EA042599B1 - COMPOUNDS - AGONISTS OF GROWTH FACTOR AND DIFFERENTIATION 15 AND METHODS OF THEIR APPLICATION - Google Patents

Description

This invention relates to biology and medicine, and in particular to compounds and compositions comprising a growth and differentiation factor 15 (GDF15) agonist compound having an extended time of action and other beneficial properties, as well as methods of using them to stimulate loss weight and for the treatment of diabetes, dyslipidemia, non-alcoholic steatohepatitis (NASH) and/or obesity. GDF15 (also known as MIC-1, NAG-1) is a cysteine (Cys, C) junction protein belonging to the transforming growth factor-beta (TGFe) superfamily. Its circulating concentration is involved in various biological functions such as cancerous depletion and metabolism (see Emmerson et al. (2017) Nat. Med. 23: 1215-1219). Mature human GDF15 (SEQ ID NO: 1) is a 112 amino acid peptide. Circulating GDF15 forms a mature protein homodimer (25 kDa) via what is reported to be a single interstrand disulfide bond between the Cys residue at position 77 of SEQ ID NO: 1 on each strand of the homodimer (corresponding to position 316 of SEQ ID NO: 2) . It is believed that the formation of a homodimer is necessary for the biological activity of GDF15 (see International Patent Application Publication No. WO 2017/147743).

Among the many known biological functions of the mature GDF15 homodimer, the regulation of energy homeostasis has attracted attention due to its potential in the treatment of obesity and type 2 diabetes (D2T). The relationship between mature GDF15 homodimer activity and energy homeostasis is based on the observation that elevated serum levels of mature GDF15 correlate with weight loss in individuals with advanced prostate cancer (Emmerson et al. (2017)).

Moreover, increasing GDF15 in mice by administration of recombinant GDF15 or by its expression and secretion from tumor xenografts has shown that GDF15 induces weight loss due to its ability to reduce food intake and increase energy expenditure. In addition, transgenic mice overexpressing GDF15 were resistant to diet-induced obesity and showed improved glucose tolerance, while GDF15 knockout mice had increased body weight and fat mass. Additionally, GDF15-stimulated weight loss has been shown to reduce inflammatory responses and increase longevity in rodents.

Glial cell-derived neurotrophic factor (GDNF) alpha-like receptor (GFRAL) has been identified as a ligand-binding receptor for GDF15. Loss of the anorexic effect of GDF15 in GFRAL-deficient mice, as well as in rats injected with anti-GFRAL neutralizing monoclonal antibodies, has shown that GFRAL is primarily responsible for the metabolic effects of GDF15 (Emmerson et al. (2017)).

The natural half-life (t _1/2 ) of a mature circulating human GDF15 homodimer agonist is relatively short (about 2-3 hours). However, International Patent Application Publication No. WO 2015/017710 generally describes the extension of t _1/2 GDF15 agonists through various conjugations, including conjugation with a crystallizing immunoglobulin (Fc) fragment. There is a need for extended acting GDF15 bioactive compounds that can be used in therapy to promote weight loss and to treat D2T, dyslipidemia, NASH and/or obesity. A therapeutically suitable compound would agonize the GFRAL receptor and provide beneficial properties. One of the useful properties would be to stimulate weight loss. Another useful property would be in vivo stability over an extended period of time in the therapeutic range to ensure a stable circulating level of the compound when dosing once a week to achieve the desired therapeutic effect. Other desirable properties also include reduced immunogenicity. Also, the desired compound could have the advantage of physical and/or chemical stability. In view of the foregoing, this invention describes biologically active GDF15 analog compounds that have an extended t _1/2 and other desirable properties. More specifically, the compounds provided herein are recombinant fusion proteins containing a hingeless monomeric (one Fc unit per GDF15) human IgG4 Fc connected via a rigid linker to the amino (N)-terminus of a GDF15 analog compound, resulting in compounds forming a covalently bonded homodimer via at least one interchain disulfide bond between the GDF15 moieties of each of the monomers. An exemplary compound, Compound 2, is a homodimer of two molecules of Compound 1 (SEQ ID NO: 2) and has surprisingly shown an increased t _1/2 time of up to 117 hours, which may provide prolonged efficacy and may allow once weekly dosing of the compound. Also, the compounds provided herein have improved physical stability. For example, compound 2 has a low 4.1% high molecular weight (HMW) soluble aggregate in the protein A uptake pool. 1 mg/ml after 4 weeks of storage at 40° C. in citrate buffer at pH 6.0.

In one embodiment, a compound is provided that contains SEQ ID NO: 2.

- 1 042599

In another embodiment, a compound is provided that includes a homodimer containing two monomers each comprising SEQ ID NO: 2, the two monomers being linked via at least one interchain disulfide bond between the Cys residue of the first monomer and the Cys residue second monomer.

In another embodiment, a pharmaceutical composition is provided that includes a compound having SEQ ID NO: 2, or a two-monomer homodimer of SEQ ID NO: 2, and one or more pharmaceutically acceptable excipients.

In another embodiment, a method is provided for promoting weight loss or for treating D2T, dyslipidemia, NASH and/or obesity in an individual, which comprises administering to the individual an effective amount of a compound having SEQ ID NO: 2, a homodimer containing two monomers of SEQ ID NO: 2, or a pharmaceutical composition containing a compound having SEQ ID NO: 2, or a homodimer containing two monomers of SEQ ID NO: 2.

In another embodiment, a compound is provided that comprises SEQ ID NO: 2 or a homodimer containing two monomers of SEQ ID NO: 2 for use in therapy. Alternatively, a compound is provided that includes SEQ ID NO: 2 or a homodimer containing two monomers of SEQ ID NO: 2 for use in promoting weight loss or for use in treating D2T, dyslipidemia, NASH and/or obesity.

In another embodiment, uses are provided for a compound comprising SEQ ID NO: 2 or a homodimer containing two monomers of SEQ ID NO: 2 in the manufacture of a medicament to promote weight loss or to treat D2T, dyslipidemia, NASH and/or obesity. .

In other embodiments, a compound is provided that is obtained by culturing a mammalian cell containing a cDNA molecule encoding a polypeptide having the amino acid sequence of SEQ ID NO: 2 under conditions under which the polypeptide is expressed and isolating the compound. Further provided is a method for inducing weight loss in an individual or for treating D2T, dyslipidemia, NASH and/or obesity, which comprises administering to the individual an effective amount of a compound provided herein or a composition provided herein once per week.

In the context of this document, about means being within a statistically significant range of a value or values, such as, for example, the declared concentration, length, molecular weight, pH, sequence identity, time frame, temperature, or volume. Such a value or range may be within an order of magnitude, typically within 20%, more typically within 10%, and even more typically within 5% of the given value or range. The tolerance covered by the term about will depend on the particular system under study and can be easily estimated by one of skill in the art.

