(12) INTERNATIONAL APPLIC ATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`(19) World Intellectual Property Organization
`International Bureau
`
`I lllll 111111111111111 IIIII IIII I II Ill lllll 111111111111111 1111111111111111111 IIII
`
`(43) International Publication Date
`13 June 2002 (13.06.2002)
`
`PCT
`
`(10) International Publication Number
`WO 02/46227 A2
`
`(51) International Patent C lassification 7: C07 K 14/605,
`14n65, C12N 15/62, A61 K 38/38, 38/26, C07K 19/00,
`A61P 3/LO
`
`(21) International Application Number: PCT/US0 l/43165
`
`(22) International Filing Date:
`29 November 2001 (29.11.2001)
`
`(25) Filing Lang uage:
`
`(26) Publication Language:
`
`English
`
`English
`
`(30) Priority Da ta:
`601251,954
`
`7 December2000 (07.12.2000) US
`
`(71) Applicant (for all designated States except US): ELI
`LILLY AND COMPANY [US/US); LiJiy Corporate
`Center, Indianapolis, TN 46285 (US).
`
`iiiiiiii
`
`::=: (72) Inventors; and
`(75) Inventors/Applicants (for US only): GLAESNER,
`-
`
`-
`= Wolfgang [DFJUS); 7512 Fieldstone Court, Indianapolis,
`=
`
`TN 46254 (US). MICANOVIC, Radmilla [US/US);
`7126 White Oak Trail, Indianapolis, IN 46236 (US).
`TSCHANG, Shen2-Hun&, Rainbow (lJS/US]; 4963
`Riley Mews, Carmel, lN 46033 (US).
`
`(74) Agents: STEWART, Mark, J. et al.; Eli LiJly and Com-
`pany, Lilly Cotporate Center, Drop Code 1104, Indianapo-
`lis, IN 46285 (US).
`
`(81) Designated States (national): AE, AG, AL, AM, AT, AT
`
`!!!!!!!
`
`-
`
`iiiiiiii
`!!!!!!!
`-
`-
`
`!!!!!!!
`iiiiiiii
`-
`
`-
`
`(84) Designa ted States (regional): ARIPO patent (GH, GM,
`KE, LS, MW, MZ, S D, 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, Fl, 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 patenJ (Rule 4. 17(i0) for the following designations AE,
`AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, BZ, CA, CH,
`CN, CO, CR, Cl!, CZ, DE, DK, DM, DZ, EC, EE, ES, FI,
`GB, GD, GE, GH, GM, HR, HU, ID, JL, JN, JS, JP, KE, KG,
`KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK,
`MN, MW, MX, MZ, NO, NZ, PH, PL, PT, RO, RU, SD, SE,
`SG, SJ, SK, SL, TJ. TM, TR, IT, TZ, VA, UG, UZ, VN, Yi!,
`ZA, ZW, ARIPO patent (GH, GM, KE, LS, MW; MZ, SD, SL,
`SZ, TZ, UG, ZA1, Zif?, Eurasian patent (4M, AZ, BY, KG,
`KZ, MD, RU, TJ, TM), European patent (4T, BE, CH, CY,
`DE, DK, ES, Fl, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE,
`TR), OAP! patent (BF, BJ, CF, CG, CI, CM, GA, GN, GQ,
`ow; ML, MR, NE, SN, TD, TG)
`as to the applicant's entitlement to claim the priority of the
`earlier application (Rule 4. I 7 (iii)) for the following des ig(cid:173)
`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, F,S, Ff, GB, GD, GE, GH, GM, HR, HU, TD, IL, JN,
`JS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV,
`MA, MD, MG, MK, AfN, MW; MX, MZ, NO, NZ, PH, PL,
`PT, RO, R[l, SD, SE, SG, SI, SK, SL, TJ, Tivl, TR, IT, TZ,
`UA, UG, UZ, VN, YU, ZA, ZW, AR/PO patent (GH, GJ'vf,
`KE, LS, Ml¥, MZ, SD. SL, SZ, TZ, UG, ZAf, Zff?, Eurasian
`patent (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), European
`patem. (AT, BE, CH, CY, DE, DK, ES, Fl, FR, GB, GR, IE,
`IT, LU, MC, NL, PT, SE, TR), OAP! patent (BF, BJ, CF, 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 tlflo-letter codes and other abbreviations, refer to the "Guid(cid:173)
`ance Notes on Codes and Abbreviations" appearing at the begin(cid:173)
`ning of each regular issue of the PCT Gazette.
