METHOD OF TREATING LIVER OR LUNG INJURY
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional Patent Application No. 63/623,01 1 , filed on January 18, 2024, the disclosure of which is hereby incorporated by reference in its entirety herein.
INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY [0002] Incorporated by reference in its entirety is a computer-readable nucleotide/amino acid sequence listing submitted concurrently herewith and identified as follows: 57588P_SeqListing.xml; Size: 14,876 bytes; Created: January 16, 2024.
FIELD OF THE INVENTION
[0003] The disclosure is related to use of FGF21 or an analog thereof (e.g., Efruxifermin (EFX)) to reduce liver or lung injury in a subject suffering from, e.g., alpha-1 antitrypsin deficiency, reduced levels of alpha-1 antitrypsin in blood or lung, and/or accumulation of alpha-1 antitrypsin in hepatocytes.
BACKGROUND
[0004] Alpha-1 antitrypsin (AAT) is a 52-kDa glycoprotein encoded by the SERPINA1 gene, and is the most abundant circulating protease inhibitor in human plasma. AAT is synthesized in hepatocytes and secreted by the liver into blood. The SERPNA1 gene is highly polymorphic, with over 100 AAT variants described in various populations. AAT variants are classified according to the protease inhibitor (Pi) system of nomenclature. Variants are assigned a letter designation, corresponding to the rate at which the variant migrates toward the anode under electrophoresis (i.e., “F” for fast, “M” for medium, “S” for slow, and “Z” for very slow). A single amino acid substitution in the Z variant AAT molecule (Glu342Lys) causes abnormal folding of AAT, resulting in less secretion of AAT protein by hepatocytes into blood, as well as polymerization and accumulation of insoluble Z-AAT aggregates within the hepatocytes, which leads to significant clinical manifestations, including liver and lung injury. For instance, patients with homozygous Z alleles have higher chances of developing liver fibrosis, cirrhosis, hepatocellular carcinoma, or liver failure. Individuals who carry a single Z allele, either MZ heterozygotes or SZ heterozygotes, also have a higher risk of developing clinically significant liver disease, particularly with an additional genetic, environmental, or other insult, including, but not limited to, excessive alcohol consumption, viral hepatitis, or hemochromatosis. Subjects homozygous for the AAT Z allele also have an increased risk of developing lung injury, e.g., early on-set emphysema, chronic obstructive pulmonary disease, and reduced capacity for physical exertion (often linked with cigarette smoking and atmospheric pollution). Current therapies and approaches under clinical development focus on either chronically augmenting blood levels of functional AAT to prevent further lung injury, or reducing levels of toxic Z-AAT in hepatocytes to reduce liver injury. There are few, if any, effective treatments for liver or lung injury caused by AAT deficiency or insoluble Z-AAT polymer aggregation. There is currently no approved therapy that addresses both liver and lung disease associated with AAT deficiency.
SUMMARY
[0005] The disclosure provides a method of reducing liver injury or lung injury in a subject in need thereof and suffering from alpha- 1 antitrypsin (AAT) deficiency, reduced levels of alpha- 1 antitrypsin in blood, or accumulation of insoluble alpha-1 antitrypsin polymers in hepatocytes. The method comprises administering an effective amount of FGF21 or an analog thereof, such as Efruxifermin (EFX), to the subject suffering from alpha-1 antitrypsin deficiency, reduced levels of alpha-1 antitrypsin in blood, or accumulation of insoluble alpha-1 antitrypsin polymers in hepatocytes. In various aspects, the subject is suffering from liver injury. Alternatively or in addition, the subject is optionally suffering from lung injury. Optionally, the subject is suffering from both liver and lung injury. In various aspects of the disclosure, the subject is suffering from alpha-1 antitrypsin deficiency and/or comprises at least one mutant SERPINA1 allele.
Optionally, the method further comprises administering to the subject a second therapeutic agent, such as an enzyme replacement therapy (e.g., AAT enzyme replacement therapy), a misfolded protein corrector small molecule, RNAi, antisense oligonucleotide, or gene editing system (or components thereof). In various aspects, the combination of EFX with the second therapeutic increases the rate and/or extent of resolution of lung and/or liver injury compared to use of the second therapeutic agent alone. Optionally, the method comprises administering about 28 mg, about 50 mg, or about 70 mg of FGF21 or an analog thereof (e.g., EFX) to the subject about once per week.
[0006] Also provided is a method of reducing liver injury or lung injury in a subject in need thereof, wherein the method comprises (a) detecting the presence of at least pathogenic SERPINA 1 allele from the subject; and (b) administering an effective amount of FGF21 or an analog thereof to a subject in need thereof. Optionally, the pathogenic SERPINA1 allele encodes an alpha-1 antitrypsin protein variant selected from Arg39Cys ( Pi*l), Phe52del (Pi*MMaiton), Glu264Val (Pi*S), Glu342Lys (Pi*Z), and Pro369Ser (Pi*MWurzbUrg). [0007] The disclosure further provides a method of increasing secretion of alpha-1 antitrypsin by hepatocytes. The method comprises contacting hepatocytes with an effective amount of FGF21 or an FGF21 analog, such as EFX. In various aspects, the contacting occurs in vivo (e.g., the FGF21 or analog is administered to a subject in an amount effective to increase secretion of alpha-1 antitrypsin from hepatocytes and/or increase circulating levels of alpha-1 antitrypsin).
