PCT
`
`WORLD INTELLECTUAL PROPERTY ORGANIZATION
`International Bureau
`
`
`
`INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`(51) International Patent Classification 5 :
`7 Egg gfiéz’cgggggiégo
`
`(11) International Publication Number:
`(43) International Publication Date:
`
`W0 93/19175
`30 September 1993 (30.09.93)
`
`(21) International Application Number:
`
`PCT/EP93/00697
`
`(22) International Filing Date:
`
`23 March 1993 (23.03.93)
`
`Published
`With international search report.
`Before the expiration of the time limit for amending the
`claims and to be republished in the event of the receipt of
`amendments.
`
`(30) Priority data:
`398/92
`
`25 March 1992 (25.03.92)
`
`DK
`
`(71x72) Applicant and Inventor: THORENS, Bernard [CH/
`CH]; 70, Grand-Chemin, CH—1066 Epalinges (CH).
`
`(74) Agent: NOVO NORDISK A/S; Patent Department, Novo
`Allé, DK-2880 Bagsvaerd (DK).
`
`(81) Designated States: AU, BB, BG, BR, CA, CZ, FI, HU, JP,
`KP, KR, KZ, LK, MG, MN, MW, NO, NZ, PL, RO,
`RU, SD, SK, UA, US, VN, European patent (AT, BE,
`CH, DE, DK, ES, FR, GB, GR, IE, IT, LU, MC, NL,
`PT, SE), OAPI patent (BF, BJ, CF, CG, CI, CM, GA,
`
`
`
`(54) Title: RECEPTOR FOR THE GLUCAGON-LIKE-PEPTIDE-l (GLP-l)
`
`25
`
`
`
`20
`
`A 3
`
`E 3
`
`,
`.5 15
`
`GN, ML, MR, NE, SN, TD, TG).
`
`
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`‘T’
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`
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`
`O
`
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`
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`
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`
`7
`
`o 20 40 60 80100120
`
`Bound (pM)
`
`(57) Abstract
`
`2
`
`4
`
`6
`
`8
`
`10
`
`GLP—l
`
`(nM)
`
`The present invention relates to a recombinant glucagon-like peptide-1 (GLP-1) receptor, to a DNA construct which com-
`prises a DNA sequence encoding a GLP-1 receptor, to methods of screening for agonists of GLP-1 activity, and to the use of the
`GLP-1 receptor for screening for agonists of GLP-1 activity.
`
`MYLANINST. EXHIBIT 1031 PAGE 1
`
`’
`
`.
`
`,,
`
`yfi
`
`o;
`
`MYLAN INST. EXHIBIT 1031 PAGE 1
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`MYLAN INST. EXHIBIT 1031 PAGE 1
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`

`

`W;
`
`FOR THE PURPOSES OF INFORMATION ONLY
`
`Codes used to identify States party to the PCI‘ on the front pages of pamphlets publishing international
`applications under the PCT.
`
`Mauritania
`Malawi
`Netherlands
`Norway
`New Zealand
`Poland
`Portugal
`Romania
`Russian Federation
`Sudan
`Sweden
`Slovak Republic
`Senegal
`Soviet Union
`Chad
`Togo
`Ukraine
`United States of America
`
`.
`
`MYLAN INST. EXHIBIT 1031 PAGE 2
`
`Viet Nam
`
`Austria
`Australia
`Barbados
`Belgium
`Burkina Faso
`Bulgaria
`Benin
`Brazil
`Canada
`Central African Republic
`(‘ongo
`Switzerland
`Cate d'lvoirc
`‘ameroon
`(Suchoslovukia
`(‘Jeeh Republic
`Germany
`Denmark
`Spain
`Finland
`
`France
`Gabon
`United Kingdom
`Guinea
`Greece
`Hungary
`Ireland
`ltaly
`Japan
`Democratic People‘s Republic
`of Korea
`Republic of Korea
`Kazakhstan
`Liechtenstein
`Sri lanka
`Luxembourg
`Monaco
`Madagascar
`Mali
`Mongolia
`
`AT
`AU
`BB
`BE
`BF
`86
`BJ
`BR
`CA
`CF
`CG
`CH
`C!
`CM
`
`$C
`
`Z
`DE
`DK
`ES
`Fl
`
`MYLAN INST. EXHIBIT 1031 PAGE 2
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`

`W0 93/ 19175
`
`PCI'/EP93/00697
`
`RECEPTOR FOR THE GLUCAGON-LIKE-PEPTIDE-l (GLP-l)
`
`FIELD OF THE INVENTION
`
`The present invention relates to a recombinant glucagon-like
`
`peptide-1 (GLP-1) receptor, to a DNA construct which comprises
`a DNA sequence encoding a GLP-1
`receptor,
`to methods of
`
`screening for agonists of GLP-1 activity, and to the use of the
`
`GLP-1 receptor for screening for agonists of GLP—1 activity.
