Ob polypeptides, modified forms and compositions thereto

• US 6,471,956 B1
• Filed: 06/07/1995
• Issued: 10/29/2002
• Est. Priority Date: 08/17/1994
• Status: Expired due to Term

• Current Assignee: Rockefeller University
• Original Assignee: Rockefeller University
• Inventors: Proenca, Ricardo, Friedman, Jeffrey M., Zhang, Yiying
• Primary Examiner(s): Saoud, Christine J.
• Application Number:
  US08/488,225
• Time in Patent Office: 2,701 Days
• Field of Search: 530/300, 530/350, 530/351, 530/402, 424/85.1, 514/2, 514/8, 514/12
• US Class Current: 424/85.1
• CPC Class Codes: A01K 2217/05 Animals comprising random inserted nucleic acids (transgenic)
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• CPC Class Codes: A61K 8/64 Proteins; Peptides; Derivatives or degradation products thereof
• CPC Class Codes: A61Q 19/06 for countering cellulitis
• CPC Class Codes: C07K 14/5759 Products of obesity genes, e.g. leptin, obese (OB), tub, fat
• CPC Class Codes: C07K 16/18 against material from animals or humans
• CPC Class Codes: C07K 16/26 against hormones ; against hormone releasing or inhibiting factors
• CPC Class Codes: C07K 2317/34 Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers
• CPC Class Codes: C12Q 2600/158 Expression markers

Abstract

The present invention relates generally to the control of body weight of animals including mammals and humans, and more particularly to materials identified herein as modulators of weight, and of the diagnostic and therapeutic uses to such modulators. In its broadest aspect, the present invention relates to the elucidation and discovery of nucleotide sequences, and proteins putatively expressed by such nucleotides or degenerate variations thereof, that demonstrate the ability to participate in the control of mammalian body weight. The nucleotide sequences in object represent the genes corresponding to the murine and human ob gene, that have been postulated to play a critical role in the regulation of body weight and adiposity. Preliminary data, presented herein, suggests that the polypeptide product of the gene in question functions as a hormone. The present invention further provides nucleic acid molecules for use as molecular probes, or as primers for polymerase chain reaction (PCR) amplification, i.e., synthetic or natural oligonucleotides. In further aspects, the present invention provides a cloning vector, which comprises the nucleic acids of the invention; and a bacterial, insect, or a mammalian expression vector, which comprises the nucleic acid molecules of the invention, operatively associated with an expression control sequence. Accordingly, the invention further relates to a bacterial or a mammalian cell transfected or transformed with an appropriate expression vector, and correspondingly, to the use of the above mentioned constructs in the preparation of the modulators of the invention. Also provided are antibodies to the ob polypeptide. Moreover, a method for modulating body weight of a mammal is provided.

Claims

1. An immunogenic fragment of an OB polypeptide, said fragment selected from the group consisting of:

(a) SEQ ID NO;

18;

(b) SEQ ID NO;

19;

(c) SEQ ID NO;

20; and

(d) SEQ ID NO;

21.

2. An OB polypeptide analog, capable of modulating body weight, comprising amino acids 22-167 of SEQ ID NOS:

2 or 4, wherein one or more amino acids selected from the group consisting of amino acids 53, 56, 71, 85, 89, 92, 95, 98, 110, 118, 121, 122, 126, 127, 128, 129, 132, 139, 157, 159, 163, and 166 is substituted with another amino acid.

9. A recombinant OB polypeptide or polypeptide analog according to any of claims 2, 3, 4, 5, 6, 7, or 8.

10. A chemically synthesized OB polypeptide or polypeptide analog according to any of claims 2, 3, 4, 5, 6, 7, or 8.

11. An OB polypeptide or polypeptide analog according to any of claims 2, 3, 4, 5, 6, 7, or 8 having one or more polymers attached thereto.

12. The polypeptide or analog of claim 11, wherein at least one of said polymers is a water soluble polymer.

13. A pharmaceutical composition comprising an OB polypeptide or polypeptide analog according to claim 12 and a pharmaceutically acceptable carrier.

