Note: Descriptions are shown in the official language in which they were submitted.
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MODIFIED CILIARY NEUROTROPHIC FACTOR, METHOD OF
MAKING AND METHODS OF USE THEREOF
This application is a continuation-in-part of U.S. Serial No.
1 0 09/373,834, filed on August 13,1999, which is a continuation-in-
part of PCT Application No. PCT/US 99/04430, filed February 26,
1999, which is a continuation-in-part of U.S. Serial .No. 09/031,693
filed February 27, 1998. Throughout this application, various
patents and publications are referenced. Those patents and
15 publications are hereby incorporated by reference in their entireties,
into this application.
BACKGROUND OF THE INVENTION
20 The present invention relates to therapeutic CNTF-related
polypeptides useful for the treatment of neurological or other
diseases or disorders.
Ciiiary neurotrophic factor (CNTF) is a protein that is required
for the survival of embryonic chick ciliary ganglion neurons in vitro
2 5 (Manthorpe et a1.,1980, J. Neurochem. 34:69-75). The ciliary
ganglion is anatomically located within the orbital cavity, lying
between the lateral rectus and the sheath of the optic nerve; it
receives parasympathetic nerve fibers from the oculomotor nerve
which innervates the ciliary muscle and sphincter pupil(ae.
3 0 Over the past decade, a number of biological effects have been
ascribed to CNTF in addition to its ability to support the survival of
ciliary ganglion neurons. CNTF is believed to induce the
differentiation of bipotential glial progenitor cells in the perinatal
rat optic nerve and brain (Hughes et al., 1988, Nature 335:70-73).
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Furthermore, it has been observed to promote the survival of
embryonic chick dorsal root ganglion sensory neurons (Skaper and
Varon, 1986, Brain Res. 389:39-46). In addition, CNTF supports the
survival and differentiation of motor neurons, hippocampal neurons
and presympathetic spinal cord neurons (Sendtner, et al., 1990,
Nature 345: 440-441; Ip; et al. 1991, J. Neurosci. 1 1 :3124-3134;
Blottner, et al. 1989, Neurosci. Lett. 105:316-320].
It has long been known that innervation of skeletal muscle
plays a critical role in the maintenance of muscle structure and
1 0 function. Skeletal muscle has been shown recently to be a target of
positive CNTF actions. Specifically, CNTF prevents both the
denervation-induced atrophy (decreased wet weight and myofiber
cross sectional area) of skeletal muscle and the reduced twitch and
tetanic tensions of denervated skeletal muscle. Helgren et al., 1994,
1 5 Cell 76:493-504. In this model, human CNTF also produces an
adverse effect that is manifested as a retardation of weight gain.
This adverse effect has also been observed in clinical trials with
rHCNTF for the treatment of ALS. Therefore, simultaneous
measurements of muscle weight and animal body weight following
20 denervation could be used as a measure of efficacy and adverse
reaction, respectively, in response to treatment with rHCNTF or
other compounds. The ratio of the potency values obtained from
these measurements is defined as the therapeutic index (T.1.),
expressed here as TD25/ED5o, so that the higher the value of T.I., the
2 5 safer the compound at a therapeutic dose.
CNTF has been cloned and synthesized in bacterial expression
systems, as described by Masiakowski, et al., 1991, J. Neurosci.
' 57:1003-1012 and in International Publication No. WO 91/04316,
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published on April 4, 1991, which are incorporated by reference in
their entirety herein.
The receptor for CNTF (termed "CNTFRa") has been cloned,
sequenced and expressed [see Davis, et al., 1991 Science 253:59-63].
CNTF and the hemopoietic factor known as leukemia inhibitory factor
(LIF) act on neuronal cells via a shared signaling pathway that
involves the IL-6 signal transducing component gp130 as well as a
second, ~-component (know as LIFR a); accordingly, the CNTF/CNTF
receptor complex can initiate signal transduction in LIF responsive
1 0 cells, or other cells which carry the gp130 and LIFR~i components [1p,
et a1.,1992, Cell 69:1 121-1 132J.
In addition to human CNTF, the corresponding rat (Stockli et
al., 1989, Nature 342:920-923), and rabbit (Lin et al., 1989, J. Biol.
Chem. 265:8942-8947) genes have been cloned and found to encode a
protein of 200 amino acids, which share about 80% sequence identity
with the human gene. Both the human and rat recombinant proteins
have been expressed at exceptionally high levels (up to 70% of total
protein) and purified to near homogeneity.
Despite their structural and functional similarity, recombinant
2 0 human and rat CNTF differ in several respects. The biological
activity of recombinant rat CNTF in supporting survival and neurite
outgrowth from embryonic chick ciliary neurons in culture is four
times better than that of recombinant human CNTF [Masiakowski et
al., 1991, J. Neurochem. 57:1003-1012]. Further, rat CNTF has a
2 5 higher affinity for the human CNTF receptor than does human CNTF.
A surprising difference in the physical properties of human and
rat CNTF, which are identical in size, is their different mobility on
SDS gels. This difference in behavior suggests the presence of an
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unusual structural feature in one of the two molecules that persists
even in the denatured state (Masiakowski et al., 1991, J. Neurochem.
57:1003-1012).
Mutagenesis by genetic engineering has been used extensively
in order to elucidate the structural organization of functional
domains of recombinant proteins. Several different approaches have
been described in the literature for carrying out deletion or
substitution mutagenesis. The most successful appear to be alanine
scanning mutagenesis [Cunningham and Wells 1989, Science 244:
1081-1085] and homolog-scanning mutagenesis [Cunningham et al.,
1989, Science 243:1330-1336]. These approaches helped identify
the receptor binding domains of growth hormone and create hybrid
proteins with altered binding properties to their cognate receptors.
To better understand the physical, biochemical and
pharmacological properties of rHCNTF, applicant undertook rational
mutagenesis of the human and rat CNTF genes based on the different
biological and physical properties of their corresponding
recombinant proteins (See Masiakowski, P., et al., 1991, J.
Neurochem., 57:1003-1012). Applicant has found that the nature of
the amino acid at position 63 could greatly enhance the affinity of
human CNTF for sCNTFRa and its biological potency in vitro
(Panayotatos, N., et al. , J. Biol. Chem., 1993, 268:19000-19003 ;
Panayotatos; N., et al., Biochemistry, 1994, 33: 5813-5818.
As described in copending U.S. Patent application Serial No.
07/570,651 filed August 20, 1990, entitled "Ciliary Neurotrophic
Factor" and International Publication Number WO 91/04316
published 4 April 1991 which are incorporated by reference in their
entireties herein, one of the uses of CNTF contemplated by
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applicants was the use of CNTF for the treatment of Huntington's
disease (Huntington's chorea). Huntington's disease (HD) is an
hereditary degenerative disorder of the central nervous system. The
pathology underlying HD is progressive, relentless degeneration of
the basal ganglia, structures deep inside the brain which are
responsible for aspects of the integration of voluntary motor and
cognitive activity. The onset of symptoms in HD is generally in
adulthood, between the ages of 20 and 40. The characteristic
manifestations of the disease are chorea and other involuntary
1 0 ' movements, dementia, and psychiatric symptoms. Choreic
movements consist of brief, involuntary, fluid movements,
predominantly affecting the distal extremities. Patients often tend
to "cover up" these movements by blending them into voluntary acts.
HD patients also, however, display a variety of other neurological
abnormalities including dystonia (sustained, abnormal posturing),
tics ("habit spasms"), ataxia (incoordination) and dysarthria (slurred
speech). The dementia of HD is characterized as the prototypical
"subcortical" dementia. Manifestations of dementia in HD include
slowness of mentation and difficulty in concentration and in
sequencing tasks. Behavioral disturbances in HD patients are
varied, and can include personality changes such as apathy and
withdrawal; agitation, impulsiveness, paranoia, depression,
aggressive behavior, delusions, psychosis, etc. The relentless
motor, cognitive and behavioral decline results in social and
functional incapacity and, ultimately death.
HD is inherited as an autosomal dominant trait. Its prevalence
in the U.S. population is estimated to be 5 to 10 per 100,000
individuals, yielding a total prevalence of 25,000 in the US
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population. However, due to the late onset of symptoms, there are a
number of "at-risk", asymptomatic individuals in the population as
well. The prevalence of asymptomatic, at-risk patients carrying the
HD gene is perhaps twice that of the symptomatic patients (W.
Koroshetz and N. Wexler, personal communication). Thus, the total
HD patient population eligible to receive a new therapy is about
75, 000.
The gene currently believed to be responsible for the
pathogenesis of HD is located at the telomeric end of the short arm
1 0 of Chromosome 4. This gene codes for a structurally novel protein of
unknown function, and the relationship of the gene product to the
pathogenesis of HD remains uncertain at the present time.
The principal anatomical lesion in HD consists of loss of the
so-called "medium spiny" neurons of the caudate nucleus and
putamen (collectively known as the striatum in rodents). These
neurons comprise the projection system whereby the
caudate/putamen projects to its output nuclei elsewhere in the
basal ganglia of the brain. The principal neurotransmitter utilized
by the medium spiny neurons is gamma-aminobutyric acid (GABA),
2 0 although many also contain neuropeptides such as enkephalins and
substance P. It is clear, however, that in HD interneurons which do
not utilize GABA as their neurotransmitter, containing instead
either acetylcholine or the neuropeptides somatostatin or
neuropeptide Y, are relatively undamaged in HD.
Pathological and neurochemical changes which mimic those
seen in HD can be mimicked by infusion of glutamatergic agonist
drugs into the striatum. Infusion of quinolinic acid under
appropriate conditions produces selective depletion of medium sized
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intrinsic striatal neurons which utilize gamma-aminobutyric acid
(GABA) as their neurotransmitter, without affecting the large,
cholinergic interneurons.
There have been no successful clinical trials of either
symptomatic or neuroprotective treatments in HD. However, useful,
validated rating instruments and neuroimaging techniques exist
which are capable of monitoring disease progress and patient
function.
The CNTF receptor complex contains 3 proteins: a specificity
1 0 determining a component that directly binds to CNTF, as well as 2
signal transducing ~i components (LIFR (3 and gp130) that cannot bind
CNTF on their own, but are required to initiate signaling in response
to CNTF. The (3 component of the CNTFR complex is more widely
distributed throughout the body than the a component. The 3
1 5 components of the CNTFR complex are normally unassociated on the
cell surface; CNTF induces the stepwise assembly of a complete
receptor complex by first binding to CNTFR a, then engaging gp130,
and finally recruiting LIFR Vii. When this final step in receptor
assembly occurs (heterodimerization of the ~3 components),
20 intracellular signaling is initiated by activating non-receptor
tyrosine kinases (JAK kinases) associated with the ~ components.
JAK kinases respond by phosphorylating each other and also tyrosine
residues on the receptor cytoplasmic domains, creating
phosphotyrosine docking sites for the Src homology 2 domains of
25 STAT proteins. After their phosphorylation, bound STAT proteins
dissociate from the receptor, dimerize, and translocate to the
nucleus where they bind DNA and activate transcription . (reviews:
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Frank, D. and Greenberg, M. (1996) Perspectives on developmental
neurobiology 4: 3-18; Stahl, N. and Yancopoulos, G. (1997) Growth
factors and cytokines in health and disease 2B, 777-809). Axokine
is a mutant CNTF molecule with improved physical and chemical
properties, which retains the ability to interact with and activate
the CNTF receptor. (Panayotatos, N., et al. (1993) J. Biol. Chem. 268:
19000-19003).
Leptin, the product of the ob gene, is secreted by adipocytes
and functions as a peripheral signal to the brain to regulate food
1 0 intake and energy metabolism (Zhang, Y., et al. (1994) Nature 372:
425-431 ). Interestingly, leptin receptor (OB-R), a single membrane-
spanning receptor has considerable sequence similarities to gp130
(Tartaglia, L., et al. (1995) Cell 83: 1263-1271 ), and like CNTF,
leptin signals through the JAK/STAT pathway (Baumann, H., et al.
1 5 (1996) Proc. Natl. Acad. Sci. USA 93: 8374-8378; Ghilardi, N., et al.
(1996) Proc. Natl. Acad. Sci. USA 93: 6231-6235). Systemic
administration of both CNTF and leptin resulted in induction of tis-
11 (Gloaguen, I., et al. (1997) Proc. Natl. Acad. Sci. USA 94: 6456-
6461 ) and STAT3 (Vaisse, C., et al. (1996) Nature Gen. 14: 95-97) in
20 the hypothalamic satiety center, indicating their roles in the
regulation of body weight and feeding behavior. Indeed,
adminstration of CNTF to humans reduced food intake and resulted in
weight loss (Group, A. C. T. S. (1996) Neurology 46:1244-1249.).
25 SUMMARY OF THE INVENTION
An object of the present invention is to provide novel CNTF-
related neurotrophic factors for the treatment of diseases or
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disorders including, but not limited to, diabetes and obesity. In a
preferred embodiment, CNTF and related molecules are utilized for
the treatment of non-insulin dependent diabetes mellitus.
A further object of the present invention is to provide a
method for identifying CNTF-related factors, other than those
specifically described herein, that have improved therapeutic
properties.
These and other objects are achieved in accordance with the
invention, whereby amino acid , substitutions in human CNTF protein
enhance its therapeutic properties. In one embodiment, alterations
in electrophoretic mobility are used to initially screen potentially
useful modified CNTF proteins.
In a preferred embodiment, the amino acid glutamine in
position 63 of human CNTF is replaced with arginine (referred to as
1 5 63Q~ R) or another amino acid which results in a modified CNTF
molecule with improved biological activity. In further embodiments,
rHCNTF variants combine the 63Q-j R mutation with three other novel
features:
1 ) Deletion of the last 13 amino acid residues (referred to as
0C13) to confer greater solubility to rHCNTF without impairing its
activity;
2 ) Substitution of the unique cysteine residue at position 17,
which results in stabilization of rHCNTF in physiological buffer, at
physiological pH and temperature conditions without affecting its
activity; or
3 ) Substitution of amino acid residue 64W, which alters the
biological activity of rHCNTF in vitro and which results in a 7-fold
improvement of its therapeutic index in vivo.
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In another preferred embodiment, a molecule designated RG297
(rHCNTF,17CA63QRoC13) combines a 63Q~R substitution (which
confers greater biological potency) with a deletion of the terminal
13 amino acid residues (which confers greater solubility under
physiological conditions) and a 17CA substitution (which confers
stability, particularly under physiological conditions at 37°C) and
shows a 2-3 fold better therapeutic index than rHCNTF in an animal
model.
In another preferred embodiment, a molecule designated RG242
is described that carries the double substitution 63QR64WA which
results in a different spectrum of biological potency and a 7-fold
higher therapeutic index.
In another preferred embodiment, a molecule designated RG290
is described that carries the double substitution 63QRoC13 which
confers greater solubility under physiological conditions.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 - Alignment of CNTF protein sequences. A. Human, rat,
2 0 rabbit mouse and chicken (Leung, _et al., 1992, Neuron 8:1045-1053)
sequences. Dots indicate residues found in the human sequence.
Panel B. Modified CNTF molecules showing human CNTF amino acid
residues (dots) and rat CNTF (residues shown). The name of the
purified recombinant protein corresponding to each sequence is
2 5 shown on the left.
Figure 2 - Mobility of human, rat and several modified CNTF
molecules on reducing SDS-15% polyacrylamide gels. Purified
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recombinant proteins were loaded as indicated. Markers of the
indicated MW were loaded on lane M.
Figure 3 - Biological activity of two modified CNTF molecules. A.
human CNTF (filled diamonds), rat CNTF (open squares), and RPN219
(filled squares). B. human CNTF (filled diamonds), rat CNTF (open
squares), and RPN228 (filled squares). Dose response of dissociated
E8 chick ciliary neurons surviving at the indicated protein
concentration, as a percentage of the number of neurons surviving in
1 0 the presence of 2 ng/ml rat CNTF. Each experimental point
represents the mean of three determinations.
Figure 4 - Competitive ligand binding towards A.) SCG neurons and
B.) MG87/huCNTFR fibroblasts. Standard deviation from the mean of
three determinations is shown by vertical bars.
Figure 5 - Mobility of human and several modified CNTF molecules on
SDS-15% polyacrylamide gels. Supernatant (A) and pellet (B)
preparations of recombinant human CNTF (designated HCNTF) and
2 0 several modified CNTF proteins were loaded as indicated. The
modified proteins shown are OC13 (also known as RG160);
17CA,oCl3 (RG162); OC13,63QR (RG290); and 17CA,oC13,63QR (RG
297). Markers of the indicated MW were loaded on lane M. Incubation
in physiological buffer at 37°C for 0, 2, 7 and 14 days is indicated in
2 5 lanes 1-4, respectively.
Figure 6 - Survival of primary dissociated E8 chick ciliary neurons
in response to increasing concentrations of various CNTF variants.
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Control concentration response curves for rat CNTF and rHCNTF
obtained with standard, untreated stock solutions, as well as with
four rHCNTF variants, RG297, RG290, RG160 and RG162.
Figure 7 - Survival of primary dissociated E8 chick ciliary neurons
in response to increasing concentrations of various CNTF variants.
Control concentration response curves for rat CNTF and rHCNTF
obtained with standard, untreated stock solutions, as well as with
rHCNTF variant RG228 (als'o known as RPN228 and having the
mutation 63QR).
Figure 8 - Survival of primary dissociated E8 chick ciliary neurons
in response to increasing concentrations of various CNTF variants.
Control concentration response curves for rat CNTF and rHCNTF
obtained with standard, untreated stock solutions, as well as with
rHCNTF variant RG242 (which has the mutation 63QR,64WA).
Figure 9 - Average plasma concentration time profiles in the rat
after intravenous (IV) administration of rHCNTF, RG228 and RG242
normalized to 100 ~,g/kg dose for all three compounds.
Figure 10 - Average plasma concentration time profiles in the rat
after subcutaneous (SC) administration of rHCNTF, RG228 and RG242
normalized to 200 wg/kg dose for all three compounds.
Figure 11 - Comparison of dose dependent rescue of rat muscle wet
weight of (A) hCNTF vs. RG228; (B) hCNTF vs. RG297 and (C) hCNTF vs.
RG242.
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Figure 12 - Comparison of in vivo toxicity for hCNTF, RG228, RG242
and RG297.
Figure 13 - Representative Nissl-stained sections (coronal plane)
from brains treated with neurotrophins and injected with quinolinic
acid. Top left: A view of an intact caudate-putamen (CPu). Adjacent
panels: Comparable views of sections from brains treated with NGF,
BDNF or NT-3 and injected with quinolinic acid. In the neurotrophin-
treated brains, a circumscribed area (indicated by open arrows) is
virtually devoid of medium-sized neurons. The two tracks in the CPu
were left by the infusion cannula (c) and the quinolinic acid
injection needle (arrowhead). ec, external capsule; LV, lateral
ventricle. Scale bar = 0.5 mm.
Figure 14 - Representative Nissl-stained sections (coronal plane)
from brains treated with CNTF or PBS and injected with quinolinic
acid. Top left: A view of an untreated, intact caudate-putamen
(CPu). Top right: A higher magnification view of the lateral CPu
showing numerous medium-sized neurons, a few of which are
indicated by arrows. Middle and bottom left: The left CPu in brains
treated with PBS or CNTF and injected with quinolinic acid. The two
tracks in the CPu were left by the PBS or CNTF infusion cannula (c)
and the quinolinic acid injection needle (arrowhead); open arrows
2 5 indicate the medial boundary of the lesion. Middle and bottom right:
Higher magnification views 250 ~,m lateral to the cannula
illustrating the virtually complete absence of medium-sized striatal
neurons in the PBS-treated brain (neuron loss score = 4), and the
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presence of numerous, normal-appearing neurons in the CNTF-treated
brain (some of the surviving neurons are indicated by arrows;
neurons loss score = 2). ec, external capsule; LV, lateral ventricle.
Left scale bar = 0.5 mm; right scale bar = 30 Vim.
Figure 15 - Effect of treatment with neurotrophic factors on
medium-sized striatal neuron loss induced by intrastriatal injection
of quinolinic acid (QA). A, B, C, D, E. Mean neuron loss scores (~SEM)
for groups treated with neurotrophic factor or PBS and injected with
quinolinic acid. The number of rats in each trophic factor-treated
group is as follows: NGF=5; BDNF=12; NT-3=10; CNTF=3; Ax1=7;
equivalent numbers were used in the PBS-treated control groups in
each experiment. Statistical comparisons were by unpaired t-test.
NT-3 treatment resulted in a significantly greater (+) mean neuron
loss score compared with the PBS-treated group: t(17)=2.75, p=0.01.
CNTF or Ax1 treatment resulted in significantly lower (*) mean
neuron loss scores compared with PBS-treated groups: t(5)=2.7,
p=0.04 and t(13)=4.2, p=0.001, respectively.
2 0 Figure 16 - Effect of treatment with Ax1 on medium-sized striatal
neuron loss induced by intrastriatal injection of quinolinic acid (QA).
Above each graph, a time line indicates the experimental scheme. A.
Mean neuron loss score (~SEM) for groups treated with Ax1 (n=6) or
PBS (n=5) in an experimental paradigm similar to that described in
2 5 figure 1 legend, except the osmotic pump was implanted for only 4
days and the injection of quinolinic acid was given 3 days after
removal of the pump. B. Mean neuron loss score (~SEM) for groups
receiving a daily intrastriatal injection of Ax1 (n=6) or PBS .(n=6)
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for 3 days before and 1 day after an injection of quinolinic acid.
*unpaired t-test, A: t(9)=2.5, p=0.03; B: t(10)=2.3, p=0.04.
Figure 17 - Effects of Axokine-15 (Ax-15) in normal mice. Normal
C57BL/6J mice were injected subcutaneously daily for 6 days with
either vehicle or Ax-15 at 0.1 mg/kg, 0.3 mg.kg, or 1.0 mg/kg.
Percent change in body weight in Ax-15-treated versus vehicle-
s treated controls is shown.
Figure 18 - Effects of Ax-15 in oblob mice. C57BL/6J oblob mice
were injected subcutaneously daily for 7 days with either vehicle,
leptin (1.0 mg/kg) or Ax-15 at 0.1 mg/kg, 0.3 mg.kg, or 1.0 mg/kg.