`
`BACKGROUND OF THE INVENTION
`
`10
`
`15
`
`As used in the present specification the designation GLP-l
`
`comprises GLP-1(7-37) as well as GLP-1(7-36)amide.
`
`Glucose-induced insulin secretion is modulated by a number of
`hormones
`and neurotransmitters.
`Inr particular,
`two
`gut
`hormones,
`glucagon—like'
`peptide-1
`(GLP-1)
`and
`_gastriC*
`
`inhibitory peptide (GIP) potentiate the effect of glucose on
`
`insulin secretion and are thus called gluco-incretins (Dupre,
`
`in The Endocrine Pancreas, E. Samois Ed.
`
`(Raven Press, New
`
`York,
`
`(1991), 253 - 281) and Ebert and Creutzfeld,
`
`(Diabetes
`
`Metab. Rev. 3,
`
`(1987)). Glucagon-like peptide-l
`
`is a gluco-
`
`20
`
`and
`and Ebert
`(Dupre
`and in man
`incretin both in rat
`(Lancet g (1987),
`Creutzfeld, vide su ra, and Kreymann et a1.
`1300)). It is part of the preproglucagon molecule (Bell et al.
`
`Nature ggg (1983), 368) which is proteolytiCally processed in
`intestinal L cells to GLP-1(1-37) and GLP-1(7-36)amide or GLP-
`1(7—37)
`(Mojsov et a1.
`(J.Biol.chem. gg;
`(1986), 11880) and
`'Habener et al.: The Endocrine Pancreas E. Samois Ed.
`(Raven
`Press, New York (1991), 53 - 71). Only the truncated forms of
`
`GLP-1 are biologically active and both have identical effects
`
`on insulin secretion in beta cells (Mojsov et a1. J.Clin.Invest
`
`12 (1987), 616) and Weir et al.
`
`(Diabetes 38 (1989), 338). They
`
`30
`
`are the most potent gluco-incretins so far described and are
`
`y')
`
`CONFIW’MQFINST. EXHIBIT 1031 PAGE 3
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`

`

`wo 93/19175
`
`PCT/EP93/00691
`
`active at concentrations as low as one to ten picomolar. The
`
`stimulatory effect of these gluco-incretin hormones requires
`
`the presence of glucose at or above the normal physiological
`
`concentration of about 5 mM and is mediated by activation of
`
`5 adenylate cyclase and a rise in the intracellular concentration
`
`of cyclic AMP
`
`(Drucker et a1. Proc.Natl.Acad.Sci. USA fl
`
`(1987), 3434) and Goke et al.
`
`(Am.J.Physiol. E (1989), G397).
`
`GLP-l
`
`has
`
`also
`
`a
`
`stimulatory effect
`
`on
`
`insulin gene
`
`transcription (Drucker et al. Proc.Natl.Acad.Sci. USA 1g
`
`10 (1987) , 3434) . In a rat model of non-insulin-dependent diabetes
`
`mellitus
`
`(NIDDM)
`
`is associated with a reduced stimulatory
`
`effect of GLP-1 on glucose-induced insulin secretion (Suzuki et
`
`al. Diabetes 3_9_
`
`(1990), 1320).
`
`In man,
`
`in one study, GLP-l
`
`levels were elevated in NIDDM patients both in the basal state
`
`15 and after glucose ingestion; however, following a glucose load
`
`there was
`
`only
`
`a
`
`very
`
`small
`
`rise
`
`in plasma
`
`insulin
`
`concentration (¢rskov et al. J .Clin.Invest. fl (1991), 415).
`
`A recent study (Nathan et al. Diabetes Care 1;;
`
`(1992) , 270)
`
`showed that GLP-l
`
`infusion could ameliorate postprandial
`
`20 insulin secretion and glucose disposal in NIDDM patients. Thus,
`
`as a further step in understanding the complex modulation of
`
`insulin secretion by gut hormones and its dysfunction in
`
`diabetes, we isolated and characterized a complementary DNA for
`
`the beta cell GLP—1 receptor and showed that it is part of a
`
`25 new family of G-coupled receptors.
`
`DESCRIPTION OF THE INVENTION
`
`The present invention relates to a recombinant glucagon-like
`
`peptide-1 (GLP-1) receptor.
`
`the invention relates to a GLP-1 receptor
`More preferably,
`30 which comprises the amino acid sequence shown in SEQ ID No. 1,
`
`or an analogue thereof binding GLP—l with an affinity constant,
`
`KD, below 100 nM, preferably below 10 nM.
