Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/18—Growth factors; Growth regulators
  • A61K38/1825—Fibroblast growth factor [FGF]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
  • A61K39/39591—Stabilisation, fragmentation
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
  • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
  • A61K47/642—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the peptide or protein in the drug conjugate being a cytokine, e.g. IL2, chemokine, growth factors or interferons being the inactive part of the conjugate
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
  • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
  • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
  • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/0012—Galenical forms characterised by the site of application
  • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/08—Solutions
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/475—Growth factors; Growth regulators
  • C07K14/50—Fibroblast growth factor [FGF]
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/575—Hormones
  • C07K14/605—Glucagons
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
  • C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
  • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
  • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
  • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/31—Fusion polypeptide fusions, other than Fc, for prolonged plasma life, e.g. albumin
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
  • C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor
  • C07K2319/75—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor containing a fusion for activation of a cell surface receptor, e.g. thrombopoeitin, NPY and other peptide hormones

Definitions

• the present disclosure relates generally to the field of therapeutic peptides, and more specifically relates to fusion polypeptides, their pharmaceutical compositions, and methods of using such to prevent and/or treat metabolic disorders such as diabetes.
• Fibroblast growth factor 21 is a hormone synthesized in several metabolically active organs and regulates glucose and lipid homeostasis.
• the biology of FGF21 is intrinsically complicated owing to its diverse metabolic functions in multiple target organs. FGF21 has been reported to function in organs such as liver, adipocytes, pancreas, hypothalamus and muscles tissues (Fisher FM, Annu Rev Physiol, 2016, 78: 223) .
• Glucagon-like-peptide-1 is a proglucagon-derived peptide that is secreted from intestinal L-cells in response to nutrient ingestion.
• GLP-1 primarily acts as an incretin, i.e. an endocrine hormone, to increase the insulin response following oral intake of food, by generally regulating the concentrations of glucagons, slowing down gastric emptying, stimulating the biosynthesis of (Pro-) insulin, increasing the sensitivity toward insulin, and stimulating the insulin-independent biosynthesis of glycogen, etc.
• GLP-1 can rapidly lower glucose levels in both normal and diabetic subjects, there has been considerable interest in developing GLP-1 based pharmaceutical agents (i.e.
• GLP-1 compounds for preventing and/or treating type 2 diabetes.
• human GLP-1 in its native form having 37 amino acid residues
• the two major naturally-occurring and biologically active truncated versions of the native GLP-1 include a 30-and 31-amino acid peptide fragment, i.e. GLP-1 (7-36) or GLP-1 (7-37) , which derive from the posttranslational processing of the proglucagon peptide, have extremely short in vivo half-lives due primarily to the N-terminal cleavage and inactivation by the dipeptidyl peptidase DPP-IV.
• Metabolic disorders or metabolic disease are a group of conditions characterized by the inability of the body to properly transform foods into energy, and to utilize and/or store energy, with the most prominent metabolic disorder being diabetes. Metabolic disorders are commonly associated with insulin resistance, visceral adiposity, atherogenic dyslipidemia, etc., which pose major and escalating public health and clinical challenge worldwide. Although tremendous efforts have been invested in developing newer and better treatment with more favorable administration regimens, existing treatment for metabolic diseases faces problems such as short half-life and/or low efficacy.
• compositions and methods of use thereof for treating/preventing metabolic disorders include pharmaceutical compositions and methods of use thereof for treating/preventing metabolic disorders.
• the present disclosure provides a pharmaceutical composition, which comprises a polypeptide and a pharmaceutically acceptable excipient.
• the polypeptide comprises a first fragment comprising a nanobody domain capable of binding to serum albumin, and a second fragment comprising a biologically active FGF21 domain, wherein the first fragment is connected to N-terminus of the second fragment via a first linker.
• the FGF21 domain comprises an amino acid sequence having at least 90%sequence identity to, while substantially retaining biological activity of, SEQ ID NO: 1.
• the FGF21 domain may comprise no more than 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 amino acid residue mutations relative to SEQ ID NO: 1, as long as such mutation-containing FGF21 variant still retains the biological activity of FGF21.
• an amino acid residue mutation can a substitution, an insertion, or a deletion.
• the FGF21 domain may comprise one or more amino acid residue mutations, each at a position selected from positions 121, 168, 171, and 180 relative to SEQ ID NO: 1.
• the one or more amino acid residue mutations in the FGF21 domain may include one, two, three or four of the following three substitutions: N121Q, M168L, P171G and A180E.
• the FGF21 domain may comprise all three substitutions of N121Q, M168L, and A180E, and the amino acid sequence of the FGF21 domain is set forth in SEQ ID NO: 5. Other amino acid residues in the FGF21 domain may be additionally mutated on top of the three mutations.
• the FGF21 domain further comprises a substitution P171G.
• the FGF21 domain comprises the amino acid sequence of SEQ ID NO: 14.
• the FGF21 domain further comprises a conjugatable residue.
• the conjugatable residue can optionally be at a position within a C-terminal fragment spanning from position 169 to position 181 relative to SEQ ID NO: 1.
• the conjugatable residue is at a position selected from the group consisting of positions 169, 170, 171, 172, 173, 174, 180 and 181 relative to SEQ ID NO: 1.
• the FGF21 domain comprises the amino acid sequence of SEQ ID NOs: 2-5, 89-91, 14, and 102-105, except for a mutation to a conjugatable residue at a position within a C-terminal fragment spanning from position 169 to position 181 relative to SEQ ID NO: 1.
• the FGF21 domain comprises the amino acid sequence of SEQ ID NOs: 6-13, 16-19, and 92.
• the polypeptide is conjugated with a functional moiety at the conjugatable residue in the FGF21 domain-containing second fragment.
• the functional moiety that is conjugated to the FGF2 domain may optionally comprise a glycosyl moiety or a synthetic chemical moiety.
• the functional moiety comprises a glycosyl moiety
• the conjugatable residue can be an introduced residue that is glycosylatable, such as Threonine (T) or Asparagine (N) residues. More specifically, the conjugatable residue may optionally be an introduced T at position 172 or position 173, or may be an introduced N residue at position 170 or position 174, relative to SEQ ID NO: 1.
• the FGF21 domain comprises the amino acid sequence selected from the group consisting of SEQ ID NOs: 16-19.
• the functional moiety comprises a synthetic chemical moiety.
• the conjugatable residue may optionally comprise an introduced cysteine (C)
• the synthetic chemical moiety may comprise a structure of *-X-Y-Z.
• X, Y, and Z are interconnected via bonds, and the *end of X is connected to the conjugatable residue on the polypeptide.
• X can be Y can be and Z can be wherein position a is linked to position a’ , position b is linked to position b’ .
• R1 can be hydrogen or -COOH; d can be 1, 2, or 3; a can be 1, 2 or 3; b can be 1, 2 or 3; c can be 1 or 2; d can be 1, 2, or 3; and e can be 1, 2, or 3.
• the synthetic chemical moiety has the below structure: also referred herein as Ac-2XADO-EDA-CO-CH 2 -*.
• the introduced cysteine can be at a position selected from a group consisting of 169, 170, 171, 172, 173, 174, 180 and 181 relative to SEQ ID NO: 1.
• the conjugatable residue is at the position 171 or 174 relative to SEQ ID NO: 1.
• the FGF21 domain comprises the amino acid sequence of SEQ ID NOs: 6-13 and 92.
• the FGF21 domain comprises the amino acid sequence of SEQ ID NO: 8, with the introduced cysteine at position 171, conjugated at the introduced cysteine residue with the synthetic chemical moiety having the below structure: (also referred herein as Ac-2XADO-EDA-CO-CH 2 *) .
• the FGF21 domain can comprise the amino acid sequence of SEQ ID NO: 92, with the introduced cysteine at position 174, conjugated at the introduced cysteine residue with the synthetic chemical moiety having the below structure: (also referred herein as Ac-2XADO-EDA-CO-CH 2 *) .
• polypeptide may alternatively include:
• the FGF21 domain comprises the amino acid sequence of SEQ ID NO: 6, and the introduced cysteine is at position 169;
• the FGF21 domain comprises the amino acid sequence of SEQ ID NO: 7, and the introduced cysteine is at position 170;
• the FGF21 domain comprises the amino acid sequence of SEQ ID NO: 8, and the introduced cysteine is at position 171;
• the FGF21 domain comprises the amino acid sequence of SEQ ID NO: 9, and the introduced cysteine is at position 172;
• the FGF21 domain comprises the amino acid sequence of SEQ ID NO: 10, and the introduced cysteine is at position 173;
• the FGF21 domain comprises the amino acid sequence of SEQ ID NO: 11, and the introduced cysteine is at position 174;
• the FGF21 domain comprises the amino acid sequence of SEQ ID NO: 12, and the introduced cysteine is at position 180; or
• the FGF21 domain comprises the amino acid sequence of SEQ ID NO: 13, and the introduced cysteine is at position 181,
• the FGF21 domain comprises the amino acid sequence of SEQ ID NO: 92, and the introduced cysteine is at position 174,
• the FGF21 domain further comprises a substitution of P171G relative to SEQ ID NO: 1.
• the FGF21 domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 14 and 92.
• the nanobody domain may optionally comprise a VHH domain, optionally a VHH domain capable of binding to human serum albumin (HSA) .
• HSA human serum albumin
• VHH domain can be humanized.
• the VHH domain may comprise a complementarity determining region 1 (CDR1) , a complementarity determining region 2 (CDR2) , and a complementarity determining region 3 (CDR3) .
• the CDR1 may comprise the sequence of SEQ ID NO: 20 or a variant thereof having up to 3, 2, or 1 amino acid mutation
• the CDR2 may comprise the sequence of SEQ ID NO: 21 or a variant thereof having up to 3, 2, or 1 amino acid mutation
• the CDR3 may comprise the sequence of SEQ ID NO: 22 or a variant thereof having up to 3, 2, or 1 amino acid mutation
• the VHH domain substantially retains the binding specificity to serum albumin, optionally to human serum albumin.
• the VHH domain comprises a complementarity determining region 1 (CDR1) comprising the sequence of SEQ ID NO: 20, a CDR2 comprising the sequence of SEQ ID NO: 21, and a CDR3 comprising the sequence of SEQ ID NO: 22.
• CDR1 complementarity determining region 1
• CDR2 comprising the sequence of SEQ ID NO: 20
• CDR3 comprising the sequence of SEQ ID NO: 22.
• the VHH domain comprises an amino acid sequence of SEQ ID NO: 23, or a variant thereof having at least 70% (e.g. at least 75%, 80%, 85%, 90%, 95%, 99%identity to SEQ ID NO: 23, and the variant substantially retains binding specificity and/or affinity to serum albumin.
• the nanobody domain may optionally further comprise an N-terminal extension attached to a N-terminus of the VHH domain, the N-terminal extension may optionally comprise amino acid residues of SG, AG, S or A.
• the nanobody domain comprises an amino acid sequence selected from SEQ ID NOs: 24-27.
• the first linker can have a length of at least four amino acid residues.
• the first linker may comprise no acidic amino acid residue, and more specifically, the first linker may not comprise any of D residue or E residue.
• the first linker may comprise one or more units of a first repeating sequence, and according to some embodiments, the first repeating sequence may consist of no more than 4 or 6 types of amino acid residues selected from the group consisting of: G, Q, A, P, T and S.
• the first repeating sequence may comprise or consist of an amino acid sequences selected from a group consisting of G f S g (herein each of f and g is independently an integer selected from 1 to 5) , SEQ ID NO: 35 (GAQP) , SEQ ID NO: 36 (GQAP) , SEQ ID NO: 37 (GPAQ) , SEQ ID NO: 38 (GPQA) , SEQ ID NO: 39 (GSQP) , SEQ ID NO: 40 (GASP) , SEQ ID NO: 41 (GPAS) , SEQ ID NO: 42 (GPSA) , SEQ ID NO: 43 (GGGS) , SEQ ID NO: 44 (GSGS) , SEQ ID NO: 45 (GGGGS) , SEQ ID NO: 46 (GSAPGSPAGSPTGSAPGSPA) , and SEQ ID NO: 110 (GS) .
• G f S g amino acid sequences selected from a group consisting of G f S g (herein each of f and
• the first linker comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 35 (GAQP) , SEQ ID NO: 49 ( (GAQP) 2 ) , SEQ ID NO: 50 ( (GAQP) 5 ) , SEQ ID NO: 51 ( (GAQP) 10 ) , and SEQ ID NO: 48 (GGGGSGGGS) .
• the polypeptide may further comprise, over an N-terminus of the first fragment, a third fragment that comprises another functional domain.
• the first fragment and the third fragment are connected via a second linker.
• the another functional domain of the third fragment may optionally comprise a biologically active protein, or a fragment thereof, of one selected from the group consisting of glucagon-like peptide-1 (GLP-1) , insulin, leptin, glucagon, gastrin, gastric inhibitory polypeptide (GIP) , amylin, calcitonin, cholecystokinin, peptide YY, neuropeptide Y, bone morphogenetic protein-6 (BMP-6) , bone morphogenetic protein-9 (BMP-9) , oxyntomodulin, oxytocin, glucagon-like peptide-2 (GLP-2) , irisin, fibronectin type III domain containing protein 5 (FNDC5) , apelin, adiponectin, Clq and tumor necrosis factor related protein (CTRP family) , resistin, visfatin, omentin, retinol binding protein-4 (RBP-4) , glicentin, GLP
• the another functional domain of the third fragment comprises a biologically active peptide, or a fragment thereof, of GLP-1, and may comprise an amino acid sequence having at least 70%sequence identity to, while retaining substantial biological activity of, SEQ ID NO: 28.
• the another functional domain may comprise one or more mutations at positions 8, 22, 26, 34, and 36, or any combination thereof, relative to SEQ ID NO: 28.
• the one or more mutations in the another functional domain of the third fragment of the polypeptide may comprise A8G, G22E, K26R, K34R, and R36G, or any combination thereof. According to some embodiments of the polypeptide, the one or more mutations in the another functional domain of the third fragment of the polypeptide may comprise A8G, G22E, and R36G, or any combination thereof.
• the another functional domain of the third fragment comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 29, and SEQ ID NOs: 31-34.
• polypeptide comprising a first fragment which connects with an N-terminus of the first fragment via a second linker.
• the second linker may have a length of at least four amino acid residues. In certain embodiments, the second linker may have a length of at least 8, 12, 16, or 20 amino acid residues.
• the second linker may comprise one or more units of a second repeating sequence, and the second repeating sequence may consist of no more than 4 or 6 types of amino acid residues selected from the group consisting of: G, Q, A, E, P, T and S.
• the second repeating sequence may comprise or consist of an amino acid sequence that is selected from a group consisting of G h S i (herein, each of h and i is independently an integer selected from 1 to 5) , SEQ ID NO: 35 (GAQP) , SEQ ID NO: 55 (GQEP) , SEQ ID NO: 56 (GEQP) , SEQ ID NO: 57 (GPQE) , SEQ ID NO: 58 (GPEQ) , SEQ ID NO: 59 (GSEP) , SEQ ID NO: 60 (GESP) , SEQ ID NO: 61 (GPSE) , SEQ ID NO: 62 (GPES) , SEQ ID NO: 36 (GQAP) , SEQ ID NO: 37 (GPAQ) , SEQ ID NO: 38 (GPQA) , SEQ ID NO: 39 (GSQP) , SEQ ID NO: 40 (GASP) , SEQ ID NO: 41 (GPAS) ,
• the second repeating sequence may have an amino acid sequence set forth in SEQ ID NO: 35 (GAQP) , and a number of the one or more units may be an integer between 1 and 15.
• the second linker comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 49 ( (GAQP) 2 ) , SEQ ID NO: 50 ( (GAQP) 5 ) , SEQ ID NO: 51 ( (GAQP) 10 ) , and SEQ ID NO: 52 ( (GAQP) 14 ) , and SEQ ID NO: 47 ( (GGGGS) 4) .
