# Patent

## Title
Klotho variant polypeptides

## Abstract

Disclosed are Klotho variant proteins in which residue Glu414 and/or residue Asp238 is substituted with an amino acid different than L-Glu or L-Asp, respectively, as well as polynucleotides encoding the variant proteins, and the use thereof in therapy, especially for the treatment of cancers, especially breast cancer and pancreatic cancer.

## Background
RELATED APPLICATION
The present invention is filed under 35 U.S.C. §371 as the U.S. national phase of International Application No. PCT/IB2013/053001, filed Apr. 16, 2013, which designated the U.S. and claims the benefit of priority to U.S. Provisional Patent Application No. 61/624,443 filed Apr. 16, 2012 and to U.S. Provisional Patent Application No. 61/752,694 filed Jan. 15, 2013, which are all hereby incorporated in their entirety including all tables, figures, and claims.
SEQUENCE LISTING
The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Oct. 14, 2014, is named GUR0015US_SeqListing.txt and is 392 kilobytes in size.


FIELD AND BACKGROUND OF THE INVENTION
The present invention, in some embodiments, relates to the field of therapeutics, and more specifically to Klotho variant polypeptides, polynucleotides encoding same, and the use thereof in therapy, especially for the treatment of cancer.
Klotho protein is a single-pass transmembrane protein.
Human Klotho protein (SEQ ID NO:1) is 1012 amino acid residues long while murine Klotho protein (SEQ ID NO:81) is 1014 amino acid residues long. All alignment programs confirm a shift of 2 amino acids between the human (1012 amino acids) and mouse (1014 amino acids) Klotho polypeptide sequences. For example, Glu414 in human Klotho protein is the equivalent of Glu416 in mouse Klotho protein and Asp238 in human Klotho protein is Asp240 in mouse Klotho protein.
Klotho protein has been implicated in a number of biological activities including forming a complex with Fibroblast Growth Factor (FGF) receptors and functioning as an obligate co-receptor for FGF23, a bone-derived hormone that induces phosphate excretion into urine. Mice lacking Klotho or FGF23 not only exhibit phosphate retention but also display a premature-aging syndrome, revealing an unexpected link between phosphate metabolism and aging.
Secreted Klotho has been implicated in a number of biological activities including a humoral factor that regulates activity of multiple glycoproteins on the cell surface, including ion channels and growth factor receptors such as Insulin/Insulin-Like Growth Factor-1 receptors.
In US 2010/0330062, the Inventors disclosed that Klotho protein and related compounds have anti-cancer properties and also disclosed the use of Klotho protein and related compounds in the treatment of cancer.
SUMMARY OF THE INVENTION
The invention, in some embodiments thereof, relates to Klotho variant polypeptides as well as to the use of such variant polypeptides as therapeutic agents, especially for the treatment of cancer.
Aspects of some embodiments of the invention relate to the Inventors' discovery that the KL1 domain of Klotho protein bears an active site, and that some of the biological activities of Klotho and of the isolated KL1 can be at least partially neutralized while retaining the anti-cancer activity of the Klotho polypeptide or of the isolated KL1 domain. In some embodiments the neutralization of the biological activities is concomitant with a reduction in adverse side effects.
According to an aspect of some embodiments of the invention, there is provided an isolated polypeptide comprising an amino-acid residue sequence having a omology of not less than 80% with a polypeptide having an amino-acid residue sequence selected from the group consisting of the amino-acid residue sequences represented by: SEQ ID NO:1 (residues 1-1012); residues 29-1012 of SEQ ID NO:1 (SEQ ID NO:2); residues 1-980 of SEQ ID NO:1 (SEQ ID NO:3); residues 29-980 of SEQ ID NO:1 (SEQ ID NO:4); residues 1-568 of SEQ ID NO:1 (SEQ ID NO:5); residues 29-568 of SEQ ID NO:1 (SEQ ID NO:6); residues 34-549 of SEQ ID NO:1 (SEQ ID NO:7); SEQ ID NO:8 (residues 1-549); residues 29-549 of SEQ ID NO:8 (SEQ ID NO:9); and residues 34-549 of SEQ ID NO:8 (SEQ ID NO:10) wherein:
the L-Glu of residue 414 is substituted with an a-amino acid residue different from L-Glu having an amino acid sequence set forth in SEQ ID NO:11-20; the L-Asp of residue 238 is substituted with an α-amino acid different from L-Asp having an amino acid sequence set forth in SEQ ID NO:21-30; or the L-Glu of residue 414 is substituted with an α-amino acid residue different from L-Glu and the L-Asp of residue 238 is substituted with an α-amino acid different from L-Asp having an amino acid sequence set forth in SEQ ID NO:31-40, numbered with reference to SEQ ID NO:1.
According to an aspect of some embodiments of the invention, there is also provided the isolated polypeptide, or a DNA encoding therefor, for use in a method of treatment of a disease.
In some embodiments provided is an isolated polynucleotide encoding a Klotho variant polypeptide selected from any one of SEQ ID NO:11-40.
In preferred embodiments the isolated polypeptide has an amino acid sequence set forth in any one of SEQ ID NO:11-40. In some embodiments the isolated polypeptide is a variant of SEQ ID NO:1 wherein the L-Glu at position 414 is substituted with an amino acid other than L-Glu and has an amino acid sequence set forth in any one of SEQ ID NO:11-20. In some embodiments the isolated polypeptide is a variant of SEQ ID NO:1 wherein the L-Asp at position 238 is substituted with an amino acid other than L-Asp and has an amino acid sequence set forth in any one of SEQ ID NO:21-30. In some embodiments the isolated polypeptide is a variant of SEQ ID NO:1 wherein the L-Glu at position 414 is substituted with an amino acid other than L-Glu; wherein the L-Asp at position 238 is substituted with an amino acid other than L-Asp and wherein the polypeptide has an amino acid sequence set forth in any one of SEQ ID NO:31-40.
In some embodiments provided is an isolated polynucleotide encoding a Klotho variant polypeptide having a polynucleotide sequence set forth in any one of SEQ ID NO :51-80.
In preferred embodiments the polynucleotide encoding the isolated polypeptide has a polynucleotide sequence set forth in any one of SEQ ID NO:51-80. In some embodiments the polynucleotide sequence comprises a variant of SEQ ID NO:41 wherein the nucleotides encoding the L-Glu at position 414 encode an amino acid other than L-Glu; and wherein the polynucleotide sequence is set forth in any one of SEQ ID NO:51-60. In some embodiments the polynucleotide sequence comprises a variant of SEQ ID NO:41 wherein the nucleotides encoding the L-Asp at position 238 encode an amino acid other than L-Asp; and wherein the polynucleotide sequence is set forth in any one of SEQ ID NO:61-70. In some embodiments the polynucleotide sequence comprises a variant of SEQ ID NO:41 wherein the nucleotides encoding L-Glu encode an amino acid other than L-Glu; wherein the nucleotides encoding L-Asp at position 238 is substituted encode an amino acid other than L-Asp; and wherein the polynucleotide sequence is set forth in any one of SEQ ID NO:71-80.
In some embodiments provided is an expression vector comprising an isolated polynucleotide encoding a Klotho variant polypeptide having a polynucleotide sequence set forth in any one of SEQ ID NO:51-80.
In some embodiments provided is a composition comprising an expression vector comprising an isolated polynucleotide encoding a variant polypeptide having a polynucleotide sequence set forth in any one of SEQ ID NO:51-80; and a carrier. In preferred embodiments the carrier is a pharmaceutically acceptable carrier.
Further provided is a host cell comprising an expression vector comprising an isolated polynucleotide encoding a variant polypeptide having a polynucleotide sequence set forth in any one of SEQ ID NO:51-80.
In some embodiments the host cell is a prokaryotic cell or a eukaryotic cell. Preferably the cell is a mammalian cell.
According to an aspect of some embodiments of the invention, there is also provided the use of the isolated polypeptide, or a DNA encoding therefor, for the preparation of a medicament for treating a disease.
According to an aspect of some embodiments of the invention, there is also provided the isolated polypeptide, or a DNA encoding therefor, for use as a medicament for treating a disease.
According to an aspect of some embodiments of the invention, there is also provided a method for the treatment of a disease, comprising, administering a pharmaceutically-acceptable effective amount of the isolated polypeptide, or a DNA encoding therefor, to a subject (human or non-human animal) in need thereof.
According to an aspect of some embodiments of the invention, there is also provided a pharmaceutical composition comprising an isolated polypeptide having an amino acid sequence set forth in any one of SEQ ID NO: 11-40; and a pharmaceutically-acceptable carrier.
According to an aspect of some embodiments of the invention, there is also provided a pharmaceutical composition comprising a DNA encoding an isolated polypeptide having an amino acid sequence set forth in any one of SEQ ID NO : 11-40, having a polynucleotide sequence set forth in any one of SEQ ID NO:51-80; and a pharmaceutically-acceptable carrier.
In some embodiments, the disease is cancer.
According to an aspect of some embodiments of the invention, there is also provided an isolated polypeptide comprising a Klotho amino acid sequence and having at least one amino acid mutation in the catalytic domain of Klotho, wherein the isolated polypeptide maintains an anti cancer activity. In some embodiments, the catalytic domain of Klotho is the KL1 domain.
In some embodiments, the amino acid mutation is in at least one amino acid residue corresponding to an amino acid coordinate selected from the group consisting of Glu414 and Asp238 of human Klotho (SEQ ID NO: 1).
In some embodiments, the isolated polypeptide comprises a reduced FGF23 mediated signaling activity as compared to that of human Klotho (SEQ ID NO: 1).
In some embodiments, the isolated polypeptide has a reduced IGF-1 signaling inhibitory activity as compared to that of human Klotho (SEQ ID NO: 1).
In some embodiments, the at least one amino acid mutation is of amino acid residues corresponding to amino acid coordinates Glu414 and Asp238 of human Klotho (SEQ ID NO: 1)
In some embodiments, the amino acid mutation is an amino acid substitution.
In some embodiments, the amino acid substitution is to an amino acid residue selected from the group consisting of a non-charged amino acid, a polar amino acid and a non-polar amino acid residue.
In some embodiments, the amino acid substitution is to a polar amino acid residue.
In some embodiments, the amino acid substitution is selected from the group consisting of Glu414Gln and Asp238Asn.
In some embodiments, the isolated polypeptide is devoid of a domain selected from the group consisting of native signal and KL2.
In some embodiments, the Klotho comprises human Klotho.
In some embodiments, the isolated polypeptide is selected from the group consisting of SEQ ID NO: 11-40.
According to an aspect of some embodiments of the invention, there is also provided an isolated polynucleotide comprising a nucleic acid sequence encoding any of the above polypeptides.
According to an aspect of some embodiments of the invention, there is also provided a nucleic acid construct comprising the nucleic acid sequence.
According to an aspect of some embodiments of the invention, there is also provided a pharmaceutical composition comprising as an active ingredient an isolated polypeptide as described above or the isolated polynucleotide described above or the nucleic acid construct described above and a pharmaceutically acceptable carrier or diluent.
According to an aspect of some embodiments of the invention, there is also provided a cell comprising the nucleic acid construct described above.
According to an aspect of some embodiments of the invention, there is also provided a method of treating cancer comprising administering to a subject in need thereof the pharmaceutical composition described above, thereby treating cancer.
According to an aspect of some embodiments of the invention, there is also provided an isolated polypeptide as described above, an isolated polynucleotide as described above or the nucleic acid construct described above for use in treating cancer, in some embodiments, breast cancer.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In case of conflict, the patent specification, including definitions, takes precedence.
As used herein, the terms “medicament” and “therapeutic agent” are used synonymously.
As used herein, the term “treating” includes curing a condition, treating a condition, preventing a condition, treating symptoms of a condition, curing symptoms of a condition, ameliorating symptoms of a condition, treating effects of a condition, ameliorating effects of a condition, and preventing results of a condition.
As used herein, the terms “comprising”, “including”, “having” and grammatical variants thereof are to be taken as specifying the stated features, integers, steps or components but do not preclude the addition of one or more additional features, integers, steps, components or groups thereof. These terms encompass the terms “consisting of” and “consisting essentially of”.
The phrase “consisting essentially of” or grammatical variants thereof when used herein are to be taken as specifying the stated features, integers, steps or components but do not preclude the addition of one or more additional features, integers, steps, components or groups thereof but only if the additional features, integers, steps, components or groups thereof do not materially alter the basic and novel characteristics of the claimed composition, device or method.
A “polynucleotide” as used herein refers to an oligonucleotide or nucleotide and fragments or portions thereof, and to DNA or RNA of genomic or synthetic origin which may be single- or double-stranded, and represent the sense or antisense strand. Similarly, “polypeptide” as used herein refers to an oligopeptide, peptide, amino acid sequence or protein sequence, and fragments or portions thereof, and to naturally occurring, synthetic or recombinant molecules.
The term “expression vector” and “recombinant expression vector” as used herein refers to a recombinant DNA molecule containing a desired coding sequence and appropriate nucleic acid sequences necessary for the expression of the operably linked coding sequence, i.e., a polynucleotide encoding a Klotho variant polypeptide, in a particular host organism. The expression vector may comprise sequences encoding heterologous domains including but not limited to protein detection, purification or cleavage sequences that may be fused at the N- or C-terminus to the desired coding sequence, to yield a fusion protein.
As used herein, the indefinite articles “a” and “an” mean “at least one” or “one or more” unless the context clearly dictates otherwise.
In some instances, especially in US 61/624,443, the term “protein” is used instead of the intended term “polypeptide”. Protein and polypeptide may be used interchangeably.
In some instances, especially in US 61/624,443, the term “mutant Klotho protein” is used instead of the intended “Klotho variant polypeptide”. The terms “mutant Klotho protein” “Klotho variant polypeptide” may be used interchangeably.
As used herein, when a numerical value is preceded by the term “about”, the term “about” is intended to indicate +/−10%.



