Therapeutic recombinant klotho protein and compositions and methods comprising 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 a CGMP-grade human recombinant soluble alpha-Klotho protein or a variant thereof, as well as methods for its 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 length that includes an extracellular region or domain (human residues 1-981) containing a short cytoplasmic tail (human residues 1003-1012), a transmembrane (TM) domain (human residues 982-1002), and two homologous (internal repeat) domains (KL1 (human residues 56-506, 450 residues in length) and KL2 (referred to as human residues 515-953, 438 residues in length), each having 20%-40% amino acid sequence homology with β-glucosidase but lacking glucosidase catalytic activity), and a signal sequence (SS) domain (human residues 1-33). The extracellular region containing the SS, KL1, and KL2 domains (human residues 1-981) 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 distinct 68 kDa (KL1+SS) and 64 kDa (KL2) proteins.

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

[0004] Klotho is highly expressed in the kidney and brain, less so in other organs, and can also be found in mammalian cerebrospinal fluid and urine. The circulating levels of soluble Klotho protein in mammals are thought to decrease with age. In addition, Klotho - deficient mice exhibit an accelerated aging phenotype, while overexpression of Klotho in mice prolongs lifespan. In addition, Klotho is involved in many cell processes related to aging. From the above perspectives, the hypothesis has been put forward that soluble Klotho may function as an anti - aging compound in the human body.

[0005] Aging is inevitable and a progressive biological process that leads to dysfunction and destruction of almost all tissues and organs and ultimately death. For example, aging in the human body is associated with a decline in cellular function that can lead to the onset of various diseases. Aging is thought to be caused by a complex interplay between genetic and environmental factors, and typical features include increased senescence, quantitative and qualitative reduction of stem cells, and abnormal structures at the tissue level.

[0006] As the so-called "baby boomer" generation ages, the global population of the elderly (e.g., 60 - 65 years old) is increasing rapidly. The increasing medical demand for this elderly population has become a significant economic burden on all healthcare systems. Recombinant Klotho protein may provide a promising therapeutic agent against age-related health conditions. Strategies and insurance interventions based on the production and (e.g., purification into substantially homogeneous forms) of soluble Klotho, as well as the administration of this protein to subjects within the increasing elderly population, may help to improve this situation and related problems.

[0007] Currently, there are no products or methods for providing exogenous forms of human Klotho protein, such as recombinant soluble human alpha-Klotho protein or protein variants, that comply with Current Good Manufacturing Practice (CGMP) regulations as determined and enforced by the US Food and Drug Administration (FDA), either alone or in combination with one or more additional active ingredients. In particular, constructing strategies and insurance interventions based on administering recombinant S-Klotho to subjects within the increasing elderly population may help to improve this situation. SUMMARY OF THE INVENTION

[0008] Embodiments of the present disclosure solve one or more of the foregoing or other problems in the art using recombinant human clotting proteins, protein fragments, and / or protein variants, expression nucleic acid constructs and / or vectors, cell lines and / or cell suspension cultures, and methods for their production, purification, and administration to a (human or non-human animal) subject.

[0009] For example, some embodiments of the present disclosure include recombinant human alpha soluble clotting protein, protein fragments, and / or protein variants, pharmaceutical (or therapeutic composition - e.g., formulation) of recombinant human alpha soluble clotting protein, composition of recombinant human alpha soluble clotting protein and at least one additional (active) ingredient, nucleic acid construct or vector encoding recombinant human alpha soluble clotting protein, (i) a nucleic acid construct or vector encoding recombinant human alpha soluble clotting protein, and / or (ii) a cell line expressing recombinant human alpha soluble clotting protein, (i) a nucleic acid construct or vector encoding recombinant human alpha soluble clotting protein, and / or (ii) a cell suspension culture of cells expressing recombinant human alpha soluble clotting protein, method for producing, and optionally purifying, recombinant human alpha soluble clotting protein, method for producing a pharmaceutical (or therapeutic composition, i.e., formulation) of recombinant human alpha soluble clotting protein, method for administering recombinant human alpha soluble clotting protein to a (human or non-human animal) subject, diagnostic method for measuring clotting protein deficiency in a subject, method for diagnosing clotting protein deficiency in a subject, A method for diagnosing a subject as in need of administration of recombinant human alpha soluble clotting protein by administration, A method for evaluating the effectiveness of a protein in a subject in need thereof and / or measuring an effective dosage, and Recombinant human alpha soluble clotting protein for use in the treatment of a specific medical condition or other pathological condition, and / or Use of recombinant human alpha soluble clotting protein in the manufacture of a medicament for the treatment of a specific medical condition or other pathological condition, may be included.

[0010] Some embodiments may include a method for producing a recombinant clotting protein, comprising producing the recombinant clotting protein in Chinese hamster ovary (CHO) cells, preferably in dihydrofolate reductase (DHFR)-deficient CHO cells, more preferably in CHO-S cells, or preferably in glutamine synthetase (GS)-deficient CHO cells, more preferably in GS- / -CHO cells, wherein the protein preferably has at least 85% amino acid sequence identity with one of SEQ ID NO: 2 to SEQ ID NO: 70.

[0011] Some embodiments may include a cell line comprising a plurality of Chinese hamster ovary (CHO) cells, preferably in dihydrofolate reductase (DHFR)-deficient CHO cells, more preferably in CHO-S cells, or preferably in glutamine synthetase (GS)-deficient cells, more preferably in GS- / -CHO cells, wherein the CHO cells comprise a promoter, preferably a strong promoter, and a polypeptide, at least a portion of the polypeptide having at least 85% amino acid sequence identity with one of SEQ ID NO: 2 to SEQ ID NO: 70, and optionally containing an exogenous nucleic acid encoding a functional dihydrofolate reductase (DHFR) enzyme or a functional glutamine synthetase (GS) enzyme.

[0012] Some embodiments include a liquid medium, preferably a serum-free and / or animal protein component-free liquid medium, preferably containing 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, and a cell line according to any one of claims 14 to 17 that grows in a liquid medium such that the CHO cells express a polypeptide encoded by a nucleic acid, wherein the polypeptide includes a recombinant clotting protein.

[0013] Some embodiments may include a recombinant clotting protein, wherein 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 an age-related or other medical condition, disease, or disorder, the method including administering to a subject in need thereof a pharmaceutically effective amount of the recombinant clotting protein described herein.

[0015] Some embodiments may include a method for treating an age-related or other medical condition, disease, or disorder, the method including administering to a subject in need thereof a pharmaceutically effective amount of a soluble recombinant clotting protein having at least 80% amino acid sequence identity with at least a subset of amino acid residues 1 to 981 of human alpha clotting isoform 1.

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

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

[0018] Some embodiments may include a method of treating or preventing acute kidney injury (AKI) or other conditions, comprising administering to a subject in need thereof a pharmaceutically effective amount of a recombinant soluble crotoxin protein, 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 the 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-crottoxin isoform 1, or at least a portion of one of SEQ ID NOs: 2-70.

[0019] Some embodiments include a therapeutic composition comprising a crotoxin protein, such as a CGMP-grade human recombinant soluble alpha-crotoxin protein, and at least one other active ingredient such as a drug, antibody, hormone, human cells, tissues, cell or tissue-derived products (HCT / Ps), and / or methods of administering them to a human or non-human subject. The combinatorial compositions and methods can be useful for treating subjects suffering from age-related disorders or conditions, metabolic disorders, chronic diseases, acute injuries, etc. Prophylactic administration of combination therapies to subjects without an apparent condition or disorder may also be useful for delaying or preventing certain conditions or disorders described herein.

[0020] Some embodiments may include a nucleic acid or nucleic acid construct. For example, an embodiment may include an expression vector or nucleic acid. The nucleic acid may encode a recombinant human alpha soluble crotoxin protein, protein fragment, or protein variant. The nucleic acid may encode a natural or non-natural signaling sequence. For example, the nucleic acid may encode a non-natural signaling sequence upstream (or N-terminus) of the encoded crotoxin protein sequence.

[0021] Some embodiments may include a method of manufacturing a recombinant human alpha soluble crotoxin protein. The manufacturing method may include growing Chinese Hamster Ovary (CHO) cells in a liquid medium, producing a recombinant soluble crotoxin protein in the CHO cells, and / or purifying a recombinant soluble crotoxin protein-containing extract from the CHO cells, the liquid medium, or both. The extract may contain at least about 98% dry weight of recombinant soluble crotoxin protein and / or less than about 1-100 ppm of CHO host cell protein (HCP). The CHO cells can be dihydrofolate reductase (DHFR)-deficient CHO cells such as CHO-S cells, or glutamine synthetase (GS)-deficient CHO cells such as GS- / -CHO cells. The produced (expressed) protein can be released (e.g., secreted) from the CHO cells into the liquid medium and / or may have one or more glycans attached thereto.

[0022] CHO cells may contain one or more exogenous nucleic acids encoding a protein and optionally a functional enzyme 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 an exogenous protein in CHO cells. The exogenous nucleic acid preferably has at least 85% nucleic acid sequence identity with one of SEQ ID NOs: 76 to 96, or any other suitable nucleic acid acidic 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 an 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 that does not contain (human, bovine (fetal), or other) serum and / or does not contain 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 method, 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 the DHFR transgene) to increase protein expression. Alternatively (or in addition), selection and / or gene amplification can be performed by adding MSX (an inhibitor of (endogenous) glutamine synthetase (GS)) 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 the liquid medium), and / or an increase in the copy number of the exogenous nucleic acid (e.g., per cell) (e.g., as compared to unselected suspension cultures or CHO cells).

[0026] Certain embodiments may include a cell line comprising a plurality of CHO cells. For example, the CHO cells can be DHFR-deficient CHO cells such as CHO-S cells. The CHO cells can contain one or more exogenous nucleic acids (including transgenes or cDNAs) encoding a polypeptide having at least 85% amino acid sequence identity with one of SEQ ID NOs: 2 to 70. The polypeptide can include a human recombinant alpha soluble clotting protein. The exogenous nucleic acid can 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 can also include a promoter (associated with the transgene), and / or can include or encode an optional (exogenous) enzyme such as a functional dihydrofolate reductase (DHFR) enzyme, glutamine synthetase (GS) enzyme, etc.

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

[0028] In some embodiments, the liquid medium can also include an effective amount of MTX and / or MSX. The suspension culture (or its CHO cells) exhibits an increase in protein production (e.g., by the CHO cells), an increase in the concentration of the protein (e.g., in the liquid medium), secretes the protein (e.g., into the liquid medium), and / or is selected to have an increase in the copy number of the exogenous nucleic acid (e.g., per cell) as compared to a suspension culture that was not preferably selected. The protein can have one or more glycans attached thereto.

[0029] Some embodiments include an extract of CHO cells, a liquid medium, or both, or an extract from a suspension cell culture thereof, which extract 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 clotting protein of CHO cells, a liquid medium, or both (e.g., a suspension cell culture), or an extract from them. 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 a method of administering a recombinant human alpha soluble clotting protein to a human or non-human animal subject in need thereof. The subject to which the clotting protein is administered may be suffering from or at risk of various pathologies (e.g., disorders, diseases, injuries, illnesses, etc.). For example, some embodiments include a method of treating one or more chronic diseases and / or pathologies associated with aging, such as physical, intellectual, neurological, or other pathologies associated with (human) aging. 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 a recombinant soluble clotting protein or protein variant to a subject in need thereof (e.g., a subject suffering from a pathology or at risk of the progression of a pathology). Administration of such a protein or protein variant may have a positive therapeutic effect on the course and outcome of a pathology, including chronic and / or age-related diseases and longevity in a human subject, and its characteristics.

[0031] A pharmaceutically effective amount may be sufficient to raise the serum soluble croton protein concentration of a subject to a predetermined level, such as about 50 to 3000 picograms or more per milliliter of serum, or a soluble croton protein therebetween. The amount may also, or alternatively, be sufficient to maintain the serum soluble croton protein concentration of the subject above a predetermined threshold for a predetermined period of time. Embodiments may also include administering the protein to a subject in need thereof so as to maintain the serum soluble croton protein concentration of the subject above a predetermined threshold for a predetermined period of time.

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

[0033] The protein may be capable of regulating the IGF-1 and / or Wnt signaling pathways (may be effective for regulating), may exhibit β-glucuronidase and / or sialidase activity, may suppress the p53 / p21 signaling pathway, and / or preferably may reduce cell senescence and apoptosis induced by H2O2 through suppression of the p53 / p21 signaling pathway. The protein preferably exhibits pleiotropic expression activity and / or preferably functions, or may function, as a humoral factor in the regulation of the activity of glycoproteins on the cell surface, such as the regulation of oxidative stress, growth factor signaling, ion homeostasis, and / or one or more ion channel proteins and / or growth factor receptors such as the insulin / insulin-like growth factor-1 receptor.

[0034] The protein may also be effective for treating one or more age-related conditions (or conditions related to (human) aging), such as debility, 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, freedom of movement, quality of life assessment, expulsion rate, decreased motor ability, learning ability, learning capacity, memory, or intelligence index, cognitive decline or amnesia, decreased cognitive ability or function, decreased synaptic plasticity or synaptic function, and cellular aging.

[0035] The protein may also be effective for treating one or more age-related conditions (or conditions related to (human) aging), such as Alzheimer's disease, Parkinson's disease, dementia or vascular dementia, amyotrophic lateral sclerosis (ALS), or motor neuron disease (MND), atrial fibrillation, chronic obstructive pulmonary disease (COPD), fibromyalgia, adult-onset diabetes, arthritis or rheumatoid arthritis, osteoarthritis, osteoporosis, glaucoma, cataract, macular degeneration and other eye diseases / disorders, multiple sclerosis (MS), lupus, and / or ulcerative colitis.

[0036] Accordingly, embodiments may also include a composition for use in treating one or more age-related conditions. The composition may include a recombinant soluble croto protein (e.g., having at least 85% amino acid sequence identity with one of SEQ ID NO: 2 to SEQ ID NO: 70), and a pharmaceutically acceptable carrier.

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

[0038] In various embodiments of the present disclosure, regardless of the product or process, the recombinant Crot protein has a sequence having 80% to 100% sequence identity with one of SEQ ID NOs: 1 to 38, preferably having a C-terminal tag having 80% to 100% sequence identity with one of the sequences of SEQ ID NO: 74 or SEQ ID NO: 75, and optionally having a linker sequence having 80% to 100% sequence identity with SEQ ID NO: 73 disposed therebetween, and may include one of the Crot proteins. The protein may optionally include, or be expressed using, a signaling sequence having 80% to 100% sequence identity with SEQ ID NO: 71 or SEQ ID NO: 72. Preferably, the protein (produced, generated, expressed or administered) has 80% to 100% sequence identity with one of SEQ ID NOs: 39 to 70.

[0039] Exemplary methods of producing a recombinant Crot protein include producing the recombinant Crot protein in Chinese hamster ovary (CHO) cells, preferably in dihydrofolate reductase (DHFR)-deficient CHO cells, more preferably in CHO-S cells, or preferably in glutamine synthetase (GS)-deficient CHO cells, more preferably in GS- / - CHO cells, wherein the protein preferably has at least 85% amino acid sequence identity with one of SEQ ID NOs: 2 to 70.

[0040] Exemplary cell lines include a plurality of Chinese hamster ovary (CHO) cells, preferably in dihydrofolate reductase (DHFR)-deficient CHO cells, more preferably in CHO-S cells, or preferably in glutamine synthetase (GS)-deficient (CHO) cells, more preferably in GS- / -CHO cells, wherein the CHO cells containing the exogenous nucleic acid contain a promoter, preferably a strong promoter, and encode a polypeptide, at least a portion of which has at least 85% amino acid sequence identity with one of SEQ ID NOs: 2 to 70, and optionally encodes a functional dihydrofolate reductase (DHFR) enzyme or a functional glutamine synthetase (GS) enzyme.

[0041] An exemplary suspension cell culture is a liquid medium, preferably a serum-free and / or animal protein component-free liquid medium, preferably containing a carbon source, a nitrogen source, and one or more vitamins, minerals, salts, amino acids, supplements, or additives, more preferably a liquid medium that lacks hypoxanthine, thymidine, and / or glutamine, and the CHO cells grow in a liquid medium such that the polypeptide encoded by the nucleic acid is expressed, and the cell line according to any one of claims 14 to 17, wherein the polypeptide includes a recombinant clotting protein.

[0042] An exemplary recombinant clotting protein includes at least 80% amino acid sequence identity with one of SEQ ID NOs: 2 to 70. An exemplary method for treating an age-related or other pathological condition, disease, or disorder, comprising administering to a subject in need thereof a pharmaceutically effective amount of a soluble recombinant clotting protein having at least 80% amino acid sequence identity with at least a subset of amino acid residues 1 to 981 of human alpha clotting isoform 1.

[0043] An exemplary method for treating age-related or other pathologies, diseases, or disorders, comprising administering to a subject in need thereof a pharmaceutically effective amount of a soluble recombinant crotoxin protein having at least 80% amino acid sequence identity to one of SEQ ID NOs: 2-70.

[0044] An exemplary pharmaceutical composition is a pharmaceutically effective amount of a recombinant soluble crotoxin protein, wherein at least a portion of the protein has at least a subset of the amino acid residues of 1-981, 29-981, 34-981, 36-981, 131-981, 1-549, 29-549, 34-549, 36-549, or 131-549 of human alpha-crottoxin isoform 1, or at least a portion of one of SEQ ID NOs: 2-70, and has at least 85% amino acid sequence identity to a protein, and a pharmaceutically acceptable carrier.

[0045] An exemplary method for treating or preventing acute kidney injury (AKI) or other pathologies, comprising administering to a subject in need thereof a pharmaceutically effective amount of a recombinant soluble crotoxin protein, wherein at least a portion of the protein has at least a subset of the amino acid residues of 1-981, 29-981, 34-981, 36-981, 131-981, 1-549, 29-549, 34-549, 36-549, or 131-549 of human alpha-crottoxin isoform 1, or at least a portion of one of SEQ ID NOs: 2-70, and has at least 85%, 86%, 88%, 90%, 92%, 95%, 98%, 99%, or preferably 100% amino acid sequence identity.

