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WO2017049205A2 - Growth hormone formulation - Google Patents

Growth hormone formulation Download PDF

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Publication number
WO2017049205A2
WO2017049205A2 PCT/US2016/052309 US2016052309W WO2017049205A2 WO 2017049205 A2 WO2017049205 A2 WO 2017049205A2 US 2016052309 W US2016052309 W US 2016052309W WO 2017049205 A2 WO2017049205 A2 WO 2017049205A2
Authority
WO
WIPO (PCT)
Prior art keywords
aqueous liquid
liquid formulation
xten
fusion protein
hgh
Prior art date
Application number
PCT/US2016/052309
Other languages
French (fr)
Other versions
WO2017049205A3 (en
Inventor
Jeffrey L. Cleland
Original Assignee
Amunix Operating Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Amunix Operating Inc. filed Critical Amunix Operating Inc.
Publication of WO2017049205A2 publication Critical patent/WO2017049205A2/en
Publication of WO2017049205A3 publication Critical patent/WO2017049205A3/en

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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
    • A61K38/22—Hormones
    • A61K38/27—Growth hormone [GH], i.e. somatotropin
    • 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
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A61K47/183—Amino acids, e.g. glycine, EDTA or aspartame
    • 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
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • 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

Definitions

  • the present invention is directed to pharmaceutical formulations containing a fusion protein comprising human growth hormone (hGH) and to methods for making and using such formulations. More particularly, this invention relates to liquid hGH formulations with increased stability.
  • hGH human growth hormone
  • hGH Native human growth hormone
  • (pit)-hGH is a single polypeptide chain protein consisting of 191 amino acids.
  • the protein is internally cross-linked by two disulphide bridges and in monomelic form has a molecular weight of 22 kDa.
  • Growth hormone (GH) of animal species is closely homologous in amino acid sequence to that of humans and is therefore very similar in its characteristics.
  • a major biological effect of GH is to promote growth throughout a range of organs and tissues in the body.
  • GH responsive organs or tissues include the liver, intestine, kidneys, muscles, connective tissue and the skeleton.
  • rhGH recombinant hGH
  • rhGH Recombinant hGH
  • rhGH Recombinant hGH
  • rhGH is used as a therapeutic and has been approved for the treatment of a number of indications.
  • rhGH is currently sold in a variety of forms worldwide including capsules, syrups, sprays, tablets, powders, vials, and injections. Numerous injectable products are currently on the market in the United States: for instance,
  • HumatropeTM (Eli Lilly & Co.), NutropinTM (Genentech), NorditropinTM (Novo-Nordisk), OmnitropeTM (Sandoz/Novartis), GenotropinTM (Pfizer), Saizen/SerostimTM (Serono), Tev- TropinTM (Teva), and ZorbtiveTM (EMD Serono).
  • hGH deficiency leads to dwarfism, for example, which has been successfully treated for decades by exogenous administration of the hormone.
  • hGH has also been approved for the treatment of several other conditions including renal failure (in children), Turner's Syndrome, and cachexia in AIDS patients.
  • FDA Food and Drug Administration
  • hGH has approved hGH for the treatment of non-GH-dependent short stature
  • hGH has been investigated for the treatment of a variety of physiological conditions, including aging, frailty in the elderly, short bowel syndrome, and congestive heart failure.
  • Target populations for hGH treatment include children with idiopathic short stature (ISS) and adults with GHD-like symptoms.
  • HumatropeTM lyophilized powder is provided in a 5 mg vial or 6-24 mg cartridge to be reconstituted with water for injection (pi.lilly.com/us/humatrope-pi.pdf; accessed September 3, 2015).
  • aqueous liquid formulations of hGH that do not require reconstitution and are chemically stable for an extended period, which have the advantages of eliminating reconstitution errors, thereby increasing dosing accuracy, as well as simplifying the use of the product clinically, thereby increasing patient compliance.
  • Such formulations also allow for improvements in the design and use of pre-filled syringes or other devices, which are sold to patients containing pre-mixed liquid formulations of rhGH (e.g., NorditropinTM).
  • Therapeutic aqueous liquid formulations of hGH preferably should be stable for 2-3 years at refrigerated temperatures and for several weeks near room temperature. For example, prior to initial use, the refrigerated shelf-life of the Norditropin FlexPro pre-filled pen is 2 years
  • hGH Agitation induced aggregation of hGH occurs due to the air-water interface denaturation of hGH (Pearlman and Bewley, supra at 36-40; Bam et al, Journal of Pharmaceutical Sciences Vol. 87, No. 12, December 1998).
  • surfactants e.g. polysorbate 20, polyethylene glycol (PEG)
  • PEG polyethylene glycol
  • This invention provides a highly stable aqueous liquid formulation of a fusion protein containing rhGH.
  • the invention provides a liquid formulation of a rhGH-XTEN fusion protein that exhibits excellent long-term stability without the use of a surfactant.
  • a stable pharmaceutically acceptable aqueous formulation containing a fusion protein comprising human growth hormone linked to an extended recombinant polypeptide (XTEN) and a buffer is disclosed, wherein the formulation is lacking a surfactant. Also disclosed are associated methods for preparing, storing, and using such formulations.
  • aspects of the invention include a storage stable aqueous liquid formulation comprising (i) about 25 mg/ml to about 150 mg/ml of a fusion protein comprising an extended recombinant polypeptide (XTEN) fused to a growth hormone (GH) sequence, wherein the GH sequence is at least 90% identical to the amino acid sequence of SEQ ID NO:43, and (ii) a buffer; characterized in that the aqueous liquid formulation is at a pH of about 4 to about 6, in the absence of a surfactant. In some embodiments, the aqueous liquid formulation is characterized in that it is free of a preservative. In some embodiments, the aqueous liquid formulation is characterized in that the pH is about 5 to about 6.
  • the aqueous liquid formulation is characterized in that the pH is 5.2 to 5.8. In some embodiments, the aqueous liquid formulation is characterized in that the pH is about 5.5. In some embodiments, the aqueous liquid formulation is characterized in that the buffer is selected from the group consisting of histidine, citrate, and succinate buffers. In some embodiments, the aqueous liquid formulation is characterized in that the buffer is a histidine buffer at a concentration of about 20 mM.
  • the aqueous liquid formulation is characterized in that the concentration of the fusion protein is selected from the group consisting of about 25 mg/ml, about 50 mg/ml, about 75 mg/ml, about 100 mg/ml, about 125 mg/ml, and about 150 mg/ml. In some embodiments, the aqueous liquid formulation is characterized in that the
  • concentration of the fusion protein is about 50 mg/ml. In some embodiments, the aqueous liquid formulation is characterized in that the concentration of the fusion protein is about 100 mg/ml. In some embodiments, the aqueous liquid formulation is characterized in that the concentration of the fusion protein is about 150 mg/ml.
  • the aqueous liquid formulation is characterized in that the aqueous liquid formulation is storage stable for at least about one month at about 23 ⁇ 3°C. In some embodiments, the aqueous liquid formulation is characterized in that the aqueous liquid formulation is storage stable for at least about 6 months to about 3 years at about 5 ⁇ 3 °C. In some embodiments, the aqueous liquid formulation is characterized in that the aqueous liquid formulation is storage stable for at least about 6 months to about 18 months at about 5 ⁇ 3 °C.
  • the aqueous liquid formulation is characterized in that the aqueous liquid formulation is storage stable at about 5 ⁇ 3 °C for at least about 3 months, for at least about 6 months, for at least about 9 months, for at least about 12 months, for at least about 18 months, for at least about 21 months, for at least about 24 months, for at least about 27 months, for at least about 30 months, for at least about 36 months, or for at least about 42 months.
  • the aqueous liquid formulation is characterized in that the oxidation of the fusion protein is reduced as compared to the oxidation of the corresponding fusion protein formulated with a surfactant. In some embodiments, the aqueous liquid formulation is characterized in that the deamidation of the fusion protein is reduced as compared to the deamidation of the corresponding fusion protein formulated at a higher pH. In some embodiments, the aqueous liquid formulation is characterized in that the isolated fusion protein does not show detectable oxidation or deamidation following incubation for about 8 hours at about 40 °C.
  • the aqueous liquid formulation is characterized in that it further comprises a tonicifying agent.
  • the aqueous liquid formulation is characterized in that the tonicifying agent is NaCl.
  • the aqueous liquid formulation is characterized as having an osmolality between about 300 mOsm to about 600 mOsm.
  • the aqueous liquid formulation is characterized in that the formulation is disposed in a container containing at least one dose.
  • the container comprises a syringe.
  • the container comprises a glass cartridge.
  • the container comprises a vial.
  • the aqueous liquid formulation is characterized in that it is for injection into a subject with a needle.
  • the needle size is selected from the group consisting of 26 gauge, 27 gauge, 29 gauge, and 30 gauge.
  • the aqueous liquid formulation is characterized in that it comprises the fusion protein according to the formula I:
  • XTEN l and XTEN 2 comprise identical or different sequences.
  • the aqueous liquid formulation is characterized in that the XTEN l comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 18-34, 38, 39, 41, and 42.
  • the aqueous liquid formulation is characterized in that the XTEN_2 comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 13-17, 35, 36, 37, and 40.
  • the aqueous liquid formulation is characterized in that the XTEN l comprises SEQ ID NO:39.
  • the aqueous liquid formulation is characterized in that the XTEN 2 comprises SEQ ID NO: 35.
  • the aqueous liquid formulation is characterizes in that XTEN l comprises SEQ ID NO: 39 and XTEN 2 comprises SEQ ID NO: 35.
  • the aqueous liquid formulation is characterized in that the fusion protein sequence is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 1.
  • the aqueous liquid formulation is characterized in that the fusion protein comprises the amino acid sequence of SEQ ID NO: 1.
  • Some aspects of the invention relate to a method of making a storage stable aqueous liquid formulation of a fusion protein comprising an extended recombinant polypeptide (XTEN) linked to a growth hormone (GH) sequence, wherein the GH sequence is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:43, comprising mixing said fusion protein and an aqueous, pharmaceutically acceptable vehicle which includes i) said fusion protein at a concentration of about 25 mg/ml to about 150 mg/ml; and ii) a buffer providing a pH of about 4 to about 6; wherein said aqueous liquid formulation is free of a surfactant.
  • the method is characterized in that it is formulated without use of a preservative.
  • Some aspects of the invention relate to a method of treating a growth-hormone related condition in a subject, comprising administering to the subject a therapeutically effective amount of the aqueous liquid formulation according to the presently described invention, wherein the growth-hormone related condition is selected from the group consisting of growth-hormone deficiency, Turner's Syndrome, Prader-Willi Syndrome, small for gestational age (SGA), idiopathic short stature, AIDS wasting, multiple sclerosis, Crohn's disease, ulcerative colitis, and muscular dystrophy.
  • the growth-hormone related condition is selected from the group consisting of growth-hormone deficiency, Turner's Syndrome, Prader-Willi Syndrome, small for gestational age (SGA), idiopathic short stature, AIDS wasting, multiple sclerosis, Crohn's disease, ulcerative colitis, and muscular dystrophy.
  • the method is characterized in that the therapeutically effective amount is administered at least 48 h, or at least 72 h, or at least about 96 h, or at least about 120 h, or at least about 7 days, or at least about 14 days, at least about 21 days, or at least about 30 days between consecutive doses.
  • the method is characterized in that the growth-hormone related condition is growth-hormone deficiency.
  • the method is characterized in that the growth-hormone related condition is Turner's Syndrome.
  • the method is characterized in that the growth-hormone related condition is Prader-Willi Syndrome.
  • the method is characterized in that the growth-hormone related condition is idiopathic short stature.
  • the method is
  • the growth-hormone related condition is small for gestational age (SGA).
  • the method is characterized in that the growth-hormone related condition is selected from the group consisting of AIDS wasting, multiple sclerosis, Crohn' s disease, ulcerative colitis, and muscular dystrophy.
  • Some aspects of the invention relate to the use of a therapeutically effective amount of the aqueous liquid formulation of according to the presently described invention in the preparation of a medicament for treating a growth-hormone related condition, wherein the growth-hormone related condition is selected from the group consisting of growth-hormone deficiency, Turner's Syndrome, Prader-Willi Syndrome, small for gestational age (SGA), idiopathic short stature, AIDS wasting, multiple sclerosis, Crohn's disease, ulcerative colitis, and muscular dystrophy.
  • the growth-hormone related condition is selected from the group consisting of growth-hormone deficiency, Turner's Syndrome, Prader-Willi Syndrome, small for gestational age (SGA), idiopathic short stature, AIDS wasting, multiple sclerosis, Crohn's disease, ulcerative colitis, and muscular dystrophy.
  • the aqueous liquid formulation is characterized in that it is for use in treating a growth-hormone related condition, wherein the growth-hormone related condition is selected from the group consisting of growth-hormone deficiency, Turner's Syndrome, Prader-Willi Syndrome, small for gestational age (SGA), idiopathic short stature, AIDS wasting, multiple sclerosis, Crohn's disease, ulcerative colitis, and muscular dystrophy.
  • the growth-hormone related condition is selected from the group consisting of growth-hormone deficiency, Turner's Syndrome, Prader-Willi Syndrome, small for gestational age (SGA), idiopathic short stature, AIDS wasting, multiple sclerosis, Crohn's disease, ulcerative colitis, and muscular dystrophy.
  • FIG. 1 provides the amino acid sequence of an hGH-XTEN fusion protein designated VRS-317 (hGH sequence is underlined and shown in bold) (SEQ ID NO: 1).
  • FIG. 2 is a strong anion exchange chromatogram of an aqueous formulation according to the present invention showing degradation of VRS-317 at 40°C via hydrolysis, or truncation, of the XTEN polypeptide in VRS-317.
  • FIG. 3 is a strong anion exchange chromatogram of an aqueous formulation according to the present invention containing different concentrations of VRS-317 showing that VRS- 317 in the formulation is not degraded.
  • FIG. 4. is a size exclusion chromatogram of an aqueous formulation according to the present invention containing different concentrations of VRS-317 showing that VRS-317 in the formulation is not aggregated.
  • FIG. 5. is a size exclusion chromatogram of an aqueous formulation according to the present invention containing different concentrations of VRS-317, showing that VRS-317 in the formulation is not aggregated after passing through a 30 gauge needle.
  • FIG. 6 is a reverse phase chromatogram of tryptic fragments of control VRS-317 (black) or VRS-317 treated with hydrogen peroxide for 72 hours (gray).
  • a & B are expanded views of the parts of the chromatogram (underlined) where degradation of VRS- 317 was observed.
  • FIG. 7 is a reverse phase chromatogram of tryptic fragments of control VRS-317 (black) or VRS-317 stressed with elevated pH and temperatures (gray).
  • Inset An expanded view of the reverse phase chromatogram, highlighting the observed degradation.
  • FIG. 8 shows SE-HPLC results comparing the degree of VRS-317 aggregation seen in aqueous formulations according to the present invention in the presence or absence of surfactant, following 4 hour agitation stress experiments at ambient temperature.
  • FIG. 9 shows graphs of integrated percent peak areas from the size exclusion chromatogram of an aqueous formulation according to the present invention containing VRS- 317 sampled periodically over up to 24 months of storage at 5°C or 25°C.
  • FIG. 10 shows a graph of integrated percent peak areas from the reverse phase chromatogram of tryptic fragments of an aqueous formulation according to the present invention containing VRS-317 sampled over 1 month of storage at 25°C.
  • FIG. 11 shows a graph of pH readings taken periodically over up to 24 months of an aqueous formulation according to the present invention containing VRS-317 stored at 5°C or 25°C.
  • polypeptide polypeptide
  • peptide protein
  • the terms “polypeptide”, “peptide”, and “protein” are used interchangeably herein to refer to polymers of amino acids of any length.
  • the polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids.
  • the terms also encompass an amino acid polymer that has been modified, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component.
  • “Growth Hormone” or “GH” means a growth hormone polypeptide, including, without limitation, native sequence human and non-human GH, and amino acid sequence variants thereof having growth hormone biological activity.
  • non-human grown hormone can be of any non-human animal species, such as a non-human mammalian species.
  • the term "native sequence human growth hormone (hGH)" or “native hGH” includes, but is not limited to, the hGH originally derived from the pituitary gland having a molecular weight of about 22, 129 daltons (22kD hGH) and a naturally occurring variant having a molecular weight of about 20,000 daltons (20kD hGH).
  • the 20kD hGH has an amino acid sequence that corresponds to that of 22kD hGH consisting of 191 amino acids except that 15 amino acid residues from the 32nd to the 46th of 22kD hGH are missing. Some reports have shown that the 20kD hGH has been found to exhibit lower risks and higher activity than 22kD hGH.
  • the invention contemplates use of the 22 kD, the 20kD hGH, as well as species and sequence variants and truncated fragments thereof as being appropriate for use as a fusion partner with XTEN disclosed herein for hGH-XTEN compositions.
  • the cloned gene for hGH has been expressed in a secreted form in Escherichia coli (United States Patent No.
  • GH human growth hormone
  • the term "storage stable”, as it relates to a pharmaceutical liquid formulation comprising hGH fusion protein, is a formulation that results in less than 5% aggregation of hGH fusion protein, less than 15% deamidation, or less than 10% oxidation of hGH fusion protein when such pharmaceutical liquid formulation comprising hGH is stored at 2° C to 8° C.
  • such liquid formulation comprising hGH is long-term stable for at least 2-3 years, and in an alternate embodiment, such liquid formulation comprising hGH is storage stable for 24 months or for 6 to 18 months, or stable at least 12 months.
  • the long-term stability of hGH may be determined directly by incubating the formulations for the above noted times, or the long-term stability may be predicted by the methods described herein, or other known methods.
  • amino acid refers to either natural and/or unnatural or synthetic amino acids, including but not limited to glycine and both the D or L optical isomers, and amino acid analogs and peptidomimetics. Standard single or three letter codes are used to designate amino acids.
  • a “fragment” is a truncated form of a native biologically active protein that retains at least a portion of the therapeutic and/or biological activity.
  • a “variant” is a protein with sequence homology to the native biologically active protein that retains at least a portion of the therapeutic and/or biological activity of the biologically active protein.
  • a variant protein may share at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity with the reference biologically active protein.
  • biologically active protein moiety includes proteins modified deliberately, as for example, by site directed mutagenesis, insertions, or accidentally through mutations.
  • GH variant comprises an amino acid sequence which differs from that of a native sequence GH, hGH or rhGH by virtue of at least one amino acid modification, preferably one or more amino acid substitution(s).
  • the variant has at least one amino acid substitution compared to a native sequence polypeptide, e.g. from about one to about ten amino acid substitutions, and preferably from about one to about five amino acid substitutions in the sequence of the parent polypeptide.
  • natural L-amino acid means the L optical isomer forms of glycine (G), proline (P), alanine (A), valine (V), leucine (L), isoleucine (I), methionine (M), cysteine (C), phenylalanine (F), tyrosine (Y), tryptophan (W), histidine (H), lysine (K), arginine (R), glutamine (Q), asparagine (N), glutamic acid (E), aspartic acid (D), serine (S), and threonine (T).
  • non-naturally occurring means polypeptide or polynucleotide sequences that do not have a counterpart to, are not complementary to, or do not have a high degree of homology with a wild-type or naturally- occurring sequence found in a mammal.
  • a non-naturally occurring polypeptide or fragment may share no more than 99%, 98%, 95%, 90%, 80%, 70%, 60%, 50% or even less amino acid sequence identity as compared to a natural sequence when suitably aligned.
  • hydrophilic and hydrophobic refer to the degree of affinity that a substance has with water.
  • a hydrophilic substance has a strong affinity for water, tending to dissolve in, mix with, or be wetted by water, while a hydrophobic substance substantially lacks affinity for water, tending to repel and not absorb water and tending not to dissolve in or mix with or be wetted by water.
  • Amino acids can be characterized based on their hydrophobicity.
  • a number of scales have been developed. An example is a scale developed by Levitt, M, et al, J Mol Biol (1976) 104:59, which is listed in Hopp, TP, et al, Proc Natl Acad Sci U S A (1981) 78:3824.
  • hydrophilic amino acids are arginine, lysine, threonine, alanine, asparagine, and glutamine. Of particular interest are the hydrophilic amino acids aspartate, glutamate, and serine, and glycine. Examples of
  • hydrophobic amino acids are tryptophan, tyrosine, phenylalanine, methionine, leucine, isoleucine, and valine.
  • hGH-XTEN is meant to encompass fusion polypeptides that comprise a payload region comprising a biologically active GH that mediates one or more biological or therapeutic activities associated with growth hormone and at least one other region comprising at least a first XTEN polypeptide that serves as a carrier.
  • the invention provides an hGH-XTEN fusion protein comprising a sequence set forth in Table 2.
  • the invention provides an hGH-XTEN fusion protein comprising SEQ ID NO: 35 and SEQ ID NO: 39.
  • buffer or “physiologically-acceptable buffer” refers to solutions of compounds that are known to be safe for pharmaceutical or veterinary use in formulations and that have the effect of maintaining or controlling the pH of a formulation in the pH range desired for the formulation. Buffers typically involve a weak acid or alkali together with one of its salts. Acceptable buffers for controlling pH at a moderately acidic pH to a moderately basic pH include, but are not limited to, such compounds as phosphate, acetate, citrate, arginine, TRIS, and histidine. "TRIS” refers to 2-amino-2-hydroxymethyl-l, 3, -propanediol, and to any pharmacologically acceptable salt thereof.
  • “Pharmaceutically acceptable” excipients are those commonly used in the art which can reasonably be administered to a subject mammal to provide an effective dose of the active ingredient employed.
  • parenteral administration e.g.
  • subcutaneous or intramuscular, or topical administration formulations may be converted into a solution, gel or emulsion, if desired, using the pharmaceutical substances customary for this purpose, such as solubilizers, thickening agents, emulsifiers, agents for tonicity, preservatives or other auxiliaries.
  • surfactant is used to refer to a surface-active agent, typically a nonionic surfactant, understood and employed by those skilled in the art.
  • surfactants include polysorbate (for example, polysorbate 20 and polysorbate 80); poloxamer (e.g. poloxamer 188); Triton; sodium dodecyl sulfate (SDS); sodium laurel sulfate; sodium octyl glycoside; lauryl-, myristyl-, linoleyl-, or stearyl- sulfobetaine; lauryl-, myristyl-, linoleyl- or stearyl-sarcosine; linoleyl-, myristyl-, or cetyl- betaine; lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-, myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine (e.g.
  • lauroamidopropyl myristamidopropyl- , palmidopropyl-, or isostearamidopropyl-dimethylamine; sodium methyl cocoyl-, or disodium methyl oleyl-taurate; polyethyl glycol, polypropyl glycol, and copolymers of ethylene and propylene glycol (e.g. Pluronics, PF68 etc).
  • a “sugar,” or “saccharide,” or “sugar alcohol,” or “polyol” as used herein refers to a substances well known in the art with multiple hydroxyl groups, and includes reducing sugars, nonreducing sugars, sugar alcohols and sugar acids.
  • a "reducing sugar” is one which contains a hemiacetal group that can reduce metal ions or react covalently with lysine and other amino groups in proteins and a "nonreducing sugar” is one which does not have these properties of a reducing sugar. Examples of reducing sugars are fructose, mannose, maltose, lactose, arabinose, xylose, ribose, rhamnose, galactose and glucose.
  • Nonreducing sugars include sucrose, trehalose, stachyose, sorbose, melezitose and raffinose.
  • Mannitol, xylitol, erythritol, threitol, sorbitol, ribitol, myoinositol, galactitol and glycerol are examples of sugar alcohols.
  • sugar acids these include L-gluconate and metallic salts thereof.
  • the polyol is preferably one which does not crystallize at freezing temperatures (e.g. -20°C.) such that it destabilizes the fusion protein composition in the formulation.
  • Polyols including mixtures of polyols, can also be used as lyoprotectants in the formulations of the present invention, which protects a protein, e.g. an fusion protein, from damage resulting from lyophilization.
  • Other stabilizing agents such as metal chelating agents, may also be useful in the invention which are generally known in the art.
  • preservative is understood in the art to be a compound which can be included in the formulation to substantially reduce bacterial action therein, thus facilitating the production of a multi-use formulation, for example.
  • preservatives include octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride (a mixture of alkylbenzyldimethylammonium chlorides in which the alkyl groups are long-chain compounds), and benzelthonium chloride.
  • preservatives include aromatic alcohols such as phenol, butyl and benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol.
  • Conjugated refers to the joining together of two or more chemical elements or components, by whatever means including chemical conjugation or recombinant means.
  • a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence.
  • operably linked means that the DNA sequences being linked are contiguous, and in reading phase or in-frame.
  • An "in-frame fusion” refers to the joining of two or more open reading frames (ORFs) to form a continuous longer ORF, in a manner that maintains the correct reading frame of the original ORFs.
  • ORFs open reading frames
  • the resulting recombinant fusion protein is a single protein containing two or more segments that correspond to polypeptides encoded by the original ORFs (which segments are not normally so joined in nature).
  • a “linear sequence” or a “sequence” is an order of amino acids in a polypeptide in an amino to carboxyl terminus direction in which residues that neighbor each other in the sequence are contiguous in the primary structure of the polypeptide.
  • a “partial sequence” is a linear sequence of part of a polypeptide that is known to comprise additional residues in one or both directions.
  • Recombinant as applied to a polynucleotide means that the polynucleotide is the product of various combinations of in vitro cloning, restriction and/or ligation steps, and other procedures that result in a construct that can potentially be expressed in a host cell.
  • percent identity and % identity refer to the percentage of residue matches between at least two polynucleotide sequences aligned using a standardized algorithm. Such an algorithm may insert, in a standardized and reproducible way, gaps in the sequences being compared in order to optimize alignment between two sequences, and therefore achieve a more meaningful comparison of the two sequences.
  • Percent identity may be measured over the length of an entire defined polynucleotide sequence, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polynucleotide sequence, for instance, a fragment of at least 45, at least 60, at least 90, at least 120, at least 150, at least 210 or at least 450 contiguous residues.
  • Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures or Sequence Listing, may be used to describe a length over which percentage identity may be measured.
  • Percent (%) amino acid sequence identity is defined as the percentage of amino acid residues in a query sequence that are identical with the amino acid residues of a second, reference polypeptide sequence or a portion thereof, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software.
  • Percent identity may be measured over the length of an entire defined polypeptide sequence, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide sequence, for instance, a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70 or at least 150 contiguous residues.
  • Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures or Sequence Listing, may be used to describe a length over which percentage identity may be measured.
  • non-repetitiveness refers to a lack or limited degree of internal homology in a peptide or polypeptide sequence.
  • substantially non-repetitive can mean, for example, that there are few or no instances of four contiguous amino acids in the sequence that are identical amino acid types or that the polypeptide has a subsequence score (defined infra) of 10 or less or that there isn't a pattern in the order, from N- to C-terminus, of the sequence motifs that constitute the polypeptide sequence.
