U.S. patent application number 13/845179 was filed with the patent office on 2013-08-22 for conjugates of insulin-like growth factor-1 and poly(ethylene glycol).
This patent application is currently assigned to HOFFMANN-LA ROCHE INC.. The applicant listed for this patent is HOFFMANN-LA ROCHE INC.. Invention is credited to BEAT AMREIN, STEFAN FOSER, FRIEDERIKE HESSE, KLAUS-PETER KUENKELE, KURT LANG, MARTIN LANZENDOERFER, FRIEDRICH METZGER, JOERG THOMAS REGULA, ANDREAS SCHAUBMAR.
Application Number | 20130217623 13/845179 |
Document ID | / |
Family ID | 34927913 |
Filed Date | 2013-08-22 |
United States Patent
Application |
20130217623 |
Kind Code |
A1 |
AMREIN; BEAT ; et
al. |
August 22, 2013 |
CONJUGATES OF INSULIN-LIKE GROWTH FACTOR-1 AND POLY(ETHYLENE
GLYCOL)
Abstract
A conjugate consisting of an insulin-like growth factor-1
(IGF-I) variant and one or two poly(ethylene glycol) group(s),
characterized in that said IGF-I variant has an amino acid
alteration at up to three amino acid positions 27, 37, 65, 68 of
the wild-type IGF-I amino acid sequence so that one or two of said
amino acids is/are lysine and amino acid 27 is a polar amino acid
but not lysine, is conjugated via the primary amino group(s) of
said lysine(s) and said poly(ethylene glycol) group(s) have an
overall molecular weight of from 20 to 100 kDa is disclosed. This
conjugate is useful for the treatment of neurodegenerative
disorders like Alzheimer's Disease.
Inventors: |
AMREIN; BEAT; (RUENENBERG,
CH) ; FOSER; STEFAN; (STEINHAUSEN, CH) ; LANG;
KURT; (PENZBERG, DE) ; METZGER; FRIEDRICH;
(FREIBURG, DE) ; REGULA; JOERG THOMAS; (MUENCHEN,
DE) ; SCHAUBMAR; ANDREAS; (PENZBERG, DE) ;
HESSE; FRIEDERIKE; (MUENCHEN, DE) ; KUENKELE;
KLAUS-PETER; (MARZLING, DE) ; LANZENDOERFER;
MARTIN; (TUTZING, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
HOFFMANN-LA ROCHE INC.; |
|
|
US |
|
|
Assignee: |
HOFFMANN-LA ROCHE INC.
NUTLEY
NJ
|
Family ID: |
34927913 |
Appl. No.: |
13/845179 |
Filed: |
March 18, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13551648 |
Jul 18, 2012 |
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13845179 |
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12791904 |
Jun 2, 2010 |
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13551648 |
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11825827 |
Jul 9, 2007 |
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12791904 |
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11313101 |
Dec 20, 2005 |
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11825827 |
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Current U.S.
Class: |
514/8.6 ;
530/399 |
Current CPC
Class: |
A61K 47/60 20170801;
A61P 43/00 20180101; C07K 14/475 20130101; A61P 25/28 20180101;
C07K 14/65 20130101 |
Class at
Publication: |
514/8.6 ;
530/399 |
International
Class: |
A61K 47/48 20060101
A61K047/48; C07K 14/475 20060101 C07K014/475 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 22, 2004 |
EP |
04030415.6 |
Claims
1. A conjugate comprising an IGF-I variant and poly(ethylene
glycol), wherein: said IGF-I variant is selected from the group
consisting of: (A) a variant which differs from wild-type IGF-I in
that the amino acid sequence of the variant has an alteration at
residue position 27 such that lysine is replaced with arginine; (B)
a variant which differs from wild-type IGF-I in that the amino acid
sequence of the variant has alterations at residue positions 27 and
65 such that both lysines are replaced with arginine; (C) a variant
which differs from wild-type IGF-I in that the amino acid sequence
of the variant has alterations at residue positions 27 and 68 such
that both lysines are replaced with arginine; and (D) a variant
which differs from wild-type IGF-I in that the amino acid sequence
of the variant has alterations at residue positions 27, 65, and 68
such that the lysines are replaced with arginine and a modification
at position 37 such that the arginine is replaced by lysine; and
said poly(ethylene glycol) is conjugated to said IGF-I variant via
one or more primary amino groups.
2. A conjugate according to claim 1 wherein said poly(ethylene
glycol) has an overall molecular weight of from 20 to 100 kDa.
3. A conjugate according to claim 1 wherein said IGF-I variant is
additionally conjugated to poly(ethylene glycol) at the N-terminal
amino acid.
4. A conjugate according to claim 1 wherein said IGF-I variant is
conjugated to poly(ethylene glycol) at a member selected from the
group consisting of lysine 65, lysine 68, and lysine 37 or is
conjugated to poly(ethylene glycol) at both K65 and K68.
5. A conjugate according to claim 1 wherein up to three amino acids
at the N-terminus are truncated.
6. A conjugate according to claim 1 wherein the poly(ethylene
glycol) group(s) is/are branched poly(ethylene glycol)
group(s).
7. A conjugate according to claim 1 wherein the poly(ethylene
glycol) group(s) have an overall molecular weight of 20 kDa to 100
kDa.
8. An IGF-I variant selected from the group consisting of: (A) a
variant which differs from wild-type IGF-I in that the amino acid
sequence of the variant has an alteration at residue position 27
such that lysine is replaced with arginine; (B) a variant which
differs from wild-type IGF-I in that the amino acid sequence of the
variant has alterations at residue positions 27 and 65 such that
both lysines are replaced with arginine; (C) a variant which
differs from wild-type IGF-I in that the amino acid sequence of the
variant has alterations at residue positions 27 and 68 such that
both lysines are replaced with arginine; and (D) a variant which
differs from wild-type IGF-I in that the amino acid sequence of the
variant has alterations at residue positions 27, 65, and 68 such
that the lysines are replaced with arginine and a modification at
position 37 such that the arginine is replaced by lysine.
9. A method for the preparation of a conjugate comprising an IGF-I
variant and one or two poly(ethylene glycol) group(s), said
poly(ethylene glycol) group(s) having an overall molecular weight
of from about 20 to about 100 kDa, said method comprising reacting
the IGF-I variant of claim 8 with activated (polyethylene) glycol
under conditions such that said (polyethylene) glycol will react
with one or two primary lysine amino groups of said IGF-I
variant.
10. The method according to claim 9 further comprising reacting
(polyethylene) glycol with said IGF-I variant via the N-terminal
amino group of said IGF-I variant to yield an IGF-I variant with
poly(ethylene glycol) bound to both the N-terminal amino group and
to one or two primary lysine amino groups.
11. A pharmaceutical composition comprising a conjugate of claim 1
and a pharmaceutically acceptable carrier.
12. A method for the treatment of Alzheimer's Disease comprising
administering to a patient in need thereof a therapeutically
effective amount of a conjugate of claim 1.
13. A composition comprising a conjugate according to claim 3.
14. A pharmaceutical composition comprising a conjugate of claim 3
and a pharmaceutically acceptable carrier.
15. A method for the treatment of Alzheimer's Disease comprising
administering to a patient in need thereof a therapeutically
effective amount of a composition of claim 13.
16. A method for the treatment of Alzheimer's Disease comprising
administering to a patient in need thereof a therapeutically
effective amount of a composition of claim 14.
17. A conjugate according to claim 1 wherein said variant is
selected from the group consisting of: (A) a variant which differs
from wild-type IGF-I in that the amino acid sequence of the variant
has alterations at residue positions 27 and 65 such that both
lysines are replaced with arginine; and (B) a variant which differs
from wild-type IGF-I in that the amino acid sequence of the variant
has alterations at residue positions 27 and 68 such that both
lysines are replaced with arginine.
18. A conjugate according to claim 1 wherein said variant differs
from wild-type IGF-I in that the amino acid sequence of the variant
has alterations at residue positions 27 and 65 such that both
lysines are replaced with arginine.
19. A pharmaceutical composition comprising a conjugate of claim 17
and a pharmaceutically-acceptable carrier.
20. A pharmaceutical composition comprising a conjugate of claim 18
and a pharmaceutically-acceptable carrier.
Description
PRIORITY TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 13/551,648, filed Jul. 18, 2012, now pending, which is a
continuation of U.S. application Ser. No. 12/791,904, filed Jun. 2,
2010, now pending; which is a continuation of U.S. application Ser.
No. 11/825,827, filed Jul. 9, 2007, now abandoned; which is a
continuation of U.S. application Ser. No. 11/313,101, filed Dec.
20, 2005, now abandoned; which claims the benefit of European
Application No. 04030415.6, filed Dec. 22, 2004. The entire
contents of the above-identified applications are hereby
incorporated by reference.
FIELD OF THE INVENTION
[0002] This invention relates to conjugates of insulin-like growth
factor-I (IGF-I) with poly(ethylene glycol) (PEG), pharmaceutical
compositions containing such conjugates, and methods for the
production and methods of use of such conjugates.
BACKGROUND OF THE INVENTION
[0003] Alzheimer's disease (AD) is an increasingly prevalent form
of neurodegeneration that accounts for approximately 50%-60% of the
overall cases of dementia among people over 65 years of age. Death
of pyramidal neurons and loss of neuronal synapses in brains
regions associated with higher mental functions results in the
typical symptoms, characterized by gross and progressive impairment
of cognitive function. Neuropathologically, the major hallmarks of
AD are the presence of two characteristic lesions: the amyloid
senile plaque and neurofibrillary tangle (NFT). While the plaque is
deposited extraneuronally, the tangle is observed intraneuronally
in the post-mortem brain. One of the major components of the
amyloid plaque core is the pathologically deposited small
amyloid-beta-peptide (A.beta.), which is cleaved by secretases from
amyloid precursor protein (APP). A.beta., a self-aggregating
peptide of 39-43 residues (MW .about.4 kDa), is synthesized as part
of the larger APP (110-120 kDa). APP is a type I integral membrane
glycoprotein with a large N-terminal extracellular domain, a single
transmembrane domain and a short cytoplasmic tail. The A.beta.
region spans portions of the extracellular and transmembrane
domains of APP. The most common hypothesis for the participation of
APP in neuronal cell death in AD is the amyloid hypothesis. This
hypothesis postulates that plaque amyloid depositions or partially
aggregated soluble A.beta. trigger a neurotoxic cascade, thereby
causing neurodegeneration similar to AD pathology.