Diet-restricted, pair-fed mice were injected with 0.3 mg/kg Ax-15
to investigate the effects of food intake reduction on weight loss.
Percent change in body weight in Ax-15-treated and leptin-treated
versus vehicle-treated controls is shown.
Figure 19 - Effects of Ax-15 in diet-induced obesity in mice. AKR/J
mice were placed on a high fat diet for seven weeks prior to
treatment with vehicle, leptin (1.0 mg/kg) or Ax-15 at 0.03 mg/kg,
0.1 mg/kg, 0.3 mg/kg, or 1.0 mg/kg. Diet-restricted, pair-fed AKR/J
mice were injected with 0.3 mg/kg Ax-15 to investigate the effects
2 0 of food-intake reduction on weight loss. Percent change in body
weight in Ax-15-treated and leptin-treated versus vehicle-treated
controls is shown.
Figures 20A and 20B - Effects of Ax-15 and diet restriction on
serum insulin and corticosterone levels in diet-induced obese AKR/J
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mice. Figure 20A- Serum insulin levels were measured in ARK/J
diet-induced obese mice following treatment with vehicle, diet
restriction and Ax-15 (0.1 mg/kg) or Ax-15 only (0.1 mg/kg) to
determine the effects of diet and/or Ax-15 treatment on obesity-
associated hyperinsulinemia. Figure 20B- Serum corticosterone
levels were measured in ARK/J diet-induced obese mice following
treatment with vehicle, diet restriction and Ax-15 (0.1 mg/kg) or
Ax-15 only (0.1 mg/kg) to determine the effects of diet and/or Ax-
treatment on obesity-associated hyperinsulinemia.
Figure 21 - 1-20-PEG Ax-15 (mono-20K-PEG-Ax-15) is 4-fold more
effective than non-pegylated Ax-15 in causing weight loss in mice
with diet induced obesity. DIO mice were given weekly
subcutaneous injections (*) of either PBS, Ax-15 (0.7 mg/kg), or 1-
1 5 20-PEG Ax-15 (0.23 and 0.7 mg/kg) for 13 days. The animals were
weighed daily and mean body weight change was expressed as
percent change from baseline +/- SEM (n=6 per group).
Figure 22 - 1-20-PEG Ax-15 decreased food intake more effectively
than non-Pegylated Ax-15 in mice with diet induced obesity. DIU
mice were given daily subcutaneous injections of either PBS, non-
pegylated Ax-15 (0.7 mg/kg), or 1-20-PEG Ax-15 (0.23 and 0.7
mg/kg) for 13 days. Food intake was recorded every 24 hours and
results expressed as mean gram weight of pellets consumed +/- SEM
2 5 (n=6 per group).
Figure 23A-23D - Figure 23A - Treatment of dbldb animals with
daily Ax-15 causes a significantly greater weight loss than does
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caloric restriction. dbldb mice or their heterozygous litter mates
(dbl?) were given daily injections (s.c.) of either Ax-15 (0.1 or 0.3
mg/kg) or vehicle for 10 days. Food intake was restricted for a
cohort of vehicle treated animals (Pair-fed) to the same amount
ingested by the highest Ax-15-treated group. The mean group
bodyweight +/- SEM (n=12) is reported for each day. Figure 23B -
The effect of 10 day Ax-15 treatment on glucose tolerance in dbldb
animals. An oral glucose tolerance test (OGTT) was performed on
vehicle (open square), pairfed-vehicle treated (filled diamond), and
Ax-15 treated (0.1 mg/kg/day, open triangle; 0.3 mg/kg/day, filled
triangle) dbldb male mice and age-matched heterozygous dbl? mice
(filled circle). Each point represents the mean of at least twelve
animals ~ SEM. Figure 23C - Treatment of dbldb animals with daily
low doses of Ax-15 causes a significant body weight loss. dbldb
mice were given daily injections (s.c.) of either Ax-15 (0.0125,
0.025 or 0.05 mg/kg) or vehicle for 10 days. The mean group
bodyweight +/- SEM (n=6) is reported for each day. Figure 23D - The
effect of 10 day low dose Ax-15 treatment on glucose tolerance in
dbldb animals. An oral glucose tolerance test (OGTT) was performed
2 0 on vehicle (open square) and Ax-15 treated (0.0125, 0.025 or 0.05
mg/kg) dbldb male mice. Each point represents the mean of at least
six animals ~ SEM.
Figure 24 Time course of effects of Ax-15 treatment (0.3
mg/kg/day; filled triangle) compared to vehicle treated (open
square), pairfed-vehicle treated (filled diamond) on non-fasting
serum blood glucose from dbldb male mice. Each point represents
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the mean of at least six animals ~ SEM 14 hour after the last
injection.
Figure 25A-25C - Physiological consequences of 10-day Ax-15
treatment in dbldb animals. Figure 25A: Fasting blood glucose
concentrations were determined with serum from dbldb male mice
treated for 10 days with Ax-15 (0.1 mg/kg/day and 0.3 mg/kg/day,
hatched bars) as compared to control groups, vehicle treated (open
bar), pairfed-vehicle treated (hatched bar) and age-matched
1 0 heterozygous db/? mice (stipled). Each bar represents the mean of
at least eight animals ~ SEM. Figure 25B Fasting insulin
concentrations were determined on serum from dbldb male mice
treated for 10 days with Ax-15 (0.1 mg/kg/day and 0.3 mg/kg/day,
hatched bars) as compared to control groups, vehicle treated (open
bar), pairfed vehicle-treated (hatched bar) and age-matched
heterozygous db/? mice (stipled). Each bar represents the mean of
at least eight animals ~ SEM. Figure 25C: Fasting free fatty acid
levels were determined on serum samples from dbldb male mice
treated for 10 days with Ax-15 (0.1 mg/kg/day and 0.3 mg/kg/day,
hatched bars) in comparison to control groups, vehicle treated (open
bar), pairfed-vehicle treated (hatched bar) and age-matched
heterozygous db/? mice (stipled). Each bar represents the mean of
at least eight animals ~ SEM. Insulin tolerance test data indicate an
improved insulin sensitivity profile from the severely impaired
2 5 vehicle treated control dbldb animals.
Figure 26A-26H - The effects of Ax-15 treatment on insulin-
stimulated phosphotyrosine immunoreactivity in the arcuate nucleus
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of dbldb mice. Immunostaining of heterozygous (dbl?) mice showed
an increase in phosphotyrosine immunoreactive staining neurons of
the arcuate nucleus (Figure 26B) following a 30 minute bolus of
insulin (1 IU v.ia the jugular vein) as compared to vehicle injected
control level (Figure 26A). Analysis of the insulin
resistant/diabetic dbldb mice (vehicle treated for 10 days) revealed
a constitutively high phosphotyrosine immunoreactive staining
pattern (Figure 26C) with no detectable change after insulin
treatment (Figure 26D). Ten day Ax-15 treatment of dbldb mice
attenuated the high basal phosphotyrosine immunoreactivity (Figure
26E and 26G) and restored insulin phosphotyrosine reponsiveness
(Figure 26F and 26H).
Figure 27A-27B - The effects of Ax-15 treatment on insulin-
1 5 stimulated signaling in the liver of dbldb mice. Male dbldb mice
were treated for 10 days with either vehicle (lanes 7 & 8), pairfed
to drug treatment levels (lanes 1 & 2) or treated with Ax-15 (0.1
mg/kg/day, lanes 5 & 6; 0.3 mg/kg/day, lanes 4 & 5). On the 11th
day animals were anaesthetized injected with either saline (-) or 1
IU of regular insulin (+) via the portal vein. The liver- was removed
and protein extracts were immunoprecipitated with an anti-
phosphotyrosine-specific antibody followed by standard Western
blot analysis with an antiserum to the p85 regulatory subunit of PI
3-kinase (Figure 27A), IRS-1-specific antisera followed by Western
blot analysis ~Nith an anti-phosphotyrosine-specific antibody (Figure
27B, upper panel), and an IRS-1-specific antiserum (Figure 27B,
bottom panel). Non-immune control immunoprecipitation (N1), no
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lysate control (NL), and 3T3-L1 lysate control for p85 (C) were run
as immunprecipitation and blotting controls.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to a method of treating
neurological or endocrine diseases and disorders in humans or
animals. It is based, in part, on the initial finding that recombinant
rat CNTF binds more efficiently to the human CNTF receptor than
1 0 does recombinant human CNTF and the subsequent discovery that
amino acid . substitutions which cause human CNTF to more closely
resemble rat CNTF result in enhanced binding of the modified CNTF
to the human CNTF receptor and concomitant enhanced biological
activity.
In a preferred embodiment, alteration of a single amino acid of
the human CNTF protein results in a significant enhancement of the
ability of the protein to promote the survival and outgrowth of
ciliary ganglion, as well as other neurons.
Recombinant human and rat CNTF have the same number of
2 0 amino acids (199) and similar mass (MW 22,798 and 22,721
respectively, after removal of the N-terminal methionine). Yet, on
reducing SDS-PAGE gels, recombinant human CNTF migrates as a
protein of MW=27,500, whereas rat CNTF migrates with the expected
mobility. In addition, human CNTF has four times lower biological
activity towards chick ciliary ganglion (CG) neurons than rat CNTF
and the human protein competes for binding to the human or the rat
receptor on cell surfaces much less effectively than rat CNTF.
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The above observation led to a directed effort to identify the
region on the CNTF molecule responsible for these differences. This
method involved the exchange, by genetic engineering methods, of
parts of the human CNTF sequence with the corresponding rat CNTF
sequence and vice versa. To achieve this, advantage was taken of
restriction sites that are common to the two CNTF genes and unique
in their corresponding expression vectors. When necessary, such
sites were engineered in one or the other of the two genes in areas
that encode the same protein sequence. With this approach,
expression vectors were obtained for each of the modified proteins
shown in Figure 1. After isolating the individual proteins to at least
60% purity, their properties, as compared to those of human and rat
CNTF were determined.
Because the electrophoretic mobilities of human and rat CNTF
differ significantly, the effect of each amino acid substitution was
monitored initially by making a determination of the effect of such
change on the mobility of the protein. As described herein,
electrophoretic mobility data indicated that all of the modified
human CNTF molecules that migrated to the same position as rat
CNTF had the single amino acid substitution GIn63-~Arg (Q63~R).
Modified human CNTF proteins that demonstrated an
electrophoretic mobility similar to that of the rat CNTF molecule
were subsequently examined for biological activity and receptor
binding.
CNTF is characterized by its capacity to support the survival
of dissociated ciliary neurons of E8 chick embryos. By this
criterion, purified recombinant rat CNTF is as active as the native
protein from rat, but four times more active than recombinant human
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CNTF -[Masiakowski, et al., 1991, J. Neurosci. 57:1003-1012 and in
International Publication No. WO 91/04316, published on April 4,
1991 ]. The same assay was utilized to determine the biological
activity of the altered molecules prepared as described above. As
described herein, all of the modified CNTF molecules that had the
Q63~R substitution exhibited an increased ability to support the
survival of ciliary ganglion neurons as compared to the parent human
CNTF protein. Such results indicated a strong correlation between
alteration of the electrophoretic mobility and enhanced biological
properties.
In addition to measuring the biological effect of modifications
made to human CNTF, an indication of the potential biological
activity of each of the molecules may also be obtained by
determining the effect of each modification on the ability of the
1 5 molecules to bind to the CNTF receptor.
In one embodiment, the ability of the modified human CNTF
proteins to compete with rat CNTF for binding to rat superior
cervical ganglia neurons (SCGs) is measured. As described herein,
human CNTF is about 90 times less potent in displacing ~25I-labelled
rat CNTF binding from these cells than unlabelled rat CNTF. Several
of the modified human CNTF proteins described herein, however, are
more potent than the human CNTF in displacing the rat protein. All
of the molecules described herein that had such increased
competitive binding ability were molecules that exhibited altered
electrophoretic mobility, wherein the molecules migrated in a
manner similar to rat CNTF.
In another embodiment, cells, such as MG87 fibroblasts, are
engineered to express the human CNTF receptor a -component and
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such cells are used to assay the binding capability of the modified
protein to the human receptor. Human CNTF is about 12 times less
potent than rat CNTF in competing with ~ 251-labelled rat CNTF for
binding to the human CNTF receptor. Several of the modified human
CNTF molecules described herein, including all of those with
electrophoretic mobility that resemble rat rather than human CNTF,
were more potent than human CNTF in competing with binding of
i251_rat CNTF to the cells expressing the human CNTF receptor.
In another embodiment, an animal model with demonstrated
utility in providing an indication of the ability of certain growth and
other factors to prevent degeneration of retinal photoreceptors may
be used to assess the therapeutic properties of the modified CNTF
molecules according to the present invention. As described in
Example 4, hCNTF (GIn63-~Arg) has a ten-fold higher ability than
1 5 recombinant human CNTF to prevent degeneration of photoreceptors
in a light-induced damage model of retinal degeneration.
Thus, according to the invention, certain amino acid
substitutions in the human CNTF protein result in modified human
CNTF proteins that exhibit enhanced binding to the human CNTF
2 0 receptor and therefore, would be expected to have enhanced
therapeutic properties.
The modified CNTF molecules useful for practicing the present
invention may be prepared by cloning and expression in a prokaryotic
or eukaryotic expression system as described, for example in
2 5 Masiakowski, et al., 1991, J. Neurosci. 57:1003-1012 and in
International Publication No. WO 91/04316, published on April 4,
1991. The recombinant neurotrophin gene may be expressed and
purified utilizing any number of methods. The gene encoding the
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factor may be subcloned into a bacterial expression vector, such as
for example, but not by way of limitation, pCP110.
The recombinant factors may be purified by any technique
which allows for the subsequent formation of a stable, biologically
active protein. For example, and not by way of limitation, the
factors may be recovered from cells either as soluble proteins or as
iriclusion bodies, from which they may be extracted quantitatively by
8M guanidinium hydrochloride and dialysis. In order to further purify
the factors, conventional ion exchange chromatography, hydrophobic
interaction chromatography, reverse phase chromatography or gel
filtration may be used.
According to the present invention, modified CNTF molecules
produced as described herein, or a hybrid or mutant thereof, may be
used to promote differentiation, proliferation or survival in vitro or
1 5 in vivo of cells that are responsive to CNTF, including cells that
express receptors of the CNTF/IL-6/LIF receptor family, or any cells
that express the appropriate signal transducing component, as
described, for example, in Davis, et a1.,1992, Cell 69:1121-1132.
Mutants or hybrids may alternatively antagonize cell differentiation
2 0 or survival.
The present invention may be used to treat disorders of any
cell responsive to CNTF or the CNTF/CNTF receptor complex. In
preferred embodiments of the invention, disorders of cells that
express members of the CNTF/IL-6/LIF receptor family may be
2 5 treated according to these methods. Examples of such disorders
include but are not limited to those involving the following cells:
leukemia cells, hematopoietic stem cells, megakaryocytes and their
progenitors, DA1 cells, osteoclasts, osteoblasts, hepatocytes,
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adipocytes, kidney epithelial cells, embryonic stem cells, renal
mesangial cells, T cells, B cells, etc.
Accordingly, the present invention provides for methods in
which a patient suffering from a CNTF-related neurological or
differentiation disorder or disease or nerve damage is treated with
an effective amount of the modified CNTF, or a hybrid or mutant
thereof. The modified CNTF molecules may be utilized to treat
disorders or diseases as. described for CNTF in International
Publication No. W091 /04316 published on April 4, 1991 by
1 0 Masiakowski, et al. and for CNTF/CNTFR complex as described in
International Publication No. W091/19009 published on December
12, 1991 by Davis, et al. both of which are incorporated by reference
in their entirety herein.
Such diseases or disorders include degenerative diseases, such
as retinal degenerations, diseases or disorders involving the spinal
cord, cholinergic neurons, hippocampal neurons or diseases or
disorders involving motorneurons, such as ar~iyotrophic lateral
sclerosis or those of the facial nerve, such as Bell's palsy. Other
diseases or disorders that may be treated include peripheral
neuropathy, Alzheimer's disease, Parkinson's disease, Huntington's
chorea (Huntington's disease or HD), or muscle atrophy resulting
from, for example, denervation, chronic disuse, metabolic stress,
and nutritional insufficiency or from a condition such as muscular
dystrophy syndrome, congenital myopathy, inflammatory disease of
2 5 muscle, toxic myopathy, nerve trauma, peripheral neuropathy, drug or
toxin-induced damage, or motor neuronopathy, or obesity, diabetic
obesity, or diabetes, including, but not limited to, non-insulin
dependent diabetes mellitus.
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In one embodiment, CNTF or CNTF-related molecules described
herein are used for the treatment of Huntington's disease.
Glutamate receptor mediated excitotoxicity has been hypothesized
to play a role in numerous neurodegenerative diseases or insults,
including Huntington's disease. The predominant neuropathological
feature of Huntington disease is a massive degeneration of the
medium-sized, GABAergic, striatal output neurons, without
substantial loss of striatal interneurons (Acheson, A. & R. Lindsay.,
1994, Seminars Neurosci: 6_:333-3410). As described in Example 7 .
1 0 below, Applicants have conducted studies, using both CNTF and the
variants described herein, in an animal model wherein the
preferential loss of striatal output neurons observed in Huntington
disease, and the resulting dyskinesia, are mimicked in rodent or
primate models in which an NMDA glutamate receptor agonist,
quinolinic acid, is injected into the striatum (DiFiglia, M. Trends
Neurosci., 1990, 13:286-289). In these studies, CNTF and its
variants afforded protection against exposure to quinolinic acid. The
close resemblance of the appearance of the quinolinic acid-lesioned
striatum to that of patients dying with HD suggests that quinolinic
2 0 acid, although it produces an acute and severe lesion in
contradistinction to the relentless and relatively slow progression
of HD, constitutes an adequate animal model for this devastating
neurological disorder.
To date, human clinical trials using recombinant human CNTF
2 5 (rHCNTF) have been limited to studies wherein subcutaneous
administration of the protein was tested for its efficacy in slowing
the progression of amyotrophic lateral sclerosis (ALS). Such
administration of rHCNTF was associated with systemic side
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effects, including cough anorexia and weight loss, and, in at least
one study, over 80% of patients receiving rHCNTF developed
neutralizing antibodies, the significance of which is uncertain.
However, despite problems with side effects and antibody formation,
a subgroup of patients in the early stages of ALS appeared to derive
benefit from rHCNTF administration in that these patients
demonstrated a reduced rate of pulmonary function loss compared to
placebo treated patients with similar disease durations.
Preliminary studies conducted by applicants, using
intermittent, compartmentalized administration of rHCNTF into the
CSF of ALS patients, have demonstrated no evidence of systemic side
effects or antibody formation. Such studies involved the use of an
infusion pump manufactured by Medtronic (SynchroMed Model
8615/Series DAA) with a side port for sampling CSF which was
implanted under general anesthesia using standard techniques (Penn,
et al., 1985, 2:125-127). The pump was attached to a subarachnoid
catheter who tip was placed at the L1 level under fluoroscopy.
Administration of 1 to 8 ~g rHCNTF per hour for 48 hours each week
was tolerated for periods up to 1 year in four patients with ALS.
2 0 These patients did not experience the range of adverse events seen
with systemic rHCNTF administration. Side effects in this patient
group consisted of sciatic pain in two patients and headaches in one.
Elevations in white blood cells and protein were seen in the CSF. In
this study, rHCNTF displayed similar distribution and
2 5 pharmacokinetic properties to small molecule drugs such as baclofen
and morphine infused into the intrathecal space. Unfortunately,
rHCNTF is too unstable for continuous CNS infusion therapy or for
local depot administration, since it tends to form covalent dimers
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through its unpaired cysteine residue, leading to aggregate
formation and precipitation. Accordingly, the need exists for stable
preparation of CNTF that can be utilized for direct infusion in the
central nervous system.
In collaboration with Aebischer, et al. (unpublished results),
Applicants have implanted encapsulated BHK cells which secrete
hCNTF into the subarachnoid space of 10 patients with ALS. Steady-
state CSF concentrations of up to 6 ng/mL have been achieved.
Although all patients complain of asthenia and fatigue, weight loss,
anorexia and activation of the acute phase response proteins were
not observed. There has been no CSF pleocytosis nor increase in
white cell counts. CNTF cannot be detected in the peripheral blood in
these patients. Results of efficacy measures to date are too sparse
to permit conclusions regarding efficacy. The lack of an
inflammatory response to hCNTF in patients receiving rHCNTF
synthesized by implanted, encapsulated cells compared to that seen
with pump-infused rHCNTF suggests that the changes seen following
pump delivery of rHCNTF may well be related to formulation and
stability issues surrounding this particular protein.
2 0 Accordingly, based on animal model data demonstrating the
efficacy of CNTF and its variants as protective agents for exitotoxic
damage of striatal neurons in an art recognized model of
Huntington's disease, combined with Applicants' discovery that the
side effects and antibody formation observed using systemic
2 5 injection of CNTF can be avoided by delivery of CNTF or its variants
directly into the CNS, applicants have discovered a useful method of
treating Huntington's disease. Accordingly, applicants invention
contemplates delivery of CNTF or its variants directly into the CNS
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via implanted cells or cellular-like vesicles, such as, for example,
liposomes, which secrete CNTF. Alternatively, CNTF variants as
described herein, which have improved stability and solubility as
compared to CNTF, provide preferred formulations for delivery of
CNTF via, for example, osmotic pumps, into the CNS as described
above. Because the instability of rHCNTF in solution at body
temperature interferes with its ability to be chronically
administered by intrathecal or intraventricular infusion, the
variants of rHCNTF described herein are preferred for such uses in
view of their improved stability, solubility, and decreased
antigenicity.
Accordingly, the present invention contemplates variants of
CNTF with improved solubility that may be used in therapeutic
applications where infusion, via, for example, osmotic pump, is used
1 5 to delivery the drug. The solubility of recombinant human CNTF
(rHCNTF) is very limited in physiological buffer, e.g., Phosphate-
Buffered-Saline, pH 7.4 (PBS). Furthermore, the solubility over at
least the 4.5-8.0 pH range depends strongly on the temperature and
on the time of incubation. At 5°C, the solubility of rHCNTF in PBS is
1 mg/ml and the solution is stable for a few hours, but at 37°C its
solubility is only 0.1 mg/ml after 2 hr and 0.05 mg/ml after 48 hrs.