[0046] An exemplary method of treating an elderly individual, wherein the elderly individual has a homozygous or heterozygous mutation in the gene encoding the Klotho protein. The method comprises administering a therapeutic concentration of a polypeptide having an amino acid sequence identity of 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% 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 the present disclosure, including other aspects or embodiments of the present disclosure. It should also be noted that each of the foregoing, following, and / or other features described herein also represents a separate embodiment of the present disclosure. Furthermore, any combination of two or more of such features represents a separate embodiment of the present disclosure. Such features or embodiments can also be combined in any suitable combination and / or order without departing from the scope of the present disclosure. Thus, each of the features described herein can be combined with any one or more of the other features described herein in any suitable combination and / or order. Accordingly, the present disclosure is not limited to the specific combinations of exemplary embodiments described in detail herein.

[0048] Additional features and advantages of exemplary embodiments of the present disclosure are described in the following description, some of which will be apparent from the description or can be learned by the practice of such exemplary embodiments. The features and advantages of such embodiments can be realized and obtained by the means and combinations particularly pointed out in the appended claims. These features and other features will become more fully apparent from the following description and the appended claims or can be learned by the practice of such exemplary embodiments as described below.

[0049] To explain the manner listed above the present disclosure and by which other advantages and features can be obtained, a more specific description of the embodiments briefly described above will be provided by referring to the specific embodiments shown in the accompanying drawings. For a better understanding, similar elements are denoted by like reference numerals throughout the drawings. It is to be understood that these drawings only show typical embodiments of the present disclosure and are therefore not to be considered as limiting its scope, and that the present disclosure will be described and explained with additional specificity and detail through the use of the accompanying drawings.

Brief Description of the Drawings

[0050]

Figure 1

Figure 2A

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Modes for Carrying Out the Invention

[0051] Before detailing various embodiments of the present disclosure, it is understood that the present disclosure is not limited to specific parameters, turns of phrase, and descriptions of specific exemplified systems, methods, and / or products that may vary in each embodiment. Thus, while certain features (such as configurations, parameters, characteristics, steps, components, ingredients, members, elements, parts, and / or portions, etc.) are referred to in the detailed description of certain embodiments of the present disclosure, the description is illustrative and should not be construed as limiting the scope of the present disclosure and / or the claimed invention. Additionally, the terms used herein are for the purpose of describing embodiments and are not necessarily intended to limit the scope of the present disclosure and / or the claimed invention.

[0052] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure pertains. Various aspects of the present disclosure, including systems, methods, and / or products, may be illustrated with reference to one or more essentially exemplary embodiments. As used herein, the term "embodiment" means "serving as an example, instance, or illustration," and should not necessarily be construed as being preferred or advantageous over other aspects disclosed herein. Additionally, references to "embodiments" of the present disclosure or the invention are intended to provide illustrative examples without limiting the scope of the invention as indicated by the appended claims.

[0053] As used in this specification and the appended claims, the singular forms "a", "the", and "thereof" are each considered to include and specifically disclose both the singular and plural referents, unless otherwise clearly indicated. For example, a reference to "a protein" contemplates and specifically discloses one, as well as multiple (e.g., two or more, three or more, etc.) proteins. Similarly, the use of plural referents does not necessarily require a plurality of such referents, but unless otherwise clearly indicated, contemplates, includes, specifically discloses, and / or provides support for both the singular and plural of such referents.

[0054] As used throughout this disclosure, the words "can" and "may" are used in an allowable sense (i.e., meaning having the possibility) rather than a mandatory sense (i.e., meaning must). Further, the terms "comprise", "have", "contain", "feature", their variants (e.g., "comprising", "having", "containing", etc.), as well as similar terms used in this specification including the claims, are to be taken as inclusive and / or modifiable, and by way of example, the words "comprise" and its variants (e.g., "comprising") are taken to have the same meaning and do not exclude additional unrecited elements or method steps.

[0055] For the sake of brevity, the present disclosure may only list a list of numerical values or ranges. However, if such a list of numerical values or a range of numerical values (e.g., greater than, less than, at most, at least, and / or about a particular value, and / or between two recited values) is disclosed or recited, it will be understood that the disclosed values or list, or any particular value or range of values within the range of values, are likewise specifically disclosed and contemplated herein. Thus, the disclosure of exemplary measured values (e.g., length, width, thickness, etc.) that are about 10 units or less or from 0 to 10 units includes, by way of example, (i) measured values of 9 units, 5 units, 1 unit, or 0 units and / or any other value from 0 to 10 units including 10 units; and / or (ii) specific disclosure of measured values of ranges of any other values from 9 units to 1 unit, 8 units to 2 units, 6 units to 4 units, and / or from 0 units to 10 units.

[0056] For ease of understanding, where possible, similar references (i.e., the same component and / or similar names of the components) are used to denote similar elements common to different embodiments of the present disclosure. Similarly, components having the same or similar functions will, where possible, be provided with similar reference notations. In this specification, a particular language will be used to describe exemplary embodiments. Nevertheless, it will be understood that this is not intended to limit the scope of the present disclosure thereby. Rather, it is understood that the language used to describe exemplary embodiments is merely illustrative (unless it is expressly stated herein that such language is essential) and is not to 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, nor to limit the description or claims. Rather, the content of each paragraph in the detailed description is intended to be read and understood as an integrated whole in which the elements of one paragraph may relate to and / or be characteristic of other paragraphs. Thus, embodiments specifically disclosed within one paragraph may also relate to and / or function in additional and / or alternative embodiments in other paragraphs having the same and / or similar products, methods, and / or terms.

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

[0059] Gene therapy may be effective in animal experiments. However, the safety of gene therapy, particularly in human therapy, remains questionable. Compared to viral delivery of the clotting gene to animals (cells), the administration of exogenous and / or recombinant clotting protein in humans can be a safer, easier, and more direct manner to restore (endocrinologically) clotting levels. Thus, similar to the administration of erythropoietin or erythropoiesis-stimulating agents to treat anemia in CKD patients and / or insulin to maintain normal glucose metabolism in type I diabetes, the administration of exogenous (human recombinant alpha-soluble) clotting protein may become a realistic and effective option for treating the elderly and / or age-related disorders in the near future. For example, the administration of exogenous (human recombinant alpha-soluble) clotting protein to humans can be an effective strategy to reverse or delay stem cell depletion and / or reduce age-related frailty or other pathological processes.

[0060] Pre-clinical data has demonstrated the potential of soluble Klotho protein for the treatment of age-related diseases and diseases associated with Klotho deficiency. Epidemiological data shows that soluble Klotho is lower in the elderly than in young adults, and that the level of soluble Klotho is inversely correlated with age, indicating that aging is associated with a decrease in soluble Klotho.

[0061] Glossary of Defined Terms To assist in the understanding of the foregoing scope and content, as well as the description and claims set forth below, several selected terms are defined below.

[0062] As used herein, the term "pathology" refers to any disorder, disease, injury or illness that appears in or is expected in a patient, as understood by one of ordinary skill in the art. The manifestation of such a pathology can be an early, intermediate or late stage manifestation known in the art, including pre-pathological symptoms, signs, or manifestations. The prediction of such a pathology can be a prediction, assumption, expectation, imagination, supposition, and / or speculation of the occurrence of the pathology, whether found on scientific or medical grounds, risk assessment, or mere unease or fear, or can include them.

[0063] As used herein, the term "patient" specifically refers to a human being under the care of a physician or other relevant medical professional, and generically refers to any animal under the care of a physician, as the term is defined herein.

[0064] As used herein, the term "physician" generally refers to a medical doctor. This term includes any medical professional, including an oncologist, surgeon, or assistant to a physician, nurse, phlebotomist, veterinarian, etc., when appropriate in the context.

[0065] The term "cancer" refers to abnormal, typically uncontrolled cell growth. As used herein, "cancer cells" include malignant cells having abnormal, typically uncontrolled growth. Thus, the term cancer is an umbrella term encompassing a plurality of different distinct diseases characterized by malignant cells that grow in a typically uncontrolled manner.

[0066] The terms "co-administration" and similar terms mean the concurrent, sequential, and / or combined administration of two or more components. For example, two components can be co-administered by administering each component separately at separate dosages either concurrently, simultaneously, or sequentially (e.g., separate administrations separated by a period of time). The period can be very short (e.g., immediately following the first administration substantially), 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 therebetween). Concurrent or simultaneous administration can include overlapping of the administration time frames of two or more components, or administration of a combination product comprising a mixture of two or more components.

[0067] As used herein, the terms "nucleic acid" and similar terms refer to natural or synthetic oligonucleotides or polynucleotides, either DNA or RNA or DNA - RNA hybrids, single-stranded or double-stranded, sense or antisense. In particular, nucleic acids can include, but are not limited to, DNA, RNA, cDNA, gDNA, ssDNA, dsDNA, or any combination thereof. Nucleic acids of the present disclosure can also include nucleotide or nucleic acid analogs known in the art (e.g., BrdU), and non-phosphodiester (internucleoside) linkages or backbones (e.g., peptide nucleic acid (PNA) or thiodiester linkages).

[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 changing codons in a nucleotide sequence to codons that preferably or more closely approximate the codon usage patterns in the organism in which expression of the molecule is desired. Thus, codons can be optimized for use in a particular organism in which expression is desired, for example, to achieve a faster translation rate and higher accuracy in order to improve the effectiveness of nucleic acid expression, based on the known codon usage in that organism. The codon usage in a particular organism is known.

[0070] The coding nucleic acid molecule can be a modified wild - type, or a codon - optimized sequence optimized for expression in a particular host cell, such as mammalian cells, for example, CHO cells or 293 cells, or in yeast, or in plant cells, eukaryotic cells, etc.

[0071] In certain examples, the nucleic acid sequence can be codon-optimized, for example, to increase the expression level of the encoded sequence. The particular codon usage depends on the host organism in which the modified polypeptide is to be expressed. One of ordinary skill in the art is familiar with the optimal codons for expression in mammalian or human cells, such as 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 for a description of the database. See also Forsburg (2004) Yeast, 10:1045-1047, Brown et al. (1991) Nucleic Acids Research, 19:4298, Sharp et al. (1988) Nucleic Acids Res., 12:8207-8211, Sharp et al. (1991) Yeast, 657-78).

[0072] Therapeutic protein Embodiments of the disclosure can include one or more therapeutic and / or recombinant human alpha soluble clotting proteins, protein fragments, and / or protein variants.

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

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

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

[0076] The protein may also include one or more (attached) glycans. For example, native human alpha - clotting isoform 1 may have glycans attached (via glycosylation) at amino acids 106, 159, 283, 344, 604, 612, and / or 694. Thus, the protein of the present disclosure or its clotting protein sequence may have one or more of the same (or similar) glycans attached (via glycosylation) thereto (e.g., at the same amino acid position(s)). In preferred embodiments, the protein includes all of the same or similar (native - type) glycans attached thereto at the same amino acid position(s).

[0077] In some embodiments, the protein may include a signal peptide or signaling sequence. For example, the protein may include a native clotting signaling sequence. The protein may include a non - native or synthetic signaling sequence. In some embodiments, the signaling sequence may be upstream (or N - terminal) of the N - terminal signaling sequence and / or the clotting protein sequence. In other embodiments, the signaling sequence may be C - terminal or otherwise arranged. Preferably, the signaling sequence has, comprises, or includes at least 80%, 85%, 90%, 95%, 98%, or 99% amino acid sequence identity with the native human alpha - clotting isoform 1 signaling sequence, the native human alpha - clotting 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 at the C-terminus and / or downstream (or C-terminus) of the clotting protein sequence. In other embodiments, the tag may be at the N-terminus or otherwise arranged. 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 be, 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 or alternatively be (e.g., as known in the art) a TEV-twinstrep protein sequence or include it. Preferably, the signaling sequence may be, include, or 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 can be cleaved from the protein. In other embodiments, the tag can be retained as part of the protein. In some embodiments, the tag can improve the solubility and / or (serum) half-life of the protein. In some embodiments, the tag can be utilized (e.g., as part of a purification mechanism) during protein purification.

[0081] In some embodiments, the protein may include a linker (e.g., an amino acid linker) disposed between the clotting protein sequence and the amino acid tag. Exemplarily, the linker may include 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 be, 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 rules determined and implemented by the US Food and Drug Administration (FDA). For example, the croto protein may be at least 95%, 96%, 97%, 98%, or 99% pure by dry weight. In some embodiments, the croto protein sample may contain CHO host cell protein (HCP), nucleic acid, and / or other cellular components at less than about 1 to less than 100 parts per million (ppm), less than about 100 to less than 1000 parts per million (ppm), or less than about 1 to 100 ppm, or any value or range of values therebetween.

[0083] Nucleic Acids and Expression Vectors Some embodiments may include a nucleic acid or nucleic acid construct. For example, an embodiment may include an expression vector or nucleic acid construct. The nucleic acid may encode a recombinant human alpha soluble croto protein, protein fragment, or protein variant as described herein. In at least one embodiment, the nucleic acid may encode a croto protein sequence, an optional (natural or non-natural) signaling sequence (e.g., of the plurality or singular N-terminus of the croto protein sequence), an optional linker sequence (e.g., 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 can express a protein comprising 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 coactosin isoform 1. At least a portion of the protein can have at least or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% amino acid sequence identity with 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 coactosin isoform 1. For example, at least a portion of the protein can have at least and / or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% amino acid sequence identity with all or a portion of one of SEQ ID NOs: 1-75, or a combination of two or more thereof. In a preferred embodiment, the protein can have at least and / or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% amino acid sequence identity with all or a portion of one of SEQ ID NOs: 2-70.

[0085] In some embodiments, at least a portion of the nucleic acid has 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 SEQ ID NOs: 76 to 101, or a combination of two or more thereof. In preferred embodiments, the nucleic acid has 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 SEQ ID NOs: 76 to 96.

[0086] The nucleic acid sequences of the present disclosure may also contain stop codons (e.g., TGA, TAG, TAA) known in the art. Cell lines and production methods One embodiment of the present disclosure may include a cell line. The cell line may include any suitable 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 comprise at least one transgene or cDNA. In some embodiments, at least a portion of the nucleic acid is one of SEQ ID NOs: 76 to 101, or a combination of two or more thereof, preferably one of SEQ ID NOs: 76 to 96, and 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. The nucleic acid may be or may comprise a plasmid or other (structural) form of nucleic acid.

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

[0089] For convenience, throughout the present disclosure, reference is made to CHO cells and / or cell lines. However, it should be noted that other cells, cell lines and / or host cells (in addition to CHO cells) are also contemplated within the scope of the present disclosure. Thus, reference to CHO cells and / or cell lines also contemplates reference to and / or use of other known cells, cell lines and / or host cells.

[0090] Transfection The method of manufacturing a CHO cell line may preferably include introducing 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 the CHO cell line was used as a host cell line for the insertion of a nucleic acid (plasmid) containing a promoter, a human alpha S-clotting 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 of the transgenes encodes amino acids 1 to 981, 29 to 981, or 34 to 981 of human alpha soluble clotting. 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 a DHFR-deficient CHO cell line (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 include growing cells (e.g., transfected cells and / or CHO cells) on a solid medium and / or in a liquid medium (e.g., in a suspension cell culture), preferably in a medium that is serum-free and / or free of animal (or animal-derived) protein (components). For example, the cells can be plated on a solid growth medium for a period of time. The cells can also, or alternatively, be grown in a suspension culture and / or in 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 (e.g., 48 hours after transfection), the cells are collected (e.g., detached), optionally centrifuged (e.g., 100×g for 5 minutes), and / or seeded (e.g., at approximately 2000 cells / well) into 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. Non-transfected cells may die within 7 to 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 contain (may be selected to contain) at least about 2 to 10 copies, at least about 10 to 20 copies, at least about 20 to 30 copies, or at least about 30 to 50 copies of exogenous nucleic acid (per cell, for example). Thus, the method may include selecting CHO cells that contain at least about 2 to 10 copies, at least about 10 to 20 copies, at least about 20 to 30 copies, or at least about 30 to 50 copies of exogenous nucleic acid (per cell, for example). For example, MTX and / or MSX may be administered to the cells (e.g., at a concentration of about 1 nM to 1 μM, about 10 to 100 nM, etc.) (while continuously increasing the level).

[0094] Amplification of the dihydrofolate reductase (DHFR) gene in DHFR-deficient CHO cells (such as the CHO-S cell line) transfected with exogenous DHFR was achieved by continuously increasing the level of methotrexate (MTX) in the growth medium. Since the plasmid contains DHFR, exposure to MTX (10 - 100 nM) enables amplification of the S-clotting gene (fragment) in the host cell. The GS gene expression system was also used to amplify CHO cells transfected with exogenous GS (e.g., upon exposure to MSX). Alternatively, GS- / - host cell lines were also used to eliminate the need for MSX. These steps resulted in the production of multiple copies of the S-clotting gene (e.g., 10 - 30 copies of the gene per cell) and high-level expression of the S-clotting protein obtained in the transgenic cell line.

[0095] In some embodiments, in suspension culture, the protein can be secreted from the CHO cells into the liquid medium. For example, certain CHO cells and / or cell lines of the present disclosure can secrete (or can be selected to secrete) protein at a concentration of 200 - 500 mg of protein per liter of liquid medium, 500 - 2000 mg of protein per liter of liquid medium, 2000 - 5000 mg of protein per liter of liquid medium, or any value or range of values therebetween (without concentrating the protein). In at least one embodiment, a high-producing cell line (or suspension culture) can be selected such that the concentration of human recombinant alpha soluble clotting protein in the medium (of the selected suspension culture or the suspension culture of the 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 concentrating the protein.

[0096] Subcloning of high S-croton production transgenic colonies obtained by limited cell dilution was performed to further produce S-croton secreting CHO cell lines that secrete S-croton in the range of 500 - 2000 mg / L into the cell conditioned medium. All cell constructs were restriction digested and the sequences were confirmed.

[0097] In some embodiments, the CHO cells can be grown in a bioreactor having a volume or working volume of at least 10 liters, preferably at least 25 liters, more preferably at least 50 liters, even more preferably at least 100 liters, still more preferably at least 250 liters, still more preferably at least 500 liters, still more preferably at least 1,000 liters, still more preferably at least 2,000 liters, still more preferably at least 2,500 liters, still more preferably at least 5,000 liters, still more preferably at least 10,000 liters.

