(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`(19) World Intellectual Property Organization
`International Bureau
`
`(43) International Publication Date
`13 June 2002 (13.06.2002)
`
` (10) International Publication Number
`
`WO 02/46227 A2
`
`C07K 14/605,
`(51) International Patent Classification7:
`14/765, C12N 15/62, A61K 38/38, 38/26, C07K 19/00,
`A61P 3/10
`
`(21) International Application Number:
`
`PCT/US01/43165
`
`(22) International Filing Date:
`29 November 2001 (29.11.2001)
`
`(25) Filing Language:
`
`(26) Publication Language:
`
`English
`
`English
`
`(30) Priority Data:
`60/251,954
`
`7 December 2000 (07.12.2000)
`
`US
`
`(71) Applicant (for all designated States except US): ELI
`LILLY AND COMPANY [US/US]; Lilly Corporate
`Center, Indianapolis, IN 46285 (US).
`
`(72) Inventors; and
`(for US only): GLAESNER,
`(75) Inventors/Applicants
`Wolfgang [DE/US]; 7512 Fieldstone Court, Indianapolis,
`IN 46254 (US).
`l\IICANOVIC, Radmilla [US/US];
`7126 White Oak Trail,
`Indianapolis,
`IN 46236 (US).
`TSCHANG, Sheng-Hung, Rainbow [US/US]; 4963
`Riley Mews, Carmel, IN 46033 (US).
`
`(74) Agents: STEWART, Mark, J. et 211.; Eli Lilly and Com—
`pany, Lilly Corporate Center, Drop Code 1104, Indianapo—
`lis, IN 46285 (US).
`
`(81) Designated States (national): AE, AG, AL, AM, AT, AT
`(utility model), AU, AZ, BA, BB, BG, BR, BY, BZ, CA,
`CH, CN, CO, CR, CU, CZ, CZ (utility model), DE, DK,
`DK (utility model), DM, DZ, EC, EE, EE (utility model),
`ES, PT, PT (utility model), GB, GD, GE, GH, GM, HR, HU,
`ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS,
`LT, LU, LV, MA, NID, MG, MK, MN, MW, MX, MZ, NO,
`NZ, PH, PL, PT, RO, RU, SD, SE, SG, SI, SK, SK (utility
`model), SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN,
`YU, ZA, ZW.
`
`(84) Designated States (regional): ARIPO patent (GH, GM,
`KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZM, ZW),
`Eurasian patent (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM),
`European patent (AT, BE, CH, CY, DE, DK, ES, FT, FR,
`GB, GR, IE, IT, LU, MC, NL, PT, SE, TR), OAPI patent
`(BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, ML, MR,
`NE, SN, TD, TG).
`
`Declarations under Rule 4.17:
`
`as to applicant’s entitlement to apply for and be granted
`a patent (Rule 4.] 7(ii)) for the following designations AE,
`AG, AL, AM, AT AU, AZ, BA, BB, BG, BR, BY, BZ, CA, CH,
`CN, CO, CR, CU, CZ, DE, DK, DM, DZ, EC, EE, ES, FI,
`GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG,
`KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV,NIA, MD, AIG, NIK,
`MN, AIW, .MX, MZ, NO, NZ, PH, PL, PT RO, RU, SD, SE,
`SG, SI, SK, SL, TJ, TM, TR, TT TZ, UA, UG, UZ, VN, YU,
`ZA, ZW, ARIPOpatent (GH, GM, KE, LS, MW, MZ, SD, SL,
`SZ, TZ, UG, ZM, ZW), Eurasian patent (AM, AZ, BY, KG,
`KZ, MD, RU, TJ, TM), European patent (AT BE, CH, CY,
`DE, DK, ES, FI, FR, GB, GR, IE IT LU, MC, NL, PT SE,
`TR), OAPI patent (BE BJ, CF, CG, CI, CM, GA, GN, GQ,
`GW, ML, MR, NE, SN, TD, TG)
`as to the applicant 's entitlement to claim the priority ofthe
`earlier application (Rule 4. I 7(iii)) for the following desig-
`nations AE, AG, AL, AM, AT AU, AZ, BA, BB, BG, BR, BY,
`BZ, CA, CH, CN, CO, CR, CU, CZ, DE, DK, DM, DZ, EC,
`EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN,
`IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT LU, LV,
`MA, MD, .MG, AIK,
`iMN, MW iMX, MZ, NO, NZ, PH, PL,
`PT R0, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, 7T TZ,
`UA, UG, UZ, VN, YU, ZA, ZW,: ARIPO patent (GH, GM,
`KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZM, ZW), Eurasian
`patent (Alli, AZ, BY, KG, KZ, MD, RU, TJ, TIM), European
`patent (AT BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE,
`IT LU, MC, NL, PT SE, TR), OAPIpatent (BF BJ, CE CG,
`CI, CM, GA, GN, GQ, GW, ML, MR, NE, SN, TD, TG)
`
`Published:
`
`without international search report and to be republished
`upon receipt of that report
`
`For two-letter codes and other abbreviations, refer to the ”Guid-
`ance Notes on Codes andAbbreviations ” appearing at the begin-
`ning ofeach regular issue ofthe PCT Gazette.
`
`WO02/46227A2
`
`(54) Title: GLP—l FUSION PROTEINS
`
`(57) Abstract: The present invention relates to glucagon—like—l compounds fused to proteins that have the effect of extending the in
`vivo half—life of the peptides. These fusion proteins can be used to treat non—insulin dependent diabetes mellitus as well as a variety
`of other conditions.
`
`MYLAN INST. EXHIBIT 1036 PAGE 1
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`WO 02/46227
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`PCT/US01/43165
`
`GLP—l FUSION PROTEINS
`
`The present invention relates to glucagon—like peptides
`
`including analogs and derivatives thereof fused to proteins
`
`that have the effect of extending the in vivo half—life of
`
`the peptides. These fusion proteins can be used to treat
`
`non—insulin dependent diabetes mellitus as well as a variety
