Methods of identifying or screening for agents that binds...

Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...

Reexamination Certificate

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C530S350000, C536S023500, C435S007210, C435S007800, C435S325000, C435S356000, C435S357000, C435S361000, C435S365000, C435S366000, C435S369000, C435S192000, C514S012200

Reexamination Certificate

active

06770444

ABSTRACT:

BACKGROUND OF THE INVENTION
Throughout this application, various references are referred to within parentheses. Disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains. Full bibliographic citations for these references may be found at the end of this application, preceding the sequence listing and the claims.
The ob gene has recently been cloned and shown to encode a 146 amino acid protein called leptin which is secreted into the blood exclusively by white fat adipocytes. The leptin receptor has also recently been cloned and shown to exist as several splice variants. The functional splice variant (Ob-Rb) is present in several tissues including hypothalamus, adipocytes and kidney. The short form splice variant (Ob-Ra) has a more ubiquitous tissue distribution and is more abundant than the functional splice variant. The role of Ob-Ra is unknown but this splice variant may serve as a leptin transport system in choroid plexus, kidney and perhaps also the lung. A third major splice variant (Ob-Re) has also been described in mice which encodes only the extracellular domain of the receptor, giving rise to a soluble protein in the circulation which may function there as a leptin binding/buffering system.
Circulating leptin acts as an antiobesity agent by restraining appetite and altering metabolic processes to burn fat. The hypothalamus appears to be the major target tissue for the hormone since leptin receptors are present there and intracerebroventricular injection of leptin leads to a reduction of food intake. Recent evidence suggests that at least part of the reduction in food intake produced by leptin may be due to a decrease in central neuropeptide Y. It is unclear whether the changes in metabolism produced by leptin are mediated by actions only on the brain or also involve direct effects upon peripheral tissues. Although the mechanism of action has not been fully elucidated, leptin may be the long sought after satiety factor released from the periphery (i.e. the adipocytes) to regulate long-term body weight. As body weight and fat mass increase, more leptin is secreted which may inhibit appetite and increase metabolism to bring the fat mass back to a certain set point. The incapacity to express a functional leptin is the cause of obesity in the ob/ob mouse. Defects in the functional leptin receptor such as those found in the db/db mouse and the fa/fa rat are responsible for the obesity observed in these animal models.
The observation that obese animals and man, although having high plasma leptin levels remain overweight, may suggest the development of ‘resistance’ to the actions of leptin. This may occur at the level of the brain and be due to saturation of the leptin uptake system. Alternatively, ‘resistance’ may be due to the presence of a circulating binding protein which by buffering leptin might reduce its actions.
The single gene defects of rodents described above may play only a minor role in human obesity. However, leptin is present in man, thus, pharmacological stimulation of the leptin pathway has the potential to reduce body weight in man by inhibiting food intake and diminishing the size of the body fat stores. Such therapeutic interventions could be achieved either by enhancing leptin release from the adipocytes, preventing the breakdown or clearance of leptin, preventing interaction of leptin binding to the soluble binding protein, by administration of leptin mimics or by stimulating events downstream of the leptin receptor. On the other hand, pharmacological inhibition of leptin action or production may have the potential to increase food intake and body weight in man. Evidence to support this comes from the ob/ob and db/db mouse and the fa/fa rat in which the actions of leptin are not apparent. These animals are obese and hyperphagic. Applicants now report the isolation of a novel human Ob-Re receptor, referred to herein as “hOb-Re” or the “polypeptide.” This discovery provides a novel approach to the treatment of eating disorders, both by therapeutic administration of the soluble human Ob-Re receptor to subjects suffering from such disorders, and through the use of heterologous expression systems to develop high-affinity compounds that could serve as therapeutic agents for such disorders.
SUMMARY OF THE INVENTION
This invention is directed to an isolated nucleic acid which encodes a polypeptide comprising the amino acid sequence shown in
FIG. 5
(Seq. I.D. No. 10) or a polypeptide having a sequence which varies therefrom by no more than 15 amino acids, such amino acid variations not involving amino acid positions 799-804 and not changing the functional properties of the polypeptide.
This invention is additionally directed to a nucleic acid which comprises the nucleic acid of above linked to a nucleic acid encoding a polypeptide corresponding to an artificial transmembrane region of a receptor which is not an Ob receptor.
This invention is additionally directed to a nucleic acid which comprises the nucleic acid of above linked to nucleic acid encoding a polypeptide corresponding to an artificial intracellular domain of a receptor which is not an Ob receptor.
This invention is additionally directed to purified polypeptides encoded by the nucleic acid of this invention.
This invention is additionally directed to vectors comprising the nucleic acid of this invention.
This invention is additionally directed to cells comprising the vector of this invention.
This invention is additionally directed to a membrane preparation isolated from the cell of this invention.
This invention is additionally directed to a nucleic acid probe comprising at least 15 nucleotides, which probe has a unique sequence corresponding to a sequence present within (a) the nucleic acid sequence from nucleotide number 2395 through nucleotide number 2412 of
FIG. 4
(Seq. I.D. No. 9) or (b) a reverse complement thereof.
This invention is additionally directed to an antisense oligonucleotide having a unique sequence corresponding to a sequence present within (a) the nucleic acid sequence from nucleotide number 2395 through nucleotide number 2412 of
FIG. 4
(Seq. I.D. No. 9) or (b) a reverse complement thereof.
This invention is additionally directed to an antibody capable of specifically binding to the polypeptide containing at least a unique sequence corresponding to a sequence present within the amino acid sequence from amino acid number 799 through amino acid number 804 of
FIG. 5
(Seq. I.D. No. 10).
This invention is additionally directed to a pharmaceutical composition comprising an amount of the oligonucleotide effective to reduce expression of a polypeptide and a pharmaceutically acceptable carrier.
This invention is additionally directed to a pharmaceutical composition which comprises an amount of the antibody effective to block binding of a ligand to the polypeptide and a pharmaceutically acceptable carrier.
This invention is additionally directed to a transgenic nonhuman mammal expressing a nucleic acid of this invention.
This invention is directed to a process for identifying a chemical compound which specifically binds to a polypeptide of this invention, which comprises contacting the polypeptide with the compound under conditions suitable for binding, and detecting specific binding of the chemical compound to the polypeptide.
This invention is additionally directed to a process involving competitive binding for identifying a chemical compound which specifically binds to a polypeptide of this invention which comprises separately contacting the polypeptide, with both the chemical compound and a second chemical compound known to bind to the polypeptide, and with only the second chemical compound, under conditions suitable for binding of both compounds, and detecting specific binding of the chemical compound to the polypeptide, a decrease in the binding of the second chemical compound to the polypeptide in the presence of the chemical compound indicating

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