Note: Descriptions are shown in the official language in which they were submitted.
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PRODUCTS AND METHODS FOR ASSESSING AND INCREASING
KLOTHO PROTEIN LEVELS
BACKGROUND
1. Technical Field
[0001] The present disclosure relates to assessing and increasing Klotho
protein levels in a
patient. In particular, the present disclosure relates to products and methods
for monitoring,
detecting, and/or qualifying the level of endogenous and/or exogenous Klotho
protein and for
diagnosing Klotho protein deficiency in a human or non-human animal (e.g.,
mammalian)
subject, and to the production, purification, and administration of
pharmaceutical or
nutraceutical compositions for increasing Klotho protein levels in the
subject.
2. Related Technology
[0002] Klotho (or alpha-Klotho, a-Klotho, etc.) is a recently characterized
protein encoded
by the KL (or klotho) gene, located on human chromosome 13. Two transcripts
that arise
from a single klotho gene through alternative RNA splicing have been
identified. See Figures
1 and 2. The first transcript is predicted to encode Klotho isoform 1 ¨ a full-
length, 1,012
amino acid, single-pass transmembrane-membrane protein, with a short
cytoplasmic tail
(human residues 1003-1012), a transmembrane (TM) domain (human residues 982-
1002),
and extracellular region or domain (human residues 1-981) comprising two
(largely)
homologous (internal repeat) domains (termed KL1 (human residues 56-506, which
is 450
residues long) and KL2 (human residues 515-953, which is 438 residues long),
which each
share 20%-40% amino acid sequence homology to P-glucosidases, but may lack
similar
levels of glucosidase catalytic activity), and a signal sequence (SS) domain
(human residues
1-33). The SS, KL1, and KL2 domain-containing extracellular region (human
residues 1-981)
may be enzymatically cleaved by a/f3-secretases, and released into the
circulatory stream as a
130 kDa circulating protein, termed soluble klotho (or sKlotho, s-Klotho,
alpha soluble
Klotho, etc.). The extracellular region can also be cleaved into separate 68
kDa protein (KL1
+ SS) and 64 kDa protein (KL2).
[0003] The second transcript, a splicing variant of alpha-klotho mRNA, encodes
a second
isoform of Klotho protein corresponding mainly to the KL1 domain. The internal
splice
donor site is thought to be located in exon 3 of the klotho gene. The
resultant alternatively
spliced transcript contains a 50 bp insertion after exon 3 (Figure 1; gray),
with an in-frame
translation stop codon at the end thereof. The expressed protein product is
secreted into the
circulation and is termed secreted Klotho (or Klotho isoform 2), which differs
from the
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canonical sequence of isoform 1 at amino acid residues 535-549:
DTTLSQFTDLNVYLW
SQLTKPISSLTKPYH, and with amino acid residues 550-1012 missing.
[0004] Accordingly, there may be a number of different Klotho proteins in the
circulation at
any given time, depending on gene expression, RNA splicing, and enzymatic
cleavage.
Despite the existence of various forms of alpha-Klotho protein, only the full
length,
membrane-bound, isoform 1 is known to form a complex with fibroblast growth
factor (FGF)
receptors and functions as an obligatory co-receptor for FGF23 ¨ a bone-
derived hormone
that induces phosphate excretion into urine and which has a regulatory role on
P, and vitamin
D metabolism.
1() [0005] Klotho is highly expressed in the kidney, brain, and to a lesser
extent in other organs,
and may also be found in the blood, cerebrospinal fluid and urine of mammals.
Circulating
levels of soluble Klotho proteins in mammals, including humans, are thought to
decrease
with age. In addition, Klotho-deficient mice exhibit accelerated aging
phenotypes, whereas
over-expression of klotho in mice has been shown to extend lifespan. In
addition, Klotho has
been implicated in a number of cellular processes related to aging. In light
of the foregoing, a
developing hypothesis states that soluble Klotho may function as an anti-aging
compound in
the human body.
[0006] Aging is an inevitable and progressive biological process resulting in
dysfunction and
destruction of almost all tissues and organs, ultimately resulting in death.
The aging of the
human body, for instance, is associated with the decline of cellular function,
which can lead
to the development of a variety of diseases. Aging is thought to be driven by
a tightly
regulated and complex interplay between genetic and acquired factors and is
typically
characterized by an increase in senescence, a quantitative and qualitative
decrease in stem
cells, and abnormal structure at tissue levels.
[0007] As the so-called "baby boomers" generation continues to advance in age,
the
population of aging individuals (e.g., age 60-65) is rapidly increasing
globally. The increased
demand for health care for this aging population places significant financial
burden on any
healthcare system. Recombinant klotho proteins may provide promising
therapeutic agents to
counter age-related health conditions. To date, all relevant treatment data
related to Klotho is
pre-clinical, research trials in animal models. Developing strategies and
health intervention
products and methods based on (i) the production and/or purification (e.g., to
substantial
homogeneity) of recombinant s-Klotho proteins or protein variants and (ii) the
administration
of recombinant s-Klotho proteins or protein variants and/or s-Klotho protein
level-increasing
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health supplements to subjects, especially humans and/or within an increasing
aging
population, may help to ameliorate this situation.
[0008] Currently, there is not a product or method for providing an exogenous
form of
human Klotho protein, such as recombinant soluble human alpha-Klotho protein
or protein
variant, suitable for human use, especially protein that is Current Good
Manufacturing
Practice (cGMP) regulation compliant, as determined and enforced by the U.S.
Food and
Drug Administration (FDA), whether alone or in combination with one or more
additional
active components.
[0009] Likewise, there is not currently a product or method for (naturally)
increasing
endogenous Klotho protein levels or protein production, in a safe and
effective manner,
especially a product that complies with the Dietary Supplement Health and
Education Act of
1994 (DSHEA) or that is Current Good Manufacturing Practice (cGMP) regulation
compliant
(e.g., as determined and enforced by the U.S. Food and Drug Administration
(FDA)),
whether alone or in combination with one or more additional active components,
drugs,
pharmaceuticals, or nutraceuticals.
[0010] Moreover, there is not currently a product (e.g., kit or system) or
method for
efficiently, reliably, reproducibly, practically, and/or (commercially or
economically) viably
monitoring Klotho protein levels, particularly a product or method for on-
demand and/or
real-time monitoring and/or quantification of endogenous and/or exogenous
Klotho protein
levels, and more particularly endogenous and/or exogenous soluble alpha Klotho
protein or
protein variant levels, or for diagnosing Klotho protein deficiency,
particularly serum soluble
Klotho protein deficiency, in human and non-human animals.
[0011] There is also not currently a suitable, large scale method for
efficiently, reliably,
reproducibly, practically, and/or (commercially or economically) viably
producing and
purifying recombinant Klotho proteins, particularly, pharmaceutical grade
recombinant
Klotho proteins, including recombinant Klotho protein fragments and
recombinant Klotho
fusion proteins.
BRIEF SUMMARY
[0012] Embodiments of the present disclosure solve one or more of the
foregoing or other
problems in the art with products and methods for assessing and increasing
Klotho protein
levels in a patient. Some Embodiments include (1) compositions and methods for
stabilizing
Klotho protein in human or non-human animal (e.g., mammalian) blood or serum,
(2)
compositions and methods for monitoring, detecting, and/or quantifying Klotho
protein
level(s), particularly endogenous and/or exogenous soluble alpha Klotho
protein level(s),
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and/or for diagnosing Klotho protein deficiency in a human or non-human animal
(e.g.,
mammalian) subject, and to products useful in performing the same, (3)
pharmaceutical or
nutraceutical compositions for increasing Klotho protein levels in the
subject. Some
embodiments include Klotho protein(s), or fragment(s) or variant(s) thereof,
as a therapeutic
agent, and more particularly, cGMP-grade, human (or non-human) recombinant
soluble
alpha-Klotho protein(s), or fragment(s) or variant(s) thereof, or a
(pharmaceutical)
composition comprising the same. Some embodiments include products and/or
methods for
manufacturing and administering (recombinant) Klotho protein(s), or
fragment(s) or
variant(s) thereof, to a human or non-human subject, and particularly for
increasing serum
.. soluble Klotho protein levels in the subject. Some embodiments include a
nutraceutical or
health supplement composition for increasing serum soluble Klotho protein
levels in the
subject. In particular, some embodiments include products that, when
administered to the
subject, cause an increase in Klotho protein (particularly endogenous soluble
alpha Klotho
protein) level(s), expression, or production, in the subject, and (4) methods
of purifying
recombinant Klotho proteins, particularly pharmaceutical grade recombinant
Klotho proteins,
recombinant Klotho protein fragments, and recombinant Klotho fusion proteins.
[0013] Various embodiments include a composition. The composition can comprise
(i) one
or more recombinant human Klotho proteins, protein fragments, and/or protein
variants,
expression nucleic acid constructs and/or vectors, cell lines and/or cell
suspension cultures,
.. and/or (ii) one or more ingredients that, when administered to the subject,
cause or induce an
increase Klotho protein level(s), expression, or production, particularly
endogenous soluble
alpha Klotho protein level(s), expression, or production, in the subject.
Certain embodiments
include methods of manufacturing, purifying, and administering a composition
of the present
disclosure to the (human or non-human animal) subjects. Some embodiments
include
.. methods for increasing Klotho protein (particularly (exogenous and/or
endogenous) soluble
alpha Klotho protein) level(s), expression, or production, in the subject.
Some embodiments
include products (e.g., diagnostic kits) and/or methods for monitoring,
detecting, and/or
quantifying Klotho protein level(s), particularly (endogenous and/or
exogenous) soluble
alpha Klotho protein level(s), and/or for diagnosing Klotho protein deficiency
in the subject.
[0014] Some embodiments of the present disclosure can include one or more of:
a
recombinant human alpha soluble Klotho protein, protein fragment, and/or
protein variant
(e.g., cGMP-grade human recombinant soluble alpha-Klotho protein, protein
fragment,
and/or protein variant); a composition (e.g., therapeutic composition,
pharmaceutical
composition, medicament, formulation, etc.) comprising a recombinant human
alpha soluble
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Klotho protein, protein fragment, and/or protein variant; a composition
comprising a
recombinant human alpha soluble Klotho protein and a (pharmaceutically-
acceptable) vehicle
(e.g., a carrier or excipient); a composition comprising a recombinant human
alpha soluble
Klotho protein and at least one additional (active) ingredient; a nucleic acid
construct or
vector that encodes a recombinant human alpha soluble Klotho protein; a cell
line that (i)
contains a nucleic acid construct or vector that encodes a recombinant human
alpha soluble
Klotho protein and/or (ii) expresses a recombinant human alpha soluble Klotho
protein; a cell
suspension culture comprising one or more cells that (each) (i) contain a
nucleic acid
construct or vector that encodes a recombinant human alpha soluble Klotho
protein and/or (ii)
express a recombinant human alpha soluble Klotho protein; a method of
manufacturing, and
optionally purifying, a recombinant human alpha soluble Klotho protein; a
method of
manufacturing a medicament (or therapeutic composition ¨ i.e., formulation) of
recombinant
human alpha soluble Klotho protein; a method of administering a recombinant
human alpha
soluble Klotho protein to a (human or non-human animal) subject; a diagnostic
method for
determining Klotho protein deficiency in a subject; a method of diagnosing
Klotho protein
deficiency in a subject; a method of diagnosing a subject as being in need of
receiving a
recombinant human alpha soluble Klotho protein by administration; a method for
evaluating
the efficacy and/or determining an effective dosage of the protein to a
subject in need thereof;
a recombinant human alpha soluble Klotho protein for use in treating a
specific medical or
other condition in a human or non-human animal (e.g., a non-human mammal); use
of a
recombinant human alpha soluble Klotho protein for the treatment of a specific
medical or
other condition in a human or non-human animal; use of a composition
comprising a
recombinant human alpha soluble Klotho protein for the treatment of a specific
medical or
other condition in a human or non-human animal; and/or use of a recombinant
human alpha
soluble Klotho protein in the manufacture of a medicament for the treatment of
a specific
medical or other condition in a human or non-human animal.
[0015] Some embodiments can include a recombinant Klotho protein, wherein at
least a
portion of the protein has at least 80% amino acid sequence identity to at
least a portion of
one of SEQ ID NO: 2 through SEQ ID NO: 70 or SEQ ID NO: 107 through SEQ ID NO:
120
or SEQ ID NO: 125 through SEQ ID NO: 128 or SEQ ID NO: 133 or SEQ ID NO: 152.
Some embodiments can include recombinant protein comprising a Klotho protein
sequence
having at least 80% amino acid sequence identity to at least a portion of one
of SEQ ID NO:
2 through SEQ ID NO: 70 or SEQ ID NO: 107 through SEQ ID NO: 120 or SEQ ID NO:
125
through SEQ ID NO: 128 or SEQ ID NO: 133 or SEQ ID NO: 152, preferably one of
SEQ ID
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NO: 52, SEQ ID NO: 54, SEQ ID NO: 66, SEQ ID NO: 125 through SEQ ID NO: 128,
SEQ
ID NO: 133, and SEQ ID NO: 152 and, optionally at least a portion of an
immunoglobulin Fe
domain sequence, human serum albumin sequence, His (e.g., 6His) tag sequence,
and/or a
Twin-Strep tag sequence, with or without an optional linker sequence
therebetween. Some
embodiments can include two or more recombinant Klotho proteins.
[0016] Some embodiments can include a pharmaceutical composition, comprising a
pharmaceutically effective amount of the recombinant Klotho protein as
described herein and
a pharmaceutically-acceptable carrier. Some embodiments can include a
pharmaceutical
composition, comprising a pharmaceutically effective amount of a recombinant
soluble
1() Klotho protein, at least a portion of the protein having at least 80%
amino acid sequence
identity to at least a subset of amino acid residues 1-981, 29-981, 34-981, 36-
981, 131-981, 1-
549, 29-549, 34-549, 36-549, or 131-549 of human alpha Klotho isoform 1 or
isoform 2, or at
least a portion of one of SEQ ID NO: 2 through SEQ ID NO: 70 or SEQ ID NO: 107
through
SEQ ID NO: 120 or SEQ ID NO: 125 through SEQ ID NO: 128 or SEQ ID NO: 133 or
SEQ
ID NO: 152, and a pharmaceutically-acceptable carrier. Some embodiments can
include a
pharmaceutical composition, comprising a pharmaceutically-acceptable carrier
or excipient
and an effective amount of a recombinant protein comprising a Klotho protein
sequence
having at least 80% amino acid sequence identity to at least a portion of one
of SEQ ID NO:
2 through SEQ ID NO: 70 or SEQ ID NO: 107 through SEQ ID NO: 120 or SEQ ID NO:
125
through SEQ ID NO: 128 or SEQ ID NO: 133 or SEQ ID NO: 152, preferably one of
SEQ ID
NO: 52, SEQ ID NO: 54, SEQ ID NO: 66, SEQ ID NO: 125 through SEQ ID NO: 128,
SEQ
ID NO: 133, and SEQ ID NO: 152 and, optionally at least a portion of an
immunoglobulin Fe
domain sequence, human serum albumin sequence, His (e.g., 6His) tag sequence,
and/or a
Twin-Strep tag sequence, with or without an optional linker sequence
therebetween. Some
embodiments can include a composition, comprising a pharmaceutically effective
amount of
two or more recombinant Klotho proteins.
[0017] Some embodiments can include compositions that comprise a therapeutic
Klotho
protein and at least one other active component, such as a drug, antibody,
hormone, human
cell, tissue, cellular or tissue-based product (HCT/Ps), etc., and/or methods
of administering
the same to human or non-human subjects. Combinatorial compositions and
methods can be
useful for treating subjects having an age-related disorder or condition, a
clinical (e.g.,
metabolic) disorder, a chronic disease, an acute injury, and so forth. The
prophylactic
administration of combination treatments to subjects with no apparent
condition or disorder
can also be useful to delay or prevent certain conditions or disorders
described herein.
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[0018] Some embodiments can include a nucleic acid or nucleic acid construct.
For instance,
embodiments can include an expression vector or nucleic acid. The nucleic acid
can encode a
recombinant human alpha soluble Klotho protein, protein fragment, or protein
variant. The
nucleic acid can encode a native or non-native signaling sequence. For
instance, the nucleic
acid can encode a non-native signaling sequence upstream of (or N-terminal to)
an encoded
Klotho protein sequence. Some embodiments can include a nucleic acid
construct,
comprising a nucleic acid sequence encoding a recombinant protein, the
recombinant protein
comprising a Klotho protein sequence having at least 80% amino acid sequence
identity to at
least a portion of one of SEQ ID NO: 2 through SEQ ID NO: 70 or SEQ ID NO: 107
through
SEQ ID NO: 120 or SEQ ID NO: 125 through SEQ ID NO: 128 or SEQ ID NO: 133 or
SEQ
ID NO: 152, preferably one of SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 66, SEQ
ID
NO: 125 through SEQ ID NO: 128, SEQ ID NO: 133, and SEQ ID NO: 152; and,
optionally
at least a portion of an immunoglobulin Fc domain sequence, human serum
albumin
sequence, His (e.g., 6His) tag sequence, and/or a Twin-Strep tag sequence,
with or without an
optional linker sequence therebetween.
[0019] Some embodiments can include a cell line. The cell line can comprise (a
plurality of)
Chinese hamster ovary (CHO) cell(s), HEK-293 cells, or other suitable (protein
expression)
cell line. In some embodiments, the cells can be dihydrofolate reductase
(DHFR)-deficient
CHO cells, such as CHO-S cells, or glutamine synthetase (GS)-deficient CHO
cells, such as
GS -/- CHO cells. The cells can contain one or more (copies of an) exogenous
nucleic acid
(comprising a transgene or cDNA) that encodes a polypeptide with at least 80%
amino acid
sequence identity to at least a portion of one of SEQ ID NO: 2 through SEQ ID
NO: 70 or
SEQ ID NO: 107 through SEQ ID NO: 120 or SEQ ID NO: 125 through SEQ ID NO: 128
or
SEQ ID NO: 133 or SEQ ID NO: 152. The polypeptide can comprise a human
recombinant
alpha soluble Klotho protein. The exogenous nucleic acid can include a
transgene or cDNA,
preferably having at least 80% nucleic acid sequence identity to one of SEQ ID
NO: 76
through SEQ ID NO: 106 or SEQ ID NO: 121 through SEQ ID NO: 124. In some
embodiments, the nucleic acid can (also) include or encode a promoter
(associated with the
transgene) and/or an optional (exogenous) enzyme, such as a (functional)
dihydrofolate
reductase (DHFR) enzyme, (functional) glutamine synthetase (GS) enzyme, etc.
[0020] At least one embodiment includes a suspension cell culture comprising
the cell line
growing in a liquid medium, preferably comprising a carbon source, a nitrogen
source, and/or
one or more vitamins, minerals, salts, amino acids, supplements, or additives,
such that the
cells express the polypeptide encoded by the nucleic acid. The liquid medium
can be (human,
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bovine, fetal bovine, or other) serum-free and/or animal (or animal-derived)
protein
(component)-free. For instance, the liquid medium can be free of bovine serum
albumin,
human serum albumin, etc. In some embodiments, the suspension cell culture can
comprise a
liquid medium, preferably a serum-free and/or animal protein component-free
liquid medium,
wherein the liquid medium preferably comprises a carbon source, a nitrogen
source, and one
or more vitamins, minerals, salts, amino acids, supplements, or additives,
more preferably
wherein the liquid medium lacks hypoxanthine, thymidine, and/or glutamine, and
the cell line
growing in the liquid medium such that the cells express the polypeptide
encoded by the
nucleic acid, the polypeptide comprising a recombinant Klotho protein.
[0021] Some embodiments include an extract of or from the cells, the liquid
medium, or both
of the suspension cell culture, the extract containing a recombinant protein
having at least
80% amino acid sequence identity to at least a portion of one of SEQ ID NO: 2
through SEQ
ID NO: 70 or SEQ ID NO: 107 through SEQ ID NO: 120 or SEQ ID NO: 125 through
SEQ
ID NO: 128 or SEQ ID NO: 133 or SEQ ID NO: 152. Certain embodiments include a
human
recombinant alpha soluble Klotho protein-containing extract of or from cells,
liquid medium,
or both (e.g., of the suspension cell culture). At least one embodiment
includes an isolated
recombinant protein having at least 80% amino acid sequence identity to at
least a portion of
one of SEQ ID NO: 2 through SEQ ID NO: 70 or SEQ ID NO: 107 through SEQ ID NO:
120
or SEQ ID NO: 125 through SEQ ID NO: 128 or SEQ ID NO: 133 or SEQ ID NO: 152.
[0022] Some embodiments can include a method of manufacturing recombinant
Klotho
protein (e.g., a recombinant human alpha soluble Klotho protein). Illustrative
methods can
comprise producing a recombinant Klotho protein in, for example, Chinese
hamster ovary
(CHO) cells or HEK-293 cells, preferably in dihydrofolate reductase (DHFR)-
deficient CHO
cells, more preferably in CHO-S cells, or preferably in glutamine synthetase
(GS)-deficient
CHO cells, more preferably in GS -/- CHO cells, the protein preferably having
at least 80%
amino acid sequence identity to at least a portion of one of SEQ ID NO: 2
through SEQ ID
NO: 70 or SEQ ID NO: 107 through SEQ ID NO: 120 or SEQ ID NO: 125 through SEQ
ID
NO: 128 or SEQ ID NO: 133 or SEQ ID NO: 152.
[0023] In some embodiments, the manufacturing method can include growing the
cells in a
liquid medium, producing the recombinant soluble Klotho protein in the cells,
and/or
purifying a recombinant soluble Klotho protein-containing extract from the
cells, liquid
medium, or both. The extract can include at least about 90%, preferably at
least about 92%,
93%, 94%, 95%, 96%, 97%, 98%, or 99% dry weight recombinant soluble Klotho
protein
and/or less than about 1-100 ppm host cell proteins (HCP). The cells can be
HEK-293 cells
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or dihydrofolate reductase (DHFR)-deficient CHO cells, such as CHO-S cells, or
glutamine
synthetase (GS)-deficient CHO cells, such as GS -/- CHO cells. The produced
(expressed)
protein can be released (e.g., secreted) from the cells into the liquid medium
and/or can have
one or more glycans attached thereto.
[0024] The cells can contain one or more exogenous nucleic acids that encode
the protein
and, optionally, a functional enzyme, such as dihydrofolate reductase enzyme,
glutamine
synthetase (GS) enzyme, etc. The exogenous nucleic acid can include a promoter
(e.g., a
strong promoter, weak promoter, etc.), such as a promoter customary or typical
for use for
expression of exogenous protein in cell. The exogenous nucleic acid can
include a transgene
or cDNA (e.g., under control of the promoter), preferably having at least 80%
nucleic acid
sequence identity to one of SEQ ID NO: 76 through SEQ ID NO: 106 or SEQ ID NO:
121
through SEQ ID NO: 124, or any other suitable nucleic acid sequence encoding a
Klotho
protein as described herein (e.g., S-Klotho variants).
[0025] The method can include introducing, such as by transfection, the
exogenous nucleic
acid into the cells. The method can include growing the cells in a liquid
medium, such as a
(human, bovine, fetal bovine, or other) serum-free and/or animal (or animal-
derived) protein
(component)-free medium. The medium preferably comprises a carbon source, a
nitrogen
source, and/or one or more vitamins, minerals, salts, amino acids,
supplements, or additives,
preferably in a bioreactor. Depending on the particular cell line, the method
can include
introducing an effective amount of methotrexate (MTX), methionine sulphoximine
(MSX), or
other agent into the liquid medium and/or selecting (e.g., by cell sub-
cloning, limited dilution,
fluorescence activated cell sorting (FACS), etc.) a suspension culture of
viable cells growing
in the liquid medium.
[0026] In some embodiments, selection and/or gene amplification can be
performed by
culturing the transfected cells in a selection medium, such as a medium
lacking hypoxanthine
and/or thymidine (e.g., -HT medium), glutamine, etc. In at least one
embodiment, a low
concentration(s) of MTX can be added or used to amplify the transfected
nucleic acid (or
gene(s) thereof) and, thereby, select for increased protein expression (e.g.,
in DHFR-deficient
cells transfected with a DHFR transgene). Alternatively (or in addition),
selection and/or gene
amplification can be performed by adding MSX (an inhibitor of (endogenous)
glutamine
synthetase (GS)) to suspension cultures of cells having at least one
(exogenous) glutamine
synthetase (GS) transgene.
[0027] The method can include sub-culturing surviving cells or cultures (e.g.,
MTX-resistant
and/or MSX-resistant cells or cultures). The selected suspension culture
and/or selected cells
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can have or exhibit an increased production of the protein (e.g., by the
cell), an increased
concentration of the protein (e.g., in the liquid medium), and/or an increased
copy number of
the exogenous nucleic acid (e.g., per cell) (e.g., as compared to a non-
selected suspension
culture or cell).
[0028] The liquid medium can also include an effective amount of MTX and/or
MSX in
some embodiments. The suspension culture (or cells thereof) can (be selected
to): exhibit an
increased production of the protein (e.g., by the cells); exhibit an increased
concentration of
the protein (e.g., in the liquid medium); secrete the protein (e.g., into the
liquid medium);
and/or have an increased copy number of the exogenous nucleic acid (e.g., per
cell),
preferably as compared to a non-selected suspension culture. The protein can
have one or
more glycans attached thereto.
[0029] Some embodiments of the present disclosure relate to compositions
configured or
formulated to augment, improve, or increase natural soluble alpha Klotho
protein production.
Some embodiments include a composition or method for increasing (endogenous)
Klotho
protein levels in a subject. Some embodiments include a (nutraceutical or
health supplement)
composition for increasing Klotho protein levels, particularly serum soluble
Klotho protein
levels, in a subject. Some embodiments include products that, when
administered to the
subject, cause an increase in Klotho protein level(s), expression, or
production, particularly
endogenous soluble alpha Klotho protein level(s), expression, or production,
in the subject.
[0030] Some embodiments of the present disclosure relate to compositions
configured or
formulated to attenuate Klotho protein damage or degradation. Some embodiments
of the
present disclosure relate to methods of manufacturing compositions of the
present disclosure.
Some embodiments of the present disclosure relate to use of compositions of
the present
disclosure or methods of using the same to treat human or non-human animal
subjects. Some
embodiments of the present disclosure relate to treatment methods involving
compositions of
the present disclosure, including administration thereof to human or non-human
animal
subjects. Such uses and treatment methods can increase endogenous Klotho
protein levels,
such as by augmenting natural soluble alpha Klotho protein production and/or
attenuating
Klotho protein damage or degradation.
[0031] Certain embodiments include compositions of matter that comprise a
health
supplement that, when administered to human or non-human animal (e.g.,
mammalian)
subjects, increases the levels of endogenous, soluble alpha-Klotho protein in
the human or
non-human animal subjects. The composition or health supplement formulation
can comprise
(synthetic) chemical and/or natural components or ingredients. In other words,
components or
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ingredients of the composition or health supplement can be or originate from
(synthetic)
chemical and/or natural sources. An exemplary composition can be adapted (or
chemical
configured), or include a plurality of components adapted (or chemical
configured), to raise
serum soluble Klotho protein levels by increasing or enhancing endogenous
production of
soluble Klotho protein by the mammalian subject.
[0032] An exemplary composition can comprise one or more components,
preferably
selected from the group consisting of, for example, Vitamin D3, Vitamin E,
Vitamin C,
Vitamin K, 3,5,4'-trihydroxy-trans-stilbene (Resveratrol), Pterostilbene, N-
acetylcysteine
(NAC), Troglitazone, Rosiglitazone, Notopterygium incisum Ting, Gentian (Root;
Extract),
Cordyceps (Cordyceps Sinensis (CS) Mycelium), Isoflavones (Soy), Genistein,
Daidzein,
Quercetin (dihydrate; dill, bay leaf, oregano), Docosahexaenoic acid (DHA),
Astaxanthin,
Sesamin (Semen Sesamin Nigrum (Black)), Sesame (Seed Extract), Rosmarinic acid
(RA;
(Marjoram)), Tetradecylthioacetic Acid (TTA), Dicalcium Phosphate (Anhydrous),
Mircrocrystalline cellulose (MCC), Croscarmellose Sodium (Primellose),
Stearing Acid,
Magnesium Stearate, Alpha Lipoic Acid, Conjugate (alpha) Linoleic Acid (CLA),
Probiotics,
Activated Charcoal, and others as known in the art.
[0033] An exemplary composition can comprise one or more components,
preferably in
suitable nutraceutical concentrations, and preferably selected from the group
consisting of,
for example, vitamin C, vitamin D3, vitamin E, N-acetylcysteine (NAC),
quercetin
(dihydrate), rosmarinic acid (RA), pterostilbene, docosahexaenoic acid (DHA),
nicotinamide
riboside (NR), nicotinamide adenine dinucleotide (NAD+), nicotinamide
mononucleotide
(NMN), and one or more probiotic. The one or more probiotic comprises an
effective amount
of the Bifidobacterium species and/or strains Bifidobacterium lactis BL-04,
Bifidobacterium
bifidum/lactis BB-02, and Bifidobacterium longum BL-05, and the Lactobacillus
species
and/or strains Lactobacillus acidophilus LA-14, Lactobacillus rhamnosus LR-32,
and
Lactobacillus paracasei LPC-37.
[0034] Certain ingredients or components can have positive benefits and very
low side
effects in treating or affecting one or more conditions that have been
associated with cellular
and tissue dysfunctions that occur with aging. Certain ingredients or
components can
(directly or indirectly) increase the circulating plasma level of soluble
alpha Klotho (s-
Klotho) levels in human or non-human animal subjects following administration
thereof.
[0035] In at least one embodiment, a composition of the present disclosure can
be or
comprise a Dietary Supplement Health and Education Act of 1994 (DSHEA)-
compliant
nutritional supplement, which may be sold OTC, marketed and/or sold direct-to-
consumer
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(DTC), and/or purchased without a prescription/physician order. In some
embodiments, a
composition of the present disclosure can be or comprise an approved Food and
Drug
Administration (FDA) pharmaceutical.
[0036] In some embodiments, ingredients can be co-administered, preferably via
at least
some co-formulation. In some embodiments, ingredients can be co-administered
in separate
formulations. For example, in some embodiments, ingredients can be co-
administered in a
multi-pill, -capsule, or dosage pack. Some ingredients can be provided in
solid, granular,
powdered, liquid, or other form.
[0037] Some embodiments can include one or more (additional) ingredients or
components,
as described herein. For instance, some embodiments can include one or more
recombinant
(e.g., human or mammalian) Klotho proteins, protein fragments, and/or protein
variants, as
described herein. Certain embodiments can include expression nucleic acid
constructs and/or
vectors, cell lines and/or cell suspension cultures, and methods of
manufacturing, purifying,
and administering the one or more recombinant (e.g., human or mammalian)
Klotho proteins,
protein fragments, and/or protein variants to (human or non-human animal)
subjects. Some
embodiments can include one or more active ingredients, such as a
pharmaceutical or
prescription medications (e.g., ARBs (e.g., Losartan, Valsartan),
testosterone, Vit. D receptor
agonists (e.g., calcitriol, paricalcitol), PPAR (gamma) agonists (e.g.,
thiazolidinediones,
troglitazone, rosiglitazone), and/or others described in the patent references
incorporated
above).
[0038] Some embodiments can include a composition, comprising a
pharmaceutically
effective amount of one or more recombinant Klotho protein and an effective
amount of a
composition (e.g., health supplement) formulated to increase endogenous Klotho
protein
levels or production (e.g., in mammalian subjects). In some embodiments, the
recombinant
Klotho protein can be co-administered with the composition. In some
embodiments, the
recombinant Klotho protein can be combined with a pharmaceutically-acceptable
carrier. In
some embodiments, the administered recombinant Klotho protein can raise serum
soluble
Klotho protein levels by providing exogenous Klotho protein, while the
composition can
raise serum soluble Klotho protein levels by increasing or enhancing (natural)
production of
Klotho protein by the patient or subject.
[0039] Some embodiments can include a treatment method. The method can
comprise
administering to a subject in need thereof a (pharmaceutically) effective
amount of a
composition of the present disclosure. The composition can include (i) a
pharmaceutically
effective amount of one or more recombinant soluble Klotho protein or protein
variant of the
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present disclosure and/or (ii) a pharmaceutically effective amount of a
composition (e.g.,
health supplement) formulated to increase endogenous Klotho protein levels or
production
(e.g., in mammalian subjects).
[0040] Some embodiments can include a method of treating Klotho deficiency
(e.g., in a
human or non-human animal (e.g., mammalian) patient or subject). The method
can comprise
administering a composition to the mammalian subject, optionally or preferably
after
diagnosing the mammalian subject with Klotho deficiency. The composition can
comprise,
for example, one or more of: a pharmaceutical composition comprising a
pharmaceutically-
acceptable carrier or excipient and a pharmaceutically effective amount of a
recombinant
Klotho protein, recombinant Klotho protein fragment, or recombinant Klotho
fusion protein
disposed in the carrier or excipient; and a nutraceutical composition
comprising a plurality of
components adapted to raise serum soluble Klotho protein levels by increasing
or enhancing
endogenous production of soluble Klotho protein by the mammalian subject.
[0041] Some embodiments can include a method of treating an aging-related or
other
condition, disease, or disorder. Some embodiments can include a method of
treating and/or
preventing acute kidney injury (AKI), chronic kidney disease (CKD), or other
condition. The
subject to whom the composition is administered can be suffering from or at
risk for a variety
of conditions (e.g., disorders, diseases, injuries, illnesses, etc.). For
example, some
embodiments include a method of treating one or more chronic diseases and/or
aging-related
condition, such as a physical, mental, neurological, or other condition
associated with
(human) aging. Some embodiments can promote healing, recovery, longevity,
and/or other
beneficial outcome through one or more mechanisms or action. Administration of
the
inventive composition(s) can have a positive therapeutic effect on the course
and outcome of
the condition, including chronic and/or age-related disease and longevity in
human subjects,
and characterization of the same.
[0042] The pharmaceutically effective amount of the composition(s) can be
sufficient to raise
or increase the serum soluble Klotho protein concentration of the subject to a
predetermined
level, such as greater than, equal to, or between about 50 to 3000 picograms
of soluble
Klotho protein per milliliter of serum. The amount can also or alternatively
be sufficient to
maintain the serum soluble Klotho protein concentration of the subject at or
above a
predetermined threshold for a predetermined period of time. Embodiments can
also include
administering the composition(s) to a subject in need thereof so as to
maintain the serum
soluble Klotho protein concentration of the subject at or above a
predetermined threshold for
a predetermined period of time.
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[0043] Embodiments can also include determining a serum soluble Klotho protein
concentration of the subject. Embodiments can also include calculating a
pharmaceutically
effective amount of a composition of the present disclosure. Embodiments can
also include
determining a rate of soluble Klotho protein decline in the serum of the
subject.
Embodiments can also include calculating a subsequent dosage time at which the
serum
soluble Klotho protein concentration of the subject will be at or below a
second
predetermined level based on the determined rate. Embodiments can also include
calculating
a subsequent dosage time and/or amount of the composition sufficient to raise
the serum
soluble Klotho protein concentration of the subject from the second
predetermined level to
1() the first predetermined level. Embodiments can also include
administering the subsequent
dosage amount of the composition to the subject. Certain embodiments can
include
prescribing a regular (e.g., daily) dose or dosage form of the composition to
the subject (e.g.,
based on determined levels of serum soluble Klotho protein concentration of
the subject).
[0044] In certain embodiments, the composition (e.g., protein or supplement)
or raised
soluble Klotho protein levels can (be effective to) modulate the IGF-1 and/or
Wnt signaling
pathways, exhibit P-glucuronidase and/or sialidase activity, suppress the
p53/p21 signaling
pathway, and/or reduce H202-induced cell senescence and apoptosis, preferably
through
suppression of the p53/p21 signaling pathway. The protein or raised protein
levels can
function or be functional as a humoral factor, preferably exhibiting
pleiotropic activity and/or
preferably in the regulation of oxidative stress, growth factor signaling, ion
homeostasis,
and/or regulation of activity of glycoproteins on the cell surface, such as
one or more ion
channel proteins and/or growth factor receptors, such as Insulin/Insulin-Like
Growth Factor-1
receptor.
[0045] In certain embodiments, the composition (e.g., protein or supplement)
or raised
soluble Klotho protein levels can (be effective to) treat one or more aging-
related condition
(or condition associated with (human) aging), such as frailty, bone density
loss or bone
mineral density loss, weight loss, muscular atrophy or degeneration, decline
in muscle mass,
decline in muscle strength, hand strength, leg strength, or physical fitness,
decline in
movement, freedom of movement, quality of life assessment, ejection fraction,
or exercise
capacity, decline in learning, learning capacity, memory, or intellectual
quotient, cognitive
deterioration or forgetfulness, decline in cognitive capacity or function,
decline in synaptic
plasticity or synaptic function, and cellular senescence.
[0046] In certain embodiments, the composition (e.g., protein or supplement)
or raised
soluble Klotho protein levels can (be effective to) treat one or more aging-
related condition
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(or condition associated with (human) aging), such as Alzheimer's disease,
Parkinson's
disease, dementia or vascular dementia, amyotrophic lateral sclerosis (ALS) or
motor neuron
disease (MND), atrial fibrillation, chronic obstructive pulmonary disease
(COPD),
fibromyalgia, adult onset diabetes, arthritis or rheumatoid arthritis,
osteoarthritis,
osteoporosis, glaucoma, cataracts, macular degeneration and other eye
diseases/disorders,
multiple sclerosis (MS), lupus, and/or ulcerative colitis.
[0047] In certain embodiments, the composition (e.g., protein or supplement)
or raised
soluble Klotho protein levels can (be effective to) treat one or more other
diseases or
conditions. For instance, some embodiments of the present disclosure can be
useful in one or
more of treating cancer, lowering serum phosphate levels in a patient,
treating diabetes or a
diabetes-related condition (e.g., Type 1 diabetes mellitus, etc.) in a subject
in need of such
treatment, treating a heart condition (e.g., cardiovascular disease, left
ventricular hypertrophy
(LVH), pathological LVH and/or congestive heart failure, etc.) in a subject,
treating acute
lung injury in a subject (e.g., using nanoparticles), protecting the lung of a
patient against
oxidant injury, detecting early acute kidney injury in critically ill
patients, attenuating
vascular calcification in a subject, improving cognition, treating renal
and/or liver ischemia,
modulating stress response in (human) senescent endothelial cells,
prophylactically and/or
therapeutically treating, preventing, attenuating, arresting, and/or reversing
acute and/or
chronic kidney injury, disease, or disease progression and/or uremic
cardiomyopathy,
.. reversing or attenuating age-related therapy resistance in melanoma or
other cancers,
targeting apoptosis of senescent cells, preferably restoring tissue
homeostasis thereby, and a
variety of other indications.
[0048] Accordingly, embodiments can also include a composition for use in
treating one or
more aging-related or other conditions. An exemplary method of treating an
aging-related or
other condition, disease, or disorder, the method comprising administering to
a subject in
need thereof an effective amount of a composition of the present disclosure.
The composition
can include (i) a supplement, as described herein, and/or (ii) a recombinant
soluble Klotho
protein (e.g., having at least 80% amino acid sequence identity to at least a
portion of one of
SEQ ID NO: 2 through SEQ ID NO: 70 or SEQ ID NO: 107 through SEQ ID NO: 120 or
SEQ ID NO: 125 through SEQ ID NO: 128 or SEQ ID NO: 133 or SEQ ID NO: 152).
Optionally, the composition can include a pharmaceutically-acceptable carrier
or excipient.
[0049] Some embodiments include a method of detecting and/or quantifying
endogenous
and/or exogenous Klotho protein, and more particularly endogenous and/or
exogenous
soluble alpha Klotho protein. Some embodiments include a method of diagnosing
Klotho
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protein deficiency in a subject. Some embodiments include a product (e.g., a
system or kit)
for detecting and quantifying Klotho protein levels, and more particularly
endogenous and/or
exogenous soluble alpha Klotho protein levels. Some embodiments include
methods of
diagnosing Klotho protein deficiency in a subject. Some embodiments can
include a method
of treating Klotho deficiency in a subject.
[0050] Certain embodiments can include or utilize a blood or serum sample of
the
(mammalian) subject, preferably capillary blood and/or blood obtained from a
finger-prick or
other non-phlebotomic (non-venesectic) blood draw technique. It will be
appreciated,
however, that phlebotomic (venesectic) and other suitable blood draw
techniques are also
1() contemplated herein.
[0051] Some embodiments include a kit. Embodiments can include, for example, a
sample
collection container. The container can have an opening configured for
receiving capillary
blood. The capillary blood can contain, or be suspected of containing, an
amount of soluble
Klotho protein in some embodiments. Embodiments can include a sample
preservative or
anti-coagulant, preferably comprising one or more of EDTA and Heparin. The
sample
preservative or anti-coagulant can be disposed in a sample collection
compartment of the
container in some embodiments. The solution can be configured to stabilize
soluble Klotho
protein for at least 24 hours, and up to 7 days or more, at room temperature
or without
freezing. Embodiments can optionally include a capillary blood sampling
device, preferably
comprising a lancing device or lancet.
[0052] Some embodiments include a method. For example, some embodiments can
include a
method of stabilizing Klotho protein in a mammalian blood sample. Embodiments
can
include, for example, drawing or obtaining capillary blood from a mammalian
subject and
storing the capillary blood in a container for at least 24 hours at room
temperature or without
freezing. The container can have a preservative or anti-coagulant disposed
therein, such that
the preservative or anti-coagulant is mixed with the capillary blood in the
container. The
preservative or anti-coagulant can stabilize the soluble Klotho protein for at
least 24 hours at
room temperature or without freezing. The preservative or anti-coagulant can
be or comprise
one or more of heparin, lithium heparin, EDTA, and K2 EDTA.
[0053] Embodiments can also include collecting the capillary blood in a
container, and/or
mixing the capillary blood with a preservative or anti-coagulant to form a
mixture. In some
embodiments, drawing capillary blood can include lancing skin of the mammal
with a lancet.
In some embodiments, the step of collecting the capillary blood in the
container can include
collecting at least or about 10-1000u1, preferably about 50-200u1 of the
capillary blood in the
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container Some embodiments can include storing the mixture or allowing the
mixture to be
stored for at least 3 days at room temperature or without freezing, the
preservative or anti-
coagulant stabilizing the soluble Klotho protein for the at least 3 days at
room temperature or
without freezing.
[0054] Some embodiments can optionally include assaying the mixture for the
presence of
the soluble Klotho protein. The step of assaying the mixture for the presence
of the soluble
Klotho protein can include detecting the soluble Klotho protein and/or
quantifying the
amount of soluble Klotho protein. Detecting the soluble Klotho protein and/or
quantifying the
amount of soluble Klotho protein can include contacting the mixture with an
antibody
configured to bind the soluble Klotho protein. Detecting the soluble Klotho
protein and/or
quantifying the amount of soluble Klotho protein can include performing ELISA
on the
mixture using an antibody configured to bind the soluble Klotho protein. Other
detection
methods may include mass spectrometry or Multi-Analyte Profiling (xMAP), such
as
Luminex technology (e.g., miniaturized liquid array bioassay, small lasers,
light emitting
diodes (LEDs), digital signal processors, photo detectors, and/or charge-
coupled device
imaging), etc.
[0055] Some embodiments include a method of diagnosing Klotho deficiency in a
mammalian subject or a method of treating Klotho deficiency in a mammalian
subject.
Embodiments can include, for example, providing or obtaining a fluid
biological sample. The
sample can include mammalian serum, preferably in the form of capillary blood.
The method
can optionally include mixing the mammalian serum with a preservative or anti-
coagulant to
form a mixture. The sample can also or alternatively include a mixture
comprising
mammalian serum, preferably in the form of capillary blood, and a preservative
or anti-
coagulant. The method can include assaying the mixture for the presence of
soluble Klotho
protein. The step of assaying the mixture for the presence of the soluble
Klotho protein can
include detecting the presence of the soluble Klotho protein, if any, in the
mixture and/or
quantifying the amount of the soluble Klotho protein, if any, in the mixture,
optionally after
storing the Klotho-stabilized mixture for a period of time at room temperature
or without
freezing. The method can include diagnosing the mammalian subject with Klotho
deficiency
when no soluble Klotho protein is detected in the mixture or when the amount
of quantified
soluble Klotho protein in the mixture is less than a predetermined amount. In
some
embodiments, diagnosing the mammalian subject with Klotho deficiency can
include
displaying the Klotho deficiency determination or diagnosis on a user
interface of the
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computer system and/or producing a file or report, in physical or electronic
form, that
displays the Klotho deficiency determination or diagnosis.
[0056] In some embodiments, providing or obtaining a fluid biological sample
can include
drawing capillary blood from the mammalian subject and/or collecting capillary
blood in a
container. In some embodiments, the step of assaying the mixture for the
presence of soluble
Klotho protein can include performing ELISA on the mixture using an antibody
configured to
bind the soluble Klotho protein. Some embodiments can include administering a
composition
to the mammalian subject when no soluble Klotho protein is detected in the
mixture or when
the amount of quantified soluble Klotho protein in the mixture is less than a
predetermined
amount. Mass spectrometry can also or alternatively be performed.
[0057] The disclosed strategies, products, and/or health intervention methods
specifically
configured to increase circulating and/or endogenous levels of soluble Klotho
may help to
ameliorate the situation and problems associated with decreased Klotho levels
in patients.
[0058] Some embodiments may include any of the features, options, and/or
possibilities set
out elsewhere in the present disclosure, including in other aspects or
embodiments of the
present disclosure. It is also noted that each of the foregoing, following,
and/or other features
described herein represent a distinct embodiment of the present disclosure.
Moreover,
combinations of any two or more of such features represent distinct
embodiments of the
present disclosure. Such features or embodiments can also be combined in any
suitable
combination and/or order without departing from the scope of this disclosure.
Thus, each of
the features described herein can be combinable with any one or more other
features
described herein in any suitable combination and/or order. Accordingly, the
present
disclosure is not limited to the specific combinations of exemplary
embodiments described in
detail herein.
[0059] Additional features and advantages of exemplary embodiments of the
present
disclosure will be set forth in the description that follows, and in part will
be obvious from
the description, or may be learned by the practice of such exemplary
embodiments. The
features and advantages of such embodiments may be realized and obtained by
means of the
instruments and combinations particularly pointed out in the appended claims.
These and
other features will become more fully apparent from the following description
and appended
claims, or may be learned by the practice of such exemplary embodiments as set
forth
hereinafter.
DETAILED DESCRIPTION
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[0060] Embodiments of the present disclosure relate to compositions and
methods for
increasing serum soluble Klotho protein levels in a human or non-human animal
(or
mammal) subject and to compositions and methods for monitoring, detecting,
and/or
quantifying Klotho protein level(s), particularly endogenous and/or exogenous
soluble alpha
Klotho protein level(s), and/or for diagnosing and/or treating Klotho protein
deficiency in the
subject. Some embodiments include one or more compositions, comprising (i)
recombinant
human Klotho proteins, protein fragments, and/or protein variants, expression
nucleic acid
constructs and/or vectors, cell lines and/or cell suspension cultures, and/or
(ii) products (e.g.,
supplements) that, when administered to the subject, cause an increase Klotho
protein
level(s), expression, or production, particularly endogenous soluble alpha
Klotho protein
level(s), expression, or production, in the subject. Certain embodiments
include methods of
manufacturing, purifying, and administering the compositions of the present
disclosure to the
(human or non-human animal) subjects and/or methods for increasing Klotho
protein level(s),
expression, or production, particularly (exogenous and/or endogenous) soluble
alpha Klotho
.. protein level(s), expression, or production, in the subject. Some
embodiments include
methods for monitoring, detecting, and/or quantifying Klotho protein level(s),
particularly
(endogenous and/or exogenous) soluble alpha Klotho protein level(s), and/or
for diagnosing
Klotho protein deficiency in the subject.
[0061] Before describing various embodiments of the present disclosure in
detail, it is to be
understood that this disclosure is not limited only to the specific
parameters, verbiage, and
description of the particularly exemplified systems, methods, and/or products
that may vary
from one embodiment to the next. Thus, while certain embodiments of the
present disclosure
will be described in detail, with reference to specific features (e.g.,
configurations,
parameters, properties, steps, components, ingredients, members, elements,
parts, and/or
.. portions, etc.), the descriptions are illustrative and are not to be
construed as limiting the
scope of the present disclosure and/or the claimed invention. In addition, the
terminology
used herein is for the purpose of describing the embodiments, and is not
necessarily intended
to limit the scope of the present disclosure and/or the claimed invention.
[0062] While the detailed description is separated into sections, the section
headers and
contents within each section are for organizational purposes only and are not
intended to be
self-contained descriptions and embodiments or to limit the scope of the
description or the
claims. Rather, the contents of each section within the detailed description
are intended to be
read and understood as a collective whole, where elements of one section may
pertain to
and/or inform other sections. Accordingly, embodiments specifically disclosed
within one
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section may also relate to and/or serve as additional and/or alternative
embodiments in
another section having the same and/or similar products, methods, and/or
terminology.
[0063] To facilitate understanding, like references (i.e., like naming of
components and/or
elements) have been used, where possible, to designate like elements common to
different
embodiments of the present disclosure. Similarly, like components, or
components with like
functions, will be provided with similar reference designations, where
possible. Specific
language will be used herein to describe the exemplary embodiments.
Nevertheless it will be
understood that no limitation of the scope of the disclosure is thereby
intended. Rather, it is to
be understood that the language used to describe the exemplary embodiments is
illustrative
only and is not to be construed as limiting the scope of the disclosure
(unless such language is
expressly described herein as essential).
[0064] For the sake of brevity, the present disclosure may recite a list or
range of numerical
values. It will be appreciated, however, that where such a list or range of
numerical values
(e.g., greater than, less than, up to, at least, and/or about a certain value,
and/or between two
recited values) is disclosed or recited, any specific value or range of values
falling within the
disclosed values or list or range of values is likewise specifically disclosed
and contemplated
herein. By way of illustrative example, disclosure of "up to 1,000 mg" of a
particular
ingredient or component includes a specific disclosure of: (i) any value
greater than zero and
less than or equal to 1,000 milligrams, including but not limited to 0.01 mg,
1 mg, 5 mg, 10
mg, 50 mg, 100 mg, 500 mg, 750 mg, 990 mg, and 1,000 mg; and/or (ii) any range
of values
from or between greater than zero and less than or equal to 1,000 milligrams,
including but
not limited to 0.01-1,000 mg, 1 mg - 990 mg, 5 mg - 750 mg, 10 mg - 500 mg,
and 50 mg -
100 mg. Similarly, disclosure of "at least 80% amino acid sequence identity"
or "80%400%
amino acid sequence identity" includes a specific disclosure of: (i) any whole
percentage
value between 80% and 100%, including 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
89%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%, as well as any
fraction
of a percent value therebetween; and/or (ii) any range of percentage values
between 80% and
100%, including, by way of non-limiting example, 81%400%, 82%400%, 83%400%,
84%-100%, 85%-100%, 86%-100%, 87%-100%, 88%-100%, 89%-100%, 90%-100%, 91%-
100%, 92%-100%, 93%-100%, 94%-100%, 95%-100%, 96%-100%, 97%-100%, 98%-100%,
and 99%-100%.
Abbreviated list of defined terms
[0065] To assist in understanding the scope and content of the foregoing and
forthcoming
written description and appended claims, a select few terms are defined
directly below.
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[0066] Unless defined otherwise, all technical and scientific terms used
herein have the
same meaning as commonly understood by one of ordinary skill in the art to
which the
present disclosure pertains.
[0067] Various aspects of the present disclosure, including systems, methods,
and/or
products may be illustrated with reference to one or more embodiments, which
are exemplary
in nature. As used herein, the terms "embodiment" mean "serving as an example,
instance, or
illustration," and should not necessarily be construed as preferred or
advantageous over other
aspects disclosed herein. In addition, reference to an" embodiment" of the
present disclosure
or invention is intended to provide an illustrative example without limiting
the scope of the
1() invention, which is indicated by the appended claims.
[0068] As used in this specification and the appended claims, the singular
forms "a," "an"
and "the" each contemplate, include, and specifically disclose both the
singular and plural
referents, unless the context clearly dictates otherwise. For example,
reference to a "protein"
contemplates and specifically discloses one, as well as a plurality of (e.g.,
two or more, three
or more, etc.) proteins. Similarly, use of a plural referent does not
necessarily require a
plurality of such referents, but contemplates, includes, specifically
discloses, and/or provides
support for a single, as well as a plurality of such referents, unless the
context clearly dictates
otherwise.
[0069] As used throughout this disclosure, the words "can" and "may" are used
in a
permissive sense (i.e., meaning having the potential to), rather than the
mandatory sense (i.e.,
meaning must). Additionally, the terms "including," "having," "involving,"
"containing,"
"characterized by," variants thereof (e.g., "includes," "has," and "involves,"
"contains," etc.),
and similar terms as used herein, including the claims, shall be inclusive
and/or open-ended,
shall have the same meaning as the word "comprising" and variants thereof
(e.g., "comprise"
and "comprises"), and do not exclude additional, un-recited elements or method
steps,
illustratively.
[0070] Except in the examples, or where otherwise expressly indicated, all
numerical
quantities in this description indicating amounts of material or conditions of
reaction and/or
use are to be understood as modified by the word "about". As used herein, the
term "about,"
with regard to a value, means +/-10% of the stated value or amount represented
thereby. For
instance, throughout the present disclosure, the term "about" is used in
connection with a
percent concentration or composition of a component or ingredient (e.g., in a
mixture, such as
a fluid or liquid mixture, aqueous mixture, solution, etc., optionally or
preferably measured as
a w/w percent, w/v percent, v/v percent, etc.). In such instance, the term
"about" and/or the
21
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term "+/-10%" implies and/or includes +/-10% of the stated numeric value, as
opposed to +/-
percentage points of the recited percent. By way of example, where 20% w/w of
a
component or ingredient reflects 20g of the component or ingredient per 100mL
of total
mixture, the term "about" and/or the term "+/-10%" implies and/or includes a
recited range
5 .. from 18g to 22g (i.e., from 18% w/w to 22% w/w), not a range of 10% w/w
to 30% w/w.
Alternatives for so-called "about" values and/or +/-10% include +/-1%, +/-2%,
+/-3%, +/-
4%, +/-5%, +/-6%, +/-7%, +/-8%, or +/-9% of the stated value, each of which is
contemplated as a suitable alternative to or substitute for the term "about"
or the use of +/-
10% herein.
10 [0071] As used herein, the terms "approximately" and "substantially"
represent or imply an
(or any) amount close to the stated amount (e.g., that still performs a
desired function or
achieves a (desired or expected) result). For example, the terms
"approximately" and
"substantially" may refer to an amount that is within, or less than, 10%, 5%,
1%, 0.1%,
0.01%, or other percent of a stated amount. As used herein, the term
"substantially devoid"
means (1) an undetectable or unquantifiable amount, (2) less than or below an
amount
generally considered by those skilled in the art to reflect a detectable or
quantifiable amount,
and/or (3) less than or below an amount generally considered by those skilled
in the art to be
functional or able to achieve a (desired or expected) result (e.g., less than
10%, 5%, 1%,
0.1%, 0.01%, or other percent).
[0072] By "Quantum satis" (also referred to as "q.s." or "qs") is meant the
amount that is
enough. Accordingly, a component or ingredient "qs 100%," "provided at qs
100%," or "qs
to 100%" indicates that the component or ingredient is provided or included in
an amount
sufficient to complete the composition or to bring the total (of all
components, whether
recited or not) to 100%. It is noted, however, that a (final) component or
ingredient "qs
100%," "provided at qs 100%," or "qs to 100%" does not indicate that the
mixture consists
of, consists essentially of, or only contains the components listed or recited
immediately
before the "qs 100%" component. In other words, "qs 100%," and similar terms,
is meant to
be an open-ended expression indicating the source of the remainder, whatever
that remainder
maybe.
[0073] As used herein, the term "product(s)" includes compositions,
formulations, and
mixtures, as well as devices, apparatus, assemblies, kits, systems, and so
forth. Similarly, the
term "method" includes processes, procedures, steps, and so forth.
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[0074] As used herein, a "feature" or "aspect" of the present disclosure or
embodiment
disclosed herein refers to a property, attribute, component, ingredient,
element, member, part,
portion, (method) step, or other facet of the subject matter at hand.
[0075] The word "or" as used herein generally means any one member of a
particular list,
but also includes any combination of two or more members of said list.
Similarly, the term
"and" can be interchangeable with the terms "or," either of which can be
understood to mean
"and/or".
[0076] The term "comprising" is synonymous with "including," "having,"
"containing," or
"characterized by." These terms are inclusive and open-ended and do not
exclude additional,
unrecited elements or method steps.
[0077] The phrase "consisting of' excludes any element, step, or ingredient
not specified in
the claim. When this phrase appears in a clause of the body of a claim, rather
than
immediately following the preamble, it limits only the element set forth in
that clause; other
elements are not excluded from the claim as a whole.
[0078] The phrase "consisting essentially of' limits the scope of a claim to
the specified
materials or steps, plus those that do not materially affect the basic and
novel characteristic(s)
of the claimed subject matter.
[0079] The terms "comprising", "consisting of', and "consisting essentially
of' can be
alternatively used. When one of these three terms is used, the presently
disclosed and claimed
subject matter can include the use of either of the other two terms.
[0080] The terms "plurality" and "at least two" are used interchangeably.
[0081] The term "condition" refers to any disorder, disease, injury, or
illness, as understood
by those skilled in the art, that is manifested or anticipated in a patient.
Manifestation of such
a condition can be an early, middle, or late stage manifestation, as known in
the art, including
pre-condition symptoms, signs, or markers. Anticipation of such a condition
can be or include
the predicted, expected, envisioned, presumed, supposed, and/or speculated
occurrence of the
same, whether founded in scientific or medical evidence, risk assessment, or
mere
apprehension or trepidation.
[0082] The term "patient," as used herein, is synonymous with the term
"subject" and
generally refers to any animal under the care of a medical professional, as
that term is defined
herein, with particular reference to (i) humans (under the care of a doctor,
nurse, or medical
assistant or volunteer) and (ii) non-human animals, such as non-human mammals
(under the
care of a veterinarian or other veterinary professional, assistant, or
volunteer).
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[0083] The terms "medical professional" as used herein, generally refers to
any individual
or entity that is responsible for or participates in providing health care to
an animal, including
human and non-human animals, such as non-human mammals, with particular
emphasis on
licensed health care providers or unlicensed providers, such as assistants,
technicians, and/or
volunteers, particularly those covered under the (blanket) license or
insurance of a health care
provider. This term may, when contextually appropriate, include an oncologist,
a surgeon, a
physician's assistant, a nurse, a phlebotomist, a veterinarian, etc.
[0084] The term "cancer" refers to an abnormal, typically uncontrolled, growth
of cells. A
"cancerous cell" as used herein comprises a malignant cell having an abnormal,
typically
uncontrolled, growth. As such, the term cancer is an umbrella term
encompassing a plurality
of different distinctive diseases characterized by malignant cells growing in
a typically
uncontrolled manner.
[0085] The term "diagnosis," or diagnosing," and similar terms, as used
herein, is not
intended to convey or require (in all cases) a certified, regulatory-
compliant, medical
diagnosis, as required or regulated by the U.S. Food and Drug Administration
(FDA) or any
other government or non-government entity, organization, or agency. Rather,
"diagnosis," or
diagnosing," and similar terms, as used herein, relates to providing
information that may be
indicative of a condition and/or helpful in making a determination related to
the condition.
[0086] The term "co-administration" and similar terms refer to concurrent,
sequential,
and/or combined administration of two or more components. For instance, two
components
can be co-administered by administering each component in a separate dosage
concurrently,
simultaneously, or sequentially (e.g., distinct administrations separated by a
period of time).
The period of time can be very small (e.g., substantially, immediately
following a first
administration) or longer (e.g., 1-60 seconds, 1-60 minutes, 1-24 hours, 1-7
days, 1-4 weeks,
1-12 months, and so forth, or any value or range of values therebetween).
Concurrent or
simultaneous administration can include overlapping administration timeframes
for the two
or more components or administration of a combination product comprising a
mixture of the
two or more components.
[0087] As used herein, "room temperature" may refer to any temperature above
freezing
(0 C, or other (equivalent) freezing temperature, depending on the presence of
freezing point
adjusting components) and below normal human body temperature (37 C, or other
(equivalent) boiling temperature, depending on the presence of boiling point
adjusting
components), preferably above about 4 C and below about 35 C, more preferably
between
about 5 C and about 32 C, between about 8 C and about 30 C, between about 10 C
and about
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30 C, between about 10 C and about 25 C, between about 10 C and about 20 C,
between
about 10 C and about 15 C, between about 15 C and about 30 C, between about 15
C and
about 25 C, between about 15 C and about 20 C, between about 20 C and about 30
C,
between about 20 C and about 25 C, between about 20 C and about 22 C, or any
value or
range of values therebetween.