`
`= (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, Fl, FI (utility model), GB, GD, GE, GH, GM, HR, HU,
`ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK1 LR, LS,
`LT, LU, LV, MA, MD, 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, TI, TZ, UA, UG, US, UZ, VN,
`YU,ZA,ZW.
`
`M
`
`<
`t(cid:173)
`M
`M
`\,,0
`~--------------------------------------
`..._
`M (54) Title: GLP-1 FUSION PROTEINS
`= 0 (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 we)] as a variety
`~ of other conditions.
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`GLP-1 FUSION PROTEINS
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`5
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`20
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`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.
`
`Glucagon-Like Peptide 1 (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
`[see, e . g., Mojsov, s., (1992) Int . J. Peptide Protein
`Research, 40:333 - 343]. · However, GLP-1 is poorly active. A
`subsequent endogenous cleavage between the 6 t h and 7 th
`position produces a more potent biologically active GLP-1(7-
`37·)OH peptide. Numerous GLP-1 analogs and derivatives are
`known and are referred to herein as "GLP-1 compounds."
`These GLP-1 analogs include the Exendins which are peptides
`found in the venom of the GILA-monster . The Exendins have
`sequence homology to native GLP- 1 and can bind the GLP-1
`receptor and initiate the signal transduction cascade
`responsible for the numerous activities that have been
`attributed to GLP-1(7-37)OH.
`GLP-1 compounds have a variety of physiologically
`significant activities. Fo_r example, GLP-1 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 al. (1992) New England J. of Med . 326:1316-
`1322; Nauck, M. A. , et al., (1993) J. Clin. Invest . 91:301-
`307) .
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`GLP-1 shows the g r eatest 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
`d rugs which stimulate the release of insulin, however, can
`also cause hypoglycemia as can the actual administration o f
`insulin . GLP-1 activi t y, 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
`hypogl ycemia associated .with treatment involving GLP - 1.
`However , the usefulness of therapy involving GLP-1
`peptides has been limited by their fast clearance and short
`half-l i ves . For example, GLP-1(7-37) has a serum half- li fe
`of only 3 to 5 minutes. GLP-1(7-36) amide h as a time action
`of about 50 minutes when administered subcutaneously. Even
`analogs and derivatives that are resistant to endogenous
`20 protease cleavage, do not have half-lives long enough to
`avoid repeated administrations over a 24 hour period. Fast
`clearance o f 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 f or diabet ic
`patients whose pas t
`treatment regimen has involved taking
`only oral medication . These patients often have an
`extremely difficult time transitioning to a regimen t hat
`involves multiple injections of medication .
`The present invention overcomes the problems associated
`with delivering a compound t h at has a short plasma half(cid:173)
`life. The compounds of the present invention encompass GLP-
`1 compounds fused to another protein with a long circulating
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`half-life such as the Fe 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-1 compounds currently in
`development. For example , GLP-1(7-37)0H has a tendency to
`under go 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-1 fusion proteins with increased
`half-lives could be developed. This was especially
`unexpected given the difficulty of working with GLP-1(7-
`37)0H alone and the large size of the fusion partner
`relative to the small GLP-1 peptide attached.
`
`5
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`20
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`25
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`Compounds of the present i nvention include heterologous
`fusion proteins c omprising a first polypeptide with a N-
`terminus and a C-terminus fused to a second pol ypeptide with
`a N-terminus and a C-terminus wherein the first polypeptide
`is a GLP- 1 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- terrninus of the second polypeptide.
`Compounds of the present invention also include a
`30 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 firs t polypeptide is a GLP-1 compound and the
`second polypeptide is selected from the group consisting of
`a) human albumin;
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`c)
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`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 via a peptide
`linker .