[0008] The disclosure further provides a method of decreasing the amount of mutant alpha-1 antitrypsin polymers (i.e., insoluble, polymerized alpha- 1 antitrypsin) within hepatocytes. Also provided is a method of increasing solubility of mutant alpha- 1 antitrypsin polymers within hepatocytes. The method comprises contacting hepatocytes with an effective amount of FGF21 or an analog thereof, such as EFX. In various aspects, the contacting occurs in vivo (e.g., FGF21 or an analog thereof is administered to a subject in an amount effective to decrease the amount of insoluble, polymerized alpha-1 antitrypsin within hepatocytes).
[0009] The foregoing summary is not intended to define every aspect of the invention, and additional aspects are described in other sections, such as the Detailed Description. Additional features and variations of the invention will be apparent to those skilled in the art from the entirety of this application, including the detailed description and figures (if present), and all such features are intended as aspects of the invention. Likewise, features of the invention described herein can be re-combined into additional aspects that also are intended as aspects of the invention, irrespective of whether the combination of features is specified as an aspect of the invention. The entire document is intended to be related as a unified disclosure, and it should be understood that all combinations of features described herein (even if described in separate sections) are contemplated, even if the combination of features is not found together in the same sentence, or paragraph, or section of this document. Also, only such limitations which are described herein as critical to the invention should be viewed as such; variations of the invention lacking limitations which have not been described herein as critical are intended as aspects of the invention. The use of section headings, if present, is merely for the convenience of reading; it should be understood that all combinations of features described herein are contemplated.
[0010] Unless otherwise defined herein, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms unless otherwise noted. If aspects of the invention are described as "comprising" a feature, aspects also are contemplated "consisting of" or "consisting essentially of" the feature. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illustrate the disclosure and does not pose a limitation on the scope of the disclosure unless otherwise claimed. No language in the specification should be construed as indicating any nonclaimed element as essential to the practice of the disclosure. Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities should be understood as modified in all instances by the term "about" as that term would be interpreted by the person skilled in the relevant art. With respect to aspects of the invention described or claimed with “a” or “an,” it should be understood that these terms mean “one or more” unless context unambiguously requires a more restricted meaning. With respect to elements described as one or more within a set, it should be understood that all combinations within the set are contemplated.
[0011] It should also be understood that when describing a range of values, the disclosure contemplates individual values found within the range. For example, “a dose from about 20 mg/kg to about 75 mg/kg,” could be, but is not limited to, 25 mg/kg, 40 mg/kg, 75 mg/kg, etc., and any value in between such values. In any of the ranges described herein, the endpoints of the range are included in the range. However, the description also contemplates the same ranges in which the lower and/or the higher endpoint is excluded. When the term “about” is used, it means the recited number plus or minus 5%, 10%, or more of that recited number. The actual variation intended is determinable from the context.
BRIEF DESCRIPTION OF THE FIGURES
[0012] FIGs. 1 A-1 B are illustrations relating to a study demonstrating that the FGF21 analog EFX increases secretion of M-AAT and Z-AAT in adult-like human hepatocytes differentiated from induced pluripotent stem cells (iPSCs) prepared from MM (normal genotype) or ZZ (homozygous Z mutation) individuals. FIG. 1 A is schematic of the study, wherein cells were cultured for 15 days at 37°C after which a subset of cells was cultured in the presence of 100 ng/mL EFX for an additional 5 days. FIG. 1 B is a bar graph illustrating levels of secreted AAT (ng/mL) produced by these differentiated hepatocytes from MM and ZZ individuals cultured in the presence or absence of EFX. EFX increased the secretion of AAT in hepatocytes differentiated from iPSCs prepared from both MM and ZZ individuals.
[0013] FIGs. 2A-2B are illustrations relating to a study demonstrating that the FGF21 analog, EFX, increases solubility of Z-AAT in vitro, reducing accumulation of Z-AAT in the insoluble fraction of cultured Chinese hamster ovary (CHO) cells which had been engineered to express receptors required for FGF21 (and EFX) signaling, and Z-AAT or M-AAT gene induced by a tetracycline-responsive promoter (respectively Tet-On-Z or Tet-On-A). CHO-Tet-On-Z cells were cultured with or without the tetracycline analog, doxycycline, for 48 hours at 37°C, and then with or without 100 ng/mL EFX for an additional 15 minutes. FIG. 2A is a bar graph showing the levels of AAT normalized to levels of GAPDH in the insoluble fraction of the cells. FIG. 2B is a bar graph showing the levels of AAT normalized to GAPDH in the soluble fraction of the cells. EFX increased the solubility of AAT in CHO-Tet-On-Z cells treated with doxycycline.
[0014] FIG. 3 includes tables of FGF analogs, which are substitution mutants wherein the amino acid positions are in reference to SEQ ID NO: 2.
