Methods of treating obesity, diabetes and liver dysfunction

Abstract

The present disclosure relates to compositions and methods for improving glycemic control, increasing lean body mass, reducing fat mass, treating obesity, diabetes, and / or treating liver dysfunction in a subject. More specifically, the present disclosure relates to compositions comprising a GDF-8 inhibitor and a GLP-1 agonist and uses thereof, as well as compositions comprising a GDF-8 inhibitor, an activin A inhibitor and a GLP-1 agonist and uses thereof.

Description

[0001] Sequence Listing XML Reference

[0002] This application contains a Sequence Listing that has been electronically submitted in XML format. The Sequence Listing XML file is incorporated herein by reference. The XML file was created on September 19, 2023, has the name 40848_0107WOU1_SL.xml, and is 24.7 kilobytes in size.

[0003] Cross - Reference to Related Applications

[0004] This application was filed as a PCT international patent application on September 21, 2023, and claims priority to U.S. Provisional Patent Application No. 63 / 376,582, filed on September 21, 2022, and U.S. Provisional Patent Application No. 63 / 508,458, filed on June 15, 2023. The entire content of each application is incorporated herein by reference. Technical Field

[0005] The present disclosure relates to compositions and methods for improving glycemic control, increasing lean body mass, reducing fat mass, treating obesity, diabetes, and / or treating liver dysfunction in a subject. More specifically, the present disclosure relates to compositions comprising a GDF - 8 inhibitor and a GLP - 1 agonist and their uses, as well as compositions comprising a GDF - 8 inhibitor, an activin A inhibitor, and a GLP - 1 agonist and their uses. Background Art

[0006] Obesity is a global problem that affects more than one - third of the world's population. In the United States, the average obesity rate exceeds 20%. The cost of obesity - related diseases is staggering, reaching up to $190.2 billion, approximately 21% of the annual U.S. healthcare costs. Obesity is an epidemic characterized by chronic low - grade inflammation associated with dysregulated (elevated) fat mass. Obesity is an important potential risk factor for other diseases such as heart disease, stroke, and diabetes. Even a modest weight loss (5 - 10% of initial body weight) can reduce the risk of developing obesity - related diseases such as heart disease and diabetes.

[0007] Diabetes is a chronic disease characterized by hyperglycemia and insulin resistance. If left untreated, hyperglycemia can lead to long - term complications, including heart disease, stroke, diabetic retinopathy, and lower - limb amputations. The treatment of diabetes includes controlling and reducing blood glucose levels, including exercise and dietary adjustments, as well as pharmacological treatments such as insulin and metformin.

[0008] Growth differentiation factor-8 (GDF8, also known as myostatin) is a secreted ligand belonging to the transforming growth factor-β (TGF-β) growth factor superfamily. GDF8 plays a central role in skeletal muscle development and maintenance and is a negative regulator of muscle mass. Although the phenotypes of myostatin-deficient mice demonstrate the importance of GDF8 in controlling muscle size during development, inhibition of GDF8 by using neutralizing antibodies, decoy receptors, or other antagonists can also induce muscle hypertrophy in adult muscle. Administration of a GDF8 neutralizing antibody has been reported to increase muscle mass by 10% to 30%. The observed increase in muscle mass is due to an increase in fiber diameter rather than myofiber hyperplasia (an increase in fiber number). Many studies have also reported improvements in muscle strength or performance commensurate with the increase in muscle size, including twitch force and tetanic force. Use of an anti-cleavage version of the GDF8 propeptide can also increase muscle size. GDF8 antibodies and methods of treating therewith are disclosed, for example, in US 8,840,894. Anti-GDF8 antibodies are also mentioned in, for example, U.S. Patent Nos. 6,096,506, 7,320,789, 7,261,893, 7,807,159, 7,888,486, 7,635,760, 7,632,499; U.S. Patent Application Publication Nos. 2006 / 0263354, 2007 / 0178095, 2008 / 0299126, 2010 / 0166764, 2009 / 0148436; and International Patent Application Publication Nos. WO2004 / 037861; WO2007 / 047112; WO 2010 / 070094.

[0009] Activin belongs to the transforming growth factor-β (TGF-β) superfamily and has broad biological effects on cell proliferation, differentiation, metabolism, homeostasis and apoptosis, as well as immune responses and tissue repair. Activin A is a disulfide-linked homodimer (two β-A chains) that binds to and activates heteromeric complexes of type I (Act RI-A and Act RI-B) and type II (Act RII-A and ActRII-B) serine-threonine kinase receptors.

[0010] Antibodies against activin A and uses thereof have been disclosed, for example, in US 8,309,082, 9,718,881 and International Patent Application Publication No. WO2008 / 031061.

[0011] Compositions and methods of treatment comprising anti-GDF8 antibodies and anti-activin A antibodies are disclosed, for example, in US 8,871,209.

[0012] One approach for treating obesity and controlling blood sugar involves glucagon-like peptide (GLP)-1 receptor agonists, which target the incretin pathway. Glucagon-like peptide (GLP)-1 is a peptide hormone secreted by intestinal endocrine cells. After oral glucose administration, GLP-1 binds to its receptor, resulting in insulin secretion and a decrease in blood sugar levels (incretin effect). However, GLP-1 is rapidly inactivated and degraded by dipeptidyl peptidase 4 (DPP4) and has a very short half-life of only 1.5 minutes. Therefore, long-acting derivatives of GLP-1 as well as GLP-1 receptor agonists (including fusion proteins containing GLP-1) have been studied for diabetes control. GLP-1 analogs, fusion proteins, and GLP-1 receptor agonists are disclosed in, for example, US7452966, US8389689, US8496149, US8497240, US8557769, US8883447, US8895694, US9409966, US20160194371, US20140024586, US20140073563, US20120148586, US20170114115, US20170112904, US20160361390, US20150313908, US20150259416, WO2017074715, WO2016127887, WO2015021871, WO2014113357, EP3034514, EP2470198, and EP2373681.

[0013] Since high fat mass is associated with serious diseases such as congestive heart failure, elevated blood pressure / hypertension, pulmonary embolism, osteoarthritis, lymphedema, gastroesophageal reflux disease, chronic renal failure, cancer, fatty liver, and even depression, there is still a need for therapies that can reduce the total fat mass and / or the fat content in the abdominal and waist regions (android fat mass) of a subject. In addition, there is still a need for drugs that can treat obesity and diabetes and simultaneously treat related liver problems. Summary of the Invention

[0015] In one aspect, the present disclosure provides a composition comprising a growth differentiation factor 8 (GDF-8) inhibitor and an incretin inhibitor. In another aspect, the present disclosure provides a composition comprising a GDF-8 inhibitor, an activin A inhibitor, and an incretin inhibitor.

[0016] In another aspect, the present disclosure provides a composition comprising a growth differentiation factor 8 (GDF-8) inhibitor and a glucagon-like peptide 1 (GLP-1) agonist. In another aspect, the present disclosure provides a composition comprising a GDF-8 inhibitor, an activin A inhibitor, and a GLP-1 agonist.

[0017] In one embodiment of the compositions of the present disclosure, the GDF-8 inhibitor is a GDF8-specific binding protein. In another embodiment, the GDF8 inhibitor is an antibody that specifically binds GDF-8 or an antigen-binding fragment thereof. In a further embodiment, the anti-GDF8 antibody or an antigen-binding fragment thereof comprises heavy chain complementarity-determining regions (HCDRs) containing a heavy chain variable region (HCVR) having SEQ ID NO:4 and light chain complementarity-determining regions (LCDRs) containing a light chain variable region (LCVR) having SEQ ID NO:5. In yet another embodiment, the anti-GDF8 antibody or an antigen-binding fragment thereof comprises: heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) respectively comprising the amino acid sequences of SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8; and three light chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3) respectively comprising the amino acid sequences of SEQ ID NO:9, TTS, and SEQ ID NO:11.

[0018] In one embodiment of the compositions of the present disclosure, the activin A inhibitor is an activin A-specific binding protein. In another embodiment, the activin A inhibitor is an antibody that specifically binds activin A or an antigen-binding fragment thereof. In yet another embodiment, the anti-activin A antibody or an antigen-binding fragment thereof comprises heavy chain complementarity-determining regions (HCDRs) containing a heavy chain variable region (HCVR) having SEQ ID NO:12 and light chain complementarity-determining regions (LCDRs) containing a light chain variable region (LCVR) having SEQ ID NO:13. In yet another embodiment, the anti-activin A antibody or an antigen-binding fragment thereof comprises: heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) respectively comprising the amino acid sequences of SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16; and three light chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3) respectively comprising the amino acid sequences of SEQ ID NO:17, GAS, and SEQ ID NO:19.

[0019] In one embodiment of the compositions of the present disclosure, the GLP-1 agonist is a GLP-1 receptor agonist. In another embodiment, the GLP-1 agonist is selected from exenatide (long-acting), dulaglutide, liraglutide, tirzepatide, and semaglutide. In yet another embodiment, the GLP-1 agonist is a GLP-1 specific binding protein. In yet another embodiment, the GLP-1 agonist is an antibody or an antigen-binding fragment thereof that specifically binds to GLP-1.

[0020] In one embodiment, the compositions of the present disclosure are used to improve glycemic control in a subject, increase lean body mass, reduce fat mass, treat obesity, treat diabetes, and / or treat liver problems associated with increased fat mass, obesity, and / or diabetes. In another embodiment, the compositions of the present disclosure are used to improve glycemic control in a subject, increase lean body mass, reduce fat mass, treat obesity, and / or treat diabetes without exacerbating liver problems associated with increased fat mass, obesity, and / or diabetes.

[0021] In one aspect, the present disclosure provides a method for improving glycemic control in a subject, increasing lean body mass, reducing fat mass, lowering total cholesterol, lowering LDL cholesterol, increasing HDL cholesterol, treating obesity, treating diabetes, and / or treating liver problems associated with increased fat mass, obesity, and / or diabetes, the method comprising administering to the subject a composition comprising a GDF8 inhibitor and a GLP-1 agonist.

[0022] In another aspect, the present disclosure provides a method for improving glycemic control in a subject, increasing lean body mass, reducing fat mass, treating obesity, treating diabetes, and / or treating liver problems associated with increased fat mass, obesity, and / or diabetes, the method comprising administering to the subject a GDF8 inhibitor, an activin A inhibitor, and a GLP-1 agonist.

[0023] In one embodiment of the methods of the present disclosure, the improvement in glycemic control is demonstrated / measured by a reduction in glycosylated hemoglobin (HbA1C). In another embodiment of the methods of the present disclosure, the GDF8 inhibitor, the GLP-1 agonist, and (if present) the activin A inhibitor are administered to the subject as a single composition. In yet another embodiment, the GDF8 inhibitor, the GLP-1 agonist, and (if present) the activin A inhibitor are administered to the subject as at least two separate compositions. In yet another embodiment, the GDF8 inhibitor, the GLP-1 agonist, and (if present) the activin A inhibitor are administered to the subject as three separate compositions.

[0024] In one embodiment of the method of the present disclosure, the GDF-8 inhibitor is a GDF8-specific binding protein. In another embodiment, the GDF8 inhibitor is an antibody or an antigen-binding fragment thereof that specifically binds to GDF-8. In yet another embodiment, the anti-GDF8 antibody or its antigen-binding fragment comprises: a heavy-chain complementarity-determining region (HCDR) comprising a heavy-chain variable region (HCVR) of SEQ ID NO: 4; and a light-chain complementarity-determining region (LCDR) comprising a light-chain variable region (LCVR) of SEQ ID NO: 5. In yet another embodiment, the anti-GDF8 antibody or its antigen-binding fragment comprises: heavy-chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) that respectively comprise SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8; and three light-chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3) that respectively comprise SEQ ID NO: 9, TTS, and SEQ ID NO: 11.

[0025] In one embodiment of the method according to the present disclosure, the activin A inhibitor is an activin A-specific binding protein. In another embodiment, the activin A inhibitor is an antibody or an antigen-binding fragment thereof that specifically binds to activin A. In yet another embodiment, the anti-activin A antibody or its antigen-binding fragment comprises: a heavy-chain complementarity-determining region (HCDR) comprising a heavy-chain variable region (HCVR) of SEQ ID NO: 12, and a light-chain complementarity-determining region (LCDR) comprising a light-chain variable region (LCVR) of SEQ ID NO: 13. In yet another embodiment, the anti-activin A antibody or its antigen-binding fragment comprises: heavy-chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) that respectively comprise SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16, and three light-chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3) that respectively comprise SEQ ID NO: 17, GAS, and SEQ ID NO: 19.

[0026] In one embodiment of the method of the present disclosure, the GLP-1 agonist is a GLP-1 receptor agonist. In another embodiment, the GLP-1 agonist is selected from exenatide (long-acting), dulaglutide, liraglutide, tirzepatide, and semaglutide. In yet another embodiment, the GLP-1 agonist is a GLP-1-specific binding protein. In yet another embodiment, the GLP-1 agonist is an antibody or an antigen-binding fragment thereof that specifically binds to GLP-1.

