Novel feline erythropoietin receptor agonists

Abstract

The present specification discloses erythropoietin receptor agonists, compositions and medicaments comprising such erythropoietin receptor agonists, methods and uses for such erythropoietin receptor agonists and compositions and medicaments, and methods and uses for erythropoietin receptor agonists and compositions and medicaments for treating an anemia.

1 . A modified non-human mammalian erythropoietin comprising a non-human mammalian erythropoietin modified to have and at least one added and/or at least one relocated glycosylation site. 
     
     
         2 . The modified non-human mammalian erythropoietin according to  claim 1 , wherein the a non-human mammalian erythropoietin comprises SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 10 or SEQ ID NO: 11, a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 5 or SEQ ID NO: 6, SEQ ID NO: 10 or SEQ ID NO: 11, wherein Xaa at position 18 of SEQ ID NO: 5 or SEQ ID NO: 6 is selected from E or G; and Xaa at position 116 of SEQ ID NO: 5 or SEQ ID NO: 6 is K or absent. 
     
     
         3 . The modified non-human mammalian erythropoietin according to  claim 2 , wherein an asparagine residue is substituted for the amino acid residue at any one or more of positions 30, 51, 57, 69, 88, 89, 137, or 139 of SEQ ID NO: 5 or SEQ ID NO: 6 or SEQ ID NO: 10; or wherein an asparagine residue is substituted for the amino acid residue at any one or more of positions 56, 77, 83, 95, 114, 115, 163, or 165 of SEQ ID NO: 11. 
     
     
         4 . The modified non-human mammalian erythropoietin according to  claim 2 , wherein in SEQ ID NO: 5 or SEQ ID NO: 6, or SEQ ID NO: 10 either a serine or a threonine residue is substituted for the amino acid residue at position 126 or wherein in SEQ ID NO: 11 either a serine or threonine residue is substituted for the amino acid residue at position 152. 
     
     
         5 . The modified non-human mammalian erythropoietin according to  claim 2 , wherein in SEQ ID NO: 5 or SEQ ID NO: 6, or SEQ ID NO: 10 one or more of the following amino acid substitution sets is present: Asn 30  and Thr 32 ; Asn 51  and Thr 53 ; Asn 57 , Thr 59  and Asn 69 ; Asn 69  and Thr 71 ; Ser 68 , Asn 69  and Thr 71 ; Val 87  and Asn 88 ; Ser 87  and Asn 88 ; Ser 87 , Asn 88  and Gly 89 ; Ser 87 , Asn 88  and Thr 92 ; Ser 87 , Asn 88  and Ala 163 ; Asn 69 , Thr 71 , Ser 87  and Asn 88 ; Asn 30 , Thr 32 , Val 87  and Asn 88 ; Asn 89 , Ile 90  and Thr 91 ; Ser 87 , Asn 89 , Ile 90  and Thr 91 ; Asn 137  and Thr 139 ; Asn 139  and Thr 141 ; Thr 126 ; Pro 125  and Thr 126 ; or any combination thereof; and wherein in SEQ ID NO: 11 one or more of the following amino acid substitution sets is present: Asn 56  and Thr 58 ; Asn 77  and Thr 79 ; Asn 83  and Thr 85 ; Asn 69 ; Asn 95  and Thr 97 ; Ser 94 , Asn 95  and Thr 97 ; Val 113  and Asn 114 ; Ser 113  and Asn 114 ; Ser 113 , Asn 114  and Gly 15 ; Ser 113 , Asn 114  and Thr 118 , Ser 113 , Asn 114  and Ala 189 ; Asn 95 , Thr 97 , Ser 113  and Asn 114 ; Asn 56 , Thr 58 , Val 113  and Asn 114 ; Asn 115 , Ile 116  and Thr 117 ; Ser 113 , Asn 115 , Ile 116  and Thr 117 ; Asn 163  and Thr 165 ; Asn 165  and Thr 167 ; Thr 152 ; and/or Pro 151  and Thr 152  or any combination thereof. 
     
     
         6 . The modified non-human mammalian erythropoietin according to  claim 5 , wherein in SEQ ID NO: 5 or SEQ ID NO: 6, or SEQ ID NO: 10 one or more of the following substitution sets is present: Ser 87  and Asn 88 ; and/or Asn 30 , Thr 32 , Val 87  and Asn 88 ; and wherein in SEQ ID NO: 11 one or more of the following substitution sets is present: Ser 113  and Asn 114  and/or Asn 56 , Thr 58 , Val 113  and Asn 114 . 
     
