WO2017210607A1 - Protéines klotho recombinées thérapeutiques et compositions et méthodes comprenant celles-ci - Google Patents

Protéines klotho recombinées thérapeutiques et compositions et méthodes comprenant celles-ci Download PDF

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WO2017210607A1
WO2017210607A1 PCT/US2017/035755 US2017035755W WO2017210607A1 WO 2017210607 A1 WO2017210607 A1 WO 2017210607A1 US 2017035755 W US2017035755 W US 2017035755W WO 2017210607 A1 WO2017210607 A1 WO 2017210607A1
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Prior art keywords
protein
klotho
seq
decline
disease
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PCT/US2017/035755
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English (en)
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Joseph F. Tarsio
Dinesh RATURI
James R. PLANTE
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Klotho Therapeutics, Inc.
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Priority to EP17807601.4A priority Critical patent/EP3464608A4/fr
Application filed by Klotho Therapeutics, Inc. filed Critical Klotho Therapeutics, Inc.
Priority to JP2019515786A priority patent/JP2019526272A/ja
Priority to MX2018014753A priority patent/MX2018014753A/es
Priority to CN202310383190.9A priority patent/CN116478907A/zh
Priority to AU2017272349A priority patent/AU2017272349B2/en
Priority to CN201780034374.7A priority patent/CN109219663A/zh
Priority to KR1020187034860A priority patent/KR102570250B1/ko
Priority to BR112018073909-0A priority patent/BR112018073909A2/pt
Priority to KR1020237028237A priority patent/KR20230125857A/ko
Priority to CA3025461A priority patent/CA3025461A1/fr
Publication of WO2017210607A1 publication Critical patent/WO2017210607A1/fr
Priority to US16/208,552 priority patent/US20190169593A1/en
Priority to JP2023096236A priority patent/JP2023123565A/ja

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    • C12N9/0026—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on CH-NH groups of donors (1.5)
    • C12N9/0028—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on CH-NH groups of donors (1.5) with NAD or NADP as acceptor (1.5.1)
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    • C12Y603/01002—Glutamate-ammonia ligase (6.3.1.2)

Definitions

  • the present disclosure relates to the production and administration of recombinant human Klotho protein compositions as therapeutic agents. Specifically, the present disclosure relates to compositions that include a CGMP-grade human recombinant soluble alpha-Klotho protein or variant thereof, and methods of manufacturing and administering the same to human or non-human subjects.
  • Klotho (or alpha-Klotho, a-Klotho, etc.) is a recently characterized protein encoded by the KL (or klotho) gene, located on human chromosome 13. Two transcripts that arise from a single klotho gene through alternative RNA splicing have been identified. See Figures 1 and 2.
  • the first transcript is predicted to encode Klotho isoform 1 - a full-length, 1,012 amino acid, single-pass transmembrane-membrane protein, with a short cytoplasmic tail (human residues 1003-1012), a transmembrane (TM) domain (human residues 982-1002), and extracellular region or domain (human residues 1-981) comprising two homologous (internal repeat) domains (termed KLl (human residues 56-506, which is 450 residues long) and KL2 (human residues 515-953, which is 438 residues long), which each share 20%-40% amino acid sequence homology to ⁇ -glucosidases, but lack glucosidase catalytic activity), and a signal sequence (SS) domain (human residues 1-33).
  • KLl human residues 56-506, which is 450 residues long
  • KL2 human residues 515-953, which is 438 residues long
  • the SS, KLl, and KL2 domain- containing extracellular region may be enzymatically cleaved by ⁇ / ⁇ - secretases, and released into the circulatory stream as a 130 kDa circulating protein, termed soluble klotho (or sKlotho, s-Klotho, alpha soluble-Klotho, etc.).
  • the extracellular region can also be cleaved into separate 68 kDa protein (KLl + SS) and 64 kDa protein (KL2).
  • the second transcript a splicing variant of alpha-klotho mRNA, encodes a second isoform of Klotho protein corresponding mainly to the KLl domain.
  • the internal splice donor site is thought to be located in exon 3 of the klotho gene.
  • the resultant alternatively spliced transcript contains a 50 bp insertion after exon 3 ( Figure 1 ; gray), with an in-frame translation stop codon at the end thereof.
  • the expressed protein product is secreted into the circulation and is termed secreted Klotho (or Klotho isoform 2).
  • Klotho is highly expressed in the kidney, brain, and to a lesser extent in other organs, and may also be found in the cerebrospinal fluid and urine of mammals. Circulating levels of soluble Klotho proteins in mammals are thought to decrease with age. In addition, Klotho-deficient mice exhibit accelerated aging phenotypes, whereas over-expression of klotho in mice has been shown to extend lifespan. In addition, Klotho has been implicated in a number of cellular processes related to aging. In light of the foregoing, a developing hypothesis states that soluble Klotho may function as an anti-aging compound in the human body.
  • Aging is an inevitable and progressive biological process resulting in dysfunction and destruction of almost all tissues and organs, ultimately resulting in death.
  • the aging of the human body for instance, is associated with the decline of cellular function, which can lead to the development of a variety of diseases.
  • Aging is thought to be driven by a tightly regulated and complex interplay between genetic and acquired factors and is typically characterized by an increase in senescence, a quantitative and qualitative decrease in stem cells, and abnormal structure at tissue levels.
  • Embodiments of the present disclosure solve one or more of the foregoing or other problems in the art with recombinant human Klotho proteins, protein fragments, and/or protein variants, expression nucleic acid constructs and/or vectors, cell lines and/or cell suspension cultures, and methods of manufacturing, purifying, and administering the same to (human or non-human animal) subjects.
  • some embodiments of the present disclosure can include:
  • a medicament or therapeutic composition - e.g., formulation) of recombinant human alpha soluble Klotho protein
  • composition of a recombinant human alpha soluble Klotho protein and at least one additional (active) ingredient is provided;
  • nucleic acid construct or vector that encodes a recombinant human alpha soluble Klotho protein encodes a recombinant human alpha soluble Klotho protein
  • a cell line that contains (i) a nucleic acid construct or vector that encodes a recombinant human alpha soluble Klotho protein and/or (ii) expresses a recombinant human alpha soluble Klotho protein;
  • a cell suspension culture of cells that contain (i) a nucleic acid construct or vector that encodes a recombinant human alpha soluble Klotho protein and/or (ii) express a recombinant human alpha soluble Klotho protein;
  • a recombinant human alpha soluble Klotho protein for use in treating a specific medical or other condition; and/or
  • Some embodiments can include method of manufacturing recombinant Klotho protein, the method comprising producing a recombinant Klotho protein in Chinese hamster ovary (CHO) cells, preferably in dihydrofolate reductase (DHFR)-deficient CHO cells, more preferably in CHO-S cells, or preferably in glutamine synthetase (GS)-deficient CHO cells, more preferably in GS -/- CHO cells, the protein preferably having at least 85% amino acid sequence identity to one of SEQ ID NO: 2 through SEQ ID NO: 70.
  • CHO Chinese hamster ovary
  • DHFR dihydrofolate reductase
  • GS glutamine synthetase
  • Some embodiments can include a cell line, comprising a plurality of Chinese hamster ovary (CHO) cells, preferably in dihydrofolate reductase (DHFR)-deficient CHO cells, more preferably in CHO-S cells, or preferably in glutamine synthetase (GS)-deficient CHO cells, more preferably in GS -/- CHO cells, the CHO cells containing an exogenous nucleic acid comprises a promoter, preferably a strong promoter, and encodes a polypeptide, at least a portion of the polypeptide having at least 85% amino acid sequence identity to one of SEQ ID NO: 2 through SEQ ID NO: 70, and optionally, a functional dihydrofolate reductase (DHFR) enzyme or a functional glutamine synthetase (GS) enzyme.
  • DHFR dihydrofolate reductase
  • GS glutamine synthetase
  • Some embodiments can include a suspension cell culture, comprising a liquid medium, preferably a serum-free and/or animal protein component-free liquid medium, wherein the liquid medium preferably comprises a carbon source, a nitrogen source, and one or more vitamins, minerals, salts, amino acids, supplements, or additives, more preferably wherein the liquid medium lacks hypoxanthine, thymidine, and/or glutamine, and the cell line of any one of claims 14-17 growing in the liquid medium such that the CHO cells express the polypeptide encoded by the nucleic acid, the polypeptide comprising a recombinant Klotho protein.
  • a suspension cell culture comprising a liquid medium, preferably a serum-free and/or animal protein component-free liquid medium, wherein the liquid medium preferably comprises a carbon source, a nitrogen source, and one or more vitamins, minerals, salts, amino acids, supplements, or additives, more preferably wherein the liquid medium lacks hypoxanthine, thymidine, and/or glutamine,
  • Some embodiments can include a recombinant Klotho protein, wherein at least a portion of the protein has at least 80% amino acid sequence identity to one of SEQ ID NO: 2 through SEQ ID NO: 70.
  • Some embodiments can include a method of treating an aging-related or other condition, disease, or disorder, the method comprising administering to a subject in need therefore a pharmaceutically effective amount of the recombinant Klotho protein as described herein.
  • Some embodiments can include a method of treating an aging-related or other condition, disease, or disorder, the method comprising administering to a subject in need therefore a pharmaceutically effective amount of a soluble recombinant Klotho protein having at least 80% amino acid sequence identity to at least a subset of amino acid residues 1-981 of human alpha Klotho isoform 1.
  • Some embodiments can include a method of treating an aging-related or other condition, disease, or disorder, the method comprising administering to a subject in need therefore a pharmaceutically effective amount of a soluble recombinant Klotho protein having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% amino acid sequence identity to one of SEQ ID NO: 2 through SEQ ID NO: 70.
  • Some embodiments can include a pharmaceutical composition, comprising a pharmaceutically effective amount of the recombinant Klotho protein as described herein and a pharmaceutically-acceptable carrier.
  • Some embodiments can include a pharmaceutical composition, comprising a pharmaceutically effective amount of a recombinant soluble Klotho protein, at least a portion of the protein having at least 85% amino acid sequence identity to at least a subset of amino acid residues 1-981, 29-981, 34-981, 36-981, 131-981, 1-549, 29-549, 34-549, 36-549, or 131-549 of human alpha Klotho isoform 1, or at least a portion of one of SEQ ID NO: 2 through SEQ ID NO: 70, and a pharmaceutically-acceptable carrier.
  • Some embodiments can include a method of treating or preventing acute kidney injury (AKI) or other condition, the method comprising administering to a subject in need therefore a pharmaceutically effective amount of a recombinant Klotho protein, at least a portion of the protein having at least 85%, 86%, 88%, 90%, 92%, 95%, 98%, 99%, or preferably 100% amino acid sequence identity to at least a subset of amino acid residues 1- 981, 29-981, 34-981, 36-981, 131-981, 1-549, 29-549, 34-549, 36-549, or 131-549 of human alpha Klotho isoform 1, or at least a portion of one of SEQ ID NO: 2 through SEQ ID NO: 70.
  • AKI acute kidney injury
  • compositions that include a therapeutic Klotho protein, for example CGMP-grade human recombinant soluble alpha-Klotho protein, and at least one other active component, such as a drug, antibody, hormone, human cell, tissue, cellular or tissue-based product (HCT/Ps), etc. and/or methods of administering the same to human or non-human subjects.
  • a therapeutic Klotho protein for example CGMP-grade human recombinant soluble alpha-Klotho protein
  • HCT/Ps tissue-based product
  • Combinatorial compositions and methods can be useful for treating subjects having an age-related disorder or condition, a metabolic disorder, a chronic disease, an acute injury, and so forth.
  • the prophylactic administration of combination treatments to subjects with no apparent condition or disorder can also be useful in to delay or prevent certain conditions or disorders described herein.
  • Some embodiments can include a nucleic acid or nucleic acid construct.
  • embodiments can include an expression vector or nucleic acid.
  • the nucleic acid can encode a recombinant human alpha soluble Klotho protein, protein fragment, or protein variant.
  • the nucleic acid can encode a native or non-native signaling sequence.
  • the nucleic acid can encode a non-native signaling sequence upstream (or N-terminal to) an encoded Klotho protein sequence.
  • Some embodiments can include a method of manufacturing a recombinant human alpha soluble Klotho protein.
  • the manufacturing method can include growing Chinese hamster ovary (CHO) cells in a liquid medium, producing the recombinant soluble Klotho protein in the CHO cells, and/or purifying a recombinant soluble Klotho protein-containing extract from the CHO cells, liquid medium, or both.
  • the extract can include at least about 98% dry weight recombinant soluble Klotho protein and/or less than about 1-100 ppm CHO host cell proteins (HCP).
  • HCP ppm CHO host cell proteins
  • the CHO cells can be dihydrofolate reductase (DHFR)-deficient CHO cells, such as CHO-S cells, or glutamine synthetase (GS)-deficient CHO cells, such as GS -/- CHO cells.
  • DHFR dihydrofolate reductase
  • GS glutamine synthetase
  • the produced (expressed) protein can be released (e.g., secreted) from the CHO cells into the liquid medium and/or can have one or more glycans attached thereto.
  • the CHO cells can contain one or more exogenous nucleic acids that encode the protein and, optionally, a functional enzyme, such as dihydrofolate reductase enzyme, glutamine synthetase (GS) enzyme, etc.
  • the exogenous nucleic acid can include a promoter (e.g., a strong promoter, weak promoter, etc.), such as a promoter customary or typical for use for expression of exogenous protein in CHO cell.
  • the exogenous nucleic acid can include a transgene or cDNA (e.g., under control of the promoter), preferably having at least 85% nucleic acid sequence identity to one of SEQ ID NO: 76 through SEQ ID NO: 96, or any other suitable nucleic acid sequence encoding a Klotho protein as described herein (e.g., S- Klotho variants).
  • a transgene or cDNA e.g., under control of the promoter
  • the method can include introducing, such as by transfection, the exogenous nucleic acid into the CHO cells.
  • the method can include growing the CHO cells in a liquid medium, such as a (human, (fetal) bovine, or other) serum-free and/or animal (or animal- derived) protein (component)-free medium.
  • the medium preferably comprises a carbon source, a nitrogen source, and/or one or more vitamins, minerals, salts, amino acids, supplements, or additives, preferably in a bioreactor.
  • the method can include introducing an effective amount of methotrexate (MTX), methionine sulphoximine (MSX), or other agent into the liquid medium and/or selecting (e.g., by CHO cell sub-cloning, limited dilution, fluorescence activated cell sorting (FACS), etc.) a suspension culture of viable CHO cells growing in the liquid medium.
  • MTX methotrexate
  • MSX methionine sulphoximine
  • FACS fluorescence activated cell sorting
  • selection and/or gene amplification can be performed by culturing the transfected cells in a selection medium, such as a medium lacking hypoxanthine and/or thymidine (e.g., -HT medium), glutamine, etc.
  • a selection medium such as a medium lacking hypoxanthine and/or thymidine (e.g., -HT medium), glutamine, etc.
  • a low concentration(s) of MTX can be added or used to amplify the transfected nucleic acid (or gene(s) thereof) and, thereby, select for increased protein expression (e.g., in DHFR-deficient CHO cells transfected with a DHFR transgene).
  • selection and/or gene amplification can be performed by adding MSX (an inhibitor of (endogenous) glutamine synthetase (GS)) to suspension cultures of CHO cells having at least one (exogenous) glutamine synthetase (GS) transgene.
  • MSX an inhibitor of (endogenous) glutamine synthetase (GS)
  • GS glutamine synthetase
  • the method can include sub-culturing surviving cells or cultures (e.g., MTX- resistant and/or MSX-resistant cells or cultures).
  • the selected suspension culture and/or selected CHO cells can have or exhibit an increased production of the protein (e.g., by the CHO cell), an increased concentration of the protein (e.g., in the liquid medium), and/or an increased copy number of the exogenous nucleic acid (e.g., per cell) (e.g., as compared to a non-selected suspension culture or CHO cell).
  • Certain embodiments can include a cell line comprising a plurality of CHO cells.
  • the CHO cells can be DHFR-deficient CHO cells, such as CHO-S cells.
  • the CHO cells can contain one or more (copies of an) exogenous nucleic acid (comprising a transgene or cDNA) that encodes a polypeptide with at least 85% amino acid sequence identity to one of SEQ ID NO: 2 through SEQ ID NO: 70.
  • the polypeptide can comprise a human recombinant alpha soluble Klotho protein.
  • the exogenous nucleic acid can include a transgene or cDNA, preferably having at least 85% nucleic acid sequence identity to one of SEQ ID NO: 76 through SEQ ID NO: 96.
  • the nucleic acid can (also) include or encode a promoter (associated with the transgene) and/or an optional (exogenous) enzyme, such as a (functional) dihydrofolate reductase (DHFR) enzyme, glutamine synthetase (GS) enzyme, etc.
  • DHFR dihydrofolate reductase
  • GS glutamine synthetase
  • At least one embodiment includes a suspension cell culture comprising the cell line growing in a liquid medium, preferably comprising a carbon source, a nitrogen source, and/or one or more vitamins, minerals, salts, amino acids, supplements, or additives, such that the CHO cells express the polypeptide encoded by the nucleic acid.
  • the liquid medium can be (human, (fetal) bovine, or other) serum-free and/or animal (or animal-derived) protein (component)-free.
  • the liquid medium can be free of bovine serum albumin, human serum albumin, etc.
  • the liquid medium can also include an effective amount of MTX and/or MSX in some embodiments.
  • the suspension culture (or CHO cells thereof) can (be selected to): exhibit an increased production of the protein (e.g., by the CHO cells); exhibit an increased concentration of the protein (e.g., in the liquid medium); secrete the protein (e.g., into the liquid medium); and/or have an increased copy number of the exogenous nucleic acid (e.g., per cell), preferably as compared to a non-selected suspension culture.
  • the protein can have one or more glycans attached thereto.
  • Some embodiments include an extract of or from the CHO cells, the liquid medium, or both of the suspension cell culture, the extract containing a recombinant protein having at least 85% amino acid sequence identity to one of SEQ ID NO: 2 through SEQ ID NO: 70.
  • Certain embodiments include a human recombinant alpha soluble Klotho protein- containing extract of or from CHO cells, liquid medium, or both (e.g., of the suspension cell culture).
  • At least one embodiment includes an isolated recombinant protein having at least 85% amino acid sequence identity to one of SEQ ID NO: 2 through SEQ ID NO: 70.
  • Some embodiments can include a method of administering a recombinant human alpha soluble Klotho protein to a human or non-human animal subject in need thereof.
  • the subject to whom the Klotho protein is administered can be suffering from or at risk for a variety of conditions (e.g., disorders, diseases, injuries, illnesses, etc.).
  • some embodiments include a method of treating one or more chronic diseases and/or aging-related condition, such as a physical, mental, neurological, or other condition associated with (human) aging.
  • Some embodiments can promote healing, recovery, longevity, and/or other beneficial outcome through one or more mechanisms or action.
  • Embodiments can include, for example, administering to a subject in need therefore (e.g., a subject having or at risk of developing a condition) a pharmaceutically effective amount of a recombinant soluble Klotho protein or protein variant.
  • Administration of such protein or protein variant can have a positive therapeutic effect on the course and outcome of the condition, including chronic and/or age-related disease and longevity in human subjects, and characterization of the same.
  • the pharmaceutically effective amount can be sufficient to raise the serum soluble Klotho protein concentration of the subject to a predetermined level, such as greater than, equal to, or between about 50 to 3000 picograms of soluble Klotho protein per milliliter of serum.
  • the amount can also or alternatively be sufficient to maintain the serum soluble Klotho protein concentration of the subject at or above a predetermined threshold for a predetermined period of time.
  • Embodiments can also include administering the protein to a subject in need therefore so as to maintain the serum soluble Klotho protein concentration of the subject at or above a predetermined threshold for a predetermined period of time.
  • Embodiments can also include determining a serum soluble Klotho protein concentration of the subject, calculating the pharmaceutically effective amount, determining a rate of soluble Klotho protein decline in the serum of the subject, calculating a subsequent dosage time at which the serum soluble Klotho protein concentration of the subj ect will be at or below a second predetermined level based on the determined rate, calculating a subsequent dosage amount of the protein sufficient to raise the serum soluble Klotho protein concentration of the subject from the second predetermined level to the first predetermined level, and/or administering the subsequent dosage amount of the protein to the subj ect.
  • the protein can (be effective to) modulate the IGF-1 and/or Wnt signaling pathways, exhibit ⁇ -glucuronidase and/or sialidase activity, suppress the p53/p21 signaling pathway, and/or reduce EhC -induced cell senescence and apoptosis, preferably through suppression of the p53/p21 signaling pathway.
  • the protein can function or be functional as a humoral factor, preferably exhibiting pleiotropic activity and/or preferably in the regulation of oxidative stress, growth factor signaling, ion homeostasis, and/or regulation of activity of glycoproteins on the cell surface, such as one or more ion channel proteins and/or growth factor receptors, such as Insulin/Insulin-Like Growth Factor-1 receptor.
  • a humoral factor preferably exhibiting pleiotropic activity and/or preferably in the regulation of oxidative stress, growth factor signaling, ion homeostasis, and/or regulation of activity of glycoproteins on the cell surface, such as one or more ion channel proteins and/or growth factor receptors, such as Insulin/Insulin-Like Growth Factor-1 receptor.
  • the protein can also be effective to treat one or more aging-related condition (or condition associated with (human) aging), such as frailty, bone density loss or bone mineral density loss, weight loss, muscular atrophy or degeneration, decline in muscle mass, decline in muscle strength, hand strength, leg strength, or physical fitness, decline in movement, freedom of movement, quality of life assessment, ejection fraction, or exercise capacity, decline in learning, learning capacity, memory, or intellectual quotient, cognitive deterioration or forgetfulness, decline in cognitive capacity or function, decline in synaptic plasticity or synaptic function, and cellular senescence.
  • one or more aging-related condition or condition associated with (human) aging
  • the protein can also be effective to treat one or more aging-related condition (or condition associated with (human) aging), such as Alzheimer's disease, Parkinson's disease, dementia or vascular dementia, amyotrophic lateral sclerosis (ALS) or motor neuron disease (MND), atrial fibrillation, chronic obstructive pulmonary disease (COPD), fibromyalgia, adult onset diabetes, arthritis or rheumatoid arthritis, osteoarthritis, osteoporosis, glaucoma, cataracts, macular degeneration and other eye diseases/disorders, multiple sclerosis (MS), lupus, and/or ulcerative colitis.
  • aging-related condition or condition associated with (human) aging
  • ALS amyotrophic lateral sclerosis
  • MND motor neuron disease
  • COPD chronic obstructive pulmonary disease
  • fibromyalgia adult onset diabetes
  • arthritis or rheumatoid arthritis osteoarthritis
  • osteoporosis osteo
  • embodiments can also include a composition for use in treating one or more aging-related conditions.
