(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`(19) World Intellectual Property
`Organization
`International Bureau
`
`\=
`
`(43) International Publication Date
`6 October 2016 (06.10.2016)
`
`WIPO!IPCT
`
`GD)
`
`International Patent Classification:
`A61K 38/17 (2006.01)
`C12N 15/867 (2006.01)
`
`(21)
`
`International Application Number:
`
`PCT/US2016/025490
`
`(74)
`
`(22)
`
`International Filing Date:
`
`(25)
`
`(26)
`
`(30)
`
`(71)
`
`Filing Language:
`
`Publication Language:
`
`1 April 2016 (01.04.2016)
`
`English
`
`English
`
`(81)
`
`Priority Data:
`62/141,598
`
`1 April 2015 (01.04.2015)
`
`US
`
`Applicant: THE SCRIPPS RESEARCH INSTITUTE
`[US/US]; 10550 North Torrey Pines Road, La Jolla, CA
`92037 (US).
`
`(10) International Publication Number
`WO 2016/161244 A2
`
`XIE, Jia; 8730 Costa Verde Blvd., Apt. 2519, San Diego,
`CA 92122 (US). STURCHLER, Emmanuel; 2614 26 Ct.,
`Jupiter, FL 33477 CUS).
`
`Agents: COOPER, Geoffrey, K. et al.; The Scripps Re-
`search Institute, 10550 North Torrey Pines Road, TPC-8,
`La Jolla, CA 92037 (US).
`
`Designated States (unless otherwise indicated, for every
`kind of national protection available): AE, AG, AL, AM,
`AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY,
`BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM,
`DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT,
`HN, HR, HU,ID,IL, IN, IR, IS, JP, KE, KG, KN, KP, KR,
`KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, MG,
`MK, MN, MW,MX, MY, MZ, NA, NG, NI, NO, NZ, OM,
`PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, SC,
`SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN,
`TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW.
`
`(72)
`
`Inventors: LERNER, Richard, A.; 7750 Roseland Drive,
`La Jolla, CA 92037 (US). ZHANG, Hongkai; 4180 Cam-
`ino Islay, San Diego, CA 92122 (US). MC DONALD,Pa-
`tricia; 13839 155th Place N., Jupiter, FL 33478 (US).
`
`(84)
`
`Designated States (unless otherwise indicated, for every
`kind of regional protection available): ARIPO (BW, GH,
`GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ,
`
`[Continued on next page]
`
`(54) Title: METHODS AND COMPOSITIONS RELATED TO GPCR AGONIST POLYPEPTIDES
`
`
`
`Roune 1 ait?ww ot
`
`
`
`a it
`.
`vg
`weal 12
`int Fo aw?
`488em Ex/?30nm Em: GFP
`488nm Ex530nm Erm GAP
`Round 2
`
`ane aed oof gt
`488rm Ex/£30nm Em: 6°F
`Round 3
`
`FIG. 1
`
`(57) Abstract: The invention provides combinat-
`orial peptide or polypeptide libraries that can be-
` LL2Signal Sequence
`Exendin-4-SL-TM NHC COOH
`come membranetethered once expressed in cells.
`
`ExendinML-TM NHK} COCH== Linker=<> Exendined
`
`
`The invention additionally provides methods for
`Exendin4-LL-TM NH==TH-( COOH Cy mChery “8 POGFRTM
`selecting peptide modulators (e.g., agonists) of
`GPCRsfrom the combinatorial libraries of the in-
`b
`
`vention. The invention also provides novel GPCR
`Bits
`Bios
`i's
`=
`=
`polypeptide modulators, e.g., biased polypeptide
`Zales
`Zi!
`| 2a
`agonists of the glucagon-like peptide 1 receptor
`(GLP-1R). The invention further provides meth-
`ods of promoting insulin sensitivity,
`lowering
`blood glucose, and reducing body weight as well
`as methods for treating various diseases such as
`diabetes and obesity.
`
`
`o| Say
`= 1a @| Zola
`‘id
`Elal 2!
`43 Eyl ae
`aoe oh aot ae
`10! aol ae a8 an 8 8 nO at
`Bint Ex63Cam Em: GFP
`488nm Ex/39Cnm Em; GFP
`488nm Ew530nn Em: GFP
`Negative
`Exend4-SLTM
`Exendir-4
`
`zit
`0408,
`aoe 8 gt
`al sgt
`48Ern Ex'830nm Em: GFP
`Exendit-4-MLTW
`
`Exendin4-LL-TM
`
`= 486m Ex/S30am Em: GFP
`
`
`
` ial)
`
`WO2016/161244A2|IHITMIINNTIINNIMNIUUMTAIREIIUAATUNITA
`
`

