Use of il6r/il6 chimera in nerve cell regeneration
United States Patent: 7,824,670
Issued: November 2, 2010
Inventors: Haggiag; Shalom
(Rehovot, IL), Revel; Michel (Rehovot, IL), Chebath; Judith (Rehovot, IL),
Levy; Alon (Ramat Hasharon, IL), Zhang; Peilin (Rehovot, IL)
Assignee: Yeda Research and
Development Co., Ltd. (Rehovot, IL)
Appl. No.: 10/498,899
Filed: December 31, 2002
PCT Filed: December 31,
PCT No.: PCT/IL02/01058
371(c)(1),(2),(4) Date: May
PCT Pub. No.: WO03/059376
PCT Pub. Date: July 24,
Training Courses --Pharm/Biotech/etc.
The present invention provides for the
use of IL6R/IL6 chimera for the manufacture of medicaments for the
treatment of neurological diseases and disorders.
Description of the
SUMMARY OF THE INVENTION
The present invention relates to the use of an IL6R/IL6 chimera, a mutein,
fused protein, functional derivative, active fraction, circularly
permutated derivative or salt thereof in the manufacture of a medicament
for inducing neuron generation, wherein progenitor cells of embryonic,
neonatal or adult origin are treated with IL6R/IL6 chimera in-vivo, or
ex-vivo and thereafter transplanted to a patient.
The present invention provides the use of an IL6R/IL6 chimera, a mutein,
fused protein, functional derivative, active fraction, circularly
permutated derivative or salt thereof, in the manufacture of a medicament
for inducing neuron generation e.g. in patients suffering from loss or
atrophy of neurons caused inter alia by aging or by a neurological
Furthermore, the invention provides the use of a cell expressing an
IL6R/IL6 chimera, a mutein, fused protein, functional derivative, active
fraction thereof, or an expression vector, preferably a lentiviral vector,
comprising the coding sequence of an IL6R/IL6 chimera, a mutein, fused
protein, active fraction thereof in the manufacture of a medicament for
inducing neuron generation.
In one embodiment, the medicament of the invention may further comprise
glial cells and/or a neurotrophic factor and/or a cytokine such as NGF,
NTFs, BDNF, IGFs, FGFs, CNTF, LIF, G-CSF, OSM, IL-11, BMP-2, GGF-2, Nrg1
The invention also provides methods for regeneration of nerve cells in
injured central nervous system comprising: administrating a
therapeutically effective amount of an IL6R/IL6 chimera, a mutein, fused
protein, functional derivative, active fraction, circularly permutated
derivative or salt thereof, or a vector encoding an IL6R/IL6, a mutein,
fused protein or an active fraction thereof, or a cell expressing an
IL6R/IL6, a mutein, fused protein or an active fraction thereof, for
restoring nerve cells to injured central nervous system (CNS). Preferably,
along with the IL6R/IL6 chimera a neural progenitor cell or embryonic stem
cell more preferably pre-stimulated with IL6R/IL6 chimera and with
neurotrophic factor and/or cytokines such as NGF, NTFs, BDNF, IGFs, FGFs,
CNTF, LIF, G-CSF, OSM, IL-11, BMP-2, GGF-2, Nrg1 and TGF is administrated.
The methods provided by the present invention may be aimed for the
treatment of patients suffering from a neurological disease e.g.
Alzheimer's disease, Parkinson's disease, multiple sclerosis and
Huntington's chorea patients or patients suffering from neurological
diseases caused by a stroke, anoxia/asphyxia, physical injury, exposure to
toxins and neoplastic disease.
In another aspect, the invention provides a method for rejuvenation
comprising the administration of a therapeutically effective amount of
IL6R/IL6 chimera, a mutein, fused protein, functional derivative, active
fraction, circularly permutated derivative or salt thereof, or a vector
encoding an IL6R/IL6, a mutein, fused protein or an active fraction
thereof, for restoring nerve cells to injured central nervous system (CNS)
preferably comprising a neural progenitor cell more preferably
pre-stimulated with IL6R/IL6 chimera and with neurotrophic factor and/or
cytokines such as NGF, NTFs, BDNF, IGFs, FGFs, CNTF, LIF, G-CSF, OSM,
IL-11, BMP-2, GGF-2, Nrg1 and TGF.