BRIEF DESCRIPTION OF THE FIGURES
Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying figures. The description, together with the figures, makes apparent how embodiments of the invention may be practiced to those skilled in the art. It is stressed that the particulars shown in the figures are by way of example and for purposes of illustrative discussion of embodiments of the invention.
In the figures:
FIG. 1A schematically depicts portions of KLrP (klotho-related protein) surrounding a glucose molecule, on which portions of native Klotho protein KL1 domain is superimposed, demonstrating the similarity of the respective active sites;
FIG. 1B schematically depicts portions of KLrP surrounding a glucose molecule, on which portions of native Klotho protein KL2 domain is superimposed, demonstrating the dissimilarity of the respective active sites;
FIG. 2 is a reproduction of a photograph of a colony formation assay, in which MCF-7 and MDA-MB-231 cells were transfected with one of the following expression vectors: an empty vector (pDNA3); wild-type full length Klotho (FL-KL); a Klotho protein variant (FL-KL (E416Q), which is a mouse full length Klotho where native L-Glu at 416 is substituted with L-Gln, equivalent to E414Q in the respective human Klotho protein variant); wild-type KL1 (KL1); KL1 variant according to an embodiment of the teachings (KL1 (E416Q), that is mouse KL1 where native L-Glu at 416 is substituted with L-Gln, equivalent to E414Q in the respective human KL1 variant); and wild-type KL2 (KL2). Transfected cells were cultured in media containing G418 for two weeks, and colonies were stained with crystal violet;
FIG. 3 is an immunoblot of MCF-7 cells transfected with an empty vector (pcDNA3); a wild type full length Klotho (FL-mKLOTHO/WT); a first Klotho variant polypeptide (FL-mKLOTHO/E416Q: mouse FL-KL E416Q, equivalent to E414Q in the respective human Klotho protein variant), wild type KL1 (ssKL1/WT; including amino acids 1-28 signal sequence); a Klotho variant polypeptide (ssKL1/WT:E416Q: mouse KL1 E416Q, equivalent to E414Q in the respective human KL1 variant) treated or untreated with IGF, and exposed to the antibodies indicated; and
FIG. 4 is an immunoblot of HEK293 cells transfected with wild type full length Klotho (FL-KL/WT); a Klotho variant polypeptide (FL-KL/E416Q) according to an embodiment of the teachings herein; wild type KL1 (KL1/WT); or a KL1 variant polypeptide (KL1/E416Q) according to an embodiment of the teachings herein, and treated with FGF23, exposed to the antibodies indicated.



