U.S. patent application number 13/757289 was filed with the patent office on 2013-09-05 for novel neurturin conjugates for pharmaceutical use.
This patent application is currently assigned to DeveloGen Aktiengesellschaft. The applicant listed for this patent is DEVELOGEN AKTIENGESELLSCHAFT. Invention is credited to Matthias Austen, Marcus Geese, Friedrich Harder, Rainer Mussmann, Martin Schneider, Thomas Siegmund.
Application Number | 20130231283 13/757289 |
Document ID | / |
Family ID | 40521395 |
Filed Date | 2013-09-05 |
United States Patent
Application |
20130231283 |
Kind Code |
A1 |
Austen; Matthias ; et
al. |
September 5, 2013 |
NOVEL NEURTURIN CONJUGATES FOR PHARMACEUTICAL USE
Abstract
The present invention relates to neurturin protein products
conjugated to polyols and to pharmaceutical compositions comprising
neurturin conjugates as active ingredients, preferably PEGylated
neurturin conjugates or variants thereof, having increased
bioavailability.
Inventors: |
Austen; Matthias;
(Goettingen, DE) ; Geese; Marcus; (Goettingen,
DE) ; Mussmann; Rainer; (Magden, CH) ; Harder;
Friedrich; (Lindau, DE) ; Siegmund; Thomas;
(Landolfshausen, DE) ; Schneider; Martin;
(Goettingen, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
DEVELOGEN AKTIENGESELLSCHAFT |
Goettingen |
|
DE |
|
|
Assignee: |
DeveloGen
Aktiengesellschaft
Goettingen
DE
|
Family ID: |
40521395 |
Appl. No.: |
13/757289 |
Filed: |
February 1, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12774235 |
May 5, 2010 |
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13757289 |
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PCT/EP2008/009320 |
Nov 5, 2008 |
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12774235 |
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Current U.S.
Class: |
514/7.6 ;
530/399 |
Current CPC
Class: |
C07K 14/4756 20130101;
A61P 1/18 20180101; A61P 3/06 20180101; A61K 38/185 20130101; A61P
25/00 20180101; A61K 9/0019 20130101; A61P 25/28 20180101; A61P
43/00 20180101; A61K 47/60 20170801; A61P 3/10 20180101 |
Class at
Publication: |
514/7.6 ;
530/399 |
International
Class: |
C07K 14/475 20060101
C07K014/475 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 5, 2007 |
EP |
07021493.7 |
Claims
1-2. (canceled)
3. The neurturin conjugate according to claim 9, wherein the polyol
moiety is a polyethylene glycol moiety.
4. The neurturin conjugate according to claim 9, wherein the polyol
moiety is a single chain polyol moiety.
5. The neurturin conjugate according to claim 9, wherein the polyol
moiety is a branched chain polyol moiety.
6. The neurturin conjugate according to claim 9, wherein the polyol
moiety is a polyalkylene glycol moiety.
7. The neurturin conjugate according to claim 9, having the same or
a higher in vivo neurturin activity as native human neurturin.
8. (canceled)
9. A neurturin conjugate comprising a polyol moiety covalently
bound to a human neurturin protein product or a biologically active
fragment thereof, wherein said neurturin protein product has an
amino acid sequence that is at least 90% identical to SEQ ID NO: 5,
wherein said neurturin protein product or biologically active
fragment thereof is capable of dimerizing with a neurturin protein
having the amino acid sequence of SEQ ID NO: 5, and wherein said
neurturin protein product or biologically active fragment thereof
has: a) at least one of the arginine residues corresponding to
amino acid positions 51, 52, 54, 56, 57, 58, 60, 61, 63 or 65 of
SEQ ID NO: 5 substituted with a lysine, and/or b) at least one of
the arginine residues corresponding to amino acid positions 51, 52,
54, 56, 57, 58, 60, 61 or 65 of SEQ ID NO: 5 substituted with a
neutral or acidic amino acid.
10. The neurturin conjugate according to claim 9, wherein at least
one of the arginine residues 51, 52, 54, 56, 57, 58, 60, 61, 63 or
65 is substituted with a lysine.
11. The neurturin conjugate according to claim 9, wherein at least
one of the arginine residues 51, 52, 54, 56, 57, 58, 60, 61, or 65
is substituted by a neutral or acidic amino acid residue.
12-13. (canceled)
14. The neurturin conjugate of claim 9, wherein the arginine
residue 63 is substituted by lysine.
15. The neurturin conjugate of claim 10, which consists of two
neurturin monomers, and which comprises 1-4 polyol moieties bound
to the N-terminus and/or bound to a lysine side chain.
16. A pharmaceutical composition comprising at least one neurturin
protein product and/or a biologically active fragment thereof
conjugated to at least one polyethylene glycol molecule as an
active ingredient together with a pharmaceutically acceptable
carrier, diluent and/or adjuvant, wherein said neurturin protein
product has an amino acid sequence that is at least 90% identical
to SEQ ID NO: 5, wherein said neurturin protein product or
biologically active fragment thereof is capable of dimerizing with
a neurturin protein having the amino acid sequence of SEQ ID NO: 5,
and wherein said neurturin protein product or biologically active
fragment thereof has: a) at least one of the arginine residues 51,
52, 54, 56, 57, 58, 60, 61, 63 or 65 substituted with a lysine,
and/or b) at least one of the arginine residues 51, 52, 54, 56, 57,
58, 60, 61, or 65 substituted with a neutral or acidic amino
acid.
17. The composition of claim 16, wherein said neurturin protein
product is a biologically active fragment thereof.
18. The composition of claim 16, wherein said neurturin protein
product is mono-PEGylated, comprising a single polyethylene glycol
molecule chain.
19. The composition of claim 16, wherein said neurturin protein
product is oligo- or poly-PEGylated, comprising two, three, four or
several polyethylene glycol molecule chains.
20. The composition of claim 16, wherein said neurturin protein
product comprises at least one linear polyethylene glycol molecule
chain.
21. The composition of claim 16, wherein said neurturin protein
product comprises at least one branched polyethylene glycol
molecule chain.
22. The composition of claim 16, wherein said polyethylene glycol
molecule has an average molecular weight of 100 to 10000 Da.
23. The composition of claim 16, wherein said polyethylene glycol
molecule is linked to the N-terminal amino acid of said neurturin
protein product.
24. The composition of claim 16, wherein said polyethylene glycol
molecule is linked to said neurturin protein product via an acyl or
alkyl linkage.
25. The composition of claim 16, wherein said polyethylene glycol
molecule is terminated by an OH-, OCH.sub.3- or OEt-group.
26. The composition of claim 16, which has an increased
bioavailability of the active ingredient compared to a composition
with an unmodified control neurturin protein product.
27. The composition of claim 16, wherein said neurturin protein is
present in an amount to provide an at least 2-fold increase in the
bioavailability of the active ingredient.
28-31. (canceled)
32. The composition of claim 16 for administration to a mammal.
33. The variant of human neurturin of claim 34, wherein said
neurturin protein product comprises 2, 3 or 4 amino acid changes
compared to wild-type human neurturin.
34. A variant of human neurturin comprising a human neurturin
protein product or a biologically active fragment thereof, wherein
said human neurturin protein product has an amino acid sequence
that is at least 90% identical to SEQ ID NO: 5, wherein said
neurturin protein product or biologically active fragment thereof
is capable of dimerizing with a neurturin protein having the amino
acid sequence of SEQ ID NO: 5, and wherein said neurturin protein
product or biologically active fragment thereof has: a) at least
one of the arginine residues 51, 52, 54, 56, 57, 58, 60, 61, 63 or
65 substituted with a lysine, and/or b) at least one of the
arginine residues 51, 52, 54, 56, 57, 58, 60, 61, or 65 substituted
with a neutral or acidic amino acid.
35. (canceled)
36. A method for administering a pharmaceutical composition to a
subject in need thereof comprising a pharmaceutically active amount
of the neurturin protein product or a biologically active fragment
thereof of claim 9, wherein said neurturin protein product or
biologically active fragment thereof is conjugated to at least one
polyethylene glycol molecule as an active ingredient together with
a pharmaceutically acceptable carrier, diluent and/or adjuvant.
37. The method of claim 36, wherein the subject is human.
38. (canceled)
39. The variant of human neurturin according to claim 34, wherein
the arginine residue at position 63 is substituted by lysine.
40. The variant of human neurturin according to claim 34, wherein
at least one arginine residue 51, 52, 54, 56, 57, 58, 60, 61, or 65
is substituted by glutamic acid.
41. The variant of human neurturin according to claim 34, wherein
said at least one amino acid change is a substitution of at least
one arginine residue with a neutral or acidic amino acid residue.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 12/774,235, filed May 5, 2010, which is a continuation-in-part
of PCT/EP2008/009320, filed Nov. 5, 2008, which claims the benefit
of European Patent Application No. 07021493.7, filed Nov. 5, 2007.
The specifications of each of the foregoing applications are
incorporated herein by reference in their entirety.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted via EFS-Web and is hereby incorporated by
reference in its entirety. Said ASCII copy, created on Feb. 1,
2013, is named 1027280040102_Seq.txt, and is 10,972 bytes in
size.
FIELD OF THE INVENTION
[0003] The present invention relates to the modification of human
neurturin to increase its serum half life and pharmacokinetic
profile. In particular, the invention relates to novel neurturin
conjugates comprising neurturin or biologically active fragments
thereof covalently attached to polyol moieties, preferably
polyethylene glycol. The invention further relates to
pharmaceutical compositions with increased bioavailability
comprising novel neurturin conjugates as active agent for therapy,
treatment, prevention and/or diagnosis of diabetes and
neurodegeneration.
BACKGROUND
[0004] Pancreatic beta-cells secrete insulin in response to
elevated blood glucose levels. Insulin amongst other hormones plays
a key role in the regulation of the fuel metabolism. Insulin leads
to the storage of glycogen and triglycerides and to the synthesis
of proteins. The entry of glucose into muscles and adipose cells is
stimulated by insulin. In patients who suffer from diabetes
mellitus type I or LADA (latent autoimmune diabetes in adults,
Pozzilli & Di Mario, 2001, Diabetes Care. 8:1460-1467)
beta-cells are being destroyed due to autoimmune attack. The amount
of insulin produced by the remaining pancreatic islet cells is too
low, resulting in elevated blood glucose levels (hyperglycemia). In
diabetes mellitus type II liver and muscle cells loose their
ability to respond to normal blood insulin levels (insulin
resistance). High blood glucose levels (and also high blood lipid
levels) in turn lead to an impairment of beta-cell function and to
an increase in beta-cell death. Interestingly, betacell
regenerative processes like neogenesis and replication do not
appear to compensate for the loss of beta cell mass in type II
diabetics, thus causing a reduction in total beta-cell mass over
time. Eventually the application of exogenous insulin becomes
necessary in type II diabetics for an adequate control of blood
glucose levels.
[0005] In type I diabetics, where beta-cells are being destroyed by
autoimmune attack, treatments have been devised which modulate the
immune system and may be able to stop or strongly reduce islet
destruction (Raz et al., 2001, Lancet 358: 1749-1753; Chatenoud et
al., 2003, Nat Rev Immunol. 3: 123-132; Homann et al., Immunity.
2002, 3:403-415). However, due to the relatively slow regeneration
of human beta-cells such treatments can be more successful if they
are combined with treatments that can stimulate beta-cell
regeneration.
[0006] Diabetes is a very disabling disease, because today's common
anti-diabetic drugs do not control blood sugar levels well enough
to completely prevent the occurrence of high and low blood sugar
levels. Frequently elevated blood sugar levels are toxic and cause
long-term complications like for example nephropathy, retinopathy,
neuropathy and peripheral vascular disease. Extensive loss of beta
cells also leads to deregulation of glucagon secretion from
pancreatic alpha cells which contributes to an increased risk of
dangerous hypoglycemic episodes. There is also a host of related
conditions, such as obesity, hypertension, heart disease and
hyperlipidemia, for which persons with diabetes are substantially
at risk.