14. The polypeptide or analog of claim 12, wherein the water soluble polymer is polyethylene glycol.

15. A pharmaceutical composition comprising an OB polypeptide or polypeptide analog according to claim 14 and a pharmaceutically acceptable carrier.

16. The polypeptide or analog of claim 14, which is mono-, di-, tri- or tetrapegylated.

17. A pharmaceutical composition comprising an OB polypeptide or polypeptide analog according to claim 16 and a pharmaceutically acceptable carrier.

18. The polypeptide or analog of claim 16, which is N-terminal monopegylated.

19. A pharmaceutical composition comprising an OB polypeptide or polypeptide analog according to claim 18 and a pharmaceutically acceptable carrier.

20. The polypeptide or analog of claim 11, wherein at least one of said polymers is a pharmaceutically acceptable polymer.

21. A pharmaceutical composition comprising an OB polypeptide or polypeptide analog according to claim 20 and a pharmaceutically acceptable carrier.

22. The polypeptide or analog of claim 20, wherein said pharmaceutically acceptable polymer is a water soluble polymer.

23. A pharmaceutical composition comprising an OB polypeptide or polypeptide analog according to claim 22 and a pharmaceutically acceptable carrier.

24. The polypeptide or analog of claim 11, wherein at least one of said polymers is a polyamino acid.

25. A pharmaceutical composition comprising an OB polypeptide or polypeptide analog according to claim 24 and a pharmaceutically acceptable carrier.

26. The polypeptide or analog of claim 11, wherein at least one of said polymers is a branched or unbranched polymer.

27. A pharmaceutical composition comprising an OB polypeptide or polypeptide analog according to claim 26 and a pharmaceutically acceptable carrier.

28. The polypeptide or analog of claim 11, wherein at least one of said polymers is N-terminally attached to said polypeptide or polypeptide analog.

29. A pharmaceutical composition comprising an OB polypeptide or polypeptide analog according to claim 28 and a pharmaceutically acceptable carrier.

30. The polypeptide or analog of claim 11, wherein at least one of said polymers is C-terminally attached to said polypeptide or polypeptide analog.

31. A pharmaceutical composition comprising an OB polypeptide or polypeptide analog according to claim 30 and a pharmaceutically acceptable carrier.

32. The polypeptide or analog of claim 11, wherein at least one of said polymers is attached by at least one covalent bond through an amino acid residue of said polypeptide or polypeptide analog via a reactive group.

33. A pharmaceutical composition comprising an OB polypeptide or polypeptide analog according to claim 32 and a pharmaceutically acceptable carrier.

34. The polypeptide or analog of claim 32, wherein said reactive group is a free amino or carboxyl group.

35. A pharmaceutical composition comprising an OB polypeptide or polypeptide analog according to claim 34 and a pharmaceutically acceptable carrier.

36. The polypeptide or analog of claim 32, wherein said amino acid residue is selected from the group consisting of lysine, aspartic acid and glutamic acid.

37. A pharmaceutical composition comprising an OB polypeptide or polypeptide analog according to claim 36 and a pharmaceutically acceptable carrier.

38. The polypeptide or analog of claim 11, wherein the number of said polymers attached is one, two, three or four.

39. A pharmaceutical composition comprising an OB polypeptide or polypeptide analog according to claim 38 and a pharmaceutically acceptable carrier.

40. The polypeptide or analog of claim 38 wherein said two, three or four polymers are the same polymer.

41. A pharmaceutical composition comprising an OB polypeptide or polypeptide analog according to claim 40 and a pharmaceutically acceptable carrier.

42. The polypeptide or analog of claim 38 wherein said two, three or four polymers are two, three or four different polymers.

43. A pharmaceutical composition comprising an OB polypeptide or polypeptide analog according to claim 42 and a pharmaceutically acceptable carrier.