[0004] Insulin-like growth factor I (IGF-I) is a circulating
hormone structurally related to insulin. IGF-I was traditionally
considered the major mediator of the actions of growth hormone on
peripheral tissues. IGF-I consists of 70 amino acids and is also
named somatomedin C and defined by SwissProt No. P01343. Use,
activity and production are mentioned in, e.g., EP 0 123 228; EP 0
128 733; U.S. Pat. No. 5,861,373; U.S. Pat. No. 5,714,460; EP 0 597
033; WO 02/32449; WO 93/02695.
[0005] The regulation of IGF-I function is quite complex. In the
circulation, only 0.2% of IGF-I exists in the free form whereas the
majority is bound to IGF-binding proteins (IGFBP's), which have
very high affinities to IGF's and modulate IGF-I function. The
factor can be locally liberated by mechanisms releasing IGF-I such
as proteolysis of IGFBPs by proteases.
[0006] IGF-I plays a paracrine role in the developing and mature
brain, and in vitro studies indicate that IGF-I is a potent
non-selective trophic agent for several types of neurons in the
CNS.
[0007] Reduction of brain and serum levels of free IGF-I has been
related to the pathogenesis of sporadic and familial forms of AD.
Furthermore, IGF-I protects neurons against A.beta.-induced
neurotoxicity. Peripherally administered IGF-I is capable of
reducing brain A.beta. levels in rats and mice and that in a
transgenic AD mouse model prolonged IGF-I treatment significantly
reduced brain amyloid plaque load. These data strongly support the
idea that IGF-I is able to reduce brain A.beta. levels and
plaque-associated brain dementia by clearing A.beta. from the
brain.
[0008] Covalent modification of proteins with poly(ethylene glycol)
(PEG) has proven to be a useful method to extend the circulating
half-lives of proteins in the body. Other advantages of PEGylation
are an increase of solubility and a decrease in protein
immunogenicity. A common method for the PEGylation of proteins is
the use of poly(ethylene glycol) activated with amino-reactive
reagents like N-hydroxysuccinimide (NHS). With such reagents
poly(ethylene glycol) is attached to the proteins at free primary
amino groups such as the N-terminal .alpha.-amino group and the
.epsilon.-amino groups of lysine residues. However, a major
limitation of this approach is that proteins typically contain a
considerable amount of lysine residues and therefore the
poly(ethylene glycol) groups are attached to the protein in a
non-specific manner at all of the free .epsilon.-amino groups,
resulting in a heterologous product mixture of random PEGylated
proteins. Therefore, many NHS-PEGylated proteins are unsuitable for
commercial use because of low specific activity. Inactivation
results from covalent modification of one or more lysine residues
or the N-terminal amino residue required for biological activity or
from covalent attachment of the poly(ethylene glycol) residues near
or at the active site of the protein.
[0009] WO 94/12219 and WO 95/32003 claim polyethylene glycol
conjugates comprising PEG and IGF or a cysteine-mutated IGF, where
the PEG is attached to said mutein at a free cysteine in the
N-terminal region of the mutein. WO 2004/60300 describes
N-terminally PEGylated IGF-I.
SUMMARY OF THE INVENTION
[0010] The invention comprises an IGF-I variant having an amino
acid alteration at at least one of amino acid positions 27, 37, 65
and 68 of the wild-type IGF-I amino acid sequence so that one or
more of amino acids 37, 65, 68 is/are lysine(K) and amino acid 27
is a polar amino acid but not lysine.
[0011] Amino acid 27 can be, for example, arginine.
[0012] Such IGF-I variants are useful as intermediates (IGF-I
intermediates) for the production of lysine-PEGylated IGF-I.
[0013] It has surprisingly been found that a lysine-PEGylated IGF-I
variant (amino-reactive PEGylated IGF-I variant), preferably a 20
kDa to 100 kDa lysine-PEGylated IGF-I variant, and especially
preferably a lysine-monoPEGylated IGF-I variant, has superior
properties in regard to therapeutic applicability.
[0014] A further embodiment of the invention is a composition
containing, inter alia, both a lysine-PEGylated IGF-I variant
according to the invention and an IGF-I variant which is
N-terminally PEGylated. The molecular ratio can be, for example,
within the range of 9:1 to 1:9, such as a composition wherein the
molar ratio is at least 1:1 (at least one part lysine-PEGylated
IGF-I variant per one part of N-terminally PEGylated IGF-I
variant), for example at least 6:4 (at least six parts
lysine-PEGylated IGF-I variant per four parts of N-terminally
PEGylated IGF-I variant). The lysine-PEGylated IGF-I variant and
the N-terminally PEGylated IGF-I variant may be monoPEGylated. The
variant may be identical in both the lysine-PEGylated IGF-I variant
and the N-terminally PEGylated IGF-I variant. The variant may be
selected from the group consisting of RRKK, RRKR, RRRK, RKRR. PEG
may typically have an average molecular weight of 30 to 45 kDa,
especially 30 kDa or 40 kDa. Lysine-PEGylated IGF-I variant and
N-terminally PEGylated IGF-I variant show comparable affinities in
binding of IGF binding proteins (e.g. BP4 and BP5), but different
activities in regard to IGF-IR phosphorylation.
[0015] The present invention provides a conjugate containing an
IGF-I variant and poly(ethylene glycol) group(s), where the IGF-I
variant has amino acid alteration(s) at at least one of amino acid
positions 27, 37, 65 and 68 of the wild-type IGF-I amino acid
sequence so that one or more of amino acids 37, 65, 68 is/are
lysine(K), amino acid 27 is a polar amino acid but not lysine and
said PEG is conjugated to said IGF-I variant via primary amino
group(s), typically via primary amino group(s) of lysine(s).
[0016] In general, the poly(ethylene glycol) group(s) may have an
overall molecular weight of at least 20 kDa, such as from about 20
to 100 kDa or such as from 20 to 80 kDa.
[0017] The poly(ethylene glycol) group(s) may be conjugated to the
IGF-I variant via the primary amino group(s) of lysine at one or
more amino acid positions 37, 65, 68 (amino-reactive PEGylation)
and are optionally PEGylated at the N-terminal amino acid. The
conjugate may be mono- or diPEGylated at lysine residue(s) at one
or more amino acid position(s) 37, 65, 68 and optionally PEGylated
at the N-terminal amino acid. Thus, the conjugate may be
monoPEGylated at K65, K68, or K37 or diPEGylated at K65 and K68 and
optionally PEGylated at the N-terminal amino acid. Preferably not
more than 20% of the PEGylated IGF-I variant is additionally
N-terminal PEGylated.
[0018] IGF-I variants are designated herein as follows: K65 means
that amino acid 65 is lysine, R27 means that amino acid 27 is
arginine etc. An IGF-I variant bearing the amino acids R27, K37,
K65, K68 is designated RKKK. IGF-I wildtype is designated KRKK.
[0019] Particular IGF-I variants and variants in the conjugates may
be, for example, RRKK, RRKR, RRRK, RKRR.
[0020] In another embodiment, the (PEGylated) IGF-I variant
according to the invention may be a variant in which up to three
(preferably all three) amino acids at the N-terminus are truncated.
The respective wild type mutant is named Des(1-3)-IGF-I and lacks
the amino acid residues gly, pro and glu from the N-terminal.
[0021] The poly(ethylene glycol) group(s) may be either linear or
branched.
[0022] The invention further comprises methods for the preparation
of a conjugate according to the invention using the IGF-I
intermediates. The method comprises the preparation of a conjugate
comprising an IGF-I variant and one or two poly(ethylene glycol)
group(s), where the poly(ethylene glycol) group(s) may have an
overall molecular weight of at least 20 kDa, or from about 20 to
100 kDa, or from 20 to 80 kDa, by reacting the IGF-I intermediate
with activated (polyethylene) glycol under conditions such that the
(polyethylene) glycol is chemically bound to the IGF-I intermediate
via primary lysine amino group(s) of IGF-I variant.
[0023] The invention further comprises pharmaceutical compositions
containing a conjugate according to the invention, which may also
include a pharmaceutically acceptable carrier.
[0024] The invention further comprises methods for the production
of pharmaceutical compositions containing a conjugate according to
the invention.
[0025] The invention further comprises methods for the treatment of
AD, comprising the administration of a pharmaceutically effective
amount of aminio-reactive PEGylated IGF-I variant to a patient in
need of such treatment, in, for example, one to two applications
per week.
BRIEF DESCRIPTION OF THE FIGURES
[0026] FIG. 1: IEC-HPLC of PEG-IGF. Pure positional isomers were
separated from the PEGylation mixture using a preparative
strong-cation exchange column (TOSOH-BIOSEP, SP-5PW). Five
different peak fractions (numbered 0-4) were isolated and processed
for further analysis.
[0027] FIG. 2: SDS-PAGE analysis of monoPEG-IGF-I peaks. The five
purified peak fractions (numbered 0-4) were electrophoresed by
4-12% Tris-glycine SDS-PAGE under non-reduced (A) and reduced (B)
conditions and gels were stained for protein with Coomassie blue.
MW, molecular weight marker.
[0028] FIG. 3: Peptide analysis of monoPEGylated IGF-I peaks 1, 2
and 3. The purified monoPEGylated peaks 1, 2 and 3 were digested
with Asp-N and the peptide fragments separated by HPLC. Peptide
sequences of the PEGylated fragments were obtained by Edman
N-terminal peptide degradation as described. A., HPLC analysis
yielded 6 different peptide fragments for rhIGF-I at retention
times between 30 and 45 minutes and a major fragment 7 (and a minor
fragment 7') for the PEGylated peaks at .about.70 minutes retention
time. Arrows indicate the major relative changes in peptide
fragments in the different peaks as compared to rhIGF-I. B.,
Peptide sequence of the 6 fragments (SEQ ID NOS: 2-7, respectively
in order of appearance) obtained from Asp-N cleavage of rhIGF-I.
The lysines (K) are marked in bold and occur in fragments 3 and 4.