`

`WO 2016/161244 A2 IfMTIU TMA ANAEM TATUAAEM TRAA
`
`TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU,
`TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE,
`DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT,
`LU, LV, MC, MK, MT, NL, NO,PL, PT, RO, RS, SE,
`SL SK, SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA,
`GN, GQ, GW, KM, ML, MR, NE, SN, TD, TG).
`
`Published:
`
`without international search report and to be republished
`upon receipt of that report (Rule 48.2(g))
`
`

`

`WO 2016/161244
`
`PCT/US2016/025490
`
`Methods and Compositions Related to GPCR Agonist
`Polypeptides
`
`CROSS-REFERENCE TO RELATED APPLICATIONS
`
`[0001]
`
`The subject patent application claims the benefit of priority to U.S. Provisional
`
`Patent Application Number 62/141,598 (filed April 1, 2015). The full disclosure of the
`
`priority application is incorporated herein by reference in its entirety and for all purposes.
`
`BACKGROUND OF THE INVENTION
`
`G-protein-coupled receptors constitute the largest family of cell surface receptor
`[0002]
`proteins. Upon activation, GPCRs couple to GTP-binding proteins that can be divided into
`
`four subclasses, Gas-, Gai/o-, Gaq and Gaj213. Gs and Gi/o regulate adenylate cyclases
`leading to an increase (Gas) or a decrease (Gai/o) in cAMP production, or to an increase in
`
`intracellular calcium concentration (Gaq). Ga(12/13) activates Rho GEFs, which in turn activate
`
`Rho. GPCRscanalso engage B-arrestins. Historically, B-arrestin-1 and B-arrestin-2 were
`
`believed to serve an exclusive role in GPCR desensitization. However, it has been shown
`
`that B-arrestins can also function to activate signaling cascades. Many humandiseasesare
`
`associated with the dysfunction of GPCRs. Thus, GPCRsrepresent some of the most
`
`attractive therapeutic or molecular targets in the pharmaceutical industry.
`
`[0003]
`
`Insulin regulates the concentration of blood sugar and bloodlipid levels through
`
`the promotion of glucose andlipid uptake into cells. Type 2 Diabetes Mellitus (12DM or
`
`T2D) is a complex metabolic disorder characterized by hyperglycemia arising from a
`
`combination of insufficient insulin secretion together with the developmentofinsulin
`
`resistance. T2D and obesity are closely linked, with obesity accounting for 80-85% ofthe
`
`risk of developing T2D. Incretin-based therapies represent a promising class of agents for
`the treatment of T2D. Incretins including the glucagon-like peptide-1 (GLP-1) and the
`
`glucose-dependentinsulinotropic polypeptide (GIP) are endogenous peptide hormones
`
`secreted from the intestine in response to food intake. GLP-1 and GIP exert their action
`
`through G-protein coupled receptor, the GLP-1 and the GIP receptors, respectively. GLP-1R
`
`is expressed in pancreatic B-cells as well as various tissues including liver, smooth muscle,
`
`heart, kidney, gastrointestinal tract, lungs, pituitary, white adiposc tissues, and the central
`1
`
`

`

`WO 2016/161244
`
`PCT/US2016/025490
`
`nervous system. GLP-1 lowers postprandial glucose excursion by potentiating glucose-
`stimulated insulin secretion from pancreatic B-cells and has recently been shown to promote
`B-cell survival in rodents.
`In addition, GLP-1 exerts extrapancreatic actions such as
`
`promoting gastric emptying, weightloss, intestinal growth and increasing insulin sensitivity
`in peripheral tissues.
`
`A need exists in the art for better and more robust meansfor identifying agonists
`[0004]
`or activators displaying novel pharmacology of various GPCRs. Thereis also needin theart
`for new compoundsand methodsfortreating diseases associated with insulin deficiency and
`high blood glucose. The instant invention addresses these and other currently unmet needs
`in the art.
`
`SUMMARYOF THE INVENTION
`
`In one aspect, the invention provides isolated or recombinant polypeptides that
`[0005]
`contain a first randomized peptide fused at its C-terminus to a second peptide capable of
`binding to the extracellular domain of glucagon-like peptide 1 receptor (GLP-1R). In these
`polypeptides, the randomized peptide typically has an amino acid sequence of XXXXXXX
`(SEQ ID NO:1), CKXXXXCXX (SEQ ID NO:2) or CKKXXCXXX (SEQ ID NO:3),
`wherein X is any aminoacid residue. In various embodiments, these polypeptides can
`
`activate glucagon-like peptide 1 receptor (GLP-1R).
`
`In some embodiments, the randomized
`
`peptide contains an amino acid sequencethat is substantially identical to a sequence selected
`
`from SEQ ID NOs:4-16 and 22-31, or an active variant or fragment thereof. In some
`
`embodiments, the randomized peptide contains an amino acid sequencethatis identical to a
`
`sequence selected from SEQ ID NOs:4-16 and 22-31, except for conservative substitution at
`
`one or more residues.
`
`In some embodiments, the randomized peptide contains an amino acid
`
`sequence shown in SEQ ID NO:4 (ACCIDSVCVI), SEQ ID NO:5 (VCPDCQV), SEQ ID
`NO:6 (ACSYMIDCVL), SEQ ID NO:8 (ELVDNAYV), SEQ ID NO:22 (CCIDSVCVD), or
`
`SEQ ID NO:23 (CSYMIDCVL).
`
`[0006]
`
`In somepolypeptides of the invention, the second peptide capable of binding to
`
`glucagon-like peptide 1 receptor contains an amino acid sequencethat is substantially
`identical to at least 8 contiguous amino acid residues of Ex4 (9-39) (SQE ID NO:17), Ex4
`(9-30) (SEQ ID NO:38), or human GLP-1 (7-37) peptide (SEQ ID NO:18).
`In some
`
`embodiments, the second peptide capable of binding to GLP-1R contains a sequencethatis
`
`2
`
`

