JP2023123565A - Therapeutic recombinant klotho protein and compositions and methods comprising same - Google Patents

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

The present disclosure relates to the production and administration of recombinant human Klotho protein compositions as therapeutic agents. Specifically, the present disclosure relates to compositions comprising CGMP grade human recombinant soluble alpha-Klotho protein or variants thereof, and methods of making and administering the same to human or non-human subjects.

Klotho (or alpha-klotho, α-klotho, etc.) is a recently characterized protein encoded by the KL (or klotho) gene located on human chromosome 13. Two transcripts have been identified that arise from a single Klotho gene through alternative RNA splicing. See Figures 1 and 2. The first transcript has a short cytoplasmic tail (human residues 1003-1012) and a transmembrane (TM) domain (human residues 982-1002) with 20%-40% amino acid sequence homology to β-glucosidase, respectively. two homologous (internal repeat) domains, KL1 (450 residues long, human residues 56-506), and KL2 (438 residues long, human residue 515). total length of 1,012 amino acids, including a signal sequence (SS) domain (human residues 1-33) and an extracellular region or domain (human residues 1-981). It is predicted to encode Klotho isoform 1, a single transmembrane transmembrane protein of the SS, KL1 and KL2 domain-containing extracellular region (human residues 1-981) enzymatically cleaved by α/β-secretase. and can be released into the circulation as a 130-kDa circulating protein termed soluble Klotho (or s-Klotho, s-Klotho, alpha-soluble Klotho, etc.) The extracellular domains are also distinct 68-kDa proteins (KL1+SS) and 64-kDa proteins (KL2) can be cleaved.

A second transcript, a splicing variant of the alpha-Klotho mRNA, encodes a second isoform of the Klotho protein that primarily corresponds to the KL1 domain. An internal splice donor site is believed to be located in exon 3 of the Klotho gene. The resulting alternatively spliced transcript contains a 50 bp insertion after exon 3 with an in-frame translation stop codon at its end (Fig. 1; grey). The expressed protein product is secreted into circulation and is referred to as secreted Klotho (or Klotho isoform 2). Therefore, there may be many different Klotho proteins in circulation at any given time, depending on gene expression, RNA splicing, and enzymatic cleavage. Despite the existence of various forms of alpha-Klotho protein, only the full-length, membrane-bound isoform 1 complexes with fibroblast growth factor (FGF) receptors and induces urinary phosphate excretion. and is known to function as an essential co-receptor for FGF23, a bone-derived hormone that has regulatory roles in _Pi and vitamin D metabolism.

Klotho is highly expressed in kidney, brain, and to a lesser extent in other organs, and can also be found in mammalian cerebrospinal fluid and urine. Circulating levels of soluble Klotho protein in mammals are thought to decrease with age. In addition, Klotho-deficient mice display an accelerated aging phenotype, whereas overexpression of Klotho in mice exhibits extended lifespan. In addition, Klotho is involved in many cellular processes associated with aging. In view of the foregoing, it has been hypothesized that soluble Klotho may function as an anti-aging compound in the human body.

Aging is an inevitable, progressive biological process that leads to the dysfunction and destruction of almost all tissues and organs, ultimately leading to death. For example, aging of the human body is associated with a decline in cell function that can lead to the development of various diseases. Aging is thought to be caused by a tightly regulated and complex interaction between genetic and acquired factors, leading to increased aging, quantitative and qualitative decline in stem cells, and abnormalities at the tissue level. structure is a typical feature.

The population of older adults (eg, 60-65 years old) is increasing rapidly worldwide as the so-called "baby boomer" generation ages. The increasing demand for health care for this aging population places a significant economic burden on all health care systems. Recombinant Klotho protein may provide a promising therapeutic agent to combat age-related health conditions. The development of strategies and interventions based on the production and purification (e.g., to substantially homogeneity) of soluble Klotho, and the administration of this protein to subjects within the growing elderly population, have implications for this situation and its implications. can help ameliorate problems with

Currently, exogenous forms of human Klotho protein, such as recombinant soluble human alpha-Klotho protein or protein variants, in particular Current Good Manufacturing Practices as determined and enforced by the US Food and Drug Administration (FDA). There is no product or method, alone or in combination with one or more additional active ingredients, to provide a protein that complies with the Practice) (CGMP) regulations. Developing strategies and insurance interventions based on administering recombinant S-Klotho to subjects, especially within the growing elderly population, could help ameliorate this situation.

Embodiments of the present disclosure provide recombinant human Klotho proteins, protein fragments, and/or protein variants, expression nucleic acid constructs and/or vectors, cell lines and/or cell suspension cultures, and methods for their production, purification, and Methods of administration to subjects (human or non-human animals) are used to solve one or more of the above or other problems in the art.

For example, some embodiments of the present disclosure provide
recombinant human alpha soluble Klotho protein, protein fragments, and/or protein variants;
a pharmaceutical product (or therapeutic composition--eg, a formulation) of recombinant human alpha-soluble Klotho protein;
a composition of recombinant human alpha soluble Klotho protein and at least one additional (active) ingredient;
a nucleic acid construct or vector encoding a recombinant human alpha soluble Klotho protein;
a cell line that (i) contains a nucleic acid construct or vector encoding a recombinant human alpha soluble Klotho protein and/or (ii) expresses a recombinant human alpha soluble Klotho protein;
a cell suspension culture of cells (i) containing a nucleic acid construct or vector encoding a recombinant human alpha soluble Klotho protein and/or (ii) expressing a recombinant human alpha soluble Klotho protein;
A method for producing and optionally purifying a recombinant human alpha soluble Klotho protein;
A method of manufacturing a pharmaceutical product (or therapeutic composition, i.e., formulation) of recombinant human alpha soluble Klotho protein;
A method of administering a recombinant human alpha soluble Klotho protein to a subject (human or non-human animal);
a diagnostic method for determining Klotho protein deficiency in a subject;
A method of diagnosing Klotho protein deficiency in a subject;
a method of diagnosing a subject in need of administration of a recombinant human alpha soluble Klotho protein;
a method of assessing the effectiveness of a protein on a subject in need thereof and/or determining an effective dosage;
Recombinant human alpha soluble Klotho protein for use in the treatment of certain medical conditions or other medical conditions and/or Recombinant human alpha soluble Klotho protein for the manufacture of pharmaceutical products for the treatment of certain medical conditions or other medical conditions and the use of

Some embodiments are methods of producing a recombinant Klotho protein in Chinese Hamster Ovary (CHO) cells, preferably in dihydrofolate reductase (DHFR) deficient CHO cells, more preferably in CHO-S cells. , or preferably in glutamine synthetase (GS)-deficient CHO cells, more preferably in GS-/- CHO cells, wherein the protein preferably comprises SEQ ID NO: 2 to SEQ ID NO: 70 have at least 85% amino acid sequence identity with one of

Some embodiments are preferably in dihydrofolate reductase (DHFR) deficient CHO cells, more preferably in CHO-S cells, or preferably in glutamine synthetase (GS) deficient cells, more preferably GS-/-. A cell line comprising a plurality of Chinese Hamster Ovary (CHO) cells in CHO cells, the CHO cells comprising a promoter, preferably a strong promoter, and a polypeptide, wherein at least a portion of the polypeptide comprises the sequence a polypeptide having at least 85% amino acid sequence identity with one of numbers 2 to SEQ ID NO: 70, and optionally a functional dihydrofolate reductase (DHFR) enzyme, or a functional glutamine synthesis (GS) Cell lines containing exogenous nucleic acids encoding enzymes can be included.

Some embodiments are liquid media, preferably serum-free and/or free of animal protein components, preferably containing a carbon source, a nitrogen source, and one or more vitamins, minerals, salts, amino acids, A liquid medium comprising supplements or additives, more preferably the liquid medium lacking hypoxanthine, thymidine and/or glutamine, and a liquid medium such that the CHO cells express the polypeptide encoded by the nucleic acid. and the cell line of any one of claims 14-17 grown in said polypeptide, wherein said polypeptide comprises a recombinant Klotho protein.

Some embodiments may include a recombinant Klotho protein, wherein at least a portion of the protein has at least 80% amino acid sequence identity with one of SEQ ID NO:2-SEQ ID NO:70.

Some embodiments are a method of treating an age-related or other condition, disease, or disorder comprising administering to a subject in need thereof a pharmaceutically effective amount of a combination described herein. A method comprising administering a recombinant Klotho protein may be included.

Some embodiments are a method of treating an age-related or other condition, disease, or disorder comprising administering to a subject in need thereof a pharmaceutically effective amount of human alpha klotho isoform 1 administering a soluble recombinant Klotho protein having at least 80% amino acid sequence identity with at least a subset of amino acid residues 1-981 of .

Some embodiments are a method of treating age-related or other conditions, diseases, or disorders comprising administering to a subject in need thereof a pharmaceutically effective amount of administering a soluble recombinant Klotho protein having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% amino acid sequence identity with one of 70. can contain.

Some embodiments may comprise a pharmaceutical composition comprising a pharmaceutically effective amount of a recombinant Klotho protein described herein and a pharmaceutically acceptable carrier.
Some embodiments are a pharmaceutical composition, wherein a pharmaceutically effective amount of a recombinant soluble Klotho protein, wherein at least a portion of the protein comprises human alpha Klotho isoform 1 1-981, 29-981, 34 at least a subset of amino acid residues from ~981, 36-981, 131-981, 1-549, 29-549, 34-549, 36-549, or 131-549, or of SEQ ID NOs:2-70 A pharmaceutical composition comprising at least a portion of one, a recombinant soluble Klotho protein having at least 85% amino acid sequence identity, and a pharmaceutically acceptable carrier can be included.

Some embodiments are methods 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 Klotho protein wherein at least a portion of the protein is human alpha klotho isoform 1 1-981, 29-981, 34-981, 36-981, 131-981, 1-549, 29-549, 34-549, 36- 549, or at least a subset of amino acid residues 131-549, or at least a portion of one of SEQ ID NOs: 2-70 and at least 85%, 86%, 88%, 90%, 92%, 95%, A method with 98%, 99%, or preferably 100% amino acid sequence identity may be included.

Some embodiments include therapeutic Klotho proteins such as CGMP grade human recombinant soluble alpha-Klotho protein and drugs, antibodies, hormones, human cell, tissue, cell or tissue lineage products (HCT/Ps), etc. and/or methods of administering them to human or non-human subjects. Combinatorial compositions and methods can be useful for treating subjects suffering from age-related disorders or conditions, metabolic disorders, chronic diseases, acute injuries, and the like. Prophylactic administration of combination therapy to subjects without overt conditions or disorders may also be useful in delaying or preventing certain conditions or disorders described herein.

Some embodiments may include nucleic acids or nucleic acid constructs. For example, embodiments can include expression vectors or nucleic acids. A nucleic acid can encode a recombinant human alpha soluble Klotho protein, protein fragment, or protein variant. Nucleic acids can encode natural or non-natural signaling sequences. For example, a nucleic acid can encode a non-natural signaling sequence upstream (or N-terminal) to the encoded Klotho protein sequence.

Some embodiments may include methods of producing recombinant human alpha soluble Klotho protein. The method of manufacture involves growing Chinese Hamster Ovary (CHO) cells in liquid medium, producing recombinant soluble Klotho protein in the CHO cells, and/or recombination from CHO cells, liquid medium, or both. purifying the soluble Klotho protein-containing extract. The extract may comprise at least about 98% dry weight recombinant soluble Klotho protein and/or less than about 1-100 ppm 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 may be released (eg, secreted) from the CHO cells into the liquid medium and/or may have one or more glycans attached thereto.

CHO cells may contain one or more exogenous nucleic acids that encode proteins and, optionally, functional enzymes such as dihydrofolate reductase, glutamine synthase (GS) enzymes, and the like. Exogenous nucleic acids can include promoters (eg, strong promoters, weak promoters, etc.), such as promoters customarily or typically used for expression of exogenous proteins in CHO cells. The exogenous nucleic acid is preferably one of SEQ ID NO:76-SEQ ID NO:96, or any other suitable nucleic acid sequence encoding a Klotho protein described herein (e.g., S-Klotho variant ) having at least 85% nucleic acid sequence identity (eg, under the control of a promoter).

Methods can include introducing exogenous nucleic acid into the CHO cells, such as by transfection. The method includes growing the CHO cells in a liquid medium, such as a serum-free (human, bovine (fetal), or other) medium and/or an animal (or animal-derived) protein (component)-free medium. can contain. 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 the bioreactor. Depending on the particular CHO cell line, the method includes introducing an effective amount of methotrexate (MTX), methionine sulfoximine (MSX), or other agent into the liquid medium and/or (e.g., CHO cell subcloning , limiting dilution, fluorescence-activated cell sorting (FACS), etc.), selecting suspension cultures of viable CHO cells growing in liquid medium.

In some embodiments, selection and/or gene amplification is performed by culturing the transfected cells in a selective medium, such as medium lacking hypoxanthine and/or thymidine (e.g., -HT medium), glutamine, etc. can be performed by In at least one embodiment, low concentrations of MTX are added or used to amplify the transfected nucleic acid (or its gene), thereby (e.g., DHFR-deficient CHO transfected with a DHFR transgene). cells) to increase protein expression. Alternatively (or in addition), selection and/or gene amplification may be performed by adding MSX ((endogenous) glutamine synthase ( GS) inhibitors).

The method can include subculturing viable cells or cultures (eg, MTX-resistant and/or MSX-resistant cells or cultures). Selected suspension cultures and/or selected CHO cells may be subjected to increased production of protein (e.g., by CHO cells), increased concentration of protein (e.g., into the liquid medium), and/or exogenous It may have or exhibit an increased copy number (eg, per cell) of the nucleic acid (eg, relative to unselected suspension cultures or CHO cells).

Certain embodiments may include cell lines comprising multiple CHO cells. For example, the CHO cells can be DHFR deficient CHO cells such as CHO-S cells. The CHO cell has one or more (copies of) exogenous nucleic acids (including transgenes or cDNAs) that encode a polypeptide having at least 85% amino acid sequence identity with one of SEQ ID NOs: 2-70. can contain The polypeptide may comprise human recombinant alpha soluble Klotho protein. An exogenous nucleic acid may comprise a transgene or cDNA, preferably having at least 85% nucleic acid sequence identity with one of SEQ ID NO:76-SEQ ID NO:96. In some embodiments, the nucleic acid is (also) a promoter (associated with the transgene), and/or (functional) an optional (functional) dihydrofolate reductive (DHFR) enzyme, glutamine synthase (GS) enzyme, etc. exogenous) enzyme.

At least one embodiment preferably comprises a carbon source, a nitrogen source, and/or one or more vitamins, minerals, salts, amino acids, supplements or additives to allow the CHO cells to express the polypeptide encoded by the nucleic acid. Includes suspension cell cultures, including cell lines grown in liquid media such as Liquid media may be serum-free (human, bovine (fetal), or otherwise) and/or free of animal (or animal-derived) protein (ingredients). For example, liquid media can be free of bovine serum albumin, human serum albumin, and the like.

In some embodiments, the liquid medium may also contain effective amounts of MTX and/or MSX. Suspension cultures (or CHO cells thereof) exhibit increased production of protein (e.g., by CHO cells), exhibit increased concentrations of protein (e.g., in liquid medium), and release protein (e.g., into liquid medium). Can (selected to) secrete and/or preferably have an increased copy number (eg, per cell) of exogenous nucleic acid compared to unselected suspension cultures. A protein may have one or more glycans attached to it.

Some embodiments include extracts of or from suspension cell cultures of CHO cells, liquid media, or both, wherein the extract comprises one of SEQ ID NO:2-SEQ ID NO:70 contains a recombinant protein that has at least 85% amino acid sequence identity with one. Certain embodiments include human recombinant alpha soluble Klotho protein-containing extracts of or from CHO cells, liquid media, or both (eg, suspension cell cultures). At least one embodiment includes an isolated recombinant protein having at least 85% amino acid sequence identity with one of SEQ ID NO:2-SEQ ID NO:70.

Some embodiments may include methods of administering recombinant human alpha soluble Klotho protein to a human or non-human animal subject in need thereof. A subject to whom a Klotho protein is administered can suffer from or be at risk of various conditions (eg, disorders, diseases, injuries, illnesses, etc.). For example, some embodiments treat one or more chronic diseases and/or age-related conditions, such as physical, intellectual, neurological, or other conditions associated with (human) aging. including how to Some embodiments may promote healing, recovery, longevity and/or other beneficial outcomes through one or more mechanisms or actions. Embodiments provide, for example, a pharmaceutically effective amount of a recombinant soluble Klotho protein or protein variant to a subject in need thereof (e.g., a subject suffering from or at risk of developing a condition) administering to. Administration of such proteins or protein variants can have positive therapeutic effects on, and characteristics of, the course and outcome of disease states, including chronic and/or age-related diseases and longevity in human subjects.

A pharmaceutically effective amount may be sufficient to raise a subject's serum soluble Klotho protein concentration to a predetermined level, such as about 50 to 3000 picograms or more of soluble Klotho protein per milliliter of serum, or therebetween. The amount may also, or alternatively, be sufficient to maintain the subject's serum soluble Klotho protein concentration at or above a predetermined threshold for a predetermined period of time. Embodiments can also include administering the protein to a subject in need thereof such that the subject's serum soluble Klotho protein concentration is maintained at or above a predetermined threshold for a predetermined period of time.

Embodiments also comprise measuring the subject's serum soluble Klotho protein concentration, calculating a pharmaceutically effective amount, measuring the rate of decline of soluble Klotho protein in the subject's serum, calculating the next dosing time at which the serum soluble Klotho protein concentration will be at or below a second predetermined level based on the measured rate; calculating a next dosage of protein sufficient to raise the level to the first predetermined level; and/or administering the next dosage of protein to the subject.

The protein can modulate (can be effective to modulate) the IGF-1 and/or Wnt signaling pathway, can exhibit β-glucuronidase and/or sialidase activity, can inhibit the p53/p21 signaling pathway, and /or preferably through inhibition of the p53/p21 signaling pathway may reduce cellular senescence and apoptosis induced by _{H2O2 .} The protein preferably exhibits pleiotropic activity and/or preferably regulates oxidative stress, growth factor signaling, ion homeostasis and/or one or more ion channel proteins and/or insulin/insulin-like growth factor-1. It may function as a humoral factor or may be functional in modulating the activity of glycoproteins on the cell surface, such as growth factor receptors.

Protein is also associated with frailty, loss of bone or bone mineral density, weight loss, muscle atrophy or wasting, decreased muscle mass, muscle strength, grip strength, decreased leg strength or strength, movement, freedom of movement, quality of life assessment, ejection fraction. , decreased motor performance, decreased learning ability, learning ability, memory, or intelligence quotient, cognitive deterioration or amnesia, decreased cognitive ability or function, decreased synaptic plasticity or function, and cellular senescence. It may be effective for treating the above age-related conditions (or (human) age-related conditions).

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

Accordingly, embodiments may also include compositions for use in treating one or more age-related conditions. A composition can include a recombinant soluble Klotho protein (eg, having at least 85% amino acid sequence identity with one of SEQ ID NOs:2-70), and a pharmaceutically acceptable carrier.

Some embodiments include therapeutic Klotho proteins, such as CGMP grade human recombinant soluble alpha-Klotho protein, and drugs, antibodies, hormones, hormones, human cell, tissue, cell or tissue system products (HCT/Ps ) and methods of administering them to human or non-human subjects. Combinatorial compositions and methods can be useful for treating subjects suffering from age-related disorders or conditions, metabolic disorders, chronic diseases, acute injuries, and the like. Prophylactic administration of combination therapy to subjects without overt conditions or disorders may also be useful in delaying or preventing certain conditions or disorders described herein.

In various embodiments of the present disclosure, the recombinant Klotho protein, regardless of product or process, has a sequence with 80% to 100% sequence identity with one of SEQ ID NOs: 1-38. preferably with 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 with a SEQ ID NO: 73 with a linker sequence that has 80%-100% sequence identity with one of the Klotho proteins. The protein may optionally comprise or be expressed with a signaling sequence having 80%-100% sequence identity with SEQ ID NO:71 or SEQ ID NO:72. Preferably, the protein (manufactured, produced, expressed or administered) has 80% to 100% sequence identity with one of SEQ ID NO:39-SEQ ID NO:70.

An exemplary method of producing a recombinant Klotho protein is in Chinese Hamster Ovary (CHO) cells, preferably in dihydrofolate reductase (DHFR)-deficient CHO cells, more preferably in CHO-S cells, or preferably in glutamine synthesis. producing a recombinant Klotho protein in enzyme (GS)-deficient CHO cells, more preferably in GS-/- CHO cells, wherein the protein preferably comprises at least one of SEQ ID NO: 2 to SEQ ID NO: 70 and at least It has 85% amino acid sequence identity.

Exemplary cell lines are 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 -/- A cell line comprising a plurality of Chinese Hamster Ovary (CHO) cells in CHO cells, the CHO cells containing the exogenous nucleic acid comprising a promoter, preferably a strong promoter, and a polypeptide, at least a portion of the polypeptide encodes a polypeptide having at least 85% amino acid sequence identity with one of SEQ ID NO: 2-SEQ ID NO: 70, optionally functional dihydrofolate reductase (DHFR ) enzyme, or a functional glutamine synthase (GS) enzyme.

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 the liquid medium lacks hypoxanthine, thymidine, and/or glutamine, and the CHO cells to express the polypeptide encoded by the nucleic acid. and the cell line of any one of claims 14-17, which grows in a liquid medium, wherein the polypeptide comprises a recombinant Klotho protein.

