WO1996001275A1 - O-glycosylated authentic igf-i and truncated variants thereof, a method of preparation thereof and pharmaceutical compositions - Google Patents
O-glycosylated authentic igf-i and truncated variants thereof, a method of preparation thereof and pharmaceutical compositions Download PDFInfo
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- WO1996001275A1 WO1996001275A1 PCT/SE1995/000774 SE9500774W WO9601275A1 WO 1996001275 A1 WO1996001275 A1 WO 1996001275A1 SE 9500774 W SE9500774 W SE 9500774W WO 9601275 A1 WO9601275 A1 WO 9601275A1
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- Prior art keywords
- igf
- glycosylated
- amino acid
- polypeptide chain
- mannose residues
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- 238000000034 method Methods 0.000 title claims abstract description 15
- 238000002360 preparation method Methods 0.000 title claims abstract description 13
- 239000008194 pharmaceutical composition Substances 0.000 title claims abstract description 8
- 108090000723 Insulin-Like Growth Factor I Proteins 0.000 claims abstract description 111
- 102000004218 Insulin-Like Growth Factor I Human genes 0.000 claims abstract description 109
- 150000001413 amino acids Chemical class 0.000 claims abstract description 33
- 229920001184 polypeptide Polymers 0.000 claims abstract description 26
- 125000000311 mannosyl group Chemical group C1([C@@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 claims abstract description 24
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 claims abstract description 22
- 102000003746 Insulin Receptor Human genes 0.000 claims abstract description 15
- 108010001127 Insulin Receptor Proteins 0.000 claims abstract description 15
- 230000012010 growth Effects 0.000 claims abstract description 14
- 102000004877 Insulin Human genes 0.000 claims abstract description 11
- 108090001061 Insulin Proteins 0.000 claims abstract description 11
- 229940125396 insulin Drugs 0.000 claims abstract description 11
- 239000003085 diluting agent Substances 0.000 claims abstract description 6
- 239000003814 drug Substances 0.000 claims abstract description 6
- 239000003937 drug carrier Substances 0.000 claims abstract description 6
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- 108090000765 processed proteins & peptides Proteins 0.000 claims description 38
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- 108091022911 insulin-like growth factor binding Proteins 0.000 claims description 10
- 208000001362 Fetal Growth Retardation Diseases 0.000 claims description 5
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- 230000016784 immunoglobulin production Effects 0.000 claims description 3
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- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 3
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- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 2
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- BTCSSZJGUNDROE-UHFFFAOYSA-N gamma-aminobutyric acid Chemical compound NCCCC(O)=O BTCSSZJGUNDROE-UHFFFAOYSA-N 0.000 description 2
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- HBOMLICNUCNMMY-KJFJCRTCSA-N 1-[(4s,5s)-4-azido-5-(hydroxymethyl)oxolan-2-yl]-5-methylpyrimidine-2,4-dione Chemical compound O=C1NC(=O)C(C)=CN1C1O[C@H](CO)[C@@H](N=[N+]=[N-])C1 HBOMLICNUCNMMY-KJFJCRTCSA-N 0.000 description 1
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- SKEFKEOTNIPLCQ-LWIQTABASA-N mating hormone Chemical compound C([C@@H](C(=O)NC(CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N1[C@@H](CCC1)C(=O)NCC(=O)N[C@@H](CCC(N)=O)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCS(C)=O)C(=O)NC(CC=1C=CC(O)=CC=1)C(O)=O)NC(=O)[C@@H](N)CC=1C2=CC=CC=C2NC=1)C1=CN=CN1 SKEFKEOTNIPLCQ-LWIQTABASA-N 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/65—Insulin-like growth factors, i.e. somatomedins, e.g. IGF-1, IGF-2
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P5/00—Drugs for disorders of the endocrine system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- the invention relates to O-glycosylated authentic IGF-I, having three or more
- the invention have substantially no insulin receptor affinity, which makes them
- the invention also relates to a method of obtaining IGF-I as defined above by
- IGF-I together with a pharmaceutically acceptable carrier, diluent or excipient. It also relates to the use of the O-glycosylated IGF-I in the preparation of a
- Insulin-like growth factor- 1 is a growth factor comprising 70 amino acid
- the endogenous human form is non-glycosylated.
- Recombinant human insulin-like growth factor I (rhIGF-I) can be produced by
- glycosylation One such modification that has been observed in proteins produced in yeast is glycosylation.
- Protein glycosylation in yeast can occur both as N- and O-linked, but since IGF-
- O-glycosylation is to be expected in IGF-I.
- One site for O-glycosylation at Thr29 is to be expected in IGF-I.
- Yeast is known to contain carboxypeptidases (JONES, E. W., (1990), "Tackling
- IGF-I polypeptide chain It is suggested that this glycosylated variant of IGF-I
- WO 93/08826 discloses (4-70) IGF -I and (54-67)IGF-I, the truncated variants
- WO 87/01038 discloses peptide analogues of IGF-I in which 1-5 amino acid
- glycosylated authentic IGF-I gIGF-I
- C-IGF-I glycosylated authentic IGF-I
- Lys68Ser69Ala70 and furthermore glycosylated at Thr29.
- therapeutical agents especially in the view of the less adverse effects such as decreased insulin-like effects although having a normal, expected IGF-I effect .
- hypoglycaemia due to decreased binding to IGF binding protein
- IGFBP-1 insulin receptor 1
- IUGR intrauterine growth retardation
- FIGS 1-6 illustrate the invention.