[0007] Apart from the impaired quality of life for the patients,
the treatment of diabetes and its long term complications presents
an enormous financial burden to our healthcare systems with rising
tendency. Thus, for the treatment of diabetes mellitus type I and
LADA, but also for the treatment of late stages of diabetes
mellitus type II there is a strong need in the art to identify
factors that induce regeneration of pancreatic insulin producing
beta-cells. These factors could restore normal function of the
endocrine pancreas once its function is impaired or event could
prevent the development or progression of diabetes type I, LADA or
diabetes type II.
[0008] Neurturin is a secreted protein which is expressed in
embryonic pancreas. Recombinant neurturin has been shown to
stimulate the differentiation of mouse embryonic stemcells into
insulin producing cells. Moreover, transgenic mice with elevated
neurturin levels in the pancreas have a substantially increased
pancreatic beta-cell mass. Based on these findings, the use of
neurturin for the treatment of pancreatic disorders such as
diabetes has been proposed (see for example WO 03/99318 and WO
2005/051415, the disclosure of which is herein incorporated by
reference).
[0009] Neurturin is a member of the GDNF family of ligands (GFL)
comprised of Glial cell line-derived neurotrophic factor (GDNF),
Neurturin, Artemin and Persephin. Mature neurturin is a homodimer
with a size of 23.6 kDa comprised of 102 amino acid monomers. Each
monomer contains 3 intrachain disulfide bonds forming a cystine
knot. An additional disulfide bridge is connecting the
monomers.
[0010] Neurturin had previously been proposed as a treatment for
neurodegenerative diseases such as Parkinsons, Alzheimers and
Huntington's disease, motor neuron disorders, spinal cord injuries
or hearing disorders (WO 97/08196, WO 99/06064; Akerud et al. J.
Neurochem. 1999; 73(1):70-78; Koeberle & Ball Neuroscience.
2002; 110(3):555-567; Bilak et al. Mol Cell Neurosci. 1999;
13(5):326-336; Perez-Navarro et al. Neuroscience. 2000;
98(1):89-96; Rosenblad et al., Eur J. Neurosci. 1999;
11(5):1554-1566, the disclosures of which are herein incorporated
by reference).
[0011] It was found, however, that upon convection-enhanced
delivery (CED) into rat brains, the distribution volume of
neurturin and the related factor GDNF was limited (Hamilton et al.,
Exp Neurol. 2001; 168(1):155-161). Proteins must typically be
administered by injection. After injection most proteins are
cleared rapidly from the body, necessitating frequent injections.
In our own studies, we found that the bioavailability after
subcutaneous and intravenous injection of neurturin in mammals is
low. Only about 10 percent of subcutaneously injected neurturin
enter the circulation. Consequently, it is difficult to achieve
therapeutically useful blood levels of the proteins in
patients.
[0012] Thus, there is a strong need to develop a neurturin variant
with enhanced availability in patients and therewith the
development of methods to prolong the circulating half-lives of
protein therapeutics in the body--the bioavailability--so that
neurturin as active agent does not have to be injected frequently
in order to satisfy the needs of patients for "user-friendly"
protein therapeutics.
[0013] Thus, the underlying problem of the present invention was to
provide new variants and formulations of neurturin that enhance its
bioavailability.
[0014] The problem is solved by providing the embodiments
characterized in the claims.
SUMMARY OF THE INVENTION
[0015] The present invention relates to a novel neurturin conjugate
comprising a polyol moiety covalently bound to a human neurturin
protein product.
[0016] The present invention further relates to a pharmaceutical
composition comprising at least one neurturin protein product
and/or a biologically active fragment thereof which is conjugated
to at least one polyethylene glycol molecule as an active
ingredient together with a pharmaceutically acceptable carrier,
diluent and/or adjuvant.
[0017] In a further embodiment, the present invention relates to a
method for administering a pharmaceutical composition to a subject
in need thereof comprising a pharmaceutically active amount of at
least one modified neurturin protein product or a biologically
active fragment thereof which is conjugated to at least one
polyethylene glycol molecule as an active ingredient together with
a pharmaceutically acceptable carrier, diluent and/or adjuvant.
[0018] It is a further aspect of the present invention to provide a
method for administering a pharmaceutical composition to a subject
in need thereof comprising a pharmaceutically active amount of at
least one modified neurturin protein product or a biologically
active fragment thereof which is conjugated to at least one
polyethylene glycol molecule as an active ingredient together with
a low-molecular weight substance together with a pharmaceutically
acceptable carrier, diluent and/or adjuvant.
[0019] The compositions of the present invention are suitable for
preventing or treating pancreatic disorders or neurodegenerative
diseases, particularly pancreatic autoimmune disorders, e.g.
autoimmune diabetes such as type I diabetes or LADA but also type
II diabetes.
DETAILED DESCRIPTION OF THE INVENTION
[0020] The present invention provides neurturin conjugates
comprising covalently attached polyol moieties such as polyethylene
glycol (PEG), as well as pharmaceutical compositions comprising
these neurturin conjugates, as well as variants, derivatives or
biologically active fragments thereof.
[0021] Unless specific definitions are provided, the nomenclature
utilized in connection with, and the laboratory procedures,
techniques and methods described herein are those known in the art
to which they pertain. Standard chemical symbols and abbreviations
are used interchangeably with the full names represented by such
symbols. Standard techniques may be used for chemical syntheses,
chemical analyses, pharmaceutical preparation, composition,
delivery, and treatment of patients. Standard techniques may be
used for recombinant DNA methodology, oligonucleotide synthesis,
tissue culture and the like. Reactions and purification techniques
may be performed e.g., using kits according to manufacturer's
specifications, as commonly accomplished in the art or as described
herein. The foregoing techniques and procedures may be generally
performed according to conventional methods well known in the art
and as described in various general or more specific references
that are cited and discussed throughout the present
specification.
[0022] The term "analog" refers to a polypeptide which resembles
neurturin in structure, having a similar function.
[0023] The term "biologically active" or "biologic activity" as
used herein means that the neurturin product induces and/or
stimulates the differentiation of insulin producing cells from
progenitor, e.g. stem cells, and/or promotes the protection,
survival, or regeneration of insulin producing cells, e.g. beta
cells in vivo or in vitro. The biologic activity of the modified
neurturin protein products as disclosed herein can easily be
determined in standard in vitro assays as disclosed for the
determination of GDNF activity, described in WO 93/06116 and in
U.S. patent application Ser. No. 08/535,681 or as described in the
present Examples.
[0024] As used herein, "fragments" of neurturin refer to portions
of neurturin that are generated by any method, including but not
limited to enzymatic digestion and chemical cleavage (e.g. CNBr) of
neurturin and physical shearing of the polypeptide. Fragments of
neurturin may also be generated, e.g. by recombinant DNA technology
and by amino acid synthesis.
[0025] As "active fragment" or "biologically active fragment" of
neurturin as used in the present invention refers to any fragment
or precursor of the polypeptidic chain of neurturin, alone or in
combination with related molecules or residues bound to it, for
example, residues of sugars or phosphates, or aggregates of the
polypeptide molecule when such fragments or precursors show the
same biological activity of neurturin. The term "biologically
active fragment" also relates to a fragment of a neurturin product
which induces and/or stimulates the differentiation of insulin
producing cells from progenitor, e.g. stem cells, and/or promotes
the protection, survival, or regeneration of insulin producing
cells, e.g. beta cells in vivo or in vitro. The biologic activity
of a fragment of the modified neurturin protein products as
disclosed herein can easily be determined in standard in vitro
assays as disclosed for the determination of GDNF activity,
described in WO 93/06116 and in U.S. patent application Ser. No.
08/535,681 or as described in the present Examples.
[0026] The term "homologue" is used herein to relate to a
polypeptide related to neurturin by descent from a common ancestral
protein sequence. The term, homolog, may apply to the relationship
between proteins separated by the event of genetic duplication.
[0027] The term "low molecular weight substance" refers to a
substance with an average molecular weight of between 100 and 12000
Da, preferably of between 200 and 8000 Da and most preferably of
about 500 and 7000 Da.
[0028] The low molecular weight substance may be a polyanionic
polymer which is natural or synthetic and which contains a
plurality of anionic groups such as carboxylate and/or sulfate
groups. For example, the polyanionic polymer may be selected from
low molecular weight sulphated saccharides, sulphated
cyclodextrins, or sulphated synthetic polymers such as acrylic
polymers, aromatic polymers, and/or polyalcohols. More
particularly, the polyanionic polymer is selected from low
molecular weight heparins or heparin derivatives, heparan
sulphates, chondroitin sulfates, dextran sulphates, chemically
modified heparin-derived oligosaccharides (list from Wang et al.
(2002), supra), heparin-like oligosaccharides, dextran sulphates,
sulphated low molecular weight glycosaminoglycans,
dextrin-2-sulphates, cellulose sulphates and naphthalene sulfonate
polymer (e.g. PRO 2000), PAVAS (a co-polymer of acrylic acid with
vinyl alcohol sulphate), the sulphonated polymer PAMPS
[poly(2-acryl-amido-2-methyl-1-propanesulfonic acid] (M.sub.w e.g.
approximately 7000-12000), chondroitin sulphates, sulphated
cyclodextrins, laminarin sulphate (Alban, S. in Carbohydrates in
Drug Design (Ed. Z. J. Witczak, K. A. Nieforth) Dekker, New York,
1997, pp 209.), polyglycerin sulphates (T.eta.rk, H., Haag, R.,
Alban, S. Bioconjugate Chem. 2004, 15, 162;), pentosan
polysulphates (PPS) and derivatives thereof such lactose-modified
pentosan polysulphates, fractionated PPS/low molecular weight PPS,
fucoidan, or derivatives or combinations thereof.
[0029] Low molecular weight heparin (LMWH) analogues such as
Enoxaparin, Dalteparin or Fragmin are preferred examples of
suitable polymers. They are obtained by fractionation and/or
limited enzymatic or chemical digestion of heparin, and have an
average molecular weight of preferably about 3000 to about 7000
Dalton (Weitz 1997 supra).
[0030] The term "modified neurturin protein product" as used herein
relates to a chemically modified neurturin protein conjugate as
well as cell-expressed conjugate comprising a covalently attached
polyol moiety.
[0031] Chemically modified neurturin conjugates may be prepared by
one of skill in the art given the disclosures herein or by any of
the methods known in the art.
[0032] The polyol moiety of the neurturin conjugate of the
invention can be any water-soluble mono- or bifunctional
poly(alkylene oxide) having a linear or branched chain. Typically,
the polyol is a poly(alkylene glycol) such as poly(ethylene glycol)
(PEG). However, those of skill in the art will recognize that other
polyols, such as, for example poly(propylene glycol) and copolymers
of polyethylene glycol and polypropylene glycol, can be suitably
used. Other neurturin conjugates can be prepared by coupling
neurturin to a water-soluble polymer. A non-limiting list of such
polymers include other polyalkylene oxide homopolymers such as
polypropylene glycols, polyoxyethylenated polyols, copolymers
thereof and block copolymers thereof. As an alternative to
polyalkylene oxide-based polymers, effectively non-antigenic
materials such as dextran, polyvinyl pyrrolidones, polyacrylamides,
polyvinyl alcohols, carbohydrate-based polymers and the like can be
used.
[0033] The protein conjugates may also be expressed in prokaryotic
or eukaryotic cells well known in the art.
[0034] The term "neurturin conjugate" relates to a neurturin
protein product which comprises a polyol moiety such as
polyethylene glycol covalently attached to an amino acid of
neurturin. The amino acid can be site-specific at the N-terminal
amino acid or at any other amino acid. PEG conjugates can contain
one or more PEG molecules covalently attached to any suitable site
of the neurturin protein molecule.