Exemplary recombinant Klotho proteins contain at least 80% amino acid sequence identity with one of SEQ ID NO:2-SEQ ID NO:70.
An exemplary method of treating age-related or other conditions, diseases, or disorders comprising administering to a subject in need thereof a pharmaceutically effective amount of amino acid residues of human alpha klotho isoform 1 A method comprising administering a soluble recombinant Klotho protein having at least 80% amino acid sequence identity with at least a subset of 1-981.

An exemplary method of treating age-related or other conditions, diseases, or disorders comprising administering to a subject in need thereof a pharmaceutically effective amount of A method comprising administering a soluble recombinant Klotho protein having at least 80% amino acid sequence identity with one.

An exemplary pharmaceutical composition is a pharmaceutically effective amount of a recombinant soluble Klotho protein, wherein at least a portion of the protein is human alpha Klotho isoform 1 1-981, 29-981, 34-981, 36-981 , 131-981, 1-549, 29-549, 34-549, 36-549, or 131-549, or at least a portion of one of SEQ ID NOS:2-70; It includes a protein having at least 85% amino acid sequence identity and a pharmaceutically acceptable carrier.

An exemplary method of treating or preventing acute kidney injury (AKI) or other condition comprises administering to a subject in need thereof a pharmaceutically effective amount of a recombinant soluble Klotho protein, comprising at least a portion of human alpha klotho isoform 1 1-981, 29-981, 34-981, 36-981, 131-981, 1-549, 29-549, 34-549, 36-549, or 131-549 or at least a portion of one of SEQ ID NOS: 2-70 with at least 85%, 86%, 88%, 90%, 92%, 95%, 98%, 99%, Or preferably have 100% amino acid sequence identity.

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 at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 98%, even more preferably at least 99%, most This includes administering therapeutic concentrations of polypeptides, preferably having 100% amino acid sequence identity.

Some embodiments may include any of the features, options, and/or possibilities described elsewhere in this disclosure, including other aspects or embodiments of this disclosure. Note also that each of the foregoing, following, and/or other features described herein also represents a separate embodiment of the present disclosure. Moreover, combinations of any two or more of such features represent separate embodiments of this disclosure. Such features or embodiments may also be combined in any suitable combination and/or order without departing from the scope of the present disclosure. Accordingly, each of the features described herein may be combined with any one or more of the other features described herein in any suitable combination and/or order. Accordingly, the disclosure is not limited to the specific combinations of exemplary embodiments detailed herein.

Additional features and advantages of exemplary embodiments of the disclosure will be set forth in the description that follows, and in part will be apparent from the description, or may be learned by practice of such exemplary embodiments. The features and advantages of such embodiments may be realized and obtained by means of the instruments and combinations particularly pointed out in the appended claims. These and other features will become more fully apparent from the following description and appended claims, or may be learned by practice of such exemplary embodiments as described below.

To illustrate the manner in which the above-enumerated and other advantages and features of the present disclosure may be obtained, a more specific description of the embodiments briefly described above is illustrated in the accompanying drawings. will be provided by reference to specific embodiments. For better understanding, like elements are designated by like reference numerals throughout the drawings. With the understanding that these drawings depict only typical embodiments of the disclosure and are, therefore, not to be considered limiting of its scope, the present disclosure is supplemented through the use of the accompanying drawings. will be described and explained with the specificity and details of

FIG. 2 is a schematic showing cellular production of various Klotho proteins, according to one embodiment of the present disclosure. Schematic structures of isoforms 1 and 2 of human α-klotho and the locations of epitopes for antibody binding used to generate CD (residues 800-900) are shown. The full-length α-Klotho protein sequence of 1012 amino acids is shown, including KL1 and KL2 shown in red and green, respectively, and TM highlighted (black). Western blot analysis of human cell lysates is shown. Western blot analysis of human tissues is shown. Shows the number of adult patients who received certain aminoglycosides in 2010. Figure 3B shows the age distribution of adult patients who received the aminoglycosides shown in Figure 3A. FIG. 3B shows treatment data collected for adult patients receiving the aminoglycosides shown in FIG. 3A.

Before describing various embodiments of the present disclosure in detail, the present disclosure provides a description of certain parameters, language, and certain exemplary systems, methods, and/or products that may be varied in each embodiment. It is understood that it is not limited to only Accordingly, certain embodiments of the present disclosure will be described in detail with reference to particular features (e.g., configurations, parameters, characteristics, steps, components, components, members, elements, parts, and/or parts, etc.) Although described, the description is exemplary and is not to be construed as limiting the scope of the present disclosure and/or the claimed invention. Additionally, the terminology used herein is for the purpose of describing embodiments and is not necessarily intended to limit the scope of the present disclosure and/or the claimed invention.

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 this disclosure pertains.
Various aspects of the disclosure, including systems, methods, and/or products, can be illustrated with reference to one or more embodiments that are exemplary in nature. As used herein, the term "embodiment" means "serving as an example, instance, or illustration" and is preferred or advantageous over other aspects disclosed herein. should not necessarily be interpreted as In addition, any reference to the disclosure or "embodiments" of the invention is intended to provide illustrative examples without limiting the scope of the invention, which is indicated by the appended claims. .

As used in this specification and the appended claims, the singular forms "one" and "said", "the" refer to both singular and plural referents unless clearly indicated otherwise. are each considered, included and specifically disclosed. For example, reference to a "protein" contemplates and specifically discloses one as well as multiple (eg, two or more, three or more, etc.) proteins. Similarly, the use of plural referents does not necessarily require pluralities of such referents, but does contemplate and includes support for the singular as well as plural of such referents, unless expressly indicated otherwise, and specifically disclose and/or provide;

As used throughout this disclosure, the words "can" and "may" have a permissive (i.e., (meaning having the potential). Further, the terms "comprising," "having," "containing," "characterized by," variations thereof (e.g., "including," "having," "containing," etc.), as well as the claims Like terms used herein, including, are intended to be inclusive and/or variable, and illustratively the word "include" and variations thereof (e.g., "include") have the same meaning. and does not exclude additional, unlisted elements or method steps.

For the sake of brevity, this disclosure may recite only lists or ranges of numerical values. However, any such list of values or ranges of values (e.g., greater, lesser, up to, at least, and/or about a particular value, and/or between two recited values) is disclosed or recited. It is to be understood that any particular value or range of values within a disclosed value or list or range of values is likewise specifically disclosed and contemplated herein. deaf. Accordingly, disclosure of exemplary measurements (e.g., length, width, thickness, etc.) that are about 10 units or less, or between 0 and 10 units, illustratively include (i) 9 units, 5 units, 1 unit, or any other value measurement from 0 to 10 units, including 0 units and/or 10 units; and/or (ii) from 9 units to 1 unit, from 8 units to 2 units, from 6 units to 4 units, and/or or include specific disclosure of measurements ranging from 0 units to any other value of 10 units.

For ease of understanding, where possible, like references (i.e. like names for components and/or elements) are used to denote like elements common to different embodiments of the present disclosure. It is Similarly, similar components, or components having similar functions, will be provided with similar reference designations where possible. Certain language will be used herein to describe the exemplary embodiments. It will nevertheless be understood that no limitation of the scope of the disclosure is thereby intended. Rather, the language used to describe the exemplary embodiments is intended to be exemplary only and to limit the scope of the disclosure (unless such language is explicitly stated to be essential). It is understood that the

Although the detailed description is divided into paragraphs, the paragraph headings and content within each paragraph are for organizational purposes only and stand alone descriptions and embodiments or otherwise limit the scope of the description or claims. not intended to Rather, the content of each paragraph in the Detailed Description should be read and understood as a collective whole in which elements of one paragraph may relate to and/or be characteristic of other paragraphs. intended to Accordingly, embodiments specifically disclosed in one paragraph may also be disclosed in additional and/or alternative embodiments in another paragraph bearing the same and/or similar products, methods, and/or terms. may relate to and/or function in

Embodiments of the present disclosure produce recombinant human Klotho protein, such as (Current Good Manufacturing Practice (CGMP) grade) human recombinant soluble alpha-Klotho protein, protein fragments, and/or protein variants. , including products, compositions and/or methods of making and/or using.

Gene therapy can be effective in animal studies. However, the safety of gene therapy, especially human therapy, remains questionable. Compared to viral delivery of the Klotho gene to animals (cells), administration of exogenous and/or recombinant Klotho protein in humans is safer, easier, and (endocrine) to restore Klotho levels. It can be in a more direct manner. Therefore, in the near future exogenous (human Administration of modified alpha soluble) Klotho protein may be a viable and effective option for treating the elderly and/or age-related disorders. For example, administration of exogenous (human recombinant alpha-soluble) Klotho protein to humans is effective in reversing or delaying stem cell depletion and/or reducing age-related frailty or other pathological processes. can be a policy.

Preclinical data demonstrate the therapeutic potential of soluble Klotho protein for age-related diseases and diseases associated with Klotho deficiency. Epidemiological data show that soluble klotho is lower in older adults than in young adults, and levels of soluble klotho are inversely correlated with age, indicating that aging is associated with decreased soluble klotho. .

SUMMARY LIST OF DEFINED TERMS To assist in understanding the scope and content of the foregoing as well as the description and claims set forth below, some selected terms are defined below.

The term "condition," as understood by those skilled in the art, refers to any disorder, disease, injury or illness that presents or is expected to occur in a patient. Such pathological manifestations may be early, intermediate or late stage manifestations known in the art, including pre-pathological symptoms, precursors or manifestations. Such predictions of conditions are predicted, assumed, expected, imagined, assumed and/or speculated occurrence of conditions, whether found on scientific or medical grounds, risk assessment, or simply anxiety or fear. can be or include them.

The term "patient" refers specifically to a human under the care of a physician or other relevant medical professional, and collectively any animal under the care of a physician as that term is defined herein. do.

As used herein, the term "physician" refers generally to a physician. The term includes any medical professional, when contextually appropriate, including any licensed medical professional such as an oncologist, surgeon, or physician's assistant, nurse, phlebotomist, veterinarian, or the like. .

The term "cancer" refers to abnormal, typically uncontrolled cell growth. As used herein, "cancer cells" include malignant cells with abnormal, typically uncontrolled growth. The term cancer is thus an umbrella term encompassing a number of different and distinct diseases, typically characterized by malignant cells that proliferate in an uncontrolled manner.

The term "co-administration" and like terms means concurrent, sequential and/or concomitant administration of two or more components. For example, two components can be co-administered by concurrent, simultaneous, or sequential administration (eg, separate administrations separated by a period of time) of each component at separate dosages. The time period can be very short (eg, substantially immediately after the first administration) or longer (eg, 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). Parallel or simultaneous administration can include overlapping administration time frames of two or more components, or administration of a combination product comprising a mixture of two or more components.

As used herein, "nucleic acid" and like terms refer to DNA or RNA or DNA-RNA hybrids, single- or double-stranded, either sense or antisense, natural or synthetic oligos. Refers to a nucleotide or polynucleotide. 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 also include nucleotides 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). can be mentioned.

As used herein, the term "standard amino acids" includes alanine-ala-A; arginine-Arg-R; asparagine-asn-N; gln-Q; glutamic acid-glu-E; glycine-gly-G; histidine-his-H; isoleucine-ile-I; leucine-leu-L; threonine-thr-T; tryptophan-trp-W; tyrosine-tyr-Y; and valine-val-V.

As used herein, "codon optimized" or "codon optimization" refers to translating the codons in a nucleotide sequence from the codons in the organism used for expression of the molecule. Refers to the process of modifying or changing codons to favor or more closely match the usage pattern. Thus, for use in specific organisms where expression is desired based on known codon usage in the organism in order to improve efficiency of expression of nucleic acids, e.g., to achieve faster translation rates and greater accuracy. In addition, the codons can be optimized. Codon usage in certain organisms is known.

Encoding nucleic acid molecules may be modified wild type, or codon optimized for expression in specific host cells, e.g., mammalian cells such as CHO cells or 293 cells, or in yeast, or in plant cells, eukaryotic cells, and the like. can be a modified, codon-optimized sequence.

In certain instances, nucleic acid sequences can be codon-optimized to, for example, increase the level of expression of the encoded sequences. Particular codon usage depends on the host organism in which the modified polypeptide is expressed. Those skilled in the art are familiar with the optimal codons for expression in mammalian or human cells such as bacteria or yeast, including E. coli or Saccharomyces cerevisiae. For example, the codon usage information is kazusa. or. jp. available from the Codon Usage Database available at codon (for a description of the database see, e.g., Richmond (2000) Genome Biology, 1:241; also Forsburg (2004) Yeast, 10:1045-1047. (1991) Nucleic Acids Research, 19:4298, Sharp et al. (1988) Nucleic Acids Res., 12:8207-8211, Sharp et al. (1991) Yeast, 657-78).

Therapeutic Proteins Embodiments of the present disclosure may comprise one or more therapeutic and/or recombinant human alpha soluble Klotho proteins, protein fragments, and/or protein variants.

The protein comprises amino acid residues 1-1012, 1-981, 29-981, 34-981, 36-981, 131-981, 1-549, 29-549, 34-549, 36 of human alpha Klotho isoform 1. -549 or all or a subset of 131-549. The protein comprises amino acid residues 1-1012, 1-981, 29-981, 34-981, 36-981, 131-981, 1-549, 29-549, 34-549, 36 of human alpha Klotho isoform 1. -549, or all or a subset of 131-549 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 can have, such as 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 have at least a portion that has an amino acid sequence identity, such as at least 85%, with any suitable portion of one of SEQ ID NOs: 1-75, or a combination of two or more thereof May contain protein.

Some embodiments may include proteins with one or more amino acid variants compared to human alpha Klotho isoform 1. Illustratively, the protein may comprise a human C370 variant. For example, the protein can contain a C370S mutation, thereby containing S370. In some embodiments, proteins may include other than human F352, or F352V. In at least one embodiment, the protein includes S370, which may include the C370S change, thereby not including the F352V variant and preferably including F352. Proteins may include H193, or other than H193R variants. All other canonical amino acid substitutions at amino acid residues (or positions) 193, 352, and/or 370 of human alpha Klotho isoform 1 are contemplated and expressly disclosed herein.

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

A protein may also include (attached to) one or more glycans. For example, native human alpha Klotho isoform 1 can have glycans attached (via glycosylation) at amino acids 106, 159, 283, 344, 604, 612 and/or 694. Thus, a protein of the disclosure or its Klotho protein sequence has one or more of the same (or similar) glycans attached thereto (via glycosylation) (e.g., at the same amino acid position(s)). can have In a preferred embodiment, the protein comprises all of the same or similar (naturally occurring) glycans attached thereto at the same amino acid position(s).

In some embodiments, proteins may include a signal peptide or signaling sequence. For example, the protein may contain the native Klotho signaling sequence. Proteins may include non-naturally occurring or synthetic signaling sequences. In some embodiments, the signaling sequence can be upstream (or N-terminal) to the N-terminal signaling sequence and/or the Klotho protein sequence. In other embodiments, the signaling sequence may be C-terminal or otherwise positioned. Preferably, the signaling sequence is the native human Alpha Klotho isoform 1 signaling sequence, the native human Alpha Klotho isoform 2 signaling sequence, SEQ ID NO: 71 or SEQ ID NO: 72 and at least 80%, 85%, 90%, 95%, 98% %, or 99% amino acid sequence identity.

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

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

In at least one embodiment, the tag can be cleaved from the protein. In other embodiments, tags may be retained as part of the protein. In some embodiments, the tag can enhance protein solubility and/or (serum) half-life. In some embodiments, tags may be utilized during protein purification (eg, as part of a purification mechanism).

In some embodiments, a protein can include a linker (eg, amino acid linker) placed between the Klotho protein sequence and the amino acid tag. Illustratively, the linker may comprise 1-40 amino acids, preferably 5-20 amino acids, more preferably 8-12 amino acids, most preferably about 10 amino acids. In some embodiments, the linker can be or include a GS linker. Preferably, the linker can be, comprise, or have at least 70%, 80%, 90%, or 100% amino acid sequence identity with SEQ ID NO:73.

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

Nucleic Acids and Expression Vectors Some embodiments may include nucleic acids or nucleic acid constructs. For example, an embodiment can include an expression vector or nucleic acid construct. A nucleic acid can encode a recombinant human alpha soluble Klotho protein, protein fragment, or protein variant, as described herein. In at least one embodiment, the nucleic acid comprises a Klotho protein sequence, optional (natural or non-natural) signaling sequences (e.g., multiple N-termini of the Klotho protein sequence or a single N-terminal), an optional linker sequence (eg, GS linker), and/or an amino acid tag (eg, IgG1-Fc or TEV-twinstrep).

In some embodiments, the nucleic acid is human alpha klotho isoform 1 amino acid residues 1-1012, 1-981, 29-981, 34-981, 36-981, 131-981, 1-549, 29- Proteins comprising all or a subset of 549, 34-549, 36-549 or 131-549 can be expressed. at least a portion of the protein comprises amino acid residues 1-1012, 1-981, 29-981, 34-981, 36-981, 131-981, 1-549, 29-549, 34- of human alpha Klotho isoform 1 549, 36-549, or all or a subset of 131-549 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 has at least and/or about 85%, 86%, 87%, 88%, all or part of one of SEQ ID NOs: 1-75, or a combination of two or more thereof, They may have 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or 100% amino acid sequence identity. In preferred embodiments, the protein is at least and/or about 85%, 86%, 87%, 88%, 89%, 90%, 91% all or part of one of SEQ ID NOs: 2-70. , 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% amino acid sequence identity.

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 can have at least a portion. In preferred embodiments, the nucleic acid is one of SEQ ID NO:76-SEQ ID NO:96 and at least and/or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, It can have 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or 100% nucleotide sequence identity.

Nucleic acid sequences of the present disclosure can also include stop codons known in the art (eg, TGA, TAG, TAA).
Cell Lines and Methods of Manufacture An embodiment of the present disclosure may include cell lines. Cell lines may comprise any suitable cell type such as CHO cells, HEK cells, HL-60 cells, or other cell lines known in the art. Illustratively, a cell line can comprise CHO cells (eg, multiple CHO cells). In some embodiments, the CHO cells may contain (each) an exogenous nucleic acid (one or more copies). The nucleic acid is one of SEQ ID NO: 1 to SEQ ID NO: 75, or a combination of two or more thereof, preferably one of SEQ ID NO: 2 to SEQ ID NO: 70, and at least and/or about 85%, 86%, A poly having 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or 100% amino acid sequence identity It can encode a peptide.

A nucleic acid can include at least one transgene or cDNA. In some embodiments, at least a portion of the nucleic acid comprises one of SEQ ID NOs:76-101, or a combination of two or more thereof, preferably one of SEQ ID NOs:76-96, and at least and/or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or Can have 100% nucleic acid sequence identity. The nucleic acid may be or include a plasmid or other (structural) form of nucleic acid.

In some embodiments, the exogenous nucleic acid can encode functional enzymes such as dihydrofolate reductase (DHFR) and/or glutamine synthetase (GS). In at least one embodiment, the CHO cells can be or comprise dihydrofolate reductase (DHFR) deficient CHO cells, such as CHO-S cells. A nucleic acid can include a promoter (eg, a weak promoter to a strong promoter, as understood by those of skill in the art). For example, in some embodiments, a nucleic acid can include a (strong) promoter associated with a transgene that has, such as at least 85%, nucleic acid sequence identity with one of SEQ ID NO:2-SEQ ID NO:70. Thus, the transgene can be under the control of a promoter.

For convenience, reference is made to CHO cells and/or cell lines throughout this disclosure. However, it should be noted that other cells, cell lines and/or host cells (besides CHO cells) are also contemplated within the scope of this disclosure. References to CHO cells and/or cell lines therefore also contemplate references to and/or uses of other known cells, cell lines and/or host cells.

Transfection A method of producing a CHO cell line may involve introducing exogenous nucleic acid into the CHO cell, preferably via transfection or other technique known in the art. In at least one embodiment, the serum-free growth-optimized cell suspension of the CHO cell line encodes a polypeptide having at least 85% amino acid sequence identity with one of the promoters, SEQ ID NO:2-SEQ ID NO:70. was used as a host cell line for the insertion of nucleic acids (plasmids) containing the human alpha S-Klotho transgene and selectable (enzymatic) markers. The transgenes encode amino acids 1-981, 29-981, or 34-981 of human alpha soluble Klotho, respectively. In certain embodiments, the transgene had a sequence corresponding to (or at least 85% nucleic acid sequence identity to) one of SEQ ID NOs:76-96. had sex). In DHFR-deficient CHO cell lines (such as the CHO-S cell line), the selectable (enzymatic) marker was exogenous DHFR. In other CHO cell lines, the selectable (enzymatic) marker was exogenous GS.

Propagation, Selection and/or Gene Amplification Some embodiments are preferably serum-free and/or animal (or animal-derived) in solid media and/or in liquid media (e.g., in suspension cell cultures). This can involve growing the cells (eg, transfected cells and/or CHO cells) in medium that does not contain proteins (components). For example, cells can be plated on solid growth medium for a period of time. Cells may also or alternatively be grown in suspension culture and/or in liquid medium. Liquid media preferably contain 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, and the like.

In at least one embodiment, after a certain period of time (e.g., 48 hours post-transfection), the cells are harvested (e.g., detached), optionally centrifuged (e.g., 100 xg for 5 minutes), and /or 96-well adherent culture plates (eg, containing serum-supplemented -HT and/or -glutamine medium), etc., may be seeded (eg, at approximately 2000 cells/well). In certain embodiments, the medium may also contain MTX and/or MSX. Non-transfected cells can die within 7-14 days after selection (eg, exposure to MTX and/or MSX in -HT and/or -glutamine medium).

