U.S. patent application number 17/504045 was filed with the patent office on 2022-05-12 for therapeutic crosslinking of cytokine receptors.
The applicant listed for this patent is Synerkine Pharma B.V.. Invention is credited to Niels EIJKELKAMP, Cornelis Erik HACK, Leendert KOENDERMAN, Judith PRADO SANCHEZ, Kris Alan REEDQUIST, Remco Henri Sebastiaan WESTERINK.
Application Number | 20220143145 17/504045 |
Document ID | / |
Family ID | 1000006165780 |
Filed Date | 2022-05-12 |
United States Patent
Application |
20220143145 |
Kind Code |
A1 |
EIJKELKAMP; Niels ; et
al. |
May 12, 2022 |
THERAPEUTIC CROSSLINKING OF CYTOKINE RECEPTORS
Abstract
The present invention relates to a compound comprising (1) a
first binding moiety that binds to an Interleukin 4 receptor; and
(2) a second binding moiety that binds to a cytokine receptor
(e.g., an Interleukin 10 receptor, Interleukin 13 receptor,
interleukin 27 receptor, interleukin 33 receptor, or transforming
growth factor beta 1/2 receptor). Compounds of the disclosure
cluster or crosslink the IL4 receptor and a cytokine receptor, and
surprisingly, elicit unique responses in the nervous system,
including unique signaling and gene expression profiles. The
signaling and gene expression profiles generated IL4-cytokine
fusion proteins of the disclosure are distinct from those observed
in response to the combination of IL4 and the cytokine, and
contribute to the superior therapeutic effects over the combination
of the component parts.
Inventors: |
EIJKELKAMP; Niels; (Utrecht,
NL) ; HACK; Cornelis Erik; (Diemen, NL) ;
PRADO SANCHEZ; Judith; (Utrecht, NL) ; WESTERINK;
Remco Henri Sebastiaan; (Odijk, NL) ; REEDQUIST; Kris
Alan; (Utrecht, NL) ; KOENDERMAN; Leendert;
(Maartensdijk, NL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Synerkine Pharma B.V. |
Naarden |
|
NL |
|
|
Family ID: |
1000006165780 |
Appl. No.: |
17/504045 |
Filed: |
October 18, 2021 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
PCT/EP2020/060914 |
Apr 17, 2020 |
|
|
|
17504045 |
|
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 38/2026 20130101;
A61P 25/04 20180101 |
International
Class: |
A61K 38/20 20060101
A61K038/20; A61P 25/04 20060101 A61P025/04 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 19, 2019 |
NL |
2022982 |
Apr 19, 2019 |
NL |
2022984 |
Claims
1-256. (canceled)
257. A method of normalizing a response of a sensitized human
neuron to a stimulus, the method comprising contacting the
sensitized human neuron with a compound that comprises an
interleukin 4 polypeptide attached to a cytokine, wherein the
compound is present at a concentration of at least 1 pM during the
contacting, wherein the response of the sensitized human neuron to
the stimulus is normalized relative to before the contacting.
258. The method of claim 257, wherein the compound normalizes the
response of the sensitized human neuron to the stimulus to a
greater degree than a ten-fold higher concentration of the
interleukin 4 polypeptide and the cytokine that are not attached to
each other.
259. The method of claim 257, wherein the compound is present at a
concentration of at most 100 .mu.M during the contacting.
260. The method of claim 257, wherein the human neuron has been
sensitized by a pro-inflammatory mediator.
261. The method of claim 260, wherein the pro-inflammatory mediator
comprises a prostaglandin.
262. The method of claim 260, wherein the pro-inflammatory mediator
comprises an inflammatory cytokine.
263. The method of claim 257, wherein the stimulus induces neuronal
action potential firing.
264. The method of claim 257, wherein the stimulus comprises a
pro-inflammatory mediator, a drug, a toxicant, or a chemical
stimulus.
265. The method of claim 257, wherein the response to the stimulus
is not significantly altered in a non-sensitized human neuron
contacted with the compound.
266. The method of claim 257, wherein the response comprises a
magnitude of a calcium flux response.
267. The method of claim 257, wherein the response comprises
depolarization.
268. The method of claim 257, wherein the response comprises action
potential frequency.
269. The method of claim 257, wherein the response comprises a
signal transduction response.
270. The method of claim 257, wherein the response is normalized as
demonstrated by a lower magnitude of a calcium flux response to a
predetermined amount of capsaicin.
271. The method of claim 257, wherein the contacting decreases
ectopic neuronal activity relative to before the contacting.
272. A method of modulating signaling in a human nervous system
cell, the method comprising contacting the human nervous system
cell with a compound that comprises an interleukin 4 polypeptide
attached to a cytokine under conditions that induce heterologous
clustering of a receptor for the interleukin 4 polypeptide and a
receptor for the cytokine.
273. The method of claim 272, wherein the heterologous clustering
of the receptor for the interleukin 4 polypeptide and the receptor
for the cytokine is as determined by a protein proximity assay.
274. The method of claim 272, wherein the heterologous clustering
of the receptor for the interleukin 4 polypeptide and the receptor
for the cytokine is as determined by a proximity ligation assay
275. The method of claim 272, wherein the receptor for the
interleukin 4 polypeptide and the receptor for the cytokine are
clustered within 51 nm of each other.
276. The method of claim 272, wherein the heterologous clustering
of the receptor for the interleukin 4 polypeptide and the receptor
for the cytokine is not induced by equivalent concentrations of the
interleukin 4 polypeptide and the cytokine that are not attached to
each other.
277. The method of claim 272, wherein the heterologous receptor
clustering reduces sensitization of the human nervous system
cell.
278. The method of claim 272, wherein the heterologous receptor
clustering protects the human nervous system cell against
neurotoxicity as determined by a neurite outgrowth assay.
279. The method of claim 272, wherein the heterologous receptor
clustering protects the human nervous system cell against
neurotoxicity as determined by an intraepidermal nerve fiber
density assay.
280. The method of claim 272, wherein the heterologous receptor
clustering decreases ectopic neuronal activity.
281. The method of claim 272, wherein the cytokine is a regulatory
cytokine.
282. The method of claim 272, wherein the heterologous receptor
clustering induces an altered kinomic profile compared to a human
nervous system cell that is contacted with equivalent amounts of
the interleukin 4 polypeptide and the cytokine that are not
attached to each other as determined by kinase array profiling.
283. The method of claim 282, wherein altered kinomic profile
comprises altered JAK-STAT signaling.
284. The method of claim 283, wherein a level of activity of JAK1,
c-Kit, or c-Met is modulated.
Description
CROSS REFERENCE
[0001] This application is a continuation of International
Application No. PCT/EP2020/60914 which claims priority to Dutch
Patent Application No. 2022982 and Dutch Patent Application No.
2022984, each of which is incorporated herein by reference in its
entirety.
SEQUENCE LISTING STATEMENT
[0002] The instant application contains a Sequence Listing which
has been submitted electronically in ASCII format and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Nov. 18, 2021, is named 56780-704_301_SL.txt and is 65,718 bytes
in size.
TECHNICAL FIELD
[0003] The present disclosure relates to the treatment of chronic
pain, osteoarthritis, inflammatory and immune disorders.
BACKGROUND OF THE DISCLOSURE
[0004] Chronic pain is the number one reason why people seek
medical advice in modern medicine. In spite of its multiple causes,
chronic pain is accompanied by a cascade of biochemical reactions
in the brain, spinal cord, dorsal root ganglia and peripheral
nerves leading to neuroinflammation and neurodegeneration.
[0005] An example of chronic pain is neuropathic pain, which is
often described as a shooting or burning pain. This type of pain
can be unrelenting and severe, and often results from nerve damage
or a malfunctioning nervous system. Neuropathic pain has multiple
causes including amputation, chemotherapy, diabetes, HIV infection,
multiple myeloma, multiple sclerosis, nerve or spinal cord
compression from herniated discs or from arthritis in the spine,
shingles, spine surgery, and other. Neuropathic pain also occurs
with no obvious cause. Unfortunately, neuropathic pain often
responds poorly to standard pain treatments and occasionally may
get worse instead of better over time. For some people, it can lead
to serious disability.
[0006] Another example of a chronic pain condition is
osteoarthritis (OA). OA is the most common joint disorder; the
majority of individuals over the age of 65 have radiographic and/or
clinical evidence of OA. The most frequently affected sites are the
hands, knees, hips, and spine. OA symptoms include chronic pain,
significant functional impairment, stiffness, and loss of mobility.
Importantly, in OA, the synovial tissue and cartilage produce
pro-inflammatory cytokines, that induce chronic pain, inflammation
and cartilage breakdown.
[0007] Regulatory cytokines such as Interleukin-4 (IL4), IL10 and
IL13 and others, have potential in the treatment of chronic pain,
inflammatory diseases, immune disorders, and other conditions.
[0008] However, cytokines as stand-alone drugs have limited
therapeutic effects in chronic pain, immune, inflammatory and other
biologic processes, and clinical application of these molecules for
inflammatory and immune diseases has been disappointing.
[0009] It is an objective of the present disclosure to overcome one
or more problems in the prior art, and the present invention
discloses a new approach to improve the therapeutic application of
regulatory cytokine therapy, in particular interleukin-4 (IL4),
IL27, IL10, and IL13.
SUMMARY OF THE DISCLOSURE
[0010] The inventors developed a novel therapeutic approach, which
relates to induction of unique signaling of cells by crosslinking
of the receptors of two different cytokines. This unique signaling
is neither induced by individual cytokines, nor by the combination
of cytokines.
[0011] Compounds of the disclosure cluster or crosslink the IL4
receptor and a cytokine receptor, and surprisingly, elicit unique
responses in the nervous system, including unique signaling and
gene expression profiles. The signaling and gene expression
profiles generated IL4-cytokine fusion proteins of the disclosure
are distinct from those observed in response to the combination of
IL4 and the cytokine, and contribute to the superior therapeutic
effects over the combination of the component parts.
[0012] Bringing a receptor binding moiety corresponding to a first
cytokine and a receptor binding moiety corresponding to a second
cytokine together to induce unique signaling (which does not occur
when both receptors are triggered simultaneously by the wild-type
cytokines), can be done with various constructs, such as with a
bispecific antibody or a fusion protein.
[0013] The approach according to the present disclosure, i.e., to
apply cross-linking of at least two or three, preferably two
receptors chosen from the group consisting of IL4R, IL10R, IL13R,
IL27R, IL33R, TGF.beta.1R, and TGF.beta.2R by incorporating two or
more moieties that bind to said receptors, into one molecule, for
example a bispecific antibody, provides a unique treatment for
chronic pain, neuro-inflammatory and neuro-degenerative diseases,
and inflammatory and immune disorders. Moreover, the compounds
disclosed herein, such as bispecific antibodies, have much better
manufacturability, pharmacokinetic and safety profiles than
cytokine-based drugs.
[0014] As an example, the first cytokine can be IL4 and the second
cytokine may be IL10 or IL13, and the resulting fusion protein can
be an IL4-10 fusion protein or an IL4/13 fusion protein,
respectively. Administration of the resulting receptor-crosslinking
agent, for example a bispecific antibody against IL4-receptor
(IL4R) and IL10-receptor (IL10R) or IL13-receptor (IL13R), may
completely resolve chronic pain, reduce neuro-inflammation and
protect against neurodegeneration in a human or animal subject,
whilst administration of a combination of the individual cytokines
IL4 and IL10 or IL13 cannot.
[0015] Previously, the inventors have reported on the therapeutic
effects of the IL4/IL10 fusion protein (N Eijkelkamp et al., J
Neuroscience, 2016, 36 (28) 7353-7363). The inventors have now
unraveled the mechanisms of action underlying the therapeutic
application of the IL4-10 fusion protein according to the prior
art, and found that the IL4-10 fusion protein surprisingly,
contrary to the combination of IL4 and IL10, promoted clustering of
the IL4 receptor (IL4R) and IL10 receptor (IL10R) on sensory
neurons, which in turn promotes unique signaling pathways and gene
expression profiles that can lead to a full resolution of
persistent pain. The inventors now further found that an IL4-13
fusion protein induces a unique protection of neurons against the
damaging effects of chemotherapeutic drugs in vitro as well as in
vivo, which was dependent on cross-linking of IL4R and IL13R, as
the combination of wild-type IL4 and wild-type IL13 does not
provide such protection.
[0016] Disclosed herein, in some aspects, is a compound that
comprises an interleukin 4 polypeptide attached to a cytokine for
use in decreasing a sensitivity threshold of a neuronal calcium
flux response of a neuron to a pro-inflammatory mediator.
[0017] Disclosed herein, in some aspects, is a compound that
comprises an interleukin 4 polypeptide attached to a cytokine for
use in decreasing a magnitude of a neuronal calcium flux response
to a pro-inflammatory mediator.
[0018] Disclosed herein, in some aspects, is a compound that
comprises an interleukin 4 polypeptide attached to a cytokine for
use in decreasing a duration of a neuronal calcium flux response of
a neuron to a pro-inflammatory mediator.
[0019] Disclosed herein, in some aspects, is a compound that
comprises an interleukin 4 polypeptide attached to a cytokine for
use in modulating a JAK-STAT signaling pathway in a nervous system
cell.
[0020] Disclosed herein, in some aspects, is a compound that
comprises an interleukin 4 polypeptide attached to a cytokine for
use in modulating a kinomic profile of a nervous system cell in a
presence of a damage associated molecular pattern (DAMP).
[0021] Disclosed herein, in some aspects, is a compound that
comprises an interleukin 4 polypeptide attached to a cytokine for
use in modulating expression of a gene involved in calcium
signaling in a nervous system cell.
[0022] Disclosed herein, in some aspects, is a compound that
comprises an interleukin 4 polypeptide attached to a cytokine for
use in decreasing ectopic neuronal activity.
[0023] Disclosed herein, in some aspects, is a compound that
comprises an interleukin 4 polypeptide attached to a cytokine
selected from the group consisting of an interleukin 13 (IL13), an
interleukin 33 (IL33), a transforming growth factor beta 1
(TGF.beta.1), and a transforming growth factor beta 2 (TGF.beta.1)
for use in crosslinking an interleukin 4 receptor with a cytokine
receptor.
[0024] Disclosed herein, in some aspects, is a compound that
comprises an interleukin 4 polypeptide attached to an interleukin
27 for use in crosslinking an interleukin 4 receptor with an
interleukin 27 receptor.
[0025] In some embodiments, the compound decreases the sensitivity
threshold of the neuronal calcium flux response relative to
equivalent amounts of the interleukin 4 polypeptide and the
cytokine. In some embodiments, decreasing the sensitivity threshold
of the neuronal calcium flux response is as determined by measuring
an amount of capsaicin required to elicit a calcium flux response
of a predetermined magnitude. In some embodiments, the sensitivity
threshold is decreased in a presence of a damage associated
molecular pattern (DAMP). In some embodiments, the compound
decreases the magnitude of the neuronal calcium flux response to
the pro-inflammatory mediator relative to equivalent amounts of the
interleukin 4 polypeptide and the cytokine. In some embodiments,
the magnitude of the neuronal calcium flux response is decreased in
a presence of a damage associated molecular pattern (DAMP). 8,
wherein the compound decreases the duration of the neuronal calcium
flux response to the pro-inflammatory mediator relative to
equivalent amounts of the interleukin 4 polypeptide and the
cytokine. In some embodiments, the duration of the neuronal calcium
flux response is decreased in a presence of a damage associated
molecular pattern (DAMP). In some embodiments, the activity of the
JAK-STAT signaling pathway is increased. In some embodiments, the
activity of the JAK-STAT signaling pathway is increased relative to
a nervous system cell contacted with equivalent amounts of the
interleukin 4 polypeptide and the cytokine. In some embodiments,
activity of the JAK-STAT signaling pathway is modulated in a
presence of a pro-inflammatory mediator. In some embodiments,
activity of the JAK-STAT signaling pathway is modulated in a
presence of a damage associated molecular pattern (DAMP). In some
embodiments, a level of activity of a kinase is modulated. In some
embodiments, the kinase is JAK1. In some embodiments, wherein the
level of activity of the kinase is increased. In some embodiments,
the level of activity of the kinase is increased relative to a
nervous system cell contacted with equivalent amounts of the
interleukin 4 polypeptide and the cytokine. In some embodiments,
the level of activity of the kinase is modulated in a presence of a
pro-inflammatory mediator. In some embodiments, the level of
activity of the kinase is modulated in a presence of a damage
associated molecular pattern (DAMP). In some embodiments, a level
of phosphorylation of Annexin 2 in the nervous system cell is
increased. In some embodiments, the level of phosphorylation is
increased relative to a nervous system cell contacted with
equivalent amounts of the interleukin 4 polypeptide and the
cytokine. In some embodiments, a level of phosphorylation of
cGMP-specific 3',5'-cyclic phosphodiesterase in the nervous system
cell is decreased. In some embodiments, a level of phosphorylation
is decreased relative to a nervous system cell contacted with
equivalent amounts of the interleukin 4 polypeptide and the
cytokine. In some embodiments, the level of phosphorylation is
modulated in a presence of a pro-inflammatory mediator. In some
embodiments, the level of phosphorylation is modulated in a
presence of a damage associated molecular pattern (DAMP). In some
embodiments, the kinomic profile is modulated as determined by
kinase array profiling relative to a nervous system cell contacted
with equivalent amounts of the interleukin 4 polypeptide and the
cytokine. In some embodiments, modulating the kinomc profile of the
nervous system cell comprises increasing an activity level of
tyrosine kinases relative to a nervous system cell contacted with
equivalent amounts of the interleukin 4 polypeptide and the
cytokine. In some embodiments, expression of the gene is
down-regulated in the nervous system cell. In some embodiments, the
expression of the gene is modulated in a presence of a
pro-inflammatory mediator. In some embodiments, the expression of
the gene is modulated in a presence of a damage associated
molecular pattern (DAMP). In some embodiments, the nervous system
cell is a central nervous system cell. In some embodiments, the
nervous system cell is a peripheral nervous system cell. In some
embodiments, the nervous system cell is a neuron. In some
embodiments, the neuron is a sensory neuron. In some embodiments,
the neuron is a somatosensory neuron. In some embodiments, the
neuron is a visceral sensory neuron. In some embodiments, the
neuron is a nociceptor. In some embodiments, the neuron is an
autonomic neuron. In some embodiments, the nervous system cell is a
glial cell. In some embodiments, the nervous system cell is a
microglial cell. In some embodiments, the nervous system cell is an
infiltrating cell. In some embodiments, the nervous system cell is
an infiltrating macrophage. In some embodiments, the compound
decreases the ectopic neuronal activity relative to equivalent
amounts of the interleukin 4 polypeptide and the cytokine. In some
embodiments, ectopic neuronal activity is decreased in a presence
of a pro-inflammatory mediator. In some embodiments, ectopic
neuronal activity is decreased in a presence of a damage associated
molecular pattern (DAMP). In some embodiments, the interleukin 4
polypeptide is a wild type interleukin 4. In some embodiments, the
interleukin 4 polypeptide comprises an interleukin 4 derivative
sequence. In some embodiments, the interleukin 4 polypeptide binds
to IL-13R.alpha.1. In some embodiments, the interleukin 4
polypeptide binds to a common gamma chain. In some embodiments, the
interleukin 4 polypeptide binds to IL-4R.alpha.. In some
embodiments, the interleukin 4 polypeptide binds to IL-13R.alpha.1,
common gamma chain, and IL-4R.alpha.. In some embodiments, the
interleukin 4 polypeptide binds to IL-13R.alpha.1 and common gamma
chain with about a comparable affinity as a wild type interleukin
4. In some embodiments, the interleukin 4 polypeptide comprises a
sequence that is either a wild type sequence or a derivative
sequence that binds to IL-13R.alpha. and common gamma chain with
about a comparable affinity as the wild type interleukin 4. In some
embodiments, the compound forms a complex with at least four
receptor polypeptide chains. In some embodiments, the interleukin 4
polypeptide is a mammalian interleukin 4. In some embodiments, the
interleukin 4 polypeptide is a human interleukin 4. In some
embodiments, the cytokine is a mammalian cytokine. In some
embodiments, the cytokine is a human cytokine. In some embodiments,
the interleukin 4 polypeptide comprises an amino acid sequence with
at least 90% sequence identity to any one of SEQ ID NOs: 11-14. In
some embodiments, the interleukin 4 polypeptide comprises an amino
acid sequence that is any one of SEQ ID NOs: 11-14. In some
embodiments, the interleukin 4 polypeptide comprises an amino acid
sequence with between 1 and 10 amino acid deletions, insertions,
substitutions, or a combination thereof relative to any one of SEQ
ID NOs: 11-14. In some embodiments, the cytokine comprises an amino
acid sequence with at least 90% sequence identity to any one of SEQ
ID NOs: 15-37. In some embodiments, the cytokine comprises an amino
acid sequence that is any one of SEQ ID NOs: 15-37. In some
embodiments, the cytokine comprises an amino acid sequence with
between 1 and 10 amino acid deletions, insertions, substitutions,
or a combination thereof relative to any one of SEQ ID NOs: 15-37.
In some embodiments, the cytokine comprises an amino acid sequence
that is a wild type cytokine sequence. In some embodiments, the
cytokine a cytokine derivative sequence. In some embodiments, the
interleukin 4 polypeptide and the cytokine are covalently linked.
In some embodiments, the compound is a fusion protein. In some
embodiments, the interleukin 4 polypeptide and the cytokine are
joined by a linker. In some embodiments, the interleukin 4
polypeptide is joined to an N-terminus of the cytokine, optionally
via a linker. In some embodiments, an N terminus of the interleukin
13 is joined to a C-terminus of the cytokine, optionally via a
linker. In some embodiments, the compound further comprises one or
more chemical modifications. In some embodiments, the chemical
modification is selected from the group consisting of
glycosylation, fucosylation, sialylation, and pegylation.
[0026] Disclosed herein, in some aspects, is a method of decreasing
a sensitivity threshold of a neuronal response to a stimulus,
comprising contacting a neuron with a compound that comprises an
interleukin 4 polypeptide attached to a cytokine in an amount
effective to decrease the sensitivity threshold of the neuronal
response to the stimulus as determined by an amount of capsaicin
required to elicit a calcium flux response of a predetermined
magnitude.
[0027] Disclosed herein, in some aspects, is a method of decreasing
a magnitude of a neuronal response to a stimulus, comprising
contacting a neuron with a compound that comprises an interleukin 4
polypeptide attached to a cytokine in an amount effective to
decrease the magnitude of the neuronal response to the stimulus as
determined by measuring calcium flux in response to a predetermined
concentration of capsaicin.
[0028] Disclosed herein, in some aspects, is a method of decreasing
a duration of a neuronal response to a stimulus, comprising
contacting a neuron with a compound that comprises an interleukin 4
polypeptide attached to a cytokine in an amount effective to
decrease the duration of the neuronal response to the stimulus as
determined by measuring calcium flux in response to a predetermined
concentration of capsaicin.
[0029] Disclosed herein, in some aspects, is a method of decreasing
ectopic neuronal activity, comprising contacting a neuron with a
compound that comprises an interleukin 4 polypeptide attached to a
cytokine in an amount effective to decrease ectopic activity of the
neuron relative to a neuron contacted with equivalent amounts of
the interleukin 4 polypeptide and the cytokine.
[0030] Disclosed herein, in some aspects, is a method of modulating
signaling in a nervous system cell, comprising contacting the
nervous system cell with a compound that comprises an interleukin 4
polypeptide attached to a cytokine in an amount effective to
modulate the signaling in the nervous system cell as determined by
kinase array profiling of dorsal root ganglia homogenates.
[0031] Disclosed herein, in some aspects, is a method of modulating
a kinomic profile of a nervous system cell, comprising contacting
the nervous system cell with a compound that comprises an
interleukin 4 polypeptide attached to a cytokine in an amount
effective to modulate the kinomic profile in the nervous system
cell as determined by kinase array profiling of dorsal root ganglia
homogenates.
[0032] Disclosed herein, in some aspects, is a method of modulating
gene expression in a nervous system cell, comprising contacting the
nervous system cell with a compound that comprises an interleukin 4
polypeptide attached to a cytokine in an amount effective to
modulate gene expression in the nervous system cell as determined
by RNA sequencing.
[0033] Disclosed herein, in some aspects, is a method of
crosslinking an interleukin 4 receptor with a cytokine receptor,
comprising contacting a cell with a compound that comprises an
interleukin 4 polypeptide attached to a cytokine selected from the
group consisting of an interleukin 13, an interleukin 33, a
transforming growth factor beta 1, and a transforming growth factor
beta 2, thereby crosslinking the interleukin 4 receptor with the
cytokine receptor, wherein the interleukin 4 polypeptide comprises
a sequence that is either a wild type sequence or a variant
sequence that binds to interleukin 4 receptor alpha (IL-4R.alpha.),
interleukin 13 receptor alpha (IL-13R.alpha.), and common gamma
chain with a comparable affinity as the wild type sequence.
[0034] Disclosed herein, in some aspects, is a method of
crosslinking an interleukin 4 receptor with a cytokine receptor,
comprising contacting a cell with a compound that comprises an
interleukin 4 and an interleukin 27.
[0035] In some embodiments, the sensitivity threshold is decreased
in a presence of a pro-inflammatory mediator. In some embodiments,
the sensitivity threshold is decreased in a presence of a damage
associated molecular pattern (DAMP). In some embodiments, the
sensitivity threshold is decreased relative to a neuron contacted
with equivalent amounts of the interleukin 4 polypeptide and the
cytokine. In some embodiments, the neuron is contacted with the
composition for a period of time sufficient to decrease the
sensitivity threshold of the neuronal response to the stimulus
relative to a neuron contacted with equivalent amounts of the
interleukin 4 polypeptide and the cytokine. In some embodiments,
the magnitude of the neuronal response is decreased in a presence
of a pro-inflammatory mediator relative to a neuron contacted with
equivalent amounts of the interleukin 4 polypeptide and the
cytokine. In some embodiments, the magnitude of the neuronal
response is decreased in a presence of a pro-inflammatory mediator.
In some embodiments, the magnitude of the neuronal response is
decreased in a presence of a damage associated molecular pattern
(DAMP). In some embodiments, the neuron is contacted with the
composition for a period of time sufficient to decrease the
magnitude of the neuronal response to the stimulus relative to a
neuron contacted with equivalent amounts of the interleukin 4
polypeptide and the cytokine. In some embodiments, the duration of
the neuronal response is decreased in a presence of a
pro-inflammatory mediator relative to a neuron contacted with
equivalent amounts of the interleukin 4 polypeptide and the
cytokine. In some embodiments, the duration of the neuronal
response is decreased in a presence of a pro-inflammatory mediator.
In some embodiments, the duration of the neuronal response is
decreased in a presence of a damage associated molecular pattern
(DAMP). In some embodiments, the neuron is contacted with the
composition for a period of time sufficient to decrease the
duration of the neuronal response to the stimulus relative to a
neuron contacted with equivalent amounts of the interleukin 4
polypeptide and the cytokine. In some embodiments, the stimulus
comprises a pro-inflammatory mediator. In some embodiments, the
stimulus comprises a pro-inflammatory cytokine, a pro-inflammatory
chemokine, a vasoactive amine, a prostaglandin, a leukotriene, a
thromboxane, an oxygen- and/or nitrogen-derived free radical,
histamine, a Th1 cytokine, a Th2 cytokine, a Th17 cytokine,
IL-1.beta., APRIL, IFN-.alpha., IFN-.beta., IFN-.gamma., IL-1a,
IL-1.beta., IL-2, IL-6, IL-8, IL-9, IL-12, IL-23, LIGHT,
TNF-.alpha., or TNF-.beta.. In some embodiments, the stimulus
comprises a damage associated molecular pattern (DAMP). In some
embodiments, the stimulus comprises an anti-inflammatory mediator.
In some embodiments, the stimulus comprises a drug. In some
embodiments, the stimulus comprises a toxin or a toxicant. In some
embodiments, the stimulus comprises a chemical stimulus. In some
embodiments, the stimulus comprises a thermal stimulus. In some
embodiments, the stimulus comprises a mechanical stimulus. In some
embodiments, the stimulus comprises capsaicin. In some embodiments,
the stimulus induces neuronal action potential firing. In some
embodiments, the neuronal response comprises depolarization. In
some embodiments, the neuronal response comprises action potential
frequency. In some embodiments, the neuronal response comprises a
transcriptional response. In some embodiments, the neuronal
response comprises a signal transduction response. In some
embodiments, the ectopic neuronal activity is decreased in a
presence of a pro-inflammatory mediator relative to a neuron
contacted with equivalent amounts of the interleukin 4 polypeptide
and the cytokine. In some embodiments, the ectopic neuronal
activity is decreased in a presence of a pro-inflammatory mediator.
In some embodiments, the ectopic neuronal activity is decreased in
a presence of a damage associated molecular pattern (DAMP). In some
embodiments, the neuron is contacted with the composition for a
period of time sufficient to decrease ectopic activity of the
neuron relative to a neuron contacted with equivalent amounts of
the interleukin 4 polypeptide and the cytokine. In some
embodiments, the nervous system cell is contacted with the
composition for a period of time sufficient to modulate signaling
in the nervous system cell. In some embodiments, the signaling
comprises a JAK-STAT signaling pathway. In some embodiments, the
signaling comprises a pro-inflammatory signaling pathway. In some
embodiments, the signaling comprises an innate immune system
pro-inflammatory signaling pathway. In some embodiments, the
signaling comprises a NOD-like receptor signaling pathway. In some
embodiments, the activity of the signaling pathway is increased. In
some embodiments, the activity of the signaling pathway is
increased relative to a nervous system cell contacted with
equivalent amounts of the interleukin 4 polypeptide and the
cytokine. In some embodiments, the activity of the signaling
pathway is decreased. In some embodiments, the activity of the
signaling pathway is decreased relative to a nervous system cell
contacted with equivalent amounts of the interleukin 4 polypeptide
and the cytokine. In some embodiments, the signaling is modulated
in a presence of a pro-inflammatory mediator. In some embodiments,
the signaling is modulated in a presence of a damage associated
molecular pattern (DAMP). In some embodiments, a level of activity
of a kinase is modulated, wherein the kinase is JAK1. In some
embodiments, a level of activity of a kinase is modulated, wherein
the kinase is c-Kit. In some embodiments, a level of activity of a
kinase is modulated, wherein the kinase is c-Met. In some
embodiments, the level of activity of the kinase is increased. In
some embodiments, the level of activity of the kinase is increased
relative to a nervous system cell contacted with equivalent amounts
of the interleukin 4 polypeptide and the cytokine. In some
embodiments, the level of activity of the kinase is decreased. In
some embodiments, the level of activity of the kinase is decreased
relative to a nervous system cell contacted with equivalent amounts
of the interleukin 4 polypeptide and the cytokine. In some
embodiments, the level of activity of the kinase is modulated in a
presence of a pro-inflammatory mediator. In some embodiments, the
level of activity of the kinase is modulated in a presence of a
damage associated molecular pattern (DAMP). In some embodiments, a
level of phosphorylation of Annexin 2 in the nervous system cell is
modulated. In some embodiments, a level of phosphorylation of
cGMP-specific 3',5'-cyclic phosphodiesterase in the nervous system
cell is modulated. In some embodiments, the level of
phosphorylation is increased. In some embodiments, the level of
phosphorylation is increased relative to a nervous system cell
contacted with equivalent amounts of the interleukin 4 polypeptide
and the cytokine. In some embodiments, the level of phosphorylation
is decreased. In some embodiments, the level of phosphorylation is
decreased relative to a nervous system cell contacted with
equivalent amounts of the interleukin 4 polypeptide and the
cytokine. In some embodiments, the level of phosphorylation is
modulated in a presence of a pro-inflammatory mediator. In some
embodiments, the level of phosphorylation is modulated in a
presence of a damage associated molecular pattern (DAMP). In some
embodiments, the nervous system cell is contacted with the
composition for a period of time sufficient to modulate the kinomic
profile in the nervous system cell. In some embodiments, the
kinomic profile is modulated as determined by kinase array
profiling relative to a nervous system cell contacted with
equivalent amounts of the interleukin 4 polypeptide and the
cytokine. In some embodiments, modulating the kinomic profile of
the nervous system cell comprises increasing an activity level of
tyrosine kinases relative to a nervous system cell contacted with
equivalent amounts of the interleukin 4 polypeptide and the
cytokine. In some embodiments, the kinomic profile is modulated in
a presence of a pro-inflammatory mediator. In some embodiments, the
kinomic profile is modulated in a presence of a damage associated
molecular pattern (DAMP). In some embodiments, the nervous system
cell is contacted with the composition for a period of time
sufficient to modulate gene expression in the nervous system cell.
In some embodiments, gene expression is modulated as determined by
RNA sequencing and hierarchical clustering of the top 500
differentially-regulated genes relative to a nervous system cell
that has not been contacted with the compound. In some embodiments,
the gene expression is modulated as determined by RNA sequencing
and hierarchical clustering of the top 500 differentially-regulated
genes relative to a nervous system cell contacted with equivalent
amounts of the interleukin 4 polypeptide and the cytokine. In some
embodiments, modulating gene expression comprises modulating a
global transcriptomic profile of the nervous system cell as
determined by principal component analysis of RNA sequencing data
relative to a nervous system cell that has not been contacted with
the compound. In some embodiments, modulating gene expression
comprises modulating a global transcriptomic profile of the nervous
system cell as determined by principal component analysis of RNA
sequencing data relative to a nervous system cell contacted with
equivalent amounts of the interleukin 4 polypeptide and the
cytokine. In some embodiments, expression of a gene encoding an
oxidative phosphorylation enzyme is down-regulated in the nervous
system cell. In some embodiments, expression of a gene involved in
calcium signaling is down-regulated in the nervous system cell. In
some embodiments, gene expression is modulated in a presence of a
pro-inflammatory mediator. In some embodiments, gene expression is
modulated in a presence of a damage associated molecular pattern
(DAMP). In some embodiments, the nervous system cell is a central
nervous system cell. In some embodiments, the nervous system cell
is a peripheral nervous system cell. In some embodiments, the
nervous system cell is a neuron. In some embodiments, the neuron is
a sensory neuron. In some embodiments, the neuron is a
somatosensory neuron. In some embodiments, the neuron is a visceral
sensory neuron. In some embodiments, the neuron is a nociceptor. In
some embodiments, the neuron is an autonomic neuron. In some
embodiments, the nervous system cell is a glial cell. In some
embodiments, the nervous system cell is a microglial cell. In some
embodiments, the nervous system cell is an infiltrating cell. In
some embodiments, the nervous system cell is an infiltrating
macrophage. In some embodiments, crosslinking is as determined by a
proximity ligation assay for the interleukin receptor and the
cytokine receptor. In some embodiments, the interleukin 4
polypeptide is a wild type interleukin 4. In some embodiments, the
interleukin 4 polypeptide comprises an interleukin 4 derivative
sequence. In some embodiments, the interleukin 4 polypeptide binds
to IL-13R.alpha.1. In some embodiments, the interleukin 4
polypeptide binds to a common gamma chain. In some embodiments, the
interleukin 4 polypeptide binds to IL-4R.alpha.. In some
embodiments, the interleukin 4 polypeptide binds to IL-13R.alpha.1,
common gamma chain, and IL-4R.alpha.. In some embodiments, the
interleukin 4 polypeptide binds to IL-13R.alpha.1 and common gamma
chain with about a comparable affinity as a wild type interleukin
4. In some embodiments, the interleukin 4 polypeptide comprises a
sequence that is either a wild type sequence or an IL4 derivative
sequence that binds to IL-13R.alpha. and common gamma chain with
about a comparable affinity as the wild type interleukin 4. In some
embodiments, the contacting, the compound form a complex with at
least four receptor polypeptide chains. In some embodiments, the
interleukin 4 polypeptide is a mammalian interleukin 4. In some
embodiments, the interleukin 4 polypeptide is a human interleukin
4. In some embodiments, the cytokine is a mammalian cytokine. In
some embodiments, the cytokine is a human cytokine. In some
embodiments, the interleukin 4 polypeptide comprises an amino acid
sequence with at least 90% sequence identity to any one of SEQ ID
NOs: 11-14. In some embodiments, the interleukin 4 polypeptide
comprises an amino acid sequence that is any one of SEQ ID NOs:
11-14. In some embodiments, the interleukin 4 polypeptide comprises
an amino acid sequence with between 1 and 10 amino acid deletions,
insertions, substitutions, or a combination thereof relative to any
one of SEQ ID NOs: 11-14. In some embodiments, the cytokine
comprises an amino acid sequence with at least 90% sequence
identity to any one of SEQ ID NOs: 15-37. In some embodiments, the
cytokine comprises an amino acid sequence that is any one of SEQ ID
NOs: 15-37. In some embodiments, the cytokine comprises an amino
acid sequence with between 1 and 10 amino acid deletions,
insertions, substitutions, or a combination thereof relative to any
one of SEQ ID NOs: 15-37. In some embodiments, the interleukin 4
polypeptide and the cytokine are covalently linked. In some
embodiments, the compound is a fusion protein. In some embodiments,
the interleukin 4 polypeptide and the cytokine are joined by a
linker. In some embodiments, a C terminus of the interleukin 4
polypeptide is joined to an N-terminus of the cytokine, optionally
via a linker. In some embodiments, an N terminus of the interleukin
13 is joined to a C-terminus of the cytokine, optionally via a
linker. In some embodiments, the compound further comprises one or
more chemical modifications. In some embodiments, the chemical
modification is selected from the group consisting of
glycosylation, fucosylation, sialylation, and pegylation. In some
embodiments, the contacting occurs in a mammalian subject. In some
embodiments, the compound is present at a concentration of at least
1 pM during the contacting. In some embodiments, the compound is
present at a concentration of at least 1 nM during the contacting.
In some embodiments, the compound is present at a concentration of
1 pM to 1 .mu.M during the contacting. In some embodiments, the
compound is present at a concentration of 100 pM to 100 nM during
the contacting.
[0036] Disclosed herein, in some aspects is a compound that
comprises an interleukin 4 polypeptide attached to a cytokine
selected from the group consisting of an interleukin 13 (IL13), an
interleukin 33 (IL33), a transforming growth factor beta 1
(TGF.beta.1), and a transforming growth factor beta 2 (TGF.beta.1),
wherein the interleukin 4 polypeptide comprises a wild type
interleukin 4 amino acid sequence.
[0037] Disclosed herein, in some aspects is a compound that
comprises an interleukin 4 polypeptide attached to a cytokine
selected from the group consisting of an interleukin 13 (IL13), an
interleukin 33 (IL33), a transforming growth factor beta 1
(TGF.beta.1), and a transforming growth factor beta 2 (TGF.beta.1),
wherein the interleukin 4 polypeptide comprises an interleukin 4
derivative that binds to interleukin 4 receptor alpha
(IL-4R.alpha.), interleukin 13 receptor alpha (IL-13R.alpha.), and
common gamma chain with a comparable affinity as a wild type
interleukin 4.
[0038] Disclosed herein, in some aspects is a compound that
comprises an interleukin 4 polypeptide and an interleukin 27
(IL27).
[0039] In some embodiments, the compound is a fusion protein. In
some embodiments, the compound comprises the interleukin 4
polypeptide and the interleukin 13. In some embodiments, the
compound comprises the interleukin 4 polypeptide and the
interleukin 33. In some embodiments, the compound comprises the
interleukin 4 polypeptide and the transforming growth factor beta
1. In some embodiments, the compound comprises the interleukin 4
polypeptide and the transforming growth factor beta 2. In some
embodiments, the interleukin 4 polypeptide is capable of binding to
IL-4R.alpha., IL-13R.alpha., and common gamma chain. In some
embodiments, the compound activates signaling by a native
configuration IL-4 receptor. In some embodiments, upon contacting
with a cell, the compound forms a complex with at least four
receptor polypeptides. In some embodiments, upon contacting a cell,
the compound crosslinks an interleukin 4 receptor and a receptor
for the interleukin 13, the interleukin 33, the transforming growth
factor beta 1, or the transforming growth factor beta 2. In some
embodiments, the compound comprises one or more chemical
modifications selected from the group consisting of glycosylation,
sialylation, fucosylation, and pegylation. In some embodiments, the
compound is a fusion protein. In some embodiments, a nucleic acid
molecule comprising a polynucleotide sequence encodes the fusion
protein. In some embodiments, the polynucleotide sequence is codon
optimized for expression in the cell. In some embodiments, the
polynucleotide sequence is present in a nucleic acid vector. In
some embodiments, the nucleic acid is present in a cell. In some
embodiments, the fusion protein or nucleic acid vector is present
in a pharmaceutical composition which also comprises a
pharmaceutically-acceptable excipient. In some embodiments, the
pharmaceutical composition is in a unit dosage form. In some
embodiments, the compound is present in the pharmaceutical
composition at about 50 .mu.g to about 100 mg per mL. In some
embodiments, the compound is formulated for administration as a
dose of between about 0.5 .mu.g to 1 mg per kg of body weight. In
some embodiments, the compound formulated for administration as a
controlled release formulation. In some embodiments, the
pharmaceutical composition is formulated for administration by a
parenteral, intravenous, intramuscular, intraarterial, intrathecal,
intracapsular, intraorbital, intracardiac, intradermal,
intraperitoneal, transtracheal, subcutaneous, subcuticular,
intraarticular, subcapsular, subarachnoid, intraspinal, epidural,
intrasternal, intracerebral, intraocular, intralesional,
intracerebroventricular, intracisternal, or intraparenchymal route.
In some embodiments, the pharmaceutical composition is used in a
method of treating a condition in need thereof, which comprises
administering the compound to the subject. In some embodiments, the
fusion protein is produced by culturing a cell under conditions
that permit production of the fusion protein, wherein the cell
comprises the polynucleotide sequence. In some embodiments, the
fusion protein is harvested. In some embodiments, the fusion
protein is purified from harvested culture medium.
[0040] Discloses herein, in some aspects, is a compound comprising:
a first binding moiety that binds to an Interleukin 4 receptor; and
a second binding moiety that binds to an Interleukin 10 receptor,
wherein the first binding moiety and/or the second binding moiety
is not a wild-type interleukin.
[0041] Disclosed herein, in some aspects, is a combination
comprising: a first binding moiety that binds to an Interleukin 4
receptor; a second binding moiety that binds to an Interleukin 10
receptor, wherein the first binding moiety has a linker that binds
the second binding moiety or wherein the second binding moiety has
a linker that binds the first binding moiety, and wherein the first
binding moiety and/or the second binding moiety is not a wild-type
interleukin.
[0042] In some embodiments, the first binding moiety and/or the
second binding moiety is a polypeptide, preferably an
immunoglobulin molecule or epitope-binding fragment thereof, more
preferably Fab, F(ab'), F(ab')2, Fv, dAb, Fd, a complementarity
determining region (CDR) fragment, a single chain antibody (scFv),
or single domain antibody. In some embodiments, the compound is a
polypeptide, preferably a bispecific antibody, a bivalent single
chain antibody, a bispecific double chain antibody, a triabody, or
a tetrabody. In some embodiments, the compound or combination is
able to cross-link an Interleukin 4 receptor to an Interleukin 10
receptor of a sensory neuron in vivo. In some embodiments, the
first binding moiety and/or second binding moiety comprises a tag;
and the linker of the first binding moiety and/or the linker of the
second binding moiety is a polypeptide that binds to the tag. In
some embodiments, the compound, first binding moiety and/or second
binding moiety comprises a polypeptide selected from the group
consisting of a signal sequence, a His-tag, and an antibody Fc
fragment. In some embodiments, the compound, first binding moiety
and/or second binding moiety comprises one or more chemical
modifications selected from the group consisting of glycosylation,
sialylation, fucosylation, and pegylation. In some embodiments, the
compound or combination is comprised in a pharmaceutical
composition comprising the compound or combination, and a
pharmaceutically acceptable carrier. In some embodiments, the
compound or combination is for use in therapeutic treatment. In
some embodiments, the compound or combination is for therapeutic
treatment by administration to a local compartment of a human or
animal body. In some embodiments, the local compartment is an
intrathecal compartment or an intraarticular compartment and/or
wherein the compound or combination is administered intrathecally
or intraarticularly. In some embodiments, the compound or
combination is for treatment of a local condition. In some
embodiments, the compound or combination is for use in the
prevention or treatment of rheumatoid arthritis; osteoarthritis;
other form of arthritis; pain; chronic pain; multiple sclerosis;
neuro-inflammatory or neuro-degenerative disease; inflammatory
bowel disease; inflammatory skin disorder; or a condition
characterized by local or systemic inflammation, immune activation,
and/or lymphoproliferation. In some embodiments, the compound or
combination is for use in the prevention or treatment of a
condition characterized by chronic pain, neuro-inflammation and/or
neuro-degeneration. In some embodiments, said condition is further
characterized by visceral or non-visceral nociceptive pain,
peripheral or central neuropathic pain, or mixed
nociceptive-neuropathic pain, neuro-inflammation, and/or
neuro-degeneration. In some embodiments, said condition is selected
from the group consisting of post-operative orthopedic surgery
pain, musculoskeletal pain, irritable bowel syndrome, inflammatory
bowel disease, rheumatoid arthritis, ankylosing spondylitis,
post-herpetic neuralgia, trigeminal neuralgia, post-traumatic or
post-operative peripheral neuropathy, diabetic peripheral
neuropathy, inflammatory peripheral neuropathy, HIV-associated
neuropathy, painful peripheral neuropathy, nerve entrapment
syndrome, chemotherapy-associated pain, chemotherapy-induced
allodynia, complex regional pain syndrome, post-spinal injury pain,
post-stroke pain, multiple sclerosis, low back pain,
osteoarthritis, cancer pain, chronic visceral pain, fibromyalgia,
polymyalgia rheumatica, myofascial pain syndrome, Alzheimer's
disease and Parkinson's disease, Huntington's disease, and/or
amyotrophic lateral sclerosis, or multiple sclerosis. In some
embodiments, the compound or composition is expressed by a cell or
a cell line. In some embodiments, a nucleotide sequence encoding
the compound or combination is present in a gene therapy vector
that is used in the prevention or treatment of a condition
characterized by chronic pain, neuro-inflammation and/or
neuro-degeneration. In some embodiments, said condition is further
characterized by visceral or non-visceral nociceptive pain,
peripheral or central neuropathic pain, or mixed
nociceptive-neuropathic pain, neuro-inflammation, and/or
neuro-degeneration. In some embodiments, said condition is selected
from the group consisting of post-operative orthopedic surgery
pain, musculoskeletal pain, irritable bowel syndrome, inflammatory
bowel disease, rheumatoid arthritis, ankylosing spondylitis,
post-herpetic neuralgia, trigeminal neuralgia, post-traumatic or
post-operative peripheral neuropathy, diabetic peripheral
neuropathy, inflammatory peripheral neuropathy, HIV-associated
neuropathy, painful peripheral neuropathy, nerve entrapment
syndrome, chemotherapy-associated pain, chemotherapy-induced
allodynia, complex regional pain syndrome, post-spinal injury pain,
post-stroke pain, multiple sclerosis, chronic widespread pain, low
back pain, osteoarthritis, cancer pain, chronic visceral pain,
fibromyalgia, polymyalgia rheumatica, myofascial pain syndrome,
Alzheimer's disease and Parkinson's disease, Huntington's disease,
and/or amyotrophic lateral sclerosis, or multiple sclerosis.
BRIEF DESCRIPTION OF THE FIGURES RELATED TO THE INVENTION
[0043] The features of the present disclosure are set forth with
particularity in the appended claims. A better understanding of the
features and advantages of the present disclosure can be obtained
by reference to the following detailed description that sets forth
illustrative embodiments, in which the principles of the disclosure
are utilized, and the accompanying drawings of which:
[0044] FIG. 1. Cytokine-receptor subunits of IL10, IL4, IL13, IL33,
IL27, TGF.beta.1, and TGF.beta.2 are expressed in the dorsal root
ganglia of human and mouse. To evaluate whether cytokine receptors
targeted by fusion proteins of the present invention are expressed
by the sensory system, RNAseq data of cytokine receptor subunits
for IL10, IL4, IL13, IL33, IL27, TGF.beta.1, and TGF.beta.2 in the
dorsal root ganglia and spinal cord were extracted from the data
base by Ray et al. (Pain 2018; 159:1325-1345) as available on
www_utdallas.edu/bbs/painneurosciencelab/sensoryomics/drgtxome/?go.
RNA sequencing data are expressed as transcripts per million. For
comparison, data for expression of the receptors in whole blood are
also given.
[0045] FIGS. 2A-2G: IL4 and IL10 receptors expressed in sensory
neurons are required for analgesia induced by cross-linking IL4R
and IL10R. (FIG. 2A) Expression of IL4R.alpha. (left) and
IL10R.alpha. (right) in murine dorsal root ganglia. Persistent
inflammatory pain was induced by an intraplantar injection of 20
.mu.l of 2% carrageenan (CAR). At days 3, 4 and 5 after
intraplantar injection of carrageenan, mice received intrathecal
injections of mismatch (mm) or IL4R antisense oligodeoxynucleotides
(asODN). At day 6 the dorsal root ganglia were obtained and
analysed. (FIG. 2B) Expression of IL4R mRNA (left panel, n=13-18)
and protein (right panel, n=4) in the DRG of IL4R asODN treated
mice. IL4R.alpha. mRNA levels were measured with qPCR and corrected
for housekeeping genes (actin, GAPDH and HPRT). Protein expression
was determined by quantifying IL4R.alpha. immunofluorescent
staining intensity. As a receptor cross-linking compound IL4-IL10
fusion protein was used. Six days after carrageenan administration
mice received an intrathecal injection of 1 .mu.g IL4-10 fusion
protein (n=5) and (FIG. 2C) thermal and (FIG. 20) mechanical
sensitivity was followed over time using Hargreaves and Von Frey
test, respectively. Right bar graphs represent the analgesic
effects of IL4-10 determined as area under the curve (AUC) between
1 and 24 hours after intrathecal injection. (FIG. 2E)
Immunofluorescence staining for IL10R expression in DRGs from wild
type (WT, left) or Na.sub.V1.8-IL10R.sup.-/- (right) mice. At six
days after induction of persistent inflammatory pain WT mice and
Na.sub.V1.8-IL10R.sup.-/- mice received an intrathecal injection of
1 .mu.g IL4-10 fusion protein (n=13-14) and (FIG. 2F) thermal and
(FIG. 2G) mechanical sensitivity was followed over time using
Hargreaves or Von Frey test, respectively. Right bar graphs
represent the analgesic effects of IL4-10 fusion protein determined
as area under the curve (AUC) for the effect of IL4-10 fusion
protein between 1 and 72 hours after intrathecal injection. Data is
represented as mean.+-.SEM. *, **, ***=p<0.05, p<0.01, and
0.001, respectively.
[0046] FIG. 3A-3C: IL4R.alpha. and IL10R.alpha. in sensory neurons
are both required for full analgesic effects of IL4-10 fusion
protein. Inflammatory pain was induced by an intraplantar injection
of 20 .mu.l of 2% carrageenan in wild type (WT) mice or
Na.sub.V1.8-IL10R.sup.-/-. At days 3, 4 and 5 after intraplantar
injection mice received intrathecal injections of mismatched (mm)
or IL4R antisense oligodeoxynucleotides (ODN). Six days after
intraplantar injection mice received an intrathecal injection of 1
.mu.g IL4-10 fusion protein to cross-link IL4R.alpha. and
IL10R.alpha. (n=5 per group) and (FIG. 3A) thermal and (FIG. 3B)
mechanical sensitivity was followed over time using Hargreaves or
Von Frey tests, respectively. Right bar graphs represent the
analgesic effects of IL4-10 fusion protein determined as area under
the curve (AUC) between 1 and 24 hours after intrathecal injection.
(FIG. 3C) Quantification of the total number of c-Fos positive
neurons in laminae I-III of the spinal cord 24 hours after IL4-10
application. Right top panel: example c-Fos staining of the
superficial dorsal horn of the spinal cord. Right bottom panels:
representative pictures of c-Fos staining of the dorsal horn of
naive (i), carrageenan-injected vehicle-treated WT mice (ii),
carrageenan-injected IL4-10-treated WT mice (iii) and
carrageenan-injected IL4R asODN and IL4-10-treated
Na.sub.V1.8-IL10R.sup.-/- mice (iv) (n=4-6 per group). Data is
represented as mean.+-.SEM. *, **, ***=p<0.05. 0.01, and 0.001,
respectively.
[0047] FIG. 4A-4C. Crosslinking IL4R and IL13R relieves
chemotherapy-induced persistent mechanical allodynia. FIG. 4A.
Paclitaxel (8 mg/kg) was administered intraperitoneally to C57BL/6
mice on days 0, 2, 4 and 6 (grey symbols on the X-axis) to induce
persistent chemotherapy-induced polyneuropathy. IL4-13 fusion
protein (0.3 [open circle], 1 [open triangle] or 3 .mu.g/mouse
[open square]; n=4/group) or vehicle (n=4) was administered
intrathecally at day 8, and the course of mechanical allodynia was
followed over time using von Frey hairs. Data is represented as
mean.+-.SEM. Statistics of the data were analysed with two-way
ANOVA followed by Tukey's multiple comparisons test. *, **,
***=p<0.05, p<0.01, and 0.001, 0.3 .mu.g IL4/IL13 fusion
protein versus vehicle respectively. &, && =p<0.05,
p<0.01 respectively, 3 .mu.g IL4/IL13 fusion protein versus
vehicle. x=p<0.05, 1 .mu.g IL4/IL13 fusion protein versus
vehicle. FIG. 4B. On day 15, the length of intraepidermal nerve
fibers in the paw skin was determined upon immunofluorescent
visualization with the neuronal marker PGP9.5. The data of mice not
treated with chemotherapeutic drug (black bar; n=4), or injected
with paclitaxel and subsequently treated with vehicle (-; n=6) or
IL4/IL3 fusion protein (IL4-13; n=4), are shown. FIG. 4C.