`
`of other conditions.
`
`10
`
`15
`
`20
`
`25
`
`30
`
`Glucagon—Like Peptide l
`
`(GLP—1) is a 37 amino acid
`
`peptide that is secreted by the L—cells of the intestine in
`
`response to food ingestion. It has been found to stimulate
`
`insulin secretion (insulinotropic action),
`
`thereby causing
`
`glucose uptake by cells and decreased serum glucose levels
`
`(1992) Int. J. Peptide Protein
`[see, e.g., Mojsov, S.,
`-However, GLP—1 is poorly active.
`Research, 40:333—343j.
`subsequent endogenous cleavage between the 6th and 7th
`
`A
`
`position produces a more potent biologically active GLP—l(7~
`
`37)OH peptide. Numerous GLP—l analogs and derivatives are
`
`known and are referred to herein as “GLP—l compounds.”
`
`These GLPel analogs include the Exendins which are peptides
`found in the venom of the GILA—monster.
`The Exendins have
`
`sequence homology to native GLP—l and can bind the GLP~l
`
`receptor and initiate the signal transduction cascade
`
`responsible for the numerous activities that have been
`
`attributed to GLP—l(7—37)OH.
`
`GLP—l compounds have a variety of physiologically
`
`significant activities.
`
`For example, GLP~l has been shown
`
`to stimulate insulin release,
`
`lower glucagon secretion,
`
`inhibit gastric emptying, and enhance glucose utilization.
`
`[Nauck, M.A., et al.
`
`(1993) Diabetologia 36:741—744;
`
`Gutniak, M., et a1.
`
`(1992) New England J. of Med. 326:1316—
`
`1322; Nauck, M.A., et a1.,
`
`(1993) J. Clin. Invest. 91:301—
`
`307].
`
`MYLAN INST. EXHIBIT 1036 PAGE 2
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`WO 02/46227
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`PCT/US01/43165
`
`GLP—l shows the greatest promise as a treatment for
`
`non—insulin dependent diabetes mellitus (NIDDM). There are
`
`numerous oral drugs on the market to treat the insulin
`
`resistance associated with NIDDM. As the disease
`
`progresses, however, patients must move to treatments that
`
`stimulate the release of insulin and eventually to
`
`treatments that involve injections of insulin. Current
`
`drugs which stimulate the release of insulin, however, can
`
`also cause hypoglycemia as can the actual administration of
`
`insulin.
`
`GLP—l activity, however,
`
`is controlled by blood
`
`glucose levels. When levels drop to a certain threshold
`
`level, GLP—1 is not active. Thus,
`
`there is no risk of
`
`hypoglycemia associated with treatment involving GLP—l.
`
`However,
`
`the usefulness of therapy involving GLP—l
`
`peptides has been limited by their fast clearance and short
`
`half—lives.
`
`For example, GLP—l(7—37) has a serum half—life
`
`of only 3 to 5 minutes.
`
`GLP—l(7—36) amide has a time action
`
`of about 50 minutes when administered subcutaneously.
`
`Even
`
`analogs and derivatives that are resistant to endogenous
`
`protease cleavage, do not have half—lives long enough to
`
`avoid repeated administrations over a 24 hour period. Fast
`
`clearance of a therapeutic agent is inconvenient in cases
`
`where it is desired to maintain a high blood level of the
`
`agent over a prolonged period of time since repeated
`
`administrations will then be necessary.
`
`Furthermore, a
`
`long—acting compound is particularly important for diabetic
`
`patients whose past treatment regimen has involved taking
`
`only oral medication. These patients often have an
`
`extremely difficult time transitioning to a regimen that
`
`involves multiple injections of medication.
`The present invention overcomes the problems associated
`
`with delivering a compound that has a short plasma half—
`
`life.
`
`The compounds of the present invention encompass GLP—
`
`1 compounds fused to another protein with a long circulating
`
`10
`
`15
`
`20
`
`25
`
`30
`
`MYLAN INST. EXHIBIT 1036 PAGE 3
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`
`half~life such as the Fc portion of an immunoglobulin or
`
`albumin.
`
`Generally, small therapeutic peptides are difficult to
`
`manipulate because even slight changes in their structure
`
`can affect stability and/or biological activity. This has
`
`been especially true for GLP—l compounds currently in
`
`development.
`
`For example, GLP—l(7—37)OH has a tendency to
`
`undergo a conformational change from a primarily alpha helix
`
`structure to a primarily beta sheet structure. This beta
`
`sheet form results in aggregated material that is thought to
`
`be inactive.
`
`It was,
`
`therefore, surprising that
`
`biologically active GLP—l fusion proteins with increased
`
`half—lives could be developed. This was especially
`
`unexpected given the difficulty of working with GLP—l(7~
`
`37)OH alone and the large size of the fusion partner
`
`relative to the small GLP—l peptide attached.
`
`Compounds of the present invention include heterologous
`
`fusion proteins comprising a first polypeptide with a N—
`
`terminus and a C—terminus fused to a second polypeptide with