`

`WO 2016/161244
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`PCT/US2016/025490
`
`In some embodiments, the
`substantially identical to SEQ ID NO:17 or SEQ ID NO:18.
`second peptide capable of binding to GLLP-1R contains a sequence shown in SQE ID NO:17
`or SEQ ID NO:18, except for conservative substitution at one or more residues.
`In some
`
`embodiments, the second peptide capable of binding to glucagon-like peptide 1 receptor
`contains a sequence shown in GGDLS KQMEE EAVRL FIEWL KNGGP SSGAP PPS
`(SEQ ID NO:21). Someof the polypeptides of the invention are G protein biased. Some
`polypeptides of the invention contain a sequencethat is substantially identical to a sequence
`selected from the group consisting of SEQ ID NOs:32-37.
`In some of these embodiments,
`the polypeptide has an amino acid sequenceselected from the group consisting of SEQ ID
`NOs:32-37,
`In some embodiments, the GLP-1R agonist polypeptides of the invention
`further contain an Fce-domain fused at its N-terminus or C-terminus. Someof these
`
`polypeptides contain an Fc-domainthatis fused at the C-terminus of SEQ ID NO:36,e.g.,
`the polypeptide shown in SEQ ID NO:45. Someofthese polypeptides can additionally
`include a signal peptide at the N-terminus.
`
`In another aspect, the invention provides methods for identifying an agonistof a
`[0007]
`G protein-coupled receptor (GPCR). These methodsentail (1) introducing a vector
`expressing a reporter gene under the control a cAMPresponse element (cAMP)into a
`population ofhost cells expressing the GPCR,(2) expressingin the hostcells a
`combinatorial library of membrane tethered candidate polypeptides to produce a population
`of host cells co-expressing a candidate polypeptide and the GPCR,(3) identifying a host cell
`that has increased expression level of the reporter gene relative to a controlcell not
`expressing any candidate polypeptide, and (4) determining the identity of the candidate
`polypeptide expressed inthe identified hostcell; thereby identifying an agonist of the G
`protein-coupled receptor.
`In some methods, each candidate polypeptide is fused at its C-
`terminusto a linker and a transmembraneprotein domain. In some methods, the employed
`transmembrane protein domain is the PDGFR transmembrane domain.
`In some methods,
`the GPCRis expressed from an expression vector in the host cells. For example, the vector
`can be a lentiviral vector.
`
`[0008]
`protein.
`
`Someof the methods of the invention employ a reporter gene that encodes a GFP
`In these methods, the reporter gene can be introducedinto the host cell via a
`
`lentiviral vector, In some embodiments, the employed host cell is the HEK 293T cell.
`some embodiments, the employed GPCR is human GLP-1R.
`In some methodsofthe
`
`In
`
`