  • the composition can include a recombinant soluble Klotho protein (e.g., having at least 85% amino acid sequence identity to one of SEQ ID NO: 2 through SEQ ID NO: 70) and a pharmaceutically-acceptable carrier.
  • compositions that include a therapeutic Klotho protein, for example CGMP-grade human recombinant soluble alpha-Klotho protein, and at least one other active component, such as a drug, antibody, hormone, hormone, human cell, tissue, cellular or tissue-based product (HCT/Ps), etc., and methods of administering the same to human or non-human subjects.
  • a therapeutic Klotho protein for example CGMP-grade human recombinant soluble alpha-Klotho protein
  • at least one other active component such as a drug, antibody, hormone, hormone, human cell, tissue, cellular or tissue-based product (HCT/Ps), etc.
  • HCT/Ps tissue-based product
  • Combinatorial compositions and methods can be useful for treating subjects having an age-related disorder or condition, a metabolic disorder, a chronic disease, an acute injury, and so forth.
  • the prophylactic administration of combination treatments to subjects with no apparent condition or disorder can also be useful in to delay or prevent certain conditions or disorders described herein.
  • the recombinant Klotho protein can include one of the Klotho protein having a sequences with 80%-100% sequence identity to one of SEQ ID NO: 1 through SEQ ID NO: 38, preferably having a C-terminal tag with 80%- 100% sequence identity to one of the sequences of SEQ ID NO: 74 or SEQ ID NO: 75, optionally with the linker sequence having 80%-100% sequence identity to SEQ ID NO: 73 disposed therebetween.
  • the protein can optionally include, or be expressed with a signaling sequence having 80%- 100% sequence identity to SEQ ID NO: 71 or SEQ ID NO: 72.
  • the (manufactured, produced, expressed or administered) protein has 80%-100% sequence identity to one of SEQ ID NO: 39 through SEQ ID NO: 70.
  • An exemplary method of manufacturing recombinant Klotho protein comprises: producing a recombinant Klotho protein in Chinese hamster ovary (CHO) cells, preferably in dihydrofolate reductase (DHFR)-deficient CHO cells, more preferably in CHO-S cells, or preferably in glutamine synthetase (GS)-deficient CHO cells, more preferably in GS -/- CHO cells, the protein preferably having at least 85% amino acid sequence identity to one of SEQ ID NO: 2 through SEQ ID NO: 70.
  • DHFR dihydrofolate reductase
  • GS glutamine synthetase
  • An exemplary cell line comprises: a plurality of Chinese hamster ovary (CHO) cells, preferably in dihydrofolate reductase (DHFR)-deficient CHO cells, more preferably in CHO-S cells, or preferably in glutamine synthetase (GS)-deficient CHO cells, more preferably in GS -/- CHO cells, the CHO cells containing an exogenous nucleic acid comprises a promoter, preferably a strong promoter, and encodes: a polypeptide, at least a portion of the polypeptide having at least 85% amino acid sequence identity to one of SEQ ID NO: 2 through SEQ ID NO: 70; and optionally, a functional dihydrofolate reductase (DHFR) enzyme or a functional glutamine synthetase (GS) enzyme.
  • DHFR dihydrofolate reductase
  • GS glutamine synthetase
  • An exemplary suspension cell culture comprises: a liquid medium, preferably a serum-free and/or animal protein component-free liquid medium, wherein the liquid medium preferably comprises a carbon source, a nitrogen source, and one or more vitamins, minerals, salts, amino acids, supplements, or additives, more preferably wherein the liquid medium lacks hypoxanthine, thymidine, and/or glutamine; and the cell line of any one of claims 14-17 growing in the liquid medium such that the CHO cells express the polypeptide encoded by the nucleic acid, the polypeptide comprising a recombinant Klotho protein.
  • An exemplary recombinant Klotho protein includes at least 80% amino acid sequence identity to one of SEQ ID NO: 2 through SEQ ID NO: 70.
  • An exemplary method of treating an aging-related or other condition, disease, or disorder comprising administering to a subject in need thereof a pharmaceutically effective amount of a soluble recombinant Klotho protein having at least 80% amino acid sequence identity to at least a subset of amino acid residues 1-981 of human alpha Klotho isoform 1.
  • An exemplary method of treating an aging-related or other condition, disease, or disorder comprising administering to a subject in need thereof a pharmaceutically effective amount of a soluble recombinant Klotho protein having at least 80% amino acid sequence identity to one of SEQ ID NO: 2 through SEQ ID NO: 70.
  • An exemplary pharmaceutical composition comprises: a pharmaceutically effective amount of a recombinant soluble Klotho protein, at least a portion of the protein having at least 85% amino acid sequence identity to: at least a subset of amino acid residues 1-981 , 29- 981, 34-981, 36-981, 131-981, 1-549, 29-549, 34-549, 36-549, or 131-549 of human alpha Klotho isoform 1; or at least a portion of one of SEQ ID NO: 2 through SEQ ID NO: 70; and a pharmaceutically-acceptable carrier.
  • An exemplary method of treating or preventing acute kidney injury (AKI) or other condition comprises: administering to a subject in need therefore a pharmaceutically effective amount of a recombinant Klotho protein, at least a portion of the protein having at least 85%, 86%, 88%, 90%, 92%, 95%, 98%, 99%, or preferably 100% amino acid sequence identity to: at least a subset of amino acid residues 1-981, 29-981, 34-981, 36-981, 131-981, 1-549, 29- 549, 34-549, 36-549, or 131-549 of human alpha Klotho isoform 1; or at least a portion of one of SEQ ID NO: 2 through SEQ ID NO: 70.
  • An exemplary method of treating an aging individual, the aging individual having a homozygous or heterozygous mutation in a gene encoding Klotho protein comprises administering a therapeutic concentration of a polypeptide having at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 98%, still more preferably at least 99%, most preferably 100% amino acid sequence identity to one of SEQ ID NO: 2 through SEQ ID NO: 70.
  • Some embodiments may include any of the features, options, and/or possibilities set out elsewhere in the present disclosure, including in other aspects or embodiments of the present disclosure. It is also noted that each of the foregoing, following, and/or other features described herein represent a distinct embodiment of the present disclosure. Moreover, combinations of any two or more of such features represent distinct embodiments of the present disclosure. Such features or embodiments can also be combined in any suitable combination and/or order without departing from the scope of this disclosure. Thus, each of the features described herein can be combinable with any one or more other features described herein in any suitable combination and/or order. Accordingly, the present disclosure is not limited to the specific combinations of exemplary embodiments described in detail herein.
  • Figure 1 depicts a schematic illustrating cellular production of various Klotho proteins according to an embodiment of the present disclosure
  • Figure 2 depicts: A) schematic structures of isoform 1 and isoform 2 of human a-Klotho, and the location of the epitope for the antibody binding used in generating C-D (residues 800 to 900); B) The full-length a-Klotho protein sequence of 1012 amino acids, with KL1 and KL2 shown in red and green, respectively, and TM highlighted (black); C) and D) Western blot analysis of human cell lysates (C) and human tissues (D);
  • Figure 3A illustrates the number of adult patients receiving certain aminoglycosides in the year 2010
  • Figure 3B illustrates the age distribution of the adult patients receiving the aminoglycosides presented in Figure 3A.
  • Figure 4 illustrates treatment data collected on the adult patients receiving the aminoglycosides presented in Figure 3A.
  • the words “can” and “may” are used in a permissive sense (i. e., meaning having the potential to), rather than the mandatory sense (i.e., meaning must).
  • the terms “including,” “having,” “involving,” “containing,” “characterized by,” variants thereof (e.g. , “includes,” “has,” and “involves,” “contains,” etc.), and similar terms as used herein, including the claims, shall be inclusive and/or open-ended, shall have the same meaning as the word “comprising” and variants thereof (e.g. , “comprise” and “comprises”), and do not exclude additional, un-recited elements or method steps, illustratively.
  • disclosure of an illustrative measurement that is less than or equal to about 10 units or between 0 and 10 units includes, illustratively, a specific disclosure of: (i) a measurement of 9 units, 5 units, 1 units, or any other value between 0 and 10 units, including 0 units and/or 10 units; and/or (ii) a measurement between 9 units and 1 units, between 8 units and 2 units, between 6 units and 4 units, and/or any other range of values between 0 and 10 units.
  • Embodiments of the present disclosure include products, compositions, and/or methods of manufacturing and/or using recombinant human Klotho proteins, such as (Current Good Manufacturing Practice (CGMP)-grade) human recombinant soluble alpha-Klotho proteins, protein fragments, and/or protein variants.
  • CGMP Current Good Manufacturing Practice
  • Gene therapy can be effective in animal studies. However, the safety of gene therapy, especially for human treatment, is still questionable. Compared to viral delivery of the klotho gene to animal (cells), the administration of exogenous and/or recombinant Klotho protein in humans may be a safer, easier, and more direct modality to restore (endocrine) klotho levels.
  • exogenous (human recombinant alpha soluble) Klotho protein may be a viable and effective option in the near future to treat aging and/or aging-related disorders.
  • administration of exogenous (human recombinant alpha soluble) Klotho protein to humans may be an effective strategy to reverse or retard stem cell depletion and/or abate age-associated frailty or other pathological processes.
  • condition refers to any disorder, disease, injury, or illness, as understood by those skilled in the art, that is manifested or anticipated in a patient. Manifestation of such a condition can be an early, middle, or late stage manifestation, as known in the art, including pre-condition symptoms, signs, or markers. Anticipation of such a condition can be or include the predicted, expected, envisioned, presumed, supposed, and/or speculated occurrence of the same, whether founded in scientific or medical evidence, risk assessment, or mere apprehension or trepidation.
  • patient generally refers to any animal under the care of a physician, as that term is defined herein, with particular reference to humans under the care of a medical doctor or other relevant medical professional.
  • the term "physician” as used herein generally refers to a medical doctor. This term may, when contextually appropriate, include any medical professional, including an oncologist, a surgeon, or any licensed medical professional, such as a physician's assistant, a nurse, a phlebotomist, a veterinarian, etc.
  • cancer refers to an abnormal, typically uncontrolled, growth of cells.
  • a "cancerous cell” as used herein comprises a malignant cell having an abnormal, typically uncontrolled, growth.
  • cancer is an umbrella term encompassing a plurality of different distinctive diseases characterized by malignant cells growing in a typically uncontrolled manner.
  • co-administration refers to concurrent, sequential, and/or combined administration of two or more components.
  • two components can be co-administered by administering each component in a separate dosage concurrently, simultaneously, or sequentially (e.g., distinct administrations separated by a period of time).
  • the period of time can be very small (e.g., substantially, immediately following a first administration) or longer (e.g., 1-60 seconds, 1-60 minutes, 1-24 hours, 1-7 days, 1 -4 weeks, 1-12 months, and so forth, or any value or range of values therebetween).
  • Concurrent or simultaneous administration can include overlapping administration timeframes for the two or more components or administration of a combination product comprising a mixture of the two or more components.
  • nucleic acid refers to a naturally occurring or synthetic oligonucleotide or polynucleotide, whether DNA or RNA or DNA-RNA hybrid, single-stranded or double-stranded, sense or antisense.
  • nucleic acids can include, without limitation, DNA, RNA, cDNA, gDNA, ssDNA, dsDNA or any combination thereof.
  • Nucleic acids of the disclosure can also include nucleotide or nucleic acid analogs (e.g., BrdU), and non-phosphodiester (intemucleoside) linkages or backbones (e.g., peptide nucleic acid (PNA) or thiodiester linkages), known in the art.
  • nucleotide or nucleic acid analogs e.g., BrdU
  • non-phosphodiester linkages or backbones e.g., peptide nucleic acid (PNA) or thiodiester linkages
  • standard amino acid includes: alanine - ala - A; arginine - arg - R; asparagine - asn - N; aspartic acid - asp - D; cysteine - cys - C; glutamine - gin - Q; glutamic acid - glu - E; glycine - gly - G; histidine - his - H; isoleucine - ile - I; leucine - leu - L; lysine - lys - K; methionine - met - M; phenylalanine - phe - F; proline - pro - P; serine - ser - S; threonine - thr - T; tryptophan - trp - W; tyrosine - tyr - Y; and valine -
  • codon optimized refers to the process of modifying or changing codons in a nucleotide sequence to codons that are preferred or more closely match the partem of codon usage in the organism used for expression of the molecule.
  • codons can be optimized for usage in a particular organism in which expression is desired based on known codon usage in the organism in order to enhance the effectiveness of expression of the nucleic acid, e.g., to achieve faster translation rates and high accuracy.
  • the codon usage in a particular organism is known.
  • the encoding nucleic acid molecule can be a modified wild-type or a codon optimized sequence, where the codons are optimized for expression in a particular host cell, such as mammalian cells, e.g. , CHO cells or 293 cells, or in a yeast, or a plant cell, eukaryotic cells.
  • a particular host cell such as mammalian cells, e.g. , CHO cells or 293 cells, or in a yeast, or a plant cell, eukaryotic cells.
  • the nucleic acid sequence can be codon optimized, for example, to increase expression levels of the encoded sequence.
  • the particular codon usage is dependent on the host organism in which the modified polypeptide is expressed.
  • codon usage information is available from the Codon Usage Database available at kazusa.or.jp. codon (see e.g., Richmond (2000) Genome Biology, 1 :241 for a description of the database. See also, Forsburg (2004) Yeast, 10: 1045-1047; Brown et al. (1991) Nucleic Acids Research, 19:4298; Sharp et al. (1988) Nucleic Acids Res., 12:8207-8211; Sharp et al. (1991) Yeast, 657-78).
  • Embodiments of the present disclosure can include one or more therapeutic and/or recombinant human alpha soluble Klotho proteins, protein fragments, and/or protein variants.
  • the protein can comprise all or a subset of amino acid residues 1-1012, 1-981, 29- 981, 34-981, 36-981, 131-981, 1-549, 29-549, 34-549, 36-549, or 131-549 of human alpha Klotho isoform 1.
  • the protein can have at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% amino acid sequence identity to all or a subset of amino acid residues 1-1012, 1-981, 29-981, 34-981, 36-981, 131-981, 1- 549, 29-549, 34-549, 36-549, or 131-549 of human alpha Klotho isoform 1.
  • at least a portion of the protein can have at least 85%, etc. amino acid sequence identity to one of SEQ ID NO: 2 through SEQ ID NO: 70, or a combination of two or more thereof.
  • some embodiments can include a protein having at least a portion with at least 85%, etc. amino acid sequence identity to any suitable portion of one of SEQ ID NOS: 1-75, or a combination of two or more thereof.
  • the protein can comprise a human C370 variant.
  • the protein can include a C370S alteration, thereby comprising S370.
  • the protein can include human F352 or other than F352V in some embodiments.
  • the protein can include the C370S alteration, thereby comprising S370, without a F352V variant, preferably with F352.
  • the protein can include H193 or other than the H193R variant. All other standard amino acid substitutions at amino acid residue (or position) 193, 352, and/or 370 of human alpha Klotho isoform 1 are contemplated and explicitly disclosed herein.
  • Some embodiments can include a variation at amino acid residue 45 of human alpha Klotho isoform 1.
  • the residue can be a valine (Val; V), a phenylalanine (Phe; F), or another amino acid.
  • the protein can also include one or more glycans (attached thereto).
  • native human alpha Klotho isoform 1 can have glycans attached (via glycosylation) at amino acids 106, 159, 283, 344, 604, 612, and/or 694.
  • the proteins of the present disclosure, or Klotho protein sequences thereof can have one or more of the same (or similar) glycans attached (via glycosylation) thereto (e.g., at the same amino acid position(s)).
  • the protein includes all of the same or similar (native-type) glycans attached thereto, at the same amino acid position(s).
  • the protein can include a signal peptide or signaling sequence.
  • the protein can include a native Klotho signaling sequence.
  • the protein can include a non-native or synthetic signaling sequence.
  • the signaling sequence can be an N-terminal signaling sequence and/or upstream (or N-terminal to) a Klotho protein sequence.
  • the signaling sequence can be C- terminal or otherwise disposed.
  • the signaling sequence can be, comprise, or have at least 80%, 85%, 90%, 95%, 98%, or 99% amino acid sequence identity to native human alpha Klotho isoform 1 signaling sequence, native human alpha Klotho isoform 2 signaling sequence, SEQ ID NO: 71 or SEQ ID NO: 72.
  • the protein can include an amino acid tag.
  • the tag can be a C-terminal tag and/or downstream of (or C-terminal to) a Klotho protein sequence.
  • the tag can be N-terminal or otherwise disposed.
  • the tag can be or comprise an Fc-fusion protein.
  • the tag can be or comprise an IgGl -Fc protein sequence.
  • the tag can be, comprise, or have at least 80%, 85%, 90%, 95%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 74.
  • the tag can also, or alternatively, be or comprise a TEV-twinstrep protein sequence (e.g., as known in the art).
  • the signaling sequence can be, comprise, or have at least 80%, 85%, 90%, 95%, 98%, or 99% amino acid sequence identity to SEQ ID NO: 75.
  • the tag can be cleaved from the protein. In other embodiments, the tag can be retained as part of the protein. In some embodiments, the tag can enhance solubility and/or (serum) half-life of the protein. In some embodiments, the tag can be utilized during protein purification (e.g., as part of a purification mechanism).
  • the protein can include a linker (e.g., amino acid linker) disposed between a Klotho protein sequence and an amino acid tag.
  • the linker can comprise between 1 and 40 amino acids, preferably between 5 and 20 amino acids, more preferably between 8 and 12 amino acids, most preferably about 10 amino acids.
  • the linker can be or comprise a GS linker in some embodiments.
  • the linker can be, comprise, or have at least 70%, 80%, 90%, or 100% amino acid sequence identity to SEQ ID NO: 73.
  • the protein can be CGMP regulation compliant, as determined and enforced by the U. S. Food and Drug Administration (FDA).
  • the Klotho protein can be at least 95% 96%, 97%, 98%, or 99% pure, dry weight.
  • the Klotho protein sample can include less than about 1-100 parts per million (ppm), less than about 100-1000 parts per billion (ppb), or less than about 1-100 ppb CHO host cell proteins (HCP), nucleic acid, and/or other cellular components, or any value or range of values disposed therebetween.
  • nucleic acid or nucleic acid construct can include an expression vector or nucleic acid construct.
  • the nucleic acid can encode a recombinant human alpha soluble Klotho protein, protein fragment, or protein variant, as described herein.
  • the nucleic acid can encode a Klotho protein sequence, an optional (native or non-native) signaling sequence (e.g., at the N-terminus or N-terminal of the Klotho protein sequence), an optional linker sequence (e.g., GS linker), and/or an amino acid tag (e.g., IgGl-Fc or TEV-twinstrep), as described herein.
  • an optional (native or non-native) signaling sequence e.g., at the N-terminus or N-terminal of the Klotho protein sequence
  • an optional linker sequence e.g., GS linker
  • an amino acid tag e.g., IgGl-Fc or TEV-twinstrep
  • the nucleic acid can express a protein that includes all or a subset of amino acid residues 1-1012, 1-981, 29-981, 34-981, 36-981, 131-981, 1-549, 29- 549, 34-549, 36-549, or 131-549 of human alpha Klotho isoform 1.
  • At least a portion of the protein can have at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% amino acid sequence identity to all or a subset of amino acid residues 1-1012, 1-981, 29-981, 34-981, 36-981, 131-981, 1-549, 29-549, 34-549, 36-549, or 131-549 of human alpha Klotho isoform 1.
  • At least a portion of the protein can have at least and/or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% amino acid sequence identity to all or a portion of one of SEQ ID NO: 1 through SEQ ID NO: 75, or a combination of two or more thereof.
  • the protein can have at least and/or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% amino acid sequence identity to all or a portion of one of SEQ ID NO: 2 through SEQ ID NO: 70.
  • At least a portion of the nucleic acid can have at least a portion of the nucleic acid can have at least and/or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% nucleotide sequence identity to one of SEQ ID NO: 76 through SEQ ID NO: 101, or a combination of two or more thereof.
  • the nucleic acid can have at least and/or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% nucleotide sequence identity to one of SEQ ID NO: 76 through SEQ ID NO: 96.
  • Nucleic acid sequences of the present disclosure can also include a stop codon, as known in the art (e.g., TGA, TAG, TAA).
  • An embodiment of the present disclosure can include a cell line.
  • the cell line can comprise any suitable cell type, such as CHO cells, HEK cells, HL-60 cells, or other cell line as known in the art.
  • the cell line can comprise CHO cells (e.g., a plurality of CHO cells).
  • the CHO cells can (each) contain (one or more copies of) an exogenous nucleic acid.
  • the nucleic acid can encode a polypeptide with at least and/or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% amino acid sequence identity to one of SEQ ID NO: 1 through SEQ ID NO: 75, or a combination of two or more thereof, preferably to one of SEQ ID NO: 2 through SEQ ID NO: 70.
  • the nucleic acid can comprise at least one transgene or cDNA. In some embodiments, at least a portion of the nucleic acid can having at least and/or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% nucleic acid sequence identity to one of SEQ ID NO: 76 through SEQ ID NO: 101, or a combination of two or more thereof, preferably one of SEQ ID NO: 76 through SEQ ID NO: 96.
  • the nucleic acid can be or comprise a plasmid or other (structural) form of nucleic acid.
  • the exogenous nucleic acid can encode a functional enzyme, such as dihydrofolate reductase (DHFR) and/or glutamine synthetase (GS).
  • the CHO cells can be or comprise dihydrofolate reductase (DHFR)- deficient CHO cells, such as CHO-S cells.
  • the nucleic acid can include a promoter (e.g., a weak up to a strong promoter, as understood by those skilled in the art).
  • the nucleic acid can include a (strong) promoter associated with the transgene having at least 85%, etc. nucleic acid sequence identity to one of SEQ ID NO: 2 through SEQ ID NO: 70.
  • the transgene can be under the control of a promoter.
  • CHO cells and/or cell lines are referred to throughout the present disclosure. It is noted, however, that other cells, cell lines and/or host cells (besides CHO cells) are also contemplated within the scope of the present disclosure. Accordingly, a reference to CHO cells and/or cell lines also contemplates reference to and/or use of other known cells, cell lines and/or host cells.
  • a method of manufacturing the CHO cell line can include introducing the exogenous nucleic acid into the CHO cells, preferably via transfection, or other technique, as known in the art.