`
`a N—terminus and a C—terminus wherein the first polypeptide
`
`is a GLP—l compound and the second polypeptide is selected
`
`from the group consisting of
`
`a) human albumin;
`
`b) human albumin analogs; and
`
`c)
`
`fragments of human albumin,
`
`and wherein the C—terminus of the first polypeptide is fused
`
`to the N—terminus of the second polypeptide.
`
`Compounds of the present invention also include a
`
`heterologous fusion protein comprising a first
`
`polypeptide with a N—terminus and a C—terminus fused to a
`
`second pOlYpeptide with a N—terminus and a C—terminus
`
`wherein the first p01Ypeptide is a GLP—l compound and the
`
`second polypeptide is selected from the group consisting of
`
`a) human albumin;
`
`10
`
`15
`
`20
`
`25
`
`3O
`
`35
`
`MYLAN INST. EXHIBIT 1036 PAGE 4
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`PCT/US01/43165
`
`lfl human albumin analogs; and
`
`c)fragments of human albumin,
`
`and wherein the C-terminus of the first polypeptide is fused
`
`to the N—terminus of the second polypeptide via a peptide
`
`5
`
`linker.
`
`It is preferred that the peptide linker is selected
`
`from the group consisting of:
`
`a) a glycine rich peptide;
`
`b) a peptide having the sequence [Gly—Gly—Gly—Gly—Ser]n
`
`where n is l, 2, 3, 4,
`
`5 or 6; and
`
`10
`
`c) a peptide having the sequence [Gly—Gly—Gly—Gly—
`
`Ser]3.
`
`Additional compounds of the present invention include a
`
`heterologous fusion protein comprising a first polypeptide
`
`with a N—terminus and a C—terminus fused to a second
`
`15
`
`polypeptide with a N—terminus and a C—terminus wherein the
`
`first polypeptide is a GLP—l compound and the second
`
`polypeptide is selected from the group consisting of
`
`a)the Fb portion of an immunoglobulin;
`
`b)an.analog of the Fc portion of an immunoglobulin;
`
`20
`
`and
`
`c) fragments of the Fc portion of an immunoglobulin,
`
`and wherein the C—terminus of the first polypeptide is fused
`
`to the N—terminus of the second polypeptide.
`
`The GLP—l
`
`compound may be fused to the second polypeptide via a
`
`25
`
`peptide linker.
`It is preferable that the peptide linker is
`selected from the group consisting of:
`'
`
`a)a glycine rich peptide;
`
`b)a peptide having the sequence [Gly—Gly—Gly~Gly—Ser]n
`
`where n is l, 2, 3, 4,
`
`5 or 6; and
`
`30
`
`cfl a peptide having the sequence [Gly—Gly—Gly—Gly—Ser]3.
`
`It is generally preferred that the GLP—l compound that
`
`is part of the heterologous fusion protein have no more than
`6 amino acids that are different from the corresponding
`
`amino acid in GLP—l(7—37)OH, GLP—l(7—36)OH,or Exendin—4.
`
`35
`
`It is even more preferred that the GLP—l compound have no
`
`MYLAN INST. EXHIBIT 1036 PAGE 5
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`MYLAN INST. EXHIBIT 1036 PAGE 5
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`WO 02/46227
`
`PCT/US01/43165
`
`more than 5 amino acids that differ from the corresponding
`
`amino acid in GLP—l(7~37)OH, GLP—l(7—36)OH, or Exendin—4.
`
`It is most preferred that the GLP—l compound have no more
`
`than 4, 3, or 2 amino acids that differ from the
`
`corresponding amino acid in GLP—l(7—37)OH, GLP—l(7—36)OH, or
`
`Exendin—4. Preferably, a GLP—l compound that is part of the
`
`heterologous fusion protein has glycine or valine at
`
`position 8.
`
`The present invention also includes polynucleotides
`
`encoding the heterologous fusion protein described herein,
`
`vectors comprising these polynucleotides and host cells
`
`transfected or transformed with the vectors described
`
`herein. Also included is a process for producing a
`
`heterologous fusion protein comprising the steps of
`
`transcribing and translating a polynucleotide described
`
`herein under conditions wherein the heterolgous fusion
`
`protein is expressed in detectable amounts.
`
`The present invention also encompasses a method for
`
`normalizing blood glucose levels in a mammal in need thereof
`
`comprising the administration of a therapeutically effective
`
`amount of a heterologous fusion protein described herein.
`
`The invention is further illustrated with reference to
`
`the following drawings:
`
`Figure l:
`
`IgGl Fc amino acid sequence encompassing the
`
`hinge region, CH2 and CH3 domains.
`
`Figure 2: Human serum albumin amino acid sequence.
`
`Figure 3: A. SDS—PAGE gel and immunoblot of same gel
`
`illustrating the molecular weight of IgGl—Fc and GLP—l—Fc
`
`fusion proteins (Lane 1, MW standards; Lane 2, Purified Fc;
`
`lane 3, Mock transfected media; Lane 4, ValB—GLP—l—Fc; Lane
`
`5, Exendin—4—Fc) B. SDS—PAGE gel and immunoblot of same gel
`
`illustrating the molecular weight of human HSA and GLP—l—HSA
`
`fusion proteins (Lane 1, MW standards; Lane 2, Purified HSA;
`
`lane 3, Mock transfected media; Lane 4, Vala—GLP—l—HSA; Lane
`
`10
`
`15
`
`20
`
`25
`
`3O
`
`35
`
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`