[0098] Maintenance of cell lines For high-producing S-croton cell lines (e.g., produced by the DHFR / MTX or GS / MSX systems) amplified prior to optional cell subcloning, the concentrated medium raw materials administered to cell line production were carefully used and carried out in a serum-free and animal protein component-free basal medium throughout scale-up and until the final bioreactor operation.

[0099] Scale-up of the high-yielding cell line was carried out by growing the cell inoculum material in a cell suspension in a shake flask or in a Wave Bag system, followed by successive inoculation of cells produced in 100 L and then 500 L volume bioreactors. The cell viability was over 85% viable cells throughout the growth cycle in the shake flask, wave bag, or bioreactor, and then the number of viable cells in the bioreactor was maintained at 80% or more during the plateau phase of CHO cell growth, with the S-croton produced concomitantly at up to 1 - 3 g / L (referred to as "high productivity").

[0100] Protein production Certain embodiments may employ recombinant DNA strategies that utilize strong promoter sequences and / or high-copy number plasmids for the production of therapeutic amounts of clotting protein in mammalian (e.g., CHO) cells. In at least one embodiment, for example, dihydrofolate reductase (DHFR) gene amplification in DHFR-deficient CHO cells may involve providing methotrexate (MTX) and / or using MTX (while continuously increasing the level). Similarly, CHO cells containing the exogenous glutamine synthetase (GS) gene can be treated with methionine sulfoximine (MSX).

[0101] The clotting protein may also contain one or more glycans (attached thereto). For example, native human alpha clotting isoform 1 (SEQ ID NO: 1) may have glycans attached (via glycosylation) at amino acids 106, 159, 283, 344, 604, 612, and / or 694. The clotting proteins of the present disclosure may have one or more of the same (or similar) glycans attached thereto (e.g., to the same amino acid(s)) (via glycosylation).

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

[0103] The glycan structures in which the produced S-Crot protein exists can be similar or identical compared to the structures of natural S-Crot structures isolated from human body fluids (i.e., blood, serum, urine, cerebrospinal fluid). In at least one embodiment, confirmation of the natural-like glycan can ensure that the correct natural post-translational modification (PTM) is generated and stably maintained in the S CHO cell-produced S-Crot protein.

[0104] Solubility and / or half-life extension of Crot protein Methods and compositions are disclosed for extending the half-life of human S-Crot protein and increasing its solubility. Also, the purification and characterization of protein constructs so produced to achieve these results are also the subject of the present disclosure. Nucleic acid changes made in the sequence of the Crot gene or nucleic acid construct (see SEQ ID NOs: 76-96), and / or changes or chemical modifications in the amino acid sequence of the Crot protein (see SEQ ID NOs: 1-70), and / or information regarding the addition or removal of chemical groups, peptides, or proteins to the amino acid sequence of the Crot protein are taught in the present disclosure to obtain resulting human Crot variant proteins (novel compositions) having an increased biological half-life or increased solubility in a biological matrix (such as blood, cerebrospinal fluid, urine, or various human tissues, etc.) compared to that of the native Crot molecule. These novel compositions can be made through the methods described herein for the modification of the S-Crot protein.

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

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

[0107] The novel S-Crot protein was produced through PEGylation. Using the foregoing and other half-life extension methods, the performance of the S-Crot protein was improved in several ways as follows: By lengthening the dosing interval of S-Crot, providing excellent convenience to patients and promising compliance, By reducing the dosing frequency, reducing the total usage of the drug and cutting the product cost, Reducing the drug amount at the same dosing interval as the parent protein, Simplifying the dosing prescription and enabling subcutaneous prescription, Using the same dosing and dosing interval as the parent protein, higher drug levels result in longer drug exposure and potentially better efficacy, Reducing the immunogenicity of S-Crot.

[0108] Production of the Fc domain fusion protein construct of S-Crot The half-life in the antibody Fc domain and human serum albumin (HSA) was extended, and the effectiveness of increasing the solubility of human S-crotatin protein was tested. Fc fusions include the fusion of a peptide, protein, or receptor ectodomain to the Fc portion of an antibody. Both Fc fusions and albumin not only achieve half-life extension by increasing the size of the peptide drug, but they also utilize the body's natural circulation mechanism through the binding of the extended protein to the neonatal Fc receptor, FcRn. After binding of the extended protein to the FcRn receptor, degradation of the fusion protein in the endosome of the cell is prevented. Fusions based on the addition of Fc or albumin can result in a biological half-life in the range of 3 to 16 days, much longer than that reported for typical PEGylated or lipidated peptides. For a review describing the use of protein fusion technologies such as Fc fusion proteins, fusions to human serum albumin, fusions to carboxy-terminal peptides, and other polypeptide fusion approaches to create biobetter drugs with more desirable pharmacokinetic profiles, see Strohl WR Fusion Proteins for Half-Life Extension of Biologics as a Strategy to Make Biobetters. Biodrugs. 2015;29(4):215-239, which is hereby incorporated by reference in its entirety.

[0109] The Fc domain was thus added to our parent protein (S-crotatin) to increase the binding affinity for the Fc receptor (FcRn). FcRn is present within the lysosomes of endothelial cells lining blood vessels and functions to rescue antibodies from degradation that would otherwise make most proteins short-lived in circulation. As a result of the interaction with FcRn, the protein has a half-life in the range of days to weeks, and the protein drug in its extended form allows for less frequent dosing than biologic agents that do not have this newly produced composition.

[0110] The main difference between Fc and albumin is the dimeric nature of Fc versus the monomeric structure of HSA, which leads to the Fc fusion peptide existing as a dimer or monomer, in contrast to HSA. The dimeric nature of the peptide Fc fusion can result in a binding activity effect if the target receptors of S-crotto are sufficiently close or separated, or if they themselves are dimeric, especially in human target organs. This can be desirable or not, depending on the target.

[0111] The fusion of the S-crotto protein to the antibody Fc is also taught in the present disclosure to improve the solubility and stability of S-crotto. The addition of the Fc domain to S-crotto also enables a reduction in the immunogenicity of the fusion protein upon administration to a human subject.

[0112] Conjugation of the S-crotto protein with human serum albumin (HSA) The 66.5 kDa protein HSA, similar to human IgG, has a long average half-life in the range of 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 maintenance. HSA, which is also the upper size limit for glomerular filtration of proteins by the kidney, is also strongly anionic and helps to further delay filtration through the kidney. Similar to IgG, HSA also binds to FcRn in a pH-dependent manner, via a site different from IgG binding and through a mechanism separate from that of IgG binding, and is recycled like IgG, resulting in an extension of its half-life. HSA also tends to accumulate in tumors and inflamed tissues, suggesting that fusion or binding to albumin may be useful for targeting proteins or peptides to those sites.

[0113] For the extension of the serum half-life of these molecules, the fusion of peptides or proteins with intrinsically short half-life characteristics to 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 potential biobetter molecules. The first HSA-peptide or protein fusion product to be approved for marketing was Tanzeum® (marketed as Eperzan® in the European Union), a 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 native GLP-1 to 4-7 days, thereby enabling once-weekly dosing. Seven other known HSA fusion protein product candidates are currently under development or have been developed in recent years. In addition, Novozyme is further developing a modified form of recombinant HSA with improved FcRn binding for the construction of "next-generation" HSA protein fusions that may have longer half-life characteristics. This was based on the use of a K573P mutant of HSA that was found to have 12-fold higher affinity for FcRn, which confers a longer half-life to HSA than the wild-type molecule in both mice and cynomolgus monkeys. These longer half-life mutants of HSA are expected to be further used as fusion proteins to improve the half-life of fusion proteins.

[0114] Accordingly, the inventors herein disclose that in order to provide the patient with excellent convenience and therapeutic advantages such as promising compliance, the inventors' Crot protein can be fused to wild-type HSA or a mutant form of HSA to produce a Crot fusion molecule having a significantly extended half-life in human blood, cerebrospinal fluid, and other human biological matrices, resulting in a reduced total drug usage and / or a reduction in product cost due to the reduced dosing frequency. Also, reducing the drug amount at the same dosing interval as the parent protein can simplify the dosing regimen, enable subcutaneous dosing, or enable a reduction in the immunogenicity of S-Crot.

[0115] Conjugation of S-Crot protein with human transferrin (TF) Transferrin is a very abundant serum glycoprotein found in serum at 3 - 4 mg / mL, which binds iron strongly and reversibly and functions to carry iron to tissues. Transferrin has 679 amino acid residues, is approximately 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 reported half-life of 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] Fusions of peptides and proteins have been made at the N-terminus and C-terminus of human transferrin, as well as in the central hinge region that connects the two major lobes of transferrin together. The N-terminus of transferrin is available and can be directly fused. The C-terminus is more buried and constrained by nearby disulfide bonds, so when a protein is fused to the C-terminus, a flexible linker is typically used. This ability has been expanded by creating libraries of peptides against specific targets and then fusing binders from those libraries to aglyco-transferrin (N-terminus, C-terminus, loop or linker regions) to construct therapeutic fusion proteins with extended half-lives.

[0117] Biotechnology company BioRexis Technologies, Inc., was founded in 2002 to develop a transferrin fusion protein platform, which it named the "Trans Body" platform as a therapeutic platform. Their lead molecule, BRX-0585, was a transferrin-GLP-1 fusion protein for the treatment of type 2 diabetes mellitus (T2DM). Fusion of GLP-1 to transferrin has been demonstrated to significantly improve the half-life of GLP-1. BioRexis was acquired by Pfizer in March 2007. As far as can be confirmed, fusion proteins derived from BioRexis are not currently in hospitals. The crotoxin protein can be fused to human transferrin to produce a crotoxin fusion molecule to significantly extend the half-life or stability in the human biological matrix in vivo and can be administered in hospitals.

[0118] Conjugation of S-crotoxin protein with Amunix's XTEN XTEN (registered trademark) is a recombinant polypeptide under exclusive ownership that extends the in vivo half-life of therapeutic payloads. XTEN consists of natural hydrophilic amino acids and is biodegradable. Pharmaceuticals such as proteins, peptides, and synthetic compounds can be XTENylated via chemical conjugation or gene fusion. The XTEN protein lacks secondary and tertiary structures, and its solution behavior resembles that of a chemically prepared polymer with a very large hydrodynamic radius. By size exclusion chromatography, the XTEN protein polymer appears much larger than typical globular proteins of similar molecular weight. The bulking effect of XTEN significantly reduces the renal clearance of the conjugated molecule and thus greatly increases the in vivo half-life. In the present invention, the length of the XTEN polymer conjugated to the clotting protein will be specified to optimize the pharmacokinetics and the in vivo distribution of the conjugated clotting protein payload.

[0119] Therefore, XTEN can be recombinantly fused to the inventors' S-clotting protein to increase the in vivo half-life of the molecule. One advantage is that the genetic S-clotting-XTEN fusion construct produces a molecule with the convenience of expression, purification, and characterization of a single molecule containing both the therapeutic and bulking portions. Recombinant fusions by therapeutic drug manufacturers that result in class-leading pharmacokinetics, as exemplified by XTENylated growth hormone (Somavaratan, Versartis) and FVIII-XTEN (Biogen), have been successfully used by enabling the attachment of multiple XTEN chains per protein at precisely defined positions. For example, pharmacokinetics conducted in pediatric patients administered different dosages of XTENylated growth hormone (Somavaratan, Versartis) demonstrated optimization of the Somavaratan molecule to reduce receptor-mediated clearance in addition to renal clearance, resulting in a class-leading half-life.

[0120] XTEN protein polymers can be produced as free intermediates for chemical conjugation to peptides, peptidomimetics, 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 XTENs containing 1-9 thiol groups at various intervals, which can be provided to partners. Thus, in the present invention, direct conjugation to amino and thiol groups in XTEN will facilitate the production of our croto-XTEN molecules.

[0121] Purification of Proteins Croto proteins can be extracted from cell suspension cultures of CHO cells (e.g., of the CHO cell line). CHO cells can produce and optionally secrete (e.g., into a liquid medium) croto proteins. Secretion of up to 200 - 500 mg / L of S-croto into the cell-consuming medium was also observed.

[0122] Purification of the recombinant proteins of the present disclosure can be carried out by any suitable method known in the art or described herein, such as any conventional procedure including extraction, precipitation, chromatography, and / or electrophoresis. Further purification procedures that can be used to purify the protein include affinity chromatography using monoclonal antibodies that bind the target protein. Some embodiments can include IgG-tagged proteins that can be purified by affinity chromatography. Generally, a crude preparation containing the recombinant protein is passed through a column to which a suitable monoclonal antibody is immobilized. The protein typically binds to the column via the 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, the spent medium from a CHO-S high-yield cell line was concentrated via tangential flow filtration, and the S-croto protein was purified by affinity chromatography followed by ion exchange cartridge or column chromatography. Size exclusion chromatography can also be used for protein purification.

[0123] In the alternative protocol, steps before and / or after one or more affinity purifications were performed. Such steps can include, for example, (ultra)centrifugation, dialysis, chromatographic separations such as ion exchange, membrane and / or tangential flow filtration, liquid-liquid extraction methods such as (aqueous) two-phase extraction methods, or other known purification steps. In certain embodiments, one or more purification post-treatment steps were performed. Such purification post-treatment steps can include, for example, tandem anion / cation flow-through chromatography (as opposed to binding and elution chromatography), virus and / or bacteria removal by 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 the croto protein Protein purity can be demonstrated by SDS-PAGE or other assays or means known in the art. For example, in at least one embodiment, a croto protein sample (50 μg) was fractionated on a precast SDS-PAGE gel (4-15%, 10 wells; catalog number 456-1083; BioRad) and stained with Coomassie blue dye. To avoid contamination between samples, all samples were placed with empty lanes in between or on separate gels. It was shown that more than 98% of S-croto was isolated from CHO S conditioned media as measured by Coomassie blue dye and densitometry transmittance, or by silver stain visualization, 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 separated by HPLC. The thus-prepared samples were then sequenced on an automated gas-phase microsequencer (Applied Biosystems model 470A, ABI, Foster City, Calif., USA) including an on-line automated HPLC PTH amino acid analyzer (Applied Biosystems model 120, ABI, see below) connected to a concentrator.

[0125] The protein was also analyzed through mass spectrometry. For sample preparation for mass spectrometry, only gel bands of 75 - 150 kDa were excised for analysis in order to limit the analysis to the correct S-crotatin protein. The gel fraction was softened with a sterile blade and subjected to in-gel digestion. The gel fraction was decolorized by washing three times with 80 μL of 50% acetonitrile (ACN) / 50 mM ammonium bicarbonate and washed with 100% ACN. The alkylation step was omitted considering the absence of cysteine residues from the target α-crotatin peptide. Trypsin digestion was carried out 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 in this process, of which 5 μL (1 μL for S-crotatin) was subjected to liquid chromatography - electrospray ionization tandem mass spectrometry (MS / MS) and PRM analysis on an Orbitrap nano-ESI Q-Exactive mass spectrometer (Thermo Scientific) attached to a nanoLC (Dionex Ultimate 3000 UHPLC). In the MS / MS analysis, it was confirmed that the human recombinant alpha-S-crotatin produced according to the embodiments of the present disclosure is substantially the same as that found in human blood, serum, urine, or cerebrospinal fluid (e.g., identical in the corresponding amino acid sequence).

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

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

[0128] The glycan structures present in S-Crot produced by CHO S cells were identical compared to the structure of native S-Crot isolated from human body fluids (i.e., blood, serum, urine, and cerebrospinal fluid). This ensured that the same native post-translational modifications (PTMs) were generated and stably maintained in the S-Crot protein produced in S CHO cells. Thus, using the manufacturing and purification methods described herein, the inventors have been successful in producing cGMP-grade human S-Crot having 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 generally may include a mixture of a therapeutically effective amount of a recombinant soluble alpha - clotting protein and a vehicle or carrier composed of one or more additional components. The components may include one or more aggregation inhibitors, buffers, tonicity agents, and additional excipients. The main solvent in the carrier may be essentially either aqueous or non - aqueous. The composition may be prepared by combining the purified clotting protein of the present disclosure with a pharmaceutically acceptable carrier.

[0130] One skilled in the art will understand that the combinations of the various components included in the composition can be carried out in any suitable order, that is, the buffer can be added first, in the middle, or last, and the tonicity agent can also be added first, in the middle, or last. Some of these chemicals may be incompatible in certain combinations and thus it will also be understood by one skilled in the art that they can be readily replaced with different chemicals that have similar properties but are compatible with the relevant mixtures.

[0131] The aggregation inhibitor reduces the tendency of the polypeptide to associate with inappropriate or undesirable three - or four - component complexes. Amino acid L - arginine, and / or L - cysteine can act to reduce the aggregation of Fc - domain - containing polypeptides in the formulation over a long period, for example, for two years or more. The concentration of the aggregation inhibitor in the formulation is preferably about 1 mM to 1 M, more preferably about 10 mM to about 200 mM, more preferably about 10 mM to about 100 mM, even more preferably about 15 mM to about 75 mM, and still more preferably about 25 mM. These compounds are available from commercial suppliers.

[0132] The compositions of the present disclosure may include a buffer. The buffer maintains the pH within a desired range. Various buffers suitable for use in the pharmaceutical compositions of the present disclosure include histidine, potassium phosphate, alkali salts, sodium phosphate or potassium phosphate or their hydrogen salts 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 buffer known in the art for maintaining the pH of the solution within the desired range. Mixtures of these buffers may also be used.

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

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

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

[0136] The formulations of the present disclosure may further comprise one or more tonicity agents (e.g., to make the solution isotonic with the patient's blood for injection). Tonicity agents are understood to be molecules that contribute to the weight osmolarity of the solution. The weight osmolarity of the pharmaceutical composition is preferably adjusted to maximize the stability of the active ingredient and also to minimize the discomfort of the patient upon administration. This is when the serum is approximately 300 + / - 50 millimoles per kilogram. It is generally preferred that the pharmaceutical composition be isotonic with the serum, i.e., have the same or a similar weight osmolarity achieved by adding a tonicity agent. Thus, while the weight osmolarity can be considered to be about 180 to about 420 milliosmoles, it is understood that the weight osmolarity can be higher or lower as required under specific conditions.