     
         7 . The modified non-human mammalian erythropoietin according to  claim 5 , wherein in SEQ ID NO: 5 or SEQ ID NO: 6, or SEQ ID NO: 10 one of the following amino acid substitution sets is present: Gln 24 , Ser 87  and Asn 88 ; Gln 38 , Ser 87  and Asn 88 ; or Gln 83 , Ser 87  and Asn 88 ; and wherein in SEQ ID NO: 11 one or more of the following substitution sets is present: Gln 50 , Ser 113  and Asn 114 ; Gln 64 , Ser 113  and Asn 114 ; or Gln 109 , Ser 113  and Asn 114 . 
     
     
         8 . The modified non-human mammalian erythropoietin according to  claim 1 , wherein SEQ ID NO: 5 or SEQ ID NO: 6, SEQ ID NO: 10 or SEQ ID NO: 11 is modified by deleting one or more of the glycosylation sites that attach to N linked carbohydrate chains. 
     
     
         7 . The modified non-human mammalian erythropoietin according to  claim 1 , wherein the at least one added and/or at least one relocated glycosylation site is a site for the attachment of an N-linked carbohydrate chain or a site for the attachment of an O-linked carbohydrate chain. 
     
     
         8 . The modified non-human mammalian erythropoietin according to  claim 1 , wherein the glycosylation site further comprises a fragment of human chorionic gonadotropin. 
     
     
         9 . The modified non-human mammalian erythropoietin according to  claim 8 , wherein the fragment of human chorionic gonadotropin is SEQ ID NO: 8 or an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 8, or an amino acid sequence having 1, 2, 3, 4, 5, 1-2, 1-3, 1-4, 1-5, 2-3, 2-4, 2-5, 3-4, 3-5, or 4-5 amino acid additions, deletions or substitutions to SEQ ID NO: 8. 
     
     
         10 . A nucleic acid sequence encoding a modified non-human mammalian erythropoietin as defined in  claim 1 . 
     
     
         11 . A vector comprising
 a) a nucleic acid sequence encoding the amino acid sequence for a modified non-human mammalian erythropoietin as defined in  claim 1 , and   b) a promoter operatively linked to the nucleic acid sequence.   
     
     
         12 . A host cell comprising the vector as defined in  claim 11 . 
     
     
         13 . A composition comprising
 a) a modified non-human mammalian erythropoietin as defined in  claim 1 , and   b) a pharmaceutically acceptable diluent, adjuvant, or carrier.   
     
     
         14 . A method of treating non-regenerative anemia (NRA) in a mammal comprising administering to a subject in need thereof an effective amount of a modified non-human mammalian erythropoietin as defined  claim 1 . 
     
     
         15 . A fusion protein, comprising a peptide, a linker and an Fc fragment, wherein:
 a) the peptide is SEQ ID NO: 1, an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 1, a feline EPO with the amino acid sequence SEQ ID NO: 5 or SEQ ID NO: 6, wherein Xaa at position 18 is selected from E or G; and Xaa at position 116 is K or absent, a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 5 or SEQ ID NO: 6, wherein Xaa at position 18 is selected from E or G; and Xaa at position 116 is K or absent, a canine EPO with the amino acid sequence SEQ ID NO: 10 or SEQ ID NO: 11, or a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 10 or SEQ ID NO: 11; and   b) the Fc fragment has an amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, or a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4;   wherein the peptide is fused, through linker, to the Fc fragment.   
     
     
         16 . The fusion protein according to  claim 15 , wherein the C terminus of the peptide is fused through the linker to the N terminus of the Fc fragment or wherein the N terminus of the peptide is fused through the linker to the C terminus of the Fc fragment. 
     
     
         17 . The fusion protein according to  claim 15 , modified to have and at least one added and/or at least one relocated glycosylation site. 
     
     
         18 . A nucleic acid sequence encoding the fusion protein as defined in  claim 15 . 
     
     
         19 . A composition comprising:
 a) a fusion protein as defined in  claim 15 , and   b) a pharmaceutically acceptable diluent, adjuvant, or carrier.   
     
     
         20 . A method of treating non-regenerative anemia (NRA) in a mammal comprising administering to a subject in need thereof an effective amount of a fusion protein as defined in  claim 15 .