`WO 02/46227
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`PCT/US01/43165
`
`_ 6 _
`
`5, ValB—GLP—l—[Gly—Gly—Gly—Gly—Ser]3—HSA; Lane 6, Exendin—4—
`
`HSA; Lane 7, Exendin—4—[Gly~Gly—Gly—Gly—Ser]3—HSA).
`
`Figure 4: SDS—PAGE gel of purified Fc, albumin, and
`
`GLP—1 fusion proteins (Lane 1, MW standards; Lane 2,
`
`purified Fc; Lane 3, ValS—GLP—l—Fc; Lane 4, Exendin—4—Fc;
`
`Lane 5, MW standard; Lane 6, ValB—GLP—l—HSA; Lane 7,
`
`Exendin—4—HSA; Lane 8, Exendin~4~[Gly—Gly—Gly—Gly—SerJ3—
`
`HSA).
`
`Figure 5: Expression cloning vector containing the Fc
`
`regions illustrated in figure 1.
`
`Figure 6: Expression cloning vector containing the
`
`albumin sequence illustrated in figure 2.
`
`Figure 7: Expression cloning vector containing DNA
`
`encoding a 15 amino acid linker fused in frame and 5’ of the
`
`albumin sequence illustrated in figure 2.
`
`Figure 8:
`
`In vitro dose response activity of GLP—1
`
`fusion proteins.
`
`Figure 9: Pharmacokinetics of GLP—1 Fc and HSA fusion
`
`proteins.
`
`Figure 10: Glucodynamic response to Exendin—Fc in two
`
`normal fasted dogs.
`
`Figure 11: Insulinotropic response to Exendin—Fc in two
`
`normal fasted dogs.
`
`Figure 12: DNA sequence encoding a human IgG1 Fc
`
`10
`
`15
`
`20
`
`25
`
`region.
`
`Figure 13: DNA sequence encoding a human albumin
`
`protein.
`
`The heterologous fusion proteins of the present
`invention comprise a GLF—l compound fused to human albumin,
`
`30
`
`a human albumin analog, a human albumin fragment,'the Fc
`
`portion of an immunoglobulin, an analog of the Fc portion of
`
`an immunoglobulin, or a fragment of the Fc portion of an
`
`immunoglobulin.
`
`The C—terminus of the GLP—l compound may be
`
`35
`
`fused directly, or fused via a peptide linker,
`
`to the N—
`
`MYLAN INST. EXHIBIT 1036 PAGE? 7
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`
`terminus of an albumin or PC protein. These heterologous
`
`fusion proteins are biologically active and have an
`
`increased half—life compared to native GLP—l.
`
`It is preferred that the GLP—l compounds that make up
`
`part of the heterologous fusion protein encompass
`
`polypeptides having from about twenty~five to about
`
`thirty—
`
`nine naturally occurring or non—naturally occurring amino
`
`acids that have sufficient homology to native GLP-l(7—37)OH
`
`such that they exhibit insulinotropic activity by binding to
`
`10
`
`the GLP—1 receptor on B—cells in the pancreas.
`
`A GLP~1
`
`compound typically comprises a polypeptide having the amino
`
`acid sequence of GLP—l(7~37)OH, an analog of GLP—1
`
`(7—37)OH,
`
`a fragment of GLP—l(7—37)OH or a fragment of a GLP—l(7—37)OH
`
`analog. GLP—l(7~37)OH has the amino acid sequence of SEQ ID
`
`15
`
`NO: 1:
`
`7
`
`8
`
`9
`
`10
`
`11
`
`12
`
`13
`
`14
`
`15
`
`l6 17
`
`His—Ala—Glu~Gly—Thr—Phe—Thr—Ser—Asp—Val—Ser—
`
`18
`
`19
`
`2O
`
`21
`
`22
`
`23
`
`24
`
`25
`
`26
`
`27
`
`28
`
`Ser—Tyr—Leu—Glu—Gly~Gln—Ala—Ala—Lys—GluwPhe—
`
`20
`
`29
`
`3O
`
`31
`
`32
`
`33
`
`34
`
`35
`
`36
`
`37
`
`Ile—Ala—Trp—Leu—Val—Lys~Gly—Arg—Gly
`
`(SEQ ID NO: 1)
`
`25
`
`30
`
`35
`
`By custom in the art,
`
`the amino terminus of GLP—l(7—
`
`37)OH has been assigned number residue 7 and the carboxy—
`
`terminus, number 37.
`
`The other amino acids in the
`
`polypeptide are numbered consecutively, as shown in SEQ
`
`ID NO: 1.
`
`For example, position 12 is phenylalanine and
`
`position 22 is glycine.
`
`GLP—l compounds also encompass "GLP—l fragments."
`
`A
`
`GLP—l fragment is a polypeptide obtained after truncation of
`
`one or more amino acids from the NLterminus and/or C—
`
`terminus of GLP—l(7—37)OH or an analog or derivative
`
`thereof.
`
`The nomenclature used to describe GLP—l(7—37)OH is
`
`also applicable to GLP—l fragments.
`
`For example, GLP—l(9—
`
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`
`36)OH denotes a GLP—l fragment obtained by truncating two
`
`amino acids from the Ntterminus and one amino acid from the
`
`C—terminus. The amino acids in the fragment are denoted by
`
`the same number as the corresponding amino acid in GLP—l(7—
`
`37)OH.
`
`For example,
`
`the Nlterminal glutamic acid in GLP—
`
`l(9—36)OH is at position 9; position 12 is occupied by
`
`phenylalanine; and position 22 is occupied by glycine, as in
`
`GLP—l(7—37)OH. For GLP—l(7—36)OH,
`
`the glycine at position 37
`
`of GLP—l(7—37)OH is deleted.
`
`GLPel compounds also include polypeptides in which one
`
`or more amino acids have been added to the NLterminus and/or
`
`C—terminus of GLP—l(7—37)OH, or fragments or analogs
`
`thereof.
`
`It is preferred that GLP—l compounds of this type
`
`have up to about thirty—nine amino acids.
`
`The amino acids
`
`in the “extended” GLP~l compound are denoted by the same
`number as the corresponding amino acid in GLP—l(7—37)OH.
`
`For example,
`
`the NLterminus amino acid of a GLP—l compound
`
`obtained by adding two amino acids to the NLterminal of GLP«
`
`l(7—37)OH is at position 5; and the C—terminus amino acid of
`
`a GLP—l compound obtained by adding one amino acid to the C—
`
`terminus of GLP—l(7—37)OH is at position 38. Thus, position
`
`12 is occupied by phenylalanine and position 22 is occupied
`
`by glycine in both of these “extended” GLP—l compounds, as
`
`in GLP—l(7—37)OH. Amino acids 1—6 of an extended GLP—l
`
`compound are preferably the same as or a conservative
`
`substitution of the amino acid at the corresponding position
`
`of GLP—l(l—37)OH. Amino acids 38—45 of an extended GLP—l
`
`compound are preferably the same as or a conservative
`
`substitution of the amino acid at the corresponding position
`
`10
`
`15
`
`20
`
`25
`
`3O
`
`of glucagon or Exendin—4.
`
`GLP—l compounds of the present invention encompass
`
`“GLP—l analogs.”
`
`A GLP—l analog has sufficient homology to
`
`GLP—l(7—37)OH or a fragment of GLP—l(7—37)OH such that the
`
`analog has insulinotropic activity. Preferably, a GLP—l
`
`35
`
`analog has the amino acid sequence of GLP—l(7—37)OH or a
`
`MYLAN INST. EXHIBIT 1036 PAGE 9
`
`MYLAN INST. EXHIBIT 1036 PAGE 9
`
`