- Figure 1 shows the IGF-I peptides after digestion with S. aureus V8 protease
- Figure 2 shows the purification step based on hydrophobic interaction of
- Figure 3 shows the elution profile of affinity-chromatography of the "fraction 8"
- Figure 4 shows the elution profile of reversed phase chromatography (RP-
- Figure 5 shows the peptide map of SAP V8 digests of the two main variants, z and x2, compared to authentic rhIGF-I (DSQ 93).
- Figure 6 shows the peptide map of SAP V8 digests of the x3, x4 and x5
- the invention relates to O-glycosylated authentic IGF-I, having three or more
- the invention have substantially no insulin receptor affinity, which makes them
- IGF- polypeptide chain amino acid of the IGF- polypeptide chain or O-glycosylated (1-69)IGF-I,
- the invention also relates to a method of obtaining IGF-I as defined above by
- IGF-I insulin growth factor-I
- IGFBP-1 IGF binding protein 1
- IGFBP1 elevated IGFBP levels, particularly IGFBP1.
- the new glycosylated IGF variants may be administrated in doses ranging
- the IGF-I gene was expressed in Saccharomyces cerevisiae using an alpha-
- the yeast fermentation medium contained authentic human IGF-I and
- IGF-I was achieved by hydrophobic interaction chromatography (HI-HPLC). The fraction before the peak of authentic rIGF-I (fraction 8) was collected for the
- the x-fraction was further purified on RPV-HPLC and the main peak was
- Figure 5 shows the peptide map of SAP V8 digests of the two
- x2 O-glycosylated authentic IGF-I, having four or five mannose
- z O-glycosylated authentic IGF-I, having three mannose residues
- x4 O-glycosylated (1-69)IGF-I, having two mannose residues to the
- x5 O-glycosylated (1-67)IGF-I, having two mannose residues to the
- SAP 5 SAP 9:1, SAP 9:2, SAP 5 and SAP 9 are shown in Figures 5 and 6.
- IGF-I receptor The structure-function relationship of the IGF-I receptor is also similar to the
- the insulin receptor and the IGF-I receptor seem to be identical at least
- IGF-I or IGF-I variants is therefore their affinity to, and the bioeffects
- IGF BP3 is the major circulating IGF BP form in adults, and normally binds
- IGF which IGF can be shuttled from the circulation to the target cells for receptor
- IGF BPl is believed to be important both for the transport of IGF-I to the target
- IGFBP-1 has been shown to inhibit the insulin-like activities of IGF-I, both in vitro and in vivo. IGFBP-1 displays a pattern of regulation
- insulin is known to regulate the production of hepatic IGFBP-1.
- IGFBP-1 not only acutely regulates IGF action with respect to blood sugar
- controlled diabetes is accompanied by a decrease in IGFBP-3 and IGF-I while
- IGFBP-1 serum levels are high. IGFBP-1 is further believed to control fetal
- IGF variants with altered affinities for binding to the IGFBPs concomitant with changes in receptor binding, may demonstrate
- the 1-69 dimannosyl Thr29 variant (x4) demonstrated a potency of 50% and
- the growth promoting effect of the glycosylated variants was thus more or less
- weight BPl was tested by a Biacore method. This method measures binding
- binding can be compensated for by other intrinsic properties of the variants e.g.
- the variants have an
- the lipogenisis assay indicates that the variants can be selective for
- IGFBPs IGF Binding proteins
- the IGF BPl binding is lower than for authentic IGF-I but not as low as for the
- the IGF BP3 binding is the same as for IGF-I indicating similarities in half-life.
- the earlier identified glycosylated variant with mannose on Thr29 has a
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Medicinal Chemistry (AREA)
- Diabetes (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- General Chemical & Material Sciences (AREA)
- Pharmacology & Pharmacy (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Animal Behavior & Ethology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Veterinary Medicine (AREA)
- Engineering & Computer Science (AREA)
- Endocrinology (AREA)
- Hematology (AREA)
- Obesity (AREA)
- Immunology (AREA)
- Emergency Medicine (AREA)
- Toxicology (AREA)
- Zoology (AREA)
- Gastroenterology & Hepatology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention relates to O-glycosylated authentic IGF-I, having three or more mannose residues to the Ser69 amino acid of the IGF-polypeptide chain or being truncated and having two mannose residues to the Thr29 amino acid of the truncated IGF-polypeptide chain. The O-glycosylated IGF-I according to the invention have substantially no insulin receptor affinity, which makes them valuable for use in therapeutic treatment. The invention also relates to a method of obtaining IGF-I as defined above by expressing IGF-I in yeast cells and isolating the claimed O-glycosylated IGF-I from the medium and to pharmaceutical composition containing these IGF-I together with a pharmaceutically acceptable carrier, diluent or excipient. It also relates to the use of the O-glycosylated IGF-I in the preparation of a medicament for the treatment of e.g. growth deficiency and insulin resistant states.
Description
O-GLYCOSYLATED AUTHENTIC IGF-I AND TRUNCATED VARIANTS
THEREOF, A METHOD OF PREPARATION THEREOF AND
PHARMACEUTICAL COMPOSITIONS.
The invention relates to O-glycosylated authentic IGF-I, having three or more
mannose residues to the Ser69 amino acid of the IGF- polypeptide chain or
being truncated and having two mannose residues to the Thr29 amino acid of
the truncated IGF- polypeptide chain. The O-glycosylated IGF-I according to
the invention have substantially no insulin receptor affinity, which makes them
valuable for use in therapeutic treatment.
