Recombinant Kroto protein for therapeutic use, and compositions and methods containing the same.

Abstract

To provide recombinant Klotho proteins and variants, nucleic acids encoding the same, cell lines and suspension cultures expressing the same, and method of manufacturing and administering the same.SOLUTION: Disclosed is a cell line comprising a plurality of Chinese hamster ovary (CHO) cells, where the CHO cells contain an exogenous nucleic acid, the exogenous nucleic acid comprises a promoter and encodes a medically effective polypeptide. The polypeptide comprises an N-terminal signal peptide, a recombinant human α soluble Klotho protein and a C-terminal polypeptide tag. The polypeptide or recombinant human α soluble Klotho protein is effective in the treatment of acute kidney injury, chronic renal diseases or aging-related conditions, diseases or disorders.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] 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 comprising CGMP-grade human recombinant soluble alpha-Klotho protein or variants thereof, as well as methods for their manufacture and methods for administration to human or non-human subjects.

Background Art

[0002] Klotho (or alpha-Klotho, α-Klotho, etc.) is a recently characterized protein encoded by the KL (or Klotho) gene located on human chromosome 13. Through alternative RNA splicing, two transcripts arising from a single Klotho gene have been identified. See FIGS. 1 and 2. The first transcript is predicted to encode Klotho isoform 1, a single-pass transmembrane protein of 1,012 amino acids in total length, comprising an extracellular region or domain (human residues 1 to 981) containing a short cytoplasmic tail (human residues 1003 to 1012), a transmembrane (TM) domain (human residues 982 to 1002), and two homologous (internal repeat) domains (KL1 (human residues 56 to 506, 450 residues in length), and KL2 (referred to as human residues 515 to 953, 438 residues in length)), each having 20% to 40% amino acid sequence homology with β-glucosidase but lacking glucosidase catalytic activity), and a signal sequence (SS) domain (human residues 1 to 33). The extracellular region containing the SS, KL1, and KL2 domains (human residues 1 to 981) can be enzymatically cleaved by α / β-secretase and released into the circulation as a 130 kDa circulating protein called soluble Klotho (or s-Klotho, s-Klotho, alpha-soluble Klotho, etc.). The extracellular region can also be cleaved into separate 68 kDa protein (KL1+SS) and 64 kDa protein (KL2).

[0003] The second transcript, a splicing variant of alpha-kroto mRNA, encodes a second isoform of the kroto protein, primarily corresponding to the KL1 domain. The internal splice donor site is thought to be located in exon 3 of the kroto gene. The resulting alternatively spliced ​​transcript contains a 50 bp insertion after exon 3, with an in-frame translation termination codon at its end (Figure 1; gray). The expressed protein product is secreted into circulation and is referred to as secretory kroto (or kroto isoform 2). Thus, depending on gene expression, RNA splicing, and enzymatic cleavage, many different kroto proteins can exist in circulation at any given time. Despite the presence of various forms of alpha-kroto protein, only the full-length membrane-bound isoform 1 complexes with the fibroblast growth factor (FGF) receptor, inducing phosphate excretion into the urine and P i It is also known to function as an essential co-receptor for FGF23, a bone-derived hormone that plays a regulatory role in vitamin D metabolism.

[0004] Klotho is highly expressed in the kidneys and brain, and less so in other organs, and can also be found in mammalian cerebrospinal fluid and urine. The circulating level of soluble klotho protein in mammals is thought to decrease with age. In addition, klotho-deficient mice exhibit an accelerated aging phenotype, while klotho overexpression in mice extends lifespan. Furthermore, klotho is involved in many cellular processes related to aging. From the above perspective, it has been hypothesized that soluble klotho may function as an anti-aging compound in the human body.

[0005] Aging is an inevitable, progressive biological process that leads to the dysfunction and destruction of almost all tissues and organs, ultimately resulting in death. For example, aging in the human body is associated with a decline in cellular function that can lead to the development of various diseases. Aging is thought to be caused by a tightly controlled, complex interaction between genetic and acquired factors, and is typically characterized by increased aging, a quantitative and qualitative decrease in stem cells, and abnormal structures at the tissue level.

[0006] As the so-called "baby boomer" generation ages, the elderly population (e.g., 60-65 years old) is rapidly increasing globally. The growing healthcare demand for this elderly population is a significant economic burden on all healthcare systems. Recombinant klotho protein may offer promising therapeutics to combat age-related health conditions. The development of strategies and insurance interventions based on the production and purification (e.g., to a substantially homogeneous form) of soluble klotho, as well as the administration of this protein to subjects within the growing elderly population, could help alleviate this situation and the problems associated with it.

[0007] Currently, there are no exogenous forms of human klotho protein, such as recombinant soluble human alpha-klotho protein or protein variants, that provide a protein compliant with Current Good Manufacturing Practice (CGMP) regulations, particularly as determined and enforced by the U.S. Food and Drug Administration (FDA), either alone or in combination with one or more additional active ingredients. Developing strategies and insurance interventions based on administering recombinant S-klotho to subjects, especially within the growing elderly population, could help improve this situation. [Overview of the Initiative]

[0008] Embodiments of the present disclosure address one or more of the aforementioned or other problems in the art by using recombinant human kroto protein, protein fragments and / or protein variants, expression nucleic acid constructs and / or vectors, cell lines and / or cell suspension cultures, as well as methods for producing, purifying, and administering the same to (human or non-human animal) subjects.

[0009] For example, some embodiments of this disclosure are Recombinant human alpha-soluble Kroto protein, protein fragments, and / or protein variants, Recombinant human alpha-soluble klotho protein as a pharmaceutical (or therapeutic composition - e.g., formulation), A composition comprising recombinant human alpha-soluble kroto protein and at least one additional (active) component, A nucleic acid construct or vector encoding recombinant human alpha-soluble kroto protein, (i) a nucleic acid construct or vector encoding recombinant human alpha-soluble kroto protein, and / or (ii) a cell line expressing recombinant human alpha-soluble kroto protein, (i) a nucleic acid construct or vector encoding recombinant human alpha-soluble kroto protein, and / or (ii) a cell suspension culture of cells expressing recombinant human alpha-soluble kroto protein, A method for producing recombinant human alpha-soluble Kroto protein, and a method for selectively purifying it, A method for producing a recombinant human alpha-soluble klotho protein pharmaceutical (or therapeutic composition, i.e., formulation), A method for administering recombinant human alpha-soluble kroto protein to a subject (human or non-human animal), A diagnostic method for measuring Kroto protein deficiency in a subject, A method for diagnosing Kroto protein deficiency in subjects, A method for diagnosing whether a subject needs to be administered recombinant human alpha-soluble klotho protein, A method for evaluating the effectiveness of a protein to a subject requiring it, and / or a method for measuring the effective dosage, Recombinant human alpha-soluble kroto protein for use in the treatment of specific medical conditions or other pathological conditions, and / or This may include the use of recombinant human alpha-soluble kroto protein in the manufacture of pharmaceuticals for the treatment of specific medical conditions or other pathological conditions.

[0010] Some embodiments may include a method for producing recombinant Kroto protein, comprising producing recombinant Kroto 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 synthase (GS)-deficient CHO cells, more preferably in GS- / -CHO cells, wherein the protein preferably has at least 85% amino acid sequence identity with one of SEQ ID NOs: 2 to 70.

[0011] Some embodiments 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 synthase (GS)-deficient cells, more preferably in GS- / -CHO cells, wherein the CHO cells comprise a promoter, preferably a strong promoter, and a polypeptide, wherein at least a portion of the polypeptide has at least 85% amino acid sequence identity with one of SEQ ID NOs: 2 to 70, and optionally, an exogenous nucleic acid encoding a functional dihydrofolate reductase (DHFR) enzyme or a functional glutamine synthase (GS) enzyme.

[0012] Some embodiments include a liquid medium, preferably serum-free and / or free of animal protein components, preferably comprising a carbon source, a nitrogen source, and one or more vitamins, minerals, salts, amino acids, supplements, or additives, more preferably comprising a liquid medium lacking hypoxanthine, thymidine, and / or glutamine, and a cell line according to any one of claims 14 to 17, wherein CHO cells are grown in the liquid medium such that they express nucleic acid-encoded polypeptides, the polypeptide comprising recombinant Kroto protein.

[0013] Some embodiments may include recombinant Kroto protein, in which at least a portion of the protein has at least 80% amino acid sequence identity with one of SEQ ID NOs: 2 to 70.

[0014] Some embodiments may include a method for treating age-related or other pathological conditions, diseases, or disorders, comprising administering a pharmaceutically effective amount of recombinant klotho protein described herein to a subject in need thereof.

[0015] Some embodiments may include a method for treating age-related or other pathological conditions, diseases, or disorders, comprising administering to a subject in need a pharmaceutically effective amount of soluble recombinant kroto protein having at least 80% amino acid sequence identity with at least a subset of amino acid residues 1-981 of human alpha kroto isoform 1.

[0016] Some embodiments may include a method for treating an age-related or other pathological condition, disease, or disorder, comprising administering to a subject in need a pharmaceutically effective amount of one of SEQ ID NOs: 2 to 70 and a soluble recombinant Kroto protein having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity.

[0017] Some embodiments may include a pharmaceutical composition comprising a pharmaceutically effective amount of recombinant Kroto protein described herein and a pharmaceutically acceptable carrier. Some embodiments may include a pharmaceutical composition comprising a pharmaceutically effective amount of recombinant soluble kroto protein, wherein at least a portion of the protein has at least 85% amino acid sequence identity with 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 kroto isoform 1, or at least a portion of one of SEQ ID NOs. 2 to SEQ ID NOs. 70, and a pharmaceutically acceptable carrier.

[0018] Some embodiments may include a method for treating or preventing acute kidney injury (AKI) or other pathological conditions, comprising administering a pharmaceutically effective amount of recombinant soluble kroto protein to a subject in need thereof, wherein at least a portion of the protein has at least 85%, 86%, 88%, 90%, 92%, 95%, 98%, 99%, or preferably 100% amino acid sequence identity with 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 kroto isoform 1, or at least a portion of one of sequence numbers 2-70.

[0019] Some embodiments include compositions comprising therapeutic kroto protein, such as CGMP-grade human recombinant soluble alpha-kroto protein, and at least one other active ingredient, such as a drug, antibody, hormone, human cell, tissue, or cell or tissue system product (HCT / Ps), and / or methods for administering them to human or non-human subjects. Combinatorial compositions and methods may be useful for treating subjects suffering from age-related disorders or conditions, metabolic disorders, chronic diseases, acute injuries, etc. Prophylactic administration of combination therapy to subjects without apparent conditions or disorders may also be useful for delaying or preventing certain conditions or disorders described herein.

[0020] Some embodiments may include nucleic acids or nucleic acid constructs. For example, embodiments may include expression vectors or nucleic acids. Nucleic acids may encode recombinant human alpha-soluble Kroto protein, protein fragments, or protein variants. Nucleic acids may encode native or non-native signaling sequences. For example, nucleic acids may encode a non-native signaling sequence upstream (or N-terminus) of the encoded Kroto protein sequence.

[0021] Some embodiments may include a method for producing recombinant human alpha-soluble Kroto protein. The method may include growing Chinese hamster ovary (CHO) cells in a liquid medium, producing recombinant soluble Kroto protein in the CHO cells, and / or purifying a recombinant soluble Kroto protein-containing extract from the CHO cells, the liquid medium, or both. The extract may contain at least about 98% by dry weight of recombinant soluble Kroto protein and / or about 1–100 ppm of CHO host cell protein (HCP). CHO cells may be dihydrofolate reductase (DHFR)-deficient CHO cells, such as CHO-S cells, or glutamine synthase (GS)-deficient CHO cells, such as GS- / -CHO cells. The produced (expressed) protein may be released (e.g., secreted) from the CHO cells into the liquid medium and / or may have one or more glycans bound to it.

[0022] CHO cells may contain one or more exogenous nucleic acids encoding a protein and optionally functional enzymes such as dihydrofolate reductase, glutamine synthetase (GS) enzyme. The exogenous nucleic acid may include a promoter (e.g., a strong promoter, a weak promoter, etc.) such as a promoter customarily or typically used for the expression of exogenous proteins in CHO cells. The exogenous nucleic acid preferably has at least 85% nucleic acid sequence identity with one of SEQ ID NO: 76 to SEQ ID NO: 96, or any other suitable nucleic acid sequence encoding the clotting protein described herein (e.g., S-clotting variant), and may include a transgene or cDNA (e.g., under the control of a promoter).

[0023] The method may include introducing the exogenous nucleic acid into CHO cells by transfection or the like. The method may include growing CHO cells in a liquid medium such as a medium free of (human, bovine (fetal), or other) serum and / or free of animal (or animal-derived) protein (components). The medium preferably contains a carbon source, a nitrogen source, and / or one or more vitamins, minerals, salts, amino acids, supplements, or additives, preferably within a bioreactor. Depending on the particular CHO cell line, the method may include introducing an effective amount of methotrexate (MTX), methionine sulfoximine (MSX), or other agent into the liquid medium, and / or selecting a suspension culture of viable CHO cells growing in the liquid medium (e.g., by CHO cell subcloning, limiting dilution, fluorescence-activated cell sorting (FACS), etc.).

[0024] In some embodiments, selection and / or gene amplification can be performed by culturing the transfected cells in a selective medium such as a medium lacking hypoxanthine and / or thymidine (e.g., -HT medium), glutamine, and the like. In at least one embodiment, a low concentration of MTX is added or used to amplify the transfected nucleic acid (or its gene), thereby selecting (e.g., in DHFR-deficient CHO cells transfected with a DHFR transgene) to increase protein expression. Alternatively (or in addition), selection and / or gene amplification can be performed by adding MSX ((endogenous) glutamine synthetase (GS) inhibitor) to a suspension culture of CHO cells having at least one (exogenous) glutamine synthetase (GS) transgene.

[0025] The method can include subculturing viable cells or cultures (e.g., MTX-resistant and / or MSX-resistant cells or cultures). The selected suspension culture and / or the selected CHO cells can have or exhibit an increase in protein production (e.g., by CHO cells), an increase in the concentration of the protein (e.g., into a liquid medium), and / or an increase in the copy number of the exogenous nucleic acid (e.g., per cell) (e.g., as compared to an unselected suspension culture or CHO cells).

[0026] Certain embodiments may include a cell line comprising multiple CHO cells. For example, the CHO cells may be DHFR-deficient CHO cells such as CHO-S cells. The CHO cells may contain one or more exogenous nucleic acids (including transgenes or cDNAs) encoding polypeptides having at least 85% amino acid sequence identity with one of SEQ ID NOs: 2 to 70. The polypeptide may include human recombinant alpha-soluble Kroto protein. The exogenous nucleic acid may preferably include a transgene or cDNA having at least 85% nucleic acid sequence identity with one of SEQ ID NOs: 76 to 96. In some embodiments, the nucleic acid may also include a promoter (related to the transgene) and / or optionally selected (exogenous) enzymes such as (functional) dihydrofolate reductase (DHFR) enzymes, glutamine synthase (GS) enzymes.

[0027] At least one embodiment includes a suspension cell culture comprising a cell line grown in a liquid medium such that CHO cells express nucleic acid-encoded polypeptides, preferably comprising a carbon source, a nitrogen source, and / or one or more vitamins, minerals, salts, amino acids, supplements, or additives. The liquid medium may be serum-free (human, bovine (fetal), or other) and / or free of animal (or animal-derived) proteins (components). For example, the liquid medium may be free of bovine serum albumin, human serum albumin, etc.

[0028] In some embodiments, the liquid medium may also contain an effective amount of MTX and / or MSX. The suspension culture (or its CHO cells) may exhibit increased protein production (e.g., by CHO cells), increased protein concentration (e.g., in the liquid medium), secretion of protein (e.g., into the liquid medium), and / or may have (or be selected to have) an increased copy number of exogenous nucleic acids (e.g., per cell) compared to suspension cultures that are not preferably selected. The protein may have one or more glycans bound to it.

[0029] Some embodiments include an extract of CHO cells, liquid medium, or both (e.g., a suspension cell culture), or an extract thereof, which contains a recombinant protein having at least 85% amino acid sequence identity with one of SEQ ID NOs: 2 to 70. Certain embodiments include an extract containing human recombinant alpha-soluble Kroto protein from or derived from CHO cells, liquid medium, or both (e.g., a suspension cell culture). At least one embodiment includes an isolated recombinant protein having at least 85% amino acid sequence identity with one of SEQ ID NOs: 2 to 70.

[0030] Some embodiments may include methods for administering recombinant human alpha-soluble Kroto protein to human or non-human animal subjects in need. Subjects to whom Kroto protein is administered may be suffering from or at risk of various pathological conditions (e.g., disabilities, diseases, injuries, illnesses, etc.). For example, some embodiments may include methods for treating one or more chronic diseases and / or age-related conditions, such as (human) aging-related physical, intellectual, neurological, or other pathological conditions. Some embodiments may promote healing, recovery, longevity, and / or other beneficial outcomes through one or more mechanisms or actions. Embodiments may include, for example, administering a pharmaceutically effective amount of recombinant soluble Kroto protein or protein variant to subjects in need (e.g., subjects suffering from or at risk of progression of a pathological condition). Administration of such protein or protein variant may have positive therapeutic effects and characteristics on the course and outcomes of pathological conditions, including chronic and / or age-related diseases and longevity, in human subjects.

[0031] A pharmaceutically effective dose may be sufficient to raise the subject's serum soluble Kroto protein concentration to a predetermined level, such as approximately 50 to 3000 picograms or more per milliliter of serum. The amount may also be sufficient to maintain the subject's serum soluble Kroto protein concentration above a predetermined threshold for a predetermined period. Embodiments may also include administering the protein to a subject requiring it in order to maintain the subject's serum soluble Kroto protein concentration above a predetermined threshold for a predetermined period.

[0032] Embodiments may also include measuring the serum soluble Kroto protein concentration of a subject, calculating a pharmaceutically effective dose, measuring the rate of decrease of soluble Kroto protein in the subject's serum, calculating the next dosing time at which the subject's serum soluble Kroto protein concentration will fall below a second predetermined level based on the measured rate, calculating the next dose of protein sufficient to raise the subject's serum soluble Kroto protein concentration from the second predetermined level to the first predetermined level, and / or administering the next dose of protein to the subject.

[0033] The protein may modulate (or be effective in modulating) the IGF-1 and / or Wnt signaling pathway, exhibit β-glucuronidase and / or sialidase activity, inhibit the p53 / p21 signaling pathway, and / or preferably reduce H2O2-induced cellular senescence and apoptosis through inhibition of the p53 / p21 signaling pathway. The protein may preferably exhibit pleiotropic activity and / or may function as a humoral factor in regulating oxidative stress, growth factor signaling, ion homeostasis, and / or the 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 receptors.

[0034] Proteins may also be effective in treating one or more age-related conditions (or (human) age-related conditions) such as weakness, bone density loss or decreased bone mineral density, weight loss, muscle atrophy or degeneration, decreased muscle mass, decreased muscle strength, grip strength, leg strength or physical strength, decreased movement, mobility, quality of life assessment, ejection fraction, decreased motor ability, decreased learning ability, memory or intelligence quotient, cognitive deterioration or amnesia, decreased cognitive ability or function, decreased synaptic plasticity or synaptic function, and cellular senescence.

[0035] Proteins may also be effective in treating one or more age-related conditions (or age-related conditions in humans) 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.

[0036] Accordingly, embodiments may also include compositions for use in the treatment of one or more age-related conditions. The compositions may comprise recombinant soluble kroto protein (for example, having at least 85% amino acid sequence identity with one of SEQ ID NOs. 2 to 70) and a pharmaceutically acceptable carrier.

[0037] Some embodiments include compositions comprising therapeutic kroto protein, such as CGMP-grade human recombinant soluble alpha-kroto protein, and at least one other active ingredient, such as a drug, antibody, hormone, human cell, tissue, or cell or tissue system product (HCT / Ps), as well as methods for administering them to human or non-human subjects. Combinatorial compositions and methods may be useful for treating subjects suffering from age-related disorders or conditions, metabolic disorders, chronic diseases, acute injuries, etc. Prophylactic administration of combination therapy to subjects without apparent conditions or disorders may also be useful for delaying or preventing certain conditions or disorders described herein.

[0038] In various embodiments of this disclosure, regardless of the product or process, the recombinant Kroto protein may include one Kroto protein having a sequence having 80% to 100% sequence identity with one of SEQ ID NOs: 1 to 38, preferably a C-terminal tag having 80% to 100% sequence identity with one of SEQ ID NOs: 74 or 75, and optionally a linker sequence having 80% to 100% sequence identity with SEQ ID NOs: 73 positioned between them. The protein may optionally include, or be expressed using, a signaling sequence having 80% to 100% sequence identity with SEQ ID NOs: 71 or 72. Preferably, the protein (to be manufactured, produced, expressed or administered) has 80% to 100% sequence identity with one of SEQ ID NOs: 39 to 70.

[0039] An exemplary method for producing recombinant Kroto protein comprises producing recombinant Kroto 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 synthase (GS)-deficient CHO cells, more preferably in GS- / -CHO cells, wherein the protein preferably has at least 85% amino acid sequence identity with one of SEQ ID NOs: 2 to SEQ ID NOs: 70.

[0040] An exemplary cell line comprises a cell line containing 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 synthase (GS)-deficient (CHO) cells, more preferably in GS- / -CHO cells, wherein the CHO cells containing exogenous nucleic acids comprise a promoter, preferably a strong promoter, and encode a polypeptide, wherein at least a portion of the polypeptide has at least 85% amino acid sequence identity with one of SEQ ID NOs: 2 to 70, and optionally encode a functional dihydrofolate reductase (DHFR) enzyme or a functional glutamine synthase (GS) enzyme.

[0041] An exemplary suspension cell culture comprises a liquid medium, preferably serum-free and / or free of animal protein components, preferably comprising a carbon source, a nitrogen source, and one or more vitamins, minerals, salts, amino acids, supplements, or additives, more preferably comprising a liquid medium lacking hypoxanthine, thymidine, and / or glutamine, and a cell line according to any one of claims 14 to 17, wherein CHO cells are grown in the liquid medium such that they express nucleic acid-encoded polypeptides, the polypeptide comprising recombinant Kroto protein.

[0042] The exemplary recombinant Kroto protein contains at least 80% amino acid sequence identity with one of SEQ ID NOs: 2 to 70. An exemplary method for treating age-related or other pathological conditions, diseases, or disorders, comprising administering to a subject in need of such treatment a pharmaceutically effective amount of soluble recombinant kroto protein having at least 80% amino acid sequence identity with at least a subset of amino acid residues 1-981 of human alpha kroto isoform 1.

[0043] An exemplary method for treating an age-related or other pathological condition, disease, or disorder, comprising administering to a subject in need of such treatment a pharmaceutically effective amount of a soluble recombinant kroto protein having at least 80% amino acid sequence identity with one of SEQ ID NOs: 2 to SEQ ID NOs: 70.

[0044] An exemplary pharmaceutical composition is a pharmaceutically effective amount of recombinant soluble kroto protein comprising at least a portion of the protein having 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 kroto isoform 1, or at least a portion of one of sequence numbers 2-70, a protein having at least 85% amino acid sequence identity, and a pharmaceutically acceptable carrier.

[0045] An exemplary method for treating or preventing acute kidney injury (AKI) or other pathological conditions comprises administering a pharmaceutically effective amount of recombinant soluble kroto protein to a subject in need thereof, wherein at least a portion of the protein has at least 85%, 86%, 88%, 90%, 92%, 95%, 98%, 99%, or preferably 100%, amino acid sequence identity with 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 kroto isoform 1, or at least a portion of one of sequence numbers 2-70.

[0046] An exemplary method for treating an aged individual, wherein the aged individual has a homozygous or heterozygous mutation in the gene encoding the Kroto protein. The method comprises administering a therapeutic concentration of 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%, and most preferably 100% amino acid sequence identity with one of SEQ ID NOs: 2 to 70.

