Fgf21 protein variant for the treatment of nash

Abstract

The present disclosure relates to methods of treating, preventing, or managing a cardiovascular and / or metabolic disorder in a human subject in need thereof by administering a human FGF21 protein variant, the metabolic disorder and / or cardiovascular disorder is selected from hypercholesterolemia, dyslipidemia, hypertriglyceridemia, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), type 2 diabetes, and obesity.

Description

[0001]Attorney Docket No.: BPH-006PC / 136177-5006 - 1 - FGF21 Protein Variant for the Treatment of NASH FIELD The present disclosure relates to methods of treating, preventing, or managing a cardiovascular and / or metabolic disorder in a human subject in need thereof by administering a human fibroblast growth factor (FGF21) protein variant which is a FGF21-Fc fusion protein. CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Application No.63 / 382,584 filed on November 07, 2022, the entire contents of which is hereby incorporated by reference in its entirety. SEQUENCE LISTING The instant application contains a sequence listing, which has been submitted in XML format via Patent Center. The contents of the XML file, named “BPH-006PC_Sequence_Listing.xml,” which was created on October 18, 2023, and is 2,301 bytes in size, are incorporated herein by reference in their entirety. BACKGROUND Fibroblast growth factor 21 (FGF21) is a member of the fibroblast growth factor (FGF) family and regulates energy balance and glucose and lipid homeostasis through a heterodimeric receptor complex comprising FGF receptor 1 (FGFR1) and β-Klotho. FGF21 is predominantly released from hepatocytes and to a lesser extent from adipocytes and pancreatic β-cells. Preclinical studies in animals have demonstrated that FGF21 may have many beneficial pleiotropic effects including a reduction in fat mass, improvements in glucose control insulin resistance, improvements in dyslipidemia, and improvements in models of Nonalcoholic fatty liver disease (NAFLD) Nonalcoholic fatty liver disease (NAFLD) is the term for a wide range of conditions caused by a build-up of fat within the liver cells as a result of caloric overburdening of the liver due to poor diet and lack of exercise, or comorbid conditions with disbalanced metabolism of lipids of glucose. NAFLD is prevalent in one-third of the United States population and is associated with other metabolic disease including obesity, type 2 diabetes mellitus (T2DM), and hyperlipidemia. NAFLD can progress to nonalcoholic steatohepatitis DB1 / 141729270.1 Attorney Docket No.: BPH-006PC / 136177-5006 - 2 - (NASH) when excessive liver fat leads to hepatocyte stress, which triggers localized inflammation and, as disease progresses, can lead to fibrosis and ultimately cirrhosis. NASH may progress to cirrhosis and liver failure which is becoming a leading cause of liver transplant, death, and the development of hepatocellular carcinoma. Additionally, people with NASH have high rates of cardiovascular-related events, such as stroke and heart attack, with cardiovascular disease being the leading cause of death in people with NASH. The prevalence of NASH in the United States is projected to increase from an estimated 17.3 million in 2016 to 27.0 million by 2030 (Estes C. et al. Modeling NAFLD disease burden in China, France, Germany, Italy, Japan, Spain, United Kingdom, and United States for the period 2016- 2030. J Hepatol.2018;69(4):896-904. There are currently no approved therapies for NASH, although there is a large number of molecules in development representing many different mechanisms of action that target different aspects of NASH pathogenesis. A number of long-acting FGF21 analogs have been developed to overcome the short t1 / 2 of wild-type FGF21 which limits its clinical use. Several FGF21 analogs and mimetics have progressed to early phases of clinical studies in subjects with obesity, T2DM, and NASH. These studies have demonstrated substantial improvements in dyslipidemia, hepatic steatosis, and serum biomarkers of liver fibrosis in subjects with NASH. More recently, Phase 2a data of the FGF21 analog efruxifermin (EFX) suggests the improvement in hepatic fat translates to improvement in liver histology (Akero Therapeutics, Inc. Corporate Presentation: A Global Disease, A Pioneering Treatment. January 2021. Available online at https: / / ir.akerotx.com / static-files / d5a2bd98-8bad-412a-97d7-0b2bce1a0f13). SUMMARY The present disclosure is based, at least in part on the protocol and results of a 12-week Phase 2a randomized, blinded, placebo-controlled study of BOS-580 (V103), a FGF21 protein variant in obese subjects at risk for nonalcoholic steatohepatitis (NASH). The purpose of this study was to evaluate the BOS-580 exposure-response relationship based on BOS-580 dose and exposure, and tolerability and efficacy biomarkers. The human FGF21 protein variant BOS-580 (V103) is described in WO 2013 / 049247, WO 2015 / 138278 and WO 2019 / 123427, the contents of which are herein incorporated by reference. BOS-580 (V103) is stabilized via introduction of a novel disulfide bond and fused at its N-terminus to human immunoglobulin G1 (IgG1) Fc fragment. DB1 / 141729270.1 Attorney Docket No.: BPH-006PC / 136177-5006 - 3 - The amino acid sequence of BOS-580 (V103) is shown in SEQ ID NO: 1. It comprises from N-terminus to C-terminus an immunoglobulin Fc fragment, a linker GS and a native mature human FGF21 variant representing amino acid positions 29-209 of the full-length FGF21 protein sequence (NCBI reference sequence number NP_061986.1) with amino acid substitutions Q55C, R105K, G148C, K150R, P158S, S195A, P199G, G202A (numbering based on the full-length sequence). A first aspect of the present disclosure relates to a human FGF21 protein variant comprising the amino acid sequence of SEQ ID NO: 1 for use in a method of treating, preventing, or managing a cardiovascular and / or metabolic disorder in a human subject in need thereof, wherein the human FGF21 protein variant is provided for administration at a dose of 75 mg, subcutaneously, once every 2 weeks. A further aspect of the present disclosure relates to the use of a human FGF21 protein variant comprising the amino acid sequence of SEQ ID NO: 1 for the manufacture of a medicament for treating, preventing, or managing a cardiovascular or metabolic disorder in a human subject in need thereof, wherein the human FGF21 protein variant is provided for administration at a dose of 75 mg, subcutaneously, once every 2 weeks. Still a further aspect of the present disclosure relates to a method of treating, preventing, or managing a cardiovascular or metabolic disorder in a human subject in need thereof, comprising administering a human FGF21 protein variant comprising the amino acid sequence of SEQ ID NO: 1 to the human subject at a dose of 75 mg, subcutaneously, once every 2 weeks. In some embodiments of any of the preceding aspects, the metabolic disorder and / or cardiovascular disorder is selected from hypercholesterolemia, dyslipidemia, hypertriglyceridemia, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), type 2 diabetes, and obesity. In some embodiments of any of the preceding aspects, the metabolic disorder and / or cardiovascular disorder is non-alcoholic fatty liver disease (NAFLD) and / or non-alcoholic steatohepatitis (NASH). In some embodiments of any of the preceding aspects, the metabolic disorder and / or cardiovascular disorder is non-alcoholic steatohepatitis (NASH). In some embodiments of any of the preceding aspects, the human subject is obese. DB1 / 141729270.1 Attorney Docket No.: BPH-006PC / 136177-5006 - 4 - In some embodiments of any of the preceding aspects, the human subject has a body mass index (BMI) of at least 30 kg / m2with ethnic adjustment of least 27.5 kg / m2for a subject of Asian descent or Asian ancestry, particularly a BMI of 30 kg / m2to 45 kg / m2. In some embodiments of any of the preceding aspects, the human subject is suffering from NASH and / or at risk of acquiring NASH. In some embodiments of any of the preceding aspects, the human subject has a liver fibrosis stage selected from F0 (no fibrosis), F1 (steatosis), F2 (early fibrosis), F3 (advanced fibrosis) and / or F4 (cirrhosis), particularly selected from F2 and / or F3. In some embodiments of any of the preceding aspects, the human subject has a liver fat assessment based on vibration controlled transient elastography (VCTE) controlled attenuation parameter (CAP) Score of more than 300 dB / m, or of more than 400 dB / m. In some embodiments of any of the preceding aspects, the human subject has a liver injury and fibrosis assessment based on VCTE liver stiffness measurement (LSM) score between 7 and 14 kPa, between 7 kPa and 12 kPa, or between 7 kPa and 9.9 kPa and / or an aspartate amino transferase (AST) activity of at least 20 U / L. In some embodiments of any of the preceding aspects, the human subject is further suffering from type 2 diabetes mellitus, hypertension and / or hyperlipidemia. In some embodiments of any of the preceding aspects, the administration is effective in improving at least one physiological parameter associated with NASH. In some embodiments of any of the preceding aspects, the at least one physiological parameter is selected from liver fibrosis stage, liver fat assessment based on VCTE CAP, liver injury and fibrosis assessment based on VCTE, LSM score, and AST activity. In some embodiments of any of the preceding aspects, the administration is effective in reducing liver fat, particularly effective in reducing the hepatic fat fraction by at least 45%, at least 50% or at least 60%. DB1 / 141729270.1 Attorney Docket No.: BPH-006PC / 136177-5006 - 5 - In some embodiments of any of the preceding aspects, the administration is effective in reducing liver injury, particularly effective in reducing adiponectin deficiency, e.g., by increasing the adiponectin level by at least 50%, 75%, 100% or 125%. In some embodiments of any of the preceding aspects, the administration is effective in a reduction of liver fibrosis, particularly effective in reducing mean alanine amino transferase (ALT) by at least 