[0035] The term "neurturin protein product", "neurturin product",
"neurturin protein" or "neurturin" which are used interchangeably
herein, relate to a protein or peptide having a degree of identity
to the biologically active human neurturin product resulting in the
cleavage of the neurturin precursor having the amino acid sequence
published as GenBank Accession Number NP.sub.--004549 (SEQ ID NO:
1, FIG. 3) or to the human neurturin precursor itself, that is in
excess of 70%, preferably in excess of 80%, more preferably in
excess of 90% and most preferably in excess of 95%, variants or
derivatives thereof. The degree of identity between the mouse and
the human protein is about 91%, and it is contemplated that
preferred mammalian neurturin proteins will have a similarly high
degree of identity. The percentage of identity of a neurturin
product and the human neurturin protein or a precursor may be
determined according to standard procedures, e.g. by using the
BLAST algorithm. Preferably, the percentage of identity is
calculated as the percentage of amino acid residues found in the
smaller of the two sequences that align with identical amino acid
residues in the sequence being compared, when four gaps in a length
of 100 amino acids may be introduced to assist in that
alignment.
[0036] The term "ortholog" relates to polypeptides in different
species that evolved from a common ancestral gene by specification.
Orthologs retain the same function in the course of evolution.
[0037] The term "polyethylene glycol" or "PEG" relates to PEG
itself as well as derivatives thereof. The polymer PEG is commonly
used as methoxy-PEG-OH, (m-PEG), in which one terminus is the
relatively inert methoxy group, while the other terminus is a
hydroxyl group that is subject to chemical modification.
[0038] Branched PEGs are also in common use. The number of
branching arms (m) can range from three to a hundred or more. The
hydroxyl groups are further subject to chemical modification.
Another branched form, such as that described in PCT patent
application WO 96/21469, has a single terminus that is subject to
chemical modification. Yet another branched form, has reactive
groups, such as carboxyl, along the PEG backbone rather than at the
end of PEG chains. In addition to these forms of PEG, the polymer
can also be prepared with weak or degradable linkages in the
backbone. For example, Harris has shown in U.S. patent application
Ser. No. 06/026,716, which is incorporated by reference herein in
its entirety, that PEG can be prepared with ester linkages in the
polymer backbone that are subject to hydrolysis. This hydrolysis
results in cleavage of the polymer into fragments of lower
molecular weight. The copolymers of ethylene oxide and propylene
oxide are closely related to PEG in their chemistry, and they can
be used instead of PEG in many of its applications.
[0039] The term "PEGylation" comprises covalent attachment of a
polyethylene glycol molecule to a substrate, which can be a
protein. PEGylation of proteins is widely known in the state of the
art and has been reviewed by, for example, Veronese, F. M.,
Biomaterials 22 (2001) 405-417. PEG can be linked using different
functional groups and polyethylene glycols with different molecular
weight, linear and branched PEGs as well as different linking
groups (see also Francis, G. E., et al., Int. J. Hematol. 68 (1998)
1-18; Delgado, C., et al., Crit. Rev. Ther. Drug Carrier Systems 9
(1992 (249-304).
[0040] PEGylation can be performed in aqueous solution with
PEGylation reagents as described, for example, in WO 00/44785,
preferably using NHS-activated linear or branched PEG molecules of
a molecular weight between 5 and 40 kDa. PEGylation can also be
performed at the solid phase according to Lu, Y. et al., Reactive
Polymers 22 (1994) 221-229.
[0041] Selective PEGylation at the N-terminal amino acid can be
performed according to Felix, A. M., et al., ACS Symp. Ser 680
(Poly(ethylene glycol)) (1997) 218-238. Selective N-terminal
PEGylation is achieved during solid-phase synthesis by coupling of
a N-PEGylated amino acid derivative to the N-terminal amino acid of
the peptide chain. Side chain PEGylation is performed during
solid-phase synthesis by coupling of N-PEGylated amino acid
derivatives to the growing chain. Combined N-terminal and side
chain PEGylation is processed either as described above within
solid-phase synthesis or by solution phase synthesis by applying
activated PEG reagents to the amino deprotected peptide.
[0042] Suitable PEG derivatives are activated PEG molecules with a
preferred average molecular weight of from about 5 to about 40 kDa,
more preferably from about 20 to about 40 kDa, and most preferably
about 30 kDa. In other embodiments, PEG derivatives with a
preferred average molecular weight of from about 1 to about 5 kDa,
more preferably from about 1.5 to about 3 kDa, and most preferably
about 2 kDa are used. The PEG derivatives can be linear or branched
PEGs.
[0043] Activated PEG derivatives are known in the art and described
in, for example, Morpurgo, M., et al., J. Bioconj. Chem. 7 (1996)
363-368, for PEG-vinylsulfone. Linear chain and branched chain PEG
species are suitable for the preparation of the PEGylated
conjugates. Examples of reactive PEG reagents have the structure
RO-(PEG).sub.m-X, e.g. iodo-acetyl-methoxy-PEG,
methoxy-PEG-vinylsulfone or methoxy-PEG-carboxylic acid active
ester, e.g. succinimidyl ester. X is a reactive group for coupling
to amino acid side chains, e.g. NH.sub.2, OH, CO.sub.2H side
chains, or to the N- or C-terminus, m is preferably an integer from
about 100 or 450 to about 900 and R is lower alkyl, linear or
branched, having one to six carbon atoms such as methyl, ethyl,
isopropyl, etc., from which methyl is preferred.
[0044] As used herein, the term "PEG" or "PEG moiety" is intended
to include, but is not limited to, linear and branched PEG, methoxy
PEG, hydrolytically or enzymatically degradable PEG, pendant PEG,
dendrimer PEG, copolymers of PEG and one or more polyols, and
copolymers of PEG and PLGA (poly(lactic/glycolic acid)).
[0045] The term "PEGylated neurturin" or "neurturin modified by
"PEGylation" or "neurturin conjugate comprising a polyethylen
glycol moiety" relates to a neurturin protein product produced by a
method by which a PEG moiety is covalently attached to form these
PEGylated proteins.
[0046] The term "pharmaceutically acceptable carrier, diluent
and/or adjuvant" relates to one and more pharmaceutically and
physiologically acceptable compounds such as excipients and
auxiliaries, which facilitate processing of the active
ingredient(s) into preparations, which can be used
pharmaceutically. Details may be found in the latest edition of
Remington's Pharmaceutical Sciences (Maack Publishing Co., Easton,
Pa.).
[0047] The term "unmodified control neurturin protein product"
relates to a neurturin protein product of the same origin as the
correlating modified neurturin protein product which is used as a
control.
[0048] The term "variant" as used herein relates to a
polynucleotide variant which may contain one or more substitutions,
additions, deletions and/or insertions, as further described below,
relative to a native polypeptide. The term "variant" also
encompasses homologous polypeptides of xenogenic origin. Typically,
a neurturin variant retains biological activity either completely,
a substantial proportion thereof, or at least partially as, for
example, can be determined using a specific assay as described
below. The term "variant" as used herein also encompasses neurturin
which was modified by altering the protein sequences by
substitutions, additions or deletions providing functionally
equivalent molecules, as is well known in the art. The latter
includes altered sequences in which functionally equivalent amino
acid residues are substituted by residues within the sequence
resulting in a biologically silent exchange.
[0049] Neurturin variants are prepared by introducing appropriate
nucleotide changes into the DNA encoding the polypeptide or by in
vitro chemical synthesis of the desired polypeptide. It will be
appreciated by those skilled in the art that deletions, insertions,
and substitutions can be made, resulting in a protein product
variant presenting neurturin biological activity.
[0050] Mutagenesis techniques for such a substitution, insertion or
deletion of one or more selected amino acid residues are well known
to one skilled in the art (e.g., U.S. Pat. No. 4,518,584, the
disclosure of which is hereby incorporated by reference.)
[0051] In a preferred embodiment, amino acid changes are introduced
into the region between amino acids 47-69, preferably 51-65, of
mature human neurturin (amino acids 141-164, preferably 147-160 as
shown in FIG. 3). These amino acid changes may comprise at least
one deletion or substitution of an arginine residue in this region,
e.g. arginine residues 51, 52, 54, 56, 57, 58, 60, 61, 63 and/or
65. Preferred are substitutions of arginine by neutral or acidic
amino acids, e.g. glycine, serine, alanine, aspartate or glutamate.
Especially preferred is a substitution by glutamate, e.g. R51E,
R52E, R54E, R56E, R57E, R58E, R60E, R61E, R63E and R65E or a
neurturin variant comprising at least 2 of these substitutions.
[0052] Alternatively and/or additionally an amino acid residue of
neurturin, e.g. in the above-indicated region, particularly an
arginine residue, may be substituted by lysine in order to
facilitate coupling to polyol groups. As there is no lysine in
mature human neurturin, such a change allows site-specific
modification with polyol groups. Especially preferred is a
substitution by lysine in the above-indicated region, e.g. R51K,
R52K, R54K, R56K, R57K, R58K, R60K, R61K, R63K and R65K or a
neurturin variant comprising at least 2 or more of these
substitutions.
[0053] In one particular embodiment, the arginine residue 63 is
deleted or substituted. The arginine residue 63 may be substituted
by neutral or acidic amino acids as described above or by lysine.
In a further particular embodiment, the arginine residue 63 is
substituted by lysine. In a further particular embodiment, this
neurturin variant is otherwise identical to human mature neurturin
or may carry an additional N-terminal methionine.
[0054] A neurturin monomer variant into which a lysine residue has
been introduced may comprise a single polyol moiety covalently
bound to the respective lysine residue or to the N-terminal amino
group or 2 polyol moieties, one of them bound to the N-terminal
amino group and one bound to the introduced lysine residue. In a
particular embodiment, a neurturin monomer conjugate is obtained
comprising (i) a single polyol moiety covalently bound to the
N-terminus or to a lysine, e.g. lysine 63 or (ii) two polyol
moieties, one of them bound to the N-terminus and one bound to or
lysine, e.g. lysine 63. These polyol moieties may, e.g. be selected
from PEG moieties as described above. These neurturin monomer
conjugates may combine to neurturin dimer conjugates which may
comprise 1-4 polyol moieties as follows: [0055] (i) a
mono-conjugated, e.g. mono-pegylated neurturin variant dimer,
consisting of 1 unconjugated monomer and 1 mono-conjugated, i.e.
N-terminal or lysine side-chain conjugated monomer, [0056] (ii) a
di-conjugated, e.g. di-pegylated neurturin variant dimer,
consisting of [0057] 1 unconjugated monomer and 1 di-conjugated,
i.e. N-terminal and lysine-side chain di-conjugated monomer, [0058]
2 N-terminal mono-conjugated monomers, [0059] 2 lysine side-chain
mono-conjugated monomers, [0060] 1 N-terminal mono-conjugated
monomer and 1 lysine side-chain mono-conjugated monomer, [0061]
(iii) a tri-conjugated, e.g. tri-pegylated neurturin variant dimer,
consisting of: [0062] 1 N-terminal mono-conjugated monomer and 1
N-terminal and lysine-side chain di-conjugated monomer, [0063] 1
lysine side-chain monoconjugated monomer and 1 N-terminal and
lysine-side chain di-conjugated monomer, and [0064] (iv) a
tetra-conjugated, e.g. tetra-pegylated neurturin variant dimer,
consisting of [0065] 2 N-terminal and lysine-side chain
di-conjugated monomers.
[0066] The present invention also encompasses mixtures of mono-,
di-, tri-, and/or tetra-conjugated neurturin dimers as described
above.