In certain embodiments, the CHO cells carry at least about 2-10 copies, at least about 10-20 copies, at least about 20-30 copies, or at least about 30-50 copies (eg, per cell) of the exogenous nucleic acid. can contain (can be selected to contain); Thus, methods select CHO cells that contain at least about 2-10 copies, at least about 10-20 copies, at least about 20-30 copies, or at least about 30-50 copies (eg, per cell) of the exogenous nucleic acid. can include doing For example, MTX and/or MSX (with serially increasing levels) can be administered to cells (eg, at a concentration of about 1 nM-1 μM, about 10-100 nM, etc.).

Dihydrofolate reductase (DHFR) gene amplification 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. Achieved. The plasmid contains DHFR, allowing amplification of the S-Klotho gene (fragment) in host cells upon exposure to MTX (10-100 nM). The GS gene expression system was also used to amplify CHO cells transfected with exogenous GS (eg upon exposure to MSX). Alternatively, a GS-/- host cell line was also used to eliminate the need for MSX. These steps resulted in the production of multiple copies of the S-Klotho gene (eg, 10-30 copies of the gene per cell) and the high levels of expression of S-Klotho protein obtained in transgenic cell lines.

In some embodiments, proteins can be secreted from CHO cells into the liquid medium in suspension cultures. For example, certain CHO cells and/or cell lines of the present disclosure have 200-500 mg protein per liter of liquid medium (without protein concentration), 500-2000 mg protein per liter of liquid medium, 1 Concentrations of 2000-5000 mg protein per liter of liquid medium, or any value or range of values therebetween, can be secreted (or selected to be secreted). In at least one embodiment, the concentration of human recombinant alpha soluble Klotho protein in the medium (of the selected suspension culture or 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, even more preferably at least 5000 mg/L object) can be selected.

High S-Klotho-producing transgene-containing colonies obtained by limited cell dilution were subcloned to create S-Klotho-secreting CHO cell lines that secrete S-Klotho in the range of 500-2000 mg/L into cell-conditioned media. produced more. All cell constructs were restriction digested and sequence verified.

In some embodiments, the CHO cells are at least 10 liters, preferably at least 25 liters, more preferably at least 50 liters, even more preferably at least 100 liters, even more preferably at least 250 liters, even more preferably at least 500 liters. liters, even more preferably at least 1,000 liters, even more preferably at least 2,000 liters, even more preferably at least 2,500 liters, even more preferably at least 5,000 liters, even more preferably at least 10,000 liters It can be grown in bioreactors having liter volumes or working volumes.

Cell Line Maintenance For high-producing S-Klotho cell lines (e.g., produced by the DHFR/MTX or GS/MSX systems) amplified prior to optional cell subcloning, bioreactors during scale-up and final Until the run of the cell line production was performed in a serum-free and animal protein component-free basal medium, with careful use of the concentrated media stocks administered.

Scale-up of high-yielding cell lines was produced in bioreactors of 100 L and then 500 L volume followed by cell inoculum growth in shake flasks or in cell suspensions in Wave Bag systems. This was done by serial inoculation of cells. Cell viability was measured at greater than 85% viable cells throughout the growth cycle in shake flasks, wavebags, or bioreactors, followed by 80% viable cell count in the bioreactor during the plateau phase of CHO cell growth. A maximum of 1-3 g/L of S-Klotho is maintained and produced concomitantly (referred to as "high productivity").

Protein Production Certain embodiments employ recombinant DNA strategies that utilize strong promoter sequences and/or high copy number plasmids for production of therapeutic amounts of Klotho protein in mammalian (e.g., CHO) cells. can. In at least one embodiment, for example, dihydrofolate reductase (DHFR) gene amplification in DHFR-deficient CHO cells provides methotrexate (MTX) and/or can include use. Similarly, CHO cells containing an exogenous glutamine synthase (GS) gene can be treated with methionine sulfoximine (MSX).

A Klotho protein may also include (attached to) one or more glycans. For example, native human alpha Klotho isoform 1 (SEQ ID NO: 1) can have glycans attached (via glycosylation) at amino acids 106, 159, 283, 344, 604, 612 and/or 694. A Klotho protein of the disclosure may have one or more of the same (or similar) glycans attached thereto (via glycosylation) (eg, to the same amino acid(s)).

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

The glycan structures present in the produced S-Klotho protein are similar or can be identical. In at least one embodiment, confirmation of native-like glycans can ensure that the correct native post-translational modifications (PTMs) are generated and stably maintained in the SCHO cell-produced S-Klotho protein.

Klotho Protein Solubility and/or Half-Life Extension Methods and compositions for extending the half-life and increasing the solubility of human S-Klotho protein are disclosed. Purification and characterization of the protein constructs so produced to achieve these results are also the subject of this disclosure. Nucleic acid changes made in the sequence of the Klotho gene or nucleic acid construct (see SEQ ID NOS: 76-96) and/or changes or chemical alterations in the amino acid sequence of the Klotho protein (see SEQ ID NOS: 1-70) and/or of the Klotho protein Relevant information regarding the addition or removal of chemical groups, peptides, or proteins to the amino acid sequence indicates increased biological half-lives or biological matrices (blood, cerebrospinal fluid, urine, or various The present disclosure teaches to obtain the resulting human Klotho variant proteins (novel compositions) with increased solubility in human tissues. These novel compositions can be made through the methods described herein for modification of S-Klotho protein.

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

A fusion protein construct was produced by combining the S-Klotho protein with human transferrin (TF).
Fusion proteins were produced by combining the S-Klotho protein with a unique recombinant polypeptide such as XTEN®.

A novel S-Klotho protein was produced through PEGylation.
Utilizing the aforementioned and other methods of half-life extension, the performance of S-Klotho protein can be evaluated in several ways, including:
longer intervals between doses of S-Klotho to provide patients with superior convenience and favorable dosing compliance;
Reducing the frequency of dosing reduces the total amount of drug used and reduces product costs;
lower doses at the same dosing interval as the parent protein,
simplifying dosing regimens and allowing subcutaneous dosing;
higher drug levels using the same dosing and dosing intervals as the parent protein, resulting in longer drug exposure and potentially better efficacy;
Reducing the immunogenicity of S-Klotho improved.

Production of Fc Domain Fusion Protein Constructs of S-Klotho The efficacy of extending the half-life in antibody Fc domains and human serum albumin (HSA) and increasing the solubility of the human S-Klotho protein was tested. Fc fusions include fusion of a peptide, protein or receptor exodomain 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 both are also able to enter the body's natural circulation through binding of the extended protein to the neonatal Fc receptor, FcRn. Also use the mechanism. After binding of the elongation protein to the FcRn receptor, degradation of the fusion protein is prevented in the endosomes of the cell. Fusions based on the addition of Fc or albumin can result in biological half-lives ranging from 3-16 days, much longer than those reported for typical pegylated or lipidated peptides. For 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. For a review stating, see Strohl WRFusion Proteins for Half-Life Extension of Biologies as a Strategy to Make Biobetters, which is hereby incorporated by specific reference in its entirety. Biodrugs. 2015;29(4):215-239.

An Fc domain was therefore added to our parental protein (S-Klotho) to increase its binding affinity to the Fc receptor (FcRn). FcRn resides within the lysosomes of endothelial cells that line blood vessels and functions to rescue antibodies from degradation that makes most proteins short-lived in circulation. As a result of its interaction with FcRn, the protein has a half-life ranging from days to weeks, making the extended form of the protein drug more efficient than biologics without this newly produced composition. allows for less frequent dosing.

The main difference between Fc and albumin is the dimeric nature of Fc versus the monomeric structure of HSA, in contrast to HSA, Fc fusion peptides exist as dimers or monomers. It leads to things. The dimeric nature of peptide Fc fusions produces an avidity effect when the target receptors for S-Klotho are spaced sufficiently closely together or are themselves dimeric, particularly in human target organs. obtain. This may or may not be desirable, depending on the target.

Fusion of S-Klotho protein to antibody Fc is also taught in this disclosure to improve the solubility and stability of S-Klotho. Addition of the Fc domain to S-Klotho also allows the fusion protein to be less immunogenic upon administration to human subjects.

Conjugation of S-Klotho Protein with Human Serum Albumin (HSA) The 66.5 kDa protein HSA, similar to human IgG, has a long mean half-life in the range of 19 days. At concentrations of about 50 mg/mL (about 600 μM), HSA is the most abundant protein in human plasma and performs several functions, including maintaining plasma pH, metabolite and fatty acid transport, and a role in maintaining blood pressure. have. HSA, the upper size limit for glomerular filtration of proteins by the kidney, is also strongly anionic, helping to further delay filtration through the kidney. Like IgG, HSA also binds to FcRn in a pH-dependent manner and is recycled like IgG, albeit at a site distinct from IgG binding and through a mechanism distinct from that of IgG binding. Prolongation of its half-life occurs. HSA also tends to accumulate in tumors and inflamed tissues, and fusion or conjugation to albumin may be of value in targeting proteins or peptides to those sites. suggesting.

Due to the increased serum half-life of these molecules, the fusion of peptides or proteins with inherently short half-life properties to HSA has been extensively studied since the early 1990's. Since then, dozens of different peptides and small proteins have been fused to HSA as both innovative and potential biobetter molecules. The first HSA-peptide or protein fusion product to be commercially approved was Tanzeum® (marketed as Eperzan® in the European Union), discovered at Human Genome Sciences, developed and marketed by GlaxoSmithKline. ) was a DPP-4-resistant GLP-1-HSA fusion protein. Tanzeum® (albiglutide) was approved by the European Medicines Agency (EMA) and FDA in March and April 2014, respectively. Thus, HSA improves the half-life of pharmacologically active GLP-1 from 1-2 minutes to 4-7 days for native GLP-1, allowing once weekly dosing. Seven other known HSA fusion protein product candidates are currently in development or have been in recent years. Novozyme is also developing modified versions of recombinant HSA with improved FcRn binding for the construction of "next generation" HSA protein fusions that may have longer half-life properties. This is based on the use of the K573P mutant of HSA, which was found to have a 12-fold higher affinity for FcRn, giving HSA a longer half-life than the wild-type molecule in both mice and cynomolgus monkeys. Ta. It is expected that these longer half-life variants of HSA could further be used as fusion proteins to improve the half-life of fusion proteins.

Therefore, we have transformed our Klotho protein into wild-type HSA or mutant forms of HSA in order to provide strategic therapeutic benefits, such as superior patient convenience and promising medication compliance. to produce Klotho fusion molecules with significantly prolonged half-lives in human blood, cerebrospinal fluid, and other human biological matrices, and reduced Frequent dosing results in lower total drug usage and/or reduced cost of goods. Also, reducing the amount of drug at the same dosing interval as the parent protein may simplify the dosing regimen, allow for subcutaneous formulation, or may allow for reduced immunogenicity of S-Klotho.

Conjugation of S-Klotho Protein with Human Transferrin (TF) Transferrin is a highly abundant serum glycoprotein, found in serum at 3-4 mg/mL, which strongly and reversibly binds iron, 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 approximately 2-8% of the total transferrin pool, has a slightly longer half-life of 14-17 days. The long-term persistence of transferrin in human serum is due to a mechanism mediated by the clathrin-dependent transferrin receptor, which recycles receptor-bound transferrin back into circulation.

Peptide and protein fusions have been made to the N- and C-termini of human transferrin and to the centrally located hinge region that links together the two major lobes of transferrin. The N-terminus of transferrin is free and can be directly fused. Because the C-terminus is more buried and constrained by a nearby disulfide bond, flexible linkers are typically used when proteins are fused to the C-terminus. This ability is extended by generating libraries of peptides against specific targets and then fusing binders from those libraries to aglyco-transferrin (N-terminus, C-terminus, loop or linker regions), It has been constructed into therapeutic fusion proteins with extended half-lives.

Biotechnology company BioRexis Technologies, Inc. was founded in 2002 to develop a transferrin fusion protein platform, which it dubbed 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 was demonstrated to significantly improve the half-life of GLP-1. BioRexis was acquired by Pfizer in March 2007. As far as can be ascertained, there are currently no BioRexis-derived fusion proteins in hospitals. Klotho proteins can be fused to human transferrin to produce Klotho fusion molecules and administered in hospitals to significantly extend half-life or stability in human biological matrices in vivo. can

Conjugation of S-Klotho Protein with Amunix's XTEN® XTEN® is a proprietary recombinant polypeptide that extends the in vivo half-life of therapeutic payloads. XTEN consists of naturally occurring hydrophilic amino acids and is biodegradable. Pharmaceuticals such as proteins, peptides, and synthetic compounds can be XTENylated through chemical conjugation or gene fusion. XTEN proteins lack secondary and tertiary structure and their solution behavior resembles chemically prepared polymers with very large hydrodynamic radii. By size exclusion chromatography, XTEN protein polymers appear much larger than typical globular proteins of similar molecular weight. The bulking effect of XTEN greatly reduces the renal clearance of bound molecules, thus greatly increasing the in vivo half-life. In the present invention, the length of the XTEN polymer attached to the Klotho protein will be specified to optimize the pharmacokinetics as well as the biodistribution of the attached Klotho protein payload.

Therefore, XTEN can be recombinantly fused to our S-Klotho protein to increase the in vivo half-life of the molecule. One advantage is that the genetic S-Klotho-XTEN fusion construct produces a molecule with the convenience of single molecule expression, purification and characterization containing both therapeutic and bulking moieties. Recombinant fusions by therapeutic drug manufacturers yielding best-in-class pharmacokinetics, exemplified by XTEN-ylated growth hormone (Somavaratan, Versartis) and FVIII-XTEN (Biogen), protein at precisely defined locations. It allows multiple XTEN strands to be attached per unit and has been successfully used. For example, pharmacokinetics performed in children administered different dosages of XTENylated growth hormone (Somavaratan, Versartis) show optimization of the Somavaratan molecule to reduce receptor-mediated excretion in addition to renal clearance. , resulting in best-in-class half-lives.

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 through the introduction of cysteine or lysine residues into the gene encoding XTEN. Amunix has developed XTEN containing 1-9 thiol groups at various intervals, which can be provided to partners. Thus, in the present invention, orthogonal conjugation to amino and thiol groups in XTEN will facilitate our production of Klotho-XTEN molecules.

Protein Purification Klotho protein can be extracted from cell suspension cultures of CHO cells (eg, of a CHO cell line). CHO cells can produce and optionally secrete (eg, into the liquid medium) the Klotho protein. Secretion of up to 200-500 mg/L of S-Klotho into cell-consumed media was also observed.

Purification of recombinant proteins of the disclosure is carried out by any conventional procedure, including any suitable method known in the art or described herein, such as extraction, precipitation, chromatography and/or electrophoresis. can be Additional purification procedures that can be used to purify the protein include affinity chromatography using monoclonal antibodies that bind the target protein. Some embodiments may include IgG tagged proteins that can be purified by affinity chromatography. Generally, a crude preparation containing the recombinant protein is passed over a column onto which a suitable monoclonal antibody is immobilized. Proteins are usually bound to the column via specific antibodies while impurities pass through. After washing the column, proteins are eluted from the gel by changing the pH or ionic strength. For example, spent medium from a CHO-S high-yielding cell line was concentrated via tangential flow filtration and the S-Klotho protein purified by affinity chromatography followed by ion exchange cartridge or column chromatography. Size exclusion chromatography can also be used for protein purification.

In an alternative protocol, one or more pre- or post-affinity purification steps were performed. Such steps include, for example, (ultra)centrifugation, dialysis, membrane and/or tangential flow filtration, chromatographic separations such as ion exchange, liquid-liquid extraction methods such as (aqueous) two-phase extraction, or other known purification step. In certain embodiments, one or more post-purification processing steps were performed. Such post-purification steps include, for example, tandem anion/cation flow-through chromatography (as opposed to bind and elute chromatography), membrane filtration (e.g., 0.2 micron, 0.1 micron, etc.), or by Removal of viruses and/or bacteria by other means known to those skilled in the art may be included.

Analysis of Klotho 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 Klotho protein sample (50 μg) was fractionated on a precast SDS-PAGE gel (4-15%, 10 well; Catalog No. 456-1083; BioRad) and stained with Coomassie blue dye. . All samples were either separated by empty lanes or placed on separate gels to avoid sample contamination. Greater than 98% of S-Klotho was isolated from CHO S-conditioned media as determined by Coomassie blue dye and densitometry transmission, by silver staining visualization, or by HPLC or RP-HPLC. was shown. To obtain sequence information, proteins (after reduction and S-carboxymethylation) can be cleaved with cyanogen bromide, trypsin, and/or proteinase K, and peptides separated by HPLC, according to known methods of protein chemistry. The samples so conditioned are then placed on an automated gas-phase microsequencing instrument (Applied Biosystems model 470A, ABI, see below) including an on-line automated HPLC PTH amino acid analyzer (Applied Biosystems model 120, ABI below), connected to a power outlet. Sequenced at ABI, Foster City, Calif., USA.

Proteins were also analyzed through mass spectrometry. For sample preparation for mass spectrometry, only the 75-150 kDa gel band was clipped for analysis in order to limit the analysis to the correct S-Klotho protein. The gel fraction was softened with a sterile blade and subjected to in-gel digestion. Gel fractions were destained 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-Klotho peptide. Tryptic digestion was performed overnight at 37° C. in 50 mm ammonium bicarbonate (0.005 μg/μL) using 60 μL trypsin (sequencing grade modified, catalog number V511A; Promega). This process yielded 25 μL, of which 5 μL (1 μL for S-Klotho) was subjected to liquid chromatography on an Orbitrap nano-ESIQ-Exactive mass spectrometer (Thermo Scientific) attached to a nanoLC (Dionex Ultimate 3000 UHPLC). - Subjected to electrospray ionization tandem mass spectrometry (MS/MS) and PRM analysis. In MS/MS analysis, human recombinant alpha-S-klotho produced by embodiments of the present disclosure is substantially similar to that found in human blood, serum, urine or cerebrospinal fluid (e.g., corresponding are identical in the amino acid sequences that correspond to each other).

Using the purification method described above, the level of contaminating CHO host cell protein (HCP) was determined to be acceptable in the purified S-Klotho protein. HCP was removed to <1-100 ppm in the final S-Klotho product. A 98+% pure S-Klotho protein product was isolated from the CHO S producing cell line (cells and/or broth) used. Specifically, CGMP grade human alpha-S-Klotho was produced and purified with an analytical profile suitable for clinical administration in human subjects. For example, an analytical profile of human recombinant alpha S-Klotho can be found at http://proteomecentral.com. proteome exchange. org/cgi/GetDataset? Reference number PXD002775 in the ProteomeXchange Database, which can be found at ID=PXD002775. The NIH's complete S-Klotho protein dataset is available at http://www. ncbi. nlm. nih. gov/protein/Q9UEF7.

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

The glycan structure present in S-Klotho produced in CHO S cells is identical compared to that of native S-Klotho isolated from human body fluids (i.e., blood, serum, urine, and cerebrospinal fluid). Met. This ensured that the same native post-translational modifications (PTMs) were generated and stably maintained in the S-Klotho protein produced in S CHO cells. Thus, using the production and purification methods described herein, the inventors have successfully produced cGMP grade human S-Klotho with analytical profiles suitable for clinical administration to human subjects. there is

Therapeutic Compositions Some embodiments of the present disclosure may include pharmaceutical compositions, such as therapeutic compositions. The pharmaceutical compositions of this disclosure may generally comprise a therapeutically effective amount of a recombinant soluble alpha Klotho protein in admixture with a vehicle or carrier composed of one or more additional ingredients. Ingredients may include one or more aggregation inhibitors, buffers, tonicity agents, and additional excipients. The primary solvent in the carrier may be either aqueous or non-aqueous in nature. A composition can be prepared by combining the purified Klotho protein of the present disclosure with a pharmaceutically acceptable carrier.

One skilled in the art will appreciate that the combination of the various components included in the composition may be done in any suitable order, i.e. the buffer may be added first, middle or last, the tonicity agent may also , may be added first, middle or last. It will also be appreciated by those skilled in the art that some of these chemicals may be incompatible in certain combinations and are therefore readily substituted with different chemicals having similar properties but compatible with the relevant mixtures. be understood.

Aggregation inhibitors reduce the tendency of a polypeptide to associate into inappropriate or undesirable ternary or quaternary complexes. The amino acids L-arginine, and/or L-cysteine can act to reduce aggregation of Fc domain-containing polypeptides in formulations for long periods of time, eg, two years or more. The concentration of 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, still more preferably about 15 mM to about 75 mM, and more preferably. is approximately 25 mM. These compounds are available from commercial sources.

Compositions of the disclosure may include a buffer. Buffers maintain the pH in the desired range. Various buffers suitable for use in the pharmaceutical compositions of the present disclosure include histidine, potassium phosphate, alkaline salts, sodium or potassium phosphate or their hydrogen or dihydrogen salts, sodium citrate or citric acid. Potassium acid/citric acid, sodium acetate/acetic acid, maleic acid, ammonium acetate, tris-(hydroxymethyl)-aminomethane (Tris), various forms of acetate and diethanolamine, and any desired range known in the art. Any other pharmaceutically acceptable pH buffer to maintain the pH of the solution within. Mixtures of these buffers can also be used.

The amount of buffer useful in the composition is highly dependent on the particular buffer used and the pH of the solution. For example, less acetate can be used in the solution at pH 5 than at pH 6 because acetate is a more efficient buffer at pH 5 than at pH 6. The preferred pH of the preferred formulations ranges from 4.0 to 5.0, and pH adjusting agents such as hydrochloric acid, citric acid, sodium hydroxide or salts thereof may also be included to obtain the desired pH.

One preferred buffer is sodium phosphate, whose buffering capacity is around pH 6.2. However, it will be appreciated that other buffers may be selected to achieve any desired pH buffering. The concentration of 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.