DETAILED DESCRIPTION
[0015] The disclosure provides a method of reducing liver injury or lung injury in a subject in need thereof. The method comprises administering an effective amount of fibroblast growth factor 21 (FGF21 ) or an analog thereof, including, for example, Efruxifermin (EFX), to a subject in need thereof. In various aspects, the subject is suffering from alpha-1 antitrypsin deficiency (AATD) or decreased AAT levels (e.g., in blood or lung) or accumulation of insoluble, polymerized alpha- 1 antitrypsin in hepatocytes. In various aspects, the subject carries at least one, and in some cases more than one, SERPINA1 mutation, including for example the Glu342Lys mutation encoding Z-AAT. Surprisingly, it has been determined that FGF21 analogs, such as EFX, modulate the endoplasmic reticulum adaptive stress response (an underlying cause of liver injury and lung injury associated with AATD), restores protein folding homeostasis, ameliorates oxidative stress, and induces protective autophagy (e.g., which degrades toxic insoluble polymers of Z-AAT). EFX also surprisingly reduces inflammation and fibrosis in lung, a major site of pathology in patients with reduced levels of AAT in circulation (e.g., subjects with less than 1 .0-2.7 g/L AAT in circulation).
[0016] FGF21 is polypeptide secreted primarily by the liver that acts as an endocrine hormone to regulate energy homeostasis, glucose metabolism, and lipid metabolism; and as a paracrine hormone to reduce the damaging effects of hepatocyte stress, including but not limited to lipotoxicity, oxidative stress, and endoplasmic reticulum stress. FGF21 activates stress response pathways including the oxidative stress response pathway, the integrated stress response pathway, endoplasmic reticulum-associated degradation (ERAD), protective autophagy to improve protein folding in and secretion from hepatocytes. The amino acid sequence of mature forms of FGF21 (i.e., lacking the signal sequence) is provided as SEQ ID NOs: 1 and 2, which are natural isoforms of FGF21. FGF21 polypeptides (i.e., naturally occurring, wild-type FGF21 or FGF21 analogs) can but need not comprise an amino-terminal methionine, which may be introduced by engineering or as a result of a bacterial expression process.
[0017] The method of the disclosure comprises administering to a subject in need thereof FGF21 or an FGF21 analog. By “FGF21 analog” is meant an FGF21 polypeptide in which a naturally occurring FGF21 amino acid sequence (e.g., SEQ ID NOs: 1 or 2) has been modified. Such modifications include, but are not limited to, one or more amino acid substitutions, including substitutions with non-naturally occurring amino acids and non-naturally-occurring amino acid analogs, and truncations. Thus, FGF21 analogs include, but are not limited to, site- directed FGF21 mutants, truncated FGF21 polypeptides, proteolysis-resistant FGF21 mutants, aggregation-reducing FGF21 mutants, FGF21 combination mutants, and FGF21 fusion proteins. For the purpose of identifying amino acid substitutions of FGF21 analogs, the numbering of amino acid residue positions herein corresponds to that of the mature 181 -residue FGF21 polypeptide (e.g., SEQ ID NO: 2). In various aspects of the disclosure, the FGF21 analog comprises an amino acid sequence that is at least about 85% identical (e.g., at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%) identical to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. Examples of amino acid positions which may be substituted and potential substitutions are provided in Fig. 3. In various aspects, the FGF21 sequence is modified at one or more of positions 4-5, 19-22, 26, 45, 52, 58-60, 78, 86-88, 98-99, 1 11 , 129, 134, 150-152, 170-173, and 178-181. Substitution at one or more of these positions can optionally increase the stability of the polypeptide by, e.g., reducing degradation or aggregation. Optionally, an FGF21 sequence is modified to provide an attachment point for an accessory molecule, such as any of the polymers described herein. For instance, position 108 of an FGF21 sequence may be modified to be para-acetyl-phenylalanine (f), which allows attachment of a PEG (e.g., a 30 kD PEG) to the sequence. In various aspects, the FGF21 comprises a substitution at position 98, 171 , and/or 180 (such as at positions 171 and 180 or at all three of positions position 98, 171 , and 180). Examples of FGF21 analogs include, but are not limited to, FGF21 analogs comprising the amino acid sequences of SEQ ID NOs: 3-6 or SEQ ID NOs: 7-10, which are SEQ ID NOs: 3-6 lacking an N-terminal methionine. In various aspects of the disclosure, the FGF21 analog comprises an amino acid sequence that is at least about 85% identical (e.g., at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%) identical to the amino acid sequence of any one of SEQ ID NOs 3-10, but wherein residues conferring a desirable property to the FGF21 polypeptide mutant (e.g., proteolysis-resistance, increased half life or aggregation-reducing properties and combinations thereof) have not been further modified. For instance, the disclosure contemplates an FGF21 analog having at least 90% identity to SEQ ID NO: 3, while maintaining the arginine at position 98, the glycine at position 171 , and the glutamic acid at position 180. The disclosure also contemplates FGF21 analogs comprising no more than five, no more than four, no more than three, no more than two, or no more than one amino acid substitution compared to SEQ ID NOs: 1 or 2 or any one of SEQ ID NOs: 3-10. The FGF21 analog is a (3-Klotho-dependent agonist of FGFRI c, FGFR2c, and/or FGFR3c.