20%, at least 30%, or at least 35%, and / or effective in reducing of aspartate amino transferase (AST) activity by at least 20% or at least 30%, and / or effective in reducing mean of procollagen amino-terminal propeptide (ProC3) by at least 15% or at least 20%. In some embodiments of any of the preceding aspects, the administration is effective in reducing the HbA1c level of a subject suffering from type 2 diabetes mellitus by at least 0.6%. In some embodiments of any of the preceding aspects, the administration has a gastro-intestinal tolerability profile of grade 1 or 2. In some embodiments of any of the preceding aspects, the human FGF21 protein variant is administered in combination with one or more additional therapeutically active agents. In some embodiments of any of the preceding aspects, the one or more additional therapeutically active agents is selected from the group consisting of compounds useful in obesity therapies, diuretics, beta- blockers, alpha-blockers, ACE inhibitors, Angiotensin II Receptor Blockers (ARBs), direct renin inhibitors, calcium channel blockers, central agonists, peripheral adrenergic blockers, vasodilators, insulin, alpha- glucosidase inhibitors, biguanides, dopamine agonist, DPP-4 inhibitors, glucagon-like peptides, e.g., GLP-1 agonists and dual GLP-1 and GLP-2 agonists, meglitinides, sodium glucose transporter (SGLT) inhibitors, sulfonylureas, thiazolidinediones, amylinomimetics, statins, fibrates, aspirin, and anticoagulants. In some embodiments of any of the preceding aspects, the human FGF21 protein variant is non- glycosylated. In some embodiments of any of the preceding aspects, the human FGF21 protein variant is glycosylated. DB1 / 141729270.1 Attorney Docket No.: BPH-006PC / 136177-5006 - 6 - In some embodiments of any of the preceding aspects, the human FGF21 protein variant consists of the amino acid sequence of SEQ ID NO: 1 DESCRIPTION OF THE DRAWINGS FIG.1A- FIG.1D show that administration BOS-580 (SEQ ID NO: 1) had no adverse effects on lipid profiles. FIG.2 shows the occurrence of TEAEs of the gastrointestinal system. FIG.3 shows the absolute change in HFF determined by Magnetic Resonance Imaging Proton Density Fat Fraction (MRI-PDFF). The normal reference range of MRI-PDFF is < 5.6%. FIG.4 shows the relative change in HFF determined by MRI-PDFF. FIG.5 shows the proportions of patients achieving specific degrees of fat reduction. FIG.6 shows the levels of alanine aminotransferase (ALT), a biomarker of liver injury. FIG.7 shows the levels of aspartate aminotransferase (AST), a biomarker of liver injury. FIG.8 shows the levels of the amino-terminal propeptide of ProCollagen 3 (ProC3), a biomarker of liver fibrosis. FIG.9 shows the degrees of adiponectin deficiency. Adiponectin is an adipose-specific adipokine that decreases insulin resistance and attenuates liver inflammation / fibrosis. FIG.10 shows the HbA1c levels in diabetic NASH patients. DETAILED DESCRIPTION DB1 / 141729270.1 Attorney Docket No.: BPH-006PC / 136177-5006 - 7 - Disclosed herein are methods, compounds, and pharmaceutical compositions for use in treating, preventing, or managing (e.g., alleviating one or more signs and / or symptoms of a disorder) a metabolic disorder and / or cardiovascular disorder, comprising administering to a human subject in need thereof a human FGF21 protein variant comprising or consisting of the amino acid sequence of SEQ ID NO: 1 at a dose of 75 mg, subcutaneously, once every 2 weeks. Typically, the human FGF21 protein variant comprising or consisting of the amino acid sequence of SEQ ID NO: 1 is obtained by expression in a recombinant host cell wherein the recombinant host cell is transfected or transformed with a nucleic acid molecule encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 1. The recombinant host cell may be cultivated in a suitable culture medium and the polypeptide may be isolated from the host cell or the culture medium. The host cell may be a prokaryotic host cell or a eukaryotic host cell, e.g., an insect or mammalian cell. Depending on the type of host cell, the human FGF21 protein variant may be glycosylated or aglycosylated / non-glycosylated. The term “managing” is understood as the management and care of a patient for the purpose of combating a disease, condition, or disorder, which does not result in a cure, but alleviation of one or more symptoms of a disease, condition, or disorder and / or reduction in hospital stay time. In embodiments, “managing,” or “management” with respect to a condition or disease includes the maintenance of symptomology, including using one or more indexes or scores (e.g., as described herein), and / or the maintenance of one or more biomarkers of disease (e.g., as described herein). The term “preventing” refers to prophylactic administration to a healthy subject or to a subject at risk of developing a disease, condition, or disorder as described herein (e.g., a subject who is considered to be prediabetic or a subject who suffers from liver fibrosis and is at risk of developing NASH) to prevent the development of one or more conditions mentioned herein. Moreover, the term “preventing” can also include prophylactic administration to patients being in a pre-stage of the conditions to be treated. In embodiments, “preventing,” or “prevention” with respect to a condition or disease includes the prevention of worsening of one or more symptoms, including using one or more indexes or scores (e.g., as described herein), and / or the maintenance of one or more biomarkers of disease (e.g., as described herein). The term “treating” is understood as the management and care of a patient for the purpose of combating a disorder, for example, the reduction or amelioration of the progression, severity, and / or duration of a disease, disorder, or condition. DB1 / 141729270.1 Attorney Docket No.: BPH-006PC / 136177-5006 - 8 - The term “metabolic disorder” refers to a disease, disorder, or condition associated with a pathological metabolic parameter such as increased blood pressure, high blood sugar, excess body and / or organ fat, an abnormal cholesterol level, an abnormal triglyceride level and any combination thereof. A metabolic disorder may increase the risk of heart failure and / or stroke. The term “cardiovascular disorder” refers to a disease, disorder, or condition of the heart and / or vascular system. Non-limiting examples of metabolic disorders and / or cardiovascular disorders to be treated, prevented or managed, by the disclosure provided herein include hypertriglyceridemia, diabetes, e.g., type 2 diabetes mellitus, obesity, type 1 diabetes mellitus, pancreatitis, dyslipidemia, nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), insulin resistance, hyperinsulinemia, glucose intolerance, hyperglycemia, metabolic syndrome, hypertension, cardiovascular disease, acute myocardial infarction, atherosclerosis, peripheral arterial disease, stroke, heart failure, coronary heart disease, kidney disease, diabetic complications, diabetic neuropathy, disorders associated with severe inactivating mutations in the insulin receptor (e.g., as described by Taylor et al., Diabetes Care 13 (1990), 257-279, the content of which is herein incorporated by reference) and / or gastroparesis. In certain embodiments, the metabolic disorder and / or cardiovascular disorder is selected from hypercholesterolemia, dyslipidemia, hypertriglyceridemia, non-alcoholic fatty liver disease (NAFLD), non- alcoholic steatohepatitis (NASH), type 2 diabetes, and obesity. In particular embodiments, the metabolic disorder and / or cardiovascular disorder is selected from non- alcoholic steatohepatitis (NASH). The term “NAFLD” or “non-alcoholic fatty liver disease” is defined as a condition in which excess fat is stored in the liver. This buildup of fat is not caused by heavy alcohol use. When heavy alcohol use causes fat to build up in the liver, this condition is called alcoholic liver disease. Typically, there are two types of NAFLD: simple fatty liver and non-alcoholic steatohepatitis (NASH). The term “NASH” or “non-alcoholic steatohepatitis” is defined as a form of nonalcoholic fatty liver disease (NAFLD) in which a patient has hepatitis - inflammation of the liver - and liver cell damage, in addition to fat in the liver. Inflammation and liver cell damage can cause fibrosis, or scarring, of the liver. In some DB1 / 141729270.1 Attorney Docket No.: BPH-006PC / 136177-5006 - 9 - cases, NASH may lead to cirrhosis or liver cancer. Cirrhosis with complications is termed ‘decompensated’ cirrhosis and it is one of the prime reasons for liver transplantation. The term “simple fatty liver,” also called nonalcoholic fatty liver (NAFL), is a form of NAFLD in which a patient has fat in the liver but little or no inflammation or liver cell damage. Simple fatty liver can in certain a percentage of individuals progress to NASH. In certain embodiments, the human subject is obese. A measure of obesity is the body mass index BMI = Body weight (kg) / [Height (m)]2), a person’s weight (in kilograms) divided by the square of his or her height (in meters). A person with a BMI of 30 or more is generally considered obese but this guideline can be adjusted for ethnic differences. For example, with ethnic adjustment, a BMI of 27.5 can be considered obese for Asian individuals (WHO Expert Consultation, 2004, Lancet, 363(9403): 157-63). Asian individuals may be, for example, subjects of Asian descent or Asian ancestry. In particular embodiments, the human subject has a body mass index (BMI) of at least 30 kg / m2with ethnic adjustment of least 27.5 kg / m2for a subject of Asian descent or Asian ancestry, e.g., a BMI of 30 kg / m2to 45 kg / m2. In embodiments, the BMI is about or at least about 20 kg / m2, about or at least about 25 kg / m2, about or at least about 30 kg / m2, about or at least about 35 kg / m2, about or at least about 40 kg / m2, about or at least about 45 kg / m2, or about or at least about 50 kg / m2. In embodiments, the BMI includes a range of about or at least about 20 kg / m2to about or at least about 25 kg / m2, about or at least about 25 kg / m2to about or at least about 30 kg / m2, about or at least about 