Oxaliplatin (3 mg/kg) was daily injected intraperitoneally in mice
for 5 days followed by 5 days no treatment and another 5 days of an
oxaliplatin treatment cycle (grey symbols on X-axis). On the day
after the last oxaliplatin injection animals received an
intrathecal injection of IL4/IL13 fusion protein (0.3 .mu.g; open
circles, n=4) or the wild-type cytokines (0.15 .mu.g; n=4,
rectangles for IL4 and triangles for IL13); or vehicle only (closed
circles). Pain was measured with von Frey test.
[0048] FIGS. 5A-5I: Cross-linking of IL4R and IL10R inhibits
inflammatory mediator-induced sensitization of sensory neurons.
Fura-2 loaded primary sensory neurons were stimulated with 30 nM
capsaicin and Ca2.sup.+ influx was measured as the ratio of
F340/F380 normalized to basal levels. Sensory neurons were
stimulated overnight with (FIG. 5A) TNF.alpha. (50 ng/ml,
n=157-244) or (FIG. 5D) PGE2 (1 .mu.M, n=119-188) in the absence or
presence of the IL4-10 fusion protein (100 ng/ml, 3 nM). Total
calcium fluxes were quantified by determining the maximal amplitude
(FIG. 5B, FIG. 5E) of capsaicin-evoked Ca2+ responses (applied at
time point 1 minute) and the area under curve (AUC; FIG. 5C, FIG.
5F) of capsaicin-evoked Ca2.sup.+ influx over 5 minutes. (FIG. 5G)
Sensory neurons were stimulated overnight with TNF.alpha. (50
ng/ml) and IL4-10 fusion protein (100 ng/ml) in the presence of
receptor-blocking antibodies (2 .mu.g/ml) against the IL4 receptor
(.alpha.IL4R) and the IL10 receptor (.alpha.IL10R). Inhibition of
TNF.alpha.-sensitization was measured as percentage of AUC of
capsaicin-evoked Ca.sup.2+ response over 5 minutes. Asterisks
represent significant differences compared to the
TNF.alpha.-sensitized neuronal response. (n=51-164) (FIG. 5H, FIG.
5I) Sensory neurons were stimulated overnight with TNF.alpha. (50
ng/ml) in combination with different concentrations (0.6, 6 and 60
nM) of IL4-10 fusion protein or equimolar doses of the combination
of both recombinant cytokines. Inhibition of
TNF.alpha.-sensitization was measured as the percentage of the
(FIG. 5H) amplitude or (FIG. 5I) AUC of capsaicin-evoked Ca2.sup.+
response over 5 minutes (n=107-244). Data is represented as
mean.+-.SEM. *, **, ***=p<0.05, 0.01 and 0.001, respectively
(n=51-244 from at least 3 different cultures).
[0049] FIG. 6. Cross-linking of IL4R and IL13R is required to
protect cultured neurons against oxaliplatin-induced damage.
Primary sensory neurons were cultured and treated overnight with
oxaliplatin (5 .mu.g/ml). Neuronal damage was then quantified by
measuring the neurite length upon .beta.3-tubulin staining. Vehicle
(-) or IL4/IL13 fusion protein or the combination of IL4 and IL13
(IL4+IL13) were added at equimolar concentrations during incubation
with the chemotherapeutic drug. Neurons cultured in absence of
oxaliplatin and cytokines are shown for comparison (black bar).
[0050] FIGS. 7A-7B: IL4-10 fusion protein induces heterologous
receptor clustering in sensory neurons. (FIG. 7A) Cultured sensory
neurons were treated for 15 minutes with IL4-10 fusion protein (100
ng/ml; right column), the combination of IL4 and IL10 (50 ng/ml
each; middle column) or vehicle (left column). After fixation, a
proximity ligation assay (PLA) for IL4R and IL10R was performed
(red) and combined with immunofluorescent staining for
anti-.beta.III-tubulin to identify sensory neurons (green).
Presence of red fluorescence indicates that IL4R and IL10R are at
less than 51 nm in proximity to each other. (FIG. 7B) Intensity of
PLA staining was quantified using Image J software. For each
condition 3 to 6 pictures of each of 2 different experiments were
analysed. Data is represented as mean.+-.SD. *=p<0.05.
[0051] FIGS. 8A-8F. Kinome activity profile in the DRG of mice with
chronic pain after cross-linking IL4R and IL10R with a fusion
protein. Mice received an intraplantar injection of 20 .mu.l of 2%
carrageenan and 6 days later received an intrathecal injection with
either vehicle, the combination of IL4+IL10 (0.5 .mu.g each), or
IL4R-IL10R cross-linking compound, i.e., IL4-10 fusion protein (1
.mu.g). One hour after intrathecal injection DRGs were isolated and
DRG homogenates were subjected to PAMGENE analysis. (FIGS. 8A-8D)
List of peptides from the PAM chips which are differentially
regulated based on one-way ANOVA analysis. Blue colour indicates
diminished phosphorylation of peptide substrates, and red colour
indicates increased phosphorylation of peptides in DRG lysates of
IL4-IL10-treated mice compared to those treated with IL4+IL10.
Black color indicates no significant changes. (A/B) Peptides that
are significantly different in the PTK chip. (FIG. 8A) List of PTK
target peptides differentially regulated between IL4-10 and
IL4+IL10-treated animals compared to control-treated animals. (FIG.
8B) List of PTK target peptides differentially regulated between
IL4-10-treated animals compared to IL4+IL10 treated animals. (C/D)
Peptides that are significantly differently phosphorylated in the
STK chip compared to vehicle. (FIG. 8C) List of STK target peptides
differentially regulated between IL4-10 and IL4+IL10 treated
animals compared to control-treated animals. (FIG. 8D) List of STK
target peptides differentially regulated between IL4-10-treated
animals and IL4+IL10 treated animals. (FIG. 8E) Predicted upstream
kinases that can be inferred from the peptide substrates
differentially phosphorylated on the PAM chips. Unpaired t-test
comparison between samples from IL4-10-treated animals compared to
IL4+IL10-treated animals (n=5 animals per group). (Illustration
reproduced courtesy of Cell Signaling Technology, Inc.
(www_cellsignal.com)). (FIG. 8F) Enriched GeneGo process pathway
analysis based on peptides that were significantly differentially
phosphorylated after t-test comparison between IL4-10 and
IL4+IL10-treated animals. The height of the histogram corresponds
to the p-values of signalling pathways that are significantly
enriched by differentially phosphorylated peptides. (FIG. 8G) Top
10 predicted Protein Tyrosine kinases differentially regulated
between samples from IL4-10-treated animals compared to
IL4+IL10-treated animals. The graph is sorted with the highest
specificity scores at the top, and the lowest specificity scores at
the bottom (FIG. 8H) top 10 predicted Serine/threonine kinases
differentially regulated between samples from IL4-10-treated
animals compared to IL4+IL10-treated animals. The graph is sorted
with the highest specificity scores at the top, and the lowest
specificity scores at the bottom
[0052] FIGS. 9A-9F. Transcriptome analysis of the DRG after
intrathecal injection of IL4-10. Persistent inflammatory pain was
induced by an intraplantar injection of 20 .mu.l of 2% carrageenan.
Six days later mice received an intrathecal injection of vehicle
(control), IL4+IL10 (0.5 .mu.g each) or IL4-10 fusion protein (1
.mu.g). Six hours after intrathecal injection, lumbar DRGs (L3-L5)
were isolated and subjected to RNA-sequencing. (FIG. 9A) Principal
component analysis (PCA) of the differentially expressed genes in
all pair-wise comparisons of different animal. (FIG. 9B)
Hierarchical clustering heat map of the expression levels of the
top 500 differentially expressed genes (based on adjusted
p-values). (FIG. 9C) Venn diagram showing the number of
differentially expressed genes in IL4-10 fusion protein or IL4+IL10
treated animals compared to vehicle-treated animals. (FIG. 9D) The
volcano plot shows the adjusted p-values and fold changes for all
transcripts in IL4+IL10-treated mice compared to IL4-10-treated
mice. Differential expression of genes (FDR corrected
p-value<0.05) was determined using DESeq2 package in R. (FIG.
9E) Top 25 pathway analysis of genes differentially regulated
between IL4-10 fusion protein and the combination of IL4+IL10
(toppgene.cchmc.org/). (FIG. 9F) Pathway enrichment analysis of the
differentially regulated transcript (IL4-10 versus IL4+II10) to
identifying potential signaling pathways upstream of the
differentially regulated genes
[0053] FIGS. 10A-10E. Interrogation of role of the role of kinases
in the superior analgesic effect of IL4-10. (FIG. 10A) Sensory
neurons were treated in vitro for 10, 30 or 60 minutes with IL4-10
fusion protein (100 ng/ml; light grey columns), the combination of
IL4 and IL10 (50 ng/ml each; dark grey columns) or vehicle (black
column). Cells were stained for pJAK1 and fluorescence intensity of
individual cells were measured. Data are obtained in 4 different
independent primary neuronal cultures. (B-E) Persistent
inflammatory pain was induced by an intraplantar injection of 20
.mu.l of 2% carrageenan. (B, C) At days 5, 6 and 7 after
intraplantar injection mice received Ruxolitinib (JAK1/2 inhibitor;
n=8) or vehicle orally (n=4). Six days after intraplantar injection
mice received an intrathecal injection of 1 .mu.g IL4-10 fusion
protein (n=8) or PBS as vehicle (n=4) and (FIG. 10B) thermal and
(FIG. 10C) mechanical sensitivity was followed overtime using
Hargreaves or Von Frey tests, respectively. Right bar graphs
represent the total analgesic effects of IL4-10 determined as area
under the curve (AUC) between 1 and 72 hours after intrathecal
injection. *p<0.05 IL4-10: Vehicle versus IL4-10:
Ruxolitinib-treated mice. #p<0.05 IL4-10: Vehicle versus
IL4+IL10: Vehicle-treated mice. (D, E) Six days after intraplantar
injection of carrageenan mice received an intrathecal injection of
1 .mu.g IL4-10 fusion protein and (FIG. 10D) thermal and (FIG. 10E)
mechanical sensitivity was followed over time using Hargreaves or
Von Frey tests, respectively. To inhibit c-kit or both c-kit and
PDGFR mice received respectively intraperitoneal injections of
Dasatinib (n=4), or Masitinib (n=4) at days 5, 6 and 7 after
intraplantar carrageenan injection. To inhibit MET mice were orally
administered with the MET inhibitor JNJ-38877605 (n=5) or vehicle.
Bar graphs represent the analgesic effects of IL4-10 determined as
area under the curve (AUC) between 1 and 72 hours after intrathecal
injection. Data is represented as mean.+-.SEM. *, **,
***=p<0.05. 0.01, and 0.001, respectively.
[0054] FIG. 11. An IL4-containing fusion protein of the disclosure
elicits a distinct kinase activity profile in dorsal root ganglia
(DRG) cells compared to a combination of unlinked cytokines.
PamGene kinase activity profiling was performed to assess global
protein tyrosine kinases (PTK) activity in homogenates of lumbar
DRGs isolated from mice with persistent paclitaxel-induced
peripheral neuropathy after IL4/IL13 fusion protein, IL4+IL13
(combination of unlinked cytokines), and vehicle administration.
Kinomic profiles were assessed at 60 minutes after intrathecal
administration of the IL4/IL13 fusion protein, the combination of
cytokines, or vehicle (PBS). Peptides are shown that were
differentially phosphorylated based on one-way ANOVA analysis
between IL4/IL13, IL4+IL13, and vehicle-treated mice compared to
naive mice (untreated; no paclitaxel, no intrathecal injection).
Black indicates no significant changes, while color indicates
decreased phosphorylation.
[0055] FIG. 12. Altered kinase activity in dorsal root ganglia
(DRG) cells of female mice treated with IL4/IL13 compared to a
combination of unlinked cytokines. PamGene kinase activity
profiling was performed to assess global protein tyrosine kinases
(PTK) activity in homogenates of lumbar DRGs isolated from female
mice with persistent paclitaxel-induced neuropathy after IL4/IL13
fusion protein or IL4+IL13 (combination of unlinked cytokines)
administration. The graph shows the predicted upstream kinases
inferred from the differentially phosphorylated peptide substrates
identified by unpaired t-test comparison between samples from
IL4/IL13 fusion protein-treated females and IL4+IL13-treated
females (n=3 animals per group). The graph is sorted with the
highest specificity scores at the top, and the lowest specificity
scores at the bottom.
[0056] FIG. 13. Altered kinase activity in dorsal root ganglia
(DRG) cells of male mice treated with IL4/IL13 compared to a
combination of unlinked cytokines. PamGene kinase activity
profiling was performed to assess global protein tyrosine kinases
(PTK) activity in homogenates of lumbar DRGs isolated from male
mice with persistent paclitaxel-induced neuropathy after IL4/IL13
fusion protein or IL4+IL13 (combination of unlinked cytokines)
administration. The graph shows the predicted upstream kinases
inferred from the differentially phosphorylated peptide substrates
identified by unpaired t test comparison between samples from
IL4/IL13 fusion protein-treated males and IL4+IL13-treated males
(n=3 animals per group). The graph is sorted with the highest
specificity scores at the top, and the lowest specificity scores at
the bottom.
DETAILED DESCRIPTION OF THE INVENTION
[0057] The present disclosure provides compounds (e.g., polypeptide
constructs, fusion proteins) that comprise an interleukin 4 (IL4)
directly or indirectly linked to an additional cytokine, for
example, a cytokine selected from the group consisting of an
interleukin 10 (IL10). an interleukin 13 (IL13), an interleukin 27
(IL27), an interleukin 33 (IL33), a transforming growth factor beta
1 (TGF.beta.1), a transforming growth factor beta 2 (TGF.beta.2),
and an additional IL4.
[0058] Compounds of the disclosure can cluster or crosslink the IL4
receptor and a cytokine receptor, and surprisingly, elicit unique
responses in the nervous system, including unique signaling and
gene expression profiles. The signaling and gene expression
profiles generated IL4-cytokine fusion proteins of the disclosure
are distinct from those observed in response to the combination of
IL4 and the cytokine, and can contribute to superior therapeutic
effects over the combination of the component parts.
[0059] In some embodiments, compounds (e.g., polypeptide
constructs, fusion proteins) disclosed herein elicit unique and
favorable responses in nervous system cells compared to equivalent
amounts of IL4 and the cytokine individually. The disclosed
compounds (e.g., polypeptide constructs, fusion proteins) can be
useful for treating a condition disclosed herein in a subject in
need thereof, including, for example, achieving superior
therapeutic effects compared to equivalent amounts of IL4 and the
cytokine administered separately or in combination (e.g., as a
combination of unlinked cytokines).
[0060] When describing a cytokine, the term "wild type" refers to a
cytokine with an amino acid sequence that is naturally occurring
and encoded by a germline genome of a given species. A species can
have one wild type sequence, or two or more wild type sequences
(for example, with one canonical wild type sequence and one or more
non-canonical wild type sequences). A wild type cytokine sequence
can include a sequence that is truncated at the N and/or C terminus
relative to the sequence encoded by an open reading frame. A wild
type cytokine sequence can be a mature form of a cytokine that has
been processed to remove N-terminal and/or C-terminal residues. A
wild type cytokine can lack a signal peptide or can include a
signal peptide (e.g., a signal peptide can be added to the
N-terminus of the wild type cytokine).
[0061] When describing a cytokine, the term "derivative" refers to
a cytokine with an amino acid sequence that differs from a wild
type sequence by one or more amino acids, for example, containing
one or more amino acid insertions, deletions, or substitutions
relative to a wild type sequence. A cytokine derivative binds to at
least one subunit of the corresponding native receptor for the wild
type cytokine and elicits signaling and/or cytokine activity. The
binding affinity, signaling, and/or cytokine activity of a cytokine
derivative can be the same or different than the corresponding wild
type cytokine.
[0062] The term "nervous system cell" refers to a cell that is
found within the central nervous system or peripheral nervous
system. A nervous system cell can be a neuron, a central nervous
system cell, a peripheral nervous system cell, a glial cell, a
microglial cell, an astrocyte, a Schwann cell, a satellite glial
cell, an oligodendrocyte, an infiltrating cell, an infiltrating
immune cell, an infiltrating myeloid cell, an infiltrating lymphoid
cell, an infiltrating macrophage, an infiltrating neutrophil, an
infiltrating lymphocyte, an infiltrating T cell, an infiltrating B
cell, or an infiltrating natural killer cell. A neuron can be, for
example, a sensory neuron, a somatosensory neuron, a visceral
sensory neuron, a nociceptor, and/or an autonomic neuron.
[0063] "Sequence identity" and "sequence similarity" can be
determined by alignment of two peptide or two nucleotide sequences
using global or local alignment algorithms. Sequences may then be
referred to as "substantially identical" or "essentially similar"
when they (when optimally aligned by for example the programs GAP
or BESTFIT using default parameters) share at least a certain
minimal percentage of sequence identity. GAP uses the Needleman and
Wunsch global alignment algorithm to align two sequences over their
entire length, maximizing the number of matches and minimizes the
number of gaps. Generally, the GAP default parameters are used,
with a gap creation penalty=50 (nucleotides)/8 (proteins) and gap
extension penalty=3 (nucleotides)/2 (proteins). For nucleotides the
default scoring matrix used is nwsgapdna and for proteins the
default scoring matrix is Blosum62 (Henikoff & Henikoff, 1992,
PNAS 89, 915-919). Sequence alignments and scores for percentage
sequence identity may be determined using computer programs, such
as the GCG Wisconsin Package, Version 10.3, available from Accelrys
Inc., 9685 Scranton Road, San Diego, Calif. 92121-3752 USA, or
EmbossWin version 2.10.0 (using the program "needle").
Alternatively, percent similarity or identity may be determined by
searching against databases, using algorithms such as FASTA, BLAST,
etc. Sequence identity can refer to the sequence identity over the
entire length of the sequence.
Interleukin 4:
[0064] A compound (e.g., polypeptide construct, fusion protein)
disclosed herein can comprise an IL4 protein, or a variant,
derivative or fragment thereof operably connected or directly or
indirectly fused to at least one additional cytokine or a variant,
derivative or fragment thereof. The IL4 protein can be a mammalian
IL4 protein, such as a human IL4, or mouse IL4. Non-limiting
examples of amino acid sequences of IL4 are set forth in SEQ ID
NOs: 11-14.
TABLE-US-00001 TABLE 1 non-limiting examples of human IL4 sequences
of the disclosure SEQ ID NO: SEQUENCE 11
HKCDITLQEIIKTLNSLTEQKTLCTELTVTDIFAASKNTTEKETFCRAATVLRQFYS
HHEKDTRCLGATAQQFHRHKQURFLKRLDRNLWGLAGLNSCPVKEANQSTLE
NFLERLKTIMREKYSKCSS 12
HKCDITLQEIIKTLNSLTEQKNTTEKETFCRAATVLRQFYSHHEKDTRCLGATAQQ
FHRHKQURFLKRLDRNLWGLAGLNSCPVKEANQSTLENFLERLKTIMREKYSKC SS 13
HKRDITLQEIIKTLNSLTEQKTLCTELTVTDIFAASKNTTEKETFCRAATVLRQFYS
HHEKDTRCLGATAQQFHRHKQURFLKRLDRNLWGLAGLNSCPVKEANQSTLE
NFLERLKTIMREKYSKCSS 14
HKRDITLQEIIKTLNSLTEQKNTTEKETFCRAATVLRQFYSHHEKDTRCLGATAQQ
FHRHKQURFLKRLDRNLWGLAGLNSCPVKEANQSTLENFLERLKTIMREKYSKC SS
[0065] Variants of IL4 include, for example, proteins having at
least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or
more, such as 100%, amino acid sequence identity to any one of SEQ
ID NOs: 11-14, for example, over the entire length. Amino acid
sequence identity can be determined by pairwise alignment using the
Needleman and Wunsch algorithm and GAP default parameters, e.g., as
disclosed herein. Variants and derivatives also include proteins
having IL4 activity, which have been derived, by way of one or more
amino acid substitutions, deletions or insertions, from the
polypeptide having the amino acid sequence of any one of SEQ ID
NOs: 11-14. Such proteins can comprise from 1, 2, 3, 4, 5, 6, 7, 8,
9, 10 or more up to about 100, 90, 80, 70, 60, 50, 45, 40, 35, 30,
25, 20, 15 amino acid substitutions, deletions or insertions.
[0066] In some embodiments, an IL4 of the disclosure (e.g., an IL4
variant, derivative, or fragment thereof) can comprise at least 1,
at least 2, at least 3, at least 4, at least 5, at least 6, at
least 7, at least 8, at least 9, at least 10, at least 11, at least
12, at least 13, at least 14, at least 15, at least 16, at least
17, at least 18, at least 19, at least 20, at least 25, at least
30, at least 35, at least 40, at least 45, at least or at least 50
amino acid substitutions, deletions, or insertions relative to an
IL4 sequence disclosed herein (e.g., a wild type IL4 sequence).
[0067] In some embodiments, an IL4 of the disclosure (e.g., an IL4
variant, derivative, or fragment thereof) can comprise at most 1,
at most 2, at most 3, at most 4, at most 5, at most 6, at most 7,
at most 8, at most 9, at most 10, at most 11, at most 12, at most
13, at most 14, at most 15, at most 16, at most 17, at most 18, at
most 19, at most 20, at most 25, at most 30, at most 35, at most
40, at most 45, or at most 50 amino acid substitutions, deletions,
or insertions relative to an IL4 sequence disclosed herein (e.g., a
wild type IL4 sequence).
[0068] In some embodiments, an IL4 sequence of the disclosure
(e.g., an IL4 variant, derivative, or fragment thereof) can
comprise 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-15, 1-20,
1-30, 1-40, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 2-15, 2-20,
2-30, 2-40, 3-3, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 3-15, 3-20,
3-30, 3-40, 5-6, 5-7, 5-8, 5-9, 5-10, 5-15, 5-20, 5-30, 5-40,
10-15, 15-20, or 20-25 amino acid substitutions, deletions, or
insertions relative to an IL4 sequence disclosed herein (e.g., a
wild type IL4 sequence).
[0069] In some embodiments, an IL4 sequence of the disclosure
(e.g., an IL4 variant, derivative, or fragment thereof) can
comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, or 20 amino acid substitutions, deletions, or insertions
relative to an IL4 sequence disclosed herein (e.g., a wild type IL4
sequence). An amino acid substitution can be a conservative or a
non-conservative substitution. The one or more amino acid
substitutions, deletions, or insertions can be at the N-terminus,
the C-terminus, within the amino acid sequence, or a combination
thereof. The amino acid substitutions, deletions, or insertions can
be contiguous, non-contiguous, or a combination thereof.
[0070] An IL4 of the disclosure can comprise a wild type IL4
sequence. Non-limiting examples of wild type IL4 sequences include
SEQ ID NOs: 11-14. SEQ ID NO: 11 can be a canonical wild type IL4
sequence of the disclosure.
[0071] An IL4 of the disclosure can comprise an IL4 variant,
derivative, or fragment thereof with one or more amino acid
substitutions. For example, an IL4 variant, derivative, or fragment
thereof can comprise an amino acid substitution at position K117,
T118, R121, E122, Y124, S125, S128, S129, or a combination thereof
of SEQ ID NO: 11. In some embodiments, an IL4 variant, derivative,
or fragment thereof comprises a substitution that is K117R, T118V,
R121Q, R121D, R121K, R121E, E122S, Y124W, Y124F, Y124D, S125F,
S128G, S125R, S129A, or a combination thereof relative to SEQ ID
NO: 11. In some embodiments, an IL4 variant, derivative, or
fragment thereof comprises the substitutions K117R, T118V, R121Q,
E122S, Y124W, S125F, S128G, and S129A relative to SEQ ID NO: 11. In
some embodiments, an IL4 variant, derivative, or fragment thereof
comprises the substitutions R121D and Y124D relative to SEQ ID NO:
11.
[0072] In some embodiments, an IL4 variant, derivative, or fragment
thereof does not contain a substitution at position K117, T118,
R121, E122, Y124, S125, S128, or S129, relative to SEQ ID NO: 11.
In some embodiments, an IL4 variant, derivative, or fragment
thereof does not contain a K117R, T118V, R121Q, R121D, R121K,
R121E, E122S, Y124W, Y124F, Y124D, S125F, S128G, S125R, or S129A
substitution.
[0073] In some embodiments, an IL4 or IL4 variant, derivative, or
fragment thereof of the disclosure binds to an IL4 receptor subunit
with about a comparable affinity as a wild type IL4 sequence. A
comparable affinity can be, for example, less than about 10, less
than about 5, less than about 2, less than about 1.9, less than
about 1.8, less than about 1.7, less than about 1.6, less than
about 1.5, less than about 1.4, less than about 1.3, less than
about 1.2, or less than about 1.1 fold increased affinity compared
to a wild type IL4 sequence. A comparable affinity can be, for
example, less than about 10, less than about 5, less than about 2,
less than about 1.9, less than about 1.8, less than about 1.7, less
than about 1.6, less than about 1.5, less than about 1.4, less than
about 1.3, less than about 1.2, or less than about 1.1 fold
decreased affinity compared to a wild type IL4 sequence.
[0074] For example, an IL4 or IL4 variant, derivative, or fragment
thereof of the disclosure can bind to an interleukin 13 receptor
alpha 1 (IL-13R.alpha.1), common gamma chain, interleukin 4
receptor alpha (IL-4R.alpha.), or a combination thereof, e.g. with
about a comparable affinity as a wild type IL4. In some
embodiments, an IL4 or IL4 variant, derivative, or fragment thereof
of the disclosure can bind to IL-13R.alpha.1 with about a
comparable affinity as a wild type IL4. In some embodiments, an IL4
or IL4 variant, derivative, or fragment thereof of the disclosure
can bind to common gamma chain with about a comparable affinity as
a wild type IL4. In some embodiments, an IL4 or IL4 variant,
derivative, or fragment thereof of the disclosure can bind to
IL-4R.alpha. with about a comparable affinity as a wild type IL4
sequence. In some embodiments, an IL4 or IL4 variant, derivative,
or fragment thereof of the disclosure can bind to IL-13R.alpha.1
and common gamma chain with about a comparable affinity as a wild
type IL4. In some embodiments, an IL4 or IL4 variant, derivative,
or fragment thereof of the disclosure can bind to IL-13R.alpha.1
and IL-4R.alpha. with about a comparable affinity as a wild type
IL4. In some embodiments, an IL4 or IL4 variant, derivative, or
fragment thereof of the disclosure can bind to common gamma chain
and IL-4R.alpha. with about a comparable affinity as a wild type
IL4. In some embodiments, an IL4 or IL4 variant, derivative, or
fragment thereof of the disclosure can bind to IL-13R.alpha.1,
common gamma chain, and IL-4R.alpha. with about a comparable
affinity as a wild type IL4.
[0075] In some embodiments, an IL4 or IL4 variant, derivative, or
fragment thereof of the disclosure binds to an IL4 receptor subunit
with at least a comparable affinity as a wild type IL4 sequence.
For example, an IL4 or IL4 variant, derivative, or fragment thereof
of the disclosure can bind to an interleukin 13 receptor alpha 1
(IL-13R.alpha.1), common gamma chain, interleukin 4 receptor alpha
(IL-4R.alpha.), or a combination thereof with at least a comparable
affinity as a wild type IL4. In some embodiments, an IL4 or IL4
variant, derivative, or fragment thereof of the disclosure can bind
to IL-13R.alpha.1 with at least a comparable affinity as a wild
type IL4. In some embodiments, an IL4 or IL4 variant, derivative,
or fragment thereof of the disclosure can bind to common gamma
chain with at least a comparable affinity as a wild type IL4. In
some embodiments, an IL4 or IL4 variant, derivative, or fragment
thereof of the disclosure can bind to IL-4R.alpha. with at least a
comparable affinity as a wild type IL4 sequence. In some
embodiments, an IL4 or IL4 variant, derivative, or fragment thereof
of the disclosure can bind to IL-13R.alpha.1 and common gamma chain
with at least a comparable affinity as a wild type IL4. In some
embodiments, an IL4 or IL4 variant, derivative, or fragment thereof
of the disclosure can bind to IL-13R.alpha.1 and IL-4R.alpha. with
at least a comparable affinity as a wild type IL4. In some
embodiments, an IL4 or IL4 variant, derivative, or fragment thereof
of the disclosure can bind to common gamma chain and IL-4R.alpha.
with at least a comparable affinity as a wild type IL4. In some
embodiments, an IL4 or IL4 variant, derivative, or fragment thereof
of the disclosure can bind to IL-13R.alpha.1, common gamma chain,
and IL-4R.alpha. with at least a comparable affinity as a wild type
IL4.
[0076] In some embodiments, an IL4 or IL4 variant, derivative, or
fragment thereof of the disclosure binds to an IL4 receptor subunit
with at most a comparable affinity as a wild type IL4 sequence. For
example, an IL4 or IL4 variant, derivative, or fragment thereof of
the disclosure can bind to an interleukin 13 receptor alpha 1
(IL-13R.alpha.1), common gamma chain, interleukin 4 receptor alpha
(IL-4R.alpha.), or a combination thereof with at most a comparable
affinity as a wild type IL4. In some embodiments, an IL4 or IL4
variant, derivative, or fragment thereof of the disclosure can bind
to IL-13R.alpha.1 with at most a comparable affinity as a wild type
IL4. In some embodiments, an IL4 or IL4 variant, derivative, or
fragment thereof of the disclosure can bind to common gamma chain
with at most a comparable affinity as a wild type IL4. In some
embodiments, an IL4 or IL4 variant, derivative, or fragment thereof
of the disclosure can bind to IL-4R.alpha. with at most a
comparable affinity as a wild type IL4 sequence. In some
embodiments, an IL4 or IL4 variant, derivative, or fragment thereof
of the disclosure can bind to IL-13R.alpha.1 and common gamma chain
with at most a comparable affinity as a wild type IL4. In some
embodiments, an IL4 or IL4 variant, derivative, or fragment thereof
of the disclosure can bind to IL-13R.alpha.1 and IL-4R.alpha. with
at most a comparable affinity as a wild type IL4. In some
embodiments, an IL4 or IL4 variant, derivative, or fragment thereof
of the disclosure can bind to common gamma chain and IL-4R.alpha.
with at most a comparable affinity as a wild type IL4. In some
embodiments, an IL4 or IL4 variant, derivative, or fragment thereof
of the disclosure can bind to IL-13R.alpha.1, common gamma chain,
and IL-4R.alpha. with at most a comparable affinity as a wild type
IL4.
[0077] In some embodiments, an IL4 or IL4 variant, derivative, or
fragment thereof can bind to an IL-13R.alpha.1 with at least about
1.5 fold, 2 fold, 5 fold, 10 fold, 15 fold, 20 fold, 30 fold, 40
fold, 50 fold, 100 fold, 200 fold, 500 fold, 1000 fold, or 10,000
fold increased affinity relative to a wild type IL4 sequence. In
some embodiments, an IL4 or IL4 variant, derivative, or fragment
thereof can bind to an IL-13R.alpha.1 with at least about 1.5 fold,
2 fold, 5 fold, 10 fold, 15 fold, 20 fold, 30 fold, 40 fold, 50
fold, 100 fold, 200 fold, 500 fold, 1000 fold, or 10,000 fold
decreased affinity relative to a wild type IL4 sequence.
[0078] In some embodiments, an IL4 or IL4 variant, derivative, or
fragment thereof can bind to a common gamma chain with at least
about 1.5 fold, 2 fold, 5 fold, 10 fold, 15 fold, 20 fold, 30 fold,
40 fold, 50 fold, 100 fold, 200 fold, 500 fold, 1000 fold, or
10,000 fold increased affinity relative to a wild type IL4
sequence. In some embodiments, an IL4 or IL4 variant, derivative,
or fragment thereof can bind to a common gamma chain with at least
about 1.5 fold, 2 fold, 5 fold, 10 fold, 15 fold, 20 fold, 30 fold,
40 fold, 50 fold, 100 fold, 200 fold, 500 fold, 1000 fold, or
10,000 fold decreased affinity relative to a wild type IL4
sequence.
[0079] In some embodiments, an IL4 or IL4 variant, derivative, or
fragment thereof can bind to an IL-4R.alpha. with at least about
1.5 fold, 2 fold, 5 fold, 10 fold, 15 fold, 20 fold, 30 fold, 40
fold, 50 fold, 100 fold, 200 fold, 500 fold, 1000 fold, or 10,000
fold increased affinity relative to a wild type IL4 sequence. In
some embodiments, an IL4 or IL4 variant, derivative, or fragment
thereof can bind to an IL-4R.alpha. with at least about 1.5 fold, 2
fold, 5 fold, 10 fold, 15 fold, 20 fold, 30 fold, 40 fold, 50 fold,
100 fold, 200 fold, 500 fold, 1000 fold, or 10,000 fold decreased
affinity relative to a wild type IL4 sequence.
[0080] In some embodiments, an IL4 or IL4 variant, derivative, or
fragment thereof of the disclosure can activate a native IL4
receptor. A native IL4 receptor can be, for example, a receptor
comprising an IL-13R.alpha.1 subunit and an IL-4R.alpha. subunit,
or a common gamma chain subunit and an IL-4R.alpha. subunit. In
some embodiments, an IL4 or IL4 variant, derivative, or fragment
thereof of the disclosure can activate a native IL4 receptor when
present in a fusion protein. In some embodiments, an IL4 or IL4
variant, derivative, or fragment thereof of the disclosure can
activate a native IL4 receptor when present as a polypeptide that
is not part of a fusion protein, but does not activate native IL4
receptor when present in a fusion protein.
[0081] In some embodiments, a polypeptide of the disclosure does
not contain IL4. In some embodiments, a polypeptide of the
disclosure does not contain SEQ ID NO: 11.
Interleukin 10:
[0082] A compound (e.g., polypeptide construct, fusion protein) may
comprise a monomeric or multimeric (for instance homodimeric) IL10
protein, or a variant, derivative, or fragment thereof operably
connected or directly or indirectly fused to an IL4 protein, or a
variant, derivative or fragment thereof. The IL10 protein can be a
mammalian IL10 protein, such as a human IL10, or mouse IL10. One
amino acid sequence representing IL10 is set forth in SEQ ID NO:
15.
TABLE-US-00002 TABLE 2 non-limiting examples of human IL10
sequences of the disclosure SEQ ID NO: SEQUENCE 15
SPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLL
EDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRR
CHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN
[0083] Variants of IL10 include, for example, proteins having at
least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or
more, such as 100%, amino acid sequence identity to SEQ ID NO: 15,
for example, over the entire length. Amino acid sequence identity
can be determined by pairwise alignment using the Needleman and
Wunsch algorithm and GAP default parameters as disclosed herein.
Variants, derivatives, and fragments thereof also include proteins
having IL10 activity, which have been derived, by way of one or
more amino acid substitutions, deletions or insertions, from the
polypeptide having the amino acid sequence of SEQ ID NO: 15. Such
proteins can comprise from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more up
to about 100, 90, 80, 70, 60, 50, 45, 40, 35, 30, 25, 20, 15 amino
acid substitutions, deletions or insertions.
[0084] In some embodiments, an IL10 of the disclosure (e.g., an
IL10 variant, derivative, or fragment thereof) can comprise at
least 1, at least 2, at least 3, at least 4, at least 5, at least
6, at least 7, at least 8, at least 9, at least 10, at least 11, at
least 12, at least 13, at least 14, at least 15, at least 16, at
least 17, at least 18, at least 19, at least 20, at least 25, at
least 30, at least 35, at least 40, at least 45, at least or at
least 50 amino acid substitutions, deletions, or insertions
relative to an IL10 sequence disclosed herein (e.g., a wild type
IL10 sequence).
[0085] In some embodiments, an IL10 of the disclosure (e.g., an
IL10 variant, derivative, or fragment thereof) can comprise at most
1, at most 2, at most 3, at most 4, at most 5, at most 6, at most
7, at most 8, at most 9, at most 10, at most 11, at most 12, at
most 13, at most 14, at most 15, at most 16, at most 17, at most
18, at most 19, at most 20, at most 25, at most 30, at most 35, at
most 40, at most 45, or at most 50 amino acid substitutions,
deletions, or insertions relative to an IL10 sequence disclosed
herein (e.g., a wild type IL10 sequence).
[0086] In some embodiments, an IL10 sequence of the disclosure
(e.g., an IL10 variant, derivative, or fragment thereof) can
comprise 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-15, 1-20,
1-30, 1-40, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 2-15, 2-20,
2-30, 2-40, 3-3, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 3-15, 3-20,
3-30, 3-40, 5-6, 5-7, 5-8, 5-9, 5-10, 5-15, 5-20, 5-30, 5-40,
10-15, 15-20, or 20-25 amino acid substitutions, deletions, or
insertions relative to an IL10 sequence disclosed herein (e.g., a
wild type IL10 sequence).
[0087] In some embodiments, an IL10 sequence of the disclosure
(e.g., an IL10 variant, derivative, or fragment thereof) can
comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, or 20 amino acid substitutions, deletions, or insertions
relative to an IL10 sequence disclosed herein (e.g., a wild type
IL10 sequence). An amino acid substitution can be a conservative or
a non-conservative substitution. The one or more amino acid
substitutions, deletions, or insertions can be at the N-terminus,
the C-terminus, within the amino acid sequence, or a combination
thereof. The amino acid substitutions, deletions, or insertions can
be contiguous, non-contiguous, or a combination thereof.
[0088] An IL10 of the disclosure can comprise a wild type IL10
sequence. A non-limiting examples of a wild type IL10 sequences is
SEQ ID NO: 15. SEQ ID NO: 15 can be a canonical wild type IL10
sequence of the disclosure.
[0089] An IL10 of the disclosure can comprise an IL10 variant,
derivative, or fragment thereof with one or more amino acid
substitutions. For example, an IL10 variant, derivative, or
fragment thereof can comprise an amino acid substitution at
position I87, A89, H109, R110, F111, Y153, M156, or a combination
thereof of SEQ ID NO: 15. In some embodiments, an IL10 variant,
derivative, or fragment thereof comprises a substitution that is
M156, F111S, I87A, I87G, A89D, H109D, R110D, Y153D, M156D, A89D,
H109E, R110E, Y153E, M156E, or a combination thereof relative to
SEQ ID NO: 15.
[0090] In some embodiments, an IL10 variant, derivative, or
fragment thereof does not contain a substitution at position I87,
A89, H109, R110, F111, Y153, or M156 relative to SEQ ID NO: 15. In
some embodiments, an IL10 variant, derivative, or fragment thereof
does not contain a M156, F111S, I87A, I87G, A89D, H109D, R110D,
Y153D, M156D, A89D, H109E, R110E, Y153E, or M156E substitution.
[0091] In some embodiments, an IL10 or IL10 variant, derivative, or
fragment thereof of the disclosure binds to an IL10 receptor
subunit with about a comparable affinity as a wild type IL10
sequence. A comparable affinity can be, for example, less than
about 10, less than about 5, less than about 2, less than about
1.9, less than about 1.8, less than about 1.7, less than about 1.6,
less than about 1.5, less than about 1.4, less than about 1.3, less
than about 1.2, or less than about 1.1 fold increased affinity
compared to a wild type IL10 sequence. A comparable affinity can
be, for example, less than about 10, less than about 5, less than
about 2, less than about 1.9, less than about 1.8, less than about
1.7, less than about 1.6, less than about 1.5, less than about 1.4,
less than about 1.3, less than about 1.2, or less than about 1.1
fold decreased affinity compared to a wild type IL10 sequence.
[0092] For example, an IL10 or IL10 variant, derivative, or
fragment thereof of the disclosure can bind to an interleukin 10
receptor 1 (IL-10R1), interleukin 10 receptor 2 (IL-10R2), or a
combination thereof with about a comparable affinity as a wild type
IL10. In some embodiments, an IL10 or IL10 variant, derivative, or
fragment thereof of the disclosure can bind to IL-10R1 with about a
comparable affinity as a wild type IL10. In some embodiments, an
IL10 or IL10 variant, derivative, or fragment thereof of the
disclosure can bind to IL-10R2 with about a comparable affinity as
a wild type IL10. In some embodiments, an IL10 or IL10 variant,
derivative, or fragment thereof of the disclosure can bind to
IL-10R1 and IL-10R2 with about a comparable affinity as a wild type
IL10.
[0093] In some embodiments, an IL10 or IL10 variant, derivative, or
fragment thereof of the disclosure can bind to an IL10 receptor
subunit with at least a comparable affinity as a wild type IL10.
For example, an IL10 or IL10 variant, derivative, or fragment
thereof of the disclosure can bind to IL-10R1, IL-10R2, or a
combination thereof with at least a comparable affinity as a wild
type IL10. In some embodiments, an IL10 or IL10 variant,
derivative, or fragment thereof of the disclosure can bind to an
IL-10R1 with at least a comparable affinity as a wild type IL10. In
some embodiments, an IL10 or IL10 variant, derivative, or fragment
thereof of the disclosure can bind to an IL-10R2 with at least a
comparable affinity as a wild type IL10. In some embodiments, an
IL10 or IL10 variant, derivative, or fragment thereof of the
disclosure can bind to an IL-10R1 and an IL-10R2 with at least a
comparable affinity as a wild type IL10.
[0094] In some embodiments, an IL10 or IL10 variant, derivative, or
fragment thereof of the disclosure can bind to an IL10 receptor
subunit with at most a comparable affinity as a wild type IL10. For
example, an IL10 or IL10 variant, derivative, or fragment thereof
of the disclosure can bind to IL-10R1, IL-10R2, or a combination
thereof with at most a comparable affinity as a wild type IL10. In
some embodiments, an IL10 or IL10 variant, derivative, or fragment
thereof of the disclosure can bind to an IL-10R1 with at most a
comparable affinity as a wild type IL10. In some embodiments, an
IL10 or IL10 variant, derivative, or fragment thereof of the
disclosure can bind to an IL-10R2 with at most a comparable
affinity as a wild type IL10. In some embodiments, an IL10 or IL10
variant, derivative, or fragment thereof of the disclosure can bind
to an IL-10R1 and an IL-10R2 with at most a comparable affinity as
a wild type IL10.
[0095] In some embodiments, an IL10 or IL10 variant, derivative, or
fragment thereof can bind to an IL-10R1 with at least about 1.5
fold, 2 fold, 5 fold, 10 fold, 15 fold, 20 fold, 30 fold, 40 fold,
50 fold, 100 fold, 200 fold, 500 fold, 1000 fold, or 10,000 fold
increased affinity relative to a wild type IL10 sequence. In some
embodiments, an IL10 or IL10 variant, derivative, or fragment
thereof can bind to an IL-10R1 with at least about 1.5 fold, 2
fold, 5 fold, 10 fold, 15 fold, 20 fold, 30 fold, 40 fold, 50 fold,
100 fold, 200 fold, 500 fold, 1000 fold, or 10,000 fold decreased
affinity relative to a wild type IL10 sequence.
[0096] In some embodiments, an IL10 or IL10 variant, derivative, or
fragment thereof can bind to an IL-10R2 with at least about 1.5
fold, 2 fold, 5 fold, 10 fold, 15 fold, 20 fold, 30 fold, 40 fold,
50 fold, 100 fold, 200 fold, 500 fold, 1000 fold, or 10,000 fold
increased affinity relative to a wild type IL10 sequence. In some
embodiments, an IL10 or IL10 variant, derivative, or fragment
thereof can bind to an IL-10R2 with at least about 1.5 fold, 2
fold, 5 fold, 10 fold, 15 fold, 20 fold, 30 fold, 40 fold, 50 fold,
100 fold, 200 fold, 500 fold, 1000 fold, or 10,000 fold decreased
affinity relative to a wild type IL10 sequence.
[0097] In some embodiments, an IL10 or IL10 variant, derivative, or
fragment thereof of the disclosure can activate a native IL10
receptor. A native IL10 receptor can be, for example, a receptor
comprising an IL-10R1 subunit and an IL-10R2 subunit. In some
embodiments, an IL10 or IL10 variant, derivative, or fragment
thereof of the disclosure can activate a native IL10 receptor when
present in a fusion protein. In some embodiments, an IL10 or IL10
variant, derivative, or fragment thereof of the disclosure can
activate a native IL10 receptor when present as a polypeptide that
is not part of a fusion protein, but does not activate native IL10
receptor when present in a fusion protein.
[0098] In some embodiments, a compound (e.g., fusion protein or
polypeptide construct) of the disclosure does not contain IL10. In
some embodiments, a polypeptide of the disclosure does not contain
SEQ ID NO: 15.
Interleukin 13:
[0099] A compound (e.g., polypeptide construct, fusion protein)
disclosed herein can comprise an IL13 protein, or a variant,
derivative, or fragment thereof operably connected or directly or
indirectly fused to an IL4 protein, or a variant, derivative or
fragment thereof. The IL13 protein can be a mammalian IL13 protein,
such as a human IL13, or mouse IL13. Non-limiting examples of amino
acid sequences representing IL13 are set forth in SEQ ID NOs
16-23.
TABLE-US-00003 TABLE 3 non-limiting examples of human IL13
sequences of the disclosure SEQ ID NO: SEQUENCE 16
PGPVPPSTALRELIEELVNITQNQKAPLCNGSMVWSINLTAGMYCAALESLINVS
GCSAIEKTQRMLSGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDLLLHLKKLFREG QFN 17
GPVPPSTALRELIEELVNITQNQKAPLCNGSMVWSINLTAGMYCAALESLINVSG
CSAIEKTQRMLSGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDLLLHLKKLFREGQ FN 18
SPGPVPPSTALRELIEELVNITQNQKAPLCNGSMVWSINLTAGMYCAALESLINV
SGCSAIEKTQRMLSGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDLLLHLKKLFRE GQFN 19
LTCLGGFASPGPVPPSTALRELIEELVNITQNQKAPLCNGSMVWSINLTAGMYCA
ALESLINVSGCSAIEKTQRMLSGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDLLL
HLKKLFREGQFN 20
PGPVPPSTALRELIEELVNITQNQKAPLCNGSMVWSINLTAGMYCAALESLINVS
GCSAIEKTQRMLSGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDLLLHLKKLFREG RFN 21
GPVPPSTALRELIEELVNITQNQKAPLCNGSMVWSINLTAGMYCAALESLINVSG
CSAIEKTQRMLSGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDLLLHLKKLFREGR FN 22
SPGPVPPSTALRELIEELVNITQNQKAPLCNGSMVWSINLTAGMYCAALESLINV
SGCSAIEKTQRMLSGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDLLLHLKKLFRE GRFN 23
LTCLGGFASPGPVPPSTALRELIEELVNITQNQKAPLCNGSMVWSINLTAGMYCA
ALESLINVSGCSAIEKTQRMLSGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDLLL
HLKKLFREGRFN
[0100] Variants of IL13 include, for example, proteins having at
least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or
more, such as 100%, amino acid sequence identity, to any one of SEQ
ID NOs: 16-23, for example, over the entire length. Amino acid
sequence identity can be determined by pairwise alignment using the
Needleman and Wunsch algorithm and GAP default parameters as
disclosed herein. Variants, derivatives, and fragments thereof also
include proteins having IL13 activity, which have been derived, by
way of one or more amino acid substitutions, deletions or
insertions, from the polypeptide having the amino acid sequence of
any one of SEQ ID NOs: 16-23. Such proteins can comprise from 1, 2,
3, 4, 5, 6, 7, 8, 9, 10 or more up to about 100, 90, 80, 70, 60,
50, 45, 40, 35, 30, 25, 20, 15 amino acid substitutions, deletions
or insertions.
[0101] In some embodiments, an IL13 (e.g., an IL13 variant,
derivative, or fragment thereof) of the disclosure can comprise at
least 1, at least 2, at least 3, at least 4, at least 5, at least
6, at least 7, at least 8, at least 9, at least 10, at least 11, at
least 12, at least 13, at least 14, at least 15, at least 16, at
least 17, at least 18, at least 19, at least 20, at least 25, at
least 30, at least 35, at least 40, at least 45, at least or at
least 50 amino acid substitutions, deletions, or insertions
relative to an IL13 sequence disclosed herein (e.g., a wild type
IL13 sequence).
[0102] In some embodiments, an IL13 (e.g., an IL13 variant,
derivative, or fragment thereof) of the disclosure can comprise at
most 1, at most 2, at most 3, at most 4, at most 5, at most 6, at
most 7, at most 8, at most 9, at most 10, at most 11, at most 12,
at most 13, at most 14, at most 15, at most 16, at most 17, at most
18, at most 19, at most 20, at most 25, at most 30, at most 35, at
most 40, at most 45, or at most 50 amino acid substitutions,
deletions, or insertions relative to an IL13 sequence disclosed
herein (e.g., a wild type IL13 sequence).
[0103] In some embodiments, an IL13 sequence (e.g., an IL13
variant, derivative, or fragment thereof) of the disclosure can
comprise 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-15, 1-20,
1-30, 1-40, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 2-15, 2-20,
2-30, 2-40, 3-3, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 3-15, 3-20,
3-30, 3-40, 5-6, 5-7, 5-8, 5-9, 5-10, 5-15, 5-20, 5-30, 5-40,
10-15, 15-20, or 20-25 amino acid substitutions, deletions, or
insertions relative to an IL13 sequence disclosed herein (e.g., a
wild type IL13 sequence).
[0104] In some embodiments, an IL13 sequence (e.g., an IL13
variant, derivative, or fragment thereof) of the disclosure can
comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, or 20 amino acid substitutions, deletions, or insertions
relative to an IL13 sequence disclosed herein (e.g., a wild type
IL13 sequence). An amino acid substitution can be a conservative or
a non-conservative substitution. The one or more amino acid
substitutions, deletions, or insertions can be at the N-terminus,
the C-terminus, within the amino acid sequence, or a combination
thereof. The amino acid substitutions, deletions, or insertions can
be contiguous, non-contiguous, or a combination thereof.
[0105] An IL13 of the disclosure can comprise a wild type IL13
sequence. Non-limiting examples of wild type IL13 sequences include
any one of SEQ ID NOs: 16-23. SEQ ID NO: 20 can be a canonical wild
type IL13 sequence of the disclosure.
[0106] An IL13 of the disclosure can comprise an IL13 variant,
derivative, or fragment thereof with one or more amino acid
substitutions. For example, an IL13 variant, derivative, or
fragment thereof can comprise an amino acid substitution at
position L10, E12, R11, I14, E15, E16, V18, R65, S68, R86, D87,
T88, K89, D98, L101, L103, K104, K105 L106, F107, R108, R111, F114,
N113, or a combination thereof of SEQ ID NO: 16 or SEQ ID NO: 20.
In some embodiments, an IL13 variant, derivative, or fragment
thereof comprises a substitution that is L10F; L10I; L10V; L10A;
L10D; L10T; L10H; R11S; R11N; R11H; R11L; R11I; I14L; I14F; I14V;
I14M; V18L; V18F; V18I; E12A; R65D; R86K; R86T; R86M; D87E; D87K;
D87R; D87G; D87S; T88S, T881; T88K; T88R; K89R; K89T; K89M; L101F;
L101I; L101Y; L101H; L101N; K104R; K104T; K104M; K105T; K105A;
K105R; K105E; F107L; F107I; F107V; F107M; R108K; R108T; R108M;
E12K, E12I, E12C, E12S, E12R, E12Y, E12D, E15K, E16K, R65D, S68D,
D98K, L101A, L103A, K104D, K105D, L106A, F107Y, R108D, R111D,
F114D, N113D, or a combination thereof relative to SEQ ID NO: 16 or
SEQ ID NO: 20. In some embodiments, an IL13 variant, derivative, or
fragment thereof comprises the substitutions L10H, R86T, D87G,
T88R, and R108K relative to SEQ ID NO: 16 or SEQ ID NO: 20. In some
embodiments, an IL13 variant, derivative, or fragment thereof
comprises the substitutions L10A, V18F, R86K, D87K, K89R, L101I,
K104R, and R108K relative to SEQ ID NO: 16 or SEQ ID NO: 20. In
some embodiments, an IL13 variant, derivative, or fragment thereof
comprises the substitutions R11S, V18I, R86K, D87G, T88S, K89M,
L101Y, K104R, and K105T relative to SEQ ID NO: 16 or SEQ ID NO: 20.
In some embodiments, an IL13 variant, derivative, or fragment
thereof comprises the substitutions L10V, K89R, L101 N, K105E, and
R108T relative to SEQ ID NO: 16 or SEQ ID NO: 20. In some
embodiments, an IL13 variant, derivative, or fragment thereof
comprises the substitutions L10D, R11I, V18I, R86K, D87K, K89R, and
R108K relative to SEQ ID NO: 16 or SEQ ID NO: 20. In some
embodiments, an IL13 variant, derivative, or fragment thereof
comprises the substitutions L10A, R86T, D87G, T88K, K89R, L101N,
K104R, K105A, and R108K relative to SEQ ID NO: 16 or SEQ ID NO: 20.
