DIABETES/METABOLISM RESEARCH AND REVIEWS
`Diabetes Metab Res Rev 2010; 26: 287–296.
`Published online 30 April 2010 in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/dmrr.1080
`
`R E S E A R C H A R T I C L E
`
`Engineering and characterization of the long-acting
`glucagon-like peptide-1 analogue LY2189265, an Fc
`fusion protein
`
`Wolfgang Glaesner*
`Andrew Mark Vick†
`Rohn Millican
`Bernice Ellis
`Sheng-Hung Tschang
`Yu Tian
`Krister Bokvist
`Martin Brenner‡
`Anja Koester
`Niels Porksen
`Garret Etgen
`Tom Bumol
`
`Lilly Research Laboratories, Division
`of Eli Lilly and Company,
`Indianapolis, IN, USA
`
`*Correspondence to:
`Wolfgang Glaesner, Lilly Research
`Labs, Lilly Biotechnology
`Center – San Diego, 10300 Campus
`Point Drive, San Diego, CA 92131,
`USA.
`E-mail: w.glaesn@lilly.com
`
`†Present address: SeventhWave
`Labs, Chesterfield, MO 63005, USA.
`
`‡Present address: Pfizer Inc.,
`Groton, CT 06340, USA.
`
`Received: 15 September 2009
`Revised: 11 February 2010
`Accepted: 25 March 2010
`
`Copyright  2010 John Wiley & Sons, Ltd.
`
`Abstract
`
`Background Glucagon-like peptide-1 (GLP-1)
`receptor agonists are
`novel agents for type 2 diabetes treatment, offering glucose-dependent
`insulinotropic effects, reduced glucagonemia and a neutral bodyweight or
`weight-reducing profile. However, a short half-life (minutes), secondary to
`rapid inactivation by dipeptidyl peptidase-IV (DPP-IV) and excretion, limits
`the therapeutic potential of the native GLP-1 hormone. Recently, the GLP-1
`receptor agonist exenatide injected subcutaneously twice daily established a
`novel therapy class. Developing long-acting and efficacious GLP-1 analogues
`represents a pivotal research goal. We developed a GLP-1 immunoglobulin G
`(IgG4) Fc fusion protein (LY2189265) with extended pharmacokinetics and
`activity.
`
`In vitro and in vivo activity of LY2189265 was characterized in
`Methods
`rodent and primate cell systems and animal models.
`
`Results LY2189265 retained full receptor activity in vitro and elicited
`insulinotropic activity in islets similar to native peptide. Half-life in rats
`and cynomolgus monkeys was 1.5–2 days, and serum immunoreactivity
`representing active compound persisted >6 days.
`In rats, LY2189265
`enhanced insulin responses during graded glucose infusion 24 h after one
`dose. LY2189265 increased glucose tolerance in diabetic mice after one dose
`and lowered weight and delayed hyperglycaemia when administered twice
`weekly for 4 weeks. In monkeys, LY2189265 significantly increased glucose-
`dependent insulin secretion for up to a week after one dose, retained efficacy
`when administered subchronically (once weekly for 4 weeks) and was well
`tolerated.
`
`Conclusions LY2189265 retains the effects of GLP-1 with increased half-life
`and efficacy, supporting further evaluation as a once-weekly treatment of type
`2 diabetes. Copyright  2010 John Wiley & Sons, Ltd.
`
`Keywords glucagon-like peptide 1; Fc fusion protein; type 2 diabetes; incretin
`mimetic
`
`Introduction
`
`The progressive nature of β-cell dysfunction in type 2 diabetes often renders
`treatment inadequate over time [1], necessitating multiple oral agents and/or
`insulin. Treatment side effects, such as weight gain and hypoglycaemia [2],
`
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`W. Glaesner et al.
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`frequently present barriers to physician administration
`and patient adherence, with resulting inadequate gly-
`caemic control [3–7]. Preserving or promoting β-cell
`function with minimal hypoglycaemia or weight gain rep-
`resents a pivotal treatment objective.
`The natural incretin hormone glucagon-like peptide-
`1 (GLP-1) supports glucose homeostasis by enhancing
`glucose-dependent insulin secretion from β-cells and sup-
`pressing inappropriately elevated postprandial glucagon
`secretion from α-cells. In addition, GLP-1 has been demon-
`strated to reduce appetite and food intake and inhibit
`gastric emptying, which may facilitate weight manage-
`ment [8,9]. Rapid enzymatic inactivation by dipeptidyl
`peptidase-IV (DPP-IV) and excretion of GLP-1 limit
`its therapeutic potential [10,11]; thus analogues and
`long-acting formulations of analogues [12] have been
`developed. For example, exenatide (Byetta; Amylin Phar-
`maceuticals Inc., San Diego, CA, USA and Eli Lilly and
`Company, Indianapolis, IN, USA), approved by the US
`Food and Drug Administration, is a GLP-1 mimetic demon-
`strating many of the beneficial effects of GLP-1 in daily
`therapeutic use. In addition, liraglutide (Novo Nordisk,
`Bagsværd, Denmark, approved on January 25, 2010 by
`the Food and Drug Administration and will be marketed
`under the proprietary name Victoza, USA) is a GLP-1
`analogue administered once-daily subcutaneously [13].