[0027] In one embodiment of the disclosed method, a subject exhibits at least one parameter change selected from the following 12 weeks after administration of the (one or more) inhibitor and the agonist:

[0028] i) a reduction in fat mass of at least about 35%;

[0029] ii) an increase in lean body mass of at least about 6%;

[0030] iii) a reduction in fasting glucose of at least about 15%;

[0031] iv) a reduction in HbA1c of at least about 6%;

[0032] v) a reduction in LDL of at least about 14%;

[0033] vi) an increase in LDL of at least about 14%;

[0034] vii) a reduction in NEFA of at least about 35%; and

[0035] viii) a reduction in TG of at least about 55%.

[0036] In another embodiment, a subject exhibits at least one parameter change selected from the following 12 weeks after administration of the (one or more) inhibitor and the agonist:

[0037] i) a reduction in fasting glucose of at least about 25%;

[0038] iv) a reduction in HbA1c of at least about 25%;

[0039] v) a reduction in LDL of at least about 50%;

[0040] vi) an increase in LDL of at least about 60%;

[0041] vii) a reduction in NEFA of at least about 50%; and

[0042] viii) a reduction in TG of at least about 65%.

[0043] In certain embodiments of the disclosed compositions or methods, a single antigen-binding molecule comprises a GDF8-specific binding domain and an activin A-specific binding domain. In one embodiment of this aspect of the disclosure, the antigen-binding molecule is a bispecific antibody that comprises a first variable domain that specifically binds GDF8 and a second variable domain that specifically binds activin A.

[0044] In one aspect, the present disclosure provides the use of a GDF-8 inhibitor and a GLP-1 agonist in the preparation of a medicament for improving glucose control, increasing lean body mass, reducing fat mass, treating obesity, treating diabetes, and / or treating liver problems associated with increased fat mass, obesity, and / or diabetes in a subject. In another aspect, the present disclosure provides the use of a GDF-8 inhibitor, an activin A inhibitor, and a GLP-1 agonist in the preparation of a medicament for improving glucose control, increasing lean body mass, reducing fat mass, treating obesity, treating diabetes, and / or treating liver problems associated with increased fat mass, obesity, and / or diabetes in a subject.

[0045] Other embodiments of the present disclosure will become apparent by reading the ensuing detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figures 1A - 1C It was shown that, as measured by MRI, body weight ( Figure 1A ), fat mass( Figure 1B ) and lean body mass ( Figure 1C ) changes over time. Semaglutide (via osmotic pump): 7 μg / day. IgG4 P (isotype control): 20 mg / kg. REGN1033 (α-GDF8): 10 mg / kg. REGN2477 (α-activin A): 10 mg / kg.

[0048] Figure 2A and 2B showed that at the end of the study, compared with the control, ( Figure 2A ) muscle and ( Figure 2B ) percentage change in fat tissue weight. For final muscle weight ( Figure 2A ), the left bar graph shows TA weight (percent change compared to control), and the right bar graph shows Quad weight (percent change compared to control). For final adipose tissue weight ( Figure 2B ), the left bar graph shows subQ fat weight (percent change compared to control), and the right bar graph shows gonadal fat weight (percent change compared to control).

[0049] Figures 3A - 3C showed that at the end of the study, other peripheral organ weights ( Figure 3A pancreas, Figure 3B heart, Figure 3C spleen).

[0050] Figures 4A - 4C Shown are changes in blood sugar and insulin levels over time after meals in the study. Figure 4A Postprandial blood sugar, Figure 4BFor postprandial insulin, their changes over time; Figure 4C Is the postprandial glucagon on the 20th day.

[0051] Figure 5A and 5B Show glucose tolerance measurement (day 23) and fasting glucose (day 23 and day 26). Figure 5A Show the change of blood glucose over time. Figure 5B Show (left bar graph) AUC glucose, (middle bar graph) 4-hour fasting blood glucose, (right bar graph) 6-hour fasting blood glucose.

[0052] Figure 6A and 6B Show, ([[]] Figure 6A ) pancreatic weight (left bar graph), β-cell mass (middle bar graph), and α-cell mass (right bar graph), measured by IHC against insulin (β-cells) or glucagon (α-cells), Figure 6B Show a representative picture of α-cell staining.

[0053] Figures 7A - 7F Show, at the end of the study, the measurements of circulating ALT ( Figure 7A ), AST ( Figure 7B ), cholesterol (Chol) ( Figure 7C ), triglyceride (Trig) ( Figure 7D ), and non-esterified fatty acid (NEFA) ( Figure 7E ), as well as the measurement of triglyceride in liver tissue ( Figure 7F ).

[0054] Figure 8A and 8B Show ( Figure 8A ) H&E staining of liver tissue and ( Figure 8B ) histological quantification of lipid droplets.

[0055] Figure 9A and 9B Show ( Figure 9A ) liver α-SMA (smooth muscle actin) staining (as an indicator of fibrosis) and ( Figure 9B ) its quantification.

[0056] Figure 10 Show the timeline of the obese NHP study, which investigated the effects of adding myostatin / activin A blockade to GLP-1R agonism on body weight, liver, and metabolism.

[0057] Figure 11A and 11B Show that adding anti-myostatin treatment to semaglutide results in greater weight loss than semaglutide monotherapy.Figure 11A Show the percentage change in body weight of each group starting from day 0 (D0); Figure 11B Show the percentage change in body weight of each group starting from the start of antibody administration (day 14 (D14)).

[0058] Figures 12A - 12C Show that adding myostatin inhibition therapy to semaglutide results in greater fat loss compared to semaglutide monotherapy, and adding activin A to myostatin inhibitor + semaglutide also increases lean body mass. Show the total body weight ( Figure 12A ), total fat mass ( Figure 12B ), and total lean body mass ( Figure 12C ) of each treatment group over time. The numbers in the line graph represent the percentage change relative to the baseline at week 12 (W12).

[0059] Figures 13A - 13C Show that after 12 weeks of treatment, the combination of semaglutide, myostatin inhibition therapy, and activin A therapy achieved the greatest reduction in HbA1c%. The fasting glucose ( Figure 13A ), HbA1c ( Figure 13B ), and insulin ( Figure 13C ) of each treatment group over time are shown in the figure. The numbers above each group represent the percentage change relative to the baseline at 12 weeks.

[0060] Figures 14A - 14E Show that after 12 weeks of treatment, the combination of semaglutide, myostatin inhibition therapy, and activin A therapy achieved the greatest reduction in LDL and the greatest increase in HDL. The total cholesterol ( Figure 14A ), LDL ( Figure 14B ), HDL ( Figure 14C ), NEFA ( Figure 14D ), and TG ( Figure 14E ) of each treatment group over time are shown in the figure. The numbers above each group are the percentage change relative to the baseline at 12 weeks.

[0061] Figure 15A and 15B Show the AST ( Figure 15A , upper figure), ALT ( Figure 15A , lower figure), and AST / ALT ratio ( Figure 15B ) of different treatment groups up to week 12.

[0062] Figure 16A and 16B Show the total energy intake (7-day average) ( Figure 16A ) and cumulative energy intake ( Figure 16B ) of different treatment groups over time (up to week 12).

[0063] Figure 17A and 17B showed that over time (up to week 12), the total water intake (7-day average) of different treatment groups ( Figure 17A ) and the cumulative water intake ( Figure 17B ).

[0064] Figure 18 showed that myostatin (GDF8) and activin A blockade synergistically increased muscle mass in mice.

[0065] Figures 19A - 19D showed the changes over time in thigh muscle volume ( Figure 19A ) in postmenopausal women, total lean body mass ( Figure 19B ) in obese non-human primates, abdominal and lumbar fat mass ( Figure 19C ) in postmenopausal women, and total fat mass ( Figure 19D ) in obese non-human primates, with each group receiving different treatments.

[0066] Figure 20A and 20B showed the basal energy expenditure ( Figure 20A ) and energy expenditure per kg of lean body mass ( Figure 20B ) in obese non-human primates receiving different treatment combinations.

[0067] Figure 21A and 21B showed energy expenditure vs. total lean body mass ( Figure 21A upper figure for the two treatment groups, lower left figure for the combination treatment group, and lower right figure for the non-combination treatment group); and energy expenditure vs. percentage change in lean body mass relative to baseline ( Figure 21B upper figure for the two treatment groups, lower left figure for the non-combination treatment group, and lower right figure for the combination treatment group) in non-combination treatment groups (vehicle, semaglutide, trevogrumab + garetosmab) and combination treatment groups (semaglutide + trevogrumab, semaglutide + trevogrumab + garetosmab).

[0068] Figures 22A - 22F showed that in the treatment groups of obese non-human primates at Figure 21A and 21B , HbA1c ( Figure 22A ), LDL-C ( Figure 22B ), ApoB ( Figure 22C ), change in HbA1c relative to baseline (Figure 22D ) Changes in LDL-C relative to baseline ( Figure 22E ) Changes in ApoB relative to baseline ( Figure 22F ) Measurement results.

[0069] Figure 23 The study design in non - human primate obese animals is schematically described for studying the effects of adding myostatin / activin A blockade on top of GLP - 1R agonism on body weight, liver, and metabolism. Detailed Description of the Invention

[0071] Before describing the present disclosure, it should be understood that the present disclosure is not limited to the specific methods and experimental conditions described, as these methods and conditions are variable. It should also be understood that the terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the invention, as the scope of the invention is limited only by the appended claims.

[0072] 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 this disclosure belongs. The term "about" as used herein, when specifically referring to a numerical value, means a value that can vary from the stated value by no more than 1%. For example, the expression "about 100" as used herein includes 99 and 101 and all values therebetween (e.g., 99.1, 99.2, 99.3, 99.4, etc.). In this document, ranges can be expressed as from "about" or "approximately" a particular value and / or to "about" or "approximately" another particular value. When expressing such ranges, embodiments covering from that particular value and / or to that other particular value are encompassed.

[0073] "Comprising" or "containing" or "including" means that at least the specified compound, element, particle, or method step is present in the composition, article, or method, but does not exclude the presence of other compounds, materials, particles, or method steps, even if such other compounds, materials, particles, or method steps have the same function as the specified compound, element, particle, or method step.

[0074] Antigen - specific binding protein

[0075] The present disclosure relates to compositions comprising an agonist and an antigen - specific binding protein. More specifically, in certain embodiments, the present disclosure provides compositions comprising a GLP - 1 agonist and a GDF - 8 - specific binding protein, and compositions comprising a GLP - 1 agonist, a GDF - 8 - specific binding protein, and an activin A - specific binding protein.

[0076] As used herein, the term "antigen - specific binding protein" refers to a protein that contains at least one domain that specifically binds to a particular antigen. Exemplary classes of antigen - specific binding proteins include antibodies, antigen - binding portions of antibodies, peptides that specifically interact with a particular antigen (e.g., peptibody), receptor molecules that specifically interact with a particular antigen, and proteins that contain the ligand - binding portion of a receptor that specifically binds to a particular antigen.

[0077] The present disclosure includes antigen - specific binding proteins that specifically bind to GDF - 8, i.e., "GDF - 8 - specific binding proteins". The term "GDF - 8" (also known as "growth differentiation factor - 8" and "myostatin") refers to a protein (mature protein) having the amino acid sequence of SEQ ID NO:25 (SEQ ID NO:1). According to the present disclosure, a GDF8 - specific binding protein specifically binds to GDF - 8, but does not bind to other ActRIIB ligands such as GDF3, BMP2, BMP4, BMP7, BMP9, BMP10, GDF11, activin A, activin B, activin AB, Nodal, etc.

[0078] The present disclosure also includes antigen - specific binding proteins that specifically bind to activin A, i.e., "activin A - specific binding proteins". Activins are homodimeric and heterodimeric molecules that contain βA and / or βB subunits. The βA subunit has the amino acid sequence of SEQ ID NO:2, and the βB subunit has the amino acid sequence of SEQ ID NO:3. Activin A is a homodimer composed of two βA subunits; activin B is a homodimer composed of two βB subunits; activin AB is a heterodimer composed of one βA subunit and one βB subunit. An activin A - specific binding protein can be an antigen - specific binding protein that specifically binds to the βA subunit. Since the βA subunit is present in activin A and activin AB molecules, an "activin A - specific binding protein" can be an antigen - specific binding protein that specifically binds to activin A and activin AB (through its interaction with the βA subunit). Thus, according to the present disclosure, an activin A - specific binding protein specifically binds to activin A, or activin A and activin AB, but does not bind to other ActRIIB ligands such as activin B, GDF3, GDF8, BMP2, BMP4, BMP7, BMP9, BMP10, GDF11, Nodal, etc.

[0079] In the context of the present disclosure, molecules that contain the ligand - binding portion of the ActRIIB receptor, such as ActRIIB - Fc (e.g., "ACE - 031"), are not considered "GDF8 - specific binding proteins" or "activin A - specific binding proteins" because such molecules bind multiple ligands in addition to GDF8, activin A, and activin AB.