[0088] As used herein, "refrigeration" may refer to any temperature above
freezing (0 C)
and below about 32 C, preferably between about 1 C and about 30 C, between
about 1 C and
about 25 C, between about 1 C and about 20 C, between about 1 C and about 15
C, between
about 2 C and about 20 C, between about 2 C and about 15 C, between about 3 C
and about
20 C, between about 3 C and about 15 C, between about 4 C and about 20 C,
between about
4 C and about 15 C, or any value or range of values therebetween.
[0089] As used herein, "nucleic acid," and similar terms, refer to a naturally
occurring or
synthetic oligonucleotide or polynucleotide, whether DNA or RNA or DNA-RNA
hybrid,
single-stranded or double-stranded, sense or antisense. In particular, nucleic
acids can
include, without limitation, DNA, RNA, cDNA, gDNA, ssDNA, dsDNA or any
combination
thereof Nucleic acids of the disclosure can also include nucleotide or nucleic
acid analogs
(e.g., BrdU), and non-phosphodiester (internucleoside) linkages or backbones
(e.g., peptide
nucleic acid (PNA) or thiodiester linkages), known in the art.
[0090] The terms "peptide" or "protein" as used herein refers to any peptide,
oligopeptide,
.. polypeptide, gene product, expression product, or protein. A peptide is
comprised of
consecutive amino acids. The terms "peptide" or "protein" encompass naturally
occurring or
synthetic molecules.
[0091] As used herein, the term "standard amino acid" includes: alanine - ala -
A; arginine
- arg ¨ R; asparagine - asn ¨ N; aspartic acid - asp ¨ D; cysteine - cys ¨ C;
glutamine - gln -
.. Q; glutamic acid - glu ¨ E; glycine - gly ¨ G; histidine - his ¨ H;
isoleucine - ile ¨ I; leucine -
leu ¨ L; lysine - lys ¨ K; methionine - met ¨ M; phenylalanine - phe ¨ F;
proline - pro ¨ P;
serine - ser ¨ S; threonine - thr ¨ T; tryptophan - trp ¨ W; tyrosine - tyr ¨
Y; and valine - val ¨
V.
[0092] As used herein, "codon optimized" or "codon optimization" refers to the
process of
modifying or changing codons in a nucleotide sequence to codons that are
preferred or more
closely match the pattern of codon usage in the organism used for expression
of the molecule.
Thus, codons can be optimized for usage in a particular organism in which
expression is
desired based on known codon usage in the organism in order to enhance the
effectiveness of
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expression of the nucleic acid, e.g., to achieve faster translation rates and
high accuracy. The
codon usage in a particular organism is known.
[0093] The encoding nucleic acid molecule can be a modified wild-type or a
codon
optimized sequence, where the codons are optimized for expression in a
particular host cell,
such as mammalian cells, e.g., CHO cells or 293 cells, or in a yeast, or a
plant cell, eukaryotic
cells.
[0094] In particular examples, the nucleic acid sequence can be codon
optimized, for
example, to increase expression levels of the encoded sequence. The particular
codon usage
is dependent on the host organism in which the modified polypeptide is
expressed. One of
skill in the art is familiar with optimal codons for expression in mammalian
or human cells,
bacteria or yeast, including for example E. coil or Saccharomyces cerevisiae.
For example,
codon usage information is available from the Codon Usage Database available
at
kazusa.orjp.codon (see e.g., Richmond (2000) Genome Biology, 1:241 for a
description of
the database. See also, Forsburg (2004) Yeast, 10:1045-1047; Brown et al.
(1991) Nucleic
Acids Research, 19:4298; Sharp et al. (1988) Nucleic Acids Res., 12:8207-8211;
Sharp et al.
(1991) Yeast, 657-78).
[0095] The first definition of an acronym or other abbreviation applies to all
subsequent
uses herein of the same abbreviation and applies mutatis mutandis to normal
grammatical
variations of the initially defined abbreviation; and, unless expressly stated
to the contrary,
measurement of a property is determined by the same technique as previously or
later
referenced for the same property.
Illustrative Embodiments
[0096] Illustrative embodiments of the present disclosure include, but are not
limited to,
compositions, compositions of matter, products, articles of manufacture,
formulations,
combination products, co-administrations, co-formulations, dosages, process,
methods,
treatment protocols, diagnostic methods, kits, and so forth.
Products and Methods for Stabilizing Klotho Protein in Blood
[0097] The stability of Klotho proteins (e.g., soluble Klotho) in blood or
serum, especially at
room temperature, and more especially for (extended) periods of time, has not
been reported
previously. Figure 3 illustrates the surprising and unexpected stability of
Klotho protein in
human serum at room temperature, as well as other temperatures, over extended
periods of
time.
[0098] Without being bound to any theory, typical blood or serum protein
diagnostics
require a venous blood draw to collect a suitable amount of blood for
monitoring, detecting,
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and/or quantifying protein (levels). Moreover, the drawn blood samples are
typically
processed soon after sampling (e.g., same day, within minutes or hours of
sampling, etc.) or
refrigerated (below room temperature) to preserve protein and/or sample
stability. The
Inventors have found, however, that Klotho protein in blood can be stable for
up to 7 days, 9
days, 14 days, or more, at room temperature, in some embodiments of the
present disclosure.
In some embodiments, for instance, adding EDTA and/or Heparin to the sample
can stabilize
Klotho protein for at least 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9
days, 10 days, 11
days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 day, 19 days,
20 days, 21 days,
22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, or 30
days at room
temperature (data not shown). By way of non-limiting example, levels of Klotho
in blood
samples collected from four donors were relatively constant over a 7-day
period (and over a
14-day period), at room temperature, with added EDTA and/or Heparin (see
Figure 3).
Additionally, both tested anticoagulants, EDTA and Heparin, showed no
significant
difference in terms of Klotho concentration. This was surprising and
unexpected to the
inventors.
[0099] Room temperature storage may be important for commercial diagnostic
applications, in which refrigeration of hundreds, thousands, tens of
thousands, etc., of
samples may not be commercially practical (logistically and/or monetarily).
With a suitable
room temperature stabilizing method, as described here, commercial diagnostic
testing of
Klotho blood or serum levels can be accomplished.
[00100] In light of the foregoing, an embodiment of the present disclosure can
include a
method and/or product that combines EDTA and/or Heparin to a biological sample
(e.g.,
blood or serum) that contains Klotho protein. Some illustrative products can
include a sample
collection kit comprising a sample collection container and a sample
preservation
composition.
[00101] Some embodiments include a method of stabilizing Klotho protein in a
mammalian
blood sample. The method can include obtaining capillary blood from a
mammalian subject.
The capillary blood can contain (or be suspected of containing) an amount of
soluble Klotho
protein. The method can include storing the capillary blood (e.g., in a
container) for at least
24 hours (or, preferably, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
18, 21, 28, or 30 days,
or more), preferably at room temperature. The capillary blood sample can be
mixed with a
preservative or anti-coagulant. For example, the container can have a
preservative or anti-
coagulant disposed therein or added thereto, such that the preservative or
anti-coagulant is
mixed with the capillary blood in the container. The preservative or anti-
coagulant can be or
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comprise one or more of heparin, lithium heparin, EDTA, and K2 EDTA. The
preservative or
anti-coagulant can stabilize the soluble Klotho protein for the at least 24
hours (or longer),
preferably at room temperature. It will be appreciated, however, that
refrigeration and/or
freezing are also contemplated herein.
[00102] In addition, the Inventors have found that measured Klotho protein
levels from
finger stick (about 10-1000u1, preferably about 50-200u1 of blood) are
consistent with Klotho
protein levels measured from venous blood draw in the same subject. Thus,
Klotho protein
can be accurately, reproducibly, and reliably measured in relatively small,
commercially
practical volumes of blood. This was surprising and unexpected to the
inventors. In
particular, the Inventors had expected that the quantity of blood collected in
a standard vial
may be required in order to accurately, reproducibly, and reliably measure
Klotho protein in
blood.
[00103] Without being bound to any theory, venous blood draws (e.g., for FDA-
approved or
other diagnostics) typically require a professional phlebotomist. Venous blood
draws are also
inconvenient and painful to the patient. A finger stick method, on the other
hand, is
convenient and low cost. Commercially, direct-to-consumer applications (e.g.,
diagnostic
kits) may require at-home blood sampling. In such cases, a venous blood draw
may be
undesirable and/or impractical. Embodiments of the present disclosure can
permit capillary
blood sampling with a simple and convenient finger prick (or other means of
sampling
capillary blood). The amount of capillary blood sampled can be, for example,
between about
10-1000u1, preferably about 20-800u1, more preferably about 30-600u1, still
more preferably
about 40-400u1, still more preferably about 50-200u1 of capillary blood.
[00104] In light of the foregoing, some embodiments of the present disclosure
can include
products and methods for capillary (non-venous, non-phlebotomic, etc.)
sampling of blood or
serum. Some embodiments can include a capillary blood sampling device.
Illustratively, the
capillary blood sampling device can be or comprise a lancing device and/or
comprise a
(blood) lancet). In some embodiments, the blood sampling or lancing device can
be or
comprise a finger stick apparatus, such as a (spring-loaded) finger pricker.
It will be
appreciated, however, that the present disclosure is not limited to fingers
and quick-prick
devices. In some embodiments, the lancing device or lancet thereof can be
disposable and/or
replaceable. Embodiments can also include written instructions for using the
kit to collect a
blood sample.
[00105] An illustrative method of stabilizing Klotho protein in a mammalian
blood sample
can comprise obtaining capillary blood from a mammalian subject, the capillary
blood
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containing an amount of soluble Klotho protein, and storing the capillary
blood in a container
for at least 24 hours at room temperature, the container having a preservative
or anti-
coagulant disposed therein, the preservative or anti-coagulant being mixed
with the capillary
blood in the container, the preservative or anti-coagulant stabilizing the
soluble Klotho
protein for at least 24 hours at room temperature, the preservative or anti-
coagulant
comprising one or more of heparin, lithium heparin, EDTA, and K2 EDTA. In some
embodiments, storing the capillary blood mixed with the preservative or anti-
coagulant for at
least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 18, 21, 28, or 30 days at
room temperature, the
preservative or anti-coagulant stabilizing the soluble Klotho protein for at
least 2, 3, 4, 5, 6, 7,
1() 8, 9, 10, 11, 12, 13, 14, 18, 21, 28, or 30 days at room temperature.
The sample of mixture
may also be refrigerated in certain embodiments.
[00106] In certain embodiments, a small amount of blood (1-4 drops, about 10-
1000u1,
preferably about 50-200u1 can be collected (e.g., at home) and sent via
unfrozen,
unrefrigerated, or uncooled (e.g., ambient or room temperature), normal
shipping methods to
a diagnostic facility. This provides a substantial benefit over existing
methods and products.
Accordingly, personal, at-home, or other non-clinic-based testing or
diagnostics (e.g., lateral
flow with quantification/measurement indicator, home ELISA kits, etc.) are
contemplated
herein. It will also be appreciated, however, that clinical or clinic-based
testing or diagnostics
are contemplated herein.
Products and Methods for Measuring Klotho Protein Levels
[00107] Some embodiments include a method of detecting and/or quantifying
endogenous
and/or exogenous Klotho protein, and more particularly endogenous and/or
exogenous
soluble alpha Klotho protein. Some embodiments include a method of diagnosing
Klotho
protein deficiency in a subject. Some embodiments include a product (e.g., a
system or kit)
for detecting and quantifying Klotho protein levels, and more particularly
endogenous and/or
exogenous soluble alpha Klotho protein levels. Some embodiments include
methods of
diagnosing Klotho protein deficiency in a subject.
[00108] It has been postulated that levels of circulating, soluble Klotho
protein in the serum
of mammals decrease with increasing age. Prior to the present disclosure,
however, reliable,
reproducible data as to what age this postulated decrease in serum Klotho
levels may begin to
take place, what the normal level (or range) of naturally-occurring,
circulating soluble Klotho
protein is in human and non-human animals, what the rate of decline in
circulating Klotho is
in human and non-human animals, and how low the level (or range) of naturally-
occurring,
circulating soluble Klotho protein gets in human and non-human animals, was
not available
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or widely accepted. Commercially-available antibodies may not be suitable for
reliable,
reproducible, and/or cost-effective detection of (both endogenous and
exogenous) Klotho
protein in a (crude and/or substantially unpurified) mammalian blood or serum
sample.
Accordingly, suitable antibodies (e.g., for blood sample ELISA detection) were
developed in
accordance with the following procedure.
[00109] Phase-1: Klotho Protein Expression: (mouse and human) alpha Klotho
proteins of
various sizes (e.g., amino acid residues 34-981, 34-549, etc. for human) with
a (C-terminal)
6xHis tag, (C-terminal) Fc tag, or (N- or C-terminal) HAS tag were
manufactured. Gene
constructs corresponding to the above proteins were synthesized and cloned
into a
commercially-available expression vector, which were then transfected into HEK-
293 cells or
CHO cells for Klotho protein production. Expressed Klotho protein was purified
by methods,
known and described in the present disclosure. Illustratively, IMAC and IE
protein
chromatography were used, yielding 0.78 mg/mL protein sample (measured by UV
absorbance at 280nm) in 330 mM NaCl, 20 mM Na-Phosphate buffer, pH 8Ø
Similarly,
Protein A chromatography followed by size exclusion chromatography, as
described herein,
yielded active protein in monomeric and multimeric (e.g., dimer and tetramer)
form.
[00110] Quality control: protein purity and integrity of purified protein was
analyzed in 4-
20% SDS-PAGE (reducing and non-reducing) stained with GelCode Blue reagent.
See e.g.,
Figure 4, in which:
[00111] Lane 1: Klotho ECD, Lot# 170918A, 2ug/lane, reduced.
[00112] Lane M: Protein molecular weight marker.
[00113] Lane 2: Klotho ECD, Lot# 170918A, 2ug/lane, non-reduced.
[00114] Lane 3: BSA standard, 2ug/lane, reduced
[00115] The theoretical, calculated molecular weight of the expressed protein
was 109.97
kDa.
[00116] Phase-2: Chicken Polyclonal (IgY) and Mouse Monoclonal (hybridoma)
development: Expressed protein was used as an antigen to immunize mice and
chickens for
development of mono- and polyclonal antibodies. Mouse monoclonal is preferred
in some
embodiments for ELISA antibodies. After the fusion process (splenocytes with
myeloma
cells), two rounds of hybridoma selection were performed, around 3,000 clones
were initially
screened and finally narrowed to 10 clones, based on activity. These 10 clones
were banked
and also cultured and purified to produce ¨5mg of purified antibody from each.
[00117] The tables below present illustrative mouse sera IgG titer evaluations
using target
antigens, by indirect ELISA (Standard ELISA protocol. TMB 5 min.).
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[00118] Antigen 1 ¨ 6xHis tagged Klotho 0.5ug/mL in 50 mM Na-bicarbonate
buffer.
[00119] Antigen 2 ¨Klotho-Fc 0.5ug/mL in 50 mM Na-bicarbonate buffer.
31
serum Mouse ID
bleed 14
,
dilution MI M2 M3
M4 MS 0
, - -
Preimmune 10000 0.068 0.065 0.056 0.062 0.063 0.059
0.059 0.065 0.061 0.06 no k-,=.õ negative Ctrl n.)
o
1-,
PreiMmune 1:10,000 0.068 0.053 0.067 0.051 0.052 0.049 0.05 0.049 0,049
0.05 o
1-,
lst bleed 1:10,0-90 2784 2,701 3.10:3
3,179 . 3.028 3,012 2.828 2.414 :3.15 3.187
1st bleed 1:100.008 0.599 0.58 0.826 0,817
0.768 0.72 0.486 0.431 0.936 1.001 --.1
2nd Heed 1.10,000 . 3,252 3.343 3,345 3,454
3,3:82 3.432 3.3:93 _ 3.361 3.39 3,398 6xl-a
tagged :Moth
2nd bieed 1:i00000 1.408 1.428 1.819 1.824 1.922
1.981 1.404 1.291 1.811 1.946
3rd bleed 1:10,000 3.475 3.404 3.39 3.446
:3.531 3.461 3.374 3.397 3.53 3.516
3rd bleed 1:100000 1.606 1.622 1.421 1.396 1.942
'1.983 1.299 1.33 2.323 2,432
,
serum mouse ID:
bleed # .
diiiition M1 M2 M3
M4 MS
P
Preimmune 1:10,000 0.049 0.048 0.047 0.045 0.05 0,048
0.045 0.046 0.045 0,045 nri Aig negative Ctri
Preimmune 1:10,000 0.054 0.040 0.051 0.047 0.047 0.045 0.046 0.047 0.045 0.049
,.µ
1st bleed 1:10,000 2.149 2.409 2.145 2.038
1.423 1.479 1.65 1.551 :3.15 3.16
'
n.)
.
1st bleed 1:100,000 0.415 0.399 0,364 0.377
0,203 0.222 0.27.3 0.228 0.72:5 078)
o
r.,
_ .
,
2nd bleed 1:10,W0 3.401 :::::: ::::::: 3 369 '3.212 3.089
:: 3.334 3.343 3.276 3.258 3.453::M M3.49'_-
Klotho-R: ,.µ
,.µ
,
, :2nd bieed 1:100,0(.0 , 1.113 1 098 _:i,.,:',09 U.919 0.9E5
1.056 a917 0.8c4 1.369 1.434
3rd bleed 1:10 000 n 3.482 3.489 2,956 3.091
3:327 3,3s 3.19 3.104 3.4n 3.493
3rd bleed 1:100,000 1.276 1.22.2 0.:. 674 0.721
1.083 .1.123 0.333 0.861 1..755 1.706
serum I mouse ID
:bleed 0
dilution M1 M2 M3
M4 MS
4th bleed 1:1,000.,000 0.053 0.059 (3,057 f).053 0.Ø55
0.053 0.055 a a52 0.056 a.059 no Ag seg3tive Uri
IV
3rd bleed L1.00,000 1D77 1,053 0.94 0.912 1.298
1.35 0.,79 0.827 1.513 L607 n
1-3
3:rd Weed 1:1fri00õ000 0.1.63 0,174 0.16 0.158 0.227 .i123
0.146 0.14 0 . 284 0.281
:
:::::: 6xHi$tagged Klotho cp
4th bleed 1:-100,000 1.137 1.143 0.9.21, 0,925
1,165 1,128 0.766 0.719 aa':1.741::::
::...1,756 " N
o
4th bleed 1:1,000,000 0,183 0.188 0,148 0.151 0.183 0.188
0,126 C.122 0.312 0,401 1-,
oe
- -1
o
.6.
c,.)
_
_______________________________________________________________________________
_______________________
. .s. . eruinq mouse
D
h.lee#
cilE.,ftion MI. M2 M3
MLI MS 0
5th. bleed . 1:1, C.00,00`.) 0Ø53 0.055 0,058. 0.051
0.052 0.051 :0.057 .0,052 0.051 0.05 , flo
Ag negative Ctri n.)
o
1-,
4th Need l:100,000 L.2(58 1.194 0..887 0.902
1.261 1.27 0.76.7 0..761 ml..807 1,81.9 o
1-,
4th Need 1: L l'rf.),C)00 0.211 :)..217 0.158 0.1.55 0..208
0.199 0127 C1134 a318 0.3=1
...*..........
,....................*:* 6xl-i4 tagged Klotho. (...)
(...)
. 5.th. bleed 1:100,0.00 1.191 1188 1.206 1.188 1.32
1.3043 0.92 0.908 ::::A.85.1:0 ::::::::: 1 87 =
= .. .::::........ ...,.......:.
:::::::::::::::...,.....,:::::::::::::õ -4
(...)
.. 5th. bleed 1:1,0.00,0a) 0,215 0..218 0207 . 0,23
0.252 0.25 Ø174 , 0,155 0..3.81 0.359
Table 1
[00120] Summary of Table 1: Mice M3 and M5 were selected for spleen
collection.
[00121] Primary screening of Klotho hybridoma: Splenocytes from mice #M3 and
#M5 collected and fused with myeloma cells according to
standard protocol.
P
[00122] Hybridoma supernatants were evaluated by indirect ELISA using target
antigens (standard indirect ELISA protocol. TMB 10 min.).
,
,
,
[00123] Antigen, Klotho protein (Lot#170918A, 0.78 mg/mL) 0.5 ug/mL in 50 mM
Na-Bicarbonate, 100 ul/well, +4 C, overnight. g
rõ
rõ
[00124] Anti-mouse HRP, Peroxidase AffiniPure Goat Anti-Mouse IgG (subclasses
1+2a+2b+3), Fcy Fragment Specific, 1:4000 (Jackson Lab., -
,
,
,
,
Cat#115-035-164) in SEDB, 100 uL/well, 1 hour, RT.
,
[00125] Selection cut-off OD450nm >0.5.
[00126] The tables below present primary Klotho hybridoma screening. Clones
with OD450nm between 0.5 and <2 are highlighted in yellow
(light grey). Clones with OD450nm >2 are highlighted in orange (dark grey).
od
n
1-i
cp
t..)
o
,-,
cio
O-
o,
.6.
(...)
(...)
(...)
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........ _____________________________________________
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:cr.) a., ir'.4) r...) 1' O.) :cf/ f.-!. g.1 :,,,,,j
c,...1 (7, tH (r'. Apr.,t,.:,..: LC) N 4.1,-
,4 0 CT) 1,1 r-1 0 :';,. C7.) r,:.) ,--ri r,--1
(_.:), r^:1 0 '...::::...ni.i:: r'l N 11
C4 C)
.... ====
==== ====
ril ,, 1--:-, -4 ,:--õI .,-..4 IS) N, . ':-..r.'
r.---. ,.....,1 r-, 00 0 1:0 õ...i =:-.-1 4 0 0 NI kll C.:57: Crq
6 6 ..- ''..'-' D .6 c-) ,==== 6 c-,-,; c-,3 6 .6 o 6 0'
r.-.4 r,P1 ID r.....,-.,f=A te:i rx-,
0 0 'µ...?. "..! (.-.:,^ 0 r.:1 r'l 0-1 --.1 ;õ-,:, r^1 ,^1 0 0 c=^1 7-^1,
0 0 r"., =,-- c,-.) 57., C:2 n c....3 6 6 c.:,.' d o 6 d
r --. c:,, ez, co
,..,....
0 ¨, (,.., 0 ,--! ,--,
r"- 0 0 C.".,, '", ,--I ,--I 0 0 r..-. ¨I =-.1 o a 0 6 7-1 q
4.! K....4 .4.1
(.0 ........
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......... .4,...,,
a 0. ,,,-, c.:-.,, ,4 cr.,
e'..l 01 1,...Itl ;7,1
Vi'll g.'r'l IC.:(`)=: :i..06H1::' Z 1:= 4:",3
= 0 ID r=-1 IN CD 0 0, 0 --,I
= . ..-r: , = ..::::::.,:::: , 4 , , .
, 4 . , , r-.4
QQ0,0 rA-1
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, r^r. 1.-. N r,K.j. 0) C'41 r--, r-D (...) ')co .q,
f'-. i4`'.,`i 6) oo
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tn
U.:.
tn.
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:.r N cr. r--... CO. (.4 r`,1 r=-=J
Kt _..-.::'' 't t'-'4 0 Crj 0 0 ,--i ,¨.1 t--4
...,4
C"3 0 ri 6 d 6' 0 c:3 r,) c:.3 c:3 d d 6 d H
IA)
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0
;;.,-1
1.Z. u...,.
W., !....0 ,71 0 al^ :...4 r---. rr
r'N 0 0, CD 0 1::::? = = 0 ,:--I N 1-1 I:::; 0 t7.) 0 e:'",) N 1-.1
C.) C.".= r.....", 0 0 '''''' 0 d o <;...) ......) c...)
.:=.;46. = '' '=
.......
== IA w., A :VI.
aj
r-, CI ..7..
r, N--= IA CD r'.1 0 CI) IX.5. i--. ri "^-I 0"1 (Cl
:::=.f..19:::= to VI 1.1") :.a
0 0 '.'4 ,=-'1 1--! 0 E.-) 0 --.7-; ','-'1 =:,-
-1 1-1 re) 0 r=.:65,I:ii:.: ....4 e=-=4 ....-1
.?..A = . r = :::::.=:*::: . . r V
0 6 d d c;) d 6 ,...j ,...). C....) o o 0
.tV%.
CL
..... 1 VI
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-4 (C ,,..) 0 w L......, rõ.v x Q.. .4 tti 4. 0 WLt, WI w
r.-,.., no
.,. ,.õ..
rn (0
34
34
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NI V) N M 0 C.
C;) ...,-.,::! r:D ri C..) 0 r0 ;...1 0 0 r>1 ÷-1 EN mi = 1
d 6 ..oi. d c-, d ,==.-
,..; d d 6 d c.-.; 0 d d d
R0r-10001c;;;P:f0, .,.....1 a a r'l CP 0 r^.1 rti .71
C . 1 0
0 41 1, .,..:6 , ,,,,, -.-..., if.:-. i..p. c, 0 (-
,;.,..,/ Cr) .4. ;7 4 :0: 4 CT,: I:4 õ,.,.4 0 ,-,1 z=-: ,--1
cp c:2 ..--i oN
d e ,i.... d = d d d d 6 6 6 .,.:,.- d 6 d
...M!!
m = 0 N. N., 0 tr.', 1,0 r- ko, r,.,1 0 ,,,.. m 7.1-
..-1 I- =.i:.
a) m. r-i 0. r.:s. .,--i Q 0 o ori 0 ,..,, 0 7--, ro c.., .,-.1 ,i,....
d ..0 ...,=,-;., :7", 0. ri 0 0 f,'. -4 0 0 r,":, d o
d co'i:-.
e--..4 ,-.1 r.;-, ,--, 0 1..0 1.0 ..<....1 c;" r...... 041 -=-.4 0 q", v-i
6,4
.2-1 :0-,) ix-J if-i v.5 0 01 r'd 1 ."... e-1 CO
.01. .m.-:.
ai .111,
al .ca
r4 Lr.1 : ,;',1 r.,,i .,-. r'l (X) "=.3 !Crl :M.
t.g: tri 0 ri"..1 r.,-4 r', r..! Of,' ,
Q .=.'-1 0 0 (.."..;, 0 e..7..! ,Z:i 0 0 S',.:) 0 0 n ,--i --i ,zt.
6 o 6 (,; p- 0 C# 0 0 = 0 C., -0 0 =-cri=
.T.
kt
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;1;
,õ c,-)-2, ::::6 p i.,,r., ,,,:-?, ,)., 1 1P- i....
tr) .i .1 i...--1 0 .:^.. r.;.." el = ..
--: --; ' "'" .. ===== ::::::;.... ',..i.
6 o ,:,- 6 C'qii.Ø.i.:.
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C.0 l', 5:.'.1 In 0 a N, 1.0 (in E":0
d d (...;) d 6 (,..::: o o VA 0 0 C.:31 d d
0 .=:
oli ....... ........
........
.......
........
....... *i.'...
.......
M r-`1 ci 6 0
.. . .. .. . " .. . __.." -
Kt) 1,-.1.,
0 Kt
. ..
F", ..gi''' to i':.:=:::VX r---,, r.-.: .4.,?, t-,,, c.,4 ci-,-.,
,.-. i??? ix., czo 0 kt.
=.--4 ,..i.:. t=-=4 ,.:.,..:4..p, r.,,, ...0 __ L. , __ r, __ ,---
i __ k-:.T __ cv __ 41.-.I __ QQ. __ C--.1 __ ...,1 __ 1-",
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=Y-1 Cr.,
c,',.:
......
¨, .,...
(1! la
0 C* in.:, N o'..:. r.r) '4:3 N .0 r, 0 (.31 cr., M 5.0
--1 ID ez) al k:'," 7=5 '....al EZ.3 0 C...) l'A `="-, q =.,1 0
73.
0 0 C..:', 0 0 0 0 0 . :4., " 0 000 r
r ' 0 eti ej 0 ',. = Ø
C7-1. ........ = = = = = = = =
= = = = = = = = ........
W Fu
:IR 63-
vs vi
.4 aj cd r:4 1.4.1 4, Z 14.4 .4:: -411 r,,I. In W
la, ct.1 X al
= 40 EX
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CA 03101100 2020-11-19
WO 2019/113373 PCT/US2018/064333
N 117) U5 r.171 ::iiM,.,A =:--1 0. M 01 t....4 11:.. 0 r, ct) rxr .71 M M
v;,1 !"! C..." rM i:M5.,,k .7'1 5=^1 .,=1 Q ;..1 v=i In O. C.',J" Q
C...... C;) C.
.........
......... ................
,M:::: =
_ til l'=1 M.. 'ml '4 ill 0 '4. if) irt CD C",1 -4 r, ey) 9-1
Q.) V..) N ''µ..) V..3 N :i .11 ,--i ki";.. it) 1,-; 1.0 1.11 .".43
rA ,I--i 0 0 '.....). I'M 0 C:::? TM rsj 1-1 M 0 r-4 0 0 0 0
C,-- .0 0: .0 :0 ..0 . 0 C.3
RN
r., '4) ".!' C.;9).i ,..4
t'.f.,3 .:;11 t,>;.) t>õ) ai co .;,... N kf..) M. N t.n tin (i)
r,4 C) --1 -'7.) '-::-.". C..' 0 ....! .;cisi ;õ--) ,4 e:?, 0 .-4 q 0 0 0
6 o 6 d d d 6
t;rt 0 0 0 ra 0 C',1 r-,2 0
.
(-4 0 0 rt, ."--4 Cri e',1 r.'i. v4. C...) 0 r.-1 .=-..-1 ?.....I V. al
11) 0 CD 1,-. 0 0 0. 1%.1
OD IA 0 4-1 0 ..7.4 0') 0 0
õ ..
t'-'4 r=-= 6,rr r- '76 . Cri :04 0/ ...,..--; 01 oh r.õ. r.., m ret m
, 0.A.cy,:,, ,,A=f co ,r1', N. t-- ,izt ..,,-;-, ..0 r).0 tY,).
F.-- e..., 0 tz-., ,-,1 0 0 ,-,1 0 r-, 0 c-...r, 0. c.,-3
, - .. . -, ='-. .-, ..,
6 (6 (6. . . . .: -. :'. .::-..; (..-j :c....:', d !,-
.:2 0 0 0 C) .0 0 C.
In VP.
4.3 f,14
4.--,-. .I...
',..1 . (9
a KI' 01) 0, N if) 0 :7,) ip :4-
. t, 0 0 ti, r.-...= 'S? ip
T., ,,Prri
0 0 1-.1 a '--,' 0 a 0 a a ....) ......) q
. = . . , . .. . ,
m T r'....j I P V 5?) P ,t--1 pi 0 +7-1 r-,, :=-=-i .zr t-...(..)
U.
to a 0 H 0 eN C..) C.) C....).
C; d. d CZ;. 6 C'.. 6 .41:1
.X
= :in:
r,or 14) CI) r=-* i.n (1) r--, C:3 NO
.d'A 'T
'1 . 5=1 C.',) r-i C.) C) 1-1 0 5',.2.0 .0 .,....) r.,...1 C.., .s.1 CD 0
=.,4
:. , , . , õ , , . ¨ . . ¨
IA, M.
1õ.1, ta=
DD..!
..,,'.' .
g1.,.., Ch. fi'l it'3 (:17). Cf:..? !fl. 14...' c.-'71 N
On 57.1 ...-4 C. CD 0 Iwi CD ..7.-$ (.4
,:...r., ,....õ ,....1 ,:.i) ,..1 :i: imt, ,..s ,i C,)
d d O. d 0 d d 6 -1 0 d d .-ci ,=,:c;),. c'. o .0 ,m
(,) *: a)
If)
r, 0-.! r, 0 m (-..r L.c, 0,5 .:',4 cõi is..) N, ,;:t I.C., a ,',..=
m, 9 o al 9 m ris-1 if) 1,0 al .....Z,J
N -1=4 V.".1 (.2:. cõ) K-'-.1 0 0 ,--1 N e,4 WI ==1 0 ,"1 0 ,4-4 C...)
00 00 C.) 05 (....) 0
. .4 .: =
. . . . . . . . .....-.. ......sfm
.,.,.,.,.,.,.,..
........ ........ ,...1 = s...1.: ........
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= = = = = = = =
.....i.
= = = = = = = :
= = = = = = = = ....v.v..: W . V
r-i LD N.,
'= 0 C.,1
: a 0.,
,..., VI
tit) rAo
I--- Jr,
36
CA 03101100 2020-11-19
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PCT/US2018/064333
........
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r;."! r.-j E',...1 ul CC:
.,=,,,,4 i:".;:v::: tf".4 (Y) al ,t, ,---i i.".0 0 t.k,4 .0 .0 eK. kr) rA ,-
--1 M ..-4ii
0 0 Q. =.-I :0 ,.-1 0 0.`=6:=
-v.:.
= = = = = = =
L) 0 t.(,) ("4 ''(:) C..) 4 r'd e'-4 Cx) i:,-2 0
,.-.,.1 r-I r-1 C.:, 0 C..) r-i r4 C....! õ,j 0 C.)
= , , . , õ =
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=,--, ,--1 c.-5-1 ...t, v.! 14? kr) *"...1' N. Op N. e'r... f.^.1 e..e.)
01. 1'0
0 1-`1 .''..1 r-- r- kr..) m ....--1 r-4 0 co V.,) =CP: 14. cf.' I-- r-,1
c.)
y,,,,i r^1 ...--1 C2 0 0 1`..) r-1 =,--1 Tri C..) 0 Cf 0 Q 0 r-1
0. 0 0 r...... .0 tp. ..i,;):.
= = = = = = = = .........
:....:.:.:.:.:.:.: = = = = = = = =
= = = = = = = = . = = = = = = = .
.................
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k=D u". i'..W..H',) is.) N- c:0 el'.;)1.4-
e'-µ4 (J=."'-i r.X) :.:.:M....: N. ( -.' V) in ki.:' ID r-, k%) m vs
r....:.f.:*.:.:.:.:. C".,) =,'"I 0 ) CZ) q c.,-; ..-.) o o a f. = = õ1
Cli C..)
: FA
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1;<1 1,1 ......4 v.-I tit; ,-1 r.:',1 0 r-1 C.K) 0 C...",) .0 0 0 0 ',-1
'Iwi
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-.1 c...1 (.7,- =,,,,i ....4
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tti.. et.
t.,--. N kr-) re! (.;....., QC., un a m rq m ln r,-.
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. ... . ...
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L.ri M
Q 0 :;::::,! V.1 CD. CD N 0 .1....;.'d V', 0 l C) !" t,.....3! c.."
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g.
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V-.'./ fõ)
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q....:
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= f 0 T. ' G. Ã9050 E6050 0,0 T. 50 TST.T.
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CA 03101100 2020-11-19
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0074 0.075 0,080 0.075 . 0:111 yD
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0.076 0,072 0,088 0,242 0,714 c,.)1-,
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0,075 0.083 0.095 0.075 0.093 --.1
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6 0,101 0,107 0,082 0,268 0 09.2 0100 0,095
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Target spedt/C.: do nets).; Non4
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r.,
8 0,111 0.057 0.087 0,087 0.076 '0.078 0.075
0,066 0,073 0,073 0_076 0.089 ?
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,
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0,081 0.035 0.090 0,072 0,081
E 0,137 0.095 9.082 0.077 0.073 0,081 0.105
0.076 0.094 0.04 0.126. 0.078
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0073 0.127 0.061 aloft.. o.105
õ:õ................................................õ õ........
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0.141 0.119 0.385 0460 0.333
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0,086 :0,063 a 069 0 079 0094
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0,104 0,117 0.110 0.098 6.303
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a 11 0 acas o299 0,111 0,107
r.,
B 0.195 0.053 0.073 0.106 .0,060,7 0.063 0..079
a 347 0a-r5 a 373 D.067 0:101 .
,
,-µ
C 0.109 0.381 0.170 0,076 Ø090 0.064 0,06,5
:0,095 a 261 0.063 0.058 iit);.573,K*f: i2-µ
0,0183 0,06.1 it,6,89:i a 081 Q.,052 0.076 :c1,071
o&. 0,089 0,131 0431
E 0.140 0.0:24 0.080 0;142 0.070 0,080
0..076 ::::::::,,i1 .4.:: 0 105 0077 0,081 0087
F 0.138 0.093 0.074 0,072 0.076 : 0.109:. .0090
0.075 0.063 0.033 0.083 0.102
ia 0.112 0,076 aan 0.080 0.081 0.168 0.131
0.280 0.122 0.080 0;399 0.1:32
H 0,132 0,093 0,090 0,104 0.092 0.092 0.131
0.089 0,091 0.091 0.104 0,117
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Target sperfficckwie4 22C:12 2204
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CA 03101100 2020-11-19
WO 2019/113373
PCT/US2018/064333
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6 a...) L.c.). c.-.) :cp. !..e) a) ''4 kr,, c-.o A
,e,:i .-1 .r.--i c) 0 c) r-1 0 0
0i V)
,N r N. r
::; . p 0 .-- ,--;
r-i "4 ,...,.,' ...., "4 P = C.: ,.... 0, .7;4 ,...4 ...;', c...,. ....;"
.;.:.! =,-I R a
6 6 c:,-; 6 ..,-... 0 6 c-,- d 6 c:',3 a 6 6 e=-; 6
,..., r.F.) r::: =st (T.) p,> r-.-... ID v.) , co t-, ..(..) r;µ, 9 o 9 H
-- - . - . - = ..
C.) 0 . C". :. i rj 0; 0 C.; 0 0 Ø..Ø Q.= .0:
r;',5 C)'= 0
9i r, 0 Oil' =:00- 0'1 eq 0 R r., F.-4 7.4. 9 !c.:-.i .4 rn
C) r) e--) C) . 0 d ci e-) a 6. 6 ':::, r," =(;) c.',1 0
1 n.4
073 1-1 If) !f.) 1,, r--, r, C.:() in ;=..A if.) =(.4
N q ,.., o ,-:i 6 6 ,-6 q r'^, r,...,-3 0 0. irk.4.. 0 0 c.') ......
e9 c; 9 c.-..:., 9 c..-..j q (7.2 0. rzi. a:A:, ci= O. Cr.) 0
mi r-A
co 0-..,
4,..., 4....
(.1) g al r
f..p r-, 1 .N. .....T 71 r-,..1 9-,
==: = Tei), 0 7, ,.!=-..j ,-. k:77.)
-"10 i -.::1.
!,=P 0 0 0 0 t...., '0 (1 0 14) eZ! 0 C:',.' 0. 0 C.' 0 C?.
6 c-3 O. ,.-.. C5 .:".:). 0 c.:'' 6 c;) r.....5 ci c;;;:.=
0 0 0 0 0
e3 6 6: c3 6 6 f.-.:5 : 0 1.
0 0 0 0 0 0 0 0
m
!N 17,4
-A CO 0 0 c.,) 0 0 0 C') 0
r,
e3 6 r.::3 c.3 6 c-,-.3 6 6 ,.x 6 c.._: fp. o 6 o 6 o o
:..5), =ci-
.:r.-4 r.....,
IN a CZ) '"i a C.: C.,) C.:, 1-'1 N 0 C) 0 0 0 0 -=-i 0
.,..
=......
:::.=====::,ii -1, 'V
_,
0
...1 C.'":;. 1-..;:i = ,....8 l':. 0 r.-...1 N
C".' cc)
6 ...pi c:3 e3 6 6õ.:0 .:A.1)
..=
.......
4 rni1.1.1 IA, Z. X. a
t,.. e,...
F15 F15
PlatF: 25
1 .7 3 4 s: 5 I
8 9 10 11. 12
0
A 0,115 0,133 0,097 0,135 0173 0.085 0,113
0:084 0,091 0,081 0,091 0,158 64
B 0..215 u ii0.:9o2 t ..6474---- 0.07$ -------4:31t
ET 0..118 0.175 4..307:iiiiiiill .0,066 0ØTh a 072.
a083
vD
1-,
C 0,09-, 0:074: A0,58 0,055 .051 0,074 0,080
0,083: 0,293 0,344 0.061 0,083 c,.)1-,
D 0.115 0.053 0.205 .... :0.55z 0.16,0 0õ.058
0.141 0.101 Qi55 0,073 0064 0071 --4
F. 0..113 0.084 0.095 Ø077 0.080.. 0..070.
0.057 0.102 .006:5 0,072 0.257 0:073.
F 0.090 0.100 0.077 0,075 0,082 0,078. 0..115
a300 0.071 0,062 0,001 0,083
6 0.080 0.084 r..WID .03--..75 0.073 0,060
0,0gi1 0.051 0.072 a a:66 0,067 0.155
H 0,075 0,113 .--L104 0.109 0,085 0.095 0.082
0,095 0.097 0.075 0.083 0.093
Target spetific: dor** 2.552 258.3:. 2515 2583
2504
P
.Pate 26.
2
,-µ
1: ,
L 4 .5 5 7
8 9 10 11 12
,-µ
.6.
o,
2
A 0.090 0.113 0.058 0.066 0.057 0..075 0.083
0,053 0.074 Ø073 04091 0,080 .
r.,
8 0,092 0.058 0,059 0.079 0,060 0.069 0t112
0.053 0.069 0.084 0.082 0.091 .
,
,-µ
C 0.070 0.072 0.089 0.059 0.072 0,087 0072
0..06.: 0,067 01:30 0,080 0,100
0 0.093 0.077 Ø072 0.Q93 a IcK 0,1,0 0..064:
0,06.8:: 0.055: '0.057 0.054: 0,072
E 0.081. 0,079 0,059 0,145 0,057 0.114 0...sz
0.033. 0.081 :0,074 0.062 0,064
F 0.062 C1075 . . 0.050 ...... 0.158 t.). 05µa
0,.365 0.054 Ø1)90 0.058 .0,052: 0,063 a071.
6 a 032 1.2
Niiiiiw:,,:::.:::,,,i:iiiiiiii: ._., .
0-.331 001C4OM 51- 0,099 0.077 0,079
11094 0,056. 421,c62 0074 9.064
H 0,082 0,095 0.115 0.077 0.102 0.074 0,127
0,109 0,081 0,084 0,099 0,090
00
T..ar.gt.t s*.dfi.O:dc,met*: .2563
n
1-i
cp
w
o
,-,
oe
-a,
c.,
.6.
c,.,
(.9)
(.9)
(.9)
.re
o
o
oo
,--1
o
el
ci)
E=1
:0040.P ?Wads
a
Layo tom). .64r0 P80'0 690 0 910'0
'9.900 020'0 6LO'D SLOD P.6".1-0 OTZ'O H
0800 9800 = TLWO ST I ' 0 ...6310' 0 lis.L
00 S00 .-3.L0. 0 590'0 = z Lcro '.3:L0'0 .V903 9
Z LOs 0 UVEREim LIT'0 = TesCf: 2LeT0 0.L0'0 'PLO.' 0
gL0'0 .6:kyr OLD'0 S 1 CIO .g90.0 A
Maox:ai:i:on .. =
S90'0, 680'0 t'390 '0 = t$.30"0 S'i60'.0 . VL 0'0
8917.:0 9L00 T90'0 LS0'0 Z90=0 13900 =-1
.8Z I '0 = Z60"0 gZIO g80'0 P60Y0 0500 LSO ' 3
L.LO'D TI=E'0 91:T."0 SLO'O ..t 800 0
,
,
EDI '..0 S'S!ITO S:60'0 7,400 t'L 0. '0
tSKY 0 E.S0"0 LL0'n De.).'0 E..4... k..1." 0 -
5'30..0 .,
,
. S'80r0 ILOT 1'790'0 :31Ø0 .i. PI '0
-e....47.8.4: &PT ==0 S.L0'.0 OLTO . 8900 EOT '0 0L00.
il
. SLD'O 8800 EID.0 . Le.;1:"0 .Z.L0'0. D90'0
OTT.E.'0 SOE'D KO 'D POO'D =OLO'D IGO'0 = =V`.
. N
.
,
IT. . OT :,.E.' g .Z. 9. .0
-V E Z 'T .7r
.
,
6
auply
:Opoloplgpads Tp2ie
ow. o E.so'o .Es o'o = 5-.7....9ty o gscra = so.
Il(30'.0 6600 T 9710 9 S.,-Y0 .7.530.0 ....--N00 I/4
g*:01-1 LVETO E.PD'O' 6S0=0 = :E:=.;0 ==D g PO
0 ,E_T0 '3 2170 "t3: LPO'D õ4v.t.....Tn 5.P0'0 . SS0'0
:0
t/cOs 0 13170s0 LP0'0 5P0'0 = 5P0'0 81700
Lt,O.' 0 LP0'0 T60'0 S WO 2.170..0 L60: 0 A
9CO`q 9.170'0 . 99.Cr0 6200 .ZOZ'O. Gscro
t co ===o 9O0E 2900 '-µ".1..00 '40.'0 a7600 7.
m
LLOT1 1,7Z. "r0 .6L0V: ZSG'D 010'0
5903 PLOD SLOTk C L Ern S90'0 t.800 0
(.9.)
(.9.)
-1 T. 60'0 L LEY 0 'Fare CL00.: WA.: 8E1'0
L'O'D 980'0 9LO'o .901:0 091'0 f.01: '0 1
,--1
--e, .E.2o,' a vEr:13 $:i..'iTtl 960'0 :0L0"0: 990..0
OLO ' 0 820 -0 St 1:.'0 ELOS i.90`0 ELTO .S.
,--1
o
el .TEZ,OsQ 1:53LTO ?WO 1.60'q. tarD. . S9Ø..0
L-30'0: Z.-{771-0 E:E4r0 .520'. 0 9E10Ø '.1Ø1-0
V
0
Z T.:. U. . :01. Is S =L 9.
.'.`: t . E. ;E: T =
.
1.Z. aqR:W
CA 03101100 2020-11-19
WO 2019/113373 PCT/US2018/064333
..4-
,-.3 ..,.: 0,3 cr, go [..;.? N.,, ::=-=I N.,
.:^1 0 r, 14.:. Cl3 0 1.-', .1-1? ::÷.1 q o .--.) .7) r.:,.." C") ,-1
=====4
or)
e-NI 01 1-4 141 u-.I C...1 r.,-;. <,-;:,1 ,:i - 0:1 r, ita r=-=.
1..r1 r, f`, (X)
=,f.1 N. ,..0 14 I,- N..! cio res <71 ,,,i q. 0 0
6 -,c.,-: co (.7.,
0 On 0 ,C1 '4'4 1+:;-, 1,1-1 1:.11 . =-=
r, ,,tt=-=-=1 RIftr5
... - ... , ., = . . - .., _õ.- 6 6 .6 6
d 6' 4 6
0 0 (3) 0 Q Q 0 Q
0 .-1 0 1;7-'1 l=, 1X:1V-1 1-11 1^, 113 1.13 14")
r., tri 1.0 M. 1---1 ef') Czi'i r,
r ...i:;?. d, c) f;::: 0 0
0 ic.,
-i. c;) 0 in c;) f.-N ,--1 c) c.-)
0 0 6 c) d 0 d 0
d d 0 . d 0 6 6 d
MI 0
(13
e,..1
4,,i Cu
m
E 1..... Lo 0 ====1 N-4 irsi in 0 ei:
r;_:.) 0 0 0 0
..,, rk=-. o.) c-41 0, 1.0 0 r.Z.,"! re) r= , k,:. 4 ,.-.. 0 ? 0
0 0 e.,-.:.. 0
r, =:--4 0 tT 01 r::,0 0 0
Kt 4r1 'CT Ca a? 1---,. 0 .M 0 9 0 k.s,?-= r,.. r, 0 ers
..,-1 0 kr. N.. .X., .H 1--, 0 in 0 s"...) 6, .L., r.:',) C.? r,=I ."..1
d 6. d 6 6 6 6 d 6 kzi 6 6 6 6 d 6
. ,--1 El's 1,-, =st Cr-, 1,D 1=115 1"--s, 1- q 6 ,-:,i. '7-1,3 q 0 -,, q 0
6 if:, 0
..........
) 0 e,:;:. 0 0
C) ,f --1 0 r.;7 0 t'...','e (..,.) 0 f,; ,,,1 õ . , .
= ' = " W
C5 C5 0 C5 0 C5 4D 0 0 0 0 0 11,)
C.,
Cr.1
"I IM CT)
f.,`,5 0 C. l'..,`,",", 0 =:-.1
r-.1 . .
0 e:.? ,Z7.) 0 c."." 00 0
.=
0
C
,ff-1. 0.3 ag t= N-. e,..1 6',) f,... C - .;.-.;-:.:
ft..). <-0 f..0 co 1..0 r, m
.: ,,.....,i ,,,....) ez.) c-,,.. c;:,,,, c,...,õ?
r.,...)
(-3 d Ã-Li 6 e....: c....7, 6 R4
a: '0
:14.t
ti.
1:ff
VI
4,
r:31.
to ,,..,
ro : i-
48 kr)
Plate :31
1 ,
4 ,
:3 4 5 6 7 8 9 10 11
12
0
A 0,095 0,082 0,074 0,081 0.059 0,079
0077 0271 007 0.071 0.087 0,090 w
=
B a057 0,061 0,068 1107 0.102 0.101
0.072 0074 0.858 0,075 0,084 0.064 vD
1-,
c a 059 0.056 0,05.3, 0.073 0.116 0,053
0185 of,:rio 0.070 0,0645 0,085 0,070
w
w
0 0,067 0,062 0..061 0.101 0.054 0,057
0,073 0,094 0,095 0,149 0.146 0,061 --,1
w
E 0,084 0,07 0,075 0.061 '0 .092 ,057
0.064 0,062 0.072 0.062 0.061 0,052
F 0.066 0.069 0.049 0.058 0,059 0.068
0,055 O. 0q1 0.0 0.062 =0,085.
CI 0,080 0,080 0.070 0,097 0,112 0,357
0.065 0,084.; =0.168 0,070 0,077 0.077
ii 0,078 0,054 0.060 0.071 0, C16, 0,074
0,051 0,057 0,074 0,058 0,1)59 0,065
Target spedfic dm** None
Table 2
P
,
[00127] Summary of Table 2: 2,976 clones isolated and analyzed. 66 target
specific clones expanded for confirmatory screening. ,
,
4,.
.
[00128] Secondary screening of Klotho hybridoma: Hybridoma supernatants were
evaluated by indirect ELISA using target antigens (standard " "
,
indirect ELISA protocol. TMB 10 min.).
,
,
,
,
[00129] Antigens, (1) Klotho-6xHis protein (Lot#170918A, 0.78 mg/mL) 0.5 ug/mL
in 50 mM Na-Bicarbonate, 100 ul/well, +4 C, overnight.
(2) Klotho-Fc protein (Vista, 0.1 mg/mL) 0.5 ug/mL in 50 mM Na-Bicarbonate,
100 ul/well, +4 C, overnight.
[00130] Detection Ab: Anti-mouse HRP, Peroxidase AffiniPure Goat Anti-Mouse
IgG (subclasses 1+2a+2b+3), Fcy Fragment Specific, 1:5000
(Jackson Lab., Cat#115-035-164) in SEDB, 100 uL/well, 1 hour, RT.
1-d
n
,-i
cp
w
=
oe
-a-,
c,
4,.
w
w
w
Nate map.:(Clarifimed itiothe-6kHis sped& clortes...anridenes suggested for
ELLA develt--ssument are.:highlighted)
1 7 3. 4 5 6 7
, 8
9 10 11 12 ..
0
...A IiiiiNiViCiIiiiiLiNgiAtMENgiill.egggi: 2F5 21412 362 EilD2MEI 4E1
:ilniiii:AiMiniiiii:Mi;Akiiii:t9Eg 4H9 Mi:i..0a4,1igi 6'
5F1 . 5H10 5612 6E3 7Al2. 768 7C3
RigiMtl`;Vi:i:ig ii::8C4iX:pi: 8H12 .10A8 .1001. o'
c i'1066:E:ii:::::::,i, 11A8
11A.9 1.1A10 1,mIte=:',i'i,,,,
=4,1Ssr:Vm ntliNa-= im-4264?:m imil2õKliuii mna...--4Wµ:tam iimaf.48i-:-
,,,iiimi 14a9 E
D.: 14611 m.446IImi 14611 sig450-ft 15612 16A11 i6C.9 iiiA.
OR:iliE 1802 .1865 16G6 21).F9: (...)
-4
...h.........õõ:õ.................
(...)
E 7107 =231612Mi 22C.12 7.2.04 22E8 .23H1 23H4.
24D7 35E17 35E3 .7565 2588
F 1'5 r) A ',,,,,,,e,ik,,,,,,,: ,s6 7
4.4''i.42.E. 29ci1.1 30(3
,=1,-- - -.., ::::::::::::,:iµ.,:,'q*.i!::::
=-,-- 4 ***=.?!.,4,4i,',...=X.,.,.:. ,
..:Plate 1.: Klothoµ*:Ks (0D450 ablVitaft.OVOlipe.,4:
1 .2 3 4 5 0 7
:6 9 10 .:11 12 i.
.A. 1:795 . 1.247 :,.. Z,21:0V: 0.331 0.071
0,694. 1.259 0.055 2Ø1 2026. 0.065 .2õ15.1
.8.= .. 0.CA 7 I 0..048 0.046 0.045 A 1404 0.045 ........
0.064 1371 .1924 0.05 0.521 0782 P C: 1.,269
i'.).055 0.049 0..05 . 2..235 2.191 H......... 1...3 0.463
0.618 0,646
0..158 7.007 0.059 0909 0.287 0048 0040
ia.,707 1.432 ... 1õ451.3 o.o73 c.o51
( li
:... r8'
it: 0,052 0.6.24 .., 0.057 0.058 0.101 0.069 0.053:
0..137 0.122 0.09 0.118 0.172 .
r.,
..F.: 0,060 iNi,:.:2.35nig. 0.07 2.005 0.073 0.081
,
#late 2'; K.I.Otho-Fc tOt."4.5.0 abS0r:bamce v,",a.ue).
... , 1 3. :7:',. 4. 5: 6 7 8
:.,9 10 11 12 .....
A iiiiiiiiii.2..068 B 0,518 .i.!iiiiiiiii:74.50;iiii*
0.414 0.03 0,821 0.401 0.066 ' 1342 ' 1,99 ' . 0.08,5
1.493
.4.. 0.057 0.049 0.046 0.051 0,'1'E.., . 0.048
0.056 0.405 1.9.13 0.045 0.747 1.356
C: 0.455 0055 0,049 _ _ __ _. 0.054
2.1a8 I 1703 0,482 0.2 0,642 0;77 1.347 0.049
D 0.135 2,076 0.058 0.216 0.287 0..049 0.048
0.612 0.61 0..205 0.046 0.047
E 0.059 0182 0045 0.053 0,052 0.062 0.05
0.052 0.051 0.048 0.053 0.061 1-d
n
..... =.... . ......
,-i
F 0,084 247-$,M, 0..046 .2..009 0,062 0.05
cp
w
o
oe'
'a
o
.6.
(...)
(...)
(...)
Nate 3: No:antigen =012'.:450 abaorbani0100s)
1 2 3 4 5 4 7 8
9 1.0 11 12
0
A 05 0.069 ni cr;
0 . 5 ,,_4, ,5 a0.55 (1.067
iiiiiiiiiiiii1.225 iiiiiii 0.054 0.055 0,051 0,053 0
,0i1,=:$ 0.06 t,.)
o
B 0,051 0,048 0,045 0,045 0,046 0,047
0,042 0,045 __ 0,045 0.045 0.046 0z-55 .
,.,
C ao53 Q.{348 0.046 0,045 . 0,055 0,044
0.0450,052 0046 0,047 0,047 0,048 1- c,.)
D 0.117 , 0.045 0.048 . 0:044 0.27 0.046 0.046
0.07 0,048 0,052 0,046 0,045 --.1
E 0.057 , 0,051 (1043 0048 0.047 0,045 0,04.9
CW47 0.043 0.044 0.048 , 0.049
F 0.059 0.057 0.046 0051 .046 0.051
Table 3
[00131] Summary of Table 3: 28 Klotho-6xHis specific clones were confirmed
(0D450 cut-off: >0.5, Klotho-6xHis plate) - 1F6, 2E1, 2E12,
3D2, 4H4, 4H5, 5D12, 7Al2, 7F9, 8G11, 10A8, 10D1, 10G3, 11C9, 11F7, 11H3,
12G4, 12H3, 14B2, 14B7, 14B12, 15E9, 16H1, 18C2, 18G5,
P
21G12, 26G3, 28F11. o
,
,
u,
[00132] 1st arrays of 9 Klotho-
6xHis specific clones recommended for native (untagged) Klotho ELISA
development (0D450 >2, Klotho- ,
,-,
.
rõ
6xHis plate) - 2E12, 4H4, 4H5, 5D12, 11C9, 11F7, 14B12, 26G3, 28F11.
2
,
,
,
[00133] 2nd array of 11 Klotho-6xHis specific clones recommended for native
(untagged) Klotho ELISA development (0D450 >1 and <2, ' ,
Klotho-6xHis plate) - 1F6, 2E1, 3D2, 7Al2, 7F9, 8G11, 10G3, 11H3, 14B7, 18C2,
18G5.
[00134] 1st array of 10 Klotho-6xHis and Klotho-Fc specific clones recommended
for the development of ELISA capable to measure both,
untagged and Klotho-Fc proteins (0D450 >1.7, Klotho-6xHis and Klotho-Fc
plates) - 1F6, 2E12, 4H4, 4H5, 8G11, 11C9, 11F7, 14B12, 26G3,
28F11.
1-d
[00135] 2nd array of 2 Klotho-6xHis and Klotho-Fc specific clones recommended
for the development of ELISA capable to measure both n
,
untagged and Klotho-Fc proteins (0D450 >1.2 and <1.7, Klotho-6xHis and Klotho-
Fc plates) - 5D12, 14B7. cp
t..)
o
[00136] mIgG isotyping: mIgG isotyping of Klotho hybridoma selected clones:
Antibody isotyping from selected hybridoma clone culture Ec
r,
.6.
supernatants.
c,.)
c,.)
[00137] Samples of hybridoma culture media were used to determine antibody iso-
types using Rapid ELISA Mouse mAb Isotyping kit
(ThermoFisher, Cat#37503) following manufacturer's instructions.
0
t..)
o
[00138] The table below presents the results of mIgG isotyping of Klotho
hybridoma selected clones.
o
,-,
,-,
1F6 2E12. 4H4 4115 I 8G11 11C9
1.1F7 14812 2.6G3 28F11 (...)
(...)
-4
IgG1 gni2,198MW 2.931.ft m 2,8113wmg2.394.mg Egg 2.7
0.085 0,085 gggg,:1326 0..081 ....õ:õ.õ:õ.õ:õ.õ:õ.õõ........õ,.
0..09 0.087
IgG2.3 0.066 0.063 0.06.3 0.062: 0.073
0.052 0,07 0..056 -0.057 0.057
õ.õ.õ.õ.õ.õ.õ.,.......,.:õ.....õ.......,õ
.õ.õ.õ.õ.õ.õ.õ_,.,õ..õõ.....,...., õ.õ.õ.õ.õ.õ.õ.: .:.,..õ..........õ..._...
..õõ....õõ
igG:2b 0, :128 , 0.081 0.236 . 0.070 0.069 mmm7,008:
. ., ..õ....õ,õõ,...õ,...õ,.....õ 0
066 mmmI-139.6 Pmm.','2.378----mmu'2.1.72
..
:::::::::::::::::::::::::::::õ_
.:::::::::::::::::::::::::::...õf . , . ,
.,........,,,,,,,,,,,,,,,,,,,,,,,,,,,.........,.........,......
igG3 0.051 , 0,046 0.049 0.C.148
0.051 0.051 th053 0,05 0Ø57 0.058
igA 0,040 , 0..058 0.070 0.050
0..049 0.1.:149 0.048 0.047 0.051 0.051
Igm 0.054 0.049 0,049 0,058 0,053
0,095 0.048 0.048 0.051 0.04:9:
::::::::::::::,::::::::::::::õ-
,::::::::::::::::::::õ...õõ:õ...õ:::::::::::::::õ.õ:õ:õ::::::::::::::::::::::::
:::::õ.õ:õ:õ:õ., ::::::::::::::::::::::::::::õ.õ,õ
:::::::::::::::::::::::::::,õ :::::::::::::::::::::::::õ.õ:õ.õ:õ.õ:õ.õ:õ.õ
P
Kappa 0 0..:42;:ami-imi::2Q1
9 m inr-233 -misiiiiiiiiI7kiiiiiiiiii
iiiiiiiiiiiiiiiiiiiiiiiiiiiAVZ iiiiiiiiiiiiiiiiiiiiiiiiii.qAW
iiiiiiiiiiiiiiiiiiiiiiii'9,::V4 iiiiiiiiiiiiiiiiiiiiiiiiii944 G..33
0.355
........ ................_.................................
...... ............,....
.
Lambda 0.049 0.051 0.05.8 0,052 0.05 0,053 0.047
0..052 0.055 0,05 ,
,
,
t..)
.
