OB fusion protein compositions and methods

• US 8,080,254 B2
• Filed: 09/22/2006
• Issued: 12/20/2011
• Est. Priority Date: 11/22/1995
• Status: Expired due to Fees

• Current Assignee: Amgen Inc.
• Original Assignee: Amgen Inc.
• Inventors: Mann, Michael B., Hecht, Randy Ira, Toombs, Christopher Francis, Pellymounter, Mary Ann
• Primary Examiner(s): HUYNH, PHUONG N
• Application Number:
  US11/525,280
• Publication Number:
  US 20070020284A1
• Time in Patent Office: 1,915 Days
• Field of Search: None
• US Class Current: 424/192.1
• CPC Class Codes: A61K 38/2264 Obesity-gene products, e.g. leptin
• CPC Class Codes: C07K 14/5759 Products of obesity genes, e.g. leptin, obese (OB), tub, fat
• CPC Class Codes: C07K 19/00 Hybrid peptides , i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/30 Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Abstract

The present invention relates to Fc-OB fusion protein compositions, methods of preparation of such compositions and uses thereof. In particular, the present invention relates to a genetic or chemical fusion protein comprising the Fc immunoglobulin region, derivative or analog fused to the N-terminal portion of the OB protein, derivative or analog.

Claims

1. A protein having a formula selected from the group consisting of:

R₁-R₂ and R₁-L-R₂, wherein;

R₁ is an Fc protein selected from the group consisting of;

(a) amino acids 1-233 of SEQ ID NO;

9, amino acids 1-233 of SEQ ID NO;

12, amino acids 1-228 of SEQ ID NO;

15, or amino acids 1-228 of SEQ ID NO;

18;

(b) the amino acid sequence of subpart (a) having a different amino acid substituted or deleted in one or more of the following positions (using the numbering according to SEQ. ID. NO.

     9);

(i) one or more cysteine residues replace by an alanine or serine residue;

(ii) one or more tyrosine residues replaced by a phenylalanine residue;

(iii) the amino acid at position 15 replaced with an glutamate;

(iv) the amino acid at position 20 replaced with glutamate;

(v) the amino acid at position 103 replaced with an alanine;

(vi) the amino acid at position 105 replaced with an alanine;

(vii) the amino acid at position 107 replaced with an alanine;

(viii) the amino acids at positions 1, 2, 3, 4, and/or 5 deleted;

(ix) the amino acid at position 15 replaced to ablate the Fc receptor binding site;

(x) one or more amino acids at positions 98, 100, and 102 replaced to ablate the complement (C1q) binding site; and

(xi) a combination of subparts i-x;

(c) the amino acid sequence of subparts (a) or (b) having a methionyl residue at the N-terminus;

(d) the amino acid sequence of subparts (a) through (c) wherein the Fc protein is connected to a water soluble polymer moiety selected from the group consisting of;

polyethylene glycol, an ethylene glycol/propylene glycol copolymer, a carboxymethylcellulose, a dextran, a polyvinyl alcohol, a polyvinyl pyrolidone, a poly-1,3-dioxolane, a poly-1,3,6-trioxane, an ethylene/maleic anhydride copolymer, a polyaminoacid homopolymer, a polyaminoacid random copolymer, an albumin, a polylysine moiety, a poly(n-vinyl pyrolidone)polyethylene glycol, a propylene glycol homopolymer, a polypropylene oxide/ethylene oxide copolymer, a polyoxyethylated polyol, a polyethylene glycol propionaldehyde, a succinate, and a styrene;

(e) the amino acid sequence of subpart (d) wherein said water soluble polymer moiety is polyethylene glycol;

(f) the amino acid sequence of subpart (d) wherein said water soluble polymer is a polyamino acid-selected from the group consisting of an albumin, and a polylysine moiety; and

(g) the amino acid sequence of subpart (d) wherein said water soluble polymer moiety is attached at solely the N-terminus of said Fc protein;

R₂ is an OB protein selected from the group consisting of;

(A) the amino acid sequence 1-146 as set forth in SEQ. ID. NO. 3 or SEQ. ID. NO. 6;

(B) the amino acid sequence 1-146 as set forth in SEQ. ID. NO. 6 having a lysine residue at position 35 and an isoleucine residue at position 74;

(C) the amino acid sequence of subpart (B) wherein one or more of the following positions (using the numbering according to SEQ. ID. NO.

