Compositions and methods for increasing serum half-life

Abstract

Provided herein are glycovariant Fc fusion proteins having increased serum half lives. Also provided are methods for increasing the serum half life of an Fc fusion protein by introducing one or more non-endogenous glycosylation sites.

1 - 33 . (canceled) 
     
     
         34 . A fusion protein comprising an immunoglobulin Fc domain and at least one heterologous polypeptide domain comprising an extracellular domain of a TNFR2 receptor, wherein the fusion protein is modified outside of the immunoglobulin Fc domain to introduce at least one non-endogenous N-linked glycosylation site, and wherein glycosylation at the one or more introduced glycosylation sites increases the serum half-life of the modified fusion protein by at least 10% relative to the serum half-life of the fusion protein lacking an introduced glycosylation site as measured in a pharmacokinetic monkey assay. 
     
     
         35 . (canceled) 
     
     
         36 . The fusion protein of  claim 34 , wherein the glycosylation site is introduced in the extracellular domain outside of the ligand binding pocket of the receptor. 
     
     
         37 . The fusion protein of  claim 34 , wherein glycosylation at the one or more introduced glycosylation sites does not affect ligand binding activity of the receptor by more than 3-fold. 
     
     
         38 . The fusion protein of  claim 34 , wherein the fusion protein is modified by addition or deletion of at least one amino acid residue to introduce at least one glycosylation site. 
     
     
         39 . The fusion protein of  claim 34 , wherein the fusion protein is modified by substitution of at least one amino acid residue to introduce at least one glycosylation site. 
     
     
         40 . The fusion protein of  claim 34 , wherein the molecular weight of the heterologous polypeptide domain is at least 25 kDa. 
     
     
         41 . The fusion protein of  claim 34 , wherein glycosylation at one or more of the introduced glycosylation sites increase the serum half-life of the fusion protein by at least 20% relative to the serum half-life of the fusion protein lacking an introduced glycosylation site. 
     
     
         42 . The fusion protein of  claim 34 , wherein the unmodified heterologous polypeptide domain comprises fewer than one N-linked glycosylation site per each 90 amino acids. 
     
     
         43 . The fusion protein of  claim 34 , wherein the unmodified heterologous polypeptide domain comprises fewer than one N-linked glycosylation site per each 125 amino acids. 
     
     
         44 . The fusion protein of  claim 34 , wherein the modified heterologous polypeptide comprises at least one N-linked glycosylation site per each 90 amino acids. 
     
     
         45 . The fusion protein of  claim 34 , wherein the modified heterologous polypeptide comprises at least one N-linked glycosylation site per each 65 amino acids. 
     
     
         46 . The fusion protein of  claim 34 , wherein each amino acid modified by an N-linked glycosylation is separated by at least 20 amino acids from any other amino acid modified by an N-linked glycosylation. 
     
     
         47 . The fusion protein of  claim 34 , wherein the fusion protein further comprises a polypeptide linker between the Fc domain and the heterologous polypeptide domain. 
     
     
         48 . The fusion protein of  claim 47 , wherein at least one of the introduced glycosylation sites is located in the linker. 
     
     
         49 . The fusion protein of  claim 34 , wherein the heterologous portion of the Fc fusion protein at least two structurally distinct α-helix and/or β-sheet domains that are connected by an unstructured polypeptide region that is surface exposed. 
     
     
         50 . The fusion protein of  claim 49 , wherein an introduced glycosylation site is positioned within the unstructured peptide region. 
     
     
         51 . (canceled) 
     
     
         52 . (canceled) 
     
     
         53 . The fusion protein of  claim 34 , wherein the extracellular domain of the TNFR2 receptor comprises an amino acid substitution at one or more amino acid residues selected from Q26, D25, A28, E133, or G231 of SEQ ID NO: 2. 
     
     
         54 . The fusion protein of  claim 53 , wherein the extracellular domain of the TNFR2 receptor comprises a Q to N substitution at amino acid 26 of SEQ ID NO: 2 and an A to S substitution at amino acid 28 of SEQ ID NO: 2. 
     
     
         55 . The fusion protein of  claim 53 , wherein the extracellular domain of the TNFR2 receptor comprises a D to N substitution at amino acid 25 of SEQ ID NO: 2 and an E to N substitution at amino acid 133 of SEQ ID NO: 2. 
     
     
         56 . The fusion protein of  claim 53 , wherein the extracellular domain of the TNFR2 receptor comprises a D to N substitution at amino acid 25 of SEQ ID NO: 2 and an G to N substitution at amino acid 231 of SEQ ID NO: 2. 
     
     
         57 . The fusion protein of  claim 53 , wherein the extracellular domain of the TNFR2 receptor is at least 90% identical to the amino acid sequence of SEQ ID NO: 2. 
     
     
         58 . The fusion protein of  claim 57 , wherein the extracellular domain of the TNFR2 receptor is at least 95% identical to the amino acid sequence of SEQ ID NO: 2. 
     
     
         59 . The fusion protein of  claim 58 , wherein the extracellular domain of the TNFR2 receptor is at least 99% identical to the amino acid sequence of SEQ ID NO: 2. 
     
     
         60 . The fusion protein of  claim 53 , wherein the fusion protein comprises a heterologous domain selected from:
 a) a polypeptide comprising amino acids 13-257 of SEQ ID NO: 5;   b) a polypeptide comprising amino acids 13-257 of SEQ ID NO: 6;   c) a polypeptide comprising amino acids 13-257 of SEQ ID NO: 7; and   d) a polypeptide comprising amino acids 13-257 of SEQ ID NO: 8.   
     
     
         61 . The fusion protein of  claim 60 , wherein the fusion protein comprises a linker and Fc portion having the amino acid sequence of SEQ ID NO: 18. 
     
     
         62 . A pharmaceutical preparation comprising the fusion protein of  claim 34  and a pharmaceutically acceptable carrier, wherein the preparation is substantially free of pyrogenic materials so as to be suitable for administration to a mammal.