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G-CSF conjugates

a granulocyte colony-stimulating factor and conjugate technology, applied in the direction of peptides/protein ingredients, animal/human proteins, peptides, etc., can solve the problems of dose-dependent bone pain, relative minor infections can be serious and even life-threatening, and patients become neutropenic after chemotherapy, etc., to reduce in vitro bioactivity, improve properties, and rapid neutrophil recovery

Inactive Publication Date: 2005-05-05
MAXYGEN HLDG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0012] 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. These conjugates have a substantially reduced in vitro bioactivity compared to that of non-conjugated hG-CSF, which surprisingly has been shown to result in a more rapid neutrophil recovery. The conjugate of the present invention thus has one or more improved properties as compared to commercially available rhG-CSF, including increased stimulation of neutrophils, 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.

Problems solved by technology

For patients with severe neutropenia, e.g. as a result of chemotherapy, even relatively minor infections can be serious and even life-threatening.
Another, more significant problem with currently available rG-CSF products is that patients become neutropenic after chemotherapy even after administration of G-CSF.
A further problem is the occurrence of dose-dependent bone pain.

Method used

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Examples

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Effect test

example 1

Construction and Cloning of Synthetic Genes Encoding hG-CSF

[0269] The following DNA fragments were synthesized following the general procedure described by Stemmer et al. (1995), Gene 164, pp. 49-53:

[0270] 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 optimized for expression in E. coli, (SEQ ID NO:2) and a Xba I digestion site.

[0271] 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 optimized for expression in E. coli, (SEQ ID NO:2) and a Xba I digestion site.

[0272] Fra...

example 2

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

[0277] 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.

[0278] 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

Generation of a Stable CHO-K1 G-CSF Producer

[0280] The day before transfection the CHO K1 cell line (ATCC #CCl-61) is seeded in a T-25 flask in 5 ml DMEM / F-12 medium (Gibco # 31330-038) supplemented with 10% FBS and penicillin / streptomycin. The following day (at nearly 100% confluency) the transfection is prepared: 90 μl DMEM medium without supplements is aliquoted into a 14 ml polypropylene tube (Corning). 10 μl Fugene 6 (Roche) is added directly into the medium and incubated for 5 min at room temperature. In the meantime 5 μg plasmid pG-CSFCHO is aliquoted into another 14 ml polypropylene tube. After incubation the Fugene 6 mix is added directly to the DNA solution and incubated for 15 min at room temperature. After incubation the whole volume is added drop-wise to the cell medium.

[0281] The next day the medium is exchanged with fresh medium containing 360 μg / ml hygromycin (Gibco). Every day hereafter the selection medium is renewed until the primary transfection pool has reach...

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Abstract

Polypeptide conjugates with G-CSF activity comprising a polypeptide having at least one introduced lysine residue and at least one removed lysine residue compared to the sequence of human G-CSF, and which are conjugated to 2-6 polyethylene glycol moieties. The conjugates have a low in vitro bioactivity, a long in vivo half-life, a reduced receptor-mediated clearance, and provide a more rapid stimulation of production of white blood cells and neutrophils than non-conjugated recombinant human G-CSF.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application is a continuation-in-part of co-pending U.S. application Ser. No. 09 / 904,196 filed Jul. 11, 2001. Pursuant to 35 U.S.C. §119(a)-(d), this application also claims priority from and benefit of Danish Patent Application No. PA 2002 00447 filed Mar. 22, 2002, and Danish Patent Application No. PA 2002 00708 filed May 8, 2002. U.S. Ser. No. 09 / 904,196 is a continuation-in-part of U.S. application Ser. No. 09 / 760,008 filed Jan. 10, 2001. U.S. Ser. No. 09 / 760,008 claims priority to and benefit of U.S. Provisional Application Ser. No. 60 / 176,376 filed Jan. 14, 2000, U.S. Provisional Application Ser. No. 60 / 189,506 filed Mar. 15, 2000, U.S. Provisional Application Ser. No. 60 / 215,644 filed Jun. 30, 2000, Danish Patent Application No. PA 2000 00024 filed Jan. 10, 2000, Danish Patent Application No. PA 2000 00341 filed Mar. 2, 2000, and Danish Patent Application No. PA 2000 00943 filed Jun. 16, 2000. The disclosure of each applicat...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K38/00A61K47/42C07K14/53C07K14/535
CPCA61K9/0019A61K38/00C07K14/535C07K14/53A61K47/42
Inventor NISSEN, TORBENANDERSEN, KIMHANSEN, CHRISTIANMIKKELSEN, JANSCHAMBYE, HANS
Owner MAXYGEN HLDG
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