`
`In the present
`
`MYLAN INST. EXHIBIT 1031 PAGE'4
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`

`W0 93/19175
`
`‘
`
`PCT/EP93/00697
`
`context,
`the term "analogue"
`is
`intended to indicate a
`naturally occurring variant (including one expressed in other
`animal species,
`in particular human) of
`the receptor or a
`"derivative" i=§b_ a polypeptide which is derived from 'the
`
`native GLP-1 receptor by suitably modifying the DNA sequence
`coding for the variant, resulting in the addition of one or
`more amino acids at either or both the C- and N-terminal ends
`
`of the native amino acid sequence, substitution of one or more
`
`amino acids at one or more sites in the native amino acid
`
`sequence, deletion of one or more amino acids at either or both
`
`ends of the native sequence or at one or more sites within the
`
`native sequence, or insertion of one or more amino acids in the
`
`native sequence.
`
`In another aspect,
`
`the present
`
`invention relates to 21 DNA
`
`construct which comprises a DNA sequence encoding the GLP-1
`
`10
`
`15
`
`receptor of the invention, as well as a recombinant expression
`
`, vector carrying the DNA construct and a cell containing said
`recombinant expression vector.
`
`In one embodiment of the invention, the GLP-1 receptor molecule
`may be provided in solubilised and/or reconstituted form.
`
`20
`
`In the present context "solubilised" is intended to indicate a
`
`'as
`receptor
`preparations.
`
`detergent-solubilised membrane
`in
`present
`'"Reconstituted"
`is
`intended to indicate
`a
`
`25
`
`receptor solubilised in the prescence of essential cofactors,
`e.g. G-protein.
`In this embodiment the receptor may be in a
`reconstituted micellar form.
`
`45:)
`
`The DNA construct of the invention encoding the GLP-1 receptor
`preferably comprises the DNA sequence shown in SEQ ID No. l, or
`at
`least. a DNA' sequence 'coding for a
`functional analogue
`thereof binding GLP-l with an affinity below 100 nM, preferably
`below 10 nM or a suitable modification thereof. Examples of
`suitable modifications of
`the DNA sequence are nucleotide
`
`30
`
`MYLAN INST. EXHIBIT 1031 PAGE 5
`
`MYLAN INST. EXHIBIT 1031 PAGE 5
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`

`W0 93/ 19175
`
`PCI'IEP93/00697
`
`substitutions which do not give rise to another amino acid
`
`sequence of the GLP-1 receptor, but which may correspond to the
`
`t.)
`
`codon usage of the host organism into which the DNA construct
`
`is introduced or nucleotide substitutions which do give rise to
`
`a different amino acid sequence and therefore, possibly,
`
`a
`
`impairing the
`different protein structure without, however,
`properties of the native variant. other examples of possible
`
`modifications are insertion of one or several nucleotides into
`
`the sequence, addition of one or several nucleotides at either
`
`end of the sequence, or deletion of one or several nucleotides
`
`at either end or within the sequence.
`
`Another example of a DNA construct of the invention is one
`
`which encodes a GLP-1 receptor variant particularly suitable
`
`for solubilisation and reconstitution.
`
`The DNA construct of the invention encoding the present GLP-1
`
`receptor may be prepared synthetically by established standard
`
`methods, e.g.
`
`the phosphoamidite method described by Beaucage
`
`and Caruthers, Tetrahedron Letters g; (1981), 1859 - 1869, or
`
`the method described by Hatthes et al., EMBO Journal 1 (1984),
`
`801
`
`-
`
`805. According
`
`to
`
`the
`
`phosphoamidite method,
`
`oligonucleotides are synthesized, e.g.
`
`in an automatic DNA
`
`synthesizer, purified, annealed, ligated and cloned in suitable
`
`vectors.
`
`The DNA construct of the invention may also be of genomic or
`
`cDNA origin, for instance obtained by preparing a genomic or
`
`cDNA library and screening for DNA sequences coding for all or
`
`part of the GLP—1 receptor of the invention by hybridization
`using synthetic oligonucleotide probes
`in accordance with
`
`standard techniques (of. Sambrook et al., Molecular Cloning: A
`
`Laboratory Manual, 2nd Ed., Cold Spring Harbor, 1989). In this
`
`case, a genomic or cDNA sequence encoding the GLP-1 receptor
`
`may be modified at a site corresponding to the site(s) at which
`
`it is desired to introduce amino acid substitutions, e.g. by
`
`10
`
`15
`
`20
`
`25
`
`30
`
`MYLAN INST. EXHIBIT 1031 PAGE 6
`
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`

`

`woos/19175
`
`PCT/EP93/00697
`
`site-directed mutagenesis using synthetic oligonucleotides
`
`fl
`
`encoding the desired amino acid sequence
`
`for homologous
`
`recombination in accordance with well-known procedures.
`
`Finally,
`
`the DNA construct may be of mixed synthetic and
`
`genomic, mixed synthetic and cDNA or mixed genomic and cDNA
`
`origin prepared by ligating fragments of synthetic, genomic or
`
`cDNA origin (as appropriate),
`
`the fragments corresponding to
`
`various parts of the entire DNA construct,
`
`in accordance with
`
`standard techniques. The DNA construct may also be prepared by
`
`1O
`
`polymerase chain reaction using specific primers, for instance
`
`as described in US 4,683,202 or Saiki et al., Science 239
`
`(1988), 437 - 491.