  • a serum-free growth optimized, cell-suspension of a CHO cell line was used as the host cell line for insertion of a nucleic acid (plasmid) containing a promoter, a human alpha S-klotho transgene encoding a polypeptide with at least 85% amino acid sequence identity to one of SEQ ID NO: 2 through SEQ ID NO: 70, and a selectable (enzyme) marker.
  • the transgenes respectively, encode amino acids 1 through 981, 29 through 981, or 34 through 981 of human alpha soluble Klotho.
  • the transgenes had sequences corresponding to one of SEQ ID NO: 76 through SEQ ID NO: 96 (or had at least 85% nucleic acid sequence identity to one of SEQ ID NO: 76 through SEQ ID NO: 96).
  • the selectable (enzyme) marker was exogenous DHFR.
  • the selectable (enzyme) marker was exogenous GS.
  • Some embodiments can include growing the cells (e.g., transfected and/or CHO cells) on a solid medium and/or in a liquid medium (e.g., in suspension cell culture), preferably in a serum-free and/or animal (or animal-derived) protein (component) free medium.
  • a liquid medium e.g., in suspension cell culture
  • cells can be plated on solid growth media for a period of time.
  • the cells can also or alternatively be grown in suspension culture and/or in liquid medium.
  • the liquid medium preferably comprises a carbon source, a nitrogen source, and one or more vitamins, minerals, salts, amino acids, supplements, or additives.
  • the medium can also lack hypoxanthine and thymidine (HT), glutamine, etc.
  • the cells after a certain period of time (e.g., 48 hours post- transfection), the cells can be gathered (e.g., detached), optionally centrifuged (e.g., at 100 x g for 5 min.), and/or seeded (e.g., at approximately 2000 cells/well), such as into a 96- well adherent culture plate (e.g., containing serum supplemented, -HT and/or -glutamine media).
  • the medium can also include MTX and/or MSX in certain embodiments.
  • Non- transfected cells may die within 7-14 days after selection (e.g., after exposure to MTX and/or MSX in -HT and/or -glutamine media.
  • the CHO cells can (be selected to) contain at least about 2 to 10 copies, at least about 10 to 20 copies, at least about 20 to 30 copies, or at least about 30 to 50 copies of the exogenous nucleic acid (e.g., per cell).
  • the method can include selecting CHO cells that contain at least about 2 to 10 copies, at least about 10 to 20 copies, at least about 20 to 30 copies, or at least about 30 to 50 copies of the exogenous nucleic acid (e.g., per cell).
  • MTX and/or MSX can be administered to the cells (e.g., at a concentration of about 1 nM - 1 uM, about 10 - 100 nM, etc.).
  • DHFR Dihydrofolate reductase gene amplification in DHFR-deficient CHO cells (such as the CHO-S cell line) transfected with exogenous DHFR, was accomplished by successively increasing levels of methotrexate (MTX) in growth medium. Because the plasmid contains DHFR, this allows for the amplification of the S-klotho gene (fragment) within the host cell upon exposure to MTX (10 - 100 nM).
  • the GS gene expression system was also used to amplify CHO cells transfected with exogenous GS (e.g., upon exposure to MSX). Alternatively, GS -/- host cell lines were also used, which removed the need for MSX.
  • the protein in suspension culture, can be secreted from the CHO cells into the liquid medium in some embodiments.
  • certain CHO cells and/or cell lines of the present disclosure can secrete (or be selected to secrete) up to a concentration of 200 - 500 mg of protein per liter of liquid medium, 500 - 2000 mg of protein per liter of liquid medium, 2000 - 5000 mg of protein per liter of liquid medium, or any value or range of values therebetween (without concentrating the protein).
  • high producing cell lines can be selected, such that the concentration of human recombinant alpha soluble Klotho protein in the medium (of the selected suspension culture or suspension cultures of the selected cell lines) is at least 200 mg/L, preferably at least 500 mg/L, more preferably at least 1000 mg/L, even more preferably at least 2000 mg/L, still more preferably at least 5000 mg/L, without concentrating the protein.
  • the CHO cells can be grown in a bioreactor having a volume or working volume of at least 10 liters, preferably at least 25 liters, more preferably at least 50 liters, even more preferably at least 100 liters, still more preferably at least 250 liters, still more preferably at least 500 liters, still more preferably at least 1,000 liters, still more preferably at least 2,000 liters, still more preferably at least 2,500 liters, still more preferably at least 5,000 liters, still more preferably at least 10,000 liters.
  • Certain embodiments can employ recombinant DNA strategies that utilize strong promoter sequences and/or high copy number plasmids for production of therapeutic amounts of the Klotho protein in mammalian (e.g., CHO) cells.
  • dihydrofolate reductase (DHFR) gene amplification in DHFR-deficient CHO cells can include providing and/or the use of (successively increasing levels of) methotrexate (MTX).
  • MTX methotrexate
  • GS glutamine synthetase
  • MSX methionine sulphoximine
  • the Klotho protein can also include one or more glycans (attached thereto).
  • the native human alpha Klotho isoform 1 (SEQ ID NO: 1) can have glycans attached (via glycosylation) at amino acids 106, 159, 283, 344, 604, 612, and/or 694.
  • the Klotho protein of the present disclosure can have one or more of the same (or similar) glycans attached (via glycosylation) thereto (e.g., at the same amino acid(s)).
  • the protein can be CGMP regulation compliant, as determined and enforced by the U.S. Food and Drug Administration (FDA).
  • the Klotho protein can be at least 95% 96%, 97%, 98%, or 99% pure, dry weight.
  • the Klotho protein sample can include less than about 1-100 parts per million (ppm), less than about 100-1000 parts per billion (ppb), or less than about 1-100 ppb CHO host cell proteins (HCP), nucleic acid, and/or other cellular components, or any value or range of values disposed therebetween.
  • Glycan structures present of the S-Klotho proteins produced can be similar or identical in comparison to the structures on native S-klotho structure isolated from human fluids (i.e., blood, sera, urine, cerebrospinal fluid).
  • confirmation of native-like glycans can ensure that the right native post-translation modifications (PTMs) are produced and stably maintained in the S CHO cell-produced S-Klotho protein. Solubility and/or half-life extension of Klotho proteins
  • nucleic acid changes made in the sequence of the klotho gene or nucleic acid construct see SEQ ID NOS: 76-96
  • changes, or chemical alterations in the amino acid sequence of a Klotho protein see SEQ ID NOS: 1-70
  • additions or subtraction of chemical groups, peptides, or proteins to the amino acid sequence of a Klotho protein is taught in the present disclosure in order to obtain resultant human Klotho variant proteins (novel compositions) with increased biological half- life or increased solubility in biological matrices (such as in blood, cerebrospinal fluid, urine, or various human tissues) than that of native Klotho molecules.
  • novel compositions can be made through the methods described herein for the modification of the S-Klotho protein.
  • Fusion protein constructs can be produced by combining the S-Klotho protein with the Fc domains of an antibody (IgG).
  • HSA human serum albumin
  • Fusion proteins produced by combining the S-Klotho protein with a proprietary recombinant polypeptide such as XTEN®.
  • Antibody Fc domain and human serum albumin were tested for effectiveness in extending the half-life and increasing the solubility of the human S-Klotho protein.
  • Fc fusions involve the fusion of peptides, proteins or receptor exodomains to the Fc portion of an antibody.
  • Both Fc and albumin fusions achieve extended half-lives not only by increasing the size of the peptide drug, but both also take advantage of the body's natural recycling mechanism through binding of the extended protein to the neonatal Fc receptor, FcRn. After binding of the extended protein to the FcRn receptor, degradation of the fusion protein in the cells' endosome is prevented.
  • Fusions based on the addition of Fc or albumin can result in biological half-lives in the range of 3-16 days, much longer than which has been reported for typical pegylated or lipidated peptides.
  • protein fusion technologies such as Fc fusion proteins, fusion to human serum albumin, fusion to carboxy-terminal peptide, and other polypeptide fusion approaches to make biobetter drugs with more desirable pharmacokinetic profiles see Strohl WR. Fusion Proteins for Half-Life Extension of Biologies as a Strategy to Make Biobetters. Biodrugs. 2015;29(4):215-239, the entirety of which is incorporated herein by specific reference.
  • Fc domains were thusly added to our parent protein (S-Klotho) to increase binding affinity to the the Fc receptor (FcRn).
  • FcRn is present inside lysosomes in endothelial cells lining the blood vessels and functions to rescue antibodies from the degradation that makes most proteins short-lived in circulation.
  • proteins have half-lives ranging from a few days to a few weeks, allowing for less frequent dosing of the extended form of the protein drugs than biologies that do not have this newly-produced composition-of-matter.
  • Fc and albumin A major difference between Fc and albumin is the dimeric nature of Fc versus the monomeric structure of HSA leading to the presentation of a Fc fused peptide as a dimer or a monomer in contrast to HSA.
  • the dimeric nature of a peptide Fc fusion can produce an avidity effect if the target receptors for S-Klotho are spaced closely enough together or are themselves dimers in particular human target organs. This may be desirable or not depending on the target.
  • Fusion of the S-Klotho protein to antibody Fc is also taught in the present disclosure to improve the solubility and stability of S-Klotho.
  • the addition of Fc domains to S-Klotho will also allow the fusion protein to be less immunogenic upon administration in human subjects.
  • HSA human serum albumin
  • HSA Like IgGs, HSA also binds FcRn in a pH-dependent manner, albeit at a site different from— and via a mechanism distinct from— that of IgG binding, and is recycled similarly to IgGs, resulting in its extended half-life. HSA also tends to accumulate in tumors and in inflamed tissues, which suggests that fusion or binding to albumin may potentially help to target proteins or peptides to those sites.
  • HSA thus improved the half-life of pharmacologically active GLP-1 from 1-2 min for native GLP-1 to 4-7 days, which allows for once weekly dosing.
  • Seven other known HSA fusion protein product candidates are either now in development or recently have been in development.
  • Novozyme has been developing modified versions of recombinant HSA with improved FcRn binding for construction of "next-generation" HSA-protein fusions that may possess even longer half-life properties. This was based on the use of a K573P mutant of HSA, which was found to possess 12-fold greater affinity for FcRn, conferred a longer half-life on HSA than the wild type molecule in both mice and cynomolgus monkeys. The expectation is that these longer-half-life mutants of HSA may of further use as fusion proteins to improve the half-life of fusion proteins.
  • the Inventors herein therefore disclose that we may fuse our Klotho protein to wild type HSA or to a mutant form of HSA to produce a Klotho fusion molecule(s) with significantly prolonged half-life in human blood, cerebral spinal fluid, and other human biological matrices in order to bring about a strategic therapeutic benefit such as superior patient convenience and likely compliance, reduced dosing frequency results in lower drug use in aggregate, and/or reduced cost of goods. Also lower drug quantities at the same dosing interval as the parent protein may simplify dosage formulation and enable subcutaneous formulation or decrease in the immunogenicity of S-Klotho.
  • Transferrin is a highly abundant serum glycoprotein, found in serum at 3-4 mg/mL, which binds iron tightly but reversibly and functions to carry iron to tissues. Transferrin has 679 amino acid residues, is about 80 kDa in size, and possesses two high- affinity Fe3+-binding sites, one in the N-terminal domain and the other in the C-terminal domain. Human transferrin has a half-life reported to be 7-10 days or 10-12 days. The aglycosylated form of human transferrin, which makes up about 2-8 % of the total transferrin pool, has a slightly longer half-life of 14-17 days. The extended persistence of transferrin in human serum is due to a clathrin-dependent transferrin receptor-mediated mechanism, which recycles receptor-bound transferrin back into the circulation.
  • the biotechnology company BioRexis Technologies, Inc. was founded in 2002 to develop the transferrin fusion protein platform, which they termed the "Trans Body” platform, as a therapeutic platform.
  • Their lead molecule, BRX-0585 was a transferrin-GLP-1 fusion protein for treatment of type 2 diabetes mellitus (T2DM). Fusion of GLP-1 to transferrin was demonstrated to significantly enhance the half-life of GLP-1.
  • BioRexis was acquired by Pfizer in March 2007. As far as can be determined, no BioRexis-derived fusion proteins are currently in the clinic.
  • Klotho protein can be fused to human transferrin to produce a Klotho fusion molecule and administer it clinically to significantly prolong half- life or stability in human biological matrices in vivo.
  • XTEN® is a proprietary recombinant polypeptide that extends the in vivo half-life of therapeutic payloads.
  • XTEN consists of naturally-occurring hydrophilic amino acids and is biodegradable.
  • Pharmaceuticals such as proteins, peptides, and synthetic compounds can be XTENylated via chemical conjugation or genetic fusion.
  • XTEN proteins lack secondary and tertiary structure and their solution behavior resembles chemically prepared polymers with very large hydrodynamic radii. By size exclusion chromatography, XTEN protein polymers appear much larger than typical globular proteins of similar molecular weight. The bulking effect of XTEN greatly reduces renal clearance of attached molecules, thus greatly increasing their in vivo half-lives.
  • the length of XTEN polymers added to a Klotho protein will be customized to optimize the pharmacokinetics as well as the bio- distribution of the attached Klotho protein payloads.
  • XTEN thusly can be recombinantly-fused to our S-Klotho protein to increase the molecules in vivo half-life.
  • One benefit is that with the genetic S-Klotho-XTEN fusion constructs, a molecule is produced that has the convenience of expression, purification and characterization of a single molecule which includes both the therapeutic and bulking moieties.
  • Recombinant fusion allows attaching multiple XTEN chains per protein in precisely-defined locations have been successfully used by therapeutic drug manufacturer that has resulting in best-in-class pharmacokinetics as exemplified by XTENylated growth hormone (Somavaratan, with the company, Versartis) and FVIII-XTEN (with the company, Biogen).
  • Somavaratan XTENylated growth hormone
  • XTEN protein polymers can be produced as free intermediates for chemical conjugation to peptides, peptidomimetics, and other synthetic molecules.
  • Reactive groups thiol, amine
  • Amunix has developed XTENs containing 1 to 9 thiol groups with various spacing which can be provided to partners.
  • orthogonal conjugation to amino and thiol groups in XTEN will facilitates the production of our Klotho-XTEN molecules.
  • the Klotho protein can be extracted from cell suspension culture of CHO cells (e.g., of a CHO cell line).
  • the CHO cells can produce and optionally secrete the Klotho protein (e.g., into liquid medium). Secretion of S-klotho into the cells spent media of up to 200 - 500 mg/L was also observed.
  • Purification of the recombinant proteins of the present disclosure can be carried out by any suitable method known in the art or described herein, for example, any conventional procedures involving extraction, precipitation, chromatography and/or electrophoresis.
  • a further purification procedure that can be used for purifying proteins includes affinity chromatography using monoclonal antibodies which bind a target protein.
  • Some embodiments can include and IgG tagged protein that can be purified by affinity chromatography.
  • crude preparations containing a recombinant protein are passed through a column on which a suitable monoclonal antibody is immobilized. The protein usually binds to the column via the specific antibody while the impurities pass through.
  • the protein is eluted from the gel by changing pH or ionic strength.
  • the spent media from the CHO-S high producer cell lines was concentrated via tangential flow filtration; and S-klotho protein was purified by affinity chromatography followed by ion exchange cartridge or column chromatography. Size exclusion chromatography can also be used purify the proteins.
  • one or more pre- or post-affinity purification steps were performed. Such steps can include, for example, (ultra) centrifugation, dialysis, membrane and/or tangential flow filtration, chromatographic separation, such as ion exchange, liquid- liquid extraction, such as (aqueous) two-phase extraction, or other known purification steps.
  • One or more post purification processing steps were performed in certain embodiments. Such post purification processing steps can include, for example, tandem anion/cation flow- through chromatography (as opposed to bind-and-elute chromatography), viral and/or bacterial removal by membrane filtration (e.g., 0.2 micron, 0.1 micron, etc.) or by other means known to one of ordinary skill in the art.
  • Protein purity can be demonstrated by SDS-PAGE or other assays or means known in the art.
  • Klotho protein samples 50 ⁇ g were fractionated on precast SDS-PAGE gels (4-15%, 10 wells; catalog no. 456-1083; BioRad) and stained with Coomassie blue stain. All samples were run with either an empty lane in between or on separate gels to avoid sample-to-sample contamination. S-klotho of greater than 98% was shown to be isolated from the CHO S conditioned media as determined by Coomaise blue stain and densitometry tracing, or by silver stain visualization, or by HPLC or RP-HPLC.
  • the proteins (after reduction and S- carboxymethylation) can be cleaved with cyanogen bromide, trypsin, and/or proteinase K and the peptides separated by HPLC according to known methods of protein chemistry.
  • cyanogen bromide, trypsin, and/or proteinase K and the peptides separated by HPLC according to known methods of protein chemistry.
  • Proteins were also be analyzed through mass spectroscopic methods. Concerning sample preparation for mass spectrometry, in order to restrict analyses to the correct S-klotho protein, only the gel band between 75 and 150 kDa was resected for analysis. Gel fractions were macerated with a sterile blade and subjected to in-gel digestion. Gel fractions were destained by three washes with 80 of 50% acetonitrile (ACN)/50 mm ammonium hydrogen carbonate and washed with 100% ACN. The alkylation step was omitted, given the absence of cysteine residues from the target a-Klotho peptides.
  • ACN acetonitrile
  • Tryptic digestion was carried out overnight at 37°C with 60 of trypsin (sequencing grade modified, catalog no. V511A; Promega) in 50 mm ammonium hydrogen carbonate (0.005 ⁇ g/ ⁇ L). This process yielded 25 ⁇ , of which 5 ⁇ (1 ⁇ for S-Klotho) was subjected to liquid chromatography-electrospray ionization tandem mass spectrometry (MS/MS) and PRM analysis in an Orbitrap nano-ESI Q-Exactive mass spectrometer (Thermo Scientific), coupled to a nanoLC (Dionex Ultimate 3000 UHPLC).
  • HCP contaminating CHO host cell proteins
  • the NIH full S-Klotho protein dataset is at http://www.ncbi.nlm.nih.gov/protein/Q9UEF7
  • An analytical profile for S-Klotho suitable for clinical administration and that obtained in an embodiment of the present disclosure included an endotoxin level less than 0.1 ng per ⁇ g (1 EU ⁇ g) of S-Klotho.
  • Glycan structures present on the CHO S cell-produced S-Klotho were identical in comparison to the structures on native S-Klotho isolated from human fluids (i.e., blood, sera, urine, and cerebrospinal fluid). This ensured that the same, native post-translation modifications (PTMs) were produced and stably maintained in the S CHO cell produced S- Klotho protein. Accordingly, using production and purification method described herein, we have been successful in producing cGMP-grade human S-klotho that had an analytical profile suitable for clinical administration in human subjects.
  • PTMs native post-translation modifications
  • compositions of the present disclosure can include a pharmaceutical composition, such as a therapeutic composition.
  • Pharmaceutical compositions of the present disclosure can generally include a therapeutically effective amount of a recombinant soluble alpha Klotho protein admixture with a vehicle or carrier comprised of one or more additional components.
  • the components can include one or more aggregation inhibitors, buffers, tonicity modifiers, and additional excipients.
  • the primary solvent in carrier can be either aqueous or non-aqueous in nature.
  • the composition can be prepared by combining a purified Klotho protein of the present disclosure with a pharmaceutically-acceptable carrier.
  • the combining of the various components to be included in the composition can be done in any appropriate order, namely, the buffer can be added first, middle or last and the tonicity modifier can also be added first, middle or last. It is also to be understood by one of ordinary skill in the art that some of these chemicals can be incompatible in certain combinations, and accordingly, are easily substituted with different chemicals that have similar properties but are compatible in the relevant mixture.
  • Aggregation inhibitors reduce a polypeptide's tendency to associate in inappropriate or unwanted ternary or quaternary complexes.
  • the amino acids L-arginine and/or, L-cysteine can act to reduce aggregation of Fc domain containing polypeptide in a formulation for long periods, e.g., two years or more.
  • the concentration of the aggregation inhibitor in the formulation is preferably between about 1 mM to 1M, more preferably about 10 mM to about 200 mM, more preferably about 10 mM to about 100 mM, even more preferably about 15 mM to about 75 mM, and yet more preferably about 25 mM. These compounds are available from commercial suppliers.
  • Compositions of the present disclosure can include a buffering agent.
  • Buffering agents maintain pH in a desired range.
  • Various buffers suitable for use in the pharmaceutical composition of the present disclosure include histidine, potassium phosphate, alkali salts, sodium or potassium phosphate or their hydrogen or dihydrogen salts, sodium citrate or potassium citrate /citric acid, sodium acetate/acetic acid, maleic acid, ammonium acetate, tris- (hydroxymethyl)-aminomethane (tris), various forms of acetate and diethanolamine, and any other pharmaceutically acceptable pH buffering agent known in the art, to maintain the pH of the solution within a desired range. Mixtures of these buffering agents may also be used.
  • the amount of buffering agent useful in the composition depends largely on the particular buffer used and the pH of the solution.
  • acetate is a more efficient buffer at pH 5 than pH 6 so less acetate may be used in a solution at pH 5 than at pH 6.
  • the preferred pH of the preferred formulations will be in the range of 4.0 to 5.0, and pH-adjusting agents such as hydrochloric acid, citric acid, sodium hydroxide, or a salt thereof, may also be included in order to obtain the desired pH.
  • One preferred buffer is sodium phosphate as its buffering capacity is at or near pH 6.2. It will be appreciated, however, that other buffers may be selected to achieve any desireable pH buffering.
  • the concentration of the buffer in the formulation is preferably between about 1 mM to about 1M, more preferably about 10 mM to about 200 mM. Buffers are well known in the art and are manufactured by known methods and available from commercial suppliers.
  • the pH of the pharmaceutical composition is set at or near physiological levels comfort of the patient upon administration is maximized.
  • the pH be within a range of pH about 5.8 to 8.4, with about 6.2 to 7.4 being preferred, however, it is to be understood that the pH can be adjusted as necessary to maximize stability and solubility of the polypeptide in a particular formulation and as such, a pH outside of physiological ranges, yet tolerable to the patient, is within the scope of the disclosure.
  • the formulations of the present disclosure may further include one or more tonicity modifiers (e.g., to render the solution isotonic with a patient's blood for inj ection).
  • a tonicity modifier is understood to be a molecule that contributes to the osmolality of a solution.
  • the osmolality of a pharmaceutical composition is preferably regulated in order to maximize the active ingredient's stability and also to minimize discomfort to the patient upon administration. Where serum is approximately 300+/-50 milliosmolals per kilogram.
  • a pharmaceutical composition be isotonic with serum, i.e., having the same or similar osmolality, which is achieved by addition of a tonicity modifier, thus it is contemplated that the osmolality can be from about 180 to about 420 milliosmolals, however, it is to be understood that the osmolality can be either higher or lower as specific conditions require.