DESCRIPTION OF SOME EMBODIMENTS OF THE INVENTION
The invention, in some embodiments thereof, relates to Klotho polypeptides as well as to the use of such polypeptides as therapeutic agents, especially for the treatment of cancer.
Klotho protein is a single pass transmembrane protein located at the cell membrane (Ito et al., 2000; Kuro-o et al., 1997; Matsumura et al., 1998; Shiraki-Iida et al., 1998) and also detected in the Golgi apparatus (Imura et al., 2007). Human Klotho protein (full-length (FL)-hKL; SEQ ID NO:1, accession no. AAQ41828) is 1012 amino acid residues long while murine Klotho protein (full-length (FL)-mKL; SEQ ID NO:81, accession no AAQ41830) is 1014 amino acid residues long. The N-terminus of human Klotho protein (residues 1-28) trails from KL1. Human Klotho protein is anchored in a cell membrane through the C-terminus (residues 981-1012).
The extracellular domain of Klotho protein is composed of two spherically-folded discrete domains, KL1 (human residues 29-568, 540 residues long) and KL2 (human residues 569-980, 411 residues long), which share amino acid sequence homology to β-glucosidase but lack glucosidase catalytic activity (Kuro-o et al., 1997). KL1 may also be transcribed through an alternative splicing. It is known that in vivo, KL1-KL2 can be cleaved together to form a single 130 kDa secreted Klotho protein, also called soluble Klotho protein, (sol-hKL or sol-mKL, residues 1-980, also called secreted Klotho protein) which is shed into the serum and acts as a circulating hormone (Imura et al., 2004), or can be cleaved separately as a 68 kDa protein (KL1) and a 64 kDa protein (KL2). It has been shown recently that A Desintegrin and Metalloproteinase (ADAM) 10 and 17 participate in this process in response to insulin stimulation (Chen et al., 2007). Only full length Klotho can function as a co-receptor for FGF23 (Kurosu et al., 2006; Wu et al., 2008).
Soluble Klotho protein has been implicated in a number of biological activities including a humoral factor that regulates activity of multiple glycoproteins on the cell surface including ion channels and growth factor receptors such as insulin/insulin-like growth factor-1 receptors.
mRNA expression analysis identified Klotho protein mainly in the distal renal tubules and the choroid plexus in the brain, but also in sex-hormone-responsive tissues including the placenta, testes and ovaries (Kuro-o et al., 1997; Ohyama et al., 1998; Shiraki-Iida et al., 1998). The expression of Klotho protein in breast or pancreatic tissue has been explored by the Inventors and disclosed in US 2010/0330062.
Klotho protein-modulated receptors include the insulin receptor (IR) (Kuro-o et al., 1997), the IGF-1 receptor (IGF-1R) and several fibroblast growth factor receptors (FGFR), but not the epidermal growth factor receptor (EGFR) (Kurosu et al., 2006; Urakawa et al., 2006). Treatment of cultured cells with soluble Klotho protein inhibited insulin-induced glucose uptake, and reduced stimulation-induced phosphorylation of the IR, the IGF-1R and the insulin receptor substrates (IRS)-1 and -2 (Kurosu et al., 2005). Moreover, Klotho protein-deficient mice are hypoglycemic and extremely sensitive to insulin (Kuro-o et al., 1997; Utsugi et al., 2000), while Klotho protein over-expressing mice are associated with insulin resistance (Kurosu et al., 2005). Klotho protein also functions as an obligate co-receptor for FGF23, a bone-derived hormone that induces phosphate excretion into urine. Mice lacking Klotho protein, and/or FGF23 not only exhibit phosphate retention but also display a premature-aging syndrome, revealing an unexpected link between phosphate metabolism and aging. Klotho protein inhibits activation of the FGFRs by bFGF in human embryonic kidney (HEK) 293 and COS7 cells (Wolf et al., 2008; Urakawa et al., 2006; Kurosu et al., 2006).
Klotho protein-induced inhibition of the IGF-1R may also affect the expression of the transcription factors CCAAT/enhancer-binding protein (C/EBP) α and β. These factors are down-regulated by the IGF-1 pathway and were recently identified as breast cancer growth suppressors (Gery et al., 2005; Gomis et al., 2006; Wolf et al., 2006a). The C/EBP family is involved in adipocyte differentiation and Klotho protein-deficient mice have barely detectable amounts of white adipose tissue (Kuro-o et al., 1997). Indeed, Klotho protein has been identified recently as an inducer of adipocyte differentiation, and this activity is mediated through up-regulation of these transcription factors (Chihara et al., 2006).
Klotho protein may play a role in human diseases, and Klotho protein polymorphism was associated with reduced life span, coronary heart disease and osteoporosis (Arking et al., 2003; Arking et al., 2002; Kim et al., 2006; Zarrabeitia et al., 2007).
Klotho protein has been shown to affect the activity of several signaling pathways, which may participate in breast cancer tumorigenesis, through modulation of ligand-dependent activation of their specific membranal receptors. Increased serum insulin levels are associated with adverse prognosis in breast cancer, high circulating IGF-1 levels are associated with increased risk of premenopausal breast cancer, and inhibition of the insulin and IGF-1 pathways inhibits growth of breast cancer cells (Bartucci et al., 2001; Wolf et al., 2006b; Yee, 2006).
In US2010/0330062 the Inventors identified high Klotho protein expression in normal breast tissue and low Klotho protein expression in breast cancer; noted inhibition of breast cancer cell growth following over-expression of Klotho protein, and growth enhancement of Klotho protein-expressing cells following Klotho protein knock-down; and revealed modulation of the IGF-1 and the insulin pathways by Klotho protein. Taken together, the results suggested Klotho protein as a novel breast cancer tumor suppressor.
Insulin like growth factor-1 (IGF-1) is also a powerful mediator of pancreatic cancer. Both IGF-1 and IGF-1 receptor (IGF-1R) are overexpressed in human pancreatic tumors as well as in pancreatic cancer cell lines (Bergmann, U., et al, 1995, Karna, E., et al, 2002). Blockage of the IGF-1R by a dominant negative inhibitor suppresses tumorigenicity both in vitro and in vivo and increases sensitivity of pancreatic tumors to radiation and chemotherapy-induced apoptosis (Edderkaoui, M., et al, 2007). Thus, the inventors consider that the IGF-1 pathway may serve as an attractive target for novel therapies against pancreatic cancer.
Klotho protein has previously been identified as an inhibitor of the IGF-1 system in hepatocytes and muscle cells. The effects of Klotho protein on inhibition of the IGF-1 system in cancer cells, such as pancreatic and breast cancer cells, have not hitherto been studied.
The appended sequence listing includes polypeptide and polynucleotide sequences encoding the polypeptides, as set forth in Table 1, infra, in the Examples.
The normal polypeptide and DNA sequences, specifically SEQ ID NO:1, 8, 41, 48 and 81-84 are fully described in PCT/JP1997/004585 published as WO1998029544.
The Inventors have since discovered and now report that some of the biological activity of human Klotho protein (e.g., formation of a complex with FGF receptors to activate FGF23 in the kidney and modulation of calcium levels in the body of an organism) is at least partially dependent on the presence of at least one of two specific amino acid residues in the KL1 domain of Klotho protein: the L-Glu of residue 414 and the L-Asp of residue 238.
The Inventors have further discovered that replacing either one or both of these amino acid residues with a different α-amino acid residue to yield a novel variant polypeptide according to some embodiments of the invention, does not necessarily adversely affect the anti-cancer activity of the polypeptide. Importantly, the Inventors have now further discovered that the IGF1 receptor activity of variant polypeptides according to some embodiments of the invention where either one or both of these amino acid residues has been replaced with a different α-amino acid residue is reduced or neutralized, indicating that the anticancer mechanism of the variants and of native Klotho is unrelated to IGF1 signaling. Therefore, the Klotho variant polypeptides in accordance with the teachings herein may serve as better candidates for therapeutic use than either full length native Klotho or KL1.
The Inventors conclude and hereby disclose that at least some polypeptides as described herein, Klotho variant polypeptides in which residue 414 and/or residue 238 is substituted with any α-amino acid different from the native L-Glu or L-Asp respectively, are not only novel, but are unexpectedly useful as therapeutic agents, especially as therapeutic agents for the treatment of cancer. Specifically, some embodiments of such polypeptides are effective therapeutic agents, yet have reduced side-effects due to the at least partial neutralization of the native biological activity affected by substitution of the native residue 414 and/or residue 238.
According to an aspect of some embodiments of the teachings herein, there is provided an isolated polypeptide comprising an amino-acid residue sequence having a homology of not less than 80% with a polypeptide having an amino-acid residue sequence selected from the group consisting of the amino-acid residue sequences represented by:
SEQ ID NO:1 (full length human Klotho protein, FL-hKL, accession no. AAQ41828);
residues 29-1012 of SEQ ID NO:1 (human Klotho protein without the N-terminal “tail”);
residues 1-980 of SEQ ID NO:1 (soluble human Klotho protein, sol-hKL);
residues 29-980 of SEQ ID NO:1 (sol-hKL without the N-terminal tail);
residues 1-568 of SEQ ID NO:1 (KL1 with N-terminal tail of human Klotho protein);
residues 29-568 of SEQ ID NO:1 (KL1 of human Klotho protein);
residues 34-549 of SEQ ID NO:1 (a shortened KL1 of human Klotho protein);
SEQ ID NO:8 (the recombinant version of KL1, with additional residues 1-33, accession no. AAQ41829);
residues 29-549 of SEQ ID NO:8 (the recombinant version of KL1, with additional
residues 29-33);
residues 34-549 of SEQ ID NO:8 (a recombinant version of KL1, commercially available from Peprotech, Rocky Hill, N.J., USA)
wherein:
the L-Glu of residue 414 is substituted with an α-amino acid residue different from L-Glu;
the L-Asp of residue 238 is substituted with an α-amino acid different from L-Asp; or
the L-Glu of residue 414 is substituted with an α-amino acid residue different from L-Glu and the L-Asp of residue 238 is substituted with an α-amino acid different from L-Asp numbered with reference to SEQ ID NO:1.
As used herein the “klotho gene” refers to the Klotho polypeptide coding sequence open reading frame, as shown in (SEQ ID NO:41), or any homologous sequence thereof preferably having at least 70% identity, more preferable 80% identity, even more preferably 90% or 95% identity. This encompasses sequences derived from SEQ ID NO:41 which have undergone mutations, alterations or modifications as described herein. Thus, in a preferred embodiment Klotho polypeptide is encoded by a nucleic acid sequence according to SEQ. ID. NO. 41. It is also to be acknowledged that based on the amino acid sequence of Klotho polypeptide (SEQ ID NO:1) or variants described herein, any nucleic acid sequence coding for such amino acid sequence can be perceived by the one skilled in the art based on the genetic code.
“Klotho polypeptide” and “KL1” refer to the polypeptides of the Klotho gene and KL1 domain of the Klotho gene respectively, and is understood to include splice variants and fragments thereof retaining biological activity, and homologs thereof, preferably having at least 70%, more preferably at least 80%, even more preferably at least 90% or 95% homology thereto. In addition, this term is understood to encompass polypeptides resulting from minor alterations in the Klotho or KL1 coding sequence, such as, inter alia, point mutations, substitutions, deletions and insertions which may cause a difference in a few amino acids between the resultant polypeptide and the naturally occurring Klotho polypeptide. Polypeptides encoded by nucleic acid sequences which bind to the Klotho coding sequence or genomic sequence under conditions of highly stringent hybridization, which are well-known in the art are also encompassed by this term. Chemically-modified Klotho polypeptide or chemically-modified fragments of Klotho polypeptide are also included in the term, so long as the biological activity is retained. Human Klotho polypeptide preferably has or comprises an amino acid sequence, amino acids 1-1012, according to SEQ I. NO:1. It is acknowledged that there might be differences in the amino acid sequence among various tissues of an organism and among different organisms of one species or among different species to which the nucleic acid according to the present invention can be applied in various embodiments of the present invention. However, based on the technical teaching provided herein, the respective sequence can be taken into consideration accordingly when designing any of the nucleic acids disclosed herein. Particular fragments of the human Klotho polypeptide include amino acid residues 29-1012 (SEQ ID NO:2), 1-980 (SEQ ID NO:3), 29-980 (SEQ ID NO:4), 1-568 (SEQ ID NO:5), 29-568 (SEQ ID NO:6) and 34-549 (SEQ ID NO:7). Particular fragments of Human KL1 (SEQ ID NO:8, residues 1-549) include amino acid residues 29-549 (SEQ ID NO:9) and 34-549 (SEQ ID NO:10).
A “variant polypeptide” is a polypeptide having one or more sequence substitutions, deletions, and/or additions as compared to the native sequence. The variants disclosed herein are artificially constructed; typically generated from the corresponding nucleic acid molecules. In preferred embodiments, the variants have 1 or 2 amino acid substitutions and retain at least some of the activity of the native polypeptide. In some embodiments the variants disclosed herein retain at least part of the anti-cancer activity of the native Klotho polypeptide and elicit reduced side effects. This unexpected property of the Klotho variant polypeptides disclosed herein warrants their use as therapeutic agents for the treatment of cancer.
In some embodiments, the isolated polypeptide has an amino acid sequence represented by SEQ ID NO:1 (full length human Klotho protein, FL-hKL), wherein:

