WO2001025797A2 - Methods of evaluating whether a test agent is an agent affecting a leptin receptor - Google Patents

Abstract

The present invention provides methods of evaluating whether a test agent is an agent affecting the activity of OBR. In said methods, a mammalian cell is cultured and exposed to the test agent. The mammalian cell has a polynucleotide encoding a chimeric receptor subunit comprising an extracellular domain of an OBR subunit, a transmembrane domain of a cytokine receptor subunit and an intracellular domain of a receptor subunit selected from a non-OBR homodimeric cytokine receptor subunit, gp130 and IL-2R β subunit; and has a proliferation dependency on OB. By measuring the proliferation of the mammalian cell, it can be evaluated whether the test agent is an agent which can affect the activity of OBR.

Description

DESCRIPTION

METHODS OF EVALUATING WHETHER A TEST AGENT

IS AN AGENT AFFECTING A LEPTIN RECEPTOR

BACKGROUND OF THE INVENTION

Field of the Invention

The present invention relates to methods of evaluating whether a test agent is an

agent affecting the activity of a leptin receptor.

Description of Related Art

A leptin receptor (OBR) is a particular homodimeric cytokine receptor that

participates in regulating mammalian body weight and hematopoiesis by binding to its

cognate ligand, leptin (OB). OB is a type of cytokine, described in Y. Zhang, 1994,

Nature 372:425-432. Typically, OB is produced in fat cells and is released into the

blood stream, in order to reach a target cell containing OBR. OBR may then be activated by binding to OB and by having particular subunits thereof assembled into a

homodimer complex. When activated, OBR can transduce a particular signal into the

cell along a particular and intricate signal transduction pathway.

It is known that chimeric receptor complexes have proven quite productive for

analyzing the mechanism of cytokine receptor activation (Tartaglia et al.,

WO97/19952; Morella et al., 1995, J. Biol. Chem. 270:8298-8310; Baumann et al.,

1994, Mol. Cell. Biol. 14:138-146 ; Vigon et al, 1993, Oncogene 8:2607-2615). Certain characteristics of cytokine receptors allow a chimeric form thereof to transduce a particular signal by having the chimeric receptor bind to a different cognate ligand. In such cases, a reporter gene may be utilized as a major target of the signal transduced

from the chimeric cytokine receptor.

The use of a reporter gene often leads to superfluous work. The reporter gene

usually requires that a cell which can properly host the reporter gene be chosen as the

host cell of the chimeric cytokine receptor. When the host cell is a mammalian host cell,

the host cell may tend to loose therefrom the plasmid containing the reporter gene after

an extended amount of time. Such properties of such host cells can often necessitate

reintroducing the reporter gene into the host cell. Such properties may also result in

inadvertently culturing a batch of the host cells, in which a significant amount of host cells in the batch may contain no reporter gene. Furthermore, a reporter gene further

distances a system utilizing a chimeric cytokine receptor away from the native system

of the cytokine receptor. A system which is less alike the native system introduces

uncertainties which may decrease the reliability of the results achieved therefrom.

SUMMARY OF THE INVENTION

The present invention provides the following.

1. A method of evaluating whether a test agent is an agent affecting the activity of

OBR, said method comprising:

culturing a mammalian cell having:

an OB bindable chimeric receptor comprising an extracellular domain of an OBR subunit, a transmembrane domain of a cytokine receptor subunit and an

intracellular domain of a receptor subunit selected from a non-OBR homodimeric

cytokine receptor subunit, gpl30 and IL-2R |3 subunit; and

a proliferation dependency on OB; exposing the test agent to the mammalian cell; and

measuring the proliferation of the mammalian cell.

2. The method according to the above 1, wherein the intracellular domain is an

intracellular domain of a non-OBR homodimeric cytokine receptor subunit.

3. The method according to the above 1, wherein the intracellular domain is an

intracellular domain of a receptor subunit selected from gpl30 and IL-2R β subunit.

4. The method according to the above 1, wherein the intracellular domain is an

intracellular domain of a receptor subunit selected from a TPOR subunit, EPOR subunit, G-CSFR subunit, GHR subunit and PRLR subunit.

5. The method according to the above 1, wherein the mammalian cell is cultured

with a culturing medium containing substantially no cytokine proteins activating the

chimeric receptor.

6. The method according to the above 1, wherein the mammalian cell is cultured

with a culturing medium comprising OB.

7. A mammalian cell having:

a polynuceotide encoding a chimeric receptor subunit comprising an extracellular

domain of an OBR subunit, a transmembrane domain of a cytokine receptor subunit and

an intracellular domain of a receptor subunit selected from a non-OBR homodimeric

cytokine receptor subunit, gpl30 and IL-2R β subunit; and

a proliferation dependency on OB.

8. The mammalian cell according to the above 7, wherein the mammalian cell is

derived from a hematopoietic mammalian cell.

9. The mammalian cell according to the above 7, wherein the mammalian cell is derived from a mammalian lymphocyte. 10. The mammalian cell according to the above 7, wherein the mammalian cell is

derived from a cell selected from BaF-3, TALL-101, OCl-Ly9, OCl-Lyl3.1, FDC-Pl,

TF-1, UT-7 and F-36-P.

11. The mammalian cell according to the above 10, wherein the mammalian cell is

derived from BaF-3.

12. The mammalian cell according to the above 7, wherein the intracellular domain is an intracellular domain of a non-OBR homodimeric cytokine receptor subunit.

13. The mammalian cell according to the above 7, wherein the intracellular domain

is an intracellular domain of a receptor subunit selected from gpl30 and IL-2R β

subunit.

14. The mammalian cell according to the above 7, wherein the intracellular domain

is an intracellular domain of a receptor subunit selected from a TPOR subunit, EPOR

subunit, G-CSFR subunit, GHR subunit and PRLR subunit.

15. The mammalian cell according to the above 7, wherein said transmembrane

domain is a transmembrane domain of a receptor subunit selected from a homodimeric

cytokine receptor subunit, gpl30 and IL-2R β subunit.

16. The mammalian cell according to the above 15, wherein the transmembrane

domain is a transmembrane domain of an OBR subunit.

17. A mammalian cell having:

a polynucleotide encoding a chimeric receptor subunit comprising an extracellular

domain of an OBR subunit and intracellular and transmembrane domains of a receptor

subunit selected from a non-OBR homodimeric cytokine receptor subunit, gpl30 and

IL-2R β subunit; and

a proliferation dependency on OB. 18. The mammalian cell according to the above 17, wherein the intracellular and

transmembrane domains are intracellular and transmembrane domains of a receptor

subunit selected from a TPOR subunit, EPOR subunit, G-CSFR subunit, GHR subunit

and PRLR subunit.

19. A mammalian cell having:

an expression vector comprising a polynucleotide encoding a chimeric receptor subunit

comprising an extracellular domain of an OBR subunit, a transmembrane domain of a

cytokine receptor subunit and an intracellular domain of a receptor subunit selected

from a non-OBR homodimeric cytokine receptor subunit, gpl30 and IL-2R β subunit;

and

a proliferation dependency on OB.

20. The mammalian cell according to the above 19, wherein the intracellular

domain is an intracellular domain of a receptor subunit selected from a TPOR subunit,

EPOR subunit, G-CSFR subunit, GHR subunit and PRLR subunit.

21. A mammalian cell having: a chimeric receptor anchored to a cell membrane thereof, wherein the chimeric receptor

comprises an extracellular domain of an OBR subunit, a transmembrane domain of a

cytokine receptor subunit and an intracellular domain of a receptor subunit selected

from a non-OBR homodimeric cytokine receptor subunit, gpl30 and IL-2R β subunit;

and

a proliferation dependency on OB.

22. A polynucleotide encoding a chimeric receptor subunit, wherein the chimeric

receptor subunit comprises an extracellular domain of an OBR subunit, a transmembrane domain of a cytokine receptor subunit and an intracellular domain of a receptor subunit selected from a TPOR subunit, EPOR subunit, GHR subunit, PRLR

subunit, gpl30 and IL-2R β subunit.

23. A chimeric receptor subunit comprising an extracellular domain of an OBR

subunit, a transmembrane domain of a cytokine receptor subunit and an intracellular

domain of a receptor subunit selected from a TPOR subunit, EPOR subunit, GHR

subunit, PRLR subunit, gpl30 and IL-2R ]3 subunit.

24. A screening method comprising:

conducting the method the above 1 on a plurality of test agents and

selecting a test agent which proliferates the mammalian cell.

25. An OBR activating agent comprising the test agent selected from the screening

method of the above 24.

26. An anti-obesity pharmaceutical comprising as an active ingredient, the OBR

activating agent of the above 25.

27. A screening method comprising:

conducting the method of the above 1 on a plurality of test agents and

selecting a test agent which proliferates the mammalian cell.

28. An OBR inhibiting agent comprising the test agent selected from the screening

method of the above 27.

29. An anti-emaciation pharmaceutical comprising as an active ingredient, the OBR

inhibiting agent of the above 28.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 illustrates expression vector pMX-LTl which encodes an extracellular domain of an OBR subunit and transmembrane and intracellular domains of a TPOR subunit. In Fig. 1, "LTR" refers to long terminal repeat, "Y" refers to a virus packaging

signal, "P" refers to a SV40 initial promoter, "NEO^r" refers to a neomyacin resistance

gene, "Ori" refers to a replication origin of pMX-LTl and "Amp^r" refers to a ampicillin

resistance gene.