The invention provides also a method for nerve regeneration in injured
central nervous system (CNS) comprising stimulating a neural progenitor
cell, in-vivo or preferably ex-vivo, prior to or during transplantation,
with a composition comprising IL6R/IL6 chimera a mutein, a derivative or
fragment thereof. Optionally, IL6R/IL6 may be supplied to the progenitor
cells by co-transplantation of the progenitor cells with cells expressing
IL6R/IL6 chimera or with an expression vector encoding the IL6R/IL6
chimera. According to the invention the progenitor cells may be derived
from embryonic neonatal or adult origin and may be co-transplanted with
glial cell, and/or with neurotrophic factor and or a cytokine such as NGF,
NTFs, BDNF, IGFs, FGFs, CNTF, LIF, G-CSF, OSM, IL-11, BMP-2, GGF-2, Nrg1
The invention provides a method for nerve regeneration in injured central
nervous system (CNS) comprising administration of IL6R/IL6 chimera, a
mutein, fused protein, functional derivative, active fraction, circularly
permutated derivative or salt thereof, or a vector encoding an IL6R/IL6, a
mutein, fused protein or an active fraction thereof or cells expressing
IL6R/IL6, a mutein, fused protein or an active fraction thereof to a
patient suffering of loss or atrophy of neurons, wherein the loss or
atrophy of neurons can be caused by aging, a neurological disease and/or
by physical damage.
DETAILED DESCRIPTION OF THE INVENTION
The invention relates to pharmaceutical compositions comprising IL6/IL6R
chimera for regenerating neural cells in injured central nervous system
(CNS). A pharmaceutical composition comprising IL6/IL6R chimera can also
be used as a rejuvenation treatment.
The invention is based on the finding that the administration of an
IL6R/IL6 chimera to normal neural stem cells, especially to neural crest
cells, leads to nerve generation. Treating CNS injury with IL6/IL6R
according to the invention has two important advantages: First,
concomitantly with nerve cell generation, it also induces Schwann cell
generation. Schwann cells are needed for myelination of both, the new
nerve cells generated, as well as of adult damaged nerve cells. Second, in
contrast to IL-6, the IL6/IL6R chimera acts in cells that lack the IL-6
receptor (gp-80) but have the GP130 receptor.
CNS injuries can be caused by neurological diseases.
A "neurological disease" is related to disorders of the central nervous
system. It may for example be a general neurodegenerative disease, such as
ageing, vascular disease, Alzheimer's disease, Parkinson's disease, or the
autoimmune disease, multiple sclerosis (MS), it may be a result of an
injury, such as a stroke, anoxia/asphyxia, or physical injury, such as
from a blow to the head, it may be a result of exposure to local (e.g.
meningitis) or systemic toxins, and it may be neoplastic. It may be
genetically based, such as Huntington's chorea, or a disorder of
metabolism such as lysosomal storage disease. There is a group of "general
neurodegenerative diseases" including AZ and others, affecting the
elderly, the usual pattern of response to acute injury (such as ischaemia),
affecting any age group including stroke victims and car accident victims,
autoimmune diseases such as MS, PD, and certain diseases, including
deficiencies of metabolism, of neonates and foetuses.
"Restorative effect" is meant to include any beneficial modification of
the disease process, including palliative, restorative, or proliferative
effects acting on neural tissue.
"Rejuvenation" is meant to include attempts to reverse changes in a brain
commonly caused by ageing, such as loss of volume, loss or atrophy of
neurones, loss of memory, and loss of ability to cope with complex sensory
Rejuvenation also comprises restorative effects on existing neurones.
One aspect of the invention relates to a method for treating injuries to
the central nervous system; the method comprising the step of
administrating a pharmaceutical composition comprising IL6R/IL6 chimera to
subjects in need. Optionally, the pharmaceutical composition may also
comprise living cells.
In another aspect this invention relates to a method for causing at least
partial rejuvenation of the brain of a mammal by treatment with IL6R/IL6
chimera with or without nerve stem cells as described below, wherein the
method may employ implantation of an implant of cells derived from,
embryonic or neonatal mammal or adult neural stem cells into the brain.