    the L-Glu of residue 414 is substituted with an α-amino acid different from L-Glu;
    the L-Asp of residue 238 is substituted with an α-amino acid different from L-Asp; or


the L-Glu of residue 414 is substituted with an α-amino acid residue different from L-Glu and the L-Asp of residue 238 is substituted with an α-amino acid different from L-Asp numbered with reference to SEQ ID NO:1.
In some embodiments, the isolated polypeptide has an amino acid sequence represented by residues 29-1012 of SEQ ID NO:1 (set forth in SEQ ID NO:2) human Klotho protein without the N-terminal “tail”), wherein:

    the L-Glu of residue 414 is substituted with an α-amino acid different from L-Glu;
    the L-Asp of residue 238 is substituted with an α-amino acid different from L-Asp; or
    the L-Glu of residue 414 is substituted with an α-amino acid residue different from L-Glu and
    the L-Asp of residue 238 is substituted with an α-amino acid different from L-Asp numbered with reference to SEQ ID NO:1.


In some embodiments, the isolated polypeptide has an amino acid sequence represented by residues 1-980 of SEQ ID NO:1 (set forth in SEQ ID NO:3) soluble human Klotho protein, sol-hKL), wherein:

    the L-Glu of residue 414 is substituted with an α-amino acid different from L-Glu;
    the L-Asp of residue 238 is substituted with an α-amino acid different from L-Asp;
    the L-Glu of residue 414 is substituted with an α-amino acid residue different from L-Glu and
    the L-Asp of residue 238 is substituted with an α-amino acid different from L-Asp numbered with reference to SEQ ID NO:1.


In some embodiments, the isolated polypeptide has an amino acid sequence represented by residues 29-980 of SEQ ID NO:1 (set forth in SEQ ID NO:4) sol-hKL without the N-terminal tail), wherein:

    the L-Glu of residue 414 is substituted with an α-amino acid different from L-Glu;
    the L-Asp of residue 238 is substituted with an α-amino acid different from L-Asp; or
    the L-Glu of residue 414 is substituted with an α-amino acid residue different from L-Glu and
    the L-Asp of residue 238 is substituted with an α-amino acid different from L-Asp numbered with reference to SEQ ID NO:1.


In some embodiments, the isolated polypeptide has an amino acid sequence represented by residues 1-568 of SEQ ID NO:1 (set forth in SEQ ID NO:5) KL1 with N-terminal tail of human Klotho protein), wherein:

    the L-Glu of residue 414 is substituted with an α-amino acid different from L-Glu;
    the L-Asp of residue 238 is substituted with an α-amino acid different from L-Asp; or
    the L-Glu of residue 414 is substituted with an α-amino acid residue different from L-Glu and
    the L-Asp of residue 238 is substituted with an α-amino acid different from L-Asp numbered with reference to SEQ ID NO:1.


In some embodiments, the isolated polypeptide has an amino acid sequence represented by residues 29-568 of SEQ ID NO:1 (set forth in SEQ ID NO:6) KL1 of human Klotho protein), wherein:

    the L-Glu of residue 414 is substituted with an α-amino acid different from L-Glu;
    the L-Asp of residue 238 is substituted with an α-amino acid different from L-Asp; or
    the L-Glu of residue 414 is substituted with an α-amino acid residue different from L-Glu and
    the L-Asp of residue 238 is substituted with an α-amino acid different from L-Asp numbered with reference to SEQ ID NO:1.


In some embodiments, the isolated polypeptide has an amino acid sequence represented by residues 34-549 of SEQ ID NO:1 (set forth in SEQ ID NO:7) a shortened KL1 of human Klotho protein), wherein:

    the L-Glu of residue 414 is substituted with an α-amino acid different from L-Glu;
    the L-Asp of residue 238 is substituted with an α-amino acid different from L-Asp; or
    the L-Glu of residue 414 is substituted with an α-amino acid residue different from L-Glu and
    the L-Asp of residue 238 is substituted with an α-amino acid different from L-Asp numbered with reference to SEQ ID NO:1.


In some embodiments, the isolated polypeptide has an amino acid sequence represented by residues 34 to 549 of SEQ ID NO:8 (set forth in SEQ ID NO:10) the recombinant version of KL1, wherein:

    the L-Glu of residue 414 is substituted with an α-amino acid different from L-Glu;
    the L-Asp of residue 238 is substituted with an α-amino acid different from L-Asp;
    the L-Glu of residue 414 is substituted with an α-amino acid residue different from L-Glu and
    the L-Asp of residue 238 is substituted with an α-amino acid different from L-Asp numbered with reference to SEQ ID NO:1.


In some embodiments, the isolated polypeptide has an amino acid sequence represented by SEQ ID NO:8 (the recombinant version of KL1, with additional residues 1-33), wherein:

    the L-Glu of residue 414 is substituted with an α-amino acid different from L-Glu;
    the L-Asp of residue 238 is substituted with an α-amino acid different from L-Asp; or
    the L-Glu of residue 414 is substituted with an α-amino acid residue different from L-Glu and
    the L-Asp of residue 238 is substituted with an α-amino acid different from L-Asp numbered with reference to SEQ ID NO:1.


In some embodiments, the isolated polypeptide has an amino acid sequence represented by residues 29 to 549 of SEQ ID NO:8 (set forth in SEQ ID NO:9) the recombinant version of KL1, with additional residues 29-33, wherein:

    the L-Glu of residue 414 is substituted with an α-amino acid different from L-Glu;
    the L-Asp of residue 238 is substituted with an α-amino acid different from L-Asp;
    the L-Glu of residue 414 is substituted with an α-amino acid residue different from L-Glu and
    the L-Asp of residue 238 is substituted with an α-amino acid different from L-Asp numbered with reference to SEQ ID NO:1.