Fig. 2 illustrates expression vector pMX-LT2 which encodes extracellular and

transmembrane domains of an OBR subunit and an intracellular domains of a TPOR

subunit. In Fig. 2, "LTR" refers to long terminal repeat, "Y" refers to a virus packaging

signal, "P" refers to a SV40 initial promoter, "NEO^r" refers to a neomyacin resistance

gene, "Ori" refers to a replication origin of pMX-LTl and "Amp^r" refers to a ampicillin resistance gene.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the following abbreviations will abbreviate the terms indicated below.

"OB" means leptin.

"OBR" means leptin receptor.

"IL-2R" means interleukin-2 receptor.

"TPOR" means trombopoietin receptor.

"EPOR" means erythropoietin receptor.

"G-CSFR" means granulocyte colony-stimulating factor receptor.

"GHR" means growth hormone receptor.

"PRLR" means prolactin receptor.

The present invention involves methods which entail utilizing a mammalian cell having a proliferation dependency which allows the mammalian cell to proliferate in

response to a stimulation from OB. Such a mammalian cell in the methods contains a polynucleotide which encodes an OB-bindable chimeric receptor subunit. A test agent

can then be exposed to the mammalian cell in order to evaluate whether the test agent is

an agent affecting OBR. When the mammalian cell is exposed to the test agent, the

affect of the test agent can be measured, based on the proliferation of the mammalian

cell.

The polynucleotide which encodes the chimeric receptor subunit (hereinafter referred to as the chimeric polynucleotide) typically is efficiently expressed in the

mammalian cell to provide for the chimeric receptor subunit. The chimeric receptor subunit may then form a chimeric receptor complex anchored to the cell membrane of

the mammalian cell. The chimeric receptor subunit in this regard has an extracellular

domain (hereinafter referred to as ECD), a transmembrane domain (hereinafter referred

to as TMD) and an intracellular domain (hereinafter referred to as ICD). The ECD,

TMD and ICD are typically arranged in the chimeric receptor subunit such that the

TMD is internally located between the ECD and ICD. Typically, the ECD is on the

N-terminal side of the TMD and the ICD is on the C-terminal side of the TMD.

The signal triggered by forming the chimeric receptor complex and by having

an appropriate ligand bind to the ECD of the chimeric receptor subunit typically

transduces through the ICD of the chimeric receptor subunit. After the signal is

transduced to the ICD of the subunit, the signal may continue to transduce along a

native signal transduction pathway of the mammalian cell.

An ECD of an OBR subunit is typically utilized for the ECD of the chimeric

receptor subunit. The ECD of the chimeric receptor may also have one or more

particular amino acids added, deleted or substituted to the ECD of the OBR subunit.

Such an amount of amino acids added, deleted or substituted to the ECD of OBR may be 1 to 3 amino acids. Typically, the addition, deletion or substitution of amino acids to the ECD of the OBR subunit is located at the extremity regions of the OBR subunit. In

having additional amino acids at the N-terminus of the ECD of the chimeric receptor,

the ECD of OBR may additionally have 1 to 30 amino acids. A mutation in the

nucleotide sequence encoding the ECD of the OBR subunit can set forth the desired

addition, deletion or substitution of an amino acid to the ECD of the OBR subunit.

A TMD of a cytokine receptor subunit is typically utilized for the TMD of the

chimeric receptor subunit. The TMD of the chimeric receptor subunit may also have one or more particular amino acids added, deleted or substituted to the TMD of the

cytokine receptor subunit. Such an amount of amino acids added, deleted or substituted

to the TMD of the cytokine receptor may be 1 to 3 amino acids. Typically, the addition,

deletion or substitution to the TMD of the cytokine receptor subunit is located at the

extremity regions of the chimeric receptor subunit. A mutation in the nucleotide

sequence encoding the cytokine receptor subunit can set forth the desired addition,

deletion or substitution of an amino acid to the TMD of the cytokine receptor subunit.

An ICD of a receptor subunit selected from a non-OBR homodimeric cytokine

receptor subunit, IL-2R β subunit and gpl30, is typically utilized for the ICD of the

chimeric receptor subunit. The ICD of the chimeric receptor subunit may have one or more particular amino acids added, deleted or substituted to such an ICD of a receptor

subunit. Such an amount of amino acids added, deleted or substituted to the ECD of the

cytokine receptor subunit may be 1 to 3 amino acids. Typically, the addition, deletion

or substitution to the ICD of the provided receptor subunit is located at the extremity

regions thereof. In having additional amino acids at the C-terminus of the ICD of the

chimeric receptor, said ICD of receptor may additionally have 1 to 30 amino acids. A

mutation in the nucleotide sequence encoding the ICD of the provided receptor subunit can set forth the desired addition, deletion or substitution of an amino acid to the ICD of

said provided receptor subunit.

The non-OBR homodimeric cytokine receptor subunit, which may be utilized

for the TMD or ICD of the chimeric receptor subunit, is a homodimeric cytokine

receptor excluding OBR. For example, when utilizing the TMD or ICD of the non-

OBR homodimeric cytokine receptor correspondingly as the TMD or ICD of the chimeric receptor, the TMD or ICD of the chimeric receptor may utilize the TMD or

ICD of a receptor selected from a TPOR subunit, EPOR subunit, G-CSF subunit, GHR

subunit and PRLR subunit.

There are cases in which the chimeric receptor subunit can have the TMD

thereof derived from the TMD of the receptor subunit utilized for the ECD or ICD of

the chimeric receptor subunit. In such cases, the TMD of the chimeric receptor

generally utilizes therein a TMD of a receptor subunit selected from an OBR subunit,

non-OBR homodimeric cytokine receptor subunit, IL-2R j3 subunit and gpl30. For

example, there includes cases in which the TMD of the chimeric receptor is the TMD of

the receptor subunit provided for the ICD of the chimeric receptor, such that the

chimeric receptor has the TMD and ICD derived from the identical receptor subunit.

Further, such cases also include cases in which the chimeric receptor has the TMD

thereof be the TMD of an OBR subunit, such that the ECD and the TMD are derived

from the OBR subunit.

The chimeric polynucleotide can be produced by PCR amplifying from a

template encoding the ECD of an OBR subunit, a template encoding the TMD of a

cytokine receptor subunit and a template encoding the ICD of the selected receptor subunit and by ligating together the amplified products. The PCR amplification utilizes

primers possessing 14 to 35 nucleotides, which can be produced with a DNA automatic synthesizer. In PCR amplifying a template encoding one of the desired domains, one of

the primers has a nucleotide sequence of the 5' terminal nucleotide sequence encoding the desired domain (hereinafter referred to as the 5' primer). Another one of the primers

in such cases has a nucleotide sequence complementary to the 3' terminal nucleotide

sequence encoding the desired domain (hereinafter referred to as the 3' primer). Such

primers may further contain a nucleotide sequence of a restriction enzyme site at the 5'

terminus thereof, so that the restriction enzyme site is added to the amplified product. The restriction enzyme site is useful to ligate together the amplified products or to insert

the constructed chimeric polynucleotide into an expression vector, as described below.

The 5' terminus of the primers may be phosphorylated.

Such primers utilized in the PCR amplification are typically designed, based on

the nucleotide sequence in the template, encoding the provided domain. For example,

when the template encodes the ECD or TMD of an OBR subunit in the PCR

amplification, the primers can be designed based on the nucleotide sequence encoding

an OBR subunit (LA. Tartaglia et al., 1995, Cell 83: 1263-1271; WO9719952), such as

a primer having a nucleotide sequence shown in SEQ ID:1, SEQ ID:2, SEQ ID:3 or SEQ ID:9 or the like. When the template encodes the TMD or ICD of a TPOR subunit

in the PCR amplification, the primers may be designed, based on the nucleotide

sequence encoding a TPOR subunit (I. Vigon et al., 1994, Genomics 20(1): 5-12 and

R.C. Skoda et al., EMBO 12(7): 2645-2653), such as a primer having a nucleotide

sequence shown in SEQ ID:4, SEQ ID:5 or SEQ ID:6 or the like. When the template

encodes the TMD or ICD of an EPOR subunit, in the PCR amplification, the primers

may be designed, based on the nucleotide sequence encoding an EPOR subunit (S.S.

Jones et al., 1990, Blood 76: 31-35; A.D. D'Andrea et al., 1989, Cell 57 (2):277-285 and S. Nagao et al., 1993, J. Biol. Chem. 268 15:11208-11216), such as a primer having a nucleotide sequence shown in SEQ ID: 10 or SEQ ID: 11 or the like. When the

template encodes the ICD of a G-CSFR subunit in the PCR amplification, the primers

can be designed, based on the nucleotide sequence encoding a G-CSFR subunit (R.

Fukunaga et al., 1990, Proc. Natl. Acad. Sci. U.S.A. 87: 8702-8706), such as the

primers having the nucleotide sequence shown in SEQ ID:12 or SEQ ID:13 or the like.

When the template encodes the TMD or ICD of a GHR subunit, the primers can be

designed, based on the nucleotide sequence encoding a GHR subunit (D.W. Leung et

al., 1987, Nature 330: 537-543; GenBank Accession No. NM_010284and LS.

Mathews et al., 1989, J. Biol. Chem. 264: 9905-9910). When the template encodes the TMD or ICD of a PRLR subunit, the primers may be designed, based on the nucleotide

sequence encoding a PRLR subunit (J.M. Boutin et al., 1989, Mol. Endocrinol. 3:

1455-1461; D.L. Clarke et al.,1993, Endocrinology 133:224-232 and R. Zhang et al.,

1990, Biochem. Biophys. Res. Commun. 168(2):415-422). When the template encodes

the TMD or ICD of gpl30, the primers may be designed, based on the nucleotide

sequence encoding gpl30 (M. Hibi et al., 1990, Cell 63: 149-1157; M. Saito, 1992, J.