[0018] FGF21 or analogs thereof may be modified by the covalent attachment of one or more polymers. Exemplary polymers each can be of any molecular weight and can be branched or unbranched. The polymers each typically have an average molecular weight of between about 2 kDa to about 100 kDa. The average molecular weight of each polymer is optionally between about 5 kDa and about 50 kDa, such as between about 12 kDa and about 40 kDa, or between about 20 kDa and about 35 kDa. In various aspects, the polymer is a water-soluble polymer, although non-water soluble polymers also are contemplated. Suitable water-soluble polymers or mixtures thereof include, but are not limited to, N-linked or O-linked carbohydrates, sugars, phosphates, polyethylene glycol (PEG) (including the forms of PEG that have been used to derivatize proteins, including mono-(Ci-Cio), alkoxy-, or aryloxy-polyethylene glycol), monomethoxy-polyethylene glycol, dextran (such as low molecular weight dextran of, for example, about 6 kD), cellulose, or other carbohydrate based polymers, poly-(N-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols (e.g., glycerol), and polyvinyl alcohol. In some aspects, an FGF21 or analog thereof is covalently, or chemically, modified to include one or more water-soluble polymers, including, but not limited to, polyethylene glycol (PEG), polyoxyethylene glycol, or polypropylene glycol. See, e.g., U.S. Patent Nos. 4,640,835;
4,496,689; 4,301 ,144; 4,670,417; 4,791 ,192; and 4,179,337. An exemplary polymer is PEG, which can be of any convenient molecular weight, and can be linear or branched. A PEG group may be attached to the FGF21 polypeptide via acylation or reductive alkylation through a reactive group on the PEG moiety (e.g., an aldehyde, amino, thiol, or ester group) to a reactive group on the FGF21 polypeptide (e.g., an aldehyde, amino, or ester group). Examples of attachment sites within the FGF21 sequence include, but are not limited to, positions 108 and 173, although attachment at the amino acid sequence termini also is contemplated. [0019] The FGF21 analog may also be a fusion protein, wherein a heterologous sequence is fused to an FGF21 sequence (or FGF21 analog sequence) at the N- or C-terminus.
Heterologous sequences can be fused directly to a parent sequence or fused via a linker, e.g., a linker peptide comprising 1 -50 amino acids in length, such as a linker comprising 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 residues. In various aspects, a linker is employed which is 15 to 35 amino acid residues in length. In various aspects, the heterologous sequence is an Fc domain (e.g., an IgG Fc domain, such as the domain of SEQ ID NO: 11. In various aspects, the FGF21 analog is in the form of a dimer or a multimer, such as a dimer comprised of two fusion proteins comprising an FGF21 analog comprising an FGF21 polypeptide domain fused to an Fc domain. Alternatively, the FG21 analog may be a heterodimer, wherein one monomer comprises an FGF21 analog sequence comprising an Fc domain (e.g., SEQ ID NO: 3) and the second monomer is an Fc domain lacking an FGF21 domain (e.g., SEQ ID NO: 12).
[0020] Efruxifermin (EFX) is a 92.1 kDa, long-acting fibroblast growth factor 21 FGF21 analog generated by the fusion of a human immunoglobulin IgG 1 Fc fragment via a polyglycine-serine linker to a variant of human FGF21 . Each molecule contains one dimeric Fc domain and two modified FGF21 polypeptide chains. EFX has eight disulfide bonds, six intra-chain and two inter-chain. Two of the intrachain disulfide bonds are in the FGF21 polypeptide sequence between Cys318 and Cys336, one for each monomer. Three modifications were introduced into the FGF21 sequence at L341 R, P414G, and A423E (corresponding to L98R, P171 G, and A180E relative to mature, human FGF21 ). These modifications 1) decrease susceptibility to in vivo proteolytic degradation, 2) increase affinity for P-Klotho, and 3) decrease the propensity to aggregate (Hecht et al., PLoS One 2012; 7(11 ): e49345; Stanislaus et aL, Endocrinology.
2017;158(5):1314-1327). EFX comprises the amino acid sequence set forth in SEQ ID NO: 3 (a dimer of monomers comprising SEQ ID NO: 3). SEQ ID NO: 7 is the EFX monomer sequence lacking the N-terminal methionine. FGF21 analogs, including EFX, have been further described in U.S. Patent Nos. 8,034,770; 8,410,051 ; 8,642,546; 8,361 ,963; 9,273,106; 10,011 ,642;
8,188,040; 8,835,385; 8,795,985; 8,618,053; and 11 ,072,640; and International Patent Publication Nos. WO 2009149171 and WO 2010129503, the disclosures of which are incorporated herein by reference in their entireties, including the sequence listings of the referenced patent publications.
[0021] Exemplary sequences are provided in Table 1 . TABLE 1
[0022] The disclosure provides a method of reducing liver injury or lung injury in a subject in need thereof. In various aspects, the method comprises reducing liver injury and lung injury in an individual. Liver injury is marked by signs or symptoms of liver dysfunction. Different primary, pathogenic insults to the liver (e.g., insults resulting from alcohol and/or viral exposure and/or steatosis) tend to have convergent downstream pathology. They cause chronic activation of hepatocyte stress response pathways, leading to injury and death of hepatocytes, activation of liver-resident pro-inflammatory Kupffer cells, finally resulting in synthesis of collagen by myofibroblasts which have differentiated from resident hepatic stellate cells (HSC). In AAT deficiency (AATD), genetic mutations lead to accumulation of insoluble protein aggregates, which induce stress pathways including the endoplasmic stress response and integrated stress response pathways in hepatocytes. In AATD, these insoluble protein aggregates reduce synthetic and secretory function of hepatocytes, directly causing hepatocyte injury and compromising liver function.
