The present invention relates to a conjugate of a biocompatible polymer and a G-CSF bonded through a thiol group of a dysteine residue in G-CSF at a 1:1 molar ratio, and methods of preparation thereof.

Description of the Invention

SUMMARY OF THE INVENTION

The present invention provides biocompatible polymer-G-CSF conjugates with 1:1 molar ratio and methods for preparation thereof.

These conjugates provide more improved stability and biological activity compared to G-CSF conjugates with several PEGs attached.

The G-CSF conjugates of the present invention provide a homogeneous product and a further purification process using the preparative column is not necessary.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the conjugates of biocompatible polymer and G-CSF bonded through a thiol group of a cysteine residue in G-CSF at a 1:1 molar ratio and methods of preparation thereof.

G-CSF

G-CSF, granulocyte colony stimulating factor, of the present invention is an important factor regulating proliferation and differentiation of hematopoietic progenitor cells (Welte et al. PNAS-USA 82: 1526-1530 (1985); Souza et al. Science 232: 61-65 (1986) and Gabrilove, J. Seminars in Hematology 26:2 1-14, 1989). In addition, G-CSF stimulates the release of mature neutrophils from bone marrow and activates their fintional states. Intracellular human G-CSF is found in the plasma (Jones et al. Bailliere's Clinical Hematology 2:1 83-111, 1989). In general, human G-CSF is produced by fibroblasts, macrophages, T cell trophoblasts, endothelial cells and epithelial cells, and is the expressed product of a single copy gene consisting of 4 exons and 5 introns in chromosome 17. It was shown that alternative splicing produced two different cDNAs that encode polypeptides of 177 amino acids and 174 amino acids (Nagata et al. EMBO J. 5: 575-581 (1986)). The Polypeptide consisting of 174 amino acids was found to possess the most characteristic biological activity. G-CSF has interspecies cross-reactivity, in other words, when G-CSF is administered to mouse, canine or other mammal such as monkey, the continuous increase of neutrophils is detected (Moore et al. PNAS-USA 84: 7134-7138, 1987). Nakata et al. reported that a human squamous carcinoma cell line (CHU-II) from human oral cavity tumor constitutively produces large quantities of G-CSF.

They isolated several clones containing G-CSF complementary DNA from the cDNA library prepared with messenger RNA from CHU-II cells. The complete nucleotide sequences of two of these cDNAs were determined and the expression of the cDNA in monkey COS cells yielded a protein showing authentic G-CSF activity (Nagata et al,. Nature, 319, 415 (1986)). Also, Sauza et al isolated cDNA of G-CSF from human bladder carcinoma cell 5637, determined the complete nucleotide sequences thereof and expressed it in E. coli (Souza et al., Science 232, 61 (1986)).

In general, G-CSF which can be used in the present invention is preferably wild type G-CSF. Also, G-CSF which has the same amino acid sequence with wild type can be preferably used. G-CSF of the present invention is the form isolated from mammalian animals, the product of organic synthesis, or the expressed product of host cell or nucleotide DNA sequences obtained by genomic and cDNA cloning or recombinant DNA. Suitable host cells include various bacteria such as E. coli, yeast such as S. cerevisiae, and mammalian cells such as CHO cells and monkey cells. Depending on host cells used, the expressed product of G-CSF is produced in glycosylated form with mammalian or other eukaryotic carbohydrates, or in non-glycosylated form. Also, the expressed product of G-CSF includes a methionine residue at position 1. The present invention includes all kinds of G-CSF, although recombinant G-CSF, especially from E. coli, is economically preferable.

It has been reported that certain G-CSF variants show biological activity. For example. G-CSF variants are described in U.S. Pat. No. 4,810,643. Other examples are described in AU-A-76380/91, EP 0,459,630, EP 0,272,703, EP 0,473,268, EP 0,335,423, AU-A-10948/92, PCT US 94/00913 and EP 0,243,153, although the activities of each variant are not described. G-CSF and their variants can be obtained from the various sources and purified for use. For example, natural human G-CSF is separated from the human carcinoma cell line culture. Also non-human G-CSF from recombinant mouse, bovine, or dog can be also used. (WO 9,105,798 and WO 8,910,932). Preferably, G-CSF of the present invention is a G-CSF having the same number of cysteine residues as wild type G-CSF.

Biocompatible Polymers to Conjugate to G-CSF

The most preferable polymer of the present invention is PEG. In general, PEG is a hydrophilic polymer and known as polyethylene oxide, and its chemical conjugation with molecules or surfaces is commonly performed. The common structure of PEG is that of a linear molecule having hydroxyl groups at both ends and expressed as follows: HO--CH.sub.2CH.sub.2O--(CH.sub.2CH.sub.2O)n-CH.sub.2CH.sub.2--OH, or HO-PEG-OH.

