Glucagon-like peptide 1 (GLP1)-growth differentiation factor 15 (GDF15) fusion protein and application thereof

Abstract

The invention relates to a GLP1-GDF15 fusion protein and an application thereof. Provided herein are GLP1-GDF15 fusion proteins comprising a GLP1 or GLP1 variant peptide, a first linker peptide, a serum albumin, a second linker peptide, and a GDF15 protein.

Description

[0001] This application is a divisional application of the invention patent with application date of October 22, 2019, application number 201980085401.2, and invention name “Glucagon-like peptide 1 (GLP1)-growth differentiation factor 15 (GDF15) fusion protein and its use”. Technical Field

[0002] The present invention as a whole relates to novel glucagon-like peptide 1 (GLP1)-growth differentiation factor 15 (GDF15) fusion proteins. GLP1-GDF15 fusion proteins regulate GLP1R and / or GDF15R. The present invention also relates to pharmaceutical compositions and methods of use thereof. The novel GLP1-GDF15 fusion proteins can be used to prevent, treat or ameliorate diseases or disorders such as obesity, type 2 diabetes, metabolic syndrome, insulin resistance and dyslipidemia, etc.

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This application claims priority to U.S. Provisional Application No. 62 / 784,603, filed on October 22, 2018, the disclosure of which is incorporated herein by reference in its entirety. Background Art

[0005] GDF15, a member of the TGFβ family, is a secreted protein that circulates in plasma as a 25 kDa homodimer and initiates its biological functions by interacting with the receptor GFRAL expressed in the brainstem (Mullican et al., Nat Med., Vol. 23, pp. 1150-1157 (2017); Yang et al., Nat Med., Vol. 23, pp. 1158-1166 (2017); Hsu et al., Nature, Vol. 550, pp. 255-259 (2017), Emmerson et al., Nat Med, Vol. 23, pp. 1215-1219 (2017)). In most individuals, the plasma level of GDF15 is between 150 pg / mL and 1150 pg / mL (Tsai et al., J Cachexia Sarcopenia Muscle, Vol. 3, pp. 239-243 (2012)). Elevated plasma levels of GDF15 are associated with weight loss due to anorexia and cachexia caused by cancer, renal failure, and heart failure. In addition, GDF15 is increased in patients who experience weight loss after Roux-en-Y gastric bypass (RYGB) (Vila et al., Clin Chem, Vol. 57, pp. 309-316 (2011)).

[0006] The correlation between weight loss and GDF15 is conserved in rodents. Overexpression of GDF15 leads to reduced food intake, weight loss, and protects mice from obesity, hepatic steatosis, and glucose intolerance when fed a high-fat diet (Baek et al., Gastroenterology, Vol. 131, pp. 1553-1560 (2006); Johnen et al., Nat Med, Vol. 13, pp. 1333-1340 (2007); Chrysovergis et al., Int JObesity, Vol. 38, pp. 1555-1564 (2014); Macia et al., PloS One, Vol. 7, p. e34868 (2012); Jones et al., Cell Reports, Vol. 22, pp. 1522-1530 (2018); Xiong et al., Sci Trans Med, Vol. 9, p. 412 (2017)). Xenografts of prostate tumor cells transfected with GDF15 also resulted in reduced food intake and weight loss (Johnen et al., Nat Med, Vol. 13, pp. 1333-1340 (2007)). In contrast, many researchers have reported that mice lacking GDF15 gain more weight and more fat mass than wild-type animals (Strelau et al., J Neurosci, Vol. 29, pp. 13640-13648 (2009); Casanovas et al., Haematologica, Vol. 98, pp. 444-447 (2013); Bonaterra et al., J Amer Heart Assoc, Vol. 1, p. e002550 (2012); Tsai et al., PloS one, Vol. 8, p. e55174 (2013)).

[0007] It has been demonstrated in mice, rats, and monkeys that pharmacological administration of GDF15 may reduce energy intake and thereby cause weight loss. Lean mice treated with recombinant GDF15 eat less and lose weight (Johnen et al., Nat Med, Vol. 13, pp. 1333-1340 (2007); Hsu et al., Nature, Vol. 550, pp. 255-259 (2017); Mullican et al., Nat Med, Vol. 23, pp. 1150-1157 (2017); Tsai et al., Int J Obesity, Vol. 42, pp. 561-571 (2018)). Following administration of GDF15, decreased food intake and weight loss were also observed in genetic and diet-induced rat and mouse obesity models (Johnen et al., Nat Med, Vol. 13, pp. 1333-1340 (2007); Hsu et al., Nature, Vol. 550, pp. 255-259 (2017); Mullican et al., Nat Med, Vol. 23, pp. 1150-1157 (2017); Yang et al., Nat Med, Vol. 23, pp. 1158-1166 (2017); Tsai et al., Int J Obesity, Vol. 42, pp. 561-571 (2018); Xiong et al., Sci Trans Med, Vol. 9, p. 412 (2017)). GDF15 treatment-mediated weight loss in diet-induced obese mice leads to metabolic improvements, including enhanced glucose homeostasis and reduced plasma triglycerides and cholesterol. These effects can be translated to higher species, because a six-week daily treatment regimen using recombinant human GDF15 in spontaneously obese non-human primates reduced food intake, body weight, and plasma triglyceride concentrations and improved glucose tolerance (Xiong et al., Sci Trans Med, Vol. 9, p. 412 (2017)). In addition, it has been previously demonstrated that the half-life of recombinant GDF15 is extended by fusion with human serum albumin (HSA), and it is predicted to be a therapeutic agent suitable for once-weekly administration in humans (Mullican et al., Nat Med, Vol. 23, p. 1150-1157 (2017) and U.S. Patent Publication 2017 / 0327560).

[0008] GLP1 is a peptide hormone derived from enteroendocrine cells in the intestine that also reduces food intake, leading to weight loss. This function is mediated by interacting with GLP1 receptors (GLP1R) in the central nervous system, and this ligand / receptor interaction in peripheral tissues has additional biological effects, including enhancing glucose-stimulated insulin secretion, inhibiting glucagon release, and slowing gastric emptying (Druker et al., Cell Met, Vol. 27, pp. 740-756 (2018)). With all these biological effects, GLP1R agonists improve glucose homeostasis in humans and drive weight loss in humans. In addition, although the exact mechanism has not yet been determined, it is reported that treatment with GLP1R agonists significantly improves cardiovascular outcomes in patients with diabetes (Lim et al., Trends Endocrinol Metab., Vol. 29, pp. 238-248 (2018)). GLP1R agonist therapeutics are peptide sequences based on native human GLP1 or exendin-4 (a peptide isolated from the saliva of the giant lizard) with modifications that prevent enzymatic cleavage. GLP1R agonists can be delivered via half-life-extending platforms such as lipidation, antibody Fc, or human serum albumin (HSA) (Cheang and Moyle, Chem Med Chem, Vol. 13, pp. 662-671 (2018)).

[0009] Therefore, it is desirable to obtain GLP1 analogs or derivatives thereof and / or GDF15 analogs or derivatives thereof having improved metabolic stability and pharmacokinetic profiles relative to GLP1 or GDF15, respectively. Such derivatives would provide GLP1 receptor and GDF15 receptor modulation with greater duration of action, making them useful as therapeutic agents for subjects in need of such modulation.

[0010] The above discussion is merely presented to provide a better understanding of the nature of the problems faced in the art and should not be construed in any way as an admission that such references constitute "prior art" to the present application. Summary of the invention

[0011] In one general aspect, the present invention relates to novel glucagon-like peptide 1 (GLP1)-growth differentiation factor 15 (GDF15) fusion proteins. The GLP1-GDF15 fusion protein regulates GLP1R and / or GDF15R (GFRAL).

[0012] Provided herein is a glucagon-like peptide 1 (GLP1)-growth differentiation factor 15 (GDF15) fusion protein. The GLP1-GDF15 fusion protein comprises a GLP1 peptide, a first linker peptide, serum albumin, a second linker peptide, and a GDF15 protein.

[0013] In certain embodiments, the GLP1 peptide comprises an amino acid sequence selected from SEQ ID NOs: 1-4.

[0014] In certain embodiments, the first linker peptide comprises an amino acid sequence selected from SEQ ID NOs: 5-25.

[0015] In certain embodiments, the serum albumin comprises an amino acid sequence selected from SEQ ID NO:26 or SEQ ID NO:27.

[0016] In certain embodiments, the second linker peptide comprises an amino acid sequence selected from SEQ ID NOs: 28-30.

[0017] In certain embodiments, the GDF15 protein comprises an amino acid sequence selected from SEQ ID NO:31 or SEQ ID NO:32.

[0018] In certain embodiments, the GLP1-GDF15 fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:33-74 and SEQ ID NO:84.

[0019] Also provided are isolated nucleic acids encoding the GLP1-GDF15 fusion proteins of the present invention. Also provided are vectors comprising the isolated nucleic acids of the present invention. Also provided are host cells comprising the isolated nucleic acids of the present invention or the vectors of the present invention.

[0020] Also provided is a pharmaceutical composition comprising the GLP1-GDF15 fusion protein of the present invention and a pharmaceutically acceptable carrier.

[0021] Also provided is a method for treating or preventing a disease or disorder in a subject in need thereof, wherein the disease or disorder is selected from the group consisting of obesity, type I or type II diabetes, metabolic syndrome, insulin resistance, impaired glucose tolerance, hyperglycemia, hyperinsulinemia, hypertriglyceridemia, hypoglycemia due to congenital hyperinsulinemia (CHI), dyslipidemia, atherosclerosis, diabetic nephropathy, and other cardiovascular risk factors such as hypertension and cardiovascular risk factors associated with uncontrolled cholesterol and / or lipid levels, osteoporosis, inflammation, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), kidney disease and eczema, the method comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition of the present invention.

[0022] Also provided is a method of reducing food intake in a subject in need thereof, the method comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition of the present invention.

[0023] Also provided is a method of modulating GLP1 receptor activity in a subject in need thereof, the method comprising administering to the subject in need thereof an effective amount of a pharmaceutical composition of the present invention.

[0024] Also provided is a method of modulating GDF15 receptor (GFRAL) activity in a subject in need thereof, the method comprising administering to the subject in need thereof an effective amount of a pharmaceutical composition of the present invention.

[0025] In certain embodiments, the pharmaceutical composition is administered via injection.

[0026] Also provided are kits comprising a GLP1-GDF15 fusion protein of the invention, an isolated nucleic acid of the invention and / or a vector of the invention. The kit may also, for example, comprise a device for injection.

[0027] Also provided is a method for preparing a pharmaceutical composition comprising the GLP1-GDF15 fusion protein of the present invention, which comprises combining the GLP1-GDF15 fusion protein with a pharmaceutically acceptable carrier to obtain a pharmaceutical composition.

[0028] Also provided is a method for preparing the GLP1-GDF15 fusion protein of the present invention, which comprises culturing cells containing a nucleic acid encoding the GLP1-GDF15 fusion protein under conditions for preparing the GLP1-GDF15 fusion protein, and recovering the GLP1-GDF15 fusion protein from the cells or culture. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The foregoing summary and the following detailed description of preferred embodiments of the present patent application can be better understood when read in conjunction with the accompanying drawings. However, it should be understood that the present patent application is not limited to the precise embodiments shown in the drawings.

[0030] Figure 1 Shown is a graph showing the percent change in body weight (starting from day 0) of DIO mice receiving daily administration of liraglutide (lira) and / or every other day administration of HSA-GDF15 (SEQ ID NO: 81).

[0031] Figure 2Shown is a graph showing the mean percent change in body weight (starting from day 0) in DIO mice receiving daily administration of GLP1-GSA-GDF15 (SEQ ID NO: 72), GLP1-GSA-GDF15 (I89R) (SEQ ID NO: 73, the I89R mutation eliminates GDF15 activity), GLP1 (9-39)-GSA-GDF15 (SEQ ID NO: 74, eliminates GLP1 activity), or GSA-GDF15 (SEQ ID NO: 75). Dulaglutide treatment served as a positive control, while vehicle treatment served as a negative control. ± SEM, n=8.

[0032] Figure 3 A schematic diagram of the GLP1-GDF15 fusion protein is shown.

[0033] Figure 4 A graph showing the effect of subcutaneous administration of SEQ ID NO: 45, 49, 50, 33, 46 and 44 on food intake in C57BL / 6 mice is shown. Data are expressed as the percentage change in food intake compared to food intake 24 hours prior to treatment.

[0034] Figure 5 Shown is a graph showing the plasma concentrations of GLP1-GDF15 fusion proteins (SEQ ID NOs: 45, 49, 50, 33, 46 and 44) ​​24 hours after administration to mice as determined by immunoaffinity capture-trypsin digestion LC-MS / MS analysis.

[0035] Figure 6 Shown is a graph demonstrating the effect of subcutaneous administration of SEQ ID NOs: 45, 50, 46 and 44 on food intake in GFRAL-deficient mice. Data are expressed as percent change in food intake (compared to vehicle treatment) relative to plasma concentration of the test article with the intact GLP1 arm at 24 hours.

[0036] Figure 7 A graph showing the effect of subcutaneous administration of SEQ ID NO: 45 on food intake in GFRAL-deficient mice is shown. Data are expressed as percent change in food intake (compared to vehicle treatment) relative to plasma concentration of the test article at 24 hours with the intact GLP1 arm.

[0037] Figure 8 A graph showing the effect of subcutaneous administration of SEQ ID NO: 33 on food intake in GFRAL-deficient mice is shown. Data are expressed as percent change in food intake (compared to vehicle treatment) relative to plasma concentration of the test article at 24 hours with the intact GLP1 arm.

[0038] Fig. 9 Shown is a graph demonstrating the effect of subcutaneous administration of SEQ ID NO: 50, 46 and 44 on glucose tolerance in mice. Data are expressed as percent change in ΔAUC relative to plasma concentration of GLP1-GDF15 fusion protein with intact GLP1 peptide or GLP1 peptide variants compared to vehicle treatment.

[0039] Fig.10 Shown is a graph demonstrating the effect of subcutaneous administration of SEQ ID NO: 45 and 49 on glucose tolerance in mice. Data are expressed as percent change in ΔAUC relative to plasma concentration of GLP1-GDF15 fusion protein with intact GLP1 peptide or GLP1 peptide variants compared to vehicle treatment.

[0040] Fig.11 Shown is a graph demonstrating the effect of subcutaneous administration of SEQ ID NO: 33 on glucose tolerance in mice. Data are expressed as percent change in ΔAUC relative to plasma concentration of GLP1-GDF15 fusion protein with intact GLP1 peptide or GLP1 peptide variants compared to vehicle treatment.

[0041] Fig.12 Shown is a graph demonstrating the effect of subcutaneous administration of SEQ ID NO: 45 and SEQ ID NO: 36 on glucose tolerance in mice. Data are expressed as percent change in ΔAUC relative to plasma concentration of GLP1-GDF15 fusion protein with intact GLP1 peptide or GLP1 peptide variants compared to vehicle treatment.

[0042] Fig.13 Shown is a graph demonstrating the effect of subcutaneous administration of SEQ ID NO: 45 and 44 on insulin secretion during graded glucose infusion in cynomolgus monkeys. Data are expressed as fold change in ISR AUC normalized to glucose AUC (compared to baseline) relative to plasma concentration of GLP1-GDF15 fusion protein with intact GLP1 peptide or GLP1 peptide variants.

[0043] Fig.14 Shown is a graph demonstrating the weight loss efficacy of daily subcutaneous administration of GLP1-GDF15 fusion proteins (SEQ ID NOs: 45 and 44) ​​in diet-induced obese mice. Data are expressed as percent body weight change from day 0.

[0044] Fig.15 Shown is a graph showing the plasma concentration of GLP1-GDF15 fusion protein (SEQ ID NOs: 45 and 44) ​​following 7 days of daily administration in diet-induced obese mice as determined by immunoaffinity capture-trypsin digestion LC-MS / MS analysis.

[0045] Fig.16 A graph showing the 3-day percent change in food intake following subcutaneous administration of GLP1-GDF15 fusion protein (SEQ ID NO: 44 and 83) every QW over 4 weeks in spontaneously obese non-human primates is shown. Data are presented for individual subjects, and individual subjects with no detectable levels of test article in their plasma are not included in the figure, as outlined below the x-axis.

[0046] Fig.17 Shown is a graph showing the plasma concentration of total test article during 4 weeks of treatment with varying concentrations of GLP1-GDF15 fusion protein (SEQ ID NO: 44 and 83) administered QW in spontaneously obese non-human primates as determined by immunoassay. Data are presented as mean (± SEM).

[0047] Fig.18 Shown is a graph showing the plasma concentration of a GLP1 portion-containing test article during 4 weeks of treatment with varying concentrations of GLP1-GDF15 fusion protein (SEQ ID NO: 44) administered QW in spontaneously obese non-human primates as determined by immunoassay. Data are presented as mean (± SEM).

[0048] Fig.19 Shown is a graph showing absolute daily food intake before, during and after 21 days of treatment with increasing concentrations of GLP1-GDF15 fusion proteins (SEQ ID NOs: 44, 83 and 84) administered Q3D in spontaneously obese non-human primates. Data are presented as means (± SEM) of ten subjects per group.

[0049] Fig. 20 Shown is a graph showing body weight change (relative to baseline) after 21 days of treatment with increasing concentrations of GLP1-GDF15 fusion proteins (SEQ ID NOs: 44, 83, and 84) administered Q3D in spontaneously obese non-human primates. Data are presented as mean values ​​(± SEM) of ten subjects per group.

[0050] Fig.21 Shown is a graph displaying plasma concentrations of total test article during and after 21 days of treatment with increasing concentrations of GLP1-GDF15 fusion protein (SEQ ID NO: 44, 83 and 84) administered Q3W in spontaneously obese non-human primates as determined by immunoassay. Data are presented for individual subjects.

