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Weight Loss Patent Abstract
This invention relates to methods for treating unwanted body weight
loss (such as with cachexia and with aging), eating disorders (such
as anorexia nervosa), or opioid-induced emesis by peripheral administration
of a trkB agonist. The invention also relates to compositions and
kits comprising a trkB agonist.
Weight Loss Patent Claims
1. A method for treating cachexia in a primate comprising peripherally
administering an effective amount of NT-4/5.
2. The method of claim 1, wherein the primate is a human.
3. The method of claim 2, wherein the human has a body mass index
(BMI) of less than about any of 25.0 kg/m.sup.2, 24.0 kg/m.sup.2,
23.0 kg/m.sup.2, 22.0 kg/m.sup.2, 21.0 kg/m.sup.2, 20 kg/m.sup.2,
19.0 kg/m.sup.2, and 18.5 kg/m.sup.2.
4. A method for treating anorexia nervosa in a primate comprising
peripherally administering an effective amount of NT-4/5.
5. The method of claim 4, wherein the primate is a human.
6. The method of claim 5, wherein the human has a BMI of less than
any of about 18.5 kg/m.sup.2, 17.5 kg/m.sup.2, or 16.5 kg/m.sup.2.
7. A method for treating opioid-induced emesis in a mammal comprising
peripherally administering an effective amount of NT-4/5.
8. The method of claim 7, wherein the mammal is a human.
9. A method for treating cachexia in a primate comprising peripherally
administering an effective amount of a trkB agonist.
10. The method of claim 9, wherein the primate is a human.
11. The method of claim 9, wherein the trkB agonist is an anti-trkB
agonist antibody.
12. The method claim 9, wherein the agonist is trkB selective.
13. The method of claim 10, wherein the human has a BMI of less
than about any of 25.0 kg/m.sup.2, 24.0 kg/m.sup.2, 23.0 kg/m.sup.2,
22.0 kg/m.sup.2, 21.0 k/m.sup.2, 20 kg/m.sup.2, 19.0 kg/m.sup.2,
and 18.5 kg/m.sup.2.
14. A method for treating anorexia nervosa in a primate comprising
peripherally administering an effective amount of a trkB agonist.
15. The method of claim 14, wherein the primate is a human.
16. The method of claim 14, wherein the trkB agonist is an anti-trkB
agonist antibody.
17. The method of claim 14, wherein the agonist is trkB selective.
18. The method of claim 15, wherein the human has a BMI of less
than any of about 18.5 kg/m.sup.2, 17.5 kg/m.sup.2, or 16.5 kg/m.sup.2.
19. A method for treating opioid-induced emesis in a mammal comprising
peripherally administering an effective amount of a trkB agonist.
20. The method of claim 19, wherein the mammal is a human.
21. The method of claim 15, wherein the trkB agonist is an anti-trkB
agonist antibody.
22. The method of claim 15, wherein the trkB agonist is trkB selective.
23. A method for treating unwanted weight loss in a primate, comprising
peripherally administering an effective amount of NT-4/5.
24. The method of claim 23, wherein the primate is a human.
25. The method of claim 23, wherein the unwanted weight loss is
associated with chronic obstructive pulmonary disease (COPD), chronic
kidney disease (CKD), chronic heart failure (CHF), aging, cancer,
or AIDS,
26. The method of claim 24, wherein the human has a BMI of less
than about any of 25.0 kg/m.sup.2, 24.0 kg/m.sup.2, 23.0 kg/m.sup.2,
22.0 kg/m.sup.2, 21.0 kg/m.sup.2, 20 kg/m.sup.2, 19.0 kg/m.sup.2,
and 18.5 kg/m.sup.2.
27. A method for treating unwanted weight loss in a primate, comprising
peripherally administering an effective amount of a trkB agonist.
28. The method of claim 27, wherein the primate is human.
29. The method of claim 27, wherein the trkB agonist is an anti-trkB
agonist antibody.
30. The method of claim 27, wherein the agonist is trkB selective.
31. The method of claim 28, wherein the human has a body mass index
of less than about any of 25.0 kg/m.sup.2, 24.0 kg/m.sup.2, 23.0
kg/m.sup.2, 22.0 kg/m.sup.2, 21.0 kg/m.sup.2, 20 kg/m.sup.219.0
kg/m.sup.2, and 18.5 kg/m.sup.2.
32. The method of claim 27, wherein the unwanted weight loss is
associated with chronic obstructive pulmonary disease (COPD), chronic
kidney disease (CKD), chronic heart failure (CHF), aging, cancer,
or AIDS.
Weight Loss Patent Description
[0001] This application claims priority, under 35 U.S.C. .sctn.119(e),
from U.S. Provisional Application Ser. No. 60/765,410, filed Feb.
2, 2006.
FIELD OF THE INVENTION
[0002] This invention concerns use of a trkB agonist in the treatment
and/or prevention of unwanted weight loss, eating disorders, or
opioid-induced emesis.
BACKGROUND OF THE INVENTION
[0003] Neurotrophins are a family of small, homodimeric proteins,
which play a crucial role in the development and maintenance of
the nervous system. Members of the neurotrophin family include nerve
growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3
(NT-3), neurotrophin-4/5 (NT-4/5), neurotrophin-6 (NT-6), and neurotrophin-7
(NT-7). Neurotrophins, similar to other polypeptide growth factors,
affect their target cells through interactions with cell surface
receptors. According to current knowledge, two kinds of transmembrane
glycoproteins serve as receptors for neurotrophins. Neurotrophin-responsive
neurons possess a common low molecular weight (65-80 kDa), low affinity
receptor (LNGFR), also known as p75NTR or p75, which binds NGF,
BDNF, NT-3 and NT-4/5 with a K.sub.D of 2.times.10.sup.-9 M; and
large molecular weight (130-150 kDa), high-affinity (K.sub.D in
the 10.sup.-11 M range) receptors, which are members of the trk
family of receptor tyrosine kinases. The identified members of the
Trk receptor family are trkA, trkB, and trkC.
[0004] Both BDNF and NT-4/5 bind to the trkB and p75NTR receptors
with similar affinity. However, NT-4/5 and BDNF mutant mice exhibit
quite contrasting phenotypes. Whereas NT-4/5.sup.-/- mice are viable
and fertile with only a mild sensory deficit, BDNF.sup.-/- mice
die during early postnatal stages with severe neuronal deficits
and behavioral symptoms. Fan et al., Nat. Neurosci. 3(4):350-7,
2000; Liu et al., Nature 375:238-241, 1995; Conover et al., Nature
375:235-238, 1995; Ernfors et al., Nature 368:147-150, 1994; Jones
et al., Cell 76:989-999, 1994. Several publications report that
NT-4/5 and BDNF have distinct biological activities in vivo and
suggest that the distinct activities may result partly from differential
activation of the trkB receptor and its down-stream signaling pathways
by NT-4/5 and BDNF. Fan et al., Nat. Neurosci. 3(4):350-7, 2000;
[0005] Minichiello et al, Neuron. 21:335-45, 1998; Wirth et al.,
Development. 130(23):5827-38, 2003; Lopez et al., Program No. 38.6,
2003 Abstract, Society for Neuroscience.
[0006] It has been shown that BDNF and NT-4/5 have blood glucose
and blood lipid controlling activity and anti-obesity activity in
type 11 diabetic model animals, such as C57db/db mice. U.S. Pat.
No. 6,391,312;
[0007] Itakura et al., Metabolism 49:129-33 (2000); U.S. Pat. Appl.
Pub. No. 2005/0209148; WO 2005/082401. It has also been shown that
BDNF has anti-obesity activity and activity in ameliorating leptin
resistance in mice fed with high fat diet. U.S. Pat. Appl. Pub.
No. 2003/0036512. Kernie et al. reported that BDNF or NT-4/5 could
transiently reverse the eating behavior and obesity in heterozygous
BDNF knock out mice in which BDNF gene expression was reduced. Kernie
et al., EMBO J. 19(6):1290-300, 2000. It has been reported that
a de novo missense mutation of Y722C substitution on human trkB
results in impaired receptor phosphorylation and signaling to MAP
kinase; and this mutation seems to result in a unique human syndrome
of hyperphagic obesity. Yeo et al., Nat. Neurosci. 7:1187-1189 (2004).
[0008] Circulating levels of BDNF in people with obesity and in
patients with anorexia nervosa have been studied. Monteleone et
al., Psychosomatic Medicine 66:744-748, 2004; Nakazato et al., Biol.
Psychiatry 54:485-490, 2003. Contrary to the prediction based on
the findings that impairments of BDNF production in mice have been
associated with increased food intake, reduced energy expenditure,
and weight gain, circulating BDNF is significantly reduced in the
anorexia nervosa patients and significantly increased in obese subjects
as compared with the non-obese healthy controls. It has been hypothesized
that in anorexia nervosa, BDNF reduction, by promoting food intake,
attempts to counterbalance the patients' altered behaviors that
lead to a negative balance; and in obesity, increased levels of
BDNF may represent an adaptive mechanism to counteract the condition
of positive energy imbalance by stimulating energy expenditure and
decreasing food ingestion. Monteleone et al., Psychosomatic Medicine
66:744-748, 2004.
[0009] All publications, patents and patent applications cited
herein are hereby incorporated by reference in their entirety for
all purposes to the same extent as if each individual publication,
patent or patent application were specifically and individually
indicated to be so incorporated by reference.
BRIEF SUMMARY OF THE INVENTION
[0010] The present invention provides methods for increasing body
weight and/or food intake by peripheral administration of a trkB
agonist, including a trkB selective agonist. These methods can be
used for treating or preventing unwanted weight loss (such as with
cachexia or with aging), eating disorders (such as anorexia nervosa),
and opioid-induced emesis.
[0011] In one aspect, the invention provides methods for increasing
body weight in a primate comprising peripherally administering to
the primate an effective amount of a trkB agonist.
[0012] In another aspect, the invention provides methods for increasing
food intake in a primate comprising peripherally administering to
the primate an effective amount of a trkB agonist.
[0013] In another aspect, the invention provides methods for treating
or preventing cachexia in a primate comprising peripherally administering
to the primate an effective amount of a trkB agonist.
[0014] In another aspect, the invention provides methods for ameliorating,
reducing incidence of, or delaying the development or progression
of cachexia in a primate comprising peripherally administering to
the primate an effective amount of a trkB agonist.
[0015] In another aspect, the invention provides methods for treating
unwanted weight loss in a primate comprising peripherally administering
to the primate an effective amount of a trkB agonist.
[0016] In another aspect the invention provides methods for ameliorating,
reducing incidence of, or delaying the development or progression
of unwanted weight loss in a primate comprising peripherally administering
to the primate an effective amount of a trkB agonist.
[0017] In another aspect, the invention provides methods for treating
or preventing anorexia nervosa in a primate comprising peripherally
administering to the primate an effective amount of a trkB agonist.
[0018] In another aspect, the invention provides methods for ameliorating,
reducing incidence of, or delaying the development or progression
of anorexia nervosa in a primate comprising peripherally administering
to the primate an effective amount of a trkB agonist.
[0019] In another aspect, the invention provides methods for treating
or preventing opioid induced emesis in an individual comprising
peripherally administering to the individual an effective amount
of a trkB agonist.
[0020] In another aspect, the invention provides methods for ameliorating,
reducing incidence of, or delaying the development or progression
of opioid-induced emesis in an individual comprising peripherally
administering to the individual an effective amount of a trkB agonist.
[0021] The trkB agonist is administered peripherally. For example,
the trkB agonist may be administered by one of the following means:
intravenously, intraperitoneally, intramuscularly, subcutaneously,
parenterally, via inhalation, intraarterially, intracardially, intraventricularly,
and transdermally.
[0022] In some embodiments, the individual is a primate. In some
embodiments, the primate is a human.