As used herein, an effective amount refers to the amount or dose of a compound provided herein, or a pharmaceutical composition containing a compound provided herein, which, when administered in single or multiple doses to a patient or subject, will produce a biological or therapeutic response, or the desired therapeutic effect in a tissue, system, body of an animal, mammal, or human under the supervision of a researcher, veterinarian, physician, or other healthcare professional. Preferably, administering to a patient in need thereof an effective amount of a compound or compositions containing a compound of this invention will result in non-specific weight loss. The dose may include a higher initial loading dose followed by a lower dose.

As used herein, individual, patient, and subject are used interchangeably and refer to an animal, in particular a human. In the case of a human, the individual is characterized by a disease, disorder, or pathology that is ameliorated by administration of a compound or composition provided herein.

As used herein, treating or treating means managing an individual and caring for an individual having a pathology for which administration of GDF15 is recommended in order to control the symptoms, or alleviate the symptoms and complications of such pathologies. Treatment includes administering to an individual in need thereof a compound or composition comprising a compound provided herein to prevent the onset of symptoms or complications, alleviate symptoms or complications, or eliminate a disease, pathology or disorder. In particular, treatment includes administering to an individual in need thereof a compound or composition comprising a compound provided herein resulting in non-specific weight loss. The individual being treated may be an animal, in particular a human.

The pharmaceutical compositions provided herein may be parenterally administered to an individual in need of such treatment. Parenteral administration may be by subcutaneous, intramuscular or intravenous injection using a syringe, optionally a syringe pen, or a mechanical injector. Alternatively, parenteral administration may be via an infusion pump. In addition, pharmaceutical compositions can

- 2 042599 may include one or more pharmaceutically acceptable excipients. Such pharmaceutical compositions can be prepared by any of a variety of methods using conventional pharmaceutical excipients that are well known in the art (see, for example, Remington's Pharmaceutical Sciences, 21st Edition, University of the Sciences in Philadelphia, Philadelphia, PA, USA ( 2006)).

The compounds and compositions provided herein may be used in simultaneous, separate or sequential combination with one or more additional therapeutic agents that are useful in promoting weight loss and in the treatment of diabetes, D2T-related pathologies, dyslipidemia, NASH, and /or obesity. Non-limiting examples of additional therapeutic agents that can be combined with the proposed compounds include, but are not limited to: insulin or insulin analogs; biguanides; sulfonylurea; thiazolidinediones; dipeptidyl peptidase-4 (DPP-4) inhibitors; sodium dependent glucose transporter (SGLT2) inhibitors; incretin compounds such as glucagon-like peptide-1 (GLP-1) or GLP-1 analogs, gastric inhibitory polypeptide (GIP) or GIP analogs, oxyntomodulin (OXM) or oxyntomodulin analogs (OXM); or combinations of any of the above agents. The compounds provided herein and the additional therapeutic agent(s) may be administered either together using the same delivery route and vehicle such as a single pill, capsule, tablet or injectable dosage form; or introduced separately or at the same time by different means of delivery or routes; or entered sequentially. The compounds described in this document can be obtained by various methods known to the person skilled in the art. For example, compounds can be obtained by obtaining a protein or precursor protein molecule using recombinant DNA techniques. DNA, including cDNA and synthetic DNA, may be double-stranded or single-stranded. The coding sequences that encode compounds may differ as a result of redundancy or degeneracy in the genetic code. DNA can be introduced into a host cell to produce a compound or a precursor thereof. Host cells can be bacterial cells such as Escherichia coli K12 or B strains, fungal cells such as yeast cells, or mammalian cells such as Chinese hamster ovary (CHO) cells.

A suitable host cell is transiently or stably transfected or transformed with an expression system, such as expression vectors, to produce a compound as described herein or a precursor thereof. Expression vectors typically replicate in hosts either as episomes or as an integral part of the host's chromosomal DNA. Typically, expression vectors will contain selection markers, for example, for tetracycline, neomycin, glutamine synthetase, and dihydrofolate reductase, to allow selection of those cells that have been transformed with the desired DNA sequences. The compounds described in this document can be obtained using various methods known to the person skilled in the art, as well as the methods described below. The specific biosynthetic or synthetic steps for each of the described steps may be used, omitted or combined in various combinations to produce the compounds described herein. GDF15 analog compounds provided herein can act as GFRAL compounds and be produced in a mammalian cell expression system using CHOK1 cell derivatives. The genes encoding the GDF15 proteins are subcloned into expression plasmid frameworks containing glutamine synthetase (GS) (plasmids based on pEE12.4). The cDNA sequence (SEQ ID NO: 16) encoding compound 1 (SEQ ID NO: 2), exemplary Fc-GDF15 protein, is fused in frame with the coding sequence of the signal peptide sequence, METDTLLLWVLLLWVPGSTG (SEQ ID NO: 3), to enhance secretion desired product into tissue culture medium.

Expression is driven by the cytomegalovirus (CMV) viral promoter. CHOK1SV cells are stably transfected using electroporation and an appropriate amount of recombinant expression plasmid, and the transfected cells are maintained in suspension culture at an appropriate cell density. The selection of transfected cells is carried out by growing in serum-free medium containing 25 μm methionine sulfoximine (MSK), and incubated at about 35-37°C and about 5-7% CO ₂ . GDF15 proteins or Fc-GDF15 fusion proteins are secreted into the medium from CHO cells. Parts of the GDF15 proteins bind to each other, creating homodimers containing two GDF15 proteins, or Fc-GDF15 fusion protein homodimers linked to each other via an interchain disulfide bond. GDF15 or Fc-GDF15 homodimers can be purified by protein A affinity chromatography followed by anion exchange and hydrophobic chromatography.

GDF15 protein homodimers from harvested media are captured on Capto Blue (GE) resin. Fc-GDF15 protein homodimers from harvested media are captured on Mab Select Protein A (GE) resin. The resin is then washed rapidly with a running buffer such as phosphate buffered saline (PBS; pH 7.4) or running buffer containing Tris to remove non-specifically bound material. The protein is eluted from the resin with a low pH solution such as 10 mM citric acid pH 3. Fractions containing GDF15 proteins are pooled and can be kept at low pH to inactivate potential viruses. pH

- 3 042599 can be neutralized by adding a base such as 0.1 M Tris pH 8.0. The GDF15 protein can be further purified by anion exchange chromatography using resins such as Poros XQ (Life Technologies). The GDF15 protein was eluted from the AEX column using a gradient of 0 to 200 mM NaCl in 50 mM Tris, pH 8.0 over 15 column volumes. The FcGDF15 fusion protein can be further purified by hydrophobic interaction chromatography using a Capto Phenyl ImpRes hydrophobic interaction chromatography (CHB) column (GE Healthcare). The column is prepared by bringing the charged solution to about 0.5 M sodium sulfate and eluting with a 10 column volume (CV) gradient going from 0.5 to 0 M sodium sulfate in 20 mM Tris pH 8. After CHB the protein can be further purified by size exclusion chromatography by loading a concentrated pool of Capto Phenyl ImpRes onto a Superdex200 (GE Healthcare) eluting isocratically in PBS pH 7.4.

