Remodeling and Glycopegylation of Fibroblast Growth Factor (Fgf)

Abstract

The present invention relates to mutants of Fibroblast Growth Factor (FGF), particularly FGF-20 and FGF-21, which contain newly introduced N-linked or O-linked glycosylation site(s). The polynucleotide coding sequences for the mutants, expression cassettes comprising the coding sequences, cells expressing the mutants, and methods for producing the mutants are also disclosed. Further disclosed are pharmaceutical compositions comprising the mutants and method for using the mutants.

1 . A Fibroblast Growth Factor (FGF) conjugate comprising:
 a FGF peptide; and   a modifying group, wherein said modifying group is covalently attached to said peptide at a preselected glycosyl or amino acid residue of said peptide via an intact glycosyl linking group.   
     
     
         2 . The FGF conjugate of  claim 1 , wherein said FGF peptide is a member selected from FGF-1, FGF-2, FGF-18, FGF-20 and FGF-21. 
     
     
         3 . The FGF conjugate of  claim 1 , wherein said FGF peptide is at least 95% homologous to at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 9-14, 18-45, 48-65, 69-109, 112-146, 161-214, 220-320 and 323-360. 
     
     
         4 . The FGF conjugate of  claim 1 , wherein said modifying group is covalently attached at said preselected glycosyl residue. 
     
     
         5 . The FGF conjugate of  claim 4 , wherein said modifying group is a non-glycosidic modifying group. 
     
     
         6 . The FGF conjugate of  claim 5 , wherein said non-glycosidic modifying group is a member selected from linear PEG and branched PEG. 
     
     
         7 . The FGF conjugate of  claim 6 , wherein said PEG moiety is linear PEG and said linear PEG has a structure according to the following formula: 
       
         
           
           
               
               
           
         
       
       in which
 R 2  is a member selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted heteroalkyl, e.g., acetal, OHC—, H 2 N—CH 2 CH 2 —, HS—CH 2 CH 2 —, and-(CH 2 ) q C(Y 1 )Z 2 ; -sugar-nucleotide, or protein; 
 n is an integer selected from 1 to 2500; 
 m, o, and q are integers independently selected from 0 to 20; 
 Z is a member selected from OH, NH 2 , halogen, S—R 3 , the alcohol portion of activated esters, —(CH 2 ) p C(Y 2 )V, —(CH 2 ) p U(CH 2 ) s C(Y 2 ) v , sugar-nucleotide, protein, and leaving groups, e.g., imidazole, p-nitrophenyl, HOBT, tetrazole, halide; 
 X, Y 1 , Y 2 , W and U are independently selected from O, S, N—R 4 ; 
 V is a member selected from OH, NH 2 , halogen, S—R 5 , the alcohol component of activated esters, the amine component of activated amides, sugar-nucleotides, and proteins; 
 p, s and v are integers independently selected from 0 to 20; and 
 R 3 , R 4  and R 5  are independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heterocycloalkyl and substituted or unsubstituted heteroaryl. 
 
     
     
         8 . The FGF conjugate of  claim 1 , wherein said glycosyl linking group has a structure according to the following formula: 
       
         
           
           
               
               
           
         
       
       wherein
 R 2  is H, CH 2 OR 7 , COOR 7  or OR 7    
 wherein
 R 7  represents H, substituted or unsubstituted alkyl or substituted or unsubstituted heteroalkyl; 
 
 R 3  and R 4  are members independently selected from H, substituted or unsubstituted alkyl, OR 8 , NHC(O)R 9    
 wherein
 R 8  and R 9  are independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl or sialic acid; 
 
 L a  is a linker selected from a bond, substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl 
 R 16  and R 17  are independently selected polymeric arms; 
 X 2  and X 4  are independently selected linkage fragments joining polymeric moieties R 16  and R 17  to C; and 
 X 5  is a non-reactive group. 
 
     
     
         9 . The FGF conjugate of  claim 8 , wherein said glycosyl linking group has a structure according to the following formula: 
       
         
           
           
               
               
           
         
       
     
     
         10 . An isolated nucleic acid comprising a polynucleotide sequence encoding a mutant Fibroblast Growth Factor, wherein the mutant Fibroblast Growth Factor comprises a newly introduced N-linked or O-linked glycosylation site that does not exist in the corresponding wild-type Fibroblast Growth Factor. 
     