In some embodiments, an IL13 variant, derivative, or fragment
thereof comprises the substitutions L10V, K89R, L101 N, K105E, and
R108T relative to SEQ ID NO: 16 or SEQ ID NO: 20. In some
embodiments, an IL13 variant, derivative, or fragment thereof
comprises the substitutions R11S, I14M, T88S, L101 N, K105A, and
R108K relative to SEQ ID NO: 16 or SEQ ID NO: 20. In some
embodiments, an IL13 variant, derivative, or fragment thereof
comprises the substitutions L10H, R11L, V18I, R86K, D87E, K89R,
L101N, K105T, and R108K relative to SEQ ID NO: 16 or SEQ ID NO: 20.
In some embodiments, an IL13 variant, derivative, or fragment
thereof comprises the substitutions L10H, R86T, D87G, T88R, and
R108K relative to SEQ ID NO: 16 or SEQ ID NO: 20. In some
embodiments, an IL13 variant, derivative, or fragment thereof
comprises the substitutions L10A, V18F, R86K, D87K, K89R, L101I,
K104R, and R108K relative to SEQ ID NO: 16 or SEQ ID NO: 20. In
some embodiments, an IL13 variant, derivative, or fragment thereof
comprises the substitutions L10T or L10D; R11I; V18I; R86K; D87K or
D87G; T88S; K89R; L101Y; K104R; K105T; and R108K relative to SEQ ID
NO: 16 or SEQ ID NO: 20. In some embodiments, an IL13 variant,
derivative, or fragment thereof comprises the substitutions L10A or
L10V; R86T; D87G; T88K; K89R; L101N; K104R; K105A or K105E; and
R108K or R108T relative to SEQ ID NO: 16 or SEQ ID NO: 20. In some
embodiments, an IL13 variant, derivative, or fragment thereof
comprises the substitutions L10V, V18I, D87S, D88S, L101F, K104R,
and K105T relative to SEQ ID NO: 16 or SEQ ID NO: 20. In some
embodiments, an IL13 variant, derivative, or fragment thereof
comprises the substitutions R11S, V18I, R86K, D87G, T88S, K89M,
L101Y, K104R, and K105T relative to SEQ ID NO: 16 or SEQ ID NO: 20.
In some embodiments, an IL13 variant, derivative, or fragment
thereof comprises the substitutions L10V, V18I, D87S, T88S, L101F,
K104R, and K105T relative to SEQ ID NO: 16 or SEQ ID NO: 20. In
some embodiments, an IL13 variant, derivative, or fragment thereof
comprises the substitutions L10V or L10I; D87S; T88S; K89R; L101H
or L101F; K104R; and K105T relative to SEQ ID NO: 16 or SEQ ID NO:
20. In some embodiments, an IL13 variant, derivative, or fragment
thereof comprises the substitutions L10I; V18I; R86T; D87G; T88S;
K89R; L101Y, L101H; K104R; and K105A relative to SEQ ID NO: 16 or
SEQ ID NO: 20. In some embodiments, an IL13 variant, derivative, or
fragment thereof comprises the substitutions L10V; V18I; D87S;
T88S; L101F; K104R; and K105T relative to SEQ ID NO: 16 or SEQ ID
NO: 20. In some embodiments, an IL13 variant, derivative, or
fragment thereof comprises the substitutions V18I, R86T, D87G,
T88S, L101Y, K104R, and K105A relative to SEQ ID NO: 16 or SEQ ID
NO: 20. In some embodiments, an IL13 variant, derivative, or
fragment thereof comprises the substitutions R11I, V18I, R86K,
D87G, T88S, L101H, K104R, K105A, and F107M relative to SEQ ID NO:
16 or SEQ ID NO: 20. In some embodiments, an IL13 variant,
derivative, or fragment thereof comprises the substitutions E12K
and S68D relative to SEQ ID NO: 16 or SEQ ID NO: 20. In some
embodiments, an IL13 variant, derivative, or fragment thereof
comprises the substitutions E12K and R108D relative to SEQ ID NO:
16 or SEQ ID NO: 20. In some embodiments, an IL13 variant,
derivative, or fragment thereof comprises the substitutions E12K
and R111D relative to SEQ ID NO: 16 or SEQ ID NO: 20. In some
embodiments, an IL13 variant, derivative, or fragment thereof
comprises the substitutions E12Y and R65D relative to SEQ ID NO: 16
or SEQ ID NO: 20. In some embodiments, an IL13 variant, derivative,
or fragment thereof comprises the substitutions E12Y and S68D
relative to SEQ ID NO: 16 or SEQ ID NO: 20. In some embodiments, an
IL13 variant, derivative, or fragment thereof comprises the
substitutions E12K, R65D and S68D relative to SEQ ID NO: 16 or SEQ
ID NO: 20. In some embodiments, an IL13 variant, derivative, or
fragment thereof comprises the substitutions E12Y, R65D and S68D
relative to SEQ ID NO: 16 or SEQ ID NO: 20. In some embodiments, an
IL13 variant, derivative, or fragment thereof comprises the
substitutions E12K, R65D, S68D and R111D relative to SEQ ID NO: 16
or SEQ ID NO: 20.
[0107] In some embodiments, an IL13 variant, derivative, or
fragment thereof does not contain a substitution at position L10,
E12, R11, I14, E15, E16, V18, R65, S68, R86, D87, T88, K89, D98,
L101, L103, K104, K105 L106, F107, R108, R111, F114, or N113
relative to SEQ ID NO: 16 or SEQ ID NO: 20. In some embodiments, an
IL13 variant, derivative, or fragment thereof does not contain a
L10F; L10I; L10V; L10A; L10D; L10T; L10H; R11S; R11N; R11H; R11L;
R11I; I14L; I14F; I14V; I14M; V18L; V18F; V18I; E12A; R65D; R86K;
R86T; R86M; D87E; D87K; D87R; D87G; D87S; T88S, T881; T88K; T88R;
K89R; K89T; K89M; L101F; L101I; L101Y; L101H; L101N; K104R; K104T;
K104M; K105T; K105A; K105R; K105E; F107L; F107I; F107V; F107M;
R108K; R108T; R108M; E12K, E12I, E12C, E12S, E12R, E12Y, E12D,
E15K, E16K, R65D, S68D, D98K, L101A, L103A, K104D, K105D, L106A,
F107Y, R108D, R111D, F114D, or N113D substitution.
[0108] In some embodiments, an IL13 or IL13 variant, derivative, or
fragment thereof of the disclosure binds to an IL13 receptor
subunit with about a comparable affinity as a wild type IL13
sequence. A comparable affinity can be, for example, less than
about 10, less than about 5, less than about 2, less than about
1.9, less than about 1.8, less than about 1.7, less than about 1.6,
less than about 1.5, less than about 1.4, less than about 1.3, less
than about 1.2, or less than about 1.1 fold increased affinity
compared to a wild type IL13 sequence. A comparable affinity can
be, for example, less than about 10, less than about 5, less than
about 2, less than about 1.9, less than about 1.8, less than about
1.7, less than about 1.6, less than about 1.5, less than about 1.4,
less than about 1.3, less than about 1.2, or less than about 1.1
fold decreased affinity compared to a wild type IL13 sequence.
[0109] For example, an IL13 or IL13 variant, derivative, or
fragment thereof of the disclosure can bind to an interleukin 13
receptor alpha 1 (IL-13R.alpha.1), interleukin 13 receptor alpha 2
(IL-13R.alpha.2), interleukin 4 receptor alpha (IL-4R.alpha.), or a
combination thereof with about a comparable affinity as a wild type
IL13. In some embodiments, an IL13 or IL13 variant, derivative, or
fragment thereof of the disclosure can bind to IL-13R.alpha.1 with
about a comparable affinity as a wild type IL13. In some
embodiments, an IL13 or IL13 variant, derivative, or fragment
thereof of the disclosure can bind to IL-13R.alpha.2 with about a
comparable affinity as a wild type IL13. In some embodiments, an
IL13 or IL13 variant, derivative, or fragment thereof of the
disclosure can bind to IL-4R.alpha. with about a comparable
affinity as a wild type IL13 sequence. In some embodiments, an IL13
or IL13 variant, derivative, or fragment thereof of the disclosure
can bind to IL-13R.alpha.1 and IL-13R.alpha.2 with about a
comparable affinity as a wild type IL13. In some embodiments, an
IL13 or IL13 variant, derivative, or fragment thereof of the
disclosure can bind to IL-13R.alpha.1 and IL-4R.alpha. with about a
comparable affinity as a wild type IL13. In some embodiments, an
IL13 or IL13 variant, derivative, or fragment thereof of the
disclosure can bind to IL-13R.alpha.2 and IL-4R.alpha. with about a
comparable affinity as a wild type IL13. In some embodiments, an
IL13 or IL13 variant, derivative, or fragment thereof of the
disclosure can bind to IL-13R.alpha.1, IL-13R.alpha.2, and
IL-4R.alpha. with about a comparable affinity as a wild type
IL13.
[0110] In some embodiments, an IL13 or IL13 variant, derivative, or
fragment thereof of the disclosure can bind to an IL13 receptor
subunit with at least a comparable affinity as a wild type IL13.
For example, an IL13 or IL13 variant, derivative, or fragment
thereof of the disclosure can bind to an interleukin 13 receptor
alpha 1 (IL-13R.alpha.1), interleukin 13 receptor alpha 2
(IL-13R.alpha.2), interleukin 4 receptor alpha (IL-4R.alpha.), or a
combination thereof with at least a comparable affinity as a wild
type IL13. In some embodiments, an IL13 or IL13 variant,
derivative, or fragment thereof of the disclosure can bind to
IL-13R.alpha.1 with at least a comparable affinity as a wild type
IL13. In some embodiments, an IL13 or IL13 variant, derivative, or
fragment thereof of the disclosure can bind to IL-13R.alpha.2 with
at least a comparable affinity as a wild type IL13. In some
embodiments, an IL13 or IL13 variant, derivative, or fragment
thereof of the disclosure can bind to IL-4R.alpha. with at least a
comparable affinity as a wild type IL13 sequence. In some
embodiments, an IL13 or IL13 variant, derivative, or fragment
thereof of the disclosure can bind to IL-13R.alpha.1 and
IL-13R.alpha.2 with at least a comparable affinity as a wild type
IL13. In some embodiments, an IL13 or IL13 variant, derivative, or
fragment thereof of the disclosure can bind to IL-13R.alpha.1 and
IL-4R.alpha. with at least a comparable affinity as a wild type
IL13. In some embodiments, an IL13 or IL13 variant, derivative, or
fragment thereof of the disclosure can bind to IL-13R.alpha.2 and
IL-4R.alpha. with at least a comparable affinity as a wild type
IL13. In some embodiments, an IL13 or IL13 variant, derivative, or
fragment thereof of the disclosure can bind to IL-13R.alpha.1,
IL-13R.alpha.2, and IL-4R.alpha. with at least a comparable
affinity as a wild type IL13.
[0111] In some embodiments, an IL13 or IL13 variant, derivative, or
fragment thereof of the disclosure can bind to an IL13 receptor
subunit with at most a comparable affinity as a wild type IL13. For
example, an IL13 or IL13 variant, derivative, or fragment thereof
of the disclosure can bind to an interleukin 13 receptor alpha 1
(IL-13R.alpha.1), interleukin 13 receptor alpha 2 (IL-13R.alpha.2),
interleukin 4 receptor alpha (IL-4R.alpha.), or a combination
thereof with at most a comparable affinity as a wild type IL13. In
some embodiments, an IL13 or IL13 variant, derivative, or fragment
thereof of the disclosure can bind to IL-13R.alpha.1 with at most a
comparable affinity as a wild type IL13. In some embodiments, an
IL13 or IL13 variant, derivative, or fragment thereof of the
disclosure can bind to IL-13R.alpha.2 with at most a comparable
affinity as a wild type IL13. In some embodiments, an IL13 or IL13
variant, derivative, or fragment thereof of the disclosure can bind
to IL-4R.alpha. with at most a comparable affinity as a wild type
IL13 sequence. In some embodiments, an IL13 or IL13 variant,
derivative, or fragment thereof of the disclosure can bind to
IL-13R.alpha.1 and IL-13R.alpha.2 with at most a comparable
affinity as a wild type IL13. In some embodiments, an IL13 or IL13
variant, derivative, or fragment thereof of the disclosure can bind
to IL-13R.alpha.1 and IL-4R.alpha. with at most a comparable
affinity as a wild type IL13. In some embodiments, an IL13 or IL13
variant, derivative, or fragment thereof of the disclosure can bind
to IL-13R.alpha.2 and IL-4R.alpha. with at most a comparable
affinity as a wild type IL13. In some embodiments, an IL13 or IL13
variant, derivative, or fragment thereof of the disclosure can bind
to IL-13R.alpha.1, IL-13R.alpha.2, and IL-4R.alpha. with at most a
comparable affinity as a wild type IL13.
[0112] In some embodiments, an IL13 or IL13 variant, derivative, or
fragment thereof can bind to an IL-13R.alpha.1 with at least about
1.5 fold, 2 fold, 5 fold, 10 fold, 15 fold, 20 fold, 30 fold, 40
fold, 50 fold, 100 fold, 200 fold, 500 fold, 1000 fold, or 10,000
fold increased affinity relative to a wild type IL13 sequence. In
some embodiments, an IL13 or IL13 variant, derivative, or fragment
thereof can bind to an IL-13R.alpha.1 with at least about 1.5 fold,
2 fold, 5 fold, 10 fold, 15 fold, 20 fold, 30 fold, 40 fold, 50
fold, 100 fold, 200 fold, 500 fold, 1000 fold, or 10,000 fold
decreased affinity relative to a wild type IL13 sequence.
[0113] In some embodiments, an IL13 or IL13 variant, derivative, or
fragment thereof can bind to an IL-13R.alpha.2 with at least about
1.5 fold, 2 fold, 5 fold, 10 fold, 15 fold, 20 fold, 30 fold, 40
fold, 50 fold, 100 fold, 200 fold, 500 fold, 1000 fold, or 10,000
fold increased affinity relative to a wild type IL13 sequence. In
some embodiments, an IL13 or IL13 variant, derivative, or fragment
thereof can bind to an IL-13R.alpha.2 with at least about 1.5 fold,
2 fold, 5 fold, 10 fold, 15 fold, 20 fold, 30 fold, 40 fold, 50
fold, 100 fold, 200 fold, 500 fold, 1000 fold, or 10,000 fold
decreased affinity relative to a wild type IL13 sequence.
[0114] In some embodiments, an IL13 or IL13 variant, derivative, or
fragment thereof can bind to an IL-4R.alpha. with at least about
1.5 fold, 2 fold, 5 fold, 10 fold, 15 fold, 20 fold, 30 fold, 40
fold, 50 fold, 100 fold, 200 fold, 500 fold, 1000 fold, or 10,000
fold increased affinity relative to a wild type IL13 sequence. In
some embodiments, an IL13 or IL13 variant, derivative, or fragment
thereof can bind to an IL-4R.alpha. with at least about 1.5 fold, 2
fold, 5 fold, 10 fold, 15 fold, 20 fold, 30 fold, 40 fold, 50 fold,
100 fold, 200 fold, 500 fold, 1000 fold, or 10,000 fold decreased
affinity relative to a wild type IL13 sequence.
[0115] In some embodiments, an IL13 or IL13 variant, derivative, or
fragment thereof of the disclosure can activate a native IL13
receptor. A native IL13 receptor can be, for example, a receptor
comprising an IL-13R.alpha.1 subunit and an IL-4R.alpha. subunit.
In some embodiments, an IL13 or IL13 variant, derivative, or
fragment thereof of the disclosure can activate a native IL13
receptor when present in a fusion protein. In some embodiments, an
IL13 or IL13 variant, derivative, or fragment thereof of the
disclosure can activate a native IL13 receptor when present as a
polypeptide that is not part of a fusion protein, but does not
activate native IL13 receptor when present in a fusion protein.
Interleukin 27:
[0116] A compound (e.g., polypeptide construct, fusion protein) may
comprise an IL27 protein, or a variant, derivative, or fragment
thereof operably connected or directly or indirectly fused to an
IL4 protein, or a variant, derivative or fragment thereof. The IL27
protein can be a mammalian IL27 protein, such as a human IL27, or
mouse IL27, or a variant, derivative, or fragment thereof. An IL27
or an IL27 variant, derivative, or fragment thereof of the
disclosure can comprise an IL27A subunit, an IL27B (EBI3) subunit,
or a combination thereof. In some embodiments, an IL27 of the
disclosure comprises an IL27A subunit. In some embodiments, an IL27
of the disclosure comprises an IL27B subunit. In some embodiments,
an IL27 of the disclosure comprises an IL27A subunit and an IL27B
subunit. In some embodiments, an IL27 of the disclosure comprises a
variant IL27A subunit as disclosed below (e.g., as provided in SEQ
ID NO: 24).
TABLE-US-00004 TABLE 4 non-limiting examples of IL27 sequences of
the disclosure SEQ ID NO: SEQUENCE 24
FPRPPGRPQLSLQELRREFTVSLHLARKLLSEVRGQAHRFAESHLPGVNLYLLP
LGEQLPDVSLTFQAWRRLSDPERLCFISTTLQPFHALLGGLGTQGRWTNMERM
QLWAMRLDLRDLQRHLRFQVLAAGFNCPEEEEEEEEEEEEERKGLLPGALGSA
LQGPAQVSWPQLLSTYRLLHSLELVLSRAVRELLLLSKAGHSVWPLGFPTLSPQ P 25
FPRPPGRPQLSLQELRREFTVSLHLARKLLSEVRGQAHRFAESHLPGVNLYLLP
LGEQLPDVSLTFQAWRRLSDPERLCFISTTLQPFHALLGGLGTQGRWTNMERM
QLWAMRLDLRDLQRHLRFQVLAAGFNLPEEEEEEEEEEEEERKGLLPGALGSA
LQGPAQVSWPQLLSTYRLLHSLELVLSRAVRELLLLSKAGHSVWPLGFPTLSPQ P 52
RKGPPAALTLPRVQCRASRYPIAVDCSWTLPPAPNSTSPVSFIATYRLGMAARG
HSWPCLQQTPTSTSCTITDVQLFSMAPYVLNVTAVHPWGSSSSFVPFITEHIIKP
DPPEGVRLSPLAERQLQVQWEPPGSWPFPEIFSLKYWIRYKRQGAARFHRVGP
IEATSFILRAVRPRARYYVQVAAQDLTDYGELSDWSLPATATMSLGK
[0117] An example of an amino acid sequence representing IL27A is
set forth in SEQ ID NO: 25. An example of an amino acid sequence
representing IL27B is set forth in SEQ ID NO: 52. Variants of IL27
include, for example, proteins having at least 70%, 75%, 80%, 85%,
90%, 92%, 95%, 96%, 97%, 98%, 99% or more, such as 100%, amino acid
sequence identity to SEQ ID NO: 25 or SEQ ID NO: 52, for example,
over the entire length. Amino acid sequence identity can be
determined by pairwise alignment using the Needleman and Wunsch
algorithm and GAP default parameters as disclosed herein. Variants,
derivatives, and fragments thereof also include proteins having
IL27 activity, which have been derived, by way of one or more amino
acid substitutions, deletions or insertions, from the polypeptide
having the amino acid sequence of SEQ ID NO: 25 or SEQ ID NO: 52.
Such proteins can comprise from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or
more up to about 100, 90, 80, 70, 60, 50, 45, 40, 35, 30, 25, 20,
15 amino acid substitutions, deletions or insertions.
[0118] In some embodiments, an IL27 of the disclosure (e.g., an
IL27 variant, derivative, or fragment thereof) can comprise at
least 1, at least 2, at least 3, at least 4, at least 5, at least
6, at least 7, at least 8, at least 9, at least 10, at least 11, at
least 12, at least 13, at least 14, at least 15, at least 16, at
least 17, at least 18, at least 19, at least 20, at least 25, at
least 30, at least 35, at least 40, at least 45, at least or at
least 50 amino acid substitutions, deletions, or insertions
relative to an IL27 sequence disclosed herein (e.g., a wild type
IL27 sequence).
[0119] In some embodiments, an IL27 of the disclosure (e.g., an
IL27 variant, derivative, or fragment thereof) can comprise at most
1, at most 2, at most 3, at most 4, at most 5, at most 6, at most
7, at most 8, at most 9, at most 10, at most 11, at most 12, at
most 13, at most 14, at most 15, at most 16, at most 17, at most
18, at most 19, at most 20, at most 25, at most 30, at most 35, at
most 40, at most 45, or at most 50 amino acid substitutions,
deletions, or insertions relative to an IL27 sequence disclosed
herein (e.g., a wild type IL27 sequence).
[0120] In some embodiments, an IL27 sequence of the disclosure
(e.g., an IL27 variant, derivative, or fragment thereof) can
comprise 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-15, 1-20,
1-30, 1-40, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 2-15, 2-20,
2-30, 2-40, 3-3, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 3-15, 3-20,
3-30, 3-40, 5-6, 5-7, 5-8, 5-9, 5-10, 5-15, 5-20, 5-30, 5-40,
10-15, 15-20, or 20-25 amino acid substitutions, deletions, or
insertions relative to an IL27 sequence disclosed herein (e.g., a
wild type IL27 sequence).
[0121] In some embodiments, an IL27 sequence of the disclosure
(e.g., an IL27 variant, derivative, or fragment thereof) can
comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, or 20 amino acid substitutions, deletions, or insertions
relative to an IL27 sequence disclosed herein (e.g., a wild type
IL27 sequence). An amino acid substitution can be a conservative or
a non-conservative substitution. The one or more amino acid
substitutions, deletions, or insertions can be at the N-terminus,
the C-terminus, within the amino acid sequence, or a combination
thereof. The amino acid substitutions, deletions, or insertions can
be contiguous, non-contiguous, or a combination thereof.
[0122] An IL27 of the disclosure can comprise a wild type IL27
sequence. A non-limiting example of a wild type IL27A sequences is
SEQ ID NO: 25, and a non-limiting example of a wild type IL27B
sequence is SEQ ID NO: 52. SEQ ID NO: 25 can be a canonical wild
type IL27 sequence. In some embodiments, an IL27 sequence of the
disclosure comprises one substitution relative to a wild type IL27
sequence.
[0123] An example of an IL27 variant, derivative, or fragment
thereof of the disclosure is an IL27 variant sequence that can be
secreted as a functional immune modulatory monomer protein, for
example, an IL27A subunit variant, derivative, or fragment thereof
that can be secreted and function as a functional immune modulatory
monomer protein without needing to associate with an IL27B (EBI3)
subunit. One or more amino acid substitutions, deletions, or
insertions can be introduced to generate such a molecule. SEQ ID
NO: 24 is an example of an IL27 variant, derivative, or fragment
thereof of the disclosure that comprises one amino acid substation
(L134C) relative to SEQ ID NO: 25 (which is L162C in the sequence
that includes the signal peptide), and can be secreted as a
functional immune modulatory monomer protein.
[0124] An IL27 of the disclosure can comprise an IL27 variant,
derivative, or fragment thereof with one or more amino acid
substitutions. For example, an IL27 variant, derivative, or
fragment thereof can comprise an amino acid substitution at
position F132, N132, L134, P135, E136, E137, L152, L153, P154, or a
combination thereof of SEQ ID NO: 25. In some embodiments, an IL27
variant, derivative, or fragment thereof comprises a substitution
that is F132C, N132C, L134C, P135C, E136C, E137C, L152C, L153C,
P154C, F132D, N132D, L134D, P135D, E136D, E137D, L152D, L153D,
P154D, F132E, N132E, L134E, P135E, E136E, E137E, L152E, L153E,
P154E, F132R, N132R, L134R, P135R, E136R, E137R, L152R, L153R,
P154R, F132K, N132K, L134K, P135K, E136K, E137K, L152K, L153K,
P154K, S31A, L91P, or a combination thereof relative to SEQ ID NO:
25.
[0125] In some embodiments, an IL27 variant, derivative, or
fragment thereof does not contain a substitution at position F132,
N132, L134, P135, E136, E137, L152, L153, or P154 relative to SEQ
ID NO: 25. In some embodiments, an IL27 variant, derivative, or
fragment thereof does not contain an F132C, N132C, L134C, P135C,
E136C, E137C, L152C, L153C, P154C, F132D, N132D, L134D, P135D,
E136D, E137D, L152D, L153D, P154D, F132E, N132E, L134E, P135E,
E136E, E137E, L152E, L153E, P154E, F132R, N132R, L134R, P135R,
E136R, E137R, L152R, L153R, P154R, F132K, N132K, L134K, P135K,
E136K, E137K, L152K, L153K, P154K, S31A, or L91P substitution.
[0126] In some embodiments, an IL27 or IL27 variant, derivative, or
fragment thereof of the disclosure binds to an IL27 receptor
subunit with about a comparable affinity as a wild type IL27
sequence. A comparable affinity can be, for example, less than
about 10, less than about 5, less than about 2, less than about
1.9, less than about 1.8, less than about 1.7, less than about 1.6,
less than about 1.5, less than about 1.4, less than about 1.3, less
than about 1.2, or less than about 1.1 fold increased affinity
compared to a wild type IL27 sequence. A comparable affinity can
be, for example, less than about 10, less than about 5, less than
about 2, less than about 1.9, less than about 1.8, less than about
1.7, less than about 1.6, less than about 1.5, less than about 1.4,
less than about 1.3, less than about 1.2, or less than about 1.1
fold decreased affinity compared to a wild type IL27 sequence.
[0127] For example, an IL27 or IL27 variant, derivative, or
fragment thereof of the disclosure can bind to an interleukin 27
receptor alpha (IL-27RA), gp130, or a combination thereof with
about a comparable affinity as a wild type IL27. In some
embodiments, an IL27 or IL27 variant, derivative, or fragment
thereof of the disclosure can bind to IL-27RA with about a
comparable affinity as a wild type IL27. In some embodiments, an
IL27 or IL27 variant, derivative, or fragment thereof of the
disclosure can bind to GP130 with about a comparable affinity as a
wild type IL27. In some embodiments, an IL27 or IL27 variant,
derivative, or fragment thereof of the disclosure can bind to
IL-27RA and GP130 with about a comparable affinity as a wild type
IL27.
[0128] In some embodiments, an IL27 or IL27 variant, derivative, or
fragment thereof of the disclosure can bind to an IL27 receptor
subunit with at least a comparable affinity as a wild type IL27.
For example, an IL27 or IL27 variant, derivative, or fragment
thereof of the disclosure can bind to IL-27RA, GP130, or a
combination thereof with at least a comparable affinity as a wild
type IL27. In some embodiments, an IL27 or IL27 variant,
derivative, or fragment thereof of the disclosure can bind to an
IL-27RA with at least a comparable affinity as a wild type IL27. In
some embodiments, an IL27 or IL27 variant, derivative, or fragment
thereof of the disclosure can bind to an GP130 with at least a
comparable affinity as a wild type IL27. In some embodiments, an
IL27 or IL27 variant, derivative, or fragment thereof of the
disclosure can bind to an IL-27RA and an GP130 with at least a
comparable affinity as a wild type IL27.
[0129] In some embodiments, an IL27 or IL27 variant, derivative, or
fragment thereof of the disclosure can bind to an IL27 receptor
subunit with at most a comparable affinity as a wild type IL27. For
example, an IL27 or IL27 variant, derivative, or fragment thereof
of the disclosure can bind to IL-27RA, GP130, or a combination
thereof with at most a comparable affinity as a wild type IL27. In
some embodiments, an IL27 or IL27 variant, derivative, or fragment
thereof of the disclosure can bind to an IL-27RA with at most a
comparable affinity as a wild type IL27. In some embodiments, an
IL27 or IL27 variant, derivative, or fragment thereof of the
disclosure can bind to an GP130 with at most a comparable affinity
as a wild type IL27. In some embodiments, an IL27 or IL27 variant,
derivative, or fragment thereof of the disclosure can bind to an
IL-27RA and an GP130 with at most a comparable affinity as a wild
type IL27.
[0130] In some embodiments, an IL27 or IL27 variant, derivative, or
fragment thereof can bind to an IL-27RA with at least about 1.5
fold, 2 fold, 5 fold, 10 fold, 15 fold, 20 fold, 30 fold, 40 fold,
50 fold, 100 fold, 200 fold, 500 fold, 1000 fold, or 10,000 fold
increased affinity relative to a wild type IL27 sequence. In some
embodiments, an IL27 or IL27 variant, derivative, or fragment
thereof can bind to an IL-27RA with at least about 1.5 fold, 2
fold, 5 fold, 10 fold, 15 fold, 20 fold, 30 fold, 40 fold, 50 fold,
100 fold, 200 fold, 500 fold, 1000 fold, or 10,000 fold decreased
affinity relative to a wild type IL27 sequence.
[0131] In some embodiments, an IL27 or IL27 variant, derivative, or
fragment thereof can bind to GP130 A with at least about 1.5 fold,
2 fold, 5 fold, 10 fold, 15 fold, 20 fold, 30 fold, 40 fold, 50
fold, 100 fold, 200 fold, 500 fold, 1000 fold, or 10,000 fold
increased affinity relative to a wild type IL27 sequence. In some
embodiments, an IL27 or IL27 variant, derivative, or fragment
thereof can bind to GP130 with at least about 1.5 fold, 2 fold, 5
fold, 10 fold, 15 fold, 20 fold, 30 fold, 40 fold, 50 fold, 100
fold, 200 fold, 500 fold, 1000 fold, or 10,000 fold decreased
affinity relative to a wild type IL27 sequence.
[0132] In some embodiments, an IL27 or IL27 variant, derivative, or
fragment thereof of the disclosure can activate a native IL27
receptor. A native IL27 receptor can be, for example, a receptor
comprising an IL-27RA subunit and an GP130 subunit. In some
embodiments, an IL27 or IL27 variant, derivative, or fragment
thereof of the disclosure can activate a native IL27 receptor when
present in a compound of the disclosure (e.g., fusion protein). In
some embodiments, an IL27 or IL27 variant, derivative, or fragment
thereof of the disclosure can activate a native IL27 receptor when
present as a polypeptide that is not part of a compound of the
disclosure (e.g., fusion protein), but does not activate native
IL27 receptor when present in a compound of the disclosure (e.g.,
fusion protein).
Interleukin 33:
[0133] A compound (e.g., polypeptide construct, fusion protein) may
comprise an IL33 protein, or a variant, derivative, or fragment
thereof operably connected or directly or indirectly fused to an
IL4 protein, or a variant, derivative or fragment thereof. The IL33
protein can be a mammalian IL33 protein, such as a human IL33, or
mouse IL33. Non-limiting examples of amino acid sequences
representing IL33 include SEQ ID NOs: 26-32.
TABLE-US-00005 TABLE 5 non-limiting examples of human IL33
sequences of the disclosure SEQ ID NO: SEQUENCE 26
MKPKMKYSTNKISTAKWKNTASKALCFKLGKSQQKAKEVCPMYFMKLRSGLMIK
KEACYFRRETTKRPSLKTGRKHKRHLVLAACQQQSTVECFAFGISGVQKYTRAL
HDSSITGISPITEYLASLSTYNDQSITFALEDESYEIYVEDLKKDEKKDKVLLSYYE
SQHPSNESGDGVDGKMLMVTLSPTKDFWLHANNKEHSVELHKCEKPLPDQAFF
VLHNMHSNCVSFECKTDPGVFIGVKDNHLALIKVDSSENLCTENILFKLSET 27
AFGISGVQKYTRALHDSSITGISPITEYLASLSTYNDQSITFALEDESYEIYVEDLKK
DEKKDKVLLSYYESQHPSNESGDGVDGKMLMVTLSPTKDFWLHANNKEHSVEL
HKCEKPLPDQAFFVLHNMHSNCVSFECKTDPGVFIGVKDNHLALIKVDSSENLC TENILFKLSET
28 SGVQKYTRALHDSSITGISPITEYLASLSTYNDQSITFALEDESYEIYVEDLKKDEK
KDKVLLSYYESQHPSNESGDGVDGKMLMVTLSPTKDFWLHANNKEHSVELHKC
EKPLPDQAFFVLHNMHSNCVSFECKTDPGVFIGVKDNHLALIKVDSSENLCTENI LFKLSET 29
HDSSITGISPITEYLASLSTYNDQSITFALEDESYEIYVEDLKKDEKKDKVLLSYYE
SQHPSNESGDGVDGKMLMVTLSPTKDFWLHANNKEHSVELHKCEKPLPDQAFF
VLHNMHSNCVSFECKTDPGVFIGVKDNHLALIKVDSSENLCTENILFKLSET 30
MKPKMKYSTNKISTAKWKNTASKALCFKLGKSQQKAKEVCPMYFMKLRSGLMIK
KEACYFRRETTKRPSLKTGRKHKRHLVLAACQQQSTVECFAFGISGVQKYTRAL
HDSSITDKVLLSYYESQHPSNESGDGVDGKMLMVTLSPTKDFWLHANNKEHSV
ELHKCEKPLPDQAFFVLHNMHSNCVSFECKTDPGVFIGVKDNHLALIKVDSSENL
CTENILFKLSET 31
MKPKMKYSTNKISTAKWKNTASKALCFKLGKSQQKAKEVCPMYFMKLRSGLMIK
KEACYFRRETTKRPSLKTGISPITEYLASLSTYNDQSITFALEDESYEIYVEDLKKD
EKKDKVLLSYYESQHPSNESGDGVDGKMLMVTLSPTKDFWLHANNKEHSVELH
KCEKPLPDQAFFVLHNMHSNCVSFECKTDPGVFIGVKDNHLALIKVDSSENLCTE NILFKLSET
32 MKPKMKYSTNKISTAKWKNTASKALCFKLGNKVLLSYYESQHPSNESGDGVDG
KMLMVTLSPTKDFWLHANNKEHSVELHKCEKPLPDQAFFVLHNMHSNCVSFEC
KTDPGVFIGVKDNHLALIKVDSSENLCTENILFKLSET
[0134] Variants of IL33 include, for example, proteins having at
least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or
more, such as 100%, amino acid sequence identity to any one of SEQ
ID NOs: 26-32, for example, over the entire length. Amino acid
sequence identity can be determined by pairwise alignment using the
Needleman and Wunsch algorithm and GAP default parameters as
disclosed herein. Variants, derivatives, and fragments thereof also
include proteins having IL33 activity, which have been derived, by
way of one or more amino acid substitutions, deletions or
insertions, from the polypeptide having the amino acid sequence of
any one of SEQ ID NOs: 26-32. Such proteins can comprise from 1, 2,
3, 4, 5, 6, 7, 8, 9, 10 or more up to about 100, 90, 80, 70, 60,
50, 45, 40, 35, 30, 25, 20, 15 amino acid substitutions, deletions
or insertions.
[0135] In some embodiments, an IL33 of the disclosure (e.g., an
IL33 variant, derivative, or fragment thereof) can comprise at
least 1, at least 2, at least 3, at least 4, at least 5, at least
6, at least 7, at least 8, at least 9, at least 10, at least 11, at
least 12, at least 13, at least 14, at least 15, at least 16, at
least 17, at least 18, at least 19, at least 20, at least 25, at
least 30, at least 35, at least 40, at least 45, at least or at
least 50 amino acid substitutions, deletions, or insertions
relative to an IL33 sequence disclosed herein (e.g., a wild type
IL33 sequence).
[0136] In some embodiments, an IL33 of the disclosure (e.g., an
IL33 variant, derivative, or fragment thereof) can comprise at most
1, at most 2, at most 3, at most 4, at most 5, at most 6, at most
7, at most 8, at most 9, at most 10, at most 11, at most 12, at
most 13, at most 14, at most 15, at most 16, at most 17, at most
18, at most 19, at most 20, at most 25, at most 30, at most 35, at
most 40, at most 45, or at most 50 amino acid substitutions,
deletions, or insertions relative to an IL33 sequence disclosed
herein (e.g., a wild type IL33 sequence). SEQ ID NO: 26 can be a
canonical wild type IL33 sequence.
[0137] In some embodiments, an IL33 sequence of the disclosure
(e.g., an IL33 variant, derivative, or fragment thereof) can
comprise 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-15, 1-20,
1-30, 1-40, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 2-15, 2-20,
2-30, 2-40, 3-3, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 3-15, 3-20,
3-30, 3-40, 5-6, 5-7, 5-8, 5-9, 5-10, 5-15, 5-20, 5-30, 5-40,
10-15, 15-20, or 20-25 amino acid substitutions, deletions, or
insertions relative to an IL33 sequence disclosed herein (e.g., a
wild type IL33 sequence).
[0138] In some embodiments, an IL33 sequence of the disclosure
(e.g., an IL33 variant, derivative, or fragment thereof) can
comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, or 20 amino acid substitutions, deletions, or insertions
relative to an IL33 sequence disclosed herein (e.g., a wild type
IL33 sequence). An amino acid substitution can be a conservative or
a non-conservative substitution. The one or more amino acid
substitutions, deletions, or insertions can be at the N-terminus,
the C-terminus, within the amino acid sequence, or a combination
thereof. The amino acid substitutions, deletions, or insertions can
be contiguous, non-contiguous, or a combination thereof.
[0139] An IL33 of the disclosure can comprise a wild type IL33
sequence. Non-limiting examples of wild type IL33 sequences include
SEQ ID NOs: 26-32. SEQ ID NO: 26 can be a canonical wild type IL33
sequence.
[0140] An IL33 of the disclosure can comprise an IL33 variant,
derivative, or fragment thereof with one or more amino acid
substitutions. For example, an IL33 variant, derivative, or
fragment thereof can comprise an amino acid substitution at
position 1263 of SEQ ID NO: 6. In some embodiments, an IL33
variant, derivative, or fragment thereof comprises a substitution
that is 1263M relative to SEQ ID NO: 26.
[0141] In some embodiments, an IL33 variant, derivative, or
fragment thereof does not contain a substitution at position 1263
relative to SEQ ID NO: 26. In some embodiments, an IL33 variant,
derivative, or fragment thereof does not contain an 1263M
substitution.
[0142] In some embodiments, an IL33 or IL33 variant, derivative, or
fragment thereof of the disclosure binds to an IL33 receptor
subunit with about a comparable affinity as a wild type IL33
sequence. A comparable affinity can be, for example, less than
about 10, less than about 5, less than about 2, less than about
1.9, less than about 1.8, less than about 1.7, less than about 1.6,
less than about 1.5, less than about 1.4, less than about 1.3, less
than about 1.2, or less than about 1.1 fold increased affinity
compared to a wild type IL33 sequence. A comparable affinity can
be, for example, less than about 10, less than about 5, less than
about 2, less than about 1.9, less than about 1.8, less than about
1.7, less than about 1.6, less than about 1.5, less than about 1.4,
less than about 1.3, less than about 1.2, or less than about 1.1
fold decreased affinity compared to a wild type IL33 sequence.
[0143] For example, an IL33 or IL33 variant, derivative, or
fragment thereof of the disclosure can bind to ST2 (IL1RL1),
IL1RAP, or a combination thereof with about a comparable affinity
as a wild type IL33. In some embodiments, an IL33 or IL33 variant,
derivative, or fragment thereof of the disclosure can bind to ST2
with about a comparable affinity as a wild type IL33. In some
embodiments, an IL33 or IL33 variant, derivative, or fragment
thereof of the disclosure can bind to IL1RAP with about a
comparable affinity as a wild type IL33. In some embodiments, an
IL33 or IL33 variant, derivative, or fragment thereof of the
disclosure can bind to ST2 and IL1RAP with about a comparable
affinity as a wild type IL33.
[0144] In some embodiments, an IL33 or IL33 variant, derivative, or
fragment thereof of the disclosure can bind to an IL33 receptor
subunit with at least a comparable affinity as a wild type IL33.
For example, an IL33 or IL33 variant, derivative, or fragment
thereof of the disclosure can bind to ST2, IL1RAP, or a combination
thereof with at least a comparable affinity as a wild type IL33. In
some embodiments, an IL33 or IL33 variant, derivative, or fragment
thereof of the disclosure can bind to an ST2 with at least a
comparable affinity as a wild type IL33. In some embodiments, an
IL33 or IL33 variant, derivative, or fragment thereof of the
disclosure can bind to an IL1RAP with at least a comparable
affinity as a wild type IL33. In some embodiments, an IL33 or IL33
variant, derivative, or fragment thereof of the disclosure can bind
to an ST2 and an IL1RAP with at least a comparable affinity as a
wild type IL33.
[0145] In some embodiments, an IL33 or IL33 variant, derivative, or
fragment thereof of the disclosure can bind to an IL33 receptor
subunit with at most a comparable affinity as a wild type IL33. For
example, an IL33 or IL33 variant, derivative, or fragment thereof
of the disclosure can bind to ST2, IL1RAP, or a combination thereof
with at most a comparable affinity as a wild type IL33. In some
embodiments, an IL33 or IL33 variant, derivative, or fragment
thereof of the disclosure can bind to an ST2 with at most a
comparable affinity as a wild type IL33. In some embodiments, an
IL33 or IL33 variant, derivative, or fragment thereof of the
disclosure can bind to an IL1RAP with at most a comparable affinity
as a wild type IL33. In some embodiments, an IL33 or IL33 variant,
derivative, or fragment thereof of the disclosure can bind to an
ST2 and an IL1RAP with at most a comparable affinity as a wild type
IL33.
[0146] In some embodiments, an IL33 or IL33 variant, derivative, or
fragment thereof can bind to an ST2 with at least about 1.5 fold, 2
fold, 5 fold, 10 fold, 15 fold, 20 fold, 30 fold, 40 fold, 50 fold,
100 fold, 200 fold, 500 fold, 1000 fold, or 10,000 fold increased
affinity relative to a wild type IL33 sequence. In some
embodiments, an IL33 or IL33 variant, derivative, or fragment
thereof can bind to an ST2 with at least about 1.5 fold, 2 fold, 5
fold, 10 fold, 15 fold, 20 fold, 30 fold, 40 fold, 50 fold, 100
fold, 200 fold, 500 fold, 1000 fold, or 10,000 fold decreased
affinity relative to a wild type IL33 sequence.
[0147] In some embodiments, an IL33 or IL33 variant, derivative, or
fragment thereof can bind to an IL1RAP with at least about 1.5
fold, 2 fold, 5 fold, 10 fold, 15 fold, 20 fold, 30 fold, 40 fold,
50 fold, 100 fold, 200 fold, 500 fold, 1000 fold, or 10,000 fold
increased affinity relative to a wild type IL33 sequence. In some
embodiments, an IL33 or IL33 variant, derivative, or fragment
thereof can bind to an IL1RAP with at least about 1.5 fold, 2 fold,
5 fold, 10 fold, 15 fold, 20 fold, 30 fold, 40 fold, 50 fold, 100
fold, 200 fold, 500 fold, 1000 fold, or 10,000 fold decreased
affinity relative to a wild type IL33 sequence.
[0148] In some embodiments, an IL33 or IL33 variant, derivative, or
fragment thereof of the disclosure can activate a native IL33
receptor. A native IL33 receptor can be, for example, a receptor
comprising an ST2 subunit and an IL1RAP subunit. In some
embodiments, an IL33 or IL33 variant, derivative, or fragment
thereof of the disclosure can activate a native IL33 receptor when
present in a fusion protein. In some embodiments, an IL33 or IL33
variant, derivative, or fragment thereof of the disclosure can
activate a native IL33 receptor when present as a polypeptide that
is not part of a fusion protein, but does not activate native IL33
receptor when present in a fusion protein.
Transforming Growth Factors (TGF):
[0149] A compound (e.g., polypeptide construct, fusion protein) may
comprise a TGF protein such as a TGF beta protein, or a variant,
derivative, or fragment thereof operatively connected or directly
or indirectly fused to an IL4 protein or variant, derivative or
fragment thereof.
[0150] A compound (e.g., polypeptide construct, fusion protein) may
comprise a TGF.beta.1 protein, or a variant, derivative, or
fragment thereof operatively connected or directly or indirectly
fused to an IL4 protein or variant, derivative or fragment thereof.
The TGF.beta.1 protein can be a mammalian TGF.beta.1 protein, such
as a human TGF.beta.1, or mouse TGF.beta.1. Non-limiting examples
of amino acid sequences representing TGF.beta.1 include SEQ ID NOs:
33 and 34.
TABLE-US-00006 TABLE 6 non-limiting examples of human TGF.beta.1
sequences of the disclosure SEQ ID NO: SEQUENCE 33
MPPSGLRLLPLLLPLLWLLVLTPGRPAAGLSTCKTIDMELVKRKRIEAIRGQILSKL
RLASPPSQGEVPPGPLPEAVLALYNSTRDRVAGESAEPEPEPEADYYAKEVTRV
LMVETHNEIYDKFKQSTHSIYMFFNTSELREAVPEPVLLSRAELRLLRLKLKVEQH
VELYQKYSNNSWRYLSNRLLAPSDSPEWLSFDVTGVVRQWLSRGGEIEGFRLS
AHCSCDSRDNTLQVDINGFTTGRRGDLATIHGMNRPFLLLMATPLERAQHLQSS
RHRRALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCP
YIWSLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYYVGRKPKVEQLSN MIVRSCKCS 34
ALDTNYCFSSTEKNCCVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSL
DTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIVYYVGRKPKVEQLSNMIVRS CKCS
[0151] Variants of TGF.beta.1 include, for example, proteins having
at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or
more, such as 100%, amino acid sequence identity to SEQ ID NO: 33
or SEQ ID NO: 34, for example, over the entire length. Amino acid
sequence identity can be determined by pairwise alignment using the
Needleman and Wunsch algorithm and GAP default parameters as
disclosed herein. Variants, derivatives, or fragments thereof also
include proteins having TGF.beta.1 activity, which have been
derived, by way of one or more amino acid substitutions, deletions
or insertions, from the polypeptide having the amino acid sequence
of SEQ ID NO: 33 or SEQ ID NO: 34. Such proteins can comprise from
1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more up to about 100, 90, 80, 70,
60, 50, 45, 40, 35, 30, 25, 20, 15 amino acid substitutions,
deletions or insertions.
[0152] In some embodiments, a TGF.beta.1 of the disclosure (e.g., a
TGF.beta.1 variant, derivative, or fragment thereof) can comprise
at least 1, at least 2, at least 3, at least 4, at least 5, at
least 6, at least 7, at least 8, at least 9, at least 10, at least
11, at least 12, at least 13, at least 14, at least 15, at least
16, at least 17, at least 18, at least 19, at least 20, at least
25, at least 30, at least 35, at least 40, at least 45, at least or
at least 50 amino acid substitutions, deletions, or insertions
relative to a TGF.beta.1 sequence disclosed herein (e.g., a wild
type TGF.beta.1 sequence).
[0153] In some embodiments, a TGF.beta.1 of the disclosure (e.g., a
TGF.beta.1 variant, derivative, or fragment thereof) can comprise
at most 1, at most 2, at most 3, at most 4, at most 5, at most 6,
at most 7, at most 8, at most 9, at most 10, at most 11, at most
12, at most 13, at most 14, at most 15, at most 16, at most 17, at
most 18, at most 19, at most 20, at most 25, at most 30, at most
35, at most 40, at most 45, or at most 50 amino acid substitutions,
deletions, or insertions relative to a TGF.beta.1 sequence
disclosed herein (e.g., a wild type TGF.beta.1 sequence).
[0154] In some embodiments, a TGF.beta.1 sequence of the disclosure
(e.g., a TGF.beta.1 variant, derivative, or fragment thereof) can
comprise 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-15, 1-20,
1-30, 1-40, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 2-15, 2-20,
2-30, 2-40, 3-3, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 3-15, 3-20,
3-30, 3-40, 5-6, 5-7, 5-8, 5-9, 5-10, 5-15, 5-20, 5-30, 5-40,
10-15, 15-20, or 20-25 amino acid substitutions, deletions, or
insertions relative to a TGF.beta.1 sequence disclosed herein
(e.g., a wild type TGF.beta.1 sequence).
[0155] In some embodiments, a TGF.beta.1 sequence of the disclosure
(e.g., a TGF.beta.1 variant, derivative, or fragment thereof) can
comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, or 20 amino acid substitutions, deletions, or insertions
relative to a TGF.beta.1 sequence disclosed herein (e.g., a wild
type TGF.beta.1 sequence). An amino acid substitution can be a
conservative or a non-conservative substitution. The one or more
amino acid substitutions, deletions, or insertions can be at the
N-terminus, the C-terminus, within the amino acid sequence, or a
combination thereof. The amino acid substitutions, deletions, or
insertions can be contiguous, non-contiguous, or a combination
thereof.
[0156] A TGF.beta.1 of the disclosure can comprise a wild type
TGF.beta.1 sequence. Non-limiting examples of wild type TGF.beta.1
sequences include SEQ ID NO: 33 and SEQ ID NO: 34. A canonical
TGF.beta.1 sequence can be SEQ ID NO: 34.
[0157] In some embodiments, a TGF.beta.1 or TGF.beta.1 variant,
derivative, or fragment thereof of the disclosure binds to a
TGF.beta.1 receptor subunit with about a comparable affinity as a
wild type TGF.beta.1 sequence. A comparable affinity can be, for
example, less than about 10, less than about 5, less than about 2,
less than about 1.9, less than about 1.8, less than about 1.7, less
than about 1.6, less than about 1.5, less than about 1.4, less than
about 1.3, less than about 1.2, or less than about 1.1 fold
increased affinity compared to a wild type TGF.beta.1 sequence. A
comparable affinity can be, for example, less than about 10, less
than about 5, less than about 2, less than about 1.9, less than
about 1.8, less than about 1.7, less than about 1.6, less than
about 1.5, less than about 1.4, less than about 1.3, less than
about 1.2, or less than about 1.1 fold decreased affinity compared
to a wild type TGF.beta.1 sequence.
[0158] For example, a TGF.beta.1 or TGF.beta.1 variant, derivative,
or fragment thereof of the disclosure can bind to a transforming
growth factor beta receptor 1 (TGF.beta.R1), a transforming growth
factor beta receptor 2 (TGF.beta.R2), an (ALK-1), an (ALK-2), or a
combination thereof with about a comparable affinity as a wild type
TGF.beta.1. In some embodiments, a TGF.beta.1 or TGF.beta.1
variant, derivative, or fragment thereof of the disclosure can bind
to TGF.beta.R1 with about a comparable affinity as a wild type
TGFB1. In some embodiments, a TGF.beta.1 or TGF.beta.1 variant,
derivative, or fragment thereof of the disclosure can bind to
TGF.beta.R2 with about a comparable affinity as a wild type
TGF.beta.1. In some embodiments, a TGF.beta.1 or TGF.beta.1
variant, derivative, or fragment thereof of the disclosure can bind
to ALK-1 with about a comparable affinity as a wild type TGF.beta.1
sequence. In some embodiments, a TGF.beta.1 or TGF.beta.1 variant,
derivative, or fragment thereof of the disclosure can bind to ALK-2
with about a comparable affinity as a wild type TGF.beta.1
sequence. In some embodiments, a TGF.beta.1 or TGF.beta.1 variant,
derivative, or fragment thereof of the disclosure can bind to a
TGF.beta.R1, TGF.beta.R2, ALK-1, and ALK-2 with about a comparable
affinity as a wild type TGF.beta.1 sequence.
[0159] In some embodiments, a TGF.beta.1 or TGF.beta.1 variant,
derivative, or fragment thereof of the disclosure can bind to a
transforming growth factor beta receptor 1 (TGF.beta.R1), a
transforming growth factor beta receptor 2 (TGF.beta.R2), an
(ALK-1), an (ALK-2), or a combination thereof with at least a
comparable affinity as a wild type TGF.beta.1. In some embodiments,
a TGF.beta.1 or TGF.beta.1 variant, derivative, or fragment thereof
of the disclosure can bind to TGF.beta.R1 with at least a
comparable affinity as a wild type TGFB1. In some embodiments, a
TGF.beta.1 or TGF.beta.1 variant, derivative, or fragment thereof
of the disclosure can bind to TGF.beta.R2 with at least a
comparable affinity as a wild type TGF.beta.1. In some embodiments,
a TGF.beta.1 or TGF.beta.1 variant, derivative, or fragment thereof
of the disclosure can bind to ALK-1 with at least a comparable
affinity as a wild type TGF.beta.1 sequence. In some embodiments, a
TGF.beta.1 or TGF.beta.1 variant, derivative, or fragment thereof
of the disclosure can bind to ALK-2 with at least a comparable
affinity as a wild type TGF.beta.1 sequence. In some embodiments, a
TGF.beta.1 or TGF.beta.1 variant, derivative, or fragment thereof
of the disclosure can bind to a TGF.beta.R1, TGF.beta.R2, ALK-1,
and ALK-2 with at least a comparable affinity as a wild type
TGF.beta.1 sequence.