The invention also relates to a method of obtaining IGF-I as defined above by
expressing IGF-I in yeast cells and isolating the claimed O-glycosylated IGF-I
from the medium, and to pharmaceutical compositions which contain these
IGF-I together with a pharmaceutically acceptable carrier, diluent or excipient.
It also relates to the use of the O-glycosylated IGF-I in the preparation of a
medicament for the treatment of e.g. growth deficiency and insulin resistant
states.
INTRODUCTION
Insulin-like growth factor- 1 (IGF-I) is a growth factor comprising 70 amino acid
as a single polypeptide chain, which displays relatively high homology with
proinsulin.
It is known to partly mediate the effect of growth hormone, in order to
promote cell growth and differentiation and also to display insulin-like
properties. The endogenous human form is non-glycosylated.
Recombinant human insulin-like growth factor I (rhIGF-I) can be produced by
expression in the yeast S. cerevisiae and is transported into the fermentation
medium by virtue of a secretion system. Expression of foreign proteins in yeast
can lead to post-translational modifications. One such modification that has been observed in proteins produced in yeast is glycosylation.
Protein glycosylation in yeast can occur both as N- and O-linked, but since IGF-
I lacks the recognition site for N-glycosylation (Asn-Xaa-Ser/Thr) only de novo
O-glycosylation is to be expected in IGF-I. One site for O-glycosylation at Thr29
has been determined, see WO 90/02198, Gellerfors, P.et al. (1989), "Isolation
and characterization of a glycosylated variant of human insulin-like growth
factor I produced in Saccharomyces cerevisiae", J. Biol. Chem., 264, 11444-11449
and Elliott, S., et al. (1990), "Yeast-derived recombinant human insulin-like
growth factor I: Production, purification, and structural characterization", J.
Prot. Chem., 9, 95-104).
Yeast is known to contain carboxypeptidases (JONES, E. W., (1990), "Tackling
the protease problem in Saccharomyces cerevisi e", Methods EnzymoL, 194, 428-
453). Hence, the expression of foreign proteins in yeast may also lead to
production of C-terminally truncated variants.
There is known from WO 90/02198 a glycosylated IGF-I variant of IGF-I which
has two or more mannose residues attached to the Thr 29 amino acid of the
IGF-I polypeptide chain. It is suggested that this glycosylated variant of IGF-I
has a more pronounced effect in lowering the blood glucose than authentic
IGF-I.
Truncated variants of IGF-I are earlier known from WO 91/18621, which
discloses des (1-3-) - IGF-I as useful in the treatment of diabetes. The truncated
variant des(l-3) IGF-I is also known to reduce the severity of CNS damage.
(WO 93/02695).
WO 93/08826 discloses (4-70) IGF -I and (54-67)IGF-I, the truncated variants
of which promote the survival of retinal neuronal cells in ophthalmic
compositions.
WO 87/01038 discloses peptide analogues of IGF-I in which 1-5 amino acid
residues are absent from the N-terminal. They are said to have increased
biological potency, which is useful in e.g. treating growth deficiency and
catabolic conditions.
The authentic IGF-I in which the glutamic acid at position 3 is replaced by
another amino acid or deleted is also known, see WO 91/10348.
We describe in this document the isolation and characterization of two other
variants of glycosylated authentic IGF-I (gIGF-I) and of two new variants of C-
terminally truncated IGF-I which have been isolated and characterized.
We have thus isolated an authentic O-glycosylated IGF-I, having four or five
mannose residues to the Ser69 amino acid of the IGF- polypeptide chain and an
O-glycosylated IGF-I, having three mannose residues to the Ser69 amino acid
of the IGF- polypeptide chain.
We have also isolated a variant lacking the C-terminal Ala70 residue and
glycosylated at Thr29. Another variant lacked the C-terminal tripeptide
Lys68Ser69Ala70, and furthermore glycosylated at Thr29.
These new O-glycosylated variants of IGF-I appear to be promising
therapeutical agents, especially in the view of the less adverse effects such as
decreased insulin-like effects although having a normal, expected IGF-I effect .
Thus, they can be given in higher doses than human authentic IGF-I in the
treatment of growth disorders, possibly without causing problems with
hypoglycaemia. Furthermore, due to decreased binding to IGF binding protein
1 (IGFBP-1) they can be used for treatment of patients suffering from insulin
resistance following e.g. trauma, insulin receptor antibody production, poorly
controlled diabetes or intrauterine growth retardation (IUGR),
Figures 1-6 illustrate the invention.
Figure 1 shows the IGF-I peptides after digestion with S. aureus V8 protease
(SAP-V8). The peptides obtained are labelled according to their appearance in
the primary structure.
Figure 2 shows the purification step based on hydrophobic interaction of
rhIGF-I. Fraction 8 was collected for the isolation of the characterized new IGF-I
variants.
Figure 3 shows the elution profile of affinity-chromatography of the "fraction 8"
on a Concanavalin A column. Two pools (x and z) were collected from repeated
preparations.
Figure 4 shows the elution profile of reversed phase chromatography (RP-
HPLC) of the fraction x pool, cf Figure 3.
Figure 5 shows the peptide map of SAP V8 digests of the two main variants, z and x2, compared to authentic rhIGF-I (DSQ 93).
Figure 6 shows the peptide map of SAP V8 digests of the x3, x4 and x5
variants, compared to authentic rhIGF-I (DSQ 93).