In some embodiments, the L-Glu of residue 414 is substituted with an α-amino acid residue different from L-Glu.
In some embodiments, the L-Glu of residue 414 is substituted with an R-α-amino acid residue. In some such embodiments, the R-α-amino acid residue is R-Glu. In some embodiments, the R-α-amino acid residue is different from R-Glu.
In some embodiments, the L-Glu of residue 414 is substituted with an L-α-amino acid residue different from L-Glu. In some such embodiments, the L-α-amino acid residue is selected from the group consisting of Ala, Arg, Asn, Asp, Cys, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, ornithine, selenocysteine (Sec), 2-aminoisobutyric acid, hydroxyproline (Hyp) and selenomethionine.
In some embodiments, the L-Glu of residue 414 is substituted with an α-amino acid residue that is devoid of an acid side chain.
In some embodiments, the L-Glu of residue 414 is substituted with an L-α-Gln. An advantage of such embodiments is that L-α-Gln is devoid of the acid group required for L-Glu activity and is of substantially the same size and shape as the native L-α-Gln, so that the change in shape of the polypeptide caused by the substitution is minimal.
In some embodiments, the L-Asp of residue 238 is substituted with an α-amino acid residue different from L-Asp.
In some embodiments, the L-Asp of residue 238 is substituted with an R-α-amino acid residue. In some such embodiments, the R-α-amino acid residue is R-Asp. In some such embodiments, the R-α-amino acid residue is different from R-Asp.
In some embodiments, the L-Asp of residue 238 is substituted with an L-α-amino acid residue different from L-Asp. In some such embodiments, the L-α-amino acid residue is selected from the group consisting of Ala, Arg, Asn, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, ornithine, selenocysteine (Sec), 2-aminoisobutyric acid, hydroxyproline (Hyp) and selenomethionine.
In some embodiments, the L-Asp of residue 238 is substituted with an α-amino acid residue that is devoid of an acid side chain.
In some embodiments, the L-Asp of residue 238 is substituted with an L-α-Asn. An advantage of such embodiments is that L-α-Asn is devoid of the acid group required for L-Asp activity and is of substantially the same size and shape as the native L-α-Asp, so that the change in shape of the polypeptide caused by the substitution is minimal.
Treatments, Such as of Cancer
As discussed herein and as described in detail in the Examples section below, it has been surprisingly found that some embodiments of a polypeptide as disclosed herein have a biological activity that renders the polypeptide useful for treatment of a disease, for example cancer. In preferred embodiments, the polypeptide having an amino acid sequence set forth in any one of SEQ ID NO:11-40 retains anti-cancer activity. In preferred embodiments, the polypeptide having an amino acid sequence set forth in any one of SEQ ID NO:11-20 retains anti-cancer activity. In preferred embodiments, the polypeptide having an amino acid sequence set forth in any one of SEQ ID NO:21-30 retains anti-cancer activity. In preferred embodiments, the polypeptide having an amino acid sequence set forth in any one of SEQ ID NO:31-40 retains anti-cancer activity. In some embodiments, such a polypeptide retains anti-cancer activity and also has one or more advantages, for example, has reduced adverse side effects compared to a polypeptide having an amino acid sequence set forth in one or more of SEQ ID NO:1-10
Thus, according to an aspect of some embodiments of the teachings herein, there is also provided an isolated polypeptide as disclosed herein, or a DNA encoding therefor, for use in a method of treatment of a disease.
According to an aspect of some embodiments of the teachings herein, there is also provided an isolated polypeptide as disclosed herein, or a DNA encoding therefor, for use as a medicament for treating a disease.
According to an aspect of some embodiments of the teachings herein, there is also provided an isolated polypeptide as disclosed herein, or a DNA encoding therefor, for use in therapy.
According to an aspect of some embodiments of the teachings herein there is also provided a pharmaceutical composition comprising: an isolated polypeptide as disclosed herein, or a DNA encoding therefor; and a pharmaceutically-acceptable carrier.
According to an aspect of some embodiments of the teachings herein there is also provided the use of an isolated polypeptide as disclosed herein, or a DNA encoding therefor, for the preparation of a medicament for treating a disease.
According to an aspect of some embodiments of the teachings herein, there is also provided a method for the treatment of a disease, comprising, administering a pharmaceutically-acceptable effective amount of an isolated polypeptide as disclosed herein, or a DNA encoding therefor, to a subject in need thereof. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human animal.
The DNA encoding for an isolated polypeptide as disclosed herein includes a DNA which hybridizes with the above-mentioned DNA under stringent conditions.
In some embodiments, it is advantageous to coadminister a polypeptide as disclosed herein or a DNA encoding therefor together with a chemotherapeutic agent. Suitable such chemotherapeutic agents are described in detail hereinbelow.
Thus, according to an aspect of some embodiments of the teachings herein there is also provided a pharmaceutical composition comprising: an isolated polypeptide as disclosed herein, or a DNA encoding therefor; a chemotherapeutic agent; and a pharmaceutically-acceptable carrier.
According to an aspect of some embodiments of the teachings herein there is also provided the use of an isolated polypeptide as disclosed herein, or a DNA encoding therefor, together with a chemotherapeutic agent for the preparation of a medicament for treating a disease.
According to an aspect of some embodiments of the teachings herein, there is also provided a method for the treatment of a disease, comprising, co-administering a pharmaceutically-acceptable effective amount of an isolated polypeptide as disclosed herein, or a DNA encoding therefor, together with a chemotherapeutic agent to a subject in need thereof. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human animal. In some embodiments, the polypeptide or DNA encoding therefor and such a co-administered chemotherapeutic agent are administered in a single dosage form or alternatively are administered sequentially or simultaneously in separate dosage forms.
In some embodiments, a polypeptide as disclosed herein is pegylated, to improve pharmacokinetics or other parameters. Various advantages of pegylation and methods for pegylation of proteins such as Klotho proteins are known in the art, see for example Ryan S M, Mantovani G, Wang X, Haddleton D M, Brayden D J “Advances in PEGylation of important biotech molecules: delivery aspects” in Expert Opin Drug Deliv. 2008, 5(4), 371-383.
Administration
For implementing the teachings herein, a polypeptide as disclosed herein is administered by any suitable route, for example as described in PCT/JP1997/004585. For example, the polypeptide may be administered by parenteral (including intravenous, intradermal, intraperitoneal, intramuscular and subcutaneous) routes. Alternatively, the polypeptide may be administered by other delivery routes, including oral, nasal, buccal, sublingual, intra-tracheal, transdermal, transmucosal, and pulmonary. The polypeptide may be administered by continuous release or delivery, using, for example, an infusion pump, continuous infusion, controlled release formulations utilizing polymer, oil or water insoluble matrices.
Carriers or excipients known in the art can also be used to facilitate administration of the polypeptide. Examples of carriers and excipients include calcium carbonate, calcium phosphate, various sugars such as lactose, or types of starch, cellulose derivatives, gelatin, vegetable oils, polyethylene glycols and physiologically compatible solvents.
According to some embodiments, the polypeptide is administered in extended release form, which is capable of releasing the polypeptide over a predetermined release period, such that a clinically effective plasma level of the polypeptide is maintained for at least 24 hours, such as at least 48 hours, at least 72 hours, at least one week, or at least one month.
According to some embodiments, the polypeptide is administered in combination with one or more chemotherapeutic agents, including, for example, alkylating agents, antimetabolites, anthracyclines, plant alkaloids, topoisomerase inhibitors, hormone receptor modulators, hormone level modulators, and other antitumour agents.
Examples of suitable alkylating agents include, without limitation, busulfan, carboplatin, carmustine, cisplatin, chloroambucil, cyclophosphamide, dacarbazine, hexamethylmelamine, ifosfamide, mechlorethamine, melphalan, oxoplatin, streptozocin, temozolomide, thiotepa, and uramustine.
Non-limiting examples of suitable antimetabolites include azathioprine, capecitabine, carmofur, cladribine, clofarabine, cytarabine, fludarabine, fluorouracil, gemcitabine, mercaptopurine, methotrexate, premetrexed, raltitrexed, tegafur, and tioguanine
Suitable anthracyclines include, for example, daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone and valrubicin.
Examples of suitable plant alkaloids include docetaxel, paclitaxel, vinblastine, vincristine, vindesine, and vinorelbine. Examples of suitable topoisomerase inhibitors include amsacrine, etoposide, etoposide phosphate, irinotecan, teniposide, and topotecan. Examples of suitable hormone receptor modulators include tamoxifen; and estrogen antagonists, such as faslodex. Examples of suitable hormone level modulators include aromatose inhibitors, such as letrozole, anastrazole and aromasin. Examples of other antitumor agents include dactinomycin, and other chemotherapeutic agents for treatment of obesity-related cancers, such as trastuzumab (herceptin), lapatinib, bevacizumab (avastin), cetuximab (erbitux), panitumumab, erlotinib, and sunitinib.
In such embodiments, the polypeptide as disclosed herein and the chemotherapeutic agent are administered by subcutaneous or intravenous injection.
The chemotherapeutic agent may optionally be provided in a combined dosage form, together with the polypeptide. Alternatively, the chemotherapeutic agent may be provided in a separate dosage form, for simultaneous or sequential co-administration, either before or after administration of the polypeptide.
The teachings herein further provide a composition comprising pharmaceutically acceptable amounts of a polypeptide as disclosed herein and a chemotherapeutic agent as an additional active pharmaceutical ingredient.
The composition may optionally be provided in extended-release form, as described above with regard to the polypeptide alone.
According to some embodiments of the teachings herein, there is provided a composition comprising a combination of at least two active pharmaceutical ingredients, at least one of which is a polypeptide as described herein and at least one of which is a chemotherapeutic agent, wherein the amount of polypeptide and amount of the chemotherapeutic agent alone is insufficient to achieve the therapeutic effect achieved by the administration of the combination of two or more of the active pharmaceutical ingredients. Such a composition comprises, in addition to the active pharmaceutical ingredients, a pharmaceutically acceptable carrier, and may optionally further comprise one or more pharmaceutically acceptable excipients.
Pharmaceutically-acceptable carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered subcutaneously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.
If desired, solutions of the above dosage compositions may be thickened with a thickening agent such as methylcellulose. They may be prepared in emulsified form, such as either water in oil or oil in water. Any of a wide variety of pharmaceutically acceptable emulsifying agents may be employed including, for example, acacia powder, a non-ionic surfactant (such as a Tween), or an ionic surfactant (such as alkali polyether alcohol sulfates or sulfonates, e.g., a Triton).
In general, a composition as described herein is prepared by mixing the ingredients following generally accepted procedures. For example, the selected components may be simply mixed in a blender or other standard device to produce a concentrated mixture which may then be adjusted to the final concentration and viscosity by the addition of water or thickening agent and possibly a buffer to control pH or an additional solute to control tonicity.
Treatment Using Gene Therapy
Discussed above are various embodiments where a polypeptide as disclosed herein is administered to a subject, for example in order to treat cancer. In some embodiments, rather than administering a polypeptide, gene therapy may be preferred, whereby a DNA encoding for a polypeptide as described herein, is administered, in the usual way, to a subject. The thus-administered DNA causes the body of the subject to produce endogenous polypeptide as disclosed herein in amounts sufficient to lead to the desired therapeutic (e.g., anti-cancer) effect. In some embodiments, the DNA is delivered inserted in a recombinant vector (e.g., a bacteria) suitable for gene therapy.
In preferred embodiments the polynucleotide encoding a variant peptide has an amino acid sequence forth in any one of SEQ ID NO:51-80. In preferred embodiments the polynucleotide encoding a variant peptide has an amino acid sequence forth in any one of SEQ ID NO:51-60. In preferred embodiments the polynucleotide encoding a variant peptide has an amino acid sequence forth in any one of SEQ ID NO:61-70. In preferred embodiments the polynucleotide encoding a variant peptide has an amino acid sequence forth in any one of SEQ ID NO:71-80.
The preferred method for producing the variants is using recombinant DNA technologies, well known to those skilled in the art. For example, the variant polynucleotide may be prepared by Polymerase Chain Reaction (PCR) using specific primers for each of the variant forms or the amino acid substitutions as disclosed herein below. The PCR fragments are purified on an agarose gel and the purified DNA fragment is cloned into an expression vector and transfected into host cells. The host cells are cultured and the protein harvested according to methods known in the art.
The term “expression vector” and “recombinant expression vector” as used herein refers to a recombinant DNA molecule containing a desired coding sequence, e.g., any one of SEQ ID NO:51-80, and appropriate nucleic acid sequences necessary for the expression of the operably linked coding sequence in a particular host organism. The expression vector may comprise sequences encoding heterologous domains including but not limited to protein detection, purification or cleavage sequences that may be fused at the N- or C-terminus to the desired coding sequence, to yield a fusion protein. It is contemplated that the present invention encompasses expression vectors that are integrated into host cell genomes, as well as vectors that remain unintegrated into the host genome.
Thus, in some embodiments of the teachings herein is provided the use of DNA encoding for a polypeptide as disclosed herein, as a medicament, for example for the treatment of cancer.
In some embodiments the teachings herein provide a method of treatment (in some embodiments, treatment of cancer) comprising administering to a subject in need thereof DNA encoding for a polypeptide as disclosed herein.
In some embodiments, the cancer is selected from the group consisting of breast cancer, pancreatic cancer, colon cancer, lung cancer, cervical cancer, ovarian cancer, and prostate cancer. In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is pancreatic cancer. In some embodiments, the cancer is metastatic cancer.
The DNA may be a DNA for encoding any suitable polypeptide as disclosed herein, and may be made, isolated, purified, and provided in any suitable fashion known to one skilled in the art, for example as described in PCT/JP1997/004585 (published, inter alia, in EP 0 945 506 B1). For example, as is well known to one skilled in the art, a preferred way of providing a protein-encoding DNA, such as DNA encoding a polypeptide as disclosed herein, is by inserting the DNA in a recombinant vector, especially a recombinant vector suitable for gene therapy.
In some embodiments, the DNA encoding for a polypeptide as disclosed herein is a DNA encoding for a polypeptide comprising an amino-acid residue sequence having a homology of not less than 80% (in some embodiments, not less than 90%, not less than 95% and even not less than 98% homology) with a polypeptide having an amino-acid residue sequence selected from the group consisting of the amino-acid residue sequences represented by:

    
    
        SEQ ID NO:1 (encoded by DNA having the sequence represented by SEQ ID NO:41, Accession nr. AR343616);
        residues 29-1012 of SEQ ID NO:1 (set-forth in SEQ ID NO:2);
        residues 1-980 of SEQ ID NO:1 (set-forth in SEQ ID NO:3);
        residues 29-980 of SEQ ID NO:1 (set-forth in SEQ ID NO:4);
        residues 1-568 of SEQ ID NO:1 (set-forth in SEQ ID NO:5);
        residues 29-568 of SEQ ID NO 1 (set-forth in SEQ ID NO:6);
        residues 34-549 of SEQ ID NO:1 (set-forth in SEQ ID NO:7);
        SEQ ID NO:8 (encoded by DNA having the sequence represented by SEQ ID NO:48, Accession nr. AR343617);
        residues 29-549 of SEQ ID NO:8 (set-forth in SEQ ID NO:9); and
        residues 34-549 of SEQ ID NO:8 (set-forth in SEQ ID NO:10),

wherein the L-Glu of residue 414 is substituted with an a-amino acid residue different from L-Glu having an amino acid sequence set forth in SEQ ID NO:11-20; the L-Asp of residue 238 is substituted with an α-amino acid different from L-Asp having an amino acid sequence set forth in SEQ ID NO:21-30; or the L-Glu of residue 414 is substituted with an α-amino acid residue different from L-Glu and the L-Asp of residue 238 is substituted with an α-amino acid different from L-Asp having an amino acid sequence set forth in SEQ ID NO:31-40, numbered with reference to SEQ ID NO:1.

    
    


In some embodiments, the DNA encoding a polypeptide according to the teachings herein is a DNA which hybridizes with a DNA described above under stringent conditions. By “DNA which hybridizes under stringent conditions” is meant DNA obtained by colony hybridization, plaque hybridization or Southern blot hybridization using DNA encoding the polypeptide, specifically including DNA identified after hybridization, using a filter on which colony- or plaque-derived DNA has been immobilized in the presence of 0.7 to 1.0 M NaCl at 65° C. and washing the resulting filter using 0.1 to 2×SSC solutions (the composition of 1×SSC solution comprises 150 mM sodium chloride and 15 mM sodium citrate) at 65° C. Hybridization can be carried out according to a method described, for example, in Molecular Cloning, A Laboratory Manual, the 2nd edition (Sambrook, Fritsch, & Maniatis eds., Cold Spring Harbor Laboratory Press, 1989). Specific examples of the DNA which hybridizes include DNA having a homology of 60% or more with a nucleotide sequence of the DNA encoding the polypeptide of an amino acid sequence selected from amino acid sequences represented by SEQ ID NO:1;

    
    
        residues 29-1012 of SEQ ID NO:1 (set-forth in SEQ ID NO:2);
        residues 1-980 of SEQ ID NO:1 (set-forth in SEQ ID NO:3);
        residues 29-980 of SEQ ID NO:1 (set-forth in SEQ ID NO:4);
        residues 1-568 of SEQ ID NO:1 (set-forth in SEQ ID NO:5);
        residues 29-568 of SEQ ID NO 1 (set-forth in SEQ ID NO:6);
        residues 34-549 of SEQ ID NO:1 (set-forth in SEQ ID NO:7);
        SEQ ID NO:8 (encoded by DNA having the sequence represented by SEQ ID NO:18, Accession nr. AR343617);
        residues 29-549 of SEQ ID NO:8 (set-forth in SEQ ID NO:9); and
        residues 34-549 of SEQ ID NO:8 (set-forth in SEQ ID NO:10), preferably DNA having a homology of 80% or more, and more preferably DNA having a homology of 95% or more.
    