Fragment 5 illustrates the N-terminal peptide. C., Peptide sequence
of the PEGylated peptide fragments 7 after Edman degradation (SEQ
ID NOS: 8-11, respectively in order of appearance). Cystein and
PEGylated lysine residues deliver breaks in the peptide
sequence.
[0029] FIG. 4: Hierarchical clustering of AD of the different
global expression profile from IGF-I and the monopegylated isomers.
The incubation time of the IGF-I and the pegylated variants was 24
h; using the mouse cell line NIH-3T3. The conditions for the data
analysis are described in the text. (A) Clusters of downregulated
genes. (B) Clusters of upregulated genes.
[0030] FIG. 5: IGF-IR phosphorylation by rhIGF-I and monoPEG-IGF-I.
NIH-3T3 cells stably transfected with human IGF-IR were
serum-starved over night and incubated with increasing doses
(0.01-10 .mu.g/ml) of rhIGF-I or monoPEG-IGF-I isomers (peak 1, 2,
3, peak mix, Des(1-3) peak 3) for 30 minutes. Subsequently, cells
were processed for Western or in-cell analysis as described in
Methods. Dose response curves with rhIGF-I (A), peak 1 (B), peak 2
(C), peak 3 (D), peak mix (E) and Des(1-3) peak 3 (F),
respectively. The phosphorylation signal was normalized for IGF-IR
levels in the individual wells. Data points show means .+-.SEM of 6
measurements from 3 independent cultures.
[0031] FIG. 6: Quantitative analysis of IGF-IR phosphorylation in
NIH-3T3 cells. Dose response curves obtained form the experiments
in Example 7 were fitted with one-site binding kinetics to obtain
specific binding (B.sub.Max), halfmaximal binding concentrations
(EC.sub.50) and Hill coefficients (n.sub.H). Data represent means
.+-.SEM of 6 measurements from 3 independent cultures.
[0032] FIG. 7: In vivo brain Abeta lowering of rhIGF-I in PS2APP
mice. Double-transgenic PS2AAP mice at 2-3 months (n=10) were
treated for 2 h with rhIGF-I (50 .mu.g/kg i.p.). Cortical extracts
were prepared and APP, C99, Abeta and control protein (albumin)
levels evaluated as described in Methods. From albumin-normalized
values, the ratios C99/APP, Abeta/APP and C99/Abeta were calculated
and are expressed as % of untreated controls and represent means
.+-.SEM. A, representative Western Blots of 2 months old cortical
extracts. Note the clear reduction in Abeta levels while other
levels were unchanged by rhIGF-I treatment. B, quantitative
analysis of cortical extracts from PS2APP mice (**, p<0.01 vs.
untreated control).
[0033] FIG. 8: Time course of in vivo Abeta lowering in PS2APP mice
by rhIGF-I. Two months old mice were treated for 2, 6 or 24 h with
rhIGF-I (50 .mu.g/kg i.p.). Subsequently, Abeta/APP and C99/Abeta
levels were evaluated and ratios calculated as described. Data are
expressed as % of untreated controls and represent means .+-.SEM
(n=10-13). A, Abeta/PP and B, C99/Abeta ratios at 2, 6 and 24 h
after injection (**, p<0.01 vs. untreated control).
[0034] FIG. 9: In vivo Abeta lowering in AAP and PS2APP mice by
peaks 1-3. Experiments were performed in mixed populations of
single-transgenic APP and double.-transgenic PS2APP mice. Abeta/APP
and C99/Abeta ratios for peak 1, 2 and 3 are shown together for
direct comparison of their relative effects on Abeta lowering and
clearance (n=8-10). A, Abeta/APP ratios showing the effects of
peaks 1-3 on brain Abeta load. B, C99/Abeta ratios of peaks 1-3
demonstrating the clearance of Abeta from the brain (*, p<0.05;
**, p<0.01; ***, p<0.001 vs. untreated control).
[0035] FIG. 10: Time course of in vivo Abeta lowering in AAP and
PS2APP mice by peak 3. Mixed populations of two months old APP and
PS2APP mice were treated for 2, 6, 24, 48 or 72 h with peak 3 (50
.mu.g/kg i.p.). Subsequently, APP, C99, Abeta and actin levels were
evaluated and ratios calculated as described. Data are expressed as
% of untreated controls and represent means .+-.SEM (n=10-15). A,
Abeta/APP and B, C99/Abeta ratios at 6, 24 and 48 h after injection
(*, p<0.05; **, p<0.01; ***, p<0.001 vs. untreated
control).
DETAILED DESCRIPTION OF THE INVENTION
[0036] "PEGylated IGF-I variant" or "amino-reactive PEGylation" as
used herein means an IGF-I variant that is covalently bonded to one
or two poly(ethylene glycol) groups by amino-reactive coupling to
one or two lysines of the IGF-I variant molecule. The PEG group(s)
is/are attached at the sites of the IGF-I variant molecule that are
the primary C-amino groups of the lysine side chains. It is further
possible that PEGylation occurs in addition on the N-terminal
.alpha.-amino group. Due to the synthesis method and variant used,
PEGylated IGF-I variant can consist of a mixture of IGF-I variants,
PEGylated at K65, K68 and/or K37 with or without N-terminal
PEGylation, whereby the sites of PEGylation can be different in
different molecules or can be substantially homogeneous in regard
to the amount of poly(ethylene glycol) side chains per molecule
and/or the site of PEGylation in the molecule. Preferably the IGF-I
variants are mono- and/or diPEGylated and especially purified from
N-terminal PEGylated IGF-I variants.
[0037] Amino-reactive PEGylation as used herein designates a method
of randomly attaching poly(ethylene glycol) chains to primary
lysine amino group(s) of the IGF-I variant by the use of reactive
(activated) poly(ethylene glycol), preferably by the use of
N-hydroxysuccinimidyl esters of, preferably, methoxypoly(ethylene
glycol). The coupling reaction attaches poly(ethylene glycol) to
reactive primary .epsilon.-amino groups of lysine residues and
optionally the .alpha.-amino group of the N-terminal amino acid of
IGF-I. Such amino group conjugation of PEG to proteins is well
known in the art. For example, review of such methods is given by
Veronese, F. M., Biomaterials 22 (2001) 405-417.
[0038] According to Veronese, the conjugation of PEG to primary
amino groups of proteins can be performed by using activated PEGs
which perform an alkylation of said primary amino groups. For such
a reaction, activated alkylating PEGs, for example PEG aldehyde,
PEG-tresyl chloride or PEG epoxide can be used. Further useful
reagents are acylating PEGs such as hydroxysuccinimidyl esters of
carboxylated PEGs or PEGs in which the terminal hydroxy group is
activated by chloroformates or carbonylimidazole. Further useful
PEG reagents are PEGs with amino acid arms. Such reagents can
contain the so-called branched PEGs, whereby at least two identical
or different PEG molecules are linked together by a peptidic spacer
(preferably lysine) and, for example, bound to IGF-I variant as
activated carboxylate of the lysine spacer. Mono-N-terminal
coupling is also described by Kinstler, O., et al., Adv. Drug
Deliv. Rev. 54 (2002) 477-485.
[0039] "PEG or poly(ethylene glycol)" as used herein means a water
soluble polymer that is commercially available or can be prepared
by ring-opening polymerization of ethylene glycol according to
methods well known in the art. The term "PEG" is used broadly to
encompass any polyethylene glycol molecule, wherein the number of
ethylene glycol units is at least 460, preferably 460 to 2300 and
especially preferably 460 to 1840 (230 PEG units refers to an
molecular weight of about 10 kDa). The upper number of PEG units is
only limited by solubility of the conjugate. Generally, PEGs which
are larger than PEGs containing 2300 units are not used.
Preferably, a PEG used in the invention terminates on one end with
hydroxy or methoxy (methoxy PEG, mPEG) and is on the other end
covalently attached to a linker moiety via an ether oxygen bond.
The polymer is either linear or branched. Branched PEGs are e.g.
described in Veronese, F. M., et al., Journal of Bioactive and
Compatible Polymers 12 (1997) 196-207. Useful PEG reagents are e.g.
available form Nektar Therapeutics.
[0040] Any molecular mass for a PEG can be used as practically
desired, e.g., from about 20 kDaltons (Da) to 100 kDa (n is 460 to
2300). The number of repeating units "n" in the PEG is approximated
for the molecular mass described in Daltons. For example, if two
PEG molecules are attached to a linker, where each PEG molecule has
the same molecular mass of 10 kDa (each n is about 230), then the
total molecular mass of PEG on the linker is about 20 kDa. The
molecular masses of the PEG attached to the linker can also be
different, e.g., of two molecules on a linker one PEG molecule can
be 5 kDa and one PEG molecule can be 15 kDa. Molecular mass means
always average molecular mass.
[0041] In the examples below, some preferred reagents for the
production of amino-reactive PEGylated IGF-I variants are
described. It is understood that modifications, for example, based
on the methods described by Veronese, F. M., Biomaterials 22 (2001)
405-417, may be made in the procedures as long as the process
results in conjugates according to the invention.
[0042] The occurrence of up to three potentially reactive primary
amino groups in the target protein (up to two lysines and one
terminal amino acid) leads to a series of PEGylated IGF-I variants
isomers that differ in the point of attachment of the poly(ethylene
glycol) chain.
[0043] The invention provides PEGylated forms of IGF-I variant with
improved properties.
[0044] Such PEGylated IGF-I variant conjugates contain one or two
PEG groups, which may be linear or branched and randomly attached
thereto, whereby the overall molecular weight of all PEG groups in
the conjugate is generally about 20 to 80 kDa. Small deviations
from this range of molecular weight are possible as long as the
PEGylated IGF-I variant does show activity in lowering Abeta
peptide levels in the brain. Also PEGylation of IGF-I variants with
PEG having molecular weights of more than 80 kDa results in higher
bioavailability. However, it is expected that activity may decrease
as the molecular weight increases due to reduced IGF-I receptor
activation and blood-brain barrier transport.
[0045] Therefore, the range of 20 to 100 kDa for the molecular
weight of PEG has been found by the inventors to be optimal range
for a conjugate of PEG and IGF-I variant useful for an efficient
treatment of a patient suffering from AD.
[0046] As used herein, "molecular weight" means the mean molecular
weight of the PEG.