The invention relates to O-glycosylated authentic IGF-I, having three or more
mannose residues to the Ser69 amino acid of the IGF- polypeptide chain or
being truncated and having two mannose residues to the Thr29 amino acid of
the truncated IGF- polypeptide chain. The O-glycosylated IGF-I according to
the invention have substantially no insulin receptor affinity, which makes them
valuable for use in therapeutic treatment.
It relates especially to those having four and /or five mannose residues to the
Ser69 amino acid of the IGF- polypeptide chain or having three mannose
residues to the Serg9 amino acid.
It also relates especially to truncated variants of O-glycosylated IGF-I which
are O-glycosylated (1-67)IGF-I, having two mannose residues to the Thr29
amino acid of the IGF- polypeptide chain or O-glycosylated (1-69)IGF-I,
having two mannose residues to the Thr29 amino acid of the IGF- polypeptide
chain.
The invention also relates to a method of obtaining IGF-I as defined above by
expressing IGF-I in yeast cells, and isolating the claimed O-glycosylated IGF-I
from the medium.
It also relates to pharmaceutical compositions containing these IGF-I together
with a pharmaceutically acceptable carrier, diluent or excipient and a method
of preparing a pharmaceutical composition by mixing the claimed IGF-I
variant with a pharmaceutically acceptable carrier, diluent or excipient.
The claimed variants of IGF-I could be useful in the preparation of a
medicament for the treatment of growth deficiency or for anabolic effects. .
Furthermore, due to deceased binding to IGF binding protein 1 (IGFBP-1) they
can be used for treatment of patients suffering from insulin resistant state
following e.g. following trauma, genetic defects in insulin receptor functions,
auto antibody production towards the insulin receptor and intrauterine growth
retardation (IUGR), and also for the treatment of patients with abnormally
elevated IGFBP levels, particularly IGFBP1.
The new glycosylated IGF variants may be administrated in doses ranging
from 20 μg/kg to 1 mg/kg, preferably between 20 and 250 μg/kg.
Isolation and characterization of glycosylated variants
The IGF-I gene was expressed in Saccharomyces cerevisiae using an alpha-
mating factor leader peptide-IGF-I expression plasmid, p539/12.
The process is described in detail in WO 90/02198 page 6 under Example to
page 7. The yeast fermentation medium contained authentic human IGF-I and
variants thereof. The first separation of the IGF-variants from authentic human
IGF-I, was achieved by hydrophobic interaction chromatography (HI-HPLC).
The fraction before the peak of authentic rIGF-I (fraction 8) was collected for the
isolation of the glycosylated variants. Se Figure 2.
Chromatography on a concanavalin A (ConA) affinity column was an effective
way of separating glycosylated IGF-I from the native form. Figure 3 shows the
elution profile of affinity-chromatography of the "fraction 8" on a Concanavalin
A column. Two partially resolved fractions were collected as two pools, x and
z. These pools were collected from repeated preparations.
Sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE)
followed by ConA blotting indicated that z was relatively homogeneous while
x consisted of two main bands.
Carbohydrate analysis of the purified variants using GC-MS (KENNE, L., and
STROMBERG, S. (1990), "A method for the microanalysis of hexoses in
glycoproteins", Carbohydr. Res., 198, 173-179.) showed that both variants x and
z contained only one type of sugar, mannose, although in different relative
quantities (data not shown).
The x-fraction was further purified on RPV-HPLC and the main peak was
further characterized. Figure 4, showing the peaks x2, x3, x4 and x5.
Immunob lotting with an antibody specific for the C-terminus of IGF-I, revealed
some further differences between the variants. Hence, modifications at the C-
terminus were to be expected.
The structural differences between the glycosylated IGF-I variants was further
delineated using a SAP V8 enzymatic mapping process.
This allowed changes to be assigned to specific peptides by comparison with
native peptides. Figure 5 shows the peptide map of SAP V8 digests of the two
main variants, z and x2, compared with authentic rhIGF-I (DSQ 93) and Figure
6 shows the peptide map of SAP V8 digests of the x3, x4 and x5 variants,
compared with authentic rhIGF-I (DSQ 93).
In the SAP V8 map, the only modified fragment of z was the C-terminal SAP 9
(Figure 1 for nomenclature). The amino acid composition of the SAP 9 fragment
was found to be correct (Table I), this Table presenting the results of amino acid
analysis of the SAP peptides in question), while the mass analysis gave a
molecular mass 484.1 units higher than the theoretical value 1279.6 (Table II,
which presents the results of mass spectrometry of the SAP peptides in
question.). This difference in mass fitted well with the suggestion of three
additional mannose residues, the molecular mass of mannose being 162.1. Mass
spectrometry of the intact polypeptide, Table III, presents the results of mass
spectrometry of the non-digested IGF-I variants and supports this suggestion.
As with variant z, the only modified SAP fragment of x2 was SAP 9, see Figure
5. Furthermore, it was observed that the fragment was not pure. The amino acid
composition was unchanged also in the case of x2, (Table I). For the SAP 9
fragment of x2, the mass analysis gave a molecular mass 645.9 units higher than
the theoretical value 1279.6 (Table III). Hence, in this case the suggested number
of additional mannose residues would be 4. On the other hand, mass analysis of
the undigested polypeptide pointed rather to 5 mannose residues (Table III),
although this value was not entirely correct. An explanation might be that x2
was a mixture of tetra- and pentasubstituted IGF-I, a heterogeneity also
indicated both by SDS-PAGE and as a shoulder on the SAP 9 peak (Figure 5).
