Polymer-modified synthetic proteins

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.

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.  
     
     
         2 . The synthetic bioactive protein of  claim 1 , wherein said synthetic bioactive protein: 
 (A) comprises a monomer molecular weight of greater than 25,000 Daltons;    (B) possesses a bioactivity that mimics a bioactivity associated with said ribosomally specified protein;    (C) is a cytokine, especially a cytokine selected from the group consisting of an interleukin, an erythropoiesis stimulating protein, an interferon, a lymphokine, a chemokine, a growth factor, a colony stimulating factor, and a signal peptide hormone;    (D) comprises a ribosomally specified glycosylation site, especially one wherein U is covalently bonded to said side chain n at a site of said polypeptide chain corresponding to a ribosomally specified glycosylation site or exclusively at a site corresponding to said ribosomally specified glycosylation site, wherein said side chain n is optionally a non-natural side chain;    (E) is a recombinantly produced natural protein or a non-natural protein optionally having: (a) one or more non-natural ribosomally specified glycosylation sites, (b) one or more irregular amino acids, optionally having a side chain group other than that of one of the 20 genetically encoded amino acids, and wherein optionally said irregular amino acid is optionally selected from the group consisting of a pseudo amino acid and a hydrophobic amino acid derivative;    (F) has one or more chemical ligation sites that comprise a bond selected from the group consisting of amide, oxime, hydrazone, thioester, thiazolidine, oxazolidine and thioether; or    (G) has U covalently bonded to said side chain n through a bond formed by chemical ligation, wherein said bond formed by chemical ligation is optionally selected from the group consisting of amide, oxime, hydrazone, thioester, thiazolidine, and oxazolidine.    
     
     
         3 . The synthetic bioactive protein of  claim 1 , wherein: 
 (A) one or more of U, B, Polymer and J* are separated by a spacer or linker, and having the formula U-s1-B-s2-Polymer-s3-J*, where s1, s2, and s3 are spacer or linker moieties that may be the same or different, and may be individually present or absent;    (B) U is a residue of a bond selected from oxime, amide, amine, urethane, ether, thioether, ester, thioester, hydrazide, oxazolidine, and thiazolidine;    (C) one arm of B is joined to U, and a second arm of B is joined to Polymer;    (D) B comprises four or more arms;    (E) at least one of said arms comprises a residue of bond selected from the group consisting of oxime, amide, amine, urethane, thioether, ester, thioester, hydrazide, oxazolidine, and thiazolidine;    (F) B comprises a branching core selected from the group consisting of amino, carboxylate and mixed amino-carboxylate, and said amino branching core optionally comprises lysine, said carboxylate branching core comprises glutamic or aspartic acid, and said mixed amino-carboxylate branching core comprises gamma-glutamic acid;    (G) Polymer is selected from the group consisting of polyalkylene oxide, polyamide alkylene oxide and derivatives thereof;    (H) said side chain n is at a chemical ligation site of said polypeptide chain;    (I) B-Polymer-J* comprises a molecular weight of between 1,500 and 80,000 Daltons;    (J) side chain n is 2 or more, and wherein one or more of B-Polymer-J* is different;    (K) B-Polymer-J* comprises a net charge under physiological conditions that is selected from the group consisting of negative, neutral and positive;    (L) B-Polymer-J* is mono-disperse; or    (M) said synthetic bioactive protein is mono-disperse.    
     
     
         4 . The synthetic bioactive protein of  claim 1 , wherein Polymer is a polyamide 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 an integer from 2 to 100, and where either or both of X and Y comprises a water-soluble repeat unit that may be linear or branched, and wherein: 
 (A) said X is optionally a divalent radical of the formula —NH—X′— or —X′—NH—, where X′ is a divalent radical comprising a water-soluble repeat unit that may be linear or branched;    (B) said Y is optionally a divalent radical of the formula —CO—Y′— or —Y′—CO—, where Y′ is a divalent radical;    (C) n is optionally a discrete integer from 2 to 50; or    (D) said water-soluble repeat unit optionally comprises an ethylene oxide repeat unit of the formula: 
 (a) —(CH 2 —CH 2 —O)— or —(O—CH 2 —CH 2 )—;  
 (b) —(CH 2 —CH 2 —O) n — or —(O—CH 2 —CH 2 ) n —, where n is a discrete integer from 2 to 50.  
   
