WO2020047505A1

WO2020047505A1 - Production and detection of bioactive soluble klotho protein

Info

Publication number: WO2020047505A1
Application number: PCT/US2019/049211
Authority: WO
Inventors: Christian FAUL; Christopher YANUCIL
Original assignee: The Uab Research Foundation
Priority date: 2018-08-30
Filing date: 2019-08-30
Publication date: 2020-03-05

Abstract

The present disclosure provides a screening assay that detects soluble klotho polypeptide in a sample. The described assay is sensitive and quantitative, can be used in a high-throughput format, and is cost-effective. The present disclosure also provides for specific uses of such an assay. Further, the present disclosure provides for kits comprising one or more components required for practicing the assay. Furthermore, the present disclosure provides a specific procedure for the synthesis and purification of bioactive recombinant sKL protein.

Description

PRODUCTION AND DETECTION OF BIOACTIVE SOLUBLE

KLOTHO PROTEIN

GOVERNMENT SUPPORT CLAUSE

This invention was made with government support under (F31DK115074) awarded by the National Institutes of Health. The government has certain rights in the invention. 37 CFR 401.14(f)(4).

FIELD OF THE DISCLOSURE

The present disclosure relates to methods of producing and detecting bioactive soluble proteins. More specifically, the present disclosure relates to methods of producing bioactive recombinant soluble klotho protein and methods of detecting bioactive soluble klotho protein in a sample.

BACKGROUND OF THE DISCLOSURE

Klotho is a hormone that protects tissues from stress and injury. Blood levels of klotho decline with age and are reduced in patients with chronic kidney disease (CKD) and other diseases and conditions. Animal studies suggest that the reduction in klotho levels causes organ damage that occurs in aging and CKD. Therefore, the administration of klotho, including recombinant klotho, has potential as a therapeutic agent in treatment of diseases and conditions in which levels of klotho are declining. For example, administration of klotho can be used to delay one or more aspects of the aging process or to treat CKD (including prolonging survival in CKD patients). However, there are currently no reliable tools to analyze the levels of klotho in a subject or to determine if recombinant klotho is biologically active and suitable for use in clinical studies and use as a therapeutic.

Therefore, the art is lacking methods to allow the therapeutic utilization of klotho, including methods for the production of recombinant, biologically active klotho and soluble klotho (sKL), methods to analyze the levels of klotho, including sKL, in a subject, and methods to determine if klotho, including recombinant klotho and recombinant sKL, is biologically active. The present disclosure provides a solution to these unmet needs in the art.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A shows an overview of one embodiment of the sKL detection assay.

FIG. 1B shows an overview of another embodiment of the sKL detection assay.

FIG. 2 shows a standard curve generated using the sKL detection assay.

FIG. 3A shows a comparison of the detection of sKL from various sources using the sKL detection assay.

FIG. 3B shows the sKL detection assay disclosed is capable of detecting elevated sKL levels in mouse serum.

FIG. 4A shows the amino acid sequence of SEQ ID NO: 1 (human FGFR1).

FIG. 4B shows the amino acid sequence of SEQ ID NO: 2 (human FGFR2).

FIG. 4C shows the amino acid sequence of SEQ ID NO: 3 (human FGFR3).

FIG. 4D shows the amino acid sequence of SEQ ID NO: 4 (human FGFR4).

FIG. 4E shows the amino acid sequence of SEQ ID NO: 5 (human FGF23).

FIG. 5 shows the domain structure of the synthesized and purified recombinant sKL protein.

DETAILED DESCRIPTION

Definitions

As used herein, the term“about” is used herein to mean approximately, roughly, around, or in the region of. When the term“about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term“about” is used herein to modify a numerical value above and below the stated value by a variance of 10 percent up or down (higher or lower).

As used herein, the term“chronic kidney disease” or“CKD” includes conditions that damage the kidneys and decrease the ability of the kidneys to function normally and keep a subject healthy. The kidneys function to clear waste products from the blood. When kidney function is impaired, the concentration of waste products increases, causing further damage to the kidneys, damage to other organs, and leads to the development of additional pathologies in the subject. Such additional pathologies include, but not limited to, high blood pressure, fluid retention, hyperkalemia, anemia (low blood count), weak bones, increased risk of bone fracture, erectile dysfunction, decreased fertility, decreased immune response, poor nutritional health, nerve damage, cardiac injury, systemic inflammation, heart disease, cardiac fibrosis, cardiac hypertrophy, fibrosis, and blood vessel disease. CKD may be caused, at least in part, by a number of conditions, including, but not limited to, diabetes (both Type I and Type II), high blood pressure, glomerulonephritis, interstitial nephritis, polycystic kidney disease, prolonged obstruction of the urinary tract (for exampl e, from conditions such as enlarged prostate, kidney stones and some cancers), vesicoureteral reflux, recurrent kidney infections (pyelonephritis) and other disorders. When CKD progresses, it may eventually lead to kidney failure, which requires dialysis or a kidney transplant to maintain life.

The term“therapeutically effective amount” denotes that amount of a compound of the disclosure that will elicit a therapeutic response of a subject that is being sought. The actual dose which comprises the therapeutically effective amount may depend upon the route of administration, the size and health of the subject, the disorder being treated, and the like. The therapeutically effective amount may be sufficient, for example, to reduce or ameliorate the severity and/or duration of a disease or condition, or one or more symptoms thereof, prevent the advancement of a disease or condition, prevent the recurrence, development, or onset of one or more symptoms associated with a disease or condition, enhance or otherwise improve the prophylactic or therapeutic effect(s) of another therapy for a disease or condition, or a combination of the foregoing. In certain embodiments, a therapeutically effective amount is an amount of the compound of the disclosure that avoids or substantially attenuates undesirable side effects. As used herein, the terms“subject” or“patient” means any mammal, including, but not limited to, humans, non-human primates, canines, felines, rodents, and the like, which is to be the recipient of a particular treatment. Typically, the term“subject” or“patient” is used in reference to a human.

As used herein, the term“sKL complex binding portion of FGFR” w th reference to any human or mouse FGFR refers to a sequen ce or portion of FGFR that binds a sKL complex containing FGF23, or a sKL binding portion of FGF23, and sKL. In certain embodiments, the ability of a“sKL complex binding portion of FGFR” to function as described is determined using the binding assay described in Example 1 where the first sKL binding agent is full length FGF23 of the same species as the“sKL complex binding portion of FGFR.”

As used herein, the term“sKL binding portion of FGF23” with reference to any human or mouse FGF23 refers to a sequence or portion of FGF23 that binds sKL. In certain embodiments, the ability of a“sKL binding portion of FGF23” to function as described is determined using the binding assay described in Example 1 where the second sKL binding agent is FGFRla(IlIC)-Fc of the same species as the“sKL binding portion of FGF23.”

As used herein, the terms“percent identity” or“percent identical”, when referring to a sequence, means that a sequence is compared to a claimed or described sequence after alignment of the sequence to be compared (the“Compared Sequence”) with the described or claimed sequence (the“Reference Sequence”). The percent identity is then determined according to the following formula:

percent identity = l00[l-(C/R)],

wherein C is the number of differences between the Reference Sequence and the Compared Sequence over the length of alignment between the Reference Sequence and the Compared Sequence, wherein (i) each base or amino acid in the Reference Sequence that does not have a corresponding aligned base or amino acid in the Compared Sequence and (ii) each gap in the Reference Sequence and (iii) each aligned base or amino acid in the Reference Sequence that is different from an aligned base or amino acid in the Compared Sequence, constitutes a difference and (iv) the alignment has to start at position 1 of the aligned sequences; and R is the number of bases or amino acids in the Reference Sequence over the length of the alignment with the Compared Sequence with any gap created in the Reference Sequence also being counted as a base or amino acid. If an alignment exists between the Compared Sequence and the Reference Sequence for which the percent identity as calculated above is about equal to or greater than a specified minimum Percent Identity then the Compared Sequence has the specified minimum percent identity to the Reference Sequence even though alignments may exist in which the herein above calculated percent identity is less than the specified percent identity.

As used herein, the term“at least 90% identical” includes sequences that range from 90 to 99.99% identity to the indicated sequences and includes all ranges in between. Thus, the term at least 90% identical thereto includes sequences that are 91, 91.5, 92, 92.5, 93, 93.5. 94, 94.5, 95, 95.5, 96, 96.5, 97, 97.5, 98, 98.5, 99, 99.5 percent identical to the indicated sequence.

As used herein, the term“klotho” as used herein refers specifically to a-Klotho (from any species, preferably human or mouse) unless otherwise indicated.

As used herein, the term“sKL” or“soluble klotho” refers to a form of klotho released from or secreted from a cell that enters the blood, urine or other bodily fluid, such as but not limited to, amino acids 1-981 of SEQ ID NO: 11, amino acids 1-982 of SEQ ID NO: 12, or SEQ ID NOS: 14 and 15. In a particular embodiment, the term“sKL” or“soluble klotho” refers to a form of klotho released from a cell that enters the blood, urine or other bodily fluid, such as but not limited to, amino acids 1-981 of SEQ ID NO: 11 or amino acids 1-982 of SEQ ID NO: 12.

As used herein, the terms“treating” or“treat” refer to improving a symptom of a disease or disorder and may comprise curing the disorder, substantially preventing the onset of the disorder, alleviating of one symptom or most of the symptoms resulting from that disorder, or improving the subject's condition. The terms refer to the full spectrum of treatments for a given disorder from which the subject is suffering. As used herein, the term“in need of treatment” as used herein refers to a judgment made by a healthcare professional that a subject requires or will benefit from treatment (including, but not limited to, the administration of a compound or composition disclosed herein). This judgment is made based on a variety of factors that are in the realm of a caregiver's expertise, but that includes the knowledge that the patient is ill, or will be ill, as the result of a disease or condition that is treatable by a method, compound, or composition of the disclosure. In a particular usage, the term“in need of treatment” indicates a patient has been diagnosed with CKD or a condition that causes, at least in part, CKD.

As used herein, the term“binding domain” means a group capable of interaction with a binding partner. Suitable binding domains are described herein.

As used herein, the term“binding partner” means a structure, molecule or substrate which interacts with a binding domain. The binding partner is not required to form a chemical bond with the binding domain. As one non-limiting example, when a binding domain is a magnetic moiety (such as a magnetic microsphere), the binding partner may be a magnet or other magnetic substrate. As another non-limiting example, when the binding domain is a biotin group (including biotin groups with reduced affinity such as desthiobiotin), the binding partner may be an avidin or streptavidin molecule. The interaction between a binding domain and its binding partner may be reversible under defined conditions. The interaction between a binding domain and its binding partner may be irreversible or maintained under defined conditions As used herein, the terms“detected,”“detection” and similar terms when referring to a method for determining the presence sKL refers to the detection of sKL in a sample through the formation of a complex between the first sKL binding agent, sKL, and the second sKL binding agent; such determining may be quantitative or non-quantitative.

Notwithstanding that the numerical ranges and parameters setting forth the broad scope of certain embodiments are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements. Moreover, all ranges disclosed herein are to be understood to encompass any and all subranges subsumed therein. For example, a stated range of“1 to 10” should be considered to include any and all subranges between (and inclusive of) the minimum value of 1 and the maximum value of 10; that is, all subranges beginning with a minimum value of 1 or more, for example 1 to 6.1, and ending with a maximum value of 10 or less, for example, 5.5 to 10

It is further noted that, as used in this specification, the singular forms“a,”“an,” and“the” include plural referents unless expressly and unequivocally limited to one referent. The term“or” is used interchangeably with the term "and/or" unless the context clearly indicates otherwise. Summary of the Disclosure

Based on biochemical characterization of klotho binding to target receptors, a detection assay to detect sKL, particularly biologically active sKL, in solution has been developed. Compared to currently available methods, the detection assay is more sensitive and provides for the more precise determination of sKL concentrations, including concentrations of biologically active sKL. Furthermore, the detection assay is suitable for use in a high- throughput format to screen multiple samples. Finally, the detection assay is cost-effective, allowing the routine use of the assay in a clinical setting. The disclosed detection assay can be used for a variety of purposes, including, but not limited to, the determination of the klotho concentration, including the concentration of sKL, in a subject and the determination of the bioactivity of klotho, including the bioactivity of sKL. Therefore, the disclosed detection assay is conveniently used to validate klotho protein, including recombinant sKL, for use in clinical studies, human treatment, and other purposes.

Using the disclosed detection assay, it is demonstrated that the currently available sKL protein which has been previously used in preclinical studies and other experiments is not bioactive. The present disclosure also provides improved methods for the production of recombinant sKL. Based on a crystal structure analysis of sKL protein, a method has been developed to synthesize and purify recombinant sKL protein. Compared to currently available sKL protein, the recombinant, sKL protein produced using the methods disclosed herein shows higher bioactivity. The use of recombinant sKL protein produced by the disclosed methods allows for the effective study of klotho in preclinical in vitro and in vivo studies and allows the use of sKL as a therapeutic agent for the treatment of human disease.

Introduction

The discovery of the Klotho (KL ) gene, which was originally identified as a putative aging-suppressor gene, has generated tremendous interest and has advanced understanding of the aging process. In mice, the overexpression of the KL gene extends the life span, whereas mutations to the KL gene shorten the life span. The human KL gene encodes the klotho protein, which is a multifunctional protein that regulates the metabolism of phosphate, calcium, and vitamin D. Klotho also may function as a hormone, although the klotho receptor(s) has not been found. Point mutations of the KL gene in humans are associated with hypertension and kidney disease, which suggests that klotho may be essential to the maintenance of normal renal function. Recent evidence suggests that klotho suppresses the insulin and Wnt signaling pathways, inhibits oxidative stress, and regulates phosphatase and calcium absorption.

Klotho is a single-pass transmembrane protein that increases the affinity of fibroblast growth factor receptors (FGFRs) for FGF23 binding. sKL can be generated by proteolytic cleavage of full-length klotho. The kidney appears to be the major source of sKL, and it has been reported that sKL can enter the circulation, cerebrospinal fluid, and the urinary space. In vitro studies have shown that sKL has pleiotropic cell-protective activities, suggesting that sKL acts as an endocrine factor that can target a variety of tissues.

CKD as well as aging have been described as a states of klotho deficiency. Low levels of klotho transcript and protein have been detected in human nephrectomy samples and in biopsies from patients with CKD, but tissue levels have limited clinical utility. While renal klotho levels seem to be uniformly reduced in CKD and in the elderly, sKL levels are extremely variable and sKL measurements have provided inconsistent results which is probably due to assay-related variance. Existing ELISA-based assays to measure sKL in solution rely on antibodies. However, currently available anti-klotho antibodies seem to be unspecific and cross- react with other proteins. A novel synthetic anti-klotho antibody, termed sbl06, has been shown to detect klotho in tissue as well as sKL in serum and urine following an immunoprecipitation/immunoblot (IP/IB) approach. However, this assay lacks sufficient sensitivity and is semi-quantitative as well as cost-intensive, and based on these limitations it is not suitable for high-throughput screens.