Different preparations may then have contained different relative amounts of
the two variants.
Regarding the amino acid sequence of the SAP 9 fragment, the only residue
known to enable O-glycosylation was Ser69, thus this was the proposed
glycosylation site.
In the SAP V8 map, the modified SAP 5 fragment common to x3, x4 and x5
(Figure 6) was caused by dimannose glycosylation at Thr 29. The respective C-
terminal SAP 9 fragments of x4 and x5 were also modified.
The SAP V8 map of variant x4 revealed two modifications of the SAP 9
fragment, labelled SAP 9:1 and SAP 9:2 (Figure 1). According to the amino acid
sequence analysis (Table I), one Ala residue was missing in both fragments,
supposedly the C-terminal Ala70. Mass analysis of fragment SAP 9:1 supported
this modification. As for the SAP 9:2 fragment, mass analysis gave an additional
26.6 units (Table II). This discrepancy was consistent when mass analysis was
performed on the undigested polypeptide (Table III). No rational explanation
has been found for this difference in molecular mass.
The fragment corresponding to SAP 9 of variant x5 had a retention time slightly
longer than x4 SAP 9:2, see Figure 4. Amino acid analysis (Table I) of the
fragment revealed the lack of one lysine, one serine, and one alanine residue,
proposed a truncation of the C-terminal tripeptide Lys68-Ser69-Ala70. This suggested modification was confirmed by mass analysis of both the SAP 9
fragment (Table II), and the undigested polypeptide (Table III).
It has thus been confirmed that:
By x2 is meant O-glycosylated authentic IGF-I, having four or five mannose
residues to the Ser69 amino acid of the IGF- polypeptide chain.
By z is meant O-glycosylated authentic IGF-I, having three mannose residues
to the Ser69 amino acid of the IGF- polypeptide chain.
By x4 is meant O-glycosylated (1-69)IGF-I, having two mannose residues to the
Thr29 amino acid of the IGF- polypeptide chain.
By x5 is meant O-glycosylated (1-67)IGF-I, having two mannose residues to the
Thr29 amino acid of the IGF- polypeptide chain.
Table I. Amino acid analysis of peptides obtained from SAP V8 digestion of
glycosylated IGF-I variants. Values are given as mol/mol. The theoretical
values as predicted from intact peptides + indicates trace amounts are given in
paran thesis.
Sample x4 x4 x4 x5 x5 z x2 peptide SAP 5 SAP 9:1 SAP 9:2 SAP 5 SAP 9 SAP9 SAP 9
Asx 1.0 (1) 1.0 (1)
Thr 1.0 (1) 0.9 (1)
Ser 1.0 (1) 0.9 (1) nd (1) 1.0 (1) 0.9(1)
Glx
Gly 3.1 (3) 3.0 (3)
Ala 2.1 (3) 2.0 (3) 1.9 (3) 3.2 (3) 2.9 (3)
Cys nd (1) nd (1) nd (1) nd (l) nd (l) Val
Met + (i) + (1) + (1) nd (l) + (1) lie
Leu 1.1 (1) 1.1 (1) 1.1 (1) 1.1 (1) 1.0 (1)
Tyr 1.9 (2) 0.9 (1) 1.0 (1) 2.0 (2) 1.1 (1) 0.9 (1) 1.0 (1) Phe 1.9 (2) 2.1 (2)
Lys 1.0 (1) 2.0 (2) 2.0 (2) 0.9 (1) 0.9 (2) 2.1 (2) 2.0 (2)
Arg 1.0 (1) 1.1 (1)
Pro 0.9 (1) 1.9 (2) 2.0 (2) 1.0 (1) 2.0 (2) 1.8 (2) 2.2 (2)
SAP 5, SAP 9:1, SAP 9:2, SAP 5 and SAP 9 are shown in Figures 5 and 6.
nd: not detected.
Table II Plasma desorption mass spectrometry of SAP V8 derived peptides of
the characterized glycosylated IGF-I variants.
Theoretical mass:
SAP 5 1406.6
SAP 9 1279.6
Sample experimental mass suggested theor. mass
mass value difference modification difference
z SAP 9 1763.7 +484.1 +3 mannos +486.4
x2 SAP 9 1925.5 +645.9 +4 mannos +648.6 x4 SAP 5 1728.5 +321.9 +2 mannos +324.3
x4 SAP 9:1 1207.4 -72.2 -Ala -71.1
x4 SAP 9:2 1235.1 -44.5 -Ala, +? -71.1
X5 SAP 9 992.4 -287.2 -LysSerAla -286.3
Table III Plasma desorption mass spectrometry of intact molecules of the
characterized glycosylated IGF-I variants.
Theoretical mass:
IGF-I 7648.7
Sample experimental mass suggested theor. mass mass value difference modification difference
z 8131.7 +483.0 +3 mannos +486.4
x2 8435.4 +786.7 +4 mannos +648.6
+5 mannos +810.7
x4 7927.6 +278.9 +2 man,-Ala,+? +253.2
x5 7683.0 +34.3 +2 mannos +38.0
-LysSerAla
BIOLOGICAL ACTIVITY
Nuclear magnetic resonance data indicate that the core of IGF-I is structurally
very similar to insulin. Structural determinants for binding IGF-I to the Insulin
and Type I receptor in this region overlap. Consequently, in addition to
binding its own receptor IGF-I crossreacts with the insulin receptor, although in
most cases with a much lower affinity.