• the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 63-68, 93, 99, 100, 101 and 107.
• the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 63, 68, and 100, and is conjugated at the introduced cysteine residue at position 171 relative to SEQ ID NO: 1 with the synthetic chemical moiety provided herein.
• the polypeptide comprises an amino acid sequence of SEQ ID NO: 93, and is conjugated at the introduced cysteine residue at position 174 relative to SEQ ID NO: 1 with the synthetic chemical moiety provided herein.
• the synthetic chemical moiety has the below structure: (also referred herein as Ac-2XADO-EDA-CO-CH 2 *) .
• the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 64-67, 93 and 107, and is not conjugated.
• the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 70, 74, 75, 79-83, 85, 94-98, and 108-109.
• the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 74, 81-83, 85, and 96-97, and is conjugated at the introduced cysteine residue at position 171 relative to SEQ ID NO: 1 with the synthetic chemical moiety provided herein.
• the polypeptide comprises an amino acid sequence of SEQ ID NO: 94, and is conjugated at the introduced cysteine residue at position 174 relative to SEQ ID NO: 1 with the synthetic chemical moiety.
• the synthetic chemical moiety has the below structure: (also referred herein as Ac-2XADO-EDA-CO-CH 2 *) .
• the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 70, 75, 79-80, 85, 94, 95, 98 and 108-109, and is not conjugated.
• a polynucleotide encoding the polypeptide portion (or fragment thereof) of the polypeptide can be designed, which can be operably arranged in a vector.
• the vector can be transferred in a host cell, such as a prokaryotic cell or eukaryotic cell, and the host cell can be cultured under a condition that allows expression of the polynucleotide, thereby obtaining the polypeptide.
• the present disclosure provides a process for producing the polypeptide as described above is further provided.
• the process comprises the steps of:
• S100 culturing the host cell as described above under a condition that allows expression of the polynucleotide as defined above or a precursor thereof further comprising a removable tag;
• S200 collecting and purifying the polypeptide or the precursor thereof from the host cell.
• the polypeptide is conjugated with a functional moiety at a conjugatable residue in the second fragment, and the process further comprises the following step after step S200 of collecting and purifying the polypeptide from the host cell:
• the host cell is E. coli
• the vector comprises an E. coli-compatible vector
• the polypeptide encoded by the polynucleotide in the vector is codon optimized for E. coli expression.
• step S200 of collecting and purifying the polypeptide from the host cell may comprise the following sub-steps:
• the process further comprises conjugating the purified polypeptide with the functional moiety.
• the functional moiety to be conjugated to the polypeptide is (Ac-2XADO-EDA-CO-CH 2 -*) .
• the pharmaceutical composition provided herein is a liquid formulation.
• the pharmaceutically acceptable excipient comprises a buffer and an isotonic agent.
• the buffer is selected from the group consisting of a phosphate buffer, citrate buffer, acetate buffer, histidine buffer, glycine buffer, carbonate buffer, borate buffer, glutamate buffer, glycylglycine buffer, lysine buffer, and arginine buffer.
• the pharmaceutical composition has a pH of about 6.0 to about 8.3 (e.g. about 6.5 to about 8.2, about 6.5 to about 8.0, about 6.5 to about 7.8, about 6.5 to about 7.4, about 6.5 to about 7.0, about 7.0 to about 7.8, about 7.0 to about 7.4, about 7.4 to about 8.2, about 7.4 to about 8.0, or about 7.4 to about 7.8) .
• the buffer is a phosphate buffer.
• the pharmaceutical composition has a pH of about 6.5 to about 8.0, about 6.5 to about 7.4, about 7.0 to about 7.8, about 7.0 to about 7.4, or about 7.4 to about 7.8.
• the phosphate buffer is present in a concentration of 0.01 -50mM of the pharmaceutical composition.
• the phosphate buffer is present in a concentration of 5 -20mM of the pharmaceutical composition, optionally about 8mM.
• the phosphate buffer is selected from the group consisting of sodium dihydrogen phosphate, disodiumhydrogen phosphate, sodium phosphate, or a hydrate thereof.
• the hydrate is dodecahydrate or dihydrate.
• the phosphate buffer is disodiumhydrogen phosphate dodecahydrate or disodium phosphate dihydrate.
• the disodiumhydrogen phosphate dodecahydrate is present in a concentration of about 0.01-17 mg/mL (e.g. about 0.01-15 mg/mL, about 0.05 -12 mg/mL, about 0.05 -10 mg/mL, about 0.1 -10 mg/mL, about 1 -5 mg/mL, or about 1 -3 mg/mL) .
• the disodiumhydrogen phosphate dodecahydrate is present in a concentration of about 2.87 mg/mL of the pharmaceutical composition.
• the disodiumhydrogen phosphate dihydrate is present in a concentration of about 0.01-8 mg/mL (e.g. about 0.01-5 mg/mL, about 0.05-4 mg/mL, about 0.05-3 mg/mL, about 0.1-3 mg/mL, or about 1-3 mg/mL) .
• the disodiumhydrogen phosphate dihydrate is present in a concentration of about 1.42 mg/mL.
• the buffer is a citrate buffer.
• the citrate buffer is present in a concentration of about 0.05 -20 mg/mL, or optionally 0.05 -10 mg/mL.
• the citrate buffer comprises a mixture of citric acid anhydrous and trisodium citrate (or a hydrate thereof, e.g. trisodium citrate dihydrate) .
• the pharmaceutical composition has a pH of about 6.0 to about 6.8, or optionally about 6.5.
• the citric acid anhydrous/trisodium citrate dihydrate is present in a concentration of about 0.05-10 mg/mL.
• the citrate buffer comprises a mixture of about 0.14 mg/mL citric acid anhydrous and about 2.74 mg/mL trisodium citrate dihydrate.
• the buffer is a histidine buffer.
• the pharmaceutical composition has a pH of about 6.0 to about 6.8, or optionally about 6.5.
• the histidine buffer is present in a concentration of about 0.5-10 mg/mL (e.g. about 1 -5 mg/mL, or about 1 -3 mg/ml) .
• the histidine buffer is present in a concentration of about 1.55 mg/mL.
• the isotonic agent is selected from the group consisting of sodium chloride, glycerol, sorbitol, sucrose, propylene glycol, mannitol, glycine, lactose monohydrate, arginine, myoinositol and dimethylsulfon. In certain embodiments, the isotonic agent is not sodium chloride.
• the isotonic agent is glycerol.
• the glycerol is about 5 -30 mg/mL.
• the glycerol is about 20 mg/mL.
• the isotonic agent is propylene glycol.
• the propylene glycol is about 1 mg/mL to about 50 mg/mL (e.g. about 5 mg/mL to about 25 mg/mL, about 8 mg/mL to about 16 mg/mL) .
• the propylene glycol is about 14 mg/mL.
• the isotonic agent is sodium chloride.
• the sodium chloride is about 5-15 mg/mL.
• the sodium chloride is about 8.25 mg/mL.
• the isotonic agent is mannitol.
• the mannitol is about 20 mg/mL to about 100 mg/mL (e.g. about 25 mg/mL to about 70 mg/mL, about 30 mg/mL to about 60 mg/mL, about 35 mg/mL to about 55 mg/mL) .
• the mannitol is about 45 mg/mL.
• the isotonic agent is sorbitol.
• the sorbitol is about 20 mg/mL to about 100 mg/mL (e.g. about 40 mg/mL to about 50 mg/mL) .
• the isotonic agent is sucrose.
• the sucrose is about 5 mg/mL to about 150 mg/mL (e.g. about 45 mg/mL to about 100 mg/mL) .
• the pharmaceutically acceptable excipient further comprises a nonionic surfactant.
• the nonionic surfactant is polysorbate 80.
• the polysorbate 80 is about 0.05 mg/mL to about 5 mg/mL (e.g. about 0.1 mg/mL to about 0.5 mg/mL) .
• the pharmaceutical excipient further comprises a preservative, a chelating agent, and/or a stabilizer.
• the pharmaceutical composition has about 1-100 mg/ml of the polypeptide provided herein (e.g. about 1-90 mg/mL, about 1-80 mg/mL, about 1-70 mg/mL, about 1-60 mg/mL, about 1-50 mg/mL, about 1-40 mg/mL, about 1-30 mg/mL, about 1-20 mg/mL, about 1-10 mg/mL) .
• the polypeptide comprises the amino acid sequence of SEQ ID NO: 85, and is conjugated at the introduced cysteine residue at position 171 relative to SEQ ID NO: 1 with the synthetic chemical moiety provided herein.
• the synthetic chemical moiety has the below structure: (also referred herein as Ac-2XADO-EDA-CO-CH 2 *) .
• the polypeptide comprises the amino acid sequence of SEQ ID NO: 96, and is conjugated at the introduced cysteine residue at position 171 relative to SEQ ID NO: 1 with the synthetic chemical moiety.
• the synthetic chemical moiety has the below structure: (also referred herein as Ac-2XADO-EDA-CO-CH 2 *) .
• compositions which comprise:
• polypeptide comprises the amino acid sequence of SEQ ID NO: 85 or SEQ ID NO: 96;
• a buffer selected from the group consisting of phosphate buffer, citrate buffer, acetate buffer, histidine buffer, glycine buffer, carbonate buffer, borate buffer, glutamate buffer, glycylglycine buffer, lysine buffer, and arginine buffer;
• an isotonic agent selected from the group consisting of sodium chloride, glycerol, sorbitol, sucrose, propylene glycol, mannitol, glycine, lactose monohydrate, arginine, myoinositol and dimethylsulfon; and
• compositions comprise:
• polypeptide comprises the amino acid sequence of SEQ ID NO: 85 or SEQ ID NO: 96;
• a buffer selected from the group consisting of phosphate buffer, citrate buffer, and histidine buffer;
• an isotonic agent selected from the group consisting of sodium chloride, glycerol, sorbitol, sucrose, propylene glycol, and mannitol;
• compositions comprise:
• polypeptide comprises the amino acid sequence of SEQ ID NO: 85 or SEQ ID NO: 96;
• an isotonic agent selected from the group consisting of sodium chloride, glycerol, sorbitol, sucrose, propylene glycol, and mannitol;
• compositions comprise:
• polypeptide comprises the amino acid sequence of SEQ ID NO: 85 or SEQ ID NO: 96;
• a buffer selected from the group consisting of phosphate buffer, citrate buffer, and histidine buffer;
• an isotonic agent selected from the group consisting of 5-15mg/ml sodium chloride, 5-30 mg/mL glycerol, 20 -100 mg/mL sorbitol, 5 -150 mg/mL sucrose, 1 -50 mg/mL propylene glycol, and 20 -100 mg/mL mannitol; and
• compositions comprise:
• polypeptide comprises the amino acid sequence of SEQ ID NO: 85 or SEQ ID NO: 96;
• an isotonic agent selected from the group consisting of 5 -15mg/ml sodium chloride, 5 -30 mg/mL glycerol, 20 -100 mg/mL sorbitol, 5 -150 mg/mL sucrose, 1 -50 mg/mL propylene glycol, and 20 -100 mg/mL mannitol; and
• compositions comprise:
• polypeptide comprises the amino acid sequence of SEQ ID NO: 85 or SEQ ID NO: 96;
• the pharmaceutical compositions further comprise a nonionic surfactant which is about 0.1-0.5 mg/mL of polysorbate 80.
• the present disclosure provides a method of preventing or treating a metabolic disorder in a subject in need thereof, comprising administering a therapeutically effective amount of the pharmaceutical composition provided herein.
• the metabolic disorder is diabetes, obesity, non-alcoholic steatohepatitis (NASH) , cardiovascular like dyslipidaemia, arteriosclerosis, alcoholic steatohepatitis (ASH) , diabeticnephropathy, gestational diabetes, metabolic syndrome such as metabolic syndrome X, nonalcoholic fatty liver disease (NAFLD) , end-stage liver disease, hepatic steatosis (fatty liver) , liver cirrhosis, primary biliary cirrhosis (PBC) or severe hypertriglyceridemia (SHTG) .
• NASH non-alcoholic steatohepatitis
• NASH nonalcoholic steatohepatitis
• NASH nonalcoholic steatohepatitis
• PBC primary biliary cirrhosis
• SHTG severe hypertriglyceridemia
• the diabetes can be any form of diabetes, including without limitation, hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, non-insulin dependent diabetes, MODY (maturity onset diabetes of the young) , gestational diabetes, and elevated level of HbA1 C.
• the present disclosure provides a method of managing body weight in a subject in need thereof, comprising administering a therapeutically effective amount of the pharmaceutical composition provided herein, thereby managing body weight of the subject.
• the present disclosure provides a method of reducing food intake in a subject in need thereof, comprising administering a therapeutically effective amount of the pharmaceutical composition provided herein, thereby reducing food intake of the subject.
• the present disclosure provides a method of reducing body weight in a subject in need thereof, comprising administering a therapeutically effective amount of the pharmaceutical composition provided herein, thereby reducing body weight of the subject.
• the subject can be human.
• the subject has a fasting blood glucose level of 125 mg/dL or greater.
• the subject has a body mass index (BMI) of at least or higher than 25.
• BMI body mass index
• the pharmaceutical composition is administered at a dosing regimen that is no more frequently than once daily, once every 3 days, once weekly, or once every two weeks.
• the pharmaceutical composition is administered twice-weekly, once-weekly, or once bi-weekly.
• the dosing regimen has a dosing interval ranging from about once every 3 days to about once bi-weekly.
• the pharmaceutical composition is administered via parenteral administration.
• the pharmaceutical composition is administered subcutaneously, intravenously or intramuscularly.
• apolypeptide means one polypeptide or more than one polypeptides.
• any of the recited numerical values may be the upper limit or lower limit of a numerical range. It is to be further understood that the invention encompasses all such numerical ranges, i.e., a range having a combination of an upper numerical limit and a lower numerical limit, wherein the numerical value for each of the upper limit and the lower limit can be any numerical value recited herein. Ranges provided herein are understood to include all values within the range. For example, 1-10 is understood to include all of the values 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10, and fractional values as appropriate. Similarly, ranges delimited by “at least” are understood to include the lower value provided and all higher numbers.
• Figures 1A to 1B illustrate a polypeptide according to two different embodiments of the present disclosure.
• Figures 2A to 2B show in vitro FGF21 activity of the Molecules MLC#9, MLC#10, MLC#14, Control #6 and a reference compound YH-dual.
• Figures 3A and 3B show efficacy of Molecules Control#2, Control#6, MLC#9 and MLC#10 on body weight reduction (Figure 3A) and food intake suppression (Figure 3B) in DIO rats.
• Figures 4A-4L show efficacy of Molecules MLC#9, MLC#14, MLC#16 and MLC#17, and reference compounds semaglutide, YH-dual, and Tirzepatide on body weight reduction (Figure 4A) , glucose control ( Figure 4B) , reduction of triglyceride (Figure 4C) , LDL-C ( Figure 4D) , total cholesterol (Figure 4E) and ALT (Figure 4F) concentration in plasma and reduction of adipose weight (Figure 4G) and liver weight (Figure 4H) , along with the improvement of liver TG ( Figure 4I) , liver TC ( Figure 4J) and insulin sensitivity (Figure 4K) , and increase in adiponectin level (Figure 4L) in DIO mice.
• Figures 5A-5M show efficacy of Molecules MLC#14 and MLC#16 at different dosages, in comparison to reference compounds semaglutide, YH-dual, and Tirzepatide on body weight reduction (Figure 5A) , glucose control ( Figure 5B) , reduction of triglyceride (Figure 5C) , LDL-C ( Figure 5D) , total cholesterol (Figure 5E) , ALT ( Figure 5F) and AST (Figure 5G) concentration in plasma and reduction of adipose weight (Figure 5H) and liver weight (Figure 5I) along with the improvement of liver TG (Figure 5J) , liver TC ( Figure 5K) and insulin sensitivity (Figure 5L) , and increase in adiponectin level (Figure 5M) in DIO mice.