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

[0138] Additional examples of suitable tonicity agents for changing the weight osmolarity include, but are not limited to, amino acids (e.g., arginine, cysteine, histidine, and glycine), salts (e.g., sodium chloride, potassium chloride, and sodium citrate), and / or saccharides (e.g., sucrose, glucose, and mannitol). The concentration of the tonicity agent in the formulation is preferably about 1 mM to 1 M, more preferably about 10 mM to about 200 mM. Tonicity agents are known in the art, manufactured by known methods, and available from commercial suppliers.

[0139] Excipients, also referred to as chemical additives, cosolutes or cosolvents that stabilize polypeptides (even in dry or frozen form) in solution, can also be added to pharmaceutical compositions. An excipient is herein defined as a non-therapeutic agent added to a pharmaceutical composition to provide a desired effect, such as stabilization, isotonicity. Common attributes of desirable excipients are water solubility, non-toxicity, non-reactivity, rapid clearance from the body, and lack of immunogenicity. In addition, an excipient should be able to stabilize the native conformation of a protein in order to maintain the efficacy and safety of the drug during processing, storage and administration to the patient. Examples include, but are not limited to, sugars / polyols such as sucrose, lactose, glycerol, xylitol, sorbitol, mannitol, maltose, inositol, trehalose, glucose; polymers such as serum albumin (bovine serum albumin (BSA), human SA or recombinant HA), dextran, PVA, hydroxypropylmethylcellulose (HPMC), polyethyleneimine, gelatin, polyvinylpyrrolidone (PVP), hydroxyethylcellulose (HEC); polyhydric alcohols (e.g., PEG, ethylene glycol, and glycerol) non-aqueous solvents such as dimethysulfoxide (DMSO), and dimethylformamide (DMF); amino acids such as proline, L-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, 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 of the above.

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

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

[0142] In one exemplary embodiment, the formulations of the present disclosure can include, at a pH of about 6.0 to about 7.0, 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 can be replaced with L-cysteine (about 1 to about 500 micromoles) in the formulation. In yet another embodiment, the pH can be about 7.0. In another specific embodiment, the formulations of the present disclosure can include, at a pH of about 6.2, 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 formulations of the present disclosure can include about 10 to about 100 mg / mL of RANK:Fc in about 10 mM to about 100 mM of L-arginine, about 10 mM to about 50 mM of sodium phosphate, about 0.75% to 1.25% of sucrose, and about 50 mM to about 150 mM of NaCl, at a pH of about 6 to about 7. In a specific embodiment, the formulations of the present disclosure include 50 mg / ml of RANK:Fc in about 25 mM of L-arginine, about 25 mM of sodium phosphate, about 98 mM of sodium chloride, and about 1% of sucrose, at a pH of about 6.2.

[0144] In yet another embodiment, the formulations of the present disclosure may contain, at a pH of about 6 to 7, an effective amount of an Fc domain-containing polypeptide, from about 10 mM to about 100 mM of L-arginine, from about 10 mM to about 50 mM of sodium phosphate, from about 0 to 5% of mannitol, and from 0 to 0.2% of Tween®-20 (polysorbate 20). In another embodiment, the formulations of the present disclosure may contain an effective amount of an antibody such as Emab (anti-CD22 specific antibody), about 25 mM of L-arginine, about 25 mM of sodium phosphate, about 4% of mannitol, about 0.02% of Tween®-20 (polysorbate 20), and have a pH of about 6.0.

[0145] In yet another embodiment, the present disclosure provides a method of treating a mammal, comprising administering a therapeutically effective amount of the pharmaceutical composition described herein, wherein the mammal has a disease or disorder that can be beneficially treated with 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 to be treated with the composition. In certain embodiments, 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 (granulomatosis), 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 disease with a mutation in one or more Crotau genes. Additional diseases or disorders that can be treated with TNFR:Fc include those described in WO00 / 62790, WO01 / 62272, and U.S. Patent Application No. 2001 / 0021380, the relevant portions of which are incorporated herein by reference.

[0146] In yet another embodiment, the present disclosure provides a method for testing the accelerated stability of the Fc domain-containing polypeptide stability in the pharmaceutical compositions of the present disclosure, the method comprising the steps of testing the activity of the polypeptide formulated according to the present disclosure prior to storage, i.e., at the start, storing the composition at 37°C for 1 month and measuring the stability of the polypeptide, and comparing the stability profile at 1 month from the start. This information helps to early remove batches or lots that initially appear to have good stability but do not store well over the long term.

[0147] Furthermore, the pharmaceutical composition provides long-term storage such that the active ingredient, e.g., the Fc domain-containing polypeptide, is stable over the storage phase, whether in liquid or frozen state. As used herein, the term "long-term" storage is understood to mean that the pharmaceutical composition can be stored for 3 months or more, 6 months or more, 1 year or more, and preferably 2 years or more. Long-term storage also means that the pharmaceutical composition is stored as a liquid at 2-8°C or frozen, e.g., at -20°C or below (e.g., -20°C or -80°C). The composition is also considered to be capable of being frozen and thawed two or more times. The term "stable" with respect to long-term storage is understood to mean that the active polypeptide of the pharmaceutical composition does not lose its activity by more than 20%, more preferably 15%, even more preferably 10%, and most preferably 5% compared to the activity of the composition at the start of storage.

[0148] One or more antioxidants can be included in the formulations of the present disclosure. Antioxidants contemplated for use in the preparation of the 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 dosage forms are also contemplated, such as, for example, parenteral sustained release formulations, mist inhalants, orally active formulations, or suppositories. Thus, the formulations may also include bulk erosion polymers (e.g., poly(lactic-co-glycolic acid) (PLGA) copolymers, PLGA polymer blends, block copolymers of PEG, and particulate preparations of polymeric compounds such as lactic and glycolic acids, poly(cyanoacrylate)); surface erosion polymers (e.g., poly(anhydrides), and poly(orthoesters)); hydrogel esters (e.g., poloxamer polyols, poly(vinyl alcohol), poly(vinyl pyrrolidone), maleic anhydride-alkyl vinyl ether copolymers, cellulose, hyaluronic acid derivatives, alginates, collagen, gelatin, albumin, and starches and dextrans), and particulate preparations of their composition systems, or preparations of liposomes or microspheres. Such formulations can affect the physical state, stability, in vivo release rate, and in vivo clearance rate of the protein and derivatives. The optimal pharmaceutical formulation for a desired protein can be determined by one of ordinary skill 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, the disclosure of which is incorporated herein by reference.

[0150] Bioactivity As a method for evaluating the effectiveness of human recombinant alpha soluble klotho and / or measuring the effective dosage (for patients, subjects, or individuals presenting with age-related or metabolic disorders), assays based on organisms such as mammals (e.g., mice, rats, primates, or any other non-human), or other animals (e.g., Xenopus laevis, zebrafish, or flies (e.g., Drosophila melanogaster) or nematodes (e.g., Caenorhabditis elegans)) can be performed. The klotho protein can be administered to the organism once or in a regimen (regular or irregular). For example, the protein can be administered a suitable number of times (e.g., once, twice, etc.) over a given period (e.g., monthly, every two weeks, weekly, every half-week, daily, etc.). Subsequently, biological parameters (e.g., age-related parameters) can be evaluated. The klotho protein of interest can be compared to a reference (e.g., the parameters of a control organism) to effect or result in a change in the parameter. Other parameters (e.g., related to toxicity, clearance, and pharmacokinetics) can also be evaluated.

[0151] The klotho proteins of the present disclosure can be evaluated using animals (models) having or presenting with specific disorders or pathologies such as age-related or age-associated disorders or pathologies, metabolic disorders or pathologies, etc. Such disorders and pathologies can also provide a sensitization system in which the physiological effects of the protein can be observed. Exemplary disorders include, for example, denervation, disuse atrophy, metabolic disorders (e.g., disorders of obese and / or diabetic animals such as db / db mice, ob / ob mice), brain disorders, hepatic ischemia or other liver disorders, cisplatin / taxol / vincristine models, various tissue (xenograft) transplants, gene delivery bone models, pain syndromes (e.g., inflammatory and neuropathic), paraquat poisoning, genotoxicity, oxidative stress models, and tumor (I) models.

[0152] To evaluate the S-crototan protein of the present disclosure, the protein may be administered to a suitable animal (for a suitable treatment period), and the parameters of the animal may be evaluated (e.g., after a suitable period such as 10 - 60 minutes, 1 - 24 hours, 1 - 30 days, 1 - 12 months, 1 - 5 years, or any value or range of values therebetween). The animals may be fed ad libitum or normally (e.g., not under calorie restriction, although some parameters may be evaluated under such conditions). Typically, a cohort of such animals is used in the assay. Generally, if a test polypeptide affects a parameter in the direction of the phenotype of similar animals subjected to calorie restriction, the test polypeptide may be shown to favorably alter the lifespan regulation of the animal. Such a test polypeptide may at least somewhat cause a lifespan regulation effect by calorie restriction (e.g., a subset of such effects), rather than causing calorie intake in the organism.

[0153] The parameters to be tested can be age-related or disease-related parameters (e.g., symptoms of a disorder relevant to an animal model). A test protein shown to be favorable may cause an improvement in symptoms as compared to similar reference animals not treated with the polypeptide. Other parameters related to disorders or aging may include antioxidant levels (e.g., antioxidant enzyme levels or activities), 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 effectiveness of the S-crototan protein of the present disclosure for treating age-related disorders, animals with reduced Klotho expression (e.g., mice with mutations or Klotho gene deficiencies) can be used. For example, the test protein may be administered to mutant mice to monitor age-related parameters. A test protein shown to be favorable may cause an improvement in symptoms as compared to similar reference animals not treated with the protein.

[0155] Parameters related to metabolic disorders or aging can be evaluated by measuring body weight, examining the acquisition of reproductive ability, measuring blood glucose levels, observing lifespan, observing the skin, observing motor functions such as walking, etc. The evaluation can also be performed by measuring thymus weight, observing the size of calcified nodules formed on the inner surface of the chest cavity, etc. Furthermore, quantitative measurement of the mRNA of the Klotho gene or Klotho protein can also be useful for the evaluation.

[0156] As other (in vivo) models and bioassays, there may be mentioned evaluating animals with metabolic parameters, for example, parameters related to insulin disorders and type II diabetes. Exemplary metabolic parameters include glucose concentration, insulin concentration, and insulin sensitivity.

[0157] When evaluating whether a test protein can change lifespan regulation, many age-related parameters or biomarkers can be monitored or evaluated. Exemplary age-related parameters include: (i) the lifespan of cells or organisms, (ii) the presence or abundance of gene transcription or gene products in cells or organisms having an expression pattern dependent on biological age, (iii) the resistance of cells or organisms to stress, (iv) one or more metabolic parameters of cells or organisms (exemplary parameters include circulating insulin level, blood glucose level, fat content, core body temperature, etc.), (v) the proliferative ability of cells or cell populations present in organisms, (vi) the physical appearance or behavior of cells or organisms.

[0158] The term "mean lifespan" refers to the average age at death of a biological cohort. In some cases, the "mean lifespan" is evaluated using a cohort of genetically identical organisms under controlled environmental conditions. Deaths due to accidents are not counted. When the mean lifespan cannot be measured under controlled environmental conditions (for example, in the case of humans), reliable statistical information for a sufficiently large population (for example, from statistical tables) can be used as the mean lifespan.

[0159] Characterization of the molecular differences between two such organisms, for example between a reference organism and an organism treated with S-crotonylated protein, can reveal differences in the physiological state of the organisms. The reference organism and the treated organism are typically of 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 preselected event such as conception, a defined embryonic or fetal period, or more preferably birth. Various criteria can be used to determine whether organisms are of "the same" chronological age for comparative analysis.

[0160] Typically, the degree of precision required is correlated 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 some control conditions, organisms of the same age can survive for the same number of days. In the case of mice, organisms of the same age can 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 chronological age can survive for a time period within 15, 10, 5, 3, 2, or 1% of the average lifespan of the wild-type organisms of that species. Preferably, the organisms are adults (e.g., organisms that have survived for at least a certain amount of time such that an average wild-type organism matures to the age of reproductive capacity).

[0161] Biological screening assays can be performed before the organisms exhibit obvious physical characteristics of aging. For example, the organisms may be adults that have only survived for 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 can be evaluated in a test subject (e.g., an assay based on an organism) or in a patient (e.g., a human or mammalian patient) before, during, or after treatment with a therapeutic agent described herein.

[0162] Markers related to calorie restriction can also be evaluated in the subject organism (or treated subject) of the screening assay. These markers may not be age-related, but may indicate a physiological state that changes when the Klotho or Klotho-related pathway is regulated. The marker can be an mRNA or protein that changes significantly in calorie-restricted animals. A cell model derived from the cells of the animals described herein, or an analog of the animal model described herein, can be used in cell-based assays.

[0163] Examples of models for evaluating the effect of a test protein on muscle atrophy include: 1) a decrease in the mass of the rat medial gastrocnemius muscle resulting from denervation, for example, by cutting the right sciatic nerve in the middle of the thigh; 2) a decrease in the mass of the rat medial gastrocnemius muscle resulting from immobilization (for example, by fixing the right ankle joint at 90-degree flexion); 3) a decrease in the mass of the rat medial gastrocnemius muscle resulting from hindlimb suspension; 4) skeletal muscle atrophy resulting from treatment with the cachectic cytokine interleukin-1 (IL-1); and 5) skeletal muscle atrophy resulting from treatment with the glucocorticoid, dexamethasone.

[0164] Administration of exogenous S-Klotho The present disclosure relates to S-Klotho preparations, clinical dosages, and administrations for increasing and / or maintaining the serum concentration of S-Klotho in, for example, normal and / or young (e.g., 18 to 30 years old) individuals without (e.g., chronic) pathological conditions.

[0165] Aspects or embodiments of the present disclosure include, for example, administering to a (human) subject in need thereof a human recombinant alpha soluble klotho protein or a protein fragment (of isoform 1) (cGMP and / or clinical grade). Embodiments may also include measuring the level or concentration of serum S-klotho in a (human) subject (e.g., by mass spectrometer (MS) or ELISA). Such measurements can be made before, during, and / or after S-klotho administration and, if necessary, repeated to measure serum S-klotho levels and / or the rate of metabolism, degradation, or reduction of serum S-klotho levels. MS is a technique known in the art. MS can be used to identify and further quantify the levels of one or more (native and / or recombinant) klotho proteins in a subject's serum.

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

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

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

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

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

[0171] Embodiments may also include measuring the preferred frequency, amount, and / or duration of S-croto administration. For example, a subject having a low S-croto serum level (e.g., as measured by MS or ELISA immunoassay quantification) may have the serum S-croto concentration (change obtained) of the subject measured (by MS or ELISA), the serum S-croto level and / or the rate of metabolism, degradation or reduction measured following a first administration, the half-life of the administered S-croto calculated, and / or the frequency and / or time frame at which a second subsequent administration of S-croto should be received (e.g., to maintain the serum S-croto level above a second predetermined level) determined, and may receive a first administration of croto configured and / or adapted to bring the serum S-croto level of the subject to a first predetermined level (e.g., about 1000 pg / mL) via (e.g., intravenous, intradermal, intraperitoneal, intramuscular, 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% of the first predetermined level, and / or in between.

[0172] Further administrations can be given over a time frame suitable to produce (chronically) in the subject an S-Croto serum level equivalent to the serum maintenance level of a normal same-sex young adult (e.g., approximately 1000 pg / ml). The total duration of S-Croto administration (to a (human) subject) can range from 1 day to 5 years or more. Measurement and / or determination of the frailty of the subject based on the use of a clinical frailty score and other measurements can also be done over the time frame.

[0173] Embodiments of the present disclosure further include increasing and maintaining the S-Croto dosage to S-Croto levels increased by 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 include administering the S-Croto protein by a single bolus injection or a long-term (IV) injection (e.g., an infusion over a long period). In at least one embodiment, 1, 2, 2.5, 2.75, 3, 3.5, 4, 4.5, 5 micrograms or more of S-Croto per subject can be administered per treatment. Suitable dosages can be calculated through one or more methods known in the art. One such method is the allometric scaling method. For example, in a rat experiment, 0.01 mg of S-Croto / body weight kg or 10 μg / kg is used per administration. Exemplarily, using 0.16 which is equivalent to the human allometric growth rate, the human equivalent dose (HED) = 10 μg / kg x 0.16 = 1.6 μg / kg. Thus, exemplarily, a 70 kg human individual would require 70 kg x 1.6 μg / kg = 112 μg, and a 60 kg human would require 60 kg x 1.6 = 96 μg.

[0175] HED was established using the standardization of body surface area, a process described by Reagan-Shaw (2008), which is incorporated herein by reference. This process, termed relative growth rate, corrects for fundamental differences in metabolic rates between different species and may be preferred over simple dosage extrapolation. Exemplarily, when HED is 0.4 mg / kg, using the relative growth rate, the human equivalent dose would be 0.4 mg / kg, i.e., 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 for the determination, measurement, and / or estimation of the amount and / or bioavailability of S-clot in humans (before and / or after administration of the recombinant protein), the total amount and / or concentration of S-clot administered, and / or the serum level response (over time) after administration of the recombinant protein. For example, such factors may include the composition of the diluent, the route of administration, the site of administration, the distribution to the tissues and organs of the subject, the metabolic or other rates of the subject, pharmacokinetics (PK), pharmacodynamics (PD), toxicology (Tox), etc.

[0177] In at least one embodiment, the (normal) concentration of S-clot (e.g., in healthy, young (18 - 30-year-old) human adults) can be approximately 1000 pg / ml in serum. A typical adult may have approximately 5 liters of blood volume, and females generally have a lower blood volume than males. Approximately 55% of human blood can be composed of serum. Thus, (5 liters of blood / adult) x (0.55 serum / blood liter) = 2.75 liters (2750 ml) of serum / adult. Assuming no endogenous serum S-clot, 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-clot would be administered per adult subject.

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

[0179] To increase the serum soluble Crotau protein concentration in a subject to any suitable level, a pharmaceutically effective and / or sufficient amount of the purified recombinant S-Crotau protein can be administered at about 50, 100, 250, 500, 750, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 11,000, 12,000, 13,000, 14,000, 15,000, 20,000, 25,000, 30,000, 40,000, 50,000, 75,000, 100,000 picograms or more per milliliter of serum, or a soluble Crotau protein such as between, or a typical healthy level of soluble Crotau protein in serum can be increased by 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%, 5000% or more, or between.

