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G-CSF polypeptides and conjugates

Inactive Publication Date: 2006-04-20
MAXYGEN HLDG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0012] More specifically, the present invention relates to specific conjugates comprising a polypeptide exhibiting G-CSF activity and a non-polypeptide moiety, methods for their preparation and their use in medical treatment and in the preparation of pharmaceuticals. Accordingly, in a first aspect the invention relates to various specific conjugates comprising a polypeptide exhibiting G-CSF activity and having an amino acid sequence that differs from the known amino acid sequence of human G-CSF as shown in SEQ ID NO:1 in at least one specified altered amino acid residue comprising an attachment group for a non-polypeptide moiety, and having at least one non-polypeptide moiety attached to an attachment group of the polypeptide. The conjugate of the present invention has one or more improved properties as compared to commercially available rhG-CSF, including increased functional in vivo half-life, increased serum half-life, reduced side effects, reduced immunogenicity and / or increased bioavailability. Consequently, medical treatment with a conjugate of the invention offers a number of advantages over the currently available G-CSF compounds.
[0014] In a further aspect the invention relates to a polypeptide conjugate comprising a polypeptide exhibiting G-CSF activity, which comprises an amino acid sequence that differs from the amino acid sequence of hG-CSF (with the amino acid sequence shown in SEQ ID NO:1) in at least one amino acid residue selected from an introduced or removed amino acid residue comprising an attachment group for a non-polypeptide moiety, and a sufficient number or type of non-polypeptide moieties to provide the conjugate with an increased half-life compared to known recombinant G-CSF products.

Problems solved by technology

Another problem with currently available RG-CSF products is the occurrence of dose-dependent bone pain.
As a result, the problem of bone pain discussed above has not been solved by this molecule.

Method used

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  • G-CSF polypeptides and conjugates
  • G-CSF polypeptides and conjugates
  • G-CSF polypeptides and conjugates

Examples

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example 1

Construction and Cloning of Synthetic Genes Encoding hG-CSF

[0280] The following DNA fragments were synthesised following the general procedure described by Stemmer et al. (1995), Gene 164, pp. 49-53:

[0281] Fragment 1, consisting of a Bam HI digestion site, a sequence encoding the YAP3 signal peptide (WO 98 / 32867), a sequence encoding the TA57 leader sequence (WO 98 / 32867), a sequence encoding a KEX2 protease recognition site (AAAAGA), a sequence encoding hG-CSF with its codon usage optimised for expression in E. coli, (SEQ ID NO:2) and a Xba I digestion site.

[0282] Fragment 2, consisting of a Bam HI digestion site, a sequence encoding the YAP3 signal peptide (WO 98 / 32867), a sequence encoding the TA57 leader sequence (WO 98 / 32867), a sequence encoding a histidine tag (SEQ ID NO:5), a sequence encoding a KEX2 protease recognition site (AAAAGA), a sequence encoding hG-CSF with its codon usage optimised for expression in E. coli, (SEQ ID NO:2) and a Xba I digestion site.

[0283] Frag...

example 2

Expression of hG-CSF in S. cerevisiae and E. coli

[0288] Transformation of Saccharomyces cerevisiae YNG318 (available from the American Type Culture Collection, VA, USA as ATCC 208973) with either plasmid pG-CSFcerevisiae or pHISG-CSFcerevisiae, isolation of transformants containing either of the two plasmids, and subsequent extracellular expression of hG-CSF without and with the HIS tag, respectively, was performed using standard techniques described in the literature. Transformation of E. coli BL21 (DE3) (Novagen, Cat. No. 69387-3) with pG-CSFcoli, isolation of transformants containing the plasmid and subsequent expression of hG-CSF in the supernatant and in the periplasm of the cell was performed as described in the pET System Manual (8th edition) from Novagen.

[0289] Expression of hG-CSF by S. cerevisiae and E. coli was verified by Western Blot analysis using the ImmunoPure Ultra-Sensitive ABC Rabbit IgG Staining kit (Pierce) and a polyclonal antibody against hG-CSF (Pepro Tech ...

example 3

Purification of hG-CSF and Variants thereof from S. cerevisiae Culture Supernatants

[0291] Purification of hG-CSF was performed as follows:

[0292] Cells are removed by centrifugation. Cell depleted supernatant is then filter sterilised through a 0.22 μm filter. Filter sterilised supernatant is diluted 5 fold in 10 mM sodium acetate pH 4.5. pH is adjusted by addition of 10 ml concentrated acetic acid per 5 liters of diluted supernatant. The ionic strength should be below 8 mS / cm before application to the cation exchange column.

[0293] Diluted supernatant is loaded at a linear flow rate of 90 cm / h onto a SP-sepharose FF (Pharmacia) column equilibrated with 50 mM sodium acetate, pH 4.5 until the effluent from the column reaches a stable UV and conductivity baseline. To remove any unbound material, the column is washed using the equilibration buffer until the effluent from the column reaches a stable level with respect to UV absorbance and conductivity. The bound G-CSF protein is eluted...

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Abstract

The invention relates to polypeptide conjugates comprising a polypeptide exhibiting G-CSF activity and having an amino acid sequence that differs from the amino acid sequence of human G-CSF in at least one specified introduced and / or removed amino acid residue comprising an attachment group for a non-polypeptide moiety, and having at least one non-polypeptide moiety attached to an attachment group of the polypeptide. The attachment group may e.g., be a lysine, cysteine, aspartic acid or glutamic acid residue or a glycosylation site, and the non-polypeptide moiety may e.g., be a polymer such as polyethylene glycol or an oligosaccharide. The conjugate has one or more improved properties such as increased biological half-life and reduced side effects.

Description

CROSS-REFERENCES TO RELATED APPLICATIONS [0001] The present application claims priority to and benefit of U.S. Ser. No. 60 / 176,376 filed on Jan. 14, 2000, U.S. Ser. No. 60 / 189,506 filed on Mar. 15, 2000, U.S. Ser. No. 60 / 215,644 filed on Jun. 30, 2000, Danish Application No. PA 2000 00024 filed on Jan. 10, 2000, Danish Application No. PA 2000 00341 filed on Mar. 2, 2000, and Danish Application No. PA 2000 00943 filed on Jun. 16, 2000.COPYRIGHT NOTIFICATION [0002] Pursuant to 37 C.F.R. 1.71(e), Applicants note that a portion of this disclosure contains material which is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or patent disclosure, as it appears in the Patent and Trademark Office patent file or records, but otherwise reserves all copyright rights whatsoever. FIELD OF THE INVENTION [0003] The present invention relates to new polypeptides exhibiting granulocyte colony-stimulating factor (G-CSF) ...

Claims

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Application Information

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IPC IPC(8): C07K14/54A61K38/00A61K47/42C07K14/535
CPCA61K9/0019A61K38/00A61K47/42C07K14/535
Inventor NISSEN, TORBENANDERSEN, KIMHANSEN, CHRISTIANMIKKELSEN, JANSCHAMBYE, HANS
Owner MAXYGEN HLDG
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