[0051] Fig. 22Shown is a graph showing plasma concentrations of a GLP1 portion-containing test article during and after 21 days of treatment with increasing concentrations of GLP1-GDF15 fusion protein (SEQ ID NOs: 44 and 84) administered Q3D in spontaneously obese nonhuman primates as determined by immunoassay. Data are presented for individual subjects. DETAILED DESCRIPTION

[0052] Various publications, articles, and patents are cited or described in the background and throughout the specification; each of these references is incorporated herein by reference in its entirety. The discussion of documents, acts, materials, devices, articles, etc. included in this specification is intended to provide context for the present invention. Such discussion is not an admission that any or all of these matters form part of the prior art with respect to any invention disclosed or claimed.

[0053] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Otherwise, certain terms used herein have the meanings described in this specification.

[0054] It must be noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0055] Unless otherwise indicated, any numerical value, such as a concentration or concentration range described herein, is understood to be modified by the term "about" in all cases. Therefore, numerical values ​​generally include the value ± 10%. For example, a concentration of 1 mg / mL includes 0.9 mg / mL to 1.1 mg / mL. Similarly, a concentration range of 1% to 10% (w / v) includes 0.9% (w / v) to 11% (w / v). Unless the context clearly indicates otherwise, as used herein, the numerical range used explicitly includes all possible subranges, all single values ​​within the range, including integers within such ranges and fractions within the range.

[0056] Unless otherwise indicated, the term "at least" preceding a series of elements should be understood to refer to each element in the series. Those skilled in the art will recognize, or will be able to ascertain using only routine experimentation, a variety of equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.

[0057] As used herein, the terms "comprises," "including," "having," or "containing," or any other variation thereof, should be understood to mean including the specified integer or group of integers but not excluding any other integer or group of integers, and are intended to be non-exclusive or open-ended. For example, a composition, mixture, process, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to these compositions, mixtures, processes, methods, articles, or apparatuses. In addition, unless expressly indicated to the contrary, "or" refers to an inclusive or and not to an exclusive or. For example, a condition A or B is satisfied by any of the following: A is true (or present) and B is false (or absent), A is false (or absent) and B is true (or present), and both A and B are true (or present).

[0058] As used herein, the connection term "and / or" between multiple listed elements is understood to include both single options and combined options. For example, where two elements are connected by "and / or", the first option refers to the application of the first element without the second element. The second option refers to the application of the second element without the first element. The third option refers to the application of the first element and the second element together. Any of these options is understood to fall within the meaning and therefore meets the requirements of the term "and / or" as used herein. The parallel applicability of more than one option is also understood to fall within the meaning and therefore meets the requirements of the term "and / or".

[0059] It should also be understood that when referring to dimensions or characteristics of components of the preferred invention, the use of "about," "approximately," "substantially," and similar terms herein indicates that the described dimensions / characteristics are not strict boundaries or parameters and do not exclude minor variations that are functionally the same or similar, as understood by those of ordinary skill in the art. At a minimum, such references including numerical parameters will include variations using mathematical and industrial principles accepted in the art (e.g., rounding, measurement or other systematic errors, manufacturing tolerances, etc.) without changing the least significant digits.

[0060] In the context of two or more nucleic acid or polypeptide sequences (e.g., GLP1 peptides, linker peptides, serum albumin, GDF15 proteins, and polynucleotides encoding peptides), the terms "same" or "percent identity" refer to two or more sequences or subsequences that are identical or have a specified percentage of identical amino acid residues or nucleotides when compared and aligned for maximum correspondence, as measured according to the present disclosure using one of the following sequence comparison algorithms or by visual inspection using methods known in the art.

[0061] For sequence comparison, usually one sequence is used as a reference sequence to which a test sequence is compared. When using a sequence comparison algorithm, the test sequence and the reference sequence are input into a computer, subsequence coordinates are specified (if necessary), and program parameters for the sequence algorithm are specified. Then, the sequence comparison algorithm calculates the sequence identity percentage for the test sequence relative to the reference sequence based on the specified program parameters.

[0062] Optimal alignment of sequences for comparison can be performed, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by using the homology alignment algorithm of Needleman & Wunsch, J Mol. Biol. 48:443 (1970), by searching the similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA, 85:2444 (1988). 85:2444 (1988)), by algorithms (GAP, BESTFIT, FASTA and TFASTA, computerized implementations of the Wisconsin Genetics Software Package, Genetics Computing Group, 575 Science Dr., Madison, WI), or by visual inspection (see generally, Current Protocols in Molecular Biology, FM Ausubel et al., eds., Laboratory Protocols, a joint venture of Greene Publishing Associates and John Wiley & Sons, Inc. (1995 supplement) (Ausubel)).

[0063] Examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1990) Journal of Molecular Biology, Vol. 215, pp. 403-410 (J. Mol. Biol. 215: 403-410) and Altschul et al. (1997) Nucleic Acids Res. Vol. 25: 3389-3402 (Nucleic Acids Res. 25: 3389-3402), respectively. Res. Vol. 25: pp. 3389-3402. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information. The algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that match or satisfy some positive threshold score T when aligned with a word of the same length in the database sequence. T is called the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence as far as the cumulative alignment score can be increased.

[0064] In addition to calculating the percentage of sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, Proc. Nat'l. Acad. Sci. USA, Vol. 90, pp. 5873-5787 (1993)). One similarity measure provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability that a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of a test nucleic acid to a reference nucleic acid is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

[0065] Another indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid immunologically cross-reacts with the polypeptide encoded by the second nucleic acid, as described below. Thus, a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions.

[0066] As used herein, "subject" refers to any animal, preferably a mammal, most preferably a human. As used herein, the term "mammal" encompasses any mammal. Examples of mammals include, but are not limited to, cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys, humans, etc., more preferably humans.

[0067] In the context of the methods of the present invention, the term "administering" means a method of therapeutically or prophylactically preventing, treating or ameliorating the syndromes, disorders or diseases described herein by using a conjugate or compound of the present invention, or a form, composition or medicament thereof. Such methods include administering an effective amount of the conjugate, compound, form, composition or medicament thereof at different times during the course of treatment, or administering simultaneously in a combined form. The methods of the present invention should be understood to encompass all known therapeutic treatment regimens.

[0068] The term "effective amount" means the amount of an active conjugate, compound or agent that elicits the biological or medicinal response in a tissue system, animal or human that is being sought by the researcher, veterinarian, medical doctor or other clinician, including the prevention, treatment or amelioration of the syndrome, disorder or disease being treated, or the prevention, treatment or amelioration of a symptom of the syndrome, disorder or disease being treated.

[0069] As used herein, the term "composition" is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combination of the specified ingredients in the specified amounts.

[0070] The term "isolated" may refer to a nucleic acid or polypeptide that is substantially free of the cellular material, bacterial material, viral material, or culture medium from which it was derived (when produced by recombinant DNA techniques), or chemical precursors or other chemicals (when chemically synthesized). In addition, an isolated polypeptide refers to a polypeptide that can be administered to a subject as an isolated polypeptide; in other words, if the polypeptide adheres to a column or is embedded in a gel, it can simply be considered not to be "isolated". In addition, an "isolated nucleic acid fragment" or "isolated peptide" is a nucleic acid or protein fragment that does not exist naturally as a fragment and / or is not usually in a functional state.

[0071] As used herein, the term "polynucleotide", which is synonymously referred to as "nucleic acid molecule", "nucleotide" or "nucleic acid", refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. "Polynucleotide" includes, but is not limited to, single-stranded and double-stranded DNA, DNA that is a mixture of single-stranded and double-stranded regions, single-stranded and double-stranded RNA, and RNA that is a mixture of single-stranded and double-stranded regions, and hybrid molecules comprising DNA and RNA that may be single-stranded or more typically double-stranded or a mixture of single-stranded and double-stranded regions. In addition, "polynucleotide" refers to a triple-stranded region comprising RNA or DNA or both RNA and DNA. The term polynucleotide also includes DNA or RNA containing one or more modified bases, and DNA or RNA having a main chain modified for stability or other reasons. "Modified" bases include, for example, tritylated bases and rare bases such as inosine. A variety of modifications can be made to DNA and RNA; thus, "polynucleotide" includes chemically, enzymatically, or metabolically modified forms of polynucleotides that commonly occur in nature, as well as chemical forms of DNA and RNA that are unique to viruses and cells."Polynucleotide" also includes relatively short nucleic acid chains, commonly referred to as oligonucleotides.

[0072] As used herein, the term "expression" refers to the biosynthesis of a gene product. The term encompasses the transcription of a gene into RNA. The term also encompasses the translation of RNA into one or more polypeptides, and also encompasses all naturally occurring post-transcriptional and post-translational modifications. The expressed polypeptide may be in the cytoplasm of the host cell, in the extracellular environment such as the growth medium of a cell culture, or anchored to the cell membrane.

[0073] As used herein, the term "peptide", "polypeptide" or "protein" may refer to a molecule composed of amino acids and may be recognized as a protein by a person skilled in the art. Conventional single-letter or three-letter codes for amino acid residues are used herein. The terms "peptide", "polypeptide" and "protein" are used interchangeably herein to refer to polymers of amino acids of any length, including those comprising linked (e.g., fused) peptides / polypeptides (e.g., fusion proteins). The polymer may be linear or branched, it may contain modified amino acids, and it may be interspersed with non-amino acids. The term also encompasses amino acid polymers that have been modified naturally or by intervention; natural or interventional modifications are, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. The definition also includes, for example, polypeptides containing one or more amino acid analogs (including, for example, non-natural amino acids, etc.) and other modifications known in the art.

[0074] The peptide sequences described herein are written according to the usual convention, wherein the N-terminal region of the peptide is on the left and the C-terminal region is on the right. Although isomeric forms of amino acids are known, unless otherwise specifically indicated, it is the L form of the amino acid represented.

[0075] Glucagon-like peptide 1 (GLP1)-growth differentiation factor 15 (GDF15) fusion protein

[0076] Although GDF15 and GLP1 signaling can reduce food intake, these effects appear to be independent of each other. For example, the GDF15 effect is maintained in the absence of GLP1R in mice, and conversely, GLP1 treatment still leads to a food intake effect in the absence of GFRAL (Hsu et al., Nature, Vol. 550, pp. 255-259 (2017); Mullican et al., Nat Med, Vol. 23, pp. 1150-1157 (2017)). Therefore, it is assumed that targeting both mechanisms simultaneously can lead to greater reductions in food intake and weight loss than either mechanism alone. Combining the independent but complementary pharmacology of GDF15 and GLP1 agonists with a single fully recombinant molecule suitable for once-weekly administration by fusion to human serum albumin will deliver beneficial effects on weight loss, insulin sensitivity, insulin secretion, and cardiovascular outcomes. The main challenge of this approach is to deliver each agonist within the molecule in a balanced manner that engages the corresponding receptor at the desired level, sufficient to achieve all desired effects, but avoids adverse effects on the target. This balance can be fine-tuned by modulating the potency and / or pharmacokinetic properties of either agonist arm of the molecule.

[0077] In one general aspect, the present invention relates to a glucagon-like peptide 1 (GLP1)-growth differentiation factor 15 (GDF15) fusion protein. The GLP1-GDF15 fusion protein comprises a GLP1 or GLP1 variant peptide, a first linker peptide (e.g., an amino (N)-terminal linker), human serum albumin (HSA), a second linker peptide (e.g., a carboxyl (C)-terminal linker), and a GDF15 or GDF15 variant protein.

[0078] Glucagon-like peptide 1 (GLP1) or GLP1 variant peptide

[0079] Glucagon-like peptide 1 (GLP1) is an insulin secretagogue synthesized in the intestine and released in response to food ingestion. It is secreted primarily in two forms: GLP1-(7-37) and GLP1-(7-36)NH2, both of which bind to specific GLP1 receptors (GLP1Rs) present in many tissues, including pancreatic β cells, where it enhances glucose-stimulated insulin secretion, and in the brainstem, where it controls satiety and food intake.

[0080] Many GLP1 analogs and derivatives are known and may be referred to herein as "GLP1 variants". These GLP1 variant peptides may include exendins, which are peptides found in the venom of the Gila monster. These exendins have sequence homology with native GLP1 and can bind to the GLP1 receptor and initiate a signal transduction cascade.

[0081] GLP1 and GLP1 variant peptides have been shown to act in a variety of ways, which may include, but are not limited to, reducing food intake, stimulating insulin release, reducing glucagon secretion, inhibiting gastric emptying, and improving glucose utilization.

[0082] GLP1R belongs to the class B family of 7-transmembrane heterotrimeric G protein-coupled receptors and is expressed in a wide range of tissues, including but not limited to pancreatic islets, heart, kidney, stomach, intestine, nodose ganglion neurons of the vagus nerve, and α-cells, β-cells, and δ-cells in several regions of the central nervous system (CNS), including the hypothalamus and brainstem. GLP1R can couple to Gα s , Gα q , Gα i and Gα o (Montrose-Rafizadeh et al., Endocrinology, Vol. 140, pp. 1132-1140 (1999); Hallbrink et al., Biochim Biophys Acta 1546:79-86 (2001)), resulting in an increase in intracellular calcium, adenylate cyclase and phospholipase C, and activation of PKA, PKC, PI-3K, Epac2 and MAPK signal transduction pathways (Drucker et al., PNAS 84:3434-8 (1987); Wheeler et al., Endrocrinology 133:57-62 (1993); and Holz et al., JBC 270:17749-57 (1995)).

[0083] Provided herein is a GLP1-GDF15 fusion protein comprising a first component, wherein the first component is a GLP1 or GLP1 variant peptide. As used herein, the terms "GLP1 peptide", "GLP1 variant peptide", "GLP1 peptide variant" and "GLP1 or GLP1 variant peptide" are used interchangeably. The GLP1 or GLP1 variant peptide may comprise one of the sequences provided in Table 1. The GLP1 or GLP1 variant peptide may have at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with one of the sequences provided in Table 1. The GLP1 or GLP1 variant peptide sequence can be selected based on at least one of the following criteria: (i) expression yield and purity, (ii) in vitro stability, (iii) in vitro potency, (iv) retention of in vitro potency for binding to GDF15 or GDF15 variant protein, (v) lack of serine xylosylation or serine xylosylation potential, and (vi) properties of the GLP1-GDF15 fusion protein (e.g., in vivo stability, in vivo potency (i.e., whether the GLP1 or GLP1 variant peptide and GDF15 or GDF15 variant protein are capable of agonist activity at the GLP1R and GDF15R (GFRAL) receptors, respectively)).

[0084] The GLP1 or GLP1 variant peptides constituting the first component of the GLP1 fusion peptide are intended to encompass peptides that have sufficient homology and functionality with natural GLP1. GLP1 or GLP1 variant peptides are designed to be able to bind to GLP1 receptors in tissues (including the pancreas and brainstem), thereby generating the same signal transduction pathways as when natural GLP1 binds to GLP1 receptors in these tissues and exhibiting the same or similar physiological activities.

[0085] Table 1: Glucagon-like peptide 1 and its variants

[0086]

[0087] First linker peptide: amino terminal linker (N-terminal linker)

[0088] Provided herein are GLP1-GDF15 fusion proteins comprising a second component, wherein the second component is a first linker peptide (i.e., an amino terminal linker peptide). The first linker peptide may comprise one of the sequences provided in Table 2. The first linker peptide may have at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with one of the sequences provided in Table 2.

[0089] The first linker peptide can, for example, comprise about 5 to about 60 amino acid residues, about 10 to about 50 amino acid residues, about 10 to about 60 amino acid residues, about 5 to about 50 amino acid residues, about 15 to about 40 amino acid residues, about 12 to about 30 amino acid residues, about 12 to about 42 amino acid residues, about 20 to about 25 amino acid residues, about 8 to about 48 amino acid residues, about 10 to about 46 amino acid residues, about 12 to about 44 amino acid residues, about 14 to about 42 amino acid residues, about 16 to about 40 amino acid residues, about 18 to about 38 amino acid residues, about 20 to about 36 amino acid residues, about 20 to about 42 amino acid residues, about 22 to about 34 amino acid residues, about 24 to about 32 amino acid residues, about 26 to about 30 amino acid residues, or any value of amino acid residues therebetween. The first linker peptide can comprise 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 amino acid residues.

[0090] In certain embodiments, the first linker peptide may comprise an alanine-proline repeat sequence (i.e., an AP repeat sequence), wherein the AP dipeptide may be referred to as an AP unit. The AP repeat sequence may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 AP units. In certain embodiments, the first linker peptide may, for example, comprise 2 to 25 AP units, 5 to 25 units, 4 to 23 AP units, 6 to 21 AP units, 8 to 19 AP units, 10 to 17 AP units, 12 to 15 AP units, 5 to 10 AP units, 5 to 15 AP units, 10 to 25 AP units, 15 to 25 AP units, 20 to 25 AP units, or any value of units therebetween. In certain embodiments, the AP repeat sequence may be inside an alanine-serine dipeptide (i.e., an AS unit) and a glycine-serine dipeptide (i.e., a GS unit). The AS unit may, for example, be located at the amino terminus of the first linker peptide. The GS unit may, for example, be located at the carboxyl terminus of the first linker peptide.

[0091] In certain embodiments, the first linker peptide may comprise a glycine-glycine-glycine-glycine-serine repeat sequence (i.e., a G4S repeat sequence), wherein the G4S pentapeptide may be referred to as a G4S unit. The G4S repeat sequence may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 G4S units. In certain embodiments, the first linker peptide may, for example, comprise 2 to 15 G4S units, 4 to 13 G4S units, 6 to 11 G4S units, 8 to 9 G4S units, 2 to 8 G4S units, 2 to 6 G4S units, 6 to 8 G4S units, 7 to 8 G4S units, 7 to 9 G4S units, 7 to 10 G4S units, or any value of units therebetween. In certain embodiments, the AS or GS unit may be located, for example, at the amino terminus of the first linker peptide.