30 kg / m2to about or at least about 35 kg / m2, about or at least about 35 kg / m2to about or at least about 40 kg / m2, about or at least about 40 kg / m2to about or at least about 45 kg / m2, or about or at least about 45 kg / m2to about or at least about 50 kg / m2. In embodiments, the subject for treatment is diagnosed with NASH, has a suspected diagnosis of NASH, and / or is at risk of acquiring NASH. In embodiments, a subject diagnosed with NASH, suspected of a diagnosis of NASH and / or at risk of NASH is pre-diabetic. In embodiments, the subject diagnosed with NASH, suspected of a diagnosis of NASH and / or at risk of NASH exhibits one or more demographic and / or lifestyle factors used to select the subject for methods of treatment herein, including being overweight, obese, or having a co-morbidity associated therewith (e.g., based on BMI, waist circumference, body weight, being pre-diabetic); age (e.g., age 45 or older); being African American, Pacific Islander, or Hispanic / Latino; having a family history of diabetes; having a sedentary lifestyle or lack of physical activity; having high blood pressure and / or abnormal cholesterol levels; having a personal DB1 / 141729270.1 Attorney Docket No.: BPH-006PC / 136177-5006 - 10 - history of gestational diabetes, heart disease, stroke, and / or polycystic ovary syndrome (PCOS). In embodiments, methods herein are intended for preventing the evolution of a disease or condition from its early stage (e.g., pre-diabetes to diabetes, NAFLD to NASH, etc.), and / or for mitigating the accumulation of damage due to lifestyle factors and / or risk factors. In embodiments, methods of prevention herein are designed for healthy individuals, or substantially healthy individuals, to stop or slow the progression of disease, or prevention of NASH into more serious stages of NASH, etc. In embodiments, the accumulation of damage, the presence of lifestyle factors and / or risk factors, and any improvements associated therewith, are measurable as described herein. In embodiments, a diagnosis, suspected diagnosis, and / or risk of NASH is ascertained by or assessed by liver biopsy results. In embodiments, NASH is diagnosed when microscopy analysis of the tissue shows fat accumulation, inflammation, and damage / or to liver cells. In embodiments, subjects with liver tissue exhibiting fat without inflammation and damage, are considered to have a diagnosis of simple fatty liver or NAFLD, and may also be at risk of developing NASH. In embodiments, the diagnosis of NASH and / or suspected diagnosis of NASH includes assessment of results from blood testing, for example with biomarkers such as liver enzymes alanine aminotransferase (ALT) and / or aspartate aminotransferase (AST), platelet count, procollagen amino-terminal propeptide (ProC3), and / or hemoglobin A1C (HbA1c). In embodiments, assessing a diagnosis of NASH, suspected diagnosis of NASH, and / or risk for NASH includes using a scoring rubric / index, such as the Fibrosis-4 (FIB-4) index for liver fibrosis, for example with the subject having a FIB-4 index of about or at least about 1.5, about or at least about 2.0, about or at least about 2.5, about or at least about 3.0, or about or at least about 3.5. In embodiments, assessing a diagnosis of NASH, suspected diagnosis of NASH, and / or risk for NASH includes using a scoring rubric / index, such as the AST to platelet ratio index (APRI), for example with the subject having an APRI of about or at least about 0.25, about or at least about 0.5, about or at least about 0.75, about or at least about 1.0, about or at least about 1.25, or about or at least about 1.5. In embodiments, a subject with a diagnosis of NASH, a suspected diagnosis of NASH, and / or a risk for NASH has one or more of an enlarged liver, enlarged spleen, or ascites (e.g., as observed from imaging of visceral organs and peripheral tissues), signs of insulin resistance (e.g., observed as darkening of skin patches over extremities), signs of cirrhosis, and / or muscle loss. In embodiments, imaging techniques to determine liver fibrosis or inflammation, enlarged organs (liver, spleen), ascites or fluid build-up, and / or muscle loss include ultrasound, computer tomography (CT), magnetic resonance imaging (MRI), and / or elastography imaging, for example, vibration-controlled transient elastography, sheer wave elastography, and / or magnetic resonance elastography. In embodiments, measuring insulin resistance includes the DB1 / 141729270.1 Attorney Docket No.: BPH-006PC / 136177-5006 - 11 - euglycemic hyperinsulinemic test, fasting plasma glucose (FPG) test (e.g., 100 to 125 mg / dL glucose or higher), A1C test (e.g., 5.7% to 6.4% or higher), and / or oral glucose tolerance test (OGTT) (e.g., 140 to 199 mg / dL glucose or higher). In certain embodiments, the human subject is suffering from NASH and / or at risk of acquiring NASH. In such embodiments, the human subject may have a liver fibrosis stage (e.g., using the Batts-Ludwig criteria) selected from F0 (no fibrosis), F1 (steatosis), F2 (early fibrosis), F3 (advanced fibrosis) and / or F4 (cirrhosis), particularly selected from F2 and / or F3. In certain embodiments, the human subject, e.g., a human subject suffering from NASH and / or at risk of acquiring NASH may be characterized by one or more of the following physiological parameters: - a liver fat assessment based on vibration controlled transient elastography (VCTE) controlled attenuation parameter (CAP) Score of more than 300 dB / m, particularly of more than 400 dB / m; - a liver injury and fibrosis assessment based on VCTE liver stiffness measurement (LSM) score of at least 7 kPa, e.g., between 7 and 14 kPa, between 7 kPa and 12 kPa, or between 7 kPa and 9.9 kPa; - an aspartate amino transferase (AST) activity of at least 20 U / L; - a NAFLD Fibrosis (NFS) score (calculated as Age, AST / ALT ratio, Platelet count, BMI, albumin (g / L), impaired fasting glucose / diabetes (+1 if Yes) of at least 1.455 indicating an intermediate or high probability for fibrosis; - a FIB-4 (fibrosis-4) score (calculated as Age X AST) / platelet count (109 / L) X square root of ALT) of at least 1.3 indicating advanced fibrosis, - an APRI index (ALT to platelet ratio index) of more than 0.5 indicating an intermediate or high probability of advanced fibrosis; - a FIBROSURE value (a serum test that combines five biomarkers: haptoglobin, α2- macroglobulin, apolipoprotein A1, total bilirubin and gamma glutamyl-transferase) of more than DB1 / 141729270.1 Attorney Docket No.: BPH-006PC / 136177-5006 - 12 - 0.31 to 0.58 indicating moderate fibrosis, or a FibroSure value of more than 0.58 indicating advanced fibrosis; - an Enhanced Liver Fibrosis (ELF) value (combining three biomarkers of fibrosis: hyaluronic acid, tissue inhibitor of metalloproteinase 1, and amino-terminal peptide of Procollagen 3) of 7.7 to less than 9.8 indicating moderate fibrosis (mild to advanced fibrosis) or at least 9.8 indicating advanced fibrosis; - a Magnetic Resonance Elastography (MRE) score of 2.97 to 3.62 indicating fibrosis stage F2 or more than 3.62 indicating advanced fibrosis (F3); - a FIBROSCAN-AST (FAST) score of more than 0.67 indicating a high risk of progression or of less than 0.35 indicating a low risk of progression. In embodiments, methods involving the prevention or management of a cardiovascular and / or metabolic disorder (e.g., NASH) include substantially maintaining the appearance of liver biopsy results, substantially maintaining the concentration and / or activity of ASL, AST, ProC3, and / or HbA1C; substantially maintaining platelet counts; substantially maintaining a scoring of the FIB-4 index, NFS score, APRI, FIBROSURE value, ELF value, MRE score, and / or FAST score; substantially maintaining an assessment or result from a Batts-Ludwig criteria, euglycemic hyperinsulinemic test, FPG test, A1C test, or OGTT; substantially maintaining a VCTE, CAP score, LSM score; and / or substantially maintaining the severity and / or frequency of one or more symptoms associated with a condition, disease, or comorbidity, as described herein. In embodiments, “substantially maintaining,” refers to a change or deviation of less than about 1%, 5%, or 10%, for example in a qualitative or quantitative measure. In certain embodiments, the human subject, e.g., a human subject suffering from NASH and / or at risk of acquiring NASH is further diagnosed with, has a suspected diagnosis of, and / or is at risk of acquiring type 2 diabetes mellitus, hypertension, and / or hyperlipidemia. In particular embodiments, the human subject, e.g., a human subject suffering from NASH and / or at risk of acquiring NASH is further suffering from type 2 diabetes mellitus. In certain embodiments, the administration of a human FGF21 protein variant comprising the amino acid sequence of SEQ ID NO: 1 is effective in improving at least one physiological parameter associated with the metabolic disorder and / or cardiovascular disorder to be treated, prevented, or managed, by the disclosure provided herein. In certain embodiments, the administration of a human FGF21 protein variant DB1 / 141729270.1 Attorney Docket No.: BPH-006PC / 136177-5006 - 13 - comprising the amino acid sequence of SEQ ID NO: 1 is effective in improving at least one physiological parameter associated with NASH. In a human subject suffering from NASH and / or at risk of acquiring NASH, the administration of a human FGF21 protein variant comprising the amino acid sequence of SEQ ID NO: 1 may be effective in improving at least one physiological parameter selected from liver fibrosis stage, liver fat assessment based on VCTE CAP, liver injury and fibrosis assessment based on VCTE, LSM score, and AST activity. In certain embodiments, the administration of the human FGF21 protein variant of SEQ ID NO: 1, particularly for a time period of 12 weeks (starting with the first administration), is effective in reducing liver fat, particularly effective reducing the hepatic fat fraction by at least 45%, at least 50% or at least 60%. In certain embodiments, the administration of the human FGF21 protein variant of SEQ ID NO: 1, particularly for a time period of 