`
`'
`
`The recombinant expression vector into Which the DNA construct
`
`15
`
`the invention is inserted3 may be any vector which may
`of
`conveniently be subjected to recombinant DNA procedures, and
`
`the choice of vector will often depend on the host cell into
`
`which it is to be introduced. Thus, the vectormay be an
`
`autonomously replicating vector, i.e. a vector which exists as
`
`20
`
`25
`
`30
`
`an extrachromosomal entity,
`
`the replication of which
`
`is
`
`independent
`
`of'
`
`chromosomal
`
`replication,
`
`e.g.
`
`a plasmid.
`
`Alternatively,
`
`the vector may be one which, when introduced
`
`into a host cell,
`
`is integrated into the host cell genome and
`
`replicated together with the chromosome(s)
`
`into which it has
`
`been integrated.
`
`In the vector,
`
`the DNA sequence encoding the GLP-1 receptor of
`
`the invention should be operably connected to a suitable pro—
`
`moter sequence. The promoter may be any DNA sequence which
`shows transcriptional activity in the host cell of choice and
`
`may be derived from genes encoding proteins either homologous
`
`Examples of suitable pro-
`or heterologous to the host cell.
`moters for directing the transcription of the DNA encoding the
`GLP-1 receptor of the invention in mammalian cells are the SV40
`
`promoter
`
`(Subramani et al., Mol. Cell Biol. ; (1981), 854 -
`
`MYLAN INST. EXHIBIT 1031 PAGE 7
`
`MYLAN INST. EXHIBIT 1031 PAGE 7
`
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`

`

`W0 93/19175
`
`'
`
`PCT/EP93/00697
`
`864),
`
`the MT-l
`
`(metallothionein gene) promoter
`
`(Palmiter et
`
`al., Science 222 (1983), 809 - 814) or the adenovirus 2 major
`
`late promoter. A suitable promoter for use in insect cells is
`
`the polyhedrin promoter
`
`(Vasuvedan et al.,
`
`FEBS Lett. 31;,
`
`5 (1992) 7 - 11). Suitable promoters for use in yeast host cells
`
`include promoters from yeast glycolytic genes (Hitzeman et al.,
`J. Biol. Chem. 255 (1980), 12073 - 12080; Alber and Kawasaki,
`
`J. Mol. Appl. Gen.
`
`1
`
`(1982),
`
`419
`
`—
`
`434)
`
`or
`
`alcohol
`
`dehydrogenase genes (Young et al. ,
`
`in Genetic Engineering of
`
`m.Microorganisms for Chemicals (Hollaender et al, eds.), Plenum
`
`Press, New York, 1982), or the E (US 4,599,311) or ADH2-4c
`
`(Russell et al., Nature 314 (1983),
`
`652 - 654) promoters.
`
`Suitable promoters for use in filamentous fungus host cells
`
`are, for instance, the A9111; promoter (McKnight et a1. , The EMBO
`
`B J. 4 (1985), 2093 - 2099) or the tpiA promoter.
`
`The DNA sequence encoding the GLP-1 receptor of the invention
`
`may also be operably connected to a suitable terminator, such
`
`as the human growth hormone terminator (Palmiter et al., 99;
`
`gig) or (for fungal hosts) the T_P;1_ (Alber and Kawasaki, opé
`
`20 Q) or m; (McKnight et a1. , pp; 92.1..) terminators. The vec-
`
`tor' may‘ further comprise elements
`
`such as polyadenylation
`
`signals
`
`(e.g.
`
`from SV40 or
`
`the adenovirus
`
`5 Elb region),
`
`transcriptional enhancer sequences (e.g. the.SV40 enhancer) and
`
`translational
`
`enhancer
`
`sequences
`
`(e.g.
`
`the ones
`
`encoding
`
`5 adenovirus VA RNAs).
`
`The recombinant expression vector of the invention may further
`
`comprise a DNA sequence enabling the vector to replicate in the
`
`host cell in question. An example of such a sequence (when the
`
`host cell
`
`is
`
`a mammalian cell)
`
`is the SV40 origin of
`
`m replication, The.vector may also comprise a selectable marker,
`
`e.g. a gene the product of which complements a defect in the
`
`host cell, such as the gene coding for dihydrofolate reductase
`
`(DHFR)
`
`or one which conferS‘
`
`resistance to a drug,
`
`e.g.
`
`neomycin,.hygromycin or methotrexate.
`
`MYLAN INST. EXHIBIT 1031 PAGE 8
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`

`W0 93/19175
`
`PCT/EP93/00697
`
`The procedures used to ligate the DNA sequences coding for the
`
`GLP-1 receptor of the invention,
`
`the promoter and the ter-
`
`minator, respectively, and to insert them into suitable vectors
`
`containing the information necessary for replication, are well
`
`known to persons skilled in the art
`
`(cf.,
`
`for
`
`instance,
`
`Sambrook et al., op.cit.).