`

`WO 2016/161244
`
`PCT/US2016/025490
`
`invention, each candidate polypeptide containsa first randomized peptidethatis fusedatits
`C-terminus to a second peptide capable of binding to the extracellular domain of glucagon-
`like peptide 1 receptor (GLP-1R), and the randomized peptide contains an amino acid
`sequence of XXXXXXX (SEQ ID NO:1), CKXXXXCXX (SEQ ID NO:2)or
`CXXXXCXXX (SEQ ID NO:3), wherein X is any aminoacid residue. In some of these
`methods, the second peptide contains an amino acid sequencethat is substantially identical
`to Ex4 (9-39) (SQE ID NO:17), Ex4 (9-30) (SEQ ID NO:38), human GLP-1 (7-37) peptide
`(SEQ ID NO:18),or an active variant thereof.
`In some methods, the transmembrane protein
`domain and the second peptide are connected via a linker peptide. For example, a linker
`peptide containing two or more tandem repeats of GGGGS (SEQ ID NO:43) can be
`employed in the methods.
`
`In some methodsof the invention, each candidate polypeptide further contains a
`[0009]
`signal peptide at the N-terminus. For example, the methods can employ a signal peptide
`containing the IL-2 signal sequence.
`In some of these methods, the signal peptide can
`contain an amino acid sequence of MYRMQLLSCIALSLALVTNS(SEQ ID NO:44).
`[0010]
`In another aspect, the invention provides methodsforactivating a signaling
`pathway mediated by the glucagon-like peptide 1 receptor (GLP-1R) in a cell. These
`methods involve contacting the cell with an effective amount of a polypeptide containing a
`first randomized peptide that is fused at its C-terminus to a second peptide capable of
`binding to the extracellular domain of glucagon-like peptide 1 receptor (GLP-1R).
`In these
`methods, the randomized peptide contains an amino acid sequence of XXXXXXX (SEQ ID
`NO:1), CKXXXXCXX (SEQ ID NO:2) or CKXXXCXXX (SEQ ID NO:3), wherein X is
`any aminoacid residue. In some methods, the second peptide contains a sequencethatis
`substantially identical to Ex4 (9-39) (SQE ID NO:17), Ex4 (9-30) (SEQ ID NO:3 8), human
`GLP-1 (7-37) peptide (SEQ ID NO:18), or an active variant thereof.
`In some embodiments,
`the randomized peptide contains an amino acid sequence as shown in SEQ ID NO:22, SEQ
`ID NO:5, or SEQ ID NO:8.
`In some methods, the employed polypeptide contains an amino
`acid sequence as shown in SEQ ID NO:36, SEQ ID NO:34,or SEQ ID NO:32.
`[0011]
`In some methods, the signaling pathwayto be activated results in insulin
`biosynthesis and release. In some embodiments, the activated cell is a pancreatic beta cell.
`For example, the methodscanbe directed to activating insulin biosynthesis and release in
`pancreatic beta cells present in a subject.
`In some of these embodiments, the subject has or
`
`4
`
`

`

`WO 2016/161244
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`PCT/US2016/025490
`
`is at risk of developing a metabolic disorder characterized by high blood glucose and low
`insulin levels, e.g., type 2 diabetes, obesity, non-alcoholic fatty liver disease (NAFLD), non-
`alcoholic steatohepatitis (NASH) and atherosclerosis.
`
`In still another aspect, the invention provides methodsfor increasing insulin
`[0012]
`level, insulin sensitivity, and decreasing blood glucoselevel, or for treating a disorder
`characterized by high blood glucose and low insulin levels in a subject. These methods
`entail administering to the subject an effective amount of a polypeptide containingafirst
`randomized peptide that is fused at its C-terminus to a second peptide capable of binding to
`the extracellular domain of glucagon-like peptide 1 receptor (GLP-1R). In various
`embodiments, the randomized peptide in the employed polypeptide contains an amino acid
`sequence of XXXXXXX (SEQ ID NO:1), CKXXXXCXX (SEQ ID NO:2) or
`
`CXXXXCXXX (SEQ ID NO:3), wherein X is any aminoacid residue.
`
`In some ofthese
`
`methods, the second peptide contains an amino acid sequence that is substantially identical
`to Ex4 (9-39) (SQE ID NO:17), Ex4 (9-30) (SEQ ID NO:38), human GLP-1 (7-37) peptide
`(SEQ ID NO:18), or an active variant thereof. In some methods, the randomized peptide
`contains an amino acid sequence as shown in SEQ ID NO:22, SEQ ID NO:5, or SEQ ID
`NO:8.
`In some methods, the employed polypeptide contains an amino acid sequence as
`shownin SEQ ID NO:36, SEQ ID NO:34, or SEQ ID NO:32. Someof these methodsare
`
`directed to increasing insulin level and decreasing blood glucose in a subject whohasoris at
`risk of developing diabetes, obesity, non-alcoholic fatty liver disease (NAFLD), non-
`alcoholic steatohepatitis (NASH), atherosclerosis, Parkinson’s disease, Alzheimer’s Disease
`
`or drug addiction.
`
`The invention also provides the use of a polypeptide containinga first
`[0013]
`randomized peptide fused at its C-terminusto a second peptide capable of binding to the
`extracellular domain of glucagon-like peptide | receptor (GLP-1R) in the preparation of a
`pharmaceutical composition for treating diabetes or other disorders characterized by high
`blood glucose and low insulin levels. The randomized peptide typically contains an amino
`acid sequence of XXXXXXX (SEQ ID NO:1), CKXXXXCXX (SEQ ID NO:2) or
`CXXXXCXXX (SEQ ID NO:3), wherein X is any aminoacid residue.
`In some
`embodiments, the second peptide contains an amino acid sequence that is substantially
`identical to Ex4 (9-39) (SQE ID NO:17), Ex4 (9-30) (SEQ ID NO:38), human GLP-1 (7-37)
`peptide (SEQ ID NO:18), or an active variant thereof. In some embodiments, the
`
`