The term "regeneration" generally means the ability of an organism to
replace lost tissue. For example after surgical removal of a hepatic lobe,
functioning new liver tissue is produced.
In accordance with the invention, dorsal root ganglia (DRG) cells were
extracted from Lewis rat E14 embryos essentially as described previously (Kleitman
et al. 1991 and Haggiag et al. 1999) and cultured 4 days on flasks at
37.degree. C. 5% CO.sub.2 in medium comprising DMEM/F12 (1:1) with 1%
chick embryo extract, bFGF 20 ng/ml, 1% N2 supplement (Gibco), 2% B27
supplement (Gibco), 50 .mu.M mercaptoethanol, 35 mg/ml (110 nM) retinoic
acid (Morrison et al, 1997). At this point, some cultures were
supplemented with pure rhIL6/RIL6 chimera produced in CHO cells (200 ng/ml;
2.3 nM), others left untreated (NT), or supplemented with Bone Morphogenic
Factor (BMP-2, 2 nM) or remaine untreated. After 9 days (day 13 from the
beginning of culture) the cultured cells showed dense axonal network
developed only after the treatment with IL6R/IL6 suggesting that IL6R/IL6
induces normal embryonic DRG cells to differentiate into neural cells.
These results were confirmed by using a more homogeneous cell population,
devoid of nerve cells, comprising mainly neural crest progenitor cells.
IL6R/IL6 chimera acted as a nerve generative factor, and not as a survival
factor, as evidenced by the fact that neuronal network can observed even
in these homogeneous neural crest populations which are devoid of nerve
cells. According to the invention, it has been also shown that in addition
to nerve cells, glial cells were generated by treatment of neural crest
enriched populations with the IL6R/IL6 chimera and that these glial cells
are associated with the newly generated nerve cells present in the
The effect of the IL6R/IL6 chimera on a population enriched with neural
crest progenitor cells, was tested in parallel to the effect of BMP2 a
factor, which is known to promote nerve differentiation. The results show
that the nerve differentiation effect promoted by the IL6R/IL6 chimera is
superior to that of BMP2, and that only the former induces, in addition to
nerve cells, Schwann cell generation. Non-treated cells (left without
cytokines) show only few, poorly developed neurons seen at 12 days and
these neurons are not associated with glial cells.
The homogeneous population used to demonstrate the nerve generation
properties of IL6R/IL6 chimera was obtained by sorting embryonic, DRG
cells by fluorescent cytometry, for LNGFR positive cells. These cells were
then cultured in defined serum free medium. The presence of nerve or glial
cells was assessed by specific immunostaining techniques as described
These results demonstrate that the IL6R/IL6 chimera can be used as a
therapeutic agent for inducing nerve replacement in injured CNS. The
IL6R/IL6 chimera may be administered locally, i.e. by direct injecting
into the damaged area within the CNS. The chimera may induce nerve
regeneration in stem cells localized within the CNS.
Neurotransplantation has been proposed as a potential treatment for the
neurodegenerative disorders, which has no effective therapy (Gage et al.
1998 and Philpott L M et al. 1997). Therefore neural cell precursors, such
as neural crest progenitor cells, can be co-implanted with IL6R/IL6
chimera or pre-treated with the IL6R/IL6 chimera prior to implantation to
regenerate neurons in damage CNS. The implanted cells may be derived
either from another mammal, from the same mammal, from a related
compatible donor or from the same organism. Neural progenitor cells may be
derived from the developing mammalian (embryonic) nervous system, from the
neonatal nervous system or from the adult nervous system of mammalian
organisms. In the present specification the terms neural progenitor cells
and neural stem cells are interchangeable.
The implant may comprise in addition to neural stem cells other cells e.g.
glial cells, known to support myelination and choroid plexus cells known
to secrete neurotrophic factors.
The pharmaceutical composition may comprise in addition to IL6R/IL6
chimera other neurotrophic factors and cytokines such as NGF, NTFs, BDNF,
IGFs, FGFs, CNTF, LIF, G-CSF, OSM, IL-11, BMP-2, GGF-2, Nrg1 and TGF.