[0160] In some embodiments, a TGF.beta.1 or TGF.beta.1 variant,
derivative, or fragment thereof of the disclosure can bind to a
transforming growth factor beta receptor 1 (TGF.beta.R1), a
transforming growth factor beta receptor 2 (TGF.beta.R2), an
(ALK-1), an (ALK-2), or a combination thereof with at most a
comparable affinity as a wild type TGF.beta.1. In some embodiments,
a TGF.beta.1 or TGF.beta.1 variant, derivative, or fragment thereof
of the disclosure can bind to TGF.beta.R1 with at most a comparable
affinity as a wild type TGF.beta.1. In some embodiments, a
TGF.beta.1 or TGF.beta.1 variant, derivative, or fragment thereof
of the disclosure can bind to TGF.beta.R2 with at most a comparable
affinity as a wild type TGF.beta.1. In some embodiments, a
TGF.beta.1 or TGF.beta.1 variant, derivative, or fragment thereof
of the disclosure can bind to ALK-1 with at most a comparable
affinity as a wild type TGF.beta.1 sequence. In some embodiments, a
TGF.beta.1 or TGF.beta.1 variant, derivative, or fragment thereof
of the disclosure can bind to ALK-2 with at most a comparable
affinity as a wild type TGF.beta.1 sequence. In some embodiments, a
TGF.beta.1 or TGF.beta.1 variant, derivative, or fragment thereof
of the disclosure can bind to a TGF.beta.R1, TGF.beta.R2, ALK-1,
and ALK-2 with at most a comparable affinity as a wild type
TGF.beta.1 sequence.
[0161] In some embodiments, an TGF.beta.1 or TGF.beta.1 variant,
derivative, or fragment thereof can bind to a TGF.beta.R1,
TGF.beta.R2, ALK-1, or ALK-2 with at least about 1.5 fold, 2 fold,
5 fold, 10 fold, 15 fold, 20 fold, 30 fold, 40 fold, 50 fold, 100
fold, 200 fold, 500 fold, 1000 fold, or 10,000 fold increased
affinity relative to a wild type TGF.beta.1 sequence. In some
embodiments, an TGF.beta.1 or TGF.beta.1 variant, derivative, or
fragment thereof can bind to a TGF.beta.R1, TGF.beta.R2, ALK-1, or
ALK-2 with at least about 1.5 fold, 2 fold, 5 fold, 10 fold, 15
fold, 20 fold, 30 fold, 40 fold, 50 fold, 100 fold, 200 fold, 500
fold, 1000 fold, or 10,000 fold decreased affinity relative to a
wild type TGF.beta.1 sequence.
[0162] In some embodiments, a TGF.beta.1 or TGF.beta.1 variant,
derivative, or fragment thereof of the disclosure can activate a
native TGF.beta.1 receptor. A native TGF.beta.1 receptor can be,
for example, a receptor comprising a TGF.beta.R1 subunit and a
TGF.beta.R2 subunit. In some embodiments, an TGF.beta.1 or
TGF.beta.1 variant, derivative, or fragment thereof of the
disclosure can activate a native TGF.beta.1 receptor when present
in a fusion protein. In some embodiments, an TGF.beta.1 or
TGF.beta.1 variant, derivative, or fragment thereof of the
disclosure can activate a native TGF.beta.1 receptor when present
as a polypeptide that is not part of a fusion protein, but does not
activate native TGF.beta.1 receptor when present in a fusion
protein.
[0163] A compound (e.g., polypeptide construct, fusion protein) may
comprise a TGF.beta.2 protein, or a variant, derivative, or
fragment thereof operatively connected or directly or indirectly
fused to an IL4 protein or variant, derivative or fragment thereof.
The TGF.beta.2 protein can be a mammalian TGF.beta.2 protein, such
as a human TGF.beta.2, or mouse TGF.beta.2. Non-limiting examples
of amino acid sequences representing TGF.beta.2 include SEQ ID NOs:
35-37.
TABLE-US-00007 TABLE 7 non-limiting examples of human TGF.beta.2
sequences of the disclosure SEQ ID NO: SEQUENCE 35
MHYCVLSAFLILHLVTVALSLSTCSTLDMDQFMRKRIEAIRGQILSKLKLTSPPED
YPEPEEVPPEVISIYNSTRDLLQEKASRRAAACERERSDEEYYAKEVYKIDMPPF
FPSETVCPVVTTPSGSVGSLCSRQSQVLCGYLDAIPPTFYRPYFRIVRFDVSAME
KNASNLVKAEFRVFRLQNPKARVPEQRIELYQILKSKDLTSPTQRYIDSKVVKTR
AEGEWLSFDVTDAVHEWLHHKDRNLGFKISLHCPCCTFVPSNNYIIPNKSEELEA
RFAGIDGTSTYTSGDQKTIKSTRKKNSGKTPHLLLMLLPSYRLESQQTNRRKKRA
LDAAYCFRNVQDNCCLRPLYIDFKRDLGWKWIHEPKGYNANFCAGACPYLWSS
DTQHSRVLSLYNTINPEASASPCCVSQDLEPLTILYYIGKTPKIEQLSNMIVKSCK CS 36
ALDAAYCFRNVQDNCCLRPLYIDFKRDLGWKWIHEPKGYNANFCAGACPYLWS
SDTQHSRVLSLYNTINPEASASPCCVSQDLEPLTILYYIGKTPKIEQLSNMIVKSCK CS 37
MHYCVLSAFLILHLVTVALSLSTCSTLDMDQFMRKRIEAIRGQILSKLKLTSPPED
YPEPEEVPPEVISIYNSTRDLLQEKASRRAAACERERSDEEYYAKEVYKIDMPPF
FPSENAIPPTFYRPYFRIVRFDVSAMEKNASNLVKAEFRVFRLQNPKARVPEQRI
ELYQILKSKDLTSPTQRYIDSKVVKTRAEGEWLSFDVTDAVHEWLHHKDRNLGF
KISLHCPCCTFVPSNNYIIPNKSEELEARFAGIDGTSTYTSGDQKTIKSTRKKNSG
KTPHLLLMLLPSYRLESQQTNRRKKRALDAAYCFRNVQDNCCLRPLYIDFKRDL
GWKWIHEPKGYNANFCAGACPYLWSSDTQHSRVLSLYNTINPEASASPCCVSQ
DLEPLTILYYIGKTPKIEQLSNMIVKSCKCS
[0164] Variants of TGF.beta.2 include, for example, proteins having
at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or
more, such as 100%, amino acid sequence identity to any one of SEQ
ID NOs: 35-37, for example, over the entire length. Amino acid
sequence identity can be determined by pairwise alignment using the
Needleman and Wunsch algorithm and GAP default parameters as
disclosed herein. Variants, derivatives, and fragments thereof also
include proteins having TGF.beta.2 activity, which have been
derived, by way of one or more amino acid substitutions, deletions
or insertions, from the polypeptide having the amino acid sequence
of any one of SEQ ID NOs: 35-37. Such proteins can comprise from 1,
2, 3, 4, 5, 6, 7, 8, 9, 10 or more up to about 100, 90, 80, 70, 60,
50, 45, 40, 35, 30, 25, 20, 15 amino acid substitutions, deletions
or insertions.
[0165] In some embodiments, a TGF.beta.2 of the disclosure (e.g., a
TGF.beta.2 variant, derivative, or fragment thereof) can comprise
at least 1, at least 2, at least 3, at least 4, at least 5, at
least 6, at least 7, at least 8, at least 9, at least 10, at least
11, at least 12, at least 13, at least 14, at least 15, at least
16, at least 17, at least 18, at least 19, at least 20, at least
25, at least 30, at least 35, at least 40, at least 45, at least or
at least 50 amino acid substitutions, deletions, or insertions
relative to a TGF.beta.2 sequence disclosed herein (e.g., a wild
type TGF.beta.2 sequence).
[0166] In some embodiments, a TGF.beta.2 of the disclosure (e.g., a
TGF.beta.2 variant, derivative, or fragment thereof) can comprise
at most 1, at most 2, at most 3, at most 4, at most 5, at most 6,
at most 7, at most 8, at most 9, at most 10, at most 11, at most
12, at most 13, at most 14, at most 15, at most 16, at most 17, at
most 18, at most 19, at most 20, at most 25, at most 30, at most
35, at most 40, at most 45, or at most 50 amino acid substitutions,
deletions, or insertions relative to a TGF.beta.2 sequence
disclosed herein (e.g., a wild type TGF.beta.2 sequence).
[0167] In some embodiments, a TGF.beta.2 sequence of the disclosure
(e.g., a TGF.beta.2 variant, derivative, or fragment thereof) can
comprise 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-15, 1-20,
1-30, 1-40, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 2-15, 2-20,
2-30, 2-40, 3-3, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 3-15, 3-20,
3-30, 3-40, 5-6, 5-7, 5-8, 5-9, 5-10, 5-15, 5-20, 5-30, 5-40,
10-15, 15-20, or 20-25 amino acid substitutions, deletions, or
insertions relative to a TGF.beta.2 sequence disclosed herein
(e.g., a wild type TGF.beta.2 sequence).
[0168] In some embodiments, a TGF.beta.2 sequence of the disclosure
(e.g., a TGF.beta.2 variant, derivative, or fragment thereof) can
comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, or 20 amino acid substitutions, deletions, or insertions
relative to a TGF.beta.2 sequence disclosed herein (e.g., a wild
type TGF.beta.2 sequence). An amino acid substitution can be a
conservative or a non-conservative substitution. The one or more
amino acid substitutions, deletions, or insertions can be at the
N-terminus, the C-terminus, within the amino acid sequence, or a
combination thereof. The amino acid substitutions, deletions, or
insertions can be contiguous, non-contiguous, or a combination
thereof.
[0169] A TGF.beta.2 of the disclosure can comprise a wild type
TGF.beta.2 sequence. Non-limiting examples of wild type TGF.beta.2
sequences include SEQ ID NOs: 35-37. SEQ ID NO: 36 can be a
canonical wild type TGF.beta.2 sequence of the disclosure.
[0170] A TGF.beta.2 of the disclosure can comprise an TGF.beta.2
variant, derivative, or fragment thereof with one or more amino
acid substitutions. For example, a TGF.beta.2 variant, derivative,
or fragment thereof can comprise an amino acid substitution at
position R18, P36, or a combination thereof of SEQ ID NO: 36. In
some embodiments, a TGF.beta.2 variant, derivative, or fragment
thereof comprises a substitution that is R18C, P36H, or a
combination thereof relative to SEQ ID NO: 22. In some embodiments,
a TGF.beta.2, fragment, or derivative thereof comprises the
substitutions R18C, and P36H relative to SEQ ID NO: 36.
[0171] In some embodiments, a TGF.beta.2 variant, derivative, or
fragment thereof does not contain a substitution at position R18,
or P36 relative to SEQ ID NO: 36. In some embodiments, a TGF.beta.2
variant, derivative, or fragment thereof does not contain a R18C or
P36H substitution.
[0172] In some embodiments, a TGF.beta.2 or TGF.beta.2 variant,
derivative, or fragment thereof of the disclosure binds to a
TGF.beta.2 receptor subunit with about a comparable affinity as a
wild type TGF.beta.2 sequence. A comparable affinity can be, for
example, less than about 10, less than about 5, less than about 2,
less than about 1.9, less than about 1.8, less than about 1.7, less
than about 1.6, less than about 1.5, less than about 1.4, less than
about 1.3, less than about 1.2, or less than about 1.1 fold
increased affinity compared to a wild type TGF.beta.2 sequence. A
comparable affinity can be, for example, less than about 10, less
than about 5, less than about 2, less than about 1.9, less than
about 1.8, less than about 1.7, less than about 1.6, less than
about 1.5, less than about 1.4, less than about 1.3, less than
about 1.2, or less than about 1.1 fold decreased affinity compared
to a wild type TGF.beta.2 sequence.
[0173] For example, a TGF.beta.2 or TGF.beta.2 variant, derivative,
or fragment thereof of the disclosure can bind to a transforming
growth factor beta receptor 1 (TGF.beta.R1), a transforming growth
factor beta receptor 2 (TGF.beta.R2), an (ALK-1), an (ALK-2), or a
combination thereof with about a comparable affinity as a wild type
TGF.beta.2. In some embodiments, a TGF.beta.2 or TGF.beta.2
variant, derivative, or fragment thereof of the disclosure can bind
to TGF.beta.R1 with about a comparable affinity as a wild type
TGF.beta.2. In some embodiments, a TGF.beta.2 or TGF.beta.2
variant, derivative, or fragment thereof of the disclosure can bind
to TGF.beta.R2 with about a comparable affinity as a wild type
TGF.beta.2. In some embodiments, a TGF.beta.2 or TGF.beta.2
variant, derivative, or fragment thereof of the disclosure can bind
to ALK-1 with about a comparable affinity as a wild type TGF.beta.2
sequence. In some embodiments, a TGF.beta.2 or TGF.beta.2 variant,
derivative, or fragment thereof of the disclosure can bind to ALK-2
with about a comparable affinity as a wild type TGF.beta.2
sequence. In some embodiments, a TGF.beta.2 or TGF.beta.2 variant,
derivative, or fragment thereof of the disclosure can bind to a
TGF.beta.R1, TGF.beta.R2, ALK-1, and ALK-2 with about a comparable
affinity as a wild type TGF.beta.2 sequence.
[0174] In some embodiments, a TGF.beta.2 or TGF.beta.2 variant,
derivative, or fragment thereof of the disclosure can bind to a
transforming growth factor beta receptor 1 (TGF.beta.R1), a
transforming growth factor beta receptor 2 (TGF.beta.R2), an
(ALK-1), an (ALK-2), or a combination thereof with at least a
comparable affinity as a wild type TGF.beta.2. In some embodiments,
a TGF.beta.2 or TGF.beta.2 variant, derivative, or fragment thereof
of the disclosure can bind to TGF.beta.R1 with at least a
comparable affinity as a wild type TGF.beta.2. In some embodiments,
a TGF.beta.2 or TGF.beta.2 variant, derivative, or fragment thereof
of the disclosure can bind to TGF.beta.R2 with at least a
comparable affinity as a wild type TGF.beta.2. In some embodiments,
a TGF.beta.2 or TGF.beta.2 variant, derivative, or fragment thereof
of the disclosure can bind to ALK-1 with at least a comparable
affinity as a wild type TGF.beta.2 sequence. In some embodiments, a
TGF.beta.2 or TGF.beta.2 variant, derivative, or fragment thereof
of the disclosure can bind to ALK-2 with at least a comparable
affinity as a wild type TGF.beta.2 sequence. In some embodiments, a
TGF.beta.2 or TGF.beta.2 variant, derivative, or fragment thereof
of the disclosure can bind to a TGF.beta.R1, TGF.beta.R2, ALK-1,
and ALK-2 with at least a comparable affinity as a wild type
TGF.beta.2 sequence.
[0175] In some embodiments, a TGF.beta.2 or TGF.beta.2 variant,
derivative, or fragment thereof of the disclosure can bind to a
transforming growth factor beta receptor 1 (TGF.beta.R1), a
transforming growth factor beta receptor 2 (TGF.beta.R2), an
(ALK-1), an (ALK-2), or a combination thereof with at most a
comparable affinity as a wild type TGF.beta.2. In some embodiments,
a TGF.beta.2 or TGF.beta.2 variant, derivative, or fragment thereof
of the disclosure can bind to TGF.beta.R1 with at most a comparable
affinity as a wild type TGF.beta.2. In some embodiments, a
TGF.beta.2 or TGF.beta.2 variant, derivative, or fragment thereof
of the disclosure can bind to TGF.beta.R2 with at most a comparable
affinity as a wild type TGF.beta.2. In some embodiments, a
TGF.beta.2 or TGF.beta.2 variant, derivative, or fragment thereof
of the disclosure can bind to ALK-1 with at most a comparable
affinity as a wild type TGF.beta.2 sequence. In some embodiments, a
TGF.beta.2 or TGF.beta.2 variant, derivative, or fragment thereof
of the disclosure can bind to ALK-2 with at most a comparable
affinity as a wild type TGF.beta.2 sequence. In some embodiments, a
TGF.beta.2 or TGF.beta.2 variant, derivative, or fragment thereof
of the disclosure can bind to a TGF.beta.R1, TGF.beta.R2, ALK-1,
and ALK-2 with at most a comparable affinity as a wild type
TGF.beta.2 sequence.
[0176] In some embodiments, a TGF.beta.2 or TGF.beta.2 variant,
derivative, or fragment thereof can bind to a TGF.beta.R1,
TGF.beta.R2, ALK-1, or ALK-2 with at least about 1.5 fold, 2 fold,
5 fold, 10 fold, 15 fold, 20 fold, 30 fold, 40 fold, 50 fold, 100
fold, 200 fold, 500 fold, 1000 fold, or 10,000 fold increased
affinity relative to a wild type TGF.beta.2 sequence. In some
embodiments, a TGF.beta.2 or TGF.beta.2 variant, derivative, or
fragment thereof can bind to a TGF.beta.R1, TGF.beta.R2, ALK-1, or
ALK-2 with at least about 1.5 fold, 2 fold, 5 fold, 10 fold, 15
fold, 20 fold, 30 fold, 40 fold, 50 fold, 100 fold, 200 fold, 500
fold, 1000 fold, or 10,000 fold decreased affinity relative to a
wild type TGF.beta.2 sequence.
[0177] In some embodiments, a TGF.beta.2 or TGF.beta.2 variant,
derivative, or fragment thereof of the disclosure can activate a
native TGF.beta.2 receptor. A native TGF.beta.2 receptor can be,
for example, a receptor comprising a TGF.beta.R1 subunit and a
TGF.beta.R2 subunit. In some embodiments, an TGF.beta.2 or
TGF.beta.2 variant, derivative, or fragment thereof of the
disclosure can activate a native TGF.beta.2 receptor when present
in a fusion protein. In some embodiments, an TGF.beta.2 or
TGF.beta.2 variant, derivative, or fragment thereof of the
disclosure can activate a native TGF.beta.2 receptor when present
as a polypeptide that is not part of a fusion protein, but does not
activate native TGF.beta.2 receptor when present in a fusion
protein.
[0178] In some embodiments, a compound (e.g., polypeptide
construct, fusion protein) of the disclosure can bind to receptors
present on the surface of a cell and form a complex with about 2,
about 3, about 4, about 5, about 6, about 7, about 8, about 9,
about 10, about 11, or about 12 receptor subunits (e.g.,
polypeptide chains). In some embodiments, a compound (e.g.,
polypeptide construct, fusion protein) of the disclosure can bind
to receptors present on the surface of a cell and form a complex
with at least 2, at least 3, at least 4, at least 5, at least 6, at
least 7, at least 8, at least 9, at least 10, at least 11, or at
least 12 receptor subunits (e.g., polypeptide chains). In some
embodiments, a compound (e.g., polypeptide construct, fusion
protein) of the disclosure can bind to receptors present on the
surface of a cell and form a complex with at most 2, at most 3, at
most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at
most 10, at most 11, or at most 12 receptor subunits (e.g.,
polypeptide chains).
[0179] Compounds (e.g., polypeptide constructs, fusion proteins) of
the disclosure can comprise two or more covalently bound cytokines.
For example, a compound (e.g., polypeptide construct, fusion
protein) of the disclosure can comprise an IL4 covalently bound to
an IL10, IL13, IL27, IL33, TGF.beta.1, TGF.beta.2, or IL4.
[0180] A compound (e.g., polypeptide construct, fusion protein) of
the disclosure can comprise a linker. For example, an IL4 sequence
can be joined to a cytokine (e.g., an IL10, IL13, IL27, IL33,
TGF.beta.1, or TGF.beta.2) by a linker.
[0181] A linker can be a peptide. A linker can comprise a linker
sequence, for example, a linker peptide sequence. A linker sequence
can be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,
32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,
49, 50, 51, 51, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,
66, 67, 68, 69, or 70 amino acid residues in length. Examples of
linker sequences and linker repeat sequences are provided in Table
8.
TABLE-US-00008 TABLE 8 Non-limiting examples of linker sequences of
the disclosure SEQ ID NO: SEQUENCE 38 GSGGGGSGT 39 GGGS 40 GGGGS 41
KESGSVSSEQLAQFRSLD 42 EGKSSGSGSESKST 43 GSAGSAAGSGEF 44 EAAAK 45
EAAAR
[0182] A linker as described herein can include a flexible or rigid
linker. A flexible linker can have a sequence containing stretches
of glycine and serine residues. The small size of the glycine and
serine residues provides flexibility, and allows for mobility of
the connected functional domains. The incorporation of serine or
threonine can maintain the stability of the linker in aqueous
solutions by forming hydrogen bonds with the water molecules,
thereby reducing unfavorable interactions between the linker and
protein moieties. Flexible linkers can also contain additional
amino acids such as threonine and alanine to maintain flexibility,
as well as polar amino acids such as lysine and glutamine to
improve solubility.
[0183] A flexible linker can comprise SEQ ID NO: 38. A flexible
linker can comprise repeats of SEQ ID NO: 39 (GGGS), for example,
1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 repeats of SEQ ID NO: 39. A
flexible linker can comprise repeats of SEQ ID NO: 40 (GGGGS), for
example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 repeats of SEQ ID NO: 40.
Several other types of flexible linkers, including SEQ ID NO: 41
(KESGSVSSEQLAQFRSLD) and SEQ ID NO: 42 (EGKSSGSGSESKST), can also
be used. The SEQ ID NO: 43 (GSAGSAAGSGEF) linker can also be used,
in which large hydrophobic residues are minimized to maintain good
solubility in aqueous solutions. The length of the flexible linkers
can be adjusted to allow for proper folding or to achieve optimal
biological activity of the fused proteins.
[0184] A rigid linker can have, for example, an alpha
helix-structure. An alpha-helical rigid linker can act as a spacer
between protein domains. A rigid linker can comprise repeats of SEQ
ID NO: 44 (EAAAK), for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10
repeats of SEQ ID NO: 44. A rigid linker can comprise repeats of
SEQ ID NO: 45 (EAAAR), for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or
10 repeats of SEQ ID NO 45. A rigid linker can have a proline-rich
sequence, (XP)n, with X designating alanine, lysine, glutamine, or
any amino acid. The presence of proline in non-helical linkers can
increase stiffness, and allow for effective separation of protein
domains.
[0185] A linker of the disclosure can include a non-peptide linker,
for example, a chemical linker. For example, two amino acid
sequences of the disclosure can be connected together by a chemical
linker. Each chemical linker of the disclosure can be alkylene,
alkenylene, alkynylene, heteroalkylene, cycloalkylene,
heterocycloalkylene, arylene, or heteroarylene, any of which is
optionally substituted. In some embodiments, a chemical linker of
the disclosure can be an ester, ether, amide, thioether, or
polyethyleneglycol (PEG). In some embodiments, a linker can reverse
the order of the amino acids sequence in a compound, for example,
so that the amino acid sequences linked by the linked are
head-to-head, rather than head-to-tail. Non-limiting examples of
such linkers include diesters of dicarboxylic acids, such as oxalyl
diester, malonyl diester, succinyl diester, glutaryl diester,
adipyl diester, pimetyl diester, fumaryl diester, maleyl diester,
phthalyl diester, isophthalyl diester, and terephthalyl diester.
Non-limiting examples of such linkers include diamides of
dicarboxylic acids, such as oxalyl diamide, malonyl diamide,
succinyl diamide, glutaryl diamide, adipyl diamide, pimetyl
diamide, fumaryl diamide, maleyl diamide, phthalyl diamide,
isophthalyl diamide, and terephthalyl diamide. Non-limiting
examples of such linkers include diamides of diamino linkers, such
as ethylene diamine, 1,2-di(methylamino)ethane, 1,3-diaminopropane,
1,3-di(methylamino)propane, 1,4-di(methylamino)butane,
1,5-di(methylamino)pentane, 1,6-di(methylamino)hexane, and
pipyrizine. Non-limiting examples of optional substituents include
hydroxyl groups, sulfhydryl groups, halogens, amino groups, nitro
groups, nitroso groups, cyano groups, azido groups, sulfoxide
groups, sulfone groups, sulfonamide groups, carboxyl groups,
carboxaldehyde groups, imine groups, alkyl groups, halo-alkyl
groups, alkenyl groups, halo-alkenyl groups, alkynyl groups,
halo-alkynyl groups, alkoxy groups, aryl groups, aryloxy groups,
aralkyl groups, arylalkoxy groups, heterocyclyl groups, acyl
groups, acyloxy groups, carbamate groups, amide groups, ureido
groups, epoxy groups, and ester groups.
[0186] In some embodiments, an N terminus of a polypeptide
disclosed herein is joined to an N terminus of another polypeptide
disclosed herein. In some embodiments, a C terminus of a
polypeptide disclosed herein is joined to a C terminus of another
polypeptide disclosed herein. In some embodiments, a compound
(e.g., polypeptide construct, fusion protein) of the disclosure
does not contain a linker.
[0187] Compounds (e.g., polypeptide constructs, fusion proteins) of
the disclosure can comprise two or more non-covalently bound
cytokines. For example, a compound (e.g., polypeptide construct,
fusion protein) of the disclosure can comprise an IL4
non-covalently bound to an IL10, IL13, IL27, IL33, TGF.beta.1,
TGF.beta.2, or IL4.
[0188] Amino acids can include genetically encoded and
non-genetically encoded occurring amino acids. Amino acids can
include naturally occurring and non-naturally occurring amino
acids. Amino acids can be L forms or D forms. Substitutions can
include conservative and/or non-conservative amino acid
substitutions. A conservative amino acid change can be, for
example, a substitution that has minimal effect on the secondary or
tertiary structure of a polypeptide. A conservative amino acid
substitution can be a substitution of one amino acid for another
amino acid of similar biochemical properties (e.g., charge, size,
and/or hydrophobicity). A non-conservative amino acid substitution
can be a substitution of one amino acid for another amino acid with
different biochemical properties (e.g., charge, size, and/or
hydrophobicity). A conservative amino acid change can be an amino
acid change from one hydrophilic amino acid to another hydrophilic
amino acid. Hydrophilic amino acids can include Thr (T), Ser (S),
His (H), Glu (E), Asn (N), Gln (Q), Asp (D), Lys (K) and Arg (R). A
conservative amino acid change can be an amino acid change from one
hydrophobic amino acid to another hydrophilic amino acid.
Hydrophobic amino acids can include Ile (I), Phe (F), Val (V), Leu
(L), Trp (W), Met (M), Ala (A), Gly (G), Tyr (Y), and Pro (P). A
conservative amino acid change can be an amino acid change from one
acidic amino acid to another acidic amino acid. Acidic amino acids
can include Glu (E) and Asp (D). A conservative amino acid change
can be an amino acid change from one basic amino acid to another
basic amino acid. Basic amino acids can include His (H), Arg (R)
and Lys (K). A conservative amino acid change can be an amino acid
change from one polar amino acid to another polar amino acid. Polar
amino acids can include Asn (N), Gln (Q), Ser (S) and Thr (T). A
conservative amino acid change can be an amino acid change from one
nonpolar amino acid to another nonpolar amino acid. Nonpolar amino
acids can include Leu (L), Val (V), Ile (I), Met (M), Gly (G) and
Ala (A). A conservative amino acid change can be an amino acid
change from one aromatic amino acid to another aromatic amino acid.
Aromatic amino acids can include Phe (F), Tyr (Y) and Trp (W). A
conservative amino acid change can be an amino acid change from one
aliphatic amino acid to another aliphatic amino acid. Aliphatic
amino acids can include Ala (A), Val (V), Leu (L) and Ile (I). In
some embodiments, a conservative amino acid substitution is an
amino acid change from one amino acid to another amino acid within
one of the following groups: Group I: ala, pro, gly, gin, asn, ser,
thr; Group II: cys, ser, tyr, thr; Group III: val, ile, leu, met,
ala, phe; Group IV: lys, arg, his; Group V: phe, tyr, trp, his; and
Group VI: asp, glu.
[0189] The IL4 can be located N-terminal of the cytokine (e.g.,
IL10, IL13, IL27, IL33, TGF.beta.1, or TGF.beta.2). The IL4 can be
located C-terminal of the cytokine (e.g., IL10, IL13, IL27, IL33,
TGF.beta.1, or TGF.beta.2). In some embodiments, the C terminus of
IL4 can be joined to the N terminus of a linker sequence, and the C
terminus of the linker sequence can be joined to the N terminus of
the cytokine (e.g., IL10, IL13, IL27, IL33, TGF.beta.1, or
TGF.beta.2). In some embodiments, the N terminus of IL4 can be
joined to the C terminus of a linker sequence, and the N terminus
of the linker sequence can be joined to the C terminus of the
cytokine (e.g., IL10, IL13, IL27, IL33, TGF.beta.1, or
TGF.beta.2).
[0190] In an embodiment, the compound (e.g., polypeptide construct,
fusion protein) consists essentially of IL4 and the cytokine (e.g.,
IL10, IL13, IL27, IL33, TGF.beta.1, or TGF.beta.2). In an
embodiment, the compound (e.g., polypeptide construct, fusion
protein) consists essentially of IL4, the cytokine (e.g., IL10,
IL13, IL27, IL33, TGF.beta.1, or TGF.beta.2), and a linker.
[0191] In an embodiment, the compound (e.g., polypeptide construct,
fusion protein) consists of IL4 and the cytokine (e.g., IL10, IL13,
IL27, IL33, TGF.beta.1, or TGF.beta.2). In an embodiment, the
compound (e.g., polypeptide construct, fusion protein) consists of
IL4, the cytokine (e.g., IL10, IL13, IL27, IL33, TGF.beta.1, or
TGF.beta.2), and a linker.
[0192] In some embodiments, a fusion protein of the disclosure is
present in, purified into, and/or used in a multimeric form, for
example, a dimeric form or a tetrameric form. In some embodiments,
a fusion protein of the disclosure is present as, purified into,
and/or used as a monomer, a dimer, a trimer, a tetramer, a
multimer, or any combination thereof. A dimer, trimer, tetramer, or
multimer can comprise subunits that are covalently or
non-covalently bound.
[0193] In some embodiments, an IL4/IL13 fusion protein of the
disclosure is present as, purified into, and/or used as a monomer.
In some embodiments, an IL4/IL13 fusion protein of the disclosure
is present as, purified into, and/or used as a dimer. In some
embodiments, an IL4/IL13 fusion protein of the disclosure is
present as, purified into, and/or used as a trimer. In some
embodiments, an IL4/IL13 fusion protein of the disclosure is
present as, purified into, and/or used as a tetramer. In some
embodiments, an IL4/IL13 fusion protein of the disclosure is
present as, purified into, and/or used as a multimer. In some
embodiments, an IL4/IL13 fusion protein is present as, purified
into, and/or used as a monomer and a dimer. In some embodiments, an
IL4/IL13 fusion protein is present as, purified into, and/or used
as a monomer, a dimer, a trimer, a tetramer, a multimer, or any
combination thereof.
[0194] In some embodiments, an IL4/IL10 fusion protein of the
disclosure is present as, purified into, and/or used as monomer. In
some embodiments, an IL4/IL10 fusion protein of the disclosure is
present as, purified into, and/or used as dimer. In some
embodiments, an IL4/IL10 fusion protein of the disclosure is
present as, purified into, and/or used as trimer. In some
embodiments, an IL4/IL10 fusion protein of the disclosure is
present as, purified into, and/or used as tetramer. In some
embodiments, an IL4/IL10 fusion protein of the disclosure is
present as, purified into, and/or used as multimer. In some
embodiments, an IL4/IL10 fusion protein is present as, purified
into, and/or used as monomer and a dimer. In some embodiments, an
IL4/IL10 fusion protein is present as, purified into, and/or used
as monomer, a dimer, a trimer, a tetramer, a multimer, or any
combination thereof.
[0195] In some embodiments, an IL4/IL4 fusion protein of the
disclosure is present as, purified into, and/or used as monomer. In
some embodiments, an IL4/IL4 fusion protein of the disclosure is
present as, purified into, and/or used as dimer. In some
embodiments, an IL4/IL4 fusion protein of the disclosure is present
as, purified into, and/or used as trimer. In some embodiments, an
IL4/IL4 fusion protein of the disclosure is present as, purified
into, and/or used as tetramer. In some embodiments, an IL4/IL4
fusion protein of the disclosure is present as, purified into,
and/or used as multimer. In some embodiments, an IL4/IL4 fusion
protein is present as, purified into, and/or used as monomer and a
dimer. In some embodiments, an IL4/IL4 fusion protein is present
as, purified into, and/or used as monomer, a dimer, a trimer, a
tetramer, a multimer, or any combination thereof.
[0196] In some embodiments, an IL4/IL27 fusion protein of the
disclosure is present as, purified into, and/or used as monomer. In
some embodiments, an IL4/IL27 fusion protein of the disclosure is
present as, purified into, and/or used as dimer. In some
embodiments, an IL4/IL27 fusion protein of the disclosure is
present as, purified into, and/or used as trimer. In some
embodiments, an IL4/IL27 fusion protein of the disclosure is
present as, purified into, and/or used as tetramer. In some
embodiments, an IL4/IL27 fusion protein of the disclosure is
present as, purified into, and/or used as multimer. In some
embodiments, an IL4/IL27 fusion protein is present as, purified
into, and/or used as monomer and a dimer. In some embodiments, an
IL4/IL27 fusion protein is present as, purified into, and/or used
as monomer, a dimer, a trimer, a tetramer, a multimer, or any
combination thereof.
[0197] In some embodiments, an IL4/IL33 fusion protein of the
disclosure is present as, purified into, and/or used as monomer. In
some embodiments, an IL4/IL33 fusion protein of the disclosure is
present as, purified into, and/or used as dimer. In some
embodiments, an IL4/IL33 fusion protein of the disclosure is
present as, purified into, and/or used as trimer. In some
embodiments, an IL4/IL33 fusion protein of the disclosure is
present as, purified into, and/or used as tetramer. In some
embodiments, an IL4/IL33 fusion protein of the disclosure is
present as, purified into, and/or used as multimer. In some
embodiments, an IL4/IL33 fusion protein is present as, purified
into, and/or used as monomer and a dimer. In some embodiments, an
IL4/IL33 fusion protein is present as, purified into, and/or used
as monomer, a dimer, a trimer, a tetramer, a multimer, or any
combination thereof.
[0198] In some embodiments, an IL4/TGF.beta.1 fusion protein of the
disclosure is present as, purified into, and/or used as monomer. In
some embodiments, a IL4/TGF.beta.1 fusion protein of the disclosure
is present as, purified into, and/or used as dimer. In some
embodiments, a IL4/TGF.beta.1 fusion protein of the disclosure is
present as, purified into, and/or used as trimer. In some
embodiments, a IL4/TGF.beta.1 fusion protein of the disclosure is
present as, purified into, and/or used as tetramer. In some
embodiments, a IL4/TGF.beta.1 fusion protein of the disclosure is
present as, purified into, and/or used as multimer. In some
embodiments, a IL4/TGF.beta.1 fusion protein is present as,
purified into, and/or used as monomer and a dimer. In some
embodiments a IL4/TGF.beta.1 fusion protein is present as, purified
into, and/or used as monomer, a dimer, a trimer, a tetramer, a
multimer, or any combination thereof.
[0199] In some embodiments, a IL4/TGF.beta.2 fusion protein of the
disclosure is present as, purified into, and/or used as monomer. In
some embodiments, a IL4/TGF.beta.2 fusion protein of the disclosure
is present as, purified into, and/or used as dimer. In some
embodiments, a IL4/TGF.beta.2 fusion protein of the disclosure is
present as, purified into, and/or used as trimer. In some
embodiments, a IL4/TGF.beta.2 fusion protein of the disclosure is
present as, purified into, and/or used as tetramer. In some
embodiments, an IL4/TGF.beta.2 fusion protein of the disclosure is
present as, purified into, and/or used as multimer. In some
embodiments, a IL4/TGF.beta.2 fusion protein is present as,
purified into, and/or used as monomer and a dimer. In some
embodiments, a IL4/TGF.beta.2 fusion protein is present as,
purified into, and/or used as monomer, a dimer, a trimer, a
tetramer, a multimer, or any combination thereof.
Exemplary Constructs:
[0200] In some embodiments, the present disclosure relates to a
compound comprising: [0201] a first binding moiety that binds to a
first interleukin receptor, preferably an interleukin 4 receptor
(IL4R), interleukin 10 receptor (IL10R), interleukin 27 receptor
(IL27R), an interleukin 13 receptor (IL13R), IL27R, or to an IL33
receptor, a TGF.beta.1 receptor, and a TGF.beta.2 receptor; and
[0202] a second binding moiety that binds to a second interleukin
receptor, preferably chosen from the group consisting of an
interleukin 4 receptor (IL4R), interleukin 10 receptor (IL10R), an
interleukin 13 receptor (IL13R), an IL33 receptor, a TGF.beta.1
receptor, and a TGF.beta.2 receptor.
[0203] For example, the first binding moiety may bind to an
interleukin 4 receptor (IL4R). Or, the first binding moiety may
bind to an interleukin 10 receptor (IL10R). Alternatively, the
first binding moiety may bind to an interleukin 13 receptor (IL13R)
or IL27R. Still alternatively, the first binding moiety may bind to
an IL33 receptor. Or, the first binding moiety may bind to a
TGF.beta.1 receptor. Further, the first binding moiety may bind to
a TGF.beta.2 receptor.
[0204] In some embodiments, the present disclosure relates to a
compound comprising: [0205] a first binding moiety that binds to a
first interleukin receptor, preferably an interleukin 4 receptor
(IL4R); and [0206] a second binding moiety that binds to a second
interleukin receptor, preferably chosen from the group consisting
of an interleukin 10 receptor (IL10R), and an interleukin 13
receptor (IL13R).
[0207] In some embodiments, preferably, the first binding moiety
and/or the second binding moiety is not a (wild-type) interleukin,
and/or does not comprise a (wild-type) interleukin-derived amino
acid sequence.
[0208] It was found that a compound according to the present
disclosure is able to induce unique signaling in neurons, glial
cells and other target cells, and can in particular be applied as a
treatment of chronic pain and other conditions disclosed
herein.
[0209] The compound according to the present disclosure may be any
kind of compound that can cross-link cytokine receptors disclosed
herein (e.g., IL4R and IL10R or IL13R). This compound can be, for
example, a complex, or a polypeptide, a fusion protein, or more
preferably a bispecific antibody, a bivalent single chain antibody,
a bispecific double chain antibody, a triabody, or a tetrabody. The
term "first" and "second" binding moiety does not refer to their
relative orientation in the compound, i.e. the first binding moiety
can be N- or C-terminal to the second binding moiety, or any
alternative configuration may be used. The term "first" and
"second" only serves to correctly refer to the two different
binding moieties in the present disclosure.
[0210] The dual binding (or multi binding) compound according to
the present disclosure can be employed to connect (e.g. cluster or
cross-link) an IL4R and an IL10R or an IL13R in vivo, preferably an
IL4R and an IL10R or an IL13R on a sensory neuron or on a glial
cell.
[0211] In an alternative embodiment, the present disclosure
provides for a combination comprising: [0212] a first binding
moiety that binds to an IL4R; [0213] a second binding moiety that
binds to an IL10R or an IL13R,
[0214] wherein the first binding moiety has a linker that binds the
second binding moiety or
[0215] wherein the second binding moiety has a linker that binds
the first binding moiety.
[0216] In some embodiments, the first binding moiety and/or the
second binding moiety is not a (wild-type) interleukin and/or does
not comprise a (wild-type) interleukin-derived amino acid sequence.
In some embodiments, a compound (e.g., polypeptide construct,
fusion protein) of the disclosure does not contain SEQ ID NO: 11.
In some embodiments, a compound (e.g., polypeptide construct,
fusion protein) of the disclosure does not contain SEQ ID NO: 15.
In some embodiments, a compound (e.g., polypeptide construct,
fusion protein) of the disclosure does not contain SEQ ID NO:
46.
[0217] The above-mentioned combination can also be employed to
connect (cluster or cross-link) cytokine receptors (e.g., an IL4R
and an IL10R IL13R, IL27R, IL33R, or TGF.beta.1R, TGF.beta.2R) in
vivo, for example, an IL4R and an IL10R or an IL13R of a sensory
neuron or a glial cell. In this embodiment, the first binding
moiety and/or second binding moiety may comprise a tag, and the
linker of the respective other binding moiety preferably is a
polypeptide that binds to the tag.
[0218] The compound or combination according to the present
disclosure is provided particularly for use as a medicament or for
use in a therapeutic treatment, preferably the treatment of
(inflammatory) pain, e.g. chronic (inflammatory) pain, or
neurodegenerative disease.
[0219] The first binding moiety, the second binding moiety, and/or
the linker according to the present disclosure may be or encompass
for example a polypeptide, either or not containing sequences of
IL4 and IL10 or IL13, respectively, or more preferably an
immunoglobulin molecule or epitope-binding fragment thereof, in
particular Fab, F(ab'), F(ab')2, Fv, dAb, Fd, or a complementarity
determining region (CDR) fragment, a single chain antibody (scFv),
or single domain antibody. A binding moiety or linker according to
the present disclosure may thus be or encompass an intact
immunoglobulin molecule such as a polyclonal or monoclonal
antibody.
[0220] A monoclonal (full) antibody comprises two light chains and
two heavy chains, each comprising three CDRs, and has a total of 12
CDRs. A CDR region is a variable sequence involved in the physical
binding of the antibody to its antigen.
[0221] It is possible to select CDR sequences from other species,
e.g. murine, and exchange these with CDR sequences in a human
immunoglobulin molecule, to obtain a human immunoglobulin molecule
having the specificity that is derived from the other species. This
may be advantageous as a human sequence may be less immunogenic to
humans as compared to the original framework sequence. Such an
exchange of CDR sequences is known as humanization. Hence, the
compound, immunoglobulin molecule, or first and/or second binding
moiety as provided by the disclosure may be humanized.
[0222] The first binding moiety, second binding moiety and/or
linker according to the present disclosure preferably specifically
binds to its respective target. With the term "specifically bind(s)
to" is meant that the binding moiety or linker has more affinity
towards its target (e.g. human IL4R or human IL10R) or human IL13R
than to other molecules present in the target environment (e.g. the
human body). The affinity of an antibody to its antigen is
expressed as K.sub.D (the equilibrium dissociation constant between
the antibody and its antigen). It is preferred that the K.sub.D of
both antigen binding sites of any of the binding moieties disclosed
here is lower than 10.sup.-5 M, more preferably lower than
10.sup.-7 M, more preferably lower than 10.sup.-9 M, more
preferably lower than 10.sup.-10 M.
[0223] The compound, first binding moiety, second binding moiety
and/or linker according to the present disclosure may be or
encompass a single domain antibody. Single domain antibodies (sdAb,
also called Nanobody, or VHH) are well known to the skilled person.
Single domain antibodies are antibodies whose complementarity
determining regions are part of a single domain polypeptide. Single
domain antibodies thus comprise a single CDR1, a single CDR2 and a
single CDR3. Examples of single domain antibodies are heavy chain
only antibodies, antibodies that do not comprise light chains,
single domain antibodies derived from conventional antibodies, and
engineered antibodies. Single domain antibodies may be derived from
any species including mouse, human, camel, llama, goat, rabbit, and
bovine. For example, naturally occurring VHH molecules can be
derived from Camelidae species, for example in camel, dromedary,
alpaca and guanaco.
[0224] Like a whole antibody, a single domain antibody is able to
bind selectively to a specific target. Single domain antibodies
contain only the variable domain of an immunoglobulin chain having
CDR1, CDR2 and CDR3 and framework regions. With a molecular weight
of only about 12-15 kDa, single domain antibodies are much smaller
than regular antibodies (150-160 kDa) which are composed of at
least two heavy chains and two light chains. The format of a single
domain antibody has the advantage of less sterical hindering when
bound to its target and may bind to epitopes not accessible to
regular antibodies. This is both advantageous for the specific
blocking and cross-linking properties as aimed for in the present
disclosure. One other advantage may be that in the case of an
allogenic source, single domain antibodies may be less immunogenic
in other species than (full) monoclonal antibodies.
[0225] In a further aspect of the present disclosure, the compound,
first binding moiety and/or second binding moiety according to the
present disclosure may comprise a polypeptide selected from the
group consisting of a signal sequence, an affinity tag (e.g., a
His-tag), and an antibody Fc fragment. Additionally, and/or
alternatively, the compound, first binding moiety and/or second
binding moiety according to the present disclosure may comprise one
or more chemical modifications selected from the group consisting
of glycosylation, sialylation, fucosylation, and pegylation.
Pharmaceutical Compositions:
[0226] In a preferred embodiment, the compound or combination
according to the present disclosure is comprised in a
pharmaceutical composition, preferably with or in a
pharmaceutically acceptable carrier.
[0227] The pharmaceutical composition may be formulated with
pharmaceutically acceptable carriers or diluents as well as any
other known adjuvants and excipients in accordance with
conventional techniques (e.g., as described in Remington: The
Science and Practice of Pharmacy, 19.sup.th Edition, Gennaro, Ed.,
Mack Publishing Co., Easton, Pa., 1995).
[0228] The term "pharmaceutically acceptable carrier" relates to
carriers or excipients, which are inherently nontoxic and
nontherapeutic. Examples of such excipients are, but are not
limited to, saline, Ringer's solution, dextrose, solution and
Hank's solution. Non-aqueous excipients such as fixed oils and
ethyl oleate may also be used. A preferred excipient is 5% dextrose
in saline. The excipient may contain minor amounts of additives
such as substances that enhance isotonicity and chemical stability,
including buffers and preservatives.
[0229] The pharmaceutical composition may be administered by any
suitable routes and mode, but preferably by intraarticular or
intrathecal administration for example by injection. As will be
appreciated by the skilled artisan, the route and/or mode of
administration will vary depending upon the desired results.
[0230] The pharmaceutical compositions according to the invention
may be formulated in accordance with routine procedures for
administration of compound or combination by injection into a local
compartment such as joint or intrathecal space. The pharmaceutical
compositions of the present invention include those suitable for
intraarticular or intrathecal administration. Or suitable for
administration to any other local compartment in the body.
[0231] As used herein, "pharmaceutically acceptable carrier"
includes any and all solvents, dispersion media, coatings,
antibacterial and antifungal agents, isotonicity agents,
antioxidants and absorption delaying agents, and the like that are
physiologically compatible.
[0232] Pharmaceutically acceptable carriers include sterile aqueous
solutions or dispersions and sterile powders for the extemporaneous
preparation of sterile injectable solutions or dispersion. The use
of such media and agents for pharmaceutically active substances is
well known in the art. Except insofar as any conventional media or
agent is incompatible with the active compound, use thereof in the
pharmaceutical composition of the disclosure is contemplated.
Preferably, the carrier is suitable for local injection in a joint
or in the intrathecal space.
[0233] A pharmaceutical composition of the disclosure can be a
combination of any pharmaceutical compounds described herein with
other chemical components, such as carriers, stabilizers, diluents,
dispersing agents, suspending agents, thickening agents, and/or
excipients. The pharmaceutical composition facilitates
administration of the compound to an organism.
[0234] Pharmaceutical formulations for administration can include
aqueous solutions of the active compounds in water soluble form.
Suspensions of the active compounds can be prepared as oily
injection suspensions. Suitable lipophilic solvents or vehicles
include fatty oils such as sesame oil, or synthetic fatty acid
esters, such as ethyl oleate or triglycerides, or liposomes.
Aqueous injection suspensions can contain substances which increase
the viscosity of the suspension, such as sodium carboxymethyl
cellulose, sorbitol, or dextran. The suspension can also contain
suitable stabilizers or agents which increase the solubility of the
compounds to allow for the preparation of highly concentrated
solutions. The active ingredient can be in powder form for
constitution with a suitable vehicle, for example, sterile
pyrogen-free water, before use.
[0235] In practicing the methods of treatment or use provided
herein, therapeutically-effective amounts of the compounds
described herein are administered in pharmaceutical compositions to
a subject having a disease or condition to be treated. In some
embodiments, the subject is a mammal such as a human. A
therapeutically-effective amount can vary widely depending on the
severity of the disease, the age and relative health of the
subject, the potency of the compounds used, and other factors.
[0236] Pharmaceutical compositions can be formulated using one or
more physiologically-acceptable carriers comprising excipients and
auxiliaries, which facilitate processing of the active compounds
into preparations that can be used pharmaceutically. Formulation
can be modified depending upon the route of administration chosen.
Pharmaceutical compositions comprising compounds described herein
can be manufactured, for example, by mixing, dissolving,
emulsifying, encapsulating, entrapping, or compression
processes.
[0237] The pharmaceutical compositions can include at least one
pharmaceutically-acceptable carrier, diluent, or excipient and
compounds described herein as free-base or
pharmaceutically-acceptable salt form. Pharmaceutical compositions
can contain solubilizers, stabilizers, tonicity enhancing agents,
buffers, and preservatives.
[0238] Methods for the preparation of compositions comprising the
compounds described herein include formulating the compounds with
one or more inert, pharmaceutically-acceptable excipients or
carriers to form a solid, semi-solid, or liquid composition. Solid
compositions include, for example, powders, dispersible granules,
and cachets. Liquid compositions include, for example, solutions in
which a compound is dissolved, emulsions comprising a compound, or
a solution containing liposomes, micelles, or nanoparticles
comprising a compound as disclosed herein. Semi-solid compositions
include, for example, gels, suspensions and creams. The
compositions can be in liquid solutions or suspensions, solid forms
suitable for solution or suspension in a liquid prior to use, or as
emulsions. These compositions can also contain minor amounts of
nontoxic, auxiliary substances, such as wetting or emulsifying
agents, pH buffering agents, and other pharmaceutically-acceptable
additives.
[0239] Non-limiting examples of dosage forms suitable for use in
the disclosure include liquid, powder, gel, nanosuspension,
nanoparticle, microgel, aqueous or oily suspensions, emulsion, and
any combination thereof.
[0240] Non-limiting examples of pharmaceutically-acceptable
excipients suitable for use in the disclosure include binding
agents, disintegrating agents, anti-adherents, anti-static agents,
surfactants, anti-oxidants, coating agents, coloring agents,
plasticizers, preservatives, suspending agents, emulsifying agents,
anti-microbial agents, spheronization agents, and any combination
thereof.
[0241] Non-limiting examples of pharmaceutically-acceptable
carriers include saline, Ringer's solution, and dextrose solution.
In some embodiments, the pH of the solution can be from about 5 to
about 8, and can be from about 7 to about 7.5. Further carriers
include sustained release preparations such as semipermeable
matrices of solid hydrophobic polymers containing the compound. The
matrices can be in the form of shaped articles, for example, films,
liposomes, microparticles, or microcapsules.
[0242] The pH of the disclosed composition can range from about 3
to about 12. The pH of the composition can be, for example, from
about 3 to about 4, from about 4 to about 5, from about 5 to about
6, from about 6 to about 7, from about 7 to about 8, from about 8
to about 9, from about 9 to about 10, from about 10 to about 11, or
from about 11 to about 12 pH units. The pH of the composition can
be, for example, about 3, about 4, about 5, about 6, about 7, about
8, about 9, about 10, about 11, or about 12 pH units. The pH of the
composition can be, for example, at least 3, at least 4, at least
5, at least 6, at least 6.2 at least 6.4, at least 6.6, at least
6.8, at least 7, at least 7.2, at least 7.4, at least 7.6, at least
7.8, at least 8, at least 9, at least 10, at least 11 or at least
12 pH units. The pH of the composition can be, for example, at most
3, at most 4, at most 5, at most 6, at most 6.2 at most 6.4, at
most 6.6, at most 6.8, at most 7, at most 7.2, at most 7.4, at most
7.6, at most 7.8, at most 8, at most 9, at most 10, at most 11, or
at most 12 pH units. A pharmaceutical formulation disclosed herein
can have a pH of from about 5.5 to about 8.5.
[0243] Formulations of the disclosure can comprise sugars,
alcohols, antioxidants, buffers, bacteriostats, solutes which
render the formulation isotonic with the blood of the intended
recipient or suspending or thickening agents. These compositions
can also contain preservatives, wetting agents, emulsifying agents
and dispersing agents. Prevention of the action of microorganisms
upon the subject compounds can be achieved by the inclusion of
various antibacterial and antifungal agents, for example, paraben,
chlorobutanol, phenol sorbic acid, and the like. It can also be
desirable to include isotonic agents, such as sugars, sodium
chloride, and the like into the compositions. In addition,
prolonged absorption of the injectable pharmaceutical form can be
brought about by the inclusion of agents which delay absorption
such as aluminum monostearate and gelatin. If desired the
formulation can be diluted prior to use with, for example, an
isotonic saline solution or a dextrose solution.
[0244] In some embodiments, the pharmaceutical composition provided
herein comprises a therapeutically effective amount of a compound
(e.g., polypeptide construct) in admixture with a
pharmaceutically-acceptable carrier and/or excipient, for example,
saline, phosphate buffered saline, phosphate and amino acids,
polymers, polyols, sugar, buffers, preservatives, and other
proteins. Illustrative agents include octylphenoxy polyethoxy
ethanol compounds, polyethylene glycol monostearate compounds,
polyoxyethylene sorbitan fatty acid esters, sucrose, fructose,
dextrose, maltose, glucose, mannitol, dextran, sorbitol, inositol,
galactitol, xylitol, lactose, trehalose, bovine or human serum
albumin, citrate, acetate, Ringer's and Hank's solutions, cysteine,
arginine, carnitine, alanine, glycine, lysine, valine, leucine,
polyvinylpyrrolidone, polyethylene, and glycol.