`
`10
`
`15
`
`The host cell into which the expression vector of the invention
`
`is introduced may be any cell which is capable of producing the
`
`GLP—1 receptor of the invention and is preferably a eukaryotic
`
`cell, such as invertebrate (insect) cells or vertebrate cells,
`
`e.g. Xenopus laevis oocytes or mammalian cells,
`
`in particular
`
`insect and mammalian cells. Examples of suitable mammalian cell
`
`lines are the COS (ATCC CRL 1650), BHK (ATCC CRL 1632, ATCC CCL
`
`10), CHL (ATCC CCL39) or CHO (ATCC CCL 61) cell lines. Methods
`
`of transfecting mammalian cells and expressing DNA sequences
`introduced in the cells are described in e.g. Kaufman and
`
`Sharp, J. Mol. Biol. 1§2 (1982), 601 — 621; Southern and Berg,
`
`20
`
`J. Mol. Appl. Genet. 1 (1982), 327 - 341; Loyter et al., Proc.
`Natl. Acad.7Sci. USA a (1932), 422 - 426; Wigler et al., Cell
`13 (1978), 725: Corsaro and Pearson, Somatic Cell Genetics 1
`(1981), 603, Graham and van der Eb, Virology §g (1973), 456;
`
`and Neumann et al., EMBO J. 1 (1982), 841 - 845.
`
`Alternatively, fungal cells (including yeast cells) may be used
`as host cells of the invention. Examples of suitable yeasts
`cells
`include
`cells
`of
`Saccharomyces
`spp.
`or Schizo-
`
`25
`
`in. particular strains of Saccharomyces
`saccharomyces spp.,
`cerevisiae. Examples of other fungal cells are cells of fila-
`
`in
`mentous fungi, e.g. Aspergillus spp. or Neurospora spp.,
`particular strains of Aspergillus oryzae or Aspergillus niger.
`The use of Aspergillus spp. for the expression of proteins is
`described in, e.g., EP 272 277.
`
`30
`
`The GLP-1 receptor according to the invention may be produced
`by a method which comprises culturing a cell as described above
`
`MYLAN INST. EXHIBIT 1031 PAGE 9
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`MYLAN INST. EXHIBIT 1031 PAGE 9
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`

`

`WO 93/19175
`
`PCI'/EP93/00697
`
`in a suitable nutrient medium under conditions which are
`
`conducive to the expression of the GLP-1 receptor, and re-
`
`covering the GLP-1 receptor from the culture. The medium used
`
`to culture the cells may be any conventional medium suitable
`
`5 for growing mammalian cells,
`
`such as a serum-containing or
`
`”A
`
`serum-free medium containing appropriate supplements. Suitable
`media are available from commercial
`suppliers or may be
`
`prepared according to published recipes (e.g.
`
`in catalogues of
`
`the American Type Culture Collection).
`
`7
`
`10 If the GLP—1 receptor has retained the transmembrane and (pos-
`
`sibly) the cytoplasmic region of the native variant, it will be
`
`anchored in the membrane of
`
`the host cell,
`
`and the cells
`
`carrying the GLP-1 receptor may be used as
`
`such in the
`
`screening or diagnostic assay. Alternatively, the receptor may
`
`15 be a component of membrane preparations, e.g.
`
`in solubilised
`
`and/ or reconstituted form as defined above.
`
`In a still further aspect,
`
`the present invention relates to a
`
`method of
`
`screening for agonists or
`
`enhancers of GLP-1
`
`activity,
`
`the method comprising incubating a GLP-1 receptor
`
`20 according to any of claims 1 - 3 with a substance suspected to
`
`be an agonist of GLP-1 activity and subsequently with a GLP-l
`
`or an analogue thereof,
`
`and detecting any effect
`
`from the
`
`suspected agonist on the binding of GLP-1
`
`to the GLP—1
`
`receptor. An enhancer being defined as a compound capable of
`
`25 stabilizing interaction between a high-affinity form of the
`
`receptor and the corresponding ligand, as described e.g.
`
`for
`
`the adenosin receptor (Bruns et al. Molecular Pharmacology 2Q
`
`(1990), 939).
`
`An alternative method of screening for agonists of GLP-1
`
`30 activity, comprises incubating GLP-l or an analogue thereof
`
`I).
`
`with a substance suspected to be an agonist of GLP—1 activity
`
`and subsequently with a GLP-1 receptor of the invention, and
`
`detecting any effect on the binding to the GLP-1 receptor. Such
`
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`
`agonists of GLP-1 activity will be substances stimulating
`
`glucose—induced insulin secretion and may be used in the
`treatment of NIDDM.
`
`The GLP-1 receptor may be immobilized on a solid support and
`
`may, as such, be used as a reagent in the screening methods of
`the invention. The GLP-1 receptor may be used in membrane-
`
`bound form,
`
`i.e. bound to whole cells or as a component of
`
`membrane preparations immobilised on a solid support.
