OB fusion protein compositions and methods

• US 20070020284A1
• Filed: 09/22/2006
• Published: 01/25/2007
• Est. Priority Date: 11/22/1995
• Status: Active Grant

• Current Assignee: Amgen Inc.
• Original Assignee: Amgen Inc.
• Inventors: Mann, Michael, Toombs, Christopher, Hecht, Randy, Pelleymounter, Mary
• Granted Patent: US 8,080,254 B2
• Time in Patent Office: Days
• US Class Current: 424/185.100
• 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 a Fc protein or analog thereof, R₂ is an OB protein or analog thereof, and L is a linker.

2. The protein according to claim 1, where in the Fc, analog or derivative is selected from the group consisting of:

(a) the Fc amino acid sequences as set forth in SEQ. ID. NOS.;

9, 12, 15 and 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 5 replaced with an alanine;

(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 or substituted;

(ix) one or more residues replaced or deleted to ablate the Fc receptor binding site;

(x) one or more residues replaced or deleted to ablate the complement (C1q) binding site; and

(ix) 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 Fc protein or analog of any of subparts (a) through (c) comprised of a chemical moiety connected to the protein moiety;

(e) a derivative of subpart (d) wherein said chemical moiety is a water soluble polymer moiety;

(f) a derivative of subpart (e) wherein said water soluble polymer moiety is polyethylene glycol;

(g) a derivative of subpart (e) wherein said water soluble polymer is a polyamino acid moiety; and

(h) a derivative of subpart (e) wherein said water soluble polymer moiety is attached at solely the N-terminus of said protein moiety.

3. The protein according to claim 1, wherein the OB protein, analog or derivative is 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) having a different amino acid substituted in 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;

(d) the amino acid sequence of subparts (a), (b), or (c) optionally lacking a glutaminyl residue at position 28;

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

(f) a truncated OB protein analog selected from among;

(using the numbering of SEQ. ID. NO. 6 having a lysine residue at position

     74);

(i) amino acids 98-146

(ii) amino acids 1-32

(iii) amino acids 40-116

(iv) amino acids 1-99 and 112-146

(v) amino acids 1-99 and 112-146 having one or more of amino acids 100-111 sequentially placed between amino acids 99 and 112; and

(vi) the truncated OB analog of subpart (f) (i) having one or more of amino acids 100, 102, 105, 106, 107, 108, 111, 112, 118, 136, 138, 142, and 145 substituted with another amino acid;

(vii) the truncated analog of subpart (f) (ii) having one or more of amino acids 4, 8, and 32 substituted with another amino acid;

(viii) the truncated analog of subpart (f) (iii) having one or more of amino acids 50, 53, 60, 64, 66, 67, 68, 71, 74, 77, 78, 89, 97, 100, 102, 105, 106, 107, 108, 111, and 112 replaced with another amino acid;

(ix) the truncated analog of subpart (f) (iv) having one or more of amino acids 4, 8, 32, 33, 35, 48, 50, 53, 60, 64, 66, 67, 68, 71, 74, 77, 78, 89, 97, 112, 118, 136, 138, 142, and 145 replaced with another amino acid;

(x) the truncated analog of subpart (f) (v) having one or more of amino acids 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 replaced with another amino acid;

(xi) the truncated analog of subparts (f) (i)-(x) having an N-terminal methionyl residue;

(g) the OB protein or analog derivative of any of subparts (a) through (f) comprised of a chemical moiety connected to the protein moiety;

(h) a derivative of subpart (g) wherein said chemical moiety is a water soluble polymer moiety;

(i) a derivative of subpart (h) wherein said water soluble polymer moiety is polyethylene glycol;

(j) a derivative of subpart (h) wherein said water soluble polymer is a polyamino acid moiety; and

(k) a derivative of subpart (h) wherein said water soluble polymer moiety is attached at solely the N-terminus of said protein moiety.

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

Glycine, Asparagine, Serine, Threonine and Alanine.

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

(a) ala-ala-ala;

(b) ala-ala-ala-ala;

(c) ala-ala-ala-ala-ala;

(d) gly-gly;

(e) gly-gly-gly;

(f) gly-gly-gly-gly-gly;

(g) gly-gly-gly-gly-gly-gly-gly;

(h) gly-pro-gly;

(i) gly-gly-pro-gly-gly;

(j) chemical moiety; and

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

16. A process for producing a protein of claim 1 comprising the steps of culturing, under suitable conditions, the host cell of claim 15, and isolating the protein produced.