[0023] The trkB agonist that can be used for the methods described
herein, includes, but is not limited to, BONF polypeptide, NT-4/5
polypeptide, and anti-trkB agonist antibodies. In some embodiments,
the trkB agonist is human NT-4/5. In some embodiments, the trkB
agonist is human BONF. In other embodiments, the trkB agonist is
an anti-trkB agonist antibody, including an anti-trkB agonist antibody
that is trkB selective. In some embodiments, the anti-trkB antibody
is human or humanized.
[0024] In another aspect, the invention provides pharmaceutical
compositions comprising an effective amount of a trkB agonist, including
a trkB selective agonist, and a pharmaceutically acceptable excipient.
The pharmaceutical compositions may be used for treating or preventing
any of the diseases described herein.
[0025] In another aspect, the invention provides kits comprising
a trkB agonist for use in any of the methods described herein. In
some embodiments, the kits comprise a container, a composition comprising
an effective amount of a trkB agonist, in combination with a pharmaceutically
acceptable excipient, and instructions for using the composition
in any of the methods described herein.
[0026] In another aspect, the invention also provides methods for
generating an agonist monoclonal antibody which specifically binds
and activates a receptor, comprising the steps of: (a) immunizing
a host mammal with an immunogenic molecule comprising an extracellular
domain of the receptor by injecting the immunogenic molecule into
the mammal at least two times within about 15 days. The methods
may further comprise the steps of fusing lymphoic cells from the
immunized mammal with an immortalized cell line to produce hybridomas
that secrete monoclonal antibodies; culturing the hybridomas under
the conditions that allow secretion of monoclonal antibodies; and
selecting a hybridoma that secretes a monoclonal antibody that binds
and activates the receptor. In some embodiments, the receptor is
a receptor which requires dimerization for the activation.
BRIEF DESCRIPTION OF THE DRAWINGS
[0027] FIG. 1 is a graph showing the effect of daily NT-4/5 infusion
on body weight in obese female baboons. The X axis corresponds to
days when body weight was measured and the Y axis corresponds to
body weight measured as a percentage of the baseline (body weight
before any treatment). Two way ANOVA was used for comparing NT-4/5
treated group and the vehicle group. Data indicated that body weight
of NT-4/5 treated group was significantly different from the vehicle
group (F-50.71, P<0.0001). Bonferroni posttests analysis showed
significant pairwise difference between NT-4/5 treated group (solid
triangles) with the vehicle group (open squares). * indicates P<0.05;
** indicates P<0.01; and indicates P<0.001 as indicated in
the graph.
[0028] FIG. 2 is a graph showing the effect of daily NT-4/5 infusion
on food intake in obese female baboons. The X axis corresponds to
days when food intake was measured and the Y axis corresponds to
number of biscuits taken by a baboon per day. Two way ANOVA was
used for comparing NT-4/5 treated group with the vehicle group.
Data indicated that food intake of NT-4/5 treated group was significantly
different from the vehicle group (F=262.5, P<0.0001). Bonferroni
posttests showed significant pairwise difference between NT-4/5
treated group (solid triangles) with the vehicle group (open squares).
The solid black bar in the graph indicates the period when the pairwise
comparison resulted in P<0.05 or less.
[0029] FIG. 3 is a graph showing the effect of twice per week NT-4/5
infusion on body weight in obese female baboons. The X axis corresponds
to days when body weight was measured and the Y axis corresponds
to body weight measured as a percentage of the baseline (body weight
before any treatment), Two way ANOVA was used for comparing NT-4/5
treated group with the vehicle group. Data indicated that body weight
of NT-4/5 treated group is significantly different from the vehicle
group (F=34.81, P<0.0001). Bonferroni posttests analysis showed
significant pairwise difference between NT-4/5 treated group (solid
triangles) with the vehicle group (open squares). * indicates P<0.05;
and ** indicates P<0.01.
[0030] FIG. 4 is a graph showing the effect of twice per week NT-4/5
infusion on food intake in obese female baboons. The X axis corresponds
to days when food intake was measured and the Y axis corresponds
to number of biscuits taken by a baboon per day.
[0031] FIG. 5 is a graph showing the effect of daily NT-4/5 and
weekly pegylated NT-4/5 infusion on body weight in lean cynomolgus
monkeys. The X axis corresponds to days when body weight was measured
and the Y axis corresponds to body weight measured as a percentage
of the baseline (body weight before any treatment). Two way ANOVA
was used for comparing NT-4/5 treated group or the pegylated NT-4/5
(PEG-NT-4/5) with the vehicle group. Data indicated that body weight
of NT-4/5 treated group, but not pegylated NT-4/5 treated group,
was significantly different from the vehicle group (F=54.98, P<0.0001).
Bonferroni posttests analysis showed significant pairwise difference
between NT-4/5 treated group (triangles) and the vehicle group (squares),
but not between the pegylated NT-4/5 group (inverted triangles)
and the vehicle group. ** indicates P<0.001 as indicated in the
graph.
[0032] FIG. 6 is a graph showing the effect of daily NT-4/5 and
weekly pegylated NT-4/5 infusion on food intake in lean cynomolgus
monkeys. The X axis corresponds to days when food intake was measured
and the Y axis corresponds to number of biscuits taken by a monkey
per day. Two way ANOVA was used for comparing NT-45 treated group
or the pegylated NT-4/5 (PEG-NT-4/5) with the vehicle group. Data
indicated that body weight of NT-4/5 treated group, but not the
pegylated NT-4/5 treated group, was significantly different from
the vehicle group (F=33.82, P<0.0001). Bonferroni posttests showed
significant pairwise difference (P<0.05 or less) between NT-4/5
treated group (triangles) and the vehicle group (squares) on day
15, 16, 17, 19, 22, 23, 25, and 30, but no significant pairwise
difference between the pegylated NT-4/5 group (inverted triangles)
and the vehicle group.
[0033] FIG. 7 is a graph showing the effect of daily NT-4/5 and
daily pegylated NT-4/5 subcutaneous injection on body weight in
lean cynomolgus monkeys. The X axis corresponds to days when body
weight was measured and the Y axis corresponds to body weight measured
as a percentage of the baseline (body weight before any treatment)
Two way ANOVA was used for comparing NT-4/5 treated group or the
pegylated NT-4/5 (PEG-NT-4/5) with the vehicle group. Data indicated
that body weight of NT-4/5 treated group was significantly different
from the vehicle group (F=19.10, P<0.0001). Bonferroni posttests
analysis showed significant pairwise difference between NT-4/5 treated
group (triangles) with the vehicle group (squares), and between
the pegylated NT-4/5 group (inverted triangles) and the vehicle
group. *** indicates P<0.001; and ** indicates P<0.01.
[0034] FIG. 8 is a graph showing effect of single injection of
NT-4/5 on morphine-induced emesis in ferrets. The X axis corresponds
to type of drug injected; and the Y axis corresponds to number of
retches and vomits over a period of 60 min post injection. One way
ANOVA with Dunnett's posttest was used for statistical analysis.
P values are indicated in the graph.
[0035] FIG. 9A and FIG. 9B show the induction of c-Fos in ferret
hindbrain by NT-4/5. FIG. 9A shows number of nuclei that are stained
by anti-c-Fos antibody in the area postrema. FIG. 9B shows number
of nuclei that are stained by anti-c-Fos antibody in the dorsal
vagal nucleus.
[0036] FIG. 10 shows level of trkB tyrosine phosphorylation in
KIRA assay by various anti-trkB antibodies (36D1, 38B8, 37D12, 19H8(1),
1F8, 23B8, 18H6) in comparison to human NT-4/5.
[0037] FIG. 11 shows a graph of the nodose neuron survival supported
by several trkB agonist antibodies. The X-axis represents the different
concentrations of anti-trkB antibodies added to the embryonic day
15 (E15) nodose neuron culture obtained from Swiss Webster mice.
The Y-axis represents the number of surviving neurons 48 hours post
plating. Each point is an average of four determinations and the
error bars show variance from that average of one standard deviation.
The data indicate that some of the trkB antibodies tested can support
nodose neuron survival and that the 50% effective concentration
(EC50) of these antibodies under this culture condition range from
less than 0.1 to over 10 pM (See table 1).
[0038] FIG. 12A and FIG. 12B are graphs showing the effect of intracranial
injections of anti-trkB agonist antibodies on body weight (FIG.
12A) and food intake (FIG. 12B) in mice. Antibodies and NT-4/5 were
injected on day 0. Body weight and food intake were measured daily
until day 15. *** indicates P<0.001 as compared to mouse IgG
control; ** indicates P<0.01 as compared to mouse IgG control;
and * indicates P<0.05 as compared to mouse IgG control.
[0039] FIG. 13A and FIG. 13B are graphs showing the effect of peripheral
intravenous injections of anti-trkB agonist antibody on body weight
(FIG. 13A) and food intake (FIG. 13B) in cynomolgus monkeys. Antibodies
were injected on day 1. Body weight was monitored weekly and food
intake was monitored daily. indicated P>0.001 as compared to
control vehicle; ** indicates P>0.01 as compared to control vehicle;
and * indicates P>0.05 as compared to control vehicle.
DETAILED DESCRIPTION OF THE INVENTION
[0040] The present invention provides methods for treating or preventing
unwanted weight loss (such as associated with cachexia or with aging),
eating disorders (such as anorexia nervosa), and opioid-induced
emesis comprising administering a trkB agonist to an individual
or a subject.
I. GENERAL TECHNIQUES
[0041] The practice of the present invention will employ, unless
otherwise indicated, conventional techniques of molecular biology
(including recombinant techniques), microbiology, cell biology,
biochemistry and immunology, which are within the skill of the art.
Such techniques are explained fully in the literature, such as,
Molecular Cloning: A Laboratory Manual, second edition (Sambrook,
et al, 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis
(M. J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press;
Cell Biology: A Laboratory Notebook (J. E. Cellis, ed., 1998) Academic
Press; Animal Cell Culture (R. I. Freshney, ed., 1987); Introduction
to Cell and Tissue Culture (J. P. Mather and P. E. Roberts, 1998)
Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A.
Doyle, J. B. Griffiths, and D. G. Newell, eds., 1993-8) J. Wiley
and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook
of Experimental Immunology (D. M. Weir and C. C. Blackwell, eds.);
Gene Transfer Vectors for Mammalian Cells (J. M. Miller and M. P.
Calos, eds., 1987); Current Protocols in Molecular Biology (F. M.
Ausubel, et al., eds., 1987); PCR: The Polymerase Chain Reaction,
(Mullis, et al., eds., 1994); Current Protocols in Immunology (J.
E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology
(Wiley and Sons, 1999); Immunobiology (C. A. Janeway and P. Travers,
1997); Antibodies (P. Finch, 1997); Antibodies: a practical approach
(D. Catty., ed., IRL Press, 1988-1989); Monoclonal antibodies: a
practical approach (P. Shepherd and C. Dean, eds., Oxford University
Press, 2000); Using antibodies: a laboratory manual (E. Harlow and
D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies
(M. Zanetti and J. D. Capra, eds., Harwood Academic Publishers,
1995).
II. DEFINITIONS
[0042] As used herein, "treatment" is an approach for
obtaining beneficial or desired clinical results. For purposes of
this invention, beneficial or desired clinical results include,
but are not limited to, one or more of the following: improving,
lessening severity, alleviation of one or more symptoms associated
with a disease. For example, for treatment of cachexia and/or unwanted
weight loss, beneficial or desired clinical results include, but
are not limited to, any improvement, lessening of severity, and/or
alleviation of any one or more of the following: weight loss, lipolysis,
loss of muscle and visceral protein, anorexia (i.e., loss of appetite),
reduced food/caloric intake, chronic nausea, fatigue and weakness.