Setting the pH of the pharmaceutical composition near physiological levels maximizes patient comfort during administration. In particular, the pH is preferably within the range of about pH 5.8 to 8.4, preferably about pH 6.2 to 7.4, however the pH may be adjusted as needed to suit the particular formulation. It is understood that it is within the scope of this disclosure to maximize the stability and solubility of the polypeptide in, for example, a pH outside the physiological range and still be acceptable to the patient.

Formulations of the present disclosure may further comprise one or more tonicity agents (eg, to make the solution isotonic with the patient's blood for injection). A tonicity agent is understood to be a molecule that contributes to the osmolality of a solution. The osmolality of the pharmaceutical composition is preferably adjusted to maximize stability of the active ingredient and also minimize patient discomfort upon administration. That is when the serum is approximately 300+/-50 millimoles per kilogram. It is generally preferred that the pharmaceutical composition is isotonic with serum, i.e., has the same or similar osmolality achieved by adding a tonicity agent, and thus has an osmolality of about It is contemplated that the osmolality can be from 180 to about 420 milliosmoles, but it is understood that the osmolality can be either higher or lower as required for particular conditions.

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 more than one tonicity agent can be used at once, for example sorbitol and glycine can be used in combination to alter the tonicity of the formulation.

Additional examples of tonicity agents suitable for altering osmolality include, but are not limited to, amino acids (eg, arginine, cysteine, histidine, and glycine), salts (eg, sodium chloride, potassium chloride). , and sodium citrate), and/or sugars (eg, 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, are manufactured by known methods, and are available from commercial suppliers.

Excipients, also referred to as chemical additives, co-solutes or co-solvents, that stabilize the polypeptide in solution (even in dried or frozen form) can also be added to the pharmaceutical composition. An excipient is defined herein as a non-therapeutic agent added to a pharmaceutical composition to provide a desired effect, eg 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, excipients should be capable of stabilizing the native conformation of the protein to maintain the efficacy and safety of the drug during processing, storage, and administration to patients. Examples include, but are not limited to sugars/polyols such as sucrose, lactose, glycerol, xylitol, sorbitol, mannitol, maltose, inositol, trehalose, glucose; serum albumin (bovine serum albumin (BSA), human SA or recombinant HA), dextran, PVA, hydroxypropylmethylcellulose (HPMC), polyethyleneimine, gelatin, polyvinylpyrrolidone (PVP), polymers such as hydroxyethylcellulose (HEC); polyhydric alcohols (e.g., PEG, ethylene glycol, and glycerol) dimethylsulfoxide (dimethylsulfoxide) (DMSO), and non-aqueous solvents such as dimethylformamide (DMF); amino acids such as proline, L-serine, sodium glutamate, alanine, glycine, lysine hydrochloride, sarcosine, and gamma-aminobutyric acid; Trademark)-80 (polysorbate 80), Tween®-20 (polysorbate 20), SDS, polysorbates, polyoxyethylene copolymers; and potassium phosphate, sodium acetate, ammonium sulfate, magnesium sulfate, sodium sulfate. , trimethylamine N-oxide, betaine, metal ions (such as zinc, copper, calcium, manganese, and magnesium), CHAPS, monolaurate, 2-O-beta-mannoglycerate, and various other excipients. , or any combination of the above.

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

In one exemplary embodiment, formulations of the present disclosure comprise HEPES, MES, or Tris-HCl and, optionally, one or more additional ingredients described herein at a pH of about 7.3 to It may contain about 150 mM NaCl buffered to 7.4.

In one exemplary embodiment, the formulation of the present disclosure contains about 25 to about 50 mg TNFR:Fc (etanercept), about 10 mM to about 100 mM L-arginine, at about pH 6.0 to about pH 7.0. , about 10 mM to about 50 mM sodium phosphate, about 0.75% to about 1.25% sucrose, about 50 mM to about 150 mM NaCl. In another embodiment, L-arginine can be replaced with L-cysteine (from about 1 to about 500 micromolar) in the formulation. In yet another embodiment, the pH can be about pH 7.0. In another specific embodiment, the formulation of the present disclosure contains about 25 mg/ml TNFR:Fc, about 25 mM L-arginine, about 25 mM sodium phosphate, about 98 mM sodium chloride, at about pH 6.2, and about 1% sucrose.

In another embodiment, the formulation of the present disclosure contains about 10 mM to about 100 mM L-arginine, about 10 mM to about 50 mM sodium phosphate, about 0.75% to 1.25, at about pH 6 to about pH 7. % sucrose, about 50 mM to about 150 mM NaCl, about 10 to about 100 mg/mL RANK:Fc. In certain embodiments, formulations of the present disclosure are 50 mg/day in about 25 mM L-arginine, about 25 mM sodium phosphate, about 98 mM sodium chloride, and about 1% sucrose at a pH of about 6.2. Contains ml of RANK:Fc.

In yet another embodiment, a formulation of the present disclosure comprises, at about pH 6-7, an effective amount of an Fc domain-containing polypeptide, about 10 mM to about 100 mM L-arginine, about 10 mM to about 50 mM sodium phosphate, about May contain 0-5% mannitol, and 0-0.2% Tween®-20 (polysorbate 20). In another embodiment, a formulation of the present disclosure contains an effective amount of an antibody such as Emab (anti-CD22 specific antibody), about 25 mM L-arginine, about 25 mM sodium phosphate, about 4% mannitol, about 0 02% Tween®-20 (polysorbate 20) and a pH of about 6.0.

In yet another embodiment, the present disclosure is a method of treating a mammal comprising administering a therapeutically effective amount of a pharmaceutical composition described herein, wherein the mammal comprises Fc in the composition Methods are provided having diseases or disorders that can be beneficially treated with domain-containing polypeptides. 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. Examples of diseases or disorders that may be treated when the Fc domain-containing polypeptide of the composition is TNFR:Fc include, but are not limited to, rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, Wegener's disease (granuloma disease), Crohn's disease (or inflammatory bowel disease), chronic obstructive pulmonary disease (COPD), hepatitis C, endometriosis, asthma, cachexia, psoriasis, and atopic dermatitis, or one or more People with inherited disorders that have mutations in the Klotho gene are included. Additional diseases or disorders that can be treated with TNFR:Fc include those described in WO00/62790, WO01/62272, and US Patent Application No. 2001/0021380, relevant portions of which are incorporated herein by reference. Incorporated.

In yet another embodiment, the present disclosure provides a method for testing accelerated stability of Fc domain-containing polypeptide stability in pharmaceutical compositions of the present disclosure, comprising: Steps for testing the activity of the peptide, i.e. at the beginning, storing the composition at 37°C for 1 month and measuring the stability of the polypeptide and comparing the stable form from the beginning to 1 month. A method is provided, comprising: This information helps in early elimination of batches or lots that appear to have good stability initially but do not store well for long periods of time.

Additionally, the pharmaceutical composition provides long-term storage such that the active ingredient, eg, the Fc domain-containing polypeptide, is stable over storage steps in either liquid or frozen conditions. As used herein, the phrase "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. be done. Long-term storage also means that the pharmaceutical composition is either stored as a liquid at 2-8°C or frozen, for example at -20°C or below (eg -20°C or -80°C). Then understood. It is also contemplated that compositions can be frozen and thawed more than once. The term "stable" with respect to long-term storage means that the active polypeptide of the pharmaceutical composition is 20%, more preferably 15%, even more preferably 10%, and most preferably is understood to mean not losing its activity by more than 5%.

One or more antioxidants may be included in the formulations of the present disclosure. Antioxidants contemplated for use in preparing 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.

Other effective dosage forms are also envisioned, such as parenteral slow release formulations, mist inhalants, orally active formulations, or suppositories. Thus, the formulation also contains bulk erosion polymers such as poly(lactic-co-glycolic acid) (PLGA) copolymers, PLGA polymer blends, block copolymers of PEG, and lactic and glycolic acid. , microparticulate preparations of polymeric compounds such as poly(cyanoacrylates)); surface eroding polymers (e.g., poly(anhydrides), and poly(orthoesters)); hydrogel esters (e.g., pluronic polyols, poly(vinyl alcohol)); , poly(vinylpyrrolidone), maleic anhydride-alkyl vinyl ether copolymers, cellulose, hyaluronic acid derivatives, alginates, collagen, gelatin, albumin, and starch and dextran), and compositions thereof based on microparticle preparations, or liposomes or microparticles. It may contain preparations of spheres. Such formulations can affect the physical state, stability, in vivo release rate, and in vivo clearance rate of the present proteins and derivatives. The optimal pharmaceutical formulation for the desired protein can be determined by one skilled in the art depending on the route of administration and desired dosage. Exemplary pharmaceutical formulations are described 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.

Biological Activity As a method for (preliminarily) assessing the efficacy and/or determining the effective dosage (for patients, subjects, or individuals presenting with age-related or metabolic disorders) of human recombinant alpha-soluble Klotho. (e.g., mammals (e.g., mice, rats, primates, or some other non-human), or other animals (e.g., Xenopus laevis, zebrafish, or flies (e.g., Drosophila melanogaster) or nematodes (e.g., (such as Caenorhabditis elegans).The Klotho protein can be administered to the organism once or according to a regimen (regular or irregular).For example, the protein may be administered a suitable number of times (eg, once, twice, etc.) for a given period of time (eg, monthly, semi-monthly, weekly, semi-weekly, daily, etc.). relevant parameters) can be assessed.The subject Klotho protein performs or produces a parameter change compared to a reference (e.g., control organism parameter).Other parameters (e.g., toxicity, clearance, and pharmacokinetics) can also be evaluated.

The Klotho proteins of the present disclosure can be evaluated using animals (models) having or exhibiting a particular disorder or condition, such as age-related or age-related disorders or conditions, metabolic disorders or conditions, such Disorders and conditions can also provide a sensitizing system in which the physiological effects of proteins can be observed. Exemplary disorders include, e.g., denervation, disuse atrophy, metabolic disorders (e.g., disorders in obese and/or diabetic animals such as db/db mice, ob/ob mice), encephalopathy, liver ischemia or Other hepatic disorders, cisplatin/taxol/vincristine models, various tissue (xenograft) transplantation, transgenic bone models, pain syndromes (e.g., inflammatory and neuropathic), paraquat poisoning, genotoxicity, oxidative stress models, and tumors (I) models.

To evaluate the S-Klotho protein of the present disclosure, the protein may be administered to a suitable animal (during a suitable treatment period) and the animal's parameters evaluated (eg, 10-60 minutes, 1-24 hours, after a suitable period of time such as 1-30 days, 1-12 months, 1-5 years, or any value or range of values therebetween). Animals may be fed ad libitum or normally (eg, not under caloric restriction, although some parameters may be assessed under such conditions). Typically a cohort of such animals is used in the assay. In general, if the test polypeptide influences a parameter towards the phenotype of similar animals subjected to caloric restriction, the test polypeptide can be shown to favorably alter the lifespan regulation of the animal. Such test polypeptides may cause at least some lifespan-modulating effects through caloric restriction (eg, a subset of such effects) rather than depriving an organism of caloric intake.

The parameters tested can be age-related or disease-related parameters (eg, symptoms of disorders associated with animal models). Advantageously indicated test proteins may cause amelioration of symptoms compared to similar reference animals not treated with the polypeptide. Other parameters related to disability or aging include antioxidant levels (e.g., antioxidant enzyme levels or activity), stress tolerance (e.g., paraquat tolerance), core body temperature, glucose levels, insulin levels, thyroid stimulating hormone levels, Prolactin levels, and luteinizing hormone levels may be included.

Animals with reduced Klotho expression (eg, mice with mutant or Klotho gene defects) can be used to measure the efficacy of the S-Klotho proteins of the present disclosure for treating age-related disorders. For example, test proteins may be administered to mutant mice to monitor age-related parameters. Advantageously indicated test proteins may cause symptomatic improvement compared to similar reference animals not treated with the protein.

Parameters related to metabolic disorders or aging can be assessed by measuring body weight, testing for acquired fertility, measuring blood sugar levels, observing lifespan, observing skin, observing motor function such as walking, and the like. Evaluation can also be performed by measuring thymus weight, observing the size of calcified nodules formed on the inner surface of the thoracic cavity, and the like. In addition, quantitative measurement of Klotho gene or Klotho protein mRNA may also be useful for evaluation.

Still other (in vivo) models and bioassays include evaluating animals with metabolic parameters, such as those associated with insulin disorders, type II diabetes. Exemplary metabolic parameters include glucose concentration, insulin concentration, and insulin sensitivity.

Many age-related parameters or biomarkers can be monitored or evaluated in assessing whether a test protein can alter lifespan regulation. Exemplary age-related parameters include (i) the lifespan of a cell or organism, (ii) the presence or abundance of gene transcripts or gene products in cells or organisms with biological age-dependent expression patterns; (iii) resistance of the cell or organism to stress, (iv) one or more metabolic parameters of the cell or organism (exemplary parameters include circulating insulin levels, blood glucose levels, fat content, core body temperature, etc.). (v) the ability of a cell or set of cells present in the organism to proliferate; (vi) the physical appearance or behavior of the cell or organism.

The term "life expectancy" refers to the average age at death of a cohort of organisms. In some cases, "life expectancy" is assessed using cohorts of genetically identical organisms under controlled environmental conditions. Accidental deaths are not counted. If life expectancy cannot be measured under controlled environmental conditions (e.g., in humans), reliable statistical information (e.g., from mathematical tables) for sufficiently large populations can be used as life expectancy.

Characterization of molecular differences between two such organisms, eg, one reference organism and one organism treated with S-Klotho 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 gender. As used herein, the term "chronological age" refers to the time that has passed since a preselected event, such as conception, the defined prenatal or fetal period, or more preferably birth. Various criteria can be used to determine if organisms are of the "same" chronological age for comparative analysis.

Typically, the degree of precision required correlates with the life expectancy of wild-type organisms. For example, in the case of the nematode Caenorhabditis elegans, where the laboratory wild-type strain N2 survives on average about 16 days under some control conditions, organisms of the same age can survive for the same number of days. Aged organisms may live for the same number of weeks or months in the case of mice, the same number of years in the case of primates or humans (ie within 2, 3 or 5 years), and so on. Generally, organisms of the same chronological age can live for an amount of time within 15, 10, 5, 3, 2 or 1% of the life expectancy of wild-type organisms of that species. Preferably, the organism is an adult (eg, the organism has survived for at least a certain amount of time for the average wild-type organism to mature to reproductive age).

Biological screening assays can be performed before the organism exhibits the overt physical hallmarks of aging. For example, an organism may be an adult that has survived only 10, 30, 40, 50, 60, or 70% of the life expectancy of a wild-type organism of the same species. Age-related changes in metabolism, immune competence, and chromosomal structure have been reported. Any of these changes may occur in a test subject (e.g., a biologically-based assay) or (e.g., in a (human or mammalian) patient) before, during, or after treatment with a therapeutic agent described herein. It can be evaluated either later.

Markers associated with caloric 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 physiological conditions that change when Klotho or Klotho-related pathways are modulated. Markers can be mRNAs or proteins that are highly altered in calorie-restricted animals. Cell models derived from the cells of the animals described herein, or analogs of the animal models described herein, can be used in cell-based assays.

Models for evaluating the effect of a test protein on muscle atrophy include: 1) medial gastrocnemius muscle mass loss in rats resulting from denervation, e.g., by cutting the right sciatic nerve in the mid-thigh, 2) (e.g., 90 3) medial gastrocnemius muscle mass loss in rats resulting from immobilization (by immobilizing the right ankle joint in flexion); 3) medial gastrocnemius muscle mass loss in rats resulting from hindlimb suspension; Skeletal muscle atrophy resulting from treatment with IL-1) and 5) skeletal muscle atrophy resulting from treatment with glucocorticoids, dexamethasone.

Administration of exogenous S-Klotho. or to maintain S-Klotho formulations, clinical dosages and administration.

Aspects or embodiments of the present disclosure, for example, administer human recombinant alpha soluble Klotho protein (cGMP and/or clinical grade) or protein fragments (of isoform 1) to a (human) subject in need thereof Including. Embodiments may also include measuring (eg, by mass spectrometry (MS) or ELISA) serum S-Klotho levels or concentrations (of the (human) subject). Such measurements can be made before, after and/or during S-Klotho administration, optionally measuring serum S-Klotho levels and/or the rate of metabolism, degradation or reduction of serum S-Klotho levels. can be iterated to MS is a technique known in the art. MS can be used to identify and quantify the levels of one or more (natural and/or recombinant) Klotho proteins in a subject's serum.

One or more additional proteins can also be measured in the subject's serum. one or more Klotho-related and/or aging-related proteins (e.g., FGF21, GDF-11, TIMP2, NAD+, CCL11, hormones testosterone, estrogen, etc.), and/or kidney function proteins (e.g., KIM- 1, cystatin-C, creatinine, BUN, creatinine, NGAL, etc.) can be measured separately or in combination with measuring Klotho in serum.

In at least one embodiment, a serum sample, such as a blood sample, is obtained. Samples may be obtained by drawing blood, as is known in the art. In preferred embodiments, a finger prick or other less invasive means to obtain a blood sample may be used. Blood samples are therefore taken more frequently (eg, throughout the day and/or every 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 hours) can be taken. Total Klotho protein serum concentration, as well as serum concentrations of various alpha Klotho protein species, such as native Klotho species (e.g., soluble Klotho, cleaved Klotho, secreted Klotho, etc.), and/or one or more Klotho of the present disclosure MS can be used to measure proteins. In some embodiments, the Klotho level is 0.5, 1, 2, 3, 4, 5, 6, 7 prior to therapeutic recombinant Klotho protein administration and again during the day after administration and/or , every 8, 9, 10, 11 or 12 hours.

Embodiments are also directed to measuring such rates and/or maintaining S-Klotho protein concentrations in the serum of such subjects within the range of normal juvenile S-Klotho serum concentrations. It may include calculating a treatment protocol (eg, including frequency of administration, dosage, and/or duration of (next) administration of S-Klotho). In at least one embodiment, the concentration of S-Klotho can be maintained at approximately 1000 picograms of (S-Klotho) protein per milliliter of serum (pg/mL).

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

Embodiments also provide any Accounting for circadian rhythm effects may include taking (baseline) S-Klotho level measurements (eg, before and after administration of S-Klotho).

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

The further administration is for a suitable time frame (long-term) to produce in the subject S-Klotho serum levels equivalent to serum maintenance levels (e.g., approximately 1000 pg/ml) in a normal young person of the same sex. can be given over The total duration of S-Klotho administration (to a (human) subject) can range from 1 day to 5 years or more. Measurements and/or determinations of a subject's frailty based on the use of a clinical frailty score and other measurements can also be made over a time frame.

Embodiments of the present disclosure reduce the S-Klotho dosage to 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or more, increasing and maintaining increased S-Klotho levels above the normal range (1000 pg/ml). include.

Certain embodiments include administering S-Klotho protein as a single bolus injection or chronic (IV) injection (eg, chronic infusion). In at least one embodiment, 1, 2, 2.5, 2.75, 3, 3.5, 4, 4.5, 5 or more micrograms of S-Klotho per subject is administered per treatment. obtain. 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 rat studies, 0.01 mg S-Klotho/kg body weight or 10 μg/kg body weight per dose is used. Illustratively, using an equivalent human relative growth rate of 0.16, Human Equivalent Dose (HED) = 10 μg/kg x 0.16 = 1.6 μg/kg. Thus, illustratively, a 70 kg human individual would need 70 kg x 1.6 ug/kg = 112 ug, and a 60 kg human would need 60 kg x 1.6 = 96 ug.

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

Amount and/or Bioavailability of S-Klotho in Humans (Before and/or After Recombinant Protein Administration), Total Amount and/or Concentration of S-Klotho Administered, and/or Recombinant Protein Administration Multiple factors may be considered or taken into account in determining, measuring and/or estimating the subsequent serum level response (over time). For example, such factors include diluent composition, route of administration, site of administration, distribution to tissues and organs of the subject, metabolic or other rates of the subject, pharmacokinetics (PK), pharmacodynamics (PD), toxicology. (Tox) and the like.

In at least one embodiment, the (normal) concentration of S-Klotho (eg, in healthy, young (18-30 year old) human adults) may be approximately 1000 pg/ml in serum. A typical adult can have approximately 5 liters of blood volume, with women generally having less blood volume than men. Approximately 55% of human blood can be made up of serum. Therefore, (5 liters of blood/adult) x (0.55 serum/liter of blood) = 2.75 liters (2750 ml) of serum/adult. Assuming no endogenous serum S-Klotho, to achieve a final concentration of 1000 pg/ml in total serum, 2750 ml X 1000 pg/ml = 2,750,000 pg (or 2750 ng or 2.75 μg ) of exogenous S-Klotho was administered.

To increase soluble Klotho by 50% over typical healthy levels (eg, 1500 pg/ml of serum), a dosage of 4.125 micrograms/subject may be administered. To increase soluble klotho 100% above typical healthy levels (eg, 2000 pg/ml serum), a dosage of 5.5 micrograms/subject may be administered, and so on.

A pharmaceutically effective and/or sufficient amount of purified recombinant S-Klotho protein is about 50% per milliliter of serum to raise the serum soluble Klotho protein concentration in a subject to any suitable level. , 100, 250, 500, 750, 1250, 1550, 1750, 2250, 2500, 2750, 2750, 3000, 3000, 3000, 45000, 55000, 55000, 65000, 65000, 7500, 7500, 8500, 8500, 8500, 8500, 8500, 8500 , 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 of soluble Klotho protein, or between about 5%, 10%, 15%, 20%, 25%, 30%, 40% of typical healthy levels of soluble Klotho protein in serum , 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 between.