     
     
         5 . The synthetic bioactive protein of  claim 1 , wherein J is: 
 (A) an ionizable group having a net charge under physiological conditions selected from the group consisting of negative and positive, said ionizable group being optionally selected from the group consisting of carboxyl, amine, and hydroxyl; or    (B) a non-ionizable group having a net neutral charge under physiological conditions.    
     
     
         6 . The synthetic bioactive protein of  claim 1 , having an increased circulating half-life in a mammal of greater compared to a corresponding synthetic bioactive protein that is devoid of said water-soluble polymer.  
     
     
         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.  
     
     
         8 . The synthetic bioactive protein of  claim 7 , wherein said synthetic bioactive protein: 
 (A) possesses a bioactivity that mimics a bioactivity associated with said ribosomally specified protein;    (B) is a cytokine, especially a cytokine selected from the group consisting of an erythropoiesis stimulating protein, Rantes, and granulocyte colony stimulating factor;    (C) comprises one or more glycosylation sites;    (D) is attached to said polypeptide chain at one or more sites corresponding to said one or more glycosylation sites, wherein said water-soluble polymer is optionally attached to said polypeptide chain exclusively at one or more sites corresponding to said one or more glycosylation sites;    (E) is a recombinantly produced natural or non-natural glycoprotein having one or more non-natural glycosylation sites or one or more irregular amino acids;    (F) has a water-soluble polymer that comprises a molecular weight of between 1,500 and 80,000 Daltons; or    (G) has water-soluble polymers that comprise a net charge under physiological conditions that is selected from the group consisting of negative, neutral and positive.    
     
     
         9 . The synthetic bioactive protein of  claim 7 , wherein: 
 (A) Polymer is selected from the group consisting of polyalkylene oxide, polyamide alkylene oxide and derivatives thereof;    (B) Polymer is a polyamide 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 an integer from 2 to 100, or from 2-50 and where either or both of X and Y comprises a water-soluble repeat unit that may be linear or branched;    (C) said water-soluble repeat unit comprises an ethylene oxide repeat unit of the formula: —(CH 2 —CH 2 —O)— or —(O—CH 2 —CH 2 )—.    
     
     
         10 . A pharmaceutical composition comprising a synthetic bioactive protein according to  claim 1 , or a pharmaceutically acceptable salt thereof, wherein said composition optionally comprises: 
 (A) one or more excipients selected from the group consisting of a buffer, a carrier protein, an amino acid, a detergent, a lipid, a water soluble polymer, and a preservative;    (B) one or more additional bioactive agents other than said synthetic bioactive protein.    
     
     
         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.  
     
     
         12 . The method of  claim 11 , wherein said synthetic bioactive protein has a bioactivity of a cytokine, wherein said cytokine is optionally selected from the group consisting of an interleukin, an erythropoiesis stimulating protein, an interferon, a lymphokine, a chemokine, a growth factor, a colony stimulating factor, and a signal peptide hormone;  
     
     
         13 . The pharmaceutical composition of  claim 10  for use in treating a human disease or condition associated with a bioactivity or lack thereof of a natural human protein receptor or fragment thereof, protein receptor ligand or fragment thereof, or a cytokine; wherein said polymer-modified synthetic bioactive protein optionally: 
 (A) has a bioactivity of a cytokine; or    (B) is selected from the group consisting of an interleukin, an erythropoiesis stimulating protein, an interferon, a lymphokine, a chemokine, a growth factor, a colony stimulating factor, and a signal peptide hormone.    
     
     
         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.  
     
     
         15 . The method of  claim 14 , wherein: 
 (A) said polypeptide chain comprises an amino acid sequence of a ribosomally specified protein;    (B) said ribosomally specified protein is a mammalian protein, optionally: (1) a cytokine, or (2) an interleukin, an erythropoiesis stimulating protein, an interferon, a lymphokine, a chemokine, a growth factor, a colony stimulating factor, and a signal peptide hormone;    (C) said method which further comprises folding said polymer-modified polypeptide chain whereby a synthetic bioactive polymer-modified protein is produced;    (D) one or more of said peptide segments are partially protected    (E) one or more of said peptide segments are unprotected;    (F) one or more of said peptide segments comprises one or more irregular amino acids;    (G) said chemically ligating comprises a chemoselective ligation chemistry selected from the group consisting of native chemical ligation, extended native chemical ligation and pseudo native chemical ligation;    (H) said water-soluble polymer comprises a molecular weight of between 1,500 and 80,000 Daltons.    
     