[0245] In some embodiments, a pharmaceutical formulation disclosed
herein can comprise: (i) a compound or polypeptide construct
disclosed herein; (ii) a buffer; (iii) a non-ionic detergent; (iv)
a tonicity agent; and (v) a stabilizer. In some embodiments, the
pharmaceutical formulation disclosed herein is a stable liquid
pharmaceutical formulation.
[0246] For solid compositions, solid carriers include, for example,
pharmaceutical grades of mannitol, lactose, starch, magnesium
stearate, sodium saccharin, talc, cellulose, glucose, sucrose, and
magnesium carbonate.
[0247] A pharmaceutical carrier or excipient can be a solvent,
dispersion media, coatings, antibacterial and antifungal agents,
isotonic and absorption delaying agents, and the like that is
physiologically compatible. The carrier can be suitable for
administration by a route disclosed herein (e.g., parenteral).
[0248] A composition of the invention can be, for example, an
immediate release form or a controlled release formulation. An
immediate release formulation can be formulated to allow the
compounds to act rapidly. Non-limiting examples of immediate
release formulations include readily dissolvable formulations. A
controlled release formulation can be a pharmaceutical formulation
that has been adapted such that release rates and release profiles
of the active agent can be matched to physiological and
chronotherapeutic requirements or, alternatively, has been
formulated to effect release of an active agent at a programmed
rate. Non-limiting examples of controlled release formulations
include granules, delayed release granules, hydrogels (e.g., of
synthetic or natural origin), other gelling agents (e.g.,
gel-forming dietary fibers), matrix-based formulations (e.g.,
formulations comprising a polymeric material having at least one
active ingredient dispersed through), granules within a matrix,
polymeric mixtures, and granular masses.
[0249] In some embodiments, a formulation of the disclosure
contains a thermal stabilizer, such as a sugar or sugar alcohol,
for example, sucrose, sorbitol, glycerol, trehalose, or mannitol,
or any combination thereof. In some embodiments, the stabilizer is
a sugar. In some embodiments, the sugar is sucrose, mannitol or
trehalose.
[0250] Non-limiting examples of pharmaceutically-acceptable
excipients can be found, for example, in Remington: The Science and
Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing
Company, 1995); Hoover, John E., Remington's Pharmaceutical
Sciences, Mack Publishing Co., Easton, Pa. 1975; Liberman, H. A.
and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker,
New York, N.Y., 1980; and Pharmaceutical Dosage Forms and Drug
Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkins
1999), each of which is incorporated by reference in its
entirety.
[0251] A pharmaceutical composition can be administered in a local
manner, for example, via injection of the compound directly into an
organ, optionally in a depot or sustained release formulation or
implant. A pharmaceutical composition can be provided in the form
of a rapid release formulation, in the form of an extended release
formulation, or in the form of an intermediate release formulation.
A rapid release form can provide an immediate release. An extended
release formulation can provide a controlled release or a sustained
delayed release.
[0252] In some embodiments, a pump can be used for delivery of the
pharmaceutical composition. In some embodiments, a pen delivery
device can be used, for example, for subcutaneous delivery of a
composition of the disclosure. Such a pen delivery device can be
reusable or disposable. A reusable pen delivery device can use a
replaceable cartridge that contains a pharmaceutical composition
disclosed herein. Once all of the pharmaceutical composition within
the cartridge has been administered and the cartridge is empty, the
empty cartridge can readily be discarded and replaced with a new
cartridge that contains the pharmaceutical composition. The pen
delivery device can then be reused. A disposable pen has no
replaceable cartridge. Rather, the disposable pen delivery device
comes prefilled with the pharmaceutical composition held in a
reservoir within the device. Once the reservoir is emptied of the
pharmaceutical composition, the entire device is discarded.
[0253] A pharmaceutical composition described herein can be in a
unit dosage form suitable for a single administration of a precise
dosage. In unit dosage form, the formulation can be divided into
unit doses containing appropriate quantities of one or more
compounds, polypeptide constructs, and/or therapeutic agents. The
unit dosage can be in the form of a package containing discrete
quantities of the formulation. Non-limiting examples are packaged
injectables, vials, and ampoules. An aqueous suspension composition
disclosed herein can be packaged in a single-dose non-reclosable
container. Multiple-dose reclosable containers can be used, for
example, in combination with or without a preservative. A
formulation for injection disclosed herein can be present in a unit
dosage form, for example, in ampoules, or in multi dose containers
with a preservative.
[0254] In some embodiments, a pharmaceutical formulation disclosed
herein is a liquid formulation that can comprise about 50 .mu.g/mL
to about 100 mg/mL of polypeptide construct. A formulation can
comprise, for example, at least 50 .mu.g/mL, at least 100 .mu.g/mL,
at least 200 .mu.g/mL, at least 300 .mu.g/mL, at least 400
.mu.g/mL, at least 500 .mu.g/mL, at least 600 .mu.g/mL, at least
700 .mu.g/mL, at least 800 .mu.g/mL, at least 900 .mu.g/mL, at
least 1 mg/mL, at least 10 mg/mL, at least 20 mg/mL, or at least 50
mg/mL. In some embodiments, a formulation can comprise at most 100
.mu.g/mL, at most 200 .mu.g/mL, at most 300 .mu.g/mL, at most 400
.mu.g/mL, at most 500 .mu.g/mL, at most 600 .mu.g/mL, at most 700
.mu.g/mL, at most 800 .mu.g/mL, at most 900 .mu.g/mL, at most 1
mg/mL, at most 10 mg/mL, at most 20 mg/mL, or at most 50 mg/mL.
[0255] Actual dosage levels of the compound according to the
present disclosure may be varied so as to obtain an amount which is
effective ("effective amount") to achieve the desired therapeutic
response for a particular patient, composition, and mode of
administration, without being toxic to the patient. The selected
dosage level will depend upon a variety of factors including the
activity of the particular compositions of the present invention
employed, the route and time of administration, the duration of the
treatment, age, sex, weight, general health and prior medical
history of the patient being treated, and like factors well known
in the medical arts.
[0256] In one embodiment, the compound or combination of the
present disclosure can be given as a bolus injection, in another
embodiment, they can be administered by slow continuous
administration via an intrathecal pump or the like, over a long
period, such as more than 24 hours, in order to reduce toxic side
effects.
[0257] In yet another embodiment, the compound or combination of
the present disclosure can be administered as maintenance therapy,
for a period of up to 6 months or more by repeated injections such
as, e.g., once a week or 1-10 times per month, for example in a
joint or the intrathecal space, or via a pump or any other
intrathecal or intraarticular delivery device.
[0258] Therapeutic agents described herein can be administered
before, during, or after the occurrence of a disease or condition,
and the timing of administering the composition containing a
therapeutic agent can vary. For example, the compositions can be
used as a prophylactic and can be administered continuously to
subjects with a propensity to conditions or diseases in order to
lessen a likelihood of the occurrence of the disease or condition.
The compositions can be administered to a subject during or as soon
as possible after the onset of the symptoms. The initial
administration can be via any route practical, such as by any route
described herein using any formulation described herein. A
therapeutic agent can be administered as soon as is practicable
after the onset of a disease or condition is detected or suspected,
and for a length of time necessary for the treatment of the
disease, such as, for example, from about 1 month to about 3
months. The length of treatment can vary for each subject.
[0259] A dose can be based on the amount of the compound (e.g.,
polypeptide construct, fusion protein) per kilogram of body weight
of a subject. A dose a of a compound can be at least about 0.5
.mu.g/kg, 1 .mu.g/kg, 25 .mu.g/kg, 50 .mu.g/kg, 75 .mu.g/kg, 100
.mu. .mu.g/kg, 125 .mu.g/kg, 150 .mu.g/kg, 175 .mu.g/kg, 200
.mu.g/kg, 225 .mu.g/kg, 250 .mu.g/kg, 275 .mu.g/kg, 300 .mu.g/kg,
325 .mu.g/kg, 350 pg/kg, 375 .mu.g/kg, 400 .mu.g/kg, 425 .mu.g/kg,
450 .mu.g/kg, 475 .mu.g/kg, 500 .mu.g/kg, 525 .mu.g/kg, 550
.mu.g/kg, 575 .mu.g/kg, 600 .mu.g/kg, 625 .mu.g/kg, 650 .mu.g/kg,
675 .mu.g/kg, 700 .mu.g/kg, 725 .mu.g/kg, 750 .mu.g/kg, 775
.mu.g/kg, 800 .mu.g/kg, 825 .mu.g/kg, 850 .mu.g/kg, 875 .mu.g/kg,
900 .mu.g/kg, 925 .mu.g/kg, 950 .mu.g/kg, 975 .mu.g/kg, or 1 mg/kg.
A dose a of a compound (e.g., polypeptide construct, fusion
protein) can be at most about 1 .mu.g/kg, 25 .mu.g/kg, 50 .mu.g/kg,
75 .mu.g/kg, 100 .mu. .mu.g/kg, 125 .mu.g/kg, 150 .mu.g/kg, 175
.mu.g/kg, 200 .mu.g/kg, 225 .mu.g/kg, 250 .mu.g/kg, 275 .mu.g/kg,
300 .mu.g/kg, 325 .mu.g/kg, 350 .mu.g/kg, 375 .mu.g/kg, 400
.mu.g/kg, 425 .mu.g/kg, 450 .mu.g/kg, 475 .mu.g/kg, 500 .mu.g/kg,
525 .mu.g/kg, 550 .mu.g/kg, 575 .mu.g/kg, 600 .mu.g/kg, 625
.mu.g/kg, 650 .mu.g/kg, 675 .mu.g/kg, 700 .mu.g/kg, 725 .mu.g/kg,
750 .mu.g/kg, 775 .mu.g/kg, 800 .mu.g/kg, 825 .mu.g/kg, 850
.mu.g/kg, 875 .mu.g/kg, 900 .mu.g/kg, 925 .mu.g/kg, 950 .mu.g/kg,
975 .mu.g/kg, or 1 mg/kg.
[0260] In some embodiments, a dose can be at least about 1 ng, at
least 10 ng, at least 100 ng, at least 500 ng, at least at least 1
.mu.g, at least 5 .mu.g, at least at least 10 .mu.g, at least 50
.mu.g, at least at least 100 .mu.g, at least 500 .mu.g, at least at
least 1 mg, at least 5 mg, at least 10 mg, at least 50 mg, or at
least 100 mg. A dose can be at most about 1 ng, at most 10 ng, at
most 100 ng, at most 500 ng, at most at most 1 .mu.g, at most 5
.mu.g, at most at most 10 .mu.g, at most 50 .mu.g, at most at most
100 .mu.g, at most 500 .mu.g, at most at most 1 mg, at most 5 mg,
at most 10 mg, at most 50 mg, or at most 100 mg.
[0261] A dose can be determined by reference to a plasma
concentration or a local concentration of the polypeptide
construct. A target plasma concentration or local concentration of
the compound (e.g., polypeptide construct, fusion protein) can be
at least about 1 pM, at least about 10 pM, at least about 20 pM, at
least about 30 pM, at least about 40 pM, at least about 50 pM, at
least about 60 pM, at least about 70 pM, at least about 80 pM, at
least about 90 pM, at least about 100 pM, at least about 200 pM, at
least about 300 pM, at least about 400 pM, at least about 500 pM,
at least about 600 pM, at least about 700 pM, at least about 800
pM, at least about 900 pM, at least about 1 nM, at least about 2
nM, at least about 3 nM, at least about 4 nM, at least about 5 nM,
at least about 6 nM, at least about 7 nM, at least about 8 nM, at
least about 9 nM, at least about 10 nM, at least about 20 nM, at
least about 30 nM, at least about 40 nM, at least about 50 nM, at
least about 60 nM, at least about 70 nM, at least about 80 nM, at
least about 90 nM, at least about 100 nM, at least about 200 nM, at
least about 300 nM, at least about 400 nM, at least about 500 nM,
at least about 600 nM, at least about 700 nM, at least about 800
nM, at least about 900 nM, at least about 1 .mu.M, at least about
10 .mu.M, or at least about 100 .mu.M. A target plasma
concentration or local concentration of the compound (e.g.,
polypeptide construct, fusion protein) can be at most about 1 nM,
at most about 10 nM, at most about 100 nM, at most about 1 .mu.M,
at most about 10 .mu.M, at most about 100 .mu.M, or at most about 1
mM.
Neuronal and Nervous System Modulation:
[0262] In some embodiments, the disclosure provides methods that
involve contacting a cell, such as a nervous system cell, with a
compound (e.g., polypeptide construct, fusion protein) of the
disclosure. A nervous system cell can be, for example, a neuron, a
central nervous system cell, a peripheral nervous system cell, a
neuron, a glial cell, a microglial cell, an astrocyte, a schwann
cell, an oligodendrocyte, an infiltrating cell, an infiltrating
immune cell, an infiltrating myeloid cell, an infiltrating lymphoid
cell, an infiltrating macrophage, an infiltrating neutrophil, an
infiltrating lymphocyte, an infiltrating T cell, an infiltrating B
cell, or an infiltrating natural killer cell. A neuron can be, for
example, a sensory neuron, a somatosensory neuron, a visceral
sensory neuron, a nociceptor, and/or an autonomic neuron
[0263] Contacting a nervous system cell with a compound (e.g.,
polypeptide construct, fusion protein) of the disclosure can
modulate a signaling pathway in the nervous system cell, for
example, compared to a nervous system cell that is contacted with
equivalent amounts of the an IL4 that is present in the polypeptide
construct, a cytokine that is present in the compound (e.g., IL10,
IL13, IL27, IL33, TGF.beta.1, TGF.beta.2), or a combination of the
IL4 and the cytokine. Non-limiting examples of signaling pathways
include those disclosed in examples 5 and 7 and FIGS. 8A-8H and
9E-9F. Non-limiting examples of kinases that can be modulated
include those disclosed in example 5, example 7, FIGS. 8A-8H, and
FIGS. 10A-10E. Non-limiting examples of phosphorylation substrates
that can be modulated include those disclosed in example 5, example
7, and FIGS. 8A-9H. Modulation of a signaling pathway can be as
determined by, for example, kinase array profiling, detection of
phosphorylated substrates via western blot, detection of
phosphorylated substrates via flow cytometry or mass cytometry,
detection of phosphorylated substrates via ELISA, or detection of
phosphorylated substrates or second messengers via mass
spectrometry. The nervous system cell can be contacted with an
amount of the compound (e.g., polypeptide construct, fusion
protein) that is sufficient to modulate the signaling pathway. The
nervous system cell can be contacted with a concentration of the
compound that is sufficient to modulate the signaling pathway. A
concentration of the compound (e.g., polypeptide construct, fusion
protein) that is sufficient to modulate the signaling pathway can
be, for example, at least about 1 pM, at least about 10 pM, at
least about 20 pM, at least about 30 pM, at least about 40 pM, at
least about 50 pM, at least about 60 pM, at least about 70 pM, at
least about 80 pM, at least about 90 pM, at least about 100 pM, at
least about 200 pM, at least about 300 pM, at least about 400 pM,
at least about 500 pM, at least about 600 pM, at least about 700
pM, at least about 800 pM, at least about 900 pM, at least about 1
nM, at least about 2 nM, at least about 3 nM, at least about 4 nM,
at least about 5 nM, at least about 6 nM, at least about 7 nM, at
least about 8 nM, at least about 9 nM, at least about 10 nM, at
least about 20 nM, at least about 30 nM, at least about 40 nM, at
least about 50 nM, at least about 60 nM, at least about 70 nM, at
least about 80 nM, at least about 90 nM, at least about 100 nM, at
least about 200 nM, at least about 300 nM, at least about 400 nM,
at least about 500 nM, at least about 600 nM, at least about 700
nM, at least about 800 nM, at least about 900 nM, at least about 1
.mu.M, at least about 10 .mu.M, or at least about 100 .mu.M. The
nervous system cell can be contacted with the compound (e.g.,
polypeptide construct, fusion protein) for a period of time that is
sufficient to modulate the signaling pathway. A period of time
sufficient to modulate the signaling pathway can be, for example,
at least about 1 second, at least about 10 seconds, at least about
30 seconds, at least about 1 minute, at least about 5 minutes, at
least about 10 minutes, at least about 30 minutes, at least about 1
hour, at least about 2 hours, at least about 3 hours, at least
about 5 hours, at least about 6 hours, at least about 10 hours, at
least about 12 hours, at least about 24 hours, at least about 2
days, at least about 3 days, at least about 5 days, or at least
about a week.
[0264] Modulating the activity of a signaling pathway can comprise
increasing the activity of the signaling pathway. Modulating the
activity of a signaling pathway can comprise decreasing the
activity of the signaling pathway. Modulating the activity of a
signaling pathway can comprise increasing the activity of a kinase,
group of kinases, or kinase class. Modulating the activity of a
signaling pathway can comprise decreasing the activity of a kinase,
group of kinases, or kinase class. Modulating the activity of a
signaling pathway can comprise increasing the phosphorylation of a
substrate, group of substrates, or substrate class. Modulating the
activity of a signaling pathway can comprise decreasing the
phosphorylation of a substrate, group of substrates, or substrate
class.
[0265] In some embodiments, increasing the activity of a signaling
pathway, kinase, group of kinases, kinase class, or increasing the
phosphorylation level of a substrate, group of substrates, or
substrate class, can comprise an increase of at least about at
least about 5%, at least about 10%, at least about 15%, at least
about 20%, at least about 30%, at least about 40%, at least about
50%, at least about 60%, at least about 70%, at least about 80%
90%, at least about or 100%. In some embodiments, increasing the
activity of a signaling pathway, kinase, group of kinases, kinase
class, or increasing the phosphorylation level of a substrate,
group of substrates, or substrate class, can comprise an increase
of at least about at least about 1.5-fold, at least about 2-fold,
at least about 3-fold, at least about 4-fold, at least about
5-fold, at least about 6-fold, at least about 7-fold, at least
about 8-fold, at least about 9-fold, at least about 10-fold, at
least about 15-fold, at least about 20-fold, at least about
25-fold, at least about 30-fold, at least about 40-fold, at least
about 50-fold, at least about 100-fold, at least about 200-fold, at
least about 300-fold, at least about 400-fold, at least about
500-fold, at least about 600-fold, at least about 700-fold, at
least about 800-fold, at least about 900-fold, at least about
1000-fold, or at least about 10,000-fold. In some embodiments,
increasing can comprise increasing from an undetectable level to a
detectable level.
[0266] In some embodiments, decreasing the activity of a signaling
pathway, kinase, group of kinases, kinase class, or decreasing the
phosphorylation level of a substrate, group of substrates, or
substrate class, can comprise a decrease of at least about at least
about 5%, at least about 10%, at least about 15%, at least about
20%, at least about 30%, at least about 40%, at least about 50%, at
least about 60%, at least about 70%, at least about 80% 90%, at
least about or 100%. In some embodiments, decreasing the activity
of a signaling pathway, kinase, group of kinases, kinase class, or
decreasing the phosphorylation level of a substrate, group of
substrates, or substrate class, can comprise a decrease of at least
about at least about 1.5-fold, at least about 2-fold, at least
about 3-fold, at least about 4-fold, at least about 5-fold, at
least about 6-fold, at least about 7-fold, at least about 8-fold,
at least about 9-fold, at least about 10-fold, at least about
15-fold, at least about 20-fold, at least about 25-fold, at least
about 30-fold, at least about 40-fold, at least about 50-fold, at
least about 100-fold, at least about 200-fold, at least about
300-fold, at least about 400-fold, at least about 500-fold, at
least about 600-fold, at least about 700-fold, at least about
800-fold, at least about 900-fold, at least about 1000-fold, or at
least about 10,000-fold. In some embodiments, decreasing can
comprise decreasing from a detectable level to an undetectable
level.
[0267] Contacting a nervous system cell with a compound (e.g.,
polypeptide construct, fusion protein) of the disclosure can
modulate a kinomic profile in the nervous system cell, for example,
compared to a nervous system cell that is contacted with equivalent
amounts of the an IL4 that is present in the polypeptide construct,
a cytokine that is present in the compound (e.g., IL10, IL13, IL27,
IL33, TGF.beta.1, TGF.beta.2), or a combination of the IL4 and the
cytokine. Modulation of a kinomic profile can be as determined by,
for example, kinase array profiling, detection of phosphorylated
substrates via mass cytometry, or detection of phosphorylated
substrates via mass spectrometry. The nervous system cell can be
contacted with an amount of the compound that is sufficient to
modulate the kinomic profile. The nervous system cell can be
contacted with a concentration of the compound that is sufficient
to modulate the kinomic profile. A concentration of the compound
that is sufficient to modulate the kinomic profile can be, for
example, at least about 1 pM, at least about 10 pM, at least about
20 pM, at least about 30 pM, at least about 40 pM, at least about
50 pM, at least about 60 pM, at least about 70 pM, at least about
80 pM, at least about 90 pM, at least about 100 pM, at least about
200 pM, at least about 300 pM, at least about 400 pM, at least
about 500 pM, at least about 600 pM, at least about 700 pM, at
least about 800 pM, at least about 900 pM, at least about 1 nM, at
least about 2 nM, at least about 3 nM, at least about 4 nM, at
least about 5 nM, at least about 6 nM, at least about 7 nM, at
least about 8 nM, at least about 9 nM, at least about 10 nM, at
least about 20 nM, at least about 30 nM, at least about 40 nM, at
least about 50 nM, at least about 60 nM, at least about 70 nM, at
least about 80 nM, at least about 90 nM, at least about 100 nM, at
least about 200 nM, at least about 300 nM, at least about 400 nM,
at least about 500 nM, at least about 600 nM, at least about 700
nM, at least about 800 nM, at least about 900 nM, at least about 1
.mu.M, at least about 10 .mu.M, or at least about 100 .mu.M. The
nervous system cell can be contacted with the compound (e.g.,
polypeptide construct, fusion protein) for a period of time that is
sufficient to modulate the kinomic profile. A period of time
sufficient to modulate the kinomic profile can be, for example, at
least about 1 second, at least about 10 seconds, at least about 30
seconds, at least about 1 minute, at least about 5 minutes, at
least about 10 minutes, at least about 30 minutes, at least about 1
hour, at least about 2 hours, at least about 3 hours, at least
about 5 hours, at least about 6 hours, at least about 10 hours, at
least about 12 hours, at least about 24 hours, at least about 2
days, at least about 3 days, at least about 5 days, or at least
about a week.
[0268] Contacting a nervous system cell with a compound (e.g.,
polypeptide construct, fusion protein) of the disclosure can
modulate a gene expression in the nervous system cell, for example,
compared to a nervous system cell that is contacted with equivalent
amounts of the an IL4 that is present in the polypeptide construct,
a cytokine that is present in the compound (e.g., IL10, IL13, IL27,
IL33, TGF.beta.1, TGF.beta.2), or a combination of the IL4 and the
cytokine. Non-limiting examples of genes that can be modulated
include those disclosed in example 5 and FIGS. 9A-9F. Modulated
gene expression can be as determined by, for example, RNA
sequencing (e.g., with principal component analysis and/or
hierarchical clustering of differentially regulated genes),
microarray profiling, gene arrays, and RT-qPCR. The nervous system
cell can be contacted with an amount of the compound (e.g.,
polypeptide construct, fusion protein) that is sufficient to
modulate gene expression. The nervous system cell can be contacted
with a concentration of the compound that is sufficient to modulate
gene expression. A concentration of the compound that is sufficient
to modulate gene expression can be, for example, at least about 1
pM, at least about 10 pM, at least about 20 pM, at least about 30
pM, at least about 40 pM, at least about 50 pM, at least about 60
pM, at least about 70 pM, at least about 80 pM, at least about 90
pM, at least about 100 pM, at least about 200 pM, at least about
300 pM, at least about 400 pM, at least about 500 pM, at least
about 600 pM, at least about 700 pM, at least about 800 pM, at
least about 900 pM, at least about 1 nM, at least about 2 nM, at
least about 3 nM, at least about 4 nM, at least about 5 nM, at
least about 6 nM, at least about 7 nM, at least about 8 nM, at
least about 9 nM, at least about 10 nM, at least about 20 nM, at
least about 30 nM, at least about 40 nM, at least about 50 nM, at
least about 60 nM, at least about 70 nM, at least about 80 nM, at
least about 90 nM, at least about 100 nM, at least about 200 nM, at
least about 300 nM, at least about 400 nM, at least about 500 nM,
at least about 600 nM, at least about 700 nM, at least about 800
nM, at least about 900 nM, at least about 1 .mu.M, at least about
10 .mu.M, or at least about 100 .mu.M. The nervous system cell can
be contacted with the compound (e.g., polypeptide construct, fusion
protein) for a period of time that is sufficient to modulate gene
expression. A period of time sufficient to modulate gene expression
can be, for example, at least about 1 second, at least about 10
seconds, at least about 30 seconds, at least about 1 minute, at
least about 5 minutes, at least about 10 minutes, at least about 30
minutes, at least about 1 hour, at least about 2 hours, at least
about 3 hours, at least about 5 hours, at least about 6 hours, at
least about 10 hours, at least about 12 hours, at least about 24
hours, at least about 2 days, at least about 3 days, at least about
5 days, or at least about a week.
[0269] Modulating gene expression in a nervous system cell can
comprise increasing expression of a gene or group of genes.
Modulating gene expression in a nervous system cell can comprise
decreasing expression of a gene or group of genes.
[0270] In some embodiments, increasing gene expression can comprise
an increase of at least about at least about 5%, at least about
10%, at least about 15%, at least about 20%, at least about 30%, at
least about 40%, at least about 50%, at least about 60%, at least
about 70%, at least about 80% 90%, at least about or 100%. In some
embodiments, increasing gene expression, can comprise an increase
of at least about at least about 1.5-fold, at least about 2-fold,
at least about 3-fold, at least about 4-fold, at least about
5-fold, at least about 6-fold, at least about 7-fold, at least
about 8-fold, at least about 9-fold, at least about 10-fold, at
least about 15-fold, at least about 20-fold, at least about
25-fold, at least about 30-fold, at least about 40-fold, at least
about 50-fold, at least about 100-fold, at least about 200-fold, at
least about 300-fold, at least about 400-fold, at least about
500-fold, at least about 600-fold, at least about 700-fold, at
least about 800-fold, at least about 900-fold, at least about
1000-fold, or at least about 10,000-fold. In some embodiments,
increasing can comprise increasing from an undetectable level to a
detectable level.
[0271] In some embodiments, decreasing gene expression can comprise
a decrease of at least about at least about 5%, at least about 10%,
at least about 15%, at least about 20%, at least about 30%, at
least about 40%, at least about 50%, at least about 60%, at least
about 70%, at least about 80% 90%, at least about or 100%. In some
embodiments, decreasing gene expression, can comprise a decrease of
at least about at least about 1.5-fold, at least about 2-fold, at
least about 3-fold, at least about 4-fold, at least about 5-fold,
at least about 6-fold, at least about 7-fold, at least about
8-fold, at least about 9-fold, at least about 10-fold, at least
about 15-fold, at least about 20-fold, at least about 25-fold, at
least about 30-fold, at least about 40-fold, at least about
50-fold, at least about 100-fold, at least about 200-fold, at least
about 300-fold, at least about 400-fold, at least about 500-fold,
at least about 600-fold, at least about 700-fold, at least about
800-fold, at least about 900-fold, at least about 1000-fold, or at
least about 10,000-fold. In some embodiments, decreasing can
comprise decreasing from a detectable level to an undetectable
level.
[0272] Contacting a neuron with a compound (e.g., polypeptide
construct, fusion protein) of the disclosure can modulate a
sensitivity threshold of a neuronal response to a stimulus, for
example, compared to a neuron that is contacted with equivalent
amounts of the an IL4 that is present in the polypeptide construct,
a cytokine that is present in the compound (e.g., IL10, IL13, IL27,
IL33, TGF.beta.1, TGF.beta.2), or a combination of the IL4 and the
cytokine. A sensitivity threshold can comprise an amount (e.g.,
concentration) of a stimulus required to elicit a pre-determined
neuronal response, for example, calcium flux above a certain level,
depolarization, alteration of depolarization frequency above or
below a certain threshold, altered expression, production, and/or
release of a neurotransmitter or neuropeptide above or below a
certain level, or altered expression/production of a gene to above
or below a certain level. A stimulus can be, for, example,
capsaicin, a pro-inflammatory mediator, an anti-inflammatory
mediator, a drug, a toxin, a toxicant, a chemical stimulus, a
mechanical stimulus, a thermal stimulus, or neuronal damage. In
some embodiments, a stimulus is not a pro-inflammatory mediator,
but the sensitivity threshold is modulated in the presence of a
pro-inflammatory mediator. Modulation of a sensitivity threshold
can be as determined by measuring a neuronal response as disclosed
herein (e.g., above) to a stimulus as disclosed herein (e.g.,
above). The neuron can be contacted with an amount of the compound
(e.g., polypeptide construct, fusion protein) that is sufficient to
modulate the sensitivity threshold. The neuron can be contacted
with a concentration of the compound that is sufficient to modulate
the sensitivity threshold. A concentration of the compound that is
sufficient to modulate the sensitivity threshold can be, for
example, at least about 1 pM, at least about 10 pM, at least about
20 pM, at least about 30 pM, at least about 40 pM, at least about
50 pM, at least about 60 pM, at least about 70 pM, at least about
80 pM, at least about 90 pM, at least about 100 pM, at least about
200 pM, at least about 300 pM, at least about 400 pM, at least
about 500 pM, at least about 600 pM, at least about 700 pM, at
least about 800 pM, at least about 900 pM, at least about 1 nM, at
least about 2 nM, at least about 3 nM, at least about 4 nM, at
least about 5 nM, at least about 6 nM, at least about 7 nM, at
least about 8 nM, at least about 9 nM, at least about 10 nM, at
least about 20 nM, at least about 30 nM, at least about 40 nM, at
least about 50 nM, at least about 60 nM, at least about 70 nM, at
least about 80 nM, at least about 90 nM, at least about 100 nM, at
least about 200 nM, at least about 300 nM, at least about 400 nM,
at least about 500 nM, at least about 600 nM, at least about 700
nM, at least about 800 nM, at least about 900 nM, at least about 1
.mu.M, at least about 10 .mu.M, or at least about 100 .mu.M. The
neuron can be contacted with the compound for a period of time that
is sufficient to modulate the sensitivity threshold. A period of
time sufficient to modulate the sensitivity threshold can be, for
example, at least about 1 second, at least about 10 seconds, at
least about 30 seconds, at least about 1 minute, at least about 5
minutes, at least about 10 minutes, at least about 30 minutes, at
least about 1 hour, at least about 2 hours, at least about 3 hours,
at least about 5 hours, at least about 6 hours, at least about 10
hours, at least about 12 hours, at least about 24 hours, at least
about 2 days, at least about 3 days, at least about 5 days, or at
least about a week.
[0273] Contacting a neuron with a compound (e.g., polypeptide
construct, fusion protein) of the disclosure can modulate a
magnitude of a neuronal response to a stimulus, for example,
compared to a neuron that is contacted with equivalent amounts of
the an IL4 that is present in the polypeptide construct, a cytokine
that is present in the compound (e.g., IL10, IL13, IL27, IL33,
TGF.beta.1, TGF.beta.2), or a combination of the IL4 and the
cytokine. A magnitude of a neuronal response can be an amount of a
response quantified upon exposure to a pre-determined stimulus. A
neuronal response can be, for example, calcium flux above a certain
level, alteration of depolarization frequency above or below a
certain threshold, altered expression, production, and/or release
of a neurotransmitter or neuropeptide above or below a certain
level, or altered expression/production of a gene to above or below
a certain level. A stimulus can be, for, example, capsaicin, a
pro-inflammatory mediator, an anti-inflammatory mediator, a drug, a
toxin, a toxicant, a chemical stimulus, a mechanical stimulus, a
thermal stimulus, or neuronal damage. In some embodiments, a
stimulus is not a pro-inflammatory mediator, but the magnitude of
the neuronal response is modulated in the presence of a
pro-inflammatory mediator. Modulation of a magnitude of a neuronal
response can be as determined by measuring a neuronal response as
disclosed herein (e.g., disclosed above) to a stimulus as disclosed
herein (e.g., disclosed above). The neuron can be contacted with an
amount of the compound that is sufficient to modulate the magnitude
of the neuronal response. The neuron can be contacted with a
concentration of the compound that is sufficient to modulate the
magnitude of the neuronal response. A concentration of the compound
that is sufficient to modulate the magnitude of the neuronal
response can be, for example, at least about 1 pM, at least about
10 pM, at least about 20 pM, at least about 30 pM, at least about
40 pM, at least about 50 pM, at least about 60 pM, at least about
70 pM, at least about 80 pM, at least about 90 pM, at least about
100 pM, at least about 200 pM, at least about 300 pM, at least
about 400 pM, at least about 500 pM, at least about 600 pM, at
least about 700 pM, at least about 800 pM, at least about 900 pM,
at least about 1 nM, at least about 2 nM, at least about 3 nM, at
least about 4 nM, at least about 5 nM, at least about 6 nM, at
least about 7 nM, at least about 8 nM, at least about 9 nM, at
least about 10 nM, at least about 20 nM, at least about 30 nM, at
least about 40 nM, at least about 50 nM, at least about 60 nM, at
least about 70 nM, at least about 80 nM, at least about 90 nM, at
least about 100 nM, at least about 200 nM, at least about 300 nM,
at least about 400 nM, at least about 500 nM, at least about 600
nM, at least about 700 nM, at least about 800 nM, at least about
900 nM, at least about 1 .mu.M, at least about 10 .mu.M, or at
least about 100 .mu.M. The neuron can be contacted with the
compound for a period of time that is sufficient to modulate the
magnitude of the neuronal response. A period of time sufficient to
modulate the magnitude of the neuronal response can be, for
example, at least about 1 second, at least about 10 seconds, at
least about 30 seconds, at least about 1 minute, at least about 5
minutes, at least about 10 minutes, at least about 30 minutes, at
least about 1 hour, at least about 2 hours, at least about 3 hours,
at least about 5 hours, at least about 6 hours, at least about 10
hours, at least about 12 hours, at least about 24 hours, at least
about 2 days, at least about 3 days, at least about 5 days, or at
least about a week.
[0274] Contacting a neuron with a compound of the disclosure can
modulate a duration of a neuronal response to a stimulus, for
example, compared to a neuron that is contacted with equivalent
amounts of the an IL4 that is present in the polypeptide construct,
a cytokine that is present in the compound (e.g., IL10, IL13, IL27,
IL33, TGF.beta.1, TGF.beta.2), or a combination of the IL4 and the
cytokine. A duration of a neuronal response can be a duration of a
response above a threshold upon exposure to a pre-determined
stimulus. A neuronal response can be, for example, calcium flux,
alteration of depolarization frequency above or below a certain
threshold, an altered depolarization threshold, an altered
frequency of action potential firing, altered expression,
production, and/or release of a neurotransmitter or neuropeptide
above or below a certain level, or altered expression/production of
a gene to above or below a certain level. A stimulus can be, for,
example, capsaicin, a pro-inflammatory mediator, an
anti-inflammatory mediator, a drug, a toxin, a toxicant, a chemical
stimulus, a mechanical stimulus, a thermal stimulus, or neuronal
damage. In some embodiments, a stimulus is not a pro-inflammatory
mediator, but the duration of the neuronal response is modulated in
the presence of a pro-inflammatory mediator. Modulation of a
duration of a neuronal response can be as determined by measuring a
duration of a neuronal response as disclosed herein (e.g.,
disclosed above) to a stimulus as disclosed herein (e.g., disclosed
above). The neuron can be contacted with an amount of the compound
that is sufficient to modulate the duration of the neuronal
response. The neuron can be contacted with a concentration of the
compound that is sufficient to modulate the duration of the
neuronal response. A concentration of the compound that is
sufficient to modulate the duration of the neuronal response can
be, for example, at least about 1 pM, at least about 10 pM, at
least about 20 pM, at least about 30 pM, at least about 40 pM, at
least about 50 pM, at least about 60 pM, at least about 70 pM, at
least about 80 pM, at least about 90 pM, at least about 100 pM, at
least about 200 pM, at least about 300 pM, at least about 400 pM,
at least about 500 pM, at least about 600 pM, at least about 700
pM, at least about 800 pM, at least about 900 pM, at least about 1
nM, at least about 2 nM, at least about 3 nM, at least about 4 nM,
at least about 5 nM, at least about 6 nM, at least about 7 nM, at
least about 8 nM, at least about 9 nM, at least about 10 nM, at
least about 20 nM, at least about 30 nM, at least about 40 nM, at
least about 50 nM, at least about 60 nM, at least about 70 nM, at
least about 80 nM, at least about 90 nM, at least about 100 nM, at
least about 200 nM, at least about 300 nM, at least about 400 nM,
at least about 500 nM, at least about 600 nM, at least about 700
nM, at least about 800 nM, at least about 900 nM, at least about 1
.mu.M, at least about 10 .mu.M, or at least about 100 .mu.M. The
neuron can be contacted with the compound for a period of time that
is sufficient to modulate the duration of the neuronal response. A
period of time sufficient to modulate the duration of the neuronal
response can be, for example, at least about 1 second, at least
about 10 seconds, at least about 30 seconds, at least about 1
minute, at least about 5 minutes, at least about 10 minutes, at
least about 30 minutes, at least about 1 hour, at least about 2
hours, at least about 3 hours, at least about 5 hours, at least
about 6 hours, at least about 10 hours, at least about 12 hours, at
least about 24 hours, at least about 2 days, at least about 3 days,
at least about 5 days, or at least about a week.
[0275] Contacting a neuron with a compound (e.g., polypeptide
construct, fusion protein) of the disclosure can modulate an
ectopic neuronal activity, for example, compared to a neuron that
is contacted with equivalent amounts of the an IL4 that is present
in the polypeptide construct, a cytokine that is present in the
compound (e.g., IL10, IL13, IL27, IL33, TGF.beta.1, TGF.beta.2), or
a combination of the IL4 and the cytokine. An ectopic neuronal
activity can comprise, for example, spontaneous afferent neuronal
activity, spontaneous sensory afferent action potential activity,
spontaneous neuronal depolarization, or a combination thereof.
Modulating ectopic neuronal activity can be as determined by, for
example, measurement of spontaneous ectopic discharge recorded from
peripheral nerve bundles (e.g., of the sciatic nerve in a rat
neuropathic pain model), measurement of spontaneous wind=up and
after-discharge activity of wide dynamic range dorsal horn neurons
recorded from the spinal cord (e.g., in a rat neuropathic pain
model), recording ectopic discharge from modified preparations of
neuropathic animals, or a multi-well multielectrode array assay
using, e.g., using neurons from dorsal root ganglia. The neuron can
be contacted with an amount of the compound that is sufficient to
modulate the ectopic neuronal activity. The neuron can be contacted
with a concentration of the compound that is sufficient to modulate
the ectopic neuronal activity. A concentration of the compound that
is sufficient to modulate the ectopic neuronal activity can be, for
example, at least about 1 pM, at least about 10 pM, at least about
20 pM, at least about 30 pM, at least about 40 pM, at least about
50 pM, at least about 60 pM, at least about 70 pM, at least about
80 pM, at least about 90 pM, at least about 100 pM, at least about
200 pM, at least about 300 pM, at least about 400 pM, at least
about 500 pM, at least about 600 pM, at least about 700 pM, at
least about 800 pM, at least about 900 pM, at least about 1 nM, at
least about 2 nM, at least about 3 nM, at least about 4 nM, at
least about 5 nM, at least about 6 nM, at least about 7 nM, at
least about 8 nM, at least about 9 nM, at least about 10 nM, at
least about 20 nM, at least about 30 nM, at least about 40 nM, at
least about 50 nM, at least about 60 nM, at least about 70 nM, at
least about 80 nM, at least about 90 nM, at least about 100 nM, at
least about 200 nM, at least about 300 nM, at least about 400 nM,
at least about 500 nM, at least about 600 nM, at least about 700
nM, at least about 800 nM, at least about 900 nM, at least about 1
.mu.M, at least about 10 .mu.M, or at least about 100 .mu.M. The
neuron can be contacted with the compound for a period of time that
is sufficient to modulate the ectopic neuronal activity. A period
of time sufficient to modulate the ectopic neuronal activity can
be, for example, at least about 1 second, at least about 10
seconds, at least about 30 seconds, at least about 1 minute, at
least about 5 minutes, at least about 10 minutes, at least about 30
minutes, at least about 1 hour, at least about 2 hours, at least
about 3 hours, at least about 5 hours, at least about 6 hours, at
least about 10 hours, at least about 12 hours, at least about 24
hours, at least about 2 days, at least about 3 days, at least about
5 days, or at least about a week.
[0276] In some embodiments, decreasing a sensitivity threshold of a
neuronal response to a stimulus, a duration of a neuronal response
to a stimulus, a magnitude of a neuronal response to a stimulus, or
ectopic neuronal activity can comprise a decrease of at least about
at least about 5%, at least about 10%, at least about 15%, at least
about 20%, at least about 30%, at least about 40%, at least about
50%, at least about 60%, at least about 70%, at least about 80%
90%, at least about or 100%. In some embodiments, decreasing a
sensitivity threshold of a neuronal response to a stimulus, a
duration of a neuronal response to a stimulus, a magnitude of a
neuronal response to a stimulus, or ectopic neuronal activity can
comprise a decrease of at least about at least about 1.5-fold, at
least about 2-fold, at least about 3-fold, at least about 4-fold,
at least about 5-fold, at least about 6-fold, at least about
7-fold, at least about 8-fold, at least about 9-fold, at least
about 10-fold, at least about 15-fold, at least about 20-fold, at
least about 25-fold, at least about 30-fold, at least about
40-fold, at least about 50-fold, at least about 100-fold, at least
about 200-fold, at least about 300-fold, at least about 400-fold,
at least about 500-fold, at least about 600-fold, at least about
700-fold, at least about 800-fold, at least about 900-fold, at
least about 1000-fold, or at least about 10,000-fold. In some
embodiments, decreasing can comprise decreasing from a detectable
level to an undetectable level.
[0277] In some embodiments, a nervous system cell can be contacted
with compound (e.g., polypeptide construct, fusion protein) of the
disclosure at a concentration that is at most about 1M, at most
about 10 nM, at most about 100 nM, at most about 1 .mu.M, at most
about 10 .mu.M, at most about 100 .mu.M, or at most about 1 mM.
[0278] A stimulus of the disclosure can comprise or can be a
pro-inflammatory mediator. In some embodiments, a stimulus is not a
pro-inflammatory mediator, but the sensitivity threshold is
modulated in the presence of a pro-inflammatory mediator.
Non-limiting examples of pro-inflammatory mediators include a
pro-inflammatory cytokine, a pro-inflammatory chemokine, a
vasoactive amine, a prostaglandin, a leukotriene, a thromboxane, an
oxygen- and/or nitrogen-derived free radical, histamine, a Th1
cytokine, a Th2 cytokine, a Th17 cytokine, IL-1.beta., APRIL,
IFN-.alpha., IFN-.beta., IFN-.gamma., IL-1a, IL-1.beta., IL-2,
IL-6, IL-8, IL-9, IL-12, IL-23, LIGHT, TNF-.alpha., and TNF-.beta..
In some embodiments, stimulus does not contain a pathogen
associated molecular pattern. In some embodiments, a stimulus does
not contain LPS.
[0279] In some embodiments, a stimulus comprises a pathogen
associated molecular pattern (PAMP) or a damage associated
molecular pattern (DAMP). In some embodiments, a signaling pathway,
activity of a kinase, kinomic profile, level of phosphorylation of
a substrate, expression of a gene, sensitivity of a neuronal
response to a stimulus, magnitude of a neuronal response to a
stimulus, duration of a neuronal response to a stimulus, ectopic
neuronal activity, or a combination thereof is modulated in a
presence of a PAMP. In some embodiments, a signaling pathway,
activity of a kinase, kinomic profile, level of phosphorylation of
a substrate, expression of a gene, sensitivity of a neuronal
response to a stimulus, magnitude of a neuronal response to a
stimulus, duration of a neuronal response to a stimulus, ectopic
neuronal activity, or a combination thereof is modulated in a
presence of a DAMP. Non-limiting examples of DAMPs include
Biglycan, Decorin, Versican, LMW hyaluronan, Heparan sulfate,
Fibronectin (EDA domain), Fibrinogen, Tenascin C, Uric acid, S100
proteins, Heat shock proteins, ATP, F-actin, Cyclophilin A,
A.beta., Histones, HMGB1, HMGN1, IL-1a, IL33, SAP130, DNA, RNA,
mtDNA, TFAM, Formyl peptide, mROS, Calreticulin, Defensins,
Cathelicidin (LL37), EDN, Granulysin, Syndecans, and Glypicans.
[0280] Therapeutic Methods:
[0281] Compounds (e.g., polypeptide constructs, fusion proteins)
disclosed herein can be useful for treating a condition (e.g.,
disease) in a subject, for example, pain, neuropathy, inflammation,
and other conditions.
[0282] Pain can include pain that is mediated by the central
nervous system, the peripheral nervous system, or a combination
thereof. Non-limiting types of pain include, nociceptive pain,
peripheral and central neuropathic pain, and mixed types of pain.
Neuropathy can contribute to pain. In some embodiments, a compound
(e.g., polypeptide construct, fusion protein) of the disclosure can
be used for treating a neuropathy. A neuropathy can be associated
with pain, numbness, weakness, or a combination thereof.
[0283] Particularly, it was found that the compound or combination
of the present disclosure has a cartilage-protective activity.
Therefore, the compound or combination may be used for prevention
and treatment of cartilage breakdown, particularly in OA. The
compound or combination may be particularly useful for prevention
or treatment of OA (prevention or treatment of cartilage
degradation) either or not associated with chronic pain.
[0284] Moreover, it was also found that the compound or combination
of the present disclosure has a neuro-protective activity.
Therefore, the compound or combination may be used for prevention
and treatment of neuro-degenerative disorders. The compound or
combination may be particularly useful for treatment of Alzheimer's
disease, Parkinson's disease, Huntington's disease, amyotrophic
lateral sclerosis, or multiple sclerosis.
[0285] The compound, combination, or nucleic acid encoding the same
according to the present disclosure can be used in therapeutic
treatment.
[0286] In one embodiment, the compound, combination, or nucleic
acid encoding the same is for, or limited to, topical
administration or administration to a local compartment of a human
or animal body, and/or wherein the compound or combination is for
treatment of a (local) part of the human or animal body, for
example a knee, hip, joint, or spine. Said local compartment
typically has a barrier function that prevents that upon
administration more than 10, 20, 30, 40, 50, 60, 70, 80, 90, 95 wt.
% of the compound or combination per day is absorbed into systemic
circulation.
[0287] Further, the compound or combination according to the
present disclosure is particularly suitable for intrathecal or
intraarticular administration, for example by injection into an
intrathecal space or intraarticular space.
[0288] In a further aspect, the present invention pertains to the
compound or combination, or a pharmaceutical composition comprising
said according to the present disclosure for use in prevention or
treatment of (chronic) pain, a condition characterized by local or
systemic inflammation, immune activation, neuro-inflammation and/or
neurodegeneration. In an embodiment, said condition characterized
by local or systemic inflammation, and/or immune activation is
selected from the group consisting of: chronic neuropathic,
nociceptive, or mixed neuropathic-nociceptive pain, sepsis, adult
respiratory distress syndrome, allo- and xenotransplantation,
dermatitis, inflammatory bowel disease, sarcoidosis, allergies,
psoriasis, ankylosing spondylarthritis, osteoarthritis, autoimmune
diseases such as systemic lupus erythematosus and rheumatoid
arthritis, glomerulonephritis, immune complex-induced and other
forms of vasculitis, Sjogren's disease, gout, burn injuries,
multiple trauma, stroke, myocardial infarction, atherosclerosis,
diabetes mellitus, extracorporeal dialysis and blood oxygenation,
ischemia-reperfusion injuries, and toxicity induced by the in vivo
administration of cytokines or other therapeutic monoclonal
antibodies.
[0289] In a further aspect, the present invention pertains to the
compound, combination, or nucleic acid encoding the same according
to the present disclosure (e.g. a fusion protein of IL4 and IL10 or
IL13 or a bispecific antibody against IL4R and IL10R or IL13R), in
a pharmaceutical composition for use in treatment a condition
characterized by neuroinflammation or neurodegeneration such as
Alzheimer's disease, Parkinson's disease, Huntington's disease,
amyotrophic lateral sclerosis, and multiple sclerosis.
[0290] In an embodiment, said condition is characterized by pain
and may be selected from pathological pain, visceral or
non-visceral inflammatory pain, visceral or non-visceral
nociceptive pain, peripheral and/or central neuropathic pain or
mixed nociceptive and neuropathic pain.
[0291] In some embodiments, a compound (e.g., polypeptide
construct, fusion protein) of the disclosure or nucleic acid
encoding the same can be used for treating Alpers' Disease,
Arachnoiditis, Arthrofibrosis, Ataxic Cerebral Palsy, Autoimmune
Atrophic Gastritis, Amyloidosis, hATTR Amyloidosis, Avascular
Necrosis, Back Pain, Batten Disease, Behcet's Disease (Syndrome),
Breakthrough Pain, Burning Mouth Syndrome, Bursitis, Central
Autosomal Dominant Arteriopathy with Subcortical Infarcts and
Leukoencephalopathy (Cadasil), Cerebral ischemia,
Cerebro-Oculo-Facio-Skeletal Syndrome (COFS), Carpal Tunnel, Cauda
Equina Syndrome, Central Pain Syndrome, Cerebral Palsy,
Cerebrospinal Fluid (CSF) Leaks, Cervical Stenosis,
Charcot-Marie-Tooth (CMT) Disease, Chronic Functional Abdominal
Pain (CFAP), Chronic Pancreatitis, Collapsed Lung (Pneumothorax),
Corticobasal Degeneration, Compression injury, Corneal Neuropathic
Pain, Crush syndrome, Degenerative Disc Disease, Dermatomyositis,
Dementia, Dystonia, Ehlers-Danlos Syndrome (EDS), Endometriosis,
Eosinophilia-Myalgia Syndrome (EMS), Erythromelalgia, Failed Back
Surgery Syndrome (FBSS), Fibromyalgia, Friedreich's Ataxia,
Frontotemporal dementia, Glossopharyngeal neuralgia, Growing Pains,
Herniated disc, Hydrocephalus, Intercostal Neuraligia, Interstitial
Cystitis, Juvenile Dermatositis, Knee Injury, Leg Pain, Lewy Body
Dementia, Loin Pain-Haematuria Syndrome, Lyme Disease, Meralgia
Paresthetica, Mitochondrial Disorders, Mixed dementia, Motor
neurone diseases (MND), Monomelic Amyotrophy, Multiple system
atrophy (MSA), Myositis, Neck Pain, Occipital Neuralgia,
Osteoporosis, Rhabdomyolysis, Paget's Disease, Parsonage Turner
Syndrome, Pelvic Pain, Peripheral Neuropathy, Phantom Limb Pain,
Pinched Nerve, Plantar Fasciitis, Polymyalgia Rhuematica,
Polymyositis, Post Herniorraphy Pain Syndrome, Post Mastectomy Pain
Syndrome, Post Stroke Pain, Post Thorocotomy Pain Syndrome,
Post-Polio Syndrome, Primary Lateral Sclerosis, Psoriatic
Arthritis, Pudendal Neuralgia, Radiculopathy, Restless Leg
Syndrome, Rheumatoid Arthritis (RA), Sacroiliac Joint Dysfunction,
Sarcoidosis, Scheuemann's Kyphosis Disease, Sciatica,
Spinocerebellar ataxia (SCA), Spinal muscular atrophy (SMA), Herpes
Zoster Shingles, Spasmodic Torticollis, Sphincter of Oddi
Dysfunction, Spinal Cord Injury, Spinal Stenosis, Syringomyelia,
Tarlov Cysts, Tethered Cord Syndrome, Thoracic Outlet Syndrome
(TOS), TMJ disorders, Transverse Myelitis, Traumatic Brain
Injuries, Vascular Pain, Vulvodynia, Whiplash, or a combination
thereof.
[0292] In some embodiments, a compound (e.g., polypeptide
construct, fusion protein) of the disclosure or nucleic acid
encoding the same can be used to treat an autoimmune disease.