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

[0181] In other embodiments, the dosage may be about 0.0001 - 10 mg / kg body weight, 0.0001 - 10 μg / kg body weight, 0.0001 - 10 ng / kg body weight, 0.0001 - 10 pg / kg body weight or more, or any value or range of values in between. Urine, and / or blood may be collected at one or more time points, for example, about 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 40 minutes, 45 minutes, 60 minutes, 90 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 9 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 hours, 2.5 days, 3 days, 5 days, 7 days, 10 days, 14 days, 21 days, 4 weeks, 1 month, 2 months, 3 months or more, less than, or in between, after a medical procedure or the first administration (dosage) of recombinant Crot protein (e.g., to measure, test, and / or determine serum S-Crot levels, as well as changes in the administration response and / or response over time).

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

[0183] Administration of exogenous S-Croto for treating frailty related to human age Exemplary embodiments of the present disclosure relate to the administration of exogenous croto protein for treating frailty related to age (e.g., of a human or non-human animal). S-Croto can rescue myogenic stem cells, improve muscle repair, and / or suppress fibrosis in animal models of human diseases. Thus, S-Croto can be a promising therapeutic agent against muscle degeneration in elderly human subjects showing signs of frailty.

[0184] For example, the present disclosure relates to S-Croto formulations, clinical dosages, and administration to frail and / or elderly individuals (e.g., 60 - 95 years old) to maintain the S-Croto serum concentration of the former subjects within the range of that of normal and / or young (e.g., 18 - 30 years old) individuals without (e.g., chronic) disease states.

[0185] Long-term croto treatment can restore and / or improve one or more age-related indicators or pathologies in the elderly, frail, or other physiological aging. Administration of exogenous S-Croto for treating (reducing) muscle atrophy in humans Exemplary embodiments of the present disclosure relate to the administration of exogenous croto protein for treating (e.g., reducing) muscle atrophy in humans as measured by skeletal muscle tissue mass and in combination with the above proteins and molecular markers, and provide guidance on the effect of croto administration in counteracting muscle atrophy.

[0186] Muscle atrophy can include a number of neuromuscular, metabolic, immune and neurological 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. Muscle atrophy can also result from reduced or disuse of muscle. 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 can include upregulation of ubiquitin ligase and a decrease in myofibrillar proteins. The breakdown of these proteins can be monitored, for example, in a specific muscle myosin (e.g., by measuring the production of 3-methyl-histidine, a specific component of actin). The release of creatine kinase (a cell damage marker) can also be an indicator.

[0187] Administration of exogenous S-croto throughout old age and / or throughout the human lifespan for treating intellectual and / or cognitive decline Exemplary embodiments of the present disclosure relate to the administration of exogenous croto protein for improving and / or suppressing cognitive function decline (related to aging). At the time of the present disclosure, it was unclear whether the administration of exogenous croto protein could suppress cognitive decline in humans. However, transgenic mice with systemic overexpression of croto had better performance than controls in multiple tests of learning and memory. The increase in croto in mice also improved long-term potentiation in the form of synaptic plasticity and enhanced synaptic GluN2B, an N-methyl-D-aspartic acid receptor (NMDAR) subunit that is important for learning and memory. Blocking GluN2B eliminated the effect mediated by croto.

[0188] The pathways that regulate Klotho may be associated with slowing the progression of Alzheimer's disease and other forms of dementia. Brain scans of over 400 healthy men and women aged 53 and older found that people who carried a single copy of a specific Klotho gene variant had larger brain regions that handle planning and decision-making. Further testing of the group found that people with an enlarged right dorsolateral prefrontal cortex (rDLPFC) performed better on a series of intellectual tasks.

[0189] Approximately one in five people inherit a single copy of a gene variant or allele known as KL-VS that improves heart and kidney function and, on average, extends human lifespan by about three years. However, with regard to brain function, having a larger rDLPFC results in only a 12% improvement in people's intellectual test scores, while, as the editors point out, retaining one copy of the KL-VS allele appears to provide ten years' worth of resilience against the expected decline in the structure and function of the rDLPFC. Thus, KL-VS heterozygosity appears to be associated with a larger volume of the right dorsolateral prefrontal cortex (rDLPFC).

[0190] Since the rDLPFC is important for executive function, the researchers also analyzed an individual's 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 Klotho gene diversity may be associated with larger brain volume and better function.

[0191] Exemplary embodiments of the present 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 improve cognitive ability and suppress cognitive deficits throughout human lifespan). Exogenous administration of clinically graded S-Klotho preserves and / or improves cognitive function (e.g., in humans).

[0192] Administration of exogenous S - croton for treating (prolonging) human longevity and / or lifespan Exemplary embodiments of the present disclosure relate to administering exogenous croton protein to human subjects of the same chronological age (e.g., the age from birth is the same) and sex to improve the average lifespan. The average lifespan results obtained with croton administration (experimental group of human subjects) can be reliably compared with those of individuals not receiving exogenous croton administration (control group) and / or statistical information for a large enough population (e.g., from mathematical tables).

[0193] Administration of exogenous S - croton for treating other clinical indicators Exemplary embodiments of the present disclosure relate to the administration of exogenous S - croton for treating morbid conditions related to or unrelated to any age, and the morbid conditions include, but are not limited to, human frailty (increase), longevity (decrease), cellular senescence (decrease), muscle strength (decrease), bone mass loss or density (decrease), cognitive ability (decrease), muscle mass (decrease), physical strength (decrease), grip strength (decrease), leg strength (decrease), etc. The present disclosure also relates to the administration of S - croton for increasing bone mineral density (BMD) (e.g., in women rather than men), increasing BMD that is reduced after menopause (e.g., in elderly women), regenerating (degenerated) skeletal muscle or reducing its degeneration, improving walking, spatial learning ability and memory, movement, freedom of movement, quality - of - life assessment, improving (or reducing the decrease of) expulsion rate, changes in exercise, improvement of exercise, etc. The present disclosure further relates to the administration of S - croton for reducing cognitive decline or amnesia, increasing cognitive ability, improving cognitive function and synaptic plasticity, reducing the decrease in learning ability, learning capacity or IQ, and improving learning ability, learning capacity, or IQ, etc.

[0194] Administration of exogenous S - croton for treating genetic defects Exemplary embodiments of the present disclosure relate to the administration of exogenous S-Klotho for treating (e.g., correcting) known human genetic defects. For example, a 13-year-old girl with familial tumoral calcinosis and a Klotho variant has been reported. Familial tumoral calcinosis is an autosomal recessive metabolic disorder characterized by ectopic calcification and hyperphosphatemia due to inactivating mutations in FGF23 or GALNT3. FGF23 is a hormone necessary for 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 the 13-year-old girl. Klotho encodes a secreted protein necessary for the transmission of signals released by FGF23 towards its receptor. Administration of exogenous human recombinant S-Klotho constitutes a highly targeted and effective treatment for addressing the dysfunctions and symptoms associated with familial tumoral calcinosis.

[0195] Administration of exogenous S-Klotho for treating acute kidney injury (AKI) Acute kidney injury (AKI), previously called acute renal failure (ARF), is often defined as the sudden onset of kidney dysfunction ranging from mild impairment to failure. AKI is a common clinical complication that occurs in approximately 4% - 7% of hospitalized patients each year and may have a poor prognosis. The mortality rate associated with AKI ranges from 20% - 35%. The expression of renal Klotho has been shown to be suppressed following AKI. Adenoviral gene transfer of Klotho may be cytoprotective in AKI.

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

[0197] AKI can occur at least in part from kidney transplantation or other surgeries, 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 surgeries can cause acute injury or damage to the kidney, leading to kidney disease and / or renal failure. Nephrotoxicity can contribute to AKI, ATN, AAIN, nephritis, etc. Drugs (e.g., clinically administered prescription drugs, illicit drugs, or other agents) 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 deterioration of renal function during hospitalization and postoperative renal insufficiency after a decrease in renal perfusion.

[0198] In some cases, drug-induced AKI can be or include nephrotoxicity induced by (resulting from) antibacterial agents. For example, certain (gram-negative) bacterial infections can be treated with one or more aminoglycosides such as paromomycin, tobramycin, gentamicin, amikacin, kanamycin, neomycin, etc. 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 more than 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, over 1.2 million adult patients of various age groups were treated with aminoglycosides in 2010. Other antibacterial agents include, for example, penicillin, ampicillin, cephalosporin, sulfonamide, ciprofloxacin, vancomycin, macrolide, tetracycline, rifampin, etc.

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

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

[0201] Drug-induced nephrotoxicity can also result from treatment with lithium, which can affect the flow of sodium through nerve and muscle cells in the body and can be used, in many cases, to treat manic episodes of bipolar disorder characterized by hyperactivity, pressured speech, decreased judgment, reduced sleep drive, aggression, and anger. Lithium can also help prevent or reduce the intensity of manic episodes. Drug-induced nephrotoxicity can also result from treatment with or exposure to gold, mercury, copper, or other elemental substances.

[0202] Drug-induced nephrotoxicity can also be caused by drugs in the chelating agent class, such as (D-)penicillamine, which can be used in the treatment of scleroderma, Wilson's disease (due to the accumulation and binding of copper removed through urine), and cystinuria (by binding to cysteine to produce mixed disulfides more soluble than cysteine); muscle relaxants that act directly on smooth muscle (e.g., hydralazine); and anticonvulsants (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, nephrotoxicity induced by contrast agents (e.g., nephropathy induced by radiocontrast agents (CIN), also known as contrast-induced nephropathy, following exposure to (iodinated) contrast agents); nephrotoxicity induced by narcotics (opiates) (e.g., after the use or abuse of certain narcotics such as cocaine, heroin, etc. (opiates)); nephrotoxicity induced by chemotherapy (e.g., cisplatin; carboplatin; oxaliplatin; alkylating agents such as bendamustine, cyclophosphamide, ifosfamide, nitrosourea, temozolomide, melphalan; antitumor antibiotics such as mitomycin C, bleomycin, anthracyclines, and related drugs; antimetabolites such as capecitabine, hydroxyurea, methotrexate, pemetrexed, pralatrexate, pentostatin, fludarabine, cladribine, gemcitabine, cytarabine; vinca alkaloids; topotecan; etoposide; taxanes; irinotecan; lenalidomide; eribulin; arsenic trioxide; ixazomib; etc. after treatment with cancer therapeutics). 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 untreated and can result in substantial treatment costs (for patients, hospitals, and insurance companies).

[0204] Embodiments of the present disclosure may include methods for treating or preventing (prophylactically) acute kidney injury (AKI) or other conditions. The methods may include administering a recombinant (soluble) Klotho protein to a subject in need thereof. For example, the methods may include administering to a subject in need thereof (e.g., so as to increase and / or maintain the subject's serum soluble Klotho protein concentration above a predetermined threshold for a predetermined period of time) a pharmaceutically effective amount of a recombinant soluble Klotho protein having at least 85% amino acid sequence identity to one of SEQ ID NOs: 2 to 70. The condition may include (i) acute tubular necrosis (ATN), acute allergic interstitial nephritis (AAIN), nephritis, glomerulonephritis, and / or nephrotoxicity, or (ii) AKI that results at least in part from kidney transplantation or other surgery, acute tubular necrosis (ATN), acute allergic interstitial nephritis (AAIN), nephritis, glomerulonephritis, nephrotoxicity, or hypotension. The condition may include drug-induced (e.g., induced by aminoglycosides) nephrotoxicity. The protein may be administered prophylactically, for example, prior to a kidney transplantation, nephrotoxin administration, or other activity, treatment, or event that is 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 a kidney transplantation or other surgery, aminoglycoside or other nephrotoxin administration, or other activity, treatment, or event that is known or expected to cause or contribute to AKI.

[0205] In some embodiments, the nephrotoxin or other agent may be, 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., (iodinated) radiocontrast agents, high osmolar contrast agents (HOCM) having an iodine to molecule ratio of about 1.5:1, low osmolar non-ionic contrast agents (LOCM) having an iodine to molecule ratio of about 3:1, isotonic (isosmotic) contrast agents (IOCM) having an iodine to molecule ratio of about 6:1, etc.); One or more antiretroviral agents (e.g., adefovir, cidofovir, tenofovir, foscarnet, etc.); One or more cancer (or chemo) therapeutic agents (e.g., cisplatin, carboplatin, oxaliplatin, alkylating agents (bendamustine, cyclophosphamide, ifosfamide, nitrosourea, temozolomide, melphalan, etc.), antitumor antibiotics (mitomycin C, bleomycin, anthracyclines and related agents, etc.), antimetabolites (capecitabine, hydroxyurea, methotrexate, pemetrexed, pralatrexate, pentostatin, fludarabine, cladribine, gemcitabine, cytarabine, etc.), vinca alkaloids, topotecan, etoposide, taxane, irinotecan, lenalidomide, eribulin, arsenic trioxide, ixazomib, etc.); One or more bisphosphonates or derivatives thereof (e.g., zoledronate / zoledronic acid, ibandronate, alendronate, alendronate / colecalciferol, etidronate, risedronate (optionally including calcium carbonate), pamidronate, tiludronic acid, etc.); and / or One or more narcotics (e.g., opioids) such as cocaine, heroin, etc., or may contain them; Embodiments of the present disclosure may include methods of administering a therapeutic recombinant (alpha-soluble) klotho protein (e.g., having at least 85% amino acid sequence identity with amino acid residues 1-981 of human alpha klotho isoform 1 or a subset thereof). The methods may include administering a therapeutic klotho protein to a human or non-human subject to (preventively) treat or prevent AKI or one or more conditions associated with AKI. The methods may include measuring the level of serum soluble klotho level in a subject, calculating a first dosage of the protein sufficient to raise the serum soluble klotho level in the subject to a predetermined level or a percentage of a normal level, administering the first dosage of the protein to the subject, such as by bolus or incremental dosing, measuring the rate of decline of soluble klotho in the serum following administration of the first dosage, calculating the next dosing time and amount, and / or administering the next dosage of the protein to the subject.

[0206] Administration of exogenous S-klotho for treating chronic kidney disease (CKD) As described in Neyra and Hu, Potential application of klotho in human chronic kidney disease, Bone (2017), which is hereby incorporated by reference in its entirety, circulating soluble klotho begins to decline early in chronic kidney disease (CKD) stage 2, and urinary klotho probably begins to decline even earlier in CKD stage 1. Thus, soluble klotho may function as an early and sensitive marker of kidney function decline. Furthermore, preclinical animal data support that klotho deficiency is not only a biomarker but also an etiology of CKD progression and extra-renal CKD complications including cardiovascular disease and mineral metabolism disorders. Prevention of klotho decline, reactivation of endogenous klotho production or supplementation with exogenous klotho are all associated with attenuation of renal fibrosis, delay of CKD progression, improvement of mineral metabolism, improvement of cardiomyopathy, and alleviation of vascular calcification due to CKD in animal models.

[0207] CKD is characterized by progressive deterioration of renal function with a high risk of ESRD. The CKD risk increases with age and occurs in approximately half of the cases of CKD stage ≧3 in subjects aged 70 years or older. CKD can be regarded as an accelerated state of aging. The relative risk of cardiovascular mortality in dialysis patients aged 25 - 34 years is the same as that in non-CKD patients aged 75 years or older. Cardiovascular disease is the main cause of death in CKD and ESRD patients. CKD and ESRD patients have low expression of renal Klotho and low levels of circulating Klotho. The renal Klotho deficiency in the early stage of CKD may be due mainly to the suppression of Klotho expression rather than the reduction of viable renal tubules. Furthermore, among dialysis patients, there are those who still have detectable circulating Klotho, the renal Klotho expression is not completely suppressed, and although the origin of Klotho has not been clarified to date, it is suggested that it may be derived from an extra-renal source. Establishing the extra-renal source of Klotho and characterizing how Klotho can be upregulated when renal production fails is of utmost importance.

[0208] Administration of the exogenous Klotho protein of the present disclosure may help prevent, delay, and reduce the burden of comorbidities in CKD. Compositions and treatments comprising S-Klotho in combination with other components Klotho may also act in additive or synergistic substances with other compounds and / or components that affect one or more aspects of human health and well-being. For example, a treatment comprising using a therapeutic human recombinant soluble alpha-Klotho (S-Klotho) protein in combination with and / or in parallel with one or more additional active ingredients may be beneficial to human patients. Such treatments may be preventive or responsive to any human pathological condition in which Klotho protein and / or other components may have a therapeutic effect. Such pathological conditions may include, for example, age-related pathological conditions, metabolic pathological conditions, chronic or acute pathological conditions, etc. Specific non-limiting examples of specific conditions are disclosed herein.

[0209] S-Clotho can exist in the human body together with other blood-borne anti-aging compounds such as growth / differentiation factor 11 (GDF-11). Thus, in certain embodiments, therapeutic S-Clotho can be co-administered with therapeutic GDF-11 (e.g., concurrently, sequentially, and / or in combination). In some embodiments, such administration can have additive or synergistic anti-aging or other effects. Similarly, co-administration of S-Clotho to a human subject using an (neutralizing) antibody against CCL11, or an inhibitor thereof, can act in concert to counteract aging or other pathologies (since CCL11, also known as eosinophil chemotactic protein-1, is understood to be a negative regulator of stem cell rejuvenation). S-Clotho can also, or alternatively, be co-administered with other eosinophils such as eosinophil chemotactic protein-2 (CCL24) and / or eosinophil chemotactic protein-3 (CCL26).

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

[0211] In some embodiments, S-Croto can be co-administered with an antibody or inhibitor of insulin growth factor-1 (IGF-1). Croto is a hormone that inhibits the intracellular insulin / IGF-1 signaling cascade, and this inhibition is understood to be a mechanism that is evolutionarily conserved to increase resistance to oxidative stress at the mammalian cell and organism levels and extend lifespan. Thus, co-administration of S-Croto with an inhibitor or antibody of insulin growth factor-1 (IGF-1) can have a synergistic or additive effect.