[0047] The following PEGylated forms of IGF-I variants are examples
of and are contemplated embodiments of the conjugates of the
invention: [0048] monoPEGylated IGF-I variant, wherein the PEG
group has a molecular weight of 20 to 80 kDa (460 to 1840 PEG
units); [0049] diPEGylated IGF-I variant, wherein the PEG groups
have a molecular weight of about 10 -50 kDa (230 to 1150 PEG units)
each; and mixtures thereof.
[0050] Of particular note is a monoPEGylated IGF-I selected from
the group consisting of RRKK, RRKR, RRRK and RKRR, wherein the
branched PEG group has a molecular weight of 30-45, preferably
40-45 kDa (about 920 PEG units). For example, based on an average
molecular weight of 44 kDa for PEG and a molecular weight of 7.6
kDa for IGF-I, the calculated average molecular weight for such a
monoPEG-IGF-I is about 51.6 kDa. Further particular embodiments are
a monoPEGylated IGF-I selected from the group consisting of RRKK,
RRKR, RRRK and RKRR, where the PEG has an average molecular weight
of 30 or 40 kDa.
[0051] "PEG or PEG group" according to the invention means a
residue containing poly(ethylene glycol) as an essential part. Such
a PEG can contain further chemical groups which are necessary for
binding reactions; which results from the chemical synthesis of the
molecule; or which is a spacer for optimal distance of the parts of
the molecule from one another. In addition, such a PEG can consist
of one or more PEG side-chains which are linked together. PEG
groups with more than one PEG chain are called multiarmed or
branched PEGs. Branched PEGs can be prepared, for example, by the
addition of polyethylene oxide to various polyols, including
glycerol, pentaerythriol, and sorbitol. For example, a four-armed
branched PEG can be prepared from pentaerythriol and ethylene
oxide. Branched PEGs usually have 2 to 8 arms and are described in,
for example, EP-A 0 473 084 and U.S. Pat. No. 5,932,462. Especially
preferred are PEGs with two PEG side-chains (PEG2) linked via the
primary amino groups of a lysine (Monfardini, C., et al.,
Bioconjugate Chem. 6 (1995) 62-69).
[0052] "Substantially homogeneous" as used herein means that the
only PEGylated IGF-I variant molecules produced, contained or used
are those having one or two PEG group(s) attached. The preparation
may contain small amounts of unreacted (i.e., lacking PEG group)
protein. As ascertained by peptide mapping and N-terminal
sequencing, one example below provides for the preparation which is
at least 90% PEG-IGF-I variant conjugate and at most 5% unreacted
protein. Isolation and purification of such homogeneous
preparations of PEGylated IGF-I variant can be performed by usual
purification methods, preferably size exclusion chromatography.
[0053] "MonoPEGylated" as used herein means that IGF-I variant is
PEGylated at only one lysine per IGF-I variant molecule, whereby
only one PEG group is attached covalently at this site. Such a
monoPEGylated IGF-I can be in addition PEGylated at the N-terminus
to a certain extent. The pure monoPEGylated IGF-I variant (without
N-terminal PEGylation) is at least 80% of the preparation,
preferably 90%, and most preferably, monoPEGylated IGF-I variant is
92%, or more, of the preparation, the remainder being e.g.
unreacted (non-PEGylated) IGF-I and/or N-terminally PEGylated IGF-I
variant. The monoPEGylated IGF-I variant preparations according to
the invention are therefore homogeneous enough to display the
advantages of a homogeneous preparation, e.g., in a pharmaceutical
application. The same applies to the diPEGylated species.
[0054] "Activated PEGs or activated PEG reagents" are well-known in
the state of the art. Preferably there are used electrophilically
activated PEGs such as alkoxybutyric acid succinimidyl esters of
poly(ethylene glycol) ("lower alkoxy-PEG-SBA") or alkoxypropionic
acid succinimidyl esters of poly(ethylene glycol) ("lower
alkoxy-PEG-SPA") or N-hydroxysuccinimide activated PEGs. Any
conventional method of reacting an activated ester with an amine to
form an amide can be utilized. In the reaction of the activated PEG
with IGF-I, the exemplified succinimidyl ester is a leaving group
causing the amide formation. The use of succinimidyl esters to
produce conjugates with proteins is disclosed in U.S. Pat. No.
5,672,662.
[0055] The reaction conditions used have an influence on the
relative amount of differently
[0056] PEGylated IGF-I variants. By manipulating the reaction
conditions (e.g., ratio of reagents, pH, temperature, protein
concentration, time of reaction etc.), the relative amounts of the
different PEGylated species can be varied. In general, the reaction
is performed in a buffered aqueous solution pH 8-10, containing
5-15% (v/v) ethanol and 0.5-4% (v/v) ethyleneglycol. The
protein:PEG ratio may be 1:0.5 to 1:2 if monoPEGylated variants are
desired and 1:2.2 to 1:5 if diPEGylated variants are desired.
Reaction temperature and reaction time can be varied according to
the knowledge of a skilled artisan, whereby high temperature and
long reaction time results in increased PEGylation. If
monoPEGylated variants are desired, it is therefore typical to work
at 4.degree. C. and for up to 30 minutes. When the PEGylation
reagent is combined with IGF-I variant in a reaction buffer which
consists of 50 mM sodium borate, 10% ethanol and 1% di(ethylene
glycol) (DEG) at a pH of about 9.0, a protein:PEG ratio of about
1:1.5, and a reaction temperature of from 4.degree. C., a mixture
of mono-, di-, and trace amounts of the tri-PEGylated species are
produced. When the protein:PEG ratio is about 1:3, primarily the
di- and oligo-PEGylated species is produced.
[0057] IGF-I variant conjugates according to the invention may be
prepared by covalently reacting a primary lysine amino group of an
IGF-I variant with a bifunctional reagent to form an intermediate
with an amide linkage and covalently reacting the intermediate
containing amide linkage with an activated poly(ethylene glycol)
derivative to form an IGF-I variant conjugate. In the foregoing
process, the bifunctional reagent is preferably
N-succinimidyl-S-acetylthiopropionate or
N-succinimidyl-S-acetylthioacetate, and the activated poly(ethylene
glycol) derivative is preferably selected from the group consisting
of iodo-acetyl-methoxy-PEG, methoxy-PEG-vinylsulfone, and
methoxy-PEG-maleimide.
[0058] Especially preferred is the use of a N-Hydroxysuccinimidyl
activated branched PEG ester (mPEG2-NHS) of a molecular weight of
40 kDa (Monfardini, C., et al., Bioconjugate Chem. 6 (1995) 62-69;
Veronese, F. M., et al., J. Bioactive Compatible Polymers 12 (1997)
197-207; U.S. Pat. No. 5,932,462).
[0059] The IGF-I variant conjugates may be prepared by
amino-reactive covalent linking of thiol groups to IGF-I variant
("activation") and coupling the resulting activated IGF-I variant
with a poly(ethylene glycol) (PEG) derivative. The first step
comprises covalent linking of thiol groups via lysine
NH.sub.2-groups of IGF-I variant. This activation of IGF-I variant
is performed with bifunctional reagents which carry a protected
thiol group and an additional reactive group, such as active esters
(e.g., a succinimidylester), anhydrides, esters of sulphonic acids,
halogenides of carboxylic acids and sulphonic acids, respectively.
The thiol group is protected by groups known in the art, e.g.,
acetyl groups. These bifunctional reagents are able to react with
the .epsilon.-amino groups of the lysine amino acids by forming an
amide linkage. The preparation of the bifunctional reagents is
known in the art. Precursors of bifunctional NHS esters are
described in DE 39 24 705, while the derivatization to the
acetylthio compound is described by March, J., Advanced Organic
Chemistry (1977) 375-376. The bifunctional reagent SATA is
commercially available (Molecular Probes, Eugene, Oreg., USA and
Pierce, Rockford, Ill.) and described in Duncan, R. J., Anal.
Biochem. 132 (1983) 68-73.
[0060] The reaction is carried out, for example, in an aqueous
buffer solution, pH 6.5-8.0, e.g., in 10 mM potassium phosphate,
300 mM NaCl, pH 7.3. The bifunctional reagent may be added in DMSO.
After completion of the reaction, preferably after 30 minutes, the
reaction is stopped by addition of lysine. Excess bifunctional
reagent may be separated by methods known in the art, e.g., by
dialysis or column filtration. The average number of thiol groups
added to IGF-I variant can be determined by photometric methods
described in, for example, Grasetti, D. R., and Murray, J. F., in
Arch. Biochem. Biophys. 119 (1967) 41-49. The above reaction is
followed by covalent coupling of an activated poly(ethylene glycol)
(PEG) derivative.
[0061] Activated PEG derivatives are known in the art and are
described in, for example, Morpurgo, M., et al., J. Bioconjugate
Chem. 7 (1996) 363-368 for PEG-vinylsulfone. Linear chain and
branched chain PEG species are suitable for the preparation of the
compounds of formula I. Examples of reactive PEG reagents are
iodo-acetyl-methoxy-PEG and methoxy-PEG-vinylsulfone. The use of
these iodo-activated substances is known in the art and is
described e.g. by Hermanson, G. T., in Bioconjugate Techniques,
Academic Press, San Diego (1996) pp. 147-148.
[0062] The further purification of the compounds according to the
invention including the separation of mono- and/or diPEGylated
IGF-I variants and preferably from N-terminally PEGylated forms may
be done by methods known in the art, e.g., column chromatography,
preferably ion exchange chromatography especially cationic exchange
chromatography.
[0063] The percentage of mono-PEG conjugates as well as the ratio
of mono- and di-PEG species can be controlled by pooling broader
fractions around the elution peak to decrease the percentage of
mono-PEG or narrower fractions to increase the percentage of
mono-PEG in the composition. About ninety percent mono-PEG
conjugates is a good balance of yield and activity. Sometimes
compositions in which, for example, at least ninety-five percent or
at least ninety-eight percent of the conjugates are mono-PEG
species may be desired. In an embodiment of this invention the
percentage of mono PEGylated conjugates is from ninety percent to
ninety-eight percent.