For treatment of anorexia nervosa, beneficial or desired clinical
results include, but are not limited to, any one or more of the
following: improvement of appetite, attenuation of food resentment,
gaining weight, maintaining normal nutritional status, hydration
and electrolyte balance, maintaining normal body weight for age
and height, reducing frequency and duration of hospitalization,
and reducing risk of death. For treatment of opioid-induced emesis,
beneficial or desired clinical results include, but are not limited
to, lessening the severity and/or shortening the duration of nausea
and/or vomiting, thereby allowing the full clinical benefits of
opioid-induced pain relief.
[0043] "Ameliorating" a disease or one or more symptoms
of the disease means a lessening or improvement of one or more symptoms
associated with the disease as compared to not administering a trkB
agonist. "Ameliorating" also includes shortening or reduction
in duration of a symptom.
[0044] "Reducing incidence" of a disease means any of
reducing severity (which can include reducing need for and/or amount
of (e.g., exposure to) other drugs and/or therapies generally used
for this condition), duration, and/or frequency (including, for
example, delaying or increasing time to next episodic attack in
an individual). As is understood by those skilled in the art, individuals
may vary in terms of their response to treatment, and, as such,
for example, a method of reducing incidence of a disease in an individual
reflects administering the trkB agonist based on a reasonable expectation
that such administration may likely cause such a reduction in incidence
in that particular individual.
[0045] As used therein, "delaying" the development of
a disease means to defer, hinder, slow, retard, stabilize, and/or
postpone progression of the disease. This delay can be of varying
lengths of time, depending on the history of the disease and/or
individuals being treated. As is evident to one skilled in the art,
a sufficient or significant delay can, in effect, encompass prevention,
in that the individual does not develop the disease (e.g., cachexia,
anorexia nervosa, and opioid-induced emesis). A method that "delays"
development of the symptom is a method that reduces probability
of developing the symptom in a given time frame and/or reduces extent
of the symptoms in a given time frame, when compared to not using
the method. Such comparisons are typically based on clinical studies,
using a statistically significant number of subjects.
[0046] "Development" or "progression" of a
disease means initial manifestations and/or ensuing progression
of the disorder. Development of a disease can be detectable and
assessed using standard clinical techniques well known in the art.
However, development also refers to progression that may be undetectable.
For purpose of this invention, development or progression refers
to the biological course of the symptoms. "Development"
includes occurrence, recurrence, and onset. As used herein "onset"
or "occurrence" of a disease includes initial onset and/or
recurrence.
[0047] As used herein, an "effective dosage" or "effective
amount" of drug, compound, or pharmaceutical composition is
an amount sufficient to effect beneficial or desired results. For
prophylactic use, beneficial or desired results include results
such as eliminating or reducing the risk, lessening the severity,
or delaying the outset of the disease, including biochemical, histological
and/or behavioral symptoms of the disease, its complications and
intermediate pathological phenotypes presenting during development
of the disease. For therapeutic use, beneficial or desired results
include clinical results such as reducing intensity, duration, or
frequency of attack of the disease, and decreasing one or more symptoms
resulting from the disease (biochemical, histological and/or behavioral),
including its complications and intermediate pathological phenotypes
presenting during development of the disease, increasing the quality
of life of those suffering from the disease, decreasing the dose
of other medications required to treat the disease, enhancing effect
of another medication, and/or delaying the progression of the disease
of patients. An effective dosage can be administered in one or more
administrations. For purposes of this invention, an effective dosage
of drug, compound, or pharmaceutical composition is an amount sufficient
to accomplish prophylactic or therapeutic treatment either directly
or indirectly. As is understood in the clinical context, an effective
dosage of a drug, compound, or pharmaceutical composition may or
may not be achieved in conjunction with another drug, compound,
or pharmaceutical composition. Thus, an "effective dosage"
may be considered in the context of administering one or more therapeutic
agents, and a single agent may be considered to be given in an effective
amount if, in conjunction with one or more other agents, a desirable
result may be or is achieved.
[0048] An "individual" or a "subject" is a
mammal, more preferably, a human. Mammals also include, but are
not limited to, farm animals, sport animals, primates (including
humans), horses, dogs, cats, mice and rats.
[0049] An "trkB agonist" refers to an agent that is able
to bind to and activate a trkB receptor and/or downstream pathway(s)
mediated by the trkB signaling function. For example, the agonist
may bind to the extracellular domain of a trkB receptor and thereby
cause dimerization of the receptor, resulting in activation of the
intracellular catalytic kinase domain. Consequently, this may result
in stimulation of growth and/or differentiation of cells expressing
the receptor in vitro and/or in vivo. In some embodiments, a trkB
agonist binds to trkB and activates a trkB biological activity.
[0050] "Biological activity", when used in conjunction
with the trkB agonist of the present invention, generally refers
to having the ability to bind and activate the trkB receptor and/or
a downstream pathway mediated by the trkB signaling function. As
used herein, "biological activity" encompasses one or
more effector functions in common with those induced by action of
NT-4/5 and/or BDNF, the native ligand of trkB, on a trkB-expressing
cell. Without limitation, biological activities include any one
or more of the following: ability to bind and activate trkB; ability
to promote trkB receptor dimerization; the ability to promote the
development, survival, function, maintenance and/or regeneration
of cells (including damaged cells), in particular neurons in vitro
or in vivo, including peripheral (sympathetic, sensory, motor, and
enteric) neurons, and central (brain and spinal cord) neurons, and
non-neuronal cells, e.g. peripheral blood leukocytes, endothelial
cells and vascular smooth muscle cells. A particular preferred biological
activity is the ability to increase body weight and/or food intake
in a primate when administered peripherally, to treat (including
prevention of) one or more symptoms of cachexia and anorexia nervosa
in a primate, and/or to treat (including prevention of) one or more
symptoms of opioid-induced emesis in a mammal.
[0051] An "agonist anti-trkB antibody" (interchangeably
termed "anti-trkB agonist antibody") refers to an antibody
that is able to bind to and activate a trkB receptor and/or downstream
pathway(s) mediated by the trkB signaling function. For example,
the agonist antibody may bind to the extracellular domain of a trkB
receptor and thereby cause dimerization of the receptor, resulting
in activation of the intracellular catalytic kinase domain. Consequently,
this may result in stimulation of growth and/or differentiation
of cells expressing the receptor in vitro and/or in vivo. In some
embodiments, an agonist anti-trkB antibody binds to trkB and activates
a trkB biological activity.
[0052] As used herein, "peripheral administration" or
"administered peripherally" refers to introducing an agent
into a subject outside of the central nervous system (CNS) or blood
brain barrier (BBB). Peripheral administration encompasses any route
of administration other than direct administration to the spine
or brain. Peripheral administration can be local or systemic.
[0053] An "antibody" is an immunoglobulin molecule capable
of specific binding to a target, such as a carbohydrate, polynucleotide,
lipid, polypeptide, etc., through at least one antigen recognition
site, located in the variable region of the immunoglobutin molecule.
As used herein, the term encompasses not only intact polyclonal
or monoclonal antibodies, but also fragments thereof (such as Fab,
Fab', F(ab').sub.2, Fv), single chain (ScFv), mutants thereof, fusion
proteins comprising an antibody portion (such as domain antibodies),
and any other modified configuration of the immunoglobulin molecule
that comprises an antigen recognition site. An antibody includes
an antibody of any class, such as IgG, IgA, or IgM (or sub-class
thereof), and the antibody need not be of any particular class.
Depending on the antibody amino acid sequence of the constant domain
of its heavy chains, immunoglobulins can be assigned to different
classes. There are five major classes of immunoglobulins: IgA, IgD,
IgE, IgG, and IgM, and several of these may be further divided into
subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2.
The heavy-chain constant domains that correspond to the different
classes of immunoglobulins are called alpha, delta, epsilon, gamma,
and mu, respectively.
[0054] The subunit structures and three-dimensional configurations
of different classes of immunoglobulins are well known.
[0055] As used herein, "monoclonal antibody" refers to
an antibody obtained from a population of substantially homogeneous
antibodies, i.e., the individual antibodies comprising the population
are identical except for possible naturally-occurring mutations
that may be present in minor amounts. Monoclonal antibodies are
highly specific, being directed against a single antigenic site.
Furthermore, in contrast to polyclonal antibody preparations, which
typically include different antibodies directed against different
determinants (epitopes), each monoclonal antibody is directed against
a single determinant on the antigen. The modifier "monoclonal"
indicates the character of the antibody as being obtained from a
substantially homogeneous population of antibodies, and is not to
be construed as requiring production of the antibody by any particular
method. For example, the monoclonal antibodies to be used in accordance
with the present invention may be made by the hybridoma method first
described by Kohler and Milstein, 1975, Nature, 256:495, or may
be made by recombinant DNA methods such as described in U.S. Pat.
No. 4,816,567. The monoclonal antibodies may also be isolated from
phage libraries generated using the techniques described in McCafferty
et al., 1990, Nature, 348:552-554, for example.
[0056] As used herein, "humanized" antibodies refer to
forms of non-human (e.g. murine) antibodies that are specific chimeric
immunoglobulins, immunoglobulin chains, or fragments thereof (such
as Fv, Fab, Fab', F(ab').sub.2 or other antigen-binding subsequences
of antibodies) that contain minimal sequence derived from non-human
immunoglobulin. For the most part, humanized antibodies are human
immunoglobulins (recipient antibody) in which residues from a complementarity
determining region (CDR) of the recipient are replaced by residues
from a CDR of a non-human species (donor antibody) such as mouse,
rat, or rabbit having the desired specificity, affinity, and biological
activity. In some instances, Fv framework region (FR) residues of
the human immunoglobulin are replaced by corresponding non-human
residues. Furthermore, the humanized antibody may comprise residues
that are found neither in the recipient antibody nor in the imported
CDR or framework sequences, but are included to further refine and
optimize antibody performance. In general, the humanized antibody
will comprise substantially all of at least one, and typically two,
variable domains, in which all or substantially all of the CDR regions
correspond to those of a non-human immunoglobulin and all or substantially
all of the FR regions are those of a human immunoglobulin consensus
sequence. The humanized antibody optimally also will comprise at
least a portion of an immunoglobulin constant region or domain (Fc),
typically that of a human immunoglobul in. Antibodies may have Fc
regions modified as described in WO 99/58572. Other forms of humanized
antibodies have one or more CDRs (one, two, three, four, five, six)
which are altered with respect to the original antibody, which are
also termed one or more CDRs "derived from" one or more
CDRs from the original antibody.
[0057] As used herein, "human antibody" means an antibody
having an amino acid sequence corresponding to that of an antibody
produced by a human and/or has been made using any of the techniques
for making human antibodies known in the art or disclosed herein.
This definition of a human antibody includes antibodies comprising
at least one human heavy chain polypeptide or at least one human
light chain polypeptide. One such example is an antibody comprising
murine light chain and human heavy chain polypeptides. Human antibodies
can be produced using various techniques known in the art. In one
embodiment, the human antibody is selected from a phage library,
where that phage library expresses human antibodies (Vaughan et
al., 1996, Nature Biotechnology, 14:309-314; Sheets et al., 1998,
PNAS, (USA) 95:6157-6162; Hoogenboom and Winter, 1991, J. Mol. Biol.,
227:381; Marks et al. 1991, J. Mol. Biol., 222:581). Human antibodies
can also be made by introducing human immunoglobulin loci into transgenic
animals, e.g., mice in which the endogenous immunoglobulin genes
have been partially or completely inactivated. This approach is
described in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126;
5,633,425; and 5,661,016. Alternatively, the human antibody may
be prepared by immortalizing human B lymphocytes that produce an
antibody directed against a target antigen (such B lymphocytes may
be recovered from an individual or may have been immunized in vitro).
See, e.g., Cole et al., Monoclonal Antibodies and Cancer Therapy,
Alan R. Liss, p. 77 (1985); Boerner et al., 1991, J. Immunol., 147
(1 ):86-95; and U.S. Pat. No. 5,750,373.