Purified GDF15 compounds can be passed through a virus-retaining filter such as Planova 20N (Asahi Kasei Medical) followed by concentration/diafiltration into 10 mM citrate, 150 mM NaCl, pH 7 using tangential flow ultrafiltration on a regenerated cellulose membrane (Millipore ).

The soluble extracellular domain (ECD) of human GFRAL is produced in a mammalian cell expression system using CHOK1 cell derivatives. The polyhistidine-tagged GFRAL EVA from the collected medium is captured on a nickel column in immobilized metal ion affinity chromatography (IMAC). The resin is then washed rapidly with a running buffer such as PBS pH 7.4 or running buffer containing Tris (pH 8.0, 500 mM NaCl) to remove non-specifically bound material. Bound HIS-tagged GFRAL EVA (SEQ ID NO: 4) was eluted using 0-250 mM imidazole in PBS or Tris (pH 8.0, 500 mM NaCl) via 10 column volumes. HIS-tagged GFRAL EVA can be further purified by size exclusion chromatography by loading a concentrated pool of IMAC onto a Superdex200 (GE Healthcare) eluting isocratically in PBS (pH 7.4).

Expression of antibodies in mammals results in glycosylation. Typically, glycosylation occurs in the Fc region of the antibody, at a highly conserved N-glycosylation site. N-glycans tend to attach to asparagine. In a typical IgG immunoglobulin constant region, there is only one site for N-glycosylation. However, this site may be modified to prevent glycosylation, or additional sites may be added for additional or different glycosylation patterns. Also, glycosylation sometimes does occur randomly in other regions of the molecules expressed in mammalian cells.

The compounds provided herein are prepared by this or a similar method, which can be readily determined by one of ordinary skill in the art.

Examples

The following non-limiting examples are offered for purposes of illustration and not limitation.

Example 1 Receptor affinity in vitro.

The in vitro binding parameters of the human GDF15 homodimer and exemplary compound 2 to the human GFRAL receptor are determined by surface plasmon resonance (SPR). The GFRAL ECD includes three Cys-rich domains (D1, D2, and D3). Affinity of human GDF15 homodimer (des-ARN SEQ ID NO: 1; i.e., GDF15 lacking Ala, Arg, and Asp at the N-terminus) and Compound 2, Fc-GDF15 Compound 1 homodimer (SEQ ID NO: 2) for HIS- the tagged human GFRAL ECP (SEQ ID NO: 4) can be measured by PPR. The kinetics are summarized in Table. 1.

To measure the binding of human GDF15 (des-ARN SEQ ID NO: 1) or Compound 2 to human GFRAL VVD, use the Biacore T200 instrument to measure the binding kinetics of human GDF15 homodimer (des-ARN SEQ ID NO: 1) and Compound 2 to GFRAL VVD. Measurements are carried out at 25°C. Samples are dissolved in HBS-EP+ pH 7.4 (GE Healthcare; 10 mM HEPES pH 7.4 + 150 mM NaCl + 3 mM EDTA + 0.05% surfactant P20). Goat anti-rabbit GDF15 (R&D) was immobilized on the flow cells of the CM5 sensor chip using amine coupling chemistry. Samples of GDF15 are prepared at a concentration of 25 μg/ml by diluting in running buffer.

Human GFRAL EVA samples are prepared at final concentrations of 1000, 500, 250, 125, 62.5, 31.3, 15.6, 7.8, 3.9, 2.0, and 0 (blank) nM by dilution in working buffer. The SPR experiment includes the following steps: (1) capture GDF15 samples on separate flow cells (Fc2, Fc3 and Fc4), (2) injection of 200 µl human GFRAL EVA into all flow cells at a rate of 100 µl/min, (3) dissociation for 600 s, (4) repair the chip surfaces with an injection of 10 µl (30 s) glycine, pH 1.5, at 20 µl/min, and (5) equilibrate the chip surfaces with an injection of 10 µl (30 s) HBS -EP+ at 20 µl/min. Each concentration of human GFRAL EVA was administered in duplicate.

Data are processed using a standard double control and fitted to a 1:1 binding model using Biacore T200 Evaluation Software to determine association rate (k _on , M ^_1 s ^_1 ), dissociation rate (kf, s ^-1 ) and maximum response (Rmax; response units (RU)). Kon

- 4 042599 equilibrium dissociation stant (K _g ) is calculated from the ratio K _d =k _on /k _off (M).

Table 1

Binding Kinetics of Human GDF15 Homodimer and Compound 2 Homodimer to Human GFRAL ECD Receptor (D1-D2-D3-ECD) at 25°C

Compound cop (1/Ms) k _O ff(l/c) CD (nM) Human GDF15 (homodimer of two compounds including des-ARN SEQ ID NO: 1) 2.92E + 05 7.21E-03 7.2 Compound 2 (homodimer of two compounds including SEQ III NO: 2) 1.05E+06 8.71Е-03 8.3

The results show that the human GDF15 homodimer (two compounds containing des-ARN SEQ ID NO: 1) had an affinity of 7.2 nM for human GFRAL EVA, and the compound 2 homodimer (two compounds containing SEQ ID NO: 2) had an affinity of 8 .3 nM for human GFRAL EVA.

Example 2 In Vitro Functional Activity GFRAL, the receptor for GDF15, is a member of the GDNF receptor family and contains a single transmembrane domain and no intracellular signaling domain. Members of the GDNF receptor family, including GFRAL, use the RET co-receptor for signal transduction. Stimulation of the GDNF/RET receptor complex is known to activate signaling networks including ERK, AKT and STAT phosphorylation.

To evaluate the potency and relative potency of mature human GDF15 variants, HEK293 (ATCC) cells are transfected with CMV-directed mammalian expression vectors containing human GFRAL (NCBI Standard Sequence No. NP_997293.2) and human RET51 (NCBI Standard Sequence No. NP_066124.1) , and grown for three weeks in the presence of geneticin and puromycin (Life Technologies) to create a pool of cells stably expressing both proteins. Measurement of ERK1/2 phosphorylation was chosen as the measured parameter for assessing RET activation using the AlphaLISA Surefire Ultra Phospho-ERK1/2 Assay Kit (Perkin Elmer), according to the manufacturer's instructions, and the EnVision Multilabel Microplate Reader Model 2102 (Perkin Elmer). Briefly, HEK293-hGFRAL/hRET cells, between passages 10-20, are plated at 20,000 cells/well in poly-D-lysine-coated 96-well plates and grown for three days at 37° C. in 5% CO ₂ / 95% O ₂ . Cells are incubated in serum-free medium for 4 hours and then treated as indicated for 10 minutes, lysed and frozen until analysis. Cell lysates are thawed by incubation for 1 hour at 4° C., mixed on an orbital plate shaker and transferred to a white 384-well plate. The AlphaLISA 2 plate/1 incubation method is used by adding the acceptor and donor mixtures supplied with the assay kit to the lysates and incubating at room temperature in the dark for 8 hours before measuring the AlphaLISA signal on the EnVision Plate Reader.