[0080] In one embodiment, myostatin (GDF8) and activin A inhibition may alternatively be provided by an ActRIIb-Fc molecule or an antibody that binds to ActRIIB. When male CB17 SCID mice were treated with an anti-activin A antibody, an anti-GDF8 antibody, an anti-activin A antibody + an anti-GDF8 antibody, or ActRIIB.hFc, the combination of anti-GDF8 + anti-activin A caused a greater increase in TA muscle than the increase in these parameters observed in subjects treated with anti-GDF8 or anti-activin A monotherapy. Animals treated with ActRIIB-Fc also showed a significantly greater increase in muscle mass ( Figure 18 ). In another embodiment, the antibody that binds to ActRIIB is bimagrumab.

[0081] The present disclosure includes antigen-specific binding proteins that specifically bind to GLP-1 and / or GLP-1R, namely "GLP-1-specific binding proteins". The term "GLP-1R" refers to the glucagon-like peptide 1 receptor, including recombinant GLP-1R protein or fragments thereof. The sequence of GLP-1R consists of 463 residues (NCBI accession number NP_002053, SEQ ID NO:20). Donnelly, 2011, Br J Pharmacol 166(1):27–41 (2011). Glucagon-like peptide 1 (GLP-1) is a peptide hormone composed of 31 amino acids that is released by intestinal L cells after nutrient intake. Binding of GLP-1 to GLP-1R enhances glucose-induced insulin secretion from pancreatic β-cells, increases insulin expression, inhibits β-cell apoptosis, promotes β-cell neogenesis, reduces glucagon secretion, delays gastric emptying, enhances satiety, and increases the peripheral glucose disposal rate.

[0082] An antigen-binding molecule having two different antigen-specific binding domains

[0083] The present disclosure also includes antigen-binding molecules comprising two different antigen-specific binding domains. Specifically, the present disclosure includes antigen-binding molecules comprising a GDF8-specific binding domain and an activin A-specific binding domain. As used herein, the term "antigen-specific binding domain" includes a polypeptide comprising or consisting of: (i) an antigen-binding fragment of an antibody molecule, (ii) a peptide that specifically interacts with a particular antigen (e.g., peptibody), and / or (iii) a ligand-binding portion of a receptor that specifically binds to a particular antigen. For example, the present disclosure includes bispecific antibodies, one arm of which comprises a first heavy chain variable region / light chain variable region (HCVR / LCVR) pair that specifically binds GDF8, and the other arm of which comprises a second HCVR / LCVR pair that specifically binds activin A. Thus, a composition comprising a GDF-8-specific binding protein and an activin A-specific binding protein (as well as a GLP-1 agonist) can in fact comprise such a single binding protein that contains both a GDF8-specific binding domain and an activin A-specific binding domain.

[0084] Specifically bind

[0085] As used herein, the term "specifically bind" or similar terms means that an antigen-specific binding protein or antigen-specific binding domain forms a complex with a particular antigen with a dissociation constant (K D ) of 500 pM or lower and does not bind to other irrelevant antigens under conventional test conditions. "Irrelevant antigens" refer to proteins, peptides or polypeptides with less than 95% amino acid identity to each other. Methods for determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, etc. For example, as used in the context of the present disclosure, an antigen-specific binding protein or antigen-specific binding domain includes a molecule that binds to a particular antigen (e.g., GDF-8, or activin protein A and / or AB, or GLP-1 / GLP-1R) or a portion thereof, as measured in a surface plasmon resonance assay, with a K D less than about 500 pM, less than about 400 pM, less than about 300 pM, less than about 200 pM, less than about 100 pM, less than about 90 pM, less than about 80 pM, less than about 70 pM, less than about 60 pM, less than about 50 pM, less than about 40 pM, less than about 30 pM, less than about 20 pM, less than about 10 pM, less than about 5 pM, less than about 4 pM, less than about 2 pM, less than about 1 pM, less than about 0.5 pM, less than about 0.2 pM, less than about 0.1 pM or less than about 0.05 pM.

[0086] As used herein, if the binding of a protein or binding domain to a particular molecule is measured at 25 °C in a surface plasmon resonance assay and the protein or binding domain exhibits a K greater than 1000 pM D , or does not exhibit any binding in that assay or an equivalent assay, the antigen-specific binding protein or antigen-specific binding domain "does not bind" the particular molecule.

[0087] The term "surface plasmon resonance" as used herein refers to an optical phenomenon that allows the analysis of real-time interactions by detecting changes in protein concentration in a biosensor matrix, for example, by using a BIAcore TM system (Biacore Life Sciences division of GE Healthcare, Piscataway, NJ).

[0088] The term "K D " as used herein refers to the equilibrium dissociation constant of a particular protein-protein interaction (e.g., an antibody-antigen interaction). Unless otherwise stated, the K D values disclosed herein refer to K D values determined by surface plasmon resonance assay at 25 °C.

[0089] Antibodies and antigen-binding fragments of antibodies

[0090] As described above, an antigen-specific binding protein can comprise or consist of an antibody or an antigen-binding fragment of an antibody. In addition, in the case where an antigen-binding molecule comprises two different antigen-specific binding domains, one or both of the antigen-specific binding domains can comprise or consist of an antigen-binding fragment of an antibody.

[0091] As used herein, the term “antibody” refers to an immunoglobulin molecule containing four polypeptide chains (two heavy chains (H) and two light chains (L) interconnected by disulfide bonds) and multimers thereof (e.g., IgM). Each heavy chain contains a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region contains three domains: CH1, CH2, and CH3. Each light chain contains a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region contains one domain (CL1). The VH and VL regions can be further subdivided into hypervariable regions, called complementarity determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In various different embodiments of the present disclosure, the FRs of the antibodies (or antigen-binding portions thereof) of the present disclosure can be the same as the human germline sequences, or can be naturally or artificially modified. Amino acid consensus sequences can be defined based on alignment analysis of two or more CDRs.

[0092] The term “antibody” as used herein also includes antigen-binding fragments of the intact antibody molecule. The terms “antigen-binding portion” of an antibody, “antigen-binding fragment” of an antibody, etc. as used herein include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex. Antigen-binding fragments of an antibody can be derived, for example, from the intact antibody molecule using any suitable standard techniques, such as proteolytic digestion or recombinant genetic engineering techniques, where the recombinant genetic engineering techniques involve manipulation and expression of DNA encoding the variable domains and optionally the constant domains of the antibody. Such DNA is known and / or can be readily obtained from, for example, commercial sources, DNA libraries (including, for example, phage antibody libraries), or can be synthesized. The DNA can be sequenced and manipulated by chemical or molecular biology techniques in order to, for example, arrange one or more variable domains and / or constant domains in a suitable configuration, or introduce codons, generate cysteine residues, modify, add, or delete amino acids, etc.

[0093] Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units composed of amino acid residues that mimic the hypervariable regions of an antibody, e.g., isolated complementarity-determining regions (CDRs), such as CDR3 peptides, or FR3-CDR3-FR4 constrained peptides. Other engineered molecules, such as domain-specific antibodies, single-domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetra-bodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small molecule immune pharmaceuticals (SMIPs), and shark variable IgNAR domains, are also encompassed by the expression "antigen-binding fragment" as used herein.

[0094] Antigen-binding fragments of antibodies generally comprise at least one variable domain. The variable domain can be of any size or amino acid composition and generally comprises at least one CDR adjacent to or in frame with one or more framework sequences. In antigen-binding fragments having a VH domain associated with a VL domain, the VH and VL domains can be positioned relative to each other in any suitable arrangement. For example, the variable regions can be dimeric and comprise VH-VH, VH-VL, or VL-VL dimers. Alternatively, the antigen-binding fragment of an antibody can comprise a monomeric VH or VL domain.

[0095] In certain embodiments, an antigen-binding fragment of an antibody can comprise at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary configurations of variable and constant domains that can be present in the antigen-binding fragments of the antibodies of the present disclosure include: (i) VH-CH1; (ii) VH-CH2; (iii) VH-CH3; (iv) VH-CH1-CH2; (v) VH-CH1-CH2-CH3; (vi) VH-CH2-CH3; (vii) VH-CL; (viii) VL-CH1; (ix) VL-CH2; (x) VL-CH3; (xi) VL-CH1-CH2; (xii) VL-CH1-CH2-CH3; (xiii) VL-CH2-CH3; and (xiv) VL-CL. In any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains can be directly linked to each other or can be linked via a full or partial hinge region or linker region. The hinge region can consist of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids, resulting in a flexible or semi-flexible linkage between adjacent variable and / or constant domains in a single polypeptide molecule. Additionally, the antigen-binding fragments of the antibodies of the present disclosure can comprise such homodimers or heterodimers (or other multimers) formed by any of the variable and constant domain configurations listed above, with each other, and / or with one or more monomeric VH or VL domains, through non-covalent association (e.g., disulfide bonds).

[0096] The molecules of the present disclosure can comprise or consist of human antibodies and / or recombinant human antibodies or fragments thereof. As used herein, the term "human antibody" includes antibodies having variable and constant regions derived from human germline immunoglobulin sequences. However, a human antibody can contain amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by in vitro random or site-directed mutagenesis or in vivo somatic mutation), such as in the CDRs, particularly CDR3. However, the term "human antibody" as used herein is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species (e.g., mouse) have been grafted onto human framework sequences.

[0097] The molecules of the present disclosure can comprise or consist of recombinant human antibodies or antigen-binding fragments thereof. As used herein, the term "recombinant human antibody" is intended to cover all human antibodies prepared, expressed, produced or isolated by recombinant methods, such as antibodies expressed using a recombinant expression vector transfected into a host cell (described further below), antibodies isolated from a recombinant combinatorial human antibody library (described further below), antibodies isolated from transgenic animals (e.g., mice) with human immunoglobulin genes (see, e.g., Taylor et al., (1992) Nucl. Acids Res. 20:6287-6295), or antibodies prepared, expressed, produced or isolated by any other method that involves splicing human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when transgenic animals with human immunoglobulin sequences are used, in vivo somatic mutagenesis), such that the amino acid sequences of the VH and VL regions of the recombinant antibody, although derived from and related to human germline VH and VL sequences, may not naturally occur in the human in vivo antibody germline repertoire.

[0098] All amino acid abbreviations used in the present disclosure are those accepted by the United States Patent and Trademark Office in 37 C.F.R. §1.822(B)(J). The amino acid sequence of an antibody or its antigen-binding fragment can be numbered using any known numbering scheme, including Kabat et al. ("Kabat" numbering scheme); Al-Lazikani et al., 1997, J. Mol. Biol. 273:927-948 ("Chothia" numbering scheme); MacCallum et al., 1996, J. Mol. Biol. 262:732-745 ("Contact" numbering scheme); Lefranc et al., 2003, Dev. Comp. Immunol. 27:55-77 ("IMGT" numbering scheme); and Honegge and Pluckthun, 2001, J. Mol. Biol. 309:657-70 ("AHo" numbering scheme).

[0099] As used herein, an "agonist" antibody or antigen-binding fragment thereof refers to an antibody or fragment that can increase or enhance at least one biological activity of an antigen (such as GLP-1 and / or GLP-1-R). This increase or enhancement can be mediated by the antibody itself, or, if the antibody is part of an antibody-drug conjugate or an antibody-tethered drug conjugate, it can be mediated by the payload or the linker-payload. For example, when the cell is a mammalian pancreatic β cell, the agonist antibody or fragment can cause stimulation of the adenylate cyclase pathway, resulting in increased cyclic AMP synthesis and insulin release. Other biological activities of GLP-1R can include cAMP-dependent activation of protein kinase A (PKA) and / or cAMP-regulated guanine nucleotide exchange factor 2 (Epac2). After administration to a subject, the agonist antibody or fragment can also lower blood glucose levels or reduce body weight.

[0100] Anti-GDF8 antibody and its antigen-binding fragment

[0101] In certain specific embodiments of the present disclosure, the GDF-8 inhibitor is a GDF8-specific binding protein, and the protein or GDF8-specific binding domain comprises or consists of an anti-GDF8 antibody or its antigen-binding fragment. Anti-GDF8 antibodies are disclosed in, for example, U.S. Patent Nos. 6,096,506, 7,320,789, 7,261,893, 7,807,159, 7,888,486, 7,635,760, 7,632,499; U.S. Patent Application Publication Nos. 2007 / 0178095, 2010 / 0166764, 2009 / 0148436; and International Patent Application Publication No. WO 2010 / 070094. Anti-GDF8 antibodies are also described in U.S. Patent Application No. 13 / 115,170, filed on May 25, 2011, the publication number of which is US20110293630, which includes antibodies named 8D12, H4H1657N2, and H4H1669P. In one embodiment, the anti-GDF8 antibody is REGN1033, also known as H4H1657N2. Any anti-GDF8 antibody or its antigen-binding fragment mentioned and / or described in any of the foregoing patents or publications can be used in the context of the present disclosure, provided that the antibody and / or antigen-binding fragment "specifically binds" to GDF8 (as defined herein).