  • the term “repetitiveness” as used herein in the context of a polypeptide refers to the degree of internal homology in a peptide or polypeptide sequence. In contrast, a
  • “repetitive” sequence may contain multiple identical copies of short amino acid sequences. For instance, a polypeptide sequence of interest may be divided into n-mer sequences and the number of identical sequences can be counted. Highly repetitive sequences contain a large fraction of identical sequences while non-repetitive sequences contain few identical sequences. In the context of a polypeptide, a sequence can contain multiple copies of shorter sequences of defined or variable length, or motifs, in which the motifs themselves have non- repetitive sequences, rendering the full-length polypeptide substantially non-repetitive. The length of polypeptide within which the non-repetitiveness is measured can vary from 3 amino acids to about 200 amino acids, about from 6 to about 50 amino acids, or from about 9 to about 14 amino acids.
  • Repetitiveness used in the context of polynucleotide sequences refers to the degree of internal homology in the sequence such as, for example, the frequency of identical nucleotide sequences of a given length. Repetitiveness can, for example, be measured by analyzing the frequency of identical sequences.
  • treatment or “treating,” or “palliating” or “ameliorating” is used interchangeably herein. These terms refer to an approach for obtaining beneficial or desired results including but not limited to a therapeutic benefit and/or a prophylactic benefit.
  • therapeutic benefit is meant eradication or amelioration of the underlying disorder being treated.
  • a therapeutic benefit is achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the pediatric subject, notwithstanding that the subject may still be afflicted with the underlying disorder.
  • the therapeutic benefits may include sustained or normalized parameters such as, but not limited to, annual height velocity (cm/yr) in the first year (12 months) of treatment, IGF-I standard deviation score (IGF-I SDS), height standard deviation score, body weight, body mass index bone age, and pubertal staging.
  • a preferred therapeutic benefit is sustained or normalized annual height velocity (cm/yr) in the first year (12 months) of treatment.
  • the compositions may be administered to a pediatric subject at risk of developing a particular disease, or to a pediatric subject reporting one or more of the physiological symptoms of a disease, even though a diagnosis of this disease may not have been made.
  • the prophylactic benefits may include sustained or normalized parameters such as, but not limited to, annual height velocity (cm/yr) in the first year (12 months) of treatment, IGF-I standard deviation score (IGF-I SDS), height standard deviation score, body weight, body mass index bone age, and pubertal staging.
  • a preferred prophylactic benefit is sustained or normalized annual height velocity (cm/yr) in the first year (12 months) of treatment.
  • a “therapeutic effect”, as used herein, refers to a physiologic effect, including but not limited to the cure, mitigation, amelioration, or prevention of disease in humans or other animals, or to otherwise enhance physical or mental wellbeing of humans or animals, caused by a fusion polypeptide of the invention other than the ability to induce the production of an antibody against an antigenic epitope possessed by the biologically active protein.
  • the therapeutic effect may include sustained or normalized parameters such as, but not limited to, annual height velocity (cm/yr) in the first year (12 months) of treatment, IGF-I standard deviation score (IGF-I SDS), height standard deviation score, body weight, body mass index bone age, and pubertal staging.
  • a preferred therapeutic effect is sustained or normalized annual height velocity (cm/yr) in the first year (12 months) of treatment.
  • terapéuticaally effective amount refers to an amount of a biologically active protein, either alone or as a part of a fusion protein composition, that is capable of having any detectable, beneficial effect on any symptom, aspect, measured parameter or characteristics of a disease state or condition when administered in one or repeated doses to a pediatric subject. Such effect need not be absolute to be beneficial.
  • the present invention is based, at least in part, on the finding that hGH covalently fused to extended recombinant polypeptides ("XTE " or "XTENs") is highly soluble and stable in aqueous liquid solution at a relatively low pH and in the absence of a surfactant.
  • XTE extended recombinant polypeptides
  • liquid aqueous formulations of hGH-XTEN according to the invention remain stable during storage over a period of at least about 24 months and longer when stored refrigerated (5 ⁇ 3°C) or stable during storage over a period of at least about 1 month and longer when stored at 25 ⁇ 3 °C.
  • hGH-XTEN can be formulated as a storage stable aqueous liquid formulation including a buffer and a tonicifying agent, free of a surfactant.
  • the formulation does not contain a preservative.
  • the hGH-XTEN of the formulation is the sequence of SEQ ID NO: 1.
  • the appearance of these solutions is a clear, pale yellow to pale reddish brown liquid, free from visible particles.
  • Suitable pH ranges, adjusted with buffer, for the aqueous hGH formulation are from about 4 to about 6, more preferably about 5 to about 6, most advantageously about 5.5.
  • a buffer concentration range is chosen to minimize deamidation, aggregation, and/or precipitation of hGH-XTEN.
  • the buffer concentration in the formulations of the present invention is in the range of about 2 mM to about 50 mM, preferably about 10 mM to about 40 mM, more preferably about 15 mM to about 25 mM, most preferably about 20 mM.
  • the buffer is a histidine buffer.
  • the buffer is L-histidine used in the formulation at a concentration of about 20 mM.
  • Biological buffers with similar effective pH ranges, for example citrate, maleate, and bis-TRIS, may be substituted for L-histidine.
  • the salt concentration is adjusted to near isotonicity, depending on the other ingredients present in the formulation.
  • concentration range of NaCl may be about 50-200 mM, depending on the other ingredients present.
  • other tonicifying agents can also be used such as, for example, MgCl 2 or CaCl 2 .
  • the concentration of hGH-XTEN in the formulations of the present invention is in the range of about 25 mg/ml to about 150 mg/ml.
  • the concentration of hGH-XTEN in the formulations is about 100 mg/ml.
  • the concentration of hGH-XTEN in the formulations is about 150 mg/ml.
  • the formulations herein have an osmolality between about 300 mOsm to about 600 mOsm, preferably between about 400 mOsm and about 500 mOsm.
  • Mannitol may optionally be included in the aqueous hGH formulations of the present invention.
  • the preferred concentration of mannitol is about 5 mg/ml to about 50 mg/ml.
  • other sugars or sugar alcohols are used, such as lactose, trehalose, stachyose, sorbitol, xylitol, ribitol, myoinositol, galactitol, and the like.
  • the formulation of the invention is storage stable for at least about one month when stored at about 23 ⁇ 3°C, and for at least about 6 months to about 18 months when stored at about 5 ⁇ 3 °C. In a preferred embodiment, the formulation is storage stable for at least about 6 months to about 3 years when stored at about 5 ⁇ 3 °C.
  • the oxidation of the fusion protein in the formulations herein is reduced as compared to the oxidation of the corresponding fusion protein formulated with a surfactant.
  • the isolated fusion protein does not show detectable oxidation or deamidation following incubation for about 8 hours at about 40 °C.
  • Another aspect of the invention provides a method of preparing (making) a storage stable aqueous liquid formulation in which a fusion protein comprising an extended recombinant polypeptide (XTEN) fused to a growth hormone (GH) sequence, wherein the GH sequence is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:43, is admixed with a buffer in the absence of a surfactant, and adjusting the pH of the mixture to obtain a pH of about 4 to about 6.
  • a fusion protein comprising an extended recombinant polypeptide (XTEN) fused to a growth hormone (GH) sequence
  • GH sequence growth hormone
  • a preferred embodiment of the present invention is prepared by dissolving about 25 mg/ml to about 150 mg/ml of hGH-XTEN in 950 ml sterile water for injection USP, 9.00 g NaCl (low endotoxin; Merck Chemicals #116224) USP (at 0.154M final volume), 3.103g L- Histidine (Avantor #2080) USP (at 0.02M final volume), adjusted to pH 5.5 +/- 0.1 at 18 +/- 1 C with 5M HC1. The final volume is adjusted to 1 L with sterile water for injection USP.
  • the conductivity of the formulation solution at 25 +/- 1 C is about 10 to about 20 mS/cm.
  • the above quantities are somewhat flexible within ranges, as set forth in more detail above, and that the materials are interchangeable within the component categories.
  • More than one buffering agent, preservative, sugar, or neutral salt may be used.
  • the formulation is isotonic and sterile.
  • the formulations of the subject invention may contain other components in amounts not detracting from the preparation of stable forms and in amounts suitable for effective, safe pharmaceutical administration.
  • other pharmaceutically acceptable excipients well known to those skilled in the art may form a part of the subject compositions. These include, for example, various bulking agents, additional buffering agents, chelating agents, antioxidants, cosolvents and the like; specific examples of these could include citrate or succinate buffers, and disodium edetate. IV).
  • the present invention concerns an improved therapeutic aqueous liquid formulation comprising hGH-XTEN fusion protein.
  • the present invention concerns a method of treating human growth hormone deficiency (GHD) in patients with a hGH-XTEN fusion protein formulation.
  • GDD human growth hormone deficiency
  • the invention contemplates hGH-XTEN in the formulations comprising sequences with homology to hGH sequences, sequence fragments that are natural, such as from humans and non-natural sequence variants which retain at least a portion of the biologic activity or biological function of hGH and/or that are useful for preventing, treating, mediating, or ameliorating a hGH-related disease, deficiency, disorder or condition in pediatric patients.
  • native sequences homologous to human GH may be found by standard homology searching techniques, such as NCBI BLAST.
  • the hGH of the subject compositions, together with their corresponding nucleic acid and amino acid sequences, are well known in the art, described in in, for example, W013/184216, WO/2014/164568, U.S.
  • Patent Nos. 8,492,530 and 8,703,717 and descriptions and sequences are available in public databases such as Chemical Abstracts Services Databases (e.g., the CAS Registry), GenBank, The Universal Protein Resource (UniProt) and subscription provided databases such as GenSeq (e.g., Derwent).
  • Chemical Abstracts Services Databases e.g., the CAS Registry
  • GenBank GenBank
  • UniProt Universal Protein Resource
  • GenSeq e.g., Derwent
  • hGH effects of hGH on the tissues of the body can generally be described as anabolic. Like most other protein hormones, native hGH acts by interacting with a specific plasma membrane receptor, referred to as growth hormone receptor. hGH acts on the liver and other tissues to stimulate production of IGF-I, which is responsible for the growth promoting effects of hGH and also reflects the amount produced. IGF-I, in turn, has stimulatory effects on osteoblast and chondrocyte activity to promote bone growth.
  • the invention provides a hGH-XTEN formulation that exhibits at least one of the properties of native hGH hereinabove described herein.
  • the hGH incorporated into the subject hGH-XTEN formulation is a recombinant polypeptide with a sequence corresponding to a protein found in nature.
  • the hGH is a sequence variant, fragment, homolog, or a mimetics of a natural sequence that retains at least a portion of the biological activity of the corresponding native GH.
  • the hGH is recombinant human GH comprising the following amino acid sequence:
  • Human GH that can be incorporated into a hGH-XTEN fusion protein can include a protein that exhibits at least about 80% sequence identity, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:43.
  • the hGH fusion proteins suitable for use in the present invention comprise a human growth hormone polypeptide and one or more XTEN sequences as described herein, and as disclosed in, for example, W013/184216, WO/2014/164568 and U.S. Patent Nos. 8,492,530 and 8,703,717, each of which is incorporated herein by reference in its entirety.
  • the hGH-XTEN fusion proteins are isolated monomelic fusion proteins of hGH comprising the full-length sequence or sequence variants of hGH covalently linked to one or more extended recombinant polypeptides ("XTEN" or "XTENs").
  • XTEN extended recombinant polypeptides
  • the hGH-XTEN fusion protein comprises an amino acid sequence shown in FIG. 1 (SEQ ID NO: l), or pharmacologically active variants thereof.
  • the hGH-XTEN fusion protein comprises an amino acid sequence selected from Table 1.
  • the hGH-XTEN fusion protein sequence exhibits at least about 80% sequence identity, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a XTEN selected from Table 1.
  • the hGH-XTEN fusion protein VRS-317 (SEQ ID NO: 1), is composed of recombinant human growth hormone (rhGH) and two recombinant polypeptides, referred to as XTEN as described in Schellenberger et al. (2009). Nat Biotechnol 27, 1186-90, Schellenberger et al. WO10/144502A2, and WO10/091122, each of which are incorporated herein by reference in their entirety.
  • the XTEN domain two unstructured hydrophilic chains of amino acids, provides half-life extension for rhGH.
  • the molecular weight of VRS-317 is 118.9 kDa, with rhGH contributing 22.1 kDa and the remaining mass contributed by the XTEN construct.
  • the mass ratio of rhGH to VRS-317 is therefore 1 :5.37.
  • Further characterization of the exemplary hGH-XTEN fusion proteins provided in Table 1 can be found in WO10/144502A2, which is incorporated herein by reference in its entirety.
  • AGSPTSTEEGTSTEPSE GTACTTCTGAAAGCGCTACCCCGGAATCTG
  • GSAPGTSTEPSEGSAPG GCCCAGGTAGCGAACCGGCTACTTCTGGTT
  • ATPESGPGSPAGSPTST ACCAGGTACTTCTACCGAACCGTCCGAGGG
  • EEGTSESATPESGPGSEP TAGCGCACCAGGTAGCCCAGCAGGTTCTCC
  • GSAPGSPAGSPTSTEEG TAGCGCACCAGGTACTTCTGAAAGCGCAAC
  • ATPESGPGSPAGSPTST TCCGGCCCAGGTACCTCTGAAAGCGCAACC
  • AGSPTSTEEGTSESATP TCTCCAACCTCCACCGAAGAAGGTACCTCT
  • GSETPGTSESATPESGP CCAGGTACCTCTGAAAGCGCTACTCCGGAG
  • PAGSPTSTEEGSPAGSP CAGGTACCTCTACCGAACCTTCTGAAGGTA
  • GSETPGTSESATPESGP GGAATCTGGTCCAGGTAGCGAACCTGCAAC
  • KVETFLRIVQCRSVEGS TGAGTCTGGCCCAGGTAGCGAACCTGCTAC
  • APGSPAGSPTSTEEGTS AAGCGCAACCCCTGAATCCGGCCCAGGTAG
  • GSAPGFPTIPLSRLFDNA CACCAGGTACTTCCCCTAGCGGTGAATCTTC
  • KDLEEGIQTLMGRLED CCGGCCCAGGTAGCGAACCGGCTACTTCCG
  • VQCRSVEGSCGF CTTCTCCTAGCGGCGAATCTTCTACCGCTCC
  • GSEGSGEGEGSEGSGEG CTGGCGAAGGTGAAGGTTCTGAAGGTGGCT
  • AGSPTSTEEGTSTEPSE GTACTTCTGAAAGCGCTACCCCGGAATCTG
  • GSAPGTSTEPSEGSAPG GCCCAGGTAGCGAACCGGCTACTTCTGGTT
  • ATPESGPGSPAGSPTST ACCAGGTACTTCTACCGAACCGTCCGAGGG
  • EEGTSESATPESGPGSEP TAGCGCACCAGGTAGCCCAGCAGGTTCTCC
  • GSAPGSPAGSPTSTEEG TAGCGCACCAGGTACTTCTGAAAGCGCAAC
  • ATPESGPGSPAGSPTST TCCGGCCCAGGTACCTCTGAAAGCGCAACC
  • AGSPTSTEEGTSESATP TCTCCAACCTCCACCGAAGAAGGTACCTCT
  • GSETPGTSESATPESGP CCAGGTACCTCTGAAAGCGCTACTCCGGAG
  • PAGSPTSTEEGSPAGSP CAGGTACCTCTACCGAACCTTCTGAAGGTA
  • GSETPGTSESATPESGP GGAATCTGGTCCAGGTAGCGAACCTGCAAC
  • KVETFLRIVQCRSVEGS TGAGTCTGGCCCAGGTAGCGAACCTGCTAC
  • AGSPTSTEEGTSTEPSE GTACTTCTGAAAGCGCTACCCCGGAATCTG
  • GSAPGTSTEPSEGSAPG GCCCAGGTAGCGAACCGGCTACTTCTGGTT
  • ATPESGPGSPAGSPTST ACCAGGTACTTCTACCGAACCGTCCGAGGG
  • GSAPGSPAGSPTSTEEG TAGCGCACCAGGTACTTCTGAAAGCGCAAC
  • ATPESGPGSPAGSPTST TCCGGCCCAGGTACCTCTGAAAGCGCAACC
  • AGSPTSTEEGTSESATP TCTCCAACCTCCACCGAAGAAGGTACCTCT
  • GSETPGTSESATPESGP CCAGGTACCTCTGAAAGCGCTACTCCGGAG
  • PAGSPTSTEEGSPAGSP CAGGTACCTCTACCGAACCTTCTGAAGGTA
  • GSETPGTSESATPESGP GGAATCTGGTCCAGGTAGCGAACCTGCAAC
  • KVETFLRIVQCRSVEGS TGAGTCTGGCCCAGGTAGCGAACCTGCTAC
  • ATSGSETPGTSESATPES TCCAACCTCCACCGAAGAAGGTACCTCTGA
  • GPGSPAGSPTSTEEGSP AAGCGCAACCCCTGAATCCGGCCCAGGTAG
  • AGSPTSTEEGTSTEPSE CGAACCGGCAACCTCCGGTTCTGAAACCCC
  • PESGPGSEPATSGSETP TTCCACCGAGGAAGGTAGCCCGGCTGGCTC
  • ATPESGPGSPAGSPTST ACCAGGTTCTACTAGCGAATCCCCGTCTGGT
  • SAPGTSTEPSEGSAPGSP AAGCGGTTCTGCATCTCCAGGTAGCGAACC
  • AGSPTSTEEGTSTEPSE GGCAACCTCCGGCTCTGAAACCCCAGGTAC
  • EGTSTEPSEGSAPGASA TCTGAAAGCGCTACTCCTGAATCCGGTCCA
  • GPGSPAGSPTSTEEGSP GCTCCAGGTACCTCTACCGAACCGTCCGAG
  • GSETPGTSESATPESGP GGAAGGTAGCTCTACCCCGTCTGGTGCTAC
  • GSSTPSGATGSPGSSPS CCACCGAGGAAGGTAGCCCGGCTGGCTCTC
  • APGFPTIPLSRLFDNAM TACTTCCCCTAGCGGTGAATCTTCTACTGCA
  • DLEEGIQTLMGRLEDGS GGTAGCGAACCGGCTACTTCCGGCTCTGAA
  • CRSVEGSCGF CGGCGAATCTTCTACCGCTCCAGGTAGCGA
  • the fusion proteins optionally include spacer sequences that further comprise cleavage sequences to release the hGH from the fusion protein when acted on by a protease, releasing hGH from the XTEN sequence(s).
  • the present invention comprises formulations comprising XTEN polypeptide compositions that are useful as a fusion protein partner to which hGH is linked, resulting in a hGH-XTEN fusion protein.
  • XTEN are generally extended length polypeptides with non- naturally occurring, substantially non-repetitive sequences that are composed mainly of small hydrophilic amino acids, with the sequence having a low degree or no secondary or tertiary structure under physiologic conditions.
  • features of XTEN sequences such as the non-repetitiveness of sequences, exemplary sequence motifs, and lengths of sequences are known in the art and are described at least in, for example,
  • the hGH-XTEN of the formulation can comprise one or more XTEN sequence wherein the sequence exhibits at least about 80% sequence identity, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a XTEN selected from Table 2.
  • XTEN selected from Table 2.
  • Examples where more than one XTEN is used in a hGH-XTEN composition include, but are not limited to constructs with an XTEN linked to both the N- and C-termini of at least one hGH. In preferred embodiments of the present invention comprising constructs with an XTEN linked to both the N- and C-termini of at least one hGH.
  • constructs with an XTEN linked to both the N- and C-termini of at least one hGH in preferred embodiments of the present invention comprising constructs with an XTEN linked to both the
  • the length of the N-terminal XTEN sequence ranges between about 500 amino acids to about 1320 amino acids and the length of the C-terminal XTEN ranges between about 40 amino acids to about 300 amino acids.

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Abstract

A stable pharmaceutically acceptable aqueous formulation containing human growth hormone linked to an extended recombinant polypeptide (XTEN) and a buffer is disclosed, wherein the formulation is lacking a surfactant. Also disclosed are associated methods for preparing, storing, and using such formulations.

Description

GROWTH HORMONE FORMULATION
FIELD OF THE INVENTION
The present invention is directed to pharmaceutical formulations containing a fusion protein comprising human growth hormone (hGH) and to methods for making and using such formulations. More particularly, this invention relates to liquid hGH formulations with increased stability.
BACKGROUND OF THE INVENTION
Native human growth hormone (hGH) in its most prominent mature form of pituitary
(pit)-hGH is a single polypeptide chain protein consisting of 191 amino acids. The protein is internally cross-linked by two disulphide bridges and in monomelic form has a molecular weight of 22 kDa. Growth hormone (GH) of animal species is closely homologous in amino acid sequence to that of humans and is therefore very similar in its characteristics. A major biological effect of GH is to promote growth throughout a range of organs and tissues in the body. GH responsive organs or tissues include the liver, intestine, kidneys, muscles, connective tissue and the skeleton. For a more detailed review of the stability and characterization of hGH, see Pearlman and Bewley, Chapter 1 in Stability and
Characterization of Protein and Peptide Drugs: Case Histories (Plenum Press, New York, 1993).
Methods for the recombinant production of recombinant hGH (rhGH) are described in U.S. Patent No. 4,601,980 and Goeddel et al, Nature 1979, 281(5732):544-8.
Recombinant hGH (rhGH) is used as a therapeutic and has been approved for the treatment of a number of indications. rhGH is currently sold in a variety of forms worldwide including capsules, syrups, sprays, tablets, powders, vials, and injections. Numerous injectable products are currently on the market in the United States: for instance,
Humatrope™ (Eli Lilly & Co.), Nutropin™ (Genentech), Norditropin™ (Novo-Nordisk), Omnitrope™ (Sandoz/Novartis), Genotropin™ (Pfizer), Saizen/Serostim™ (Serono), Tev- Tropin™ (Teva), and Zorbtive™ (EMD Serono). hGH deficiency leads to dwarfism, for example, which has been successfully treated for decades by exogenous administration of the hormone. In addition to hGH deficiency, hGH has also been approved for the treatment of several other conditions including renal failure (in children), Turner's Syndrome, and cachexia in AIDS patients. The Food and Drug Administration (FDA) has approved hGH for the treatment of non-GH-dependent short stature and hGH has been investigated for the treatment of a variety of physiological conditions, including aging, frailty in the elderly, short bowel syndrome, and congestive heart failure. Target populations for hGH treatment include children with idiopathic short stature (ISS) and adults with GHD-like symptoms.
A long appreciated problem with aqueous liquid formulations of pharmaceutical proteins, not just hGH, has been that of instability during storage over a period of time.
While liquid hGH formulations are described in the patent literature (see, e.g. U.S. Patent Nos. 5,763,394; 7,662,393; 8,338,374 and 8,409,586), many of the major injectable hGH products are formulated as lyophilized preparations requiring reconstitution prior to use. For example, in the United States, Genotropin™ lyophilized powder is provided in a 5 mg or 12 mg two-chamber cartridge to be reconstituted with water for injection
(www.pfizermedicalinformation.com/en-us/genotropin; accessed September 3, 2015);
Humatrope™ lyophilized powder is provided in a 5 mg vial or 6-24 mg cartridge to be reconstituted with water for injection (pi.lilly.com/us/humatrope-pi.pdf; accessed September 3, 2015).
Thus, there remains a need to develop aqueous liquid formulations of hGH that do not require reconstitution and are chemically stable for an extended period, which have the advantages of eliminating reconstitution errors, thereby increasing dosing accuracy, as well as simplifying the use of the product clinically, thereby increasing patient compliance. Such formulations also allow for improvements in the design and use of pre-filled syringes or other devices, which are sold to patients containing pre-mixed liquid formulations of rhGH (e.g., Norditropin™). Therapeutic aqueous liquid formulations of hGH preferably should be stable for 2-3 years at refrigerated temperatures and for several weeks near room temperature. For example, prior to initial use, the refrigerated shelf-life of the Norditropin FlexPro pre-filled pen is 2 years
(www. novonordi sk. com/ content/ dam/Denmark/HQ/hcp/Documents/Norditropin%20FlexPro- SmPC_August2012.pdf; accessed September 3, 2015). However, a major challenge facing the development of such viable aqueous liquid formulations of hGH is that hGH in aqueous solution is known to undergo a variety of degradative changes.
Human growth hormone degrades by three primary pathways: deamidation, oxidation and aggregation (Pearlman and Bewley, supra at 26-42; Cleland et al., Crit Rev Ther Drug Carrier Syst. 1993; 10(4):307-77). Deamidation is minimized between pH 5 and 6 and more preferably near pH 5.5 (Pearlman and Bewley, supra at 27-29). However, solubility of hGH decreases substantially at this pH because the pi of hGH is 5.2 {Id at 17). Formulations have therefore been developed to balance solubility and deamidation. Agitation induced aggregation of hGH occurs due to the air-water interface denaturation of hGH (Pearlman and Bewley, supra at 36-40; Bam et al, Journal of Pharmaceutical Sciences Vol. 87, No. 12, December 1998). To inhibit aggregation in hGH formulation, surfactants have been added and have been shown to bind to hGH blocking aggregation (Bam et al., Pharmaceutical Research, Vol. 12, No. 1, 1995). These surfactants (e.g. polysorbate 20, polyethylene glycol (PEG)) generate free radical oxygen upon storage causing oxidation of exposed methionine residues in hGH. Overall, hGH formulations have been developed to balance the degradation pathways to enable sufficient stability for commercial application (e.g., 2 years at refrigerated conditions).
This invention provides a highly stable aqueous liquid formulation of a fusion protein containing rhGH. In particular, the invention provides a liquid formulation of a rhGH-XTEN fusion protein that exhibits excellent long-term stability without the use of a surfactant.
SUMMARY OF THE INVENTION
A stable pharmaceutically acceptable aqueous formulation containing a fusion protein comprising human growth hormone linked to an extended recombinant polypeptide (XTEN) and a buffer is disclosed, wherein the formulation is lacking a surfactant. Also disclosed are associated methods for preparing, storing, and using such formulations.
Aspects of the invention include a storage stable aqueous liquid formulation comprising (i) about 25 mg/ml to about 150 mg/ml of a fusion protein comprising an extended recombinant polypeptide (XTEN) fused to a growth hormone (GH) sequence, wherein the GH sequence is at least 90% identical to the amino acid sequence of SEQ ID NO:43, and (ii) a buffer; characterized in that the aqueous liquid formulation is at a pH of about 4 to about 6, in the absence of a surfactant. In some embodiments, the aqueous liquid formulation is characterized in that it is free of a preservative. In some embodiments, the aqueous liquid formulation is characterized in that the pH is about 5 to about 6. In some embodiments, the aqueous liquid formulation is characterized in that the pH is 5.2 to 5.8. In some embodiments, the aqueous liquid formulation is characterized in that the pH is about 5.5. In some embodiments, the aqueous liquid formulation is characterized in that the buffer is selected from the group consisting of histidine, citrate, and succinate buffers. In some embodiments, the aqueous liquid formulation is characterized in that the buffer is a histidine buffer at a concentration of about 20 mM.