Animal studies indicate that the overexpression of klotho as well as the injection of recombinant sKL protein have tissue-protective effects and prolong survival in pathologic scenarios, such as CKD. Furthermore, overexpression of klotho also prolongs survival of healthy wild-type mice, indicating that sKL might act as an anti-aging hormone. sKL seems to be able to target several tissues and cell types and affect a variety of cellular functions. However, the molecular mechanisms underlying sKL’s pleiotropic actions are not known and a receptor for sKL has not been identified to date.

Klotho mRNA levels also are decreased in the distal convoluted tubules under several physiological and disease states, such as diabetic nephropathy and dehydration. Most factors that decrease klotho mRNA expression are associated with changes in the aging process, which suggests that klotho plays important roles in aging.

Furthermore, recombinant sKL is being produced for use as a drug based on its tissue- protective effects in CKD as well as a general anti-aging agent. However, based on the lack of mechanistic insights, the bioactivity of the recombinant sKL cannot be determined.

The art is lacking a reliable assay to detect levels of sKL in samples, including human samples. Such an assay is needed in order to fully assess the role of sKL in various disease process (such as, but not limited to, the potential of sKL as a biomarker and/or protective circulating factor).

Klotho. sKL. and secreted Klotho

Three klotho protein types with potentially different functions have been identified: a full- length transmembrane klotho, sKL, and a secreted klotho. The klotho gene is highly conserved in humans, mice, and rats and is also found in Danio rerio and Caenorhabditis elegans. The klotho protein is also highly homologous across species. In particular, the klotho protein sequence is 98% identical between humans and mice.

The amino acid sequence of human (SEQ ID NO: 11) and mouse (SEQ ID NO: 12) klotho are 98% identical. The membrane-bound full-length klotho protein can be cleaved by the membrane-anchored proteases ADAM10, ADAM17, and BACE1, resulting in sKL. The truncated klotho protein, which is also known as the sKL, is released from the cell membrane. After entering the urine and blood, sKL functions as a hormone. sKL from human contains amino acids 1-981 of SEQ ID NO: 11. sKL from mouse contains amino acids 1 to 982 of SEQ ID NO: 12. The sequence of secreted klotho from human and mouse is provided in SEQ ID NOS: 14 and

15, respectively.

The klotho detected by the methods of the present disclosure may be from any species expressing klotho. In preferred aspects, the klotho detected by the methods of the present disclosure is from human or mouse. Furthermore, any variant of klotho may be detected by the methods of the present disclosure, including but not limited to, sKL and secreted klotho. In a preferred aspect, sKL is detected by the methods of the present disclosure. Still further, a variant of sKL containing a naturally occurring mutation or a sKL polypeptide derivative may also be detected by the methods of the present disclosure. Such naturally occurring mutations are known to those in the art. Such naturally occurring variants for human sKL include, but are not limited to, P15Q, F45V, H193R, F352V, C370S, P514S, and P954L.

Methods of Detection In co-immunoprecipitations assay using purified recombinant mouse proteins, it was determined that FGF23 and sKL can bind to each other in the absence of an FGFR. Furthermore, it was shown sKL binds the ectodomain of different FGFR isoforms with different affinities, with highest binding affinity being determined for the FGFR isoform 1 (FGFR1). Based on these findings, an assay was developed to detect biologically active sKL by its ability to sequentially bind FGF23 and a FGFR.

In a general descripti on of the detection method disclosed, a sample containing (or suspected of containing) sKL is provided, a first sKL binding agent is provided (such as, but not limited to, FGF23, or a sKL binding portion of FGF23), and a second sKL binding agent (such as, but not limited to, FGFR and a sKL binding portion of FGFR) is provided that binds a complex of sKL and the first sKL binding agent, and the components are incubated together to form a complex comprising sKL and the first and second sKL binding agents. The presence of the complex is detected via a detectable label incorporated on, or specifically bound to, one of the first or the second sKL binding agents. In a preferred embodiment, the presence of sKL bound to the first sKL binding agent is required for the efficient binding of the second sKL binding agent to the first sKL complex. The higher the levels of the detectable label detected, the higher the levels of sKL in the sample. A negative control value may also be determined by omitting sKL and incubating the first and second sKL binding agents together directly. In such an embodiment, an increase over the control value may be used to determine the presence of sKL in the sample. Furthermore, the concentration of sKL in a sample from the value obtained by referencing the value obtained to a standard curve.

In one embodiment, the level of binding of FGFR to the first sKL complex is 90% or greater than the negative control value, 80% or greater than the negative control value, 70% or greater than the negative control value, 60% or greater than the negative control value, 50% or greater than the negative control value, 40% or greater than the negative control value, 30% or greater than the negative control value, 20% or greater than the negative control value, 10% or greater than the negative control value, or 5% or greater than the negative control value. In a first embodiment, the method for detecting sKL comprises: i) contacting a sample containing sKL with a first sKL binding agent; ii) incubating the sample containing sKL with the first sKL binding agent for a period of time to form a first sKL complex; iii) contacting the first sKL complex with a second sKL binding agent; iv) incubating the first sKL complex with the second sKL binding agent for a second period of time to form a second sKL complex; and v) detecting the presence of the second sKL complex. In such an embodiment, the amount of the second sKL complex detected is directly correlated with the amount of sKL in the sample.

In a second embodiment, the first sKL binding agent is FGF23, or a sKL binding portion of FGF23, and the second sKL binding agent is a FGFR, or a sKL complex binding portion of a FGFR.

Therefore, the present disclosure provides a method for detecting sKL comprising: i) contacting a sample containing sKL with FGF23, or a sKL binding portion of FGF23; ii) incubating the sample containing sKL with the FGF23, or a sKL binding portion of FGF23, for a period of time to form a first sKL complex; iii) contacting the first sKL complex with a FGFR, or a sKL complex binding portion of a FGFR; iv) incubating the first sKL complex with the FGFR, or a sKL complex binding portion of a FGFR, for a period of time to form a second sKL complex; and v) detecting the presence of the second sKL complex. In such an embodiment, the amount of the second sKL complex detected is directly correlated with the amount of sKL in the sample.

In a third embodiment, the first sKL binding agent is bound to a solid support. Therefore, the present disclosure provides a method for detecting sKL comprising: i) contacting a sample containing sKL with a first sKL binding agent, wherein the first sKL binding agent is bound to a solid support; ii) incubating the sample containing sKL with the first sKL binding agent for a period of time to form a first sKL complex bound to a solid support; iii) contacting the first sKL complex with a second sKL binding agent; iv) incubating the second sKL complex with the second sKL binding agent for a period of time to form a second sKL complex bound to a solid support; and v) detecting the presence of the second sKL complex. In such an embodiment, the amount of the second sKL complex detected is directly correlated with the amount of sKL in the sample.

In an exemplary application of this embodiment, the first sKL binding agent (for example, FGF23; SEQ ID NO: 5) is immobilized on a solid support (such as a well of a 96 well plate). Non-specific binding sites are blocked by incubation for 1 hour at room temperature with a blocking solution (such as PBS with 5% BSA, 0.1% Tween 20, and 0.25% nonidet-P40). The plate is washed with washing solution (such as 10 mM Tris (pH 7.2), 25 mM sodium chloride, 0.05% Tween 20, and 0.25% nonidet-P40). The sample (such as a plasma sample) containing sKL is added to the wells of the plate and incubated for 1 hour at room temperature to form the first sKL complex. The plate is washed with washing solution and the second sKL binding agent (such as human FGFR1, SEQ ID NO: 1, conjugated to a binding domain) is added in binding buffer and the plate incubated for 1 hour at room temperature to form the second sKL complex. The plate is washed with washing solution and a detectable label capable of binding the binding domain (for example, an anti-body conjugated to horseradish peroxidase, HRP) is added in blocking buffer and the plate is incubated for 1 hour at room temperature. The plate is washed with PBS and the detectable label substrate (such as TMB, 3,3',5,5'-tetramethylbenzidine when the detectable label utilizes HRP) and the plate incubated for 30 minutes at room temperature. The reaction is stopped by the addition of stopping solution (such as sulfuric acid for TMB/HRP) and absorbance determined using a standard plate reader via absorption at an appropriate wavelength (450 nM with TMB).

In a fourth embodiment, the second sKL binding agent is bound to a solid support. Therefore, the present disclosure provides a method for detecting sKL comprising: i) contacting a sample containing sKL with a first sKL binding agent; ii) incubating the sample containing sKL with the first sKL binding agent for a period of time to form a first sKL complex; iii) contacting the first sKL complex with a second sKL binding agent, wherein the second sKL binding agent is bound to a solid support; iv) incubating the first sKL complex with the second sKL binding agent for a period of time to form a second sKL complex bound to a solid support; and v) detecting the presence of the second sKL complex. In such an embodiment, the amount of the second sKL complex detected is directly correlated with the amount of sKL in the sample.

In an exemplary application of this embodiment, the first sKL binding agent (for example,

FGF23, SEQ ID NO: 5) is added to a sample (such as a plasma sample) containing sKL protein and incubated for 1 hour at room temperature to form the first sKL complex. The second sKL binding agent (such as human FGFR1, SEQ ID NO: 1, conjugated to a binding domain) is immobilized on a solid support (such as a well of a 96 well plate) and non-specific binding sites are blocked by incubation for 1 hour at room temperature with a blocking solution (such as PBS with 5% BSA, 0.1% Tween 20, and 0.25% nonidet-P40). The plate is washed with washing solution (such as 10 mM Tris (pH 7.2), 25 mM sodium chloride, 0.05% Tween 20, and 0.25% nonidet-P40). The sample containing the first sKL complex is added to the plate and incubated for 1 hour at room temperature to form the second sKL complex. The plate is washed with washing solution and a detectable label capable of binding the binding domain (for example, an antibody conjugated to HRP) is added in blocking buffer and the plates incubated for 1 hour at room temperature. The plate is washed with PBS and the substrate for the detectable label (such as TMB when the detectable label utilizes HRP) and the plate incubated for 30 minutes at room temperature. The reaction is stopped by the addition of stopping solution (such as sulfuric acid for TMB/HRP) and absorbance determined using a standard plate reader via absorption at an appropriate wavelength (450 nM with TMB).

In a fifth embodiment, the first and second sKL binding agents are bound to a solid support. Therefore, the present disclosure provides a method for detecting sKL comprising: i) contacting a sample containing sKL with a first sKL binding agent wherein the first sKL binding agent is linked to a solid support; ii) incubating the sample containing sKL with the first sKL binding agent for a period of time to form a first sKL complex bound to a solid support; iii) contacting the first sKL complex with a second sKL binding agent, wherein the second sKL binding agent is bound to a solid support; iv) incubating the first sKL complex with the second sKL binding reagent for a period of time to form a second sKL complex bound to a solid support; and v) detecting the presence of the second sKL complex. In such an embodiment, the amount of the second sKL complex detected is directly correlated with the amount of sKL in the sample.

In an exemplary application of this embodiment, the first sKL binding agent (for example, FGF23, SEQ ID NO: 5, conjugated to a magnetic component) is added to a reaction vessel containing a sample (such as a plasma sample) containing sKL protein and incubated for 1 hour at room temperature. The first sKL complex is isolated (such as by magnetic isolation), washed with washing buffer and resuspended in an appropriate solution (such as blocking buffer). The second sKL binding agent (such as human FGFR1, SEQ ID NO: 1, conjugated to a binding domain) is immobilized on a solid support (such as a well of a 96 well plate) and non-specific binding sites are blocked by incubation for 1 hour at room temperature with a blocking solution (such as PBS with 5% BSA, 0.1% Tween 20, and 0.25% nonidet-P40). The plate is washed with washing solution (such as 10 mM Tris (pH 7.2), 25 mM sodium chloride, 0.05% Tween 20, and 0.25% nonidet-P40). The isolated first sKL complex is added to the wells of the plate and incubated for 1 hour at room temperature. The plate is washed with washing solution and a detectable label capable of binding the binding domain (for example, an antibody conjugated to HRP) is added in blocking buffer and the plates incubated for 1 hour at room temperature. The plate is washed with PBS and the substrate for the detectable label (such as TMB when the detectable label utilizes HRP) and the plate incubated for 30 minutes at room temperature. The reaction is stopped by the addition of stopping solution (such as sulfuric acid for TMB and HRP) and absorbance determined using a standard plate reader via absorption at an appropriate wavelength (450 nM with TMB).

In any embodiments of the method of detection disclosed herein, the formation of the second sKL complex is dependent on the presence of biologically active sKL in the sample. Therefore, the amount of the second sKL complex detected is directly correlated with the amount of biologically active sKL in the sample.

In any embodiments of the method of detection disclosed herein, the first sKL binding agent binds sKL in the absence of any other protein or factor.

In any embodiments of the method of detection disclosed herein, the second sKL binding agent binds the first sKL complex in the absence of any other protein or factor. In any embodiments of the method of detection disclosed herein, the second sKL binding agent binds the sKL and/or the first sKL binding agent in the first sKL complex in the absence of any other protein or factor.

In any embodiments of the method of detection disclosed herein, the first sKL binding agent binds sKL in the absence of any other protein or factor and the second sKL binding agent binds the sKL and/or the first sKL binding agent in the first sKL complex in the absence of any other protein or factor.

In any embodiments of the method of detection disclosed herein, the first sKL binding agent is FGF23, or a sKL binding portion of FGF23, and the second sKL binding agent is FGFR, or a sKL complex binding portion of FGFR.

In any embodiments of the method of detection disclosed herein, the first and second sKL binding agents are each from the same species. In any embodiments of the method of detection disclosed herein, the first and second sKL binding agents are each human sequences. In any embodiments of the method of detection disclosed herein, the first and second sKL binding agents are each mouse sequences.

In any embodiments of the method of detection disclosed herein, the first sKL binding agent is human FGF23, or a sKL binding portion of human FGF23, and the second sKL binding agent is human FGFR, or a sKL complex binding portion of human FGFR. In any embodiments of the method of detection disclosed herein, the first sKL binding agent is mouse FGF23, or a sKL binding portion of mouse FGF23, and the second sKL binding agent is mouse FGFR, or a sKL complex binding portion of mouse FGFR.

In the detection methods described herein, the first and/or second sKL binding agents may be linked to a binding domain that is capable of interacting with a binding partner. The binding domain may be used to bind a detectable label or to aid in the isolation of a complex of which the first and/or second sKL binding agent is a member. A number of suitable binding domains and binding partners are known in the art and may be used in conjunction with the first and second sKL binding agents. Exemplary binding domains and binding partners include, but are not limited to, a magnetic moiety and a magnetic substrate, bi otin/des thi obiotin and avidin/ streptavidin, protein, (such as, but not limited to, immunoglobulin) domains and antibodies, histidine and nickel, FITC and anti-FITC, strep-tag II and strep-tactin, and digoxigenin and anti-digoxigenin. When the binding partner is used in detection, the binding partner may further comprises a detectable label.

A“detectable label” refers to molecule, protein, or compound which may be detected either directly or indirectly through the use of a suitable substrate or detection device. A

“substrate” for a detectable label refers to a composition providing conditions suitable for detecting a detectable label. Such compositions may, for example, allow the generation and/or observation of a detectable signal, such as, but not limited to, a colorimetric, fluorescent, or chemiluminescent signal, when the detectable label is contacted with the substrate. Suitably detectable labels include, but are not limited to, a protein, an enzyme, a radioisotope, a nucleic acid segment, a fluorophore, and a fluorescent protein. For example, the he first and/or second sKL binding agents may be biotinylated to bind a binding partner, such as a streptavidin-enzyme conjugate.