Each assay cell plate contains native mature human GDF15 as an assay control. ERK1/2 (pERK1/2) phosphorylation data from multiple plates are assembled into one plot by converting the raw AlphaLISA Y-axis signal data to %Maxima relative to the human GDF15 reference standard using the following formula:

%Maxima=100*(x-min)/(max-min) where min=0 and represents no stimulation, max=100 and represents the stimulation seen with a loading dose of the compound, and x=any given treatment. EC ₅₀ for each compound is determined using a sigmoidal dose-response curve with a variable slope with four-parameter logistic regression in GraphPad Prism version 7.00. EC ₅₀ specified for each connection in the table. 2 is determined by calculating the geometric mean EC ₅₀ for 3 independent analyzes of each compound using the following formula:

HEK293 cells expressing both human GFRAL and human RET51 were used to evaluate the potency of compound 2. Native mature human GDF15 (SEQ ID NO: 1) as a homodimer was used as a control for each cell plate. The test samples are analyzed in quadruplicate in three independent experiments.

- 5 042599

table 2

Efficacy Comparison of Compound 2 with Human GDF15

Test sample EU ₅ about (pM) cos (standard error of the mean) N Mature GDF 15 man (homodimer of two compounds including SEQ III NO: 1) 27.9 2.9 3 Compound 2 (homodimer of two compounds including 86.2 5.8 3 SEQ ID NO: 2)

Data from a representative experiment are shown as mean ± standard error. N is the number of independent experiments.

Example 3 In vivo efficacy in conventional rat models.

Obesity is a common comorbidity associated with diabetes and insulin resistance. GDF15 has been reported to reduce food intake, induce weight loss, and improve glucose homeostasis. Therefore, the GDF15 compound may be effective in therapy to promote weight loss and pathologies such as D2T, dyslipidemia, NASH and/or obesity. Compound 2 was administered to male Sprague Dawley rats for 15 days with daily measurements of body weight and food intake.

Conventional male Sprague-Dawley rats (Envigo; 14 weeks of age) are maintained on their normal diet upon arrival at the facility (TD2014; Teklad, Madison, Wisconsin) and used in further studies. Animals were housed separately in a temperature-controlled room (24°C) with a 12-hour light/dark cycle (lights on at 06:00) and with free access to food (TD2014) (Teklad) and water.

After at least one week of indoor adaptation, the rats were randomized according to their body weight, so that each experimental group of animals had a similar body weight. Body weight ranged from 332 to 369 grams.

All groups contain five rats. Vehicle or compound 2 (dose range 0.01 to 0.3 mg/g) dissolved in vehicle (PBS) administered by subcutaneous (s/c) injection (1 ml/kg) every 3 days for 15 days ordinary rats that are fed at their discretion.

SC injections are given on days 1, 4, 7, 10 and 13. Daily measurements of body weight and food intake are made throughout the study period.

The percentage body weight is calculated daily from the starting body weight of each animal as follows:

% Body weight = (daily body weight / initial body weight) x100.

All data are presented as mean±SOS for five animals per group. Effective dose concentrations for percent weight loss and cumulative food intake are determined in GraphPad Prism using a non-linear fitting tool. Statistical analysis is performed using repeated ANOVA evaluations, followed by Dunnett's test for multiple comparisons. A statistically significant difference is determined at *-p<0.05, ** - p<0.01, *** - p<0.001, **** - p<0.0001.

Table 3

Effect of compound 2 treatment on percentage body weight and cumulative food intake in conventional DM rats (Sprague-Dawley)

Treatment Body mass (%) Cumulative food intake (g) Carrier (1 ml/kg, s.c.) 106.69±0.54 264.48±7.28 Compound 2 (0.01 mg/kg) 93.34±2.13* 193.04±8.54* Compound 2 (0.03 mg/kg) 85.18±2.60* 155.42±11.13* Compound 2 (0.06 mg/kg) 81.80±1.66* 122.54±14.05* Compound 2 (0.1 mg/kg) 78.30±3.71* 115.74±15.75* Compound 2 (0.3 mg/kg) 73.57±1.56* 91.22±8.57*

All data are presented as mean±SOS of 5 animals/group from day 14. Statistical analysis was performed using repeated estimates of ANOVA followed by Dunnett's test for multiple comparisons. Statistically significant differences were found at *p<0.05.

Compound 2 systemic treatment in normal male DM rats resulted in a statistically significant decrease in food intake and a statistically significant decrease in percent body weight at the end of the study compared to dose-dependent vehicle treated animals. Cumulative food intake decreases from 264.48 to 91.22 at maximum test

- 6 042599 dose, and the decrease in body weight ranges from 7 to 26%. The data appear to indicate that Compound 2 has a statistically significant effect on weight loss and food intake in normal DM rats in a dose-dependent manner.

Example 4 In vivo efficacy in alimentary obese (AO) mice.

To investigate the effect of Compound 2 on weight loss, metabolism, body composition, and hepatic steatosis, Compound 2 was systematically administered to C57/B16 AO mice. Male C57/B16 AO mice (Taconic) aged 24 to 25 weeks are maintained on a high energy diet (TD95217; Teklad, Madison, Wisconsin) upon arrival at the facility and used in further studies. Animals are housed separately in a temperature-controlled room (24° C.) with a 12-hour light/dark cycle (lights on at 22:00) and with free access to food (TD95217) (Teklad) and water. After a minimum of 2 weeks of house acclimatization, mice are randomized according to their body weight so that each experimental group of animals has a similar body weight. Body weight ranges from 40 to 50 g. All groups contain five mice. Vehicle or Compound 2 (0.002, 0.006, 0.02, 0.06 and 0.2 mg/kg), dissolved in vehicle (PBS) and administered by subcutaneous (s/c) injection (10 ml/kg) to AO mice who are fed ad libitum every 3 days for 21 days 30-90 minutes before the start of the dark cycle. Sc injections are given on days 1, 4, 7, 10, 13, 16, 19 and 22. Body weight and food intake are measured daily throughout the study.