Immunol. 148(12):4066-4071 and Y. Wang et al., 1992, Genomics, 14:666-672).

When the template encodes the TMD or ICD of an IL-2 β subunit, the primers may be

designed, based on the nucleotide sequence encoding an IL-2 β subunit (M.

Hatakeyama et al, 1989, Science 244: 551-556; A. Shimizu et al., 1985, Nucleic Acid

Res. 13:1505-1516 and T.H. Page et al., 1991, Eur. J. Immunol. 21: 2133-2138).

When producing the chimeric polynucleotide encoding the chimeric receptor

subunit in which the TMD thereof is derived from the identical receptor subunit utilized

for the ECD or ICD of the chimeric receptor subunit, the PCR amplification can

continuously amplify a template encoding such domains in connection to provide an amplified product in which the nucleotide sequence of the ECD is connected with the

nucleotide sequence of the TMD or in which the nucleotide sequence of TMD is

connected with the nucleotide sequence of the ICD. Further, the template in such cases

are typically arranged such that the nucleotide sequence encoding the ECD is upstream

from the nucleotide sequence encoding the TMD or that the nucleotide sequence

encoding the TMD is upstream from the nucleotide sequence encoding ICD.

Various forms of the template can be utilized in the PCR amplification. For

example, the template may be a plasmid which encodes the desired domain, a cDNA or

a chromosomal DNA prepared from mammalian tissues such as the brain, liver and

lungs and the like. Such templates can be produced according to conventional methods

(such as the methods described in J. Sambrook, E.F. Frisch, T. Maniatis, Molecular

Cloning 2^nd Edition, Cold Springs Harbor Laboratory press). Further, a commercially

available library, such as a human brain cDNA library (Stratagene), human bone

marrow library (Clonetech) and the like, may be utilized to provide the template in the

PCR amplification .

To PCR amplify the templates encoding the desired domains, an appropriate

PCR mixture is prepared and is incubated. The PCR mixture may be prepared by

mixing in a reaction buffer, the 5' primer, 3' primer, the template, a heat-resistant DNA

polymerase and a dNTP mixture. The 5' primer and 3' primer in the PCR mixture

correspond to the template in the PCR mixture, in that said 5' primer and 3' primer can

anneal to the template. The heat-resistant DNA polymerase in the PCR amplification

mixture may be a Takara Ex Taq (Takara Shuzo), pyrobest DNA polymerase (Takara

Shuzo) or the like.

The incubations in the PCR amplification can be conducted by repeating 25 to

40 times an incubation cycle including heat denaturation, annealing and elongation. The heat denaturation can be, for example, conducted under the conditions of 94°C for

10 seconds to 1 minute. The annealing can be, for example, conducted under the

conditions of a temperature of from 35°C to the meting point of the primers (Tm value)

for 1 minute. The elongation can be, for example, conducted under the conditions of a

72°C for 1 to 5 minutes. After repeating the incubation cycle in the PCR amplification,

an incubation for a elongation reaction of 72°C for 10 minutes may be added. Such

incubations can be conducted with a thermal cycler (PE Applied Biosystems).

In the above PCR amplification, a polynucleotide encoding the ECD of the OBR

subunit can amplified when utilizing the primer having the nucleotide sequence as

shown in SEQ ID: 1 and the primer having the nucleotide sequence as shown in SEQ

ID: 2. The polynucleotide encoding the ECD of the OBR subunit can also be amplified

when utilizing the primer having the nucleotide sequence as shown in SEQ ID: 1 and

the primer having the nucleotide sequence as shown in SEQ ID: 9. The polynucleotide

encoding the ECD and TMD of the OBR subunit can be amplified when utilizing the

primer having the nucleotide sequence as shown in SEQ ID: 1 and the primer having the

nucleotide sequence as shown in SEQ ID: 3. The polynucleotide encoding the ICD and TMD of the TPOR subunit can be amplified when utilizing the primer having the

nucleotide sequence as shown in SEQ ID: 4 and the primer having the nucleotide

sequence as shown in SEQ ID: 6. The polynucleotide encoding the ICD of the TPOR

subunit can be amplified when utilizing the primer having the nucleotide sequence as

shown in SEQ ID: 5 and the primer having the nucleotide sequence as shown in SEQ

ID: 6. The polynucleotide encoding the ICD and TMD of the EPOR subunit can be

amplified when utilizing the primer having the nucleotide sequence as shown in SEQ

ID: 10 and the primer having the nucleotide sequence as shown in SEQ ID: 11. The polynucleotide encoding the ICD of the G-CSFR subunit can be amplified when

utilizing the primer having the nucleotide sequence as shown in SEQ ID: 12 and the

primer having the nucleotide sequence as shown in SEQ ID: 13.

The amplified products can then be ligated together to produce the chimeric

polynucleotide. The amplified products can also be ligated together with a vector in one

pot to construct a chimeric vector.

The vector is useful for introducing the chimeric polynucleotide into a cytokine dependent mammalian host cell. The vector comprises a promoter which is functional

in the mammalian cell and a selective marker gene. It is preferable that the promoter

can also functional in conventional host organisms such as Escherichia coli and yeast.

As an example of the selective marker gene, there is mentioned an antibiotic resistance

gene such as a neomyacin resistance gene, puromyacin resistance gene and the like.

Such a selective marker gene can be utilized to confirm that the chimeric polynucleotide

has been introduced into the mammalian cell. Examples of such vectors include a

retroviruses vector such as pMX (M. Onishi et al., 1996, Experimental Hematology

24:324-329), pBabe-neo (J. P. Morgenstern et al. Nucleic Acid Res. 18: 3587-3596) and pBabe-puro (J. P. Morgenstern et al. Nucleic Acid Res. 18: 3587-3596), an

adenovirus vector such as pREP (Invitrogen), an EB vims vector such as pQBI

(QUANTUM BIOTECHNOLOGIES), a plasmid vector such as pcDNA (Invitrogen)

and pRc (Invitrogen) and the like. The retrovirus vector, for example, can be introduced

into the mammalian cell to provide a high efficacy rate with cell infection methods.

Further, the retrovirus vector can provide with the mammalian cell, a stable expression

of the chimeric polynucleotide therein.

The insertion of the chimeric polynucleotide or amplified products into the vector can be conducted according to conventional methods (for example the methods described in J. Sambrook, E.F. Frisch, T. Maniatis, Molecular Cloning 2^nd Edition,

Cold Springs Harbor Laboratory press). In this regard, a chimeric vector can be

constructed by inserting operably downstream from the promoter the polynucleotides

encoding the ECD of the OBR subunit, the TMD of the cytokine receptor subunit and

the ICD of receptor subunit selected from a non-OBR homodimeric cytokine receptor

subunit, IL-2R j3 subunit and gρl30. An operably downstream site in the vector is

usually located therein so that the promoter and the chimeric polynucleotide are in a

relationship permitting the promoter to induce the expression of the chimeric polynucleotide therein. Such polynucleotides are generally arranged so that from

upstream to downstream, respectively, there is said polynucleotide encoding the ECD

of the OBR subunit, the polynucleotide encoding the TMD of the cytokine receptor and

then the polynucleotide encoding the ICD of selected subunit.

For example, a chimeric vector can be constructed as follows. The amplified

products which contain the restriction enzyme sites added by the primers, as described

above, are restriction digested with the appropriate restriction enzyme. Further, the

vector is restriction digested with a restriction enzyme at a restriction enzyme site

operably downstream from the promoter therein. Such restriction digested fragments

can be then subjected to agarose gel electrophoresis to obtain the desired

polynucleotides with Gene Clean Kit (BIO 101 Inc.). Such obtained polynucleotides

are ligated together with a commercially available ligation reaction kit, such as Ligation

Kit Ver.l (Takara Shuzo). The resulting ligation product can be have its nucleotide

sequence analyzed to confirm that the polynucleotide encoding the desired chimeric

receptor is in an expressible form in the constructed chimeric vector. Analysis of the

nucleotide sequence can be conducted by, for example, using a Thermo Sequenase II Die Terminator kit (Amerscham Pharmacia Biotech) with a DNA sequencer (PE

Applied Biosystems).

The mammalian cell of the present invention can be produced by introducing

into a cytokine dependent mammalian host cell, the chimeric polynucleotide. The

chimeric vector can be introduced into the cytokine dependent mammalian host cell to

provide for the chimeric polynucleotide. As methods for introducing the chimeric

polynucleotide into the cytokine dependent mammalian host cells, there can be

mentioned a retrovirus infection method (M. Onishi et al., 1996, Experimental

Hematology 24:324-329), electroporation method (A. ting et al. EMBO J. 15:6189-

6196) potassium phosphate method (S. Grimm et al. Proc. Natl. Acad. Sci. USA

93:10923-10927) and the like. A successful introduction of the chimeric

polynucleotide into the cytokine dependent mammalian host cell can be confirmed by

selecting a transformed cell which comprises the characteristics provided by the

selective marker gene. When the selective marker gene is an antibiotic resistance gene,

such a transformed cell can be selected by targeting for the antibiotic resistance.

