METHODS OF INCREASING LEAN TISSUE MASS USING OB PROTEIN COMPOSITIONS

• US 20070224193A1
• Filed: 03/16/2007
• Published: 09/27/2007
• Est. Priority Date: 11/22/1995
• Status: Active Grant

• Current Assignee: Amgen Inc.
• Original Assignee: Amgen Inc.
• Inventors: Mann, Michael, Pelleymounter, Mary, Toombs, Christopher
• Granted Patent: US 7,718,400 B2
• Time in Patent Office: Days
• US Class Current: 424/134.100
• 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 2319/30 Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Abstract

DNA sequences comprising nucleic acids encoding fusion proteins comprising an Fc portion of an antibody attached at the N-terminus of an OB protein moiety, vectors comprising such DNA sequences, host cells comprising such vectors or DNA sequences, and processes for preparing such fusion proteins, and pharmaceutical compositions comprising such fusion proteins, are described. The DNA sequences, vectors comprising such DNA sequences, host cells comprising such vectors or DNA sequences, and processes for preparing such fusion proteins, and pharmaceutical compositions comprising such fusion proteins are useful, for example, in providing therapeutically effective amounts of compositions useful for, for example, increasing insulin sensitivity, decreasing the dose of insulin required for the treatment of diabetes, controlling serum glucose levels, increasing lean tissue mass, increasing overall body strength, or regulating bone resorption, or effecting any combination of such, in subjects in need or desirous thereof to which the compositions are administered.

Claims

1. A process of producing a fusion protein comprising an Fc portion of an antibody attached at the N-terminus of an OB protein moiety, wherein the process comprises expressing a DNA sequence comprising:

(a) a first nucleic acid encoding said Fc portion of an antibody; and

(b) a second nucleic acid sequence encoding said OB protein moiety selected from the group consisting of;

(i) a nucleic acid sequence encoding an amino acid sequence set forth in SEQ ID. NO;

2 or an amino acid sequence set forth in SEQ ID NO;

4;

(ii) a nucleic acid sequence encoding an amino acid sequence set forth in SEQ. ID. NO;

4 having a lysine residue at position 35 and an isoleucine residue at position 74 (according to the position numbering designation set forth in item identifier <

223>

of SEQ. ID. NO;

4);

(iii) a nucleic acid sequence encoding an amino acid sequence of subpart (ii) having a different amino acid substituted in one or more of the following positions (according to the position numbering designation set forth in item identifier <

223>

of SEQ. ID. NO;

4);

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;

(iv) a nucleic acid sequence encoding an amino acid sequence of subparts (i), (ii), or (iii) optionally lacking a glutaminyl residue at position 28;

(v) a nucleic acid sequence encoding an amino acid sequence of subparts (i), (ii), (iii), or (iv) having a methionyl residue at the N-terminus,

(vi) a nucleic acid sequence encoding a truncated OB protein analog selected from among;

(according to the position numbering designation set forth in item identifier <

223>

of SEQ. ID. NO;

4 and having a lysine residue at position

     74);

(1) amino acids 98-146;

(2) amino acids 1-32;

(3) amino acids 40-116;

(4) amino acids 1-99 and 112-146;

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

(6) the truncated OB analog of subpart (vi)(1) 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;

(7) the truncated analog of subpart (vi)(2) having one or more of amino acids 4, 8, and 32 substituted with another amino acid;

(8) the truncated analog of subpart (vi)(3) 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;

(9) the truncated analog of subpart (vi)(4) 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;

(10) the truncated analog of subpart (vi)(5) 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

(11) the truncated analog of subparts (vi)(1)-(10) having an N-terminal methionyl residue;

(vii) a nucleic acid sequence encoding an OB protein derivative of any of subparts (i) through (vi) comprised of a polyamino acid connected to the protein moiety; and

(viii) a derivative of subpart (vii) wherein said polyamino acid is attached at solely the N-terminus of said protein moiety.

2. A process according to claim 1, wherein said first nucleic acid sequence and said second amino acid sequence are attached via a third nucleic acid sequence encoding a linker moiety.

3. A process according to claim 2, wherein said linker moiety 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) a chemical moiety; and

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

7. An isolated host cell transformed or transfected with a DNA sequence according to claims 1, 2, or 3.

9. A host cell according to claim 7 or claim 8, wherein said host cell produces the fusion protein for a use selected from the group consisting of:

increasing insulin sensitivity;

decreasing the dose of insulin required for the treatment of diabetes;

controlling serum glucose levels;

increasing lean tissue mass;

increasing overall body strength; and

regulating bone resorption.

10. A host cell according to claim 9, wherein said host cell produces the fusion protein for use in increasing insulin sensitivity.

11. A host cell according to claim 9, wherein said host cell produces the fusion protein for use in decreasing the dose of insulin required for the treatment of diabetes.

12. A host cell according to claim 9, wherein said host cell produces the fusion protein for use in controlling serum glucose levels.

13. A host cell according to claim 9, wherein said host cell produces the fusion protein for use in increasing lean tissue mass.

14. A host cell according to claim 9, wherein said host cell produces the fusion protein for use in increasing overall body strength.

15. A host cell according to claim 9, wherein said host cell produces the fusion protein for use in regulating bone resorption.