The invention therefore relates to the use of an IL6R/IL6 chimera, a
mutein, isoform, fused protein, functional derivative, active fraction,
circularly permutated derivative or salt thereof for the manufacture of a
medicament for the treatment of CNS injuries.
An "IL6R/IL6 chimera" (also called "IL6R/IL6" or "IL-6 chimera"), as used
herein, is a chimeric molecule comprising a soluble part of the
interleukin-6 receptor fused to all or a biologically active fraction of
interleukin-6. The moieties of the chimeric protein can be fused directly,
or they can be linked by any suitable linker, such as a disulfide bridge
or a polypeptide linker. The linker may be a short linker peptide, which
can be as short as 1 to 3 amino acid residues in length or longer, for
example, 13 or 18 amino acid residues in length. Said linker may be a
tripeptide of the sequence E-F-M (Glu-Phe-Met), for example, or a 13-amino
acid linker sequence comprising
Glu-Phe-Gly-Ala-Gly-Leu-Val-Leu-Gly-Gly-Gln-Phe-Met introduced between the
amino acid sequence of the soluble IL-6 receptor and the IL-6 sequence.
Examples of IL6R/IL6 chimeric molecules are known in the art and have been
described in detail e.g. in WO 99/02552 or WO 97/32891.
The terms "treating" as used herein should be understood as preventing,
inhibiting, attenuating, ameliorating or reversing any or all symptoms or
cause(s) of neurodegenerative diseases or aging.
The invention provides for a new possibility of treating CNS injuries
since IL6R/IL6 chimera exhibits a pronounced beneficial effect over BMP-2
in terms of neurogeneration and has also the capability of inducing
myelinating cells and is able to act in the absence of IL-6 receptor.
As used herein the term "muteins" refers to analogs of an IL6R/IL6
chimera, in which one or more of the amino acid residues of the naturally
occurring components of IL-6R/IL6 are replaced by different amino acid
residues, or are deleted, or one or more amino acid residues are added to
the original sequence of an IL6R/IL6, without changing considerably the
activity of the resulting products as compared with the original IL6R/IL6.
These muteins are prepared by known synthesis and/or by site-directed
mutagenesis techniques, or any other known technique suitable therefore.
Muteins in accordance with the present invention include proteins encoded
by a nucleic acid, such as DNA or RNA, which hybridizes to DNA or RNA,
which encodes an IL6R/IL6, in accordance with the present invention, under
stringent conditions. The term "stringent conditions" refers to
hybridization and subsequent washing conditions, which those of ordinary
skill in the art conventionally refer to as "stringent". See Ausubel et
al., Current Protocols in Molecular Biology, supra, Interscience, N.Y., .sctn..sctn.63
and 6.4 (1987, 1992), and Sambrook et al. (Sambrook, J. C., Fritsch, E.
F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).
Without limitation, examples of stringent conditions include washing
conditions 12-20.degree. C. below the calculated Tm of the hybrid under
study in, e.g., 2.times.SSC and 05% SDS for 5 minutes, 2.times.SSC and
0.1% SDS for 15 minutes; 0.1.times.SSC and 0.5% SDS at 37.degree. C. for
30-60 minutes and then, a 0.1.times.SSC and 0.5% SDS at 68.degree. C. for
30-60 minutes. Those of ordinary skill in this art understand that
stringency conditions also depend on the length of the DNA sequences,
oligonucleotide probes (such as 10-40 bases) or mixed oligonucleotide
probes. If mixed probes are used, it is preferable to use tetramethyl
ammonium chloride (TMAC) instead of SSC. See Ausubel, supra.
Any such mutein preferably has a sequence of amino acids sufficiently
duplicative of that of an IL6R/IL6, such as to have substantially similar,
or even better, activity to IL6R/IL6.