`
`The solid Support employed in the screening methods of the
`
`invention preferably comprises a polymer. The support may in
`
`itself be composed of the polymer or may be composed of a
`
`matrix coated with the polymer. The matrix may be of any
`
`suitable material such as glass, paper or plastic. The polymer
`
`may be selected from the group consisting of a plastic (e.g.
`
`latex,
`
`a
`
`polystyrene,
`
`polyvinylchloride,
`
`polyurethane,
`
`polyacrylamide, polyvinylalcohol, nylon, polyvinylacetate, and
`
`any suitable copolymer thereof), cellulose (e.g. various types
`
`of paper, such as nitrocellulose paper and the like), a silicon
`
`polymer
`
`(e.g. siloxane),
`
`a polysaccharide (e.g. agarose or
`
`dextran), an ion exchange resin (e.g. conventional anion or
`
`cation exchange resins), a polypeptide such as polylysine, or
`
`a ceramic material such as glass (e.g. controlled pore glass).
`
`The physical shape of the solid support is not critical,
`
`al—
`
`though some shapes may be more convenient than others for the
`
`present purpose. Thus, the solid support may be in the shape of
`
`a plate, e.g.
`
`a thin layer or microtiter plate, or a film,
`
`strip, membrane (e.g. a nylon membrane or a cellulose filter)
`or solid particles (e.g.
`latex beads or dextran or agarose
`
`beads). In a preferred embodiment, the solid support is in the
`
`form 'of wheat
`
`germ agglutinin—coated SPA beads
`
`(of.
`
`US
`
`4,568,649).
`
`10
`
`15
`
`20
`
`25
`
`30
`
`é!
`
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`
`Alternatively, screening for GLP-l agonists can also be carried
`
`out using a cell
`
`line expressing the cloned GLP—1 receptor
`
`ll
`
`functionally coupled to a G-protein. In living cells, exposure
`
`in the
`increase
`rise to an
`agonist will give
`to an
`intracellular CAMP concentration. The cAMP concentration can
`
`then be measured directly. Changes in CAMP levels may also be
`
`monitored indirectly using appropriate cell lines in which a
`measurable signal is generated in response to an increase in
`
`intracellular CAMP.
`
`10
`
`15
`
`20
`
`25
`
`30
`
`It is furthermore contemplated to locate the ligand-binding
`
`site on the GLP-1 receptor of the invention, for instance by
`
`preparing deletion or substitution derivatives of the native
`
`GLP-1 receptor (as described above) and incubating these with
`
`ligands known to bind the full-length GLP-1
`
`receptor and
`
`detecting any binding of the ligand to the GLP-1 receptor
`
`deletion derivative. Once the ligand-binding site has been
`
`located, this may be used to aquire further information about
`
`the three-dimensional structure of
`
`the ligand-binding site.
`
`Such three-dimensional
`
`structures may,
`
`for
`
`instance,
`
`be
`
`established by means
`
`of protein engineering,
`
`computer
`
`modelling, NMR technology and/or crystallographic techniques.
`
`Based on the three-dimensional structure of the ligand-binding
`
`site,
`
`it may be possible to design substances which are
`
`agonists to the GLP-l molecule.
`
`The characterization of the GLP—1 receptor is of considerable
`
`physiological and pathological importance. It will help study
`
`a fundamental aspect of the entero-insular axis (Unger and
`
`the potentiating
`Eisentraut, Arch.Int.Med. lg; (1969), 261):
`effect of gut hormones on glucoseeinduced insulin secretion,
`
`the role of
`
`these hormones
`
`in the control of glucose
`
`homeostasis and also the possible therapeutic use of GLP-1 to
`
`'I
`
`stimulate insulin secretion in NIDDM patients (mathan et al.
`
`Diabetes Care ;5 (1992), 270). Investigation of the regulated
`
`expression and desensitization of the receptor in the normal
`
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`
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`
`11
`
`state and during the development of diabetes will contribute to
`
`a)
`
`a better understanding of the modulation of insulin secretion
`
`in normal
`
`and pathological
`
`situations. Availability of
`
`antibodies against this receptor may also allow an analysis of
`
`5 the surface localization of this receptor and its distribution
`
`relative to the beta cell glucose transporter GLUT2 (Thorens et
`al. Cell 5; (1988), 281 and Orci et al. Science 23;
`(1989),
`295). This aspect pertains to the hypothesis that the beta cell
`
`membrane has
`
`a "regulatory" domain which contains hormone
`
`m receptors (Bonner-Weir Diabetes 81 (1988), 616), and which may
`
`be distinct from GLUT2—containing membrane domains previously
`
`identified (Thorens et al. Cell §§ (1988), 281 and Orci et al.