  • Typical tonicity modifiers are well known in the art and include but are not limited to various salts, amino acids or polysaccharides.
  • suitable amino acids include glycine.
  • suitable polysaccharides include sucrose, mannitol and sorbitol. It is understood that more than one tonicity modifier may be used at once, for example, sorbitol and glycine can be used in combination to modify a formulation's tonicity.
  • tonicity modifiers suitable for modifying osmolality include, but are not limited to amino acids (e.g., arginine, cysteine, histidine and glycine), salts (e.g., sodium chloride, potassium chloride and sodium citrate) and/or saccharides (e.g., sucrose, glucose and mannitol).
  • concentration of the tonicity modifier in the formulation is preferably between about 1 mM to 1M, more preferably about 10 mM to about 200 mM.
  • Tonicity modifiers are well known in the art and are manufactured by known methods and available from commercial suppliers.
  • Excipients also referred to as chemical additives, co-solutes, or co-solvents, that stabilize the polypeptide while in solution (also in dried or frozen forms) can also be added to a pharmaceutical composition.
  • Excipient is defined herein as a non-therapeutic agent added to a pharmaceutical composition to provide a desired effect, e.g. stabilization, isotonicity.
  • a desired effect e.g. stabilization, isotonicity.
  • Common attributes of desirable excipients are aqueous solubility, non-toxicity, non- reactivity, rapid clearance from the body, and the absence of immunogenicity.
  • the excipients should be capable of stabilizing the native conformation of the protein so as to maintain the efficacy and safety of the drug during processing, storage and administration to the patient.
  • sugars/polyols such as: sucrose, lactose, glycerol, xylitol, sorbitol, mannitol, maltose, inositol, trehalose, glucose; polymers such as: serum albumin (bovine serum albumin (BSA), human SA or recombinant HA), dextran, PVA, hydroxypropyl methylcellulose (HPMC), polyethyleneimine, gelatin, polyvinylpyrrolidone (PVP), hydroxyethylcellulose (HEC); non-aqueous solvents such as: polyhydric alcohols, (e.g., PEG, ethylene glycol and glycerol) dimethysulfoxide (DMSO) and dimethylformamide (DMF); amino acids such as: proline, L-serine, sodium glutamic acid, alanine, glycine, lysine hydrochloride, sarcosine and
  • concentration of one or more excipients in a formulation of the disclosure is/are preferably between about 0.001 to 5 weight percent, more preferably about 0.1 to 2 weight percent.
  • Excipients are well known in the art and are manufactured by known methods and available from commercial suppliers.
  • a formulation of the disclosure can comprise about 150 mM NaCl buffered to pH 7.3 to 7.4 with HEPES, MES, or Tris-HCl, and, optionally, one or more additional components as described herein.
  • a formulation of the disclosure can comprise about 25 to about 50 mg TNFR:Fc (etanercept), about 10 mM to about 100 mM L-arginine, about 10 mM to about 50 mM sodium phosphate, about 0.75% to about 1.25% sucrose, about 50 mM to about 150 mM NaCl, at about pH 6.0 to about pH 7.0.
  • L- arginine can be replaced with L-cysteine (at about 1 to about 500 micromolar) in the formulation.
  • the pH can be about pH 7.0.
  • a formulation of the disclosure can comprise about 25 mg/ml TNFR:Fc, about 25 mM L-arginine, about 25 mM sodium phosphate, about 98 mM sodium chloride, and about 1 % sucrose at about pH 6.2.
  • a formulation of the disclosure can comprise about 10 to about 100 mg/mL of RANK: Fc in about 10 mM to about 100 mM L-arginine, about 10 mM to about 50 mM sodium phosphate, about 0.75% to about 1.25% sucrose, about 50 mM to about 150 mM NaCl, at about pH 6 to about pH 7.
  • the formulation of the disclosure comprises 50 mg/ml RANK:Fc in about 25 mM L-arginine, about 25 mM sodium phosphate, about 98 mM sodium chloride, and about 1 % sucrose at about pH 6.2.
  • a formulation of the disclosure can comprise an effective amount of an Fc domain containing polypeptide, about 10 mM to about 100 mM L - arginine, about 10 mM to about 50 mM sodium phosphate, about 0 to 5% Mannitol and 0 to 0.2% Tween-20TM (polysorbate 20) at about pH 6 to 7.
  • a formulation of the disclosure can comprise an effective amount of an antibody, such as Emab (an anti- CD22 specific antibody), about 25 mM L-arginine, about 25 mM sodium phosphate, about 4% Mannitol, about 0.02% Tween-20TM (polysorbate 20) and at about pH 6.0.
  • the disclosure provides a method of treating a mammal comprising administering a therapeutically effective amount of the pharmaceutical composition described herein, wherein the mammal has a disease or disorder that can be beneficially treated with a Fc domain containing polypeptide in the composition.
  • the Fc domain containing polypeptide is derived from the same species of mammal as is to be treated with the composition.
  • the mammal is a human patient in need of treatment.
  • examples of diseases or disorders that can be treated include but are not limited to rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, Wegener's disease (granulomatosis), Crohn's disease (or inflammatory bowel disease), chronic obstructive pulmonary disease (COPD), Hepatitis C, endometriosis, asthma, cachexia, psoriasis, and atopic dermatitis, or persons with a genetic disorder with mutations in one or more Klotho genes.
  • Additional diseases or disorders that can be treated with TNFR:Fc include those described in WO 00/62790, WO 01/62272 and U.S. Patent Application No. 2001/0021380, the relevant portions of which are incorporated herein by reference.
  • the disclosure provides a method for accelerated stability testing of the stability an Fc domain containing polypeptide in a pharmaceutical composition of the disclosure comprising the steps of testing the activity of the polypeptide formulated according to the disclosure prior to storage, i.e., time zero, storing the composition at 37° C. for one month and measuring the stability of the polypeptide, and comparing the stability form time zero to the one month time point.
  • the pharmaceutical composition provides long-term storage such that the active ingredient, e.g., an Fc domain containing polypeptide, is stable over the course of storage either in liquid or frozen states.
  • the phrase "long-term" storage is understood to mean that the pharmaceutical composition can be stored for three months or more, for six months or more, for one year or more, and preferably for two year or more.
  • Long term storage is also understood to mean that the pharmaceutical composition is stored either as a liquid at 2-8° C. or is frozen, e.g., at -20° C. or colder (e.g., -20 ° C or -80° C). It is also contemplated that the composition can be frozen and thawed more than once.
  • stable with respect to long-term storage is understood to mean that the active polypeptide of the pharmaceutical composition does not lose more than 20%, or more preferably 15%, or even more preferably 10%, and most preferably 5% of its activity relative to activity of the composition at the beginning of storage.
  • Anti-oxidants contemplated for use in the preparation of the formulations include amino acids such as glycine and lysine, chelating agents such as EDTA and DTP A, and free- radical scavengers such as sorbitol and mannitol.
  • compositions may also involve particulate preparations of polymeric compounds such as bulk erosion polymers (e.g., poly(lactic-co-glycolic acid) (PLGA) copolymers, PLGA polymer blends, block copolymers of PEG, and lactic and gly colic acid, poly(cyanoacrylates)); surface erosion polymers (e.g., poly(anhydrides) and poly(ortho esters)); hydrogel esters (e.g., pluronic polyols, poly(vinyl alcohol), poly(vinylpyrrolidone), maleic anhydride-alkyl vinyl ether copolymers, cellulose, hyaluronic acid derivatives, alginate, collagen, gelatin, albumin, and starches and dextrans) and composition systems thereof; or preparations of lipo
  • bulk erosion polymers e.g., poly(lactic-co-glycolic acid) (PLGA) copolymers, PLGA polymer blends, block copolymers of P
  • Such formulations may influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the present proteins and derivatives.
  • the optimal pharmaceutical formulation for a desired protein can be determined by one skilled in the art depending upon the route of administration and desired dosage. Exemplary pharmaceutical formulations are disclosed in Remington's Pharmaceutical Sciences, 18th Ed. (1990), Mack Publishing Co., Easton, Pa. 18042, pages 1435-1712, the disclosure of which is incorporated herein by reference.
  • Methods for evaluating the efficacy and/or determining an effective dose of human recombinant alpha soluble Klotho can (preliminarily) include performing an organismal-based assays (e.g., using a mammal (e.g., a mouse, rat, primate, or some other non-human), or other animal (e.g., Xenopus, zebrafish, or an invertebrate such as a fly (e.g., Drosophila melanogaster) or nematode (e.g., Caenorhabditis elegans).
  • an organismal-based assays e.g., using a mammal (e.g., a mouse, rat, primate, or some other non-human), or other animal (e.g., Xenopus, zebrafish, or an invertebrate such as a fly (e.g., Drosophila melanogaster) or nematode (
  • the Klotho protein can be administered to the organism once or as a regimen (regular or irregular). For instance, the protein can be administered a suitable number of times (e.g., once, twice, etc.) in a given period of time (e.g., monthly, semi-monthly, weekly, semi-weekly, daily, etc.). A parameter of the organism (e.g., an age-associated parameter) can then be evaluated. Klotho proteins of interest can effectuate or result in a change in the parameter relative to a reference (e.g., a parameter of a control organism). Other parameters (e.g., related to toxicity, clearance, and pharmacokinetics) can also be evaluated.
  • a suitable number of times e.g., once, twice, etc.
  • a given period of time e.g., monthly, semi-monthly, weekly, semi-weekly, daily, etc.
  • a parameter of the organism e.g., an age-associated parameter
  • Klotho proteins of interest can effectuate or
  • the klotho proteins of the present disclosure may be evaluated using an animal (model) that has or exhibits a particular disorder or condition, such as an age-related or age- associated disorder or condition, a metabolic disorder or condition, etc.
  • a particular disorder or condition such as an age-related or age- associated disorder or condition, a metabolic disorder or condition, etc.
  • Such disorders and conditions can also provide a sensitized system in which the effect of the protein on physiology can be observed.
  • Exemplary disorders include, for example, denervation, disuse atrophy, metabolic disorders (e.g., disorder of obese and/or diabetic animals such as db/db mouse, ob/ob mouse, etc.), cerebral disorders, liver ischemia or other liver disorders, cisplatin/taxol/vincristine models, various tissue (xenograft) transplants, transgenic bone models, pain syndromes (e.g., inflammatory and neuropathic disorders), Paraquat, genotoxic, oxidative stress models, and tumor (I) models.
  • metabolic disorders e.g., disorder of obese and/or diabetic animals such as db/db mouse, ob/ob mouse, etc.
  • cerebral disorders e.g., liver ischemia or other liver disorders
  • cisplatin/taxol/vincristine models e.g., various tissue (xenograft) transplants, transgenic bone models, pain syndromes (e.g., inflammatory and neuropathic
  • the protein can be administered to a suitable animal (for a suitable treatment period) and the parameter of the animal is evaluated (e.g., after a suitable period of time, such as 10 to 60 minutes, 1 to 24 hours, 1 to 30 days, 1 to 12 months, 1 to 5 years, or any value or range of values therebetween.
  • the animal can be fed ad libitum or normally (e.g., not under caloric restriction, although some parameters can be evaluated under such conditions).
  • a cohort of such animals is used for the assay.
  • test polypeptide can be indicated as favorably altering lifespan regulation in the animal if the test polypeptide affects the parameter in the direction of the phenotype of a similar animal subject to caloric restriction.
  • test polypeptides may cause at least some of the lifespan regulatory effects of caloric restriction (e.g., a subset of such effects) without having to deprive the organism of caloric intake.
  • the parameter(s) to be tested may be age-associated or disease associated parameter(s) (e.g., a symptom of the disorder associated with the animal model).
  • a test protein that is favorably indicated can cause an amelioration of the symptom relative to a similar reference animal not treated with the polypeptide.
  • Other parameters relevant to a disorder or to aging can include: antioxidant levels (e.g. antioxidant enzyme levels or activity), stress resistance (e.g., paraquat resistance), core body temperature, glucose levels, insulin levels, thyroid-stimulating hormone levels, prolactin levels, and leutinizing hormone levels.
  • an animal having decreased Klotho expression may be used (e.g., a mouse with a mutant or deleted klotho gene).
  • the test protein can be administered to the mutant mouse and age-related parameters are monitored.
  • a test protein that is favorably indicated can cause an amelioration of the symptom relative to a similar reference animal not treated with the protein.
  • a parameter relevant to a metabolic disorder or to aging can be assessed by measurement of body weight, examination on the acquisition of reproductive ability, measurement of blood sugar level, observation of life span, observation of skin, observation of motor functions such as walking, and the like.
  • the assessment can also be made by measurement of thymus weight, observation of the size of calcified nodules formed on the inner surface of thoracic cavity, and the like. Further, quantitative determination of mRNA for the klotho gene or the Klotho protein can also be useful for the assessment.
  • Still other (in vivo) models and organismal assays include evaluating an animal for a metabolic parameter, e.g., a parameter relevant to an insulin disorder, type II diabetes.
  • a metabolic parameter e.g., a parameter relevant to an insulin disorder, type II diabetes.
  • Exemplary metabolic parameters include: glucose concentration, insulin concentration, and insulin sensitivity.
  • age-associated parameters include: (i) lifespan of the cell or the organism; (ii) presence or abundance of a gene transcript or gene product in the cell or organism that has a biological age-dependent expression partem; (iii) resistance of the cell or organism to stress; (iv) one or more metabolic parameters of the cell or organism (exemplary parameters include circulating insulin levels, blood glucose levels; fat content; core body temperature and so forth); (v) proliferative capacity of the cell or a set of cells present in the organism; and (vi) physical appearance or behavior of the cell or organism.
  • the term "average lifespan” refers to the average of the age of death of a cohort of organisms. In some cases, the “average lifespan” is assessed using a cohort of genetically identical organisms under controlled environmental conditions. Deaths due to mishap are discarded. Where average lifespan cannot be determined (e.g., for humans) under controlled environmental conditions, reliable statistical information (e.g., from actuarial tables) for a sufficiently large population can be used as the average lifespan.
  • the degree of accuracy required is a function of the average lifespan of a wildtype organism.
  • organisms of the same age may have lived for the same number of days.
  • organism of the same age may have lived for the same number of weeks or months; for primates or humans, the same number of years (or within 2, 3, or 5 years); and so forth.
  • organisms of the same chronological age may have lived for an amount of time within 15, 10, 5, 3, 2 or 1 % of the average lifespan of a wildtype organism of that species.
  • the organisms are adult organisms (e.g., the organisms have lived for at least an amount of time in which the average wildtype organism has matured to an age at which it is competent to reproduce).
  • the organismal screening assay can be performed before the organisms exhibit overt physical features of aging.
  • the organisms may be adults that have lived only 10, 30, 40, 50, 60, or 70% of the average lifespan of a wildtype organism of the same species.
  • Age-associated changes in metabolism, immune competence, and chromosomal structure have been reported. Any of these changes can be evaluated, either in a test subject (e.g., for an organism based assay), or prior, during or after treatment with a therapeutic described herein (e.g., for a (human, or mammal) patient).
  • a marker associated with caloric restriction can also be evaluated in a subject organism of a screening assay (or a treated subj ect). Although these markers may not be age- associated, they may be indicative of a physiological state that is altered when a Klotho or Klotho-related pathway is modulated.
  • the marker can be an mRNA or protein whose abundance changes in calorically restricted animals.
  • Cellular models derived from cells of an animal described herein or analogous to an animal model described herein can be used for a cell-based assay.
  • Models for evaluating the effect of a test protein on muscle atrophy include: 1) rat medial gastrocnemius muscle mass loss resulting from denervation, e.g., by severing the right sciatic nerve at mid-thigh; 2) rat medial gastrocnemius muscle mass loss resulting from immobilization (e.g., by fixed the right ankle j oint at 90 degrees of flexion); 3) rat medial gastrocnemius muscle mass loss resulting from hind limb suspension; 4) skeletal muscle atrophy resulting from treatment with the cachectic cytokine, interleukin-1 (IL-1); and 5) skeletal muscle atrophy resulting from treatment with the glucocorticoid, dexamethasone.
  • the present disclosure relates to S-Klotho formulations, clinical dosages, and administration (e.g., to increase and/or maintain serum concentrations of S-Klotho within the range of a normal and/or young (e.g., 18 - 30 years of age) person (e.g., without any chronic conditions).
  • Aspects or embodiments of the present disclosure include, for example, administering a (cGMP- and/or clinical grade) human recombinant alpha soluble Klotho protein or protein fragment (of isoform 1) to a (human) subject in need thereof.
  • Embodiments can also include measuring (in the (human) subject) serum S-Klotho levels or concentration (e.g., by Mass Spectroscopy (MS) or ELISA). Such measuring can occur before, after, and/or during S-Klotho administration, and can be repeated, as necessary, to determine serum S- Klotho levels and/or a rate of metabolism, degradation, or reduction of serum S-Klotho levels.
  • MS is a technique well known in the art. MS can be used to identify and even quantify the level of one or more (native and/or recombinant) Klotho proteins in the serum of a subject.
  • One or more additional proteins can also be measured in the subject's serum.
  • one or more Klotho-related and/or aging-related proteins e.g., FGF21, GDF-11, TIMP2, NAD+, CCL11, hormones testosterone, estrogen, etc.
  • kidney function proteins e.g., KIM-1, Cystatin-C, creatinine, BUN, creatinine, NGAL, etc.
  • a serum sample is obtained, such as a blood sample.
  • the sample can be obtained by a blood draw, as known in the art.
  • a finger prick or other less invasive means of obtaining a blood sample can be used. Accordingly, blood samples can be taken more frequently (e.g., throughout the day and/or every 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 hours).
  • MS can be used to measure the total Klotho protein serum concentration, as well as the serum concentration of various alpha Klotho protein species, such as native Klotho species (e.g., soluble Klotho, cleaved Klotho, secreted Klotho, etc.) and/or one or more Klotho proteins of the present disclosure.
  • Klotho levels can be measured before therapeutic recombinant Klotho protein administration and again throughout the day and/or every 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 hours post-administration.
  • Embodiments can also include determining such a rate and/or calculating a treatment protocol (e.g., including frequency, amount, and/or duration of (subsequent) administration(s) of S-Klotho) for maintaining within the serum of such subject a S-Klotho concentration within the range of a normal young person's sera concentration of S-Klotho.
  • a treatment protocol e.g., including frequency, amount, and/or duration of (subsequent) administration(s) of S-Klotho
  • the concentration of S-Klotho can be maintained at approximately 1000 picograms of (the S-Klotho) protein per milliliter of serum (pg/mL).
  • a S-Klotho administration strategy in humans can include the measurement of one or more pharmacokinetic parameters of S-Klotho. For instance, measurements can be made of resultant changes in vivo of S-Klotho levels in the sera, urine and cerebrospinal fluid in response to S-Klotho administration. Some embodiments can include measuring the effectiveness of S-Klotho administration on one or more clinical indicators. Clinical indicators for various conditions, diseases, and disorders are known in the art and described further herein.
  • Embodiments can also include taking a (baseline) S-Klotho level measurement(s) (e.g., at zero time (before any administration of exogenous S-Klotho) and/or at different times of day before and/or throughout the treatment protocol (e.g., before and after administration of S-Klotho) in order to account for any circadian rhythm effects in the (human) subject(s)).
  • a (baseline) S-Klotho level measurement(s) e.g., at zero time (before any administration of exogenous S-Klotho) and/or at different times of day before and/or throughout the treatment protocol (e.g., before and after administration of S-Klotho) in order to account for any circadian rhythm effects in the (human) subject(s)).
  • Embodiments can also include determining a suitable frequency, amount, and/or duration of S-Klotho administration. For instance, subjects with low S-Klotho serum levels (e.g., as determined by MS or ELISA immunoassay quantification), can be given (e.g., via intravenous, intradermal, intraperitoneal, intramuscular, intracutaneous, and/or subcutaneous injection or other administration) a first administration of klotho configured and/or adapted to bring the subjects serum S-Klotho levels to a first predetermined level (e.g., about 1000 pg/mL), measuring (the resultant change in) serum S-Klotho concentration in the subject (e.g., by MS or ELISA), measuring the levels and/or a rate of metabolism, degradation, or reduction of serum S-Klotho levels (following the first administration), calculating a half-life of the administered S-Klotho, and/or determining a frequency and/or timeframe in which
  • the second predetermined level can be between and/or about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, and/or 5% of the first predetermined level.
  • Further administrations can be given over a timeframe suitable to produce in the subject (long-term) S-Klotho serum level equivalent to that of normal sex-matched young person's serum maintenance level (e.g., approximately 1000 pg/ml).
  • the total timespan of the S-Klotho administration can range from 1 day to 5 years, or more. Measurements and/or determinations of frailty in the subject based on the use of the clinical frailty score and other measurements can also be made over the timeframe.
  • Embodiments of the present disclosure further include increasing the S-Klotho dosage to maintain a 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or more increase in S-Klotho levels above the normal range (of 1000 pg/ml).
  • Certain embodiments include administering the S-Klotho protein in a single bolus or extended (IV) injection (e.g., drip over an extended period of time). In at least one embodiment, 1, 2, 2.5, 2.75, 3, 3.5, 4, 4.5, 5, or more micrograms of S-Klotho per subject can be administered per treatment.
  • a suitable administration amount can be calculated through one or more methods known in the art.
  • One such method is the allometric scaling method.
  • 0.01 mg of S-Klotho /kg body weight per administration was used, or 10 ug/kg.
  • HED human equivalent dose
  • the HED was established using normalization to body surface area, a process described by Reagan-Shaw (2008), incorporated herein by reference. This process, termed allometric scaling, corrects for basal differences in metabolism rate between different species, and may be preferred over simple dose extrapolation. Illustratively, where the HED is 0.4 mg/kg, using allometric scaling, the human equivalent dose would be 0.4 mg/kg or 28 mg for an individual of 70 kg in weight, or 24 mg for an individual of 60 kg in weight.
  • Multiple factors can be considered or taking in account in determining, measuring, and/or estimating the amount and/or bioavailability of S-Klotho in humans (before and/or after recombinant protein administration), the total amount and/or concentration of S-Klotho to be administered, and/or the serum level response (over time) after recombinant protein administration.
  • Such factors can include, for example, composition of the diluent, route of administration, site of administration, distribution into the tissues and organs of the subject, metabolic or other rate of the subject, pharmacokinetics (PK), pharmacodynamics (PD), toxicology (Tox), and so forth.
  • a (normal) concentration of S-Klotho (e.g., in a healthy, young (18-30 years old) human adult) can be approximately 1000 pg/ml in sera.