17. The process of claim 16 further comprising the step of purifying the protein produced.

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

19. A method of treatment of a disorder selected from the group consisting of excess weight, diabetes, high blood lipid level, arterial sclerosis, arterial plaque, the reduction or prevention of gall stones formation, insufficient lean tissue mass, insufficient sensitivity to insulin, and stroke, wherein the method consists of administering a therapeutically effective amount of protein according to claim 1.

6. A fusion protein comprising a Fc protein, analog or derivative thereof, fused to the N-terminus of an OB protein, analog or derivative thereof.

7. A nucleic acid sequence encoding for a protein having a formula selected from the group consisting of:

R₁—

R₂ and R₁-L-R₂, wherein R₁ is a Fc protein or analog thereof, R₂ is an OB protein or analog thereof, and L is a linker.

8. The nucleic acid sequence according to claim 7 encoding for a protein having a Fc, analog or derivative portion selected from the group consisting of:

(a) amino acids 1-233 of SEQ ID NOS;

9 and 12 and amino acids 1-228 of SEQ ID NOS;

15 and 18;

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

9 having a different amino acid substituted or deleted in one or more of the following positions;

(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 5 replaced with an alanine;

(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 or substituted;

(ix) one or more residues replaced or deleted to ablate the Fc receptor binding site;

(x) one or more residues replaced or deleted to ablate the complement (C1q) binding site; and

(xi) a combination of subparts i-x; and

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

9. The nucleic acid sequence according to claim 7, encoding for a protein having an OB protein, analog or derivative portion 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) having a different amino acid substituted in 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;

(d) the amino acid sequence of subparts (a), (b), or (c) optionally lacking a glutaminyl residue at position 28;

(e) the amino acid sequence of subparts (a), (b), (c), or (d) having a methionyl residue at the N-terminus, and

(f) a truncated OB protein analog selected from among;

(using the numbering of SEQ. ID. NO. 6 having a lysine residue at position

     74);

(i) amino acids 98-146

(ii) amino acids 1-32

(iii) amino acids 40-116

(iv) amino acids 1-99 and 112-146

(v) amino acids 1-99 and 112-146 having one or more of amino acids 100-111 sequentially placed between amino acids 99 and 112; and

(vi) the truncated OB analog of subpart (f) (i) having one or more of amino acids 100, 102, 105, 106, 107, 108, 111, 112, 118, 136, 138, 142, and 145 substituted with another amino acid;

(vii) the truncated analog of subpart (f) (ii) having one or more of amino acids 4, 8, and 32 substituted with another amino acid;

(viii) the truncated analog of subpart (f) (iii) having one or more of amino acids 50, 53, 60, 64, 66, 67, 68, 71, 74, 77, 78, 89, 97, 100, 102, 105, 106, 107, 108, 111, and 112 replaced with another amino acid;

(ix) the truncated analog of subpart (f) (iv) having one or more of amino acids 4, 8, 32, 33, 35, 48, 50, 53, 60, 64, 66, 67, 68, 71, 74, 77, 78, 89, 97, 112, 118, 136, 138, 142, and 145 replaced with another amino acid;

(x) the truncated analog of subpart (f) (v) having one or more of amino acids 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 replaced with another amino acid; and

(xi) the truncated analog of subparts (f) (i)-(x) having an N-terminal methionyl residue.

10. The nucleic acid sequence of claim 7 encoding for a protein with a linker sequence of one or more amino acids selected from the group consisting of:

Gly, Asn, Ser, Thr and Ala.

11. The nucleic acid sequence of claim 7 encoding for a protein with a linker selected from the group consisting of:

(a) ala-ala-ala;

(b) ala-ala-ala-ala;

(c) ala-ala-ala-ala-ala;

(d) gly-gly;

(e) gly-gly-gly;

(f) gly-gly-gly-gly-gly;

(g) gly-gly-gly-gly-gly-gly-gly;

(h) gly-pro-gly;

(i) gly-gly-pro-gly-gly;

(j) a chemical moiety, and

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

13. A vector containing a nucleic acid sequence according to claim 7.

14. The vector of claim 13 wherein the vector is pAMG21.

15. A prokaryotic or eukaryotic host cell containing the vector of claim 13.

12. A nucleic acid sequence encoding for a fusion protein having a Fc protein, analog or derivative thereof, fused to the N-terminus of an OB protein, analog or derivative thereof.