`

`WO 02/46227
`
`PCT/US01/43165
`
`fragment thereof, modified so that from.one,
`
`two,
`
`three,
`
`four or five amino acids differ from the amino acid in the
`
`corresponding position of GLP—l(7—37)OH or a fragment of
`
`GLP—l(7—37)OH.
`
`In the nonmenclature used herein to
`
`designate GLP—l compounds,
`
`the substituting amino acid and
`
`its position is indicated prior to the parent structure.
`
`For example, GluZZ—GLP—l(7—37)OH designates a GLP—l compound
`
`in which the glycine normally found at position 22 of GLP—
`
`l(7—37)OH has been replaced with glutamic acid; va18—Glu22—
`
`10
`
`GLP—l(7—37)OH designates a GLP—l compound in which alanine
`
`normally found at position 8 and glycine normally found at
`
`position 22 of GLP—l(7—37)OH have been replaced with valine
`
`and glutamic acid, respectively.
`
`GLP—l compounds of the present invention also include
`
`15
`
`"GLP~l derivatives."
`
`A GLP—l derivative is defined as a
`
`~20
`
`25
`
`30
`
`molecule having the amino acid sequence of GLP—1 or of a
`
`GLP—l analog, but additionally having chemical modification
`
`of one or more of its amino acid side groups, a—carbon
`
`atoms,
`
`terminal amino group, or terminal carboxylic acid
`
`group.
`
`A chemical modification includes, but is not limited
`
`to, adding chemical moieties, creating new bonds, and
`
`removing chemical moieties. Modifications at amino acid
`
`side groups include, without limitation, acylation of lysine
`
`E—amino groups, N—alkylation of arginine, histidine, or
`
`lysine, alkylation of glutamic or aspartic carboxylic acid
`
`groups, and deamidation of glutamine or asparagine.
`
`Modifications of the terminal amino group include, without
`
`limitation,
`
`the des~amino, N—lower alkyl, N—di~lower alkyl,
`
`and N—acyl modifications. Modifications of the terminal
`
`carboxy group include, without limitation,
`
`the amide,
`
`lower
`
`alkyl amide, dialkyl amide, and lower alkyl ester
`
`modifications.
`
`Lower alkyl is C1—C4 alkyl.
`
`Furthermore,
`
`one or more side groups, or terminal groups, may be
`
`protected by protective groups known to the ordinarily~
`
`MYLAN INST. EXHIBIT 1036 PAGE 10
`
`MYLAN INST. EXHIBIT 1036 PAGE 10
`
`

`

`WO 02/46227
`
`PCT/US01/43165
`
`_ 10‘_
`
`skilled protein chemist.
`
`The d—carbon of an amino acid may
`
`be mono— or dimethylated.
`
`Any GLP—l compound can be part of the heterologous
`
`fusion proteins of the present invention as long as the GLP—
`
`5
`
`1 compound itself is able to bind and induce signaling
`
`through the GLP—1 receptor.
`
`GLP—1 receptor binding and
`
`signal transduction can be assessed using in vitro assays
`
`such as those described in EP 619,322 and U.S. Patent No.
`5,120,712, respectively.
`2
`
`10
`
`Numerous active GLP—l fragments, analogs and
`
`derivatives are known in the art and any of these analogs
`
`and derivatives can also be part of the heterologous fnsion
`
`proteins of the present invention.
`
`Some examples of novel
`
`GLP—l analogs as well as GLP—l analogs and derivatives known
`
`15
`
`in the art are provided herein.
`
`Some GLP—l analogs and GLP—1 fragments known in the art
`
`include, for example, GLP—1(7—34) and GLP—1(7—35), GLP—1(7—
`
`36), Glng—GLP—1(7—37), D—G1n9—GLP—1(7—37), Thrl6—Lys18~GLP—
`
`1(7—37), and LyslB—GLP—1(7—37).
`
`GLP—l analogs such as GLP—
`
`2O
`
`1(7—34) and GLP—1(7—35) are disclosed in U.S. Patent No.
`
`5,118,666. Biologically processed forms of GLP—1 which have
`
`insulinotropic properties, such as GLP—1(7—36) are also
`
`known. Other known biologically active GLP—l compounds are
`
`disclosed in U.S. Patent No 5,977,071 to Hoffmann, et al.,
`
`25 U.S. Patent No. 5,545,618 to Buckley, et a1., and Adelhorst,
`
`et al., J1 Biol. Chem. 269:6275 (1994).
`
`MYLAN INST. EXHIBIT 1036 PAGE 11
`
`MYLAN INST. EXHIBIT 1036 PAGE 11
`
`