[0058] A "variable region" of an antibody refers to the
variable region of the antibody light chain or the variable region
of the antibody heavy chain, either alone or in combination. The
variable regions of the heavy and light chain each consist of four
framework regions (FR) connected by three complementarity determining
regions (CDRs) also known as hypervariable regions. The CDRs in
each chain are held together in close proximity by the FRs and,
with the CDRs from the other chain, contribute to the formation
of the antigen-binding site of antibodies. There are at least two
techniques for determining CDRs: (1) an approach based on cross-species
sequence variability (i.e., Kabat et al. Sequences of Proteins of
Immunological Interest, (5th ed., 1991, National Institutes of Health,
Bethesda Md.)); and (2) an approach based on crystallographic studies
of antigen-antibody complexes (Al-lazikani et al (1997) J. Molec.
Biol. 273:927-948)). As used herein, a CDR may refer to CDRs defined
by either approach or by a combination of both approaches.
[0059] A "constant region" of an antibody refers to the
constant region of the antibody light chain or the constant region
of the antibody heavy chain, either alone or in combination.
[0060] An epitope that "preferentially binds" or "specifically
binds" (used interchangeably herein) to an antibody or a polypeptide
is a term well understood in the art, and methods to determine such
specific or preferential binding are also well known in the art.
A molecule is said to exhibit specific binding"or "preferential
binding" if it reacts or associates more frequently, more rapidly,
with greater duration and/or with greater affinity with a particular
cell or substance than it does with alternative cells or substances.
An antibody "specifically" binds or "preferentially"
binds or "selectively" binds to a target if it binds with
greater affinity, avidity, more readily, and/or with greater duration
than it binds to other substances. For example, an antibody that
specifically or preferentially binds to a trkB epitope is an antibody
that binds this epitope with greater affinity, avidity, more readily,
and/or with greater duration than it binds to other trkB epitopes
or non-trkB epitopes. It is also understood by reading this definition
that, for example, an antibody (or moiety or epitope) that specifically
or preferentially binds to a first target may or may not specifically
or preferentially bind to a second target. As such, "specific"
binding or "preferential" binding or "selective"
binding of an antibody to trkB does not necessarily require (although
it can include) exclusive binding. Generally, but not necessarily,
reference to selective trkB binding means preferential binding (e.g.,
binding with an IC50 at a concentration at least 3, 5, or, preferably,
at least 10-old or 100-fold lower for trkB as compared to other
receptors).
[0061] The term "Fc region" is used to define a C-terminal
region of an immunoglobulin heavy chain. The "Fc region"
may be a native sequence Fc region or a variant Fc region. Although
the boundaries of the Fc region of an immunoglobulin heavy chain
might vary, the human IgG heavy chain Fc region is usually defined
to stretch from an amino acid residue at position Cys226, or from
Pro230, to the carboxyl-terminus thereof. The numbering of the residues
in the Fc region is that of the EU index as in Kabat. Kabat et al.,
Sequences of Proteins of Imunological Interest, 5th Ed. Public Health
Service, National institutes of Health, Bethesda, Md., 1991. The
Fc region of an immunoglobulin generally comprises two constant
domains, CH2 and CH3.
[0062] As used herein, "Fc receptor" and "FcR"
describe a receptor that binds to the Fc region of an antibody.
The preferred FcR is a native sequence human FcR. Moreover, a preferred
FcR is one which binds an IgG antibody (a gamma receptor) and includes
receptors of the Fc.gamma.RI, Fc.gamma.RII, Fc.gamma.RIII, and Fc.gamma.RIV
subclasses, including allelic variants and alternatively spliced
forms of these receptors. Fc.gamma.RlI receptors include Fc.gamma.RIIA
(an "activating receptor") and Fc.gamma.RIIB (an "inhibiting
receptor"), which have similar amino acid sequences that differ
primarily in the cytoplasmic domains thereof. FcRs are reviewed
in Ravetch and Kinet, 1991, Ann. Rev. Immunol., 9:457-92; Capel
et al., 1994, Immunomethods, 4:25-34; de Haas et al., 1995, J. Lab.
Clin. Med., 126:330-41; Nimmerjahn et al., 2005, Immunity 23:2-4.
"FcR" also includes the neonatal receptor, FcRn, which
is responsible for the transfer of maternal IgGs to the fetus (Guyer
et al., 1976, J. Immunol., 1 1 7:587; and Kim et al., 1994, J. Immunol.,
24:249). "Complement dependent cytotoxicity" and "CDC"
refer to the lysing of a target in the presence of complement. The
complement activation pathway is initiated by the binding of the
first component of the complement system (C1q) to a molecule (e.g.
an antibody) complexed with a cognate antigen. To assess complement
activation, a CDC assay, e.g. as described in Gazzano-Santoro et
at., J. Immunol. Methods, 202:163 (1996), may be performed.
[0063] A "functional Fc region" possesses at least one
effector function of a native sequence Fc region. Exemplary "effector
functions" include C1q binding; complement dependent cytotoxicity
(CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity
(ADCC); phagocytosis; downregulation of cell surface receptors (e.g.
B cell receptor; BCR), etc. Such effector functions generally require
the Fc region to be combined with a binding domain (e.g. an antibody
variable domain) and can be assessed using various assays known
in the art for evaluating such antibody effector functions.
[0064] A "native sequence Fc region" comprises an amino
acid sequence identical to the amino acid sequence of an Fc region
found in nature. A "variant Fc region" comprises an amino
acid sequence which differs from that of a native sequence Fc region
by virtue of at least one amino acid modification, yet retains at
least one effector function of the native sequence Fc region. Preferably,
the variant Fc region has at least one amino acid substitution compared
to a native sequence Fc region or to the Fc region of a parent polypeptide,
e.g. from about one to about ten amino acid substitutions, and preferably
from about one to about five amino acid substitutions in a native
sequence Fc region or in the Fc region of the parent polypeptide.
The variant Fc region herein will preferably possess at least about
80% sequence identity with a native sequence Fc region and/or with
an Fc region of a parent polypeptide, and most preferably at least
about 90% sequence identity therewith, more preferably, at least
about 95%, at least about 96%, at least about 97%, at least about
98%, at least about 99% sequence identity therewith.
[0065] As used herein "antibody-dependent cell-mediated cytotoxicity"
and "ADCC" refer to a cell-mediated reaction in which
nonspecific cytotoxic cells that express Fc receptors (FcRs) (e.g.
natural killer (NK) cells, neutrophils, and macrophages) recognize
bound antibody on a target cell and subsequently cause lysis of
the target cell. ADCC activity of a molecule of interest can be
assessed using an in vitro ADCC assay, such as that described in
U.S. Pat. Nos. 5,500,362 or 5,821,337. Useful effector cells for
such assays include peripheral blood mononuclear cells (PBMC) and
NK cells. Alternatively, or additionally, ADCC activity of the molecule
of interest may be assessed in vivo, e.g., in a animal model such
as that disclosed in Clynes et al., 1998, PNAS (USA), 95:652-656.
[0066] As used herein, "pharmaceutically acceptable carrier"
or "pharmaceutical acceptable excipient" includes any
material which, when combined with an active ingredient, allows
the ingredient to retain biological activity and is non-reactive
with the subject's immune system. Examples include, but are not
limited to, any of the standard pharmaceutical carriers such as
a phosphate buffered saline solution, water, emulsions such as oil/water
emulsion, and various types of wetting agents. Preferred diluents
for aerosol or parenteral administration are phosphate buffered
saline or normal (0.9%) saline. Compositions comprising such carriers
are formulated by well known conventional methods (see, for example,
Remington's Pharmaceutical Sciences, 18th edition, A. Gennaro, ed.,
Mack Publishing Co., Easton, Pa., 1990; and Remington, The Science
and Practice of Pharmacy 20th Ed. Mack Publishing, 2000).
[0067] The terms "polypeptide", "oligopeptide",
"peptide" and "protein" are used interchangeably
herein to refer to polymers of amino acids of any length. The polymer
may be linear or branched, it may comprise modified amino acids,
and it may be interrupted by non-amino acids. The terms also encompass
an amino acid polymer that has been modified naturally or by intervention;
for example, disulfide bond formation, glycosylation, lipidation,
acetylation, phosphorylation, or any other manipulation or modification,
such as conjugation with a labeling component. Also included within
the definition are, for example, polypeptides containing one or
more analogs of an amino acid (including, for example, unnatural
amino acids, etc.), as well as other modifications known in the
art. It is understood that, because the polypeptides of this invention
are based upon an antibody, the polypeptides can occur as single
chains or associated chains.
[0068] "Polynucleotide," or "nucleic acid,"
as used interchangeably herein, refer to polymers of nucleotides
of any length, and include DNA and RNA. The nucleotides can be deoxyribonucleotides,
ribonucleotides, modified nucleotides or bases, and/or their analogs,
or any substrate that can be incorporated into a polymer by DNA
or RNA polymerase. A polynucleotide may comprise modified nucleotides,
such as methylated nucleotides and their analogs. If present, modification
to the nucleotide structure may be imparted before or after assembly
of the polymer. The sequence of nucleotides may be interrupted by
non-nucleotide components. A polynucleotide may be further modified
after polymerization, such as by conjugation with a labeling component.
Other types of modifications include, for example, "caps",
substitution of one or more of the naturally occurring nucleotides
with an analog, internucleotide modifications such as, for example,
those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters,
phosphoamidates, carbamates, etc.) and with charged linkages (e.g.,
phosphorothioates, phosphorodithioates, etc.), those containing
pendant moieties, such as, for example, proteins (e.g., nucleases,
toxins, antibodies, signal peptides, ply-L-lysine, etc.), those
with intercalators (e.g., acridine, psoralen, etc.), those containing
chelators (e.g., metals, radioactive metals, boron, oxidative metals,
etc.), those containing alkylators, those with modified linkages
(e.g., alpha anomeric nucleic acids, etc.), as well as unmodified
forms of the polynucleotide(s). Further, any of the hydroxyl groups
ordinarily present in the sugars may be replaced, for example, by
phosphonate groups, phosphate groups, protected by standard protecting
groups, or activated to prepare additional linkages to additional
nucleotides, or may be conjugated to solid supports. The 5' and
3' terminal OH can be phosphorylated or substituted with amines
or organic capping group moieties of from 1 to 20 carbon atoms.
Other hydroxyls may also be derivatized to standard protecting groups.
Polynucleotides can also contain analogous forms of ribose or deoxyribose
sugars that are generally known in the art, including, for example,
2'-O-methyl-, 2'-O-allyl, 2'-fluoro- or 2'-azido-ribose, carbocyclic
sugar analogs, .quadrature.-anomeric sugars, epimeric sugars such
as arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars,
sedoheptuloses, acyclic analogs and a basic nucleoside analogs such
as methyl riboside. One or more phosphodiester linkages may be replaced
by alternative linking groups. These alternative linking groups
include, but are not limited to, embodiments wherein phosphate is
replaced by P(O)S("thioate"), P(S)S ("dithioate"),
(O)NR.sub.2 ("amidate"), P(O)R, P(O)OR', CO or CH.sub.2
( formacetal"), in which each R or R' is independently H or
substituted or unsubstituted alkyl (1-20 C) optionally containing
an ether (--O--) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl
or araldyl. Not all linkages in a polynucleotide need be identical.
The preceding description applies to all polynucleotides referred
to herein, including RNA and DNA.
[0069] As used herein, "substantially pure" refers to
material which is at least 50% pure (i.e., free from contaminants),
more preferably, at least 90% pure, more preferably, at least 95%
pure, more preferably, at least 98% pure, more preferably, at least
99% pure.