12 weeks (starting with the first administration), is effective in reducing liver injury, particularly effective in reducing adiponectin deficiency, e.g., by increasing the adiponectin level by at least 50%, at least 75%, at least 100% or at least 125%. In certain embodiments, the administration of the human FGF21 protein variant of SEQ ID NO: 1, particularly for a time period of 12 weeks (starting with the first administration), is effective in a reduction of liver injury, particularly effective in reducing mean alanine amino transferase (ALT) activity by at least 20%, at least 30%, or at least 35%, and / or effective in reducing of aspartate amino transferase (AST) activity by at least 20% or at least 30%, and / or effective in reducing mean of procollagen amino-terminal propeptide (Pro-C3) by at least 15% or at least 20%. In a subject suffering from NASH and / or at risk of acquiring NASH, the administration of the human FGF21 protein variant of SEQ ID NO 1, particularly for a time period of 12 weeks (starting with the first administration), may be effective in reducing the HbA1c level of a subject suffering from type 2 diabetes mellitus, e.g., by at least 0.6%. In certain embodiments, the administration of the human FGF21 protein variant of SEQ ID NO: 1, particularly for a time period of 12 weeks (starting with the first administration), has a gastro-intestinal tolerability profile of grade 1 or 2. DB1 / 141729270.1 Attorney Docket No.: BPH-006PC / 136177-5006 - 14 - The human FGF21 protein variant of SEQ ID NO: 1 may be administered as a monotherapy or in combination with one or more additional therapeutically active agents. In some examples, the one or more additional therapeutically active agents may be selected from the group consisting of compounds useful in obesity therapies (e.g., phentermine / topiramate, orlistat, lorcaserin, liraglutide, bupropion / naltrexone and combinations thereof), compounds useful in high blood pressure therapies (e.g., diuretics, beta- blockers, alpha-blockers, ACE inhibitors, Angiotensin II Receptor Blockers (ARBs), direct renin inhibitors, calcium channel blockers, central agonists, peripheral adrenergic blockers, vasodilators and combinations thereof), compounds useful in diabetic therapies (e.g., insulin, alpha-glucosidase inhibitors, biguanides, dopamine agonists, DPP-4 inhibitors, glucagon-like peptides, e.g., GPL-1 agonists such as Semaglutide, and GLP-2 agonists, meglitinides, sodium glucose transporter (SGLT) inhibitors, sulfonylureas, thiazolidinediones, amylinomimetics and combinations thereof), compounds useful in NAFLD / NASH and cardiovascular therapies (e.g., statins, fibrates, aspirin, anticoagulant and / or combinations thereof). In one aspect, provided herein are combination therapies for treating, preventing, or managing a metabolic disorder and / or or a cardiovascular disorder comprising administering the human FGF21 protein variant of SEQ ID NO: 1 and one or more therapeutically active agents selected from the following: amiloride (Midamor), bumetanide (Bumex), chlorthalidone (Hygroton), chlorothiazide (Diuril), furosemide (Lasix), hydrochlorothiazide or HCTZ (Esidrix, Hydrodiuril, Microzide), indapamide (Lozol), metolazone (Mykrox, Zaroxolyn), spironolactone (Aldactone), triamterene (Dyrenium), Acebutolol (Sectral), Atenolol (Tenormin), Betaxolol (Kerlone), Bisoprolol (Zebeta), Carteolol (Cartrol), Metoprolol (Lopressor, Toprol XL), Nadolol (Corgard), Nebivolol (Bystolic), Penbutolol (Levatol), Pindolol (Visken), Propranolol (Inderal), Sotalol (Betapace), Timolol (Blocadren), Doxazosin (Cardura), Prazosin (Minipress), Terazosin (Hytrin), Benazepril (Lotensin), Captopril (Capoten), Enalapril (Vasotec), Fosinopril (Monopril), Lisinopril (Prinivil, Zestril), Moexipril (Univasc), Perindopril (Aceon), Quinapril (Accupril), Ramipril (Altace), Trandolapril (Mavik), Norvasc (amlodipine), Plendil (felodipine), DynaCirc (isradipine), Cardene (nicardipine), Procardia XL, Adalat (nifedipine), Cardizem, Dilacor, Tiazac, Diltia XL (diltiazem), Sular (Nisoldipine), Isoptin, Calan, Verelan, Covera-HS (verapamil), Capoten (captopril), Vasotec (enalapril), Prinivil, Zestril (lisinopril), Lotensin (benazepril), Monopril (fosinopril), Altace (ramipril), Accupril (quinapril), Aceon (perindopril), Mavik (trandolapril), Univasc (moexipril), Atacand (candesartan), Avapro (irbesartan), Benicar (olmesartan), Cozaar (losartan), Diovan (valsartan), Micardis (telmisartan), Teveten (eprosartan), Chlorthalidone (Hygroton), Chlorothiazide (Diuril), Hydrochlorothiazide or HCTZ (Esidrix, Hydrodiuril, Microzide), Indapamide (Lozol), Metolazone (Mykrox, Zaroxolyn), Amiloride (Midamor), Bumetanide (Bumex), Furosemide (Lasix), Spironolactone (Aldactone), Triamterene (Dyrenium), Acebutolol (Sectral), Atenolol (Tenormin), Betaxolol (Kerlone), Bisoprolol (Zebeta, Ziac), Carteolol (Cartrol), Carvedilol (Coreg), DB1 / 141729270.1 Attorney Docket No.: BPH-006PC / 136177-5006 - 15 - Labetalol (Normodyne, Trandate), Metoprolol (Lopressor, Toprol-XL), Nadolol (Corgard), Nebivolol (Bystolic), Penbutolol (Levatol), Pindolol (Visken), Propanolol (Inderal), Sotalol (Betapace), Timolol (Blocadren), fibric acid derivatives, niacin, and omega-3 fatty acids, fenofibrate, gemfibrozil, atorvastatin, fluvastatin, lovastatin, pitavastatin, pravastatin, rosuvastatin, simvastatin, pramlintide, acarbose (Precose), miglitol (Glyset), metformin, bromocriptine, alogliptin, linagliptin, saxagliptin, sitagliptin, albiglutide (Tanzeum), dulaglutide (Trulicity), exenatide (Byetta), exenatide extended-release (Bydureon), liraglutide (Victoza), nateglinide (Starlix), repaglinide (Prandin), repaglinide- metformin (Prandimet), dapagliflozin (Farxiga), dapagliflozin-metformin (Xigduo XR), canagliflozin (Invokana), canagliflozin- metformin (Invokamet), empagliflozin (Jardiance), empagliflozin-linagliptin (Glyxambi), empagliflozin- metformin (Synjardy), sotagliflozin, tofogliflozin, remogliflozin, luseogliflozin, ipragliflozin, atigliflozin, bexagliflozin, henagliflozin, licogliflozin, glimepiride (Amaryl), glimepiride-pioglitazone (Duetact), glimeperide-rosiglitazone (Avandaryl), gliclazide, glipizide-metformin (Metaglip), glyburide (DiaBeta, Glynase, Micronase), glyburide-metformin (Glucovance), chlorpropamide (Diabinese), tolazamide (Tolinase), tolbutamide (Orinase, Tol-Tab), rosiglitazone (Avandia), rosiglitazone-glimepiride (Avandaryl), rosiglitizone-metformin (Amaryl M),pioglitazone (Actos), pioglitazone-alogliptin (Oseni), pioglitazone- glimepiride (Duetact), and pioglitazone -metformin (Actoplus Met, Actoplus Met XR). In some embodiments, the sodium glucose transporter (SGLT) inhibitor is selected from dapagliflozin, empagliflozin, canagliflozin, ertugliflozin, sotagliflozin, tofogliflozin, remogliflozin, luseogliflozin, ipragliflozin, atigliflozin, bexagliflozin, henagliflozin, licogliflozin, and a pharmaceutically acceptable salt of any of these. In some embodiments, the sodium glucose transporter (SGLT) inhibitor is dapagliflozin. In some embodiments, the sodium glucose transporter (SGLT) inhibitor is empagliflozin. In some embodiments, the sodium glucose transporter (SGLT) inhibitor is canagliflozin. In some embodiments, the sodium glucose transporter (SGLT) inhibitor is ertugliflozin. In some embodiments, the sodium glucose transporter (SGLT) inhibitor is licogliflozin. According to certain embodiments, the administration of the human FGF21 protein variant of SEQ ID NO: 1 is accompanied by monitoring the FGF21 bioactivity, as described for example, in WO 2013 / 049247, which is incorporated by reference herewith in its entirety. In certain embodiments, at least one biomarker of liver function, is assessed to determine the therapeutic success. Examples of biomarkers of liver function include but are not limited to alanine aminotransferase (ALT), aspartate aminotransferase (AST), an amino-terminal propeptide of ProCollagen 3 (ProC3), GGT, DB1 / 141729270.1 Attorney Docket No.: BPH-006PC / 136177-5006 - 16 - and alkaline phosphatase. In further embodiments, at least one biomarker of glucose metabolism, e.g., HbA1c is assessed. In certain embodiments, the disclosure provides pharmaceutical compositions comprising the human FGF21 protein variant of SEQ ID NO: 1 for use in methods of treating, preventing, and / or managing metabolic and / or cardiovascular disorders, for example hypertriglyceridemia and cardiac risk, insulin resistance such as in patients with genetic mutations of insulin receptor and lipodystrophy, diabetes, obesity, and nonalcoholic fatty liver disease (NAFLD) / nonalcoholic steatohepatitis (NASH). A pharmaceutical composition described herein comprises a therapeutically effective amount of the human FGF21 protein variant of SEQ ID NO: 1 and a pharmaceutically acceptable carrier suitable for subcutaneous administration. Usually, the pharmaceutical composition is in the form of a sterile, pyrogen- free, parenterally acceptable composition. A particularly suitable vehicle for parenteral injection is a sterile, isotonic solution, properly preserved. The pharmaceutical composition can be in the form of a lyophilizate, such as a lyophilized cake. Suitable methods and components for pharmaceutical formulations are well known (c.f., e.g., Allen, Lloyd V. Ed, (2012) Remington's Pharmaceutical Sciences, 22nd Edition, which is incorporated by reference for this purpose). In particular embodiments, the pharmaceutical composition comprises at least one unit dose of 75 mg of the human FGF21 protein variant of SEQ ID NO: 1 to be administered subcutaneously every two weeks. The pharmaceutical composition provided herein is for subcutaneous administration. Suitable formulation components and methods for subcutaneous administration of polypeptide therapeutics (e.g., antibodies, fusion proteins and the like) are known in the art (c.f. e.g., US 2011 / 0044977 A1, US 8,465,739 B2 and US 8,476,239 B2, each of which is incorporated by reference for this purpose). Typically, pharmaceutical compositions for subcutaneous administration contain suitable stabilizers (e.g., amino acids, such as methionine, and / or saccharides such as sucrose), buffering agents, and / or tonicifying