In the detection methods described herein, the first and/or second sKL binding agents may be a fusion protein comprising the binding domain. In a particular aspect, the binding domain is a defined protein domain. In a particular aspect, the binding domain is an immunoglobulin domain, such as, but not limited to, an immunoglobulin domain comprising a constant portion of a light chain or heavy chain immunoglobulin region. In a particular aspect, the immunoglobulin domain comprises a CH1 domain, a hinge domain, a CH2 domain, and a CH3 domain; a hinge domain, a CH2 domain, and a CH3 domain; a CH2 domain and a CH3 domain; a CH2 domain; or a CH3 domain from a light chain or heavy chain immunoglobulin. An exemplary fusion protein comprises a FGF23 ligand binding portion of FGFR4 (for example, amino acids 1 to 369 of SEQ ID NO: 1) linked to a heavy chain constant region from human IgGl comprising all or a portion of the hinge domain, the CH2 domain, and the CH3 domain. For example, the FGF23 and/or FGFR4 ligands may contain an immunoglobulin domain that binds a binding partner, such as an antibody-enzyme conjugate.

In the detection methods described herein, at least one of the first and/or second sKL binding agents comprises a detectable label or a binding domain capable of binding a detectable label. In a particular aspect, the first or second sKL binding agent that is not bound to a solid support comprises the detectable label or a binding domain capable of binding a detectable label. For example, at least one of the first or second sKL binding agents may be directly labeled with an enzyme (such as alkaline phosphatase or HRP) or a fluorophore (such as AlexaFluor532). In one embodiment, at least one of the first or second sKL binding agents may be provided as a fusion protein with a binding domain or be modified to contain a binding domain. When the binding domain is used to bind a detectable label, additional steps of the method may comprise incubating the first or second sKL binding agent with a detectable label that binds the binding domain for a period of time, removing unbound detectable label, adding a substrate for the detectable label, and measuring the detectable signal (for example, obtaining an absorbance reading). Preferably, the detectable signal detected is directly proportional to the formation of a second sKL complex.

In any embodiments of the method of detection disclosed herein, the method may further comprise a washing step between one or more or all of steps of the method. In any embodiments of the method of detection disclosed herein, the method may further comprise obtaining the sample from a subject. In such an aspect the sample may be any sample taken from a bodily fluid or a tissue of a subject. In any embodiments of the method of detection disclosed herein, the sample is a liquid sample. Suitable samples include, but are not limited to, blood, plasma, serum, cerebrospinal fluid, urine, an extract/lysate from a cell, and an extract/lysate from a tissue. Suitable sample volumes are from 50 pi to 5 mis. The sample may be processed prior to use in the methods, for example to remove, intact cells, cellular components and/or debris. Furthermore, the sample may be stored prior to use in the methods described (for example at temperatures at or less than 0°C, -30°C, or -80°C.

In any embodiments of the method of detection disclosed herein, the method are performed ex vivo (for example, in vitro).

In any embodiments of the method of detection disclosed herein, the period of time in the various steps is independently selected from 5 minutes to 5 hours, preferably 15 minutes to 1 hour.

In any embodiments of the method of detection disclosed herein where a solid support is employed, the solid support may be any solid support used in the art for the detection of proteins. In certain aspects, the solid support is manufactured from glass nitrocellulose, polyvinylidene difluoride, polyamide, polycarbonate, polyether, polymethyl methacrylate, nitrocellulose, nylon, polystyrene or polypropylene. Examples of solid support include, but are not limited to, plastic beads, agarose beads, magnetic beads, an antigen microarray or a microwell plate. Antigen microarray is a form of protein microarray, which is also known as a protein chip. A microarray is a solid support (typically glass) on which thousands of different proteins (for example, a first sKL binding agent) are immobilized in discrete spatial locations, forming a high density protein dot matrix. A microwell plate is a flat plate with multiple "wells", where each well is used for one specific sample. The microwell plate is a standard tool in clinical diagnostic testing laboratories. A very common usage is in the enzyme-linked immunosorbent assay (ELISA). In certain aspects, the solid support is a bead. In certain aspects, the solid support is fluorescent bead. In certain aspects, the solid support is a chromatography resin.

The described method of detection provides significant advances over the prior art. The assay has high sensitivity, provides quantitative read-outs with precise concentrations rather than absolute values, does not require the use of anti-klotho antibodies (which are not specific), is applicable to a high-throughput format, and is cost-effective. Based on these advantages, the disclosed sKL detection assay can be easily used to screen large sample numbers.

First and Second sKL Binding Agents

In the discussion herein, the first sKL binding agent is sometimes described as FGF23, or a FGFR binding portion of FGF23, and the second sKL binding agent is described as FGFR, or a FGF23 binding portion of FGFR. The identity of the first and second sKL binding agents may be reversed as would be understood by the person of ordinary skill in the art.

In the detection methods described herein where the second sKL binding agent is a FGFR, the FGFR may be FGFR1, FGFR2, FGFR3, or FGFR4. Alternatively, in the detection methods described herein where the second sKL binding agent is a FGFR, the FGFR may be FGFR1, FGFR2, or FGFR3. The FGFR receptor from mouse or human is preferably used.

In a particular aspect of the detection methods described herein where the second sKL binding agent is a FGFR, the FGFR is human FGFR1. Any isoform of human FGFR1 may be used, including for use in generating a sKL complex binding portion of FGFR1. In a particular aspect, when an isoform is used the isoform is selected from isoforms 2-5, 14, 16, 19, and 20-21 of human FGFR1. In certain aspects, the human FGFR1 has the sequence of SEQ ID NO: 1. In alternative aspects, the human FGFR1 has a sequence that has 50% or greater, 60% or greater, 70% or greater, 80% or greater, 90% or greater, 95% or greater, or 99% or greater sequence identity to SEQ ID NO: 1, preferably 90% or greater or 95% or greater sequence identity.

In a particular aspect of the detection methods described herein where the second sKL binding agent is a FGFR, the FGFR is human FGFR2. Any isoform of human FGFR2 may be used, including for use in generating a sKL complex binding portion of FGFR2. In a particular aspect, when an isoform is used the isoform is selected from isoforms 2-3, 5-13, and 16-20 of human FGFR2. In certain aspects, the human FGFR2 has the sequence of SEQ ID NO: 2. In alternative aspects, the human FGFR2 has a sequence that has 50% or greater, 60% or greater, 70% or greater, 80% or greater, 90% or greater, 95% or greater, or 99% or greater sequence identity to SEQ ID NO: 2, preferably 90% or greater or 95% or greater sequence identity.

In a particular aspect of the detection methods described herein where the second sKL binding agent is a FGFR, the FGFR is human FGFR3. Any isoform of human FGFR3 may be used, including for use in generating a sKL complex binding portion of FGFR3. In a particular aspect, when an isoform is used the isoform is selected from isoforms 2 or 4 of human FGFR3. In certain aspects, the human FGFR3 has the sequence of SEQ ID NO: 3. In alternative aspects, the human FGFR3 has a sequence that has 50% or greater, 60% or greater, 70% or greater, 80% or greater, 90% or greater, 95% or greater, or 99% or greater sequence identity to SEQ ID NO: 3, preferably 90% or greater or 95% or greater sequence identity.

In a particular aspect of the detection methods described herein where the second sKL binding agent is a FGFR, the FGFR is human FGFR4. Any isoform of human FGFR4 may be used, including for use in generating a sKL complex binding portion of FGFR4. In a particular aspect, when an isoform is used the isoform is isoform 3 of human FGFR4. In certain aspects, the human FGFR4 has the sequence of SEQ ID NO: 4. In alternative aspects, the human FGFR4 has a sequence that has 50% or greater, 60% or greater, 70% or greater, 80% or greater, 90% or greater, 95% or greater, or 99% or greater sequence identity to SEQ ID NO: 4, preferably 90% or greater or 95% or greater sequence identity.

In a particular aspect of the detection methods described herein where the second sKL binding agent is a FGFR, the FGFR is mouse FGFR1. Any isoform of mouse FGFR1 may be used, including for use in generating a sKL complex binding portion ofFGFRl . In certain aspects, the mouse FGFR1 has the sequence of SEQ ID NO: 6. In alternative aspects, the mouse FGFR1 has a sequence that has 50% or greater, 60% or greater, 70% or greater, 80% or greater, 90% or greater, 95% or greater, or 99% or greater sequence identity to SEQ ID NO: 6, preferably 90% or greater or 95% or greater sequence identity.

In a particular aspect of the detection methods described herein where the second sKL binding agent is a FGFR, the FGFR is mouse FGFR2. Any isoform of mouse FGFR2 may be used, including for use in generating a sKL complex binding portion of FGFR2. In certain aspects, the mouse FGFR2 has the sequence of SEQ ID NO: 7. In alternative aspects, the mouse FGFR2 has a sequence that has 50% or greater, 60% or greater, 70% or greater, 80% or greater, 90% or greater, 95% or greater, or 99% or greater sequence identity to SEQ ID NO: 7, preferably 90% or greater or 95% or greater sequence identity.

In a particular aspect of the detection methods described herein where the second sKL binding agent is a FGFR, the FGFR is mouse FGFR3. Any isoform of mouse FGFR3 may be used, including for use in generating a sKL complex binding portion of FGFR3. In a particular aspect, when an isoform is used the isoform is isoform 2 of mouse FGFR3. In certain aspects, the mouse FGFR3 has the sequence of SEQ ID NO: 8. In alternative aspects, the mouse FGFR3 has a sequence that has 50% or greater, 60% or greater, 70% or greater, 80% or greater, 90% or greater, 95% or greater, or 99% or greater sequence identity to SEQ ID NO: 8, preferably 90% or greater or 95% or greater sequence identity.

In a particular aspect of the detection methods described herein where the second sKL binding agent is a FGFR, the FGFR is mouse FGFR4. Any isoform of mouse FGFR4 may be used, including for use in generating a sKL complex binding portion of FGFR4. In a particular aspect, when an isoform is used the isoform is isoform 2 of mouse FGFR4. In certain aspects, the mouse FGFR4 has the sequence of SEQ ID NO: 9. In alternative aspects, the mouse FGFR4 has a sequence that has 50% or greater, 60% or greater, 70% or greater, 80% or greater, 90% or greater, 95% or greater, or 99% or greater sequence identity to SEQ ID NO: 9, preferably 90% or greater or 95% or greater sequence identity. In the detection methods described herein where the second sKL binding agent is a sKL complex binding portion of a FGFR, the sKL complex binding portion of a FGFR may be derived from FGFR1, FGFR2, FGFR3, or FGFR4. Alternatively, in the detection methods described herein where the second sKL binding agent is a sKL complex binding portion of a FGFR, the sKL complex binding portion of a FGFR may be derived from FGFR1, FGFR2, or FGFR3. The FGFR receptor from mouse or human is preferably used.

In a particular aspect of the detection methods described herein where the second sKL binding agent is a sKL complex binding portion of a FGFR, the sKL complex binding portion of FGFR is derived from human FGFR1. In certain aspects, the human FGFR1 has the sequence of SEQ ID NO: 1. In certain aspects, the sKL complex binding portion of human FGFR1 is an ectodomain portion of FGFR1 containing all or a portion of IgG domains I-III or IgG domains I-II. In certain aspects, the ectodomain portion does not contain the signal peptide (amino acids 1-21 of SEQ ID NO: 1). In certain aspects, the ectodomain portion does not contain the transmembrane portion (amino acids 377-397 of SEQ ID NO: 1).

In certain aspects, the ectodomain portion comprises a sequence containing all or a portion of IgG domains I-III, lacks the signal peptide and the transmembrane portion, and optionally 1 to 20 amino acids added to the amino and/or carboxy terminus of such sequence. The 1 to 20 amino acids added to the amino terminal end may correspond, completely or partially, to the amino acid sequence of the signal sequence. The 1 to 20 amino acids added to the carboxy terminal end may correspond, completely or partially, to the amino acid sequence of the transmembrane sequence. In certain aspects, the ectodomain portion comprises a sequence containing all or a portion of IgG domains I- II, lacks the signal peptide and the transmembrane portion, and optionally 1 to 20 amino acids added to the amino and/or carboxy terminus of such sequence. The 1 to 20 amino acids added to the amino terminal end may correspond, completely or partially, to the amino acid sequence of the signal sequence. The 1 to 20 amino acids added to the carboxy terminal end may correspond, completely or partially, to the amino acid sequence of the transmembrane sequence. In certain aspects, the sKL complex binding portion of human FGFR1 is a portion of human FGFR1 selected from Tables 1 A and 1B, or a sequence that has 50% or greater, 60% or greater, 70% or greater, 80% or greater, 90% or greater, 95% or greater, or 99% or greater sequence identity to any of the sequences in Table 1A, preferably 90% or greater or 95% or greater sequence identity. In Table 1B, the 1 to 20 amino acids added to the amino terminal end may be any amino acid or may correspond, completely or partially, to the amino acid sequence of the signal peptide. The 1 to 20 amino acids added to the carboxy terminal end may be any amino acid or may correspond, completely or partially, to the amino acid sequence of the transmembrane portion. When the additional amino acids correspond partially to a recited sequence, any substitutions are preferably selected from the substitutions for polypeptide derivatives described herein.

In a particular aspect of the detection methods described herein where the second sKL binding agent is a sKL binding portion of a FGFR, the sKL complex binding portion of FGFR is derived from human FGFR2. In certain aspects, the human FGFR2 has the sequence of SEQ ID NO: 2. In certain aspects, the sKL complex binding portion of human FGFR2 is an ectodomain portion of FGFR2 containing all or a portion of IgG domains I-III or IgG domains I-II. In certain aspects, the ectodomain portion does not contain the signal peptide (amino acids 1-21 of SEQ ID NO: 2). In certain aspects, the ectodomain portion does not contain the transmembrane portion (amino acids 378-398 of SEQ ID NO: 2).

In certain aspects, the ectodomain portion comprises a sequence containing all or a portion of IgG domains I-III, lacks the signal peptide and the transmembrane portion, and optionally 1 to 20 amino acids added to the amino and/or carboxy terminus of such sequence. The 1 to 20 amino acids added to the amino terminal end may correspond, completely or partially, to the amino acid sequence of the signal sequence. The 1 to 20 amino acids added to the carboxy terminal end may correspond, completely or partially, to the amino acid sequence of the transmembrane sequence. In certain aspects, the ectodomain portion comprises a sequence containing all or a portion of IgG domains I- II, lacks the signal peptide and the transmembrane portion, and optionally 1 to 20 amino acids added to the amino and/or carboxy terminus of such sequence. The 1 to 20 amino acids added to the amino terminal end may correspond, completely or partially, to the amino acid sequence of the signal sequence. The 1 to 20 amino acids added to the carboxy terminal end may correspond, completely or partially, to the amino acid sequence of the transmembrane sequence.