`It i s preferr ed 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- Serln
`where n is 1, 2, 3, 4, 5 or 6; and
`a peptide having the sequence [Gly-Gly-Gly-Gl y(cid:173)
`Serb .
`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-1 compound a n d the second
`pol ypeptide is selected from the group consisting of
`a) the F"c portion of an immunoglobulin;
`b) an analog of the Fe portion of an immunoglobulin;
`and
`c) fragments of the Fe portion of an immunoglobulin,
`and wherein the C-terminu s of the first polypeptide is fused
`to the N-terminus of the second polypeptide . The GLP-1
`compound may be fused to the second polypeptide via a
`25 peptide linker. It is preferable that the pe~tide linker is
`selected from the group consi sting of:
`a) a glycine rich peptide;
`[Gly-Gly-Gly- Gly-Ser]n
`b) a peptide ~aving the sequenc e
`where n is 1, 2, 3, 4, 5 or 6; and
`c) a peptide having the sequence [Gly-Gly-Gly-Gly-Ser] 3 •
`It is generally preferred that the GLP-1 compound that
`is part of the heterologous f usion protein have no more than
`6 amino acids that are different from the corresponding
`amino acid i n GLP-1(7-37)OH, GLP-1(7-36)OH, or Exendin-4.
`It is even more preferred that the GLP-1 compound have no
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`more than 5 amino acids that differ from the corresponding
`amino acid in GLP-1(7-37)OH, GLP-1(7-36)OH , or Exendin-4.
`It is most preferred that the GLP-1 compound have no more
`than 4, 3, or 2 amino acids that differ from the
`corresponding amino acid in GLP-1(7-37)OH, GLP-1(7-36)OH, or
`Exendin-4. Preferably, a GLP- 1 compound tha~ is part of the
`heterologous fusion protein has glycine or valine at
`position 8.
`The present invention also includes polynuc l eotides
`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 1: IgGl Fe 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- 1- Fc
`fusion proteins (Lane 1, MW standards; Lane 2, Purified Fe;
`lane 3, Mock transfected media; Lane 4, Val 8 -GLP-1-Fc; Lane
`5, Exendin-4-Fc) B. SDS-PAGE gel and immunoblot of same gel
`illustrating the molecular weight of human HSA and GLP-1-HSA
`fusion proteins (Lane 1, MW standards; Lane 2, Purified HSA;
`lane 3, Mock transfected media; Lane 4, Val 8- GLP-l-HSA; Lane
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`5, Val8 -GLP-1-[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 Fe, albumin, and
`GLP-1 fusion proteins (Lane 1 , MW standards; Lane 2,
`5 purified Fe; Lane 3, Val8-GLP-1-Fc; Lane 4, Exendin-4-Fc;
`Lane 5, MW standard; Lane 6, Val8-GLP-1-HSA; Lane 7,
`Exendin-4-HSA; Lane 8, Exendin- 4-[Gly-Gly-Gly-Gly-Ser] 3 -
`HSA).
`Figure 5 : Expression cloning vector containing the Fe
`r egions illustrat ed in f igure 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 -il lustrated in figure 2 .
`Figure 8: In vitro dose response activity of GLP-1
`fusion protei ns.
`Figure 9: Pharmacokinetics o f GLP-1 Fe and HSA fusion
`proteins.
`Figure 10 : Glucodynamic response to Exendin-Fc in two
`normal f asted dogs .
`Figure 11 : Insulinotropic response to Exendin-Fc in two
`normal fasted dogs .
`Figure 12: DNA sequence encoding a human IgGl Fe
`region.
`Figur e 1 3: DNA sequence encoding a human albumin
`protein .
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`20
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`25
`
`The heterologous fusion proteins of the present
`invention comprise a GLP-1 compound fused to human albumin,
`a human albumin analog, a human a l bumin fragment, the Fe
`portion of an immunoglobulin, an analog of the Fe porti on of
`an immunoglobulin , or a fragment of the Fe portion of an
`immunoglobulin. The C-terminus of the GLP-1 compound may be
`fused directl y, or fused via a peptide linker, to the N-
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`terminus of an albumin or Fe protein . These heterologous
`fusion proteins are biologically active and have an
`increased half-life compared to native GLP-1.