`Fusing GLP-1 to a larger
`‘carrier’ moiety, hence
`in vivo clearance, might also enhance
`slowing its
`pharmacokinetics. In pre-clinical studies,
`linking GLP-
`1 to albumin substantially prolonged the half-life (to
`∼10–12 h) [14,15]. When GLP-1 was fused to the
`Fc domain of
`immunoglobulin,
`the plasma half-life
`of GLP-1 was substantially prolonged (∼30 h) [16].
`We describe the engineering and characterization of
`LY2189265, a DPP-IV-protected GLP-1(7–37) analogue
`fused to a modified immunoglobulin G (IgG4) Fc
`fragment; the fusion protein maintains the insulinotropic
`activity of the native peptide with substantially improved
`plasma half-life, decreased clearance and a flat profile
`with no burst effect, potentially allowing once-weekly
`dosing. The potential safety of LY2189265 was enhanced
`by engineering to reduce Fcγ receptor binding and
`immunogenic potential.
`
`Materials and methods
`
`Expression and purification of GLP-1-Fc
`
`(HEK) 293-EBNA cells
`Human embryonic kidney
`were maintained in Dulbecco modified Eagle medium
`(DMEM)/Ham F-12 medium (Invitrogen, Carlsbad,
`CA, USA) supplemented with 20 mM HEPES (Invit-
`rogen), 5 µg/mL nucellin (Eli Lilly and Company),
`0.4 µg/mL tropolone (Sigma–Aldrich, St Louis, MO,
`USA), 0.075% (w/v) F68 (Invitrogen), and 50 µg/mL
`geneticin (Sigma–Aldrich) (37 ◦C; 5–8% CO2). DNA was
`added to FuGene6 transfection reagent (Roche Molec-
`ular Biochemicals, Indianapolis, IN, USA) in OptiMEM
`
`Copyright  2010 John Wiley & Sons, Ltd.
`
`(Gibco/BRL, Gaithersburg, MD, USA) and incubated
`(15 min, 37 ◦C). Concentrated expression media was
`loaded directly onto a Hi-Trap Protein A column
`(GE Healthcare, Piscataway, NJ, USA), equilibrated in
`phosphate-buffered saline (PBS; 3 mL/min flow rate)
`and washed. Pooled fractions of bound GLP-1-Fc (pH
`7.4), eluted with a step gradient of 100% 50 mM Na-
`citrate (pH 2.2), were concentrated and loaded onto
`a Superdex 200 (26/60, GE Healthcare) column (PBS-
`equilibrated; 3 mL/min flow rate). The GLP-1-Fc fractions
`were characterized by SDS-PAGE and mass spectrome-
`try, sterile-filtered (0.22 µm), assessed for concentration
`(absorption at 280 nm) and stored at −20 ◦C.
`
`Evaluation of T-cell epitopes
`
`Potential T-cell epitopes were identified in silico using
`EpiMatrix, a matrix-based algorithm for T-cell epitope
`mapping [17]. The N-terminal 64 amino acids of GLP-1-Fc
`were parsed into 9-mer frames overlapping by eight amino
`acids. Binding was then predicted to eight major histocom-
`patibility complex class II alleles representative of human
`populations
`(DRB1*0101, DRB1*0301, DRB1*0401,
`DRB1*0701, DRB1*0801, DRB1*1101, DRB1*1301 and
`DRB1*1501) and reported as Z-scores uniformly scaled
`for direct comparison across alleles. All scores in the top
`5% (Z-score ≥1.64) were considered ‘hits’, and scores
`in the top 1% (Z-score ≥2.32) were considered highly
`likely for major histocompatibility complex binding. An
`overall EpiMatrix score for immunogenicity was calcu-
`lated by determining the deviation from the number of
`potential T-cell epitopes predicted for random-sequence
`pseudoproteins per 1000 amino acid assessments. EpiMa-
`trix Z-scores ≥1.64 (303/5664 total assessments; ∼5%)
`were investigated further.
`
`Antibody-dependent, cell-mediated
`cytotoxicity assays
`
`Jurkat Fcγ III cells (V) were created by cotransducing
`Jurkat T cells with the murine Maloney leukemia virus
`(MMLV)-based vector pLHCX (Clontech, Mountain View,
`CA, USA) expressing the 158V allotype of human
`Fcγ RIIIa with a hygromycin resistance cassette and the
`MMLV vector pLNX2, expressing human FcεRI with
`a neomycin resistance cassette. Dual-resistant colonies
`were screened by fluorescence-activated cell sorting for
`high Fcγ RIIIa expression and confirmed by anti-FcR
`crosslinking-induced interleukin 2 release. The reporter
`line Jurkat Fcγ RIII (V) NFAT Luc was created by co-
`electroporating the luciferase reporter under the control
`of the NFAT promoter (Stratagene, La Jolla, CA, USA)
`and pPUR (Clontech) containing the puromycin resistance
`cassette. Puromycin-resistant colonies were screened by
`anti-FcR-induced luciferase expression.