[0092] In certain embodiments, the first linker peptide may comprise a glycine-glycine-glycine-glycine-alanine repeat sequence (i.e., a G4A repeat sequence), wherein the G4A pentapeptide may be referred to as a G4A unit. The G4A repeat sequence may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 G4A units. In certain embodiments, the first linker peptide may, for example, comprise 2 to 15 G4A units, 4 to 13 G4A units, 6 to 11 G4A units, 8 to 9 G4A units, 2 to 8 G4A units, 2 to 4 G4A units, 2 to 6 G4A units, 4 to 6 G4A units, 4 to 8 G4A units, 6 to 8 G4A units, 6 to 9 G4A units, 6 to 10 G4A units, or any value thereof. In certain embodiments, a glycine-alanine dipeptide unit (i.e., a GA unit) may, for example, be located at the amino terminus of the first linker peptide.

[0093] In certain embodiments, the first linker peptide can be a polyglycine peptide. The polyglycine peptide can include about 6 to about 50 glycine residues, about 10 to about 45 glycine residues, about 15 to about 40 glycine residues, about 20 to about 35 glycine residues, about 25 to about 30 glycine residues, about 20 to about 25 glycine residues, or any number of glycine residues therebetween. The poly-glycine first linker peptide can comprise 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 glycine residues.

[0094] The first linker peptide sequence can be selected based on at least one of the following criteria: (i) expression yield and purity, (ii) in vitro potency, (iii) in vitro stability, (iv) lack of serine xylosylation or serine xylosylation potential, and (v) properties of the GLP1-GDF15 fusion protein (e.g., in vivo stability, in vivo potency (i.e., whether GLP1 or GLP1 variant peptide and GDF15 or GDF15 variant protein are able to have agonist activity towards GLP1R and GDF15R (GFRAL), respectively)).

[0095] Table 2: First linker peptide (N-terminal linker peptide)

[0096]

[0097]

[0098] Serum albumin

[0099] Provided herein is a GLP1-GDF15 fusion protein comprising a third component, wherein the third component is serum albumin (e.g., human serum albumin (HSA) or gorilla serum albumin (GSA)). Natural human serum albumin contains 35 cysteine ​​(Cys, C) residues, which form 17 disulfide bonds, of which the Cys-34 residue is the only free cysteine ​​in the molecule. By capturing a variety of reactive oxygen species (ROS) and reactive nitrogen species (RNS), it has been shown that this free Cys-34 acts as a free radical scavenger (Taverna et al., Ann. Intensive Care, Vol. 3, p. 4 (2013)). This free Cys is mutated to serine (Ser) to minimize the risk of heterogeneity due to oxidation.

[0100] The serum albumin may comprise one of the sequences provided in Table 3. The serum albumin may have at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to one of the sequences provided in Table 3. The serum albumin sequence may be selected based on at least one of the following criteria: (i) in vitro stability, (ii) in vitro potency, and (iii) properties of the GLP1-GDF15 fusion protein (e.g., in vivo stability and in vivo potency (i.e., whether the GLP1 or GLP1 variant peptide and GDF15 or GDF15 variant protein are capable of agonist activity at the GLP1R and GDF15R receptors, respectively)).

[0101] Table 3: Serum Albumin

[0102]

[0103] Second linker peptide: Carboxyl terminal linker (C-terminal linker)

[0104] Provided herein are GLP1-GDF15 fusion proteins comprising a fourth component, wherein the fourth component is a second linker peptide (i.e., a carboxyl terminal linker peptide). The second linker peptide may comprise one of the sequences provided in Table 4. The second linker peptide may have at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with one of the sequences provided in Table 4.

[0105] The second linker peptide can, for example, comprise about 5 to about 60 amino acid residues, about 10 to about 50 amino acid residues, about 10 to about 60 amino acid residues, about 5 to about 50 amino acid residues, about 15 to about 40 amino acid residues, about 12 to about 30 amino acid residues, about 12 to about 42 amino acid residues, about 20 to about 25 amino acid residues, about 8 to about 48 amino acid residues, about 10 to about 46 amino acid residues, about 12 to about 44 amino acid residues, about 14 to about 42 amino acid residues, about 16 to about 40 amino acid residues, about 18 to about 38 amino acid residues, about 20 to about 36 amino acid residues, about 20 to about 42 amino acid residues, about 22 to about 34 amino acid residues, about 24 to about 32 amino acid residues, about 26 to about 30 amino acid residues, or any value of amino acid residues therebetween. The second linker peptide can comprise 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 amino acid residues.

[0106] In certain embodiments, the second linker peptide may comprise an alanine-proline repeat sequence (i.e., an AP repeat sequence), wherein the AP dipeptide may be referred to as an AP unit. The AP repeat sequence may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 AP units. In certain embodiments, the second linker peptide may, for example, comprise 2 to 25 AP units, 5 to 25 units, 4 to 23 AP units, 6 to 21 AP units, 8 to 19 AP units, 10 to 17 AP units, 12 to 15 AP units, 5 to 10 units, 5 to 15 units, 10 to 25 units, 15 to 25 units, 20 to 25 units, or any value of units therebetween. In certain embodiments, the AP repeat sequence may be inside an alanine-serine dipeptide (i.e., an AS unit) and a glycine-serine dipeptide (i.e., a GS unit). The AS unit may, for example, be located at the amino terminus of the second linker peptide. The GS unit may, for example, be located at the carboxyl terminus of the second linker peptide.

[0107] In certain embodiments, the second linker peptide may comprise a glycine-glycine-glycine-glycine-serine repeat sequence (i.e., a G4S repeat sequence), wherein the G4S pentapeptide may be referred to as a G4S unit. The G4S repeat sequence may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 G4S units. In certain embodiments, the second linker peptide may, for example, comprise 2 to 15 G4S units, 4 to 13 G4S units, 6 to 11 G4S units, 8 to 9 G4S units, 2 to 8 G4S units, 2 to 6 G4S units, 6 to 8 G4S units, 7 to 8 G4S units, 7 to 9 G4S units, 7 to 10 G4S units, or any value of units therebetween. In certain embodiments, the AS or GS unit may be located, for example, at the amino terminus of the second linker peptide.

[0108] In certain embodiments, the second linker peptide may comprise a glycine-glycine-glycine-glycine-alanine repeat sequence (i.e., a G4A repeat sequence), wherein the G4A pentapeptide may be referred to as a G4A unit. The G4A repeat sequence may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 G4A units. In certain embodiments, the second linker peptide may, for example, comprise 2 to 15 G4A units, 4 to 13 G4A units, 6 to 11 G4A units, 8 to 9 G4A units, 2 to 8 G4A units, 2 to 4 G4A units, 2 to 6 G4A units, 4 to 6 G4A units, 4 to 8 G4A units, 6 to 8 G4A units, 6 to 9 G4A units, 6 to 10 G4A units, or any value thereof. In certain embodiments, a glycine-alanine dipeptide unit (i.e., a GA unit) may, for example, be located at the amino terminus of the second linker peptide.

[0109] In certain embodiments, the second linker peptide can be a polyglycine peptide. The polyglycine peptide can include about 6 to about 50 glycine residues, about 10 to about 45 glycine residues, about 15 to about 40 glycine residues, about 20 to about 35 glycine residues, about 25 to about 30 glycine residues, about 20 to about 25 glycine residues, or any number of glycine residues therebetween. The poly-glycine second linker peptide can comprise 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 glycine residues.

[0110] The second linker peptide sequence can be selected based on at least one of the following criteria: (i) expression yield and purity, (ii) in vitro potency, (iii) in vitro stability, (iv) lack of serine xylosylation or serine xylosylation potential, and (v) properties of the GLP1-GDF15 fusion protein (e.g., in vivo stability, in vivo potency (i.e., whether the GLP1 or GLP1 variant peptide and GDF15 or GDF15 variant protein are able to have agonist activity towards GLP1R and GDF15R (GFRAL), respectively)).

[0111] Table 4: Second linker peptide (C-terminal linker peptide)

[0112]

[0113] GDF15 or GDF15 variant protein

[0114] Growth differentiation factor 15 (GDF15) is a protein that belongs to the transforming growth factor beta (TGF-β) superfamily. GDF15 is a secreted protein that circulates as a 25-kDa dimer. GDF15 is also known as prostate-derived factor (PDF), macrophage inhibitory cytokine-1 (MIC-1), NSAID (nonsteroidal anti-inflammatory drug) activated gene (NAG-1), and placental TGF-β (PTGFβ).

[0115] The function of GDF15 has not been fully elucidated, but it is involved in a variety of biological processes, including but not limited to energy homeostasis, weight regulation, and cachexia driven by cancer and chronic diseases. It has been confirmed in mice, rats and monkeys that pharmacological administration of GDF15 may reduce energy intake and thereby cause weight loss. (Johnen et al., Nat Med, Vol. 13, pp. 1333-1340 (2007); Hsu et al., Nature, Vol. 550, pp. 255-259 (2017); Mullican et al., Nat Med, Vol. 23, pp. 1150-1157 (2017); Tsai et al., Int JObesity, Vol. 42, pp. 561-571 (2018)). GDF15 treatment-mediated weight loss resulted in metabolic improvements, including enhanced glucose homeostasis and reduced plasma triglycerides and cholesterol (Xiong et al., Sci Trans Med, Vol. 9, p. 412 (2017)).

[0116] GDF15 binds to the GDNF family alpha-like receptor (GFRAL), a transmembrane receptor located exclusively in neurons of the brainstem (Mullican et al., Hsu et al., Yang et al., Emmerson et al.). Upon GDF15 binding, GFRAL complexes with RET, a tyrosine kinase gamma that stimulates a downstream intracellular phosphorylation cascade including post-translational modification of AKT, ERK, and PLC. Although additional molecular and cellular components of this cascade remain to be elucidated, the ultimate effect of GDF15 / GFRAL signaling is reduced food intake and weight loss (Mullican and Rangwala, 2018).

[0117] Provided herein are GLP1-GDF15 fusion proteins comprising a fifth component, wherein the fifth component is a GDF15 or GDF15 variant protein. The GDF15 protein may comprise a sequence provided in Table 5, and the GDF15 variant protein may comprise a variant of the sequence provided in Table 5. The GDF15 or GDF15 variant peptide may have at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with one of the sequences provided in Table 5. GDF15 and / or GDF15 variant protein sequences can be selected based on at least one of the following criteria: (i) expression yield and purity, (ii) in vitro stability, (iii) in vitro potency, (iv) retention of in vitro potency for binding to GLP1 or GLP1 variant proteins, (v) lack of serine xylosylation or serine xylosylation potential, and (vi) properties of the GLP1-GDF15 fusion protein (e.g., in vivo stability, in vivo potency (i.e., whether the GLP1 or GLP1 variant peptide and GDF15 or GDF15 variant protein are capable of agonist activity at the GLP1R and / or GDF15R (GFRAL) receptor, respectively)).

[0118] The GDF15 or GDF15 variant protein that constitutes the fifth component of the GLP1-GDF15 fusion protein is intended to include peptides that have sufficient homology and functionality to activate the native GDF15R (GFRAL). GDF15 or GDF15 variant proteins are designed to bind to GFRAL in the brainstem, thereby generating the same signaling pathway as when native GDF15 binds to GFRAL in these neurons and exhibiting the same effects on food intake.

[0119] Table 5: GDF15 or GDF15 variant proteins

[0120]

[0121] GLP1-GDF15 fusion protein

[0122] Provided herein is a GLP1-GDF15 fusion protein comprising a first component, a second component, a third component, a fourth component, and a fifth component as described above. The first component is a GLP1 or a GLP1 variant peptide, the second component is a first linker peptide, the third component is serum albumin, the fourth component is a second linker peptide, and the fifth component is a GDF15 or a GDF15 variant protein. The GLP1-GDF15 fusion protein may comprise one of the sequences provided in Table 6. The GLP1-GDF15 fusion protein may have at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with one of the sequences provided in Table 6. The GLP1-GDF15 fusion protein can be selected based on at least one of the following criteria: (i) expression yield and purity, (ii) in vitro potency, (iii) in vitro stability, (iv) lack of serine xylosylation, and (v) physical properties of the GLP1-GDF15 fusion protein (e.g., in vivo stability, in vivo potency (i.e., whether the GLP1 or GLP1 variant peptide and GDF15 or GDF15 variant protein are capable of agonist activity at GLP1R and GDF15R, respectively)), and (vi) the desired balance of in vivo dual agonist pharmacology.

[0123] For fusion protein therapeutics that deliver dual pharmacology in one molecule, it is important to appropriately adjust the pharmacokinetic / pharmacodynamic (PK / PD) properties of each part so that both agonists are within the therapeutic range of the intended dose. Extensive studies of various glucagon-like peptide-1 (GLP1)-growth differentiation factor 15 (GDF15) fusion proteins have led to the discovery of novel molecules with unique properties of delivering optimal balanced doses of both GLP1 and GDF15 agonists, the fusion protein comprising various combinations of a GLP1 peptide or GLP1 variant peptide sequence, a first linker peptide sequence, a serum albumin sequence, a second linker peptide sequence, and a GDF15 or GDF15 variant protein sequence. These novel GLP1-GDF15 fusion proteins have been shown to be as effective as HSA-GDF15 in engaging GDF15R (GFRAL) in vivo when used in a specific dose range (see Example 11). Furthermore, the GLP1-GDF15 fusion protein used in the same dose range (see Example 11) was unexpectedly as effective as dulaglutide (GLP1-Fc) in engaging the GLP1R and without adverse side effects, but the GLP1 moiety was exposed at concentrations 10-30 times greater than in dulaglutide. Delivery of the GLP1 peptide as a fusion peptide with HSA-GDF15 unexpectedly altered the in vivo potency of the GLP1 moiety, but enabled a novel glucagon-like peptide-1 (GLP1)-growth differentiation factor 15 (GDF15) fusion protein to have an optimal balance of the two agonists.

[0124] Table 6: GLP1-GDF15 fusion protein

[0125]

[0126]

[0127]

[0128]

[0129]

[0130]

[0131]

[0132]

[0133]

[0134]

[0135]

[0136]

[0137]

[0138]

[0139]

[0140]

[0141]

[0142]

[0143]

[0144]

[0145]

[0146] GLP1-GDF15 fusion polynucleotides and vectors

[0147] In another general aspect, the present invention relates to an isolated nucleic acid encoding a GLP1-GDF15 fusion protein of the present invention. One skilled in the art will appreciate that the coding sequence of a protein may be altered (e.g., substituted, deleted, inserted, etc.) without changing the amino acid sequence of the protein. Thus, one skilled in the art will appreciate that the nucleic acid sequence encoding the fusion protein of the present invention may be altered without changing the amino acid sequence of the protein.

[0148] In another general aspect, the present invention relates to a vector comprising a nucleic acid separation encoding the fusion protein of the present invention. According to the present disclosure, any vector known to those skilled in the art, such as a plasmid, a cosmid, a phage vector or a viral vector, can be used. In some embodiments, the vector is a recombinant expression vector, such as a plasmid. The vector may include any element of the conventional function of establishing an expression vector, such as a promoter, a ribosome binding element, a terminator, an enhancer, a selective marker and an origin of replication. The promoter may be a constitutive, inducible or repressible promoter. A variety of expression vectors capable of delivering nucleic acids to cells are known in the art and can be used herein to prepare fusion proteins in cells. Conventional cloning techniques or artificial gene synthesis can be used to generate recombinant expression vectors according to embodiments of the present invention.

[0149] In another general aspect, the present invention relates to a host cell comprising an isolated nucleic acid encoding a fusion protein of the present invention. In view of the present disclosure, any host cell known to those skilled in the art can be used for recombinantly expressing the fusion protein of the present invention. In some embodiments, the host cell is Escherichia coli (E.coli) TG1 or BL21 cell, CHO-DG44 or CHO-K1 cell or HEK293 cell. According to a specific embodiment, the recombinant expression vector is transformed into a host cell by conventional methods such as chemical transfection, heat shock or electroporation, wherein the recombinant expression vector is stably integrated into the host cell genome so that the recombinant nucleic acid is effectively expressed.

[0150] In another general aspect, the present invention relates to a method for preparing a fusion protein of the present invention, the method comprising culturing a cell comprising a nucleic acid encoding a fusion protein under conditions for preparing the fusion protein of the present invention, and recovering the fusion protein from the cell or cell culture (e.g., from the supernatant). The expressed fusion protein can be harvested from the cell and purified according to conventional techniques known in the art and as described herein.

[0151] Pharmaceutical composition

[0152] In another general aspect, the present invention relates to a pharmaceutical composition comprising a GLP1-GDF15 fusion protein and / or a GLP1-GDF15 fusion polynucleotide of the present invention and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutical composition" means a product comprising a GLP1-GDF15 fusion protein and / or a GLP1-GDF15 fusion polynucleotide of the present invention together with a pharmaceutically acceptable carrier. The GLP1-GDF15 fusion protein and / or GLP1-GDF15 fusion polynucleotide of the present invention and compositions comprising them can also be used to manufacture medicaments for therapeutic applications described herein.

[0153] As used herein, the term "carrier" refers to any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, oil, lipid, lipid-containing vesicle, microsphere, liposome encapsulation, or other materials known in the art for use in pharmaceutical preparations. It should be understood that the characteristics of the carrier, excipient or diluent will depend on the route of administration of the specific application. As used herein, the term "pharmaceutically acceptable carrier" refers to a non-toxic material that does not interfere with the effect of the composition according to the present invention or the biological activity of the composition according to the present invention. According to the present disclosure, according to specific embodiments, any pharmaceutically acceptable carrier suitable for peptide pharmaceutical compositions can be used in the present invention.