One characteristic activity of IL6R/IL6 is its capability of binding to
gp130. An ELSA type assay for measuring the binding of IL6R/IL6 to gp130
has been described in detail in example 7 on page 39 of WO 99/02552, which
is fully incorporated by reference herein. As long as the mutein has
substantial binding activity to gp130, it can be considered to have
substantially similar activity to IL6R/IL6. Thus, it can be determined
whether any given mutein has at least substantially the same activity as
IL6R/IL6 by means of routine experimentation comprising subjecting such a
mutein, e.g., to a simple sandwich binding assay to determine whether or
not it binds to an immobilized gp130, as described in example 7 of WO
In a preferred embodiment, any such mutein has at least 40% identity or
homology with the amino acid sequence of IL6R/IL6 comprised in WO
99/02552. More preferably, it has at least 50%, at least 60%, at least
70%, at least 80% or, most preferably, at least 90% identity or homology
Identity reflects a relationship between two or more polypeptide sequences
or two or more polynucleotide sequences, determined by comparing the
sequences. In general, identity refers to an exact nucleotide to
nucleotide or amino acid to amino acid correspondence of the two
polynucleotides or two polypeptide sequences, respectively, over the
length of the sequences being compared.
For sequences where there is not an exact correspondence, a "percent
identity" may be determined. In general, the two sequences to be compared
are aligned to give a maximum correlation between the sequences. This may
include inserting "gaps" in either one or both sequences, to enhance the
degree of alignment. A percent identity may be determined over the whole
length of each of the sequences being compared (so-called global
alignment), that is particularly suitable for sequences of the same or
very similar length, or over shorter, defined lengths (so-called local
alignment), that is more suitable for sequences of unequal length.
Methods for comparing the identity and homology of two or more sequences
are well known in the art. Thus for instance, programs available in the
Wisconsin Sequence Analysis Package, version 9.1 (Devereux J et al 1984),
for example the programs BESTFIT and GAP, may be used to determine the %
identity between two polynucleotides and the % identity and the % homology
between two polypeptide sequences. BESTFIT uses the "local homology"
algorithm of Smith and Waterman (1981) and finds the best single region of
similarity between two sequences. Other programs for determining identity
and/or similarity between sequences are also known in the art, for
instance the BLAST family of programs (Altschul S F et al, 1990, Altschul
S F et al, 1997, accessible through the home page of the NCBI at
www.ncbi.nlm.nih.gov) and FASTA (Pearson W R, 1990; Pearson 1988).
Muteins of IL6R/IL6, which can be used in accordance with the present
invention, or nucleic acid coding therefore, include a finite set of
substantially corresponding sequences as substitution peptides or
polynucleotides which can be routinely obtained by one of ordinary skill
in the art, without undue experimentation, based on the teachings and
guidance presented herein.
Preferred changes for muteins in accordance with the present invention are
what are known as "conservative" substitutions. Conservative amino acid
substitutions of IL6R/IL6 may include synonymous amino acids within a
group which have sufficiently similar physicochemical properties that
substitution between members of the group will preserve the biological
function of the molecule (Grantham, 1974). It is clear that insertions and
deletions of amino acids may also be made in the above-defined sequences
without altering their function, particularly if the insertions or
deletions only involve a few amino acids, e.g., under thirty, and
preferably under ten, and do not remove or displace amino acids which are
critical to a functional conformation, e.g., cysteine residues. Proteins
and muteins produced by such deletions and/or insertions come within the
purview of the present invention.
Preferably, the synonymous amino acid groups are those defined in Table 1 (see Original Patent).
More preferably, the synonymous amino acid groups are those defined in
Table 2 (see Original Patent); and most preferably the synonymous amino
acid groups are those defined in Table 3 (see Original Patent).
Examples of production of amino acid substitutions in proteins which can
be used for obtaining muteins of IL6R/IL6 polypeptides, for use in the
present invention include any known method steps, such as presented in
U.S. Pat. Nos. 4,959,314, 4,588,585 and 4,737,462, to Mark et al; U.S.
Pat. No. 5,116,943 to Koths et al., U.S. Pat. No. 4,965,195 to Namen et
al; U.S. Pat. No. 4,879,111 to Chong et al; and U.S. Pat. No. 5,017,691 to
Lee et al; and lysine substituted proteins presented in U.S. Pat. No.
4,904,584 (Shaw et al).
The term "fused protein" refers to a polypeptide comprising an IL6R/IL6,
or a mutein or fragment thereof, fused with another protein, which, e.g.,
has an extended residence time in body fluids. An IL6R/IL6 may thus be
fused to another protein, polypeptide or the like, e.g., an immunoglobulin
or a fragment thereof.