[0067] Neurturin substitution variants have at least one amino acid
residue of the human or mouse neurturin amino acid sequence removed
and a different residue inserted in its place. Such substitution
variants include allelic variants, which are characterized by
naturally occurring nucleotide sequence changes in the species
population that may or may not result in an amino acid change.
[0068] Surprisingly, it was found by the present inventors that a
pharmaceutical composition as disclosed has an increased
bioavailability of its active ingredient, said conjugated neurturin
and/or biologically active fragment thereof, compared to a
pharmaceutical composition comprising no conjugated neurturin. The
invention demonstrates that the conjugation of neurturin protein to
a polyol such as PEG improves significantly the serum half life and
bioavailability of a neurturin product. Improvement of the
bioavailability of a neurturin product in turn may reduce
accumulation or processing of the agent at the injection site if
applied subcutaneously and may improve local tolerance this
way.
[0069] The present invention provides for a method of applying the
inventive neurturin conjugates as active agents to patients in a
much lower dose compared to unmodified control neurturin in order
to have a therapeutic effect. This complies with the need for
"user-friendly protein therapeutics" and may also result in lower
costs for producing active agent and/or pharmaceutical
composition.
[0070] The present neurturin conjugates also have a higher
solubility in aqueous solutions compared to unmodified neurturin
which may result in a simplification of formulating the active
agent.
[0071] As a further advantage, coupling polyols such as PEG to
neurturin may reduce immunogenicity to neurturin and therefore the
formation of neutralizing antibodies or allergic reactions against
the active agent.
[0072] Preferably, the neurturin protein product of the present
invention is a human neurturin or a biologically active fragment
thereof. Neurturin protein products are preferably produced via
recombinant techniques because such methods are capable of
achieving high amounts of protein at a great purity, and are not
limited to products expressed in bacterial, plant, mammalian, or
insect cell systems.
[0073] Recombinant neurturin protein products include glycosylated
and non-glycosylated forms of the protein. In general, recombinant
techniques involve isolating the genes encoding a neurturin protein
product, cloning the gene in a suitable vector and/or cell type,
modifying the gene if necessary to encode a desired variant, and
expressing the gene in order to produce the neurturin protein
product.
[0074] Alternatively, a nucleotide sequence encoding the desired
neurturin product may be chemically synthesized. It is contemplated
that a neurturin product may be expressed using nucleotide
sequences that vary in codon usage due to the degeneration of the
genetic code or allelic variations or alterations made to
facilitate production of the protein product by the selected
cell.
[0075] Kotzbauer et al., Nature 384:467-470, describe the
identification of a mouse cDNA and amino acid sequence and a human
cDNA and amino acid sequence for a neurturin protein, identified
herein as SEQ ID NO: 1.
[0076] The neurturin products according to the present invention
may be isolated or generated by a variety of means. Exemplary
methods for producing neurturin products are described in patent
application WO 97/08196, which is hereby incorporated by reference.
Also described therein are a variety of vectors, host cells, and
culture growth conditions for the expression of neurturin protein,
as well as methods to synthesize variants of neurturin protein
product. Additional vectors suitable for the expression of
neurturin protein product in E. coli are disclosed in Patent No. EP
0 423 980, the disclosure of which is hereby incorporated by
reference.
[0077] The biologically active form of the protein neurturin is a
disulfide-bonded dimer which can be determined by the molecular
weight of purified neurturin. The material isolated after
expression in a bacterial system is essentially biologically
inactive, and exists as a monomer. Refolding is necessary to
produce the biologically active disulfide-bonded dimer. Processes
suitable for the refolding and maturation of the neurturin
expressed in bacterial systems are substantially similar to those
described in WO 93/06116. Standard in vitro assays for the
determination of neurturin activity are also substantially similar
to those determining GDNF activity as described in WO 93/06116 and
in U.S. patent application Ser. No. 08/535,681, and are hereby
incorporated by reference.
[0078] Covalent attachment of the inert, non-toxic, big-degradable
polymer polyethylene glycol (PEG), also known as polyethylene oxide
(PEO), to neurturin has important applications in biotechnology and
medicine. PEGylation of neurturin results in improved
pharmacokinetics resulting in sustained duration, improve safety
(e.g. lower toxicity, immunogenicity and antigenicity), increase
efficacy, decrease dosing frequency, improve drug solubility and
stability, reduce proteolysis, and facilitate controlled drug
release.
[0079] The present invention contemplates use of neurturin protein
products, e.g. derivatives which are prokaryote-expressed
neurturin, or variants thereof, linked to at least one polyethylene
glycol molecule, as well as use of neurturin, or variants thereof,
attached to one or more polyethylene glycol molecules via an acyl
or alkyl linkage. PEGylation may be carried out by any of the
pegylation reactions known in the art. See, for example: Focus on
Growth Factors, 3 (2):4-10, 1992; EP 0 154 316, the disclosure of
which is hereby incorporated by reference; EP 0 401 384; and the
other publications cited herein that relate to PEGylation.
[0080] The pharmaceutical composition of the present invention
preferably comprises a neurturin conjugate as described above,
which is present as a dimer, preferably as a dimer of two neurturin
monomer conjugate molecules comprising 1-4 polyol, e.g.
polyethylene glycol, modifications, e.g. as described above.
[0081] The pharmaceutical composition of the present invention has
an increased bioavailability of the active ingredient in a mammal,
e.g. a human, compared to a pharmaceutical composition which does
not contain the PEGylated neurturin (see FIG. 2). Preferably, the
PEGylated neurturin is present in an amount to provide an at least
2-fold, preferably at least 5-fold and more preferably at least
10-fold increase in the bioavailability of the active
ingredient.
[0082] The increase of bioavailability may be determined as shown
in the Examples of the present application. More particularly, a
composition containing the active ingredient in a given amount or
dose is compared to a pharmaceutical composition containing the
active ingredient in the same dose but without PEGylation. The
bioavailability of both compositions may be determined from the
plasma concentration after subcutaneous administration to
experimental animals, e.g. mice. Preferably, the
plasma-concentration is measured over a time period of at least 120
min, preferably of 240 min and most preferably of 24 h (see FIG.
2).
[0083] The pharmaceutical composition of the present invention may
be adapted for administration by any effective route, e.g. by oral,
nasal, rectal, pulmonal, topical, transdermal or parenteral routes
of administration. Thus, the composition may be a solid or liquid
composition, e.g. a tablet, capsule, powder, cream, gel, ointment,
solution, emulsion, suspension, lyophilisate etc. Preferably,
however, the composition is administered by injection or infusion,
more preferably by injection, e.g. by subcutaneous or intravenous
injection. The pharmaceutical composition is preferably an aqueous
solution.
[0084] In addition to the active ingredient and optionally low
molecular weight substance such as a polyanionic polymer, the
pharmaceutical composition may comprise a pharmaceutically
acceptable carrier, diluent and/or adjuvant, such as buffers,
agents for adjusting tonicity, stabilizers, fillers, disintegrants,
thickeners, etc.
[0085] The pharmaceutical composition contains the active
ingredient in a therapeutically effective amount or dose. The
therapeutically effective dose depends on the type of the active
ingredient, the type and the variety of the disease to be treated
and the type of administration. For parenteral compositions
containing neurturin as an active ingredient, the therapeutically
effective dose is preferably in the range of about 0.001 mg to 500
mg, more preferably from about 0.05 to about 100 mg, most
preferably from about 0.01 to about 5 mg per day.
[0086] The composition is preferably administered to a mammal,
particularly a human. Thus, the composition is suitable for human
and veterinary medicine. The composition is particularly suitable
for the prevention and/or treatment of neurodegenerative or
pancreatic disorders, particularly pancreatic autoimmune disorders
such as diabetes type I and LADA, or diabetes type II.
[0087] The neurturin conjugate as described herein may be
administered by any suitable means, preferably enterally or
parenterally or topically directly to the pancreas, as known to
those skilled in the art. The specific dose may be calculated
according to considerations of body weight, body surface area or
organ size. Further refinement of the calculations necessary to
determine the appropriate dosage for treatment involving each of
the above mentioned compositions is routinely made by those of
ordinary skill in the art and is within the ambit of tasks
routinely performed. Appropriate dosages may be ascertained through
use of the established assays for determining dosages utilized in
conjunction with appropriate dose-response data. The final dosage
regimen involved in a method for treating the above described
conditions will be determined by the attending physician,
considering various factors which modify the action of drugs, e.g.,
the age, condition, body weight, sex and diet of the patient, the
severity of any infection, time of administration and other
clinical factors. As studies are conducted, further information
will emerge regarding the appropriate dosage levels for the
treatment of various diseases and conditions.
[0088] It is envisioned that the continuous administration or
sustained delivery of a neurturin conjugate as described herein may
be advantageous for a given treatment. While continuous
administration may be accomplished via a mechanical means, such as
with an infusion pump, it is contemplated that other modes of
continuous or near continuous administration may be practiced. For
example, chemical derivatization or encapsulation may result in
sustained release forms of the protein having the effect of
continuous presence, in predictable amounts, based on a determined
dosage regimen. Thus, neurturin protein products include proteins
derivatized or otherwise formulated to effectuate such continuous
administration.
[0089] In a further preferred embodiment, neurturin conjugate can
be delivered directly to progenitor, e.g. stem cells in order to
stimulate the differentiation of insulin producing cells in vitro
or in vivo. In this embodiment of the invention, neurturin
conjugate may be added preferably at concentrations between 0.1
ng/ml and 500 ng/ml, more preferably between 1 ng/ml and 100 ng/ml,
even more preferably between 20 and 80 ng/ml and most preferably 50
ng/ml.
[0090] The neurturin conjugate as disclosed herein may be used
either in a monotherapy or in a combination therapy with other
pharmaceutical agents. For example, it may be administered together
with other pharmaceutical agents suitable for the treatment or
prevention of pancreatic diseases and/or obesity and/or metabolic
syndrome, particularly with other pharmaceutical agents suitable
for stimulating and/or inducing the differentiation of insulin
producing cells from progenitor cells. Further, it may be used
together with pharmaceutical agents which have an immunosuppressive
activity, e.g. antibodies, polypeptides and/or peptidic or
non-peptidic low molecular weight substances as disclosed in WO
2005/51415.
[0091] A further embodiment of the present invention is a variant
of human neurturin comprising at least one, e.g. 1, 2, 3 or 4 amino
acid changes compared to wild-type human neurturin. The variant of
human neurturin may contain one or more substitutions, additions,
deletions and/or insertions, as described in detail above, relative
to the native human polypeptide. Neurturin variants can be prepared
as described above.
[0092] In a preferred embodiment, amino acid changes are present in
the region between amino acids 47-69, preferably 51-65 of mature
human neurturin (amino acids 141-164, preferably 147-160 as shown
in FIG. 3). These amino acid changes may comprise at least one
deletion or substitution of an arginine residue in this region,
e.g. arginine residues 51, 52, 54, 56, 57, 58, 60, 61 and/or 65.
Preferred are substitutions of arginine by neutral or acidic amino
acids, e.g. glycine, serine, alanine, aspartate or glutamate.
Especially preferred is a substitution by glutamate, e.g. R51E,
R52E, R54E, R56E, R57E, R58E, R60E, R61E, R63E and R65E, or a
neurturin variant comprising at least two or more of these
substitutions.
[0093] Also preferred are variants of human neurturin, wherein
alternatively and/or additionally an amino acid residue, e.g. in
the above-indicated region, particularly an arginine residue, is
substituted by lysine. Thereby, site-specific coupling of the
neurturin variant to polyol groups is facilitated. Especially
preferred is a substitution by lysine in the above-indicated
region, e.g. R51K, R52K, R54K, R56K, R57K, R58K, R60K, R61K, R63K
and R65K or a neurturin variant comprising at least 2 of these
substitutions.