The structure-function relationship of the IGF-I receptor is also similar to the
insulin receptor. Apart from the different binding affinities for their respective
ligands, the insulin receptor and the IGF-I receptor seem to be identical at least
with regard to the initial steps in signal transduction following ligand binding.
This suggests that bioeffect differences between IGF-I and insulin are mainly
due to differential expression of their receptors and signalling pathways in
different target cells. An important determinant for the target organ bioeffects
of IGF-I or IGF-I variants is therefore their affinity to, and the bioeffects
mediated by binding to the two receptor types.
Bioavailability and the actions of the IGFs are also influenced by their
association with a group of specific binding proteins (IGF BP 1-6). IGF BP3 is the major circulating IGF BP form in adults, and normally binds
more than 95% of IGF-I in serum in a 150 kDa ternary complex. The ternary
complex has a relatively long half-life and is unable to leave circulation intact.
Since only the free, uncomplexed, forms of IGF are considered to be
biologically active, it is necessary to provide specific mechanisms by means of
which IGF can be shuttled from the circulation to the target cells for receptor
binding. IGF BPl is believed to be important both for the transport of IGF-I to the target
cell and for regulating the IGF responsiveness of target cells. Studies support a
physiological role for IGFBP-1 in modulating IGF availability for glucose
homeostasis. IGFBP-1 has been shown to inhibit the insulin-like activities of
IGF-I, both in vitro and in vivo. IGFBP-1 displays a pattern of regulation
characteristic for glucose counterregulators, and is the only IGFBP which
displays rapid fluctuations in serum with a pronounced diurnal rhythm. There
exists an inverse relationship between circulating insulin and IGFBP-1 and
insulin is known to regulate the production of hepatic IGFBP-1.
IGFBP-1 not only acutely regulates IGF action with respect to blood sugar
control (as well as other insulin-like actions), but also appears to be highly
important in controlling growth in the long term. Clinical observations point to
an inverse correlation between circulating IGFBP-1 levels and various growth
parameters. Low levels are found at puberty concomitant with peak IGF-I
levels and accelerated growth. Conversely, the stunted growth of poorly
controlled diabetes is accompanied by a decrease in IGFBP-3 and IGF-I while
IGFBP-1 serum levels are high. IGFBP-1 is further believed to control fetal
growth, and elevated levels correlate with low weight at birth.
It is therefore obvious that the distribution of IGF between the various IGFBP
forms is an important determinant for clearance, tissue distribution and
bioactivity of IGF. It follows that IGF variants with altered affinities for binding to the IGFBPs concomitant with changes in receptor binding, may demonstrate
dramatically different bioprofiles.
The ability of the glycosylated IGF-I variants to bind to the IGF-I receptor was
tested in a receptor binding assay using a human placenta membrane
preparation (Hall K et al., A. J. Clin. Endocrinol. Metabol. 39:973-976, 1974).
The z and x2 variants both demonstrated a reduction of IGF-I receptor binding
(60 - 70%, compared to the non-glycosylated authentic IGF-I preparation.
The 1-69 dimannosyl Thr29 variant (x4) demonstrated a potency of 50% and
the 1-67 dimannosyl Thr29 variant (x5) demonstrated a potency of 30% relative
to authentic human IGF-I. See Table IV.
Thus, type I receptor binding was not significantly reduced by glycosylation in
pos Thr 29, but when combined with further C-terminal truncations produced a
graded and significant loss of receptor binding potency.
The in vitro bone growth stimulatory effects of the glycosylated rh IGF-I
variants were tested in an embryonal chick femur assay (Endo H et al, Nature
(London), 286:262-264, 1980). The stimulatory effects of IGF-I in this model are
primarily mediated by the chick IGF-I receptor. All glycosylated rhIGF-I
variants were found to be equipotent to the non-glycosylated authentic IGF-I
regarding bone growth stimulation. See Table IV.
This either suggest that the ability to bind the chick IGF-I receptor is intact, or
that the binding potency for this receptor is marginally affected, as for the
human IGF-I receptor, but that this effect is compensated for e.g. by an altered
affinity to the IGF-I binding proteins. Local binding protein for IGF-I in the
bone are identified.
The growth promoting effect of the glycosylated variants was thus more or less
unchanged.
When the insulin-like properties of the IGF-I variants was tested in a lipogenic
assay using primary rat adipocytes (Small J et al., J. Biol. Chem. 262:11071-79,
1987) a major reduction of lipogenic activity was revealed. The lipogenic
potency of all variants was reduced to 30% or less compared to non-
glycosylated authentic rhIGF-I. See Table IV.
This indicates that the ability of these IGF-I analogues to bind the insulin
receptor is severely impaired.
Since it is known that primary rat adipocytes carry few, if any, functional IGF-I
receptors, it is suggested that O-glycosylation leads to a reduction in insulin-
receptor binding, and hence the reduced insulin-like potency. This is
advantageous to patients in need of IGF-I for growth effects.
Affinities to the large BP3 (BP = binding protein) and the small molecular
weight BPl was tested by a Biacore method. This method measures binding
of IGF to IGFBPs immobilized on a biosensor chip surface by a surface plasmon
resonance technique (Jonsson U, Bio Techniques 11:5, 1991). The z and x2
variants demonstrated a reduced association with the IGFBPl (approximately
60% of the non-glycosylated authentic IGF-I), while the IGFBP3 affinity of the
two variants was unchanged. See Table IV.