  • a typical adult can have a blood volume of approximately 5 liters, with females generally having less blood volume than males.
  • Approximately 55% of human blood can be comprised of serum.
  • (5 liters of blood / adult) x (0.55 sera / liter blood) 2.75 liters (2750 ml) of sera/adult.
  • a dose of 4.125 micrograms/subject can be administered.
  • a dose of 5.5 micrograms/subject can be administered, and so forth.
  • a pharmaceutically effective and/or sufficient amount of purified recombinant S- klotho protein can be administered so as to raise the serum soluble Klotho protein concentration of the subject to any suitable level, such as greater than, equal to, or between about 50, 100, 250, 500, 750, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 1 1,000, 12,000, 13,000, 14,000, 15,000 20,000, 25,000, 30,000 40,000, 50,000, 75,000, 100,000 or more picograms of soluble Klotho protein per milliliter of serum, or greater than, equal to, or between about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 400%, 500%, 600%, 700%, 800%
  • subj ects can be administered one or more (bolus) intravenous, intradermal, intraperitoneal, intramuscular, intracutaneous, subcutaneous, and/or other inj ection of recombinant human S-Klotho protein at a dosage of greater than, equal to, or between about 0.01 mg/kg body weight in a suitable volume of Klotho buffer (e.g., 150 mM NaCl and 10 mM HEPES pH 7.4) or other pharmaceutically acceptable carrier.
  • a suitable volume of Klotho buffer e.g., 150 mM NaCl and 10 mM HEPES pH 7.4
  • a 160 pound subject i.e., body weight of 72.57 kg
  • a 170 pound person can receive a 0.77 mg of S-Klotho per administration.
  • the total number and frequency of administrations can be determined based on achieving and maintaining in sera, a concentration of, for example, 1000 pg/ml of S-Klotho (equivalent to 0.000001 mg/ml sera). The latter can be ascertained as measured by MS or by a human S-Klotho ELISA assay.
  • the dosage can be greater than, equal to, or between about 0.0001 - 10 mg/kg body weight, 0.0001 - 10 ⁇ g/kg body weight, 0.0001 - 10 ng/kg body weight, 0.0001 - 10 pg/kg body weight, or any value or range of values therebetween.
  • Urine and/or blood can be collected at one or more time points, such as greater than, less than, equal to, between, and/or about 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 40 minutes, 45 minutes, 60 minutes, 90 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 9 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 hours, 2.5 days, 3 days, 5 days, 7 days, 10 days, 14 days, 21 days, 4 weeks, 1 month, 2 months, 3 months, or more after a medical procedure or a first administration (dose) of recombinant Klotho protein (e.g., to measure, test, and/or determine serum S-Klotho levels and changes in response to administration and/or over time).
  • a first administration e.g., to measure, test, and/or determine serum S-Klotho levels and changes in response to administration and/or over time.
  • One or more embodiments include production and/or (subsequent) administration of a unique formulation of the S-Klotho active drug product and/or in combination with a pharmaceutically-acceptable carrier.
  • the carrier can be suitable for IV and/or bolus injection.
  • Embodiments can also include production and/or (subsequent) administration of a unique inactive prodrug formulation of the S-Klotho and/or in combination with a pharmaceutically- acceptable carrier (so that an inactive S-Klotho can be activated in vivo to release biologically effective S-Klotho in animals or in human subjects.
  • Such prodrug formulation can include a coating or time-release formulation.
  • An exemplary embodiment of the present disclosure relates to the administration of exogenous Klotho protein to treat age-related frailty (e.g., in humans or non-human animals).
  • S-Klotho may rescue myogenic stem cells, improve muscle repair, and/or suppress fibrosis in animal models of human disease.
  • S-Klotho may, therefore, be a promising therapeutic agent to counter muscle degeneration in aging human subjects showing signs of frailty.
  • the present disclosure relates to S-Klotho formulations, clinical dosages, and administration to a fragile and/or elderly (e.g., 60 - 95 years of age) person in order to maintain in the former subject a maintenance sera concentration of S-Klotho within the range of a normal and/or young (e.g., 18 - 30 years of age) person (e.g., without any chronic conditions).
  • a fragile and/or elderly e.g., 60 - 95 years of age
  • a maintenance sera concentration of S-Klotho within the range of a normal and/or young (e.g., 18 - 30 years of age) person (e.g., without any chronic conditions).
  • Klotho treatment over an extended period of time can recover and/or improve one or more aging-related indicator or conditions in the elderly, frail, or otherwise physiologically aging.
  • An exemplary embodiment of the present disclosure relates to the administration of exogenous Klotho protein to treat (e.g., decrease (the rate of)) muscle atrophy in human as measured by skeletal muscle tissue mass and the concurrent use of the above protein and molecular indicators to provide guidance on the effects of Klotho administration in countering muscle atrophy.
  • Muscle atrophy can include numerous neuromuscular, metabolic, immunological and neurological disorders and diseases as well as starvation, nutritional deficiency, metabolic stress, diabetes, aging, muscular dystrophy, or myopathy. Muscle atrophy can occur during the aging process. Muscle atrophy can also result from reduced use or disuse of the muscle. Symptoms include a decline in skeletal muscle tissue mass. In human males, muscle mass declines by one-third between the ages of 50 and 80. Some molecular features of muscle atrophy can include the upregulation of ubiquitin ligases, and the loss of myofibrillar proteins.
  • An exemplary embodiment of the present disclosure relates to the administration of exogenous Klotho protein to improve and/or counteract (aging-related) decline in cognitive function(s).
  • exogenous Klotho proteins may counteract cognitive decline in humans.
  • transgenic mice with systemic over-expression of klotho performed better than controls in multiple tests of learning and memory. Elevating klotho in mice also enhanced long-term potentiation, a form of synaptic plasticity, and enriched synaptic GluN2B, an N- methyl-D-aspartate receptor (NMDAR) subunit with key functions in learning and memory. Blockade of GluN2B abolished klotho-mediated effects.
  • NMDAR N- methyl-D-aspartate receptor
  • Klotho-regulated pathways may be relevant to slowing the progression of Alzheimer's disease and other forms of dementia.
  • rDLPFC right dorsolateral prefrontal cortex
  • KL-VS gene variant, or allele
  • rDLPFC right dorsolateral prefrontal cortex
  • KL-VS heterozygosity may be associated with enhanced executive function across the lifespan examined.
  • results may suggest that variation in the klotho gene may be associated with bigger brain volume and better function.
  • An exemplary embodiment of the present disclosure relates to the administration of exogenous Klotho protein to augment in vivo klotho levels and/or (cellular, molecular, and/or downstream) effects (e.g., in order to enhance cognition and counteract cognitive deficits throughout the human lifespan).
  • Exogenous administration of clinical grade S-Klotho preserves and/or improves cognitive function (e.g., in humans).
  • An exemplary embodiment of the present disclosure relates to the administration of exogenous Klotho protein to chronological (e.g., age-matched from birth) and sex-matched human subjects to improve average lifespan.
  • the results on average lifespan obtained with Klotho administration can be compared reliably to those individuals not receiving Klotho exogenous administration (the control group) and/or with statistical information (e.g., from actuarial tables) for a sufficiently large human population.
  • An exemplary embodiment of the present disclosure relates to the administration of exogenous S-Klotho to treat any age-associated or otherwise non-age associated condition, including, but not limited to (increase in) human frailty, (decrease in) longevity, (decrease in) cellular senescence, (decline in) muscle strength, (decrease in) bone loss or density, (decrease in) cognition, (decline in) muscle mass, (decline in) physical fitness, (decline in) hand strength, (decline in) leg strength, and so forth.
  • the present disclosure also relates to the administration of S-Klotho to increase bone mineral density (BMD) (e.g., in women but not in men), to increase BMD (e.g., in elderly women), which is reduced after menopause, to regenerate or reduce the degeneration of (degenerative) skeletal muscle, to improve walking gait, to improve (or reduce the decline in) spatial learning and memory, movement, freedom of movement, quality of life assessment, ejection fraction, exercise change, exercise improvement, and so forth.
  • BMD bone mineral density
  • BMD bone mineral density
  • the present disclosure further relates to the administration of S- Klotho to decrease cognitive deterioration or forgetfulness, to increase cognitive capacity, to improve cognitive function and synaptic plasticity, to decrease a decline in learning, learning capacity or IQ, to improve learning, learning capacity or IQ, and so forth.
  • An exemplary embodiment of the present disclosure relates to the administration of exogenous S-Klotho to treat (e.g., correct) known human genetic defects.
  • a 13-year-old girl with familial tumoral calcinosis and a Klotho mutation has been reported.
  • Familial tumoral calcinosis is an autosomal recessive metabolic disease characterized by ectopic calcifications and hyperphosphatemia due to inactivating mutations in FGF23 or GALNT3.
  • FGF23 is a hormone necessary for the renal excretion of phosphate, while GALNT is an enzyme contributing to the maturation and secretion of FGF23.
  • a homozygous mutation in the KLOTHO gene has been identified in the 13-year old girl.
  • KLOTHO encodes a secreted protein necessary to the transmission of the signal emitted by FGF23 toward its receptors.
  • the administration of exogenous human recombinant S-Klotho constitutes a highly targeted and effective therapy to address the malfunction and symptoms associated with familial tumoral calcinosis.
  • Acute kidney injury (AKI), previously called acute renal failure (ARF), is often defined as an abrupt onset of renal dysfunction ranging from minor loss of function to failure.
  • AKI is a common clinical complication that develops in approximately 4%-7% of hospitalized patients each year and the prognosis can be poor. Mortality rates associated with AKI range from 20%-35%. Renal Klotho expression has been shown to be suppressed following AKI. Adenoviral gene transfer of Klotho can be cytoprotective in AKI.
  • Acute Kidney Injury has been report in approximately 4.9% - 7% of hospitalized patients each year.
  • the rate of AKI may be as high as 60% in (hospitalized) elderly persons and 20-30% in elderly or critically ill patients.
  • AKI is also associated with increased mortality, length of stay (LOS), and hospital cost.
  • AKI may result at least in part from kidney transplant or other surgery, acute tubular necrosis (ATN), acute allergic interstitial nephritis (AAIN), nephritis (e.g., glomerulonephritis), nephrotoxicity (e.g., drug-induced nephrotoxicity), low blood pressure, or other contributing factor(s).
  • Kidney transplant and other surgeries can cause acute damage or injury to the kidneys, cause renal disease and/or failure.
  • Nephrotoxicity can contribute to AKI, ATN, AAIN, nephritis, etc. It has been reported that drugs (e.g., clinically- administered, prescription, illegal, or other drugs) are associated with 15% to 25% of all cases of AKI.
  • Contrast media alone accounts for 10% of all causes of hospital-acquired acute renal failure (e.g., via contrast-induced acute kidney injury CIAKI) and represents the third leading cause of in-hospital renal function deterioration after decreased renal perfusion and postoperative renal insufficiency.
  • drug-induced AKI can be or comprise anti-microbial -induced nephrotoxicity (resulting from anti-microbial treatment).
  • certain (gram- negative) bacterial infections can be treated with one or more aminoglycosides, such as paromomycin, tobramycin, gentamicin, amikacin, kanamycin, neomycin, and so forth.
  • Aminoglycosides have been shown to be nephrotoxic. As illustrated in Figure 4, for example, nearly 23% of patients treated with amikacin, a common aminoglycoside, developed acute kidney disease and over 17% of patients treated with amikacin dies prior to being discharged.
  • aminoglycosides including gentamicin, and tobramycin also induced (or were associated with or contributed to) kidney disease. As illustrated in Figures 3A and 3B, over 1.2 million adult patients, in various age groups, were treated with aminoglycosides in the year 2010.
  • Other anti-microbial agents include, for example, penicillins, ampicillin, cephalosporins, sulfonamides, ciprofloxacin, vancomycin, macrolides, tetracyclines, rifampin, and so forth.
  • Drug-induced nephrotoxicity can also result from treatment with one or more nonsteroid anti-inflammatory drugs (NSAIDs), such as aspirin (acetylsalicylic acid), celecoxib, diclofenac, diflunisal, etodolac, ibuprofen, indomethacin, ketoprofen, ketorolac, nabumetone, naproxen, oxaprozin, piroxicam, salsalate, sulindac, tolmetin, and so forth.
  • NSAIDs nonsteroid anti-inflammatory drugs
  • Drug-induced nephrotoxicity can also result from treatment with one or more cyclooxygenase-2 (COX-2) inhibitors (e.g., valdecoxib, rofecoxib, celecoxib, etc.), proton pump inhibitors (e.g., omeprazole, lansoprazole, etc.), anticonvulsants (e.g., phenytoin, valproic acid, etc.), histamine H2 receptor antagonist (e.g., nizatidine, ranitidine, famotidine, cimetidine, etc.), diuretics (e.g., carbonic anhydrase inhibitors, loop diuretics (e.g., bumetanide, ethacrynic acid, torsemide, furosemide, etc.), potassium-sparing diuretics (e.g., triamterene, spironolactone, amiloride, etc.), thiazide di
  • Drug-induced nephrotoxicity can also result from treatment with lithium, which can affect the flow of sodium through nerve and muscle cells in the body and can be used to treat manic episodes of bipolar disorder, often characterized by hyperactivity, rushed speech, poor judgment, reduced need for sleep, aggression, and anger. Lithium can also help to prevent or lessen the intensity of manic episodes. Drug-induced nephrotoxicity can also result from treatment with or exposure to gold, mercury, copper, or other elemental matter.
  • Drug-induced nephrotoxicity can also result from treatment with (D-) penicillamine; a medication of the chelator class, which can be used to treat scleroderma, Wilson's disease (by binding to accumulated copper for elimination through urine), cystinuria (by binding with cysteine to yield a mixed disulfide which is more soluble than cysteine), direct-acting smooth muscle relaxant (e.g., hydralazine), spasmolytics (e.g., carisoprodol, cyclobenzaprine, metaxalone, methocarbamol, benzodiazepines, such as diazepam, clonidine and other imidazoline compounds, tizanidine, baclofen, hydantoin derivatives, dantrolene, and so forth.
  • smooth muscle relaxant e.g., hydralazine
  • spasmolytics e.g., carisoprodol, cyclobenza
  • contrast- induced nephrotoxicity e.g., following exposure to (iodinated) contrast media, also known as radiocontrast-induced nephropathy (ON)
  • narcotic- (opioid-) induced nephrotoxicity e.g., following use or abuse of certain narcotics (e.g., opioids), such as cocaine, heroin, etc.
  • chemotherapy-induced nephrotoxicity e.g., following treatment with a cancer therapeutic, such as: cisplatin; carboplatin; oxaliplatin; alkylating agents, such as bendamustine, cyclophosphamide, ifosfamide, nitrosoureas, temozolomide, melphalan, etc.
  • antitumor antibiotics such as mitomycin C, bleomycin, anthracyclines and related agents, etc.
  • antimetabolites such as capecitabine, hydroxyurea, methotrexate, pemetrexed, pralatrexate, pentostatin, fludarabine, cladribine, gemcitabine, cytarabine, etc.
  • vinca alkaloids topotecan; etoposide; taxanes; irinotecan; lenalidomide; eribulin; arsenic trioxide; ixazomib; etc.
  • nephrotoxic drugs can induce nephrotoxicity, leading to AKI. Drug-induced nephrotoxicity (and other forms of AKI) can be life-threatening if untreated and can incur enormous costs (to patients, hospitals, and insurers) to treat.
  • Embodiments of the present disclosure can include a method of treating or preventing (prophylactically) acute kidney injury (AKI) or other condition.
  • the method can comprise administering a recombinant (soluble) Klotho protein to a subject in need thereof.
  • the method can comprise administering to a subject in need thereof, a pharmaceutically-effective amount of a recombinant soluble Klotho protein having at least 85% amino acid sequence identity to one of SEQ ID NO: 2 through SEQ ID NO: 70 (e.g., so as to raise and/or maintain a serum soluble Klotho protein concentration of the subject at or above a predetermined threshold for a predetermined period of time).
  • the condition can comprises: (i) acute tubular necrosis (ATN), acute allergic interstitial nephritis (AAIN), nephritis, glomerulonephritis, and/or nephrotoxicity; or (ii) AKI resulting at least in part from kidney transplant or other surgery, acute tubular necrosis (ATN), acute allergic interstitial nephritis (AAIN), nephritis, glomerulonephritis, nephrotoxicity, or low blood pressure.
  • the condition can comprise a drug-induced (e.g., aminoglycosides-induced) nephrotoxicity.
  • the protein can be administered prophylactically, for example, prior to kidney transplant, nephrotoxin administration, or other activity, treatment, or event known or anticipated to cause or contribute to AKI.
  • the protein can be administered in response to AKI, such as after kidney transplant or other surgery, aminoglycoside or other nephrotoxin administration, or other activity, treatment, or event known or anticipated to cause or contribute to AKI.
  • the nephrotoxin or other drug can be or comprise, for example:
  • one or more aminoglycosides e.g., paromomycin, tobramycin, gentamicin, amikacin, kanamycin, neomycin, etc.
  • aminoglycosides e.g., paromomycin, tobramycin, gentamicin, amikacin, kanamycin, neomycin, etc.
  • one or more anti-fungal agents e.g., amphotericin B, flucytosine, etc.
  • anti-fungal agents e.g., amphotericin B, flucytosine, etc.
  • one or more contrast agents e.g., (iodinated) radiocontrast media, high-osmolality contrast media (HOCM) having an iodine to molecule ratio of about 1.5 : 1, low-osmolality, nonionic contrast media (LOCM) having an iodine to molecule ratio of about 3 : 1, isosmolar (isoosmolality) contrast media (IOCM) having an iodine to molecule ratio of about 6 : 1), etc.);
  • contrast agents e.g., (iodinated) radiocontrast media, high-osmolality contrast media (HOCM) having an iodine to molecule ratio of about 1.5 : 1, low-osmolality, nonionic contrast media (LOCM) having an iodine to molecule ratio of about 3 : 1, isosmolar (isoosmolality) contrast media (IOCM) having an iodine to molecule ratio of about 6 : 1), etc.);
  • one or more antiretro viral agents e.g., adefovir, cidofovir, tenofovir, foscarnet, etc.
  • antiretro viral agents e.g., adefovir, cidofovir, tenofovir, foscarnet, etc.
  • one or more cancer (or chemo-) therapeutics e.g., cisplatin, carboplatin, oxaliplatin, alkylating agents (such as bendamustine, cyclophosphamide, ifosfamide, nitrosoureas, temozolomide, melphalan, etc.), antitumor antibiotics (such as mitomycin C, bleomycin, anthracyclines and related agents, etc.), antimetabolites (such as capecitabine, hydroxyurea, methotrexate, pemetrexed, pralatrexate, pentostatin, fludarabine, cladribine, gemcitabine, cytarabine, etc.), vinca alkaloids, topotecan, etoposide, taxanes, irinotecan, lenalidomide, eribulin, arsenic trioxide, ixazomib, etc.);
  • alkylating agents such as benda
  • one or more bisphosphonates, or derivatives thereof e.g., zoledronate / zoledronic acid, ibandronate, alendronate, alendronate/cholecalciferol, etidronate, risedronate (optionally, with calcium carbonate), pamidronate, tiludronate, etc.); and/or
  • one or more narcotics e.g., opioids
  • opioids such as cocaine, heroin, etc.
  • Embodiments of the present disclosure can include a method of administering a therapeutic recombinant (alpha soluble) Klotho protein (e.g., with at least 85% amino acid sequence identity to amino acid residues 1-981, or a subset thereof, of human alpha Klotho isoform 1).
  • the method can include administering the therapeutic Klotho protein to a human or non-human subj ect to treat or prevent (prophylactically) AKI or one or more conditions associated with AKI.
  • the method can include determining a level of serum soluble klotho level in the subj ect, calculating a first dosage of protein sufficient to raise the serum soluble klotho level in the subject to a predetermined level or percent of normal levels, administering the first dosage of protein to the subject, such as by bolus or gradual administration, determining a rate of soluble Klotho decline in the serum of the subject, such as following administration of the first dosage, calculating a subsequent dosage time and amount, and/or administering the subsequent dosage of protein to the subject.
  • soluble Klotho in the circulation starts to decline early in chronic kidney disease (CKD) stage 2 and urinary Klotho possibly even earlier in CKD stage 1. Therefore, soluble Klotho could serve as an early and sensitive marker of kidney function decline.
  • preclinical animal data support Klotho deficiency is not just merely a biomarker, but a pathogenic factor for CKD progression and extrarenal CKD complications including cardiovascular disease and disturbed mineral metabolism.
  • CKD is characterized by progressive deterioration of renal function with high risk of ESRD.
  • CKD risk increases with age, and about half of the CKD stage > 3 cases occurs in subjects N 70 years old.
  • CKD can be viewed as a state of accelerated aging.
  • the relative risk for cardiovascular mortality of a 25 to 34-year-old dialysis patient is similar to a non-CKD patient of N75 years of age.
  • Cardiovascular disease is the principal killer in CKD and ESRD patients.
  • CKD and ESRD patients have low renal Klotho expression and low levels of circulating Klotho. Renal Klotho deficiency in early stages of CKD may be attributed mainly to suppression of Klotho expression rather than loss of viable renal tubules.
  • compositions and treatments including S-Klotho in combination with other component(s) including S-Klotho in combination with other component(s)
  • Klotho can also act in an additive or synergistic matter with other compounds and/or components to influence one or more aspects of human health and well-being.
  • treatments that include a therapeutic human recombinant soluble alpha Klotho (S- Klotho) protein in combination and/or concurrently with one or more additional active components can benefit human patients.
  • Such treatments can be prophylactic or responsive to any human condition on which the Klotho protein and/or other component(s) can have a therapeutic effect.
  • Such conditions can include, for example, an age-related condition, a metabolic condition, a chronic or acute condition, and so forth. Specific, non-limiting examples of specific conditions are disclosed herein.
  • S-klotho may exist in the human body along with other blood borne anti-aging compound such as growth/differentiation factor 11 (GDF-11). Accordingly, in certain embodiments, therapeutic S-klotho can be co-administered (e.g., concurrently, sequentially, and/or in combination) with therapeutic GDF-11. Such administration can have additive or synergistic anti-aging or other effects in some embodiments. Likewise the co-administration S-klotho to human subjects with (neutralizing) antibodies to or suppressor of CCL11 may work in unison to counter aging or other condition (as CCL11 (also known as eotaxin-1) is understood to be a negative regulator of stem cell rejuvenation). S-klotho can also or alternatively be co-administered with other eotaxins, such as eotaxin-2 (CCL24) and/or eotaxin-3 (CCL26).