     
         16 . A method of producing a polymer-modified polypeptide chain of a synthetic bioactive polymer-modified protein, said method comprising the steps: 
 (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.    
     
     
         17 . A method of producing a synthetic bioactive polymer-modified protein, said method comprising the steps: 
 (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.    
     
     
         18 . A molecularly homogeneous water-soluble polymer of the formula:  
         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.  
     
     
         19 . The water-soluble polymer of  claim 18 , wherein: 
 (A) U is a residue of a functional group selected from the group consisting of acrylate, aldehyde, ketone, aminooxy, amine, carboxylic acid, ester, thioester, halogen, thiol, cyanoacetate, dipalmitoyl phosphatidylethanolamine, distearoyl phosphatidylethanolamine, epoxide, hydrazide, azide, isocyanate, maleimide, methacrylate, nitrophenyl carbonate, orthopyridyl disulfide, silane, sulfhydryl, vinyl sulfone, succinimidyl glutarate, succimidyl succinate, succinic acid, tresylate and an activatable functional group;    (B) U is a residue of a functional group capable of forming a bond selected from the group consisting of carbonate, ester, urethane, orthoester, amide, amine, oxime, imide, urea, thiourea, thioether, thiourethane, thioester, ether, thiazolidine, hydrazone, and oxazolidine;    (C) U is protected;\   (D) one or more of s1, s2 and s3 are present and are selected comprise from 1 to 18 carbons;    (E) one or more of s1, s2 and s3 are present and are selected and comprise Polymer;    (F) s1 comprise Polymer;    (G) B comprises four or more arms;    (H) said molecular weight is greater than about 10 kDa;    (I) said molecular weight is greater than about 15 kDa;    (J) B comprises a branching core joined to s2 or Polymer through one or more amide bonds;    (K) B comprises a branching core joined to Polymer through amide bonds.    
     
     
         20 . The water-soluble polymer of  claim 18 , wherein: 
 (A) said repeat unit comprises an ethylene oxide of the formula —(CH 2 —CH 2 —O)— or —(CH 2 —CH 2 —O)—;    (B) X, Y, or X and Y are selected from: —((CH 2 ) n1 —(CH 2 —CH 2 —O) n2 —(CH 2 ) n1 —)— or —(CH 2 ) n1 —(O—CH 2 —CH 2 ) n2 —(CH 2 ) n1 —), where n1 is a discrete integer from 1 to 6, and n2 is a discrete integer from 2 to 50;    (C) one of X or Y is selected from the group consisting of phenyl, a C 1 -C 10  alkylene moiety, a C 1 -C 10  alkyl group, a heteroatom-containing phenyl, a heteroatom-containing C 1 -C 10  alkylene moiety, a heteroatom-containing C 1 -C 10  alkyl group, and a combination thereof;    (D) X is —(CH 2 —CH 2 )—; and Y is optionally —(CH 2 —(CH 2 —CH 2 —O) 3 —CH 2 —CH 2 —CH 2 ) n — or —(CH 2 —CH 2 —CH 2 —(O—CH 2 —CH 2 ) 3 —CH 2 )-n, where n is 6 to 36;    (E) J* is protected;    (F) J* comprises a non-ionizable group having a net neutral charge under physiological conditions;    (G) J* comprises an ionizable group that comprises a net positive charge under physiological conditions;    (H) J* comprises an ionizable group that comprises a net negative charge under physiological conditions;    (I) X is —X′—NH— or —NH—X′—;    (J) Y is —Y′—NH— or —NH—Y′—, and optionally wherein at least one of X′ and Y′ comprise a polyethylene oxide of formula (—CH2-CH 2 —O—) n  or (O—CH 2 —CH 2 -) n  where n is 2 to 50; or    (K) said water-soluble polymer is mono-disperse.