`
`Science 22;
`
`(1989), 295); Finally,
`
`the identification of an
`
`additional member of this new family of G-coupled receptors
`
`5 will help design experiments to probe the structure—function
`
`relationship of these new molecules.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`The present invention is further illustrated in the following
`
`examples with reference to the appended drawings in which
`
`n Fig. 1A and Fig. 1B which is a continuation of Fig. 1A together
`
`show the amino acid sequence of the rat GLP—1 receptor in a
`
`comparison with the sequence of
`
`the rat secretin receptor
`
`(SECR),
`
`the opossium parathyroid hormone receptor
`
`(PTHR) and
`
`the porcine calcitonin receptor (CTRl). The GLP-1 receptor has
`
`5 three N glycosylation sites
`
`in the extracellular domain
`
`(arrows). Four cysteines are conserved at identical places in
`the four receptor (boxeS). Note the otherwise very divergent
`
`sequences in this part of the molecules as well as in the COOH-
`
`terminal cytoplasmic tail. Sequence identities are denoted by
`
`.3ostars and homologies by dots. The location of the putative
`
`transmembrane domains are indicated by horizontal bars above
`the sequences.
`I
`
`SUBSTITUTE SHEET
`MYLAN INST. EXHIBIT 1031 PAGE 13
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`wo 93/19175
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`
`12
`
`Fig.
`
`2
`
`shows binding Of 125I-GLP-l to COS cells transfected
`
`with the pGLPR-16 plasmid. Specific binding reaches saturation
`
`at
`
`1
`
`to 10 nM GLP-l.
`
`Insert: Scatchard analysis of GLP-1
`
`binding.
`
`5- Fig.
`
`3
`
`shows binding of 1275I-GLP--1 to INS-l cells. Specific
`
`binding reaches saturation at
`
`1
`
`to 10
`
`nM GLP—l.
`
`Insert:
`
`Scatchard analysis of GLP—1 binding.
`
`Fitting of the curves in Figs. 2 and 3 were performed with the
`
`LIGAND program (McPherson, Kinetic, EBDA, Ligand, Lowry. A
`
`10 Collection
`
`of
`
`radioligand
`
`analysis
`
`programs
`
`(Elsevier,
`
`Amsterdam, 1985)).
`
`Fig.
`
`4 shows displacement of 12SI-GLP-l binding to COS cells
`
`transfected with the rat GLP-l receptor cDNA. Transfected cells
`were incubated with 20
`pM 125I---GLP-1
`in the presence of
`
`1s increasing concentrations of cold peptides. Each point was
`
`measured in duplicate and the experiments repeated three times
`
`for GLP-l, GIP and glucagon and once for VIP and secretin.
`
`Fig. 5 shows stimulation of cyclic AMP formation in COS cells
`
`transfected with the rat GLP-1 receptor cDNA. COS cells were
`
`20 transfected with the pcDNA-l vector alone (open bars) or the
`
`pGLPR—l plasmid (stripped bar) and incubated in the absence or
`
`the presence of GLP-1 at the indicated concentration. CAMP
`
`production was measured in triplicate with a radioimmunoassay
`
`(Amersham) .
`
`25 Fig.
`
`6 shows tissue specificity of GLP-1 receptor expression
`
`assessed by Northern blotting of RNA from different tissues and
`
`from the INS-l cell
`
`line. Ten micrograms of total RNA was
`
`analyzed on each lane. Two major RNA species of 2.7 and 3.6 kb
`
`were detected in all
`
`tissues
`
`in' which the receptor was
`
`30 detected. The position of the migration of the ribosomal RNAs
`
`is indicated to the left of the picture.
`
`MYLAN INST. EXHIBIT 1031 PAGE 14
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`W0 93/19175 '
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`
`13
`
`Fig.
`
`7 is a comparison of the rat GLP-1 receptor amino acid
`
`:1)
`
`sequence (rat) and a partial amino acid sequence of the human
`
`GLP-1 receptor (human).
`
`The present invention is further illustrated in the following
`examples which is not intended to be in any way limiting to the
`scope of the invention as claimed.
`
`EXAMPLE 1
`
`Molecular Cloning and Characterisation of the Rat Islet GLP-l
`
`Receptor cDNA.
`
`10
`
`A rat pancreatic islet cDNA library was constructed in the
`
`pcDNA-l expression vector (Rat pancreatic islets were prepared
`according to Gotoh et a1.
`(Transplantation g;
`(1985), 725).
`PolyA+ RNA was prepared and the cDNA library was constructed in
`
`the pcDNA—l vector (In Vitrogen) as described by Aruffo and
`Seed (Proc.Natl.Acad.Sci. USA 83 (1987), 8573) and Lin et al.
`
`15
`
`(Proc.Nat1.Acad.Sci. USA Q (1991), 3185). Plasmid DNA was
`prepared from pools of five to eight thousands bacterial clones
`(Maniatis et al., Molecular Cloning. A Laboratory Manual. Cold
`Spring Harbor Laboratory, 1982) and transfected into COS cells
`
`(Sompayrac and Dana, Proc.Natl.Acad.Sci. USA 18 (1981), 7575).