[0047] Some embodiments may include any of the features, options, and / or possibilities described elsewhere in this disclosure, including other aspects or embodiments of this disclosure. It should also be noted that each of the aforementioned, hereafter, and / or other features described herein also represents a distinct embodiment of this disclosure. Furthermore, any combination of two or more such features also represents a distinct embodiment of this disclosure. Such features or embodiments may also be combined in any preferred combination and / or order without departing from the scope of this disclosure. Thus, each of the features described herein may be combined with any one or more other features described herein in any preferred combination and / or order. Therefore, this disclosure is not limited to any particular combination of the exemplary embodiments described herein in detail.

[0048] Additional features and advantages of the exemplary embodiments of this disclosure are described below, some of which will become apparent from the description or may be acquired by carrying out such exemplary embodiments. The features and advantages of such embodiments may be realized and acquired by the equipment and combinations specifically indicated in the appended claims. These and other features will become more fully apparent from the following description and the appended claims or may be acquired by carrying out such exemplary embodiments as described below.

[0049] To illustrate the methods enumerated above and from which other advantages and features may be obtained, a more specific description of the embodiments briefly described above will be provided by reference to the particular embodiments shown in the accompanying drawings. For better understanding, similar elements are denoted by the same reference numerals throughout the drawings. This disclosure will be described and explained with additional specificities and details through the use of the accompanying drawings, with the understanding that these drawings show only typical embodiments of this disclosure and are therefore not intended to limit its scope. [Brief explanation of the drawing]

[0050] [Figure 1] This is a schematic diagram illustrating the cellular production of various kroto proteins according to one embodiment of the present disclosure. [Figure 2A] The schematic structures of human α-Kroto isoform 1 and isoform 2 are shown, along with the locations of the epitopes for antibody binding used in the generation of C-D (residues 800-900). [Figure 2B] The full-length α-Kroto protein sequence of 1012 amino acids is shown, including KL1 and KL2, indicated in red and green respectively, as well as the highlighted (black) TM. [Figure 2C] This shows the Western blot analysis of human cell lysates. [Figure 2D] This shows a Western blot analysis of human tissue. [Figure 3A] This shows the number of adult patients who received a specific aminoglycoside in 2010. [Figure 3B] Figure 3A shows the age distribution of adult patients who were administered aminoglycosides. [Figure 4] Figure 3A shows treatment data collected from adult patients who were administered aminoglycosides. [Modes for carrying out the invention]

[0051] Before describing in detail the various embodiments of this disclosure, it should be understood that this disclosure is not limited to descriptions of specific parameters, wording, and specific illustrated systems, methods, and / or products that may vary in each embodiment. Therefore, while certain embodiments of this disclosure will be described in detail with reference to specific features (e.g., configuration, parameters, characteristics, steps, components, ingredients, members, elements, parts, and / or parts), the descriptions are illustrative and should not be construed as limiting the scope of the inventions described herein and / or claimed. Furthermore, the terms used herein are for illustrative purposes only and are not necessarily intended to limit the scope of the inventions described herein and / or claimed.

[0052] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to whom this disclosure relates. Various aspects of this disclosure, including systems, methods, and / or products, may be illustrated by reference to one or more essentially exemplary embodiments. Where used herein, the term “embodiment” means “serving as an example, case, or illustration” and should not necessarily be construed as being preferable or advantageous to other aspects disclosed herein. In addition, references to “embodiments” in this disclosure or to the present invention are intended to provide exemplary examples without limiting the scope of the invention as set forth by the appended claims.

[0053] As used herein and in the appended claims, the singular “one” and “the said” and “it” are, unless otherwise explicitly indicated, considered, included, and specifically disclosed to be both singular and plural references, respectively. For example, a reference to “protein” considers and specifically discloses one and multiple (e.g., two or more, three or more, etc.) proteins. Similarly, the use of plural references does not necessarily require multiple such references, but unless otherwise explicitly indicated, considers, included, specifically discloses, and / or provides singular and plural references to such references.

[0054] As used throughout this disclosure, the words “may” and “may” are used in a permissive sense (i.e., meaning to have a possibility) rather than a mandatory sense (i.e., meaning to have a requirement). Furthermore, the terms “include,” “have,” “contain,” “characterize,” and their variations (e.g., “include,” “have,” “contain,” etc.), as well as similar terms used herein, including in the claims, are inclusive and / or modifiable, and exemplarily, the words “include” and its variations (e.g., “contain”) have the same meaning and do not exclude additional unlisted elements or method steps.

[0055] For the sake of brevity, this disclosure may enumerate only lists or ranges of numerical values. However, where such lists or ranges of numerical values ​​(e.g., greater than, less than, maximum, minimum, and / or about a particular value, and / or between two enumerated values) are disclosed or enumerated, it will be understood that any particular value or range of values ​​within the disclosed values ​​or list, or within the range of values, is also specifically opened and considered herein. Thus, a disclosure of exemplary measurements (e.g., length, width, thickness, etc.) that are about 10 units or less or between 0 and 10 units includes, exemplary, specific disclosures of measurements of any other values ​​between 0 and 10 units, including 9 units, 5 units, 1 unit, or 0 units and / or 10 units; and / or (ii) measurements in any other ranges of values ​​between 9 units and 1 unit, 8 units and 2 units, 6 units and 4 units, and / or 0 units and / or 10 units.

[0056] For ease of understanding, where possible, similar references (i.e., components and / or similar names of components) are used to indicate similar elements common to different embodiments of this disclosure. Similarly, similar components, or components having similar functions, will be provided with similar reference notation where possible. Certain languages ​​will be used in this specification to describe exemplary embodiments. Nevertheless, it will be understood that this is not intended to limit the scope of this disclosure. Rather, it will be understood that the languages ​​used to describe exemplary embodiments are merely illustrative (unless such languages ​​are explicitly stated herein as essential) and should not be construed as limiting the scope of the disclosure.

[0057] The detailed description is divided into paragraphs, but the paragraph headings and content within each paragraph are for structural purposes only and are not intended to be independent descriptions and embodiments, or to limit the scope of the description or claims. Rather, the content of each paragraph within the detailed description is intended to be read and understood as a collective whole in which elements of one paragraph relate to and / or characterize other paragraphs. Accordingly, embodiments specifically disclosed within one paragraph may also relate to and / or function as additional and / or alternative embodiments in other paragraphs having the same and / or similar products, methods, and / or terminology.

[0058] Embodiments of this disclosure include products, compositions, and / or methods for producing and / or using recombinant human kroto proteins, such as human recombinant soluble alpha-kroto proteins, protein fragments, and / or protein variants (of Current Good Manufacturing Practice (CGMP) grade).

[0059] Gene therapy may be effective in animal studies. However, the safety of gene therapy, particularly in human treatment, remains questionable. Compared to viral delivery of the Klotho gene to animals (cells), administration of exogenous and / or recombinant Klotho protein in humans may be a safer, simpler, and more direct mode of action for restoring Klotho levels (endocrine). Therefore, similar to the administration of erythropoietin or erythropoiesis-stimulating agents to treat anemia in patients with chronic kidney disease (CKD), and / or insulin to maintain normal glucose metabolism in type 1 diabetes, the administration of exogenous (human recombinant alpha-soluble) Klotho protein may become a realistic and effective option for treating aging and / or age-related disorders in the near future. For example, the administration of exogenous (human recombinant alpha-soluble) Klotho protein in humans may be an effective strategy for reversing or delaying stem cell depletion and / or mitigating age-related weakness or other pathological processes.

[0060] Preclinical data demonstrate the potential of soluble klotho protein therapy for age-related diseases and diseases associated with klotho deficiency. Epidemiological data show that soluble klotho is lower in older adults than in younger adults, and that soluble klotho levels are inversely correlated with age, indicating that aging is associated with a decline in soluble klotho.

[0061] Summary list of defined terms To aid in understanding the aforementioned scope and content, as well as the following descriptions and claims, several selected terms are defined below.

[0062] The term “pathological condition” means any disorder, disease, injury, or illness that appears or is expected to appear in a patient, as understood by those skilled in the art. Such manifestations of a pathological condition may include early, mid, or late-stage manifestations known in the art, including pre-pathological symptoms, signs, or appearances. Predictions of such a pathological condition may include, or may include, predictions, assumptions, expectations, imaginations, assumptions, and / or speculated occurrences of a pathological condition, whether found in scientific or medical evidence, risk assessments, or mere anxiety or fear.

[0063] The term “patient” refers specifically to any animal under the care of a physician, as defined herein, with a particular reference to a human being under the care of a physician or other relevant medical professional.

[0064] As used herein, the term “physician” generally refers to a physician. The term also includes, when appropriate to the context, any licensed medical professional, including oncologists, surgeons, or physician’s assistants, nurses, phlebotomists, veterinarians, and other medical professionals.

[0065] The term "cancer" refers to abnormal, typically uncontrolled, cell proliferation. As used herein, "cancer cells" include malignant cells that have abnormal, typically uncontrolled proliferation. Thus, the term cancer is a comprehensive term encompassing several distinct diseases characterized by malignant cells that proliferate in a typically uncontrolled manner.

[0066] The term “co-administration” and similar terms refer to the parallel, sequential, and / or concomitant administration of two or more components. For example, two components may be co-administered by administering each component in separate doses in parallel, simultaneously, or sequentially (e.g., separate doses separated by a period of time). The period may be very short (e.g., substantially immediately after the first dose) or longer (e.g., 1–60 seconds, 1–60 minutes, 1–24 hours, 1–7 days, 1–4 weeks, 1–12 months, etc., or any value or range of values ​​in between). Parallel or concomitant administration may include overlapping administration timeframes for two or more components, or administration of a combination product containing a mixture of two or more components.

[0067] As used herein, “nucleic acid” and similar terms refer to native or synthetic oligonucleotides or polynucleotides, whether DNA or RNA or DNA-RNA hybrids, single-stranded or double-stranded, sense or antisense. Nucleic acids may, but are not limited to, DNA, RNA, cDNA, gDNA, ssDNA, dsDNA, or any combination thereof. Nucleic acids of this disclosure may also include nucleotides or nucleic acid analogs known in the art (e.g., BrdU), and non-phosphodiester (internucleoside) bonds or backbone structures (e.g., peptide nucleic acids (PNA) or thiodiester bonds).

[0068] As used herein, the term “standard amino acid” includes alanine-ala-A; arginine-Arg-R; asparagine-asn-N; aspartic acid-asp-D; cysteine-cys-C; glutamine-gln-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-val-V.

[0069] As used herein, “codon optimized” or “codon optimization” refers to the process of modifying or altering codons in a nucleotide sequence to codons that are preferred or more closely approximate to the codon usage patterns in the organism in which the molecule is expressed. Therefore, codons can be optimized for use in a specific organism where expression is desired, based on known codon usage in that organism, for example, to improve the effectiveness of nucleic acid expression, to achieve faster translation rates and higher accuracy. The codon usage in a specific organism is known.

[0070] The coding nucleic acid molecule may be a modified wild type, or a codon-optimized sequence in which the codon is optimized for expression in a specific host cell, such as mammalian cells like CHO cells or 293 cells, or in yeast, or in plant cells, eukaryotic cells, etc.

[0071] In certain cases, nucleic acid sequences may be codon-optimized, for example, to increase the expression level of the encoded sequence. Specific codon usage depends on the host organism in which the modified polypeptide is expressed. Those skilled in the art are familiar with the optimal codons for expression in mammalian or human cells, and in bacteria or yeast, including Escherichia coli or Saccharomyces cerevisiae. For example, codon usage information is available from the Codon Usage Database, available at kazusa.or.jp.codon (see, for example, Richmond (2000) Genome Biology, 1:241; also see 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; and Sharp et al. (1991) Yeast, 657-78).

[0072] Therapeutic protein Embodiments of the present disclosure may include one or more therapeutic and / or recombinant human alpha-soluble kroto protein, protein fragments, and / or protein variants.

[0073] The protein may contain 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-kroto isoform 1. The protein may 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 with all or a subset of amino acid residues 1-1012, 1-981, 29-981, 36-981, 131-981, 1-549, 29-549, 34-549, 36-549, or 131-549 of human alpha-kroto isoform 1. For example, at least a portion of the protein may have at least 85% amino acid sequence identity with one of SEQ ID NOs. 2-70, or a combination of two or more of them. Other portions or fragments of the protein sequences described in this application are also considered herein. For example, some embodiments may include a protein having at least one preferred portion of SEQ ID NOs: 1 to 75, or a combination of two or more thereof, and at least one portion having amino acid sequence identity such as at least 85%.

[0074] Some embodiments may include proteins having one or more amino acid variants compared to human alpha-kroto isoform 1. Exemplarily, a protein may include a human C370 variant. For example, a protein may include a C370S variant, thereby including S370. In some embodiments, a protein may include something other than human F352 or F352V. In at least one embodiment, a protein may include a C370S variant, thereby including S370, preferably including F352 but not the F352V variant. A protein may include something other than H193 or H193R variants. All other standard amino acid substitutions at amino acid residues (or positions) 193, 352, and / or 370 of human alpha-kroto isoform 1 are considered and expressly disclosed herein.

[0075] Some embodiments may involve a mutation at amino acid residue 45 of human alpha-kroto isoform 1. At position 45, the residue may be valine (Val; V), phenylalanine (Phe; F), or another amino acid.

[0076] A protein may also contain one or more glycans (bound to it). For example, natural human alpha-kroto isoform 1 may have glycans bound (via glycosylation) at amino acids 106, 159, 283, 344, 604, 612 and / or 694. Thus, a protein of this disclosure or its kroto protein sequence may have one or more of the same (or similar) glycans bound there (via glycosylation) (e.g., at the same amino acid position). In a preferred embodiment, the protein contains all of the same or similar (natural) glycans bound there at the same amino acid position.

[0077] In some embodiments, the protein may include a signal peptide or signaling sequence. For example, the protein may include a native Kroto signaling sequence. The protein may include a non-native or synthetic signaling sequence. In some embodiments, the signaling sequence may be an N-terminal signaling sequence and / or upstream (or N-terminal) of the Kroto protein sequence. In other embodiments, the signaling sequence may be C-terminal or otherwise positioned. Preferably, the signaling sequence may have, include, or have at least 80%, 85%, 90%, 95%, 98%, or 99% amino acid sequence identity with the native human alpha Kroto isoform 1 signaling sequence, the native human alpha Kroto isoform 2 signaling sequence, SEQ ID NO: 71, or SEQ ID NO: 72.

[0078] In some embodiments, the protein may include an amino acid tag. The tag may be a C-terminal tag and / or downstream (or C-terminal) of the Kroto protein sequence. In other embodiments, the tag may be N-terminal or otherwise positioned. The tag may be or include an Fc fusion protein. For example, the tag may be or include an IgG1-Fc protein sequence. Preferably, the tag may have, include, or have at least 80%, 85%, 90%, 95%, 98%, or 99% amino acid sequence identity with SEQ ID NO: 74.

[0079] The tag may also be, or may include, a TEV-twinstrep protein sequence (as known, for example, in the art). Preferably, the signaling sequence may be, may include, or may have at least 80%, 85%, 90%, 95%, 98%, or 99% amino acid sequence identity with SEQ ID NO: 75.

[0080] In at least one embodiment, the tag may be cleaved from the protein. In other embodiments, the tag may be retained as part of the protein. In some embodiments, the tag may improve the solubility and / or (serum) half-life of the protein. In some embodiments, the tag may be utilized during protein purification (e.g., as part of a purification mechanism).

[0081] In some embodiments, the protein may include a linker (e.g., an amino acid linker) positioned between the Kroto protein sequence and the amino acid tag. Exemplarily, the linker may contain 1 to 40 amino acids, preferably 5 to 20 amino acids, more preferably 8 to 12 amino acids, and most preferably about 10 amino acids. In some embodiments, the linker may be or include a GS linker. Preferably, the linker may have, include, or have at least 70%, 80%, 90%, or 100% amino acid sequence identity with SEQ ID NO: 73.

[0082] In at least one embodiment, the protein may comply with CGMP regulations as determined and enforced by the U.S. Food and Drug Administration (FDA). For example, the kroto protein may be at least 95%, 96%, 97%, 98%, or 99% pure by dry weight. In some embodiments, the kroto protein sample may contain CHO host cell proteins (HCPs), nucleic acids, and / or other cellular components in parts per ten million (ppm) of about 1 to less than 100, about 100 to less than 1000, or about 1 to less than 100 ppm, or any value or range of values ​​placed in between.

[0083] Nucleic acids and expression vectors Some embodiments may include nucleic acids or nucleic acid constructs. For example, embodiments may include expression vectors or nucleic acid constructs. The nucleic acids may encode recombinant human alpha-soluble Kroto protein, protein fragments, or protein variants as described herein. In at least one embodiment, the nucleic acids may encode a Kroto protein sequence, an optional (natural or non-natural) signaling sequence (e.g., of multiple or single N-terminuses of the Kroto protein sequence), an optional linker sequence (e.g., a GS linker), and / or an amino acid tag (e.g., IgG1-Fc or TEV-twinstrep), as described herein.

[0084] In some embodiments, the nucleic acid may express a protein containing 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-kroto isoform 1. At least a portion of the protein may 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 with all or a subset of amino acid residues 1-1012, 1-981, 29-981, 36-981, 131-981, 1-549, 29-549, 34-549, 36-549, or 131-549 of human alpha-kroto isoform 1. For example, at least a portion of a protein may 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 with all or part of one of SEQ ID NOs: 1 to 75, or a combination of two or more such SEQ ID NOs: 75. In a preferred embodiment, a protein may 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 with all or part of one of SEQ ID NOs: 2 to 70.

[0085] In some embodiments, at least a portion of the nucleic acid may 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 with one of sequence numbers 76 to 96. In preferred embodiments, the nucleic acid may 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 with one of sequence numbers 76 to 96.

[0086] The nucleic acid sequences of this disclosure may also include stop codons known in the art (e.g., TGA, TAG, TAA). Cell lines and manufacturing methods One embodiment of the present disclosure may include a cell line. The cell line may include any preferred cell type, such as CHO cells, HEK cells, HL-60 cells, or other cell lines known in the art. Exemplarily, the cell line may include CHO cells (e.g., multiple CHO cells). In some embodiments, the CHO cells may each contain an exogenous nucleic acid (one or more copies). The nucleic acid may encode a polypeptide having 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 with one of SEQ ID NOs: 1 to 75, or a combination of two or more thereof, preferably one of SEQ ID NOs: 2 to 70.

[0087] The nucleic acid may contain at least one transgene or cDNA. In some embodiments, at least a portion of the nucleic acid may have 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 with one or more of sequence numbers from SEQ ID NOs: 76 to SEQ ID NOs: 101, preferably one of sequence numbers from SEQ ID NOs: 76 to SEQ ID NOs: 96. The nucleic acid may be or may contain plasmids or other (structural) forms of nucleic acids.

[0088] In some embodiments, the exogenous nucleic acid may encode a functional enzyme such as dihydrofolate reductase (DHFR) and / or glutamine synthase (GS). In at least one embodiment, the CHO cell may be or include a dihydrofolate reductase (DHFR)-deficient CHO cell, such as a CHO-S cell. The nucleic acid may include a promoter (e.g., from a weak promoter to a strong promoter, as understood by those skilled in the art). For example, in some embodiments, the nucleic acid may include one of SEQ ID NOs: 2 to 70 and a (strong) promoter associated with the transgene having nucleic acid sequence identity such as at least 85%. Thus, the transgene may be under the control of the promoter.

[0089] For convenience, CHO cells and / or cell lines are referred to throughout this disclosure. However, it should be noted that other cells, cell lines, and / or host cells (in addition to CHO cells) are also considered within the scope of this disclosure. Accordingly, references to CHO cells and / or cell lines also consider references to and / or use of other known cells, cell lines, and / or host cells.

[0090] Transfection A method for producing a CHO cell line may preferably involve introducing an exogenous nucleic acid into CHO cells via transfection or other techniques known in the art. In at least one embodiment, a serum-free growth-optimized cell suspension of a CHO cell line was used as a host cell line for insertion of a nucleic acid (plasmid) containing a promoter, a human alpha-S-Kroto transgene encoding a polypeptide having at least 85% amino acid sequence identity with one of SEQ ID NOs. 2 to 70, and a selectable (enzyme) marker. Each transgene encodes amino acids 1-981, 29-981, or 34-981 of human alpha-soluble Kroto, respectively. In certain embodiments, the transgene had a sequence corresponding to one of SEQ ID NOs. 76 to 96 (or had at least 85% nucleic acid sequence identity with one of SEQ ID NOs. 76 to 96). In DHFR-deficient CHO cell lines (such as the CHO-S cell line), the selectable (enzyme) marker was exogenous DHFR. In other CHO cell lines, the selectable (enzyme) marker was exogenous GS.

[0091] Proliferation, selection, and / or gene amplification Some embodiments may involve growing cells (e.g., transfected cells and / or CHO cells) in a solid medium and / or a liquid medium (e.g., a suspension cell culture), preferably in a serum-free and / or animal (or animal-derived) protein (component-free) medium. For example, cells may be plated on a solid growth medium for a period of time. Cells may also be grown in a suspension culture and / or a liquid medium. The liquid medium preferably contains a carbon source, a nitrogen source, and one or more vitamins, minerals, salts, amino acids, supplements, or additives. In some embodiments, the medium may also lack hypoxanthine and thymidine (HT), glutamine, etc.

[0092] In at least one embodiment, after a certain period of time (e.g., 48 hours after transfection), cells may be collected (e.g., detached), optionally centrifuged (e.g., 100 × g for 5 minutes), and / or seeded (e.g., approximately 2000 cells / well) in a 96-well adherent culture plate (e.g., containing serum-supplemented -HT and / or -glutamine medium). In certain embodiments, the medium may also contain MTX and / or MSX. Untransfected cells may be killed within 7–14 days after selection (e.g., after exposure to MTX and / or MSX in -HT and / or -glutamine medium).

[0093] In certain embodiments, CHO cells may (and may be selected to) contain at least about 2–10 copies, at least about 10–20 copies, at least about 20–30 copies, or at least about 30–50 copies of exogenous nucleic acids (e.g., per cell). Therefore, the method may involve selecting CHO cells containing at least about 2–10 copies, at least about 10–20 copies, at least about 20–30 copies, or at least about 30–50 copies of exogenous nucleic acids (e.g., per cell). For example, MTX and / or MSX may be administered to the cells (at concentrations such as about 1 nM–1 μM, about 10–100 nM, etc.) (while continuously increasing the level).

[0094] Dihydrofolate reductase (DHFR) gene amplification in DHFR-deficient CHO cells transfected with exogenous DHFR (e.g., CHO-S cell lines) was achieved by successively increasing the level of methotrexate (MTX) in the growth medium. Because the plasmid contains DHFR, exposure to MTX (10–100 nM) enables amplification of the S-Kroto gene (fragment) within the host cells. GS gene expression systems were also used to amplify CHO cells transfected with exogenous GS (e.g., upon exposure to MSX). Alternatively, GS- / - host cell lines were also used, eliminating the need for MSX. These steps resulted in the production of numerous copies of the S-Kroto gene (e.g., 10–30 copies per cell) and high levels of S-Kroto protein expression in the transgenic cell lines.

[0095] In some embodiments, the protein may be secreted from CHO cells into a liquid medium in a suspension culture. For example, certain CHO cells and / or cell lines of the present disclosure may secrete (or be selected to secrete) at concentrations 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 ​​in between (without protein concentration). In at least one embodiment, a highly productive cell line (or suspension culture) may be selected such that the concentration of human recombinant alpha-soluble Kroto protein in the medium (of a selected suspension culture or suspension culture of a selected cell line) 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, and still more preferably at least 5000 mg / L, without protein concentration.