"Functional derivatives" as used herein cover derivatives of IL6R/IL6, and
their muteins and fused proteins, which may be prepared from the
functional groups which occur as side chains on the residues or are
additions to the N- or C-terminal groups, by means known in the art, and
are included in the invention as long as they remain pharmaceutically
acceptable, i.e. they do not destroy the activity of the protein which is
substantially similar to the activity of IL6R/IL6, and do not confer toxic
properties on compositions containing it.
These derivatives may, for example, include polyethylene glycol
side-chains, which may mask antigenic sites and extend the residence of an
IL6R/IL6 in body fluids. Other derivatives include aliphatic esters of the
carboxyl groups, amides of the carboxyl groups by reaction with ammonia or
with primary or secondary amines, N-acyl derivatives of free amino groups
of the amino acid residues formed with acyl moieties (e.g. alkanoyl or
carboxylic aroyl groups) or O-acyl derivatives of free hydroxyl groups
(for example that of seryl or threonyl residues) formed with acyl
An "active fraction" according to the present invention may e.g. be a
fragment of IL6R/IL6. The term fragment refers to any subset of the
molecule, that is, a shorter peptide that retains the desired biological
activity. Fragments may readily be prepared by removing amino acids from
either end of the IL6R/IL6 molecule and testing the resultant fragment for
its properties to bind to gp130. Proteases for removing one amino acid at
a time from either the N-terminal or the C-terminal of a polypeptide are
known, and so determining fragments, which retain the desired biological
activity, involves only routine experimentation.
As active fractions of an IL6R/IL6, muteins and fused proteins thereof,
the present invention further covers any fragment or precursors of the
polypeptide chain of the protein molecule alone or together with
associated molecules or residues linked thereto, e.g., sugar or phosphate
residues, or aggregates of the protein molecule or the sugar residues by
themselves, provided said fraction has substantially similar activity to
The term "salts" herein refers to both salts of carboxyl groups and to
acid addition salts of amino groups of the IL6R/IL6 molecule or analogs
thereof. Salts of a carboxyl group may be formed by means known in the art
and include inorganic salts, for example, sodium, calcium, ammonium,
ferric or zinc salts, and the like, and salts with organic bases as those
formed, for example, with amines, such as triethanolamine, arginine or
lysine, piperidine, procaine and the like. Acid addition salts include,
for example, salts with mineral acids, such as, for example, hydrochloric
acid or sulfuric acid, and salts with organic acids, such as, for example,
acetic acid or oxalic acid. Of course, any such salts must retain the
biological activity of IL6R/IL6, i.e., the ability to bind to gp130.
In a preferred embodiment of the invention, the IL6R/IL6 chimera is
glycosylated at one or more sites.
A glycosylated form of an IL6R/IL6 chimera has been described in WO
99/02552 (PCT/IL98/00321), which is the chimeric molecule highly preferred
according to the invention. The IL6R/IL6 chimera described therein is a
recombinant glycoprotein, which was obtained fusing the entire coding
sequence of the naturally occurring soluble IL-6 receptor .delta.-Val (Novick
et al., 1990) to the entire coding sequence of mature naturally occurring
IL-6, both from human origin.
The IL6R/IL6 chimera may be produced in any adequate eucaryotic or
procaryotic cell type, like yeast cells, insect cells, bacteria, and the
like. It is preferably produced in mammalian cells, most preferably in
genetically engineered CHO cells as described in WO 99/02552. Whilst the
protein from human origin is preferred, it will be appreciated by the
person skilled in the art that a similar fusion protein of any other
origin may be used according to the invention, as long as it retains the
biological activity described herein.
The chimeric molecule can then be produced in bacterial cells, which are
not capable of synthesizing glycosyl residues, but usually have a high
yield of produced recombinant protein.
The IL6R/IL6 chimera may comprises an immunoglobulin fusion, i.e. the
IL6R/IL6 according to the invention may be fused to all or a portion of an
immunoglobulin. Methods for making immunoglobulin fusion proteins are well
known in the art, such as the ones described in WO 01/03737, for example.
