Stabilized insulin-like growth factor polypeptides

a growth factor and polypeptide technology, applied in the direction of peptide/protein ingredients, extracellular fluid disorder, metabolism disorder, etc., can solve the problems that igf-1 and igf-2 appear to be poor drug candidates, and achieve the effect of avoiding or reducing this cleavag

Inactive Publication Date: 2010-07-08
NOVARTIS AG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0004]The invention is based on the fact that a precursor IGF-1 or IGF-2 protein containing substantially its E-peptide is bioactive and stabilized in the presence of serum, resulting in an IGF-1 or IGF-2 polypeptide that is useful as a pharmaceutical. In the compositions of the invention, the normal cleavage of the E-peptide from IGF-1 is avoided, for example, by mutating or deleting either of the arginine at position 1 or the serine at position 2 of the E-peptides (corresponding to positions 71 and 72 in the wild-type precursor IGF-1). In IGF-2, the cleavage is avoided, for example, by mutating or deleting either the arginine at position 1 or the aspartic acid at position 2 of the E-peptide (corresponding to positions 68 and 69 in the wild-type precursor IGF-2). Other modifications of an IGF precursor protein can avoid or reduce this cleavage.
[0005]In addition, further modifications of the IGF-1 precursor amino acid sequence can confer additional pharmaceutical benefits. For example, the polypeptides of the invention can exhibit increased affinity for the IGF-1 receptor or decreased binding ability to an inhibitory IGF-1 or IGF-2 binding protein.
[0012]Similarly, the invention includes a IGF-2 precursor protein where the cleavage of the E-peptide from IGF-2 by a protease is reduced by modification of the precursor protein. In particular, deletion or mutation of R68 or D69 can be an effective means of avoiding protease digestion of the IGF-2 precursor protein.
[0013]In addition, any E-peptide of IGF-1 can be combined with an IGF-2 and any E-peptide of IGF-2 can be combined with IGF-1 to provide the benefits described herein.
[0017]The veterinary uses include (i) enhancing the rate and / or extent of growth in an animal, (ii) enhancing the efficiency of their conversion of feed into body tissue, (iii) enhancing milk production in lactating animals, (iv) treating animal wasting symptoms associated with cachexia, trauma, or other consumption diseases, and (v) treating lactating animals for improvement in neonatal health.

Problems solved by technology

Both IGF-1 and IGF-2 appear to be poor drug candidates, since these proteins are quickly degraded by endogenous proteases in the serum of patients.

Method used

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  • Stabilized insulin-like growth factor polypeptides
  • Stabilized insulin-like growth factor polypeptides
  • Stabilized insulin-like growth factor polypeptides

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0111]A DNA expression vector encoding the hIGF-1-Ea precursor polypeptide containing the following modifications was constructed: deletion of G1, deletion of P2, and deletion of E3; mutation of R37 to A; and deletion of R71 and deletion of S72. These mutations are sometimes referred to as “3mut” throughout the present disclosure. This results in the following secreted protein sequence:

(SEQ ID NO: 8)tlcgaelvdalqfvcgdrgfyfnkptgygsssraapqtgivdeccfrscdlrrlemycaplkpaksavraqrhtdmpktqkevhlknasrgsagnknyrm

[0112]Cos7 cells (available from ATCC) were maintained in DMEM containing 10% fetal bovine serum, 100 U / ml penicillin, and 100 μg / ml streptomycin and plated at a density of 1×106 cells per 10-cm plate. These cell cultures were transfected with 8 μg of expression plasmid using Fugene (Roche) according to manufacturer's instructions. Twenty-four hours post-transfection, cells were washed once and cultured in serum-free medium for 48 hours. Supernatants were collected and stored at −80° C.

[01...

example 2

[0116]A DNA expression vector encoding the hIGF-1-Eb precursor polypeptide containing the following mutations was constructed: deletion of G1, deletion of P2, and deletion of E3; mutation of R37 to A; and deletion of R71 and deletion of S72 (i.e., the “3mut”). This results in the following secreted protein sequence:

(SEQ ID NO: 9)tlcgaelvdalqfvcgdrgfyfnkptgygsssarapqtgivdeccfrscdlrrlemycaplkpaksavraqrhtdmpktqkyqppstnkntksqrrkgwpkthpggeqkegteaslqirgkkkeqrreigsrnaecrgkkgk

[0117]The polypeptide was assayed in accordance with the procedures described in Example 1 above. FIG. 1B and use of densitometry indicated that the ratio of uncleaved to cleaved IGF-1 was about 1:9, while the ratio for hIGF-1Eb3mut was about 1:1, showing that these modifications result in a stabilized polypeptide. FIG. 2B indicates that the hIGF-1Eb3mut was able to activate the IGF-1R cellular pathway to a similar extent as the long-R3-IGF-1 positive control reagent and the recombinant IGF-1. In addition, the data in ...

example 3

[0118]A DNA expression vector encoding the hIGF-1-Ec precursor polypeptide containing the following mutations was constructed: deletion of G1, deletion of P2, and deletion of E3; mutation of R37 to A; and deletion of R71 and deletion of S72 (i.e., the “3mut”). This results in the following secreted protein sequence:

(SEQ ID NO: 10)tlcgaelvdalqfvcgdrgfyfnkptgygsssrapqtgivdeccfrscdlrrlemycaplkpaksavraqrhtdmpktqkyqppstnkntksqrrkgstfeerk

[0119]The polypeptide was assayed in accordance with the procedures described in Example 1 above. FIG. 1C and use of densitometry indicated that the ratio of uncleaved to cleaved IGF-1 was about 1:5, while the ratio for hIGF-1Ec3mut was about 1:0.96, showing that these modifications result in a stabilized polypeptide. FIG. 2D indicates that the hIGF-1Ec3mut was able to activate the IGF-1R cellular pathway to a similar extent as the long-R3-IGF-1 positive control reagent and the recombinant IGF-1. In addition, the data in FIG. 5 directly shows that hIGF-1E...

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Abstract

The invention relates to stabilized polypeptides having an IGF-1 or IGF-2 sequence and an E-peptide sequence, where the natural physiological cleavage of the E-peptide from the IGF is prevented.

Description

BACKGROUND OF THE INVENTION[0001]Insulin-like growth factors (IGFs) are part of a complex system that cells use to communicate with their physiologic environment. This complex system (often referred to as the insulin-like growth factor axis) consists of two cell-surface receptors (IGF-1R and IGF-2R), two ligands (IGF-1 and IGF-2), a family of six high-affinity IGF-binding proteins (IGFBP 1-6), and associated IGFBP degrading enzymes (proteases). This system is important not only for the regulation of normal physiology but also for a number of pathological states (Glass, Nat Cell Biol 5:87-90, 2003).[0002]The IGF axis has been shown to play roles in the promotion of cell proliferation and the inhibition of cell death (apoptosis). IGF-1 is mainly secreted by the liver as a result of stimulation by human growth hormone (hGH). Almost every cell in the human body is affected by IGF-1, especially cells in muscles, cartilage, bones, liver, kidney, nerves, skin and lungs. In addition to the ...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K38/16C07K14/00A61P43/00
CPCA61K38/30C07K14/475C07K14/65A61P11/00A61P17/02A61P19/00A61P19/08A61P19/10A61P21/00A61P25/00A61P25/28A61P27/02A61P3/10A61P43/00A61P5/48A61P7/06
Inventor GLASS, DAVID JONATHAN
Owner NOVARTIS AG
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