`

`WO 2016/161244
`
`PCT/US2016/025490
`
`randomized peptide contains an amino acid sequence as shown in SEQ ID NO:22, SEQ ID
`NO:5, or SEQ ID NO:8.
`In some embodiments, the employed polypeptide contains an
`amino acid sequence as shown in SEQ ID NO:36, SEQ ID NO:34, or SEQ ID NO:32.
`
`[0014]
`In a related aspect, the invention provides isolated or recombinant
`polynucleotides that encode a polypeptide containing a randomized peptide fusedat its C-
`terminus to a second peptide capable of bindingto the extracellular domain of glucagon-like
`peptide | receptor (GLP-1R). The randomized peptide in the encoded polypeptide typically
`contains an amino acid sequence of XXXXXXX (SEQ ID NO:1), CKXXXKCXX (SEQ ID
`NO:2) or CXXXXCXXX (SEQ ID NO:3), wherein X is any amino acid residue. In some
`embodiments, the second peptide contains a sequencethat is substantially identical to Ex4
`(9-39) (SQE ID NO:17), Ex4 (9-30) (SEQ ID NO:38), human GLP-1 (7-37) peptide (SEQ
`ID NO:18), or an active variant thereof.
`In some embodiments, the randomized peptide
`contains an amino acid sequence as shown in SEQ ID NO:22, SEQ ID NO:5, or SEQ ID
`NO:8. Some polynucleotides of the invention encode a polypeptide containing an amino
`acid sequence as shown in SEQ ID NO:36, SEQ ID NO:34, or SEQ ID NO:32.
`[0015]
`In some embodiments, the polypeptide encoded by the polynucleotide further
`contains a transmembraneprotein domain at the C-terminus of the second peptide. For
`example, the encoded polypeptide can contain the PDGFR transmembrane domain. In some
`
`embodiments,the transmembraneprotein and the second peptide in the encoded polypeptide
`are connected via a linker peptide. For example, a linker peptide containing two or more
`tandem repeats of GGGGS (SEQ ID NO:43) can be employed. In some embodiments, the
`encoded polypeptide can further contain a signal peptide at the N-terminus. For example,
`the IL-2 signal sequence can be used.
`In some specific embodiments, the employed signal
`peptide contains a sequence of MYRMQLLSCIALSLALVTNS(SEQ ID NO:44).
`[0016]
`In somerelated aspects, the invention additionally provides fusion polypeptides
`encoded bythe isolated or recombinant polynucleotides, as well as expression vectors
`harboring the isolated or recombinant polynucleotide of the invention.
`[0017]
`A further understanding of the nature and advantagesofthe present invention
`mayberealized by reference to the remaining portions of the specification and claims.
`
`[0018]
`
`Figure 1 shows autocrine-based system for selection of agonists from large
`
`DESCRIPTION OF THE DRAWINGS
`
`6
`
`