[0212] Due to the revelation of a comprehensive regulatory scheme for mineral homeostasis that includes the mutually regulated positive / negative feedback actions of croto alpha-K1, FGF23, and 1,25(OH)2D, and / or a similar regulatory network composed of croto beta-K1, FGF15 / human FGF19, and bile acids that regulate bile acid / cholesterol metabolism, in some embodiments, S-Croto 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, FGF-15, FGF-19, and / or croto beta. Such co-administration can have a synergistic or additive effect on numerous pathologies and / or processes in the body. In some embodiments, S-Croto can be administered in combination with FGF-21. In some embodiments, S-Croto can be co-administered with a carbonic anhydrase inhibitor such as acetazolamide, methazolamide, dichlorphenamide, dorzolamide, brinzolamide and / or topiramate. Such combinatorial administration can be useful for the treatment of ankylosing spondylitis (AS), rheumatoid arthritis (RA), and various other conditions. Various investigations have shown that increased bone resorption is characteristic of AS and RA, and that carbonic anhydrase inhibitors play an anti-arthritic role by inhibiting bone resorption. At the bone level, through a different mechanism acting on TRPV5, an osteoclast function regulator identified in recent years, S-Croto stimulates bone resorption and phosphate release. The increase in the level of 1,25(OH) + The increase in the level of D3 also stimulates osteoclast differentiation and bone resorption, thereby releasing phosphate. Thus, co-administration of S-Croto and a carbonic anhydrase inhibitor can have additive or synergistic effects, particularly in AS and RA, especially in promoting bone health.

[0213] For the treatment of severe active rheumatoid arthritis, S-Croto can be administered in combination with one or more disease-modifying anti-rheumatic drugs (DMARDs). Since cyclosporine decreases Croto mRNA and protein and increases oxidative stress leading to cyclosporine-induced kidney injury (CsA), S-Croto can be co-administered with cyclosporine. The associated decrease in Croto mRNA and protein and increase in oxidative stress can be counteracted by exogenous co-administration of S-Croto.

[0214] In some embodiments, S-Croto can be co-administered with losartan, and / or cyclosporine. Treatment with losartan, an angiotensin II type 1 (AT1) receptor blocker, reversed the decrease in Croto expression seen with cyclosporine. Losartan also resulted in a parallel improvement in kidney tissue (using losartan which decreases tubulointerstitial fibrosis caused by cyclosporine).

[0215] In some embodiments, S-clotrim can be co-administered with one or more aminoglycosides such as amikacin, gentamicin, tobramycin, etc. When aminoglycosides are used to treat infectious diseases, which can significantly expand the use of aminoglycosides in the treatment of (Gram-negative) pathogen infections, such treatment can be useful for preventing nephrotoxicity and / or acute kidney injury (AKI). Administering S-clotrim together with verapamil and / or diltiazem, which have been used to block AKI, can be therapeutic for the treatment and / or prevention of renal dysfunction from AKI.

[0216] In some embodiments, S-clotrim can be co-administered with testosterone or androgen receptor (AR) upregulation compounds. Recent reports have shown that no beneficial effects have been observed in testosterone treatment of men regarding 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 was considered not to be supported by randomized clinical trials. However, testosterone replacement has consistently been found to increase muscle strength, but no beneficial effects on physical function have been found. Administration of S-clotrim in combination with testosterone and / or androgen receptor (AR) upregulation compounds can significantly increase muscle strength and / or physical function in elderly, frail, or low T men beyond any effects that testosterone or S-clotrim may have alone on these treatment groups.

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

[0218] In some embodiments, S-Croto can be co-administered with one or more nootropics, also referred to as smart drugs or cognitive enhancers. Nootropic agents, supplements, and / or other substances can improve cognitive function, particularly executive function, memory, creativity, motivation, task saliency (the motivation to perform a task), performance (particularly of tedious tasks that require high effort), and can be useful in treating cognitive or motor dysfunctions that cause disorders such as Alzheimer's disease, Parkinson's disease, Huntington's disease, and ADHD. The most commonly used classes of drugs known to improve some aspects of cognitive ability are stimulants, particularly those that act as direct or indirect agonists of dopamine receptor D1, adrenergic receptor A2, or both receptors in the prefrontal cortex, and thus show a cognitive enhancing effect in humans. Examples of stimulants include, for example, amphetamines (e.g., amphetamine, dextroamphetamine, lisdexamfetamine, etc.) which can be beneficial in the range of cognitive functions (e.g., inhibitory control, episodic memory, working memory, and attention aspects) particularly in individuals with ADHD; dimethylamylamine (DMAA) such as 1,3-dimethylamylamine which can improve physical ability, alertness, reaction time, etc.

[0219] Methylphenidate - a substituted phenethylamine that can improve the range of cognitive functions (e.g., working memory, episodic memory, and aspects of inhibitory control, attention, and planning latency); eugeroics (e.g., armodafinil, modafinil, etc.) which can function as wake-promoting agents that can particularly increase the alertness of sleep-deprived individuals, facilitate logical thinking and problem-solving, and treat daytime sleepiness that remains after treatment of narcolepsy, sleep disorders due to shift work, and sleep apnea, etc.; xanthines (e.g., caffeine, etc.) that can increase alertness, performance, and / or memory; nicotine, etc.

[0220] In some embodiments, S-Croto can be co-administered with one or more osteoporosis and / or osteopenia pharmaceuticals, as is known in the art. Croto can play a role in regulating bone mineral density, and the absence of Croto can lead to a reduction in bone mineral density in animals. For example, Croto knockout mice show a reduction in bone mineral density over time. Croto expression can rescue bone defects in Croto knockout animals, such as when Croto knockout mice show a reduction in bone mineral density over time. Epidemiological studies have shown an association between various Croto gene polymorphisms and changes in bone mineral density and the spread of hand osteoarthritis.

[0221] S-Croto can be administered in combination with one or more anti-cancer treatments and / or preventions, such as chemotherapy. In lung cancer, such as non-small cell lung cancer (NSCLC), for example, S-Croto administration can affect the resistance of lung cancer cells to cisplatin and / or other chemotherapy. In addition, S-Croto can function as a potential tumor suppressor in lung cancer, gastric cancer, pancreatic cancer (adenocarcinoma), and other forms of cancer. S-Croto can be administered in combination with sorafenib chemotherapy for the treatment of hepatocellular carcinoma (HCC). Overexpression of Croto and treatment with soluble Croto protein can reduce the growth of liver cancer cells in vitro and in vivo. Other types of cancer that can be treated with S-Croto co-administration include hepatocellular carcinoma (HCC), central nervous system (CNS) cancer (such as brain (e.g., glioma, craniopharyngioma, medulloblastoma, and meningioma), spinal cord and other tumors, lymphoma, etc.), (metastatic) colon cancer, and the like.

[0222] S-Croto can also be administered in combination with chemotherapy to treat the frailty induced by chemotherapy in cancer patients. S-Croto can also be administered following other known treatments to treat the frailty induced by cancer in cancer patients.

[0223] S-Croto can be administered in combination with kidney dialysis or other treatments. Since frailty is associated with poor prognosis in dialysis patients, S-Croto can be administered to treat frailty in dialysis patients.

[0224] Since an appropriate amount of BDNF can help develop and maintain normal neuronal circuits in the brain, S-Croto can be administered in combination with one or more Alzheimer's disease treatments or preventions, or in combination with brain-derived neurotrophic factor (BDNF).

[0225] To enhance the ability of croto to enter the central nervous system (CNS) for treating or preventing CNS-related pathologies, S-Croto can be administered in combination with one or more molecules that enhance the ability of croto to cross the blood-brain barrier. For example, it is known that neither S-Croto nor BDNF can cross the blood-brain barrier. Embodiments of the present disclosure include utilizing blood-brain barrier delivery techniques for administering S-Croto, including S-Croto and / or BDNF, to the CNS for treating Alzheimer's disease and / or improving the cognitive ability of individuals not suffering from Alzheimer's disease.

[0226] S-Croto can be administered in combination with a component that positively regulates the 5'-adenosine monophosphate-activated protein kinase (AMPK), or an AMPK-activating agent, or a signaling pathway that replenishes the ATP supply of cells including fatty acid oxidation and autophagy, or negatively regulates ATP-consuming biosynthetic processes including gluconeogenesis, lipid, and protein synthesis.

[0227] S-Croto can be administered in combination with one or more anti-diabetic agents such as insulin, phlorizin, or the antioxidant tyron, and these combination treatments may have merit in preventing kidney damage due to oxidative stress occurring in diabetic disorders. Co-administration of S-Croto and other anti-diabetic agents for type 1 diabetes can 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-Croto can be administered in combination with one or more type 2 anti-diabetic agents, such as metformin, to improve glycemic control and vascular function in overweight and obese diabetic subjects. S-Croto can be administered in combination with the treatment or prevention of one or more blood pressure pharmaceuticals, calcium regulators, or chronic kidney disease (CKD). For example, soft tissue calcification is a prominent feature of CKD, and croto can improve vascular calcification by improving phosphate excretion, maintaining glomerular filtration rate, and directly inhibiting phosphate uptake by vascular smooth muscle.

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

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

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

[0232] Embodiments of the present disclosure further include a combination product comprising a therapeutic recombinant crotor protein in combination with human stem cells. The composition can also include a pharmaceutically acceptable carrier as described herein. Such composition can be included or classified as a regenerative medicine for treating, modifying, reversing, or curing a severe or life-threatening disease or condition that has been approved by the FDA. Preliminary clinical evidence indicates that the composition (agent) has the potential to address unmet medical needs for such diseases or conditions.

[0233] Exemplarily, the stem cells can be or can include mesenchymal stem cells (MSCs) such as from the human umbilical cord or placenta. In at least one embodiment, the composition comprising huMSC and the therapeutic recombinant crotor protein of the present disclosure attenuated the inflammatory and oxidative stress responses that occur in AKI and / or decreased the expression of proteins and microRNAs associated with aging.

[0234] S-Clotho can be administered in combination with one or more anti-aging agents. For example, the Clotho protein, in combination with Pin1-FOXM1 and / or other anti-aging agents, can improve the outcomes in patients undergoing such treatment.

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

[0236] Any of the foregoing or other treatments or co-administrations can have an additive or synergistic effect compared to any one treatment alone. For example, co-administration of the Clotho protein with one or more of the foregoing can produce a greater therapeutic outcome than the sum of the individual outcomes of administering the individual components at the same concentration. In addition, the synergistic effect can include a therapeutic outcome similar to that of the individual outcomes of administering the individual components at a lower concentration. The synergistic effect can also include an increase in the maximum effective dosage of one or more of the components, a reduction in the toxicity of one or more of the components, or any other beneficial outcome beyond the mere additive effect of the individual therapeutic outcomes. In addition, the additive effect of the individual therapeutic outcomes can include a synergistic effect where such an additive effect is not predicted or expected considering the nature and understanding of the individual components.

[0237] As used herein, combination therapy or co - administration may include the treatment or administration of a combination product, composition, or formulation that includes a Crot protein and one or more additional active ingredients. The one or more additional active ingredients may be selected from among the ingredients, agents, substances, therapeutic compositions, etc. described herein, or others known in the art. For example, the Crot protein and one or more additional active ingredients may be co - formulated into injectable (e.g., intramuscular, intravenous, etc.), ingestible, transdermal, inhalable, topical, or other formulations.

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

[0239] Also, 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 period of time. In some embodiments, the period may be very short. For example, immediately following administration of a first component of a Crot protein and one or more additional active ingredients, a second component of the Crot protein and one or more additional active ingredients may be substantially administered (e.g., injected). Alternatively, the first and second administrations may be separated by a period such as 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 therebetween. Similarly, simultaneous administration may include an overlap in the administration time frames of two or more components.

[0240] Crot protein variants Therapeutic S-Crot proteins of various lengths (e.g., S-Crot 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 various beneficial effects and / or results not exhibited by native Crot proteins. Exemplarily, the QuickChangeXL Site-Directed Mutagenesis Kit (Stratagene) can be used to vary the nucleic acid sequences of various S-Crot constructs. Other mutagenesis methods and kits known in the art can also be used. For example, various subcloning methods and kits are known in the art and are commercially available.

[0241] In at least one embodiment of the present disclosure, the protein is modified with one or more C-terminal tags and / or N-terminal tags. Such tags may function to extend the serum and / or soluble half-life of the protein (in one or more therapeutic or other settings). 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, and the like. In at least one embodiment, the C-terminus of the protein may be tagged with a TEV-TwinStrep and / or an Fc fusion, as known in the art and further described herein. Additional explanations may 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 its entirety. In certain embodiments, a linker or linker peptide may be inserted and / or positioned between the (native or variant) clotting protein sequence and the tag.

[0242] In some embodiments, the modified clotting protein may include an alternative (e.g., natural, non-natural, and / or synthetic) signal peptide. For example, in some embodiments, the natural signal peptide sequence may be replaced and / or supplemented with an alternative signal peptide or signaling sequence (SS). In some embodiments, the native methionine residue of the clotting protein may be removed and a methionine residue may be included at the N-terminus of the SS. Additional 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 hereby incorporated by reference in its entirety. In certain embodiments, a linker or linker peptide may be inserted and / or positioned between the (native or variant) clotting protein sequence and the alternative SS.

[0243] Some embodiments may include one or more amino acid variants. It will be understood that the present disclosure contemplates diversifying any one or more of any of the native amino acids of the disclosed clotting proteins to any other amino acid, regardless of whether natural, synthetic, or otherwise configured.

[0244] S-Clotting 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 is composed 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 are reduced 6-fold in the V352 variant, while they are increased nearly 3-fold in the S370 form. However, these two variants in the human Klotho gene co-segregate and form the KL-VS haplotype that increases Klotho secretion in the range of 1.6-fold. For example, screening of over 300 individuals collected from geographically and / or ethnically distinct cohorts has reported that no individual has been found to carry only one of the V352 or S370 variants.

[0245] One embodiment of the present disclosure includes a recombinant S-Klotho protein having the C370S homozygous variant (i.e., the F352V variant is absent (or with its deletion)). The C370S variant can be produced and / or expressed in relation to any protein construct described herein. For example, the C370S variant can be produced or expressed in relation to S-Klotho 1-981, 29-981, 34-981, 36-981, 131-981, 1-549, 29-549, 34-549, 36-549, 131-549, etc., regardless of the presence or absence of the Fc fusion and / or TEV-TwinStrep. Thus, the nucleic acid construct or cDNA in which the protein is expressed can be of the corresponding length.

[0246] Embodiments can include producing an S370 heterozygous or homozygous polymorphic construct, introducing the resulting construct encoding the S-croto C370S protein (e.g., via transfection) into a suitable expression system (e.g., CHO cells), and / or transiently expressing the S-croto S370 protein. The S370 croto protein can be expressed at a higher level than the F352V / C370S protein and / or the wild-type F352 / C370 protein. Embodiments can include purifying the protein expressed for therapeutic administration (and optionally conducting quality control tests). Embodiments can include administering a therapeutic or therapeutically effective amount of the S-croto C370S protein to a subject in need thereof. The subject can, for example, carry or express the KL-VS polymorph. Alternatively, the subject can be wild-type for other variants or polymorphs. Administration of the recombinant S-croto C370S protein can lead to a beneficial increase in blood S-croto levels. Thus, the circulating concentration of S-croto in a subject administered the therapeutic recombinant S-croto C370S protein may not be subject to the dilution effect observed when the F352V polymorph is present.

[0247] Therapeutic treatment of hyperphosphatemic familial tumoral calcinosis (HFTC) In affected human individuals, HFTC is caused by a mutation from histidine (H) to arginine (R) at amino acid (AA) position 193 of S-croto-rs121908423. Without being bound by any theory, the H193R mutation in HFTC individuals is thought to impair the ability of S-croto to form a three-component complex with FGF23 and FGFR1c that impairs KL-dependent FGF23 signaling. As a result, affected subjects exhibit a severe metabolic disorder with hyperphosphatemia and massive calcium deposition in the skin and subcutaneous tissue. In some patients, there is a recurring transient painful swelling of the long bones associated with radiological findings of periosteal reaction and cortical hyperostosis and absence of skin infiltration.

[0248] One embodiment of the present disclosure includes an S-crotan protein having H193. The H193 protein can be produced or expressed in relation to any protein construct described herein. For example, the H193 variant can be produced or expressed in relation to S-croto 1-981, 29-981, 34-981, 36-981, 131-981, 1-549, 29-549, 34-549, 36-549, 131-549, etc., regardless of the presence or absence of Fc fusion and / or TEV-TwinStrep. Thus, the nucleic acid construct or cDNA in which the protein is expressed can be in the corresponding conformation.

[0249] Embodiments can include producing an H193 heterozygous or homozygous variant construct, introducing the resulting construct encoding the S-croto H193 protein (e.g., via transfection) into a suitable expression system (e.g., CHO cells), and / or transiently expressing the S-croto H193 protein. The H193 crotan protein can also be expressed at a higher level than the R193 (or H193R) protein. Embodiments can include purifying the protein expressed for therapeutic administration (and optionally conducting quality control tests). Embodiments can include administering a therapeutic or therapeutically effective amount of the S-croto H193 protein to a subject in need thereof (e.g., an HFTC individual, an individual diagnosed with HFTC, or a patient carrying H193R (rs121908423) or other variants). Alternatively, the subject can be wild-type for other variants or variants. Administration of the recombinant S-croto H193 protein can lead to a beneficial increase in blood S-croto levels. The administration can reverse or suppress the adverse effects of the R193 variant transcribed and circulated in HFTC individuals as a result of the H-~-R193 point mutation found in the human crotan gene of individuals suffering from HFTC. Thus, the circulating concentration of S-croto H193 can help counteract the effects observed in H193R or HFTC individuals.

