Modulators of body weight, corresponding nucleic acids and proteins, and diagnostic and therapeutic uses thereof

• US 6,309,853 B1
• Filed: 06/07/1995
• Issued: 10/30/2001
• 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): Yucel, Remy
• Application Number:
  US08/483,211
• Time in Patent Office: 2,337 Days
• Field of Search: 536/23.5, 536/23.1, 536/23.51, 536/24.3, 435/320.1, 435/325, 435/252.3, 435/252.31, 435/252.33, 435/252.34, 435/252.35, 435/255.1, 435/358, 435/365, 435/354, 435/355, 435/69.1
• US Class Current: 435/69.1
• 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: A61P 3/00 Drugs for disorders of the metabolism of the blood or the extracellular fluid A61P7/00
• CPC Class Codes: A61P 3/04 Anorexiants; Antiobesity agents
• CPC Class Codes: A61P 3/06 Antihyperlipidemics
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 9/12 Antihypertensives
• 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 body weight, and to diagnostic and therapeutic uses of such modulators. In its broadest aspect, the present invention relates to nucleotide sequences corresponding to the murine and human OB gene, and two isoforms thereof, and proteins expressed by such nucleotides or degenerate variations thereof, that demonstrate the ability to participate in the control of mammalian body weight and that have been postulated to play a critical role in the regulation of body weight and adiposity. The present invention further provides nucleic acid molecules for use as molecular probes or as primers for polymerase chain reaction (PCR) amplification. In further aspects, the present invention provides cloning vectors and mammalian expression vectors comprising the nucleic acid molecules of the invention. The invention further relates to host cells transfected or transformed with an appropriate expression vector and to their use 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 isolated nucleic acid encoding an analog of an OB polypeptide, 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.

12. The isolated nucleic acid according to claim 1, 2, 3, 4-6 or 7 which is a DNA molecule.

13. A detectably labeled nucleic acid molecule of at least 10 nucleotides hybridizable under moderate stringency hybridization conditions to a DNA molecule according to claim 12.

14. A vector which comprises a DNA molecule according to claim 12.

15. An expression vector which comprises a DNA molecule according to claim 12 operatively associated with an expression control sequence.

16. An unicellular host transformed or transfected with an expression vector of claim 15.

17. A unicellular host according to claim 16, wherein the unicellular host is selected from the group consisting of bacteria, yeast, mammalian cells, plant cells, insect cells, and human cells in tissue culture.

18. The unicellular host of claim 16, wherein the unicellular host is selected from the group consisting of E. coli, Pseudomonas, Bacillus, Streptomyces, yeast, CHO, R1.1, B-W, LM, COS 1. COS 7, BSC1, BSC40, BMT10, and Sf9 cells.

19. A unicellular host according to claim 16 wherein the unicellular host is a yeast host selected from the group consisting of Saccharomyces, Pichia, Candida, Hansenula and Torulopsis.

20. A method for preparing an OB polypeptide comprising:

(a) culturing a cell according to claim 16 under conditions that provide for expression of the OB polypeptide; and

(b) recovering the expressed OB polypeptide.

21. The method according to claim 20 wherein the cell is a bacterium or a yeast.

2. An isolated nucleic acid encoding an analog of an OB polypeptide, 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, valine, 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.

3. An isolated nucleic acid encoding an analog of an OB polypeptide, capable of modulating body weight, comprising amino acids 22-167 of SEQ ID NOS:

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

(a) one or more aspartic acid residues is substituted with glutamic acid;

(b) one or more isoleucine residues is substituted with leucine;

(c) one or more glycine or valine residues is substituted with alanine;

(d) one or more arginine residues is substituted with histidine;

(e) one or more tyrosine or phenylalanine residues is substituted with tryptophan;

(f) one or more of residues 121 through 128 is substituted with glycine or alanine; and

(g) one or more residues at positions 54 through 60 or 118 through 166 is substituted with lysine, glutamic acid, cysteine, or proline.

4. An isolated nucleic acid encoding an OB polypeptide comprising amino acids 22-167 of SEQ ID Nos. 2 or 4 or amino acids 22-166 of SEQ ID Nos. 5 or 6 and capable of modulating body weight said OB polypeptide having 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) a glycine-serine-proline sequence.

5. An isolated nucleic acid encoding an analog of an OB polypeptide, 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) a glycine-serine-proline sequence.

6. An isolated nucleic acid encoding an analog of an OB polypeptide comprising an amino acid sequence of SEQ ID NOS:

2 or 4, wherein the analog is a truncated analog selected from the group consisting of polypeptides wherein;

(a) one or more residues at positions 121 to 128 are deleted;

(b) residues 1-116 are deleted;

(c) residues 1-21 and 54 to 167 are deleted;

(d) residues 1-60 and 117 to 167 are deleted;

(e) residues 1-60 are deleted;

(f) residues 1-53 are deleted;

(g) an analog of subpart (a) wherein residues 1-21 are deleted; and

(h) an analog of subpart (a) through (g) having an N-terminal amino acid or amino acid sequence selected from the group consisting of;

(1) methionine,

(2) SEQ ID NO;

38,

(3) SEQ ID NO;

98,

(4) SEQ ID NO;

26,

(5) SEQ ID NO;

27,

(6) SEQ ID NO;

28,

(7) SEQ ID NO;

99, and

(8) a glycine-serine-proline sequence.

7. An isolated nucleic acid encoding an analog of an OB polypeptide, 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.

8. An isolated nucleic acid encoding an analog of an OB polypeptide, 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.

9. An isolated nucleic acid encoding an analog of an OB polypeptide, capable of modulating body weight, comprising amino acids 22-166 of SEQ ID NOS:

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

(a) one or more aspartic acid residues is substituted with glutamic acid;

(b) one or more isoleucine residues is substituted with leucine;

(c) one or more glycine or valine residues is substituted with alanine;

(d) one or more arginine residues is substituted with histidine;

(e) one or more tyrosine or phenylalanine residues is substituted with tryptophan;

(f) one or more of residues 120 through 127 is substituted with glycine or alanine; and

(g) one or more residues at positions 53 through 59 or 117 through 165 is substituted with lysine, glutamic acid, cysteine, or proline.

10. An isolated nucleic acid encoding an analog of an OB polypeptide comprising an amino acid sequence of SEQ ID NOS:

5 or 6, wherein the analog is a truncated analog selected from the group consisting of polypeptides wherein;

(a) one or more residues at positions 120 to 127 are deleted;

(b) residues 1-115 are deleted;

(c) residues 1-21 and 53 to 166 are deleted;

(d) residues 1-59 and 116 to 166 are deleted;

(e) residues 1-59 are deleted;

(f) residues 1-52 are deleted;

(g) an analog of subpart (a) wherein residues 1-21 are deleted; and

(h) an analog of subpart (a) through (g) having an N-terminal amino acid or amino acid sequence selected from the group consisting of;

(1) methionine,

(2) SEQ ID NO;

38,

(3) SEQ ID NO;

98,

(4) SEQ ID NO;

26,

(5) SEQ ID NO;

27,

(6) SEQ ID NO;

28,

(7) SEQ ID NO;

99, and

(8) a glycine-serine-proline sequence.

11. An isolated nucleic acid encoding an analog of an OB polypeptide, 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 and wherein said analog has an N-terminal amino acid sequence selected from the group consisting;

(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) a glycine-serine-proline sequence.