In some embodiments, the aqueous liquid formulation is characterized in that the concentration of the fusion protein is selected from the group consisting of about 25 mg/ml, about 50 mg/ml, about 75 mg/ml, about 100 mg/ml, about 125 mg/ml, and about 150 mg/ml. In some embodiments, the aqueous liquid formulation is characterized in that the
concentration of the fusion protein is about 50 mg/ml. In some embodiments, the aqueous liquid formulation is characterized in that the concentration of the fusion protein is about 100 mg/ml. In some embodiments, the aqueous liquid formulation is characterized in that the concentration of the fusion protein is about 150 mg/ml.
In some embodiments, the aqueous liquid formulation is characterized in that the aqueous liquid formulation is storage stable for at least about one month at about 23±3°C. In some embodiments, the aqueous liquid formulation is characterized in that the aqueous liquid formulation is storage stable for at least about 6 months to about 3 years at about 5±3 °C. In some embodiments, the aqueous liquid formulation is characterized in that the aqueous liquid formulation is storage stable for at least about 6 months to about 18 months at about 5±3 °C. In some embodiments, the aqueous liquid formulation is characterized in that the aqueous liquid formulation is storage stable at about 5±3 °C for at least about 3 months, for at least about 6 months, for at least about 9 months, for at least about 12 months, for at least about 18 months, for at least about 21 months, for at least about 24 months, for at least about 27 months, for at least about 30 months, for at least about 36 months, or for at least about 42 months.
In some embodiments, the aqueous liquid formulation is characterized in that the oxidation of the fusion protein is reduced as compared to the oxidation of the corresponding fusion protein formulated with a surfactant. In some embodiments, the aqueous liquid formulation is characterized in that the deamidation of the fusion protein is reduced as compared to the deamidation of the corresponding fusion protein formulated at a higher pH. In some embodiments, the aqueous liquid formulation is characterized in that the isolated fusion protein does not show detectable oxidation or deamidation following incubation for about 8 hours at about 40 °C.
In some embodiments, the aqueous liquid formulation is characterized in that it further comprises a tonicifying agent. In some embodiments, the aqueous liquid formulation is characterized in that the tonicifying agent is NaCl. In some embodiments, the aqueous liquid formulation is characterized as having an osmolality between about 300 mOsm to about 600 mOsm.
In some embodiments, the aqueous liquid formulation is characterized in that the formulation is disposed in a container containing at least one dose. In some embodiments, the container comprises a syringe. In some embodiment, the container comprises a glass cartridge. In some embodiments, the container comprises a vial.
In some embodiments, the aqueous liquid formulation is characterized in that it is for injection into a subject with a needle. In some embodiments, the needle size is selected from the group consisting of 26 gauge, 27 gauge, 29 gauge, and 30 gauge.
In some embodiments, the aqueous liquid formulation is characterized in that it comprises the fusion protein according to the formula I:
(XTEN l )x-GH-(XTEN_2)y
wherein independently for each occurrence:
(a) x is either 0 or 1; and
(b) y is either 0 or 1, wherein x+y>l;
wherein XTEN l and XTEN 2 comprise identical or different sequences. In some embodiments, the aqueous liquid formulation is characterized in that the XTEN l comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 18-34, 38, 39, 41, and 42. In some embodiments, the aqueous liquid formulation is characterized in that the XTEN_2 comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 13-17, 35, 36, 37, and 40. In some embodiments, the aqueous liquid formulation is characterized in that the XTEN l comprises SEQ ID NO:39. In some embodiments, the aqueous liquid formulation is characterized in that the XTEN 2 comprises SEQ ID NO: 35. In some embodiments, the aqueous liquid formulation is characterizes in that XTEN l comprises SEQ ID NO: 39 and XTEN 2 comprises SEQ ID NO: 35. In some embodiments, the aqueous liquid formulation is characterized in that the fusion protein sequence is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the aqueous liquid formulation is characterized in that the fusion protein comprises the amino acid sequence of SEQ ID NO: 1. Some aspects of the invention relate to a method of making a storage stable aqueous liquid formulation of a fusion protein comprising an extended recombinant polypeptide (XTEN) linked to a growth hormone (GH) sequence, wherein the GH sequence is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:43, comprising mixing said fusion protein and an aqueous, pharmaceutically acceptable vehicle which includes i) said fusion protein at a concentration of about 25 mg/ml to about 150 mg/ml; and ii) a buffer providing a pH of about 4 to about 6; wherein said aqueous liquid formulation is free of a surfactant. In some embodiments, the method is characterized in that it is formulated without use of a preservative.
Some aspects of the invention relate to a method of treating a growth-hormone related condition in a subject, comprising administering to the subject a therapeutically effective amount of the aqueous liquid formulation according to the presently described invention, wherein the growth-hormone related condition is selected from the group consisting of growth-hormone deficiency, Turner's Syndrome, Prader-Willi Syndrome, small for gestational age (SGA), idiopathic short stature, AIDS wasting, multiple sclerosis, Crohn's disease, ulcerative colitis, and muscular dystrophy. In some embodiments, the method is characterized in that the therapeutically effective amount is administered at least 48 h, or at least 72 h, or at least about 96 h, or at least about 120 h, or at least about 7 days, or at least about 14 days, at least about 21 days, or at least about 30 days between consecutive doses. In some embodiments, the method is characterized in that the growth-hormone related condition is growth-hormone deficiency. In some embodiments, the method is characterized in that the growth-hormone related condition is Turner's Syndrome. In some embodiments, the method the method is characterized in that the growth-hormone related condition is Prader-Willi Syndrome. In some embodiments, the method is characterized in that the growth-hormone related condition is idiopathic short stature. In some embodiments, the method is
characterized in that the growth-hormone related condition is small for gestational age (SGA). In some embodiments, the method is characterized in that the growth-hormone related condition is selected from the group consisting of AIDS wasting, multiple sclerosis, Crohn' s disease, ulcerative colitis, and muscular dystrophy.
Some aspects of the invention relate to the use of a therapeutically effective amount of the aqueous liquid formulation of according to the presently described invention in the preparation of a medicament for treating a growth-hormone related condition, wherein the growth-hormone related condition is selected from the group consisting of growth-hormone deficiency, Turner's Syndrome, Prader-Willi Syndrome, small for gestational age (SGA), idiopathic short stature, AIDS wasting, multiple sclerosis, Crohn's disease, ulcerative colitis, and muscular dystrophy.
In some embodiments, the aqueous liquid formulation is characterized in that it is for use in treating a growth-hormone related condition, wherein the growth-hormone related condition is selected from the group consisting of growth-hormone deficiency, Turner's Syndrome, Prader-Willi Syndrome, small for gestational age (SGA), idiopathic short stature, AIDS wasting, multiple sclerosis, Crohn's disease, ulcerative colitis, and muscular dystrophy.
INCORPORATION BY REFERENCE
All publications, patent and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application were specifically and individually indicated to be incorporated by reference.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 provides the amino acid sequence of an hGH-XTEN fusion protein designated VRS-317 (hGH sequence is underlined and shown in bold) (SEQ ID NO: 1).
FIG. 2 is a strong anion exchange chromatogram of an aqueous formulation according to the present invention showing degradation of VRS-317 at 40°C via hydrolysis, or truncation, of the XTEN polypeptide in VRS-317.
FIG. 3 is a strong anion exchange chromatogram of an aqueous formulation according to the present invention containing different concentrations of VRS-317 showing that VRS- 317 in the formulation is not degraded.
FIG. 4. is a size exclusion chromatogram of an aqueous formulation according to the present invention containing different concentrations of VRS-317 showing that VRS-317 in the formulation is not aggregated.
FIG. 5. is a size exclusion chromatogram of an aqueous formulation according to the present invention containing different concentrations of VRS-317, showing that VRS-317 in the formulation is not aggregated after passing through a 30 gauge needle.
FIG. 6 is a reverse phase chromatogram of tryptic fragments of control VRS-317 (black) or VRS-317 treated with hydrogen peroxide for 72 hours (gray). Inset: A & B are expanded views of the parts of the chromatogram (underlined) where degradation of VRS- 317 was observed.
FIG. 7 is a reverse phase chromatogram of tryptic fragments of control VRS-317 (black) or VRS-317 stressed with elevated pH and temperatures (gray). Inset: An expanded view of the reverse phase chromatogram, highlighting the observed degradation.
FIG. 8 shows SE-HPLC results comparing the degree of VRS-317 aggregation seen in aqueous formulations according to the present invention in the presence or absence of surfactant, following 4 hour agitation stress experiments at ambient temperature.
FIG. 9 shows graphs of integrated percent peak areas from the size exclusion chromatogram of an aqueous formulation according to the present invention containing VRS- 317 sampled periodically over up to 24 months of storage at 5°C or 25°C.
FIG. 10 shows a graph of integrated percent peak areas from the reverse phase chromatogram of tryptic fragments of an aqueous formulation according to the present invention containing VRS-317 sampled over 1 month of storage at 25°C.
FIG. 11 shows a graph of pH readings taken periodically over up to 24 months of an aqueous formulation according to the present invention containing VRS-317 stored at 5°C or 25°C.
DETAILED DESCRIPTION OF THE INVENTION
I) DEFINITIONS
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which the inventions described herein belong. For purposes of interpreting this specification, the following definitions will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. In the event that any definition set forth conflicts with any document incorporated herein by reference, the definition set forth below shall control.
The terms "polypeptide", "peptide", and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component. "Growth Hormone" or "GH" means a growth hormone polypeptide, including, without limitation, native sequence human and non-human GH, and amino acid sequence variants thereof having growth hormone biological activity. The term "native sequence" specifically encompasses naturally occurring truncated or variant forms, including alternatively spliced forms and naturally occurring allelic variants. A non-human grown hormone can be of any non-human animal species, such as a non-human mammalian species. The term "native sequence human growth hormone (hGH)" or "native hGH" includes, but is not limited to, the hGH originally derived from the pituitary gland having a molecular weight of about 22, 129 daltons (22kD hGH) and a naturally occurring variant having a molecular weight of about 20,000 daltons (20kD hGH). The 20kD hGH has an amino acid sequence that corresponds to that of 22kD hGH consisting of 191 amino acids except that 15 amino acid residues from the 32nd to the 46th of 22kD hGH are missing. Some reports have shown that the 20kD hGH has been found to exhibit lower risks and higher activity than 22kD hGH. The invention contemplates use of the 22 kD, the 20kD hGH, as well as species and sequence variants and truncated fragments thereof as being appropriate for use as a fusion partner with XTEN disclosed herein for hGH-XTEN compositions. The cloned gene for hGH has been expressed in a secreted form in Escherichia coli (United States Patent No. 4,898,830; Chang, C. N., et al., Gene 55: 189 [1987]) and its DNA and amino acid sequence has been reported (Goeddel, et al. Nature ,281 :544 [1979]); Gray, et al., Gene 39: 247[1985]). The terms "GH" and "hGH" specifically include recombinant human growth hormone (rhGH) produced in any recombinant host cell, including prokaryotic and eukaryotic hosts.
The term "storage stable", as it relates to a pharmaceutical liquid formulation comprising hGH fusion protein, is a formulation that results in less than 5% aggregation of hGH fusion protein, less than 15% deamidation, or less than 10% oxidation of hGH fusion protein when such pharmaceutical liquid formulation comprising hGH is stored at 2° C to 8° C. In one embodiment, such liquid formulation comprising hGH is long-term stable for at least 2-3 years, and in an alternate embodiment, such liquid formulation comprising hGH is storage stable for 24 months or for 6 to 18 months, or stable at least 12 months. As will be obvious to one skilled in the art, the long-term stability of hGH may be determined directly by incubating the formulations for the above noted times, or the long-term stability may be predicted by the methods described herein, or other known methods.
As used herein the term "amino acid" refers to either natural and/or unnatural or synthetic amino acids, including but not limited to glycine and both the D or L optical isomers, and amino acid analogs and peptidomimetics. Standard single or three letter codes are used to designate amino acids.
A "fragment" is a truncated form of a native biologically active protein that retains at least a portion of the therapeutic and/or biological activity. A "variant" is a protein with sequence homology to the native biologically active protein that retains at least a portion of the therapeutic and/or biological activity of the biologically active protein. For example, a variant protein may share at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity with the reference biologically active protein. As used herein, the term "biologically active protein moiety" includes proteins modified deliberately, as for example, by site directed mutagenesis, insertions, or accidentally through mutations. Thus, "GH variant," "hGH variant" or "rhGH variant" comprises an amino acid sequence which differs from that of a native sequence GH, hGH or rhGH by virtue of at least one amino acid modification, preferably one or more amino acid substitution(s). Preferably, the variant has at least one amino acid substitution compared to a native sequence polypeptide, e.g. from about one to about ten amino acid substitutions, and preferably from about one to about five amino acid substitutions in the sequence of the parent polypeptide.
The term "natural L-amino acid" means the L optical isomer forms of glycine (G), proline (P), alanine (A), valine (V), leucine (L), isoleucine (I), methionine (M), cysteine (C), phenylalanine (F), tyrosine (Y), tryptophan (W), histidine (H), lysine (K), arginine (R), glutamine (Q), asparagine (N), glutamic acid (E), aspartic acid (D), serine (S), and threonine (T).
The term "non-naturally occurring," as applied to sequences and as used herein, means polypeptide or polynucleotide sequences that do not have a counterpart to, are not complementary to, or do not have a high degree of homology with a wild-type or naturally- occurring sequence found in a mammal. For example, a non-naturally occurring polypeptide or fragment may share no more than 99%, 98%, 95%, 90%, 80%, 70%, 60%, 50% or even less amino acid sequence identity as compared to a natural sequence when suitably aligned.
The terms "hydrophilic" and "hydrophobic" refer to the degree of affinity that a substance has with water. A hydrophilic substance has a strong affinity for water, tending to dissolve in, mix with, or be wetted by water, while a hydrophobic substance substantially lacks affinity for water, tending to repel and not absorb water and tending not to dissolve in or mix with or be wetted by water. Amino acids can be characterized based on their hydrophobicity. A number of scales have been developed. An example is a scale developed by Levitt, M, et al, J Mol Biol (1976) 104:59, which is listed in Hopp, TP, et al, Proc Natl Acad Sci U S A (1981) 78:3824. Examples of "hydrophilic amino acids" are arginine, lysine, threonine, alanine, asparagine, and glutamine. Of particular interest are the hydrophilic amino acids aspartate, glutamate, and serine, and glycine. Examples of
"hydrophobic amino acids" are tryptophan, tyrosine, phenylalanine, methionine, leucine, isoleucine, and valine.
The term "hGH-XTEN", as used herein, is meant to encompass fusion polypeptides that comprise a payload region comprising a biologically active GH that mediates one or more biological or therapeutic activities associated with growth hormone and at least one other region comprising at least a first XTEN polypeptide that serves as a carrier. In one embodiment, the invention provides an hGH-XTEN fusion protein comprising a sequence set forth in Table 2. In a preferred embodiment, the invention provides an hGH-XTEN fusion protein comprising SEQ ID NO: 35 and SEQ ID NO: 39.
The term "buffer" or "physiologically-acceptable buffer" refers to solutions of compounds that are known to be safe for pharmaceutical or veterinary use in formulations and that have the effect of maintaining or controlling the pH of a formulation in the pH range desired for the formulation. Buffers typically involve a weak acid or alkali together with one of its salts. Acceptable buffers for controlling pH at a moderately acidic pH to a moderately basic pH include, but are not limited to, such compounds as phosphate, acetate, citrate, arginine, TRIS, and histidine. "TRIS" refers to 2-amino-2-hydroxymethyl-l, 3, -propanediol, and to any pharmacologically acceptable salt thereof.
"Pharmaceutically acceptable" excipients (vehicles, additives) are those commonly used in the art which can reasonably be administered to a subject mammal to provide an effective dose of the active ingredient employed. For parenteral administration (e.g.
subcutaneous or intramuscular, or topical administration) formulations may be converted into a solution, gel or emulsion, if desired, using the pharmaceutical substances customary for this purpose, such as solubilizers, thickening agents, emulsifiers, agents for tonicity, preservatives or other auxiliaries.
As used herein, the term "surfactant" is used to refer to a surface-active agent, typically a nonionic surfactant, understood and employed by those skilled in the art.
Examples of suitable surfactants include polysorbate (for example, polysorbate 20 and polysorbate 80); poloxamer (e.g. poloxamer 188); Triton; sodium dodecyl sulfate (SDS); sodium laurel sulfate; sodium octyl glycoside; lauryl-, myristyl-, linoleyl-, or stearyl- sulfobetaine; lauryl-, myristyl-, linoleyl- or stearyl-sarcosine; linoleyl-, myristyl-, or cetyl- betaine; lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-, myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine (e.g. lauroamidopropyl); myristamidopropyl- , palmidopropyl-, or isostearamidopropyl-dimethylamine; sodium methyl cocoyl-, or disodium methyl oleyl-taurate; polyethyl glycol, polypropyl glycol, and copolymers of ethylene and propylene glycol (e.g. Pluronics, PF68 etc).
A "sugar," or "saccharide," or "sugar alcohol," or "polyol" as used herein refers to a substances well known in the art with multiple hydroxyl groups, and includes reducing sugars, nonreducing sugars, sugar alcohols and sugar acids. A "reducing sugar" is one which contains a hemiacetal group that can reduce metal ions or react covalently with lysine and other amino groups in proteins and a "nonreducing sugar" is one which does not have these properties of a reducing sugar. Examples of reducing sugars are fructose, mannose, maltose, lactose, arabinose, xylose, ribose, rhamnose, galactose and glucose. Nonreducing sugars include sucrose, trehalose, stachyose, sorbose, melezitose and raffinose. Mannitol, xylitol, erythritol, threitol, sorbitol, ribitol, myoinositol, galactitol and glycerol are examples of sugar alcohols. As to sugar acids, these include L-gluconate and metallic salts thereof. Where it is desired that the formulation is freeze-thaw stable, the polyol is preferably one which does not crystallize at freezing temperatures (e.g. -20°C.) such that it destabilizes the fusion protein composition in the formulation. Polyols, including mixtures of polyols, can also be used as lyoprotectants in the formulations of the present invention, which protects a protein, e.g. an fusion protein, from damage resulting from lyophilization. Other stabilizing agents, such as metal chelating agents, may also be useful in the invention which are generally known in the art.
A "preservative" is understood in the art to be a compound which can be included in the formulation to substantially reduce bacterial action therein, thus facilitating the production of a multi-use formulation, for example. Examples of preservatives include octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride (a mixture of alkylbenzyldimethylammonium chlorides in which the alkyl groups are long-chain compounds), and benzelthonium chloride. Other types of preservatives include aromatic alcohols such as phenol, butyl and benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol.
"Conjugated", "linked," "fused," and "fusion" are used interchangeably herein. These terms refer to the joining together of two or more chemical elements or components, by whatever means including chemical conjugation or recombinant means. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and in reading phase or in-frame. An "in-frame fusion" refers to the joining of two or more open reading frames (ORFs) to form a continuous longer ORF, in a manner that maintains the correct reading frame of the original ORFs. Thus, the resulting recombinant fusion protein is a single protein containing two or more segments that correspond to polypeptides encoded by the original ORFs (which segments are not normally so joined in nature).
In the context of polypeptides, a "linear sequence" or a "sequence" is an order of amino acids in a polypeptide in an amino to carboxyl terminus direction in which residues that neighbor each other in the sequence are contiguous in the primary structure of the polypeptide. A "partial sequence" is a linear sequence of part of a polypeptide that is known to comprise additional residues in one or both directions.
"Recombinant" as applied to a polynucleotide means that the polynucleotide is the product of various combinations of in vitro cloning, restriction and/or ligation steps, and other procedures that result in a construct that can potentially be expressed in a host cell.
The terms "percent identity" and "% identity," as applied to polynucleotide sequences, refer to the percentage of residue matches between at least two polynucleotide sequences aligned using a standardized algorithm. Such an algorithm may insert, in a standardized and reproducible way, gaps in the sequences being compared in order to optimize alignment between two sequences, and therefore achieve a more meaningful comparison of the two sequences. Percent identity may be measured over the length of an entire defined polynucleotide sequence, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polynucleotide sequence, for instance, a fragment of at least 45, at least 60, at least 90, at least 120, at least 150, at least 210 or at least 450 contiguous residues. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures or Sequence Listing, may be used to describe a length over which percentage identity may be measured.
"Percent (%) amino acid sequence identity," with respect to the polypeptide sequences identified herein, is defined as the percentage of amino acid residues in a query sequence that are identical with the amino acid residues of a second, reference polypeptide sequence or a portion thereof, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. Percent identity may be measured over the length of an entire defined polypeptide sequence, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide sequence, for instance, a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70 or at least 150 contiguous residues. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures or Sequence Listing, may be used to describe a length over which percentage identity may be measured.
The term "non-repetitiveness" as used herein in the context of a polypeptide refers to a lack or limited degree of internal homology in a peptide or polypeptide sequence. The term "substantially non-repetitive" can mean, for example, that there are few or no instances of four contiguous amino acids in the sequence that are identical amino acid types or that the polypeptide has a subsequence score (defined infra) of 10 or less or that there isn't a pattern in the order, from N- to C-terminus, of the sequence motifs that constitute the polypeptide sequence. The term "repetitiveness" as used herein in the context of a polypeptide refers to the degree of internal homology in a peptide or polypeptide sequence. In contrast, a
"repetitive" sequence may contain multiple identical copies of short amino acid sequences. For instance, a polypeptide sequence of interest may be divided into n-mer sequences and the number of identical sequences can be counted. Highly repetitive sequences contain a large fraction of identical sequences while non-repetitive sequences contain few identical sequences. In the context of a polypeptide, a sequence can contain multiple copies of shorter sequences of defined or variable length, or motifs, in which the motifs themselves have non- repetitive sequences, rendering the full-length polypeptide substantially non-repetitive. The length of polypeptide within which the non-repetitiveness is measured can vary from 3 amino acids to about 200 amino acids, about from 6 to about 50 amino acids, or from about 9 to about 14 amino acids. "Repetitiveness" used in the context of polynucleotide sequences refers to the degree of internal homology in the sequence such as, for example, the frequency of identical nucleotide sequences of a given length. Repetitiveness can, for example, be measured by analyzing the frequency of identical sequences.
As used herein, "treatment" or "treating," or "palliating" or "ameliorating" is used interchangeably herein. These terms refer to an approach for obtaining beneficial or desired results including but not limited to a therapeutic benefit and/or a prophylactic benefit. By therapeutic benefit is meant eradication or amelioration of the underlying disorder being treated. Also, a therapeutic benefit is achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the pediatric subject, notwithstanding that the subject may still be afflicted with the underlying disorder. The therapeutic benefits may include sustained or normalized parameters such as, but not limited to, annual height velocity (cm/yr) in the first year (12 months) of treatment, IGF-I standard deviation score (IGF-I SDS), height standard deviation score, body weight, body mass index bone age, and pubertal staging. A preferred therapeutic benefit is sustained or normalized annual height velocity (cm/yr) in the first year (12 months) of treatment. For prophylactic benefit, the compositions may be administered to a pediatric subject at risk of developing a particular disease, or to a pediatric subject reporting one or more of the physiological symptoms of a disease, even though a diagnosis of this disease may not have been made. The prophylactic benefits may include sustained or normalized parameters such as, but not limited to, annual height velocity (cm/yr) in the first year (12 months) of treatment, IGF-I standard deviation score (IGF-I SDS), height standard deviation score, body weight, body mass index bone age, and pubertal staging. A preferred prophylactic benefit is sustained or normalized annual height velocity (cm/yr) in the first year (12 months) of treatment.
A "therapeutic effect", as used herein, refers to a physiologic effect, including but not limited to the cure, mitigation, amelioration, or prevention of disease in humans or other animals, or to otherwise enhance physical or mental wellbeing of humans or animals, caused by a fusion polypeptide of the invention other than the ability to induce the production of an antibody against an antigenic epitope possessed by the biologically active protein.
Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein. The therapeutic effect may include sustained or normalized parameters such as, but not limited to, annual height velocity (cm/yr) in the first year (12 months) of treatment, IGF-I standard deviation score (IGF-I SDS), height standard deviation score, body weight, body mass index bone age, and pubertal staging. A preferred therapeutic effect is sustained or normalized annual height velocity (cm/yr) in the first year (12 months) of treatment.
The terms "therapeutically effective amount" and "therapeutically effective dose" and "pharmaceutically effective amount", as used herein, refers to an amount of a biologically active protein, either alone or as a part of a fusion protein composition, that is capable of having any detectable, beneficial effect on any symptom, aspect, measured parameter or characteristics of a disease state or condition when administered in one or repeated doses to a pediatric subject. Such effect need not be absolute to be beneficial.
II). GENERAL TECHNIQUES
The practice of the present invention employs, unless otherwise indicated,
conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics and recombinant DNA, which are within the skill of the art. See Sambrook, J. et al., "Molecular Cloning: A Laboratory Manual," 3rd edition, Cold Spring Harbor Laboratory Press, 2001; "Current protocols in molecular biology", F. M.
Ausubel, et al. eds.,1987; the series "Methods in Enzymology," Academic Press, San Diego, CA.; "PCR 2: a practical approach", M.J. MacPherson, B.D. Hames and G.R. Taylor eds., Oxford University Press, 1995; "Antibodies, a laboratory manual" Harlow, E. and Lane, D. eds., Cold Spring Harbor Laboratory, 1988; "Goodman & Gilman's The Pharmacological Basis of Therapeutics," 11th Edition, McGraw-Hill, 2005; and Freshney, R.I., "Culture of Animal Cells: A Manual of Basic Technique," 4th edition, John Wiley & Sons, Somerset, NJ, 2000, the contents of which are incorporated in their entirety herein by reference. III). hGH FORMULATIONS
The present invention is based, at least in part, on the finding that hGH covalently fused to extended recombinant polypeptides ("XTE " or "XTENs") is highly soluble and stable in aqueous liquid solution at a relatively low pH and in the absence of a surfactant. Specifically, liquid aqueous formulations of hGH-XTEN according to the invention remain stable during storage over a period of at least about 24 months and longer when stored refrigerated (5±3°C) or stable during storage over a period of at least about 1 month and longer when stored at 25±3 °C. According to the present invention, hGH-XTEN can be formulated as a storage stable aqueous liquid formulation including a buffer and a tonicifying agent, free of a surfactant. In one embodiment, the formulation does not contain a preservative. In a preferred
embodiment, the hGH-XTEN of the formulation is the sequence of SEQ ID NO: 1. In some embodiments, the appearance of these solutions is a clear, pale yellow to pale reddish brown liquid, free from visible particles.
Suitable pH ranges, adjusted with buffer, for the aqueous hGH formulation are from about 4 to about 6, more preferably about 5 to about 6, most advantageously about 5.5.
Preferably, a buffer concentration range is chosen to minimize deamidation, aggregation, and/or precipitation of hGH-XTEN.
The buffer concentration in the formulations of the present invention is in the range of about 2 mM to about 50 mM, preferably about 10 mM to about 40 mM, more preferably about 15 mM to about 25 mM, most preferably about 20 mM. Preferably, the buffer is a histidine buffer. In yet another preferred embodiment, the buffer is L-histidine used in the formulation at a concentration of about 20 mM. Biological buffers with similar effective pH ranges, for example citrate, maleate, and bis-TRIS, may be substituted for L-histidine.