Absolute changes in body weight are calculated by subtracting the body weight of the same animal before the first injection of the molecule. On day 17, animals are not fed at 06:00 and at 13:00, blood is collected to measure fasting blood glucose and insulin levels. Blood glucose levels are measured with AccuChek® glucometers (Roche Diabetes Care, Inc.; Indianapolis, IN). Insulin is measured by ELISA (MSD, Rockville, MD). The insulin resistance index is calculated using the formula: HOMA IR = fasting plasma insulin (mIU/l) * (fasting blood glucose (mmol/l)/22.5. On day 24, animals are sacrificed before the onset of the dark photoperiod and the liver is removed and frozen. Triglyceride levels determined in liver homogenates collected at sacrifice and the test substance in plasma are measured on a Hitachi Modular P clinical analyzer.

Table 4

Effect of Compound 2 Treatment on Body Weight and Cumulative Food Intake in AO Mice at Day 16

Treatment Change in body weight from initial measurement (g) Cumulative food intake (g) Carrier (10 ml/kg, s.c.) -0.68±0.14 41.30±0.94 Compound 2 (0.002 mg/kg) -4.58±0.71* 33.22±0.99* Compound 2 (0.006 mg/kg) -8.64±0.41* 29.24±1.41* Compound 2 (0.02 mg/kg) -11.82±0.57* 23.00±1.86* Compound 2 (0.06 mg/kg) -9.02±0.57* 23.86±0.96* Compound 2 (0.2 mg/kg) -9.08±0.59* 27.52±1.30*

All data are presented as mean±SOS of 5 animals/group.

Values are presented as mean±SOS with n=5. *p<0.05 compared to the vehicle group; one-way ANOVA followed by Dunnett's test for multiple comparisons.

Table 5

Effect of Compound 2 Treatment on Serum Glucose, Cholesterol, Alanine Aminotransferase (ALT), and Liver Triglycerides in AO Mice at Day 24

Treatment Glucose (mg/dl) Cholesterol (mg/dl) ALT (IU/L) Liver triglycerides (mg/g tissue) Carrier (10 ml/kg, s.c.) 339.18±30.58 271.80±12.02 310.80±34.92 210.10±11.82 Compound 2 (0.002 mg/kg) 317.52±17.95 220.80±4.89* 231.60±25.39 69.88±18.88* Compound 2 (0.006 mg/kg) 261.00±20.43* 177.00±7.82* 159.60±73.32* 46.56±13.85* Compound 2 (0.02 mg/kg) 223.38±16.02* 172.80±18.51* 87.00±13.04* 24.82±8.80* Compound 2 (0.06 mg/kg) 245.94±10.10* 199.20±15.34* 114.60±22.72* 58.90±14.45* Compound 2 (0.2 mg/kg) 241.02±8.88* 167.40±8.72* 112.80±19.15* 34.62±10.88*

Values are presented as mean±SOS with n=5. *p<0.05 compared to the vehicle group; one-way analysis of variance, followed by Dunnett's test.

- 7 042599

Table 6

Effect of compound 2 treatment on glucose, fasting insulin, and

HOMA-IR, in AO mice on day 17

Treatment Fasting glucose (mmol/l) Fasting insulin (mIU/l/ml) HOMA-IR Carrier (10 ml/kg, s.c.) 6.80±0.28 85.64±13.68 25.78±4.13 Compound 2 (0.002 mg/kg) 6.64±0.28 45.94±4.66* 13.51±1.32* Compound 2 (0.06 mg/kg) 5.16±0.53* 26.44±2.95* 6.22±1.15*

All data are presented as mean±SOS of 5 animals/group from day 17. Statistical analysis was performed using repeated estimates of ANOVA followed by Dunnett's test for multiple comparisons. Statistically significant differences were found at p<0.05.

Compound 2 dose-dependently reduces body weight and food intake in male AO mice, indicating that it can be used in therapy to promote weight loss and obesity (Table 4). Compound 2 also lowers serum glucose, indicating that it may be useful in the treatment of diabetes (Table 5). Compound 2 further lowers cholesterol, indicating that it can be used in the treatment of dyslipidemia (Table 5). Compound 2 improves liver health (demonstrated by a decrease in serum ALT levels) and lowers hepatic triglycerides, indicating that it may be useful in NASH therapy (Table 5). The study of fasting blood glucose and insulin levels performed on day 17 demonstrates increased insulin sensitivity and a reduced insulin resistance index, further supporting the use of compound 2 in the treatment of diabetes (Table 6).

Example 5 Stability of various Fc-GDF15 constructs.

Protein aggregation in a pharmaceutical product is undesirable because it reduces the overall yield and may elicit an immune response when administered to individuals. Thus, maintaining the compound in a monomeric state, or in this case as a covalently bonded homodimer, is preferred. To test the propensity of various GDF15 fusion proteins to aggregate and/or degrade, various Fc-GDF15 fusion proteins can be expressed in mammalian CHO cells. Fc-GDF15 fusion proteins can be captured by Mab Select Protein A (GE; Piscataway, NJ) with PBS running buffer (pH 7.4); can be washed quickly with running buffer to remove non-specifically bound material; and can be eluted with 20 mM acetic acid and 5 mM citric acid, pH 3. Fractions containing Fc-GDF15 proteins are pooled and the pH is neutralized by adding 1/10 volume of 1 M Tris pH 8.0.

The protein A-entrapped pool is then analyzed by size exclusion chromatography (SEC) to determine the % high molecular weight aggregate (HMW) indicating the degree of aggregation that has occurred. The SEC separation method is performed on a Tosoh Bioscience 3000SWXL, 5 µm column with dimensions of 30 cm x 0.78 cm. The mobile phase is PSB x 1, 350 mM NaCl (pH 7.4) at a flow rate of 0.5 ml/min. Samples with an initial low concentration are administered as injections of 10 μl and monitored at an absorption wavelength of 214 nm. The % HMM of the components indicates the chemical stability, or low molecular weight degradation products of the compound.

Table 7

Comparison of physical and chemical stability of various Fc-GDF15 fusion proteins

Compound % WMM % nmm Compound 2 (including monomeric Fc) 4.1 2.3 Compound 3 (including tandem Fc) 60.8 3.6 Compound 4 (including heterodimeric Fc) 2.0 49.1

Fc-GDF15 fusion protein tandem construct (two identical Fc antibody moieties end-to-end, including the hinge region, and then attached to a GDF15 molecule, where the GDF15 moieties then self-assemble to create a dimer), Compound 3 (homodimer of two molecules , each containing SEQ ID NO: 5) has a MMW of 60.8% compared to 4.1% MMW of compound 2. This suggests that compound 2 has better physical stability, less aggregation, and less risk of immunogenic aggregates than the tandem fusion protein construct Fc-GDF15.