Non-limiting examples of autoimmune diseases include Acute
disseminated encephalomyelitis, Acute motor axonal neuropathy,
Addison's disease, Adiposis dolorosa, Adult-onset Still's disease,
Alopecia areata, Ankylosing Spondylitis, Anti-Glomerular Basement
Membrane nephritis, Anti-neutrophil cytoplasmic antibody-associated
vasculitis, Anti-N-Methyl-D-Aspartate Receptor Encephalitis,
Antiphospholipid syndrome, Antisynthetase syndrome, Aplastic
anemia, Autoimmune Angioedema, Autoimmune Encephalitis, Autoimmune
enteropathy, Autoimmune hemolytic anemia, Autoimmune hepatitis,
Autoimmune inner ear disease, Autoimmune lymphoproliferative
syndrome, Autoimmune neutropenia, Autoimmune oophoritis, Autoimmune
orchitis, Autoimmune pancreatitis, Autoimmune polyendocrine
syndrome, Autoimmune polyendocrine syndrome type 2, Autoimmune
polyendocrine syndrome type 3, Autoimmune progesterone dermatitis,
Autoimmune retinopathy, Autoimmune thrombocytopenic purpura,
Autoimmune thyroiditis, Autoimmune urticaria, Autoimmune uveitis,
Balo concentric sclerosis, Behcet's disease, Bickerstaffs
encephalitis, Bullous pemphigoid, Celiac disease, Chronic fatigue
syndrome, Chronic inflammatory demyelinating polyneuropathy,
Churg-Strauss syndrome, Cicatricial pemphigoid, Cogan syndrome,
Cold agglutinin disease, Complex regional pain syndrome, CREST
syndrome, Crohn's disease, Dermatitis herpetiformis,
Dermatomyositis, Diabetes mellitus type 1, Discoid lupus
erythematosus, Endometriosis, Enthesitis, Enthesitis-related
arthritis, Eosinophilic esophagitis, Eosinophilic fasciitis,
Epidermolysis bullosa acquisita, Erythema nodosum, Essential mixed
cryoglobulinemia, Evans syndrome, Felty syndrome, Fibromyalgia,
Gastritis, Gestational pemphigoid, Giant cell arteritis,
Goodpasture syndrome, Graves' disease, Graves ophthalmopathy,
Guillain-Barre syndrome, Hashimoto's Encephalopathy, Hashimoto
Thyroiditis, Henoch-Schonlein purpura, Hidradenitis suppurativa,
Idiopathic dilated cardiomyopathy, Idiopathic inflammatory
demyelinating diseases, IgA nephropathy, IgG4-related systemic
disease, Inclusion body myositis, Inflamatory Bowel Disease (IBD),
Intermediate uveitis, Interstitial cystitis, Juvenile Arthritis,
Kawasaki's disease, Lambert-Eaton myasthenic syndrome,
Leukocytoclastic vasculitis, Lichen planus, Lichen sclerosus,
Ligneous conjunctivitis, Linear IgA disease, Lupus nephritis, Lupus
vasculitis, Lyme disease, Meniere's disease, Microscopic colitis,
Microscopic polyangiitis, Mixed connective tissue disease, Mooren's
ulcer, Morphea, Mucha-Habermann disease, Multiple sclerosis,
Myasthenia gravis, Myocarditis, Myositis, Neuromyelitis optica,
Neuromyotonia, Opsoclonus myoclonus syndrome, Optic neuritis, Ord's
thyroiditis, Palindromic rheumatism, Paraneoplastic cerebellar
degeneration, Parry Romberg syndrome, Parsonage-Turner syndrome,
Pediatric Autoimmune Neuropsychiatric Disorder Associated with
Streptococcus, Pemphigus vulgaris, Pernicious anemia, Pityriasis
lichenoides et varioliformis acuta, POEMS syndrome, Polyarteritis
nodosa, Polymyalgia rheumatica, Polymyositis, Postmyocardial
infarction syndrome, Postpericardiotomy syndrome, Primary biliary
cirrhosis, Primary immunodeficiency, Primary sclerosing
cholangitis, Progressive inflammatory neuropathy, Psoriasis,
Psoriatic arthritis, Pure red cell aplasia, Pyoderma gangrenosum,
Raynaud's phenomenon, Reactive arthritis, Relapsing polychondritis,
Restless leg syndrome, Retroperitoneal fibrosis, Rheumatic fever,
Rheumatoid arthritis, Rheumatoid vasculitis, Sarcoidosis,
Schnitzler syndrome, Scleroderma, Sjogren's syndrome, Stiff person
syndrome, Subacute bacterial endocarditis, Susac's syndrome,
Sydenham chorea, Sympathetic ophthalmia, Systemic Lupus
Erythematosus, Systemic scleroderma, Thrombocytopenia, Tolosa-Hunt
syndrome, Transverse myelitis, Ulcerative colitis, Undifferentiated
connective tissue disease, Urticaria, Urticarial vasculitis,
Vasculitis, and Vitiligo.
[0293] In some embodiments, a compound (e.g., polypeptide
construct, fusion protein) of the disclosure or nucleic acid
encoding the same can be used for treating neuropathy. Non-limiting
examples of neuropathy include post-traumatic peripheral
neuropathy, post-operative peripheral neuropathy, diabetic
peripheral neuropathy, inflammatory peripheral neuropathy,
HIV-associated neuropathy, chemotherapy-induced neuropathy,
polyneuropathy, mononeuropathy, multiple mononeuropathy, cranial
neuropathy, predominantly motor neuropathy, predominantly sensory
neuropathy, sensory-motor neuropathy, autonomic neuropathy,
idiopathic neuropathy, post-herpetic neuralgia, trigeminal
neuralgia, glossopharyngeal neuralgia, occipital neuralgia, pudenal
neuralgia, atypical trigeminal neuralgia, sciatica, brachial
plexopathy, or intercostal neuralgia. A neuropathy can be
associated with, for example, pain, numbness, weakness, burning,
atrophy, tingling, twitching, or a combination thereof.
[0294] In some embodiments, a compound (e.g., polypeptide
construct, fusion protein) of the disclosure can be used to treat a
condition associated with cancer or chemotherapy, for example,
chemotherapy-induced neuropathy, chemotherapy-associated pain,
chemo brain, cancer-related cognitive impairment, cancer-related
cognitive dysfunction. In some embodiments, the condition is
associated with a chemotherapy that is being used to treat acute
leukemia, astrocytomas, biliary cancer (cholangiocarcinoma), bone
cancer, breast cancer, brain stem glioma, bronchioloalveolar cell
lung cancer, cancer of the adrenal gland, cancer of the anal
region, cancer of the bladder, cancer of the endocrine system,
cancer of the esophagus, cancer of the head or neck, cancer of the
kidney, cancer of the parathyroid gland, cancer of the penis,
cancer of the pleural/peritoneal membranes, cancer of the salivary
gland, cancer of the small intestine, cancer of the thyroid gland,
cancer of the ureter, cancer of the urethra, carcinoma of the
cervix, carcinoma of the endometrium, carcinoma of the fallopian
tubes, carcinoma of the renal pelvis, carcinoma of the vagina,
carcinoma of the vulva, cervical cancer, chronic leukemia, colon
cancer, colorectal cancer, cutaneous melanoma, ependymoma,
epidermoid tumors, Ewings sarcoma, gastric cancer, glioblastoma,
glioblastoma multiforme, glioma, hematologic malignancies,
hepatocellular (liver) carcinoma, hepatoma, Hodgkin's Disease,
intraocular melanoma, Kaposi sarcoma, lung cancer, lymphomas,
medulloblastoma, melanoma, meningioma, mesothelioma, multiple
myeloma, muscle cancer, neoplasms of the central nervous system
(CNS), neuronal cancer, small cell lung cancer, non-small cell lung
cancer, osteosarcoma, ovarian cancer, pancreatic cancer, pediatric
malignancies, pituitary adenoma, prostate cancer, rectal cancer,
renal cell carcinoma, sarcoma of soft tissue, schwanoma, skin
cancer, spinal axis tumors, squamous cell carcinomas, stomach
cancer, synovial sarcoma, testicular cancer, uterine cancer, or
tumors and their metastases, including refractory versions of any
of the above cancers, or any combination thereof.
[0295] In some embodiments, a compound (e.g., fusion protein,
polypeptide construct) is used to treat a condition, wherein the
condition is not a cancer.
[0296] The present disclosure further provides a gene therapy
vector containing nucleotide sequence(s) coding for the compound or
combination according to the disclosure, for use in the prevention
or treatment of a condition characterized by chronic pain,
neuro-inflammation and/or neuro-degeneration.
[0297] Preferably said condition is further characterized by
pathological pain, visceral or non-visceral inflammatory pain,
visceral or non-visceral nociceptive pain, peripheral or central
neuropathic pain, or mixed nociceptive-neuropathic pain,
neuro-inflammation, and/or neuro-degeneration.
[0298] Alternatively, said condition is selected from the group
consisting of post-operative orthopedic surgery pain,
musculoskeletal pain, irritable bowel syndrome, inflammatory bowel
disease, rheumatoid arthritis, ankylosing spondylitis,
post-herpetic neuralgia, trigeminal neuralgia, post-traumatic or
post-operative peripheral neuropathy, diabetic peripheral
neuropathy, inflammatory peripheral neuropathy, HIV-associated
neuropathy, painful peripheral neuropathy, nerve entrapment
syndrome, chemotherapy-associated pain, chemotherapy-induced
allodynia, complex regional pain syndrome, post-spinal injury pain,
post-stroke pain, multiple sclerosis, chronic widespread pain, low
back pain, osteoarthritis, cancer pain, chronic visceral pain,
fibromyalgia, polymyalgia rheumatica, Alpers' Disease,
Arachnoiditis, Arthrofibrosis, Ataxic Cerebral Palsy, Autoimmune
Atrophic Gastritis, Amyloidosis, hATTR Amyloidosis, Avascular
Necrosis, Back Pain, Batten Disease, Behcet's Disease (Syndrome),
Breakthrough Pain, Burning Mouth Syndrome, Bursitis, Central
Autosomal Dominant Arteriopathy with Subcortical Infarcts and
Leukoencephalopathy (Cadasil), Cerebral ischemia,
Cerebro-Oculo-Facio-Skeletal Syndrome (COFS), Carpal Tunnel, Cauda
Equina Syndrome, Central Pain Syndrome, Cerebral Palsy,
Cerebrospinal Fluid (CSF) Leaks, Cervical Stenosis,
Charcot-Marie-Tooth (CMT) Disease, Chronic Functional Abdominal
Pain (CFAP), Chronic Pancreatitis, Collapsed Lung (Pneumothorax),
Corticobasal Degeneration, Compression injury, Corneal Neuropathic
Pain, Crush syndrome, Degenerative Disc Disease, Dermatomyositis,
Dementia, Dystonia, Ehlers-Danlos Syndrome (EDS), Endometriosis,
Eosinophilia-Myalgia Syndrome (EMS), Erythromelalgia, Failed Back
Surgery Syndrome (FBSS), Fibromyalgia, Friedreich's Ataxia,
Frontotemporal dementia, Glossopharyngeal neuralgia, Growing Pains,
Herniated disc, Hydrocephalus, Intercostal Neuraligia, Interstitial
Cystitis, Juvenile Dermatositis, Knee Injury, Leg Pain, Lewy Body
Dementia, Loin Pain-Haematuria Syndrome, Lyme Disease, Meralgia
Paresthetica, Mitochondrial Disorders, Mixed dementia, Motor
neurone diseases (MND), Monomelic Amyotrophy, Multiple system
atrophy (MSA), Myositis, Neck Pain, Occipital Neuralgia,
Osteoporosis, Rhabdomyolysis, Paget's Disease, Parsonage Turner
Syndrome, Pelvic Pain, Peripheral Neuropathy, Phantom Limb Pain,
Pinched Nerve, Plantar Fasciitis, Polymyalgia Rhuematica,
Polymyositis, Post Herniorraphy Pain Syndrome, Post Mastectomy Pain
Syndrome, Post Stroke Pain, Post Thorocotomy Pain Syndrome,
Post-Polio Syndrome, Primary Lateral Sclerosis, Psoriatic
Arthritis, Pudendal Neuralgia, Radiculopathy, Restless Leg
Syndrome, Rheumatoid Arthritis (RA), Sacroiliac Joint Dysfunction,
Sarcoidosis, Scheuemann's Kyphosis Disease, Sciatica,
Spinocerebellar ataxia (SCA), Spinal muscular atrophy (SMA), Herpes
Zoster Shingles, Spasmodic Torticollis, Sphincter of Oddi
Dysfunction, Spinal Cord Injury, Spinal Stenosis, Syringomyelia,
Tarlov Cysts, Tethered Cord Syndrome, Thoracic Outlet Syndrome
(TOS), TMJ disorders, Transverse Myelitis, Traumatic Brain
Injuries, Vascular Pain, Vulvodynia, Whiplash, and any other
condition disclosed herein.
[0299] According to one embodiment, the compound or combination
taught herein can be used for inhibiting production and release of
cytokines and other inflammatory mediators by cells, such as
macrophages, monocytes, T-lymphocytes, glial cells and other cells.
As a result, the compound or combination of the present disclosure
can be used for the preparation of a medicament for attenuating
inflammatory reactions by inhibiting the release of cytokines and
other inflammatory mediators by these cells in vivo. The compound
or combination of the present disclosure can be used as stand-alone
drug or in combination with other drugs. In some embodiments, a
compound (e.g., polypeptide construct, fusion protein) of the
disclosure is administered in combination with an analgesic, for
example, acetaminophen/paracetamol, a non-steroidal
anti-inflammatory drug (NSAID), or an opioid. In some embodiments,
a compound (e.g., polypeptide construct, fusion protein) of the
disclosure can be used in combination with NSAIDs, such as aspirin,
ibuprofen, naproxen, celecoxib, ketorolac, or diclofenac. In some
embodiments, a compound (e.g., polypeptide construct, fusion
protein) of the disclosure can be used in combination with specific
COX-2 inhibitors, such as celecoxib (Celebrex.RTM.), rofecoxib, or
etoricoxib. In some embodiments, a compound (e.g., polypeptide
construct, fusion protein) of the disclosure can be used in
combination with corticosteroids, such as dexamethasone or
glucosteroids (e.g., hydrocortisone and prednisone). In some
embodiments, a compound (e.g., polypeptide construct, fusion
protein) of the disclosure is administered in combination with an
antagonist of a pro-inflammatory cytokine (e.g., and antibody
derivative, or other molecule thereof that binds to TNF-.alpha.
(e.g., adalimumab, etanercept), IL-17 (e.g., secukinumab), IL-23
(e.g., guselkumab, ildrakizumab), or IL-12 and IL-23 (e.g.,
ustekinumab). In some embodiments, a fusion protein is administered
in combination with hyaluronic acid. Multiple therapeutic agents
can be administered in any order or simultaneously. In some
embodiments, a compound of the invention is administered in
combination with, before, or after another drug.
[0300] Treatment (prophylactic or therapeutic) may comprise of
administering the compound or combination of the present disclosure
systemically, intraarticularly, intrathecally, epidurally, or
spinally, by injection or by a drug delivery system suitable for
local administration. In some embodiments, a pharmaceutical
composition of the disclosure can be administered parenterally, for
example, by intravenous, intramuscular, intraarterial, intrathecal,
intracapsular, intraorbital, intracardiac, intradermal,
intraperitoneal, transtracheal, subcutaneous, subcuticular,
intraarticular, subcapsular, subarachnoid, intraspinal, epidural,
intrasternal, intracerebral, intraocular, intralesional,
intracerebroventricular, intracisternal, or intraparenchymal, e.g.,
injection and infusion. However, other administration routes as set
forth above with respect to pharmaceutical compositions comprising
the compound or combination recited above may also be employed. The
dose and administration regimen may depend on the pharmacodynamic
effect aimed at. Typically, the amount of the compound or
combination of first and second binding moieties given will be in
the range of 0.5 .mu.g to 1 mg per kg of body weight. The dosage
can be determined or adjusted by measuring the amount of
circulating or local level of the compound or combination upon
administration in a biological compartment or space.
[0301] Typically, the compound or combination of the present
disclosure may be formulated in such vehicles at a concentration of
from about 50 .mu.g to about 100 mg per ml.
[0302] In an embodiment, the compound or combination according to
the present disclosure is biologically active and/or able to signal
cells to downregulate the production of at least one inflammatory
cytokine or mediator such as IL1.beta., IL6, IL8, TNF.alpha..
Preferably, at least TNF.alpha., IL6, and IL8 are
downregulated.
[0303] In particular, the compound or combination according to the
present disclosure can be biologically active and/or able to signal
neurons and other cells in the dorsal root ganglion and posterior
horn of the spinal cord to activate unique kinase profiles and
express unique sets of genes leading to resistance to the damaging
effects of e.g. chemotherapeutic drugs.
[0304] In another embodiment, the compound or combination according
to the present disclosure is biologically active and/or able to
signal neurons to normalize hypersensitivity induced by
inflammatory mediators such as proinflammatory cytokines or
prostaglandins.
[0305] In another embodiment, the compound or combination according
to the present disclosure is biologically active and/or able to
de-activate glial cells in the dorsal root ganglion, the spinal
cord and/or the central nervous system.
[0306] Administration of the compound (e.g., polypeptide construct,
fusion protein) can continue for as long as clinically necessary.
In some embodiments, a compound of the disclosure can be
administered for more than 1 day, more than 1 week, more than 1
month, more than 2 months, more than 3 months, more than 4 months,
more than 5 months, more than 6 months, more than 7 months, more
than 8 months, more than 9 months, more than 10 months, more than
11 months, more than 12 months, more than 13 months, more than 14
months, more than 15 months, more than 16 months, more than 17
months, more than 18 months, more than 19 months, more than 20
months, more than 21 months, more than 22 months, more than 23
months, or more than 24 months. In some embodiments, a compound of
the disclosure is administered for less than 1 week, less than 1
month, less than 2 months, less than 3 months, less than 4 months,
less than 5 months, less than 6 months, less than 7 months, less
than 8 months, less than 9 months, less than 10 months, less than
11 months, less than 12 months, less than 13 months, less than 14
months, less than 15 months, less than 16 months, less than 17
months, less than 18 months, less than 19 months, less than 20
months, less than 21 months, less than 22 months, less than 23
months, or less than 24 months.
[0307] In some embodiments, a compound (e.g., polypeptide
construct, fusion protein) can be administered to a subject 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20
times over a treatment cycle. In some embodiments, a treatment
cycle is 7 days, 14 days, 21 days, or 28 days long. In some
embodiments, a treatment cycle is 1 month, 2 months, 3 months, 4
months, 5 months, 6 months, 7 months, 8 months, 9 months, 10
months, 11 months, 12 months, 13 months, 14 months, 15 months, 16
months, 17 months, 18 months, 19 months, 20 months, 11 months, 22
months, 23 months, 24 months, 25 months, 26 months, 27 months, 28
months, 29 months, 30 months, 31 months, 32 months, 33 months, 34
months, 35 months, 36 months, 37 months, 38 months, 39 months, 40
months, 41 months, 42 months, 43 months, 44 months, 45 months, 46
months, 47 months, 48 months, 49 months, 50 months, 51 months, 52
months, 53 months, 54 months, 55 months, 56 months, 57 months, 58
months, 59 months, or 60 months.
[0308] In some embodiments, a compound is administered to a subject
once every 1, 2, 3, 4, 5, 6, 7, or 8 weeks.
[0309] In a further aspect, the present disclosure relates to the
compound or combination, or a pharmaceutical composition comprising
said according to the present disclosure for use as a medicament.
In an aspect, the present disclosure pertains to the compound or
combination, or a pharmaceutical composition comprising the said
for use in preventing or treating neuro-inflammation and/or
(chronic) pain, for example chronic neuropathic pain experienced
during and after chemotherapy, or postherpetic neuralgia, or
post-traumatic neuropathic pain, or painful diabetes neuropathy,
and other forms of chronic pain, including nociceptive pain and
mixed nociceptive and neuropathic pain. The term "chronic" can be
defined at persisting for at least 1, 2, 3, 4, 5, 6, 8, 10, or 12
weeks, at least 1, 2, 3, 4, 5, 6, 8, 10, or 12 months, and/or at
least 1, 2, 3, 4, or 5 years.
Methods of Making:
[0310] The compound or combination of the present disclosure may be
prepared by techniques which are routine to the skilled person. For
example, it may be prepared using a technique which provides for
the production of recombinant proteins by continuous cell lines in
culture. For example, the compound or combination of the present
invention can be produced in a host cell using a combination of
recombinant DNA techniques and gene transfection methods.
[0311] For example, to express the compound or combination
according to the present disclosure, a nucleic acid molecule
encoding the compound or (components of the) combination can be
prepared by standard molecular biology techniques. The nucleic acid
molecule of the disclosure is preferably operably linked to
transcription regulatory sequences such as a promoter, and
optionally a 3' untranslated region. The nucleic acid molecule of
the present disclosure may be inserted into a vector, such as an
expression vector, such that the genes are operatively linked to
transcriptional and translational control sequences. The expression
vector and transcription regulatory sequences are selected to be
compatible with the expression host cell used. The nucleic acid
molecule encoding a compound or (component(s) of the) combination
of the present disclosure may be inserted into the expression
vector by routine methods.
[0312] Additional amino acid sequences may be present at the N-
and/or C-terminus of a compound or polypeptide construct (e.g.,
fusion protein) for example, at any one or both of the first and
second binding moieties according to the present disclosure, e.g.,
to facilitate purification. For example, a poly-histidine-tag,
GST-tag, FLAG-tag, CBP tag, HA tag, or Myc tag may be present at
the C- or N-terminus to facilitate purification. In some
embodiments, an affinity tag is removed from a compound (e.g.,
polypeptide construct, fusion protein) of the disclosure, e.g.,
after purification. In some embodiments, a compound (e.g.,
polypeptide construct, fusion protein) of the disclosure does not
contain an affinity tag, (e.g., the compound can be purified by
other methods). Alternatively or additionally, the compound,
polypeptide construct (e.g., fusion protein) or combination
according to the present disclosure may optionally comprise
additional protein moieties, such as moieties capable of targeting,
e.g., a protein moiety comprising one or more antibody Fc regions.
In some embodiments, a compound (e.g., fusion protein, polypeptide
construct) comprises an antibody Fc region. In some embodiments, a
compound (e.g., fusion protein, polypeptide construct) comprises an
extracellular matrix-binding polypeptide.
[0313] The compound or combination according to the present
disclosure is preferably an isolated (combination of) protein(s)
which can be seen as a protein which is no longer in its natural
environment, for example in vitro or in a recombinant host
cell.
[0314] The present disclosure also encompasses a cell (line)
expressing the compound or (first and/or second binding moiety of
the) combination as disclosed herein, preferably a Chinese hamster
ovary (CHO) cell line, which provides a popular mammalian host for
large-scale commercial production of compound or combinations as
these cells are safe and allow high volumetric yields.
[0315] In another aspect, the present disclosure relates to a host
cell comprising a nucleic acid sequence of the present disclosure,
or a nucleic acid construct or vector comprising a nucleic acid
sequence of the present disclosure. The host cell may be any host
cell that can transiently or permanently express the compound or
(components of the) combination. The host cell is preferably an
animal cell or cell line, such as a mammalian cell or cell
line.
[0316] In one embodiment the compound or (components of the)
combination of the present disclosure is expressed in eukaryotic
cell, such as mammalian host cell. Preferred mammalian host cells
for expressing include CHO cells (including dhfr-CHO cells,
described in (Urlaub et al., 1980), used with a DHFR selectable
marker, NS/0 myeloma cells, COS cells, HEK293 cells and SP2.0
cells. When recombinant expression vectors comprising nucleic acid
sequences encoding the compound or (components of the) combination
are introduced into mammalian host cells, they may be produced by
culturing the host cells for a period of time sufficient to allow
for expression of the compound or (components of the) combination
in the host cells or, more preferably, secretion of thereof into
the culture medium in which the host cells are grown. The compound
or combination of the present disclosure may be recovered from the
culture medium in which the host cells are grown and/or may be
purified from the culture medium using standard protein
purification methods.
[0317] Alternatively, the nucleic acid sequences encoding the
compound or (components of the) combination of the disclosure can
be expressed in other expression systems, such as e.g. algae, as
well as insect cells. Furthermore, the compound or combination can
be produced in transgenic non-human animals, such as in milk from
sheep and rabbits or eggs from hens, or in transgenic plants.
[0318] Introduction of the nucleic acid sequence of the present
disclosure into a host cell may be carried out by any standard
technique known in the art. For expression of the compound or
combination of the present disclosure, the expression vector(s)
encoding the compound or combination may be transfected into a host
cell by standard techniques. The various forms of the term
"transfection" are intended to encompass a wide variety of
techniques commonly used for the introduction of exogenous DNA into
a prokaryotic or eukaryotic host cell, e.g., electroporation,
calcium-phosphate precipitation, DEAE-dextran transfection,
lipofectamine transfection, and freeze-dry method transfection, and
the like. Cell lines that secrete the compound or combination of
the present disclosure can be identified by assaying culture
supernatants for the presence of the compound or combination. The
preferred screening procedure comprises two sequential steps, the
first being identification of cell lines that secrete the compound
or combination, the second being determination of the quality of
the compound or combination such as the ability thereof to inhibit
cytokine production by blood cells stimulated with LPS or other
Toll-like receptor agonists, and others.
[0319] In an aspect, the present invention is concerned with a
method for producing the compound or combination according to the
present disclosure, said method comprising the steps of: culturing
a host cell of the present invention under conditions permitting
the production of the compound or combination; and optionally,
recovering the compound or combination. The skilled person will be
capable of routinely selecting conditions permitting production of
the compound or combination of the present disclosure.
Additionally, a person skilled in the art will be capable of
recovering the compound or combination produced using routine
methods, which include, without limitation, chromatographic methods
(including, without limitation, size exclusion chromatography,
hydrophobic interaction chromatography, ion exchange
chromatography, affinity chromatography, immunoaffinity
chromatography, metal binding, and the like), immunoprecipitation,
HPLC, ultracentrifugation, precipitation and differential
solubilisation, and extraction. As said above, recovery or
purification of the compound or combination of the present
disclosure may be facilitated by adding, for example, a
Histidine-tag to the fusion protein.
[0320] In some cases, compounds (e.g., polypeptide constructs,
fusion proteins) disclosed herein contain two cytokines, C1 and C2,
and are referred to in the format C1/C2 or C1-C2. The order in
which the cytokines are presented is not limiting and does not
necessarily infer the orientation of the cytokines. For example,
C1/C2 or C1-C2 can contain cytokine C1 on the C-terminal side of C2
or on the N-terminal side of C2. Similarly, a cytokine referred to
as C1/C2 can be the same as a cytokine referred to as C2/C1 unless
otherwise specified.
[0321] In this document and in its claims, the verb "to comprise"
and its conjugations is used in its non-limiting sense to mean that
items following the word are included, but items not specifically
mentioned are not excluded. In addition, reference to an element by
the indefinite article "a" or "an" does not exclude the possibility
that more than one of the element is present, unless the context
clearly requires that there be one and only one of the elements.
The indefinite article "a" or "an" thus usually means "at least
one".
EXAMPLES
[0322] To improve the therapeutic potency of regulatory cytokines
such as IL4, IL10 and IL13, the present invention discloses that
unique signalling of intracellular processes is induced by
crosslinking of receptors for these regulatory cytokines and that
this unique signalling in cells in the dorsal root ganglia and the
dorsal horn of the spinal cord results in an unprecedented
analgesic effect.
[0323] Moreover, the present invention provides several compounds
that can be used to crosslink regulatory cytokine receptors to
induce such a unique cell signalling, which can be used as a
medicament to treat chronic pain, and neuro-inflammatory and
neurodegenerative conditions. These compounds include genetically
engineered bispecific antibody that binds to two different
receptors for regulatory cytokines, such as the IL4 receptor (IL4R)
and the IL10 receptor (IL10R), or the IL4R and the IL13 receptor
(IL13R). Such bispecific antibodies have potent analgesic
effects--surprisingly they inhibit chronic pain more effectively
than the mere sum of the combination of the two cytokine moieties.
Moreover, injection of the anti-IL4R-IL10R bispecific antibody or
anti-IL4R-IL13R bispecific antibody may completely resolve chronic
pain in animal models.
[0324] In order to understand how a bispecific antibody against
regulatory cytokine receptors exert its unique analgesic and
neuroprotective effects, the inventors considered the mechanisms of
action of a fusion protein that binds to both IL4R and IL10R and
displays potent analgesic properties.
[0325] Similarly, they analysed the potency and mechanisms of a
fusion protein that binds to both IL4R and IL13R and which has
superior analgesic activity compared to the combination of
wild-type IL4 and IL10. To that end, the inventors looked at the
way in which cross-linking of IL4R and IL10R may transduce signals
to the sensory nervous system. The inventors envisage that the
superior analgesic effects resulting from cross-linking IL4R and
IL10R result from a unique signalling process that has a unique
effect on pain resolving pathways in sensory neurons. To confirm
the general applicability of cross-linking of regulatory cytokine
receptors as a therapeutic approach for chronic pain,
neuroinflammation and neuro-degeneration, the inventors also
evaluated the effects of cross-linking IL4 and IL13 receptors.
Materials and Methods
Animals
[0326] All animal experiments are performed in accordance with
international guidelines and with prior approval from the
University Medical Center Utrecht experimental animal committee.
Experiments were conducted using both male and female mice, all of
which were between 8-14 weeks old when tested. Observers who
performed the behavioral experiments were blind to the mouse
genotype and treatment. The following mice were used: wild type
(WT) C57BL/6 mice (Envigo, The Netherlands). For generation of
nociceptor-specific IL10R knockout mouse strains we used the
Cre-loxP system (Sauer B & Henderson N (1988) Site-specific DNA
recombination in mammalian cells by the Cre recombinase of
bacteriophage P1. Proc Natl Acad Sci USA 85(14):5166-5170). Floxed
IL10R mice (Pils M C, et al. (2010) Monocytes/macrophages and/or
neutrophils are the target of IL-10 in the LPS endotoxemia model.
Eur J Immunol 40(2):443-448) were crossed with Na.sub.v1.8-Cre mice
(Nassar M A, et al. (2004) Nociceptor-specific gene deletion
reveals a major role for Nav1.7 (PN1) in acute and inflammatory
pain. Proc Natl Acad Sci USA 101(34):12706-12711) in which Cre
expression is driven by the Na.sub.v1.8 promoter that is expressed
in >90% of nociceptors (Shields S D, et al. (2012) Nav1.8
expression is not restricted to nociceptors in mouse peripheral
nervous system. Pain 153(10):2017-2030).
Hyperalgesia Models
[0327] Mice received an intraplantar injection of 20 .mu.l
.lamda.-carrageenan (2% (w/v), Sigma-Aldrich) dissolved in saline
solution (NaCl 0.9%) in both hind paws. To induce transient
chemotherapy-induced polyneuropathy (CIPN), paclitaxel (2 mg/kg,
Cayman Chemical Company) was injected intraperitoneally on days 0
and 2. To induce persistent paclitaxel-induced CIPN paclitaxel (8
mg/kg, Cayman Chemical Company) was injected intraperitoneal on day
0, 2, 4 and 6. To induce persistent oxaliplatin-induced
polyneuropathy, mice received two treatment cycles, each consisting
of 5 daily intraperitoneal injections of 3 mg/kg oxaliplatin
(Tocris) with a 5 days free interval.
[0328] To induce transient chemotherapy-induced polyneuropathy
(CIPN), paclitaxel (2 mg/kg, Cayman Chemical Company) was injected
intraperitoneally on days 0 and 2. To induce persistent
paclitaxel-induced CIPN paclitaxel (8 mg/kg, Cayman Chemical
Company) was injected intraperitoneal on day 0, 2, 4 and 6. To
induce persistent oxaliplatin-induced polyneuropathy, mice received
two treatment cycles, each consisting of 5 daily intraperitoneal
injections of 3 mg/kg oxaliplatin (Tocris) with a 5 days free
interval.
[0329] Thermal hyperalgesia was assessed by determining the heat
withdrawal latency times using the Hargreaves test (IITC Life
Science) (Hargreaves K, Dubner R, Brown F, Flores C, & Joris J
(1988) A new and sensitive method for measuring thermal nociception
in cutaneous hyperalgesia. Pain 32(1):77-88). Mechanical thresholds
were determined using the von Frey test with the up-and-down method
(Chaplan S R, Bach F W, Pogrel J W, Chung J M, & Yaksh T L
(1994) Quantitative assessment of tactile allodynia in the rat paw.
J Neurosci Methods 53(1):55-63). All experimenters were blind to
genotype and/or treatment.
Drugs & Administration
[0330] IL4-10 protein was produced in HEK293 cells and purified as
described previously (Eijkelkamp N, et al. (2016) IL4-10 Fusion
Protein Is a Novel Drug to Treat Persistent Inflammatory Pain. J
Neurosci 36(28):7353-7363). Similarly, a fusion protein of IL4 and
IL13 (IL4-13) was produced. IL4-10 and IL4-13 fusion protein
concentrations were determined based on IL10 ELISA (IL4 Pelipair
ELISA kit, Sanquin; IL13 and IL10, DuoSet ELISAs, R&D Systems),
as well as on Bicinchoninic Acid Protein Assay (BCA Pierce Protein
Assay Kit, ThermoFisher Scientific). Intrathecal (i.t.) injections
of different compounds (5 .mu.l/mouse) were performed as described
before (Eijkelkamp N, et al. (2010) GRK2: a novel cell-specific
regulator of severity and duration of inflammatory pain. J Neurosci
30(6):2138-2149) under light isoflurane/O2 anaesthesia. The IL4-10
fusion protein (1 .mu.g/mouse) or equimolar doses of recombinant
human IL4 and IL10 (Sigma) were injected intrathecally at day 6
after intraplantar .lamda.-carrageenan injection.
[0331] IL4R.alpha. expression in sensory neurons was knocked down
by intrathecal injections of antisense oligodeoxynucleotides
(asODN) directed against IL4R.alpha. mRNA (Ripple M J, et al.
(2010) Immunomodulation with IL-4R alpha antisense oligonucleotide
prevents respiratory syncytial virus-mediated pulmonary disease. J
Immunol 185:4804-4811). This approach has been shown to
successfully inhibit the expression of several proteins in dorsal
root ganglia (DRG) neurons (Stone L S & Vulchanova L (2003) The
pain of antisense: in vivo application of antisense
oligonucleotides for functional genomics in pain and analgesia. Adv
Drug Deliv Rev 55(8):1081-1112). AsODN were dissolved in saline (15
.mu.g per 5 .mu.l) and injected intrathecally at day 3, 4 and 5
after intraplantar .lamda.-carrageenan injection. AsODN had a
phosphorothioate backbone. The following ODN were used: Mismatch
ODN (mmODN): TGGAAAGGCTTATACCCCTC (SEQ ID NO:1); IL4R asODN:
CCGCTGTTCTCAGGTGACAT (SEQ ID NO:2).
Tissue Preparation & Immunochemistry
[0332] Mice were deeply anaesthetised with an injection of
pentobarbital (60 mg/kg; intraperitoneal) and transcardially
perfused with Phosphate-buffered saline (PBS; 140 mM NaCl.sub.2, 20
mM Na.sub.2HPO.sub.4, 2.4 mM NaH.sub.2PO.sub.4) followed by 4%
paraformaldehyde (PFA) in PBS (Klinipath). Lumbar DRGs and spinal
cords (lumbar L3-L5 section) were isolated and postfixed in 4% PFA
cryoprotected in sucrose, and embedded and frozen in optimal
cutting temperature (OCT) compound (Tissue-Tek, Sakura). For IL4R
and IL10R stainings, DRGs were directly frozen in OCT without prior
fixation. Spinal cords and DRGs were cut in 20 .mu.m and 10 .mu.m
thick sections respectively using a cryostat (CM 3050S; Leica).
Sections were collected on SuperFrost plus microscope slides (VWR
International).
[0333] IL4R and IL10R: Sections were fixed with 10% neutral buffer
formalin for 10 min and incubated in PBS containing 0.3% Triton
X-100 (PBS-T) and 5% normal donkey serum (NDS)). Cultured primary
sensory neurons were fixed with 4% PFA for 10 minutes and incubated
in PBS containing 0.05% Tween-20, 1% bovine serum albumin (BSA) and
5% NDS). Sections and cells were incubated overnight at 4.degree.
C. with rabbit anti-IL4R.alpha. (sc-686, Santa Cruz; 1:100), rabbit
anti-IL10R.alpha. (sc-985, Santa Cruz; 1:100), mouse
anti-Neurofilament 200 (NO142, Sigma; 1:500), mouse anti-Peripherin
(MAB1527, Millipore; 1:100) or Isolectin B4 (B1205, Vector; 1:50)
diluted in antibody diluent (PBS-T with 2% BSA). Subsequently,
sections were incubated with alexafluor 488- or 594-conjugated
secondary antibodies (Thermofisher, 1:1000) followed by DAPI
(1:1000, Sigma) staining before sections were mounted on slides
with FluorSave reagent (Millipore).
[0334] For staining of c-Fos, a marker for neuronal activation, we
performed a colorimetric staining as described previously (64) with
some modifications. Serial spinal cord sections were incubated in
PBS-T containing 0.1% BSA and 2% NDS. Subsequently, sections were
incubated with rabbit anti c-Fos (sc-52, Santa Cruz; 1:500)
overnight at 4.degree. C. After washing, sections were incubated
with Biotin SP conjugated donkey anti-rabbit IgG (1:250, Jackson IR
Laboratories) for 90 minutes. Subsequently, sections were incubated
with Vector ABC-Elite (1:50, Vector Laboratories) for 90 minutes.
Thereafter, sections were developed with diaminobenzidine
(DAB)-Nikel solution (0.05M Tris HCl pH 7.6; 3 mg/ml
(NH.sub.4).sub.2Ni(SO.sub.4).sub.2; 25 mg/ml DAB (1:100, Sigma),
hydrogen peroxide (0.1 .mu.l/ml). Finally, the sections were
dehydrated and mounted with DePeX (Serva).
[0335] Images were taken using a Zeiss Axio Lab A1 (Zeiss).
Pictures were analysed with ImageJ (NIH).
Culture of DRG Neurons
[0336] DRGs were cultured as described previously (Eijkelkamp N, et
al. (2013) A role for Piezo2 in EPAC1-dependent mechanical
allodynia. Nat Commun 4:1682). Briefly, DRGs were dissected and
placed on ice-cold dissection medium (HBSS w/o Ca.sup.2+ and
Mg.sup.2+, 5 mM HEPES, and 10 mM glucose). After dissection, axons
were cut and dissection medium was replaced by filtered enzyme mix
(HBSS w/o Ca.sup.2+ and Mg.sup.2+, 5 mM HEPES, 10 mM glucose, 5
mg/ml collagenase type XI (Sigma), and 10 mg/ml Dispase (Gibco)).
The DRGs were incubated in enzyme mix for 30 minutes at 37.degree.
C. and 5% CO.sub.2. Subsequently, enzyme mix was inactivated with
heat-inactivated foetal bovine serum (FBS, Sigma). Cells were
cultured in Dulbecco's modified Eagle's medium (Gibco) containing
10% FBS (Gibco), 2 mmol/L glutamine (Gibco), 10,000 IU/ml
penicillin-streptomycin (Gibco) on poly-L-lysine (0.01 mg/ml,
Sigma) and laminin (0.02 mg/ml, Sigma)-coated glass coverslips in a
5% CO.sub.2 incubator at 37.degree. C. Cells were used the
following 1-2 days.
[0337] To evaluate the effect of cross-linking IL4R, IL10R and
IL13R on the damage to neurons induced by chemotherapeutic drugs,
cells were incubated in 24 wells plates for 24 hours in presence of
paclitaxel (1 uM) or oxaliplatin (5 ug/ml) to induce neurotoxicity.
IL4-10 fusion protein (100 ng/mL), IL4-13 fusion protein (100
ng/mL), IL4 an IL13 (50 ng/mL each), IL4 (50 ng/mL), IL10 (50
ng/mL), and IL13 (50 ng/mL) were added together with the
chemotherapeutic agent. As controls cells were also cultured in
absence of chemotherapeutic drugs or cytokines, and in presence of
chemotherapeutic drugs only. After fixation with 4%
paraformaldehyde, cells were stained with rabbit anti-mouse
.beta.III-tubulin (ab18207, 1:1000; Abcam). Neurites were
visualized with a Zeiss Axio Lab A1 microscope (Zeiss-Oberkochen,
Germany) and using a random sampling method, at least 10 images per
glass slide were made at a magnification of 10.times.. The length
of neurites was measured with the ImageJ plugin Simple Neurite
Tracer76. The averages of neurite length per neuron for a minimum
of five neurons per condition were compared between groups for the
three individual primary sensory cultures.
Calcium Imaging
[0338] DRG neurons used for calcium imaging experiments were
stimulated overnight with TNF.alpha. (50 ng/ml, Peprotech) with or
without the IL4-10 fusion protein (100 ng/ml; 3 nM), recombinant
IL4 and IL10 (50 ng/ml each; 3.3 and 2.9 nm, respectively) and/or
receptor blocking antibodies targeted against mouse IL4R or IL10R
(2 .mu.g/ml, BD pharmingen).
[0339] To measure changes in the capsaicin-evoked calcium response,
cells were loaded with 5 .mu.M Fura-2-AM (Invitrogen) for 25
minutes in 140 mM NaCl, 4 mM KCl, 1 mM MgCl.sub.2, 2 mM CaCl.sub.2,
10 mM HEPES, and 10 mM Glucose; pH 7.4. Cells were excited at 340
and 380 nm wavelengths and fluorescence was collected every 3
seconds at 510 nm using an Axio Observer A1 inverted microscope
(X20 objective, Zeiss). The ratio 340/380 is directly correlated
with the amount of intracellular calcium.
[0340] Recordings were performed as previously described (66) with
some modifications. Briefly, every experiment included a 5 minutes
baseline measurement followed by a stimulation of the cells by
superfusion with capsaicin (0.03 .mu.M) for 21 seconds followed by
superfusion of medium. A subsequent 5 minutes of superfusion with
high K*-buffer (4 mM NaCl, 140 mM KCl, 1 mM MgCl.sub.2, 2 mM
CaCl.sub.2, 10 mM HEPES, and 10 mM Glucose; pH 7.4) was added at
the end of each experiment to depolarize the neurons to confirm
cell viability and functionality.
RNA Extraction and Quantitative PCR
[0341] Lumbar DRGs were homogenized using TRIzol (Invitrogen).
Total RNA was extracted using the RNeasy Mini Kit (Qiagen) and 1
.mu.g of total RNA was used to synthesize cDNA. cDNA was
synthesized using SuperScript reverse transcriptase (Invitrogen).
RNA concentrations were determined using a NanoDrop 2000 (Thermo
Scientific).
[0342] The real-time PCR reaction using SYBRgreen master mix
(BioRad) was performed on an iQ5 Real-Time PCR Detection System
(BioRad). Primers used for qPCR are listed in table 9. The mRNA
expression levels were normalized for GAPDH, HRPT and actin.
TABLE-US-00009 TABLE 9 List of primers used Gene Forward Reverse
GAPDH 5'-TGAAGCAGGCATCTGAGGG-3' 5'-CGAAGGTGGAAGAGTGGGAG- (SEQ ID
NO: 3) 3' (SEQ ID NO: 4) HRPT 5'-TCCTCCTCAGACCGCTTTT-3'
5'-CCTGGTTCATCATCGCTAATC-3' (SEQ ID NO: 5) (SEQ ID NO: 6) Actin 5'-
5'-CACTCAGGGCAGGTGAAACT-3' GATGCACAGTAGGTCTAAGTGGAG- (SEQ ID NO: 8)
3' (SEQ ID NO: 7) IL4RA 5'-TCTGCATCCCGTTGTTTTGC-3'
5'-GCACCTGTGCATCCTGAATG-3' (SEQ ID NO: 9) (SEQ ID NO: 10)
In Situ Proximity Ligation Assay (PLA)
[0343] IL4R.alpha. antibody was labelled to thiol-MINUS-oligo with
the dual-crosslinker sulfo-SMCC (ThermoFisher). IL10R.alpha.
antibody was labelled with biotin using EZ-Link.TM.
Sulfo-NHS-LC-Biotin SMCC (ThermoFischer). The Biotin-PLUS-probe was
later bound to the antibody with streptavidin.
[0344] Primary DRG cultures were treated with IL4-10 fusion protein
(100 ng/ml; 3 nM) or the combination of IL4 and IL10 (50 ng/ml
each; 3.3 and 2.9 nM, respectively) for 15 minutes and fixed with
4% PFA for 10 minutes. In situ PLA was performed as described
(Soderberg O, et al. (2008) Characterizing proteins and their
interactions in cells and tissues using the in situ proximity
ligation assay. Methods 45(3):227-232) with some modifications.
Samples were blocked with blocking buffer (PBS containing 0.05%
Tween-20, 1% BSA and polyA 1:100). Subsequently, samples were
incubated overnight at 4.degree. C. with the following antibodies:
MINUS probe-labelled rabbit-anti IL4R.alpha. and biotin-labelled
rabbit-anti-IL10R.alpha.. For controls biotin-labelled rat
anti-CD200 (Serotec) was used instead of anti-IL10R.alpha.
antibody. Next, samples were incubated with streptavidin (5
.mu.g/ml) for 25 minutes at room temperature and incubated with
biotin linked to a PLUS probe for 30 minutes at room temperature.
T4 DNA ligase (5 Weis units/.mu.l) and the circle and linker probes
were added (in the presence of 0.1 M DTT and 3 mM ATP) and
incubated at 37.degree. C. for 50 minutes in PHI buffer (50 mM
Tris-HCl, 10 mM MgCl.sub.2, 10 mM (NH.sub.4)2SO.sub.4, pH 7.5+0.05%
Tween20). After washing, samples were incubated with PHI polymerase
(kindly donated by Toshiro Kobori), in the presence of 1 mM dNTPs
and 0.1 M DTT for 90 minutes at 32.degree. C. Next, samples were
stained with antibody against .beta.III-tubulin, to stain neurons,
for 1 hour at RT. After washing, samples were incubated with
Cy5-oligonucleotide probe to label amplified DNA and secondary
anti-rabbit antibody conjugated with Alexa488. For the minimal
(Min) and Maximal (Max) staining control only MINUS probe
antibodies were used and the incubation with streptavidin was
skipped. For the Max control Biotin-PLUS was added after the ligase
incubation. Images were taken using a Zeiss LSM confocal microscope
(Zeiss).
Kinase Activity Profiling
[0345] Lumbar DRGs were homogenized using M-PER mammalian
Extraction buffer (Pierce) supplemented with phosphatase and
protease inhibitor cocktails (Pierce). Protein concentration was
determined using the Bradford assay (Bio-Rad). Kinase activity
profiling was performed using the Tyrosine Kinase PamChip.RTM.
(PTK) Array and the Serine/Threonine Kinase PamChip.RTM. (STK)
Array for Pamstation.RTM. 12 (PamGene International B.V.). For the
PTK arrays 7.5 .mu.g of protein lysate per array were used while
for the STK Arrays 2 .mu.g of protein lysate per array were used.
Image quantification and statistical analysis were performed using
BioNavigator.RTM. Software (PamGene International B.V.). Upstream
kinase analysis was performed using BioNavigator.RTM. Software with
peptide-kinase mapping using Kinexus phosphonet enrichment files
(www_phosphonet.ca/). Kinome tree illustration was constructed
using data from the interactive PamGene BioNaviagor Upstream Kinase
Tool (www_kinhub.org/kinmap/index.html). For pathway analyses
peptides found to be significantly differentially phosphorylated
(p<0.05) between IL4-10 and the combination of the individual
cytokines were subjected to pathway analysis using the GeneGo
pathway analysis package
RNA Sequencing
[0346] RNA libraries were prepared with the poly A selection method
followed by multiplexing and sequencing on the Illumina NextSeq500@
platform in a 1.times.75 bp single-read and 350 million reads per
lane (Utrecht Sequencing Facility). All samples passed the read
quality checks performed using FastQC (Andrews S (2010) FastQC: a
quality control tool for high throughput sequence data. Available
online at: www_bioinformatics.babraham.ac.uk/projects/fastqc.). The
sequencing reads from each sample were aligned to the recent
reference human genome GRCh38 build 79 assembly from Ensembl
(Genome Reference Consortium Human Build 38) using STAR aligner
(Cunningham F, et al. (2015) Ensembl 2015. Nucleic Acids Res
43(Database issue):D662-669; Anders S, Pyl P T, & Huber W
(2015) HTSeq--a Python framework to work with high-throughput
sequencing data. Bioinformatics 31(2):166-169). Gene expression
data for the annotated genes was generated using HTSeq-count
(Anders S, Pyl PT, & Huber W (2015) HTSeq--a Python framework
to work with high-throughput sequencing data. Bioinformatics
31(2):166-169). The samples exhibited batch effect due to different
days of isolation and library preparation. The batch effect was
corrected using R package RUVSeq (Risso D, Ngai J, Speed T P, &
Dudoit S (2014) Normalization of RNA-seq data using factor analysis
of control genes or samples. Nat Biotechnol 32(9):896-90).
Differential gene expression analysis and variance-stabilizing
transformation (to obtain normalized read counts) were performed
using R/Bioconductor package DESeq2 (Love M I, Huber W, &
Anders S (2014) Moderated estimation of fold change and dispersion
for RNA-seq data with DESeq2. Genome Biol 15(12):550).
Results
Example 1: Expression of Receptors for Regulatory Cytokines by
Sensory Neurons and Glial Cells in the Dorsal Ganglia and Spinal
Cord
[0347] To identify whether sensory neurons are able to respond to
regulatory cytokines, it was analyzed which receptors for
regulatory cytokines are expressed by sensory neurons and glial
cells. RNAseq data of receptors for IL10, IL4, IL13, and
TGF.beta.1/2 in the dorsal root ganglia and spinal cord were
extracted from the data base by Ray et al. (Pain 2018;
159:1325-1345) as available on
www_utdallas.edu/bbs/painneurosciencelab/sensoryomics/drgtxome/?go.
RNA sequencing revealed expression of receptors for IL10, IL4,
IL33, TGF.beta.1 and TGF.beta.2 in the dorsal root ganglia and
spinal cord of human and mouse (FIG. 1; data are expressed as
transcripts per million).
[0348] Next, expression of the alpha chain of both cytokine
receptors, IL4R (IL4R.alpha.) and IL10R (IL10R.alpha.) in vivo in
mice was studied. IL10R.alpha. was expressed in almost all sensory
neurons of the DRG, whilst IL4R.alpha. is expressed in subsets of
sensory neurons in the DRG (FIG. 2A). In culture, both IL4R.alpha.
and IL10R.alpha. are expressed in NF200-, peripherin-expressing
neurons and non-peptidergic 1B4+ neurons, often by the same
cells.
Example 2: Cross-Linking Regulatory Cytokine Receptors in Dorsal
Root Ganglion and Spinal Cord Results in a Potent Analgesic
Effect
[0349] Intrathecal administration is a well-known method to deliver
drugs and other compounds in the direct environment of cells in the
dorsal root ganglion and spinal cord. To test the requirement of
IL4R.alpha. expression by sensory neurons for the analgesic
activity of IL4R-IL10R cross-linking compound, the inventors used
intrathecal administration of antisense oligodeoxynucleotides
(asODN) targeting IL4R.alpha. mRNA. Three daily intrathecal
injections of IL4R.alpha. asODN significantly reduced IL4R.alpha.
mRNA and protein expression in the DRG by .about.70% compared to
mismatched (mm) ODN-treated animals (FIG. 2B). IL4-10 fusion
protein was used to cross-link IL4R and IL10R. Intrathecal
injection of IL4-IL10 fusion protein (1 .mu.g) at 6 days after the
induction of persistent inflammatory pain completely inhibited
carrageenan induced mechanical and thermal hyperalgesia in mice
treated with mmODN confirming the potent analgesic properties of
crosslinking IL4R and IL10R. It is to be noted that cross linking
IL4R and IL10R may not completely abolish ability to detect sensory
stimuli, but rather normalizes aberrant pain sensation. IL4R.alpha.
knockdown in the DRG markedly reduced the analgesic effect of the
IL4R-IL10R cross-linking compound compared to animals treated with
mmODN (FIGS. 2C and 2D). Knockdown of IL4R.alpha. during
established carrageenan-induced hyperalgesia did not affect the
magnitude of mechanical hypersensitivity.
[0350] Nav1.8+ sensory neurons mediate inflammatory pain
(Abrahamsen et al. Science. 2008 Aug. 1; 321(5889):702-5). To
identify the role of IL10R.alpha. expression in the analgesic
properties of the IL4R-IL10R cross-linking compound, IL10R.alpha.
was selectively ablated in Nav1.8+ neurons (FIG. 2E). The course of
carrageenan-induced persistent inflammatory hyperalgesia was
indistinguishable between wild-type and Nav1.8-IL10R.sup.-/-
animals.
[0351] Intrathecal injection of the IL4R-IL10R crosslinking
compound (1 .mu.g) at day 6 after induction of inflammatory pain
attenuated thermal and mechanical hyperalgesia in wild type animals
(FIGS. 2F and 2G). In contrast, deletion of IL10R.alpha. in Nav1.8+
nociceptors partially ablated the analgesic effect of the
IL4R-IL10R cross-linking compound (FIGS. 2F and 2G), indicating
that nociceptor IL10R.alpha. may be required in part for the pain
inhibiting effects of IL4R-IL10R cross-linking compound.
[0352] Since both receptors are required for full analgesic effect
of cross-linking IL4R and IL10R, the inventors next considered if
ablation of both receptors in sensory neurons would completely
prevent the analgesic actions of the IL4R-IL10R cross-linking
compound. To that end, IL4R expression was knocked down in
Nav1.8-IL10R-/- mice with intrathecal IL4R.alpha. asODN injections.
Knockdown of both IL4R.alpha. and IL10R.alpha. itself does not
affect the course of persistent inflammatory pain. Importantly,
knockdown of both IL4R.alpha. and IL10R.alpha. expression in the
DRGs completely prevented resolution of pain by the IL4R-IL10R
cross-linking compound (FIGS. 3A-D).