[0064] A "polar amino acid" as used herein refers to an amino acid
selected from the group consisting of cysteine (C), aspartic acid
(D), glutamic acid (E), histidine (H), asparagine (N), glutamine
(Q), arginine (R), serine (S), and threonine (T). Lysine is also a
polar amino acid, but excluded, as lysine is replaced according to
the invention. Arginine is preferably used as polar amono acid.
Pharmaceutical Formulations
[0065] The PEGylated IGF-I according to the invention provides
improved stability in the circulation enabling a sustained access
to IGF-I receptors throughout the body with low application
intervals.
[0066] PEGylated IGF-I variants can be administered as a mixture,
or as the ion exchange chromatography or size exclusion
chromatography separated different PEGylated species. The compounds
of the present invention can be formulated according to methods for
the preparation of pharmaceutical compositions which methods are
known to the person skilled in the art. For the production of such
compositions, a PEGylated IGF-I variant according to the invention
is combined in a mixture with a pharmaceutically acceptable
carrier, preferably by dialysis against an aqueous solution
containing the desired ingredients of the pharmaceutical
compositions. Such acceptable carriers are described, for example,
in Remington's Pharmaceutical Sciences, 18.sup.th edition, 1990,
Mack Publishing Company, edited by Oslo et al. (e.g. pp.
1435-1712). Typical compositions contain an effective amount of the
substance according to the invention, for example from about 0.1 to
100 mg/ml, together with a suitable amount of a carrier. The
compositions may be administered parenterally. The PEGylated IGF-I
according to the invention is administered preferably via
intraperitoneal, subcutaneous, intravenous or intranasal
application.
[0067] The pharmaceutical formulations according to the invention
can be prepared according to known methods in the art. Usually,
solutions of PEGylated IGF-I variant are dialyzed against the
buffer intended to be used in the pharmaceutical composition and
the desired final protein concentration is adjusted by
concentration or dilution.
[0068] Such pharmaceutical compositions may be used for
administration for injection or infusion, preferably via
intraperitoneal, subcutaneous, intravenous or intranasal
application and contain an effective amount of the PEGylated IGF-I
variant together with pharmaceutically acceptable diluents,
preservatives, solubilizers, emulsifiers, adjuvants and/or
carriers. Such compositions include diluents of various buffer
contents (e.g. arginine, acetate, phosphate), pH and ionic
strength, additives such as detergents and solubilizing agents
(e.g. Tween.TM. 80/polysorbate, pluronic.TM. F68), antioxidants
(e.g. ascorbic acid, sodium metabisulfite), preservatives
(Timersol.TM., benzyl alcohol) and bulking substances (e.g.
saccharose, mannitol), incorporation of the material into
particulate preparations of polymeric compounds such as polylactic
acid, polyglycolic acid, etc. or into liposomes. Such compositions
may influence the physical state stability rate of release and
clearance of the monoPEGylated IGF-I according to the
invention.
Dosages and Drug Concentrations
[0069] Typically, in a standard treatment regimen, patients are
treated with dosages in the range between 0.001 to 3 mg, preferably
0.01 to 3 mg of PEGylated IGF-I variant per kg per day over a
certain period of time, lasting from one day to about 30 days or
even longer. Drug is applied as a single daily subcutaneous or i.v.
or i.p. (intraperitoneal) bolus injection or infusion of a
pharmaceutical formulation containing 0.1 to 100 mg PEGylated IGF-I
per ml. This treatment can be combined with any standard (e.g.
chemotherapeutic) treatment, by applying PEGylated IGF-I before,
during or after the standard treatment. This results in an improved
outcome compared to standard treatment alone.
[0070] PEGylated IGF-I according to the invention may be
administered only one or two times per week for successful
treatment. A further embodiment of the invention is therefore a
method for the treatment of Alzheimer's Disease comprising
administering to a patient in need thereof a therapeutically
effective amount of a PEGylated IGF-I according to the invention
with one dosage each in the range between 0.001 to 3 mg, preferably
0.01 to 3 mg of PEGylated IGF-I variant per kg and per 3-8 days,
preferably per 7 days. The PEGylated IGF-I used is preferably
monoPEGylated IGF-I, preferably as a composition of a
lysine-PEGylated IGF-I variant according to the invention and an
IGF-I variant which is N-terminally PEGylated wherein the molar
ratio is at least 1:1.
[0071] The following examples, references and figures are provided
to aid the understanding of the present invention, the true scope
of which is set forth in the appended claims. It is understood that
modifications can be made in the procedures set forth without
departing from the spirit of the invention.
Sequence Listing
[0072] SEQ ID NO: 1 amino acid sequence of human IGF-I (amino acids
1-70 IGF-I; amino acids 71-105 propeptide according to SwissProt
P01343).
EXAMPLES
[0073] Recombinant human insulin-like growth factor (rhIGF-I) was
purchased from PeproTech (Rocky Hill, N.J., USA) via Cell Concepts
(Umkirch, Germany) and Des(1-3)-IGF-I was obtained from GroPep
(Adelaide, Australia). The polyethylene glycol (PEG) reagent was
delivered from Nektar Ltd. (San Carlos, Calif., USA). All other
chemicals and solvents for this investigation were of the highest
purity available. IGF-I variants can be produced recombinantly
according to the state of the art by e.g. using site directed
mutagenesis in combination with expression methods preferably in E.
coli as e.g. described in U.S. Pat. No. 6,509,443 or U.S. Pat. No.
6,403,764.
PEGylation of IGF-I
[0074] IGF-I isomers with PEGylation at a single site
(monoPEG-IGF-I) were produced.
[0075] MonoPEG-IGF-I was prepared by the conjugation of lysine
.epsilon.-amino groups at the surface or on the N-terminus of the
IGF-I molecule with an activated branched PEG moiety of 40 kDa
molecular weight. PEGylation reaction mixture contained rhIGF-I and
40 kDa PEG-NHS reagent at 1:1.5 molar ratio in 50 mM sodium borate
buffer (10% ethanol and 1% DEG), pH 9.0. The reaction was performed
for 30 min at 4.degree. C. Based on the average molecular weight of
44 kDa for the used PEG moiety and a molecular weight of 7.6 kDa
for IGF-I, the calculated average molecular weight for
monoPEG-IGF-I was expected around 51.6 kDa.
Separation of Positional PEG-IGF-I Isomers by Ion Exchange
Chromatography
[0076] A purification scheme with a preparative strong-cation
exchange column (TOSOH-BIOSEP, SP-5PW, 30 mm i.d. and 75 mm length)
was used to prepare pure monoPEG-IGF-I isoforms. The buffer system
consisted of 7.5 mM sodium acetate, 10% ethanol and 1% diethylene
glycol, adjusted to pH 4.5 (buffer A) and 20 mM potassium
phosphate, 10% ethanol and 1% diethylene glycol, adjusted to pH 6.5
(buffer B).
[0077] The column was pre-equilibrated with 25% buffer B. After
loading the PEG-IGF-I samples, the column was washed with 35%
buffer B, followed by an ascending linear gradient to 65% buffer B
to separate the isomers. For the elution of the nonPEGylated IGF-I
the system was switched to modified buffer B with a pH. 8.0. The
flow rate was 8 ml/min and the detection was performed at 218 nm.
The resulting protein samples were collected manually and stored
aliquoted at -20.degree. C. to be analyzed by a variety of protein
chemical and biological assays (see below).
[0078] An analytical strong-cation exchange column (TOSOH-BIOSEP,
SP-NPR, 2.5 .mu.m particle size, 4.6 mm diameter, 3.5 cm length)
was used to investigate the purity of the separated positional
isomers. For this analytical column we used the same mobile phases
as for the preparative one but with reduced flow rate and running
time. The protein concentration of the monoPEG-IGF-I isomers was
determined by spectrophotometry, based on the 280 nm absorption
(E.sub.280.sup.1mg/ml=0.584) of the protein moiety of
monoPEG-IGF-I.
Analysis of monoPEG-IGF-I Purity
[0079] Individual monoPEG-IGF-I isomers were analyzed by 4-12%
Tris-glycine SDS-PAGE under non-reduced or reduced conditions. The
proteins were fixed and stained using the Simple Blue SaveStain
(Invitrogen, Basel, Switzerland).
Mass Spectroscopy Identification of monoPEG-IGF-I Isomers
[0080] The purified monoPEG-IGF isomers were cleaved with Asp-N to
identify the four possible PEGylation sites at the N-terminus, at
K27, K65 or K68. The cleavage buffer consisted of 100 mM Tris/HCl
pH 8.0 with 0.04 .mu.g/.mu.l Asp-N (Roche Diagnostics GmbH, DE). 20
.mu.g monoPEG-IGF were incubated in cleavage buffer for 16 h at 37
.degree. C. with 1 .mu.g Asp-N. After 16 h the reaction mixture was
reduced by addition of TCEP (10 mM) for 1.5 h at 37.degree. C.
Subsequently the reaction solution was quenched by addition of 1/20
volume of 10% TFA to finalize the cleavage reaction. The obtained
peptide mixtures were either directly analyzed by online-HPLC ESI
mass spectrometry, HPLC or stored at -80.degree. C.
[0081] For ESI-LC-MS analysis, the peptide mixtures were separated
on an Agilent 1100 HPLC system equipped with a Phenomenex Jupiter
C18 reversed phase column (1.times.250 mm, 5 .mu.m, 300 .ANG.) with
a flow rate of 40 .mu.l/min. The UV signal was also recorded at 220
nm. A Q-ToF II or LCT mass spectrometer (Micromass) was directly
coupled to the HPLC system. ESI-ToF spectra were recorded with 1
scan/s in the mass range from 200-2000 m/z. UV and TIC spectra were
evaluated and each peptide could be assigned to a single peak in
the chromatogram.
[0082] For HPLC analysis, peptide mixtures were separated on an
Agilent 1100 HPLC system equipped with a Phenomenex Jupiter C18
reversed phase column (1.times.250 mm, 5 .mu.m, 300 .ANG.) with a
flow rate of 40 .mu.l/min. The UV signal was also recorded at 220
nm. PEG peptides were shifted in retention time towards higher
acetonitrile concentrations and were manually collected and
submitted to N-terminal Edman degradation.
