Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Lymphokines – e.g. – interferons – interlukins – etc.
Reexamination Certificate
1996-12-05
2001-05-01
Scheiner, Laurie (Department: 1648)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Lymphokines, e.g., interferons, interlukins, etc.
C530S350000, C530S324000, C530S399000, C424S085200, C514S002600
Reexamination Certificate
active
06225446
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to compositions which include variants of human Ob, a protein exhibiting cytokine—like structure, which functions in regulating metabolism and weight. In particular, it provides agonists and/or antagonists of said protein.
BACKGROUND OF THE INVENTION
Recent studies have identified a protein product, designated Ob, which when injected into a mouse causes that animal to lose weight. See, e.g., Barinaga (1995)
Science
269:475-476; Zhang, et al. (1994)
Nature
372:425-432; Pelleymounter, et al. (1995)
Science
269:540-543; Halaas, et al. (1995)
Science
269:543-546; and Campfield, et al. (1995)
Science
269:546-548. Mice deficient in active Ob gene product are grossly obese, but injection of the Ob protein causes the mice to curb their eating and shed fat.
Physiologically, it appears that the Ob protein has two activities, it decreases appetite and increases energy use, leading to metabolic deficit and loss of weight.
In the United States, up to one third of all Americans are overweight. Obesity is the cause of a myriad of serious health problems, including, e.g., sleep apnea, adult-onset diabetes, and heart disease. See, e.g., The Merck Manual. The availability of agonists and antagonists will be used to modulate these processes. The present invention provides these, as well as other proteins, useful, e.g., in determining the structure and mechanisms of weight and appetite regulation.
SUMMARY OF THE INVENTION
The present invention provides molecules which can serve as an agonist or antagonist for the Ob proteins. These agonists and antagonists will be useful in regulating weight and appetite regulation, and may be important in other hematopoietic or immunological function. In certain circumstances, these molecules will also have in vitro or in vivo therapeutic effects.
The present invention is based, in part, upon the recognition by structural analysis of the Ob sequence that it shares structural homology with a subclass of proteins known as cytokines. Cytokines are molecules which mediate differentiation or other signals, typically between he circulating component of the mammalian circulatory system. Ob belongs to the subgroup of hematopoietic cytokines, which includes IL-2, IL-4, IL-5, GM-CSF, and growth hormone.
In particular, this insight leads to recognition of which specific amino acid residues of a cytokine—like protein are important in receptor binding. It embraces various mutein agonists and antagonists of natural ligands, e.g., specific mutations (muteins) of the natural sequences, fusion proteins, and chemical mimetics. It is also directed to DNAs encoding such variant proteins. Various uses of these different protein or nucleic acid compositions are also provided.
The present invention provides a mutein of a mammalian Ob which comprises a variation in sequence at a position in a solvent exposed residue in helix A; in a solvent exposed residue in helix C; in extruded loop from 100 to 108; or in a solvent exposed residue in helix D. In preferred embodiments, the position is in helix A; the mammalian Ob is mouse Ob, rat Ob, or human Ob; the Ob has a sequence of SEQ ID NO: 1, 2, or 3; the variation is a non-conservative substitution; the substitution is at a position corresponding to D
8
, D
9
, K
11
, T
12
, K
15
, T
16
, V
18
, T
19
, I
21
, N
22
, N
78
, H
78
, D
79
, E
81
, N
82
, R
84
, D
85
, L
86
, V
89
, V
123
, V
124
, S
127
, R
128
, Q
130
, G
131
, S
132
, Q
134
, D
135
, or W
138
; the substitution is at a position corresponding to D
8
, D
9
, K
11
, T
12
, K
15
, T
16
, T
19
, E
81
, N
82
, R
84
, D
85
, R
128
, Q
130
, Q
134
, or D
135
; the substitution is selected from D
8
K, D
9
K, K
11
E, T
12
E, K
15
E, T
16
E, T
19
E, E
81
K, N
82
D, R
84
E, D
85
K, R
128
E, Q
130
K, Q
134
K, or D
135
K; and/or the variation is in the extruded loop at a position between 100 and 108, including deletion of amino acid residues
121
-
129
in SEQ ID NO: 1, 2, or 3.
In other embodiments, the mutein exhibits at least about a 30% decrease in biological activity; exhibits less than about 80% maximal agonist activity; or exhibits at a 100-fold excess antagonist activity of said mammalian Ob by at least about 50%; will have a sequence variation which disrupts helical structure of helix A, C, or D; or will competitively compete with mammalian OB.
The invention also embraces a pharmaceutical composition comprising the mutein a pharmaceutically acceptable carrier or excipient. It also embraces a nucleic acid encoding these muteins.
In other embodiments, the invention provides a method of antagonizing the biological activity of a mammalian Ob on a cell comprising contacting the cell with a described mutein. Preferably the biological activity is receptor binding and the method includes decreasing appetite or increasing metabolic rate in a mammal.
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Naoko Arai, et al., Gen Bank Accession No. M23442, pp. 1-4, Jan. 1995.
Marcia Barinaga,Science,269:475-476, Jul. 28, 1995, “‘Obese’ Protein Slims Mice”.
J. Fernando Bazan,Immunol. Today,11:350-354, 1990, “Haemopoietic receptors and helical cytokines”.
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Streamson C. Chua Jr., et al.,Science,271:994-996, Feb. 16, 1996 “Phenotypes of Mouse diabetes and Rat fatty Due to Mutations in the OB (Leptin) Receptor”.
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Mary Ann Pelleymounter, et al.,Science,269:540-543, Jul. 28, 1995 “Effects of the obese Gene Product on Body Weight Regulation in ob/ob Mice”.
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P.H. Seeburg, Gen Bank Accession No. M13438, pp. 1-3, Apr. 1996.
Stephen R. Sprang, et al.,Curr. Opin. Struct. Biol.3:815-827, 1993 “Cytokine structural taxonomy and mechanisms of receptor engagement”.
T. Tanabe, et al., Gen Bank Accession No. J03478, pp. 1-2, Aug. 1995.
G.G. Wong, et al., Gen Bank Accession No. M37435, pp. 1-2, Nov. 1994.
Y. Zhang, et al., Gen Bank Accession No. U18812, pp. 1-2, Mar. 1995.
Y. Zhang et al., Gen Bank Accession No. U18915, p. 1, Jan. 1995.
Yiying Zhang, et al.,Nature372:425-432, Dec. 1, 1994 “Positional cloning of the mouse obese gene and its human homologue”.
Sandra M. Zurawski, et al.,J. Immunol.137:3354-3360, Nov. 15, 1986 “Alternations in the Amino-Terminal Third of Mouse Interleukin 2: Effects on Biological Activity and Immunoreactivity”.
Sandra M. Zurawski, et al.,EMBO J.7:1061-1069, 1988 “Identification for three critical regions within mouse interleukin 2 by fine structural deletion analysis”.
Sandra M. Zurawaki, et al.,EMBO J.8:2583-2590, 1989, “Mouse interleukin-2 structure-function studies: substitutions in the first &agr;-helix can specifically inactivate p70 receptor binding and mutations in the fifth &agr;-helix can specifically inactivate p55 receptor binding”.
Sandra M. Zurawski, et al.,EMBO J.12:2663-2670, 1993 “Receptors for interleukin-13 and interleukin-4 are complex and share a novel component that functions in signal transduct
Altmann Scott W.
Bazan J. Fernando
Kastelein Robert A.
Rock Fernando L.
Ching Edwin P.
Scheiner Laurie
Schering Corporation
Wang Hugh
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