Table 4 rõ
0
rõ
0
,
[00139] Summary of Table 4: mIgG isotype determination:
,
,
,
,
[00140] 1F6, 2E12, 4H4, 4H5, 8G11 and 11F7: IgG1
[00141] 11C9, 14B12, 26G3 and 28F11: IgG2b
[00142] Quality Control: Indirect ELISA was performed for quality control: An
ELISA plate was coated with unconjugated 6xHis tagged
Klotho protein or Klotho-Fc protein at 0.5 ug/ml overnight at +4 C in 50 mM Na-
bicarbonate buffer. Purified 1F6, 2E12, 4H4, 4H5, 8G11,
11C9, 11F7, 14B12, 26G3, and 28F11 antibodies were applied in 5-fold serial
dilutions starting at 500 ng/mL and the secondary antibody, goat A
1-i
anti-mouse HRP, was applied at 0.16 [tg/ml. Reaction was developed for 7
minutes. Raw absorbance data at 450 nm and corresponding antibody
ctfJ
o
titration curve.
cio
O-
o
.6.
(...)
(...)
(...)
CA 03101100 2020-11-19
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1. laxHis tagged. Moth . protein
[01, 1F6õ 2E12, 484, 4.135, 8611: 1159,
.. nil/m.0 ..Lot41.8N10.A i..ot#1.8041.08 Lot/1410C , lot#18041,0D Lot#180410E
tO1-4180410F ,
500 Ø051 0,048 0.047 .. 02047 0.048, 0,048 0.048 0,045 0.046 0.046
0.048 0.048 NO i ntigen.
500 1.782 1 793 2.533 2531 1.82241.662 1.759 1.721 1.801 1.9
22577 2,993
. 100 0.827 0.852 1.398 1.327 0.681 I 0.M4 L0.712 0.57 I 109D 0.789
1.994 2,207
20 0..260. 0.271 0,457 0425 0.209 0 194 0.199 1 0.217 I 0.259 0.223..
0.862 0.945
4 I 0.096 0.102 0.152 0.14:3 0.09 I 0.04 0.082 I 0.086 0,091 0Ø87
0.251 0.32
.. 0.8 I 0..06.4 0.061 0.065 0.074 0.058 I 0.061 I 0.06 I 0.06 I 0.062 . 0.05
0,105 0,124
0.16 0.056 0.055 0062 0.058 0.052 I 0.059 I75Ø56 I 0-05.210'055 0-062 0-064
0-067
0 0.051 0.049 0.052 0.05 0,051 Ø049 0.05 0.052 . 0.052_0,052 0,054
0.051
IAN, 111:9, 1167, 14812, 2653, 28F11.,
ngifrn L. 01)1:#180410F Lai:#.1804106 Lot#190410H Lc.,t#1804.101
Lotff1804101=
500 0,071 0,017 0.059 0.058 0,06 0.00.2 0.08 02172 0.076 0.073 NO
antigen.
500 3.023 2,816 1.754 1.779 1.953 I 1.851 2.9!'..) I 2.92 2.572
2.586
100 2.171 1.859 0.472 0.506 0.28 0.857 2.103 2.145 1.44 1.565
20 0,988. 0,864 0.154 0.137 0.29 0,253 I 0.994 1.W1 I 0.51240.586
. 4 I 020 0232 0.075 0072 0.122 1 0.102 1 0.291! 0.341 0 175 0491..
08 0,114 Ø104 0 -059 0.06 0.066 0.06 0.107 0,126 0.08.2 0.081
0.16 0,951 0.068 0.057 0.054 0.053 9.055J.075 02161 ' 0.057 0.059
0 I 0,072 0.057- 0.06 0.06 0,057 I
0,059 1 0.Ø5 I 0. 0 i.,--3 1 0.068 1 0:062
2, Klotho-Fc protein
1,Abb . 1F6, 2E12, 4134, 4135., 8511, 1169,
.
nern0 .10:4180410A 0otg1804108 L01741804100 Lot:4180410D 0ot#180410E
1ot#1.80410F
500 I 0.065 0.058 0.055 0.059 0.058 0.053 0.059 0.069 0.055 0.056 :-1.05
0.065 No antigen
.500 I 1.328 1,307 I 1.78 1.838 1.333 I 1,37 1.366 1.25 1.325
1.352 2.74 2.755
10i.) I 0.505 0.462 L0749 9.155 0.466 0.44 0.412 0.42.3 0.43j0.455 1.801
1.787
20 I 0.176 0,146 0.241 0.223 0.151 0.147 0.14 0.135 0.15 0.144 0.742 0.732
4 I 0.087 0.088 0.014 0.093 0.075 0.076 I 0.071 0.078 0,019 I 0.072
0.231 0.322
0.8 I 0.073 0.065d 0.062 0.063 0Ø57 0.057 I 0.058 0.061 . 0.06 I 0.058 0
101 0.127
0,16 0.068 0.065 I 0.061 0,0:58 0,055 . 0.057 0.057 0.058 0.055 0.055 0.067
0.148
p 0.07 0,07 0,065 0.057 0.07 0.059 0.050 0.015 0.06 0.063 0 096 0.112
IAN, 11.99õ .1.1F7,. 14812, 2653, 28F11õ
nginii_.. 6ai-4180410F 0ot#180410Ã 0ot4I80410H 0ot41804101 Lot41804105.
.500 .0,012 1-0.065 I 0.058 0.057 0,06- . 0.06 0.0650.063 1 0.063 0.06.5 No
antigen .
500 2,623 2.613 0.798 0.82 2.044 .2.085 2.641 2.607 I .2.417 2.325
100 '1.886 1.686 , 0.291 0.288 1:041 1.012 1.82.2 1.784 1.337 '.1.421
20 0,816 , 0.717 T 0.104 0.108 0.33 0.316 0.821 0.787 0.54 0.558
4 0.23 0.74 0.067 0.066 0.125 0.135 0.255 1.-217 1.165 0:171
0.8 0.183 0.101 0.055 0.051 0.071 0.069 I 0.106 0.104 0,083 0,083
0.16 0.078 0,058 0.059 0,058 . 0.057 0.05 0.06.3 0.052 0.059 0.059
. 0 0.016 0.066 I 0.056 .0061 0.051 0.06 Ø057 0.057 0,059 0.066
Table 5
[00143] See also, dose responsive curve illustrated in Figures 5A, 5B, and 6
respectively.
[00144] Quality control: Purity and integrity of purified antibody were
analyzed in 4-20%
SDS-PAGE (reducing and non-reducing) stained with GelCode Blue reagent. See
Figures 7A
and 7B, respectively.
53
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[00145] Polyclonal IgY antibodies: Polyclonal IgY antibodies were also
developed. The
above design was adapted as necessary for production of polyclonal IgY
antibodies in
chickens.
[00146] Chicken serum IgY titer evaluation using target antigens, by indirect
ELISA (TMB:
5 min). Read at 450nm
[00147] Antigen: Klotho 1 ug/mL (0.78 mg/mL, Lot# 170918A) in 50 mM Na-
bicarbonate.
[00148] Samples: Chicken #1, #2, RT, lhr (5% milk in PBS).
imniuhizatiori antigen: Klotho
serum C.hken ID:
bleed
dilution 1 2
Pre-bieed 1:1.000 0.05 0.048 0,051 0.051 No
Antigen
Pre-bleed 1:1000 0,123 0.06 0.064 0.06.2
Bleed 1 1:1,000 2,686 2,464 3.166 3.101
110,000 1.125 0.931 2,689 .2.76
1:100,000 0.156 0.153 0,927 0.924 is:lotho
Bleed 2 1:1,000 2.278 2..215 2.895 2.963
1:10,0-00 0.985 0.858 2,679 ;.543
1:100,000 0.171 0,144 1.02 L065
Table 6
[00149] An ELISA plate was coated with 6xHis tagged Klotho protein or Klotho-
Fc protein
at 0.5 ug/ml overnight at +4 C in 50 mM Na-bicarbonate buffer. Purified anti-
Klotho IgY
antibody (lot# 180327A) was applied in 5-fold serial dilutions starting at
1000 ng/mL and the
secondary antibody, goat anti-chicken HRP, was applied at 0.16 pg/ml. Reaction
was
developed for 5 minutes. Raw absorbance data at 450 nm and corresponding
antibody
titration curve. See also, Figures 8A-8B.
Antigen
fIgn
Moth Kiotha-Fc
neml __________________________________________________________
1000 0,047 0,046 0,046 0,054 No Antigen
100.0 2.192 2.179 2,137 2,171
200 1.916 1.795 1.797 1942.
40 1,076 1.032 0,865 0,9
8. 0.354 0.337 0.328 0.293
1.6 0.123 0.118 0.106 0.109
0.32 0.063 0.073 0.053 0,062
0: 0,046 0.047 0.047 0,045
Table 7
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[00150] Summary: Both HRP and Biotin-anti-Klotho IgY show significant signal
in indirect
ELISA. Bio-IgY has stronger signal than HRP-IgY.
[00151] As illustrated in Figures 9A and 9B, there is a relatively weak signal
in IgY/HRP-
IgY pair. IgY/Bio-IgY shows relatively high background, even at low
concentration (0.25
i.tg/mL). IgY/Bio-4H5 pair appears to work better than IgY/labled-IgY pair.
[00152] It has been postulated that levels of circulating, soluble Klotho
protein in the serum
of mammals decrease with increasing age. Baseline measurement for naturally-
occurring,
circulating soluble Klotho protein in a human subject has been reported in
various
publications. A thorough literature review yielded the following average
range(s) for
circulating soluble Klotho protein in the indicated human subject (see Tables
8-9).
Age
group Sex
Healthy male Healthy female
0-15 1692 (372, 5694) 2487 (964, 5866)
15-35 459.4 (335.1-617.2)- 508 (448-661) 459.4 (335.1-617.2)- 508
(448-661)
35-50 476.9 (416.9-545.5)-692 (618-866) 476.9 (416.9-545.5)-692 (618-
866)
141.8 (94.7-189.2)-377.17 (359.46-
50-65 394.90) 141.8 (94.7-189.2)-377.17
(359.46-394.90)
>65 354 (264-410)-999.5 (887.7-1186.4) 354 (264-410)-999.5 (887.7-
1186.4)
Age
group Disease
GH deficiency CKD all
0-15 340 (433,7826) 1746 (671-3438)
15-35
35-50 396.3 (306.3-558.4)-478 (348-
658)
50-65 613.0 (504.0 to 811.0)-809
(674-981)
Men 596.0 (464.3 to 790.3); Women 614.0
>65 (558.0 to 811.0)
Age
group
CKD stage 1-2 CKD stage 3
0-15 1418 (501-3314)-1736.5 (1272-2737) 1418 (501-3314)-1736.5
(1272-2737)
15-35
35-50 540.5 (371.3-824.8)-482.7 (360.2-683.1) 386.4 (319.2-456.7)
50-65 388 (254-532) 316 (199-464)-337 (217-489)
>65
Age
group
CKD stage 4 CKD stage 5
0-15 1019 (502-1592)-1418 (501-3314) 1019 (502-1592)- 1418 (501-
3314)
15-35
35-50 337.8 (251.0-444.2) 295.6 (207.7-387.5)
50-65 215 (135-331) 249 (100-452)
>65 Mean SEM 473.9 121.4 Mean SEM 546 164.3
Age
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group
Kidney transplant T1D
0-15 1698 (1110-2123)
15-35 454 (420-492)
35-50 659.3 (525.3, 827.6)- 787.7 (629.5,
1007)
50-65 550 (304-651) -605 (506-784)
>65
Age
group
T2D CAD
0-15
15-35
35-50 572.4 (541.9-604.6)
Men 67.3 (52.8-69.3); Women 65.7 (51.6-
50-65 71.6) 490.5 (349.0-549.4)
Men 45.1 (33.7-54.1); Women 40.7 (31-
>65 493.7 (287.8-777.7) 56.4)
Age
group
COPD MS
0-15
new cases Mean SEM 585.56 153.99;
15-35 chronic cases Mean SEM 696.94 170.52
35-50
50-65
>65 250 (168-330)
Table 8
Age group KL-VS genotype
Non carrier Heterozygous Homozygous
>65 Mean SEM 780.9 19.6 Mean SEM 891.7 45.3
Mean SEM 599.1 75.7
Table 9
[00153] In the table, above, CKD: chronic kidney disease; GH: growth hormone;
T1D: type
1 diabetes; T2D: type 2 diabetes; CAD: coronary artery disease; COPD: chronic
obstructive
pulmonary disease; MS: multiple sclerosis.
[00154] In light of the variability observed in the literature, a more robust,
more consistent,
and/or more accurate means of measuring serum Klotho levels is needed.
Embodiments of
the present disclosure include a Klotho diagnostic kit and method of using the
same, as
described in further detail, below.
[00155] Table 10 presents a baseline measurement for naturally-occurring,
circulating
soluble Klotho protein in a human subject (prior to receiving any treatment,
e.g., health
supplement, pharmaceutical, therapeutic protein, etc.). Figure 10 is a
graphical representation
of the (daily) change in circulating Klotho protein levels in a human subject
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Time Collected [Klotho] (ng/mL)
0650 0.579
0750 0.782
0850 0.66
0950 0.717
1050 0.611
1150 0.596
1255 0.597
1645 0.56
1745 0.556
1845 0.588
1945 0.592
2045 0.638
2155 0.551
Table 10
[00156] Once per hour, beginning at 0650 hours, a blood sample was taken from
the human
subject and Klotho protein levels were measured by ELISA.
[00157] Quantitative ELISA according to an embodiment of the present
disclosure is able to
detect (1) Native (e.g., endogenous) Klotho, (2) recombinant Klotho-Fc fusion,
and (3)
recombinant 6xHis Klotho in human plasma samples.
[00158] Further measurements of Klotho protein are illustrated and presented
in Figures
11A-11B and Table 11, below.
1.ft** 00:00iii$04tm
: _______________________
Neat _______________________________________________ _=_. __________ 233.
301 __
1:2 3&74 2E.3.23O .92.946
394..257
= 1:4 859.529 _ 585.1564 2.1.05
511..514
B.13.7.52 1 Out of Range .580329 700.209
man-1.La.i
Table 11
Klotho Stability in Human Plasma Using ELISA Kit
[00159] Samples of venous and capillary ("finger") blood were collected from
four donors
(A, B, C, D) and stored in (i) EDTA or (ii) heparin containing tubes. 1.5
ng/mL of Klotho
(lot# 170918A ¨ human soluble alpha Klotho) was spiked into a set of venous
and capillary
samples collected from donor A. Collected blood (blood samples) were stored
at room
temperature for 0, 1, 4, 16, 24, 48, 72, 96, or 168 h, then spun-down. Plasma
from each
sample was stored at -80 C prior to ELISA test. ELISA plate maps, raw data,
standard curve
data, and concentrations are presented in Tables 12-21, below.
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ELISA plate map 1:
STD, 6 nemL A (0 h) EDTA A(1 h) EDTA A(4 h) EDTA A(16 h)
EDTA A (24 h) EDTA
STD, 3 nemL A(48 h) EDTA A(72 h) EDTA A(96 h) EDTA
A(168 h) EDTA A (Finger) EDTA
STD, 1.5 nemL A (0 h) Heparin A (1 h) Heparin A (4 h) Heparin
A (16 h) Heparin A (24 h) Heparin
STD, 0.75 nemL A (48 h) Heparin A (72 h) Heparin A (96 h) Heparin A (168 h)
Heparin A (Finger) Heparin
STD, 0.375 nemL B (0 h) EDTA B (1 h) EDTA B (4 h) EDTA B (16
h) EDTA B (24 h) EDTA
STD, 0.188 nemL B (48 h) EDTA B (72 h) EDTA B (96 h) EDTA B
(168 h) EDTA B (Finger) EDTA
STD, 0.094 nemL B (0 h) Heparin B (1 h) Heparin B (4 h) Heparin
B (16 h) Heparin B (24 h) Heparin
STD, 0 nemL B (48 h) Heparin B (72 h) Heparin B (96 h) Heparin B (168 h)
Heparin B (Finger) Heparin
Table 12
ELISA plate map 2:
STD, 6 nemL C (0 h) EDTA C(1 h) EDTA C (4 h) EDTA C(16 h)
EDTA C (24 h) EDTA
STD, 3 nemL C (48 h) EDTA C (72 h) EDTA C (96 h) EDTA C
(168 h) EDTA C (Finger) EDTA
STD, 1.5 nemL C (0 h) Heparin C (1 h) Heparin C (4 h) Heparin
C (16 h) Heparin C (24 h) Heparin
STD, 0.75 nemL C (48 h) Heparin C (72 h) Heparin C (96 h) Heparin C (168 h)
Heparin C (Finger) Heparin
STD, 0.375 nemL D (0 h) EDTA D (1 h) EDTA D (4 h) EDTA D (16
h) EDTA D (24 h) EDTA
STD, 0.188 nemL D (48 h) EDTA D (72 h) EDTA D (96 h) EDTA D
(168 h) EDTA D (Finger) EDTA
STD, 0.094 nemL D (0 h) Heparin D (1 h) Heparin D (4 h) Heparin
D (16 h) Heparin D (24 h) Heparin
STD, 0 nemL D (48 h) Heparin D (72 h) Heparin D (96 h) Heparin D (168 h)
Heparin D (Finger) Heparin
Table 13
ELISA plate map 3:
STD, 6 nemL Spike (0 h) EDTA Spike (168 h) EDTA Spike (72 h)
Heparin
STD, 3 nemL Spike (1 h) EDTA Spike (Finger) EDTA Spike (96 h)
Heparin
STD, 1.5 nemL Spike (4 h) EDTA Spike (0 h) Heparin Spike (168 h)
EDTA
STD, 0.75 nemL Spike (16 h) EDTA Spike (1 h) Heparin
Spike (Finger) Heparin
STD, 0.375 nemL Spike (24 h) EDTA Spike (4 h) Heparin
STD, 0.188 nemL Spike (48 h) EDTA Spike (16 h) Heparin
STD, 0.094 nemL Spike (72 h) EDTA Spike (24 h) Heparin
STD, 0 nemL Spike (96 h) EDTA Spike (48 h) Heparin
Table 14
ELISA 450 nm absorbance raw data (plate 1):
3.44 3.39 0.238 0.231 0.231 0.247 0.235 0.233 0.248 0.244 0.261 0.276
2.03 2.005 0.235 0.235 0.242 0.235 0.247 0.248 0.254 0.264 0.283 0.282
1.117 1.077 0.239 0.241 0.229 0.236 0.236 0.248 0.253 0.264 0.256 0.283
0.593 0.573 0.237 0.23 0.241 0.248 0.239 0.26 0.252 0.248 0.254 0.258
0.31 0.328 0.162 0.157 0.16 0.163 0.162 0.161 0.173 0.173 0.184 0.181
0.206 0.202 0.167 0.178 0.17 0.172 0.173 0.177 0.18 0.174 0.182 0.199
0.133 0.131 0.162 0.173 0.175 0.172 0.174 0.172 0.173 0.176 0.176 0.185
0.059 0.059 0.177 0.18 0.181 0.182 0.176 0.163 0.165 0.185 0.182 0.19
Table 15
ELISA 450 nm absorbance raw data (plate 2):
L_3.409 3.302 0.228 0.223 0.237 0.237 0.232 0.233 0.24 0.233 0.26 0.275
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r----- ------
1.986 1.904 0.246 0.239 0.242 0.247 0.244 0.242 0.251 0.256 0.227 0.251
1.066 1.052 0.229 0.226 0.222 0.228 0.226 0.236 0.234 0.246 0.244 0.251
0.589 0.56 0.225 0.233 0.242 0.238 0.266 0.239 0.25 0.251 0.244 0.23
0.329 0.327 0.192 0.181 0.165 0.174 0.187 0.174 0.196 0.168 0.174 0.163
0.195 0.195 0.174 0.176 0.184 0.188 0.196 0.184 0.205 0.172 0.169 0.17
0.136 0.129 0.136 0.165 0.177 0.165 0.163 0.171 0.191 0.168 0.177 0.186
0.064 0.072 0.19 0.177 0.184 0.177 0.198 0.188 0.197 0.18 0.17 0.167
Table 16
ELISA 450 nm absorbance raw data (plate 3):
7.:
i. 3.482 3.393 0.449 0.442 0.553 0.558 0.51 0.506
2.04 1.976 0.438 0.429 0.587 0.603 0.501 0.509
1.11 1.087 0.426 0.427 0.506 0.498 0.481 0.503
0.6 0.577 0.442 0.441 0.514 0.518 0.568 0.524
0.341 0.331 0.471 0.453 0.506 0.522
0.202 0.201 0.47 0.462 0.537 0.548
0.137 0.137 0.514 0.517 0.54 0.537
0.062 0.064 0.558 0.539 0.564 0.57
Table 17
Calculation: Standard curve 1:
Concentration Back Conc.
Sample Wells Values Mean Value Std. Dev.
CV%
(ng/mL) Calc.
St01 6 Al 6.062 3.44 3.415 0.035 1
A2 5.934 3.39
5t02 3 B1 3.03 2.03 2.018 0.018 0.9
B2 2.985 2.005
5t03 1.5 Cl 1.525 1.117 1.097 0.028 2.6
C2 1.464 1.077
5t04 0.75 D1 0.758 0.593 0.583 0.014 2.4
D2 0.73 0.573
5t05 0.375 El 0.358 0.31 0.319 0.013 4
E2 0.384 0.328
5t06 0.188 Fl 0.21 0.206 0.204 0.003 1.4
F2 0.204 0.202
5t07 0.094 G1 0.102 0.133 0.132 0.001 1.1
G2 0.099 0.131
5t08 0 H1 Range 0.059 0.059 0 0
H2 Range 0.059
Table 18
Concentration: ly = ((A - D)/(1 + (x/C)^13)) + D: A B C D RA2
Std (Standards: Concentration vs MeanValue) 0.069 1.0781 0.627 9.614 1
Calculation: Standard curve 2:
Concentration Back Conc.
Sample Wells Values Mean Value Std. Dev. CV%
(ng/mL) Calc.
St01 6 Al 6.13 3.409 3.356 0.076
2.3
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A2 5.868 3.302
St02 3 B1 3.082 1.986 1.945 0.058 3
B2 2.931 1.904
St03 1.5 Cl 1.504 1.066 1.059 0.01 0.9
C2 1.482 1.052
St04 0.75 D1 0.769 0.589 0.575 0.021 3.6
D2 0.726 0.56
St05 0.375 El 0.385 0.329 0.328 0.001 0.4
E2 0.382 0.327
St06 0.188 Fl 0.188 0.195 0.195 0 0
F2 0.188 0.195
St07 0.094 G1 0.1 0.136 0.133 0.005 3.7
G2 0.09 0.129
St08 0 H1 Range 0.064 0.068 0.006 8.3
H2 0.002 0.072
Table 19
Concentration: ly = ((A - D)/(1 + (x/C)^13)) + D: A B C D RA2
Std (Standards: Concentration vs MeanValue) 0.071 1.039 14.67 11.673 1
Calculation: Standard curve 3:
Concentration Back Conc.
Sample Wells Values Mean Value Std. Dev. CV%
(ng/mL) Calc.
St01 6 Al 6.108 3.482 3.438 0.063 1.8
A2 5.89 3.393
5t02 3 B1 3.063 2.04 2.008 0.045 2.3
B2 2.948 1.976
5t03 1.5 Cl 1.514 1.11 1.099 0.016 1.5
C2 1.479 1.087
5t04 0.75 D1 0.757 0.6 0.589 0.016 2.8
D2 0.724 0.577
5t05 0.375 El 0.389 0.341 0.336 0.007 2.1
E2 0.375 0.331
5t06 0.188 Fl 0.193 0.202 0.202 0.001 0.4
F2 0.192 0.201
5t07 0.094 G1 0.1 0.137 0.137 0 0
G2 0.1 0.137
5t08 0 H1 Range 0.062 0.063 0.001 2.2
H2 Range 0.064
Table 20
Concentration: ly = ((A - D)/(1 + (x/C)^13)) + D: A B C D RA2
Std (Standards: Concentration vs MeanValue) 0.07 1.043 13.427 11.242 1
Calculation: human plasma samples:
Mean
Klotho
Sample Wells Values Result Std.Dev. CV% Dilution
Result
(ng/m14
A (0 h) A3 0.238 0.256 0.251 0.007 2.8 4
1.003
EDTA A4 0.231 0.246
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A (1 h) A5 0.231 0.246 0.257 0.016 6.3 4 1.028
EDTA A6 0.247 0.269
A (4 h) A7 0.235 0.251 0.25 0.002 0.8 4 1
EDTA A8 0.233 0.249
A (16 h) A9 0.248 0.27 0.267 0.004 1.5 4 1.068
EDTA A10 0.244 0.264
A (24 h) All 0.261 0.289 0.299 0.015 5 4 1.197
EDTA Al2 0.276 0.31
A (48 h) B3 0.235 0.251 0.251 0 0 4 1.006
EDTA B4 0.235 0.251
A (72 h) B5 0.242 0.261 0.256 0.007 2.8 4 1.026
EDTA B6 0.235 0.251
A (96 h) B7 0.247 0.269 0.269 0.001 0.4 4 1.077
EDTA B8 0.248 0.27
A (168 h) B9 0.254 0.279 0.286 0.01 3.5 4
1.143
EDTA B10 0.264 0.293
A (Finger) B11 0.283 0.32 0.319 0.001 0.3 4 1.276
EDTA B12 0.282 0.318
A (0 h) C3 0.239 0.257 0.259 0.002 0.8 4 1.034
Heparin C4 0.241 0.26
A (1 h) C5 0.229 0.243 0.248 0.007 2.9 4 0.991
Heparin C6 0.236 0.253
A (4 h) C7 0.236 0.253 0.261 0.012 4.6 4 1.046
Heparin C8 0.248 0.27
A (16 h) C9 0.253 0.277 0.285 0.011 3.9 4 1.14
Heparin C10 0.264 0.293
A (24 h) C11 0.256 0.281 0.301 0.027 9 4 1.202
Heparin C12 0.283 0.32
A (48 h) D3 0.237 0.254 0.249 0.007 2.8 4 0.997
Heparin D4 0.23 0.244
A (72 h) D5 0.241 0.26 0.265 0.007 2.7 4 1.06
Heparin D6 0.248 0.27
A (96 h) D7 0.239 0.257 0.272 0.021 7.8 4 1.088
Heparin D8 0.26 0.287
A (168 h) D9 0.252 0.276 0.273 0.004 1.5 4
1.091
Heparin D10 0.248 0.27
A (Finger) Dll 0.254 0.279 0.279 0 0 4 1.114
Heparin D12 0.066 Range
B (0 h) E3 0.162 0.146 0.142 0.005 3.7 4 0.568
EDTA E4 0.157 0.138
B (1 h) E5 0.16 0.143 0.145 0.003 2.2 4 0.579
EDTA E6 0.163 0.147
B (4 h) E7 0.162 0.146 0.145 0.001 0.7 4 0.579
EDTA E8 0.161 0.144
B (16 h) E9 0.173 0.162 0.162 0 0 4 0.647
EDTA E10 0.173 0.162
B (24 h) Ell 0.184 0.178 0.176 0.003 1.8 4 0.702
EDTA E12 0.181 0.173
B (48 h) F3 0.167 0.153 0.161 0.011 7.1 4 0.644
EDTA F4 0.178 0.169
61
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B (72 h) F5 0.17 0.157 0.159 0.002 1.3 4 0.635
EDTA F6 0.172 0.16
B (96 h) F7 0.173 0.162 0.165 0.004 2.5 4
0.659
EDTA F8 0.177 0.168
B (168 h) F9 0.18 0.172 0.168 0.006 3.7 4
0.67
EDTA F10 0.174 0.163
B (Finger) Eli 0.182 0.175 0.187 0.017 9.3 4
0.749
EDTA F12 0.199 0.2
B (0 h) G3 0.162 0.146 0.154 0.011 7.4 4 0.615
Heparin G4 0.173 0.162
B (1 h) G5 0.175 0.165 0.162 0.003 1.9 4 0.65
Heparin G6 0.172 0.16
B (4 h) G7 0.174 0.163 0.162 0.002 1.3 4 0.647
Heparin G8 0.172 0.16
B (16 h) G9 0.173 0.162 0.164 0.003 1.9 4
0.656
Heparin G10 0.176 0.166
B (24 h) G11 0.176 0.166 0.173 0.009 5.4 4
0.691
Heparin G12 0.185 0.179
B (48 h) H3 0.177 0.168 0.17 0.003 1.8 4 0.679
Heparin H4 0.18 0.172
B (72 h) H5 0.181 0.173 0.174 0.001 0.6 4
0.697
Heparin H6 0.182 0.175
B (96 h) H7 0.176 0.166 0.157 0.013 8.6 4
0.626
Heparin H8 0.163 0.147
B (168 h) H9 0.165 0.15 0.165 0.021 12.6 4
0.658
Heparin H10 0.185 0.179
B (Finger) H11 0.182 0.175 0.181 0.008 4.6 4 0.723
Heparin H12 0.19 0.187
C (0 h) A3 0.228 0.236 0.233 0.005 2.2 4 0.931
EDTA A4 0.223 0.229
C (1 h) AS 0.237 0.25 0.25 0 0 4 0.998
EDTA A6 0.237 0.25
C (4 h) A7 0.232 0.242 0.243 0.001 0.4 4 0.972
EDTA A8 0.233 0.244
C (16 h) A9 0.24 0.254 0.249 0.007 2.9 4 0.996
EDTA A10 0.233 0.244
C (24 h) All 0.26 0.283 0.294 0.016 5.3 4
1.177
EDTA Al2 0.275 0.305
C (48 h) B3 0.246 0.263 0.258 0.007 2.8 4
1.031
EDTA B4 0.239 0.253
C (72 h) B5 0.242 0.257 0.261 0.005 2 4
1.042
EDTA B6 0.247 0.264
C (96 h) B7 0.244 0.26 0.258 0.002 0.8 4 1.034
EDTA B8 0.242 0.257
C (168 h) B9 0.251 0.27 0.274 0.005 1.9 4
1.095
EDTA B10 0.256 0.277
C (Finger) B11 0.227 0.235 0.253 0.025 9.9 4
1.01
EDTA B12 0.251 0.27
C (0 h) C3 0.229 0.238 0.236 0.003 1.3 4 0.943
Heparin C4 0.226 0.233
62
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C (1 h) C5 0.222 0.228 0.232 0.006 2.7 4 0.928
Heparin C6 0.228 0.236
C (4 h) C7 0.226 0.233 0.241 0.01 4.3 4 0.963
Heparin C8 0.236 0.248
C (16 h) C9 0.234 0.245 0.254 0.012 4.9 4 1.016
Heparin C10 0.246 0.263
C (24 h) C11 0.244 0.26 0.265 0.007 2.7 4 1.06
Heparin C12 0.251 0.27
C (48 h) D3 0.225 0.232 0.238 0.008 3.5 4 0.951
Heparin D4 0.233 0.244
C (72 h) D5 0.242 0.257 0.254 0.004 1.6 4 1.016
Heparin D6 0.238 0.251
C (96 h) D7 0.266 0.292 0.272 0.028 10.3 4 1.089
Heparin D8 0.239 0.253
C (168 h) D9 0.25 0.269 0.269 0.001 0.4 4 1.078
Heparin D10 0.251 0.27
C (Finger) Dll 0.244 0.26 0.25 0.015 5.8 4 0.998
Heparin D12 0.23 0.239
D (Oh) E3 0.192 0.183 0.175 0.011 6.5 4 0.701
EDTA E4 0.181 0.167
D (1 h) E5 0.165 0.144 0.15 0.009 6.3 4 0.601
EDTA E6 0.174 0.157
D (4 h) E7 0.187 0.176 0.166 0.014 8.2 4 0.666
EDTA E8 0.174 0.157
D (16 h) E9 0.196 0.189 0.169 0.029 17.3 4 0.675
EDTA E10 0.168 0.148
D (24 h) Ell 0.174 0.157 0.149 0.012 7.7 4 0.595
EDTA E12 0.163 0.141
D (48 h) F3 0.174 0.157 0.158 0.002 1.3 4 0.633
EDTA F4 0.176 0.16
D (72 h) F5 0.184 0.172 0.175 0.004 2.4 4 0.698
EDTA F6 0.188 0.178
D (96 h) F7 0.196 0.189 0.18 0.013 6.9 4 0.722
EDTA F8 0.184 0.172
D (168 h) F9 0.205 0.203 0.178 0.034 19.3 4
0.713
EDTA F10 0.172 0.154
D (Finger) Fll 0.169 0.149 0.15 0.001 0.7 4 0.601
EDTA F12 0.17 0.151
D (0 h) G3 0.136 0.1 0.122 0.03 25 4 0.488
Heparin G4 0.165 0.144
D (1 h) G5 0.177 0.161 0.152 0.013 8.2 4 0.61
Heparin G6 0.165 0.144
D (4 h) G7 0.163 0.141 0.147 0.008 5.7 4 0.586
Heparin G8 0.171 0.152
D (16 h) G9 0.191 0.182 0.165 0.024 14.6 4 0.66
Heparin G10 0.168 0.148
D (24 h) G11 0.177 0.161 0.168 0.009 5.6 4 0.672
Heparin G12 0.186 0.175
D (48 h) H3 0.19 0.181 0.171 0.014 7.9 4 0.684
Heparin H4 0.177 0.161
63
CA 03101100 2020-11-19
WO 2019/113373 PCT/US2018/064333
D (72 h) H5 0.184 0.172 0.166 0.007 4.4 4 0.666
Heparin H6 0.177 0.161
D (96 h) H7 0.198 0.192 0.185 0.01 5.6 4 0.74
Heparin H8 0.188 0.178
D (168 h) H9 0.197 0.191 0.178 0.018 9.9 4 0.713
Heparin H10 0.18 0.166
D (Finger) H11 0.17 0.151 0.149 0.003 2.1 4 0.595
Heparin H12 0.167 0.147
Spike (0 h) A3 0.449 0.542 0.537 0.007 1.3 4 2.147
EDTA A4 0.442 0.532
Spike (1 h) B3 0.438 0.526 0.52 0.009 1.7 4 2.079
EDTA B4 0.429 0.513
Spike (4 h) C3 0.426 0.509 0.51 0.001 0.2 4 2.039
EDTA C4 0.427 0.511
Spike (16 D3 0.442 0.532 0.531 0.001 0.2 4 2.124
h)
EDTA D4 0.441 0.53
Spike (24 E3 0.471 0.573 0.56 0.018 3.2 4 2.24
h)
EDTA E4 0.453 0.547
Spike (48 F3 0.47 0.571 0.566 0.008 1.4 4 2.263
h)
EDTA F4 0.462 0.56
Spike (72 G3 0.514 0.634 0.636 0.003 0.5 4 2.545
h)
EDTA G4 0.517 0.638
Spike (96
H3 0.558 0.697 0.683 0.019 2.8 4 2.733
h)
EDTA H4 0.539 0.67
Spike (168 AS 0.553 0.69 0.693 0.005 0.7 4 2.773
h)
EDTA A6 0.558 0.697
Spike
B5 0.587 0.738 0.75 0.016 2.2 4 3
(Finger)
EDTA B6 0.603 0.761
Spike (0 h) C5 0.506 0.623 0.617 0.008 1.3 4
2.468
Heparin C6 0.498 0.611
Spike (1 h) D5 0.514 0.634 0.637 0.004 0.6 4 2.548
Heparin D6 0.518 0.64
Spike (4 h) E5 0.506 0.623 0.634 0.016 2.5 4
2.536
Heparin E6 0.522 0.645
Spike (16
F5 0.537 0.667 0.675 0.011 1.6 4 2.699
h)
Heparin F6 0.548 0.683
Spike (24
G5 0.54 0.671 0.669 0.003 0.5 4 2.676
h)
Heparin G6 0.537 0.667
Spike (48
H5 0.564 0.705 0.71 0.006 0.9 4 2.839
h)
Heparin H6 0.57 0.714
64
CA 03101100 2020-11-19
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PCT/US2018/064333
Spike (72
A7 0.51 0.628 0.625 0.004 0.6 4
2.502
h)
Heparin A8 0.506 0.623
Spike (96
B7 0.501 0.616 0.621 0.008 1.3 4
2.485
h)
Heparin B8 0.509 0.627
Spike (168
C7 0.481 0.587 0.603 0.022 3.7 4
2.411
h)
Heparin C8 0.503 0.618
Spike
D7 0.568 0.711 0.68 0.044 6.5 4 2.719
(Finger)
Heparin D8 0.524 0.648
Table 21
[00160] Figure 12 illustrates Klotho level at different incubation time at RT.
[00161] Table 22, below presents average Klotho protein level (ng/mL) in
plasma:
Blood sample Venous Capillary (finger)
Anticoagulant EDIA Heparin EDIA Heparin
A 1.06 1.07 1.28 1.11
0.63 0.66 0.75 0.72
1.03 1.08 1.01 1.00
0.67 0.65 0.60 0.60
A, Spiked 2.33 2.57 3.00 2.72
Table 22
[00162] Table 23, below presents spiked Klotho recovery rate (%)
Blood sample Venous Capillary
(finger)
Anticoagulant EDIA Heparin EDIA Heparin
A, Spiked 84% 100% 115% 107%
Table 23
Klotho Stability Testing at Various Temperatures Emulating Shipping Stress
[00163] Phlebotomist(s) collected >12 mL blood (sample) from each of four
donors into
heparinized blood tubes. Technician(s) immediately dispensed 300-400uL of
blood from the
collected sample of each donor into separate heparinized Micro Tubes (from
BD). Tubes
were stored and processed according to the study design (see table below).
Blood was
processed at each timepoint or centrifuged and stored (plasma) at -80 C. All
samples were
tested at approximately day 17.
[00164] For the study, 120 data points were collected, representing 6
conditions, over 14
days (see below).
_______________________________________________________________________________
________________________________________ .=
n.)
------------------------------------------------------------------------
..
.::iiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiii
= i:::i
.===
.= 1-,
Donor 4
=
......................................................, ........... .i. ...
.i.i.i.i.i.i.i.i.i.i.i.i.i.i.i.i.i.i.i.,.i.i.i.i.,
....................................................
...............................................................................
.................
.......................................,.............................
......................
= ------------------------ ---
------------------------ --------------- ========= =============
4i:i:n :i:i:i:i:i:li:i:niMii.Di:4.=:n ":0:::::.""4::: 7 ----
---------- ------
..,=========::::""=1::21:=:".=:=:::"::6:::=::=:=:"=:::=4:::".
7 6......... ........::'.4:%.......-.. c.:. 4 7
0=14 .=== 1-,
...................................................................
1-,
Temperature Group '
' -gpayg -,..--:-..m4,,,,,,,,,,::-.*DO:::::::::::-.-.i,i,i-
.13iyi,i,i, ..i:Iiiri:i:i :::..i:Davi:.::::.A;,.ay.::. ::.:EGV:.:.:
:...::.0a.y:.. ::.11-1,.:-:: ..]:Qtay.:.:.:::...ilDiayi:. ::.4...)Vcf:.:
:......:Etayi.:....:..::::1-4.r Day Ci:iziy Day Cifay ....i:
c=ii")
:.:.
,te. c=i") .
.......õ................. ............................ ...............
.._..._. _._._._._ _._._._.. _._._._._ _._._._._.. ._._._..
_._._._._._._._._.. _._._._._ _._._._._.. ._.
20'il= the entire time .-,--.-..i............... i:i:i:i:Aii:i:i:i:i
:im:=:Ximii:i:i:i:Ammi:i:Xt:i:i:i :X :ii i:i .. ii.X:: ii i: :Xii
::: :i:X:: :: i: ::X: ::: ::: i.:Xi iii iii :X:: iiiii X
iii X i::: ilfi:: :iiii X X X
i,:.:...-...-...-...-...-...,..-.......-...-...-...i.:...i.:...-...i.:...-...-
...,..-...-...-...-...i.:...i.:...iii.:...-...-...:-...i.:...i.:...-...i.:...-
...i.:...-...-...,....-...-...i.:...-...i.:...-...-...i.:...-...-...i.:.
.:=:=:::::::=:=:=:::::::=:=:=:::::::::::=:=:=:::::=:=:=:=:::::::::=:=:=:=:=:::=
:=:=:::::::::::=:=:=:::::=:=:=:=:=:::::::::::=:=:=:=:::::::=:=:=:=:=:=:=:=:::::
::=:=:=:::::::::::=:=:=::::::=:=:=:=:::::::::=:=:=:,
:::=:=:=:::::::=:::=:=:=:::::=:=:=:=:=:::::::::::=:=:=:=::::::=:=:=: ::::
=:=:=:=::::
8 ii-trs. =<-22.0,,-.., 20'C remaining time . kii.:*XiNi iiiiNiXin
i:i:i:AM..:iNi:iX=i:M:i:XM ..... . X....:..i:i iii... . . . i.X.i.i......ii
ii..... . . . X. . . . .... iii..... . :X. . ....i: ii:....... . . . iiik .
......iii iii... . ::X:::..::::ii... . . . :X. . ....iiiii..... . . . X. .
...... :ii... . . . iii:Xii......i.i. ii......... . i.X.i........iiiii.... A
X X X ==>: iii
24 hrs @iii=C, 20 C remaining time
......................................... ki.:::::-.X:M.::::::::::::::::ikiN
i:i:i:i:i:i.i.X.iii:Mai:i:XiiMiM:i: iiii. . . . iigi:::. . iii iii... . .
. :X. . ....ii ::..... . . . X. . . . .... :::..... . . . X. . ....::::.......
. . . :X........::: iii....iiXi::..::: iii... . . . i:X. . ....:::::..... .
. . iX. . ...... :::... . . . :::Xii.......ii ::....... . . . :Xi:. .
......iiii...... A. X X X i.gi:ii........iii
8 hrs Ca.:=-15C, 20rC remaining time . ii:i:=.:.-X:i:i:i:
i:i:i:i:i:i:X:i.:i:i:i:i
::::::::::::::::=)ti:iMi:i:i:i:i:i:X:=::::::::::::::::::::::::X:i:::::::: __
:ii..X.:=::i :::...1C......ii ii........:X....... __ ......:X..... __
..........4 __ ....==.X.::.. __ ......4...............=:.=XL __
:.:.:.:::.).(U:::.:.:.:.:4:.:.:.>c-. __ 1 __ x __ x __ x __ )(a.õ.
---------- ------------- -------------- ----------------------------- '
" ' "
........... .............. ............................ ............... ..
. .. . .
24 itti,s. ii10-15C, 20 C remaining One
J:i:::::).(.0
i:EXM:iii:iii:i:iXiiiiMi:iii:i:iii:iii:iii:i:A:i:i2 iii.. X. iii iii... X ..
ii ii....iiX.... ii:i... X .. ii ii.....X...iii iii.. XX... iii... X .. ii
ii.....X...iiiii... X: H X X
.---'.----.:.::=
:-:---.---..-:---.'"".--'-:--:--:-'-'-'""""--.-'-'-'-'
...:'..........:.'1.n...:'.....:F..........:.r . I .
8 hrs i9,,/:"...õ 20 C remaining time ..................
.. ::::::-.=..tt:-.:::::-
..:::::::::::iX:::::::::::::::::::::::Xiyi::::::::::::::::::::::::::::::::::::X
::::::::: :::... . :A:::..::::::... . . . :::X.:::. . ....::::..... . . .
::).g.::........ :::..... . . ... ....::::....... . . .
:...X.::........::::::... . A:::.......::::... . . . :=A::. .
....:::::..... . . . X:. . ...... :::... . . . :0::.:. . ....: :....... . . .
:..k........:: :.... x I X X X N;ii........iii
24 br. (P37C, 20'"C remaining time
ii.:::Aiiii:::iLiii:::::iiiXii:::::iii:ii:::::ii2i:::::::iiiii:::::iiiiXii:::::
ni:::::::iiii&:::::::=M....iX:i..iiiii......iXii......ii ii........X........
iii......:X......ii
ii..........X........iii,iii....iiXiii..iii,iii......iXii......iiiii........X..
...... iii......:Xii......ii
ii..........X.............X...iiiii.......X......ii ii........X.....iii
iii......iXii......ii ii..........iiXii........iii .
P
Table 24
.
,
,
o,
[00165] The plate layout (see Table
25) was the same for all four donors (see Tables 26-29, below). ,
c7,
.
A STD, 6ngimL Oh @-15C, 8hr, Day 7 @-
0C, 24hr, Day 4 @37C, 8hr, Day 9
,
,
B STD, 3ngimL @RT Day 4 @-15C, 8hr, Day 9 @-
0C, 24hr, Day 5 @37Cõ 8hr, Day 14 ,
,
,
C STD, 1.5 ng/mL @RT Day 5 @-15C, 8hr, Day 14 @-
0C, 24hr, Day 7 @37C, 24hr, Day 4
D STD, 0.75ngirni_ @RT Day 7 @OC, 8hr, Day 4 @-0C, 24hr, Day 9
@37C, 24hr, Day 5
E STD, 0.375 ng/mL @RT Day 9 @OC, 8hr, Day 5
@-0C, 24hr, Day 14 @37C, 24hr, Day 7
F STD, 0.188 ng/mL @RT Day 14 @OC, 8hr, Day 7
@37C, 8hr, Day 4 @37C, 24hr, Day 9
G STD, 0.094 ng/mL @-15C, 8hr, Day 4 @-0C,
8hr, Day 9 @37C, 8hr, Day 5 @37C, 24hr, Day 14
Iv
H STD, OngirnL @-15C, 8hr, Day 5 @-0C, 8hr, Day 14
@37C, 8hr, Day 7 rn
..i
cp
Table 25
t..)
=
oe
-a-,
c,
4,.
c,.,
CA 03101100 2020-11-19
WO 2019/113373
PCT/US2018/064333
[00166] ELISA protocol was conducted according to instructions of soluble
Klotho ELISA
kit (human), IBL, Cat# 27998). Raw data are given below in Tables 26-29:
Plate 1, Donor 1:
2.689 2.577 0.138 0.141 0.09 0.088 0.133 0.125 0.107 0.109 0.047
1.481 1.423 0.174 0.162 0.107 0.177 0.169 0.161 0.142 0.159 0.053
0.801 0.833 0.128 0.131 0.081 0.085 0.125 0.124 0.114 0.11 0.128
0.443 0.414 0.129 0.123 0.123 0.126 0.121 0.12 0.111 0.111 0.065
0.26 0.253 0.111 0.128 0.132 0.129 0.116 0.118 0.111 0.115 0.06
0162 0.154 0.109 0.116 0.128 0.131 0.135 0.127 0.113 0.111 0.062
0.15 0.114 0.087 0.092 0.126 0.131 0.137 0.137 0.099 0.124 0.062
0.148 0.058 0.219 0.098 0.115 0.277 0.243 0.128 0.052 0.056 0.087
Table 26
Plate 2, Donor 2:
2.799 2.887 0.218 0.223 0.124 0.122 0.217 0.201 0.174 0.167 0.088
1394 1.596 0.222 0.218 0.1.12 0.105 0.208 0.208 0.161 0.153 0.092
0.91 0.898 0.223 0.219 0.108 0.109 0.202 0.202 0.172 0.168 0.154
0.49 0.475 0.207 0.21 0.208 0.201 0.199 0.203 0.17 0.175 0.067
0.288 0.284 0.217 0.212 0.21 0.208 0.162 0.165 0.163 0.167 0.086
0.175 0.162 0.174 0.173 0.203 0.201 0.203 0.202 0.161 0.157 0.105
0.114 0.114 0.116 0.11 0.208 0.206 0.211 0.219 0.173 0.147 0.486
0.08 0.064 0.175 0.123 0.175 0.186 0.237 0.203 0.058 0.051 0.082
Table 27
Plate 3, Donor 3:
2.768 2.8
0.155 0.156 0.108 0.102 0.161 0,159 0.14 0.137 0.132
1.629 1.642 0.161 0.158 0.1
0.093 0.156 0.16 0.131 0.13 0.057
0.926 0.911 0.159 0.159 0.099 0.105
0.15 0,152 0.139 0.145 0.053
0.51 0.484 0.15 0.151 0.15 0.146 0.154 0,149 0.137 0.138 0.057
0.288 0.284 0.16 0.162 0.156 0.157 0.132 0,133 0.134 0.134 0.111
0.188 0.165 0.131 0.136 0.151 0.152 0.15 ft
152 0.133 0.129 0.068
0.119 0.121 0.114 0.115 0.154 0.156 0.164 0.164 0.122 0.123 0.066
0.092 0.071 0.158 0.113 0.149 0.155 0.17 0.164
0.059 0.061 0.078
Table 28
Plate 4, Donor 4:
67
CA 03101100 2020-11-19
WO 2019/113373 PCT/US2018/064333
2.787 2.699 0.177 0.207 0.137 0.128 0.196 0.185 0.144 0.14 0.132
1.614 1.622 0.199 0.186 0.112 0.104 0.18 0.178 0.139 0.133 0.057
0.911 0.884 0.199 0.187 0.109 0.104 0.168 0.17 0.139 0.135 0,053
0.494 0.475 0.177 0.173 0.182 0.178 0.166 0.168 0.147 0.148 0.058
0.302 0.287 0.164 0õ17 0.19 0.188 0.15 0.156 0.14 0.137 0.111
0.179 0.175 0.164 0.169 0.183 0.179 0.187 0.191 0.144 0.14 0.068
0.122 0.122 0.125 0.122 0.171 0.164 0.174 0.172 0.133 0.133 0.067
0.083 0.062 0.165 0.128 0.165 0.162 0.176 0.173 0.049 0.048 0,079
Table 29
[00167] Data analysis is given below in Tables 30-41:
Plate 1, Donor 1:
Standard calculations
11Ø4C9DIC#Lc Values !fttlYalUe. 5.',tAge1Y- Citt
St01 6 Al 5.965 2.611 2.555 0.079
3.1
A2 5.7 2.499
5t02 3 B1 3.133 1.403 1.374 0.041
3
B2 2.998 1.345
5t03 1.5 (1 1.575 0.723 0.739 0.023
3.1
Ã2 1.647 0.755
5t04 0.75 D1 0.775 0.365 0.35 0.021
5.9
02 0.711 0.336
5t05 0.375 El 0.376 0.182 0.178 0.005
2.8
2 0.361 0.175
St06 0.188 Fl 0.168 0.084 0.08 0.006
7.1
F2 0.151 0.076
St07 0.094 61 0.143 0.072 0.054 0.025
47.6
62 0.069 0.036
Table 30
Sample calculations
68
CA 03101100 2020-11-19
WO 2019/113373
PCT/US2018/064333
S.M10:40-AV:ME MM-EReatiaNkfteklAtiiItilVattei --
,i0A0Diktt:&.VgAtiatUrm.A.V2INU
0,...'1 43 0.06 0.115 0121 0.004 3.E 4 1435 100
.44 Ø-053 0.1.24
F7 0,a
4 33 0.03:5. 0.133 0.1.R1 0.013 9,..3 4
0.723 1.49
34 0.1094 0.15.."--
RT 0,a';,,
.,-,--:,.,
, 0.05 0.093 0.101 0.004 43. 4
0.403 a2.3
04 0.053 0.104
FC7.1...`t.a.
7 03 0M1 at 0.094 0.005 33. 4 0.37C
0,77
04 0:045- 0.035
RI- 9. E3 0:033 0.053 008 0.024 30.4 4 0.32-2
0E5
34 0.05 0.095
RI- 0,-,,Ey
14 F3 0031. 0.053 0.055 0_01 15.1 4
0.255 0.55
F4 0:032: 0.073
-.1_0,,
.5}%rõ Day
4 µ,3 4-,_,-.
0.009 0.015 0.021 0.007 333. 4 0.032 0.17
434 0014 0.023
-15.0õ
8Ã-,:rõ Day
5 HS 0.143._ 0.228 0.153 0.17.8 103.1 4 ,-k.
,..,
,.,_,.g.a.:_ 1.34
H4 0.02 0.037
-15.0õ
8Ã-,:rõ Day
7 .45 0.022_ 0.022 0z2: 0.003 .14 4
0_075 0,15
AS 0.01 0.013
8#1:r,
9 35 0023 0.05.5 0.127 0.102 802 4
0.509 1 .0S
3.5 0.l0.39. 0.2
Str,. Day
14 CS 0.:a33. 0.004 0.008 0.C42.5 .543 4
0.033 007
CS 0.1007 0.012
00, 8r,
Day 4 DS 00,19 0.028 0.091 0.004 43 4
0.352 0.7-4
06 0:045 0.054
00, aitr,.
Day S ES 0:054 0.109 0.103 0.004 4.2 4
0.412 0.35
ES 0.051 al
00, 8r,
Ds? FS 0.05 0.035 (nal, 0.0a4 43 4 0_403
0.33
FS 0053 0.104
00., 8r.
Day 9 r'T.,
..a., 0.042. 0.094 0.095 0.007 73. 4 0.395
0.2.1
06 0.a.53. 0.104
Da.,y 14 HS 0037 0.071 0.242 0.241 933 4
0.958 1,39
H6 0.199 0.413
69
CA 03101100 2020-11-19
WO 2019/113373
PCT/US2018/064333
0C, 24hr,.
Day 4 47 0.055 0102 0.1 0.012 ILE. 4 0393
:1122
43 11047 0.052
0C, 24h-,
Day 5 67 11091 0.163 0.174 0.012 62 A
0:655. 1.44
ES' 11053 0.155
00, 24h,
Day 7 C7 11047 0.092 0.091 0.001 LS 4
1136.2 ,0..74
CS 0.046 0.05
00, 24h-.
Day 9 07 11043 0.057 0.052 0.001 1.7 4 0.33
.1163.
0.5 0042, 0.021
00, 2.4ihr,.
Cs-.ay 14 E7 0.032 11.073 0.075 0.003: 3...3
4 0.301 Ø62
ES 0.04 0.077
370õ Ehs-.õ
Day 4 F7 1105,7 0.112 0.10$ 0.012 11.2 4 :11416
F.2 11049. 0.056
37'.7."-, Shc.
Day 5 137 acsa 0.116 0.116 0 0 4 0.4E5 .1196
GE 0.056 0.116
3711. $h,
Day 7 H7 11165 0.34 0.215 0_171 75.2 4 0275
1.50
HE 1105 0.052
37Z, ShF,.
Day 9 45 agis 0.095 0.057 0.003 5 4 11229
.1147
410 0.031 0.055
3711. Shs-,.
Day 14 69 11064 0.127 0.144 0.025 17.3 4 0377-
1.15
&ID 11081 0.162
D.
-,,,,,,S--, . i
241-Or,
Day 4 CS 11036 0.069 0.065 0.006 &E 4 0.262
,11.54
010 0_032. 0.061
37,,:-,
24hr,
Elay 5 03 11033 0.063 0.0E3 0 0 4 11753. :1152
010 11033 0.063
24-:hr,
Eia,y '7 ES 0.033 0.063 0.067 0.0:36 ,2:.5
4 11269 0.55
E10 0.037 0.071
37C,
24hEr.,
Elay 9 F9 11035 0.067 0.065 0.003 4.4. 4 0.2E2
;1154
F10 01.033 0.063
,--,
-,..r,
7 i
241-Or,
Day 14 09 11021. 0.039 0.064 0.036 55.3 4
0,295. 0.53
010 0.046 0.06
Table 31
Klotho Concentration Changes in Time Course
Room Temp -15 C 8hr 0 C 8hr 0 C 24hr 37 C 8hr 37 C 24hr
Day 4 149% 17% 74% 82% 86% 54%
Day 5 83% 134% 85% 144% 96% 52%
CA 03101100 2020-11-19
WO 2019/113373
PCT/US2018/064333
Day 7 77% 16% 83% 74% 180% 55%
Day 9 66% 105% 81% 68% 47% 54%
Day 14 55% 7% 199% 62% 119% 53%
Table 32
Plate 2, Donor 2:
Standard calculations
Sample Concentration Wells BackConcCalc Values MeanValue Std.Dev. CV%
St01 6 Al 5.451 2.736 2.78 0.062
2.2
A2 5.633 2.824
5t02 3 B1 2.983 1.531 1.532
0.001 0.1
B2 2.987 1.533
5t03 1.5 Cl 1.613 0.847 0.841 0.008
1
C2 1.589 0.835
5t04 0.75 D1 0.792 0.427 0.419
0.011 2.5
D2 0.763 0.412
5t05 0.375 El 0.407 0.225 0.223
0.003 1.3
E2 0.399 0.221
5t06 0.188 Fl 0.197 0.112 0.105
0.009 8.7
F2 0.173 0.099
5t07 0.094 G1 0.087 0.051 0.051 0
0
G2 0.087 0.051
Table 33
Sample calculations
71
CA 03101100 2020-11-19
WO 2019/113373
PCT/US2018/064333
iSVtgiN. IM.-er-
MMV.g1I:8=RAWItE41Wti.gt0Atl...M..ali.^V -,Cg.'4iMMEM.It AgjAfukv -
:',tv..,71-=
C. AS 0.155 0.275 0281 0.&27 23 4 2.124
.44 025 0.286
.R7 tia',s
4 23 12155 0.284 0.28 3005 1.9. 4 112
1.00
E4 0.255 0.275
R7. Day.
C3 0.16 0.285 0282 0.D.a5 13 4 1227 1.00
04 a .156 0272
87 EiBy
7 03 0.144 0256 es -,,,,:ci 0.1:>24 LS 4
1.035 032
04 0..147 0.261
RT Day 3, E3 13.15$ (....., .274 027 0Ø27 2.4 4
1.079 0.96
E4 0149 0.265
gi" Day
14 F3 11121 0.195 0.164 a Wil. 1-=...,, ,r, 4
0,777 0.63
F4 02:1 0.193
4;0;
z hT, Day
4 Ci3 0053. 0.09 0.065 0.D38 3.5. 4 0.34
0.7:0
134 0.047 0.0g
-15C0
Sh:r; Els,
C 1-13 .0,112 :::,.1.97 0.15 0.067 44.4 4 0.6
0.33
1-14 1206 0.103
4;0;
7 AS 0.061 0.105 0.203 0.303 2.5 4 0.411
0.37
AS :0..055 0.01
6.1rõ Da,;
g ES 0.045 0.033 0277 0.039 11.4 4 0.303
0.27
E6 0.042 0.071
-150,
8, Day
24 CS 0.045 0.076 0.077 &Dal 1.6 4 12308
0.27
OS 0.046 0.078
00, am.,
Day 4 DS 0245 0.258 0.251 0005 3.6 4 2.005
0.E9
DE 0.138 0.245
0112 .3 w.,
Day 5 ES -0147 0.261 026 aiXR 1 4 1.033
0.92
ES 0145 0.258
0i2,, .a:r,
Bay? FS 0.24, 0.*745. 0.247 0.003 1.1 4 0.937
1233
F6 [j3 0.245
00., &.-sr,
Day 9 .95 0245 0.252 0.255 0.033 :1 4 1224
0.51
.GE Ø143 0.2.54
OC,
Bay :14 HS 0.122 0.197 0.207 0.024 6,5 4 0_823
0.74
0123 0.217
72
CA 03101100 2020-11-19
WO 2019/113373
PCT/US2018/064333
010õ 24iv,
Day 4 .47 9,154 9.274 0.26 a 021. 8:_t 4
1.099. 0.92
.48 9.139 0.245
00,, 241-13-,
Day 5 97 0,145 a zszi 0.255 0 0 4
1.031 0.92
as 9.1.45 9256
00, 241v,
Da y 7 0:7 91.39 0.247 0.247 0 9 4
0:987 058
CZ 9,139 9.247
00, 24,
Day 9 07 9.139 9.241 0.245 0.039 2..1 .4
9.979 097
DE 0,14 9.249
00, 24iv.,.
Day 14 E7 9.095 9.173 0175 0.004 2.2 .4
9704 0.93
ES an2 0.173
370.
Day 4 F7 0.14 9.249 3.245 0.031 95 4
959 0.88
F5 9.139 9.247
.37D 3iv,
Day 5 57 9,145 9.253 0,271 0.01 3.5 4
Lass 0.36
,-..z. .9
..,z. 9.159 278
.,,
37D 3.
Day 7 H7 9,174 9.312 0.25 9.049 15.:5 4
1.12 Loa
HS 0.14 9.245
37D Eiw,
...Da:i." 9 .49 9.111 9.195 0.183 0,009 4.3 4
r...-,cr. 0.57
.410 9,104 0.152
37D.
Day 14 99 9.099 9.171 0.164 051 9,3 4
0.557 0.93
919 0.09 0.157
370.
24hr.
0:109. .0,192. 0.183. 0.005 .25 4 .,.,:-..,-, _,;,,
,.µ 957
000 9.105. 9.184
37.
.24hr,
Day 5 E:.'3 9,107 9.158 3.192 0.035 3_4 4
07? 0.59
an 9.112. 9.197
37D
24:hr,.
Day 7 E9 92 0175 9.179 9.005 .25 4 0.715
0.54
E.10 9104 01:32
.37D
.24hr,
Day 9 F9 0,995 0171. 0169 9.005 3,1 4
9571 0.90
F1.9 9,994 0164
370..