agents. In certain embodiments, the pharmaceutical composition comprises the human FGF21 protein variant of SEQ ID NO: 1 as a dry formulation, e.g., a lyophilizate which may be reconstituted as a solution prior to subcutaneous administration. DB1 / 141729270.1 Attorney Docket No.: BPH-006PC / 136177-5006 - 17 - In certain embodiments, the pharmaceutical composition is in the form of a solution (e.g., after reconstitution of a lyophilizate) of about 50 mg / mL to about 150 mg / mL or about 100 mg / mL to about 150 mg / mL of the FGF21 protein variant. In certain embodiments, the pharmaceutical composition comprises (e.g., after reconstitution of a lyophilizate) a trometamol buffer, for example, at a concentration of 10 mM to or 50 mM trometamol buffer, for example 30 mM trometamol buffer. In certain embodiments, the pharmaceutical composition is in the form of a solution (e.g., after reconstitution of a lyophilizate) comprising sucrose, for example, about 200 mM to about 300 mM sucrose, for example 270 mM sucrose. In certain embodiments, the pharmaceutical composition is in the form of a solution (e.g., after reconstitution of a lyophilizate) comprising a polysorbate, such as Polysorbate 20, e.g., about 0.01% (w / w) to about 0.10% (w / w) Polysorbate 20, for example 0.06% (w / w) Polysorbate 20. In certain embodiments, the pharmaceutical composition is in the form of a solution (e.g., after reconstitution of a lyophilizate) at a pH in the range of 6.5 to 9, for example a pH of about 8.0. In certain embodiments, the pharmaceutical composition is in the form of a solution (e.g., after reconstitution of a lyophilizate) comprising trometamol buffer, sucrose, and Polysorbate 20, e.g., comprising about 80 mg / mL to 100 mg / mL of the FGF21 protein variant, about 30 mM of trometamol buffer, 270 mM sucrose, and 0.06% Polysorbate 20, at pH 7.5 to 8.5, for example pH 8.0. In particular embodiments, the pharmaceutical composition is in the form of a solution (e.g., after reconstitution of a lyophilizate) comprising about 100 mg / mL of the FGF21 protein variant, about 30 mM of trometamol buffer, 270 mM sucrose, and 0.06% polysorbate 20, at pH 8.0. Further, the invention shall be illustrated in more detail by the following examples. DB1 / 141729270.1 Attorney Docket No.: BPH-006PC / 136177-5006 - 18 - EXAMPLES Example 1: Study Protocol A 12-week Phase 2a randomized, blinded, placebo-controlled study of BOS-580 in obese subjects at risk for nonalcoholic steatohepatitis (NASH) 1.1 Abbreviations and Definitions ACTH adrenocorticotropic hormone ADA anti-drug antibody ADL activities of daily living AE adverse event AESI adverse event of special interest ALT alanine aminotransferase ApoB apolipoprotein B AST aspartate aminotransferase AUC area under the serum concentration-time curve AUClast area under the serum concentration-time curve from time zero to time of last measurable concentration AUCtauarea under the serum concentration-time curve during a dosage interval AV atrioventricular BG blood glucose BL baseline BMI body mass index BP blood pressure BSAP bone specific alkaline phosphatase CAP controlled attenuation parameter Cavg average serum drug concentration CGM continuous glucose monitoring CKD-EPI Chronic Kidney Disease Epidemiology Collaboration Cmax maximum serum drug concentration Cmin minimum serum drug concentration COVID-19 coronavirus disease 2019 CRO Contract Research Organization CRP C-reactive protein CSR clinical study report CTCAE Common Terminology Criteria for Adverse Events CTX-1 type I collagen C-telopeptide DHEAS dehydroepiandrosterone sulfate DB1 / 141729270.1 Attorney Docket No.: BPH-006PC / 136177-5006 - 19 - DM diabetes mellitus DPP-4 dipeptidyl peptidase-4 ECG electrocardiogram eCRF electronic Case Report Form EDC electronic data capture EFX efruxifermin eGFR estimated glomerular filtration rate ELF enhanced liver fibrosis ELISA enzyme-linked immunosorbent assay FAS full analysis set Fc fragment crystallizable FGF fibroblast growth factor FGF21 fibroblast growth factor 21 FGFR1 fibroblast growth factor receptor 1 FIB-4 fibrosis-4 FPG fasting plasma glucose FSH follicle-stimulating hormone GAD glutamic acid decarboxylase GCP Good Clinical Practice GI gastrointestinal GLP Good Laboratory Practice GLP-1 glucagon-like peptide 1 HBsAg hepatitis B virus surface antigen HBV hepatitis B virus hCG human chorionic gonadotropin HCV hepatitis C virus HDL-C high density lipoprotein C HFF hepatic fat fraction HIV human immunodeficiency virus HOMA-IR homeostatic model assessment of insulin resistance HRT hormonal replacement therapy IB Investigator’s Brochure ICF Informed Consent Form ICH International Council for Harmonisation IEC Independent Ethics Committee IFN gamma interferon gamma IgG1 immunoglobulin G1 IL interleukin IMP investigational medicinal product DB1 / 141729270.1 Attorney Docket No.: BPH-006PC / 136177-5006 - 20 - INR international normalized ratio IRB Institutional Review Board IRT Interactive Response Technology LDL-C low density lipoprotein C LSM liver stiffness measurement MCP-1 monocyte chemoattractant protein-1 Meds medications MELD Model for End-stage Liver Disease MRI magnetic resonance imaging MRI-PDFF magnetic resonance imaging-derived proton density fat fraction NAFLD nonalcoholic fatty liver disease NASH nonalcoholic steatohepatitis NCI National Cancer Institute NIMP non-investigational medicinal product P1NP procollagen type 1 N-terminal pro-peptide PBO placebo PCR polymerase chain reaction PCSK9 proprotein convertase subtilisin kexin 9 PD pharmacodynamic pDILI potential drug induced liver injury PEF peak expiratory flow PK pharmacokinetic Post-Tx post-treatment Pro-C3 type III collagen pro-peptide PT prothrombin time Q2W every 2 weeks Q4W every 4 weeks QTcB QT interval corrected for heart rate using Bazett's formula QTcF QT interval corrected for heart rate using Fridericia's formula QW every week SAE serious adverse event SAP Statistical Analysis Plan SC subcutaneous SD standard deviation SoA Schedule of Activities SOP standard operating procedure SRT Safety Review Team t1 / 2 Half-life T2DM type 2 diabetes mellitus DB1 / 141729270.1 Attorney Docket No.: BPH-006PC / 136177-5006 - 21 - TEAE treatment-emergent adverse event TESAE treatment-emergent serious adverse event Tmaxtime to reach maximum serum concentration TNF tumor necrosis factor TSH thyroid stimulating hormone ULN upper limit of normal VCAM-1 vascular cell adhesion molecule-1 VCTE vibration controlled transient elastography Wk week WOCBP woman of childbearing potential 1.2 Rationale The purpose of this study was to evaluate the BOS-580 (SEQ ID NO: 1) exposure-response relationship based on BOS-580 dose and exposure, and tolerability and efficacy biomarkers. These data were utilized to confirm the benefit / risk profile and dose rationale of BOS-580 to support advancing to late-stage development for the treatment of NASH. 1.3 Objectives and Endpoints Objectives Endpoints E) se lic art ry DB1 / 141729270.1 Attorney Docket No.: BPH-006PC / 136177-5006 - 22 - ug ug ug m he m le m ge or to to ce on ed hy III ne te LT D) 6), is 0, te ls, DB1 / 141729270.1 Attorney Docket No.: BPH-006PC / 136177-5006 - 23 - a el ), c, nd tal ), nd B C ne ne ne N- in es nd 80 be ns er This was a 2-part, multicenter, randomized, double-blind (sponsor unblinded), placebo (PBO)-controlled, safety study to evaluate BOS-580 administered subcutaneously with repeated dosing over 12 weeks. The study included up to 105 male and / or female subjects, 18 to 75 years of age inclusive, with a body mass DB1 / 141729270.1 Attorney Docket No.: BPH-006PC / 136177-5006 - 24 - index (BMI) of 30 to 45 kg / m2inclusive, MRI-PDFF ≥ 10%, a VCTE LSM between 7 and 9.9 kPa inclusive, and AST > 20 U / L. Part A consisted of 5 cohorts (A1 through A4, with A5 randomized separately) for a total of approximately 75 subjects. Subjects were initially randomized to cohorts A1 through A4, with approximately 15 subjects per cohort. Subjects in cohorts A1 through A4 were then be randomized within each cohort in a 4:1 ratio (BOS-580 vs PBO). Once cohorts A1 through A4 had been randomized, approximately 15 subjects will be randomized to Cohort A5 in a 4:1 ratio (BOS-580 vs PBO). Subjects returned to the study center for regular visits over 12 weeks for the study drug administration, pharmacokinetic (PK), biomarker, and safety assessments. In Part A, BOS-580 or PBO was administered by subcutaneous (SC) injection as follows: • Cohort A1: 300 mg once every 4 weeks (Q4W) (300 mg / month) or PBO. •Cohort A3: 75 mg Q2W (150 mg / month) or PBO.• Cohort A4: 75 mg Q4W (75 mg / month) or PBO. Additional cohorts may be initiated for the purpose of exploring additional doses, frequencies of administration, or up-titration of doses in Part B, which would occur after data from the Week 6 interim analysis for cohorts A1, A3, and A4. In Parts A and B, subjects will return to the study center for a follow-up visit approximately 4 weeks after receiving the last study dose. 1.5 Number of Subjects Subjects were enrolled such that approximately 75 evaluable subjects complete Part A. Approximately 30 subjects may be enrolled in Part B (optional). 1.6 Inclusion / Exclusion Criteria Inclusion Subjects were eligible to be included in the study only if all of the following criteria apply: Criteria 1. Subject is either male or female and 18 to 75 years of age inclusive, at the time DB1 / 141729270.1 Attorney Docket No.: BPH-006PC / 136177-5006 - 25 - P) nd nd ly y: ed s, nt .). d. e e, be ds ts D st ed DB1 / 141729270.1 Attorney Docket No.: BPH-006PC / 136177-5006 - 26 - cid tic on . sis ult int ss se es ng ity BP , a cid re ho an nd 73 n. ng ed ee D . Attorney Docket No.: BPH-006PC / 136177-5006 - 27 - de F) val le te ell he of g, on he ty of or . in or ar s), ts tor he ith a of of ch DB1 / 141729270.1 Attorney Docket No.: BPH-006PC / 136177-5006 - 28 - nd a 1- ay. g, or he he to 4) lin ct in be e, es cal tat e- tor de ss ior . he he D B1 / 141729270.1 Attorney Docket No.: BPH-006PC / 136177-5006 - 29 - al in ed 1.7 Study Periods and Duration The study consisted of 2 screening periods. Subjects meeting the eligibility criteria specified in Screening I (Day -42 to Day -10) proceeded to the imaging evaluation specified in Screening II (Day -28 to Day -7). The study treatment period consisted of bi-weekly or monthly dosing (weekly regimens may be considered in optional Part B). Post-treatment follow-up included a visit approximately 4 weeks after receiving the last study dose. The total duration of involvement for each subject was approximately 22 weeks. 