`Chinese hamster ovary-K1 GLPR1 cells were plated at
`2 × 104 cells/well in Costar 96-well white luminescence
`
`Diabetes Metab Res Rev 2010; 26: 287–296.
`DOI: 10.1002/dmrr
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`Development/Characterization of GLP-1-Fc
`plates and incubated for 1 h at 37 ◦C,
`followed by
`incubation with different concentrations of GLP-1-Fc for
`1 h. A total of 1 × 105 Jurkat Fcγ RIII (V) NFAT Luc cells
`were added to each well and incubated for an additional
`5 h. Luciferase activity was assayed by incubation with
`Pierce Steady-Glo luminescence reagent (Thermo Fisher,
`Rockford, IL, USA) and analysed on a Molecular Devices
`LMaxII luminometer (Sunnyvale, CA, USA).
`
`Reporter gene activity with β-luciferase
`
`HEK 293 cells (<passage 5) expressing human GLP-1
`receptor and a cyclic AMP (cAMP)-responsive CRE4-
`luciferase system were seeded (80 000 cells/well
`in
`80 µL) and incubated overnight in DMEM/F12 (3 : 1)
`medium (Gibco; no. 93-0152-DK) containing 0.25% foetal
`bovine serum (FBS), 50 µg/mL gentamicin and 2 mM
`L-glutamine. Subsequently, GLP-1-Fc or Val8-GLP-1 (in
`0.5% bovine serum albumin [BSA]) was added to result
`in final concentrations of 0.0003 nM to 3 nM (5 h, 37 ◦C
`in 5% CO2). Plates were read after the addition of
`LucLite luciferase reagent (100 µL; Packard Bioscience,
`Groningen, the Netherlands) and mixing in a TriLux
`instrument (TRILUX GmbH & Co., Arnsberg, Germany).
`
`Reporter gene activity with β-lactamase
`
`HEK 293 cells expressing human GLP-1 receptor and a
`cAMP-responsive CRE-BLAM reporter system were seeded
`in 100 µL DMEM plus 10%
`(20 000–40 000 cells/well
`FBS) and incubated overnight at 37 ◦C. Medium was
`subsequently replaced with plasma-free DMEM (80 µL).
`One day later, serum-free DMEM (20 µL) plus 0.5%
`BSA containing the GLP-1 agonist was added. Half-
`maximal effective concentration values were determined
`from a dose–response curve (0.00003–30 nM dilutions).
`After incubation (5 h), 20 µL lactamase substrate (CCF2-
`AM; PanVera LLC, Madison, WI, USA) was added, and
`fluorescence was measured 1 h later (Cytofluor Plate
`Reader, Applied Biosystems Inc., Foster City, CA, USA).
`
`Insulin secretion in rat islets
`
`After common bile duct cannulation in male Sprague–
`Dawley rats (250–280 g), the pancreas was distended
`with Hank buffer (10 mL, containing 2% BSA and
`1 mg/mL Sigma type V collagenase or 0.15 mg/mL lib-
`erase [Roche]). Subsequently, tissues were digested in
`Hank buffer at 37 ◦C for 10–12 min (or 19–21 min
`for liberase). Purified islets (Histopaque-1077 gradi-
`ent [Sigma–Aldrich], 18 min at 750 g) were cultured
`overnight in RPMI-1640 medium (Invitrogen) containing
`10% FBS, 100 U/mL penicillin, and 100 µg/mL strepto-
`mycin and preconditioned by starvation in Earle balanced
`salt solution (EBSS) supplemented with 0.1% BSA and
`2.8 mM glucose. Subsequently, islets were incubated in
`EBSS (Invitrogen) supplemented with 0.1% BSA, 2.8 mM
`
`Copyright  2010 John Wiley & Sons, Ltd.
`
`289
`
`to 16.8 mM glucose, and increasing levels of LY2189265
`(3–6 batches of 3–4 islets/condition) with or without
`addition of 1 µM exendin 9–39 (Ex[9–39]; Bachem
`Americas Inc., Torrance, CA, USA). Insulin was measured
`over 90 min in supernatant using the Meso Scale Insulin
`Assay (Meso Scale, Gaithersburg, MD, USA).
`
`Insulin secretion in monkey islets
`
`Two halved cynomolgus monkey pancreata (Covance Inc.,
`Princeton, NJ, USA) distended with Hank buffer (contain-
`ing 2% BSA and 1 mg/mL collagenase [Sigma–Aldrich])
`were digested (37 ◦C) and purified on a discontinu-
`ous Histopaque-1077 gradient (750 g for 18 min). After
`overnight culture in RPMI-1640 medium (containing 10%
`FBS, 100 U/mL penicillin and 100 µg/mL streptomycin
`[all Invitrogen]), islets were starved in EBSS contain-
`ing 2.8 mM glucose (30 min; 37 ◦C), and batches of three
`islets were then incubated for 90 min in 0.3 mL EBSS con-
`taining 16.7 mM glucose and compounds, as indicated.