Biocompatible polymers of the present invention are intended to include not only linear polymers but also polymers as follows. Biocompatible polymers of the present invention include soluble, non-antigenic polymers linked to an activated functional group that is capable of being nucleophilically substituted through an aliphatic linker residue (U.S. Pat. Nos. 5,643,575 and 5,919,455). Biocompatible polymers of the present invention also include multi-armed, mono-functional and hydrolytically stable polymers, having two linker fragments which have polymer arms in central carbon atom, a residue which is capable of being activated for attachment to biologically active materials such as proteins, and side chains which can be hydrogen or methyl group, or other linker fragment (U.S. Pat. No. 5,932,462). In addition, biocompatible polymers of the present invention also include polymers of branched PEG in which the functional groups such as PEGs are attached to biologically active materials via linker arms having reporter residues (WO 00/33881).

Also, biocompatible polymers of the present invention include biocompatible polymers having the structural formula PEI-P-A, wherein PEI is ethyleneimine, P is a biocompatible polymer and A is a reactive functional group or methoxy (Korean patent application No. 10-2001-0074728). In this Korean patent application, the reactive functional group (A) includes (I) functional groups able to react with amino group, for example, carbonate (for example, p-nitrophenyl or succinimidyl), carbonyl imidazole, azlactone, cyclic imide thione and isocyanate or isothiocyanate; (II) functional groups able to react with carboxylic acid and reactive carbonyl group, for example, primary amine, or hydrazine and hydrazide functional group (such as acyl hydrazide, carbazate, semicarbazate, thiocarbazate etc.); (III) functional groups able to react with mercapto or sulfhydryl group, for example phenyl glyoxal; (IV) functional groups able to react with hydroxyl group, for example carboxylic acid; and (V) other nucleophiles able to react with electrophilic centers.

Also, biocompatible polymers of the present invention include activated biocompatible polymers with a peptide spacer having structural formula [P--OCH.sub.2CO--Y].sub.n-(L).sub.s-(Q).sub.t-(Y').sub.k-A, wherein P and Q are the same or different, and independently a biocompatible polymer, t is 0 or 1, Y and Y' are the same or different, and each is a peptide consisting of 2 to 18 amino acids in any combination, k is an integer of 0 or 1, L is an aliphatic linker residue or diaminocarboxylic acid, s is 0 or 1, A is a reactive functional group, and n is 1 or 2 (Korean patent application No. 10-2001-0067369). In this Korean patent application, the reactive functional group (A) includes (I) functional groups able to react with amino groups, for example, carbonate (for example, p-nitrophenyl or succinimidyl), carbonyl imidazole, azlactone, cyclic imide thione, or isocyanate or isothiocyanate; (II) functional groups able to react with carboxylic acid and reactive carbonyl groups, for example, primary amine, or hydrazine and hydrazide functional groups (such as acyl hydrazide, carbazate, semicarbazate, thiocarbazate etc.); (III) functional groups able to react with mercapto or sulfhydryl groups, for example phenyl glyoxal; and (IV) functional groups able to react with hydroxyl groups, for example (carboxylic) acid, for example hydroxyl, amino, carboxyl, thiol group, and active methylene etc.

Preferred biocompatible polymers of the present invention include, but are not limited to, polyethylene glycol (PEG) and derivatives thereof, polypropylene glycol (PPG), polyoxyethylene (POE), polytrimethylene glycol, polylactic acid and derivatives thereof, polyacrylic acid and derivatives thereof, polyamino acid, poly (L-lysine), polyurethane, polyphosphazene, polyalkylene oxide (PAO), polysaccharide, dextran, polyvinyl pyrrolidone, polyvinyl alcohol (PVA), polyacrylamide and similar non-antigenic polymers. The polymers of the present invention have a molecular weight of between about 300 and 100,000 daltons and preferably between about 2,000 and 40,000 daltons.

Functional Group of Biocompatible Polymer

The biocompatible polymer needs to be activated to react with G-CSF through a thiol group of a cysteine residue of G-CSF by activating its functional group. The functional group is the activated group or moiety for linking to the biologically active materials. To conjugate the biologically active molecules to biocompatible polymers, one of the end groups of biocompatible polymers is converted to a reactive functional group suitable for conjugation. This process is referred to as "activation" and the product is called an "activated" polymer. For instance, to conjugate poly(alkylene oxides) to biologically active materials, one of hydroxyl end groups of the polymer is converted to a reactive functional group such as carbonate and the product is called an activated poly(alkylene oxide).

A preferred reactive functional group of the present invention includes, but is not limited to, maleimide, acetamide, pentenoic amide, butenoic amide, isocyanate, isothiocyanate, cyanuric chloride, 1,4-benzoquinone, and disulfide.