[0094] Neurturin substitution variants according to the present
invention have at least one amino acid residue of the human
neurturin amino acid sequence removed and a different residue
inserted in its place. Such substitution variants include allelic
variants which are characterized by naturally occurring nucleotide
sequence changes in the species population that may or may not
result in an amino acid change.
[0095] The present invention further provides a method for
administering a pharmaceutical composition to a subject in need
thereof, wherein said composition comprises a pharmaceutically
active amount or dose of at least one neurturin protein product
conjugated with at least one polyol such as PEG or a biologically
active fragment of such conjugated neurturin as an active
ingredient together with a pharmaceutically acceptable carrier,
diluent and/or adjuvant.
[0096] The invention even further provides a method for
administering a pharmaceutical composition to a subject in need
thereof, wherein said composition comprises a pharmaceutically
active amount or dose of at least one modified neurturin protein
product or a biologically active fragment thereof as an active
ingredient together with a low molecular weight substance together
with a pharmaceutically acceptable carrier, diluent and/or
adjuvant.
FIGURES
[0097] The following Figures and Examples illustrate the invention
and are non-limiting embodiments of the invention as claimed below.
Numerous additional aspects and advantages of the invention will
become apparent to those skilled in the art upon consideration of
the following description of the Figures.
[0098] FIG. 1: Biological activity of PEGylated neurturin in cell
culture
[0099] The reporter cell line was subjected to increasing
concentrations of neurturin or neurturin conjugated with PEG
(PEGylated neurturin). The resulting reporter gene activity
(relative light units (RLU) given on the y-axis) was plotted
against the final concentration of the test agent (given on the
x-axis in ng/ml; FIGS. 1A and B). Two independently produced
batches of unmodified neurturin were used as experimental controls
(PeproTech; Lot0805112 and Lot0106112) and their relative activity
was compared to those of the three PEGylated conjugates
(mono-mPEG-NHS-neurturin, mono-mPEG-CHO-neurturin and
mono-CES0310-neurturin). Given values are means of at least 3
wells.+-.S.D. The calculated EC.sub.50 of the various tests are:
Neurturin (Lot0805112, unmodified protein): EC.sub.50=2.2 ng/ml,
neurturin (Lot0106112, unmodified protein): EC.sub.50=1.2 ng/ml,
mono-CES0310-neurturin: EC.sub.50=12 ng/ml (FIG. 1A), and neurturin
(Lot0106112, unmodified protein): EC.sub.50=3.8 ng/ml,
mono-mPEG-NHS-neurturin: EC.sub.50=9.7 ng/ml, and
mono-mPEG-CHO-neurturin: EC.sub.50=7.4 ng/ml (FIG. 1B).
[0100] FIG. 2: Pharmacokinetic (PK) studies after subcutaneous
administration of test agents in mice
[0101] FIGS. 2A and B show the neurturin plasma concentrations in
ng/ml at various time points after the administration of 0,050
mg/kg BW (FIG. 2A) or 0.50 mg/kg BW (FIG. 2B) neurturin or
PEGylated neurturin to mice. Resulting plasma levels were
determined by a neurturin ELISA assay. Time point zero shows the
average background signal for neurturin in mouse serum using this
ELISA assay. The background values were calculated from
independently measured values from sera of 12 (FIG. 2A) or from 11
(FIG. 2B) untreated mice. The other indicated values are means of
measured serum levels from three animals .+-.SD. FIG. 2A shows PK
data for the neurturin conjugate mono-NHS-neurturin stemming from
two independent experiments (mono-NHS-neurturin_Exp.1 and -_Exp.2).
FIG. 2B shows data derived from yet another experiment where mice
were treated with 0.50 mg/kg BW mono-NHS-neurturin.
[0102] For area under the curve (AUC) analysis covering all
measured time points, the baseline was set to 0.6 ng/ml (the
average background value at the time point 0): Neurturin unmodified
protein: 18.8 ng*min/ml, mono-mPEG-NHS-neurturin_Exp1: 409
ng*min/ml, mono-mPEG-NHS-neurturin_Exp2: 918 ng*min/ml,
mono-CES0310-neurturin: EC.sub.50=142 ng*min/ml (FIG. 2A), and
neurturin unmodified protein: 324 ng*min/ml,
mono-mPEG-NHS-neurturin: 1157 ng*min/ml, and
mono-mPEG-CHO-neurturin: 845 ng*min/ml (FIG. 2B).
[0103] For the area under the curve (AUC) analysis (FIGS. 2A and B)
of neurturin serum levels the baseline was set to 0.6 ng/ml
neurturin which corresponds to the average background level
determined in the serum of untreated mice.
[0104] FIG. 3:
[0105] Protein sequence of human neurturin precursor (SEQ ID NO: 1)
as published by the National Center for Biotechnology Information
(NCBI) (accession number: NP.sub.--004549). The sequence of the
mature protein (SEQ ID NO: 5) which corresponds to the biologically
as well as pharmacologically active form is highlighted in
bold.
[0106] FIG. 4:
[0107] FIG. 4A. Expression of human neurturin and mutated neurturin
variants. Native-PAGE/Western-blot of concentrated supernatant from
HEK-293 cells expressing neurturin and neurturin variants
(numbering of the positions according to the sequence of mature
human neurturin, R: arginine, E: glutamic acid).
[0108] FIG. 4B. Neurturin ELISA: Representative standard curve
using human recombinant neurturin expressed from E. coli.
[0109] FIG. 5: Quantification of biological activity of
neurturin
[0110] FIG. 5A. Cellular neurturin biological activity assay:
Tyrosine hydroxylase reporter gene activity stimulated by exogenous
human recombinant neurturin from E. coli in increasing
concentrations combined with increasing serum concentrations.
[0111] FIG. 5B. Biological activity of neurturin variants (n.d.:
not detected)
[0112] FIG. 6: Alignment of neurturin sequences from different
vertebrate species
[0113] Amino acids 47-69 of mature human neurturin (SEQ ID NO: 8)
are shown (numbering of the positions according to the sequence of
mature human neurturin peptide, genbank accession number
NP.sub.--004549, mature peptide as 96-197). Amino acid variations
different to human neurturin are printed bold and underlined
(GenBank Accession Numbers in brackets). Several non-human
neurturin amino acid sequences corresponding to amino acids 47-69
of mature human neurturin are depicted, including the sequences
from Macaca mulatta (SEQ ID NO: 9); Bos tauros (SEQ ID NO: 10);
Canis familiaris (SEQ ID NO: 11); Mus musculus (SEQ ID NO: 12);
rattus norvegicus (SEQ ID NO: 13); Ornithorhnchus anatinus (SEQ ID
NO: 14); Monodelphis domestica (SEQ ID NO: 15) and Gallus gallus
(SEQ ID NO: 16).
[0114] FIG. 7: DNA Sequence of a neurturin expression construct:
Kr-G3-H6-G3-Xa-rhneurturin (wt)
[0115] Kr-G3-H6-G3-Xa-rhneurturin(wt) cloned into pMA vector. FIGS.
7A and 7B depict the DNA-sequence of the sense (SEQ ID NO: 17) and
the anti-sense strand (SEQ ID NO: 18) with restriction sites and
the resulting amino-acid sequence (SEQ ID NO: 19) of the coding
region.
[0116] FIG. 8: Anti-neurturin antibody ELISA
[0117] Anti-neurturin antibody ELISA: left: Standard curve using
anti-neurturin IgG, right: Standard curve using anti-neurturin
IgG/IgM
[0118] FIG. 9: Capture and purification of
G.sub.3H.sub.6G.sub.3Xa-rhNTN(R63K)
[0119] HEK293 cells were transfected with the pcDNA
3.1+Kr-G.sub.3H.sub.6G.sub.3Xa-rhNTN(R63K) expression vector and
cultured for 4 days in a volume of 1 L. The expressed
G.sub.3H.sub.6G.sub.3Xa-rhNTN(R63K) was captured and purified by
immobilized nickel ion chromatography. Non-reducing SDS-PAGE and
coomassie blue protein stain of the load (lane 02), the flow
through (lane 03) and the eluted fractions (lanes 04-24) containing
G.sub.3H.sub.6G.sub.3Xa-rhNTN(R63K) in dimeric (a) or monomeric (b)
form with an apparent MW of 23.6 and 13.3 kDa, respectively, are
shown. The apparent molecular mass is judged by comparison with a
molecular weight marker (lane 1).
[0120] FIG. 10: FIG. 2: PEGylation of
G.sub.3H.sub.6G.sub.3Xa-rhNTN(R63K)
[0121] Purified G.sub.3H.sub.6G.sub.3Xa-rhNTN(R63K) was PEGylated
with NHS-PEG with a MW of 2 kDa and subsequently cleaved with
Protease Xa to remove the G.sub.3H.sub.6G.sub.3Xa-tag leaving
PEGylated rhNTN(R63K). Non-reducing SDS-PAGE and Coomassie blue
protein stain of the PEG-free control reaction (lane 2) and the
PEG-containing reaction (lane 3) are shown. The apparent molecular
mass is judged by comparison with a molecular weight marker (lane
1). The different rhNTN(R63K) fractions are indicated:
non-PEGylated rhNTN(R63K) dimer (a), mono-PEGylated rhNTN(R63K-PEG)
dimer (b), and di-PEGylated rhNTN dimer (R63K-PEG.sub.2) (c). It
should be noted that all preferred embodiments discussed for one or
several aspects of the invention also relate to all other
aspects.
[0122] The versatility of the present invention is illustrated, but
not limited, by the following Examples.
EXAMPLES
Example 1
PEGylation of Neurturin
[0123] Polyethylenglycol groups (PEG) were conjugated to neurturin
through a process called PEGylation. This technology is widely used
for the modification of therapeutic proteins and the procedure is
familiar to anyone skilled in the art. In the present invention,
mono-PEGylated neurturin conjugates were produced by linking a
single PEG molecule to the N-terminal amino acid of one of the two
subunits of the homodimer. Two different conjugates of
mono-PEGylated neurturin were produced that differ in size and
structure of the attached PEG as well as in the method of
production to conjugate the PEG to the protein. The conjugate
disclosed as "mono-mPEG-NHS-neurturin" herein was produced using an
about 5 kDa linear PEG-reagent (mPEG-succinimidyl succinat; NOF,
Japan). This so-called "NHS-method" is the most commonly used
method for PEGylation of soluble proteins. Further, a linear 5 kDa
polyethylene glycol butyraldehyde (Nektar, 082M0H01) was used to
generate the conjugate disclosed as "mono-mPEG-CHO-neurturin".
Under specific experimental conditions NHS esters or aldehydes
efficiently react with free amino groups of proteins and peptides.
In yet another conjugate disclosed as "mono-CES0310-neurturin", the
N-terminal amino acid of one chain of the neurturin homodimer was
conjugated to a six-arm branched PEG of about 5 kDa using a
PEGylation methodology known in the prior art (see, for example,
but not limited to, DE 2005 100 04 157.0, EP 1 631 545 A2; WO
04/108634; WO 07/025,763; CA 2528667). Neurturin does not contain
lysine residues, therefore the PEGylation reactions described above
are expected to primarily lead to a N-terminal PEGylation via the
reactive amino groups of the N-terminal amino acids of the
neurturin homodimer. The quality (size and purity) of the PEGylated
products was confirmed using standard biochemical methodology known
to anyone skilled in the art.
Example 2
Biological Activity of PEGylated Neurturin Conjugates or Variants
Thereof
[0124] The biological activity of the neurturin conjugates was
evaluated using a cell based reporter assay. It was determined how
strong neurturin or its PEGylated conjugates activate a reporter
gene construct in a cell line expressing neurturin receptors on the
surface. In this assay the activity of both PEGylated neurturin
conjugates was found to be reduced 3 to 10 fold relative to that of
unmodified neurturin (FIGS. 1A and B).