Heterodimeric Fc-GDF15 (two different Fc portions of an antibody that are linked to each other side by side, and where one Fc portion (strand B, SEQ ID NO: 17) is linked to another Fc portion that is attached to a GDF15 molecule (strand A, SEQ ID NO: 6) and where the GDF15 portions then self-assemble to form

- 8 042599 homodimer), compound 4 (homodimer of two molecules, each molecule contains a heterodimer of SEQ ID NO: 6 and SEQ ID NO: 17) has a high 49.1% HMM components compared to compound 2, which has only 2.3% HMM, suggesting that compound 2 has better chemical stability and a lower propensity for chemical or proteolytic degradation. Chemical stability and failure prevention help maintain the effectiveness of the compound for extended periods of time. In addition, less degradation minimizes the risk that degradation by-products cause additional performance problems due to interfering interaction with the active ingredient in the dosage form, or that degradation by-products will aggregate and cause immunogenicity problems.

Example 6 Comparison of Fc Stability with Double and Single Mutation.

The unique structure of the Fc portion of the compounds provided herein with two specific mutations surprisingly demonstrates better stability and higher solubility at high concentrations than an Fc construct with only one of any mutation. Get two Fc with a single mutation to disrupt the combination of CH3/CH3 in IgG4-Fc and to create a monomeric Fc construct. The first single mutant is compound 7 in which glutamine is replaced by phenylalanine at position 405 in the full-length human IgG4 heavy chain corresponding to position 173 in SEQ ID NO: 2 (position 175 in SEQ ID NO: 9). The second single mutant generated is compound 8, Fc with glutamic acid substituted with tyrosine at position 407 in the full-length human IgG4 heavy chain corresponding to position 175 in SEQ ID NO: 2 (position 177 in SEQ ID NO: 10). An Fc double mutant, Compound 5 (SEQ ID NO: 7), was also generated and contains both Fc mutations from Compounds 7 and 8. Compound 5 has the same sequence as Compound 2, except that two additional amino acids, AP are present at the N-terminus of the Fc region in compound 5. Compounds 2, 5, 7 and 8 lack the ESKYGPPCPPCP hinge region (SEQ ID NO: 8) to allow monomeric IgG4 Fc formation.

Compounds in this example are expressed in mammalian CHO cells and captured from harvested medium with Mab Select (GE) protein A with PBS working buffer (pH 7.4); rinse quickly with running buffer to remove non-specifically bound material; and eluted with 20 mM acetic acid and 5 mM citric acid, pH 3.0. Fractions containing GDF15 proteins are pooled and the pH is neutralized by adding 1/10 volume of 1 M Tris pH 8.0.

The neutralized, protein A-entrapped pools are then buffered in 10 mM citrate, pH 5.0, in a 0.5 ml Amicon Ultra centrifuge concentrator with 10 MWCO. Fc-GDF15 single and double mutants are then attempted to be concentrated up to or above 10 mg/ml using a 0.5 ml Amicon Ultra centrifuge concentrator with 10 MWCO.

Table 8

Maximum solubility of fused GDF15 at pH 5.0

Compound Maximum solubility at pH 5.0 (mg/ml) visual observations Compound 7 homodimer (SEQ W NO: 9) 1.7 Unable to reach 10 mg/mL; precipitate observed during concentration Compound 8 homodimer (SEQIDNO: 10) 6.7 Unable to reach 10 mg/mL, precipitate noted during concentration Compound 5 homodimer (SEQ W NO: 7) 9.8 Clear solution Control - only monomeric Fc IgG4 12.0 Clear solution

The results show that single Fc mutants, Compound 7 and Compound 8 homodimers, have reduced solubility compared to the Fc double mutant, Compound 5 homodimer. Compound 7 homodimer failed to reach 10 mg/mL due to precipitation and loss of material. Compound 8 homodimer showed improved solubility compared to Compound 7 homodimer; however, it was not possible to reach 10 mg/ml due to loss of material during the concentration process. Compound 5 homodimer with the new Fc double mutation retained a solubility of 9.8 mg/mL. The control, monomeric IgG4 Fc alone, was soluble up to 12 mg/ml.

Example 7 Comparison of chemical stability for flexible or rigid linkers.

To test the chemical stability of the GDF15 compounds provided herein, such compounds can be dialyzed overnight at 4°C into the following buffers: 10 mM citrate, pH 6 (C6), 10 mM citrate, pH 7 (C7), 10 mM Tris , pH 7.0 (T7), 10 mM Tris, pH 8.0 (T8) or 1xPBS, pH 7.4 (PBS). To evaluate the long-term stability of Fc-GDF15 fusions, 1.0 mg/mL protein solutions in appropriate buffers can be stored at 4, 25 and 40°C for 4 weeks, and % LMW determined by analytical SEC.

- 9 042599

The EC separation method can be performed on a Tosoh Bioscience 3000SWXL, 5 µm 30 cm x 0.78 cm column. The mobile phase can be PBS, 350 mM NaCl, pH 7.4, at a flow rate of 0.5 ml/min. Samples with an initial low concentration can be administered as injections of 10 μl and monitored at an absorption wavelength of 214 nm.

Changes in chemical stability, as measured by % LMW, are evaluated relative to time point samples 0. As determined by studies conducted essentially as described above, the increase in % LMW for GDF15 compounds is summarized in Table 9.

Test compounds are Compound 2, Compound 1 homodimer (SEQ ID NO: 2), with G4-AP10-G4 rigid linker, Compound 5 homodimer (SEQ ID NO: 7) with L5Q flexible linker (GGGGQGGGGQGGGGQGGGGQGGGGQ (SEQ ID NO: 12) ) and compound 6 homodimer (SEQ ID NO: 11) with a G4S-AP10-G4S rigid linker.

Table 9

Growth in % LMW for GDF15 compounds at 1 mg/mL as determined by analytical SEC

Growth in % LMW at 1 mg/ml after 4 weeks of storage at 4°C, 25°C and 40°C Citrate pH 6.0 Citrate pH 7.0 or Tris pH 7.0 Tris pH 8.0 or FSB pH 7.4 4°C 25°C 40°C 4°C 25°C 40°C 4°C 25°C 40°C Compound 2 Linker: GGGGAP10-GGGG 0 0 0.75 0 0.36 3.45 0.15 0.99 7.09 Homodimer Connections 5 Linker: L5Q 0.37 0.81 3.99 0.56 1.8 6.63 0.71 2.8 10.5 Homodimer Compounds 6 Linker: GGGGS-AP10GGGGS 1.65 0.76 2.7 1.52 1.2 5.76 1.83 1.52 5.65