The person skilled in the art will understand that the resulting fusion
protein of the invention retains the biological activity of the IL6R/IL6
chimera. The resulting fusion protein ideally has improved properties,
such as an extended residence time in body fluids (half-life), increased
specific activity, increased expression level, or facilitated purification
of the fusion protein.
The IL6R/IL6 chimera may be fused to the constant region of an Ig
molecule. Preferably, to heavy chain regions, like the CH2 and CH3 domains
of human IgG1, for example. Other isoforms of Ig molecules are also
suitable for the generation of fusion proteins according to the present
invention, such as isoforms IgG.sub.2 or IgG.sub.4, or other Ig classes,
like IgM or IgA, for example. Fusion proteins may be monomeric or
multimeric, hetero- or homomultimeric.
Functional derivatives of IL6R/IL6 chimera may be conjugated to polymers
in order to improve the properties of the protein, such as the stability,
half-life, bioavailability, tolerance by the human body, or immunogenicity.
Therefore, a preferred embodiment of the invention relates to a functional
derivative of the IL6R/IL6 chimera comprising at least one moiety attached
to one or more functional groups, which occur as one or more side chains
on the amino acid residues.
A highly preferred embodiment relates to an IL6R/IL6 linked to
Polyethlyenglycol (PEG). PEGylation may be carried out by known methods,
such as the ones described in WO 92/13095, for example.
The IL6R/IL6 chimera may be delivered to the brain in any adequate
formulation. It may also be delivered in form of cells expressing and/or
secreting an IL6R/IL6 chimera, a mutein, fused protein, active fraction or
circularly permutated derivative thereof.
The invention therefore further relates to the use of a neural stem cell
and IL6R/IL6 chimera, a mutein, fused protein, active fraction or
circularly permutated derivative thereof, for manufacture of a medicament
for the treatment of CNS injuries. The cells may be administered in any
suitable form. However, a polymer-encapsulated cell is a highly preferred
mode of delivery of the cells. The encapsulation procedure is described in
detail e.g. by Emerich et al (1994) or U.S. Pat. No. 5,853,385. Suitable
cell lines and stable expression systems are well known in the art.
The delivery of IL6R/IL6 chimera to the brain may also be carried out
using a vector comprising the coding sequence or an IL6R/IL6 chimera, a
mutein, fused protein, active fraction or circularly permutated derivative
thereof. The vector comprises all regulatory sequences needed for
expression of the desired protein in the human body, preferably in the
brain, more preferably in the striatum. Regulatory sequences for
expression vectors are known by the person skilled in the art. The
invention thus also relates to the use of a vector comprising the coding
sequence of IL6R/IL6 chimera for manufacture of a medicament for the
treatment of CNS injuries.
Any expression vector known in the art may be used according to the
invention. However, a lentivirally-derived vector may be particularly
useful for the delivery of IL6R/IL6 chimera directly into the striatum.
Such lentiviral vectors are known in the art. They are specifically
described e.g. in Kordower et al. (1999) or Deglon et al. (2000).
It is a further object of the present invention to provide a
pharmaceutical composition comprising IL6R/IL6 chimera, a mutein, fused
protein, functional derivative, active fraction, circularly permutated
derivative or salt thereof, optionally together with one or more
pharmaceutically acceptable carriers, diluents or excipients, for the
treatment of CNS injury. The IL6/IL6 chimera used may be either from
eukaryotic origin (glycosylated) or from bacterial origin (non-glycosylated).
The invention further relates to a pharmaceutical composition comprising
IL6R/IL6 chimera, to a pharmaceutical composition comprising an expression
vector, in particular a lentiviral gene therapy vector expressing IL6R/mL
chimera and to pharmaceutical composition comprising in addition to the
IL6R/IL6 chimera (in the form of protein or cells producing the chimera or
an expression vector encoding the chimera) neural stem cells optionally
together with one or more pharmaceutically acceptable carriers, diluents
or excipients, for the treatment of CNS injury.