     6);

4, 8, 32, 33, 35, 48, 50, 53, 60, 64, 66, 67, 68, 71, 74, 77, 78, 89, 97, 100, 102, 105, 106, 107, 108, 111, 112, 118, 136, 138, 142, and 145;

is substituted with the corresponding amino acid at the corresponding position as set forth in SEQ ID NO;

3;

(D) the amino acid sequence of subpart (B) wherein one or more of the following positions (using the numbering according to SEQ. ID. NO.

     6) is substituted according to the following;

(i) the amino acid at position 8 replaced with a serine;

(ii) the amino acid at position 35 replaced with an arginine;

(iii) the amino acid at position 48 replaced with a valine;

(iv) the amino acid at position 53 replaced with a glutamine;

(v) the amino acid at position 60 replaced with an isoleucine;

(vi) the amino acid at position 66 replaced with an isoleucine;

(vii) the amino acid at position 67 replaced with an asparagine;

(viii) the amino acid at position 68 replaced with a leucine;

(ix) the amino acid at position 89 replaced with a leucine;

(x) the amino acid at position 100 replaced with a leucine;

(xi) the amino acid at position 108 replaced with a glutamate;

(xii) the amino acid at position 112 replaced with an aspartate; and

(xiii) the amino acid at position 118 replaced with a leucine;

(E) the amino acid sequence of subparts (A), (B), (C), or (D) optionally lacking a glutaminyl residue at position 28;

(F) the amino acid sequence of subparts (A), (B), (C), (D), or (E) having a methionyl residue at the N-terminus,

(G) the OB protein of subpart (A) through (F) wherein the OB protein comprises a water soluble polymer moiety selected from the group consisting of;

polyethylene glycol, an ethylene glycol/propylene glycol copolymer, a carboxymethylcellulose, a dextran, a polyvinyl alcohol, a polyvinyl pyrolidone, a poly-1,3-dioxolane, a poly-1,3,6-trioxane, an ethylene/maleic anhydride copolymer, a polyaminoacid homopolymer, a polyaminoacid random copolymer, an albumin, a polylysine moiety, a poly(n-vinyl pyrolidone)polyethylene glycol, a propylene glycol homopolymer, a polypropylene oxide/ethylene oxide copolymer, a polyoxyethylated polyol, a polyethylene glycol propionaldehyde, a succinate, and a styrene;

(H) the OB protein of subpart (G) wherein said water soluble polymer moiety is polyethylene glycol;

(I) the OB protein of subpart (G) wherein said water soluble polymer is a polyamino acid selected from the group consisting of an albumin, and a polylysine moiety; and

(J) the OB protein of subpart (G) wherein said water soluble polymer moiety is attached at solely the N-terminus of said OB protein; and

L is a linker.

2. The protein of claim 1 wherein the linker is one or more amino acids selected from the group consisting of:

Glycine, Asparagine, Serine, Threonine and Alanine.

3. The protein of claim 1 wherein the linker is selected from the group consisting of:

(a) ala-ala-ala;

(b) ala-ala-ala-ala (SEQ ID NO;

19);

(c) ala-ala-ala-ala-ala (SEQ ID NO;

20);

(d) gly-gly;

(e) gly-gly-gly;

(f) gly-gly-gly-gly-gly (SEQ ID NO;

21);

(g) gly-gly-gly-gly-gly-gly-gly (SEQ ID NO;

22);

(h) gly-pro-gly;

(i) gly-gly-pro-gly-gly (SEQ ID NO;

23);

(j) a chemical moiety; and

(k) any combination of subparts (a) through (j).

4. A pharmaceutical composition comprising an effective amount of the protein according to claim 1 in a pharmaceutically acceptable diluent, adjuvant or carrier.