24:hc
D3,*t- 24 5.9 0.11 0.1.93 1017 0.033 19,7 4
0.675 0.99
4319 9.984 9.143
Table 34
Klotho Concentration Changes in Time Course
Room Temp -15 C 8hr 0 C 8hr 0 C 24hr 37 C 8hr 37 C 24hr
Day 4 100% 30% 89% 92% 88% 67%
Day 5 100% 53% 92% 92% 96% 69%
Day 7 92% 37% 88% 88% 100% 64%
73
CA 03101100 2020-11-19
WO 2019/113373
PCT/US2018/064333
Day 9 96% 27% 91% 87% 67% 60%
Day 14 69% 27% 74% 63% 58% 60%
Table 35
Plate 3, Donor 3:
Standard calculations
Sample Concentration Wells BackConcCalc Values MeanValue Std.Dev. CV%
St01 6 Al 6.24 2.768 2.784 0.023
0.8
A2 6.333 2.8
5t02 3 B1 3.15 1.629 1.636
0.009 0.6
B2 3.182 1.642
5t03 1.5 Cl 1.52 0.926 0.919 0.011
1.2
C2 1.489 0.911
5t04 0.75 D1 0.705 0.51 0.497
0.018 3.7
D2 0.659 0.484
5t05 0.375 El 0.337 0.288 0.286
0.003 1
E2 0.331 0.284
5t06 0.188 Fl 0.195 0.188 0.177
0.016 9.2
F2 0.164 0.165
5t07 0.094 G1 0.108 0.119 0.12 0.001
1.2
G2 0.11 0.121
Table 36
Sample calculations
74
CA 03101100 2020-11-19
WO 2019/113373 PCT/US2018/064333
W6R.M.WiiittMAt,-..W.tMAItti,ta.eatt ft0,-00,,.=Eg AWmoDikiti-wv -,--
AdiA.m.,1:v -,to:.:Gbi*Ki
a-4 43 0.155 0.152 0.152 awl 05: 4 0509
LCD
.k4 0.156 0153
FT DEY
4 53 0161 0.155 0.157 0.003 1.7 4 063
1.0E.,
54 0.150 0156
RT. Day
03 0.1.59 0157 0157 0 0 4 0627 1,;03
04 0155 0.1.57
.P.7 Dav
7 03 015 0145 0.146 0.001 0.6 4 0524 096
04 0.151 0.147
51- Day 5, 53 0.16 0l5,3 0:.159 0.002 1,1 4 0.537
LOS
54 03.:52 0.161
RI Dav
14 F3 0d31 0.122 0.125 0004 3.4 4 0501
052
F4 0.135 0125
-15C,
Sh:f , Day
4 03 0.114 0.102 0.103 0001. 05 4
0.411 0.57
124 0 115 0.103
150,
8hr,, iDow
5 H3 0.155 0.156 0.128 0.035 30.1 4
0513 084
H4 0,113 0101
-150,
Stv,. Day
7 .45 0.100 0.095 am aces 5.2 4 0367
050
.46 0.102 0.052
f.h:fõ Day
9 55 ,111 0.056. 0052 0.00.; 6.6 4
0329
6.5 0.093 0.079 0.54
-150,
al-, Day
14 CS 0.095 0.055 0.0S3 0005 5.4 4 0354
06 0. 105 0.092 0.55
00, 817:r,
0;Tg 4 09 0.15 0145 0..143 0004 2.5 4 0.572
06 0.146 0.14 054
00, D.r,
Day 5 ES 0,156 0.153 0.154 0.0121 116 4
11514
ES 0.157 0.154 1,01
oc, 517,e,
Day 7 F5 0,151 0:147 0.147 0001 OS 4 0.539
FS 03.52 0148 11 6.7
012, 2.-71-,
Day 5 GS 0.154 1135 0.152 0.002 1.2 4 0507
125 0.156 0.153
00, al-,
Day 14 HS 0145 0.144 01148 0.00.4 3.6 4 0.592
HE 03.55 0.1.52 0.57
CA 03101100 2020-11-19
WO 2019/113373
PCT/US2018/064333
Or, .24h .-,.
Day 4 A7 0.151 12.1.59 0..158 0.002 Li 4
0.532
A8 0.129 0.157
1.04
00, 24hc
Day 5 B.7 1,156 :1153 0.155 0.004 2..7. 4
0.522
ES 0..16 alsa
1.02
OS, 24hc.
Day 7 07 0.15 1145 0.147 0.002 1_2 4 1537
02 :0.1.52. 0.148
0.35
00,
Day 9 07 0154 0..15 0.147 0.004 7:,., 4
1589
08 0,149 1144
027
00, 24h
D8:14 E7 .1.132 111.23 0,124 0.1201 117 4
11.495
E8 1133. 0.125
0.21
3712-, She,
03:V4 F7 0_15 1:143 13.147 0.002 1.2 4 0.597
F8. .1.152 1145
0.95
3712, 2h;7.,.
Da!,$k 5 G7 0..154 :1153 0.13 0 0: 4 a 553
Ci 8 0.154 1153
2.:07
3712, 81-p:,.
Day 7 H7 0.17 1171 1167 0.005 3,3 4 15E8
HS 1154 111E3
1.10
37C. tih;7õ
Day 9 AS 0.14 a1.33 0,131 11003 2 4
10.523
410 41137 1125
10.86
37Cõ Sh;7õ
Day 14 E9 0131 :12122 0.122 a Dal 0:7 4 11426
BIG 0..13 0.121
0,80
37C.õ
.24hr,
Day 4 CS 02139 1132 0.135 0,009 3.9 4 0.542
010: 11145 :121.33
0.29
37C,
.24hrt
Day 5 09 1,137 0.1.-23 '0.23 0.001 117 4
0.92
010 013a 1131
1185
37c
-24hF,.
0i7 ES 111..34 1125: 0,125 0 0 4 0,503
E 10 13.13$ 0.126
0.23
37C,
.24hr..
Day :9 F9 0,133 1125 0,122 a 003 2.8 4 0.459
F20 ,11129 0.12
1280
37C.,
.24hr,
Day .14 .G9 13..1.22 am 0.112 0.001 117 4 a 445
G.10 aua 0113
0.74 .:
Table 37
Klotho Concentration Changes in Time Course
76
CA 03101100 2020-11-19
WO 2019/113373
PCT/US2018/064333
Room Temp -15 C 8hr 0 C 8hr 0 C 24hr 37 C 8hr 37 C 24hr
Day 4 100% 67% 94% 104% 96% 89%
Day 5 103% 84% 101% 102% 107% 85%
Day 7 103% 60% 97% 96% 110% 83%
Day 9 96% 54% 100% 97% 86% 80%
Day 14 105% 58% 97% 81% 80% 74%
Table 38
Plate 4, Donor 4:
Standard calculations
Sample Concentration Wells BackConcCalc Values MeanValue Std.Dev. CV%
St01 6 Al 5.507 2.715 2.671 0.062
2.3
A2 5.321 2.627
5t02 3 B1 3.058 1.542 1.546
0.006 0.4
B2 3.074 1.55
5t03 1.5 Cl 1.623 0.839 0.825 0.019
2.3
C2 1.569 0.812
5t04 0.75 D1 0.794 0.422 0.412
0.013 3.3
D2 0.757 0.403
5t05 0.375 El 0.422 0.23 0.222
0.011 4.8
E2 0.393 0.215
5t06 0.188 Fl 0.19 0.107 0.105
0.003 2.7
F2 0.182 0.103
5t07 0.094 G1 0.086 0.05 0.05 0
0
G2 0.086 0.05
Table 39
Sample calculations
77
CA 03101100 2020-11-19
WO 2019/113373 PC T/US2018/064333
...............................................................................
...............................................................................
...............................................................................
........
...............................................................................
...............................................................................
...............................................................................
......
3iiittei ii.W;gt.iliipinA,iiiii-iiimii =kfin Afi.*Igt(VhV ,11.Viba
=tl'ii=Uiininiiti-ii:J. fiii. ===iii =AitiltiNk i.-,-,.t.6i.C. 6iniMil
011 .43 0.105 Ø186 0.214. 0.04 205 4
137 1.03
.44 0.135 0.242
RT Day
4 e.3 0,127 0.127 0215 0017 8. 4 ass
Lim
34 0114. 0.203
RT Day
S ,---,
...._.z. 0.1.27 0.277 0.215. 0.0' 6 7.3 4
0..364 1.01
4 :0115 0.205
RI Day
7 03 0.105 0.116 0103 12.005. 2..9. 4
0.73 0.35
04 0101 0.1.79:
RT DE4 .9 E3 0.092. 0.152 0238 0008 4.7 .4 0.671
0.78
54 0_093. 0173
RT Day
1$ F3 0.092 0.162 r.,-.3,--.
,:;..,......,,ri,e 0.007 3,9 4 0.657
0.78
FA 0.097 0.171.
-1SC,
31-$1õ Day
4. r_...3 0.053 0.091 0.008 0.034 4.3 .4 0353
0.41
124 025 0256
-15Cõ
Shr, Day
H.S.' 0.053 0.164 0.13 0.043 36_7 4 0.521 061
H4 0.055 0.11:96
-16Cõ
si-a, Day
7 AS aoss 0.1.13 010.6 0_012 2.1 4 04.23
0:49
.46 .0,056 aoss
-isc
Sh:rõ Day
9 ES 0.0A. 0053 0.061 031. 2E. 6 4 3242
0.23
96 0.032 0.054
-ISO,
81.v, Day
14 CF, 0.037 0.052 Ø.06:3 0_036 -102. 4
0232 0.27
CS .0,032 0.054
00, 8r,
pari,,. 4 DS 01.1.. 0195 0.197 0.035. 2.7 .4 07'67
0.39
06 0106 :0138
0Cõ ".&r,,
Day S ES 0.113. 0.21. 0205 0.033 1,:3 4
0.334 097
56 0.1.16 0.207
0Cõ. 8r.
Da.:,, 7 FS :0.111 0.197 01.94. 0Ø06 2.7
4 ,,_.:,-, . i7 , -TA L-F 0.. 53
Fs 0,107 0.19
00.
aoss 0175 0.159 0009 5,4 4 0675 0.79
06 acs 2 0.152
0C., 8r,
C14 HS 0.053 0.164 01.51 0.0114 2.4 4 0.545
0.7;
He 0.09 0.153
78
CA 03101100 2020-11-19
WO 2019/113373 PCT/US2018/064333
O0, 24h;7õ
Day 4 47 0_124 0222. 0.211 0014 6.9 4 0.2.45
093
AS 01 13 0201
O0, .24h7õ
Day S. 57 010E 0192. 0.29 0.033 14 4 0.7s
0E9
ES 0106 0.152.
oc, 241-B-,
Day- 7- 07 0:096 017 n .. 71, 0.D33 IS 4
0.,6,96 050
CS 0.095. 0173
oc, 241v,
0 3 V 9 07 0094, 0156 0165 0.M3 1.6 4 0.571
0.75
DS 0036 0.:17'
00, 241-13-,
Day- 14 E7 0.075. 0136 01 42 0.0)0 5.5 4
056.8 0.66
Ea 0034 0.147
370, 61-1
4 F7 0.115 020r5 azim a DIS 2,5 4 0934
0.97
RS ans 0217
370õ Stoc
Day S 417 0102 0.181. 0179 0033 15 4 aTts
023
02; 0,1 0.177
370. Shc
Day 7 H7 0104 015.4 alK 0.004 2,2 4 0726
025
HS 0.101 0179
370. Zi-q.
Day 3 49 0072. 0.125 0122 0,039 42 4 0437
0,57
.410 0068 alla
370,. 91-o;
Day 14 E9 0067 0.116. 0111 CMS 6,9 4 0444
0,92
s'.o 0051. 0:109
370.
=24h r,,
Day- 4 09 0057 0126 0.113 0035 4.6 4 0452
093
010 0.063 :0.105
24hr,
Day. S 09 0075 0231 0.132 0.031 1 4 0522
0.52
Die 0:076 0133
370.
24hr,
Day '7 E9 aosa allS 0.115 0,034 13 4 0.462
0.94
Elf) 0065 am
370,
24hr.,.
0y9 F9 0072 0125, 0_122 00JS, 4.2 4 045.7
0.97
F1C 0066 ai la
370,
24,
Day 14 C4.9 :C....061 01,05 0.105 0 0 4
0.422 0,49
-...1..,... 0051 0.105.
Table 40
Klotho Concentration Changes in Time Course
Room Temp -15 C 8hr 0 C 8hr 0 C 24hr 37 C 8hr 37 C 24hr
79
CA 03101100 2020-11-19
WO 2019/113373
PCT/US2018/064333
Day 4 100% 41% 89% 99% 97% 53%
Day 5 101% 61% 97% 89% 83% 62%
Day 7 85% 49% 90% 80% 85% 54%
Day 9 78% 28% 79% 78% 57% 57%
Day 14 78% 27% 75% 66% 52% 49%
Table 41
[00168] In light of the foregoing, at least one embodiment includes a Klotho
detection kit
and/or assay. The inventive kit and/or assay may be suitable for CLIA lab
testing of clinical
samples. The kit can be configured (specifically) for use in monitoring,
detecting, and/or
quantifying, or diagnosing deficiency of one or more proteins selected from
(1) Native (e.g.,
endogenous) Klotho, (2) recombinant Klotho-Fc fusion, and (3) recombinant
6xHis Klotho.
The Klotho protein can be detected in a biological sample. The sample can be
(substantially)
unpurified in some embodiments. For example, the sample can be or comprise
mammalian
(e.g., human) blood and/or plasma.
[00169] In at least one embodiment, a kit can comprise one or more lancets
adapted,
configured, or suitable for a finger prick, a sample collection container
adapted, configured,
or suitable for collecting blood from a finger prick, one or more antiseptic
elements, such as
disposable (e.g., alcohol) wipe(s), one or more adhesive bandages, a foil
envelope or other
biopackaging, and a shipping container, such as an envelope.
[00170] Some embodiments can include a method of diagnosing Klotho deficiency
in a
mammalian subject. The method can include obtaining a fluid sample mixture
selected from
the group consisting of: (i) mammalian serum, optionally in the form of
capillary blood,
mixed with a preservative or anti-coagulant comprising one or more of heparin,
lithium
heparin, EDTA, and K2 EDTA, the mammalian serum having an amount of soluble
Klotho
protein; and (ii) mammalian serum, optionally in the form of capillary blood,
the mammalian
serum having an amount of soluble Klotho protein, the method further
comprising mixing the
mammalian serum with a preservative or anti-coagulant comprising one or more
of heparin,
lithium heparin, EDTA, and K2 EDTA to form the mixture. The method can also
include
storing the mixture for at least 24 hours, or more, at room temperature, the
preservative or
anti-coagulant stabilizing the soluble Klotho protein for the at least 24
hours, or more, at
room temperature. After the storing step, the method can include quantifying
the amount of
the soluble Klotho protein in the mixture. The method can also include
diagnosing the
mammalian subject with Klotho deficiency when the quantified amount of soluble
Klotho
protein in the mixture is less than a predetermined threshold amount.
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[00171] As described above, the fluid sample mixture comprises about 10-
1000u1,
preferably about 50-200u1 of capillary blood from the mammalian subject, or
wherein the
step of obtaining the fluid sample mixture comprises obtaining about 10-
1000u1, preferably
about 50-200u1 of capillary blood from the mammalian subject. The step of
quantifying the
amount of the soluble Klotho protein in the mixture comprises detecting the
soluble Klotho
protein using a first antibody that binds to a portion of the soluble Klotho
protein, and
optionally, detecting the soluble Klotho protein using a detection antibody
that binds to a
portion of the first antibody or performing an enzyme-linked immunosorbent
assay (ELISA)
to detect and optionally quantify the soluble Klotho protein. Mass
spectrometry can also be
used to detect proteins.
[00172] In some embodiments, a diagnostic or informational report can be
prepared. The
report can include an indication or display of measured Klotho level(s) for
one or more
Klotho proteins. In some embodiments, the report can display a comparison
chart or graph
depicting the measured level overlaid on an age-based graph illustrating
average or median
Klotho levels for various ages and, optionally, indication of the 25th
percentile and/or 75th
percentile for the various ages. Figure 13 illustrates an example report graph
or chart. Thus,
in some embodiments, diagnosing the mammalian subject with Klotho deficiency
comprises
displaying a Klotho deficiency determination, indication, or diagnosis on a
user interface of a
computer system and/or producing a file or report, in physical or electronic
form, that
displays the Klotho deficiency determination, indication, or diagnosis, the
Klotho deficiency
determination, indication, or diagnosis optionally including the quantified
amount of the
soluble Klotho protein and/or the predetermined threshold amount.
[00173] It will be appreciated that the term "diagnosis," or diagnosing," and
similar terms,
as used herein, is not intended to convey or require (in all cases) a
certified, regulatory-
.. compliant, medical diagnosis, as required or regulated by the U.S. Food and
Drug
Administration (FDA) or any other government or non-government entity,
organization, or
agency. Rather, "diagnosis," or diagnosing," and similar terms relates to
providing
information that may be indicative of a condition and/or helpful in making a
determination
related to the condition.
.. [00174] In some embodiments, one or more additional analytes can be
monitored, detected,
and/or quantified. Illustratively, the additional analytes can be related to
Kidney disease,
fibrotic diseases, and/or aging. In some embodiments, the one or more
additional analytes can
be selected from the group consisting of FGF, PTH, non-oxidized PTH, Vit D,
Vit E, KIM-1,
NGAL, Interleukins and other inflammatory bio-markers, testosterone, estrogen,
etc.
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Therapeutic Proteins
[00175] Embodiments of the present disclosure can include one or more
therapeutic and/or
recombinant human alpha soluble Klotho proteins, protein fragments, and/or
protein variants.
[00176] The protein can comprise all or a subset of amino acid residues 1-
1012, 1-981, 29-
981, 34-981, 36-981, 131-981, 1-549, 29-549, 34-549, 36-549, or 131-549 of
human alpha
Klotho isoform 1 or 2. The protein can have at least and/or about 80% amino
acid sequence
identity to all or a subset of amino acid residues 1-1012, 1-981, 29-981, 34-
981, 36-981, 131-
981, 1-549, 29-549, 34-549, 36-549, or 131-549 of human alpha Klotho isoform 1
or 2. For
instance, at least a portion of the protein can have at least 80%, etc. amino
acid sequence
1() identity to at least a portion of one of SEQ ID NO: 2 through SEQ ID
NO: 70 or SEQ ID NO:
107 through SEQ ID NO: 120 or SEQ ID NO: 125 through SEQ ID NO: 128 or SEQ ID
NO:
133 or SEQ ID NO: 152, or a combination of two or more thereof Other portions
or
fragments of the protein sequences described in the present application are
also contemplated
herein. For instance, some embodiments can include a protein having at least a
portion with
at least 80%, etc. amino acid sequence identity to any suitable portion of one
of SEQ ID
NOS: 1-75, or a combination of two or more thereof.
[00177] Some embodiments can include a protein having one or more amino acid
variations
as compared to human alpha Klotho isoform 1. Illustratively, the protein can
comprise a
human C370 variant. For instance, the protein can include a C3705 alteration,
thereby
comprising S370. The protein can include human F352 or other than F352V in
some
embodiments. In at least one embodiment, the protein can include the C3705
alteration,
thereby comprising S370, without a F352V variant, preferably with F352. The
protein can
include H193 or other than the H193R variant. All other standard amino acid
substitutions at
amino acid residue (or position) 193, 352, and/or 370 of human alpha Klotho
isoform 1 are
.. contemplated and explicitly disclosed herein.
[00178] Some embodiments can include a variation at amino acid residue 45 of
human alpha
Klotho isoform 1. At position 45, the residue can be a valine (Val; V), a
phenylalanine (Phe;
F), or another amino acid.
[00179] The protein can also include one or more glycans (attached thereto).
For instance,
.. native human alpha Klotho isoform 1 can have glycans attached (via
glycosylation) at amino
acids 106, 159, 283, 344, 604, 612, and/or 694. Accordingly, the proteins of
the present
disclosure, or Klotho protein sequences thereof, can have one or more of the
same (or
similar) glycans attached (via glycosylation) thereto (e.g., at the same amino
acid position(s)).
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In a preferred embodiment, the protein includes all of the same or similar
(native-type)
glycans attached thereto, at the same amino acid position(s).
[00180] In some embodiments, the protein can include a signal peptide or
signaling
sequence. For example, the protein can include a native Klotho signaling
sequence. The
protein can include a non-native or synthetic signaling sequence. In some
embodiments, the
signaling sequence can be an N-terminal signaling sequence and/or upstream of
(or N-
terminal to) a Klotho protein sequence. In other embodiments, the signaling
sequence can be
C-terminal or otherwise disposed. Preferably, the signaling sequence can be,
comprise, or
have at least 80% amino acid sequence identity to native human alpha Klotho
isoform 1
signaling sequence, native human alpha Klotho isoform 2 signaling sequence,
SEQ ID NO:
71 or SEQ ID NO: 72.
[00181] In some embodiments, the protein can include an amino acid tag. The
tag can be a
C-terminal tag and/or downstream of (or C-terminal to) a Klotho protein
sequence. In other
embodiments, the tag can be N-terminal or otherwise disposed. The tag can be
or comprise an
Fc-peptide (or Fc-fusion tag, Fc domain, etc.). For instance, the tag can be
or comprise an
IgGl-Fc protein sequence. Preferably, the tag can be, comprise, or have at
least 80% amino
acid sequence identity to SEQ ID NO: 74.
[00182] The tag can also, or alternatively, be or comprise a peptide
comprising a Twin-Strep
protein sequence, such as a Twin-Strep tag or protein sequence (e.g., as known
in the art).
Preferably, the signaling sequence can be, comprise, or have at least 80%
amino acid
sequence identity to SEQ ID NO: 75, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO:
104.
[00183] The tag can also, or alternatively, be or comprise a polysialic acid
(PSA). In at least
one embodiment, the PSA tag can render the conjugated (Klotho) protein
resistant to one or
more proteases. In some embodiments, maintaining an amount (e.g., even a
relatively small
amount) of sialic acid in the protein conjugate can prevent protease attack
and subsequent
targeting, breakdown, and clearance of the molecule (e.g., through the liver).
[00184] In at least one embodiment, the tag can be cleaved from the protein.
In other
embodiments, the tag can be retained as part of the protein. In some
embodiments, the tag can
enhance solubility and/or (serum) half-life of the protein. In some
embodiments, the tag can
be utilized during protein purification (e.g., as part of a purification
mechanism).
[00185] In some embodiments, the protein can include a linker (e.g., amino
acid linker)
disposed between a Klotho protein sequence and an amino acid tag.
Illustratively, the linker
can comprise between 1 and 40 amino acids, preferably between 5 and 20 amino
acids, more
preferably between 6 and 12 amino acids, most preferably between about 8 to 10
amino
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acids. The linker can be or comprise a GS linker (e.g., a (G4S)2 linker) or
other linker (e.g., a
GGENLYFQ linker) in some embodiments. Preferably, the linker can be, comprise,
or have
at least 80% amino acid sequence identity to SEQ ID NO: 73 or SEQ ID NO: 105.
[00186] In at least one embodiment, the protein can be cGMP regulation
compliant, as
determined and enforced by the U.S. Food and Drug Administration (FDA). For
instance, the
Klotho protein can be at least 90%, 92%, 95%, 96%, 97%, 98%, or 99% pure, dry
weight. In
some embodiments, the Klotho protein sample can include less than about 1-100
parts per
million (ppm), less than about 100-1000 parts per billion (ppb), or less than
about 1-100 ppb
CHO host cell proteins (HCP), nucleic acid, and/or other cellular components,
or any value or
range of values disposed therebetween.
Klotho Protein Variants
[00187] Therapeutic S-Klotho proteins of various lengths (e.g., S-Klotho,
isoform 1 or 2, 1-
981, 29-981, 34-981, 36-981, 131-981, 1-549, 29-549, 34-549, 36-549, 131-549,
and so forth)
can be modified in a variety of ways to achieve various beneficial effects
and/or results not
exhibited in native Klotho proteins. Illustratively, the QuickChange XL Site-
Directed
Mutagenesis Kit (Stratagene) can be used to alter the nucleic acid sequence of
various 5-
Klotho constructs. Other mutagenesis methods and kits as known in the art can
also be used.
For instance, various sub-cloning methods and kits are known in the art and
commercially
available. Amino acid changes may affect (e.g., improve, enhance, decrease,
etc.) protein
expression, protein stability, protein solubility, protein binding, protein
specificity, protein
activity, protein function, immunogenicity, toxicity, etc.
[00188] In at least one embodiment of the present disclosure, a protein is
modified with one
or more C-terminal tags and/or N-terminal tags. Such tags can function to
extend the serum
and/or soluble half-life of the protein (in one or more therapeutic or other
environments).
Tags can also be useful as markers for the presence or diagnostic localization
of the protein,
isolation or removal of the protein, delivery or transport of the protein,
binding of the protein
to one or more targets (e.g., protein, nucleic acid, organelle, cellular
structural component,
etc.), enzymatic processing or cleavage, and so forth. In at least one
embodiment, the C-
terminus of the protein can be tagged with a TEV-Twin-Strep tag or sequence,
an
immunoglobulin (IgG1) Fc domain tag or sequence, or other tag or sequence as
known in the
art and/or described further herein. Additional description can be found in
the articles "Fusion
Proteins for Half-Life Extension of Biologics as a Strategy to Make
Biobetters," "What is the
future of PEGylated therapies?" and "Strategies for extended serum half-life
of protein
therapeutics," the entirety of each of which is incorporated herein by
specific reference. In
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certain embodiments, a linker or linker peptide can be inserted and/or
disposed between the
(native or variant) Klotho protein sequence and the tag.
[00189] In some embodiments, the modified Klotho protein can include an
alternative (e.g.,
native, non-native, and/or synthetic) signal peptide. For instance, in some
embodiments, the
native signal peptide sequences can be replaced and/or supplemented with an
alternative
signal peptide or signaling sequence (SS). In some embodiments, a native
Methionine residue
of a Klotho protein can be removed and a Methionine residue at the N-terminus
of the SS can
be included. Additional description can be found in the thesis "Generation of
high expressing
CHO cell lines for the production of recombinant antibodies using optimized
signal peptides
1() and a novel ER stress based selection system," the entirety of which is
incorporated herein by
specific reference. In certain embodiments, a linker or linker peptide can be
inserted and/or
disposed between the (native or variant) Klotho protein sequence and the
alternative SS.
[00190] Some embodiments can include one or more amino acid variants. It will
be
appreciated that the present disclosure contemplates variation of any one or
more of the
native amino acids in any of the disclosed Klotho proteins to any other amino
acid, whether
naturally-occurring, synthetic, or otherwise configured.
S-Klotho V45F Protein Variant
[00191] Substituting a Phenylalanine in the place of the Valine at (native)
position 45 of
human alpha Klotho (V45F) has been found by the Inventors to have surprising
and
unexpected benefits. For example, V45F soluble Klotho, and fragments or fusion
proteins
thereof, express at higher levels in CHO and HEK-293 cells than does the
native, wild type
V45 protein. Commercially, achieving higher levels of soluble protein
expression can be
desirable. Accordingly, some embodiments of the present disclosure include a
Klotho protein
having the V45F substitution.
S-Klotho C3705 Protein Variant
[00192] In humans, the Klotho gene maps on chromosome 13q12. A variant, known
as KL-
VS, is present in approximately 15-25% of Caucasians. The variant is composed
of six single
nucleotide polymorphisms (SNPs), two of which cause amino acid substitutions
(i.e. F352V
and C3705 ¨ Phenylalanine 352 changed to Valine and Cysteine 370 changed to
Serine). In
vitro transfection assays have shown that secreted levels of klotho are
reduced 6-fold for the
V352 variant, while they are increased almost 3-fold for the S370 form.
However, these two
variants in the human KLOTHO gene segregate together and form the KL-VS
haplotype that
increases klotho secretion in the range of 1.6-fold. For instance, it has been
reported that in a
screening of over 300 individuals taken from the geographically and/or
ethnically distinct
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cohorts, not a single individual was found to harbor only one of the V352
variant or the S370
variant.
[00193] An embodiment of the present disclosure includes a recombinant S-
Klotho protein
having the C370S homovariant (i.e., without the presence (or with deletion) of
the F352V
variant). The C370S variant can be produced and/or expressed in the context of
any protein
construct described herein. For instance, the C370S variant can be produced or
expressed in
the context of S-Klotho, isoform 1 or 2, 1-981, 29-981, 34-981, 36-981, 131-
981, 1-549, 29-
549, 34-549, 36-549, 131-549, and so forth, with or without a tag (e.g., (IgG)
Fc tag, TEV-
Twin-Strep tag, TEV sequence, etc.). Accordingly, the nucleic acid construct
or cDNA from
which the protein is expressed can be of corresponding length.
[00194] Embodiments can include producing a S370 heterozygous or homozygous
variant
construct, transferring (e.g., via transfection) the resulting construct,
which encodes the 5-
Klotho C370S protein, into an appropriate expression system (e.g., CHO cells),
and/or
transiently expressing the S-Klotho S370 protein. The S370 Klotho protein may
be expressed
at higher levels than the F352V/C3705 protein and/or wild-type F352/C370
protein.
Embodiments can include purifying (and optionally quality-control testing) the
expressed
protein for therapeutic administration. Embodiments can include administering
a therapeutic
or therapeutically-effective amount of the S-Klotho C3705 protein to a subject
in need
thereof The subject may, for example, harbor or express the KL-VS variant.
Alternatively,
the subject may be of wild-type of other mutant or variant type.
Administration of the
recombinant S-Klotho C3705 protein can lead to a beneficial increase in blood
S-Klotho
levels. Accordingly, the circulating concentration of S-Klotho in subjects
that receive the
therapeutic, recombinant, S-Klotho C3705 protein may not be subjected to the
dilutive effects
that are observed when the F352V variant is present.
[00195] Nucleic Acids and Expression Vectors
[00196] Some embodiments can include a nucleic acid or nucleic acid construct.
For
instance, embodiments can include an expression vector or nucleic acid
construct. The
nucleic acid can encode a recombinant human alpha soluble Klotho protein,
protein fragment,
or protein variant, as described herein. In at least one embodiment, the
nucleic acid can
encode a Klotho protein sequence, an optional (native or non-native) signaling
sequence (e.g.,
at the N-terminus or N-terminal of the Klotho protein sequence), an optional
linker sequence
(e.g., GS linker), and/or an amino acid tag (e.g., IgGl-Fc or TEV-Twin-Strep),
as described
herein.
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[00197] In some embodiments, the nucleic acid can express a protein that
includes all or a
subset of amino acid residues 1-1012, 1-981, 29-981, 34-981, 36-981, 131-981,
1-549, 29-
549, 34-549, 36-549, or 131-549 of human alpha Klotho isoform 1 or 2. At least
a portion of
the protein can have at least or about 80% amino acid sequence identity to all
or a subset of
amino acid residues 1-1012, 1-981, 29-981, 34-981, 36-981, 131-981, 1-549, 29-
549, 34-549,
36-549, or 131-549 of human alpha Klotho (isoform 1 or 2). For instance, at
least a portion of
the protein can have at least and/or about 80% amino acid sequence identity to
all or a portion
of one of SEQ ID NO: 1 through SEQ ID NO: 75, or a combination of two or more
thereof
In a preferred embodiment, the protein can have at least and/or about 80%
amino acid
sequence identity to all or a portion of one of SEQ ID NO: 2 through SEQ ID
NO: 70 or SEQ
ID NO: 107 through SEQ ID NO: 120 or SEQ ID NO: 125 through SEQ ID NO: 128 or
SEQ
ID NO: 133 or SEQ ID NO: 152.
[00198] In some embodiments, at least a portion of the nucleic acid can have
at least and/or
about 80% (e.g., at least about 82%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%,
94%, 95%, 96%, 97%, 98%, or 99%, or 100% nucleotide sequence identity to one
of SEQ ID
NO: 76 through SEQ ID NO: 106 or SEQ ID NO: 121 through SEQ ID NO: 124, or a
combination of two or more thereof In a preferred embodiment, the nucleic acid
can have at
least and/or about 80% nucleotide sequence identity to one of SEQ ID NO: 76
through SEQ
ID NO: 106 or SEQ ID NO: 121 through SEQ ID NO: 124.
[0199] Nucleic acid sequences of the present disclosure can also include a
stop codon, as
known in the art (e.g., TGA, TAG, TAA).
Cell Lines and Methods of Manufacture
[0200] An embodiment of the present disclosure can include a cell line. The
cell line can
comprise any suitable cell type, such as CHO cells, HEK cells, HL-60 cells, or
other cell line
as known in the art. Illustratively, the cell line can comprise CHO cells
(e.g., a plurality of
CHO cells). For the sake of brevity, reference to CHO cells contemplates a
specific reference
to HEK cells (e.g., HEK-293 cells), HL-60 cells, and/or any other (protein
expression) cell(s)
or cell line(s) known in the art. In some embodiments, the cells can (each)
contain (one or
more copies of) an exogenous nucleic acid. The nucleic acid can encode a
polypeptide with at
least and/or about 80% amino acid sequence identity to at least a portion of
one of SEQ ID
NO: 1 through SEQ ID NO: 75, or a combination of two or more thereof,
preferably to one of
SEQ ID NO: 2 through SEQ ID NO: 70.
[0201] The nucleic acid can comprise at least one transgene or cDNA. In some
embodiments, at least a portion of the nucleic acid can have at least and/or
about 80% nucleic
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acid sequence identity to one of SEQ ID NO: 76 through SEQ ID NO: 106 or SEQ
ID NO:
121 through SEQ ID NO: 124, or a combination of two or more thereof,
preferably one of
SEQ ID NO: 76 through SEQ ID NO: 106 or SEQ ID NO: 121 through SEQ ID NO: 124.
The nucleic acid can be or comprise a plasmid or other (structural) form of
nucleic acid.
[0202] In some embodiments, the exogenous nucleic acid can encode a functional
enzyme,
such as dihydrofolate reductase (DHFR) and/or glutamine synthetase (GS). In at
least one
embodiment, the cells can be or comprise dihydrofolate reductase (DHFR)-
deficient CHO
cells, such as CHO-S cells or CHO-M cells. The nucleic acid can include a
promoter (e.g., a
weak up to a strong promoter, as understood by those skilled in the art). For
instance, in some
1() .. embodiments, the nucleic acid can include a (strong) promoter
associated with the transgene
having at least 80%, etc. nucleic acid sequence identity to one of SEQ ID NO:
2 through SEQ
ID NO: 70 or SEQ ID NO: 107 through SEQ ID NO: 120 or SEQ ID NO: 125 through
SEQ
ID NO: 128 or SEQ ID NO: 133 or SEQ ID NO: 152. Thus, the transgene can be
under the
control of a promoter.
[0203] For convenience, CHO cells and/or cell lines are referred to throughout
the present
disclosure. It is noted, however, that other cells, cell lines and/or host
cells (besides CHO
cells) are also contemplated within the scope of the present disclosure.
Accordingly, a
reference to CHO cells and/or cell lines also contemplates reference to and/or
use of other
known cells, cell lines and/or host cells (e.g., HEK cells, such as HEK-293
cells, HL-60 cells,
and so forth).
Transfection
[0204] A method of manufacturing the cell line can include introducing the
exogenous
nucleic acid into the cells, preferably via transfection, or other technique,
as known in the art.
In at least one embodiment, a serum-free growth optimized, cell-suspension of
a cell line was
used as the host cell line for insertion of a nucleic acid (plasmid)
containing a promoter, a
human alpha S-klotho transgene encoding a polypeptide with at least 80% amino
acid
sequence identity to at least a portion of one of SEQ ID NO: 2 through SEQ ID
NO: 70 or
SEQ ID NO: 107 through SEQ ID NO: 120 or SEQ ID NO: 125 through SEQ ID NO: 128
or
SEQ ID NO: 133 or SEQ ID NO: 152, and a selectable (enzyme) marker. The
transgenes,
respectively, encode amino acids 1 through 981, 29 through 981, or 34 through
981 of human
alpha soluble Klotho. In particular embodiments, the transgenes had sequence
portion(s)
corresponding to one or more of SEQ ID NO: 76 through SEQ ID NO: 106 or SEQ ID
NO:
121 through SEQ ID NO: 124 (or had at least 80% nucleic acid sequence identity
to one of
SEQ ID NO: 76 through SEQ ID NO: 106 or SEQ ID NO: 121 through SEQ ID NO:
124).
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For DHFR-deficient CHO cell lines (such as the CHO-S cell line), the
selectable (enzyme)
marker was exogenous DHFR. For other cell lines, the selectable (enzyme)
marker was
exogenous GS.
Growth, Selection and/or Gene Amplification
[0205] Some embodiments can include growing the cells (e.g., transfected
cells) on a solid
medium and/or in a liquid medium (e.g., in suspension cell culture),
preferably in a serum-
free and/or animal (or animal-derived) protein (component) free medium. For
instance, cells
can be plated on solid growth media for a period of time. The cells can also
or alternatively
be grown in suspension culture and/or in liquid medium. The liquid medium
preferably
comprises a carbon source, a nitrogen source, and one or more vitamins,
minerals, salts,
amino acids, supplements, or additives. In some embodiments, the medium can
also lack
hypoxanthine and thymidine (HT), glutamine, etc.
[0206] In at least one embodiment, after a certain period of time (e.g., 48
hours post-
transfection), the cells can be gathered (e.g., detached), optionally
centrifuged (e.g., at
100 x g for 5 min.), and/or seeded (e.g., at approximately 2000 cells/well),
such as into a 96-
well adherent culture plate (e.g., containing serum supplemented, -HT and/or -
glutamine
media). The medium can also include MTX and/or MSX in certain embodiments. Non-
transfected cells may die within 7-14 days after selection (e.g., after
exposure to MTX and/or
MSX in -HT and/or -glutamine media.
[0207] In certain embodiments, the cells can (be selected to) contain at least
about 2 to 10
copies, at least about 10 to 20 copies, at least about 20 to 30 copies, or at
least about 30 to 50
copies of the exogenous nucleic acid (e.g., per cell). Accordingly, the method
can include
selecting cells that contain at least about 2 to 10 copies, at least about 10
to 20 copies, at least
about 20 to 30 copies, or at least about 30 to 50 copies of the exogenous
nucleic acid (e.g.,
per cell). For instance, (successively increasing levels of) MTX and/or MSX
can be
administered to the cells (e.g., at a concentration of about 1 nM ¨ 1 p,M,
about 10 ¨ 100 nM,
etc.).
[0208] Dihydrofolate reductase (DHFR) gene amplification in DHFR-deficient CHO
cells
(such as the CHO-S cell line) transfected with exogenous DHFR, was
accomplished by
successively increasing levels of methotrexate (MTX) in growth medium. Because
the
plasmid contains DHFR, this allows for the amplification of the S-klotho gene
(fragment)
within the host cell upon exposure to MTX (10 ¨ 100 nM). The GS gene
expression system
was also used to amplify cells transfected with exogenous GS (e.g., upon
exposure to MSX).
Alternatively, GS -/- host cell lines were also used, which removed the need
for MSX. These
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steps resulted in the production of numerous copies of the S-klotho gene
(e.g., between 10 to
30 copies of the gene per cell) and resultant high levels of expression of the
S-klotho protein
in the transgenic cell line.
[0209] In suspension culture, the protein can be secreted from the cells into
the liquid
medium in some embodiments. For instance, certain cells and/or cell lines of
the present
disclosure can secrete (or be selected to secrete) up to a concentration of
200 - 500 mg of
protein per liter of liquid medium, 500 - 2000 mg of protein per liter of
liquid medium, 2000 -
5000 mg of protein per liter of liquid medium, or any value or range of values
therebetween
(without concentrating the protein). In at least one embodiment, high
producing cell lines (or
suspension cultures) can be selected, such that the concentration of human
recombinant alpha
soluble Klotho protein in the medium (of the selected suspension culture or
suspension
cultures of the selected cell lines) is at least 200 mg/L, preferably at least
500 mg/L, more
preferably at least 1000 mg/L, even more preferably at least 2000 mg/L, still
more preferably
at least 5000 mg/L, without concentrating the protein.
[0210] Sub-cloning of the resultant high S-klotho producing, transgene-
containing colonies
by limited cell dilution was conducted to further produce S-klotho-secreting
cell lines that
secrete in the range of 500 to 2000 mg/L S-klotho into the cells conditioned
media. All cell
constructs were restriction digested and sequence verified.
[0211] In some embodiments, the cells can be grown in a bioreactor having a
volume or
working volume of at least 10 liters, preferably at least 25 liters, more
preferably at least 50
liters, even more preferably at least 100 liters, still more preferably at
least 250 liters, still
more preferably at least 500 liters, still more preferably at least 1,000
liters, still more
preferably at least 2,000 liters, still more preferably at least 2,500 liters,
still more preferably
at least 5,000 liters, still more preferably at least 10,000 liters.
Cell Line Maintenance
[0212] For the amplified high-producing S-Klotho cell lines (e.g., produced by
DHFR/MTX or GS/MSX systems), optionally followed by cell subcloning, judicious
use of
concentrated media feeds administered to the production cell lines during
scale-up and up to
the final bioreactor run were done in serum-free and animal protein component-
free basal
media.
[0213] Scale-up of high producer cell lines was done by cell inoculum
expansion in cell
suspension in shake flasks or in the Wave Bag systems followed by the
successive
inoculation of the cells produced in 100L and then 500L capacity bioreactors.
Cell viability
was maintained at greater than 85% viable cells throughout the growth cycle in
shake flasks,
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Wave Bags, or bioreactors; and then at 80% or greater viable cell counts in
bioreactors during
the plateau phase of CHO S cellular growth with the concomitant production of
up to 1-3 g/L
of S-klotho produced (designated as the "high producers").
Protein Production
[0214] Certain embodiments can employ recombinant DNA strategies that utilize
strong
promoter sequences and/or high copy number plasmids for production of
therapeutic amounts
of the Klotho protein in mammalian (e.g., CHO) cells. In at least one
embodiment, for
example, dihydrofolate reductase (DHFR) gene amplification in DHFR-deficient
CHO cells
can include providing and/or the use of (successively increasing levels of)
methotrexate
.. (MTX). Similarly, exogenous glutamine synthetase (GS) gene-containing cells
can be treated
with methionine sulphoximine (MSX).
[0215] The Klotho protein can also include one or more glycans (attached
thereto). For
instance, the native human alpha Klotho isoform 1 (SEQ ID NO: 1) can have
glycans
attached (via glycosylation) at amino acids 106, 159, 283, 344, 604, 612,
and/or 694. The
Klotho protein of the present disclosure can have one or more of the same (or
similar)
glycans attached (via glycosylation) thereto (e.g., at the same amino
acid(s)).
[0216] In at least one embodiment, the protein can be cGMP regulation
compliant, as
determined and enforced by the U.S. Food and Drug Administration (FDA). For
instance, the
Klotho protein can be at least 90%, 92%, 95%, 96%, 97%, 98%, or 99% pure, dry
weight. In
some embodiments, the Klotho protein sample can include less than about 1-100
parts per
million (ppm), less than about 100-1000 parts per billion (ppb), or less than
about 1-100 ppb
CHO host cell proteins (HCP), nucleic acid, and/or other cellular components,
or any value or
range of values disposed therebetween.
[0217] Glycan structures present on the S-Klotho proteins produced can be
similar or
identical in comparison to the structures on native S-klotho proteins isolated
from human
fluids (i.e., blood, serum, urine, cerebrospinal fluid). In at least one
embodiment,
confirmation of native-like glycans can ensure that the right native post-
translation
modifications (PTMs) are produced and stably maintained in the cell-produced S-
Klotho
protein.
Solubility and/or half-life extension of Klotho proteins
[0218] Disclosed are methods and compositions for extending the half-life and
increasing
the solubility of the human S-Klotho protein. Also, the purification and
characterization of
the protein constructs so produced to achieve these results are also the
subject matter of the
present disclosure. Relevant information regarding nucleic acid changes made
in the
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sequence of the klotho gene or nucleic acid construct (see SEQ ID NOS: 76-96)
and/or of
changes, or chemical alterations in the amino acid sequence of a Klotho
protein (see SEQ ID
NOS: 1-70), and/or additions or subtraction of chemical groups, peptides, or
proteins to the
amino acid sequence of a Klotho protein is taught in the present disclosure in
order to obtain
resultant human Klotho variant proteins (novel compositions) with increased
biological half-
life or increased solubility in biological matrices (such as in blood,
cerebrospinal fluid, urine,
or various human tissues) than that of native Klotho molecules. These novel
compositions
can be made through the methods described herein for the modification of the S-
Klotho
protein.
[0219] Illustratively, fusion protein constructs can be produced by combining
the S-Klotho
protein with the Fc domains of an antibody (IgG), an albumin, such as human
serum albumin
(HSA), a transferrin, such as human transferrin (TF), and/or a proprietary
recombinant
polypeptide such as XTEN . The novel proteins can also be produced by
conjugating (or
modifying) the S-Klotho protein with a posttranslational modification, such as
polysialic acid
(PSA) or pegylation. Moreover, a novel S-Klotho protein can be produced
through
pegylation. The foregoing and other (half-life extension) methodologies can
improve the
performance of the S-Klotho protein by one or more of: increasing the S-Klotho
dosing
interval; providing superior patient convenience and likely compliance;
reducing dosing
frequency requirement, resulting in lower drug use in the aggregate; reducing
the cost of the
drug; lowering drug quantities at the same dosing interval as the parent
protein; simplifying
dosage formulation and enabling subcutaneous formulation; providing higher
drug levels
using the same dose and dosing interval as the parent protein resulting in
longer drug
exposure and potentially better efficacy; and decreasing the immunogenicity of
S-Klotho.
Production of Fc domain fusion protein constructs of S-Klotho
[0220] Antibody Fc domain, human serum albumin (HSA), and polysialic acid
(PSA) were
tested for effectiveness in extending the half-life and increasing the
solubility of the human 5-
Klotho protein. Fc fusions involve the fusion of peptides, proteins or
receptor exodomains to
the Fc portion of an antibody. Both Fc and albumin fusions achieve extended
half-lives not
only by increasing the size of the peptide drug, but both also take advantage
of the body's
.. natural recycling mechanism through binding of the extended protein to the
neonatal Fc
receptor, FcRn. After binding of the extended protein to the FcRn receptor,
degradation of the
fusion protein in the cells' endosome is prevented. Fusions based on the
addition of Fc or
albumin can result in biological half-lives in the range of 3-16 days, much
longer than which
has been reported for typical pegylated or lipidated peptides. For a review
that describes the
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use of protein fusion technologies such as Fc fusion proteins, fusion to human
serum
albumin, fusion to carboxy-terminal peptide, and other polypeptide fusion
approaches to
make biobetter drugs with more desirable pharmacokinetic profiles see Strohl
WR. Fusion
Proteins for Half-Life Extension of Biologics as a Strategy to Make
Biobetters. Biodrugs.
2015;29(4):215-239, the entirety of which is incorporated herein by specific
reference.
[0221] Fc domains were thusly added to the parent protein (S-Klotho) to
increase binding
affinity to the Fc receptor (FcRn). FcRn is present inside lysosomes in
endothelial cells lining
the blood vessels and functions to rescue antibodies from the degradation that
makes most
proteins short-lived in circulation. As a result of interactions with FcRn,
proteins have half-
lives ranging from a few days to a few weeks, allowing for less frequent
dosing of the
extended form of the protein drugs than biologics that do not have this newly-
produced
composition-of-matter.
[0222] The dimeric nature of Fc versus the monomeric structure of HSA can lead
to the
presentation of a Fc fused peptide as a dimer or a monomer in contrast to HSA.
The dimeric
nature of a peptide Fc fusion can produce an avidity effect if the target
receptors for S-Klotho
are spaced closely enough together or are themselves dimers in particular
human target
organs. This may be desirable or not depending on the target. Fusion of the S-
Klotho protein
to antibody Fc is also taught in the present disclosure to improve the
solubility and stability of
S-Klotho. Surprisingly, Fc-fusion Klotho proteins appear to have improved
binding, activity,
and/or immunogenicity. The addition of Fc domains to S-Klotho will also allow
the fusion
protein to be less immunogenic upon administration in human subjects.
Conjugation of the S-Klotho protein with human serum albumin (HSA)
[0223] The 66.5 kDa protein HSA, similar to human IgGs, has a long average
half-life in
the 19-day range. At a concentration of ¨50 mg/mL (-600
HSA is the most abundant
protein in human plasma, where it has several functions, including maintenance
of plasma
pH, metabolite and fatty acid transport, and a role in maintaining blood
pressure. HSA, which
is at the upper limit of size for glomerular filtration of proteins by the
kidney, is also strongly
anionic, which helps even more to retard its filtration via the kidney. Like
IgGs, HSA also
binds FcRn in a pH-dependent manner, albeit at a site different from¨and via a
mechanism
distinct from¨that of IgG binding, and is recycled similarly to IgGs,
resulting in its extended
half-life. HSA also tends to accumulate in tumors and in inflamed tissues,
which suggests that
fusion or binding to albumin may potentially help to target proteins or
peptides to those sites.
[0224] The fusion of peptides or proteins with inherently short-half-life
properties to HSA
for prolongation of the serum half-life of these molecules has been
investigated broadly since
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the early 1990s. Since then, dozens of different peptides and small proteins
have been fused
to HSA as both innovative and potential biobetter molecules. The first HSA-
peptide or
protein fusion product to be approved for marketing was Tanzeumg (marketed as
Eperzang
in the European Union) a DPP-4-resistant GLP-1-HSA fusion protein discovered
at Human
Genome Sciences and developed and marketed by GlaxoSmithKline. Tanzeumg
(albiglutide)
was approved by the European Medicines Agency (EMA) and the FDA in March and
April
of 2014, respectively. HSA thus improved the half-life of pharmacologically
active GLP-1
from 1-2 min for native GLP-1 to 4-7 days, which allows for once weekly
dosing. Seven
other known HSA fusion protein product candidates are either now in
development or
recently have been in development. Also, Novozyme, has been developing
modified versions
of recombinant HSA with improved FcRn binding for construction of "next-
generation"
HSA-protein fusions that may possess even longer half-life properties. This
was based on the
use of a K573P mutant of HSA, which was found to possess 12-fold greater
affinity for FcRn,
conferred a longer half-life on HSA than the wild type molecule in both mice
and
cynomolgus monkeys. The expectation is that these longer-half-life mutants of
HSA may be
of further use as fusion proteins to improve the half-life of fusion proteins.
[0225] The Inventors herein therefore disclose the fusion of Klotho proteins
to wild type
HSA or to a mutant form of HSA to produce a Klotho fusion molecule(s) with
significantly
prolonged half-life in human blood, cerebral spinal fluid, and other human
biological
matrices in order to bring about a strategic therapeutic benefit such as
superior patient
convenience and likely compliance, reduced dosing frequency resulting in lower
drug use in
aggregate, and/or reduced cost of goods. Also, lower drug quantities at the
same dosing
interval as the parent protein may simplify dosage formulation and enable
subcutaneous
formulation or decrease in the immunogenicity of S-Klotho. Surprisingly, HAS-
fusion Klotho
proteins appear to have improved binding, activity, and/or immunogenicity.
Conjugation of the S-Klotho protein with human transferrin (TF)
[0226] Transferrin is a highly abundant serum glycoprotein, found in serum at
3-4 mg/mL,
which binds iron tightly but reversibly and functions to carry iron to
tissues. Transferrin has
679 amino acid residues, is about 80 kDa in size, and possesses two high-
affinity Fe3+-
binding sites, one in the N-terminal domain and the other in the C-terminal
domain. Human
transferrin has a half-life reported to be 7-10 days or 10-12 days. The
aglycosylated form of
human transferrin, which makes up about 2-8 % of the total transferrin pool,
has a slightly
longer half-life of 14-17 days. The extended persistence of transferrin in
human serum is due
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to a clathrin-dependent transferrin receptor-mediated mechanism, which
recycles receptor-
bound transferrin back into the circulation.
[0227] Fusions of peptide and proteins have been made to human transferrin to
the N- and
C- termini, as well as to the centrally located hinge region that links the
two major lobes of
transferrin together. The N terminus of transferrin is free and can be fused
directly. The C
terminus is more buried and is constrained by a nearby disulfide bond, so
flexible linkers are
typically used when proteins are fused to the C terminus. This capability was
extended by
making libraries of peptides against specific targets and then fusing binders
from those
libraries into aglyco-transferrin (N-terminal, C-terminal, loops, or linker
region) to be
developed into therapeutic fusion proteins with extended half-lives.
[0228] The biotechnology company BioRexis Technologies, Inc., was founded in
2002 to
develop the transferrin fusion protein platform, which they termed the "Trans
Body"
platform, as a therapeutic platform. Their lead molecule, BRX-0585, was a
transferrin-GLP-1
fusion protein for treatment of type 2 diabetes mellitus (T2DM). Fusion of GLP-
1 to
transferrin was demonstrated to significantly enhance the half-life of GLP-1.
BioRexis was
acquired by Pfizer in March 2007. As far as can be determined, no BioRexis-
derived fusion
proteins are currently in the clinic. Klotho protein can be fused to human
transferrin to
produce a Klotho fusion molecule, which can be administered clinically to
significantly
prolong half-life or stability in human biological matrices in vivo.
Conjugation of the S-Klotho protein with XTEN from Amunix
[0229] XTEN is a proprietary recombinant polypeptide that extends the in vivo
half-life
of therapeutic payloads. XTEN consists of naturally-occurring hydrophilic
amino acids and is
biodegradable. Pharmaceuticals such as proteins, peptides, and synthetic
compounds can be
XTENylated via chemical conjugation or genetic fusion. XTEN proteins lack
secondary and
tertiary structure and their solution behavior resembles chemically prepared
polymers with
very large hydrodynamic radii. By size exclusion chromatography, XTEN protein
polymers
appear much larger than typical globular proteins of similar molecular weight.
The bulking
effect of XTEN greatly reduces renal clearance of attached molecules, thus
greatly increasing
their in vivo half-lives. In the current invention, the length of XTEN
polymers added to a
Klotho protein will be customized to optimize the pharmacokinetics as well as
the bio-
distribution of the attached Klotho protein payloads.
[0230] XTEN thusly can be recombinantly-fused to S-Klotho protein(s) to
increase the
molecules in vivo half-life. One benefit is that with the genetic S-Klotho-
XTEN fusion
constructs, a molecule is produced that has the convenience of expression,
purification and
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characterization of a single molecule which includes both the therapeutic and
bulking
moieties. Recombinant fusion allows the attaching of multiple XTEN chains per
protein in
precisely-defined locations and has been successfully used by therapeutic drug
manufacturers, resulting in best-in-class pharmacokinetics as exemplified by
XTENylated
growth hormone (Somavaratan, from the company, Versartis) and FVIII-XTEN (from
Biogen). For example, pharmacokinetic studies conducted in children receiving
different
doses of Somavaratan) have shown that XTENylation resulted in best-in-class
half-life due to
reduced receptor-mediated elimination in addition to kidney clearance.
[0231] XTEN protein polymers can be produced as free intermediates for
chemical
conjugation to peptides, peptidomimetics, and other synthetic molecules.
Reactive groups
(thiol, amine) are inserted in precisely-defined positions via introduction of
cysteine or lysine
residues into XTEN-encoding genes. Amunix has developed XTENs containing 1 to
9 thiol
groups with various spacing which can be provided to partners. Thus, in the
present
invention, orthogonal conjugation to amino and thiol groups in XTEN
facilitates the
production of Klotho-XTEN molecules.
Purification of Proteins
[0232] The Klotho protein can be extracted from cell suspension culture of
cells (e.g., of a
CHO cell line). The cells can produce and optionally secrete the Klotho
protein (e.g., into
liquid medium). Secretion of S-klotho into the spent media was also observed.
[0233] Purification of the recombinant proteins of the present disclosure can
be carried out
by any suitable method known in the art or described herein, for example, any
conventional
procedures involving extraction, precipitation, chromatography and/or
electrophoresis. A
further purification procedure that can be used for purifying proteins
includes affinity
chromatography using monoclonal antibodies which bind to target protein(s).
Some
embodiments can include an IgG-tagged protein, Fc-tagged protein, His-tagged
protein,
HSA-tagged protein, GST-tagged protein, etc., that can be purified by affinity
chromatography. For instance, some embodiments can include at least a portion
of an Fc
domain of an IgG, such as IgG1 . Generally, crude preparations containing a
recombinant
protein are passed through a column on which a suitable monoclonal antibody is
immobilized. For other tags, a corresponding, tag-binding affinity entity can
be disposed in
the column. After washing the column, the protein is eluted from the gel by
changing pH or
ionic strength. For example, the spent media from the CHO-S high producer cell
lines were
concentrated via tangential flow filtration; and S-klotho protein was purified
by affinity
chromatography followed by ion exchange cartridge or column chromatography.
Size
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exclusion chromatography can also be used to purify the proteins. It will be
appreciated that
various ordering of chromatography types can be applied. Any suitable
combination of
chromatography steps or columns is contemplated herein.
[0234] In alternative protocols, one or more pre- or post-affinity
purification steps were
performed. Such steps can include, for example, (ultra) centrifugation,
dialysis, membrane
and/or tangential flow filtration, chromatographic separation, such as ion
exchange, liquid-
liquid extraction, such as (aqueous) two-phase extraction, or other known
purification steps.
One or more post purification processing steps were performed in certain
embodiments. Such
post purification processing steps can include, for example, tandem
anion/cation flow-
through chromatography (as opposed to bind-and-elute chromatography), viral
and/or
bacterial removal by membrane filtration (e.g., 0.2 micron, 0.1 micron, etc.)
or by other
means known to one of ordinary skill in the art.
[0235] Certain embodiments include a method of purifying a recombinant Klotho
protein.
The method can include expressing the recombinant Klotho protein in suspension
cell
culture. The method can include harvesting the Klotho protein, such as by
collecting
suspension culture medium and/or cells. In at least one embodiment, the method
can include
a first purification step. It will be appreciated that the "first"
purification step need not occur
first or prior to any other purification step(s), or after any other
purification step(s). The first
purification step can comprise, involve, or use chromatography, such as High
Performance
Liquid Chromatography (HPLC), Fast Protein Liquid Chromatography (FPLC), ion
exchange
chromatography, affinity chromatography, etc. The first purification step can
comprise,
involve, or use affinity chromatography. The affinity chromatography can be
based on a tag
or other protein fusion entity of the Klotho protein. For example, a His-
tagged protein can be
purified by His-tag affininty chromatography (e.g., HisTrap or nickel column
chromatography). Similarly, a GST-conjugated protein can be purified by GST-
affinity
chromatography. Depending on the specific tag(s), a corresponding, tag-binding
affinity
entity can be disposed in a chromatography column for use in protein
purification.
[0236] In at least one embodiment, the first purification step can comprise,
involve, or use
affinity chromatography using, for example, Protein A or Protein A-conjugated
particles or
beads (e.g., Protein A Sepharose 4 Fast Flow column (0.75 ml) (0.5 cm x 3 cm)
for
purification of Ig-tagged or Fc-fusion Klotho proteins. The column can be
equilibrated in an
appropriate solution. The solution can include a buffering agent at a suitable
concentration
(e.g., 10 mM Tris-HC1, pH 8.0). A suitable amount of protein-containing sample
(e.g.,
suspension cell culture supernatant, optionally adjusted to pH 8.0, with 1.5 M
Tris-HC1, pH
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8.0) can be loaded on to the column (e.g., at a flow rate of 0.75 ml/min),
optionally washed
with a suitable wash buffer (e.g., 10 mM Tris/Arginine-HC1, pH 8.0), and
eluted from the
column with a suitable elution buffer (e.g., 4 M MgCl2, pH 3 (i.e., low pH,
high salt)), at a
suitable flow rate (e.g., about 0.50-0.25 ml/min, or equivalent), and/or to
produce a
concentrated sample.
[0237] In at least one embodiment, the elution buffer can have a pH of about
3. In some
embodiments, the elution buffer can have a pH between about pH 2 and about pH
6,
preferably between about pH 2 and about pH 5, more preferably between about pH
2 and
about pH 4, still more preferably between about pH 2.5 and about pH 3.5.
.. [0238] In at least one embodiment, the elution buffer can include at least
one salt.
Illustratively, the salt can be or comprise MgCl2, preferably at a
concentration of about 4 M.
In some embodiments, the elution buffer can have a salt concentration between
about 1 M
and about 6 M, preferably between about 2 M and about 5 M, more preferably
between about
3 M and about 5 M, still more preferably between about 3.5 M and about 4.5 M.
.. [0239] Additional purification step(s) can comprise, involve, or use
chromatography, such
as High Performance Liquid Chromatography (HPLC), Fast Protein Liquid
Chromatography
(FPLC), ion exchange chromatography, affinity chromatography, size exclusion
(or gel
filtration) chromatography, etc. In at least one embodiment, the method can
include a second
purification step. It will be appreciated that the "second" purification step
need not occur
second, prior to, or before any other purification step(s). The second
purification step can
comprise, involve, or use size exclusion chromatography (and a suitable mobile
phase buffer)
to fractionate, resolve, clarify, or purify the protein-containing sample(s).