• Figure 5A glucose control
• Figure 5C reduction of triglyceride
• Figure 5D LDL-C
• Figure 5E total cholesterol
• ALT Figure 5F
• AST Figure 5G
• Figures 6A-6H show efficacy of Molecules MLC#16, MLC#19, MLC#6, and MLC#23 at different dosages, in comparison to reference compounds semaglutide, serum triglyceride (Figure 6C) , LDL-C ( Figure 6D) and total cholesterol (Figure 6E) concentration in plasma and reduction of adipose weight (Figure 6F) and liver weight ( Figure 6G) , along with the improvement of liver TG ( Figure 6H) .
• FIG. 7 shows all the sequences disclosed in the present disclosure.
• protein protein
• peptide and “polypeptide” are used interchangeably herein and refer a polymer of amino acid residues linked by covalent bonds such as peptide bonds.
• a protein or polypeptide as provided herein can comprise naturally occurring or non-natural amino acid residues, or both.
• Polypeptides, peptides and proteins provided herein can comprise any suitable length of amino acid residues, for example, from at least 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or more amino acid residues in length.
• amino acid refers to an organic compound containing amine (-NH 2 ) and carboxyl (-COOH) functional groups, along with a side chain specific to each amino acid.
• naturally occurring amino acid residue refers to an amino acid residue found in native proteins or peptides, all in their D and L stereoisomers if their structure allows such stereoisomeric forms.
• naturally occurring amino acid residues include, but not limited to, 20 standard amino acids, including, glycine (Gly or G) , alanine (Ala or A) , valine (Val or V) , leucine (Leu or L) , isoleucine (Ile or I) , serine (Ser or S) , cysteine (Cys or C) , threonine (Thr or T) , methionine (Met or M) , proline (Pro or P) , phenylalanine (Phe or F) , tyrosine (Tyr or Y) , tryptophan (Trp or W) , histidine (His or H) , lysine (Lys or K) ,
• amino acid analog is a compound that has the same basic chemical structure as a naturally occurring amino acid, i.e., a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium.
• R groups e.g., norleucine
• modified peptide backbones but will retain the same basic chemical structure as a naturally occurring amino acid.
• non-natural amino acid residue refers to any amino acid residues that are not found in nature, including without limitation, a modified amino acid residue, and/or an amino acid mimetic, which is not one of the known naturally occurring amino acids, yet functions in a manner similar to the naturally occurring amino acids.
• Modified amino acid or a mimetic can be generated by addition of a chemical entity such as a carbohydrate group, a phosphate group, a farnesyl group, an isofamesyl group, a fatty acid group, a linker for conjugation, functionalization, or other modification, etc.
• a non-natural amino acid can also refer to an amino acid manufactured by chemical synthesis.
• non-natural amino acids include, but not limited to, 2-Aminoisobutyric acid (Aib) , imidazole-4-acetate (IA) , imidazolepropionic acid (IPA) , a-aminobutyric acid (Abu) , tert-butylglycine (Tle) , b-alanine, 3-aminomethyl benzoic acid, anthranilic acid, des-amino-histidine (abbreviated DesaminoHis, alternative name imidazopropionic acid, abbreviated lmpr) , the beta analogues of amino acids such as ⁇ -alanine, 2-amino-histidine, ⁇ -hydroxy-histidine, homohistidine, N ⁇ -acetyl-histidine, ⁇ -fluoro-methyl-histidine, ⁇ -methyl-histidine, ⁇ , ⁇ -dimethyl-glutamic acid, m-CF3-phenylalan
• codons such as a four-base codon (e.g. AGGA, AGGU, CGGU, CGCU, CGAU, CCCU, CUCU, CUAU, and GGGU) , a five-base codon, a six-base codon, etc. can also be introduced into the expression systems for non-natural amino acids.
• Such expression vectors are then introduced into a host that can utilize a tRNA specific for the introduced stop codon or other codons and carried with the non-natural amino acid of choice.
• non- natural amino acid can be chemically synthesized and inserted into or attached to a polypeptide by chemical reaction such as acylation.
• fusion refers to combination of two or more amino acid sequences, for example by chemical bonding or recombinant means, into a single amino acid sequence which does not exist naturally.
• a fusion amino acid sequence may be produced by genetic recombination of two encoding polynucleotide sequences, and can be expressed by a method of introducing a construct containing the recombinant polynucleotides into a host cell.
• Percent (%) sequence identity is defined as the percentage of amino acid (or nucleic acid) residues in a candidate sequence that are identical to the amino acid (or nucleic acid) residues in a reference sequence, after aligning the sequences and, if necessary, introducing gaps, to achieve the maximum number of identical amino acids (or nucleic acids) .
• percent (%) sequence identity of an amino acid sequence (or nucleic acid sequence) can be calculated by dividing the number of amino acid residues (or bases) that are identical relative to the reference sequence to which it is being compared by the total number of the amino acid residues (or bases) in the candidate sequence or in the reference sequence, whichever is shorter. Conservative substitution of the amino acid residues is not considered as identical residues.
• Alignment for purposes of determining percent amino acid (or nucleic acid) sequence identity can be achieved, for example, using publicly available tools such as BLASTN, BLASTp (available on the website of U.S. National Center for Biotechnology Information (NCBI) , see also, Altschul S.F. et al, J. Mol. Biol., 215: 403-410 (1990) ; Stephen F. et al, Nucleic Acids Res., 25: 3389-3402 (1997) ) , ClustalW2 (available on the website of European Bioinformatics Institute, see also, Higgins D.G. et al, Methods in Enzymology, 266: 383-402 (1996) ; Larkin M.A.
• BLASTN Altschul S.F. et al, J. Mol. Biol., 215: 403-410 (1990) ; Stephen F. et al, Nucleic Acids Res., 25: 3389-3402 (1997)
• ClustalW2
• a “conservative substitution” with reference to amino acid sequence refers to replacing an amino acid residue with a different amino acid residue having a side chain with similar physiochemical properties.
• conservative substitutions can be made among amino acid residues with hydrophobic side chains (e.g. Met, Ala, Val, Leu, and Ile) , among residues with neutral hydrophilic side chains (e.g. Cys, Ser, Thr, Asn and Gln) , among residues with acidic side chains (e.g. Asp, Glu) , among amino acids with basic side chains (e.g. His, Lys, and Arg) , or among residues with aromatic side chains (e.g. Trp, Tyr, and Phe) .
• conservative substitution usually does not cause significant change in the protein conformational structure, and therefore could retain the biological activity of a protein.
• substantial biological activity means exhibiting at least part of (for example, no less than about 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%) or all of the biological activity of the parent molecule.
• a functional form of a parent polypeptide may include both naturally-occurring variant forms and non-naturally occurring forms such as those obtained by recombinant methods or chemical synthesis.
• the functional forms may contain non-natural amino acid residues.
• variant refers to a polypeptide having at least 70%sequence identity to the parent polypeptide and retain at least partial function of the parent polypeptide.
• a variant may differ from the parent peptide by one or more amino acid residues.
• a variant may have substitutions, additions, deletions, insertions, or truncations of one more amino acid residue of the parent polypeptide.
• fragment refers to partial sequence of the parent polypeptide of any length. A fragment can still retain at least partial function of the parent polypeptide.
• derivative refers to a chemically modified polypeptide or fusion polypeptide, in which one or more well-defined number of substituent groups have been covalently attached to one or more specific amino acid residues of the polypeptide or fusion polypeptide.
• exemplary chemical modification can be, e.g. alkylation, acylation, esterification, amidation, phosphorylation, glycosylation, labeling, methylation of one or more amino acids, or conjugation with one or more moieties.
• amino acid mimetics refers to molecular structures that serve as substitutes for amino acids, peptides, polypeptides, or fusion polypeptide.
• amino acid mimetics can be synthetic structures (either known or yet unknown) , which may or may not be an amino acid, but retain the functional features of the parent amino acids while the structure of the amino acid mimetic is different from the structure of the parent amino acid. Examples include a methacryloyl or acryloyl derivative of an amide, ⁇ -, ⁇ -, ⁇ -imino acids (such as piperidine-4-carboxylic acid) and the like.
• Treating” or “treatment” of a condition as used herein includes preventing or alleviating a condition, slowing the onset or rate of development of a condition, reducing the risk of developing a condition, preventing or delaying the development of symptoms associated with a condition, reducing or ending symptoms associated with a condition, generating a complete or partial regression of a condition, curing a condition, or some combination thereof.
• vector refers to a vehicle into which a polynucleotide encoding a protein may be operably inserted so as to bring about the expression of that protein.
• a vector may be used to transform, transduce, or transfect a host cell so as to bring about expression of the genetic element it carries within the host cell.
• vectors include plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosome (YAC) , bacterial artificial chromosome (BAC) , or P1-derived artificial chromosome (PAC) , bacteriophages such as lambda phage or M13 phage, and animal viruses.
• a vector may contain a variety of elements for controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selectable elements, and reporter genes. In addition, the vector may contain an origin of replication.
• a vector may also include materials to aid in its entry into the cell, including but not limited to a viral particle, a liposome, or a protein coating.
• a vector can be an expression vector or a cloning vector.
• the present disclosure provides vectors (e.g., expression vectors) containing the nucleic acid sequence provided herein encoding the fusion polypeptide, at least one promoter (e.g., SV40, CMV, EF-1 ⁇ ) operably linked to the nucleic acid sequence, and at least one selection marker.
• promoter e.g., SV40, CMV, EF-1 ⁇
• vectors include, but are not limited to, retrovirus (including lentivirus) , adenovirus, adeno-associated virus, herpesvirus (e.g., herpes simplex virus) , poxvirus, baculovirus, papillomavirus, papovavirus (e.g., SV40) , lambda phage, and M13 phage, plasmid pcDNA3.3, pMD18-T, pOptivec, pCMV, pEGFP, pIRES, pQD-Hyg-GSeu, pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX, pGEX, pCI, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBA
• RTM. pCDM8, pCDNA1.1/amp, pcDNA3.1, pRc/RSV, PCR 2.1, pEF-1, pFB, pSG5, pXT1, pCDEF3, pSVSPORT, pEF-Bos etc.
• host cell refers to a cell into which an exogenous polynucleotide and/or a vector has been introduced.
• pharmaceutically acceptable indicates that the designated carrier, vehicle, diluent, excipient (s) , and/or salt is generally chemically and/or physically compatible with the other ingredients comprising the formulation, and physiologically compatible with the recipient thereof.
• subject or “individual” or “animal” or “patient” as used herein refers to human or non-human animal, including a mammal or a primate, in need of diagnosis, prognosis, amelioration, prevention and/or treatment of a disease or disorder.
• Mammalian subjects include humans, domestic animals, farm animals, and zoo, sports, or pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, swine, cows, bears, and so on.
• polypeptides disclosed herein comprises the FGF21 domain.
• each of the first and second embodiments of the polypeptide disclosed herein and illustrated in Figures. 1A and 1B comprises the FGF21 domain.
• Figures. 1A and 1B illustrate two major embodiments of the polypeptide provided in this present disclosure.
• the first embodiment of the polypeptide 001A comprises, in an N-terminus to C-terminus direction, a first fragment 100 and a second fragment 200, which are connected via a first linker 10.
• the first fragment 100 substantially comprises a nanobody domain that is capable of binding to serum albumin, such as human serum albumin (HSA) .
• the second fragment 200 comprises a biologically active FGF21 domain, which substantially comprises a functional form of FGF21, and according to certain specific embodiments of the disclosure, comprises an amino acid sequence having at least 90%sequence identity to, while substantially retaining biological activity of, SEQ ID NO: 1.
• the polypeptide may further comprise yet one additional functional domain besides the nanobody domain in the first fragment 100 and the FGF21 domain in the second fragment 200.
• this second embodiment of the polypeptide 001B further includes a third fragment 300 over an N-terminus of the first fragment 100.
• the third fragment 300 comprises an additional functional domain that can exert a biological activity that is additive to, or in synergy with, the FGF21 domain in the second fragment 200.
• the first fragment 100 and the third fragment 300 are connected via a second linker 20.
• this additional functional domain in the third fragment 300 is a biologically active glucagon-like peptide-1 (GLP-1) domain, which substantially comprises a functional form of GLP-1.
• GLP-1 biologically active glucagon-like peptide-1
• this additional functional domain in the third fragment 300 is a biologically active glucagon-like peptide-1 (GLP-1) domain, which substantially comprises a functional form of GLP-1.
• GLP-1 biologically active glucagon-like peptide-1
• FGF21 refers to, and is short for, “Fibroblast Growth Factor 21” , and is intended to broadly encompasses all functional forms including the native human FGF21, as well as functional variants, fragments, fusions, derivatives or mimetics thereof.
• the native human FGF21 consists of 209 amino acid residues (Uniprot datebase with access no. Q9NSA1) , with amino acid residues 1-28 being a signal polypeptide and amino acid residues 29-209 being a mature polypeptide of 181 residues.
• biologically active FGF21 domain refers to a functional form of FGF21, which can be the mature polypeptide, or functional variants, fragments, fusions, derivatives or mimetics thereof.
• the mature polypeptide of human FGF21 is included herein as SEQ ID NO: 1.
• the numbering of the residues in FGF21 is referred to the sequence of SEQ ID NO: 1, which begins with His at position 1 and Ser at position 181.
• a functional form of the mature polypeptide of FGF21 is capable of activating the FGF21 receptor at a level comparable to, or no less than about 20% (or no less than 30%, 40%, 50%, 60%, 70%, 80%, 90%) of, that of the mature polypeptide of the native human FGF21.
• Activation of the FGF21 receptor can lead to biological activities such as, for example, the ability to activate glucose uptake in adipocytes, the ability to lower blood glucose and triglyceride levels, or the ability to lower bodyweight (Tezze C et al, Front Physiol. 2019, 10: 419) .
• the FGF21 domain provided herein comprises an amino acid sequence having at least 90%sequence identity to, while retaining substantial biological activity of, SEQ ID NO: 1.
• the FGF21 domain comprises no more than 12, 11, 10, 9, 8, 7 amino acid mutations (e.g. substitutions, insertions, or deletions) relative to SEQ ID NO: 1 while retaining substantial biological activity of SEQ ID NO: 1.
• the FGF21 domain comprises the amino acid sequence of SEQ ID NOs: 2-14, 16-19, 89-92, and 102-105.
• the FGF21 comprises one or more mutations.
• the one or more mutations comprise a conservative substitution.
• the one or more mutations is at a position selected from positions consisting of 121, 168, 171 and 180 relative to SEQ ID NO: 1.
• the one or more mutations in the FGF21 domain are selected from N121Q, M168L, P171G and A180E, or any combination thereof.
• the FGF21 domain in the polypeptide disclosed herein may optionally contain one of the four mutations, two of the four mutations, three of the four mutations or four of the mutations.
• the FGF21 domain comprises of a combination of mutations relative to SEQ ID NO: 1 selected from the group consisting of:
• the FGF21 domain may comprise an amino acid sequence set forth in SEQ ID NO: 2 (N121Q) , SEQ ID NO: 3 (M168L) , SEQ ID NO: 4 (A180E) , SEQ ID NO: 5 (N121Q, M168L, and A180E) , SEQ ID NO: 89 (N121Q and M168L) , SEQ ID NO: 90 (N121Q and A180E) , SEQ ID NO: 91 (M168L and A180E) , or SEQ ID NO: 14 (N121Q, M168L, P171G and A180E) .
• the FGF21 domain in the polypeptide further comprises a conjugatable residue or up to one conjugatable residue, to which the functional moiety is conjugated.