[0154] Pharmaceutically acceptable acid salts / anionic salts used in the present invention include, but are not limited to, acetate, benzenesulfonate, benzoate, bicarbonate, bitartrate, bromide, calcium edetate, camphorsulfonate, carbonate, chloride, citrate, dihydrochloride, edetate, edisylate, etopate, esylate, fumarate, glucoheptonate, gluconate, glutamate, p-hydroxyacetamidophenylarsonic acid salt, hexylresorcinol salt, hydrazine, hydrobromide, Hydrochloride, hydroxynaphthoate, iodide, isethionate, lactate, lactobionate, malate, maleate, mandelate, methanesulfonate, methyl bromide, methyl nitrate, methyl sulfate, mucate, naphthenate, nitrate, pamoate, pantothenate, phosphate / diphosphate, polygalacturonate, salicylate, stearate, subacetate, succinate, sulfate, tannate, tartrate, theochlorate, toluenesulfonate and triethiodide. Organic or inorganic acids also include, but are not limited to, hydroiodic acid, perchloric acid, sulfuric acid, phosphoric acid, propionic acid, glycolic acid, methanesulfonic acid, isethionic acid, oxalic acid, 2-naphthalenesulfonic acid, p-toluenesulfonic acid, cyclohexanesulfamic acid, saccharinic acid or trifluoroacetic acid.

[0155] Pharmaceutically acceptable base / cationic salts include, but are not limited to, aluminum, 2-amino-2-hydroxymethyl-propane-1,3-diol (also known as tris(hydroxymethyl)aminomethane, tromethamine or "TRIS"), ammonia, benzathine penicillin, tert-butylamine, chloroprocaine, choline, cyclohexylamine, diethanolamine, ethylenediamine, lithium, L-lysine, magnesium, meglumine, N-methyl-D-glucamine, piperidine, potassium, procaine, quinine, sodium, triethanolamine or zinc.

[0156] In some embodiments of the present invention, a pharmaceutical preparation is provided, which comprises a GLP1-GDF15 fusion protein and / or a GLP1-GDF15 fusion polynucleotide of the present invention in an amount of about 0.001 mg / mL to about 100 mg / mL, about 0.01 mg / mL to about 50 mg / mL, or about 0.1 mg / mL to about 25 mg / mL. The pharmaceutical preparation may have a pH of about 3.0 to about 10, such as about 3 to about 7, or about 5 to about 9. The preparation may also include at least one component selected from the following: a buffer system, a preservative, a tonicity agent, a chelating agent, a stabilizer, and a surfactant.

[0157] The preparation of pharmaceutically active ingredients and pharmaceutically acceptable carriers is known in the art, such as Remington: The Science and Practice of Pharmacy (e.g., 21st edition (2005) and any subsequent editions). Non-limiting examples of additional ingredients include: buffers, diluents, solvents, tonicity regulators, preservatives, stabilizers and chelating agents. One or more pharmaceutically acceptable carriers can be used to formulate the pharmaceutical compositions of the present invention.

[0158] In one embodiment of the invention, the pharmaceutical composition is a liquid formulation. A preferred example of a liquid formulation is an aqueous formulation, i.e. a formulation comprising water. The liquid formulation may comprise a solution, a suspension, an emulsion, a microemulsion, a gel, etc. The aqueous formulation typically comprises at least 50% w / w of water, or at least 60% w / w, 70% w / w, 75% w / w, 80% w / w, 85% w / w, 90% w / w, or at least 95% w / w of water.

[0159] In one embodiment, the pharmaceutical composition can be formulated as an injection that can be injected, for example, via an injection device (eg, a syringe or an infusion pump). The injection can be delivered, for example, subcutaneously, intramuscularly, intraperitoneally, or intravenously.

[0160] In another embodiment, the pharmaceutical composition is a solid preparation, such as a freeze-dried or spray-dried composition, which can be used as is, or a solvent and / or diluent can be added by a physician or patient before use. Solid dosage forms can include tablets, such as compressed and / or coated tablets, and capsules (e.g., hard gelatin capsules or soft gelatin capsules). The pharmaceutical composition can also be in the form of a sachet, dragee, powder, granule, lozenge, or powder, for example, for reconstitution.

[0161] Dosage forms may be immediate release, in which case they may contain a water soluble or dispersible carrier, or they may be delayed, sustained or modified release, in which case they may contain a water insoluble polymer that modulates the rate of dissolution of the dosage form in the gastrointestinal tract.

[0162] In other embodiments, the pharmaceutical composition may be delivered intranasally, intrabuccally, or sublingually.

[0163] The pH in the aqueous formulation may be between pH 3 and pH 10. In one embodiment of the invention, the pH of the formulation is from about 7.0 to about 9.5. In another embodiment of the invention, the pH of the formulation is from about 3.0 to about 7.0.

[0164] In another embodiment of the present invention, the pharmaceutical composition comprises a buffer. Non-limiting examples of buffers include: arginine, aspartic acid, bicine, citrate, sodium dihydrogen phosphate, fumaric acid, glycine, glycine, histidine, lysine, maleic acid, malic acid, sodium acetate, sodium carbonate, sodium dihydrogen phosphate, sodium phosphate, succinate, tartaric acid, triazine and tris (hydroxymethyl) aminomethane and mixtures thereof. Buffers may exist alone or in aggregates with a concentration of about 0.01 mg / ml to about 50 mg / ml, for example, about 0.1 mg / ml to about 20 mg / ml. Pharmaceutical compositions comprising each of these specific buffers constitute alternative embodiments of the present invention.

[0165] In another embodiment of the present invention, pharmaceutical composition comprises preservative.The non-limiting example of preservative comprises: benzethonium chloride, benzoic acid, benzyl alcohol, bromonitropropylene glycol, 4-hydroxy butyl benzoate, chlorobutanol, chlorocresol, chlorhexidine, chlorphenesin ether, o-cresol, meta-cresol, p-cresol, 4-hydroxy ethyl benzoate, imidourea, 4-hydroxy methyl benzoate, phenol, 2-phenoxyethanol, 2-phenylethanol, 4-hydroxy propyl benzoate, sodium dehydroacetate, thimerosal and their mixture.Preservative can exist alone or in aggregate with about 0.01mg / ml to about 50mg / ml, for example, about 0.1mg / ml to about 20mg / ml concentration exists.The pharmaceutical composition comprising each in these specific preservatives constitutes the alternative embodiment of the present invention.

[0166] In another embodiment of the present invention, the pharmaceutical composition comprises an isotonic agent. Non-limiting examples of this embodiment include salts (such as sodium chloride), amino acids (such as glycine, histidine, arginine, lysine, isoleucine, aspartic acid, tryptophan and threonine), sugar alcohols (such as glycerol, 1,2-propylene glycol, propylene glycol), 1,3-propylene glycol and 1,3-butylene glycol), polyethylene glycol (e.g., PEG400) and mixtures thereof. Another example of an isotonic agent includes sugar. Non-limiting examples of sugars can be monosaccharides, disaccharides or polysaccharides, or water-soluble glucans, including, for example, fructose, glucose, mannose, sorbose, xylose, maltose, lactose, sucrose, trehalose, glucan, amylopectin, dextrin, cyclodextrin, α and β-HPCD, soluble starch, hydroxyethyl starch and sodium carboxymethylcellulose. Another example of an isotonic agent is sugar alcohol, wherein the term "sugar alcohol" is defined as a C (4-8) hydrocarbon with at least one -OH group. The non-limiting example of sugar alcohol comprises mannitol, sorbitol, inositol, galactitol, hexadecene, xylitol and arabitol.The pharmaceutical composition comprising every kind of isotonic agent listed in this paragraph constitutes alternative embodiment of the present invention.Isotonic agent can exist alone or in aggregate with about 0.01mg / ml to about 50mg / ml, for example, about 0.1mg / ml to about 20mg / ml concentration exists.The pharmaceutical composition comprising each in these specific isotonic agents constitutes alternative embodiment of the present invention.

[0167] In another embodiment of the present invention, the pharmaceutical composition comprises a chelating agent. Non-limiting examples of chelating agents include salts of citric acid, aspartic acid, ethylenediaminetetraacetic acid (EDTA), and mixtures thereof. Chelating agents may exist alone or in aggregates at a concentration of about 0.01 mg / ml to about 50 mg / ml, for example, about 0.1 mg / ml to about 20 mg / ml. Pharmaceutical compositions comprising each of these specific chelating agents constitute alternative embodiments of the present invention.

[0168] In another embodiment of the present invention, the pharmaceutical composition comprises a stabilizer. Non-limiting examples of stabilizers include one or more aggregation inhibitors, one or more oxidation inhibitors, one or more surfactants and / or one or more protease inhibitors.

[0169] In another embodiment of the invention, the pharmaceutical composition comprises a stabilizer, wherein the stabilizer is carboxy- / hydroxycellulose and its derivatives (such as HPC, HPC-SL, HPC-L and HPMC), cyclodextrin, 2-methylthioethanol, polyethylene glycol (such as PEG 3350), polyvinyl alcohol (PVA), polyvinyl pyrrolidone, salts (such as sodium chloride), sulfur-containing substances such as thioglycerol or thioglycolic acid. The stabilizer may be present alone or in aggregates at a concentration of about 0.01 mg / ml to about 50 mg / ml, for example, about 0.1 mg / ml to about 20 mg / ml. Pharmaceutical compositions comprising each of these specific stabilizers constitute alternative embodiments of the present invention.

[0170] In another embodiment of the present invention, the pharmaceutical composition comprises one or more surfactants, preferably a surfactant, at least one surfactant or two different surfactants. The term "surfactant" refers to any molecule or ion consisting of a water-soluble portion (hydrophilicity) and a fat-soluble and part (lipophilicity). For example, the surfactant can be selected from: anionic surfactants, cationic surfactants, nonionic surfactants and / or zwitterionic surfactants. Surfactants can exist alone or in aggregates with a concentration of about 0.1mg / ml to about 20mg / ml. Pharmaceutical compositions comprising each of these specific surfactants constitute alternative embodiments of the present invention.

[0171] In another embodiment of the invention, the pharmaceutical composition comprises one or more protease inhibitors, such as, for example, EDTA and / or benzamidine hydrochloride (HCl). The protease inhibitors may be present alone or in aggregates at a concentration of about 0.1 mg / ml to about 20 mg / ml. Pharmaceutical compositions comprising each of these specific protease inhibitors constitute alternative embodiments of the invention.

[0172] Pharmaceutical composition of the present invention may include a certain amount of amino acid bases that are enough to reduce the aggregation formation of polypeptide during storage of the composition.Term " amino acid base " refers to one or more amino acids (such as methionine, histidine, imidazole, arginine, lysine, isoleucine, aspartic acid, tryptophan, threonine) or their analogs.Any amino acid can exist in its free alkali form or its salt form.Any stereoisomer (i.e. L, D or their mixture) of amino acid base may be present.Amino acid base may exist alone or in combination with other amino acid bases with a concentration of about 0.01mg / ml to about 50mg / ml, for example, about 0.1mg / ml to about 20mg / ml.The pharmaceutical composition comprising each of these specific amino acid bases constitutes an alternative embodiment of the present invention.

[0173] Pharmaceutically acceptable salts of the GLP1-GDF15 fusion protein and / or GLP1-GDF15 fusion polynucleotide of the present invention include conventional non-toxic salts or quaternary ammonium salts formed from inorganic or organic acids or bases. Examples of such acid addition salts include acetates, adipates, benzoates, benzenesulfonates, citrates, camphorates, dodecyl sulfates, hydrochlorides, hydrobromides, lactates, maleates, methanesulfonates, nitrates, oxalates, pivalates, propionates, succinates, sulfates, and tartrates. Alkali salts include ammonium salts, alkali metal salts such as sodium and potassium salts, alkaline earth metal salts such as calcium and magnesium salts, organic alkali salts such as dicyclohexylamine salts, and amino acids such as arginine. In addition, basic nitrogen-containing groups can be quaternized with, for example, alkyl halides.

[0174] The pharmaceutical compositions of the present invention can be administered in any manner that achieves its intended purpose. Examples include administration by parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, oral or ocular routes. Administration can be by oral route. Formulations suitable for parenteral administration include aqueous solutions of water-soluble forms of the active conjugate (e.g., water-soluble salts), acidic solutions, alkaline solutions, aqueous dextrose solutions, isotonic carbohydrate solutions, and cyclodextrin inclusion complexes.

[0175] The present invention also encompasses a method for preparing a pharmaceutical composition, the method comprising mixing a pharmaceutically acceptable carrier with any of the GLP1-GDF15 fusion proteins and / or GLP1-GDF15 fusion polynucleotides of the present invention. In addition, the present invention includes a pharmaceutical composition prepared by mixing one or more pharmaceutically acceptable carriers with any of the GLP1-GDF15 fusion proteins and / or GLP1-GDF15 fusion polynucleotides of the present invention.

[0176] In addition, the GLP1-GDF15 fusion protein and / or GLP1-GDF15 fusion polynucleotide of the present invention may have one or more polymorphs or amorphous crystalline forms, and are therefore intended to be included within the scope of the present invention. In addition, the GLP1-GDF15 fusion protein and / or the GLP1-GDF15 fusion polynucleotide may form a solvate (i.e., a hydrate) with, for example, water or a solvate with a commonly used organic solvent. The term "solvate" as used herein means the physical association of the GLP1-GDF15 fusion protein and / or the GLP1-GDF15 fusion polynucleotide of the present invention with one or more solvent molecules. The physical association involves varying degrees of ionic bonding and covalent bonding, including hydrogen bonding. In some cases, the solvate will be able to be separated when, for example, one or more solvent molecules are incorporated into the crystal lattice of the crystalline solid. The term "solvate" is intended to cover both solution-phase solvates and separable solvates. Non-limiting examples of suitable solvates include ethanolates, methanolates, and the like.

[0177] The present invention is intended to include within its scope the polymorphs and solvates of the GLP1-GDF15 fusion protein and / or GLP1-GDF15 fusion polynucleotide of the present invention. Therefore, in the therapeutic methods of the present invention, the term "administering" should cover methods for treating, ameliorating or preventing the syndromes, disorders or diseases described herein using the GLP1-GDF15 fusion protein and / or GLP1-GDF15 fusion polynucleotide of the present invention or their polymorphs or solvates, which, although not specifically disclosed, will be clearly included within the scope of the present invention.

[0178] In another embodiment, the present invention relates to a GLP1-GDF15 fusion protein and / or a GLP1-GDF15 fusion polynucleotide of the present invention for use as a medicament.

[0179] The present invention includes within its scope prodrugs of the GLP1-GDF15 fusion proteins and / or GLP1-GDF15 fusion polynucleotides of the present invention. Generally speaking, such prodrugs will be functional derivatives of the GLP1-GDF15 fusion proteins and / or GLP1-GDF15 fusion polynucleotides that can be easily converted in vivo into the desired GLP1-GDF15 fusion proteins and / or GLP1-GDF15 fusion polynucleotides. Therefore, in the treatment methods of the present invention, the term "administering" shall cover the treatment of the various disorders described with the specifically disclosed GLP1-GDF15 fusion proteins and / or GLP1-GDF15 fusion polynucleotides or with GLP1-GDF15 fusion proteins and / or GLP1-GDF15 fusion polynucleotides that may not be specifically disclosed but are converted in vivo into the specified GLP1-GDF15 fusion proteins and / or GLP1-GDF15 fusion polynucleotides after administration to the patient. Conventional methods for the selection and preparation of suitable prodrug derivatives are described, for example, in "Design of Prodrugs", ed. H. Bundgaard, Elsevier, 1985.

[0180] In any of the methods for preparing the GLP1-GDF15 fusion proteins and / or GLP1-GDF15 fusion polynucleotides of the invention, it may be necessary and / or desirable to protect sensitive or reactive groups on any of the molecules involved. This can be achieved by conventional protecting groups, such as Protective Groups in Organic Chemistry, ed. JFW McOmie, Plenum Press, 1973; and TW Greene and PGM Wuts, Protective Groups in Organic Synthesis, John Wiley & Sons, 1991, each of which is incorporated herein by reference in its entirety for all purposes. Protecting groups can be removed at a convenient subsequent stage using methods known in the art.

[0181] How to use

[0182] The present invention relates to a method for preventing, treating or ameliorating a GDF15 receptor (GDF15R, GFRAL)-mediated syndrome and / or a GLP1 receptor-mediated syndrome, disorder or disease in a subject in need thereof, the method comprising administering an effective amount of the GLP1-GDF15 fusion protein, GLP1-GDF15 fusion polynucleotide and / or pharmaceutical composition of the present invention to a subject in need thereof.

[0183] The present invention also provides a method for preventing, treating or ameliorating a disorder, disease or condition or any one or more symptoms of the disorder, disease or condition in a subject in need thereof, or delaying the onset thereof, the method comprising administering to a subject in need thereof an effective amount of the GLP1-GDF15 fusion protein, GLP1-GDF15 fusion polynucleotide and / or pharmaceutical composition of the present invention.

[0184] According to specific embodiments, the disease, disorder or condition is selected from obesity, type I or type II diabetes, metabolic syndrome (i.e., syndrome X), insulin resistance, impaired glucose tolerance (e.g., glucose intolerance), hyperglycemia, hyperinsulinemia, hypertriglyceridemia, hypoglycemia due to congenital hyperinsulinemia (CHI), dyslipidemia, atherosclerosis, diabetic nephropathy, and other cardiovascular risk factors such as hypertension and cardiovascular risk factors associated with uncontrolled cholesterol and / or lipid levels, osteoporosis, inflammation, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), kidney disease, eczema, sleep apnea, osteoarthritis, polycystic ovary syndrome, chronic renal syndrome, depression and / or cancer.