[0096] Subcloning of colonies containing high S-Klotho-producing transgenes obtained by restricted cell dilution further produced S-Klotho-secreting CHO cell lines that secrete S-Klotho in the range of 500-2000 mg / L into cell conditioning medium. All cell constructs were restricted digested and their sequences were confirmed.

[0097] In some embodiments, 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, even more preferably at least 250 liters, even more preferably at least 500 liters, even more preferably at least 1,000 liters, even more preferably at least 2,000 liters, even more preferably at least 2,500 liters, even more preferably at least 5,000 liters, and even more preferably at least 10,000 liters.

[0098] Maintenance of cell lines For highly productive S-Kroto cell lines amplified prior to optional cell subcloning (e.g., those produced by DHFR / MTX or GS / MSX systems), the process was carried out in serum-free and animal protein-free basal media, using carefully selected concentrates supplied for cell line production, throughout scale-up and until final bioreactor operation.

[0099] Scaling up of high-yielding cell lines was performed by growing cell inoculation material in cell suspension in shaking flasks or wave bag systems, followed by continuous inoculation of cells produced in 100 L and then 500 L volume bioreactors. Cell viability was over 85% throughout the growth cycle in shaking flasks, wave bags, or bioreactors, and then maintained at over 80% viable cells in the bioreactor during the plateau phase of CHO cell proliferation, with accompanying S-klotho production at up to 1-3 g / L (referred to as "high-yielding").

[0100] Protein production Certain embodiments may employ recombinant DNA strategies utilizing potent promoter sequences and / or high copy number plasmids for the production of therapeutic levels of Kroto protein in mammalian (e.g., CHO) cells. In at least one embodiment, for example, amplification of the dihydrofolate reductase (DHFR) gene in DHFR-deficient CHO cells may include providing methotrexate (MTX) and / or the use of MTX (with continuously increasing levels). Similarly, CHO cells containing the exogenous glutamine synthase (GS) gene may be treated with methionine sulfoximine (MSX).

[0101] Kroto proteins may also contain one or more glycans (bound to them). For example, natural human alpha-kroto isoform 1 (SEQ ID NO: 1) may have glycans bound (via glycosylation) at amino acids 106, 159, 283, 344, 604, 612 and / or 694. Kroto proteins of this disclosure may have one or more of the same (or similar) glycans bound thereto (for example, to the same amino acid(s)) (via glycosylation).

[0102] In at least one embodiment, the protein may comply with CGMP regulations as determined and enforced by the U.S. Food and Drug Administration (FDA). For example, the kroto protein may be at least 95%, 96%, 97%, 98%, or 99% pure by dry weight. In some embodiments, the kroto protein sample may contain CHO host cell proteins (HCPs), nucleic acids, and / or other cellular components in parts per ten million (ppm) of about 1 to less than 100, about 100 to less than 1000, or about 1 to less than 100 ppm, or any value or range of values ​​placed in between.

[0103] The glycan structure in which the produced S-Kroto protein exists may be similar to or identical to the structure of native S-Kroto structures isolated from human bodily fluids (i.e., blood, serum, urine, cerebrospinal fluid). In at least one embodiment, confirmation of a native-like glycan can ensure that the correct native post-translational modifications (PTMs) are produced and stably maintained in S-CHO cell-produced S-Kroto protein.

[0104] Extension of the solubility and / or half-life of Kroto protein Methods and compositions for extending the half-life and increasing the solubility of human S-Kroto protein are disclosed. Purification and characterization of protein constructs thus produced to achieve these results are also the subject of this disclosure. Relevant information regarding nucleic acid changes made in the sequence of the Kroto gene or nucleic acid construct (see SEQ ID NOs. 76-96), and / or changes or chemical alterations in the amino acid sequence of the Kroto protein (see SEQ ID NOs. 1-70), and / or the addition or removal of chemical groups, peptides, or proteins to the amino acid sequence of the Kroto protein is taught in this disclosure to obtain human Kroto variant proteins (novel compositions) having an increased biological half-life or increased solubility in biological matrices (such as blood, cerebrospinal fluid, urine, or various human tissues) compared to that of the natural Kroto molecule. These novel compositions can be prepared through the methods described herein for the modification of S-Kroto protein.

[0105] Fusion protein constructs can be produced by combining the S-Kroto protein with the Fc domain of an antibody (IgG). The fusion protein construct was produced by combining the S-Kroto protein with human serum albumin (HSA).

[0106] The fusion protein construct was produced by combining the S-Kroto protein with human transferrin (TF). The fusion protein was produced by combining the S-Kroto protein with a proprietary recombinant polypeptide such as XTEN®.

[0107] The novel S-Kroto protein was produced through PEGylation. Using the aforementioned and other half-life extension methods, the performance of S-Kroto protein can be measured in several ways, as follows: By extending the dosing interval of S-Klotho, we can provide patients with superior convenience and promising compliance with medication. By reducing the frequency of medication, the total amount of medication used is reduced, and product costs are lowered. Reduce the drug dose while maintaining the same dosage interval as the parent protein. To simplify medication prescriptions and enable subcutaneous administration, Using the same dosage and interval as the parent protein, higher drug levels result in longer drug exposure and potentially better efficacy. The condition improved by reducing the immunogenicity of S-Klotho.

[0108] Production of S-Kroto's Fc domain fusion protein construct The efficacy of extending the half-life of antibody Fc domains and human serum albumin (HSA) and increasing the solubility of human S-Kroto protein was tested. Fc fusions involve the fusion of a peptide, protein, or receptor exodomain to the Fc portion of an antibody. Both Fc fusions and albumin achieve half-life extension not only by increasing the size of the peptide drug, but also by utilizing the body's natural circulatory mechanisms through the binding of the elongation protein to the neonatal Fc receptor, FcRn. After the binding of the elongation protein to the FcRn receptor, degradation of the fusion protein in cellular endosomes is prevented. Fusions based on the addition of Fc or albumin can result in biological half-lives ranging from 3 to 16 days, much longer than those reported for typical PEGylated or lipidized peptides. For a review describing the use of protein fusion techniques, such as Fc fusion proteins, fusion to human serum albumin, fusion to carboxy-terminal peptides, and other polypeptide fusion approaches for creating biobetter drugs with more desirable pharmacokinetic profiles, see Strohl WRFusion Proteins for Half-Life Extension of Biologics as a Strategy to Make Biobetters. Biodrugs. 2015;29(4):215-239, which is incorporated herein by specific reference.

[0109] The Fc domain was therefore added to our parent protein (S-Klotho) to increase its binding affinity to the Fc receptor (FcRn). FcRn resides within the lysosomes of endothelial cells lining blood vessels and functions to rescue antibodies from degradation that shortens the lifespan of most proteins in circulation. As a result of interaction with FcRn, the protein has a half-life ranging from several days to several weeks, allowing the extended form of the protein agent to be administered at a lower frequency than biologics without this newly produced composition.

[0110] The main difference between Fc and albumin is the dimerization of Fc versus the monomeric structure of HSA, which, in contrast to HSA, leads to Fc fusion peptides existing as either dimers or monomers. The dimerization of peptide Fc fusions can produce binding activity effects if the target receptors of S-Klotho are sufficiently close and separated, or if they themselves are dimers, particularly in human target organs. This may or may not be desirable, depending on the target.

[0111] Fusion of the antibody Fc with the S-Kroto protein is also taught in this disclosure to improve the solubility and stability of S-Kroto. The addition of the Fc domain to S-Kroto also allows for a reduction in the immunogenicity of the fusion protein upon administration to human subjects.

[0112] Conjugation of S-Klotho protein with human serum albumin (HSA) The 66.5 kDa protein HSA, similar in size to human IgG, has a long mean half-life ranging from 19 days. At a concentration of approximately 50 mg / mL (approximately 600 μM), HSA is the most abundant protein in human plasma and has several functions, including maintaining plasma pH, metabolite and fatty acid transport, and blood pressure. As the upper limit of protein size for glomerular filtration by the kidney, HSA is also strongly anionic, which helps to further slow filtration via the kidney. Like IgG, HSA also binds to FcRn in a pH-dependent manner, albeit at a different site than IgG binding and through a mechanism distinct from that of IgG binding, and is recycled similarly to IgG, resulting in an extended half-life. HSA also tends to accumulate in tumor and inflamed tissues, suggesting that fusion or binding to albumin may help target proteins or peptides to those sites.

[0113] Due to the extension of the serum half-lives of these molecules, the fusion of peptides or proteins with inherently short half-life properties into HSA has been widely studied since the early 1990s. Since then, dozens of different peptides and small proteins have been fused to HSA, both as innovative and potentially biobetter molecules. The first commercially approved HSA-peptide or protein fusion product was Tanzeum® (marketed as Eperzan® in the European Union) DPP-4 resistant GLP-1-HSA fusion protein, discovered by Human Genome Sciences and developed and marketed by GlaxoSmithKline. Tanzeum® (albiglutide) was approved by the European Medicines Agency (EMA) and the FDA in March and April 2014, respectively. Thus, HSA improves the half-life of pharmacologically active GLP-1 from 1-2 minutes for natural GLP-1 to 4-7 days, enabling weekly dosing. Seven other known HSA fusion protein candidate products are currently under development or have been under development in the near future. Novozyme is also developing modified recombinant HSAs with improved FcRn binding for the construction of “next-generation” HSA protein fusions that may have longer half-lives. This was based on the use of the K573P mutant of HSA, which was found to have a 12-fold higher affinity for FcRn, giving HSA a longer half-life than the wild-type molecule in both mouse and cynomolgus monkeys. These longer-half-life HSA mutants are expected to be further used as fusion proteins to improve the half-life of fusion proteins.

[0114] Accordingly, we hereby disclose that our Klotho protein can be fused to wild-type HSA or a variant form of HSA to produce Klotho fusion molecules with a significantly extended half-life in human blood, cerebrospinal fluid, and other human biological matrices, providing patients with strategic therapeutic benefits such as superior convenience and promising medication compliance, resulting in reduced dosing frequency, lower total drug usage, and / or reduced commodity costs. Furthermore, by reducing the drug dose at the same dosing interval as the parent protein, drug prescriptions can be simplified, subcutaneous administration may be possible, or the immunogenicity of S-Klotho may be reduced.

[0115] Conjugation of S-Kroto protein with human transferrin (TF) Transferrin is a very abundant serum glycoprotein, found in serum at 3–4 mg / mL, and functions to strongly and reversibly bind iron and transport it to tissues. Transferrin has 679 amino acid residues, is about 80 kDa in size, and has 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 that has been reported to be 7–10 days, or 10–12 days. The aglycosylated form of human transferrin, which accounts for about 2–8% of the total transferrin pool, has a slightly longer half-life of 14–17 days. The long persistence of transferrin in human serum is due to a mechanism mediated by the clathrin-dependent transferrin receptor (which returns receptor-bound transferrin to the circulation for reuse).

[0116] Peptide-protein fusions are created at the N-terminus and C-terminus of human transferrin, as well as at the centrally located hinge region that links the two main protrusions of transferrin together. The N-terminus of transferrin is open and can be fused directly. The C-terminus is more embedded and constrained by a nearby disulfide bond, so a flexible linker is typically used when a protein is fused to the C-terminus. This ability has been expanded by creating libraries of peptides for specific targets and then fusing binders from those libraries to aglyco-transferrin (N-terminus, C-terminus, loop, or linker region) to construct therapeutic fusion proteins with extended half-lives.

[0117] BioRexis Technologies, Inc., a biotechnology company founded in 2002, developed a transferrin fusion protein platform, which it called the "Trans Body" platform for therapeutic use. Its lead molecule, BRX-0585, was a transferrin-GLP-1 fusion protein for the treatment of type 2 diabetes mellitus (T2DM). The fusion of GLP-1 to transferrin was demonstrated to significantly improve the half-life of GLP-1. BioRexis was acquired by Pfizer in March 2007. To the best of our knowledge, no BioRexis-derived fusion proteins are currently available in hospitals. Klotho protein can be fused to human transferrin to produce Klotho fusion molecules that can be administered in hospitals, significantly extending the half-life or stability in the human biological matrix in vivo.

[0118] The binding of S-Croto protein to Amunix's XTEN. XTEN® is a proprietary recombinant polypeptide that extends the in vivo half-life of therapeutic payloads. XTEN is composed of naturally occurring hydrophilic amino acids and is biodegradable. Pharmaceuticals such as proteins, peptides, and synthetic compounds can be XTENized via chemical conjugation or gene fusion. XTEN proteins lack secondary and tertiary structures, and their solution behavior is similar to that of a chemically prepared polymer with a very large hydrodynamic radius. By size exclusion chromatography, XTEN protein polymers appear much larger than typical globular proteins of similar molecular weight. The bulking effect of XTEN significantly reduces the renal clearance of the bound molecule and therefore significantly increases the in vivo half-life. In this invention, the length of the XTEN polymer bound to the Kroto protein will be specified to optimize pharmacokinetics, as well as the in vivo distribution of the bound Kroto protein payload.

[0119] Therefore, XTEN can be recombinantly fused to the inventors' S-Kroto protein to increase the molecule's in vivo half-life. One advantage is that the genetic S-Kroto-XTEN fusion construct produces a molecule with the convenience of single-molecule expression, purification, and characterization, containing both the therapeutic and bulking portions. Recombination fusions by therapeutic drug manufacturers that produce best-in-class pharmacokinetics, such as XTEN-modified growth hormone (Somavaratan, Versartis) and FVIII-XTEN (Biogen), have been successfully utilized, allowing for the attachment of multiple XTEN chains per protein to precisely defined positions. For example, pharmacokinetic studies conducted in children administered different doses of XTEN-modified growth hormone (Somavaratan, Versartis) have shown optimization of the Somavaratan molecule, reducing receptor-mediated efflux in addition to renal clearance, resulting in a best-in-class half-life.

[0120] XTEN protein polymers can be produced as free intermediates for chemical conjugation to peptides, peptide mimes, and other synthetic molecules. Reactive groups (thiols, amines) are inserted at precisely defined positions by introducing cysteine ​​or lysine residues into the gene encoding XTEN. Amunix has developed XTEN containing 1 to 9 thiol groups at various intervals, which can be provided to partners. Thus, in this invention, orthogonal conjugation to amino and thiol groups in XTEN will facilitate the production of our Kroto-XTEN molecules.

[0121] Protein purification Kroto protein can be extracted from cell suspension cultures of CHO cells (e.g., CHO cell lines). CHO cells can produce and selectively secrete Kroto protein (e.g., into liquid medium). Secretion of up to 200–500 mg / L of S-Kroto into cell-consuming medium has also been observed.

[0122] The purification of recombinant proteins in this disclosure may be carried out by any preferred method known in the art or described herein, such as any conventional procedure including extraction, precipitation, chromatography and / or electrophoresis. Further purification procedures that may be used to purify proteins include affinity chromatography using a monoclonal antibody that conjugates the target protein. Some embodiments may include IgG-tagged proteins that can be purified by affinity chromatography. Generally, a crude preparation containing recombinant protein is passed through a column immobilized with a preferred monoclonal antibody. The protein usually conjugates to the column via a specific antibody while impurities pass through. After washing the column, the protein is eluted from the gel by changing the pH or ionic strength. For example, consumption medium from a CHO-S high-productivity cell line was concentrated via tangential flow filtration, and S-Kroto protein was purified by affinity chromatography, followed by ion-exchange cartridge or column chromatography. Size exclusion chromatography may also be used for protein purification.

[0123] In alternative protocols, one or more steps were performed before or after affinity purification. Such steps may 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. In certain embodiments, one or more post-purification treatment steps were performed. Such post-purification treatment steps may include, for example, removal of viruses and / or bacteria by tandem anion / cation flow-through chromatography (in contrast to binding and elution chromatography), membrane filtration (e.g., 0.2 micron, 0.1 micron, etc.), or by other means known to those skilled in the art.

[0124] Analysis of Kroto protein Protein purity can be demonstrated by SDS-PAGE or other assays or by means known in the art. For example, in at least one embodiment, a kroto protein sample (50 μg) was fractionated on a precast SDS-PAGE gel (4–15%, 10 wells; catalog no. 456-1083; BioRad) and stained with Coomassie blue dye. To avoid contamination between samples, all samples were placed on separate gels or with empty lanes in between. It was shown that more than 98% of S-kroto was isolated from the CHO S condition medium, as measured by Coomassie blue dye and densitometry, or by visualization with silver staining, or by HPLC or RP-HPLC. To obtain sequence information, the protein (after reduction and S-carboxymethylation) can be cleaved with cyanogen bromide, trypsin, and / or proteinase K according to known methods of protein chemistry, and the peptides can be separated by HPLC. Next, the prepared samples were sequenced using an automated vapor-phase microsequencing system (Applied Biosystems Model 470A, ABI, Foster City, California, USA) which included an online automated HPLC PTH amino acid analyzer (Applied Biosystems Model 120, ABI see below) connected to a power outlet.

[0125] The protein was also analyzed by mass spectrometry. For sample preparation for mass spectrometry, only gel bands between 75 and 150 kDa were cut out for analysis to limit the analysis to the correct S-Kroto protein. The gel fraction was softened with a sterile blade and subjected to in-gel digestion. The gel fraction was destained by washing three times with 80 μL of 50% acetonitrile (ACN) / 50 mm ammonium bicarbonate and then washed with 100% ACN. The alkylation step was omitted, assuming that no cysteine ​​residues from the target α-Kroto peptide were present. Trypsin digestion was performed overnight at 37°C in 50 mm ammonium bicarbonate (0.005 μg / μL) using 60 μL of trypsin (sequencing grade modified, catalog number V511A; Promega). 25 μL was obtained from this process, of which 5 μL (1 μL for S-Kroto) was subjected to liquid chromatography-electrospray ionization tandem mass spectrometry (MS / MS) and PRM analysis using an Orbitrap nano-ESIQ-Exactive mass spectrometer (Thermo Scientific) attached to a nanoLC (Dionex Ultimate 3000 UHPLC). MS / MS analysis confirmed that the human recombinant alpha-S-Kroto produced by the embodiments of this disclosure was substantially identical to that found in human blood, serum, urine, or cerebrospinal fluid (e.g., identical in the corresponding amino acid sequence).

[0126] Using the purification method described above, the level of contaminating CHO host cell protein (HCP) was determined to be acceptable in the purified S-Kroto protein. In the final S-Kroto product, HCP was removed to <1–100 ppm. 98+% pure S-Kroto protein products were isolated from the CHO S-producing cell lines (cells and / or liquid media) used. Specifically, CGMP-grade human alpha-S-Kroto with an analytical profile suitable for clinical administration in human subjects was produced and purified. For example, the analytical profile of human recombinant alpha-S-Kroto is described in ProteomeXchangeDatabase reference number PXD002775, which can be found at http: / / proteomecentral.proteomexchange.org / cgi / GetDataset?ID=PXD002775. The complete NIH S-Kroto protein dataset is available at http: / / www.ncbi.nlm.nih.gov / protein / Q9UEF7.

[0127] The analytical profiles of S-Klotho suitable for clinical administration, and the analytical profiles obtained in one embodiment of this disclosure, contained endotoxin levels of less than 0.1 ng (1 EU / μg) per 1 μg of S-Klotho. In addition, purified human recombinant S-Klotho was also shown to have a purity of > or = 98% by SDS-PAGE.

[0128] The glycan structure present in S-klotho produced in CHO S cells was identical to that of native S-klotho isolated from human bodily fluids (i.e., blood, serum, urine, and cerebrospinal fluid). This confirmed that the same native post-translational modifications (PTMs) were generated and stably maintained in the S-klotho protein produced in S CHO cells. Therefore, using the manufacturing and purification methods described herein, the inventors have successfully produced cGMP-grade human S-klotho with an analytical profile suitable for clinical administration to human subjects.

[0129] therapeutic composition Some embodiments of the present disclosure may include pharmaceutical compositions, such as therapeutic compositions. The pharmaceutical compositions of the present disclosure may generally comprise a mixture of a therapeutically effective amount of recombinant soluble alpha-kroto protein with a vehicle or carrier comprising one or more additional components. These components may include one or more aggregation inhibitors, buffers, isotonic agents, and additional excipients. The main solvent in the carrier may be either essentially aqueous or non-aqueous. The compositions may be prepared by combining the purified kroto protein of the present disclosure with a pharmaceutically acceptable carrier.

[0130] Those skilled in the art will understand that the various combinations of components in a composition can be arranged in any suitable order; that is, buffers can be added first, in the middle, or last, and isotonic agents can also be added first, in the middle, or last. It will also be understood that some of these chemicals may be incompatible in certain combinations and can therefore be readily substituted with different chemicals having similar properties but compatible with the relevant mixture.

[0131] Aggregation inhibitors reduce the tendency of polypeptides to associate into inappropriate or undesirable three- or four-component complexes. The amino acids L-arginine and / or L-cysteine ​​may act to reduce the aggregation of Fc domain-containing polypeptides in the formulation for a long period, e.g., more than two years. The concentration of the aggregation inhibitor in the formulation is preferably about 1 mM to 1 M, more preferably about 10 mM to 200 mM, more preferably about 10 mM to 100 mM, even more preferably about 15 mM to 75 mM, and also more preferably about 25 mM. These compounds are available from commercial suppliers.

[0132] The compositions of this disclosure may include buffers that maintain the pH within a desired range. Various buffers suitable for use in the pharmaceutical compositions of this disclosure include histidine, potassium phosphate, alkali salts, sodium phosphate 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 acetates and diethanolamine, and any other pharmaceutically acceptable pH buffers known in the art for maintaining the pH of a solution within a desired range. Mixtures of these buffers may also be used.

[0133] The amount of buffer useful in a composition depends largely on the specific buffer used and the pH of the solution. For example, since acetate is a more efficient buffer at pH 5 than at pH 6, less acetate may be used in the solution at pH 5 than at pH 6. The preferred pH of a preferred formulation is in the range of 4.0 to 5.0, and pH adjusters such as hydrochloric acid, citric acid, sodium hydroxide or salts thereof may also be included to obtain the desired pH.

[0134] One preferred buffer is sodium phosphate with a buffering capacity of around pH 6.2. However, it will be understood that other buffers may be selected to achieve any desired pH buffer. The concentration of the buffer in the formulation is preferably about 1 mM to about 1 M, more preferably about 10 mM to about 200 mM. Buffers are known in the art, can be manufactured by known methods, and are available from commercial suppliers.

[0135] Setting the pH of a pharmaceutical composition near physiological levels maximizes patient comfort during administration. In particular, the pH is preferably in the range of approximately 5.8 to 8.4, and preferably approximately 6.2 to 7.4. However, the pH can be adjusted as needed to maximize the stability and solubility of polypeptides in certain formulations, and it is understood that pH outside the physiological range that is acceptable to the patient is within the scope of this disclosure.

[0136] The formulations of this disclosure may further comprise one or more isotonic agents (for example, to make the solution isotonic with the patient's blood for injection). An isotonic agent is understood to be a molecule that contributes to the gravimetric osmolality of the solution. The gravimetric osmolality of the pharmaceutical composition is preferably adjusted to maximize the stability of the active ingredient and also minimize patient discomfort at administration. This is when serum is approximately 300 ± 50 millimoles per kilogram. It is generally preferred that the pharmaceutical composition is isotonic with serum, i.e., has the same or similar gravimetric osmolality achieved by adding an isotonic agent, and therefore it is considered that the gravimetric osmolality may be about 180 to about 420 millimoles, although it is understood that the gravimetric osmolality may be higher or lower as required under particular conditions.

[0137] Typical isotonic agents are known in the art and include, but are not limited to, various salts, amino acids, or polysaccharides. A non-limiting example of a suitable amino acid is glycine. Non-limiting examples of suitable polysaccharides include sucrose, mannitol, and sorbitol. It is understood that two or more isotonic agents may be used at once; for example, sorbitol and glycine may be used in combination to alter the tonicity of the formulation.

[0138] Suitable additional isotonic agents for changing the osmolality by weight 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 sugars (e.g., sucrose, glucose, and mannitol). The concentration of the isotonic agent in the formulation is preferably about 1 mM to 1 M, more preferably about 10 mM to about 200 mM. Isotonic agents are known in the art, can be manufactured by known methods, and are available from commercial suppliers.