The results suggest that administered Ser69 variants will form the BP complex
similar to authentic IGF-I in contrast to the earlier characterized 1-70 Thr29
variant, which will not. For all variants a reduced IGFBPl association suggests
a higher bioavailability and less inhibition of bioactivity.
In conclusion, in vitro data indicate an altered pharmacological profile resulting
from O-glycosylation on position Ser69 and from O-glycosylation on position
Thr29 when truncated. The reduced insulin-like activity together with the
altered IGFBP 1 affinity are expected to lead to changes in bioeffect selectivity
and availability that may be used to advantage in clinical situation.
TABLE IV
Comparison of Biological effects in vitro. The potency of gIGF-I variants are
given relative to the non-glycosylated authentic rhIGF-I.
BIOLOGICAL EFFECT
authentic 1-70 IGF x2 X4 X5
IGF-I Thr 29
IGF-I Receptor binding
(hplacenta) 1 0.9 0.6 0.7 0.5 0.3
Growth of chick embryo
femora 1 1 1 1 1 1
Lipogenesis in primary rat
adipocytes 1 0.3 0.3 0.3 <0.2* <0.3
IGF BPl binding 1 0.1 0.6 0.6 n.d. n.d.
IGF BP3 binding 1 0.25 ' 1 1 n.d. n.d.
n.d.= not determined
Not statistically different from nonstimulated controls.
CONCLUSIONS
A second site for O-glycosylation of rhIGF-I has been localized in fragment SAP
9, at residue Ser69. The degree of glycosylation appeared to range from three to
five mannose residues. The mannosylated(Ser69) rhIGF-I variants characterized
were not glycosylated at other sites.
The biological results show that all of the described variants of IGF-I have
normal, expected IGF-I effect on in vitro growth of chick embryo femora,
despite of IGF-I receptor binding being either similar to authentic IGF-I (1-70
Thr29 IGF) or reduced (z x2, x4, x5 variants). Although this was unexpected, it
is nevertheless of considerable interest, since it indicates that a loss of receptor
binding can be compensated for by other intrinsic properties of the variants e.g.
the demonstrated changes in IGFBP associations. The variants have an
increased selectivity for IGF-I receptors over insulin receptors as indicated by
the lipogenisis assay. This assay indicates that the variants can be selective for
bone and muscles but not for fat or liver, since the latter tissues are known to
contain low concentration of receptors for IGF-I and significant concentrations
of insulin receptors.
As the insulin-like effect is reduced, less adverse effects such as hypoglycaemia
are expected and the dose may be higher.
All variants are of potential interest clinically as they
1/ offer a graded receptor selectivity which may be used to achieve selective
strengthening of the classical IGF-I effects on cellproliferation and
differentiation at the expense of insulin receptor mediated "insulin-like" effects,
2/ are likely to demonstrate differences in kinetics and bioavailability as a
result of either the presence or absence of glycosylation and due to changed
affinities to IGF Binding proteins (IGFBPs).
Both C-terminal truncation and glycosylation important. Decreased
lipogenesis versus authentic IGF-I has been shown for all variants.
The IGF BPl binding is lower than for authentic IGF-I but not as low as for the
glycosylated variant with mannose on Thr29-
The IGF BP3 binding is the same as for IGF-I indicating similarities in half-life.
The earlier identified glycosylated variant with mannose on Thr29, has a
much lower IGFBP3 binding.
Claims
1. O-glycosylated authentic IGF-I, having three or more mannose
residues to the Ser69 amino acid of the IGF- polypeptide chain or
being truncated and having two mannose residues to the Thr29
amino acid of the truncated IGF- polypeptide chain.
2. O-glycosylated IGF-I according to claim 1 having substantially
no insulin receptor affinity.
3. O-glycosylated IGF-I according to claim 1 or 2 having four or
five mannose residues to the Ser69 amino acid of the IGF-
polypeptide chain.
4. O-glycosylated IGF-I according to claim 1 or 2 having three
mannose residues to the Ser69 amino acid of the IGF- polypeptide
chain.
5. Truncated variant of O-glycosylated IGF-I according to claim 1
or 2, which is an O-glycosylated (1-67)IGF-I, having two mannose
residues to the Thr29 amino acid of the IGF- polypeptide chain.
6. Truncated variant of O-glycosylated IGF-I according to claim 1
or 2, which is an O-glycosylated (1-69)IGF-I, having two mannose
residues to the Thr29 amino acid of the IGF- polypeptide chain.
7. A method of obtaining O-glycosylated IGF-I according to any
of claims 1 to 6 by expressing IGF-I in yeast cells, and isolating the
claimed O-glycosylated IGF-I from the medium.
8. A pharmaceutical composition containing O-glycosylated IGF-I
according to any of claims 1 to 6 together with a pharmaceutically
acceptable carrier, diluent or excipient.
9. A method of preparing a pharmaceutical composition
comprising mixing O-glycosylated IGF-I according to any of
claims 1 to 6 with a pharmaceutically acceptable carrier, diluent
or excipient.
10. Use of O-glycosylated IGF-I according to any of claims 1-6 in
the preparation of a medicament for the treatment of growth
deficiency.
11. Use of O-glycosylated IGF-I according to any of claims 1-6 in
the preparation of a medicament for the treatment of insulin
resistant states e.g. following trauma, genetic defects in insulin
receptor functions, auto antibody production towards the insulin
receptor and intrauterine growth retardation (IUGR).