[0070] A "host cell" includes an individual cell or cell
culture that can be or has been a recipient for vector(s) for incorporation
of polynucleotide inserts. Host cells include progeny of a single
host cell, and the progeny may not necessarily be completely identical
(in morphology or in genomic DNA complement) to the original parent
cell due to natural, accidental, or deliberate mutation. A host
cell includes cells transfected in vivo with a polynucleotide(s)
of this invention.
[0071] As used herein, "vector" means a construct, which
is capable of delivering, and preferably expressing, one or more
gene(s) or sequence(s) of interest in a host cell. Examples of vectors
include, but are not limited to, viral vectors, naked DNA or RNA
expression vectors, plasmid, cosmid or phage vectors, DNA or RNA
expression vectors associated with cationic condensing agents, DNA
or RNA expression vectors encapsulated in liposomes, and certain
eukaryotic cells, such as producer cells.
[0072] As used herein, "expression control sequence"
means a nucleic acid sequence that directs transcription of a nucleic
acid. An expression control sequence can be a promoter, such as
a constitutive or an inducible promoter, or an enhancer. The expression
control sequence is operably linked to the nucleic acid sequence
to be transcribed.
[0073] The term "k.sub.on", as used herein, is intended
to refer to the rate constant for association of an antibody to
an antigen.
[0074] The term "k.sub.off", as used herein, is intended
to refer to the rate constant for dissociation of an antibody from
the antibody/antigen complex.
[0075] The term "K.sub.D", as used herein, is intended
to refer to the equilibrium dissociation constant of an antibody-antigen
interaction.
[0076] As used herein, the singular form "a", "an",
and "the" includes plural references unless indicated
otherwise.
Ill. METHODS OF THE INVENTION
[0077] The present invention encompasses methods for increasing
body weight and/or food intake by peripheral administration of a
trkB agonist. These methods can be used for treating or preventing
unwanted weight loss (such as with cachexia and with aging) and
eating disorders (such as anorexia nervosa) in primates, and opioid-induced
emesis in mammals. The methods entail peripheral administration
of an effective amount of one or more trkB agonists to an individual
in need thereof (various indications and aspects are described herein).
[0078] With respect to all methods described herein, reference
to trkB agonists also includes compositions comprising one or more
of these agents. These compositions may further comprise suitable
excipients, such as pharmaceutically acceptable excipients including
buffers, which are well known in the art. The present invention
can be used alone or in combination with other conventional methods
of treatment.
[0079] Cachexia and unwanted weight loss that can be treated and/or
prevented by the methods described herein may be caused and/or associated
with one or more of the following: chronic obstructive pulmonary
disease (COPD), chronic kidney disease (CKD), chronic heart failure
(CH F), aging, cancer, and AIDS.
[0080] In some embodiments, the human patients having cachexia
treated or having unwanted weight loss treated have a Body Mass
Index (BMI), calculated as body weight per height in meters squared
(kg/m.sup.2)) less than about any of 25.0 kg/m.sup.2, 24.0 kg/m.sup.2,
23.0 kg/m.sup.2, 22.0 kg/m.sup.2, 21.0 kg/m.sup.2, 20.0 kg/m.sup.2,
19.0 kg/m.sup.2, and 18.5 kg/,.sup.2. In some embodiments, the human
patients having cachexia treated or unwanted weight loss treated
have a daily food intake less than about 90%, about 80%, about 70%,
about 60%, about 50%, about 40%, about 30%, about 20% or about 10%
of the normal recommended daily intake level or pre-morbid level.
In some embodiments in which patients are having cachexia treated
or having unwanted weight loss treated, the cachexia or unwanted
weight loss is associated with chronic obstructive pulmonary disease
(COPD), chronic kidney disease (CKD), chronic heart failure (CHF),
aging, cancer, or AIDS.
[0081] In some embodiments, the human patients having anorexia
nervosa treated by the methods described herein have a BMI less
than any of about 18.5 kg/m.sup.2, 17.5 kg/m.sup.2, and 16.5 kg/m.sup.2.
In some embodiments, the human patients having anorexia nervosa
treated have a daily food intake less than about 90%, about 80%,
about 70%, about 60%, about 50%, about 40%, about 30%, about 20%,
or about 10% of the normal recommended daily intake level or pre-morbid
level.
[0082] The trkB agonist is administered peripherally. It is understood
that although the agent is administered peripherally, a small percentage
of the agent may pass blood brain barrier and result in delivery
to the central nervous system depending on the properties of the
agent. In some embodiments, less than any of about 1%, about 0.5%,
about 0.25%, and about 0.1% of peripherally administered trkB agonist
(for example, trkB agonist antibody) gains access to the CNS.
[0083] The trkB agonist can be administered to an individual via
any suitable peripheral route. It should be apparent to a person
skilled in the art that the examples described herein are not intended
to be limiting but to be illustrative of the techniques available.
Accordingly, in some embodiments, the trkB agonist is administered
to an individual in accord with known methods, such as intravenous
administration, e.g., as a bolus or by continuous infusion over
a period of time, by intramuscular, intraperitoneal, subcutaneous,
intra-articular, sublingually, intrasynovial, via insufflation,
oral, inhalation or topical routes. Administration can be systemic,
e.g., intravenous administration, or localized. Commercially available
nebulizers for liquid formulations, including jet nebulizers and
ultrasonic nebulizers are useful for administration. Liquid formulations
can be directly nebulized and lyophilized powder can be nebulized
after reconstitution. Alternatively, trkB agonist can be aerosolized
using a fluorocarbon formulation and a metered dose inhaler, or
inhaled as a lyophilized and milled powder.
[0084] A trkB agonist may be administered via site-specific or
targeted local delivery techniques outside of the CNS or the blood
brain barrier. Examples of site-specific or targeted local delivery
techniques include various implantable depot sources of the trkB
agonist or local delivery catheters, such as infusion catheters,
an indwelling catheter, or a needle catheter, synthetic grafts,
adventitial wraps, shunts and stents or other implantable devices,
site specific carriers, direct injection, or direct application.
See, e.g., PCT Publication No. WO 00/53211 and U.S. Pat. No. 5,981,568.
[0085] Various formulations of trkB agonists may be used for administration.
In some embodiments, a trkB agonist may be administered neat. In
other embodiments, a trkB agonist and a pharmaceutically acceptable
excipient are administered, and may be in various formulations.
Pharmaceutically acceptable excipients are known in the art, and
are relatively inert substances that facilitate administration of
a pharmacologically effective substance. For example, an excipient
can give form or consistency, or act as a diluent. Suitable excipients
include but are not limited to stabilizing agents, wetting and emulsifying
agents, salts for varying osmolarity, encapsulating agents, buffers,
and skin penetration enhancers. Excipients as well as formulations
for parenteral and nonparenteral drug delivery are set forth in
Remington, The Science and Practice of Pharmacy 20th Ed. Mack Publishing
(2000). Generally, these agents are formulated for administration
by injection (e.g., intraperitoneally, intravenously, subcutaneously,
intramuscularly, etc.), although other forms of administration (e.g.,
oral, mucosal, transdermal, inhalation, etc) can be also used.
[0086] The particular dosage regimen, i.e., dose, timing and repetition,
will depend on the particular individual and that individual's medical
history, the particular disease (e.g., cachexia, unwanted weight
loss, anorexia nervosa, and opioid-induced emesis) to be treated,
and the particular trkB agonist. Generally, any of the following
doses of trkB agonist (e.g., NT-4/5, BDNF, and anti-trkB agonist
antibody) may be used: a dose of at least about 50 mg/kg body weight;
at least about 20 mg/kg body weight; at least about 10 mg/kg body
weight; at least about 5 mg/kg body weight; at least about 3 mg/kg
body weight; at least about 2 mg/kg body weight; at least about
1 mg/kg body weight; at least about 750 pg/kg body weight; at least
about 500 .mu.g/kg body weight; at least about 250 ug/kg body weight;
at least about 100 pg /kg body weight; at least about 50 .mu.g/kg
body weight; at least about 10 ug/kg body weight; at least about
1 .mu.g/kg body weight or more, is administered. Empirical considerations,
such as the half-life, generally will contribute to determination
of the dosage. For repeated administrations over several days or
longer, depending on the condition, the treatment is sustained until
a desired suppression of disease symptoms occurs or until sufficient
therapeutic levels are achieved. For example, dosing from one to
five times a week is contemplated. Other dosing regimens include
a regimen of up to 1 time per day, 1 to 5 times per week, or less
frequently. In some embodiments, the trkB agonist is administered
about once per week, about 1 to 4 times per month. Intermittent
dosing regime with staggered dosages spaced by 2 days up to 7 days
or even 14 days may be used. In some embodiments, treatment may
start with a daily dosing and later change to weekly even monthly
dosing. The progress of this therapy is easily monitored by conventional
techniques and assays.
[0087] In some individuals, more than one dose may be required.
Frequency of administration may be determined and adjusted over
the course of therapy. For example, frequency of administration
may be determined or adjusted based on the type and severity of
the disease to be treated, whether the agent is administered for
preventive or therapeutic purposes, previous therapy, the patient's
clinical history and response to the agent, and the discretion of
the attending physician. Typically the clinician will administer
a trkB agonist until a dosage is reached that achieves the desired
result. In some cases, sustained continuous release formulations
of trkB agonist may be appropriate. Various formulations and devices
for achieving sustained release are known in the art. For example,
trkB agonist may be administered through a mechanical pump or embedded
in a matrix bed for sustained or slow release.
[0088] In one embodiment, dosages for a trkB agonist may be determined
empirically in individuals who have been given one or more administration(s).
Individuals are given incremental dosages of a trkB agonist. To
assess efficacy of a trkB agonist, markers of the disease state
can be monitored. It will be apparent to one of skill in the art
that the dosage will vary depending on the individual, the stage
of the disease (such as cachexia, anorexia nervosa, and opioid-induced
emesis), and the past and concurrent treatments being used.
[0089] Administration of a trkB agonist in accordance with the
method in the present invention can be continuous or intermittent,
depending, for example, upon the recipient's physiological condition,
whether the purpose of the administration is therapeutic or prophylactic,
and other factors known to skilled practitioners. The administration
of a trkB agonist may be essentially continuous over a preselected
period of time or may be in a series of spaced doses.
[0090] Other formulations include suitable delivery forms known
in the art including, but not limited to, carriers such as liposomes.
See, for example, Mahato et al. (1997) Pharm. Res. 14:853-859. Liposomal
preparations include, but are not limited to, cytofectins, multilamellar
vesicles and unilamellar vesicles.
[0091] Assessment of disease is performed using standard methods
known in the arts, for example, by monitoring appropriate marker(s).
For example, for cachexia, the following markers may be monitored:
body weight, plasma albumin, body fat, body lean mass, fatigue,
weakness, and appetite. For anorexia nervosa, the following markers
may be monitored: body weight, appetite, and fear of gaining weight.
For opioid-induced emesis, the following markers may be monitored:
nausea, vomiting, appetite, body weight, and other associated medical
complications.
IV. COMPOSITIONS AND METHODS OF MAKING THE COMPOSITIONS
[0092] The methods of the invention use a trkB agonist, which refers
to any molecule that binds and activates a native trkB receptor
and/or downstream pathways mediated by the trkB signaling function.
The trkB agonist includes any native ligand of a trkB receptor,
such as NT-4/5 and BDNF. The trkB agonist also includes non-native
ligand (e.g., polypeptides, peptide-derived compound, cyclic peptide-derived
or non peptide derived molecules) of a trkB receptor that binds
to and activates a native trkB receptor, thereby mimicking a biological
activity of a native ligand of the receptor. An example of non-native
ligands of a trkB receptor is an anti-trkB agonist antibody. TrkB
agonists also include small molecules or peptide mimetics (e.g.,
peptide mimetics of BDNF). See, e.g., O'Leary et al., J. Biol. Chem.
278:25738-44, 2003. In some embodiments, the small molecule trkB
agonist does not significantly pass blood brain barrier when administered
peripherally.