Alternatively, the successful introduction of the chimeric polynucleotide can be confirmed by selecting a transformed cell which can proliferate in a culturing medium

containing OB and substantially no cytokines.

Such a cytokine dependent mammalian host cell, to which the chimeric

polynucleotide is inserted, is a mammalian cell which dependently needs to be exposed

to a cytokine to proliferate, with the proviso that the cytokine is not OB. Examples of

such cytokine dependent mammalian host cells include a mammalian hematopoietic

cell, mammalian epithelial cell and the like. Examples of the mammalian

hematopoietic cell include pluripotent mammalian hematopietic stem cells, cells which

differentiate therefrom and the like. Examples of cells which differentiate from pluripotent mammalian hematopietic cells include mammalian lymphocytes,

mammalian bone marrow cells, blood cells differentiating therefrom and the like.

Examples of such mammalian lymphocytes include mammalian lymphocyte stem cells,

cells differentiating therefrom and the like. Examples of such cells differentiating from

mammalian lymphocyte stem cells include mammalian T cells, mammalian B cells,

mammalian NK cells, blood cells differentiating therefrom and the like. In this regard,

there can be utilized as such lymphocytes, BaF-3 cells (RIKEN RCB0805 R. Palacios et al., 1985, Cell 41: 727-734), TALL-101 cells (B. Lange et al., 1987, Blood, 74:192-

199), OCl-Ly9 cells (N. Tweeddale et al., 1989, Blood, 74: 572-578), OCl-Lyl3.1

cells (N. Tweeddale, 1989, Blood 74: 572-578) and the like. As the bone marrow cells,

there may be utilized FDC-Pl cells (ATCC No. CRL-12103), TF-1 cells (ATCC No.

CRL-2003, T. Kitamura et al., 1989, J. Cell. Physiol. 140:323-334), Ut-7 cells (N.

Komatsu et al., 1991, Cancer Res. 51:341-348), F36-P cells (RIKEN RCB0775 S.

Chiba et al., 1991, Blood 78: 2261-2268) and the like. As the epithelial cells, there is

mentioned HK-2 (ATCC No. CRL-2190).

Further, the mammalian cell of the present invention can be cloned by culturing

the mammalian cell containing the chimeric polynucleotide in a culturing medium

containing OB and substantially no cytokines and then by utilizing conventional cloning methods such as the methylcellulose method, penicillin cup method and the

like. When cloning, the concentration of OB in the culturing medium can be from 5pM

to InM. In such cases, there is cloned, a mammalian cell which receives an efficient

level of stimulation from the OB and which has an efficient level of proliferation

dependency on OB. The level of stimulation from the OB or level of the proliferation dependency on OB can be measured by having the concentration of the OB in the

culturing medium at various levels. Such a level of stimulation from OB is a level based on the concentration of OB which provides a proliferating speed of 50% of the maximum proliferating speed thereof, in the culturing medium containing OB and

substantially no cytokines. An inverse relationship is present between the level of

stimulation from OB and the concentration of OB in said culturing medium containing

OB and substantially no cytokines, such that the level of stimulation is higher as the

concentration of OB is lower.

In this regard, the mammalian cell can be utilized to evaluate whether a test

agent is an agent affecting OBR. Such an evaluation may be provided by culturing the

mammalian cell and measuring the proliferation of the mammalian cell when exposed

to the test agent.

The mammalian cell of the present invention is typically cultured under normal

conditions in a basic culturing medium to which OB or a particular cytokine has been

added. Such a basic culturing medium can be a culturing medium in which fetal bovine

serum is added to RPMI1640 (GibcoBRL) to amount to 10% (v/v). The particular

cytokine which is added to the basic culturing medium is a cytokine needed to

proliferate the cytokine dependent host cell that is utilized to derive the mammalian

cells of the present invention. Such a particular cytokine may be interleukin-3 when

BaF-3 is utilized as the cytokine dependent mammalian host cell. The normal

conditions to culture the mammalian cell is typically provided by an incubator in which

the temperature is set to provide a temperature of 37°C with 5% (v/v) CO₂ gas.

To expose the test agent to the mammalian cell, the test agent is typically added

to a basic culturing medium utilized for the mammalian cells. For example, a basic

culturing medium containing the test agent may be fractioned into a 96 well plate. The cultured mammalian cells can then be fractioned into each of the wells and be further

cultured under the normal culturing conditions. The test agent is usually added to the basic culturing medium so that the concentration of the test agent therein is InM to

ImM, and preferably 50nM to 50μM. The concentration of the cultured cells in the

basic culturing medium containing the test agent, varies with the proliferating

capabilities of said mammalian cell, but may generally be 5x10 cells/ml to 5x10

cells/ml, and preferably 1x10 cells/ml to lxlO⁵ cell/ml.

If so desired, the cultured mammalian cells can be washed with the basic

culturing medium before being exposed to the test agent. Typically, such cells are

washed a plurality of times, such as 2 to 5 times. Such a process of washing the cultured mammalian cells substantially avoids carrying over to the step of being exposed to the

test agent, the OB or the particular cytokine utilized to culture said mammalian cells.

The proliferation of the mammalian cells can be measured by utilizing

conventional methods which can measure whether the mammalian cell has proliferated

or conventional methods which can measure the amount of proliferation of a

mammalian cell. Examples of such conventional methods include an alamar blue

assay, MTT assay, various DNA quantification methods and the like. Further, to

measure the proliferation of the mammalian cell, the proliferation value achieved from

exposing the mammalian cell with the test agent is compared to a control culture. As

such a control culture, there is mentioned a first control culture in which the mammalian

cell of the present invention is unexposed to a test agent, a second control culture in

which the cytokine dependent mammalian host cell is exposed to the test agent and a

third control culture in which the cytokine dependent mammalian host cell is

unexposed to the test agent. The first control culture can be cultured in the basic

culturing medium to which the particular cytokine needed to proliferate the cytokine dependent mammalian host cell proliferate is added. The second and third controls can be cultured by using the basic culturing medium to which the particular cytokine needed to proliferate the cytokine dependent mammalian host cell is added.

The methods of the present invention can be utilized to screen a test agent as a

OB-like activating agent. The OB-like activating abilities of the test agent can be

measured when the mammalian cell is exposed to the test agent and to substantially no

cytokines activating the chimeric receptor. In such cases, there is selected as the OB-

like activating agent, a test agent which can provide a significantly higher proliferation

with the mammalian cell than that of the first control culture. Further, such a test agent usually provides no significant difference of proliferation between the second control

culture and the third control culture. Such a OB-like activating agent is a substance other than OB, which has a physiological function substantially identical to OB, such as

binding to OBR and transducing the triggered signal into the cell.

The test agent having the OB-like activating agent, which can be screened from

the present invention, can be, for example, N-(2-methoxyethyl) O-(5-methoxy-2-

nitrophenyl) methylsulfonate thiomide.

Additionally, the methods of the present invention can be utilized to screen a

test agent as an OB inhibiting agent. The OB inhibiting abilities of the test agent can be

measured when mammalian cell is exposed to the test agent and OB. When exposing

the mammalian cell with OB, the concentration of OB in the utilized culturing medium

is typically from InM to ImM, and preferably 50nM to 50 M. In such cases, there is

selected as the OB inhibiting agent, a test agent which can provide a significantly lower

proliferation with the mammalian cell than that of the first control culture. The second

control in screening for the OB inhibiting agent is usually cultured with a corresponding

amount of OB in its culturing medium. Further, such a test agent usually provides no significant difference of proliferation between the second control culture and the third

control culture.

A pharmaceutical composition comprising as an active ingredient, the OB-like

activating agent, which is selected from the screening methods above, can be used as an

anti-obesity pharmaceutical compositions. Further, a pharmaceutical composition

comprising as an active ingredient, the OB inhibiting agent, which is selected from the screening methods above, can be used as an anti-emaciation pharmaceutical

compositions. Further, such pharmaceutical compositions may comprise as the active

ingredient a pharmaceutically acceptable salt of the OB-like activating agent or OB

inhibiting agent.

A mammal, such as a human, can be applied orally or non-orally with an

effective amount of the pharmaceutical compositions. For example, when applying

orally, the pharmaceutical compositions can be used as a typical formulation such as

tablet, capsule, syrup, suspension and the like. Further, when applying non-orally, such

compositions can be used as a typical liquid formulation such as a solution, emulsion,

suspension and the like. As methods of non-orally applying the above formulations, for

example, there is mentioned injection methods, methods of directly applying as a

suppository to the intestine, and the like.

The formulations can be combined with an acceptable carrier, vehicle, adhesive,

stabilizer, diluent or the like to produce the pharmaceutical compositions. Further, there

may be added to the formulations when used as a injection formulation, a buffer,

dilution auxiliary, isotoning agent and the like, to produce the pharmaceutical

compositions.

Effective amounts of the applying the compositions may differ according to the

age, sex, weight, severity of symptoms and the like, but may be for an average adult mammal when orally applying the composition, in an amount of from lmg to 2g. When

injecting the compositions to an average adult mammal, the effective amount of the

provided composition may be O.lmg to 500mg. Further, such an effective amount may

be applied 1 to several times a day.

EXAMPLES

Example 1: Production of the mammalian cells of the present invention containing a

polynucleotide encoding the ECD of an OBR subunit and the ICD of a TPOR subunit

In the present Example 1, polynucleotides encoding the ECD of an OBR subunit

and the ICD of a TPOR subunit are introduced respectively to BaF-3 cells. The first of

the polynucleotides encodes the ECD of an OBR subunit as well as the TMD and ICD

of a TPOR subunit. The second of the polynucleotides encodes the ECD and TMD of

an OBR subunit and the ICD of a TPOR subunit. This can be accomplished according

to the following subsections (1.1) to (1.5).