[0185] According to specific embodiments, a therapeutically effective amount refers to a therapeutic amount sufficient to achieve one, two, three, four, or more of the following effects: (i) reduce or ameliorate the severity of the disease, disorder, or condition being treated or symptoms associated therewith; (ii) reduce the duration of the disease, disorder, or condition being treated or symptoms associated therewith; (iii) prevent the development of the disease, disorder, or condition being treated or symptoms associated therewith; (iv) cause the regression of the disease, disorder, or condition being treated or symptoms associated therewith; (v) prevent the development or onset of the disease, disorder, or condition being treated or symptoms associated therewith; (vi) prevent the recurrence of the disease, disorder or condition being treated or symptoms associated therewith; (vii) reduce hospitalization of subjects suffering from the disease, disorder or condition being treated or symptoms associated therewith; (viii) reduce the length of hospital stay of subjects suffering from the disease, disorder or condition being treated or symptoms associated therewith; (ix) improve the survival of subjects suffering from the disease, disorder or condition being treated or symptoms associated therewith; (xi) inhibit or reduce the symptoms of the disease, disorder or condition being treated or symptoms associated therewith in a subject; (xii) enhance or improve the preventive or therapeutic effect of another therapy. And / or (xiii) improve the quality of life of subjects suffering from the disease, disorder or condition being treated.

[0186] The therapeutically effective amount or dosage may vary depending on various factors, such as the disease, disorder or condition to be treated, the mode of administration, the target site, the physiological state of the subject (including, for example, age, weight, health), whether the subject is human or animal, other drugs administered, and whether the treatment is prophylactic or therapeutic. The therapeutic dose is optimally titrated to optimize safety and efficacy.

[0187] As used herein, the terms "treat," "treatment," and "therapy" are all intended to mean an improvement or reversal of at least one measurable physical parameter associated with a disease, disorder, or condition that is not necessarily identifiable in a subject, but is capable of being identified in a subject. The terms "treatment" and "treatment" may also refer to causing regression, preventing development, or at least delaying the development of a disease, disorder, or condition. In a specific embodiment, "treatment," "treatment," and "therapy" refer to mitigation, prevention of development or onset, or shortening the duration of one or more symptoms associated with a disease, disorder, or condition. In specific embodiments, "treatment" and "treatment" refer to preventing the recurrence of a disease, disorder, or condition. In specific embodiments, "treatment" and "treatment" refer to improved survival of a subject suffering from a disease, disorder, or condition. In specific embodiments, "treatment" and "treatment" refer to the elimination of a disease, disorder, or condition in a subject.

[0188] In one embodiment, the present invention provides a method for preventing, treating or ameliorating obesity or any one or more symptoms of obesity in a subject in need thereof, or delaying its onset, the method comprising administering to a subject in need thereof an effective amount of a GLP1-GDF15 fusion protein, a GLP1-GDF15 fusion polynucleotide and / or a pharmaceutical composition of the present invention. In some embodiments, the subject's weight loss is, for example, between about 0.01% and about 0.1%, relative to the subject's weight before administration of any of the GLP1-GDF15 fusion proteins, GLP1-GDF15 fusion polynucleotides, pharmaceutical compositions, forms or drugs of the present invention described herein, or compared to a control subject who has not received any of the GLP1-GDF15 fusion proteins, GLP1-GDF15 fusion polynucleotides, compositions, forms or drugs of the present invention described herein. Between about 0.1% to about 0.5%, between about 0.5% to about 1%, between about 1% to about 5%, between about 2% to about 3%, between about 5% to about 10%, between about 10% to about 15%, between about 15% to about 20%, between about 20% to about 25%, between about 25% to about 30%, between about 30% to about 35%, between about 35% to about 40%, between about 40% to about 45%, or between about 45% to about 50%.

[0189] In some embodiments, the reduction in body weight lasts, for example, for about 1 week, about 2 weeks, about 3 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 1 year, about 1.5 years, about 2 years, about 2.5 years, about 3 years, about 3.5 years, about 4 years, about 4.5 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, about 10 years, about 15 years, or about 20 years.

[0190] The present invention provides a method for preventing, treating or ameliorating a syndrome, disorder or disease or any one or more symptoms of the syndrome, disorder or disease in a subject in need thereof, or delaying the onset thereof, wherein the syndrome, disorder or disease is selected from: obesity, type I or type II diabetes, metabolic syndrome (i.e., syndrome X), insulin resistance, impaired glucose tolerance (e.g., glucose intolerance), hyperglycemia, hyperinsulinemia, hypertriglyceridemia, hypoglycemia due to congenital hyperinsulinemia (CHI), dyslipidemia, atherosclerosis, diabetic nephropathy, and other cardiovascular risk factors such as hypertension and cardiovascular risk factors associated with uncontrolled cholesterol and / or lipid levels, osteoporosis, inflammation, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), kidney disease and eczema, the method comprising administering to a subject in need thereof an effective amount of a GLP1-GDF15 fusion protein, GLP1-GDF15 fusion polynucleotide and / or pharmaceutical composition of the present invention.

[0191] As used herein, metabolic syndrome refers to a subject having any one or more of the following: high blood sugar (e.g., high fasting glucose), high blood pressure, abnormal cholesterol levels (e.g., low HDL levels), abnormal triglyceride levels (e.g., high triglycerides), large waist circumference (i.e., waist circumference), increased fat in the abdominal area, insulin resistance, glucose intolerance, elevated C-reactive protein levels (i.e., a pro-inflammatory state), and increased plasma plasminogen activator inhibitor-1 and fibrinogen content (i.e., a pro-thrombotic state).

[0192] The present invention provides a method for reducing food intake in a subject in need thereof, the method comprising administering to a subject in need thereof an effective amount of a GLP1-GDF15 fusion protein, GLP1-GDF15 fusion polynucleotide and / or pharmaceutical composition of the present invention. In some embodiments, the subject's food intake is reduced, for example, by between about 0.01% and about 0.1% relative to the subject's food intake prior to administration of any of the GLP1-GDF15 fusion proteins, GLP1-GDF15 fusion polynucleotides, compositions, forms, drugs, or combinations of the present invention described herein, or compared to a control subject who has not received any of the GLP1-GDF15 fusion proteins, GLP1-GDF15 fusion polynucleotides, compositions, forms, drugs, or combinations of the present invention described herein. %, between about 0.1% to about 0.5%, between about 0.5% to about 1%, between about 1% to about 5%, between about 2% to about 3%, between about 5% to about 10%, between about 10% to about 15%, between about 15% to about 20%, between about 20% to about 25%, between about 25% to about 30%, between about 30% to about 35%, between about 35% to about 40%, between about 40% to about 45%, or between about 45% to about 50%.

[0193] In some embodiments, the reduction in food intake lasts, for example, for about 1 week, about 2 weeks, about 3 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 1 year, about 1.5 years, about 2 years, about 2.5 years, about 3 years, about 3.5 years, about 4 years, about 4.5 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, about 10 years, about 15 years, or about 20 years.

[0194] The present invention provides a method for reducing glycated hemoglobin (A1C) in a subject in need thereof, the method comprising administering to a subject in need thereof an effective amount of a GLP1-GDF15 fusion protein, a GLP1-GDF15 fusion polynucleotide, and / or a pharmaceutical composition of the present invention. In some embodiments, the subject's A1C is reduced, for example, by about 0.001% and about 0.01%, between about 0.01% and about 0.1%, between ... and about 0.2%, between about 0.2% and about 0.3%, between about 0.3% and about 0.4%, between about 0.4% and about 0.5%, between about 0.5% and about 1%, between about 1% and about 1.5%, between about 1.5% and about 2%, between about 2% and about 2.5%, between about 2.5% and about 3%, between about 3% and about 4%, between about 4% and about 5%, between about 5% and about 6%, between about 6% and about 7%, between about 7% and about 8%, between about 8% and about 9%, or between about 9% and about 10%.

[0195] In other embodiments, methods for reducing fasting blood glucose levels in a subject in need thereof are provided, comprising administering to a subject in need thereof an effective amount of a GLP1-GDF15 fusion protein, GLP1-GDF15 fusion polynucleotide and / or pharmaceutical composition of the present invention. Relative to the subject's fasting blood glucose level before administration of any of the GLP1-GDF15 fusion proteins, GLP1-GDF15 fusion polynucleotides, compositions, forms, medicaments, or combinations of the invention described herein, or compared to a control subject not receiving any of the GLP1-GDF15 fusion proteins, GLP1-GDF15 fusion polynucleotides, compositions, forms, medicaments, or combinations of the invention described herein, fasting blood glucose levels may be reduced to less than about 140 mg / dL to about 150 mg / dL, less than about 140 mg / dL to about 130 mg / dL, less than about 130 mg / dL to about 120 mg / dL, less than about 120 mg / dL to about 110 mg / dL, less than about 110 mg / dL to about 100 mg / dL, less than about 100 mg / dL to about 90 mg / dL, or less than about 90 mg / dL to about 80 mg / dL.

[0196] The present invention provides a method for regulating the GLP1 receptor activity and GDF15 receptor activity of a subject in need thereof, the method comprising administering an effective amount of the GLP1-GDF15 fusion protein, GLP1-GDF15 fusion polynucleotide and / or pharmaceutical composition of the present invention to a subject in need thereof. As used herein, "regulating" refers to increasing or decreasing receptor activity.

[0197] In some embodiments, an effective amount of the GLP1-GDF15 fusion protein and / or GLP1-GDF15 fusion polynucleotide of the present invention or a form, composition or drug thereof is administered once a day, twice a day, three times a day, four times a day, five times a day, six times a day, seven times a day, or eight times a day to a subject in need thereof. In other embodiments, an effective amount of the GLP1-GDF15 fusion protein and / or GLP1-GDF15 fusion polynucleotide of the present invention or a form, composition or drug thereof is administered once every other day, once a week, twice a week, three times a week, four times a week, five times a week, six times a week, twice a month, three times a month, or four times a month to a subject in need thereof.

[0198] Another embodiment of the present invention includes a method for preventing, treating or ameliorating a disease, disorder or syndrome or one or more symptoms of any of the diseases, disorders or syndromes in a subject in need thereof, or delaying the onset thereof, the method comprising administering an effective amount of the GLP1-GDF15 fusion protein, GLP1-GDF15 fusion polynucleotide and / or pharmaceutical composition of the present invention to a subject in need thereof in the form of a combination therapy. In certain embodiments, the combination therapy is a second therapeutic agent. In certain embodiments, the combination therapy is surgical therapy.

[0199] As used herein, the term "in combination with" in the context of administering two or more therapies to a subject refers to the use of more than one therapy.

[0200] As used herein, combination therapy refers to administering an effective amount of the GLP1-GDF15 fusion protein and / or GLP1-GDF15 fusion polynucleotide of the present invention, or a form, composition or medicament thereof, to a subject in need thereof, and concurrently administering one or more additional therapeutic agents, or one or more surgical therapies. In some embodiments, one or more additional therapeutic agents or surgical therapies may be administered on the same day as an effective amount of the GLP1-GDF15 fusion protein and / or GLP1-GDF15 fusion polynucleotide of the present invention, and in other embodiments, one or more additional therapeutic agents or surgical therapies may be administered in the same week or month as an effective amount of the GLP1-GDF15 fusion protein and / or GLP1-GDF15 fusion polynucleotide of the present invention.

[0201] The present invention also contemplates the use of combination therapy to prevent, treat or improve any of the diseases, disorders, syndromes or symptoms described herein in a subject in need thereof, or to delay the onset thereof, the combination therapy comprising administering to a subject in need thereof an effective amount of the GLP1-GDF15 fusion protein, GLP1-GDF15 fusion polynucleotide and / or pharmaceutical composition of the present invention in combination with any one or more of the following therapeutic agents: dipeptidyl peptidase-4 (DPP-4) inhibitors (e.g., sitagliptin, saxagliptin, linagliptin, alogliptin, etc.); GLP1 receptor agonists (e.g., short-acting GLP1 receptor agonists, such as exenatide and lixisenatide; intermediate-acting GLP1 receptor agonists, such as liraglutide; long-acting GLP1 receptor agonists, such as extended-release exenatide, albiglutide, dulaglutide); sodium-glucose co-administration; SGLT-2 inhibitors (e.g., canaglifozin, dapaglifozin, empaglifozin); bile acid sequestrants (e.g., colesevelam, etc.); dopamine receptor agonists (e.g., immediate-release bromocriptine); biguanides (e.g., metformin, etc.); insulin; oxyntomodulin; sulfonylureas (e.g., chlorpromazine, glimepiride, glipizide, glyburide, glibenclamide, gliprexa, gliprexa, glyburide, gliprexa, gliprexa, glyburide, glyburide, tolsulfuronamide, tolbutamide, acetophenonesulfonylcyclohexylamide, amoxicillin, etc.); and thiazolidinediones (e.g., pioglitazone, rosiglitazone, lobeglitazone, ciglitazone, daglitazone, englitazone, neglitazone, rivoglitazone, troglitazone, etc.). In some embodiments, the dosage of the additional therapeutic agent is reduced when provided in combination with the GLP1-GDF15 fusion protein and / or GLP1-GDF15 fusion polynucleotide of the present invention. In some embodiments, when used in combination with the GLP1-GDF15 fusion protein and / or GLP1-GDF15 fusion polynucleotide of the present invention, the additional therapeutic agent can be used at a lower dose than when each is used alone.

[0202] The present invention contemplates the use of combined therapy to prevent, treat or ameliorate any of the diseases, disorders, syndromes or symptoms described herein in a subject in need thereof, or to delay the onset thereof, the combined therapy comprising administering to a subject in need thereof an effective amount of a GLP1-GDF15 fusion protein, a GLP1-GDF15 fusion polynucleotide and / or a pharmaceutical composition of the present invention in combination with surgical therapy. In certain embodiments, the surgical therapy may be obesity surgery (e.g., gastric bypass surgery, such as biliojejunal gastric bypass surgery; sleeve gastric reduction surgery; adjustable gastric banding surgery; biliopancreatic diversion duodenal switch surgery; indwelling gastric balloon; gastric plication; and combinations thereof).

[0203] In embodiments where one or more additional therapeutic agents or surgical therapies are administered on the same day as an effective amount of a GLP1-GDF15 fusion protein and / or a GLP1-GDF15 fusion polynucleotide of the invention, the GLP1-GDF15 fusion protein and / or a GLP1-GDF15 fusion polynucleotide of the invention may be administered before, after, or simultaneously with the additional therapeutic agent or surgical therapy. The use of the term "in combination" does not limit the order in which the therapies are administered to a subject. For example, a first therapy (e.g., a composition described herein) can be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 16 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concurrently with, or after (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 16 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks later) the second therapy is administered to the subject.

[0204] Implementation

[0205] The present invention also provides the following non-limiting embodiments.

[0206] Embodiment 1 is a glucagon-like peptide-1 (GLP1) / growth differentiation factor 15 (GDF15) fusion protein, wherein the GLP1-GDF15 fusion protein comprises a GLP1 peptide, a first linker peptide, serum albumin, a second linker peptide and a GDF15 protein.

[0207] Embodiment 2 is the GLP1-GDF15 fusion protein according to embodiment 1, wherein the GLP1 peptide comprises an amino acid sequence selected from SEQ ID NOs: 1-4.

[0208] Embodiment 3 is the GLP1-GDF15 fusion protein according to embodiment 1 or 2, wherein the first linker peptide comprises an amino acid sequence selected from SEQ ID NO: 5-25.

[0209] Embodiment 4 is the GLP1-GDF15 fusion protein according to any one of embodiments 1-3, wherein the serum albumin comprises an amino acid sequence selected from SEQ ID NO:26 or SEQ ID NO:27.

[0210] Embodiment 5 is the GLP1-GDF15 fusion protein according to any one of embodiments 1-4, wherein the second linker peptide comprises an amino acid sequence selected from SEQ ID NOs: 28-30.

[0211] Embodiment 6 is the GLP1-GDF15 fusion protein according to any one of embodiments 1-5, wherein the GDF15 protein comprises an amino acid sequence selected from SEQ ID NO:31 or SEQ ID NO:32.

[0212] Embodiment 7 is a GLP1-GDF15 fusion protein, wherein the GLP1-GDF15 fusion protein comprises an amino acid sequence selected from SEQ ID NO: 33-74 and 84.

[0213] Embodiment 8 is an isolated nucleic acid encoding the GLP1-GDF15 fusion protein according to any one of embodiments 1-7.

[0214] Embodiment 9 is a vector comprising the isolated nucleic acid according to embodiment 8.

[0215] Embodiment 10 is a host cell comprising the isolated nucleic acid according to claim 8 or the vector according to claim 9.

[0216] Embodiment 11 is a pharmaceutical composition comprising the GLP1-GDF15 fusion protein according to any one of Embodiments 1-7 and a pharmaceutically acceptable carrier.

[0217] Embodiment 12 is a method for treating or preventing obesity in a subject in need thereof, comprising administering an effective amount of the pharmaceutical composition according to embodiment 11 to the subject in need thereof.

[0218] Embodiment 13 is a method according to embodiment 12, wherein administering an effective amount of the pharmaceutical composition to a subject in need thereof results in a reduction in body weight of about 5% to about 10%, about 10% to about 15%, about 15% to about 20%, or about 20% to about 25% compared to the subject's body weight before administration of the pharmaceutical composition.