[0093] A trkB agonist should exhibit any one or more of the following
characteristics: (a) bind to trkB receptor; (b) bind to trkB receptor
and activate trkB biological activity(ies) and/or one or more downstream
pathways mediated by trkB signaling function(s); (c) bind to trkB
receptor and increase body weight and/or food intake in a primate
when administered peripherally; (d) bind to trkB receptor and treat,
prevent, reverse, or ameliorate one or more symptoms of cachexia
or unwanted weight loss in a primate when administered peripherally;
(e) bind to trkB receptor and treat, prevent, reverse, or ameliorate
one or more symptoms of anorexia nervosa in a primate when administered
peripherally; (f) bind to trkB receptor and treat, prevent, reverse,
or ameliorate one or more symptoms of opioid-induced emesis in a
mammal when administered peripherally; (g) promote trkB receptor
dimerization and activation; and (h) increase trkB receptor-dependent
neuronal survival and/or neurite outgrowth. In some embodiments,
the trkB agonist binds and activates trkB receptor, but does not
significantly or preferentally activate one or more other trk receptors,
such as trkA and/or trkC.
[0094] The trkB agonist may be in the form of a composition for
use in any of the methods described herein. The composition used
in the methods of the invention comprises an effective amount of
a trkB agonist. The composition can further comprise pharmaceutically
acceptable carriers, excipients, or stabilizers (Remington: The
Science and practice of Pharmacy 20th Ed. (2000) Lippincott Williams
and Wilkins, Ed. K. E. Hoover.), in the form of lyophilized formulations
or aqueous solutions. Acceptable carriers, excipients, or stabilizers
are nontoxic to recipients at the dosages and concentrations, and
may comprise buffers such as phosphate, citrate, and other organic
acids; antioxidants including ascorbic acid and methionine; preservatives
(such as octadecyidimethylbenzyl ammonium chloride; hexamethonium
chloride; benzalkonium chloride, benzethonium chloride; phenol,
butyl or benzyl alcohol; alkyl parabens such as methyl or propyl
paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol);
low molecular weight (less than about 10 residues) polypeptides;
proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic
polymers such as polyvinylpyrrolidone; amino acids such as glycine,
glutamine, asparagine, histidine, arginine, or lysine; monosaccharides,
disaccharides, and other carbohydrates including glucose, mannose,
or dextrans; chelating agents such as EDTA; sugars such as sucrose,
mannitol, trehalose or sorbitol; salt-forming counter-ions such
as sodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionic
surfactants such as TWEEN.TM., PLURONICS.TM. or polyethylene glycol
(PEG). Pharmaceutically acceptable excipients are further described
herein.
[0095] TrkB agonists described herein can be formulated for sustained-release.
Suitable examples of sustained-release preparations include semipermeable
matrices of solid hydrophobic polymers containing trkB agonist,
which matrices are in the form of shaped articles, e.g. films, or
microcapsules. Examples of sustained-release matrices include polyesters,
hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(v
nylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers
of L-glutamic acid and 7 ethyl-L-glutamate, non-degradable ethylene-vinyl
acetate, degradable lactic acid-glycolic acid copolymers such as
the LUPRON DEPOT.TM. (injectable microspheres composed of lactic
acid-glycolic acid copolymer and leuprolide acetate), sucrose acetate
isobutyrate, and poly-D-(-)-3-hydroxybutyric acid. Another example
of sustained release drug-delivery system that can be used is the
ATRIGEL.RTM. made by Atrix Laboratories. See, for example, U.S.
Pat. No. 6,565,874. The ATRIGEL.RTM. drug delivery system consists
of biodegradable polymers, similar to those used in biodegradable
sutures, dissolved in biocompatible carriers. TrkB agonists may
be blended into this liquid delivery system at the time of manufacturing
or, depending upon the product, may be added later by the physician
at the time of use. When the liquid product is injected subcutaneously
or intramuscularly through a small gauge needle or placed into accessible
tissue sites through a cannula, displacement of the carrier with
water in the tissue fluids causes the polymer to precipitate to
form a solid film or implant. TrkB agonists encapsulated within
the implant are then released in a controlled manner as the polymer
matrix biodegrades with time. Depending upon the patient's medical
needs, the Atrigel system can deliver proteins over a period ranging
from days to months. Injectable sustained release systems, such
as ProLease.RTM., Medisorb.RTM., manufactured by Alkermes may also
be used.
[0096] In some embodiments, the invention provides compositions
(described herein) for use in any of the methods described herein,
whether in the context of use as a medicament and/or use for manufacture
of a medicament.
[0097] NT-4/5 Polypeptides
[0098] The trkB agonist used in the methods of the invention includes
NT-4/5 polypeptides. As used herein, "NT-4/5 polypeptide"
includes naturally-occurring mature protein (interchangeably termed
"NT-4/5") such as mature human NT-4/5 shown in Table 1
below, and in U.S. Pat. Appl. Pub. No. 2005/0209148 and PCT Pub.
No. WO 2005/08240 and FIG. 1 in U.S. Pat. Appl. Pub. No. 20030203383
and naturally occurring amino acid sequence variants of NT-4/5;
amino acid sequence variants of NT-4/5; peptide fragments of mature
NT-4/5 (such as human) and amino acid sequence variants; and modified
forms of mature NT-4/5 and said amino acid sequence variants and
peptide fragments wherein the polypeptide or peptide has been covalently
modified by substitution with a moiety other than a naturally occurring
amino acid, as long as the amino acid sequence variant, peptide
fragment, and the modified form thereof show one or more biological
activities of a trkB agonist and/or of naturally occurring mature
NT-4/5 protein. The trkB agonist also includes fusion proteins and
conjugates comprising any of the NT-4/5 polypeptide embodiments
described herein, e.g., an NT-4/5 polypeptide conjugated or fused
to a half life extending moiety, such as a PEG, IgG Fc region, albumin,
or a peptide. The amino acid sequence variants, peptide fragments
(including fragments of variants), or modified forms thereof under
consideration do not include NGF, BDNF, or NT-3 of any animal species.
Variants, peptide fragments, and modified forms of naturally occurring
NT-4/5 are described in U.S. Pat. Appl. Pub. Nos. 2003/0203383;
200210045576; 2005/0209148; U.S. Pat. Nos. 5,702,906; 6,506,728;
6,566,091; 5,830,858. NT-4/5 polypeptides include any one or more
embodiments described herein. For example, NT-4/5 polypeptide comprises
a naturally occurring sequence with one or more amino acid insertions,
deletions, or substitutions. TABLE-US-00001 TABLE 1 Amino acid sequence
of mature human NT-4/5 and the human nucleotide sequence encoding
the mature human NT-415 Amino acid sequence (SEQ ID NO:1): GVSETAPASRRGELAVCDAVSGWVTDRRTAVDLRGREVEVLGEVPAAGGS
PLRQYFFETRCKADNAEEGGPGAGGGGCRGVDRRHWVSECKAKQSYVRAL TADAQGRVGWRWIRIDTACVCTLLSRTGRA
Nucleotide sequence (SEQ ID NO:2) GGGGTGAGCGAAACTGCACCAGCGAGTCGTCGGGGTGAGCTGGCTGTGTG
CGATGCAGTCAGTGGCTGGGTGACAGACCGCCGGACCGCTGTGGACTTGC GTGGGCGCGAGGTGGAGGTGTTGGGCGAGGTGCCTGCAGCTGGCGGCAGT
CCCCTCCGCCAGTACTTCTTTGAAACCCGCTGCAAGGCTGATAACGCTGA GGAAGGTGGCCCGGGGGCAGGTGGAGGGGGCTGCCGGGGAGTGGACAGGA
GGCACTGGGTATCTGAGTGCAAGGCCAAGCAGTCCTATGTGCGGGCATTG ACCGCTGATGCCCAGGGCCGTGTGGGCTGGCGATGGATTCGAATTGACAC
TGCCTGCGTCTGCACACTCCTCAGCCGGACTGGCCGGGCCTGAG
[0099] In some embodiments, the NT-4/5 polypeptide is a mammalian
NT-4/5 polypeptide which may be a naturally occurring mammalian
NT-4/5, or NT-4/5 polypeptide derived from a naturally occurring
mammalian NT-4/5 and having a sequence that does not match any part
of a naturally occurring non-mammalian NT-4/5.
[0100] In some embodiments, the NT-4/5 polypeptide is a human NT-4/5
polypeptide which may be a naturally occurring human NT-4/5, or
NT-4/5 polypeptide derived from a naturally occurring human NT-4/5
and having a sequence that does not match any part of a naturally
occurring non-human NT-4/5.
[0101] NT-4/5 polypeptides, including variants, peptide fragments,
modified forms of NT-4/5 polypeptides (including naturally occurring
NT-4/5), fusion protein and conjugate of the invention are characterized
by any (one or more) of the following characteristics: (a) bind
to trkB receptor; (b) bind to trkB receptor and activate trkB biological
activity(ies) and/or one or more downstream pathways mediated by
trkB signaling function(s); (c) bind to trkB receptor and increase
body weight and/or food intake in a primate when administered peripherally;
(d) bind to trkB receptor and treat, prevent, reverse, or ameliorate
one or more symptoms of cachexia in a primate when administered
peripherally; (e) bind to trkB receptor and treat, prevent, reverse,
or ameliorate one or more symptoms of anorexia nervosa in a primate
when administered peripherally; (f) bind to trkB receptor and treat,
prevent, reverse, or ameliorate one or more symptoms of opioid-induced
emesis in a mammal when administered peripherally; (g) promote trkB
receptor dimerization and activation; and (h) increase trkB receptor-dependent
neuronal survival and/or neurite outgrowth. Thus all NT-4/5 polypeptides
(including variants, fragments, and modified forms) are functional
as described above.
[0102] Biological activity of variants may be tested in vitro and
in vivo using methods known in the art and methods described herein.
Methods described herein for identifying anti-trkB agonist may also
be used. NT-4/5 polypeptides may have an enhanced activity or reduced
activity as compared to a naturally occurring NT-4/5 protein. In
some embodiments, functionally equivalent variants have at least
about any of 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% of activity
as compared to the native NT-4/5 protein from which the NT-4/5 polypeptide
is derived with respect to one or more of the biological assays
described above (or known in the art). In some embodiments, functionally
equivalent variants have an EC.sub.50 (half of the maximal effective
concentration) of less than about any of 0.01 nM, 0.1 nM, 1 nM,
10 nM, or 100 nM in TrkB receptor activation in vitro (e.g., assays
described in Example 6, and in US 200510209148 and PCT Pub. No.
WO 2005/082401).
[0103] Amino acid sequence variants of NT-4/5 include polypeptides
having an amino acid sequence which differs from naturally occurring
NT-4/5 by virtue of the insertion, deletion, and/or substitution
of one or more amino acid residues within the sequence of naturally
occurring NT-4/5 (for example, mature human NT-4 shown in Table
1). Amino acid sequence variants generally will be at least about
65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical
to any naturally occurring NT-4/5 (such as mature human NT-4/5 shown
in SEQ ID NO:1). In some embodiments, the variant is at least about
70% identical to the amino acid sequence of SEQ ID NO:1. In some
embodiments, the variant is at least about 85% identical to the
amino acid sequence of SEQ ID NO:1. In some embodiments, the variant
is at least about 90% identical to the amino acid sequence of SEQ
ID NO:1, In some embodiments, the variant is at least about 95%
identical to the amino acid sequence of SEQ ID NO:1.
[0104] Amino acid sequence variants of NT-4/5 can be generated
by making predetermined mutations in a previously isolated NT-4/5
DNA. Amino acid variants may be designed and generated based on
crystal structure of NT-4/5 and TrkB receptor. Banfield et al.,
Structure 9: 1191-9 (2001) For example, amino acids that are not
directly involved in interaction between monomers of NT-4/5 and
between NT-4/5 and the TrkB receptor may be mutated, for example,
to introduce PEG attaching site. Methods known in the art may be
used to design variants of NT-4/5 polypeptide that have enhanced
or reduced one or more biological activities as compared to the
naturally occurring NT-4/5 protein.