[0102] In one embodiment, the anti-GDF8 antibody or antigen-binding fragment thereof comprises: heavy chain complementarity determining regions (HCDRs) comprising the heavy chain variable region (HCVR) of SEQ ID NO:4; and light chain complementarity determining regions (LCDRs) comprising the light chain variable region (LCVR) of SEQ ID NO:5. In another embodiment, the anti-GDF8 antibody or antigen-binding fragment thereof comprises: heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) comprising SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8, respectively; and three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) comprising SEQ ID NO:9, TTS, and SEQ ID NO:11, respectively.

[0103] Anti-Activin A antibodies and antigen-binding fragments thereof

[0104] In certain specific embodiments of the present disclosure, the Activin A inhibitor is an Activin A-specific binding protein, and the protein or Activin A-specific binding domain comprises or consists of an antibody or antigen-binding fragment thereof that specifically binds Activin A. In certain embodiments, the Activin A-specific binding protein specifically binds to the βA subunit. The antigen-specific binding protein that specifically binds to the βA subunit can recognize both Activin A (βA / βA homodimer) and Activin AB (βA / βB heterodimer). Thus, according to the present disclosure, the Activin A-specific binding protein can bind Activin A and Activin AB (but not Activin B). Anti-Activin A antibodies are described, for example, in U.S. Patent Application Publication No. 2009 / 0234106. In one embodiment, the anti-Activin A antibody is REGN2477, also known as H4H10446P2. In another embodiment, the anti-Activin A antibody is REGN2376, also known as H4H10430P. Another anti-Activin A antibody is named "MAB3381" and is available from R&D Systems, Inc, Minneapolis, MN. MAB3381 specifically binds Activin A (homodimer) as well as Activin AB (heterodimer). Any of the above anti-Activin A antibodies or antigen-binding fragments thereof can be used in the present disclosure, provided that the antibody and / or antigen-binding fragment "specifically binds" Activin A and / or Activin AB as defined herein.

[0105] In one embodiment, the anti-Activin A antibody or antigen-binding fragment thereof comprises: a heavy chain complementarity determining region (HCDR) comprising a heavy chain variable region (HCVR) of SEQ ID NO:12; and a light chain complementarity determining region (LCDR) comprising a light chain variable region (LCVR) of SEQ ID NO:13. In another embodiment, the anti-Activin A antibody or antigen-binding fragment thereof comprises: heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) comprising SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16, respectively; and three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) comprising SEQ ID NO:17, GAS, and SEQ ID NO:19, respectively.

[0106] Glucagon-like peptide (GLP)-1 agonist / glucagon-like peptide (GLP)-1 receptor agonist

[0107] Incretin is an intestine-derived hormone that is released in response to nutrient intake and stimulates insulin secretion and hyperglycemia. In some embodiments of the compositions and methods of the present disclosure, an incretin inhibitor is used in combination with other active agents (e.g., a GDF-8 inhibitor, or a GDF-8 inhibitor + an Activin A inhibitor). In further embodiments of the compositions and methods of the present disclosure, a GLP-1 agonist or a dipeptidyl peptidase-IV (DPP-4) inhibitor is used in combination with other active agents (e.g., a GDF-8 inhibitor, or a GDF-8 inhibitor + an Activin A inhibitor).

[0108] The term "GLP-1", also known as "glucagon-like peptide 1", refers to a 31-amino acid peptide hormone released by intestinal L cells after nutrient intake. GLP-1 binds to the GLP-1 receptor, enhances glucose-induced insulin secretion by pancreatic β cells, increases insulin expression, inhibits β cell apoptosis, promotes β cell neogenesis, reduces glucagon secretion, delays gastric emptying, promotes satiety, and increases peripheral glucose disposal.

[0109] As used herein, the term "GLP-1 agonist" refers to a compound that promotes, upregulates, or stimulates GLP-1 activity. GLP-1 agonists can activate GLP-1R and include GLP-1 analogs, peptide variants, antibodies (including ligand-tethered antibodies), and fusion proteins. GLP-1 agonists include GLP-1 receptor agonists (GLP-1RA). The GLP-1 agonists described / used herein can be GLP-1 receptor agonists. In fact, for the purposes of this disclosure, the terms "GLP-1 agonist" and "GLP-1R agonist" are used interchangeably. As used herein, the term "GLP-1 receptor agonist" refers to a compound that binds to the GLP-1 receptor. GLP-1 receptor agonists can increase glucose-dependent insulin secretion, reduce inappropriate glucagon secretion, delay gastric emptying, and increase satiety (Trujillo et al., 2021, Ther Adv Endocrinol Metab, 12:1-15). GLP-1 agonists can be selected, for example, from small molecule and peptide GLP-1R agonists and allosteric modulators (Graaf et al., 2016, Pharmacol Rev 68:954-1013).

[0110] GLP-1 agonists used in this disclosure include currently marketed peptide agonists. In certain embodiments, the GLP-1 agonist mimics the action of glucagon-like peptide 1. Known GLP-1 receptor agonists include Albiglutide, Exenatide (short-acting and long-acting), Efpeglenatide, ITCA650, Lixisenatide, Liraglutide, Dulaglutide, and Semaglutide. In certain embodiments, the GLP-1 agonist is selected from Exenatide (long-acting), Dulaglutide, Liraglutide, and Semaglutide. In another embodiment of the compositions or methods of this disclosure, the GLP-1 agonist is Semaglutide. Semaglutide (sold under trade names such as Ozempic) is a glucagon-like peptide-1 receptor agonist that increases the production and secretion of insulin, thereby promoting glucose metabolism. In one embodiment, the GLP-1 agonist used in the methods or compositions of the present invention is a modified peptide drug, such as Tirzepatide, which activates both glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) receptors.

[0111] In another embodiment, the GPL-1 agonist / receptor agonist used in the compositions or methods of the present invention is an antibody or an antigen-binding fragment thereof that specifically binds to GLP-1.

[0112] In certain embodiments, the GLP-1 agonist for use in the compositions and methods of the present invention is an antibody-drug conjugate (ADC) that specifically binds to the glucagon-like peptide 1 receptor (GLP-1R) protein. In another embodiment, the antibody or antigen-binding fragment thereof of the ADC specifically targets the extracellular domain of GLP-1R, wherein the GLP-1 peptide mimetic functionally activates GLP-1R.

[0113] An antibody tethered drug conjugate (ATDC) or antibody-drug conjugate (ADC) refers to an antibody or antigen-binding fragment thereof that is tethered, with or without a linker, to a payload (e.g., a GLP-1 peptide mimetic). An antibody-payload conjugate refers to an antibody or fragment that is conjugated to a payload, while an antibody-linker-payload conjugate refers to an antibody or fragment that is conjugated to a payload through a linker. Antibodies or antigen-binding fragments referred to herein include embodiments in which the antibody or fragment is conjugated to a payload or linker-payload.

[0114] Bioequivalent

[0115] In certain embodiments, the GDF-8 inhibitors, activin A inhibitors, and / or GLP-1 agonists of the present disclosure encompass proteins having an amino acid sequence different from the described GDF-8 inhibitors, activin A inhibitors, and / or GLP-1 agonists, but retaining the ability to bind GDF-8, activin A, or GLP-1. Such variants contain one or more amino acid additions, deletions, or substitutions compared to the parental sequence, but exhibit substantially equivalent biological activity to the described GDF-8 inhibitors, activin A inhibitors, and / or GLP-1 agonists.

[0116] Two proteins are considered bioequivalent if they are, for example, pharmaceutical equivalents or pharmaceutical alternatives with no significant difference in the rate and extent of absorption when administered at the same molar dose (single or multiple doses) under similar experimental conditions. If certain proteins are equivalent in extent of absorption but not in rate of absorption, they will be considered equivalents or pharmaceutical alternatives, and if such difference in rate of absorption is intentional and reflected in the label, is not critical for achieving an effective body drug concentration (e.g., for long-term use), and is considered medically insignificant for the particular pharmaceutical product under study, they may still be considered bioequivalent.

[0117] In one embodiment, two GDF8 inhibitors, activin A inhibitors, or GLP-1 agonist proteins are bioequivalent if there are no clinically significant differences in safety, purity, or potency.

[0118] In one embodiment, two GDF8 inhibitors, activin A inhibitors, or GLP-1 agonist proteins are bioequivalent if a patient can switch between a reference product and a biological product one or more times and the expected risk of adverse reactions (including clinically significant changes in immunogenicity) does not increase or the efficacy does not decrease compared to continuous treatment without such switching.

[0119] In one embodiment, two GDF8 inhibitors, activin A inhibitors, or GLP-1 agonist proteins are bioequivalent if, for the (one or more) indications for which they are used, their mechanisms of action, to the extent known for such mechanisms, are common.

[0120] Bioequivalence can be demonstrated by in vivo and / or in vitro methods. Bioequivalence measurements include, for example: (a) in vivo tests in humans or other mammals in which the concentration of the protein or its metabolite in blood, plasma, serum, or other biological fluids is measured over time; (b) in vitro tests in which the test has been correlated with and can reasonably predict human in vivo bioavailability data; (c) in vivo tests in humans or other mammals in which the appropriate acute pharmacological effect of the protein (or its target) is measured over time; and (d) tightly controlled clinical trials in which the clinical trial establishes the safety, efficacy, bioavailability, or bioequivalence of an antigen-binding protein.

[0121] Bioequivalent variants of the GDF8 inhibitors, activin A inhibitors, and / or GLP-1 agonist proteins of the present disclosure can be constructed, for example, by making various substitutions of residues or sequences, or deleting terminal or internal residues or sequences that are not required for biological activity. For example, cysteine residues that are not necessary for biological activity can be deleted or replaced with other amino acids to prevent the formation of unwanted or incorrect intramolecular disulfide bonds upon refolding. In other cases, bioequivalent proteins can include variants that contain amino acid changes that alter the glycosylation properties of the protein, such as mutations that eliminate or remove glycosylation.

[0122] Pharmaceutical Compositions and Methods of Administration

[0123] The present disclosure includes pharmaceutical compositions comprising a GDF8 inhibitor and a GLP-1 agonist. The present disclosure also includes pharmaceutical compositions comprising a GDF8 inhibitor, an activin A inhibitor, and a GLP-1 agonist. The pharmaceutical compositions of the present disclosure are formulated with suitable carriers, excipients, and other agents that provide for appropriate transfer, delivery, tolerance, etc. A variety of suitable formulations can be found, for example, in Remington's Pharmaceutical Sciences (Mack Publishing Company, Easton, PA). Suitable formulations include, for example, powders, pastes, ointments, gels, waxes, oils, lipids, lipid-containing (cationic or anionic) vesicles (such as LIPOFECTIN TM ), DNA conjugates, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, emulsions carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. Other suitable formulations are also described in Powell et al., “Compendium of excipients for parenteral formulations”, PDA (1998) J Pharm Sci Technol 52:238-311.

[0124] A variety of delivery systems are known and can be used to administer the pharmaceutical compositions of the present disclosure. For example, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, receptor-mediated endocytosis (see, for example, Wu et al., 1987, J. Biol. Chem. 262:4429-4432). Administration methods include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compositions can be administered by any convenient route, such as by infusion or bolus injection, absorption through epithelial or skin mucosa (such as oral mucosa, rectal and intestinal mucosa, etc.), and can be administered together with other bioactive agents.

[0125] The pharmaceutical compositions of the present disclosure can be administered subcutaneously or intravenously using standard needles and syringes. In addition, for subcutaneous delivery, pen-type delivery devices can be readily applied to deliver the pharmaceutical compositions of the present disclosure. Such pen-type delivery devices can be reusable or disposable. Reusable pen-type delivery devices typically employ a replaceable cartridge containing the pharmaceutical composition. Once all of the pharmaceutical composition in the cartridge has been administered and the cartridge is empty, the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. Thereafter, the pen-type delivery device can be reused. Disposable pen-type delivery devices do not have a replaceable cartridge. Instead, disposable pen-type delivery devices are pre-filled with the pharmaceutical composition, which is stored in a reservoir within the device. Once the pharmaceutical composition in the reservoir has been used up, the entire device can be discarded.

[0126] A variety of reusable pen and autoinjector delivery devices can be used to subcutaneously deliver the pharmaceutical compositions of the present disclosure. Examples include, but are not limited to, AUTOPEN TM (Owen Mumford, Inc., Woodstock, UK), DISSETRONIC TM Pen (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75 / 25 TM Pen, HUMALOG TM Pen, HUMALIN 70 / 30 TM Pen (Eli Lilly and Co., Indianapolis, IN), NOVOPEN TM I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR TM (Novo Nordisk, Copenhagen, Denmark), BD TM Pen (Becton Dickinson, Franklin Lakes, NJ), OPTIPEN TM 、OPTIPEN PRO TM 、OPTIPEN STARLET TM and OPTICLIK TM (sanofi-aventis, Frankfurt, Germany), etc. Examples of disposable pen delivery devices that can be used for subcutaneous delivery of the pharmaceutical compositions of the present disclosure include, but are not limited to, SOLOSTAR TM Pen (sanofi-aventis), FLEXPEN TM (Novo Nordisk),KWIKPEN TM (Eli Lilly), SURECLICK TM Autoinjector (Amgen, Thousand Oaks, CA), PENLET TM (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, LP), and HUMIRA TM pen (Abbott Labs, Abbott Park IL), etc.