`

`WO 2016/161244
`
`PCT/US2016/025490
`
`combinatorial peptide libraries, a, Schematic representation of membrane tethered exendin-4.
`
`b, FACS analysis of mCherry and GFP expression 2 of days after transduction of HEK293-
`
`GLP-1R-GFP cells with the membrane tethered exendin-4 displaying different linkersize. c,
`
`Schematic representation of the autocrine based selection of combinatorial peptide library.
`Thelentivirus peptide library is prepared from lentiviral plasmids (step 1). The CRE-
`
`responsive GLP-1R reportercell line is transduced with lentiviral library (step 2). GFP
`
`expressing cells are sorted (step 3) and peptide encoding genes are amplified from genomic
`
`DNAofsorted cells to make the library for the next selection round(step 4). After iterative
`
`roundsofselection, enriched peptide sequences are analyzed by deep sequencing(step 5). d,
`
`Enrichment of GFPpositive cells during three rounds of FACS selection.
`
`[0019]
`
`Figure 2 shows in vitro pharmacological characterization of agonist peptide P5.
`
`a-b, Concentration response curves for P5- and Ex4-induced increase in cAMPproduction in
`
`CHOcells expressing the human GLP-1R (a) or in HEK293 expressing the mouse GLP-1IR
`
`(b). c, Concentration response curves for P5- and Exe4-induced calcium mobilization in
`
`CHOcells expressing the human GLP-1R.d-e, Concentration response curves for P5- and
`
`Exe4-induced B-arrestin-1 (d) and B-arrestin-2 (e) recruitment in CHO cells expressing the
`human GLP-IR.f, Biased factors (8) from an equiactive comparison indicate bias for PS.
`
`The data are mean + SEM oftypical experiment that was performed three times.
`
`[0020]
`
`Figure 3 shows acute P5 injection lowers glucose in diabetic mice. a-i,
`
`Intraperitoneal glucose tolerance (“insulin disposal’) test; a single intraperitoneal injection of
`
`saline, Ex4 or P5, co-injected with an intraperitoneal glucose challenge (n=5). Glucose
`
`tolerance in lean mice treated with 10 pg/kg (a), 1 ug/kg (b) or 0.1ug/kg peptides(c),in
`
`GLP-1-R KO treated with 1 g/kg peptides (d), in ob/ob mice treated with 1 g/kg peptides
`
`(e) and in DIO micetreated with 1 pg/kg peptides (f). g-i, Plasma insulin levels was
`
`analyzed after a single intraperitoneal co-injection of saline, 1 pg/kg of Ex4 or 1 pg/kg of P5
`
`G-protein biased agonist with glucose challenge (n=5) in lean mice (g), in ob/ob mice (h)
`
`and in DIO mice(i). Data are mean + s.e.m. Statistic by two-tailed t-test: *P < 0.05; **P <
`0.01, comparingsaline to peptide injection; *P < 0.05; *P < 0.01, comparing Ex4 to PS
`injection. AUC, area under the curve.
`
`10021]
`
`Figure 4 showsthat chronic administration of the G-protein biased GLP-1R
`
`agonist peptide P5 improves glycemic status in diabctic mice. a-g, four week of treatment of
`
`DIO male mice with daily dose of Ex4 and P5 biased agonist. Effect on body weight(a),
`
`7
`
`

