Polymer-modified synthetic proteins
First Claim
1. A composition comprising an isolated synthetic bioactive protein having the formula
Protein-hkUkPolymer-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 h of one or more internal amino acids of one or more of said non-overlapping peptide segments, where k is a discrete integer selected from 1 to 6,Polymer is a substantially non-antigenic water-soluble polymer, andJ* is a pendant group having a net charge under physiological conditions selected from the group consisting of negative, neutral and positive, andwherein the composition is uniform with respect to the attachment sites of the Polymer to the Protein;
- andwherein said synthetic bioactive protein comprises a monomer molecular weight of greater than 25,000 Daltons.
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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 (FIG. 1D), and of pharmaceutical compositions that contain such proteins.
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Citations
62 Claims
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1. A composition comprising an isolated synthetic bioactive protein having the formula
Protein-hkUkPolymer-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 h of one or more internal amino acids of one or more of said non-overlapping peptide segments, where k is a discrete integer selected from 1 to 6, 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, and wherein the composition is uniform with respect to the attachment sites of the Polymer to the Protein; - and
wherein said synthetic bioactive protein comprises a monomer molecular weight of greater than 25,000 Daltons. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24)
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- 25. A composition comprising 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 polypeptide molecular weight of greater than 25 kDa, wherein the composition is uniform with regard to the amino acid attachment sites of the polymer, and wherein the polymer is attached to at least one internal amino acid of the protein.
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28. A composition comprising 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, wherein said polypeptide chain together with said attached one or more water-soluble polymers has a molecular weight greater than 25 kDa, wherein the composition is uniform with regard to the amino acid attachment sites of the polymer, and wherein the polymer is attached to at least one internal amino acid of the protein, and wherein said synthetic bioactive protein possesses a bioactivity that mimics a bioactivity associated with a cytokine selected from the group consisting of an erythropoiesis stimulating protein, Rantes, and granulocyte colony stimulating factor.
- 42. 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 a polymer-modified bioactive protein having a monomer polypeptide molecular weight greater than about 25 kDa, wherein the polymer-modified bioactive protein 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, wherein the composition is uniform with regard to the attachment sites of the polymer to the protein, and wherein the polymer is attached to at least one internal amino acid of the protein.
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45. A method of producing a polymer-modified polypeptide chain of a synthetic bioactive polymer-modified protein, said method comprising
providing 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 comprises an irregular amino acid having a first chemoselective functional group attached thereto, and reacting said irregular amino acid with a water-soluble polymer having a second chemoselective functional group attached thereto, wherein the second chemoselective group is uniquely and mutually reactive with the first chemoselective group, to obtain at least one of said peptide segments having a polymer attached thereto, chemically ligating said peptide segments to obtain a polymer-modified polypeptide chain of said synthetic bioactive polymer-modified protein.
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55. A method of producing a polymer-modified polypeptide chain of a synthetic bioactive polymer-modified protein, said method comprising:
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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 an irregular amino acid having a first unique chemoselective group attached thereto; 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. - View Dependent Claims (56)
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57. A method of producing a synthetic bioactive polymer-modified protein, said method comprising
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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58. A molecularly homogeneous water-soluble polymer of the formula:
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-U-s1-B-s2-Polymer-s3-J*wherein; U is a residue of a unique functional group covalently bonded to a mutually reactive unique functional group of a side chain of an internal amino acid of a peptide, B comprises a branching core joined to Polymer through amide bonds, and Y is —
(CH2—
(CH2—
CH2—
O)3—
CH2—
CH2—
CH2)z—
or —
(CH2—
CH2—
CH2—
(O—
CH2—
CH2)3—
CH2)z, where z is 6 to 36,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 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 (59, 60, 61, 62)
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Specification