Glycoproteins produced in plants and methods of their use

Abstract

Methods of increasing the yield in plant expression of recombinant proteins comprising: engineering glycosylation sites into cloned genes or cDNAs for proteins using codons that drive post-translational modifications in plants; and engineering the cloned genes or cDNAs to contain a plant secretory signal sequence that targets the gene products (protein) for secretion. The methods result in increased recombinant glycosylated protein yields. Proteins produced according to these methods are disclosed.

1 . An injectable pharmaceutical formulation comprising at least one O-hydroxyproline glycosylated glycomodule covalently linked to human growth hormone, and excluding at least one excipient chosen from mannitol, sorbitol, trehalose, glucose, glycine, leucine, trileucine, histidine, and phospholipid, wherein the at least one O-hydroxyproline glycosylated glycomodule that is covalently linked to human growth hormone exhibits a pharmacokinetic plasma half-life that is increased as compared with human growth hormone that lacks a covalently linked O-hydroxyproline glycosylated glycomodule. 
     
     
         2 . The injectable pharmaceutical formulation according to  claim 1 , wherein the at least one O-hydroxyproline glycosylated glycomodule comprises (Ser-Hyp) n , where n is from 2 to 1000. 
     
     
         3 . The injectable pharmaceutical formulation according to  claim 2 , wherein the at least one O-hydroxyproline glycosylated glycomodule comprises (Ser-Hyp) n , where n is from 6 to 100. 
     
     
         4 . The injectable pharmaceutical formulation according to  claim 3 , wherein the at least one O-hydroxyproline glycosylated glycomodule comprises (Ser-Hyp) n , where n is from 6 to 20. 
     
     
         5 . A lyophilized powder formulation of glycosylated human growth hormone exhibiting a solubility of greater than or equal to about 10 mg/ml, wherein the formulation excludes at least one excipient chosen from mannitol, sorbitol, trehalose, glucose, glycine, leucine, trileucine, histidine, and phospholipid. 
     
     
         6 . The lyophilized powder formulation according to  claim 5 , wherein the formulation excludes mannitol, sorbitol, trehalose, glucose, glycine, leucine, trileucine, and phospholipid. 
     
     
         7 . A human growth hormone antagonist molecule covalently attached to an amino acid sequence comprising at least one glycomodule, wherein the glycomodule is chosen from i) X-Hyp n  (SEQ ID NO: 169) or X-Pro-Hyp n  (SEQ ID NO: 170), where n is from 4 to about 100, ii) Hyp n -X (SEQ ID NO: 171), where n is from 4 to about 100, iii) (Hyp-X) n  (SEQ ID NO: 172), where n is from 4 to about 100, and iv) (X-Hyp) n  (SEQ ID NO: 173), where n is from 4 to about 100; wherein X is any amino acid in the glycomodule X-Pro-Hyp n , and wherein X is chosen from Ser, Ala, Thr, and Val for the glycomodules X-Hyp n , Hyp n -X, (Hyp-X) n , and (X-Hyp) n . 
     
     
         8 . A method of treating a patient suffering from growth hormone deficiency or insufficiency comprising administering a therapeutically effective amount of glycosylated human growth hormone. 
     
     
         9 . The method of treating a patient according to  claim 8 , wherein the growth hormone is a human growth hormone molecule covalently attached to an amino acid sequence comprising at least one glycomodule, wherein the glycomodule is chosen from i) X-Hyp n  (SEQ ID NO: 169) or X-Pro-Hyp n  (SEQ ID NO: 170), where n is from 4 to about 100, ii) Hyp n -X (SEQ ID NO: 171), where n is from 4 to about 100, iii) (Hyp-X) n  (SEQ ID NO: 172), where n is from 4 to about 100, and iv) (X-Hyp) n  (SEQ ID NO: 173), where n is from 4 to about 100; wherein X is any amino acid in the glycomodule X-Pro-Hyp n , and wherein X is chosen from Ser, Ala, Thr, and Val for the glycomodules X-Hyp n , Hypn-X, (Hyp-X) n , and (X-Hyp) n . 
     
     
         10 . A method of treating a patient suffering from excess human growth hormone or growth hormone activity comprising administering a therapeutically effective amount of glycosylated growth hormone antagonist. 
     
     
         11 . The method of treating a patient according to  claim 10 , wherein the growth hormone is according to  claim 8 . 
     
     
         12 . A method of treating a patient suffering from type I or type II diabetes comprising administering a therapeutically effective amount of glycosylated insulin. 
     