17. A method of increasing insulin sensitivity, decreasing the dose of insulin required for the treatment of diabetes, controlling serum glucose levels, increasing lean tissue mass, increasing overall body strength, or regulating bone resorption, or effecting any combination of such, in a subject in need or desirous thereof, said method comprising administering to said subject a therapeutically effect amount of a pharmaceutical composition according to claim 15.

4. A vector comprising a DNA sequence, wherein said DNA sequence comprises:

(a) a first nucleic acid encoding said Fc portion of an antibody; and

(b) a second nucleic acid sequence encoding said OB protein moiety selected from the group consisting of;

(i) a nucleic acid sequence encoding an amino acid sequence set forth in SEQ ID. NO;

2 or an amino acid sequence set forth in SEQ ID NO;

4;

(ii) a nucleic acid sequence encoding an amino acid sequence set forth in SEQ. ID. NO;

4 having a lysine residue at position 35 and an isoleucine residue at position 74 (according to the position numbering designation set forth in item identifier <

223>

of SEQ. ID. NO;

4);

(iii) a nucleic acid sequence encoding an amino acid sequence of subpart (ii) having a different amino acid substituted in one or more of the following positions (according to the position numbering designation set forth in item identifier <

223>

of SEQ. ID. NO;

4);

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;

(iv) a nucleic acid sequence encoding an amino acid sequence of subparts (i), (ii), or (iii) optionally lacking a glutaminyl residue at position 28;

(v) a nucleic acid sequence encoding an amino acid sequence of subparts (i), (ii), (iii), or (iv) having a methionyl residue at the N-terminus,

(vi) a nucleic acid sequence encoding a truncated OB protein analog selected from among;

(according to the position numbering designation set forth in item identifier <

223>

of SEQ. ID. NO;

4 and having a lysine residue at position

     74);

(1) amino acids 98-146;

(2) amino acids 1-32;

(3) amino acids 40-116;

(4) amino acids 1-99 and 112-146;

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

(6) the truncated OB analog of subpart (vi)(1) 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;

(7) the truncated analog of subpart (vi)(2) having one or more of amino acids 4, 8, and 32 substituted with another amino acid;

(8) the truncated analog of subpart (vi)(3) 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;

(9) the truncated analog of subpart (vi)(4) 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;

(10) the truncated analog of subpart (vi)(5) 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

(11) the truncated analog of subparts (vi)(1)-(10) having an N-terminal methionyl residue;

(vii) a nucleic acid sequence encoding an OB protein derivative of any of subparts (i) through (vi) comprised of a polyamino acid connected to the protein moiety; and

(viii) a derivative of subpart (vii) wherein said polyamino acid is attached at solely the N-terminus of said protein moiety.

5. A vector according to claim 4, wherein said first nucleic acid sequence and said second amino acid sequence are attached via a third nucleic acid sequence encoding a linker moiety.

6. A process according to claim 5, wherein said linker moiety 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) a chemical moiety; and

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

8. An isolated host cell transformed or transfected with a vector according to claims 4, 5, or 6.

16. A pharmaceutical composition comprising a fusion protein and a pharmaceutically acceptable carrier, wherein said fusion protein comprises an Fc portion of an antibody attached at the N-terminus of an OB protein moiety, and further wherein said fusion protein is encoded by a DNA sequence comprising:

(a) a first nucleic acid encoding said Fc portion of an antibody; and

(b) a second nucleic acid sequence encoding said OB protein moiety selected from the group consisting of;

(i) a nucleic acid sequence encoding an amino acid sequence set forth in SEQ ID. NO;

2 or an amino acid sequence set forth in SEQ ID NO;

4;

(ii) a nucleic acid sequence encoding an amino acid sequence set forth in SEQ. ID. NO;

4 having a lysine residue at position 35 and an isoleucine residue at position 74 (according to the position numbering designation set forth in item identifier <

223>

of SEQ. ID. NO;

4);

(iii) a nucleic acid sequence encoding an amino acid sequence of subpart (ii) having a different amino acid substituted in one or more of the following positions (according to the position numbering designation set forth in item identifier <

223>

of SEQ. ID. NO;

4);

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;

(iv) a nucleic acid sequence encoding an amino acid sequence of subparts (i), (ii), or (iii) optionally lacking a glutaminyl residue at position 28;

(v) a nucleic acid sequence encoding an amino acid sequence of subparts (i), (ii), (iii), or (iv) having a methionyl residue at the N-terminus,

(vi) a nucleic acid sequence encoding a truncated OB protein analog selected from among;

(according to the position numbering designation set forth in item identifier <

223>

of SEQ. ID. NO;

4 and having a lysine residue at position

     74);

(1) amino acids 98-146;

(2) amino acids 1-32;

(3) amino acids 40-116;

(4) amino acids 1-99 and 112-146;

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

(6) the truncated OB analog of subpart (vi)(1) 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;

(7) the truncated analog of subpart (vi)(2) having one or more of amino acids 4, 8, and 32 substituted with another amino acid;

(8) the truncated analog of subpart (vi)(3) 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;

(9) the truncated analog of subpart (vi)(4) 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;

(10) the truncated analog of subpart (vi)(5) 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

(11) the truncated analog of subparts (vi)(1)-(10) having an N-terminal methionyl residue;

(vii) a nucleic acid sequence encoding an OB protein derivative of any of subparts (i) through (vi) comprised of a polyamino acid connected to the protein moiety; and

(viii) a derivative of subpart (vii) wherein said polyamino acid is attached at solely the N-terminus of said protein moiety.