The salt concentration is adjusted to near isotonicity, depending on the other ingredients present in the formulation. For example, the concentration range of NaCl may be about 50-200 mM, depending on the other ingredients present. Of course, other tonicifying agents can also be used such as, for example, MgCl2 or CaCl2.
The concentration of hGH-XTEN in the formulations of the present invention is in the range of about 25 mg/ml to about 150 mg/ml. Preferably, the concentration of hGH-XTEN in the formulations is about 100 mg/ml. In yet another preferred embodiment, the concentration of hGH-XTEN in the formulations is about 150 mg/ml. The formulations herein have an osmolality between about 300 mOsm to about 600 mOsm, preferably between about 400 mOsm and about 500 mOsm.
Mannitol may optionally be included in the aqueous hGH formulations of the present invention. The preferred concentration of mannitol is about 5 mg/ml to about 50 mg/ml. As an alternative to mannitol, other sugars or sugar alcohols are used, such as lactose, trehalose, stachyose, sorbitol, xylitol, ribitol, myoinositol, galactitol, and the like.
The formulation of the invention is storage stable for at least about one month when stored at about 23±3°C, and for at least about 6 months to about 18 months when stored at about 5±3 °C. In a preferred embodiment, the formulation is storage stable for at least about 6 months to about 3 years when stored at about 5±3 °C. The oxidation of the fusion protein in the formulations herein is reduced as compared to the oxidation of the corresponding fusion protein formulated with a surfactant. The deamidation of the fusion protein in the
formulations is reduced as compared to the deamidation of the corresponding fusion protein formulated at a higher pH. Preferably, the isolated fusion protein does not show detectable oxidation or deamidation following incubation for about 8 hours at about 40 °C.
Another aspect of the invention provides a method of preparing (making) a storage stable aqueous liquid formulation in which a fusion protein comprising an extended recombinant polypeptide (XTEN) fused to a growth hormone (GH) sequence, wherein the GH sequence is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:43, is admixed with a buffer in the absence of a surfactant, and adjusting the pH of the mixture to obtain a pH of about 4 to about 6.
A preferred embodiment of the present invention is prepared by dissolving about 25 mg/ml to about 150 mg/ml of hGH-XTEN in 950 ml sterile water for injection USP, 9.00 g NaCl (low endotoxin; Merck Chemicals #116224) USP (at 0.154M final volume), 3.103g L- Histidine (Avantor #2080) USP (at 0.02M final volume), adjusted to pH 5.5 +/- 0.1 at 18 +/- 1 C with 5M HC1. The final volume is adjusted to 1 L with sterile water for injection USP. The conductivity of the formulation solution at 25 +/- 1 C is about 10 to about 20 mS/cm.
It will be understood that the above quantities are somewhat flexible within ranges, as set forth in more detail above, and that the materials are interchangeable within the component categories. More than one buffering agent, preservative, sugar, or neutral salt may be used. Preferably, the formulation is isotonic and sterile.
In general, the formulations of the subject invention may contain other components in amounts not detracting from the preparation of stable forms and in amounts suitable for effective, safe pharmaceutical administration. For example, other pharmaceutically acceptable excipients well known to those skilled in the art may form a part of the subject compositions. These include, for example, various bulking agents, additional buffering agents, chelating agents, antioxidants, cosolvents and the like; specific examples of these could include citrate or succinate buffers, and disodium edetate. IV). GROWTH HORMONE
The present invention concerns an improved therapeutic aqueous liquid formulation comprising hGH-XTEN fusion protein. In addition, in one aspect, the present invention concerns a method of treating human growth hormone deficiency (GHD) in patients with a hGH-XTEN fusion protein formulation.
The invention contemplates hGH-XTEN in the formulations comprising sequences with homology to hGH sequences, sequence fragments that are natural, such as from humans and non-natural sequence variants which retain at least a portion of the biologic activity or biological function of hGH and/or that are useful for preventing, treating, mediating, or ameliorating a hGH-related disease, deficiency, disorder or condition in pediatric patients. In addition, native sequences homologous to human GH may be found by standard homology searching techniques, such as NCBI BLAST. The hGH of the subject compositions, together with their corresponding nucleic acid and amino acid sequences, are well known in the art, described in in, for example, W013/184216, WO/2014/164568, U.S. Patent Nos. 8,492,530 and 8,703,717, and descriptions and sequences are available in public databases such as Chemical Abstracts Services Databases (e.g., the CAS Registry), GenBank, The Universal Protein Resource (UniProt) and subscription provided databases such as GenSeq (e.g., Derwent).
Effects of hGH on the tissues of the body can generally be described as anabolic. Like most other protein hormones, native hGH acts by interacting with a specific plasma membrane receptor, referred to as growth hormone receptor. hGH acts on the liver and other tissues to stimulate production of IGF-I, which is responsible for the growth promoting effects of hGH and also reflects the amount produced. IGF-I, in turn, has stimulatory effects on osteoblast and chondrocyte activity to promote bone growth. In one embodiment, the invention provides a hGH-XTEN formulation that exhibits at least one of the properties of native hGH hereinabove described herein.
In one embodiment, the hGH incorporated into the subject hGH-XTEN formulation is a recombinant polypeptide with a sequence corresponding to a protein found in nature. In another embodiment, the hGH is a sequence variant, fragment, homolog, or a mimetics of a natural sequence that retains at least a portion of the biological activity of the corresponding native GH. In one other embodiment, the hGH is recombinant human GH comprising the following amino acid sequence:
FPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIP TPS REETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDL EEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKV ETFLRIVQCRSVEGSCGF (SEQ ID NO:43). Any human GH sequences or homologous derivatives constructed by shuffling individual mutations between families that retain at least a portion of the biological activity of the native GH may be useful for the fusion proteins of this invention. Human GH that can be incorporated into a hGH-XTEN fusion protein can include a protein that exhibits at least about 80% sequence identity, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:43.
V). HUMAN GROWTH HORMONE-XTEN FUSION PROTEIN COMPOSITIONS
In one aspect, the hGH fusion proteins suitable for use in the present invention comprise a human growth hormone polypeptide and one or more XTEN sequences as described herein, and as disclosed in, for example, W013/184216, WO/2014/164568 and U.S. Patent Nos. 8,492,530 and 8,703,717, each of which is incorporated herein by reference in its entirety.
In one other aspect, the hGH-XTEN fusion proteins are isolated monomelic fusion proteins of hGH comprising the full-length sequence or sequence variants of hGH covalently linked to one or more extended recombinant polypeptides ("XTEN" or "XTENs"). In one embodiment, the hGH-XTEN fusion protein comprises an amino acid sequence shown in FIG. 1 (SEQ ID NO: l), or pharmacologically active variants thereof. In another
embodiment, the hGH-XTEN fusion protein comprises an amino acid sequence selected from Table 1. In another embodiment, the hGH-XTEN fusion protein sequence exhibits at least about 80% sequence identity, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a XTEN selected from Table 1.
For example, the hGH-XTEN fusion protein VRS-317 (SEQ ID NO: 1), is composed of recombinant human growth hormone (rhGH) and two recombinant polypeptides, referred to as XTEN as described in Schellenberger et al. (2009). Nat Biotechnol 27, 1186-90, Schellenberger et al. WO10/144502A2, and WO10/091122, each of which are incorporated herein by reference in their entirety. The XTEN domain, two unstructured hydrophilic chains of amino acids, provides half-life extension for rhGH. The molecular weight of VRS-317 is 118.9 kDa, with rhGH contributing 22.1 kDa and the remaining mass contributed by the XTEN construct. The mass ratio of rhGH to VRS-317 is therefore 1 :5.37. Further characterization of the exemplary hGH-XTEN fusion proteins provided in Table 1 can be found in WO10/144502A2, which is incorporated herein by reference in its entirety.
Table 1 - Exemplary hGH-XTEN fusion proteins
hGH- SEQ SEQ XTEN Amino Acid Sequence ID DNA Nucleotide Sequence ID Name* NO: NO:
AE912- AEPAGSPTSTEEGTPGS 1 ATGGCTGAACCTGCTGGCTCTCCAACCTCCA 7 hGH- GTASSSPGSSTPSGATG CTGAGGAAGGTACCCCGGGTAGCGGTACTG
AE144 SPGASPGTSSTGSPGSP CTTCTTCCTCTCCAGGTAGCTCTACCCCTTC
AGSPTSTEEGTSESATP TGGTGCAACCGGCTCTCCAGGTGCTTCTCCG
ESGPGTSTEPSEGSAPG GGCACCAGCTCTACCGGTTCTCCAGGTAGC
SPAGSPTSTEEGTSTEPS CCGGCTGGCTCTCCTACCTCTACTGAGGAA
EGSAPGTSTEPSEGSAP GGTACTTCTGAAAGCGCTACTCCTGAGTCTG
GTSESATPESGPGSEPA GTCCAGGTACCTCTACTGAACCGTCCGAAG
TSGSETPGSEPATSGSET GTAGCGCTCCAGGTAGCCCAGCAGGCTCTC
PGSPAGSPTSTEEGTSES CGACTTCCACTGAGGAAGGTACTTCTACTG
ATPESGPGTSTEPSEGS AACCTTCCGAAGGCAGCGCACCAGGTACCT
APGTSTEPSEGSAPGSP CTACTGAACCTTCTGAGGGCAGCGCTCCAG
AGSPTSTEEGTSTEPSE GTACTTCTGAAAGCGCTACCCCGGAATCTG
GSAPGTSTEPSEGSAPG GCCCAGGTAGCGAACCGGCTACTTCTGGTT
TSESATPESGPGTSTEPS CTGAAACCCCAGGTAGCGAACCGGCTACCT
EGSAPGTSESATPESGP CCGGTTCTGAAACTCCAGGTAGCCCGGCAG
GSEPATSGSETPGTSTEP GCTCTCCGACCTCTACTGAGGAAGGTACTTC
SEGSAPGTSTEPSEGSA TGAAAGCGCAACCCCGGAGTCCGGCCCAGG
PGTSESATPESGPGTSES TACCTCTACCGAACCGTCTGAGGGCAGCGC
ATPESGPGSPAGSPTST ACCAGGTACTTCTACCGAACCGTCCGAGGG
EEGTSESATPESGPGSEP TAGCGCACCAGGTAGCCCAGCAGGTTCTCC
ATSGSETPGTSESATPES TACCTCCACCGAGGAAGGTACTTCTACCGA
GPGTSTEPSEGSAPGTS ACCGTCCGAGGGTAGCGCACCAGGTACCTC
TEPSEGSAPGTSTEPSEG TACTGAACCTTCTGAGGGCAGCGCTCCAGG
SAPGTSTEPSEGSAPGT TACTTCTGAAAGCGCTACCCCGGAGTCCGG
STEPSEGSAPGTSTEPSE TCCAGGTACTTCTACTGAACCGTCCGAAGG
GSAPGSPAGSPTSTEEG TAGCGCACCAGGTACTTCTGAAAGCGCAAC
TSTEPSEGSAPGTSESAT CCCTGAATCCGGTCCAGGTAGCGAACCGGC
PESGPGSEPATSGSETP TACTTCTGGCTCTGAGACTCCAGGTACTTCT
GTSESATPESGPGSEPA ACCGAACCGTCCGAAGGTAGCGCACCAGGT
TSGSETPGTSESATPESG ACTTCTACTGAACCGTCTGAAGGTAGCGCA
PGTSTEPSEGSAPGTSES CCAGGTACTTCTGAAAGCGCAACCCCGGAA
ATPESGPGSPAGSPTST TCCGGCCCAGGTACCTCTGAAAGCGCAACC
EEGSPAGSPTSTEEGSP CCGGAGTCCGGCCCAGGTAGCCCTGCTGGC
AGSPTSTEEGTSESATP TCTCCAACCTCCACCGAAGAAGGTACCTCT
ESGPGTSTEPSEGSAPG GAAAGCGCAACCCCTGAATCCGGCCCAGGT
TSESATPESGPGSEPATS AGCGAACCGGCAACCTCCGGTTCTGAAACC
GSETPGTSESATPESGP CCAGGTACCTCTGAAAGCGCTACTCCGGAG
GSEPATSGSETPGTSES TCTGGCCCAGGTACCTCTACTGAACCGTCTG
ATPESGPGTSTEPSEGS AGGGTAGCGCTCCAGGTACTTCTACTGAAC
APGSPAGSPTSTEEGTS CGTCCGAAGGTAGCGCACCAGGTACTTCTA
ESATPESGPGSEPATSG CCGAACCGTCCGAAGGCAGCGCTCCAGGTA
SETPGTSESATPESGPGS CCTCTACTGAACCTTCCGAGGGCAGCGCTC
PAGSPTSTEEGSPAGSP CAGGTACCTCTACCGAACCTTCTGAAGGTA
TSTEEGTSTEPSEGSAP GCGCACCAGGTACTTCTACCGAACCGTCCG
GTSESATPESGPGTSES AGGGTAGCGCACCAGGTAGCCCAGCAGGTT
ATPESGPGTSESATPES CTCCTACCTCCACCGAGGAAGGTACTTCTAC hGH- SEQ SEQ XTEN Amino Acid Sequence ID DNA Nucleotide Sequence ID Name* NO: NO:
GPGSEPATSGSETPGSE CGAACCGTCCGAGGGTAGCGCACCAGGTAC
PATSGSETPGSPAGSPTS CTCTGAAAGCGCAACTCCTGAGTCTGGCCC
TEEGTSTEPSEGSAPGT AGGTAGCGAACCTGCTACCTCCGGCTCTGA
STEPSEGSAPGSEPATS GACTCCAGGTACCTCTGAAAGCGCAACCCC
GSETPGTSESATPESGP GGAATCTGGTCCAGGTAGCGAACCTGCAAC
GTSTEPSEGSAPGFPTIP CTCTGGCTCTGAAACCCCAGGTACCTCTGA
LSRLFDNAMLRAHRLH AAGCGCTACTCCTGAATCTGGCCCAGGTAC
QLAFDTYQEFEEAYIPK TTCTACTGAACCGTCCGAGGGCAGCGCACC
EQKYSFLQNPQTSLCFS AGGTACTTCTGAAAGCGCTACTCCTGAGTC
ESIPTP SNREETQQKSNL CGGCCCAGGTAGCCCGGCTGGCTCTCCGAC
ELLPJSLLLIQSWLEPVQ TTCCACCGAGGAAGGTAGCCCGGCTGGCTC
FLRS VF ANSL VYG ASD S TCCAACTTCTACTGAAGAAGGTAGCCCGGC
NVYDLLKDLEEGIQTL AGGCTCTCCGACCTCTACTGAGGAAGGTAC
MGRLEDGSPRTGQIFK TTCTGAAAGCGCAACCCCGGAGTCCGGCCC
QTYSKFDTNSHNDDAL AGGTACCTCTACCGAACCGTCTGAGGGCAG
LKNYGLLYCFRKDMD CGCACCAGGTACCTCTGAAAGCGCAACTCC
KVETFLRIVQCRSVEGS TGAGTCTGGCCCAGGTAGCGAACCTGCTAC
CGFGGTSESATPESGPG CTCCGGCTCTGAGACTCCAGGTACCTCTGA
TSTEPSEGSAPGTSTEPS AAGCGCAACCCCGGAATCTGGTCCAGGTAG
EGSAPGTSESATPESGP CGAACCTGCAACCTCTGGCTCTGAAACCCC
GTSTEPSEGSAPGTSTEP AGGTACCTCTGAAAGCGCTACTCCTGAATC
SEGSAPGTSESATPESG TGGCCCAGGTACTTCTACTGAACCGTCCGA
PGTSTEPSEGSAPGTSTE GGGCAGCGCACCAGGTAGCCCTGCTGGCTC
PSEGSAPGTSTEPSEGS TCCAACCTCCACCGAAGAAGGTACCTCTGA
APGSPAGSPTSTEEGTS AAGCGCAACCCCTGAATCCGGCCCAGGTAG
TEPSEGSAPG CGAACCGGCAACCTCCGGTTCTGAAACCCC
AGGTACTTCTGAAAGCGCTACTCCTGAGTC
CGGCCCAGGTAGCCCGGCTGGCTCTCCGAC
TTCCACCGAGGAAGGTAGCCCGGCTGGCTC
TCCAACTTCTACTGAAGAAGGTACTTCTACC
GAACCTTCCGAGGGCAGCGCACCAGGTACT
TCTGAAAGCGCTACCCCTGAGTCCGGCCCA
GGTACTTCTGAAAGCGCTACTCCTGAATCC
GGTCCAGGTACTTCTGAAAGCGCTACCCCG
GAATCTGGCCCAGGTAGCGAACCGGCTACT
TCTGGTTCTGAAACCCCAGGTAGCGAACCG
GCTACCTCCGGTTCTGAAACTCCAGGTAGC
CCAGCAGGCTCTCCGACTTCCACTGAGGAA
GGTACTTCTACTGAACCTTCCGAAGGCAGC
GCACCAGGTACCTCTACTGAACCTTCTGAG
GGCAGCGCTCCAGGTAGCGAACCTGCAACC
TCTGGCTCTGAAACCCCAGGTACCTCTGAA
AGCGCTACTCCTGAATCTGGCCCAGGTACTT
CTACTGAACCGTCCGAGGGCAGCGCACCAG
GTTTTCCGACTATTCCGCTGTCTCGTCTGTTT
GATAATGCTATGCTGCGTGCGCACCGTCTG
CACCAGCTGGCCTTTGATACTTACCAGGAA
TTTGAAGAAGCcTACATTCCTAAAGAGCAG
AAGTACTCTTTCCTGCAAAACCCACAGACTT
CTCTCTGCTTCAGCGAATCTATTCCGACGCC
TTCCAATCGCGAGGAAACTCAGCAAAAGTC
CAATCTGGAACTACTCCGCATTTCTCTGCTT
CTGATTCAGAGCTGGCTAGAACCAGTGCAA
TTTCTGCGTTCCGTCTTCGCCAATAGCCTAG
TTTATGGCGCATCCGACAGCAACGTATACG
ATCTCCTGAAAGATCTCGAGGAAGGCATTC hGH- SEQ SEQ XTEN Amino Acid Sequence ID DNA Nucleotide Sequence ID Name* NO: NO:
AGACCCTGATGGGTCGTCTCGAGGATGGCT
CTCCGCGTACTGGTCAGATCTTCAAGCAGA
CTTACTCTAAATTTGATACTAACAGCCACAA
TGACGATGCGCTTCTAAAAAACTATGGTCT
GCTGTATTGTTTTCGTAAAGATATGGACAA
AGTTGAAACCTTCCTGCGTATTGTTCAGTGT
CGTTCCGTTGAGGGCAGCTGTGGTTTCTAAG
GTGGTAGCGAACCGGCAACTTCCGGCTCTG
AAACCCCAGGTACTTCTGAAAGCGCTACTC
CTGAGTCTGGCCCAGGTAGCGAACCTGCTA
CCTCTGGCTCTGAAACCCCAGGTAGCCCGG
CAGGCTCTCCGACTTCCACCGAGGAAGGTA
CCTCTACTGAACCTTCTGAGGGTAGCGCTCC
AGGTAGCGAACCGGCAACCTCTGGCTCTGA
AACCCCAGGTAGCGAACCTGCTACCTCCGG
CTCTGAAACTCCAGGTAGCGAACCGGCTAC
TTCCGGTTCTGAAACTCCAGGTACCTCTACC
GAACCTTCCGAAGGCAGCGCACCAGGTACT
TCTGAAAGCGCAACCCCTGAATCCGGTCCA
GGTAGCGAACCGGCTACTTCTGGCTCTGAG
ACTCCAGGTACTTCTACCGAACCGTCCGAA
GGTAGCGCACCA
AM864- GGSPGTSTEPSEGSAPG 2 ggtGGGTCTCCAGGTACTTCTACTGAACCGTC 8 hGH SEPATSGSETPGSPAGSP TGAAGGCAGCGCACCAGGTAGCGAACCGGC
TSTEEGSTSSTAESPGPG TACTTCCGGTTCTGAAACCCCAGGTAGCCC
TSTPESGSASPGSTSESP AGCAGGTTCTCCAACTTCTACTGAAGAAGG
SGTAPGSTSESPSGTAP TTCTACCAGCTCTACCGCAGAATCTCCTGGT
GTSTPESGSASPGTSTPE CCAGGTACCTCTACTCCGGAAAGCGGCTCT
SGSASPGSEPATSGSETP GCATCTCCAGGTTCTACTAGCGAATCTCCTT
GTSESATPESGPGSPAG CTGGCACTGCACCAGGTTCTACTAGCGAAT
SPTSTEEGTSTEPSEGSA CCCCGTCTGGTACTGCTCCAGGTACTTCTAC
PGTSESATPESGPGTSTE TCCTGAAAGCGGTTCCGCTTCTCCAGGTACC
PSEGSAPGTSTEPSEGS TCTACTCCGGAAAGCGGTTCTGCATCTCCAG
APGSPAGSPTSTEEGTS GTAGCGAACCGGCAACCTCCGGCTCTGAAA
TEPSEGSAPGTSTEPSEG CCCCAGGTACCTCTGAAAGCGCTACTCCTG
SAPGTSESATPESGPGT AATCCGGCCCAGGTAGCCCGGCAGGTTCTC
SESATPESGPGTSTEPSE CGACTTCCACTGAGGAAGGTACCTCTACTG
GSAPGTSTEPSEGSAPG AACCTTCTGAGGGCAGCGCTCCAGGTACTT
TSESATPESGPGTSTEPS CTGAAAGCGCTACCCCGGAGTCCGGTCCAG
EGSAPGSEPATSGSETP GTACTTCTACTGAACCGTCCGAAGGTAGCG
GSPAGSPTSTEEGS STPS CACCAGGTACTTCTACCGAACCGTCCGAGG
GATGSPGTPGSGTASSS GTAGCGCACCAGGTAGCCCAGCAGGTTCTC
PGSSTPSGATGSPGTST CTACCTCCACCGAGGAAGGTACTTCTACCG
EPSEGSAPGTSTEPSEGS AACCGTCCGAGGGTAGCGCACCAGGTACTT
APGSEPATSGSETPGSP CTACCGAACCTTCCGAGGGCAGCGCACCAG
AGSPTSTEEGSPAGSPT GTACTTCTGAAAGCGCTACCCCTGAGTCCG
STEEGTSTEPSEGSAPG GCCCAGGTACTTCTGAAAGCGCTACTCCTG
ASASGAPSTGGTSESAT AATCCGGTCCAGGTACCTCTACTGAACCTTC
PESGPGSPAGSPTSTEE CGAAGGCAGCGCTCCAGGTACCTCTACCGA
GSPAGSPTSTEEGSTSST ACCGTCCGAGGGCAGCGCACCAGGTACTTC
AESPGPGSTSESPSGTAP TGAAAGCGCAACCCCTGAATCCGGTCCAGG
GTSPSGESSTAPGTPGS TACTTCTACTGAACCTTCCGAAGGTAGCGCT
GTASSSPGSSTPSGATG CCAGGTAGCGAACCTGCTACTTCTGGTTCTG
SPGSSPSASTGTGPGSEP AAACCCCAGGTAGCCCGGCTGGCTCTCCGA
ATSGSETPGTSESATPES CCTCCACCGAGGAAGGTAGCTCTACCCCGT
GPGSEPATSGSETPGST CTGGTGCTACTGGTTCTCCAGGTACTCCGGG hGH- SEQ SEQ XTEN Amino Acid Sequence ID DNA Nucleotide Sequence ID Name* NO: NO:
SSTAESPGPGSTSSTAES CAGCGGTACTGCTTCTTCCTCTCCAGGTAGC
PGPGTSPSGESSTAPGSE TCTACCCCTTCTGGTGCTACTGGCTCTCCAG
PATSGSETPGSEPATSG GTACCTCTACCGAACCGTCCGAGGGTAGCG
SETPGTSTEPSEGSAPGS CACCAGGTACCTCTACTGAACCGTCTGAGG
TSSTAESPGPGTSTPESG GTAGCGCTCCAGGTAGCGAACCGGCAACCT
SASPGSTSESPSGTAPGT CCGGTTCTGAAACTCCAGGTAGCCCTGCTG
STEPSEGSAPGTSTEPSE GCTCTCCGACTTCTACTGAGGAAGGTAGCC
GSAPGTSTEPSEGSAPG CGGCTGGTTCTCCGACTTCTACTGAGGAAG
SSTPSGATGSPGSSPSAS GTACTTCTACCGAACCTTCCGAAGGTAGCG
TGTGPGASPGTSSTGSP CTCCAGGTGCAAGCGCAAGCGGCGCGCCAA
GSEPATSGSETPGTSES GCACGGGAGGTACTTCTGAAAGCGCTACTC
ATPESGPGSPAGSPTST CTGAGTCCGGCCCAGGTAGCCCGGCTGGCT
EEGSSTPSGATGSPGSSP CTCCGACTTCCACCGAGGAAGGTAGCCCGG
SASTGTGPGASPGTSST CTGGCTCTCCAACTTCTACTGAAGAAGGTTC
GSPGTSESATPESGPGT TACCAGCTCTACCGCTGAATCTCCTGGCCCA
STEPSEGSAPGTSTEPSE GGTTCTACTAGCGAATCTCCGTCTGGCACCG
GSAPGFPTIPLSRLFDNA CACCAGGTACTTCCCCTAGCGGTGAATCTTC
MLRAHRLHQLAFDTYQ TACTGCACCAGGTACCCCTGGCAGCGGTAC
EFEE AYIPKEQKY SFLQ CGCTTCTTCCTCTCCAGGTAGCTCTACCCCG
NPQTSLCFSESIPTPSNR TCTGGTGCTACTGGCTCTCCAGGTTCTAGCC
EETQQKSNLELLPJSLL CGTCTGCATCTACCGGTACCGGCCCAGGTA
LIQSWLEPVQFLRSVFA GCGAACCGGCAACCTCCGGCTCTGAAACTC
NSL VYG ASD SNVYDLL CAGGTACTTCTGAAAGCGCTACTCCGGAAT
KDLEEGIQTLMGRLED CCGGCCCAGGTAGCGAACCGGCTACTTCCG
GSPRTGQIFKQTYSKFD GCTCTGAAACCCCAGGTTCCACCAGCTCTA
TNSHNDDALLKNYGLL CTGCAGAATCTCCGGGCCCAGGTTCTACTA
YCFRKDMDKVETFLRI GCTCTACTGCAGAATCTCCGGGTCCAGGTA
VQCRSVEGSCGF CTTCTCCTAGCGGCGAATCTTCTACCGCTCC
AGGTAGCGAACCGGCAACCTCTGGCTCTGA
AACTCCAGGTAGCGAACCTGCAACCTCCGG
CTCTGAAACCCCAGGTACTTCTACTGAACCT
TCTGAGGGCAGCGCACCAGGTTCTACCAGC
TCTACCGCAGAATCTCCTGGTCCAGGTACCT
CTACTCCGGAAAGCGGCTCTGCATCTCCAG
GTTCTACTAGCGAATCTCCTTCTGGCACTGC
ACCAGGTACTTCTACCGAACCGTCCGAAGG
CAGCGCTCCAGGTACCTCTACTGAACCTTCC
GAGGGCAGCGCTCCAGGTACCTCTACCGAA
CCTTCTGAAGGTAGCGCACCAGGTAGCTCT
ACTCCGTCTGGTGCAACCGGCTCCCCAGGTT
CTAGCCCGTCTGCTTCCACTGGTACTGGCCC
AGGTGCTTCCCCGGGCACCAGCTCTACTGG
TTCTCCAGGTAGCGAACCTGCTACCTCCGGT
TCTGAAACCCCAGGTACCTCTGAAAGCGCA
ACTCCGGAGTCTGGTCCAGGTAGCCCTGCA
GGTTCTCCTACCTCCACTGAGGAAGGTAGC
TCTACTCCGTCTGGTGCAACCGGCTCCCCAG
GTTCTAGCCCGTCTGCTTCCACTGGTACTGG
CCCAGGTGCTTCCCCGGGCACCAGCTCTACT
GGTTCTCCAGGTACCTCTGAAAGCGCTACTC
CGGAGTCTGGCCCAGGTACCTCTACTGAAC
CGTCTGAGGGTAGCGCTCCAGGTACTTCTA
CTGAACCGTCCGAAGGTAGCGCACCAGGTT
TTCCGACTATTCCGCTGTCTCGTCTGTTTGA
TAATGCTATGCTGCGTGCGCACCGTCTGCAC
CAGCTGGCCTTTGATACTTACCAGGAATTTG hGH- SEQ SEQ XTEN Amino Acid Sequence ID DNA Nucleotide Sequence ID Name* NO: NO:
AAGAAGCcTACATTCCTAAAGAGCAGAAGT
ACTCTTTCCTGCAAAACCCACAGACTTCTCT
CTGCTTCAGCGAATCTATTCCGACGCCTTCC
AATCGCGAGGAAACTCAGCAAAAGTCCAAT
CTGGAACTACTCCGCATTTCTCTGCTTCTGA
TTCAGAGCTGGCTAGAACCAGTGCAATTTC
TGCGTTCCGTCTTCGCCAATAGCCTAGTTTA
TGGCGCATCCGACAGCAACGTATACGATCT
CCTGAAAGATCTCGAGGAAGGCATTCAGAC
CCTGATGGGTCGTCTCGAGGATGGCTCTCC
GCGTACTGGTCAGATCTTCAAGCAGACTTA
CTCTAAATTTGATACTAACAGCCACAATGA
CGATGCGCTTCTAAAAAACTATGGTCTGCT
GTATTGTTTTCGTAAAGATATGGACAAAGTT
GAAACCTTCCTGCGTATTGTTCAGTGTCGTT
CCGTTGAGGGCAGCTGTGGTTTC
Y576- GEGSGEGSEGEGSEGSG 3 GGTGAGGGTTCTGGCGAAGGTTCCGAAGGT 9 hGH EGEGSEGSGEGEGGSE GAGGGCTCCGAAGGATCTGGCGAAGGTGAG
GSEGEGSEGSGEGEGG GGTTCCGAAGGTTCTGGCGAAGGTGAAGGC
EGSGEGEGSGEGSEGE GGTTCTGAGGGATCCGAAGGTGAAGGCTCC
GGGEGSEGEGSGEGGE GAAGGATCTGGCGAAGGTGAAGGTGGTGA
GEGSEGGSEGEGGSEG AGGTTCTGGCGAAGGTGAGGGATCTGGCGA