• conjugation residue refers to an amino acid residue at a particular position within the polypeptide, which not only has a functional group capable of being chemically or enzymatically conjugated to a chemical moiety, but its position in the polypeptide allows it to be prone to such conjugation in a conjugating reaction for that particular functional group.
• Conjugation refers to a reaction that joins two molecules together to form one physical entity. For example, a covalent bond linking the two molecules can be formed in conjugation.
• a conjugatable residue can be a natural amino acid residue, a non-natural amino acid residue, modified amino acid residue or an amino acid mimetic. Examples of conjugatable residues include, without limitation, lysine, cysteine, or a non-natural amino acid residue.
• a residue is conjugatable residue would depend on the given conjugating reaction, and/or the functional group to be conjugated.
• the function group to be conjugated is a thiol group and/or the conjugation reaction is specific or selective for a thiol group, then lysine (i.e. having no functional thiol group in its side chain) would not be the conjugatable residue regardless of its position, whereas an unpaired cysteine residue that does not form a disulfide bond (whether intrachain bond or interchain bond) , could be a conjugatable residue.
• the polypeptide comprises a conjugatable residue for a conjugation reaction specific or selective for thiol group (for example, a maleimide reaction, or a reaction for disulfide bond formation) .
• conjugation reaction specific or selective for thiol group for example, a maleimide reaction, or a reaction for disulfide bond formation.
• conjugatable when referred to an amino acid residue is intended to mean that, the residue at a particular position in the polypeptide is sufficiently accessible for conjugation.
• a cysteine residue as part of a disulfide bridge is not a conjugatable residue in the present disclosure.
• the conjugatable residue is cysteine residue, preferably a free cysteine residue which is not part of a disulfide bridge.
• the conjugatable residue CR in the FGF21 domain of the polypeptide provided herein can be at a position within a C-terminal fragment spanning of the FGF21 domain, e.g. from position 169 to position 181 relative to SEQ ID NO: 1. Further optionally, the conjugatable residue can be at a position selected from the group consisting of positions 169, 170, 171, 172, 173, 174, 180 and 181 relative to SEQ ID NO: 1. Without wishing to be bound by any theory, it is found by the present inventors that conjugation at C-terminal fragment of the FGF21 domain reduces the C-terminal degradation of the FGF21 domain.
• the FGF21 domain in the polypeptide has a combination of mutations of: 1) N121Q and M168L, 2) N121Q, M168L and P171G, 3) N121Q, M168L and A180E, or 4) N121Q, M168L, P171G and A180E and further comprises a conjugatable residue (e.g. a cysteine residue) at position 169, 170, 171, 172, 173, 174, or 180, 181 relative to SEQ ID NO: 1.
• a conjugatable residue e.g. a cysteine residue
• the FGF21 domain in the polypeptide has a combination of mutations of N121Q and M168L, or a combination of mutations of N121Q, M168L and P171G, and further comprises a conjugatable residue (e.g. a cysteine residue) at position 180 relative to SEQ ID NO: 1.
• a conjugatable residue e.g. a cysteine residue
• the FGF21 domain in the polypeptide has a combination of mutations of N121Q, P171G and M168L, or a combination of mutations of N121Q, M168L, P171G and A180E, and further comprises a conjugatable residue at a position relative to SEQ ID NO: 1, listed as follows: introduced cysteine residue (e.g. via substitution) at position 169, position 170, position 172, position 173, or position 174.
• the FGF21 domain in the polypeptide has an amino acid sequence comprising has an introduced cysteine residue at position 169 (e.g. SEQ ID NO: 6) , position 170 (e.g. SEQ ID NO: 7) , position 171 (e.g. SEQ ID NO: 8) , position 172 (e.g. SEQ ID NO: 9) , position 173 (e.g. SEQ ID NO: 10) , position 174 (e.g. SEQ ID NO: 11, or SEQ ID NO: 92) , position 180 (e.g. SEQ ID NO: 12) , or position 181 (e.g. SEQ ID NO: 13) .
• position 169 e.g. SEQ ID NO: 6
• position 170 e.g. SEQ ID NO: 7
• position 171 e.g. SEQ ID NO: 8
• position 172 e.g. SEQ ID NO: 9
• position 173 e.g. SEQ ID NO: 10
• position 174
• the FGF21 comprises the amino acid sequence of SEQ ID NOs: 2-5, 89-91, 14, and 102-105, except for one or more amino acid residue mutations, each at a position selected from positions 121, 168, 171, and 180 relative to SEQ ID NO: 1.
• the FGF21 domain in the polypeptide has an introduced G at position 171 (e.g. SEQ ID NO: 14) .
• introduction of 171G in the FGF21 domain could also be useful to reduce the C-terminal degradation of the FGF21 domain.
• the FGF21 domain in the polypeptide have a combination of mutations of N121Q, P171G and M168L, or a combination mutations of N121Q, M168L, P171G and A180E, and further comprises a conjugatable residue at a position relative to SEQ ID NO: 1, listed as follows: an introduced T at position 172, position 173, position 174, wherein the position is relative to SEQ ID NO: 1; or introduced N at position 170 or position 174, wherein the position is relative to SEQ ID NO: 1.
• the FGF21 domain in the polypeptide has an introduced T at position 172 (e.g. SEQ ID NO: 16) or at position 173 (e.g. SEQ ID NO: 17) ; or an introduced N at position 170 (e.g. SEQ ID NO: 18) or at position 174 (e.g. SEQ ID NO: 19) , relative to SEQ ID NO: 1.
• the FGF21 domain in the polypeptide has the amino acid sequence selected from the group consisting of SEQ ID NOs: 16-19.
• either or both of the polypeptide 001A and 001B can optionally be conjugated with a functional moiety 800 at a conjugatable residue CR (as illustrated by the “*”in both figures) in the second fragment 200 (i.e. the FGF21 domain) to thereby form a polypeptide conjugate.
• the polypeptide is not conjugated with the functional moiety, for example, when the polypeptide has an introduced G at position 171.
• conjugate refers to a compound as a result of two or more molecules joined together to form one physical entity.
• the polypeptide of the present disclosure can, according to certain embodiments, form a polypeptide conjugate, which is substantially a compound resulting from the polypeptide and a functional moiety being joined together.
• the molecules e.g. the functional moiety and the polypeptide
• the molecules may attach together by covalent bonds.
• the attachment may be permanent or reversible.
• certain cleavable or non-cleavable linkers may be included.
• the term “functional moiety” as used herein refers to a moiety that can functionally alter the biological, pharmacokinetic (PK) , pharmacodynamic (PD) properties (e.g. to enhance the biological activity, to increase the stability in vitro, to increase the half-life in vivo, or to enhance the binding to target receptor, etc. ) .
• the functional moiety that is conjugated to the FGF2 domain may optionally comprise a glycosyl moiety or a synthetic chemical moiety.
• the functional moiety 800 can comprise a synthetic chemical moiety
• the conjugatable residue CR can be an introduced residue that can be conjugated with the synthetic chemical moiety.
• the conjugatable residue CR can be an introduced cysteine residue, in any of the above mentioned positions 169, 170, 171, 172, 173, 174, 180 and 181 relative to SEQ ID NO: 1.
• the synthetic chemical moiety can optionally comprise a structure of *-X-Y-Z, wherein X, Y, and Z are interconnected via bonds, and the *end of X is connected to the conjugatable residue on the polypeptide.
• X can be Y can be or and Z can be wherein position a is linked to position a’, position b is linked to position b’ .
• R1 can be hydrogen or -COOH; d can be 1, 2, or 3; a can be 1, 2 or 3; b can be 1, 2 or 3; c can be 1 or 2; and d can be 1, 2, or 3, and e can be 1, 2, or 3.
• the synthetic chemical moiety can be Ac-2XADO-EDA-CO-CH 2 *, where the *end is connected to the conjugatable cysteine residue on the polypeptide, having a structure of:
• a synthetic chemical moiety such as Ac-2XADO-EDA-CO-CH 2 -*can be conjugated at the introduced cysteine residue (e.g. via substitution) to thereby obtain a polypeptide conjugate carrying the synthetic chemical moiety.
• the functional moiety 800 can optionally comprise a glycosyl moiety
• the conjugatable residue CR can be an introduced residue that is glycosylatable (i.e. can be glycosylated) , which can be an introduced T at position 172 or position 173, or an introduced N residue at position 170 or position 174, relative to SEQ ID NO: 1.
• a glycosyl moiety can be conjugated at the introduced cysteine residue to thereby obtain a polypeptide conjugate carrying the glycosyl moiety.
• FGF21 domain comprise an introduced G at position 171 relative to SEQ ID NO: 1, and is not conjugated with the functional moiety.
• FGF21 domain can comprise an amino acid sequence of SEQ ID NO: 14.
• Each of the first and second embodiments of the polypeptide disclosed herein and illustrated in Figures 1A and 1B comprises the nanobody domain.
• nanobody as used herein is considered to be exchangeable with “single-domain antibody” , which refers to an antibody fragment containing a single variable domain of a heavy chain or a single variable domain of a light chain.
• a nanobody contains three complementarity determining regions (CDRs) .
• the nanobody is capable of binding to a specific antigen (e.g., serum albumin) .
• serum albumin refers to an albumin (atype of globular protein) found in vertebrate blood. Serum albumin is produced by the liver, occurs dissolved in blood plasma and is the most abundant blood protein in mammals. Serum albumin typically has a half-life of around three weeks, which is mainly regulated by neonatal Fc receptor (FcRn) . FcRn protects serum albumin from intracellular degradation by binding it with high affinity and diverting it from a lysosomal pathway, returning it to the extracellular compartment.
• the serum albumin is selected from human serum albumin (HSA) , cynomolgus monkey serum albumin and mouse serum albumin.
• the serum albumin provided herein is HSA.
• antibody as used herein includes any immunoglobulin that binds to a specific antigen.
• Conventional antibody e.g., antibodies from human or mice
• the heavy chains are classified as ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , each heavy chain consists of a variable domain (VH domain) and a first, second, third, and optionally fourth constant region (CH1, CH2, CH3, CH4 respectively) ;
• the light chains are classified as ⁇ or ⁇ , while each light chain consists of a variable domain (VL domain) and a constant domain.
• the antibody has a “Y” shape, with the stem of the Y consisting of the second and third constant domain of two heavy chains bound together via disulfide bonding.
• Each arm of the Y includes the variable domain and first constant domain of a single heavy chain bound to the variable and constant domains of a single light chain.
• the variable domains of the light and heavy chains are responsible for antigen binding.
• the variable domains in both chains generally contain three hypervariable regions called the complementarity determining regions (CDRs, namely CDR1, CDR2, and CDR3 of light chain or heavy chain) .
• CDRs complementarity determining regions
• CDR boundaries for the antibodies and antigen-binding fragments disclosed herein may be defined or identified by the conventions of Kabat, IMGT, Chothia, or Al-Lazikani (Al-Lazikani, B., Chothia, C., Lesk, A.M., J. Mol. Biol., 273 (4) , 927 (1997) ; Chothia, C. et al., J Mol Biol. Dec 5; 186 (3) : 651-63 (1985) ; Chothia, C. and Lesk, A.M., J. Mol. Biol., 196, 901 (1987) ; Chothia, C. et al., Nature.
• the three CDRs are interposed between flanking stretches known as framework regions (FRs, namely FR1, FR2, FR3 and FR4 of light chain or heavy chain) , which are more highly conserved than the CDRs and form a scaffold to support the highly variable loops.
• FRs flanking stretches known as framework regions (FRs, namely FR1, FR2, FR3 and FR4 of light chain or heavy chain)
• FRs flanking stretches known as framework regions (FRs, namely FR1, FR2, FR3 and FR4 of light chain or heavy chain)
• FRs flanking stretches known as framework regions (FRs, namely FR1, FR2, FR3 and FR4 of light chain or heavy chain)
• FRs flanking stretches known as framework regions (FRs, namely FR1, FR2, FR3 and FR4 of light chain or heavy chain)
• FRs flanking stretches known as framework regions (FRs, namely FR1, FR2, FR3 and
• the single variable domain can be derived from the variable domain of a camelid antibody (VHH domain) , or the variable domain of cartilaginous fish antibody (VNAR domain) . Both camelid antibodies and cartilaginous fish antibodies naturally lack light chains and consist of a pair of heavy chains.
• the single variable domain can be derived from the variable domain of a conventional antibody (e.g., from humans or mice) heavy chain (VH domain) or the variable domain of a common antibody light chain (VL domain) . It is contemplated that a single domain antibody is fairly small in size, for example, has a molecular weight of no more than 25 kD, no more than 20 kD, no more than 15 kD, or no more than 10 kD.
• a single domain antibody can be obtained, for example (1) by isolating the VHH domain or VNAR domain of a naturally occurring heavy chain antibody; (2) by expression of a nucleotide sequence encoding a naturally occurring VHH domain or VNAR domain; (3) by “humanization” (as described below) of a naturally occurring VHH domain or VNAR domain or by expression of a nucleic acid encoding a such humanized VHH domain or VNAR domain; (4) by “camelization” of a naturally occurring VH domain from any animal species, in particular a species of mammal, such as from a human being, or by expression of a nucleic acid encoding such a camelized VH domain; 5) using synthetic or semi-synthetic techniques for preparing proteins, polypeptides or other amino
• the single domain antibody described herein comprises a VHH domain derived from antibodies raised in Camelidae species, for example in camel, dromedary, alpaca and guanaco.
• a single domain antibody comprising a VHH domain are highly soluble and highly stable to heat, pH, proteases and other denaturing agents or conditions.
• the first polypeptide fragment comprises one or more single domain antibody that is capable of specifically binding to serum albumin.
• binding specificity in reference to the interaction of a binding molecule, e.g., an antibody, and its binding partner, e.g., an antigen, means that the interaction is dependent upon the presence of a particular structure, e.g., an antigenic determinant or epitope, on the binding partner. In other words, the antibody preferentially binds or recognizes the binding partner even when the binding partner is present in a mixture of other molecules or organisms.
• Antibodies or fragments thereof that immunospecifically bind to an antigen may be cross-reactive with related antigens, carrying the same epitope.
• the “binding specificity” is generally measured against nonspecific background binding. Typically, an antibody is considered specific when it binds to the target antigen at least 10 times above background binding.
• the serum binding single domain antibody contemplated herein can bind to or otherwise associate with serum albumin in such a way that the binding of said serum albumin molecule to FcRn is not (significantly) reduced or inhibited (i.e. compared to the binding of said serum albumin molecule to FcRn when the single domain antibody is not bound thereto) .
• by “not significantly reduced or inhibited” is meant that the binding affinity for serum albumin to FcRn (as measured using a suitable assay, such as SPR) is not reduced by more than 50%, preferably not reduced by more than 30%, even more preferably not reduced by more than 10%, such as not reduced by more than 5%, or essentially not reduced at all.
• not significantly reduced or inhibited may also mean that the half-life of the serum albumin molecule is not significantly reduced (e.g., is not reduced by more than 50%, preferably not reduced by more than 30%, even more preferably not reduced by more than 10%, such as not reduced by more than 5%, or essentially not reduced at all, as measured using a suitable technique known per se) .
• the single domain antibody is capable of binding to amino acid residues on serum albumin that are not involved in binding of serum albumin to FcRn.
• the single domain antibody described herein binds to serum albumin selected from HSA, cynomolgus monkey serum albumin and mouse serum albumin. In some embodiments, the binding affinity towards mouse serum albumin is about weaker than that towards human or cynomolgus serum albumin. In some embodiments, the single domain antibody specifically binds to HSA.
• the single domain antibody described herein binds to serum albumin with sufficient binding affinity.
• affinity refers to the strength of non-covalent interaction between an immunoglobulin molecule (i.e. antibody) or fragment thereof and an antigen. Affinity can be expressed numerically using “Kd” values. In general, a lower Kd value corresponds to a stronger binding. Kd may be determined by using any conventional method known in the art, including but are not limited to radioimmunoassays (RIA) , enzyme-linked immunosorbent assays (ELISA) , surface plasmon resonance (SPR) method, microscale thermophoresis method, HPLC-MS method and flow cytometry (such as FACS) method.