For example, in an
illustrative second purification step, protein-containing elution sample(s)
(from the first
purification step) can be optionally buffer exchanged, and purified using size
exclusion
chromatography (e.g., Superdex 200 (1 cm x 30 cm) column) in a suitable mobile
phase
buffer. In at least one embodiment, the mobile phase buffer can include a
suitable buffering
agent (e.g., 100 mM Tris-HC1) and one or more optional reducing agent (e.g., 5
mM L-
Methionine and/or 0.6% Sodium Thioglycolate), at a suitable pH (e.g., about pH
8). In at
least one embodiment, the buffering agent can be other than a phosphate-
containing buffer
(e.g., Tris, Citrate, etc.). In at least one embodiment, the reducing agent
can be or comprise L-
Methionine and/or Sodium Thioglycolate, preferably at effective reducing
concentration(s).
Alternative reducing agents may include an acetylcysteine (i.e., N-
acetylcysteine (NAC),
including N-acetyl-L-cysteine, N-acetyl-D-cysteine, and racemic N-
acetylcysteine or a
(racemic) mixture of N-acetyl-L-cysteine and N-acetyl-D-cysteine), ascorbic
acid, dithionite,
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erythiorbate, cysteine, glutathione, dithiothreitol, 2-mercaptoethanol,
dierythritol, a resin-
supported thiol, a resin-supported phosphine, vitamin E, and/or trolox, or
salts thereof,
sodium citrate, potassium citrate, potassium iodide, ammonium chloride,
guaiphenesin (or
guaifenesin), Tolu balsam, Vasaka, ambroxol, carbocisteine, erdosteine,
mecysteine, dornase
alfa, and so forth.
[0240] In at least one embodiment, the mobile phase buffer can have a pH of
about 8. In
some embodiments, the elution buffer can have a pH between about pH 6 and
about pH 10,
preferably between about pH 7 and about pH 9, more preferably between about pH
7.2 and
about pH 8.8, still more preferably between about pH 7.5 and about pH 8.5.
[0241] In at least one embodiment, the eluted protein can be or include
monomeric Klotho
protein. In at least one embodiment, the eluted protein can be or include
multimeric Klotho
protein. In some embodiments, the eluted protein can be or include dimeric
Klotho protein. In
some embodiments, the eluted protein can be or include tetrameric Klotho
protein. In some
embodiments, the eluted protein can be or include hexameric Klotho protein. In
some
embodiments, the eluted protein can be or include octameric Klotho protein. In
at least one
embodiment, the eluted protein can be or include both monomeric and multimeric
Klotho
proteins. In at least one embodiment, the eluted protein can be or include a
single species of
either monomeric or multimeric Klotho protein. In at least one embodiment, the
eluted
protein can be or include greater than or equal to about 80% of the single
species of either
.. monomeric or multimeric Klotho protein. In at least one embodiment, the
eluted protein can
be or include greater than or equal to about 82%, 85%, 88%, 90%, 92%, 95%,
96%, 97%,
98%, or 99% of the single species of either monomeric or multimeric Klotho
protein.
[0242] Additional purification step(s) can comprise, involve, or use
chromatography, such
as High Performance Liquid Chromatography (HPLC), Fast Protein Liquid
Chromatography
(FPLC), ion exchange chromatography, affinity chromatography, size exclusion
(or gel
filtration) chromatography, etc.
Analysis of Klotho proteins
[0243] Protein purity can be demonstrated by SDS-PAGE or other assays or means
known
in the art. For instance, in at least one embodiment, Klotho protein samples
(50 1.tg) were
fractionated on precast SDS-PAGE gels (4-15%, 10 wells; catalog no. 456-1083;
BioRad)
and stained with Coomassie blue stain. All samples were run with either an
empty lane in
between or on separate gels to avoid sample-to-sample contamination. S-klotho
of greater
than 98% was shown to be isolated from the CHO S conditioned media as
determined by
Coomaise blue stain and densitometry tracing, or by silver stain
visualization, or by HPLC or
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RP-HPLC. In order to obtain sequence information, the proteins (after
reduction and S-
carboxymethylation) can be cleaved with cyanogen bromide, trypsin, and/or
proteinase K and
the peptides separated by HPLC according to known methods of protein
chemistry. Thus-
prepared samples were then sequenced in an automatic gas phase microsequencing
apparatus
(Applied Biosystems Model 470A, ABI, Foster City, Calif., USA) with an on-line
automatic
HPLC PTH amino acid analyzer (Applied Biosystems Model 120, ABI see above)
connected
to the outlet.
[0244] Proteins were also analyzed through mass spectroscopic methods.
Concerning
sample preparation for mass spectrometry, in order to restrict analyses to the
correct S-klotho
protein, only the gel band between 75 and 150 kDa was resected for analysis.
Gel fractions
were macerated with a sterile blade and subjected to in-gel digestion. Gel
fractions were de-
stained by three washes with 80 [IL of 50% acetonitrile (ACN)/50 mM ammonium
hydrogen
carbonate and washed with 100% ACN. The alkylation step was omitted, given the
absence
of cysteine residues from the target a-Klotho peptides. Tryptic digestion was
carried out
overnight at 37 C with 60 [IL of trypsin (sequencing grade modified, catalog
no. V511A;
Promega) in 50 mM ammonium hydrogen carbonate (0.005 1.tg/IlL). This process
yielded 25
[IL, of which 5 [IL (1 [IL for S-Klotho) was subjected to liquid
chromatography-electrospray
ionization tandem mass spectrometry (MS/MS) and PRIM analysis in an Orbitrap
nano-ESI
Q-Exactive mass spectrometer (Thermo Scientific), coupled to a nanoLC (Dionex
Ultimate
3000 UHPLC). MS/MS analysis confirmed that the human recombinant alpha S-
klotho
produced by embodiments of the present disclosure was substantially similar
(e.g., identical
in corresponding amino acid sequence) to that found in human blood, serum,
urine or
cerebrospinal fluid.
[0245] Using the above purification methods, the level of contaminating CHO
host cell
proteins (HCP) was determined to be acceptable in the purified S-Klotho
protein. In the final
S-Klotho product, HCP was removed to < 1-100 ppm. S-klotho protein products of
at least
90%, and up to 98% purity were isolated from the spent CHO S production cell
line (cells
and/or liquid medium). Specifically, cGMP-grade human alpha S-klotho that had
an
analytical profile suitable for clinical administration in human subjects was
produced and
purified. For example, the analytical profile of human recombinant alpha S-
Klotho is
designated by reference number PXD002775 in the ProteomeXchange Database,
which can
be found at
http://proteomecentral.proteomexchange.org/cgi/GetDataset?ID=PXDO02775.
The NIH full S-Klotho protein dataset is at
http://www[dot]ncbi[dot]nlm[dot]nih[dot]gov/
protein/Q9UEF7.
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[0246] An analytical profile for S-Klotho suitable for clinical administration
and that
obtained in an embodiment of the present disclosure included an endotoxin
level less than 0.1
ng per 1.tg (1 EU/I.tg) of S-Klotho. In addition, the purified human
recombinant S-Klotho was
also shown to have > or = 98% purity by SDS PAGE.
[0247] Glycan structures present on the CHO S cell-produced S-Klotho were
identical in
comparison to the structures on native S-Klotho isolated from human fluids
(i.e., blood,
serum, urine, and cerebrospinal fluid). This ensured that the same, native
post-translation
modifications (PTMs) were produced and stably maintained in the cell produced
S-Klotho
protein. Accordingly, using production and purification method described
herein, the
1() Inventors have been successful in producing cGMP-grade human S-klotho that
had an
analytical profile suitable for clinical administration in human subjects.
Therapeutic compositions
[0248] Some embodiments of the present disclosure can include a pharmaceutical
composition, such as a therapeutic composition. Pharmaceutical compositions of
the present
disclosure can generally include a therapeutically effective amount of a
recombinant soluble
alpha Klotho protein admixture with a (pharmaceutically-acceptable) vehicle,
carrier, or
excipient comprised of one or more additional components. The Klotho protein
can be
included in any suitable amount, such as about 0.001-1000 mg, about 0.01-100
mg, about
0.1-10 mg, about 1-5 mg, or any amount or range of amounts therebetween. In
some
embodiments, the composition can include a pharmaceutically or therapeutically
effect
amount of Klotho protein (e.g., to raise serum Klotho levels to a
predetermined level in a
subject to which the composition is administered).
[0249] The components can include one or more aggregation inhibitors, buffers,
tonicity
modifiers, and additional excipients. The primary solvent in the carrier can
be either aqueous
or non-aqueous in nature. The composition can be prepared by combining a
purified Klotho
protein of the present disclosure with a pharmaceutically-acceptable carrier.
[0250] It will be understood by one of ordinary skill in the art that the
combining of the
various components to be included in the composition can be done in any
appropriate order,
namely, the buffer can be added first, middle or last and the tonicity
modifier can also be
added first, middle or last. It is also to be understood by one of ordinary
skill in the art that
some of these chemicals can be incompatible in certain combinations, and
accordingly, are
easily substituted with different chemicals that have similar properties but
are compatible in
the relevant mixture.
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[0251] Aggregation inhibitors reduce a polypeptide's tendency to associate in
inappropriate
or unwanted ternary or quaternary complexes. The amino acids L-arginine
and/or, L-cysteine,
can act to reduce aggregation of Fc domain containing polypeptides in a
formulation for long
periods, e.g., two years or more. The concentration of the aggregation
inhibitor in the
formulation is preferably between about 1 mM to 1 M, more preferably about 10
mM to
about 200 mM, more preferably about 10 mM to about 100 mM, even more
preferably about
mM to about 75 mM, and yet more preferably about 25 mM. These compounds are
available from commercial suppliers.
[0252] Compositions of the present disclosure can include a buffering agent.
Buffering
10 agents maintain pH in a desired range. Various buffers suitable for use
in the pharmaceutical
composition of the present disclosure include histidine, potassium phosphate,
alkali salts,
sodium or potassium phosphate or their hydrogen or dihydrogen salts, sodium
citrate or
potassium citrate /citric acid, sodium acetate/acetic acid, maleic acid,
ammonium acetate, tris-
(hydroxymethyl)-aminomethane (tris), various forms of acetate and
diethanolamine, and any
15 other pharmaceutically acceptable pH buffering agent known in the art,
to maintain the pH of
the solution within a desired range. Mixtures of these buffering agents may
also be used.
[0253] The amount of buffering agent useful in the composition depends largely
on the
particular buffer used and the pH of the solution. For example, acetate is a
more efficient
buffer at pH 5 than pH 6 so less acetate may be used in a solution at pH 5
than at pH 6. The
preferred pH of the preferred formulations will be in the range of 4.0 to 5.0,
and pH-adjusting
agents such as hydrochloric acid, citric acid, sodium hydroxide, or a salt
thereof, may also be
included in order to obtain the desired pH.
[0254] One preferred buffer is sodium phosphate as its buffering capacity is
at or near pH
6.2. It will be appreciated, however, that other buffers may be selected to
achieve any
desirable pH buffering. The concentration of the buffer in the formulation is
preferably
between about 1 mM to about 1 M, more preferably about 10 mM to about 200 mM.
Buffers
are well known in the art and are manufactured by known methods and available
from
commercial suppliers.
[0255] When the pH of the pharmaceutical composition is set at or near
physiological
levels comfort of the patient upon administration is maximized. In particular,
it is preferred
that the pH be within a range of pH about 5.8 to 8.4, with about 6.2 to 7.4
being preferred,
however, it is to be understood that the pH can be adjusted as necessary to
maximize stability
and solubility of the polypeptide in a particular formulation and as such, a
pH outside of
physiological ranges, yet tolerable to the patient, is within the scope of the
disclosure.
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[0256] The formulations of the present disclosure may further include one or
more tonicity
modifiers (e.g., to render the solution isotonic with a patient's blood for
injection). A tonicity
modifier is understood to be a molecule that contributes to the osmolality of
a solution. The
osmolality of a pharmaceutical composition is preferably regulated in order to
maximize the
active ingredient's stability and also to minimize discomfort to the patient
upon
administration. Where serum is approximately 300+/-50 milliosmoles per
kilogram. It is
generally preferred that a pharmaceutical composition be isotonic with serum,
i.e., having the
same or similar osmolality, which is achieved by addition of a tonicity
modifier, thus it is
contemplated that the osmolality can be from about 180 to about 420
milliosmoles, however,
.. it is to be understood that the osmolality can be either higher or lower as
specific conditions
require.
[0257] Typical tonicity modifiers are well known in the art and include but
are not limited
to various salts, amino acids or polysaccharides. Non-limiting examples of
suitable amino
acids include glycine. Non-limiting examples of suitable polysaccharides
include sucrose,
mannitol and sorbitol. It is understood that more than one tonicity modifier
may be used at
once, for example, sorbitol and glycine can be used in combination to modify a
formulation's
tonicity.
[0258] Additional examples of tonicity modifiers suitable for modifying
osmolality
include, but are not limited to amino acids (e.g., arginine, cysteine,
histidine and glycine),
salts (e.g., sodium chloride, potassium chloride and sodium citrate) and/or
saccharides (e.g.,
sucrose, glucose and mannitol). The concentration of the tonicity modifier in
the formulation
is preferably between about 1 mM to 1 M, more preferably about 10 mM to about
200 mM.
Tonicity modifiers are well known in the art and are manufactured by known
methods and
available from commercial suppliers.
[0259] Excipients, also referred to as chemical additives, co-solutes, or co-
solvents, that
stabilize the polypeptide while in solution (also in dried or frozen forms)
can also be added to
a pharmaceutical composition. Excipient is defined herein as a non-therapeutic
agent added
to a pharmaceutical composition to provide a desired effect, e.g.
stabilization, isotonicity.
Common attributes of desirable excipients are aqueous solubility, non-
toxicity, non-
reactivity, rapid clearance from the body, and the absence of immunogenicity.
In addition, the
excipients should be capable of stabilizing the native conformation of the
protein so as to
maintain the efficacy and safety of the drug during processing, storage and
administration to
the patient. Examples include but are not limited to sugars/polyols such as:
sucrose, lactose,
glycerol, xylitol, sorbitol, mannitol, maltose, inositol, trehalose, glucose;
polymers such as:
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serum albumin (bovine serum albumin (BSA), human SA or recombinant HA),
dextran,
PVA, hydroxypropyl methylcellulose (HPMC), polyethyleneimine, gelatin,
polyvinylpyrrolidone (PVP), hydroxyethylcellulose (HEC); non-aqueous solvents
such as:
polyhydric alcohols, (e.g., PEG, ethylene glycol and glycerol)
dimethylsulfoxide (DMSO)
and dimethylformamide (DIVIF); amino acids such as: proline, L-serine, sodium
glutamic
acid, alanine, glycine, lysine hydrochloride, sarcosine and gamma-aminobutyric
acid;
surfactants such as: Tween-80Tm (polysorbate 80), Tween-20Tm (polysorbate 20),
SDS,
polysorbate, polyoxyethylene copolymer; and miscellaneous excipients such as:
potassium
phosphate, sodium acetate, ammonium sulfate, magnesium sulfate, sodium
sulfate,
trimethylamine N-oxide, betaine, metal ions (e.g., zinc, copper, calcium,
manganese, and
magnesium), CHAPS, monolaurate, 2-0-beta-mannoglycerate or any combination of
the
above. As a matter of caution, it is noted that the highly toxic white
hellebore (Veratrum
album) can be mistaken for gentian.
[0260] The concentration of one or more excipients in a formulation of the
disclosure is/are
preferably between about 0.001 to 5 weight percent, more preferably about 0.1
to 2 weight
percent. Excipients are well known in the art and are manufactured by known
methods and
available from commercial suppliers.
[0261] In one illustrative embodiment, a formulation of the disclosure can
comprise about
150 mM NaCl buffered to pH 7.3 to 7.4 with HEPES, IVIES, or Tris-HC1, and,
optionally, one
or more additional components as described herein.
[0262] In one illustrative embodiment, a formulation of the disclosure can
comprise a
(therapeutically-effective) amount of Klotho protein in about 10 mM to about
100 mM L-
arginine, about 10 mM to about 50 mM sodium phosphate, about 0.75% to about
1.25%
sucrose, and/or about 50 mM to about 150 mM NaCl, at about pH 6.0 to about pH
7Ø In
another embodiment, L-arginine can be replaced with L-cysteine (at about 1 to
about 500
micromolar) in the formulation. In yet another embodiment, the pH can be about
pH 7Ø In
another specific embodiment, a formulation of the disclosure can comprise a
(therapeutically-
effective) amount of Klotho protein in about 25 mM L-arginine, about 25 mM
sodium
phosphate, about 98 mM sodium chloride, and/or about 1% sucrose at about pH
6.2.
[0263] In another embodiment, a formulation of the disclosure can comprise a
(therapeutically-effective) amount of Klotho protein in about 10 mM to about
100 mM L-
arginine, about 10 mM to about 50 mM sodium phosphate, about 0.75% to about
1.25%
sucrose, about 50 mM to about 150 mM NaCl, at about pH 6 to about pH 7. In a
specific
embodiment, the formulation of the disclosure comprises a (therapeutically-
effective) amount
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of Klotho protein in about 25 mM sodium phosphate, about 98 mM sodium
chloride, and/or
about 1% sucrose at about pH 6.2.
[0264] In yet another embodiment, a formulation of the disclosure can comprise
an
effective amount of an Fc domain containing Klotho fusion protein, about 10 mM
to about
100 mM L -arginine, about 10 mM to about 50 mM sodium phosphate, about 0 to 5%
Mannitol and/or 0 to 0.2% Tween-20Tm (polysorbate 20) at about pH 6 to 7. In
another
embodiment, a formulation of the disclosure can comprise an effective amount
of a Klotho
protein, about 25 mM L-arginine, about 25 mM sodium phosphate, about 4%
Mannitol, about
0.02% Tween-20Tm (polysorbate 20), and/or at about pH 6Ø
[0265] In yet another embodiment, the disclosure provides a method of treating
a mammal
comprising administering a therapeutically effective amount of the
pharmaceutical
composition described herein, wherein the mammal has a disease or disorder
that can be
beneficially treated with a Fc domain containing polypeptide in the
composition. In yet
another embodiment, the Fc domain containing polypeptide is derived from the
same species
of mammal as is to be treated with the composition. In a particular
embodiment, the mammal
is a human patient in need of treatment. When the Fc domain containing
polypeptide of the
composition is Klotho:Fc, examples of diseases or disorders that can be
treated include but
are not limited to rheumatoid arthritis, psoriatic arthritis, ankylosing
spondylitis, Wegener's
disease (granulomatosis), Crohn's disease (or inflammatory bowel disease),
chronic
obstructive pulmonary disease (COPD), Hepatitis C, endometriosis, asthma,
cachexia,
psoriasis, and atopic dermatitis, or persons with a genetic disorder with
mutations in one or
more Klotho genes. Additional diseases or disorders that can be treated with
Klotho:Fc
include those described in WO 00/62790, WO 01/62272 and U.S. Patent
Application No.
2001/0021380, the entirety of each of which is incorporated herein by
reference.
[0266] In yet another embodiment, the disclosure provides a method for
accelerated
stability testing of the stability an Fc domain containing polypeptide in a
pharmaceutical
composition of the disclosure comprising the steps of testing the activity of
the polypeptide
formulated according to the disclosure prior to storage, i.e., time zero,
storing the
composition at 37 C. for one month and measuring the stability of the
polypeptide, and
comparing the stability form time zero to the one month time point. This
information is
helpful for early elimination of batches or lots that appear to have good
stability initially, yet
do not store well for longer periods.
[0267] Moreover, the pharmaceutical composition provides long-term storage
such that the
active ingredient, e.g., an Fc domain containing polypeptide, is stable over
the course of
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storage either in liquid or frozen states. As used herein, the phrase "long-
term" storage is
understood to mean that the pharmaceutical composition can be stored for three
months or
more, for six months or more, for one year or more, and preferably for two
year or more.
Long term storage is also understood to mean that the pharmaceutical
composition is stored
either as a liquid at 2-8 C. or is frozen, e.g., at -20 C. or colder (e.g., -
20 C or -80 C). It is
also contemplated that the composition can be frozen and thawed more than
once. The term
"stable" with respect to long-term storage is understood to mean that the
active polypeptide
of the pharmaceutical composition does not lose more than 20%, or more
preferably 15%, or
even more preferably 10%, and most preferably 5% of its activity relative to
activity of the
composition at the beginning of storage.
[0268] One or more anti-oxidants can be included in the formulations of the
present
disclosure. Anti-oxidants contemplated for use in the preparation of the
formulations include
amino acids such as glycine and lysine, chelating agents such as EDTA and
DTPA, and free-
radical scavengers such as sorbitol and mannitol.
[0269] Other effective administration forms such as (an oral or parenteral)
modified release
formulations (e.g., coated, encapsulated, slow-release, controlled-release,
extended-release,
delayed-release, sustained-release, time-release, depot, etc.), inhalant
mists, orally-active
formulations, suppositories, and implants or implantable medical devices are
also
contemplated herein. As such, the formulations may also involve particulate
preparations of
polymeric compounds such as bulk erosion polymers (e.g., poly(lactic-co-
glycolic acid)
(PLGA) copolymers, PLGA polymer blends, block copolymers of PEG, and lactic
and
glycolic acid, poly(cyanoacrylates)); surface erosion polymers (e.g.,
poly(anhydrides) and
poly(ortho esters)); hydrogel esters (e.g., pluronic polyols, poly(vinyl
alcohol),
poly(vinylpyrrolidone), maleic anhydride-alkyl vinyl ether copolymers,
cellulose, hyaluronic
acid derivatives, alginate, collagen, gelatin, albumin, and starches and
dextrans) and
composition systems thereof; or preparations of liposomes or microspheres.
Such
formulations may influence the physical state, stability, rate of in vivo
release, and rate of in
vivo clearance of the present proteins and derivatives. The optimal
pharmaceutical
formulation for a desired protein can be determined by one skilled in the art
depending upon
the route of administration and desired dosage. Exemplary pharmaceutical
formulations are
disclosed in Remington's Pharmaceutical Sciences, 18th Ed. (1990), Mack
Publishing Co.,
Easton, Pa. 18042, pages 1435-1712, the disclosure of which is incorporated
herein by
reference.
Biological activity
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[0270] Methods for evaluating the efficacy and/or determining an effective
dose of human
recombinant alpha soluble Klotho (to a patient, subject, or individual
exhibiting an age-
related disorder or clinical (e.g., metabolic) disorder) can (preliminarily)
include performing
organismal-based assays (e.g., using a mammal (e.g., a mouse, rat, primate, or
some other
non-human). The Klotho protein can be administered to the organism once or as
a regimen
(regular or irregular). For instance, the protein can be administered a
suitable number of times
(e.g., once, twice, etc.) in a given period of time (e.g., monthly, semi-
monthly, weekly, semi-
weekly, daily, etc.). A parameter of the organism (e.g., an age-associated
parameter) can then
be evaluated. Klotho proteins of interest can effectuate or result in a change
in the parameter
relative to a reference (e.g., a parameter of a control organism). Other
parameters (e.g.,
related to toxicity, clearance, and pharmacokinetics) can also be evaluated.
[0271] The klotho proteins of the present disclosure may be evaluated using an
animal
(model) that has or exhibits a particular disorder or condition, such as an
age-related or age-
associated disorder or condition, a clinical (e.g., metabolic) disorder or
condition, etc. Such
disorders and conditions can also provide a sensitized system in which the
effect of the
protein on physiology can be observed. Exemplary disorders include, for
example,
denervation, disuse atrophy, clinical (e.g., metabolic) disorders (e.g.,
disorder of obese and/or
diabetic animals such as db/db mouse, ob/ob mouse, etc.), cerebral disorders,
liver ischemia
or other liver disorders, cisplatin/taxol/vincristine models, kidney
idschemia/reperfusion
models, various tissue (xenograft) transplants, transgenic bone models, pain
syndromes (e.g.,
inflammatory and neuropathic disorders), Paraquat, genotoxic, oxidative stress
models, and
tumor (I) models.
[0272] To evaluate the S-Klotho protein of the present disclosure, the protein
can be
administered to a suitable animal (for a suitable treatment period) and the
parameter of the
animal is evaluated (e.g., after a suitable period of time, such as 10 to 60
minutes, 1 to 24
hours, 1 to 30 days, 1 to 12 months, 1 to 5 years, or any value or range of
values
therebetween). The animal can be fed ad libitum or normally (e.g., not under
caloric
restriction, although some parameters can be evaluated under such conditions).
Typically, a
cohort of such animals is used for the assay. Generally, a test polypeptide
can be indicated as
favorably altering lifespan regulation in the animal if the test polypeptide
affects the
parameter in the direction of the phenotype of a similar animal subject to
caloric restriction.
Such test polypeptides may cause at least some of the lifespan regulatory
effects of caloric
restriction (e.g., a subset of such effects) without having to deprive the
organism of caloric
intake.
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[0273] The parameter(s) to be tested may be age-associated or disease
associated
parameter(s) (e.g., a symptom of the disorder associated with the animal
model). A test
protein that is favorably indicated can cause an amelioration of the symptom
relative to a
similar reference animal not treated with the polypeptide. Other parameters
relevant to a
disorder or to aging can include: antioxidant levels (e.g. antioxidant enzyme
levels or
activity), stress resistance (e.g., paraquat resistance), core body
temperature, glucose levels,
insulin levels, thyroid-stimulating hormone levels, prolactin levels, and
luteinizing hormone
levels.
[0274] To measure the effectiveness of the S-Klotho protein of the present
disclosure for
treating an age-related disorder, an animal having decreased Klotho expression
may be used
(e.g., a mouse with a mutant or deleted klotho gene). For example, the test
protein can be
administered to the mutant mouse and age-related parameters are monitored. A
test protein
that is favorably indicated can cause an amelioration of the symptom relative
to a similar
reference animal not treated with the protein.
[0275] A parameter relevant to a clinical (e.g., metabolic) disorder or to
aging can be
assessed by measurement of body weight, examination on the acquisition of
reproductive
ability, measurement of blood sugar level, observation of life span,
observation of skin,
observation of motor functions such as walking, and the like. The assessment
can also be
made by measurement of thymus weight, observation of the size of calcified
nodules formed
on the inner surface of thoracic cavity, and the like. Further, quantitative
determination of
mRNA for the klotho gene or the Klotho protein can also be useful for the
assessment.
[0276] Still other (in vivo) models and organismal assays include evaluating
an animal for a
clinical (e.g., metabolic) parameter, e.g., a parameter relevant to an insulin
disorder, type II
diabetes. Exemplary metabolic parameters include: glucose concentration,
insulin
concentration, and insulin sensitivity.
[0277] In assessing whether a test protein is capable of altering life span
regulation, a
number of age-associated parameters or biomarkers can be monitored or
evaluated.
Exemplary age associated parameters include: (i) lifespan of the cell or the
organism; (ii)
presence or abundance of a gene transcript or gene product in the cell or
organism that has a
biological age-dependent expression pattern; (iii) resistance of the cell or
organism to stress;
(iv) one or more metabolic parameters of the cell or organism (exemplary
parameters include
circulating insulin levels, blood glucose levels; fat content; core body
temperature and so
forth); (v) proliferative capacity of the cell or a set of cells present in
the organism; and (vi)
physical appearance or behavior of the cell or organism.
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[0278] The term "average lifespan" refers to the average of the age of death
of a cohort of
organisms. In some cases, the "average lifespan" is assessed using a cohort of
genetically
identical organisms under controlled environmental conditions. Deaths due to
mishap are
discarded. Where average lifespan cannot be determined (e.g., for humans)
under controlled
.. environmental conditions, reliable statistical information (e.g., from
actuarial tables) for a
sufficiently large population can be used as the average lifespan.
[0279] Characterization of molecular differences between two such organisms,
e.g., one
reference organism and one organism treated with a S-Klotho protein can reveal
a difference
in the physiological state of the organisms. The reference organism and the
treated organism
are typically the same (or substantially the same) chronological age and/or
sex. The term
"chronological age" as used herein refers to time elapsed since a preselected
event, such as
conception, a defined embryological or fetal stage, or, more preferably,
birth. A variety of
criteria can be used to determine whether organisms are of the "same"
chronological age for
the comparative analysis.
.. [0280] Typically, the degree of accuracy required is a function of the
average lifespan of a
wildtype organism. For example, for the nematode C. elegans, for which the
laboratory
wildtype strain N2 lives an average of about 16 days under some controlled
conditions,
organisms of the same age may have lived for the same number of days. For
mice, organism
of the same age may have lived for the same number of weeks or months; for
primates or
humans, the same number of years (or within 2, 3, or 5 years); and so forth.
Generally,
organisms of the same chronological age may have lived for an amount of time
within 15, 10,
5, 3, 2 or 1% of the average lifespan of a wildtype organism of that species.
Preferably, the
organisms are adult organisms (e.g., the organisms have lived for at least an
amount of time
in which the average wildtype organism has matured to an age at which it is
competent to
reproduce).
[0281] The organismal screening assay can be performed before the organisms
exhibit
overt physical features of aging. For example, the organisms may be adults
that have lived
only 10, 30, 40, 50, 60, or 70% of the average lifespan of a wildtype organism
of the same
species. Age-associated changes in metabolism, immune competence, and
chromosomal
structure have been reported. Any of these changes can be evaluated, either in
a test subject
(e.g., for an organism based assay), or prior, during or after treatment with
a therapeutic
described herein (e.g., for a (human, or mammal) patient).
[0282] A marker associated with caloric restriction can also be evaluated in a
subject
organism of a screening assay (or a treated subject). Although these markers
may not be age-
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associated, they may be indicative of a physiological state that is altered
when a Klotho or
Klotho-related pathway is modulated. The marker can be an mRNA or protein
whose
abundance changes in calorically restricted animals. Cellular models derived
from cells of an
animal described herein or analogous to an animal model described herein can
be used for a
cell-based assay.
[0283] Models for evaluating the effect of a test protein on muscle atrophy
include: 1) rat
medial gastrocnemius muscle mass loss resulting from denervation, e.g., by
severing the right
sciatic nerve at mid-thigh; 2) rat medial gastrocnemius muscle mass loss
resulting from
immobilization (e.g., by fixed the right ankle joint at 90 degrees of
flexion); 3) rat medial
gastrocnemius muscle mass loss resulting from hind limb suspension; 4)
skeletal muscle
atrophy resulting from treatment with the cachectic cytokine, interleukin-1
(IL-1); and 5)
skeletal muscle atrophy resulting from treatment with the glucocorticoid,
dexamethasone.
Products, Compositions, Formulations, Supplements, Etc.
[00284] Some embodiments of the present disclosure relate to compositions
configured or
formulated to augment natural soluble alpha Klotho protein production and/or
attenuate
Klotho protein damage or degradation, and to methods, kits, formulations,
combination
products, co-administrations, co-formulations, dosages, process, methods,
treatment
protocols, and diagnostic methods related to the same. Some embodiments of the
present
disclosure relate to methods of manufacturing compositions of the present
disclosure. Some
embodiments of the present disclosure relate to use of compositions of the
present disclosure
or methods of using the same to treat human or non-human animal subjects. Some
embodiments of the present disclosure relate to treatment methods involving
compositions of
the present disclosure, including administration thereof to human or non-human
animal
subjects. Such uses and treatment methods can increase endogenous Klotho
protein levels,
such as by augmenting natural soluble alpha Klotho protein production and/or
attenuating
Klotho protein damage or degradation.
[00285] Certain embodiments include compositions of matter that comprise a
health
supplement that, when administered to human or non-human animal (e.g.,
mammalian)
subjects, increases the levels of endogenous, soluble alpha-Klotho protein in
the human or
non-human animal subjects. The composition or health supplement formulation
can comprise
(synthetic) chemical and/or natural components or ingredients. In other words,
components or
ingredients of the composition or health supplement can be or originate from
(synthetic)
chemical and/or natural sources.
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[00286] An exemplary composition can comprise one or more components,
preferably
selected from the group consisting of, for example, Vitamin D3, Vitamin E,
Vitamin C,
Vitamin K, 3,5,4'-trihydroxy-trans-stilbene (Resveratrol), Pterostilbene, N-
acetylcysteine
(NAC), Troglitazone, Rosiglitazone, Notopterygium incisum Ting, Gentian (Root;
Extract),
Cordyceps (Cordyceps Sinensis (CS) Mycelium), Isoflavones (Soy), Genistein,
Daidzein,
Quercetin (dihydrate; dill, bay leaf, oregano), Docosahexaenoic acid (DHA),
Astaxanthin,
Sesamin (Semen Sesamin Nigrum (Black)), Sesame (Seed Extract), Rosmarinic acid
(RA;
(Marjoram)), Tetradecylthioacetic Acid (TTA), Dicalcium Phosphate (Anhydrous),
Mircrocrystalline cellulose (MCC), Croscarmellose Sodium (Primellose),
Stearing Acid,
Magnesium Stearate, Alpha Lipoic Acid, Conjugate (alpha) Linoleic Acid (CLA),
Probiotics,
Activated Charcoal, and others as known in the art.
[00287] Certain ingredients or components can have positive benefits and very
low side
effects in treating or affecting one or more conditions that have been
associated with cellular
and tissue dysfunctions that occur with aging. Certain ingredients or
components can
(directly or indirectly) increase the circulating plasma level of soluble
alpha Klotho (s-
Klotho) levels in human or non-human animal subjects following administration
thereof.
[00288] The following examples are illustrative only. The following examples
illustrate
various optional ingredients, components, method steps, etc. that may be
useful in the
practice of the embodiments of the present disclosure.
[00289] Embodiments of the present disclosure include formulations
(compositions, co-
administrations, etc.) of several components (or ingredients) that,
individually, can have a
potential for positive benefits and/or very low side effects in addressing,
treating, affecting,
and/or countering one or more conditions that have been associated with
cellular and tissue
dysfunctions and/or that can occur with aging.
[00290] Without being bound to any theory, some of the components or
ingredients of the
present disclosure may activate the promoter of the klotho gene (e.g., in the
human
HEK293/k1 cell line), or reverse age-related declines in expression of Klotho
in hepatic,
kidney, and other tissues. Some of the components may attenuate, decrease,
inhibit, and/or
prevent oxidative or other damage to Klotho protein or the klotho gene.
[00291] Without being bound to any theory, some of the components have been
shown by
one or more animal and human clinical studies to have a potential for positive
benefits and/or
very low side effects in addressing, treating, affecting, and/or countering
one or more
conditions that have been associated with cellular and tissue dysfunctions
and/or that can
occur with aging. The present disclosure utilizes these various components in
compositions
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and methods for increasing endogenous Klotho protein levels. Specifically, the
present
disclosure utilizes these various components in compositions configured or
formulated to
augment natural soluble alpha Klotho protein production and/or attenuate
Klotho protein
damage or degradation.
[00292] Without being bound to any theory, certain of the individual
ingredients in some
embodiments can increase the circulating plasma level of soluble alpha-Klotho
in mammalian
(e.g., human or non-human) animals. Example ingredients may include, but are
not limited
to: N-acetylcysteine (NAC), an antioxidant; D vitamins, such as Vitamin D3
(1,25-
dihydroxyvitamin D3 [1,25(OH)2D3]); and/or vitamin D receptor agonist (e.g.,
calcitriol,
paricalcitol), which are thought to increase Klotho expression, increase
phosphaturia, correct
hyperphosphatemia, and lower serum fibroblast growth factor-23 (FGF-23)
resulting in
associated decreasing aortic calcification in mice suffering from chronic
kidney disease; and
Vitamin C and/or Vitamin E, which are thought to ameliorate age-related
crystalluria and
kidney damage at the proximal tubule.
[00293] Further example ingredients may include, but are not limited to non-
prescription
ingredients that may supplement the anti-aging properties of alpha Klotho,
such as, for
example: docosahexaenoic acid (DHA), linoleic acid, conjugated linoleic acid
(CLA),
isoflavones (e.g., genistein, daidzein, quercetin), rosmarinic acid, sesamin
and astaxanthin
(food-derived antioxidants), and tetradecylthioacetic acid (TTA). Without
being bound to any
.. theory, the aging brain is particularly prone to inflammatory and oxidative
alterations which
may underlie decreases in learning and memory and omega-3 polyunsaturated
fatty acids
(PUFAs) such as DHA, protect the aging brain from the development of
neurodegenerative
diseases. Accordingly, certain formulaE can include alpha linoleic acid or
conjugated alpha
linoleic acid because alpha linoleic acid can serve as a precursor molecule
used in the human
body in the manufacture of DHA.
[00294] In at least one embodiment, one or more DHA precursors or omega-3
fatty acids,
such as LA or CLA can be omitted from the formulation. Without being bound to
any theory,
omega-3 fatty acids such as LA or CLA may increase the effects of blood-
thinning
medications and raise the risk of bleeding. Example medications include,
without limitation,
warfarin (Coumadin), clopidogrel (Plavix), and aspirin, among others. In some
embodiments,
DHA can be included in the formulation. Without being bound to any theory, DHA
may not
directly cause blood thinning.
[00295] Without being bound to any theory, mitochondrial damage can be the
cause and
outcome of cell injury resulting from a variety of toxic insults, hypoxia, or
trauma. Increasing
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mitochondrial biogenesis after renal proximal tubular cell (RPTC) injury may
accelerate the
recovery of mitochondrial and cellular functions after such injury. Relevant
to these
observations, isoflavones, such as daidzein and genistein, may increase
peroxisome
proliferator-activated receptor gamma coactivator (PGC)-1 alpha expression and
result in
mitochondrial biogenesis as indicated by increased expression of ATP synthase
beta and
NADH:ubiquinone oxidoreductase core subunit 6 (ND6), which may result in a 1.5-
fold
increases in respiration and ATP in RPTC and which benefits recovery in
injured RPTC.
Without being bound to any theory, daidzein may be metabolized within the gut,
by particular
intestinal flora, to produce the bioactive compound equol. Equol can also be
included in
certain embodiments. The isoflavone, quercetin, on the other hand, may counter
detrimental
effects of aging by increasing oxidative stress resistance in the proteasome -
large protein
complexes responsible for the proper regulation of the cellular protein load
that play a major
role in the maintenance of redox balance by recognizing and removing
oxidatively modified
or damaged proteins.
[00296] Rosmarinic acid can also be added as an ingredient in some
formulations.
Rosmarinic acid (RA) is a naturally occurring phenolic compound, which may
contribute to
the beneficial and health-promoting effects of herbs, spices and medicinal
plants.
Administration of RA at a dosage of 200 mg per kg, once a day for 30 days to
aging mice,
can significantly (p < 0.05 or p < 0.01) increase the activity of superoxide
dismutase (SOD)
catalase (CAT) and glutathione peroxidase (GSH-Px), with a decrease in
malondialdehyde
(MDA), in comparison to an aging control that did not receive RA. Thus, RA may
promote
significant antioxidant enzyme activity in liver and kidney tissues of aging
mice.
[00297] Some embodiments may also contain the food-derived antioxidants, such
as
astaxanthin (A) and/or sesamin (S). Without being bound to any theory, when
both of these
ingredients (astaxanthin is a red carotenoid found in salmon, shrimp, crab,
and microalgae
while sesamin is a major lignan found in sesame extract) were combined (i.e.,
AS) and
administered daily to subjects with mild cognitive impairment (MCI),
significant
improvements in psychomotor speed and processing speed was observed in the AS
group
compared with a placebo group. Accordingly, daily supplementation of AS may
improve
cognitive functions related to the ability to comprehend and perform complex
tasks quickly
and accurately.
[00298] One or more peroxisome proliferator-activated receptor (PPAR-y)
agonists, such as
Tetradecylthioacetic Acid (TTA), may also be added to certain embodiments.
PPAR-y
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agonists may have beneficial effects on heart muscle through the regulation of
amino acids
metabolism and L-carnitine biosynthesis.
[00299] In addition to the purified chemical compounds described above, some
embodiments may contain one or more (e.g., several) natural ingredients or
extracts. These
include, but are not limited to, extracts of gentian root extract, Cordyceps
mushroom, and
Notopterygium inc/sum Ting.
[00300] Certain embodiments can include one or more (selective) strains of
(probiotic)
bacteria, such as: Lactobacillus sp. (e.g., acidophilus, brevis, bulgaricus,
casei, gasseri,
johnsonii, lactis, plantarum, paracasei, rhamnosus, salivarius, sake, etc.),
Bifidobacterium
sp. (e.g., bifidum, breve, infantis, lactis, longum, etc.), Streptococcus sp.
(e.g., thermophiles,
etc.), Bacillus sp. (e.g., laterosporus, etc.), Pediococcus sp. (e.g.,
acidilactici, etc.), and others
as known in the art. Without being bound to any theory, probiotics may
alleviate age-inflicted
oxidative stress and improve expression of biomarkers of aging (e.g., in
mice). Additional
information regarding probiotics, which may be included in certain embodiments
of the
present disclosure, can be found in a literature by Sabina Fijan,
"Microorganisms with
Claimed Probiotic Properties: An Overview of Recent Literature," published in
Int JEnviron
Res Public Health, 2014 May; 11(5): 4745-4767, the entirety of which is
incorporated herein
by specific reference.
[00301] Table 42 provides a list of ingredients that can be formulated into a
particular
embodiment (designated KSF 101).
KSF 101 Formulation
Ingredient Amount/ Dose
Vitamin D3 1,000 IU
Vitamin E 2,000 IU
Vitamin C 1000 mg
Resveratrol
(3,5,4'-trihydroxy-trans-stilbene) 1000 mg
Pterostilbene 5 mg
NAC (N-acetylcysteine) 600 mg
Troglitazone 1360 mg
Rosiglitazone 136 mg
Notopterygium incisum Ting 3000 mg
Gentian Root Extract 500 mg
Cordyceps 1000 mg
Genistein 10 mg
Daidzein 40 mg
Quercetin 150 mg
Docosahexaenoic acid (DHA) 1000 mg
Astaxanthin 3 mg
Sesamin (S) 5 mg
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Rosmarinic acid (RA) 500 mg
Tetradecylthioacetic Acid (TA) 500 mg
Alpha Linoleic Acid 3200 mg
Conjugate Alpha Linoleic Acid (CLA) 1000 mg
Pro biotics 8 g
Activated Charcoal 5000 mg
Table 42
[00302] By way of non-limiting example, the KSF 101 formulation can be or
comprise an
orally administered, DSHEA-compliant formulation or composition. Accordingly,
in some
instances, the KSF 101 supplement may be marketed and/or sold directly-to-
consumer (DTC)
.. and/or without requiring a prescription/physician order. It will be
appreciated, however, that
KSF 101 can be formulated in other dosage forms and/or as an FDA-approved drug
without
departing from the scope of this disclosure. The concentration of ingredients
in KSF 101 are
based on a standard adult reference of a 150-pound person (68 kilograms).
Alternative
dosage(s), as understood by those skilled in the art, are also contemplated
herein.
[00303] Table 43 lists potential (e.g., preferred and optional) ingredients
that can be
formulated into another embodiment of the present disclosure.
Illustrative Ingredients
Ingredient (potential max.) Amount/ Dose
Preferred Ingredients
Vitamin D3 1,000 IU
Vitamin E 2,000 IU
Vitamin C 1,250 mg
Pterostilbene 5 mg
NAC (N-acetylcysteine) 600 mg
Notopterygium incisum Ting 3,000 mg
Gentian Root Extract 500 mg
Cordyceps 1,000 mg
Optional or Alternative Ingredients
2,3,5,4'-Tetrahydroxystilbene-2-0-13-D-glucoside (THSG)
4,000 mg
Resveratrol
(3,5,4'-trihydroxy-trans-stilbene) 1,000 mg
Rosiglitazone 136 mg
Troglitazone 1,360 mg
Genistein 10 mg
Daidzein 40 mg
Quercetin
(from dill, bay leaves, and oregano) 500 mg
Isoflavones (Genistein, Daidzein, Equol) 10 mg
Docosahexaenoic acid (DHA) 1,000 mg
Astaxanthin 3 mg
Sesamin (S) 200 mg
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Illustrative Ingredients
Rosmarinic acid (RA) (marjoram) 500 mg
Tetradecylthioacetic Acid (TA) 500 mg
Conjugate Linoleic Acid (CLA) 3,200 mg
Alpha Lipoic Acid (ALA) 1000 mg
Probiotics 10 ¨ 20 billion CFU
Activated Charcoal 5,000 mg
Vitamin K 90 ¨ 120 mcg
Table 43
[00304] Table 44 lists potential (e.g., preferred and optional) ingredients
that can be
formulated into another embodiment of the present disclosure.
Label Claim Optimized
Ingredient mg Potency mg/Tab Amount/Day
**9 TABLETS (excluding Probiotics and
Charcoal) 1303.22
Vitamin D3 (100,000 lu/Gm 1000IU 1.22 1000 IU
Vitamin E 1210 lu (D-Alpha Tocopherol
Succinate 1162Iu 2000IU 202.02 2000 IU
Vitamin C 97% (Ascorbic Acid) Granular 1250mg 97% 157.5 1000 mg
Pterostilbene Powder 5mg 0.58 5 mg
N-Acetyl L-Cysteine (NAC) 600mg 70 600 mg
Notopterygium Incisum Ting 3000mg 350 0
Gentian Root 10:1 (Powder) 500mg 58.33 500 mg
Cordyceps Sinensis (CS) Mycelium Powder 1000mg 116.67 0 to 300
mg
Soy Isoflavones 40% 10mg 40% 2.92 10 mg
Dha Powder 17% from Algae 170mg 17% 116.67 170 mg
Semen Sesamin Nigrum (Black) Powder 200mg 23.33 100 - 150
mg
Quercetin From Dill, Bay Leaves and
Oregano 500mg 98% 59.52 150 mg
Rosmarinic Acid (RA)(Marjoram) 500mg 58.33 500 mg
Tetradecylthioacetic acid (TA) 500mg 58.33 500 mg
Dicalcium Phosphate Anhydrous Powder 5.56
Mcc (Mircrocrystalline Cellulose) Ph 102 5.56
Stearing Acid Nf 5.56
Croscarmellose Sodium (Primellose) 5.56
Magnesium Stearate 5.56
Probiotics 0
Charcoal (organic coconut) 260mg
Table 44
[00305] The ingredients of Tables 42-44 can be optionally combined in any
suitable
combination. By way of non-limiting example, the alternative formulation(s)
can be or
comprise an orally administered, DSHEA-compliant formulation or composition.
It will be
appreciated, however, that alternative formulation(s) can be formulated in
other dosage forms
and/or as an FDA-approved drug without departing from the scope of this
disclosure. The
respective concentrations of ingredients in the alternative formulation(s) are
based on a
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standard adult reference of a 150-pound person (68 kilograms). Alternative
dosage(s), as
understood by those skilled in the art, are also contemplated herein.
[00306] An illustrative composition (e.g., a nutraceutical composition) can
comprise two or
more components selected from the group consisting of vitamin C, vitamin D3,
vitamin E, N-
acetylcysteine (NAC), quercetin (dihydrate), rosmarinic acid (RA),
pterostilbene,
docosahexaenoic acid (DHA), nicotinamide riboside (NR), nicotinamide adenine
dinucleotide
(NAD+), nicotinamide mononucleotide (NMN), and one or more probiotic. The one
or more
probiotic can include an effective amount of one or more of: (i) one or more
Bifidobacterium
species and/or strains selected from Bifidobacterium lactis BL-04,
Bifidobacterium
bifidum/lactis BB-02, and Bifidobacterium longum BL-05, and/or (ii) one or
more
Lactobacillus species and/or strains selected from Lactobacillus acidophilus
LA-14,
Lactobacillus rhamnosus LR-32, and Lactobacillus paracasei LPC-37.
[00307] Illustratively, the composition (or formulation) includes up to, at
least, and/or about
50 billion CFU (per daily dose) of a Probiotic ingredient. Alternative
embodiments can
include up to, at least, between, and/or about 20 billion CFU (per daily
dose), 30 billion CFU
(per daily dose), 40 billion CFU (per daily dose), 60 billion CFU (per daily
dose), 70 billion
CFU (per daily dose), and/or 80 billion CFU (per daily dose) of the Probiotic
ingredient.
Illustratively, the Probiotic ingredient can comprise or include a single
probiotic (bacterial)
species or strain, or up to, at least, and/or between 2, 3, 4, 5, 6, 7, 8, 9,
and 10 probiotic
(bacterial) species or strains. In at least one embodiment, the Probiotic
ingredient comprises
the following probiotic (bacterial) species or strains: 30 billion CFU/day
each of the
Bifidobacterium species and/or strains Bifidobacterium lactis BL-04,
Bifidobacterium
bifidum/lactis BB-02, and Bifidobacterium longum BL-05. It will be
appreciated, however,
that certain embodiments may include one or two of the foregoing
Bifidobacterium species
and/or strains and/or in an amount of up to, at least, between, and/or about
10 billion CFU
(per daily dose), 20 billion CFU (per daily dose), 30 billion CFU (per daily
dose), 40 billion
CFU (per daily dose), and/or 50 billion CFU (per daily dose). In at least one
embodiment, the
Probiotic ingredient comprises the following probiotic (bacterial) species or
strains: 20 billion
CFU/day each of the Lactobacillus species and/or strains Lactobacillus
acidophilus LA-14,
Lactobacillus rhamnosus LR-32, and Lactobacillus paracasei LPC-37. It will be
appreciated,
however, that certain embodiments may include one or two of the foregoing
Lactobacillus
species and/or strains and/or in an amount of up to, at least, between, and/or
about 5 billion
CFU (per daily dose), 10 billion CFU (per daily dose), 20 billion CFU (per
daily dose), 30
billion CFU (per daily dose), and/or 40 billion CFU (per daily dose).
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[00308] In at least one embodiment, vitamin C can be included in a daily dose
and/or
concentration of about 500 mg, or between about 100-2000 mg, preferably about
200-1500
mg, more preferably about 250-1200 mg, still more preferably about 400-1000
mg, still more
preferably about 500-1000 mg, still more preferably about 500-750 mg (per day
or per
dosage). In at least one embodiment, vitamin C can be included in a daily dose
and/or
concentration of at least about 100 mg, 200 mg, 250 mg, 300 mg, 400 mg, 500
mg, 750 mg,
or 1000 mg (per day or per dosage). In at least one embodiment, vitamin C can
be included in
a daily dose and/or concentration of less than or equal to about 2500 mg, 2000
mg, 1500 mg,
1000 mg, or 750 mg (per day or per dosage).
[00309] In at least one embodiment, vitamin D3 can be included in a daily dose
and/or
concentration of about 1000 mg, or between about 100-2500 mg, preferably about
200-2000
mg, more preferably about 250-1800 mg, still more preferably about 400-1500
mg, still more
preferably about 500-1500 mg, still more preferably about 750-1250 mg (per day
or per
dosage). In at least one embodiment, vitamin D3 can be included in a daily
dose and/or
concentration of at least about 100 mg, 200 mg, 250 mg, 300 mg, 400 mg, 500
mg, 750 mg,
1000 mg, 1250 mg, 1500 mg, or 2000 mg (per day or per dosage). In at least one
embodiment, vitamin D3 can be included in a daily dose and/or concentration of
less than or
equal to about 3000 mg, 2500 mg, 2000 mg, or 1500 mg (per day or per dosage).
[00310] In at least one embodiment, vitamin E can be included in a daily dose
and/or
concentration of about 400 mg, or between about 100-2000 mg, preferably about
150-1500
mg, more preferably about 200-1200 mg, still more preferably about 250-1000
mg, still more
preferably about 250-750 mg, still more preferably about 250-500 mg, still
more preferably
about 300-750 mg, still more preferably about 300-500 mg (per day or per
dosage). In at least
one embodiment, vitamin E can be included in a daily dose and/or concentration
of at least
about 100 mg, 200 mg, 250 mg, 300 mg, 400 mg, 500 mg, 750 mg, or 1000 mg (per
day or
per dosage). In at least one embodiment, vitamin E can be included in a daily
dose and/or
concentration of less than or equal to about 2000 mg, 1500 mg, 1000 mg, 750
mg, or 500 mg
(per day or per dosage).
[00311] In at least one embodiment, N-acetylcysteine (NAC) can be included in
a daily dose
and/or concentration of about 600 mg, or between about 100-2000 mg, preferably
about 150-
1500 mg, more preferably about 200-1200 mg, still more preferably about 250-
1000 mg, still
more preferably about 250-750 mg, still more preferably about 500-750 mg. In
at least one
embodiment, N-acetylcysteine (NAC) can be included in a daily dose and/or
concentration of
at least about 100 mg, 200 mg, 250 mg, 300 mg, 400 mg, 500 mg, 750 mg, or 1000
mg (per
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day or per dosage). In at least one embodiment, N-acetylcysteine (NAC) can be
included in a
daily dose and/or concentration of less than or equal to about 2000 mg, 1500
mg, 1000 mg, or
750 mg (per day or per dosage).
[00312] In at least one embodiment, quercetin (dihydrate) can be included in a
daily dose
and/or concentration of about 150 mg, or between about 10-1000 mg, preferably
about 25-
750 mg, more preferably about 50-500 mg, still more preferably about 100-250
mg, still more
preferably about 100-200 mg (per day or per dosage). In at least one
embodiment, quercetin
(dihydrate) can be included in a daily dose and/or concentration of at least
about 10 mg, 20
mg, 25 mg, 30 mg, 40 mg, 50 mg, 75 mg, 100 mg, 150 mg, 200 mg, or 250 mg (per
day or
per dosage). In at least one embodiment, quercetin (dihydrate) can be included
in a daily dose
and/or concentration of less than or equal to about 1000 mg, 750 mg, 500 mg,
400 mg, 300
mg, 250 mg, or 200 mg (per day or per dosage).
[00313] In at least one embodiment, rosmarinic acid (RA) can be included in a
daily dose
and/or concentration of about 500 mg, or between about 100-2000 mg, preferably
about 200-
1500 mg, more preferably about 250-1200 mg, still more preferably about 400-
1000 mg, still
more preferably about 500-1000 mg, still more preferably about 500-750 mg (per
day or per
dosage). In at least one embodiment, rosmarinic acid (RA) can be included in a
daily dose
and/or concentration of at least about 100 mg, 200 mg, 250 mg, 300 mg, 400 mg,
500 mg,
750 mg, or 1000 mg (per day or per dosage). In at least one embodiment,
rosmarinic acid
(RA) can be included in a daily dose and/or concentration of less than or
equal to about 2500
mg, 2000 mg, 1500 mg, 1000 mg, or 750 mg (per day or per dosage).
[00314] In at least one embodiment, pterostilbene can be included in a daily
dose and/or
concentration of about 50 mg, or between about 10-200 mg, preferably about 20-
150 mg,
more preferably about 25-120 mg, still more preferably about 25-120 mg, still
more
preferably about 40-100 mg, still more preferably about 50-100 mg, still more
preferably
about 50-75 mg (per day or per dosage). In at least one embodiment,
pterostilbene can be
included in a daily dose and/or concentration of at least about 10 mg, 20 mg,
25 mg, 30 mg,
40 mg, 50 mg, 75 mg, or 100 mg (per day or per dosage). In at least one
embodiment,
pterostilbene can be included in a daily dose and/or concentration of less
than or equal to
about 250 mg, 200 mg, 150 mg, 100 mg, or 75 mg (per day or per dosage).
[00315] In at least one embodiment, DHA (preferably vegetarian DHA) can be
included in a
daily dose and/or concentration of about 200 mg, or between about 10-1000 mg,
preferably
about 25-750 mg, more preferably about 50-500 mg, still more preferably about
100-250 mg,
still more preferably about 150-250 mg, still more preferably about 100-200 mg
(per day or
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per dosage). In at least one embodiment, quercetin (dihydrate) can be included
in a daily dose
and/or concentration of at least about 10 mg, 20 mg, 25 mg, 30 mg, 40 mg, 50
mg, 75 mg,
100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 400 mg, or 500 mg (per day or per
dosage). In at
least one embodiment, quercetin (dihydrate) can be included in a daily dose
and/or
concentration of less than or equal to about 2000 mg, 1500 mg, 1000 mg, 750
mg, 500 mg,
400 mg, 300 mg, or 250 mg (per day or per dosage).
[00316] In at least one embodiment, Nicotinamide Riboside (NR) can be included
in a daily
dose and/or concentration of about 500 mg, or between about 100-2000 mg,
preferably about
200-1500 mg, more preferably about 250-1200 mg, still more preferably about
400-1000 mg,
still more preferably about 500-1000 mg, still more preferably about 500-750
mg (per day or
per dosage). In at least one embodiment, Nicotinamide Riboside (NR) can be
included in a
daily dose and/or concentration of at least about 100 mg, 200 mg, 250 mg, 300
mg, 400 mg,
500 mg, 750 mg, or 1000 mg (per day or per dosage). In at least one
embodiment,
Nicotinamide Riboside (NR) can be included in a daily dose and/or
concentration of less than
or equal to about 2500 mg, 2000 mg, 1500 mg, 1000 mg, or 750 mg (per day or
per dosage).
[00317] In at least one embodiment, Nicotinamide adenine dinucleotide (NAD+)
can be
included in a daily dose and/or concentration of about 300 mg, or between
about 50-2000 mg,
preferably about 100-1000 mg, more preferably about 150-750 mg, more
preferably about
200-600 mg, still more preferably about 200-500 mg, still more preferably
about 200-400 mg,
still more preferably about 250-500 mg, still more preferably about 250-400
mg, still more
preferably about 300-500 mg, still more preferably about 300-400 mg (per day
or per
dosage). In at least one embodiment, Nicotinamide adenine dinucleotide (NAD+)
can be
included in a daily dose and/or concentration of at least about 50 mg, 100 mg,
200 mg, 250
mg, 300 mg, 400 mg, 500 mg, 750 mg, or 1000 mg (per day or per dosage). In at
least one
embodiment, Nicotinamide adenine dinucleotide (NAD+) can be included in a
daily dose
and/or concentration of less than or equal to about 2000 mg, 1500 mg, 1000 mg,
750 mg, 500
mg, 400 mg, or 350 mg (per day or per dosage).
[00318] In at least one embodiment, Nicotinamide Mononucleotide (NMN) can be
included
in a daily dose and/or concentration of about 200 mg, or between about 10-1000
mg,
preferably about 25-750 mg, more preferably about 50-500 mg, still more
preferably about
100-250 mg, still more preferably about 150-250 mg, still more preferably
about 100-200 mg
(per day or per dosage). In at least one embodiment, Nicotinamide
Mononucleotide (NMN)
can be included in a daily dose and/or concentration of at least about 10 mg,
20 mg, 25 mg,
30 mg, 40 mg, 50 mg, 75 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 400 mg, or
500 mg
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(per day or per dosage). In at least one embodiment, Nicotinamide
Mononucleotide (NMN)
can be included in a daily dose and/or concentration of less than or equal to
about 2000 mg,
1500 mg, 1000 mg, 750 mg, 500 mg, 400 mg, 300 mg, or 250 mg (per day or per
dosage).
[00319] Without being bound to any theory, in embodiments that include NAD+
and/or
NAD+ precursor(s) (NR and/or NMN), the NAD+ and/or NAD+ precursor(s) may be in
the
form of a Lozenge for sublingual application (e.g., to get into the
bloodstream, initially
bypassing the liver and extending the time until the liver converts them into
NAM). Other
components may also be administered separately or co-administered. In some
embodiments,
ingredients can be co-administered, preferably via at least some co-
formulation, or
formulated as a multi-dosage composition. For example, some embodiments can
be,
comprise, or be provided as a multi-pill, -capsule, or other dosage kit or
pack. Embodiments
can include one, two, three, four, five, six, seven, eight, nine, ten, or more
dosed components,
such as pills, capsule, etc. Some ingredients can be provided in solid,
granular, powdered,
liquid, or other form.
[00320] Table 45 presents an illustrative formulation according to an
embodiment of the
present disclosure.
Ingredient for Capsules Amount/Dose
Unit
vitamin C 500 mg
vitamin D3 1000 IU
Vitamin E 400 IU
N-acetylcysteine (NAC) 600 mg
quercetin dihydrate 150 mg
rosmarinic acid (RA) 500 mg
pterostilbene 50 mg
DHA (Vegetarian) 200 mg
Nicotinamide Riboside (NR) 500 mg
Nicotinamide adenine dinucleotide (NAD+) 300 mg
Nicotinamide Mononucleotide (NMN) 200 mg
Probiotics (50B CFU - 6 Strains, below) 50 Billion CFU
Table 45
[00321] The ingredients of Table 45 can be optionally combined in any suitable
combination. By way of non-limiting example, the alternative formulation(s)
can be or
comprise an orally administered, DSHEA-compliant formulation or
composition. It will be
appreciated, however, that alternative formulation(s) can be formulated in
other dosage forms
and/or as an FDA-approved drug without departing from the scope of this
disclosure. The
respective concentrations of ingredients in the alternative formulation(s) are
based on a
standard adult reference of a 150-pound person (68 kilograms). Alternative
dosage(s), as
understood by those skilled in the art, are also contemplated herein.