This limited solubility and thermal stability preclude stable
formulation of rHCNTF in physiological buffer. Such formulations
are particularly desirable for continuous administration into the
2 5 CNS.
It was discovered that rHCNTF lacking the last 13 amino acid
residues from the carboxyl end (rHCNTF,oCl3 also designated
RPN160 or RG160) retains full biological activity and is soluble at
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low temperatures (5-10°C) to at least 12 mg/ml. Yet, despite this
far greater solubility, rHCNTF,oCl3 still falls out of a PBS solution
upon incubation at 37°C over a period of several hours, even at
concentrations as low as 0.1 mg/ml.
It was determined that the thermal instability of rHCNTF and
rHCNTF,oCl3 was the result of aggregation that was initiated by
intermolecular disulfide bond formation and depended strongly on
protein concentration and temperature. By replacing the single
cysteine residue at position 17 of human CNTF with an alanine
residue, proteins were obtained that show far greater stability and
maintain their biological activity after incubation for at least 7
days in PBS at 37°C: This property is maintained in rHCNTF,63QR
variants which have higher potency due to the substitution of the
glutamine residue at position 63 by arginine. In a particular
1 5 example, rHCNTF,17CA,63QR,oCl3 (also designated RG297) shows
greater biological potency than rHCNTF because of the 63QR
substitution, greater solubility because of the OC13 deletion and
greater stability because of the 17CA substitution.
The present invention contemplates treatment of a patient
2 0 having HD with a therapeutically effective amount of CNTF or the
variants described herein. Effective amounts of CNTF or its variants
are amounts which result in the slowing of the progression of the
disease, or of a reduction in the side-effects associated with the
disease. The efficacy of the treatment may be measured by
2 5 comparing the effect of the treatment as compared to controls
which receive no treatment. The clinical course and natural history
of HD have been extensively characterized both in field studies
(Young et a1.,1996, Ann Neurol. 20:296-303; Penney and Young, 1990,
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Movement Disorders 5:93-99), the development of clinical rating
instruments (Shoulson and Fahn, 1979, Neurology 29:1-3; Shoulson et
al, 1989, Quantification of Neurologic Deficit, TL Munsat (ed)
Butterworths 271-284.; Feigin et al., 1995, Movement Disorders
10:211-214), and radiographic correlates of disease progression
using computed X-ray tomography (Terrence et al., 1977,
Neuroradiology 13:173-175; Barr et al., 1978, Neurology 28:1196-
1200; Neophytides et al., 1979, 23:185-191; Stober et al., 1984,
Neuroradiology 26:25-28); magnetic resonance imaging (Grafton et
al., 1992, Arch. Neurol. 49:1161-1167) and positron emission
tomographic techniques (Harris, et al., 1996, Arch. Neurol. 53:316-
324).
Clinical rating of the progression of Huntington's disease has
been assessed using the HD Functional Capacity Scale (HDFC)
1 5 developed by Shoulson -and Fahn (1979, Neurology 29:1-3). A fully
functional patient receives a score of 13 on this scale; a score of 0
reflects total disability Shoulson et al., 1989, Quantification of
Neurologic Deficit, TL Munsat (ed) Butterworths 271-284. The
average rate of progression of patients using this scale is
2 0 approximately 0.65 units/year. Shoulson et al., 1989, Quantification
of Neurologic Deficit, TL Munsat (ed) Butterworths 271-284; Feigin
et al.; 1995, Movement Disorders 10:211-214. If this scale is truly
linear (an hypothesis which has not been tested) this rate of
progression would correspond nicely with the average 20 year
2 5 duration of symptomatic HD in patients. HDFC scores can be roughly
grouped into 5 clinical stages (Shoulson et al., 1989, Quantification
of Neurologic Deficit, TL Munsat (ed) Butterworths 271-284).
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Neuroimaging studies have focused on the gross pathological
consequences of neuronal loss and consequent atrophy of basal
ganglia structures. As HD progresses, the caudate nuclei shrink,
giving a characteristic "box-car" appearance to the lateral
ventricles. The degree of caudate atrophy can be quantified using a
"bicaudate index".
Magnetic resonance imaging may be used to generate similar
indices to those given by CT. A relatively new technique, in vivo NMR
spectroscopy, however, offers the ability to assess metabolic
processes within the living brain. One preliminary study (Jenkins, et
al., 1993, Neurology 43:2689-2695 has detected an increased amount
of lactic acid, presumably reflecting either neuronal cell loss or a
defect in intermediary metabolism, in the brains of HD patients.
Positron Emission tomographic (PET) permits functional
imaging to be performed in living patients. Changes in metabolic
state can be assessed using 2-deoxyglucose (which reflects synaptic
activity), or selective radioligands which mark selected neuronal
populations. To determine the rate of change of glucose metabolism
and caudate size in persons at risk for Huntington's disease, Grafton
et al., (1992, Arch Neurol. 49:1161-1167) evaluated 18 persons at
risk for Huntington's disease with two positron emission
tomographic glucose metabolic studies and two magnetic resonance
imaging scans separated by 42 (+/- 9) months. Seven of the
individuals were Huntington' disease gene negative; the remainder
2 5 were gene positive by genetic testing or onset of chorea after study
entry. The gene-positive group demonstrated a significant 3.1 % loss
of glucose metabolic rate per year in the caudate nucleus (95%
confidence interval [CI], -4.64, -1.48) compared with the gene-
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negative group. There was a 3.6% per year increase in the magnetic
resonance imaging bicaudate ratio (95% CI, 1.81, 5.37), a linear
measure of caudate atrophy. However, the rate of change in caudate
size did not correlate with the rate of change in caudate
metabolism, suggesting that metabolic loss and atrophy may develop
independently. Thus serial positron emission tomographic or
magnetic resonance imaging yield rates of loss not too different
from those observed in clinical rating scales (approximately 5% per
year, vide supra), and thus may be useful means by which to monitor
experimental pharmacologic interventions in presymptomatic
individuals at risk for HD should clinical trials be designed to
incorporate such a patient population.
In addition to glucose metabolic mapping, other radioligands
may be used to monitor striatal integrity in HD. For example, since
intrinsic striatal neurons which are lost in HD uniformly bear
dopamine receptors, ligands for the dopamine receptor have been
used to monitor the progression of HD. These studies do indeed show
a parallel reduction of both striatal D1 and D2 receptors in HD
patients (Turjanski et al., 1995, Brain 118:689-696).
Similar metabolic and neurochemical findings have been
obtained in PET studies of primates treated with quinolinic acid in
the striatum. Brownell et al., (1994, Exp. Neurol.l 25:41-51 ),
reported that, following a quinolinate lesion of the striata of 3 non-
human primates, symptoms similar to those of Huntington's disease
2 5 could be induced by dopamine agonist treatment. All animals showed
a long-term 40-50% decrease in glucose utilization in the caudate by
[l9F~fluoro-2-deoxy-D-glucose positron emission tomography (PET).
Caudate-putamen uptake rate constants for D1 receptors reflected
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neuronal loss and decreased by an average 40 to 48%. Dopamine
reuptake sites and fibers assessed by PET showed a temporary
decrease in areas with mild neuronal loss and a long-term decrease
in striatal regions with severe destruction. These results, which
were consistent with behavioral changes and neuropathology seen at
postmortem examination, are similar to those observed in clinical
studies of Huntington's disease patients, and serve to additionally
validate the quinolinic acid model, and suggest that these measures
may be of use in human clinical trials.
Clinical trials in HD have largely been limited to the
assessment of palliative symptomatic therapies for psychiatric
symptoms and involuntary movements (Shoulson et al., 1981,
Neurology 29:1-3). However, there has been one attempt to examine
a potential neuroprotective agent. This trial involved the use of
baclofen, a GABA-B receptor antagonist, on the theory that this
agent would reduce glutamate release from corticostriatal
terminals in the striatum, thereby retarding the progression of HD
(Shoulson et al., 1989, Quantification of Neurologic Deficit, TL
Munsat (ed) Butterworths 271-284). The outcome of this trial was
2 0 negative, in that baclofen-treated patients fared no better than
controls over the 30-month duration of the trial. Nonetheless, this
trial provided the proving ground for the use and validation of the
HDFC. One important outcome of the study was that the intrinsic
rate of disease progression in the study subjects was only one-half
of that originally estimated by the investigators. This information
may now be used in the design of future clinical trials using this
rating instrument.
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Currently, there are no major ongoing clinical trials in HD.
However, a clinical trials organization, the Huntington's disease
Study Group, has been organized and has the infrastructure in place
for the conduct of clinical trials in HD. This group is currently
investigating a variety of clinical trial options including 1 ) the use
of Coenzyme Q to enhance intermediary metabolism and 2) the use of
glutamate antagonists and/or glutamate release blockers (W.
Koroshetz, personal communication). A parallel group has been
established in Europe, and this group will be using PET methodology
to examine the potential efficacy of fetal striatal implants and,
eventually, the use of xenograft transplants as well.
The availability of a validated clinical rating instrument, and
the existence of correlative radiographic measures to assess
disease progression in HD, combined with the existence of 2 large,
organized multicenter clinical trials consortia will make
implementation of clinical trials in HD straightforward.
Applicants describe herein the production of a modified CNTF
molecule, known as Ax-13 or Ax-1, (designated
rHCNTF,17CA63QROC13) which combines a 63Q-~R substitution
(which confers greater biological potency) with a deletion of the
terminal 13 amino acid residues (which confers greater solubility
under physiological conditions) and a 17CA substitution (which
confers stability, particularly under physiological conditions at
37°C) and shows a 2-3 fold better therapeutic index than rHCNTF in
2 5 an animal model. However, when expressed in E. coli, a substantial
portion of the expressed protein produced is tagged with a
decapeptide at the C-terminus. Because of this, purification of Ax-
13 is difficult and results in a low yield of purified, untagged
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product. This decapeptide tagging likely does not occur when the
Ax-13 is expressed in a mammalian expression system. In addition,
it is possible that the decapeptide tag could contribute to increased
immunogenicity of the molecule and may also possibly cause
problems with stability.
However, use of the E. coli expression system would be
preferable from the standpoint of cost and efficiency: Therefore,
applicants undertook to develop a truncated CNTF molecule that
would retain the improved potency, solubility and stability
properties of Ax-13, while avoiding the problem of decapeptide
tagging when expressed in E. coli. As described herein, applicants
have succeeded in producing a molecule known as Ax-15, (designated
rHCNTF,17CA63QROC15), which retains the improved properties of
Ax-13, but which also has the added advantage of being expressed by
1 5 E. coli with reduced amino acid tag being added. The new molecule,
Ax-15, therefore has the advantage of being more easily purified
with a greater yield. Additionally, because there is greatly reduced
bacterial amino acid tagging, Ax-15 does not raise the concern with
regard to the immunogenicity or stability of the molecule that could
2 0 be raised by Ax-13.
Therefore the object of the present invention is to provide an
improved modified ciliary neurotrophic factor molecule.
Specifically, one embodiment of the invention is a modified human
ciliary neurotrophic factor having the modification Cysl7-~Ala,
25 GIn63~Arg, and a deletion of the terminally amino acid residues.
The present invention also provides for an isolated nucleic acid
molecule encoding the modified human ciliary neurotrophic factor of
the invention. Also contemplated by the invention is a recombinant
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DNA molecule that encodes the modified human ciliary neurotrophic
factor of the invention and which is operatively linked to an
expression control sequence, as well as a host cell transformed with
the recombinant DNA molecule. The host cell may be prokaryotic or
eukaryotic; and therefore may be, for example, a bacterium such as E .
coli, a yeast cell such as Pichia pastoris, an insect cell such as
Spodoptera frugiperda, or a mammalian cell such as a COS or CHO
cell. Said host cell may be used in a method for producing the
modified ciliary neurotrophic factor molecule comprising: (a)
growing the host cell transformed with the recombinant DNA
molecule of the invention so that the DNA molecule is expressed by
the host cell to produce the modified ciliary neurotrophic factor
molecule of the invention and (b) isolating the expressed, modified
ciliary neurotrophic factor molecule.
The subject invention further contemplates a composition
comprising the modified ciliary neurotrophic factor molecule of the
invention (Ax-15), and a carrier.
Another object of the present invention is to provide a method
of treating a disease or disorder of the nervous system comprising
administering the modified ciliary neurotrophic factor described
herein as Ax-15. The disease or disorder treated may be a
degenerative disease and/or involve the spinal cord, motor neurons,
cholinergic neurons or cells of the hippocampus. Alternatively, the
method of treatment may be for treating a disease or disorder of the
2 5 nervous system which comprises damage to the nervous system
caused by an event selected from the group consisting of trauma,
surgery, infarction, infection, malignancy and exposure to a toxic
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agent. Also contemplated by the present invention is a method of
treating a disease or disorder involving muscle atrophy.
A further object of the present invention is to provide a
method of protecting striatal neurons from degeneration comprising
treating said striatal neurons with an effective amount of the
modified ciliary neurotrophic factor described herein as Ax-15.
Also contemplated by the present invention is a method of
treating Huntington's disease comprising direct administration to
the central nervous system of the modified ciliary neurotrophic
factor described herein as Ax-15.
A further object of the present invention is to provide a
method of inducing weight loss in a mammal comprising
administration to the mammal of the modified ciliary neurotrophic
factor described herein as Ax-15. A specific embodiment of this
invention involves inducing weight loss in a human.
The method of administering Ax-15 may be used in the
treatment of morbid obesity or obesity of a genetically determined
origin. The Ax-15 described herein may also be used in a method of
preventing and/or treating the occurrence of gestational or adult
2 0 onset diabetes in a human.
Any of the above-described methods involving the
administration of Ax-15 may be practiced by administering the Ax-
15 via a route of delivery selected from the group consisting of
intravenous, intramuscular, subcutaneous, intrathecal,
intracerebroventricular and intraparenchymal.
Alternatively, the Ax-15 may be administered via the
implantation of cells that release the modified ciliary neurotrophic
factor.
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The present invention also contemplates diseases or disorders
resulting from damage to the nervous system, wherein such damage
may be caused by trauma, surgery, infarction, infection and
malignancy or by exposure to a toxic agent.
The present invention also provides for pharmaceutical
compositions comprising a modified CNTF molecule or hybrid or
mutant thereof, as described herein, as the sole therapeutic agent or
in a complex with the CNTF receptor, in a suitable pharmacologic
carrier.
1 0 The active ingredient, which may comprise CNTF or the
modified CNTF molecules described herein should be formulated in a
suitable pharmaceutical carrier for administration in vivo by any
appropriate route including, but not limited to intraparenchymal,
intraventricular or intracerebroventricular delivery, or by a
sustained release implant, including a cellular or tissue implant
such as is described, for example, in published application
W096/02646 published on February 1, 1996, W095/28166 published
on October 26, 1995, or W095/505452 published February 23, 1995.
Depending upon the mode of administration, the active
ingredient may be formulated in a liquid carrier such as saline,
incorporated into liposomes, microcapsules, polymer or wax-based
and controlled release preparations, In preferred embodiments,
modified CNTF preparations which are stable, or formulated into
tablet, pill or capsule forms.
The concentration of the active ingredient used in the
formulation will depend upon the effective dose required and the
mode of administration used. The dose used should be sufficient to
achieve circulating plasma concentrations of active ingredient that
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are efficacious. Effective doses may be extrapolated from dose-
response curves derived from in vitro or animal model test systems.
Effective doses are expected to be within the range of from about
001 to about 1 mg/day.
EXAMPLES
Example 1' Electrophoretic Mobility of Modified Human
CNTF Molecules
Materials and Methods
Preparation of Modified CNTF molecules
Bacterial Strains and Plasmids
E. coli K-12 RFJ26 is a strain that overproduces the lactose
operon repressor.
The expression vectors pRPN33, which carries the human CNTF
gene and pRPN110 which carries the rat CNTF gene are nearly
identical (Masiakowski, et al., 1991, J. Neurosci. 57:1003-1012 and
in International Publication No. WO 91/04316, published on April 4,
1991 .)
Plasmid pRPN219 was constructed by first digesting pRPN33
with the restriction enzymes Nhe1 plus Hind3 and gel purifying the
4,081 by fragment. The second, much smaller fragment which codes
2 5 for part of the human CNTF gene was subsequently replaced with an
167 by Nhe1-Hind3 fragment that was obtained by PCR amplification
from the rat gene using the primers RAT-III-dniH: 5' ACGGTAAGCT
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TGGAGGTTCTC 3'; and RAT-Nhe-I-M: 5' TCTATCTGGC TAGCAAGGAA
GATTCGTTCA GACCTGACTG CTCTTACG 3'.
Plasmid pRPN228 was constructed in the same manner as
pRPN219, except that the 167 by replacement fragment was
amplified using the DNA primers Rat-III-dniH-L-R : 5' AAG GTA CGA
TAA GCT TGG AGG TTC TCT TGG AGT CGC TCT GCC TCA GTC AGC TCA
CTC CAA CGA TCA GTG 3' and Rat-Nhe-I: 5' TCT ATC TGG CTA GCA
AGG AAG 3'.
Plasmids pRPN186, pRPN187, pRPN188, pRPN189, pRPN192,
1 0 pRPN218, and pRPN222 were generated by similar means or by direct
exchange of DNA fragments using the unique restriction sites shown
in Figure 1.
The identity of all plasmids was confirmed by restriction
analysis and DNA sequencing.
Protein Purification
Induction of protein synthesis, selective extraction,
solubilization and purification from inclusion bodies were as
described for rat and human CNTF (Masiakowski, et al., 1991, J.
Neurosci. 57:1003-1012 and in International Publication No. WO
91/04316, published on April 4, 1991) except that gel filtration was
occasionally used instead or in addition to ion exchange
chromatography. Alternatively, proteins were purified from the
supernatants of cell lysates by streptomycin and ammonium sulfate
2 5 fractionation, followed by column chromatography, as described for
other proteins (Panayotatos et al., 1989, J. Biol. Chem. 264:15066-
15069). All proteins were isolated to at least 60% purity.
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Conditions for enzymatic reactions, DNA electrophoresis and
other techniques used in these studies have been described in detail
(Panayotatos, N. 1987, Engineering an Efficient Expression System in
Plasmids: A practical Approach (Hardy, K.G. ed.) pp 163-176, IRL
Press, Oxford, U.K.).
Results
The mobilities of human, rat and several chimeric CNTF
molecules on reducing SDS-polyacrylamide gels are shown in Figure
1 0 2. The chimeric molecules RPN186, RPN189, RPN218 and RPN228
exhibit mobilities comparable to rat CNTF, whereas RPN187,
RPN188, RPN192 and RPN222 exhibit mobilities comparable to human
CNTF. Cross-reference of these results to the aligned sequences of
these proteins in Figure 1 reveals that all proteins carrying an
arginine residue at position 63 (R63) display the mobility of rat
CNTF. In the case of RPN228, this single amino acid substitution
(Q63->R) is sufficient to confer to human CNTF the normal mobility
of rat CNTF.
Figure 2 also provides a measure of the purity of the different
recombinant proteins. By visual inspection, purity varies from 60%
for RPN189 to better than 90% for RPN228.
Example 2' Measurement of Binding Activity of Modified
CNTF Molecules
Materials and Methods
Preparation of ~?51-CNTF
Recombinant rat CNTF (28 fig) in 37 ~,I 0.2 M sodium borate
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buffer, pH 8.5 was transferred to a vial containing 4 mCi, (2,000
Ci/mmole; NEN) of 1251 and reagent (Bolton and Hunter,1973, Biochem
J. 133: 529-539) which had been dried under a gentle stream of
nitrogen. Reactions were incubated for 45 min at 0°C followed by 15
min at room temperature and terminated by the addition of 30 ml of
0.2 M glycine solution. After 15 min, 0.2 ml PBS containing 0.08
gelatin was also added and the mixture was passed through a
Superdex-75 column (Pharmacia) to separate the labelled monomeric
CNTF from dimeric and other multimeric derivatives. Percentage of
incorporation was typically 20%, as determined by thin layer
chromatography and the specific activity was typically around 1,000
Ci/mmole. The monomeric ~ 251-CNTF was stored at 4°C and used up
to one week after preparation. As a test of structural and
conformational integrity, X251-CNTF (approximately 10,000 cpm) was
1 5 mixed with a 5 wg unlabelled CNTF and analyzed by native gel
electrophoresis. One major band was visible by either Coomassie
staining or autoradiography. ~251_CNTF also showed comparable
activity to native CNTF in supporting survival of E8 chick ciliary
neurons in culture.
Tissue Culture Techniaues
Superior cervical ganglia (SCG) from neonatal rats were
treated with trypsin (0.1 %), mechanically dissociated and plated on a
poly-ornithine (30 ~g/ml) substratum. Growth medium consisted of
Ham's nutrient mixture F12 with 10% heat-inactivated fetal bovine
serum (Hyclone), nerve growth factor (NGF) (100 ng/ml), penicillin
(50 U/ml) and streptomycin (50 ~g/ml). Cultures were maintained at
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37°C in a humidified 95% air/5% C02 atmosphere. Ganglion non-
neuronal cells were eliminated by treatment with araC (10 wM) on
days 1 and 3 of culture. Cultures were fed 3 times/week and were
routinely used for binding assays within 2 weeks.
MG87/CNTFR is a fibroblast cell line transfected with the
human CNTFa receptor gene (Squinto, et a1.,1990, Neuron 5:757-766;
Davis et al., 1991, Science 253:59-63).
Bindina Assays
Binding was performed directly on cell monolayers. Cells in
culture wells were washed once with assay buffer consisting of
phosphate buffered saline (PBS; pH 7.4), 0.1 mM bacitracin, 1 mM
PMSF, 1 ~g/ml leupeptin, and 1 mg/ml BSA. After incubation with
1251-CNTF for 2 hours at room temperature, cells were quickly
1 5 washed twice with assay buffer, lysed with PBS containing 1 % SDS
and counted in a Packard Gamma Counter. Non-specific binding was
determined in the presence of 1,000-fold excess of unlabelled CNTF.
Specific binding towards MG87/CNTFR was 80-90%. Data were
analyzed using the GRAPHPAD program (1S1, Philadelphia, PA).