[0139] Excipients, also called co-solubilates or co-solvents, are chemical additives that stabilize polypeptides in solution (or in dry or frozen form) and may also be added to pharmaceutical compositions. Excipients are defined herein as non-therapeutic agents added to pharmaceutical compositions to provide desired effects, such as stabilization or isotonicity. Common desirable attributes of excipients include water solubility, non-toxicity, non-reactivity, rapid clearance from the body, and lack of immunogenicity. In addition, excipients should be capable of stabilizing the natural conformation of proteins to maintain the efficacy and safety of the drug during processing, storage, and administration to patients. Examples include, but are not limited to, sugars / polyols such as sucrose, lactose, glycerol, xylitol, sorbitol, mannitol, maltose, inositol, trehalose, and glucose; polymers such as serum albumin (bovine serum albumin (BSA), human SA, or recombinant HA), dextran, PVA, hydroxypropyl methylcellulose (HPMC), polyethyleneimine, gelatin, polyvinylpyrrolidone (PVP), and hydroxyethylcellulose (HEC); polyhydric alcohols (e.g., PEG, ethylene glycol, and glycerol), dimethysulfoxide (DMSO), and dimethylformamide (DMF); proline, L- Examples include amino acids such as serine, sodium glutamate, alanine, glycine, lysine hydrochloride, sarcosine, and gamma-aminobutyric acid; surfactants such as Tween®-80 (polysorbate 80), Tween®-20 (polysorbate 20), SDS, polysorbate, and polyoxyethylene copolymer; and various excipients such as potassium phosphate, sodium acetate, ammonium sulfate, magnesium sulfate, sodium sulfate, trimethylamine N-oxide, betaine, metal ions (e.g., zinc, copper, calcium, manganese, and magnesium), CHAPS, monolaurate, 2-O-beta-mannoglycerate, or any combination thereof.

[0140] The concentration of one or more excipients in the formulations of this disclosure is preferably about 0.001 to 5 weight percent, more preferably about 0.1 to 2 weight percent. The excipients are known in the art, can be manufactured by known methods, and are available from commercial suppliers.

[0141] In one exemplary embodiment, the formulation of the present disclosure may comprise HEPES, MES, or Tris-HCl, and optionally, about 150 mM NaCl buffered to pH about 7.3–7.4 with one or more additional components described herein.

[0142] In one exemplary embodiment, a formulation of the present disclosure may have a pH of about 6.0 to about 7.0 and contain about 25 to about 50 mg of TNFR:Fc (etanercept), about 10 mM to about 100 mM of L-arginine, about 10 mM to about 50 mM of sodium phosphate, about 0.75% to about 1.25% of sucrose, and about 50 mM to about 150 mM of NaCl. In another embodiment, L-arginine may be replaced with L-cysteine ​​(about 1 to about 500 micromoles) in the formulation. In yet another embodiment, the pH may be about 7.0. In another specific embodiment, a formulation of the present disclosure may have a pH of about 6.2 and contain about 25 mg / ml of TNFR:Fc, about 25 mM of L-arginine, about 25 mM of sodium phosphate, about 98 mM of sodium chloride, and about 1% of sucrose.

[0143] In another embodiment, the formulation of the present disclosure may contain 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 1.25% sucrose, and about 50 mM to about 150 mM NaCl at a pH of about 6 to about 7. In a particular embodiment, the formulation of the present disclosure may contain 50 mg / mL of RANK:Fc in about 25 mM L-arginine, about 25 mM sodium phosphate, about 98 mM sodium chloride, and about 1% sucrose at a pH of about 6.2.

[0144] In yet another embodiment, the formulation of the present disclosure may have a pH of about 6 to 7 and contain an effective amount of 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®-20 (polysorbate 20). In yet another embodiment, the formulation of the present disclosure may contain an effective amount of antibody such as Emab (anti-CD22 specific antibody), about 25 mM L-arginine, about 25 mM sodium phosphate, about 4% mannitol, about 0.02% Tween®-20 (polysorbate 20), and have a pH of about 6.0.

[0145] In yet another embodiment, the Disclosure provides a method for treating a mammal, comprising administering a therapeutically effective amount of a pharmaceutical composition described herein, wherein the mammal has a disease or disorder that can be beneficially treated with the Fc domain-containing polypeptide in the composition. In yet another embodiment, the Fc domain-containing polypeptide is derived from the same species as the mammal that will be treated with the composition. In a particular embodiment, the mammal is a human patient in need of treatment. When the Fc domain-containing polypeptide of the composition is TNFR:Fc, examples of diseases or disorders that can be treated include, but are not limited to, rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, Wegener's disease (granulomatous disease), Crohn's disease (or inflammatory bowel disease), chronic obstructive pulmonary disease (COPD), hepatitis C, endometriosis, asthma, cachexia, psoriasis, and atopic dermatitis, or a person having a genetic disorder in which one or more Klotho genes are mutated. Additional diseases or disorders that can be treated with TNFR:Fc are those described in WO00 / 62790, WO01 / 62272, and U.S. Patent Application No. 2001 / 0021380, the relevant portions thereof incorporated herein by reference.

[0146] In yet another embodiment, the Disclosure provides a method for testing the accelerated stability of an Fc domain-containing polypeptide in a pharmaceutical composition of the Disclosure, comprising the steps of testing the activity of a polypeptide formulated according to the Disclosure before storage, i.e., storing the composition at 37°C for one month at a starting point and measuring the stability of the polypeptide, and comparing the stability form from the starting point to one month later. This information helps to remove batches or lots that initially appear to have good stability but do not store well for long periods.

[0147] Furthermore, the pharmaceutical composition provides long-term storage such that the active ingredient, e.g., the Fc domain-containing polypeptide, remains stable across either liquid or frozen storage stages. As used herein, the term “long-term storage” is understood to mean that the pharmaceutical composition can be stored for three months or more, six months or more, one year or more, and preferably two years or more. Long-term storage is also understood to mean that the pharmaceutical composition is stored as a liquid at 2–8°C or frozen at, for example, -20°C or below (e.g., -20°C or -80°C). It is also considered that the composition may be frozen and thawed two or more times. The term “stable” in relation to long-term storage is understood to mean that the active polypeptide of the pharmaceutical composition does not lose more than 20%, more preferably 15%, even more preferably 10%, and most preferably 5% of its activity compared to the activity of the composition at the start of storage.

[0148] One or more antioxidants may be included in the formulations of this disclosure. Antioxidants that may be considered for use in the preparation of formulations include amino acids such as glycine and lysine, chelating agents such as EDTA and DTPA, and free radical scavengers such as sorbitol and mannitol.

[0149] Other effective forms of administration are also envisioned, such as parenteral sustained-release formulations, mist inhalations, orally active formulations, or suppositories. Thus, formulations may also include bulk erosion polymers (e.g., poly(lactic acid-glycolic acid copolymer) (PLGA) copolymers, PLGA polymer blends, PEG block copolymers, and particulate preparations of polymer compounds such as lactic acid and glycolic acid, poly(cyanoacrylate); surface erosion polymers (e.g., poly(anhydride), and poly(orthoesters)); hydrogel esters (e.g., pluronic polyols, poly(vinyl alcohol), poly(vinylpyrrolidone), maleic anhydride-alkyl vinyl ether copolymers, cellulose, hyaluronic acid derivatives, alginates, collagen, gelatin, albumin, and starch and dextran), and particulate preparations of these composition systems, or preparations of liposomes or microspheres. Such formulations may affect the physical state, stability, in vivo release rate, and in vivo clearance rate of the protein and its derivatives. The optimal pharmaceutical formulation for a desired protein can be determined by those skilled in the art, depending on the route of administration and the desired dosage. Exemplary pharmaceutical formulations are disclosed in Remington's Pharmaceutical Sciences, 18th Ed. (1990), Mack Publishing Co., Easton, Pa. 18042, pages 1435-1712, which are incorporated herein by reference.

[0150] biological activity Methods for evaluating the efficacy of (prior to) human recombinant alpha-soluble klotho and / or determining the effective dose (for patients, subjects, or individuals exhibiting age-related disorders or metabolic disorders) include performing organism-based assays (e.g., mammals (e.g., mice, rats, primates, or any other non-human), or other animals (e.g., African clawed frogs, zebrafish, or flies (e.g., Drosophila melanogaster) or nematodes (e.g., Caenorhabditis elegans)) for klotho protein The substance may be administered to the organism once or according to a regimen (regular or irregular). For example, a protein may be administered a suitable number of times (e.g., once, twice, etc.) over a given period (e.g., monthly, bi-monthly, weekly, bi-weekly, daily, etc.). Then, biological parameters (e.g., age-related parameters) may be evaluated. The Klotho protein in question will perform or produce parameter changes compared to a reference (e.g., parameters of a control organism). Other parameters (e.g., related to toxicity, clearance, and pharmacokinetics) may also be evaluated.

[0151] The Kroto protein of this disclosure may be evaluated using animals (models) that have or exhibit specific disorders or conditions, such as age-related or age-associated disorders or conditions, metabolic disorders or conditions, and may also provide sensitization systems in which the physiological effects of the protein can be observed. Exemplary disorders include, for example, denervation, disuse atrophy, metabolic disorders (e.g., disorders in obese and / or diabetic animals such as db / db mice and ob / ob mice), brain disorders, hepatic ischemia or other hepatic disorders, cisplatin / taxol / vincristine models, various tissue (xenograft) transplants, genetically modified bone models, pain syndromes (e.g., inflammatory and neuropathy), paraquat poisoning, genotoxicity, oxidative stress models, and tumor (I) models.

[0152] To evaluate the S-Kroto protein of this disclosure, the protein may be administered to suitable animals (for a suitable treatment period), and the parameters of the animals may be evaluated (after a suitable period, e.g., 10–60 minutes, 1–24 hours, 1–30 days, 1–12 months, 1–5 years, or any value or range of values ​​in between). The animals may be nutritioned as needed or as usual (e.g., not under calorie restriction, although some parameters may be evaluated under such conditions). Typically, a cohort of such animals is used for the assay. Generally, if the test polypeptide affects the parameters toward the phenotype of similar animals subjected to calorie restriction, the test polypeptide may be shown to favorably alter the lifespan regulation of the animals. Such test polypeptide may induce at least some lifespan regulation effects by calorie restriction (e.g., a subset of such effects) rather than by withholding calories from the organism.

[0153] The parameters tested may be age-related or disease-related (e.g., symptoms of a disorder related to an animal model). A test protein that shows favorable results may evoke improvement in symptoms compared to a similar reference animal not treated with the polypeptide. Other parameters related to disorder or aging may include antioxidant levels (e.g., antioxidant enzyme levels or activity), stress tolerance (e.g., paraquat tolerance), core body temperature, glucose levels, insulin levels, thyroid-stimulating hormone levels, prolactin levels, and luteinizing hormone levels.

[0154] To measure the efficacy of the S-Kroto protein of this disclosure for treating age-related disorders, animals with reduced Kroto expression (e.g., mutant or Kroto gene-deficient mice) may be used. For example, the test protein may be administered to mutant mice to monitor age-related parameters. The test protein, if favorably demonstrated, may evoke improvement in symptoms compared to similar reference animals not treated with the protein.

[0155] Parameters associated with metabolic disorders or aging can be assessed by measuring body weight, testing for fertility, measuring blood glucose levels, observing lifespan, examining skin, and observing motor function such as walking. Assessment can also be performed by measuring thymic weight and observing the size of calcified nodules formed on the inner surface of the pleural cavity. Furthermore, quantitative measurement of Klotho gene or Klotho protein mRNA may also be useful for assessment.

[0156] Further (in vivo) models and bioassays include evaluating animals for metabolic parameters, such as those related to insulin impairment and type II diabetes. Exemplary metabolic parameters include glucose concentration, insulin concentration, and insulin sensitivity.

[0157] When evaluating whether a test protein can alter lifespan regulation, many age-related parameters or biomarkers may be monitored or evaluated. Exemplary age-related parameters include: (i) the lifespan of a cell or organism; (ii) the presence or abundance of gene transcriptions or gene products in a cell or organism that have an expression pattern dependent on biological age; (iii) the tolerance of a cell or organism to stress; (iv) one or more metabolic parameters of a cell or organism (exemplary parameters include circulating insulin levels, blood glucose levels, fat content, and core body temperature); (v) the proliferative capacity of cells or groups of cells present in an organism; and (vi) the physical appearance or behavior of a cell or organism.

[0158] The term "life expectancy" refers to the average age at death in a cohort of organisms. In some cases, "life expectancy" is assessed using a cohort of genetically identical organisms under controlled environmental conditions. Deaths due to accidents are not included. If life expectancy cannot be measured under controlled environmental conditions (e.g., in the case of humans), reliable statistical information from a sufficiently large population (e.g., from mathematical tables) may be used as life expectancy.

[0159] Characterizing the molecular differences between two such organisms, for example, one reference organism and one organism treated with S-Kroto protein, can reveal differences in the physiological state of the organisms. The reference organism and the treated organism are typically the same (or substantially the same) chronological age and / or sex. As used herein, the term “chronological age” refers to the time elapsed since a pre-selected event, such as conception, a defined gestational or fetal period, or more preferably, birth. Various criteria may be used to determine whether organisms are of the “same” chronological age for comparative analysis.

[0160] Typically, the required degree of precision correlates with the average lifespan of the wild-type organism. For example, in the case of the nematode Caenorhabditis elegans, where the laboratory wild-type strain N2 survives for an average of about 16 days under several control conditions, organisms of the same age may survive for the same number of days. In the case of mice, organisms of the same age may survive for the same number of weeks or months, and in the case of primates or humans, for the same number of years (i.e., within 2, 3, or 5 years), etc. Generally, organisms of the same actual age may survive for a time period within 15, 10, 5, 3, 2, or 1% of the average lifespan of the wild-type organism of that species. Preferably, the organism is an adult (for example, the organism has survived for at least a certain amount of time, until the average wild-type organism matures to reproductive age).

[0161] Biological screening assays may be performed before an organism exhibits obvious physical characteristics of aging. For example, the organism may be an adult that has survived only 10, 30, 40, 50, 60, or 70% of the average lifespan of wild-type organisms of the same species. Age-related changes in metabolism, immune capacity, and chromosomal structure have been reported. Any of these changes may be evaluated in test subjects (e.g., in organism-based assays) or (e.g., in patients (human or mammalian)) before, during, or after treatment with the therapeutic agents described herein.

[0162] Markers associated with calorie restriction may also be evaluated in test organisms (or treated subjects) in screening assays. These markers may not be age-dependent but may indicate physiological states that change when Klotho or Klotho-related pathways are regulated. The markers may be mRNA or proteins that change significantly in calorie-restricted animals. Cell models derived from the animal cells described herein, or analogues of the animal models described herein, may be used in cell-based assays.

[0163] Models for evaluating the effects of test proteins on muscle atrophy include: 1) decreased medial gastrocnemius muscle mass in rats resulting from denervation, for example, by severing the right sciatic nerve in the mid-thigh; 2) decreased medial gastrocnemius muscle mass in rats resulting from immobilization (for example, by fixing the right ankle joint at a 90-degree flexion); 3) decreased medial gastrocnemius muscle mass in rats resulting from hindlimb suspension; 4) skeletal muscle atrophy resulting from treatment with the cachexic cytokine interleukin-1 (IL-1); and 5) skeletal muscle atrophy resulting from treatment with glucocorticoids and dexamethasone.

[0164] Administration of exogenous S-clotah This disclosure relates to S-klotho formulations, clinical dosages, and administration for increasing and / or maintaining serum S-klotho concentrations in the normal and / or young (e.g., 18-30 years of age) range (e.g., without chronic conditions).

[0165] Aspects or embodiments of the present disclosure include, for example, administering (cGMP and / or clinical-grade) human recombinant alpha-soluble kroto protein or a protein fragment (of isoform 1) to a (human) subject requiring it. Embodiments may also include measuring (for example, by mass spectrometry (MS) or ELISA) the level or concentration of serum S-kroto (of the (human) subject). Such measurements may be performed before, during, and / or after S-kroto administration and may be repeated as needed to measure the rate of metabolism, degradation, or reduction of serum S-kroto levels and / or serum S-kroto levels. MS is a technique known in the art. MS may be used to identify and further quantify the levels of one or more (natural and / or recombinant) kroto proteins in the serum of a subject.

[0166] One or more additional proteins may also be measured in the subject's serum. One or more Klotho-related and / or age-related proteins (e.g., FGF21, GDF-11, TIMP2, NAD+, CCL11, hormone testosterone, estrogen, etc.) and / or renal function proteins (e.g., KIM-1, cystatin-C, creatinine, BUN, creatinine, NGAL, etc.) may be measured separately from or in combination with the measurement of Klotho in serum.

[0167] In at least one embodiment, a serum sample, such as a blood sample, is obtained. The sample may be obtained by blood collection, as is known in the art. In a preferred embodiment, a finger prick or other less invasive means may be used to obtain a blood sample. Thus, blood samples may be collected 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 may be used to measure the total krotho protein serum concentration, as well as the serum concentrations of various alpha krotho protein species, such as natural krotho species (e.g., soluble krotho, cleaved krotho, secreted krotho, etc.), and / or one or more krotho proteins of the present disclosure. In some embodiments, krotho levels may be measured before and after administration of therapeutic recombinant krotho protein, throughout the day and / or every 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 hours.

[0168] Embodiments may also include measuring such rate and / or calculating a treatment protocol (e.g., including the frequency, dosage, and / or duration of the next administration of S-Klotho) for maintaining the S-Klotho protein concentration in the serum of such subject within the range of normal serum S-Klotho concentrations in a young person. In at least one embodiment, the S-Klotho concentration may be maintained at approximately 1000 picograms (pg / mL) of (S-Klotho) protein per milliliter of serum.

[0169] In at least one embodiment, the S-Klotho administration strategy (in humans) may include the measurement of one or more pharmacokinetic parameters of S-Klotho. For example, in vivo changes in S-Klotho levels in serum, urine, and cerebrospinal fluid in response to S-Klotho administration may be measured. Some embodiments may include measuring the efficacy of S-Klotho administration against one or more clinical indicators. Clinical indicators for various pathological conditions, diseases, and disorders are known in the art and are further described herein.

[0170] Embodiments may also include measuring (reference value) S-Klotho levels (e.g., before and after S-Klotho administration) to account for any circadian rhythm effects in (human) subjects (e.g., at initiation (before administration of any exogenous S-Klotho)) and / or at different times before and / or throughout the treatment protocol.

[0171] Embodiments may also include measuring a preferred frequency, amount, and / or duration of S-Klotho administration. For example, a subject with low S-Klotho serum levels (measured, e.g., by MS or ELISA immunoassay quantification) may receive a first dose of Klotho configured and / or adapted to bring the subject's serum S-Klotho level to a first predetermined level (e.g., about 1000 pg / mL) by measuring the subject's serum S-Klotho concentration (the change obtained) (by MS or ELISA), measuring the serum S-Klotho level and / or the rate of metabolism, degradation, or reduction (following the first dose), calculating the half-life of the administered S-Klotho, and / or determining the frequency and / or time frame in which a second subsequent dose of S-Klotho should be received (e.g., to maintain the serum S-Klotho level above a second predetermined level), by (e.g., via intravenous, intradermal, intraperitoneal, intramuscular, intradermal, and / or subcutaneous injection or other administration). In at least one embodiment, the second predetermined level may be about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, and / or 5%, and / or in between, of the first predetermined level.

[0172] Further administration may be given over a timeframe suitable for producing S-Klotho serum levels equivalent to the maintenance serum level of a normal young person of the same sex (e.g., approximately 1000 pg / ml) in the subject (over the long term). The total duration of S-Klotho administration (to human subjects) may range from 1 day to more than 5 years. Measurement and / or determination of subject frailty based on the use of a clinical frality score and other measures may also be performed over the timeframe.

[0173] Embodiments of the present disclosure further include increasing and maintaining the S-Klotho dosage to an increased S-Klotho level of 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or more, above the normal range (1000 pg / ml).

[0174] Certain embodiments involve administering S-Klotho protein in a single bolus injection or a long-term (IV) injection (e.g., a long-term intravenous infusion). In at least one embodiment, 1, 2, 2.5, 2.75, 3, 3.5, 4, 4.5, or more micrograms of S-Klotho per subject may be administered per treatment. A preferred dose may be calculated through one or more methods known in the art. One such method is the relative growth (allometric scaling) method. For example, in rat experiments, 0.01 mg of S-Klotho / kg body weight or 10 μg / kg is used per dose. Exemplarily, using 0.16, which is equivalent to the human relative growth rate, the human equivalent dose (HED) = 10 μg / kg x 0.16 = 1.6 μg / kg. Therefore, as an example, a 70kg human individual would require 70kg x 1.6ug / kg = 112ug, and a 60kg human individual would require 60kg x 1.6 = 96ug.

[0175] The HED was established using a process described by Reagan-Shaw (2008), which is incorporated herein by reference, for standardization of body surface area. This process, referred to as the relative growth rate, corrects for underlying differences in metabolic rates between different species and may be preferable to simple dose extrapolation. Exemplaryly, if the HED is 0.4 mg / kg, using the relative growth rate, the human equivalent dose would be 0.4 mg / kg, or 28 mg, for an individual weighing 70 kg, and 24 mg for an individual weighing 60 kg.

[0176] Multiple factors may be considered or taken into 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 administered, and / or the serum level response (over time) after recombinant protein administration. Such factors may include, for example, the composition of the diluent, the route of administration, the site of administration, the distribution to the subject's tissues and organs, the subject's metabolism or other rates, pharmacokinetics (PK), pharmacodynamics (PD), and toxicology (Tox).

[0177] In at least one embodiment, the (normal) concentration of S-klotho (in, for example, a healthy, young (18-30 years) human adult) may be approximately 1000 pg / ml in serum. A typical adult may have a blood volume of approximately 5 liters, and women generally have less blood volume than men. Approximately 55% of human blood may consist of serum. Therefore, (5 liters of blood / adult) x (0.55 serum / liter of blood) = 2.75 liters (2750 ml) of serum / adult. Assuming no endogenous serum S-klotho is present, to achieve a final concentration of 1000 pg / ml in total serum, 2750 ml x 1000 pg / ml = 2,750,000 pg (or 2750 ng or 2.75 μg) of exogenous S-klotho was administered per adult subject.

[0178] To increase soluble klotho by 50% above typical healthy levels (e.g., 1500 pg / serum ml), a dose of 4.125 micrograms / subject may be administered. To increase soluble klotho by 100% above typical healthy levels (e.g., 2000 pg / serum ml), a dose of 5.5 micrograms / subject may be administered, and so on.

[0179] To increase the serum soluble kroto protein concentration in a subject to any preferred level, a pharmaceutically effective and / or sufficient amount of purified recombinant S-kroto protein is approximately 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, 11,000, 12,000, 13,000, 14,000, 15,000, 20,000, 25,000, 30,000 per milliliter of serum. Soluble Kroto protein may be administered in amounts exceeding 40,000, 50,000, 75,000, 100,000 picograms or more, or in between, or exceeding typical healthy levels of soluble Kroto protein in serum by approximately 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%, and 5000%, or in between.

[0180] In some embodiments, subjects may be administered recombinant human S-Klotho protein in one or more (bolus) intravenous, intradermal, intraperitoneal, intramuscular, intracutaneous, subcutaneous, and / or other injections in a suitable amount of Klotho buffer (e.g., 150 mM NaCl and 10 mM HEPES pH 7.4) or other pharmaceutically acceptable carrier at doses of about 0.01 mg / kg body weight or more, or in between. Thus, a subject weighing 160 pounds (i.e., 72.57 kg body weight) may be administered a (bolus) injection of about 0.73 mg of S-Klotho per dose (based on the calculation of 0.01 mg of S-Klotho / kg x body weight 72.57 kg). Similarly, a person weighing 170 pounds may receive 0.77 mg of S-Klotho per dose. The total number and frequency of administrations may be determined, for example, based on achieving and maintaining a serum concentration of 1000 pg / ml of S-Klotho (equivalent to 0.000001 mg / ml serum). The latter may be confirmed by measurement by MS or by a human S-Klotho ELISA assay.