`Supernatants were frozen (−20 ◦C) until electrochemi-
`luminescent insulin assay (Meso Scale). For all in vitro
`assays, molar concentrations of GLP-1-Fc were calculated
`by dividing the molecular weight of the fusion protein by
`two because of its homodimeric nature.
`
`Animals
`
`For all in vivo studies, animals were maintained in a
`controlled environment (20 ± 2 ◦C, 50–60% humidity,
`12-h light–dark cycle,
`lights on at 6 : 00 AM) and
`fed a standard chow (Mice: Purina 5008, LabDiets, St
`Louis, MO, USA; Rats: Purina 5001 LabDiets; cynomolgus
`monkeys: Certified Global Primate Diet #2055C, Harlan
`Laboratories, Indianapolis, IN, USA). All procedures were
`performed in accordance with the National Institutes of
`Health Guide for the Care and Use of Laboratory Animals
`and with approval of the Eli Lilly Research Laboratories
`and Covance Inc. Institutional Animal Care and Use
`Committees.
`
`Blood glucose and insulin level
`measurement
`
`Blood glucose levels were determined by Precision-G
`Blood Glucose Testing System (Abbott Diagnostics, Abbott
`Park, IL, USA), and insulin levels were determined by
`radioimmunoassay (Linco Diagnostics, St Charles, MO,
`USA).
`
`Pharmacokinetics in Sprague–Dawley
`rats and cynomolgus monkeys
`Adult male rats (n = 3/group) received a single subcuta-
`neous (SC) dose of 0.1 mg/kg LY2189265, and blood was
`collected 1, 2, 4, and 6 days later. Monkeys (n = 3/group)
`
`Diabetes Metab Res Rev 2010; 26: 287–296.
`DOI: 10.1002/dmrr
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`290
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`W. Glaesner et al.
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`received a single SC dose of 0.1 mg/kg LY2189265, and
`blood (2 mL) was collected at 0 (pre-administration), 2,
`4, 8, 12, 48, 72, 96, 192, 240, 288, and 336 h after admin-
`istration. Plasma samples were stabilized with 10 µL
`DPP-IV inhibitor/mL (Millipore, St Charles, MO, USA),
`and immunoreactive GLP-1-Fc concentration was deter-
`mined by enzyme-linked immunosorbent assay (ELISA)
`using antibodies recognizing the N-terminus of GLP-1-
`Fc (Eli Lilly and Company) and the Fc domain (mouse
`anti-human IgG4; Southern Biotech, Birmingham, AL,
`USA). Plasma samples were diluted with equal amounts of
`casein/PBS and incubated for 1.5 h. Secondary antibody
`(1 : 2000 in blocking buffer) was added for 1 h. Optical
`density (450–630 nm) of 3,3(cid:4),5,5(cid:4)-tetramethylbenzidine
`development was determined, concentrations of GLP-1-
`Fc were calculated using a four-parameter algorithm, and
`standard curves were prepared for GLP-1-Fc in rat plasma.
`The ELISA assay range was approximately 0.9–80 ng/mL.
`
`Graded glucose infusion in rats
`
`Adult male Sprague–Dawley rats (420–460 g) with
`femoral artery and vein cannulation were acclimated to
`study boxes and subsequently treated with SC vehicle
`(saline; n = 18) or LY2189265 (0.3 nmol/kg [n = 4],
`1 nmol/kg [n = 3], 3 nmol/kg [n = 7], or 30 nmol/kg
`[n = 4]). After 24 h, fasted rats (16 h) were infused
`with saline (20 min),
`followed by low-dose glucose
`(50 mg/kg/min, 30 min) and finally high-dose glucose
`(150 mg/kg/min, 30 min). Blood samples (250 µL) were
`collected at −20, −10, 0, 10, 20, 30, 40, 50, and 60 min.
`Statistical significance was evaluated using the paired
`Student’s t-test (JMP 4.04 statistical software).
`
`Graded glucose infusion in cynomolgus
`monkeys
`
`Sedated and fasted (16–18 h) cynomolgus monkeys
`(n = 6) were infused with glucose immediately after SC
`administration of vehicle control (PBS) or LY2189265
`(1.7 nmol/kg) and 1, 5, and 7 days later. Glucose solution
`(20% dextrose solution, 200 mg/mL, intravenous) was
`infused at 10 mg/kg/min (3.0 mL/kg/h) for 20 min and
`then at 25 mg/kg/min (7.5 mL/kg/h) for 20 min. Blood
`was collected at −10, 0, 10, 20, 30, and 40 min.
`In a separate experiment, monkeys (n = 6) receiving
`SC vehicle or LY2189265 (1.7 nmol/kg) once weekly
`for 4 weeks were evaluated using this graded glucose
`infusion paradigm 4 days after the last LY2189265
`dose.