(1.1) Preparation of polynucleotides encoding specified domains of an OBR subunit

and a TPOR subunit

A human brain cDNA library (CLONTECH) and a TF-1 cell cDNA library are

utilized to PCR amplify specified domains of an OBR subunit and a TPOR subunit. A

first set of the primers is designed, based on the nucleotide sequence of a OBR subunit

(GenBank Accession No. U43168). As such primers in the first set, Primers 1, 2 and 3

are synthesized with a DNA automatic synthesizer (PE Applied Biosystems) so that

Primer 1 has the nucleotide sequence shown in SEQ ID:1, Primer 2 has the nucleotide

sequence shown in SEQ ID:2 and Primer 3 has the nucleotide sequence shown in SEQ ID:3. A second set of primers is designed, based on the nucleotide sequence of a TPOR subunit (GenBank Accession No. M90102). As such primers in the second set, Primers

4, 5 and 6 are synthesized with a DNA automatic synthesizer (PE Applied Biosystems)

so that Primer 4 has the nucleotide sequence shown in SEQ ID: 4, Primer 5 has the

nucleotide sequence shown in SEQ ID:5 and Primer 6 has the nucleotide sequence

shown in SEQ ID: 6. Further, the first and second sets of the primers are synthesized to

additionally contain a particular restriction enzyme site, so that restriction enzyme sites

are added to the amplified product.

A PCR amplification is conducted with 50μl of a PCR mixture containing lOng

of the human brain cDNA library, lOOpmol of Primer 1, lOOpmol of Primer 2, 1.25U of Pyrobest DNA polymerase (Takara Shuzo), 4μl (2.5mM) of the dNTP mixture

provided with the Pyrobest DNA polymerase (Takara Shuzo) and 5μl of the buffer

provided with the Pyrobest DNA polymerase (Takara Shuzo). In the PCR

amplification, there is repeated 30 times, after an initial incubation of 94"C for 1

minute, an incubation cycle including incubations at 94°C for 30 seconds followed by

55T for 1 minute and then 72°C for 3 minutes. After repeating the incubation cycle,

the PCR amplification has an incubation at 72°C for 10 minutes. The resulting PCR

product thereof is subjected to agarose gel electrophoresis to obtain the amplified DNA

thereof (hereinafter referred to as DNA 1), which moves in the agarose gel as a size

corresponding to about 2.5kbp.

Another PCR amplification is similarly conducted under the conditions above

with a PCR mixture containing lOng of the human brain cDNA library, lOOpmol of

Primer 1, lOOpmol of Primer 3, 1.25U of the Pyrobest DNA polymerase, 4μl (2.5mM)

of the dNTP mixture provided with the Pyrobest DNA polymerase and 5μl of the buffer provided with the Pyrobest DNA polymerase. The resulting PCR product thereof is subjected to agarose gel electrophoresis to obtain that the amplified DNA (hereinafter

referred to as DNA 2), which moves in the agarose gel as a size corresponding to about

2.5kbp.

A cDNA library is prepared from TF-1 cells (ATCC No. CRL-2003 T.

Kitamura et al., 1989, J. Cell. Physiol. 140:323-334) according to a method described in

J. Sambrook, E.F. Frisch, T. Maniatis, Molecular Cloning 2^nd Edition (Cold Springs

Harbor Laboratory press). A PCR amplification is similarly conducted under the

conditions above with a PCR mixture containing lOng of the cDNA library obtained

from the TF-1 cells, lOOpmol of Primer 4, lOOpmol of Primer 6, 1.25U of the Pyrobest

DNA polymerase, 4μl (2.5mM) of the dNTP mixture provided with the Pyrobest DNA

polymerase and 5μl of the buffer provided with the Pyrobest DNA polymerase. The

resulting PCR product thereof is subjected to agarose gel electrophoresis to obtain the

amplified DNA (hereinafter referred to as DNA 3), which moves in the agarose gel as a

size corresponding to about 440bp. Further, another PCR amplification is similarly conducted under the conditions

above with a PCR mixture containing lOng of the cDNA library obtained from the TF-1

cells, lOOpmol of Primer 5, lOOpmol of Primer 6, 1.25U of the Pyrobest DNA

polymerase, 4μl (2.5mM) of the dNTP mixture provided with the Pyrobest DNA

polymerase and 5μl of the buffer provided with the Pyrobest DNA polymerase. The

resulting PCR product thereof is subjected to agarose gel electrophoresis to obtain the amplified DNA (hereinafter referred to as DNA 4), which moves in the agarose gel as a

size corresponding to 370bp.

Each of the amplified DNAs 1, 2, 3 and 4 are phenol-chloroform extracted and

collected through ethanol precipitation. Each of the DNAs 1, 2, 3 and 4 are then dried and have added thereto 20μl of water to produce respectively aqueous solutions of the

DNAs 1, 2, 3 and 4.

(1.2) Preparation of vector pMX-neo

Based on the nucleotide sequence of pMAM-neo (GenBank Accession No.

U02432), Primer 7 having the sequence shown in SEQ ID:7 and Primer 8 having the

nucleotide sequence shown in SEQ ID:8 are synthesized with a DNA automatic

synthesizer (PE applied Biosystems). Further, Primers 7 and 8 are synthesized to additionally contain a particular restriction enzyme site.

A PCR amplification is then conducted with 50 l of a PCR solution containing

50pg of pMAM-neo (Clontech), lOOpmol of Primer 7, lOOpmol of Primer 8, 1.25U of

Pyrobest DNA Polymerase (Takara Shuzo), 4μl (2.5mM) of dNTP mixture provided

with the Pyrobest DNA Polymerase (Takara Shuzo) and 5μl of the buffer provided with

the Pyrobest DNA Polymerase (Takara Shuzo). In the PCR amplification, there is

repeated 30 times, after an initial incubation at 94°C for 1 minute, an incubation cycle

including incubations at 94°C for 30 seconds followed by 55°C for 1 minute and then

72°C for 3 minutes. After repeating the incubation cycle, the PCR amplification has an

incubation at 72°C for 10 minutes. The resulting PCR product thereof is subjected to

agarose gel electrophoresis to confirm that the amplified DNA (hereinafter referred to

as DNA 5) moves in the agarose gel as a size corresponding to about 1.5kbp. DNA 5 is

phenol-chloroform extracted and collected through ethanol precipitation. DNA 5 is

then dried and has added thereto 20μl of water.

Ten micro-liters (lOμl) of the resulting DNA 5 solution is restriction digested

for 2 hours at 37°C in a 50 fi L reaction buffer with 5U of Sal 1. One micro-gram (lμg) of vector pMX is constructed according to the method

disclosed in Exp. Hematol. 24:324-329 (1996). Vector pMX is then restriction digested

for 2 hours at 37°C in a lOμl reaction buffer with 5U Sal I.

The restriction digest products of DNA 5 and pMX are subjected to agarose gel

electrophoresis to separate, respectively, the DNA fragments of the restriction digest

products. The desired DNA fragments are obtained with Gene Clean Kit (BIO 101

Inc.). Ten micro-liters (lOμl) of water is added, respectively, to the fragment from DNA 5. Twenty micro-liters (20μl) of water is added to the fragment from pMX. For 1

hour, lμl of the solution containing the fragment from DNA 5 and lμl of the solution

containing the fragment from pMX are allowed to ligate together atl6°C in 30μl of a

ligation reaction mixture. A competent E. coli JM 109 strain is transformed with lOμl

of the resulting ligation reaction mixture. The E. coli JM 109 transformant is cultured

in 100ml of LB additionally containing 50μg/ml of ampicillin. Vector pMX-neo is then

prepared from the E. coli JM109 transformants with a QLAGEN Plasmid Maxi Prep.

(1.3) Construction of the chimeric receptor subunit expression vector

Ten micro-liters (lOμl) of each of the aqueous solutions of DNA 1, DNA 2,

DNA 3 and DNA 4 (prepared in (1.1)) are subjected, respectively, to a restriction digest

in 50μl of a reaction mixture for 2 hours at 37°C with the particular restriction enzymes

shown in Table 1. Further, lμg of vector pMX-neo is subjected to a restriction digest at

37°C for 2 hours in lOμl of a reaction mixture with 2U of each of BamH I and EcoR I.

The resulting products from the restriction digest are subjected to agarose gel

electrophoresis. The desired DNA fragments from DNA 1, DNA 2, DNA 3, DNA 4 and pMX-neo are then prepared from the agarose gel with Gene Clean Kit (BIO 101 Inc.). Ten micro-liters (lOμl) of water is added, respectively, to each of the prepared

DNA fragments to produce aqueous solutions of the prepared DNAs 1, 2, 3, and 4.

Twenty micro-liters (20μl) of water is added to the prepared pMX-neo fragment to

produce an aqueous solution of the prepared pMX-neo fragment.

One micro-liter (lμl) of the aqueous solution of the prepared pMX-neo

fragment, 2μl of the aqueous solution of the prepared DNA 1 fragment and 2μl of the

aqueous solution of the prepared DNA 3 fragment are allowed to ligate together in 50μl

of a ligation reaction mixture by utilizing Ligation Kit ver.l (Takara Shuzo) at 16°C for

1 hour. Ten micro-liters (lOμl) of the resulting reaction mixture is then utilized to transform a competent E. coli JM109 strain. The transformants are cultured in 100ml of

LB additionally containing 50μg/ml of ampicillin. The expression vector pMX-LTl

(Fig. 1) is then prepared from colonies of the transformant with QIAGEN Plasmid Maxi

Prep.

