Gly Leu Arg Phe Val Ala Pro Asp Ala
Phe His 100 105 110Phe Thr Pro Arg Leu Ser Arg Leu Asn Leu Ser Phe
Asn Ala Leu Glu 115 120 125Ser Leu Ser Trp Lys Thr Val Gln Gly Leu
Ser Leu Gln Glu Leu Val 130 135 140Leu Ser Gly Asn Pro Leu His Cys
Ser Cys Ala Leu Arg Trp Leu Gln145 150 155 160Arg Trp Glu Glu Glu
Gly Leu Gly Gly Val Pro Glu Gln Lys Leu Gln 165 170 175Cys His Gly
Gln Gly Pro Leu Ala His Met Pro Asn Ala Ser Cys Gly 180 185 190Val
Pro Thr Leu Lys Val Gln Val Pro Asn Ala Ser Val Asp Val Gly 195 200
205Asp Asp Val Leu Leu Arg Cys Gln Val Glu Gly Arg Gly Leu Glu Gln
210 215 220Ala Gly Trp Ile Leu Thr Glu Leu Glu Gln Ser Ala Thr Val
Met Lys225 230 235 240Ser Gly Gly Leu Pro Ser Leu Gly Leu Thr Leu
Ala Asn Val Thr Ser 245 250 255Asp Leu Asn Arg Lys Asn Val Thr Cys
Trp Ala Glu Asn Asp Val Gly 260 265 270Arg Ala Glu Val Ser Val Gln
Val Asn Val Ser Phe Pro Ala Ser Val 275 280 285Gln Leu His Thr Ala
Val Glu Met His His Trp Cys Ile Pro Phe Ser 290 295 300Val Asp Gly
Gln Pro Ala Pro Ser Leu Arg Trp Leu Phe Asn Gly Ser305 310 315
320Val Leu Asn Glu Thr Ser Phe Ile Phe Thr Glu Phe Leu Glu Pro Ala
325 330 335Ala Asn Glu Thr Val Arg His Gly Cys Leu Arg Leu Asn Gln
Pro Thr 340 345 350His Val Asn Asn Gly Asn Tyr Thr Leu Leu Ala Ala
Asn Pro Phe Gly 355 360 365Gln Ala Ser Ala Ser Ile Met Ala Ala Phe
Met Asp Asn Pro Phe Glu 370 375 380Phe Asn Pro Glu Asp Pro Ile Pro
Val Ser Phe Ser Pro Val Asp Thr385 390 395 400Asn Ser Thr Ser Gly
Asp Pro Val Glu Lys Lys Asp Glu Thr Pro Phe 405 410 415Gly Val Ser
Val Ala Val Gly Leu Ala Val Phe Ala Cys Leu Phe Leu 420 425 430Ser
Thr Leu Leu Leu Val Leu Asn Lys Cys Gly Arg Arg Asn Lys Phe 435 440
445Gly Ile Asn Arg Pro Ala Val Leu Ala Pro Glu Asp Gly Leu Ala Met
450 455 460Ser Leu His Phe Met Thr Leu Gly Gly Ser Ser Leu Ser Pro
Thr Glu465 470 475 480Gly Lys Gly Ser Gly Leu Gln Gly His Ile Ile
Glu Asn Pro Gln Tyr 485 490 495Phe Ser Asp Ala Cys Val His His Ile
Lys Arg Arg Asp Ile Val Leu 500 505 510Lys Trp Glu Leu Gly Glu Gly
Ala Phe Gly Lys Val Phe Leu Ala Glu 515 520 525Cys His Asn Leu Leu
Pro Glu Gln Asp Lys Met Leu Val Ala Val Lys 530 535 540Ala Leu Lys
Glu Ala Ser Glu Ser Ala Arg Gln Asp Phe Gln Arg Glu545 550 555
560Ala Glu Leu Leu Thr Met Leu Gln His Gln His Ile Val Arg Phe Phe
565 570 575Gly Val Cys Thr Glu Gly Arg Pro Leu Leu Met Val Phe Glu
Tyr Met 580 585 590Arg His Gly Asp Leu Asn Arg Phe Leu Arg Ser His
Gly Pro Asp Ala 595 600 605Lys Leu Leu Ala Gly Gly Glu Asp Val Ala
Pro Gly Pro Leu Gly Leu 610 615 620Gly Gln Leu Leu Ala Val Ala Ser
Gln Val Ala Ala Gly Met Val Tyr625 630 635 640Leu Ala Gly Leu His
Phe Val His Arg Asp Leu Ala Thr Arg Asn Cys 645 650 655Leu Val Gly
Gln Gly Leu Val Val Lys Ile Gly Asp Phe Gly Met Ser 660 665 670Arg
Asp Ile Tyr Ser Thr Asp Tyr Tyr Arg Val Gly Gly Arg Thr Met 675 680