`
`Subchronic dosing of diabetic db/db
`mice for 4 weeks
`
`Five-week-old female diabetic db/db mice (C57BL/
`KsOlaHsd-Leprdb, Harlan Laboratories) were randomly
`grouped (n = 10/group) according to body weight, and
`
`Copyright  2010 John Wiley & Sons, Ltd.
`
`LY2189265 (10 nmol/kg) was administered subcuta-
`neously once weekly for 4 weeks. Blood glucose was
`measured in conscious mice just before dosing by tail clip
`at each weekly injection, except for the first week, when
`glucose was measured 1 h after administration. Fasted
`insulin levels were measured on day 0 and day 26 after
`an overnight fast.
`
`Statistical analysis
`
`Unless otherwise noted, groups were compared by one-
`way analysis of variance followed by Dunnett test with
`JMP 5.1.1 statistical software (SAS Institute).
`
`Results
`
`The GLP-1-Fc fusion protein LY2189265
`has preserved in vitro activity and an
`extended in vivo half-life
`
`Direct fusion of DPP-IV-protected GLP-1 analogue (V8-
`GLP-1) to the human G-type immunoglobulin (IgG1)
`hinge region dramatically reduced in vitro activity (by
`∼95%) compared to that of free V8-GLP-1 (Figure 1A).
`To restore and optimize in vitro activity, linker sequences
`were added between the C-terminus of further modified
`GLP-1 analogues and the N-terminus of the IgG hinge,
`and these were tested in the in vitro assay. A molecule
`with optimal linker length and sequence was identified
`that demonstrated approximately 4-fold greater in vitro
`potency over that of free V8-GLP-1 (Figure 1B and C). To
`reduce potential complement-dependent and antibody-
`dependent cell-mediated cytotoxicity (ADCC), IgG1 was
`replaced with a modified IgG4 isotype, optimized at
`two selected positions (F234A and L235A) to reduce
`interaction with high-affinity Fc receptors, which resulted
`in significant reduction of dose-dependent cytotoxicity of
`two IgG4 versions over the IgG1 version in an ADCC
`assay (Figure 1D). In addition, S228 was mutated to
`proline to eliminate half-antibody formation, the GLP-
`1 R36G mutation was introduced to de-immunize the
`fusion protein based on the results of
`the EpiVax
`algorithm (Figure S1, Supporting information), and the
`C-terminal
`lysine of the IgG-Fc was removed. In its
`final version, LY2189265 had 4-fold greater GLP-1
`receptor activation compared to that of V8-GLP-1 peptide
`(Figure 1C).
`Insulin secretion from isolated rat islets was potently
`(2.5–3-fold) enhanced by the inclusion of 3 nM or
`30 nM LY2189265 in the extracellular medium at
`high (16.8 mM) glucose with no significant enhance-
`ment of
`insulin secretion seen in the presence of
`low (2.8 mM) glucose (Figure 2A). Unmodified human
`GLP-1 (3 nM) produced a 4-fold enhancement of
`insulin secretion elicited by 16.8 mM glucose. Half-
`maximal stimulation of insulin secretion by LY2189265
`
`Diabetes Metab Res Rev 2010; 26: 287–296.
`DOI: 10.1002/dmrr
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`Development/Characterization of GLP-1-Fc
`
`291
`
`V8: A8V
`G8: A8G
`CEX: SSGAPPPS
`C2: SSGASSGA
`L: (GGGGS)3
`IgG4v: S228P, F234A, L235A
`
`700
`
`600
`
`500
`
`400
`
`300
`
`200
`
`100
`
`B
`
`Expression (mean ± SEM)
`
`Relative Reporter Gene
`
`Standard
`(n = 565)
`
`GLP-IgG1
`(n = 9)
`
`0
`
`V8E22 CEX IgG1 (n = 3)
`V8 CEX IgG1 (n = 3)
`G8E22 C2 IgG1 (n = 3)
`G8E22 CEX L IgG4 (n = 5)
`G8E22 L IgG4v (n = 10)
`V8GLP-1 (n = 565)
`V8 IgG1 (n = 6)
`
`--+-
`-----
`···•···
`
`V8E22CEX (IgG1)
`G8E22CEX L (IgG4)
`LY2189265
`
`250
`
`125.00
`
`15.63
`31.25
`62.50
`0.98
`1.95
`3.91
`7.81
`Antibody Concentration (µg/mL)
`
`0.49
`
`D
`
`0.350
`
`0.300
`
`0.250
`
`0.200
`
`0.150
`
`0.100
`
`0.050
`
`0.000
`
`(mean luminescence ± SEM)
`
`Cytotoxicity
`
`Standard
`(n = 19)
`
`LY2189265
`(n = 19)
`
`120
`
`100
`
`80
`
`60
`
`40
`
`20
`
`0
`
`600
`
`500
`
`400
`
`300
`
`200
`
`100
`
`0
`
`A
`
`Expression (mean ± SEM)
`
`Relative Reporter Gene
`
`C
`
`Expression (mean ± SEM)
`
`Relative Reporter Gene
`
`Figure 1. LY2189265 shows increased in vitro activity over dipeptidyl peptidase-IV (DPP-IV)-protected glucagon-like peptide-1
`(GLP-1) analogue human G-type immunoglobulin (IgG1) fusion proteins without linker sequences and attenuated cytotoxicity.