[0250] Therapeutic Treatments in CC Genotype Patients with End-Stage Renal Disease (ESRD) Approximately 350,000 patients with end-stage renal disease (ESRD) suffer from a very high mortality rate during the first year of regular hemodialysis. Both vitamin D and fibroblast growth factor (FGF)-23 levels are correlated with the survival of these patients. Although not bound by any theory, klotho is a protein in the vitamin D / FGF-23 signaling pathway and is associated with accelerated aging and premature death in animal models. A hypothesis has been proposed that genetic mutations in the klotho gene may be associated with the survival of subjects with ESRD. The researchers examined the association between 12 single nucleotide polymorphisms (SNPs) of the klotho gene and the mortality rate of an ESRD patient cohort (n = 1,307, white and Asian) during the first year of hemodialysis. A significant association was found between the CC genotype of one tag SNP, rs577912 (a common HapMap variant with a minor allele frequency [MAF] > 0.05 within the klotho 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). Among patients not treated with active vitamin D supplements (HR, 2.51; 95% CI, 1.18 - 5.34; p = 0.005), this effect was even more pronounced in individuals with the CC genotype. In lymphoblastoid cell lines derived from HapMap subjects, the CC genotype was associated with 16 - 21% lower klotho expression compared to the AA or AC genotypes. However, none of the above rs577912 SNP nucleotide changes result in amino acid changes in the klotho protein. Therefore, this functional SNP (rs577912) may quantitatively affect klotho gene expression at the mRNA level.

[0251] One embodiment of the present disclosure includes an S-Crot protein expressed from the AA or AC genotype. The protein can be produced or expressed in relation to any protein construct described herein. For example, the protein can be produced or expressed in relation to S-Crot 1-981, 29-981, 34-981, 36-981, 131-981, 1-549, 29-549, 34-549, 36-549, 131-549, etc., regardless of the presence or absence of an Fc fusion and / or TEV-TwinStrep. Thus, the nucleic acid construct or cDNA from which the protein is expressed can be of the corresponding length.

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

[0253] Treatment of Hand Osteoarthritis (OA) and Bone Spurs by X-ray Osteoarthritis (OA) is a common complex disease with a strong genetic component. Researchers studied the association between four putative functional gene polymorphisms of the klotho gene and hand osteoarthritis OA in a large population of white women. The researchers found a significant association between SNP G-395A and the presence or absence of hand OA and bone spur formation by X-ray. Allele G significantly increased the risk of hand OA and bone spurs by X-ray with odds ratios (OR) of 1.44 (P = 0.008, 95% confidence interval (CI) 1.09 - 1.91) and 1.36 (P = 0.006, 95% CI 1.09 - 1.70), respectively. From logistic regression modeling, genotype GG showed more than a three-fold increase in the risk of both hand OA (OR = 3.10, 95% CI 1.10 - 8.76) and bone spurs (OR = 3.10, 95% CI 1.10 - 8.75) by X-ray compared to genotype AA. After age adjustment, the OR for genotype GG further increased to 4.39 (P = 0.006, 95% CI 1.51 - 12.74) for hand OA by X-ray and 4.47 (P = 0.005, 95% CI 1.56 - 12.77) for bone spurs. The researchers also suggested that one polymorphism (SNP G-395A) of the klotho gene is associated with the susceptibility to hand OA and appears to act through bone spur formation rather than cartilage damage.

[0254] One embodiment of the present disclosure includes an S-crotan protein expressed from a construct having SNP G395A. The resulting protein can be produced or expressed in relation to any protein construct described herein. For example, the A395 variant can be produced or expressed in relation to S-croto 1-981, 29-981, 34-981, 36-981, 131-981, 1-549, 29-549, 34-549, 36-549, 131-549, etc., regardless of the presence or absence of an Fc fusion and / or TEV-TwinStrep. Thus, the nucleic acid construct or cDNA from which the protein is expressed can be of the corresponding length.

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

[0256] Therapeutic treatment of metabolic syndrome The risk and / or incidence of metabolic syndrome (MetS), a cluster of cardiometabolic risk factors including abdominal obesity, hyperglycemia, dyslipidemia, and hypertension, increases with age. In older adults, MetS not only increases the risk of cardiovascular disease and type 2 diabetes but is also associated with cognitive decline and physical disability. Current evidence suggests that MetS is, in part, hereditary and that genetic factors play a greater role in the incidence of MetS than environmental factors. Researchers discovered an association between the G-395A polymorphism and metabolic syndrome (MetS) among the Chinese nonagenarian and centenarian populations. The subjects were from the Project of Longevity and Aging in Dujiangyan City (PLAD). Genotyping of G-395A (rs1207568) in the promoter region of the Klotho gene was performed using the TaqMan allelic discrimination assay. MetS was diagnosed according to the criteria of the International Diabetes Federation. Six hundred and ninety-five subjects aged 93.5 ± 3.2 years were included. The allelic frequencies of 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). -395A allele carriers 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 women (OR 0.51, 95% CI 0.24–0.97), but not in men (OR 0.42, 95% CI 0.05–3.85). In the overall population and in women, the relationship between the Klotho G-395A SNP and MetS may be due to its effects 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 may be due to its effects 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 -395A allele of the Klotho gene are correlated with a low risk of MetS among Chinese, especially women in their 90s and 100s.

[0257] One embodiment of the present disclosure includes an S-Klotho protein expressed from a construct having the -395A allele. The resulting protein can be produced or expressed in relation to any protein construct described herein. For example, the A395 allele can be produced or expressed in relation to S-Klotho 1-981, 29-981, 34-981, 36-981, 131-981, 1-549, 29-549, 34-549, 36-549, 131-549, etc., regardless of the presence or absence of an Fc fusion and / or TEV-TwinStrep. Accordingly, the nucleic acid construct or cDNA from which the protein is expressed can be of the corresponding length.

[0258] Embodiments can include producing a -395A heterozygous or homozygous construct, introducing the resulting construct encoding the S-crototan protein (e.g., via transfection) into a suitable expression system (e.g., CHO cells), and / or transiently expressing the S-crototan protein. The crototan protein expressed in A395 heterozygous or homozygous cells can be expressed at a higher level than in G396 cells. Embodiments can include purifying the protein expressed for therapeutic administration (and optionally conducting quality control tests). Embodiments can include administering a therapeutic or therapeutically effective amount of the S-crototan protein to a subject in need thereof (e.g., an individual or patient having or at risk of developing G395 SNP and / or metabolic syndrome (MetS)). Alternatively, the subject can be a wild-type of other variants or polymorphs. Administration of the recombinant S-crototan protein can lead to a beneficial increase in blood S-crot levels. Administration can reverse or suppress the adverse effects of the G395 SNP transcribed and circulating in affected individuals. Thus, the circulating concentration of S-crot can help counteract the effects observed in G395 individuals, particularly those at risk of developing metabolic syndrome (MetS). Thus, administration of the G-395A S-crototan protein can reduce the risk of metabolic syndrome (MetS) in patients (e.g., elderly human and / or female patients having the G395 SNP).

[0259] Therapeutic treatment of cancer S-Crotoin inhibits basal Wnt signaling activity and is thus thought to function as a tumor suppressor in colorectal cancer (CRC). In addition, crotoin gene variants associated with lifespan differences suppress the overactivation of Wnt mediated by butyrate and can thus increase the risk of CRC. In such a manner, the hypothesis has been put forward that the type of crotoin variant present and its relative expression interact with the levels of butyrate derived from the diet and can modify the CRC risk. Furthermore, mTOR signaling is also associated with human aging, and the crosstalk between Wnt and mTOR signaling can affect colon tumorigenesis.

[0260] The KL-VS variant or other constructs can function as vehicles to investigate which SNPs (e.g., within KL-VS) are factors that affect S-Crotoin to result in a decrease in basal Wnt signaling and / or suppression of the overactivation of Wnt mediated by butyrate, the latter being an activity related to Wnt associated with S-Crotoin tumor suppression. Embodiments include modifying appropriate amino acids (e.g., the KL-VS stretch of S-Crotoin) that have been shown to affect the tumor suppressor effect of the KL-VS variant. One embodiment of the present disclosure includes a recombinant S-Crotoin protein having one or more amino acid changes in the KL-VS stretch of six SNPs. The protein can be produced and / or expressed in relation to any protein construct described herein. For example, the protein can be produced or expressed in relation to S-Crotoin 1-981, 29-981, 34-981, 36-981, 131-981, 1-549, 29-549, 34-549, 36-549, 131-549, etc., regardless of the presence or absence of an Fc fusion and / or TEV-TwinStrep. Thus, the nucleic acid construct or cDNA in which the protein is expressed can be of the corresponding length.

[0261] Embodiments can include producing a heterozygous or homozygous polymorphic construct, introducing the resulting construct encoding the S-Crototan protein (e.g., via transfection) into a suitable expression system (e.g., CHO cells), and / or transiently expressing the S-Crototan protein. The Crototan protein can be expressed at a higher level than other Crototan proteins including the wild type. Embodiments can include purifying the protein expressed for therapeutic administration (and optionally conducting quality control tests). Embodiments can include administering a therapeutic or therapeutically effective amount of the S-Crototan protein to a subject in need thereof (e.g., a patient having or at risk of developing colorectal cancer (CRC) or another tumor). The subject can, for example, harbor or express a Crototan polymorph having decreased Wnt inhibitory activity. Alternatively, the subject can be wild type for other variants or polymorphisms. Administration of the recombinant S-Crototan protein can lead to a beneficial increase in blood S-Croto levels.

[0262] Therapeutic treatment of age-related pathologies The researchers discovered the association between Klotho and commonly accepted biological parameters as indicators of the clinical conditions of hospitalized elderly patients. The researchers genotyped single nucleotide polymorphisms (SNPs) rs9536314, rs1207568, and rs564481 at the KL locus in 594 hospitalized elderly patients (65 - 99 years old), who visited the geriatric ward continuously, and tested the association with KL variants with these biological quantitative traits using co-variance and genetic risk score model analysis. A significant association was observed between rs9536314 and serum levels of hemoglobin, albumin, and high-density lipoprotein cholesterol (HDL-C), and between rs564481 and serum levels of hemoglobin, fasting insulin, and fasting glucose. Gender analysis confirmed these associations, suggesting that the associations between KL genotype and HDL-C, fasting glucose, and fasting insulin levels may be dominated by female gender, while the association with serum level of hemoglobin may be dominated by male gender. The association between KL genotype and creatinine level was found in females, while the association between insulin-like growth factor-1 (IGF-1) and lymphocyte count (LC) was found in males. The genetic risk score (GRS) model further confirmed the significant association between KL SNP and hemoglobin, total cholesterol, and HDL-C. Gender analysis using the GRS tagging approach confirmed the associations with HDL-C, fasting glucose levels, and fasting insulin levels in females and with hemoglobin and LC in males. These findings suggest that the KL locus may affect quantitative traits such as serum lipid levels, fasting glucose, albumin, and hemoglobin in hospitalized elderly patients, with some gender differences suggested in creatinine, IGF-1 levels, and LC, and thus one of the genetic factors may contribute to age-related diseases and lifespan.

[0263] One embodiment of the present disclosure includes the S-Croto protein described herein. Embodiments can include producing a suitable Croto construct, introducing a construct encoding the S-Croto protein (e.g., via transfection) into a suitable expression system (e.g., CHO cells), and / or transiently expressing the S-Croto protein. Embodiments can include purifying the protein expressed for therapeutic administration (and optionally conducting quality control tests). Embodiments can include administering a therapeutic or therapeutically effective amount of the S-Croto protein to a subject in need thereof (e.g., an individual or patient, optionally an elderly person, and / or a condition related to aging, low endogenous S-Croto protein expression, and / or a condition related to age, or an individual or patient suffering from symptoms of decreased lifespan). Administration of the recombinant S-Croto protein can lead to a beneficial increase in blood S-Croto levels. In a positive treatment modality that is a quantitative trait such as serum levels of total cholesterol, HDL-C, fasting glucose, fasting insulin, albumin, creatinine, IGF-1, hemoglobin, and lymphocyte count (e.g., in-hospital patients and / or elderly patients), administration can reverse or suppress conditions and / or adverse effects related to aging.

[0264] In one or more of the treatment 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 treatment regimen may include administering a therapeutic dosage of a peptide comprising wild-type Klotho and / or any one or more variants of Klotho disclosed herein, including variants similar to the mutation expressed by the individual. Alternatively, the individual being treated may encode / express wild-type Klotho, and the treatment regimen may include administering a therapeutic dosage of a peptide comprising wild-type Klotho and / or any one or more variants of Klotho disclosed herein. In some embodiments, regardless of the native wild-type or mutant form of Klotho expressed by the individual, the treatment method includes measuring the low level of circulating and / or cell-bound Klotho (e.g., compared to a control group), and administering a therapeutic dosage that can operably restore the concentration of circulating and / or cell-bound Klotho protein to at least a homeostatic level. In some embodiments, this may include measuring the level of Klotho (e.g., gene expression level, protein expression level, circulating level, etc.) prior to administration of the therapeutic concentrate. Additionally, the therapeutic dosage may depend on the level of Klotho measured in the individual. In some embodiments, the therapeutic dosage exceeds the homeostatic level, e.g., by a scalar multiple of the homeostatic level (e.g., 1.5-fold more, 2-fold more, 3-fold more, 4-fold more, 5-fold more, 6-fold more, 7-fold more, 8-fold more, 9-fold more, 10-fold more, 15-fold more, 20-fold more, 25-fold more, 30-fold more, 40-fold more, 50-fold more, 75-fold more, 100-fold more, 500-fold more, 1,000-fold more, 10,000-fold 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 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 protein at constitutive 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 S-croto protein to an individual or subject in need thereof for prophylactic purposes and / or for the maintenance of certain health attributes. For example, administration of a particular S-croto protein may help maintain the youth 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 prophylactic 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 results of expression and purification of the listed Crotau variants in HEK and / or CHO cell lines. The results provided in Table 1 below were according to the following simplified protocol.

[0268] For Fc fusion proteins, the protein expression vector was transfected into HEK293.sus or CHO using the standard ATUM method. Briefly, cells were grown for 7 days and harvested. The cell counts are described in the annotation column. The supernatant pH was adjusted with 1M Hepes pH7.4 and sodium azide was added. The protein was 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. The protein was eluted with 50 mM citrate pH3.5, 100 mM NaCl. The protein was immediately neutralized with 1M Tris pH8, 0.5M arginine. SDS PAGE gel samples were removed at this stage. The protein was buffer exchanged into PBS. The protein was quantified by OD280 and the amount and concentration were measured using the calculated extinction coefficient. Purity and molecular weight were determined using reducing and non-reducing SDS-PAGE (Biorad standard Tris / glycine / SDS, 4-20%). The aggregation state was measured by HPLC using a Sepax Zenix-C SEC-300, 3um, 300Å, 4.6×150mm size exclusion column and PBS running buffer with detection at 280 nm. After filter sterilization and rapid freezing in liquid nitrogen, the protein was shipped as aliquots. As in previous purification rounds, for Fc-tagged proteins, it should be noted that protein loss was observed during desalting into PBS as measured from samples run on SDS-PAGE before and after purification. These proteins were expressed from HPLC but problems occurred during desalting or during assays on silica HPLC columns in PBS. Therefore, buffer selection can be optimized.

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

[0270] [Table 1]

[0271] In addition to one or more other CrotA variants disclosed herein but not shown in Table 1, the expression and / or purification of the CrotA variants disclosed in Table 1, in some embodiments, is understood to result in advantages over the expression and / or purification of native CrotA. For example, when expressing and / or purifying a CrotA variant, the number and / or type of alternative products can be reduced. Additionally, or alternatively, the expression level of the desired CrotA variant can be increased compared to the expression level of native CrotA. Additionally, or alternatively, the desired CrotA variant is expressed and / or purified in a more pure form under equivalent conditions and methods (e.g., the concentration of the desired CrotA variant increases with an accompanying decrease in the expressed and / or purified by-products).

[0272] Example 2 The following examples include a set of exemplary claims that define the scope of the disclosed invention. However, as provided herein, the scope of the invention is indicated by the appended claims, not by the following examples or the foregoing description.

[0273] 1. An exemplary method of producing a recombinant CrotA protein, comprising producing the recombinant CrotA protein in Chinese hamster ovary (CHO) cells, preferably in dihydrofolate reductase (DHFR)-deficient CHO cells, more preferably in CHO-S cells, or preferably in glutamine synthetase (GS)-deficient CHO cells, more preferably in GS- / -CHO cells, wherein the protein preferably has at least 85% amino acid sequence identity with one of SEQ ID NO: 2 to SEQ ID NO: 70.

[0274] 2. The method of claim 1, wherein the protein comprises one or more glycans attached thereto. 3. The method according to claim 1 or 2, wherein the method of producing the recombinant clotting protein comprises expressing the polypeptide encoded by the nucleic acid, wherein the CHO cell contains an exogenous nucleic acid encoding a promoter, preferably a strong promoter, and 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 synthetase (GS) enzyme.

[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 cell, preferably via transfection, and growing the CHO cell in a liquid medium, preferably a medium free of serum and / or animal protein components, wherein the liquid medium preferably contains 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 cell 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 per liter of liquid medium, 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, preferably to a concentration of about 1 nM to 1 μM, more preferably to a concentration of about 10 to 100 nM.

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

[0279] 8. The viable CHO cells of the selected suspension culture contain at least about 2 to 10 copies, preferably at least about 10 to 20 copies, more preferably at least about 20 to 30 copies, even more preferably at least about 30 to 50 copies of the exogenous nucleic acid per cell, the method according to claim 7.

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

[0281] 10. The method according to claim 9, wherein purifying the extract maintains the glycosylation of the protein. 11. Growing the CHO cells includes culturing the CHO cells in a bioreactor having a volume or working volume of at least 10 liters, preferably at least 25 liters, more preferably at least 50 liters, even more preferably at least 100 liters, still more preferably at least 250 liters, still more preferably at least 500 liters, still more preferably at least 1,000 liters, still more preferably at least 2,000 liters, still more preferably at least 2,500 liters, still more preferably at least 5,000 liters, still more preferably at least 10,000 liters, the method according to claim 4.

[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 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 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 comprise a promoter, preferably a strong promoter, and a polypeptide, at least a portion of the polypeptide having an amino acid sequence identity of at least 85% with one of SEQ ID NOs: 2 to 70, and optionally a functional dihydrofolate reductase (DHFR) enzyme or a functional glutamine synthetase (GS) enzyme, and containing an exogenous nucleic acid encoding the same.

[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 same.

[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 contains a transgene or cDNA having at least 85%, preferably at least 90%, 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 SEQ ID NOs: 76 to 96.