[0181] In other embodiments, the dosage may be approximately 0.0001 to 10 mg / kg body weight, 0.0001 to 10 μg / kg body weight, 0.0001 to 10 ng / kg body weight, 0.0001 to 10 pg / kg body weight or more, or any value or range in between. Urine and / or blood may be collected at one or more time points in between approximately 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, or less, after the medical procedure or first dose (dosage) of recombinant Klotho protein (for example, to measure, test, and / or determine serum S-Klotho levels, as well as changes in administration response and / or response over time).

[0182] One or more embodiments include the manufacture and / or administration of a unique formulation of the S-Klotho active drug product and / or a combination with a pharmaceutically acceptable carrier. The carrier may be suitable for IV and / or bolus injection. The embodiments may also include the manufacture and / or administration of a unique inactive prodrug formulation in combination with S-Klotho and / or a pharmaceutically acceptable carrier, such that inactive S-Klotho can be activated in vivo to release a bioactive S-Klotho in an animal or human subject. Such prodrug formulations may include coated or sustained-release formulations.

[0183] Administration of exogenous S-Klotho to treat age-related weakness in humans. Exemplary embodiments of this disclosure relate to the administration of exogenous klotho protein for the treatment of age-related frailty (e.g., in human or non-human animals). S-klotho may rescue myogenic stem cells, improve muscle repair, and / or suppress fibrosis in animal models of human diseases. Thus, S-klotho may be a promising therapeutic agent for combating muscle degeneration in aged human subjects exhibiting signs of frailty.

[0184] For example, this disclosure relates to S-Klotho formulations, clinical dosages, and administration to vulnerable and / or elderly subjects (e.g., 60–95 years) to maintain S-Klotho serum concentrations in the former subjects within the range of normal and / or young persons (e.g., 18–30 years) (e.g., without chronic conditions).

[0185] Long-term Klotho therapy may restore and / or improve one or more age-related indicators or conditions in older adults, frail individuals, or other physiologically aged individuals. Administration of exogenous S-Klotho for the treatment (reduction) of muscle atrophy in humans Exemplary embodiments of this disclosure relate to the administration of exogenous klotho protein to treat (e.g., reduce) muscle atrophy in humans, as measured by skeletal muscle tissue mass and by a combination of the above-mentioned protein and molecular indicators, and provide guidance on the effects of klotho administration on combating muscle atrophy.

[0186] Muscle atrophy can be caused by a number of neuromuscular, metabolic, immune, and neuropathy-related disorders and diseases, as well as starvation, nutritional deficiencies, metabolic stress, diabetes, aging, muscular dystrophy, or myopathy. Muscle atrophy can occur during the aging process. It can also result from reduced or disuse of muscles. Symptoms include a decrease in skeletal muscle tissue mass. In human males, muscle mass decreases by one-third between the ages of 50 and 80. Some molecular features of muscle atrophy include upregulation of ubiquitin ligases and a decrease in myofibrild proteins. The breakdown of these proteins can be monitored, for example, by myosin in a particular muscle (e.g., by measuring the production of 3-methylhistidine, a specific component of actin). The release of creatine kinase (a cell damage marker) can also be an indicator.

[0187] Administration of exogenous S-Klotho to treat intellectual and / or cognitive decline in old age and / or throughout human lifespan. Exemplary embodiments of this disclosure relate to the administration of exogenous klotho protein to improve and / or suppress cognitive decline (related to aging). At the time of this disclosure, it was unclear whether the administration of exogenous klotho protein could suppress cognitive decline in humans. However, transgenic mice with systemic overexpression of klotho performed better than controls in several learning and memory tests. Klotho elevation in mice also enhanced synaptic GluN2B, an N-methyl-D-aspartate receptor (NMDAR) subunit that enhances long-term potentiation and plays a crucial role in learning and memory, in the form of synaptic plasticity. Blockade of GluN2B resulted in the loss of the klotho-mediated effects.

[0188] Pathways that modulate Kloto may be associated with slowing the progression of Alzheimer's disease and other forms of dementia. Brain scans of more than 400 healthy men and women aged 53 and older found that those who possessed a single copy of a specific Kloto gene variant had larger brain regions associated with planning and decision-making. Further testing of the group found that those with an enlarged right dorsolateral prefrontal cortex (rDLPFC) performed better on a range of intellectual tasks.

[0189] Approximately one in five people inherit a single copy of a genetic variant or allele known as KL-VS, which improves heart and kidney function and extends human lifespan by an average of about three years. However, when it comes to brain function, having a larger rDLPFC accounts for only a 12% improvement in people's intelligence test scores. On the other hand, editors note that retaining one copy of the KL-VS allele appears to provide a 10-year resilience against the expected decline in the structure and function of the rDLPFC. Therefore, KL-VS heterozygosity appears to be associated with a larger volume of the right dorsolateral prefrontal cortex (rDLPFC).

[0190] Because rDLPFC is important for executive function, the researchers also analyzed individuals' working memory and processing speed. KL-VS heterozygosity may be associated with improved executive function over the lifespan tested. In short, the results suggest that Kroto gene diversity may be associated with larger brain volume and better function.

[0191] Exemplary embodiments of this disclosure relate to the administration of exogenous klotho protein to complement in vivo klotho levels and / or (cellular, molecular, and / or downstream) effects (e.g., to enhance cognitive function and suppress cognitive decline throughout the human lifespan). Exogenous administration of clinical-grade S-klotho preserves and / or improves cognitive function (e.g., in humans).

[0192] Administration of exogenous S-Klotho to treat (extend) human longevity and / or lifespan. Exemplary embodiments of this disclosure relate to improving life expectancy by administering exogenous Klotho protein to human subjects matched for actual age (e.g., age from birth) and sex. Life expectancy results obtained with Klotho administration (experimental group of human subjects) can be reliably compared with statistical information (e.g., from mathematical tables) for individuals not receiving exogenous Klotho administration (control group) and / or for a sufficiently large population.

[0193] Administration of exogenous S-Klotho to treat other clinical indicators Exemplary embodiments of the present disclosure relate to the administration of exogenous S-klotho for the treatment of any age-related or age-independent conditions, including, but not limited to, human frailty (increased), longevity (decreased), cellular senescence (decreased), muscle strength (decreased), bone loss or density (decreased), cognitive ability (decreased), muscle mass (decreased), physical fitness (decreased), grip strength (decreased), leg strength (decreased). The disclosure also relates to the administration of S-klotho for increasing bone mineral density (BMD) (e.g., in women, not men), increasing BMD that is reduced after menopause (e.g., in elderly women), regenerating (degenerated) skeletal muscle or reducing its degeneration, improving gait, spatial learning and memory, movement, freedom of movement, quality of life assessment, improving (or reducing) ejection fraction, changes in movement, and improvements in movement. This disclosure further relates to the administration of S-Klotho for reducing cognitive deterioration or amnesia, increasing cognitive abilities, improving cognitive function and synaptic plasticity, reducing declines in learning ability, learning ability or IQ, and improving learning ability, learning ability or IQ.

[0194] Administration of exogenous S-Klotho to treat genetic defects Exemplary embodiments of this disclosure relate to the administration of exogenous S-Klotho for the treatment (e.g., correction) of known human genetic defects. For example, a 13-year-old girl with familial neoplastic calcification and a Klotho variant has been reported. Familial neoplastic calcification is an autosomal recessive metabolic disorder characterized by ectopic calcification and hyperphosphatemia due to inactivating mutations in FGF23 or GALNT3. FGF23 is a hormone required for the renal excretion of phosphate, while GALNT is an enzyme that contributes to the maturation and secretion of FGF23. A homozygous mutation in the Klotho gene has been identified in a 13-year-old girl. Klotho encodes a secretory protein required for the transmission of signals that FGF23 releases toward its receptor. Administration of exogenous human recombinant S-Klotho constitutes a highly targeted and effective therapeutic approach to address the dysfunctions and symptoms associated with familial neoplastic calcification.

[0195] Administration of exogenous S-Klotho to treat acute kidney injury (AKI) Acute kidney injury (AKI), formerly known as acute renal failure (ARF), is often defined as the sudden onset of renal dysfunction ranging from mild impairment to complete failure. AKI is a common clinical complication, occurring in approximately 4%–7% of hospitalized patients each year, and the prognosis may not be favorable. The mortality rate associated with AKI ranges from 20% to 35%. Renal Klotho expression has been shown to be suppressed following AKI. Adenovirus gene transfection of Klotho may be cytoprotective in AKI.

[0196] Acute kidney injury (AKI) is reported in approximately 4.9% to 7% of hospitalized patients each year. The rate of AKI can be as high as 60% in older patients (hospitalized) and 20-30% in older or critically ill patients. AKI is also associated with increased mortality, length of stay (LOS), and hospital costs.

[0197] Acute kidney injury (AKI) can result at least partially from kidney transplantation or other surgical procedures, acute tubular necrosis (ATN), acute allergic interstitial nephritis (AAIN), nephritis (e.g., glomerulonephritis), nephrotoxicity (e.g., drug-induced nephrotoxicity), hypotension, or other contributing factors. Kidney transplantation and other surgical procedures can cause acute injury or damage to the kidney, leading to renal disease and / or renal failure. Nephrotoxicity can contribute to AKI, ATN, AAIN, nephritis, etc. Drugs (e.g., clinically administered prescription drugs, illegal drugs, or other drugs) have been reported to be associated with 15%–25% of all cases of AKI. Contrast agents alone account for 10% of all causes of hospital-acquired acute renal failure (e.g., contrast-induced acute kidney injury (CIAKI)) and are the third leading cause of hospitalization-induced deterioration of renal function and postoperative renal insufficiency after reduced renal perfusion.

[0198] In some cases, drug-induced AKI may be, or may include, nephrotoxicity induced by antimicrobial agents (resulting from antimicrobial treatment). For example, certain (Gram-negative) bacterial infections may be treated with one or more aminoglycosides, such as paromomycin, tobramycin, gentamicin, amikacin, kanamycin, and neomycin. Aminoglycosides have been shown to be nephrotoxic. For example, as shown in Figure 4, nearly 23% of patients treated with amikacin, a common aminoglycoside, developed acute kidney disease, and over 17% of patients treated with amikacin died before discharge. Other aminoglycosides, including gentamicin and tobramycin, also induced (or were associated with or contributed to) kidney disease. As shown in Figures 3A and 3B, more than 1.2 million adult patients of various age groups were treated with aminoglycosides in 2010. Other antibacterial agents include, for example, penicillin, ampicillin, cephalosporins, sulfonamides, ciprofloxacin, vancomycin, macrolides, tetracyclines, and rifampin.

[0199] Drug-induced nephrotoxicity can also result from treatment with one or more nonsteroidal anti-inflammatory drugs (NSAIDs), such as aspirin (acetylsalicylic acid), celecoxib, diclofenac, diflunisal, etodolac, ibuprofen, indomethacin, ketoprofen, ketorolac, nabumetone, naproxen, oxaprozin, piroxicam, sarsalate, sulindac, and tolmetin.

[0200] Drug-induced nephrotoxicity can also be caused by one or more cyclooxygenase-2 (COX-2) inhibitors (e.g., valdecoxib, rofecoxib, celecoxib), proton pump inhibitors (e.g., omeprazole, lansoprazole), anticonvulsants (e.g., phenytoin, valproic acid), histamine H2 receptor antagonists (e.g., nizatidine, ranitidine, famotidine, cimetidine), diuretics (e.g., carbonic anhydrase inhibitors, loop diuretics (e.g., etc.)). It can result from treatment with other diuretics such as bumetanide, ethacrine, torasemide, furosemide, potassium-sparing diuretics (e.g., triamterene, spironolactone, amiloride, etc.), thiazide diuretics (e.g., indapamide, chlorthalidone, metrazon, meticlothiazide, hydrochlorothiazide, chlorothiazide, bendroflumethiazide, polythiazide, hydroflumethiazide, etc.), or pamabrom, mannitol, etc.

[0201] Drug-induced nephrotoxicity can also arise from treatment with lithium, which can affect the flow of sodium through nerve and muscle cells in the body, and is often used to treat manic episodes of bipolar disorder, characterized by hyperactivity, urgency, impaired judgment, reduced need for sleep, aggression, and anger. Lithium may also help prevent or reduce the severity of manic episodes. Drug-induced nephrotoxicity can also arise from treatment with or exposure to gold, mercury, copper, or other elemental substances.

[0202] Drug-induced nephrotoxicity can also arise from treatment with chelating agents such as (D-)penicillamine, which may be used to treat scleroderma, Wilson's disease (due to the accumulation and binding of copper to remove it through urine), and cysteinuria (due to the binding of copper with cysteine ​​to form a mixed disulfide that is more soluble than cysteine); muscle relaxants that act directly on smooth muscle (e.g., hydralazine); antispasmodics (e.g., benzodiazepines such as carisoprodol, cyclobenzaprine, metaxalone, methocarbamol, diazepam, clonidine and other imidazoline compounds, tizanidine, baclofen, hydantoin derivatives, dantrolene, etc.).

[0203] Other forms of drug-induced nephrotoxicity include, for example, contrast-induced nephrotoxicity (e.g., nephrotoxicity induced by radioactive contrast agents following exposure to (iodinated) contrast agents, also known as CIN); opioid-induced nephrotoxicity (e.g., after use or abuse of certain opioids such as cocaine and heroin); and chemotherapy-induced nephrotoxicity (e.g., cisplatin; carboplatin; oxaliplatin; bendamustine, cyclophosphamide, ifosfamide, nitrosourea, temozolam). Examples include alkylating agents such as zolomid and melphalan; antitumor antibiotics such as mitomycin C, bleomycin, anthracyclines, and related drugs; antimetabolites such as capecitabine, hydroxyurea, methotrexate, pemetrexed, pralatrexate, pentostatin, fludarabine, cladribine, gemcitabine, and cytarabine; vinca alkaloids; topotecan; etoposide; taxanes; irinotecan; lenalidomide; eribulin; arsenic trioxide; and ixazomib (after treatment with these cancer drugs). In fact, a wide variety of nephrotoxic drugs can induce nephrotoxicity and lead to AKI. Drug-induced nephrotoxicity (and other forms of AKI) can be life-threatening if left untreated and can incur enormous treatment costs (for patients, hospitals, and insurance companies).

[0204] Embodiments of the present disclosure may include methods for treating or preventing (prophylactic) acute kidney injury (AKI) or other conditions. The methods may include administering recombinant (soluble) Kroto protein to a subject in need. For example, the methods may include administering a pharmaceutically effective amount of recombinant soluble Kroto protein having at least 85% amino acid sequence identity with one of SEQ ID NOs: 2 to SEQ ID NOs: 70 to a subject in need (e.g., to increase and / or maintain the subject's serum soluble Kroto protein concentration above a predetermined threshold for a predetermined period). The conditions may include (i) acute tubular necrosis (ATN), acute allergic interstitial nephritis (AAIN), nephritis, glomerulonephritis, and / or nephrotoxicity, or (ii) AKI resulting at least partially from kidney transplantation or other surgical procedures, acute tubular necrosis (ATN), acute allergic interstitial nephritis (AAIN), nephritis, glomerulonephritis, nephrotoxicity, or hypotension. This condition may include drug-induced (e.g., aminoglycoside-induced) nephrotoxicity. The protein may be administered prophylactically, for example, before kidney transplantation, administration of nephrotoxicides, or other activities, treatments, or events known or expected to cause or contribute to AKI. Alternatively, or in addition, the protein may be administered in response to AKI, such as after kidney transplantation or other surgical procedures, administration of aminoglycosides or other nephrotoxicides, or other activities, treatments, or events known or expected to cause or contribute to AKI.

[0205] In some embodiments, nephrotoxins or other agents are, for example: One or more aminoglycosides (e.g., paromomycin, tobramycin, gentamicin, amikacin, kanamycin, neomycin, etc.); One or more antifungal agents (e.g., amphotericin B, flucytosine, etc.); One or more contrast agents (e.g., (iodized) radiocontrast agents, hyperosmolar contrast agents (HOCM) with an iodine-to-molecular ratio of approximately 1.5:1, hypoosmolar nonionic contrast agents (LOCM) with an iodine-to-molecular ratio of approximately 3:1, isotonic (isoosmolar) contrast agents (IOCM) with an iodine-to-molecular ratio of approximately 6:1), etc.); One or more antiretroviral agents (e.g., adefovir, cidofovir, tenofovir, foscanet, etc.); One or more cancer (or chemotherapy) drugs (e.g., cytoplatin, carboplatin, oxaliplatin, alkylating agents (bendamustine, cyclophosphamide, ifosfamide, nitrosourea, temozolomide, melphalan, etc.), antitumor antibiotics (mitomycin C, bleomycin, anthracyclines and related drugs, etc.), antimetabolites (capecitabine, hydroxyurea, methotrexate, pemetrexed, pralatrexate, pentostatin, fludarabine, cladribine, gemcitabine, cytarabine, etc.), vinca alkaloids, topotecan, etoposide, taxanes, irinotecan, lenalidomide, eribulin, arsenic trioxide, ixazomib, etc.); One or more bisphosphonates or derivatives thereof (e.g., zoledronate / zoledronic acid, ibandronate, alendronate, alendronate / cholecalciferol, etidronate, risedronate (optionally containing calcium carbonate), pamidronate, chydronic acid, etc.); and / or It may be, or may contain, one or more narcotics (e.g., opioids) such as cocaine and heroin; Embodiments of the present disclosure may include a method for administering a therapeutic recombinant (alpha-soluble) Kroto protein (e.g., having at least 85% amino acid sequence identity with amino acid residues 1-981 or a subset thereof of human alpha-Kroto isoform 1). The method may include administering the therapeutic Kroto protein to a human or non-human subject to (prophylactically) treat or prevent one or more conditions associated with AKI or AKI. The method may include measuring the level of serum soluble Kroto in the subject; calculating a first dose of protein sufficient to raise the serum soluble Kroto level in the subject to a predetermined level or a percentage of a normal level; administering the first dose of protein to the subject by means of a bolus or stepwise administration; measuring the rate of decrease of serum soluble Kroto, such as following the administration of the first dose; calculating the timing and amount of the next dose; and / or administering the next dose of protein to the subject.

[0206] Administration of exogenous S-Klotho for the treatment of chronic kidney disease (CKD) As described in Neyra and Hu, Potential application of klotho in human chronic kidney disease, Bone (2017), the entirety of which is incorporated herein by reference, circulating soluble klotho begins to decline early in chronic kidney disease (CKD) stage 2, and urinary klotho likely begins to decline even earlier in CKD stage 1. Therefore, soluble klotho may serve as an early and highly sensitive marker of renal decline. Furthermore, preclinical animal data support that klotho deficiency is not merely a biomarker but also a pathogenesis of CKD progression and extrarenal CKD complications, including cardiovascular disease and mineralogy impairment. Prevention of klotho decline, reactivation of endogenous klotho production, or supplementation of exogenous klotho are all associated in animal models with attenuation of renal fibrosis, delay of CKD progression, improvement of mineralogy, improvement of cardiomyopathy, and mitigation of vascular calcification due to CKD.

[0207] CKD is characterized by progressive deterioration of renal function, which carries a high risk of ESRD. The risk of CKD increases with age, occurring in approximately half of cases with CKD stage ≥3 in subjects N70 years of age. CKD can be considered an accelerated state of aging. The relative risk of cardiovascular mortality in dialysis patients aged 25-34 years is similar to that of non-CKD patients N75 years of age. Cardiovascular disease is the leading cause of death in patients with CKD and ESRD. Patients with CKD and ESRD have low renal Klotho expression and low levels of circulating Klotho. Renal Klotho deficiency in the early stages of CKD may be primarily due to suppression of Klotho expression rather than a decrease in viable renal tubules. Furthermore, some dialysis patients still have detectable circulating Klotho, and renal Klotho expression is not completely suppressed, suggesting that the origin of Klotho may be extrarenal, although the origin is not yet clear. Establishing extrarenal sources of Klotho and characterizing how Klotho may be upregulated when renal production fails is of paramount importance.

[0208] Administration of the exogenous klotho protein disclosed herein may help prevent, delay, and reduce the burden of comorbidities in CKD. Compositions and treatments containing S-Klotho in combination with other components Kroto may also act additively or synergistically with other compounds and / or components that affect one or more aspects of human health and well-being. For example, a treatment involving the use of therapeutic human recombinant soluble alpha-kroto (S-kroto) protein in combination with and / or in parallel with one or more additional active ingredients may be beneficial to a human patient. Such a treatment may be prophylactic or responsive to any human condition in which kroto protein and / or other components may have a therapeutic effect. Such conditions may include, for example, age-related conditions, metabolic conditions, chronic or acute conditions. Certain non-limiting examples of specific conditions are disclosed herein.

[0209] S-Klotho can be present in the human body alongside other blood-borne anti-aging compounds such as growth / differentiation factor 11 (GDF-11). Therefore, in certain embodiments, therapeutic S-Klotho may be co-administered with therapeutic GDF-11 (e.g., in parallel, sequentially, and / or in combination). In some embodiments, such administration may have additive or synergistic anti-aging or other effects. Similarly, co-administration of S-Klotho to human subjects using (neutralizing) antibodies against CCL11 or its inhibitors may act in sync to counteract aging or other pathological conditions (since CCL11 (also known as eotaxin-1) is understood to be a negative regulator of stem cell rejuvenation). S-Klotho may also be co-administered with other eotaxins, such as eotaxin-2 (CCL24) and / or eotaxin-3 (CCL26).

[0210] In some embodiments, S-Klotho can be co-administered with a transforming growth factor β-1 (TGF-β1) inhibitor or antibody. S-Klotho administration may counteract the action of the TGF-β1 signaling pathway involved in endogenous anti-epithelial-mesenchymal transition (anti-EMT), which leads to renal fibrosis and fibrosis of other tissues. Anti-EMT is also associated with cancer cells, and inhibition of EMT may confer metastatic ability to cancer cells; this latter process is understood to be counteracted by Klotho. Therefore, co-administration of S-Klotho with a transforming growth factor β-1 (TGF-β1) inhibitor or antibody may have synergistic or additive effects.

[0211] In some embodiments, S-Klotho can be co-administered with an antibody or inhibitor of insulin growth factor-1 (IGF-1). Klotho is a hormone that inhibits the intracellular insulin / IGF-1 signaling cascade, and this inhibition is understood to be an evolutionarily conserved mechanism that increases resistance to oxidative stress at the cellular and biological levels in mammals and extends lifespan. Therefore, co-administration of S-Klotho with an inhibitor or antibody of insulin growth factor-1 (IGF-1) may have synergistic or additive effects.

[0212] Numerous studies have revealed a comprehensive regulatory scheme of mineral homeostasis, including the mutually regulated positive / negative feedback action of klotho-alpha-K1, FGF23, and 1,25(OH)2D, as well as a similar regulatory network comprising klotho-β-K1, FGF15 / human FGF19, and bile acids that regulate bile acid / cholesterol metabolism. Therefore, in some embodiments, S-klotho can be administered in combination with vitamin D (e.g., vitamin D3), or 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], FGF-15, FGF-19, and / or klotho-β. Such co-administration may have synergistic or additive effects on numerous pathological conditions and / or processes in the body. In some embodiments, S-klotho can be administered in combination with FGF-21. In some embodiments, S-Klotho can be co-administered with carbonic anhydrase inhibitors such as acetazolamide, metazolamide, dichlorfenamide, dorzolamide, brinzolamide, and / or topiramate. Such combinatorial administration may be useful in the treatment of ankylosing spondylitis (AS), rheumatoid arthritis (RA), and various other conditions. Various studies have shown that increased bone resorption is characteristic of AS and RA, and that carbonic anhydrase inhibitors play an anti-arthritis role by inhibiting bone resorption. At the bone level, S-Klotho stimulates bone resorption and phosphate release through a different mechanism by which it acts on TRPV5, an osteoclast function regulator recently identified. + Increased levels of D3 also stimulate osteoclast differentiation and bone resorption, thereby releasing phosphate. Therefore, co-administration of S-Klotho with carbonic anhydrase inhibitors may have additive or synergistic effects, particularly in promoting bone health, especially in AS and RA.