An expression vector pMX-LT2 (Fig. 2) is similarly prepared with lμl of the

aqueous solution of the prepared pMX-neo fragment and 2μl of the aqueous solution of

the prepared DNA 2 fragment and 2μl of the aqueous solution of the prepared DNA 4

fragment.

The expression vectors pMX-LTl and pMX-LT2 are analyzed with a DNA

automatic sequencer (PE Applied Biosystems) using a Thermo Sequenase II Die

terminator kit (Amersham Pharmacia Biotech). As a result, pMX-LTl is confirmed to

contain a polynucleotide encoding amino acid 1 to 841 (from the N-terminus) of a

human OBR subunit (GenBank Accession No. AAA93015) and amino acid 492 to 635

(from the N-terminus) of a human TPOR subunit (GenBank Accession No.

AAA69971). As such, it is confirmed that pMX-LTl contains a polynucleotide encoding the ECD of an OBR subunit as well as the TMD and ICD of a TPOR subunit. PMX-LT2 is confirmed to contain a polynucleotide encoding amino acid 1 to 863 (from

the N-terminus) of a human OBR subunit (GenBank Accession No. AAA93015) and

amino acid 514 to 635 (from the N-terminus) of a human TPOR subunit (GenBank

Accession No. AAA69971). As such, it is confirmed that pMX-LT2 contains a

polynucleotide encoding the ECD and TMD of an OBR subunit as well as ICD of a

TPOR subunit.

Table 1

Primers Restriction Enzymes

DNA 1 SEQ ID:1 and SEQ ID:2 BamH \ and Hind \\\

DNA 2 SEQ ID:1 and SEQ ID:3 BamH I and Xba I

DNA 3 SEQ ID:4 and SEQ ID:6 Hind \\\ and EcoR \

DNA 4 SEQ ID:6 and SEQ ID:6 Xba I and EcoR I

(1.4) The preparation of a viral medium

The expression vectors prepared in (1.3), pMX-LTl and pMX-LT2, are transfected into virus packaging BOSC23 cells by using lipofectamine (GibcoBRL).

For pMX-LTl, a mixture is produced by combining a solution containing 3μl of

pMX-LTl and 200μl of OPTI-MEM (GibcoBRL) with a solution containing 18μl of

lipofectamine and 200μl of the OPTI-MEM and is then incubated. Subsequently,

1600μl of the OPTI-MEM is added and mixed into the resulting mixture.

In a 60mm dish, 2xl0⁶ cells of BOSC23 cells are placed therein and are cultured

overnight with 5% CO at 37°C in Dulbecco^'s modified Eagle's medium (GibcoBRL;

hereinafter referred to as D-MEM) additionally containing 10% (v/v) of fetal bovine serum (GibcoBRL; hereinafter referred to as FBS). After removing the supernatant D-

MEM additionally containing 10% (v/v) of FBS, the BOSC23 cells are washed with the

OPTI-MEM. The above mixture containing pMX-LTl, OPTI-MEM and lipofectamine

is added to the BOSC23 cells. The BOSC23 cells are then cultured for 5 hours in

similar culturing conditions as above. Subsequently, 2ml of D-MEM additionally

containing 20% (v/v) of FBS is added to the BOSC23 cells to further culture the

BOSC23 cells for 19 hours. The culturing medium is then replaced with 3ml of D-

MEM additionally containing 10% (v/v) of FBS. The BOSC23 cells are cultured for 24

hours under similar culturing conditions as above, to produce an innoculation medium. The innoculation medium is then centrifuged at 3000rpm for 5 minutes to obtain a first

viral medium. The expression vector pMX-LT2 is subjected under similar conditions

to obtain a second viral medium.

(1.5) Cloning of the mammalian cell of the present invention

BaF-3 cells are cultured in a basic culturing medium additionally containing

lng/ml of mouse IL-3 (R&D System) at 37°C with 5% CO₂ gas. The cultured BaF-3

cells are centrifuged at lOOOrpm for 3 minutes to collect the cultured BaF-3 cells.

Polyprene (hexadimethrine bromide, Sigma) is added to the concentration of lOμg/ml

and mouse IL-3 is added to the concentration of lng/ml to the first and second viral

mediums prepared above in (1.4). The collected BaF-3 cells are suspended,

respectively, in 1ml of the resulting first and second viral mediums and are then

transferred to a 24 well plate. After 5 hours of culturing, 1 ml of a basic culturing medium additionally containing lng/ml of mouse IL-3 is added thereto. The cultured

BaF-3 cells are then further cultured for 1 hour to produce first infected BaF-3 cells (hereinafter referred to as cl cells) and second infected BaF-3 cells (hereinafter referred

to as c2 cells).

The cl cells and c2 cells are cultured, respectively, in a basic culturing medium

additionally containing lng/ml of mouse IL-3 and lmg/ml of GENETICIN

(GibcoBRL). The cl cells and c2 cells are suspended to 5 cells/ml with a basic culturing

medium additionally containing lOpM of OB, are transferred to a 96 well plate at

lOOμl/well and are cultured for 5 days at 37°C with 5% CO gas. The cl cells and c2

cells are then observed under a microscope to select the successfully cloned cl and c2 cells. The clones are then scaled up, respectively, to obtain 7 clones of the cl cells and

11 clones of the c2 cells.

Example 2: Production of the mammalian cells of the present invention containing a

polynucleotide encoding the ECD of an OBR subunit as well as the TMD and ICD of an

EPOR subunit

A human brain cDNA library (CLONTECH) and a TF-1 cell cDNA library is

utilized to PCR amplify specified domains of an OBR subunit and a TPOR subunit.

Two sets of primers are designed to amplify the desired nucleotide sequence in the

human brain cDNA library. A first set of the primers is designed, based on the

nucleotide sequence of an OBR subunit (GenBank Accession No. U43168). As such

primers, Primers 1 and 9 are synthesized with a DNA automatic synthesizer (PE

Applied Biosystems) so that Primer 1 has the nucleotide sequence shown in SEQ ID:1

and Primer 9 has the nucleotide sequence shown in SEQ ID:9. A second set of primers

is designed, based on the nucleotide sequence of an EPOR subunit (GenBank Accession No. M34986). As such primers, Primers 10 and 11 are synthesized with a DNA automatic synthesizer (PE Applied Biosystems) so that Primer 10 has the nucleotide sequence shown in SEQ ID: 10 and Primer 11 has the nucleotide sequence

shown in SEQ ID: 11. Further, the first and second sets of the primers are synthesized to

additionally contain a particular restriction enzyme site, so that restriction enzyme sites

are added to the amplified product.

A PCR amplification is conducted similarly to the conditions of (1.1) with 50μl

of a PCR mixture containing lOng of the human brain cDNA library, lOOpmol of

Primer 1, lOOpmol of Primer 9, 1.25U of Pyrobest DNA polymerase (Takara Shuzo),

4μl (2.5mM) of the dNTP mixture provided with the Pyrobest DNA polymerase

(Takara Shuzo) and 5μl of the buffer provided with the Pyrobest DNA polymerase (Takara Shuzo). The resulting PCR product thereof is subjected to agarose gel

electrophoresis to obtain the amplified DNA (hereinafter referred to as DNA 6), which

moves to a length corresponding to a length of about 2.5kbp.

Another PCR is conducted similarly to the conditions of (1.1) with 50μl of a

PCR mixture containing lOng of the TF-1 cell cDNA library, lOOpmol of Primer 10,

lOOpmol of Primer 11, 1.25U of Pyrobest DNA polymerase (Takara Shuzo), 4μl

(2.5mM) of the dNTP mixture provided with the Pyrobest DNA polymerase (Takara

Shuzo) and 5μl of the buffer provided with the Pyrobest DNA polymerase (Takara

Shuzo). The resulting PCR product thereof is subjected to agarose gel electrophoresis

and Gene Clean Kit (BIO 101 Inc.) to obtain the amplified DNA (hereinafter referred to

as DNA 7), which moves in the agarose gel to a size corresponding to about 780bp.

Forty-five micro-liters (45μl) of each of the PCR products for DNA 6 and

DNA7 are phenol-chloroform extracted and are collected by ethanol precipitation. Each

of the DNAs 6 and 7 are then dried and have 20μl of water added, to produce,

respectively aqueous solutions of the DNAs 6 and 7. Ten micro-liters (lOμl) of the aqueous solution of DNA 6 and lOμl of the aqueous solution of DNA 7 are restriction digested for 2 hours at 37°C in a 50μl reaction mixture with 5U of the particular

restriction enzyme shown in Table 2. Further, lμg of vector pMX-neo is subjected to a

restriction digest at 37°C for 2 hours in lOμl of a reaction mixture with 2U of each of

BamH I and EcoR I. The resulting products from the restriction digest are subjected to

agarose gel electrophoresis. The desired DNA fragments from DNA 6, DNA 7 and

pMX-neo are then prepared from the agarose gel with Gene Clean Kit (BIO 101 Inc.).

Ten microliters (lOμl) of water is added, respectively, to each of the prepared DNA 6

and DNA 7 fragments to produce, respectively, aqueous solutions of the prepared

DNAs 6 and 7. Twenty microliters (20μl) of water is added to the prepared pMX-neo fragment to produce an aqueous solution of the pMX-neo fragment.