[0125] The human neuroblastoma cell line TGW (JCRB0618) which
expresses the receptors for neurturin on the cell surface was
stably transfected with a neurturin reporter gene construct. The
reporter gene constructs contains a luciferase gene which is among
other regulatory elements under the transcriptional control of
repetitive serum response elements (SRE). In these cells the
expression of luciferase is stimulated in response to the
activation of the MAPK pathway, for instance through binding of
neurturin to its surface receptors. The assay is carried out in 96
well plates. The luciferase activity was measured by addition of
luciferin substrate to the wells and read-out was generated on a
Analyst reader (Molecular Devices) operating in the luminescence
mode.
Example 3
Improved Bioavailability of PEGylated Neurturin Conjugates or
Variants Thereof
[0126] The relative bioavailability of the PEGylated conjugates of
neurturin was estimated with the help of PK studies in mice (FIGS.
2A and B). In these experiments the protein was subcutaneously
injected into the neck region of mice. Following, the serum
concentration of neurturin was determined at defined time points
after the delivery of the protein using a specific neurturin ELISA
assay (FIGS. 2A and B). The sensitivity of this neurturin ELISA
assay was limited because of relatively high background signals in
the presence of mouse serum, as detected by the use of serum from
mice that were not treated with neurturin (point zero values).
After the injection of 0,050 mg/kg body weight (BW) unmodified
neurturin, the neurturin serum levels did not significantly exceed
this background level at any time point. On the contrary, when the
same amount of PEGylated neurturin was applied a significant
increase in serum levels of the injected protein was detected,
irrespective of the kind of the PEG modification (FIG. 2A). When
the amount of injected unmodified neurturin was increased 10 times
(0.5 mg/kg BW) the protein was significantly detected in the serum
at time points 60, 90, 120 and 180 minutes (FIG. 2B). Significance
of this observation was confirmed using the T-test and p-values
<0.05 were calculated. Also, the serum levels of the PEGylated
neurturin derivatives given at the 0.5 mg/kg BW dose were a
few-fold higher than these of the unmodified protein (FIGS. 2A and
B).
[0127] The neurturin conjugates presented herein consistently
demonstrate higher serum levels relative to those of the unmodified
protein. In conclusion, PEGylation of neurturin significantly
improves the bioavailability of neurturin.
Example 4
Neurturin Variants
[0128] Basic arginine residues that are located within the basic
patch of neurturin (amino acids 51-65 of the mature protein) were
replaced by acidic glutamic acid residues through site directed
PCR-based mutagenesis. Briefly, the coding sequence of mature human
neurturin with an additional N-terminal eukaryotic secretion signal
peptide was modified by PCR-based mutagenesis using mismatch
primers introducing the desired mutations:
[0129] R51E, R52E, R54E, R56E, R57E, R58E, R60E, R61E, R63E, or
R65E
[0130] (numbering of the positions according to the sequence of
mature human neurturin peptide, Genbank Accession Number
NP.sub.--004549, mature peptide aa 96-197, this sequence is also
shown in FIG. 3; R: arginine; E: glutamic acid).
[0131] The resulting plasmids were purified and the correctness of
the coding sequence was checked by sequencing. The coding sequences
were cloned via flanking HindIII and XhoI cloning sites into
pcDNA3.1+ vector (Invitrogen Cat. No. V790-20) for eukaryotic
expression.
[0132] For eukaryotic expression, these vectors as well as empty
control vector and GFP expression vector as transfection control
were transiently transfected in HEK-293 cells cultured on 175
cm.sup.2 tissue culture vessels with 25 ml medium, each. Media
harvest was performed 48 hours after transfection and the media
supernatant was concentrated by the use of ultra-filtration columns
to app. 0.4 ml, each.
[0133] The expression of wt human neurturin and the mutated
variants was first controlled by native-PAGE/Western-blot using
neurturin-specific antibodies for detection (see FIG. 4). As
control human recombinant neurturin expressed from E. coli was
used.
[0134] FIG. 4A shows that human wild-type neurturin (wt) as well as
all modified neurturin variants except R63E were expressed in
similar amounts. The apparent mass of the neurturin variants ranged
between 20 and 25 kDa similar to wt neurturin.
[0135] The yield of expressed neurturin was determined by an ELISA
specific for neurturin. Briefly, rabbit anti-neurturin-antibody was
coated onto micro-wells for neurturin capture from the neurturin
containing concentrated media supernatant. After washing, the
captured neurturin was quantified by a luminescence read-out using
biotin-conjugated goat anti-neurturin-antibody followed by the use
of peroxidase-conjugated streptavidin. Human recombinant neurturin
expressed from E. coli was used to establish a standard curve (see
FIG. 4B).
[0136] Furthermore, the biological activity of the expressed
neurturin variants were quantified in a specific cellular assay.
The assay principle is based on experiments by Tanaka et al., 2002,
2003 (Tanaka M, Xiao H, Kiuchi K. Heparin facilitates glial cell
line-derived neurotrophic factor signal transduction. Neuroreport.
2002 Oct. 28; 13(15):1913-6; Tanaka M, Xiao H, Hirata Y, Kiuchi K.
A rapid assay for glial cell line-derived neurotrophic factor and
neurturin based on transfection of cells with tyrosine hydroxylase
promoter-luciferase construct. Brain Res Brain Res Protoc. 2003
May; 11(2): 119-22.) showing the induction of a tyrosine
hydroxylase (TH) reporter gene construct by neurturin stably
expressed in the human TGW neuroblastoma cell line. Briefly, TGW
cells over-expressing the tyrosine hydroxylase (TH) reporter gene
construct were treated with the neurturin containing samples
leading to induction of MAPK pathway by neurturin signaling. The
reporter gene constructs contains a luciferase gene under control
of repetitive serum response elements (SRE). The expression of
luciferase is depending of MAPK pathway activation. The assay is
carried out in 96 well plates, neurturin mediated luciferase
expression is measured by addition of luciferin and analyzed with a
luminescence reader.
[0137] A representative experiment demonstrating the quantification
of the biological activity of neurturin using human recombinant
neurturin expressed in E. coli as standard in combination with
increasing serum concentrations is shown in FIG. 5A.
[0138] FIG. 5B shows that wild-type recombinant human neurturin as
well as all neurturin variants except R63E were expressed in
similar amounts resulting in protein concentrations between 1.2-1.7
g/l, and all expressed variants displayed a similar biological
activity in vitro compared to wild-type recombinant human neurturin
with an EC.sub.50 between 0.32-0.89 ng/ml (see FIG. 5B). The human
recombinant neurturin from E. coli used for comparison showed a
weaker potency with an EC.sub.50 of 2.20 ng/ml probably resulting
from a portion of not properly refolded neurturin in the sample
(see FIG. 5B).
[0139] Thus, FIG. 5 shows that modifications in neurturin's "heel"
region such as an introduction of a negatively charged, acidic
amino acid into the basic patch does not compromise the biological
activity of neurturin in general. This is surprising that the prior
art shows that a modification from basic to acidic amino acids in
the heel region would interfere with the biological activity of
neurturin (see sequence and structure comparisons with other GFL,
as discussed above).
[0140] The failure of expressing neurturin variant R63E shows that
at this position a change from arginine to glutamic acid is not
tolerated. Nevertheless, other modifications not resulting in a
complete change of amino acid charge are functional. A sequence
comparison of known neurturin homologs shows that a R63E variant
exists in the human neurturin homolog of Ornithorhynchus anatinus,
Monodelphis domestica, and Gallus gallus. Also natural variants of
the arginin residues at position 52, 57, 58, and 61 exist in
different neurturin homologs (see FIG. 6).
[0141] In order to create biologically active neurturin variants
with improved bioavailability, the inventors replaced basic
arginines in the "heel" region with Lysine. As arginine and lysine
are structurally related, such exchanges are expected to affect the
structure of the neurturin molecule only minimally. Such changes
are enabling the site-specific directed PEGylation of neurturin via
the primary amine of the lysine side chain in a region of the
protein shown to tolerate substantial amino acid changes, and which
is therefore not important for biological activity. This reaction
is highly specific since no lysine is present in the mature native
human neurturin sequence. Reactions of activated PEG with other
amines are minimized by optimizing reaction conditions, e.g. pH,
temperature and reaction time. By placing a PEG modification within
the basic patch of the "heel" region, two objectives are achieved:
(i) the benefits of protein PEGylation (improved bioavailability
via reduced renal clearance, reduced immunogenicity, improved
protein stability) can be obtained while minimizing effects on
protein activity and (ii) PEGylation in this region is expected to
shield the basic patch by preventing interactions with negatively
charged cell surface proteoglycans, which is also expected to
improve protein bioavailabiiity.
[0142] In a first step, the coding sequence of mature human
neurturin which is optimized for human codon usage (with an
additional N-terminal signal peptide for eukaryotic secretion) and
an additional purification tag (repeat of six histidine residues
with an additional site for cleavage of this purification tag with
factor-Xa) is modified by PCR-based site directed mutagenesis.
Here, codons are exchanged at arginine residues 51, 52, 54, 56, 57,
58, 60, 61, 63, or 65 with the codons for lysine residues. [0143]
The resulting expression construct referred to as
Kr-G3-H6-G3-Xa-rhneurturin in this invention containing the
following features: [0144] Kr: DNA-sequence of mouse
kringle-containing transmembrane protein 2, amino acids 1-26,
signal peptide (GenBank Accession Number NP.sub.--082692) for
cytoplasmic secretion optimized for human codon usage (amino acid
sequence: MGTPHLQGFLLLFPLLLRLHGASAGS; SEQ ID NO: 2). [0145] G3:
DNA-sequence of a glycine spacer optimized for human codon usage
(amino acid sequence: GGG). [0146] H6: DNA-sequence of a
6-histidine tag for purification purposes optimized for human codon
usage (amino acid sequence: HHHHHH; SEQ ID NO: 3). [0147] Xa:
DNA-sequence of Factor-Xa protease recognition site optimized for
human codon usage (amino acid sequence: IEGR; SEQ ID NO: 4).
Factor-Xa protease cuts C-terminal after the arginine. [0148]
rhneurturin: DNA-sequence of mature recombinant human neurturin
optimized for human codon usage (amino acid sequence:
ARLGARPCGLRELEVRVSELGLGYASDETVLFRYCAGACEAAARVYDLGL
RRLRQRRRLRRERVRAQPCCRPTAYEDEVSFLDAHSRYHTVHELSARECA CV; SEQ ID NO:
5). Single arginine codons at positions 51, 52, 54, 56, 57, 58, 60,
61, 63, or 65 are replaced by the lysine codon AAG in the
respective neurturin variant expression construct. [0149] The
complete DNA sequence of Kr-G3-H6-G3-Xa-rhneurturin(wt) cloned into
pMA vector is shown in FIG. 7; SEQ ID NO: 6.
Example 5
Peglyation of Neurturin Variants
[0150] The resulting constructs are cloned in the eukaryotic
expression vector pcDNA3.1+ which are transfected into HEK-293
cells for the expression of the modified neurturin variants as
describe above.
[0151] The expressed neurturin variants are purified by
ultra-filtration and by affinity chromatography by means of the
introduced purification tag.
[0152] The structure, purity and biological activity of the
resulting neurturin variant preparations are checked by
native-PAGE/Western-blot and neurturin activity assay as shown
above.
[0153] Biological active neurturin variants which are expressed
with sufficient yield and are purified to acceptable purity are
used for covalent modification with different mono-disperse PEGs.
This PEGs have a preferably size of 2.5-30 kDa, are linear or
branched and activated with N-hydroxysuccinimide (NHS) ester. The
N-hydroxysuccinimide (NHS) ester is spontaneously reactive with
primary amines, providing for efficient PEGylation of proteins.
Other PEG derivatives are for example but not limited to molecules
with different sizes, not mono-disperse or with different branching
patterns and coupling chemistries may be applied as well.