`

`WO 02/46227
`
`PCT/US01/43165
`
`_ 11 _
`
`A preferred group of GLP—1 analogs is composed of
`
`GLP—
`
`1 analogs of formula I
`
`(SEQ ID NO: 2)
`m
`
`7
`
`8
`
`9
`
`10
`
`11
`
`12
`
`13
`
`14
`
`15
`
`16
`
`17
`
`His—Xaa—Xaa—Gly—Xaa—Phe—Thr—Xaa—Asp—Xaa*Xaa—
`
`18
`
`19
`
`20
`
`21
`
`22
`
`23
`
`24
`
`25
`
`26
`
`27
`
`28
`
`Xaa—Xaa—Xaa—Xaa—Xaa—Xaa—Xaa—Xaa—Xaa—Xaa~Phe—
`
`29
`
`3O
`
`31
`
`32
`
`33
`
`34
`
`35
`
`36
`
`37
`
`38
`
`39
`
`‘10
`
`Ile—Xaa—Xaa—Xaa—Xaa—Xaa—Xaa—Xaa—Xaa—Xaa—Xaa—
`
`4O
`
`41
`
`42
`
`43
`
`44
`
`45
`
`Xaa—Xaa—Xaa—Xaa—Xaa—Xaa
`
`Formula I (SEQ ID NO: 2)
`
`15
`
`wherein:
`
`Xaa
`
`at position 8 is Ala, Gly, Ser, Thr, Leu, Ile, Val,
`
`20
`
`Xaa
`
`Xaa
`
`Xaa
`
`Xaa
`
`Glu, Asp, or Lys;
`
`at position 9 is Glu, Asp, or Lys;
`
`at position 11 is Thr, Ala, Gly, Ser,
`
`Leu,
`
`Ile, Val,
`
`Glu, Asp, or Lys;
`
`at position 14 is Ser, Ala, Gly, Thr,
`
`Leu,
`
`Ile, Val,
`
`Glu, Asp, or Lys;
`
`at position 16 is Val, Ala, Gly, Ser,
`
`Thr,
`
`Leu, Ile,
`
`Tyr, Glu, Asp, Trp, or Lys;
`
`25
`
`Xaa
`
`at position 17 is Ser, Ala, Gly, Thr,
`
`Leu,
`
`Ile, Val,
`
`Glu, Asp, or Lys;
`
`Xaa
`
`Xaa
`
`at position 18 is Ser, Ala, Gly, Thr,
`
`Leu,
`
`Ile, Val,
`
`Glu, Asp, Trp, Tyr, or Lys;
`
`at position 19 is Tyr, Phe, Trp, Glu,
`
`ASP,
`
`Gln,
`
`or Lys;
`
`3O
`
`Xaa
`
`at position 20 is Leu, Ala, Gly, Ser,
`
`Thr,
`
`Ile,
`
`Val,‘
`
`Xaa
`
`Xaa
`
`Glu, Asp, Met, Trp, Tyr, or Lys;
`
`'at position 21 is Glu, Asp, or Lys;
`
`at position 22 is Gly, Ala, Ser, Thr,
`
`Leu,
`
`Ile, Val,
`
`Glu,
`
`Asp, or Lys;
`
`35
`
`Xaa
`
`at position 23 is Gln, Asn, Arg, Glu,
`
`ASP,
`
`or Lys;
`
`MYLAN INST. EXHIBIT 1036 PAGE 12
`
`MYLAN INST. EXHIBIT 1036 PAGE 12
`
`

`

`WO 02/46227
`
`PCT/US01/43165
`
`Xaa
`
`Xaa
`
`Xaa
`
`Xaa
`
`Xaa
`
`Xaa
`
`Xaa
`
`Xaa
`
`Xaa
`
`Xaa
`
`Xaa
`
`Xaa
`
`10
`
`15
`
`20
`
`Xaa
`
`Xaa
`
`Xaa
`
`Xaa
`
`25
`
`_ 12 _
`
`at position 24 is Ala,
`
`Gly, Ser, Thr, Leu, Ile, Val,
`
`Arg,
`
`Glu,
`
`Asp,
`
`or Lys;
`
`at position 25 is Ala,
`
`Gly, Ser, Thr, Leu, Ile, Val,
`
`Glu, Asp,
`
`or Lys;
`
`at position 26 is Lys,
`
`Arg, Gln, Glu, Asp, or His;
`
`at position 27 is Leu,
`
`Glu, Asp, or Lys;
`
`at position 30 is Ala,
`
`Gly, Ser, Thr, Leu, Ile, Val,
`
`Glu, Asp,
`
`0r LYS;
`
`at position 31 is Trp,
`
`Phe, Tyr, Glu, Asp, or Lys;
`
`at position 32 is Leu,
`
`Gly, Ala, Ser, Thr, Ile, Val,
`
`Glu,
`
`Asp, or Lys;
`
`at position 33 is Val,
`
`Gly, Ala, Ser, Thr, Leu, Ile,
`
`Glu,
`
`Asp, or Lys;
`
`at position 34 is Asn,
`
`Lys, Arg, Glu, Asp, or His;
`
`at position 35 is Gly,
`
`Ala, Ser, Thr, Leu, Ile, Val,
`
`Glu,
`
`Asp, or Lys;
`
`at position 36 is Gly,
`
`Arg, Lys, Glu, Asp, or His;
`
`at position 37 is Pro,
`
`Gly, Ala, Ser, Thr, Leu, Ile,
`
`Val, Glu,
`
`Asp, or Lys,
`
`or is deleted;
`
`at position 38 is Ser,
`
`Arg, Lys, Glu, Asp, or His, or is
`
`deleted;
`
`at position 39 is Ser,
`
`Arg, Lys, Glu, Asp, or His, or is
`
`deleted;
`
`at position 40 is
`
`Gly,
`
`Asp, Glu, or Lys, or is deleted;
`
`at position 41 is Ala,
`
`Phe, Trp, Tyr, Glu, Asp, or Lys,
`
`or is deleted;
`
`Xaa at position 42 is
`
`Ser,
`
`Pro, Lys, Glu, or Asp, or is
`
`deleted;
`
`Xaa at position 43 is
`
`Ser,
`
`Pro, Glu, Asp, or Lys, or is
`
`30
`
`deleted;
`
`Xaa at position 44 is
`
`Gly,
`
`Pro, Glu, Asp, or Lys, or is
`
`deleted;
`
`and
`
`Xaa at position 45 is
`
`Ala,
`
`Ser, Val, Glu, Asp, or Lys, or is
`
`35
`
`deleted;
`
`MYLAN INST. EXHIBIT 1036 PAGE 13
`
`MYLAN INST. EXHIBIT 1036 PAGE 13
`
`