`In vitro activity was assessed with a cyclic AMP (cAMP) response element transcriptional reporter (β-lactamase in (A) and (B),
`luciferase in (C)) in human embryonic kidney (HEK) 293 cells expressing the GLP-1 receptor. (A) Compared to the V8-GLP-1
`peptide analogue that we used as a standard, DPP-IV-protected analogues fused to the hinge region of human IgG (GLP-IgG1)
`lost most of the in vitro activity. (B) The addition of linker sequences between GLP-1 and the IgG moiety increased activity
`significantly up to 4-fold that of standard peptide. (C) The fully engineered GLP-Fc molecule LY2189265 demonstrated 4-fold
`increased potency compared to that of the standard peptide V8-GLP-1. The amino acid sequence of GLP-1 including the linker is
`as follows: HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGGGGGGSGGGGSGGGGSA. (D) Antibody-dependent cell-mediated cytotoxicity
`(ADCC) associated with IgG1-Fc was attenuated by the incorporation of IgG4-Fc isotype into the GLP-1-Fc fusion protein and by
`mutations in the Fcγ R binding site to reduce its receptor-binding affinity. HEK 293 cells expressing the human GLP-1 receptor (target
`cells) and human peripheral blood mononuclear cells (effector cells) were exposed to several doses of LY2189265 (engineered IgG4
`isotype) or GLP-1-Fc analogues V8E22CEX (IgG1 isotype) or G8E22CEXL (IgG4 isotype). Cytotoxicity was estimated by the amount
`of luminescence, reflecting the release of lactate dehydrogenase from lysed target cells. Results are the mean of three experiments
`performed in duplicate. Gene reporter activity is represented as percent activity compared to that of the standard peptide V8-GLP-1.
`Data points are shown as mean ± standard error of the mean (SEM). n = the number of independent experiments carried out in
`triplicate (GLP-1-Fc) or duplicate (standard)
`
`was observed at 2.7 nM. The maximal efficacy, 4-
`fold stimulation, was observed at 300 nM LY2189265
`(Figure 2B). The inclusion of 1 µM of
`the GLP-1
`receptor antagonist Ex(9–39) reversed the glucose-
`dependent stimulation of
`insulin secretion observed
`with LY2189265, suggesting that LY2189265 acts via
`the islet GLP-1 receptor (Figure 2C). In islets isolated
`from cynomolgus monkeys, LY2189265 also increased
`insulin secretion in the presence of a high glucose
`
`concentration in a concentration-dependent manner
`(Figure 2D).
`The pharmacokinetic profile of LY2189265 in rats and
`cynomolgus monkeys is summarized in Table 1. The half-
`life of LY2189265 after a single dose of 0.1 mg/kg was
`approximately 1.5 days in rats and >2 days in monkeys
`(Figure 3 and Table 1). Immunoreactivity for LY2189265
`after a single dose remained detectable for several days
`(>6 days in rats and >14 days in monkeys) (Figure 3).
`
`Copyright  2010 John Wiley & Sons, Ltd.
`
`Diabetes Metab Res Rev 2010; 26: 287–296.
`DOI: 10.1002/dmrr
`
`
`
`MPI EXHIBIT 1025 PAGE 5
`
`MPI EXHIBIT 1025 PAGE 5
`
`Apotex v. Novo - IPR2024-00631
`Petitioner Apotex Exhibit 1025-0005
`
`

`

`292
`
`A
`
`16.EI mM Qlucoso
`
`B
`
`W. Glaesner et al.
`
`2.8 m'M Gh.UJO$e
`
`Basal
`
`l \'2111-9265
`
`0 ... - - . - - . - - . - - - - - - - -
`o.1
`'3
`0.3,
`1
`10
`'JO 1'00 '301>
`[tl"2189265] )nM)
`
`,oo
`
`C
`
`3
`E
`tn
`C
`
`= 2,ll riM _gruc°"" .. o µM emridln 9,as
`=
`
`18.B nM 11lucase • · !l µNI l!lf!!r clln B--39
`11i,6 nM glUQ0$!l + 1 /lllt ll~•ndlll S-39
`
`-
`
`D
`
`Glucose
`only
`
`lllM
`30nM
`L V218921i5 LY2189l!95
`
`3 nM
`GLP-1
`
`16 18 mM Gluc9se
`Forskolin tuMI
`LY2189265 (nM!
`
`+
`
`+
`
`-;--
`
`+-
`
`10 30
`
`f 00 3011
`
`Figure 2. LY2189265 induces glucose-dependent insulin secretion via activation of the GLP-1 receptor. (A) LY2189265 increased
`insulin secretion in rat islets up to 3-fold in the presence of high glucose but was not insulinotropic at low glucose concentration.