[0213] For the treatment of severe, active rheumatoid arthritis, S-Klotho may be administered in combination with one or more disease-modifying antirheumatic drugs (DMARDs). Since cyclosporine reduces klotho mRNA and protein and increases oxidative stress, which can lead to cyclosporine-induced renal injury (CsA), S-klotho may be administered in combination with cyclosporine. The associated reduction in klotho mRNA and protein and the increase in oxidative stress can be counteracted by exogenous co-administration of S-klotho.

[0214] In some embodiments, S-Klotho can be co-administered with losartan and / or cyclosporine. Treatment with losartan, an angiotensin type II1 (AT1) receptor blocker, reversed the reduction in Klotho expression seen with cyclosporine. Losartan also resulted in parallel improvements in renal tissue (with losartan reducing tubulointerstitial fibrosis caused by cyclosporine).

[0215] In some embodiments, S-Klotho can be co-administered with one or more aminoglycosides, such as amikacin, gentamicin, and tobramycin. The use of aminoglycosides in the treatment of infections can be greatly expanded. When aminoglycosides are used to treat infections of (Gram-negative) pathogens, such treatment may be useful in preventing nephrotoxicity and / or acute kidney injury (AKI). Administering S-Klotho together with verapamil and / or diltiazem, which have been used to block AKI, may be therapeutic in treating and / or preventing renal impairment from AKI.

[0216] In some embodiments, S-Klotho can be co-administered with testosterone or androgen receptor (AR) upregulation compounds. Recent reports have shown that testosterone treatment in men has no beneficial effects on personality, psychological well-being, or mood. In addition, the prescription of testosterone replacement for low T as cardiovascular health, sexual function, physical function, mood, or cognitive function has been considered to lack support from randomized clinical trials. However, while testosterone replacement has consistently been found to increase muscle strength, no beneficial effects on physical function have been found. S-Klotho administration in combination with testosterone and / or androgen receptor (AR) upregulation compounds may significantly increase muscle strength and / or physical function in older, frail, or low T men beyond any effects that testosterone or S-Klotho alone may have on these treatment groups.

[0217] In some embodiments, S-Klotho may be co-administered with estrogen or estrogen hormones (e.g., estradiol, estriol, estrone, etc.). Such co-administration may improve women's health indicators (e.g., menstruation, menopause, or women transitioning to menopause) and / or treat infertility, polycystic ovary disease or disorder, obesity, hormonal imbalances and related conditions, and / or other women's health conditions.

[0218] In some embodiments, S-klotho may be co-administered with one or more nootropics, also known as smart drugs or cognitive enhancers. Nootropic drugs, supplements, and / or other substances may improve cognitive function, particularly executive ability, memory, creativity, motivation, task saliency (motivation to perform tasks), and performance (especially tedious tasks requiring significant effort), and may be useful in treating cognitive or motor impairments that cause disorders such as Alzheimer's disease, Parkinson's disease, Huntington's disease, and ADHD. The most commonly used class of drugs known to improve several aspects of cognition is the class of stimulants, which exert cognitive-enhancing effects on humans by acting as direct or indirect agonists on dopamine receptor D1, adrenergic receptor A2, or both of these receptors in the prefrontal cortex. Examples of stimulants include amphetamines (e.g., amphetamine, dextroamphetamine, lisdexamfetamine, etc.), which may be beneficial to a range of cognitive functions (e.g., inhibitory control, episodic memory, working memory, and attention) particularly in individuals with ADHD; and dimethylamylamines (DMAAs), such as 1,3-dimethylamylamine, which may improve physical ability, alertness, and reaction time.

[0219] Examples include methylphenidate-substituted phenethylamines that can improve a range of cognitive functions (e.g., working memory, episodic memory, and inhibitory control, attention, and planning latency); eugeroics (e.g., almodafinil, modafinil, etc.) that can act as wakefulness enhancers, particularly in sleep-deprived individuals, to facilitate logical thinking and problem-solving, and to treat narcolepsy, sleep disorders due to shift work, and daytime sleepiness that persists after treatment for sleep apnea; xanthines (e.g., caffeine, etc.) that can enhance alertness, performance, and / or memory; and nicotine.

[0220] In some embodiments, S-Klotho may be co-administered with one or more osteoporosis and / or osteopenia drugs, as is known in the art. Klotho may play a role in regulating bone mineral density, and the absence of Klotho may lead to a decrease in bone mineral density in animals. For example, Klotho knockout mice show a decrease in bone mineral density over time. Klotho expression may rescue bone defects in Klotho knockout animals, as Klotho knockout mice show a decrease in bone mineral density over time. Epidemiological studies have shown associations between various Klotho gene variants and changes in bone mineral density and the prevalence of osteoarthritis of the hand.

[0221] S-Klotho can be administered in combination with one or more anticancer therapies and / or preventive measures, such as chemotherapy. In lung cancers, such as non-small cell lung cancer (NSCLC), S-Klotho administration may, for example, affect the resistance of lung cancer cells to cisplatin and / or other chemotherapy. In addition, S-Klotho may act 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). Overexpression of Klotho and treatment with soluble Klotho protein may reduce hepatocellular carcinoma cell proliferation in vitro and in vivo. Other types of cancer that can be treated with S-Klotho include hepatocellular carcinoma (HCC), central nervous system (CNS) cancers (e.g., brain (e.g., glioma, craniopharyngioma, medulloblastoma, and meningioma), spinal cord and other tumors, lymphoma, etc.), and (metastatic) colon cancer.

[0222] S-Kloto may also be administered in combination with chemotherapy to treat chemotherapy-induced weakness in cancer patients. S-Kloto may also be administered following other known treatments to treat cancer-induced weakness in cancer patients.

[0223] S-Klotho may be administered in combination with renal dialysis or other treatments. Since weakness is associated with a poor prognosis in dialysis patients, S-Klotho may be administered to treat weakness in dialysis patients.

[0224] Because appropriate amounts of BDNF may help develop and maintain normal neuronal circuits in the brain, S-Klotho may be administered in combination with one or more treatments or preventative measures for Alzheimer's disease, or in combination with brain-derived neurotrophic factor (BDNF).

[0225] To enhance klotho's ability to enter the central nervous system (CNS) in order to treat or prevent CNS-related pathological conditions, S-klotho may be administered in combination with one or more molecules that enhance klotho's ability to cross the blood-brain barrier. For example, both S-klotho and BDNF are known not to cross the blood-brain barrier. Embodiments of this disclosure include utilizing blood-brain barrier delivery techniques for administering S-klotho, including S-klotho and / or BDNF, to the CNS in order to treat Alzheimer's disease and / or to improve cognitive function in individuals not suffering from Alzheimer's disease.

[0226] S-Klotho may be administered in combination with 5'-adenosine monophosphate-activated protein kinase (AMPK), or AMPK activators, or components that positively modulate signaling pathways that replenish cellular ATP supply, including fatty acid oxidation and autophagy, or negatively modulate ATP-consuming biosynthetic processes, including gluconeogenesis, lipid and protein synthesis.

[0227] S-Klotho may be administered in combination with one or more antidiabetic agents such as insulin, phlorizin, or the antioxidant tyron, and these combination therapies may have benefits in preventing renal damage caused by oxidative stress resulting from diabetic complications. Co-administration of S-Klotho with other antidiabetic agents for type 1 diabetes may protect β-cells by inhibiting β-cell apoptosis through activation of the integrin β1-FAK / Akt pathway, leading to inhibition of caspase 3 cleavage.

[0228] S-Klotho may be administered in combination with one or more type 2 antidiabetic agents, such as metformin, to improve glycemic control and vascular function in overweight and obese diabetic subjects. S-Klotho may be administered in combination with one or more antihypertensive drugs, calcium regulators, or for the treatment or prevention of chronic kidney disease (CKD). For example, soft tissue calcification is a prominent feature of CKD, and Klotho may improve vascular calcification by improving phosphateuria, maintaining glomerular filtration rate, and directly inhibiting phosphate uptake by vascular smooth muscle.

[0229] Since PAI-1 inhibition or deficiency is thought to slow the progression of aging and extend the lifespan of Klotho-deficient (kl / kl) mice while protecting organ structure and function, S-Klotho may be administered in combination with TM5441 or other PAI-1 inhibitors (plasminogen activator 1).

[0230] S-Klotho can be administered in combination with sirtuin 1 (SIRT1) or SIRT1 activating compounds (STACs) such as resveratrol. SIRT1, a type III protein deacetylase, is considered a novel anti-aging protein involved in the modulation of cellular senescence / aging and inflammation. SIRT1 levels and activity decrease during lung inflammation caused by oxidative stress. SIRT1-mediated defense mechanisms against inflammation are involved in the modulation of inflammation, premature aging, telomere shortening, aging-related secretory phenotypes, and DNA damage responses. Various dietary polyphenols and pharmacological activators have been shown to modulate SIRT1 to intervene in the progression of type 2 diabetes, cancer, cardiovascular disease, and chronic obstructive pulmonary disease associated with inflammation. Therefore, some or all of the health benefits of SIRT-1 may be complemented by co-administration of SIRT1 and / or SIRT-activating compounds administered with S-Klotho.

[0231] S-Kroto may be administered in combination with one or more human cells, tissues, or cell and tissue lineage products (HCT / Ps) approved by the FDA. Such products may include, for example, one or more bones (including demineralized bone, ligaments, tendons, fascia, cartilage, ocular tissue (cornea and sclera), skin, and vascular grafts (veins and arteries)), excluding preserved umbilical veins, pericardium, amniotic membrane (when used alone for eye repair (without added cells)), dura mater, cardiac valve allogeneic grafts, peripheral blood or umbilical cord blood, semen, and hematopoietic stem cells derived from oocytes or embryos. In at least one embodiment, the HCT / P may be or contain one or more stem cells. Stem cell therapy for damaged body tissues and organs is becoming common. Administration of recombinant Kroto protein for therapy in combination with stem cells has provided surprising, unexpected, and even synergistic results for subjects in need.

[0232] Embodiments of this disclosure further include combination products comprising recombinant Kroto protein for therapeutic use in combination with human stem cells. The compositions may also include pharmaceutically acceptable carriers as described herein. Such compositions may be included, or may be classified as, FDA-approved regenerative agents for treating, modifying, reversing, or curing serious or life-threatening diseases or conditions. Preliminary clinical evidence indicates that the compositions (agents) have the potential to address medical needs that are not adequately addressed for such diseases or conditions.

[0233] Exemplary, stem cells may be or include mesenchymal stem cells (MSCs), such as those derived from human umbilical cord or placenta. In at least one embodiment, compositions comprising huMSCs and recombinant Kroto protein for therapeutic purposes of this disclosure attenuated the inflammatory and oxidative stress responses occurring in AKI and / or reduced the expression of proteins and microRNAs associated with aging.

[0234] S-Kroto can be administered in combination with one or more anti-aging inhibitors. For example, Kroto protein may improve outcomes in patients receiving such treatment when combined with Pin1-FOXM1 and / or other anti-aging inhibitors.

[0235] S-Klotho may be administered in combination with one or more of the following: Klotho stimulants (Vit.D, losartan, testosterone), GDF-11, trichostatin A antifungal agents (GDF-11 stimulants), TIMP-2, CCL-11 inhibitors / antibodies, dasatinib, nicotinamide riboside (NAD+), nicotinamide mononucleotide (NMN) (NDA+), AMPK stimulants (resveratrol, aspirin, salicylate, phytochemicals, DR), C60 fullerene, rapamycin, FGF inhibitors, senolytic agents / compounds such as FOXO4-p53 interfering peptides (e.g., FOXO4-DRI), and inhibitors of the anti-apoptotic proteins BCL-2 and BCL-xL.

[0236] Any of the aforementioned or other treatments or co-administrations may have additive or synergistic effects compared to any treatment alone. For example, co-administration of Klotho protein with one or more of the aforementioned may produce greater therapeutic results than the sum of the individual results of administering the individual components at similar concentrations. In addition, synergistic effects may include therapeutic results similar to those of administering the individual components at lower concentrations. Synergistic effects may also include an increase in the maximum effective dose of one or more components, a reduction in the toxicity of one or more components, or any other beneficial results beyond the mere additive effect of the individual therapeutic results. Furthermore, the additive effect of individual therapeutic results may include synergistic effects that, given the nature and understanding of the individual components, would not have been predicted or expected.

[0237] As used herein, combination therapy or co-administration may include the treatment or administration of a combination product, composition, or formulation comprising Kroto protein and one or more additional active ingredients. One or more additional active ingredients may be selected from the ingredients, drugs, substances, therapeutic compositions, etc., described herein, or from others known in the art. For example, Kroto protein and one or more additional active ingredients may be co-composed in an injectable (e.g., intramuscular, intravenous, etc.), ingestible, transdermal, inhalable, topical, or other formulation.

[0238] Alternatively, combination therapy or co-administration may involve the treatment or administration of Klotho protein and one or more additional active ingredients, which are not combined or formulated in a combination product, composition, or formulation. For example, Klotho protein and one or more additional active ingredients each comprise or may be separately injectable (e.g., intramuscular, intravenous, etc.), ingestible, transdermal, inhalable, topical, or other formulations.

[0239] Furthermore, co-administration may include simultaneous administration of two or more components, or separate administration of two or more components, and it will be understood that separate administrations are preferably separated by a certain period of time. In some embodiments, the period may be very short. For example, immediately after administration of a first component of Klotho protein and one or more additional active ingredients, a second component of Klotho protein and one or more additional active ingredients may be substantially administered (e.g., by injection). Alternatively, the first and second administrations may be separated by a period of time such as 1 to 60 seconds, 1 to 60 minutes, 1 to 24 hours, 1 to 7 days, 1 to 4 weeks, 1 to 12 months, or any value or a range of values ​​in between. Similarly, co-administration may include overlapping administration timeframes for two or more components.

[0240] Kroto protein variant Therapeutic S-Kroto proteins of various lengths (e.g., S-Kroto 1-981, 29-981, 34-981, 36-981, 131-981, 1-549, 29-549, 34-549, 36-549, 131-549, etc.) can be modified in various ways to achieve a variety of beneficial effects and / or results not exhibited by native Kroto proteins. Exemplarily, the nucleic acid sequences of various S-Kroto constructs can be altered using the QuickChangeXL site-directed mutagenesis kit (Stratagene). Other mutagenesis methods and kits known in the art may also be used. For example, various subcloning methods and kits are known and commercially available in the art.

[0241] In at least one embodiment of this disclosure, a protein is modified with one or more C-terminal tags and / or N-terminal tags. Such tags may function to extend the soluble half-life of the serum and / or protein (in one or more therapeutic or other settings). The tags may 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., proteins, nucleic acids, organelles, cellular structural components, etc.), enzymatic processing or cleavage, etc. In at least one embodiment, the C-terminus of the protein may be tagged with a TEV-TwinStrep and / or Fc fusion, as is known in the art and further described herein. Additional descriptions can be found in the literature “Fusion Proteins for Half-Life Extension of Biologics as a Strategy to Make Biobetters”, “What is the future of PEGylated therapies?” and “Strategies for extended serum half-life of protein therapeutics,” each of which is incorporated herein by specific reference. In certain embodiments, a linker or linker peptide may be inserted and / or positioned between the (natural or variant) Kroto protein sequence and the tag.

[0242] In some embodiments, the modified Kroto protein may include alternative (e.g., native, non-native, and / or synthetic) signal peptides. For example, in some embodiments, the native signal peptide sequence may be replaced and / or supplemented with the alternative signal peptide or signaling sequence (SS). In some embodiments, the native methionine residue of the Kroto protein may be removed, and a methionine residue may be included at the N-terminus of the SS. Further explanation can be found in the paper “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,” which is invoked in its entirety by specific reference. In certain embodiments, a linker or linker peptide may be inserted and / or positioned between the (native or variant) Kroto protein sequence and the alternative SS.

[0243] Some embodiments may include one or more amino acid variants. It will be understood that this disclosure considers diversifying any one or more of the native amino acids of any of the disclosed Kroto proteins to any other amino acids, whether native, synthetic, or otherwise.

[0244] S-Kroto C370S protein variant In humans, the Klotho gene is located on chromosome 13q12. A variant known as KL-VS is present in approximately 15% of Caucasians. This variant consists of six single nucleotide polymorphisms (SNPs), two of which cause amino acid substitutions (i.e., F352V and C370S - phenylalanine 352 is changed to valine, and cysteine ​​370 is changed to serine). In vitro transfection assays have shown that Klotho secretion levels were reduced sixfold in the V352 variant, while they were increased nearly threefold in the S370 variant. However, these two variants in the human Klotho gene segregate together, forming a KL-VS haplotype that increases Klotho secretion by a range of 1.6 times. For example, screening of more than 300 individuals taken from geographically and / or ethnically separate cohorts has reported that no individuals possessing only one of the V352 or S370 variants have been found.

[0245] One embodiment of the present disclosure comprises a recombinant S-Kroto protein having a C370S homogeneous variant (i.e., the absence (or deletion) of the F352V variant). The C370S variant may be produced and / or expressed in relation to any protein construct described herein. For example, the C370S variant may be produced or expressed in relation to S-Kroto 1-981, 29-981, 34-981, 36-981, 131-981, 1-549, 29-549, 34-549, 36-549, 131-549, etc., with or without the Fc fusion and / or TEV-TwinStrep. Thus, the nucleic acid construct or cDNA on which the protein is expressed may be of the corresponding length.

[0246] Embodiments may include producing a heterozygous or homozygous S370 variant construct, introducing the resulting construct encoding the S-Kroto C370S protein into a suitable expression system (e.g., CHO cells) (e.g., via transfection), and / or transiently expressing the S-Kroto S370 protein. The S370 Kroto protein may be expressed at higher levels than the F352V / C370S protein and / or the wild-type F352 / C370 protein. Embodiments may include purifying the expressed protein for therapeutic administration (and optionally performing quality control testing). Embodiments may include administering a therapeutic or therapeutically effective amount of the S-Kroto C370S protein to a subject in need. The subject may, for example, harbor or express the KL-VS variant. Alternatively, the subject may be wild-type of another variant or variant. Administration of recombinant S-Klotho C370S protein may lead to a beneficial increase in blood S-Klotho levels. Therefore, the circulating concentration of S-Klotho in subjects administered with therapeutic recombinant S-Klotho C370S protein may not be subject to the dilution effect observed in the presence of the F352V variant.

[0247] Therapeutic treatment of hyperphosphatemic familial neoplastic calcification (HFTC) In affected human individuals, HFTC is caused by a mutation in the amino acid (AA) 193 position of S-Klotho-rs121908423, from histidine (H) to arginine (R). While not bound by any theory, it is thought that the H193R mutation in HFTC individuals impairs S-Klotho's ability to form a triplicate complex with FGF23 and FGFR1c, thereby impairing KL-dependent FGF23 signaling. As a result, affected subjects exhibit severe metabolic disorders, manifesting as hyperphosphatemia and massive calcium deposition in the skin and subcutaneous tissue. Some patients present with recurrent, transient, painful swelling of long bones, associated with radiological findings of periosteal reaction and cortical hyperostosis and lack of skin infiltration.

[0248] One embodiment of this disclosure comprises an S-Kroto protein having H193. The H193 protein may be produced or expressed in relation to any protein construct described herein. For example, the H193 variant may be produced or expressed in relation to S-Kroto 1-981, 29-981, 34-981, 36-981, 131-981, 1-549, 29-549, 34-549, 36-549, 131-549, etc., with or without Fc fusions and / or TEV-TwinStrep. Thus, the nucleic acid construct or cDNA on which the protein is expressed may have the corresponding configuration.

[0249] Embodiments may include producing an H193 heterozygous or homozygous variant construct, introducing the resulting construct encoding the S-Krotoh H193 protein into a suitable expression system (e.g., CHO cells) (e.g., via transfection), and / or transiently expressing the S-Krotoh H193 protein. The H193 Krotoh protein may also be expressed at higher levels than the R193 (or H193R) protein. Embodiments may include purifying the expressed protein for therapeutic administration (and optionally performing quality control testing). Embodiments may include administering a therapeutic or therapeutically effective dose of the S-Krotoh H193 protein to a subject in need (e.g., an HFTC individual, an individual diagnosed with HFTC, or a patient harboring H193R (rs121908423) or another variant). Alternatively, the subject may be a wild-type of another variant or variant. Administration of recombinant S-Klotho H193 protein may lead to a beneficial increase in blood S-Klotho levels. The administration may reverse or suppress the adverse effects of the R193 variant, which is transcribed and circulated in HFTC individuals as a result of the H-~-R193 point mutation found in the human Klotho gene of individuals affected by HFTC. Therefore, circulating concentrations of S-Klotho H193 may help counteract the effects observed in H193R or HFTC individuals.

[0250] Therapeutic treatment for patients with the CC genotype and end-stage renal disease (ESRD) Approximately 350,000 patients with end-stage renal disease (ESRD) suffer a very high mortality rate in the first year of habitual hemodialysis. Both vitamin D and fibroblast growth factor (FGF)-23 levels correlate with survival in these patients. While not bound by any theory, klotho is a protein in the vitamin D / FGF-23 signaling pathway and has been associated with accelerated aging and premature death in animal models. It has been hypothesized that genetic variations in the klotho gene may be associated with survival in subjects with ESRD. Researchers tested the association between 12 single nucleotide polymorphisms (SNPs) in the klotho gene and mortality in a cohort of ESRD patients (n=1307, Caucasian and Asian) during the first year of hemodialysis. A significant association was found between the CC genotype of a single tag SNP, rs577912 (a common HapMap variant with a minor allele frequency [MAF] > 0.05 in the Kroto gene sequence located in intron 1) and an increased risk of 1-year mortality (RR, 1.76; 95% CI, 1.19–2.59; p=0.003). This effect was even more pronounced in individuals with the CC genotype among patients not treated with active vitamin D supplementation (HR, 2.51; 95% CI, 1.18–5.34; p=0.005). In lymphoblastoid cell lines derived from HapMap subjects, the CC genotype was associated with 16–21% lower Kroto expression compared to the AA or AC genotypes. However, none of the rs577912 SNP nucleotide alterations described above result in amino acid alterations of the Kroto protein. Therefore, this functional SNP (rs577912) may quantitatively affect Kroto gene expression at the mRNA level.

[0251] One embodiment of this disclosure comprises an S-Kroto protein expressed from an AA or AC genotype. The protein may be produced or expressed in relation to any protein construct described herein. For example, the protein may be produced or expressed in relation to S-Kroto 1-981, 29-981, 34-981, 36-981, 131-981, 1-549, 29-549, 34-549, 36-549, 131-549, etc., with or without Fc fusions and / or TEV-TwinStrep. Thus, the nucleic acid construct or cDNA on which the protein is expressed may be of the corresponding length.