`

`WO 02/46227
`
`PCT/US01/43165
`
`_ 13 _
`
`provided that when the amino acid at position 37, 38,
`
`39, 40, 41, 42, 43, or 44 is deleted,
`
`then each amino acid
`
`downstream of that amino acid is also deleted.
`
`It is preferred that the GLP—l compound of formula I
`
`contain less than six amino acids that differ from the
`
`corresponding amino acid in GLP—1(7—37)OH or Exendin—4. It
`
`is more preferred that less than five amino acids differ
`
`from the corresponding amino acid in GLP—1(7—37)OH or
`
`Exendin—4.
`
`It is even more preferred that less than four
`
`amino acids differ from the corresponding amino acid in GLP—
`
`1(7—37)OH or Exendin—4.
`
`GLP—l compounds of the present invention include
`
`derivatives of formula I such as a C—1—6—ester, or amide, or
`
`C—1—6—alkylamide, or C—l—6—dialkylamide thereof. WO99/43706
`
`describes derivatives of GLP—1 compounds of formula I and is
`
`incorporated by reference herein in its entirety.
`
`The
`
`compounds of formula I derivatized as described in
`
`WO99/43706 and underivatized are encompassed by the present
`
`invention.
`
`Another preferred group of GLP—1 compounds is composed
`
`of GLP—1 analogs of formula II (SEQ ID NO: 3):
`
`7
`
`8
`
`9
`
`10
`
`11
`
`12
`
`13
`
`14
`
`15
`
`16
`
`17
`
`Xaa—Xaa~Xaa—Gly—Xaa—Xaa—Thr—Ser—Asp—Xaa—Ser—
`
`18
`
`19
`
`20
`
`21
`
`22
`
`23
`
`24
`
`25
`
`26
`
`27
`
`28
`
`Xaa—Xaa—Leu—Glu—Gly~Xaa—Xaa—Ala—Xaa~Xaa—Phe—
`
`29
`
`3O
`
`31
`
`32
`
`33
`
`34
`
`35
`
`36 37
`
`Ile—Xaa—Xaa—Leu—Xaa—Xaa—Xaa—Xaa—R
`
`Formula II (SEQ ID NO: 3)
`
`10
`
`15
`
`20
`
`25
`
`3O
`
`wherein:
`
`Xaa at position-7 is: L—histidine, D—histidine, desamino—
`
`histidine, 2~amino—histidine, B—hydroxy—histidine,
`
`homohistidine, a—fluoromethyl~histidine or a—methyl—
`
`35
`
`histidine;
`
`MYLAN INST. EXHIBIT 1036 PAGE 14
`
`MYLAN INST. EXHIBIT 1036 PAGE 14
`
`

`

`WO 02/46227
`
`PCT/US01/43165
`
`._l4_
`
`Xaa at position 8 is: Gly, Ala, Val, Leu, Ile, Ser, or Thr;
`
`Xaa at position 9 is: Thr, Ser, Arg, Lys, Trp, Phe, Tyr,
`
`Glu, or His;
`
`Xaa at position 11 is: Asp, Glu, Arg, Thr, Ala, Lys, or His;
`
`5 Xaa at position 12 is: His, Trp, Phe, or Tyr;
`
`Xaa at position 16 is: Leu, Ser, Thr, Trp, His, Phe, Asp,
`
`Val, Tyr, Glu, or Ala;
`
`Xaa at position 18 is: His, Pro, Asp, Glu, Arg, Ser, Ala, or
`
`Lys;
`
`10
`
`Xaa at position 19 is: Gly, Asp, Glu, Gln, Asn, Lys, Arg, or
`
`CYS;
`
`,
`
`Xaa at position 23 is: His, Asp, Lys, Glu, Gln, or Arg;
`
`Xaa at position 24 1s: Glu, Arg, Ala, or Lys;
`
`Xaa at position 26 is: Trp, Tyr, Phe, Asp, Lys, Glu, or His;
`
`15
`
`Xaa at position 27 is: Ala, Glu, His, Phe, Tyr, Trp, Arg, or
`
`Lys;
`
`Xaa at position 30 is: Ala, Glu, Asp, Ser, or His;
`
`Xaa at position 31 is: Asp, Glu, Ser, Thr, Arg, Trp, or Lys;
`
`Xaa at position 33 is: Asp, Arg, val, Lys, Ala, Gly, or Glu;
`
`20
`
`Xaa at position 34 is: Glu, Lys, or Asp;
`
`Xaa at position 35 is: Thr, Ser, Lys, Arg, Trp, Tyr, Phe,
`
`Asp, Gly, Pro, His, or Glu;
`
`Xaa at position 36 is: Thr, Ser, Asp, Trp, Tyr, Phe, Arg,
`
`Glu, or His;
`
`25
`
`R at position 37 is: Lys, Arg, Thr, Ser, Glu, Asp, Trp, Tyr,
`
`Phe, His, Gly, Gly—Pro, or is deleted.
`
`MYLAN INST. EXHIBIT 1036 PAGE 15
`
`MYLAN INST. EXHIBIT 1036 PAGE 15
`
`