In certain aspects, the sKL complex binding portion of human FGFR2 is a portion of human FGFR2 selected from Tables 2A and 2B, or a sequence that has 50% or greater, 60% or greater, 70% or greater, 80% or greater, 90% or greater, 95% or greater, or 99% or greater sequence identity to any of the sequences in Table 2 A, preferably 90% or greater or 95% or greater sequence identity. In Table 2B, the 1 to 20 amino acids added to the amino terminal end may be any amino acid or may correspond, completely or partially, to the amino acid sequence of the signal peptide. The 1 to 20 amino acids added to the carboxy terminal end may be any amino acid or may correspond, completely or partially, to the amino acid sequence of the transmembrane portion. When the additional amino acids correspond partially to a recited sequence, any substitutions are preferably selected from the substitutions for polypeptide derivatives described herein.

In a particular aspect of the detection methods described herein where the second sKL binding agent is a sKL complex binding portion of a FGFR, the sKL complex binding portion of FGFR is derived from human FGFR3. In certain aspects, the human FGFR3 has the sequence of SEQ ID NO: 3. In certain aspects, the sKL complex binding portion of human FGFR3 is an ectodomain portion of FGFR3 containing all or a portion of IgG domains I-III or IgG domains I-II. In certain aspects, the ectodomain portion does not contain the signal peptide (amino acids 1-22 of SEQ ID NO: 3). In certain aspects, the ectodomain portion does not contain the transmembrane portion (amino acids 376-396 of SEQ ID NO: 3).

In certain aspects, the ectodomain portion comprises a sequence containing all or a portion of IgG domains I-III, lacks the signal peptide and the transmembrane portion, and opti onally 1 to 20 amino acids added to the amino and/or carboxy terminus of such sequence. The 1 to 20 amino acids added to the amino terminal end may correspond, completely or partially, to the amino acid sequence of the signal sequence. The 1 to 20 amino acids added to the carboxy terminal end may correspond, completely or partially, to the amino acid sequence of the transmembrane sequence. In certain aspects, the ectodomain portion comprises a sequence containing all or a portion of IgG domains I- II, lacks the signal peptide and the transmembrane portion, and optionally 1 to 20 amino acids added to the amino and/or carboxy terminus of such sequence. The 1 to 20 amino acids added to the amino terminal end may correspond, completely or partially, to the amino acid sequence of the signal sequence. The 1 to 20 amino acids added to the carboxy terminal end may correspond, completely or partially, to the amino acid sequence of the transmembrane sequence.

In certain aspects, the sKL complex binding portion of human FGFR3 is a portion of hum an

FGFR3 selected from Tables 3A and 3B, or a sequence that has 50% or greater, 60% or greater, 70% or greater, 80% or greater, 90% or greater, 95% or greater, or 99% or greater sequence identity to any of the sequences in Table 3 A, preferably 90% or greater or 95% or greater sequence identity. In Table 3B, the 1 to 20 amino acids added to the amino terminal end may be any amino acid or may correspond, completely or partially , to the amino acid sequence of the signal peptide. The 1 to 20 amino acids added to the carboxy terminal end may be any amino acid or may correspond, completely or partially, to the amino acid sequence of the transmembrane portion. When the additional amino acids correspond partially to a recited sequence, any substitutions are preferably selected from the substitutions for polypeptide derivatives described herein.

In a particular aspect of the detection methods described herein where the second sKL binding agent is a sKL complex binding portion of a FGFR, the sKL complex binding portion of FGFR is derived from human FGFR4. In certain aspects, the human FGFR4 has the sequence of SEQ ID NO: 4. In certain aspects, the sKL complex binding portion of human FGFR4 is an ectodomain portion of FGFR4 containing all or a portion of IgG domains I-II1 (amino acids 22-349 of SEQ 1D NO: 4) or IgG domains I-II (amino acids 22-240 of SEQ ID NO: 4). In certain aspects, the ectodomain portion does not contain the signal peptide (amino acids 1-21 of SEQ ID NO: 4). In certain aspects, the ectodomain portion does not contain the transmembrane portion (amino acids 370-390 of SEQ ID NO: 4).

In certain aspects, the sKL complex binding portion of human FGFR4 is a portion of human FGFR4 selected from Tables 4A and 4B, or a sequence that has 50% or greater, 60% or greater, 70% or greater, 80% or greater, 90% or greater, 95% or greater, or 99% or greater sequence identity to any of the sequences in Table 4A, preferably 90% or greater or 95% or greater sequence identity . In Table 4B, the 1 to 20 amino acids added to the amino terminal end may be any amino acid or may correspond, completely or partially, to the amino acid sequence of the signal peptide. The 1 to 20 amino acids added to the carboxy terminal end may be any amino acid or may correspond, completely or partially, to the amino acid sequence of the transmembrane portion. When the additional amino acids correspond partially to a recited sequence, any substitutions are preferably selected from the substitutions for polypeptide derivatives described herein.

In a particular aspect of the detection methods described herein where the second sKL binding agent is a sKL complex binding portion of a FGFR, the sKL complex binding portion of FGFR is derived from mouse FGFR1. In certain aspects, the mouse FGFR1 has the sequence of SEQ ID NO: 6. In certain aspects, the sKL complex binding portion of mouse FGFR1 is an ectodomain portion of FGFR1 containing all or a portion of IgG domains I-III or IgG domains I-II. In certain aspects, the ectodomain portion does not contain the signal peptide (amino acids 1-21 of SEQ ID NO: 6). In certain aspects, the ectodomain portion does not contain the transmembrane portion (amino acids 377-397 of SEQ ID NO: 6).

In certain aspects, the sKL complex binding portion of mouse FGFR1 is a portion of mouse FGFR1 selected from Tables 5A and 5B, or a sequence that has 50% or greater, 60% or greater, 70% or greater, 80% or greater, 90% or greater, 95% or greater, or 99% or greater sequence identity to any of the sequences in Table 5 A, preferably 90% or greater or 95% or greater sequence identity. In Table 5B, the 1 to 20 amino acids added to the amino terminal end may be any amino acid or may correspond, completely or partially, to the amino acid sequence of the signal peptide. The 1 to 20 amino acids added to the carboxy terminal end may be any amino acid or may correspond, completely or partially, to the amino acid sequence of the transmembrane portion. When the additional amino acids correspond partially to a recited sequence, any substitutions are preferably selected from the substitutions for polypeptide derivatives described herein.

In a particular aspect of the detection methods described herein where the second sKL binding agent is a sKL complex binding portion of a FGFR, the sKL complex binding portion of FGFR is derived from mouse FGFR2. In certain aspects, the mouse FGFR2 has the sequence of SEQ ID NO: 7. In certain aspects, the sKL complex binding portion of mouse FGFR2 is an ectodomain portion of FGFR2 containing all or a portion of IgG domains I-II1 or IgG domains I-II. In certain aspects, the ectodomain portion does not contain the signal peptide (amino acids 1-21 of SEQ ID NO: 7). In certain aspects, the ectodomain portion does not contain the transmembrane portion (amino acids 378-398 of SEQ ID NO: 7).

In certain aspects, the sKL complex binding portion of mouse FGFR2 is a portion of mouse FGFR2 selected from Tables 6A and 6B, or a sequence that has 50% or greater, 60% or greater, 70% or greater, 80% or greater, 90% or greater, 95% or greater, or 99% or greater sequence identity to any of the sequences in Table 6 A, preferably 90% or greater or 95% or greater sequence identity. In Table 6B, the 1 to 20 amino acids added to the amino terminal end may be any amino acid or may correspond, completely or partially , to the amino acid sequence of the signal peptide. The 1 to 20 amino acids added to the carboxy terminal end may be any amino acid or may correspond, completely or partially, to the amino acid sequence of the transmembrane portion. When the additional amino acids correspond partially to a recited sequence, any substitutions are preferably selected from the substitutions for polypeptide derivatives described herein.

In a particular aspect of the detection methods described herein where the second sKL binding agent is a sKL complex binding portion of a FGFR, the sKL complex binding portion of FGFR is derived from mouse FGFR3. In certain aspects, the mouse FGFR3 has the sequence of SEQ ID NO: 8. In certain aspects, the sKL complex binding portion of mouse FGFR3 is an ectodomain portion of FGFR3 containing all or a portion of IgG domains I-II1 or IgG domains I-II. In certain aspects, the ectodomain portion does not contain the signal peptide (amino acids 1-20 of SEQ ID NO: 8). In certain aspects, the ectodomain portion does not contain the transmembrane portion (amino acids 370-390 of SEQ ID NO: 8).

In certain aspects, the ectodomain portion comprises a sequence containing all or a portion of IgG domains I-III, lacks the signal peptide and the transmembrane portion, and optionally 1 to 20 amino acids added to the amino and/or carboxy terminus of such sequence. The 1 to 20 amino acids added to the amino terminal end may correspond, completely or partially, to the amino acid sequence of the signal sequence. The 1 to 20 amino acids added to the carboxy terminal end may correspond, completely or partially, to the amino acid sequence of the transmembrane sequence. In certain aspects, the ectodomain portion comprises a sequence containing all or a portion of IgG domains I- II, lacks the signal peptide and the transmembrane portion, and optionally 1 to 20 amino acids added to the amino and'or carboxy terminus of such sequence. The 1 to 20 amino acids added to the amino terminal end may correspond, completely or partially, to the amino acid sequence of the signal sequence. The 1 to 20 amino acids added to the carboxy terminal end may correspond, completely or partially, to the amino acid sequence of the transmembrane sequence.

In certain aspects, the sKL complex binding portion of mouse FGFR3 is a portion of mouse FGFR3 selected from Tables 7A and 7B, or a sequence that has 50% or greater, 60% or greater, 70% or greater, 80% or greater, 90% or greater, 95% or greater, or 99% or greater sequence identity to any of the sequences in Table 7 A, preferably 90% or greater or 95% or greater sequence identity. In Table 7B, the 1 to 20 amino acids added to the amino terminal end may be any amino acid or may correspond, completely or partially, to the amino acid sequence of the signal peptide. The 1 to 20 amino acids added to the carboxy terminal end may be any amino acid or may correspond, completely or partially, to the amino acid sequence of the transmembrane portion. When the additional amino acids correspond partially to a recited sequence, any substitutions are preferably- selected from the substitutions for polypeptide derivatives described herein.

In a particular aspect of the detection methods described herein where the second sKL binding agent is a sKL complex binding portion of a FGFR, the sKL complex binding portion of FGFR is derived from mouse FGFR4. In certain aspects, the mouse FGFR4 has the sequence of SEQ ID NO: 9. In certain aspects, the sKL complex binding portion of mouse FGFR4 is an ectodomain portion of FGFR4 containing all or a portion of IgG domains I-III or IgG domains I-II. In certain aspects, the ectodomain portion does not contain the signal peptide (amino acids 1-16 of SEQ ID NO: 9). In certain aspects, the ectodomain portion does not contain the transmembrane portion (amino acids 367-387 of SEQ ID NO: 9).

In certain aspects, the ectodomain portion comprises a sequence containing all or a portion oflgG domains I-III, lacks the signal peptide and the transmembrane portion, and optionally 1 to 20 amino acids added to the amino and/or carboxy terminus of such sequence. The 1 to 20 amino acids added to the amino terminal end may correspond, completely or partially, to the amino acid sequence of the signal sequence. The 1 to 16 amino acids added to the carboxy terminal end may correspond, completely or partially, to the amino acid sequence of the transmembrane sequence. In certain aspects, the ectodomain portion comprises a sequence containing all or a portion of IgG domains I- II, lacks the signal peptide and the transmembrane portion, and optionally 1 to 20 amino acids added to the amino and/or carboxy terminus of such sequence. The 1 to 16 amino acids added to the amino terminal end may correspond, completely or partially, to the amino acid sequence of the signal sequence. The 1 to 20 amino acids added to the carboxy terminal end may correspond, completely or partially, to the amino acid sequence of the transmembrane sequence.

In certain aspects, the sKL complex binding portion of mouse FGFR4 is a portion of mouse

FGFR4 selected from Tables 8A and 8B, or a sequence that has 50% or greater, 60% or greater, 70% or greater, 80% or greater, 90% or greater, 95% or greater, or 99% or greater sequence identity to any of the sequences in Table 8 A, preferably 90% or greater or 95% or greater sequence identity. In Table 8B, the 1 to 16 amino acids added to the amino terminal end may be any amino acid or may correspond, completely or partially, to the amino acid sequence of the signal peptide. The 1 to 20 amino acids added to the carboxy terminal end may be any amino acid or may correspond, completely or partially, to the amino acid sequence of the transmembrane portion. When the additional amino acids correspond partially to a recited sequence, any substitutions are preferably selected from the substitutions for polypeptide derivatives described herein.

In the detection methods described herein where the first sKL binding agent is FGF23, FGF23 from mouse or human is preferably used.

In a particular aspect of the detection methods described herein where the first sKL binding agent is FGF23, the FGF23 is human FGF23. In certain aspects, the human FGF23 has the sequence of SEQ ID NO: 5. In alternative aspects, the human FGF23 has a sequence that has 50% or greater, 60% or greater, 70% or greater, 80% or greater, 90% or greater, 95% or greater, or 99% or greater sequence identity to SEQ ID NO: 5, preferably 90% or greater or 95% or greater sequence identity.

In a particular aspect of the detection methods described herein where the first sKL binding agent is a FGF23, the FGF23 is mouse FGF23. In certain aspects, the mouse FGF23 has the sequence of SEQ ID NO: 10. In alternative aspects, the mouse FGF23 has a sequence that has 50% or greater, 60% or greater, 70% or greater, 80% or greater, 90% or greater, 95% or greater, or 99% or greater sequence identity to SEQ ID NO: 10, preferably 90% or greater or 95% or greater sequence identity. In the detection methods described herein where the first sKL binding agent is a sKL binding portion of a FGF23, the FGF23 from mouse or human is preferably used.

In a particular aspect of the detection methods described herein where the first sKL binding agent is a sKL binding portion of a FGF23, the sKL binding portion of FGF23 is derived from human FGF23. In certain aspects, the human FGF23 has the sequence of SEQ ID NO: 5. In certain aspects, the sKL binding portion of human FGF23 does not contain the signal peptide (amino acids 1-24 of SEQ ID NO: 5). In certain embodiments, the sKL binding portion of human FGF23 is a portion of human FGF23 selected from Tables 9A or 9B, or a sequence that has 50% or greater, 60% or greater, 70% or greater, 80% or greater, 90% or greater, 95% or greater, or 99% or greater sequence identity to any of the sequences in Table 9A, preferably 90% or greater or 95% or greater sequence identity. In Table 9B, the 1 to 20 amino acids added to the amino terminal and/or carboxy terminal end may be any amino acid or may correspond, completely or partially, to the amino acid sequence of the wild type polypeptide. When the additional amino acids correspond partially to a recited sequence, any substitutions are preferably selected from the substitutions for polypeptide derivatives described herein.