`It is preferred that the GLP-1 compounds that make up
`5 part of the heterologous fusion protein encompass
`polypeptides having from about twenty-five to about thirty(cid:173)
`nine naturally occurring or non-naturally occurring amino
`acids that have sufficient homology to native GLP- 1(7 - 37)OH
`such that they exhibit insulinotropic activity by binding to
`the GLP-1 receptor on ~-cells in the pancreas . A GLP-1
`compound t ypically comprises a polypeptide having the amino
`acid sequence of GLP-1(7-37)OH, an analog of GLP-1 (7-37)OH,
`a fragment of GLP-1(7-37)OH or a fragment of a GLP-1(7 - 37)OH
`analog. GLP- 1(7-37)OH has the amino acid sequence of SEQ ID
`15 NO: 1 :
`
`10
`
`15 16 17
`1 4
`i3
`10 11 12
`9
`7
`8
`His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-
`18 19 20 21 22 23 24 25
`26 27
`28
`Ser- Tyr -Leu- Glu- Gly-Gln- Ala - Ala-Lys - Glu- Phe-
`29 30 31
`32
`33 34 35 36 37
`Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Gly
`(SEQ ID NO: 1)
`
`By custom in the art, the amino terminus of GLP- 1(7-
`37)OH has been assigned number residue 7 and the carboxy(cid:173)
`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- 1 compounds also encompass "GLP- 1 fragments . " A
`GLP-1 fragment is a polypeptide obtained after truncation of
`one or more amino acids f r om the N-terminus and/or C(cid:173)
`terminus of GLP-1(7-37)OH or an a nal og or derivative
`thereof . The nomenclature used to describe GLP-1(7-37)OH is
`also applicable to GLP-1 fragments . For example, GLP- 1(9-
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`36)0H denotes a GLP-1 fragment obtained by truncating two
`amino acids from the N-terminus 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-1(7-
`37)0H. For example, the N-terminal glutamic acid in GLP-
`1(9- 36)0H is at position 9; position 12 is occupied by
`phenylalanine; and position 22 is occupied by glycine, as in
`GLP-1(7-37)0H. For GLP-1(7-36)0H, the glycine at position 37
`of GLP-1(7-37)0H is deleted.
`GLP-1 compounds also include polypeptides in which one
`or. more amino acids have been added to the N- terminus and/or
`C-terminus of GLP-1(7-37)0H, or fragments or analogs
`thereof. It is preferred that GLP-1 compounds of this type
`have up to about thirty-nine amino acids. The amino acids
`in the "extended" GLP-1 compound are denoted by the same
`number as the correspohding amino acid in GLP- 1(7-37)0H .
`For example, the N- terminus ·amino acid of a GLP-1 compound
`obtained by adding two amino acids to the N-termi nal of GLP-
`1(7-37)0H is at position 5 ; and the C-terminus amino acid of
`a GLP-1 compound obtained by adding one amino acid to the c(cid:173)
`terminus of GLP-1(7-37)0H is at position 38 . Thus, position
`12 is occupied by phenylalanine and position 22 is occupied
`by glycine in both of these "extended" GLP-1 compounds, as
`in GLP-1(7-37)0H. Amino acids 1-6 of an extended GLP-1
`compound are preferably the same as or a conservative
`substitution of the amino acid at the corresponding position
`of GLP-1(1-37)0H . Amino acids 38-45 of an extended GLP-1
`compound are preferably the same as or a conservative
`substitution of the amino acid at the correspondi ng pos i tion
`30 of glucagon or Exendin-4 .
`GLP-1 compounds of the present invention encompass
`"GLP-1 analogs." A GLP-1 analog has sufficient homology to
`GLP- 1(7 - 37)0H or a fragment of GLP- 1(7 - 37)0H such that the
`analog has insulinotropic activity. Preferably, a GLP-1
`analog has the amino acid sequence of GLP-1(7-37)0H or a
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`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- 1(7-37)OH or a fragment of
`GLP-1(7-37)OH.