[0219] Embodiment 14 is a method for treating or preventing a disease or disorder in a subject in need thereof, wherein the disease or disorder is selected from: obesity, type I or type II diabetes, metabolic syndrome, insulin resistance, impaired glucose tolerance, hyperglycemia, hyperinsulinemia, hypertriglyceridemia, hypoglycemia due to congenital hyperinsulinemia (CHI), dyslipidemia, atherosclerosis, diabetic nephropathy, and other cardiovascular risk factors such as hypertension and cardiovascular risk factors associated with uncontrolled cholesterol and / or lipid levels, osteoporosis, inflammation, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), kidney disease and eczema, the method comprising administering to a subject in need thereof an effective amount of the pharmaceutical composition according to embodiment 11.

[0220] Embodiment 15 is a method according to embodiment 14, wherein the disease or disorder is obesity.

[0221] Embodiment 16 is a method according to embodiment 14, wherein the disease or disorder is type 1 diabetes.

[0222] Embodiment 17 is a method according to embodiment 14, wherein the disease or disorder is type II diabetes.

[0223] Embodiment 18 is a method according to embodiment 14, wherein the disease or disorder is metabolic syndrome.

[0224] Embodiment 19 is a method according to embodiment 14, wherein the disease or disorder is kidney disease.

[0225] Embodiment 20 is a method according to embodiment 14, wherein the disease or disorder is non-alcoholic steatohepatitis (NASH).

[0226] Embodiment 21 is a method according to embodiment 14, wherein the disease or disorder is non-alcoholic fatty liver disease (NAFLD).

[0227] Embodiment 22 is a method of reducing food intake in a subject in need thereof, comprising administering to the subject in need thereof an effective amount of the pharmaceutical composition according to embodiment 11.

[0228] Embodiment 23 is a method according to embodiment 22, wherein administering an effective amount of the pharmaceutical composition to a subject in need thereof results in a decrease in food intake of about 5% to about 10%, about 10% to about 15%, about 15% to about 20%, about 20% to about 25%, about 25% to about 30%, about 30% to about 35%, about 35% to about 40%, about 40% to about 45%, or about 45% to about 50% compared to the subject's food intake prior to administration of the pharmaceutical composition.

[0229] Embodiment 24 is a method of modulating GLP1 receptor activity in a subject in need thereof, the method comprising administering an effective amount of the pharmaceutical composition according to embodiment 11 to the subject in need thereof.

[0230] Embodiment 25 is a method of modulating GDF15 receptor activity in a subject in need thereof, the method comprising administering an effective amount of the pharmaceutical composition according to embodiment 11 to the subject in need thereof.

[0231] Embodiment 26 is a method according to any one of embodiments 11-25, wherein the pharmaceutical composition is administered via injection.

[0232] Embodiment 27 is a method according to embodiment 26, wherein the injection is delivered subcutaneously, intramuscularly, intraperitoneally, or intravenously.

[0233] Embodiment 28 is a method according to any one of embodiments 11-27, wherein the pharmaceutical composition is administered in combination with a second therapeutic agent.

[0234] Embodiment 29 is the method according to any one of embodiments 11-28, wherein the pharmaceutical composition is administered to a subject in need thereof daily, weekly or monthly.

[0235] Embodiment 30 is the method according to embodiment 29, wherein the pharmaceutical composition is administered once, twice, three times, four times, five times or six times per day.

[0236] Embodiment 31 is a method according to embodiment 29, wherein the pharmaceutical composition is administered once, twice, three times, four times, five times or six times per week.

[0237] Embodiment 32 is the method according to embodiment 29, wherein the pharmaceutical composition is administered once, twice, three times or four times per month.

[0238] Embodiment 33 is a kit comprising the GLP1-GDF15 fusion protein according to any one of embodiments 1-7, the isolated nucleic acid according to embodiment 8 and / or the vector according to embodiment 9.

[0239] Embodiment 34 is a kit according to embodiment 33, wherein the kit further comprises a device for injection.

[0240] Embodiment 35 is a method for preparing a pharmaceutical composition comprising the GLP1-GDF15 fusion protein according to any one of Embodiments 1-7, the method comprising combining the GLP1-GDF15 fusion protein with a pharmaceutically acceptable carrier to obtain the pharmaceutical composition.

[0241] Embodiment 36 is a method for preparing the GLP1-GDF15 fusion protein according to any one of embodiments 1-7, the method comprising culturing cells containing a nucleic acid encoding the GLP1-GDF15 fusion protein under conditions for preparing the GLP1-GDF15 fusion protein, and recovering the GLP1-GDF15 fusion protein from the cells or culture.

[0242] Example

[0243] Example 1: Combination of GLP1 and GDF15 agonists

[0244] The potential additive weight loss effect of combining GLP1 and GDF15 agonists was tested in diet-induced obese (DIO) mice. Liraglutide, a GLP1 agonist, was administered alone or in combination with the HSA-GDF15 (SEQ ID NO: 81) molecule for 8 days. Liraglutide was administered subcutaneously daily while HSA-GDF15 was fed every two days. Compared to either single agent, weight loss was greater in animals receiving both agonists, suggesting that there is potential for additive weight loss when these independent mechanisms are combined ( Figure 1 ).

[0245] Example 2: Design of GLP1-GDF15 fusion protein

[0246] The recombinant fusion protein composed of GLP1 peptide or GLP1 variant peptide, serum albumin and GDF15 protein or GDF15 variant protein is designed as follows: GLP1 peptide or GLP1 variant peptide is connected to the N-terminus of human serum albumin via the first linker peptide at the C-terminus of GLP1 peptide or GLP1 variant peptide. Human serum albumin is connected to the N-terminus at the C-terminus of GDF15 protein or GDF15 protein variant via the second linker peptide. The design is intended to undisturb the GDF15 dimerization interface and allow the formation of native interchain disulfides, thereby obtaining a homodimer of approximately 170KDa. The entire molecule is designed to be prepared in a recombinant manner, requiring only one gene ( Figure 3 ).

[0247] GLP1 peptides or GLP1 variant peptides include exendin 4 (9-39) peptide (which is a venom peptide from the Gila monster that agonizes the human GLP1 receptor) or a human GLP1 (7-36) peptide with a mutation. SEQ ID NO: 1 includes (A8S, A30E) mutations (the numbering of the mutation position refers to the GLP1 peptide mutations in Table 1, not starting from the fusion protein), and SEQ ID NO: 2 includes (A8G, G22E, R36G) mutations from a human GLP1 peptide (UniProtKB-P0127598-127). SEQ ID NO: 3 includes exendin 4 peptide (UniProtKB-P2634948-86).

[0248] Native human serum albumin (UniProtKB-P0276825-609) contains 35 cysteine ​​(Cys, C) residues that form 17 intramolecular disulfide bonds, leaving Cys-34 as the only free cysteine. This free Cys-34 has been shown to act as a free radical scavenger by capturing a variety of reactive oxygen species (ROS) and reactive nitrogen species (RNS) (Taverna et al., Ann. Intensive Care, Vol. 3, p. 4 (2013)). Therefore, this free Cys was mutated to Ser to generate SEQ ID NO: 26, thereby minimizing chemical reactivity and the risk of heterogeneity due to oxidation.

[0249] The N-terminus of mature GDF15 (UniProtKB-Q99988197-308) contains a proteolytically susceptible site (R198) and a deamidation susceptible site (N199). Thus, the fusion protein comprises GDF15 (201-308) (SEQ ID NO: 31), in which those susceptible sites are deleted.

[0250] Example 3: Expression and purification methods

[0251] The GLP1-GDF15 fusion protein, GLP1-first linker-serum albumin (eg, HSA, GSA), serum albumin (eg, HSA, GSA)-second linker-GDF15 protein and / or GDF15 protein used in the above examples are grown in HEKExpi293F TM (ThermoFisher Scientific, catalog number A14527) or ExpiCHO-S TM (ThermoFisher Scientific, Waltham, MA, catalog number A29127). TMThe plasmid encoding the GLP1-GDF15 fusion protein was transfected into the cells by transient transfection according to the manufacturer's recommendations. Briefly, Expi293F cells were cultured in an Expi293 shaking incubator set at 37°C, 8% CO2, and 125 RPM. TM The cells were kept suspended in expression medium (ThermoFisher Scientific). The cells were passaged so that on the day of transfection, a dilution of 2.5 × 10 per ml could be achieved. 6 cells and maintained cell viability at 95% or better. TM Transient transfection was performed using the OptiMEM 293 transfection kit (ThermoFisher Scientific). For each ml of diluted cells to be transfected, one milligram of plasmid DNA was diluted into OptiMEM TM ExpiFectamine was used at a ratio of 1:2.6 (v / v, DNA:reagent) in SFM complex medium. TM 293 reagent, and also diluted this reagent into OptiMEM TM 20 minutes, allowing the DNA / lipid complex to form, and then added to the cells. TM Feed and ExpiFectamine TM 293 enhancer was added to the cells. The cells were cultured for four days at 37°C with shaking, after which the culture supernatant was harvested.

[0252] The ExpiCHO-S was placed in a shaking incubator set at 37°C, 8% CO2, and 125 RPM. TM Cells in ExpiCHO TM The cells were kept suspended in expression medium (ThermoFisher Scientific). The cells were passaged so that on the day of transfection, a dilution of 6.0 × 10 per ml could be achieved. 6 cells and maintained cell viability at 98% or better. TM Transient transfection was performed using the CHO Transfection Kit (ThermoFisher Scientific). For each ml of diluted cells to be transfected, one milligram of plasmid DNA was diluted into OptiPRO TM In SFM complex medium. ExpiFectamine was used at a 1:3 ratio (v / v, DNA:reagent) TM CHO reagent and also diluted into OptiPRO TMThe diluted DNA and transfection reagent were combined for one minute to allow DNA / lipid complexes to form and then added to the cells. After overnight incubation, the ExpiCHO TM Feed and ExpiFectamine TM The CHO enhancer was added to the cells. The cells were cultured at 32° C. with shaking for five days, after which the culture supernatant was harvested.

[0253] GLP1-GDF15 fusion protein was purified from the harvested culture supernatant by either a single-step affinity capture or a two-step approach using affinity capture followed by a preparative size exclusion chromatography (SEC) polishing step. TM The cell supernatant of the cells was loaded onto a pre-equilibrated (dPBS, pH 7.2) HSA CaptureSelect column (CaptureSelect human albumin affinity matrix from ThermoFisher Scientific) with an approximate capacity of 10 mg protein per ml of resin. After loading, unbound proteins and impurities were removed by washing the column with up to 12 column volumes (CV) of dPBS (pH 7.2), followed by washing the column with 3 CVs of 1M NaCl in 50 mM sodium phosphate (pH 7.4). The GLP1-GDF15 fusion protein bound to the column was eluted into a fraction tube containing 10% (by volume) 1M Tris (untitrated) with up to 10 CVs of 0.1M sodium acetate (pH 3.5). The peak fractions were combined and filtered on a 0.2 μm membrane, and then the buffer was exchanged for dPBS at pH 7.2 or continued to the SEC step at 4°C.

[0254] For the SEC step, the protein from the capture step was concentrated to an appropriate volume and then loaded onto a 26 / 60 superdex 200 column (GE Healthcare, Little Chalfont, UK). The high purity (determined by SDS-PAGE) protein fractions eluted from the SEC column were combined. Protein concentration (derived from either method) was determined by absorbance at 280 nm on a BioTek Synergy HTTM spectrophotometer. The quality of the purified protein was assessed by SDS-PAGE and analytical size exclusion HPLC (SE-HPLC, Dionex HPLC system). The LAL assay ( Endotoxin levels were measured by Associates of Cape Cod, Inc., East Falmouth, MA.

[0255] Example 4: Dual target engagement and additive effects of delivering two agonists, GLP1 and GDF15, on one molecule

[0256] The potential for additive effects by delivering two agonists, GLP1 and GDF15, on one molecule was tested by assessing weight loss in DIO mice treated with agonists fused to gorilla serum albumin (GSA) over eight days. GSA is used as a surrogate for HSA in these tool molecules. Mice received subcutaneous administration of 8 nmol / kg of GLP1-GSA-GDF15 (SEQ ID NO: 72), GLP1-GSA-GDF15 (I89R) (SEQ ID NO: 73, the I89R mutation eliminates GDF15 activity), GLP1 (9-36) -GSA-GDF15 (SEQ ID NO: 74) or GSA-GDF15 (SEQ ID NO: 75), both of which were designed to represent degraded GLP1. Two additional groups of animals were used as controls and references, one group receiving vehicle alone or the other group receiving the GLP1 agonist dulaglutide. Mice treated with GLP1-GSA-GDF15 (SEQ ID NO: 72) had greater weight loss than mice treated with GLP1(9-36)-GSA-GDF15 (SEQ ID NO: 74), GLP1-GSA-GDF15(I89R) (SEQ ID NO: 73), and GSA-GDF15 (SEQ ID NO: 75), providing proof of concept that both GLP1R and GFRAL can be agonized with a single molecule, resulting in additive efficacy ( Figure 2 ).

[0257] Example 5: Effects of GLP1 peptide or GLP1 peptide variant and first linker peptide on human GLP1R potency in vitro, in vitro and in vivo Effect of mouse plasma stability

[0258] Different GLP1 peptides or GLP1 peptide variants and different first linker peptides have different effects on in vitro hGLP1 receptor potency in an assay measuring cyclic adenosine monophosphate (cAMP) levels in hGLP1-overexpressing HEK cells. Fusions were screened for in vitro GLP1R potency in a cell-based assay measuring intracellular cAMP production using the Lance competitive cAMP immunoassay (PerkinElmer, Waltham, Massachusetts) according to the kit instructions. Clonal HEK293 cells stably expressing mouse or human GLP1R were used in the assay. The resulting data were used to calculate compound EC using Prism statistical software (GraphPad Software, San Diego, CA). 50 In general, the GLP1 variant (SEQ ID NO: 1) was less potent than the other two GLP1 variants (SEQ ID NO: 2 and SEQ ID NO: 3) in the in vitro hGLP1R assay.

[0259] Evaluation of ex vivo stability in human plasma. Briefly, fresh, non-frozen human plasma was generated from heparinized blood by centrifugation. The fusion protein was incubated in the matrix at 37°C for 0 hours, 4 hours, 24 hours, 48 ​​hours, 72 hours and 96 hours while gently mixing. The stability of the GLP1 region of the fusion molecule in human plasma over time was monitored by immunoaffinity capture-trypsin digestion LC-MS / MS analysis. Selected tryptic peptides, namely HSE (HSEGTFTSDVSSYLEGQAAK) (SEQ ID NO: 76), HGE-1 (HGEGTFTSDVSSYLEEQAAK) (SEQ ID NO: 77) and HGE-2 (HGEGTFTSDLSK) (SEQ ID NO: 78) are located at the N-terminus of the GLP1 peptide or GLP1 variant peptide (SEQ ID NO: 1, 2 and 3), respectively. These selected tryptic peptides were monitored by LC-MS / MS after anti-GDF15 immunoaffinity capture and trypsin digestion and used as a surrogate measure of the concentration of fusion molecules with intact N-terminal GLP1. Using this approach, all molecules tested showed reasonable GLP1 stability in human plasma over time.

[0260] The stability in mice was evaluated. The fusion protein was subcutaneously administered to male C57Bl / 6 mice at a dose of 2 mg / kg PBS solution (pH 7). Blood samples were collected into K3E Sarstedt blood collection tubes containing protease inhibitors at 0 hours, 4 hours, 24 hours and 48 hours after administration. Plasma was prepared by centrifugation. The stability of the GLP1 region of the fusion protein in mice over time in vivo was monitored by immunoaffinity capture-trypsin digestion LC-MS / MS analysis. Selected tryptic peptides, i.e., HSE (HSEGTFTSDVSSYLEGQAAK) (SEQ ID NO: 76), HGE-1 (HGEGTFTSDVSSYLEEQAAK) (SEQ ID NO: 77) and HGE-2 (HGEGTFTSDLSK) (SEQ ID NO: 78) were located at the N-terminus of SEQ IDNO: 1, 2 and 3 comprising the fusion protein, respectively. These selected tryptic peptides were monitored by LC-MS / MS after anti-GDF15 immunoaffinity capture and trypsin digestion and used as a surrogate measure of the concentration of fusion proteins with intact N-terminal GLP1. Using this approach, all molecules tested in mice exhibited reasonable GLP1 stability in vivo over time.

[0261] Table 7 provides the results of in vitro GLP1 receptor potency, measuring cAMP levels in GLP1R overexpressing cells, ex vivo human plasma stability, and in vivo mouse plasma stability (both assessed by mass spectrometry) for the indicated GLP1-GDF15 fusion proteins.

[0262] Table 7: In vitro hGLP1R potency, ex vivo human plasma stability and in vivo mouse plasma stability of GLP1-GDF15 fusion proteins stability

[0263]

[0264]

[0265] Example 6: Removal of O-linked xylosyl groups by eliminating serine residues in the first linker peptide and the second linker peptide change

[0266] The O-linked xylose level of the molecule containing the glycine-serine linker is tested because it is reported that xyloglucan is attached to the glycine-serine linker (Spahr et al., mAbs, Vol. 6, No. 4, pp. 904-914). In short, the sample is prepared by diluting in a guanidine-HCl buffer at a pH of 8.0 for denaturation, then adding DTT for reduction and incubating at 37°C for 1 hour. After reduction, the sample is alkylated at room temperature in the dark for 60 minutes using the iodoacetamide just prepared. Then, DTT is added to the sample to chelate the unreacted iodoacetamide. Next, the sample is desalted using a Zeba spin desalting column according to the manufacturer's protocol in 50mM Tris, 1mM CaCl2 at a pH of 8.0, and digested for 4 hours at 37°C with trypsin (Promega). After digestion, TFA is added to each sample to quench the digestion reaction. The digested sample is kept at 4°C and injected into the LC / MS within 24 hours.