[0252] Embodiments may include producing AA or AC heterozygous or homozygous constructs, introducing the resulting constructs encoding the S-Kroto protein into a suitable expression system (e.g., CHO cells) (e.g., via transfection), and / or transiently expressing the S-Kroto protein. The Kroto protein expressed in AA or AC heterozygous or homozygous cells may be expressed at higher levels than in CC cells. Embodiments may include purifying the expressed protein for therapeutic administration (and optionally performing quality control testing). Embodiments may include administering a therapeutic or therapeutically effective amount of the S-Kroto protein to a subject in need (e.g., an individual or patient with low endogenous S-Kroto protein expression, carrying the CC mutation at one tagged SNP, rs577912, and / or having end-stage renal disease (ESRD)). Alternatively, the subject may be a wild-type of another variant or cultivar. Administration of recombinant S-Klotho protein may lead to a beneficial increase in blood S-Klotho levels. The administration may reverse or suppress the adverse effects of CC variants transcribed and circulating in individuals as a result of point mutations found in the human Klotho gene of affected individuals. Therefore, circulating concentrations of S-Klotho may help counteract the effects observed in CC individuals, particularly in patients with end-stage renal disease (ESRD), namely the first-year mortality rate in ESRD patients undergoing habitual hemodialysis.

[0253] Therapeutic treatment of osteoarthritis (OA) and bone spurs of the hand using X-rays. Osteoarthritis (OA) is a common and complex disease with a strong genetic component. Researchers studied the association between four putative functional variants of the Kroto gene and osteoarthritis of the hand (OA) in a large population of Caucasian women. The researchers found a significant association between SNP G-395A and the presence or absence of radiographic hand OA and osteophyte formation. Allele G significantly increased the risk of radiographic hand OA and osteophyte formation, with odds ratios (OR) of 1.44 (P=0.008, 95% CI 1.09–1.91) and 1.36 (P=0.006, 95% CI 1.09–1.70), respectively. Logistic regression modeling showed that genotype GG was associated with more than a three-fold increased risk of both radiographic hand osteoarthritis (OR=3.10, 95%CI 1.10–8.76) and osteophytes (OR=3.10, 95%CI 1.10–8.75) compared to genotype AA. After age-adjusted, the OR for genotype GG further increased to 4.39 (P=0.006, 95%CI 1.51–12.74) for radiographic hand osteoarthritis and 4.47 (P=0.005, 95%CI 1.56–12.77) for osteophytes. The researchers also suggested that one variant of the Klotho gene (SNP G-395A) is associated with susceptibility to hand osteoarthritis and appears to act through osteophyte formation rather than cartilage damage.

[0254] One embodiment of this disclosure includes an S-Kroto protein expressed from a construct having SNP G395A. The resulting protein may be produced or expressed in relation to any protein construct described herein. For example, the A395 variant may be produced or expressed in relation to S-Kroto 1-981, 29-981, 34-981, 36-981, 131-981, 1-549, 29-549, 34-549, 36-549, 131-549, etc., with or without Fc fusions and / or TEV-TwinStrep. Thus, the nucleic acid construct or cDNA on which the protein is expressed may be of the corresponding length.

[0255] Embodiments may include producing a G395A heterozygous or homozygous construct, introducing the resulting construct encoding the S-Kroto protein into a suitable expression system (e.g., CHO cells) (e.g., via transfection), and / or transiently expressing the S-Kroto protein. The Kroto protein expressed in A395 heterozygous or homozygous cells may be expressed at higher levels than in G396 cells. Embodiments may include purifying the expressed protein for therapeutic administration (and optionally performing quality control testing). Embodiments may include administering a therapeutic or therapeutically effective amount of the S-Kroto protein to a subject in need (e.g., an individual or patient with a G395 SNP, and / or radiographic osteoarthritis (OA) and / or osteophytes of the hand (or at risk of developing them)). Alternatively, the subject may be a wild-type of another variant or cultivar. Administration of recombinant S-Klotho protein may lead to a beneficial increase in blood S-Klotho levels. Administration may reverse or suppress the adverse effects of G395 SNPs transcribed and circulating in affected individuals. Therefore, circulating concentrations of S-Klotho may help counteract the effects observed in G395 individuals, particularly those at risk of developing radiographic osteoarthritis (OA) and / or osteophytes of the hand. Thus, administration of G-395A S-Klotho protein may reduce the risk of radiographic osteoarthritis (OA) and osteophyte formation in patients (e.g., patients carrying G395 SNPs).

[0256] Therapeutic treatment for metabolic syndrome The risk and / or incidence of metabolic syndrome (MetS), a group of cardiovascular metabolic risk factors including abdominal obesity, hyperglycemia, dyslipidemia, and hypertension, increases with age. In older adults, MetS is associated not only with an increased risk of cardiovascular disease and type 2 diabetes, but also with cognitive decline and physical disability. Current evidence suggests that MetS is partially hereditary, with genetic factors playing a greater role than environmental factors in its incidence. Researchers found an association between the G-395A polymorphism and metabolic syndrome (MetS) among the 90s and 100s population in China. Subjects were from the Longevity and Aging Project in Dujiangyan City (PLAD). Genotyping of G-395A (rs1207568) in the promoter region of the Klotho gene was performed using the TaqMan allele recognition assay. MetS was diagnosed according to the International Diabetes Federation criteria. 695 subjects aged 93.5 ± 3.2 years were included. The allele frequencies for G and A were 0.852 and 0.148, respectively. In the overall population, the frequencies of MetS were 10.8% and 5.9% in the GG and GA+AA genotype groups, respectively (p=0.004). Carriers of the -395A allele had a significantly lower risk of MetS in the overall population (odds ratio [OR] 0.50, 95% confidence interval [CI] 0.25–0.98) and in females (OR 0.51, 95% CI 0.24–0.97), but in males (OR 0.42, 95% CI 0.05–3.85). In the overall population and in women, the relationship between Klotho G-395A SNP and MetS was likely due to its effect on hypertension (OR 0.48, 95% CI 0.34–0.67; OR 0.47, 95% CI 0.31–0.71, respectively) and hypertriglyceridemia (OR 0.66, 95% CI 0.39–0.95; OR 0.54, 95% CI 0.31–0.98, respectively). In men, this relationship was likely due to its effect on hypertension (OR 0.47, 95% CI 0.25–0.90) and low HDL-C (OR 0.69, 95% CI 0.27–0.93).The researchers concluded that carriers of the Klotho gene's -395A allele are associated with a lower risk of MetS among Chinese women, particularly those in their 90s and 100s.

[0257] One embodiment of this disclosure comprises an S-Kroto protein expressed from a construct having the -395A allele. The resulting protein may be produced or expressed in relation to any protein construct described herein. For example, the A395 allele may be produced or expressed in relation to S-Kroto 1-981, 29-981, 34-981, 36-981, 131-981, 1-549, 29-549, 34-549, 36-549, 131-549, etc., with or without Fc fusions and / or TEV-TwinStrep. Thus, the nucleic acid construct or cDNA on which the protein is expressed may be of the corresponding length.

[0258] Embodiments may include producing a -395A heterozygous or homozygous construct, introducing the resulting construct encoding the S-Kroto protein into a suitable expression system (e.g., CHO cells) (e.g., via transfection), and / or transiently expressing the S-Kroto protein. The Kroto protein expressed in A395 heterozygous or homozygous cells may be expressed at higher levels than in G396 cells. Embodiments may include purifying the expressed protein for therapeutic administration (and optionally performing quality control testing). Embodiments may involve administering a therapeutic or therapeutically effective dose of S-Klotho protein to a subject (e.g., an individual or patient harboring a G395 SNP and / or metabolic syndrome (MetS)) or a patient at risk of developing these conditions. Alternatively, the subject may be a wild-type variant or other variant. Administration of recombinant S-Klotho protein may lead to a beneficial increase in blood S-Klotho levels. The administration may reverse or suppress the adverse effects of G395 SNPs transcribed and circulating in affected individuals. Thus, circulating concentrations of S-Klotho may help counteract the effects observed in G395 individuals, particularly those at risk of developing metabolic syndrome (MetS). Therefore, administration of G-395A S-Klotho protein may reduce the risk of metabolic syndrome (MetS) in patients (e.g., elderly human and / or female patients harboring a G395 SNP).

[0259] Therapeutic treatment for cancer S-Klotho is thought to inhibit basal Wnt signaling activity, thereby functioning as a tumor suppressor in colorectal cancer (CRC). In addition, Klotho gene variants associated with lifespan differences may suppress butyrate-mediated Wnt hyperactivation, and thus increase the risk of CRC. In this manner, it is hypothesized that the type of Klotho variant present and its relative expression interact with dietary butyrate levels to modify the risk of CRC. Furthermore, mTOR signaling is also associated with human aging, and crosstalk between Wnt and mTOR signaling may influence colonic tumorigenesis.

[0260] KL-VS variants or other constructs may serve as vehicles for investigating which SNPs (e.g., within KL-VS) affect S-Kroto to result in a decrease in basal Wnt signaling and / or suppression of butyrate-mediated Wnt hyperactivation, the latter being Wnt-related activity associated with S-Kroto tumor suppression. Embodiments include modifying appropriate amino acids (e.g., the KL-VS stretch of S-Kroto) that have been shown to affect the tumor suppressor effect of KL-VS variants. One embodiment of this disclosure comprises a recombinant S-Kroto protein having one or more amino acid changes in the KL-VS stretch of six SNPs. The protein may be produced and / or expressed in relation to any protein construct described herein. For example, proteins can be produced or expressed in relation to S-Kroto 1-981, 29-981, 34-981, 36-981, 131-981, 1-549, 29-549, 34-549, 36-549, 131-549, etc., with or without Fc fusions and / or TEV-TwinStrep. Therefore, the nucleic acid construct or cDNA on which the protein is expressed may be of the corresponding length.

[0261] Embodiments may include producing heterozygous or homozygous variant constructs, introducing the resulting constructs encoding the S-Kroto protein into a suitable expression system (e.g., CHO cells) (e.g., via transfection), and / or transiently expressing the S-Kroto protein. The Kroto protein may be expressed at higher levels than other Kroto proteins, including the wild type. Embodiments may include purifying the expressed protein for therapeutic administration (and optionally performing quality control testing). Embodiments may include administering a therapeutic or therapeutically effective dose of the S-Kroto protein to a subject in need (e.g., a patient with or at risk of developing colorectal cancer (CRC) or another tumor). The subject may possess or express a Kroto variant with reduced Wnt inhibitory activity, for example. Alternatively, the subject may be the wild type of another variant or variant. Administration of recombinant S-Kroto protein may lead to a beneficial increase in blood S-Kroto levels.

[0262] Therapeutic treatment for age-related conditions Researchers discovered associations between Klotho and commonly accepted biological parameters as indicators of clinical condition in hospitalized elderly patients. The researchers genotyped single nucleotide polymorphisms (SNPs) rs9536314, rs1207568, and rs564481 at the KL locus of 594 hospitalized elderly patients (65–99 years old), followed them in a geriatric ward, and tested the associations with KL variants possessing these biological quantitative traits using covariant and genetic risk score model analysis. Significant associations were observed between rs9536314 and serum levels of hemoglobin, albumin, and high-density lipoprotein cholesterol (HDL-C), as well as between rs564481 and serum levels of hemoglobin, fasting insulin, and fasting glucose. Sex analysis confirmed these associations, suggesting that the association between KL genotype and HDL-C, fasting glucose, and fasting insulin levels may be governed by sex in women, while the association with serum hemoglobin levels may be governed by sex in men. An association between KL genotype and creatine levels was found in women, while an association between insulin-like growth factor-1 (IGF-1) and lymphocyte count (LC) was found in men. Genetic risk score (GRS) models further confirmed significant associations between KL SNPs and hemoglobin, total cholesterol, and HDL-C. Sex analysis using a GRS-tagged approach confirmed associations with HDL-C, fasting glucose levels, and fasting insulin levels in women, and with hemoglobin and LC in men. These findings suggest that the KL locus may influence quantitative traits such as serum lipid levels, fasting glucose, albumin, and hemoglobin in hospitalized elderly patients, and that some sex differences are suggested in creatine, IGF-1 levels, and LC, thus suggesting that one of the genetic factors may contribute to age-related disease and lifespan.

[0263] One embodiment of the present disclosure comprises the S-Kroto protein described herein. The embodiment may include producing a suitable Kroto construct, introducing the construct encoding the S-Kroto protein into a suitable expression system (e.g., CHO cells) (e.g., via transfection), and / or transiently expressing the S-Kroto protein. The embodiment may include purifying the expressed protein for therapeutic administration (and optionally performing quality control testing). The embodiment may include administering a therapeutic or therapeutically effective amount of the S-Kroto protein to a subject in need (e.g., an individual or patient, optionally the elderly, and / or an individual or patient suffering from age-related conditions, low endogenous S-Kroto protein expression, and / or age-related conditions, or symptoms of reduced lifespan). Administration of recombinant S-Kroto protein may result in a beneficial increase in blood S-Kroto levels. Aggressive therapeutic approaches, such as administering quantitative traits like serum levels of total cholesterol, HDL-C, fasting glucose, fasting insulin, albumin, creatine, IGF-1, hemoglobin, and lymphocyte count (e.g., in hospitalized and / or elderly patients), may reverse or suppress age-related conditions and / or adverse effects.

[0264] In one or more therapeutic methods described herein, the individual being treated may have a mutation in the klotho gene (e.g., a genomically encoded heterozygous or homozygous mutation), and the therapeutic regimen may include administering a therapeutic dose of a peptide containing any one or more variants of wild-type klotho and / or klotho disclosed herein, for example, a variant similar to the mutation expressed by the individual. Alternatively, the individual being treated may encode / express wild-type klotho, and the therapeutic regimen may include administering a therapeutic dose of a peptide containing any one or more variants of wild-type klotho and / or klotho disclosed herein. In some embodiments, regardless of whether the klotho expressed by the individual is the natural wild-type or mutant form, the therapeutic method may include measuring the low level of circulating and / or cell-bound klotho (e.g., compared to a control group) and administering a therapeutic dose that restores the concentration of circulating and / or cell-bound klotho protein to at least a homeostatic level. In some embodiments, this may include measuring Klotho levels (e.g., gene expression levels, protein expression levels, circulating levels, etc.) before administering the therapeutic concentrate. In addition, the therapeutic dose may depend on the Klotho levels measured in the individual. In some embodiments, the therapeutic dose exceeds the homeostatic level, 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.).

[0265] More specifically, it should be understood that therapeutic treatment of age-related conditions may include administering one or more crotoid variants at a therapeutic concentration. In some embodiments, this may include treating a patient with a crotoid variant (or combination of crotoid variants) disclosed herein at a therapeutic concentration, such as a peptide selected from any one or more of SEQ ID NOs: 2 to SEQ ID NO: 70. It should be understood that age-related conditions may be treated with the same or different crotoid variants than those expressed and / or encoded by an individual having the age-related condition. For example, an individual suffering from an age-related condition may genetically encode one or more wild-type or mutant forms of croto, and this individual may express the wild-type and / or mutant croto proteins at constitutive levels, at levels below constitutive levels, or not at all (compared to a control group). A treatment regimen aimed at treating an individual's age-related condition may include administering one or more crotoid variants disclosed herein.

[0266] Preventive S-croto administration In addition to the foregoing, embodiments of the present disclosure may include administering a therapeutic or therapeutically effective amount of an S-croto protein to an individual or subject who needs it for preventive purposes and / or maintenance of certain health attributes. For example, administration of a particular S-croto protein may help maintain the youthfulness of an optional aging patient who has not yet suffered from a diagnosed age-related condition. Thus, certain embodiments of the present disclosure may relate to and / or include treatment of a patient's condition, while other embodiments may relate to and / or include prevention, inhibition of onset, and / or preventive approaches to one or more conditions. For example, S-croto may be administered to a person having a genetic disorder with a mutation in one or more croto genes.

Examples

[0267] Example 1 Table 1 shows the expression and purification results of the listed Kroto variants in HEK and / or CHO cell lines. The results provided in Table 1 below followed the following simplified protocol.

[0268] For Fc fusion proteins, the protein expression vector was transfected to HEK293.sus or CHO using the standard ATUM method. In summary, cells were grown for 7 days and harvested. Cell counts are noted in the notes section. The supernatant pH was adjusted with 1M Hepes pH 7.4 and sodium azide was added. Proteins were captured using KanCap A resin. The resin was washed with PBS. The resin was washed with PBS plus 1M NaCl. The resin was washed with PBS. Proteins were eluted with 50mM citrate pH 3.5 and 100mM NaCl. Proteins were immediately neutralized with 1M Tris pH 8 and 0.5M arginine. The SDS-PAGE gel sample was removed at this stage. The proteins were buffered with PBS. Proteins were quantified by OD280, and the volume and concentration were measured using the calculated extinction coefficient. Purity and molecular weight were determined using reducing and non-reducing SDS-PAGE (Biorad reference Tris / glycine / SDS, 4–20%). Aggregation status was measured by HPLC with detection at 280 nm using a Sepax Zenix-C SEC-300, 3 μm, 300 Å, 4.6 × 150 mm size exclusion column and PBS running buffer. After filter sterilization and rapid freezing in liquid nitrogen, proteins were shipped as aliquots. It should be noted that, as in previous purification rounds, protein loss was observed during desalting to PBS for Fc-tagged proteins, as measured from samples that underwent SDS-PAGE before and after purification. These proteins were expressed by HPLC, but problems occurred during assays on silica HPLC columns during desalting or in PBS. Therefore, buffer selection may be optimized.

[0269] For Strep-tagged proteins, the protein expression vector was transfected to HEK293.sus or CHO using the standard ATUM method. In summary, cells were grown for 7 days and harvested. The supernatant pH was adjusted with 1M Hepes pH 7.4 and sodium azide was added. BioLock biotin isolation reagent was added. Proteins were captured using StrepTactin superflow resin. The resin was washed with 100 mM Tris pH 8, 150 mM NaCl, and 1 mM EDTA. Proteins were eluted with 100 mM Tris pH 8, 150 mM NaCl, 1 mM EDTA plus 2.5 mM desthiovin. Proteins were quantified by OD280, and the volume and concentration were determined using the calculated extinction coefficient. Purity and molecular weight were determined using reduced and unreduced SDS-PAGE (Biorad reference Tris / glycine / SDS, 4–20%). Aggregation was measured by HPLC at 280 nm using a Sepax Zenix-C SEC-300, 3 μm, 300 Å, 4.6 × 150 mm size exclusion column and PBS running buffer. After filter sterilization and rapid freezing in liquid nitrogen, proteins were shipped as aliquots. For Strep-tagged proteins, it should be noted that the samples were assayed in elution buffer. This elution buffer is very "neutral" and not harmful to the proteins. Also, since the running buffer for the SEC column was PBS, the proteins underwent buffer exchange during the assay. Absorbance at times exceeding 7 minutes indicates small molecules.

[0270] [Table 1]

[0271] In addition to one or more other Klotho manifolds disclosed herein but not shown in Table 1, it should be understood that the expression and / or purification of the Klotho manifolds disclosed in Table 1 may, in some embodiments, yield advantages over the expression and / or purification of natural Klotho. For example, the expression and / or purification of Klotho manifolds may reduce the number and / or types of alternative products. In addition, or / or, the expression level of the desired Klotho manifold may increase compared to the expression level of natural Klotho. In addition, or / or, the desired Klotho manifold may be expressed and / or purified in a purer form under equivalent conditions and methods (e.g., the concentration of the desired Klotho manifold increases with the accompanying decrease in expressed and / or purified byproducts).

[0272] Example 2 The following examples include an exemplary set of claims that define the scope of the invention as disclosed. However, as provided herein, the scope of the invention is indicated by the appended claims rather than by the following examples or the preceding description.

[0273] 1. An exemplary method for producing recombinant Kroto protein, comprising producing recombinant Kroto 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 synthase (GS)-deficient CHO cells, more preferably in GS- / -CHO cells, wherein the protein preferably has at least 85% amino acid sequence identity with one of SEQ ID NOs. 2 to SEQ ID NOs. 70.

[0274] 2. The method according to claim 1, wherein the protein comprises one or more glycans bound thereto. 3. The method according to claim 1 or 2, wherein the CHO cells contain an exogenous nucleic acid encoding a promoter, preferably a strong promoter, a polypeptide having at least 85% amino acid sequence identity with one of SEQ ID NOs: 2 to 70, and optionally a functional dihydrofolate reductase (DHFR) enzyme or a functional glutamine synthase (GS) enzyme, and the production of the recombinant Kroto protein is comprised of expressing the polypeptide encoded by the nucleic acid.

[0275] 4. The method according to claim 3, further comprising one or more steps selected from the steps of introducing the exogenous nucleic acid into the CHO cells, preferably via transfection, and growing the CHO cells in a liquid medium, preferably a serum-free and / or animal protein-free 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, and more preferably the liquid medium lacks hypoxanthine, thymidine, and / or glutamine.

[0276] 5. The method according to claim 4, wherein the protein is secreted from the CHO cells into the liquid medium at a concentration of preferably 200 to 500 mg of protein per liter of liquid medium, more preferably 500 to 2000 mg of protein, and even more preferably 2000 to 5000 mg of protein per liter of liquid medium, without concentrating the protein.

[0277] 6. The method according to claim 4, further comprising introducing an effective amount of methotrexate (MTX) and / or methionine sulfoximine (MSX) into the liquid medium to a concentration preferably of about 1 nM to 1 μM, more preferably of about 10 to 100 nM.

[0278] 7. The method according to claim 4, further comprising selecting a suspension culture of viable CHO cells growing in the liquid medium, wherein the concentration of the selected suspension culture in the medium 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, and still more preferably at least 5000 mg / L, without concentrating the protein.

[0279] 8. The method according to claim 7, wherein the viable CHO cells in 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, and even more preferably at least 30 to 50 copies of the exogenous nucleic acid per cell.

[0280] 9. The method according to claim 4, further comprising purifying a recombinant Kroto protein-containing extract from the CHO cells, liquid culture medium, or both thereof, wherein the extract preferably contains at least about 98% protein and / or less than about 1 to 100 ppm of CHO host cell protein (HCP) by dry weight.

[0281] 10. The method according to claim 9, wherein the purification of the extract maintains the glycosylation of the protein. 11. The method according to claim 4, wherein the CHO cells are cultured 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, even more preferably at least 250 liters, even more preferably at least 500 liters, even more preferably at least 1,000 liters, even more preferably at least 2,000 liters, even more preferably at least 2,500 liters, even more preferably at least 5,000 liters, and even more preferably at least 10,000 liters.

[0282] 12. The method according to any one of claims 1 to 11, wherein the nucleic acid comprises a transgene or cDNA having a nucleic acid sequence identity of 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%, and most preferably 100% with one of SEQ ID NOs: 76 to SEQ ID NOs: 96.

[0283] 13. The method according to any one of claims 1 to 12, wherein the protein has an amino acid sequence identity of at least 90%, preferably at least 95%, more preferably at least 98%, even more preferably at least 99%, and most preferably 100% with one of SEQ ID NOs: 2 to SEQ ID NOs: 70.

[0284] 14. 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 cells, more preferably in GS- / -CHO cells, wherein the CHO cells contain a promoter, preferably a strong promoter, and a polypeptide, wherein at least a portion of the polypeptide has an amino acid sequence identity of at least 85% with one of SEQ ID NOs: 2 to SEQ ID NOs: 70, and optionally contains an exogenous nucleic acid encoding a functional dihydrofolate reductase (DHFR) enzyme or a functional glutamine synthetase (GS) enzyme.

[0285] 15. The cell line according to claim 14, wherein the CHO cells 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, or are selected to contain the exogenous nucleic acid.

[0286] 16. The cell line according to claim 14, wherein the nucleic acid encodes a polypeptide having at least 90%, preferably at least 95%, more preferably at least 98%, even more preferably at least 99%, and most preferably 100% amino acid sequence identity with one of SEQ ID NOs: 2 to 70.

[0287] 17. The cell line according to claim 14, wherein the nucleic acid comprises a transgene or cDNA 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%, and most preferably 100% nucleic acid sequence identity with one of sequence numbers 76 to 96.

[0288] 18. A suspension cell culture comprising a liquid medium, preferably serum-free and / or free of animal protein components, preferably comprising a carbon source, a nitrogen source, and one or more vitamins, minerals, salts, amino acids, supplements, or additives, and more preferably the liquid medium lacking hypoxanthine, thymidine, and / or glutamine, and a cell line according to any one of claims 14 to 17, wherein the CHO cells are grown in the liquid medium such that they express the polypeptide encoded by the nucleic acid, the polypeptide comprising recombinant Kroto protein.