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[00322] Some embodiments can include a composition, comprising an effective
amount of a
composition (e.g., health supplement), as described herein, formulated or co-
formulated to
increase endogenous Klotho protein levels or production (e.g., in mammalian
subjects). The
pharmaceutically effective amount of the composition can be sufficient to
increase or raise
the serum soluble Klotho protein concentration of the subject (e.g., to a
predetermined level,
such as greater than, equal to, or between about 50 to 3000 picograms of
soluble Klotho
protein per milliliter of serum). The pharmaceutically effective amount of the
composition
can also or alternatively be sufficient to maintain the serum soluble Klotho
protein
concentration of the subject at or above a predetermined threshold (e.g., for
a predetermined
period of time).
[00323] In certain embodiments, sufficient or suitable increase in Klotho
protein levels can
(be effective to) modulate the IGF-1 and/or Wnt signaling pathways, exhibit P-
glucuronidase
and/or sialidase activity, suppress the p53/p21 signaling pathway, and/or
reduce H202-
induced cell senescence and apoptosis, preferably through suppression of the
p53/p21
signaling pathway. The (raised) protein can function or be functional as a
humoral factor,
preferably exhibiting pleiotropic activity and/or preferably in the regulation
of oxidative
stress, growth factor signaling, ion homeostasis, and/or regulation of
activity of glycoproteins
on the cell surface, such as one or more ion channel proteins and/or growth
factor receptors,
such as Insulin/Insulin-Like Growth Factor-1 receptor.
[00324] The formulated composition (e.g., health supplement) of the present
disclosure can
raise serum soluble Klotho protein levels by increasing or enhancing (natural)
production of
Klotho protein by the patient or subject or attenuating (e.g., decreasing,
inhibiting, and/or
preventing) oxidative or other damage or degradation to/of Klotho protein or
the klotho gene.
[00325] Some embodiments of the present disclosure can include a
pharmaceutical
composition, such as a therapeutic composition or drug. For example, some
embodiments can
include one or more active ingredients, such as a pharmaceutical or
prescription medications
(e.g., ARBs (e.g., Losartan, Valsartan), testosterone, Vit. D receptor
agonists (e.g., calcitriol,
paricalcitol), PPAR (gamma) agonists (e.g., thiazolidinediones, troglitazone,
rosiglitazone),
or others. Some embodiments can include a formulation or co-formulation, in
combination
with prescribed medications such as angiotension receptor blockers (ARBs)
(i.e., losartan,
valsartan) or Vitamin D receptor agonists (VDRAs) (i.e., calcitriol or
paricalcitol). Other
prescription medications that may be administered with formulations include
troglitazone (at
200 mg/kg/day) and rosiglitazone (at 20 mg/kg/day), both Peroxidase
Proliferator-Activated
Receptor (PPAR-y) agonists. Without being bound to any theory, klotho is
thought to be a
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target gene of PPAR-y in the cultured human kidney cells, as well as in mouse
kidneys in
vivo. Moreover, Klotho protein expression may be enhanced (or upregulated) by
treatment
with PPAR-y agonists (e.g., at the described oral dosages).
[00326] Without being bound to any theory, losartan may (significantly)
increase circulating
a-Klotho levels in human type 2 diabetics by an average of 23% (from 542 pg/ml
to 668
pg/ml, p=0.001). Also, linear regression analysis suggests that the increment
in plasma cc-
Klotho achieved enhanced healthy renal function as seen by an associated
decrease in urine
albumin/creatinine ratio (0=-0.263, p=0.029). Similarly, valsartan may
significantly increase
cc-Klotho levels (from 432.7 179 to 506.4 226.8 pg/ml).
[00327] Also, in regard to the above, it is thought that treatment of mice
with chronic kidney
disease (CKD) with calcitriol or paricalcitrol (at VDRAs doses extrapolated
from human
subjects that are currently approved for the treatment of secondary
hyperparathyroidism)
result in half the amount of aortic calcification as compared to no VDRAs. In
particular, mice
given i.p. calcitriol or paricalcitol 3x per week for 3 weeks (dosing:
calcitriol at 30 ng/kg
(C30), or paricalcitol at 100 ng/kg (P100), or paricalcitol at 300 ng/kg
(P300)) may exhibit
increased serum and urine Klotho levels, increased phosphaturia, correction of
hyperphosphatemia, and lowering of serum fibroblast growth factor-23. Klotho
and
osteopontin may also be upregulated by VDRA therapy independent of changes in
serum
parathyroid hormone and calcium.
[00328] Some embodiments, or pharmaceutical components thereof, can include
one or
more recombinant (e.g., human or mammalian) Klotho proteins, protein
fragments, and/or
protein variants. Such active ingredients or pharmaceutical components can
contribute to
increased levels of endogenous Klotho protein (production) in the patient or
subject.
Additional description of (various and/or additional) active ingredients, such
as therapeutics,
recombinant proteins, pharmaceutical, and/or prescription medications, are
described in the
patent references incorporated above (i.e., (i) PCT/U52017/035755 filed June
2, 2017, (ii)
PCT/US2017/063149 filed November 22, 2017, and (iii) U.S. Provisional
Application Serial
No. 62/595,567 filed December 6, 2017). Such active ingredients can contribute
to increased
levels of endogenous Klotho protein (production) in the patient or subject.
[00329] Pharmaceutical compositions or components, regardless of type, can,
generally,
include a drug component, optionally admixed with a (pharmaceutically-
acceptable) vehicle,
carrier, or excipient comprised of one or more additional components. The
components can
include one or more aggregation inhibitors, buffers, tonicity modifiers, and
additional
excipients. The primary solvent in the carrier can be either aqueous or non-
aqueous in nature.
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[00330] Some embodiments can include a combination product, composition, or
formulation
comprising a recombinant Klotho protein and one or more additional active
ingredients. The
one or more additional active ingredients can be selected from among the
components, drugs,
substances, treatment compositions, etc. described herein or others known in
the art. For
instance, the Klotho protein and one or more additional active ingredients can
be co-
formulated into an injectable (e.g., intramuscular, intravenous, etc.),
ingestible, transdermal,
inhalable, topical, or other formulation. Alternatively, a combination
treatment or co-
administration can include treatment or administration of a Klotho protein and
one or more
additional active ingredients, but without the Klotho protein and one or more
additional
active ingredients being combined or formulated into a combination product,
composition, or
formulation. For instance, the Klotho protein and one or more additional
active ingredients
can each comprise or be in a separate injectable (e.g., intramuscular,
intravenous, etc.),
ingestible, transdermal, inhalable, topical, or other formulation.
[00331] Some embodiments can include a composition, comprising an effective
amount of a
composition (e.g., health supplement) and, optionally, an effective (e.g., a
pharmaceutically
effective) amount of the recombinant Klotho protein and/or pharmaceutical or
prescription
medications, as described herein, formulated or co-formulated to increase
endogenous Klotho
protein levels or production (e.g., in mammalian subjects). In some
embodiments, the
recombinant Klotho protein and/or pharmaceutical or prescription medications
can be co-
administered with the composition. In some embodiments, the recombinant Klotho
protein
and/or pharmaceutical or prescription medications can be combined with a
pharmaceutically-
acceptable carrier. In some embodiments, the optionally administered, optional
recombinant
Klotho protein can raise serum soluble Klotho protein levels by providing
exogenous Klotho
protein, while the optionally administered, optional pharmaceutical or
prescription
medications can raise serum soluble Klotho protein levels by increasing or
enhancing
(natural) production of Klotho protein by the patient or subject or
attenuating (e.g.,
decreasing, inhibiting, and/or preventing) oxidative or other damage or
degradation to/of
Klotho protein or the klotho gene.
[00332] It will also be appreciated that co-administration can comprise
simultaneous
administration of two or more components or distinct administrations of two or
more
components, the distinct administrations preferably being separated by a
period of time. The
period of time can be very small, in some embodiments. For instance, a second
component
can be administered substantially, immediately following administration of a
first component.
Alternatively, the first and second administrations can be separated by a time
period of 1-60
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seconds, 1-60 minutes, 1-24 hours, 1-7 days, 1-4 weeks, 1-12 months, and so
forth, or any
value or range of values therebetween. Similarly, simultaneous administration
can include
overlapping administration timeframes for the two or more components.
[00333] Any of the foregoing or other compositions, formulations, and/or co-
administrations
can have additive or synergistic effects over those of any of the individual
components, alone.
For instance, co-administration of various health supplement ingredients or a
health
supplement composition with an active component (e.g., a recombinant Klotho
protein,
pharmaceutical drug, etc.) can result in treatment outcomes greater than the
sum of the
individual outcomes of administering the individual components alone at
similar
concentrations. In addition, synergistic effects can include treatment
outcomes similar to
those of the individual outcomes of administering the components alone, but at
lower
concentrations. Synergistic effects can also include increasing the maximum
effective dose of
one or more of the components, reducing toxicity of one or more of the
components, or any
other beneficial result that is more than a mere additive effect of the
individual treatment
outcomes. In addition, additive effects of the individual treatment outcomes
can comprise one
or more synergistic effects. Such additive/synergistic effects may not be
predicted or
expected given the nature and understanding of the individual components.
[00334] Certain embodiments can include expression nucleic acid constructs
and/or vectors,
cell lines and/or cell suspension cultures, and methods of manufacturing,
purifying, and
administering the one or more recombinant (e.g., human or mammalian) Klotho
proteins,
protein fragments, and/or protein variants to (human or non-human animal)
subjects.
[00335] It will be appreciated that each of the forging, including but not
limited to health
supplements, formulations, co-formulations, co-administrations, combination
products, and
so forth, can be referred to as a "composition" or "composition of the present
disclosure".
Administration of exogenous S-Klotho
[0336] The present disclosure relates to S-Klotho formulations, clinical
dosages, and
administration (e.g., to increase and/or maintain serum concentrations of S-
Klotho within the
range of a normal and/or young (e.g., 18 ¨ 30 years of age) person (e.g.,
without any chronic
conditions).
[0337] Aspects or embodiments of the present disclosure include, for example,
administering a (cGMP- and/or clinical grade) human recombinant alpha soluble
Klotho
protein or protein fragment (of isoform 1) to a (human) subject in need
thereof. Embodiments
can also include measuring (in the (human) subject) serum S-Klotho levels or
concentration
(e.g., by Mass Spectroscopy (MS) or ELISA). Such measuring can occur before,
after, and/or
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during S-Klotho administration, and can be repeated, as necessary, to
determine serum 5-
Klotho levels and/or a rate of metabolism, degradation, or reduction of serum
S-Klotho
levels. MS is a technique well known in the art. MS can be used to identify
and even quantify
the level of one or more (native and/or recombinant) Klotho proteins in the
serum of a
subject.
[0338] One or more additional proteins can also be measured in the subject's
serum. For
instance, one or more Klotho-related and/or aging-related proteins (e.g.,
FGF21, GDF-11,
TIMP2, NAD+, CCL11, hormones testosterone, estrogen, etc.) and/or kidney
function
proteins (e.g., KIM-1, Cystatin-C, creatinine, BUN, creatinine, NGAL, etc.)
can be measured
separate from or in combination with measuring Klotho in the serum.
[0339] In at least one embodiment, a serum sample is obtained, such as a blood
sample.
The sample can be obtained by a blood draw, as known in the art. In a
preferred embodiment,
a finger prick or other less invasive means of obtaining a blood sample can be
used.
Accordingly, blood samples can be taken more frequently (e.g., throughout the
day and/or
every 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 hours). MS can be used to
measure the total
Klotho protein serum concentration, as well as the serum concentration of
various alpha
Klotho protein species, such as native Klotho species (e.g., soluble Klotho,
cleaved Klotho,
secreted Klotho, etc.) and/or one or more Klotho proteins of the present
disclosure. In some
embodiments, Klotho levels can be measured before therapeutic recombinant
Klotho protein
administration and again throughout the day and/or every (or one or more of)
0.5, 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, or 12 hours post-administration. Alternatively, or in
addition, Klotho
levels can be measured every (or one or more of) 0.5, 1, 2, 3, 4, 5, 6, 7, 8,
9, 10, 11, 12, 13,
14, 16, 18, 20, 21, 25, 28, 30, 36, 40, 45, 50, 60, or 90 days (or weeks) post-
administration.
[0340] Embodiments can also include determining such a rate and/or calculating
a
treatment protocol (e.g., including frequency, amount, and/or duration of
(subsequent)
administration(s) of S-Klotho) for maintaining within the serum of such
subject a S-Klotho
concentration within the range of a normal young person's serum concentration
of S-Klotho.
In at least one embodiment, the concentration of S-Klotho can be maintained at
approximately 1000 picograms of (the S-Klotho) protein per milliliter of serum
(pg/mL).
[0341] In at least one embodiment, a S-Klotho administration strategy (in
humans) can
include the measurement of one or more pharmacokinetic parameters of S-Klotho.
For
instance, measurements can be made of resultant changes in vivo of S-Klotho
levels in the
serum, urine and cerebrospinal fluid in response to S-Klotho administration.
Some
embodiments can include measuring the effectiveness of S-Klotho administration
on one or
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more clinical indicators. Clinical indicators for various conditions,
diseases, and disorders are
known in the art and described further herein.
[0342] Embodiments can also include taking a (baseline) S-Klotho level
measurement(s)
(e.g., at zero time (before any administration of exogenous S-Klotho) and/or
at different times
of day before and/or throughout the treatment protocol (e.g., before and after
administration
of S-Klotho) in order to account for any circadian rhythm effects in the
(human) subject(s)).
[0343] Embodiments can also include determining a suitable frequency, amount,
and/or
duration of S-Klotho administration. For instance, subjects with low S-Klotho
serum levels
(e.g., as determined by MS or ELISA immunoassay quantification), can be given
(e.g., via
any suitable route of administration) a first administration of klotho
configured and/or
adapted to bring the subjects serum S-Klotho levels to a first predetermined
level (e.g., about
1000 pg/mL), measuring (the resultant change in) serum S-Klotho concentration
in the
subject (e.g., by MS or ELISA), measuring the levels and/or a rate of
metabolism,
degradation, or reduction of serum S-Klotho levels (following the first
administration),
calculating a half-life of the administered S-Klotho, and/or determining a
frequency and/or
timeframe in which a second, subsequent administration of S-Klotho should be
given (e.g., in
order to maintain serum S-Klotho levels above a second predetermined level).
In at least one
embodiment, the second predetermined level can be between and/or about 95%,
90%, 85%,
80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%,
and/or
5% of the first predetermined level.
[0344] Further administrations can be given over a timeframe suitable to
produce in the
subject (long-term) S-Klotho serum level equivalent to that of normal sex-
matched young
person's serum maintenance level (e.g., approximately 1000 pg/ml). The total
timespan of the
S-Klotho administration (to (human) subjects) can range from 1 day to 5 years,
or more.
Measurements and/or determinations of frailty in the subject based on the use
of the clinical
frailty score and other measurements can also be made over the timeframe.
[0345] Embodiments of the present disclosure further include increasing the S-
Klotho
dosage to maintain a 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,
60%,
65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or more increase in S-Klotho levels
above the
normal range (e.g., at least about 500-1000 pg/ml serum, illustratively).
[0346] Certain embodiments include administering the S-Klotho protein. Non-
limiting
examples of suitable routes of administration include injection (e.g., bolus,
gradual,
intravenous, intradermal, intraperitoneal, intramuscular, intracutaneous,
and/or subcutaneous
injection), oral or oral-related administration (e.g., ingestion, buccal
administration, and/or
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sublingual administration), topical administration, transdermal
administration, rectal
administration, vaginal administration, inhalation, and so forth.
[0347] An illustrative embodiment can include administering the S-Klotho
protein or
composition in a single bolus or extended (IV) injection (e.g., drip over an
extended period of
time). In at least one embodiment, 0.01, 0.05, 0.1, 0.5, 1, 1.5, 2, 2.5, 2.75,
3, 3.5, 4, 4.5, 5, 6,
7, 8, 9, 10, 12, 15, 18, 20, or more micrograms of S-Klotho per kilogram of
subject body
weight can be administered per treatment. A suitable administration amount can
be calculated
through one or more methods known in the art. One such method is the
allometric scaling
method. For example, in rat experiments, 0.01 mg of S-Klotho /kg body weight
per
administration was used, or 10 ug/kg. Using the human allometric scaled
equivalent of 0.16,
illustratively, the human equivalent dose (HED) = 10 ug/kg x 0.16 = 1.6 ug/kg.
Hence a 70
kg human individual would require, 70 kg x 1.6 ug/kg = 112 ug and a 60 kg
human would
require, 60 kg x 1.6 = 96 ug, illustratively. Table 46, below, demonstrates
conversion of
various animal doses to human equivalent doses (HED) based on body surface
area. The
conversions assume a 60kg human.
[0348] The HED was established using normalization to body surface area, a
process
described by Reagan-Shaw (2008), incorporated herein by reference. This
process, termed
allometric scaling, corrects for basal differences in metabolism rate between
different species,
and may be preferred over simple dose extrapolation. Illustratively, when the
animal species
dose is known, then the HED can be calculated as: HED = animal dose in mg/kg x
allometric
scaling factor for that appropriate animal species. The actual dose to be
administered to a
patient can then be calculated by multiplying the HED times the patient's
weight.
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1 Coamt. Coovat Amimfl Dow In mg":
tokoimal Dow in p kumAgs
Ew.r:õõõõ,
Spooks 41, IgAs to Dim Di i?iik Maktply
37
ztuou': 1:aply Dow
liy1Mittul 11Xmo By
Him=
Child (20 tie
Wow 3 12.3.
laroget. 5 7.4 0:12
6 62 6,16
Ftvel: 7 0,19
1w ma pig 4,6 022
Rabbit 2,3 032
Dog 20 L6 664
Nitadtv:
Moat:It' 0,32
Mannowt 62 0,16
Sq-airM roalizey 7 0.19
12Oimazi 044
Mims*. 27 1.4 073
Mini*fit 6.15
Table 46
[0349] Multiple factors can be considered or taking in account in determining,
measuring,
and/or estimating the amount and/or bioavailability of S-Klotho in humans
(before and/or
after recombinant protein administration), the total amount and/or
concentration of S-Klotho
to be administered, and/or the serum level response (over time) after
recombinant protein
administration. Such factors can include, for example, composition of the
diluent, route of
administration, site of administration, distribution into the tissues and
organs of the subject,
metabolic or other rate of the subject, pharmacokinetics (PK),
pharmacodynamics (PD),
toxicology (Tox), and so forth.
[0350] In at least one embodiment, a (normal) concentration of S-Klotho (e.g.,
in a healthy,
young (18-30 years old) human adult) can be approximately 1000 pg/ml in serum.
A typical
adult can have a blood volume of approximately 5 liters, with females
generally having less
blood volume than males. Approximately 55% of human blood can be comprised of
serum.
Thus, (5 liters of blood / adult) x (0.55 serum/liter blood) = 2.75 liters
(2750 ml) of
serum/adult. Assuming no endogenous serum S-klotho, to achieve a final
concentration of
1000 pg/ml in the total blood serum; 2750 ml X 1000 pg/ml = 2,750,000 pg (or
2750 ng, or
2.75 [tg) exogenous S-Klotho can be administered per adult subject.
[0351] To increase soluble Klotho to 50% greater than typical healthy levels
(e.g., to 1500
pg/ml serum), a dose of 4.125 micrograms/subject can be administered. To
increase soluble
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Klotho to 100% greater than typical healthy levels (e.g., to 2000 pg/ml serum)
a dose of 5.5
micrograms/subject can be administered, and so forth.
[0352] A pharmaceutically effective and/or sufficient amount of purified
recombinant S-
klotho protein can be administered so as to raise the serum soluble Klotho
protein
concentration of the subject to any suitable level, such as greater than,
equal to, or between
about 50, 100, 250, 500, 750, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750,
3000, 3500,
4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500,
10,000, 11,000,
12,000, 13,000, 14,000, 15,000 20,000, 25,000, 30,000 40,000, 50,000, 75,000,
100,000 or
more picograms of soluble Klotho protein per milliliter of serum, or greater
than, equal to, or
between about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%,
150%, 200%, 250%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, 1200%,
1500%, 2000%, 2500%, 3000%, 4000%, 5000%, or more greater than typical healthy
levels
of soluble Klotho protein in serum.
[0353] In some embodiments, subjects can be administered one or more (bolus)
intravenous, intradermal, intraperitoneal, intramuscular, intracutaneous,
subcutaneous, and/or
other injection of recombinant human S-Klotho protein at a dosage of greater
than, equal to,
or between about 0.01 mg/kg body weight in a suitable volume of Klotho buffer
(e.g., 150
mM NaCl and 10 mM HEPES pH 7.4) or other pharmaceutically acceptable carrier.
Thus, a
160 pound subject (i.e., body weight of 72.57 kg) can be administered a
(bolus) injection of
about 0.73 mg of S-Klotho per administration (based on the calculation of 0.01
mg of 5-
Klotho/kg X 72.57 kg body weight). Likewise, a 170 pound person can receive a
0.77 mg of
S-Klotho per administration. The total number and frequency of administrations
can be
determined based on achieving and maintaining in serum, a concentration of,
for example,
1000 pg/ml of S-Klotho (equivalent to 0.000001 mg/ml serum). The latter can be
ascertained
as measured by MS or by a human S-Klotho ELISA assay.
[0354] In other embodiments, the dosage can be greater than, equal to, or
between about
0.0001 - 10 mg/kg body weight, 0.0001 - 10 [tg/kg body weight, 0.0001 - 10
ng/kg body
weight, 0.0001 - 10 pg/kg body weight, or any value or range of values
therebetween. Urine
and/or blood can be collected at one or more time points, such as greater
than, less than, equal
to, between, and/or about 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25
minutes, 30
minutes, 40 minutes, 45 minutes, 60 minutes, 90 minutes, 2 hours, 3 hours, 4
hours, 5 hours,
6 hours, 9 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 hours, 2.5 days,
3 days, 5 days, 7
days, 10 days, 14 days, 21 days, 4 weeks, 1 month, 2 months, 3 months, or more
after a
medical procedure or a first administration (dose) of recombinant Klotho
protein (e.g., to
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measure, test, and/or determine serum S-Klotho levels and changes in response
to
administration and/or over time).
[0355] One or more embodiments include production and/or (subsequent)
administration of
a unique formulation of the S-Klotho active drug product and/or in combination
with a
pharmaceutically-acceptable carrier. The carrier can be suitable for IV and/or
bolus injection.
Embodiments can also include production and/or (subsequent) administration of
a unique
inactive prodrug formulation of the S-Klotho and/or in combination with a
pharmaceutically-
acceptable carrier (so that an inactive S-Klotho can be activated in vivo to
release biologically
effective S-Klotho in animals or in human subjects. Such prodrug formulation
can include a
coating or time-release formulation.
Treatment Methods, Uses, Etc.
[00356] Some embodiments include a method or treatment method (e.g.,
comprising one or
more method steps). Some embodiments can include administering one or more
compositions, as described herein, to a subject (e.g., a subject in need
thereof).
Administration of the inventive composition(s) can be sufficient to raise the
serum soluble
Klotho protein concentration of the subject to or above a predetermined
threshold, or to
maintain the serum soluble Klotho protein concentration of the subject at or
above a
predetermined threshold, for a period of time.
[00357] The composition can comprise, for example, (i) a therapeutic
recombinant Klotho
protein or fragment or fusion protein thereof, as described in the present
disclosure, and/or
(ii) a product that increases, enhances, augments, and/or supports endogenous
Klotho protein
production and/or level(s) in a patient to which the product is administered,
optionally with
(iii) one or more additional (active or inactive) ingredients or compositions.
Those skilled in
the art will appreciate that each of the therapeutic effects, indications,
and/or treatments
outlined herein in relation to recombinant, therapeutic Klotho proteins and
products,
compositions, and methods related thereto can be achieved by (naturally)
increasing,
enhancing, augmenting, and/or supporting endogenous Klotho protein production
and/or
level(s). Those skilled in the art will also appreciate that each of the
therapeutic effects,
indications, and/or treatments outlined herein in relation to (naturally)
increasing, enhancing,
augmenting, and/or supporting endogenous Klotho protein production and/or
level(s) can be
achieved by administering recombinant, therapeutic Klotho proteins and
products,
compositions, and through methods related thereto. Accordingly, and for the
sake of brevity,
the present disclosure need not rehearse each of the disclosed therapeutic
effects, indications,
and/or treatments with reference to both (i) the administration of
recombinant, therapeutic
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Klotho proteins, and (ii) the administration of the health supplement or
nutraceutical
compositions of the present disclosure, as such are clearly contemplated
herein.
[00358] Certain embodiments can include a composition, as described herein,
for use in
treating one or more diseases, disorders, injuries, and/or (medical)
conditions. Similarly,
embodiments can include a method of treating (or alleviating, addressing,
preventing,
inhibiting, etc.) one or more diseases, disorders, injuries, and/or (medical)
conditions. An
exemplary treatment method comprises administering to a subject in need
thereof an effective
amount of a composition of the present disclosure. Without being bound to any
theory,
administering an effective amount of the composition to a subject in need
thereof can cause
or result in elevated (or raised) serum soluble Klotho protein concentration
in the subject.
Accordingly, the "effective amount" of a composition of the present disclosure
can be an
amount effective to raise or elevate the serum soluble Klotho protein
concentration of the
subject to or above a predetermined threshold, or to maintain the serum
soluble Klotho
protein concentration of the subject at or above a predetermined threshold,
for a
predetermined period of time. Alternatively, or in addition, the "effective
amount" of a
composition of the present disclosure can be an amount effective to achieve a
beneficial
therapeutic effect in a patient to which the composition is administered.
[00359] The method, composition or (raised) protein can also be effective to
treat one or
more diseases, disorders, injuries, and/or (medical) conditions.
Illustratively, the condition
can be or comprise an age-related or aging-related condition (or condition
associated with
(human) aging), such as frailty, bone density loss or bone mineral density
loss, weight loss,
muscular atrophy or degeneration, decline in muscle mass, decline in muscle
strength, hand
strength, leg strength, or physical fitness, decline in movement, freedom of
movement,
quality of life assessment, ejection fraction, or exercise capacity, decline
in learning, learning
capacity, memory, or intellectual quotient, cognitive deterioration or
forgetfulness, decline in
cognitive capacity or function, decline in synaptic plasticity or synaptic
function, and cellular
senescence. It will be appreciated, however, that the condition can also, or
alternatively, be or
comprise any physical, mental, emotional, or other deficiency, need, or desire
that increased
serum Klotho levels can provide a beneficial therapeutic effect, including
those described in
the present disclosure and those known to those skilled in the art.
[00360] The method, composition or (raised) protein can also be effective to
treat one or
more aging-related condition (or condition associated with (human) aging),
such as
Alzheimer's disease, Parkinson's disease, dementia or vascular dementia,
amyotrophic lateral
sclerosis (ALS) or motor neuron disease (MND), atrial fibrillation, chronic
obstructive
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pulmonary disease (COPD), fibromyalgia, adult onset diabetes, arthritis or
rheumatoid
arthritis, osteoarthritis, osteoporosis, glaucoma, cataracts, macular
degeneration and other eye
diseases/disorders, multiple sclerosis (MS), lupus, and/or ulcerative colitis.
[00361] Some embodiments of the present disclosure can be useful in one or
more of
treating cancer, lowering serum phosphate levels in a patient, treating
diabetes or a diabetes-
related condition (e.g., Type 1 diabetes mellitus, etc.) in a subject in need
of such treatment,
treating a heart condition (e.g., cardiovascular disease, left ventricular
hypertrophy (LVH),
pathological LVH and/or congestive heart failure, etc.) in a subject, treating
acute lung injury
in a subject (e.g., using nanoparticles), protecting the lung of a patient
against oxidant injury,
detecting early acute kidney injury in critically ill patients, attenuating
vascular calcification
in a subject, improving cognition, treating renal and/or liver ischemia,
modulating stress
response in (human) senescent endothelial cells, prophylactically and/or
therapeutically
treating, preventing, attenuating, arresting, and/or reversing acute and/or
chronic kidney
injury, disease, or disease progression and/or uremic cardiomyopathy,
reversing or
attenuating age-related therapy resistance in melanoma and other cancers, as
well as muscular
dystrophy, including DMD, targeting apoptosis of senescent cells, preferably
restoring tissue
homeostasis thereby, and a variety of other indications.
[00362] Some embodiments of the present disclosure can be useful for treating
one or more
other conditions. Such conditions may be described or disclosed herein. Such
conditions may
be known by those skilled in the art to be related to Klotho serum levels.
[00363] As described briefly, above, the present disclosure includes and
contemplates
various dosage forms and/or methods of administration of compositions of the
present
disclosure. Some embodiments can be dosed in an oral dosage form and/or
administrated
orally (e.g., in one or more convenient forms including tinctures, tablets,
capsules, powders,
teas, etc.). For instance, a tincture, comprised of an alcohol- or glycerin-
based extract of a
herb or natural ingredient can be packaged in a glass dropper bottle. Glycerin-
based tinctures
can provide a sweet taste, making the formulation more easily to swallow.
Tinctures may also
be administered in a dropper. Tinctures may include a dosage range on the
label (e.g., 20 to
40 drops per adult dose).
[00364] Capsules and tablets are also popular form for the oral administration
of
supplements. For example, a dried herb can be powdered and pressed into
gelatin capsules.
The supplement may be supplied as a capsule and/or tablet. The supplement may
be supplied
as a powder within a bottle or envelope with dosage amounts to be used listed
on the
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container. An appropriate amount of powder per dose may also be obtained by
opening up a
dosage capsule and sprinkling the content into a drink or on food.
[00365] Teas (e.g., herbal infusions) are common forms of herbal remedy and a
soothing
way to take herbs. An herbal infusion can be made by placing a small amount of
the herb in a
cup, filling the cup with boiling water and letting it steep. In a tea form,
herbal ingredients of
the supplements may increase in potency with extended steeping time and the
tea is best left
to steep for at least 10 minutes. Covering the cup (a small saucer works well)
keeps the steam
in and keeps the tea hot longer.
[00366] Some embodiments can include a method of treating an aging-related or
other
condition, disease, or disorder, the method comprising administering to a
subject in need
thereof an effective amount of a composition of the present disclosure. Some
embodiments
can include a method of treating and/or preventing a condition, disease, or
disorder,
preferably a condition, disease, or disorder associated with aging, in a
mammalian subject,
the method comprising administering to a subject in need thereof an effective
amount of a
composition of the present disclosure. Some embodiments can include a method
of treating or
preventing acute kidney injury (AKI), chronic kidney disease (CKD), or other
condition, the
method comprising administering to a subject in need thereof an effective
amount of a
composition of the present disclosure. Some embodiments can include a method
of treating
and/or preventing kidney injury in a mammalian subject, the method comprising
administering to the subject, optionally prophylactically, prior to a medical
procedure or
administration of a nephrotoxin and/or following a medical procedure or
administration of a
nephrotoxin, an effective amount of a composition of the present disclosure.
Some
embodiments can include a method of administering a composition of the present
disclosure
to a human or non-human animal (e.g., mammalian) subject in need thereof. The
subject to
whom the composition is administered can be suffering from or at risk for a
variety of
conditions (e.g., disorders, diseases, injuries, illnesses, etc.). For
example, some embodiments
include a method of treating one or more chronic diseases and/or aging-related
condition,
such as a physical, mental, neurological, or other condition associated with
(human) aging.
Some embodiments can promote healing, recovery, longevity, and/or other
beneficial
outcome through one or more mechanisms or action.
[00367] The pharmaceutically effective amount of the composition can be
sufficient to
increase or raise the serum soluble Klotho protein concentration of the
subject (e.g., to a
predetermined level, such as greater than, equal to, or between about 50 to
3000 picograms of
soluble Klotho protein per milliliter of serum). The pharmaceutically
effective amount of the
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composition can also or alternatively be sufficient to maintain the serum
soluble Klotho
protein concentration of the subject at or above a predetermined threshold
(e.g., for a
predetermined period of time).
[00368] In certain embodiments, suitable increase in protein levels can (be
effective to)
modulate the IGF-1 and/or Wnt signaling pathways, exhibit P-glucuronidase
and/or sialidase
activity, suppress the p53/p21 signaling pathway, and/or reduce H202-induced
cell
senescence and apoptosis, preferably through suppression of the p53/p21
signaling pathway.
The (raised) protein can function or be functional as a humoral factor,
preferably exhibiting
pleiotropic activity and/or preferably in the regulation of oxidative stress,
growth factor
signaling, ion homeostasis, and/or regulation of activity of glycoproteins on
the cell surface,
such as one or more ion channel proteins and/or growth factor receptors, such
as
Insulin/Insulin-Like Growth Factor-1 receptor.
[00369] Certain embodiments can include determining a serum soluble Klotho
protein
concentration of the subject, calculating the effective amount, determining a
rate of soluble
Klotho protein decline in the serum of the subject, calculating a subsequent
dosage time at
which the serum soluble Klotho protein concentration of the subject will be at
or below a
second predetermined level based on the determined rate, calculating a
subsequent dosage
amount of the composition sufficient to raise the serum soluble Klotho protein
concentration
of the subject from the second predetermined level to the first predetermined
level, and/or
administering the subsequent dosage amount of the composition to the subject.
Certain
embodiments can include prescribing a regular (e.g., daily) dose or dosage
form of the
composition to the subject (e.g., based on determined levels of serum soluble
Klotho protein
concentration of the subject).
[0370] An illustrative embodiment comprises a (treatment) method. The
treatment method
can be similar to and/or modeled after a study of the therapeutic effect or
health benefits of
various compositions of the present disclosure. The results of administration
of various
compositions are monitored in human subjects. Once a subject has voluntarily
agreed to be in
the study, and signed the Patient Consent Form, they are given a physical
examination and
blood and saliva tests. Upon meeting the Inclusion Criteria in the Study
Protocol, the subject
is then enrolled in the study. Illustratively, the study may include 100
healthy individuals
that are not currently on vitamin D supplementation and that do not live in a
sunny
environment. Subjects with a KLVS variant (Klotho over-expressor) and/or other
genetic
variant of klotho gene(s) may be excluded from the study. Following
enrollment, the subjects
receive a 3-month supply of a Klotho supplement formulation of the present
disclosure or a
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placebo in a double-blind study. Subjects receive unmarked bottles of
supplement
formulation or placebo at no cost. Subjects take this supplement daily for 3
months.
[0371] At the end of this 3-month period, blood is drawn for a fasting blood
test, and the
subject's weight, blood pressure is measured and recorded along with the
concentration of
plasma alpha soluble Klotho determined by an Alpha Klotho (a - Klotho) Human
Soluble
ELISA Assay Kit (from for example, Catalog No. 27998, from IBL America,
Minneapolis,
MN, USA).
[0372] The Study/Design Procedures regarding the human subjects in this study
is given
below.
Study Design/Procedures:
[0373] Up to 100 patients
[0374] Duration: 3 months to first endpoint, crossover, second endpoint at 6
months
including placebo patients that elected to take the supplement.
[0375] Baseline Questionnaire to include: Current medications (statins, ARBs),
nutritional
supplements, blood pressure, exercise frequency, alcohol consumption.
[0376] Prior to enrolling:
[0377] Collect blood sample and cheek swab (PI office or mobile phlebotomist)
[0378] Determine KLVS variant (KLOTHO overexpressor) ¨ send cheek swab to lab
to
determine KLVS variant (Klotho over-expressor) status
[0379] Lab analysis of Klotho level, Vitamins D & E, and other analyses for
pre-treatment
baseline
[0380] Supplementation: (3-month supply) shipped to participant. (Active
supplement and
placebo controls, equal # of active and placebo controls)
[0381] Outcomes measure: Participant downloads mobile application to monitor
compliance and questionnaires (exercise, blood pressure)
[0382] Follow up blood sample collection at 3 months and 6 months
[0383] The ingredients in the supplement are comprised of nutritional
components that
pose very low risk. If subject experiences any discomfort or adverse reaction
to the
formulation he or she will be instructed to stop taking the product and
contact the study
doctor.
[0384] Any significant adverse reactions will be reported to the study's IRCM
Institutional
review Board (IRE) within 48 hours.
Example Conditions
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[0385] In some embodiments, the condition, disease, or disorder treated with
the
composition(s) and/or method(s) of the present disclosure can, preferably, be
selected from
the group consisting of, for example: frailty; bone density loss; bone mineral
density loss;
weight loss; muscular atrophy; muscular degeneration; decline in muscle mass;
decline in
.. muscle strength; decline in hand strength; decline in leg strength; decline
in physical fitness;
decline in movement; decline in freedom of movement; decline in quality of
life assessment;
decline in ejection fraction; decline in exercise capacity; decline in
learning; decline in
learning capacity; decline in memory; decline in intellectual quotient;
cognitive deterioration;
forgetfulness; decline in cognitive capacity; decline in cognitive function;
decline in synaptic
.. plasticity; decline in synaptic function; cellular senescence; chronic
kidney disease (CKD);
chronic kidney disease ¨ mineral and bone disorder (CKD-MBD); polycystic
kidney disease
(PKD); autosomal dominant polycystic kidney disease (ADPKD); acute kidney
injury (AKI);
acute tubular necrosis (ATN); acute allergic interstitial nephritis (AAIN);
glomerulonephritis;
kidney disease; renal failure; Alport Syndrome; nonoliguric renal failure;
alcoholism;
hyperphosphatemia; muscular dystrophy (MS); type 1 diabetes; type 2 diabetes;
cardiovascular disease (CVD); cardiovascular calcification; cerebrovascular
insufficiency;
vascular calcification; coronary artery disease; heart failure; left
ventricular hypertrophy;
uremic cardiomyopathy; abnormalities in blood pressure; salt-sensitive
hypertension; tissue
calcification; calcific atherosclerotic plaque burden; calcinosis; familial
tumoral calcinosis;
cancer; one or more tumors; myelin-related diseases; demyelinating diseases;
neurodegenerative disease; neurovascular diseases; progressive supranuclear
palsy (PSP);
Pompe disease; Niemann-Pick disease; microgliosis; Farber disease (FD); bone
mass
diseases; osteoporosis; osteopenia; osteopenia (particularly loss of BMD of
cortical bone);
pulmonary emphysema; pulmonary fibrosis; cystic fibrosis, idiopathic (i.e.,
cause unknown)
pulmonary fibrosis, radiation-induced lung injury, cirrhosis, biliary atresia,
atrial fibrosis,
endomyocardial fibrosis, (old) myocardial infarction, glial scar, arterial
stiffness,
arthrofibrosis, Crohn's disease, Dupuytren's contracture, keloid, mediastinal
fibrosis,
myelofibrosis, Peyronie's disease, nephrogenic systemic fibrosis, progressive
massive
fibrosis, retroperitoneal fibrosis, scleroderma/systemic sclerosis, adhesive
capsulitis, skin
.. atrophy; thymic atrophy; accumulation of renal interstitial matrix;
glomerulosclerosis;
anemia; albuminuria; proteinuria; infertility; Alzheimer's disease;
Parkinson's Disease;
dementia; vascular dementia; amyotrophic lateral sclerosis (ALS); motor neuron
disease
(MND); atrial fibrillation; chronic obstructive pulmonary disease (COPD);
fibromyalgia;
adult onset diabetes; arthritis; rheumatoid arthritis; osteoarthritis;
glaucoma; cataracts;
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macular degeneration; multiple sclerosis (MS); lupus; ulcerative colitis;
cachexia; obesity;
vitamin D-related conditions; bone diseases; bone diseases through bone
remodeling; gut
diseases and related conditions; stem cell depletion; sea sickness; space
adaptation syndrome
(SAS); nausea; vertigo; non-alcoholic steatohepatitis (NASH), cirrhosis of the
liver and
alcoholic steatohepatitis.
[0386] Klotho serum levels have also been reported as being associated with
muscle
recovery, such as postinjury muscle healing. In particular, and without being
bound to any
theory, age-related declines in a-Klotho may drive progenitor cell
mitochondrial dysfunction
and impaired muscle regeneration. Clinically, this could translate to
treatment of older adults
who either sustained a muscle injury or underwent muscle-damaging surgery.
Embodiments
of the present disclosure can include administering a composition, as
described herein,
prophylactically and/or post-operatively. Administration at the appropriate
timepoint(s) can
enhance or support muscle regeneration and lead to a more complete recovery.
Serum Klotho
protein levels may also enhance or support muscle regeneration in athletes and
other patients.
Accordingly, embodiments of the present disclosure can include administering a
composition,
as described herein, prophylactically (pre-workout) and/or following exercise.
Administration of compositions to treat age-related frailty in humans and non-
human
mammals
[0387] An exemplary embodiment of the present disclosure relates to the
administration of
a composition to treat age-related frailty (e.g., in humans or non-human
animals). s-Klotho
may rescue myogenic stem cells, improve muscle repair, and/or suppress
fibrosis in animal
models of human disease. Compositions that increase s-Klotho levels may,
therefore, be a
promising approach to counter muscle degeneration in aging human subjects
showing signs
of frailty.
[0388] For instance, the present disclosure relates to s-Klotho level-
increasing
compositions, formulations, and dosages, and administration thereof to a
fragile and/or
elderly (e.g., 60 ¨ 95 years of age) person in order to maintain in the
subject a maintenance
serum concentration of s-Klotho within the range of a normal and/or young
(e.g., 18 ¨ 30
years of age) person (e.g., without any chronic conditions).
[0389] Such Klotho treatment over an extended period of time can recover
and/or improve
one or more aging-related indicator or conditions in the elderly, frail, or
otherwise
physiologically aging.
Administration of compositions to treat (decrease) muscle atrophy in humans
and non-human
mammals
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[0390] An exemplary embodiment of the present disclosure relates to the
administration of
compositions of the present disclosure to treat (e.g., decrease (the rate of))
muscle atrophy in
human, as measured by skeletal muscle tissue mass and, preferably, the
concurrent use of the
above compositions and molecular indicators to provide guidance on the effects
of
administration in countering muscle atrophy.
[0391] Muscle atrophy can include numerous neuromuscular, metabolic,
immunological
and neurological disorders and diseases as well as starvation, nutritional
deficiency,
metabolic stress, diabetes, aging, muscular dystrophy, or myopathy. Muscle
atrophy can
occur during the aging process. Muscle atrophy can also result from reduced
use or disuse of
1() the muscle. Symptoms include a decline in skeletal muscle tissue mass.
In human males,
muscle mass declines by one-third between the ages of 50 and 80. Some
molecular features
of muscle atrophy can include the upregulation of ubiquitin ligases, and the
loss of
myofibrillar proteins. The breakdown of these proteins, which can be monitored
(e.g., by
measuring 3-methyl-histidine production, which is a specific constituent of
actin), for
example, in certain muscles of myosin. Release of creatine kinase (a cell
damage marker) can
also be indicative.
Administration of compositions to treat (improve) muscle repair or recovery in
humans and
non-human mammals
[0392] Klotho serum levels have also been reported as being associated with
muscle
recovery, such as postinjury muscle healing. In particular, and without being
bound to any
theory, age-related declines in a-Klotho may drive progenitor cell
mitochondrial dysfunction
and impaired muscle regeneration. Clinically, this could translate to
treatment of older adults
who either sustained a muscle injury or underwent muscle-damaging surgery.
Embodiments
of the present disclosure can include administering a composition, as
described herein,
prophylactically and/or post-operatively. Administration at the appropriate
timepoint(s) can
enhance or support muscle regeneration and lead to a more complete recovery.
Serum Klotho
protein levels may also enhance or support muscle regeneration in athletes and
other patients.
Accordingly, embodiments of the present disclosure can include administering a
composition,
as described herein, prophylactically (pre-workout) and/or following exercise.
[0393]
Administration of compositions to treat mental and/or cognitive decline in old
age and/or
throughout the human lifespan
[0394] An exemplary embodiment of the present disclosure relates to the
administration of
compositions of the present disclosure to improve and/or counteract (aging-
related) decline in
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cognitive function(s). At the time of the present disclosure, it was unknown
whether
administration of compositions of the present disclosure may counteract
cognitive decline in
humans. However, transgenic mice with systemic over-expression of klotho
performed better
than controls in multiple tests of learning and memory. Elevating klotho in
mice also
enhanced long-term potentiation, a form of synaptic plasticity, and enriched
synaptic
GluN2B, an N-methyl-D-aspartate receptor (NMDAR) subunit with key functions in
learning
and memory. Blockade of GluN2B abolished klotho-mediated effects.
[0395] Klotho-regulated pathways may be relevant to slowing the progression of
Alzheimer's disease and other forms of dementia. Brain scans of more than 400
healthy men
and women aged 53 and over found that those who carried a single copy of a
particular
Klotho gene variant had a larger brain region that deals with planning and
decision making.
Further tests on the group found that those with an enlarged right
dorsolateral prefrontal
cortex (rDLPFC) fared better on a series of mental tasks.
[0396] About one in five people inherits a single copy of the gene variant, or
allele, known
.. as KL-VS, which improves heart and kidney function, and on average adds
about three years
to human lifespan. However as to the brain function, having a larger rDLPFC
accounted for
only 12% of the improvement in people's mental test scores while, on the other
hand, as the
editorial points out, carrying one copy of the KL-VS allele appears to confer
a decade of
resilience against the expected decline in structure and function of the
rDLPFC. Thus, KL-VS
.. heterozygosity appears to be associated with greater volume in right
dorsolateral prefrontal
cortex (rDLPFC).
[0397] Because rDLPFC is important for executive function, the investigators
also
analyzed working memory and processing speed in individuals. KL-VS
heterozygosity may
be associated with enhanced executive function across the lifespan examined.
In short, results
may suggest that variation in the klotho gene may be associated with bigger
brain volume and
better function.
[0398] An exemplary embodiment of the present disclosure relates to the
administration of
compositions of the present disclosure to augment in vivo Klotho protein
levels and/or
(cellular, molecular, and/or downstream) effects (e.g., in order to enhance
cognition and
counteract cognitive deficits throughout the human lifespan). Administration
of s-Klotho
level-increasing compositions can preserve and/or improve cognitive function
(e.g., in
humans).
Administration of compositions to treat (prolong) human longevity and/or
lifespan
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[0399] An exemplary embodiment relates to the administration of compositions
of the
present disclosure to chronological (e.g., age-matched from birth) and sex-
matched human
subjects to improve average lifespan. The results on average lifespan obtained
with
composition administration (the experimental group of human subjects) can be
compared
reliably to those individuals not receiving a composition (the control group)
and/or with
statistical information (e.g., from actuarial tables) for a sufficiently large
human population.
Administration of compositions to treat other clinical indications
[0400] An exemplary embodiment of the present disclosure relates to the
administration of
compositions of the present disclosure to treat any age-associated or
otherwise non-age
associated condition, including, but not limited to (increase in) human
frailty, (decrease in)
longevity, (decrease in) cellular senescence, (decline in) muscle strength,
(decrease in) bone
loss or density, (decrease in) cognition, (decline in) muscle mass, (decline
in) physical
fitness, (decline in) hand strength, (decline in) leg strength, and so forth.
The present
disclosure also relates to the administration of compositions of the present
disclosure to
increase bone mineral density (BMD) (e.g., in women but not in men), to
increase BMD (e.g.,
in elderly women), which is reduced after menopause, to regenerate or reduce
the
degeneration of (degenerative) skeletal muscle, to improve walking gait, to
improve (or
reduce the decline in) spatial learning and memory, movement, freedom of
movement,
quality of life assessment, ejection fraction, exercise change, exercise
improvement, and so
forth. The present disclosure further relates to the administration of
compositions of the
present disclosure to decrease cognitive deterioration or forgetfulness, to
increase cognitive
capacity, to improve cognitive function and synaptic plasticity, to decrease a
decline in
learning, learning capacity or IQ, to improve learning, learning capacity or
IQ, and so forth.
Administration of compositions to treat Acute Kidney Injury (AKI)
[0401] Acute kidney injury (AKI), previously called acute renal failure (ARF),
is often
defined as an abrupt onset of renal dysfunction ranging from minor loss of
function to failure.
AKI is a common clinical complication that develops in approximately 4%-7% of
hospitalized patients each year and the prognosis can be poor. Mortality rates
associated with
AKI range from 5%-35%. Renal Klotho expression has been shown to be suppressed
following AKI. Adenoviral gene transfer of Klotho can be cytoprotective in
AKI.
[0402] Acute Kidney Injury (AKI) has been report in approximately 4.9%-7% of
hospitalized patients each year. The rate of AKI may be as high as 60% in
(hospitalized)
elderly persons and 20-30% in elderly or critically ill patients. AKI is also
associated with
increased mortality, length of stay (LOS), 30 day re-administration rates and
hospital cost.
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[0403] AKI may result at least in part from kidney transplant or other
surgery, acute tubular
necrosis (ATN), acute allergic interstitial nephritis (AAIN), nephritis (e.g.,
glomerulonephritis), nephrotoxicity (e.g., drug-induced nephrotoxicity), low
blood pressure,
sepsis or septic condition, or other contributing factor(s). Kidney transplant
and other
surgeries can cause acute damage or injury to the kidneys, cause renal disease
and/or failure.
Nephrotoxicity can contribute to AKI, ATN, AAIN, nephritis, etc. It has been
reported that
drugs (e.g., clinically-administered, prescription, illegal, or other drugs)
are associated with
15% to 25% of all cases of AKI. Contrast media alone accounts for 10% of all
causes of
hospital-acquired acute renal failure (e.g., via contrast-induced acute kidney
injury CIAKI)
and represents the third leading cause of in-hospital renal function
deterioration after
decreased renal perfusion and postoperative renal insufficiency.
[0404] In some cases, drug-induced AKI can be or comprise anti-microbial-
induced
nephrotoxicity (resulting from anti-microbial treatment). For instance,
certain (gram-
negative) bacterial infections can be treated with one or more
aminoglycosides, such as
paromomycin, tobramycin, gentamicin, amikacin, kanamycin, neomycin, and so
forth.
Aminoglycosides have been shown to be nephrotoxic. Table 47 presents treatment
data
collected on the adult patients receiving the aminoglycosides. As illustrated
in Table 47,
nearly 23% of patients treated with amikacin, a common aminoglycoside,
developed acute
kidney disease and over 17% of patients treated with amikacin died prior to
being discharged.
Other aminoglycosides, including gentamicin, and tobramycin also induced (or
were
associated with or contributed to) kidney disease.
Length of Stay U. 8.7 10.4 9.2.
Discharged. Home 55.1% 72.0% 62.6% 68,9%
Died 1.7:1% 4,1%. 6.3% 5.8%
ICU 21,8% [18...2% 23_8%
Pediatric Patient (<1,8 yrs.) 0.0% 21.6% 6.1% 16.5%
Nursing Horne .Patient 22.1%= 12.5% 20..2% 14,6%
Kidney Disease 22.9% 13,5% 1.6.5%
UTItcy.stitis LM/c45=tit1s Pneumonia
till/cystitis
Sepsis Sepsis UTI,/cy.stftit.. Sepsis
Top S Dx Preurnona Skin nfectio n Sqa51.5
Pneumnia
Skin nfectic n Pneumonia Skin infection
Skriitife.ction
, Pelvic infection , Rio Pelvic
infection PIvk nfection
Table 47
[0405] As illustrated in Figures 13A and 13B, over 1.2 million adult patients,
in various age
groups, were treated with aminoglycosides in the year 2010. Other anti-
microbial agents
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include, for example, penicillins, ampicillin, cephalosporins, sulfonamides,
ciprofloxacin,
vancomycin, macrolides, tetracyclines, rifampin, and so forth.
[0406] Drug-induced nephrotoxicity can also result from treatment with one or
more non-
steroid anti-inflammatory drugs (NSAIDs), such as aspirin (acetylsalicylic
acid), celecoxib,
diclofenac, diflunisal, etodolac, ibuprofen, indomethacin, ketoprofen,
ketorolac, nabumetone,
naproxen, oxaprozin, piroxicam, salsalate, sulindac, tolmetin, and so forth.
[0407] Drug-induced nephrotoxicity can also result from treatment with one or
more
cyclooxygenase-2 (COX-2) inhibitors (e.g., valdecoxib, rofecoxib, celecoxib,
etc.), proton
pump inhibitors (e.g., omeprazole, lansoprazole, etc.), anti convul sants
(e.g., phenytoin,
valproic acid, etc.), histamine H2 receptor antagonist (e.g., nizatidine,
ranitidine, famotidine,
cim eti dine, etc.), diuretics (e.g., carbonic anhydrase inhibitors, loop
diuretics (e.g.,
bumetanide, ethacrynic acid, torsemide, furosemide, etc.), potassium-sparing
diuretics (e.g.,
triamterene, spironolactone, amil ori de, etc.), thi azi de diuretics (e.g.,
indap ami de,
chl orthali done, metolazone, m ethycl othi azi de, hydrochl orothi azi de,
chl orothi azi de,
bendroflumethiazide, polythiazide, hydroflumethiazide, etc.), or other
diuretics, such as
pamabrom, mannitol, and so forth.
[0408] Drug-induced nephrotoxicity can also result from treatment with
lithium, which can
affect the flow of sodium through nerve and muscle cells in the body and can
be used to treat
manic episodes of bipolar disorder, often characterized by hyperactivity,
rushed speech, poor
judgment, reduced need for sleep, aggression, and anger. Lithium can also help
to prevent or
lessen the intensity of manic episodes. Drug-induced nephrotoxicity can also
result from
treatment with or exposure to gold, mercury, copper, or other elemental
matter.
[0409] Drug-induced nephrotoxicity can also result from treatment with (D-)
penicillamine;
a medication of the chelator class, which can be used to treat scleroderma,
Wilson's disease
(by binding to accumulated copper for elimination through urine), cystinuria
(by binding with
cysteine to yield a mixed disulfide which is more soluble than cysteine),
direct-acting smooth
muscle relaxant (e.g., hydralazine), spasmolytics (e.g., carisoprodol,
cyclobenzaprine,
metaxalone, methocarbamol, benzodiazepines, such as diazepam, clonidine and
other
imidazoline compounds, tizanidine, baclofen, hydantoin derivatives,
dantrolene, and so forth.
[0410] Other forms of drug-induced nephrotoxicity can include, for example:
contrast-
induced nephrotoxicity (e.g., following exposure to (iodinated) contrast
media, also known as
radiocontrast-induced nephropathy (CIN)); narcotic- (opioid-) induced
nephrotoxicity (e.g.,
following use or abuse of certain narcotics (e.g., opioids), such as cocaine,
heroin, etc.);
chemotherapy-induced nephrotoxicity (e.g., following treatment with a cancer
therapeutic,
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such as: cisplatin; carboplatin; oxaliplatin; alkylating agents, such as
bendamustine,
cyclophosphamide, ifosfamide, nitrosoureas, temozolomide, melphalan, etc.;
antitumor
antibiotics, such as mitomycin C, bleomycin, anthracyclines and related
agents, etc.;
antimetabolites, such as capecitabine, hydroxyurea, methotrexate, pemetrexed,
pralatrexate,
pentostatin, fludarabine, cladribine, gemcitabine, cytarabine, etc.; vinca
alkaloids; topotecan;
etoposide; taxanes; irinotecan; lenalidomide; eribulin; arsenic trioxide;
ixazomib; etc.); and
so forth. Indeed, a wide variety of nephrotoxic drugs can induce
nephrotoxicity, leading to
AKI. Drug-induced nephrotoxicity (and other forms of AKI) can be life-
threatening if
untreated and can incur enormous costs (to patients, hospitals, and insurers)
to treat.
[0411] Embodiments of the present disclosure can include a method of treating
or
preventing (e.g., prophylactically or in response to at least one event) acute
kidney injury
(AKI), chronic kidney disease (CKD), or other condition. The method can
comprise
administering a composition of the present disclosure to a subject in need
thereof For
instance, the method can comprise administering to a subject in need thereof,
an effective
amount of a composition of the present disclosure (e.g., so as to raise and/or
maintain a serum
soluble Klotho protein concentration of the subject at or above a
predetermined threshold for
a predetermined period of time). The composition can comprise (i) a
therapeutic recombinant
Klotho protein or fragment or fusion protein thereof and/or (ii) a product
that increases,
enhances, augments, and/or supports endogenous Klotho protein production
and/or level(s) in
a patient to which the product is administered, optionally with one or more
additional
ingredients or compositions.
[0412] In some embodiments, the condition can comprise: (i) acute tubular
necrosis
(ATN), acute allergic interstitial nephritis (AAIN), nephritis,
glomerulonephritis, and/or
nephrotoxicity; or (ii) AKI resulting at least in part from sepsis, kidney
transplant or other
surgery or medical procedure, acute tubular necrosis (ATN), acute allergic
interstitial
nephritis (AAIN), nephritis, glomerulonephritis, nephrotoxicity, or low blood
pressure. The
condition can comprise a drug-induced (e.g., aminoglycosides-induced)
nephrotoxicity. The
protein can be administered prophylactically, for example, prior to kidney
transplant,
nephrotoxin administration, or other activity, treatment, or event known or
anticipated to
cause or contribute to AKI. Alternatively, or in addition, the protein can be
administered in
response to AKI, such as after kidney transplant or other surgery,
aminoglycoside or other
nephrotoxin administration, or other activity, treatment, or event known or
anticipated to
cause or contribute to AKI.
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[0413] In some embodiments, the nephrotoxin or other drug can be or comprise,
for
example: one or more aminoglycosides (e.g., paromomycin, tobramycin,
gentamicin,
amikacin, kanamycin, neomycin, etc.); one or more anti-fungal agents (e.g.,
amphotericin B,
flucytosine, etc.); one or more contrast agents (e.g., (iodinated)
radiocontrast media, high-
osmolality contrast media (HOCM) having an iodine to molecule ratio of about
1.5: 1, low-
osmolality, nonionic contrast media (LOCM) having an iodine to molecule ratio
of about
3 : 1, isosmolar (isoosmolality) contrast media (I0CM) having an iodine to
molecule ratio of
about 6: 1), etc.); one or more antiretroviral agents (e.g., adefovir,
cidofovir, tenofovir,
foscarnet, etc.); one or more cancer (or chemo-) therapeutics (e.g.,
cisplatin, carboplatin,
oxaliplatin, alkylating agents (such as bendamustine, cyclophosphamide,
ifosfamide,
nitrosoureas, temozolomide, melphalan, etc.), antitumor antibiotics (such as
mitomycin C,
bleomycin, anthracyclines and related agents, etc.), antimetabolites (such as
capecitabine,
hydroxyurea, methotrexate, pemetrexed, pralatrexate, pentostatin, fludarabine,
cladribine,
gemcitabine, cytarabine, etc.), vinca alkaloids, topotecan, etoposide,
taxanes, irinotecan,
lenalidomide, eribulin, arsenic trioxide, ixazomib, etc.); one or more
bisphosphonates, or
derivatives thereof (e.g., zoledronate / zoledronic acid, ibandronate,
alendronate,
alendronate/cholecalciferol, etidronate, risedronate (optionally, with calcium
carbonate),
pamidronate, tiludronate, etc.); and/or one or more narcotics (e.g., opioids),
such as cocaine,
heroin, etc.);
[0414] Embodiments of the present disclosure can include a method of
administering a
composition of the present disclosure. The method can include administering
the composition
of the present disclosure to a human or non-human subject to treat or prevent
(prophylactically) AKI or one or more conditions associated with AKI. The
method can
include determining a level of serum soluble klotho level in the subject,
calculating a first
dosage of a composition of the present disclosure sufficient to raise the
serum soluble klotho
level in the subject to a predetermined level or percent of normal levels,
administering the
first dosage to the subject, such as by oral, bolus or gradual administration,
determining a rate
of soluble Klotho decline in the serum of the subject, such as following
administration of the
first dosage, calculating a subsequent dosage time and amount, and/or
administering the
subsequent dosage of protein to the subject.
Administration of compositions to treat Chronic Kidney disease (CKD)
[0415] Without being bound to any theory, klotho may be involved in human
chronic
kidney disease. Soluble Klotho in the circulation starts to decline early in
chronic kidney
disease (CKD) stage 2 and urinary Klotho possibly even earlier in CKD stage 1.
Therefore,
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soluble Klotho could serve as an early and sensitive marker of kidney function
decline.
Moreover, preclinical animal data support Klotho deficiency is not just merely
a biomarker,
but a pathogenic factor for CKD progression and extrarenal CKD complications
including
cardiovascular disease and disturbed mineral metabolism. Prevention of Klotho
decline, re-
activation of endogenous Klotho production, or supplementation of exogenous
Klotho could
all contribute to attenuation of renal fibrosis, retardation of CKD
progression, improvement
of mineral metabolism, amelioration of cardiomyopathy, and alleviation of
vascular
calcification in CKD.
