Polymer conjugates with decreased antigenicity, methods of preparation and uses thereof

Abstract

Methods are provided for the preparation of conjugates of a variety of bioactive components, especially proteins, with water-soluble polymers (e.g., poly(ethylene glycol) and derivatives thereof), which conjugates have reduced antigenicity and immunogenicity compared to similar conjugates prepared using poly(ethylene glycol) containing a methoxyl or another alkoxyl group. The invention also provides conjugates prepared by such methods, compositions comprising such conjugates, kits containing such conjugates or compositions and methods of use of the conjugates and compositions in diagnostic and therapeutic protocols.

1 - 100 . (canceled) 
     
     
         101 . A method of reducing the immunoreactivity of a bioactive component, the method comprising conjugating the bioactive component to a linear or branched polyalkylene glycol, wherein the linear or branched polyalkylene glycol is attached to the bioactive component at a single attachment site on the linear or branched polyalkylene glycol, wherein 95% or more of the linear or branched polyalkylene glycol has an unsubstituted hydroxyl group at the terminal of the linear or termini of the branched polyalkylene glycol distal to the attachment site, and wherein the conjugated bioactive component has reduced immunoreactivity. 
     
     
         102 . The method of  claim 101 , wherein the bioactive component is a lipid. 
     
     
         103 . The method of  claim 101 , wherein the lipid is incorporated into a lipsome. 
     
     
         104 . The method of  claim 102 , wherein the lipid is phosphatidyl ethanolamine and the attachment site is the amino group of the phosphatidyl ethanolamine. 
     
     
         105 . The method of  claim 102 , wherein the lipid is diacylglycerol and the attachment site is the hydroxyl group of the diacylglycerol. 
     
     
         106 . The method of  claim 101 , wherein the bioactive component is a protein. 
     
     
         107 . The method of  claim 106 , wherein the protein is a glycoprotein. 
     
     
         108 . The method of  claim 101 , wherein the bioactive component is a peptide. 
     
     
         109 . The method of  claim 101 , wherein the bioactive component is an organic compound. 
     
     
         110 . The method of  claim 101 , wherein the bioactive component is a therapeutic virus. 
     
     
         111 . The method of  claim 101 , wherein the reduced immunoreactivity of the conjugated bioactive component is reduced compared to a second conjugated bioactive component comprising the same bioactive component linked at the same attachment site to a polyalkylene glycol of the same size and the same linear or branched structure having an alkoxyl or an aryloxyl group at the terminal of the linear or termini of the branched polyalkylene glycol distal to the attachment site. 
     
     
         112 . The method of  claim 101 , wherein the linear or branched polyalkylene glycol has a nominal molecular weight of from about 1,000 Daltons (1 kDa) to about 100,000 Daltons (100 kDa). 
     
     
         113 . The method of  claim 101 , wherein the linear or branched polyalkylene glycol is selected from the group consisting of a poly(ethylene glycol) and a copolymer of ethylene oxide and propylene oxide. 
     
     
         114 . The method of  claim 101 , wherein the linear or branched polyalkylene glycol is a poly(ethylene glycol) (“PEG”). 
     
     
         115 . The method of  claim 114 , wherein the conjugation of the PEG to the bioactive component is carried out using a monofunctionally activated derivative of a PEG selected from the group consisting of a linear dihydroxyPEG, a hydroxyPEG-monoacetal and a hydroxyPEG-monoacid. 
     
     
         116 . The method of  claim 101 , wherein the conjugating comprises the steps of:
 (a) obtaining a polyalkylene glycol that comprises only hydroxyl end groups;   (b) protecting all except one of the hydroxyl groups in the polyalkylene glycol by the addition of one or more removable blocking groups to obtain a protected monohydroxy polyalkylene glycol;   (c) producing a monofunctionally activated derivative of the protected polyalkylene glycol of (b) by reacting the protected polyalkylene glycol of (b) with a derivatizing compound or compounds under conditions such that the protected polyalkylene glycol of (b) is derivatized with a single derivatizing group at a hydroxyl group that does not contain the removable blocking group or groups of (b);   (d) purifying the monofunctionally activated derivative of (c);   (e) removing the blocking group or groups of (b) from the monofunctionally activated derivative of (d) without removing the derivatizing group attached in (c), to produce a monofunctionally activated polyalkylene glycol wherein the distal terminus is or distal termini are hydroxyl group(s); and   (f) contacting the monofunctionally activated polyalkylene glycol of (e) with the bioactive component under conditions that favor the covalent attachment of the monofunctionally activated polyalkylene glycol to the bioactive component.   
     
     
         117 . The method of  claim 116 , wherein the one or more removable blocking groups is or are selected from the group consisting of a t-butoxyl group, an aryloxyl group and a triphenylmethyl group. 
     
     
         118 . The method of  claim 116 , wherein the polyalkylene glycol of (a) is selected from the group consisting of a poly(ethylene glycol) and a copolymer of ethylene oxide and propylene oxide. 
     
     
         119 . The method of  claim 101 , wherein 98% or more of the linear or branched polyalkylene glycol has an unsubstituted hydroxyl group at the terminal of the linear or termini of the branched polyalkylene glycol distal to the attachment site. 
     
     
         120 . The method of  claim 101 , wherein 99% or more of the linear or branched polyalkylene glycol has an unsubstituted hydroxyl group at the terminal of the linear or termini of the branched polyalkylene glycol distal to the attachment site.