Results
Competition curves of purified recombinant human, rat and
CNTF RPN219 towards X251-rat CNTF for binding on rat SCG neurons
are shown in Figure 4a. Both rat and human CNTF compete with X251_
2 5 rat CNTF for binding to SCG neurons, but human CNTF (1C50 = 25 nM)
is 90 times less potent in displacing 1251-rat CNTF binding than
unlabelled rat CNTF (1C50 = 0.28 nM). In contrast, RPN219 is almost
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as potent as rat CNTF and clearly more potent than human CNTF (1C50
= 0.3 nM).
Similar results were obtained from competition experiments
with mouse fibroblasts transfected with a plasmid directing the
expression of the human CNTF receptor (Figure 4b). Both rat, human
and RPN228 compete with X251-rat CNTF for binding to MG87/CNTFR
cells. Human CNTF (1C50 = 30 nM) is 12 times less potent than rat
CNTF (1C50 = 2.8 nM), whereas RPN228 is clearly more potent than
the human protein (1C50 = 5.6 nM).
Competition binding experiments with the other modified CNTF
proteins shown in Figure 1 also demonstrated that proteins having
R63 displayed the biological activity of rat CNTF, whereas proteins
having Q63 displayed the binding properties of human CNTF (data not
shown). These results indicate that the single amino acid
substitution (Q63->R) is sufficient to confer to human CNTF the
receptor binding properties characteristic of rat CNTF.
Example 3' Measurement of Biological activity of Modified
CNTF Molecules
Materials and Methods
Recombinant CNTF was assayed on dissociated cultures of
chick ciliary ganglion (CG) neurons as described (Masiakowski, et al.,
1991, J. Neurosci: 57:1003-1012 and in International Publication No.
2 5 WO 91 /04316, published on April 4, 1991 ), except that surviving
cells were stained with MTT (Mosmann, T. 1983; J. Immunol. Methods
65:55-63).
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Results
Figure 3 shows dose-response curves of dissociated, neuron-
enriched cultures of E8 chick embryo ciliary ganglia for purified
recombinant human, rat and the modified CNTF proteins RPN219 and
RPN228. By this assay, the biological activity of the chimeric
proteins is indistinguishable from that of purified recombinant rat
CNTF and clearly higher than that of recombinant human CNTF.
Comparison of the dose-response curves in Figure 3 also shows that
the maximal levels of surviving neurons obtained with RPN219,
1 0 RPN228 or rat CNTF are higher than those obtained with human CNTF.
These results suggest that RPN219 and RPN228, like rat CNTF, are
active towards a larger population of neurons than human CNTF. In
parallel experiments, the biological activity of the other modified
CNTF proteins shown in Figure 1 was examined. In every case,
1 5 modified CNTF proteins carrying the (Q63-~ R) substitution displayed
the biological activity of rat CNTF whereas proteins having Q63
displayed the activity of human CNTF (data not shown).
Overall, these results indicate that the single amino acid
substitution (Q63--~ R) is sufficient to confer to human CNTF the
2 0 biological activity of rat CNTF.
Example 4~ Use of Modified CNTF To Prevent Liaht Induced
Photoreceptor Iniury
2 5 Albino rats of either the F344 or Sprague-Dawley strain were
used at 2-5 months of age. The rats were maintained in a cyclic
light environment (12 hr on: 12 hr off at an in-cage illuminance of
less than 25 ft-c) for 9 or more days before being exposed to
constant light. The rats were exposed to 1 or 2 weeks of constant
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light at an illuminance level of 115-200 ft-c (most rats received
125-170 ft-c) provided by two 40 watt General Electric
"cool-white" fluorescent bulbs with a white reflector that was
suspended 60cm above the floor of the cage. During light exposure,
rats were maintained in transparent polycarbonate cages with
stainless steel wire-bar covers.
Two days before constant light exposure, rats anesthetized
with a ketamine-xylazine mixture were injected intravitreally with
1 ~,I of rat CNTF, human CNTF or modified CNTF [hCNTF (Q63-jR)]
dissolved in phosphate buffered saline (PBS) at a concentration of
0.1 to 500 ng/~,I. The injections were made with the insertion of a
32 gauge needle through the sclera, choroid and retina approximately
midway between the ora serrata and equator of the eye. In all cases,
the injections were made into the superior hemisphere of the eye.
Immediately following constant light exposure, the rats were
sacrificed by overdose of carbon dioxide followed immediately by
vascular perfusion of mixed aldehydes. The eyes were embedded in
epoxy resin for sectioning at 1 ~.m thickness to provide sections of
the entire retina along the vertical meridian of the eye. The degree
of light-induced retinal degeneration was quantified by assessing
the degree of photoreceptor rescue by a 0-4+ pathologist's scale of
rescue, 4+ being maximal rescue and almost normal retinal integrity.
The degree of photoreceptor rescue in each section, as based on
comparison to the control eye in the same rat, was scored by four
2 5 individuals. This method has the advantage of considering not only
the ONL thickness, but also more subtle degenerative changes to the
photoreceptor inner and outer segments, as well as spatial
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degenerative gradients within the eye. Three eyes were examined
for each time point to generate a dose response curve.
Results
The degree of rescue was measured for human, rat and hCNTF
(Q63~ R). The data indicated that both rat and hCNTF (Q63-~ R) had
ten-fold greater ability to rescue photoreceptors in the light damage
model than did recombinant human CNTF.
It is to be understood that while the invention has been
described above in conjunction with preferred specific embodiments,
the description and examples are intended to illustrate and not limit
the scope of the invention, which is defined by the scope of the
appended claims.
Example 5
Materials and Methods
Recombinant human CNTF variants were genetically engineered,
expressed in E. coli and recovered at greater than 90% purity, as
2 0 described previously (Masiakowski, et al., 1991, J. Neurosci.
57:1003-1012 and in International Publication No. WO 91/04316,
published on April 4, 1991; Panayotatos et al., 1993, J. Biol. Chem.
268:19000-19003).
The following stock solutions were prepared freshly in PBS at
2 5 5°C:
rHCNTF..............................................................Ø5 mg/ml
RG160 (rHCNTF,OC13)................................Ø5 mg/ml
RG162 (rHCNTF,17CA,OC13)....................Ø5 mg/ml
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RG290 (rHCNTF,63QR,oCl3)....................1.2 mg/ml
RG297 (rHCNTF,17CA,63QR,oCl3)........Ø4 mg/ml
To determine the stability of rHCNTF and several derivatives in
physiological buffer at 37°C, stock solutions were dialyzed
exhaustively against PBS at 5°C, diluted with PBS to 0.1 mg/ml and
sterilized by filtration. Aliquots (0.2 ml), were transferred into 0.5
ml capacity polypropylene centrifugation tubes. The tubes were
placed in a 37°C incubator and, at the indicated times, individual
tubes were removed and centrifuged at 15,000 rpm for 3 min at room
temperature to separate soluble protein from insoluble precipitates.
Supernatants were pipetted off into clean tubes containing an equal
volume of 2X protein gel sample buffer, placed in a 85°C bath for 2
min, mixed and stored at -20°C until analysis by 15% SDS-PAGE.
Pellets were resuspended in 1/10 original volume of water, mixed
with an equal volume of 2X protein gel sample buffer and treated as
above.
Methods for biological activity assays on E8 chick ciliary
neurons and for protein gel electrophoresis have been described
(Masiakowski; et al., 1991, J. Neurosci. 57:1003-1012 and in
International Publication No. WO 91/04316, published on April 4,
1991; Panayotatos et al., 1993, J. BioI.Chem. 268:19000-19003).
Protein gel sample buffer (2X) consists of 12.5 ml TrisHCl, pH 6.8 -
20 ml glycerol - 40 ml 10% SDS and 5 mg Bromophenol Blue per 100
ml.
Results
The solubility of rHCNTF is particularly limited in
physiological buffer at neutral pH. Furthermore, the solubility over a
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broad pH range (4.5-8.0) depends strongly on the temperature and on
the time of incubation. At 5°C, the solubility of rHCNTF in PBS is 1.4
mg/ml and the protein remains in solution for a few hours. In sharp
contrast to the limited solubility of rHCNTF, the variant
rHCNTF,oCl3 can be concentrated to at least 12 mg/ml at 5°C.
Despite this greater solubility, however, rHCNTF,OC13 still shows
strong instability in physiological buffer, pH and temperature
conditions. Upon incubation at 37°C, rHCNTF,oCl3 falls out of
solution at a rate that depends on the initial concentration.
To determine the cause of this instability, we analyzed the
physical integrity of rHCNTF and several variants in parallel
experiments. Figure 5 shows that incubation of rHCNTF in
physiological buffer at 37°C for 0, 2, 7 and 14 days (lanes 1-4,
respectively) caused progressive disappearance of the protein from
the supernatants, accompanied by concomitant progressive
appearance in the pellets. Furthermore, a good proportion of rHCNTF
in the pellets appeared as a 48kD species that corresponded to the
size of dimeric rHCNTF (Fig. 5, double arrow). At longer incubation
times, a small proportion of higher order aggregates was also
2 0 evident. However, when the same samples were analyzed on the
same type of gel but in the presence of disulfide reducing agents, the
48 kD species was converted to monomeric rHCNTF, evidence that
the 48 kD species represents rHCNTF dimers covalently linked by
disulfide bonds. Such dimers would be expected to form through the
2 5 unique cysteine residue of rHCNTF. Therefore, these results
indicated that the instability of rHCNTF at 37°C is caused by
aggregation initiated by intermolecular disulfide bond formation.
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Similar results were obtained with two rHCNTF variants,
rHCNTF,OC13 and rHCNTF,63QR,oCl3, except that the appearance of
insoluble aggregates in the pellets was somehow slower in the case
of rHCNTF,OC13 (Figure 5). Given the fact that the 0C13 deletion
confers to rHCNTF much greater solubility in physiological buffer,
the improved stability of rHCNTF,oCl3 is most likely an indirect
consequence of its greater solubility.
To further test the possibility that the instability of rHCNTF
at 37°C is caused . by aggregation initiated by intermolecular
disulfide bond formation, the unique cysteine residue at position 17
was substituted by alanine, using established genetic engineering
methodology. The two rHCNTF variants, rHCNTF,17CA,OC13 and
rHCNTF,17CA,63QR,OC13 generated by this process were subjected
to the same analysis by non-reducing 15% SDS-PAGE. Figure 5 shows
that even after incubation for 14 days at 37°C both proteins
remained soluble with no evidence of dimerization or aggregate
formation. Even in the small proportion of protein found in the
pellets, which represented mostly the small amount of soluble
protein remaining in the centrifuge tubes after removal of the
2 0 supernatant, there was little evidence of dimerization. These
results confirmed the conclusion that the instability of rHCNTF is
caused by aggregation initiated by intermolecular disulfide bond
formation, and demonstrated that elimination of the free -SH
functional group in other rHCNTF variants, e.g. RG297, also result in
greater stability.
To test whether the proteins remaining in solution after
incubation at 37°C were still biologically active, samples were
analyzed for neuronal survival activity. Figure 6 shows control
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concentration response curves for rat CNTF and rHCNTF obtained
with standard, untreated stock solutions, as well as with four
rHCNTF variants incubated for 7 days at 37°C. Of the latter, the
proteins carrying the 17CA mutation, RG297 and RG162, were
assayed at their nominal concentrations, whereas RG290 and RG160
were assayed after correcting their concentrations for the amount
of protein remaining in solution. Figure 6 shows that the
concentration response curves displayed by these compounds are
those expected from these proteins in their fully active form:
1 0 RG160 and RG162 show the same potency as rHCNTF within
experimental error, whereas RG290 and RG297 that carry the 63QR
substitution show 4-5 fold higher potency than rHCNTF, as
previously observed (Panayotatos, N., et al., 1993, J. Biol. Chem.
268:19000-19003) and as shown in Fig. 7. Therefore, incubation of
1 5 rHCNTF and its derivatives at 37°C for 7 days does not cause loss
of
biological activity, only loss of protein through dimerization
followed by precipitation.
Example 6
Materials and Methods
Protein Engineerina and Purification - The following rHCNTF
variants were compared to rHCNTF:
RG228 (rHCNTF,63QR);
RG297 (rHCNTF,17CA,63QR,~C13)
RG242 (rHCNTF,63QR64WA)
These proteins were genetically engineered, expressed in E.
coli and recovered at greater than 90% purity by the methodology
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described for rHCNTF (Masiakowski, et al., 1991, J. Neurosci.
57:1003-1012 and in International Publication No. WO 91/04316,
published on April 4, 1991; Panayotatos et al., 1993, J. Biol. Chem.
268:19000-19003).
Biological Activit~r Assays - Methods for biological activity
assays on E8 chick ciliary neurons have been described (Panayotatos
et al., 1993, J. Biol. Chem. 268:19000-19003).
Pharmacokinetic Determinations - Rats were injected
intravenously (i.v.) with rHCNTF (n=1 ) and RG242 (n=2) at 100 wg/kg
and with RG228 (n=1 ) at 200 ~g/kg. Rats were also injected
subcutaneously (s.c.) with rHCNTF (n=2), RG242 (n=2) and RG228
(n=1 ) at 200 ~,g/kg. Blood specimens were collected prior to dosing
and at various times after dosing and were processed to obtain
plasma. The plasma specimens were analyzed using the rHCNTF
ELISA method for rodent plasma (D.B. Lakings, et al. DSER
93/DMAP/006, "Dose Proportionality and Absolute Bioavailability of
rHCNTF in the Rat Following Subcutaneous Administration at Eight
Dose Levels" (Phoenix International Project No. 920847) 10
November 1993).
The plasma concentrations were evaluated using non
compartment techniques. A standard curve for each compound was
included on each assay plate and was used to calculate the amount of
2 5 that compound present in the specimens analyzed on the plate. The
sensitivity of the assay varied among compounds by less than
twofold.
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Efficacy and Toxicity Determinations In Vivo - Male Sprague-
Dawley rats weighing 220 g were anesthetized before surgery. The
right sciatic nerve was transected at the level of the knee and a 5
mm segment of nerve was removed. Sham surgeries were performed
on the left side of each animal. Starting the morning after surgery,
rats were weighed and administered vehicle (either PBS or
lactate/phosphate/mannitol, pH 4.5) or the rHCNTF compound to be
tested, dissolved in the same vehicle at doses ranging from 0.01-1.0
mg/kg, s.c. Rats were weighed and injected daily for 1 week, at
which time they were sacrificed and the soleus muscles dissected
and weighed. The ratio of the right (denervated) to left (sham)
soleus wet weights for each animal was calculated to assess the
degree of atrophy caused by denervation and the prevention thereof
by treatment with each compound. For assessment of toxicity, the
body weights were calculated as a percent of the weight gain of
vehicle-treated rats. Both vehicle solutions produced similar
results in atrophy and body weight gain.
Results
Biological Activity In Vitro - To characterize the activity of rHCNTF
in vitro, we measured its effect on mediating the survival of
primary dissociated E8 chick ciliary neurons. Neuronal survival in
response to increasing concentrations of various human CNTF
variants is shown in Figures 6, 7 and 8. The variants RG228 (Fig. 7)
2 5 and RG297 (Fig. 8) that carry the 63QR substitution show 4-5 times
greater potency than rHCNTF but the variant RG242 showed a 10-fold
weaker potency than rHCNTF, despite the fact that it carries the
63QR substitution. Thus, introduction of various amino acid side
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chains at various positions of the CNTF sequence has very different
effects on the survival of primary neurons in vitro that vary from
great loss to strong gain of activity relative to rHCNTF.
Pharmacokinetics - Before attempting to correlate the in vitro
biological potency of a set of compounds to their pharmacological
efficacy in vivo, it is useful to determine their absolute
bioavailability in the same animal model. In the experiments
described below, the disposition kinetics after i.v. administration
and the absolute bioavailability after s.c. administration of RG228
and RG242 were determined and compared to those of rHCNTF.
The average plasma concentration time profiles in the rat
after IV administration of rHCNTF, RG228 and RG242 are shown in
Figure 9, normalized to 100 ~g/kg dose for all three compounds. The
average pharmacokinetic parameters are summarized in Table 1.
After i.v. administration to rats, RG242 had a distribution
phase a somewhat faster than that of rHCNTF and RG228. The
disposition phase ~i for RG242 and RG228 was faster than that of
rHCNTF. Thus, RG242 appeared to be distributed into the body and
2 0 cleared from systemic circulation somewhat more rapidly than
rHCNTF, whereas RG228 appeared to be distributed into the body as
fast as rHCNTF and cleared from systemic circulation somewhat
faster. The area under the concentration time curve (AUC) for RG242
was comparable to that of rHCNTF, indicating that the total body
clearance (CIT) was about the same for the two compounds. A twice
larger area was observed with RG228. However, the apparent volume
of distribution (Varea~~ which is a function of both a and AUC, was
approximately twofold smaller for both RG228 and RG242 relative to
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rHCNTF, suggesting that these variants are distributed less widely.
The limited number of animals used in these evaluations did not
allow the quantitative distinction of these values. However, these
results clearly indicate that the distribution and disposition
kinetics of RG228 and RG242 after i.v. administration are not
substantially different from those of rHCNTF.
After s.c. administration, RG228 and RG242 had a 2-3 fold
longer absorption phase (Ka) relative to rHCNTF (Fig. 10 and Table 2).
The disposition phase of RG242 was also somewhat longer. The
longer apparent terminal disposition phase of RG242 after s.c. dosing
compared to i.v. administration may be attributed to the incomplete
characterization of the terminal phase after the i.v. injection.
Overall, the absolute bioavailability of RG228 (13.7 %) and RG242
(10.9%) were comparable to that of rHCNTF (6.0%), in view of the
fact that in two previous independent studies, the absolute
bioavailability of rHCNTF was found to be 14.2 % (n=18) and 7.5%
(n=8) (D.B. Lakings, et al., DSER 93/DMAP/006, "Dose Proportionality
and Absolute Bioavailability of rHCNTF in the Rat Following
Subcutaneous Administration at Eight Dose Levels" (Phoenix
International Project No. 920847) 10 November 1993;
D.B. Lakings; et al., Dose Proportionality and Absolute Bioavailability
of rHCNTF Administered Subcutaneously to Rats. AAPS Ninth Annual
Meeting, San Diego, CA, November, 1994). Therefore, the
bioavailabilities of rHCNTF, RG228 and RG242 are not significantly
2 5 different within experimental error.
Efficacy and Toxicity In Vivo - In control experiments, denervation
of the soleus muscle resulted in a loss of 40% of muscle wet weight
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by 7 days. This value is very accurate and reproducible, since it
varies by only 3% among independent experiments. Daily
administration of rHCNTF resulted in a dose-dependent rescue of
muscle wet weight at an ED5o = 0.12 mg/kg and a maximal effect at
0.3 mg/kg (Figure 11 ). At the same time, even though animals
continued to gain weight during the course of these experiments,
they clearly did not gain as much as their vehicle-treated
counterparts (p<0.01; Figure 12), especially at the maximally
efficacious doses.
In the course of several experiments conducted in parallel with
rHCNTF, it was determined that the 63QR substitution resulted in a
2-fold increase in potency in vivo (Figure 11 ) but, also, a
concomitant 2 fold increase in toxicity (Figure 12). In contrast,
RG297, which carries the additional C17A and OC13 modifications,
shows a 2.6 fold greater potency but the same toxicity relative .to
rHCNTF. Finally, RG242 produced a 2.8 fold increased potency and an
2.4 fold decreased toxicity relative to rHCNTF. These results are
summarized in Table 3.
The relative therapeutic index (T.1.) for each of these
2 0 compounds was calculated as the ratio of the TD25 and EDSO values,
normalized to that of rHCNTF. While the T.I. of RG228 is equal to that
of rHCNTF, the T.I. of RG297 and RG242 is 2.5 and 6.8 fold superior to
that of rHCNTF, respectively.
Therefore, RG297 and RG242 have superior pharmacological
2 5 properties than rHCNTF. This is of great relevance to the clinical
situation where decreased body weight is observed upon rHCNTF
treatment in humans.
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One skilled in the art will recognize that other alterations in
the amino acid sequence of CNTF can result in a biologically active
molecule which may have enhanced properties. For example,
applicant has prepared a 17CS mutant which has a serine residue in
place of the cysteine residue at position 17 and is biologically
active. Applicant has also prepared a biologically active quadruple
mutant, 17CA,oC13,63QR,64WA. Further CNTF mutants, all of which
retain biological activity, are set forth in Table 4.
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Table 1. Average Pharmacokinetic Parameters for rHCNTF, RG228 and RG242
after Intravenous
Administration to
Rats at 100 wg/kg.
Pharmacokinetic Compound
Parameter rHCNTF RG242 RG228*
n 1 2 1
Co (ng/ml) 726 2,175 NC
AUCo_~(ng~min/ml) 20,230 22,890 55,800
a (min-1) 0.0492 0.0856 0.041
to2a (min) 14 8 17
~i (min-~) 0.0106 0.0200 0.0176
tii2p (min) 65 35 39
Varea (ml/kg) 470 220 204
CIT (ml/min/kg) 4.9 4.4 3.6
*RG228 values normalized to ~.g/kg i.v.
a 100 dose to
be comparable
to the
other two compounds that were 100 ~,g/kg.
administered
at
Co: Estimated by the first plasma concentrations
extrapolation of two to
time zero.
NC: Not calculated
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Table 2. Average Pharmacokinetic Parameters for rHCNTF, RG228 and RG242
After Subcutaneous Rats at wg/kg
Administration to 200
Pharmacokinetic Compound
Parameter rHCNTF RG242 RG228
n 2 2 1
Cmax (ng~ml) 18 32 50
Tmax (min) 30-45 30-45 60
AUCo_~(ng~min/ml) 2,425 4,980 7,620
Absolute
Bioavailability 6.0 10.9 13.7
ke (min-) 0.0133 0.0083 NC
t1/2ke (min) 52 82 NC
ka (min-1 ) 0.0401 0.0180 0.0102
tii2ka (min) 1 7 39 68
2 NC: Not calculated.
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Table 3. Efficacy, Toxicity and Therapeutic Index of rHCNTF and
Derivatives
Compound ED5o TD25 Therapeutic Index Relative
Therapeutic
(mg/kg) (mg/kg) (TD25/ED5o) Index
rHCNTF 0.12 0.087 0.72 1.0
RG228 0.065 0.047 0.72 1.0
RG297 0.045 0.080 1.78 2.5
RG242 0.043 0.21 4.88 6.8
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Table 4 - Biological activity of rHCNTF variants on E8 chick ciliary
neurons. Potency units (1/EC5o) are shown relative to human CNTF
which is assigned a value of 100. One potency unit is defined as the
reciprocal ligand concentration showing the same biological activity
as 1 ng/ml rHCNTF.