  • CCL24 eotaxin-2
  • CCL26 eo
  • S-klotho can be co-administered with suppressors of or antibodies to Transforming Growth Factor ⁇ -1 (TGF- ⁇ ).
  • TGF- ⁇ Transforming Growth Factor ⁇ -1
  • S-klotho administration may oppose the action of the TGF- ⁇ signal pathways involved in an endogenous anti-cellular epithelial-to-mesenchymal transition (anti-EMT) that leads to renal and other tissue fibrosis).
  • anti-EMT is also relevant in cancer cells where inhibiting EMT may confer on cancer cells the ability to metastasize - this latter process is understood to be opposed by klotho.
  • co-administration of S-klotho and a suppressor of or antibody to Transforming Growth Factor ⁇ -1 (TGF- ⁇ ) can have synergistic or additive effects.
  • S-klotho can be co-administered with antibodies to or suppressors of Insulin Growth Factor-1 (IGF-1).
  • IGF-1 Insulin Growth Factor-1
  • Klotho is understood to be a hormone that inhibits the intracellular insulin/IGF-1 signaling cascade, and this inhibition increases resistance to oxidative stress at the cellular and organismal level in mammals; a mechanism which is considered to be evolutionarily conserved for extending life span.
  • coadministration of S-klotho and a suppressor of or antibody to Insulin Growth Factor- 1 (IGF- 1) can have synergistic or additive effects.
  • S-klotho can be administered in combinations with vitamin D (e.g., Vitamin D3) or 1,25-dihydroxy vitamin D 3 [l,25(OH) 2 D 3 ], FGF-15, FGF-19, and/or Klotho ⁇ ; since numerous studies have revealed a comprehensive regulatory scheme of mineral homeostasis involving the mutually regulated positive/negative feedback actions of Klotho a-Kl, FGF23, and l,25(OH)2D and/or an analogous regulatory network composed of Klotho ⁇ - ⁇ , FGF15/humanFGF19, and bile acids that regulate bile acid/cholesterol metabolism. Such co-administration can have synergistic or additive effects on numerous conditions and/or processes in the body.
  • S-klotho can be administered in combinations with FGF-21,
  • S-klotho can be co-administered with carbonic anhydrase inhibitors, such as acetazolamide, methazolamide, dichlorphenamide, dorzolamide, brinzolamide, and/or topiramate.
  • carbonic anhydrase inhibitors such as acetazolamide, methazolamide, dichlorphenamide, dorzolamide, brinzolamide, and/or topiramate.
  • Such combinatorial administration can be useful in the treatment of ankylosing spondylitis (AS), rheumatoid arthritis (RA), and a variety of other conditions.
  • AS ankylosing spondylitis
  • RA rheumatoid arthritis
  • Various investigations have shown that increased bone resorption is a characteristic of AS and RA and that carbonic anhydrase inhibitors play an antiarthritic role by inhibiting bone resorption.
  • S-klotho stimulates bone resorption and phosphate release by acting on TRPV5, which is a recently identified osteoclast function modulator.
  • Increased levels of l,25(OH)2D3 caused by S-Klotho administration can also stimulate osteoclast differentiation and bone resorption and, thereby, phosphate release.
  • co-administration of S-Klotho and carbonic anhydrase inhibitors can have an additive or synergistic effect, especially in promoting bone health, particularly in AS and RA.
  • S-Klotho can be administered in combination with one or more disease-modifying antirheumatic drugs (DMARDs) - for the treatment of severe active rheumatoid arthritis.
  • DMARDs disease-modifying antirheumatic drugs
  • S-Klotho can be administered in combination with cyclosporine, since cyclosporine decreasing klotho mRNA and protein and increases oxidative stress leading to cyclosporine induced-renal injury (CsA). The associated decrease of klotho mRNA and protein and increases oxidative stress can be countered by exogenous co-administration of S- Klotho.
  • S-klotho can be co-administered with losartan and/or cyclosporine. Treatment with losartan, an angiotensin II type 1 (ATI) receptor blocker, reversed the decrease in klotho expression seen with cyclosporine. Losartan also produced a concurrent improvement in renal histology (with losartan decreasing the tubulointerstitial fibrosis that is caused by cyclosporine).
  • ATI angiotensin II type 1
  • S-klotho can be co-administered with one or more aminoglycosides, such as amikacin, gentamicin, tobramycin, etc.
  • aminoglycosides such as amikacin, gentamicin, tobramycin, etc.
  • Such treatment can be useful to prevent nephrotoxicity and/or acute kidney injury (AKI) when aminoglycosides are used to treat (gram-negative) pathogen infection, which may greatly expand the use of aminoglycosides to treat infections.
  • the administration of S-Klotho with verapamil and/or diltiazem which have been used to block AKI, can be therapeutic in the treatment and/or prevention of renal dysfunction from AKI.
  • S-klotho can be co-administered with testosterone or androgen receptor (AR) up-regulating compounds.
  • AR androgen receptor
  • S-Klotho administration in combination with testosterone and/or androgen receptor (AR) up-regulating compounds can significantly increase muscle strength and/or physical function in elderly, frail, or low-T men beyond any effect that testosterone or S-Klotho may have alone in these treatment groups.
  • AR androgen receptor
  • S-klotho can be co-administered with estrogen or an estrogen hormone (e.g., estradiol, estriol, estrone, etc.).
  • estrogen or an estrogen hormone e.g., estradiol, estriol, estrone, etc.
  • Such co-administration can improve health indicators in women (e.g., menstrual, menopausal, or menopausal transitioning women) and/or treat infertility, polycystic ovarian disease or disorder, obesity, hormone imbalance and related conditions, and/or other female health conditions.
  • S-klotho can be co-administered with one or more nootropics - also called smart drugs or cognitive enhancers.
  • nootropics drugs, supplements, and/or other substances can improve cognitive function, particularly executive functions, memory, creativity, motivation, task saliency (motivation to perform a task), performance (especially on tedious tasks that require a high degree of effort), and can be useful in treating cognitive or motor function difficulties attributable to disorders such as Alzheimer's disease, Parkinson's disease, Huntington's disease, and ADHD.
  • stimulants especially the classes of stimulants that demonstrate cognition-enhancing effects in humans by acting as direct agonists or indirect agonists of dopamine receptor Dl, adrenoceptor A2, or both receptors in the prefrontal cortex.
  • Stimulants include, for example: amphetamines (e.g., amphetamine, dextroamphetamine, lisdexamfetamine, etc.), which can benefit a range of cognitive functions (e.g., inhibitory control, episodic memory, working memory, and aspects of attention), especially in individuals with ADHD; dimethylamylamine (DMAA), such as 1,3- dimethylamylamine, which can improve physical performance, alertness, reaction time, etc. ;
  • amphetamines e.g., amphetamine, dextroamphetamine, lisdexamfetamine, etc.
  • cognitive functions e.g., inhibitory control, episodic memory, working memory, and aspects of attention
  • DMAA dimethylamylamine
  • 1,3- dimethylamylamine which can improve physical performance, alertness, reaction time, etc.
  • methylphenidate - a substituted phenethyl amine that can improve a range of cognitive functions (e.g., working memory, episodic memory, and inhibitory control, aspects of attention, and planning latency); eugeroics (e.g., armodafinil, modafinil, etc.), which can function as wakefulness promoting agents that can increase alertness, particularly in sleep deprived individuals, facilitate reasoning and problem solving, treat narcolepsy, shift work sleep disorder, and daytime sleepiness remaining after sleep apnea treatments, and so forth; xanthines (e.g., caffeine, etc.), which can increase alertness, performance, and/or memory; nicotine, and so forth.
  • cognitive functions e.g., working memory, episodic memory, and inhibitory control, aspects of attention, and planning latency
  • eugeroics e.g., armodafinil, modafinil, etc.
  • xanthines e.g., caffeine, etc.
  • S-klotho can be co-administered with one or more osteoporosis and/or osteopenia medications, as known ion the art.
  • Klotho may play a role in regulating bone mineral density, as the absence of Klotho can lead to reduced bone mineral density in animals.
  • Klotho knockout mice show reduced bone mineral density over time.
  • Klotho expression can rescue bone defect, such as Klotho knockout mice show reduced bone mineral density over time, in Klotho knockout animals.
  • Epidemiological studies have shown associations between various Klotho gene variants and changes in bone mineral density and prevalence of hand osteoarthritis.
  • S-Klotho can be administered in combination with one or more anti-cancer treatments and/or preventions, such as a chemotherapeutic.
  • lung cancers such as non- small cell lung cancer (NSCLC)
  • S-klotho administration can affect the resistance of lung cancer cells to cisplatin and/or other chemotherapy.
  • S-klotho can function as a potential tumor suppressor in lung cancer, gastric cancer, pancreatic cancer (adenocarcinoma), and other forms of cancer.
  • S-Klotho can be administered in combination with sorafenib chemotherapy for the treatment of hepatocellular carcinoma (HCC).
  • HCC hepatocellular carcinoma
  • HCC hepatocellular carcinoma
  • CNS central nervous system
  • S-Klotho can also be administered in combination with chemotherapeutic to treat chemotherapy-induced frailty in cancer patients.
  • S-Klotho can also be administered to treat cancer-induced frailty in cancer patients following other known treatments.
  • S-Klotho can be administered in combination with kidney dialysis or other procedures.
  • S-Klotho can be administered to treat frailty in dialysis patients, as frailty is associated with poor outcomes for patients on dialysis.
  • S-Klotho can be administered in combination with one or more Alzheimer's Disease treatment or preventions, or with brain-derived neurotrophic factor (BDNF), as an adequate amount of BDNF can help to develop and maintain normal neuronal circuits in the brain.
  • BDNF brain-derived neurotrophic factor
  • S-Klotho can be administered in combination with one or more molecules that increase the ability of klotho to cross the blood-brain barrier in order to increase the ability of klotho to enter the central nervous system (CNS) in order to treat or prevent CNS-relates conditions.
  • CNS central nervous system
  • S-Klotho nor BDNF are known to cross the blood-brain barrier.
  • Embodiments of the present discloser include utilizing blood-brain barrier delivery techniques for the administration of S-klotho and/or S-klotho with BDNF to the CNS to treat Alzheimer's Disease and/or improve cognition in individuals not affected by Alzheimer's Disease.
  • S-Klotho can be administered in combination with 5' adenosine monophosphate- activated protein kinase (AMPK) or AMPK activating drugs or ingredients that positively regulates signaling pathways that replenish cellular ATP supplies, including fatty acid oxidation and autophagy; or that negatively regulates ATP-consuming biosynthetic processes including gluconeogenesis, lipid and protein synthesis.
  • AMPK 5' adenosine monophosphate- activated protein kinase
  • AMPK activating drugs or ingredients that positively regulates signaling pathways that replenish cellular ATP supplies, including fatty acid oxidation and autophagy; or that negatively regulates ATP-consuming biosynthetic processes including gluconeogenesis, lipid and protein synthesis.
  • S-Klotho can be administered in combination with one or more anti-diabetic drugs such as insulin, phloridzin or the antioxidant tiron and these combination treatments may have merit in the prevention of renal damage from oxidative stress produced in diabetic disorders.
  • the co-administration of S-Klotho with other antidiabetic drugs for type 1 diabetes can protect ⁇ -cells by inhibiting ⁇ -cell apoptosis through activation of the integrin ⁇ - FAK/Akt pathway, leading to inhibition of caspase 3 cleavage.
  • S-Klotho can be administered in combination with one or more type 2 anti- diabetes drugs such as metformin for improving glycemic control and vascular function in overweight and obese diabetic subjects.
  • S-Klotho can be administered in combination with one or more blood pressure medications, calcium regulators, or treatments or preventions of chronic kidney disease (CKD).
  • CKD chronic kidney disease
  • soft-tissue calcification is a prominent feature in CKD and Klotho can ameliorate vascular calcification by enhancing phosphaturia, preserving glomerular filtration, and directly inhibiting phosphate uptake by vascular smooth muscle.
  • S-Klotho can be administered in combination with TM5441 or other inhibitors of PAI-1 (plasminogen activator 1), since it is thought that PAI-1 inhibition or deficiency retards the development of senescence and protects organ structure and function while prolonging the lifespan of Klotho-deficient (kl/kl) mice.
  • PAI-1 plasmaogen activator 1
  • SIRT1 sirtuinl
  • STACs SIRT1- activating compounds
  • SIRT1 a type III protein deacetylase
  • SIRT1 level and activity are decreased during lung inflammation caused by oxidative stress.
  • the mechanism of SIRT1 -mediated protection against inflammation is associated with the regulation of inflammation, premature senescence, telomere attrition, senescence associated secretory phenotype, and DNA damage response.
  • SIRT1 A variety of dietary polyphenols and pharmacological activators are shown to regulate SIRT1 so as to intervene the progression of type 2 diabetes, cancer, cardiovascular diseases, and chronic obstructive pulmonary disease associated with inflammation.
  • SIRT-1 may be augmented by co-administration of SIRT1 and/or SIRTI-activating compounds given with S-Klotho.
  • S-Klotho can be administered in combination with one or more human Cells, Tissues, and Cellular and Tissue-Based Products (HCT/Ps), as recognized by the FDA.
  • Such products can include, for example, one or more of bone (including demineralized bone, ligaments, tendons, fascia, cartilage, ocular tissues (corneas & sclera), skin, vascular grafts (veins & arteries), except preserved umbilical cord veins, pericardium, amniotic membrane (when used alone (-without added cells-) for ocular repair), dura mater, heart valve allografts, hematopoietic stem cells derived from peripheral or umbilical cord blood, semen, oocytes, or embryos.
  • bone including demineralized bone, ligaments, tendons, fascia, cartilage, ocular tissues (corneas & sclera), skin, vascular grafts (veins & arteries), except preserved um
  • the HCT/P can be or comprise one or more stem cells.
  • Stem cell treatment for damaged bodily tissues and organs continues to grow in popularity.
  • Administration of therapeutic, recombinant Klotho protein in combination with stem cells provided surprising, unexpected, and even synergistic outcomes for subjects in need thereof.
  • Embodiments of the present disclosure further include a combination product, comprising a therapeutic, recombinant Klotho protein in combination with human stem cells.
  • the composition can also include a pharmaceutically-acceptable carrier as described herein.
  • Such compositions can comprise or be classified as regenerative medicines, to treat, modify, reverse, or cure a serious or life-threatening disease or condition, as recognized by the FDA. Preliminary clinical evidence indicates that the composition (drug) has the potential to address unmet medical needs for such disease or condition.
  • stem cells can be or comprise mesenchymal stem cells (MSCs), such as from human umbilical cords or placentas.
  • MSCs mesenchymal stem cells
  • the composition comprising huMSCs and a therapeutic, recombinant Klotho protein of the present disclosure attenuated the inflammatory and oxidative stress responses occurring in AKI, and/or reduced the expression of senescence-related proteins and microRNAs.
  • S-Klotho can be administered in combination with one or more senescence inhibitors.
  • Klotho protein can be combined with Pinl-FOXMl and/or other senescence inhibitors to enhance outcomes in patients receiving such treatment.
  • S-Klotho can be administered in combination with one or more of the following: Klotho stimulators (Vit. D, Losartan, Testosterone), GDF-11, Trichostatin A anti-fungal (GDF-11 Stimulator), TIMP-2, CCL-11 Inhibitor/antibodies, Dasatinib, Nicotinamide Riboside (NAD+), nicotinamide mononucleotide (NMN) (NDA+), AMPK Stimulators ( Resveratrol, Aspirin, Salicylate, phytochemicals, DR), C60 Fullerene, Rapamycin, FGF inhibitor, Senolytics drugs/compounds such as FOX04- p53 interfering peptides (e.g. FOX04-DRI), inhibitors of the anti-apoptotic proteins BCL-2 and BCL-xL.
  • Klotho stimulators Vit. D, Losartan, Testosterone
  • GDF-11 Trichostatin A anti-fungal (
  • any of the foregoing or other treatments or co-administrations can have additive or synergistic effects over those of any of the treatments alone.
  • coadministration of a Klotho protein with one or more of the foregoing can result in treatment outcomes greater than the sum of the individual outcomes of administering the components alone at similar concentrations.
  • synergistic effects can include treatment outcomes similar to those of the individual outcomes of administering the components alone, but at lower concentrations.
  • Synergistic effects can also include increasing the maximum effective dose of one or more of the components, reducing toxicity of one or more of the components, or any other beneficial result that is more than a mere additive effect of the individual treatment outcomes.
  • additive effects of the individual treatment outcomes can comprise synergistic effects where such additive effects is not predicted or expected given the nature and understanding of the individual components.
  • a combination treatment or co-administration can include treatment or administration of a combination product, composition, or formulation comprising a Klotho protein and one or more additional active ingredients.
  • the one or more additional active ingredients can be selected from among the components, drugs, substances, treatment compositions, etc. described herein or others known in the art.
  • the Klotho protein and one or more additional active ingredients can be co-formulated into an inj ectable (e.g., intramuscular, intravenous, etc.), ingestible, transdermal, inhalable, topical, or other formulation.
  • a combination treatment or co-administration can include treatment or administration of a Klotho protein and one or more additional active ingredients, but without the Klotho protein and one or more additional active ingredients being combined or formulated into a combination product, composition, or formulation.
  • the Klotho protein and one or more additional active ingredients can each comprise or be in a separate inj ectable (e.g., intramuscular, intravenous, etc.), ingestible, transdermal, inhalable, topical, or other formulation.
  • co-administration can comprise simultaneous administration of two or more components or distinct administrations of two or more components, the distinct administrations preferably being separated by a period of time.
  • the period of time can be very small, in some embodiments.
  • a second component of the Klotho protein and one or more additional active ingredients can be administered (e.g., inj ected) substantially, immediately following administration of a first component of the Klotho protein and one or more additional active ingredients.
  • the first and second administrations can be separated by a time period of 1-60 seconds, 1 -60 minutes, 1-24 hours, 1 -7 days, 1-4 weeks, 1-12 months, and so forth, or any value or range of values therebetween.
  • simultaneous administration can include overlapping administration timeframes for the two or more components.
  • Therapeutic S-Klotho proteins of various lengths can be modified in a variety of ways to achieve various beneficial effects and/or results not exhibited in native Klotho proteins.
  • the QuickChange XL Site-Directed Mutagenesis Kit (Stratagene) can be used to alter the nucleic acid sequence of various S-Klotho constructs.
  • Other mutagenesis methods and kits as known in the art can also be used. For instance, various sub-cloning methods and kits are known in the art and commercially available.
  • a protein is modified with one or more C-terminal tags and/or N-terminal tags.
  • tags can function to extend the serum and/or soluble half-life of the protein (in one or more therapeutic or other environments).
  • Tags can also be useful as markers for the presence or diagnostic localization of the protein, isolation or removal of the protein, delivery or transport of the protein, binding of the protein to one or more targets (e.g., protein, nucleic acid, organelle, cellular structural component, etc.), enzymatic processing or cleavage, and so forth.
  • the C-terminus of the protein can be tagged with TEV-TwinStrep and/or Fc-fusion, as known in the art and described further herein. Additional description can be found in the articles "Fusion Proteins for Half-Life Extension of Biologies as a Strategy to Make Biobetters," "What is the future of PEGylated therapies?" and "Strategies for extended serum half-life of protein therapeutics," the entirety of each of which is incorporated herein by specific reference.
  • a linker or linker peptide can be inserted and/or disposed between the (native or variant) Klotho protein sequence and the tag.
  • the modified Klotho protein can include an alternative (e.g., native, non-native, and/or synthetic) signal peptide.
  • the native signal peptide sequences can be replaced and/or supplemented with an altemative signal peptide or signaling sequence (SS).
  • SS an altemative signal peptide or signaling sequence
  • a native Methionine residue of a Klotho protein can be removed and a Methionine residue at the N-terminus of the SS can be included. Additional description can be found in the thesis "Generation of high expressing CHO cell lines for the production of recombinant antibodies using optimized signal peptides and a novel ER stress based selection system," the entirety of which is incorporated herein by specific reference.
  • a linker or linker peptide can be inserted and/or disposed between the (native or variant) Klotho protein sequence and the altemative SS.
  • Some embodiments can include one or more amino acid variants. It will be appreciated that the present disclosure contemplates variation of any one or more of the native amino acids in any of the disclosed Klotho proteins to any other amino acid, whether naturally-occurring, synthetic, or otherwise configured.
  • the Klotho gene maps on chromosome 13ql2.
  • a variant known as KL-VS, is present in approximately 15% of Caucasians.
  • the variant is composed of six single nucleotide polymorphisms (SNPs), two of which cause amino acid substitutions (i.e. F352V and C370S - Phenylalanine 352 changed to Valine and Cysteine 370 changed to Serine).
  • SNPs single nucleotide polymorphisms
  • F352V and C370S - Phenylalanine 352 changed to Valine and Cysteine 370 changed to Serine In vitro transfection assays have shown that secreted levels of klotho are reduced 6- fold for the V352 variant, while they are increased almost 3-fold for the S370 form.
  • An embodiment of the present disclosure includes a recombinant S-Klotho protein having the C370S homovariant (i.e., without the presence (or with deletion) of the F352V variant).
  • the C370S variant can be produced and/or expressed in the context of any protein construct described herein.
  • the C370S variant can be produced or expressed in the context of S-Klotho 1-981, 29-981, 34-981, 36-981, 131-981, 1-549, 29-549, 34-549, 36- 549, 131-549, and so forth, with or without Fc fusion and/or TEV-TwinStrep.
  • the nucleic acid construct or cDNA from which the protein is expressed can be of corresponding length.
  • Embodiments can include producing a S370 heterozygous or homozygous variant construct, transferring (e.g., via transfection) the resulting construct, which encodes the S- Klotho C370S protein, into an appropriate expression system (e.g., CHO cells), and/or transiently expressing the S-Klotho S370 protein.
  • the S370 Klotho protein may be expressed at higher levels than the F352V/C370S protein and/or wild-type F352/C370 protein.
  • Embodiments can include purifying (and optionally quality-control testing) the expressed protein for therapeutic administration.
  • Embodiments can include administering a therapeutic or therapeutically-effective amount of the S-Klotho C370S protein to a subject in need thereof.
  • the subject may, for example, harbor or express the KL-VS variant.
  • the subject may be of wild-type of other mutant or variant type.
  • Administration of the recombinant S-Klotho C370S protein can lead to a beneficial increase in blood S-Klotho levels. Accordingly, the circulating concentration of S-Klotho in subjects that receive the therapeutic, recombinant, S-Klotho C370S protein may not be subjected to the dilutive effects that are observed when the F352V variant is present.
  • HFTC is caused by a histidine (H) to arginine (R) mutation at amino acid (AA) position 193 of S-Klotho - rsl21908423.