• RIA radioimmunoassays
• ELISA enzyme-linked immunosorbent assays
• SPR surface plasmon resonance
• HPLC-MS HPLC-MS method
• flow cytometry such as FACS
• the antibody disclosed here has a K d value of ⁇ 10 -6 M (e.g. ⁇ 5x10 -7 M, ⁇ 2x10 -7 M, ⁇ 10 -7 M, ⁇ 5x10 -8 M, ⁇ 2x10 - 8 M, ⁇ 10 -8 M, ⁇ 5x10 -9 M, ⁇ 4x10 -9 M, ⁇ 3x10 -9 M, ⁇ 2x10 -9 M, or ⁇ 10 -9 M) with the specific antigen.
• ⁇ 10 -6 M e.g. ⁇ 5x10 -7 M, ⁇ 2x10 -7 M, ⁇ 10 -7 M, ⁇ 5x10 -8 M, ⁇ 2x10 - 8 M, ⁇ 10 -8 M, ⁇ 5x10 -9 M, ⁇ 4x10 -9 M, ⁇ 3x10 -9 M, ⁇ 2x10 -9 M, or ⁇ 10 -9 M
• the single domain antibody provided herein are capable of binding to HSA at a Kd value of 10 -5 M to 1x10 -12 M or less, of 10 -7 M to 1x10 -12 M or less, or of 10 -8 M to 1x10 -12 M or less.
• the Kd is no more than 1x10 - 7 M (e.g.
• the single domain antibody provided herein is a humanized antibody.
• humanized as used herein means that the single domain antibody comprises CDRs derived from a non-human animal, and FR regions derived from human.
• a humanized antibody polypeptide is desirable in its reduced immunogenicity in human.
• a humanized antibody polypeptide is chimeric in its variable regions, as non-human CDR sequences are grafted to human or substantially human FR sequences.
• Humanization of an antibody polypeptide can be essentially performed by substituting the non-human (such as camelid) CDR genes for the corresponding human CDR genes in a human immunoglobulin gene (see, for example, Jones et al. (1986) Nature 321: 522-525; Riechmann et al. (1988) Nature 332: 323-327; Verhoeyen et al. (1988) Science 239: 1534-1536) .
• HSA-binding single domain antibodies are disclosed in US8188223B2, US9067991B2, US9321832B2, PCT application WO2008028977A2, WO2008043822A2, WO2020099871A1, G. Winter, et al, Annu. Rev. Immunol., 1994, 12, 433-455., L.J. Holt, et al., Protein Eng., Des.
• the single domain antibody comprises a VHH domain.
• the VHH domain is humanized.
• the VHH domain comprises a complementarity determining region 1 (CDR1) , CDR2 and CDR3, wherein the CDR1 comprises the sequence of SEQ ID NO: 20 (SFGMS) or a variant thereof having up to 3, 2, or 1 amino acid mutation, a CDR2 comprising the sequence of SEQ ID NO: 21 (SISGSGSDTLYADSVKG) or a variant thereof having up to 3, 2, or 1 amino acid mutation, and/or a CDR3 comprising the sequence of SEQ ID NO: 22 (GGSLSR) or a variant thereof having up to 3, 2, or 1 amino acid mutation, wherein the VHH domain retains the binding specificity to serum albumin, optionally to human serum albumin.
• CDR1 comprises the sequence of SEQ ID NO: 20 (SFGMS) or a variant thereof having up to 3, 2, or 1 amino acid mutation
• a CDR2 comprising the sequence of SEQ ID NO: 21 (SISGSGSDTLYADSVKG) or a variant thereof having up to 3, 2, or 1 amino acid mutation
• the VHH domain comprises a complementarity determining region 1 (CDR1) comprising the sequence of SEQ ID NO: 20, a CDR2 comprising the sequence of SEQ ID NO: 21, and a CDR3 comprising the sequence of SEQ ID NO: 22.
• CDR1 complementarity determining region 1
• CDR2 comprising the sequence of SEQ ID NO: 20
• CDR3 comprising the sequence of SEQ ID NO: 22.
• the VHH domain comprises a complementarity determining region 1 (CDR1) consisting of the sequence of SEQ ID NO: 20, a CDR2 consisting of the sequence of SEQ ID NO: 21, and a CDR3 consisting of the sequence of SEQ ID NO: 22.
• CDR1 complementarity determining region 1
• CDR2 complementarity determining region 2
• CDR3 complementarity determining region 3
• the VHH domain comprises the sequence of SEQ ID NO: 23, or a variant thereof having at least 70% (e.g. at least 75%, 80%, 85%, 90%, 95%, 99%identity to SEQ ID NO: 23, wherein the variant retains the binding specificity and/or affinity to serum albumin.
• the variant of SEQ ID NO: 23 has up to 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutation relative to SEQ ID NO: 23.
• the serum albumin binding nanobody domain further comprises an N-terminal extension attached to the VHH domain.
• the N-terminal extension comprises amino acid residues of SG, AG, S or A, and thus may comprise an amino acid sequence selected from SEQ ID NOs: 24-27.
• the N-terminal extension comprises a tag, optionally a cleavable tag. Without wishing to be bound by any theory, it is believed that certain N-terminal extension can be useful for expression and post-translational processing.
• the serum albumin-binding nanobody domain does not comprise an N-terminal extension attached to the VHH domain.
• the VHH domain can be attached to a cleavable tag which, after cleavage, is no longer present in the final product.
• the first fragment may comprise one or more serum albumin binding nanobody domains.
• the second embodiments of the polypeptide disclosed herein and illustrated in Figure 1B further comprises, in addition to the above described FGF21 domain in the second fragment 200 and the serum albumin binding nanobody domain in the first fragment 100, a third fragment 300 comprising an additional functional domain.
• the additional functional domain comprises a biologically active GLP-1 domain.
• Glucagon-like peptide-1 or “GLP-1” as used herein is intended to broadly encompasses native GLP-1 peptide and all its functional forms such as its functional variants, fragments, fusions, derivatives and mimetics.
• GLP-1 peptide refers to the native human Glucagon-Like Peptide-1 (GLP-1 (7-37) ) , the sequence of which is set forth in SEQ ID NO: 28. The numbering of the residues in GLP-1 is referred to the sequence of SEQ ID NO:28, which begins with the H residue at position 7 and ends with the G residue at position 37.
• a functional form of the native GLP-1 peptide is capable of activating the GLP-1 receptor at a level comparable to, or no less than about 20% (or no less than 30%, 40%, 50%, 60%, 70%, 80%, 90%) of, that of the native GLP-1 peptide.
• Activation of the GLP-1 receptor typically initiates signal transduction pathway resulting in insulinotropic action or other physiological effects as is known in the art.
• GLP-1 peptide Many functional forms of GLP-1 peptide are known in the art, for example, without limitation, liraglutide, semaglutide, dulaglutide, albiglutide, and those disclosed in WO2000055203A1, WO 98/08871, WO 2006/097537, WO2007139589A1, WO1998019698A1, WO2001098331A2, WO2003040309A2, WO2005000892A2, WO2015000942A1, WO2016083499A1 the disclosure of which is incorporated herein to its entirety.
• the GLP-1 domain provided herein comprises an amino acid sequence having at least 70% (e.g. at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%) sequence identity to SEQ ID NO: 28 while retaining substantial biological activity of SEQ ID NO: 28.
• the GLP-1 domain comprises no more than 9, 8, 7, 6, 5, 4, 3, or 2 substitutions relative to SEQ ID NO: 28 while retaining substantial biological activity of SEQ ID NO: 28. In certain embodiments, the GLP-1 domain comprises at least 2, 3, 4, 5, 6, 7, 8, or 9 substitutions relative to SEQ ID NO: 28 while retaining substantial biological activity of SEQ ID NO: 28.
• the GLP-1 domain further comprises one or more mutations.
• mutations One of ordinary skill in the art will appreciate that various amino acid substitutions, e.g., conservative amino acid substitutions, may be made in the sequence of any of the polypeptide described herein, without necessarily decreasing its activity.
• amino acid substitutions include substituting an L-amino acid for its corresponding D-amino acid, substituting cysteine for homocysteine or other non-natural amino acids having a thiol-containing side chain, substituting a lysine for homolysine, diaminobutyric acid, diaminopropionic acid, ornithine or other non-national amino acids having an amino containing side chain, or substituting an alanine for norvaline or the like.
• the GLP-1 comprises one or more mutations at a position selected from the group consisting of: A8, G22, K34, R36, and H7, and or any combination thereof, relative to SEQ ID NO: 28.
• substitution at A8 is useful to prevent DPP4 enzymatic cleavage at the residue
• substitution at G22 is desirable to improve activity and solubility
• substitution at R36 is useful to reduce immunogenicity.
• the GLP-1 comprises one or more mutations at a position selected from the group consisting of: A8, G22, K26, K34, and R36, and or any combination thereof, relative to SEQ ID NO: 28.
• substitutions at these positions include, without limitation, A8G, A8S, A8V, A8Aib, A8T, A8I, A8L, G22E, K26R, K34R, R36G, or any combination thereof, as well as the substitutions described in U. S. Patent Nos. 8, 273, 854, which are incorporated herein by its entirety.
• the one or more additional substitutions comprises a conservative substitution.
• the GLP-1 domain comprises a substitution of A8 which is selected from the group consisting of: A8G, A8S, A8V, A8Aib, A8T, and A8L.
• the GLP-1 domain comprises a substitution of G22E.
• the GLP-1 domain comprises n substitution of R36G.
• the GLP-1 domain comprises a substitution of K26 which is K26R.
• the GLP-1 domain comprises a substitution of K34 which is K34R.
• the GLP-1 comprises or consists of one or more substitutions selected from the group consisting of A8G, K26R, K34R, G22E, and R36G. In certain embodiments, the GLP-1 comprises or consists of one or more substitutions selected from the group consisting of A8G, G22E, and R36G.
• SEQ ID NO: 28 representing a wildtype functional form of GLP-1
• SEQ ID NO: 29 representing a three-substitution functional form of GLP-1 comprising substitutions A8G, G22E, and R36G;
• SEQ ID NO: 31 representing a four-substitution functional form of GLP-1 comprising substitutions A8G, G22E, K34R, and R36G;
• SEQ ID NO: 32 representing a five-substitution functional form of GLP-1 comprising substitutions A8G, G22E, K26R, K34R, and R36G;
• SEQ ID NO: 33 representing a five-substitution functional form of GLP-1 comprising substitutions A8G, G22E, K26R, K34R, and R36K;
• SEQ ID NO: 34 representing a four-substitution functional form of GLP-1 comprising substitutions A8G, G22E, K26R, and R36G.
• the additional functional domain of the third fragment 300 as illustrated in Figure 1B may alternatively comprise a biologically active form (i.e. functional form) of insulin, C-peptide, leptin, glucagon, gastrin, gastric inhibitory polypeptide (GIP) , amylin, calcitonin, cholecystokinin, peptide YY, neuropeptide Y, bone morphogenetic protein-6 (BMP-6) , bone morphogenetic protein-9 (BMP-9) , oxyntomodulin, oxytocin, glucagon-like peptide-2 (GLP-2) , irisin, fibronectin type III domain containing protein 5 (FNDC5) , apelin, adiponectin, Clq and tumor necrosis factor related protein (CTRP family) , resistin, visfatin, omentin, retinol binding protein-4 (RBP-4) , glicentin, angio
• first and second embodiments of the polypeptide disclosed herein and illustrated in Figures 1A and 1B utilizes the first linker 10 to connect the first fragment 100 (comprising the nanobody domain capable of binding to serum albumin) and the second fragment 200 (comprising the biologically active FGF21 domain)
• the second embodiments of the polypeptide illustrated in Figure 1B utilizes the second linker 20 to connect the first fragment 100 and the third fragment 300 (comprising a biologically active additional functional domain that has additive or synergistic effect on FGF21 domain; e.g. GLP-1 domain) .
• linker refers generally to a “polypeptide linker” , which can be any suitable polypeptide capable of bonding two entities to thereby form one molecule, or maintaining association of the two entities in sufficiently close proximity, yet without substantially interference to the respective biological activities of the two entities.
• the linker can be integrated in the resulting linked molecule or structure.
• the first linker 10 operably separates the nanobody domain 100 and the FGF21 domain 200 without substantial interference to the respective biological activities of the two functional domains
• the second linker 20 operably separates the GLP-1 domain and the nanobody domain 100 without substantial interference to the respective biological activities of the two functional domains.
• the linker can be made up of amino acid residues linked together by peptide bonds, yet may optionally further comprise one or more non-natural amino acids.
• each of the first linker 10 and the second linker 20 has a length of at least four amino acid residues.
• each linker has a length of at least 4, 8, 10, 20, 24, 28, 30, 40, 48, 50, 60, 70, 80, 90, 100, 110, 120 or more amino acid resides.
• a suitable length of a linker can further improve the biological activity, stability, or pharmacokinetic parameters of the each of the two functional domains such linked thereby in the whole polypeptide molecule.
• the polypeptide linker may comprise or consist of amino acid residues selected from the amino acids glycine (G) , serine (S) , alanine (A) , methionine (M) , asparagine (N) , glutamine (Q) , cysteine (C) , Proline (P) , Glutamate (E) , Threonine (T) and lysine (K) .
• the polypeptide linker can be made up of a majority of amino acids that are sterically unhindered, such as glycine and alanine.
• linkers are polyglycines, polyalanines, combinations of glycine and alanine (such as poly (Gly-Ala) ) , or combinations of glycine and serine (such as poly (Gly-Ser) ) .
• each of the first linker and the second linker comprises or consists of one or more repeats of a repeating sequence.
• the polypeptide linker comprises or consists of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 repeats of a repeating sequence, or within any numerical range defined by any two numbers listed above.
• first linker it may comprise no acidic amino acid residue (e.g. D or E) according to some embodiments.
• the first linker may optionally comprise one or more units of a first repeating sequence, and the first repeating sequence may consist of no more than 4 or 6 types of amino acid residues, which can be selected from the group consisting of: G, Q, A, P, T and S.
• the first repeating sequence comprises or consists of an amino acid sequences selected from a group consisting of G f S g (each of f and g is independently an integer selected from 1 to 5) , SEQ ID NO: 35 (GAQP) , SEQ ID NO: 36 (GQAP) , SEQ ID NO: 37 (GPAQ) , SEQ ID NO: 38 (GPQA) , SEQ ID NO: 39 (GSQP) , SEQ ID NO: 40 (GASP) , SEQ ID NO: 41 (GPAS) , SEQ ID NO: 42 (GPSA) , SEQ ID NO: 43 (GGGS) , SEQ ID NO: 44 (GSGS) , SEQ ID NO: 45 (GGGGS) , SEQ ID NO: 46 (GSAPGSPAGSPTGSAPGSPA) and SEQ ID NO: 110 (GS) .
• G f S g each of f and g is independently an integer selected from 1 to 5
• SEQ ID NO: 35 GQP
• the first repeating sequence has an amino acid sequence set forth in SEQ ID NO: 35 (GAQP) , and a number of the one or more units is an integer between 1 and 10.
• the first linker comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 35 (GAQP) , SEQ ID NO: 49 ( (GAQP) 2 ) , SEQ ID NO: 50 ( (GAQP) 5 ) , SEQ ID NO: 51 ( (GAQP) 10 ) , and SEQ ID NO: 48 (GGGGSGGGS) .
• the second linker may similarly comprise one or more units of a second repeating sequence, and the second repeating sequence may consist of no more than 4 or 6 types of amino acid residues selected from the group consisting of: G, Q, A, E, P, T and S.