[0083] Conditions for HPLC runs (Solvent A: 0.1% TFA in water,
solvent B: 0.1% TFA in Acetonitrile) are shown in table 1:
TABLE-US-00001 TABLE 1 Gradient: Time % B 0 0 10 0 30 20 60 28 70
48 80 100 85 100 86 0 96 0
[0084] N-terminal Edman degradation sequencing was conducted on an
Applied Biosystems Procise system according to N-terminal Edman
degradation sequencing was conducted on an Applied Biosystems
Proteinsequencer Procise 492 with the procise system control
software according to the manufacturers instructions. 20 .mu.l of
fractions collected from HPLC runs were directly applied to
BioBrene Plus.TM. conditioned micro TFA filters. Filters were dried
under argon covered with cartridge seals and introduced into the
proteinsequencer. Automatic standard programs were used to conduct
the sequential degradation of the polypeptide. Analysis of the HPLC
chromatograms from each degradation cycle with Applied Biosystems
data evaluation software 610A revealed the positions each amino
acid. Cysteins as well as modified amino acids like pegylated
lysine appeared as a gap in the chromatogram.
Oligonucleotide Array Transcriptional Analysis
[0085] For in vitro transcriptional profiling of different
monoPEG-IGF-I isomers, IGF-IR stably transfected NIH-3T3 cells were
serum-starved for 2 h and incubated in the absence of serum over 24
h with 0.1 or 1 .mu.g/ml rhIGF and 1 .mu.g/ml of the respective
monoPEG-IGF-I isomers or the mixture of all isomers obtained
without separation. Subsequently, the cultured cells were harvested
and the total cellular RNA was extracted with RNA-BeeTM. From each
sample 10 .mu.g RNA were reversely transcribed, labelled and
processed by using commercial kits according to the supplier's
instructions (Invitrogen, Basel, Switzerland; Ambion, Huntingdon,
UK). The methods for alkaline heat fragmentation and the
hybridization conditions for MOE 430A GeneChip arrays were standard
procedures provided by the manufacturer (Affymetrix, US).
Fluorescence (cell intensities) of the arrays was recorded with a
confocal laser scanner and data were analyzed using MAS 5.0
software (Affymetrix, US). The expression level for each gene was
calculated as normalized average difference of fluorescence
intensity as compared to hybridization to mismatched
oligonucleotides, expressed as average difference (AD). Each
experiment was performed in triplicate in order to account for
biological variation.
[0086] The following two criteria were chosen for the selection of
differentially expressed genes: i) the mRNA levels value of the
treated cells had to be at least five fold higher or five fold
lower as compared to the untreated cells. ii) The standard
deviation must be significantly smaller than the absolute change in
average difference and the calculated confidence level of a gene
was set greater than 97% (p<0.03).
IGF-IR Phosphorylation Assay
[0087] NIH-3T3 cells stably transfected with human IGF-IR were used
for these experiments between passages 2 and 4. Cells were
cultivated either in uncoated 24- or in 96-well plates and grown
until 70% confluency. Subsequently, cells were serum-starved over
night and then incubated with rhIGF-I or the respective PEG-IGF-I
peaks (or peak mix) for 30 minutes. Cells were then either lysed in
Laemmli buffer for Western or fixed with 4% paraformaldehyde for 30
minutes for in-cell analysis of IGF-IR phosphorylation. For Western
analysis, protein extracts were separated by 10% Bis-Tris SDS-PAGE
and blotted onto nitrocellulose membranes. Blots were co-incubated
with mouse-anti-phosphotyrosine (4G10, 1:1000; Upstate) and
rabbit-anti-IGF-IR (C-20, 1:1000; Santa Cruz) primary antibodies
and labeled with anti-mouse-Alexa680 (1:10000; Molecular Probes)
and anti-rabbit-IRDye800 secondary antibodies (1:10000; Jackson).
For in-cell analysis, fixed cells were blocked and permeabilized
with 2% goat serum and Triton X-100 (0.1%) and incubated with the
same primary and secondary antibodies. Fluorescence detection of
protein bands was performed with the Odyssey imaging system (Licor
Biosciences). From digital images, pixel intensity of protein bands
was quantified and dose response curves were analyzed using
GraphPad Prism software. Phosphotyrosine levels were normalized
with IGF-IR values obtained from the same regions of interest to
obtain real IGF-IR activation changes. Experiments were performed
in duplicate and repeated 3 times to obtain 6 independent
investigations per dose. Data were expressed as means .+-.SEM.
In vivo Experiments with rhIGF-I and PEG-IGF-I Isomers
[0088] A mouse model for Alzheimer's disease consisting of
single-transgenic AAP and double-transgenic PS2AAP mice (Richards,
J. G., et al., J. Neurosci. 23 (2003) 8989-9003) was used to
investigate the effect of rhIGF-I (and monoPEG-IGF-I) on brain
amyloidosis that has been recently shown in other mouse and rat
models (Carro, E., et al., Nat. Med. 8 (2002) 1390-1397). The
PS2APP mouse model has been shown to develop an amyloidosis with
measurable Abeta levels at 2 months and onset of plaque deposition
at 8 months of age (Richards, J. G., et al., J. Neurosci. 23 (2003)
8989-9003). Single-transgenic APP mice show very similar Abeta
levels at this young age and therefore were included into the
groups. We analyzed a pre-plaque age (2-3 months) to investigate
the effects of rhIGF-I and PEG-IGF-I isomers on soluble brain Abeta
levels. All experiments were performed in accordance with Swiss
animal protection rights and suffering of the animals was kept to a
minimum. From stock solutions in 1 mM HCl (rhIGF-I) or PBS with 10%
glycerol (monoPEG-IGF-I isomers) injection solutions were prepared
in 0.9% NaCl with solvent less than 1%. Controls were injected with
0.9% NaCl. Injections were performed i.p. under slight isoflurane
anesthesia. At different times points after injection (2 h, 6 h, 24
h, 48 h or 72 h) animals were sacrificed under isoflurane
anesthesia. Mice were decapitated and brains were removed for
isolation of the telencephalon (cortex including hippocampus).
Cortical protein extracts were prepared in hypotonic lysis buffer
containing 4 mM Tris pH 7.4 and 320 mM Sucrose (both Fluka) with
protease and phosphatase inhibitor cocktails (both Sigma). Sample
buffer was Laemmli containing 8 M urea (Fluka). Proteins were
separated by 4-12% Bis-Tris SDS-PAGE and blotted onto
nitrocellulose membranes. Blots were co-incubated with
mouse-anti-amyloid precursor protein (APP) (WO-2 clone, 1:5000; The
Genetics Company) detecting APP, the C99 fragment and A.beta. and
goat-anti-Actin (C-11, 1:5000; Santa Cruz) primary antibodies and
labeled with anti-mouse-Alexa680 (1:10000; Molecular Probes) and
anti-goat-IRDye800 secondary antibodies (1:10000; Jackson).
Fluorescent detection of protein bands was performed with the
Odyssey imaging system (Licor Biosciences). From digital images,
pixel intensity of protein bands was analyzed using GraphPad Prism
software. Data were expressed as means .+-.SEM.
[0089] All values were normalized for actin (or albumin as control
protein) and the specific ratios (C99/APP, A.beta./APP,
C99/A.beta.) were calculated. The C99/APP ratio gives information
about the activity state of .beta.- and .gamma.-secretase because
C99 is the product of .beta.- and the substrate of
.gamma.-secretase; alterations in this measure would indicate a
modulatory effect on one of these secretases independent on the
later fate of A.beta.. With constant C99/APP levels after a
particular treatment, the C99/A.beta. ratio monitors the clearance
of A.beta. independent on its production, being higher with
increased and lower with decreased clearance rate. Furthermore,
A.beta. was normalized for APP because its production depends on
transgenic APP expression which varied between individual mice. The
ratio calculations were performed for every individual animal. All
obtained data are expressed in % of an untreated control group
included in every experiment. Individual experiments with 2-5
animals per dose/time interval were repeated 2-4 times. Statistical
differences were assessed from means .+-.SEM by unpaired t tests
with p<0.05 considered statistically significant.
Example 1
Chromatographical Separation of monoPEG-IGF-I Positional
Isomers
[0090] IGF-I contains 4 amino groups as potential PEGylation sites
and 4 possible monoPEGylated IGF-I (monoPEG-IGF-I) isomers were
expected. Further derivates were expected to be oligoPEGylated
depending on the reaction conditions. A strong-cation high pressure
liquid chromatography method (IEC-HPLC) was developed for the
separation of PEG-IGF-I or -Des(1-3)-IGF-I isomers based on their
local charge differences. A preparative elution profile of 5 mg
PEG-IGF-I is shown in FIG. 1. The result of this method was a
separation into 5 major peaks, 3 peaks with baseline separation and
2 with partial separation. The decrease of the baseline absorption
towards the end of the chromatogram suggests no additional
monoPEGylated IGF-I species eluting at higher retention time. An
additional peak appears between peak 3 and 4 resulting from the
switch to the modified buffer B with different pH. Analytical
IEC-HPLC was used to estimate the purity of the individual isomers
and contamination by other positional isomers in the IEC fractions.
All monoPEGylated peaks had a purity of >99%.
Example 2
SDS-PAGE Analysis of monoPEG-IGF-I Isomers
[0091] SDS-PAGE was performed both under non-reducing and reducing
conditions to evaluate potential unwanted cross-linking of
different IGF-I molecules through intermolecular disulfide bridges.
Both conditions yielded similar results indicating that no
significant amount of the protein was abnormally cross-linked (FIG.
2). The SDS-PAGE analysis showed that peak 0 had an apparent
molecular weight of >100 kDa whereas the major detected bands
were peaks 1-3 at .about.70 kDa; additionally, peak 4 was detected
at .about.7 kDa, a size expected for unPEGylated IGF-I (FIG. 2).
From this running profile and the retention times obtained in the
HPLC we concluded that peak 0 consists most probably of di- and
oligoPEG-IGF-I. In contrast, peaks 1-3 were designated as 3
different monoPEG-IGF-I isomers. There was a discrepancy between
the expected molecular weight for monoPEG-IGF-I (51.6 kDa) and the
apparent size of .about.70 kDa; however, the observed higher
apparent molecular weight can be explained by the larger
hydrodynamic volume of PEG due to water binding considerably
slowing the electrophoretic motility of PEG-IGF-I and increasing
the apparent molecular weight (Foser, S., et al., Pharmacogenomic
J. 3 (2003) 319).
[0092] Taken together, IEC-HPLC and SDS-PAGE experiments indicate
that the purity of the IEC fractions can be considered sufficiently
pure for further characterization.