[0023] Liver injury can present itself clinically in a variety of ways, including jaundice, scleral icterus, hepatosplenomegaly, lobular hepatitis, fibrosis, cirrhosis, hepatocellular necrosis, bile duct paucity, decline of liver function and liver failure, bile duct proliferation, or hepatocellular carcinoma. Liver injury also may be detected via measurement of elevated liver enzymes (e.g., gamma-glutamyl transpeptidase (GGT), serum alanine transaminase (ALT), and aspartate transaminase (AST), and/or alkaline phosphatase (ALP)), uric acid, and/or serum bilirubin, and of biomarkers of active remodeling of the extracellular matrix consistent with fibrosis and scarring (including, but not limited to, P3NP, Pro-C3 (markers of fibrinogenesis), CTX-III (a marker of fibrolysis), Tissue Inhibitor of Metalloproteinase-1 (TIMP-1 ), Pro-C4, C4M, and Pro- 06). Liver injury, including liver injury associated with apha-1 antitrypsin deficiency or accumulation of alpha-1 antitrypsin in hepatocytes, can also be detected via biopsy to determine levels of misfolded protein (e.g., accumulation of AAT mutant Z protein and/or periodic acid- Schiff-staining with diastase (PAS-D)-positive globules), as well as presence of inflammatory cells, presence and activation state of collagen-secreting myofibroblasts, and extent of fibrosis (e.g., fibrosis observed via histopathology). Liver injury further may be characterized using non- invasive imaging, e.g., cT1 MRI (iron-corrected T1 magnetic resonance imaging, measure of liver fibroinflammation), transient elastography (FibroScan®, measure of liver stiffness due to fibrosis), or magnetic resonance elastography (MRE, measure of liver stiffness due to fibrosis).
[0024] In various aspects, the disclosure provides a method of reducing lung injury in a subject in need thereof. AATD predisposes subjects to the development of emphysema or chronic obstructive pulmonary disease (COPD), which typically manifest at a younger age with severe lung dysfunction. Lung injury is often associated with inflammatory changes and/or fibrosis in the lung. Inflammation causes damage and loss of type II pneumocytes, resulting in reduced surfactant production. In subjects with AATD, lung injury arises from decreased inhibition of neutrophil elastase, which results from reduced levels of AAT (a natural inhibitor of elastase) in circulation, optionally due to misfolding of AAT protein in hepatocytes and inability of hepatocytes to successfully process the misfolded protein through the secretory pathway. Lung injury may be determined in a variety of ways. An increased elastase activity, which correlates to the severity of the lung injury, can be measured in bronchoalveolar lavage fluid. Lung (pulmonary) function tests, six-minute walk test, arterial blood gas analysis, chest X-ray, and CT scan also are suitable for characterizing lung injury.
[0025] Administration of FGF21 or an FGF21 analog (such as EFX) to the subject reduces liver injury or lung injury (and, in some instances, liver injury and lung injury) in the subject. “Reducing” or ‘treating” liver and/or lung injury does not necessarily imply 100% or completely effective treatment or remission. Rather, there are varying degrees of effectiveness of treatment of which one of ordinary skill in the art recognizes as having a meaningful clinical benefit or therapeutic effect. In this respect, the method of reducing liver injury and/or lung injury can provide any amount or any level of beneficial biological response, which ideally results in a positive clinical response. The method of the disclosure may improve or inhibit one or more signs or symptoms of liver injury and/or lung injury in the subject, or slow, halt, or reverse the progression of liver injury and/or lung injury.
[0026] As merely one example of reducing of liver injury, the method may result in regression of a fibrosis score of the subject, such as a that described in Kleiner et al., Hepatology, 41 , 1313 (2005) (e.g., regression from F4 (cirrhosis) to F3 (advanced fibrosis) or less) or Ishak et al., J Hepatol., 22(6), 696-9 (1995). In other embodiments, liver enzymes that are elevated upon liver injury, e.g., ALT, AST, ALP, or GGT, are reduced, optionally to within their normal ranges. Alternatively or in addition, Pro-C3, a marker of type-l 11 collagen synthesis, is reduced to <9.2 pg/L based on the first-generation ELISA format. Pro-C3 levels <9.2 pg/L are within the 90% confidence interval of the mean value for healthy subjects (see, for example, Erhardtsen et aL, JHEP Reports, 3(4), 100317 (2021)). Alternatively or in addition, ELF score, a composite biomarker of liver fibrosis, is reduced to <9.8, which is below the threshold suggestive of advanced fibrosis (see, for example, Sanyal et aL, Hepatology, 6(70), 1913-1927 (2019)). In other embodiments, the number or area of globules positive for periodic acid-Schiff staining with diastase (PAS-D) is reduced on sections of a liver biopsy, which indicates decreased intrahepatocyte aggregation of insoluble, polymerized AAT. Other measures associated with reducing liver injury include, but are not limited to, reduced liver stiffness by ultrasound imaging (FibroScan), reduced corrected T1 by MRI (e.g., below a threshold of 875 ms), normalized liver function tests, or resolution of hepatitis and/or steatohepatitis or reduced collagen proportionate area of liver biopsy section (determined by, e.g., digital pathology using artificial intelligencebased quantitative algorithms), and the like. Improvements in liver function may also be demonstrated by, e.g., increased secretion of AAT protein from hepatocytes into circulation, i.e., blood, in subjects with AATD, or by reduced levels of serum bilirubin.