[0127] In certain cases, the pharmaceutical compositions of the present disclosure can be delivered via controlled release systems. In one embodiment, a pump can be used (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201). In another embodiment, a polymeric material can be used; see Medical Applications of Controlled Release, edited by Langer and Wise, 1974, CRC Pres., Boca Raton, Florida. In another embodiment, the controlled release system can be placed near the target site of the composition, whereby only a fraction of the systemic dose is required (e.g., see Goodson, 1984, Medical Applications of Controlled Release, supra, Volume 2, pp. 115-138). Other controlled release systems have been discussed in Langer's review (1990, Science 249:1527-1533).

[0128] Injectable preparations can include dosage forms such as intravenous, subcutaneous, intradermal, and intramuscular injections, infusions, etc. These injectable preparations can be prepared by known methods. For example, they can be prepared by dissolving, suspending, or emulsifying the above-mentioned antibody or its salt in a sterile aqueous medium or an oily medium commonly used for injection. Examples of injectable aqueous media include physiological saline, isotonic solutions containing glucose and other adjuvants, etc., and can be used in combination with suitable solubilizers such as alcohols (e.g., ethanol), polyhydric alcohols (e.g., propylene glycol, polyethylene glycol), nonionic surfactants [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 moles) adduct of hydrogenated castor oil)], etc. Examples of oily media include sesame oil, soybean oil, etc., and can be used in combination with suitable solubilizers such as benzyl benzoate, benzyl alcohol, etc. The injectable preparations thus prepared are preferably filled into appropriate ampoules.

[0129] Advantageously, the above-mentioned orally or parenterally administered pharmaceutical compositions are prepared into unit dosage forms suitable for placing a dose of an active ingredient. Such unit dosage forms include, for example, tablets, pills, capsules, injectables (ampoules), suppositories, etc.

[0130] Dose

[0131] The amount of the active ingredient (e.g., GDF8 inhibitor, activin A inhibitor, GLP-1 agonist) that can be administered to a subject is generally a therapeutically effective amount. As used herein, the expression "therapeutically effective amount" refers to the dose of an inhibitor (e.g., an antigen-specific binding protein and / or an antigen-binding molecule) and / or an agonist that can cause a detectable change in one or more of the following parameters: lean body mass (increase), fat mass (decrease), body weight (decrease), skeletal muscle mass (increase), plasma ALT and / or AST (decrease), liver triglyceride content / fatty change (decrease), and liver smooth muscle actin (decrease). In a specific embodiment, the therapeutically effective amount of a GDF8 inhibitor vs. an activin A inhibitor vs. a GLP-1 agonist refers to the amount that achieves different effects. For example, in one embodiment, the therapeutically effective amount of a GLP-1 agonist refers to the amount that causes one or more of the following results: (a) a high blood glucose level is reduced to a normal level; and / or (b) one or more symptoms or indicators of diabetes are significantly improved; and / or (c) the liver dysfunction associated with NASH is improved and / or NASH is treated; and / or (d) cholesterol, LDL cholesterol, and / or HDL cholesterol are improved.

[0132] The dose of the active ingredient (e.g., GDF8 inhibitor, activin A inhibitor, GLP-1 agonist) can vary depending on the age and body size of the subject to be administered, the target disease, condition, route of administration, etc.

[0133] For the antibodies of the present disclosure (e.g., anti-GDF8 antibody, anti-Activin A antibody, anti-GLP-1 antibody, anti-GLP-1R antibody, or bispecific antibody), a therapeutically effective amount can be from about 0.05 mg to about 600 mg; for example, about 0.05 mg, about 0.1 mg, about 1.0 mg, about 1.5 mg, about 2.0 mg, about 10 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, about 150 mg, about 160 mg, about 170 mg, about 180 mg, about 190 mg, about 200 mg, about 210 mg, about 220 mg, about 230 mg, about 240 mg, about 250 mg, about 260 mg, about 270 mg, about 280 mg, about 290 mg, about 300 mg, 310 mg, about 320 mg, about 330 mg, about 340 mg, about 350 mg, about 360 mg, about 370 mg, about 380 mg, about 390 mg, about 400 mg, about 410 mg, about 420 mg, about 430 mg, about 440 mg, about 450 mg, about 460 mg, about 470 mg, about 480 mg, about 490 mg, about 500 mg, about 510 mg, about 520 mg, about 530 mg, about 540 mg, about 550 mg, about 560 mg, about 570 mg, about 580 mg, about 590 mg, or about 600 mg of the corresponding antibody.

[0134] The amount of the antibody of the present disclosure (e.g., anti-GDF8 antibody, anti-Activin A antibody, anti-GLP-1 antibody, anti-GLP-1R antibody, or bispecific antibody) contained in a single dose can be expressed as milligrams of antibody per kilogram of patient body weight (i.e., mg / kg). For example, a dose of from about 0.0001 to about 50 mg / kg of patient body weight (such as 0.0001 mg / kg, 0.001 mg / kg, 0.01 mg / kg, 0.1 mg / kg, 0.5 mg / kg, 1.0 mg / kg, 1.5 mg / kg, 2.0 mg / kg, 2.5 mg / kg, 3.0 mg / kg, 3.5 mg / kg, 4.0 mg / kg, 4.5 mg / kg, 5.0 mg / kg, 5.5 mg / kg, 6.0 mg / kg, 6.5 mg / kg, 7.0 mg / kg, 7.5 mg / kg, 8.0 mg / kg, 8.5 mg / kg, 9.0 mg / kg, 9.5 mg / kg, 10.0 mg / kg, 10.5 mg / kg, 11.0 mg / kg, 11.5 mg / kg, etc.) can be administered to a patient for the anti-GDF8 antibody, anti-Activin A antibody, anti-GDF8 / anti-Activin A bispecific antibody, and / or anti-GLP-1 antibody of the present disclosure.

[0135] In certain embodiments, the GLP-1 agonists of the present disclosure can be administered to a subject (patient) at a dose of from about 0.05 mg / mL to about 5 mg / mL (e.g., about: 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1.0 mg / mL, 1.1 mg / mL, 1.2 mg / mL, 1.3 mg / mL, 1.4 mg / mL, 1.5 mg / mL, 1.6 mg / mL, 1.7 mg / mL, 2.0 mg / mL, 2.5 mg / mL, 3.0 mg / mL, 3.5 mg / mL, 4.0 mg / mL, 4.5 mg / mL or 5 mg / mL).

[0136] In certain embodiments, the GLP-1 agonists of the present disclosure can be administered to a subject (patient) at a dose of from about 0.000001 mg / kg body weight to about 50 mg / kg body weight. In additional embodiments, the GLP-1 agonists (GLP-1R agonists) of the present disclosure are administered to a subject (patient) at a dose of from about 0.00001 mg / kg body weight to about 10 mg / kg body weight. In other embodiments, the GLP-1 agonists (GLP-1R agonists) of the present disclosure are administered to a subject (patient) at a dose of about 1 mg / kg body weight. In yet some embodiments, the GLP-1 agonists (GLP-1R agonists) of the present disclosure are administered to a subject (patient) at a dose of about 0.1 mg / kg subject body weight. In yet some embodiments, the GLP-1 agonists (GLP-1R agonists) of the present disclosure are administered to a subject (patient) at a dose of about 10 μg / kg subject body weight. In certain embodiments, the GLP-1 receptor agonists of the present disclosure can be administered in one or more doses comprising from about 0.01 mg to about 60 mg. In other embodiments, the GLP-1 receptor agonists of the present disclosure can be administered in one or more doses comprising from about 0.1 mg to about 6 mg.

[0137] In one specific embodiment, the anti-GDF8 antibody or its antigen-binding fragment is administered at a concentration of about 10 mg / kg to about 100 mg / kg. In another embodiment, the anti-GDF8 antibody or its antigen-binding fragment is administered at a concentration of about 50 mg / kg. In another specific embodiment, the anti-Activin A antibody or its antigen-binding fragment is administered at a concentration of about 10 mg / kg to about 100 mg / kg. In another embodiment, the anti-Activin A antibody or its antigen-binding fragment is administered at a concentration of about 50 mg / kg. In another specific embodiment, the GLP-1 agonist is administered at a concentration of about 1 μg / kg to about 100 μg / kg. In another specific embodiment, the GLP-1 agonist is administered at a concentration of about 10 μg / kg. In yet another embodiment, the GLP-1 agonist is administered at a concentration of about 0.833 mg / mL or about 7 μg / day (infusion). In yet another embodiment, the anti-GDF8 antibody or its antigen-binding fragment is administered at a concentration of about 50 mg / kg, the anti-Activin A antibody or its antigen-binding fragment is administered at a concentration of about 50 mg / kg, and the GLP-1 agonist is administered at a concentration of about 10 μg / kg.

[0138] The compositions of the present disclosure can comprise equal amounts of a GDF8-specific binding protein and an Activin A-specific binding protein. Alternatively, the amount of the GDF8-specific binding protein in the composition can be less than or greater than the amount of the Activin A-specific binding protein. Alternatively, the compositions of the present disclosure can not comprise an Activin A-specific binding protein. A person of ordinary skill in the art will be able to determine the appropriate amounts of the individual components of the compositions of the present disclosure necessary to produce the desired therapeutic effect through routine experimentation and based on the present disclosure.

[0139] Methods of treatment

[0140] As used herein, the terms "treatment" or "therapy" refer to reducing or ameliorating the severity of at least one symptom or indication of a disease or disorder associated with GDF-8, Activin A, and / or GLP-1. In some embodiments, this disease or disorder is obesity, diabetes, liver dysfunction, or other disorders associated with hyperglycemia. In one embodiment, the diabetes is type 2 diabetes. These terms can also refer to inhibiting disease progression or symptom worsening. These terms can also refer to a positive prognosis of the disease, i.e., after administration of a therapeutic agent (e.g., a composition according to the present disclosure, i.e., a composition comprising a GDF8 inhibitor and a GLP-1 agonist, or a composition comprising a GDF8 inhibitor, an Activin A inhibitor, and a GLP-1 agonist), the subject can be asymptomatic or have no indication, or the intensity of the symptoms or indications can be reduced. The therapeutic agent can be administered to the subject at a therapeutic dose.

[0141] The term "prevention" refers to inhibiting the manifestation of any symptoms or indicia of a disease or disorder associated with GDF-8, activin A, and / or GLP-1. These terms can also refer to inhibiting the manifestation of symptoms or indicia of such a disease or disorder in a subject at risk of developing a disease or disorder associated with GDF-8, activin A, and / or GLP-1. In some embodiments, such a disease or disorder is obesity, diabetes, liver dysfunction, or other conditions associated with hyperglycemia. In one embodiment, the diabetes is type 2 diabetes.

[0142] The present disclosure includes compositions and methods for treating a condition or disease that can be cured, alleviated, or improved by increasing an individual's lean body mass and / or reducing fat mass, or by favorably altering blood glucose control, by specifically binding to GDF8 and activin A and exerting agonist activity on GLP-1. For example, the present disclosure includes compositions and methods for improving glucose control, increasing lean body mass, reducing fat mass, treating diabetes, and / or treating obesity and / or treating liver problems associated with increased fat mass, obesity, and / or diabetes in a subject, the method including, in certain embodiments, administering to the subject a composition comprising a GDF-8 inhibitor and a GLP-1 agonist, and in further embodiments, administering to the subject a composition comprising a GDF-8 inhibitor, an activin A inhibitor, and a GLP-1 agonist. The present disclosure also includes compositions and methods for improving glucose control, increasing lean body mass, reducing fat mass, treating diabetes, and / or treating obesity and / or treating liver problems associated with increased fat mass, obesity, and / or diabetes in a subject, the method including in certain embodiments administering to the subject a composition comprising a GDF-8 inhibitor and a GLP-1 agonist, and in further embodiments, administering to the subject a composition comprising a GDF-8 inhibitor, an activin A inhibitor, and a GLP-1 agonist, wherein the administration is carried out with a single composition or with more than one composition (e.g., where each inhibitor and agonist is in a separate composition, or where the two inhibitors are in one composition and the agonist is in another composition).

[0143] Treatment of liver problems can include alleviating signs / symptoms of liver injury (e.g., ALT / AST), reducing liver triglycerides, reducing steatosis (in the liver) and / or reducing fibrosis. In other embodiments, treatment of liver problems includes treating liver dysfunction, such as hepatitis (hepatitis A, B, C, D, and E), fatty liver disease (alcoholic and non-alcoholic), autoimmune diseases (autoimmune hepatitis, primary biliary cirrhosis, primary sclerosing cholangitis), genetic diseases (hemochromatosis, Wilson's disease, α-1 antitrypsin deficiency), drug-induced liver disease, cancer (e.g., hepatocellular carcinoma), cirrhosis, and liver failure. Any GDF-8 inhibitor (e.g., GDF8-specific binding protein), activin A inhibitor (e.g., activin A-specific binding protein), and GLP-1 agonist (e.g., GLP-1 receptor agonist) disclosed or mentioned herein can be used in these aspects of the present disclosure. For example, the treatment methods of the present disclosure include administering to a subject an anti-GDF8 antibody and a GLP-1 agonist, or an anti-GDF8 antibody and an anti-activin A antibody and a GLP-1 agonist.