In some embodiments, the subject administers recombinant human S-Klotho protein by one or more (bolus) intravenous, intradermal, intraperitoneal, intramuscular, intracutaneous, subcutaneous and or other injections of about 0.01 mg/kg body weight or greater in a suitable amount of Klotho's buffer (e.g., 150 mM NaCl and 10 mM HEPES pH 7.4) or other pharmaceutically acceptable carrier; or dosages therebetween may be administered. Thus, a 160-pound (ie, 72.57 kg body weight) subject would receive approximately 0.73 mg S-Klotho per dose (based on a calculation of 0.01 mg S-Klotho/kg X 72.57 kg body weight). (bolus) injections of Similarly, a 170 pound person could receive 0.77 mg of S-Klotho per dose. The total number and frequency of administrations can be determined, for example, based on achieving and maintaining a concentration of 1000 pg/ml S-Klotho (equivalent to 0.000001 mg/ml serum) in serum. The latter can be measured and confirmed by MS or by the human S-Klotho ELISA assay.

In other embodiments, the dosage is 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 between , or any value or range of values therebetween. Urine, and/or blood may be used for recombinant Klotho protein for medical procedures or About 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 40 minutes, 45 minutes, 60 minutes, 90 minutes, 2 hours, 3 hours, 4 hours, 5 minutes after the first administration (dose) 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 It can be collected at one or more time points such as months, two months, three months or more, less than or between.

One or more embodiments involve the manufacture and/or (subsequent) administration of unique formulations of S-Klotho active drug products and/or combinations with pharmaceutically acceptable carriers. Carriers may be suitable for IV and/or bolus injection. Embodiments also include S-Klotho and/or pharmaceutical manufacturing and/or (subsequent) administration of unique inactive prodrug formulations in combination with acceptable carriers, such prodrug formulations may include coatings or sustained release formulations.

Administration of Exogenous S-Klotho to Treat Age-Related Frailty in Humans administration of sex Klotho protein. S-Klotho can rescue myogenic stem cells, improve muscle repair, and/or suppress fibrosis in animal models of human disease. Therefore, S-Klotho may be a promising therapeutic agent against muscle wasting in elderly human subjects showing signs of frailty.

For example, the present disclosure provides S-Klotho formulations, clinical dosages, and administration to frail and/or elderly subjects (eg, 60-95 years old) to maintain S-Klotho serum concentrations in the former subject, compared to normal. and/or (eg, without chronic medical conditions) within the range of young people (eg, 18-30 years old).

Long-term Klotho treatment can reverse and/or improve one or more age-related indicators or conditions in the elderly, infirm, or other physiological aging.
Administration of Exogenous S-Klotho for the Treatment (Reduction) of Muscle Atrophy in Humans With respect to administration of exogenous Klotho protein to treat (eg, slow) muscle atrophy, guidance is provided on the effects of administration of Klotho against muscle atrophy.

Muscular wasting can include numerous 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 use or disuse of muscles. Symptoms include decreased skeletal muscle tissue mass. In human males, muscle mass declines by a third between the ages of 50 and 80. Some molecular hallmarks of muscle atrophy may include upregulation of ubiquitin ligases and reduction of myofibrillar proteins. The degradation of these proteins can be monitored, for example, in certain muscle myosins (eg, by measuring 3-methyl-histidine production, a specific component of actin). Release of creatine kinase (a cell damage marker) can also be an indicator.

Administration of Exogenous S-Klotho to Treat Intellectual and/or Cognitive Declining in Old Age and/or Throughout Human Lifespan It relates to administration of exogenous Klotho protein for improvement and/or suppression. At the time of this disclosure, it was unknown whether administration of exogenous Klotho protein could prevent cognitive decline in humans. However, transgenic mice with systemic overexpression of Klotho performed better than controls in multiple tests of learning and memory. Klotho elevation in mice also enhanced long-term potentiation, a form of synaptic plasticity, and synaptic GluN2B, an N-methyl-D-aspartate receptor (NMDAR) subunit with important functions in learning and memory. increased. GluN2B blockade abolished the Klotho-mediated effect.

Klotho-regulated pathways may be involved in slowing the progression of Alzheimer's disease and other forms of dementia. Brain scans of more than 400 healthy men and women over the age of 53 found that those who carried a single copy of a particular Klotho gene variant had larger brain regions involved in planning and decision-making. It was found that Further testing on the group found that people with enlarged right dorsolateral prefrontal cortex (rDLPFC) performed better on a range of intellectual tasks.

About 1 in 5 people inherit a single copy of a genetic variant or allele known as KL-VS that improves heart and kidney function and increases human lifespan by about 3 years on average. However, with respect to brain function, having a larger rDLPFC accounts for only a 12% improvement in people's intelligence test scores, while the editors note that one copy of the KL-VS allele Retention appears to provide 10 years of resilience against the expected decline in rDLPFC structure and function. Thus, KL-VS heterozygosity appears to be associated with a larger volume of the right dorsolateral prefrontal cortex (rDLPFC).

Since the rDLPFC is important for executive function, the researchers also analyzed individuals' working memory and processing speed. KL-VS heterozygosity may be associated with improved executive function over the life span tested. In sum, the results suggest that Klotho gene diversity may be associated with greater brain volume and better function.

Exemplary embodiments of the present disclosure provide in vivo clot levels and/or (cellular, molecular and/or downstream) effects (e.g., to enhance cognition and inhibit cognitive deficits throughout the human lifespan). administration of exogenous Klotho protein to complement Exogenous administration of clinical grade S-Klotho preserves and/or improves cognitive function (eg, in humans).

Administration of Exogenous S-Klotho to Treat (Prolong) Longevity and/or Lifespan in Humans It relates to administering exogenous Klotho protein to a human subject to improve life expectancy. Life expectancy results obtained with Klotho administration (experimental group of human subjects) are statistical information (e.g., from mathematical tables) for individuals not receiving exogenous Klotho administration (control group) and/or for a sufficiently large population. can be reliably compared with

Administration of Exogenous S-Klotho to Treat Other Clinical Indicators With respect to the administration of, pathologies include, but are not limited to, human frailty (increase), longevity (decrease), cellular senescence (decrease), muscle strength (decrease), bone mass loss or density (decrease) ), (decreased) cognitive ability, (decreased) muscle mass, (decreased) physical strength, (decreased) grip strength, (decreased) leg strength, etc. The disclosure also provides for increased bone mineral density (BMD) (e.g., in women as opposed to men), increased BMD that is reduced after menopause (e.g., in older women), regenerates (degenerate) skeletal muscle, or S for reduction of its degeneration, improvement of ambulation, spatial learning and memory, movement, freedom of movement, quality of life assessment, improvement of ejection fraction (or reduction of decline), alteration of movement, improvement of movement, etc. - Concerning the administration of Klotho. The disclosure further provides a reduction in cognitive deterioration or amnesia, an increase in cognitive performance, an improvement in cognitive function and synaptic plasticity, a reduction in learning ability, a decline in learning ability or IQ, an improvement in learning ability, learning ability, or IQ, etc. administration of S-Klotho for

Administration of Exogenous S-Klotho to Treat Genetic Defects Exemplary embodiments of the present disclosure relate to administration of exogenous S-Klotho to treat (eg, correct) known human genetic defects . For example, a 13-year-old girl with familial neoplastic calcification and the Klotho variant has been reported. Familial neoplastic calcification is an autosomal recessive metabolic disease characterized by ectopic calcification and hyperphosphatemia due to inactivating mutations in FGF23 or GALNT3. FGF23 is a hormone required 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 a 13-year-old girl. Klotho encodes a secreted protein that is required for transduction of the signal released by FGF23 towards its receptor. Administration of exogenous human recombinant S-Klotho constitutes a highly targeted and effective therapy to address the dysfunctions and symptoms associated with familial neoplastic calcification.

Administration of Exogenous S-Klotho to Treat Acute Kidney Injury (AKI) Acute Kidney Injury (AKI), formerly called acute renal failure (ARF), often results in mild to dysfunctional defined as the sudden onset of renal dysfunction ranging from AKI is a common clinical complication occurring in approximately 4% to 7% of hospitalized patients each year and can have a poor prognosis. Mortality rates associated with AKI range from 20% to 35%. Renal Klotho expression has been shown to be suppressed following AKI. Adenoviral gene transfer of Klotho may be cytoprotective in AKI.

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

AKI is associated with kidney transplantation or other surgery, acute tubular necrosis (ATN), acute allergic interstitial nephritis (AAIN), nephritis (eg glomerulonephritis), nephrotoxicity (eg drug-induced nephrotoxicity), low It may arise, at least in part, from blood pressure, or other contributing factors. Kidney transplantation and other surgeries cause acute damage or injury to the kidneys and can lead to renal disease and/or renal failure. Nephrotoxicity can contribute to AKI, ATN, AAIN, nephritis, and the like. Drugs (eg, clinically administered prescription drugs, illicit drugs, or other drugs) have been reported to be involved in 15% to 25% of all cases of AKI. Contrast agents alone account for 10% of all causes of hospital-acquired acute renal failure (e.g., contrast agent-induced acute kidney injury CIAKI), deterioration of renal function during hospitalization after decreased renal perfusion and postoperative It is the third leading cause of renal insufficiency.

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

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

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

Drug-induced nephrotoxicity also results from treatment with lithium, which can affect the flow of sodium through nerve and muscle cells in the body, often resulting in hyperactivity, urgent speech, poor judgment, It can be used to treat manic episodes of bipolar disorder characterized by reduced sleep desire, 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.

Drug-induced nephrotoxicity is also associated with scleroderma, Wilson's disease (due to accumulation and binding of copper for removal through the urine), cysteinuria (due to binding with cysteine to produce mixed disulfides that are more soluble than cysteine). (D-)penicillamine, a chelating agent class of drugs, which may be used in the treatment of , smooth muscle relaxants (e.g. hydralazine), antispasmodics (e.g. carisoprodol, cyclobenzaprine , metaxalone, methocarbamol, diazepam, benzodiazepines such as clonidine and other imidazoline compounds, tizanidine, baclofen, hydantoin derivatives, dantrolene, and the like.

Other forms of drug-induced nephrotoxicity include, for example, contrast agent-induced nephrotoxicity (e.g., radiocontrast agent-induced nephropathy (CIN) following exposure to an (iodinated) contrast agent). narcotic (opioid)-induced nephrotoxicity (e.g., after use or abuse of certain narcotics (e.g., opioids) such as cocaine, heroin); chemotherapy-induced nephrotoxicity ( cisplatin; carboplatin; oxaliplatin; alkylating agents such as bendamustine, cyclophosphamide, ifosfamide, nitrosourea, temozolomide, melphalan; antitumor antibiotics such as mitomycin C, bleomycin, anthracyclines, and related agents; antimetabolites such as capecitabine, hydroxyurea, methotrexate, pemetrexed, pralatrexate, pentostatin, fludarabine, cladribine, gemcitabine, cytarabine; vinca alkaloids; topotecan; etoposide; taxanes; irinotecan; after treatment with a cancer therapeutic drug such as cancer). In fact, a wide variety of nephrotoxic agents 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 enormous treatment costs (to patients, hospitals, and insurance companies).

Embodiments of the present disclosure may include methods of (prophylactically) treating or preventing acute kidney injury (AKI) or other conditions. The method may comprise administering the recombinant (soluble) Klotho protein to a subject in need thereof. For example, a method provides a subject in need thereof (eg, to elevate and/or maintain the subject's serum soluble Klotho protein concentration above a predetermined threshold for a predetermined period of time) by providing SEQ ID NOS:2 to administering a pharmaceutically effective amount of a recombinant soluble Klotho protein having at least 85% amino acid sequence identity with one of SEQ ID NO:70. This condition is characterized by (i) acute tubular necrosis (ATN), acute allergic interstitial nephritis (AAIN), nephritis, glomerulonephritis, and/or nephrotoxicity, or (ii) kidney transplantation or other surgery, It may include AKI resulting at least in part from acute tubular necrosis (ATN), acute allergic interstitial nephritis (AAIN), nephritis, glomerulonephritis, nephrotoxicity, or hypotension. This condition can include drug-induced (eg, aminoglycoside-induced) nephrotoxicity. The protein is administered prophylactically, e.g., prior to kidney transplantation, nephrotoxin administration, or other activity, treatment, or event known or expected to cause or contribute to AKI. obtain. Alternatively, or in addition, the protein may be used for kidney transplantation or other surgery, aminoglycoside or other nephrotoxin administration, or other activities, treatments known or expected to cause or contribute to AKI. or in response to AKI, such as after an event.

In some embodiments, the nephrotoxin or other agent is, 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, such as an (iodinated) radiocontrast agent, a hyperosmotic contrast agent (HOCM) having an iodine to molecule ratio of about 1.5:1, having an iodine to molecule ratio of about 3:1 hypotonic non-ionic contrast media (LOCM), isotonic (iso-osmotic) contrast media (IOCM) with an iodine to molecular ratio of about 6:1, etc.);
one or more antiretroviral agents (e.g., adefovir, cidofovir, tenofovir, foscanet, etc.);
One or more cancer (or chemical) therapeutic agents (e.g., cytoplatin, carboplatin, oxaliplatin, alkylating agents (bendamustine, cyclophosphamide, ifosfamide, nitrosourea, temozolomide, melphalan, etc.), antitumor antibiotics (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.);
one or more bisphosphonates or derivatives thereof (e.g. zoledronate/zoledronic acid, ibandronate, alendronate, alendronate/cholecalciferol, etidronate, risedronate (optionally with calcium carbonate), pamidronate, tiludron acid, etc.); and/or one or more narcotics (e.g., opioids) such as cocaine, heroin, etc.);
Embodiments of the present disclosure administer a therapeutic recombinant (alpha soluble) Klotho protein (eg, having at least 85% amino acid sequence identity with amino acid residues 1-981 of human alpha Klotho isoform 1, or a subset thereof). can include a method of A method may comprise administering a therapeutic Klotho protein to a human or non-human subject to (prophylactically) treat or prevent AKI or one or more conditions associated with AKI. The method comprises measuring the level of serum soluble klotho level in the subject and calculating a first dosage of protein sufficient to raise the serum soluble klotho level in the subject to a predetermined level or percent of normal level. administering a first dosage of the protein to the subject, such as by bolus or stepwise administration, and measuring the rate of soluble klotho reduction in the serum, such as following administration of the first dosage and calculating the next dosing time and amount, and/or administering the next dose of protein to the subject.

Administration of Exogenous S-Klotho to Treat Chronic Kidney Disease (CKD) Neyra and Hu, Potential application of klotho in human chronic kidney disease, Bone (2017), herein incorporated by reference in its entirety As noted, 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. Therefore, soluble Klotho may serve as an early and sensitive marker of renal decline. Furthermore, preclinical animal data support that Klotho deficiency is not just a biomarker, but a pathogenesis of CKD progression and extrarenal CKD complications, including cardiovascular disease and mineralization disturbances. . Prevention of Klotho decline, reactivation of endogenous Klotho production, or exogenous Klotho supplementation all attenuated renal fibrosis, slowed progression of CKD, improved mineral metabolism, ameliorated cardiomyopathy, and CKD in animal models. associated with mitigation of vascular calcification by

CKD is characterized by progressive deterioration of renal function with a high risk of ESRD. CKD risk increases with age, occurring in about half of CKD stage ≥3 cases in N70 year old subjects. CKD can be considered a condition of accelerated aging. The relative risk of cardiovascular mortality in dialysis patients aged 25-34 years is similar to N75 year old non-CKD patients. Cardiovascular disease is the leading cause of death in CKD and ESRD patients. CKD and ESRD patients have low expression of renal Klotho and low levels of circulating Klotho. Renal Klotho deficiency in early stages of CKD may be primarily due to suppression of Klotho expression rather than loss of viable renal tubules. Moreover, some dialysis patients still have detectable circulating Klotho, renal Klotho expression is not completely suppressed, and although the origin of Klotho is unclear to date, it may be derived from an extrarenal source. It is suggested to obtain Establishing an extrarenal source of Klotho and characterizing how Klotho can be upregulated when renal production fails is of paramount importance.

Administration of exogenous Klotho protein of the present disclosure can help prevent, delay, or reduce the burden of comorbidities in CKD.
Compositions and Treatments Comprising S-Klotho in Combination with Other Ingredients Klotho can also be used additively or synergistically with other compounds and/or ingredients that affect one or more aspects of human health and well-being. It can act in matter. For example, a therapy involving the use of therapeutic human recombinant soluble alpha klotho (S-klotho) protein in combination and/or in parallel with one or more additional active ingredients may benefit human patients. Such treatment may be prophylactic or responsive to any human condition in which the Klotho protein and/or other components may have a therapeutic effect. Such conditions can include, for example, age-related conditions, metabolic conditions, chronic or acute conditions, and the like. Certain non-limiting examples of particular conditions are disclosed herein.

S-Klotho may be present in the human body along with other blood-borne anti-aging compounds such as growth/differentiation factor 11 (GDF-11). Thus, in certain embodiments, therapeutic S-Klotho may be co-administered (eg, in parallel, sequentially, and/or in combination) with therapeutic GDF-11. In some embodiments, such administration may have additive or synergistic anti-aging or other effects. Similarly, co-administration of S-Klotho to human subjects with (neutralizing) antibodies to CCL11, or inhibitors thereof, may act in concert to combat aging or other disease states (CCL11 ( eotaxin-1) is understood to be a negative regulator of stem cell rejuvenation). S-Klotho may also or alternatively be co-administered with other eotaxin such as eotaxin-2 (CCL24) and/or eotaxin-3 (CCL26).

In some embodiments, S-Klotho may be co-administered with inhibitors or antibodies of transforming growth factor beta-1 (TGF-β1). S-Klotho administration can counteract the effects of the TGF-β1 signaling pathway, which is involved in endogenous anti-cell 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 may confer metastatic potential on cancer cells, and it is understood that this latter process is countered by Klotho. Thus, co-administration of S-Klotho with inhibitors or antibodies of transforming growth factor beta-1 (TGF-β1) may have synergistic or additive effects.

In some embodiments, S-Klotho may be co-administered with an antibody or inhibitor of insulin growth factor-1 (IGF-1). Klotho is a hormone that inhibits the intracellular insulin/IGF-1 signaling cascade, and this inhibition is evolutionarily conserved to increase resistance to oxidative stress and extend lifespan at the cellular and organismal level in mammals. It is understood that the mechanism is thought to be Thus, co-administration of S-Klotho with inhibitors or antibodies of insulin growth factor-1 (IGF-1) may have synergistic or additive effects.

Numerous studies have demonstrated the mutually regulated positive/negative feedback effects of Klotho alpha-K1, FGF23, and 1,25(OH) ₂ D, and/or Klotho β-K1, which regulates bile acid/cholesterol metabolism. Since a comprehensive regulatory scheme of mineral homeostasis has been revealed involving a similar regulatory network composed of FGF15/human FGF19, and bile acids, in some embodiments S-Klotho may be added to vitamin D (e.g., vitamin D3), or in combination with 1,25-dihydroxyvitamin D ₃ [1,25(OH) ₂ D ₃ ], FGF-15, FGF-19, FGF-15, FGF-19, and/or Klotho-β obtain. Such co-administration can have synergistic or additive effects on numerous disease states and/or processes in the body. In some embodiments, S-Klotho may be administered in combination with FGF-21
In some embodiments, S-Klotho may be co-administered with a carbonic anhydrase inhibitor such as acetazolamide, methazolamide, dichlorphenamide, dorzolamide, brinzolamide and/or topiramate. Such combinatorial administration may be useful in treating ankylosing spondylitis (AS), rheumatoid arthritis (RA), and various other conditions. Various studies have shown that increased bone resorption is a hallmark of AS and RA, and that carbonic anhydrase inhibitors play an anti-arthritic role by inhibiting bone resorption. At the bone level, S-Klotho stimulates bone resorption and phosphate release through distinct mechanisms by acting on TRPV5, a recently identified regulator of osteoclast function. Elevated levels of 1,25(OH) _{+ D3} caused by S-Klotho administration also stimulate osteoclast differentiation and bone resorption, thereby releasing phosphate. Co-administration of S-Klotho with a carbonic anhydrase inhibitor may therefore have additive or synergistic effects, particularly in promoting bone health, particularly in AS and RA.

For the treatment of severe active rheumatoid arthritis, S-Klotho may be administered in combination with one or more disease-modifying antirheumatic drugs (DMARDs).
S-Klotho can be administered in combination with cyclosporine because cyclosporine decreases klotho mRNA and protein and increases oxidative stress leading to cyclosporine-induced renal injury (CsA). Associated decreases in Klotho mRNA and protein and increases in oxidative stress can be counteracted by exogenous co-administration of S-Klotho.

In some embodiments, S-Klotho may be co-administered with losartan and/or cyclosporine. Treatment with the angiotensin II type II (AT1) receptor blocker losartan reversed the decrease in Klotho expression seen with cyclosporine. Losartan also produced parallel improvements in renal tissue (with losartan reducing tubulointerstitial fibrosis caused by cyclosporine).

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

In some embodiments, S-Klotho may be co-administered with testosterone or androgen receptor (AR) upregulating compounds. Recent reports indicate that no beneficial effects of testosterone treatment in men on personality, psychological well-being, or mood are observed. In addition, the prescribing of testosterone supplementation for low T as cardiovascular health, sexual function, physical function, mood, or cognitive function was considered not supported by randomized clinical trials. However, while testosterone supplementation was consistently found to increase muscle strength, no beneficial effects on physical function were found. S-Klotho administration in combination with testosterone and/or androgen receptor (AR) upregulating compounds exceeds any effect that testosterone or S-Klotho alone may have on these treatment groups, in the elderly, It can significantly increase muscle strength and/or physical function in frail or low T men.

In some embodiments, S-Klotho may be co-administered with estrogen or estrogenic hormones (eg, estradiol, estriol, estrone, etc.). Such co-administration may improve female health indicators (e.g., menstruation, menopause, or women transitioning to menopause) and/or infertility, polycystic ovary disease or disorder, obesity, hormonal imbalance and Related conditions and/or other female health conditions may be treated.