[0289] 19. The suspended cell culture according to claim 18, wherein the CHO cells secrete the protein into the liquid medium at a concentration of preferably 200 to 500 mg of protein per liter of liquid medium, more preferably 500 to 2000 mg of protein, and even more preferably 2000 to 5000 mg of protein per liter of liquid medium, and / or the protein is present in the liquid medium at a concentration of 200 to 500 mg of protein per liter of liquid medium, more preferably 500 to 2000 mg of protein, and even more preferably 2000 to 5000 mg of protein per liter of liquid medium, without concentrating the protein.

[0290] 20. The suspension cell culture according to claim 18 or 19, wherein the protein comprises one or more glycans bound thereto. 21. The suspended cell culture according to any one of claims 18 to 20, wherein the liquid medium further comprises an effective amount of methotrexate (MTX) and / or methionine sulfoximine (MSX), preferably at a concentration of about 1 nM to 1 μM, more preferably at a concentration of about 10 nM to 100 nM.

[0291] 22. The suspended cell culture according to any one of claims 18 to 21, wherein the protein has at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 98%, still more preferably at least 99%, and most preferably 100% amino acid sequence identity with one of SEQ ID NOs: 2 to 70.

[0292] 23. A recombinant Kroto protein in which at least a portion of the protein has at least 80% amino acid sequence identity with one of SEQ ID NOs. 2 to 70. 24. The recombinant protein according to claim 23, wherein the protein inhibits the p53 / p21 signaling pathway, exhibits β-glucuronidase and / or sialidase activity that modulates the IGF-1 and / or Wnt signaling pathway, and / or preferably reduces H2O2-induced cellular senescence and apoptosis through the inhibition of the p53 / p21 signaling pathway.

[0293] 25. The recombinant Kroto protein according to claim 23 or 24, wherein the protein preferably functions as a humoral factor exhibiting pleiotropic activity in oxidative stress, growth factor signaling, regulation of ion homeostasis, and / or regulation of the activity of one or more glycoproteins on the cell surface, preferably one or more ion channel proteins and / or growth factor receptors, preferably insulin / insulin-like growth factor-1 receptors.

[0294] 26. The recombinant Kroto protein according to any one of claims 23 to 25, wherein at least a portion of the protein has at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 98%, still more preferably at least 99%, and most preferably 100% amino acid sequence identity with one of SEQ ID NOs: 2 to 70.

[0295] 27. A method for treating an age-related or other pathological condition, disease, or disorder, comprising administering a pharmaceutically effective amount of recombinant klotho protein according to any one of claims 23 to 26 to a subject in need thereof.

[0296] 28. A method for treating an age-related or other pathological condition, disease, or disorder, comprising administering to a subject in need of such treatment a pharmaceutically effective amount of soluble recombinant kroto protein having at least 80% amino acid sequence identity with at least a subset of amino acid residues 1-981 of human alpha kroto isoform 1.

[0297] 29. A method for treating an age-related or other pathological condition, disease, or disorder, comprising administering to a subject in need of such treatment a pharmaceutically effective amount of a soluble recombinant kroto protein having at least 80% amino acid sequence identity with one of SEQ ID NOs: 2 to SEQ ID NOs: 70.

[0298] 30. The method according to any one of claims 27 to 29, wherein the protein has at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 98%, still more preferably at least 99%, and most preferably 100% amino acid sequence identity with one of SEQ ID NOs: 2 to 70.

[0299] 31. The method according to claim 30, wherein the pharmaceutically effective amount is sufficient to raise the serum soluble Kroto protein concentration of the subject to a predetermined level, and preferably to maintain the serum soluble Kroto protein concentration of the subject above a predetermined threshold for a predetermined period of time.

[0300] 32. The method according to claim 31, wherein the predetermined level is approximately 1,000 picograms or more of soluble krotho protein per milliliter of serum. 33. The specified levels are approximately 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, 11,000, 12,000, 13,000, 14,000, 15,000, 20,000, 25,000, and 30,000 per milliliter of serum. The method according to claim 31, wherein the amount of soluble krotho protein is 40,000, 50,000, 75,000, or 100,000 picograms or more, or in between, and / or is about 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% or more, or in between, compared to a typical healthy level of soluble krotho protein in serum.

[0301] 34. The method according to claim 31, further comprising one or more of the following: measuring the serum soluble Kroto protein concentration of the subject; calculating a pharmaceutically effective amount of protein sufficient to raise the serum soluble Kroto protein concentration of the subject to a first predetermined level, wherein the first predetermined level is preferably about 1000 picograms or more of soluble Kroto protein per milliliter of serum; measuring the rate of decrease and / or metabolic rate of soluble Kroto protein in the subject's serum; calculating the next dosing time such that the serum soluble Kroto protein concentration of the subject falls below a second predetermined level based on the measured rate; and calculating the next dosing amount of protein sufficient to raise the serum soluble Kroto protein concentration of the subject from the second predetermined level to the first predetermined level.

[0302] 35. The method according to claim 34, further comprising administering the following dose of the protein to the subject. 36. Preferably, introduce an exogenous nucleic acid into Chinese hamster ovary (CHO) cells via transfection, wherein the nucleic acid preferably comprises a transgene or cDNA, wherein the nucleic acid encodes a polypeptide having at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 98%, even more preferably at least 99%, most preferably 100%, amino acid sequence identity with one of SEQ ID NOs. 2 to SEQ ID NOs. 70, and wherein the nucleic acid has nucleic acid sequence identity with one of SEQ ID NOs. 76 to SEQ ID NOs. 96, preferably at least 85%, more preferably at least 90%, even more preferably at least 95%, even more preferably at least 98%, even more preferably at least 99%, most preferably 100%, and grow the CHO cells in a liquid medium, preferably a serum-free and / or animal protein-free liquid medium, wherein the liquid medium preferably contains a carbon source, a nitrogen source, and one or more vitamins, minerals, salts, amino acids, supplements, or additives. More preferably, the liquid medium lacks hypoxanthine, thymidine, and / or glutamine, and the CHO cells are preferably dihydrofolate reductase (DHFR) deficient CHO cells, more preferably CHO-S cells, or glutamine synthase (GS) deficient CHO cells, more preferably GS- / -CHO cells, and the cells are grown; and an effective amount of methotrexate (MTX) and / or methionine sulfoximine (MSX) is introduced into the liquid medium to a concentration preferably of about 1 nM to 1 μM, more preferably of about 10 to 100 nM. The process involves selecting a suspension culture of viable CHO cells growing in the liquid medium through a cell selection process, wherein the concentration of the protein in the medium of the selected suspension culture 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, and still more preferably at least 5000 mg / L, without concentrating the protein, and producing the recombinant soluble Kroto protein in the CHO cells.The method according to claim 31, further comprising one or more of the following: producing the protein, preferably secreted from the CHO cells into the liquid medium at a protein concentration of preferably 200-500 mg, more preferably 500-2000 mg, and even more preferably 2000-5000 mg per liter of liquid medium; and purifying a recombinant soluble kroto protein-containing extract from the CHO cells, the liquid medium, or both thereof, wherein the extract preferably contains at least about 98% by dry weight of the recombinant soluble kroto protein and / or less than about 1-100 ppm of CHO host cell protein (HCP); and purifying the extract preferably maintains the glycosylation of the protein and has one or more glycans bound to it.

[0303] 37. The method according to claim 31, wherein the predetermined period 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, and still more preferably at least about 72 hours.

[0304] 38. The method according to claim 31, wherein the prescribed period is approximately 1 to 120 days or more. 39. The method according to claim 31, wherein the prescribed period is approximately 6 months, 9 months, or 1 year or more.

[0305] 40. The method according to any one of claims 30 to 39, wherein the subject is a human, a non-human animal, or a non-human mammal. 41. The method according to any one of claims 30 to 40, wherein the protein is administered in or together with a pharmaceutically acceptable carrier.

[0306] 42. The method according to any one of claims 30 to 41, wherein the age-related or other pathological condition, disease, or disorder includes one or more of the following: frailty, decreased bone density, decreased bone mineral density, weight loss, muscle atrophy, muscle degeneration, decreased muscle mass, decreased muscle strength, decreased grip strength, decreased leg strength, decreased physical fitness, decreased movement, decreased freedom of movement, decreased quality of life assessment, decreased ejection fraction, decreased motor ability, decreased learning ability, decreased memory, decreased intelligence quotient, cognitive deterioration, amnesia, decreased cognitive ability, decreased cognitive function, decreased synaptic plasticity, decreased synaptic function, and cellular senescence.

[0307] 43. The age-related or other pathological conditions, diseases, or disorders include 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, non-oliguric renal failure, alcoholism, hyperphosphatemia, muscular dystrophy (MS), type 1 diabetes, type 2 diabetes, cardiovascular disease (CVD), cardiovascular calcification, cerebrovascular failure, vascular calcification, ischemic heart disease, abnormal blood pressure, salt-sensitive hypertension, tissue calcification, calcified atherosclerotic plaque load, calcification, familial neoplastic calcification, cancer, one or more tumors, myelin-related diseases, demyelinating diseases, neurodegenerative diseases, neurovascular diseases, progressive supranuclear palsy (PSP), Pompe disease, Niemann-Pick disease, microgliosis, and FAVA. - Diseases (FD), bone density disorders, osteoporosis, osteopenia, osteopenia (especially decreased BMD of cortical bone), emphysema, pulmonary fibrosis, skin atrophy, thymic atrophy, accumulation of renal interstitial matrix, glomerulosclerosis, anemia, albuminuria, proteinuria, infertility, Alzheimer's disease, Parkinson's disease, dementia, vascular dementia, amyotrophic lateral sclerosis (ALS), motor neuron disease (MND), atrial fibrillation, chronic obstructive pulmonary disease (CO2). The method according to any one of claims 30 to 41, comprising one or more of the following: PD), fibromyalgia, adult-onset diabetes, arthritis, rheumatoid arthritis, osteoarthritis, glaucoma, cataract, macular degeneration, multiple sclerosis (MS), lupus, ulcerative colitis, cachexia, obesity, vitamin D-related conditions, bone diseases, bone diseases through bone regeneration, stem cell depletion, seasickness, space maladjustment syndrome (SAS), nausea, and dizziness.

[0308] 44. The method according to any one of claims 30 to 43, further comprising administering or co-administering one or more additional active ingredients. 45. The method according to claim 44, wherein the one or more additional active ingredients are selected from the group consisting of drugs, antibodies, hormones, contrast agents, pharmaceuticals, natural compounds, synthetic compounds, or pharmaceutical compositions.

[0309] 46. ​​A pharmaceutical composition comprising a pharmaceutically effective amount of recombinant kroto protein according to any one of claims 23 to 25 and a pharmaceutically acceptable carrier. 47. A pharmaceutical composition comprising a pharmaceutically effective amount of recombinant soluble kroto protein, wherein at least a portion of the protein has at least 85% amino acid sequence identity with 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 kroto isoform 1, or at least a portion of one of sequence numbers 2-70, and a pharmaceutically acceptable carrier.

[0310] 48. The pharmaceutical composition according to claim 46 or 47, wherein at least a portion of the protein has 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%, and most preferably 100% amino acid sequence identity with at least a portion of one of SEQ ID NOs: 2 to 70.

[0311] 49. The pharmaceutical composition according to any one of claims 46 to 48, further comprising one or more additional active ingredients. 50. Weakness, decreased bone density, decreased bone mineral density, weight loss, muscle atrophy, muscle degeneration, decreased muscle mass, decreased muscle strength, decreased grip strength, decreased leg strength, decreased physical fitness, decreased movement, decreased freedom of movement, decreased quality of life assessment, decreased ejection fraction, decreased motor ability, decreased learning ability, decreased memory, decreased intelligence quotient, cognitive deterioration, amnesia, decreased cognitive ability, decreased cognitive function, decreased synaptic plasticity, decreased synaptic function, cellular senescence, chronic kidney disease (CKD), polycystic kidney disease (PKD), autosomal dominant polycystic kidney disease (ADPKD), acute kidney Acute kidney injury (AKI), acute tubular necrosis (ATN), acute allergic interstitial nephritis (AAIN), glomerulonephritis, kidney disease, renal failure, non-oliguric renal failure, alcoholism, hyperphosphatemia, muscular dystrophy (MS), type 1 diabetes, type 2 diabetes, cardiovascular disease (CVD), cardiovascular calcification, cerebrovascular failure, vascular calcification, ischemic heart disease, abnormal blood pressure, salt-sensitive hypertension, tissue calcification, calcified atherosclerotic plaque load, calcification, familial neoplastic calcification, cancer, one or more tumors, myelin-related diseases, demyelinating diseases Patients, neurodegenerative diseases, neurovascular diseases, progressive supranuclear palsy (PSP), Pompe disease, Niemann-Pick disease, microgliosis, Faber's disease (FD), bone density disorders, osteoporosis, osteopenia, osteopenia (especially loss of BMD in cortical bone), emphysema, pulmonary fibrosis, skin atrophy, thymic atrophy, accumulation of renal interstitial matrix, glomerulosclerosis, anemia, albuminuria, proteinuria, infertility, Alzheimer's disease, Parkinson's disease, dementia, vascular dementia, amyotrophic lateral sclerosis (ALS), motor neuron disease (MND), atrial fibrillation, chronic obstructive pulmonary disease A pharmaceutical composition according to any one of claims 46 to 49, for use in the treatment of one or more age-related or other conditions, diseases, or disorders, including lung disease (COPD), fibromyalgia, adult-onset diabetes, arthritis, rheumatoid arthritis, osteoarthritis, glaucoma, cataract, macular degeneration, multiple sclerosis (MS), lupus, ulcerative colitis, cachexia, obesity, vitamin D-related conditions, bone diseases, bone diseases through bone regeneration, stem cell depletion, seasickness, space maladjustment syndrome (SAS), nausea, and dizziness.

[0312] 51. A pharmaceutical composition according to any one of claims 46 to 49, for use in the treatment or prevention of acute kidney injury (AKI). 52. A method for treating or preventing acute kidney injury (AKI) or other pathological conditions, comprising administering a pharmaceutically effective amount of recombinant soluble kroto protein to a subject in need thereof, wherein at least a portion of the protein has at least 85%, 86%, 88%, 90%, 92%, 95%, 98%, 99%, or preferably 100% amino acid sequence identity with 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 kroto isoform 1, or at least a portion of one of sequence numbers 2-70.

[0313] 53. The method according to claim 52, further comprising co-administering one or more additional active ingredients together with the pharmaceutically effective amount of recombinant soluble Kroto protein. 54. The method according to claim 53, wherein the protein and one or more additional active ingredients are incorporated into a combination product or composition.

[0314] 55. The method according to claim 53, wherein the protein and one or more additional active ingredients are separate compositions. 56. The method according to claim 53, wherein the protein and one or more additional active ingredients are mixed together.

[0315] 57. The method according to claim 53, wherein the protein and one or more additional active ingredients are configured for co-administration, and the co-administration comprises simultaneous administration or separate administrations preferably separated by a certain period of time.

[0316] 58. The method according to claim 53, wherein the one or more additional active ingredients are selected from the group consisting of drugs, antibodies, hormones, contrast agents, pharmaceuticals, or compositions. 59. The method according to claim 52 or 53, wherein the symptoms include acute tubular necrosis (ATN), nephritis, acute allergic interstitial nephritis (AAIN), glomerulonephritis and / or nephrotoxicity, or AKI resulting at least partially from kidney transplantation or other surgical procedure, acute tubular necrosis (ATN), nephritis, acute allergic interstitial nephritis (AAIN), glomerulonephritis, nephrotoxicity, or hypotension.

[0317] 60. The method according to claim 52 or 53, wherein the nephrotoxicity includes drug-induced nephrotoxicity. 61. The method according to claim 60, wherein the drug-induced nephrotoxicity includes nephrotoxicity induced by an antimicrobial agent.

[0318] 62. The method according to claim 60, wherein the drug-induced nephrotoxicity includes nephrotoxicity induced by an aminoglycoside. 63. The method according to claim 52 or 53, wherein the administration step comprises one or more steps selected from the group consisting of: measuring the serum soluble Klotho level in the subject; calculating a first dose of protein sufficient to raise the serum soluble Klotho level in the subject to a predetermined level or a percentage of the normal level; administering the first dose of protein to the subject, preferably by bolus or stepwise administration, more preferably by injection; preferably measuring the rate of decrease of soluble Klotho in the subject's serum following the administration of the first dose; calculating the time and / or amount of the next dose of protein; and administering the next dose of protein to the subject according to the calculated time and / or amount.

[0319] 64. The method according to claim 52 or 53, wherein the administration step is sufficient to selectively increase and / or maintain the serum soluble klotho protein concentration of the subject above a predetermined level or threshold for a predetermined period of time.

[0320] 65. The specified level or threshold is approximately 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, 11,000, 12,000, 13,000, 14,000, 15,000, 20,000, 25,000, 30,000 per milliliter of serum. The method according to claim 64, wherein soluble krotoh protein is present in amounts of 40,000, 50,000, 75,000, or 100,000 picograms or more and / or in between, or approximately 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%, exceeding typical healthy levels of soluble krotoh protein.

[0321] 66. The method according to claim 64, wherein the prescribed period is approximately 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 or more.

[0322] 67. The method according to claim 52 or 53, wherein the protein is administered prophylactically before kidney transplantation or administration of nephrotoxin, and / or following kidney transplantation or administration of nephrotoxin. 68. The nephrotoxin is preferably one or more aminoglycosides selected from the group consisting of paromomycin, tobramycin, gentamicin, amikacin, kanamycin, and neomycin; preferably one or more antifungal agents selected from the group consisting of amphotericin B and flucytosine; preferably a hyperosmolar modulating medium (HOCM) having an iodine-to-molecular ratio of about 1.5:1, a hypoosmolar nonionic contrast agent (LOCM) having an iodine-to-molecular ratio of about 3:1, and One or more contrast agents selected from the group consisting of isotonic (isoosmolar) contrast agents (IOCM) having an iodine-to-molecular ratio of approximately 6:1; preferably one or more antiretroviral agents selected from the group consisting of adefovir, cidofovir, tenofovir, and foscanet; preferably cisplatin, carboplatin, oxaliplatin, alkylating agents, bendamustine, cyclophosphamide, ifosfamide, nitrosourea, temozolomide, melphalan, antitumor antibiotics One or more cancer (or chemo) therapeutic agents selected from the group consisting of substances, mitomycin C, bleomycin, anthracyclines, antimetabolites, capecitabine, hydroxyurea, methotrexate, pemetrexed, pralatrexate, pentostatin, fludarabine, cladribine, gemcitabine, cytarabine, vinca alkaloids, topotecan, etoposide, taxanes, irinotecan, lenalidomide, eribulin, arsenic trioxide, or ixazomib; preferred The method according to claim 67, further comprising one or more bisphosphonates or derivatives thereof selected from the group consisting of zoledronate / zoledronic acid, ibandronate, alendronate, alendronate / cholecalciferol, etidronate, risedronate, calcium carbonate risedronate, pamidronate, and tildronate; and / or preferably one or more anesthetics or opioids selected from the group consisting of cocaine and heroin.

[0323] 69. The method according to claim 52 or 53, wherein the protein preferably does not contain F352V, more preferably contains F352, C370S, and / or a variant other than H193 or H193R.

[0324] 70. A method for treating an aged individual, comprising administering a therapeutic concentration of polypeptide to the aged individual, which has a homozygous or heterozygous mutation in the gene encoding the Kroto protein and has at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 98%, still more preferably at least 99%, and most preferably 100% amino acid sequence identity with one of SEQ ID NOs: 2 to 70.

[0325] 71. The method according to claim 70, further comprising measuring the expression level of the gene. 72. The method according to claim 71, wherein the step of administering the therapeutic agent concentrate depends on the expression level of the gene.

[0326] conclusion While the above-mentioned detailed description refers to specific exemplary embodiments, this disclosure can be embodied in other specific forms without departing from its spirit or essential features. Therefore, the embodiments described should be considered in all respects as exemplary and not restrictive. For example, various substitutions, changes, and / or modifications of the features of the invention described and / or illustrated herein, as well as additional applications of the principles described and / or illustrated herein, which may be conceivable to those skilled in the art and the owners of this disclosure, can be made to the described and / or illustrated embodiments without departing from the spirit and scope of this disclosure as set forth by the appended claims. Such substitutions, changes, and / or modifications are considered to be within the scope of this disclosure.

[0327] Accordingly, the scope of the present invention is indicated not by the foregoing description but by the appended claims. The limitations enumerated in the claims are to be interpreted broadly based on the language used in the claims and are to be interpreted as non-exclusive and non-limiting, and not limited to the specific examples set forth in the preceding detailed description. All modifications that fall within the scope and meaning of the claims are included within that scope.

[0328] It will also be understood that various features of a particular embodiment may be compatible with, combined with, included in and / or incorporated into other embodiments of the present disclosure. For example, a system, method, and / or product according to a particular embodiment of the present disclosure may include, incorporate, or include features described in other embodiments disclosed and / or described herein. Accordingly, the disclosure of a particular feature relating to a particular embodiment of the present disclosure should not be construed as limiting the application or inclusion of such feature to a particular embodiment.

[0329] In addition, unless otherwise stated that a feature is required in a particular embodiment, the features described in various embodiments are optional and may not be included in other embodiments of this disclosure. Furthermore, unless otherwise stated that a feature requires another feature in combination with it, any feature in this specification may be combined with any other feature in the same or different embodiments disclosed herein. In a particular embodiment, a feature may be optional, but when such an embodiment includes a feature, it will be understood that they may be required to have a particular configuration as described herein.

[0330] Similarly, the steps listed in any method or process described herein and / or enumerated in the claims may be performed in any preferred order and are not necessarily limited to the order described and / or enumerated unless otherwise specifically stated (expressly or implicitly). However, such steps may also be required in certain embodiments of the present disclosure to be performed in a particular order or any preferred order.

[0331] Furthermore, various well-known embodiments of exemplary systems, methods, products, etc., are not described in detail herein in order to avoid obscuring the embodiments of the exemplary models. However, such embodiments are also considered herein.

Claims

1. A recombinant protein represented by Sequence ID No.

40.

2. The recombinant protein according to claim 1 for use in the treatment of acute kidney injury.

3. A recombinant protein according to claim 1 for use in the treatment of acute kidney injury, wherein the acute kidney injury is (i) Kidney injury resulting from surgery, (ii) nephrotoxicity; (iii) Drug-induced nephrotoxicity (iv) Nephrotoxicity induced by antibacterial agents, and (v) Nephrotoxicity induced by aminoglycosides Recombinant proteins selected from the group consisting of the following.

4. A pharmaceutical composition comprising a pharmaceutically acceptable carrier or excipient and an effective amount of the recombinant protein described in Claim 1.

5. The pharmaceutical composition according to claim 4 for use in the treatment of acute kidney injury.

6. A pharmaceutical composition according to claim 4 for use in the treatment of acute kidney injury, wherein the acute kidney injury is (i) Kidney injury resulting from surgery, (ii) nephrotoxicity; (iii) Drug-induced nephrotoxicity (iv) Nephrotoxicity induced by antibacterial agents, and (v) Nephrotoxicity induced by aminoglycosides A pharmaceutical composition selected from the group consisting of the following.

7. A nucleic acid construct comprising a nucleic acid sequence encoding the recombinant protein described in Claim 1.

8. The nucleic acid construct according to claim 7, further comprising a promoter, wherein the nucleic acid sequence encoding the recombinant protein is under the control of the promoter.

9. A cell line comprising Chinese hamster ovary (CHO) cells comprising the nucleic acid construct according to claim 7 or 8.

10. Liquid culture medium, and The cell line according to claim 9, wherein the CHO cells are grown in the liquid medium so as to express the recombinant protein described in claim 1. A suspension cell culture containing the above.

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