Table 2

Primers Restriction Enzymes

DNA 6 SEQ ID:1 and SEQ ID:9 BamH \

DNA 7 SEQ ID:10 and SEQ ID:1 1 EcoR \

One micro-liter (lμl) of the aqueous solution of the prepared pMX-neo

fragment, 2μl of the aqueous solution of the prepared DNA 6 fragment, and 2μl of the

aqueous solution of the prepared DNA 7 fragment are allowed to ligate together in 50μl

of a ligation reaction mixture by utilizing Ligation Kit ver.l (Takara Shuzo) at 16°C for

1 hour. Ten micro-liters (lOμl) of the resulting reaction mixture is then utilized to

transform a competent E. coli JM109 strain. The transformants are cultured in 100ml of

LB additionally containing 50μg/ml of ampicillin. The expression vector pMX-LE is then prepared from colonies of the transformant with QIAGEN Plasmid Maxi Prep. The expression vector pMX-LE is analyzed with a DNA automatic sequencer

(PE Applied Biosystems) using a Thermo Sequenase II Die terminator kit (Amersham

Pharmacia Biotech). As a result, pMX-LE is confirmed to contain a polynucleotide

encoding amino acid 1 to 841 (from the N-terminus) of a human OBR subunit

(GenBank Accession No. AAA93015) and amino acid 251 to 508 (from the N-

terminus) of a human EPOR subunit (GenBank Accession No. AAA52401). As such, it

is confirmed that pMX-LE contains a polynucleotide encoding the ECD of an OBR

subunit as well as the TMD and ICD of an EPOR subunit.

A viral medium is produced similarly to the procedures of (1.4) with pMX-LE.

TF-1 cells are cultured in a basic culturing medium additionally containing

lng/ml of human granulocyte macrophage colony-stimulating factor receptor

(hereinafter referred to as human GM-CSF) with 5% C0 gas and at 37°C . The cultured

TF-1 cells are centrifuged at lOOOrpm for 3 minutes to collect 1 x 10⁵ cells thereof.

Polyprene (hexadimethrine bromide, Sigma) is added to the concentration of lOμg/ml and human GM-CSF is added to the concentration of lng/ml to the viral medium. The

TF-1 cells are suspended in 1ml of the resulting viral medium and are then transferred

to a 24 well plate. After 5 hours of culturing, the TF-1 cells have added thereto a basic

culturing medium additionally containing lng/ml of human GM-CSF and are cultured overnight to obtain infected TF-1 cells (hereinafter referred to as c3 cells).

The c3 cells are cultured in a basic culturing medium additionally containing

lng/ml of human GM-CSF and lmg/ml of GENETICIN (GibcoBRL). The c3 cells are

suspended to 5 cells/ml in a basic culturing medium additionally containing lOpM of

OB, are transferred to a 96 well plate at lOOμl/well and are cultured for 5 days at 37°C

with 5% C0₂ gas. The c3 cells are then observed under a microscope to select the successfully cloned c3cells. The clones are then scaled up, respectively, to obtain a c3

cell clone.

Example 3: Proliferation dependency of the mammalian cells of the present invention

In a 100mm dish, the 7 clones of the cl cells and the 11 clones of the c2 cells,

prepared in (1.5), are cultured to 4xl0⁵ to 8xl0⁵ cells/ml at 37°C with 5% C0₂ gas in

10ml of a basic culturing medium additionally containing lng/ml of mouse IL-3 and

lmg/ml of GENEΗCIN. The cl cells and c2 cells are collected by centrifuging said cl

cells and c2 cells for 3 minutes at lOOOrpm. After the cl cells and c2 cells are washed 3

times with the basic culturing medium, the cl cells and the c2 cells are suspended in a

basic culturing medium.

The basic culturing medium and a basic culturing medium additionally

containing lOnM of OB are added, respectively, to the wells of a 96 well plate ([3603]

Costar) at lOμl/well. The suspensions of the cl cells are then added, respectively, at

90μl/well (5xl0³ cells/well) to the basic culturing medium (final OB concentration of

OpM) and to the basic culturing medium additionally containing OB (final OB

concentration of InM). The suspensions of the c2 cells are also added, respectively, at

90μl/well (5xl0³ cells/well) to the basic culturing medium (final OB concentration of OpM) and to the basic culturing medium additionally containing OB (final OB

concentration of InM). The cl cells and the c2 cells are cultured for 48 hours with 5%

C0₂ gas at 37°C. Ten micro-liters (lOμl) of Alamar blue (BIOSOURCE) are added to

each of the wells. After the cl cells and the c2 cells are then further cultured for 24

hours, the fluorescence thereof are measured with spectrofluoro (TECAN) under the

conditions in which the gain is set to 50, the excited wavelength is set to 595nm and the detection wavelength is set to 595nm. The experiment is repeated 40 or 80 times. When

the fluorescence value provided by the cultures containing no OB is subtracted from the

fluorescence value provided by the cultures containing OB, the resulting value for clone

8 of the cl cells (hereinafter referred to as the cl-8 cell) is 13,000 and the for clone 7 of

the c2 cells (hereinafter referred to as the c2-7 cell) is 11,000.

Subsequently, the cl-8 cells in a 100mm dish are cultured in lOμl of a basic

culturing medium additionally containing lng/nl of mouse IL-3 and lmg/ml of

GENETICIN with 5% C0₂ gas at 37°C. After the cl-8 cells are washed 3 times with

basic culturing medium, the cl-8 cells are suspended in basic culturing medium.

Culturing mediums are prepared by adding OB to basic culturing medium to

amount to, respectively, lOnM, InM, 500pM, lOOpM and 50pM. Subsequently, basic

culturing medium and the prepared mediums containing specified amounts of OB are

added, respectively to the wells of a 96 well plate at lOμl/well. The suspension of the

cl-8 cells is added at 90μl/well (5x10 cells/well) to the basic culturing medium (final

OB concentration of OpM) and to the basic culturing medium additionally containing

OB (final OB concentration of InM, 100pm, 50pM, lOpM and 5pM). The cl-8 cells

are then cultured for 48 hours at 37°C with 5% C0 gas. Ten microliters (lOμl) of

Alamar blue is added to each of the wells. After further culturing the cl-8 cells for 24

hours, the fluorescence thereof is measured similarly to the above.

Additionally, the procedures above of exposing OB is repeated with BaF-3

cells, instead of the cl-8 cells. The results of the BaF-3 cells and cl-8 cells are shown in

Table 4.The values in Table 4 illustrate the resulting value from correspondingly

subtracting fluorescence value provided by the cultures containing no OB from the fluorescence value provided by the cultures containing OB. Table 4 c1-8 cells BaF-3 cells

1 nM OB 13,000 30

100pM OB 12,900 -6

50pM OB 12,700 20

10pM OB 12,000 2

5pM OB 9,900 4 no OB 0 0

Example 4: Screening of a OB-like activating agent

(4.1) The screening method

In a 100mm dish, cl-8 cells are cultured with 5% C0₂ gas at 37°C in lOμl of a

basic culturing medium additionally containing lng/nl of mouse IL-3 and lmg/ml of

GENETICIN. After the cl-8 cells are washed 3 times with a basic culturing medium,

the cl-8 cells are suspended in a basic culturing medium.

Culturing mediums are prepared by adding a test agent to a basic culturing

mediums to amount to lOOμM or lOμM. As the test agent, there is utilized the

compound shown in the following general formula (I), N-(2-methoxyethyl) 0-(5- methoxy-2-nitrophenyl) methylsulfonatethioamide.

(I)

Subsequently, the basic culturing medium and the prepared mediums containing

specified amounts of the test agent are added, respectively, to the wells of a 96 well

plate at lOμl/well. The suspension of the cl-8 cells is added at 90μl/well (5xl0³

cells/well) to the basic culturing medium (final test agent concentration of OpM) and to

the basic culturing mediums additionally containing the test agent (final test agent

concentration of lOμM and lμM). The cl-8 cells are then cultured for 48 hours at 37 C

with 5% C0 gas. Ten micro-liters (lOμl) of Alamar blue is added to each of the wells.

After further culturing the cl-8 cells for 24 hours, the fluorescence thereof is measured

similarly to the above. The resulting value from subtracting fluorescence value

provided by the cultures containing no test agent from the fluorescence value provided

by the cultures containing lμM of the test agent is 880.