[0416] CKD is characterized by progressive deterioration of renal function
with high risk
of ESRD. CKD risk increases with age, and about half of the CKD stage > 3
cases occurs in
subjects > or = 70 years old. CKD can be viewed as a state of accelerated
aging. The relative
risk for cardiovascular mortality of a 25 to 34-year-old dialysis patient is
similar to a non-
CKD patient of > or = 75 years of age. Cardiovascular disease is the principal
killer in CKD
and ESRD patients. CKD and ESRD patients have low renal Klotho expression and
low
levels of circulating Klotho. Renal Klotho deficiency in early stages of CKD
may be
attributed mainly to suppression of Klotho expression rather than loss of
viable renal tubules.
Furthermore, some dialysis patients still have detectable circulating Klotho,
suggesting that
renal Klotho expression is not completely suppressed, and Klotho may come from
extra-renal
source(s), although its origin is not clear to date. Establishing extra-renal
sources of Klotho
.. and characterizing how these can be up-regulated when renal production
fails is of paramount
importance.
[0417] Administration of compositions of the present disclosure can help to
prevent, retard,
and decrease the burden of comorbidity in CKD.
Administration of compositions to treat gut diseases and related conditions
.. [0418] Without being bound to any theory, Interstitial Cells of Cajal
(ICCs) are interstitial
cells that are between other cells in the GI tract with an ongoing function in
controlling
peristalsis. ICCs (main) function may be to create the electrical signals that
control the
rhythmic smooth muscle contraction in the gut (different regions of the
gastrointestinal
system have different frequencies of contraction called peristalsis). ICCs may
guide the
migration of mesenchymal stem cells in the gut (and many other organs).
[0419] In the gut, ICCs may produce and/or become activated by klotho, leading
to
optimized regenerative processes. In Klotho knockout mice, ICCs are
diminished, leading to
impairment of the peristaltic process, mimicking gut disorders that occur in
older humans.
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[0420] Administration of compositions of the present disclosure can help to
prevent, retard,
and decrease impairment of the peristaltic process and/or augment, support,
and/or optimize
the regenerative processes, at least in the gut. These effects may further
support gut health
and/or prevent, retard, and decrease the occurrence and/or severity of gut
diseases and related
conditions.
Therapeutic Treatment of Age-related Conditions
[0421] Investigators have discovered an association between Klotho and
biological
parameters commonly accepted as indicators of the clinical status in
hospitalized older
patients. The investigators genotyped the single-nucleotide polymorphisms
(SNPs)
rs9536314, rs1207568, and rs564481 at the KL locus in 594 hospitalized older
patients (65-
99 years), consecutively attending a geriatric ward, and tested the
association of these KL
variants with biological quantitative traits using analyses of covariance and
genetic risk score
models. Significant associations of rs9536314 with serum levels of hemoglobin,
albumin, and
high-density lipoprotein cholesterol (HDL-C) as well as significant
associations of rs564481
with serum levels of hemoglobin, fasting insulin, and fasting glucose were
observed. Gender-
segregated analyses confirmed these associations and suggests that the
associations of KL
genotypes with HDL-C, fasting glucose and fasting insulin levels may be driven
by the
female gender, while the association with serum levels of hemoglobin may be
driven by the
male gender. The association of KL genotypes with creatine levels was found in
females,
while the association with insulin-like growth factor-1 (IGF-1) and
lymphocytes count (LC)
was found in males. The genetic risk score (GRS) models further confirmed
significant
associations among KL SNPs and hemoglobin, total cholesterol, and HDL-C.
Gender-
segregated analyses with the GRS-tagged approach confirmed the associations
with HDL-C,
fasting glucose, and fasting insulin levels in females, and with hemoglobin
and LC in males.
The findings suggested that KL locus may influence quantitative traits such as
serum levels
of lipid, fasting glucose, albumin and hemoglobin in hospitalized older
patients, with some
gender differences suggested for creatine, IGF-1 levels, and LC, thus being
one of the genetic
factors possibly contributing to age-related diseases and longevity.
[0422] Embodiments of the present disclosure can include administering a
composition of
the present disclosure to a subject in need thereof (e.g., an individual or
patient, optionally
elderly and/or suffering from an aging-related condition, low endogenous s-
Klotho protein
expression, and/or symptoms of age-related condition or decreased longevity).
Administration of the composition(s) of the present disclosure can lead to a
beneficial
increase in blood s-Klotho levels. The administration may reverse or
counteract the
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deleterious effects of the aging-related condition and/or influence, in a
positive therapeutic
manner, quantitative traits such as serum levels of total cholesterol, HDL-C,
fasting glucose,
fasting insulin, albumin, creatine, IGF-1, hemoglobin, and lymphocytes count
(e.g., in
hospitalized and/or elderly patients).
Prophylactic Composition Administration
[0423] In addition to the foregoing, embodiments of the present disclosure can
include
administering a composition of the present disclosure to an individual or
subject in need
thereof for prophylactic purposes and/or maintenance of certain health
attributes. For
example, administration of certain compositions of the present disclosure can
help maintain
1() youthfulness in optionally aging patients not yet suffering from a
diagnosed aging-related
condition. Accordingly, while certain embodiments of the present disclosure
can relate to
and/or comprise treating a condition in a patient, other embodiments can
relate to and/or
comprise preventing, inhibiting development of, and/or prophylactically
addressing one or
more conditions.
.. Administration of exogenous S-Klotho to treat genetic defects
[0424] An exemplary embodiment of the present disclosure relates to the
administration of
exogenous S-Klotho to treat (e.g., correct) known human genetic defects. For
example, a 13-
year-old girl with familial tumoral calcinosis and a Klotho mutation has been
reported.
Familial tumoral calcinosis is an autosomal recessive metabolic disease
characterized by
ectopic calcifications and hyperphosphatemia due to inactivating mutations in
FGF23 or
GALNT3. FGF23 is a hormone necessary for the renal excretion of phosphate,
while GALNT
is an enzyme contributing to the maturation and secretion of FGF23. A
homozygous mutation
in the klotho gene has been identified in the 13-year old girl. Klotho encodes
a secreted
protein necessary to the transmission of the signal emitted by FGF23 toward
its receptors.
The administration of exogenous human recombinant S-Klotho constitutes a
highly targeted
and effective therapy to address the malfunction and symptoms associated with
familial
tumoral calcinosis.
Compositions and treatments including S-Klotho and/or Supplement in
combination with
other component(s)
.. [0425] In at least one embodiment, a composition, medicament, dosage, or
therapeutic
treatment can include a therapeutic human recombinant soluble alpha Klotho (S-
Klotho)
protein and a natural supplement composition that increases endogenous Klotho
production
or one or more component thereof Klotho, whether endogenous or exogenous
(recombinant)
can also act in an additive or synergistic matter with other compounds and/or
components to
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influence one or more aspects of human health and well-being. For instance,
treatments that
include a therapeutic human recombinant soluble alpha Klotho (S-Klotho)
protein, and/or
natural supplement composition that increases endogenous Klotho production, in
combination and/or concurrently with one or more additional active components
can benefit
human patients. Such treatments can be prophylactic or responsive to any human
condition
on which the Klotho protein and/or other component(s) can have a therapeutic
effect. Such
conditions can include, for example, an age-related condition, a clinical
(e.g., metabolic)
condition, a chronic or acute condition, and so forth. Specific, non-limiting
examples of
specific conditions are disclosed herein.
[0426] S-klotho may exist in the human body along with other blood borne anti-
aging
compound such as growth/differentiation factor 11 (GDF-11). Accordingly, in
certain
embodiments, therapeutic S-klotho can be co-administered (e.g., concurrently,
sequentially,
and/or in combination) with therapeutic GDF-11. Such administration can have
additive or
synergistic anti-aging or other effects in some embodiments. Likewise, the co-
administration
S-klotho to human subjects with (neutralizing) antibodies to or suppressor of
CCL11 may
work in unison to counter aging or other condition (as CCL11 (also known as
eotaxin-1) is
understood to be a negative regulator of stem cell rejuvenation). S-klotho can
also or
alternatively be co-administered with other eotaxins, such as eotaxin-2
(CCL24) and/or
eotaxin-3 (CCL26).
[0427] In some embodiments, S-klotho can be co-administered with suppressors
of or
antibodies to Transforming Growth Factor 13-1 (TGF-(31). S-klotho
administration may
oppose the action of the TGF-(31 signal pathways involved in an endogenous
anti-cellular
epithelial-to-mesenchymal transition (anti-EMT) that leads to renal and other
tissue fibrosis.
Anti-EMT is also relevant in cancer cells where inhibiting EMT may confer on
cancer cells
the ability to metastasize ¨ this latter process is understood to be opposed
by klotho.
Accordingly, co-administration of S-klotho and a suppressor of or antibody to
Transforming
Growth Factor 13-1 (TGF-(31) can have synergistic or additive effects.
[0428] In some embodiments, S-klotho can be co-administered with antibodies to
or
suppressors of Insulin Growth Factor-1 (IGF-1). Klotho is understood to be a
hormone that
inhibits the intracellular insulin/IGF-1 signaling cascade, and this
inhibition increases
resistance to oxidative stress at the cellular and organismal level in
mammals; a mechanism
which is considered to be evolutionarily conserved for extending life span.
Accordingly, co-
administration of S-klotho and a suppressor of or antibody to Insulin Growth
Factor-1 (IGF-
1) can have synergistic or additive effects.
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[0429] In some embodiments, S-klotho can be administered in combinations with
vitamin
D (e.g., Vitamin D3) or 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], FGF-15, FGF-
19, and/or
Klotho (3; since numerous studies have revealed a comprehensive regulatory
scheme of
mineral homeostasis involving the mutually regulated positive/negative
feedback actions of
Klotho a-K1, FGF23, and 1,25(OH)213 and/or an analogous regulatory network
composed of
Klotho f3-K1, FGF15/humanFGF19, and bile acids that regulate bile
acid/cholesterol
metabolism. Such co-administration can have synergistic or additive effects on
numerous
conditions and/or processes in the body. In some embodiments, S-klotho can be
administered
in combinations with FGF-21,
[0430] In some embodiments, S-klotho can be co-administered with carbonic
anhydrase
inhibitors, such as acetazolamide, methazolamide, dichlorphenamide,
dorzolamide,
brinzolamide, and/or topiramate. Such combinatorial administration can be
useful in the
treatment of ankylosing spondylitis (AS), rheumatoid arthritis (RA), and a
variety of other
conditions. Various investigations have shown that increased bone resorption
is a
characteristic of AS and RA and that carbonic anhydrase inhibitors play an
antiarthritic role
by inhibiting bone resorption. At the bone level, through a different
mechanism, S-klotho
stimulates bone resorption and phosphate release by acting on TRPV5, which is
a recently
identified osteoclast function modulator. Increased levels of 1,25(OH)2D3
caused by S-Klotho
administration can also stimulate osteoclast differentiation and bone
resorption and, thereby,
phosphate release. Thus, co-administration of S-Klotho and carbonic anhydrase
inhibitors can
have an additive or synergistic effect, especially in promoting bone health,
particularly in AS
and RA.
[0431] S-Klotho can be administered in combination with one or more disease-
modifying
antirheumatic drugs (DMARDs) ¨ for the treatment of severe active rheumatoid
arthritis.
[0432] S-Klotho can be administered in combination with cyclosporine, since
cyclosporine
decreases klotho mRNA and protein and increases oxidative stress leading to
cyclosporine
induced-renal injury (CsA). The associated decrease of klotho mRNA and protein
and
increases oxidative stress can be countered by exogenous co-administration of
S-Klotho.
[0433] In some embodiments, S-klotho can be co-administered with losartan
and/or
cyclosporine. Treatment with losartan, an angiotensin II type 1 (AT1) receptor
blocker,
reversed the decrease in klotho expression seen with cyclosporine. Losartan
also produced a
concurrent improvement in renal histology (with losartan decreasing the
tubulointerstitial
fibrosis that is caused by cyclosporine).
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[0434] In some embodiments, S-klotho can be co-administered with one or more
aminoglycosides, such as amikacin, gentamicin, tobramycin, etc. Such treatment
can be
useful to prevent nephrotoxicity and/or acute kidney injury (AKI) when
aminoglycosides are
used to treat (gram-negative) pathogen infection, which may greatly expand the
use of
aminoglycosides to treat infections. The administration of S-Klotho with
verapamil and/or
diltiazem, which have been used to block AKI, can be therapeutic in the
treatment and/or
prevention of renal dysfunction from AKI.
[0435] In some embodiments, S-klotho can be co-administered with testosterone
or
androgen receptor (AR) up-regulating compounds. Recent reports indicate no
beneficial
effect of testosterone treatment in men in regards to personality,
psychological well-being, or
mood is observed. In addition, the prescription of testosterone
supplementation for low-T for
cardiovascular health, sexual function, physical function, mood, or cognitive
function was
considered to be without support from randomized clinical trials. However,
testosterone
supplementation was consistently found to increase muscle strength but did not
have
beneficial effects on physical function. S-Klotho administration in
combination with
testosterone and/or androgen receptor (AR) up-regulating compounds can
significantly
increase muscle strength and/or physical function in elderly, frail, or low-T
men beyond any
effect that testosterone or S-Klotho may have alone in these treatment groups.
[0436] In some embodiments, S-klotho can be co-administered with estrogen or
an
estrogen hormone (e.g., estradiol, estriol, estrone, etc.). Such co-
administration can improve
health indicators in women (e.g., menstrual, menopausal, or menopausal
transitioning
women) and/or treat infertility, polycystic ovarian disease or disorder,
obesity, hormone
imbalance and related conditions, and/or other female health conditions.
[0437] In some embodiments, S-klotho can be co-administered with one or more
nootropics ¨ also called smart drugs or cognitive enhancers. Nootropics drugs,
supplements,
and/or other substances can improve cognitive function, particularly executive
functions,
memory, creativity, motivation, task saliency (motivation to perform a task),
performance
(especially on tedious tasks that require a high degree of effort), and can be
useful in treating
cognitive or motor function difficulties attributable to disorders such as
Alzheimer's disease,
Parkinson's disease, Huntington's disease, and ADHD. The most commonly used
class of
drug that is known to improve some aspect of cognition is stimulants,
especially the classes
of stimulants that demonstrate cognition-enhancing effects in humans by acting
as direct
agonists or indirect agonists of dopamine receptor D1, adrenoceptor A2, or
both receptors in
the prefrontal cortex. Stimulants include, for example: amphetamines (e.g.,
amphetamine,
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dextroamphetamine, lisdexamfetamine, etc.), which can benefit a range of
cognitive functions
(e.g., inhibitory control, episodic memory, working memory, and aspects of
attention),
especially in individuals with ADHD; dimethylamylamine (DMAA), such as 1,3-
dimethylamylamine, which can improve physical performance, alertness, reaction
time, etc.;
methylphenidate ¨ a substituted phenethylamine that can improve a range of
cognitive
functions (e.g., working memory, episodic memory, and inhibitory control,
aspects of
attention, and planning latency); eugeroics (e.g., armodafinil, modafinil,
etc.), which can
function as wakefulness promoting agents that can increase alertness,
particularly in sleep
deprived individuals, facilitate reasoning and problem solving, treat
narcolepsy, shift work
sleep disorder, and daytime sleepiness remaining after sleep apnea treatments,
and so forth;
xanthines (e.g., caffeine, etc.), which can increase alertness, performance,
and/or memory;
nicotine, and so forth.
[0438] In some embodiments, S-klotho can be co-administered with one or more
osteoporosis and/or osteopenia medications, as known in the art. Klotho may
play a role in
regulating bone mineral density, as the absence of Klotho can lead to reduced
bone mineral
density in animals. For instance, Klotho knockout mice show reduced bone
mineral density
over time. Klotho expression can rescue bone defect, such as reduced bone
mineral density
shown by Klotho knockout mice over time. Epidemiological studies have shown
associations
between various Klotho gene variants and changes in bone mineral density and
prevalence of
hand osteoarthritis.
[0439] S-Klotho can be administered in combination with one or more anti-
cancer
treatments and/or preventions, such as a chemotherapeutic. In lung cancers,
such as non-
small cell lung cancer (NSCLC), for example, S-klotho administration can
affect the
resistance of lung cancer cells to cisplatin and/or other chemotherapy. In
addition, S-klotho
can function as a potential tumor suppressor in lung cancer, gastric cancer,
pancreatic cancer
(adenocarcinoma), and other forms of cancer. S-Klotho can be administered in
combination
with sorafenib chemotherapy for the treatment of hepatocellular carcinoma
(HCC).
Overexpression of klotho as well as treatment with soluble klotho protein can
reduce
hepatoma cell growth in vitro and in vivo. Other cancer types that can be
treated with S-
klotho co-administration include hepatocellular carcinoma (HCC), central
nervous system
(CNS) cancers (e.g., brain (e.g., glioma, craniopharyngioma, medulloblastoma
and
meningioma), spinal cord, and other tumors, lymphoma, etc.), (metastatic)
colon cancer, and
so forth.
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[0440] S-Klotho can also be administered in combination with
chemotherapeutic(s) to treat
chemotherapy-induced frailty in cancer patients. S-Klotho can also be
administered to treat
cancer-induced frailty in cancer patients following other known treatments.
[0441] S-Klotho can be administered in combination with kidney dialysis or
other
procedures. S-Klotho can be administered to treat frailty in dialysis
patients, as frailty is
associated with poor outcomes for patients on dialysis.
[0442] S-Klotho can be administered in combination with one or more
Alzheimer's Disease
treatment or preventions, or with brain-derived neurotrophic factor (BDNF), as
an adequate
amount of BDNF can help to develop and maintain normal neuronal circuits in
the brain.
1() [0443] S-Klotho can be administered in combination with one or more
molecules that
increase the ability of klotho to cross the blood¨brain barrier in order to
increase the ability of
klotho to enter the central nervous system (CNS) to treat or prevent CNS-
relates conditions.
For example, neither S-Klotho nor BDNF are known to cross the blood-brain
barrier.
Embodiments of the present disclosure include utilizing blood-brain barrier
delivery
.. techniques for the administration of S-klotho and/or S-klotho with BDNF to
the CNS to treat
Alzheimer's Disease and/or improve cognition in individuals not affected by
Alzheimer's
Disease.
[0444] S-Klotho can be administered in combination with 5' adenosine
monophosphate-
activated protein kinase (AMPK) or AMPK activating drugs or ingredients that
positively
regulate signaling pathways that replenish cellular ATP supplies, including
fatty acid
oxidation and autophagy; or that negatively regulate ATP-consuming
biosynthetic processes
including gluconeogenesis, lipid and protein synthesis.
[0445] S-Klotho can be administered in combination with one or more anti-
diabetic drugs
such as insulin, phloridzin or the antioxidant tiron and these combination
treatments may
have merit in the prevention of renal damage from oxidative stress produced in
diabetic
disorders. The co-administration of S-Klotho with other antidiabetic drugs for
type 1 diabetes
can protect 13-cells by inhibiting 13-cell apoptosis through activation of the
integrin 131-
FAK/Akt pathway, leading to inhibition of caspase 3 cleavage.
[0446] S-Klotho can be administered in combination with one or more type 2
anti-diabetes
drugs such as metformin for improving glycemic control and vascular function
in overweight
and obese diabetic subjects.
[0447] S-Klotho can be administered in combination with one or more blood
pressure
medications, calcium regulators, or treatments or preventions of chronic
kidney disease
(CKD). For example, soft-tissue calcification is a prominent feature in CKD
and Klotho can
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ameliorate vascular calcification by enhancing phosphaturia, preserving
glomerular filtration,
and directly inhibiting phosphate uptake by vascular smooth muscle.
[0448] S-Klotho can be administered in combination with TM5441 or other
inhibitors of
PAT-1 (plasminogen activator 1), since it is thought that PAT-1 inhibition or
deficiency retards
the development of senescence and protects organ structure and function while
prolonging
the lifespan of Klotho-deficient (kl/kl) mice.
[0449] S-Klotho can be administered in combination with sirtuinl (SIRT1) or
SIRT1-
activating compounds (STACs) such as resveratrol. SIRT1, a type III protein
deacetylase, is
thought to be a novel anti-aging protein involved in regulation of cellular
senescence/aging
and inflammation. SIRT1 level and activity are decreased during lung
inflammation caused
by oxidative stress. The mechanism of SIRT1-mediated protection against
inflammation is
associated with the regulation of inflammation, premature senescence, telomere
attrition,
senescence associated secretory phenotype, and DNA damage response. A variety
of dietary
polyphenols and pharmacological activators are shown to regulate SIRT1 so as
to intervene
the progression of type 2 diabetes, cancer, cardiovascular diseases, and
chronic obstructive
pulmonary disease associated with inflammation. Thus some or all of the health
benefits of
SIRT-1 may be augmented by co-administration of SIRT1 and/or SIRTI-activating
compounds given with S-Klotho.
[0450] S-Klotho can be administered in combination with one or more Human
Cells,
Tissues, and Cellular and Tissue-Based Products (HCT/Ps), as recognized by the
FDA. Such
products can include, for example, one or more of bone (including
demineralized bone,
ligaments, tendons, fascia, cartilage, ocular tissues (corneas & sclera),
skin, vascular grafts
(veins and/or arteries), optionally (or other than) preserved umbilical cord
veins, pericardium,
amniotic membrane (when used alone (-without added cells-) for ocular repair),
dura mater,
heart valve allografts, hematopoietic stem cells derived from peripheral or
umbilical cord
blood, semen, oocytes, or embryos. In at least one embodiment, the HCT/P can
be or
comprise one or more stem cells. Stem cell treatment for damaged bodily
tissues and organs
continues to grow in popularity. Administration of therapeutic, recombinant
Klotho protein in
combination with stem cells provided surprising, unexpected, and even
synergistic outcomes
for subjects in need thereof.
[0451] Embodiments of the present disclosure further include a combination
product,
comprising a therapeutic, recombinant Klotho protein in combination with human
stem cells.
The composition can also include a pharmaceutically-acceptable carrier as
described herein.
Such compositions can comprise or be classified as regenerative medicines, to
treat, modify,
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reverse, or cure a serious or life-threatening disease or condition, as
recognized by the FDA.
Preliminary clinical evidence indicates that the composition (drug) has the
potential to
address unmet medical needs for such disease or condition.
[0452] Illustratively, stem cells can be or comprise mesenchymal stem cells
(MSCs), such
as from human umbilical cords or placentas. In at least one embodiment, the
composition
comprising huMSCs and a therapeutic, recombinant Klotho protein of the present
disclosure
attenuated the inflammatory and oxidative stress responses occurring in AKI,
and/or reduced
the expression of senescence-related proteins and microRNAs.
[0453] S-Klotho can be administered in combination with one or more senescence
inhibitors. For instance, Klotho protein can be combined with Pinl-FOXM1
and/or other
senescence inhibitors to enhance outcomes in patients receiving such
treatment.
[0454] S-Klotho can be administered in combination with one or more of the
following:
Klotho stimulators (e.g., Vit. D, Losartan, Testosterone), GDF-11,
Trichostatin A anti-fungal
(e.g., GDF -11 Stimulator), TIMP-2,
CCL-11 Inhibitor/antibodies, D as atinib,
Nicotinamide Riboside (e.g., NAD+), nicotinamide mononucleotide (NMN) (e.g.,
NDA+),
AMPK Stimulators (e.g., Resveratrol, Aspirin, Salicylate, phytochemicals, DR),
C60 Fullerene, Rapamycin, FGF inhibitor, Senolytics drugs/compounds such as
FOX04-
p53 interfering peptides (e.g. FOX04-DRI), inhibitors of the anti-apoptotic
proteins BCL-2
and BCL-xL.
[0455] Any of the foregoing or other treatments or co-administrations can have
additive or
synergistic effects over those of any of the treatments alone. For instance,
co-administration
of a Klotho protein with one or more of the foregoing can result in treatment
outcomes
greater than the sum of the individual outcomes of administering the
components alone at
similar concentrations. In addition, synergistic effects can include treatment
outcomes similar
to those of the individual outcomes of administering the components alone, but
at lower
concentrations. Synergistic effects can also include increasing the maximum
effective dose of
one or more of the components, reducing toxicity of one or more of the
components, or any
other beneficial result that is more than a mere additive effect of the
individual treatment
outcomes. In addition, additive effects of the individual treatment outcomes
can comprise one
or more synergistic effects. Such additive/synergistic effects may not be
predicted or
expected given the nature and understanding of the individual components.
[0456] As used herein, a combination treatment or co-administration can
include treatment
or administration of a combination product, composition, or formulation
comprising a Klotho
protein and one or more additional active ingredients. The one or more
additional active
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ingredients can be selected from among the components, drugs, substances,
treatment
compositions, etc. described herein or others known in the art. For instance,
the Klotho
protein and one or more additional active ingredients can be co-formulated
into an injectable
(e.g., intramuscular, intravenous, etc.), ingestible, transdermal, inhalable,
topical, or other
formulation.
[0457] Alternatively, a combination treatment or co-administration can include
treatment or
administration of a Klotho protein and one or more additional active
ingredients, but without
the Klotho protein and one or more additional active ingredients being
combined or
formulated into a combination product, composition, or formulation. For
instance, the Klotho
protein and one or more additional active ingredients can each comprise or be
in a separate
injectable (e.g., intramuscular, intravenous, etc.), ingestible, transdermal,
inhalable, topical,
or other formulation.
[0458] It will also be appreciated that co-administration can comprise
simultaneous
administration of two or more components or distinct administrations of two or
more
components, the distinct administrations preferably being separated by a
period of time. The
period of time can be very small, in some embodiments. For instance, a second
component of
the Klotho protein and one or more additional active ingredients can be
administered (e.g.,
injected) substantially, immediately following administration of a first
component of the
Klotho protein and one or more additional active ingredients. Alternatively,
the first and
second administrations can be separated by a time period of 1-60 seconds, 1-60
minutes, 1-24
hours, 1-7 days, 1-4 weeks, 1-12 months, and so forth, or any value or range
of values
therebetween. Similarly, simultaneous administration can include overlapping
administration
timeframes for the two or more components.
Therapeutic Treatment of Hyperphosphatemic Familial Tumoral Calcinosis (HFTC)
[0459] In affected human individuals, HFTC is caused by a histidine (H) to
arginine (R)
mutation at amino acid (AA) position 193 of S-Klotho ¨ rs121908423. Without
being bound
to any theory, it is thought that the H193R mutation in HFTC individuals
impairs the ability
of S-Klotho to form a ternary complex with FGF23 and FGFR1c, which impairs KL-
dependent FGF23 signaling. As a result, affected subjects present a severe
metabolic disorder
that manifests with hyperphosphatemia and massive calcium deposits in the skin
and
subcutaneous tissues. Some patients manifest recurrent, transient, painful
swellings of the
long bones associated with the radiographic findings of periosteal reaction
and cortical
hyperostosis and absence of skin involvement.
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[0460] An embodiment of the present disclosure includes a S-Klotho protein
having H193.
The H193 protein can be produced or expressed in the context of any protein
construct
described herein. For instance, the H193 variant can be produced or expressed
in the context
of S-Klotho, isoform 1 or 2, 1-981, 29-981, 34-981, 36-981, 131-981, 1-549, 29-
549, 34-549,
36-549, 131-549, and so forth, with or without a tag (e.g., Fc tag, TEV-Twin-
Strep tag, TEV
sequence, etc.). Accordingly, the nucleic acid construct or cDNA from which
the protein is
expressed can be of corresponding configuration.
[0461] Embodiments can include producing a H193 heterozygous or homozygous
variant
construct, transferring (e.g., via transfection) the resulting construct,
which encodes the 5-
Klotho H193 protein, into an appropriate expression system (e.g., CHO cells),
and/or
transiently expressing the S-Klotho H193 protein. The H193 Klotho protein may
also be
expressed at higher levels than the R193 (or H193R) protein. Embodiments can
include
purifying (and optionally quality-control testing) the expressed protein for
therapeutic
administration. Embodiments can include administering a therapeutic or
therapeutically-
effective amount of the S-Klotho H193 protein to a subject in need thereof
(e.g., HFTC
individuals, individual diagnosed with HFTC, or patient harboring the H193R
(r5121908423)
or other variant). Alternatively, the subject may be of wild-type of other
mutant or variant
type. Administration of the recombinant S-Klotho H193 protein can lead to a
beneficial
increase in blood S-Klotho levels. The administration may reverse or
counteract the
deleterious effects of the R193 mutation that is transcribed and circulated in
HFTC
individuals as a result of the H-to-R193 point mutation found in the human
klotho gene in
individuals affected with HFTC. Accordingly, the circulating concentration of
S-Klotho
H193 may help to counter the effects observed in H193R or HFTC individuals.
Therapeutic Treatment in CC Genotype Patients with End-Stage Renal Disease
(ESRD)
[0462] Approximately 350,000 patients with end-stage renal disease (ESRD)
suffer
exceptionally high mortality rates in their first year of chronic
hemodialysis. Both vitamin D
and fibroblast growth factor (FGF)-23 levels correlate with survival in these
patients. Without
being bound to any theory, Klotho is a protein in the vitamin D/FGF-23
signaling pathway
that has been linked with accelerated aging and early mortality in animal
models. It has been
hypothesized that genetic variation in the Klotho gene may be associated with
survival in
subjects with ESRD. Investigators tested the association between 12 single
nucleotide
polymorphisms (SNPs) in the Klotho gene and mortality in a cohort of ESRD
patients during
their first year on hemodialysis (n = 1307 white and Asian). A significant
association was
discovered between the CC genotype of one tag SNP, rs577912 (a common HapMap
variant
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with a minor allele frequency [MAF] > 0.05 within the Klotho gene sequence
located in
intron 1), and increased risk for 1-yr mortality (RR, 1.76; 95% CI, 1.19-2.59;
p = 0.003).
This effect in individuals with the CC genotype was even more marked among
patients who
were not treated with activated vitamin D supplementation (HR, 2.51; 95% CI,
1.18-5.34; p
= 0.005). In lymphoblastoid cell lines derived from HapMap subjects, the CC
genotype was
associated with a 16-21% lower Klotho expression compared with the AA or AC
genotypes.
However, none of the rs577912 SNPs nucleotide change described above result in
an amino
acid change in the Klotho protein. Accordingly, this functional SNP (r5577912)
may
quantitatively affect Klotho gene expression at the mRNA level.
[0463] An embodiment of the present disclosure includes a S-Klotho protein
expressed
from the AA or AC genotype. The protein can be produced or expressed in the
context of any
protein construct described herein. For instance, the protein can be produced
or expressed in
the context of S-Klotho, isoform 1 or 2. 1-981, 29-981, 34-981, 36-981, 131-
981, 1-549, 29-
549, 34-549, 36-549, 131-549, and so forth, with or without a tag (e.g., Fc
tag, TEV-Twin-
Strep tag, TEV sequence, etc.). Accordingly, the nucleic acid construct or
cDNA from which
the protein is expressed can be of corresponding length.
[0464] Embodiments can include producing a AA or AC heterozygous or homozygous
construct, transferring (e.g., via transfection) the resulting construct,
which encodes the 5-
Klotho protein, into an appropriate expression system (e.g., CHO cells),
and/or transiently
expressing the S-Klotho protein. Klotho protein expressed in AA or AC
heterozygous or
homozygous cells may be expressed at higher levels than in CC cells.
Embodiments can
include purifying (and optionally quality-control testing) the expressed
protein for therapeutic
administration. Embodiments can include administering a therapeutic or
therapeutically-
effective amount of the S-Klotho protein to a subject in need thereof (e.g.,
an individual or
patient harboring the CC mutation at the one tag SNP, rs577912, having low
endogenous 5-
Klotho protein expression, and/or with end-stage renal disease (ESRD)).
Alternatively, the
subject may be of wild-type of other mutant or variant type. Administration of
the
recombinant S-Klotho protein can lead to a beneficial increase in blood S-
Klotho levels. The
administration may reverse or counteract the deleterious effects of the CC
mutation that is
.. transcribed and circulated in individuals as a result of the point
mutation(s) found in the
human klotho gene in affected individuals. Accordingly, the circulating
concentration of 5-
Klotho may help to counter the effects observed in CC individuals, especially
those with end-
stage renal disease (ESRD), namely mortality in the first year in ESRD
patients undergoing
chronic hemodialysis.
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Therapeutic Treatment of Radiographic Hand Osteoarthritis (OA) and Osteophyte
[0465] Osteoarthritis (OA) is a common complex disease with strong heritable
components.
Investigators studied the association between four putatively functional
genetic variants in
klotho gene and hand OA in a large female Caucasian population. The
investigators found
significant association between SNP G-395A and the presence/absence of
radiographic hand
OA and osteophyte formation. Allele G significantly increased the risk for
radiographic hand
OA and osteophytes with odds ratios (ORs) of 1.44 (P=0.008, 95% confidence
interval (CI)
1.09-1.91) and 1.36 (P=0.006, 95% CI 1.09-1.70), respectively. From logistic
regression
modelling, genotype GG showed more than three-fold increased risk for both
radiographic
hand OA (OR=3.10, 95% CI 1.10-8.76) and osteophyte (OR=3.10, 95% CI 1.10-8.75)
when
compared to genotype AA. After adjustment for age, ORs for genotype GG further
increased
to 4.39 (P=0.006, 95% CI 1.51-12.74) for radiographic hand OA and to 4.47
(P=0.005, 95%
CI 1.56-12.77) for osteophytes. The investigators also suggested that one
variant (SNP G-
395A) in the klotho gene is associated with the susceptibility of hand OA and
appears to act
through osteophyte formation rather than cartilage damage.
[0466] An embodiment of the present disclosure includes a S-Klotho protein
expressed
from a construct having SNP G395A. The resulting protein can be produced or
expressed in
the context of any protein construct described herein. For instance, the A395
variant can be
produced or expressed in the context of S-Klotho, isoform 1 or 2, 1-981, 29-
981, 34-981, 36-
981, 131-981, 1-549, 29-549, 34-549, 36-549, 131-549, and so forth, with or
without a tag
(e.g., Fc tag, TEV-Twin-Strep tag, TEV sequence, etc.). Accordingly, the
nucleic acid
construct or cDNA from which the protein is expressed can be of corresponding
length.
[0467] Embodiments can include producing a G395A heterozygous or homozygous
construct, transferring (e.g., via transfection) the resulting construct,
which encodes the S-
Klotho protein, into an appropriate expression system (e.g., CHO cells),
and/or transiently
expressing the S-Klotho protein. Klotho protein expressed in A395 heterozygous
or
homozygous cells may be expressed at higher levels than in G396 cells.
Embodiments can
include purifying (and optionally quality-control testing) the expressed
protein for therapeutic
administration. Embodiments can include administering a therapeutic or
therapeutically-
effective amount of the S-Klotho protein to a subject in need thereof (e.g.,
an individual or
patient harboring the G395 SNP and/or (at risk of developing) radiographic
hand
osteoarthritis (OA) and/or osteophytes). Alternatively, the subject may be of
wild-type of
other mutant or variant type. Administration of the recombinant S-Klotho
protein can lead to
a beneficial increase in blood S-Klotho levels. The administration may reverse
or counteract
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the deleterious effects of the G395 SNP that is transcribed and circulated in
affected
individuals. Accordingly, the circulating concentration of S-Klotho may help
to counter the
effects observed in G395 individuals, especially those at risk of developing
radiographic hand
osteoarthritis (OA) and/or osteophytes. Administration of the G-395A S-Klotho
protein may,
therefore, decrease the risk of radiographic hand osteoarthritis (OA) and
osteophyte
formation in patients (e.g., patients harboring the G395 SNP.
Therapeutic Treatment of Metabolic Syndrome
[0468] The risk and/or incidence of metabolic syndrome (MetS), a cluster of
cardiometabolic risk factors including abdominal obesity, hyperglycemia,
dyslipidemia and
hypertension, increases with age. In elderly adults, MetS not only increases
the risk of
cardiovascular diseases and type 2 diabetes, but also is associated with
cognitive decline and
disability. Current evidence suggests that MetS is partly heritable, and
genetic factors play a
greater role than environment factors on the incidence of MetS. Investigators
discovered an
association between the G-395A polymorphism and metabolic syndrome (MetS)
among a
population of Chinese nonagenarians and centenarians. Subjects were from the
Project of
Longevity and Aging in Dujiangyan (PLAD). The genotyping of G-395A (r51207568)
in the
promoter region of the Klotho gene was performed using the TaqMan allelic
discrimination
assay. MetS was diagnosed according to the International Diabetes Federation
criteria.
Included were 695 subjects aged 93.5 3.2 years. G and A allele frequencies
were 0.852 and
0.148, respectively. In the whole population, the frequency of MetS was 10.8%
and 5.9% in
the GG and GA + AA genotype group, respectively (p = 0.004). The -395A allele
carriers had
significantly lower risk of MetS in the whole population (odds ratio [OR]
0.50, 95 %
confidential interval [CI] 0.25 to 0.98) and in women (OR 0.51, 95 % CI 0.24
to 0.97), but
not in men (OR 0.42, 95 % CI 0.05 to 3.85). In the whole population and women,
the
relationship between the Klotho G-395A SNP and MetS might be due to its
influence on high
blood pressure (OR 0.48, 95 % CI 0.34 to 0.67; OR 0.47, 95 % CI 0.31 to 0.71,
respectively)
and hypertriglyceridemia (OR 0.66, 95 % CI 0.39 to 0.95; OR 0.54, 95 % CI 0.31
to 0.98,
respectively). In men, this relationship might be due to its influence on high
blood pressure
(OR 0.47, 95 % CI 0.25 to 0.90) and low HDL-C (OR 0.69, 95 % CI 0.27 to 0.93).
Investigators concluded that the -395A allele carriers of the Klotho gene were
correlated with
lower risk of MetS among Chinese nonagenarians and centenarians, especially in
women.
[0469] An embodiment of the present disclosure includes a S-Klotho protein
expressed
from a construct having the -395A allele. The resulting protein can be
produced or expressed
in the context of any protein construct described herein. For instance, the
A395 allele can be
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produced or expressed in the context of S-Klotho, isoform 1 or 2, 1-981, 29-
981, 34-981, 36-
981, 131-981, 1-549, 29-549, 34-549, 36-549, 131-549, and so forth, with or
without a tag
(e.g., Fc tag, TEV-Twin-Strep tag, TEV sequence, etc.). Accordingly, the
nucleic acid
construct or cDNA from which the protein is expressed can be of corresponding
length.
[0470] Embodiments can include producing a -395A heterozygous or homozygous
construct, transferring (e.g., via transfection) the resulting construct,
which encodes the S-
Klotho protein, into an appropriate expression system (e.g., CHO cells),
and/or transiently
expressing the S-Klotho protein. Klotho protein expressed in A395 heterozygous
or
homozygous cells may be expressed at higher levels than in G396 cells.
Embodiments can
.. include purifying (and optionally quality-control testing) the expressed
protein for therapeutic
administration. Embodiments can include administering a therapeutic or
therapeutically-
effective amount of the S-Klotho protein to a subject in need thereof (e.g.,
an individual or
patient harboring the G395 SNP and/or (at risk of developing) metabolic
syndrome (MetS).
Alternatively, the subject may be of wild-type of other mutant or variant
type. Administration
of the recombinant S-Klotho protein can lead to a beneficial increase in blood
S-Klotho
levels. The administration may reverse or counteract the deleterious effects
of the G395 SNP
that is transcribed and circulated in affected individuals. Accordingly, the
circulating
concentration of S-Klotho may help to counter the effects observed in G395
individuals,
especially those at risk of developing metabolic syndrome (MetS).
Administration of the G-
395A S-Klotho protein may, therefore, decrease the risk of metabolic syndrome
(MetS) in
patients (e.g., patients harboring the G395 SNP, in elderly human, and/or in
women).
Therapeutic Treatment of Cancer
[0471] S-Klotho is thought to inhibit basal Wnt signaling activity, thereby
functioning as a
tumor suppressor for colorectal cancer (CRC). In addition, klotho gene
variants associated
with lifespan differences may repress butyrate-mediated Wnt hyperactivation,
and thus
increase the risk of CRC. It has been hypothesized that in this manner, the
type of klotho
variant present, and its relative expression, can interact with levels of
butyrate derived from
diet to modify CRC risk. Moreover, mTOR signaling has also been linked to
human aging,
and crosstalk between Wnt and mTOR signaling may influence colonic
tumorigenesis
[0472] The KL-VS variant or other construct can serve as a vehicle for
investigating which
SNPs (e.g., within KL-VS) are responsible for influencing S-Klotho to produce
a decrease in
basal Wnt signaling and/or the suppression of butyrate-mediated Wnt
hyperactivation ¨ the
latter Wnt-related activities which have been associated with S-Klotho tumor
suppression.
Embodiments include modifying the appropriate amino acids (e.g., in the KL-VS
stretch of 5-
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Klotho) shown to influence the tumor suppressing action of the KL-VS variant.
An
embodiment of the present disclosure includes a recombinant S-Klotho protein
having one or
more amino acid alterations in the KL-VS stretch of 6 SNPs. The proteins can
be produced
and/or expressed in the context of any protein construct described herein. For
instance, the
proteins can be produced or expressed in the context of S-Klotho, isoform 1 or
2, 1-981, 29-
981, 34-981, 36-981, 131-981, 1-549, 29-549, 34-549, 36-549, 131-549, and so
forth, with or
without a tag (e.g., Fc tag, TEV-Twin-Strep tag, TEV sequence, etc.).
Accordingly, the
nucleic acid construct or cDNA from which the protein is expressed can be of
corresponding
length.
[0473] Embodiments can include producing a heterozygous or homozygous variant
construct, transferring (e.g., via transfection) the resulting construct,
which encodes the 5-
Klotho protein, into an appropriate expression system (e.g., CHO cells),
and/or transiently
expressing the S-Klotho protein. The Klotho protein may be expressed at higher
levels than
the other Klotho proteins, including wild-type. Embodiments can include
purifying (and
optionally quality-control testing) the expressed protein for therapeutic
administration.
Embodiments can include administering a therapeutic or therapeutically-
effective amount of
the S-Klotho protein to a subject in need thereof (e.g., a patient with or at
risk of developing
colorectal cancer (CRC) or another tumor). The subject may, for example,
harbor or express a
Klotho variant with decreased Wnt inhibition activity. Alternatively, the
subject may be of
wild-type of other mutant or variant type. Administration of the recombinant S-
Klotho
protein can lead to a beneficial increase in blood S-Klotho levels.
Therapeutic Treatment of Non-human Mammals
[0474] In addition to the foregoing, one or more non-human, mammalian Klotho
proteins
or protein sequences (or portion(s) thereof) can be useful in implementing
certain
embodiments of the present disclosure. SEQ ID NOS: 121-124 present DNA
sequences for
encoding various non-human mammalian Klotho proteins. SEQ ID NOS: 111-120
present
amino acid sequences for various non-human mammals. These Klotho protein
sequences (or
portion(s) thereof) can be expressed, purified, formulated into compositions,
and/or
administered to animals in a manner similar to that described above.
Therapeutics
administration of recombinant Klotho (fusion) proteins can be effective in
treating the non-
human mammal version of the medical and other conditions outlined herein. In
particular,
embodiments of the present disclosure can include veterinary treatment method,
proteins, and
compositions, as will be understood by those skilled in the art.
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Examples
Example 1
[0475] Table 48 illustrates the results from transient expression and
purification of the
recited Klotho variants in HEK and/or CHO cell lines. In the results provided
in Table 48,
below, the following abbreviated protocols were followed.
[0476] For Fc fusion proteins, protein expression vectors transfected into
HEK293.sus or
CHO using standard methods. Briefly, cells were grown for 7 days and
harvested. Cell counts
are given in notes section. Supernatant pH was adjusted with 1 M Hepes pH 7.4
and sodium
azide added. KanCap A resin was used to capture proteins. Resin was washed
with PBS.
1() Resin was washed with PBS plus 1 M NaCl. Resin was washed with PBS.
Proteins were
eluted with 50 mM Citrate pH 3.5, 100 mM NaCl. Proteins were immediately
neutralized
with 1 M Tris pH 8, 0.5M Arginine. SDS PAGE gel samples were removed at this
stage.
Proteins were buffer exchanged into PBS. Protein was quantified by 0D280,
quantity and
concentration was determined using calculated extinction coefficient. Reduced
and non-
reduced SDS-PAGE (Biorad criterion Tris/Glycine/SDS, 4-20%) were used to
determine
purity and approximate molecular mass. Aggregation status was determined by
HPLC, with
detection at 280nm using a Sepax Zenix-C SEC-300, 3um, 300A, 4.6*150 mm size
exclusion
column and PBS running buffer. Proteins were shipped as aliquots after filter
sterilization,
snap frozen in liquid nitrogen. Loss of protein was observed for some Fc
proteins during
desalting into PBS, as determined from samples run on SDS-PAGE before and
after
purification. These proteins are expressed but from the HPLC, problems arose
during
desalting or assay on the silica HPLC column in PBS. Accordingly, buffer
selection was
optimized to reduce protein loss.
[0477] For Strep tagged proteins, protein expression vectors transfected into
HEK293.sus
or CHO using standard methods. Briefly, cells were grown for 7 days and
harvested.
Supernatant pH was adjusted with 1 M Hepes pH 7.4 and sodium azide added.
BioLock
biotin sequestration reagent was added. StrepTactin superflow resin was used
to capture
proteins. Resin was washed with 100 mM Tris pH 8, 150 mM NaCl, 1 mM EDTA.
Proteins
were eluted with 100 mM Tris pH 8, 150 mM NaCl, 1 mM EDTA plus 2.5 mM
Desthiobitin.
Protein was quantified by 0D280, quantity and concentration was determined
using
calculated extinction coefficient. Reduced and non-reduced SDS-PAGE (Biorad
criterion
Tris/Glycine/SDS, 4-20%) were used to determine purity and approximate
molecular mass.
Aggregation status was determined by HPLC, with detection at 280nm using a
Sepax Zenix-
C SEC-300, 3 um, 300A, 4.6*150 mm size exclusion column and PBS running
buffer.
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Proteins were shipped as aliquots after filter sterilization, snap frozen in
liquid nitrogen. It
should be noted that for the Strep tagged proteins, samples were assayed in
elution buffer.
This elution buffer is very 'neutral' and is not detrimental to proteins.
Also, the running buffer
for the SEC column was PBS, so the proteins underwent a buffer exchange during
assay.
Absorbance at times above 7 minutes are small molecules.
OD280n Conc. Extinction Molecular
Titer
Protein Name
m (mg/mL) Coefficient
Weight (Da) (mg/L)
Native Klotho [CD (34-981)-
2.01 0.95 246780 116615 6.18
nativeSS-strep-HEK
Native Klotho [CD (34-981)-
1.73 0.79 246780 113119 5.16
nonNativeSS-strep-HEK
Klotho [CD variant-1(36-981)-
0.89 0.41 246780 112893 2.64
nonNativeSS-strep-HEK
Native secreted/Isoform 2 (34-
1.33 0.63 140510 66255 4.07
549)-nativestrep-HEK
Native secreted/Isoform 2 (34-
2.84 1.27 140510 62760 8.24
549)-SS-strep-HEK
Secreted variant (36-549)-
1.40 0.62 140510 62534 4.04
nonNativeSS-strep-HEK
Klotho [CD variant-2 (131-981)-
1.12 0.52 221800 103079 3.40
nonNativeSS-strep-HEK
Native Klotho [CD (34-981)-
0.02 0.01 270075 138668 0.27
nativeSS-Fc-HEK
Native Klotho [CD (34-981)-
0.01 0.00 270075 135172 0.12
nonNativeSS-Fc-HEK
Klotho [CD variant-1(36-981)-
-0.04 -0.02 270075
134946 -0.48
nonNativeSS-Fc-HEK
Native secreted/Isoform 2 (34-
0.03 0.02 163805 88308 0.43
549)-nativeSS-Fc-HEK
Native secreted/Isoform 2
0.05 0.03 163805 84813 0.76
(34-549)-nonNativeSS-Fc-HEK
Secreted variant (36-549)-
-0.01 -0.00 163805
84587 -0.08
nonNativeSS-Fc-HEK
Klotho [CD variant-2 (131-981)-
-0.01 -0.01 245095
125132 -0.15
nonNativeSS-Fc-HEK
Native Klotho [CD (34-981)-
0.42 0.20 246780 116615 1.97
nativeSS-strep-CHO
Native Klotho [CD (34-981)-
0.28 0.13 246780 113119 1.30
nonNativeSS-strep-CHO
Klotho [CD variant-1(36-981)-
0.15 0.07 246780 112893 0.68
nonNativeSS-strep-CHO
Native secreted/Isoform 2
0.26 0.12 140510 66255 1.24
(34-549)-nativeSS-strep-CHO
Native secreted/Isoform 2
0.36 0.16 140510 62760 1.61
(34-549)-nonNativeSS-strep-CHO
Secreted variant (36-549)-
0.22 0.10 140510 62534 0.98
nonNativeSS-strep-CHO
Klotho [CD variant-2 (131-981)- 0.19 0.09 221800 103079
0.86
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non NativeSS-strep-CHO
Native secreted/lsoform 2 (34-
0.12 0.06 163805 88308 1.54
549)-nativeSS-Fc-CHO
Native secreted/lsoform 2 (34-
0.16 0.08 163805 84813 2.05
549)-non NativeSS-Fc-CHO
Table 48
[0478] In certain embodiments, EDTA and/or other metal chelating agent(s) were
removed
from (or not included in) one or more (e.g., all) of the purification steps
and/or buffers. In
some embodiments, EDTA and/or other metal chelating agent(s) may contribute to
or cause
deactivation of the Klotho protein.
[0479] In a follow up study, transiently-expressed Fc-Klotho proteins were
purified using
Aquity UPLC BEH200 SEC analytical column: 1.7 um, 4.6 X 150 mm; Mobile Phase:
100
mM Sodium Phosphate, 200 mM Sodium Chloride, pH 6.8; Flow Rate: 0.35 mL/min;
Run
Time: 9 minutes; Detection: A280, MALS & RI detector; Ambient sample
compartment, 30C
column compartment; 100 uL of sample injected (37 ug). Figure 14 illustrates a
chromatograph of Klotho-Fc protein. The majority of Fc Klotho protein appears
to overlay
with the first peak of MW markers near the void volume. This corresponds to a
very high
molecular weight seen for the main peak (in mega daltons), which is much
higher than native
Klotho. By SDS-PAGE, non-reducing, the peak (fractions) contains protein the
size of a
monomer, suggesting non-covalent association in solution. A minor shoulder is
observed on
the tail of the main Klotho peak (marked with dashed oval); this is <1% of
total Klotho peak
area with a MW larger than IgG by elution time on the SEC column relative to
the MW
markers and ¨700 kDa by light scattering (the LS signal is very low and has
tailing of the
aggregates, so might be over-estimated). This peak corresponds to a multimer,
perhaps a
dimer or tetramer.
[0480] Various alternative purification schemes, including different columns,
buffers,
conditions, etc. were used to address the issue with protein aggregation and
multimer
formation. However, in at least one embodiment, a multimer (dimeric,
tetrameric, etc.)
Klotho protein can be purified.
Example 2
[0481] Stable expression of the (human) Klotho derived proteins represented in
SEQ ID
NO: 52 (N'-human alpha Klotho 34-981 isoform 1(G45)2 linker human IgG1 Fc-C'),
SEQ
ID NO: 54 (N'-human alpha Klotho 34-549 isoform 2(G45)2 linker human IgG1 Fc-
C'),
and SEQ ID NO: 66 (N'-human alpha Klotho 34-981 isoform 1 Twin-Strep cleavage
site
residues) was performed in CHO cells. Each of the three Klotho protein
constructs was
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expressed with a non-naturally-occurring N-terminal signal sequence, which was
then
cleaved during the stable expression/purification process to yield the N-
terminal (Klotho
protein) amino acid. C-terminal to the Klotho protein sequence in SEQ ID NOS:
52 and 54 is
a GS linker (GGGGSGGGGS) and an Fc-fusion tag (human IgG1 Fc domain), which
are (or
appear to be) retained in the expressed protein. C-terminal to the Klotho
protein sequence in
SEQ ID NOS: 52 is a TEV-Twin-Strep tag (with GS linker)
(GGENLYFQ/SSAWSHPQFEK-GGGSGGGSGGS-SAWSHPQFEK), which is cleaved
following the glutamine 8 (Q8) of the TEV-Twin-Strep tag (i.e., GGENLYFQ--),
to release
the Twin- Strep tag portion (SSAWSHPQFEK-GGGSGGGSGGS-SAWSHPQFEK) and
retain at least a portion of the TEV (protease recognition or consensus)
sequence
(GGENLYF Q-C ' ).
[0482] The coding sequences were codon optimized using DNA2.0 proprietary
algorithms.
The genes were synthesized and Pd3600 transposon backbone-based expression
constructs
were assembled.
[0483] A GS knockout CHOK1 derivative host cell line was used to express the
human
Klotho variants. The cells have been co-transfected, with Leap-In transposon
based
expression plasmids coding for the three different hKlotho designs and Leap-In
transposases.
The transfections were performed by electroporation using a Neon apparatus
(Thermo).
Transfected cells were selected under glutamine free conditions and the
resulting stable pools
were cryopreserved. Cells from the established stable pools were inoculated
into shake flasks
and fed batch production studies were conducted using small scale established
feeding and
cell culture conditions. Clarified harvest samples were collected and
submitted for Western
blot analysis using anti-Fc antibody- (SEQ ID NOS: 52 and 54) or Strep-Tactin-
(SEQ ID
NO: 66) HRP conjugate based detection. Example Western blots are shown in
Figures 15A-
16B.
[0484] Stable pool 291645 expressing the FL-ECD (34-981)-Fc Klotho derivative
(SEQ ID
NO: 52) produces the protein predominantly at the expected size (see Figure
15A). The
predicted size of the full-length extracellular domain-Fc fusion protein
monomer is 139 kDa.
The predominant anti-Fc positive band is at the predicted ¨140kDa size (arrow)
indicating
that expression construct 291645 expresses the correct size fusion protein.
The protein
dimerizes under native and non-reducing conditions (see Figure 15B). Under non-
reducing
conditions the full-length extracellular domain-Fc fusion protein may form
homodimers. The
size of the predominant band detected by anti Fc antibody on a non-reducing
Western blot
(arrow) is consistent with the formation of the predicted homodimers.
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[0485] The FL-ECD (34-981)-TEV-Twin-Strep molecule expressed by stable pool
291647
(SEQ ID NO: 66), also shows the correct size on Western blots and remains a
monomer
under native and non-reducing conditions (see Figures 16A-16B). The expected
size isoform-
2 -Fc variant represents the secreted human Klotho protein. The predicted
molecular mass of
the mature full length ECD (34-981)-twin-strep tagged Klotho protein (SEQ ID
NO: 66), is ¨
109 kDa. Non-reduced Western blot analysis demonstrates the successful
expression of the
correct size protein from CHO cells coded by expression construct 291647 (see
Figure 16A).
Non-reduced denatured Western blot analysis indicates that the full-length
extracellular
domain of hKlotho does not form covalent homodimers when secreted from CHO
cells (see
Figure 16B). Other protein constructs represented in the accompanying Sequence
Listing
were also prepared. Figure 17 is a gel of the various indicated recombinant
Klotho protein
constructs. Figure 18 is a series of gels for the respective indicated
recombinant Klotho
protein constructs.
[0486] It should be appreciated that the expression and/or purification of the
Klotho
variants disclosed in Table 48 and/or shown in Figures 15A through 18, in
addition to one or
more other Klotho variants disclosed herein but not shown in Table 48, can, in
some
embodiments, result in advantages over the expression and/or purification of
native Klotho.
For example, there can be a reduction in the number and/or types of
alternative products
when expressing and/or purifying Klotho variants. Additionally, or
alternatively, there can be
an increase in expression level of the desired Klotho variant compared to the
expression level
of native Klotho. Additionally, or alternatively, the desired Klotho variant
is expressed and/or
purified in a purer form under comparable conditions and methods (e.g., the
concentration of
the desired Klotho variant is increased with a concomitant decrease in side
products
expressed and/or purified). Preliminary results indicated that designed
recombinant fusion
proteins are being expressed and purified in suitable titer ranges and are
soluble in acceptable
buffers, carriers, and excipients.
[0487] Further cell lines were developed and assessed for density, viability
and production.
Data from the top 10 clones are illustrated in Figures 19-22. Data from the
top 4 clones are
illustrated in Figures 23-26. Based on fed batch ranking, the top two clones
appear to be 138
D9 and 145 G8. Based on pseudo-perfusion cumulative productiveity, 138 D9
appears to be
the top performer. 140 F6 reached the highest VCD. Copy number stability was
also assessed
as illustrated in Figure 27. Total RNA was extracted and RNA isolation was
performed using
RNeasy Mini Kit (Qiagen, catalog # 74104). Following RNA isolation,
amplification of
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templates was carried out using Omniscript RT Kit (Qiagen, catalog # 205111).
All cDNA
sequences were as expected.
[0488] Stability of five of the clones was further tested as illustrated in
Figures 28-32.
These clones were grown in growth media with and without glutamine, and shown
to have
higher growth rates in the presence of 4 mM glutamine, compared to the
glutamine-free
condition. No growth rate change was observed during >60 population doublings
in either
sets. Viable cell density and percent viability of clones over a 7 day time
course is illustrated
in Figure 30-31. Volumetric productivity of clones in 7 day fed batch cultures
of Time 0,
60PD in the presence (+) and in the absence (-) of 4 mM glutamine is
illustrated in Figure 32.
The volumetric productivity by the TO and PD60 no glutamine cells are
comparable. The
PD60 + glutamine volumetric productivities are either comparable or somewhat
lower, but
never lower than the ¨70% of the TO values.
[0489] Cells were adapted to 100% for EX-CELL, ActiPro, and CD OptiCHO media,
and
nearly 100% adapted to CD CHO, CD FortiCHO, BalanCD CHO Growth A, and Select
CD1000 media.
Example 3
[0490] Purified Klotho proteins were observed on gel following ELISA, as shown
in Figure
33. Table 49 presents an ELISA plate map key (upper two segments) and heat map-
labeled
ELISA 450nm data (lower two segments). Table 50 presents the ELISA data for
the Klotho
proteins from cell culture supernatants and human serum samples. Table 51
presents the
average concentration of Klotho in each sample group.
[0491] Table 52 presents an ELISA plate map key for human serum Klotho levels
before
(upper two segments) and after (lower two segments) recombinant Klotho protein
injection.
Table 53 presents heat map-labeled ELISA 450nm data for Klotho protein levels
in human
serum samples before (upper two segments) and after (lower two segments)
recombinant
Klotho protein injection as measured by ELISA. Human serum samples were
diluted 2-fold
and cell culture supernatants were diluted 5,000-fold using Klotho ELISA assay
sample
dilution buffer. Serum B1 and cell culture supernatants were included in both
ELISA plates
as controls. TMB 30 minutes. Table 54 presents a summary of average
concentration of
Klotho protein in the samples indicated.
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.. 1 . .2, . . 3. 4 5 5. .
4 = 1 =. I = =
A STD, 5 nerrall. Kkith0 n StikM'', 5 inwrr 1,. i
Mr,,,tbet.FC, 6 PierriL.
.11 STD, ::. nernt.. ..:ith:o ri.3:0w, 314-v'tni,
0.z<tbct. FC, 1 ngirni.
1
C = 'STD, :1..5ML Mot ho .l7C, 1.5
n.Fjml.
.D . = STO., 0,75 P.v/ 111, K lot hi:.),ii:iais,x.,. 075
tv,,,W.fl, KRA ho.. FC, 0.75
F 5;170., 0.:375 nglmL ......................................
)10tho...nat!ve, 0..:375 z-Wmt.. klf-Ahcp rf..õ. 0.3 IS ngityi,. 1
F STR, 0:71M g:ii:i.nL .. iMot.ho 0.:4.1,..,03, 0.,IM aerE:11..
Klutbo.F:(:õ. 0,1 =,%F.,
G STDõ Ø0q4 1,7,1ral.,.. . Mothr., natime, 0.094 .rq-frni..1 Ktath a. F
Cõ. 0.:C.04 inglmi.
,
14. F7.),. .c. ngfra. Krok4o r!atkoa, 0 pg/tOt 1
.10otho-FC, 0
,
;.3 = 9 10 11 12 .. .=
Moto -3W, ,6 rlerrit. KLA-645 .fi:i õow) KLA-4347 (L.1,000),
- ;=
Motho takziV, 3 NOT.I.L. .:LA-f4 5 (1aY)) KLA-Ã47 f1.10,000)
lactho GW.1õ.5 :'i:g.,,WL _ MA-645 (1:10), )00) .g..U\--64.1 (1:100,X0)
60,,= 0,75 ish-nL KLA-6.45 U''0 11 (1:.1.,000.0O
Ktotho V.. (1.175 nerni_ Sf....turr:; 1 A
Sw.,:rt.la.;.. 11A
Ktot ho GW, O. '1.88 nelmi, Seni:m. 1A i L '2 .. 5e ru m
laa. (1'.2) = .
k^ ......................................... T ...............