[0154] After the reaction, the purification tag is cleaved at the
introduced proteinase site by factor-Xa and PEG-neurturin variants
are purified by size exclusion and/or affinity chromatography.
[0155] A number of additional purification tags and protease
site/recombinant protease combinations are well known in the art
and may replace the His-Tag and/or factor-Xa site/factor Xa
cleavage. Alternatively, the neurturin protein containing the
introduced PEGylation site(s) may be purified from the supernatant
of eukaryotic expression systems or from the lysate or supernatant
of overexpressing bacteria without purification tag by
chromatographic methods.
[0156] The structure, purity and biological activity of the
resulting PEG-neurturin variant preparations are checked by
SDS-PAGE/Western-blot, native-PAGE/Western-blot, and neurturin
activity assay as shown above.
[0157] Purified PEG-neurturin variants showing sufficient
biological activity in vitro are assayed in vivo for their
pharmacokinetic behavior after subcutaneous administration of
50:mol/kg s.c. to mice and rats. Plasma neurturin concentration is
determined by neurturin ELISA at baseline, and at 0.25, 0.5, 1,
1.5, 2, 3, 4, 6 and 8 hours after administration.
[0158] Selected PEG-neurturin variants showing improved
bioavailability compared to wt neurturin are tested for their
efficacy in vivo by subcutaneous administration to neo STZ rats
once daily 50:mol/kg for 6 days. To assay beta-cell function, blood
glucose is determined every day, and pancreatic insulin content is
determined after 6 days treatment compared to vehicle control and
wt neurturin groups.
Example 6
Immunogenic Potential of Pegylated Neurturin
[0159] Human recombinant wt neurturin has an immunogenic potential
as demonstrated by the appearance of anti-neurturin antibodies
after administration to adult mice for more than 2 weeks. The
anti-neurturin antibodies have been detected with a specific assay.
Briefly, recombinant human neurturin was coated on microplates to
capture serum antibodies. After washing, the captured
anti-neurturin antibodies were quantified by a luminescence
read-out using biotin-conjugated goat anti-IgG antibody or goat
anti-IgG/IgM antibody followed by the use of peroxidase conjugated
streptavidin. Anti-neurturin IgG and anti-neurturin IgM diluted in
serum was used to establish standard curves (see FIG. 8).
[0160] As the PEGylation of pharmacologically active proteins
reduces their immunogenic potential, selected PEG-neurturin
variants with improved bioavailability and sustained potency are
tested for their immunogenic potential by subcutaneous
administration of once daily 50:mol/kg for 28 days in adult mice.
The appearance of anti-neurturin antibodies is determined by
anti-neurturin antibody ELISA every weak compared to vehicle
control and wt neurturin groups.
Example 7
Synthesis of a PEGylated Neurturin Variant with an R63K Amino Acid
Substitution
[0161] A neurturin fusion protein with the structure
Kr-G.sub.3H.sub.6G.sub.3Xa-rhNTN was designed as a basis for
recombinant human neurturin (rhNTN) variants to be secreted from
cultured eukaryotic cells, recovered from the cell culture medium
supernatant and for subsequent purification and chemical
modification with PEG (PEGylation). [0162] The full length human
neurturin (amino acid sequence:
[0163] ARLGARPCGLRELEVRVSELGLGYASDETVLFRYCAGACEAAARVYDLGL
RRLRQRRRLRRERVRAQPCCRPTAYEDEVSFLDAHSRYHTVHELSARECA CV, SEQ ID NO:5)
is fused N-terminally to the following components (from N- to
C-terminus): [0164] 1. Secretion signal peptide consisting of the
amino acids 1-26 of the Mus musculus kringle-containing
transmembrane protein 2 (NP.sub.--082692) (Kr; amino acid sequence
MGTPHLQGFLLLFPLLLRLHGASAGS, SEQ ID NO:2) for the secretion of the
expressed protein into the cell culture medium. [0165] 2. Linker
made of three glycine residues (G3; amino acid sequence GGG) for
the separation of the signal peptide from the following
purification tag. [0166] 3. 6-histidine tag for the capture and
purification by immobilized metal ion chromatography (H6; amino
acid sequence HHHHHH, SEQ ID NO:3). [0167] 4. Linker made of three
glycine residues (G3; amino acid sequence GGG) for the separation
of the 6-histidine tag from the following protease site. [0168] 5.
Recognition site of Factor-Xa protease (Xa; amino acid sequence
IEGR, SEQ ID NO:4) for cleavage at the C-terminus of its arginine
leaving neurturin with the natural N-terminus [0169] The complete
coding nucleotide sequence of Kr-G3H6G3Xa-rhNTN was synthesized in
vitro using an optimized codon usage for higher eukaryotes. The
synthesized DNA was ligated into the pcDNA 3.1+ vector (Invitrogen)
by means of standard molecular cloning techniques and the integrity
of the sequence was controlled by nucleotide sequencing. The coding
nucleotide sequence of Kr-G3H6G3Xa-rhNTN (SEQ ID NO:6) is:
TABLE-US-00001 [0169]
ATGGGCACCCCACACCTGCAGGGCTTTCTGCTGCTGTTCCCCCTGCTGC
TGCGGCTGCACGGCGCCTCTGCCGGCTCTGGCGGCGGACACCACCACC
ATCACCACGGCGGAGGGATCGAGGGCAGAGCCAGACTGGGCGCCAGA
CCCTGCGGCCTGCGGGAGCTGGAAGTGCGGGTGTCCGAGCTGGGCCTG
GGCTACGCCAGCGACGAGACAGTGCTGTTCCGGTACTGCGCTGGCGCC
TGCGAGGCCGCTGCCCGGGTGTACGACCTGGGGCTCCGGAGACTGAGA
CAGCGGCGGAGACTGAGGCGGGAGAGAGTGCGCGCCCAGCCCTGCTGC
AGACCCACCGCCTACGAGGACGAGGTGTCCTTCCTGGACGCCCACAGC
AGATACCACACCGTGCACGAGCTGTCCGCCAGAGAATGCGCCTGCGTG TGA
[0170] 7.2 Design of a Neurturin Variant R63K Fusion Protein
[0171] In order to generate a specific site for the
posttranslational chemical attachment of PEG molecules within the
basic patch of neurturin, the arginine at amino acid position 63
within the basic patch of rhNTN encoded by AGA was changed by means
of PCR mediated mutagenesis to lysine encoded by AAG (R63K). Since
the neurturin variant fusion protein only contains a single lysine
residue, the primary amino group containing side chain of the
introduced lysine will serve as a specific PEGylation site for PEG
molecules activated with a carboxylic acid succinimidyl ester group
(NHS-PEG). Neurturin is a homo-dimer consisting of two identical
amino acid chains. Thus one rhNTN(R63K) protein will now harbor two
lysine residues serving as PEGylation sites. The integrity of the
sequence was controlled by nucleotide sequencing. The coding
nucleotide sequence of Kr-G.sub.3H.sub.6G.sub.3Xa-rhNTN(R63K) is
(nucleotide exchanges in small letters) (SEQ ID NO:8):
TABLE-US-00002 ATGGGCACCCCACACCTGCAGGGCTTTCTGCTGCTGTTCCCCCTGCTG
CTGCGGCTGCACGGCGCCTCTGCCGGCTCTGGCGGCGGACACCACCA
CCATCACCACGGCGGAGGGATCGAGGGCAGAGCCAGACTGGGCGCCA
GACCCTGCGGCCTGCGGGAGCTGGAAGTGCGGGTGTCCGAGCTGGGC
CTGGGCTACGCCAGCGACGAGACAGTGCTGTTCCGGTACTGCGCTGG
CGCCTGCGAGGCCGCTGCCCGGGTGTACGACCTGGGGCTCCGGAGAC
TGAGACAGCGGCGGAGACTGAGGCGGGAGAagGTGCGCGCCCAGCCC
TGCTGCAGACCCACCGCCTACGAGGACGAGGTGTCCTTCCTGGACGCC
CACAGCAGATACCACACCGTGCACGAGCTGTCCGCCAGAGAATGCGCC TGCGTGTGA
[0172] 7.3 Recombinant Expression of the Neurturin Variant R63K
Fusion Protein
[0173] The resulting plasmid pcDNA 3.1+
Kr-G.sub.3H.sub.6G.sub.3Xa-rhNTN(R63K) was transfected into HEK293
cells by means of PEI-plasmid complexes (Backliwal et al.,
Biotechnol. Bioeng. 2008 Feb. 15; 99(3):721-7) in order to obtain
G.sub.3H.sub.6G.sub.3Xa-rhNTN(R63K) protein secreted into the
culture medium. The cells were cultured for 4 days in shaker flasks
on a horizontal shaker with 70 rpm agitation in Ex-Cell 293 SF
medium (Sigma-Aldrich) supplemented with 6 mM L-glutamine
(Invitrogen) at 37.degree. C. in a humidified atmosphere containing
5% CO.sub.2. The cell culture supernatant was harvested and cleared
from cellular contaminations by centrifugation and microfiltration
through a 0.2:m polyethersulfone membrane. The cleared cell culture
supernatant was supplemented with 1.5 M NaCl, 20 mM imidazole, 0.1%
Tween-20, and 25% glycerol (final concentrations). The pH was
adjusted to pH 7.4 with 1 M HCl.
G.sub.3H.sub.6G.sub.3Xa-rhNTN(R63K) was captured from the
supplemented cell culture supernatant on a HisTrap Ni Sepharose
Fast Flow column (GE Healthcare) loaded with nickel ions, washed
with 50 mM NaH.sub.2PO.sub.4, 1.5 M NaCl, 20 mM imidazole, 0.1%
Tween-20, 25% glycerol (final concentrations), pH 7.4 and eluted
with an increasing imidazole gradient from 20-250 mM in the same
buffer. The eluted fractions containing
G.sub.3H.sub.6G.sub.3Xa-rhNTN(R63K) in the dimeric (MW: 23.6 kDa)
or monomeric (MW: 13.3 kDa) form were identified by non reducing
SDS-PAGE and Coomassie blue staining (FIG. 9) and pooled. The
eluted protein was concentrated by means of ultrafiltration with 10
KDa cut-off until a concentration of 1 pmol/:L was achieved and
dialyzed against 30 mM Na.sub.2B.sub.4O.sub.7, 150 mM NaCl, pH
9.0.
[0174] 7.4 PEGylation of the Neurturin Variant R63K Fusion
Protein
[0175] In order to PEGylate the prepared
G.sub.3H.sub.6G.sub.3Xa-rhNTN(R63K), NHS-PEG
(alpha-methoxy-omega-carboxylic acid succinimidyl ester
poly(ethylene glycol)) with a molecular weight of 2 kDa (IRIS
Biotech) was added to the protein solution to a final concentration
of 30 mM. A control reaction mix without the addition of NHS-PEG
was prepared in parallel. The reaction mixes were incubated at
4.degree. C. with 500 rpm agitation for 2 h until termination by
addition of glycine to quench residual reactive NHS-moieties. 2.5
mU/:L Factor Xa protease (Novagen) was added to the reaction mixes
followed by incubation at 37.degree. C. with 500 rpm agitation for
20 h to cleave off the G.sub.3H.sub.6G.sub.3Xa-tag.
[0176] The PEGylation and the control reaction mixes were diluted
1:2 with non-reducing Laemmli SDS-PAGE loading buffer. SDS-PAGE and
subsequent protein staining with Coomassie blue was performed (FIG.