CNTF POTENCY
rat 500.0
1 0 human 100.0
17CS 100.0
63QA 87.0
1 5 63QN 100 . 0
630H 2.5
63OE ~1
63QK 1.1
63QR 400.0
64WA 2.0
63QR64WA
63QR64WF 250.0
63QR64WH 25.0
2 5 63OR64WO 10.0
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Exam 1e 7: Efficac of CNTF and variants in animal models
of Huntington's disease
Background
Glutamate receptor-mediated excitotoxicity has been
hypothesized to play a role in numerous neurodegenerative diseases,
including Huntington disease and motor neuron disease (DiFiglia, M. ,
1990, Trends Neurosci. 13:286-289; Rothstein, et al., 1995, J.
Neurochem. 65:643-651 ). The predominant neuropathological feature
of Huntington disease is a massive degeneration of the medium-
sized, GABAergic, striatal output neurons, without substantial loss
of striatal interneurons (Albin, et al., 1989, Trends Neurosci.
12:366-375; Harrington, et al., 1991, J. Neuropathol. Exp. Neurol.
50:309). The preferential loss of striatal output neurons observed in
Huntington disease, and the resulting dyskinesia, are mimicked in
rodent or primate models in which an NMDA glutamate receptor
agonist, quinoliriic acid, is injected into the striatum (DiFiglia, M.,
1990, Trends Neurosci. 13:286-289).
In the absence of a genetic animal model for HD,
neuroscientists continue to rely on acute lesion models for
investigation of the HD phenotype. The classic animal model of HD
involves production of an excitotoxic lesion of the rat striatum
using a glutamate agonist of the NMDA-receptor class. In such
lesion paradigms, injection of the neurotoxin directly into the
striatum results in loss of the medium sized intrinsic striatal
neurons which utilize gamma-aminobutyric acid (GABA) as their
neurotransmitter, with relative preservation of the two classes of
striatal interneurons which utilize either acetylcholine or
somatostatin and neuropeptide Y as their neurotransmitters. Most
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recent studies have relied upon intrastriatal injection of quinolinic
acid, which seems to most faithfully reproduce the appearance of
the HD striatum.
Figueredo-Cardenas et al. (1994, Exp. Neurol 129:37-56)
injected quinolinic acid (QA), into the striatum in adult rats and 2-4
months post lesion explored the relative patterns of survival for the
various different types of striatal projection neurons and
interneurons as well as the striatal efferent fibers in the different
striatal projection areas. The perikarya of all projection neuron
types (striatopallidal, striatonigral, and striato-entopeduncular)
were more vulnerable than the cholinergic interneurons. Among
projection neuron perikarya, there was evidence of differential
vulnerability, with striatonigral neurons appearing to be the most
vulnerable. Examination of immunolabeled striatal fibers in the
striatal target areas indicated that striato-entopeduncular fibers
better survived intrastriatal QA than did striatopallidal or
striatonigral fibers. The apparent order of vulnerability observed in
this study among projection neurons and/or their efferent fiber
plexuses and the invulnerability observed in this study of cholinergic
interneurons is similar to that observed in HD.
In another animal model, systemic administration of 3-
nitropropionic acid (3-NP) leads to neuropathological changes
similar to those seen in Huntington's disease (HD). Although the
behavioral hypoactivity seen in these animals differs from the
observed hyperactivity in most excitotoxic models of HD, 3-NP is
considered by some to provide a better model of juvenile onset and
advanced HD. The neuropathological effects of 3-NP include loss of
intrinsic striatal cholinergic neurons, but some sparing of large
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AChE positive neurons, minimal damage of NADPH-diaphorase-
containing neurons, and glial infiltration (Borlongan et al., 1995,
Brain Res. Bull. 365:49-56). There have been relatively few studies
with 3-NP as a neurotoxic model of HD. Its faithfulness and utility
remain to be explored.
Recent studies have begun to explore the relationship between
excitotoxic injury and the role of Huntingtin in the striatum.
Striatal injection of quinolinic acid in mice induces increased
immunoreactivity for Huntingtin in some remaining neurons but not
in glial cells. This increase is apparent in both neuronal cell bodies
and in cell processes in the white matter six hours after excitotoxic
challenge. Thus Huntington may be involved in the response to
excitotoxic stress in these neurons Tatter, et al., 1995, Neuroreport
6:1125-1129). Following an initial increase between 1 h and 6 h,
IT15 mRNA levels declined in a pattern homologous to a group of
neuron-specific genes. Decreased mRNA levels after 24 h
demonstrated that glial transcription is not activated by
neurodegeneration or gliosis. The 1 h and 24 h mRNA levels strongly
suggest that IT15 transcription preferentially localizes to
2 0 degenerating neurons. Oarlock et al., _1995, Neuroreport 6:1 121-
1 124.
Excitotoxic injury to the striatum also mimics certain of the
aspects of cell death seen in HD brain (Beal et al., 1986, Nature
321 :168-171 ). In the neostriatum of individuals with HD, patterns
of distribution of TUNEL-positive neurons and glia were reminiscent
of those seen in apoptotic cell death during normal development of
the nervous system; in the same areas, nonrandom DNA
fragmentation was detected occasionally. Following excitotoxic
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injury of the rat striatum, internucleosomal DNA fragmentation
(evidence of apoptosis) was seen at early time intervals and random
DNA fragmentation (evidence of necrosis) at later time points. In
addition, EM detected necrotic profiles of medium spiny neurons in
the lesioned rats. Thus, apoptosis occurs in both HD and excitotoxic
animal models. Furthermore, apoptotic and necrotic mechanisms of
neuronal death may occur simultaneously within individual dying
cells in the excitotoxically injured brain. (Portera et al., 1995, J.
Neuroscience 15:3775-3787).
The Tdt-mediated dUTP-biotin nick end labeling (TUNEL)
technique has been 'investigated in preliminary studies of a variety
of pathologic conditions of the human brain (e.g., gliomas, traumatic
brain injury, Parkinson's .disease, Parkinson's-Alzheimer's complex,
multisystem atrophy, striatonigral degeneration). Only Huntington's
disease revealed significant and consistent labeling with this
method. Thomas et al., 1995, Experimental Neurology 133:265-272).
c-fos expression increases soon after quinolinic acid injection, is
widespread in rat brain, but is effectively absent by 24 h
postinjection. DNA fragmentation, however, is limited to striatum
2 0 and is maximal at 24 h after injection. These results demonstrate
the sensitivity of in situ nick translation for the detection of
regional neuropathology and illustrate the temporal and spatial
relationship of c-fos expression to excitotoxic neuronal death (Dure
et al., 1995, Exp. Neurol. 133:207-214 ).
2 5 Excitotoxic lesions have also been used to explore possible
therapeutic avenues in HD. Excitotoxic striatal lesions induced by
quinolinic acid, a model for Huntington's disease, have been used to
test for neuroprotective actions of nerve growth factor (NGF) on
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striatal cholinergic and GABAergic neurons in adult rats following
quinolinic acid lesion (150 nmol). Daily intrastriatal NGF
administration for one week increased the cellular expression of
choline acetyltransferase messenger RNA three times above control
levels and restored the levels of Trk A messenger RNA expression to
control levels. In contrast to the protective effects on cholinergic
cells, NGF treatment failed to attenuate the quinoliriic acid-induced
decrease in glutamate decarboxylase messenger RNA levels. Thus,
striatal glutamate decarboxylase messenger RNA-expressing
GABAergic neurons which degenerate in Huntington's disease are not
responsive to NGF.
Frim; et al. (1993, J. Neurosurg. 78:267-273) implanted
fibroblasts secreting NGF into quinolinic-acid lesioned rat striata.
They found that preimplantation of NGF-secreting fibroblasts placed
within the corpus callosum reduced the maximum cross-sectional
area of a subsequent excitotoxic lesion in the ipsilateral striatum by
80% when compared to the effects of a non-NGF-secreting fibroblast
graft, and by 83% when compared to excitotoxic lesions in ungrafted
animals (p < 0.003).
Materials and Methods
Trophic Factors. Recombinant human BDNF, nerve growth
factor (NGF) and NT-3, and recombinant rat CNTF were prepared in E.
coli and characterized as described (Maisonpierre, et al., 1990,
2 5 Science 247:1446-1451; Masiakowski, et al., 1991, J. Neurochem.
57:1003-1012). Axokinel (Ax1 ) is the designation for recombinant
human CNTF with the following modifications: substitutions of
alanine for cysteine at position 17 and arginine for glutamine at
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position 63, and deletion of the 13 C-terminal amino acids. This
CNTF analog has enhanced solubility, is stable for at least a week at
37°C in physiological buffer, and exhibits 4-5-fold greater potency
in vitro relative to native human CNTF (Panayotatos et al., 1993, J.
Biol. Chem. 268:19000-19003).
Animal Treatments. All animal procedures were conducted in
strict compliance with protocols approved by the institutional
animal care and use committee.
Trophic factor delivery by osmotic pump. A 30-gauge osmotic
pump infusion cannula and a 22-gauge guide cannula (5.0 and 2.2 mm
long, respectively) were chronically implanted side-by-side into the
left hemisphere (stereotaxic coordinates AP 0.7, ML 3.2 relative to
bregma; incisor bar 3.3 mm below the interaural line) in 250-300 g
male, Sprague-Dawley rats under deep chloral hydrate (170 mg/kg)
and pentobarbital (35 mg/kg) anesthesia. Thirty days later, the rats
were again anesthetized and an Alzet osmotic minipump 2002 (two-
week capacity at a delivery rate of 0.5 ~,I/hr), containing 0.1 M
2 0 phosphate buffered saline (PBS) (pH 7.4), or PBS solutions of
recombinant human NGF (0.9 mg/ml), human BDNF (1 mg/ml), human
NT-3 (1 mg/ml), rat CNTF (0.78 mg/ml), or Ax1 (0.4 mg/ml) was
connected by plastic tubing to the infusion cannula and implanted
subcutaneously (Anderson, et al., 1995, J. Comp. Neurol. 357:296-
2 5 317). Due to the dead volume of the infusion cannula and tubing, the
delivery of neurotrophic factor into the brain began about 1 day after
pump implantation. Neurotrophins maintained in osmotic pumps at
37°C for 12 days were completely stable, as determined by bioassay,
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and effective intrastriatal delivery of the neurotrophins was
verified by immunohistochemical staining of sections for the
appropriate factor (Anderson, et al., 1995, J. Comp. Neurol. 357:296-
317). Three or four days after pump implantation, anesthetized rats
received an injection of quinolinic acid (50 nmol in 1 ~.I phosphate
buffer, pH 7.2, over 10 minutes) through the guide cannula using a
10-~I Hamilton syringe with a 28-gauge blunt-tipped needle.
Trophic factor delivery by daily infection. A 22-gauge guide
cannula (2.2 mm long) was chronically implanted into the left
hemisphere (stereotaxic coordinates AP 0.5, ML 3.0) of anesthetized
rats, as described above. Beginning 1 week later, anesthetized rats
received a daily intrastriatal injection of Ax1 (0.4 ~.g in 1 ~I, over
10 minutes) or vehicle through the guide cannula using a Hamilton
syringe. Ax1 was injected for 3 consecutive days before and 1 day
after injection of quinolinic acid, which was injected as described
above.
Histological Procedures and Analysis. Brains perfusion-fixed
in 4% paraformaldehyde were collected 8 or 9 days after the
quinolinic acid injection, and cut in the coronal plane into forty-
micron thick sections ~ that were stained with thionin. In each
experiment, a series of 1 in 12 Nissl-stained sections was evaluated
by an investigator unaware of treatment conditions, and the relative
2 5 loss of medium-sized striatal neurons was rated on the following
scale: 0 (no neuron loss), 1 (clear but slight neuron loss), 2
(moderate neuron loss), 3 (severe but not total neuron loss), 4 (total
loss of medium-sized neurons within the field of the quinolinic acid
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injection). In cases where neuron loss appeared intermediate to two
criteria, a half score between the two closest scores was assigned.
Neuron loss scores that were assigned independently by two
different observers in the experiments using BDNF and NT-3 were
within 0-0.5 points of each other for 40 of 42 rats (correlation
coefficient = 0.8; p=.0001 ).
In the experiment using CNTF, neuron loss also'was evaluated
by counting neurons in sections taken 0.5 mm rostral to the infusion
cannula. For each section, neurons were counted that intersected
1 0 every vertical line of a 10 x 10 sampling grid placed over seven
fields, 0.4 x 0.4 mm, within the treated striatum. The first field
was located slightly lateral to the center of the striatum, at the
center of a typical quinolinic acid-induced lesion (i.e. immediately
rostral to the tip of the infusion cannula). The six other fields were
selected by moving diagonally from the first field, twice each in the
dorsomedial and the ventromedial directions, and once each in the
dorsolateral and the ventrolateral directions. To control for
possible variation in section thickness, seven fields in equivalent
locations were sampled in the contralateral striatum (approximately
600 neurons counted per 7 fields), and neuron survival was
expressed as a percentage of neurons on the treated side relative to
the intact side. The results of actual neuron counts (31 and 61
neuron loss for CNTF- and PBS-treated groups, respectively) showed
close agreement with the results of the neuron loss scoring system
2 5 (mean neuron loss scores of 1.67 and 3.25, respectively), as
assessed by regression analysis (Spearman rank correlation
coefficient=0.82, p<0.05).
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Differences between experimental groups and their respective
control groups were evaluated by unpaired t-test.
Results
In a series of experiments,quinolinic
acid (50
nmol) was
injected into the left striatumadult rats or 4 days after the
of 3
start of intrastriatal infusionneurotrophicfactor ' by osmotic
of
pump (nominal ~g/day; human BDNF
delivery rates:
human NGF,
10.8
or NT-3, 12.0 ~,g/day; rat CNTF,9.4 ~,g/day).This dose of quinolinic
acid is toxic to medium-sized neurons, which
striatal output
constitute over 90% of all striatal neurons, yet leaves the striatal
populations of cholinergic interneurons and parvalbumin/GABAergic
interneurons largely intact (Qin, et al., 1992, Experimental Neurology
1 15:200-211; Figueredo-Cardenas, et al., 1994, Exp. Neurol. 129:37-
56). Microscope analysis of Nissl-stained sections from brains
collected 8-9 days after injection of quinolinic acid demonstrated
no significant sparing of medium-sized striatal neurons in BDNF-,
NGF-, or NT-3-treated brains (Fig. 13). In an additional set of
experiments, no neuron sparing was apparent when quinolinic acid
was injected 7 days after the start of BDNF or NGF infusion.
In striking contrast, neuron survival was significantly greater
in rats treated with CNTF compared to rats treated with vehicle
alone (Fig. 14), as determined by neuron counts that demonstrated a
mean percent survival (~ SEM) of 69 ~ 17 and 29 ~ 11 %, respectively
2 5 (unpaired t-test, t(5)=2.12, p=0.04), or as assessed by assignment of
semi-quantitative neuron loss scores (Fig. 15). Surviving neurons in
CNTF-treated brains were disseminated throughout the striatal area
affected by the quinolinic acid injection
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Given the favorable effect demonstrated by CNTF, a similar
experiment was conducted using a polypeptide CNTF receptor
agonist, Axokine 1 (Ax-1 ) (24). As observed after administration of
CNTF, infusion of Ax-1 (4.8 ~g/day) resulted in significant sparing
of medium-sized striatal neurons exposed to quinolinic acid (Figure
15). This result supports the conclusion that CNTF receptor-
mediated mechanisms effect protection of striatal neurons from
NMDA receptor-mediated excitotoxicity.
The neuroprotective effect of CNTF or Ax-1 was achieved
without apparent adverse effects on behavior or health, as indicated,
for example, by body weight. Body weights measurea at me ena or
the experiments were not significantly affected by CNTF or Ax-1
treatment (unpaired t-test). The mean body weights (~ SEM) of the
trophic factor-treated and the vehicle-treated groups in the CNTF
1 5 experiment were 369 ~ 20 g and 331 ~ 15 g, respectively, (p = 0.21 );
mean body weights in the Ax-1 experiment were 431 ~ 26 g and 453
~ 14 g, respectively, (p = 0.44).
Two additional experiments were performed to determine
whether the neuroprotective effect of CNTF receptor ligands might
persist after termination of neurotrophic factor administration, and
whether treatment is effective when a lower dose of trophic factor
is delivered intermittently. In the tirst experiment, rats were
infused intrastriatally with Ax-1 (4.8 ~g/day) or vehicle for 3 days
and then delivery was terminated by removal of the osmotic pump.
2 5 Quinolinic acid was injected into the striatum 3 days thereafter (Fig
16A). In the second experiment, rats received a daily intrastriatal
injection of Ax-1 (0.4 ~,g/day) or vehicle for 3 days before and 1 day
after intrastriatal - injection of quinolinic acid (Fig. 16B); thus these
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rats received a total of only 1.6 wg Ax-1. In both experiments,
microscope analysis of Nissl-stained sections demonstrated
significant sparing of medium-sized striatal neurons in Ax-1-
treated brains that was comparable to sparing seen when CNTF or
Ax-1 were infused continuously for the duration of the experiment
(Figure 16).
Discussion
Since over 90% of the neurons in the striatum are medium-
sized, GABAergic, striatonigral and striatopallidal projection
neurons (Graybiel, A.M., 1990, TINS 13:244-254), the present results
show that treatment with CNTF or a CNTF receptor agonist protects
striatal output neurons against excitotoxic insult. Thus, CNTF is one
of the first purified trophic factors demonstrated to protect striatal
output neurons after pharmacological application in an adult animal
model of Huntington disease. Among other factors characterized,
only treatment with basic fibroblast growth factor has been
reported to diminish the size of a striatal lesion induced by
injection of N-methyl-D-aspartate (NMDA) or malonic acid in adult
2 0 and neonatal rats (Nozaki, et al., 1993, J. Cereb. Blood Flow Metab.
13:221-228; Kirschner, et al., 1995, J. Cereb. Blood Flow Metab.
15:619-623). Although NGF-secreting fibroblasts implanted near the
striatum have been shown to protect medium-sized striatal neurons
from quinolinic -acid in rats (Frim, et al., 1993, NeuroReport 4:367-
370; Emerich, et al., 1994, Exp. Neurol. 130:141-150), we obtained no
survival-promoting effect on these neurons with purified NGF, in
agreement with several earlier studies (Davies, et al., 1992,
Neurosci. Lett. 140:161-164; Venero, et al., 1994, Neuroscience
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61:257-268; Kordower, et al., 1994, Proc. Natl. Acad. Sci. USA
91:9077-9080). This finding suggests that NGF is not the sole
mediator of the neuroprotection provided by NGF-secreting
fibroblasts. We did, however, observe that the large, darkly
staining, presumably cholinergic interneurons were more prominent
in NGF-treated brains, as previously reported (Davies, et al., 1992,
Neurosci. Lett. 140:161-164; Kordower, et al., 1994, Proc. Natl. Acad.
Sci. USA 91:9077-9080; Perez-Navarro, et al., 1994, Eur. J. Neurosci.
6:706-711). Striatal expression of the high-affinity NGF receptor,
TrkA, is restricted to cholinergic interneurons (Steininger, et al.,
1993, Brain Res. 612:330-335), consistent with the finding of a
selective action of NGF on these neurons, whereas the high-affinity
receptors for BDNF and NT-3 (TrkB and TrkC) are expressed by
numerous medium-sized striatal neurons (Altar, et al., 1994, Eur. J.
1 5 Neurosci. 6:1389-1405). BDNF and NT-3 (unlike NGF) promote the
survival and phenotypic differentiation of embryonic, GABAergic,
striatal output neurons in vitro (Mizuno, et al., 1994 Dev. Biol.
165:243-256; Ventimiglia, et al., 1995, Eur. J. Neurosci). Moreover,
these neurotrophins can protect certain neuron populations from
glutamate toxicity in vitro (Lindholm, et al., 1993, Eur. J. Neurosci.
5:1455-1464; Shimohama, et al., 1993, Neurosci. Lett. 164:55-58;
Cheng, et al., 1994, Brain Res. 640:56-67). Nevertheless, infusion of
BDNF or NT-3 does not appear to protect striatal output neurons
against NMDA receptor-mediated excitotoxicity in vivo, although
intracerebral infusion of BDNF or NT-3 at comparable doses elicits
pronounced biological effects in the striatum and elsewhere in the
brain (Lindsay, et al., 1994, TINS 17:182-190). The contrasting
results between in vivo and in vitro studies may be explained by
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differences in neuron type (striatal vs. hippocampal, cortical or
cerebellar), a difference in the developmental stage of the neurons
(adult vs. embryonic), or the presence of glutamatergic synaptic
input in vivo.
The neuroprotective effect displayed by CNTF receptor ligands
may occur through direct action on medium-sized striatal neurons,
since there is abundant expression of mRNA for components of the
CNTF receptor (CNTFRa, LIFR(3, gp130) in the striatum (1p, et al.,
1993, Neuron 10:89-102; Rudge; et al., 1994, Eur. J. Neurosci. 6:693-
705). Potential mechanisms might include alteration of the
expression or function of glutamate receptors, thereby modifying
neuron sensitivity to glutamatergic stimulation, or enhancement of
the neuron's capacity to regulate the cytosolic concentration of
calcium ion, an increase in which is thought to be a critical event
initiating the neurodegenerative process (Choi, D. W., 1988, Neuron
1:623-634). The possibility that CNTF acts as a glutamate receptor
antagonist to block quinolinic acid toxicity is unlikely, since CNTF
does not block the toxic effects of glutamate in vitro (Mattson, et
al., 1995, J. Neurochem. 65:1740-1751 ).