[0027] As a non-limiting example of reducing lung injury, inflammation, or fibrosis, the method may result in improvements in the six-minute walk test, a measure of pulmonary function. The method may result in slowing or reversal of lung density loss measured by CT. The method may also result in an improvement in, or a slowing or reversal of decline in, forced expiratory volume in one second (FEVi), another measure of pulmonary function. In various embodiments, the method results in improved oxygenation of peripheral arterial blood as measured by transcutaneous pC>2 sensors. The method may also delay the progression to requiring supplemental oxygen in a subject with COPD or emphysema. In various embodiments the method may result in reduction in inflammatory cytokines reflecting lung inflammation, including, e.g., IL-8 levels in blood, or reduced IL-8 levels or reduced neutrophil elastase activity levels in lung sputum.
[0028] Improvement of quality of life of a subject can be measured by determining one or more quality of life parameters using, for instance, the European Quality of Life 5 questions tool (EQ-5D) to determine mobility, mood, holistic impact on patients’ quality of life as reported by patients. The EQ-5D questionnaire also includes a Visual Analog Scale (VAS), by which respondents can report their perceived health status. See, for example, Balestroni et aL, Monaldi Arch Chest Dis., 78(3), 155-9 (2012), which is incorporated by reference in its entirety. Treatment may also be monitored using a Liver Disease Questionnaire. See, for example, Younossi et aL, Clin Gastroenterol Hepatol., 17(10), 2093-2100. e3 (2019), which is incorporated by reference in its entirety.
[0029] A “subject in need thereof” is a subject, such as a human, that would benefit from the administration of a pharmaceutical composition comprising FGF21 or an FGF21 analog, such as EFX. The subject may be diagnosed with or suffering from symptoms of liver injury or lung injury (or both liver injury and lung injury). In aspects of the disclosure, the subject is a human suffering from alpha-1 antitrypsin deficiency (AATD), or reduced AAT levels in blood or lung, or accumulation of insoluble AAT polymers in hepatocytes. AATD is typically diagnosed using a combination of different laboratory methods, such as detecting or measuring protein (e.g., AAT) concentration in serum and/or genetic testing. AAT accumulation in hepatocytes may be detected by biopsy and/or imaging. AAT accumulation may entail, e.g., polymerization of Z-AAT protein in a “globule” conformation in hepatocytes, which is a histopathological feature characteristic of AATD liver disease. Detection methodologies to identify a protein or nucleic acid of interest in a sample are known in the art and include, e.g., radioimmunoassay (RIA), magnetic immunoassay (MIA), immunocytochemical (ICC) assays, immunohistochemical (IHC) assays, immunofluorescent assays, ELISA, EIA, ELISPOT, ECLIA, enzyme multiplied immunoassay, radiobinding assay, Western blotting, immunoprecipitation, dot blots, flow cytometry, immunoquantitative real-time PCR, next-gen sequencing, protein microarrays, and the like. In various aspects of the disclosure, the subject comprises one or more mutant SERPINA 1 alleles. In various aspects, the subject is suffering from liver injury. Alternatively or in addition, the subject is optionally suffering from lung injury. Optionally, the subject is suffering from both liver and lung injury. In various aspects of the disclosure, the subject is not suffering from non-alcoholic steatohepatitis (NASH) or metabolic dysfunction-associated steatohepatitis (MASH).
[0030] The disclosure further provides a method of reducing liver injury or lung injury in a subject in need thereof, the method comprising (a) detecting the presence of at least pathogenic SERPINA 1 allele from the subject; and (b) administering an effective amount of FGF21 or an analog thereof to a subject in need thereof. Pathogenic SERPINA 1 alleles (i.e., SERPINA1 variants or mutants that are associated with, e.g., quantitative and/or qualitative changes in AAT) are known in the art. See, e.g., Foil, Ther Adv Chronic Dis. 2021 Jul 29;12_suppl:20406223211015954 (doi: 10.1177/20406223211015954); and Seixas et al., Appl Clin Genet. 2021 ;14:173-194. In various aspects, the pathogenic SERPINA1 allele encodes an alpha-1 antitrypsin protein variant selected from Arg39Cys (Pi*l), Phe52del (Pi*MMaiton), Glu264Val (Pi*S), Glu342Lys (Pi*Z), and Pro369Ser (Pi*MwUrzt>urg) (see, e.g., NM 001127701.1 (SERPINA1 ):c.187C>T (p.Arg63Cys);
NM 000295.5(SERPINA1 ):c.221TCT[2] (p.Phe76del); NM 001127701 .1 (SERPINA1 ):c.863A>T (p.Glu288Val); NM_001127701.1 (SERPINA1 ):c.1096G>A (p.Glu366Lys); and NM_000295.4(SERPINA1 ):c.1177C>T (p.Pro393Ser)). The presence of a pathogenic SERPINA 1 allele may be detected in any suitable biological sample obtained from the subject, such as a blood or tissue (e.g., liver or lung) sample. Detection methodologies to identify a protein or nucleic acid of interest in a sample are known in the art and include, e.g., radioimmunoassay (RIA), magnetic immunoassay (MIA), immunocytochemical (ICC) assays, immunohistochemical (IHC) assays, immunofluorescent assays, ELISA, EIA, ELISPOT, ECLIA, enzyme multiplied immunoassay, radiobinding assay, Western blotting, immunoprecipitation, dot blots, flow cytometry, immunoquantitative real-time PCR, next-gen sequencing, protein microarrays, and the like. FGF21 and analogs thereof are described above. In various aspects, the subject exhibits symptoms of AAT deficiency. In alternative aspects, the subject does not exhibit symptoms of AAT deficiency.
