US2016250340A1PendingUtilityA1
POLYMER CONJUGATES OF INTERFERON BETA-1a AND USES
Est. expiryOct 16, 2018(expired)· nominal 20-yr term from priority
A61P 9/00A61P 37/00A61P 37/02A61P 9/10A61P 37/06A61P 43/00A61P 31/00A61P 35/02A61P 35/00A61P 31/12A61P 31/16A61P 29/00A61P 27/02A61P 31/14A61P 25/00A61P 1/16A61K 47/61A61K 47/60A61K 47/59Y10S930/142A61K 38/215A61K 47/48215A61K 47/50
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Claims
Abstract
An interferon beta polypeptide comprising interferon-beta 1a coupled to a polymer containing a polyalkylene glycol moiety wherein the interferon-beta-1a and the polyalkylene glycol moiety are arranged such that the interferon-beta-1a has an enhanced activity relative to another therapeutic form of interferon beta (interferon-beta-1b) and exhibits no decrease in activity as compared to non-conjugated interferon-beta-1a. The conjugates of the invention are usefully employed in therapeutic as well as non-therapeutic, e.g., diagnostic, applications.
Claims
exact text as granted — not AI-modified1 . A method of preparing a glycosylated interferon beta-1a molecule coupled to a polyethylene glycol polymer at an N-terminal end of the glycosylated interferon beta-1a molecule comprising steps of:
a) preparing an activated polyethylene glycol polymer; b) reacting said glycosylated interferon beta-1a molecule with said activated polyethylene glycol polymer under reductive alkylation conditions in a reaction mixture; c) coupling said activated polyethylene glycol polymer to the N-terminal end of the glycosylated interferon beta-1a molecule; and d) purifying said reaction mixture to obtain purified glycosylated interferon beta-1a molecule comprising [one or more of the amino acid sequences recited in the specification] coupled to a polyethylene glycol polymer at an N-terminal end of the glycosylated interferon beta-1a molecule, wherein said glycosylated interferon beta-1a molecule has an amino acid sequence selected from:
MSYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMNFDIPEE
IKQLQQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVEN
LLANVYHQINHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRI
LHYLKAKEYSHCAWTIVRVEILRNFYFINRLTGYLRN;
MAYAALGALQASSNFQCQKLLWQLNGRLEYCLKDRMNFDIPEE
IKQLQQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVEN
LLANVYHQINHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRI
LHYLKAKEYSHCAWTIVRVEILRNFYFINRLTGYLRN;
MSYNLLGFLQRSSNAACAALLAALNGRLEYCLKDRMNFDIPEE
IKQLQQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVEN
LLANVYHQINHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRI
LHYLKAKEYSHCAWTIVRVEILRNFYFINRLTGYLRN;
MSYNLLGFLQRSSNFQCQKLLWQLNGRAAACAADRMNFDIPEE
IKQLQQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVEN
LLANVYHQINHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRI
LHYLKAKEYSHCAWTIVRVEILRNFYFINRLTGYLRN;
MSYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRAAFAIPAE
IKQLQQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVEN
LLANVYHQINHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRI
LHYLKAKEYSHCAWTIVRVEILRNFYFINRLTGYLRN;
MSYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMNFDIPEE
IAAAAAFAAADAALTIYEMLONIFAIFRQDSSSTGWNETIVEN
LLANVYHQINHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRI
LHYLKAKEYSHCAWTIVRVEILRNFYFINRLTGYLRN;
MSYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMNFDIPEE
IKQLQQFQKEDAALTIYEMLANIASIFRQDSSSTGWNETIVEN
LLANVYHQINHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRI
LHYLKAKEYSHCAWTIVRVEILRNFYFINRLTGYLRN;
MSYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMNFDIPEE
IKQLQQFQKEDAALTIYEMLQNIFAIFAAASSSTGWNETIVEN
LLANVYHQINHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRI
LHYLKAKEYSHCAWTIVRVEILRNFYFINRLTGYLRN;
MSYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMNFDIPEE
IKQLQQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNASIVAA
LLSNVYHQINHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRI
LHYLKAKEYSHCAWTIVRVEILRNFYFINRLTGYLRN;
MSYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMNFDIPEE
IKQLQQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVEN
LLANVAHQIAHLAAVLEEKLEKEDFTRGKLMSSLHLKRYYGRI
LHYLKAKEYSHCAWTIVRVEILRNFYFINRLTGYLRN;
MSYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMNFDIPEE
IKQLQQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVEN
LLANVYHQINHLKTVLAAKLAAADFTRGKLMSSLHLKRYYGRI
LHYLKAKEYSHCAWTIVRVEILRNFYFINRLTGYLRN;
MSYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMNFDIPEE
IKQLQQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVEN
LLANVYHQINHLKTVLEEKLEKEAATAGKAMSALHLKRYYGRI
LHYLKAKEYSHCAWTIVRVEILRNFYFINRLTGYLRN;
MSYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMNFDIPEE
IKQLQQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVEN
LLANVYHQINHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGAI
AAYLAAKEYSHCAWTIVRVEILRNFYFINRLTGYLRN;
MSYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMNFDIPEE
IKQLQQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVEN
LLANVYHQINHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRI
LHYLKAAAYSHCAWTIVRVEILRNFYFINRLTGYLRN;
MSYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMNFDIPEE
IKQLQQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVEN
LLANVYHQINHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRI
LHYLKAKEYAACAWTIVRVEILRNFYFINRLTGYLRN;
or
MSYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMNFDIPEE
IKQLQQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVEN
LLANVYHQINHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRI
LHYLKAKEYSHCAWTIVRAEILANFAFIARLTGYLRN.