The L5Q linker has a higher % LMW after storage at 25 and 40°C than either of the AP10 linkers, suggesting a higher propensity for chemical degradation of GDF15 fusion proteins containing the flexible L5Q linker compared to the protein containing the rigid AP10 linker ( ARARARARARARARARAR, SEQ ID NO: 13). Also, the homodimer of compound 6 with a linker containing G4S (GGGGS; SEQ ID NO: 14) has a higher % HMW than compound 2 with a linker containing G4 (GGGG; SEQ ID NO: 15) under most conditions, which indicates greater chemical stability of compound 2 with its G4-AP10-G4 linker. The chemical and physical stability of GDF15 compounds with rigid (compound 2 and compound 6) and flexible linkers (compound 5) can be assessed at a high concentration of 10 mg/ml. Compounds may first be dialyzed O/N at 4° C. into the following buffers: (a) 10 mM citrate, pH 6.5; (b) 10 mM citrate, pH 6.5, 150 mM NaCl; and (c) 10 mM citrate, pH 6.5, 0.02% polysorbate 80. Excipients such as 150 mM NaCl and 0.02% polysorbate are added to the formulation to test their effect on the chemical and physical stability of the GDF15 hybrids. Compounds can then be concentrated to 10 mg/mL using Millipore Spin 10,000 MWCO, 15 mL concentrators. After concentration, % HMW and % LMW can be determined by SEC using concentrated protein (t=0). The EC separation method can be performed on a 5 μm column with dimensions of 30 cm x 0.78 cm, Tosoh Bioscience 3000SWXL. The mobile phase was PBS, 350 mM NaCl (pH 7.4) at a flow rate of 0.5 ml/min. Samples of 10 mg/ml are diluted to 1 mg/ml in appropriate buffers and then injected at 10 µl and monitored at an absorbance wavelength of 214 nm.

Formulations of 10 mg/ml can be incubated for 4 weeks at 4, 25 and 40°C to assess long-term stability under stress conditions. % LMW and % HMW can be determined again after 4 weeks (t=4 weeks) using SEC. Changes in chemical stability (% NMW) can be assessed against time point 0 samples.

Table 10

Chemical stability reported as % LMW growth for GDF15 compounds at 10 mg/mL

Growth in % LMW at 10 mg/ml after 4 weeks of storage at 4°C, 25°C and 40°C Citrate pH 6.5 Citrate pH 6.5 + 150 MMNaCl Citrate pH 6.5 + 0.02% Polysorbate 80

- 10 042599

4°C 25°C 40°C 4°C 25°C 40°C 4°C 25°C 40°C Homodimer Connections 5 Linker: L5Q 0.52 1.03 6.39 0.25 0.86 5.11 0.27 1.04 6.2 Homodimer Compounds 6 Linker: GGGGS-AP10GGGGS 0 0.2 2.73 0 oh, s 2.52 0 0.31 2.8

Table 11

Physical stability reported as % HMW growth for GDF15 compounds at 10 mg/mL

Growth in % HMW at 10 mg/ml after 4 weeks of storage at 4, 25 and 40°C Citrate pH 6.5 Citrate pH 6.5 + 150mMKaS1 Citrate pH 6.5 + 0.02% polysorbate 80 4°C 25°C 40°C 4°C 25°C 40°C 4°C 25°C 40°C Homodimer Compounds 5 Linker: L5Q 0.06 0.23 3.04 0.05 0.19 7.3 0.1 0.24 3.13 Homodimer Compounds 6 Linker: GGGGSAP10-GGGGS 0 0.1 2.98 0 0.33 7.18 0 0 2.85

As shown in Table. 10, the compound 5 homodimer at 10 mg/mL containing the flexible linker showed a higher % LMW level than the compound 6 homodimer with the rigid linker at 25°C and 40°C, indicating lower chemical stability.

As shown in Table. 11, the compound 5 homodimer at 10 mg/mL containing the flexible linker showed a higher % HMW than the rigid linker compound 6 homodimer, indicating lower physical stability. Poor chemical and/or physical stability may cause precipitation and possible immunogenicity problems.

Example 8 Pharmacokinetics of GDF15 compounds.

The pharmacokinetics of Compound 2 was studied in monkeys. Plasma GDF15 compound concentrations were determined by ELISA at Eli Lilly and Company (Indianapolis, Ind.). Sandwich ELISA uses polyclonal goat anti-human GDF15 (R&D Systems/AF957) or monoclonal mouse anti-human/primate GDF15 (R&D Systems/MAB957) coated at 1 or 3 µg/mL as the capture reagent. The GDF15 compound is then detected with mouse anti-human IgG4 pFc-HRP (Southern Biotech/9190-05) or mouse anti-human IgG Fc-HRP (Southern Biotech/9200-05). The plates are developed using a tetramethylbenzidine (TMB) substrate system (SeraCare/5120) and the enzymatic reaction is quenched with TMB Stop Solution (KPL/50-85-04). Male cynomolgus monkeys are given a single subcutaneous dose (0.3 mg/kg) of compound 2 in PBS (pH 7.4) in a volume of 0.5 ml/kg. Blood is collected from each animal before dosing and 6, 24, 48, 72, 96, 168, 240, 336, 408, and 504 hours post-dose for pharmacokinetic characterization.

Table 12

Individual and mean pharmacokinetic parameters after administration of a single subcutaneous dose of 0.3 mg/kg to male cynomolgus monkeys

Compound (Dose) Animal tl/2 (h) Tmax (h) Stax (mcg/ml) AUCo-inf (h*mcg/ml) CL/F (ml/h/kg) Compound 1 118 48 1.96 206 1.45 2 2 116 48 2.44 184 1.63 (0.3 mg/kg) Average 117 48 2.20 195 1.54

Abbreviations: AUC ₀ . _inf = area under the curve from time point 0 h to infinity, CL/F = clearance/bioavailability, T _max = time to maximum concentration, C _max = maximum observed plasma concentration, t _1/2 = half-life.

The data in the table. 12 show that t _1/2 in macaques is 117 hours. This is an unexpectedly long t _1/2 for a monomeric Fc molecule. Also, the pharmacokinetic profile in macaques is stable over a long period of time, suggesting that the pharmacokinetic profile of Compound 2 in humans may also be clearly stable over a long period of time within the therapeutic range, allowing stable circulating drug levels upon dosing. once a week.

- 11 042599

Sequences

Reference is made to the following nucleic and/or amino acid sequences in the disclosure of this invention and is provided below for reference.