     
         11 . The nucleic acid of  claim 10 , wherein the nucleic acid encodes a mutant Fibroblast Growth Factor-20 that has a corresponding wild-type Fibroblast Growth Factor-20 amino acid sequence that is at least 95% homologous to SEQ ID NO:1. 
     
     
         12 . The nucleic acid of  claim 10 , wherein the newly introduced glycosylation site is near a proline residue. 
     
     
         13 . The nucleic acid of  claim 12 , wherein the proline residue is located at position 3, 28, 29, 34, 35, 52, 81, 175, 192, 197, or 201 of SEQ ID NO:1. 
     
     
         14 . The nucleic acid of  claim 10 , wherein the mutant Fibroblast Growth Factor is at least 95% homologous to at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 9-14, 18-45, 48-65, 69-109, 112-145, 161-214, 220-320 and 323-360. 
     
     
         15 . The nucleic acid of  claim 10 , wherein the mutant Fibroblast Growth Factor comprises more than one newly introduced glycosylation site. 
     
     
         16 . An expression cassette comprising the nucleic acid of  claim 10 . 
     
     
         17 . A cell comprising the nucleic acid of  claim 10 . 
     
     
         18 . A mutant Fibroblast Growth Factor, comprising a newly introduced N-linked or O-linked glycosylation site that does not exist in the corresponding wild-type Fibroblast Growth Factor. 
     
     
         19 . The mutant Fibroblast Growth Factor of  claim 18 , wherein the corresponding wild-type Fibroblast Growth Factor has the amino acid sequence that is at least 95% homologous to SEQ ID NO:1. 
     
     
         20 . The mutant Fibroblast Growth Factor of  claim 18 , wherein the newly introduced glycosylation site is proximate to a proline residue. 
     
     
         21 . The mutant Fibroblast Growth Factor of  claim 20 , wherein the proline residue is located at position 3, 28, 29, 34, 35, 52, 81, 175, 192, 197, or 201 of SEQ ID NO:1. 
     
     
         22 . The mutant Fibroblast Growth Factor of  claim 18 , wherein said mutant FGF is at least 95% homologous to at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 9-14, 18-45, 48-65, 69-109, 112-145, 161-214, 220-320 and 323-360. 
     
     
         23 . The mutant Fibroblast Growth Factor of  claim 18 , wherein the mutant Fibroblast Growth Factor comprises more than one newly introduced glycosylation site. 
     
     
         24 . The mutant Fibroblast Growth Factor of  claim 18 , comprising a water-soluble polymer attached to a glycosylation site through a glycosyl linker. 
     
     
         25 . The mutant Fibroblast Growth Factor of  claim 24 , wherein said glycosyl linker is an intact glycosyl linker. 
     
     
         26 . A method for making a mutant Fibroblast Growth Factor, which comprises a newly introduced N-linked or O-linked glycosylation that does not exist in the corresponding wild-type Fibroblast Growth Factor, comprising the steps of:
 (a) recombinantly producing the mutant Fibroblast Growth Factor; and   (b) glycosylating the mutant Fibroblast Growth Factor at the newly introduced glycosylation site   wherein
 said glycosylating is a cell free, in vitro process. 
   
     
     
         27 . The method of  claim 26 , wherein the corresponding wild-type Fibroblast Growth Factor has the amino acid sequence that is at least 95% homologous to SEQ ID NO:1. 
     
     
         28 . The method of  claim 26 , wherein the newly introduced glycosylation site is near a proline residue. 
     
     
         29 . The method of  claim 28 , wherein the proline residue is located at position 3, 28, 29, 34, 35, 52, 81, 175, 192, 197, or 201 of SEQ ID NO:1. 
     
     
         30 . The method of  claim 26 , wherein the mutant Fibroblast Growth Factor comprises at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 9-14, 18-45, 48-65, 69-109, 112-145, 161-214, 220-320 and 323-360. 
     
     
         31 . The method of  claim 26 , wherein the mutant Fibroblast Growth Factor comprises more than one newly introduced glycosylation site. 
     