In a particular aspect of the detection methods described herein where the first sKL binding agent is a sKL binding portion of a FGF23, the sKL binding portion of FGF23 is derived from mouse FGF23. In certain aspects, the mouse FGF23 has the sequence of SEQ ID NO: 10. In certain aspects, the sKL binding portion of mouse FGF23 does not contain the signal peptide (amino acids 1-24 of SEQ ID NO: 10). In certain embodiments, the sKL binding portion of mouse FGF23 is a portion of mouse FGF23 selected from Tables 10A or 10B, or a sequence that has 50% or greater, 60% or greater, 70% or greater, 80% or greater, 90% or greater, 95% or greater, or 99% or greater sequence identity to any of the sequences in Table 10 A, preferably 90% or greater or 95% or greater sequence identity. In Table 10B, the 1 to 20 amino acids added to the amino terminal end may be any amino acid or may correspond, completely or partially, to the amino acid sequence of the signal peptide. When the additional amino acids correspond partially to a recited sequence, any substitutions are preferably selected from the substitutions for polypeptide derivatives described herein.

Any of the FGFR1, FGFR2, FGFR3, and FGFR4 polypeptides, or a sKL complex binding portion of any of the FGFR1, FGFR2, FGFR3, and FGFR4 polypeptides, or any of the FGF23 polypeptides, or a sKL binding portion of FGF23 of any of the FGF23 polypeptides, may contain any natural mutation or variation known to occur in such polypeptide, preferably a human or mouse polypeptide. Such naturally occurring mutations/variations are known to those in the art.

Any of the first or second sKL binding agents described above may be used in conjunction with the embodiments of the detection assay described herein, particularly the first through fifth embodiments of the detection assay described herein.

Methods of Production

The present disclosure provides for producing sKL polypeptide, a sKL polypeptide derivative, or a sKL polypeptide precursor. A nucleic acid encoding polypeptide of interest (for example, sKL, a sKL polypeptide derivative, or a sKL precursor) can be incorporated into a suitable vector. The vector may be used to replicate the nucleic acid in a compatible host cell line. Therefore, the present disclosure also provides a method of making a nucleic acid encoding sKL, a sKL polypeptide derivative, or a polypeptide containing sKL and introducing such nucleic acid (i.e., coding sequence) encoding the relevant polypeptide into a vector, introducing the vector into a compatible host cell line, and growing the host cell line under conditions favorable for the expression of the encoded polypeptide. In one embodiment, the host cell line is a mammalian cell line. In another embodiment, the host cell line is an insect cell line. In one embodiment, the host cell line is HEK293T. Nucleic acids coding for human and mouse klotho polypeptide are provided in SEQ ID NOS; 19 and 20, respectively. The present disclosure also provides for a nucleic acid sequence coding for any of the sKL polypeptides, sKL polypeptide derivatives, and sKL polypeptides precursors. Such nucleic acids may be generated by appropriate modification of the nucleic acid sequence of SEQ ID NOS; 11 and 12.

As used herein, the term“vector” means any mechanism for the transfer of a nucleic acid into a host cell of a subject. The term vector includes both viral and non-viral mechanisms for introducing the nucleic acid into a cell. Non-viral vectors include but are not limited to plasmids, liposomes, electrically charged lipids (such as cytofectins), DNA-protein complexes, and biopolymers. Viral vectors include but are not limited to vectors derived from adenoviral vectors, retroviral vectors, lentiviral vectors, bovine papilloma viruses, Epstein-Barr virus, adeno associated, viruses, pox viruses, baculovirus, vaccinia virus, herpes simplex virus, and hybrids of two or more viral vector types. A vector may contain regulatory sequences required for expression of the nucleic acid encoding sKL, a sKL polypeptide derivative, or a sKL precursor polypeptide, such as, but not limited to, promoters and the like. The regulatory sequences may be optimized based on a particular host cell line. In addition, a vector may contain one or more regulatory regions, and/or selectable markers useful in selecting, measuring, and monitoring nucleic acid transfer results. In some embodiments, the vector is an expression plasmid. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is a recombinant adeno-associated viral (rAAV) vector. In some embodiments, a rAAV nucleic acid encodes a polypeptide of interest and is packaged in a rAAV particle. In some embodiments, the vector used is a vector well known in the art (for example, a vector as described in Wang et al, Diabetes, 55(4), 875-884, 2006, or Wang et al, Gene Ther 10, 2105-2111, 2003, or Wang et al, Nat Biotechnol, 23, 321-328, 2005).

Preferably, a polynucleotide in a vector is operably linked to a control/regulatory sequence that is capable of providing for the expression of the coding sequence by the host cell line, i.e. the vector is an expression vector. The term“operably linked” means that the components described are in a relationship permitting them to function in their intended manner. A regulatory sequence“operably linked” to a coding sequence is arranged in such a way that expression of the coding sequence is achieved under condition compatible with the control sequences. The vectors may be for example, plasmid or virus vectors provided with an origin of replication, optionally a promoter for the expression of the said polynucleotide and optionally a regulator of the promoter. The vectors may contain one or more selectable marker genes, for example an ampicillin resistance gene in the case of a bacterial plasmid. Vectors may be used, for example, to transfect or transform a host cell. Control sequences operably linked to sequences encoding the protein of the invention include promoters/enhancers and other expression regulation signals. These control sequences may be selected to be compatible with the host cell for which the expression vector is designed to be used in. The term“promoter” is well-known in the art and encompasses nucleic acid regions ranging in size and complexity from minimal promoters to promoters including upstream elements and enhancers.

Vectors of the invention may be transformed or transfected into a suitable host cell line as described to provide for expression of sKL, a sKL polypeptide derivative, or a sKL polypeptide precursor. This process may comprise culturing a host cell line transformed with a vector containing a coding sequence encoding a desired polypeptide as described above under conditions to provide for expression by the vector of a coding sequence encoding the polypeptide, and optionally recovering the expressed polypeptide. Expression of the desired polypeptides may be constitutive such that they are continually produced, or inducible, requiring a stimulus to initiate expression. In the case of inducible expression, polypeptide production can be initiated when required by, for example, addition of an inducer substance to the culture medium, for example dexamethasone or IPTG. Polypeptides produced by the described host cell lines can be extracted from host cells by a variety of techniques known in the art, including enzymatic, chemical and/or osmotic lysis and physical disruption.

In certain embodiments, a sKL polypeptide precursor is expressed through the use of a nucleic acid encoding full length klotho (for example, having the sequence of SEQ ID NOS: 11 or 12). The expressed full length klotho polypeptide is cleaved by a protease to produce sKL (for example, sKL having the sequence amino acids 1-981 of SEQ ID NO: 11 or amino acids 1-982 of SEQ ID NO: 12).

In certain embodiments, a sKL polypeptide precursor is expressed through the use of a nucleic acid encoding a polypeptide at least 80% or at least 90% identical to full length klotho (for example, at least 80% or at least 90% identical to the sequence of SEQ ID NOS: 11 or 12). The expressed polypeptide is cleaved by a protease to produce sKL (for example, sKL having the sequence amino acids 1-981 of SEQ ID NO: 11 or amino acids 1-982 of SEQ ID NO: 12).

In certain embodiments, a sKL polypeptide precursor is expressed through the use of a nucleic acid encoding a polypeptide precursor (for example, sKL having the sequence amino acids 1-981 or 34-981 of SEQ ID NO: 11 or amino acids 1-982 or 35-982 of SEQ ID NO: 12 or a sequence that is at least 80% or at least 90% identical to any of the foregoing sequences) that also contains 1 to 20 additional amino acids c-terminal to amino acids 981 and 982 of SEQ ID NOS: 11 or 12, respectively, to optionally form a protease cleavage site and the polypeptide is cleaved by a protease to produce sKL. Suitable proteases for cleavage of a sKL polypeptide precursor include, but are not limited to, ADAM 10, ADAM 17, and BACE1; suitably the protease is ADAM17. The 1 to 20 amino acids added to the carboxy terminal end may be any amino acid or may correspond, completely or partially, to the amino acid sequence of the transmembrane portion.

In certain embodiments, sKL is produced directly through the use of a nucleic acid encoding sKL (for example, a nucleic acid encoding amino acids 1-981 or 34-981 of SEQ ID NO: 11 or amino acids 1-982 or 35-982 of SEQ ID NO: 12 or a sequence that is at least 80% or at least 90% identical to any of the foregoing sequences).

In certain embodiments, sKL is produced directly through the use of a nucleic acid encoding a sKL polypeptide derivative. As defined herein, a sKL polypeptide derivative refers to a sKL polypeptide that includes one or more insertions, deletions and/or substitutions as determined from the amino acid sequence of the human or mouse polypeptide sequence of SEQ ID NOS: 11 and 12 and that is capable of forming a complex with FGF23 and FGFR (such as FGFR1, FGFR2, FGFR3, and FGFR4), in the absence of any additional polypeptides. The methods disclosed herein may be used to determine if a polypeptide produced by the methods disclosed herein meets the definition of a sKL polypeptide derivative. The exact cleavage site in klotho that generates sKL has not been precisely identified and it is postulated that sKL may be produced from klotho by several proteases, resulting in forms of sKL that differ in amino acid length. A sKL polypeptide derivative encompasses all known natural variants of sKL differing in amino acid length. Such a sKL polypeptide derivative may be generated by deleting one or more sKL amino acids from the n-terminal portion and/or the c- terminal portion (amino acid 981 of SEQ ID NO: 11 or amino acid 982 of SEQ ID NO: 12).

In certain embodiments, a sKL polypeptide derivative does not contain the signal sequence (amino acids 1-33 of SEQ ID NO: 11 or amino acids 1-34 of SEQ ID NO: 12). In certain embodiment, a sKL polypeptide derivative is as set forth in Table 11 A or 11B, or a sequence that has 50% or greater, 60% or greater, 70% or greater, 80% or greater, 90% or greater, 95% or greater, or 99% or greater sequence identity to any of the sequences in Table 11 A, preferably 90% or greater or 95% or greater sequence identity ln Table 11B, the additional amino acids added to the amino terminal end may be any amino acid or may correspond, completely or partially, to the amino acid sequence of the signal peptide. The additional amino acids added to the carboxy terminal end may be any amino acid or may correspond, completely or partially, to the amino acid sequence of the transmembrane portion or intracellular portion. When the additional amino acids correspond partially to a recited sequence, any substitutions are preferably selected from the substitutions for polypeptide derivatives described herein.

The present disclosure provides methods for producing a recombinant, biologically active sKL polypeptide.

In a first embodiment, a method for producing a recombinant, biologically active sKL polypeptide comprises growing a host cell line containing a vector comprising a nucleic acid coding sequence directing the expression of a sKL polypeptide or a sKL polypeptide derivative under conditions favorable or the expression of the polypeptide; and ii) isolating said polypeptide.

In a second embodiment, a method for producing a recombinant, biologically active sKL polypeptide precursor comprises growing a host cell line containing a vector comprising a nucleic acid coding sequence directing the expression of a sKL polypeptide precursor under conditions favorable or the expression of the polypeptide; ii) isolating said polypeptide; and iii) cleaving the sKL polypeptide precursor with a protease to provide sKL.

In any of the embodiments for producing a sKL, a sKL derivative, or a sKL precursor, when the cells have reached a desired state of growth and/or when the cells have expressed a desired amount of polypeptide, the expressed polypeptide is isolated. In one aspect, the expressed polypeptide is secreted into culture medium and the culture medium is collected and the expressed polypeptide isolated. In another aspect, the host cell line is lysed in an appropriate EDTA-free lysis buffer comprising one or more protease inhibitors and the expressed polypeptide isolated. A suitable EDTA-free lysis buffer is RIPA buffer (50 mM Tris-HCl. pH 7.5, 200 mM NaCl, 1% Triton, 0.25% DOC, EDTA-free protease inhibitor cocktail). The isolation preferably involves one or more purification steps. In any of the embodiments for producing a sKL, a sKL derivative, or a sKL precursor, the isolated polypeptide may be subject to one or more purification steps. The method of purification used may depend on the nature of the polypeptide expressed and/or the nature of the purification tag on the expressed polypeptide. In a preferred aspect, all buffers and solutions used in the one or more isolation and purification steps are EDTA-free. In a further preferred aspect, the expressed polypeptide is not incubated with or exposed to a solution containing EDTA. In a further preferred aspect, the growth medium for the host cell line is EDTA-free.

In any of the embodiments for producing a sKL, a sKL derivative, or a sKL precursor, the method may further comprise providing the host cell line containing a vector comprising a nucleic acid coding sequence directing the expression of a desired polypeptide. In any of the embodiments for producing a sKL, a sKL derivative, or a sKL precursor, the sKL, sKL derivative, or sKL precursor may contain any natural mutation or variation known to occur in a klotho polypeptide, preferably a human or mouse klotho polypeptide. Such naturally occurring mutations/variations are known to those in the art. Such naturally occurring mutations/variants for human klotho include, but are not limited to, P15Q, F45V, H193R, F352V, C370S, P514S, and P954L.

In any of the embodiments for producing a sKL, a sKL derivative, or a sKL precursor, the polypeptide produced contains a purification tag. Suitable purification tags include, but are not limited to, a hexa-histidine tag and a Strep tag. The identity of the purification tag used may be selected based on the purification method employed.

In any of the embodiments for producing a sKL, a sKL derivative, or a sKL precursor, the polypeptide produced comprises a targeting sequence to allow for increased secretion efficiency of the polypeptide from the host cell line. Preferably, such targeting sequence is not associated with klotho in nature and such targeting sequence is used as a replacement for the signal peptide/targeting sequence naturally found associated with klotho. Suitable targeting sequences for enhanced sKL secretion are known in the art and include, but are not limited to, the targeting sequence from human CD33 (MPLLLLLPLLWAGALA; SEQ ID NO: 16), the targeting sequence from mouse IgG (METDTLLLWVLLLWVPGSTG; SEQ ID NO: 17), and the targeting sequence from human tissue plasminogen activator (MDAMKRGLCCVLLLCGAVFVSPS; SEQ ID NO: 18). The identity of the targeting sequence used may be selected based on the host cell line employed.

In any of the embodiments for producing a sKL, a sKL derivative, or a sKL precursor, cell lines expressing any of the foregoing polypeptides may be grown under a variety of conditions as is known in the art. In a preferred embodiment, host cell lines are grown in liquid culture format.

In a specific embodiment for isolating the expressed polypeptide, the clarified extract is applied to a cobalt talon column (GE Healthcare Life Sciences) using an ATKA Start Protein Purification System (GE Healthcare Life Sciences), and run and washed in running buffer (50 mM sodium phosphate, 300 mM NaCl). Polypeptide is eluted in running buffer containing 100 mM imidazole. No EDTA is added to eluted samples to bind leached metal ions. Eluates containing polypeptide are diluted 1:7 in buffer XT (100 mM Tris, 150 mM NaCl, without EDTA) and applied to a Streptactin XT column (IBA Life Sciences) via the AKTA System. Polypeptide is eluted using buffer XT containing 50 mM biotin without EDTA (as per manufacturer’s protocol). Aliquots containing the desired polypeptide are diluted 1 : 10 in heparin buffer (10 mM sodium phosphate) and applied to a heparin column via the AKTA System. Polypeptide is eluted using heparin buffer containing 500 mM NaCl. If the polypeptide is a polypeptide containing sKL, the polypeptide may be subject a cleavage step to release sKL, optionally followed by one or more additional purification steps (such as those disclosed herein, particularly where all buffers and solution used are EDTA-free).

