Recombinant Glycoproteins and Uses Thereof

Abstract

Provided herein are recombinant glycoproteins (e.g., recombinant human α-galactosidase-A proteins) with an altered (e.g., improved) glycosylation profile, and pharmaceutical compositions and kits including one or more of these proteins. Also provided are methods of generating a mammalian cell useful for recombinant expression of a glycoprotein (e.g., recombinant human α-galactosidase-A), methods of producing recombinant glycoproteins, and methods of treatment that include administering to a subject at least one of the recombinant glycoproteins (e.g., recombinant human α-galactosidase-A protein).

1 - 109 . (canceled) 
     
     
         110 . An expression vector comprising:
 a sequence that encodes recombinant human α-galactosidase-A protein;   a promoter sequence operably linked to a 5′ end of the sequence encoding human α-galactosidase-A protein;   a sequence encoding a peptide of human CD52 protein with a TTG start codon operably linked to the 5′ end of the sequence encoding human α-galactosidase-A protein; and   a sequence encoding a poly(A) recognition site operably linked to a 3′ end of the sequence encoding human α-galactosidase-A protein.   
     
     
         111 . A recombinant human α-galactosidase-A (rhAGA) protein produced by a method comprising:
 (a) providing a Chinese hamster ovary (CHO) cell; 
 (b) generating a single-cell clone from the CHO cell wherein the single-cell clone grows in a protein-free, animal derived component free medium; 
 (c) stably transfecting the single-cell clone from step (b) with an expression vector comprising a sequence encoding the rhAGA glycoprotein; 
 (d) selecting a clone that produces rhAGA; 
 (e) culturing the clone in protein-free, animal derived component free medium; and 
 (f) harvesting the rhAGA protein from the CHO cell, 
 wherein the rhAGA protein comprises a percentage of total N-linked oligosaccharides that are bis-mannose-6-phosphorylated oligosaccharides greater than 9.0%. 
 
     
     
         112 . The rhAGA protein according to  claim 111 , wherein the sequence encoding the glycoprotein is at least 90% identical to SEQ ID NO: 1. 
     
     
         113 . The rhAGA protein of  claim 111 , wherein the amino acid sequence of the rhAGA protein comprises SEQ ID NO:3. 
     
     
         114 . The rhAGA protein of  claim 111 , wherein the amino acid sequence of the rhAGA protein consists of SEQ ID NO:3. 
     
     
         115 . The rhAGA protein of  claim 111 , wherein the amino acid sequence of the rhAGA protein consists of SEQ ID NO:2. 
     
     
         116 . A pharmaceutical composition comprising the rhAGA protein of  claim 111 , and a pharmaceutically acceptable carrier. 
     
     
         117 . The pharmaceutical composition of  claim 116 , wherein the composition is formulated for intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal administration. 
     
     
         118 . The pharmaceutical composition of  claim 117 , wherein the pharmaceutical composition comprises a concentration of 4 mg/mL to 6 mg/mL recombinant human α-galactosidase-A protein. 
     
     
         119 . The pharmaceutical composition of  claim 118 , wherein the pharmaceutical composition comprises a concentration of 5 mg/mL recombinant human α-galactosidase-A protein. 
     
     
         120 . The pharmaceutical composition of  claim 116 , wherein the pharmaceutical composition is a sterile, lyophilized powder. 
     
     
         121 . A recombinant human α-galactosidase-A (rhAGA) protein comprising:
 a percentage of total N-linked oligosaccharides that are bis-mannose-6-phosphorylated oligosaccharides that is greater than 9%; and 
 
       a mole/mole ratio of mannose-6-phosphate to protein that is between 1.8-3.0. 
     
     
         122 . The rhAGA protein of  claim 121 , wherein the rhAGA protein further comprises a percentage of total N-linked oligosaccharides that are monophosphosphorylated oligosaccharides that is greater than 14.8%. 
     
     
         123 . The rhAGA protein of  claim 121 , wherein the rhAGA protein further comprises a percentage of total N-linked oligosaccharides that are tetrasialylated oligosaccharides that is greater than 4.9%. 
     
     
         124 . The rhAGA protein of  claim 122 , wherein the rhAGA protein has a percentage of total N-linked oligosaccharides that are monophosphosphorylated oligosaccharides that is greater than 16%. 
     
     
         125 . The rhAGA protein of  claim 123 , wherein the rhAGA protein has a percentage of total N-linked oligosaccharides that are tetrasialylated oligosaccharides that is greater than 6%. 
     
     
         126 . A pharmaceutical composition comprising the rhAGA protein of  claim 121 , and a pharmaceutically acceptable carrier. 
     
     
         127 . The pharmaceutical composition of  claim 126 , wherein the composition is formulated for intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal administration. 
     
     
         128 . The pharmaceutical composition of  claim 127 , wherein the pharmaceutical composition comprises a concentration of rhAGA of 4 mg/mL to 6 mg/mL. 
     
     
         129 . The pharmaceutical composition of  claim 128 , wherein the pharmaceutical composition comprises a concentration of rhAGA of 5 mg/mL. 
     
     
         130 . The pharmaceutical composition of  claim 126 , wherein the pharmaceutical composition is a sterile, lyophilized powder. 
     
     
         131 . The pharmaceutical composition of  claim 126 , wherein the pharmaceutically acceptable carrier is one or more agents selected from the group consisting of: mannitol, sodium phosphate monobasic, monohydrate, sodium phosphate dibasic, and heptahydrate. 
     
     
         132 . A method of treating of Fabry disease in a subject, the method comprising administering to a subject having Fabry disease a therapeutically effective amount of rhAGA protein of  claim 121 .