    


The DNA encoding for a polypeptide as described herein may be administered by any suitable route, for example as described in PCT/JP1997/004585. For example, in some embodiments the DNA is provided inserted in a recombinant vector (e.g., a bacteria), and the vector administered by parenteral routes. According to some embodiments, the DNA encoding for a polypeptide as disclosed herein is administered in combination with one or more chemotherapeutic agents, as discussed above for administration of Klotho protein as a polypeptide
As noted above, in some embodiments, the teachings herein (e.g., administration of a polypeptide or of DNA encoding therefor) are directed to treating cancer for example, to reduce or eliminate cancerous tumors and metastatic cells and tumors.
In some embodiments, the teachings herein (e.g., administration of a polypeptide or of DNA encoding therefor) are implemented to treat cancer as an adjuvant treatment, that is to say together with another modality of cancer treatment, such as a known modality of cancer treatment, for example, together with radiotherapy, brachiotherapy, surgery and the like.
In some embodiments, the teachings herein (e.g., administration of a polypeptide or of DNA encoding therefor) are implemented to treat cancer as a neo-adjuvant treatment, for example to reduce the size of a tumor prior to surgical excision thereof.
In some embodiments, the teachings herein are implemented prophylactically (e.g., administration of a polypeptide or of DNA encoding therefor). For example, in some embodiments, the teachings herein are implemented on a person who has not yet been diagnosed with cancer but is a member of a group at high risk of being diagnosed with cancer, for example has a genetic inclination to cancer (family history), a pathological indication of pre-cancer (e.g., pre breast cancer), DCIS (ductal carcinoma in situ), clinically significant alcohol use, age or use of HRT (hormone replacement therapy). For example, in some embodiments, the teachings herein are implemented on a person whose cancer is in remission (complete or partial) but may be susceptible to a return of the disease.
Exemplary embodiments of the teachings herein are discussed hereinbelow with reference to specific materials, methods and examples. The material, methods and examples discussed herein are illustrative and not intended to be limiting. In some embodiments, methods and materials similar or equivalent to those described herein are used in the practice or testing of embodiments of the invention. It is to be understood that the invention is not necessarily limited in its application to the details of construction and the arrangement of the components and/or methods set forth in the following description and/or illustrated in the drawings. The invention is capable of other embodiments or of being practiced or carried out in various ways.
EXAMPLES
Materials and Methods
Chemicals, Antibodies and Constructs:
bFGF was obtained from Biological Industries (Kibbutz Beit Haemek, Israel). IGF-1 was obtained from PeproTech Inc (Rocky Hill, N.J.), G418 from Invitrogen (Carlsbad, Calif.). Antibodies used in this study: anti-IGF-1β receptor (Santa Cruz Biotechnology, Santa Cruz, Calif.), anti-phospho-AKT1 (S473), phospho-IGF-1R (Y1131), total pan-AKT (Cell Signaling Technology, Danvers, Mass.), anti-diphosphorylated and -total ERK 1/2 (Sigma), total IRS1 from Upstate Chemicon (Temecula, Calif., USA), phospho-IRS1 (Invitrogen), anti-HA (Covance, Princeton, N.J., USA). The Klotho protein (for murine Klotho having SEQ ID NO. 81) expression vector was a generous gift of Y. Nabeshima (Kyoto University, Japan). Soluble human Klotho protein (hKL) was obtained from R&D Systems (Minneapolis, Minn. USA). Human KL1 (hKL1, SEQ ID NO: 8) and human KL1 (residues 34-549 of SEQ ID NO: 10) were obtained from PeproTech Inc. (Rocky Hill, N.J., USA). Klotho variant construction:
Point mutations were inserted to the DNA (using full length mouse klotho or ssKL1 (KL1 domain with 1-28 amino acid signal sequence) in pcDNA3) to generate amino acid substitutions in positions 416 and 238 of the polypeptide (corresponding to residues 414 and 238 of human Klotho of SEQ ID NO:1 and SEQ ID NO:5) using QuikChange® Site-Directed Mutagenesis Kit (Stratagene, La Jolla, Calif., USA). Accuracy of plasmid sequences was verified by sequencing following mutagenesis. All examples hereinbelow utilized mouse Klotho protein, in which the amino acid sequence is shifted by 2 amino acids from the human Klotho protein (SEQ ID NO:1). For example, Glu at position 416 (E416) in mouse Klotho protein is the equivalent of Glu 414 (E414) in human Klotho protein and variants (i.e., Glu 414 substituted in SEQ ID NO:11-20), and likewise Asp at position 240 in mouse Klotho protein is the equivalent of Asp 238 in human Klotho protein and variants (i.e., Asp238 substituted in SEQ ID NO:21-30).
Cells and transfections: Breast cancer cell lines and HEK293 cells were obtained from the American Type Culture Collection (Manassas, Va.). All transfections used Lipofectamine® 2000 (Invitrogen). Stable clones were generated by selection in complete culture medium containing 750 g/ml G418.

Western blot analysis: For breast cancer cell lines, cells were harvested and lysed for total protein extraction in RIPA buffer (50 mM Tris-Cl pH 7.4, 150 mM NaCl, 1% NP-40, 0.25% Na-deoxycholate, 1 mM EDTA, 1 mM NaF) together with a protease inhibitor cocktail (Sigma). 50 μg protein extracts were loaded on 10% polyacrylamide gels, separated electrophoretically and blotted from the gel onto nitrocellulose membrane (Schleicher & Schuell Bioscience GmbH, Dassel, Del.). The membranes were then immunoblotted with the indicated antibodies.

Colony Assays:

Breast cancer cells: Two days following transfection with the indicated plasmids, G418 (750 μg/ml) was added to the culture media; and at day 14, the cells were stained using gentian violet. Untransfected cells were treated similarly, and all died within the 2 weeks of culture in the selection media. Quantification of the results was performed using AlphaImager 2000 (Alpha Innotech, CA).

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) Viability Assay

Breast cancer cells: 7.5×103 cells/well were plated in 96-well plates, cultured in the appropriate culture media, and transfected with either control plasmid or plasmid expressing Klotho protein; and at indicated times, cells were cultured for two hours with 500 g/ml MTT reagent (Sigma-Aldrich, St. Louis, Mo.). The medium was aspirated, and the cells were resuspended in dimethyl sulfoxide (DMSO). Absorbance of the formazan product was measured by an enzyme-linked immunosorbent assay reader. Optical density is directly correlated with cell quantity.

Statistical Analysis:

    Results are presented as mean±standard deviation (SD). Categorized variables were compared between the study groups using Fisher's exact test and continuous variables were compared using t-test. All significance tests were two-tailed and a P-value of <0.05 was considered as statistically significant. The nature of interaction between Klotho protein and 5-FU or Gemcitabine was analyzed using the additive model (Jonsson et al., 1998). A ratio between the observed and the predicted viability was calculated for all combinations and a ratio <0.8 for the interaction was considered to be synergistic.

Superimposition of KLrP on KL1 and KL2 to Identify Active Site



The classic family-1 glycosidase (glycoside hydrolase) KLrP ((klotho-related protein, Hyashi 2007 JBC 282(42):30889) was crystallized using methods known in the art, with part of its product, glucose and the three-dimensional structure determined. In FIGS. 1A and 1B, the side chains of the active-site amino-acid residues of KLrP are presented in white (outline, identity marked with an asterisk) surrounding the glucose molecule in black.
In FIG. 1A, superimposed on the KLrP side chains are the side chains of amino-acid residues of a portion of native Klotho protein KL1 domain that constitutes an active site of the KL1 domain, where the side-chains are depicted filled and identity designated without an asterisk.
In FIG. 1B, superimposed on the KLrP side chains are the side chains of amino-acid residues of a portion of native Klotho protein KL2 domain that constitutes an active site of the KL2 domain, where the side-chains are depicted filled and identity designated without an asterisk.
KLrP displays the Koshland retaining mechanism. In similar manner to other family 1 glycoside hydrolases, two glutamic acid residues are identified as the catalytic residues. The KL1 Glu-373 is found to be the nucleophile and Glu-165 is found to be the acid/base catalyst.
As seen in FIG. 1A, the active site of the Klotho protein KL1 domain has an arrangement of amino-acid residue side-chains similar to that of KLrP including the Klotho protein Glu-414 superimposed on the KLrP nucleophilic residue Glu-373. The residue corresponding to the KLrP acid/base catalyst Glu-165 is the Klotho protein Asn-239. Asn-239 does not serve as an acid/base catalyst. The Inventors believe that the KL1 domain of Klotho protein may function as a glycoside hydrolase with Asp-238 as the acid/base catalyst, since it is positioned at the “correct” distance from Glu-414 and therefore might replace Asn-239.
In contrast, the active site of the Klotho protein KL2 domain does not share homology with the active site of KLrP, as shown in FIG. 1B. Almost all the residues are different except for Klotho protein Glu-689 corresponding to the KLrP Glu-165 (the acid/base catalyst in KLrP) and the Klotho protein Asn-688 corresponding to the KLrP Asn-688. In Klotho protein KL2 domain Ser-872 replaces the nucleophilic KLrP Glu-373.
Anti-Tumorigenic Activity of Klotho Variant Polypeptides
MCF-7 and MDA-MB-231 cells were transfected with:


    
    
        a. an empty vector (pDNA3);
        b. wild-type full length Klotho (FL-KL) equivalent to SEQ ID NO:1;
        c. a Klotho protein variant according to an embodiment of the teachings herein (FL-KL (E416Q), that is mouse full length Klotho where native L-Glu at 416 is substituted with L-Gln, equivalent to E414Q in the respective human Klotho protein variant) equivalent to SEQ ID NO:11;
        d. wild-type KL1 domain (KL1) equivalent to SEQ ID NO:8;
        e. a KL1 domain variant protein according to an embodiment of the teachings herein (KL1 (E416Q), that is mouse KL1 where native L-Glu at 416 is substituted with L-Gln, equivalent to E414Q in the respective human KL1 variant) equivalent to SEQ ID NO:18; and
        f. wild-type KL2 (KL2) equivalent to SEQ ID NO:85.
    