[0353] An increase of spinal immediate-early gene c-Fos expression
can be used as a proxy of spinal neuronal activation in the dorsal
horn of the spinal cord. At day 7 after intraplantar carrageenan
injection the number of c-Fos positive neurons in the superficial
layers of the dorsal horn was significantly increased compared to
naive animals (FIG. 3C). Intrathecal administration of IL4-10
fusion protein significantly reduces the number of dorsal horn
spinal cord c-Fos positive neurons. Knockdown of IL4R.alpha. and
IL10R.alpha. in sensory neurons completely prevents IL4R-IL10R
cross linking compound mediated attenuation of the c-Fos expression
(FIG. 3C). Overall this indicates that both IL4R.alpha. and
IL10R.alpha. in sensory neurons are required for the full
inhibition of persistent inflammatory pain resulting from
cross-linking IL4R and IL10R.
[0354] The potential of cross-linking IL4R and IL13R to inhibit
pain was investigated in chemotherapy-induced neuropathy models.
Mice received 4 injections of paclitaxel (8 mg/kg) every other day
from day 0 to 6. Paclitaxel induced mechanical hyperalgesia that
started on the first day after the first injection and that
persisted at least 3 weeks after chemotherapy-treatment was
stopped. Two days after the last paclitaxel injection, mice were
injected intrathecally with 3 different doses of IL4-13 fusion
protein (0.3, 1 and 3 .mu.g/mouse) to cross-link IL4R and IL13R on
cells in the dorsal root ganglion and the spinal cord (FIG. 4A). An
almost normalization of mechanical hyperalgesia lasting for at
least a week was observed, demonstrating the potential of
cross-linking IL4R and IL13R for long-lasting resolution of
chemotherapy-induced polyneuropathy. Importantly, cross-linking
IL4R and IL13R also reduced paclitaxel-induced intra-epidermal
nerve fibre loss in the paw skin (FIG. 4B), demonstrating that this
intervention prevents neuronal damage in vivo.
[0355] To confirm that ross-linking IL4R and IL13R provides
neuroprotection against a broader spectrum of chemotherapy-induced
polyneuropathy, toxic neuropathy was also induced in mice using a
platinum-based chemotherapeutic drug, oxaliplatin. Two cycles of 5
times a daily injection of oxaliplatin, separated by 5 days without
intraperitoneal injection, induced mechanical allodynia that
persisted for at least 3 weeks (FIG. 4C). Intrathecal injection of
IL4-13 fusion protein on the second day after the last oxaliplatin
injection reduced mechanical allodynia significantly for 4 days
(FIG. 4C). Intrathecal injection of either wild-type IL4 or
wild-type IL13 transiently inhibited oxaliplatin-induced mechanical
allodynia for about 1 day, which effect was significantly shorter
than that of cross-linking IL4R and IL13R by the fusion
protein.
Example 3: Cross-Linking IL4R and IL10R or IL4R and IL13R has
Unique Effects on Neurons
[0356] Pro-inflammatory mediators sensitize sensory neurons for
noxious and innocuous stimuli. To test whether cross-linking of
IL4R and IL10R inhibits inflammatory mediator-induced sensitization
of capsaicin-induced calcium responses in sensory neurons, cultured
neurons were treated with TNF (50 ng/ml) with and without IL4-10
fusion protein, which was used to cross-link IL4R and IL10R (100
ng/ml, 3 nM). TNF significantly increased the magnitude of
capsaicin-induced calcium influxes compared to untreated cells
(FIG. 5A-C). Co-treatment with IL4-10 fusion protein completely
prevents the sensitization of capsaicin-evoked calcium influx by
TNF.alpha.. In a control experiment, it was excluded that IL4-10
fusion protein affects capsaicin-induced calcium responses in
non-sensitized neurons (not shown). Similarly, cross-linking IL4R
and IL10R inhibits PGE2-induced sensitization of capsaicin-induced
calcium responses (FIG. 5D-F), without affecting normal
capsaicin-induced calcium fluxes (not shown). This indicates that
cross-linking IL4R and IL10R prevents neuronal sensitization by
inflammatory mediators.
[0357] To identify whether the inhibitory effects of IL4-10 fusion
protein on neuronal sensitization requires both cytokine binding
moieties, separate receptor blocking antibodies were added.
Blocking either IL4R or IL10R slightly reduced IL4-10 fusion
protein-induced inhibition of TNF.alpha.-induced sensitization of
capsaicin evoked calcium responses. Interestingly, blocking both
IL4R and IL10R receptors completely abrogates inhibition of
neuronal sensitization induced by IL4-10 fusion protein (FIG.
5G).
[0358] Next the inventors considered to what extent cross-linking
IL4R and IL10R to normalize neuronal sensitization by inflammatory
mediators is superior to those of the combination of the wild-type
cytokines. IL4-10 fusion protein dependently inhibits
TNF.alpha.-induced sensitization of capsaicin-evoked calcium
influx. At a concentration of 3 nM, it completely reversed
TNF.alpha.-induced sensitization, whilst the combination of
individual cytokines at the highest dose tested (30 nM) inhibited
the TNF.alpha.-induced sensitization to a maximum of 50%. This
indicates that cross-linking IL4R and IL10R has a superior potency
over the combination of wild-type IL4 and IL10 to reduce neuronal
sensitization by inflammatory mediators.
[0359] Next, the inventors investigated whether such unique effects
of crosslinking IL4R and IL10R on neurons also could be found for
cross-linking IL4R with IL13R. To evaluate this, mouse sensory
neurons were cultured in presence of oxaliplatin with or without
IL4-13 fusion to cross-link IL4R and IL13R, or equimolar
concentrations of wild-type IL4 and IL13 protein. Neurite length
was measured to assess neurotoxicity. Oxaliplatin had a significant
negative effect on neurite length when compared to the control
group (FIG. 6). IL4-13 fusion protein completely protected against
oxaliplatin-induced neurotoxicity, whilst the combination of
equimolar concentrations of wild-type IL4 and IL13 did not. An
additional assay with two IL4-IL13 fusion proteins showed
similar
[0360] Thus, these data together demonstrate that cross-linking
IL4R and IL10R, or IL4R and IL13R induces effects in neurons that
normalize the hypersensitization by inflammatory mediators and
protect the cells against damage. Importantly, these effects are
unique to cross-linking involved receptors, since they are not
induced by combinations of the wild-type cytokines tested at
concentrations equimolar to that of the fusion proteins.
Example 4: IL4-10 Fusion Protein Crosslinks IL4R and IL10R in
Sensory Neurons
[0361] To further investigate the mechanisms of the unique effects
of IL4-10 fusion protein and IL4-13 fusion proteins, the inventors
decided to analyse the cellular processes induced by IL4-10 fusion
protein in more detail. The inventors hypothesized that the IL4-10
fusion protein causes heterologous clustering of IL4R and IL10R,
thereby inducing unique signalling in sensory neurons, i.e.
signalling that is not induced by wild-type cytokines, and even not
by the combination of wild-type cytokines. First, cross-linking of
IL4R and IL10R by IL4-10 fusion proteins on neurons was
demonstrated. Sensory neurons were incubated IL4-10 fusion protein
or the combination of the respective interleukins for 15 minutes in
vitro followed by a proximity ligation assay (PLA) to assess
clustering of IL4R and IL10R. This method enables the inventors to
detect clustering of the 2 receptors within a 51 nm range. IL4-10
fusion protein (100 ng/ml, 3 nM) treatment indeed cluster IL4R and
IL10R receptors in sensory neurons, whilst receptor clustering did
not occur after treatment with equimolar concentration of the
combination of the respective cytokines or after vehicle (FIG. 7).
Clustering of IL4R and IL10R is specifically induced by the fusion
protein as clustering of IL4R and another highly expressed membrane
protein, CD200, was not observed (data not shown).
Example 5: Cross-Linking IL4R and IL10R Induces a Distinct Kinase
Activity Profile Compared to the Combination of Wild-Type
Cytokines
[0362] The ability of IL4-10 fusion protein to cross-link IL4 and
IL10 receptors raises the possibility that this drives unique
downstream signaling events. To elucidate downstream signaling in
sensory neurons in an unbiased manner, the inventors performed
PAMgene kinase activity profiling to assess global protein tyrosine
kinases (PTK) and serine/threonine kinases (STK) activity in
homogenates of lumbar DRGs of mice with persistent inflammatory
pain after administration of IL4-10 fusion protein. Kinomic
profiles were assessed at 30, 60 and 240 minutes after intrathecal
administration with either the anti-IL4R-IL10R bispecific antibody,
the combination of cytokines or PBS. Analyses of the 3 different
time points indicated that the most prominent changes in kinome
profiles were found at 60 minutes after intrathecal injection,
whilst differences are less pronounced at the other time points
examined (FIG. 8). Analyses of the peptides that are differentially
phosphorylated by PTK present in the DRG homogenates at 60 minutes
after treatment shows that in total 38 peptides were differentially
phosphorylated when in IL4-10 fusion protein-treated mice compared
to vehicle treated mice. Cross-linking IL4R and IL10R induces
stronger phosphorylation of 5 peptide substrates for PTKs that were
also activated by the combination of cytokines (FIG. 8A).
Interestingly, 33 peptides are only phosphorylated by homogenates
from mice treated with IL4-10 fusion protein and from mice treated
with the combination of IL4 and IL10, indicating that
anti-IL4R-IL10R bispecific antibody activates a unique set of PTK
(FIG. 8A). Peptides were identified that exhibited different
phosphorylation between IL4-IL10 treatment and treatment with the
combination of unlinked cytokines (FIG. 8B). Next the inventors
evaluated the differentially phosphorylated STK peptides.
Intriguingly DRG homogenates of mice treated with the combination
of the cytokines phosphorylate 14 STK peptides to a lesser extent
compared to vehicle-treated mice, whilst the homogenates from mice
treated with IL4-10 fusion protein did not exhibit differences for
most peptides, and induced increased phosphorylation in some (FIG.
8C). Peptides were identified that exhibited different
phosphorylation between IL4-IL10 treatment and treatment with the
combination of unlinked cytokines (FIG. 8D). These data indicate
that both PTK and STK activity are differentially regulated upon
cross-linking of IL4R and IL10R in vivo compared to the effects of
the combination of wild-type cytokines. Each of the peptides
present in the kinase activity array can be substrates for
different kinases, and one kinase or family of kinases can
phosphorylate different peptides. To predict the putative upstream
kinases activated specifically by cross-linking IL4R and IL10R, the
differential phosphorylation profiles are loaded into PhosphoNET.
These analyses predict that several kinases are activated at 60
minutes after injection based on the observed phosphorylation
patterns of the peptides (FIG. 8E-H). The inventors identified
kinases, including the PTK platelet-derived growth factor receptor
A (PDGFRa), KIT, fms like tyrosine kinase 3 (FLT3), MER or RET
tyrosine kinases; the calcium calmodulin kinases (CAMK) such as
CHK2, CAMK4 or DCAMKL2; AMPK, JAK1, and the Protein kinase A/G/C
family such as AKT3, PKCb or NDR1/2. Examples of differentially
activated kinases between IL4-IL10 and the combination of unlinked
cytokines are provided in FIG. 8G and FIG. 8H, and include the PTKs
KIT, PDGFRA, FER, MET and JAK1, and the STKs AKT3, LATS2, NDR2 or
CAMK2. Pathway analysis of these predicted differentially can
activate kinases indicates that the most active cellular processes
induced by anti-IL4R-IL10R bispecific antibody treatment are mapped
to immune responses, altered transcription, cell adhesion or cell
cycle. The top 5 kinase substrates ranked by occurrence in the top
50 of the pathway analysis were NAPDH oxidase P47-phox, p120GAP, Rb
protein, CDC25A and ZAP70. Overall this indicates that
cross-linking IL4R and IL10R drives different signalling pathways,
differential activation of kinases, different phosphorylation of
kinase substrates, and a different kinomic profile compared to
vehicle and compared to treatment with equivalent amounts of
unlinked cytokines. For example, the activation of sets of kinases
not activated by the combination of IL4 and IL10 and drives
stronger activation of several kinases also activated by the
combination of IL4 and IL10.
Example 6: Cross-Linking IL4R and IL10R In Vivo Induces a Unique
Transcriptome
[0363] To further explore the potential capacity of cross-linking
IL4R and IL10R to elicit downstream effects that differ from that
of the combination of IL4 and IL10, the inventors performed RNA
sequencing of DRGs, six hours after a single intrathecal injection
of IL4-10 fusion protein, the combination of IL4 and IL10, or PBS
alone in mice with persistent nociceptive pain. Principal component
analysis (PCA) indicates that 75% of the variance in gene
expression can be explained by the first two principal components.
The first principal component captures 55% variance of the data and
separates the mice with cross-linking of IL4R and IL10R from mice
that received vehicle or the combination of unfused cytokines,
while the second principal component may capture 20% variance of
the data and separates vehicle treated animals, indicating that
IL4-10 fusion protein induces different transcriptome changes (FIG.
9A). Hierarchical clustering of the top 500 differentially
regulated genes showed that based on their transcriptional profile
the individual animals clustered based on the three different
treatments (FIG. 9B). Thus, cross-linking IL4R and IL10R induces a
different transcriptional profile compared to mice treated with IL4
and IL10 or vehicle. Treatment of mice with the combination of IL4
and IL10 resulted in 4905 genes differentially expressed (FDR
corrected p-value<0.05) compared to those injected with PBS
(FIG. 9C). KEGG Pathway analysis indicated that these genes
aggregate in pathways affecting TLR signaling, oxidative
phosphorylation, ribosomal proteins and lipid metabolism. In the
mice injected intrathecally with IL4-10 fusion protein, 3995 genes
are differentially expressed (FDR corrected p-value<0.05)
compared to those injected with vehicle. Pathway analysis of the
differentially expressed genes reveals that neuronal-related genes
such as axon guidance and calcium signaling or energy production
such as oxidative phosphorylation are downregulated in mice upon
cross-linking IL4R and IL10R. Moreover, genes involved in
inflammatory pathways like interferon signaling, antigen processing
and presentation, complement and interleukin signalling are
affected. Interestingly, when genes induced by cross-linking of
IL4R and IL10R are compared with those induced by the combination
of the cytokines, 3025 genes are differentially expressed (FIG.
9C). Analysis of these genes indicates that the expression of 1650
genes is increased, whilst 1375 are downregulated upon
cross-linking IL4R and IL10R, as compared to the combination of
cytokines. Importantly, 1675 genes are uniquely regulated by IL4-10
fusion protein. From these uniquely regulated genes, the expression
of 981 genes is increased by the fusion protein, whilst 694 are
downregulated. Pathway analysis of the genes that are
differentially expressed upon crosslinking of IL4R and IL10R as
compared to the transcriptome of animals receiving the combination
of wild-type IL4 and IL10 indicates that these genes belong to
pathways including cytokine signaling, neurotrophin signaling, and
pathways affecting innate and adaptative immune system indicating
potential DRG-infiltrating immune cells (FIG. 9E). Additional
analysis of upstream kinases based on KEGG pathways indicated that
signalling pathways including cytokine and chemokine signaling,
NOD-like receptor signaling or JAK-STAT signaling were affected
(FIG. 9F). Focal and cell adhesion molecules and pathways affecting
innate and adaptive immune system, such as TLR, T and B cells
receptor pathways were also affected, which may reflect potential
DRG-infiltrating immune cells. Overall, RNAseq data demonstrates
that cross-linking IL4R and IL10R induces a unique transcriptome
change as compared to animals treated with the combination of IL4
and IL10.
Example 7: JAK1 Activation Contributes to the Superior Analgesic
Effect of IL4-10
[0364] Based on the data obtained from PAMgene analysis, KIT,
PDGFRA, FER, MET and JAK1 were identified as the 5 highest ranking
potential Tyrosine kinases that could be linked to the superior
effects of IL4-10. Whether IL4-10 differentially activates JAK1 in
sensory neurons in vitro by was investigated by determining
phosphorylation of JAK1 after stimulation of sensory neurons in
vitro with IL4-10 or equimolar concentration of IL4 and IL10 for
10, 30 or 60 minutes. After 10 minutes, IL4-10 significantly
increased pJAK1 in sensory neurons compared to vehicle and IL4+IL10
(FIG. 10A). At 30 minutes after stimulation both IL4-10 and
IL4+IL10 increased pJAK1. To investigate whether JAK1 signaling is
required for the superior IL4-10 mediated pain inhibition,
carrageenan-injected animals were treated with Ruxolitinib (JAK1/2
inhibitor) for 3 consecutive days, starting one day before
intrathecal administration of IL4-10. Ruxolitinib almost completely
prevented the IL4-10-induced inhibition of thermal hyperalgesia
(FIG. 10B) and shortened the duration of IL4-10-induced inhibition
of mechanical (FIG. 10C) hyperalgesia. Inhibition of JAK1 with
Ruxolitinob did not affect IL4+IL10-mediated inhibition of
hyperalgesia. To verify whether other kinases identified based on
PAMgene and RNAseq data (c-Kit, PDGFR or c-Met) also contribute to
the pain-inhibiting effects of IL4-10, these were inhibited using
Dasatinib (Kit inhibitor), Masitinib (Kit and PDGFR inhibitor) and
JNJ-38877605 (c-Met inhibitor). Inhibition of c-Kit or combined
inhibition of c-kit and PDGFR did not affect IL4-10-induced
inhibition of thermal hyperalgesia (FIG. 10D). In contrast,
inhibition of c-Kit and c-Kit and PDGFR enhanced the IL4-10-induced
reduction in mechanical hyperalgesia by IL4-10 (FIG. 10E).
Pharmacological inhibition of c-Met partially attenuated the
pain-inhibiting effects of IL4-10 (FIG. 10 D, E).
[0365] These data suggest that pJAK1 and c-MET signaling contribute
to the superior pain-alleviating effect of IL4-10.
Example 8: Additional Polypeptide Constructs of the Disclosure
[0366] This example demonstrates design and generation of
non-limiting examples of IL4-containing compounds (e.g.,
polypeptide constructs, fusion proteins) of the disclosure.
[0367] An IL10/IL4 compound (e.g., polypeptide construct, fusion
protein) of the disclosure is designed. SEQ ID NO: 11 is joined to
SEQ ID NO: 15 using the SEQ ID NO: 38 linker, resulting in SEQ ID
NO: 46. A poly-histidine tag is added to the N-terminus, and the
construct is produced by transient transfection of HEK293E cells as
disclosed below.
[0368] Additional IL10/IL4 compounds (e.g., polypeptide constructs,
fusion proteins) are designed wherein any one of SEQ ID NOs: 11-14
(or a variant, derivative, or fragment thereof) is joined to SEQ ID
NO: 15 (or a variant, derivative, or fragment thereof), either
directly or via a linker as disclosed herein (for example, any one
of SEQ ID NOs: 38-45, or a multiple thereof). The compounds (e.g.,
polypeptide constructs, fusion proteins) are designed in both
orientations, e.g., with IL4 located on the C-terminal side of
IL10, or with IL4 located on the N-terminal side of IL10. An
affinity tag is added to the N-terminus and/or the C-terminus of
each construct, and the constructs are produced by transient
transfection of HEK293E cells as disclosed below.
[0369] An IL13/IL4 compound (e.g., polypeptide construct, fusion
protein) of the disclosure is designed. SEQ ID NO: 11 is joined to
SEQ ID NO: 22 using the SEQ ID NO: 38 linker, resulting in SEQ ID
NO: 47. A poly-histidine tag is added to the N-terminus, and the
construct is produced by transient transfection of HEK293E cells as
disclosed below.
[0370] Additional IL13/IL4 compounds (e.g., polypeptide constructs,
fusion proteins) are designed wherein any one of SEQ ID NOs: 11-14
(or a variant, derivative, or fragment thereof) is joined to any
one of SEQ ID NOs: 16-23 (or a variant, derivative, or fragment
thereof), either directly or via a linker as disclosed herein (for
example, any one of SEQ ID NOs: 38-45, or a multiple thereof). The
compounds (e.g., polypeptide constructs, fusion proteins) are
designed in both orientations, e.g., with IL4 located on the
C-terminal side of IL13, or with IL4 located on the N-terminal side
of IL13. An affinity tag is added to the N-terminus and/or the
C-terminus of each construct, and the constructs are produced by
transient transfection of HEK293E cells as disclosed below.
[0371] An IL27/IL4 compound (e.g., polypeptide construct, fusion
protein) of the disclosure is designed. SEQ ID NO: 11 is joined to
SEQ ID NO: 24 using the SEQ ID NO: 38 linker, resulting in SEQ ID
NO: 48. A poly-histidine tag is added to the N-terminus, and the
construct is produced by transient transfection of HEK293E cells as
disclosed below.
[0372] Additional IL27/IL4 compounds (e.g., polypeptide constructs,
fusion proteins) are designed wherein any one of SEQ ID NOs: 11-14
(or a variant, derivative, or fragment thereof) is joined to any
one of SEQ ID NOs: 24-45 (or a variant, derivative, or fragment
thereof), either directly or via a linker as disclosed herein (for
example, any one of SEQ ID NOs: 38-45, or a multiple thereof). The
polypeptide constructs (e.g., fusion proteins) are designed in both
orientations, e.g., with IL4 located on the C-terminal side of
IL27, or with IL4 located on the N-terminal side of IL27. An
affinity tag is added to the N-terminus and/or the C-terminus of
each construct, and the constructs are produced by transient
transfection of HEK293E cells as disclosed below.
[0373] An IL33/IL4 compound (e.g., polypeptide construct, fusion
protein) of the disclosure is designed. SEQ ID NO: 11 is joined to
SEQ ID NO: 26 using the SEQ ID NO: 38 linker, resulting in SEQ ID
NO: 49. A poly-histidine tag is added to the N-terminus, and the
construct is produced by transient transfection of HEK293E cells as
disclosed below.
[0374] Additional IL33/IL4 compounds (e.g., polypeptide constructs,
fusion proteins) are designed wherein any one of SEQ ID NOs: 11-14
(or a variant, derivative, or fragment thereof) is joined to any
one of SEQ ID NOs: 26-32 (or a variant, derivative, or fragment
thereof), either directly or via a linker as disclosed herein (for
example, any one of SEQ ID NOs: 38-45, or a multiple thereof). The
polypeptide constructs (e.g., fusion proteins) are designed in both
orientations, e.g., with IL4 located on the C-terminal side of
IL33, or with IL4 located on the N-terminal side of IL33. An
affinity tag is added to the N-terminus and/or the C-terminus of
each construct, and the constructs are produced by transient
transfection of HEK293E cells as disclosed below.
[0375] An TGF.beta.1/IL4 compound (e.g., polypeptide construct,
fusion protein) of the disclosure is designed. SEQ ID NO: 11 is
joined to SEQ ID NO: 34 using the SEQ ID NO: 38 linker, resulting
in SEQ ID NO: 50. A poly-histidine tag is added to the N-terminus,
and the construct is produced by transient transfection of HEK293E
cells as disclosed below.
[0376] Additional TGF.beta.1/IL4 compounds (e.g., polypeptide
constructs, fusion proteins) are designed wherein any one of SEQ ID
NOs: 11-14 (or a variant, derivative, or fragment thereof) is
joined to any one of SEQ ID NOs: 33-34 (or a variant, derivative,
or fragment thereof), either directly or via a linker as disclosed
herein (for example, any one of SEQ ID NOs: 38-45, or a multiple
thereof). The polypeptide constructs (e.g., fusion proteins) are
designed in both orientations, e.g., with IL4 located on the
C-terminal side of TGF.beta.1, or with IL4 located on the
N-terminal side of TGF.beta.1. An affinity tag is added to the
N-terminus and/or the C-terminus of each construct, and the
constructs are produced by transient transfection of HEK293E cells
as disclosed below.
[0377] An TGF.beta.2/IL4 compound (e.g., polypeptide construct,
fusion protein) of the disclosure is designed. SEQ ID NO: 11 is
joined to SEQ ID NO: 36 using the SEQ ID NO: 38 linker, resulting
in SEQ ID NO: 51. A poly-histidine tag is added to the N-terminus,
and the construct is produced by transient transfection of HEK293E
cells as disclosed below.
[0378] Additional TGF.beta.2/IL4 compounds (e.g., polypeptide
constructs, fusion proteins) are designed wherein any one of SEQ ID
NOs: 11-14 (or a variant, derivative, or fragment thereof) is
joined to any one of SEQ ID NOs: 35-37 (or a variant, derivative,
or fragment thereof), either directly or via a linker as disclosed
herein (for example, any one of SEQ ID NOs: 38-45, or a multiple
thereof). The polypeptide constructs (e.g., fusion proteins) are
designed in both orientations, e.g., with IL4 located on the
C-terminal side of TGF.beta.2, or with IL4 located on the
N-terminal side of TGF.beta.2. An affinity tag is added to the
N-terminus and/or the C-terminus of each construct, and the
constructs are produced by transient transfection of HEK293E cells
as disclosed below.
TABLE-US-00010 TABLE 10 Examples of compounds (e.g., polypeptide
constructs, fusion proteins) of the disclosure SEQ ID NO: SEQUENCE
46 HKCDITLQEIIKTLNSLTEQKTLCTELTVTDIFAASKNTTEKETFCRAATVLR
QFYSHHEKDTRCLGATAQQFHRHKQLIRFLKRLDRNLWGLAGLNSCPVKE
ANQSTLENFLERLKTIMREKYSKCSSGSGGGGSGTSPGQGTQSENSCTH
FPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQA
LSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCE
NKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN 47
HKCDITLQEIIKTLNSLTEQKTLCTELTVTDIFAASKNTTEKETFCRAATVLR
QFYSHHEKDTRCLGATAQQFHRHKQLIRFLKRLDRNLWGLAGLNSCPVKE
ANQSTLENFLERLKTIMREKYSKCSSGSGGGGSGTSPGPVPPSTALRELIE
ELVNITQNQKAPLCNGSMVWSINLTAGMYCAALESLINVSGCSAIEKTQRM
LSGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDLLLHLKKLFREGRFN 48
HKCDITLQEIIKTLNSLTEQKTLCTELTVTDIFAASKNTTEKETFCRAATVLR
QFYSHHEKDTRCLGATAQQFHRHKQLIRFLKRLDRNLWGLAGLNSCPVKE
ANQSTLENFLERLKTIMREKYSKCSSGSGGGGSGTFPRPPGRPQLSLQEL
RREFTVSLHLARKLLSEVRGQAHRFAESHLPGVNLYLLPLGEQLPDVSLTF
QAWRRLSDPERLCFISTTLQPFHALLGGLGTQGRWTNMERMQLWAMRLD
LRDLQRHLRFQVLAAGFNCPEEEEEEEEEEEEERKGLLPGALGSALQGPA
QVSWPQLLSTYRLLHSLELVLSRAVRELLLLSKAGHSVWPLGFPTLSPQP 49
HKCDITLQEIIKTLNSLTEQKTLCTELTVTDIFAASKNTTEKETFCRAATVLR
QFYSHHEKDTRCLGATAQQFHRHKQLIRFLKRLDRNLWGLAGLNSCPVKE
ANQSTLENFLERLKTIMREKYSKCSSGSGGGGSGTMKPKMKYSTNKISTA
KWKNTASKALCFKLGKSQQKAKEVCPMYFMKLRSGLMIKKEACYFRRETT
KRPSLKTGRKHKRHLVLAACQQQSTVECFAFGISGVQKYTRALHDSSITGI
SPITEYLASLSTYNDQSITFALEDESYEIYVEDLKKDEKKDKVLLSYYESQHP
SNESGDGVDGKMLMVTLSPTKDFWLHANNKEHSVELHKCEKPLPDQAFF
VLHNMHSNCVSFECKTDPGVFIGVKDNHLALIKVDSSENLCTENILFKLSET 50
HKCDITLQEIIKTLNSLTEQKTLCTELTVTDIFAASKNTTEKETFCRAATVLR
QFYSHHEKDTRCLGATAQQFHRHKQLIRFLKRLDRNLWGLAGLNSCPVKE
ANQSTLENFLERLKTIMREKYSKCSSGSGGGGSGTALDTNYCFSSTEKNC
CVRQLYIDFRKDLGWKWIHEPKGYHANFCLGPCPYIWSLDTQYSKVLALY
NQHNPGASAAPCCVPQALEPLPIVYYVGRKPKVEQLSNMIVRSCKCS 51
HKCDITLQEIIKTLNSLTEQKTLCTELTVTDIFAASKNTTEKETFCRAATVLR
QFYSHHEKDTRCLGATAQQFHRHKQLIRFLKRLDRNLWGLAGLNSCPVKE
ANQSTLENFLERLKTIMREKYSKCSSGSGGGGSGTALDAAYCFRNVQDN
CCLRPLYIDFKRDLGWKWIHEPKGYNANFCAGACPYLWSSDTQHSRVLSL
YNTINPEASASPCCVSQDLEPLTILYYIGKTPKIEQLSNMIVKSCKCS
[0379] Additional IL4/IL4 compounds (e.g., polypeptide constructs,
fusion proteins) are designed wherein any one of SEQ ID NOs: 11-14
(or a variant, derivative, or fragment thereof) is joined to anyone
of SEQ ID NOs: 11-14 (or a variant, derivative, or fragment
thereof), either directly or via a linker as disclosed herein (for
example, any one of SEQ ID NOs: 38-45, or a multiple thereof). The
polypeptide constructs (e.g., fusion proteins) are designed in both
orientations, e.g., with IL4 located on the C-terminal side of
TGF.beta.2, or with IL4 located on the N-terminal side of
TGF.beta.2. An affinity tag is added to the N-terminus and/or the
C-terminus of each construct, and the constructs are produced by
transient transfection of HEK293E cells as disclosed below.
[0380] IL4-containing fusion proteins of the disclosure are
produced by transient transfection of HEK293E cells. Cells are
transfected with a pUPE expression vector containing a transgene
coding one of the IL4-containing fusion protein sequences. To
enable purification, a hexa-histidine affinity tag is cloned at the
N-terminus of each IL4-containing protein. Six days post
transfection, conditioned medium containing recombinant protein is
harvested by low-speed centrifugation (10 minutes, 1000.times.g)
followed by high-speed centrifugation (10 minutes,
4000.times.g).
[0381] Proteins are purified via His-tag by Immobilized Metal
Affinity Chromatography (IMAC). In short, the recombinant protein
is bound to 0.5 ml Nickel Sepharose.RTM. excel at 20.degree. C.
Nickel Sepharose.RTM. excel containing bound protein is harvested
by centrifugation and transferred into a gravity flow column.
Non-specifically bound proteins are removed by washing the column
with IMAC buffer (500 mM Sodium Chloride, 25 mM Tris, pH=8.2)
containing 0 and 10 mM imidazol. The proteins are eluted with IMAC
buffer containing 500 mM imidazol. Fractions of 2.5 ml are
collected. Recombinant protein-containing fractions are pooled.
Conditioned medium and the unbound IMAC fraction are analyzed by
LabChip.RTM. capillary electrophoresis. The IMAC pool is
concentrated to 2-4 ml using an Amicon 10 kDa spin filter.
Aggregates are removed by centrifugation (10 minutes 18000.times.g,
4.degree. C.).
[0382] The proteins are purified further by gel filtration using a
Superdex200 16/600 column that has been equilibrated in PBS.
Protein containing fractions are analyzed by LabChip.RTM. capillary
electrophoresis and recombinant protein containing fractions are
pooled. Protein pools are sterilized by filtration using a 0.22
.mu.m syringe filter and the product stored in 1 ml vials at
80.degree. C.
[0383] Protein assays: Protein concentration in batches is
determined spectrophotometrically by measuring the absorbance at
280 nm (DropSense16, Trinean) and using a BCA (Thermo Scientific)
protein assay.
[0384] SDS-Page: Purified proteins are analysed on 12%
polyacrylamide SDS-Page gels (Bio-Rad) and bands are visualized by
InstantBlue.RTM. protein stain (Expedeon; Cambridge).
Example 9: Effects of Compounds (e.g., Polypeptide Constructs,
Fusion Proteins) of the Disclosure on Neuronal Sensitization
[0385] Experiments are conducted to determine whether
IL4-containing compounds (e.g., polypeptide constructs, fusion
proteins) of the disclosure affect neuronal sensitization. Cultured
neurons are treated with TNF.alpha. (50 ng/ml) with and without a
compound (e.g., polypeptide construct, fusion protein) of the
disclosure, (e.g., at approximately 3 nM). TNF.alpha. increases the
magnitude of capsaicin-induced calcium influxes compared to
untreated cells. Co-treatment with a compound (e.g., polypeptide
construct, fusion protein) of the disclosure reduces the
sensitization of capsaicin-evoked calcium influx by TNF.alpha..
Similar experiments are conducted to determine how sensitivity to
PGE2 is affected. Additional experiments are conducted to determine
whether the compound of the disclosure affects capsaicin-induced
calcium responses in non-sensitized neurons. Constructs are
identified that reduce neuronal sensitization by inflammatory
mediators. For each polypeptide construct, controls are included to
determine the effect of the individual cytokines, and the
combination of the cytokines, that the compound (e.g., polypeptide
construct, fusion protein) comprises. Constructs are identified
that exhibit superiority over individual cytokines, and the
combination of cytokines.
Example 10: Compounds (e.g., Polypeptide Constructs, Fusion
Proteins) of the Disclosure Crosslink Cytokine Receptors
[0386] Experiments are conducted to determine whether compounds
(e.g., polypeptide constructs, fusion proteins) of the disclosure
crosslink cytokine receptors. Sensory neurons are incubated with
polypeptide constructs of the disclosure or the combination of the
respective cytokines for 15 minutes in vitro followed by a
proximity ligation assay (PLA) to assess receptor clustering.
Constructs are identified that induce crosslinking of cytokine
receptors.
Example 11: Compounds (e.g., Polypeptide Constructs, Fusion
Proteins) of the Disclosure Elicit Distinct Kinase Activity
Profiles Compared to the Combination of Unlinked Cytokines
[0387] Experiments are conducted to determine whether compounds
(e.g., polypeptide constructs, fusion proteins) of the disclosure
elicit distinct kinase activity profiles compared to the
combination of unlinked cytokines.
[0388] Persistent inflammatory pain is induced in mice as using
carrageenan as disclosed herein. PAMgene kinase activity profiling
is performed to assess global protein tyrosine kinases (PTK) and
serine/threonine kinases (STK) activity in homogenates of lumbar
DRGs of mice after administration of protein constructs of the
disclosure, or their component cytokines in combination. Kinomic
profiles are assessed at 30, 60 and 240 minutes after intrathecal
administration with either the polypeptide construct, the
combination of cytokines, or PBS. To predict the putative upstream
kinases, differential phosphorylation profiles are loaded into
PhosphoNET, and pathway analysis is conducted. Compounds (e.g.,
polypeptide constructs, fusion proteins) are identified that elicit
different kinomic profiles to the combination of their component
cytokines. Differentially phosphorylated substrates, differentially
active upstream kinases, and differentially active pathways are
identified.
Example 12: Compounds (e.g., Polypeptide Constructs, Fusion
Proteins) of the Disclosure Induces Distinct Transcriptomes
Compared to the Combination of Unlinked Cytokines
[0389] Experiments are conducted to determine whether compounds
(e.g., polypeptide constructs, fusion proteins) of the disclosure
elicit distinct transcriptomic profiles compared to the combination
of unlinked cytokines.
[0390] Persistent inflammatory pain is induced in mice as using
carrageenan as disclosed herein. RNA sequencing of DRGs is done six
hours after administering a compound of the disclosure, or their
component cytokines in combination. Principal component analysis
(PCA) and hierarchical clustering of the top differentially
regulated genes, and pathway analysis are conducted. Compounds
(e.g., polypeptide constructs, fusion proteins) are identified that
elicit different transcriptomic profiles to the combination of
their component cytokines. Differentially expressed genes and
pathways are identified.
Example 13: In Vivo Assessment of Compounds (e.g., Polypeptide
Constructs, Fusion Proteins) of the Disclosure
[0391] Assays are conducted to assess whether the IL4-containing
compounds (e.g., polypeptide constructs, fusion proteins) can
treat, for example, pain, chemotherapy-induced polyneuropathy
(CIPN) pain, nerve fiber loss, and allodynia. The assays are
conducted for any compound (e.g., polypeptide construct, fusion
protein) disclosed herein, for example, polypeptide constructs
comprising an IL4 and a regulatory cytokine, e.g., an IL10/IL4,
IL13/IL4, IL27/IL4, IL33/IL4, TGF.beta.1/IL4, TGF.beta.2/IL4, or
IL4/IL4 of the disclosure.
[0392] To induce transient chemotherapy-induced polyneuropathy
(CIPN), paclitaxel (2 mg/kg, Cayman Chemical Company) is injected
intraperitoneally into C57BL/6 mice on days 0 and 2. To induce
persistent paclitaxel-induced CIPN, paclitaxel (8 mg/kg, Cayman
Chemical Company) is injected intraperitoneally on day 0, 2, 4 and
6. To induce persistent oxaliplatin-induced polyneuropathy, mice
receive two treatment cycles, each consisting of 5 daily
intraperitoneal injections of 3 mg/kg oxaliplatin (Tocris) with a 5
days free interval. To induce inflammatory hyperalgesia, mice
receive an intraplantar injection of 20 .mu.l .lamda.-carrageenan
(2% (w/v), Sigma-Aldrich) dissolved in saline solution (NaCl 0.9%)
in both hind paws.
[0393] Noxious mechanical sensitivity in the hind paws is measured
using von Frey hairs (Stoelting, Wood Dale, USA). Results are
expressed as the 50% paw-withdrawal threshold using the up-and-down
method. Thermal hyperalgesia is assessed by determining the heat
withdrawal latency times using the Hargreaves test (IITC Life
Science). In some experiments the length of intraepidermal nerve
fibers in the paw skin at day 15 is determined by immunofluorescent
staining of skin biopsies with the neuronal marker PGP9.5. All
experiments are performed in a blinded manner.
[0394] IL4-containing compounds (e.g., polypeptide constructs,
fusion proteins) are administered to mice, e.g., via intrathecal
injection under light isoflurane/O2 anaesthesia, or by another
route as disclosed herein. The ability of IL4-containing compounds
(e.g., polypeptide constructs, fusion proteins) of the disclosure
to inhibit paclitaxel or oxaliplatin-induced neuropathy,
carrageenan-induced inflammatory hyperalgesia, and intra-epidermal
nerve fibre loss, is determined and compared to the combination of
unlinked cytokines for each construct.
Example 14: Neuroprotective Effects of Compounds (e.g., Polypeptide
Constructs, Fusion Proteins) of the Disclosure
[0395] Assays are conducted to assess whether the IL4-containing
compounds (e.g., polypeptide constructs, fusion proteins) possess
neuroprotective properties, e.g., inhibit paclitaxel-induced
reduction of neurite length. The assays are conducted for any
compound (e.g., polypeptide construct, fusion protein) disclosed
herein, for example, polypeptide constructs comprising an IL4 and a
regulatory cytokine, e.g., an IL13/IL4, IL10/IL4, IL13/IL4,
IL27/IL4, IL33/IL4, TGF.beta.1/IL4, TGF.beta.2/IL4, or IL4/IL4 of
the disclosure.
[0396] Culture of DRG neurons: DRGs are cultured as described
previously (Nat. Commun. 4, 1682 (2013)). Briefly, DRGs are
dissected and placed on ice-cold dissection medium (HBSS w/o Ca2+
and Mg2+, 5 mM HEPES, and 10 mM glucose). After dissection, axons
are cut and dissection medium is replaced by filtered enzyme mix
(HBSS without Ca2+ and Mg2+, 5 mM HEPES, 10 mM glucose, 5 mg/ml
collagenase type XI (Sigma), and 10 mg/ml Dispase (Gibco)). The
DRGs are incubated in enzyme mix for 30 minutes at 37.degree. C.
and 5% C02. Subsequently, enzyme mix is inactivated with
heat-inactivated fetal bovine serum (FBS, Sigma). Cells are
cultured in Dulbecco's modified Eagle's medium (Gibco) containing
10% FBS (Gibco), 2 mmol/L glutamine (Gibco), 10,000 IU/ml
penicillin-streptomycin (Gibco) on poly-L-lysine (0.01 mg/ml,
Sigma) and laminin (0.02 mg/ml, Sigma)-coated glass coverslips in a
5% CO2 incubator at 37.degree. C. Cells are used the following 1-2
days.
[0397] Treatments and neurite length measurement: After 24 h in
culture, DRG neurons are treated with Paclitaxel (1 .mu.M) alone,
or in the presence of different concentrations of IL4 compounds
(e.g., polypeptide constructs, fusion proteins) (e.g., 0.12 nM, 0.6
nM, or 3 nM), or equimolar doses of the individual cytokines that
are present in the constructs for 24h. Neurites are visualised
using p3-tubulin staining, whilst the number of sensory neurons is
determined using NeuN staining. Pictures are taken and for each
picture, the neurite length per sensory neuron is determined. The
neurite length is averaged to a single value for each animal and
condition. The percentage inhibition of paclitaxel-induced neurite
length loss per mouse culture of 1 mouse is calculated according
the following formula
((.mu..sub.control-.mu..sub.paclitaxel)-(.mu..sub.control-X.sub.cytokine)-
)/(.mu..sub.control-.mu..sub.paclitaxel)*100 (where p is the
average neurite length per neuron averaged over all samples and X
is the neurite length of each individual sample).
[0398] .beta.3-tubulin and NeuN staining: cells are fixed in 4% PFA
for 10 minutes, permeabilized with PBS with 0.05% Tween-20,
followed by incubation in blocking buffer (1% BSA and 5% Normal
donkey serum in PBS with 0.05% Tween-20 and 0.01% triton) for 1
hour. Cells are incubated with rabbit anti-.beta.3 tubulin
(Anti-beta III Tubulin antibody-Neuronal Marker, ab18207, 1:1500,
Abcam, Cambridge, UK) and NeuN (Anti-NeuN Antibody clone A60,
MAB377, 1:500) Sigma Aldrich (Merck), Darmstadt, Germany) overnight
at 4.degree. C., followed by washes and incubation with
AF488-conjugated donkey anti-rabbit and 568-conjugated donkey
anti-mouse secondary antibodies (Thermofisher, 1:500) followed by
DAPI (1:5000, Sigma) staining before sections are mounted on slides
with FluorSave reagent (Millipore).
[0399] Images are taken using an Olympus IX83 microscope (Olympus).
Pictures are analysed using CellSens software (Olympus) and ImageJ
(NIH), using the NeuralNetrics macro kindly provided by prof. dr.
Winnok de Vos (University of Antwerp) (Pani G, De Vos W H, Samari
N, de Saint-Georges L, Baatout S, Van Oostveldt P, et al.
MorphoNeuroNet: An automated method for dense neurite network
analysis. Cytom Part A. 2014 February; 85(2):188-99). Other plugins
used are Olympus Viewer plugin (Olympus). Cell sense software is
used to automatically count number of neurons based on NeuN
staining. Neurite length is determined using the NeuralNetric macro
in ImageJ (Fuij).
Example 15. An IL4-Containing Fusion Protein of the Disclosure
Elicits a Distinct Kinase Activity Profile Compared to a
Combination of Unlinked Cytokines
[0400] Animals: All animal experiments were performed in accordance
with international guidelines and with prior approval from the
University Medical Center Utrecht experimental animal committee.
Experiments were conducted with 8-14 weeks old male and female wild
type (WT) C57BL/6 mice.
[0401] Paclitaxel-induced CIPN: At day 0, 2, 4 and 6 animals were
injected intraperitoneally with 8 mg/kg of Paclitaxel (diluted in
Cremophor:EtOH 1:1; volume of injection 40 .mu.l/10 g of
bodyweight. At day 8, animals received i.t. injections of: IL4/IL13
fusion protein (0.3 .mu.g), IL4+IL13 (0.15 .mu.g each) or
Vehicle.
[0402] Drugs and administration: The IL4/IL13 was produced by
transient transfection of HEK293F cells with the pcDNA3.1-neo
expression vector (Invitrogen; Carlsbad, Calif.) with dual CMV
promotor. The vector contained two transgenes: cDNA coding for
IL4/IL13 fusion protein and cDNA coding beta-galactoside-2,
3-sialyl-transferase to optimize glycan capping with sialic acid.
The IL4/IL13 contained a 6-His tag at the N terminus and was
purified through HIS-Select Nickel Affinity gel (Sigma). IL4/IL13
concentrations were determined with an IL4 ELISA kit (IL-4 Pelipair
ELISA kit; Sanquin) and Bicinchoninic Acid Protein Assay (BCA
Pierce Protein Assay Kit, ThermoFisher Scientific). Intrathecal
(i.t.) injections of different compounds (5 .mu.l/mouse) were
performed as described before (J Neurosci 30, 2138-2149, 2010)
under light isoflurane/O.sub.2 anesthesia. The IL4/IL13 (0.3
.mu.g/mouse) or equimolar doses (0.15 .mu.g each/mouse) of
recombinant human HEK-produced IL4 (Sigma) and IL13 (2bsciences)
were injected intrathecally at day 8 after the first paclitaxel
injection.
[0403] Kinase Activity Profiling: Animals were killed an hour after
intrathecal injection of the IL4/IL13 fusion protein, IL4+IL13 or
vehicle, followed by immediate DRG isolation. Lumbar DRGs were
homogenized using M-PER mammalian Extraction buffer (Pierce)
supplemented with phosphatase and protease inhibitor cocktails
(Pierce). Protein concentration was determined using the Bradford
assay (Bio-Rad). Kinase activity profiling was performed using the
Tyrosine Kinase PamChip.RTM. (PTK) Array for Pamstation.RTM.12
(PamGene International B.V.). For the PTK array, 7.5 .mu.g of
protein lysate per array was used. Image quantification and
statistical analysis were performed using BioNavigator.RTM.
Software (PamGene International B.V.). Upstream kinase analysis was
performed using BioNavigator.RTM. Software with peptide-kinase
mapping using Kinexus phosphonet enrichment files
(www_phosphonet.ca/).
Results
[0404] To elucidate downstream signaling in sensory neurons in an
unbiased manner, PamGene kinase activity profiling was performed to
assess global protein tyrosine kinases (PTK) activity in
homogenates of lumbar DRGs isolated from mice with persistent
paclitaxel-induced CIPN after IL4/IL13 fusion protein, IL4+IL13
(combination of unlinked cytokines), and vehicle administration.
Kinomic profiles were assessed at 60 minutes after intrathecal
administration of the IL4/IL13 fusion protein, the combination of
cytokines, or vehicle (PBS). Naive mice (i.e. not treated with
paclitaxel or IL4/IL13 fusion protein) were also included.
[0405] FIG. 11 illustrates peptides that were differentially
phosphorylated based on one-way ANOVA analysis between IL4/IL13,
IL4+IL13, and vehicle-treated mice compared to naive mice
(untreated; no paclitaxel, no intrathecal injection). Black
indicates no significant changes, while color indicates decreased
phosphorylation. Analyses of the peptides that were differentially
phosphorylated by PTK in the DRG homogenates of IL4/IL13-treated
versus IL4 plus IL13-treated mice and vehicle-treated mice,
indicated that in total 19 peptides were uniquely phosphorylated
upon treatment with the fusion protein.
[0406] Analyses of the peptides that were differentially
phosphorylated by PTK in the DRG homogenates of IL4/IL13-treated
male and female mice versus IL4 plus IL13-treated mice, indicated
that in both sexes the activity of different kinases is
differentially affected by the IL4/IL13 compared to the combination
of cytokines (FIG. 12 and FIG. 13).
[0407] In DRGs from female mice treated with the IL4/IL13 fusion
protein, the activity of several kinases was reduced when compared
female mice treated with the combination of IL4 and IL13 (FIG. 12).
The graph shows the predicted upstream kinases inferred from the
differentially phosphorylated peptide substrates on the
PamChips.RTM. identified by unpaired t-test comparison between
samples from IL4/IL13 fusion protein-treated females and
IL4+IL13-treated females (n=3 animals per group). The top 5
predicted putative kinases affected (according to summation of
sensitivity and specificity score) were: ITK, RET, TYK2, FER and
ERBB4.
[0408] In male mice, unique kinase activity was predominantly
increased (FIG. 13). The graph shows predicted upstream kinases
that can be inferred from the differentially phosphorylated peptide
substrates on the PamChips.RTM. identified by unpaired t-test
comparison between samples from IL4/IL13 fusion protein-treated
males and IL4+IL13-treated males (n=3 animals per group). The top 5
predicted putative kinases affected were: LTK, RYK, ALK, AXL and
BLK.
[0409] These data show that IL4/IL13 uniquely regulates sets of
kinases compared to the combination of unlinked IL4 plus IL13.
CONCLUSIONS
[0410] In the present work the inventors consider that the coupling
of IL4R binding region and a cytokine receptor (e.g., IL10R, IL13R,
IL27R, IL33R, TGF.beta.1R, TGF.beta.2R) binding region in a single
molecule/protein creates a novel molecule that is able to target
regulatory cytokine receptors in sensory neurons to trigger unique
signalling pathways (e.g., pain resolution pathways) that are not
activated by the combination of both wild-type cytokines. Sensory
neurons and other cells in the dorsal root ganglion and spinal cord
express functional cytokine receptors (e.g., IL4R, IL10R IL13R,
IL10R, IL33R, TGF.beta.1R, and TGF.beta.2R), and cross-linking
these receptors by a compound (e.g., polypeptide construct, fusion
protein) or a bispecific antibody or any other compound that can
bind at least two of these receptors in sensory neurons, can be
used to stop, for example, chronic pain, either nociceptive,
neuropathic or combined nociceptive-neuropathic pain. Furthermore,
this unique signalling induced by cross-linking of receptors of
regulatory cytokines on neurons and glial cells, prevents neuronal
damage.