KtOth0 ti:K. 0.094 rierni S.,7-.1-um lA(I : 4) Ser um 13A11A):
.
Motto tr.Li:.A:', 0 nerrit= SerumIA:(1:8) Se rtg rri 1.3A (IS)
t.., _________________________________________________________ .,
1 1 . A. 4 ..b= 6. . .
' A r3 .138 3,102. 007:3 0,075 0,06s5 0;075 .
,
1.7.17 1351 0 .0(it-3. 0.012 . 0..07..7 .0
.07. ..
C. 0.963 0.952 0:011 0,044 0,)19
Di 0.54 0_532 0:073 0,07 0:064 0..071 .
d
E: a. 3C4.3 Ø..7119 0.08.3: ..
01145 0..074 . :Li:1.74 .
F: 0.201 0.14 0..074 0..074. . 0.075 0,068
G . 0143 . .. 0133 . 0..071 0:058 0..0a5 cL084
H 0.071 0.075 . 0:078 = 0,075 0,071 0.068 .
7. .8 P, 10. 11 12
3.253 - 0,g7 Ø.90 15.21 1383 ''=
'-..õ 7,17 1 271 ',i7 tRcy co R3 ,c.., 548
r1:574
1..,.....õõ:Sa:.L'aZ-- ,,,,,, - ' '. '.- 4 v. ...:
1 1 . Mb 1..92 0.08.8 0.1)9 0.. 1..:. 0. 144 .
,..!
1.,&-.6 1128 0,084 0,08.9 0,041
.
1 e.t.,596 0;615 0.197 0405 0,449 0.122
0,2 -:?.5 0,. 79 0. 301.
,
G. 2 16 a.207 0167 0.11:1 0. :t.4. '.) 195
..
0,071 Ø081 013 0,13. .0,142. 0..143.
Table 49
169
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r
k k
*.k1k3k7i
ilW:.i. '?, V::k.An7; R: Re.,.1:A M&=.4.5t..,:,.;t1
.,%..t.E)t. 1 C.':'4%. t4i4õtit.:41 1,Adi-,.:4&.;.:i.t. .
. ...:
;',Is=',1:
i i = t
.õ4" -7..-n 7.775 :
. 1
,
,
.'.'.3:7 1 1.124.7 g 6,11. 6,355 41542. i 57
2 12,FS .`
,
i....................,............. ,
''<µ. 1 I,S5 12% : ..,:-
4.,\.4v::::::....g ::::=.:.:".o.o.* &
:::::=::=:r.:õL'...$:.:.:::;:1',....='=::=::=.:7''t. .1.:4:,10..:.,
..4...s.w
=.c,.., 1 1,:...ii,. .=.:::,1n,
:::::::::::,:,........,........::::::::::::::::::::::::::::::::::: :::::
7 1 1:4NS :::::54,..1:1 11...764.:::::::. ::::::: ,o,t.4.-
.1., ,;...:=.......4..:,õ:,:,:,,,..,:,:,:,:,:,:.::,4.....,::: 144,o::
:,:,:,:,:,:,:,:,:,:,:,:,:,:,:
, ::::: ::::.: ,
....................õ............................
..........================
t:IIS
::: . . ....... ,
....................................
...... 1 ,.=,,144.S. ..4%....t,t1,..::
z..........;..........;....= .................:::::::',..4:H:
H..........',...',...........Q.;...;..........m..... :::,..aHH0 HHHHHHH
,:,:, 7,if.r'--.- ::...:,...:,.......;:.,................................
::7:: :...::. ::H,,: .::........;......,...:..............................
:,:
.:F7 1 4.134, :=,5...Q.4., ,54,1$:..g......::: 5ikaa1:: ;
08::::::::::?:::::.,...1.:1.:,..:,..:,=U ISM: :H:::::::::::
::::::::, ... = .:.,.......:.:.:.....:.: ::::: ,
. , ..:......,...:::::::::::::: ..:,:,:::::. 7:
41. 1 5,5X H.....C.g%i* .0:"::.:.=:..::::::::::::..:.i:40I
1 A.4i,::::.õ..:::::: : 114 15.:5*.i.: M::::H
:g ...:
3Sf7 i akm
,
, __
1; ::....W..4tV:
, ... ...... . ,
.........,........... . : ...:
:4='.10 1 0.56' HH...11-=.40 :::::::::::::.:...:
.......:::::::::::.:...:::::::::: ;
::::::::..........::.:...::.::.:...:...:...::.:::...:...:...:...:...:...:...:::
:.::.::.::.::::.::::::
1 ..4P,i ::::::ica::;'4'.: *4':7::::::::::::2:::.4"Pkv.
1 H::=:......=:..',...:::,t..."..i:,...õ.....õ:::õR :: IMIV: t7.4,a
:H:::n:
. .... . ....
, 5 Ci
..-VO ..0itii.:, HH:,ta .......... :: =: : =
=.;.11 i= o.kw,-, C.0'7. 04,V5 . 0.KKV
1 1,.Z..2 .:1X41(1 1 Ug.:=AI41,1. .
k
k ,
k ,
:i:n 1.," 0E.M4 545 5A.:1\4. 1
i'4505 1 -M:.:5 5:41111ta 1 143%. TZ.. .
,
. . ,
5.15 ; 5A1.15 i.15.1Z., 1 .
= j - ....... - ............. . . -
4 - I
A. 1,,: :...:2:. g ' ==*,?.V. '''.:&t,4 11: 01.14 ;
.1.fi ,14XV i MtV=':41
,
:Ok:0,
.....51.1. ; =.:I%:15,
H.......tt. ; .a...s-,4
4 ...,.µ,77.Z
..441.6
.41n
0,:i56
attli
012. 1 aagg 41.M'.5. 1 Ø,73j.C..:::M:HH *tat 35' a:...: H::
1,0...11)- ..s,µ=X,9t.S .:5.:.1.114.
. ........... . . . ,
... ... .. . . . . .. . . .
...........
::::::::::.............::.:. :
:
tIttg> ; 16.1. 3541.1.X4.1 1 277N37
, .
:
:
:
:
:
:
,
,
.. .. 1 ...4 I ant. :::...ct.'*.,..s.::
OVit.:Miii:... I: 0:00.7 1 %H....!....!,!...A.4@in
.............i....i.............i....i.....ZH Ø..W HHH
k . : .. ,........ :,,,::
::HHH.,................::::::::::... :...................................mm
fat. ; :WM.. :.=*IirtI ........... ,
................................................
................. ,
................................................ ,
................. ,
................................................ ,
, ::: : :::::::. -...=== = ===============
==================
::=:4,!.,.. 1 ..0:47::.:: :::::.: 114.i
k -
'@: Iz" Q.1 0.M.k.= ama 1 a a 5.Kg :
. ,
4:143 k 0 :13 x k . t7iiM
k =
=
=
. .
<14,=<,,,F55,5=5=5,5=5=Fvv...t.
Is ::::::::::...:::::::: :::::::::::: =.t.
: eiml ,:::::,::4,7... ;
.
fti.'.. ; =0:,11 5,N, ::::::::?:::::::::::=:::::::: :
; " ::::::::::;,.....:.:.:.:.:.:.:.,.:.......,... :
::::::::::::: ::::::::::::::::: ,
: . . H.....:::::::::::.....M :
m:::=:::i...:::=...:?:....:?:....:....:?:....:....:?:....:?:::: gHHH H
;
. '411:: 0...i.tU t:11 alt:N:.....i. :H: itsf.4.
...:tij..::.:H:T... :.=...1.:.:...:.i.:.i1.4:' 4A,ii..*:
HHH:::::::::::::
:::::: ::: , """" """"""" =
,
- = ...
.;`,.4...M ::: === : 0:4":',H 0
s
...4,,.................õ...... ...... k,..........:õ.,.....:, ..
......?,..y. .. -4
Table 50
170
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,
[Klot.hcb
Samples
n giniL
, ,
Kotho trona en Way 13.58
,
Supernatant, KLA-645 I59446
Supernatant. ?::: LA -647 8700,99
Human serum, E:ase, IA 0.56
. , .
Human serum, Prmefr), 13A .
0:71
Table 51
A. 1 :
* 3
. ::: '
4.- 5 6:
----
A sq-D, 6 Ninit. IIIIIIIEIIIIIIIIIIIIIMIIIIII
a IIIISIBZIZIIIMIIINIIIIIIZMIIIIIMIIIIIIIIZIIIIIIIII
C ..................... :51Iii., 1.5 ng,:fillt. 11A .12A
...,,,,,................,,,,,,A,:,,,,,,........,,,,,
,,,,,....,,,,,,......................,,,,,,,,,,,,,,,
,,,,,......,,,,,,,,,,,.........................,,,,,
11k:f545 awg.) itaA-645
,..,.,....,___,.,..,..õ...,. " ,..._,...
n :-q .r.3...::: .........41,...i
k.,,,, :v. ::,õ IIIIIIIIIIIIIIIIIIIIIIIIIIIIII
,,,*
:.--
k ciTtl g,aq; no ?mi,.
H INMEMOMINIUM111111111111111111111111111
y g 9 ID 1.I .;..,=:. ,
m .,......: x...
;=-i.A ,
,
. i
SA ...=. IN.
v.:
KLA-.64:7 1140):
= i ------ " L.
; ,=
, ,
$ ,
,
, ,=
4
1 -4
... $
\.=
Ai 57,..), :=',3 :;,V)T-4, 1 LA :ZA
8,I, T. 3 :nkitr4L :6A :7A
1
CI STD: LS l
El _____________________ STD. 0 375 ng,h-a qi: 4:
.................................... ,...,.,..,,,s .,...z...,,,
41',
.,.. . .,..:.:4:::,z twn., :`,.itip, .y=-. Z.
GI ..STr '.,. tY-M realt. XLA--.645 fDaY.4.1
KL.M.4.540aVID)
c.-----...-:-.-- =
;. :
it3L.SED, IL) :rTimL_ ,
õõ.
..... ¨........ _____
.4...=
,....,,
&A SA
. = = . .
&A &A MA
:
kw.;,..
= -
,-,,,,, 13A ,=
,=
,,,,,,..4. =
= l'
MA ::::.3 ¨
. _i_
=
ZIA :MA ,,ti.-4
MA . -, .. -= A
:: .. =.:,\-, 61 ---
ia,4.4547:K.4'1.) gLA-K1 (Dir-i4
.q
Table 52
171
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I 2. 3 4 5 fi
Arliiii.7 77"771:1. ..37Miiiiiiii 03 . 0290 a.2:92 ases-
0 2 149 2 343 = 0230 0292 ] 0...%.,:\ 023t5
CI 1184 1.193 argairms k3.312 0.324
Di 0..060 0.643 0343 O3:35 am oiN :
El 0.32 030
Fi 024. OIEEMIIIIIIIIIIIIIIIIIINIIIIIIIIIMIIIIIIII
Gi 01.163 01% IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
U 12
7 A 9 10 ,
¨ _____ . . ...._...... .
0_302 0.319 * 0.311 =1.V1
la,...A.:* o...311 -03.31
0A11 11130MINEEM 0.341 . 3354 0363 .
111133111111=1111 034 11113211111111111111111111111111
REM -0,685. INEE1111610111111111111111111111111:
IIINIIIMNIIIIIMIMIIIIIIIIMIIIIIIIIIIIIIIMIIIIIIII
IIIIIIIIIIIII 1111
, õõõõõõ ZIIIEIIIIMIIMIIIIIIN
..2 4 5 6
A p3.52.2. 1 3A28 :=1 03:,12 0.337 0323 . 0.342
Et 2.%3 2.t*6 3.313 334 0303 0..3D.
t-- , .
C.' 71.1.c3 1125 l a 92 a .302 0.288 0_276
i
rs 0.i&68 4 0.603 i ..... 0174 0.265 0235 0186
E 0364 02,W,1 1.1314 9.315 0301 6305
, ,
F 0224 0.21C .0232 0.292. 0.2kri 0.285
_ ..... õ .. ... ....
6 .0148 0444 . 6924: 0.339 . 6276 0.272. i
11 22 ..
1 0321 632.1 0316 0.183 0341
;
a316 0,286 0369 a9e1 0334
, (XVY2 0304 0.:29 0299 0233 0115. ,
0,1Vi 0301 0.30 ts'.315- 0136 0:32
.. . . - - - - i
0.234 0231 019 0.2q4 I 0205 011
- . . ... .
029 0.314 . 004 0,3()3 0.323 -- 0.31
.. .... . .
TM 0.1Z2 1069 1112
..
Table 53
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i Svro e4.:. i
giottvt.
rigo.mik. aft?.r Nor':.
24 /-)m-,=/ /
,
v k; =:, e ,
= :
4A =:>..592 .
v. ,....- 4.
SS I i &A
..k
' .
6.:?..1 ...
,
&A 0..7K 11 :Uk 1 i..5=47 ...
- __________________________________ - - i.= __ 1' __ ¨
..'. 717
.. _________________________________ i: _____
12.4 0-.5M I2.A I
,
1 .. 4 0.'+:*". __ 7 IAA
i
rA ; 1..%7 ...: ISA
Sz'Us.-6, it) I':-
KLAW. EA S2.W. = 17A
.....
4:7 'V4
:
:
:
: .21A.
: k
:
:
:
: ,
:
:
:
: -.1'!',.= a ,all
:
:
,
: ,.......................õ........... .....,.õ--
: 27J :
:
:
:
: = '+''''''''77`rs's;
,
:
:
:
=1 0.673 1
..k
: 11 KLA-64.S. i 17n,
,
:
:
: 11 A.'. 0
,
:
: i: :c=LA - 647; DR 5a.12:3 :
___________________________________ fl
Table 54
[0492] Table 55 presents the concentration and monoclonality of the replicate
Klotho
protein samples.
Clone ID Gel Conc. (mg/L) Conc. (mg/L) Monoclonality
Lane Method 1 Method 2
1 KL0-145-G8 B8 127.2 154.0 Likely
2 KLO-138-138 A7 108.9 79.4 yes
3 KL0-138-D9 D5 106.54 133.9 yes
4 KL0-139-C6 Al2 102.0 64.6 yes
KL0-138-D9 D4 98.33 138.1 yes
6 KL0-145-E11 B3 94.1 62.3 yes
7 KL0-140-E8 A4 91.4 55.5 yes
173
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8 KL0-137-G6 Al 88.7 50.9 yes
9 KL0-139-G5 A5 83.9 53.7 yes
KL0-137-D8 A6 83.4 51.2 yes
11 KL0-139-G17 A9 81.7 49.1 yes
12 KL0-140-D6 B6 76.88 63.2 yes
13 KL0-138-E2 A3 76.8 33.0 yes
14 KL0-140-G3 All 74.4 44.9 yes
KL0-138-E11 B5 73.04 53.2 questionable
16 KL0-143-G6 B2 70.6 40.5 yes
17 KL0-140-G4 A2 68.2 36.2 no
18 KL0-137-D5 A10 66.7 38.7 yes
19 KL0-137-D6 A8 66.1 36.8 yes
KL0-144-C4 B4 60.5 30.3 very likely
21 KL0-138-G8 B7 58.11 40.1 no
22 KL0-139-D11 C5 49.99 40.4 questionable
23 KL0-139-F11 C3 49.37 41.4 yes
24 KL0-138-G2 C4 47.5 37.6 Unknown
KLO-645-146-132 C8 47.39 34.4 Unknown
26 KL0-138-G4 Cl 35.59 24.3 Unknown
27 KLO-138-1310 B10 35.27 38.8 Unknown
28 KL0-144-G6 B1 34.8 20.4 Unknown
29 KLO-139-1311 B12 34.66 32.7 Unknown
KL0-140-F6 B11 34.53 34.6 Unknown
31 KL0-645-143-D7 C7 30.28 15.7 Unknown
32 KL0-645-143-F9 D1 28.04 22.1 Unknown
33 KL0-645-168-A8 C10 26.92 16.3 Unknown
34 KL0-645-142-G2 B9 22.71 18.8 Unknown
KL0-645-169-D7 D3 21.08 17.1 Unknown
36 KL0-645-168-G12 C12 18.46 11.7 Unknown
37 KLO-645-167-1310 C9 8.49 0.0 Unknown
38 KL0-645-146-C10 D2 7.71 17.4 Unknown
39 KL0-645-168-F10 C11 0 43.0 Unknown
Table 55
[0493] An illustrative method for estimation of concentration involved
quantitation
(quantification) by PAGE against a BSA standard. An illustrative method for
estimation of
concentration involved measurement against purified Klotho standard.
5 [0494] One interpretation of the data could be that native-like,
recombinant (TwinStrep-
TEV tag-removed) Klotho protein expresses better, purifies cleaner, and/or
retains more
activity than does the recombinant Fc-tagged Klotho protein. This was
surprising and
unexpected. An alternative interpretation is that neither protein, or the Fc-
fusion protein more
so than the native-like protein, showed levels of klotho protein reaction to
the ELISA
1() antibody. Accordingly, we may have identified that expression,
purification, storage, and/or
administration of native-like recombinant Klotho protein and Fc-fusion Klotho
protein was
not as simple as expected and that standard techniques did not work well.
Proceeding based
174
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on the later interpretation, procedures were modified to obtain more active
protein, while
continuing to process the data for further interpretation.
[0495] Three commercially-available ELISA detection kits for Klotho protein
were also
tested for suitability in accordance with certain embodiments of the present
disclosure. See
Tables 57 and 58, below. The IBL Kits shows low background, liner dilution of
serum
samples, acceptable spike recovery after 1:4 dilution. The Cloud-clone Kits
shows high
background, no liner dilution of serum samples, no acceptable spike recovery.
The EIAab
Kits shows low background, very low Klotho concentration in serum sample, no
liner
dilution of serum samples, no acceptable spike recovery.
[0496] Total 8 human serum samples were diluted with each kit's dilution
buffer at 1:2,
1:4, 1:8. 4 of these 8 serum samples were spiked with top concentration of
Standard from
each kit at 1:20 dilution. The protocol from each kit was followed per
instructions provided.
[0497] Figures 34A-34C illustrate Klotho protein concentration measured and
spike
recovery observed in the respective samples.
õ\\
mumumum03-I6Ø6.01:5(g,t.ititemmmom
.c:jouti:40.600 1.;"=]6.4000 pg/ffiti
monomom::::7-A45.6.6-p.g.irriivommmmom
------------------------------------------------- --------------------------
-------------------------------------------------------------------------------
-
Table 57
. = . , '
= , = = = ..S.; =
= z-ke \
BL 300
Ø):013d4Cier.o.*
EMb
...............................................................................
...............................................................................
........................................................
...............................................................................
...............................................................................
........................................................,
Table 58
175
Example 4
[0498] Preliminary global glycan analysis was also performed as illustrated in
Figures 35A-35B. Experimental conditions and results for the 0
glycan analysis are presented in Table 59.
= N-Glycans were released and labeled using the GIyXTM N-Glyean rapid
release and labeling with
I nstant PC`m Kit.
= LC analysis of 1 uL of sample was performed on a Advance Giycan Map
2.1x100 mm column.
= To gain additional insight into the core glycan structures, the sialic
add content of the sample was
released using Sialidase A
Experimental conditions
= Each peak was assigned a GU unit that was referenced to a in-house
database generated from Glycan
Standards.
= Area under the peak was calculated for peaks with retention times from 43
- 25 mins and a minimum
peak height of 0.2 and area of 10 or greater,
= Percentage area of individual peaks was also calculated.
= Only the glycaris GOF, MS, Gl, G1F, G2F, G2 and M7 were assigned with
confidence.
= Both samples were treated with Sialidase A to try and understand ail of
the core structures in the
Results sample. This approached provided no further
insight into the unknown peaks,
= The sample appears to contain several glycans that are not represented by
the glycan standards that
are commercially available. Further characterization either by exoglycosidase
treatment or by LC/MS is
recommended.
cio
Table 59
[0499] Tables 60 and 61, below present the data illustrated in Figures 35A-
35B, respectively.
õ-----------,õõõõõ,õõõ:õõ=,õõõõõõõõ
õõõõõõõõõõõõõõ,.......,
...............................................................................
.........................,..............õ...õ....õ..õ,,õ,.,õõ..,.,.,.,.õ
MEMENWPMENEMEMEW MM MFEIMM30140 wwwwwwwww minimagnimmim
mi4eitott ,'.',i,,õ: WOE 0
EmiktittiKOMMinatatittAt4iM MagniiiiiimigmAigg gfflatiiiikKOME isigfetailowagg
Egi:kiia:::.k.miwi.:::ixi::6i.:: aini ,..,
=
w
-4
GOF 5.18 5,38
GOF
2.68 1.91
M5 5.46 4.18
EV15
1,62 1.45
GlF 6.46 530
G1
2.98 2.68
G2 4.54 931
GlF
2.11 233
G2F 13.36 36.23
G2
3.87 7,33
* G2S1 a2-3 4.32
* G2FS1 a 2-3 18.57 5,84 *
G1S1 a 2-3 4.06 3.947 .. P
* G2S2 4.03 *
G1F51 a 2-3 8.50 8.85
,
,
--.1 * G2FS2 a 2-3 17.66 Man7
17.96 16.97 .
--.1
rõ
Unknown 20A3 33.79
Unknown 55,21 52.86 rõc'
,
,
,
Table 60
Table 61 ,
,
[0500] An additional pool of 9 clones (see Table 62, below) was assessed for
suitability.
,-o
n
,-i
cp
w
o
re ,
-a-,
.6.
w
w
w
Plant
Comstruct
0
:Du B-1+ X_WtS P_Se Sc4
..6i=Vz3P._10ot:403 EC.D1: 1-981
WE '4-
.tSP 2 1 - S 49 . . _
=
X.C.= LutiSP
50ASPLKI0V-to ..1.- 981 ).- Pc VAST
_
.
MNM:ggM:g:MggE:gMgg:gggM:MgggMM.
XE PLEO_BT
/.3,1bSp_._secretedi Iscs,torm2 (17549)-Pc.
rj
oe
Table 62
[0501] Growth, viability, and titer of each cell line is presented Table 63,
below.
oe
day 3 day
7.: Oft. 9,
cen cic vhy oubj:
Viatiii'ity rite:ft ctc ThtTitet 0
011 iL
V.44B 6,57E+N 9.9.3 16.2 0 2.18E+0?
H5...45õ0: 2:63E+97 85.2
.................,
0.4ME.4iNin
WE 6.03E+06 99.8 16,1 20 1..0E+07'
,.15.8 70 I .09E+i07 VA
gggg4ggg
MgMilieMEingl$4*Em
xe ,o5.E44.:01, 99.0 17.8 1 85E+07'
97.6.. 9.58E+ 01 .9fl
p
XE 8.29E406 99,,1 150 13 2..35EtØ7
.g4.6 49 1.37E+97 89.2 32
M2.5.1E*07i:::UMMZEM
U424ENIENNEOVIMMINNiIIMiiS
Table 63
[0502] Figures 36A-36C illustrate growth, viability, and titer curves,
respectively, for each cell line.
[0503] An additional pool of 3 clones (see Table 64, below) was assessed for
suitability.
. .
Plasmid Comstruct
.
........................... ..................................... . .
....... . . ....... . . . . . ... . . . . . . ............... . .
......... . . . ..... . . ..............
.gMMMM.4ti..t'iisgiST4.g.fik.)t4:419iLt3cttSPU.4a:i::iStiMMMM
. . . . . . . . . . . ..... . . . .... . . ....
. . . ..... . . . . .. . .
33;_wn PL5eq6.__Sir.
iiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiii
iiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiii
Table 64
[0504] Growth, viability, and production of each cell line is presented Table
65, below.
day 3 -
d,Ity 7 day .9
.K=Oric viability doubling Titer
teH Ceg CO.EaC:bity Titer
jc.4tiLl ti e ittg:itriLl
ivtai 1'41 Rtg.mL.1
5E+06 9aS :124 132E+07 555 87,2
:MMRMRMWMRMMM
c,.)w
Table 65
[0505] Figures 37A-37C illustrate growth, viability, and titer curves,
respectively, for each cell line.
[0506] One of the promising clones (see Table 66, below) was re-assessed for
growth, viability, and production.
day day 7
day IQ
be-4i bartt daiiNing time Titer cleft
Tiler cbric Titer
terti:q
..f%1
=
5.
_______________________________________________________________________________
____________________________________
Table 66
[0507] Figures 38A-38C illustrate growth, viability, and titer curves,
respectively, for the cell line.
oe'"
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Example 5
[0508] Western blot analysis was performed on expressed protein pools as
illustrated in
Figures 39A-39B. Figure 39A illustrates Klotho ECD (34-981) 6x His Protein,
and Figure 39B
illustrates the indicated Klotho protein. Unexpectedly, Klotho proteins appear
to aggregate into
higher molecular weight multimers.
[0509] A 5L bioreactor expression run was carried out, followed by adapter
purification
protocols. In a first purification step, Protein A Sepharose 4 Fast Flow (0.75
ml) (0.5 cm x 3 cm)
column was equilibrated in 10 mM Tris-HC1, pH 8Ø Protein sample (100 ml of
Cell Supernatant
adjusted to pH 8.0, with 1.5 M Tris-HC1, pH 8.0 (¨ 1 m1)) was loaded at a flow
rate of 0.75
ml/min, washed with 10 mM Tris/Arginine-HC1, pH 8.0 buffer, and eluted with 4
M MgCl2, pH
3 (i.e., low pH, high salt) at 0.50-0.25 ml/min to produce a concentrated
sample. See Figure 40A.
The column was re-equilibrated with 10 mM Tris-HC1, pH 8.0 (to prevent salt
precipitate), and
sanitized with 0.1 M NaOH: 0.5-0.3 ml/ml to avoid over pressurization. Minimal
Klotho protein
was eluted during re-equilibration and sanitation.
[0510] Peak #1 and Peak #2 from the Protein A elution were analyzed
separately, buffer
exchanged, and fractionated using Superdex 200 (1 cm x 30 cm) size exclusion
chromatography
column with 100 mM Tris-HC1/5 mM L-Methionine/0.6% Sodium Thioglycolate, pH
8.0 mobile
phase at 0.5 mL/min flow rate. See Figure 40B. Tris was used to buffer the
mobile phase instead
of a phosphate-containing buffer to prevent the precipitation of magnesium
salts. Reducing agent
(L-methionine) was used to prevent the oxidation of Klotho-Fc. Sodium
Thioglycolate was used
to prevent the scrambling of free sulfhydryl cysteine groups of Klotho-Fc.
Buffer components
are GRAS listed by the FDA.
[0511] Superdex 200 fractions from single isolated Peak #1 and Peak #2 each
show different
MW species of klotho-Fc protein. See Figure 41A and 41B. In summary, analysis
of harvested
proteins (in media) by SDS-PAGE, suggests that Klotho-Fc exists in various
states of
aggregation. The purification produced two populations of multimeric Klotho-Fc
corresponding
to a dimer (< 300 kDa) and a tetramer ( < 600 kDa).The binding/elution
protocol from Protein A
shows far superior product elution profile and produces minimal protein after
sanitation. As the
formulation has thioglycolate as a reducing agent, the product separates as
monomer after
heating, and monomer and dimer without heating in gel electrophoresis. These
new purification
procedures resulted in non-aggregated Fc-klotho protein.
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Example 6
[0512] A bioassay was developed to test the activity of both native and Fc-
Klotho proteins, and
particularly to test the binding affinity of Klotho proteins to FGF 23 using
BLITZ by ForteBio.
Initially, the binding affinity of Recombinant Mouse Klotho to Mouse FGF-23
using Surface
Plasmon Resonance (SPR) binding methods was assessed. SPR is an optical
technique utilized
for detecting molecular interactions between an analyte, a mobile molecule,
and a ligand, an
immobilized molecule bound to the sensor, which upon interaction change the
refractive index of
the sensor. In this study, the Inventors compared the affinity of six
concentrations of
Recombinant Mouse Klotho (2-fold serial dilutions from 400 ug/mL to 12.5
ug/mL) antibody to
Mouse FGF-23. Using anti-HIS (HI52) sensors, Mouse FGF-23 is bound to the
sensors and then
subsequently these sensors are used to produce binding curves for six
different concentrations of
the Mouse Klotho. From the six association and disassociation curves produced
for the antibody,
the disassociation constant (Kd) value of the antibody is determined. Assays
were performed and
data verified (data not shown).
[0513] For human recombinant Klotho protein, HIS-Tag biosensor tips were
hydrated in PBS
for 10 minutes at RT. HIS-Tag labeled FGF23 (ProSpec, Cat#CYT-374) at 300ug/m1
in PBS was
coupled to biosensor for 2 minutes. Base line was established for 30 seconds
on BLITZ
instrument. Then, the binding of native and Fc-sKlotho proteins at five two-
fold serial dilutions
(from 400ug/m1 to 12.5 ug/ml) to immobilized FGF23 was tested for 2 minutes in
PBD, and
subsequently dissociated for 2 minutes in PBS. The Km (association), Koff
(dissociation), and KD
(Koff/Kon) was analyzed using global analysis. See Figure 42.
[0514] Further assays were performed to test the bioactivity of Klotho
proteins using cell based
functional assay, and particularly to test the bioactivity of both native and
Fc-Klotho proteins by
measuring their effect on stimulating NIH/3T3 cell proliferation. Based on
biology of Klotho, the
rationale of this assay development is to test the effect of Klotho on
stimulating the proliferation
of NIH-3T3 cells that expressed FGFR.
[0515] Growing phase cells were seeded in culture media (10%FBS-DMEM) at a
density of
5000 cells/well/96-well plate, and cultured overnight, followed by a media
change to 0.5% FBS
and overnight culture. Cells were treated with 10 doses of respective Klotho
protein starting at 30
ug/ml, 2-fold serial dilution, each dose in triplicate, and incubated for 48
hours. Cell proliferation
was measured using Cell Titer-Glu assay kit, and data was analyzed using 4
parameter-fit curve
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with GraphPad Prism. The data indicate that purified Klotho proteins, both
Native-His and Fc-
Klotho, stimulated cell proliferation in a dose-dependent manner. See Figures
43A-43E.
Example 7
[0516] A study to determine the Acute Tolerated Dose (ATD) of Alpha Human
Native Klotho
and Alpha Human Fc-Fusion Klotho proteins following intravenous administration
in male
Sprague-Dawley rats was conducted. In the acute tolerated dose study, the
safety of the test
articles Alpha Human Native Klotho and Alpha Human Fc-Fusion Klotho proteins
was assessed
in male Sprague-Dawley rats. Prior to dose administration, 21 animals were
subcutaneously
implanted with transponders. On Study Day -1, animals were randomized into
seven groups of
three, based on body weights. Alpha Human Native Klotho protein was tested at
three dose
concentrations of 10 [tg/kg, 30 [tg/kg, and 100 [tg/kg (based on individual
body weight)
administered intravenously at a dose volume of 1 ml/kg (based on individual
body weight) in
vehicle (PBS, pH 7.2) on Study Day 0.
[0517] Alpha Human Fc-Fusion Klotho protein was also tested at three dose
concentrations of
3 [tg/kg, 10 [tg/kg and 30 [tg/kg (based on individual body weight)
administered intravenously at
a dose volume of 1 ml/kg (based on individual body weight) in vehicle (PBS, pH
7.2) on Study
Day 0. Dosing began with a vehicle-only (PBS, pH 7.2) Group, 10 [tg/kg Alpha
Human Native
Klotho protein Group, and 3 [tg/kg Alpha Human Fc-Fusion Klotho protein Group
on Study Day
0 and were staggered by at least 24 hours between each test article dose level
group. All dosing
was given once on the respective start date. Animals were observed for any
adverse effects for
maximum of 14 days following treatment.
[0518] In-life measurements included twice-daily checks for morbidity and
mortality, and
clinical observations were noted daily. Body weight measurements were recorded
daily.
Following completion of the observation phase, all animals were euthanized via
cardiac
exsanguination. Terminal measurements included blood hematology, serum
biochemistry and
coagulation. Gross pathology changes were recorded for all animals at necropsy
and weights of
ten organs (adrenals, brain, epididymides, heart, kidneys, liver, spleen,
testes, thyroid/parathyroid
and thymus) were taken.
[0519] Table 67 presents a summary of study animals.
Species/Strain/Sex Age Range Total Number Number of Animals Number
of
of Animals per Group Groups
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Rat/Sprague Dawley/M 8 weeks 21 3 7
Table 67
[0520] Table 68 presents a summary of study design.
Group Test Article Dose Level lig/kg
1 PBS, pH 7.2
2 Alpha Human Native Klotho 10
3 Alpha Human Native Klotho 30
4 Alpha Human Native Klotho 100
Alpha Human Fc- Fusion Klotho 3
6 Alpha Human Fc- Fusion Klotho 10
7 Alpha Human Fc- Fusion Klotho 30
Table 68
[0521] For Group Assignment: Matched Pair Distribution, an optimized
distribution method
5 using an algorithm that matches the individual measurement values with
the mean of all selected
animals was performed. The method begins by taking matching pairs that average
close to the
mean of all selected animals and then distributes them into groups that will
then match the
average (or as close as possible) of the mean of all selected animals. Animals
are distributed so
that the average of the measurement for each group will be as close to the
mean of all selected
animals as possible.
[0522] For Statistical Analyses, descriptive statistics were generated from
Study data. Data
was assessed to determine whether parametric or non- parametric analysis was
appropriate. For
parametric data, analysis of variance (ANOVA) followed by post-hoc tests was
performed to
determine significant differences between treatments, time points, and/or
groups. For non-
parametric data, appropriate statistical analyses were performed (e.g., Kaplan-
Meier Survival,
Kruskal-Wallis One-way ANOVA, Mann- Whitney or Wilcoxon Rank Sum, etc.).
[0523] Table 69 summarizes sample collection procedure.
Task Method & Collection Sample
Info/Special
Instructions
Blood for clinical biochemistry collected into non-heparinized stored at
-80 C and
tubes for serum preparation shipped on dry ice
Blood for coagulation analyses collected into sodium citrate stored at -
80 C and
tubes shipped on dry ice
Whole blood for CBC count collected into K2EDTA tubes stored at 2-8
C and
hematology analysis shipped
Table 69
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[0524] Table 70 summarizes sample collection tasks.
Task Frequency
Body weight Daily
Adrenals, brain, epididymides, heart, kidneys, liver, spleen, testes, At
termination
thyroid/parathyroid and thymus to be excised and weighted
Clinical Observation Daily
Morbidity Check 2x daily
Mortality Observation 2x daily
Table 70
[0525] Any animal whose body weight dropped below 85% of initial body weight,
or displays
severe, prolonged adverse clinical signs was to be euthanized. Animals were to
be euthanized via
cardiac bleed for collection of plasma, serum and whole blood as indicated
above.
[0526] Preliminary results indicated that the rats tolerate relatively high
doses of recombinant
Klotho proteins administered thereto.
Example 8
[0527] A study to determine Pharmacokinetics of Alpha Human Native Klotho and
Alpha
Human Fc-Fusion Klotho Proteins Following IV Dosing in Male Sprague Dawley
Rats was
conducted. Prior to Study Day 0, animals were subcutaneously implanted with
transponders. On
Study Day -1 body weights were acquired and animals were randomized based on
body weight.
On Study Day 0, animals in Groups 5, 6, and 7 (10 animals per group) received
a single dose of
Alpha Human Fc-Fusion Klotho protein intravenously via the tail vein (see
detailed dosing
schedule, below) at 3, 10, and 30 pig/kg in 1 ml/kg vehicle (PBS, pH 7.2),
respectively. On Study
Day 1, animals in Groups 2, 3 and 4 (10 animals per group) received a single
dose of Alpha
Human Native Klotho protein intravenously via the tail vein (see detailed
dosing schedule,
below) at 10, 30 and 100 rig/kg protein in 1 ml/kg vehicle (PBS, pH 7.2),
respectively. Animals
in the vehicle Group 1 (3 animals) was also dosed on Study Day 1 at 1 ml/kg
vehicle (PBS, pH
7.2) without protein. The volume of dosing solution administered to each
animal was calculated
and adjusted based on individual body weight measured acquired on Study Day -
1.
[0528] Three blood samples were obtained from each group (2-7) for all time
points, equating
to three blood samples per rat (see detailed blood collection schedule,
below). Time-point blood
samples were obtained pre-dose, 3 min, 10 min, 20 min, 30 min, 1 hr, 2 hr, 4
hr, 8 hr and 24 hr
post-dose (Groups 2, 3 and 4), as presented in Table 71, below.
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___________________________________________________________________________ :
R GrOUpS 2.3 and 4 :
at LD
0 min 3 min ICI min 20 min 30 min 1 lir 21n 4 hr 8
Isr .. 2,1 hr
I X , X
¨
2 X X X
3 X X ' X i
, 4 , X X X ,
. ¨ .
X X
6 X X X
I X X X
, 8 X , X X
9 , X X v X ,
0 ..
Table 71
[0529] Time-point blood samples were obtained pre-dose, 3 min, 1 hr, 4 hr, 8
hr, 24 hr, 48 hr,
72 hr, 96 hr and 7 days post-dose (Groups 5, 6, and 7), as presented in Table
72, below. An
5 additional blood collection at 14 days post dosing was obtained in Groups
5, 6, and 7.
Groups 5, 6 and I .
_ ,
Rai D 14
day (to he
0 mitt 3 mim lhr 4hr 8hr 24hr 48 hr 72 hr % lir ?day
determined)
a
1_ I X X X
_ 4 .4 A 4 A
2 X X X
3 , X X X X
. .
4 X X X
5 X X X
, ,
6 , X X X X
_
7
,
8 , X X X
4 , a ... A
9 X X X
_ - . ,
, X X X X
a
Table 72
[0530] Vehicle Group 1 animals were bled terminally once, 1 hr post dose
administration, as
presented in Table 73, below.
,
Group 1
Rat .ED
1 hr ,
1 X
2 X
3 10 X
Table 73
[0531] Resulting serum samples were analyzed via Alpha Klotho Human Soluble
ELISA kit.
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[0532] Table 74 presents a summary of study animals.
Species/Strain/Sex Age Range
Total Number Number of Animals Number of
of Animals per Group Groups
Rat/Sprague Dawley/M 8-11 weeks 63 3-10 7
Table 74
[0533] Table 75 presents a summary of study design.
Group Test Article Dose Level lig/kg
1 PBS, pH 7.2
2 Alpha Human Native Klotho 10
3 Alpha Human Native Klotho 30
4 Alpha Human Native Klotho 100
Alpha Human Fc- Fusion Klotho 3
6 Alpha Human Fc- Fusion Klotho 10
7 Alpha Human Fc- Fusion Klotho 30
Table 75
5 [0534] For Group Assignment: Stratified Sampling, a non-optimized
randomization method
that uses "binning" of the animals with measurement values of similar size was
used. By this
method, the pool of animals is stratified by sorting them by the measurement
value of interest in
ascending order. Animals are then organized into "bins" or strata according to
the size of the
measurement value. The number of strata is determined by the number of animals
that are to be
in assigned per group. For example, if there are three animals per group,
then three bins would be
created: small, medium and large. One animal from each size bin is assigned
randomly to each
group. Stratified sampling ensures that each group receives one animal
randomly from each size
bin and the means of each group will remain similar to the other groups. This
method balances
differences between group means and standard deviations and controls for pool
order bias.
[0535] For Statistical Analyses, Descriptive statistics were generated from
Study data. Data
were assessed to determine whether parametric or non-parametric analysis was
appropriate. For
parametric data, analysis of variance (ANOVA) followed by post-hoc tests was
performed to
determine significant differences between treatments, time points, and/or
groups. For non-
parametric data, appropriate statistical analyses were performed (e.g., Kaplan-
Meier Survival,
Kruskal-Wallis One-way ANOVA, Mann-Whitney or Wilcoxon Rank Sum, etc.).
[0536] Table 76 summarizes sample collection procedure.
Task Method & Collection
Sample Info/Special
Instructions
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Serum preparation for PK Blood collected via saphenous Samples
processed
sampling vein or cardiac puncture into non- to serum
and stored
heparinized tubes for serum at -80C prior to
ELISA
analysis
Table 76
[0537] Table 77 summarizes sample collection tasks.
Task Frequency
Body weight On Study Day -1 and twice weekly thereafter
Clinical Observation 2x weekly
Mortality Observation As required
Table 77
[0538] Any animal whose body weight dropped below 85% of initial body weight
or displays
severe, prolonged adverse clinical signs was euthanized (data not shown).
Animals were
euthanized via cardiac bleed for collection of plasma, serum and whole blood
as indicated above.
Example 9
[0539] A study to determine Dose-Dependent Effects of Alpha Human Fc-Klotho
and Alpha
Human Native Klotho on I/R Induced-AKI and Renal Biomarkers (by measuring
renal I/R-
induced increases in plasma renal injury biomarkers) in Wistar Rats was
performed. Specifically,
the objective of this study was to evaluate the dose-dependent effects of
alpha human Fc-Klotho
and alpha human native Klotho on renal FR-induced increases in plasma renal
injury biomarkers
in rats. The study was designed to provide data from or related to bilateral
renal ischemia-
reperfusion (I/R) injury (30' ischemia duration) surgery (n=63) verses sham
surgery (n=4),
Compound dosing (one i.v. injection/rat), serial urine collections via
metabolic cage housing (3
collections/rat; D1, D2 & D7), clinical chemistry analysis of serial urine
specimens for creatinine
and protein (D1, D2 & D7), serial blood collections (3 collections/rat; D1, D2
& D7), clinical
chemistry analysis of serial plasma specimens for creatinine and BUN (D1, D2 &
D7), daily
body weights and health observations post-surgery through endpoint, and
endpoint tissue
collection and processing, with data presented in tabular and graphical
format.
[0540] In addition, analysis of serial urine specimens for Kim-1 via species
specific ELISA
(D1, D2 & D7), analysis of serial urine specimens for NGAL via species
specific ELISA (D1,
D2 & D7), left and/or right kidney mRNA expression analysis (1-2
kidney/animal: n = 67
samples; 10 analytes, includes 3 normalization genes; performed using the
Affymetrix
Quantigene Plex 2.0 platform); MCP-1, a-SMA, Co11 Al, Col3A1, Fn-1, CTGF),
and
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left and/or right renal cortical immunohistochemistry and quantitation of
macrophage infiltration
via specific staining and quantitation of ED-1+ macrophage staining were also
optionally
performed.
[0541] Table 78 presents that study parameters.
Compounds: Alpha Human Fc- Klotho: 10 or 30 lig/kg
Alpha human Native Klotho: 10 or 30 lig/kg
Route / Frequency: Alpha Human Fc-Klotho: i.v. / dose once at
either t-0.5 hour
or t+1 hour relative to time of ischemia inception
Native Klotho: i.v. / dose once at either t-0.5 hour or t+1
hour relative to time of ischemia inception
Vehicle! Dose Volume: -- Alpha Human Fc-Klotho: PBS! 1 ml/kg i.v.
Native Klotho: PBS! 1 ml/kg i.v.
Compound Requirement: Alpha Human Fc-Klotho:
0.221 mg
Native Klotho: 0.221 mg
Table 78
[0542] Table 79 presents a summary of study design.
Compound &
N/group Administration Interval -- Dose
Volume &
Group Challenge
(Animal #s) (0 hr = time of ischemia Route
inception)
1 N = 4 Vehicle 1 ml/kg Sham
operated
(4528 - 4531) +1 Hr. i.v. 7 Days Post-Sx
2 N = 9 Vehicle 1 ml/kg 30'
lschemia
(4532 - 4540) +1 Hr. i.v. 7 Days
Reperfusion
N = 9 10 lig/kg Fc-Klotho 1 ml/kg 30' lschemia
3
(4541 - 4549) +1 Hr. i.v. 7 Days
Reperfusion
N = 9 30 lig/kg Fc-Klotho 1 ml/kg 30' lschemia
4
(4550 - 4558) +1 Hr. i.v. 7 Days
Reperfusion
N = 9 10 lig/kg Native Klotho 1 ml/kg 30' lschemia
5
(4559 - 4567) +1 Hr. i.v. 7 Days
Reperfusion
6 N = 9 30 lig/kg Native Klotho 1
ml/kg 30' lschemia
(4568 - 4576) +1 Hr. i.v. 7 Days
Reperfusion
N = 9 30 lig/kg Fc-Klotho 1 ml/kg 30' lschemia
7
(4577 - 4585) -0.5 Hr. i.v. 7 Days
Reperfusion
8 N = 9 30 lig/kg Native Klotho 1
ml/kg 30' lschemia
(4586 - 4594) -0.5 Hr. i.v. 7 Days
Reperfusion
Table 79
[0543] Sixty-seven (67) male Wistar rats weighing approximately 151-175g were
acquired and
fed a standard chow diet (Harlan 8640), group housed under standard
conditions, and allowed to
-- acclimate for at least 7 days before enrolling in study. Prior to study
inception, the study rats
were placed into weight-matched treatment groups. Animals were enrolled to
study using a
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balanced design, such that approximately equal numbers of animals were
enrolled per group per
surgical day, and the heaviest animals enrolled first to minimize differences
in animal age. When
not in metabolic cage housing, rats were group housed under standard
conditions in static caging.
[0544] At t-0.5 hours, study rats were anesthetized with isoflurane anesthesia
on a nosecone,
prepared for surgery and core temperature allowed to equilibrate prior to
renal I/R operative
procedure. Rats were administered 10 ml/kg warm, sterile saline (s.c.; 25% pre-
and 75% post-
surgery) to minimize perturbations in body fluid volume. Core temperatures
were maintained
normative (37 0.2 C) throughout ischemia duration. At t-0.5 hours, animals in
Groups 7 and 8
also received an i.v. injection of compound as appropriate.
[0545] A laparotomy was then performed. At tO, rats were subjected to either
sham (Group 1,
n=4) or bilateral renal arterial ischemia (Groups 2-8; n=9/grp) using heat
sterilized instruments,
vascular occlusive clamps, and aseptic surgical technique. Following thirty
(30) minutes of
continuous ischemia time (t+0.5), the occlusive clamps were removed, the
kidneys reperfused
and the abdominal wound closed with sterile silk suture. Rats were
administered 0.01 mg/kg
buprenorphine as post-operative analgesic and allowed to recover briefly in a
clean, heated cage.
At t+1 hour, rats in Groups 1-6 were briefly restrained and received an i.v.
injection of vehicle or
Klotho compound in vehicle as appropriate. Rats in Groups 7 and 8 were also
briefly restrained
(but not injected) to control for handling stress. Rats were then immediately
placed into
metabolic cages.
[0546] Twenty-four (24) hours following reperfusion (D1), all animals had
urine volumes
determined and sampled. All animals then had blood (500 [11) collected on Li-
Heparin by direct
tail vein stick. Immediately post-bleed, rats received 500 IA warm saline.
Urine and blood
collection was repeated again on D2 (48 hours post-reperfusion) and D7 (168
hours post-
reperfusion). On D2-D5, all rats were grouped housed under standard conditions
in static caging
and weighed, and underwent health observations daily. Whole blood (500 ill)
was processed
appropriately for the production of plasma and processed as described.
[0547] Clinical observations were conducted once daily following inception of
dosing. With
regard to data/tissues to be collected/calculated, animal health was observed
daily following
surgical procedure, and kidney mass (left and right kidney weights) was
indexed to tibia length
and body weight. Urine was collected at D1, D2 and D7 (targeting n=3
samples/animal; 500
ill/sample). At least one sample was subjected to clinical chemistry analysis
(creatinine, protein)
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and at least one sample was optionally used for Kim-1 and NGAL analysis.
Plasma was collected
on Li-Heparin at D1, D2 and D7 (targeting n=2 samples/animal; 125 ill/sample).
At least one
sample was subjected to clinical chemistry analysis (creatinine, BUN).
[0548] On D7, rats were anesthetized with isoflurane immediately following
bleed and tissues
were harvested. Both left and right kidneys were immediately placed in ice-
cold 0.9% NaCl, de-
encapsulated and weighed. After tissue harvest (for histological and other
analysis), all
remaining inner cortical/outer medullary (i.e. S3/THAL enriched) from each
sham and ischemic
kidney (left and right kidney from each animal; 1 tube/kidney/animal) were
snap frozen in
appropriately labeled tube and stored at -80 C for future potential
biochemical analysis. Prior to
any analyses being performed, cortical tissue was optionally cryogenically
powdered and mixed
to enable better homogeneity of the tissue sample across biochemical assays as
well as limit
introduction of sampling bias that may occur with cortical biopsies. At least
one mid-transverse
section of each sham and ischemic kidney (left and right from each animal) was
optionally
collected and immersion fixed in 10% neutral buffered formalin for
histological analysis. The
rats were then sacrificed.
Example 10
[0549] The body weight of study rats were measured to test for the effect of
Klotho protein
administration on subjects. Table 80 presents a summary of study animals.
Group Test Article
Group 01 Vehicle only, PBS, pH 7.2
Group 02 Alpha Human Native Klotho,10 lig/kg
Group 03 Alpha Human Native Klotho, 30 lig/kg
Group 04 Alpha Human Native Klotho, 100 lig/kg
Group 05 Alpha Human Fc-Fusion Klotho, 3 lig/kg
Group 06 Alpha Human Fc-Fusion Klotho, 10 lig/kg
Group 07 Alpha Human Fc-Fusion Klotho, 30 lig/kg
Table 80
[0550] Figure 44 illustrates, graphically, the average (mean) body weight (+/-
standard error of
the mean; SEM bars) of rats in each group over the course of the 12-day study
(daily). Table 81,
below, presents the corresponding data for mean body weight illustrated in
Figure 44, Table 82,
below, presents the corresponding SEM illustrated in Figure 44, and Table 83,
below, presents
the percent change in mean body weight of rats in each group over the course
of the 12-day study
(daily).
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Example 11
[0551] To study the therapeutic effect of Klotho administration on Acute
Kidney Injury (AKI)
following renal ischaemia reperfusion injury (IRI), Sprague¨Dawley rats were
randomly divided
into either Sham or AM groups, and the animals in each group were randomly
allocated into
vehicle or Klotho treatment. In humans, AM is a formidable clinical problem
resulting primarily
from ischemia or nephrotoxins, with outcomes ranging from full recovery to the
development of
chronic kidney disease with dialysis dependence, or death. Animals in the
Klotho group received
one bolus intraperitoneal injection of recombinant mouse Klotho protein (0.01
mg/kg body
weight) 30 or 60 min after reperfusion; rats in vehicle group received the
same volume of Klotho
buffer (150 mM NaCl and 10 mM HEPES pH 7.4). Twenty-four-hour urine and blood
were
collected on days 1, 2, and 7 after surgery. This study showed that a single
bolus injection of
murine S-Klotho 30 min post-injury was effective in attenuating (AKI) in
animal model of the
human condition.
Example 12
[0552] To determine whether Klotho protects the lung against oxidant injury,
Sprague-Dawley
rats (body weight ¨300 g) were exposed to normoxia (N: 21% inspired 0 ) or
hyperoxia (H: 90%
0 ) for 3 days while receiving intra-peritoneal injections of one of 3
preparations: Dulbeco's
modified Eagle's Medium (DMEM) or conditioned medium (CM) from CHO cells
overexpressing either Klotho (Klotho CM; ¨60 pmol) or empty vector (Control
CM) (n=6-8
animals each). Injections were given 12h before, and repeated at 24h and 48h
during exposure.
[0553] Also, 3LL cells were transplanted at the flanks of athymic mice by
subcutaneous
injection (2 x 106 cells per mouse) and treated with Klotho protein (0.01
mg/kg, intraperitoneal)
or vehicle every other day for 10 days. Lungs were harvested 21 days after
transplantation and
the number of metastatic nodules was counted.
192
Group Day 0 Day 1 Day 2 Day 3 Day 4 Day 5 Day 6
Day 7 Day 8 Day 9 Day 10 Day 11 Day 12
1 280.67 283.93 287.87 288.93 292.63 295.00
298.30 302.90 305.03 307.30 312.27 313.63 316.47
0
2 280.40 284.77 286.40 290.77 294.77 298.07
301.20 303.23 306.33 307.00 311.97 317.33 318.80
t,.)
o
3 287.23 289.47 294.67 299.60 302.83 305.23
308.37 312.47 313.47 317.77 322.17 323.93 325.27
1-
o
4 285.83 290.43 293.50 296.73 302.30 304.50 307.40 309.27 311.93 315.53 322.17
322.43 320.70 1-
1-
283.10 285.50 287.10 290.07 296.93 300.00 304.53
305.77 309.17 312.63 313.30 316.67 319.53 c,.)
--4
6 290.23 291.37 296.53 301.50 306.17 309.33
312.30 316.93 319.03 322.17 329.43 331.30 332.30
7 291.10 294.23 300.10 300.57 307.30 309.77 314.53 312.83 319.03 322.40 327.30
327.27 328.83
Table 81
Group Day 0 Day 1 Day 2 Day 3 Day 4 Day 5 Day 6
Day 7 Day 8 Day 9 Day 10 Day 11 Day 12
1 5.72 4.64 4.98 4.40 6.57 4.62 5.28 5.70
6.82 7.07 7.74 7.33 9.08
2 4.07 5.06 6.35 6.70 6.70 7.92 7.09 9.12
8.13 8.21 8.56 8.06 9.33
3 5.55 3.71 4.83 4.41 4.56 5.05 6.27 4.94
6.20 6.20 5.87 5.52 6.57 P
4 2.97 5.81 5.20 5.44 5.78 5.88 6.53 4.92
4.91 4.90 5.23 5.47 5.69
,
1- 5 6.92 5.68 6.08 6.45 6.03 6.53 6.72 7.11
6.48 7.00 6.81 6.32 7.31 ,
,
o .
6 4.72 3.84 3.51 4.15 4.72 5.34 4.48 4.54
4.25 5.86 4.78 5.40 4.47
r.,
7 4.91 4.62 3.64 4.56 5.39 5.67 5.85 5.24
4.95 4.60 5.14 5.39 6.98 0
,
,
,
,
Table 82
,
Group Day 0 Day 1 Day 2 Day 3 Day 4 Day 5 Day 6
Day 7 Day 8 Day 9 Day 10 Day 11 Day 12
1 0.00% 1.18% 2.58% 2.97% 4.26% 5.13% 6.29% 7.93% 8.67% 9.48% 11.24% 11.73%
12.72%
2 0.00% 1.55% 2.12% 3.68% 5.11% 6.28% 7.40% 8.12% 9.23% 9.46% 11.23% 13.15%
13.66%
3 0.00% 0.81% 2.60% 4.32% 5.45% 6.28% 7.36% 8.80% 9.14% 10.63% 12.17% 12.79%
13.25%
4 0.00% 1.59% 2.67% 3.80% 5.74% 6.51% 7.52% 8.19% 9.12% 10.38% 12.70% 12.79%
12.18%
5 0.00% 0.87% 1.43% 2.47% 4.91% 5.98% 7.58% 8.01% 9.23% 10.45% 10.68% 11.88%
12.88% 1-d
n
6 0.00% 0.40% 2.19% 3.89% 5.49% 6.58% 7.61% 9.21% 9.93% 11.00% 13.51% 14.15%
14.50% 1-3
7 0.00% 1.08% 3.11% 3.26% 5.56% 6.41% 8.05% 7.47% 9.60% 10.77% 12.45% 12.43%
12.95% cp
o
Table 83
oe
O-
o,
.6.
c,.)
CA 03101100 2020-11-19
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Conclusion
[0554] Existing Klotho protein measurement may be prohibitively expensive
and/or
technically challenging for at-home collection and/or quantitative diagnostic.
Embodiments
of the present disclosure provide methods for monitoring, detecting and/or
quantifying
Klotho protein level(s), particularly endogenous and/or exogenous soluble
alpha Klotho
protein level(s), and more particularly soluble alpha Klotho protein levels,
diagnosing Klotho
protein deficiency, and/or increasing Klotho protein level(s), expression, or
production,
particularly endogenous and/or exogenous soluble alpha Klotho protein
level(s), expression,
or production, in a subject, and products useful in performing the same,
including diagnostic
kits and compositions for treating Klotho protein deficiency.
[0555] In addition, while gene therapy can be effective to increase certain
protein levels in
animal models or studies, the safety of gene therapy, especially for human
treatment, is still
questionable. Compared to viral delivery of the klotho gene to animal (cells),
the
administration of exogenous and/or recombinant protein in humans may be a
safer, easier,
and more direct modality to restore (endocrine) klotho levels. Preclinical
data supports the
therapeutic potential of soluble Klotho protein for age-related disorders and
klotho
deficiency-associated diseases. Epidemiological data has shown that soluble
Klotho is lower
in the elderly than in young adults, and that levels of soluble Klotho are
inversely correlated
with age, indicating that aging may be associated with soluble Klotho decline.
Generally,
however, the production and administration of exogenous and/or recombinant
protein to
humans is tightly regulated by government and other entities. Because of the
time and
expense associated with regulatory compliance, the cost of such treatments can
be prohibitive
for developers, manufactures, and patients alike. Compositions and methods of
the present
disclosure, however, can provide a viable and effective option for increasing
endogenous
Klotho protein levels in humans and non-human animals without one or more of
the
drawbacks associated with other solutions. In particular, some embodiments of
the present
disclosure include products, compositions, and/or methods of manufacturing
and/or using
recombinant human Klotho proteins, such as (Current Good Manufacturing
Practice (cGMP)-
grade) human recombinant soluble alpha-Klotho proteins, protein fragments,
and/or protein
variants. In addition, some embodiments of the present disclosure include
(nutraceutical or
health supplement) products, compositions, and/or methods for (naturally)
increasing
(endogenous and/or serum soluble) Klotho protein level(s), expression, or
production in a
patient or subject.
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[0556] Those skilled in the art will appreciate that each of the therapeutic
effects,
indications, and/or treatments outlined herein in relation to recombinant,
therapeutic Klotho
proteins and products, compositions, and methods related thereto can be
achieved by
(naturally) increasing, enhancing, augmenting, and/or supporting endogenous
Klotho protein
production and/or level(s). Those skilled in the art will also appreciate that
each of the
therapeutic effects, indications, and/or treatments outlined herein in
relation to (naturally)
increasing, enhancing, augmenting, and/or supporting endogenous Klotho protein
production
and/or level(s) can be achieved by administering recombinant, therapeutic
Klotho proteins
and products, compositions, and through methods related thereto. Accordingly,
and for the
sake of brevity, the present disclosure need not rehearse each of the
disclosed therapeutic
effects, indications, and/or treatments with reference to both (i) the
administration of
recombinant, therapeutic Klotho proteins, and (ii) the administration of the
health supplement
or nutraceutical compositions of the present disclosure, as such are clearly
contemplated
herein.
[0557] While the foregoing detailed description makes reference to specific
exemplary
embodiments, the present disclosure may be embodied in other specific forms
without
departing from its spirit or essential characteristics. Accordingly, the
described embodiments
are to be considered in all respects only as illustrative and not restrictive.
For instance,
various substitutions, alterations, and/or modifications of the inventive
features described
and/or illustrated herein, and additional applications of the principles
described and/or
illustrated herein, which would occur to one skilled in the relevant art and
having possession
of this disclosure, can be made to the described and/or illustrated
embodiments without
departing from the spirit and scope of the disclosure as defined by the
appended claims. Such
substitutions, alterations, and/or modifications are to be considered within
the scope of this
disclosure.
[0558] The scope of the invention is, therefore, indicated by the appended
claims rather
than by the foregoing description. The limitations recited in the claims are
to be interpreted
broadly based on the language employed in the claims and not limited to
specific examples
described in the foregoing detailed description, which examples are to be
construed as non-
exclusive and non-exhaustive. All changes which come within the meaning and
range of
equivalency of the claims are to be embraced within their scope.
[0559] It will also be appreciated that various features of certain
embodiments can be
compatible with, combined with, included in, and/or incorporated into other
embodiments of
the present disclosure. For instance, systems, methods, and/or products
according to certain
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embodiments of the present disclosure may include, incorporate, or otherwise
comprise
features described in other embodiments disclosed and/or described herein.
Thus, disclosure
of certain features relative to a specific embodiment of the present
disclosure should not be
construed as limiting application or inclusion of said features to the
specific embodiment.
[0560] In addition, unless a feature is described as being required in a
particular
embodiment, features described in the various embodiments can be optional and
may not be
included in other embodiments of the present disclosure. Moreover, unless a
feature is
described as requiring another feature in combination therewith, any feature
herein may be
combined with any other feature of a same or different embodiment disclosed
herein. It will
1() be appreciated that while features may be optional in certain
embodiments, when features are
included in such embodiments, they can be required to have a specific
configuration as
described in the present disclosure.
[0561] Likewise, any steps recited in any method or process described herein
and/or recited
in the claims can be executed in any suitable order and are not necessarily
limited to the order
described and/or recited, unless otherwise stated (explicitly or implicitly).
Such steps can,
however, also be required to be performed in a specific order or any suitable
order in certain
embodiments of the present disclosure.
[0562] Furthermore, various well-known aspects of illustrative systems,
methods, products,
and the like are not described herein in particular detail in order to avoid
obscuring aspects of
the example embodiments. Such aspects are, however, also contemplated herein.
196