GEGEGSEGSGEGEGSE AGGCTCTGAAGGTGAAGGTGGTGGTGAAGG
GGSEGEGSEGGSEGEGS CTCTGAAGGTGAAGGATCTGGTGAAGGTGG
EGSGEGEGSEGSGEGE CGAAGGTGAGGGATCTGAAGGCGGCTCCGA
GSEGSGEGEGSEGSGEG AGGTGAAGGCGGATCTGAAGGCGGCGAAG
EGSEGGSEGEGGSEGSE GTGAAGGTTCCGAAGGTTCTGGTGAAGGTG
GEGSGEGSEGEGGSEGS AAGGATCTGAAGGTGGCTCCGAAGGTGAAG
EGEGGGEGSEGEGSGE GATCTGAAGGCGGTTCCGAAGGTGAGGGCT
GSEGEGGSEGSEGEGGS CTGAAGGTTCTGGCGAAGGTGAAGGCTCTG
EGSEGEGGEGSGEGEG AAGGATCTGGTGAAGGTGAAGGTTCCGAAG
SEGSGEGEGSGEGSEGE GTTCTGGTGAAGGTGAAGGTTCCGAAGGTT
GSEGSGEGEGSEGSGEG CTGGCGAAGGTGAAGGTTCTGAAGGTGGCT
EGGSEGSEGEGSGEGSE CTGAAGGTGAAGGCGGCTCTGAAGGATCCG
GEGSEGSGEGEGSEGSG AAGGTGAAGGTTCTGGTGAAGGCTCTGAAG
EGEGGSEGSEGEGGSE GTGAAGGCGGCTCTGAGGGTTCCGAAGGTG
GSEGEGGSEGSEGEGG AAGGCGGAGGCGAAGGTTCTGAAGGTGAG
EGSGEGEGSEGSGEGE GGATCTGGTGAAGGTTCTGAAGGTGAAGGC
GSGEGSEGEGSEGSGEG GGTTCTGAAGGTTCCGAAGGTGAAGGTGGC
EGSEGSGEGEGGSEGSE TCTGAGGGATCCGAAGGTGAAGGTGGCGAA
GEGSEGSGEGEGGEGS GGATCTGGTGAAGGTGAAGGTTCTGAAGGT
GEGEGSGEGSEGEGGG TCTGGCGAAGGTGAGGGTTCTGGCGAAGGT
EGSEGEGSEGSGEGEGS TCCGAAGGTGAGGGCTCCGAAGGATCTGGC
EGSGEGEGSEGGSEGE GAAGGTGAGGGTTCCGAAGGTTCTGGCGAA
GGSEGSEGEGSEGGSEG GGTGAAGGCGGTTCTGAGGGATCCGAAGGT
EGSEGGSEGEGSEGSGE GAGGGTTCTGGCGAAGGTTCCGAAGGTGAG
GEGSEGSGEGEGSGEGS GGCTCCGAAGGATCTGGCGAAGGTGAGGGT
EGEGGSEGGEGEGSEG TCCGAAGGTTCTGGCGAAGGTGAAGGCGGT
GSEGEGSEGGSEGEGG TCTGAGGGATCCGAAGGTGAAGGCGGTTCT
EGSGEGEGGGEGSEGE GAAGGTTCCGAAGGTGAAGGTGGCTCTGAG
GSEGSGEGEGSGEGSEG GGATCCGAAGGTGAAGGTGGCGAAGGATCT
FPTIPLSRLFDNAMLRA GGTGAAGGTGAAGGTTCTGAAGGTTCTGGC
HRLHQLAFDTYQEFEE GAAGGTGAGGGTTCTGGCGAAGGTTCCGAA
AYIPKEQKYSFLQNPQT GGTGAGGGCTCCGAAGGATCTGGCGAAGGT
SLCFSESIPTPSNREETQ GAGGGTTCCGAAGGTTCTGGCGAAGGTGAA
QKSNLELLRISLLLIQS GGCGGTTCTGAGGGATCCGAAGGTGAAGGC hGH- SEQ SEQ XTEN Amino Acid Sequence ID DNA Nucleotide Sequence ID Name* NO: NO:
WLEPVQFLRSVFANSL TCCGAAGGATCTGGCGAAGGTGAAGGTGGT
VYGASDSNVYDLLKDL GAAGGTTCTGGCGAAGGTGAGGGATCTGGC
EEGIQTLMGRLEDGSPR GAAGGCTCTGAAGGTGAAGGTGGTGGTGAA
TGQIFKQTYSKFDTNSH GGCTCTGAAGGTGAAGGTTCCGAAGGTTCT
NDDALLKNYGLLYCFR GGTGAAGGTGAAGGTTCCGAAGGTTCTGGC
KDMDKVETFLRIVQCR GAAGGTGAAGGTTCTGAAGGTGGCTCTGAA
SVEGSCGF GGTGAAGGCGGCTCTGAAGGATCCGAAGGT
GAAGGATCTGAAGGTGGCTCCGAAGGTGAA
GGATCTGAAGGCGGTTCCGAAGGTGAGGGC
TCTGAAGGTTCTGGCGAAGGTGAAGGCTCT
GAAGGATCTGGTGAAGGTGAAGGATCTGGC
GAAGGCTCCGAAGGTGAAGGCGGTTCTGAA
GGTGGCGAAGGTGAAGGATCTGAAGGTGGT
TCCGAAGGTGAGGGATCTGAAGGTGGCTCT
GAAGGTGAAGGTGGCGAAGGTTCTGGCGAA
GGTGAAGGTGGAGGCGAAGGTTCTGAAGGT
GAAGGTTCCGAAGGTTCTGGTGAAGGTGAG
GGATCTGGCGAAGGTTCTGAAGGTTTTCCG
ACTATTCCGCTGTCTCGTCTGTTTGATAACG
CTATGCTGCGTGCGCACCGTCTGCACCAGCT
GGCGTTCGACACTTACCAGGAATTTGAAGA
AGCGTACATTCCGAAGGAACAGAAGTACTC
TTTCCTGCAAAACCCGCAGACCTCCCTGTGC
TTCAGCGAATCTATTCCGACTCCGTCCAATC
GTGAAGAAACTCAGCAAAAGTCCAATCTGG
AGCTGCTGCGCATCTCTCTGCTGCTGATTCA
GAGCTGGCTGGAGCCTGTTCAGTTTCTGCGT
TCCGTCTTCGCCAACAGCCTGGTTTATGGTG
CTTCCGACAGCAACGTATACGATCTGCTGA
AAGATCTGGAAGAAGGCATTCAGACCCTGA
TGGGTCGTCTGGAAGATGGTTCTCCGCGTA
CTGGTCAGATCTTCAAACAAACTTACTCCA
AATTTGATACTAACAGCCATAACGACGATG
CTCTGCTGAAAAACTATGGTCTGCTGTATTG
CTTCCGCAAGGATATGGACAAAGTTGAAAC
CTTCCTGCGTATTGTGCAGTGTCGTTCCGTT
GAGGGCAGCTGTGGTTTC
AE912- AEPAGSPTSTEEGTPGS 4 ATGGCTGAACCTGCTGGCTCTCCAACCTCCA 10 hGH GTASSSPGSSTPSGATG CTGAGGAAGGTACCCCGGGTAGCGGTACTG
SPGASPGTSSTGSPGSP CTTCTTCCTCTCCAGGTAGCTCTACCCCTTC
AGSPTSTEEGTSESATP TGGTGCAACCGGCTCTCCAGGTGCTTCTCCG
ESGPGTSTEPSEGSAPG GGCACCAGCTCTACCGGTTCTCCAGGTAGC
SPAGSPTSTEEGTSTEPS CCGGCTGGCTCTCCTACCTCTACTGAGGAA
EGSAPGTSTEPSEGSAP GGTACTTCTGAAAGCGCTACTCCTGAGTCTG
GTSESATPESGPGSEPA GTCCAGGTACCTCTACTGAACCGTCCGAAG
TSGSETPGSEPATSGSET GTAGCGCTCCAGGTAGCCCAGCAGGCTCTC
PGSPAGSPTSTEEGTSES CGACTTCCACTGAGGAAGGTACTTCTACTG
ATPESGPGTSTEPSEGS AACCTTCCGAAGGCAGCGCACCAGGTACCT
APGTSTEPSEGSAPGSP CTACTGAACCTTCTGAGGGCAGCGCTCCAG
AGSPTSTEEGTSTEPSE GTACTTCTGAAAGCGCTACCCCGGAATCTG
GSAPGTSTEPSEGSAPG GCCCAGGTAGCGAACCGGCTACTTCTGGTT
TSESATPESGPGTSTEPS CTGAAACCCCAGGTAGCGAACCGGCTACCT
EGSAPGTSESATPESGP CCGGTTCTGAAACTCCAGGTAGCCCGGCAG
GSEPATSGSETPGTSTEP GCTCTCCGACCTCTACTGAGGAAGGTACTTC
SEGSAPGTSTEPSEGSA TGAAAGCGCAACCCCGGAGTCCGGCCCAGG
PGTSESATPESGPGTSES TACCTCTACCGAACCGTCTGAGGGCAGCGC hGH- SEQ SEQ XTEN Amino Acid Sequence ID DNA Nucleotide Sequence ID Name* NO: NO:
ATPESGPGSPAGSPTST ACCAGGTACTTCTACCGAACCGTCCGAGGG
EEGTSESATPESGPGSEP TAGCGCACCAGGTAGCCCAGCAGGTTCTCC
ATSGSETPGTSESATPES TACCTCCACCGAGGAAGGTACTTCTACCGA
GPGTSTEPSEGSAPGTS ACCGTCCGAGGGTAGCGCACCAGGTACCTC
TEPSEGSAPGTSTEPSEG TACTGAACCTTCTGAGGGCAGCGCTCCAGG
SAPGTSTEPSEGSAPGT TACTTCTGAAAGCGCTACCCCGGAGTCCGG
STEPSEGSAPGTSTEPSE TCCAGGTACTTCTACTGAACCGTCCGAAGG
GSAPGSPAGSPTSTEEG TAGCGCACCAGGTACTTCTGAAAGCGCAAC
TSTEPSEGSAPGTSESAT CCCTGAATCCGGTCCAGGTAGCGAACCGGC
PESGPGSEPATSGSETP TACTTCTGGCTCTGAGACTCCAGGTACTTCT
GTSESATPESGPGSEPA ACCGAACCGTCCGAAGGTAGCGCACCAGGT
TSGSETPGTSESATPESG ACTTCTACTGAACCGTCTGAAGGTAGCGCA
PGTSTEPSEGSAPGTSES CCAGGTACTTCTGAAAGCGCAACCCCGGAA
ATPESGPGSPAGSPTST TCCGGCCCAGGTACCTCTGAAAGCGCAACC
EEGSPAGSPTSTEEGSP CCGGAGTCCGGCCCAGGTAGCCCTGCTGGC
AGSPTSTEEGTSESATP TCTCCAACCTCCACCGAAGAAGGTACCTCT
ESGPGTSTEPSEGSAPG GAAAGCGCAACCCCTGAATCCGGCCCAGGT
TSESATPESGPGSEPATS AGCGAACCGGCAACCTCCGGTTCTGAAACC
GSETPGTSESATPESGP CCAGGTACCTCTGAAAGCGCTACTCCGGAG
GSEPATSGSETPGTSES TCTGGCCCAGGTACCTCTACTGAACCGTCTG
ATPESGPGTSTEPSEGS AGGGTAGCGCTCCAGGTACTTCTACTGAAC
APGSPAGSPTSTEEGTS CGTCCGAAGGTAGCGCACCAGGTACTTCTA
ESATPESGPGSEPATSG CCGAACCGTCCGAAGGCAGCGCTCCAGGTA
SETPGTSESATPESGPGS CCTCTACTGAACCTTCCGAGGGCAGCGCTC
PAGSPTSTEEGSPAGSP CAGGTACCTCTACCGAACCTTCTGAAGGTA
TSTEEGTSTEPSEGSAP GCGCACCAGGTACTTCTACCGAACCGTCCG
GTSESATPESGPGTSES AGGGTAGCGCACCAGGTAGCCCAGCAGGTT
ATPESGPGTSESATPES CTCCTACCTCCACCGAGGAAGGTACTTCTAC
GPGSEPATSGSETPGSE CGAACCGTCCGAGGGTAGCGCACCAGGTAC
PATSGSETPGSPAGSPTS CTCTGAAAGCGCAACTCCTGAGTCTGGCCC
TEEGTSTEPSEGSAPGT AGGTAGCGAACCTGCTACCTCCGGCTCTGA
STEPSEGSAPGSEPATS GACTCCAGGTACCTCTGAAAGCGCAACCCC
GSETPGTSESATPESGP GGAATCTGGTCCAGGTAGCGAACCTGCAAC
GTSTEPSEGSAPGFPTIP CTCTGGCTCTGAAACCCCAGGTACCTCTGA
LSRLFDNAMLRAHRLH AAGCGCTACTCCTGAATCTGGCCCAGGTAC
QLAFDTYQEFEEAYIPK TTCTACTGAACCGTCCGAGGGCAGCGCACC
EQKYSFLQNPQTSLCFS AGGTACTTCTGAAAGCGCTACTCCTGAGTC
ESIPTP SNREETQQKSNL CGGCCCAGGTAGCCCGGCTGGCTCTCCGAC
ELLPJSLLLIQSWLEPVQ TTCCACCGAGGAAGGTAGCCCGGCTGGCTC
FLRS VF ANSL VYG ASD S TCCAACTTCTACTGAAGAAGGTAGCCCGGC
NVYDLLKDLEEGIQTL AGGCTCTCCGACCTCTACTGAGGAAGGTAC
MGRLEDGSPRTGQIFK TTCTGAAAGCGCAACCCCGGAGTCCGGCCC
QTYSKFDTNSHNDDAL AGGTACCTCTACCGAACCGTCTGAGGGCAG
LKNYGLLYCFRKDMD CGCACCAGGTACCTCTGAAAGCGCAACTCC
KVETFLRIVQCRSVEGS TGAGTCTGGCCCAGGTAGCGAACCTGCTAC
CGF CTCCGGCTCTGAGACTCCAGGTACCTCTGA
AAGCGCAACCCCGGAATCTGGTCCAGGTAG
CGAACCTGCAACCTCTGGCTCTGAAACCCC
AGGTACCTCTGAAAGCGCTACTCCTGAATC
TGGCCCAGGTACTTCTACTGAACCGTCCGA
GGGCAGCGCACCAGGTAGCCCTGCTGGCTC
TCCAACCTCCACCGAAGAAGGTACCTCTGA
AAGCGCAACCCCTGAATCCGGCCCAGGTAG
CGAACCGGCAACCTCCGGTTCTGAAACCCC
AGGTACTTCTGAAAGCGCTACTCCTGAGTC
CGGCCCAGGTAGCCCGGCTGGCTCTCCGAC hGH- SEQ SEQ XTEN Amino Acid Sequence ID DNA Nucleotide Sequence ID Name* NO: NO:
TTCCACCGAGGAAGGTAGCCCGGCTGGCTC
TCCAACTTCTACTGAAGAAGGTACTTCTACC
GAACCTTCCGAGGGCAGCGCACCAGGTACT
TCTGAAAGCGCTACCCCTGAGTCCGGCCCA
GGTACTTCTGAAAGCGCTACTCCTGAATCC
GGTCCAGGTACTTCTGAAAGCGCTACCCCG
GAATCTGGCCCAGGTAGCGAACCGGCTACT
TCTGGTTCTGAAACCCCAGGTAGCGAACCG
GCTACCTCCGGTTCTGAAACTCCAGGTAGC
CCAGCAGGCTCTCCGACTTCCACTGAGGAA
GGTACTTCTACTGAACCTTCCGAAGGCAGC
GCACCAGGTACCTCTACTGAACCTTCTGAG
GGCAGCGCTCCAGGTAGCGAACCTGCAACC
TCTGGCTCTGAAACCCCAGGTACCTCTGAA
AGCGCTACTCCTGAATCTGGCCCAGGTACTT
CTACTGAACCGTCCGAGGGCAGCGCACCAG
GTTTTCCGACTATTCCGCTGTCTCGTCTGTTT
GATAATGCTATGCTGCGTGCGCACCGTCTG
CACCAGCTGGCCTTTGATACTTACCAGGAA
TTTGAAGAAGCcTACATTCCTAAAGAGCAG
AAGTACTCTTTCCTGCAAAACCCACAGACTT
CTCTCTGCTTCAGCGAATCTATTCCGACGCC
TTCCAATCGCGAGGAAACTCAGCAAAAGTC
CAATCTGGAACTACTCCGCATTTCTCTGCTT
CTGATTCAGAGCTGGCTAGAACCAGTGCAA
TTTCTGCGTTCCGTCTTCGCCAATAGCCTAG
TTTATGGCGCATCCGACAGCAACGTATACG
ATCTCCTGAAAGATCTCGAGGAAGGCATTC
AGACCCTGATGGGTCGTCTCGAGGATGGCT
CTCCGCGTACTGGTCAGATCTTCAAGCAGA
CTTACTCTAAATTTGATACTAACAGCCACAA
TGACGATGCGCTTCTAAAAAACTATGGTCT
GCTGTATTGTTTTCGTAAAGATATGGACAA
AGTTGAAACCTTCCTGCGTATTGTTCAGTGT
CGTTCCGTTGAGGGCAGCTGTGGTTTCTAA
AE912- AEPAGSPTSTEEGTPGS 5 ATGGCTGAACCTGCTGGCTCTCCAACCTCCA 11 hGH- GTASSSPGSSTPSGATG CTGAGGAAGGTACCCCGGGTAGCGGTACTG
AE288 SPGASPGTSSTGSPGSP CTTCTTCCTCTCCAGGTAGCTCTACCCCTTC
AGSPTSTEEGTSESATP TGGTGCAACCGGCTCTCCAGGTGCTTCTCCG
ESGPGTSTEPSEGSAPG GGCACCAGCTCTACCGGTTCTCCAGGTAGC
SPAGSPTSTEEGTSTEPS CCGGCTGGCTCTCCTACCTCTACTGAGGAA
EGSAPGTSTEPSEGSAP GGTACTTCTGAAAGCGCTACTCCTGAGTCTG
GTSESATPESGPGSEPA GTCCAGGTACCTCTACTGAACCGTCCGAAG
TSGSETPGSEPATSGSET GTAGCGCTCCAGGTAGCCCAGCAGGCTCTC
PGSPAGSPTSTEEGTSES CGACTTCCACTGAGGAAGGTACTTCTACTG
ATPESGPGTSTEPSEGS AACCTTCCGAAGGCAGCGCACCAGGTACCT
APGTSTEPSEGSAPGSP CTACTGAACCTTCTGAGGGCAGCGCTCCAG
AGSPTSTEEGTSTEPSE GTACTTCTGAAAGCGCTACCCCGGAATCTG
GSAPGTSTEPSEGSAPG GCCCAGGTAGCGAACCGGCTACTTCTGGTT
TSESATPESGPGTSTEPS CTGAAACCCCAGGTAGCGAACCGGCTACCT
EGSAPGTSESATPESGP CCGGTTCTGAAACTCCAGGTAGCCCGGCAG
GSEPATSGSETPGTSTEP GCTCTCCGACCTCTACTGAGGAAGGTACTTC
SEGSAPGTSTEPSEGSA TGAAAGCGCAACCCCGGAGTCCGGCCCAGG
PGTSESATPESGPGTSES TACCTCTACCGAACCGTCTGAGGGCAGCGC
ATPESGPGSPAGSPTST ACCAGGTACTTCTACCGAACCGTCCGAGGG
EEGTSESATPESGPGSEP TAGCGCACCAGGTAGCCCAGCAGGTTCTCC hGH- SEQ SEQ XTEN Amino Acid Sequence ID DNA Nucleotide Sequence ID Name* NO: NO:
ATSGSETPGTSESATPES TACCTCCACCGAGGAAGGTACTTCTACCGA
GPGTSTEPSEGSAPGTS ACCGTCCGAGGGTAGCGCACCAGGTACCTC
TEPSEGSAPGTSTEPSEG TACTGAACCTTCTGAGGGCAGCGCTCCAGG
SAPGTSTEPSEGSAPGT TACTTCTGAAAGCGCTACCCCGGAGTCCGG
STEPSEGSAPGTSTEPSE TCCAGGTACTTCTACTGAACCGTCCGAAGG
GSAPGSPAGSPTSTEEG TAGCGCACCAGGTACTTCTGAAAGCGCAAC
TSTEPSEGSAPGTSESAT CCCTGAATCCGGTCCAGGTAGCGAACCGGC
PESGPGSEPATSGSETP TACTTCTGGCTCTGAGACTCCAGGTACTTCT
GTSESATPESGPGSEPA ACCGAACCGTCCGAAGGTAGCGCACCAGGT
TSGSETPGTSESATPESG ACTTCTACTGAACCGTCTGAAGGTAGCGCA
PGTSTEPSEGSAPGTSES CCAGGTACTTCTGAAAGCGCAACCCCGGAA
ATPESGPGSPAGSPTST TCCGGCCCAGGTACCTCTGAAAGCGCAACC
EEGSPAGSPTSTEEGSP CCGGAGTCCGGCCCAGGTAGCCCTGCTGGC
AGSPTSTEEGTSESATP TCTCCAACCTCCACCGAAGAAGGTACCTCT
ESGPGTSTEPSEGSAPG GAAAGCGCAACCCCTGAATCCGGCCCAGGT
TSESATPESGPGSEPATS AGCGAACCGGCAACCTCCGGTTCTGAAACC
GSETPGTSESATPESGP CCAGGTACCTCTGAAAGCGCTACTCCGGAG
GSEPATSGSETPGTSES TCTGGCCCAGGTACCTCTACTGAACCGTCTG
ATPESGPGTSTEPSEGS AGGGTAGCGCTCCAGGTACTTCTACTGAAC
APGSPAGSPTSTEEGTS CGTCCGAAGGTAGCGCACCAGGTACTTCTA
ESATPESGPGSEPATSG CCGAACCGTCCGAAGGCAGCGCTCCAGGTA
SETPGTSESATPESGPGS CCTCTACTGAACCTTCCGAGGGCAGCGCTC
PAGSPTSTEEGSPAGSP CAGGTACCTCTACCGAACCTTCTGAAGGTA
TSTEEGTSTEPSEGSAP GCGCACCAGGTACTTCTACCGAACCGTCCG
GTSESATPESGPGTSES AGGGTAGCGCACCAGGTAGCCCAGCAGGTT
ATPESGPGTSESATPES CTCCTACCTCCACCGAGGAAGGTACTTCTAC
GPGSEPATSGSETPGSE CGAACCGTCCGAGGGTAGCGCACCAGGTAC
PATSGSETPGSPAGSPTS CTCTGAAAGCGCAACTCCTGAGTCTGGCCC
TEEGTSTEPSEGSAPGT AGGTAGCGAACCTGCTACCTCCGGCTCTGA
STEPSEGSAPGSEPATS GACTCCAGGTACCTCTGAAAGCGCAACCCC
GSETPGTSESATPESGP GGAATCTGGTCCAGGTAGCGAACCTGCAAC
GTSTEPSEGSAPGFPTIP CTCTGGCTCTGAAACCCCAGGTACCTCTGA
LSRLFDNAMLRAHRLH AAGCGCTACTCCTGAATCTGGCCCAGGTAC
QLAFDTYQEFEEAYIPK TTCTACTGAACCGTCCGAGGGCAGCGCACC
EQKYSFLQNPQTSLCFS AGGTACTTCTGAAAGCGCTACTCCTGAGTC
ESIPTP SNREETQQKSNL CGGCCCAGGTAGCCCGGCTGGCTCTCCGAC
ELLPJSLLLIQSWLEPVQ TTCCACCGAGGAAGGTAGCCCGGCTGGCTC
FLRS VF ANSL VYG ASD S TCCAACTTCTACTGAAGAAGGTAGCCCGGC
NVYDLLKDLEEGIQTL AGGCTCTCCGACCTCTACTGAGGAAGGTAC
MGRLEDGSPRTGQIFK TTCTGAAAGCGCAACCCCGGAGTCCGGCCC
QTYSKFDTNSHNDDAL AGGTACCTCTACCGAACCGTCTGAGGGCAG
LKNYGLLYCFRKDMD CGCACCAGGTACCTCTGAAAGCGCAACTCC
KVETFLRIVQCRSVEGS TGAGTCTGGCCCAGGTAGCGAACCTGCTAC
CGFGGTSESATPESGPG CTCCGGCTCTGAGACTCCAGGTACCTCTGA
SEPATSGSETPGTSESAT AAGCGCAACCCCGGAATCTGGTCCAGGTAG
PESGPGSEPATSGSETP CGAACCTGCAACCTCTGGCTCTGAAACCCC
GTSESATPESGPGTSTEP AGGTACCTCTGAAAGCGCTACTCCTGAATC
SEGSAPGSPAGSPTSTE TGGCCCAGGTACTTCTACTGAACCGTCCGA
EGTSESATPESGPGSEP GGGCAGCGCACCAGGTAGCCCTGCTGGCTC
ATSGSETPGTSESATPES TCCAACCTCCACCGAAGAAGGTACCTCTGA
GPGSPAGSPTSTEEGSP AAGCGCAACCCCTGAATCCGGCCCAGGTAG
AGSPTSTEEGTSTEPSE CGAACCGGCAACCTCCGGTTCTGAAACCCC
GSAPGTSESATPESGPG AGGTACTTCTGAAAGCGCTACTCCTGAGTC
TSESATPESGPGTSESAT CGGCCCAGGTAGCCCGGCTGGCTCTCCGAC
PESGPGSEPATSGSETP TTCCACCGAGGAAGGTAGCCCGGCTGGCTC
GSEPATSGSETPGSPAG TCCAACTTCTACTGAAGAAGGTACTTCTACC hGH- SEQ SEQ XTEN Amino Acid Sequence ID DNA Nucleotide Sequence ID Name* NO: NO:
SPTSTEEGTSTEPSEGSA GAACCTTCCGAGGGCAGCGCACCAGGTACT PGTSTEPSEGSAPGSEP TCTGAAAGCGCTACCCCTGAGTCCGGCCCA ATSGSETPGTSESATPES GGTACTTCTGAAAGCGCTACTCCTGAATCC GPGTSTEPSEGSAPG GGTCCAGGTACTTCTGAAAGCGCTACCCCG
GAATCTGGCCCAGGTAGCGAACCGGCTACT
TCTGGTTCTGAAACCCCAGGTAGCGAACCG
GCTACCTCCGGTTCTGAAACTCCAGGTAGC
CCAGCAGGCTCTCCGACTTCCACTGAGGAA
GGTACTTCTACTGAACCTTCCGAAGGCAGC
GCACCAGGTACCTCTACTGAACCTTCTGAG
GGCAGCGCTCCAGGTAGCGAACCTGCAACC
TCTGGCTCTGAAACCCCAGGTACCTCTGAA
AGCGCTACTCCTGAATCTGGCCCAGGTACTT
CTACTGAACCGTCCGAGGGCAGCGCACCAG
GTTTTCCGACTATTCCGCTGTCTCGTCTGTTT
GATAATGCTATGCTGCGTGCGCACCGTCTG
CACCAGCTGGCCTTTGATACTTACCAGGAA
TTTGAAGAAGCcTACATTCCTAAAGAGCAG
AAGTACTCTTTCCTGCAAAACCCACAGACTT
CTCTCTGCTTCAGCGAATCTATTCCGACGCC
TTCCAATCGCGAGGAAACTCAGCAAAAGTC
CAATCTGGAACTACTCCGCATTTCTCTGCTT
CTGATTCAGAGCTGGCTAGAACCAGTGCAA
TTTCTGCGTTCCGTCTTCGCCAATAGCCTAG
TTTATGGCGCATCCGACAGCAACGTATACG
ATCTCCTGAAAGATCTCGAGGAAGGCATTC
AGACCCTGATGGGTCGTCTCGAGGATGGCT
CTCCGCGTACTGGTCAGATCTTCAAGCAGA
CTTACTCTAAATTTGATACTAACAGCCACAA
TGACGATGCGCTTCTAAAAAACTATGGTCT
GCTGTATTGTTTTCGTAAAGATATGGACAA
AGTTGAAACCTTCCTGCGTATTGTTCAGTGT
CGTTCCGTTGAGGGCAGCTGTGGTTTCTAAG
GTGGTACCTCTGAAAGCGCAACTCCTGAGT
CTGGCCCAGGTAGCGAACCTGCTACCTCCG
GCTCTGAGACTCCAGGTACCTCTGAAAGCG
CAACCCCGGAATCTGGTCCAGGTAGCGAAC
CTGCAACCTCTGGCTCTGAAACCCCAGGTA
CCTCTGAAAGCGCTACTCCTGAATCTGGCCC
AGGTACTTCTACTGAACCGTCCGAGGGCAG
CGCACCAGGTAGCCCTGCTGGCTCTCCAAC
CTCCACCGAAGAAGGTACCTCTGAAAGCGC
AACCCCTGAATCCGGCCCAGGTAGCGAACC
GGCAACCTCCGGTTCTGAAACCCCAGGTAC
TTCTGAAAGCGCTACTCCTGAGTCCGGCCC
AGGTAGCCCGGCTGGCTCTCCGACTTCCAC
CGAGGAAGGTAGCCCGGCTGGCTCTCCAAC
TTCTACTGAAGAAGGTACTTCTACCGAACCT
TCCGAGGGCAGCGCACCAGGTACTTCTGAA
AGCGCTACCCCTGAGTCCGGCCCAGGTACT
TCTGAAAGCGCTACTCCTGAATCCGGTCCA
GGTACTTCTGAAAGCGCTACCCCGGAATCT
GGCCCAGGTAGCGAACCGGCTACTTCTGGT
TCTGAAACCCCAGGTAGCGAACCGGCTACC
TCCGGTTCTGAAACTCCAGGTAGCCCAGCA
GGCTCTCCGACTTCCACTGAGGAAGGTACT hGH- SEQ SEQ XTEN Amino Acid Sequence ID DNA Nucleotide Sequence ID Name* NO: NO:
TCTACTGAACCTTCCGAAGGCAGCGCACCA
GGTACCTCTACTGAACCTTCTGAGGGCAGC
GCTCCAGGTAGCGAACCTGCAACCTCTGGC
TCTGAAACCCCAGGTACCTCTGAAAGCGCT
ACTCCTGAATCTGGCCCAGGTACTTCTACTG
AACCGTCCGAGGGCAGCGCACCA
AM875- GTSTEPSEGSAPGSEPA 6 GGTACTTCTACTGAACCGTCTGAAGGCAGC 12 hGH TSGSETPGSPAGSPTSTE GCACCAGGTAGCGAACCGGCTACTTCCGGT
EGSTSSTAESPGPGTSTP TCTGAAACCCCAGGTAGCCCAGCAGGTTCT
ESGSASPGSTSESPSGTA CCAACTTCTACTGAAGAAGGTTCTACCAGC
PGSTSESPSGTAPGTSTP TCTACCGCAGAATCTCCTGGTCCAGGTACCT
ESGSASPGTSTPESGSAS CTACTCCGGAAAGCGGCTCTGCATCTCCAG
PGSEPATSGSETPGTSES GTTCTACTAGCGAATCTCCTTCTGGCACTGC
ATPESGPGSPAGSPTST ACCAGGTTCTACTAGCGAATCCCCGTCTGGT
EEGTSTEPSEGSAPGTS ACTGCTCCAGGTACTTCTACTCCTGAAAGCG
ESATPESGPGTSTEPSEG GTTCCGCTTCTCCAGGTACCTCTACTCCGGA
SAPGTSTEPSEGSAPGSP AAGCGGTTCTGCATCTCCAGGTAGCGAACC
AGSPTSTEEGTSTEPSE GGCAACCTCCGGCTCTGAAACCCCAGGTAC
GSAPGTSTEPSEGSAPG CTCTGAAAGCGCTACTCCTGAATCCGGCCC
TSESATPESGPGTSESAT AGGTAGCCCGGCAGGTTCTCCGACTTCCAC
PESGPGTSTEPSEGSAP TGAGGAAGGTACCTCTACTGAACCTTCTGA
GTSTEPSEGSAPGTSES GGGCAGCGCTCCAGGTACTTCTGAAAGCGC
ATPESGPGTSTEPSEGS TACCCCGGAGTCCGGTCCAGGTACTTCTACT
APGSEPATSGSETPGSP GAACCGTCCGAAGGTAGCGCACCAGGTACT
AGSPTSTEEGSSTPSGA TCTACCGAACCGTCCGAGGGTAGCGCACCA
TGSPGTPGSGTASSSPG GGTAGCCCAGCAGGTTCTCCTACCTCCACC
SSTPSGATGSPGTSTEPS GAGGAAGGTACTTCTACCGAACCGTCCGAG
EGSAPGTSTEPSEGSAP GGTAGCGCACCAGGTACTTCTACCGAACCT
GSEPATSGSETPGSPAG TCCGAGGGCAGCGCACCAGGTACTTCTGAA
SPTSTEEGSPAGSPTSTE AGCGCTACCCCTGAGTCCGGCCCAGGTACT
EGTSTEPSEGSAPGASA TCTGAAAGCGCTACTCCTGAATCCGGTCCA
SGAPSTGGTSESATPES GGTACCTCTACTGAACCTTCCGAAGGCAGC
GPGSPAGSPTSTEEGSP GCTCCAGGTACCTCTACCGAACCGTCCGAG
AGSPTSTEEGSTSSTAES GGCAGCGCACCAGGTACTTCTGAAAGCGCA
PGPGSTSESPSGTAPGTS ACCCCTGAATCCGGTCCAGGTACTTCTACTG
PSGESSTAPGTPGSGTA AACCTTCCGAAGGTAGCGCTCCAGGTAGCG
SSSPGSSTPSGATGSPGS AACCTGCTACTTCTGGTTCTGAAACCCCAGG
SPSASTGTGPGSEPATS TAGCCCGGCTGGCTCTCCGACCTCCACCGA
GSETPGTSESATPESGP GGAAGGTAGCTCTACCCCGTCTGGTGCTAC
GSEPATSGSETPGSTSST TGGTTCTCCAGGTACTCCGGGCAGCGGTAC
AESPGPGSTSSTAESPGP TGCTTCTTCCTCTCCAGGTAGCTCTACCCCT
GTSPSGESSTAPGSEPA TCTGGTGCTACTGGCTCTCCAGGTACCTCTA
TSGSETPGSEPATSGSET CCGAACCGTCCGAGGGTAGCGCACCAGGTA
PGTSTEPSEGSAPGSTSS CCTCTACTGAACCGTCTGAGGGTAGCGCTC
TAESPGPGTSTPESGSA CAGGTAGCGAACCGGCAACCTCCGGTTCTG
SPGSTSESPSGTAPGTST AAACTCCAGGTAGCCCTGCTGGCTCTCCGA
EPSEGSAPGTSTEPSEGS CTTCTACTGAGGAAGGTAGCCCGGCTGGTT
APGTSTEPSEGSAPGSS CTCCGACTTCTACTGAGGAAGGTACTTCTAC
TPSGATGSPGSSPSAST CGAACCTTCCGAAGGTAGCGCTCCAGGTGC
GTGPGASPGTSSTGSPG AAGCGCAAGCGGCGCGCCAAGCACGGGAG
SEPATSGSETPGTSESAT GTACTTCTGAAAGCGCTACTCCTGAGTCCG
PESGPGSPAGSPTSTEE GCCCAGGTAGCCCGGCTGGCTCTCCGACTT
GSSTPSGATGSPGSSPS CCACCGAGGAAGGTAGCCCGGCTGGCTCTC
ASTGTGPGASPGTSSTG CAACTTCTACTGAAGAAGGTTCTACCAGCT
SPGTSESATPESGPGTST CTACCGCTGAATCTCCTGGCCCAGGTTCTAC
EPSEGSAPGTSTEPSEGS TAGCGAATCTCCGTCTGGCACCGCACCAGG hGH- SEQ SEQ XTEN Amino Acid Sequence ID DNA Nucleotide Sequence ID Name* NO: NO:
APGFPTIPLSRLFDNAM TACTTCCCCTAGCGGTGAATCTTCTACTGCA
LRAHRLHQLAFDTYQE CCAGGTACCCCTGGCAGCGGTACCGCTTCTT
FEEAYIPKEQKYSFLQN CCTCTCCAGGTAGCTCTACCCCGTCTGGTGC
PQTSLCFSESIPTPSNRE TACTGGCTCTCCAGGTTCTAGCCCGTCTGCA
ETQQKSNLELLRISLLLI TCTACCGGTACCGGCCCAGGTAGCGAACCG
QSWLEPVQFLRSVFAN GCAACCTCCGGCTCTGAAACTCCAGGTACT
SL VYG ASD SNVYDLLK TCTGAAAGCGCTACTCCGGAATCCGGCCCA
DLEEGIQTLMGRLEDGS GGTAGCGAACCGGCTACTTCCGGCTCTGAA
PRTGQIFKQTYSKFDTN ACCCCAGGTTCCACCAGCTCTACTGCAGAA
SHNDDALLKNYGLLYC TCTCCGGGCCCAGGTTCTACTAGCTCTACTG
FRKDMDKVETFLRIVQ CAGAATCTCCGGGTCCAGGTACTTCTCCTAG
CRSVEGSCGF CGGCGAATCTTCTACCGCTCCAGGTAGCGA
ACCGGCAACCTCTGGCTCTGAAACTCCAGG
TAGCGAACCTGCAACCTCCGGCTCTGAAAC
CCCAGGTACTTCTACTGAACCTTCTGAGGGC
AGCGCACCAGGTTCTACCAGCTCTACCGCA
GAATCTCCTGGTCCAGGTACCTCTACTCCGG
AAAGCGGCTCTGCATCTCCAGGTTCTACTA
GCGAATCTCCTTCTGGCACTGCACCAGGTA
CTTCTACCGAACCGTCCGAAGGCAGCGCTC
CAGGTACCTCTACTGAACCTTCCGAGGGCA
GCGCTCCAGGTACCTCTACCGAACCTTCTGA
AGGTAGCGCACCAGGTAGCTCTACTCCGTC
TGGTGCAACCGGCTCCCCAGGTTCTAGCCC
GTCTGCTTCCACTGGTACTGGCCCAGGTGCT
TCCCCGGGCACCAGCTCTACTGGTTCTCCAG
GTAGCGAACCTGCTACCTCCGGTTCTGAAA
CCCCAGGTACCTCTGAAAGCGCAACTCCGG
AGTCTGGTCCAGGTAGCCCTGCAGGTTCTCC
TACCTCCACTGAGGAAGGTAGCTCTACTCC
GTCTGGTGCAACCGGCTCCCCAGGTTCTAG
CCCGTCTGCTTCCACTGGTACTGGCCCAGGT
GCTTCCCCGGGCACCAGCTCTACTGGTTCTC
CAGGTACCTCTGAAAGCGCTACTCCGGAGT
CTGGCCCAGGTACCTCTACTGAACCGTCTG
AGGGTAGCGCTCCAGGTACTTCTACTGAAC
CGTCCGAAGGTAGCGCACCAGGTTTTCCGA
CTATTCCGCTGTCTCGTCTGTTTGATAATGC
TATGCTGCGTGCGCACCGTCTGCACCAGCT
GGCCTTTGATACTTACCAGGAATTTGAAGA
AGCcTACATTCCTAAAGAGCAGAAGTACTCT
TTCCTGCAAAACCCACAGACTTCTCTCTGCT
TCAGCGAATCTATTCCGACGCCTTCCAATCG
CGAGGAAACTCAGCAAAAGTCCAATCTGGA
ACTACTCCGCATTTCTCTGCTTCTGATTCAG
AGCTGGCTAGAACCAGTGCAATTTCTGCGT
TCCGTCTTCGCCAATAGCCTAGTTTATGGCG
CATCCGACAGCAACGTATACGATCTCCTGA
AAGATCTCGAGGAAGGCATTCAGACCCTGA
TGGGTCGTCTCGAGGATGGCTCTCCGCGTA
CTGGTCAGATCTTCAAGCAGACTTACTCTAA
ATTTGATACTAACAGCCACAATGACGATGC
GCTTCTAAAAAACTATGGTCTGCTGTATTGT
TTTCGTAAAGATATGGACAAAGTTGAAACC
TTCCTGCGTATTGTTCAGTGTCGTTCCGTTG
AGGGCAGCTGTGGTTTCTAA As described more fully in, for example, W013/184216, WO/2014/164568, U.S. Patent Nos. 8,492,530 and 8,703,717, each of which is incorporated herein by reference in its entirety, the fusion proteins optionally include spacer sequences that further comprise cleavage sequences to release the hGH from the fusion protein when acted on by a protease, releasing hGH from the XTEN sequence(s).
VI). EXTENDED RECOMBINANT POLYPEPTIDES
The present invention comprises formulations comprising XTEN polypeptide compositions that are useful as a fusion protein partner to which hGH is linked, resulting in a hGH-XTEN fusion protein. XTEN are generally extended length polypeptides with non- naturally occurring, substantially non-repetitive sequences that are composed mainly of small hydrophilic amino acids, with the sequence having a low degree or no secondary or tertiary structure under physiologic conditions. Among other characteristics, features of XTEN sequences such as the non-repetitiveness of sequences, exemplary sequence motifs, and lengths of sequences are known in the art and are described at least in, for example,