[0267] Agilent Infinity 1290UHPLC (Agilent Technologies) was used to inject the digested samples into the Agilent AdvanceBio Peptide Map micropore high resolution fast column at a flow rate of 0.1mL / min. The column temperature was maintained at 65°C. Mass spectrometry grade HPLC solvents (0.1% formic acid, and B: 0.1% formic acid solution of 100% ACN) were purchased from VWR. A 50-minute gradient of 0.1% formic acid solution of 2% to 40% ACN was used to elute proteolytic peptides from the column. Using a spray voltage of 3.5kV, a sheath gas of 20, an auxiliary gas of 7, an ion transfer tube of 299°C and a vaporizer of 100°C, the column effluent was introduced into a Thermo Orbitrap Q-Exactive mass spectrometer via a heated electrospray ionization probe (HESI).

[0268] The first two data-dependent experiments were performed with precursor scan settings of Orbitrap detection, 70,000 resolution, mass range 150m / z-2000m / z, AGC target 1.0e6, maximum injection time 50ms, 1 microscan, positive polarity. The precursor decision criteria were monoisotopic precursor selection peptide, charge state: 2-7, dynamic exclusion: 6.0 seconds and precursor intensity threshold of 5e4. The precursor peptides were separated by a quadrupole with an isolation window of 1.6m / z and sent to the collision cell. A collision energy of 28 resulted in high energy collisional dissociation (HCD) of the peptides. These fragments were then transferred to the orbitrap for mass measurement. The orbitrap was set to 17,500 resolution, 200-2000m / z range, AGC target 5e5, maximum injection time 100ms, 1 microscan, and spectra were collected in centroid mode.

[0269] The peptide map data were processed using the Byonic (Protein Metrics) search algorithm. A custom library of core xylose glycosaminoglycans and their monoisotopic masses was added to the search parameters as a Ser-specific modification. The Byonic search results were imported into Byologic (Protein Metrics) for quantification based on the extracted ion chromatogram (XIC) areas of the modified and unmodified peptide species.

[0270] GLP1-GDF15 fusion proteins containing a G4S first linker peptide and a second linker peptide showed various levels of O-linked xylosylation on the serine residues of the first linker peptide and the second linker peptide. Table 8 shows the results of three such molecules transiently expressed from ExpiCHO cells. The level of xylose is protein environment dependent and ranges from "not detected" to 1% in the first linker peptide that connects the GLP1 peptide or GLP1 peptide variant to the HSA protein, and ranges from 11.61% to 56.2% in the second linker peptide that connects the HSA protein to the GDF15 protein or the GDF15 protein variant. In order to avoid the risk of potential O-linked xylosylation on the linker peptide, GLP1-GDF15 fusion proteins that do not contain serine residues in the first linker peptide and / or the second linker peptide were designed and generated. These GLP1-GDF15 fusion proteins include a linker that includes an AP repeat sequence, a G4A repeat sequence, or a polyglycine repeat sequence.

[0271] Table 8: Silk in the first linker peptide or the second linker peptide of the GLP1-GDF15 fusion protein containing the G4S linker peptide Xylose levels detected on amino acid residues .

[0272]

[0273] Example 7: In vitro potency of GLP1R and GDF15R

[0274] The in vitro activity of the GLP1-GDF15 fusion protein was tested to obtain the potency of both GLP1R and GDF15R in a cell-based assay. GDF15R (GFRAL) activity was determined by measuring the level of phosphorylated AKT (Ser473) in SK-N-AS human neuroblastoma cells stably transfected to overexpress human or cynomolgus monkey GFRAL. The phosphorylation of AKT after treatment of GFRAL expressing cells with various concentrations of the fusion molecule was measured using the phosphorylated AKT (Ser473) assay kit (Cisbio, Beford, MA) according to the manufacturer's instructions. The resulting data were used to calculate compound EC using Prism statistical software (GraphPad Software, San Diego). 50 The GLP1-GDF15 fusion proteins tested in Table 9 have similar potency to GDF15R, where EC 50 In the range of 4.6nM-6.9nM.

[0275] The GLP1R potency of the fusion proteins was determined by measuring cAMP levels in cloned HEK293 cells stably transfected to overexpress human, mouse, or cynomolgus monkey GLP1R. Intracellular cAMP production after treatment of cells with different concentrations of the fusion molecule was measured using the Lance competitive cAMP immunoassay (Perkin Elmer, Waltham, Massachusetts) according to the kit instructions. The resulting data were used to calculate compound EC using Prism statistical software (GraphPad Software, San Diego). 50 value.

[0276] When the serine residue in the first linker peptide connecting the GLP1 peptide or GLP1 peptide variant to the HSA protein was replaced or removed by alanine (converted from G4S to G4A or a poly-G linker of similar length), GLP1 activity was not affected when tested in the human or mouse GLP1R assay. In addition, changing the second linker peptide connecting the HSA protein to the GDF15 protein or GDF15 protein variant from GS-8x(G4S) to GA-8x(G4A) did not affect GLP1R potency. In addition, fusion proteins with an increasing number of repeat sequences in the first linker (5X(G4A), 8X(G4A), 5X(AP), 10X(AP), 20X(AP), 25X(AP)) were prepared and tested in the human GLP1R assay. The results show that these fusion proteins activate GLP1R signaling with different potencies.

[0277] Table 9: In vitro potency of GLP1-GDF15 fusion protein against GLP1R and GDF15R

[0278]

[0279]

[0280]

[0281] Example 8: Effects of the first linker peptide and the second linker peptide on protein purity and stability

[0282] Use size exclusion high performance liquid chromatography (SE-HPLC), by quantitative main material and high molecular weight (HMW) material representing aggregation and the percentage ratio of low molecular weight (LMW) material representing fragment is checked for purity and stability of molecule.In brief, in order to measure protein purity, 20 μ g protein is injected on Tosoh TSKgel BioAssist G3SWXL (catalog number 20026) post of 1x DPBS mobile phase (Gibco catalog number 14190-136) that adopts pH to be 7.2.At room temperature with the flow velocity elution protein material of 1mL / min, and use the Dionex Ultimate3000 HPLC system equipped with variable wavelength detector to monitor UV-280nm absorbance value.In order to determine protein stability, 100 μ g protein is injected on Tosoh TSKgel BioAssist G3SWXL (catalog number 20026) post of 0.2M sodium phosphate mobile phase that adopts pH to be 7.0. The protein material was eluted at a flow rate of 0.7 mL / min at room temperature and the UV-280 nm absorbance value was monitored using a Dionex Ultimate 3000 HPLC system equipped with a variable wavelength detector. The data were analyzed using Chromeleon software.

[0283] When the GLP1-GDF15 fusion protein was made by transient expression in ExpiCHO cells and purified by affinity capture as described in Example 2, the GLP1-GDF15 fusion protein with a 10x (AP) second linker peptide (SEQ ID NO: 28) (linking the HSA protein to the GDF15 protein or GDF15 protein variant) consistently resulted in a lower percentage of low molecular weight (LMW) species (Table 10) compared to the molecule containing a GA-8x (G4A) second linker peptide (SEQ ID NO: 29). LMW species were removed by a polishing step during purification, and the purity of the resulting final GLP1-GDF15 fusion protein was greater than 97% as determined by SE-HPLC.

[0284] The stability of these GLP1-GDF15 fusion proteins under defined stress conditions was tested. To force chemically induced oxidative stress, samples were exposed to hydrogen peroxide at a final concentration of 0.1% and incubated in the dark for 6 hours before the addition of catalase to terminate the reaction. Under metal-induced oxidative conditions, ferrous iron was added to the samples at a final concentration of 30 μM. After two weeks of incubation in the dark, EDTA was added to terminate the reaction. To test stability at low pH, samples were dialyzed into 50 mM acetate buffer at pH 3.5 for 6 hours and dialyzed back into 0.1 M sodium phosphate at pH 7.4. To test the stability of samples under heat stress, they were concentrated to approximately 10 mg / mL using an ultracentrifugal filter with a molecular weight cutoff of 30 KDal and kept in PBS at 40°C for two weeks. These samples subjected to the above chemical or metal-induced oxidation and low pH conditions or thermal stress conditions were analyzed in analytical size exclusion chromatography (TOSOH column) at a flow rate of 1 mL / min and run at room temperature for 20 minutes. The signal at UV-280 nm (Agilent 1100LC system) was collected and data analysis was performed using Chemstation (Agilent).

[0285] Table 11 shows the effect of the second linker on the stability of the GLP1-GDF15 fusion protein under heat stress (2 weeks at 40°C). The GLP1-GDF15 fusion protein containing a 10x (AP) second linker peptide (SEQ ID NO: 28) resulted in the formation of lower levels of LMW species than the GLP1-GDF15 fusion protein containing a GA-8x (G4A) second linker peptide (SEQ ID NO: 29), indicating that the AP second linker peptide is more thermally stable than the G4A second linker peptide. Under other tested stress conditions (low pH, metal-induced or chemically induced oxidation), the level of LMW species of the sample remained at the lowest level, which was similar to the non-stressed sample.

[0286] Differential scanning calorimetry (DSC) was performed to determine the thermal stability of the GLP1-HSA-GDF15 protein. Samples were evaluated at approximately 0.5 mg / mL-1 mg / mL in PBS buffer at pH 7.4 using an automated MicroCal VP-capillary DSC instrument. The thermal scan spanned from 25°C to 95°C at a linear rate of 1°C / min. A 15-minute prescan time and a 10-second filtration cycle were used for each run. The data were processed using the non-2 state fitting function in the Origin7 software package. The DSC results showed that these GLP1-GDF15 fusion proteins had a melting temperature (T m ) (Table 12), similar to HSA-GDF15 fusion protein without GLP1 peptide or GLP1 peptide variants.

[0287] Table 10: GLP1-GDF15 fusions with different first and second linker peptides as determined by SE-HPLC Protein shows various levels of low molecular weight species after affinity purification .

[0288]

[0289] Table 11: GLP1-GDF15 fusion proteins with different first linker peptides and second linker peptides under heat stress (40 ℃ for 2 weeks) showed various levels of low molecular weight substances .

[0290]

[0291] Table 12: Tm of GLP1-GDF15 fusion protein determined by differential scanning calorimetry (DSC) .

[0292]

[0293] Example 9: Ex vivo human plasma studies

[0294] The stability of the GLP1-GDF15 fusion protein was evaluated in vitro in human plasma. In brief, fresh, non-frozen human plasma was generated from heparinized blood by centrifugation. The fusion protein was incubated in the matrix at 37°C for 0 hours, 4 hours, 24 hours, 48 ​​hours, 72 hours and 96 hours while gently mixing. The concentration of the fusion molecule in human plasma over time was monitored by immunoaffinity capture with anti-GDF15 antibodies and immunodetection with anti-HSA antibodies ("total form") or anti-GLP1 N-terminal specific antibodies ("GLP1 active form"). Tables 13 and 14 show the results of these two immunoassays.

[0295] Table 13: Ex vivo immunoassay determined by anti-GDF15 capture antibody and anti-HSA detection antibody Human plasma stability .

[0296]

[0297] Table 14: Immunoassay using anti-GDF15 capture antibody and anti-GLP1 detection antibody (recognizing GLP1) Ex vivo human plasma stability of the active form with specific N-terminal GLP1 .

[0298]

[0299] Example 10: Multi-species Pharmacokinetics

[0300] Mouse Pharmacokinetics

[0301] GLP1-GDF15 fusion proteins derived from SEQ ID NOs: 50, 45, 46 and 44 were administered IV and SC to female C57B1 / 6 mice at a dose of 5 mg / kg in PBS at pH 7. Blood samples were collected, plasma was processed and drug concentrations were measured up to 4 days after both routes of administration. Analyte concentrations in plasma after IV and SC administration were measured by immunoaffinity capture-trypsin digestion LC-MS / MS analysis. Tryptic peptides were selected, namely ALV (ALVLIAFAQYLQQSPFEDHVK) (SEQ ID NO: 79), HGE-1 (HGEGTFTSDVSSYLEEQAAK) (SEQ ID NO: 77), HGE-2 (HGEGTFTSDLSK) (SEQ ID NO: 78) and TDT (TDTGVSLQTYDDLLAK) (SEQ ID NO: 80), which are located near the N-terminus of the HSA protein, the N-terminus of the GLP1 peptide or GLP1 peptide variant, and the C-terminus of the GDF15 protein or GDF15 protein variant, respectively. Monitoring these surrogate peptides enabled pharmacokinetic assessment of each region (GLP1, HSA, GDF15) of the GLP1-GDF15 fusion protein. Plasma drug concentration-time profiles are summarized in Tables 15-22.

[0302] Table 15: Expression of SEQ ID NO: 50 over time following a single subcutaneous (SC) administration in C57BL / 6 female mice Plasma concentrations (μg / mL) and standard error of the mean (SEM, n=3).

[0303]

[0304] Table 16: SEQ ID NO: 50 over time following a single intravenous (IV) administration in C57BL / 6 female mice Plasma concentrations (μg / mL) and standard error of the mean (SEM, n=3).

[0305]

[0306] Table 17: Expression of SEQ ID NO: 45 over time following a single subcutaneous (SC) administration in C57BL / 6 female mice Plasma concentrations (μg / mL) and standard error of the mean (SEM, n=3).

[0307]

[0308] Table 18: SEQ ID NO: 45 over time following a single intravenous (IV) administration in C57BL / 6 female mice Plasma concentrations (μg / mL) and standard error of the mean (SEM, n=3).

[0309]

[0310] Table 19: Expression of SEQ ID NO: 46 over time following a single subcutaneous (SC) administration in C57BL / 6 female mice Plasma concentrations (μg / mL) and standard error of the mean (SEM, n=3).

[0311]

[0312] Table 20: Plasma concentrations (μ) of SEQ ID NO: 46 following a single intravenous (IV) administration in C57BL / 6 female mice g / mL) and standard error of the mean (SEM, n=3).

[0313]

[0314] Table 21: Plasma concentrations (μg / mL) of SEQ ID NO: 44 following a single subcutaneous (SC) administration in C57BL / 6 female mice mL) and standard error of the mean (SEM, n=3).

[0315]

[0316] Table 22: Plasma concentrations (μ) of SEQ ID NO: 44 following a single intravenous (IV) administration in C57BL / 6 female mice g / mL) and standard error of the mean (SEM, n=3) .

[0317]

[0318] Non-compartmental pharmacokinetic analysis (NCA) using the data in Tables 15-22 indicated that following SC and IV administration of the four GLP1-GDF15 fusion proteins, the terminal half-life of the GLP1 peptide or GLP1 peptide variant tested in C57BL / 6 mice was 14-23 hours, the terminal half-life of the GDF15 protein or GDF15 protein variant was 22-49 hours, and the terminal half-life of the HSA protein was 22-47 hours (Table 23).

[0319] Table 23: Estimated terminal responses of GLP1-GDF15 fusion protein at 5 mg / kg IV and SC administration in female C57BL / 6 mice Terminal half-life .

[0320]

[0321] Monkey pharmacokinetics

[0322] GLP1-GDF15 fusion proteins derived from SEQ ID NO: 33, 50, 45, 46 and 44 were administered SC to male cynomolgus monkeys (Macaca fascicularis) that had not been administered the fusion proteins at a dose of 0.5 mg / kg in PBS at pH 7. Blood samples were collected for up to 21 days, plasma was processed and drug concentrations were measured using an immunoassay bioassay. The immunoassay strategy included an anti-GDF15 capture antibody and detection with an antibody that recognizes intact GLP1 ("active" form) or with an antibody that recognizes HSA ("total" form). The plasma drug concentration-time curves are summarized in Tables 24 and 25.

[0323] Table 24: Activity of a single SC administration in cynomolgus monkeys as determined by immunoassay SEQ ID NO: 33, Its mean (mean, n=3) plasma concentration (ng / mL) over time after 50, 45, 46 and 44 .

[0324]

[0325] Table 25: Single SC administration of total SEQ ID NO: 33, 50, Its mean (mean, n=3) plasma concentration (ng / mL) over time after 45, 46 and 44.

[0326]

[0327] Pharmacokinetic analysis of the data in Tables 24 and 25 indicated that the terminal half-life of the active molecule in cynomolgus monkeys following SC administration was 2-3 days and the terminal half-life of the total molecule was 5-8 days (Table 26).

[0328] Table 26: Terminal half-life of SEQ ID NO: 50, 33, 45, 46 and 44 following SC administration of 0.5 mg / kg in cynomolgus monkeys .

[0329]

[0330] Example 11: In vivo efficacy of GLP1-GDF15 fusion protein

[0331] Assessment of food intake in C57BL / 6 male mice :The purpose of this experiment is to demonstrate the in vivo pharmacokinetic effects of molecules corresponding to SEQ ID NO: 50, 33, 49, 45, 46 and 44 on inhibiting food intake in C57BL / 6 mice. Food intake was measured between 4:00 pm and 5:00 pm before and after subcutaneous administration of GLP1-GDF15 fusion protein (test article) or control (PBS vehicle or dulaglutide) for 24 hours. Changes in food weight per cage were recorded every 24 hours. Results are expressed as a percentage of change in food intake compared to 24 hours before treatment and depend in part on the circulating concentration of each test article used in the study ( Figure 4 ). Circulating concentrations of the test article were determined 24 hours after administration by immunoaffinity capture-trypsin digestion LC-MS / MS analysis. Selected tryptic peptides, ALV (ALVLIAFAQYLQQSPFEDHVK) (SEQ ID NO: 79), HGE-1 (HGEGTFTSDVSSYLEEQAAK) (SEQ ID NO: 77), HGE-2 (HGEGTFTSDLSK) (SEQ ID NO: 78), and TDT (TDTGVSLQTYDDLLAK) (SEQ ID NO: 80), were monitored, which are located near the N-terminus of the HSA protein, the N-terminus of the GLP1 peptide or GLP1 peptide variant, and the C-terminus of the GDF15 protein or GDF15 protein variant, respectively. The VVS peptide (VVSVLTVLHQDWLNGK) (SEQ ID NO: 82) is located in the Fc portion of dulaglutide. Monitoring these alternative peptides enables pharmacokinetic assessment of each region of the GLP1-GDF15 fusion protein (GLP1, HSA, GDF15). Plasma drug concentrations are shown in Figure 5 The results showed that subcutaneous administration of SEQ ID NOs: 50, 33, 49, 45, 46 and 44 to C57BL / 6 mice significantly inhibited food intake relative to vehicle-treated animals.