[0144] The present disclosure also includes methods of managing or treating liver disorders or problems associated with increased fat mass, obesity, and diabetes by administering to a subject in need a GDF8 inhibitor and a GLP-1 agonist, or a GDF8 inhibitor, an activin A inhibitor, and a GLP-1 agonist. In certain embodiments, the management or treatment of the liver disorder or problem is manifested as reducing plasma ALT and / or AST (liver injury markers), reducing liver triglyceride content / steatosis, and / or reducing liver smooth muscle activity (fibrosis marker). Thus, in certain embodiments, administering the compositions of the present disclosure alleviates liver injury or its risk, reduces steatosis or its risk, and / or reduces liver fibrosis or its risk in a subject.

[0145] In methods that include administering to a subject a GDF-8 inhibitor and a GLP-1 agonist, or a GDF-8 inhibitor and an activin A inhibitor and a GLP-1 agonist, the GDF-8 inhibitor and the activin A inhibitor (if present) and the GLP-1 agonist can be administered to the subject simultaneously or substantially simultaneously, such as in a single therapeutic dose, or as two or more separate doses simultaneously or sequentially, such as in separate therapeutic doses that are separated in time from each other, or as two or three separate doses simultaneously or sequentially.

[0146] The compositions of the present disclosure can be administered to a subject in combination with one or more other therapeutic agents, including, for example, growth factor inhibitors, immunosuppressants, anti-inflammatory agents, metabolic inhibitors, enzyme inhibitors, cytotoxic / cytostatic agents, and drugs for controlling blood glucose levels (e.g., metformin). In some embodiments, the other therapeutic agent is selected from insulin or insulin analogs, biguanides (e.g., metformin), thiazolidinediones, sulfonylureas (e.g., chlorpropamide), glinides (e.g., nateglinide), α-glucosidase inhibitors, DPP4 inhibitors (e.g., sitagliptin), pramlintide, bromocriptine, SGLT2 inhibitors (e.g., canagliflozin), anti-hypertensive drugs, statins, aspirin, dietary modifications, exercise, and dietary supplements. The other therapeutic agent(s) can be administered before, simultaneously with, or after administration of the GDF-8 inhibitor, activin A inhibitor (if present), and GLP-1 agonist (or composition comprising the same) of the present disclosure.

[0147] Exemplary diseases, disorders, and conditions treatable with the compositions of the present disclosure include, but are not limited to, sarcopenia, cachexia (idiopathic or secondary to other conditions such as cancer, chronic renal failure, or chronic obstructive pulmonary disease), muscle injury, muscle wasting, and muscle atrophy, such as muscle wasting or atrophy caused by or associated with disuse, immobilization, bed rest, injury, drug therapy, or surgical intervention (such as hip fracture, hip replacement, knee replacement, etc.) or the need for mechanical ventilation. The compositions of the present invention can also be used to treat, prevent, or improve diseases such as cancer, obesity, diabetes, arthritis, multiple sclerosis, muscular dystrophy, amyotrophic lateral sclerosis, Parkinson's disease, osteoporosis, osteoarthritis, osteopenia, metabolic syndrome (including but not limited to diabetes, obesity, dystrophy, organ atrophy, chronic obstructive pulmonary disease, and anorexia), etc. The compositions of the present invention can also be used to treat, prevent, or improve diseases such as diabetes mellitus, obesity, insulin resistance, hypertension, dyslipidemia, type 2 diabetes, type 1 diabetes, prediabetes, cardiovascular disease, atherosclerosis, congestive heart failure, coronary heart disease, arteriosclerosis, peripheral artery disease, stroke, respiratory dysfunction, kidney disease, fatty liver, non-alcoholic steatohepatitis (NASH), and metabolic syndrome. NASH is a non-alcoholic fatty liver disease (NAFLD) characterized by hepatic steatosis with inflammation and hepatocyte ballooning, which can lead to advanced fibrosis, cirrhosis, and hepatocellular carcinoma (Paternostro and Trauner, 2022, J Intern Med 0:1-15).

[0148] The compositions of the present disclosure can also be used to treat, prevent, or improve diseases such as liver dysfunction, such as hepatitis (A, B, C, D, and E), fatty liver diseases (alcoholic and non-alcoholic), autoimmune diseases (autoimmune hepatitis, primary biliary cirrhosis, primary sclerosing cholangitis), genetic diseases (hemochromatosis, Wilson's disease, α-1 antitrypsin deficiency), drug-induced liver disease, cancer (such as hepatocellular carcinoma), cirrhosis, and liver failure.

[0149] Administration Regimen

[0150] In certain embodiments of the present invention, multiple doses of the disclosed compositions (e.g., a composition comprising a GDF8 inhibitor and a GLP-1 agonist, or a composition comprising a GDF8 inhibitor, an activin A inhibitor, and a GLP-1 agonist) can be administered to a subject within a defined time period. The methods according to this aspect of the present invention include sequentially administering multiple doses of the disclosed compositions to a subject. As used herein, "sequential administration" means administering each dose of the disclosed composition to a subject at different time points (e.g., on different days separated by a predetermined interval (e.g., hours, days, weeks, or months)). The present disclosure includes methods of sequentially administering an initial dose of the disclosed composition to a patient, followed by one or more second doses of the composition, and optionally followed by one or more third doses of the composition.

[0151] The terms "initial dose", "second dose", and "third dose" refer to the chronological order of administering the disclosed composition. Thus, an "initial dose" is the dose administered at the start of a treatment regimen (also referred to as the "baseline dose"); a "second dose" is the dose administered after the initial dose; a "third dose" refers to the dose administered after the second dose. The initial dose, second dose, and third dose can all contain the same amount of the active ingredient(s), but typically differ in the frequency of administration. In certain embodiments, however, the amount of the active ingredient(s) contained in the initial dose, second dose, and / or third dose can vary relative to each other during the course of treatment (e.g., increased or decreased as needed).

[0152] In an exemplary embodiment of the present disclosure, each second dose and / or third dose is administered 1 to 30 days (e.g., 1, 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, 27, 28, 29, 30 days or longer) after the administration of the immediately preceding dose. As used herein, the phrase "immediately preceding dose" means that, in a series of multiple administrations, this dose of the composition of the present invention is administered to the subject immediately before the administration of the next dose in sequence, with no intervening dose administrations.

[0153] The methods according to this aspect of the present invention can include administering any number of second and / or third doses of the composition of the present invention to a patient. For example, in certain embodiments, only a single second dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8 or more) second doses are administered to the patient. Similarly, in certain embodiments, only a single third dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8 or more) third doses are administered to the patient.

[0154] In embodiments involving multiple second doses, each second dose may be administered to the patient at the same frequency as the other second doses. For example, each second dose may be administered to the patient 1 to 29 days after the immediately preceding dose. Similarly, in embodiments involving multiple third doses, each third dose may be administered to the patient at the same frequency as the other third doses. For example, each third dose may be administered to the patient 1 to 60 days after the immediately preceding dose. Alternatively, the frequency of administration of the second dose and / or the third dose to the patient may vary during the course of the treatment regimen. The frequency of administration may also be adjusted by a physician during the course of treatment based on the needs of the individual patient following a clinical examination.

[0155] Sequence

[0156] The sequences mentioned herein have the SEQ ID NO and sequences shown in the following informal sequence table:

[0157]

[0158]

[0159]

[0160]

[0161]

[0162] * = H4H10446P2; ** = H4H10430P Example

[0163] The following examples are provided to give a complete disclosure and description to those of ordinary skill in the art of how to make and use the methods and compositions of the present disclosure. These examples are not intended to limit the scope of what the inventors regard as their disclosure. Although efforts have been made to ensure the accuracy of the numbers used (e.g., amounts, temperatures, etc.), some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weights are average molecular weights, temperatures are in degrees Celsius, and pressures are at or near atmospheric pressure.

[0164] Example 1: In Vivo Efficacy Study to Examine the Effects of REGN1033 & REGN2477 Alone and in Combination with the GLP-1 Agonist Semaglutide on Body Composition and Glucose Metabolism

[0165] To study the effects of inhibiting GDF8 and activin A, with and without GLP-1 agonism, on body composition, a treatment study was conducted using a diet-induced obesity (DIO) mouse model. DIO mice that had been fed a high-fat diet for at least 20 weeks were evenly divided into 4 different groups based on body fat content and blood glucose measured by MRI. On day 0, all mice were implanted with an osmotic pump (Alzet, Cat#2004), in which the stainless-steel flow regulator was replaced with a PEEK tube (DURECT Corporation, Cat#0002496) to allow for MRI measurements during the experiment. The pumps used in groups 1 and 2 were filled with PBS, and the pumps used in groups 3 and 4 were filled with semaglutide (0.833 mg / mL, 7 μg infusion per day). The next day (day 1), the mice were injected with the corresponding antibodies: groups 1 and 3: isotype control (20 mg / kg); groups 2 and 4: anti-GDF8 antibody (REGN1033) & anti-activin A antibody (REGN2477) (10 mg / kg each). The antibody injections were repeated on days 4 and 7, and then once a week thereafter.

[0166] Once a week, body composition was measured using MRI in the morning and postprandial blood glucose was measured by taking blood from the tail of the mice using a blood glucose meter. Mouse blood was also taken weekly for insulin measurement. An oral glucose tolerance test (oGTT) was performed on day 23. In the oGTT, the mice were fasted for 4 hours and baseline glucose was measured. Subsequently, 2 g / kg glucose was administered to the mice by oral gavage, and glucose was measured again at 30, 60, 90, and 120 minutes. On day 26, 6-hour fasting blood glucose was measured and mouse blood was taken for insulin assessment as described above. At the end of the study (days 28 / 29), the mice were sacrificed and tissues (skeletal muscle, fat, pancreas, heart, spleen, liver) were collected for weighing and further analysis. The circulating levels of ALT, AST, non-esterified fatty acids (NEFA), triglycerides (trigs), and cholesterol (chol) were measured using mouse plasma on a Siemens Advia biochemical analyzer. One lobe of the liver was freshly frozen to extract and quantify triglycerides using standard protocols. Another lobe of the liver and the pancreas were fixed in 10% formalin, paraffin-embedded, and sectioned for histological analysis of the liver. H&E and smooth muscle actin (SMA) staining were performed, and the sections were quantitatively analyzed using Halo software. Pancreatic paraffin sections were stained for insulin and glucagon to determine the β-cell and α-cell amounts, respectively, using Halo software.

[0167] Results

[0168] Compared with the control group (Group 1), treatment of DIO mice with REGN1033®N2477 resulted in an approximately 10% increase in lean body mass and an approximately 15% reduction in fat mass. Overall, there was no change in body weight in the isotype group. Semaglutide treatment reduced body weight (by approximately 14% compared with the control group), and this weight loss was associated with a reduction in fat mass (by approximately 18% compared with the control group) and a smaller reduction in lean body mass (by approximately 3 - 4% compared with the control group)( Figures 1A - 1C ). The combination treatment of REGN1033®N2477 and semaglutide (Group 4) appeared to have an additive effect on fat reduction (by approximately 50% compared with the control group), while maintaining the increase in lean body mass observed with REGN1033 and REGN2477 alone (8% compared with the control group). Due to the increase in lean body mass, the weight loss in Group 4 was similar to that observed in mice treated with semaglutide alone (Group 3). The increase in skeletal muscle weight at the end of the study (by approximately 40% compared with the control group) confirmed that the increase in lean body mass mediated by REGN1033®N2477 was due to an increase in muscle mass rather than an increase in heart or spleen mass( Figure 2A ). In addition, only in Group 4, when REGN1033®N2477 was combined with semaglutide treatment, the subcutaneous and gonadal fat pads were significantly reduced (by 50% compared with the control group)( Figure 2B ).

[0169] Table 1. Changes in body weight over time

[0170]

[0171] Table 2. Changes in lean body mass over time

[0172]

[0173] Table 3. Changes in fat mass over time

[0174]

[0175] Table 4. Final organ weights

[0176]

[0177] During the entire study, no major changes in glucose metabolism were observed. All mice treated with semaglutide (Groups 3 and 4) showed a transient decrease in postprandial blood glucose on day 7, but this was not observed in subsequent days( Figure 4A ). Although fasting blood glucose was lower in the semaglutide treatment group in the later stage of the study, no major changes in glucose tolerance were observed. However, a decrease in circulating insulin levels was observed in all treatment groups compared with the control group, indicating that blood glucose could be controlled with less insulin(Figure 4B )。 Additionally, glucagon levels appeared lower in the REGN1033 & REGN2477 treatment group ( Figure 4C ). In this study, when REGN1033 & REGN2477 was combined with semaglutide treatment, pancreatic mass increased significantly ( Figure 6A ). This effect was thought to be driven by semaglutide and has been reported previously with other GLP-1 agonists or compounds that activate this pathway (PMID: 19509017, 22266668). To examine whether the changes in pancreatic mass, insulin, and glucagon were driven by changes in β-cell mass or α-cell mass, pancreatic histology was performed ( Figure 6B ). Although there was no significant change in β-cell mass, a trend towards a decrease in β-cell mass was observed in Groups 2 and 4 when REGN1033 & REGN2477 was administered. Additionally, in mice treated with REGN1033 & REGN2477, α-cell mass decreased significantly (by approximately 50% compared to the control group), which could explain the decrease in glucagon observed above.