     
         32 . A pharmaceutical composition comprising an effective amount of a mutant Fibroblast Growth Factor, which comprises a newly introduced N-linked or O-linked glycosylation that does not exist in the corresponding wild-type Fibroblast Growth Factor. 
     
     
         33 . The composition of  claim 32 , wherein the corresponding wild-type Fibroblast Growth Factor has the amino acid sequence that is at least 95% homologous to SEQ ID NO:1. 
     
     
         34 . The composition of  claim 32 , wherein the newly introduced glycosylation site is near a proline residue. 
     
     
         35 . The composition of  claim 34 , wherein the proline residue is located at position 3, 28, 29, 34, 35, 52, 81, 175, 192, 197, or 201 of SEQ ID NO:1. 
     
     
         36 . The composition of  claim 32 , wherein the mutant Fibroblast Growth Factor is at least 95% homologous to at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 9-14, 18-45, 48-65, 69-109, 112-145, 161-214, 220-320 and 323-360. 
     
     
         37 . The composition of  claim 32 , wherein the mutant Fibroblast Growth Factor comprises more than one newly introduced glycosylation site. 
     
     
         38 . A method for treating Fibroblast Growth Factor deficiency in a patient, comprising the step of administering an effective amount of a mutant Fibroblast Growth Factor to the patient, wherein the mutant Fibroblast Growth Factor comprises a newly introduced N-linked or O-linked glycosylation that does not exist in the corresponding wild-type Fibroblast Growth Factor. 
     
     
         39 . The method of  claim 38 , wherein the corresponding wild-type Fibroblast Growth Factor has the amino acid sequence of SEQ ID NO:1. 
     
     
         40 . The method of  claim 38 , wherein the newly introduced glycosylation site is near a proline residue. 
     
     
         41 . The method of  claim 40 , wherein the proline residue is located at position 3, 28, 29, 34, 35, 52, 81, 175, 192, 197, or 201 of SEQ ID NO:1. 
     
     
         42 . The method of  claim 38 , wherein the mutant Fibroblast Growth Factor is at least 95% homologous to at least one amino acid sequence selected from the group consisting of SEQ ID NOs:9-14, 18-45, 48-65, 69-109, 112-145, 161-214, 220-320 and 323-360. 
     
     
         43 . The method of  claim 38 , wherein the mutant Fibroblast Growth Factor comprises more than one newly introduced glycosylation site. 
     
     
         44 . A method for making a glycoconjugate of a mutant Fibroblast Growth Factor, which comprises a newly introduced N-linked or O-linked glycosylation that does not exist in the corresponding wild-type Fibroblast Growth Factor, comprising the steps of:
 (a) recombinantly producing the mutant Fibroblast Growth Factor, and   (b) enzymatically glycosylating the mutant Fibroblast Growth Factor with a modified sugar at the newly introduced glycosylation site   wherein
 said glycosylating is a cell free, in vitro process. 
   
     
     
         45 . The method of  claim 44 , wherein the modified sugar is modified with a water-soluble polymer. 
     
     
         46 . The method of  claim 45 , wherein the modified sugar is modified with a water soluble polymer selected from the group consisting of poly(ethylene glycol) and m-poly(ethylene glycol). 
     
     
         47 . The method of  claim 44  wherein the corresponding wild-type Fibroblast Growth Factor has the amino acid sequence of SEQ ID NO:1. 
     
     
         48 . The method of  claim 45 , wherein the newly introduced glycosylation site is near a proline residue. 
     
     
         49 . The method of  claim 44 , wherein the proline residue is located at position 3, 28, 29, 34, 35, 52, 81, 175, 192, 197, or 201 of SEQ ID NO:1. 
     
     
         50 . The method of  claim 44 , wherein the mutant Fibroblast Growth Factor comprises at least one amino acid sequence selected from the group consisting of SEQ ID NOs: SEQ ID NOs: 9-14, 18-45, 48-65, 69-109, 112-145, 161-214, 220-320 and 323-360. 
     
     
         51 . The method of  claim 44 , wherein the mutant Fibroblast Growth Factor comprises more than one newly introduced glycosylation site.