[0031] In various aspects, the method comprises administering FGF21 or an FGF21 analog (such as EFX) to the subject as part of a therapeutic regimen which also includes administration of a second therapeutic agent. For example, the method of the disclosure may further comprise administering, e.g., enzyme replacement therapy (e.g., AAT enzyme replacement therapy), a neutrophil elastase inhibitor, a misfolded protein corrector small molecule (e.g., a chaperone), RNAi, antisense oligonucleotide, or gene editing system (or components thereof) to the subject. Misfolded protein correctors (e.g., chaperones) are further described in, e.g., Gamez et aL, Clinical Genetics, 93(3), 450-458 (2018); and Lomas et aL, EMBO Mol Med, 13(3), e13167 (2021 ). Without limitation, examples of second therapeutic agents include alpha-1 antitrypsin protein, ALN-AAT02, alvelestat, APB-101 , APB-103, belcesiran, BMN-349, carbamazepine, CB- 151 , fazirsiran, GSK716, HY1003, INBRX-101 , KB-408, LB-201 , NTLA-2003, NTLA-3001 , PHP- 303, SHP-221 , VX-814, VX-864, VX-634, ZF-874, ZF-887, ABBV-576, elexacaftor, deutivacaftor, galicaftor, icenticaftor, ivacaftor, lumacaftor, navocaftor, SION-109, SION-638, tezacaftor, VX-121 , VXc-522, acoramidis, ALN-TTRsc04, eplontersen, inotersen, NI-006, patisiran, PRX-004, tafamidis, vutrisiran, WVE-006, and BEAM-302. Examples of gene editing systems (or components thereof) include, but are not limited to, those based on a zinc finger (ZF), transcription activator-like effector nucleases (TALEN), meganuclease, and clustered regularly interspaced short palindromic repeat (CRISPR)-associated proteins (e.g., Cas9, xCas9, Cas12a (Cpf1 ), Cas13a, Cas14, CasX, CasY, a Class 1 Cas protein, a Class 2 Cas protein, MAD7, and gRNA complexes thereof).
[0032] FGF21 or an FGF21 analog (such as EFX) is provided in a pharmaceutically acceptable composition comprising physiological acceptable excipients, such as those which are GRAS (generally recognized as safe). In various aspects, EFX (or FGF21 or a different FGF21 analog) is provided in a formulation comprising a sugar (e.g., sucrose, fructose, glucose, or maltose), one or more amino acids (e.g., arginine/arginine-HCI or arginine/glutamic acid), and/or a surfactant (e.g., polysorbate-20 or polysorbate-80). Examples of buffers which may be present include, e.g., Tris-HCI, sodium phosphate, glycylglycine/glycylglycine-HCI, or histidine buffer. FGF21 or an analog thereof (e.g., EFX) may be present at any suitable concentration (e.g., about 20-150 mg/mL).
[0033] With regard to the foregoing methods, FGF21 or an analog thereof (e.g., EFX) may be administered by any suitable route of administration, including intravenous, intraperitoneal, intracerebral (intra-parenchymal), intramuscular, intra-ocular, intraarterial, intraportal, intramedullary, intrathecal, intraventricular, intradermal, transdermal, subcutaneous, intranasal, inhalation (e.g., upper and/or lower airways), enteral, epidural, urethral, vaginal, or rectal routes of administration. In various instances, FGF21 or an analog thereof (e.g., EFX) is administered to the subject intravenously, intramuscularly, or subcutaneously. For example, in some aspects, FGF21 or an analog thereof (e.g., EFX) is administered subcutaneously.
[0034] The amount or dose of FGF21 or an analog thereof (e.g., EFX) (i.e., the "effective amount") administered should be sufficient to achieve a desired biological effect in the subject over a clinically reasonable time frame. In various aspects, the amount of FGF21 or an analog thereof (e.g., EFX) administered is about 25 mg to about 75 mg per administration (e.g., about 28 mg, 50 mg, or 70 mg). The amount of FGF21 or an analog thereof (e.g., EFX) administered may be about 25 mg to about 50 mg per administration, or about 50 mg to about 75 mg per administration. One or more doses of FGF21 or an analog thereof (e.g., EFX) may be administered over a treatment period lasting one week, two weeks, three weeks, four weeks, six weeks, eight weeks, 10 weeks, 12 weeks, 14 weeks, 16 weeks, 18 weeks, 20 weeks, 24 weeks, or more. Two or more doses of FGF21 or an analog thereof (e.g., EFX) may be administered at any suitable interval to achieve a desired biological response. For example, a dose may be administered daily, every three days, every five days, weekly, every 10 days, every two weeks, every three weeks, once a month, once every two months, and the like. In various aspects, FGF21 or an analog thereof (e.g., EFX) is administered at a dose of about 28 mg, about 50 mg, or about 70 mg, once a week or once a month, optionally via subcutaneous injection.