`(B) LY2189265 increased insulin secretion in rat islets with a half-maximal effective concentration of 2.7 nM. (C) LY2189265 acts
`via the GLP-1 receptor. The effect of LY2189265 on insulin secretion in rat islets was abolished by the addition of the specific
`GLP-1 receptor blocker Ex(9–39) (insulin concentrations were determined after a 90-min incubation, n = 3–6 batches of 3–4
`islets/condition tested, values are represented as mean ± SEM). (D) LY2189265 increased insulin secretion of islets prepared from
`cynomolgus monkeys in a concentration-dependent manner (insulin concentrations were determined after a 90-min incubation,
`n = 6 batches of 3–4 islets/condition tested, values are shown as mean ± SEM)
`
`Table 1. Pharmacokinetic parameters in Sprague–Dawley rats and cynomolgus monkeys (mean ± standard deviation)
`AUC0−∞ (ng/h/mL)
`10 537 ± 1103
`15 207 ± 5565
`
`Cmax (ng/mL)
`179.7 ± 15.3
`292.2 ± 21.9
`
`Tmax (h)
`24.0 ± 0.0
`16.7 ± 12.7
`
`Rat
`Monkey
`
`T1/2 (h)
`38.2 ± 2.0
`51.6 ± 3.2
`
`CL/F (mL/h/kg)
`9.6 ± 1.0
`7.3 ± 3.2
`
`Vss/F (mL/kg)
`525.0 ± 46.2
`557.5 ± 281.6
`
`Plasma concentrations of GLP-Fc (0.1 mg/kg dose) were determined with a sandwich ELISA recognizing the N-terminus of the GLP-1 portion and the
`Fc portion. Pharmacokinetic parameters were determined from the mean plasma concentration data from three animals per time point. Cmax indicates
`maximal observed plasma concentration; Tmax indicates time of maximal observed plasma concentration; AUC0−∞ indicates area under the plasma
`concentration curve from zero to infinity; T1/2 indicates elimination half-life; CL/F indicates clearance as a function of bioavailability; Vss/F indicates
`volume of distribution at steady state as a function of bioavailability.
`GLP-1, glucagon-like peptide-1; ELISA, enzyme-linked immunosorbent assay.
`
`LY2189265 enhances glucose-induced
`insulin secretion in rats
`
`doses of LY2189265 demonstrated statistical significance,
`enhancing secretion by up to 4-fold (p < 0.05).
`
`A graded glucose infusion assay was used to define the
`dose relation of LY2189265 to glucose-dependent insulin
`release. Graded glucose infusion in conscious rats 24 h
`after a single SC dose of LY2189265 (0.03–30 nmol/kg)
`demonstrated dose-dependent increases in insulin levels
`for all glucose infusion rates (Figure 4). However,
`compared to vehicle, only the 3 and 30 nmol/kg
`
`Insulinotropic effects of LY2189265
`in cynomolgus monkeys
`
`Insulin secretion in response to graded glucose infusion
`was evaluated in monkeys 1, 5, and 7 days after one
`SC dose of LY2189265 (1.7 nmol/kg). Administration
`
`Copyright  2010 John Wiley & Sons, Ltd.
`
`Diabetes Metab Res Rev 2010; 26: 287–296.
`DOI: 10.1002/dmrr
`
`MPI EXHIBIT 1025 PAGE 6
`
`MPI EXHIBIT 1025 PAGE 6
`
`Apotex v. Novo - IPR2024-00631
`Petitioner Apotex Exhibit 1025-0006
`
`

`

`Development/Characterization of GLP-1-Fc
`
`293
`
`LY2189265 improves glucose tolerance
`and produces sustained reductions
`in blood glucose in diabetic mice
`
`Twice-weekly dosing of db/db mice with 10 nmol/kg
`LY2189265 for 4 weeks starting at 5 weeks of age resulted
`in consistently lowered plasma glucose over the 4-
`week period compared to that of controls (p < 0.001)
`(Figure 6A). Four-week treatment of db/db mice with
`LY2189265 also resulted in a small but statistically
`significant reduction in weight (vehicle: 38.5 ± 0.9 g vs.
`LY2189265: 35.5 ± 0.9 g; p < 0.02) (Figure 6B).
`
`Discussion
`
`We describe the engineering of LY2189265, a recombi-
`nant fusion protein linking a human GLP-1 peptide ana-
`logue and a variant of a human IgG4-Fc domain, for the
`potential treatment of type 2 diabetes. Despite increased
`molecular weight, LY2189265 exhibits activities similar
`to native GLP-1. In the present study, LY2189265 bound
`to the GLP-1 receptor, dose-dependently increased cAMP
`production in vitro, augmented glucose-dependent insulin
`secretion in vitro and demonstrated glucose-regulatory
`activity in vivo.
`We engineered LY2189265 to be a potent GLP-
`1 receptor agonist. Adding a DPP-IV-protected GLP-1
`variant to the hinge region of an IgG-Fc domain resulted
`in a >95% loss of in vitro potency. Introducing optimized
`linker sequences as spacers between the GLP-1 moiety
`and the IgG-Fc hinge restored the full potency of the free
`peptide to LY2189265, presumably by allowing sufficient
`conformational freedom and distance from the carrier
`domain for receptor interaction. Retaining full activity
`of GLP-1 is critical for in vivo efficacy at doses that
`meet manufacturing and supply requirements. We also
`demonstrated that LY2189265 exerted its actions via the
`GLP-1 receptor because its insulinotropic effect on rat
`islets was completely abolished by adding the specific
`GLP-1 receptor inhibitor Ex(9–39).