    


The transfected cells were cultured in media containing G418 for two weeks. Colonies were stained with crystal violet and photographed.
As seen in FIG. 2, the Klotho protein variants were shown to retain at least some of the anti-tumorigenic activity of the comparable native Klotho protein.
Effect of a Klotho Variant Polypeptide on the IGF-1 Pathway


    
    
        MCF-7 cells were transfected with:
        a. a wild type full length Klotho (FL-mKLOTHO/WT) equivalent to SEQ ID NO:1;
        b. a first Klotho variant polypeptide according to an embodiment of the teachings herein (FL-mKLOTHO/E416Q: mouse FL-KL E416Q, equivalent to E414Q in the respective human Klotho protein variant) equivalent to SEQ ID NO:11;
        c. wild type KL1 domain (ssKL1/WT; KL1 domain with 1-28 signal sequence) equivalent to SEQ ID NO:8;
        d. KL1 domain variant polypeptide according to an embodiment of the teachings herein (ssKL1/WT:E416Q: mouse KL1 E416Q, equivalent to E414Q in the respective human KL1 variant) or
        e. an empty vector (pcDNA3).
    
    


After 24 hours, cells were serum starved for 48 hours and treated with IGF-1. Following treatment, cells were harvested and proteins were resolved and immunoblotted using antibodies as indicated.
Results are presented in FIG. 3. FIG. 3 shows that while wild type full length Klotho protein inhibits IGF-1 signaling, as evidenced by inhibition of IGF-1R and ERK1/2 phosphorylation (pIGFR and pERK1/2), E416Q-variant Klotho and KL1 according to the teachings herein do not retain this activity. This suggests that mechanisms other than IGF-1 inhibition may be responsible to the reported anti-cancer activity. Total ERK (tERK) and total IGF-1 (tIGF1) refer to phosphorylated and non-phosphorylated ERK1/2 and phosphorylated and non-phosphorylated IGF-1, respectively.
Effect of Klotho Variant Polypeptides on FGF23 Activity
HEK293 cells were treated as for the previous example, with the addition of FGF23. The two Klotho variant polypeptides (FL-KL/E416Q and KL1/E416Q, equivalent to E414Q in the respective human Klotho protein variants) were found not to function as a cofactor for FGF23. Results are presented in FIG. 4. The variant polypeptides exhibit less FGFR co-activation with FGF23 than the native polypeptides, as determined by phosphorylation of ERK (pERK) compared to total ERK (tERK).
Results and Discussion
The present Inventors have established previously that Klotho protein expression is reduced in breast and pancreatic cancers, and that treatment with soluble Klotho protein inhibits growth of breast and pancreatic cancer cell in vitro and in vivo.
The Inventors have now demonstrated that anti-cancer activities of Klotho are mediated by the KL1 domain. KL1 appears to be a more potent inhibitor of the IGF-1 pathway than full-length Klotho protein. Yet, unlike full length Klotho protein, KL1 does not control phosphate homeostasis and does not interfere with serum phosphate levels. As KL1 is 560 amino acids long (compared to 1012 amino acids of full length Klotho protein), production of KL1 is expected to be easier than that of full length Klotho protein. Even at this size, which is similar to that of antibodies, the KL1 is expected to be stable. Therefore, KL1 may serve as a better candidate than full length Klotho protein for therapeutic use.
Interestingly, variation of a putative enzymatically active site of Klotho protein (as demonstrated with mouse FL-KL E416Q and KL1 E416Q (equivalent to human FL-KL E414Q and KL1 E414Q, respectively) yields a protein that exhibits better anti-cancer properties. These variants do not mediate FGF23 signaling and seem to be more stable than the native protein. Interestingly, the variants do not inhibit IGF-1 signaling, as opposed to the wild type proteins, suggesting IGF-1 plays a modest role, if any, in mediating Klotho protein anti-cancer activities. Therefore, the Klotho variant polypeptides in accordance with the teachings herein may serve as better candidates for therapeutic use than either full length native Klotho or KL1.
SEQUENCE LISTING
This application incorporates-by-reference nucleotide and/or amino acid sequences which are present in the file named “Klotho_SEQ_listing_15APR2013.ST25.txt”, which is 392 kilobytes in size, and which was created on Apr. 15, 2013 in the IBM-PC machine format, having an operating system compatibility with MS-Windows, and is submitted herewith.
Key to SEQ ID NO:




















encoded
L-Glu pos
encoded
L-Asp pos
encoded
L-Glu pos




Poly--
by poly-
414 poly-
by poly-
238 poly-
by poly-
414 + L-Asp
encoded



peptide
nucleotide
peptide
nucleotide
peptide
nucleotide
pos 238
by poly-



SEQ ID
SEQ ID
SEQ ID
SEQ ID
SEQ ID
SEQ ID
poly-peptide
nucleotide



NO:
NO:
NO:
NO:
NO:
NO:
SEQ ID NO:
SEQ ID NO:























AAQ41828
1
41
11
51
21
61
31
71


aa 1-1012


AAQ41828
2
42
12
52
22
62
32
72


aa 29-1012


AAQ41828
3
43
13
53
23
63
33
73


aa 1-980


AAQ41828
4
44
14
54
24
64
34
74


aa 29-980


AAQ41828
5
45
15
55
25
65
35
75


aa 1-568


AAQ41828
6
46
16
56
26
66
36
76


aa 29-568


AAQ41828
7
47
17
57
27
67
37
77


aa 34-549


AAQ41829
8
48
18
58
28
68
38
78


aa 1-549


AAQ41829
9
49
19
59
29
69
39
79


aa 29-549


AAQ41829
10
50
20
60
30
70
40
80


aa 34-549





(pos = position)


SEQ ID NO: 81 mouse kidney AAQ41830 FL mouse Klotho polypeptide


SEQ ID NO: 82 mouse kidney AAQ41831 mouse KL1 polypeptide


SEQ ID NO: 83 AR343618 polynucleotide encoding SEQ ID NO: 81


SEQ ID NO: 84 AR343619 polynucleotide encoding SEQ ID NO: 82


SEQ ID NO: 85: amino acid sequence of Human KL2 domain (amino acids 569-980 of klotho)






It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.
Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.
Citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention.
To the extent that section headings are used, they should not be construed as necessarily limiting.
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## Claims

1. An isolated polypeptide comprising an amino-acid residue sequence selected from the group consisting of:
SEQ ID NO: 1 or a sequence at least 95% identical thereto;
residues 29-1012 of SEQ ID NO:1 or a sequence at least 95% identical thereto;
residues 1-980 of SEQ ID NO:1 or a sequence at least 95% identical thereto;
residues 29-980 of SEQ ID NO:1 or a sequence at least 95% identical thereto;
residues 1-568 of SEQ ID NO:1 or a sequence at least 95% identical thereto;
residues 29-568 of SEQ ID NO:1 or a sequence at least 95% identical thereto;
residues 34-549 of SEQ ID NO:1 or a sequence at least 95% identical thereto;
SEQ ID NO:8 or a sequence at least 95% identical thereto;
residues 29-549 of SEQ ID NO:8 or a sequence at least 95% identical thereto; and
residues 34-549 of SEQ ID NO:8 or a sequence at least 95% identical thereto,
wherein:
the L-Glu of residue 414 is substituted with L-α-Gln.

2. An isolated polypeptide comprising a Klotho amino acid sequence selected from the group consisting of:
SEQ ID NO:1 or a sequence at least 95% identical thereto;
residues 29-1012 of SEQ ID NO:1 or a sequence at least 95% identical thereto;
residues 1-980 of SEQ ID NO:1 or a sequence at least 95% identical thereto;
residues 29-980 of SEQ ID NO:1 or a sequence at least 95% identical thereto;
residues 1-568 of SEQ ID NO:1 or a sequence at least 95% identical thereto;
residues 29-568 of SEQ ID NO:1 or a sequence at least 95% identical thereto;
residues 34-549 of SEQ ID NO:1 or a sequence at least 95% identical thereto;
SEQ ID NO:8 or a sequence at least 95% identical thereto;
residues 29-549 of SEQ ID NO:8 or a sequence at least 95% identical thereto; and
residues 34-549 of SEQ ID NO:8 or a sequence at least 95% identical thereto,
and having at least one amino acid mutation in the catalytic domain of Klotho, wherein the isolated polypeptide inhibits IGF-1 signaling when expressed in MCF-7 cells in vitro, wherein said amino acid mutation is an amino acid substitution selected from the group consisting of Glu414Gln and Asp238Asn.