`

`WO 02/46227
`
`PCT/US01/43165
`
`— 15
`
`Another preferred group of GLP—1 compounds is composed
`
`of GLP~1 analogs of formula III
`
`(SEQ ID NO: 4):
`
`7
`
`8
`
`9
`
`10
`
`ll
`
`12
`
`l3
`
`14
`
`15
`
`16
`
`17
`
`Xaa—Xaa—Glu—Gly—Xaa—Xaa—Thr—Ser—Asp—Xaa—Ser—
`
`l8
`
`19
`
`20
`
`21
`
`22
`
`23
`
`24
`
`25
`
`26
`
`27
`
`28
`
`Ser—Tyr—Leu—Glu—Xaa—Xaa—Xaa—Xaa—Lys—Xaa—Phe—
`
`29
`
`3O
`
`31
`
`32
`
`33
`
`34
`
`35
`
`36
`
`37
`
`Ile—Xaa—Trp—Leu—Xaa~Xaa—Xaa—Xaa—R
`
`formula III
`
`(SEQ ID NO: 4)
`
`wherein:
`
`Xaa at position 7 is: L—histidine,
`
`D—histidine,
`
`desamino—
`
`histidine, 2—amino—histidine, B—hydroxy~histidine,
`
`homohistidine, a—fluoromethyl—histidine or a—methyl—
`
`histidine;
`
`Xaa
`
`Xaa
`
`Xaa
`
`Xaa
`
`Xaa
`
`Xaa
`
`at position 8 is: Gly, Ala, Val, Leu, Ile, Ser, or Thr;
`
`at position 11 is: Asp, Glu, Arg, Thr, Ala, Lys, or His;
`
`at position 12 is: His, Trp, Phe, or Tyr;
`
`at position 16 is: Leu, Ser, Thr, Trp, His, Phe, Asp,
`
`Val, Glu, or Ala;
`
`at position 22: Gly, Asp, Glu, Gln, Asn, Lys, Arg, or
`
`Cys;
`at position 23 is: His, Asp, Lys, Glu, or Gln;
`
`10
`
`15
`
`20
`
`25
`
`Xaa
`
`at position 24 is: Glu, His, Ala, or Lys;
`
`30
`
`Xaa
`
`Xaa
`
`Xaa
`
`Xaa
`
`Xaa
`
`Xaa
`
`Xaa
`
`at position 25 is: Asp, Lys, Glu, or His;
`
`at position 27 is: Ala, Glu, His, Phe, Tyr, Trp, Arg, or
`
`Lys;
`
`at position 30 is: Ala, Glu, Asp, Ser, or His;
`
`at position 33 is: Asp, Arg, Val, Lys, Ala, Gly, or Glu;
`
`at position 34 is: Glu, Lys, or Asp;
`
`at position 35 is: Thr, Ser, Lys, Arg, Trp, Tyr, Phe,
`
`Asp, Gly, Pro, His, or Glu;
`
`at position 36 is: Arg, Glu, or His;
`
`MYLAN INST. EXHIBIT 1036 PAGE 16
`
`MYLAN INST. EXHIBIT 1036 PAGE 16
`
`

`

`WO 02/46227
`
`PCT/US01/43165
`
`-16-
`
`R at position 37 is: Lys, Arg, Thr, Ser, Glu, Asp, Trp, Tyr,
`
`Phe, His, Gly, Gly—Pro, or is deleted.
`
`Another preferred group of GLP—1 compounds is composed
`
`of GLP~1 analogs of formula IV (SEQ ID NO: 5):
`
`7
`
`8
`
`9
`
`10
`
`ll
`
`l2
`
`l3
`
`14
`
`15
`
`l6
`
`l7
`
`Xaa—Xaa—Glu—Gly—Thr—Xaa—ThrwSer—Asp—Xaa—Ser—
`
`18
`
`19
`
`20
`
`21
`
`22
`
`23
`
`24
`
`25
`
`26
`
`27
`
`28
`
`Ser—Tyr—Leu—Glu—Xaa—Xaa—Ala—Ala—Xaa—Glu—Phe—
`
`29
`
`30
`
`31
`
`32
`
`33
`
`34
`
`35
`
`36
`
`37
`
`Ile—Xaa—Trp—Leu—Val—Lys—XaauArg—R
`
`formula IV (SEQ ID NO: 5)
`
`wherein:
`
`Xaa at position 7 is: L—histidine, D—histidine, desamino—
`
`histidine, 2—amino~histidine, B—hydroxy—histidine,
`
`homohistidine, a—fluoromethylwhistidine or a—methyl—
`
`histidine;
`
`Xaa at position 8 is: Gly, Ala, Val, Leu, Ile, Ser, Met, or
`
`10
`
`15
`
`20
`
`Thr;
`
`Xaa at position 12 is: His, Trp, Phe, or Tyr;
`
`Xaa at position 16 is: Leu, Ser, Thr, Trp, His, Phe, Asp,
`
`Val, Glu, or Ala;
`
`Xaa at position 22 is: Gly, Asp, Glu, Gln, Asn, Lys, Arg, or
`
`25
`
`Cys;
`
`Xaa at position 23 is: His, Asp, Lys, Glu, or Gln;
`
`Xaa at position 26 is: Asp, Lys, Glu, or His;
`
`Xaa at position 30 is: Ala, Glu, Asp, Ser, or His;
`
`Xaa at position 35 is: Thr, Ser, Lys, Arg, Trp, Tyr, Phe,
`
`30
`
`Asp, Gly, Pro, His, or Glu;
`
`R at position 37 is: Lys, Arg, Thr, Ser, Glu, Asp, Trp, Tyr,
`
`Phe, His, Gly, Gly—Pro, or is deleted.
`
`MYLAN INST. EXHIBIT 1036 PAGE 17
`
`MYLAN INST. EXHIBIT 1036 PAGE 17
`
`

`

`WO 02/46227
`
`PCT/US01/43165
`
`_ 17 _
`
`Another preferred group of GLP—1 compounds is composed
`
`of GLP—1 analogs of formula V (SEQ ID NO: 6):
`
`7
`
`8
`
`9
`
`10
`
`11
`
`12
`
`13
`
`14
`
`15
`
`16
`
`17
`
`Xaa~Xaa—Glu—Gly—Thr—Phe—Thr—Ser—Asp—Val~Ser-
`
`18
`
`19
`
`20
`
`21
`
`22
`
`23
`
`24
`
`25
`
`26
`
`27
`
`28
`
`Ser—Tyr—Leu—Glu—Xaa—Xaa—Xaa—Ala—Lys—Glu—Phe—
`
`29
`
`3O
`
`31
`
`32
`
`33
`
`34
`
`35
`
`36
`
`37
`
`Ile—Xaa—Trp—Leu~Val—Lys—Gly—Arg—R
`
`formula V (SEQ ID NO: 6)
`
`10
`
`wherein:
`
`Xaa at position 7 is: L—histidine, D—histidine, desamino—
`histidine, 2—amino—histidine, B—hydroxy—histidine,
`
`homohistidine, a—fluoromethyl—histidine or a—methyl—
`
`15
`
`histidine;
`
`Xaa at position 8 is: Gly, Ala,