On the other hand, CNTF receptor ligands could potentially act
indirectly, via other components of the striatum. For example,
elimination of nigral or cortical input to the striatum prior to
exposure to quinolinic acid results in a significant reduction in the
loss of striatal -neurons (DiFiglia, M., 1990, Trends Neurosci. 13:286-
289; Buisson, et al., 1991, Neurosci. Lett. 131:257-259) indicating
that the combined actions of exogenous toxin and endogenous
neurotransmitters are required to induce cell death. Thus, a
reduction in synaptic transmission at either glutamatergic or .
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dopaminergic synapses would likely protect striatal neurons from an
injection of quinolinic acid. Although astrocytes do not normally
express detectable CNTFRa -in vivo (1p, et al., 1993, Neuron 10:89-
102), astrocytes do express all CNTF receptor components when
activated by brain injury or when maintained in vitro (Budge, et al.,
1994, Eur. J. Neurosci. 6:693-705). Furthermore, intracerebral
delivery of CNTF appears to activate astrocytes 10-4.8 hours after
exposure, as indicated by increased content of glial fibrillary acidic
protein and its mRNA (Levison, et al., 1995, Soc. Neurosci. Abst.
1 0 21:497; Winter, et al.; 1995, Proc. Natl. Acad. Sci. USA 92:5865-
5869). Whether activated indirectly or directly by CNTF, astrocytes
might promote neuron survival through enhanced sequestration of
excitatory amino acids or by release of substances that protect
neurons.
The striatal neuron populations protected from excitotoxic
damage by CNTF receptor-mediated events in the present study are
the same types selectively lost in Huntington disease (Albin, et al.,
1989, Trends Neurosci. 12: 366-375). A potential link between
excitotoxic stimulation and increased expression of the Huntington
2 0 disease gene has recently been suggested (Carlock, et al., 1995,
NeuroReport 6:1121-1124; Tatter, et al., 1995, NeuroReport 6:1125-
1129). While extensive studies are in progress to identify the
mechanisms which lead to Huntington disease, existing lines of
evidence clearly implicate a role for NMDA receptor-mediated
excitotoxicity -(DiFiglia, M., 1990, Trends Neurosci. 13:286-289).
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EXAMPLE 8: PEGyrlation of Axokine Protein
Pegylation of proteins has been shown to increase their in vivo
potency by enhancing stability and bioavailability while minimizing
immunogenicity. It is known that the properties of certain proteins
can be modulated by attachment of polyethylene glycol (PEG)
polymers, which increases the hydrodynamic volume of the protein
and thereby slows its clearance by kidney filtration. (See. eTa. Clark,
R., et al., 1996, J. Biol. Chem. 271: 21969-21977). We have
generated PEGylated Axokine by covalently linking polyethylene
glycol (PEG) to Ax-13. We have also developed a purification
methodology to separate different PEGylated forms of Axokine from
unmodified molecules. PEGylated Ax-13 has better solubility and
stability properties, at physiological pH, than unPEGylated Ax-13.
PEGylation has been shown to greatly enhance pharmacokinetic
properties of Ax-13 and would be expected to similarly enhance the
properties of other Axokine molecules.
Purified Ax-13 derived from E. coli was used for these studies.
20kD mPEG-SPA was obtained from Shearwater Polymers, Bicine
2 0 from Sigma, and Tris-Glycine precast gels from Novex, CA. A small
scale reaction study was set up to determine reaction conditions.
20kD mPEG SPA was reacted with purified Ax-13 at a final
concentration of 0.6 mg/ml, at 4°C in an amine-free buffer at a pH of
8.1. Molar ratios of PEG to protein were varied and two reaction
2 5 times were used. The reaction was stopped by the addition of a
primary amine in large excess. Reaction products were analyzed by
reducing SDS-PAGE. The predominant modified species ran at a
molecular weight of approximately 60 kD. Higher order modified
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bands that ran at higher molecular weights were also seen. Based on
this study, an overnight reaction at a PEG-to-protein ratio of 4 was
chosen.
Ax-13 at 0.6 mg/mL was reacted with 20 kD mPEG SPA in a
Bicine buffer overnight at 4°C at a pH of 8.1. The reaction was
stopped by the addition of a primary amine in large excess. The
reaction product was diluted with a low salt buffer and applied to an
ion-exchange column. The column was washed with a low salt buffer
and eluted with a NaCI gradient. A good separation between higher
1 0 order forms (apparent MW >66kD on SDS-PAGE), a distinct modified
species that ran at about 60kD and unmodified Ax-13 was obtained.
Fractions corresponding to the 60kD band were tested in a Bioassay.
A very faint band of unmodified Ax-13 was noticed in the fractions
corresponding to the 60kD band. To ensure that the bioassay results
were not influenced significantly by this material, the 60kD band
was further purified by Size exclusion chromatography (SEC) that
resulted in baseline separation between unmodified Ax-13 and the
60kD band. The purified modified Ax-13 was tested in a Bioassay and
the results were indistinguishable from those obtained with the
2 0 material prior to SEC.
EXAMPLE 9: Construction of Ax-15 Expression Plasmid
pRG643
2 5 The expression plasmid pRG632 is a high copy plasmid that
encodes ampicillin resistance and the gene for human CNTF-
C17A,Q63R,OC13 (also referred to herein as either Ax1 or Ax-13)
with a unique Eag I restriction enzyme recognition sequence 3' to the
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stop codon. This plasmid was used to construct a human CNTF
mutation C17A,Q63R,oC15 (designated Ax-15) by PCR amplification
of a 187 by BseR I-Eag1 DNA fragment that incorporates the DC 15
mutation. The 5' primer {0C15- 5' (5'-
CCAGATAGAGGAGTTAATGATACTCCT-3')} encodes the BseR I site and
the 3' primer OC15-3' {(5'-
GCGTCGGCCGCGGACCACGCTCATTACCCAGTCTGTGA GAAGAAATG-3')}
encodes the C-terminus of the Ax-15 gene ending at GIy185 followed
by two stop codons and an Eag I restriction enzyme recognition
1 0 sequence. This DNA fragment was digested with BseR I and Eag I and
ligated into the same sites in pRG632. The resulting plasmid,
pRG639, encodes the gene for Ax-15 (human CNTF C17A,Q63R,OC15).
The OC15 mutation was then transferred as a 339 by Hind III-Eag I
DNA fragment into the corresponding sites within pRG421, a high
1 5 copy number expression plasmid encoding the gene for kanamycin
resistance and human CNTF C17A,Q63R,oC13. The resulting plasmid,
pRG643, encodes the gene for Ax-15 under transcriptional control of
the IacUVS promoter, and confers kanamycin resistance. The Ax-15
gene DNA sequence was confirmed by sequence analysis.
EXAMPLE 10: Small Scale Expression and Purification of
Ax-15 Protein
E. coli strain RFJ141 containing pRG639 was grown in LB
medium and expression of Ax-15 protein was induced by the addition
of lactose to 1 % (w/v). Induced cells were harvested by
centrifugation, resuspended in 20 mM Tris-HCI, pH 8.3, 5 mM EDTA, 1
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mM DTT, and lysed by passage through a French pressure cell at
10,000 psi. The cell lysate was centrifuged and the pellet was
resuspended in 8 M guanidinium-HCI, 50 mM Tris-HCI, pH 8.3, 0.05
mM EDTA then diluted with 5 volumes of 50 mM Tris-HCI, pH 8.3,
0.05 mM EDTA (Buffer A) followed by dialysis against Buffer A. The
dialysate was loaded onto a Q-sepharose column equilibrated with
Buffer A. The Ax-15 protein was eluted by a linear gradient to 1 M
NaCI in 10 column volumes of buffer. Fractions containing Ax-15
were pooled and brought to 1 M (NH4)2S04 by the slow addition of
solid (NH4)2S04 while maintaining the pH at 8.3 by the addition of
NaOH. The pool was loaded onto a phenyl-sepharose column
equilibrated with 1 M (NH4)2S04 in Buffer A. The column was washed
with 0:5 M (NH4)2S04 in Buffer A, and the Ax-15 protein was eluted
by a linear gradient of decreasing (NH4)2S04 concentration.
Fractions containing Ax-15 protein were pooled, dialyzed against 5
mM NaP04, pH 8.3, then concentrated by ultrafiltration. The
concentrated pool was fractionated on an Sephacryl S-100 column
equilibrated with 5 mM NaP04, pH 8.3.
EXAMPLE 11: La_ rae Scale Expression and Purification of
Ax-15 protein
A recombinant, kanamycin resistant E. Coli strain RFJ141
expressing the Ax-15 protein under lac promoter control (pRG643)
was grown to an intermediate density of 30-35 AU55o (Absorbance C
550 nM) in a minimal salts, glucose medium containing 20 ~g/ml
Kanamycin. Expression of Ax-15 protein was induced by addition of
IPTG (isopropyl thiogalactoside) to 1.0 mM and the fermentation
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was continued for an additional 8 hr. Ax-15 protein was expressed
as insoluble inclusion bodies following IPTG induction. Post-
induction, cells were harvested, cell paste concentrated, and buffer
exchanged to 20 mM Tris, 1.0 mM DTT, 5.0 mM EDTA, pH 8.5 via AGT
500,000 molecular weight cut off (mwco) hollow fiber diafiltration
(ACG Technologies, Inc.). Inclusion bodies were released from the
harvested cells by disruption via repeated passage of cooled (0-
10~C) cell paste suspension through a continuous flow, high pressure
(>8,000 psi) Niro Soavi homogenizes. The homogenate was subjected
to two passages through a cooled (4-8~C) continuous flow, high
speed (>17,000 x G) Sharpies centrifuge (source) to recover inclusion
bodies. Recovered inclusion bodies were extracted in 8.0 M
Guanidine HCL with 1.0 mM DTT. The Ax-15 protein/guanidine
solution was diluted into 50 mM Tris-HCI, 1.0 mM DTT, 0.05 mM
1 5 EDTA, pH 8.0-8.3, and diafiltered versus diluent buffer with AGT
5,000 mwco hollow fiber filters (ACG Technologies, Inc.). The
resulting solution, containing refolded Ax-15, was filtered through a
Microgon 0.22 ~.m hollow fiber filter (ACG Technologies, Inc.) prior
to chromatographic purification.
EXAMPLE 12: Column Chromatographic Purification of
Refolded Ax-15
The filtered Ax-15 solution described above was loaded onto a
2 5 16.4 L DEAE Sepharose (Pharmacia) column at 10.9 mg/ml resin and
washed with 50 L of 50 mM Tris, pH 8.0-8.3, 1.0 mM DTT, and 0.05
mM EDTA buffer. The Ax-15 protein was eluted from the column
with a 120 mM NaCI step in the same Tris buffer. Eluate exceeding a
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previously established 280 nM absorbance criteria of 40% maximum
A28o on the ascending portion of the peak and 20% of maximum A28o
on the descending portion of the peak was pooled and either stored
frozen (-30~C) or used in the next step of the purification procedure.
Pooled eluted Ax-15 protein was adjusted to 1.0 M ammonium
sulfate by gradual addition of the solid compound, maintaining the pH
at 8.0-8.3. The solution was filtered through a 0.22 ~,m Sartorious
capsule filter, loaded onto a 12.5 L phenyl Sepharose HP (Pharmacia)
column at 8.24 mg/ml of resin, and washed with 55 L of 1.0 M
1 0 ammonium sulfate in 50 mM Tris buffer with 0.05 mM EDTA, pH 8.0-
8.3. Following a 12.0 L wash with 250 mM ammonium sulfate in the
same Tris buffer, the Ax-15 protein was eluted with a 125 mM
ammonium sulfate, Tris buffer wash step. Eluate exceeaing
previously established 280 nM absorbance criteria of 100% maximum
1 5 A28o on the ascending portion of the peak and 20% of maximum A28o
on the descending portion of the peak was pooled. Eluate was
simultaneously diluted 1:4 into 50 mM Tris, pH 8.0-8.3 buffer
without salt to reduce its conductivity. Pooled material was stored
frozen (-30~C) or used in the following step. Pooled hydrophobic
20 interaction chromatography (HIC) material was concentrated to 25 L
and diafiltered versus 5.0 mM sodium phosphate buffer pH 8.0-8.3
using a 5,000 mwco AGT hollow fiber filter (ACG Technologies, Inc.).
The pH was adjusted to 7.0-7.2 immediately prior to sulfyl propyl
fast flow (SP FF) sepharose chromatography by gradual addition of
2 5 concentrated (85%) phosphoric acid. The pH-adjusted pooled
material was loaded onto a 7.7 L SP FF sepharose (Pharmacia)
column to 9.0 mg/ml of resin and washed with a minimum of 25 L of
5.0 mM sodium phosphate buffer, pH 7Ø The Ax-15 protein was
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eluted with a 77.0 L step of 5.0 mM sodium phosphate, 130 mM NaCI,
pH 7.0-7.2. The eluate was simultaneously diluted 1:5 into 10.0 mM
sodium phosphate, pH 9.0-9.2 buffer without salt to reduce
conductivity and increase pH. Peak material exceeding 20%
maximum A28o on the ascending portion of the peak and 20% of the
maximum A28o on the descending portion of the peak was pooled.
Pooled Ax-15 protein was stored frozen (-30~C) or used in the
following step. Pooled SP FF sepharose Ax-15 protein was
concentrated and diafiltered versus 5.0 mM sodium phosphate, pH
1 0 8.0-8.3 buffer with a 5,000 mwco AGT hollow fiber filter (ACG
Technologies, Inc.): The pool (24.66 g) was concentrated to <5.0 L.
Concentrated, diafiltered Ax-15 protein was loaded onto a 50 L S-
100 Sephacryl (Pharmacia) sizing column and eluted with 250 L of
the same 5.0 mM sodium phosphate buffer, pH 8.0-8.3. Peak material
1 5 exceeding 40% maximum A28o on the ascending portion of the peak
and 40% of the maximum A28o on the descending portion of the peak
was pooled. The pooled Ax-15 protein was filtered through Millipak
0.22 ~.m filters and stored at -80~C prior to dispensing or
formulation. The amino acid sequence of Ax-15 produced follows.
2 0 Alternatively, one could produce a sequence which contains a
Methionine residue before the initial Alanine.
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9 . 19 29 39 49 S
* * * * * * k k k k' k
AFTEHSPLT PHRRDLASRS IWLARKIRSD LTALTESYVK fiQGLNKNINL DSADGMPVAS
69 79 89 99 109 119
* ik k * * k k * k k * *
TDRWSELTEA ERLQErILQAY RTFHVLLARL LEDQQVHFTP TEGDFHQAIH TLLLQVAAFA
1 0 129 139 149 159 169 179
* * k * * * * * k k * *
YQIEELMILL EYKIPRNEAD GMPINVGDGG LFEKKLWGLK VLQELSQWTV RSIHDLRFIS
SHQTG
Methioninei-
20 30 40 50 60
k * * * k k * k k k' * x
MAFTEHSPLTPHRRDLASRSIWLARKIRSDLTALTESYVKHQGLI3~CNINLDSADGMPVAS
70 80 90 100 110 120
k k' k k k k k k k k k' k
TDRWSELTEAERLQEL~ITlQAYRTFI~VLLARLLEDQQVHFTPTEGDFHQAIHTLLLQVAAFA
130 140 150 160 170 180
k' * * k k k' k' k
* k k k GMPINVGDGGLFEKKLWGLKVLQELSQWTVRSIHDLRFIS
YQIEELMILLEYKIPRNEAD
k
SHQTG
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EXAMPLE 13: Use of Ax-15 to treat obesity
Animal models
Normal mice
Normal (8weeks) C57BL/6J mice were obtained from Taconic.
The mice received daily subcutaneous injections of vehicle or Ax-15.
The animals were weighed daily and food intake over 24-hours was
determined between days 3 and 4.
ob/ob mice
1 0 As a result of a single gene mutation on chromosome 6, ob/ob
mice produce a truncated, non-functional gene product (Leptin).
These mice are hyperphagic, hyperinsulinemic, and markedly obese.
C57BL/6J ob/ob mice were obtained from Jackson Laboratory
and used for experiments at 12-14 weeks of age. The mice received
daily subcutaneous injection of vehicle, Ax-15, or leptin. Pair-fed
group was given the average amount (g) of food consumed by animals
treated with Ax-15 (0.3 mg/kg). Body weights were obtained daily
and food intake over 24-hours was determined between days 3 and 4.
On day 8, the animals were sacrificed and carcass analysis was
performed.
Diet-induced obesit DIO mice
AKR/J mice have been shown to be very susceptable to diet
induced obesity by increasing body fat content. Although the gene-
environment(diet) interaction is not completely known regarding
2 5 this kind of dietary obesity, like in human obesity, the genotype is
polygenic.
AKR/J mice were obtained from Jackson Laboratory and put on
a high fat diet (45% fat; Research Diets) at age 10-12 weeks old. All
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experiments commenced after 7 weeks on high fat diet. The mice
received daily subcutaneous injection of vehicle, Ax-15, or Leptin.
Pair-fed group was given the average amount (g) of food consumed by
animals treated with Ax-15 (0.1 mg/kg). The animals were weighed
daily and food intake over 24-hours was determined between days 3
and 4. On day 8, the animals were sacrificed and sera were obtained
for insulin and corticosterone measurements.
Reagents
Recombinant human Ax-15 was manufactured as set forth above and
Leptin was purchased from R & D Systems.
Results
Normal mice
1 5 Ax-15 reduced body weight in normal mice in a dose dependent
manner. In 6 days, the animals lost approximately 4%, 11 %, and 16%
of their body weight at 0.1 mg/kg, 0.3 mg/kg, and 1 mg/kg,
respectively (Figure 17).
ob/ob mice
2 0 There was a dose related (0.1 mg/kg - 3 mg/kg) decrease in
body weight after Ax-15 treatment in ob/ob mice (figure 18). At a
dose range of 0.1 mg/kg to 3 mg/kg, there was a 8%-25% reduction
of body weight. Animals pair-fed to a specific dose of Ax-15 (0.3
mg/kg) showed equivalent loss of body weight as the mice given that
25 dose of Ax-15, suggesting food intake is the primary cause of weight
reduction.
Leptin was also effective in decreasing body weight in ob/ob
mice. At 1 mg/kg, leptin decreased body weight 6% in 7 days,
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following a course almost identical to that of Ax-15 given at 0.1
mg/kg (figure 18).
Carcass analysis showed that there was a significant
reduction of total body fat with Ax-15 and Leptin treatments as
well as in pair-fed controls (Table 5). There was a small but non-
significant loss of lean mass in all groups as compared to vehicle
control animals. Mice receiving only food restriction (pair-fed) had
a fat/lean mass ratio no different from vehicle controls, indicating
that they lost fat and lean mass equally. However, the Ax-15 and
Leptin treated animals showed preferential loss of body fat as
reflected by a decrease in fat/lean mass ratio (Table 5).
DIO mice
Ax-15 reduced body weight in DIO mice dose dependently.
Within one week, the animals lost approximately 14%, 26%, and 33%
of their body weight when given Ax-15 at 0.1 mg/kg, 0.3 mg/kg, and
1 mg/kg, respectively (Figure 19). Comparing the effects of the Ax-
15 treatment and the pair-fed control animals, there was a small
but significant difference between the 2 groups, suggesting that
decreased food intake was probably the primary, although not the
only, cause of weight loss with Ax-15 treatment. Indeed, Ax-15
significantly attenuated the obesity associated hyperinsulinemia in
DIO mice, whereas merely reducing food intake (pair-fed) did not
(Figure 20A). In addition, Ax-15 did not cause elevation of
corticosterone levels, which is a common effect of food restriction
(Figure 20B).
It is of interest to note that when Ax-15 was administered in
the same dose range (0.1-1 mg/kg), DIO mice lost more than twice
the body weight when compared to normal mice (see Figure 17). This
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higher sensitivity of diet-induced obese animals to Ax-15 suggests
that adiposity may regulate the efficacy of Ax-15 such that Ax-15
will not cause continuous weight loss after adiposity is normalized.
DIO mice are leptin resistant; no weight loss effect was
observed in these animals with daily injection of leptin (1 mg/kg;
Figure 19).
We conclude as follows:
1. Ax-15 caused weight loss in normal mice in a dose
dependent manner.
1 0 2. Ax-15 induced weight loss in ob/ob mice in a dose
dependent manner. Ax-15 (0.1 mg/kg) was as effective as Leptin (1
mg/kg) in causing weight loss in ob/ob mice. Both Ax-15 and Leptin
treatments, but not pair-fed, preferentially reduced total body fat
over lean mass.
3. Ax-15 caused weight loss in diet-induced obesity mice in a
dose dependent manner, whereas Leptin was ineffective. Ax-15
treatment attenuated obesity associated hyperinsulinemia in DIO
mice; this effect was not observed in pair-fed control animals. In
addition, Ax-15 was more effective in inducing weight loss in DIO
2 0 mice than normal or ob/ob mice. Taken together, our results suggest
a specific useful application of Ax-15 in the treatment of leptin
resistant obesity, such as type II diabetes associated obesity.
4. The effectiveness of Ax-15 in reducing body weight in leptin
resistant mouse model suggests that Ax-15 may also be effective in
2 5 reducing body weight in obese humans who are resistant or
unresponsive to Leptin.
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Tables: Results from carcass anafysls of ob/ob mice
Fat g Lean mass g Fat:Lean mass
Vehicle Mean 34.77 4.79 7.26
sem 1 .41 0.24
1 0 Palr-fed to Ax-15 0.3 mg/kg 29.36 4.03 7.28
0.93 0.07
Ax-15 0.1 mg/kg 30.22 4.38 6.9
0.59 0.13
Ax-15 0.3 mg/kg 26.77 4.03 6.64
0.66 0.08
Ax-15 1 mg/kg 23.29 3.35 6.95
0.87 0.12
Ax-15 3 mg/kg 23 3.5 6.57
1 5 0.53 0.12
l_eptln 1 mg/kg 28.89 4.73 6.11
0.89 0.1
EXAMPLE 14: Peg_~ lad ted Ax-15
Applicants have generated several different pegylated Ax-15
molecules by covalently linking polyethylene glycol chains of
2 5 different lengths and types to Ax-15 polypeptide molecules.
Applicants have also developed purification methodologies to
separate different pegylated forms of Ax-15 from unmodified Ax-15
molecules.
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Materials and Methods
Purified Ax-15 derived from E. coli (supra) was used for these
studies. PEG chains of various molecular weights functionalized
with amine-specific terminal moieties were obtained from
Shearwater Polymers, AL. Bicine was obtained from Sigma, MO, and
Bis-Tris precast gels were obtained from Novex, CA. Small-scale
reaction studies were set-up to test various reaction conditions.