The definition of "pharmaceutically acceptable" is meant to encompass any
carrier, which does not interfere with effectiveness of the biological
activity of the active ingredient and that is not toxic to the host to
which it is administered. For example, for parenteral administration,
IL6R/IL6 chimera may be formulated in a unit dosage form for injection in
vehicles such as saline, dextrose solution, serum albumin and Ringer's
The IL6R/IL6 chimera can be administered to a patient in need of
administration thereof in a variety of ways. The routes of administration
include intracranial, intradermal, transdermal (e.g. in slow release
formulations), intramuscular, intraperitoneal, intravenous, subcutaneous,
oral, epidural, topical, and intranasal routes. Any other therapeutically
efficacious route of administration can be used, for example by gene
therapy wherein a DNA molecule encoding the IL6R/IL6 chimera is
administered to the patient (e.g. via a vector), which causes the IL6R/IL6
chimera to be expressed and secreted in vivo. In addition the IL6R/IL6
chimera can be administered together with other components of biologically
active agents such as pharmaceutically acceptable surfactants, excipients,
carriers, diluents and vehicles.
A "therapeutically effective amount" is such that when administered, the
MIFNAR2, or a functional derivative, analog, fusion protein or fragments
thereof results in modulation of the biological activity of IFN-.beta..
The dosage administered, as single or multiple doses, to an individual may
vary depending upon a variety of factors, including the route of
administration, patient conditions and characteristics (sex, age, body
weight, health, size), extent of symptoms, concurrent treatments,
frequency of treatment and the effect desired. Adjustment and manipulation
of established dosage ranges are well within the ability of those skilled
in the art, as well as in vitro and in vivo methods of determining the
activity of MIFNAR2.
For parenteral (e.g. intravenous, subcutaneous, intramuscular)
administration, IL6R/IL6 chimera can be formulated as a solution,
suspension, emulsion or lyophilized powder in association with a
pharmaceutically acceptable parenteral vehicle (e.g. water, saline,
dextrose solution) and additives that maintain isotonicity (e.g. mannitol)
or chemical stability (e.g. preservatives and buffers). The formulation is
sterilized by commonly used techniques.
It is a further object of the present invention to provide for a method
for treating CNS injury, comprising administering to a patient in need
thereof an effective amount of IL6R/IL6 chimera, a mutein, fused protein,
functional derivative, active fraction, circularly permutated derivative
or salt thereof optionally together with a pharmaceutically acceptable
An "effective amount" refers to an amount of the active ingredients that
is sufficient to affect the course and the severity of the diseases
described above, leading to the reduction or remission of such pathology.
The effective amount will depend on the route of administration and the
condition of the patient.
The dosage administered, as single or multiple doses, to an individual
will vary depending upon a variety of factor, including IL6R/IL6 chimera
pharmacokinetic properties, the route of administration, patient
conditions and characteristics (sex, age, body weight, health, size),
extent of symptoms, concurrent treatments, frequency of treatment and the
effect desired. Adjustment and manipulation of established dosage ranges
are well within the ability of those skilled.
A method for treating CNS injury, comprising administering to a patient in
need thereof an effective amount of IL6R/IL6 chimera, a mutein, fused
protein, active fraction or circularly permutated derivative thereof, or
comprising administering to a patient in need thereof an expression vector
comprising the coding sequence of IL6R/IL6 chimera, a mutein, fused
protein, active fraction or circularly permutated derivative thereof, are
further objects of the present invention and or neural stem cells.
It is a further object of the present invention to provide for a method
for the preparation of differentiated nerve and associate glial cells for
transplantation into patients in order to repair damage to nervous
tissues. The IL6R/IL6 chimera will in this case be used ex-vivo to
stimulate the development of neural cells from either embryonic
neuro-glial progenitor cells or from human embryonic Stem cell lines (ES
cells). Such stimulation can greatly improve the yield of nerve cells from
in vitro cultures, facilitating the use of these tissues for subsequent
Claim 1 of 6 Claims
1. A method of increasing the number of
neurons in a patient, comprising stimulating neural progenitor cells by
culturing the cells ex vivo with a composition comprising IL6R/IL6
chimera, fused protein, functional derivative, circularly permutated
derivative or salt thereof prior to transplantation, and then subsequently
transplanting the cells to a patient suffering from loss or atrophy of
neurons caused by aging or by a neurological disease.
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