2 . The method of claim 1 , wherein the reacting step (b) involves reaction 1.0 to 10 moles of activated polymer per mole of the protein.
3 . The method of claim 1 , wherein reductive alkylation is performed in the presence of sodium cyanoborohydride.
4 . The method of claim 1 , wherein the molecular weight of the polyalkylene glycol polymer is from 5 to 40 kilodaltons.
5 . The method of claim 1 , wherein the polyethylene glycol polymer is coupled to the interferon-beta by way of a group selected from an aldehyde group, a maleimide group, a vinylsulfone group, a haloacetate group, plurality of histidine residues, a hydrazine group, or an aminothiol group.
6 . The method of claim 1 , wherein the polyalkylene glycol polymer is a polyethylene glycol polymer.
7 . The method of claim 6 , wherein the polyethylene glycol polymer is 20 kilodalton PEG aldehyde.
8 . The method of claim 1 , wherein the method further comprises the step of preparing a composition comprising the polyethylene glycol polymer coupled glycosylated interferon beta-1a molecule and a pharmaceutically acceptable carrier suitable for subcutaneous or intravenous administration.
9 . The method of claim 1 , wherein the purifying step involves sequential chromatography.
10 . The method of claim 9 , wherein the sequential chromatography comprises using sizing columns.
11 . The method of claim 1 , wherein reductive alkylation is performed at a pH of 5 to 7.
12 . The method of claim 1 , wherein said glycosylated interferon beta-1a molecule has amino acid sequence:
MSYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMNFDIPEE
IKQLQQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVEN
LLANVYHQINHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRI
LHYLKAKEYSHCAWTIVRVEILRNFYFINRLTGYLRN.
13 . A method of decreasing stress-induced aggregation of interferon beta-1a, comprising coupling glycosylated interferon-beta-1a having the amino acid sequence of SEQ ID NO: 25 to a polyalkylene glycol moiety at the N-terminal end of said glycosylated interferon-beta-1a to form an aggregation-resistant interferon-beta-1a conjugate.
14 . The method of claim 13 , wherein the polyalkylene glycol moiety is a polyethylene glycol moiety.
15 . The method of claim 13 , wherein the polyalkylene glycol moiety has a molecular weight of about 20 kilodaltons.
16 . The method of claim 13 , wherein the aggregation-resistant interferon-beta-1a molecule does not aggregate when subjected to thermal denaturation at 60° C.
17 . A subcutaneous unit dosage form filled with a composition comprising a glycosylated interferon-beta-1a having the amino acid sequence set forth in SEQ ID NO: 25 coupled to a non-naturally occurring polymer at the N-terminal end of said glycosylated interferon-beta-1a, said polymer comprising a polyalkylene glycol moiety having a molecular weight of about 20 kilodaltons, and a carrier suitable for subcutaneous administration to a mammalian subject.
18 . The subcutaneous unit dosage of claim 17 , wherein the polyalkylene glycol moiety is a polyethylene glycol moiety.
19 . The subcutaneous unit dosage of claim 17 , where in the composition is in the form of a sterile aqueous formulation.
20 . The subcutaneous unit dosage of claim 17 , further comprising one or more diluents, buffers, surface active agents, thickeners, or preservatives.
21 . The subcutaneous unit dosage of claim 17 , wherein said composition does not contain a preservative.Join the waitlist — get patent alerts
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