`

`WO 2016/161244
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`PCT/US2016/025490
`
`intraperitoneal glucose tolerance (b), ad-libitum-fed plasma glucose level (c), insulin plasma
`
`level (d), HbAj¢ (e), islet morphology (f) and GIP plasmalevel (g) after daily subcutaneous
`
`injections ofsaline, Ex4 and P5 G-protein biased agonist at] ug/kg (lighter shade) or 10
`
`ug/kg (darker shade) . Data are mean + s.e.m. Statistic by two-tailed ¢-test: *P < 0.05; **P <
`
`0.01, comparing saline to peptide injection. AUC, area underthe curve.
`
`[0022]
`
`Figure 5 showsadditional in vitro pharmacological characterization of Peptide P5
`
`selectivity. a-b, Concentration response curves for Ex9-induced decrease in cAMP
`
`production in presence of an Ec90 of P5 in CHOcells expressing the human GLP-1R (a) or
`
`in HEK293 expressing the mouse GLP-1R (b).
`
`c, Concentration response curves for
`
`glucagon- P5- and Ex4-induced calcium mobilization in CHO cells expressing the human
`
`glucagon receptor. The data are mean + SEM of typical experiment that was performed three
`
`times.
`
`[0023]
`
`Figure 6 displays additional results indicating effect of acute P5 injection on
`
`glycemia and insulin level in ob/ob mice. a-i, Acute treatment of male mice with P5 G-
`
`protein biased agonist. a-c, Effect on intraperitoneal glucose tolerance and plasma insulin
`
`levels following a single intraperitoneal co-injection with glucose challenge (n=5) of saline,
`
`Ex4 and P5 G-protein biased agonist. Glucose tolerance in ob/ob mice treated with 10 pg/kg
`
`(a), 0.1 pg/kg (b) (n=5). c, Plasma insulin level in ob/ob mice were analyzed after a single
`
`intraperitoneal co-injection of saline, 10 we/kg of Ex4 or 10 ug/kg of P5 G-protein biased
`
`agonist with glucose challenge (n=5). Data are mean + s.e.m., Statistic by two-tailed f-test:
`*P <0.05; **P < 0.01, comparing saline to peptide injection; #p < 0.05; "P <0.01,
`
`comparing Ex4 to P5 injection. AUC, area under the curve.
`
`[0024]
`
`Figure 7 displays additional results indicating that chronic administration of
`
`Peptide P5 improves glycemic status in diabetic mice independently of body weight and fat
`
`mass. a-b Four week of treatment of DIO male mice with daily dose of Ex4 and P5 biased
`
`agonist. MRI analyses of fat (a) and body weight (b) following daily subcutaneousinjections
`
`of saline, Ex4 and P5 G-protein biased agonist at 10 ng/kg (n=8). Data are mean + s.e.m.
`
`Statistic by two-tailed r-test: *P < 0.05; **P <0.01, comparing saline to peptide injection.
`
`[0025]
`
`Figure 8 displays additional results indicating effect of chronic administration of
`
`Peptide P5 on metabolic hormones in DIO mice. Four week oftreatment of DIO male mice
`
`with daily dose of Ex4 and P5 biased agonist. Effects on c-peptide plasma level (a) and
`
`glucagon plasma level (b) (n=8) after daily subcutaneousinjections of saline, Ex4 and P5 G-
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`protein biased agonist at 10 pg/kg. Data are mean + s.e.m. Statistic by two-tailed ¢-test: *P <
`
`0.05; **P < 0.01, comparing saline to peptide injection.
`
`[0026]
`
`Figure 9 displays additional results indicating effect of chronic administration of
`
`P5 G-protein biased agonist on hepatoxicity and steatosis in DIO mice. Four week of
`
`treatment of DIO male mice with daily dose of Ex4 and P5 biased agonist. Effects on plasma
`
`ALP(a), plasma ALT (b), plasma AST (c) and hepatic steatosis (n=8) following daily
`
`subcutaneousinjections of saline (control), Ex4 and P5 G-protein biased agonist. Data are
`
`mean + s.e.m. Statistic by two-tailed t-test: *P < 0.05; **P <0.01, comparing saline to
`
`peptide injection.
`
`[0027]
`
`Figure 10 shows schematically the construction of the HEK293-GLP-1R-GFP
`
`reporter cell line.
`
`[0028]
`
`Figure 11 shows G-protein bias of other agonist peptides in activating GLP-1R
`
`signaling. (A) Classical paradigm of signal transduction by GPCR. The agonist binding to
`
`the receptor stimulates heterotrimeric G proteins and is rapidly phosphorylated by G protein-
`
`coupled receptor kinases (GRKsthat recruit B-arrestin to desensitize the receptor; CRE-
`
`responsive GLP-1R reporter cell line (B) and Tango GLP-1R-Bla U2OScell line (C) are
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`used to monitor the cAMP increase and B-arrestin recruitment after GPCR activation.
`
`[0029]
`
`Figure 12 shows in vitro characterization of the long acting G-protein biased
`
`agonist fusion polypeptide P5-Fc. (a) Schematic representation of P5-Fc (b) Concentration
`
`response curves for P5- and P5-Fc-induced increase in cAMP production in CHO cells
`
`expressing the human GLP-1R. (c) Concentration response curves for P5, and Ex4-induced
`
`B-arrestin-2 recruitment in CHO cells expressing the human GLP-1R.
`
`[0030]
`
`Figure 13 shows that GLP-1R agonist fusion polypeptide P5-Fc displays
`
`enhanced pharmacodynamics. (a) Glucose tolerance tests performed either directly following
`
`a single injection of P5 (20nmol/kg) or P5-fe (5 nmo!/kg) (day 0) or two and seven dayslater.
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`(b} Effect of a single injection of P5-fc (50 nmoi/kg) on fed blood glucoselevel.
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`[0031]
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`Figure 14 showsthat P5-Fc decreases body weight and fat mass in diabetic mice.
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`(a) Effect of once-weekly administration of P5-Fc on body weight in DIO mice.
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`(b) Effect
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`of once-weekly administration of P5-Fc on fat mass, fluid mass and lean mass and in DIO