  • H histidine
  • R arginine
  • AA amino acid
  • An embodiment of the present disclosure includes a S-Klotho protein having H193.
  • the H193 protein can be produced or expressed in the context of any protein construct described herein.
  • the HI 93 variant can be produced or expressed in the context of S-Klotho 1-981, 29-981, 34-981, 36-981, 131-981, 1-549, 29-549, 34-549, 36-549, 131- 549, and so forth, with or without Fc fusion and/or TEV-TwinStrep.
  • the nucleic acid construct or cDNA from which the protein is expressed can be of corresponding configuration.
  • Embodiments can include producing a HI 93 heterozygous or homozygous variant construct, transferring (e.g., via transfection) the resulting construct, which encodes the S- Klotho H193 protein, into an appropriate expression system (e.g., CHO cells), and/or transiently expressing the S-Klotho H193 protein.
  • the H193 Klotho protein may also be expressed at higher levels than the R193 (or H193R) protein.
  • Embodiments can include purifying (and optionally quality-control testing) the expressed protein for therapeutic administration.
  • Embodiments can include administering a therapeutic or therapeutically- effective amount of the S-Klotho H193 protein to a subject in need thereof (e.g., a HFTC individuals, individual diagnosed with HFTC, or patient harboring the H193R (rsl21908423) or other variant).
  • a subject in need thereof e.g., a HFTC individuals, individual diagnosed with HFTC, or patient harboring the H193R (rsl21908423) or other variant.
  • the subject may be of wild-type of other mutant or variant type.
  • Administration of the recombinant S-Klotho HI 93 protein can lead to a beneficial increase in blood S-Klotho levels.
  • the administration may reverse or counteract the deleterious effects of the R193 mutation that is transcribed and circulated in HFTC individuals as a result of the H-to-R193 point mutation found in the human klotho gene in individuals affected with HFTC. Accordingly, the circulating concentration of S-Klotho H193 may help to counter the effects observed in H193R or HFTC individuals.
  • ESRD end-stage renal disease
  • FGF fibroblast growth factor
  • Klotho is a protein in the vitamin D/FGF-23 signaling pathway that has been linked with accelerated aging and early mortality in animal models. It has been hypothesized that genetic variation in the Klotho gene may be associated with survival in subjects with ESRD.
  • SNPs single nucleotide polymorphisms
  • rs577912 SNPs nucleotide change described above result in an amino acid change in the Klotho protein. Accordingly, this functional SNP (rs577912) may quantitatively affect Klotho gene expression at the mRNA level.
  • An embodiment of the present disclosure includes a S-Klotho protein expressed from the AA or AC genotype.
  • the protein can be produced or expressed in the context of any protein construct described herein.
  • the protein can be produced or expressed in the context of S-Klotho 1-981, 29-981, 34-981, 36-981, 131-981, 1-549, 29-549, 34-549, 36- 549, 131-549, and so forth, with or without Fc fusion and/or TEV-TwinStrep.
  • the nucleic acid construct or cDNA from which the protein is expressed can be of corresponding length.
  • Embodiments can include producing a AA or AC heterozygous or homozygous construct, transferring (e.g., via transfection) the resulting construct, which encodes the S- Klotho protein, into an appropriate expression system (e.g., CHO cells), and/or transiently expressing the S-Klotho protein.
  • Klotho protein expressed in AA or AC heterozygous or homozygous cells may be expressed at higher levels than in CC cells.
  • Embodiments can include purifying (and optionally quality-control testing) the expressed protein for therapeutic administration.
  • Embodiments can include administering a therapeutic or therapeutically- effective amount of the S-Klotho protein to a subject in need thereof (e.g., an individual or patient harboring the CC mutation at the one tag SNP, rs577912, having low endogenous S- Klotho protein expression, and/or with end-stage renal disease (ESRD)).
  • a subject in need thereof e.g., an individual or patient harboring the CC mutation at the one tag SNP, rs577912, having low endogenous S- Klotho protein expression, and/or with end-stage renal disease (ESRD)
  • the subject may be of wild-type of other mutant or variant type.
  • Administration of the recombinant S-Klotho protein can lead to a beneficial increase in blood S-Klotho levels.
  • the administration may reverse or counteract the deleterious effects of the CC mutation that is transcribed and circulated in individuals as a result of the point mutation(s) found in the human klotho
  • Osteoarthritis is a common complex disease with strong heritable components.
  • SNP G-395A one variant in the klotho gene is associated with the susceptibility of hand OA and appears to act through osteophyte formation rather than cartilage damage.
  • An embodiment of the present disclosure includes a S-Klotho protein expressed from a construct having SNP G395A.
  • the resulting protein can be produced or expressed in the context of any protein construct described herein.
  • the A395 variant can be produced or expressed in the context of S-Klotho 1-981, 29-981, 34-981, 36-981, 131-981, 1- 549, 29-549, 34-549, 36-549, 131-549, and so forth, with or without Fc fusion and/or TEV- TwinStrep.
  • the nucleic acid construct or cDNA from which the protein is expressed can be of corresponding length.
  • Embodiments can include producing a G395A heterozygous or homozygous construct, transferring (e.g., via transfection) the resulting construct, which encodes the S- Klotho protein, into an appropriate expression system (e.g., CHO cells), and/or transiently expressing the S-Klotho protein.
  • Klotho protein expressed in A395 heterozygous or homozygous cells may be expressed at higher levels than in G396 cells.
  • Embodiments can include purifying (and optionally quality-control testing) the expressed protein for therapeutic administration.
  • Embodiments can include administering a therapeutic or therapeutically- effective amount of the S-Klotho protein to a subject in need thereof (e.g., an individual or patient harboring the G395 SNP and/or (at risk of developing) radiographic hand osteoarthritis (OA) and/or osteophytes.
  • a subject in need thereof e.g., an individual or patient harboring the G395 SNP and/or (at risk of developing) radiographic hand osteoarthritis (OA) and/or osteophytes.
  • the subject may be of wild-type of other mutant or variant type.
  • Administration of the recombinant S-Klotho protein can lead to a beneficial increase in blood S-Klotho levels.
  • the administration may reverse or counteract the deleterious effects of the G395 SNP that is transcribed and circulated in affected individuals.
  • the circulating concentration of S-Klotho may help to counter the effects observed in G395 individuals, especially those at risk of developing radiographic hand osteoarthritis (OA) and/or osteophytes.
  • Administration of the G-395A S-Klotho protein may, therefore, decrease the risk of radiographic hand osteoarthritis (OA) and osteophyte formation in patients (e.g., patients harboring the G395 SNP.
  • MetS metabolic syndrome
  • PLAD Dujiangyan
  • G-395A rsl207568
  • the -395A allele carriers had significantly lower risk of MetS in the whole population (odd ratio [OR] 0.50, 95 % confidential interval [CI] 0.25 to 0.98) and in women (OR 0.51, 95 % CI 0.24 to 0.97), but not in men (OR 0.42, 95 % CI 0.05 to 3.85).
  • the relationship between the Klotho G-395A SNP and MetS might be due to its influence on high blood pressure (OR 0.48, 95 % CI 0.34 to 0.67; OR 0.47, 95 % CI 0.31 to 0.71, respectively) and hypertriglyceridemia (OR 0.66, 95 % CI 0.39 to 0.95; OR 0.54, 95 % CI 0.31 to 0.98, respectively).
  • An embodiment of the present disclosure includes a S-Klotho protein expressed from a construct having the -395A allele.
  • the resulting protein can be produced or expressed in the context of any protein construct described herein.
  • the A395 allele can be produced or expressed in the context of S-Klotho 1-981, 29-981, 34-981, 36-981, 131-981, 1- 549, 29-549, 34-549, 36-549, 131-549, and so forth, with or without Fc fusion and/or TEV- TwinStrep.
  • the nucleic acid construct or cDNA from which the protein is expressed can be of corresponding length.
  • Embodiments can include producing a -395A heterozygous or homozygous construct, transferring (e.g., via transfection) the resulting construct, which encodes the S- Klotho protein, into an appropriate expression system (e.g., CHO cells), and/or transiently expressing the S-Klotho protein.
  • Klotho protein expressed in A395 heterozygous or homozygous cells may be expressed at higher levels than in G396 cells.
  • Embodiments can include purifying (and optionally quality-control testing) the expressed protein for therapeutic administration.
  • Embodiments can include administering a therapeutic or therapeutically- effective amount of the S-Klotho protein to a subject in need thereof (e.g., an individual or patient harboring the G395 SNP and/or (at risk of developing) metabolic syndrome (MetS).
  • a subject in need thereof e.g., an individual or patient harboring the G395 SNP and/or (at risk of developing) metabolic syndrome (MetS).
  • the subject may be of wild-type of other mutant or variant type.
  • Administration of the recombinant S-Klotho protein can lead to a beneficial increase in blood S-Klotho levels.
  • the administration may reverse or counteract the deleterious effects of the G395 SNP that is transcribed and circulated in affected individuals. Accordingly, the circulating concentration of S-Klotho may help to counter the effects observed in G395 individuals, especially those at risk of developing metabolic syndrome (MetS).
  • Administration of the G- 395A S-Klotho protein may, therefore, decrease the risk
  • S-Klotho is thought to inhibit basal Wnt signaling activity, thereby functioning as a tumor suppressor for colorectal cancer (CRC).
  • CRC colorectal cancer
  • klotho gene variants associated with lifespan differences may repress butyrate-mediated Wnt hyperactivation, and thus increase the risk of CRC. It has been hypothesized that in this manner, the type of klotho variant present, and its relative expression, can interact with levels of butyrate derived from diet to modify CRC risk.
  • mTOR signaling has also been linked to human aging, and crosstalk between Wnt and mTOR signaling may influence colonic tumorigenesis
  • the KL-VS variant or other construct can serve as a vehicle for investigating which SNPs (e.g., within KL-VS) are responsible for influencing S-Klotho to produce a decrease in basal Wnt signaling and/or the suppression of butyrate-mediated Wnt hyperactivation - the latter Wnt-related activities which have been associated with S-Klotho tumor suppression.
  • Embodiments include modifying the appropriate amino acids (e.g., in the KL-VS stretch of S-Klotho) shown to influence the tumor suppressing action of the KL-VS variant.
  • An embodiment of the present disclosure includes a recombinant S-Klotho protein having one or more amino acid alterations in the KL-VS stretch of 6 SNPs.
  • the proteins can be produced and/or expressed in the context of any protein construct described herein.
  • the proteins can be produced or expressed in the context of S-Klotho 1-981, 29-981, 34-981, 36-981, 131-981, 1-549, 29-549, 34-549, 36-549, 131-549, and so forth, with or without Fc fusion and/or TEV-TwinStrep.
  • the nucleic acid construct or cDNA from which the protein is expressed can be of corresponding length.
  • Embodiments can include producing a heterozygous or homozygous variant construct, transferring (e.g., via transfection) the resulting construct, which encodes the S- Klotho protein, into an appropriate expression system (e.g., CHO cells), and/or transiently expressing the S-Klotho protein.
  • the Klotho protein may be expressed at higher levels than the other Klotho proteins, including wild-type.
  • Embodiments can include purifying (and optionally quality-control testing) the expressed protein for therapeutic administration.
  • Embodiments can include administering a therapeutic or therapeutically-effective amount of the S-Klotho protein to a subject in need thereof (e.g., a patient with or at risk of developing colorectal cancer (CRC) or another tumor).
  • a subject in need thereof e.g., a patient with or at risk of developing colorectal cancer (CRC) or another tumor.
  • the subject may, for example, harbor or express a Klotho variant with decreased Wnt inhibition activity.
  • the subject may be of wild-type of other mutant or variant type.
  • Administration of the recombinant S-Klotho protein can lead to a beneficial increase in blood S-Klotho levels.
  • the genetic risk score (GRS) models further confirmed significant associations among KL SNPs and hemoglobin, total cholesterol, and HDL-C.
  • Gender- segregated analyses with the GRS-tagged approach confirmed the associations with HDL-C, fasting glucose, and fasting insulin levels in females, and with hemoglobin and LC in males.
  • An embodiment of the present disclosure includes a S-Klotho protein as described herein.
  • Embodiments can include producing a suitable klotho construct, transferring (e.g., via transfection) the construct, which encodes the S-Klotho protein, into an appropriate expression system (e.g., CHO cells), and/or transiently expressing the S-Klotho protein.
  • an appropriate expression system e.g., CHO cells
  • Embodiments can include purifying (and optionally quality-control testing) the expressed protein for therapeutic administration.
  • Embodiments can include administering a therapeutic or therapeutically-effective amount of the S-Klotho protein to a subject in need thereof (e.g., an individual or patient, optionally elderly and/or suffering from an aging-related condition, low endogenous S-Klotho protein expression, and/or symptoms of age-related condition or decreased longevity).
  • a subject in need thereof e.g., an individual or patient, optionally elderly and/or suffering from an aging-related condition, low endogenous S-Klotho protein expression, and/or symptoms of age-related condition or decreased longevity.
  • Administration of the recombinant S-Klotho protein can lead to a beneficial increase in blood S-Klotho levels.
  • the administration may reverse or counteract the deleterious effects of the aging-related condition and/or influence, in a positive therapeutic manner, quantitative traits such as serum levels of total cholesterol, HDL-C, fasting glucose, fasting insulin, albumin, creatine, IGF-1, hemoglobin, and lymphocytes count (e.g., in hospitalized and/or elderly patients).
  • quantitative traits such as serum levels of total cholesterol, HDL-C, fasting glucose, fasting insulin, albumin, creatine, IGF-1, hemoglobin, and lymphocytes count (e.g., in hospitalized and/or elderly patients).
  • an individual who is to be treated can have a mutation in the Klotho gene (e.g., genomically encoded heterozygous mutation(s) or homozygous mutation), and the treatment regimen can include administering a therapeutic dose of a peptide comprising wild-type Klotho and/or any one or more variants of Klotho disclosed herein, including, for example, a variant similar to the mutation expressed by the individual.
  • an individual to be treated can encode/express wild-type Klotho, and the treatment regimen can include administering a therapeutic dose of a peptide comprising wild-type Klotho and/or any one or more variants of Klotho disclosed herein.
  • a treatment method can include determining a low-level (e.g., compared to a control group) of circulating and/or cell- bound Klotho protein and administering a therapeutic dose that operatively restores concentrations of circulating and/or cell-bound Klotho to at least a homeostatic level. In some embodiments, this may include determining a level of Klotho (e.g., a gene expression level, a protein expression level, circulating levels, etc.) before administering the therapeutic concentration. Additionally, the therapeutic dose can be dependent upon the level of Klotho determined in the individual.
  • a level of Klotho e.g., a gene expression level, a protein expression level, circulating levels, etc.
  • the therapeutic dose exceeds the homeostatic level, such as, for example, by a scalar multiple of the homeostatic level (e.g., 1.5 times more, 2 times more, 3 times more, 4 times more, 5 times more, 6 times more, 7 times more, 8 times more, 9 times more, 10 times more, 15 times more, 20 times more 25 times more, 30 times more, 40 times more, 50 times more, 75 times more, 100 times more, 500 times more 1,000 times more, 10,000 times more, etc.).
  • a scalar multiple of the homeostatic level e.g., 1.5 times more, 2 times more, 3 times more, 4 times more, 5 times more, 6 times more, 7 times more, 8 times more, 9 times more, 10 times more, 15 times more, 20 times more 25 times more, 30 times more, 40 times more, 50 times more, 75 times more, 100 times more, 500 times more 1,000 times more, 10,000 times more, etc.
  • therapeutic treatments for age- related conditions can include administering a therapeutic concentration of one or more Klotho variants.
  • this can include treating a patient with a therapeutic concentration of a Klotho variant (or combination of Klotho variants) disclosed herein, such as, for example, peptides selected from any one or more of SEQ ID NO: 2 through SEQ ID NO: 70.
  • a Klotho variant or combination of Klotho variants disclosed herein, such as, for example, peptides selected from any one or more of SEQ ID NO: 2 through SEQ ID NO: 70.
  • the age-related condition can be treated with a Klotho variant that is the same or different than that expressed and/or encoded by the individual having an age-related condition.
  • an individual suffering from an age- related condition may genetically encode one or more wild-type or variants of Klotho, and the individual may express the wild-type and/or variant Klotho proteins at homeostatic levels (as compared to a control group), less than homeostatic levels, or not at all.
  • a therapeutic regimen aimed at treating the individual's age-related condition can include administering one or more Klotho variants disclosed herein.
  • embodiments of the present disclosure can include administering a therapeutic or therapeutically-effective amount of an S-Klotho protein to an individual or subject in need thereof for prophylactic purposes and/or maintenance of certain health attributes.
  • administration of certain S-Klotho proteins can help maintain youthfulness in optionally aging patients not yet suffering from a diagnosed aging-related condition.
  • certain embodiments of the present disclosure can relate to and/or comprise treating a condition in a patient, other embodiments can relate to and/or comprise preventing, inhibiting development of, and/or prophylactically addressing one or more conditions.
  • S-Klotho can be administered to persons with a genetic disorder with mutations in one or more Klotho genes.
  • Table 1 illustrates the results from expression and purification of the recited Klotho variants in HEK and/or CHO cell lines. In the results provided in Table 1, below, the following abbreviated protocols were followed.
  • Fc fusion proteins protein expression vectors transfected into HEK293.sus or CHO using standard ATUM methods. Briefly, cells were grown for 7 days and harvested. Cell counts are given in notes section. Supernatant pH was adjusted with 1M Hepes pH 7.4 and sodium azide added. KanCap A resin was used to capture proteins. Resin was washed with PBS. Resin was washed with PBS plus 1M NaCl. Resin was washed with PBS. Proteins were eluted with 50mM Citrate pH 3.5, lOOmM NaCl. Proteins were immediately neutralized with 1M Tris pH 8, 0.5M Arginine. SDS PAGE gel samples were removed at this stage. Proteins were buffer exchanged into PBS.
  • Protein was quantified by OD280, quantity and concentration was determined using calculated extinction coefficient. Reduced and non- reduced SDS-PAGE (Biorad criterion Tris/Glycine/SDS, 4-20%) were used to determine purity and approximate molecular mass. Aggregation status was determined by HPLC, with detection at 280nm using a Sepax Zenix-C SEC-300, 3um, 300 A, 4.6*150mm size exclusion column and PBS running buffer. Proteins were shipped as aliquots after filter sterilization, snap frozen in liquid nitrogen. It should be noted that for the Fc tagged proteins, as in a prior round of purification, losses of protein were observed during desalting into PBS, as determined from samples run on SDS-PAGE before and after purification.
  • Strep tagged proteins protein expression vectors transfected into HEK293.sus or CHO using standard ATUM methods. Briefly, cells were grown for 7 days and harvested. Supernatant pH was adjusted with 1M Hepes pH 7.4 and sodium azide added. BioLock biotin sequestration reagent was added. StrepTactin superflow resin was used to capture proteins. Resin was washed with lOOmM Tris pH 8, 150mM NaCl, ImM EDTA.
  • Proteins were eluted with lOOmM Tris pH 8, 150mM NaCl, ImM EDTA plus 2.5mM Desthiobitin. Protein was quantified by OD280, quantity and concentration was determined using calculated extinction coefficient. Reduced and non-reduced SDS-PAGE (Biorad criterion Tris/Glycine/SDS, 4- 20%) were used to determine purity and approximate molecular mass. Aggregation status was determined by HPLC, with detection at 280nm using a Sepax Zenix-C SEC-300, 3um, 300A, 4.6* 150mm size exclusion column and PBS running buffer. Proteins were shipped as aliquots after filter sterilization, snap frozen in liquid nitrogen.
  • the expression and/or purification of the Klotho variants disclosed in Table 1, in addition to one or more other Klotho variants disclosed herein but not shown in Table 1, can, in some embodiments, result in advantages over the expression and/or purification of native Klotho. For example, there can be a reduction in the number and/or types of alternative products when expressing and/or purifying Klotho variants. Additionally, or altematively, there can be an increase in expression level of the desired Klotho variant compared to the expression level of native Klotho.
  • the desired Klotho variant is expressed and/or purified in a purer form under comparable conditions and methods (e.g., the concentration of the desired Klotho variant is increased with a concomitant decrease in side products expressed and/or purified).
  • An exemplary method of manufacturing recombinant Klotho protein comprising: producing a recombinant Klotho protein in Chinese hamster ovary (CHO) cells, preferably in dihydrofolate reductase (DHFR)-deficient CHO cells, more preferably in CHO- S cells, or preferably in glutamine synthetase (GS)-deficient CHO cells, more preferably in GS -/- CHO cells, the protein preferably having at least 85% amino acid sequence identity to one of SEQ ID NO: 2 through SEQ ID NO: 70.
  • CHO Chinese hamster ovary
  • DHFR dihydrofolate reductase
  • GS glutamine synthetase
  • the CHO cells contain an exogenous nucleic acid that encodes: a promoter, preferably a strong promoter; a polypeptide with at least 85% amino acid sequence identity to one of SEQ ID NO: 2 through SEQ ID NO: 70; and optionally, a functional dihydrofolate reductase (DHFR) enzyme or a functional glutamine synthetase (GS) enzyme, wherein producing the recombinant Klotho protein comprises expressing the polypeptide encoded by the nucleic acid.
  • a promoter preferably a strong promoter
  • a polypeptide with at least 85% amino acid sequence identity to one of SEQ ID NO: 2 through SEQ ID NO: 70 and optionally, a functional dihydrofolate reductase (DHFR) enzyme or a functional glutamine synthetase (GS) enzyme
  • DHFR dihydrofolate reductase
  • GS glutamine synthetase
  • the method of claim 4, further comprising introducing an effective amount of methotrexate (MTX) and/or methionine sulphoximine (MSX) into the liquid medium, preferably to a concentration of about 1 nM - 1 ⁇ , more preferably to a concentration of about 10 - 100 nM.
  • MTX methotrexate
  • MSX methionine sulphoximine
  • the viable CHO cells of the selected suspension culture contain at least about 2 to 10 copies, preferably at least about 10 to 20 copies, more preferably at least about 20 to 30 copies, even more preferably at least about 30 to 50 copies of the exogenous nucleic acid per cell.
  • growing the CHO cells comprises culturing the CHO cells in a bioreactor having a volume or working volume of at least 10 liters, preferably at least 25 liters, more preferably at least 50 liters, even more preferably at least 100 liters, still more preferably at least 250 liters, still more preferably at least 500 liters, still more preferably at least 1,000 liters, still more preferably at least 2,000 liters, still more preferably at least 2,500 liters, still more preferably at least 5,000 liters, still more preferably at least 10,000 liters.