Sequence CWU 1
1
52120DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 1tggaaaggct tatacccctc 20220DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 2ccgctgttct caggtgacat 20319DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 3tgaagcaggc atctgaggg 19420DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 4cgaaggtgga agagtgggag 20519DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 5tcctcctcag accgctttt 19621DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 6cctggttcat catcgctaat c 21724DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 7gatgcacagt aggtctaagt ggag 24820DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 8cactcagggc aggtgaaact 20920DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 9tctgcatccc gttgttttgc 201020DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 10gcacctgtgc atcctgaatg 2011129PRTHomo sapiens
11His Lys Cys Asp Ile Thr Leu Gln Glu Ile Ile Lys Thr Leu Asn Ser1
5 10 15Leu Thr Glu Gln Lys Thr Leu Cys Thr Glu Leu Thr Val Thr Asp
Ile 20 25 30Phe Ala Ala Ser Lys Asn Thr Thr Glu Lys Glu Thr Phe Cys
Arg Ala 35 40 45Ala Thr Val Leu Arg Gln Phe Tyr Ser His His Glu Lys
Asp Thr Arg 50 55 60Cys Leu Gly Ala Thr Ala Gln Gln Phe His Arg His
Lys Gln Leu Ile65 70 75 80Arg Phe Leu Lys Arg Leu Asp Arg Asn Leu
Trp Gly Leu Ala Gly Leu 85 90 95Asn Ser Cys Pro Val Lys Glu Ala Asn
Gln Ser Thr Leu Glu Asn Phe 100 105 110Leu Glu Arg Leu Lys Thr Ile
Met Arg Glu Lys Tyr Ser Lys Cys Ser 115 120 125Ser12113PRTHomo
sapiens 12His Lys Cys Asp Ile Thr Leu Gln Glu Ile Ile Lys Thr Leu
Asn Ser1 5 10 15Leu Thr Glu Gln Lys Asn Thr Thr Glu Lys Glu Thr Phe
Cys Arg Ala 20 25 30Ala Thr Val Leu Arg Gln Phe Tyr Ser His His Glu
Lys Asp Thr Arg 35 40 45Cys Leu Gly Ala Thr Ala Gln Gln Phe His Arg
His Lys Gln Leu Ile 50 55 60Arg Phe Leu Lys Arg Leu Asp Arg Asn Leu
Trp Gly Leu Ala Gly Leu65 70 75 80Asn Ser Cys Pro Val Lys Glu Ala
Asn Gln Ser Thr Leu Glu Asn Phe 85 90 95Leu Glu Arg Leu Lys Thr Ile
Met Arg Glu Lys Tyr Ser Lys Cys Ser 100 105 110Ser13129PRTHomo
sapiens 13His Lys Arg Asp Ile Thr Leu Gln Glu Ile Ile Lys Thr Leu
Asn Ser1 5 10 15Leu Thr Glu Gln Lys Thr Leu Cys Thr Glu Leu Thr Val
Thr Asp Ile 20 25 30Phe Ala Ala Ser Lys Asn Thr Thr Glu Lys Glu Thr
Phe Cys Arg Ala 35 40 45Ala Thr Val Leu Arg Gln Phe Tyr Ser His His
Glu Lys Asp Thr Arg 50 55 60Cys Leu Gly Ala Thr Ala Gln Gln Phe His
Arg His Lys Gln Leu Ile65 70 75 80Arg Phe Leu Lys Arg Leu Asp Arg
Asn Leu Trp Gly Leu Ala Gly Leu 85 90 95Asn Ser Cys Pro Val Lys Glu
Ala Asn Gln Ser Thr Leu Glu Asn Phe 100 105 110Leu Glu Arg Leu Lys
Thr Ile Met Arg Glu Lys Tyr Ser Lys Cys Ser 115 120
125Ser14113PRTHomo sapiens 14His Lys Arg Asp Ile Thr Leu Gln Glu
Ile Ile Lys Thr Leu Asn Ser1 5 10 15Leu Thr Glu Gln Lys Asn Thr Thr
Glu Lys Glu Thr Phe Cys Arg Ala 20 25 30Ala Thr Val Leu Arg Gln Phe
Tyr Ser His His Glu Lys Asp Thr Arg 35 40 45Cys Leu Gly Ala Thr Ala
Gln Gln Phe His Arg His Lys Gln Leu Ile 50 55 60Arg Phe Leu Lys Arg
Leu Asp Arg Asn Leu Trp Gly Leu Ala Gly Leu65 70 75 80Asn Ser Cys
Pro Val Lys Glu Ala Asn Gln Ser Thr Leu Glu Asn Phe 85 90 95Leu Glu
Arg Leu Lys Thr Ile Met Arg Glu Lys Tyr Ser Lys Cys Ser 100 105
110Ser15160PRTHomo sapiens 15Ser Pro Gly Gln Gly Thr Gln Ser Glu
Asn Ser Cys Thr His Phe Pro1 5 10 15Gly Asn Leu Pro Asn Met Leu Arg
Asp Leu Arg Asp Ala Phe Ser Arg 20 25 30Val Lys Thr Phe Phe Gln Met
Lys Asp Gln Leu Asp Asn Leu Leu Leu 35 40 45Lys Glu Ser Leu Leu Glu
Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala 50 55 60Leu Ser Glu Met Ile
Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala65 70 75 80Glu Asn Gln
Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly Glu 85 90 95Asn Leu
Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu 100 105
110Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala Phe
115 120 125Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu
Phe Asp 130 135 140Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met
Lys Ile Arg Asn145 150 155 16016113PRTHomo sapiens 16Pro Gly Pro
Val Pro Pro Ser Thr Ala Leu Arg Glu Leu Ile Glu Glu1 5 10 15Leu Val
Asn Ile Thr Gln Asn Gln Lys Ala Pro Leu Cys Asn Gly Ser 20 25 30Met
Val Trp Ser Ile Asn Leu Thr Ala Gly Met Tyr Cys Ala Ala Leu 35 40
45Glu Ser Leu Ile Asn Val Ser Gly Cys Ser Ala Ile Glu Lys Thr Gln
50 55 60Arg Met Leu Ser Gly Phe Cys Pro His Lys Val Ser Ala Gly Gln
Phe65 70 75 80Ser Ser Leu His Val Arg Asp Thr Lys Ile Glu Val Ala
Gln Phe Val 85 90 95Lys Asp Leu Leu Leu His Leu Lys Lys Leu Phe Arg
Glu Gly Gln Phe 100 105 110Asn17112PRTHomo sapiens 17Gly Pro Val
Pro Pro Ser Thr Ala Leu Arg Glu Leu Ile Glu Glu Leu1 5 10 15Val Asn
Ile Thr Gln Asn Gln Lys Ala Pro Leu Cys Asn Gly Ser Met 20 25 30Val
Trp Ser Ile Asn Leu Thr Ala Gly Met Tyr Cys Ala Ala Leu Glu 35 40
45Ser Leu Ile Asn Val Ser Gly Cys Ser Ala Ile Glu Lys Thr Gln Arg
50 55 60Met Leu Ser Gly Phe Cys Pro His Lys Val Ser Ala Gly Gln Phe
Ser65 70 75 80Ser Leu His Val Arg Asp Thr Lys Ile Glu Val Ala Gln
Phe Val Lys 85 90 95Asp Leu Leu Leu His Leu Lys Lys Leu Phe Arg Glu
Gly Gln Phe Asn 100 105 11018114PRTHomo sapiens 18Ser Pro Gly Pro
Val Pro Pro Ser Thr Ala Leu Arg Glu Leu Ile Glu1 5 10 15Glu Leu Val
Asn Ile Thr Gln Asn Gln Lys Ala Pro Leu Cys Asn Gly 20 25 30Ser Met
Val Trp Ser Ile Asn Leu Thr Ala Gly Met Tyr Cys Ala Ala 35 40 45Leu
Glu Ser Leu Ile Asn Val Ser Gly Cys Ser Ala Ile Glu Lys Thr 50 55
60Gln Arg Met Leu Ser Gly Phe Cys Pro His Lys Val Ser Ala Gly Gln65
70 75 80Phe Ser Ser Leu His Val Arg Asp Thr Lys Ile Glu Val Ala Gln
Phe 85 90 95Val Lys Asp Leu Leu Leu His Leu Lys Lys Leu Phe Arg Glu
Gly Gln 100 105 110Phe Asn19122PRTHomo sapiens 19Leu Thr Cys Leu
Gly Gly Phe Ala Ser Pro Gly Pro Val Pro Pro Ser1 5 10 15Thr Ala Leu
Arg Glu Leu Ile Glu Glu Leu Val Asn Ile Thr Gln Asn 20 25 30Gln Lys
Ala Pro Leu Cys Asn Gly Ser Met Val Trp Ser Ile Asn Leu 35 40 45Thr
Ala Gly Met Tyr Cys Ala Ala Leu Glu Ser Leu Ile Asn Val Ser 50 55
60Gly Cys Ser Ala Ile Glu Lys Thr Gln Arg Met Leu Ser Gly Phe Cys65
70 75 80Pro His Lys Val Ser Ala Gly Gln Phe Ser Ser Leu His Val Arg
Asp 85 90 95Thr Lys Ile Glu Val Ala Gln Phe Val Lys Asp Leu Leu Leu
His Leu 100 105 110Lys Lys Leu Phe Arg Glu Gly Gln Phe Asn 115
12020113PRTHomo sapiens 20Pro Gly Pro Val Pro Pro Ser Thr Ala Leu
Arg Glu Leu Ile Glu Glu1 5 10 15Leu Val Asn Ile Thr Gln Asn Gln Lys
Ala Pro Leu Cys Asn Gly Ser 20 25 30Met Val Trp Ser Ile Asn Leu Thr
Ala Gly Met Tyr Cys Ala Ala Leu 35 40 45Glu Ser Leu Ile Asn Val Ser
Gly Cys Ser Ala Ile Glu Lys Thr Gln 50 55 60Arg Met Leu Ser Gly Phe
Cys Pro His Lys Val Ser Ala Gly Gln Phe65 70 75 80Ser Ser Leu His
Val Arg Asp Thr Lys Ile Glu Val Ala Gln Phe Val 85 90 95Lys Asp Leu
Leu Leu His Leu Lys Lys Leu Phe Arg Glu Gly Arg Phe 100 105
110Asn21112PRTHomo sapiens 21Gly Pro Val Pro Pro Ser Thr Ala Leu
Arg Glu Leu Ile Glu Glu Leu1 5 10 15Val Asn Ile Thr Gln Asn Gln Lys
Ala Pro Leu Cys Asn Gly Ser Met 20 25 30Val Trp Ser Ile Asn Leu Thr
Ala Gly Met Tyr Cys Ala Ala Leu Glu 35 40 45Ser Leu Ile Asn Val Ser
Gly Cys Ser Ala Ile Glu Lys Thr Gln Arg 50 55 60Met Leu Ser Gly Phe
Cys Pro His Lys Val Ser Ala Gly Gln Phe Ser65 70 75 80Ser Leu His
Val Arg Asp Thr Lys Ile Glu Val Ala Gln Phe Val Lys 85 90 95Asp Leu
Leu Leu His Leu Lys Lys Leu Phe Arg Glu Gly Arg Phe Asn 100 105
11022114PRTHomo sapiens 22Ser Pro Gly Pro Val Pro Pro Ser Thr Ala
Leu Arg Glu Leu Ile Glu1 5 10 15Glu Leu Val Asn Ile Thr Gln Asn Gln
Lys Ala Pro Leu Cys Asn Gly 20 25 30Ser Met Val Trp Ser Ile Asn Leu
Thr Ala Gly Met Tyr Cys Ala Ala 35 40 45Leu Glu Ser Leu Ile Asn Val
Ser Gly Cys Ser Ala Ile Glu Lys Thr 50 55 60Gln Arg Met Leu Ser Gly
Phe Cys Pro His Lys Val Ser Ala Gly Gln65 70 75 80Phe Ser Ser Leu
His Val Arg Asp Thr Lys Ile Glu Val Ala Gln Phe 85 90 95Val Lys Asp
Leu Leu Leu His Leu Lys Lys Leu Phe Arg Glu Gly Arg 100 105 110Phe
Asn23122PRTHomo sapiens 23Leu Thr Cys Leu Gly Gly Phe Ala Ser Pro
Gly Pro Val Pro Pro Ser1 5 10 15Thr Ala Leu Arg Glu Leu Ile Glu Glu
Leu Val Asn Ile Thr Gln Asn 20 25 30Gln Lys Ala Pro Leu Cys Asn Gly
Ser Met Val Trp Ser Ile Asn Leu 35 40 45Thr Ala Gly Met Tyr Cys Ala
Ala Leu Glu Ser Leu Ile Asn Val Ser 50 55 60Gly Cys Ser Ala Ile Glu
Lys Thr Gln Arg Met Leu Ser Gly Phe Cys65 70 75 80Pro His Lys Val
Ser Ala Gly Gln Phe Ser Ser Leu His Val Arg Asp 85 90 95Thr Lys Ile
Glu Val Ala Gln Phe Val Lys Asp Leu Leu Leu His Leu 100 105 110Lys
Lys Leu Phe Arg Glu Gly Arg Phe Asn 115 12024215PRTHomo sapiens
24Phe Pro Arg Pro Pro Gly Arg Pro Gln Leu Ser Leu Gln Glu Leu Arg1
5 10 15Arg Glu Phe Thr Val Ser Leu His Leu Ala Arg Lys Leu Leu Ser
Glu 20 25 30Val Arg Gly Gln Ala His Arg Phe Ala Glu Ser His Leu Pro
Gly Val 35 40 45Asn Leu Tyr Leu Leu Pro Leu Gly Glu Gln Leu Pro Asp
Val Ser Leu 50 55 60Thr Phe Gln Ala Trp Arg Arg Leu Ser Asp Pro Glu
Arg Leu Cys Phe65 70 75 80Ile Ser Thr Thr Leu Gln Pro Phe His Ala
Leu Leu Gly Gly Leu Gly 85 90 95Thr Gln Gly Arg Trp Thr Asn Met Glu
Arg Met Gln Leu Trp Ala Met 100 105 110Arg Leu Asp Leu Arg Asp Leu
Gln Arg His Leu Arg Phe Gln Val Leu 115 120 125Ala Ala Gly Phe Asn
Cys Pro Glu Glu Glu Glu Glu Glu Glu Glu Glu 130 135 140Glu Glu Glu
Glu Arg Lys Gly Leu Leu Pro Gly Ala Leu Gly Ser Ala145 150 155
160Leu Gln Gly Pro Ala Gln Val Ser Trp Pro Gln Leu Leu Ser Thr Tyr
165 170 175Arg Leu Leu His Ser Leu Glu Leu Val Leu Ser Arg Ala Val
Arg Glu 180 185 190Leu Leu Leu Leu Ser Lys Ala Gly His Ser Val Trp
Pro Leu Gly Phe 195 200 205Pro Thr Leu Ser Pro Gln Pro 210
21525215PRTHomo sapiens 25Phe Pro Arg Pro Pro Gly Arg Pro Gln Leu
Ser Leu Gln Glu Leu Arg1 5 10 15Arg Glu Phe Thr Val Ser Leu His Leu
Ala Arg Lys Leu Leu Ser Glu 20 25 30Val Arg Gly Gln Ala His Arg Phe
Ala Glu Ser His Leu Pro Gly Val 35 40 45Asn Leu Tyr Leu Leu Pro Leu
Gly Glu Gln Leu Pro Asp Val Ser Leu 50 55 60Thr Phe Gln Ala Trp Arg
Arg Leu Ser Asp Pro Glu Arg Leu Cys Phe65 70 75 80Ile Ser Thr Thr
Leu Gln Pro Phe His Ala Leu Leu Gly Gly Leu Gly 85 90 95Thr Gln Gly
Arg Trp Thr Asn Met Glu Arg Met Gln Leu Trp Ala Met 100 105 110Arg
Leu Asp Leu Arg Asp Leu Gln Arg His Leu Arg Phe Gln Val Leu 115 120
125Ala Ala Gly Phe Asn Leu Pro Glu Glu Glu Glu Glu Glu Glu Glu Glu
130 135 140Glu Glu Glu Glu Arg Lys Gly Leu Leu Pro Gly Ala Leu Gly
Ser Ala145 150 155 160Leu Gln Gly Pro Ala Gln Val Ser Trp Pro Gln
Leu Leu Ser Thr Tyr 165 170 175Arg Leu Leu His Ser Leu Glu Leu Val
Leu Ser Arg Ala Val Arg Glu 180 185 190Leu Leu Leu Leu Ser Lys Ala
Gly His Ser Val Trp Pro Leu Gly Phe 195 200 205Pro Thr Leu Ser Pro
Gln Pro 210 21526270PRTHomo sapiens 26Met Lys Pro Lys Met Lys Tyr
Ser Thr Asn Lys Ile Ser Thr Ala Lys1 5 10 15Trp Lys Asn Thr Ala Ser
Lys Ala Leu Cys Phe Lys Leu Gly Lys Ser 20 25 30Gln Gln Lys Ala Lys
Glu Val Cys Pro Met Tyr Phe Met Lys Leu Arg 35 40 45Ser Gly Leu Met
Ile Lys Lys Glu Ala Cys Tyr Phe Arg Arg Glu Thr 50 55 60Thr Lys Arg
Pro Ser Leu Lys Thr Gly Arg Lys His Lys Arg His Leu65 70 75 80Val
Leu Ala Ala Cys Gln Gln Gln Ser Thr Val Glu Cys Phe Ala Phe 85 90
95Gly Ile Ser Gly Val Gln Lys Tyr Thr Arg Ala Leu His Asp Ser Ser
100 105 110Ile Thr Gly Ile Ser Pro Ile Thr Glu Tyr Leu Ala Ser Leu
Ser Thr 115 120 125Tyr Asn Asp Gln Ser Ile Thr Phe Ala Leu Glu Asp
Glu Ser Tyr Glu 130 135 140Ile Tyr Val Glu Asp Leu Lys Lys Asp Glu
Lys Lys Asp Lys Val Leu145 150 155 160Leu Ser Tyr Tyr Glu Ser Gln
His Pro Ser Asn Glu Ser Gly Asp Gly 165 170 175Val Asp Gly Lys Met
Leu Met Val Thr Leu Ser Pro Thr Lys Asp Phe 180 185 190Trp Leu His
Ala Asn Asn Lys Glu His Ser Val Glu Leu His Lys Cys 195 200 205Glu
Lys Pro Leu Pro Asp Gln Ala Phe Phe Val Leu His Asn Met His 210
215 220Ser Asn Cys Val Ser Phe Glu Cys Lys Thr Asp Pro Gly Val Phe
Ile225 230 235 240Gly Val Lys Asp Asn His Leu Ala Leu Ile Lys Val
Asp Ser Ser Glu 245 250 255Asn Leu Cys Thr Glu Asn Ile Leu Phe Lys
Leu Ser Glu Thr 260 265 27027176PRTHomo sapiens 27Ala Phe Gly Ile
Ser Gly Val Gln Lys Tyr Thr Arg Ala Leu His Asp1 5 10 15Ser Ser Ile
Thr Gly Ile Ser Pro Ile Thr Glu Tyr Leu Ala Ser Leu 20 25 30Ser Thr
Tyr Asn Asp Gln Ser Ile Thr Phe Ala Leu Glu Asp Glu Ser 35 40 45Tyr
Glu Ile Tyr Val Glu Asp Leu Lys Lys Asp Glu Lys Lys Asp Lys 50 55
60Val Leu Leu Ser Tyr Tyr Glu Ser Gln His Pro Ser Asn Glu Ser Gly65
70 75 80Asp Gly Val Asp Gly Lys Met Leu Met Val Thr Leu Ser Pro Thr
Lys 85 90 95Asp Phe Trp Leu His Ala Asn Asn Lys Glu His Ser Val Glu
Leu His 100 105 110Lys Cys Glu Lys Pro Leu Pro Asp Gln Ala Phe Phe
Val Leu His Asn 115 120 125Met His Ser Asn Cys Val Ser Phe Glu Cys
Lys Thr Asp Pro Gly Val 130 135 140Phe Ile Gly Val Lys Asp Asn His
Leu Ala Leu Ile Lys Val Asp Ser145 150 155 160Ser Glu Asn Leu Cys
Thr Glu Asn Ile Leu Phe Lys Leu Ser Glu Thr 165 170 17528172PRTHomo
sapiens 28Ser Gly Val Gln Lys Tyr Thr Arg Ala Leu His Asp Ser Ser
Ile Thr1 5 10 15Gly Ile Ser Pro Ile Thr Glu Tyr Leu Ala Ser Leu Ser
Thr Tyr Asn 20 25 30Asp Gln Ser Ile Thr Phe Ala Leu Glu Asp Glu Ser
Tyr Glu Ile Tyr 35 40 45Val Glu Asp Leu Lys Lys Asp Glu Lys Lys Asp
Lys Val Leu Leu Ser 50 55 60Tyr Tyr Glu Ser Gln His Pro Ser Asn Glu
Ser Gly Asp Gly Val Asp65 70 75 80Gly Lys Met Leu Met Val Thr Leu
Ser Pro Thr Lys Asp Phe Trp Leu 85 90 95His Ala Asn Asn Lys Glu His
Ser Val Glu Leu His Lys Cys Glu Lys 100 105 110Pro Leu Pro Asp Gln
Ala Phe Phe Val Leu His Asn Met His Ser Asn 115 120 125Cys Val Ser
Phe Glu Cys Lys Thr Asp Pro Gly Val Phe Ile Gly Val 130 135 140Lys
Asp Asn His Leu Ala Leu Ile Lys Val Asp Ser Ser Glu Asn Leu145 150
155 160Cys Thr Glu Asn Ile Leu Phe Lys Leu Ser Glu Thr 165
17029162PRTHomo sapiens 29His Asp Ser Ser Ile Thr Gly Ile Ser Pro
Ile Thr Glu Tyr Leu Ala1 5 10 15Ser Leu Ser Thr Tyr Asn Asp Gln Ser
Ile Thr Phe Ala Leu Glu Asp 20 25 30Glu Ser Tyr Glu Ile Tyr Val Glu
Asp Leu Lys Lys Asp Glu Lys Lys 35 40 45Asp Lys Val Leu Leu Ser Tyr
Tyr Glu Ser Gln His Pro Ser Asn Glu 50 55 60Ser Gly Asp Gly Val Asp
Gly Lys Met Leu Met Val Thr Leu Ser Pro65 70 75 80Thr Lys Asp Phe
Trp Leu His Ala Asn Asn Lys Glu His Ser Val Glu 85 90 95Leu His Lys
Cys Glu Lys Pro Leu Pro Asp Gln Ala Phe Phe Val Leu 100 105 110His
Asn Met His Ser Asn Cys Val Ser Phe Glu Cys Lys Thr Asp Pro 115 120
125Gly Val Phe Ile Gly Val Lys Asp Asn His Leu Ala Leu Ile Lys Val
130 135 140Asp Ser Ser Glu Asn Leu Cys Thr Glu Asn Ile Leu Phe Lys
Leu Ser145 150 155 160Glu Thr30228PRTHomo sapiens 30Met Lys Pro Lys
Met Lys Tyr Ser Thr Asn Lys Ile Ser Thr Ala Lys1 5 10 15Trp Lys Asn
Thr Ala Ser Lys Ala Leu Cys Phe Lys Leu Gly Lys Ser 20 25 30Gln Gln
Lys Ala Lys Glu Val Cys Pro Met Tyr Phe Met Lys Leu Arg 35 40 45Ser
Gly Leu Met Ile Lys Lys Glu Ala Cys Tyr Phe Arg Arg Glu Thr 50 55
60Thr Lys Arg Pro Ser Leu Lys Thr Gly Arg Lys His Lys Arg His Leu65
70 75 80Val Leu Ala Ala Cys Gln Gln Gln Ser Thr Val Glu Cys Phe Ala
Phe 85 90 95Gly Ile Ser Gly Val Gln Lys Tyr Thr Arg Ala Leu His Asp
Ser Ser 100 105 110Ile Thr Asp Lys Val Leu Leu Ser Tyr Tyr Glu Ser
Gln His Pro Ser 115 120 125Asn Glu Ser Gly Asp Gly Val Asp Gly Lys
Met Leu Met Val Thr Leu 130 135 140Ser Pro Thr Lys Asp Phe Trp Leu
His Ala Asn Asn Lys Glu His Ser145 150 155 160Val Glu Leu His Lys
Cys Glu Lys Pro Leu Pro Asp Gln Ala Phe Phe 165 170 175Val Leu His
Asn Met His Ser Asn Cys Val Ser Phe Glu Cys Lys Thr 180 185 190Asp
Pro Gly Val Phe Ile Gly Val Lys Asp Asn His Leu Ala Leu Ile 195 200
205Lys Val Asp Ser Ser Glu Asn Leu Cys Thr Glu Asn Ile Leu Phe Lys
210 215 220Leu Ser Glu Thr22531228PRTHomo sapiens 31Met Lys Pro Lys
Met Lys Tyr Ser Thr Asn Lys Ile Ser Thr Ala Lys1 5 10 15Trp Lys Asn
Thr Ala Ser Lys Ala Leu Cys Phe Lys Leu Gly Lys Ser 20 25 30Gln Gln
Lys Ala Lys Glu Val Cys Pro Met Tyr Phe Met Lys Leu Arg 35 40 45Ser
Gly Leu Met Ile Lys Lys Glu Ala Cys Tyr Phe Arg Arg Glu Thr 50 55
60Thr Lys Arg Pro Ser Leu Lys Thr Gly Ile Ser Pro Ile Thr Glu Tyr65
70 75 80Leu Ala Ser Leu Ser Thr Tyr Asn Asp Gln Ser Ile Thr Phe Ala
Leu 85 90 95Glu Asp Glu Ser Tyr Glu Ile Tyr Val Glu Asp Leu Lys Lys
Asp Glu 100 105 110Lys Lys Asp Lys Val Leu Leu Ser Tyr Tyr Glu Ser
Gln His Pro Ser 115 120 125Asn Glu Ser Gly Asp Gly Val Asp Gly Lys
Met Leu Met Val Thr Leu 130 135 140Ser Pro Thr Lys Asp Phe Trp Leu
His Ala Asn Asn Lys Glu His Ser145 150 155 160Val Glu Leu His Lys
Cys Glu Lys Pro Leu Pro Asp Gln Ala Phe Phe 165 170 175Val Leu His
Asn Met His Ser Asn Cys Val Ser Phe Glu Cys Lys Thr 180 185 190Asp
Pro Gly Val Phe Ile Gly Val Lys Asp Asn His Leu Ala Leu Ile 195 200
205Lys Val Asp Ser Ser Glu Asn Leu Cys Thr Glu Asn Ile Leu Phe Lys
210 215 220Leu Ser Glu Thr22532144PRTHomo sapiens 32Met Lys Pro Lys
Met Lys Tyr Ser Thr Asn Lys Ile Ser Thr Ala Lys1 5 10 15Trp Lys Asn
Thr Ala Ser Lys Ala Leu Cys Phe Lys Leu Gly Asn Lys 20 25 30Val Leu
Leu Ser Tyr Tyr Glu Ser Gln His Pro Ser Asn Glu Ser Gly 35 40 45Asp
Gly Val Asp Gly Lys Met Leu Met Val Thr Leu Ser Pro Thr Lys 50 55
60Asp Phe Trp Leu His Ala Asn Asn Lys Glu His Ser Val Glu Leu His65
70 75 80Lys Cys Glu Lys Pro Leu Pro Asp Gln Ala Phe Phe Val Leu His
Asn 85 90 95Met His Ser Asn Cys Val Ser Phe Glu Cys Lys Thr Asp Pro
Gly Val 100 105 110Phe Ile Gly Val Lys Asp Asn His Leu Ala Leu Ile
Lys Val Asp Ser 115 120 125Ser Glu Asn Leu Cys Thr Glu Asn Ile Leu
Phe Lys Leu Ser Glu Thr 130 135 14033390PRTHomo sapiens 33Met Pro
Pro Ser Gly Leu Arg Leu Leu Pro Leu Leu Leu Pro Leu Leu1 5 10 15Trp
Leu Leu Val Leu Thr Pro Gly Arg Pro Ala Ala Gly Leu Ser Thr 20 25
30Cys Lys Thr Ile Asp Met Glu Leu Val Lys Arg Lys Arg Ile Glu Ala
35 40 45Ile Arg Gly Gln Ile Leu Ser Lys Leu Arg Leu Ala Ser Pro Pro
Ser 50 55 60Gln Gly Glu Val Pro Pro Gly Pro Leu Pro Glu Ala Val Leu
Ala Leu65 70 75 80Tyr Asn Ser Thr Arg Asp Arg Val Ala Gly Glu Ser
Ala Glu Pro Glu 85 90 95Pro Glu Pro Glu Ala Asp Tyr Tyr Ala Lys Glu
Val Thr Arg Val Leu 100 105 110Met Val Glu Thr His Asn Glu Ile Tyr
Asp Lys Phe Lys Gln Ser Thr 115 120 125His Ser Ile Tyr Met Phe Phe
Asn Thr Ser Glu Leu Arg Glu Ala Val 130 135 140Pro Glu Pro Val Leu
Leu Ser Arg Ala Glu Leu Arg Leu Leu Arg Leu145 150 155 160Lys Leu
Lys Val Glu Gln His Val Glu Leu Tyr Gln Lys Tyr Ser Asn 165 170
175Asn Ser Trp Arg Tyr Leu Ser Asn Arg Leu Leu Ala Pro Ser Asp Ser
180 185 190Pro Glu Trp Leu Ser Phe Asp Val Thr Gly Val Val Arg Gln
Trp Leu 195 200 205Ser Arg Gly Gly Glu Ile Glu Gly Phe Arg Leu Ser
Ala His Cys Ser 210 215 220Cys Asp Ser Arg Asp Asn Thr Leu Gln Val
Asp Ile Asn Gly Phe Thr225 230 235 240Thr Gly Arg Arg Gly Asp Leu
Ala Thr Ile His Gly Met Asn Arg Pro 245 250 255Phe Leu Leu Leu Met
Ala Thr Pro Leu Glu Arg Ala Gln His Leu Gln 260 265 270Ser Ser Arg
His Arg Arg Ala Leu Asp Thr Asn Tyr Cys Phe Ser Ser 275 280 285Thr
Glu Lys Asn Cys Cys Val Arg Gln Leu Tyr Ile Asp Phe Arg Lys 290 295
300Asp Leu Gly Trp Lys Trp Ile His Glu Pro Lys Gly Tyr His Ala
Asn305 310 315 320Phe Cys Leu Gly Pro Cys Pro Tyr Ile Trp Ser Leu
Asp Thr Gln Tyr 325 330 335Ser Lys Val Leu Ala Leu Tyr Asn Gln His
Asn Pro Gly Ala Ser Ala 340 345 350Ala Pro Cys Cys Val Pro Gln Ala
Leu Glu Pro Leu Pro Ile Val Tyr 355 360 365Tyr Val Gly Arg Lys Pro
Lys Val Glu Gln Leu Ser Asn Met Ile Val 370 375 380Arg Ser Cys Lys
Cys Ser385 39034112PRTHomo sapiens 34Ala Leu Asp Thr Asn Tyr Cys
Phe Ser Ser Thr Glu Lys Asn Cys Cys1 5 10 15Val Arg Gln Leu Tyr Ile
Asp Phe Arg Lys Asp Leu Gly Trp Lys Trp 20 25 30Ile His Glu Pro Lys
Gly Tyr His Ala Asn Phe Cys Leu Gly Pro Cys 35 40 45Pro Tyr Ile Trp
Ser Leu Asp Thr Gln Tyr Ser Lys Val Leu Ala Leu 50 55 60Tyr Asn Gln
His Asn Pro Gly Ala Ser Ala Ala Pro Cys Cys Val Pro65 70 75 80Gln
Ala Leu Glu Pro Leu Pro Ile Val Tyr Tyr Val Gly Arg Lys Pro 85 90
95Lys Val Glu Gln Leu Ser Asn Met Ile Val Arg Ser Cys Lys Cys Ser
100 105 11035442PRTHomo sapiens 35Met His Tyr Cys Val Leu Ser Ala
Phe Leu Ile Leu His Leu Val Thr1 5 10 15Val Ala Leu Ser Leu Ser Thr
Cys Ser Thr Leu Asp Met Asp Gln Phe 20 25 30Met Arg Lys Arg Ile Glu
Ala Ile Arg Gly Gln Ile Leu Ser Lys Leu 35 40 45Lys Leu Thr Ser Pro
Pro Glu Asp Tyr Pro Glu Pro Glu Glu Val Pro 50 55 60Pro Glu Val Ile
Ser Ile Tyr Asn Ser Thr Arg Asp Leu Leu Gln Glu65 70 75 80Lys Ala
Ser Arg Arg Ala Ala Ala Cys Glu Arg Glu Arg Ser Asp Glu 85 90 95Glu
Tyr Tyr Ala Lys Glu Val Tyr Lys Ile Asp Met Pro Pro Phe Phe 100 105
110Pro Ser Glu Thr Val Cys Pro Val Val Thr Thr Pro Ser Gly Ser Val
115 120 125Gly Ser Leu Cys Ser Arg Gln Ser Gln Val Leu Cys Gly Tyr
Leu Asp 130 135 140Ala Ile Pro Pro Thr Phe Tyr Arg Pro Tyr Phe Arg
Ile Val Arg Phe145 150 155 160Asp Val Ser Ala Met Glu Lys Asn Ala
Ser Asn Leu Val Lys Ala Glu 165 170 175Phe Arg Val Phe Arg Leu Gln
Asn Pro Lys Ala Arg Val Pro Glu Gln 180 185 190Arg Ile Glu Leu Tyr
Gln Ile Leu Lys Ser Lys Asp Leu Thr Ser Pro 195 200 205Thr Gln Arg
Tyr Ile Asp Ser Lys Val Val Lys Thr Arg Ala Glu Gly 210 215 220Glu
Trp Leu Ser Phe Asp Val Thr Asp Ala Val His Glu Trp Leu His225 230
235 240His Lys Asp Arg Asn Leu Gly Phe Lys Ile Ser Leu His Cys Pro
Cys 245 250 255Cys Thr Phe Val Pro Ser Asn Asn Tyr Ile Ile Pro Asn
Lys Ser Glu 260 265 270Glu Leu Glu Ala Arg Phe Ala Gly Ile Asp Gly
Thr Ser Thr Tyr Thr 275 280 285Ser Gly Asp Gln Lys Thr Ile Lys Ser
Thr Arg Lys Lys Asn Ser Gly 290 295 300Lys Thr Pro His Leu Leu Leu
Met Leu Leu Pro Ser Tyr Arg Leu Glu305 310 315 320Ser Gln Gln Thr
Asn Arg Arg Lys Lys Arg Ala Leu Asp Ala Ala Tyr 325 330 335Cys Phe
Arg Asn Val Gln Asp Asn Cys Cys Leu Arg Pro Leu Tyr Ile 340 345
350Asp Phe Lys Arg Asp Leu Gly Trp Lys Trp Ile His Glu Pro Lys Gly
355 360 365Tyr Asn Ala Asn Phe Cys Ala Gly Ala Cys Pro Tyr Leu Trp
Ser Ser 370 375 380Asp Thr Gln His Ser Arg Val Leu Ser Leu Tyr Asn
Thr Ile Asn Pro385 390 395 400Glu Ala Ser Ala Ser Pro Cys Cys Val
Ser Gln Asp Leu Glu Pro Leu 405 410 415Thr Ile Leu Tyr Tyr Ile Gly
Lys Thr Pro Lys Ile Glu Gln Leu Ser 420 425 430Asn Met Ile Val Lys
Ser Cys Lys Cys Ser 435 44036112PRTHomo sapiens 36Ala Leu Asp Ala
Ala Tyr Cys Phe Arg Asn Val Gln Asp Asn Cys Cys1 5 10 15Leu Arg Pro
Leu Tyr Ile Asp Phe Lys Arg Asp Leu Gly Trp Lys Trp 20 25 30Ile His
Glu Pro Lys Gly Tyr Asn Ala Asn Phe Cys Ala Gly Ala Cys 35 40 45Pro
Tyr Leu Trp Ser Ser Asp Thr Gln His Ser Arg Val Leu Ser Leu 50 55
60Tyr Asn Thr Ile Asn Pro Glu Ala Ser Ala Ser Pro Cys Cys Val Ser65
70 75 80Gln Asp Leu Glu Pro Leu Thr Ile Leu Tyr Tyr Ile Gly Lys Thr
Pro 85 90 95Lys Ile Glu Gln Leu Ser Asn Met Ile Val Lys Ser Cys Lys
Cys Ser 100 105 11037414PRTHomo sapiens 37Met His Tyr Cys Val Leu
Ser Ala Phe Leu Ile Leu His Leu Val Thr1 5 10 15Val Ala Leu Ser Leu
Ser Thr Cys Ser Thr Leu Asp Met Asp Gln Phe 20 25 30Met Arg Lys Arg
Ile Glu Ala Ile Arg Gly Gln Ile Leu Ser Lys Leu 35 40 45Lys Leu Thr
Ser Pro Pro Glu Asp Tyr Pro Glu Pro Glu Glu Val Pro 50 55 60Pro Glu
Val Ile Ser Ile Tyr Asn Ser Thr Arg Asp Leu Leu Gln Glu65 70 75
80Lys Ala Ser Arg Arg Ala Ala Ala Cys Glu Arg Glu Arg Ser Asp Glu
85 90 95Glu Tyr Tyr Ala Lys Glu Val Tyr Lys Ile Asp Met Pro Pro Phe
Phe 100 105 110Pro Ser Glu Asn Ala Ile Pro Pro Thr Phe Tyr Arg Pro
Tyr Phe Arg 115 120 125Ile Val Arg Phe Asp Val Ser Ala Met Glu Lys
Asn Ala Ser Asn Leu 130 135 140Val Lys Ala Glu Phe Arg Val Phe Arg
Leu Gln Asn Pro Lys Ala Arg145 150 155 160Val Pro Glu Gln Arg Ile
Glu Leu Tyr Gln Ile Leu Lys Ser Lys Asp 165 170 175Leu Thr Ser Pro
Thr Gln Arg Tyr Ile Asp Ser Lys Val Val Lys Thr 180 185 190Arg Ala
Glu Gly Glu Trp Leu Ser Phe Asp Val Thr Asp Ala Val His 195 200
205Glu Trp Leu His His Lys Asp Arg Asn Leu Gly Phe Lys Ile Ser Leu
210 215 220His Cys Pro Cys Cys Thr Phe Val Pro Ser Asn Asn Tyr Ile
Ile Pro225
230 235 240Asn Lys Ser Glu Glu Leu Glu Ala Arg Phe Ala Gly Ile Asp
Gly Thr 245 250 255Ser Thr Tyr Thr Ser Gly Asp Gln Lys Thr Ile Lys
Ser Thr Arg Lys 260 265 270Lys Asn Ser Gly Lys Thr Pro His Leu Leu
Leu Met Leu Leu Pro Ser 275 280 285Tyr Arg Leu Glu Ser Gln Gln Thr
Asn Arg Arg Lys Lys Arg Ala Leu 290 295 300Asp Ala Ala Tyr Cys Phe
Arg Asn Val Gln Asp Asn Cys Cys Leu Arg305 310 315 320Pro Leu Tyr
Ile Asp Phe Lys Arg Asp Leu Gly Trp Lys Trp Ile His 325 330 335Glu
Pro Lys Gly Tyr Asn Ala Asn Phe Cys Ala Gly Ala Cys Pro Tyr 340 345
350Leu Trp Ser Ser Asp Thr Gln His Ser Arg Val Leu Ser Leu Tyr Asn
355 360 365Thr Ile Asn Pro Glu Ala Ser Ala Ser Pro Cys Cys Val Ser
Gln Asp 370 375 380Leu Glu Pro Leu Thr Ile Leu Tyr Tyr Ile Gly Lys
Thr Pro Lys Ile385 390 395 400Glu Gln Leu Ser Asn Met Ile Val Lys
Ser Cys Lys Cys Ser 405 410389PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 38Gly Ser Gly Gly Gly Gly Ser
Gly Thr1 5394PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 39Gly Gly Gly Ser1405PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 40Gly
Gly Gly Gly Ser1 54118PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 41Lys Glu Ser Gly Ser Val Ser
Ser Glu Gln Leu Ala Gln Phe Arg Ser1 5 10 15Leu
Asp4214PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 42Glu Gly Lys Ser Ser Gly Ser Gly Ser Glu Ser Lys
Ser Thr1 5 104312PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 43Gly Ser Ala Gly Ser Ala Ala Gly Ser
Gly Glu Phe1 5 10445PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 44Glu Ala Ala Ala Lys1 5455PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 45Glu
Ala Ala Ala Arg1 546298PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 46His Lys Cys Asp Ile Thr
Leu Gln Glu Ile Ile Lys Thr Leu Asn Ser1 5 10 15Leu Thr Glu Gln Lys
Thr Leu Cys Thr Glu Leu Thr Val Thr Asp Ile 20 25 30Phe Ala Ala Ser
Lys Asn Thr Thr Glu Lys Glu Thr Phe Cys Arg Ala 35 40 45Ala Thr Val
Leu Arg Gln Phe Tyr Ser His His Glu Lys Asp Thr Arg 50 55 60Cys Leu
Gly Ala Thr Ala Gln Gln Phe His Arg His Lys Gln Leu Ile65 70 75
80Arg Phe Leu Lys Arg Leu Asp Arg Asn Leu Trp Gly Leu Ala Gly Leu
85 90 95Asn Ser Cys Pro Val Lys Glu Ala Asn Gln Ser Thr Leu Glu Asn
Phe 100 105 110Leu Glu Arg Leu Lys Thr Ile Met Arg Glu Lys Tyr Ser
Lys Cys Ser 115 120 125Ser Gly Ser Gly Gly Gly Gly Ser Gly Thr Ser
Pro Gly Gln Gly Thr 130 135 140Gln Ser Glu Asn Ser Cys Thr His Phe
Pro Gly Asn Leu Pro Asn Met145 150 155 160Leu Arg Asp Leu Arg Asp
Ala Phe Ser Arg Val Lys Thr Phe Phe Gln 165 170 175Met Lys Asp Gln
Leu Asp Asn Leu Leu Leu Lys Glu Ser Leu Leu Glu 180 185 190Asp Phe
Lys Gly Tyr Leu Gly Cys Gln Ala Leu Ser Glu Met Ile Gln 195 200
205Phe Tyr Leu Glu Glu Val Met Pro Gln Ala Glu Asn Gln Asp Pro Asp
210 215 220Ile Lys Ala His Val Asn Ser Leu Gly Glu Asn Leu Lys Thr
Leu Arg225 230 235 240Leu Arg Leu Arg Arg Cys His Arg Phe Leu Pro
Cys Glu Asn Lys Ser 245 250 255Lys Ala Val Glu Gln Val Lys Asn Ala
Phe Asn Lys Leu Gln Glu Lys 260 265 270Gly Ile Tyr Lys Ala Met Ser
Glu Phe Asp Ile Phe Ile Asn Tyr Ile 275 280 285Glu Ala Tyr Met Thr
Met Lys Ile Arg Asn 290 29547252PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 47His Lys Cys Asp Ile
Thr Leu Gln Glu Ile Ile Lys Thr Leu Asn Ser1 5 10 15Leu Thr Glu Gln
Lys Thr Leu Cys Thr Glu Leu Thr Val Thr Asp Ile 20 25 30Phe Ala Ala
Ser Lys Asn Thr Thr Glu Lys Glu Thr Phe Cys Arg Ala 35 40 45Ala Thr
Val Leu Arg Gln Phe Tyr Ser His His Glu Lys Asp Thr Arg 50 55 60Cys
Leu Gly Ala Thr Ala Gln Gln Phe His Arg His Lys Gln Leu Ile65 70 75
80Arg Phe Leu Lys Arg Leu Asp Arg Asn Leu Trp Gly Leu Ala Gly Leu
85 90 95Asn Ser Cys Pro Val Lys Glu Ala Asn Gln Ser Thr Leu Glu Asn
Phe 100 105 110Leu Glu Arg Leu Lys Thr Ile Met Arg Glu Lys Tyr Ser
Lys Cys Ser 115 120 125Ser Gly Ser Gly Gly Gly Gly Ser Gly Thr Ser
Pro Gly Pro Val Pro 130 135 140Pro Ser Thr Ala Leu Arg Glu Leu Ile
Glu Glu Leu Val Asn Ile Thr145 150 155 160Gln Asn Gln Lys Ala Pro
Leu Cys Asn Gly Ser Met Val Trp Ser Ile 165 170 175Asn Leu Thr Ala
Gly Met Tyr Cys Ala Ala Leu Glu Ser Leu Ile Asn 180 185 190Val Ser
Gly Cys Ser Ala Ile Glu Lys Thr Gln Arg Met Leu Ser Gly 195 200
205Phe Cys Pro His Lys Val Ser Ala Gly Gln Phe Ser Ser Leu His Val
210 215 220Arg Asp Thr Lys Ile Glu Val Ala Gln Phe Val Lys Asp Leu
Leu Leu225 230 235 240His Leu Lys Lys Leu Phe Arg Glu Gly Arg Phe
Asn 245 25048353PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 48His Lys Cys Asp Ile Thr Leu Gln
Glu Ile Ile Lys Thr Leu Asn Ser1 5 10 15Leu Thr Glu Gln Lys Thr Leu
Cys Thr Glu Leu Thr Val Thr Asp Ile 20 25 30Phe Ala Ala Ser Lys Asn
Thr Thr Glu Lys Glu Thr Phe Cys Arg Ala 35 40 45Ala Thr Val Leu Arg
Gln Phe Tyr Ser His His Glu Lys Asp Thr Arg 50 55 60Cys Leu Gly Ala
Thr Ala Gln Gln Phe His Arg His Lys Gln Leu Ile65 70 75 80Arg Phe
Leu Lys Arg Leu Asp Arg Asn Leu Trp Gly Leu Ala Gly Leu 85 90 95Asn
Ser Cys Pro Val Lys Glu Ala Asn Gln Ser Thr Leu Glu Asn Phe 100 105
110Leu Glu Arg Leu Lys Thr Ile Met Arg Glu Lys Tyr Ser Lys Cys Ser
115 120 125Ser Gly Ser Gly Gly Gly Gly Ser Gly Thr Phe Pro Arg Pro
Pro Gly 130 135 140Arg Pro Gln Leu Ser Leu Gln Glu Leu Arg Arg Glu
Phe Thr Val Ser145 150 155 160Leu His Leu Ala Arg Lys Leu Leu Ser
Glu Val Arg Gly Gln Ala His 165 170 175Arg Phe Ala Glu Ser His Leu
Pro Gly Val Asn Leu Tyr Leu Leu Pro 180 185 190Leu Gly Glu Gln Leu
Pro Asp Val Ser Leu Thr Phe Gln Ala Trp Arg 195 200 205Arg Leu Ser
Asp Pro Glu Arg Leu Cys Phe Ile Ser Thr Thr Leu Gln 210 215 220Pro
Phe His Ala Leu Leu Gly Gly Leu Gly Thr Gln Gly Arg Trp Thr225 230
235 240Asn Met Glu Arg Met Gln Leu Trp Ala Met Arg Leu Asp Leu Arg
Asp 245 250 255Leu Gln Arg His Leu Arg Phe Gln Val Leu Ala Ala Gly
Phe Asn Cys 260 265 270Pro Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu
Glu Glu Glu Arg Lys 275 280 285Gly Leu Leu Pro Gly Ala Leu Gly Ser
Ala Leu Gln Gly Pro Ala Gln 290 295 300Val Ser Trp Pro Gln Leu Leu
Ser Thr Tyr Arg Leu Leu His Ser Leu305 310 315 320Glu Leu Val Leu
Ser Arg Ala Val Arg Glu Leu Leu Leu Leu Ser Lys 325 330 335Ala Gly
His Ser Val Trp Pro Leu Gly Phe Pro Thr Leu Ser Pro Gln 340 345
350Pro49408PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 49His Lys Cys Asp Ile Thr Leu Gln Glu Ile Ile
Lys Thr Leu Asn Ser1 5 10 15Leu Thr Glu Gln Lys Thr Leu Cys Thr Glu
Leu Thr Val Thr Asp Ile 20 25 30Phe Ala Ala Ser Lys Asn Thr Thr Glu
Lys Glu Thr Phe Cys Arg Ala 35 40 45Ala Thr Val Leu Arg Gln Phe Tyr
Ser His His Glu Lys Asp Thr Arg 50 55 60Cys Leu Gly Ala Thr Ala Gln
Gln Phe His Arg His Lys Gln Leu Ile65 70 75 80Arg Phe Leu Lys Arg
Leu Asp Arg Asn Leu Trp Gly Leu Ala Gly Leu 85 90 95Asn Ser Cys Pro
Val Lys Glu Ala Asn Gln Ser Thr Leu Glu Asn Phe 100 105 110Leu Glu
Arg Leu Lys Thr Ile Met Arg Glu Lys Tyr Ser Lys Cys Ser 115 120
125Ser Gly Ser Gly Gly Gly Gly Ser Gly Thr Met Lys Pro Lys Met Lys
130 135 140Tyr Ser Thr Asn Lys Ile Ser Thr Ala Lys Trp Lys Asn Thr
Ala Ser145 150 155 160Lys Ala Leu Cys Phe Lys Leu Gly Lys Ser Gln
Gln Lys Ala Lys Glu 165 170 175Val Cys Pro Met Tyr Phe Met Lys Leu
Arg Ser Gly Leu Met Ile Lys 180 185 190Lys Glu Ala Cys Tyr Phe Arg
Arg Glu Thr Thr Lys Arg Pro Ser Leu 195 200 205Lys Thr Gly Arg Lys
His Lys Arg His Leu Val Leu Ala Ala Cys Gln 210 215 220Gln Gln Ser
Thr Val Glu Cys Phe Ala Phe Gly Ile Ser Gly Val Gln225 230 235
240Lys Tyr Thr Arg Ala Leu His Asp Ser Ser Ile Thr Gly Ile Ser Pro
245 250 255Ile Thr Glu Tyr Leu Ala Ser Leu Ser Thr Tyr Asn Asp Gln
Ser Ile 260 265 270Thr Phe Ala Leu Glu Asp Glu Ser Tyr Glu Ile Tyr
Val Glu Asp Leu 275 280 285Lys Lys Asp Glu Lys Lys Asp Lys Val Leu
Leu Ser Tyr Tyr Glu Ser 290 295 300Gln His Pro Ser Asn Glu Ser Gly
Asp Gly Val Asp Gly Lys Met Leu305 310 315 320Met Val Thr Leu Ser
Pro Thr Lys Asp Phe Trp Leu His Ala Asn Asn 325 330 335Lys Glu His
Ser Val Glu Leu His Lys Cys Glu Lys Pro Leu Pro Asp 340 345 350Gln
Ala Phe Phe Val Leu His Asn Met His Ser Asn Cys Val Ser Phe 355 360
365Glu Cys Lys Thr Asp Pro Gly Val Phe Ile Gly Val Lys Asp Asn His
370 375 380Leu Ala Leu Ile Lys Val Asp Ser Ser Glu Asn Leu Cys Thr
Glu Asn385 390 395 400Ile Leu Phe Lys Leu Ser Glu Thr
40550250PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 50His Lys Cys Asp Ile Thr Leu Gln Glu Ile Ile
Lys Thr Leu Asn Ser1 5 10 15Leu Thr Glu Gln Lys Thr Leu Cys Thr Glu
Leu Thr Val Thr Asp Ile 20 25 30Phe Ala Ala Ser Lys Asn Thr Thr Glu
Lys Glu Thr Phe Cys Arg Ala 35 40 45Ala Thr Val Leu Arg Gln Phe Tyr
Ser His His Glu Lys Asp Thr Arg 50 55 60Cys Leu Gly Ala Thr Ala Gln
Gln Phe His Arg His Lys Gln Leu Ile65 70 75 80Arg Phe Leu Lys Arg
Leu Asp Arg Asn Leu Trp Gly Leu Ala Gly Leu 85 90 95Asn Ser Cys Pro
Val Lys Glu Ala Asn Gln Ser Thr Leu Glu Asn Phe 100 105 110Leu Glu
Arg Leu Lys Thr Ile Met Arg Glu Lys Tyr Ser Lys Cys Ser 115 120
125Ser Gly Ser Gly Gly Gly Gly Ser Gly Thr Ala Leu Asp Thr Asn Tyr
130 135 140Cys Phe Ser Ser Thr Glu Lys Asn Cys Cys Val Arg Gln Leu
Tyr Ile145 150 155 160Asp Phe Arg Lys Asp Leu Gly Trp Lys Trp Ile
His Glu Pro Lys Gly 165 170 175Tyr His Ala Asn Phe Cys Leu Gly Pro
Cys Pro Tyr Ile Trp Ser Leu 180 185 190Asp Thr Gln Tyr Ser Lys Val
Leu Ala Leu Tyr Asn Gln His Asn Pro 195 200 205Gly Ala Ser Ala Ala
Pro Cys Cys Val Pro Gln Ala Leu Glu Pro Leu 210 215 220Pro Ile Val
Tyr Tyr Val Gly Arg Lys Pro Lys Val Glu Gln Leu Ser225 230 235
240Asn Met Ile Val Arg Ser Cys Lys Cys Ser 245
25051250PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 51His Lys Cys Asp Ile Thr Leu Gln Glu Ile Ile
Lys Thr Leu Asn Ser1 5 10 15Leu Thr Glu Gln Lys Thr Leu Cys Thr Glu
Leu Thr Val Thr Asp Ile 20 25 30Phe Ala Ala Ser Lys Asn Thr Thr Glu
Lys Glu Thr Phe Cys Arg Ala 35 40 45Ala Thr Val Leu Arg Gln Phe Tyr
Ser His His Glu Lys Asp Thr Arg 50 55 60Cys Leu Gly Ala Thr Ala Gln
Gln Phe His Arg His Lys Gln Leu Ile65 70 75 80Arg Phe Leu Lys Arg
Leu Asp Arg Asn Leu Trp Gly Leu Ala Gly Leu 85 90 95Asn Ser Cys Pro
Val Lys Glu Ala Asn Gln Ser Thr Leu Glu Asn Phe 100 105 110Leu Glu
Arg Leu Lys Thr Ile Met Arg Glu Lys Tyr Ser Lys Cys Ser 115 120
125Ser Gly Ser Gly Gly Gly Gly Ser Gly Thr Ala Leu Asp Ala Ala Tyr
130 135 140Cys Phe Arg Asn Val Gln Asp Asn Cys Cys Leu Arg Pro Leu
Tyr Ile145 150 155 160Asp Phe Lys Arg Asp Leu Gly Trp Lys Trp Ile
His Glu Pro Lys Gly 165 170 175Tyr Asn Ala Asn Phe Cys Ala Gly Ala
Cys Pro Tyr Leu Trp Ser Ser 180 185 190Asp Thr Gln His Ser Arg Val
Leu Ser Leu Tyr Asn Thr Ile Asn Pro 195 200 205Glu Ala Ser Ala Ser
Pro Cys Cys Val Ser Gln Asp Leu Glu Pro Leu 210 215 220Thr Ile Leu
Tyr Tyr Ile Gly Lys Thr Pro Lys Ile Glu Gln Leu Ser225 230 235
240Asn Met Ile Val Lys Ser Cys Lys Cys Ser 245 25052209PRTHomo
sapiens 52Arg Lys Gly Pro Pro Ala Ala Leu Thr Leu Pro Arg Val Gln
Cys Arg1 5 10 15Ala Ser Arg Tyr Pro Ile Ala Val Asp Cys Ser Trp Thr
Leu Pro Pro 20 25 30Ala Pro Asn Ser Thr Ser Pro Val Ser Phe Ile Ala
Thr Tyr Arg Leu 35 40 45Gly Met Ala Ala Arg Gly His Ser Trp Pro Cys
Leu Gln Gln Thr Pro 50 55 60Thr Ser Thr Ser Cys Thr Ile Thr Asp Val
Gln Leu Phe Ser Met Ala65 70 75 80Pro Tyr Val Leu Asn Val Thr Ala
Val His Pro Trp Gly Ser Ser Ser 85 90 95Ser Phe Val Pro Phe Ile Thr
Glu His Ile Ile Lys Pro Asp Pro Pro 100 105 110Glu Gly Val Arg Leu
Ser Pro Leu Ala Glu Arg Gln Leu Gln Val Gln 115 120 125Trp Glu Pro
Pro Gly Ser Trp Pro Phe Pro Glu Ile Phe Ser Leu Lys 130 135 140Tyr
Trp Ile Arg Tyr Lys Arg Gln Gly Ala Ala Arg Phe His Arg Val145 150
155 160Gly Pro Ile Glu Ala Thr Ser Phe Ile Leu Arg Ala Val Arg Pro
Arg 165 170 175Ala Arg Tyr Tyr Val Gln Val Ala Ala Gln Asp Leu Thr
Asp Tyr Gly 180 185 190Glu Leu Ser Asp Trp Ser Leu Pro Ala Thr Ala
Thr Met Ser Leu Gly 195 200 205Lys
* * * * *