`The half-life of LY2189265 was >1.5 days in rats
`and >2 days in monkeys and the molecule showed
`dramatically reduced clearance relative to unconjugated
`peptide. After one dose, we detected plasma levels
`for >6 days in rodents and >14 days in primates
`using antibodies
`specific to the intact N-terminus
`of GLP-1,
`indicating that we detected the active
`portion of GLP-1. Indeed, we demonstrated prolonged
`activity of LY2189265 in a graded glucose infusion
`experiment in rats 24 h after single SC administration,
`and in cynomolgus monkeys up to 7 days after one
`administration.
`To eliminate the potential confound of food intake
`and potential delay of gastric emptying on glucose
`in acute in vivo experiments
`and insulin levels
`in
`rodents, animals were fasted for 16–24 h before glucose
`challenge. Treatment of db/db mice with LY2189265 for
`
`Diabetes Metab Res Rev 2010; 26: 287–296.
`DOI: 10.1002/dmrr
`
`Rat
`t1/2 = 1.5 d
`
`1
`
`4
`3
`2
`Time after administration (d)
`
`5
`
`6
`
`Monkey
`t1/2 = 2 d
`
`2
`
`10
`8
`6
`4
`Time after administration (d)
`
`12
`
`14
`
`A
`
`1000
`
`100
`
`10
`
`1
`
`0
`
`Immunoreactivity (ng/mL)
`
`(mean ± SEM)
`
`B
`
`1000
`
`100
`
`10
`
`1
`
`0
`
`Immunoreactivity (ng/mL)
`
`(mean ± SEM)
`
`Figure 3. Pharmacokinetic profile and terminal half-life of
`LY2189265 in rats (A) and cynomolgus monkeys (B). LY2189265
`was administered as a single subcutaneous (SC) dose of
`0.1 mg/kg. Data are the concentrations of LY2189265 deter-
`mined from plasma samples by enzyme-linked immunosorbent
`assay (ELISA). Data are shown as mean ± standard deviation
`(n = 3/group)
`
`of LY2189265 demonstrated significant enhancement of
`glucose-elicited insulin release for at least 7 days (2-fold
`vs. vehicle; p < 0.0001) (Figure 5A and B). The mean
`serum concentration of LY2189265 was 324.7 ng/mL on
`the day after administration and remained detectable
`for 7 days after administration (Figure 5B). Levels of C-
`peptide were significantly elevated in LY2189265-treated
`monkeys on days 1, 5, and 7; no effect on glucose, GLP-1
`or glucagon levels was observed (data not shown).
`A second experiment was performed in which cynomol-
`gus monkeys received LY2189265 (1.7 nmol/kg) once
`weekly for 4 weeks before evaluation of insulinotropic
`activity with graded glucose infusion 4 days after the
`final dose. Augmented glucose-stimulated insulin release
`persisted for at least 4 days in monkeys dosed subchron-
`ically with LY2189265 (>2-fold vs. vehicle; p < 0.0002)
`(Figure 5C). The degree of insulin secretion in repeat-
`dose monkeys was comparable to that observed 5 days
`after one dose of LY2189265. Repeated dosing was also
`associated with a significantly increased C-peptide level
`and significantly reduced triglyceride level but unchanged
`glucose and glucagon levels (data not shown).
`
`Copyright  2010 John Wiley & Sons, Ltd.
`
`MPI EXHIBIT 1025 PAGE 7
`
`MPI EXHIBIT 1025 PAGE 7
`
`Apotex v. Novo - IPR2024-00631
`Petitioner Apotex Exhibit 1025-0007
`
`

`

`294
`
`W. Glaesner et al.
`
`c=J
`Vehicle (n = 18)
`I =::=1
`0.3 nmol/kg (n = 4) LY2189265
`
`filIIIIIIIIlill -~
`
`1 nmol/kg (n = 3) LY2189265
`
`3 nmol/kg (n = 7) LY2189265
`
`30 nmol/kg (n = 4) LY2189265
`
`*
`
`*
`
`*
`
`*
`
`70
`
`60
`
`50
`
`40
`
`30
`
`20
`
`10
`
`0
`
`Serum Insulin (ng/mL) (mean ± SEM)
`
`Baseline
`
`50 mg/kg/min
`
`150 mg/kg/min
`
`Glucose Infusion Rate
`
`Figure 4. LY2189265 significantly and dose-dependently increases insulin secretion in rats in response to a graded hyperglycaemic
`stimulus. A single SC dose of LY2189265 or saline was administered 24 h prior to a graded glucose infusion. Glucose infusion
`resulted in dose-dependent increases in insulin levels in all groups compared to baseline. Statistical s