In some embodiments, S-Klotho may be co-administered with one or more nootropics, also called smart drugs or cognitive enhancers. Nootropic agents, supplements, and/or other substances may improve cognitive function, particularly executive performance, memory, creativity, motivation, task saliency (motivation to perform tasks), performance (especially high effort ) and may be useful in treating cognitive or motor impairments that lead to disorders such as Alzheimer's disease, Parkinson's disease, Huntington's disease and ADHD. The most commonly used class of drugs known to improve some aspect of cognition are stimulants, particularly dopamine receptor D1, adrenergic receptor A2, or both, in the prefrontal cortex. A class of stimulants that exhibit cognitive enhancing effects in humans by acting as direct or indirect agonists of receptors for Stimulants include, for example, amphetamines (e.g., amphetamine, dextroamphetamine, lisdexamphetamine); dimethylamylamines (DMAA) such as 1,3-dimethylamylamine, which can improve physical performance, alertness, reaction time, and the like.

Methylphenidate - a substituted phenethylamine that can improve a range of cognitive functions such as working memory, episodic memory, and inhibitory control, aspects of attention, and planning latency; especially alert for sleep-deprived individuals It functions as a wakefulness-promoting agent that increases mental alertness, facilitates reasoning and problem-solving, and can treat conditions such as narcolepsy, shift work sleep disturbances, and daytime sleepiness that persists after treatment for sleep apnea. eugeroics (eg, armodafinil, modafinil, etc.) that may be obtained; xanthines (eg, caffeine, etc.) that may enhance alertness, performance, and/or memory; nicotine, and the like.

In some embodiments, S-Klotho may be co-administered with one or more osteoporosis and/or osteopenia medications, as known in the art. Klotho may play a role in regulating bone mineral density and a lack of Klotho may lead to reduced bone mineral density in animals. For example, Klotho knockout mice exhibit decreased bone mineral density over time. Klotho expression can rescue bone defects in Klotho knockout animals, such as Klotho knockout mice exhibit reduced bone mineral density over time. Epidemiological studies have shown associations between various Klotho gene variants and alterations in bone mineral density and prevalence of hand osteoarthritis.

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

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

S-Klotho may be administered in combination with renal dialysis or other treatments. S-Klotho may be administered to treat frailty in dialysis patients, as frailty is associated with poor prognosis in dialysis patients.

S-Klotho may be used in combination with one or more Alzheimer's disease treatments or preventions, or in combination with brain-derived neurotrophic factor (BDNF), because adequate amounts of BDNF can help develop and maintain normal neuronal circuits in the brain. ) in combination with

To enhance the ability of Klotho to enter the central nervous system (CNS) to treat or prevent CNS-related conditions, S-Klotho is combined with one or more molecules that enhance the ability of Klotho to cross the blood-brain barrier. Can be administered in combination. For example, neither S-Klotho nor BDNF are known to cross the blood-brain barrier. Embodiments of the present disclosure use S-Klotho and/or S-Klotho, including BDNF, to the CNS to treat Alzheimer's disease and/or to improve cognition in individuals not suffering from Alzheimer's disease. including utilizing blood-brain barrier delivery technology for administration.

S-Klotho positively regulates 5′ adenosine monophosphate-activated protein kinase (AMPK), or AMPK activating agents, or signaling pathways that replenish the cellular ATP supply, including fatty acid oxidation and autophagy, or sugars. It can be administered in combination with ingredients that negatively regulate ATP-consuming biosynthetic processes, including neogenesis, lipid and protein synthesis.

S-Klotho can be administered in combination with one or more antidiabetic agents such as insulin, phlorizin, or the antioxidant tiron, and these combination treatments may help prevent renal damage due to oxidative stress that occurs in diabetic disorders. can have benefits. Co-administration of S-Klotho with other anti-diabetic agents for type 1 diabetes inhibits β-cell apoptosis through activation of the integrin β1-FAK/Akt pathway, leading to inhibition of caspase 3 cleavage, thereby inhibiting β- - can protect cells;

S-Klotho can be administered in combination with one or more type 2 antidiabetic agents, such as metformin, to improve glycemic control and vascular function in overweight and obese diabetic subjects.
S-Klotho can be administered in combination with one or more blood pressure medications, calcium regulating agents, or treatment or prevention of chronic kidney disease (CKD). For example, soft tissue calcification is a prominent feature of CKD, and Klotho ameliorates vascular calcification by enhancing phosphouria, maintaining glomerular filtration rate, and directly inhibiting phosphate uptake by vascular smooth muscle. obtain.

S-Klotho, TM5441 or other of PAI-1 inhibitors (plasminogen activator 1).

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

S-Klotho can be administered in combination with one or more FDA-approved human cell, tissue, cell and tissue lineage products (HCT/Ps). Such products include, for example, preserved umbilical veins, pericardium, amniotic membrane (when used alone (no cells added) for eye repair), dura mater, heart valve allografts, Except peripheral blood or cord blood, semen, oocytes or embryonic hematopoietic stem cells (demineralized bone, ligaments, tendons, fascia, cartilage, eye tissue (cornea and sclera), skin, vascular grafts (venous and arteries)). In at least one embodiment, the HCT/P can be or include one or more stem cells. Stem cell therapy of damaged body tissues and organs is becoming popular. Administration of therapeutic recombinant Klotho protein in combination with stem cells has provided surprising, unexpected and even synergistic results for subjects in need thereof.

Embodiments of the present disclosure further include combination products comprising therapeutic recombinant Klotho protein in combination with human stem cells. Compositions can also include a pharmaceutically acceptable carrier as described herein. Such compositions may be included as or classified as regenerative medicines approved by the FDA to treat, alter, reverse, or cure serious or life-threatening diseases or conditions. Preliminary clinical evidence indicates that a composition (drug) has the potential to address a medical need not matched to such disease or condition.

Illustratively, stem cells can be or include mesenchymal stem cells (MSCs), such as from human umbilical cord or placenta. In at least one embodiment, compositions comprising huMSCs and therapeutic recombinant Klotho proteins of the present disclosure attenuate the inflammatory and oxidative stress responses that occur in AKI and/or decrease the expression of proteins and microRNAs associated with aging. decreased.

S-Klotho can be administered in combination with one or more anti-aging agents. For example, Klotho protein may be combined with Pin1-FOXM1 and/or other anti-aging agents to improve outcomes in patients undergoing such treatment.

S-Klotho is a Klotho stimulator (Vit. D, Losartan, Testosterone), GDF-11, Trichostatin A antifungal (GDF-11 stimulator), TIMP-2, CCL-11 inhibitor/antibody , dasatinib, nicotinamide riboside (NAD+), nicotinamide mononucleotide (NMN) (NDA+), AMPK stimulants (resveratrol, aspirin, salicylates, phytochemicals, DR), C60 fullerenes, rapamycin, FGF inhibitors, FOXO4 - can be administered in combination with one or more of senolytic agents/compounds such as p53 interfering peptides (eg FOXO4-DRI), inhibitors of the anti-apoptotic proteins BCL-2 and BCL-xL.

Any of the foregoing or other treatments or co-administration may have additive or synergistic effects over either treatment alone. For example, co-administration of a Klotho protein with one or more of the foregoing may produce therapeutic results greater than the sum of the individual results of administering the single components at similar concentrations. In addition, synergistic effects may include therapeutic results similar to those of the individual results of administering the single components at lower concentrations. A synergistic effect can also be an increase in the maximum effective dosage of one or more of the components, a reduction in toxicity of one or more of the components, or any other effect that is more than just an additive effect of the individual therapeutic outcomes. May contain beneficial results. In addition, additive effects of individual therapeutic outcomes may include synergistic effects where such additive effects would not be expected or expected given the nature and understanding of the individual components.

As used herein, combination therapy or co-administration can include treatment or administration of a combination product, composition, or formulation comprising the Klotho protein and one or more additional active ingredients. The one or more additional active ingredients may be selected from among the ingredients, drugs, substances, therapeutic compositions, etc. described herein, or others known in the art. For example, the Klotho protein and one or more additional active ingredients may be injectable (e.g., intramuscular, intravenous, etc.), ingestible, transdermal, inhalable, topical, or other formulations. can be co-blended with.

Alternatively, combination therapy or co-administration may include Klotho protein and one or more additional active ingredients, wherein the Klotho protein and one or more additional active ingredients are not combined or incorporated into a combination product, composition, formulation. may include treatment or administration of the active ingredient of For example, Klotho protein and one or more additional active ingredients may be separately injectable (e.g., intramuscular, intravenous, etc.), ingestible, transdermal, inhalable, topical, or other each comprising or may be such a formulation.

It is also understood that co-administration can involve simultaneous administration of two or more components, or separate administration of two or more components, the separate administrations preferably being separated by a period of time. deaf. In some embodiments, the period of time may be very short. For example, a second component of Klotho protein and one or more additional active ingredients is substantially administered (e.g., injected) immediately after administration of the first component of Klotho protein and one or more additional active ingredients. can be Alternatively, the first and second administrations are 1-60 seconds, 1-60 minutes, 1-24 hours, 1-7 days, 1-4 weeks, 1-12 months, etc., or any value or values in between. may be separated by a period of time, such as a range of . Similarly, co-administration can involve overlapping dosing windows of two or more components.

Klotho protein variants Therapeutic S-Klotho proteins of varying lengths (eg, S-Klotho 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 that the native Klotho protein does not exhibit. Illustratively, the QuickChange XL Site-Directed Mutagenesis Kit (Stratagene) can be used to alter the nucleic acid sequences of various S-Klotho 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 commercially available.

In at least one embodiment of the present disclosure, proteins are modified with one or more C-terminal and/or N-terminal tags. Such tags may function to extend serum and/or protein soluble half-life (in one or more therapeutic or other settings). Tags may also be used for the presence or diagnostic localization of proteins, isolation or removal of proteins, delivery or transport of proteins, targeting one or more of proteins (e.g., proteins, nucleic acids, organelles, cellular structural components, etc.). It can be useful as a marker for binding to, enzymatic processing or cleavage, or the like. In at least one embodiment, the C-terminus of the protein may be tagged with TEV-TwinStrep and/or Fc fusions, as known in the art and further described herein. Additional description can be found in the articles "Fusion Proteins for Half-Life Extension of Biologies as a Strategy to Make Biobetters," "What is the future of PEGylated therapy," each of which is hereby incorporated by specific reference in its entirety. ies?” , and in "Strategies for extended serum half-life of protein therapeutics". In certain embodiments, a linker or linker peptide may be inserted and/or positioned between the (naturally occurring or variant) Klotho protein sequence and the tag.

In some embodiments, a modified Klotho protein may include an alternative (eg, natural, non-natural and/or synthetic) signal peptide. For example, in some embodiments the native 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 Klotho protein can be removed and a methionine residue can be included at the N-terminus of the SS. Additional description can be found in the article "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". In certain embodiments, a linker or linker peptide may be inserted and/or positioned between the (naturally occurring or variant) Klotho protein sequence and the alternative SS.

Some embodiments may contain one or more amino acid variants. The present disclosure contemplates diversification of any one or more of any of the naturally occurring amino acids of any of the disclosed Klotho proteins to any other amino acid, whether natural, synthetic, or otherwise constructed. will be understood.

S-Klotho C370S Protein Variant In humans, the Klotho gene is located on chromosome 13q12. A variant known as KL-VS is present in approximately 15% of Caucasians. This variant consists of six single nucleotide polymorphisms (SNPs), two of which cause amino acid substitutions (ie, F352V and C370S-Phenylalanine 352 changed to valine and cysteine 370 changed to serine). . In vitro transfection assays showed that the secretion level of Klotho was reduced 6-fold with the V352 variant, while increased nearly 3-fold with the S370 form. However, these two variants in the human Klotho gene segregate together and form a KL-VS haplotype that increases Klotho secretion by a range of 1.6-fold. For example, screening of over 300 individuals from geographically and/or ethnically distinct cohorts did not find a single individual carrying only one of the V352 or S370 variants. reportedly not.

One embodiment of the present disclosure includes a recombinant S-Klotho protein with a C370S homovariant (ie, absent (or with deletion) of the F352V variant). The C370S variant can be produced and/or expressed in the context of any protein construct described herein. For example, the C370S variant is S-Klotho 1-981, 29-981, 34-981, 36-981, 131-981, 1-549, 29 with or without an Fc fusion and/or TEV-TwinStrep. -549, 34-549, 36-549, 131-549, etc. Thus, the nucleic acid construct or cDNA from which the protein is expressed can be of corresponding length.

An embodiment involves producing an S370 heterozygous or homozygous variant construct and inserting the resulting construct encoding the S-Klotho C370S protein (e.g., via transfection) into an appropriate expression system (e.g., CHO cells) and/or transiently expressing the S-Klotho S370 protein. The S370 Klotho protein may be expressed at higher levels than the F352V/C370S protein and/or the wild-type F352/C370 protein. Embodiments may include purifying (and optionally quality control testing) the expressed protein for therapeutic administration. Embodiments may comprise administering a therapeutic or therapeutically effective amount of S-Klotho C370S protein to a subject in need thereof. A subject may, for example, carry or express a KL-VS variant. Alternatively, the subject may be of other mutant or variant wild type. Administration of recombinant S-Klotho C370S protein can lead to beneficial increases in blood S-Klotho levels. Therefore, circulating concentrations of S-Klotho in subjects administered therapeutic recombinant S-Klotho C370S protein may not be subject to the dilution effect observed when the F352V variant is present.

Therapeutic treatment of hyperphosphatemic familial neoplastic calcification (HFTC) In affected human individuals, HFTC is transformed from histidine (H) at amino acid (AA) position 193 of S-Klotho-rs121908423. Caused by mutations to arginine (R). Without being bound by any theory, it is believed that the H193R mutation in HFTC individuals impairs the ability of S-Klotho to form a ternary complex with FGF23 and FGFR1c that impairs KL-dependent FGF23 signaling. It is As a result, afflicted subjects exhibit a severe metabolic disorder manifested by hyperphosphatemia and massive calcium deposits in the skin and subcutaneous tissue. Some patients present with recurrent, transient, painful swelling of long bones associated with periosteal reaction and radiological findings of cortical osteophysis and lack of skin involvement.

One embodiment of the disclosure includes an S-Klotho protein with H193. H193 proteins can be produced or expressed in the context of any of the protein constructs described herein. For example, H193 variants are S-Klotho 1-981, 29-981, 34-981, 36-981, 131-981, 1-549, 29 with or without Fc fusions and/or TEV-TwinStrep. -549, 34-549, 36-549, 131-549, etc. Thus, the nucleic acid construct or cDNA from which the protein is expressed can be in the corresponding configuration.

Embodiments involve producing H193 heterozygous or homozygous variant constructs and converting the resulting constructs encoding the S-Klotho H193 protein (eg, via transfection) into a suitable expression system (eg, CHO cells) and/or transiently expressing the S-Klotho H193 protein. The H193 Klotho protein can also be expressed at higher levels than the R193 (or H193R) protein. Embodiments may include purifying (and optionally quality control testing) the expressed protein for therapeutic administration. Embodiments provide a subject in need thereof (eg, an HFTC individual, an individual diagnosed with HFTC, or a patient carrying H193R (rs121908423) or other variant) with a therapeutic or therapeutically effective amount of S- administering Klotho H193 protein. Alternatively, the subject may be of other mutant or variant wild type. Administration of recombinant S-Klotho H193 protein can lead to beneficial increases in blood S-Klotho levels. Administration may reverse or suppress the deleterious effects of the R193 variant transcribed and circulated in HFTC individuals as a result of the H- to -R193 point mutation found in the human Klotho gene in individuals with HFTC. Therefore, circulating concentrations of S-Klotho H193 may help counteract the effects observed in H193R or HFTC individuals.

Therapeutic treatment in CC genotype patients with end-stage renal disease (ESRD) Approximately 350,000 patients with end-stage renal disease (ESRD) suffer a very high mortality rate in their first year of chronic hemodialysis. I'm in. Both vitamin D and fibroblast growth factor (FGF)-23 levels correlate with survival in these patients. Without being bound by any theory, Klotho is a protein in the vitamin D/FGF-23 signaling pathway and is associated with accelerated aging and premature mortality in animal models. It has been hypothesized that genetic mutations in the Klotho gene may be associated with survival in subjects with ESRD. The researchers found an association between 12 single nucleotide polymorphisms (SNPs) in the Klotho gene and mortality in a cohort of ESRD patients (n=1307, Caucasian and Asian) during the first year of hemodialysis. was tested. CC genotype for one tagging SNP, rs577912 (a common HapMap variant with a minor allele frequency [MAF]>0.05 within the Klotho gene sequence located in intron 1) and 1-year mortality risk A significant association was found between the increase (RR, 1.76; 95% CI, 1.19-2.59; p=0.003). Among patients who were not treated with active vitamin D supplements (HR, 2.51; 95% CI, 1.18-5.34; p=0.005), this increase in individuals with the CC genotype The effect was even more pronounced. In lymphoblastoid cell lines 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 rs577912 SNP nucleotide changes described above result in amino acid changes in the Klotho protein. Therefore, this functional SNP (rs577912) can quantitatively affect Klotho gene expression at the mRNA level.

One embodiment of the present disclosure includes S-Klotho protein expressed from AA or AC genotypes. Proteins can be produced or expressed in the context of any of the protein constructs described herein. For example, the protein is S-Klotho 1-981, 29-981, 34-981, 36-981, 131-981, 1-549, 29-549, with or without Fc fusions and/or TEV-TwinStrep. , 34-549, 36-549, 131-549, etc. Thus, the nucleic acid construct or cDNA from which the protein is expressed can be of corresponding length.

Embodiments involve producing an AA or AC heterozygous or homozygous construct and transferring the resulting construct encoding the S-Klotho protein (e.g., via transfection) into a suitable expression system (e.g., CHO cells) and/or transiently expressing the S-Klotho protein. Klotho protein expressed in AA or AC heterozygous or homozygous cells may be expressed at higher levels than in CC cells. Embodiments may include purifying (and optionally quality control testing) the expressed protein for therapeutic administration. Embodiments provide a therapeutic or therapeutically effective amount of S-Klotho protein to a subject in need thereof (e.g., low endogenous S-Klotho protein expression, carrying a CC mutation at one tagging SNP, rs577912, and/or an individual or patient with end stage renal disease (ESRD). Alternatively, the subject may be of other mutant or variant wild type. Administration of recombinant S-Klotho protein can lead to beneficial increases in blood S-Klotho levels. Administration may reverse or suppress the adverse effects of CC variants that are transcribed and circulated in affected individuals as a result of point mutations found in the human Klotho gene of affected individuals. Thus, circulating concentrations of S-Klotho counteract the effects observed in CC individuals, particularly those with end-stage renal disease (ESRD), namely first-year mortality in ESRD patients chronically undergoing hemodialysis. can help to

Therapeutic Treatment of Radiographic Hand Osteoarthritis (OA) and Osteophytes Osteoarthritis (OA) is a common and complex disease with a strong hereditary component. Researchers studied the association between four putative functional genetic variants of the Klotho gene and OA of the hands in a large population of Caucasian women. Investigators found a significant association between SNP G-395A and radiographic hand OA and presence or absence of osteophyte formation. Allele G was 1.44 (P=0.008, 95% confidence interval (CI) 1.09-1.91) and 1.36 (P=0.006, 95% CI 1.09-1.91), respectively. significantly increased the risk of radiographic hand OA and osteophytes with an odds ratio (OR) of 1.70). From logistic regression modeling, genotype GG was associated with radiographic hand OA (OR=3.10, 95% CI 1.10-8.76) and osteophytes (OR=3.10) when compared with genotype AA. 10, 95% CI 1.10-8.75) showed a more than 3-fold increase in both risks. After age adjustment, the OR for genotype GG was 4.39 (P=0.006, 95% CI 1.51-12.74) for radiographic hand OA and 4.47 (P=0 .005, 95% CI 1.56-12.77). The researchers also suggested that one variant in the Klotho gene (SNP G-395A) is associated with susceptibility to OA of the hand and appears to act through osteophyte formation rather than cartilage damage.

One embodiment of the present disclosure includes S-Klotho protein expressed from a construct with SNP G395A. The resulting protein can be produced or expressed in the context of any protein construct described herein. For example, A395 variants are S-Klotho 1-981, 29-981, 34-981, 36-981, 131-981, 1-549, 29 with or without Fc fusions and/or TEV-TwinStrep -549, 34-549, 36-549, 131-549, etc. Thus, the nucleic acid construct or cDNA from which the protein is expressed can be of corresponding length.

An embodiment involves producing a G395A heterozygous or homozygous construct and transferring the resulting construct encoding the S-Klotho protein (e.g., via transfection) to a suitable expression system (e.g., CHO cells). and/or transiently expressing the S-Klotho protein. Klotho protein expressed in A395 heterozygous or homozygous cells may be expressed at higher levels than in G396 cells. Embodiments may include purifying (and optionally quality control testing) the expressed protein for therapeutic administration. Embodiments are directed to subjects in need thereof (e.g., individuals carrying (at risk of developing) osteoarthritis (OA) and/or radiographic hand osteoarthritis (OA) and/or bone spurs in the G395 SNP and/or or patient) in a therapeutic or therapeutically effective amount of S-Klotho protein. Alternatively, the subject may be of other mutant or variant wild type. Administration of recombinant S-Klotho protein can lead to beneficial increases in blood S-Klotho levels. Administration may reverse or suppress the adverse effects of the G395 SNP that is transcribed and circulated in afflicted individuals. Thus, circulating concentrations of S-Klotho may help counter 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-Klotho protein may reduce the risk of radiographic hand osteoarthritis (OA) and osteophyte formation in patients (eg, patients carrying the G395 SNP).