The bioactivity of sKL aliquots may be confirmed by testing in a number of in vitro assays, including the sKL detection assay disclosed herein. Positive aliquots are pooled and flash frozen for storage at -80° C.

Polypeptide Derivatives

As discussed herein, polypeptide disclosed herein may be polypeptide derivatives containing one or more deletions, additions, and/or substitutions as compared to the corresponding wild-type sequence of the polypeptide. The deletions, additions and substitutions can be selected, as would be known to one of ordinary skill in the art, to generate a desired polypeptide derivative. With regard to deletions, a polypeptide derivative may be generated by deleting one or more amino acids from the n-terminal portion and/or the c-terminal portion of the amino acid sequence. With regard to substitutions, conservative substitutions or substitutions of amino acids with similar properties are expected to be well tolerated. Conservative modifications to the amino acid sequence is expected to produce a polypeptide derivative having functional and chemical characteristics similar to those of the wild-type polypeptide. For example, a“conservative amino acid substitution” may involve a substitution of a native amino acid residue with a normative residue such that there is little or no effect on the polarity or charge of the amino acid residue at that position. Furthermore, any native residue in the polypeptide may also be substituted with alanine.

Naturally occurring amino acid residues may be divided into classes based on common side chain properties: 1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; 2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; 3) acidic: Asp, Glu; 4) basic: His, Lys, Arg; 5) residues that influence chain orientation: Gly, Pro; and 6) aromatic: Trp, Tyr, Phe. For example, non-conservative substitutions may involve the exchange of a member of one of these classes for a member from another class.

In making such changes, the hydropathic index of amino acids may be considered. Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics, these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cy stine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5). The importance of the hydropathic amino acid index in conferring interactive biological function on a protein is understood in the art (Kyte et al, J. Mol. Biol., 157: 105-131, 1982). It is known that certain amino acids may be substituted for other amino acids having a similar hydropathic index or score and still retain a similar biological activity. In making changes based upon the hydropathic index, the substitution of amino acids whose hydropathic indices are within +1-2 may be used; in an alternate embodiment, the hydropathic indices are with +/-1; in yet another alternate embodiment, the hydropathic indices are within +/-0.5.

It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity. The greatest local average hydrophilicity of a polypeptide as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein. The following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0.+-.1); glutamate (+3.0.+-.1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5.+- .1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). In making changes based upon similar hydrophilicity values, the substitution of amino acids whose hydrophilicity values are within +1-2 may be used; in an alternate embodiment, the hydrophilicity values are with +/- 1; in yet another alternate embodiment, the hydrophilicity values are within +/-0.5.

Exemplary amino acid substitutions are set forth below.

Numerous scientific publications have been devoted to the prediction of secondary structure from analyses of amino acid sequences (see Chou et al., Biochemistry, l3(2):222-245, 1974; Chou et al, Biochemistry, 113(2):211-222, 1974; Chou et al, Adv. Enzymol. Relat. Areas Mol. Biol., 47:45-148, 1978; Chou et al, Ann. Rev. Biochem, 47:251-276, 1979; and Chou et al, Biophys. I, 26:367-384, 1979). Moreover, computer programs are currently available to assist with predicting secondary structure of polypeptides. Examples include those programs based upon the Jameson-Wolf analysis (Jameson et al., Comput. Appl. Biosci., 4(l): l8l-l 86, 1998; and Wolf et al, Comput. Appl. Biosci., 4(1): 187-191; 1988), the program PepPlot.RTM. (Brutlag et al, CABS, 6:237-245, 1990; and Weinberger et al., Science, 228:740-742, 1985), and other new programs for protein tertiary structure prediction (Fetrow. et al, Biotechnology, 11 :479-483, 1993). Moreover, computer programs are currently available to assist with predicting secondary structure. One method of predicting secondary structure is based upon homology modeling. For example, two polypeptides or proteins which have a sequence identity or greater than 30%, or similarity greater than 40% often have similar structural topologies. The recent growth of the protein structural data base (PDB) has provided enhanced predictability of secondary structure, including the potential number of folds within a polypeptide's or protein's structure (see Holm et al, Nucl. Acid. Res., 27(l):244-247, 1999).

Uses

The methods of detection of sKL as described herein may be used for a variety of purposes.

In one embodiment, the methods are used to assess the potential of sKL as a biomarker for CKD, aging, and pathologies related to CKD and/or aging. Such additional pathologies include, but not limited to, high blood pressure, fluid retention, hyperkalemia, anemia (low blood count), weak bones, increased risk of bone fracture, erectile dysfunction, decreased fertility, decreased immune response, poor nutritional health, nerve damage, cardiac injury, systemic inflammation, heart disease, cardiac fibrosis, cardiac hypertrophy, fibrosis, and blood vessel disease.

In one embodiment, the present disclosure provides a method for determining if a subject is suffering from a particular disease or condition. In another embodiment, the present disclosure provides a method for determining if a subject is at risk for suffering from a particular disease or condition. Such methods comprise obtaining or having obtained a sample from a subject containing or suspected of containing sKL, determining the level or concentration of sKL in the sample, comparing the level or concentration of sKL in the sample to a threshold value and determining if the subject is at risk for a disease or condition based on the comparison of the level or concentration of sKL in the sample and the threshold value. Such method may further comprise administering or having administered a pharmaceutical compound or therapy to the subject based on the level or concentration of sKL in the sample or based on the comparison of the level or concentration of sKL in the sample to the threshold value. Such pharmaceutical compound may be administered in a therapeutically effective amount. In an embodiment where the disease or condition is caused, at least in part, by increased sKL concentrations, if the concentration or level of sKL is determined to be below the threshold level (which may be determined empirically for a given disease or pathology and/or subject), the subject is determined not to be at risk for a given disease or condition and if the concentration or level of sKL is determined to be above the threshold level (which may be determined empirically for a given disease or pathology and/or subject), the subject is determined to be at risk for a given disease or condition. In an embodiment where the disease or condition is caused, at least in part, by decreased sKL concentrations, if the concentration or level of sKL is determined to be below the threshold level (which may be determined empirically for a given disease or pathology and/or subject), the subject is determined to be at risk for a given disease or condition and if the concentration or level of sKL is determined to be above the threshold level (which may be determined empirically for a given disease or pathology and/or subject), the subject is determined not to be at risk for a given disease or condition. In any of the foregoing, the level or concentration of bioactive sKL may be determined specifically.

The threshold value may be determined in a variety of ways. In one embodiment, the threshold value is determined empirically. In another embodiment, the threshold value is determined from a value established by a competent agency or body. In still another embodiment, the threshold value is determined from a comparative database, which may be an existing database or a specifically designed database.

When a comparative database is used, the individuals in the comparative database may be appropriately selected for comparison to the subject. In other words, the individuals selected for comparison to the subject may be selected from the comparative database based on an inclusion criteria. Exemplary inclusion criteria are selected as described below. For example, the individuals in the comparative database may be matched to the subject in one or more criteria, non-matched to the subject in one or more criteria, or matched to the subject in one or more criteria and non-matched to the subject in one or more criteria. The individuals in the database may be selected based on age. For example, the individuals in the comparative database may be matched to the subject by aged or may be non-aged matched to the subject. The use of a comparative database comprising a younger population may offer certain advantages since a younger population may be more likely to be free the disease state and other condition of interest.

The individuals in the comparative database may be selected based on a health status. For example, the individuals in the comparative database may be defined as healthy or free of a particular disease or condition and the subject defined as having a particular disease or condition. If healthy individuals are selected for inclusion in the comparative database, the value determined from the subject can be compared with the corresponding values from the comparative database. If individuals with a diagnosed disease state are selected for inclusion in the comparative database, the value determined from the subject can be compared with the corresponding value from the comparative database. In this manner, the comparison can predict if the subject has a particular disease or condition, is suffering from or likely to suffer from a particular disease state or condition, or to diagnose the severity of a particular disease state or condition. For example, if the disease state or condition is CKD, the value determined for the subject may be compared to a corresponding value from individuals in the comparative database who are diagnosed with CKD, or a particular stage of CKD; the value may also be obtained from individuals in the comparative database that are age matched to the subject.

The individuals in the comparative database may be selected based on other statuses as well, such as, but not limited to, risk factors (including genetic risk factors), demographic factors, other relevant factors or a combination of the preceding. Examples, of risk factors include, but are not limited to, age, smoking status, body mass index, genetic predisposition, and status with regard to health conditions. Demographic factors include, but are not limited to, gender and ethnicity. The individuals in the comparative database may be tagged or otherwise identified, such that the appropriate population of individuals in the comparative database may be selected for the comparison to the subject.

Furthermore, the comparative database may be refined over time. The individuals in the database may be followed over time and a status (for example, health status) monitored. If an individual no longer meets an inclusion criterion for the comparative database, the individual may be removed or the reclassified. In this manner the quality of the comparative database may be improved over time, resulting in a database with improved sensitivity and specificity.

The value from the subject is then compared to a value determined from the comparative database from appropriately selected individuals or all individuals. Appropriately selected means that the values from a defined group of individuals in the comparative database is selected for comparison to the value from the subject. The defined group may be all the individuals in the database or less than all the individuals in the comparative database. The defined group may be selected on the basis of status as discussed herein. The healthcare provider may select the defined group, with such selection based on one or more defining characteristics of the subject. For example, if the subject is a 60 year old, non-smoking, Caucasian male suspected of having early stage CKD, the defined group from the comparative database for the comparison step may selected based on ethnicity (Caucasian), gender (male), health status (disease free or diagnosed with CKD), and age (20-45 years of age of 50-70 years or age). Furthermore, the comparison may be carried out multiple times for any given subject to various iterations of the comparative database. For example, given the same 60 year old, non-smoking, Caucasian male subject suspected of having early stage CKD, a second comparison could be made using a defined group from the database selected based on gender (male) and health status (diagnosed with any form of CKD), and age (20-45 years of age of 50-70 years or age) or selected to include all individuals in the comparative database. The comparison may be made to the absolute value or to a normal reference range of the appropriate value from the comparative database. The normal reference range is a statistical range about a value. In one embodiment, the statistical range is the mean of the values for the selected value from the comparative database ± two standard deviations of the mean; other statistical ranges may also be used. If the value determined for the subject satisfies a threshold value, the subject is considered to have a particular disease or condition and vice versa.

If a comparison is made to a defined group of healthy individuals from the comparative database, and the value determined for the subject fall outside of the normal reference range for the corresponding value in the comparative database (the threshold value), the subject is determined to have or be at risk for a disease or condition. If a comparison is made to individuals from the comparative database having a diagnosed disease state and/or a specific stage of a disease state, and the value determined for the subject fall inside of the normal reference range for the corresponding value in the comparative database (the threshold value), the subject is determined to have or be at risk for a disease or condition.

In another embodiment, the methods of detection of sKL is used to determine the bioactivity of sKL. In another embodiment, the methods of detection of sKL is used to determine the bioactivity of sKL in a sample isolated from a subject. In another embodiment, the methods of detection of sKL is used to determine the bioactivity of recombinant sKL. In another embodiment, the methods of detection of sKL is used to validate sKL, including recombinant sKL, for use in a subject, such as, but not limited to, a human. In a particular embodiment, the methods of detection of sKL is used to validate recombinant sKL produced by the methods described in the present disclosure (including by the methods of Example 4), for use in a subject, such as, but not limited to, a human. In another embodiment, the methods of detection of sKL is used to identify an inhibitor of sKL binding to FGF23 or the binding of an FGFR, including FGFR1-4, with a complex of FGF23 and sKL. In another embodiment, the sKL produced according to the methods of the present disclosure may be used to treat a subject suffering from a disease or condition mediated, at least in part, by decreased klotho expression or decreased sKL concentrations in the blood. Such method comprises the step of administering to the subject an amount of a sKL polypeptide produced by the methods of the present disclosure.

Such disease and conditions include, but are not limited to, cancer, decreased cognitive function, decreased synaptic plasticity, aging and CKD and pathologies associated with the aging and/or CKD. Such pathologies include, but are not limited to, high blood pressure, fluid retention, hyperkalemia, anemia (low blood count), weak bones, increased risk of bone fracture, erectile dysfunction, decreased fertility, decreased immune response, poor nutritional health, nerve damage, cardiac injury, systemic inflammation, heart disease, cardiac fibrosis, cardiac hypertrophy, fibrosis, and blood vessel disease.

In any of the foregoing methods of treatment, the sKL polypeptide may be administered as a part of a pharmaceutical composition. In any of the foregoing methods of treatment, the sKL polypeptide may be validated as biologically active through the use of the methods described herein prior to administration to the subject. In any of the foregoing methods of treatment, the sKL polypeptide may be administered in a therapeutically effective amount.

In any of the foregoing methods of treatment, the sKL polypeptide is administered alone or as a part of a pharmaceutical composition. In any of the foregoing methods of treatment, the subject may be a mammal. In any of the foregoing methods of treatment, the subject is a human.

In any of the foregoing methods of treatment, sKL polypeptide either alone or as a part of a pharmaceutical composition, may be administered to a subject by any route, including, but not limited to, intravenously, intraperitoneally, parenterally, intramuscularly or orally. In any of the foregoing methods of treatment, the subjects treated can be further treated with one or more additional active agents that is known for the treatment of a disease or condition (for example, CKD) or the additional active agent may increase the effectiveness of the sKL polypeptide (such as by slowing degradation for example) or increase the production of endogenous klotho. In a specific aspect, the additional active agent is gamma-aminobutyric acid. The one or more additional active agents and the sKL polypeptide described herein or pharmaceutically acceptable salts or prodrugs thereof, can be administered together in a single composition or in separate compositions in any order, including simultaneous administration, as well as temporally spaced order of minutes to several days apart. The methods can also include more than a single administration of the one or more additional active agents and/or the sKL polypeptide described herein or pharmaceutically acceptable salts or prodrugs thereof. The administration of the one or more additional agents and the sKL polypeptide described herein or pharmaceutically acceptable salts or prodrugs thereof can be by the same or different routes and concurrently or sequentially. Kits

The present disclosure provides a kit comprising, consisting essentially of or consisting of at least one of a first sKL binding agent, a second sKL binding agent, and optionally one or more of the following: packaging material and instructions for carrying out an assay to identify the presence of sKL in a sample.

In one embodiment, the present disclosure provides a kit comprising, consisting essentially of or consisting of FGF23, or a sKL binding portion of FGF23 and a FGFRl, or a sKL complex binding portion of a FGFR1, and optionally one or more of the following: packaging material and instructions for carrying out an assay to identify the presence of sKL in a sample.

In one embodiment, the present disclosure provides a kit comprising, consisting essentially of or consisting of FGF23, or a sKL binding portion of FGF23 and a FGFR2, or a sKL complex binding portion of a FGFR2, and optionally one or more of the following: packaging material and instructions for carrying out an assay to identify the presence of sKL in a sample.

In one embodiment, the present disclosure provides a kit comprising, consisting essentially of or consisting of FGF23, or a sKL binding portion of FGF23, a FGFR3, or a sKL complex binding portion of a FGFR3, and optionally one or more of the following: packaging material and instructions for carrying out an assay to identify the presence of sKL in a sample.