12. Use of O-glycosylated IGF-I according to any of claims 1-6 in
the preparation of a medicament for the treatment of patients with abnormally elevated IGFBP levels, particularly IGFBPl.
Priority Applications (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AU29406/95A AU2940695A (en) | 1994-07-01 | 1995-06-22 | O-glycosylated authentic igf-i and truncated variants thereof, a method of preparation thereof and pharmaceutical compositions |
| JP8503823A JPH10502623A (en) | 1994-07-01 | 1995-06-22 | O-Glycosylated Authentic IGF-I and Its Truncated Variants, Methods for Producing the Same, and Pharmaceutical Compositions |
| EP95925201A EP0769022A1 (en) | 1994-07-01 | 1995-06-22 | O-glycosylated authentic igf-i and truncated variants thereof, a method of preparation thereof and pharmaceutical compositions |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| SE9402332-2 | 1994-07-01 | ||
| SE9402332A SE9402332D0 (en) | 1994-07-01 | 1994-07-01 | IGF-1 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO1996001275A1 true WO1996001275A1 (en) | 1996-01-18 |
Family
ID=20394599
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/SE1995/000774 WO1996001275A1 (en) | 1994-07-01 | 1995-06-22 | O-glycosylated authentic igf-i and truncated variants thereof, a method of preparation thereof and pharmaceutical compositions |
Country Status (7)
| Country | Link |
|---|---|
| EP (1) | EP0769022A1 (en) |
| JP (1) | JPH10502623A (en) |
| AU (1) | AU2940695A (en) |
| CA (1) | CA2193399A1 (en) |
| IL (1) | IL114270A0 (en) |
| SE (1) | SE9402332D0 (en) |
| WO (1) | WO1996001275A1 (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1996033216A1 (en) * | 1995-04-21 | 1996-10-24 | Pharmacia & Upjohn Ab | Truncated igf-i |
| US6660279B2 (en) * | 1998-09-01 | 2003-12-09 | Astrazeneca Ab | Stability for injection solutions |
| EP2241575A1 (en) * | 2005-01-07 | 2010-10-20 | Regeneron Pharmaceuticals, Inc. | Methods of treating obesity with combination therapeautics of igf-1 fusion polypeptides |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1990002198A1 (en) * | 1988-08-20 | 1990-03-08 | Kabi Pharmacia Ab | O-glycosylated igf-1 |
| WO1993002695A1 (en) * | 1991-08-01 | 1993-02-18 | Genentech, Inc. | Igf-1 to improve the neural condition |
-
1994
- 1994-07-01 SE SE9402332A patent/SE9402332D0/en unknown
-
1995
- 1995-06-22 AU AU29406/95A patent/AU2940695A/en not_active Abandoned
- 1995-06-22 IL IL11427095A patent/IL114270A0/en unknown
- 1995-06-22 WO PCT/SE1995/000774 patent/WO1996001275A1/en not_active Application Discontinuation
- 1995-06-22 EP EP95925201A patent/EP0769022A1/en not_active Withdrawn
- 1995-06-22 CA CA002193399A patent/CA2193399A1/en not_active Abandoned
- 1995-06-22 JP JP8503823A patent/JPH10502623A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1990002198A1 (en) * | 1988-08-20 | 1990-03-08 | Kabi Pharmacia Ab | O-glycosylated igf-1 |
| WO1993002695A1 (en) * | 1991-08-01 | 1993-02-18 | Genentech, Inc. | Igf-1 to improve the neural condition |
Non-Patent Citations (2)
| Title |
|---|
| CHEMICAL ABSTRACTS, Volume 111, No. 11, 11 Sept. 1989, (Columbus, Ohio, USA), GELLERFORS PAER et al., "Isolation and Characterization of A Glycosylated Form of Human Insulin-Like Growth Factor I Produced in Saccharomyces Cerevisiae", page 90588, The Abstract No. 90592g; & J. BIOL. CHEM., 1989, 264(19), 1144. * |
| FEBS LETTERS, Volume 248, No. 1,2, May 1989, KARL HARD et al., "O-Mannosylation of Recombinant Human Insulin-Like Growth Factor I (IGF-I) Produced in Saccharomyces Cerevisiae", pages 111-114. * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1996033216A1 (en) * | 1995-04-21 | 1996-10-24 | Pharmacia & Upjohn Ab | Truncated igf-i |
| US6660279B2 (en) * | 1998-09-01 | 2003-12-09 | Astrazeneca Ab | Stability for injection solutions |
| US6998136B2 (en) | 1998-09-01 | 2006-02-14 | Astrazeneca Ab | Stability for injection solutions |
| EP2241575A1 (en) * | 2005-01-07 | 2010-10-20 | Regeneron Pharmaceuticals, Inc. | Methods of treating obesity with combination therapeautics of igf-1 fusion polypeptides |
| US8158581B2 (en) | 2005-01-07 | 2012-04-17 | Regeneron Pharmaceuticals, Inc. | IGF-1 fusion polypeptides and therapeutic uses thereof |
| US8445434B2 (en) | 2005-01-07 | 2013-05-21 | Regeneron Pharmaceuticals, Inc. | IGF-1 fusion polypeptides and therapeutic uses thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| IL114270A0 (en) | 1995-10-31 |
| CA2193399A1 (en) | 1996-01-18 |
| AU2940695A (en) | 1996-01-25 |
| SE9402332D0 (en) | 1994-07-01 |
| JPH10502623A (en) | 1998-03-10 |
| EP0769022A1 (en) | 1997-04-23 |
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