W013/184216, WO/2014/164568 and U.S. Patent Nos. 8,492,530 and 8,703,717, each of which is incorporated herein by reference in its entirety.
In one embodiment, the hGH-XTEN of the formulation can comprise one or more XTEN sequence wherein the sequence exhibits at least about 80% sequence identity, or alternatively 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a XTEN selected from Table 2. Examples where more than one XTEN is used in a hGH-XTEN composition include, but are not limited to constructs with an XTEN linked to both the N- and C-termini of at least one hGH. In preferred embodiments of the present invention comprising constructs with an
XTEN linked to both the N- and C-termini of at least one hGH, the length of the N-terminal XTEN sequence ranges between about 500 amino acids to about 1320 amino acids and the length of the C-terminal XTEN ranges between about 40 amino acids to about 300 amino acids.
Table 2: XTEN Polypeptides
Figure imgf000034_0001
SEQ
XTEN ID Amino Acid Sequence
Name
NO:
AM48 14 MAEPAGSPTSTEEGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGS
AE144 15 GSEPATSGSETPGTSESATPESGPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGS
APGSEPATSGSETPGSEPATSGSETPGSEPATSGSETPGTSTEPSEGSAPGTSESATP ESGPGSEPATSGSETPGTSTEPSEGSAP
AF144 16 GTSTPESGSASPGTSPSGESSTAPGTSPSGESSTAPGSTSSTAESPGPGSTSESPSGTA
PGSTSSTAESPGPGTSPSGESSTAPGTSTPESGSASPGSTSSTAESPGPGTSPSGESST APGTSPSGESSTAPGTSPSGESSTAP
AE288 17 GTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPES
GPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATP ESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESA TPESGPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTST EPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP
AF504 18 GASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATG
SPGSXPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGT
ASSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPG
TSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSXPSASTGTGPGSSPSASTGTGPGSS
TPSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPG
TPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTG
PGTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGAT
GSPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSG
ATGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSP
AF540 19 GSTSSTAESPGPGSTSSTAESPGPGSTSESPSGTAPGSTSSTAESPGPGSTSSTAESPG
PGTSTPESGSASPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGT
APGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSG
TAPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGSTSESPSGTAPGTSTPESG
SASPGSTSSTAESPGPGSTSSTAESPGPGTSTPESGSASPGTSTPESGSASPGSTSESP
SGTAPGTSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGSTSES
PSGTAPGSTSSTAESPGPGTSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGSTSE
SPSGTAPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSP
SGESSTAPGSTSSTAESPGPGTSPSGESSTAPGSTSSTAESPGPGTSTPESGSASPGST
SESPSGTAP
AD576 20 GSSESGSSEGGPGSGGEPSESGSSGSSESGSSEGGPGSSESGSSEGGPGSSESGSSEG
GPGSSESGSSEGGPGSSESGSSEGGPGESPGGSSGSESGSEGSSGPGESSGSSESGSS
EGGPGSSESGSSEGGPGSSESGSSEGGPGSGGEPSESGSSGESPGGSSGSESGESPG
GSSGSESGSGGEPSESGSSGSSESGSSEGGPGSGGEPSESGSSGSGGEPSESGSSGSE
GSSGPGESSGESPGGSSGSESGSGGEPSESGSSGSGGEPSESGSSGSGGEPSESGSSG
SSESGSSEGGPGESPGGSSGSESGESPGGSSGSESGESPGGSSGSESGESPGGSSGSE
SGESPGGSSGSESGSSESGSSEGGPGSGGEPSESGSSGSEGSSGPGESSGSSESGSSE
GGPGSGGEPSESGSSGSSESGSSEGGPGSGGEPSESGSSGESPGGSSGSESGESPGG
SSGSESGSSESGSSEGGPGSGGEPSESGSSGSSESGSSEGGPGSGGEPSESGSSGSGG
EPSESGSSGESPGGSSGSESGSEGSSGPGESSGSSESGSSEGGPGSEGSSGPGESS
AE576 21 GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGS
APGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPT
STEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPS
EGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPA
TSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSP
AGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPG
TSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSA
PGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPE
SGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSP
TSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAP
AF576 22 GSTSSTAESPGPGSTSSTAESPGPGSTSESPSGTAPGSTSSTAESPGPGSTSSTAESPG
PGTSTPESGSASPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGT APGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSG TAPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGSTSESPSGTAPGTSTPESG SASPGSTSSTAESPGPGSTSSTAESPGPGTSTPESGSASPGTSTPESGSASPGSTSESP SEQ
XTEN ID Amino Acid Sequence
Name
NO:
SGTAPGTSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGSTSES PSGTAPGSTSSTAESPGPGTSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGSTSE SPSGTAPGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSP SGESSTAPGSTSSTAESPGPGTSPSGESSTAPGSTSSTAESPGPGTSTPESGSASPGST SESPSGTAPGSTSSTAESPGPGTSTPESGSASPGTSTPESGSASP
AE624 23 MAEPAGSPTSTEEGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGSPAGSPT
STEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPS
EGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSES
ATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTS
TEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPG
TSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTE
EGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEG
SAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSP
TSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPA
TSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSP
AGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAP
AD836 24 GSSESGSSEGGPGSSESGSSEGGPGESPGGSSGSESGSGGEPSESGSSGESPGGSSGS
ESGESPGGSSGSESGSSESGSSEGGPGSSESGSSEGGPGSSESGSSEGGPGESPGGSS
GSESGESPGGSSGSESGESPGGSSGSESGSSESGSSEGGPGSSESGSSEGGPGSSESG
SSEGGPGSSESGSSEGGPGSSESGSSEGGPGSSESGSSEGGPGSGGEPSESGSSGESP
GGSSGSESGESPGGSSGSESGSGGEPSESGSSGSEGSSGPGESSGSSESGSSEGGPGS
GGEPSESGSSGSEGSSGPGESSGSSESGSSEGGPGSGGEPSESGSSGESPGGSSGSES
GSGGEPSESGSSGSGGEPSESGSSGSSESGSSEGGPGSGGEPSESGSSGSGGEPSESG
SSGSEGSSGPGESSGESPGGSSGSESGSEGSSGPGESSGSEGSSGPGESSGSGGEPSE
SGSSGSSESGSSEGGPGSSESGSSEGGPGESPGGSSGSESGSGGEPSESGSSGSEGSS
GPGESSGESPGGSSGSESGSEGSSGPGSSESGSSEGGPGSGGEPSESGSSGSEGSSGP
GESSGSEGSSGPGESSGSEGSSGPGESSGSGGEPSESGSSGSGGEPSESGSSGESPGG
SSGSESGESPGGSSGSESGSGGEPSESGSSGSEGSSGPGESSGESPGGSSGSESGSSE
SGSSEGGPGSSESGSSEGGPGSSESGSSEGGPGSGGEPSESGSSGSSESGSSEGGPGE
SPGGSSGSESGSGGEPSESGSSGSSESGSSEGGPGESPGGSSGSESGSGGEPSESGSS
GESPGGSSGSESGSGGEPSESGSS
AE864 25 GSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGS
APGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPT
STEEGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPS
EGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPA
TSGSETPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSP
AGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPG
TSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSA
PGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPE
SGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSP
TSTEEGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSES
ATPESGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTS
TEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPG
SPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESG
PGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEG
SAPGTSTEPSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP
AF864 26 GSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSAS
PGTSTPESGSASPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGT
APGTSPSGESSTAPGTSPSGESSTAPGSTSSTAESPGPGTSPSGESSTAPGTSPSGESS
TAPGSTSSTAESPGPGTSTPESGSASPGTSTPESGSASPGSTSESPSGTAPGSTSESPS
GTAPGTSTPESGSASPGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPGTSPSG
ESSTAPGSTSSTAESPGPGTSPSGESSTAPGTSTPESGSASPGSTSSTAESPGPGSTSS
TAESPGPGSTSSTAESPGPGSTSSTAESPGPGTSPSGESSTAPGSTSESPSGTAPGSTS
ESPSGTAPGTSTPESGPXXXGASASGAPSTXXXXSESPSGTAPGSTSESPSGTAPGS
TSESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPG
TSPSGESSTAPGTSPSGESSTAPGSTSSTAESPGPGTSPSGESSTAPGTSTPESGSASP
GSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGSTSESPSGTAPGTSTPESGSAS SEQ
XTEN ID Amino Acid Sequence
Name
NO:
PGTSTPESGSASPGSTSESPSGTAPGTSTPESGSASPGSTSSTAESPGPGSTSESPSGT APGSTSESPSGTAPGTSPSGESSTAPGSTSSTAESPGPGTSPSGESSTAPGTSTPESGS ASPGTSPSGESSTAPGTSPSGESSTAPGTSPSGESSTAPGSTSSTAESPGPGSTSSTAE SPGPGTSPSGESSTAPGSSPSASTGTGPGSSTPSGATGSPGSSTPSGATGSP
AG864 27 GASPGTSSTGSPGSSPSASTGTGPGSSPSASTGTGPGTPGSGTASSSPGSSTPSGATG
SPGSSPSASTGTGPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTA
SSSPGASPGTSSTGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGASPGT
SSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGSST
PSGATGSPGSSTPSGATGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGT
PGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSPSASTGTGP
GTPGSGTASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGSSTPSGATG
SPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGA
TGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGASPGTSSTGSPGTPGSG
TASSSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGASPGTSSTGSPGTPG
SGTASSSPGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATGSPGTPGSGTASSSPGS
STPSGATGSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTGSP
GTPGSGTASSSPGSSTPSGATGSPGSSPSASTGTGPGSSPSASTGTGPGASPGTSSTG
SPGASPGTSSTGSPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTGSPGSSPSAST
GTGPGTPGSGTASSSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTGSP
AM87 28 GTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSTSSTAESPGPGTSTPESGSA 5 SPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSEPATSGS
ETPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSTEPSE
GSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESA
TPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTST
EPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSSTPSGATGSPGTPGSGTASSSPGS
STPSGATGSPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEE
GSPAGSPTSTEEGTSTEPSEGSAPGASASGAPSTGGTSESATPESGPGSPAGSPTSTE
EGSPAGSPTSTEEGSTSSTAESPGPGSTSESPSGTAPGTSPSGESSTAPGTPGSGTAS
SSPGSSTPSGATGSPGSSPSASTGTGPGSEPATSGSETPGTSESATPESGPGSEPATS
GSETPGSTSSTAESPGPGSTSSTAESPGPGTSPSGESSTAPGSEPATSGSETPGSEPA
TSGSETPGTSTEPSEGSAPGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPGTST
EPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSSTPSGATGSPGSSPSASTGTGPGA
SPGTSSTGSPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSSTPSGATGSP
GSSPSASTGTGPGASPGTSSTGSPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGS
AP
AE912 29 MAEPAGSPTSTEEGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGSPAGSPT
STEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPS
EGSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSES
ATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTS
TEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPG
TSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTE
EGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEG
SAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSP
TSTEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPA
TSGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSP
AGSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPG
SEPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSA
PGSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTS
TEEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESAT
PESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTE
PSEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP
AM92 30 MAEPAGSPTSTEEGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGSPGTSTEPSE 3 GSAPGSEPATSGSETPGSPAGSPTSTEEGSTSSTAESPGPGTSTPESGSASPGSTSESP
SGTAPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSEPATSGSETPGTSES
ATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTS
TEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPG
TSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSA SEQ
XTEN ID Amino Acid Sequence
Name
NO:
PGSEPATSGSETPGSPAGSPTSTEEGSSTPSGATGSPGTPGSGTASSSPGSSTPSGAT
GSPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSPAGSP
TSTEEGTSTEPSEGSAPGASASGAPSTGGTSESATPESGPGSPAGSPTSTEEGSPAGS
PTSTEEGSTSSTAESPGPGSTSESPSGTAPGTSPSGESSTAPGTPGSGTASSSPGSSTP
SGATGSPGSSPSASTGTGPGSEPATSGSETPGTSESATPESGPGSEPATSGSETPGST
SSTAESPGPGSTSSTAESPGPGTSPSGESSTAPGSEPATSGSETPGSEPATSGSETPG
TSTEPSEGSAPGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPGTSTEPSEGSAP
GTSTEPSEGSAPGTSTEPSEGSAPGSSTPSGATGSPGSSPSASTGTGPGASPGTSSTG
SPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSSTPSGATGSPGSSPSAST
GTGPGASPGTSSTGSPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAP
AM13 31 GTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSTSSTAESPGPGTSTPESGSA 18 SPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSEPATSGS
ETPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSTEPSE
GSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESA
TPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTST
EPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSSTPSGATGSPGTPGSGTASSSPGS
STPSGATGSPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEE
GSPAGSPTSTEEGTSTEPSEGSAPGPEPTGPAPSGGSEPATSGSETPGTSESATPESG
PGSPAGSPTSTEEGTSESATPESGPGSPAGSPTSTEEGSPAGSPTSTEEGTSESATPE
SGPGSPAGSPTSTEEGSPAGSPTSTEEGSTSSTAESPGPGSTSESPSGTAPGTSPSGES
STAPGSTSESPSGTAPGSTSESPSGTAPGTSPSGESSTAPGTSTEPSEGSAPGTSESA
TPESGPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSESATPESGPGTST
EPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSPSGESSTAPGTSPSGESSTAPGT
SPSGESSTAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGSSPSASTGTGP
GSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGSSTPSGATGSPGASPGTSSTG
SPGASASGAPSTGGTSPSGESSTAPGSTSSTAESPGPGTSPSGESSTAPGTSESATPE
SGPGTSTEPSEGSAPGTSTEPSEGSAPGSSPSASTGTGPGSSTPSGATGSPGASPGTS
STGSPGTSTPESGSASPGTSPSGESSTAPGTSPSGESSTAPGTSESATPESGPGSEPAT
SGSETPGTSTEPSEGSAPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGSPA
GSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSESATPESGPGS
EPATSGSETPGSSTPSGATGSPGASPGTSSTGSPGSSTPSGATGSPGSTSESPSGTAP
GTSPSGESSTAPGSTSSTAESPGPGSSTPSGATGSPGASPGTSSTGSPGTPGSGTASS
SPGSPAGSPTSTEEGSPAGSPTSTEEGTSTEPSEGSAP
BC 32 GTSTEPSEPGSAGTSTEPSEPGSAGSEPATSGTEPSGSGASEPTSTEPGSEPATSGTE 864 PSGSEPATSGTEPSGSEPATSGTEPSGSGASEPTSTEPGTSTEPSEPGSAGSEPATSG
TEPSGTSTEPSEPGSAGSEPATSGTEPSGSEPATSGTEPSGTSTEPSEPGSAGTSTEPS
EPGSAGSEPATSGTEPSGSEPATSGTEPSGTSEPSTSEPGAGSGASEPTSTEPGTSEP
STSEPGAGSEPATSGTEPSGSEPATSGTEPSGTSTEPSEPGSAGTSTEPSEPGSAGSG
ASEPTSTEPGSEPATSGTEPSGSEPATSGTEPSGSEPATSGTEPSGSEPATSGTEPSG
TSTEPSEPGSAGSEPATSGTEPSGSGASEPTSTEPGTSTEPSEPGSAGSEPATSGTEP
SGSGASEPTSTEPGTSTEPSEPGSAGSGASEPTSTEPGSEPATSGTEPSGSGASEPTS
TEPGSEPATSGTEPSGSGASEPTSTEPGTSTEPSEPGSAGSEPATSGTEPSGSGASEP
TSTEPGTSTEPSEPGSAGSEPATSGTEPSGTSTEPSEPGSAGSEPATSGTEPSGTSTEP
SEPGSAGTSTEPSEPGSAGTSTEPSEPGSAGTSTEPSEPGSAGTSTEPSEPGSAGTST
EPSEPGSAGTSEPSTSEPGAGSGASEPTSTEPGTSTEPSEPGSAGTSTEPSEPGSAGT
STEPSEPGSAGSEPATSGTEPSGSGASEPTSTEPGSEPATSGTEPSGSEPATSGTEPS
GSEPATSGTEPSGSEPATSGTEPSGTSEPSTSEPGAGSEPATSGTEPSGSGASEPTST
EPGTSTEPSEPGSAGSEPATSGTEPSGSGASEPTSTEPGTSTEPSEPGSA
BD864 33 GSETATSGSETAGTSESATSESGAGSTAGSETSTEAGTSESATSESGAGSETATSGS
ETAGSETATSGSETAGTSTEASEGSASGTSTEASEGSASGTSESATSESGAGSETAT SGSETAGTSTEASEGSASGSTAGSETSTEAGTSESATSESGAGTSESATSESGAGSE TATSGSETAGTSESATSESGAGTSTEASEGSASGSETATSGSETAGSETATSGSETA GTSTEASEGSASGSTAGSETSTEAGTSESATSESGAGTSTEASEGSASGSETATSGS ETAGSTAGSETSTEAGSTAGSETSTEAGSETATSGSETAGTSESATSESGAGTSESA TSESGAGSETATSGSETAGTSESATSESGAGTSESATSESGAGSETATSGSETAGSE TATSGSETAGTSTEASEGSASGSTAGSETSTEAGSETATSGSETAGTSESATSESGA GSTAGSETSTEAGSTAGSETSTEAGSTAGSETSTEAGTSTEASEGSASGSTAGSETS SEQ
XTEN ID Amino Acid Sequence
Name
NO:
TEAGSTAGSETSTEAGTSTEASEGSASGSTAGSETSTEAGSETATSGSETAGTSTEA
SEGSASGTSESATSESGAGSETATSGSETAGTSESATSESGAGTSESATSESGAGSE
TATSGSETAGTSESATSESGAGSETATSGSETAGTSTEASEGSASGTSTEASEGSAS
GSTAGSETSTEAGSTAGSETSTEAGSETATSGSETAGTSESATSESGAGTSESATSE
SGAGSETATSGSETAGSETATSGSETAGSETATSGSETAGTSTEASEGSASGTSESA
TSESGAGSETATSGSETAGSETATSGSETAGTSESATSESGAGTSESATSESGAGSE
TATSGSETA
AE911 34 AEPAGSPTSTEEGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGSPAGSPTST
EEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSE
GSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESA
TPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTST
EPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGT
STEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEE
GTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGS
APGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPT
STEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPAT
SGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPA
GSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGS
EPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP
GSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTST
EEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATP
ESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEP
SEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP
AE146 35 GGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEG
SAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPS EGSAPGSPAGSPTSTEEGTSTEPSEGSAPG
AE48. 36 AEPAGSPTSTEEGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGS
1
AM48. 37 AEPAGSPTSTEEGASPGTSSTGSPGSSTPSGATGSPGSSTPSGATGS
1
AE912 38 AEPAGSPTSTEEGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGSPAGSPTST .1 EEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSE
GSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESA
TPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTST
EPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGT
STEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEE
GTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGS
APGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPT
STEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPAT
SGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPA
GSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGS
EPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP
GSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTST
EEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATP
ESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEP
SEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP
AE912 39 AEPAGSPTSTEEGTPGSGTASSSPGSSTPSGATGSPGASPGTSSTGSPGSPAGSPTST .2 EEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSE
GSAPGTSESATPESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSESA
TPESGPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTST
EPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGT
STEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGSPAGSPTSTEE
GTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGS
APGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSPAGSPT
STEEGTSTEPSEGSAPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSEPAT
SGSETPGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGSPAGSPTSTEEGSPA SEQ
XTEN ID Amino Acid Sequence
Name
NO:
GSPTSTEEGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGTSESATPESGPGS EPATSGSETPGTSESATPESGPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAP GSPAGSPTSTEEGTSESATPESGPGSEPATSGSETPGTSESATPESGPGSPAGSPTST EEGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSESATPESGPGTSESATP ESGPGSEPATSGSETPGSEPATSGSETPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEP SEGSAPGSEPATSGSETPGTSESATPESGPGTSTEPSEGSAPG
AE146 40 TSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSA .1 PGTSTEPSEGSAPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSTEPSEG
SAPGSPAGSPTSTEEGTSTEPSEGSAPG
AM87 41 GTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSTSSTAESPGPGTSTPESGSA 5.1 SPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSEPATSGS
ETPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSTEPSE
GSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESA
TPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTST
EPSEGSAPGSEPPTSTEEGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATATSGSET
PGSPAGSGSPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEE
GSPAGSPTSTEEGTSTEPSEGSAPGASASGAPSTGGTSESATPESGPGSPAGSPTSTE
EGSPAGSPTSTEEGSTSSTAESPGPGSTSESPSGTAPGTSPSGESSTAPGTPGSGTAS
SSPGSSTPSGATGSPGSSPSASTGTGPGSEPATSGSETPGTSESATPESGPGSEPATS
GSETPGSTSSTAESPGPGSTSSTAESPGPGTSPSGESSTAPGSEPATSGSETPGSEPA
TSGSETPGTSTEPSEGSAPGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPGTST
EPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSSTPSGATGSPGSSPSASTGTGPGA
SPGTSSTGSPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSSTPSGATGSP
GSSPSASTGTGPGASPGTSSTGSPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGS
AP
AM87 42 GTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEEGSTSSTAESPGPGTSTPESGSA 5.2 SPGSTSESPSGTAPGSTSESPSGTAPGTSTPESGSASPGTSTPESGSASPGSEPATSGS
ETPGTSESATPESGPGSPAGSPTSTEEGTSTEPSEGSAPGTSESATPESGPGTSTEPSE
GSAPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESA
TPESGPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGTST
EPSEGSAPGSEPPTSTEEGSSTPSGATGSPGTPGSGTASSSPGSSTPSGATATSGSET
PGSPAGSGSPGTSTEPSEGSAPGTSTEPSEGSAPGSEPATSGSETPGSPAGSPTSTEE
GSPAGSPTSTEEGTSTEPSEGSAPGASASGAPSTGGTSESATPESGPGSPAGSPTSTE
EGSPAGSPTSTEEGSTSSTAESPGPGSTSESPSGTAPGTSPSGESSTAPGTPGSGTAS
SSPGSSTPSGATGSPGSSPSASTGTGPGSEPATSGSETPGTSESATPESGPGSEPATS
GSETPGSTSSTAESPGPGSTSSTAESPGPGTSPSGESSTAPGSEPATSGSETPGSEPA
TSGSETPGTSTEPSEGSAPGSTSSTAESPGPGTSTPESGSASPGSTSESPSGTAPGTST
EPSEGSAPGTSTEPSEGSAPGTSTEPSEGSAPGSSTPSGATGSPGSSPSASTGTGPGA
SPGTSSTGSPGSEPATSGSETPGTSESATPESGPGSPAGSPTSTEEGSSTPSGATGSP
GSSPSASTGTGPGASPGTSSTGSPGTSESATPESGPGTSTEPSEGSAPGTSTEPSEGS
APG
In those embodiments wherein the XTEN component of the hGH-XTEN fusion protein has less than 100% of its amino acids consisting of 4, 5, or 6 types of amino acid selected from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P), or less than 100% of the sequence consisting of the XTEN sequences of Table 2, the other amino acid residues of the XTEN are selected from any of the other 14 natural L-amino acids, but are preferentially selected from hydrophilic amino acids such that the XTEN sequence contains at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99% hydrophilic amino acids. The XTEN amino acids that are not glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P) are either interspersed throughout the XTEN sequence, are located within or between the sequence motifs, or are concentrated in one or more short stretches of the XTEN sequence, e.g., to create a linker between the XTEN and the hGH components. In such cases where the XTEN component of the hGH-XTEN comprises amino acids other than glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P), it is preferred that less than about 2% or less than about 1% of the amino acids be hydrophobic residues such that the resulting sequences generally lack secondary structure, e.g., not having more than 2% alpha helices or 2% beta- sheets, as determined by the methods disclosed herein. Hydrophobic residues that are less favored in construction of XTEN include tryptophan, phenylalanine, tyrosine, leucine, isoleucine, valine, and methionine. Additionally, one can design the XTEN sequences to contain less than 5% or less than 4% or less than 3% or less than 2% or less than 1% or none of the following amino acids: cysteine (to avoid disulfide formation and oxidation), methionine (to avoid oxidation), asparagine and glutamine (to avoid deamidation). Thus, in some embodiments, the XTEN component of the hGH-XTEN fusion protein comprising other amino acids in addition to glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P) have a sequence with less than 5% of the residues contributing to alpha- helices and beta-sheets as measured by the Chou-Fasman algorithm and have at least 90%, or at least about 95% or more random coil formation as measured by the GOR algorithm.
In some embodiments, the hGH-XTEN of the formulation comprises i) one or more
XTEN sequence wherein at least about 80% the XTEN sequence consist of non-overlapping sequence motifs, wherein each sequence motif has 9 to 14 amino acid residues, wherein each motif consist of 4 to 6 types of amino acids selected from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and praline (P), the sequence of any two contiguous amino acid residues in any one sequence motif does not occur more than twice in the sequence motif and the XTEN sequence contains no three contiguous amino acids that are identical unless the amino acids are serine residues, ii) where at least one XTEN is linked to either the N- and C- termini of at least one hGH, and wherein when more than one XTEN is used in a hGH-XTEN composition an XTEN can be linked to both the N- and C-termini of at least one hGH, iii) the length of the N-terminal XTEN sequence ranges between about 500 amino acids to about 1320 amino acids and the length of the C-terminal XTEN ranges between about 40 amino acids to about 300 amino acids, iv) when the XTEN component of the hGH-XTEN fusion protein has less than 100% of its amino acids consisting of 4, 5, or 6 types of amino acid selected from glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P), the other amino acid residues of the XTEN are selected from any of the other 14 natural L-amino acids, but are preferentially selected from hydrophilic amino acids such that the XTEN sequence contains at least about 90% to at least about 99% hydrophilic amino acids, v) the XTEN amino acids that are not glycine (G), alanine (A), serine (S), threonine (T), glutamate (E) and proline (P) are either interspersed throughout the XTEN sequence, are located within or between the sequence motifs, or are concentrated in one or more short stretches of the XTEN sequence, e.g., to create a linker between the XTEN and the hGH components, vi) less than about 2% or less than about 1% of the amino acids are hydrophobic residues such that the resulting sequences generally lack secondary structure, e.g., not having more than 2% alpha helices or 2% beta-sheets, and vii) the XTEN sequences are designed to contain less than 5% or less than 4% or less than 3% or less than 2% or less than 1% or none of the following amino acids: cysteine, methionine, asparagine and glutamine. In some embodiments, the XTEN sequence exhibits at least about 80% to about 100% sequence identity to an XTEN selected from Table 2.
VII) . hGH-XTEN STRUCTURAL CONFIGURATIONS AND PROPERTIES
The human growth hormone (hGH) of the subject formulations are not limited to native, full-length polypeptides, but also include recombinant versions as well as biologically and/or pharmacologically active variants or fragments thereof. For example, it will be appreciated that various amino acid deletions, insertions and substitutions can be made in the hGH to create variants without departing from the spirit of the invention with respect to the biological activity or pharmacologic properties of the hGH. For a more detailed discussion of possible structural configurations and properties of hGH-XTEN sequences in the subject formulations, including amino acid substitutions and fusion protein configurations, see
W013/184216, WO/2014/164568, U.S. Patent Nos. 8,492,530 and 8,703,717, each of which is incorporated herein by reference in its entirety.
VIII) . USES OF THE FORMULATIONS OF THE PRESENT INVENTION
In another aspect, the invention provides a method for achieving a beneficial therapeutic effect in a disease, disorder or condition mediated by hGH by therapeutic treatment with a storage stable aqueous liquid formulation comprising an hGH-XTEN fusion protein. The present invention addresses disadvantages and/or limitations of prior aqueous liquid hGH formulations, which are unstable in aqueous solution over time, have a relatively short terminal half-life and/or a narrow therapeutic window. The fact that growth hormone has a short half-life, necessitates frequent dosing in order to achieve clinical benefit, which results in difficulties in the management of such patients. Methods suitable for treating a hGH-related condition in a subject using the hGH-XTEN formulation of the present invention are known in the art and are described in, for example, in U.S. Patent No. 8,703,717, in WO/2010/144502, in WO/2013/184216 and WO/2014/164568. All embodiments of the methods of treatment described or incorporated by reference herein apply to all method and use claims recited in this application.
In one embodiment, the invention provides a method for achieving a beneficial effect in a subject with a growth hormone-related disease, disorder or condition comprising the step of administering to the subject a therapeutically- or prophylactically-effective amount of a hGH-XTEN formulation, wherein said administration results in the improvement of one or more biochemical or physiological parameters or clinical endpoints associated with a growth hormone-related disease, disorder or condition. The effective amount produces a beneficial effect in helping to treat (e.g., cure or reduce the severity) or prevent (e.g., reduce the likelihood of onset or severity) a growth hormone-related disease, disorder or condition. In some cases, the method for achieving a beneficial effect includes administering a
therapeutically effective amount of a hGH-XTEN fusion protein composition to treat a subject with a growth hormone-related disease, disorder, or condition, including, but not limited to, congenital or acquired GH deficiency in adults and children, Turner's Syndrome, Prader-Willi Syndrome, chronic renal failure, intrauterine growth retardation, idiopathic short stature, AIDS wasting, obesity, multiple sclerosis, aging, fibromyalgia, Crohn's disease, ulcerative colitis, muscular dystrophy, low muscle mass (e.g. bodybuilding), low bone density, or any other indication for which GH can be utilized (but for which endogenous growth hormone levels in a subject are not necessarily deficient).
In a preferred embodiment, the therapeutic effect is a measured parameter selected from, for example, IGF-1 concentrations, IGFBP3 concentration, height velocity, lean body mass, total body fat, trunk fat, response to insulin challenge, rate of division of chondrocytes, chondrocyte numbers, bone density, bone growth, and increase in epiphyseal plate width.
Determination of a therapeutically effective amount is well within the capability of those skilled in the art. In a preferred embodiment, therapeutically effective amounts result in a sustained beneficial effect on any clinical sign or symptom, aspect, measured parameter or characteristic of a metabolic disease state or condition, including, but not limited to, those described herein. In one embodiment, the parameters include but are not limited to mean (SD) height standard deviation score (HT-SDS), changes in height velocity, IGF-I concentration, ratio of IGF-I/IGFBP-3, IGFBP3 concentration, change in weight, lean body mass, change in body mass index, total body fat (adipose fat/tissue), trunk fat, response to insulin challenge, rate of division of chondrocytes, chondrocyte numbers, bone density, bone age, bone growth, bone turnover, increase in epiphyseal plate width, reduction in cholesterol, reduction in triglycerides, and reduction in LDL. In another embodiment of the method, the administration to a human pediatric patient of successive or consecutive doses of a therapeutically effective amount of the hGH-XTEN results in a beneficial effect in two or more of the parameters including, but not limited to mean (SD) height standard deviation score (HT-SDS), changes in height velocity, IGF-I concentration, ratio of IGF-I/IGFBP-3, IGFBP3 concentration, change in weight, lean body mass, change in body mass index, total body fat (adipose fat/tissue), trunk fat, response to insulin challenge, rate of division of chondrocytes, chondrocyte numbers, bone density, bone age, bone growth, bone turnover, increase in epiphyseal plate width, reduction in cholesterol, reduction in triglycerides, and reduction in LDL.
In another embodiment, the invention provides a method of stimulating IGF- 1 production in individuals with GH deficiency. The method comprises the step of
administering therapeutically effective amount of an hGH-XTEN to a subject that results in the increased blood levels and/or duration in increased blood levels of IGF-I compared to a subject receiving a GH not linked to an XTEN and administered at a comparable dose. In another embodiment, the invention provides a method of stimulating the division and numbers of chrondrocytes. In another embodiment, the invention provides a method comprising the step of administering therapeutically effective amount of hGH-XTEN that results in increased bone growth as measured by increase in epiphyseal plate width compared to a subject receiving a GH not linked to an XTEN and administered at a comparable dose.
As a result of the enhanced pharmacokinetic parameters of the hGH-XTEN formulation, the hGH can be administered using longer intervals between doses compared to the corresponding GH not linked to XTEN to prevent, treat, alleviate, reverse or ameliorate symptoms or clinical abnormalities of the growth hormone- related disease, disorder or condition or prolong the survival of the subject being treated. The methods of the invention includes administration of consecutive doses of a therapeutically effective amount of the hGH-XTEN for a period of time sufficient to achieve and/or maintain the desired parameter or clinical effect, and such consecutive doses of a therapeutically effective amount establishes the therapeutically effective dose regimen for the hGH-XTEN; i.e., the schedule for consecutively administered doses of the fusion protein composition, wherein the doses are given in therapeutically effective amounts to result in a sustained beneficial effect on any clinical sign or symptom, aspect, measured parameter or characteristic of a metabolic disease state or condition, including, but not limited to, those described herein. In one embodiment, the method comprises administering a therapeutically- effective amount of a pharmaceutical composition comprising a hGH-XTEN fusion protein composition comprising a GH linked to an XTEN sequence(s) and at least one
pharmaceutically acceptable carrier to a subject in need thereof that results in greater improvement in at least one parameter, physiologic condition, or clinical outcome mediated by the GH component(s) (non- limiting examples of which are described above) compared to the effect mediated by administration of a pharmaceutical composition comprising a GH not linked to XTEN and administered at a comparable dose. In one embodiment, the
pharmaceutical composition is administered at a therapeutically effective dose. In another embodiment, the pharmaceutical composition is administered using multiple consecutive doses using a therapeutically effective dose regimen (as defined herein) for the length of the dosing period.
A therapeutically effective amount of the hGH-XTEN varies according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the hGH to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the hGH-XTEN are outweighed by the therapeutically beneficial effects. A prophylactically effective amount refers to an amount of hGH-XTEN required for the period of time necessary to achieve the desired prophylactic result.
For the inventive methods, longer acting hGH-XTEN compositions are preferred, so as to improve patient convenience, to increase the interval between doses and to reduce the amount of drug required to achieve a sustained effect. In one embodiment, a method of treatment comprises administration of a therapeutically effective dose of a hGH-XTEN to a subject in need thereof that results in a gain in time spent within a therapeutic window established for the fusion protein of the composition compared to the corresponding GH component(s) not linked to the fusion protein and administered at a comparable dose to a subject. In some cases, the gain in time spent within the therapeutic window is at least about three-fold to at least about 100-fold longer, compared to the corresponding GH component not linked to the fusion protein and administered at a comparable dose to a subject.
The methods further provide that administration of multiple consecutive doses of a hGH-XTEN administered using a therapeutically effective dose regimen to a subject in need thereof results in a gain in time between consecutive Cmax peaks and/or Cmin troughs for blood levels of the fusion protein compared to the corresponding GH not linked to the fusion protein and administered using a dose regimen established for that GH. In the foregoing embodiment, the gain in time spent between consecutive Cmax peaks and/or Cmin troughs is at least about three-fold to at least about 100-fold longer, compared to the corresponding GH component not linked to the fusion protein and administered using a dose regimen established for that GH. In the embodiments hereinabove described in this paragraph the administration of the fusion protein results in an improvement in at least one of the parameters (disclosed herein as being useful for assessing the subject diseases, conditions or disorders) using a lower unit dose in moles of fusion protein compared to the corresponding GH component not linked to the fusion protein and administered at a comparable unit dose or dose regimen to a subject.
The method of treatment comprises administration of a hGH-XTEN formulation using a therapeutically effective dose regimen to effect improvements in one or more parameters associated with growth hormone diseases, disorders or conditions. In some embodiments, administration of the hGH-XTEN formulation to a subject results in an improvement in one or more of the biochemical, physiologic, or clinical parameters that is of greater magnitude than that of the corresponding GH component not linked to XTEN, determined using the same assay or based on a measured clinical parameter. In other embodiments, administration of the hGH-XTEN formulation to a subject using a therapeutically effective dose regimen results in activity in one or more of the biochemical, physiologic, or clinical parameters that is of longer duration than the activity of one of the single GH components not linked to XTEN, determined using that same assay or based on a measured clinical parameter. In one embodiment of the foregoing, the administration of the hGH-XTEN formulation to a subject using a therapeutically effective dose regimen results in an improvement in peak
concentrations and area under the curve of blood IGF-I levels of at least about 10% to about 100% or more in the subject compared to a comparable dose of GH not linked to XTEN administered to a subject. In another embodiment of the foregoing, the administration of the hGH-XTEN formulation to a subject using a therapeutically effective dose regimen results in increased weight gain in the subject of at least about 10% to about 50% or more compared to a comparable dose regimen of GH not linked to XTEN administered to a subject.