In one embodiment, the present disclosure provides a kit comprising, consisting essentially of or consisting of FGF23, or a sKL binding portion of FGF23 and a FGFR4, or a sKL complex binding portion of a FGFR4, and optionally one or more of the following: packaging material and instructions for carrying out an assay to identify the presence of sKL in a sample.

In any of the described kits, the first and second sKL binding agents may be any first and second binding agents described herein. Furthermore, such first and second sKL binding agents may be from mouse or human.

In any of the kits described above, the kit may further comprise: i) a solid support and or reagents for associating the first and/or second sKL binding agents to the solid support; ii) additional reagent(s) to determine a level of binding (such as but not limited to a detectable label and a substrate for the detectable label); iii) a positive control compound; iv) a negative control compound; v) an sKL polypeptide (any sKL polypeptide described in the present disclosure may be included, such as, but not limited to, a sKL polypeptide having the sequence of amino acids 1- 981 of SEQ ID NO: 11 or amino acids 1-982 of SEQ ID NO: 12); vi) an sKL prepared as described in Example 4; or vii) a combination of any of the foregoing.

All patent applications, patents, and printed publications which are cited to be incorporated by reference are incorporated herein by reference in the entireties (unless specifically noted), except for any definitions, subject matter disclaimers or disavowals, and except to the extent that the incorporated material is inconsistent with the express disclosure herein, in which case the language in this disclosure controls.

EXAMPLES

Example 1- sKL Detection Assay The following is a description of one implementation of the sKL detection assay disclosed herein. This embodiment is meant to illustrate the operation of the methods disclosed herein only and not to be limiting to the materials and other limitations disclosed.

Briefly, a commercially available ELISA plates (Maxisorp) was coated with 5 pg/ml of recombinant human FGF23 protein (R&D Systems). Non-specific binding sites were blocked by incubation with blocking buffer (PBS with 5% BSA, 0.1% Tween 20). Wells were incubated with a sample obtained from a human subject containing sKL in solution (in this case a plasma sample) for one hour, washed and incubated for one hour with 1.5 pg/ml of human recombinant FGFRla(IIIc)-Fc (R&D Systems). Human recombinant FGFRla(IIIc)-Fc is a chimeric protein consisting of the FGFR1 ectodomain (amino acids 1 to 374 of SEQ ID NO: 1) linked to the Fc region of a human IgG (amino acids 100 to 330) an amino acid linker (IEGRDDMD; SEQ ID NO: 13). FGFRla(IIIc)-Fc only binds to FGF23 in the presence of sKL. The formation of this trimeric sandwich complex is then measured by detecting the presence of the Fc domain of FGFRla(IIIc)-Fc. The wells were washed and incubated with 270 ng/ml of anti-human IgG conjugated to HRP (Jackson Labs). Wells were washed again and incubated with HRP substrate for about 30 minutes and the reaction stopped with sulfuric acid. Each well was analyzed on a standard plate reader via absorptions at 450 nM.

To demonstrate the efficacy of the described sKL detection assay, purified recombinant sKL (obtained as described in Example 4) in PBS/BSA at various concentrations was used to construct a standard curve (FIG. 2). As shown in FIG. 2, the detection method described resulted in the accurate and consistent determination of sKL concentrations. Such a standard curve may be used to provide a reference value to determine sKL concentrations in a studied sample.

Example 2- Comparison of Detection of sKL by Source of sKL

The sKL detection assay described in Example 2 was used to compare sKL obtained from various commercial sources to the sKL purified according to Example 4. The sKL samples were diluted in buffer (such as PBS/BSA) to a concentration of 500 ng/ml and the detection assay described in Example 1 was used to detect the presence of sKL from the various sources. The results are shown in FIG. 3A. As can be seen the sKL obtained from two commercial suppliers A and B demonstrated significantly decreased binding to FGF23 as compared to the sKL prepared as described in Example 4.

Example 3- Detection of sKL in Mouse Plasma

The sKL detection assay of Example 1 is effective in detecting sKL from plasma. In this example, sKL levels were determined from C57BL/6 mice injected with an adeno-associated virus (AAV) expressing sKL (resulting in overexpression of sKL) and from C57BL/6 mice injected with a vehicle control. Blood samples were taken from mice that overexpressed sKL and control mice that did not overexpress sKL. Serum samples were prepared and the level of sKL binding determined as shown in Example 1. The results are shown in FIG. 3B. The sKL detection assay described showed significantly increased sKL in the blood from mice overexpressing sKL as expected. Therefore, the detection assay of Example 1 was effective in detecting elevated sKL levels in a mouse model.

Example 4- Methods of Producing sKL

HEK293T cells were stably transfected with an adenovirus associated vector expressing Strep/His-tagged sKL (FIG. 5; amino acids 1-981 of SEQ ID NO: 11) from the thyroxine binding globulin promoter (AAV-8/TBG; as described in Lock M, et al, Hum Gene Ther 21, 1259-1271 2010). Transfected HEK293T cells were grown in liquid culture format according to standard protocols. Cells were lyzed in RIPA buffer (50 mM Tris-HCl. pH 7.5, 200 mM NaCl, 1% Triton, 0.25% DOC, EDTA-free protease inhibitor cocktail).

Clarified extract was applied to a cobalt talon column (GE Healthcare Life Sciences) using an ATKA Start Protein Purification System (GE Healthcare Life Sciences), ran and washed in running buffer (50 mM sodium phosphate, 300 mM NaCl) according to manufacturer’s instructions. sKL was eluted in running buffer containing 100 mM imidazole. No EDTA is added to eluted samples to bind leached metal ions.

sKL-containing eluates were diluted 1 :7 in buffer XT (100 mM Tris, 150 mM NaCl, without EDTA) and applied to a Streptactin XT column (IBA Life Sciences) via the AKTA System according to manufacturer’s instructions. sKL was eluted using buffer XT containing 50 mM biotin without EDTA (as per manufacturer’s protocol).

sKL-containing aliquots were diluted 1 : 10 in heparin buffer (10 mM sodium phosphate) and applied to a heparin column via the AKTA System according to manufacturer’s instructions. sKL was eluted using heparin buffer containing 500 mM NaCl.

The bioactivity of sKL aliquots was confirmed by testing in a number of in vitro assays, including the sKL detection assay disclosed herein (see FIG. 2). Positive aliquots are pooled and flash frozen for storage at -80° C.

Claims

What is claimed:

1. A method for detecting soluble klotho (sKL) in a sample, the method comprising:

a. contacting a sample containing sKL with a first sKL binding agent; b. incubating the sample with the first sKL binding agent for a period of time to form a first sKL complex;

c. contacting the first sKL complex with a second sKL binding agent; d. incubating the first sKL complex with the second sKL binding agent for a

second period of time to form a second sKL complex; and

e. detecting the presence of the second sKL complex.

2. The method of claim 1, wherein the amount of the second sKL complex detected is directly correlated with the amount of biologically active sKL in the sample.

3. The method of claim 1, wherein the first sKL binding agent is bound to a solid support, the second sKL binding agent is bound to a solid support, or both the first and the second sKL binding agents are bound to a solid support.

4. The method of claim 1, wherein the first sKL binding agent binds sKL in the absence of any other protein or factor.

5. The method of claim 1, wherein the second sKL binding agent binds the first sKL

complex in the absence of any other protein or factor.

6. The method of claim 1, wherein the first and second sKL binding agents are each from the same species.

7. The method of claim 1, wherein the first sKL binding agent is human FGF23, or a sKL binding portion of human FGF23, and the second sKL binding agent is human FGFR, or a sKL complex binding portion of human FGFR.

8. The method of claim 1, wherein the first sKL binding agent is mouse FGF23, or a sKL binding portion of mouse FGF23, and the second sKL binding agent is mouse FGFR, or a sKL complex binding portion of mouse FGFR.

9. The method of claim 1, wherein at least one of the first sKL binding agent or the second sKL binding agent comprises a binding domain.

10. The method of claim 1, wherein at least one of the first sKL binding agent or the second sKL binding agent comprises a detectable label or a binding domain that binds a detectable label and the second sKL complex is detected using the detectable label.

11. The method of claim 1, further comprising obtaining the sample from a subject.

12. The method of claim 1, wherein the sample is a liquid sample.

13. The method of claim 1, wherein the sample is a blood sample, a plasma sample, a serum sample, a cerebrospinal fluid sample, a urine sample, an extract from a cell, or an extract from a tissue.

14. The method of claim 1, wherein the method is performed ex vivo.

15. The method of claim 1, wherein the first sKL binding agent is FGF23, or a sKL binding portion of FGF23 and the second sKL binding agent is a FGFR, or a sKL complex binding portion of a FGFR.

16. The method of claim 15, wherein the first sKL binding agent is bound to a solid support.

17. The method of claim 16, wherein the second sKL binding agent comprises a detectable label or a binding domain that binds a detectable label and the second sKL complex is detected using the detectable label.

18. The method of claim 15, wherein the second sKL binding agent is FGFR1 or a sKL complex binding portion of FGFR1.

19. The method of claim 18, wherein the second sKL binding agent has an amino acid

sequence of SEQ ID NO: 1 or SEQ ID NO: 6 or a sequence that has 90% or greater sequence identity to SEQ ID NO: 1 or SEQ ID NO: 6.

20. The method of claim 18, wherein the second sKL binding agent is an ectodomain portion of FGFR1 containing IgG domains I-III or IgG domains I-II.

21. The method of claim 18, wherein the second sKL binding agent has a sequence of: a. amino acids 1 to 376 of SEQ ID NO: 1 or SEQ ID NO: 6;

b. amino acids 22 to 376 of SEQ ID NO: 1 or SEQ ID NO: 6;

c. amino acids 1 to 310 of SEQ ID NO: 1 ;

d. amino acids 22 to 310 of SEQ ID NO: 1;

e. amino acids 1 to 374 of SEQ ID NO: 1;

f. amino acids 22 to 374 of SEQ ID NO: 1

g. amino acids 1 to 246 of SEQ ID NO: 1 or SEQ ID NO: 6;

h. amino acids 22 to 246 of SEQ ID NO: 1 or SEQ ID NO: 6;

i. amino acids 1 to 357 of SEQ ID NO: 1 or SEQ ID NO: 6;

j. amino acids 22 to 357 of SEQ ID NO: 1 or SEQ ID NO: 6; or

a sequence at least 90% identical to any of the foregoing sequences.

22. The method of claim 18, wherein the second sKL binding agent has a sequence:

a. at least 90% identical to amino acids 22 to 374 of SEQ ID NO: 1 or SEQ ID NO: 6, optionally comprising 1 to 20 amino acid residues n-terminal to amino acid 22, 1 to 20 amino acids c-terminal to amino acid 374, or both 1 to 20 amino acid residues n- terminal to amino acid 22 and 1 to 20 amino acids c-terminal to amino acid 374; b. at least 90% identical to amino acids 22 to 357 of SEQ ID NO: 1 or SEQ ID NO: 6, optionally comprising 1 to 20 amino acid residues n-terminal to amino acid 22, 1 to 20 amino acids c-terminal to amino acid 357, or both 1 to 20 amino acid residues n- terminal to amino acid 22 and 1 to 20 amino acids c-terminal to amino acid 357; or c. at least 90% identical to amino acids 22 to 246 of SEQ ID NO: 1 or SEQ ID NO: 6, optionally comprising 1 to 20 amino acid residues n-terminal to amino acid 22, 1 to 20 amino acids c-terminal to amino acid 246, or both 1 to 20 amino acid residues n- terminal to amino acid 22 and 1 to 20 amino acids c-terminal to amino acid 246.

23. The method of claim 11, wherein the second sKL binding agent is FGFR2 or a sKL complex binding portion of FGFR2.

24. The method of claim 23, wherein the second sKL binding agent has an amino acid

sequence of SEQ ID NO: 2 or SEQ ID NO: 7 or a sequence that has 90% or greater sequence identity to SEQ ID NO: 2 or SEQ ID NO: 7.

25. The method of claim 23, wherein the second sKL binding agent is an ectodomain portion of FGFR2 containing IgG domains I-III or IgG domains I-II.

26. The method of claim 23, wherein the second sKL binding agent has an amino acid

sequence of:

a. amino acids 1 to 377 of SEQ ID NO: 2 or SEQ ID NO: 7;

b. amino acids 22 to 377 of SEQ ID NO: 2 or SEQ ID NO: 7;

c. amino acids 1 to 247 of SEQ ID NO: 2 or SEQ ID NO: 7;

d. amino acids 22 to 247 of SEQ ID NO: 2 or SEQ ID NO: 7;

e. amino acids 1 to 358 of SEQ ID NO: 2 or SEQ ID NO: 7;

f. amino acids 22 to 358 of SEQ ID NO: 2 or SEQ ID NO: 7; or

a sequence at least 90% identical to any of the foregoing sequences.

27. The method of claim 23, wherein the second sKL binding agent has an amino acid

sequence:

a. at least 90% identical to amino acids 22 to 377 of SEQ ID NO: 2 or SEQ ID NO: 7, optionally comprising 1 to 20 amino acid residues n-terminal to amino acid 22, 1 to 20 amino acids c-terminal to amino acid 377, or both 1 to 20 amino acid residues n- terminal to amino acid 22 and 1 to 20 amino acids c-terminal to amino acid 377; b. a sequence at least 90% identical to amino acids 22 to 247 of SEQ ID NO: 2 or SEQ ID NO: 7, optionally comprising 1 to 20 amino acid residues n-terminal to amino acid 22, 1 to 20 amino acids c-terminal to amino acid 247, or both 1 to 20 amino acid residues n-terminal to amino acid 22 and 1 to 20 amino acids c-terminal to amino acid 247; or

c. a sequence at least 90% identical to amino acids 22 to 358 of SEQ ID NO: 2 or SEQ ID NO: 7, optionally comprising 1 to 20 amino acid residues n-terminal to amino acid 22, 1 to 20 amino acids c-terminal to amino acid 358, or both 1 to 20 amino acid residues n-terminal to amino acid 22 and 1 to 20 amino acids c-terminal to amino acid 358.

28. The method of claim 11, wherein the second sKL binding agent is FGFR3 or a sKL complex binding portion of FGFR3.

29. The method of claim 28, wherein the second sKL binding agent has an amino acid

sequence of SEQ ID NO: 3 or SEQ ID NO: 8 or a sequence that has 90% or greater sequence identity to SEQ ID NO: 3 or SEQ ID NO: 8.

30. The method of claim 28, wherein the second sKL binding agent is an ectodomain portion of FGFR3 containing IgG domains I-I1I or IgG domains I-II.

31. The method of claim 28, wherein the second sKL binding agent has an amino acid

sequence of:

a. amino acids 1 to 375 of SEQ ID NO: 3;

b. amino acids 23 to 375 of SEQ ID NO: 3;

c. amino acids 1 to 244 of SEQ ID NO: 3;

d. amino acids 23 to 244 of SEQ ID NO: 3

e. amino acids 1 to 355 of SEQ ID NO: 3;

f. amino acids 23 to 355 of SEQ ID NO: 3;

g. amino acids 1 to 369 of SEQ ID NO: 8;

h. amino acids 21 to 369 of SEQ ID NO: 8;

i. amino acids 1 to 238 of SEQ ID NO: 8;

j. amino acids 21 to 238 of SEQ ID NO: 8

k. amino acids 1 to 349 of SEQ ID NO: 8;

l. amino acids 21 to 349 of SEQ ID NO: 8; or

a sequence at least 90% identical to any of the foregoing sequences.