[0093] PEGylation and separation of monoPEG-Des(1-3)-IGF-I was
performed correspondingly to rhIGF-I and yielded similarly 3 major
monoPEG-Des(1-3)-IGF-I peaks.
Example 3
Analysis of monoPEG-IGF-I Isomers
[0094] Cleavage of rhIGF-I with Asp-N delivered 6 independent
peptide fragments that were separated by HPLC between 30 and 45
minutes retention time (FIG. 3A, upper panel). After cleavage of
the purified peaks 1 to 3 (FIG. 3A, lower panels), different
distributions of the 6 fragments and the occurrence of an
additional fragment (fragment 7) was observed eluting at .about.70
minutes retention time. For peak 1, specifically peptide fragment 4
was diminished with a concomitant increase of fragment 7. For peak
2, we observed a clear decrease in fragment 3 and a slight decrease
in fragment 5 and the appearance of a major and a minor PEG
fragment (7 and 7'). Similarly, HPLC analysis of peak 3 yielded a
clear decrease in fragments 3 and 5 with the concomitant occurrence
of fragments 7 and 7'. FIG. 11B shows the peptide sequences of the
respective fragments as obtained by Asp-N cleavage of rhIGF-I.
Using Edman N-terminal peptide degradation we analyzed the peptide
sequences of the major fragments 7 obtained with the PEGylated
peaks 1, 2 and 3. Thereby, cysteins and PEGylated amino acids
delivered breaks in the peptide sequence. Using this analysis peak
1 could be clearly mapped as rhIGF-I being PEGylated at K27 (FIG.
3C). For peak 2, the major fragment 7 (>90%) was mapped as K65
being PEGylated since K68 was confirmed as unmodified lysine (K) by
Edman degradation. In contrast, the fraction of peak 3 delivered a
PEGylation at K68 (gap in the sequence) with K65 resulting in a
signal in the Edman degradation HPLC chromatogram (FIG. 3C). The
minor peak 7' could not be sequenced by Edman degradation
indicating that the N-terminus was not accessible to the reaction,
most probably by PEGylation. Taken together, these data indicate
that peak 1 consists of the K27 PEGylated isomer, peak 2 is IGF-I
PEGylated at K65 and peak 3 is K68 PEGylated with a significant
amount of N-terminally PEGylated IGF-I.
Example 4
Transcriptional Profiling of rhIGF-I and the monoPEG-IGF-I
Isomers
[0095] Using DNA micorarrays we determined the transcriptional
response of NIH-3T3 cells stably transfected with human IGF-IR over
a 24 h treatment with rhIGF-I or the PEG-IGF-I isomers. Therefore,
we stimulated the cells with 0.1 and 1 .mu.g/ml of IGF-I or with 1
.mu.g/ml of monoPEG-IGF-I peaks or the peak mixture obtained from
the PEGylation reaction. We compared the global transcriptional
activity of stimulated cells with control cultures using a
commercial chip type (MOE 430A; Affymetrix Inc.) containing
probesets for about 14,000 mouse genes including all known IGF-I
response genes. The mRNA abundance was expressed as average
difference (AD) between perfect match oligonucleotide probes and a
corresponding probe with mismatch in the center position. We
considered only genes with a change factor of greater than five and
97% reproducibility (p-value<0.03) within three biological
replicates. This analysis yielded to a total of 162 genes, 86
upregulated and 76 downregulated by all IGF-I variants. A general
correlation profile for transcriptional activity of different
monoPEG-IGF-I isomers is illustrated in form of a hierarchical
cluster of the up- and downregulated genes in FIG. 4. An inspection
of the induction levels of individual gene of 0.1 .mu.g/ml and 1.0
.mu.g/ml IGF-I shows that the selected clusters are very similar.
Peak 3 generates a similar expression profile as unpegylated IGF-I
at the same concentration and it is more potent than the PEG-IGF
mixture and the other peaks. Interestingly, peak 2 triggers a
similar transcriptional response like the PEG-IGF mixture.
Consistent with the biological activity Peak 1 shows the weakest
transcriptional response, indicating that pegylation interferes
with receptor interaction.
Examples 5 and 6
In vitro IGF-IR Phosphorylation by rhIGF-I and monoPEG-IGF-I
[0096] For the in vitro analysis of IGF-IR activation, NIH-3T3
cells stably expressing the human IGF-IR were used. After serum
starvation over night, cells were treated with increasing doses of
rhIGF-I or the respective PEG-IGF-I isomer (0.003-10 .mu.g/ml).
Western analysis of phosphorylated IGF-IR was performed according
to described above and the obtained dose response curves were
fitted with a one-site binding kinetics including the Hill
coefficient (n.sub.H); quantitative data of the association curves
are shown in the table in FIG. 6. The dose response of rhIGF-I
(FIG. 5A) yielded an EC50 of 6.3 nM and nearly occurred in an
all-or-nothing fashion with an n.sub.H of 2.27 (FIG. 6). In
contrast, peak 1 of monoPEG-IGF-I did not show a clear dose
response with no saturation, an n.sub.H of 0.34 and an estimated
EC.sub.50 of 91.5 nM (FIG. 5B, 6). The peaks 2 and 3 (FIGS. 5C and
5D) showed similar binding affinities with EC.sub.50 values of 13.4
and 21.5 nM, respectively (FIG. 6). In both cases, n.sub.H was
regular with 1.27 and 1.19, respectively. The peak mixture
demonstrated a similar IGF-IR activation pattern with slightly
lower affinity with an EC50 of 28.8 nM and regular n.sub.H of 1.28
(FIG. 5E, 7). Finally, PEG-Des(1-3)-IGF-I peak 3 had the highest
affinity of all PEG isomers with an EC.sub.50 of 10.8 nM and
regular n.sub.H of 1.08 (FIG. 5F, 7). The data indicate that all
peaks with exception of peak 1 specifically activated the human
IGF-IR with a 2-5 fold loss of affinity as compared to rhIGF-I.
Example 7
In vivo Abeta Lowering in 2 Months Old PS2APP Mice by rhIGF-I
[0097] Double-transgenic PS2APP mice were used to analyze the
short-term effects of rhIGF-I on brain Abeta load. We treated these
mice with 50 .mu.g/kg rhIGF-I i.p. and analyzed cortical A.beta.2 h
after injection. FIG. 7A shows Western Blots of brain extracts from
2 months old mice. Whereas APP, C99 and control protein (albumin)
levels appear unchanged by rhIGF-I, Abeta levels were reduced 2 h
after rhIGF-I injection. Quantitative analysis was performed and
ratios of the respective pixel intensities were calculated to
obtain information about potential effects of rhIGF-I on Abeta
production. This analysis revealed that the APP/control protein
(97.5.+-.5.7% of control) and C99/APP (87.2.+-.9.8% of control)
ratios were not changed indicating that neither transgenic APP
expression nor APP processing was altered 2 h after treatment with
rhIGF-I (FIG. 7B). In contrast, Abeta/APP significantly dropped to
68.4.+-.7.1% of control (p<0.01) and C99/Abeta increased to
157.9.+-.16.6% of control (p<0.01) indicating that rhIGF-I
lowered Abeta. Taken together, these data suggest that treatment of
young PS2APP mice for 2 h with rhIGF-I mainly increases Abeta
clearance from the brain.
Example 8
Time Course of in vivo Abeta Lowering in PS2APP Mice by rhIGF-I
[0098] For evaluation of the time course of this short-term effect
of rhIGF-I on soluble brain Abeta, young PS2APP mice devoid of
brain plaques (2 months old) were treated by i.p. injection of 50
.mu.g/kg rhIGF-I and cortical APP, C99, Abeta and actin levels were
detected 2, 6 or 24 h later. APP/albumin and C99/APP ratios were
not significantly changed by rhIGF-I at any time point
investigated. Lowering of Abeta/APP by rhIGF-I was observed at 2 h
after injection whereas the effect was absent after 6 and 24 h
(FIG. 8A). Similarly, the increase in Abeta reduction monitored by
the C99/Abeta ratio was only detectable at 2 h and disappeared at 6
and 24 h (FIG. 8B). This indicates that the effect of rhIGF-I on
Abeta clearance was of short duration probably due to the short
half-life of isolated IGF-I in the blood stream.
Example 9
Comparative Analysis of Peaks 1-3 for in vivo Abeta Clearance
Potency in 2 Months Old APP and PS2APP Mice
[0099] In this example, data for PEG-IGF-I peaks 1-3 are shown
together for direct comparison of the potencies for Abeta lowering
and Abeta clearance. Similarly to rhIGF-I, APP/Actin (actin was
used here as control protein) or C99/APP ratios were not altered by
any concentration of peak 1, 2 or 3. Peak 3 had the highest potency
in reducing Abeta/APP at 6 h after i.p. injection (FIG. 9A). In
contrast, peak 1 was without activity over the whole concentration
range tested and peak 2 only exerts a significant effect on
lowering Abeta/APP at the highest concentration used (500
.mu.g/kg). Similarly, as shown in FIG. 9B, peak 3 was the only
compound active at low doses (15-50 .mu.g/kg) in increasing the
C99/Abeta ratio representative for an increased Abeta clearance.
Also in this evaluation, peak 1 was inactive and peak 2 only
exerted significant effects at 500 .mu.g/kg. Taken together, these
data suggest that peak 3 is the most active monoPEG-IGF-I isomer in
this in vivo experimental paradigm.
Example 10
Time Course of in vivo Abeta Lowering in PS2APP Mice by Peak 3
[0100] At 6 h after i.p. injection, 50 .mu.g/kg peak 3 exerted a
significant effect in reduction of brain Abeta levels. To test how
long this effect is maintained we analyzed brain extracts from
PS2APP mice treated for 2, 6, 24, 48 and 72 h with 50 .mu.g/kg peak
3. No significant changes either in APP/Actin or C99/APP ratios
were observed over the whole time period. In contrast, Abeta/APP
levels were significantly reduced at 6, 24 and 48 h after i.p.
injection (p<0.05, p<0.001 and p<0.01, respectively)
indicating an Abeta lowering effect of peak 3 over at least 48 h
(FIG. 10A). C99/Abeta ratios, representing Abeta clearance
independent of Abeta production (since C99/APP remained constant),
were significantly increased in a very similar time course being
well maintained over at least 24 h after injection (FIG. 10B).