[0105] There are two principal variables to consider in making
such predetermined mutations: the location of the mutation site
and the nature of the mutation. In general, the location and nature
of the mutation chosen generally depends upon the NT-4/5 characteristic
to be modified. For example, candidate NT-4/5 antagonists or super
agonists initially can be selected by locating amino acid residues
that are identical or highly conserved among NGF, BDNF, NT-3, and
NT-4. Those residues can then be modified in series, e.g., by (1)
substituting first with conservative choices and then with more
radical selections depending upon the results achieved, (2) deleting
the target residue, or (3) inserting residues of the same or different
class adjacent to the located site, or combinations of options 1-3.
[0106] One helpful technique is called "ala scanning".
Here, an amino acid residue or group of target residues are identified
and substituted by alanine or polyalanine. Those domains demonstrating
functional sensitivity to the alanine substitutions then are refined
by introducing further or other variants at or for the sites of
alanine substitution.
[0107] Obviously, such variations which, for example, convert NT-4/5
into NGF, BDNF, or NT-3 are not included within the scope of this
invention. Thus, while the site for introducing an amino acid sequence
variation is predetermined, the nature of the mutation per se need
not be predetermined. For example, to optimize the performance of
a mutation at a given site, ala scanning or random mutagenesis is
conducted at the target codon or region and the expressed NT-4/5
variants are screened for the optimal desired activity.
[0108] Amino acid sequence deletions generally range from about
1 to 30 residues, more preferably, about 1 to 10 residues, and typically
are contiguous. Deletions may be introduced into regions of low
homology among BDNF, NGF, NT-3, and NT-4/5 to modify the activity
of NT-4/5. Deletions from NT-4/5 in areas of substantial homology
with BDNF, NT-3, and NGF may be more likely to modify the biological
activity of NT-4/5 more significantly. The number of consecutive
deletions may be selected so as to preserve the tertiary structure
of NT-4/5 in the affected domain, e.g., beta-pleated sheet or alpha
helix.
[0109] Amino acid sequence insertions include amino- and/or carboxyl-terminal
fusions ranging in length from one residue to polypeptides containing
a thousand or more residues, as well as intrasequence insertions
of single or multiple amino acid residues. Intrasequence insertions
(i.e., insertions within the mature NT-4/5 sequence) may range generally
from about 1 to 10 residues, more preferably, 1 to 5, most preferably
1 to 3. An example of a terminal insertion includes fusion of a
heterologous N-terminal signal sequence to the N-terminus of the
NT-4/5 molecule to facilitate the secretion of mature NT-4/5 from
recombinant host. Such signals generally will be homologous to the
intended host cell and include STII or lpp for E. coli, alpha factor
for yeast, and viral signals such as herpes gD for mammalian cells.
Other insertions include the fusion of a polypeptide to the N- or
C-termini of NT-4/5.
[0110] Another group of variants includes those in which at least
one amino acid residue in NT-4/5, and preferably only one, has been
removed and a different residue inserted in its place. An example
is the replacement of arginine and lysine by other amino acids to
render the NT-4/5 resistant to proteolysis by serine proteases,
thereby creating a variant of NT-4/5 that is more stable. The sites
of greatest interest for substitutional mutagenesis include sites
where the amino acids found in BDNF, NGF, NT-3, and NT-4 are substantially
different in terms of side chain bulk, charge or hydrophobicity,
but where there also is a high degree of homology at the selected
site within various animal analogues of NGF, NT-3, and BDNF (e.g.
among all the animal NGFs, all the animal NT-3, and all the BDNFs).
This analysis will highlight residues that may be involved in the
differentiation of activity of the trophic factors, and therefore,
variants at these sites may affect such activities. Examples of
such sites in mature human NT-4/5, numbered from the N-terminal
end, and exemplary substitutions include G77 to K, H, Q or R and
R84 to E, F, P, Y or W of NT-4/5 of SEQ ID NO:1, respectively. Other
sites of interest are those in which the residues are identical
among all animal species BDNF, NGF, NT-3, and NT-4/5, this degree
of conformation suggesting importance in achieving biological activity
common to all four factors.
[0111] For example, substitution of one or more amino acids includes
conservative substitutions. Methods of making conservative substitutions
are known in the art. For example, ala (A) may be substituted by
val, leu, ile, preferably by val; arg (R) may be substituted by
lys, gin, asn, preferably by lys; asn (N) may be substituted by
gln, his, lys, arg, preferably by gin; asp (D)may be substituted
by glu; cys (C) may be substituted by ser; gin (Q) may be substituted
by asn; glu (E) may be substituted by asp; gly (G) may be substituted
by pro; his (H) may be substituted by asn, gin, lys, arg; preferably
by arg; ile (I) may be substituted by leu, val, met, ala, phe, norleucine,
preferably by leu; leu (L) may be substituted by norleucine, ile,
val, met, ala; phe, preferably by ile; lys (K) may be substituted
by arg; gin, asn, preferably by arg; met (M) may be substituted
by leu; phe; ile, preferably by leu; phe (F) may be substituted
by leu, val, ile, ala, preferably by leu; pro (P) may be substituted
by gly; ser (S) may be substituted by thr; thr (T) may be substituted
by ser; trp (W) may be substituted by tyr; tyr (Y) may be substituted
by trp, phe, thr, ser, preferably by phe; val (V) may be substituted
by ile; leu; met; phe, ala; norieucine, preferably by leu.
[0112] Sites particularly suited for conservative substitutions
include, numbered from the N-terminus of the mature human NT-4 (SEQ
ID NO:1), R11, G12, E13, V16, D18, W23, V24, D26, V40, L41, Q54,
Y55, F56, E58, T59, G77, R79, G80, H85, W86, A99, L100, T101, W110,
R111, W112, I113, R114, I115, D116, and A118. Cysteine residues
not involved in maintaining the proper conformation of NT-4/5 also
may be substituted, generally with serine, in order to improve the
oxidative stability of the molecule and prevent aberrant crosslinking.
Sites other than those set forth in this paragraph are suitable
for deletional or insertional studies generally described above.
[0113] Substantial modifications in function may be accomplished
by selecting substitutions that differ significantly in their effect
on maintaining (a) the structure of the polypeptide backbone in
the area of the substitution, for example, as a sheet or helical
conformation, (b) the charge or hydrophobicity of the molecule at
the target site, or (c) the bulk of the side chain. Naturally occurring
residues are divided into groups based on common side chain properties
(some of these may fall into several functional groups): [0114]
(1) hydrophobic: norieucine, met, ala, val, leu, ile; [0115] (2)
neutral hydrophilic: cys, ser, thr; [0116] (3) acidic: asp, glu;
[0117] (4) basic: asn, gln, his, lys, arg; [0118] (5) residues that
influence chain orientation: gly, pro; and [0119] (6) aromatic:
trp, tyr, phe.
[0120] Non-conservative substitutions will entail exchanging a
member of one of these classes for another.
[0121] Examples of NT-4 variants include: polypeptide of SEQ ID
NO:1 with mutation of E67 to S or T (this adds an N-linked glycosylation
site); polypeptide from amino acid residue R83 to Q94, GI to C61,
G1 to C17, C17 to C61, C17 to C78, C17 to C90, C17 to C119, C17
to C121, R11 to R27, R11 to R34, R34 to R53, C61 to C78, R53 to
C61, C61 to C 19, C61 to C78, C78 to C119, C61 to C90, R60 to C78,
K62 to C119, K62 to K91, R79 to R98, R83 to K93, T101 to R111, G1
to C121 of SEQ ID NO:1; polypeptide comprises V40-C121 of SEQ ID
NO:1, for example, V40-C121 of SEQ ID NO:1 fused to a polypeptide
at the N-terminal and/or C-terminal; polypeptide comprises SEQ ID
NO:1 with deletion of C78, C61, Q54-T59, R60-D82, H85-S88, W86-T101
(deletions of the indicated span of residues, inclusive); SEQ ID
NO:1 with mutation from R53 to H, from K91 to H, from V108 to F,
from R84 to Q, H, N, T, Y or W, and from D116 to E, N, Q, Y, S or
T. Also included is NT-4/5 (SEQ ID NO:1 ) wherein position 70 is
substituted with an amino acid residue other than G, E, D or P;
position 71 with other than A, P or M; and/or position 83 with other
than R, D, S or K; as well as cyclized NT-4 fragments.
[0122] Two polynucleotide or polypeptide sequences are said to
be "identical" if the sequence of nucleotides or amino
acids in the two sequences is the same when aligned for maximum
correspondence as described below. Comparisons between two sequences
are typically performed by comparing the sequences over a comparison
window to identify and compare local regions of sequence similarity.
A "comparison window" as used herein, refers to a segment
of at least about 20 contiguous positions, usually 30 to about 75,
40 to about 50, in which a sequence may be compared to a reference
sequence of the same number of contiguous positions after the two
sequences are optimally aligned.
[0123] Optimal alignment of sequences for comparison may be conducted
using the Megalign program in the Lasergene suite of bioinformatics
software (DNASTAR, Inc., Madison, Wis.), using default parameters.
This program embodies several alignment schemes described in the
following references: Dayhoff, M. O. (1978) A model of evolutionary
change in proteins--Matrices for detecting distant relationships.
In Dayhoff, M. O. (ed.) Atlas of Protein Sequence and Structure,
National Biomedical Research Foundation, Washington D.C. Vol. 5,
Suppl. 3, pp. 345-358; Hein J., 1990, Unified Approach to Alignment
and Phylogenes pp. 626-645 Methods in Enzymology vol. 183, Academic
Press, Inc., San Diego, Calif.; Higgins, D. G. and Sharp, P. M.,
1989, CABIOS 5:151-153; Myers, E. W. and Muller W., 1988, CABIOS
4:11-17; Robinson, E. D., 1971, Comb. Theor. 11:105; Santou, N.,
Nes, M., 1987, Mol. Biol. Evol. 4:406-425; Sneath, P. H. A. and
Sokal, R. R., 1973, Numerical Taxonomy the Principles and Practice
of Numerical Taxonomy, Freeman Press, San Francisco, Calif.; Wilbur,
W. J. and Lipman, D. J., 1983, Proc. Natl. Acad. Sci. USA 80:726-730.
[0124] Preferably, the "percentage of sequence identity"
is determined by comparing two optimally aligned sequences over
a window of comparison of at least 20 positions, wherein the portion
of the polynucleotide or polypeptide sequence in the comparison
window may comprise additions or deletions (i.e. gaps) of 20 percent
or less, usually 5 to 15 percent, or 10 to 12 percent, as compared
to the reference sequences (which does not comprise additions or
deletions) for optimal alignment of the two sequences. The percentage
is calculated by determining the number of positions at which the
identical nucleic acid bases or amino acid residue occurs in both
sequences to yield the number of matched positions, dividing the
number of matched positions by the total number of positions in
the reference sequence (i.e., the window size) and multiplying the
results by 100 to yield the percentage of sequence identity.
[0125] Amino acid sequence variants of NT-4/5 may be naturally
occurring or may be prepared synthetically, such as by introducing
appropriate nucleotide changes into a previously isolated NT-4/5
DNA, or by in vitro synthesis of the desired variant polypeptide.
As indicated above, such variants may comprise deletions from, or
insertions or substitutions of, one or more amino acid residues
within the amino acid sequence of mature NT-4/5 (e.g., sequence
shown in Table 1). Any combination of deletion, insertion, and substitution
is made to arrive at an amino acid sequence variant of NT-4/5, provided
that the resulting variant polypeptide possesses a desired characteristic.