Different reaction conditions were used and the following were
varied:
1. Ax-15 protein concentration: ranging from 0.6 mg/ml to 6.0
mg/ml.
2. PEG/Ax-15 protein molar ratios up to 30:1.
3. Temperature: 4°C to room temperature
Additionally, in instances where an aldehyde chemistry was
used, different concentrations of a reducing agent (for example
sodium cyanoborohydride from Aldrich Chemicals, Milwaukee, WI)
was used to reduce the Schiff base. The reactions were stopped by
2 0 the addition Tris-HCI, pH 7.5 in large excess from a 1 M stock
solution obtained from Life Technologies, Gaithersburg, MD.
Typically, 50 mM Tris-HCI, pH 7.5 is used as the protein
concentration was in the ~,M range.
For purification, the reaction products were typically diluted
with a low salt buffer and applied to an ion-exchange column. The
column was washed with a low salt buffer and eluted with a NaCI
gradient ranging from 0 to 300mM NaCI in a l5mM Bicine buffer over
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a column packed with Q-HP anion exchange resin obtained from
Pharmacia, Piscataway, NJ. A good separation between unmodified
Ax-15 and pegylated forms corresponding to different numbers of
attached PEG chains was observed. Different pools from the ion-
s exchange purification were concentrated and further purified by
standard preparative size-exclusion chromatography. In some cases,
two close forms of PEG Ax-15 protein were pooled together and
treated as one sample (e.g.: a sample marked PEG 5K (3,4)- 2°Amine-
Ax 15 would consist predominantly of Ax-15 molecules attached
1 0 with 3 or 4 chains of approximately 5KD PEG molecules using
2°amine linkages.)
Reaction products and samples from purification runs were
analyzed by any or all of the following standard methods:
1. SDS-PAGE under reducing and non-reducing conditions
15 2. Analytical ion-exchange chromatography
3. Analytical size exclusion chromatography
The number of chains attached to each Ax-15 molecule was
initially assigned to samples based on band patterns on SDS-PAGE
gels. Confirmation was obtained using a free amine assay to detect
2 0 primary amines based on published techniques (Karr, L.J. et. al.,
Methods in Enzymology 228: 377-390 (1994)) or by using an
analytical size exclusion column coupled to a MALLS ( Multi-angle
laser light scattering) system with UV, RI (Refractive Index) and
MALLS detectors in series. Light scattering is a function of mass
2 5 and concentration of a macromolecule. To determine molecular
weight, the protein sample is injected onto a gel filtration column
and the effluent is monitored with an on line light scattering
detector and a refractive index and/or a UV detector. The light
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scattering detector is a MiniDawn laser light scattering detector
was from Wyatt Technology Corporation (Santa Barbara, CA). This
instrument measures static light at three different angles. The on
line refractive index detector or UV detector serve to measure
protein concentration. Astra 4.7 Software (Wyatt Technology
Corporation, Santa Barbara, CA) is used to calculate the protein
concentration based on either dn/dc (dn = change of refractive index;
do = concentration) or the extinction coefficient of the protein. The
SEC-MALLS system was also used to detect the purity and molecular
weights of PEG Ax-15 preparations.
The various PEG Ax-15 molecules were tested in in vivo
experiments as follows.
EXAMPLE 15: In vivo -experiments usin PEG Ax-15 to treat
obesity.
AKR/J mice have been shown to be susceptible to diet-induced
obesity by increasing body fat content. Although the gene-
environment (diet) interaction is not completely understood
regarding this kind of dietary obesity, as in human obesity, the
genotype is polygenic. The following experiments were performed to
test the effects of PEG Ax-15 on body weight and food intake in this
experimental animal model of diet-induced obesity. The particular
molecule that is described in the experiments is called 1-20-PEG
2 5 Ax-15 and is just one of the many pegylated Ax-15 molecules
produced by the procedures described above and tested in in vivo
experiments. This molecule is mono-pegylated with a 20 KD PEG
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chain via a 2°. amine linkage. Table 6 shows a Comparison of the In
Vivo Activity of Various Pegylated Ax-15 Preparations.
10
20 Experimental procedure
Male AKR/J mice (The Jackson Laboratory, Bar Harbor, ME)
were fed a high fat diet (with 45 kcal% from fat) starting at 10
weeks of age. By 17 weeks of age, the mice weighed about 30% more
than lean littermates that were fed a normal chow diet and were
termed diet-induced obesity (D10) mice. Four groups of six DIO mice
received weekly subcutaneous injections of either vehicle (PBS),
non-pegylated Ax-15 (0.7 mg/kg), or 1-20-PEG Ax-15 (0.23 or 0.7
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mg/kg). During the treatment period body weight and 24-hour food
intake measurements were recorded daily for 13 days.
Results
1-20-PEG Ax-15 treatment reduced body weight in DIO mice in
a dose-dependent manner (Figure 21). At 0.7 mg/kg, 1-20-PEG Ax-
caused a nearly 32% weight loss, where as non-pegylated Ax-15
at the same dose decreased body weight by only 8%. In addition,
weight loss was closely correlated to a decrease in food intake,
1 0 with the greatest loss of appetite observed in the high dose (0.7
mg/kg 1-20-PEG Ax-15) treatment group (Figure 22). The duration
of appetite suppression was longest in this treatment group as well
(Figure 22). These findings suggest that pegylation enhances the
efficacy of Ax-15 in reducing body weight in DIO mice by 4-fold
1 5 (Figure 21 ). Thus, pegylation of Ax-15 may allow for lower doses
and less frequent dosing regimens.
Example 16: The use of Ax-15 to treat non-insulin
dependent Diabetes Mellitus ~NIDDM).
Background
Non Insulin Dependent Diabetes Mellitus (NIDDM or Type II
diabetes) affects about 5% of the population and is characterized by
elevated blood glucose which arises primarily due to resistance to
insulin's action in peripheral tissue. NIDDM is one of the most
common metabolic diseases and is determined by both environmental
and genetic factors. Attempts to uncover the molecular identity of
specific NIDDM susceptibility genes has led to the identification of
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several abnormalities which may contribute to the disease in small
subsets of individuals. However, the molecular identity of the genes
involved in the most common, late-onset form of NIDDM have yet to
be identified.
C57BL/KsJ dbldb (dbldb) mice are the best studied animal
model of NIDDM. These mice are insulin resistant and also exhibit a
myriad of metabolic and hormonal abnormalities such as massive
obesity, hyperphagia, and low energy expenditure (Kodama, H., et al.,
1994 Diabetologia 37:739-744). In dbldb, as well as in human
NIDDM, there is a diminished homeostatic control of glucose
metabolism, highlighted by high plasma glucose levels as well as
delayed glucose disappearance as evaluated by oral glucose tolerance
testing (OGTT). Systemic administration of ciliary neurotrophic
factor (CNTF) is known to reduce the obesity in mice which lack
either functional leptin (ob/ob mice) or the leptin receptor (dbldb
mice) (Gloaguen; I. et al., 1997, Proc Natl Acad Sci 94:6456-6461 ).
Our studies with this model have shown a dramatic effect of Ax-15
treatment on food intake and bodyweight regulation (described in
detail infra), as well as a dramatic effect on glucose tolerance,
2 0 which can not be ascribed to weight loss alone. Treatment of
animals for 10 days with Ax-15 significantly improves the oral
glucose profile in a dose-related fashion as compared to pair-fed
and vehicle-treated diabetic mice (described in detail infra). This
suggests an improvement in the animal's ability to dispose of an
injected glucose bolus either in an insulin-dependent or insulin-
independent manner. Importantly, fasting plasma glucose and insulin
levels (described in detail infra) are significantly reduced to near
normal, non-diabetic levels in mice treated with Ax-15. As there is
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a strong correlation between a high fasting serum insulin levels and
insulin resistance in NIDDM, these results suggest that Ax-15
treatment produces a significant reduction in insulin resistance in
this experimental model. There is also a significant reduction in
free fatty acid levels in Ax-15-treated mice vehicle-treated control
dbldb mice (described in detail infra).
These combined data suggests that Ax-15 treatment results in
an improvement in disposal of glucose and an increased sensitivity
to insulin, which can not be attributed to decreased food intake and
consequent weight loss. At a biochemical level it is known that
insulin signaling involves a cascade of events initiated by insulin
binding to its cell surface receptor, followed by autophosphorylation
and activation of receptor tyrosine kinases, which result in tyrosine
phosphorylation _of insulin receptor substrates (IRSs) (Avruch, J.,
1998, Molecular Cell Biochem 182:31-48). While the majority of
insulin's action is thought to be mediated by its receptors in the
periphery, it is also known that neurons in the arcuate nucleus
express the insulin receptor and IRSs (Baskin, D.G., et al., 1993, Reg
Peptides 48:257-266; Schwartz, M.W., et al., 1992, Endocr Rev
13:387-414). Our assessment of p(tyr) (pTyr) staining proteins in
the arcuate nucleus of dbldb animals surprisingly revealed
constitutive activation of proteins, presumably IRSs, when compared
to heterozygous litter mates (db/? ). This aberration is attenuated
by both Ax-15 doses tested in these experiments and suggests
restoration of normal signaling to the insulin signaling pathway in
this region.
Another well defined action of insulin is the binding of IRSs to
the regulatory subunit of phosphoinositide (PI) 3-kinase, which has
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been shown to be necessary for many of insulin actions (glucose
transport, protein synthesis, and glycogen synthesis) (Shepard, P.R.,
et al., 1996, J. Mol Endocr 17:175-184.). The only P13-kinases that
are currently known to be stimulated by insulin are the class I
heterodimeric p85/p110 catalytic P13 kinases. The p85 subunit acts
as an adaptor which links the p110 catalytic subunit to the
appropriate signalling complex. All of the forms of this adaptor
subunit contain SH2 domains which bind to tyrosine phosphorylated
motifs on IRS-1, IRS-2, and growth factor receptors (see Shepard,
ibid.). Analysis of liver tissue from Ax-15-treated dbldb mice
reveals a restoration of the ability of insulin to promote p85
association with p(Tyr) proteins in response to insulin. These
combined results suggest that Ax-15 treatment can (1 ) improve the
ability of dbldb animal to dispose of glucose and (2) that
assessment of individual tissues suggests an increased sensitivity
to insulin.
The object of this study was to characterize the effects of Ax-
15, a modified CNTF, on the diabetic profile in the dbldb mouse
model of NIDDM.
Experimental Procedures
,(~ Animals
Male dbldb C57BUKsJ mice (Jackson Laboratories, Bar Harbor,
ME), aged 6-8 weeks, were housed in a room maintained at 69-75oC
2 5 with lights on for 12 hours per day. All animal procedures were
conducted in compliance with protocols approved by the Institutional
Animal Care and Use Committee (IACUC). Starting at 10 weeks of
age, mice were individually housed, received standard mouse chow
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(Purina Mills, Richmond, IN ) ad libitum and had free access to water.
"Pair-fed" animals were provided with the same amount of food on a
daily basis as the average amount ingested by the highest dose of
Ax-15'in all studies reported. Ax-15 (0.1 and 0.3 mg/kg, s.c.) and
vehicle (10 mM Sodium Phosphate, 0.05% Tween 80, 3% PEG 3350,
20% Sucrose pH 7.5) were injected daily at approximately the same
time each day. Animal body weights were recorded daily and, where
indicated, blood samples collected from tail veins into capillary
tubes. For an oral glucose tolerance test (OGTT) all animals were
fasted for 18-20 hours and were tail bled for baseline (time 0)
measurements starting at approximately 10:00 AM. Subsequent to
the tail bleed, animals were administered 89mg D-glucose (Sigma,
St. Louis, MO) dissolved in 0.2m1 distilled water (~2.2g/kg body
weight) through a feeding needle (VWR, Plainfield, NJ). Blood was
drawn from the tail at 20, 60, and 210 minutes after the glucose
administration. Serum was stored at -20oC until time of assay for
blood glucose, insulin, free fatty acids, triglycerides (Linco
Research Immunoassay, St Charles, MO) as previously outlined
(Tonra; J.R., et al., 1999, Diabetes 48:588-594).
Tissue samplina. homogenation and immunoprecipitation
In a separate group of experiments, mice were studied to
examine the effect of Ax-15 treatment on receptor signaling
components. After the indicated times and doses of Ax-15 (see
2 5 above), liver tissue was isolated and snap frozen for subsequent
analysis. Tissue samples (100mg) were homogenized on ice in
Buffer A (1 % NP-40, 50 mM Hepes pH 7.4, 150mM NaCI, 1 mM EDTA,
30mM sodium pyrophosphate, 50mM Sodium Fluoride, 0.5mM sodium
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orthovanadate, 5~,g/ml aprotinin, 5~g/ml leupeptin, 1 mM PMSF) and
centrifuged for 10 minutes at 14,000g. Lysate protein (2mg) was
immunoprecipitated overnight at 4oC with either 5~,1 of anti-p(tyr)
antibody (4610) or anti-IRS-1 antibody coupled to Protein A
sepharose (Upstate Biotechnology, NY). The immunoprecipitates
were washed three times with Buffer A, resuspended in standard
Laemmli sample buffer and heated for approximately 5 minutes at
65°C. The protein samples were resolved by standard SDS-PAGE
analysis on 6 or 8°/a precast gels and transferred to nitrocellulose
1 0 membranes (Novex, CA) using a Trans Blot system (Hoeffer
Transblotter, Pharmacia, NJ). Nitrocellulose membranes were
blocked with 5% BSA (for 4610 blots) or 3% Blotto/0.5% BSA for at
least 1 hour at room temperature and then incubated with the
primary antibody overnight at 4oC. Antibodies used included anti-
p(tyr) 4610 (1:5000; Upstate Biotechnology Inc); anti-IRS-1 and anti
p85 (New England Biolabs, Beverley, MA).
Immunohistochemistry
Animals to be assessed by immunohistochemistry were
2 0 perfused transcardially with 4% paraformaldehyde and the brains
were removed and frozen until processed. Forty ~,m sections were
cut at the level of the arcuate nucleus, washed in KPBS (potassium
buffer saline, pH 7.2) and blocked for 20 minutes at room
temperature (4 % normal serum in KPBS/0.4% Triton X100/1 % Bovine
2 5 Serum Albumin, Fraction V, Sigma). The free floating sections were
incubated overnight at 4oC with mouse anti-p(tyr) (4610) at a
1:1000 dilution to detect p(tyr) protein, washed, then incubated with
biotinylated horse anti-mouse antibody diluted in buffer
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(KPBS/0.02% Triton X-100/1.0% BSA) at 1:1500 dilution followed by
avidin-biotin peroxidase (1:500 in PBS; Vector Elite Kit, Vector
Laboratories, Burlington, CA) both for 60 minutes and at room
temperature. Between each step sections were washed thoroughly in
PBS and the tissue-bound peroxidase was visualized by a
diaminobenzidine (Sigma St Louis, MO) reaction mounted on gelatin-
coated slides, dehydrated, and coverslipped.
Results
Treatment of dbldb animals with dail Ax-15 causes a
significantly greater weight loss than does caloric
restriction. dbldb mice or their heterozygous litter mates (dbl?)
were given daily injections (s.c.) of either Ax-15 (0.1 or 0.3 mg/kg)
1 5 or vehicle for 10 days. Food intake was restricted for a cohort of
vehicle treated animals (Pair-fed) to the same amount ingested by
the highest Ax-15-treated group. Figure 23 shows the results of
this experiment. The mean group bodyweight +/- SEM (n=12) is
reported for each day. Peripheral administration of Ax-15 (0.1 & 0.3
2 0 mg/kg/day for 10 days) produced a significant reduction in food
intake and dose dependent reduction in bodyweight (BW). For the
highest dose tested, the effect on BW was greater than attributable
to caloric restriction (c.f pairfed vehicle dbldb; PF) and was
associated with a reduction in the mass of epididymal fat (by 25%)
2 5 and liver (35%) with no effect on muscle mass.
The effect of 10 da~Ax-15 treatment on Glucose
tolerance in dbldb animals. An oral glucose tolerance test
(OGTT) was performed on vehicle (open square), pairfed-vehicle
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treated (filled diamond), and Ax-15 treated (0.1 mg/kg/day, open
triangle; 0.3 mg/kg/day, filled triangle) dbldb male mice and age-
matched heterozygous db/? mice (filled circle). Figure 24 shows the
results of this experiment. Each point represents the mean of at
least twelve animals ~ SEM. There was a reduction in fasting
plasma glucose (by 65%), insulin (by 53%) and NEFA (23%) compared
to vehicle treated levels. Oral glucose tolerance tests revealed a
dose dependent improvement in glucose tolerance, with the area
under the curve significantly different from PF and vehicle controls.
(3) Treatment of dbldb animals with d_aily low doses of
Ax-15 causes a significant body weictht loss. dbldb mice
were given daily injections (s.c.) of either Ax-15 (0.0125, 0.025 or
1 5 0.05 mg/kg) or vehicle for 10 days. The mean group bodyweight +/-
SEM (n=6) is reported for each day. As shown in Figure 23C, body
weight is reduced in a dose-dependent manner.
(4) The effect of 1 0 d_ay low dose Ax-15 treatment o n
glucose tolerance in dbldb animals. An oral glucose tolerance
test (OGTT) was performed on vehicle (open square) and Ax-15
treated (0.0125, 0.025 or 0.05 mg/kg) dbldb male mice. Each point
represents the mean of at least six animals ~ SEM. As shown in
Figure 23D, plasma glucose is reduced in a dose-dependent manner,
2 5 with the 0.05 mg/kg dose exhibiting the greatest reduction is
plasma glucose.
(5) Time course of effects of Ax-15 treatment. Time course
of effects of Ax-15 treatment (0.3 mg/kg/day; filled triangle)
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compared to vehicle treated (open square), pairfed-vehicle treated
(filled diamond) on non-fasting serum blood glucose from dbldb male
mice. Each point represents the mean of at least six animals ~ SEM
14 hour after the last injection. As shown in Figure 24, Ax-15
significantly reduces non-fasting serum blood glucose by the third
day of treatment as compared to vehicle treated or pairfed-vehicle
treated mice.
'6 , Physiological consequences of 10-day Ax-15 treatment
1 0 in dbldb animals. Figure 25A-25C shows the results of an
experiment that was designed to evaluate the physiological
consequences of 10-day Ax-15 treatment on db/db mice. Figure 25A:
Fasting blood glucose concentrations were determined with serum
from dbldb male mice treated for 10 days with Ax-15 (0.1
mg/kg/day and 0.3 mg/kg/day, hatched bars) as compared to control
groups, vehicle treated (open bar), pairfed-vehicle treated (hatched
bar) and age-matched heterozygous db/? mice (stippled). Each bar
represents the mean of at least eight animals ~ SEM. Figure 25B
Fasting insulin concentrations were determined on serum from dbldb
male mice treated for 10 days with Ax-15 (0.1 mg/kg/day and 0.3
mg/kg/day, hatched bars) as compared to control groups, vehicle
treated (open bar), pairfed vehicle-treated (hatched bar) and age-
matched heterozygous db/? mice (stippled). Each bar represents the
mean of at least eight animals ~ SEM. Figure 25C: Fasting free fatty
2 5 acid levels were determined on serum samples from dbldb male mice
treated for 10 days with Ax-15 (0.1 mg/kg/day and 0.3 mg/kg/day,
hatched bars) in comparison to control groups, vehicle treated (open
bar), pairfed-vehicle treated (hatched bar) and age-matched
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heterozygous db/? mice (stippled). Each bar represents the mean of
at least eight animals ~ SEM.
The effects of Ax-15 treatment on insulin-stimulated
t r immunoreactivity -in the arcuate nucleus of dbldb
mice. Immunostaining of heterozygous (db/?) mice showed an
increase in p(tyr) immunoreactive staining neurons of the arcuate
nucleus (Figure 26B) following a 30 minute bolus of insulin (1 IU via
the jugular vein) as compared to vehicle injected control level
(Figure 26A). This result presumably reflects neurons in the arcuate
nucleus that express insulin receptors and its substrates (eg. IRS-1),
both of which are phosphorylated after insulin binding. Analysis of
the insulin resistant/diabetic dbldb mice (vehicle treated for 10
days) revealed a constitutively high p(tyr) immunoreactive staining
pattern (Figure 26C) with no detectable change after insulin
treatment (Figure 26D). Ten day Ax-15 treatment of dbldb mice
attenuated the high basal p(tyr) immunoreactivity (Figure 26E and
26G) and restored insulin p(tyr) responsiveness (Figure 26F and 26H).
,(~ The effects of Ax-15 treatment on insulin-stimulated
signaling in the liver of dbldb mice. Figure 27A-27B shows
the results of an experiment designed to evaluate the effects of Ax-
15 treatment on insulin-stimulated signaling in the liver of dbldb
mice. Male dbldb mice were treated for 10 days with either vehicle
2 5 (lanes 7 & 8), pairfed to drug treatment levels (lanes 1 & 2) or
treated with Ax-15 (0.1 mg/kg/day, lanes 5 & 6; 0.3 mg/kg/day,
lanes 4 & 5). On the 11 th day animals were anaesthetized with
pentobarbital and injected with either saline (-) or 1 IU of regular
insulin (+) via the portal vein. The liver was removed after 1 min,
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and protein extracts were subjected to immunoprecipitation with an
anti-p(tyr) specific antibody 4G 10 followed by standard Western
blot analysis with an antiserum to the p85 regulatory subunit of
P13-kinase (Figure 27A), IRS-1-specific antisera followed by
Western blot analysis with an anti-p(tyr)-specific antibody (Figure
27B, upper panel), and an IRS-1-specific antiserum (Figure 27B,
bottom panel). Non-immune control immunoprecipitation (N1), no
lysate control (NL), and 3T3-L1 lysate control for p85 (C) were run
as immunoprecipitation and blotting controls.
Analysis of insulin action in peripheral tissues from Ax-15
treated mice indicated enhanced tyrosine phosphorylation (ptyr) of
specific substrates (IRS-1) and increased p(tyr) associated P13
kinase in response to an acute i.v. insulin bolus.