Taken together, these data demonstrate that peak 3 is able to
reduce brain Abeta in PS2APP.
Example 11
Binding to IGF Binding Proteins IGFBP4 (BP4) and IGFBP5(BP5)
[0101] IGFBP4 (SwissProt 22692) was identified and cloned by
Shimasaki, S., Mol. Endocrinol. 4 (1990) 1451-1458. IGFBP5
(SwissProt 24593) was identified and cloned by Kiefer, M. C.,
Biochem. Biophys. Res. Commun. 176 (1991) 219-225. Both binding
proteins were produced recombinantly in E. coli.
[0102] For Surface Plasmon Resonance (SPR) analysis of the protein
interaction a Biacore 3000 instrument was used. Running and
reaction buffer was HBS-P (10 mM HEPES, 150 mM NaCl, 0.005%
polysurfactant, ph 7.4) at 25.degree. C. All samples were
pre-cooled at 12.degree. C. IGFBP4 and IGFBP5 were amine-coupled at
concentrations of 5 .mu.g/ml. The coupling on a CM5 chip resulted
in a loading signal of .about.700 RUs. Pegylated IGF-1 samples
(analytes) were injected at 5 concentrations between 1.23 nM and
100 nM for 5 minutes (association phase) and washed with HBS-P for
five minutes at a flow rate of 50 .mu.l/min. The chip surface was
regenerated by one injection of 100 mM HCl for 1 min.
[0103] The chip, assay format and sequence of injections correspond
to the description in table 2. Data evaluation was done by using a
1:1 Langmuir binding model.
TABLE-US-00002 TABLE 2 Chip Ligand Analyte ka (1/Ms) kd (1/s) KD
(M) CM5 BP4 IGF-1 4.0 .times. 10.sup.6 7.2 .times. 10.sup.-4 .sup.
1.8 .times. 10.sup.-10 CM5 BP4 40 kDa/NT- 5.4 .times. 10.sup.4 1.5
.times. 10.sup.-3 2.8 .times. 10.sup.-8 RRRK CM5 BP4 40 kDa/RRRK
7.1 .times. 10.sup.4 6.8 .times. 10.sup.-4 9.5 .times. 10.sup.-9
CM5 BP4 composition 6.9 .times. 10.sup.4 5.3 .times. 10.sup.-4 7.7
.times. 10.sup.-9 CM5 BP5 IGF-1 9.6 .times. 10.sup.6 1.6 .times.
10.sup.-3 .sup. 1.7 .times. 10.sup.-10 CM5 BP5 40 kDa/NT- 1.1
.times. 10.sup.5 2.1 .times. 10.sup.-3 2.0 .times. 10.sup.-8 RRRK
CM5 BP5 40 kDa/RRRK 1.5 .times. 10.sup.5 2.0 .times. 10.sup.-3 1.3
.times. 10.sup.-8 CM5 BP5 composition 1.1 .times. 10.sup.5 2.5
.times. 10.sup.-3 2.3 .times. 10.sup.-8
Abbreviations:
[0104] 40 kDa: 40 kDa PEG branched [0105] 40 kDa/NT-RRRK: RRRK
which is N-terminally PEGylated with 40 kDa PEG [0106] 40 kDa/RRRK:
RRRK which is lysine PEGylated at K68 with 40 kDa PEG [0107]
composition: composition of 40 kDa/NT-RRRK and 40 kDa/RRRK
(1:1)
[0108] These results show, that all PEGylated IGF samples were
actively binding to IGFBP4 and IGFBP5 in a similar range. All
pegylated samples showed a significantly reduced association rate
constant in comparison to non-PEGylated IGF.
[0109] Negative control data (e.g. buffer curves) were subtracted
from sample curves for correction of system intrinsic baseline
drift and for signal noise reduction.
Example 12
Autophosphorylation of IGF-IR by Ligands
[0110] In order to determine the ability of the polypeptides of the
invention to activate IGF-IR and induce IGF-IR phosphorylation,
IGF-IR overexpressing cells were stimulated by the polypeptides of
the invention and IGF-IR phosphorylation status was analyzed
subsequently by ELISA.
[0111] For ELISA, 96-Well streptavidin coated polystyrene plates
(Nunc) were coated with 100 .mu.l monoclonal antibody against human
IGF-1R.alpha. (0.5 mg/ml) diluted 1:350 in PBST with 3% BSA. After
incubation for 1 hour at room temperature on a plate shaker, the
coating solution was removed and plates were washed thrice with 200
.mu.l PBST per well.
[0112] IGF-IR transfected NIH-3T3 cells were plated in MEM Dulbecco
medium (DMEM) with high glucose (PAA, Cat No. E15-009) supplemented
with 2 mM L-Glutamin (Gibco, Cat No. 25030-024) and 0.5% heat
inactivated FCS (PAA, Cat No. A15-771). For determination of
EC.sub.50 values, 96 well plates inoculated with 1.3*10.sup.4 cells
per well were cultivated for two days at 37.degree. C. and 5%
CO2.
[0113] After 48 hours of cultivation with low serum medium, the
medium was carefully removed and replaced by different
concentrations of the polypeptides of the invention diluted in 50
.mu.l of the respective medium. After 10 minutes of incubation at
37.degree. C. and 5% CO2 the medium was carefully removed by
aspiration and 120 .mu.l of cold lysis buffer was added per well
(50 mM Tris pH 7.5, 1 mM EDTA, 1 mM EGTA, 20% glycerol, 1%
Triton-X100, 100 mM NaF, 1 mM NaVO.sub.4, Complete.TM. protease
inhibitor). The plates were incubated with lysis buffer for 15
minutes at 4.degree. C. on a plate shaker and 100 .mu.l of the well
contents were transferred afterwards to the ELISA plates coated
with monoclonal antibody against human IGF-1R.alpha.. The lysates
were incubated for 1 h at room temperature on a plate shaker to
allow IGF-IR binding to the capture antibody and were carefully
aspirated afterwards. Unbound material was removed by three washing
steps with 200 .mu.l PBST/well each.
[0114] To detect the bound phosphorylated IGF-1R, 100 .mu.l of
polyclonal IgG rabbit antibody against human IGF-1R.alpha. diluted
1:12650 in 3% BSA/PBST were added to each well followed by another
incubation period for 1 hour at room temperature on the plate
shaker. The well contents were again carefully removed and the
wells washed three times with 200 .mu.l PBST/well. For the
detection of polyclonal rabbit antibody, 100 .mu.l of a polyclonal
antibody against rabbit IgG coupled to HRP (Cell Signaling
Technology Inc. USA) diluted 1:6000 in 3% BSA/PBST were added to
each well. After incubation for 1 hour at room temperature on a
shaker, unbound detection antibody was removed by washing the
plates six times with 200 .mu.l PBST/well. As a substrate for the
antibody-coupled HRP, 100 .mu.l of 3,3'-5,5'-tetramethylbenzidine
were added to each well followed by another incubation for 0.5
hours at room temperature on a shaker.
[0115] Quantification occurred after stopping the reaction with 25
.mu.l/well 1M H.sub.2SO.sub.4 by measuring absorption at a
wavelength of 450 nm.
[0116] The obtained OD450 values of the samples were transformed
into percent activation using 10 nM IGF-1 as 100% (max) and w/o
IGF-1 as 0% (min) controls by the following formula: percent
activation=(sample-min)/(max-min). The resulting EC50 (polypeptide
concentrations at half maximum activation of IGF-1R)values are
summarized in table 3.
TABLE-US-00003 TABLE 3 EC50 Sample [nM] "STDEV" K68-RRRK 20 kDa
linear 2.1 0.2 NT-RRRK 20 kDa linear 29.6 1.9 composition 20 kDa
linear 7.6 K68-RRRK 30 kDa linear 4.7 0.9 NT-RRRK 30 kDa linear
44.5 5.0 composition 30 kDa linear 9.2 1.1 composition 40 kD
branched 16.4 0.7 K68-RRRK 20 kD branched 1.5 1.1 composition 20 kD
branched 5.1 0.5 NT-RRRK 40 kD branched 19.9 1.1
[0117] Abbreviations are as in example 11.
Sequence CWU 1
1
111105PRTHomo sapiens 1Gly Pro Glu Thr Leu Cys Gly Ala Glu Leu Val
Asp Ala Leu Gln Phe1 5 10 15Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn
Lys Pro Thr Gly Tyr Gly 20 25 30Ser Ser Ser Arg Arg Ala Pro Gln Thr
Gly Ile Val Asp Glu Cys Cys 35 40 45Phe Arg Ser Cys Asp Leu Arg Arg
Leu Glu Met Tyr Cys Ala Pro Leu 50 55 60Lys Pro Ala Lys Ser Ala Arg
Ser Val Arg Ala Gln Arg His Thr Asp65 70 75 80Met Pro Lys Thr Gln
Lys Glu Val His Leu Lys Asn Ala Ser Arg Gly 85 90 95Ser Ala Gly Asn
Lys Asn Tyr Arg Met 100 10525PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 2Asp Leu Arg Arg Leu1
538PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 3Asp Glu Cys Cys Phe Arg Ser Cys1
5413PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 4Glu Met Tyr Cys Ala Pro Leu Lys Pro Ala Lys Ser
Ala1 5 10525PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 5Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr
Gly Tyr Gly Ser Ser Ser1 5 10 15Arg Arg Ala Pro Gln Thr Gly Ile Val
20 25611PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 6Gly Pro Glu Thr Leu Cys Gly Ala Glu Leu Val1 5
1078PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 7Asp Ala Leu Gln Phe Val Cys Gly1
587PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 8Asp Arg Gly Phe Tyr Phe Asn1 5914PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 9Pro
Thr Gly Tyr Gly Ser Ser Ser Arg Arg Ala Pro Gln Thr1 5
10105PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 10Pro Ala Lys Ser Ala1 5116PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 11Ala
Pro Leu Lys Pro Ala1 5
* * * * *