  • nucleic acid comprises a transgene or cDNA, preferably having at least 85%, more preferably at least 90%, even more preferably at least 95%, still more preferably at least 98%, still more preferably at least 99%, most preferably 100% nucleic acid sequence identity to one of SEQ ID NO: 76 through SEQ ID NO: 96.
  • a cell line comprising: a plurality of Chinese hamster ovary (CHO) cells, preferably in dihydrofolate reductase (DHFR)-deficient CHO cells, more preferably in CHO-S cells, or preferably in glutamine synthetase (GS)-deficient CHO cells, more preferably in GS -/- CHO cells, the CHO cells containing an exogenous nucleic acid comprises a promoter, preferably a strong promoter, and encodes: a polypeptide, at least a portion of the polypeptide having at least 85% amino acid sequence identity to one of SEQ ID NO: 2 through SEQ ID NO: 70; and optionally, a functional dihydrofolate reductase (DHFR) enzyme or a functional glutamine synthetase (GS) enzyme.
  • DHFR dihydrofolate reductase
  • GS glutamine synthetase
  • nucleic acid encodes a polypeptide with at least 90%, preferably at least 95%, more preferably at least 98%, even more preferably at least 99%, most preferably 100% amino acid sequence identity to one of SEQ ID NO: 2 through SEQ ID NO: 70.
  • nucleic acid comprises a transgene or cDNA, preferably having at least 85%, more preferably at least 90%, even more preferably at least 95%, still more preferably at least 98%, still more preferably at least 99%, most preferably 100% nucleic acid sequence identity to one of SEQ ID NO: 76 through SEQ ID NO: 96.
  • a suspension cell culture comprising: a liquid medium, preferably a serum-free and/or animal protein component-free liquid medium, wherein the liquid medium preferably comprises a carbon source, a nitrogen source, and one or more vitamins, minerals, salts, amino acids, supplements, or additives, more preferably wherein the liquid medium lacks hypoxanthine, thymidine, and/or glutamine; and the cell line of any one of claims 14-17 growing in the liquid medium such that the CHO cells express the polypeptide encoded by the nucleic acid, the polypeptide comprising a recombinant Klotho protein.
  • liquid medium further comprises an effective amount of methotrexate (MTX) and/or methionine sulphoximine (MSX), preferably at a concentration of about 1 nM - 1 ⁇ , more preferably at a concentration of about 10 nM - 100 nM.
  • MTX methotrexate
  • MSX methionine sulphoximine
  • a recombinant Klotho protein wherein at least a portion of the protein has at least 80% amino acid sequence identity to one of SEQ ID NO: 2 through SEQ ID NO: 70.
  • a method of treating an aging-related or other condition, disease, or disorder comprising administering to a subj ect in need therefore a pharmaceutically effective amount of the recombinant Klotho protein of any one of claims 23 to 26.
  • a method of treating an aging-related or other condition, disease, or disorder comprising administering to a subject in need thereof a pharmaceutically effective amount of a soluble recombinant Klotho protein having at least 80% amino acid sequence identity to at least a subset of amino acid residues 1 -981 of human alpha Klotho isoform 1.
  • a method of treating an aging-related or other condition, disease, or disorder comprising administering to a subject in need thereof a pharmaceutically effective amount of a soluble recombinant Klotho protein having at least 80% amino acid sequence identity to one of SEQ ID NO: 2 through SEQ ID NO: 70.
  • the pharmaceutically effective amount is sufficient to: raise the serum soluble Klotho protein concentration of the subject to a predetermined level; and preferably maintain the serum soluble Klotho protein concentration of the subject at or above a predetermined threshold for a predetermined period of time.
  • the predetermined level is greater than, equal to, or between about: 50, 100, 250, 500, 750, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750,
  • the method of claim 31 further comprising one or more of: determining a serum soluble Klotho protein concentration of the subject; calculating a pharmaceutically effective amount of the protein sufficient to raise the serum soluble Klotho protein concentration of the subject to a first predetermined level, wherein the first predetermined level is preferably greater than or equal to about 1000 picograms of soluble Klotho protein per milliliter of serum; determining a rate of soluble Klotho protein decline and/or metabolism in the serum of the subject; calculating a subsequent dosage time at which the serum soluble Klotho protein concentration of the subject will be at or below a second predetermined level based on the determined rate; and calculating a subsequent dosage amount of the protein sufficient to raise the serum soluble Klotho protein concentration of the subject from the second predetermined level to the first predetermined level.
  • the method of claim 31, further comprising one or more of: introducing an exogenous nucleic acid into Chinese hamster ovary (CHO) cells, preferably via transfection, the nucleic acid preferably comprising a transgene or cDNA, the nucleic acid encoding a polypeptide with at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 98%, still more preferably at least 99%, most preferably 100% amino acid sequence identity to one of SEQ ID NO: 2 through SEQ ID NO: 70, the nucleic acid preferably having at least 85%, more preferably at least 90%, even more preferably at least 95%, still more preferably at least 98%, still more preferably at least 99%, most preferably 100% nucleic acid sequence identity to one of SEQ ID NO: 76 through SEQ ID NO: 96; growing the CHO cells in a liquid medium, preferably in a serum-free and/or animal protein component-free liquid medium, wherein the liquid medium preferably comprises a
  • the predetermined period of time is at least about 6 hours, preferably at least about 12 hours, more preferably at least about 18 hours, even more preferably at least about 24 hours, still more preferably at least about 30 hours, still more preferably at least about 36 hours, still more preferably at least about 42 hours, still more preferably at least about 48 hours, still more preferably at least about 54 hours, still more preferably at least about 60 hours, still more preferably at least about 66 hours, still more preferably at least about 72 hours.
  • the aging-related or other condition, disease, or disorder comprises one or more of: frailty; bone density loss; bone mineral density loss; weight loss; muscular atrophy; muscular degeneration; decline in muscle mass; decline in muscle strength; decline in hand strength; decline in leg strength; decline in physical fitness; decline in movement; decline in freedom of movement; decline in quality of life assessment; decline in ejection fraction; decline in exercise capacity; decline in learning; decline in learning capacity; decline in memory; decline in intellectual quotient; cognitive deterioration; forgetfulness; decline in cognitive capacity; decline in cognitive function; decline in synaptic plasticity; decline in synaptic function; and cellular senescence.
  • the aging-related or other condition, disease, or disorder comprises one or more of: chronic kidney disease (CKD); polycystic kidney disease (PKD); autosomal dominant polycystic kidney disease (ADPKD); acute kidney injury (AKI); acute tubular necrosis (ATN); acute allergic interstitial nephritis (AAIN); glomerulonephritis; kidney disease; renal failure; nonoliguric renal failure; alcoholism; hyperphosphatemia; muscular dystrophy (MS); type 1 diabetes; type 2 diabetes; cardiovascular disease (CVD); cardiovascular calcification; cerebrovascular insufficiency; vascular calcification; coronary artery disease; abnormalities in blood pressure; salt-sensitive hypertension; tissue calcification; calcific atherosclerotic plaque burden; calcinosis; familial tumoral calcinosis; cancer; one or more tumors; myelin-related diseases; demyelinating diseases; neurodegenerative disease; neurovascular diseases; progressive
  • the one or more additional active ingredients are selected from the group consisting of a drug, antibody, hormone, radiocontrast agent, medicament, natural compound, synthetic compound, or pharmaceutical composition.
  • a pharmaceutical composition comprising: a pharmaceutically effective amount of the recombinant Klotho protein of any one of claims 23-25; and a pharmaceutically - acceptable carrier.
  • a pharmaceutical composition comprising: a pharmaceutically effective amount of a recombinant soluble Klotho protein, at least a portion of the protein having at least 85% amino acid sequence identity to: at least a subset of amino acid residues 1-981 , 29-981, 34- 981, 36-981 , 131 -981, 1-549, 29-549, 34-549, 36-549, or 131-549 of human alpha Klotho isoform 1 ; or at least a portion of one of SEQ ID NO: 2 through SEQ ID NO: 70; and a pharmaceutically-acceptable carrier.
  • composition of claim 46 or 47 wherein at least a portion of the protein having at least 85%, preferably at least 88%, more preferably at least 90%, even more preferably at least 92%, still more preferably at least 95%, still more preferably at least 98%, still more preferably at least 99%, most preferably 100% amino acid sequence identity to at least a portion of one of SEQ ID NO: 2 through SEQ ID NO: 70.
  • any one of claims 46 to 49 for use in treating an aging-related or other condition, disease, or disorder comprising one or more of: frailty; bone density loss; bone mineral density loss; weight loss; muscular atrophy; muscular degeneration; decline in muscle mass; decline in muscle strength; decline in hand strength; decline in leg strength; decline in physical fitness; decline in movement; decline in freedom of movement; decline in quality of life assessment; decline in ej ection fraction; decline in exercise capacity; decline in learning; decline in learning capacity; decline in memory; decline in intellectual quotient; cognitive deterioration; forgetfulness; decline in cognitive capacity; decline in cognitive function; decline in synaptic plasticity; decline in synaptic function; cellular senescence; chronic kidney disease (CKD); polycystic kidney disease (PKD); autosomal dominant polycystic kidney disease (ADPKD); acute kidney injury (AKI); acute tubular necrosis (ATN); acute allergic interstitial nephritis (AAIN); glomerulonephritis
  • Pompe disease Niemann-Pick disease; microgliosis; Farber disease (FD); bone mass diseases; osteoporosis; osteopenia; osteopenia (particularly loss of BMD of cortical bone); pulmonary emphysema; pulmonary fibrosis; skin atrophy; thymic atrophy; accumulation of renal interstitial matrix; glomerulosclerosis; anemia; albuminuria; proteinuria; infertility;
  • ALS amyotrophic lateral sclerosis
  • MND motor neuron disease
  • COPD chronic obstructive pulmonary disease
  • fibromyalgia adult onset diabetes; arthritis; rheumatoid arthritis; osteoarthritis; glaucoma; cataracts; macular degeneration; multiple sclerosis (MS); lupus; ulcerative co
  • a method of treating or preventing acute kidney injury (AKI) or other condition comprising: administering to a subject in need therefore a pharmaceutically effective amount of a recombinant Klotho protein, at least a portion of the protein having at least 85%, 86%, 88%, 90%, 92%, 95%, 98%, 99%, or preferably 100% amino acid sequence identity to: at least a subset of amino acid residues 1-981, 29-981 , 34-981 , 36-981, 131-981, 1-549, 29-
  • the one or more additional active ingredients are selected from the group consisting of a drug, antibody, hormone, radiocontrast, medicament, or composition.
  • ATN acute tubular necrosis
  • AAIN acute allergic interstitial nephritis
  • AAIN glomerulonephritis
  • nephrotoxicity or low blood pressure.
  • nephrotoxicity comprises drug-induced nephrotoxicity.
  • step of administering comprises one or more steps select from the group consisting of: determining a serum soluble Klotho level in the subject; calculating a first dosage of the protein sufficient to raise the serum soluble Klotho level in the subject to a predetermined level or percent of normal levels; administering the first dosage of protein to the subject, preferably by bolus or gradual administration, more preferably by injection; determining a rate of soluble Klotho decline in the serum of the subject, preferably following administration of the first dosage; calculating a time and/or amount of a subsequent dosage of the protein; and administering the subsequent dosage of the protein to the subj ect in accordance with the calculated time and/or amount.
  • step of administering is sufficient to raise and/or maintain a serum soluble Klotho protein concentration of the subject at or above a predetermined level or threshold, optionally for a predetermined period of time.
  • the predetermined level or threshold is greater than, equal to, and/or between about: 50, 100, 250, 500, 750, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 1 1,000, 12,000, 13,000, 14,000, 15,000 20,000, 25,000, 30,000 40,000, 50,000, 75,000, or 100,000 picograms of soluble Klotho protein per milliliter of serum; or 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, 1200%, 1500%, 2000%, 2500%, 3000%, 4000%, or 5000%greater than typical healthy levels of soluble Klotho protein in serum.
  • the predetermined period of time is greater than or equal to about 6 hours, 12 hours, 18 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 12 days, 14 days, 21 days, 30 days, 45 days, 60 days, 90 days, 120 days, 6 months, 9 months, 1 year, 2 years, 3 years, 4 years, or 5 years.
  • the nephrotoxin comprises: one or more aminoglycosides, preferably selected from the group consisting of paromomycin, tobramycin, gentamicin, amikacin, kanamycin, and neomycin; one or more anti-fungal agents, preferably selected from the group consisting of amphotericin B and flucytosine; one or more contrast agents, preferably selected from the group consisting of iodinated radiocontrast media, high- osmolality contrast media (HOCM) having an iodine to molecule ratio of about 1.5 : 1, low- osmolality, nonionic contrast media (LOCM) having an iodine to molecule ratio of about 3 : 1, and isosmolar (isoosmolality) contrast media (IOCM) having an iodine to molecule ratio of about 6 : 1); one or more antiretroviral agents, preferably selected from the group consisting of adef
  • a method of treating an aging individual, the aging individual having a homozygous or heterozygous mutation in a gene encoding Klotho protein comprising: administering a therapeutic concentration of a polypeptide having at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 98%, still more preferably at least 99%, most preferably 100% amino acid sequence identity to one of SEQ ID NO: 2 through SEQ ID NO: 70.
  • any steps recited in any method or process described herein and/or recited in the claims can be executed in any suitable order and are not necessarily limited to the order described and/or recited, unless otherwise stated (explicitly or implicitly). Such steps can, however, also be required to be performed in a specific order or any suitable order in certain embodiments of the present disclosure.

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Abstract

L'invention concerne des protéines Klotho recombinées et des variants de celles-ci, des acides nucléiques codant pour celles-ci, des lignées cellulaires et des cultures en suspension exprimant celles-ci, ainsi qu'un procédé de fabrication et d'administration de celles-ci. Les protéines comprennent des éléments permettant de prolonger la solubilité ou la demi-vie comme des marqueurs de glycosylation et de protéines de fusion. Les protéines présentent au moins 85 % d'identité de séquences d'acides aminés avec une partie de l'isoforme 1 de l'alpha-Klotho humaine. Les protocoles de traitement comprennent la détermination du taux de Klotho soluble dans le sérum chez un sujet, le calcul d'une dose de la protéine qui soit suffisante pour accroître le taux de Klotho soluble dans le sérum chez le sujet jusqu'à un taux prédéfini, l'administration au sujet de la dose de protéine, tel que par une seule injection ou une injection progressive, la détermination d'une vitesse de diminution du taux de protéine Klotho dans le sérum du sujet après l'administration de la première dose, le calcul d'un instant et d'une quantité d'une dose suivante de la protéine Klotho, et l'administration au sujet de la dose suivante de la protéine Klotho.
PCT/US2017/035755 2016-06-02 2017-06-02 Protéines klotho recombinées thérapeutiques et compositions et méthodes comprenant celles-ci WO2017210607A1 (fr)

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CN201780034374.7A CN109219663A (zh) 2016-06-02 2017-06-02 治疗性重组klotho蛋白及其组合物和方法
JP2019515786A JP2019526272A (ja) 2016-06-02 2017-06-02 治療用組換えクロトータンパク質ならびにそれを含む組成物および方法
MX2018014753A MX2018014753A (es) 2016-06-02 2017-06-02 Proteinas klotho recombinantes terapeuticas y composiciones y metodos que involucran a las mismas.
CN202310383190.9A CN116478907A (zh) 2016-06-02 2017-06-02 治疗性重组klotho蛋白及其组合物和方法
AU2017272349A AU2017272349B2 (en) 2016-06-02 2017-06-02 Therapeutic recombinant klotho proteins and compositions and methods involving the same
EP17807601.4A EP3464608A4 (fr) 2016-06-02 2017-06-02 Protéines klotho recombinées thérapeutiques et compositions et méthodes comprenant celles-ci
KR1020187034860A KR102570250B1 (ko) 2016-06-02 2017-06-02 치료학적 재조합 클로토 단백질 및 이를 수반하는 조성물 및 방법
CA3025461A CA3025461A1 (fr) 2016-06-02 2017-06-02 Proteines klotho recombinees therapeutiques et compositions et methodes comprenant celles-ci
KR1020237028237A KR20230125857A (ko) 2016-06-02 2017-06-02 치료학적 재조합 클로토 단백질 및 이를 수반하는 조성물및 방법
BR112018073909-0A BR112018073909A2 (pt) 2016-06-02 2017-06-02 proteínas klotho recombinantes terapêuticas e composições e métodos envolvendo as mesmas
US16/208,552 US20190169593A1 (en) 2016-06-02 2018-12-03 Products and methods for assessing and increasing klotho protein levels
JP2023096236A JP2023123565A (ja) 2016-06-02 2023-06-12 治療用組換えクロトータンパク質ならびにそれを含む組成物および方法

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US201662401600P 2016-09-29 2016-09-29
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WO2018098375A1 (fr) * 2016-11-22 2018-05-31 Klotho Therapeutics, Inc. Nouvelles protéines klotho recombinées et compositions et méthodes les comprenant
CN108333355A (zh) * 2018-02-01 2018-07-27 黄曙 Klotho蛋白在制备诊断胃肠道间质瘤危险度的试剂中的用途
CN108384747A (zh) * 2018-03-05 2018-08-10 安徽省农业科学院园艺研究所 表达狂犬抗体的cho细胞无血清悬浮培养方法
RU2712770C1 (ru) * 2019-05-21 2020-01-31 Всеволод Викторович Мелехин Способ ингибирования роста опухолевых клеток
WO2020160617A1 (fr) * 2019-02-05 2020-08-13 Helium 3 Biotech Pty Ltd Composition de modulation de l'expression et de la distribution de protéines membranaires et son procédé d'utilisation
CN112915193A (zh) * 2021-03-05 2021-06-08 南方医科大学南方医院 Kp-1在制备治疗慢性肺病的药物中的用途
CN113409306A (zh) * 2021-07-15 2021-09-17 推想医疗科技股份有限公司 一种检测装置、训练方法、训练装置、设备和介质
CN114487218A (zh) * 2020-10-23 2022-05-13 北京红惠新医药科技有限公司 β-烟酰胺单核苷酸的分析方法
WO2022243519A1 (fr) * 2021-05-21 2022-11-24 Universitat Autònoma De Barcelona Variant d'épissage sécrété de klotho pour le traitement de troubles osseux
WO2023218445A1 (fr) * 2022-05-08 2023-11-16 Ichilov Tech Ltd. Dérivés de klotho à structure modifiée
WO2023172444A3 (fr) * 2022-03-08 2023-11-16 Mayo Foundation For Medical Education And Research Agents sénothérapeutiques et polypeptides alpha-klotho
US11891615B2 (en) 2020-06-10 2024-02-06 Gail Marion Humble Process to produce Klotho protein in vitro
WO2024115728A1 (fr) * 2022-12-02 2024-06-06 Universitat Autònoma De Barcelona Variant d'épissage sécrété de klotho pour le traitement de troubles musculaires

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IT201900007446A1 (it) 2019-05-29 2020-11-29 Giuseppe Castellano Composizione comprendente citrato e carnitina in grado di attivare la produzione della proteina klotho
CN112195165A (zh) * 2020-10-15 2021-01-08 广东药科大学 一种抗衰老分泌型Klotho蛋白及其编码基因、重组表达载体与应用
CN113444730A (zh) * 2021-03-17 2021-09-28 昆明市延安医院 一种原发性肝细胞klotho基因转导干细胞筛选构建方法
CN113215199A (zh) * 2021-04-29 2021-08-06 广州博识生物科技有限公司 一种Klotho+/-基因缺失斑马鱼

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2017363321B2 (en) * 2016-11-22 2023-09-28 Klotho Therapeutics, Inc. Novel recombinant Klotho proteins and compositions and methods involving the same
US11932676B2 (en) 2016-11-22 2024-03-19 Klotho Therapeutics, Inc. Recombinant klotho proteins and compositions and methods involving same
WO2018098375A1 (fr) * 2016-11-22 2018-05-31 Klotho Therapeutics, Inc. Nouvelles protéines klotho recombinées et compositions et méthodes les comprenant
CN108333355A (zh) * 2018-02-01 2018-07-27 黄曙 Klotho蛋白在制备诊断胃肠道间质瘤危险度的试剂中的用途
CN108333355B (zh) * 2018-02-01 2020-05-26 黄曙 Klotho蛋白在制备诊断胃肠道间质瘤危险度的试剂中的用途
CN108384747A (zh) * 2018-03-05 2018-08-10 安徽省农业科学院园艺研究所 表达狂犬抗体的cho细胞无血清悬浮培养方法
WO2020160617A1 (fr) * 2019-02-05 2020-08-13 Helium 3 Biotech Pty Ltd Composition de modulation de l'expression et de la distribution de protéines membranaires et son procédé d'utilisation
RU2712770C1 (ru) * 2019-05-21 2020-01-31 Всеволод Викторович Мелехин Способ ингибирования роста опухолевых клеток
US11891615B2 (en) 2020-06-10 2024-02-06 Gail Marion Humble Process to produce Klotho protein in vitro
CN114487218A (zh) * 2020-10-23 2022-05-13 北京红惠新医药科技有限公司 β-烟酰胺单核苷酸的分析方法
CN114487218B (zh) * 2020-10-23 2023-11-14 北京红惠新医药科技有限公司 β-烟酰胺单核苷酸的分析方法
CN112915193B (zh) * 2021-03-05 2022-09-13 南方医科大学南方医院 Kp-1在制备治疗慢性肺病的药物中的用途
CN112915193A (zh) * 2021-03-05 2021-06-08 南方医科大学南方医院 Kp-1在制备治疗慢性肺病的药物中的用途
WO2022243519A1 (fr) * 2021-05-21 2022-11-24 Universitat Autònoma De Barcelona Variant d'épissage sécrété de klotho pour le traitement de troubles osseux
CN113409306A (zh) * 2021-07-15 2021-09-17 推想医疗科技股份有限公司 一种检测装置、训练方法、训练装置、设备和介质
WO2023172444A3 (fr) * 2022-03-08 2023-11-16 Mayo Foundation For Medical Education And Research Agents sénothérapeutiques et polypeptides alpha-klotho
WO2023218445A1 (fr) * 2022-05-08 2023-11-16 Ichilov Tech Ltd. Dérivés de klotho à structure modifiée
WO2024115728A1 (fr) * 2022-12-02 2024-06-06 Universitat Autònoma De Barcelona Variant d'épissage sécrété de klotho pour le traitement de troubles musculaires

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AU2017272349A1 (en) 2018-11-22
CN116478907A (zh) 2023-07-25
EP3464608A1 (fr) 2019-04-10
AU2017272349B2 (en) 2022-12-01
CN109219663A (zh) 2019-01-15

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