[0332] Assessment of GLP1R-mediated food intake effects in GFRAL KO mice: Inhibition of food intake in C57BL / 6 mice is a result of engagement of both GLP1R and GDF15R (GFRAL). To determine the GLP1R-specific effects of SEQ ID NOs: 50, 33, 45, 46, and 44 on food intake, mice lacking GFRAL were used. The purpose of these experiments was to determine the extent to which the HSA-GDF15 fusion protein engages GLP1R when administered at doses predicted to be effective for the fusion protein in mice ( Figure 1 and Figure 2 and US20170327560A1). Food intake was measured for 24 hours between 4:00 and 5:00 pm before and after subcutaneous administration of GLP1-GDF15 fusion protein (test article: 3 nmol / kg and 10 nmol / kg) or control (PBS vehicle or dulaglutide). Changes in food weight per cage were recorded every 24 hours. Circulating concentrations of fusion molecules comprising intact GLP1 peptide or GLP1 peptide variants were determined 24 hours after administration by immunoaffinity capture-trypsin digestion LC-MS / MS analysis and selective monitoring of tryptic peptides HGE-1 (HGEGTFTSDVSSYLEEQAAK) (SEQ ID NO: 77) or HGE-2 (HGEGTFTSDLSK) (SEQ ID NO: 78) located near the N-terminus of the GLP1 peptide or GLP1 peptide variant. The results are plotted as the percentage change in food intake relative to the plasma concentration of HGE peptide (intact GLP1 peptide or GLP1 peptide variant) ( Figure 6 , Figure 7 and Figure 8 ). The results showed that delivery of fusion proteins having SEQ ID NOs: 50, 33, 45, 46, and 44 resulted in GLP1R engagement at doses previously demonstrated to be effective for HSA-GDF15. Surprisingly, GLP1R engagement elicited the expected pharmacodynamic response similar to the Fc-GLP1 agonist dulaglutide, despite the exposure concentration of its GLP1 moiety being 10-30 times greater than dulaglutide. If the GLP1 component of the fusion protein was as effective as dulaglutide in vivo, GLP1R engagement would be much higher than expected, potentially leading to predicted adverse events in humans, such as nausea and vomiting. Therefore, delivery of the GLP1 peptide as a fusion peptide with HSA-GDF15 affects the in vivo efficacy of the GLP1R in a manner that achieves a desired balance of the two agonists.

[0333] Assessment of GLP1R-mediated glucose tolerance in fasting DIO mice: In diet-induced obese mice, GLP1R stimulation in the pancreas leads to enhanced insulin secretion and increased glucose uptake, and can be measured using an intraperitoneal glucose tolerance test. The purpose of these experiments was to determine the extent to which the HSA-GDF15 fusion protein engages the GLP1R when the fusion protein is administered at doses predicted to be effective for the fusion protein in mice ( Figure 1 and Figure 2 And US20170327560A1). Male C57BL / 6 mice were fed with research diet D12492 from 6 weeks of age until dosing began at 20 weeks of age to induce obesity. Mice were randomly divided into treatment groups using blood glucose and body weight measurements after eating. At 4:00 p.m., mice were transferred to clean cages and supplied with water; food supply was stopped during the study. Controls (PBS vehicle and dulaglutide) and test articles (SEQ ID NO: 33, 50, 49, 45, 46, 44 and 36) (1nmol / kg, 3nmol / kg and 10nmol / kg) were subcutaneously administered when feeding was stopped. 17 hours after administration, baseline glucose was collected and 1g of glucose was administered intraperitoneally per kg of body weight. Blood glucose was measured at 10 minutes, 30 minutes, 60 minutes and 120 minutes after glucose bolus. Circulating plasma concentrations of fusion molecules comprising intact GLP1 peptide or GLP1 peptide variants were determined immediately after the 120 minute time point by immunoaffinity capture-trypsin digestion LC-MS / MS analysis and selective monitoring of the tryptic peptides HGE-1 (HGEGTFTSDVSSYLEEQAAK) (SEQ ID NO: 77) or HGE-2 (HGEGTFTSDLSK) (SEQ ID NO: 78) located near the N-terminus of the GLP1 peptide or GLP1 peptide variant. Glucose levels were plotted as a function of time and the area under the curve (ΔAUC) normalized to baseline glucose levels was calculated for each animal. Results were plotted as percent change in ΔAUC (compared to vehicle treatment) relative to plasma concentration of HGE peptide (intact GLP1 agonist) ( Fig. 9 , Fig.10 , Fig.11 and Fig.12). The results showed that delivery of fusion proteins having SEQ ID NO: 33, 50, 49, 45, 46, 44 and 36 at doses previously demonstrated to be effective against HSA-GDF15 resulted in GLP1R engagement. Surprisingly, GLP1R engagement elicited the expected pharmacodynamic response similar to the Fc-GLP1 agonist dulaglutide, despite the exposure concentration of its GLP1 moiety being 10-30 times greater than dulaglutide. If the GLP1 component of the fusion protein was as effective as dulaglutide in vivo, GLP1R engagement would be much higher than expected, potentially leading to predicted adverse events in humans, such as nausea and vomiting. Therefore, delivery of the GLP1 peptide as a fusion peptide with HSA-GDF15 affects the in vivo efficacy of the GLP1R in a manner that achieves a desired balance of the two agonists.

[0334] Assessing GLP1R engagement in primates using gradient glucose infusion :Insulin secretion during intravenous gradient glucose infusion (GGI) in non-human primates was used to evaluate the GLP1R engagement of SEQ ID NO: 45 and 44. The purpose of these experiments was to determine the extent to which the fusion protein engaged the GLP1R when the fusion protein was administered at a dose predicted to be effective for the HSA-GDF15 fusion protein in non-human primates (Mullican et al., 2017 and US20170327560A1). After a 16-hour overnight fast, the GGI process was performed in sedated cynomolgus monkeys to compare the baseline and treatment response with the 14-day recovery period between the two GGI processes. On day 1, the animals were given the vehicle immediately after the food supply was stopped. On day 2, the animals were anesthetized and a baseline was established with two blood samples collected 10 minutes apart. GGI1 was started at 0 minutes with a glucose infusion rate of 8 mg / kg / min, followed by infusions of 12 mg / kg / min, 16 mg / kg / min, and 24 mg / kg / min. Each infusion rate was administered for 40 minutes. Blood samples were collected at 20-minute intervals to measure glucose, insulin, and C-peptide, the results were plotted as a function of time, and the area under the curve (AUC) for each analyte measured in each animal was determined. The animals were then randomized into test article treatment groups based on their baseline insulin secretion rate (ISR) from GGI1. On day 15, the animals were fed the test article at different dose concentrations (0.1 mg / kg to 1.1 mg / kg fusion protein and 0.016 mg / kg to 0.1 mg / kg dulaglutide) before an overnight fast. A second GGI (GGI2) was performed on day 16 as described on day 2. Compound exposure was assessed on plasma samples collected at time point 0 before glucose infusion by an immunoassay specifically designed to monitor test articles with intact GLP1 peptide or peptide variants. Fig.13The data in are reported as treatment-induced fold changes in ISR AUC normalized to glucose AUC compared to baseline relative to plasma concentrations of the test article with intact GLP1. Dulaglutide was used as an Fc-GLP1 agonist reference. The results indicate that delivery of fusion proteins with SEQ ID NOs: 45 and 44 resulted in GLP1R engagement at doses previously demonstrated to be effective for HSA-GDF15. Surprisingly, GLP1R engagement elicited an expected pharmacodynamic response similar to that of the Fc-GLP1 agonist dulaglutide, despite the exposure concentration of its GLP1 moiety being 10-30 times greater than dulaglutide. If the GLP1 component of the fusion protein is as effective as dulaglutide in vivo, GLP1R engagement would be much higher than expected, potentially leading to predicted adverse events in humans, such as nausea and vomiting. Therefore, the delivery of the GLP1 peptide as a fusion peptide with HSA-GDF15 affects the in vivo efficacy of the GLP1R in a manner that achieves the desired balance of the two agonists.

[0335] Figure 6-Figure 13 The results shown in show that the in vivo activity of GLP1 in the homodimeric GLP1-GDF15 dual agonist or the potency in preclinical material is reduced compared to the Fc-fused GLP1 peptide of dulaglutide. This enables the two agonists (GLP1 peptide or GLP1 peptide variant and GDF15 protein or GDF15 protein variant) to be balanced on one molecule; therefore, allowing the delivery of GLP1 peptide or GLP1 peptide variant at an effective concentration that also achieves exposure of GDF15 protein or GDF15 protein variant within the predicted therapeutic range.

[0336] Evaluation of the weight loss efficacy of GLP1-GDF15 fusion protein after repeated dosing in mice: The ability of GLP1-GDF15 fusion protein to cause weight loss was tested in diet-induced obese mice. Mice were subcutaneously administered different concentrations of the test article (SEQ ID NO: 45 and 44), dulaglutide or vehicle control daily for seven days. Body weight was monitored throughout the study, and Fig.14The percent change in body weight over time relative to day 0 (just before the first dose) is shown. After seven days of treatment, circulating concentrations of the test article were determined by immunoaffinity capture-trypsin digestion LC-MS / MS analysis. Selected tryptic peptides, ALV (ALVLIAFAQYLQQSPFEDHVK) (SEQ ID NO: 79), HGE-1 (HGEGTFTSDVSSYLEEQAAK) (SEQ ID NO: 77), HGE-2 (HGEGTFTSDLSK) (SEQ ID NO: 78), and TDT (TDTGVSLQTYDDLLAK) (SEQ ID NO: 80), were monitored, which are located near the N-terminus of the HSA protein, the N-terminus of the GLP1 peptide or GLP1 peptide variant, and the C-terminus of the GDF15 protein or GDF15 protein variant, respectively. The VVS peptide (VVSVLTVLHQDWLNGK) (SEQ ID NO: 82) is located in the Fc portion of dulaglutide. Monitoring these surrogate peptides enables pharmacokinetic assessment of each region of the GLP1-GDF15 fusion protein (GLP1, HSA, GDF15). Plasma drug concentrations are shown in Fig.15 The results showed that repeated administration of SEQ ID NO: 45 and 44 resulted in weight loss in diet-induced obese mice in a concentration-dependent manner.

[0337] Evaluation of the effect of GLP1-GDF15 fusion protein on food intake in non-human primates.

[0338] The ability of GLP1-GDF15 fusion proteins to reduce food intake was tested in spontaneously obese cynomolgus monkeys. Subjects were administered different concentrations of the test article with and without an active GLP1 portion (SEQ ID NO: 44 and 83, respectively; SEQ ID NO: 83, which comprises an HSA peptide fused to an AP10 linker fused to a GDF15 peptide, previously disclosed in U.S. Pat. No. 10,336,812) or vehicle subcutaneously QW for a period of 4 weeks. Food intake was monitored prior to dosing and throughout the study. The 3-day percent change in food intake at baseline and after each administration of the test article is shown in Fig.16 The concentrations of SEQ ID NOs: 44 and 83 in the plasma of cynomolgus monkeys during the study were determined using two separate immunoassays to detect the "total" molecule (HSA assay) and the "active" GLP1 portion (N-terminal GLP1 peptide assay). Plasma drug concentrations are shown in Fig.17 and Fig.18 The results show that administration of SEQ ID NOs: 44 and 83 reduces food intake in spontaneously obese non-human primates.

[0339] Evaluation of the effects of GLP1-GDF15 fusion protein on food intake and body weight following 21 days of dose escalation in nonhuman primates.

[0340] The ability of the GLP1-GDF15 fusion protein to reduce food intake and cause weight loss was tested in spontaneously obese cynomolgus monkeys. Subjects were subcutaneously administered a test article containing active GDF15 and GLP1, active GDF15 alone, or active GLP1 portion alone (SEQ ID NOs: 44, 83, 84, respectively; SEQ ID NO: 84 comprises a GLP1 peptide fused to an AP5 linker peptide fused to an HSA peptide fused to an AP10 linker peptide fused to a GDF15 peptide with an I89R mutation; a GDF15 peptide with an I89R mutation was previously disclosed in U.S. Pat. No. 10,336,812) every three days for a period of 21 days. An initial dose of 0.7 nmol / kg was given on day 0 and was gradually increased to 20 nmol / kg at each administration. Fig.19 Daily food intake before dosing, during the 21-day treatment period, and during the test article washout period is shown in FIG. Body weight was monitored throughout the study, and Fig. 20 The percent change over time from baseline body weight is shown. The concentration of the test article in the plasma of cynomolgus monkeys during the study was determined using two separate immunoassays to detect the "total" molecule (HSA assay; SEQ ID NOS: 44, 83, 84) and the "active" GLP1 portion (N-terminal GLP1 peptide assay; for SEQ ID NOS: 44 and 84 only). Plasma drug concentrations are shown in Fig.21 and Fig. 22 The results show that repeated administration of SEQ ID NO: 44, 83, 84 leads to reduced food intake and subsequent weight loss in spontaneously obese non-human primates.

[0341] It will be appreciated by those skilled in the art that modifications may be made to the above embodiments without departing from the broad inventive concept of the present invention. It will therefore be appreciated that the present invention is not limited to the particular embodiments disclosed, but is intended to cover modifications within the spirit and scope of the present invention, as defined in this specification.

[0342] All documents cited herein are incorporated by reference.

Claims

1. A glucagon-like peptide-1 (GLP1) / growth differentiation factor 15 (GDF15) fusion protein, wherein the GLP1-GDF15 fusion protein comprises a GLP1 peptide, a first linker peptide, serum albumin, a second linker peptide and a GDF15 protein. 2 . The GLP1-GDF15 fusion protein according to claim 1 , wherein the GLP1 peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-4.

3. The GLP1-GDF15 fusion protein according to claim 1, wherein the first linker peptide comprises an amino acid sequence selected from SEQ ID NO: 5-25.

4. The GLP1-GDF15 fusion protein according to claim 1, wherein the serum albumin comprises an amino acid sequence selected from SEQ ID NO: 26 or SEQ ID NO:

27. 5 . The GLP1-GDF15 fusion protein according to claim 1 , wherein the second linker peptide comprises an amino acid sequence selected from SEQ ID NO: 28-30. 6 . The GLP1-GDF15 fusion protein according to claim 1 , wherein the GDF15 protein comprises an amino acid sequence selected from SEQ ID NO: 31 or SEQ ID NO:

32.

7. The GLP1-GDF15 fusion protein according to claim 1, wherein the GLP1-GDF15 fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 34-43, 47, 48, 51-74 and 84.

8. An isolated nucleic acid encoding the GLP1-GDF15 fusion protein according to claim 1.

9. A vector comprising the isolated nucleic acid according to claim 8.

10. A host cell comprising the isolated nucleic acid according to claim 8 or the vector according to claim 9. 11 . A pharmaceutical composition comprising the GLP1-GDF15 fusion protein according to claim 1 and a pharmaceutically acceptable carrier.

12. A method for treating or preventing a disease or disorder in a subject in need thereof, wherein the disease or disorder is selected from the group consisting of obesity, type I or type II diabetes, metabolic syndrome, insulin resistance, impaired glucose tolerance, hyperglycemia, hyperinsulinemia, hypertriglyceridemia, hypoglycemia due to congenital hyperinsulinemia (CHI), dyslipidemia, atherosclerosis, diabetic nephropathy, and other cardiovascular risk factors such as hypertension and cardiovascular risk factors associated with uncontrolled cholesterol and / or lipid levels, osteoporosis, inflammation, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), kidney disease and eczema, the method comprising administering to the subject in need thereof an effective amount of the pharmaceutical composition according to claim 11.

13. A method of reducing food intake in a subject in need thereof, the method comprising administering to the subject in need thereof an effective amount of the pharmaceutical composition of claim 11.

14. A method of modulating GLP1 receptor activity in a subject in need thereof, the method comprising administering to the subject in need thereof an effective amount of the pharmaceutical composition of claim 11.

15. A method of modulating GDF15 receptor (GFRAL) activity in a subject in need thereof, the method comprising administering to the subject in need thereof an effective amount of the pharmaceutical composition of claim 11.

16. The method of claim 12, wherein the pharmaceutical composition is administered via injection. 17 . A kit comprising the GLP1-GDF15 fusion protein according to claim 1 , the isolated nucleic acid according to claim 8 and / or the vector according to claim 9 .

18. The kit of claim 17, wherein the kit further comprises a device for injection.

19. A method for preparing a pharmaceutical composition, the pharmaceutical composition comprising the GLP1-GDF15 fusion protein according to claim 1, the method comprising combining the GLP1-GDF15 fusion protein with a pharmaceutically acceptable carrier to obtain the pharmaceutical composition.

20. A method for preparing the GLP1-GDF15 fusion protein according to claim 1, the method comprising culturing cells containing a nucleic acid encoding the GLP1-GDF15 fusion protein under conditions for preparing the GLP1-GDF15 fusion protein, and recovering the GLP1-GDF15 fusion protein from the cells or culture.

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