1.8 Study Analyses The following analyses were performed: • An interim analysis was conducted once approximately 90% of subjects randomized in cohorts A1 to A4 have completed the Week 6 visit. Data were used to guide Sponsor-driven decisions regarding the clinical program. •Based on the results of the Week 6 interim analysis, an additional interim analysis may beconducted once all subjects in cohorts A1 to A4 have completed the Week 12 end of treatment. Available data for any subjects enrolled in Part B may be included in the Week 12 end of treatment analysis listed above. Depending on timing of when all subjects complete the End of Study visit (Week 16) in Part A relative to when all subjects complete the End of Study visit in Part B, the final analyses for Part A and B may be completed at the same time or separately. Additional analyses may also be conducted if required. Example 2 Preliminary Study Results 2.1 Demographics and baseline characteristics • Mean age 53.37, ~75% pts Hispanic DB1 / 141729270.1 Attorney Docket No.: BPH-006PC / 136177-5006 - 30 - • BMI Median about 34-36 kg / m2(30-45) • 40 females, 48 males • 50% hypertension, 30% hyperlipidemia • ~40% T2DM; Mean Baseline HbA1c ~6 (5-9.9) • Baseline Hepatic Fat Fraction (HFF), lowest in placebo group (PBO), but all >10% • F2 by Vibration Controlled Transient Elastography (VCTE) – mean score 8.1-8.7 (F2= 7.1-9.9) except for 3 subjects with VCTE 7.0. 2.2 Compliance • At the time of interim analysis, 85% of A3 cohort and 44% of PBO cohort reached 12 weeks • Study compliance was good. Missed / delayed visits / dosing were highest in A2 and PBO cohorts • Duration of individual patients on study could be longer than 16 weeks, due to “out of window” visits (in A3 in particular) 2.3 Treatment Emergent Adverse Events (TEAEs) • The rate of any TEAEs was the lowest in the A4 cohort (50%) to highest in A1 cohort (84.6%) • Placebo TEAE rate was 35.3% • Majority of events grade 1, grade 2 the rarest in A4; similar in A1, and A3 • Rate of “related” TEAEs by frequency: A1>A3, while A4 and PBO were similar (~25%) o Majority of “related events” were gastrointestinal (A1>A3>PBO>A4) > metabolism and nutritional disorders > general disorders & injection site • No significant dose dependent changes were found in lipid, hematology parameters, or lipase levels. • No significant pruritis TEAEs • No weight gain • A1 and A3 had the highest rate of study discontinuations. • Adverse Events of Special Interest (Nausea, Vomiting, Diarrhea) o Rates were similar in A1, A3; Highest in A3 (57%) and <20% in A4 and PBO o Most frequent: Grade 2 Nausea in A1 and A3, and Grade 2 Vomiting in A1 o Diarrhea most frequent in A3 DB1 / 141729270.1 Attorney Docket No.: BPH-006PC / 136177-5006 - 31 - Figs.1A, 1B, 1C and 1D show that administration of BOS-580 (SEQ ID NO: 1) had no adverse effects on lipid profiles. BOS-580 treatment positively impacted levels of HDL-cholesterol (increased) and Triglycerides (lowered) in all tested dose schedules, which is known to decrease the risk of cardiovascular disease. The LDL cholesterol was not significantly changed over time in any direction, in particular, it did not increase (which would increase the risk of cardiovascular disease). Overall cholesterol levels did not significantly change in any direction, upon treatment with BOS-580. Fig.2 shows the occurrence of TEAEs of the gastrointestinal system. Most frequently occurring TEAEs were adverse events of special interest (AESIs) from the Gastrointestinal System Organ class (e.g., nausea, vomiting, diarrhea, abdominal pain), followed by conditions from the Metabolism and Nutrition class (e.g., increased appetite, hyperphagia and food craving). The increase in appetite did not cause an increase in body weight. Most of these events were mild and transient. The highest number of events occurred in cohorts A1 and A3. 2.4 Therapeutic effects Administration of BOS-580 resulted in a reduction of the hepatic fat fraction (HFF) in cohorts A1 and A3 as shown in Fig.3 and 4. Fig.3 shows the absolute change in HFF determined by Magnetic Resonance Imaging Proton Density Fat Fraction (MRI-PDFF). The normal reference range of MRI-PDFF is < 5.6%. Absolute change in the liver fat fraction was significant to changes observed in placebo, in cohorts A1 and A3. The absolute changes were from mean value of >20% at baseline to less than 10% in only 12 weeks of treatment. The effect was significant to placebo, for cohorts A1 and A3 but was not significant for cohort A4. Most of the effect was observed as soon as 6 weeks of treatment with some additional increment by week 12. Fig.4 shows the relative change in HFF determined by MRI-PDFF. Relative change in hepatic fraction was >60% in cohorts A1 and A3 at week 12 and significant over placebo. Most of the effect was observed as soon as week 6, with some additional increment by week 12. Fig.5 shows the proportions of patients achieving specific degrees of fat reduction. The relative reduction of ≥ 30% in HFF is established to be predictive of improvement in the disease based on the histopathology evaluation, therefore the percentage of patients achieving this level of reduction (responders) was used DB1 / 141729270.1 Attorney Docket No.: BPH-006PC / 136177-5006 - 32 - as one of the measures of efficacy. More recently the reduction ≥50% was considered predictive of fibrosis improvement, therefore the percentage of patients achieving this level of reduction (super-responders) was utilized as more stringent criteria. In the interim analysis 100% of patients in cohort A1 were responders and more than 90% in A3 cohort. The placebo rate was 28.6%. In the cohorts A1 and A3, the rate of super-responders was also very high (≥75%), while the rate of placebo was 14.3%. In the 12 week treatment period there were also some patients that normalized liver fat (<=5%), again relative similar within cohorts A1 and A3. 2.5 Biomarkers Fig.6 shows the levels of alanine aminotransferase (ALT), a biomarker of liver injury. Increased liver enzymes are a sign of liver injury, therefore the normalization or trend towards normal values (decrease) is a sign of liver function improvement. In the interim analysis ALT decreased from the median of 40-55 U / L at baseline more than 35% as early as week 6 and was sustained until the last assessment at week 12, in cohorts A1 and A3, and was significantly different from placebo. Cohort A4 did not show a significantly different effect to placebo treated patients. Fig.7 shows the levels of aspartate aminotransferase (AST), a biomarker of liver injury. The decrease in AST from the median baseline levels of 23-34 U / L was more than 40% at the last study measurement and was already achieved in cohort A1 as early as week 6. This change was significantly different from placebo at weeks 6 and the last study assessment at 12 weeks for cohorts A1 and A3. Fig.8 shows the levels of the amino-terminal propeptide of ProCollagen 3 (ProC3), a biomarker of liver fibrosis. Collagen 3 is the predominant collagen type deposited in the liver during the pathogenesis of liver fibrosis. In order for the collagen to be deposited in the extracellular space of liver tissue, the N-terminal peptide of Pro-collagen (Pro-C3) is removed enzymatically. This N-terminal peptide can be measured in the circulation and its levels are correlated to the extent of the fibrosis. The lowering of Pro-C3 for 15% or more or 20% or more is predictive of the improvement in the fibrosis. In cohort A1, the treatment with 300 mg Q4W decreased Pro-C3 by at least 20% as early as week 6 and the effect was sustained until the last study assessment. The effect was significantly different to placebo. In cohorts A3 and A4, the treatment with 75 mg Q2W or 75 mg Q4W decreased Pro-C3 levels for >15% at week 6, but the effect was not sustained with the longer treatment. Fig.9 shows the degrees of adiponectin deficiency. Adiponectin is an adipose-specific adipokine that decreases insulin resistance and attenuates liver inflammation / fibrosis. Adiponectin levels are known to DB1 / 141729270.1 Attorney Docket No.: BPH-006PC / 136177-5006 - 33 - be low in NASH patients. Upon FGF21 administration the levels of adiponectin are expected to improve as binding on the respective receptor for FGF21 on adipocytes directly stimulates production of adiponectin. Treatment with all schedules of BOS-580 caused prompt effect as early as week 4, with >50% improvement as week 2. All treatment schedules remained significant to placebo until week 12 of treatment. Fig.10 shows the HbA1c levels in diabetic NASH patients for cohorts A1 and A3. In the sub-population of patients with baseline values of HbA1c >6.5% (diabetic patients) a decline of -1.0% and -0.7% respectively in cohorts A1 and A3 was observed, compared to -0.5% in the Placebo group. It is worth noting that only one of the placebo patients reached week 12 at the time of the analysis, therefore the true median change in placebo patients over time is not possible to assess. It is however known that metformin in 12-24 weeks may decrease HbA1c for 0.6%, which can be used as the benchmark. Treatment with BOS-580 showed improvement in HbA1c levels better than this benchmark. Overall, the data demonstrated, inter alia, that the A3 cohort 75 mg Q2W (150 mg / month) exhibited better efficacy relative to safety considerations in comparison to cohorts A1300 mg Q4W (300 mg / month) and A475 mg Q4W (75 mg / month). SEQ ID NO: 1 as disclosed herein is defined as follows: DKTHTCPPCP APEAAGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPGKGSD SSPLLQFGGQ VRQRYLYTDD ACQTEAHLEI REDGTVGGAA DQSPESLLQL KALKPGVIQI LGVKTSRFLC QKPDGALYGS LHFDPEACSF RELLLEDGYN VYQSEAHGLP LHLPCNRSPH RDPASRGPAR FLPLPGLPPA LPEPPGILAP QPPDVGSSDP LAMVGGSQAR SPSYAS DB1 / 141729270.1