`In the nonmenclatur e used herein to
`designate GLP-1 compounds, the substituting amino acid and
`its position is indicated prior to the parent structure.
`For example , Glu22 - GLP-1(7-37)OH designates a GLP-1 compound
`in which the glycine normally found at position 22 of GLP-
`1(7-37)OH has been replaced with glutamic acid; Val8-Glu22-
`10 GLP-1(7-37)OH designates a GLP-1 compound in which alanine
`normally found at position 8 and glycine normally found at
`position 22 of GLP-1(7-37)OH have been replaced with valine
`and glutamic acid, respectively.
`GLP-1 compounds of the present invention also include
`"GLP-1 derivatives." A GLP-1 deri vative is defined as a
`molecule having the amino acid sequence of GLP-1 or of a
`GLP-1 analog, but additionally having chemical modification
`of one or more of its amino acid side groups , a - carbon
`atoms, terminal amino group, or termina l carboxylic acid
`group . A chemical modification includes, but is not limited
`to , adding c h emical moieties, creating new bonds, and
`removing chemical moieties. Modifications at amino acid
`side groups include, withou t limitation, acylation of lysine
`£- 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 g roup include, without
`limitation, the des-amino, N-lower alkyl, N-di-lower alkyl,
`and N-acyl modifications . Modifications of the terminal
`carboxy group include, withou t limitation, the amide, lower
`alkyl amide, dialkyl ami de, and lower alkyl ester
`modifications. Lower alkyl is C1- C4 a l kyl . Furthermore,
`one or more side groups, or terminal groups, may be
`protected by protective groups known to the ordinarily-
`
`20
`
`25
`
`30
`
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`5
`
`s k illed protein chemist. The a-carbon of an amino acid may
`be mono- or dimethylated .
`Any GLP- 1 compound can be part of the heterologous
`fusion pro teins of the present i nven t ion as lon g as the GLP-
`1 compound itself is able to bind and induce signal ing
`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 61 9,322 and U. S . Patent No.
`5,120 , 712, respectively .
`Numerous active GLP-1 fragments , analogs and
`derivatives are known in the art and any of these anal ogs
`and derivatives can also be part of the heterologous f~sion
`proteins of the present invent ion .
`Some examples of novel
`GLP-1 analogs as well as GLP-1 analogs and derivatives known
`in the art a r e provided herein .
`Some GLP-1 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), Gln9-GLP- 1(7 - 37), D- Gln9 - GLP-1 (7 - 37), Thr16-Lysl 8_GLP-
`1 (7-37) , and LyslB_GLP-1(7-37) . GLP-1 analogs such as GLP-
`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 a c tive GLP-1 compounds are
`disclosed in U. S . Patent No 5,977 , 071 to Hoffmann, et al.,
`25 U.S . Patent No . 5,545 , 618 to Buckl ey, et al . , and Adelhorst ,
`et al., J. Biol . Chem . 269:6275 (1994) .