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 disability. Current evidence suggests that MetS is partly heritable and that genetic factors play a greater role than environmental factors on the incidence of MetS. Researchers have discovered an association between the G-395A polymorphism and metabolic syndrome (MetS) among the Chinese 90s and 100s population. Subjects were from the Longevity and Aging Project of 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 International Diabetes Federation criteria. 695 subjects aged 93.5±3.2 years were included. The allele frequencies of G and A were 0.852 and 0.148, respectively. In the population as a whole, the frequency of MetS was 10.8% and 5.9% in the GG and GA+AA genotype groups, respectively (p=0.004). −395A allele carriers 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 had a significantly lower risk of MetS of ∼0.97), whereas in men it was (OR 0.42, 95% CI 0.05-3.85). Across the population and in women, the relationship between the Klotho G-395A SNP and MetS was associated with higher blood pressure (OR 0.48, 95% CI 0.34-0.67; OR 0.47, 95% CI 0.31-0.67, respectively). 71), and possibly due to its effect on hypertriglyceridemia (OR 0.66, 95% CI 0.39-0.95; OR 0.54, 95% CI 0.31-0.98, respectively). In men, this relationship is due to its effect on hypertension (OR 0.47, 95% CI 0.25-0.90) and low HDL-C (OR 0.69, 95% CI 0.27-0.93) It was possible. The researchers concluded that carriers of the -395A allele of the Klotho gene correlated with a lower risk of MetS during the 90s and 100s in China, especially among women.

One embodiment of the present disclosure includes S-Klotho protein expressed from a construct having the -395A allele. The resulting protein can be produced or expressed in the context of any protein construct described herein. For example, the A395 allele is S-Klotho 1-981, 29-981, 34-981, 36-981, 131-981, 1-549, 29 with or without Fc fusions and/or TEV-TwinStrep. -549, 34-549, 36-549, 131-549, etc. Thus, the nucleic acid construct or cDNA from which the protein is expressed can be of corresponding length.

An embodiment involves producing a -395A heterozygous or homozygous construct and transferring the resulting construct encoding the S-Klotho protein (e.g., via transfection) into a suitable expression system (e.g., CHO cells). ) and/or transiently expressing the S-Klotho protein. Klotho protein expressed in A395 heterozygous or homozygous cells may be expressed at higher levels than in G396 cells. Embodiments may include purifying (and optionally quality control testing) the expressed protein for therapeutic administration. Embodiments provide a therapeutic or therapeutically effective amount of Alternatively, the subject may be wild-type of another mutant or variant Administration of recombinant S-Klotho protein may comprise administering S-Klotho protein to reduce blood S-Klotho levels Administration may reverse or suppress the adverse effects of G395 SNPs that are transcribed and circulated in affected individuals.Therefore, circulating concentrations of S-Klotho may be associated with G395 individuals, particularly those with metabolic syndrome ( MetS), administration of the G-395A S-Klotho protein may help counteract the effects observed in individuals at risk of developing G395 SNP. may reduce the risk of metabolic syndrome (MetS) in female patients).

Therapeutic Treatment of Cancer S-Klotho is believed to inhibit basal Wnt signaling activity, thereby functioning as a tumor suppressor in colorectal cancer (CRC). In addition, Klotho gene variants associated with lifespan differences may suppress butyrate-mediated Wnt hyperactivation, thus increasing the risk of CRC. In this manner, it has been hypothesized that the type of Klotho variant present and its relative expression may interact with dietary-derived butyrate levels to modify CRC risk. In addition, mTOR signaling is also associated with human aging, and cross-talk between Wnt and mTOR signaling may affect colon tumorigenesis.

KL-VS variants or other constructs are used to determine which SNPs (eg, within KL-VS) affect S-Klotho to reduce basal Wnt signaling and/or butyrate-mediated Wnt hyperactivation. It can serve as a vehicle to examine whether it is a factor that causes suppression, the latter being a Wnt-associated activity associated with S-Klotho tumor suppression. Embodiments include modifying appropriate amino acids (eg, KL-VS stretch of S-Klotho) that have been shown to affect the tumor suppressor action of KL-VS variants. One embodiment of the present disclosure includes a recombinant S-Klotho protein with one or more amino acid changes in the KL-VS stretch of 6 SNPs. Proteins can be produced and/or expressed in the context of any of the protein constructs described herein. For example, the protein is S-Klotho 1-981, 29-981, 34-981, 36-981, 131-981, 1-549, 29-549, with or without Fc fusions and/or TEV-TwinStrep. , 34-549, 36-549, 131-549, etc. Thus, the nucleic acid construct or cDNA from which the protein is expressed can be of corresponding length.

Embodiments include producing heterozygous or homozygous variant constructs and transferring the resulting constructs encoding S-Klotho protein (eg, via transfection) into a suitable expression system (eg, CHO cells). ) and/or transiently expressing the S-Klotho protein. Klotho proteins can be expressed at higher levels than other Klotho proteins, including wild-type. Embodiments may include purifying (and optionally quality control testing) the expressed protein for therapeutic administration. Embodiments administer a therapeutic or therapeutically effective amount of S-Klotho protein to a subject in need thereof (e.g., a patient having or at risk of developing colorectal cancer (CRC) or another tumor) can include doing A subject can, for example, carry or express a Klotho variant with reduced Wnt inhibitory activity. Alternatively, the subject may be of other mutant or variant wild type. Administration of recombinant S-Klotho protein can lead to beneficial increases in blood S-Klotho levels.

Therapeutic Treatment of Age-Related Conditions Investigators have discovered associations between Klotho and biological parameters that are generally accepted as indicators of clinical condition in hospitalized elderly patients. The investigators genotyped the single nucleotide polymorphisms (SNPs) rs9536314, rs1207568, and rs564481 at the KL locus in 594 elderly patients (ages 65-99) who were hospitalized, followed by consecutive geriatric visits and co-operative studies. Analysis of the dispersive and genetic risk score models was used to test the association of KL variants with these bioquantitative traits. Significant associations of rs9536314 with serum levels of hemoglobin, albumin, and high density lipoprotein cholesterol (HDL-C) and rs564481 with serum levels of hemoglobin, fasting insulin and fasting glucose were observed. was done. Gender analysis confirmed these associations, and while the associations between KL genotype and HDL-C, fasting glucose, and fasting insulin levels may be dominated by female gender, serum levels of hemoglobin and associations suggest that they may be dominated by male gender. An association between KL genotype and creatine levels was found in females, while an association between insulin-like growth factor-1 (IGF-1) and lymphocyte count (LC) was found in males. A genetic risk score (GRS) model further confirmed the significant association between the KL SNPs and hemoglobin, total cholesterol, and HDL-C. Gender analysis using the GRS-tagged approach confirmed associations with HDL-C, fasting glucose levels, and fasting insulin levels in females, and hemoglobin and LC in males. This finding suggests that the KL locus can affect quantitative traits such as serum levels of lipids, fasting glucose, albumin, and hemoglobin in hospitalized elderly patients, with creatine, IGF-1 levels, and some sex differences in LC. has been suggested, thus suggesting that one of the genetic factors may contribute to age-related diseases and longevity.

One embodiment of the disclosure includes the S-Klotho protein described herein. Embodiments involve producing a suitable Klotho construct, introducing the construct encoding the S-Klotho protein (eg, via transfection) into a suitable expression system (eg, CHO cells), and/ or transiently expressing the S-Klotho protein. Embodiments may include purifying (and optionally quality control testing) the expressed protein for therapeutic administration. Embodiments provide a therapeutic or therapeutically effective amount of S-Klotho protein to a subject in need thereof (eg, an individual or patient, optionally elderly and/or age-related conditions, low endogenous individuals or patients suffering from S-Klotho protein expression, and/or age-related conditions, or symptoms of decreased lifespan). Administration of recombinant S-Klotho protein can lead to beneficial increases in blood S-Klotho levels. Quantitative traits such as serum levels of total cholesterol, HDL-C, fasting glucose, fasting insulin, albumin, creatine, IGF-1, hemoglobin, and lymphocyte counts (e.g., in hospitalized and/or geriatric patients) In some active therapeutic modalities, administration may reverse or inhibit age-related conditions and/or adverse effects.

In one or more of the treatment methods described herein, the individual to be treated can have a mutation in the Klotho gene (e.g., a genomically encoded heterozygous or homozygous mutation), and the treatment regimen is 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 mutations expressed by an individual, for example administering the Alternatively, the individual to be treated may encode/express wild-type Klotho, and the treatment regimen is a therapeutic regimen comprising wild-type Klotho and/or any one or more variants of Klotho disclosed herein. It can include administering a dosage of the peptide. In some embodiments, the method of treatment reduces the level of circulating and/or cell-bound Klotho (e.g., compared to a control group), regardless of whether the native wild-type or mutant form of Klotho expressed by the individual. and administering a therapeutic dosage that operatively restores the concentration of circulating and/or cell-bound Klotho protein to at least homeostatic levels. In some embodiments, this may involve measuring levels of Klotho (eg, gene expression levels, protein expression levels, circulating levels, etc.) prior to administration of the therapeutic concentrate. In addition, therapeutic dosages may depend on the level of Klotho measured in an individual. In some embodiments, the therapeutic dosage is, for example, a scalar multiple of the homeostatic level (e.g., 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7 times more, 8 times more, 9 times more, 10 times more, 15 times more, 20 times more, 25 times more, 30 times more, 40 times more, 50 times more, 75 times more, 100 times more, 500 times more , 1,000 times more, 10,000 times more, etc.).

More specifically, it should be understood that therapeutic treatment of age-related conditions may involve administering therapeutic concentrations of one or more Klotho variants. In some embodiments, this is a therapeutic concentration of a Klotho variant disclosed herein (or Klotho treatment of the patient with a combination of variants). It should be understood that an age-related condition can be treated with a Klotho variant that is the same as or different from the Klotho variant expressed and/or encoded by an individual with 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 Klotho, and the individual may be a wild-type and/or mutant Klotho. The protein may be expressed at constitutive levels (compared to control groups), below constitutive levels, or not expressed at all. A therapeutic regimen aimed at treating age-related conditions in an individual can include administering one or more Klotho variants disclosed herein.

Prophylactic S-Klotho Administration In addition to the foregoing, embodiments of the present disclosure provide therapeutic or therapeutically effective amounts of S-Klotho protein to those in need thereof for prophylactic purposes and/or for the maintenance of certain health attributes. administering to an individual or subject. For example, administration of certain S-Klotho proteins can help maintain youthfulness in optionally aging patients who are not yet suffering from a diagnosed age-related condition. Accordingly, while certain embodiments of the present disclosure may relate to and/or involve treatment of a condition in a patient, other embodiments may involve the prevention, inhibition of development, and treatment of one or more conditions. /or may relate to and/or include prophylactic measures. For example, S-Klotho can be administered to people with inherited disorders who have mutations in one or more Klotho genes.

Example 1
Table 1 shows the results of expression and purification of the listed Klotho variants in HEK and/or CHO cell lines. For the results provided in Table 1 below, the following simplified protocol was followed.

For Fc fusion proteins, the protein expression vector was transformed into HEK293. sus or CHO. Briefly, cells were grown for 7 days and harvested. Cell numbers are given in the notes column. Supernatant pH was adjusted with 1M Hepes pH 7.4 and sodium azide was added. Proteins were captured using KanCap A resin. The resin was washed with PBS. The resin was washed with PBS plus 1M NaCl. The resin was washed with PBS. Proteins were eluted with 50 mM citrate pH 3.5, 100 mM NaCl. Proteins were immediately neutralized with 1 M Tris pH 8, 0.5 M Arginine. SDS PAGE gel samples were removed at this stage. Proteins were buffer exchanged into PBS. Proteins were quantified by OD280 and calculated extinction coefficients were used to measure quantity and concentration. Purity and molecular weight were determined using reducing and non-reducing SDS-PAGE (Biorad standard Tris/glycine/SDS, 4-20%). Aggregation status was determined 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 quick freezing in liquid nitrogen, the protein was shipped in aliquots. Note that for Fc-tagged proteins, as in previous purification rounds, protein loss was observed during desalting into PBS, as measured from samples run on SDS-PAGE before and after purification. Should. These proteins were expressed from HPLC but encountered problems during desalting or assay on silica HPLC columns in PBS. Therefore, buffer selection can be optimized.

For Strep-tagged proteins, protein expression vectors were transfected into HEK293. sus or CHO. Briefly, cells were grown for 7 days and harvested. Supernatant pH was adjusted with 1M Hepes pH 7.4 and sodium azide was added. BioLock Biotin Sequestration Reagent was added. Proteins were captured using StrepTactin superflow resin. The resin was washed with 100 mM Tris pH 8, 150 mM NaCl, 1 mM EDTA. Proteins were eluted with 100 mM Tris pH 8, 150 mM NaCl, 1 mM EDTA plus 2.5 mM desthiobin. Proteins were quantified by OD280 and calculated extinction coefficients were used to measure quantity and concentration. Purity and molecular weight were determined using reducing and non-reducing SDS-PAGE (Biorad standard Tris/glycine/SDS, 4-20%). Aggregation status was determined 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 quick freezing in liquid nitrogen, the protein was shipped in aliquots. Note that for Strep-tagged proteins, samples were assayed in elution buffer. This elution buffer is very "neutral" and non-toxic to proteins. Also, since the running buffer for the SEC column was PBS, the protein underwent a buffer exchange during the assay. Absorbance at times greater than 7 minutes are small molecules.

Expression and/or purification of the Klotho variants disclosed in Table 1, in addition to one or more other Klotho variants disclosed herein but not shown in Table 1, may be used in some embodiments. provides advantages over the expression and/or purification of native Klotho. For example, expressing and/or purifying a Klotho variant can reduce the number and/or type of surrogate products. Additionally or alternatively, the expression level of the desired Klotho variant may be increased compared to the expression level of native Klotho. In addition, or alternatively, the desired Klotho variant is purified under comparable conditions and methods (e.g., the concentration of the desired Klotho variant is increased with a concomitant decrease in expressed and/or purified by-products). expressed and/or purified in a more pure form.

Example 2
The following example includes an exemplary series of claims that define the scope of the disclosed invention. As provided herein, however, the scope of the invention is indicated by the appended claims rather than by the following examples or foregoing description.

1. An exemplary method of producing a recombinant Klotho protein in Chinese Hamster Ovary (CHO) cells, preferably in dihydrofolate reductase (DHFR) deficient CHO cells, more preferably in CHO-S cells, or preferably producing a recombinant Klotho protein in glutamine synthetase (GS)-deficient CHO cells, more preferably in GS-/- CHO cells, wherein the protein is preferably one of SEQ ID NO: 2 to SEQ ID NO: 70 having at least 85% amino acid sequence identity with one.

2. 2. The method of claim 1, wherein said protein comprises one or more glycans attached thereto.
3. optionally, said CHO cell is conjugated with 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 NO:2 to SEQ ID NO:70; , a functional dihydrofolate reductive (DHFR) enzyme, or a functional glutamine synthase (GS) enzyme to produce said recombinant Klotho protein expressing said polypeptide encoded by said nucleic acid 3. The method of claim 1 or 2, comprising:

4. introducing said exogenous nucleic acid into said CHO cells, preferably via transfection, and growing said CHO cells in liquid medium, preferably serum-free and/or animal protein component free medium, , said liquid medium preferably comprises a carbon source, a nitrogen source and one or more vitamins, minerals, salts, amino acids, supplements or additives, more preferably said liquid medium comprises hypoxanthine, thymidine, and/or growing without glutamine.

5. The protein preferably reaches a concentration of 200-500 mg protein, more preferably 500-2000 mg protein, even more preferably 2000-5000 mg protein per liter of liquid medium without concentrating the protein. 5. The method of claim 4, wherein up to a concentration of is secreted from said CHO cells into said liquid medium.

6. 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; 5. The method of claim 4.

7. further comprising selecting a suspension culture of viable CHO cells growing in said liquid medium, wherein said protein concentration in said medium of said selected suspension culture is at least 5. The method of claim 4, wherein 200 mg/L, preferably at least 500 mg/L, more preferably at least 1000 mg/L, even more preferably at least 2000 mg/L, even more preferably at least 5000 mg/L.

8. The viable CHO cells of the selected suspension culture are at least about 2-10 copies, preferably at least about 10-20 copies, more preferably at least about 20-30 copies, even more preferably at least about 8. The method of claim 7, containing 30-50 copies of said exogenous nucleic acid.

9. further comprising purifying the recombinant Klotho protein-containing extract from said CHO cells, the liquid medium, or both, wherein said extract preferably comprises at least about 98% protein by dry weight, and/or about 5. The method of claim 4, comprising less than 1-100 ppm CHO host cell protein (HCP).

10. 10. The method of claim 9, wherein purifying said extract maintains glycosylation of said protein.
11. growing said CHO cells in at least 10 liters, preferably at least 25 liters, more preferably at least 50 liters, even more preferably at least 100 liters, even more preferably at least 250 liters, even more preferably at least 500 liters; Even more preferably at least 1,000 liters, even more preferably at least 2,000 liters, even more preferably at least 2,500 liters, even more preferably at least 5,000 liters, even more preferably at least 10,000 liters 5. The method of claim 4, comprising culturing said CHO cells in a bioreactor having a volume or working volume.

12. preferably at least 85%, more preferably at least 90%, even more preferably at least 95%, even more preferably at least 98%, even more preferably A method according to any one of claims 1 to 11, comprising a transgene or cDNA with at least 99%, most preferably 100% nucleic acid sequence identity.

13. The protein is at least 90%, preferably at least 95%, more preferably at least 98%, even more preferably at least 99%, most preferably 100% of the amino acid sequence of one of SEQ ID NOs: 2-70 13. The method of any one of claims 1-12 having identity.

14. 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. A cell line comprising Hamster Ovary (CHO) cells, said CHO cells comprising a promoter, preferably a strong promoter, and a polypeptide, wherein at least a portion of said polypeptide comprises SEQ ID NO: 2 to SEQ ID NO: 70 and optionally a functional dihydrofolate reductase (DHFR) enzyme or a functional glutamine synthase (GS) enzyme. A cell line containing a human nucleic acid.

15. The CHO cells carry at least about 2-10 copies, preferably at least about 10-20 copies, more preferably at least about 20-30 copies, even more preferably at least about 30-50 copies per cell of the exogenous nucleic acid. 15. A cell line according to claim 14 which contains or is selected to contain.

16. The nucleic acid is at least 90%, preferably at least 95%, more preferably at least 98%, even more preferably at least 99%, most preferably 100% of the amino acid sequence of one of SEQ ID NOs: 2-70 15. The cell line of claim 14, encoding a polypeptide of identity.

17. preferably at least 85%, more preferably at least 90%, even more preferably at least 95%, even more preferably at least 98%, even more preferably 15. A cell line according to claim 14, comprising a transgene or cDNA with at least 99%, most preferably 100% nucleic acid sequence identity.

18. A liquid medium, preferably serum-free and/or free of animal protein components, preferably containing a carbon source, a nitrogen source, and one or more vitamins, minerals, salts, amino acids, supplements, or additives. and more preferably a liquid medium lacking hypoxanthine, thymidine and/or glutamine, and in said liquid medium such that said CHO cells express said polypeptide encoded by said nucleic acid. and the cell line of any one of claims 14-17, wherein said polypeptide comprises a recombinant Klotho protein.

19. Preferably, the CHO cells can be grown to a concentration of 200-500 mg protein, more preferably 500-2000 mg protein, even more preferably 2000-5000 mg protein per liter of liquid medium without concentrating the protein. The protein is secreted into the liquid medium to a concentration of protein and/or the protein is secreted into the liquid medium at a concentration of 200-500 mg protein per liter of liquid medium, more preferably 500-500 mg, without concentrating the protein. 19. A suspension cell culture according to claim 18, present in said liquid medium up to a concentration of 2000 mg protein, even more preferably up to a concentration of 2000-5000 mg protein.

20. 20. The suspension cell culture of claim 18 or 19, wherein said protein comprises one or more glycans attached thereto.
21. 18. Claim 18, wherein said 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. 21. The suspension cell culture of any one of paragraphs 1-20.

22. the protein is at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 98%, even more preferably at least 99% with one of SEQ ID NO: 2 to SEQ ID NO: 70; The suspension cell culture of any one of claims 18-21, most preferably having 100% amino acid sequence identity.

23. A recombinant Klotho protein, wherein at least a portion of said protein has at least 80% amino acid sequence identity with one of SEQ ID NO:2-SEQ ID NO:70.
24. The protein modulates the IGF-1 and/or Wnt signaling pathway, exhibits β-glucuronidase and/or sialidase activity, inhibits the p53/p21 signaling pathway, and/or, preferably, the p53/p21 signaling pathway. 24. The recombinant protein of claim 23, which, through inhibition, reduces H2O2-induced cellular senescence and apoptosis.

25. Said protein preferably modulates oxidative stress, growth factor signaling, regulation of ion homeostasis and/or said cell surface, preferably one or more ion channel proteins and/or growth factor receptors, preferably insulin/insulin-like proliferation 25. A recombinant Klotho protein according to claims 23 or 24, which functions as a humoral factor, preferably exhibiting pleiotropic activity, in modulating the activity of one or more glycoproteins on the factor-1 receptor.

26. at least a portion of said protein is at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 98%, even more preferably at least A recombinant Klotho protein according to any one of claims 23 to 25, having an amino acid sequence identity of 99%, most preferably 100%.

27. 27. A method of treating age-related or other conditions, diseases or disorders comprising administering to a subject in need thereof a pharmaceutically effective amount of the combination of any one of claims 23-26. A method comprising administering a recombinant Klotho protein.