Claims

Attorney Docket No.: BPH-006PC / 136177-5006 - 34 - CLAIMS What is claimed is:

1. A method of treating, preventing, or managing a cardiovascular and / or metabolic disorder in a human subject in need thereof, comprising administering a human FGF21 protein variant comprising the amino acid sequence of SEQ ID NO: 1 to the human subject at a dose of 75 mg, subcutaneously, once every 2 weeks.

2. The method of claim 1, wherein the metabolic disorder and / or cardiovascular disorder is selected from non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), hypercholesterolemia, dyslipidemia, hypertriglyceridemia, type 2 diabetes, and obesity.

3. The method of claim 2, wherein the metabolic disorder and / or cardiovascular disorder is non- alcoholic steatohepatitis (NASH) or non-alcoholic fatty liver disease (NAFLD).

4. The method of claim 3, wherein the metabolic disorder and / or cardiovascular disorder is non- alcoholic steatohepatitis (NASH).

5. The method of any one of claims 1-4, wherein the human subject is obese.

6. The method of any one of claims 1-5, wherein the human subject has a body mass index (BMI) of at least 30 kg / m2with ethnic adjustment of least 27.5 kg / m2for a subject of Asian descent or Asian ancestry, particularly a BMI of 30 kg / m2to 45 kg / m2.

7. The method of any one of claims 1-6, wherein the human subject is diagnosed with NASH, has a suspected diagnosis of NASH, and / or is at risk of acquiring NASH.

8. The method of any one of claims 1-7, wherein the human subject has a liver fibrosis stage selected from F0 (no fibrosis), F1 (steatosis), F2 (early fibrosis), F3 (advanced fibrosis) and / or F4 (cirrhosis), particularly selected from F2 and / or F3.

9. The method of any one of claims 1-8, wherein the human subject has a liver fat assessment based on vibration controlled transient elastography (VCTE) or a controlled attenuation parameter (CAP) score of more than 300 dB / m, or of more than 400 dB / m. DB1 / 141729270.1Attorney Docket No.: BPH-006PC / 136177-5006 - 35 - 10. The method of any one of claims 1-9, wherein the human subject has a liver injury and fibrosis assessment based on VCTE liver stiffness measurement (LSM) score between 7 and 14 kPa, between 7 kPa and 12 kPa, or between 7 kPa and 9.9 kPa and / or an aspartate amino transferase (AST) activity of at least 20 U / L.

11. The method of any one of claims 1-10, wherein the human subject is diagnosed with, has a suspected diagnosis of, and / or is at risk of acquiring type 2 diabetes mellitus, hypertension, and / or hyperlipidemia.