`
`1 0
`
`15
`
`20
`
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`A preferred group of GLP-1 analogs is composed of
`GLP-1 analogs of formula I
`(SEQ ID NO: 2)
`
`5
`
`10
`
`7
`1 0 11 12 13 14 15 16 17
`8
`9
`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 30 31 32
`33
`34 35 36
`37 38 39
`Ile-Xaa-Xaa-Xaa- Xaa-Xaa-Xaa-Xaa-Xaa- Xaa-Xaa-
`40 41
`42 43
`44 45
`Xaa-xaa-Xaa-Xaa-Xaa-Xaa
`Formula I
`(SEQ ID NO : 2)
`
`Leu, Ile, Val,
`
`Thr, Leu, Ile,
`
`wherein :
`Xaa at position 8 is Al a, Gly, Ser, Thr, Leu, Ile, Val,
`Glu, Asp, or Lys;
`Xaa at position 9 is Glu, Asp, or Lys ;
`Xaa at position 11 is Thr, Ala, Gly, Ser, Leu, Ile, Val,
`Glu, Asp, or Lys;
`Xaa at position 14 is Ser, Ala, Gly, Thr,
`Glu, Asp , or Lys;
`Xaa at position 16 is Val, Ala , Gly, Ser ,
`Tyr, Glu , Asp , Trp, or Lys ;
`Xaa at position 17 is Ser, Ala, Gly, Thr,
`Glu, Asp, or Lys;
`Xaa at position 18 is Ser, Ala, Gly, Thr ,
`Glu, Asp, Trp, Tyr, or Lys;
`Xaa at position 19 is Tyr, Phe , Trp, Glu,
`Xaa at position 20 is Leu, Ala, Gly, Ser ,
`Glu, Asp, Met, Trp, Tyr, or Lys ;
`Xaa ·at position 21 is Glu, Asp , o r Lys ;
`Xaa at position 22 is Gly, Ala, Ser, Thr , Leu, Ile, Val,
`Glu , Asp, o r Lys ;
`35 Xaa at position 23 is Gln, Asn, Arg, Glu, Asp , or Lys;
`
`15
`
`20
`
`25
`
`30
`
`Leu , I l e, Val,
`
`Leu, Ile, Val,
`
`Asp, Gln , or Lys ;
`Thr , Ile, Val,
`
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`
`Xaa at position 24 is Ala, Gly, Ser, Thr, Leu, Ile , Val,
`Arg, Glu, Asp, or Lys;
`Xaa at position 25 is Ala, Gly, Ser, Thr, Leu, Ile, Val,
`Glu, Asp, or Lys;
`5 Xaa at position 26 is Lys, Arg, Gln , Glu , Asp, or His;
`Xaa at position 27 is Leu, Glu, Asp, or Lys;
`Xaa at position 30 is Ala, Gly, Ser, Thr, Leu, Ile, Val,
`Glu, Asp, or _Lys ;
`Xaa at position 31 is Trp, Phe, Tyr, Glu, Asp, or Lys ;
`10 Xaa at position 32 is Leu, Gly, Ala, Ser, Thr, Ile, Val,
`Glu , Asp, or Lys;
`Xaa at position 33 is Val, Gly, Ala, Ser, Thr, Leu, Ile,
`Glu, Asp, or Lys;
`Xaa at position 34 is Asn, Lys, Arg , Glu, Asp, or His;
`15 Xaa at position 35 is Gly, Ala, Ser, Thr, Leu, Ile, Val,
`Glu , Asp, or Lys;
`Xaa at position 36 is Gly, Arg, Lys, Glu, Asp, or His;
`Xaa at position 37 is Pro, Gly, Ala, Ser, Thr, Leu, Ile,
`Val, Glu, Asp, or Lys, or is deleted;
`20 Xaa at position 38 is Ser, Arg, Lys, Glu, Asp, or His, or is
`deleted;
`Xaa at position 39 is Ser, Arg, Lys, Glu, ·Asp, or His, or is
`deleted;
`xaa at position 4 0 is Gly, Asp, Glu , or Lys, or is deleted;
`xaa 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, Gl u, Asp, or Lys, or is
`deleted;
`
`35
`
`25
`
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`
`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-1 compound of formu la I
`contain l ess t han six amino acids that dif f er 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 fou r
`amino acids differ from the corresponding amino acid in GLP-
`1(7-37)OH or Exendi n-4 .
`GLP-1 compounds of the present invention inc lude
`derivatives of formula I such as a C- 1-6- ester , or amide, or
`C- 1-6-alkylamide , or C-1 - 6- dialkylamide thereof . WO99/43706
`describes derivatives of GLP-1 compou nds of formul a I and is
`i ncorporated by ref erence herein in i ts entirety. The
`compounds o f 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
`
`15 16 17
`10 11 12 13 14
`8
`9
`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-Al a-Xaa-Xaa-Phe-
`29 30 31 32
`33 34 35 36 37
`Ile-Xaa-Xaa-Leu-Xaa-xaa-Xa a -Xaa-R
`Formula II (SEQ ID NO: 3}
`
`wherein :
`Xaa at position ·7 is : L-histidine, D-histidi ne, desamino(cid:173)
`histidine , 2-amino-histidine , ~-hydr oxy-histidine,
`homohistidine, a-fluoromethyl-hi stidine or a -methyl(cid:173)
`histi dine;
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
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`

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`
`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 posi t ion 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 is: 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 3.1 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: ~lu, 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 Rat position 37 is: Lys, Arg, Thr, Ser, Glu, Asp, Trp, Tyr,
`Phe, His, Gly, Gly-Pro, or is deleted.
