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 - 24 . (canceled) 
     
     
         25 . A method for preparing a modified nucleic acid encoding a modified Fc fusion protein that has an extended serum half-life relative to an initial Fc fusion protein, wherein the modified and initial Fc fusion proteins each comprise an Fc portion and a heterologous portion, the method comprising: modifying the nucleic acid encoding the heterologous portion of the initial Fc fusion protein to code for one or more additional N-linked glycosylation sites, wherein (a) the modified nucleic acid encodes a modified Fc fusion protein that, when expressed in a suitable cell culture, has a serum half-life that is at least 10% longer than the serum half-life of the initial Fc fusion protein as measured in a pharmacokinetic monkey assay, and (b) the modified Fc fusion protein has substantially the same or greater in vivo biological activity relative to the unmodified Fc fusion protein. 
     
     
         26 . The method of  claim 25 , wherein the one or more additional N-linked glycosylation sites are introduced into one or more amino acid sequences of the heterologous portion that are surface exposed. 
     
     
         27 . The method of  claim 26 , wherein the one or more additional N-linked glycosylation sites are introduced into one or more amino acid sequences of the heterologous portion that are surface exposed and not incorporated into a β-sheet or α-helix. 
     
     
         28 . The method of  claim 25 , wherein the heterologous portion comprises a ligand binding domain. 
     
     
         29 . The method of  claim 28 , wherein the ligand binding domain is derived from the extracellular domain of a transmembrane receptor. 
     
     
         30 . The method of  claim 28 , wherein the one or more additional N-linked glycosylation sites are introduced at positions in the heterologous portion such that any additional sugar moieties do not substantially interfere with the ligand binding domain. 
     
     
         31 . The method of  claim 28 , wherein the one or more additional N-linked glycosylation sites are introduced at positions such that the amino acid sequence of the ligand binding domain is not modified. 
     
     
         32 . The method of  claim 28 , wherein the one or more additional N-linked glycosylation sites are introduced at positions such that any sugar moieties attached to the additional N-linked glycosylation sites are predicted not to interfere substantially with the ligand binding interface. 
     
     
         33 . The method of  claim 28 , wherein the modified Fc fusion protein binds to a ligand with half maximal inhibitory concentration (IC 50 ) that is no more than two-fold less than that of the initial Fc fusion protein. 
     
     
         34 . The method of  claim 25 , wherein the initial Fc fusion protein is modified by addition or deletion of at least one amino acid residue to introduce at least one glycosylation site. 
     
     
         35 . The method of  claim 25 , wherein the initial Fc fusion protein is modified by substitution of at least one amino acid residue to introduce at least one glycosylation site. 
     
     
         36 . The method of  claim 25 , wherein the heterologous portion of the initial Fc fusion protein comprises at least two structurally distinct α-helix and/or β-sheet domains that are connected by an unstructured polypeptide region that is surface exposed. 
     
     
         37 . The method of  claim 36 , wherein an additional N-linked glycosylation site is positioned within the unstructured peptide region. 
     
     
         38 . The method of  claim 25 , wherein the heterologous portion of the initial Fc fusion protein comprises fewer than one N-linked glycosylation site per each 90 amino acids. 
     
     
         39 . The method of  claim 25 , wherein the heterologous portion of the initial Fc fusion protein comprises fewer than one N-linked glycosylation site per each 125 amino acids. 
     
     
         40 . The method of  claim 25 , wherein the heterologous portion of the modified Fc fusion protein comprises at least one N-linked glycosylation site per each 90 amino acids. 
     
     
         41 . The method of  claim 25 , wherein the heterologous portion of the modified Fc fusion protein comprises at least one N-linked glycosylation site per each 65 amino acids. 
     
     
         42 . The method of  claim 25 , wherein each amino acid of the heterologous portion of the modified Fc fusion protein that is attached to an N-linked glycosylation is separated by at least 20 amino acids from any other amino acid modified by an N-linked glycosylation. 
     
     
         43 . The method of  claim 25 , wherein the initial Fc fusion protein further comprises a polypeptide linker between the Fc portion and the heterologous portion that does not contain an N-linked glycosylation site. 
     
     
         44 . The method of  claim 25 , wherein the modified Fc fusion protein comprises an N-linked glycosylation site positioned within the linker. 
     
     
         45 . The method of  claim 25 , wherein the heterologous portion comprises the extracellular domain of a TNFR2 receptor that includes a ligand binding domain. 
     
     
         46 . The method of  claim 45 , wherein the extracellular domain of the TNFR2 receptor comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 2. 
     
     
         47 . A method for preparing a modified Fc fusion protein that has an extended serum half-life relative to an initial Fc fusion protein, the method comprising:
 (a) expressing a modified nucleic acid prepared according to the method of any of  claims 25  to  46  in a cell culture that provides mammalian or mammalian-like glycosylation; and   (b) recovering the modified Fc fusion protein from the cell culture.   
     
     
         48 . The method of  claim 47 , further comprising a step of purifying the modified Fc fusion protein. 
     
     
         49 . The method of  claim 48 , wherein a step of purifying the modified Fc fusion protein comprises exposing the modified Fc fusion protein to protein A and recovering the modified Fc fusion protein that is bound to the protein A. 
     
     
         50 . The method of  claim 47 , further comprising formulating the modified Fc fusion protein for administration to a patient. 
     
     
         51 . The method of  claim 47 , wherein the modified nucleic acid is expressed by a mammalian cell line. 
     
     
         52 . The method of  claim 51 , wherein the mammalian cell line is selected from the group consisting of: a CHO cell line, an NSO cell line, a COS cell line and an HEK236 cell line. 
     
     
         53 . The method of  claim 26 , wherein the modified nucleic acid is expressed in a cell line that generates N-linked sugar moieties that comprise sialic acid. 
     
     
         54 . The method of  claim 47 , wherein the modified nucleic acid is expressed by a non-mammalian cell that has been engineered to provide mammalian or mammalian-like glycosylation. 
     
     
         55 . The method of  claim 54 , wherein the cell line derives from a fungal cell, an insect cell or a plant cell. 
     
     
         56 . A cell line comprising a modified nucleic acid prepared according to a method of  claim 25 . 
     
     
         57 . A modified Fc fusion protein prepared according to a method of  claim 47 . 
     
     
         58 - 96 . (canceled)