12. The method of any one of claims 1-11, wherein the administration is effective in improving at least one physiological parameter associated with NASH.

13. The method of claim 12, wherein the at least one physiological parameter is selected from liver fibrosis stage, liver fat assessment based on VCTE or CAP, liver injury and / or fibrosis assessment based on VCTE, LSM score, and / or AST activity.

14. The method of any one of claims 1-13, wherein the administration is effective in reducing liver fat, particularly effective in reducing the hepatic fat fraction by at least 45%, at least 50% or at least 60%.

15. The method of any one of claims 1-14, wherein the administration is effective in reducing liver injury, particularly effective in reducing adiponectin deficiency, optionally by increasing the adiponectin level by at least 50%, 75%, 100% or 125%.

16. The method of any one of claims 1-5, wherein the administration is effective in a reduction of liver injury, effective in reducing mean alanine amino transferase (ALT) activity by at least 20%, at least 30%, or at least 35%, and / or effective in reducing aspartate amino transferase (AST) activity by at least 20% or at least 30%, and / or effective in reducing mean of procollagen amino-terminal propeptide (ProC3) by at least 15% or at least 20%.

17. The method of any one of claims 1-16, wherein the administration is effective in reducing the HbA1c level of a subject with type 2 diabetes mellitus by at least 0.6%.

18. The method of any one of claims 1-17, wherein the administration has a gastro-intestinal tolerability profile of grade 1 or 2. DB1 / 141729270.1Attorney Docket No.: BPH-006PC / 136177-5006 - 36 - 19. The method of any one of claims 1-18, wherein the human FGF21 protein variant is administered in combination with one or more additional therapeutically active agents.

20. The method of any one of claims 1-19, wherein the one or more additional therapeutically active agents is selected from the group consisting of compounds useful in obesity therapies, diuretics, beta-blockers, alpha-blockers, ACE inhibitors, Angiotensin II Receptor Blockers (ARBs), direct renin inhibitors, calcium channel blockers, central agonists, peripheral adrenergic blockers, vasodilators, insulin, alpha-glucosidase inhibitors, biguanides, dopamine agonist, DPP-4 inhibitors, glucagon-like peptides, GPL-1 agonists, dual GLP-1 and GLP-2 agonists, meglitinides, sodium glucose transporter (SGLT) inhibitors, sulfonylureas, thiazolidinediones, amylinomimetics, statins, fibrates, aspirin, and anticoagulants.

21. The method of any one of claims 1-20, wherein the human FGF21 protein variant consists of the amino acid sequence of SEQ ID NO:

1.

22. The method of any one of claims 1-21, wherein the human FGF21 protein variant is glycosylated.

23. The method of any one of claims 1-22, wherein the human FGF21 protein variant is non- glycosylated.

24. A human FGF21 protein variant comprising the amino acid sequence of SEQ ID NO: 1 for use in a method of treating, preventing, or managing a cardiovascular and / or metabolic disorder in a human subject, wherein the human FGF21 protein variant is provided for administration at a dose of 75 mg, subcutaneously, once every 2 weeks.

25. The human FGF21 protein variant for use according to claim 24, wherein the metabolic disorder and / or cardiovascular disorder is selected from non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), hypercholesterolemia, dyslipidemia, hypertriglyceridemia, type 2 diabetes, and obesity. DB1 / 141729270.1Attorney Docket No.: BPH-006PC / 136177-5006 - 37 - 26. The human FGF21 protein variant for use according to claim 24 or 25, wherein the metabolic disorder and / or cardiovascular disorder is non-alcoholic steatohepatitis (NASH) or non- alcoholic fatty liver disease (NAFLD).

27. The human FGF21 protein variant for use according to claim 24 or 25, wherein the metabolic disorder and / or cardiovascular disorder is non-alcoholic steatohepatitis (NASH).

28. The human FGF21 protein variant for use according to any one of claims 24-27, wherein the human subject is obese.

29. The human FGF21 protein variant for use according to any one of claims 24-28, wherein the human subject has a body mass index (BMI) of at least 30 kg / m2with ethnic adjustment of least 27.5 kg / m2for a subject of Asian descent or Asian ancestry, particularly a BMI of 30 kg / m2to 45 kg / m2.

30. The human FGF21 protein variant for use according to any one of claims 24-29, wherein the human subject is diagnosed with NASH, has a suspected diagnosis of NASH, and / or is at risk of acquiring NASH.

31. The human FGF21 protein variant for use according to claim 30, wherein the human subject has a liver fibrosis stage selected from F0 (no fibrosis), F1 (steatosis), F2 (early fibrosis), F3 (advanced fibrosis) and / or F4 (cirrhosis), particularly selected from F2 and / or F3.

32. The human FGF21 protein variant for use according to any one of claims 24-31, wherein the human subject has a liver fat assessment based on vibration controlled transient elastography (VCTE) controlled attenuation parameter (CAP) Score of more than 300 dB / m, or of more than 400 dB / m.

33. The human FGF21 protein variant for use according to any one of claims 24-32, wherein the human subject has a liver injury and fibrosis assessment based on VCTE liver stiffness measurement (LSM) score between 7 and 14 kPa, between 7 kPa and 12 kPa, or between 7 kPa and 9.9 kPa and / or an aspartate amino transferase (AST) activity of at least 20 U / L.

34. The human FGF21 protein variant for use according to any one of claims 24-33, wherein the human subject is diagnosed with, has a suspected diagnosis of, and / or is at risk of acquiring type 2 diabetes mellitus, hypertension, and / or hyperlipidemia. DB1 / 141729270.1Attorney Docket No.: BPH-006PC / 136177-5006 - 38 - 35. The human FGF21 protein variant for use according to any one of claims 24-34, wherein the administration is effective in improving at least one physiological parameter associated with NASH.

36. The human FGF21 protein variant for use according to claim 35, wherein the at least one physiological parameter is selected from liver fibrosis stage, liver fat assessment based on VCTE CAP, liver injury and fibrosis assessment based on VCTE LSM score and AST activity.

37. The human FGF21 protein variant for use according to any one of claims 24-36, wherein the administration is effective in reducing liver fat, particularly effective reducing the hepatic fat fraction by at least 45%, at least 50% or at least 60%.

38. The human FGF21 protein variant for use according to any one of claims 24-37, wherein the administration is effective in reducing liver injury, particularly effective in reducing adiponectin deficiency, optionally by increasing the adiponectin level by at least 50%, 75%, 100% or 125%.

39. The human FGF21 protein variant for use according to any one of claims 24-38, wherein the administration is effective in a reduction of liver injury, effective in reducing mean alanine amino transferase (ALT) activity by at least 20%, at least 30%, or at least 35%, and / or effective in reducing aspartate amino transferase (AST) activity by at least 20% or at least 30%, and / or effective in reducing mean of procollagen amino-terminal propeptide (ProC3) by at least 15% or at least 20%.

40. The human FGF21 protein variant for use according to any one of claims 24-39, wherein the administration is effective in reducing the HbA1c level of a subject with type 2 diabetes mellitus by at least 0.6%.

41. The human FGF21 protein variant for use according to any one of claims 24-40, wherein the administration has a gastro-intestinal tolerability profile of grade 1 or 2.

42. The human FGF21 protein variant for use according to any one of claims 24-41, wherein the human FGF21 protein variant is administered in combination with one or more additional therapeutically active agents. DB1 / 141729270.1Attorney Docket No.: BPH-006PC / 136177-5006 - 39 - 43. The human FGF21 protein variant for use according to claim 42, wherein the one or more additional therapeutically active agents is selected from the group consisting of compounds useful in obesity therapies, diuretics, beta-blockers, alpha-blockers, ACE inhibitors, Angiotensin II Receptor Blockers (ARBs), direct renin inhibitors, calcium channel blockers, central agonists, peripheral adrenergic blockers, vasodilators, insulin, alpha-glucosidase inhibitors, biguanides, dopamine agonist, DPP-4 inhibitors, glucagon-like peptides, GLP-1 agonists, dual GLP-1 and GLP-2 agonists, meglitinides, sodium glucose transporter (SGLT) inhibitors, sulfonylureas, thiazolidinediones, amylinomimetics, statins, fibrates, aspirin, and anticoagulants.

44. The human FGF21 protein variant for use according to any one of claims 24-43, which consists of the amino acid sequence of SEQ ID NO:

1.

45. The human FGF21 protein variant for use according to any one of claims 24-44, wherein the human FGF21 protein variant is glycosylated.

46. The human FGF21 protein variant for use according to any one of claims 24-44, wherein the human FGF21 protein variant is non-glycosylated.

47. Use of a human FGF21 protein variant comprising the amino acid sequence of SEQ ID NO: 1 for the manufacture of a medicament for treating, preventing, or managing a cardiovascular and / or metabolic disorder in a human subject, wherein the human FGF21 protein variant is provided for administration at a dose of 75 mg, subcutaneously, once every 2 weeks.

48. The use of claim 47, wherein the human FGF21 protein variant consists of the amino acid sequence of SEQ ID NO:

1. DB1 / 141729270.1

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