[0178] Table 5: Changes in postprandial glucose over time

[0179]

[0180] Table 6: Changes in postprandial insulin over time

[0181]

[0182] Table 7: Postprandial glucagon levels on Day 20

[0183]

[0184] Table 8: 6-hour fasting glucose levels on Day 26

[0185] <![CDATA[PBS / IgG4 P > PBS / REGN1033 & REGN2477 <![CDATA[Semaglutide / IgG4 P > Semaglutide / REGN1033 & REGN2477 N 7 7 8 8 Mean 228 237 172.6 201.5 Standard Deviation 30.2 41.97 43.05 41.55 Standard Error of the Mean 11.42 15.86 15.22 14.69

[0186] Table 9: 4-hour fasting glucose levels on Day 23

[0187] <![CDATA[PBS / IgG4 P > PBS / REGN1033 & REGN2477 <![CDATA[Semaglutide / IgG4 P > Semaglutide / REGN1033 & REGN2477 N 8 7 8 8 Mean 225 212.6 192.1 161.9 Standard Deviation 28.7 23.51 17.11 36.73 Standard Error of the Mean 10.15 8.888 6.049 12.99

[0188] Table 10: Area under the oGTT curve on Day 23

[0189] <![CDATA[PBS / lgG4 P > PBS / REGN1033 & REGN2477 <![CDATA[Semaglutide / IgG4 P > Semaglutide / REGN1033 & REGN2477 N 8 7 8 9 Mean 28997 32633 27698 27963 Standard Deviation 1791 1679 2489 3809 Standard Error of the Mean 633.1 634.6 879.9 1270

[0190] Table 11: oGTT measurements on Day 23

[0191]

[0192] Table 12: Changes in pancreatic weight

[0193] <![CDATA[PBS / lgG4 P > PBS / REGN1033 & REGN2477 <![CDATA[Semaglutide / IgG4 P > Semaglutide / REGN1033 & REGN2477 N 8 7 8 9 Mean -7.143E-09 9.21 22.02 31.9 Standard Deviation 10.26 22.07 20.34 24.56 Standard Error of the Mean 3.876 8.342 7.191 8.682

[0194] Table 13: Changes in β-cell mass

[0195] <![CDATA[PBS / lgG4 P > PBS / REGN1033 & REGN2477 <![CDATA[Semaglutide / IgG4 P > Semaglutide / REGN1033 & REGN2477 N 8 7 8 9 Mean F -45.74 -16.12 -38.83 Standard Deviation 49.35 32.04 50.64 29.02 Standard Error of the Mean 18.65 12.11 17.9 10.26

[0196] Table 14: Changes in α-cell mass

[0197] <![CDATA[PBS / lgG4 P > PBS / REGN1033 & REGN2477 <![CDATA[Semaglutide / IgG4 P > [Semaglutide / REGN1033 & REGN2477 N 8 7 8 9 Mean -1.286E-09 -60.56 -28.15 -46.58 Standard Deviation 27.69 21.72 35.44 34.32 Standard Error of the Mean 10.47 8.209 12.53 12.14

[0198] Since the reduction in adipose tissue was associated with improved liver phenotypes, it was also evaluated. Plasma ALT and AST (markers of liver injury) measurements showed significant decreases in all treatment groups, but the greatest decrease occurred when REGN1033®N2477 was combined with semaglutide treatment (Group 4) ( Figure 7A and 7B ). In addition, circulating cholesterol was reduced in Group 4 ( Figure 7C ). Associated with the improvement in ALT and AST, liver triglyceride content was reduced in all treatment groups ( Figure 7F ). The combination of REGN1033®N2477 and semaglutide again appeared to have an additive effect in reducing liver triglycerides and ALT. Histological analysis of liver tissue confirmed these results on liver triglycerides. In addition, the two groups receiving REGN1033®N2477 treatment had a decrease in smooth muscle actin staining (a marker of early fibrosis) ( Figure 9B ), indicating that blocking activin A and GDF8 can prevent liver fibrosis. None of the treatments led to an increase in circulating free fatty acids or triglycerides, indicating that fat was burned rather than simply entering the circulation.

[0199] Table 15: Plasma measurements - liver group

[0200]

[0201] Table 16: Liver triglycerides

[0202] <![CDATA[PBS / IgG4 P > PBS / REGN1033 & REGN2477 <![CDATA[Semaglutide / IgG4 P > Semaglutide / REGN1033 & REGN2477 N 8 7 8 9 Mean 260.8 171.4 153.6 119.6 Standard Deviation 58.29 21.75 56.12 50.77 Standard Error of the Mean 22.03 8.219 19.84 16.92

[0203] Table 17: Liver fat area

[0204] <![CDATA[PBS / IgG4 P > PBS / REGN1033 & REGN2477 <![CDATA[Semaglutide / IgG4 P > Semaglutide / REGN1033 & REGN2477 N 8 7 8 9 Mean 31.52 30.99 28.34 20.68 Standard Deviation 7.101 3.946 3.16 5.187 Standard Error of the Mean 2.684 1.492 1.117 1.834

[0205] Table 18: SMA-positive area (percentage of fat-free area)

[0206] <![CDATA[PBS / IgG4 P > PBS / REGN1033 & REGN2477 <![CDATA[Semaglutide / IgG4 P > Semaglutide / REGN1033 & REGN2477 N 8 7 8 9 Mean 5.888 4.318 5.942 3.962 Standard Deviation 2.809 1.014 2.438 1.473 Standard Error of the Mean 1.062 0.3834 0.8621 0.4909

[0207] Table 19: SMA positive area (normalized by the number of cell nuclei)

[0208] <![CDATA[PBs / IgG4 P > PBS7REGN1033 & REGN2477 <![CDATA[Semaglutide / IgG4 P > Semaglutide / REGN1033 & REGN2477 N 8 7 8 9 Mean 14.72 10.94 14.32 8.79 Standard Deviation 6.62 2.425 6.237 1.767 Standard Error of the Mean 2.502 0.9166 2.205 0.6249

[0209] Overall, these data indicate that the combination of REGN1033 & REGN2477 with semaglutide has an additive effect in reducing body fat and improving liver disease (reducing signs of liver injury (ALT / AST), reducing liver triglycerides, and decreasing steatosis and fibrosis).

[0210] Example 2: Efficacy study to examine the effects of adding myostatin inhibition or adding myostatin and activin A inhibition to GLP-1 agonist therapy

[0211] Obese NHP studies were conducted to examine the effects of adding myostatin / activin A blockade to GLP-1R agonism on body weight, liver, and metabolism. Figure 10 Shown are the studies and schedules of obese adult male cynomolgus non-human primates (NHP) with NASH, stratified by body weight and percent body fat, n = 10 per arm. It has been demonstrated that NHP spontaneously develop obesity, metabolic syndrome, and type 2 diabetes, with metabolic characteristics similar to humans (Canter et al., 2018, J Leukoc Biol 104(3):487-497).

[0212] Treatment groups were:[[]]

[0213] · Vehicle;

[0214] · Semaglutide;

[0215] · Myostatin + activin A;

[0216] · Semaglutide + myostatin; and

[0217] · Semaglutide + myostatin + activin A.

[0218] REGN1033 is an anti-myostatin antibody and REGN2477 is an anti-activin A antibody. The GLP-1 agonist used was semaglutide. Semaglutide was administered at 10 μg / kg. The anti-myostatin antibody was administered at 50 mg / kg. The anti-activin A antibody was administered at 50 mg / kg.

[0219] Results

[0220] Compared to semaglutide monotherapy, adding myostatin inhibition to semaglutide resulted in increased weight loss ( Figure 11A and 11B ).

[0221] Compared with semaglutide monotherapy, adding myostatin inhibition to semaglutide resulted in increased fat loss, and adding activin A to myostatin inhibition + semaglutide further increased lean body mass( Figures 12A - 12C ). At week 12, adding myostatin inhibition (REGN1033) to semaglutide increased the amount of fat loss compared to semaglutide alone( Figure 12B ). Further adding activin A (REGN2477) also restored the lean body mass loss observed with semaglutide and further increased lean body mass beyond baseline at week 12( Figure 12C ).

[0222] After 12 weeks of treatment, the triple combination of semaglutide, myostatin antibody, and activin A antibody showed the greatest reduction in HbA1c%( Figures 13A - 13C ).

[0223] After 12 weeks of treatment, the triple combination of semaglutide, myostatin antibody, and activin A antibody showed the greatest reduction in LDL and the greatest increase in HDL( Figures 14A - 14E ).

[0224] The AST / ALT ratio improved in all groups at week 12( Figure 15A and 15B ).

[0225] All groups receiving semaglutide had lower food intake after the initial dose, but the semaglutide control group returned to baseline more quickly( Figure 16A and 16B ). Myostatin inhibition and activin A had a slightly increased food intake at the start of dosing, but as seen at week 12, this change did not expand over time. However, as observed at week 12, adding myostatin inhibition to semaglutide helped delay the recovery of the appetite-suppressing effect of semaglutide to baseline.

[0226] All groups receiving semaglutide had lower water intake after the initial dose, but the triple combination group was much less inhibited than the myostatin inhibition + semaglutide group( Figure 17A ). All semaglutide treatment groups had lower water intake from the start of dosing (up to week 12). However, adding activin A to myostatin inhibition and semaglutide alleviated this thirst inhibition.

[0227] Therefore, the results indicate that:

[0228] · Semaglutide and myostatin inhibition have a synergistic effect on fat loss;

[0229] · Adding activin A antagonist can further increase lean body mass and further improve fasting blood glucose, HbA1c, LDL, and HDL;

[0230] · Using myostatin antagonist and activin A antagonist alone shows certain fat reduction and lean body mass increase similar to that of the triple combination, but does not improve circulating lipids and glucose; and

[0231] · Food intake decreased in all semaglutide treatment groups.

[0232] Example 3. Blocking myostatin and activin A in non-human primates and humans

[0233] Postmenopausal women received 10 mg / kg (myostatin antagonist + activin A antagonist) IV, Q2W. Thigh muscle volume was measured by MRI, and abdominal fat mass was measured by iDXA, both for 30 weeks. Obese non-human primates received 50 mg / kg myostatin antagonist + 50 mg / kg activin A antagonist (QW). Total lean body mass was measured by iDXA, and total fat mass was measured by iDXA, both for 28 weeks.

[0234] Inhibition of myostatin and activin A led to a stable increase in thigh muscle volume (MRI) in postmenopausal women during the first ten weeks of treatment ( Figure 19A ), and also led to a (relative) stable increase in total lean body mass (iDXA) in obese non-human primates ( Figure 19B ). At the same time, inhibition of myostatin and activin A led to a stable decrease in abdominal fat mass (iDXA) in postmenopausal women during 30 weeks of treatment ( Figure 19C ), and also led to a stable decrease in total fat mass (iDXA) in obese non-human primates during 28 weeks of treatment ( Figure 19D ).

[0235] Inhibition of myostatin and activin A led to significant fat reduction in both humans and non-human primates, but this phenomenon occurred after a significant increase in lean body mass. However, the effect on fat persisted after lean body mass returned to baseline.

[0236] Example 4. Adding blockade of myostatin and activin A to semaglutide (GLP-1R agonist) can increase energy expenditure in obese non-human primates

[0237] In this diet-induced obesity non-human primate study, 64 male obese monkeys that had not received human immunoglobulin were initially screened, including physical examination, metabolic and safety clinical chemistry analysis, hematology examination, body composition examination by iDEXA (using a GE Lunar iDXA scanner), and liver biopsy to score non-alcoholic fatty liver disease plus fibrosis (NAS+). Animals were required to have a body fat > 25%, fasting blood glucose ≤ 350 mg / dL and ≥ 100 mg / dL, a liver NAS+ score greater than 4, and a steatosis score greater than 2. From this cohort, 55 monkeys were selected for a diet transition from a high-fat diet to a high-fat, high-fructose diet that would exacerbate any liver disease. The animals were maintained on this diet for six weeks, after which they underwent baseline screening for the study, including another physical examination, metabolic and safety clinical chemistry analysis, hematology examination, and iDEXA (using a GE Lunar iDXA scanner) to detect body composition. In addition, food and water intake were monitored daily. The average of the daily food and water intake over six days was taken as the baseline reading for these parameters.