685Leu Pro Ile Arg Trp Met Pro Pro Glu Ser Ile Leu Tyr Arg Lys Phe
690 695 700Thr Thr Glu Ser Asp Val Trp Ser Phe Gly Val Val Leu Trp
Glu Ile705 710 715 720Phe Thr Tyr Gly Lys Gln Pro Trp Tyr Gln Leu
Ser Asn Thr Glu Ala 725 730 735Ile Asp Cys Ile Thr Gln Gly Arg Glu
Leu Glu Arg Pro Arg Ala Cys 740 745 750Pro Pro Glu Val Tyr Ala Ile
Met Arg Gly Cys Trp Gln Arg Glu Pro 755 760 765Gln Gln Arg His Ser
Ile Lys Asp Val His Ala Arg Leu Gln Ala Leu 770 775 780Ala Gln Ala
Pro Pro Val Tyr Leu Asp Val Leu Gly785 790
7956839PRTArtificialhuman (Homo sapiens) TrkC 6Met Asp Val Ser Leu
Cys Pro Ala Lys Cys Ser Phe Trp Arg Ile Phe1 5 10 15Leu Leu Gly Ser
Val Trp Leu Asp Tyr Val Gly Ser Val Leu Ala Cys 20 25 30Pro Ala Asn
Cys Val Cys Ser Lys Thr Glu Ile Asn Cys Arg Arg Pro 35 40 45Asp Asp
Gly Asn Leu Phe Pro Leu Leu Glu Gly Gln Asp Ser Gly Asn 50 55 60Ser
Asn Gly Asn Ala Asn Ile Asn Ile Thr Asp Ile Ser Arg Asn Ile65 70 75
80Thr Ser Ile His Ile Glu Asn Trp Arg Ser Leu His Thr Leu Asn Ala
85 90 95Val Asp Met Glu Leu Tyr Thr Gly Leu Gln Lys Leu Thr Ile Lys
Asn 100 105 110Ser Gly Leu Arg Ser Ile Gln Pro Arg Ala Phe Ala Lys
Asn Pro His 115 120 125Leu Arg Tyr Ile Asn Leu Ser Ser Asn Arg Leu
Thr Thr Leu Ser Trp 130 135 140Gln Leu Phe Gln Thr Leu Ser Leu Arg
Glu Leu Gln Leu Glu Gln Asn145 150 155 160Phe Phe Asn Cys Ser Cys
Asp Ile Arg Trp Met Gln Leu Trp Gln Glu 165 170 175Gln Gly Glu Ala
Lys Leu Asn Ser Gln Asn Leu Tyr Cys Ile Asn Ala 180 185 190Asp Gly
Ser Gln Leu Pro Leu Phe Arg Met Asn Ile Ser Gln Cys Asp 195 200
205Leu Pro Glu Ile Ser Val Ser His Val Asn Leu Thr Val Arg Glu Gly
210 215 220Asp Asn Ala Val Ile Thr Cys Asn Gly Ser Gly Ser Pro Leu
Pro Asp225 230 235 240Val Asp Trp Ile Val Thr Gly Leu Gln Ser Ile
Asn Thr His Gln Thr 245 250 255Asn Leu Asn Trp Thr Asn Val His Ala
Ile Asn Leu Thr Leu Val Asn 260 265 270Val Thr Ser Glu Asp Asn Gly
Phe Thr Leu Thr Cys Ile Ala Glu Asn 275 280 285Val Val Gly Met Ser
Asn Ala Ser Val Ala Leu Thr Val Tyr Tyr Pro 290 295 300Pro Arg Val
Val Ser Leu Glu Glu Pro Glu Leu Arg Leu Glu His Cys305 310 315
320Ile Glu Phe Val Val Arg Gly Asn Pro Pro Pro Thr Leu His Trp Leu
325 330 335His Asn Gly Gln Pro Leu Arg Glu Ser Lys Ile Ile His Val
Glu Tyr 340 345 350Tyr Gln Glu Gly Glu Ile Ser Glu Gly Cys Leu Leu
Phe Asn Lys Pro 355 360 365Thr His Tyr Asn Asn Gly Asn Tyr Thr Leu
Ile Ala Lys Asn Pro Leu 370 375 380Gly Thr Ala Asn Gln Thr Ile Asn
Gly His Phe Leu Lys Glu Pro Phe385 390 395 400Pro Glu Ser Thr Asp
Asn Phe Ile Leu Phe Asp Glu Val Ser Pro Thr 405 410 415Pro Pro Ile
Thr Val Thr His Lys Pro Glu Glu Asp Thr Phe Gly Val 420 425 430Ser
Ile Ala Val Gly Leu Ala Ala Phe Ala Cys Val Leu Leu Val Val 435 440
445Leu Phe Val Met Ile Asn Lys Tyr Gly Arg Arg Ser Lys Phe Gly Met
450 455 460Lys Gly Pro Val Ala Val Ile Ser Gly Glu Glu Asp Ser Ala
Ser Pro465 470 475 480Leu His His Ile Asn His Gly Ile Thr Thr Pro