• the second repeating sequence comprises or consists of an amino acid sequences selected from a group consisting of G h S i (each of h and i is independently an integer selected from 1 to 5) , SEQ ID NO: 35 (GAQP) , SEQ ID NO: 55 (GQEP) , SEQ ID NO: 56 (GEQP) , SEQ ID NO: 57 (GPQE) , SEQ ID NO: 58 (GPEQ) , SEQ ID NO: 59 (GSEP) , SEQ ID NO: 60 (GESP) , SEQ ID NO: 61 (GPSE) , SEQ ID NO: 62 (GPES) , SEQ ID NO: 36 (GQAP) , SEQ ID NO: 37 (GPAQ) , SEQ ID NO: 38 (GPQA) , SEQ ID NO: 39 (GSQP) , SEQ ID NO: 40 (GASP) , SEQ ID NO: 41 (GPAS) , SEQ ID NO: 34 (GASP) , S
• the second repeating sequence has an amino acid sequence set forth in SEQ ID NO: 35 (GAQP) , and a number of the one or more units is an integer between 1 and 15, optionally 1, 2, 5, 10, or 14.
• the second linker comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 49 ( (GAQP) 2 ) , SEQ ID NO: 50 ( (GAQP) 5 ) , SEQ ID NO: 51 ( (GAQP) 10 ) , and SEQ ID NO: 52 ( (GAQP) 14 ) , and SEQ ID NO: 47 ( (GGGGS) 4 ) .
• each of the first linker and the second linker may comprise or consist of more than one repeating sequence.
• one such linker may comprise or consist of 2, 3, or 4 different repeating sequences.
• one such linker may comprise or consist of sequential or tandem repeats of the different repeating sequences. The number of repeats of each repeating sequence more be independently selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more.
• amino acid substitutions include substituting an L-amino acid for its corresponding D-amino acid, substituting cysteine for homocysteine or other non-natural amino acids having a thiol-containing side chain, substituting a lysine for homolysine, diaminobutyric acid, diaminopropionic acid, ornithine or other non-national amino acids having an amino containing side chain, or substituting an alanine for norvaline or the like.
• polypeptides are substantially a fusion polypeptide between a serum albumin-binding nanobody domain and an FGF21 domain in an N-terminus-to-C-terminus direction, and may have an amino acid sequence that is selected from SEQ ID NOS: 63-68, 93, 99-101 and 107.
• the polypeptide is conjugated to a synthetic chemical moiety provided herein (see, e.g. based on Figure 1A) .
• the synthetic chemical moiety for example, can be Ac-2XADO-EDA- CO-CH 2 -*, which is conjugated with an introduced cysteine residue at position 171 or position 174 relative to SEQ ID NO: 1 for a polypeptide with a sequence selected from the group consisting of SEQ ID NOS: 63, 68, 93 and 100.
• the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 64-67, 69, 99 and 108, and is not conjugated.
• Table 1A shows the SEQ ID NOs of the exemplary fusion polypeptide sequences, and the SEQ ID NOs for the nanobody, the FGF21, and the first polypeptide linker and the second polypeptide linker contained in the fusion polypeptides.
• Table 1A Exemplary fusion polypeptide sequences # :the mutation positions are relative to SEQ ID NO: 1.
• Moiety A refers to Ac-2XADO-EDA-CO-CH 2 *, where the *end is connected to the conjugatable cysteine residue on the polypeptide, having a structure of:
• polypeptides are substantially a fusion polypeptide among a GLP-1 domain, a serum albumin-binding nanobody domain, and an FGF21 domain in an N-terminus-to-C-terminus direction, which may have an amino acid sequence that is selected from SEQ ID NOS: 70, 74, 75, 79-83, 85, 94-98 and 108-109.
• the polypeptide is conjugated to a synthetic chemical moiety as provided herein (see, e.g., based on Figure 1B) .
• the synthetic chemical moiety for example, can be Ac-2XADO-EDA-CO-CH 2 -*, which is conjugated with an introduced cysteine residue at position 171 or position 174 relative to SEQ ID NO: 1 for the amino acid sequences selected from the group consisting of SEQ ID NOs: 74, 81-83, 85, 94, 96 and 97.
• the polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 70, 75, 79, 80, 95, 98, and 108-109, and is not conjugated.
• Table 1B shows the SEQ ID NOs of the exemplary fusion polypeptide sequences, and the SEQ ID NOs for the GLP-1 domain, the first polypeptide linker, nanobody, the FGF21 domain, and the first polypeptide linker and the second polypeptide linker contained in the fusion polypeptides. Mutations in the FGF21 and GLP-1, as well as the repeating sequences and number of repeats in the peptide linker sequences are also shown.
• Moiety A refers to Ac-2XADO-EDA-CO-CH 2 *, where the *end is connected to the conjugatable cysteine residue on the polypeptide, having a structure of:
• the present disclosure further provides methods for preparing the polypeptide (or optionally the polypeptide conjugate) as described above. To this end, the following are provided in the present disclosure.
• the present disclosure provides isolated nucleic acids or polynucleotides that encode the fusion polypeptide as described herein.
• nucleic acid or “polynucleotide” as used herein refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in either single-or double-stranded form. Unless specifically limited, the term encompasses polynucleotides containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides.
• a particular polynucleotide sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) , alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated.
• degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (see Batzer et al., Nucleic Acid Res. 19: 5081 (1991) ; Ohtsuka et al., J. Biol. Chem. 260: 2605-2608 (1985) ; and Rossolini et al., Mol. Cell. Probes 8: 91-98 (1994) ) .
• nucleic acids or polynucleotides encoding the fusion polypeptide described herein can be constructed using recombinant techniques.
• DNA encoding the serum albumin-binding nanobody, DNA encoding the FGF21 domain, and optionally DNA encoding the GLP-1 domain can be obtained and operably linked to allow transcription and expression in a host cell to produce the polypeptide.
• Polynucleotide sequences encoding for the polypeptide linkers are also operably linked to allow expression of the desired product.
• the encoding polynucleotide sequences can be further operably linked to one or more regulatory sequences, optionally in an expression vector, such that the expression or production of the fusion polypeptides is feasible and under proper control.
• the encoding polynucleotide sequence (s) can be inserted into a vector for further cloning (amplification of the DNA) or for expression, using recombinant techniques known in the art.
• Many vectors are available.
• the vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter (e.g.
• prokaryotic promoters such as T7, T7lac, Sp6, araBAD, trp, lac, tac, pLm, A3, lac, lpp, npr, pac, syn, trc and T3, or eukaryotic promoters such as SV40, CMV, and EF-1 ⁇ ) , and a transcription termination sequence.
• the present disclosure further provides a vector comprising the polynucleotide of provided above, and a host cell that comprises the vector described herein.
• vector refers to a vehicle into which a polynucleotide encoding a protein may be operably inserted so as to bring about the expression of that protein.
• a vector may be used to transform, transduce, or transfect a host cell so as to bring about expression of the genetic element it carries within the host cell.
• Non-limiting examples of vectors include plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosome (YAC) , bacterial artificial chromosome (BAC) , or P1-derived artificial chromosome (PAC) , bacteriophages such as lambda phage or M13 phage, and animal viruses.
• a vector may contain a variety of elements for controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selectable elements, and reporter genes. In addition, the vector may contain an origin of replication.
• a vector may also include materials to aid in its entry into the cell, including but not limited to a viral particle, a liposome, or a protein coating.
• a vector can be an expression vector or a cloning vector.
• the present disclosure provides vectors (e.g., expression vectors) containing the nucleic acid sequence provided herein encoding the fusion polypeptide, at least one promoter (e.g., SV40, CMV, EF-1 ⁇ ) operably linked to the nucleic acid sequence, and at least one selection marker.
• promoter e.g., SV40, CMV, EF-1 ⁇
• vectors include, but are not limited to, retrovirus (including lentivirus) , adenovirus, adeno-associated virus, herpesvirus (e.g., herpes simplex virus) , poxvirus, baculovirus, papillomavirus, papovavirus (e.g., SV40) , lambda phage, and M13 phage, plasmid pcDNA3.3, pMD18-T, pOptivec, pCMV, pEGFP, pIRES, pQD-Hyg-GSeu, pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX, pGEX, pCI, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBA
• RTM. pCDM8, pCDNA1.1/amp, pcDNA3.1, pRc/RSV, PCR 2.1, pEF-1, pFB, pSG5, pXT1, pCDEF3, pSVSPORT, pEF-Bos etc.
• host cell refers to a cell into which an exogenous polynucleotide and/or a vector has been introduced.
• Vectors comprising the polynucleotide sequence (s) provided herein can be introduced to a host cell for cloning or gene expression.
• the phrase “host cell” as used herein refers to a cell into which an exogenous polynucleotide and/or a vector has been introduced. In other embodiments, the vectors are extra-chromosomal.
• the host cells can be isolated if desired. In certain embodiments, the host cell is a prokaryotic cell, and in some other embodiments, the host cell is an eukaryotic cell.
• Suitable host cells for cloning or expressing the DNA in the vectors herein are mainly prokaryotes.
• Suitable prokaryotes for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacilli such as B. subtilis and B. licheniformis, Pseudomonas such as P. aeruginosa, and Streptomyces.
• host cells are eukaryotes, such as yeast and mammalian cells (e.g., immortalized mammalian cells) .
• a vector comprising the polynucleotide sequence (s) provided herein can be introduced into a host cell using any suitable method known to a skilled person in the art, e.g., transformation, transfection or transduction.
• the polynucleotide sequence encoding the fusion polypeptide can be subcloned into an expression vector, which is expressed as inclusion bodies in the host cells.
• the vector can be a viral vector, and any suitable viral vector can be used in this capacity.
• the host cell can be a prokaryotic cell or an eukaryotic cell.
• Host cells transformed with the above-described expression or cloning vectors can be cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the cloning vectors.
• the present disclosure provides a method of producing the fusion polypeptide as described herein, comprising culturing the host cell provided herein under a condition that allows expression of the fusion polypeptide as described herein.
• the host cells transformed with the expression vector may be cultured in a variety of media.
• bacteria growth media such as Terrific Broth, LB Broth, LB Agar, M9 minimal media, MagiaMedia Medium, and ImMedia Medium (ThermoFisher) are suitable for culturing the bacterial host cells.
• Commercially available media such as Ham's F10 (Sigma) , Minimal Essential Medium (MEM) , (Sigma) , RPMI-1640 (Sigma) , and Dulbecco's Modified Eagle's Medium (DMEM) , Sigma) are suitable for culturing the eukaryotic host cells.
• any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor) , salts (such as sodium chloride, calcium, magnesium, and phosphate) , buffers (such as HEPES) , nucleotides (such as adenosine and thymidine) , antibiotics (such as GENTAMYCIN TM drug) , trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range) , and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art.
• the culture conditions such as temperature, pH, and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.
• the present disclosure provides a method of expressing the fusion polypeptide as described herein, comprising culturing the host cell provided herein under the condition at which the fusion polypeptide as described herein is expressed.
• the fusion polypeptide is expressed as inclusion bodies. In certain embodiments, the method further comprises renaturing the fusion polypeptide from the inclusion bodies.
• the fusion polypeptide as described herein can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the product is produced intracellularly, as a first step, the particulate debris, either host cells or lysed fragments, is removed, for example, by centrifugation or ultrafiltration. Carter et al., Bio/Technology 10: 163-167 (1992) describe a procedure for isolating proteins which are secreted to the periplasmic space of E. coli.
• cell paste is thawed in the presence of sodium acetate (pH 3.5) , EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30 min.
• PMSF phenylmethylsulfonylfluoride
• Cell debris can be removed by centrifugation.
• supernatants from such expression systems are generally first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit.
• a protease inhibitor such as PMSF may be included in any of the foregoing steps to inhibit proteolysis and antibiotics may be included to prevent the growth of adventitious contaminants.
• the method further comprises isolating the fusion polypeptide.
• the fusion polypeptide as described herein prepared from the cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, DEAE-cellulose ion exchange chromatography, ammonium sulfate precipitation, salting out, and affinity chromatography.
• a method or process for producing the polypeptide as described above is also provided in the present disclosure, which substantially comprises the following two steps:
• S100 culturing the host cell as described above under a condition that allows expression of the polynucleotide as defined above or a precursor thereof further comprising a removable tag;
• S200 collecting and purifying the polypeptide or the precursor thereof from the host cell.
• the polypeptide is expressed as inclusion bodies. In certain embodiments, the method further comprises renaturing the polypeptide from the inclusion bodies to allow a refolding thereof. Further according to some embodiments, the host cell is E. coli, the vector comprises an E. coli-compatible vector, and the polypeptide encoded by the polynucleotide in the vector is codon optimized for E. coli expression.
• the polypeptide as described herein can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the product is produced intracellularly, as a first step, the particulate debris, either host cells or lysed fragments, is removed, for example, by centrifugation or ultrafiltration.
• step S200 of collecting and purifying the polypeptide from the host cell may comprise the following sub-steps:
• the host cell is lysed and the polypeptide or the precursor of the polypeptide is obtained from the insoluble fraction containing the polypeptide or the precursor of the polypeptide.
• the process further comprises conjugating the purified polypeptide with the functional moiety.
• the functional moiety to be conjugated to the polypeptide is (Ac-2XADO-EDA-CO-CH 2 -*) .
• the present disclosure also provides a pharmaceutical composition comprising the polypeptide provided herein.
• the pharmaceutical composition is liquid composition for parenteral administration.
• the polypeptide substantially serves as the active pharmaceutical ingredient (API) in the pharmaceutical composition.
• the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients.
• pharmaceutically acceptable indicates that the designated excipient (s) is generally chemically and/or physically compatible with the other ingredients comprising the formulation, and physiologically compatible with the recipient thereof.
• a “pharmaceutically acceptable excipient” refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is bioactivity acceptable and nontoxic to a subject.
• Examples of pharmaceutical acceptable excipients for liquid formulation may include, pharmaceutically acceptable liquid, gel, solid carriers, aqueous vehicles, nonaqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending/dispending agents, sequestering or chelating agents, diluents, adjuvants, excipients, or non-toxic auxiliary substances, other components known in the art, or various combinations thereof.
• Suitable pharmaceutically-acceptable excipients for a liquid formulation include, for example, aqueous vehicles such as sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection, or dextrose and lactated Ringer's injection, nonaqueous vehicles such as fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil, or peanut oil, antimicrobial agents at bacteriostatic or fungistatic concentrations, isotonic agents such as sodium chloride or dextrose, buffers such as phosphate or citrate buffers, antioxidants such as sodium bisulfate, local anesthetics such as procaine hydrochloride, suspending and dispersing agents such as sodium carboxymethylcelluose, hydroxypropyl methylcellulose, or polyvinylpyrrolidone, emulsifying agents such as Polysorbate 80 (TWEEN-80) , sequestering or chelating agents such as EDTA (ethylenediaminetetra
• Antimicrobial agents utilized as carriers may be added to pharmaceutical compositions in multiple-dose containers that include phenols or cresols, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride and benzethonium chloride.
• Suitable excipients may include, for example, water, saline, dextrose, glycerol, or ethanol.
• Suitable non-toxic auxiliary substances may include, for example, wetting or emulsifying agents, pH buffering agents, stabilizers, solubility enhancers, or agents such as sodium acetate, sorbitan monolaurate, triethanolamine oleate, or cyclodextrin.
• the pharmaceutical compositions are formulated into an injectable composition.
• the injectable pharmaceutical compositions may be prepared in any conventional form, such as for example liquid solution, suspension, emulsion, or solid forms suitable for generating liquid solution, suspension, or emulsion.
• Preparations for injection may include sterile and/or non-pyretic solutions ready for injection, sterile dry soluble products, such as lyophilized powders, ready to be combined with a solvent just prior to use, including hypodermic tablets, sterile suspensions ready for injection, sterile dry insoluble products ready to be combined with a vehicle just prior to use, and sterile and/or non-pyretic emulsions.
