Polymer-modified synthetic proteins
First Claim
1. An isolated synthetic bioactive protein having the formula Protein-Un-B-Polymer-J*, wherein Protein comprises a polypeptide chain of a ribosomally specified protein, said polypeptide chain comprising one or more non-overlapping peptide segments covalently bonded by one or more chemical ligation sites, U is a residue of a unique functional group covalently bonded to a mutually reactive unique functional group of a side chain n of one or more amino acids of one or more of said non-overlapping peptide segments, where n is a discrete integer selected from 1 to 6, B is a branching core having three or more arms that may be the same or different and may be present or absent, Polymer is a substantially non-antigenic water-soluble polymer, and J* is a pendant group having a net charge under physiological conditions selected from the group consisting of negative, neutral and positive.
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Abstract
The present invention relates to methods and compositions for modifying peptides, polypeptides and proteins with polymers, especially glyco-mimetic polymers, so as to improve their biological activity or pharmacokinetic properties. The invention further provides methods and uses for such polymer-modified peptides, polypeptides and proteins. The invention is particularly suitable for use in the synthesis of polymer-modified, synthetic bioactive proteins, and of pharmaceutical compositions that contain such proteins.
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Citations
20 Claims
- 1. An isolated synthetic bioactive protein having the formula Protein-Un-B-Polymer-J*, wherein Protein comprises a polypeptide chain of a ribosomally specified protein, said polypeptide chain comprising one or more non-overlapping peptide segments covalently bonded by one or more chemical ligation sites, U is a residue of a unique functional group covalently bonded to a mutually reactive unique functional group of a side chain n of one or more amino acids of one or more of said non-overlapping peptide segments, where n is a discrete integer selected from 1 to 6, B is a branching core having three or more arms that may be the same or different and may be present or absent, Polymer is a substantially non-antigenic water-soluble polymer, and J* is a pendant group having a net charge under physiological conditions selected from the group consisting of negative, neutral and positive.
- 7. A synthetic bioactive protein comprising a polypeptide chain comprising an amino acid sequence of a ribosomally specified glycoprotein, said polypeptide chain having one or more water-soluble polymers attached thereto and a monomer molecular weight of greater than 25 kDa.
- 11. A method of treating a human disease or condition that comprises administering to an individual in need of such treatment an effective amount of a pharmaceutical composition comprising one or more molecularly homogeneous pharmaceutical compositions of a polymer-modified synthetic bioactive protein having a monomer molecular weight greater than about 25 kDa and that possesses a bioactivity that mimics a bioactivity associated with a natural human protein receptor or fragment thereof, protein receptor ligand or fragment thereof, or a cytokine.
- 14. A method of producing a polymer-modified polypeptide chain of a synthetic bioactive polymer-modified protein, said method comprising chemically ligating peptide segments comprising non-overlapping amino acid sequences of a polypeptide chain of said synthetic bioactive polymer-modified protein, wherein one or more of said peptide segments has a water-soluble polymer attached thereto, whereby a polymer-modified polypeptide chain of said synthetic bioactive polymer-modified protein is produced.
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16. A method of producing a polymer-modified polypeptide chain of a synthetic bioactive polymer-modified protein, said method comprising the steps:
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(A) chemically ligating peptide segments comprising non-overlapping amino acid sequences of said synthetic bioactive polymer-modified protein to form a full length polypeptide chain corresponding to the amino acid sequence of said synthetic bioactive polymer-modified protein, wherein one or more of said peptide segments comprises a first unique chemoselective group; and
(B) chemically ligating a water-soluble polymer to said full length polypeptide chain, said water-soluble polymer comprising a second chemoselective group that is mutually and uniquely reactive with said first unique chemoselective group, whereby a polymer-modified polypeptide chain of said synthetic bioactive polymer-modified protein is produced;
wherein said method optionally further comprises folding said polymer-modified polypeptide chain whereby a synthetic bioactive polymer-modified protein is produced.
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17. A method of producing a synthetic bioactive polymer-modified protein, said method comprising the steps:
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(A) chemically ligating peptide segments comprising non-overlapping amino acid sequences of said synthetic bioactive polymer-modified protein to form a full length polypeptide chain corresponding to the amino acid sequence of said synthetic bioactive polymer-modified protein, wherein one or more of said peptide segments comprises a non-native side chain comprising a first unique chemoselective group that is unreactive with a side chain functional group of an amino acid of a genetically encoded amino acid;
(B) folding said full length polypeptide chain to form a folded polypeptide chain; and
(C) chemically ligating a water-soluble polymer to said folded polypeptide chain, said water-soluble polymer comprising a second chemoselective group that is mutually and uniquely reactive with said first unique chemoselective group, whereby a polymer-modified polypeptide chain of said synthetic bioactive polymer-modified protein is produced.
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18. A molecularly homogeneous water-soluble polymer of the formula:
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U-s1-B-s2-Polymer-s3-J*where U is a residue of a unique functional group, B is a branching core having three or more arms that may be the same or different and may be present or absent, Polymer is a polyamide having a molecular weight greater than about 5,000 Da of the formula —
[C(O)—
X—
C(O)—
NH—
Y—
NH]n- or —
[NH—
Y—
NH—
C(O)—
X—
C(O)]n-, where X and Y are divalent radicals that may be the same or different and may be branched or linear, and n is a discrete integer from 2 to 50, and where either or both of X and Y comprises a substantially non-antigenic water-soluble repeat unit that may be linear or branched, J* is a residue of a substantially non-antigenic pendant group having a net charge under physiological conditions selected from the group consisting of negative, positive and neutral, and where s1, s2, and s3 are spacer or linker moieties that may be the same or different, and may be individually present or absent.- View Dependent Claims (19, 20)
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Specification