     
         13 . The method of treating a patient according to  claim 12 , wherein the glycosylated insulin comprises at least one glycomodule chosen from i) X-Hyp n  (SEQ ID NO: 169) or X-Pro-Hyp n  (SEQ ID NO: 170), where n is from 4 to about 100, ii) Hyp n -X (SEQ ID NO: 171), where n is from 4 to about 100, iii) (Hyp-X) n  (SEQ ID NO: 172), where n is from 4 to about 100, and iv) (X-Hyp) n  (SEQ ID NO: 173), where n is from 4 to about 100; wherein X is any amino acid in the glycomodule X-Pro-Hyp n , and wherein X is chosen from Ser, Ala, Thr, and Val for the glycomodules X-Hyp n , Hyp n -X, (Hyp-X) n , and (X-Hyp) n . 
     
     
         14 . The injectable pharmaceutical formulation according to  claim 1 , wherein the at least one O-hydroxyproline glycosylated glycomodule covalently linked to human growth hormone exhibits an increased bioavailability as compared to wild-type human growth hormone that lacks a covalently linked O-hydroxyproline glycosylated glycomodule. 
     
     
         15 . The injectable pharmaceutical formulation according to  claim 14 , wherein the at least one O-hydroxyproline glycosylated glycomodule comprises (Ser-Hyp) n , where n is from 2 to 1000. 
     
     
         16 . The injectable pharmaceutical formulation according to  claim 15 , wherein the at least one O-hydroxyproline glycosylated glycomodule comprises (Ser-Hyp) n , where n is from 6 to 100. 
     
     
         17 . The injectable pharmaceutical formulation according to  claim 16 , wherein the at least one O-hydroxyproline glycosylated glycomodule comprises (Ser-Hyp) n , where n is from 6 to 20. 
     
     
         18 . The injectable pharmaceutical formulation according to  claim 1 , wherein the at least one O-hydroxyproline glycosylated glycomodule covalently linked to human growth hormone exhibits immunogenicity that is not greater than that of wild-type human growth hormone that lacks a covalently linked O-hydroxyproline glycosylated glycomodule. 
     
     
         19 . The injectable pharmaceutical formulation according to  claim 18 , wherein the at least one O-hydroxyproline glycosylated glycomodule comprises (Ser-Hyp) n , where n is from 2 to 1000. 
     
     
         20 . The injectable pharmaceutical formulation according to  claim 19 , wherein the at least one O-hydroxyproline glycosylated glycomodule comprises (Ser-Hyp) n , where n is from 6 to 100. 
     
     
         21 . The injectable pharmaceutical formulation according to  claim 20 , wherein the at least one O-hydroxyproline glycosylated glycomodule comprises (Ser-Hyp) n , where n is from 6 to 20. 
     
     
         22 . An injectable pharmaceutical formulation comprising at least one O-hydroxyproline glycosylated glycomodule covalently linked to human growth hormone, and excluding at least one excipient chosen from mannitol, sorbitol, trehalose, glucose, glycine, leucine, trileucine, histidine, and phospholipid, wherein the at least one O-hydroxyproline glycosylated glycomodule that is covalently linked to human growth hormone exhibits increased bioavailability as compared with wild-type human growth hormone that lacks a covalently linked O-hydroxyproline glycosylated glycomodule. 
     
     
         23 . An injectable pharmaceutical formulation comprising at least one O-hydroxyproline glycosylated glycomodule covalently linked to human growth hormone, and excluding at least one excipient chosen from mannitol, sorbitol, trehalose, glucose, glycine, leucine, trileucine, histidine, and phospholipid, wherein the at least one O-hydroxyproline glycosylated glycomodule that is covalently linked to human growth hormone exhibits immunogenicity that is not greater than that of wild-type human growth hormone that lacks a covalently linked O-hydroxyproline glycosylated glycomodule. 
     
     
         24 . An injectable pharmaceutical formulation comprising at least one O-hydroxyproline glycosylated glycomodule covalently linked to human growth hormone, and excluding at least one excipient chosen from mannitol, sorbitol, trehalose, glucose, glycine, leucine, trileucine, histidine, and phospholipid, wherein the at least one O-hydroxyproline glycosylated glycomodule that is covalently linked to human growth hormone exhibits a pharmacokinetic plasma half-life and bioavailability that is increased as compared with wild-type human growth hormone that lacks a covalently linked O-hydroxyproline glycosylated glycomodule, and immunogenicity that is not greater than wild-type human growth hormone that lacks a covalently linked O-hydroxyproline glycosylated glycomodule.