Ser Ser Leu Asp Ala 485 490 495Gly Pro Asp Thr Val Val Ile Gly Met
Thr Arg Ile Pro Val Ile Glu 500 505 510Asn Pro Gln Tyr Phe Arg Gln
Gly His Asn Cys His Lys Pro Asp Thr 515 520 525Tyr Val Gln His Ile
Lys Arg Arg Asp Ile Val Leu Lys Arg Glu Leu 530 535 540Gly Glu Gly
Ala Phe Gly Lys Val Phe Leu Ala Glu Cys Tyr Asn Leu545 550 555
560Ser Pro Thr Lys Asp Lys Met Leu Val Ala Val Lys Ala Leu Lys Asp
565 570 575Pro Thr Leu Ala Ala Arg Lys Asp Phe Gln Arg Glu Ala Glu
Leu Leu 580 585 590Thr Asn Leu Gln His Glu His Ile Val Lys Phe Tyr
Gly Val Cys Gly 595 600 605Asp Gly Asp Pro Leu Ile Met Val Phe Glu
Tyr Met Lys His Gly Asp 610 615 620Leu Asn Lys Phe Leu Arg Ala His
Gly Pro Asp Ala Met Ile Leu Val625 630 635 640Asp Gly Gln Pro Arg
Gln Ala Lys Gly Glu Leu Gly Leu Ser Gln Met 645 650 655Leu His Ile
Ala Ser Gln Ile Ala Ser Gly Met Val Tyr Leu Ala Ser 660 665 670Gln
His Phe Val His Arg Asp Leu Ala Thr Arg Asn Cys Leu Val Gly 675 680
685Ala Asn Leu Leu Val Lys Ile Gly Asp Phe Gly Met Ser Arg Asp Val
690 695 700Tyr Ser Thr Asp Tyr Tyr Arg Leu Phe Asn Pro Ser Gly Asn
Asp Phe705 710 715 720Cys Ile Trp Cys Glu Val Gly Gly His Thr Met
Leu Pro Ile Arg Trp 725 730 735Met Pro Pro Glu Ser Ile Met Tyr Arg
Lys Phe Thr Thr Glu Ser Asp 740 745 750Val Trp Ser Phe Gly Val Ile
Leu Trp Glu Ile Phe Thr Tyr Gly Lys 755 760 765Gln Pro Trp Phe Gln
Leu Ser Asn Thr Glu Val Ile Glu Cys Ile Thr 770 775 780Gln Gly Arg
Val Leu Glu Arg Pro Arg Val Cys Pro Lys Glu Val Tyr785 790 795
800Asp Val Met Leu Gly Cys Trp Gln Arg Glu Pro Gln Gln Arg Leu Asn
805 810 815Ile Lys Glu Ile
Tyr Lys Ile Leu His Ala Leu Gly Lys Ala Thr Pro 820 825 830Ile Tyr
Leu Asp Ile Leu Gly 83577PRTArtificialhuman (Homo sapiens) 7Lys Asn
Glu Tyr Gly Lys Asp1 586PRTArtificialHuman (Homo sapiens) 8Lys Gly
Asn Pro Lys Pro1 596PRTArtificialmurine (Mus musculus) 9Arg Gly Asn
Pro Lys Pro1 5107PRTArtificialhuman (Homo sapiens) 10Glu Asn Leu
Val Gly Glu Asp1 5116PRTArtificialhuman (Homo sapiens) 11Ala Gly
Asp Pro Val Pro1 51271PRTArtificial SequenceHuman - (homo sapiens)
TrkB IGG-1 domain 12Gly Lys Ser Ile Thr Leu Ser Cys Ser Val Ala Gly
Asp Pro Val Pro1 5 10 15Asn Met Tyr Trp Asp Val Gly Asn Leu Val Ser
Lys His Met Asn Glu 20 25 30Thr Ser His Thr Gln Gly Ser Leu Arg Ile
Thr Asn Ile Ser Ser Asp 35 40 45Asp Ser Gly Lys Gln Ile Ser Cys Val
Ala Glu Asn Leu Val Gly Glu 50 55 60Asp Gln Asp Ser Val Asn Leu65
701377PRTArtificial SequenceHuman - (homo sapiens) TrkB - IGG-2
domain 13Pro Thr Ser Asp His His Trp Cys Ile Pro Phe Thr Val Lys
Gly Asn1 5 10 15Pro Lys Pro Ala Leu Gln Trp Phe Tyr Asn Gly Ala Ile
Leu Asn Glu 20 25 30Ser Lys Tyr Ile Cys Thr Lys Ile His Val Thr Asn
His Thr Glu Tyr 35 40 45His Gly Cys Leu Gln Leu Asp Asn Pro Thr His
Met Asn Asn Gly Asp 50 55 60Tyr Thr Leu Ile Ala Lys Asn Glu Tyr Gly
Lys Asp Glu65 70 75
* * * * *