SEQ W NO: 1 - Mature human GDF15

ARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRAANM HAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI

SEQ III NO: 2 - Fusion protein

EAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTK PREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVY TLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFQLESR LTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGAPAPAPAPAPAPAPAPA PAPGGGGGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFRA ANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI

SEQ W NO: 3 - Leader sequence

METDTLLLWVLLLWVPGSTG

SEQ W NO: 4 - Artificial sequence

QTNNCTYLREQCLRDANGCKHAWRVMEDACNDSDPGDPCKMRNSSYCNLSIQYLVES NFQFKECLCTDDFYCTVNKLLGKKCINKSDNVKEDKFKWNLTTRSHHGFKGMWSCLE VAEACVGDVVCNAQLASYLKACSANGNPCDLKQCQAAIRFFYQNIPFNIAQMLAFCDC AQSDIPCQQSKEALHSKTCAVNMVPPPTCLSVIRSCQNDELCRRHYRTFQSKCWQRVTR KCHEDENCISTLSKQDLTCSGSDDCKAAYIDILGTVLQVQCTCRTITQSEESLCKIFQHML HRKSCFNYPTLSNVKGMALYTRKHANKHHHHHH

- 12 042599

SEQ ID NO: 5 - Artificial sequence

ERKSSVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYV DGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTIS KTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP MLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGG GGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSERKSSVECPPCPAPPVAGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVV SVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGGGGSGGGGASARNGDHCPLGPGRCCRLHTVRA SLEDLGWADWVLSPREVQVTMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVP ASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI

SEQ ID NO: 6 - Artificial sequence

DKTHTCPPCPAPALGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGG GGSGGGGSGGGGSGGGGSNGTHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQV TMCIGACPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQT YDDLLAKDCHCI

SEQ ID NO: 7 - Artificial sequence

APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAK TKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQ VYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFQL ESRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGQGGGGQGGGGQGG GGQGGGGQGGGGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACP SQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAK DCHCI

- 13 042599

SEQ ID NO: 8 - Fc fragment

ESKYGPPCPPCP

SEQ ID NO: 9 - Artificial sequence

APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAK TKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQ VYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFQL YSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGQGGGGQGGGGQG GGGQGGGGQGGGGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGA CPSQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLA KDCHCI

SEQ ID NO: 10 - Artificial sequence

APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAK TKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQ VYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL ESRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGQGGGGQGGGGQGG GGQGGGGQGGGGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACP SQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAK DCHCI

SEQ ID NO: 11 - Artificial sequence

APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAK TKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQ VYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFQL ESRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGGGGGSAPAPAPAP АРАРА PAPAPAPGGGGSGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACP SQFRAANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAK DCHCI

SEQ ID NO: 12 - Artificial sequence GGGGQGGGGQGGGGQGGGGQGGGGQ

-

Claims (21)

SEQ ID NO: 13 - Artificial sequence AR AR AR AR AR AR AR AR AR AR SEQ W NO: 14 - Artificial sequence GGGGS SEQ W NO: 15 - Artificial sequence GGGG SEQ Ш NO: 16 - Искусственная последовательность gaggccgccgggggaccatcagtcttcctgttccccccaaaacccaaggacactctcatgatctcccggacccctgaggtcacgtgcgtg gtggtggacgtgagccaggaagaccccgaggtccagttcaactggtacgtggatggcgtggaggtgcataatgccaagacaaagccgc gggaggagcagttcaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaacggcaaggagtacaagtgc aaggtctccaacaaaggcctcccgtcctccatcgagaaaaccatctccaaagccaaagggcagccccgagagccacaggtgtacaccct gcccccatcccaggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctaccccagcgacatcgccgtggagt gggaaagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttccagctcgagagcaggct aaccgtggacaagagcaggtggcaggaggggaatgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacacagaagag cctctccctgtctctgggtggcggtggtggagctccagccccagctcctgctcctgcaccagcacctgcccccgctccagcacccgcacct ggaggagggggcggtgaccactgtcccctggggcctggccggtgttgcagactgcatacagtccgggcctccctggaagacctgggttg ggcagattgggtgctctcacctcgcgaggttcaggtgaccatgtgcattggggcttgtccctcacaatttcgcgcagctaacatgcacgccc aaatcaaaacctccctgcaccggcttaaaccggatactgttcctgctccctgctgcgtgccagcatcctacaaccccatggtgctgatccaga agacggatacaggggtgtcactgcagacctatgatgacctcctggccaaagattgccactgtatc SEQ ID NO: 17 - Artificial sequence DKTHTCPPCPAPALAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDDNCSHFLVSKLTVDKHEKW CLAIM 1. Compound containing the sequence of SEQ ID NO: 2. 2. Compound consisting of the sequence SEQ ID NO: 2. 3. A homodimer containing two compounds according to claim 1 or 2, characterized in that the compounds are linked by at least one interchain disulfide bond between a cysteine residue of the first compound and a cysteine residue of the second compound. 4. A pharmaceutical composition containing a compound according to claims 1 to 3 and one or more pharmaceutically acceptable excipients. 5. A method of inducing weight loss in a patient, comprising administering to a patient in need thereof an effective amount of the compounds of claims 1 to 3 or the composition of claim 4. 6. A method of treating type 2 diabetes in a patient, comprising administering to a patient in need thereof an effective amount of the compounds of claims 1 to 3 or a composition of claim 4. 7. A method of treating obesity in a patient, comprising administering to a patient in need thereof an effective amount of the compounds of claims 1 to 3 or the composition of claim 4. 8. A method of treating NASH in a patient, comprising administering to a patient in need thereof an effective amount of the compounds of claims 1 to 3 or the composition of claim 4. 9. A method of treating dyslipidemia in a patient, comprising administering to a patient in need thereof an effective amount of the compounds of claims 1 to 3 or the composition of claim 4. 10. Application of connection according to pp.1-3 to promote weight loss. 11. Application of connection according to pp.1-3 for the treatment of type 2 diabetes. 12. Application of connection according to pp.1-3 for the treatment of obesity. 13. Application of connection according to pp.1-3 for the treatment of dyslipidemia. 14. Application of connection according to pp.1-3 for the treatment of NASH. 15. Application of connection according to pp.1-3 in the manufacture of a stimulant drug - 15 042599 weight loss. 16. Use of a compound according to claims 1 to 3 in the manufacture of a medicament for the treatment of type 2 diabetes. 17. Use of a compound according to claims 1 to 3 in the manufacture of a medicament for the treatment of obesity. 18. Use of a compound according to claims 1 to 3 in the manufacture of a medicament for the treatment of dyslipidemia. 19. Use of a compound according to claims 1 to 3 in the manufacture of a medicament for the treatment of NASH. 20. A compound comprising the sequence of SEQ ID NO: 2, wherein the compound is obtained by culturing a mammalian cell containing a cDNA molecule encoding a polypeptide containing the amino acid sequence of SEQ ID NO: 2 under conditions under which the polypeptide is expressed and isolating the compound. 21. A method for promoting weight loss in a patient, treating type 2 diabetes, obesity, NASH and/or dyslipidemia, comprising administering to a patient in need thereof an effective amount of the compounds of claims 1 to 3 or the composition of claim 4 once a week. Eurasian Patent Organization, EAPO Russia, 109012, Moscow, Maly Cherkassky per., 2