[0238] A cohort of 50 monkeys was finally selected and evenly divided into 5 groups of 10 each based on the following parameters: body weight, total body fat, and NAS+ score. The secondary parameters considered included fasting blood glucose, total lean body mass, and fasting triglycerides. The baseline characteristics of the 5 groups are listed in Table 20.

[0239] Table 20. Baseline Characteristics

[0240]

[0241] Mean ± SEM

[0242] Compound and Treatment Group

[0243] The grouping and dosing regimens for this study are listed in Table 21 below and are designed as shown in Figure 23 This study was divided into five groups: vehicle control group, GLP-1 group, α-MSTN and α-ActA group, GLP-1 + α-MSTN group, and GLP-1 + α-MSTN + α-ActA group. All compounds were administered subcutaneously. GLP-1 was administered twice a week starting from day 0 of the study at a dose of 10 μg / kg for a total of 20 weeks, and due to this low-dose regimen, GLP-1 titration was not performed. The groups receiving α-MSTN and α-ActA were administered once a week starting from week 2 of the study at a dose of 50 mg / kg until week 12. All administrations were completed by week 20, after which the cohort underwent an additional 8-week washout period follow-up. The number of animals listed in Table 21 represents the final number at the end of the study. Four monkeys died during the study; unless otherwise stated, all data from these monkeys were not included in the mean.

[0244] Table 21. Study Groups and Doses

[0245]

[0246]

[0247] Liver Histology and Scoring

[0248] Two liver biopsies were performed during the study: one during animal screening and one at the end of week 12. Under ultrasound guidance, liver tissue samples (each sample approximately 0.5 to 1.0 cm) were obtained by biopsy from animals after an overnight fast under sedation / anesthesia (ketamine, 5 - 10 mg / kg, IM). For all samples (each time), after accurately positioning the right lobe of the liver by ultrasound, a 16 - 18G biopsy needle was used and slowly advanced under ultrasound guidance through the skin, muscle tissue, and liver capsule into the interior of the liver tissue to collect the biopsy specimen. The biopsy specimen was fixed in 10% neutral buffered formalin and embedded in paraffin. The paraffin - embedded tissue samples were sectioned and stained simultaneously with hematoxylin - eosin (H&E) and Sirius red. A KBI pathologist evaluated the slides for NASH (steatosis, ballooning, inflammation, and fibrosis).

[0249] Doubly - labeled Water

[0250] Total energy expenditure (TEE) was measured using the doubly - labeled water method. Subjects received DLW at a dose of 1.51 g of estimated total body water (TBW) per kg. This dose consisted of 0.17 g / kg of estimated TBW of 99% 2H2O (Sigma - Aldrich 151882) and 0.3 g / kg of estimated TBW of 97% H218O (Sigma - Aldrich 329878) to achieve initial enrichment for their body weight. The dose was administered intravenously via a saphenous vein catheter or similar device, and the syringe was weighed and flushed to ensure all compounds had been injected. Blood samples were collected at the following time points: 1) before dosing (baseline), 2) 4 - 6 hours after dosing (equilibration), 3) 1 week and 2 weeks after dosing.

[0251] 1.5 ml of blood samples were collected, transferred to non - liquid heparin sodium anticoagulant tubes, inverted 5 times, immediately placed on ice, and then centrifuged at 1300 x g for 10 minutes at 4°C. Aliquots were stored at - 80°C and sent to Metabolic Solutions, Inc. for tracer measurement and analysis.

[0252] In summary, obese non-human primates were treated with i) vehicle, ii) semaglutide, iii) anti-myostatin antibody + anti-activin A antibody, iv) semaglutide + anti-myostatin antibody, or v) semaglutide + anti-myostatin antibody + anti-activin A antibody. Energy expenditure was evaluated by DLW at weeks 18 to 20 of the study (end of the dosing period).

[0253] Original energy expenditure measurements showed that the group of primates receiving gatortumab (anti-activin A antibody) + semaglutide + anti-myostatin antibody expended more energy ( Figure 20A ). Energy expenditure measurements per kg of lean body mass showed that even after normalizing by per kg of lean body mass, semaglutide + zoglumizumab (anti-myostatin antibody REGN1033) also led to an increase in energy ( Figure 20B ).

[0254] Data graphs were plotted to determine whether energy expenditure was related to lean body mass or changes in lean body mass. The combination treatment groups (semaglutide + zoglumizumab, semaglutide + zoglumizumab + gatortumab (REGN2477)) expended more energy than the non-combination treatment groups (vehicle, semaglutide, zoglumizumab + gatortumab) ( Figure 21A ). In addition, the combination treatment groups with greater increases in lean body mass (semaglutide + zoglumizumab, semaglutide + zoglumizumab + gatortumab) had higher energy expenditure ( Figure 21B ). Therefore, these primates treated with combination therapies had higher energy expenditure per gram of lean body mass, and the increase in their energy expenditure was proportional to the increase in lean body mass. However, the "boost" in energy expenditure only occurred when both semaglutide and zoglumizumab were used.

[0255] On the basis of semaglutide and anti-myostatin antibody, the combined use of activin A blockade also caused further improvement in HbA1c and cholesterol. In the treatment groups of obese non-human primates, HbA1c ( Figure 22A ), LDL-C ( Figure 22B ), ApoB ( Figure 22C ), change in HbA1c relative to baseline ( Figure 22D ), change in LDL-C relative to baseline ( Figure 22E ), and change in ApoB relative to baseline ( Figure 22F ) were measured.

[0256] Combination treatment not only increased the lean body mass of obese non-human primates but also improved HbA1c and cholesterol.

[0257] The scope of the present disclosure is not limited to the specific embodiments described herein. In fact, various other modifications of the present disclosure will be apparent to those skilled in the art from the foregoing description and the accompanying drawings. Such modifications are intended to fall within the scope of the appended claims.

Claims

1. A composition comprising a growth differentiation factor 8 (GDF-8) inhibitor and a glucagon-like peptide 1 (GLP-1) agonist.

2. A composition comprising a growth differentiation factor 8 (GDF-8) inhibitor, an activin A inhibitor, and a glucagon-like peptide 1 (GLP-1) agonist.

3. The composition according to claim 1 or 2, wherein the GDF-8 inhibitor is a GDF8 specific binding protein.

4. The composition according to any one of claims 1-3, wherein the GDF-8 inhibitor is an antibody or an antigen-binding fragment thereof that specifically binds to GDF-8.

5. The composition according to claim 4, wherein the anti-GDF8 antibody or its antigen-binding fragment comprises the heavy chain complementarity-determining regions (HCDRs) of the heavy chain variable region (HCVR) of SEQ ID NO:4, and the light chain complementarity-determining regions (LCDRs) of the light chain variable region (LCVR) of SEQ ID NO:

5.

6. The composition according to claim 4 or 5, wherein the anti-GDF8 antibody or its antigen-binding fragment comprises the heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) respectively comprising SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8, and three light chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3) respectively comprising SEQ ID NO:9, TTS, and SEQ ID NO:

11.

7. The composition according to any one of claims 2-6, wherein the activin A inhibitor, if present, is an activin A specific binding protein.

8. The composition according to any one of claims 2-7, wherein the activin A inhibitor, if present, is an antibody or an antigen-binding fragment thereof that specifically binds to activin A.

9. The composition according to claim 8, wherein the anti-activin A antibody or its antigen-binding fragment comprises the heavy chain complementarity-determining region (HCDR) of the heavy chain variable region (HCVR) of SEQ ID NO:12 and the light chain complementarity-determining region (LCDR) of the light chain variable region (LCVR) of SEQ ID NO:

13.

10. The composition according to claim 8 or 9, wherein the anti - activin A antibody or its antigen - binding fragment comprises heavy - chain complementarity - determining regions (HCDR1, HCDR2, and HCDR3) comprising SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16, respectively, and three light - chain complementarity - determining regions (LCDR1, LCDR2, and LCDR3) comprising SEQ ID NO:17, GAS, and SEQ ID NO:19, respectively.

11. The composition according to any one of claims 1 - 10, wherein the GLP - 1 agonist is a GLP - 1 receptor agonist.

12. The composition according to any one of claims 1 - 10, wherein the GLP - 1 agonist is selected from exenatide (extended - release), dulaglutide, liraglutide, tirzepatide, and semaglutide.

13. The composition according to claim 12, wherein the GLP - 1 agonist is semaglutide.

14. The composition according to any one of claims 1 - 10, wherein the GLP - 1 agonist is a GLP - 1 - specific binding protein.

15. The composition according to claim 14, wherein the GLP - 1 agonist is an antibody or its antigen - binding fragment that specifically binds to GLP - 1.

16. The composition according to any one of claims 1 - 15, which is used for improving glucose control in a subject, increasing lean body mass, reducing fat mass, treating obesity, treating diabetes, and / or treating liver problems associated with increased fat mass, obesity, and / or diabetes.

17. A method for improving glucose control in a subject, increasing lean body mass, reducing fat mass, treating obesity, treating diabetes, and / or treating liver problems associated with increased fat mass, obesity, and / or diabetes, comprising administering a GDF8 inhibitor and a GLP - 1 agonist to the subject.

18. A method for improving glucose control in a subject, increasing lean body mass, reducing fat mass, treating obesity, treating diabetes, and / or treating liver problems associated with increased fat mass, obesity, and / or diabetes, comprising administering a GDF8 inhibitor, an activin A inhibitor, and a GLP - 1 agonist to the subject.

19. The method according to claim 17 or 18, wherein the GDF8 inhibitor, the GLP - 1 agonist, and the activin A inhibitor (if present) are administered to the subject as a single composition.

20. The method of claim 17 or 18, wherein the GDF8 inhibitor, the GLP-1 agonist, and the activin A inhibitor (if present) are administered to the subject as at least two separate compositions.

21. The method according to any one of claims 17-20, wherein the GDF-8 inhibitor is a GDF8-specific binding protein.

22. The method according to any one of claims 17-21, wherein the GDF-8 inhibitor is an antibody that specifically binds GDF-8 or an antigen-binding fragment thereof.

23. The method according to claim 22, wherein the anti-GDF8 antibody or an antigen-binding fragment thereof comprises: a heavy-chain complementarity-determining region (HCDR) comprising a heavy-chain variable region (HCVR) of SEQ ID NO:4, and a light-chain complementarity-determining region (LCDR) comprising a light-chain variable region (LCVR) of SEQ ID NO:

5.

24. The method according to claim 22 or 23, wherein the anti-GDF8 antibody or an antigen-binding fragment thereof comprises heavy-chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) comprising SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8, respectively, and three light-chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3) comprising SEQ ID NO:9, TTS, and SEQ ID NO:11, respectively.

25. The method according to any one of claims 18-24, wherein the activin A inhibitor, if present, is an activin A-specific binding protein.

26. The method according to any one of claims 18-25, wherein the activin A inhibitor, if present, is an antibody that specifically binds activin A or an antigen-binding fragment thereof.

27. The method according to claim 26, wherein the anti-activin A antibody or an antigen-binding fragment thereof comprises: a heavy-chain complementarity-determining region (HCDR) comprising a heavy-chain variable region (HCVR) of SEQ ID NO:12, and a light-chain complementarity-determining region (LCDR) comprising a light-chain variable region (LCVR) of SEQ ID NO:

13.

28. The method according to claim 26 or 27, wherein the anti-activin A antibody or an antigen-binding fragment thereof comprises heavy-chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) comprising SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16, respectively, and three light-chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3) comprising SEQ ID NO:17, GAS, and SEQ ID NO:19, respectively.

29. The method according to any one of claims 17 - 28, wherein the GLP-1 agonist is a GLP-1 receptor agonist.

30. The method according to any one of claims 17 - 28, wherein the GLP-1 agonist is selected from exenatide (long-acting), dulaglutide, liraglutide, tirzepatide, and semaglutide.

31. The method according to claim 30, wherein the GLP-1 agonist is semaglutide.

32. The method according to any one of claims 17 - 28, wherein the GLP-1 agonist is a GLP-1 specific binding protein.

33. The method according to claim 32, wherein the GLP-1 agonist is an antibody that specifically binds GLP-1 or an antigen-binding fragment thereof.

34. The method according to any one of claims 17 - 33, wherein, At 12 weeks after administration of the (one or more) inhibitor and the agonist, the subject exhibits at least one parameter change selected from the following group: i) A reduction in fat mass of at least about 35%; ii) An increase in lean body mass of at least about 6%; iii) A reduction in fasting glucose of at least about 15%; iv) A reduction in HbA1c of at least about 6%; v) A reduction in LDL of at least about 14%; vi) An increase in LDL of at least about 14%; vii) A reduction in NEFA of at least about 35%; and viii) A reduction in TG of at least about 55%.

35. The method according to claim 34, wherein, At 12 weeks after administration of the (one or more) inhibitor and the agonist, the subject exhibits at least one parameter change selected from the following group: i) A decrease in fasting glucose of at least about 25%; iv) A decrease in HbA1c of at least about 25%; v) A decrease in LDL of at least about 50%; vi) An increase in LDL of at least about 60%; vii) A decrease in NEFA of at least about 50%; and viii) A decrease in TG of at least about 65%.

Images