[0288] 18. A liquid medium, preferably a serum-free and / or animal protein component-free liquid medium, preferably containing a carbon source, a nitrogen source, and one or more vitamins, minerals, salts, amino acids, supplements, or additives, more preferably the liquid medium lacking hypoxanthine, thymidine, and / or glutamine, and the cell line according to any one of claims 14 to 17, wherein the CHO cell grows in the liquid medium such that the polypeptide encoded by the nucleic acid is expressed, and the polypeptide contains a recombinant clotting protein, a suspension cell culture.

[0289] 19. The suspension cell culture according to claim 18, wherein the CHO cell secretes the protein into the liquid medium to a concentration of preferably 200 to 500 mg, more preferably 500 to 2000 mg, still more preferably 2000 to 5000 mg of protein per liter of the liquid medium without concentrating the protein, and / or the protein is present in the liquid medium to a concentration of 200 to 500 mg, more preferably 500 to 2000 mg, still more preferably 2000 to 5000 mg of protein per liter of the 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 suspension 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 suspension 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%, most preferably 100% amino acid sequence identity with one of SEQ ID NOs: 2 to 70.

[0292] 23. A recombinant clotting protein, wherein 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 regulates the IGF-1 and / or Wnt signaling pathways, exhibits β-glucuronidase and / or sialidase activity, suppresses the p53 / p21 signaling pathway, and / or preferably reduces cell senescence and apoptosis induced by H2O2 through suppression of the p53 / p21 signaling pathway.

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

[0294] 26. The recombinant clotting protein according to any one of claims 23 to 25, wherein at least a part of the protein has an amino acid sequence identity of 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% with one of SEQ ID NO: 2 to SEQ ID NO: 70.

[0295] 27. A method for treating age-related or other pathological conditions, diseases, or disorders, comprising administering to a subject in need thereof a pharmaceutically effective amount of the recombinant clotting protein according to any one of claims 23 to 26.

[0296] 28. A method for treating age-related or other pathological conditions, diseases, or disorders, comprising administering to a subject in need thereof a pharmaceutically effective amount of a soluble recombinant clotting protein having at least 80% amino acid sequence identity with at least a subset of amino acid residues 1 to 981 of human alpha clotting isoform 1.

[0297] 29. A method for treating age-related or other pathological conditions, diseases, or disorders, comprising administering to a subject in need thereof a pharmaceutically effective amount of a soluble recombinant clotting protein having at least 80% amino acid sequence identity with one of SEQ ID NO: 2 to SEQ ID NO: 70.

[0298] 30. The method according to any one of claims 27 to 29, wherein the protein has an amino acid sequence identity of 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% with one of SEQ ID NO: 2 to SEQ ID NO: 70.

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

[0300] 32. The method according to claim 31, wherein the predetermined level is at least about 1000 picograms of soluble croto - tannin protein per milliliter of serum. 33. The predetermined level is at least about 50, 100, 250, 500, 750, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 11,000, 12,000, 13,000, 14,000, 15,000, 20,000, 25,000, 30,000, 40,000, 50,000, 75,000, or 100,000 picograms or more of soluble croto - tannin protein per milliliter of serum and / or is at least 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 between, higher than the typical healthy level of soluble croto - tannin protein in serum. The method according to claim 31.

[0301] 34. Measuring the serum soluble croto - tatin protein concentration of the subject, calculating a pharmaceutically effective amount of protein sufficient to increase the serum soluble croto - tatin protein concentration of the subject to a first predetermined level, wherein the first predetermined level is preferably soluble croto - tatin protein of about 1000 picograms or more per milliliter of serum, calculating, measuring the rate of decrease and / or metabolic rate of the soluble croto - tatin protein in the serum of the subject, calculating the next dosing time when the serum soluble croto - tatin protein concentration of the subject reaches a second predetermined level or below based on the measured rate, and calculating the next dosing amount of the protein sufficient to increase the serum soluble croto - tatin protein concentration of the subject from the second predetermined level to the first predetermined level, the method according to claim 31, further comprising one or more of the above.

[0302] 35. The method according to claim 34, further comprising administering the next dosing amount of the protein to the subject. 36. Preferably, exogenous nucleic acid is introduced into Chinese hamster ovary (CHO) cells via transfection, wherein the nucleic acid preferably contains a transgene or cDNA, and 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%, still more preferably at least 99%, most preferably 100% amino acid sequence identity with one of SEQ ID NOs: 2 to 70, and the nucleic acid has nucleic acid sequence identity with one of SEQ ID NOs: 76 to 96 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%, most preferably 100%, introducing; growing the CHO cells in a liquid medium, preferably in a liquid medium free of serum and / or animal protein components, 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 in dihydrofolate reductase (DHFR)-deficient CHO cells, more preferably in CHO-S cells, or glutamine synthetase (GS)-deficient CHO cells, more preferably in GS- / -CHO cells, growing; introducing an effective amount of methotrexate (MTX) and / or methionine sulfoximine (MSX) into the liquid medium, preferably to a concentration of about 1 nM to 1 μM, more preferably to a concentration of about 10 to 100 nM; 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, still more preferably at least 5000 mg / L without concentrating the protein, selecting; producing the recombinant soluble clotting protein in the CHO cells,The method according to claim 31, further comprising one or more of: producing the protein such that the protein is secreted, preferably from the CHO cells into the liquid medium, to a protein concentration of preferably 200 to 500 mg per liter of the liquid medium, more preferably 500 to 2000 mg of protein concentration, still more preferably 2000 to 5000 mg of protein concentration; and purifying a recombinant soluble clotting protein-containing extract from the CHO cells, the liquid medium, or both, wherein the extract preferably contains at least about 98% of the recombinant soluble clotting protein by dry weight and / or less than about 1 to 100 ppm of CHO host cell protein (HCP), and purifying the extract preferably maintains the glycosylation of the protein and the protein has one or more glycans attached thereto.

[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, still more preferably at least about 72 hours.

[0304] 38. The method according to claim 31, wherein the predetermined period is about 1 to 120 days or more. 39. The method according to claim 31, wherein the predetermined period is about 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 a pharmaceutically acceptable carrier or with a pharmaceutically acceptable carrier.

[0306] 42. The method according to any one of claims 30 to 41, wherein the aging-related or other pathological conditions, diseases, or disorders include one or more of 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 strength, decreased movement, decreased freedom of movement, decreased quality of life assessment, decreased expulsion rate, decreased exercise ability, decreased learning ability, decreased learning power, decreased memory, decreased intelligence index, cognitive decline, amnesia, decreased cognitive ability, decreased cognitive function, decreased synaptic plasticity, decreased synaptic function, and cellular aging.

[0307] 43. The method according to any one of claims 30 to 41, wherein the aging-related or other pathological conditions, diseases, or disorders include one or more of chronic kidney disease (CKD), polycystic kidney disease (PKD), autosomal dominant polycystic kidney (ADPKD), acute kidney injury (AKI), acute tubular necrosis (ATN), acute allergic interstitial nephritis (AAIN), glomerulonephritis, kidney disease, renal insufficiency, non-oliguric renal insufficiency, alcohol dependence, hyperphosphatemia, muscular dystrophy (MS), type 1 diabetes, type 2 diabetes, cardiovascular disease (CVD), cardiovascular calcification, cerebrovascular insufficiency, vascular calcification, ischemic heart disease, blood pressure abnormality, salt-sensitive hypertension, tissue calcification, calcified atherosclerotic plaque burden, calcinosis, familial tumoral calcinosis, cancer, one or more tumors, diseases related to myelin, demyelinating diseases, neurodegenerative diseases, neurovascular diseases, progressive supranuclear palsy (PSP), Pompe disease, Niemann-Pick disease, microgliosis, Faber disease (FD), bone mass diseases, 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 (COPD), fibromyalgia, adult-onset diabetes, arthritis, rheumatoid arthritis, osteoarthritis, glaucoma, cataract, macular degeneration, multiple sclerosis (MS), lupus, ulcerative colitis, cachexia, obesity, conditions related to vitamin D, bone diseases, bone diseases through bone remodeling, stem cell depletion, space sickness, space adaptation 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 the recombinant clotting protein according to any one of claims 23 to 25 and a pharmaceutically acceptable carrier. 47. A pharmaceutically effective amount of a recombinant soluble clotting protein, wherein at least a portion of the protein has at least 85% amino acid sequence identity with at least a subset of the amino acid residues 1 to 981, 29 to 981, 34 to 981, 36 to 981, 131 to 981, 1 to 549, 29 to 549, 34 to 549, 36 to 549, or 131 to 549 of human alpha clotting isoform 1, or at least a portion of one of SEQ ID NOs: 2 to SEQ ID NOs: 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 SEQ ID NOs: 70.

[0311] 49. The pharmaceutical composition according to any one of claims 46 to 48, further comprising one or more additional active ingredients. 50. For use in the treatment of one or more of the following age-related or other conditions, diseases, or disorders: weakness, low bone density, reduced bone mineral density, weight loss, muscle atrophy, muscle degeneration, reduced muscle mass, reduced muscle strength, reduced grip strength, reduced leg strength, reduced physical strength, reduced motor function, reduced freedom of movement, reduced quality of life assessment, reduced expulsion rate, reduced exercise capacity, reduced learning ability, reduced learning power, reduced memory, reduced intelligence quotient, cognitive decline, amnesia, reduced cognitive ability, reduced cognitive function, reduced synaptic plasticity, reduced synaptic function, cellular aging, chronic kidney disease (CKD), polycystic kidney disease (PKD), autosomal dominant polycystic kidney (ADPKD), acute kidney injury (AKI), acute tubular necrosis (ATN), acute allergic interstitial nephritis (AAIN), glomerulonephritis, kidney disease, renal failure, non-oliguric renal failure, alcohol dependence, hyperphosphatemia, muscular dystrophy (MS), type 1 diabetes, type 2 diabetes, cardiovascular disease (CVD), cardiovascular calcification, cerebrovascular insufficiency, vascular calcification, ischemic heart disease, blood pressure abnormalities, salt-sensitive hypertension, tissue calcification, calcified atherosclerotic plaque burden, calcinosis, familial tumoral calcinosis, cancer, one or more tumors, diseases related to myelin, demyelinating diseases, neurodegenerative diseases, neurovascular diseases, progressive supranuclear palsy (PSP), Pompe disease, Niemann-Pick disease, microgliosis, Farber disease (FD), bone mass diseases, osteoporosis, osteopenia, osteopenia (especially loss of cortical bone BMD), 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 (COPD), fibromyalgia, adult-onset diabetes, arthritis, rheumatoid arthritis, osteoarthritis, glaucoma, cataract, macular degeneration, multiple sclerosis (MS), lupus, ulcerative colitis, cachexia, obesity, conditions related to vitamin D, bone diseases, bone diseases through bone remodeling, stem cell depletion, space sickness, space adaptation syndrome (SAS), nausea, and dizziness, the pharmaceutical composition according to any one of claims 46 to 49.

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

[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 the recombinant soluble clotting protein. 54. The method according to claim 53, wherein the protein and one or more additional active ingredients are formulated in 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.

[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, preferably, separate administrations separated by a 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 symptom comprises acute tubular necrosis (ATN), nephritis, acute allergic interstitial nephritis (AAIN), glomerulonephritis and / or nephrotoxicity, or AKI that results at least in part from kidney transplantation or other surgery, 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 comprises drug-induced nephrotoxicity. 61. The method according to claim 60, wherein the drug-induced nephrotoxicity comprises nephrotoxicity induced by an antibacterial agent.

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

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

[0320] 65. The method according to claim 64, wherein the predetermined level or threshold is about 50, 100, 250, 500, 750, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 11,000, 12,000, 13,000, 14,000, 15,000, 20,000, 25,000, 30,000, 40,000, 50,000, 75,000, or 100,000 picograms or more per milliliter of serum, and / or soluble croto - tannin protein therebetween, or is above the typical healthy level of soluble croto - tannin protein of 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%.

[0321] 66. The method according to claim 64, wherein the predetermined period is about 6 hours, 12 hours, 18 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 12 days, 14 days, 21 days, 30 days, 45 days, 60 days, 90 days, 120 days, 6 months, 9 months, 1 year, 2 years, 3 years, 4 years, or 5 years or more.

[0322] 67. The method according to claim 52 or 53, wherein the protein is administered prophylactically before kidney transplantation or administration of a nephrotoxin and / or subsequent to kidney transplantation or administration of a nephrotoxin. 68. The method according to claim 67, comprising the renal toxin being 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 one or more contrast agents selected from the group consisting of a hyperosmolar contrast medium (HOCM) having an iodine-to-molecule ratio of about 1.5:1, a low osmolar non-ionic contrast agent (LOCM) having an iodine-to-molecule ratio of about 3:1, and an isotonic (isosmolar) contrast agent (IOCM) having an iodine-to-molecule ratio of about 6:1; preferably one or more antiretroviral agents selected from the group consisting of adefovir, cidofovir, tenofovir, and phosphonoformate; preferably one or more cancer (or chemical) therapeutic agents selected from the group consisting of cisplatin, carboplatin, oxaliplatin, alkylating agents, bendamustine, cyclophosphamide, ifosfamide, nitrosourea, temozolomide, melphalan, antitumor antibiotics, 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; preferably 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 tiludronate; 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 C370S that contains F352, and / or contains variants other than H193 or H193R.

[0324] 70. A method for treating an elderly individual, wherein the elderly individual has a homozygous or heterozygous mutation in the gene encoding the Klotho protein and is administered a therapeutic concentration of a polypeptide having at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 98%, still more preferably at least 99%, most preferably 100% amino acid sequence identity 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 concentrate is dependent on the expression level of the gene.

[0326] Conclusion The foregoing detailed description refers to specific exemplary embodiments, but the present disclosure may be embodied in other specific forms without departing from its spirit or essential characteristics. Accordingly, the described embodiments should be considered in all respects as illustrative 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 contemplated by those skilled in the relevant art and the owner of the present disclosure, may be made to the described and / or illustrated embodiments without departing from the spirit and scope of the present disclosure as defined by the appended claims. Such substitutions, changes and / or modifications are considered to be within the scope of the present disclosure.

[0327] Accordingly, the scope of the present invention is shown not by the foregoing description but by the appended claims. The limitations recited in the claims are to be broadly construed based on the language employed in the claims and are not limited to the specific examples described in the foregoing detailed description, and are to be construed as non-exclusive and non-limiting. All changes that come within the scope and meaning equivalent to the claims are included within their scope.

[0328] It will also be understood that the various features of certain embodiments 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 certain embodiments of the present disclosure may include, incorporate, or include features described in other embodiments disclosed and / or described herein. Thus, the disclosure of certain features related to a particular embodiment of the present disclosure should not be construed as limiting the application or inclusion of such features to that particular embodiment.

[0329] In addition, unless a feature is described as required in a particular embodiment, the features described in the various embodiments are optional and may not be included in other embodiments of the present disclosure. Further, unless a feature is described as requiring another feature in combination therewith, any feature herein may be combined with any other feature, whether the same or different, of the embodiments disclosed herein. In certain embodiments, features may be optional, but it will be understood that when such features are included in such embodiments, they may be required to have a particular configuration as described in the present disclosure.

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

[0331] Furthermore, various well-known aspects of exemplary systems, methods, products, etc. are not described in detail herein so as not to obscure aspects of the exemplary embodiments. However, such aspects are also considered herein.

Claims

1. 1. A cell line comprising: The invention relates to a method for producing a human hamster ovary (CHO) cell, the method comprising the steps of:

1. A cell line comprising a recombinant protein having the amino acid sequence of SEQ ID NO:40, said recombinant protein being effective in treating acute kidney injury or chronic kidney disease.

2. The CHO cells a dihydrofolate reductase (DHFR)-deficient CHO cell, wherein the exogenous nucleic acid further encodes a functional dihydrofolate reductase (DHFR); or a glutamine synthetase (GS)-deficient CHO cell, wherein the exogenous nucleic acid further encodes a functional glutamine synthetase (GS); The cell line of claim 1.

3. 3. The cell line of claim 1 or 2, wherein the CHO cells contain, or are selected to contain, at least 10 copies of the exogenous nucleic acid per cell.

4. 1. A suspension cell culture comprising: A liquid medium, a serum-free and / or animal protein component-free liquid medium; The cell line according to any one of claims 1 to 3, wherein said CHO cells are grown in said liquid medium such that they express said polypeptide encoded by said nucleic acid. the liquid medium comprising a carbon source, a nitrogen source, and one or more vitamins, minerals, salts, amino acids, supplements, or additives; The suspension cell culture, wherein the polypeptide comprises a recombinant Klotho protein.

5. the CHO cells secrete the polypeptide or protein into the liquid medium to a concentration of 200-500 mg, 500-2000 mg, or 2000-5000 mg of polypeptide or protein per liter of liquid medium without concentrating the polypeptide or protein; and / or 5. The suspension cell culture of claim 4, wherein the polypeptide or protein is present in the liquid medium at a concentration of 200-500 mg, 500-2000 mg, or 2000-5000 mg of polypeptide or protein per liter of liquid medium without concentrating the polypeptide or protein.

6. 6. The suspension cell culture of claim 4 or claim 5, wherein the liquid medium further comprises an effective amount of methotrexate (MTX) at a concentration of 1 nM to 1 μM or 10 nM to 100 nM.

7. A method for producing a recombinant Klotho protein, comprising the steps of: Growing the cell line of any one of claims 1 to 3 in a serum-free and / or animal protein component-free liquid medium such that the CHO cells express the polypeptide encoded by the nucleic acid, wherein the liquid medium comprises a carbon source, a nitrogen source, and one or more vitamins, minerals, salts, amino acids, supplements, or additives, and wherein the polypeptide comprises a recombinant Klotho protein. A method comprising:

8. the CHO cells secrete the polypeptide or protein into the liquid medium to a concentration of 200-500 mg, 500-2000 mg, or 2000-5000 mg of polypeptide or protein per liter of liquid medium without concentrating the polypeptide or protein; and / or 8. The method of claim 7, wherein the polypeptide or protein is present in the liquid medium at a concentration of 200-500 mg, 500-2000 mg, or 2000-5000 mg of polypeptide or protein per liter of liquid medium without concentrating the polypeptide or protein.

9. A recombinant protein having the amino acid sequence of SEQ ID NO: 40 for use in the treatment of acute kidney injury or chronic kidney disease, comprising: The recombinant protein is effective in treating acute kidney injury or chronic kidney disease.

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