32. The method of claim 28, wherein the second sKL binding agent has an amino acid

sequence:

a. at least 90% identical to amino acids 22 to 375 of SEQ ID NO: 3, optionally

comprising 1 to 20 amino acid residues n-terminal to amino acid 22, 1 to 20 amino acids c-terminal to amino acid 375, or both 1 to 20 amino acid residues n-terminal to amino acid 22 and 1 to 20 amino acids c-terminal to amino acid 375; b. a sequence at least 90% identical to amino acids 22 to 355 of SEQ ID NO: 3, optionally comprising 1 to 20 amino acid residues n-terminal to amino acid 22, 1 to 20 amino acids c-terminal to amino acid 355, or both 1 to 20 amino acid residues n- terminal to amino acid 22 and 1 to 20 amino acids c-terminal to amino acid 355; or c. a sequence at least 90% identical to amino acids 22 to 244 of SEQ ID NO: 3, optionally comprising 1 to 20 amino acid residues n-terminal to amino acid 22, 1 to 20 amino acids c-terminal to amino acid 244, or both 1 to 20 amino acid residues n- terminal to amino acid 22 and 1 to 20 amino acids c-terminal to amino acid 244; d. at least 90% identical to amino acids 21 to 369 of SEQ ID NO: 8, optionally

comprising 1 to 20 amino acid residues n-terminal to amino acid 21 , 1 to 20 amino acids c-terminal to amino acid 369, or both 1 to 20 amino acid residues n-terminal to amino acid 21 and 1 to 20 amino acids c-terminal to amino acid 369; e. at least 90% identical to amino acids 21 to 349 of SEQ ID NO: 8, optionally

comprising 1 to 20 amino acid residues n-terminal to amino acid 21, 1 to 20 amino acids c-terminal to amino acid 349, or both 1 to 20 amino acid residues n-terminal to amino acid 21 and 1 to 20 amino acids c-terminal to amino acid 349; or f. at least 90% identical to amino acids 21 to 238 of SEQ ID NO: 8, optionally

comprising 1 to 20 amino acid residues n-terminal to amino acid 21 , 1 to 20 amino acids c-terminal to amino acid 238, or both 1 to 20 amino acid residues n-terminal to amino acid 21 and 1 to 20 amino acids c-terminal to amino acid 238.

33. The method of claim 11, wherein the second sKL binding agent is FGFR4 or a sKL complex binding portion of FGFR4.

34. The method of claim 33, wherein the second sKL binding agent has an amino acid

sequence of SEQ ID NO: 4 or SEQ ID NO: 9 or a sequence that has 90% or greater sequence identity to SEQ ID NO: 4 or SEQ ID NO: 9.

35. The method of claim 33, wherein the second sKL binding agent is an ectodomain portion of FGFR4 containing IgG domains I-I1I or IgG domains I-II.

36. The method of claim 33, wherein the second sKL binding agent has an amino acid

sequence of:

a. amino acids 1 to 369 of SEQ ID NO: 4;

b. amino acids 22 to 369 of SEQ ID NO: 4;

c. amino acids 1 to 240 of SEQ ID NO: 4; d. amino acids 22 to 240 of SEQ ID NO: 4;

e. amino acids 1 to 349 of SEQ ID NO: 4;

f. amino acids 22 to 349 of SEQ ID NO: 4

g. amino acids 1 to 366 of SEQ ID NO: 9

h. amino acids 17 to 366 of SEQ ID NO: 9;

i. amino acids 1 to 237 of SEQ ID NO: 9;

j. amino acids 17 to 237 of SEQ ID NO: 9

k. amino acids 1 to 346 of SEQ ID NO: 9;

l. amino acids 17 to 346 of SEQ ID NO: 9; or

or a sequence at least 90% identical to any of the foregoing sequences.

37. The method of claim 33, wherein the second sKL binding agent has an amino acid

sequence:

a. at least 90% identical to amino acids 22 to 369 of SEQ ID NO: 4, optionally

comprising 1 to 20 amino acid residues n-terminal to amino acid 22, 1 to 20 amino acids c-terminal to amino acid 369, or both 1 to 20 amino acid residues n-terminal to amino acid 22 and 1 to 20 amino acids c-terminal to amino acid 369; b. a sequence at least 90% identical to amino acids 22 to 349 of SEQ ID NO: 4, optionally comprising 1 to 20 amino acid residues n-terminal to amino acid 22, 1 to 20 amino acids c-terminal to amino acid 349, or both 1 to 20 amino acid residues n- terminal to amino acid 22 and 1 to 20 amino acids c-terminal to amino acid 349; c. a sequence at least 90% identical to amino acids 22 to 240 of SEQ ID NO: 4, optionally comprising 1 to 20 amino acid residues n-terminal to amino acid 22, 1 to 20 amino acids c-terminal to amino acid 240, or both 1 to 20 amino acid residues n- terminal to amino acid 22 and 1 to 20 amino acids c-terminal to amino acid 240; d. at least 90% identical to amino acids 17 to 366 of SEQ ID NO: 9, optionally

comprising 1 to 16 amino acid residues n-terminal to amino acid 17, 1 to 20 amino acids c-terminal to amino acid 366, or both 1 to 16 amino acid residues n-terminal to amino acid 17 and 1 to 20 amino acids c-terminal to amino acid 366; e. at least 90% identical to amino acids 17 to 237 of SEQ ID NO: 9, optionally

comprising 1 to 16 amino acid residues n-terminal to amino acid 17, 1 to 20 amino acids c-terminal to amino acid 237, or both 1 to 16 amino acid residues n-terminal to amino acid 17 and 1 to 20 amino acids c-terminal to amino acid 237; or f. at least 90% identical to amino acids 17 to 346 of SEQ ID NO: 9, optionally

comprising 1 to 16 amino acid residues n-terminal to amino acid 17, 1-20 amino acids c-terminal to amino acid 346, or both 1 to 16 amino acid residues n-terminal to amino acid 17 and 1-20 amino acids c-terminal to amino acid 346.

38. The method of claim 11, wherein the first sKL binding agent is FGF23 or a sKL binding portion of FGF23.

39. The method of claim 38, wherein the first sKL binding agent has an amino acid

sequence of SEQ ID NO: 5 or SEQ ID NO: 10 or a sequence that has 90% or greater sequence identity to SEQ ID NO: 5 or SEQ ID NO: 10.

40. The method of claim 38, wherein the first sKL binding agent has a sequence of:

a. amino acids 1 to 251 of SEQ ID NO: 5 or SEQ ID NO: 10;

b. amino acids 25 to 251 of SEQ ID NO: 5 or SEQ ID NO: 10;

c. amino acids 1 to 179 of SEQ ID NO: 5 or SEQ ID NO: 10;

d. amino acids 25 to 179 of SEQ ID NO: 5 or SEQ ID NO: 10;

e. amino acids 180 to 251 of SEQ ID NO: 5 or SEQ ID NO: 10, or

a sequence at least 90% identical to any of the foregoing sequences.

41. The method of claim 38, wherein the first sKL binding agent has a sequence:

a. at least 90% identical to amino acids 25 to 251 of SEQ ID NO: 10, optionally comprising 1 to 20 amino acid residues n-terminal to amino acid 25, 1 to 20 amino acids c-terminal to amino acid 251, or both 1 to 20 amino acid residues n-terminal to amino acid 25 and 1 to 20 amino acids c-terminal to amino acid 251; b. at least 90% identical to amino acids 25 to 179 of SEQ ID NO: 10, optionally comprising 1 to 20 amino acid residues n-terminal to amino acid 25, 1 to 20 amino acids c-terminal to amino acid 179, or both 1 to 20 amino acid residues n-terminal to amino acid 25 and 1 to 20 amino acids c-terminal to amino acid 179; or c. at least 90% identical to amino acids 180 to 251 of SEQ ID NO: 10, optionally comprising 1 to 20 amino acid residues n-terminal to amino acid 180, 1 to 20 amino acids c-terminal to amino acid 251, or both 1 to 20 amino acid residues n- terminal to amino acid 180 and 1 to 20 amino acids c-terminal to amino acid 251.

42. A method for producing a recombinant, biologically active sKL polypeptide or sKL polypeptide derivative, the method comprising:

a. growing a host cell line containing a vector comprising a coding sequence

directing the expression of the sKL polypeptide or the sKL polypeptide derivative under conditions favorable or the expression of the sKL polypeptide or the sKL polypeptide derivative; and

b. isolating the sKL polypeptide or the sKL polypeptide derivative, wherein the sKL polypeptide or the sKL polypeptide derivative is not exposed to EDTA during step (b).

43. The method of claim 42, wherein the vector is a non-viral vector or a viral vector.

44. The method of claim 42, wherein the vector is an adeno associated viral vector.

45. The method of claim 42, wherein the sKL polypeptide or the sKL polypeptide derivative comprises a targeting sequence for enhanced secretion.

46. The method of claim 42, wherein the sKL polypeptide or the sKL polypeptide derivative comprises a purification tag.

47. The method of claim 42, wherein the isolation step comprises at least one purification step.

48. The method of claim 42, wherein the sKL polypeptide has a sequence of:

a. amino acids 1-981 of SEQ ID NO: 11:

b. amino acids 34-981 of SEQ ID NO: 11;

c. amino acids 1-982 of SEQ ID NO: 12;

d. amino acids 35-982 of SEQ ID NO: 12; or

a sequence at least 90% identical to any of the foregoing sequences.

49. The method of claim 42, wherein the sKL polypeptide derivative has a sequence of: a. amino acids 1 to 954 of SEQ ID NO: 11 ;

b. amino acids 34 to 954 of SEQ ID NO: 11 ;

c. amino acids 1 to 955 of SEQ ID NO: 12;

d. amino acids 35 to 955 of SEQ ID NO: 12;

e. amino acids 45 to 950 of SEQ ID NO: 11 or SEQ ID NO: 12;

f. amino acids 50 to 940 of SEQ ID NO: 11 or SEQ ID NO: 12;

g. amino acids 55 to 930 of SEQ ID NO: 11 or SEQ ID NO: 12;

h. amino acids 60 to 920 of SEQ ID NO: 11 or SEQ ID NO: 12; or

a sequence at least 90% identical to any of the foregoing sequences.

50. The method of claim 42, wherein the sKL polypeptide derivative has a sequence:

a. at least 90% identical to amino acids 1 to 954 of SEQ ID NO: 11, optionally comprising 1 to 20 amino acids c-terminal to amino acid 954;

b. at least 90% identical to amino acids 34 to 954 of SEQ ID NO: 11, optionally comprising 1 to 20 amino acid residues n-terminal to amino acid 34, 1 to 20 amino acids c-terminal to amino acid 954, or both 1 to 20 amino acid residues n-terminal to amino acid 34 and 1 to 20 amino acids c-terminal to amino acid 954; c. at least 90% identical to amino acids 1 to 955 of SEQ ID NO: 11, optionally comprising 1 to 20 amino acids c-terminal to amino acid 954;

d. at least 90% identical to amino acids 35 to 955 of SEQ ID NO: 11, optionally comprising 1 to 20 amino acid residues n-terminal to amino acid 35, 1 to 20 amino acids c-terminal to amino acid 955, or both 1 to 20 amino acid residues n-terminal to amino acid 35 and 1 to 20 amino acids c-terminal to amino acid 955; e. at least 90% identical to amino acids 45 to 950 of SEQ ID NO: 11 or SEQ ID NO: 12, optionally comprising 1 to 20 amino acid residues n-terminal to amino acid 45, 1-20 amino acids c-terminal to amino acid 950, or both 1 to 20 amino acid residues n-terminal to amino acid 45 and 1 to 20 amino acids c-terminal to amino acid 950;

f. at least 90% identical to amino acids 50 to 940 of SEQ ID NO: 11 or SEQ ID NO: 12, optionally comprising 1 to 20 amino acid residues n-terminal to amino acid 50, 1-20 amino acids c-terminal to amino acid 940, or both 1 to 20 amino acid residues n-terminal to amino acid 50 and 1 to 20 amino acids c-terminal to amino acid 940;

g. at least 90% identical to amino acids 55 to 930 of SEQ ID NO: 11 or SEQ ID NO: 12, optionally comprising 1 to 20 amino acid residues n-terminal to amino acid 55, 1 to 20 amino acids c-terminal to amino acid 930, or both 1 to 20 amino acid residues n-terminal to amino acid 55 and 1 to 20 amino acids c-terminal to amino acid 930; or

h. at least 90% identical to amino acids 60 to 920 of SEQ ID NO: 11 or SEQ ID NO: 12, optionally comprising 1 to 20 amino acid residues n-terminal to amino acid 60, 1-20 amino acids c-terminal to amino acid 920, or both 1 to 20 amino acid residues n-terminal to amino acid 60 and 1-20 amino acids c-terminal to amino acid

920.

51. The method of claim 42, wherein the sKL polypeptide or the sKL polypeptide derivative is not exposed to EDTA during steps (a) or (b).

52. A kit comprising, a first sKL binding agent and a second sKL binding agent, and

optionally one or more of the following: packaging material and instructions for carrying out an assay to determine the presence of sKL..

53. The kit of claim 52, wherein the first sKL binding agent is FGF23, or a sKL binding portion of FGF23 and the second sKL binding agent is:

a. FGFR1 , or a sKL complex binding portion of FGFR1 ; b. FGFR2, or a sKL complex binding portion of FGFR2;

c. FGFR3, or a sKL complex binding portion of FGFR3;

d. FGFR4, or a sKL complex binding portion of FGFR4; or

e. any combination of the foregoing.

54. The kit of claim 52 or claim 53, wherein the kit further comprises:

a. a solid support;

b. reagents for associating the first, second, or both first and second sKL binding agents to the solid support;

c. a detectable label;

d. a substrate for the detectable label;

e. a positive control compound;

f. a negative control compound;

g. a sKL polypeptide;

h. a sKL prepared as described in claim 42; or

i. a combination of any of the foregoing.

55. A method for determining if a subject is suffering from a disease or condition, the

method comprising the steps of:

a. obtaining or having obtained a sample from a subject containing or suspected of containing sKL:

b. determining the concentration of sKL in the sample;

c. comparing the concentration of sKL in the sample to a threshold value; and d. determining if the subject is at risk for the disease or condition based on the comparison of the concentration of sKL in the sample and the threshold value.

56. The method of claim 56, wherein the method further comprises administering or having administered a medication or therapy to the subject based on the concentration of sKL in the sample or based on the comparison of the concentration of sKL in the sample to the threshold value.

57. The method of claim 56, wherein the threshold value is a value determined from a comparative database comprising a corresponding value from a plurality of individuals.

58. The method of claim 57, wherein, the plurality of individuals are selected for

comparison based on an inclusion criteria.