• the solutions may be either aqueous or nonaqueous.
• unit-dose parenteral preparations are packaged in an ampoule, a vial or a syringe with a needle. All preparations for parenteral administration should be sterile and not pyretic, as is known and practiced in the art.
• a sterile, lyophilized powder is prepared by dissolving the polypeptide as disclosed herein in a suitable solvent.
• the solvent may contain an excipient which improves the stability or other pharmacological components of the powder or reconstituted solution, prepared from the powder. Excipients that may be used include, but are not limited to, water, dextrose, sorbital, fructose, corn syrup, xylitol, glycerin, glucose, sucrose or other suitable agent.
• the solvent may contain a buffer, such as citrate, sodium or potassium phosphate or other such buffer known to those of skill in the art at, in one embodiment, about neutral pH.
• the resulting solution will be apportioned into vials for lyophilization.
• Each vial can contain a single dosage or multiple dosages of the polypeptide (e.g. conjugated polypeptide) provided herein or composition thereof. Overfilling vials with a small amount above that needed for a dose or set of doses (e.g., about 10%) is acceptable so as to facilitate accurate sample withdrawal and accurate dosing.
• the lyophilized powder can be stored under appropriate conditions, such as at about 4 °C to room temperature.
• Reconstitution of a lyophilized powder with water for injection provides a formulation for use in parenteral administration.
• the sterile and/or non-pyretic water or other liquid suitable carrier is added to lyophilized powder. The precise amount depends upon the selected therapy being given, and can be empirically determined.
• peripheral parenteral administration by a sterile syringe or some other mechanical device such as an infusion pump.
• peripheral parenteral route is intravenous, intramuscular, or subcutaneous routes of administration.
• the polypeptide described herein is formulated in a solid formulation such as lyophilization or spray drying, which is then reconstituted in a suitable diluent solution prior to administration.
• Standard pharmaceutical formulation techniques such as those described in Remington: The Science and Practice of Pharmacy (D. B. Troy, Editor, 21st Edition, Lippincott, Williams &Wilkins, 2006) , may be employed.
• the pharmaceutical composition provided herein is a liquid formulation.
• the liquid formulation is an aqeous solution.
• An aqeous solution can contain, for example, at least 70%w/w, at least 75%w/w, at least 80%, at least 85%w/w, at least 90%w/w, or at least 95%w/w water.
• the pharmaceutical composition provided herein comprises a polypeptide provided herein and a pharmaceutically acceptable excipient.
• the pharmaceutically acceptable excipient comprises a buffer and an isotonic agent.
• the buffer is selected from the group consisting of a phosphate buffer, citrate buffer, acetate buffer, histidine buffer, glycine buffer, carbonate buffer, borate buffer, glutamate buffer, glycylglycine buffer, lysine buffer, and arginine buffer.
• the pharmaceutical composition has a pH of about 6.0 to about 8.3 (e.g. about 6.5 to about 8.2, about 6.5 to about 8.0, about 6.5 to about 7.8, about 6.5 to about 7.4, about 6.5 to about 7.0, about 7.0 to about 7.8, about 7.0 to about 7.4, about 7.4 to about 8.2, about 7.4 to about 8.0, or about 7.4 to about 7.8) .
• the buffer is a phosphate buffer.
• the pharmaceutical composition has a pH of about 6.5 to about 8.0, about 6.5 to about 7.4, about 7.0 to about 7.8, about 7.0 to about 7.4, or about 7.4 to about 7.8.
• the phosphate buffer is present in a concentration of 0.01-50mM of the pharmaceutical composition.
• the phosphate buffer is present in a concentration of 5-20mM of the pharmaceutical composition, optionally about 8mM.
• the phosphate buffer is selected from the group consisting of sodium dihydrogen phosphate, disodiumhydrogen phosphate, sodium phosphate, or a hydrate thereof.
• the hydrate is dodecahydrate or dihydrate.
• the phosphate buffer is disodiumhydrogen phosphate dodecahydrate or disodium phosphate dihydrate.
• the disodiumhydrogen phosphate dodecahydrate is present in a concentration of about 0.01-17 mg/mL (e.g. about 0.01-15 mg/mL, about 0.05-12 mg/mL, about 0.05-10 mg/mL, about 0.1-10 mg/mL, about 1-5 mg/mL, or about 1-3 mg/mL) .
• the disodiumhydrogen phosphate dodecahydrate is present in a concentration of about 2.87 mg/mL of the pharmaceutical composition.
• the disodiumhydrogen phosphate dihydrate is present in a concentration of about 0.01-8 mg/mL (e.g. about 0.01-5 mg/mL, about 0.05-4 mg/mL, about 0.05-3 mg/mL, about 0.1-3 mg/mL, or about 1-3 mg/mL) .
• the disodiumhydrogen phosphate dihydrate is present in a concentration of about 1.42 mg/mL.
• the buffer is a citrate buffer.
• the citrate buffer is present in a concentration of about 0.05-20 mg/mL, or optionally 0.05-10 mg/mL.
• the citrate buffer comprises a mixture of citric acid anhydrous and trisodium citrate (or a hydrate thereof, e.g. trisodium citrate dihydrate) .
• the pharmaceutical composition has a pH of about 6.0 to about 6.5, or optionally about 6.5.
• the citric acid anhydrous/trisodium citrate dihydrate is present in a concentration of about 0.05-10 mg/mL.
• the citrate buffer comprises a mixture of about 0.14 mg/mL citric acid anhydrous and about 2.74 mg/mL trisodium citrate dihydrate.
• the buffer is a histidine buffer.
• the pharmaceutical composition has a pH of about 6.0 to about 6.8, or optionally about 6.5.
• the histidine buffer is present in a concentration of about 0.5-10 mg/mL (e.g. about 1-5 mg/mL, or about 1-3 mg/ml) .
• the histidine buffer is present in a concentration of about 1.55 mg/mL.
• the isotonic agent is selected from the group consisting of sodium chloride, glycerol, sorbitol, sucrose, propylene glycol, mannitol, glycine, lactose monohydrate, arginine, myoinositol and dimethylsulfon. In certain embodiments, the isotonic agent is not sodium chloride.
• the isotonic agent is glycerol.
• the glycerol is about 5-30 mg/mL.
• the glycerol is about 20 mg/mL.
• the isotonic agent is propylene glycol.
• the propylene glycol is about 1 mg/mL to about 50 mg/mL (e.g. about 5 mg/mL to about 25 mg/mL, about 8 mg/mL to about 16 mg/mL) .
• the propylene glycol is about 14 mg/mL.
• the isotonic agent is sodium chloride.
• the sodium chloride is about 5-15 mg/mL.
• the sodium chloride is about 8.25 mg/mL.
• the isotonic agent is mannitol.
• the mannitol is about 20 mg/mL to about 100 mg/mL (e.g. about 25 mg/mL to about 70 mg/mL, about 30 mg/mL to about 60 mg/mL, about 35 mg/mL to about 55 mg/mL) .
• the mannitol is about 45 mg/mL.
• the isotonic agent is sorbitol.
• the sorbitol is about 20 mg/mL to about 100 mg/mL (e.g. about 40 mg/mL to about 50 mg/mL) .
• the isotonic agent is sucrose.
• the sucrose is about 5 mg/mL to about 150 mg/mL (e.g. about 45 mg/mL to about 100 mg/mL) .
• the pharmaceutically acceptable excipient further comprises a nonionic surfactant.
• the nonionic surfactant is polysorbate 80.
• the polysorbate 80 is about 0.05 mg/mL to about 5 mg/mL (e.g. about 0.1 mg/mL to about 0.5 mg/mL) .
• the pharmaceutical excipient further comprises a preservative, a chelating agent, and/or a stabilizer.
• the pharmaceutical composition has about 1-100 mg/ml of the polypeptide provided herein (e.g. about 1-90 mg/mL, about 1-80 mg/mL, about 1-70 mg/mL, about 1-60 mg/mL, about 1-50 mg/mL, about 1-40 mg/mL, about 1-30 mg/mL, about 1-20 mg/mL, about 1-10 mg/mL) .
• the polypeptide comprises the amino acid sequence of SEQ ID NO: 85, and is conjugated at the introduced cysteine residue at position 171 relative to SEQ ID NO: 1 with the synthetic chemical moiety provided herein.
• the synthetic chemical moiety has the below structure: (also referred herein as Ac-2XADO-EDA-CO-CH 2 *) .
• the polypeptide comprises the amino acid sequence of SEQ ID NO: 96, and is conjugated at the introduced cysteine residue at position 171 relative to SEQ ID NO: 1 with the synthetic chemical moiety.
• the synthetic chemical moiety has the below structure: (also referred herein as Ac-2XADO-EDA-CO-CH 2 *) .
• compositions which comprise:
• a buffer selected from the group consisting of phosphate buffer, citrate buffer, acetate buffer, histidine buffer, glycine buffer, carbonate buffer, borate buffer, glutamate buffer, glycylglycine buffer, lysine buffer, and arginine buffer;
• an isotonic agent selected from the group consisting of sodium chloride, glycerol, sorbitol, sucrose, propylene glycol, mannitol, glycine, lactose monohydrate, arginine, myoinositol and dimethylsulfon; and
• compositions comprise:
• a buffer selected from the group consisting of phosphate buffer, citrate buffer, and histidine buffer;
• an isotonic agent selected from the group consisting of sodium chloride, glycerol, sorbitol, sucrose, propylene glycol, and mannitol;
• compositions comprise:
• an isotonic agent selected from the group consisting of sodium chloride, glycerol, sorbitol, sucrose, propylene glycol, and mannitol;
• compositions comprise:
• a buffer selected from the group consisting of phosphate buffer, citrate buffer, and histidine buffer;
• an isotonic agent selected from the group consisting of 5-15mg/ml sodium chloride, 5-30 mg/mL glycerol, 20 -100 mg/mL sorbitol, 5 -150 mg/mL sucrose, 1 -50 mg/mL propylene glycol, and 20 -100 mg/mL mannitol; and
• compositions comprise:
• an isotonic agent selected from the group consisting of 5 -15mg/ml sodium chloride, 5 -30 mg/mL glycerol, 20 -100 mg/mL sorbitol, 5 -150 mg/mL sucrose, 1 -50 mg/mL propylene glycol, and 20 -100 mg/mL mannitol; and
• compositions comprise:
• the pharmaceutical compositions further comprise a nonionic surfactant which is about 0.1-0.5 mg/mL of polysorbate 80.
• the present disclosure provides a method of preventing or treating a metabolic disorder in a subject in need thereof, comprising administering a therapeutically effective amount of the pharmaceutical composition provided herein.
• Therapeutic methods comprising: administering a therapeutically effective amount of the pharmaceutical composition provided herein to a subject in need thereof, thereby treating or preventing a condition or a disorder.
• the subject has been identified as having a disorder or condition likely to respond to the pharmaceutical composition provided herein.
• the metabolic disorder is diabetes, obesity, non-alcoholic steatohepatitis (NASH) , cardiovascular like dyslipidaemia, artheroscelerosis, alcoholic steatohepatitis (ASH) , diabeticnephropathy, gestational diabetes, metabolic syndrome such as metabolic syndrome X, nonalcoholic fatty liver disease (NAFLD) , end-stage liver disease, hepatic steatosis (fatty liver) , liver cirrhosis, primary biliary cirrhosis (PBC) , or severe hypertriglyceridemia (SHTG) .
• the condition diabetes includes all forms of diabetes, such as hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, non-insulin dependent diabetes, MODY (maturity onset diabetes of the young) , gestational diabetes, and/or an elevated level of HbA1 C.
• diabetes such as hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, non-insulin dependent diabetes, MODY (maturity onset diabetes of the young) , gestational diabetes, and/or an elevated level of HbA1 C.
• the present disclosure provides a method of managing body weight in a subject in need thereof, comprising administering a therapeutically effective amount of the pharmaceutical composition provided herein.
• the present disclosure provides a method of reducing food intake in a subject in need thereof, comprising administering a therapeutically effective amount of the pharmaceutical composition provided herein.
• the present disclosure provides a method of reducing body weight in a subject in need thereof, comprising administering a therapeutically effective amount of the pharmaceutical composition provided herein.
• the condition or metabolic disorder that can be treated or ameliorated using the pharmaceutical composition provided herein includes a condition where a human subject has a fasting blood glucose level of 125 mg/dL or greater, for example 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or greater than 200 mg/dL.
• Blood glucose levels can be determined in the fed or fasted state, or at random.
• the metabolic condition or disorder can also comprise a condition in which a subject is at increased risk of developing a metabolic condition. For a human subject, such conditions include a fasting blood glucose level of 100 mg/dL.
• the condition or metabolic disorder that can be treated or ameliorated using the pharmaceutical composition provided herein includes a condition where a human subject has a body mass index (BMI) of at least or higher than 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40.
• BMI body mass index
• the human subject has an BMI ranging from 25 to 30, 26 to 30, 27 to 30, 28 to 30, 25 to 29, or 25 to 28.
• the therapeutically effective amount of the pharmaceutical composition provided herein will depend on various factors known in the art, such as for example body weight, age, past medical history, present medications, state of health of the subject and potential for cross-reaction, allergies, sensitivities and adverse side-effects, as well as the administration route and extent of disease development. Dosages may be proportionally reduced or increased by one of ordinary skill in the art (e.g., physician or veterinarian) as indicated by these and other circumstances or requirements.
• the therapeutically effective amount can be an amount of the fusion polypeptide and the pharmaceutical composition herein, that elicits a biological or medicinal response in a tissue system, animal, or human being sought by a researcher, medical doctor, or other clinician, which includes alleviation or amelioration of the symptoms of the disease or disorder being treated, i.e., an amount that supports an observable level of one or more desired biological or medicinal response, for example lowering blood glucose, insulin, triglyceride, or cholesterol levels; reducing body weight; or improving glucose tolerance, energy expenditure, or insulin sensitivity.
• the polypeptide or the pharmaceutical composition provided herein may be administered at a therapeutically effective dosage of about 0.01 mg/kg to about 100 mg/kg (e.g., about 0.01 mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 2 mg/kg, about 5 mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, about 30 mg/kg, about 35 mg/kg, about 40 mg/kg, about 45 mg/kg, about 50 mg/kg, about 55 mg/kg, about 60 mg/kg, about 65 mg/kg, about 70 mg/kg, about 75 mg/kg, about 80 mg/kg, about 85 mg/kg, about 90 mg/kg, about 95 mg/kg, or about 100 mg/kg) .
• the polypeptide or the pharmaceutical composition provided herein is administered at a dosage of about 50 mg/kg or less, and in certain of these embodiments the dosage is 10 mg/kg or less, 5 mg/kg or less, 1 mg/kg or less, 0.5 mg/kg or less, or 0.1 mg/kg or less.
• the administration dosage may change over the course of treatment. For example, in certain embodiments the initial administration dosage may be higher than subsequent administration dosages. In certain embodiments, the administration dosage may vary over the course of treatment depending on the reaction of the subject.
• the polypeptide provided herein may be administered to the subject (e.g. human) at a dosing regimen that is no more frequently than once daily, once every 3 days, once weekly, once every two weeks. In certain embodiments, the polypeptide provided herein may be administered to the subject (e.g. human) at a dosing interval of twice weekly, once weekly, or once every two weeks. Therapeutic efficacy with low dosing frequency have the potential to improve a patient's adherence and long-term treatment success. The currently available treatment semaglutide is dosed once weekly.
• the dosing regimen is a continuous dosing regimen selected from twice-weekly dosing, once-weekly dosing, or once bi-weekly dosing.
• the dosing regimen has a dosing interval ranging from about once every 3 days to about once bi-weekly.
• Dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic response) .
• a single dose may be administered, or several divided doses may be administered over time.