Immunohistochemical assessment at the level of the arcuate nucleus
in the CNS revealed that Ax-15 treatment attenuates the elevated
basal p(tyr) levels seen in vehicle treated dbldb and restores
insulin-stimulated p(tyr). These data suggest improved peripheral
glucose tolerance and restoration of both peripheral and central
insulin-dependent signaling events with Ax-15 treatment in animals
that lack the functional long form of the leptin receptor (i.e. dbldb).
These results indicate that Ax-15 has the ability to normalize
glucose metabolism over and above the effect caused by weight loss
alone, suggesting the utility of CNTF or its variants for the
normalization of glucose metabolism in patients having abnormal
glucose metabolism such as hyperinsulinemics, hypoglycemics, or
diabetics, especially Type II or non-insulin dependent (NIDDM)
diabetics.
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Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity of
understanding, it will be readily apparent to those of ordinary skill
in the art in light of the teachings of this invention that certain
changes and modifications may be made thereto without departing
from the spirit or scope of the appended claims.
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SEQUENCE LISTING
<110> Regeneron Pharmaceuticals, Inc.
<120> MODIFIED CILIARY NEUROTROPHIC FACTOR, METHOD OF
MAKING AND METHODS OF USE THEREOF
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<140> PCT/US00/20432
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<151> 1999-12-03
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His Phe Thr Pro Thr Glu Gly Asp Phe His Gln Ala Ile His Thr Leu
100 105 110
Met Leu Gln Val Ser Ala Phe Ala Tyr Gln Leu Glu Glu Leu Met Val
115 120 125
Leu Leu Glu Gln Lys Ile Pro Glu Asn Glu Ala Asp Gly Met Pro Ala
130 135 140
Thr Val Gly Asp Gly Gly Leu Phe Glu Lys Lys Leu Trp Gly Leu Lys
145 150 155 160
Val Leu Gln Glu Leu Ser Gln Trp Thr Val Arg Ser Ile His Asp Leu
165 170 175
Arg Val Ile Ser Ser His Gln Met Gly Ile Ser Ala Leu Glu Ser His
180 185 190
Tyr Gly Ala Lys Asp Lys Gln Met
195 200
<210> 7
<211> 200
<212> PRT
<213> Artificial Sequence
<220>
<223> Modified CNTF
<400> 7
Met Ala Phe Ala Glu Gln Thr Pro Leu Thr Leu His Arg Arg Asp Leu
1 5 10 15
Cys Ser Arg Ser Ile Trp Leu Ala Arg Lys Ile Arg Ser Asp Leu Thr
20 25 30
Ala Leu Met Glu Ser Tyr Val Lys His Gln Gly Leu Asn Lys Asn Ile
35 40 45
Asn Leu Asp Ser Ala Asp Gly Met Pro Val Ala Ser Thr Asp Gln Trp
50 55 60
Ser Glu Leu Thr Glu Ala Glu Arg Leu Gln Glu Asn Leu Gln Ala Tyr
65 70 75 80
Arg Thr Phe His Val Leu Leu Ala Arg Leu Leu Glu Asp Gln Gln Val
85 90 95
His Phe Thr Pro Thr Glu Gly Asp Phe His Gln Ala Il.e His Thr Leu
100 105 110
Leu Leu Gln Val Ala Ala Phe Ala Tyr Gln Ile Glu Gl.u Leu Met Ile
115 120 125
Leu Leu Glu Tyr Lys Ile Pro Arg Asn Glu Ala Asp Gly Met Pro Ile
130 135 140
Asn Val Gly Asp Gly Gly Leu Phe Glu Lys Lys Leu Trp Gly Leu Lys
105/4
Glu Ser Gln
195
<210> 6
CA 02379940 2002-O1-18
145 150 155 160
Val Leu Gln Glu Leu Ser Gln Trp Thr Val Arg Ser Ile His Asp Leu
165 170 175
Arg Phe Ile Ser Ser His Gln Thr Gly Ile Pro Ala Arg Gly Ser His
180 185 190
Tyr Ile Ala Asn Asn Lys Lys Met
195 200
<210> 8
<211> 200
<212> PRT
<213> Artificial Sequence
<220>
<223> Modified CNTF
<400> 8
Met Ala Phe Thr Glu His Ser Pro Leu Thr Pro His Arg Arg Asp Leu
1 5 10 15
Cys Ser Arg Ser Ile Trp Leu Ala Arg Lys Ile Arg Ser Asp Leu Thr
20 25 30
Ala Leu Thr Glu Ser Tyr Val Lys His Gln Gly Leu Asn Lys Asn Ile
35 40 45
Asn Leu Asp Ser Ala Asp Gly Met Pro Val Ala Ser Thr Asp Gln Trp
50 55 60
Ser Glu Leu Thr Glu Ala Glu Arg Leu Gln Glu Asn Leu Gln Ala Tyr
65 70 75 80
Arg Thr Phe His Val Leu Leu Ala Arg Leu Leu Glu Asp Gln Gln Val
85 90 95
His Phe Thr Pro Thr Glu Gly Asp Phe His Gln Ala Ile His Thr Leu
100 105 110
Leu Leu Gln Val Ala Ala Phe Ala Tyr Gln Ile Glu Glu Leu Met Ile
115 120 125
Leu Leu Glu Tyr Lys Ile Pro Arg Asn Glu Ala Asp Gly Met Pro Ile
130 135 140
Asn Val Gly Asp Gly Gly Leu Phe Glu Lys Lys Leu Trp Gly Leu Lys
145 150 155 160
Val Leu Gln Glu Leu Ser Gln Trp Thr Val Arg Ser Ile His Asp Leu
165 170 175
Arg Val Ile Ser Ser His Gln Met Gly Ile Ser Ala Leu Glu Ser His
180 185 190
Tyr Glu Ala Lys Asp Lys Gln Met
195 200
<210> 9
<211> 200
<212> PRT
<213> Artificial Sequence
<220>
<223> Modified CNTF
<400> 9
Met Ala Phe Ala Glu Gln Thr Pro Leu Thr Leu His Arg Arg Asp Leu
1 5 10 15
Cys Ser Arg Ser Ile Trp Leu Ala Arg Lys Ile Arg Ser Asp Leu Thr
20 25 30
Ala Leu Met Glu Ser Tyr Val Lys His Gln Gly Leu Asn Lys Asn Ile
35 40 45
Asn Leu Asp Ser Val Asp Gly Val Pro Val Ala Ser Thr Asp Arg Trp
50 55 60
Ser Glu Met Thr Glu Ala Glu Arg Leu Gln Glu Asn Leu Gln Ala Tyr
65 70 75 80
105/5
CA 02379940 2002-O1-18
Arg Thr Phe Gln Gly Met Leu Thr Lys Leu Leu Glu Asp Gln Arg Val
85 90 95
His Phe Thr Pro Thr Glu Gly Asp Phe His Gln Ala Ile His Thr Leu
100 105 110
Met Leu Gln Val Ser Ala Phe Ala Tyr Gln Leu Glu Glu Leu Met Val
115 120 125
Leu Leu Glu Gln Lys Ile Pro Glu Asn Glu Ala Asp Gly Met Pro Ala
130 135 140
Thr Val Gly Asp Gly Gly Leu Phe Glu Lys Lys Leu Trp Gly Leu Lys
145 150 155 160
Val Leu Gln Glu Leu Ser Gln Trp Thr Val Arg Ser Ile His Asp Leu
165 170 175
Arg Phe Ile Ser Ser His Gln Thr Gly Ile Pro Ala Arg Gly Ser His
180 185 190
Tyr Ile Ala Asn Asn Lys Lys Met
195 200
<210> 10
<211> 200
<212> PRT
<213> Artificial Sequence
<220>
<223> Modified CNTF
<400> 10
Met Ala Phe Thr Glu His Ser Pro Leu Thr Pro His Arg Arg Asp Leu
1 5 10 15
Cys Ser Arg Ser Ile Trp Leu Ala Arg Lys Ile Arg Ser Asp Leu Thr
20 25 30
Ala Leu Thr Glu Ser Tyr Val Lys His Gln Gly Leu Asn Lys Asn Ile
35 40 45
Asn Leu Asp Ser Ala Asp Gly Met Pro Val Ala Ser Thr Asp Gln Trp
50 55 60
Ser Glu Leu Thr Glu Ala Glu Arg Leu Gln Glu Asn Leu Gln Ala Tyr
65 70 75 80
Arg Thr Phe Gln Gly Met Leu Thr Lys Leu Leu Glu Asp Gln Arg Val
85 90 95
His Phe Thr Pro Thr Glu Gly Asp Phe His Gln Ala I:Le His Thr Leu
100 105 110
Met Leu Gln Val Ser Ala Phe Ala Tyr Gln Leu Glu Glu Leu Met Val
115 120 125
Leu Leu Glu Gln Lys Ile Pro Glu Asn Glu Ala Asp Gly Met Pro Ala
130 135 140
Thr Val Gly Asp Gly Gly Leu Phe Glu Lys Lys Leu Trp Gly Leu Lys
145 150 155 160
Val Leu Gln Glu Leu Ser Gln Trp Thr Val Arg Ser Ile His Asp Leu
165 170 175
Arg Phe Ile Ser Ser His Gln Thr Gly Ile Pro Ala Arg Gly Ser His
180 185 190
Tyr Ile Ala Asn Asn Lys Lys Met
195 200
<210> 11
<211> 200
<212> PRT
<213> Artificial Sequence
<220>
<223> Modified CNTF
<400> 11
Met Ala Phe Thr Glu His Ser Pro Leu Thr Pro His Arg Arg Asp Leu
105/6
CA 02379940 2002-O1-18
1 5 10 15
Cys Ser Arg Ser Ile Trp Leu Ala Arg Lys Ile Arg Ser Asp Leu Thr
20 25 30
Ala Leu Met Glu Ser Tyr Val Lys His Gln Gly Leu Asn Lys Asn Ile
35 40 45
Asn Leu Asp Ser Val Asp Gly Val Pro Val Ala Ser Thr Asp Arg Trp
50 55 60
Ser Glu Met Thr Glu Ala Glu Arg Leu Gln Glu Asn Leu Gln Ala Tyr
65 70 75 80
Arg Thr Phe His Val Leu Leu Ala Arg Leu Leu Glu Asp Gln Gln Val
85 90 95
His Phe Thr Pro Thr Glu Gly Asp Phe His Gln Ala Ile His Thr Leu
100 105 110
Leu Leu Gln Val Ala Ala Phe Ala Tyr Gln Ile Glu Glu Leu Met Ile
115 120 125
Leu Leu Glu Tyr Lys Ile Pro Arg Asn Glu Ala Asp Gly Met Pro Ile
130 135 140
Asn Val Gly Asp Gly Gly Leu Phe Glu Lys Lys Leu Trp Gly Leu Lys
145 150 155 160
Val Leu Gln Glu Leu Ser Gln Trp Thr Val Arg Ser Ile His Asp Leu
165 170 175
Arg Phe Ile Ser Ser His Gln Thr Gly Ile Pro Ala Arg Gly Ser His
180 185 190
Tyr Ile Ala Asn Asn Lys Lys Met
195 200
<210> 12
<211> 200
<212> PRT
<213> Artificial Sequence
<220>
<223> Modified CNTF
<400> 12
Met Ala Phe Thr Glu His Ser Pro Leu Thr Pro His Arg Arg Asp Leu
1 5 10 15
Cys Ser Arg Ser Ile Trp Leu Ala Arg Lys Ile Arg Ser Asp Leu Thr
20 25 30
Ala Leu Thr Glu Ser Tyr Val Lys His Gln Gly Leu Asn Lys Asn Ile
35 40 45
Asn Leu Asp Ser Val Asp Gly Val Pro Val Ala Ser Thr Asp Arg Trp
50 55 60
Ser Glu Met Thr Glu Ala Glu Arg Leu Gln Glu Asn Leu Gln Ala Tyr
65 70 75 80
Arg Thr Phe His Val Leu Leu Ala Arg Leu Leu Glu Asp Gln Gln Val
85 90 95
His Phe Thr Pro Thr Glu Gly Asp Phe His Gln Ala Ile His Thr Leu
100 105 110
Leu Leu Gln Val Ala Ala Phe Ala Tyr Gln Ile Glu Glu Leu Met Ile
115 120 12.5
Leu Leu Glu Tyr Lys Ile Pro Arg Asn Glu Ala Asp Gl.y Met Pro Ile
130 135 140
Asn Val Gly Asp Gly Gly Leu Phe Glu Lys Lys Leu Trp Gly Leu Lys
145 150 155 160
Val Leu Gln Glu Leu Ser Gln Trp Thr Val Arg Ser Ile His Asp Leu
165 170 175
Arg Phe Ile Ser Ser His Gln Thr Gly Ile Pro Ala Arg Gly Ser His
180 185 190
Tyr Ile Ala Asn Asn Lys Lys Met
195 200
<210> 13
105/7
CA 02379940 2002-O1-18
<211> 200
<212> PRT
<213> Artificial Sequence
<220>
<223> Modified CNTF
<400> 13
Met Ala Phe Thr Glu His Ser Pro Leu Thr Pro His Arg Arg Asp Leu
1 5 10 15
Cys Ser Arg Ser Ile Trp Leu Ala Arg Lys Ile Arg Ser Asp Leu Thr
20 25 30
Ala Leu Thr Glu Ser Tyr Val Lys His Gln Gly Leu Asn Lys Asn Ile
35 40 95
Asn Leu Asp Ser Val Asp Gly Met Pro Val Ala Ser Thr Asp Gln Trp
50 55 60
Ser Glu Met Thr Glu Ala Glu Arg Leu Gln Glu Asn Leu Gln Ala Tyr
65 70 75 80
Arg Thr Phe His Val Leu Leu Ala Arg Leu Leu Glu Asp Gln Gln Val
85 90 95
His Phe Thr Pro Thr Glu Gly Asp Phe His Gln Ala Ile His Thr Leu
100 105 110
Leu Leu Gln Val Ala Ala Phe Ala Tyr Gln Ile Glu Glu Leu Met Ile
115 120 125
Leu Leu Glu Tyr Lys Ile Pro Arg Asn Glu Ala Asp Gly Met Pro Ile
130 135 140
Asn Val Gly Asp Gly Gly Leu Phe Glu Lys Lys Leu Trp Gly Leu Lys
145 150 155 160
Val Leu Gln Glu Leu Ser Gln Trp Thr Val Arg Ser Ile His Asp Leu
165 170 175
Arg Phe Ile Ser Ser His Gln Thr Gly Ile Pro Ala A:rg Gly Ser His
180 185 190
Tyr Ile Ala Asn Asn Lys Lys Met
195 200
<210> 14
<211> 200
<212> PRT
<213> Artificial Sequence
<220>
<223> Modified CNTF
<400> 14
Met Ala Phe Thr Glu His Ser Pro Leu Thr Pro His Arg Arg Asp Leu
1 5 10 15
Cys Ser Arg Ser Ile Trp Leu Ala Arg Lys Ile Arg Ser Asp Leu Thr
20 25 30
Ala Leu Thr Glu Ser Tyr Val Lys His Gln Gly Leu Asn Lys Asn Ile
35 40 45
Asn Leu Asp Ser Val Asp Gly Val Pro Val Ala Ser Thr Asp Gln Trp
50 55 60
Ser Glu Leu Thr Glu Ala Glu Arg Leu Gln Glu Asn Leu Gln Ala Tyr
65 70 75 80
Arg Thr Phe His Val Leu Leu Ala Arg Leu Leu Glu Asp Gln Gln Val
85 90 95
His Phe Thr Pro Thr Glu Gly Asp Phe His Gln Ala Il.e His Thr Leu
100 105 110
Leu Leu Gln Val Ala Ala Phe Ala Tyr Gln Ile Glu Glu Leu Met Ile
115 120 125
Leu Leu Glu Tyr Lys Ile Pro Arg Asn Glu Ala Asp Gly Met Pro Ile
130 135 140
Asn Val Gly Asp Gly Gly Leu Phe Glu Lys Lys Leu Trp Gly Leu Lys
105/8
CA 02379940 2002-O1-18
145 150 155 160
Val Leu Gln Glu Leu Ser Gln Trp Thr Val Arg Ser Ile His Asp Leu
165 170 175
Arg Phe Ile Ser Ser His Gln Thr Gly Ile Pro Ala Arg Gly Ser His
180 185 190
Tyr Ile Ala Asn Asn Lys Lys Met
195 200
<210> 15
<211> 200
<212> PRT
<213> Artificial Sequence
<220>
<223> Modified CNTF
<400> 15
Met Ala Phe Thr Glu His Ser Pro Leu Thr Pro His Arg Arg Asp Leu
1 5 10 15
Cys Ser Arg Ser Ile Trp Leu Ala Arg Lys Ile Arg Ser Asp Leu Thr
20 25 30
Ala Leu Thr Glu Ser Tyr Val Lys His Gln Gly Leu Asn Lys Asn Ile
35 40 45
Asn Leu Asp Ser Ala Asp Gly Met Pro Val Ala Ser Thr Asp Arg Trp
50 55 60
Ser Glu Leu Thr Glu Ala Glu Arg Leu Gln Glu Asn Leu Gln Ala Tyr
65 70 75 g0
Arg Thr Phe His Val Leu Leu Ala Arg Leu Leu Glu Asp Gln Gln Val
85 90 95
His Phe Thr Pro Thr Glu Gly Asp Phe His Gln Ala Ile His Thr Leu
100 105 110
Leu Leu Gln Val Ala Ala Phe Ala Tyr Gln Ile Glu Glu Leu Met Ile
115 120 125
Leu Leu Glu Tyr Lys Ile Pro Arg Asn Glu Ala Asp Gly Met Pro Ile
130 135 190
Asn Val Gly Asp Gly Gly Leu Phe Glu Lys Lys Leu Trp Gly Leu Lys
145 150 155 160
Val Leu Gln Glu Leu Ser Gln Trp Thr Val Arg Ser Ile His Asp Leu
165 170 175
Arg Phe Ile Ser Ser His Gln Thr Gly Ile Pro Ala Arg Gly Ser His
180 185 190
Tyr Ile Ala Asn Asn Lys Lys Met
195 200
<210> 16
<211> 184
<212> PRT
<213> Artificial Sequence
<220>
<223> Ax-15 protein
<400> 16
Ala Phe Thr Glu His Ser Pro Leu Thr Pro His Arg Arg Asp Leu Ala
1 5 10 15
Ser Arg Ser Ile Trp Leu Ala Arg Lys Ile Arg Ser Asp Leu Thr Ala
20 25 30
Leu Thr Glu Ser Tyr Val Lys His Gln Gly Leu Asn Lys Asn Ile Asn
35 40 45
Leu Asp Ser Ala Asp Gly Met Pro Val Ala Ser Thr Asp Arg Trp Ser
50 55 60
Glu Leu Thr Glu Ala Glu Arg Leu Gln Glu Asn Leu Gln Ala Tyr Arg
65 70 75 80
105/9
CA 02379940 2002-O1-18
Thr Phe His Val Leu Leu Ala Arg Leu Leu Glu Asp C~ln Gln Val His
85 90 g5
Phe Thr Pro Thr Glu Gly Asp Phe His Gln Ala Ile His Thr Leu Leu
100 105 110
Leu Gln Val Ala Ala Phe Ala Tyr Gln Ile Glu Glu Leu Met Ile Leu
115 120 1.25
Leu Glu Tyr Lys Ile Pro Arg Asn Glu Ala Asp Gly Met Pro Ile Asn
130 135 140
Val Gly Asp Gly Gly Leu Phe Glu Lys Lys Leu Trp Gly Leu Lys Val
145 150 155 160
Leu Gln Glu Leu Ser Gln Trp Thr Val Arg Ser Ile His Asp Leu Arg
165 170 175
Phe Ile Ser Ser His Gln Thr Gly
180
<210> 17
<211> 185
<212> PRT
<213> Artificial Sequence
<220>
<223> Methionine+ Ax-15 protein
<400> 17
Met Ala Phe Thr Glu His Ser Pro Leu Thr Pro His Arg Arg Asp Leu
1 5 10 15
Ala Ser Arg Ser Ile Trp Leu Ala Arg Lys Ile Arg Ser Asp Leu Thr
20 25 30
Ala Leu Thr Glu Ser Tyr Val Lys His Gln Gly Leu Asn Lys Asn Ile
35 40 45
Asn Leu Asp Ser Ala Asp Gly Met Pro Val Ala Ser Thr Asp Arg Trp
50 55 60
Ser Glu Leu Thr Glu Ala Glu Arg Leu Gln Glu Asn Leu Gln Ala Tyr
65 70 75 g0
Arg Thr Phe His Val Leu Leu Ala Arg Leu Leu Glu Asp Gln Gln Val
85 90 95
His Phe Thr Pro Thr Glu Gly Asp Phe His Gln Ala Ile His Thr Leu
100 105 110
Leu Leu Gln Val Ala Ala Phe Ala Tyr Gln Ile Glu Glu Leu Met Ile
115 120 125
Leu Leu Glu Tyr Lys Ile Pro Arg Asn Glu Ala Asp G1y Met Pro Ile
130 135 140
Asn Val Gly Asp Gly Gly Leu Phe Glu Lys Lys Leu Trp Gly Leu Lys
145 150 155 160
Val Leu Gln Glu Leu Ser Gln Trp Thr Val Arg Ser Ile His Asp Leu
165 170 175
Arg Phe Ile Ser Ser His Gln Thr Gly
180 185
<210> 18
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer
<400> 18
acggtaagct tggaggttct c 21
<210> 19
<211> 48
<212> DNA
105/10
CA 02379940 2002-O1-18
<213> Artificial Sequence
<220>
<223> Primer
<400> 19
tctatctggc tagcaaggaa gattcgttca gacctgactg ctcttacg 4g
<210> 20
<211> 69
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer
<400> 20
aaggtacgat aagcttggag gttctcttgg agtcgctctg cctcagtcag ctcactccaa 60
cgatcagtg 69
<210> 21
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer
<400> 21
tctatctggc tagcaaggaa g 21
<210> 22
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer
<400> 22
ccagatagag gagttaatga tactcct 27
<210> 23
<211> 47
<212> DNA
<213> Artificial Sequence
<220>
<223> Primer
<400> 23
gcgtcggccg cggaccacgc tcattaccca gtctgtgaga agaaatg 47
1
1
105/11