`The presence of GLP-1 receptor expressed in COS cells was
`
`followed by
`assessed by binding of the radioiodinated peptide
`photographic emulsion a'utoradiography and screening by dark
`field microscopy (Gearing et al. EMBO J. g (1989), 3667). GLP-
`l(7-36)amide, as well as the other peptides, were purchased
`from Peninsula Laboratories.
`Iodination was performed by the
`iodine monochloride method (Contreras et a1. Meth.Enzymol. 9g
`(1983), 277), the peptide was purified by passage over Sephadex
`G-10
`followed by CM-Sepharose
`and
`specific activity was
`determined by the self displacement
`technique (Calvo et al.
`Biochem. gig (1983), 259). A 1.6 kb cDNA clone (pGLPR—l) was
`isolated by subfractionation of an original positive pool and
`
`20
`
`25
`
`30
`
`MYLAN INST. EXHIBIT 1031 PAGE 15
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`MYLAN INST. EXHIBIT 1031 PAGE 15
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`W0 93/19175
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`14
`
`was used to isolate,
`
`by* DNA hybridization. screening,
`
`two
`
`additional clones from primary positive pools. These plasmids
`
`(pGLPR-16 and -87) had inserts of 3.0 and 2.0 kb, respectively.
`
`Transfection of these clones into COS cells generated high
`
`affinity (KD = 0.6 nM) binding sites for GLP—l (Fig. 2). This
`
`.affinity is comparable to that seen for binding of GLP-1 to the
`
`rat insulinoma cell line INS-1 (Asfari et a1. Endocrinology 139
`
`(1992) , 167)
`
`(KD
`
`0.12 nM; Fig. 3).
`
`In both cases a single
`
`high affinity binding component was detected. The binding to
`
`GLP-1 receptor transfected COS cells reached a plateau between
`
`1 and 10 nM. At concentrations above 10 nM a second, high
`
`capacity,
`
`low affinity,
`
`binding component was detected.
`
`Although specifically displacable by cold GLP—l, this binding
`
`was also present in COS cells transfected with the expression
`
`vector alone and was therefore not further characterized.
`
`Binding of GLP-1 to the receptor expressed in COS cells was
`
`displaced.by cold GLP-l with.a 50 percent displacement achieved
`
`at 0.5 to 1 nM (Fig. 4). other peptide hormones of related
`
`structure such as secretin, gastric inhibitory peptide (GIP)
`
`and vasoactive intestinal peptide (VIP)
`
`(Dupre in The Endocrine
`
`Pancreas, E. Samois Ed.
`
`(Raven Press, New York,
`
`(1991), 253 -
`
`281) and Ebert and Creutzfeld, Diabetes Metab. Rev. 3,
`
`(1987)
`
`did not displace binding. Glucagon could displace the binding
`
`by 50 percent but only at a concentration of one micromolar
`
`(Fig. 4). The addition of subnanomolar concentrations of GLP—1
`
`to transfected COS cells stimulated the production of cyclic
`
`AMP indicating that the receptor was functionally coupled to
`
`activation of adenylate cyclase (Fig. 5).
`
`DNA sequence analysis of the rat GLP—1 receptor cDNA revealed
`
`a major open reading'
`
`frame coding for a 463
`
`amino acid
`
`polypeptide (SEQ ID No. 1). Hydrophaphy plot analysis indicated
`
`[1'
`
`the. presence of an amino-terminal hydrophobic.
`
`region ‘most
`
`probably‘
`
`representing a leader
`
`sequence. This hydrophobic
`
`segment is followed by a hydrophilic domain of about 120 amino
`
`1D
`
`15
`
`20
`
`30
`
`MYLAN INST. EXHIBIT 1031 PAGE 16
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`
`acids which contains three N-linked glycosylation sites. Seven
`
`hydrophobic segments are present which may form transmembrane
`
`domains. Search for
`
`sequence
`
`identities showed the GLP-1
`
`-receptor to be homologous to the secretin receptor (Ishihara et
`al.
`EMBO J. 19 (1991),7 1635)
`(40 percent
`identity),
`the
`
`parathyroid. hormone
`
`receptor
`
`(Juppner et al.
`
`(Science ggg
`
`(1991),
`1024)
`(32.4 percent
`identity)
`and the calcitonin
`receptor (Lin et al. Science ggg (1991), 1022)
`(27.5 percent
`
`identity)
`
`(Fig. 1). These four receptors do not share any
`
`significant sequence homology with other known members of the
`
`G—coupled receptor family and are characterized by a relatively
`
`long amino
`
`terminal, probably extracellular,
`
`domain. The
`
`sequence of
`
`the extracellular domain
`
`is unique
`
`for
`
`each
`
`receptor, yet four cysteines are perfectly conserved (boxes in
`
`Fig. 1). A fifth cysteine at position 12