Preferable Conjugates of the Present Invention and Preparation Method Thereof

The preferable biocompatible polymer-G-CSF conjugate of the present invention relates to the conjugates of PEG and G-CSF through a thiol group of a cysteine residue of G-CSF at a 1:1 molar ratio. The conjugate with such as 1:1 binding ratio retains the biological activity of G-CSF and increased stability in vivo.

Preparation of biocompatible polymer-G-CSF conjugates through a thiol group of G-CSF.

The conjugation of PEG with a protein through thiol groups of a protein can be performed by a general method.

For example, a thiol group can bind to an activated double bond of PEG-maleimide by the Michael reaction (Ishii et al., Biophys J. 1986, 50:75-80). Another example of such reaction can be performed by using PEG-iodoacetoamide, as is well known in protein chemistry. This method has an advantage of producing a stable PEG-cysteine derivative, carboxymethyl cysteine, which is readily analyzed by amino acid analysis (Gard FRN. Carboxymethylation. Method Enzymol 1972; B25: 424-49). other examples are as follows: the method to obtain stable symmetric disulfide bonds using PEG-ortho-pyridyl-disulfide (Woghiren et al. Bioconjgate Chem 1993, 50:75-80); the method to react activated PEG, sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylated PEG, with a thiol groups of cysteine residues to form covalent bonds (U.S. Pat. No. 5,166,322); the method to react activated PEG, maleimido-6-aminocaproyl ester PEG4000 with substituted cysteine residues of IL-2 mutant by a mutagenesis method (U.S. Pat. No. 5,206,344); the method to chemically alter thiol groups of proteins using gamma-maleimido butyric acid and beta-maleimido propionic acid (Rich et al., J. med. Chem. 18, 1004, 1975) etc.

The present invention has used a method of reacting activated polymers, mPEG-Gly-Gly-maleimide, mPEG-maleimide and mPEG-chloroacetamide, respectively, with G-CSF through a thiol group of a cysteine residue of G-CSF. Briefly, G-CSF was reacted with mPEG(5000 or 20000)-Gly-Gly-maleimide, mPEG(5000 or 20000)-malemide or mPEG(5000 or 12000)-chloroacetamide to produce conjugates of biologically active material, G-CSF and biocompatible polymers, i.e., MPEG(5000 or 20000)-Gly-Gly-maleimide-G-CSF, mPEG(5000 or 20000)-maleimide-G-CSF and MPEG(5000 or 12000)-Gly-Gly-acetamide-G-CSF.

As mentioned hereinbefore, G-CSF is a protein consisting of 174 or 177 amino acids, having 5 cysteine residues in which four of them form disulfide bonds and only one is free. In contrast to conventional method in which PEG was conjugated to proteins through amino groups of the proteins with the result that several PEGs were conjugated to one protein, when biocompatible PEG is conjugated to G-CSF through the a thiol group of a cysteine residue of G-CSF according to the present invention, conjugates of PEG and G-CSF at a 1:1 molar ratio were obtained, i.e., one molecule of PEGs was reacted with a thiol group of only a free cysteine residue to produce PEG-conjugated G-CSF at a 1:1 molar ratio.

In general, the pH of reaction buffer for protein/peptide conjugation is preferably between 6 and 10. The suitable temperature for the conjugation reaction is in the range of 0 to 60.degree. C. and preferably in the range of 4 to 30.degree. C. Also, the reaction time of 5 minutes to 10 hours is preferable in this preparation.

The present inventors found that when PEG was conjugated to cysteine of G-CSF, aggregates having no biological activity of PEG-G-CSF were immediately formed. Therefore, the present inventors confirmed that treatment with a small amount of SDS, tween20, tween80 detergent etc. is necessary to prevent the aggregates from being formed. In other words, the present inventors confirmed that if not treated with any of such detergents, active PEG-G-CSF conjugates could not be obtained. In addition, if treated with reducing agent DTT instead of a detergent, the aggregates remained and any PEG-G-CSF conjugate was also inactive.

Therefore, to prevent aggregation, the present inventors have kept PEG-G-CSF conjugates of the present invention in cold storage with a small amount, i.e., 0.001% to 1%, preferably 0.003 to 0.5%, and more preferably 0.005% to 0.1% of SDS detergent.

In addition, the present inventors confirmed that SDS can be removed from PEG-G-CSF conjugates treated with SDS solution by ultra-filtration using centricon-30 (Milipore, USA) before administration in vivo. The present inventors also confirmed that after monomeric PEG-G-CSF conjugates (not forming aggregates) were isolated via treatment with SDS, such conjugates can be administrated in vivo without adding further SDS solution.
 

Claim 1 of 10 Claims

1. A conjugate of a biocompatible polymer and wild type G-CSF, wherein the activated biocompatible polymer is bonded to wild type G-CSF at a 1:1 molar ratio through a thiol group of a cysteine residue free of disulfide bonds corresponding to the position 17 (C17) of the mature human wild-type G-CSF.

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