The invention further contemplates that hGH-XTEN used in accordance with the methods provided herein is administered in conjunction with other treatment methods and pharmaceutical compositions useful for treating growth hormone-related diseases, disorders, and conditions, or conditions for which growth hormone is adjunctive therapy; e.g., insulin resistance and poor glycemic control. Such compositions, include for example, DPP-IV inhibitors, insulin, insulin analogues, PPAR gamma agonists, dual-acting PPAR agonists, GLP-1 agonists or analogues, PTPIB inhibitors, SGLT inhibitors, insulin secretagogues, RXR agonists, glycogen synthase kinase-3 inhibitors, insulin sensitizers, immune modulators, beta- 3 adrenergic receptor agonists, pan-PPAR agonists, 11 beta-HSDl inhibitors, biguanides, alpha-glucosidase inhibitors, meglitinides, thiazolidinediones, sulfonylureas and other diabetes medicants known in the art, or anti-hypertensive drugs, calcium channel blockers, and related products. In some embodiments, the administration of a hGH-XTEN formulation permits use of lower dosages of the co-administered pharmaceutical composition to achieve a comparable clinical effect or measured parameter for the disease, disorder or condition in the subject. IX). PHARMACEUTICAL COMPOSITIONS
The present invention provides pharmaceutical compositions comprising hGH-XTEN. In one embodiment, the pharmaceutical composition comprises the hGH-XTEN fusion protein and at least one pharmaceutically acceptable carrier. hGH-XTEN polypeptides of the present invention can be formulated according to known methods to prepare
pharmaceutically useful compositions, whereby the polypeptide is combined in admixture with a pharmaceutically acceptable carrier vehicle, such as aqueous solutions or buffers, pharmaceutically acceptable suspensions and emulsions. Examples of non-aqueous solvents include propyl ethylene glycol, polyethylene glycol and vegetable oils. Therapeutic formulations are prepared for storage by mixing the active ingredient having the desired degree of purity with optional physiologically acceptable carriers, excipients or stabilizers, as described in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980), in the form of lyophilized formulations or aqueous solutions. In addition, the pharmaceutical compositions can also contain other pharmaceutically active compounds or a plurality of compounds of the invention.
The present pharmaceutical compositions may be administered for therapy by any suitable route established in the art. It will also be appreciated that the preferred route will vary with the condition and age of the recipient, and the disease being treated. In one embodiment, the pharmaceutical composition is administered subcutaneously. In this embodiment, the composition may be supplied as a lyophilized powder to be reconstituted prior to administration. The composition may also be supplied in a liquid form, which can be administered directly to a patient. In one embodiment, the composition is supplied as a liquid in a pre-filled syringe such that a patient can easily self-administer the composition.
The compositions of the invention may also be formulated using a variety of excipients known in the art. Suitable excipients include microcrystalline cellulose (e.g. Avicel® PH-102, Avicel® PH-101), polymethacrylate, poly(ethyl acrylate, methyl methacrylate, trimethylammomoethyl methacrylate chloride) (such as Eudragit® RS-30D), hydroxypropyl methylcellulose (Methocel™ K100M, Premium CR Methocel™ K100M, Methocel™ E5, Opadry®), magnesium stearate, talc, triethyl citrate, aqueous ethylcellulose dispersion (Surelease®), and protamine sulfate. The compositions may also comprise a carrier, which can comprise, for example, solvents, dispersion media, antibacterial and antifungal agents, isotonic and absorption delaying agents. Pharmaceutically acceptable salts may also be used in these compositions, for example, mineral salts such as hydrochlorides, hydrobromides, phosphates, or sulfates, as well as the salts of organic acids such as acetates, proprionates, malonates, or benzoates. The composition may also contain liquids, such as water, saline, glycerol, and ethanol, as well as substances such as wetting agents, emulsifying agents, or pH buffering agents. Liposomes may also be used as a carrier.
For liquid formulations, a desired property is that the formulation be supplied in a form that can pass through a 25, 28, 30, 31, 32 gauge needle for intravenous, intramuscular, intraarticular, or subcutaneous administration.
X). PHARMACEUTICAL KITS
In another aspect, the invention provides a kit to facilitate the use of the hGH-XTEN formulation. The kit comprises the pharmaceutical composition provided herein, a label identifying the pharmaceutical composition, and an instruction for storage, reconstitution and/or administration of the pharmaceutical compositions to a subject. In some embodiments, the kit comprises, preferably: (a) an amount of a hGH-XTEN fusion protein composition sufficient to treat a disease, condition or disorder upon administration to a subject in need thereof; and (b) an amount of a pharmaceutically acceptable carrier; together in a formulation ready for injection or for reconstitution with sterile water, buffer, or dextrose; together with a label identifying the hGH-XTEN drug and storage and handling conditions, and a sheet of the approved indications for the drug, instructions for the reconstitution and/or administration of the hGH-XTEN drug for the use for the prevention and/or treatment of approved indication, appropriate dosage and safety information, and information identifying the lot and expiration of the drug. In another embodiment of the foregoing, the kit can comprise a second container that can carry a suitable diluent for the hGH-XTEN composition, which will provide the user with the appropriate concentration of hGH-XTEN to be delivered to the subject.
EXPERIMENTAL EXAMPLES
Example I - Reference Assay Methods
Reverse-phase high-performance liquid chromatography (RP-HPLC) was run on the Agilent® 1100 HPLC system using a Kinetex® reversed phase C18 column (2.6 μπι, 150 x 4.6mm, Phenomenex®, P/N 00F-4462-E0) at a column temperature of 40° C with a flow rate of 1 ml/min. Mobile phase A was 0.1% TFA in water and mobile phase B was 0.1% TFA in acetonitrile. Elution was performed using a 15 minute gradient from 60-90%) mobile phase B beginning at 95 minutes. The total run time was 105 minutes. The column load was 40 μg of protein. Detection was at 214 nm.
Size exclusion high-performance liquid chromatography (SE-HPLC) was run on the Agilent® 1100 HPLC system using a Phenomenex® Bio-Sep-SEC-S4000 column
(7.8x600mm, part # 00K-2147-KO) at ambient column temperature with a Phenomenex®
SecurityGuard with GFC 4000 cartridge (Part # KJO-4282) guard column with a flow rate of 0.5 ml/min. The mobile phase was 20mM Tris, 300mM NaCl at pH 7.5. The total run time was 75 minutes. The column load was 25 μg of protein. Detection was at 215 nm.
Strong anion exchange high-performance liquid chromatography (SAX-HPLC) was run on the Agilent 1100 HPLC system using a ProSwift® SAX-1 S column (4.6 x 50 mm, Thermo®, P/N 64293) at a column temperature of 25° C with a flow rate of 1 ml/min.
Mobile phase A was 20mM Tris at pH 8.0 and mobile phase B was Mobile A + 1.0 M NaCl. Elution was performed using a 28 minute gradient from 5 (at 4 min), 50 (at 25 min), 100 (at 25.01 min), 100 (at 30 min), 5 (at 30.01 min), and 5% (at 32 min) mobile phase B beginning at 4 minutes. The total run time was 32 minutes. The column load was 25 μg of protein. Detection was at 215 nm. Example II - Solubility of the Aqueous Formulation
VRS-317 contains rhGH and two XTEN domains consisting of hydrophilic amino acids and a large number of glutamate residues. The high glutamate content of VRS-317 results in an isoelectric point (pi) of approximately 3.6 compared to the rhGH pi of 5.2. This lower pi allows for the aqueous formulation of VRS-317 at lower pH than rhGH.
The aqueous formulation prepared according to the present invention containing 54 mg/ml of VRS-317 (SEQ ID NO: 1) in 20 mM L-histidine and 154 mM NaCl at pH 5.5 (the reference standard), was concentrated in a step-wise manner up to 200 mg/mL via centrifugal filtration (3 OK MWCO PES) to assess the osmolality and solubility of formulations containing various concentrations of VRS-317. Accordingly, VRS-317 was suspended at 125 mg/ml, 150 mg/ml, 175 mg/ml, and 200 mg/ml. The osmolality of these solutions is shown in Table 3. Compared to a reference standard, the measured concentration of VRS-317 suspended at 125 mg/ml, 150 mg/ml, 175 mg/ml, and 200 mg/ml was 125.0±1.0 , 154.3±4.0, 170.7±3.1, and 208.3±15.7, respectively (n=3, ± Std Dev). VRS-317 was found to be soluble in the aqueous formulation at the concentrations tested. All samples remained clear and free of particulates.
Table 3
Figure imgf000050_0001
Example III - Stability of VRS-317 in the Aqueous Formulation
Hydrolysis, Aggregation, Oxidation, and Deamidation
To assess the suitability of several possible formulations, a formulation screen, which included accelerated stability at 40°C, was performed on batches of VRS-317. The primary mode of degradation that was observed under accelerated stability conditions of VRS-317 formulated in 20 mM L-histidine, 154 mM sodium chloride, pH 5.5 at 40°C was the hydrolysis, or truncation, of the XTEN polypeptide. The truncated products are resolved by anion exchange HPLC as shown in FIG. 2. An increase in the peak area of the leading peak (RRT < main product peak) was observed over a period of four weeks. Several buffers and excipients were evaluated and the results are summarized in Table 4.
These results suggested that VRS-317 formulated according to the present invention was less truncated at higher pH (up to 6.0) in 154 mM sodium chloride as compared to other formulations tested.
Table 4. Summary of VRS-317 Accelerated Stability (40°C) Data. Anion exchange analysis of VRS-317 was performed as described.
Figure imgf000051_0001
2 82.8
4 66.2
0 90.4
1 82.7
4% mannitol
2 79.6
4 59.7
0 90.5
1 81.6
8% sucrose
2 65.2
4 43.8
0 90.9
154 mM sodium 1 85.1
20 mM L-histidine, pH 6.0
chloride 2 76.7
4 68.3
0 90.7
1 85.1
4% mannitol
2 78.4
4 55.3
SE-HPLC and SAX-HPLC analyses were performed on the aqueous formulation containing VRS-317 suspended at 5.0 mg/ml of VRS-317 in 20 mM L-histidine and 154 mM NaCl at pH 5.5 (the reference standard), concentrated in a step-wise manner to 50.0, 125.0, 150.0, 175.0, or 200 mg/mL via centrifugal filtration (30K MWCO PES). No significant change in percent main peak was detected for any of the concentrations tested based on the chromatograms obtained from SAX-HPLC (FIG. 3) and SE-HPLC (FIG. 4) experiments. These SE-HPLC and SAX-HPLC results indicate, respectively, that VRS-317 is not degraded or aggregated in the aqueous liquid formulation according to the present invention. To test whether injection of the formulation through a needle would cause aggregation, the samples were also loaded into 1 ml Luer-lok® tip syringes (BD lot #3234079), passed through 0.5" inch 26 gauge, 27 gauge, or 30 gauge needles, and assayed via SE-HPLC. SE-HPLC results for the samples passed through the 30 gauge needle are shown in FIG. 5, and similar results were obtained for the other wider-diameters tested (data not shown). No aggregation of VRS-317 was detected after the formulation according to the present invention was passed through the various needles.
To demonstrate the ability to detect degradation of VRS-317, oxidation or
deamidation of VRS-317 was induced with hydrogen peroxide or basic pH, respectively. The reverse phase HPLC analysis of tryptic fragments of control VRS-317 (black) or VRS-317 treated with hydrogen peroxide for 72 hours (gray) is shown in FIG. 6. The predicted tryptic fragments for VRS-317 (shown with the N-terminal methionine encoded by the start codon) are displayed in Table 5. Oxidation is observed at two methionine residues, Met928
(equivalent to Metl4, rhGH sequence) and Metl039 (equivalent to Metl25, rhGH sequence). Incubation of VRS-317 with 0.1% hydrogen peroxide for 72 hours at room temperature resulted in a complete loss of the HPLC peaks corresponding to the Met928 and Metl039. Two additional peaks, likely the methionine sulfoxide and sulfone degradation products of Metl039, were observed (FIG. 6, inset B). Similar peaks were not detected for Met928; however, any degradation products may co-elute with the intense peaks that elute a few minutes prior to the native Met928 peptide (FIG. 6, inset A).
To induce deamidation, VRS-317 was subjected to elevated pH (-10) and temperature (40°C) for one week. The reverse phase HPLC analysis of tryptic fragments of control VRS- 317 (black) or VRS-317 stressed with elevated pH and temperatures (gray) is shown in FIG. 7. A significant loss of peak area at 36 minutes was observed (FIG. 7). This peak
corresponds to the tryptic fragment containing Asnl063 and Asnl066 (equivalent to Asnl49 and Asnl52, respectively, in the rhGH sequence), which is prone to deamidation. One or more peaks, with retention times greater than 36 minutes, showed increased peak area in the stressed sample (FIG. 7, inset). Synthetic peptides corresponding to the native and monodesamido forms (Asnl063→Asp) of the tryptic fragment can be resolved using the HPLC method employed here (data not shown). The monodesamido peptide elutes after the native peptide consistent with the observations in FIG. 7. Table 5: Predicted Tryptic Peptides for VRS-317
Figure imgf000054_0001
Deamidation of rhGH is known to occur at elevated temperature with increasing pH and truncation of the XTEN domain of VRS-317, as shown in the above example, increases with decreasing pH (from pH 6 to pH 5). A formulation of pH 5.5 was selected to minimize both deamidation and truncation of the XTEN domain. At 40°C storage for periods of up to 1 month or 25°C storage for periods of up to 3 months, no deamidation of the rhGH domain of VRS-317 was detected by tryptic mapping. rhGH stored under these conditions has been observed to undergo extensive deamidation (Pearlman and Bewley, supra at 27-29).
As described earlier, another degradation pathway of rhGH is aggregation. Typically, rhGH aggregation is reduced or prevented by the addition of surfactants such as polysorbate 20 (Tween® 20) or Poloxamer 188 (Pluronic® F68). To evaluate the need for surfactants in the VRS-317 formulation, 122 mg/ml of VRS-317 was formulated in 20 mM L-histidine, 154 mM sodium chloride, pH 5.5 with and without Tween® 20 or Pluronic® F68 and stored for 4 hours while agitated at room temperature. Non-agitated and surfactant free samples served as controls. Samples were analyzed by visual inspection, absorbance at 600 nm (to monitor for turbidity), and SE-HPLC (to monitor for soluble aggregation). VRS-317 samples at 122 mg/mL remained clear and particle free after agitation with and without surfactant. No turbidity was observed (A600 < 0.01) for 122 mg/mL VRS-317 following agitation in the presence and absence of surfactant. As shown in FIG. 8, a slight increase in pre-peak species was observed for agitated VRS-317 in the absence of surfactant, slightly lower pre-peaks were seen following agitation for the product in the presence of 0.1% Pluronic® F68 and 0.1% Tween® 20, and a significant pre-peak species was seen in the presence of 0.01% Tween® 20 after agitation. Taken together, these results indicate that VRS-317 formulated as described did not significantly aggregate even in the absence of a surfactant and following agitation stress for 4 hours at room temperature.
To further evaluate the need for surfactants in the VRS-317 formulation, VRS-317 was formulated in 20 mM L-histidine, 154 mM sodium chloride, pH 5.5 with and without Tween® 20 and stored at 40°C for up to 4 weeks. As shown in Table 6, the addition of Tween® 20 was not necessary to prevent aggregation since VRS-317 did not aggregate under these conditions, likely due to the hydrophilic XTEN domains. In contrast, rhGH aggregates under similar conditions as previously reported (Cleland et al 1993, supra).
Table 6: SEC HPLC analysis of VRS-317 formulated with and without Tween® 20 and stored at 40°C for up to 4 weeks.
Figure imgf000055_0001
0.01% Tween® 20, 40°C, week 1 1.29 98.71
0.1% Tween® 20, 40°C, week 1 0.68 99.32
No Tween® 20, 40°C, week 2 0.56 98.75
0.01% Tween® 20, 40°C, week 2 0.65 99.35
0.1% Tween® 20, 40°C, week 2 0.68 98.68
No Tween® 20, 40°C, week 4 0.63 98.39
0.01% Tween® 20, 40°C, week 4 0.63 98.1
0.1% Tween® 20, 40°C, week 4 0.61 97.92
As noted above, oxidation of VRS-317 may be induced with the addition of peroxide and changes in the tryptic digest profile of methionine containing tryptic peptides can be detected after oxidation. Polysorbate and other polyether containing surfactants used in rhGH formulations to prevent aggregation are a source of oxygen free radicals that cause oxidation or rhGH (Pearlman and Bewley, supra at 26-42; Cleland et al., supra at 307-77). The optimal VRS-317 formulations do not require polysorbate (e.g. Tween® 20) or other surfactants.
VRS-317 formulated in 20 mM L-histidine, 154 mM sodium chloride, pH 5.5 stored for 24 months at 5°C, 3 months at 25°C or 1 month at 40°C does not contain detectable amounts of oxidized rhGH as measured by the tryptic mapping method noted above. In contrast, rhGH stored under these conditions with the typical surfactant required for prevention of aggregation (e.g. Tween® 20) undergoes extensive oxidation (Pearlman and Bewley, supra at 26-42; Cleland et al., supra at 307-77).
Long-Term Stability of VRS-317 Formulation Dispensed into Syringes, Cartridges, and Vials
The aqueous formulation was prepared according to the present invention containing 125 mg/ml of VRS-317 in 20 mM L-histidine and 154 mM NaCl at pH 5.5, and filled into syringes, cartridges, and vials to determine the stability of VRS-317 in solution over time.
The formulation containing VRS-317 according to present invention was dispensed into 0.1 ml and 1.0 ml syringes and stored for several months at 5°C or 25°C to determine the chemical stability of VRS-317 in the formulation over time. At the intervals shown in Table 7, samples were analyzed using SE-HPLC as described. The initial chromatogram of the formulation at month 0 showed three distinct peaks, two small pre-peaks, and a much larger main peak corresponding to VRS-317. Table 7 shows integrated percent peak areas corresponding to these three peaks, which are also plotted in FIG. 9, sampled from syringes at various times over 24 months at 5°C or 25°C. The limited degree to which the percent peak areas changed over time indicate that VRS-317 is stable and does not aggregate as an aqueous liquid formulation when stored at 5°C over a period of at least 24 months or at 25°C over a period of at least 6 months. Similar stability has been achieved up to 30 months and is expected to be achieved up to 42 months.
Table 7: SE-HPLC analysis of VRS-317 formulated according to the present invention and stored for several months at 5°C or 25°C
Figure imgf000057_0001
The formulation containing VRS-317 according to present invention was dispensed into 1.0 ml glass cartridges and stored for one month at 25°C to determine the chemical stability of VRS-317 in the formulation over time. Samples were analyzed using RP-HPLC, as described, at 2 weeks and one month as shown in Table 8. Table 8 and FIG. 10 show integrated percent peak areas corresponding to the RP-HPLC chromatogram of VRS-317 aqueous formulations sampled at 2 weeks and one month stored at 25°C. These data indicate that VRS-317 is stable as an aqueous liquid formulation for a period of weeks, even when stored only at 25°C. Table 8: RP-HPLC analysis of VRS-317 formulated according to the present invention and stored for several months at 25°C
Figure imgf000058_0001
The formulation containing VRS-317 according to present invention was dispensed into 1.0 ml vials and stored for several months at 5°C or 25°C to determine the stability of the pH of the formulation over time. The pH of these samples was tested at the intervals shown in Table 9, data also plotted in FIG. 11. As these measurements show, the pH of the formulation remained stable during storage over a period of at least 24 months, when the formulation was stored at either 5°C, or at least 6 months when the formulation was stored at 25°C. Similar stability has been achieved up to 30 months and is expected to be achieved up to 42 months.
Table 9: pH of the VRS-317 formulation according to the present invention after storage for several months at 5°C or 25°C
Figure imgf000058_0002

Claims

WHAT IS CLAIMED IS:
1. A storage stable aqueous liquid formulation comprising:
(i) about 25 mg/ml to about 150 mg/ml of a fusion protein comprising an extended recombinant polypeptide (XTEN) fused to a growth hormone (GH) sequence, wherein the GH sequence is at least 90% identical to the amino acid sequence of SEQ ID NO: 43, and
(ii) a buffer;
at a pH of about 4 to about 6, in the absence of a surfactant.
2. The aqueous liquid formulation of claim 1, wherein the formulation is free of a preservative.
3. The aqueous liquid formulation of claim 1, wherein the pH is about 5 to about
6.
4. The aqueous liquid formulation of claim 3, wherein the pH is 5.2 to 5.8.
5. The aqueous liquid formulation of claim 4, wherein the pH is about 5.5.
6. The aqueous liquid formulation of claim 1, wherein said buffer is selected from the group consisting of histidine, citrate, and succinate buffers.
7. The aqueous liquid formulation of claim 6, wherein the buffer is a histidine buffer at a concentration of about 20 mM.
8. The aqueous liquid formulation of claim 1, wherein the concentration of the fusion protein is selected from the group consisting of about 25 mg/ml, about 50 mg/ml, about 75 mg/ml, about 100 mg/ml, about 125 mg/ml, and about 150 mg/ml.
9. The aqueous liquid formulation of claim 8, wherein the concentration of the fusion protein is about 50 mg/ml.
10. The aqueous liquid formulation of claim 8, wherein the concentration of the fusion protein is about 100 mg/ml.
11. The aqueous liquid formulation of claim 8, wherein the concentration of the fusion protein is about 150 mg/ml.
12. The aqueous liquid formulation of claim 1, wherein the aqueous liquid formulation is storage stable for at least about one month at about 23±3°C.
13. The aqueous liquid formulation of claim 1, wherein the aqueous liquid formulation is storage stable for at least about 6 months to about 3 years at about 5±3°C.
14. The aqueous liquid formulation of claim 13, wherein the aqueous liquid formulation is storage stable for at least about 6 months to about 18 months at about 5±3°C.
15. The aqueous liquid formulation of claim 1, wherein the oxidation of the fusion protein is reduced as compared to the oxidation of the corresponding fusion protein formulated with a surfactant.
16. The aqueous liquid formulation of claim 1, wherein the deamidation of the fusion protein is reduced as compared to the deamidation of the corresponding fusion protein formulated at a higher pH.
17. The aqueous liquid formulation of claim 1, wherein the fusion protein does not show detectable oxidation or deamidation following incubation for about 8 hours at about 40°C.
18. The aqueous liquid formulation of claim 1, further comprising a tonicif ing agent.
19. The aqueous liquid formulation of claim 18, wherein the tonicifying agent is
NaCl.
20. The aqueous liquid formulation of claim 1 having an osmolality between about 300 mOsm to about 600 mOsm.
21. The aqueous liquid formulation of claim 1, wherein the formulation is disposed in a container containing at least one dose.
22. The aqueous liquid formulation of claim 21, wherein the container comprises a syringe.
23. The aqueous liquid formulation of claim 21, wherein the container comprises a glass cartridge.
24. The aqueous liquid formulation of claim 21, wherein the container comprises a vial.
25. The aqueous liquid formulation of claim 1 for injection into a subject with a needle.
26. The aqueous liquid formulation of claim 25, wherein the needle size is selected from the group consisting of 26 gauge, 27 gauge, 29 gauge, and 30 gauge.
27. The aqueous liquid formulation of claim 1, comprising the fusion protein according to the formula I:
(XTEN_l)x-GH-(XTEN_2)y
wherein independently for each occurrence:
(a) x is either 0 or 1; and
(b) y is either 0 or 1, wherein x+y>l; wherein XTEN l and XTEN 2 comprise identical or different sequences.
28. The aqueous liquid formulation of claim 27, wherein the XTEN l comprises a sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 18-34, 38, 39, 41, and 42.
29. The aqueous liquid formulation of claim 27, wherein the XTEN 2 comprises a sequence having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 13-17, 35, 36, 37, and 40.
30. The aqueous liquid formulation of claim 28, wherein the XTEN l comprises SEQ ID NO:39.
31. The aqueous liquid formulation of claim 29, wherein the XTEN 2 comprises
SEQ ID NO: 35.
32. The aqueous liquid formulation of claim 27, wherein XTEN l comprises SEQ ID NO: 39 and XTEN 2 comprises SEQ ID NO: 35.
33. The aqueous liquid formulation of claim l,wherein the fusion protein is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 1.
34. The aqueous liquid formulation of claim 33, wherein the fusion protein comprises the amino acid sequence of SEQ ID NO: 1.
35. A method of making a storage stable aqueous liquid formulation of a fusion protein comprising an extended recombinant polypeptide (XTEN) linked to a growth hormone (GH) sequence, wherein the GH sequence is at least 90% identical to the amino acid sequence of SEQ ID NO:43, comprising mixing said fusion protein and an aqueous, pharmaceutically acceptable vehicle which includes:
i) said fusion protein at a concentration of about 25 mg/ml to about 150 mg/ml; and
ii) a buffer providing a pH of about 4 to about 6;
wherein said aqueous liquid formulation is free of a surfactant.
36. The method of claim 35 without use of a preservative.
37. A method of treating a growth-hormone related condition in a subject, comprising administering to the subject a therapeutically effective amount of the aqueous liquid formulation of claim 1, wherein the growth-hormone related condition is selected from the group consisting of growth-hormone deficiency, Turner's Syndrome, Prader-Willi Syndrome, small for gestational age (SGA), idiopathic short stature, AIDS wasting, multiple sclerosis, Crohn's disease, ulcerative colitis, and muscular dystrophy.
38. The method of claim 37, wherein the therapeutically effective amount is administered at least 48 h, or at least 72 h, or at least about 96 h, or at least about 120 h, or at least about 7 days, or at least about 14 days, at least about 21 days, or at least about 30 days between consecutive doses.
39. The method of claim 37, wherein growth-hormone related condition is growth-hormone deficiency.
40. The method of claim 37, wherein growth-hormone related condition is Turner's Syndrome.
41. The method of claim 37, wherein growth-hormone related condition is Prader- Willi Syndrome.
42. The method of claim 37, wherein growth-hormone related condition is idiopathic short stature.
43. The method of claim 37, wherein growth-hormone related condition is small for gestational age (SGA).
44. The method of claim 37, wherein the growth-hormone related condition is selected from the group consisting of AIDS wasting, multiple sclerosis, Crohn' s disease, ulcerative colitis, and muscular dystrophy.
45. The use of a therapeutically effective amount of the aqueous liquid
formulation of claim 1 in the preparation of a medicament for treating a growth-hormone related condition, wherein the growth-hormone related condition is selected from the group consisting of growth-hormone deficiency, Turner's Syndrome, Prader-Willi Syndrome, small for gestational age (SGA), idiopathic short stature, AIDS wasting, multiple sclerosis, Crohn's disease, ulcerative colitis, and muscular dystrophy.
46. The aqueous liquid formulation of claim 1 for use in treating a growth- hormone related condition, wherein the growth-hormone related condition is selected from the group consisting of growth-hormone deficiency, Turner's Syndrome, Prader-Willi Syndrome, small for gestational age (SGA), idiopathic short stature, AIDS wasting, multiple sclerosis, Crohn's disease, ulcerative colitis, and muscular dystrophy.
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