The amino acid changes also may result in further modifications
of NT-4/5 upon expression in recombinant hosts, e.g., introducing
or moving sites of glycosylation, or introducing membrane anchor
sequences (see, e.g., PCT Pub. No. WO 89/01041).
[0126] In some embodiments, NT-4/5 polypeptide comprises an amino
acid sequence encoded by a nucleic acid that hybridizes under stringent
conditions to a nucleic acid sequence (e.g., SEQ ID NO:2) encoding
mature human NT-4/5.
[0127] Variants polynucleotides may also, or alternatively, be
substantially homologous to a native gene, or a portion or complement
thereof. Such polynucleotide variants are capable of hybridizing
under moderately stringent conditions to a naturally occurring DNA
sequence encoding a the polypeptide (or a complementary sequence).
[0128] Suitable "moderately stringent conditions" include
prewashing in a solution of 5.times.SSC, 0.5% SDS, 1.0 mM EDTA (pH
8.0); hybridizing at 50.degree. C.-65.degree. C., 5.times.SSC, overnight;
followed by washing twice at 65.degree. C. for 20 minutes with each
of 2.times., 0.5.times. and 0.2.times.SSC containing 0.1% SDS.
[0129] As used herein, "highly stringent conditions"
or "high stringency conditions" are those that: (1) employ
low ionic strength and high temperature for washing, for example
0.015 M sodium chloride/0.001 5 M sodium citrate/0.1% sodium dodecyl
sulfate at 50.degree. C.; (2) employ during hybridization a denaturing
agent, such as formamide, for example, 50% (v/v) formamide with
0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50
mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride,
75 mM sodium citrate at 42.degree. C.; or (3) employ 50% formamide,
5.times.SS (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate
(pH 6.8), 0.1% sodium pyrophosphate, 5.times. Denhardt's solution,
sonicated salmon sperm DNA (50 .mu.g/ml), 0.1% SDS, and 10% dextran
sulfate at 42.degree. C., with washes at 42.degree. C. in 0.2.times.SSC
(sodium chloride/sodium citrate) and 50% formamide at 55.degree.
C., followed by a high-stringency wash consisting of 0.1.times.SSC
containing EDTA at 55.degree. C. Another exemplary stringent condition
hybridization in 50% formamide, 5.times.SSC, 0.1% sodium dodecyl
sulfate, 0.1% sodium pyrophosphate, 50 mM sodium phosphate pH 6.8,
2.times. Denhardt's solution, and 10% dextran sulfate at 42.degree.
C., followed by a wash in 0,1.times.SSC and 0.1% SDS at 42.degree.
C. The skilled artisan will recognize how to adjust the temperature,
ionic strength, etc. as necessary to accommodate factors such as
probe length and the like.
[0130] It will be appreciated by those of ordinary skill in the
art that, as a result of the degeneracy of the genetic code, there
are many nucleotide sequences that encode a polypeptide as described
herein. Some of these polynucleotides bear minimal homology to the
nucleotide sequence of any native gene. Nonetheless, polynucleotides
that vary due to differences in codon usage are specifically contemplated
by the present invention. Further, alleles of the genes comprising
the polynucleotide sequences provided herein are within the scope
of the present invention. Alleles are endogenous genes that are
altered as a result of one or more mutations, such as deletions,
additions and/or substitutions of nucleotides. The resulting mRNA
and protein may, but need not, have an altered structure or function.
Alleles may be identified using standard techniques (such as hybridization,
amplification and/or database sequence comparison).
[0131] TrkB agonists used in the methods of the invention also
include fusion proteins comprising the amino acid sequence of NT-4/5
(e.g., human NT-4/5 shown in Table 1) or a functional peptide fragment
thereof.
[0132] Biologically active NT-4/5 polypeptides can be fused with
sequences, such as sequences that enhance immunological reactivity,
facilitate the coupling of the polypeptide to a support or a carrier,
or facilitate refolding and/or purification (e.g., sequences encoding
epitopes such as Myc, HA derived from influenza virus hemagglutinin,
His-6, FLAG). These sequences may be fused to NT-4/5 polypeptide
at the N-terminal end or at the C-terminal end. In addition the
protein or polynucleotide can be fused to other or polypeptides
which increase its function, or specify its localization in the
cell, such as a secretion sequence. Methods for producing recombinant
fusion proteins described above are known in the art. The recombinant
fusion protein can be produced, refolded and isolated by methods
well known in the art.
[0133] NT-4/5 polypeptides described herein may be modified to
increase their half lives in an individual. For example, NT-4/5
polypeptide may be pegylated to reduce systemic clearance with minimal
loss of biological activity. The invention also provides compositions
(including pharmaceutical compositions) comprising an NT-4/5 polypeptide
linked to a PEG molecule. In some embodiments, the PEG molecule
is linked to the NT-4/5 polypeptide through a reversible linkage.
The half life of a pegylated NT-4/5 polypeptide may be extended
by more than about any of 2-fold, 5-fold, 10-fold, 15-fold, 20-fold,
and 30-fold of the half life of the non-pegylated NT-4/5 polypeptide.
[0134] Polyethylene glycol polymers (PEG) may be linked to various
functional groups of the NT-4/5 polypeptide using methods known
in the art. See, e.g., Roberts et al., Advanced Drug Delivery Reviews
54:459-476 (2002); Sakaane et al. Pharm. Res. 14 1085-91 (1997).
PEG may be linked to the following functional groups on the polypeptide:
amino groups, carboxyl groups, modified or natural N-termini, amine
groups, and thiol groups. In some embodiments, one or more surface
amino acid residues are modified with PEG molecules. PEG molecules
may be of various sizes (e.g., ranging from about 2 to 40 KDa).
PEG molecules linked to NT-4/5 polypeptide may have a molecular
weight about any of 2000, 10,000, 15,000, 20,000, 25,000, 30,000,
35,000, 40,000 Da. PEG molecule may be a single or branched chain.
To link PEG to NT-4/5 polypeptide, a derivative of the PEG having
a functional group at one or both termini may be used. The functional
group is chosen based on the type of available reactive group on
NT-4/5 polypeptide. Methods of linking derivatives to polypeptides
are known in the art. Roberts et al., Advanced Drug Delivery Reviews
54:459-476 (2002). The linkage between the NT-4/5 polypeptide and
the PEG may also be such that it can be cleaved or naturally degrades
(reversible or degradable linkage) in an individual which may improve
the half-life but minimize loss of activity. PEG linking site on
NT-4/5 polypeptide may also be created by mutating surface residues
to an amino acid residue having a PEG reactive group, such as, a
cysteine. For example, the following amino acids of human NT-4/5
(SEQ ID NO:1) may be mutated for PEG attachment: G1, V2, S3, E4,
T5, S9, R10, T25, D26, R28, T29, V31, E37, E39, L41, E43, A46, A47,
G48, G49, S50, R53, D64, N65, A66, E67, E68, G69, D82, R83, R84,
H85, A104, Q105, G106, R107, V108, S125, and T127. These may be
applied to the corresponding residues in other species.
[0135] Several pegylated NT-4/5 have been generated and are shown
in Examples 6 and 7 of US Pat. Appl Pub. No. 2005/0209148 and PCT
Pub. No. WO 2005/082401. Serine residue at position 50 of the mature
human NT-4/5 may be changed to cysteine to generate NT-4-S50C which
is then pegylated, wherein the PEG is linked to the cysteine at
position 50. One example of an N-terminal specific attachment for
PEG is to mutate the residue at position I to a serine or threonine,
then followed with pegylation, wherein the PEG is linked to the
serine at position 1.
[0136] NT-4/5 polypeptide can be produced by recombinant means,
that is, by expression of nucleic acid encoding the NT-4/5 polypeptide.
In recombinant cell culture, and, optionally, purification of the
variant polypeptide from the cell culture, for example, by bioassay
of the variant's activity or by adsorption on an immunoaffinity
column comprising rabbit anti-NT-4/5 polyclonal antibodies (which
will bind to at least one immune epitope of the variant which is
also present in native NT-4/5). Small peptide fragments, on the
order of 40 residues or less, are conveniently made by in vitro
methods.
[0137] DNA encoding NT-4/5 polypeptide may be cloned into an expression
vector for expressing the protein in a host cell. Examples of nucleic
acids encoding NT-4/5 polypeptide are described in U.S. Pat. Appl.
Pub. No. 2003/0203383. The DNA encoding NT-4/5 polypeptide in its
mature form may be linked at its amino terminus to a secretion signal.
This secretion signal preferably is the NT-4/5 presequence that
normally directs the secretion of NT-4/5 from human cells in vivo.
However, suitable secretion signals also include signals from other
animal NT-4/5, signals from NGF, NT-2.sub.1 or NT-3, viral signals,
or signals from secreted polypeptides of the same or related species.
Any host cell (such as E. coli) may be used for expressing the protein
or polypeptide.
[0138] NT-4/5 polypeptide expressed may be purified. NT-4/5 polypeptide
may be recovered from the culture medium as a secreted protein,
although it also may be recovered from host cell lysates when directly
expressed without a secretory signal. Protein purification methods
known in the art may be used. Methods of producing NT-4/5 polypeptide
and purifying the expressed NT-4/5 polypeptide are described in
U.S. Pat. Appl. Pub. No. 2003/0203383, and U.S. Pat. No. 6,184,360.
NT-4/5 polypeptide can be expressed in E. coli and refolded according
to methods known in the art. Mature human NT-4/5 may also be obtained
commercially (for example, from R&D Systems, Minneapolis, Minn.,
Sigma, St. Louis, Mo., and Upstate Biotech., Temecula, Calif.).
[0139] Anti-trkB Agonist Polypeptides and Antibodies
[0140] The trkB agonist used in the methods of the invention also
includes anti-trkB agonist polypeptides, including anti-trkB agonist
antibodies. An anti-trkB agonist polypeptide (e.g., an antibody)
should exhibit any one or more of the following characteristics:
(a) bind to trkB receptor; (b) bind to trkB receptor and activate
trkB biological activity(ies) and/or one or more downstream pathways
mediated by trkB signaling function(s); (c) bind to trkB receptor
and increase body weight and/or food intake in a primate when administered
peripherally; (d) bind to trkB receptor and treat, prevent, reverse,
or ameliorate one or more symptoms of cachexia in a primate when
administered peripherally; (e) bind to trkB receptor and treat,
prevent, reverse, or ameliorate one or more symptoms of anorexia
nervosa in a primate when administered peripherally; (f) bind to
trkB receptor and treat, prevent, reverse, or ameliorate one or
more symptoms of opioid-induced emesis in a mammal when administered
peripherally; (g) promote trkB receptor dimerization and activation;
and (h) increase trkB receptor-dependent neuronal survival and/or
neurite outgrowth.
[0141] In some embodiments, the anti-trkB agonist polypeptide (e.g.,
antibody) is multivalent and binds to the extracellular domain of
a trkB receptor. It has been shown that immunoglobulins that are
able to bind and cross-link or dimerize the trk family of neurotrophin-receptors
activate these receptors and produce consequences in neurons that
are similar to exposure to a neurotrophin. See, U.S. Pat. No. 6,656,465;
and PCT Pub. No. WO 01/98361.
[0142] The trkB agonist antibodies can encompass monoclonal antibodies,
polyclonal antibodies, antibody fragments (e.g., Fab, Fab', F(ab').sub.2,
Fv, Fc, etc.), chimeric antibodies, single chain (ScFv), mutants
thereof, fusion proteins comprising an antibody portion, and any
other modified configuration of the immunoglobulin molecule that
comprises an antigen recognition site of the required specificity.
The antibodies may be murine, rat, human, or any other origin (including
humanized antibodies).
[0143] In some embodiments, the polypeptide (including the antibody)
binds trkB and does not significantly cross-react (bind) with other
neurotrophin receptors (such as the related neurotrophin receptors,
trkA and/or |