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

29. A method of treating age-related or other conditions, diseases, or disorders comprising administering to a subject in need thereof a pharmaceutically effective amount of one of SEQ ID NOs: 2-70 and at least A method comprising administering a soluble recombinant Klotho protein having 80% amino acid sequence identity.

30. the protein is at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 98%, even more preferably at least 99% with one of SEQ ID NO: 2 to SEQ ID NO: 70; A method according to any one of claims 27-29, most preferably having 100% amino acid sequence identity.

31. The pharmaceutically effective amount increases the concentration of the serum soluble Klotho protein in the subject to a predetermined level, and preferably increases the concentration of the serum soluble Klotho protein in the subject above a predetermined threshold for a predetermined period of time 31. The method of claim 30, wherein maintaining concentration is sufficient.

32. 32. The method of claim 31, wherein said predetermined level is about 1000 picograms or more of soluble Klotho protein per milliliter of serum.
33. the predetermined level is about 50, 100, 250, 500, 750, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750, 3000, 3500, 4000, 4500, 5000, 5500 per milliliter of serum; 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 greater than or between 40,000, 50,000, 75,000, or 100,000 picograms of soluble Klotho protein and/or about 5%, 10% above typical healthy levels of soluble Klotho protein in serum %, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 400%, 32. 500%, 600%, 700%, 800%, 900%, 1000%, 1200%, 1500%, 2000%, 2500%, 3000%, 4000%, or 5000% or more, or higher therebetween. the method of.

34. measuring the serum soluble Klotho protein concentration of the subject; and calculating a pharmaceutically effective amount of protein sufficient to raise the serum soluble Klotho protein concentration of the subject to a first predetermined level. calculating that said first predetermined level is preferably greater than or equal to about 1000 picograms of soluble Klotho protein per milliliter of serum; measuring the metabolic rate; calculating the next dosing time at which the subject's serum soluble Klotho protein concentration is at or below a second predetermined level based on the measured rate; calculating a next dosage of said protein sufficient to raise said serum soluble Klotho protein concentration from said second predetermined level to said first predetermined level. 32. The method of claim 31, comprising:

35. 35. The method of claim 34, further comprising administering said next dosage of said protein to said subject.
36. Introducing an exogenous nucleic acid into Chinese Hamster Ovary (CHO) cells, preferably via transfection, said nucleic acid preferably comprising a transgene or cDNA, said nucleic acid comprising the sequence at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 98%, even more preferably at least 99%, most preferably 100% with one of No. 2 to SEQ ID No. 70 Preferably, the nucleic acid encodes a polypeptide with amino acid sequence identity, preferably at least 85%, more preferably at least 90%, even more preferably at least 95%, even more preferably at least 98%, even more preferably at least having 99%, most preferably 100% nucleic acid sequence identity with one of SEQ ID NO: 76 to SEQ ID NO: 96; Growing in a liquid medium that is free of biological protein components, which liquid medium preferably contains a carbon source, a nitrogen source, and one or more vitamins, minerals, salts, amino acids, supplements, or additives. more preferably said liquid medium lacks hypoxanthine, thymidine and/or glutamine and said CHO cells are preferably dihydrofolate reductase (DHFR) deficient CHO cells, more preferably in CHO-S cells or in glutamine synthetase (GS)-deficient CHO cells, more preferably GS-/- CHO cells, and an effective amount of methotrexate (MTX) and/or methionine sulfoximine (MSX), preferably is introduced into the liquid medium to a concentration of about 1 nM to 1 μM, more preferably to a concentration of about 10-100 nM, and a suspension culture of viable CHO cells growing in the liquid medium is selected through a cell selection process. wherein the concentration of said protein in said medium of said selected suspension culture is at least 200 mg/L, preferably at least 500 mg/L, more preferably at least 1000 mg/L without concentrating said protein. L, even more preferably at least 2000 mg/L, even more preferably at least 5000 mg/L, and producing said recombinant soluble Klotho protein in said CHO cells, wherein said protein is Preferably secreted from said CHO cells into said liquid medium, preferably to a protein concentration of 200-500 mg, more preferably 500-2000 mg, even more preferably 2000-5000 mg protein per liter of liquid medium. and purifying a recombinant soluble Klotho protein-containing extract from the CHO cells, the liquid medium, or both, wherein the extract preferably contains at least about 98% by dry weight. % of the recombinant soluble Klotho protein, and/or less than about 1-100 ppm CHO host cell proteins (HCPs), and purifying the extract preferably maintains the glycosylation of the protein, 32. The method of claim 31, further comprising one or more of purifying, wherein the protein has one or more glycans attached thereto.

37. The predetermined time 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, even more preferably at least about 30 hours, even more preferably at least about 36 hours, even more preferably at least about 42 hours, even more preferably at least about 48 hours, even more preferably at least about 54 hours, even more preferably at least about 60 hours, even more preferably at least about 66 hours, even more 32. The method of claim 31, preferably at least about 72 hours.

38. 32. The method of claim 31, wherein said predetermined period of time is about 1-120 days or more.
39. 32. The method of claim 31, wherein said predetermined period of time is about 6 months, 9 months, or 1 year or more.

40. 40. The method of any one of claims 30-39, wherein said subject is a human, non-human animal, or non-human mammal.
41. 41. The method of any one of claims 30-40, wherein said protein is administered in or with a pharmaceutically acceptable carrier.

42. The age-related or other condition, disease or disorder is frailty, low bone density, low bone mineral density, weight loss, muscle atrophy, muscle wasting, muscle mass, muscle weakness, grip strength, leg weakness , decreased physical strength, decreased movement, decreased freedom of movement, decreased quality of life evaluation, decreased ejection fraction, decreased motor ability, decreased learning ability, decreased learning ability, decreased memory, decreased IQ, decreased cognitive ability, amnesia, 42. The method of any one of claims 30-41, comprising one or more of cognitive decline, cognitive decline, synaptic plasticity decline, synaptic dysfunction, and cellular senescence.

43. The age-related or other condition, disease, or disorder is chronic kidney disease (CKD), polycystic kidney disease (PKD), autosomal dominant polycystic kidney disease (ADPKD), acute kidney injury (AKI), acute renal tubular necrosis (ATN), acute allergic interstitial nephritis (AAIN), glomerulonephritis, renal disease, renal failure, nonoliguric renal failure, alcoholism, hyperphosphatemia, muscular dystrophy (MS), Type 1 diabetes, type 2 diabetes, cardiovascular disease (CVD), cardiovascular calcification, cerebrovascular insufficiency, vascular calcification, ischemic heart disease, blood pressure abnormality, salt-sensitive hypertension, tissue calcification, calcified atherosclerotic plaque burden, calcification, familial neoplastic calcification, cancer, one or more tumors, myelin-related disease, demyelinating disease, neurodegenerative disease, neurovascular disease, progressive supranuclear palsy (PSP), Pompe disease , Niemann-Pick disease, microgliosis, Farber's disease (FD), bone mass disease, 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), exercise Neuronal disease (MND), atrial fibrillation, chronic obstructive pulmonary disease (COPD), fibromyalgia, adult-onset diabetes, arthritis, rheumatoid arthritis, osteoarthritis, glaucoma, cataracts, macular degeneration, multiple sclerosis ( MS), lupus, ulcerative colitis, cachexia, obesity, vitamin D-related conditions, bone disease, bone remodeling-mediated bone disease, stem cell depletion, seasickness, space maladaptation syndrome (SAS), nausea, and dizziness.

44. The method of any one of claims 30-43, further comprising administering or co-administering one or more additional active ingredients.
45. 45. The method of claim 44, wherein said 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.

46. A pharmaceutical composition comprising a pharmaceutically effective amount of the recombinant Klotho protein of any one of claims 23-25 and a pharmaceutically acceptable carrier.
47. a pharmaceutically effective amount of a recombinant soluble Klotho protein, wherein at least a portion of said protein is human alpha Klotho isoform 1 1-981, 29-981, 34-981, 36-981, 131-981, 1- at least 85% amino acid sequence identity with at least a subset of amino acid residues 549, 29-549, 34-549, 36-549, or 131-549, or at least a portion of one of SEQ ID NOs:2-70 A pharmaceutical composition comprising the protein having a specific property and a pharmaceutically acceptable carrier.

48. at least a portion of said protein is at least 85%, preferably at least 88%, more preferably at least 90%, even more preferably at least 92%, and even more preferably at least a portion of one of SEQ ID NOs: 2-70 48. A pharmaceutical composition according to claim 46 or 47, preferably having at least 95%, even more preferably at least 98%, even more preferably at least 99%, most preferably 100% amino acid sequence identity.

49. The pharmaceutical composition of any one of claims 46-48, further comprising one or more additional active ingredients.
50. Weakness, decreased bone density, decreased bone mineral density, weight loss, muscle atrophy, muscle atrophy, 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 evaluation , Decreased ejection fraction, Decreased motor capacity, Decreased learning ability, Decreased learning ability, Decreased memory, Decreased intelligence quotient, Cognitive deterioration, Amnesia, Decreased cognitive ability, Decreased cognitive function, Decreased synaptic plasticity, Decreased synaptic function, Cell aging, chronic kidney disease (CKD), polycystic kidney disease (PKD), autosomal dominant polycystic kidney disease (ADPKD), acute kidney injury (AKI), acute tubular necrosis (ATN), acute allergic interstitial nephritis ( AAIN), glomerulonephritis, renal disease, renal failure, nonoliguric renal failure, alcoholism, hyperphosphatemia, muscular dystrophy (MS), type 1 diabetes, type 2 diabetes, cardiovascular disease (CVD), Cardiovascular calcification, cerebrovascular insufficiency, vascular calcification, ischemic heart disease, abnormal blood pressure, salt-sensitive hypertension, tissue calcification, calcified atherosclerotic plaque burden, calcification, familial neoplastic calcification, cancer , one or more tumors, myelin-related disease, demyelinating disease, neurodegenerative disease, neurovascular disease, progressive supranuclear palsy (PSP), Pompe disease, Niemann-Pick disease, microgliosis, Farber disease (FD) , bone mass disease, osteoporosis, osteopenia, osteopenia (especially loss of cortical BMD), emphysema, pulmonary fibrosis, skin atrophy, thymic atrophy, accumulation of renal interstitial matrix, glomerulosclerosis, anemia, albumin Urinary disease, 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 disease (COPD), fibromyalgia, adult-onset diabetes, arthritis, rheumatoid arthritis, osteoarthritis, glaucoma, cataracts, macular degeneration, multiple sclerosis (MS), lupus, ulcerative colitis, cachexia, Aging, including one or more of obesity, vitamin D-related conditions, bone disease, bone remodeling-mediated bone disease, stem cell depletion, seasickness, space maladaptation syndrome (SAS), nausea, and dizziness. 50. A pharmaceutical composition according to any one of claims 46-49 for use in the treatment of conditions, diseases or disorders associated with or other conditions, diseases or disorders.

51. A pharmaceutical composition according to any one of claims 46 to 49 for use in treating or preventing acute kidney injury (AKI).
52. A method of treating or preventing acute kidney injury (AKI) or other condition comprising administering to a subject in need thereof a pharmaceutically effective amount of a recombinant soluble Klotho protein, comprising at least a portion of human alpha klotho isoform 1 1-981, 29-981, 34-981, 36-981, 131-981, 1-549, 29-549, 34-549, 36-549, or 131-549 or at least a portion of one of SEQ ID NOS: 2-70 with at least 85%, 86%, 88%, 90%, 92%, 95%, 98%, 99%, Or preferably having 100% amino acid sequence identity.

53. 53. The method of claim 52, further comprising co-administering one or more additional active ingredients with said pharmaceutically effective amount of recombinant soluble Klotho protein.
54. 54. The method of claim 53, wherein said protein and one or more additional active ingredients are formulated into a combination product or composition.

55. 54. The method of claim 53, wherein said protein and one or more additional active ingredients are separate compositions.
56. 54. The method of claim 53, wherein said protein and one or more additional active ingredients are mixed.

57. 54. A method according to claim 53, wherein said protein and one or more additional active ingredients are configured for co-administration, co-administration comprising simultaneous administration or separate administration, preferably separated by a period of time. .

58. 54. The method of Claim 53, wherein said one or more additional active ingredients are selected from the group consisting of drugs, antibodies, hormones, imaging agents, pharmaceuticals, or compositions.
59. The condition is acute tubular necrosis (ATN), nephritis, acute allergic interstitial nephritis (AAIN), glomerulonephritis and/or nephrotoxicity, or kidney transplant or other surgery, acute tubular necrosis (ATN) 54. The method of claim 52 or 53, comprising AKI resulting at least in part from , nephritis, acute allergic interstitial nephritis (AAIN), glomerulonephritis, nephrotoxicity, or hypotension.

60. 54. The method of claim 52 or 53, wherein said nephrotoxicity comprises drug-induced nephrotoxicity.
61. 61. The method of claim 60, wherein said drug-induced nephrotoxicity comprises antibacterial agent-induced nephrotoxicity.

62. 61. The method of claim 60, wherein said drug-induced nephrotoxicity comprises aminoglycoside-induced nephrotoxicity.
63. wherein said administering comprises measuring a serum soluble klotho level in said subject; and a first dosage sufficient to raise said serum soluble klotho level in said subject to a predetermined level or percent of normal level and administering to said subject a first dose of said protein, preferably by bolus or stepwise administration, more preferably by injection, preferably said first dose of measuring the rate of soluble klotho reduction in the serum of the subject following administration; calculating the next dosage time and/or amount of the protein; and according to the calculated time and/or amount 54. The method of claim 52 or 53, comprising one or more steps selected from the group consisting of: administering the next dosage of said protein to said subject.

64. 52. wherein said administering step is sufficient to raise and/or maintain said subject's serum soluble Klotho protein concentration above a predetermined level or threshold, optionally for a predetermined period of time, or 53. The method according to 53.

65. 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 per milliliter of serum , 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 and/or between soluble Klotho protein, or 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% above typical healthy levels of soluble Klotho protein.

66. 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 65 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. the method of.

67. 54. The method of claim 52 or 53, wherein said protein is administered prophylactically prior to kidney transplantation or administration of nephrotoxin and/or subsequent to kidney transplantation or administration of nephrotoxin.
68. The nephrotoxin is preferably one or more aminoglycosides selected from the group consisting of paromomycin, tobramycin, gentamicin, amikacin, kanamycin and neomycin; fungal agents; preferably hyperosmotic media (HOCM) having an iodine to molecular ratio of about 1.5:1; hypotonic non-ionic contrast media (LOCM) having an iodine to molecular ratio of about 3:1; ), and an isotonic (iso-osmotic) contrast agent (IOCM) having an iodine to molecule ratio of about 6:1); preferably adefovir, cidofovir, one or more antiretroviral agents selected from the group consisting of tenofovir and foscanet; preferably 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 zoledronate/zoledronic acid, ibandronate, alendronate, alendrone one or more bisphosphonates or derivatives thereof selected from the group consisting of to/cholecalciferol, etidronate, risedronate, calcium carbonate risedronate, pamidronate, and tiludronate; and/or one preferably selected from the group consisting of cocaine and heroin. 68. The method of claim 67, comprising one or more anesthetics or opioids.

69. 54. A method according to claim 52 or 53, wherein said protein preferably does not contain F352V, more preferably comprises C370S containing F352, and/or variants other than H193 or H193R.

70. A method of treating an elderly individual, wherein the elderly individual has a homozygous or heterozygous mutation in the gene encoding the Klotho protein, wherein one of SEQ ID NO: 2 to SEQ ID NO: 70 and at least 85% , preferably at least 90%, more preferably at least 95%, even more preferably at least 98%, even more preferably at least 99%, and most preferably 100% amino acid sequence identity A method comprising:

71. 71. The method of claim 70, further comprising measuring the expression level of said gene.
72. 72. The method of claim 71, wherein said step of administering said therapeutic agent concentration is dependent on said expression level of said gene.

CONCLUSION While the foregoing detailed description refers to specific exemplary embodiments, the disclosure may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. Accordingly, the described embodiments are to 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 exemplified herein, and the Additional applications of the illustrated principles 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 intended to be within the scope of this disclosure.

The scope of the invention is, therefore, indicated by the appended claims rather than by the foregoing description. Limitations recited in the claims are to be interpreted broadly based on the language employed in the claims, and are not limited to the specific examples set forth in the foregoing Detailed Description, and are non-exclusive. and shall be construed as non-limiting. All changes that come within the range and meaning of the claims are to be embraced within their scope.

It will also be understood that various features of certain embodiments may be compatible, combined, included, and/or incorporated with other embodiments of the present disclosure. For example, systems, methods, and/or products according to certain embodiments of the present disclosure may include or incorporate features described in other embodiments disclosed and/or described herein. or may include Accordingly, disclosure of certain features in connection with particular embodiments of the present disclosure should not be construed as limiting the application or inclusion of such features to particular embodiments.

Additionally, features described in various embodiments are optional and may not be included in other embodiments of the present disclosure, unless a feature is stated to be required in a particular embodiment. Further, unless a feature is described as requiring another feature in combination with it, any feature herein may be combined with any other feature of the same or different embodiments disclosed herein. can be combined. In certain embodiments, features may be optional, but it is understood that when features are included in such embodiments, they may be required to have certain configurations as described in this disclosure. would be

Similarly, the steps recited in any method or process described herein and/or recited in the claims may be performed in any suitable order, particularly (whether express or implied) ) are not necessarily limited to the order in which they are described and/or listed, unless 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.

Moreover, various well-known aspects of exemplary systems, methods, products, etc. are not described in detail herein to avoid obscuring aspects of the exemplary embodiments. However, such aspects are also contemplated herein.

Claims (10)

a cell line,
comprising a plurality of Chinese Hamster Ovary (CHO) cells, said CHO cells comprising an exogenous nucleic acid, said exogenous nucleic acid comprising a promoter and encoding a medically effective polypeptide, said polypeptide comprising:
an N-terminal signal peptide having at least 98% amino acid sequence identity with SEQ ID NO: 72;
a recombinant human alpha soluble Klotho protein having at least 98% amino acid sequence identity with SEQ ID NO: 25;
a C-terminal polypeptide tag having at least 98% amino acid sequence identity with one of SEQ ID NO: 74 or SEQ ID NO: 75, wherein said polypeptide, or recombinant human alpha soluble Klotho protein thereof, is effective for acute kidney injury, chronic kidney disease A cell line that is effective in treating a disease, or an age-related condition, disease, or disorder. The CHO cells are
a dihydrofolate reductase (DHFR)-deficient CHO cell, wherein said exogenous nucleic acid further encodes a functional dihydrofolate reductase (DHFR) enzyme; or a glutamine synthase (GS)-deficient CHO cell, wherein said exogenous the nucleic acid further encodes a functional glutamine synthase (GS) enzyme;
A cell line according to claim 1 . said N-terminal signal peptide has at least 99% amino acid sequence identity with SEQ ID NO: 72;
said recombinant human alpha soluble Klotho protein has at least 99% amino acid sequence identity with SEQ ID NO:25, and said C-terminal polypeptide tag has at least 99% amino acid sequence identity with one of SEQ ID NO:74 or SEQ ID NO:75 having sex
3. A cell line according to claim 1 or 2. The cell line of any one of claims 1-3, wherein said CHO cells contain or are selected to contain at least 10 copies of said exogenous nucleic acid per cell. A suspension cell culture,
a liquid medium, serum-free and/or a liquid medium free of animal protein components;
5. The cell line of any one of claims 1-4, wherein the CHO cells are grown in the liquid medium so as to express the polypeptide encoded by the nucleic acid, wherein the liquid medium contains carbon. source, nitrogen source, and one or more vitamins, minerals, salts, amino acids, supplements, or additives;
A suspension cell culture, wherein said polypeptide comprises a recombinant Klotho protein. The CHO cells are capable of enriching the polypeptide or protein to a concentration of 200-500 mg, 500-2000 mg, or 2000-5000 mg of the polypeptide or protein per liter of liquid medium without enriching the polypeptide or protein. 200-500 mg, 500-2000 mg, or 2000-5000 mg of polypeptide or protein per liter of liquid medium without secreting into the medium and/or said polypeptide or protein concentrating said polypeptide or protein 6. The suspension cell culture of claim 5, which is present in said liquid medium at a concentration of . 7. The suspension cell culture of claim 5 or claim 6, wherein said 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. A method for producing a recombinant Klotho protein, comprising:
A cell line according to any one of claims 1 to 4 in a liquid medium free of serum and/or animal protein components, such that said CHO cells express said polypeptide encoded by said nucleic acid. growing, 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 the recombinant Klotho protein , a method comprising the step of propagating. The CHO cells are capable of enriching the polypeptide or protein to a concentration of 200-500 mg, 500-2000 mg, or 2000-5000 mg of the polypeptide or protein per liter of liquid medium without enriching the polypeptide or protein. 200-500 mg, 500-2000 mg, or 2000-5000 mg of polypeptide or protein per liter of liquid medium without secreting into the medium and/or said polypeptide or protein concentrating said polypeptide or protein 9. The method of claim 8, wherein is present in the liquid medium at a concentration of 1. A recombinant Klotho protein for use in treating acute kidney injury, chronic kidney disease, or an age-related condition, disease, or disorder, comprising:
an N-terminal signal peptide having at least 98% amino acid sequence identity with SEQ ID NO: 72;
a recombinant human alpha soluble Klotho protein having at least 98% amino acid sequence identity with SEQ ID NO: 25;
a C-terminal polypeptide tag having at least 98% amino acid sequence identity with one of SEQ ID NO: 74 or SEQ ID NO: 75, wherein said polypeptide, or recombinant human alpha soluble Klotho protein thereof, is effective for acute kidney injury, chronic kidney disease A recombinant Klotho protein that is effective in treating a disease, or an age-related condition, disease, or disorder.