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`mice ( after injection 6). n=5; *, p<0.05; **, p<0.01.
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`[0032]
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`Figure 15 shows that P5-Fe decreases food intake in diabetic mice. (a) and daily
`
`food intake (b). P5-Fc significantly decreases food intake for 2-3 days post injection. n=5; *,
`
`p<0.05; **, p<0.01.
`
`[0033]
`
`Figure 16 shows effect of PS and P5-Fc on reducing re-feeding of fasting mice.
`
`Male C57BL/6 mice (12-14 wk; 28-30g) fasted for 16-18h were injected ip with P5, P5-Fc
`
`and Ex4, Cumulative FI was measured at 1, 2, 4, 8, 24 and 31 h post-injection. n=3; *,
`
`p<0.05
`
`[0034]
`
`Figure 17 showsanti-hyperglycemia efficacy of P5-Fe in treating diabetic mice,
`
`(a) Effect of once-weekly administration of P5-Fc on glucose tolerance in response to
`
`glucose challenge (one week after injection 5 and before injection 6). (b) Effect of once-
`
`weekly administration of P5-Fc on fed plasma glucose levels (one weekafter injection 5 and
`
`before injection 6) and (c) on HbAIc (after injection 5). n=5; *, p<0.05; **, p<0.01.
`
`[0035]
`
`Figure 18 shows that P5-Fe enhancesinsulin sensitivity in diabetic mice. (a)
`
`Effect of once-weekly administration of P5-Fc on glucose level in response to insulin
`
`injection (0.75 U/kg) (one weekafter injection 4 and before injection 5), (b) Data are
`
`represented as a % of basal blood glucose level for each individual group. n=5; *, p<0.05;
`
`** p<0.01.
`
`[6036]
`
`Figure 19 showsthat P5-Fc induces intestinal growth. Effect of once-weekly
`
`administration of P5-Fc on (a) small bowel (SB) length, and (b) large bowcl (LB) length,
`
`n=5; *p<0.05, **, p<0.01.
`
`[0037]
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`Figure 20 showsthat-chronic administration of a G-protein biased agonist
`
`improves hepatic steatosis in DIO mice.
`
`[00338]
`
`Figure 21 showsthat chronic administration of the G-protein biased agonist
`
`modulates adipogenesis and insulin sensitivity in DIO mice.
`
`DETAILED DESCRIPTION OF THE INVENTION
`
`I.
`
`Overview
`
`[0039]
`
`The invention is predicated in part on the development by the present inventors of
`
`an autocrine-based system for selection of agonists from large intracellular combinatorial
`peptide libraries. As detailed herein, this system allows one out of about 10° different
`
`peptides and a receptor to be co-localized in the plasma membrane of cells. When the co-
`
`localized peptide activates the neighboring receptor a fluorescent signal is generated such
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`that each cell becomesa reporter unto itself. The system was validated by selection of highly
`
`potent agonists for the GLP-1 GPCR receptor that activated signaling pathways and induced
`
`insulin secretion and lowering of blood glucose in mice. Unlike the balanced agonists,
`
`endogenous GLP-1 or the synthetic peptide Exendin4 that activate both Go-protein and f-
`
`arrestin signaling pathways, the sclected molecules werebiased in that they activated both
`
`Gas and Gaq but not the B-arrestin mediated signal transduction pathways. Such studies
`
`open the way to select new agonists for GPCRs and other receptors. Agonists selected by
`
`these methodsare useful for de-convolution of signal transduction pathways and discovery
`
`of new mechanisms of action.
`
`[0040]
`
`As exemplified herein, the inventors examined the glucagon-like peptide-1 (GLP-
`
`1) system. GLP-1 carboxamide is an important incretin hormonethat activates the GPCR
`
`GLP-1 receptor (GLP-1-R) resulting in a lowering of blood glucose levels in a glucose-
`
`dependent manner. When the endogenous GLP-| bindsto the receptor, a complex network
`
`of downstream signaling pathways are activated that include, amongst others, G-proteins, B-
`
`arrestin and a variety of kinases. Specifically, the inventors constructed combinatorial
`
`peptide libraries and expressed them in reporter cells such that each cell expresses the GLP-
`
`1R and a different peptide that are co-localized on the plasma membrane.
`
`In onelibrary, a
`
`sequence of 7 randomized seven amino acids was coupled to the Exentin-4 C-terminus
`
`(amino acids 9-39) as an anchor sequence. Exendin-4 from the Gila monsterlizard
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`Heloderma suspectum was chosen as the anchor sequenceso as not to be confused by the
`
`agonist activity of the C-terminal GLP-1 domain. When the amino terminus of exendin-4 is
`
`truncated by the first eight amino acid residues (Ex9-39), it acts as an antagonist of the GLP-
`
`1R. This autocrine selection system is linked to a fluorescent signal such that receptor
`
`activation can be monitored and selected in the cell sorter.
`
`[0041]
`
`Some of the GLP-1R agonists identified by the inventors are G-protein biased
`
`It is known that GLP-1R coupling to G-protein upon activation can lead
`(e.g., peptide P5).
`to an increasein intracellular 3’,5’ cyclic AMP (cAMP)andelevation of intracellular Ca**
`
`concentration, which are critical events in promoting glucose-dependentinsulin secretion in
`
`the pancreatic B-cells. GLP-1R mediated physiological consequencesalso include
`
`engagementof B-arrestins which regulates insulin secretion. Current treatments with GLP-
`
`1R agonists have demonstrated anti-diabetic effects, but also shown various adverse side
`
`effects and concerns. Optimization of these GLP-1R agonist therapies often focuses on
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`increasing the half-life of GLP-1 and GLP-1 mimetics which signal via both G-protein and
`
`B-arrestin coupling.
`
`[0042]
`
`G-protein bias of some peptides ofthe invention refers to the ability of the
`
`p