10). The PEG-free control reaction loaded on lane 2 harbors
non-PEGylated rhNTN(R63K) dimer with an apparent MW of 23.4 kDa
(a). The PEGylation reaction loaded on lane 3 harbors three
fractions: Non-PEGylated rhNTN(R63K) dimer with an apparent MW of
23.4 kDa (a), rhNTN(R63K-PEG) dimer PEGylated at one polypeptide
chain with an apparent MW of 25.4 kDa (b), and
rhNTN(R63K-PEG.sub.2) PEGylated dimer at both polypeptide chains
with an apparent MW of 27.4 kDa (c). Thus the protein band c
represents the desired di-PEGylated neurturin variant with an
additional mass of 4 kDa resulting from the attachment of two 2 kDa
PEG moieties. Comparing the relative abundance of the three
fractions resulting from the PEGylation reaction shown in lane 3 by
measuring the integrated density of the protein bands using ImageJ
Software shows that 17% of neurturin is non-PEGylated dimer (a),
28% is mono-PEGylated rhNTN(R63K-PEG) dimer (b) and 55% is the
di-PEGylated rhNTN(R63K-PEG.sub.2) dimer (c).
[0177] As an alternative, the protocol can be changed such that the
N-terminal tag is removed from rhNRTN R63K variant protein before
the PEGylation reaction, or to perform the PEGylation reaction on
untagged rhNTN R63K variant protein purified by means other than
Ni-affinity chromatography. This may result in reaction products
carrying PEG groups on the introduced lysine as well as on one or
both N-terminal amino groups, depending on the reaction conditions.
Sequence CWU 1
1
191197PRTHomo sapiens 1Met Gln Arg Trp Lys Ala Ala Ala Leu Ala Ser
Val Leu Cys Ser Ser 1 5 10 15 Val Leu Ser Ile Trp Met Cys Arg Glu
Gly Leu Leu Leu Ser His Arg 20 25 30 Leu Gly Pro Ala Leu Val Pro
Leu His Arg Leu Pro Arg Thr Leu Asp 35 40 45 Ala Arg Ile Ala Arg
Leu Ala Gln Tyr Arg Ala Leu Leu Gln Gly Ala 50 55 60 Pro Asp Ala
Met Glu Leu Arg Glu Leu Thr Pro Trp Ala Gly Arg Pro 65 70 75 80 Pro
Gly Pro Arg Arg Arg Ala Gly Pro Arg Arg Arg Arg Ala Arg Ala 85 90
95 Arg Leu Gly Ala Arg Pro Cys Gly Leu Arg Glu Leu Glu Val Arg Val
100 105 110 Ser Glu Leu Gly Leu Gly Tyr Ala Ser Asp Glu Thr Val Leu
Phe Arg 115 120 125 Tyr Cys Ala Gly Ala Cys Glu Ala Ala Ala Arg Val
Tyr Asp Leu Gly 130 135 140 Leu Arg Arg Leu Arg Gln Arg Arg Arg Leu
Arg Arg Glu Arg Val Arg 145 150 155 160 Ala Gln Pro Cys Cys Arg Pro
Thr Ala Tyr Glu Asp Glu Val Ser Phe 165 170 175 Leu Asp Ala His Ser
Arg Tyr His Thr Val His Glu Leu Ser Ala Arg 180 185 190 Glu Cys Ala
Cys Val 195 226PRTMus musculus 2Met Gly Thr Pro His Leu Gln Gly Phe
Leu Leu Leu Phe Pro Leu Leu 1 5 10 15 Leu Arg Leu His Gly Ala Ser
Ala Gly Ser 20 25 36PRTArtificial Sequence6 - Histidine Tag 3His
His His His His His 1 5 44PRTArtificial SequenceFactor Xa protease
recognition site 4Ile Glu Gly Arg 1 5102PRTArtificial
Sequencemature recombinant human neurturin 5Ala Arg Leu Gly Ala Arg
Pro Cys Gly Leu Arg Glu Leu Glu Val Arg 1 5 10 15 Val Ser Glu Leu
Gly Leu Gly Tyr Ala Ser Asp Glu Thr Val Leu Phe 20 25 30 Arg Tyr
Cys Ala Gly Ala Cys Glu Ala Ala Ala Arg Val Tyr Asp Leu 35 40 45
Gly Leu Arg Arg Leu Arg Gln Arg Arg Arg Leu Arg Arg Glu Arg Val 50
55 60 Arg Ala Gln Pro Cys Cys Arg Pro Thr Ala Tyr Glu Asp Glu Val
Ser 65 70 75 80 Phe Leu Asp Ala His Ser Arg Tyr His Thr Val His Glu
Leu Ser Ala 85 90 95 Arg Glu Cys Ala Cys Val 100 6438DNAArtificial
Sequencecoding nucleotide sequence of Kr-G3H6G3Xa-rhNTN 6atgggcaccc
cacacctgca gggctttctg ctgctgttcc ccctgctgct gcggctgcac 60ggcgcctctg
ccggctctgg cggcggacac caccaccatc accacggcgg agggatcgag
120ggcagagcca gactgggcgc cagaccctgc ggcctgcggg agctggaagt
gcgggtgtcc 180gagctgggcc tgggctacgc cagcgacgag acagtgctgt
tccggtactg cgctggcgcc 240tgcgaggccg ctgcccgggt gtacgacctg
gggctccgga gactgagaca gcggcggaga 300ctgaggcggg agagagtgcg
cgcccagccc tgctgcagac ccaccgccta cgaggacgag 360gtgtccttcc
tggacgccca cagcagatac cacaccgtgc acgagctgtc cgccagagaa
420tgcgcctgcg tgtgatga 4387435DNAArtificial SequenceCoding
nucleotide sequence of Kr-G3H6G3Xa-rhNTN (R63K) 7atgggcaccc
cacacctgca gggctttctg ctgctgttcc ccctgctgct gcggctgcac 60ggcgcctctg
ccggctctgg cggcggacac caccaccatc accacggcgg agggatcgag
120ggcagagcca gactgggcgc cagaccctgc ggcctgcggg agctggaagt
gcgggtgtcc 180gagctgggcc tgggctacgc cagcgacgag acagtgctgt
tccggtactg cgctggcgcc 240tgcgaggccg ctgcccgggt gtacgacctg
gggctccgga gactgagaca gcggcggaga 300ctgaggcggg agagagtgcg
cgcccagccc tgctgcagac ccaccgccta cgaggacgag 360gtgtccttcc
tggacgccca cagcagatac cacaccgtgc acgagctgtc cgccagagaa
420tgcgcctgcg tgtga 435823PRTHomo sapiens 8Asp Leu Gly Leu Arg Arg
Leu Arg Gln Arg Arg Arg Leu Arg Arg Glu 1 5 10 15 Arg Val Arg Ala
Gln Pro Cys 20 923PRTMacaca mulatta 9Asp Leu Gly Leu Arg Arg Leu
Arg Gln Arg Arg Arg Leu Arg Arg Glu 1 5 10 15 Arg Val Arg Ala Gln
Pro Cys 20 1023PRTBos taurus 10Asp Leu Gly Leu Arg Arg Leu Arg Gln
Arg Arg Arg Val Arg Arg Glu 1 5 10 15 Arg Val Arg Ala Gln Pro Cys
20 1123PRTCanis familiaris 11Asp Leu Gly Leu Arg Arg Leu Arg Gln
Arg Arg Arg Val Arg Arg Glu 1 5 10 15 Arg Val Arg Ala Gln Pro Cys
20 1223PRTMus musculus 12Asp Leu Gly Leu Arg Arg Leu Arg Gln Arg
Arg Arg Val Arg Arg Glu 1 5 10 15 Arg Ala Arg Ala His Pro Cys 20
1323PRTRattus norvegicus 13Asp Leu Gly Leu Arg Arg Leu Arg Gln Arg
Arg Arg Val Arg Lys Glu 1 5 10 15 Arg Val Arg Ala His Pro Cys 20
1423PRTOrnithorhynchus anatinus 14Asp Leu Gly Leu Arg Arg Leu Arg
Gln Arg Arg Arg Val Arg Lys Glu 1 5 10 15 Lys Val Arg Ala Gln Pro
Cys 20 1523PRTMonodelphis domestica 15Asp Leu Gly Leu Arg Arg Leu
Arg Gln Arg Arg Arg Val Arg Lys Glu 1 5 10 15 Lys Ile Arg Ala Arg
Pro Cys 20 1623PRTGallus gallus 16Asp Leu Ser Leu Lys Ser Val Arg
Ser Arg Lys Lys Ile Arg Lys Glu 1 5 10 15 Lys Val Arg Ala Arg Pro
Cys 20 17531DNAArtificial SequencepMA vector +
KrG3H6G3Xa-rhneurturin - Forward Strand 17gagcggaagg cccatgaggc
cagttaatta agaggtacca agcttgccac catgggcacc 60ccacacctgc agggctttct
gctgctgttc cccctgctgc tgcggctgca cggcgcctct 120gccggctctg
gcggcggaca ccaccaccat caccacggcg gagggatcga gggcagagcc
180agactgggcg ccagaccctg cggcctgcgg gagctggaag tgcgggtgtc
cgagctgggc 240ctgggctacg ccagcgacga gacagtgctg ttccggtact
gcgctggcgc ctgcgaggcc 300gctgcccggg tgtacgacct ggggctccgg
agactgagac agcggcggag actgaggcgg 360gagagagtgc gcgcccagcc
ctgctgcaga cccaccgcct acgaggacga ggtgtccttc 420ctggacgccc
acagcagata ccacaccgtg cacgagctgt ccgccagaga atgcgcctgc
480gtgtgatgac tcgagctcat ggcgcgccta ggccttgacg gccttccgcc a
53118531DNAArtificial SequencepMA vector + KrG3H6G3Xa-rhneurturin -
Reverse Complement 18tggcggaagg ccgtcaaggc ctaggcgcgc catgagctcg
agtcatcaca cgcaggcgca 60ttctctggcg gacagctcgt gcacggtgtg gtatctgctg
tgggcgtcca ggaaggacac 120ctcgtcctcg taggcggtgg gtctgcagca
gggctgggcg cgcactctct cccgcctcag 180tctccgccgc tgtctcagtc
tccggagccc caggtcgtac acccgggcag cggcctcgca 240ggcgccagcg
cagtaccgga acagcactgt ctcgtcgctg gcgtagccca ggcccagctc
300ggacacccgc acttccagct cccgcaggcc gcagggtctg gcgcccagtc
tggctctgcc 360ctcgatccct ccgccgtggt gatggtggtg gtgtccgccg
ccagagccgg cagaggcgcc 420gtgcagccgc agcagcaggg ggaacagcag
cagaaagccc tgcaggtgtg gggtgcccat 480ggtggcaagc ttggtacctc
ttaattaact ggcctcatgg gccttccgct c 53119144PRTArtificial
SequenceKr-G3H6G3Xa-rhNTN - fusion protein 19Met Gly Thr Pro His
Leu Gln Gly Phe Leu Leu Leu Phe Pro Leu Leu 1 5 10 15 Leu Arg Leu
His Gly Ala Ser Ala Gly Ser Gly Gly Gly His His His 20 25 30 His
His His Gly Gly Gly Ile Glu Gly Arg Ala Arg Leu Gly Ala Arg 35 40
45 Pro Cys Gly Leu Arg Glu Leu Glu Val Arg Val Ser Glu Leu Gly Leu
50 55 60 Gly Tyr Ala Ser Asp Glu Thr Val Leu Phe Arg Tyr Cys Ala
Gly Ala 65 70 75 80 Cys Glu Ala Ala Ala Arg Val Tyr Asp Leu Gly Leu
Arg Arg Leu Arg 85 90 95 Gln Arg Arg Arg Leu Arg Arg Glu Arg Val
Arg Ala Gln Pro Cys Cys 100 105 110 Arg Pro Thr Ala Tyr Glu Asp Glu
Val Ser Phe Leu Asp Ala His Ser 115 120 125 Arg Tyr His Thr Val His
Glu Leu Ser Ala Arg Glu Cys Ala Cys Val 130 135 140
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