• polypeptide or the pharmaceutical composition provided herein may be administered by any route known in the art, such as for example parenteral (e.g., subcutaneous, intraperitoneal, intravenous, including intravenous infusion, intramuscular, or intradermal injection) or non-parenteral (e.g., oral, intranasal, intraocular, sublingual, rectal, or topical) routes.
• parenteral e.g., subcutaneous, intraperitoneal, intravenous, including intravenous infusion, intramuscular, or intradermal injection
• non-parenteral e.g., oral, intranasal, intraocular, sublingual, rectal, or topical routes.
• polypeptide or the pharmaceutical composition provided herein provided herein may be administered to the subject via parenteral administration.
• the polypeptide may be administered alone or in combination with one or more additional therapeutic means or agents.
• the polypeptide provided herein when used for treating a metabolic disease, may be administered in combination with any other therapeutic agent for use in the treatment of a metabolic disease or any medical disorder that related.
• administering in combination includes administration simultaneously as part of the same pharmaceutical composition, simultaneously as separate compositions, or at different timings as separate compositions. A composition administered prior to or after another agent is considered to be administered “in combination” with that agent as the phrase is used herein, even if the composition and the second agent are administered via different routes.
• additional therapeutic agents administered in combination with the polypeptide provided herein are administered according to the schedule listed in the product information sheet of the additional therapeutic agent, or according to the Physicians'Desk Reference (Physicians'Desk Reference, 70th Ed (2016) ) or protocols well known in the art.
• kits for practicing the methods for administering the pharmaceutical composition as described above can comprise a pharmaceutical composition such as those described herein, which can be provided in a sterile container.
• instructions on how to employ the provided pharmaceutical composition in the treatment of a metabolic disorder can also be included or be made available to a patient or a medical service provider.
• kit may comprise: (a) a pharmaceutical composition comprising a therapeutically effective amount of the polypeptide provided herein; and (b) one or more containers for the pharmaceutical composition.
• a kit can also comprise instructions for the use thereof; the instructions can be tailored to the precise metabolic disorder being treated. The instructions can describe the use and nature of the materials provided in the kit.
• kits include instructions for a patient to carry out administration to treat a metabolic disorder, such as elevated glucose levels, elevated insulin levels, diabetes, obesity, non-alcoholic steatohepatitis (NASH) , cardiovascular like dyslipidaemia, artherosclerosis, alcoholic steatohepatitis (ASH) , diabeticnephropathy, metabolic syndrome such as metabolic syndrome X, nonalcoholic fatty liver disease (NAFLD) , end-stage liver disease, hepatic steatosis (fatty liver) , liver cirrhosis, primary biliary cirrhosis (PBC) or severe hypertriglyceridemia (SHTG) .
• a metabolic disorder such as elevated glucose levels, elevated insulin levels, diabetes, obesity, non-alcoholic steatohepatitis (NASH) , cardiovascular like dyslipidaemia, artherosclerosis, alcoholic steatohepatitis (ASH) , diabeticnephropathy, metabolic syndrome such as metabolic syndrome X, nonalcoholic fatty liver disease (NAFLD)
• Instructions can be printed on a substrate, such as paper or plastic, etc, and can be present in the kits as a package insert, in the labeling of the container of the kit or components thereof (e.g., associated with the packaging) , etc.
• the instructions are present as an electronic storage data file present on a suitable computer readable storage medium, e.g. CD-ROM, diskette, etc.
• the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source, such as over the internet, are provided.
• An example of this embodiment is a kit that includes a web address where the instructions can be viewed and/or from which the instructions can be downloaded.
• kits are packaged in suitable packaging to maintain sterility.
• the components of a kit can be packaged in a kit containment element to make a single, easily handled unit, where the kit containment element, e.g., box or analogous structure, may or may not be an airtight container, e.g., to further preserve the sterility of some or all of the components of the kit.
• Example 1 Recombinant expression of nanobody-FGF21 proteins and GLP-1-nanobody-FGF21
• the GLP-1-nanobody-FGF21 proteins or nanobody-FGF21 fusion proteins listed in Tables 1A, 1B, and Table 1C were produced from bacterial E. coli expression system, using BL21 (DE3) derivative strain.
• the DNA coding for the GLP-1-nanobody-FGF21 fusion precursor or nanobody-FGF21 fusion proteins was codon optimized for E. coli expression, de novo synthesized and subcloned into PET derivative expression vectors (Novagen) . Amino acid substitutions were accomplished by modification of the corresponding genetic codes.
• the conjugated fusion proteins were detected and characterized by LC-MS method with Waters BioAccord LC-MS system, or by UPLC with Waters Acquity UPLC system, using conditions optimized for different conjugates, following the supplier’s manuals.
• the in vitro GLP-1 activities of the fusion protein were measured using a BHK cell line overexpressing human GLP-1 receptor and CRE luciferase reporter with or without 1%human serum albumin (HSA) .
• Tested fusion proteins were measured at 100 nM as top concentration in the presence of 1%HSA with 3-fold serial dilutions. After cells were treated with molecules for 4 hours, luciferase activities were measured by Steadylite plus kit (Perkin Elmer, 6066751) .
• each fusion protein was represented by EC50, derived from non-linear regression analysis.
• the in vitro FGF21 activities of the fusion protein were assessed using a HEK293 cell line overexpressing human beta-Klotho. Tested fusion protein conjugates were measured at 400 nM top concentration with 4-fold serial dilutions in the presence of 1%HSA. After cells were treated with fusion protein conjugates for 12 mins, p-ERK levels were measured by p-ERK kit (Cisbio, 64ERKPEH) .
• each fusion protein was represented by EC 50, derived from non-linear regression analysis.
• the fusion molecules (MLC#9, MLC#10, control#6 and MLC#14) showed significantly higher FGF21 efficacy than YH-dual molecule (i.e. the molecule disclosed as SEQ ID NO. 66 in WO 2017/074123) .
• Selected molecules are assessed in disease animal models (such db/db mice, Diet-Induced-Obesity (DIO) mice) to determine body weight, food intake, glucose efficacy with dose responses in chronic studies. Some biomarkers are also measured, including plasma insulin, plasma triglyceride, plasma cholesterol, plasma LDL-c, plasma adiponectin, liver triglyceride, liver cholesterol and liver function indexes (ALT, AST) .
• DIO Diet-Induced-Obesity
• Body weight reduction on Day 25 is calculated by -1* (%BW loss -%BW loss of vehicle group) ; Cumulative food intake reduction is calculated by -100* (cumulative food intake-cumulative food intake of vehicle) /cumulative food intake of vehicle.
• Terminal blood was collected and EDTA-K3 plasma was prepared and frozen at -80 °C for biomarker measurement (LDL-C, TC, TG, ALT, insulin, adiponectin) .
• Liver and adipose tissue were also collected and frozen in liquid nitrogen and store at -80 C. Data are indicated as mean values and standard error (SEM) or pooled values.
• Statistical analysis was performed by One-way ANOVA. Body weight reduction on Day 21 is calculated by -1* (%BW loss -%BW loss of vehicle group) ; Cumulative food intake reduction is calculated by -100* (cumulative food intake-cumulative food intake of vehicle) /cumulative food intake of vehicle.
• MLC#9, MLC#14, MLC#16 and MLC#17 groups showed better glucose control than Semaglutide, Tirzepatide and YH-dual.
• MLC#9, MLC#14, MLC#16 and MLC#17 decreased non-fasting glucose level to lower than 7 mmol/L.
• Semaglutide decreased non-fasting glucose to about 7.5-10 mmol/L.
• MLC#9, MLC#14, MLC#16 and MLC#17 also induced the reduction of triglyceride (Figure 4C) , LDL-C ( Figure 4D) , total cholesterol (Figure 4E) and ALT (Figure 4F) concentration in plasma and reduction of adipose weight (Figure 4G) and liver weight (Figure 4H) , along with the improvement of liver TG ( Figure 4I) , liver TC ( Figure 4J) and insulin sensitivity (Figure 4K) .
• FGF21 can induce secretion of adiponectin which is reported to be an insulin sensitizer.
• the fusion molecules MLC#9, MLC#14, MLC#16 and MLC#17 showed better effect on increase of adiponectin level than YH-dual indicating the better FGF21 activity ( Figure 4L) .
• mice 10 week old ob/ob male mice (42 ⁇ 55g) were injected once daily (QD) subcutaneously with designated GLP-1/FGF21conjugates (i.e., MLC#14 and MLC#16) for 14 days. Food intake and body weight were measured once everyday. Non-fasting blood glucose was measured once every three days. Plasma triglyceride (TG) levels was measured once every week. Five or six animals were used for each treatment group. Body weight and blood glucose was monitored for each individual animal, but food intake for each group animals was measured together. Day 1 and Day 14 are first day and last day of molecule dosage. On the Day 14 after treatment, mice were sacrificed via cardiac puncture under anesthesia.
• GLP-1/FGF21conjugates i.e., MLC#14 and MLC#16
• Terminal blood was collected and frozen at -80 °C for biomarker measurement (LDL-C, TC, TG, ALT/AST, insulin, adiponectin) .
• Liver and adipose tissue were also collected and frozen in liquid nitrogen and store at -80 °C. Data are indicated as mean values and standard error (SEM) or pooled values.
• Statistical analysis was performed by One-way ANOVA. Body weight reduction on Day 14 is calculated by -1* (%BW loss -%BW loss of vehicle group) ; Cumulative food intake reduction is calculated by -100* (cumulative food intake-cumulative food intake of vehicle) /cumulative food intake of vehicle.
• MLC#14 and MLC#16 induced better reduction of plasma triglyceride compared to Semaglutide, Tirzepatide and YH-dual at the same dosage.
• MLC#14 and MLC#16 also reduced the level of LDL-C (Figure 5D) , total cholesterol (Figure 5E) , ALT ( Figure 5F) and AST (Figure 5G) concentration in plasma and reduction of adipose weight (Figure 5H) and liver weight (Figure 5I) along with the improvement of liver TG ( Figure 5J) , liver TC ( Figure 5K) and insulin sensitivity (Figure 5L) .
• the fusion molecule MLC#14 and MLC#16 showed significantly better effect on increase of adiponectin level than YH-dual at the same dosage indicating the better FGF21 activity (Figure 5M) .
• MLC#16, MLC#19, MLC#6 and MLC#23 groups showed better glucose control than Semaglutide, and also induced the reduction of serum triglyceride (Figure 6C) , LDL-C ( Figure 6D) and total cholesterol (Figure 6E) concentration in plasma and reduction of adipose weight (Figure 6F) and liver weight ( Figure 6G) , along with the improvement of liver TG ( Figure 6H) .
• Plasma samples were collected pre-dose (-5 min) , 0.5 hr, 1 hr, 2 hr, 4 hr, 6 hr, 8 hr, 12 hr, 24 hr, 32 hr, 48 hr, 72 hr, 96 hr, after subcutaneous administration.
• the concentrations of the polypeptide conjugates in the plasma were measured by ELISA method. Based on the graph showing plasma concentration of each polypeptide conjugate versus time after subcutaneous injection, the pharmacokinetic parameters were calculated by WinNonlin.
• the MLC#9 has half-life T 1/2 of 14.3 hrs in rat which shows longer half life than the MLC#10 with half-life T 1/2 of 9.5 hrs.
• Example 8 PK study in minipig
• Pharmacokinetics of selected molecules are assessed in minipig. Both subcutaneous and intravenous injections are performed.
• Pharmacokinetics of selected molecules are assessed in monkeys. Both subcutaneous and intravenous injections are performed.
• Selected GLP-1 polypeptide conjugates are also assessed for immunogenicity by in silico (iTope and TCED methods) and ex vivo (EpiScreen) methods.
• the different GLP-1 polypeptide conjugates are formulated in buffers with different compositions (pH6, 7, 7.4 and 8.0) and stored at different temperatures (such as 4 °C and 25 °C) for 2-4 weeks.
• the %HMWP and %LMW are analyzed by size exclusion chromatography (SEC) -HPLC.
• the concentration and modifications were analyzed by reverse phase (RP) -UPLC and LC/MS.
• Example 12 Human serum albumin binding.
• Binding of molecules to serum albumin was characterized by surface plasmon resonance in a Biacore 8K instrument. Human serum albumin was covalently bound to CM5 sensor chips surface until 4000 RU was reached. The chip was blocked by 1M ethanolamine with flowrate of 10 ⁇ L/min for 420 s. Each molecule sample was diluted and injected at a flow rate of 30 ⁇ L/min to allow for binding to chip-bound albumin for 120 s and for dissociation for 300 s. Binding buffer without molecule was sent over the chip at the flow rate of 20 seconds to allow spontaneous dissociation of bound molecule for 30 seconds.
• Example 13 FAP enzyme cleavage on the fusion proteins.
• Fibroblast activation protein is a serine protease. This enzyme has been reported to regulate degradation of FGF21.
• FAP enzyme In order to test the C terminal degradation of the fusion protein by FAP enzyme, the fusion protein (MLC#23, MLC#25) and FAP enzyme were incubated with ration 200: 1 at 37 °C for 20 hours. LC-MS were performed to analyze the degradation percentage of the fusion protein.
• the fusion protein without conjugation (MLC#25, MLC#18 and MLC#21) showed 50.6-65.2%C-terminal degradation when incubation with FAP enzyme at 37 °C for 20 hours.
• the fusion protein conjugate (MLC#23, MLC#16 and MLC#19) and the fusion protein with 171G substitution (MLC#10) showed resistance to FAP enzyme.
• MLC#16 or MLC#19 was prepared by diluting MLC#16 or MLC#19 into the formulation buffer, which was composed of buffer agent (e.g. disodiumhydrogen phosphate dodecahydrate or disodiumhydrogen phosphate dihydrate was used for pH 7.4-8.2 and histidine or citric acid anhydrous/trisodium citrate dihydrate was used for pH6.5) and isotonic agent (e.g. sodium chloride, glycerol, sorbitol, sucrose, propylene glycol or mannitol) .
• Nonionic surfactant e.g. polysorbate 80
• pH was adjusted to the relevant value by sodium hydroxide and/or hydrochloric acid.
• the composition was filtrated by a 0.22 ⁇ m sterile filter.
• compositions comprising MLC#16 or MLC#19 were tested in this experiment.
• the tested compositions were with glycerol (20 mg/ml) , propylene glycol (14 mg/ml) , sodium chloride (8.25 mg/ml) , mannitol (40-50 mg/ml) , sorbitol (40-50 mg/mL) , or sucrose (45-100 mg/mL) as isotonic agent, combined with varieties of pH as shown in Table 8.
• glycerol 20 mg/ml
• propylene glycol 14 mg/ml
• sodium chloride 8.25 mg/ml
• mannitol 40-50 mg/ml
• sorbitol 40-50 mg/mL
• sucrose 45-100 mg/mL
• Example 14 The 48 compositions listed in Example 14 were prepared and stored at 25°C for up to 6 months. Impurities and high molecular weight products were investigated with LC-MS and SEC-HPLC at 0, 3, 6 months.
• the LC-MS was performed with Acquity BEH C4 column (Waters) and UPLC I-class (Waters) .
• SEC-HPLC was performed with Xbridge BEH 200A SEC 3.5 ⁇ m 7.8 ⁇ 150 mm (Waters) and Arc-HPLC (Waters) .
• the results for some compositions i.e. No. 1-5, 7, 18-19, 21, 24-30, 37, 40-41, 43-45 and47-48) were shown in Table 9.
• compositions in Table 9 are tested and results show that they are stable over the tested period except compositions 25 and 48.
• Compositions with a pH higher than 8.0 i.e. No. 7, 24, 25 and 48
• Compositions with sodium chloride as isotonic agent showed relatively higher HMWP and total impurites than compositions with other isotonic agents (e.g. No. 24 vs 25) , indicating that isotonic agents glycerol, propylene glycol, mannitol, sorbitol, sucrose are more beneficial for the stability of the molecules.

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