[0035] In jurisdictions that forbid the patenting of methods that are practiced on the human body, the meaning of “administering” a composition to a human subject may be restricted to prescribing a controlled substance that a human subject can self-administer by any technique (e.g., injection, insertion, etc.). The disclosure contemplates use of FGF21 or an analog thereof (e.g., EFX) to reduce liver injury and/or lung injury as disclosed herein. The disclosure further contemplates use of FGF21 or an analog thereof (e.g., EFX) in the preparation of a medicament for reducing liver injury and/or lung injury as disclosed herein. The disclosure further provides FGF21 or an analog thereof (e.g., EFX) for use in the treatment of liver injury and/or lung injury as disclosed herein. In jurisdictions that do not forbid the patenting of methods that are practiced on the human body, the “administering” of compositions includes both methods practiced on the human body and also the foregoing activities.
[0036] As an additional aspect, kits are provided which comprise FGF21 or an analog thereof (e.g., EFX) packaged in a manner which facilitates administration to subjects for the treating of liver injury associated with protein-misfolding. In one aspect, the kit includes a pharmaceutical composition/formulation comprising FGF21 or an analog thereof (e.g., EFX) packaged in a container such as a sealed bottle, vessel, single-use or multi-use vial, prefilled device (e.g. syringe, such as a dual chamber syringe), or prefilled injection device, optionally with a label affixed to the container or included in the package that describes use of EFX in practicing the method. In one aspect, the composition comprising EFX is packaged in a unit dosage form. The kit may include a device suitable for administering the composition according to a specific route of administration, although this is not required.
[0037] The disclosure also provides a method of increasing secretion of alpha- 1 antitrypsin (AAT) by hepatocytes. Also contemplated is a method of increasing the solubility of mutant AAT polymers (e.g., Z-AAT) within hepatocytes, which may or may not be accompanied by increased secretion. Also provided is a method of reducing the amount of mutant AAT polymers within hepatocytes (optionally by increasing degradation of polymerized AAT). The method comprises contacting hepatocytes with an effective amount of FGF21 or an analog thereof (e.g., EFX). The hepatocytes may be contacted in vitro or in vivo. When the contacting occurs in vivo, FGF21 or an analog thereof (e.g., EFX) may be administered to a subject via any of the routes of administration and doses disclosed herein. [0038] In various aspects, contacting the hepatocytes with FGF21 or an analog thereof (e.g., EFX) results in an increase in circulating AAT in a subject (i.e. , the amount of AAT detected in circulation following administration of FGF21 or an analog thereof (e.g., EFX) is greater than the amount of AAT detected in the subject prior to administration of EFX, or in a biologically matched subject or population which has not received EFX). Thus, the disclosure further contemplates a method of increasing the amount of circulating AAT (i.e., the amount of AAT in the bloodstream) in a subject in need thereof, the method comprising administering FGF21 or an analog thereof (e.g., EFX) to the subject in an amount effective to increase the amount of circulating AAT.
[0039] In various aspects, contacting the hepatocytes with FGF21 or an analog thereof (e.g., EFX) results in increasing the solubility of mutant AAT polymers (e.g., Z-AAT) within hepatocytes and/or reducing the amount of mutant AAT polymers within hepatocytes. Mutant AAT polymers within hepatocytes may be detected and quantified using any suitable method, such has, but not limited to, periodic acid-Schiff staining with diastase (PAS-D). With respect to this detection method, the number and/or size of mutant AAT polymer globules from hepatocyte samples is preferably reduced.
[0040] The Examples below illustrate representative features of the disclosure. From the description of these aspects, other aspects of the invention can be made and/or practiced based on the description provided below.
EXAMPLES
Example 1
[0041] Surprisingly, it was determined that EFX increases secretion of AAT from human hepatocytes. An in vitro study was conducted as illustrated in FIG. 1 A. Human-derived induced pluripotent stem cells (iPSCs) were differentiated to hepatocytes and cultured for 15 days at 37°C, after which a subset of cells was cultured in the presence of 100 ng/mL EFX and another subset was cultured in the absence of EFX for an additional 5 days. Secretion of AAT was measured at day 20. As illustrated in FIG. 1 B, EFX increased the secretion of AAT in adult-like hepatocytes differentiated from iPSCs derived from either MM or ZZ individuals.
Example 2
[0042] It was determined that EFX surprisingly increases the solubility of Z-AAT within cells in vitro. Chinese hamster ovary (CHO) cells were engineered to express transgenes encoding human sequences of FGFRIc and KLB, the receptors required to transduce FGF21 and EFX signaling into the cell, as well as a tetracycline-inducible M-AAT or Z-AAT gene (Tet-On-M or Tet-On-Z). When exposed to tetracycline or its analogs, including doxycycline (dox), Tet-On-Z cells but not Tet-On-M cells accumulate insoluble Z-AAT polymers within their cytoplasm. CHO- Tet-On-M or CHO-Tet-On-Z cells were cultured with or without doxycycline for 48 hours at 37°C, then some were treated with 100 ng/mL EFX for 15 minutes while others were not. Levels of AAT protein in the soluble and insoluble cell fractions were determined by Western blot, and band intensities of AAT were normalized to GAPDH. As illustrated in FIG. 2A, while doxycycline increased the amount of insoluble AAT retained in the CHO-Tet-On-Z cells, EFX decreased the amount of insoluble AAT retained in the CHO-Tet-On-Z cells exposed to doxycycline. At the same time, more AAT was detected in the soluble fraction of CHO-Tet-On-Z cells exposed to doxycycline when EFX was added, as illustrated in FIG. 2B.
[0043] All publications, patents and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this disclosure that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.