Additionally, the procedures above of exposing the test agent is repeated with

BaF-3 cells, instead of the cl-8 cells. The resulting value from subtracting fluorescence

value provided by the cultures containing no test agent from the fluorescence value provided by the cultures containing lμM of the test agent is 35. (4.2) Production of N-(2-methoxyethyl) 0-(5-methoxy-2-nitrophenyl)

methylsulfonatethioamide

One and eighty-eight hundredths milliliters (1.88ml) of triethylamine and 7ml

of a toluene solution of 5-methoxy-2-nitrophenol (2.30g, 13.6 millimoles) is added at

drop increments to a toluene solution of methylphosphonothiophosphate dichloride (2.02g, 13.6 millimoles). The resulting solution is then allowed to react by being stirred

at room temperature for 12 hours. After lowering the temperature of the reaction

solution to 10°C or less, there is added thereto in drop increments, 2-

methoxyethylamine (2.04g, 27.2 millimoles) to produce a second reaction solution. The

second reaction solution is allowed to react by being stirred at room temperature for 12

hours. The second reaction solution is poured into water and is extracted with ethyl

acetate. The organic layer therefrom is washed with saturated salt water and dried with

anhydrous magnesium sulfate. The organic layer is then concentrated under pressurized

conditions and the resulting residue is subjected to silica gel chromatography to produce 2.37g of N-(2-methoxyethyl) 0-(5-methoxy-2-nitrophenyl)

methylsulfonatethioamide .

n_D ^25'8 1.5784

1H-NMR (300MHz, CDC1₃/TMS) : <5 (ppm)= 8.02 (d, J=9.2Hz, IH), 7.31 (d, J=3.1Hz,

IH), 6.75 (dd, J=9.2,3.1Hz, IH), 3.89 (s, 3H), 3.62-3.75 (m, IH), 3.30-3.40 (m, 2H),

3.19-3.28 (m, 2H), 3.26 (s, 3H), 2.07 (d, J=15.3Hz, 3H)

Example 5: Screening of an OB inhibiting agent In a 100mm dish, cl-8 cells are cultured with 5% C0 gas at 37°C in lOμl of a

basic culturing medium additionally containing lng/nl of mouse IL-3 and lmg/ml of

GENETICIN. After the cl-8 cells are washed 3 times with basic culturing medium, the

cl-8 cells are suspended in a basic culturing medium additionally containing 50pM of

OB.

Culturing mediums are prepared by adding to basic culturing mediums, OB to

amount to 50pM and a test agent to amount to lOOμM or lOμM. Subsequently, the basic culturing medium and the prepared mediums containing specified amounts of OB

and the test agent are added, respectively, to the wells of a 96 well plate at lOμl/well. The suspension of the cl-8 cells is also added at 90μl/well (5xl0³ cells/well) to the

basic culturing medium (final test agent concentration of OpM) and to the basic

culturing mediums additionally containing OB and the test agent (final test agent

concentration of lOμM and lμM). The cl-8 cells are then cultured for 48 hours at 37°C

with 5% C0 gas. Ten microliters (lOμl) of Alamar blue is added to each of the wells.

After further culturing the cl-8 cells for 24 hours, the fluorescence thereof is measured

similarly to the above.

Additionally, the procedures above of exposing OB and the test agent are

repeated with BaF-3 cells, instead of the cl-8 cells.

An OB inhibiting agent can be then screened by selecting a test agent which

provides a low level of proliferation and no substantial differences between the BaF-3

cells cultured with the test agent and the BaF-3 cells cultured with no test agent. In

providing the low level of proliferation, the test agent provides a level of proliferation

which is lower than the cl-8 cells cultured with no test agent. All of the documents, patents, patent applications and publications mentioned above are incorporated herein by reference in their entirety.

Sequence Free Text

SEQ ID:1

An oligonucleotide primer designed for PCR amplification.

SEQ ID:2

An oligonucleotide primer designed for PCR amplification.

SEQ ID:3

An oligonucleotide primer designed for PCR amplification.

SEQ ID:4

An oligonucleotide primer designed for PCR amplification.

SEQ ID:5

An oligonucleotide primer designed for PCR amplification.

SEQ ID:6

An oligonucleotide primer designed for PCR amplification.

SEQ ID:7

An oligonucleotide primer designed for PCR amplification.

SEQ ID:8

An oligonucleotide primer designed for PCR amplification. SEQ ID:9

An oligonucleotide primer designed for PCR amplification.

SEQ ID: 10

An oligonucleotide primer designed for PCR amplification.

SEQ ID:11

An oligonucleotide primer designed for PCR amplification.

SEQ ID: 12

An oligonucleotide primer designed for PCR amplification.

SEQ ID: 13

An oligonucleotide primer designed for PCR amplification.

Claims

CLAIMS:

1. A method of evaluating whether a test agent is an agent affecting the activity of

OBR, said method comprising:

culturing a mammalian cell having:

an OB bindable chimeric receptor comprising an extracellular domain of an OBR

subunit, a transmembrane domain of a cytokine receptor subunit and an

intracellular domain of a receptor subunit selected from a non-OBR homodimeric

cytokine receptor subunit, gpl30 and IL-2R β subunit; and

a proliferation dependency on OB;

exposing the test agent to the mammalian cell; and

measuring the proliferation of the mammalian cell.

2. The method according to claim 1, wherein the intracellular domain is an

intracellular domain of a non-OBR homodimeric cytokine receptor subunit.

3. The method according to claim 1, wherein the intracellular domain is an

intracellular domain of a receptor subunit selected from gpl30 and IL-2R β subunit.

4. The method according to claim 1, wherein the intracellular domain is an

intracellular domain of a receptor subunit selected from a TPOR subunit, EPOR

subunit, G-CSFR subunit, GHR subunit and PRLR subunit.

5. The method according to claim 1 , wherein the mammalian cell is cultured with a culturing medium containing substantially no cytokine activating the chimeric receptor.

6. The method according to claim 1, wherein the mammalian cell is cultured with a

culturing medium comprising OB.

7. A mammalian cell having:

a polynuceotide encoding a chimeric receptor subunit comprising an extracellular

domain of an OBR subunit, a transmembrane domain of a cytokine receptor subunit and

an intracellular domain of a receptor subunit selected from a non-OBR homodimeric

cytokine receptor subunit, gpl30 and IL-2R β subunit; and

a proliferation dependency on OB.

8. The mammalian cell according to claim 7, wherein the mammalian cell is

derived from a hematopoietic mammalian cell.

9. The mammalian cell according to claim 7, wherein the mammalian cell is

derived from a mammalian lymphocyte.

10. The mammalian cell according to claim 7, wherein the mammalian cell is

derived from a cell selected from BaF-3, TALL-101, OCl-Ly9, OCl-Lyl3.1, FDC-Pl,

TF-1, UT-7 and F-36-P.

11. The mammalian cell according to claim 10, wherein the mammalian cell is

derived from BaF-3.

12. The mammalian cell according to claim 7, wherein the intracellular domain is an intracellular domain of a non-OBR homodimeric cytokine receptor subunit.

13. The mammalian cell according to claim 7, wherein the intracellular domain is

an intracellular domain of a receptor subunit selected from gpl30 and IL-2R β subunit.

14. The mammalian cell according to claim 7, wherein the intracellular domain is

an intracellular domain of a receptor subunit selected from a TPOR subunit, EPOR subunit, G-CSFR subunit, GHR subunit and PRLR subunit.

15. The mammalian cell according to claim 7, wherein said transmembrane domain

is a transmembrane domain of a receptor subunit selected from a homodimeric cytokine

receptor subunit, gpl30 and IL-2R β subunit.

16. The mammalian cell according to claim 7, wherein the transmembrane domain

is a transmembrane domain of an OBR subunit.

17. A mammalian cell having:

a polynucleotide encoding a chimeric receptor subunit comprising an extracellular

domain of an OBR subunit and intracellular and transmembrane domains of a receptor

subunit selected from a non-OBR homodimeric cytokine receptor subunit, gpl30 and

IL-2R j3 subunit; and

a proliferation dependency on OB.

18. The mammalian cell according to claim 17, wherein the intracellular and

transmembrane domains are intracellular and transmembrane domains of a receptor

subunit selected from a TPOR subunit, EPOR subunit, G-CSFR subunit, GHR subunit

and PRLR subunit.

19. A mammalian cell having:

an expression vector comprising a polynucleotide encoding a chimeric receptor subunit

comprising an extracellular domain of an OBR subunit, a transmembrane domain of a cytokine receptor subunit and an intracellular domain of a receptor subunit selected

from a non-OBR homodimeric cytokine receptor subunit, gpl30 and IL-2R β subunit;

and

a proliferation dependency on OB.

20. The mammalian cell according to claim 19, wherein the intracellular domain is

an intracellular domain of a receptor subunit selected from a TPOR subunit, EPOR

subunit, G-CSFR subunit, GHR subunit and PRLR subunit.

21. A mammalian cell having:

a chimeric receptor anchored to a cell membrane thereof, wherein the chimeric receptor

comprises an extracellular domain of an OBR subunit, a transmembrane domain of a

cytokine receptor subunit and an intracellular domain of a receptor subunit selected

from a non-OBR homodimeric cytokine receptor subunit, gρl30 and IL-2R β subunit;

and

a proliferation dependency on OB.

22. A polynucleotide encoding a chimeric receptor subunit, wherein the chimeric

receptor subunit comprises an extracellular domain of an OBR subunit, a

transmembrane domain of a cytokine receptor subunit and an intracellular domain of a

receptor subunit selected from a TPOR subunit, EPOR subunit, GHR subunit, PRLR

subunit, gpl30 and IL-2R β subunit.

23. A chimeric receptor subunit comprising an extracellular domain of an OBR

subunit, a transmembrane domain of a cytokine receptor subunit and an intracellular domain of a receptor subunit selected from a TPOR subunit, EPOR subunit, GHR

subunit, PRLR subunit, gpl30 and IL-2R β subunit.

24. A screening method comprising:

conducting the method claim 1 and

selecting a test agent which proliferates the mammalian cell.

25. An OBR activating agent comprising the test agent selected from the screening

method of claim 24.

26. An anti-obesity pharmaceutical comprising as an active ingredient, the OBR

activating agent of claim 25.

27. A screening method comprising:

conducting the method of claim 1 and selecting a test agent which proliferates the mammalian cell.

28. An OBR inhibiting agent comprising the test agent selected from the screening

method of claim 27.

29. An anti-emaciation pharmaceutical comprising as an active ingredient, the OBR

inhibiting agent of claim 28.