`
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`
`Another preferred group of GLP-1 compounds is composed
`
`of GLP- 1 analogs o f formula III (SEQ ID NO: 4) :
`
`7
`
`8
`
`9
`
`10 11 12 13 14 15 16 17
`
`5
`
`Xaa-Xaa-Glu-Gly-Xaa-Xaa-Thr-Ser-Asp-Xaa-Ser -
`
`18 19
`
`20 21 22 23 24 25
`
`26 27 28
`
`Ser-Tyr-Leu-Glu-Xaa-Xaa-Xaa-Xaa-Lys -Xaa-Phe-
`29 30 31 32 33 34 35 36 37
`
`Ile-Xaa-Trp-Leu-Xaa-Xaa-Xaa-Xaa-R
`
`10
`
`formula I I I
`
`(SEQ ID NO : 4)
`
`wherein:
`
`15
`
`Xaa at position 7 i s: L-histidine, D-histidine, desamino(cid:173)
`histidine, 2-arnino-histidine, ~-hydroxy-histidine,
`homohistidine, a - fluoromethyl - histidine or a -methyl-
`histid ine ;
`Xaa at position 8 is : G_ly, Ala , Val , Leu , Ile, Ser, or Thr;
`Xaa at position 11 is : Asp, Glu, Arg, Thr , Ala , Lys, o r His;
`Xaa at position 12 is: His, Trp, Phe, or Tyr ;
`20 Xaa at position 16 is: Leu, Ser, Thr, Trp, His , Phe, Asp,
`Val, Glu, or Ala ;
`Xaa at position 22 : Gly, Asp, Glu, Gln, Asn, Lys, Arg, or
`Cys;
`Xaa at position 23 is: His, Asp, Lys, Glu, or Gln ;
`
`25 Xaa at position 24 is: Glu, His, Ala, or Lys ;
`
`Xaa at position 25 is : Asp, Lys , Glu, or His;
`
`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 ;
`
`30 Xaa at position 33 is: Asp, Arg, Val, Lys, Ala, Gly, or Glu;
`
`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 : Arg, Gl u, or His ;
`
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`

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`
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`
`Rat position 37 is: Lys, Arg, Thr, Ser, Glu, Asp, Trp , Tyr ,
`Phe , His, Gly, Gly-Pro, or is d eleted.
`
`5
`
`10
`
`Another preferred group of GLP-1 compounds is composed
`of GLP-1 analogs of formula IV (SEQ ID NO : 5) :
`7
`8
`9
`10 11 12 13 14 15 16 17
`Xaa-Xaa-Glu-Gl y-Thr-Xaa-Thr-Ser-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-Xaa-Arg- R
`formula IV (SEQ ID NO: 5)
`
`20
`
`wherein:
`15 Xaa at position 7 is : L-histidine, D-histidine, desarnino(cid:173)
`histidine, 2 - arnino- histidine, ~- hydroxy- histidine,
`hornohistidine, a-fluorometh yl-histidi ne or a-methyl-
`histidine;
`Xaa a t position 8 is : Gly, Ala, Val, Leu, Ile , Ser, Met, or
`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 a t position 22 is : Gl y, Asp , Glu, Gln , Asn , Lys, Arg, or
`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 ,
`Asp , Gly, Pro, His, or Glu ;
`Rat position 37 is : Lys, Arg, Thr, Ser, Glu, Asp, Trp, Tyr,
`Phe, His, Gly, Gly- Pro, or is deleted.
`
`25
`
`30
`
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`
`compounds is composed
`Another preferred group of GLP-1
`of GLP-1 analogs of formula V (SEQ ID
`NO: 6) :
`10 11 12
`7
`8
`9
`13 14 15 16