44. A pharmaceutical composition comprising an OB polypeptide or polypeptide analog according to claim 11 and a pharmaceutically acceptable carrier.

45. A pharmaceutical composition comprising an OB polypeptide or polypeptide analog according to any of claims 2, 3, 4, 5, 6, 7, or 8 and a pharmaceutically acceptable carrier.

3. An OB polypeptide analog, capable of modulating body weight, comprising amino acids 22-167 of SEQ ID NO:

4 wherein the analog is selected from the group consisting of polypeptides wherein;

(a) the serine residue at position 53 is substituted with glycine, alanine, valine, cysteine, methionine, or threonine;

(b) the serine residue at position 98 is substituted with glycine, alanine, saline, cysteine, methionine, or threonine; and

(c) the arginine residue at position number 92 is substituted with asparagine, lysine, histidine, glutamine, glutamic acid, aspartic acid, serine, threonine, methionine, or cysteine.

4. An OB polypeptide capable of modulating body weight, comprising amino acids 22-167 of SEQ ID NOS:

2 or 4 or amino acids 22-166 of SEQ ID NO;

5 or 6, wherein said polypeptide has an N-terminal amino acid sequence selected from the group consisting of;

(a) SEQ ID NO;

38;

(b) SEQ ID NO;

98;

(c) SEQ ID NO;

26;

(d) SEQ ID NO;

27;

(e) SEQ ID NO;

28;

(f) SEQ ID NO;

99; and

(g) glycine-serine-proline.

5. An OB polypeptide analog, capable of modulating body weight, comprising amino acids 22-167 of SEQ ID NOS:

2 or 4, wherein one or more amino acids selected from the group consisting of amino acids 53, 56, 71, 85, 89, 92, 95, 98, 110, 118, 121, 122, 126, 127, 128, 129, 132, 139, 157, 159, 163, and 166 is substituted with another amino acid and wherein said analog has an N-terminal amino acid sequence selected from the group consisting of;

(a) SEQ ID NO;

38;

(b) SEQ ID NO;

98;

(c) SEQ ID NO;

26;

(d) SEQ ID NO;

27;

(e) SEQ ID NO;

28;

(f) SEQ ID NO;

99; and

(g) glycine-serine-proline.

6. An OB polypeptide analog, capable of modulating body weight, comprising amino acids 22-166 of SEQ ID NOS:

5 or 6, wherein one or more amino acids selected from the group consisting of amino acids 52, 55, 70, 84, 88, 91, 94, 97, 109, 117, 120, 121, 125, 126, 127, 128, 131, 138, 156, 158, 162, and 165 is substituted with another amino acid.

7. An OB polypeptide analog, capable of modulating body weight, comprising amino acids 22-166 of SEQ ID NO:

6 wherein the analog is selected from the group consisting of polypeptides wherein;

(a) the serine residue at position 52 is substituted with glycine, alanine, valine, cysteine, methionine, or threonine;

(b) the serine residue at position 97 is substituted with glycine, alanine, valine, cysteine, methionine, or threonine; and

(c) the arginine residue at position number 91 is substituted with asparagine, lysine, histidine, glutamine, glutamic acid, aspartic acid, serine, threonine, methionine, or cysteine.

8. An OB polypeptide analog, capable of modulating body weight, comprising an amino acids 22-166 of SEQ ID NOS:

5 or 6, wherein one or more amino acids selected from the group consisting of amino acids 52, 55, 70, 84, 88, 91, 94, 97, 109, 117, 120, 121, 125, 126, 127, 128, 131, 138, 156, 158, 162, and 165 is substituted with another amino acid and wherein said analog has an N-terminal amino acid sequence selected from the group consisting of;

(a) SEQ ID NO;

38;

(b) SEQ ID NO;

98;

(c) SEQ ID NO;

26;

(d) SEQ ID NO;

27;

(e) SEQ ID NO;

28;

(f) SEQ ID NO;

99; and

(g) glycine-serine-proline.