Note: Descriptions are shown in the official language in which they were submitted.
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METHODS AND COMPOSITIONS FOR TREATING
AGING-ASSOCIATED IMPAIRMENTS WITH TREFOIL FACTOR FAMILY
MEMBER 2 MODULATORS
1. CROSS REFERNCE TO RELATED APPLICATIONS
This application claims priority to United States Patent Application Serial
No. 17/318,875,
filed May 12, 2021, which application is a continuation-in-part of United
States Application Serial
No. 17/104,344, filed November 25, 2020, which application, pursuant to 35
U.S.C. 119 (e),
claims priority to the filing date of United States Provisional Patent
Application Serial No.
62/940,477, filed November 26. 2019; and United States Provisional Patent
Application Serial No.
63/071,515, filed August 28, 2020; the disclosures of which applications are
herein incorporated
by reference.
2. FIELD OF THE INVENTION
This invention pertains to the prevention and treatment of aging-related
conditions. The
invention relates to the use of agents modulating the biological
concentrations of trefoil factor
family member 2 (TFF2) with efficacy in treating and/or preventing aging-
related conditions such
as neurocognitive and neurudegenerative disorders.
3. SUMMARY
Aging in an organism is accompanied by an accumulation of changes over time.
In the
nervous system, aging is accompanied by structural and neurophysiological
changes that drive
cognitive decline and susceptibility to degenerative disorders in healthy
individuals. (Heeden &
Gabrieli, "Insights into the ageing mind: a view from cognitive neuroscience,"
Nat. Rev. Neurosci.
(2004) 5: 87-96; Raz et al., "Neuroanatomical correlates of cognitive aging:
evidence from
structural magnetic resonance imaging," Neuropsychology (1998) 12:95-114;
Mattson & Magnus,
"Ageing and neuronal vulnerability," Nat. Rev. Neurosci. (2006) 7: 278-294;
and Rapp & Heindel,
"Memory systems in normal and pathological aging," Curr. Opin. Neurol. (1994)
7:294-298).
Included in these changes are synapse loss and the loss of neuronal function
that results. Thus,
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although significant neuronal death is typically not observed during the
natural aging process,
neurons in the aging brain are vulnerable to sub-lethal age-related
alterations in structure, synaptic
integrity, and molecular processing at the synapse, all of which impair
cognitive function.
In addition to the normal synapse loss during natural aging, synapse loss is
an early
pathological event common to many neurodegenerative conditions and is the best
correlate to the
neuronal and cognitive impairment associated with these conditions. Indeed,
aging remains the
single most dominant risk factor for dementia-related neurodegenerative
diseases such as
Alzheimer's disease (AD) (Bishop et al., "Neural mechanisms of ageing and
cognitive decline,"
Nature (2010) 464: 529-535 (2010); Heeden & Gabrieli, "Insights into the
ageing mind: a view
from cognitive neuroscience," Nat. Rev. Neurosci. (2004) 5:87-96; Mattson &
Magnus, "Ageing
and neuronal vulnerability," Nat. Rev. Neurosci. (2006) 7:278-294).
As human lifespan increases, a greater fraction of the population suffers from
aging
associated cognitive impairments, making it crucial to elucidate means by
which to maintain
cognitive integrity by protecting against, or even counteracting, the effects
of aging (Hebert et al.,
"Alzheimer disease in the US population: prevalence estimates using the 2000
census," Arch.
Neurol. (2003) 60:1119-1122; Bishop et al., "Neural mechanisms of ageing and
cognitive decline,"
Nature (2010) 464:529-535).
Trefoil factor family member 2 (TFF2, also known as spasmolytic polypeptide)
is a small
peptide member of the trefoil family of peptides. The trefoil family of
peptides are small (7-12
kDa) protease-resistant proteins secreted by the gastrointestinal mucosa. TFF2
is predominantly
found in the epithelium of the gut, but also found in immune cells, lymphoid
tissues, the central
nervous system, specifically the hypothalamus, and the endocrine system,
specifically the anterior
pituitary. In its primary area of expression, the gastric epithelium and
duodenal Brunner' s glands,
it is usually expressed with the mucin MUC6, and together they work in the
formation and
stabilization of the mucus barrier. TFF2 is also present in the human gastric
juice at concentrations
between 1 and 20 rig/nil (May, et al., "The human two domain trefoil protein,
TFF2, is glycosylated
in vivo in the stomach," Gut (2000) 46:454-459).
Mammalian TFF2 contains two trefoil or P domains, unlike the other mammalian
trefoil
peptides. These domains contain multiple secondary structural elements, which
suggests multiple
pharmacophores and matches with the multiple observed functions of TFF.
However, little is
currently known about the molecular mechanisms of TFF2, and all attempts have
so far failed to
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convincingly demonstrate a typical transmembrane receptor. TFF2 has also been
reported to
activate PAR4, which likely contributes to mucosal healing (Zhang Y, et al.,
"Activation of
protease-activated receptor (PAR) 1 by frog trefoil factor (TFF) 2 and PAR4 by
human TFF2,"
Cell Mol Life Sci. (2011) 68:3771-3780). Porcine TFF2 binds non-covalently to
integrin 131,
which plays an important role in cell migration that is enhanced by TFF
peptides (Hoffmann W.,
-TFF2, a MUC6-binding lectin stabilizing the gastric mucus barrier and more,-
Int J Oncol. (2015)
47:806-816; Otto W, Thim L., -Trefoil factor family-interacting proteins,"
Cell Mol Life Sci.
(2005) 62:2939-2946). Porcine TFF2 has also been found to bind non-covalently
to the cysteine-
rich repetitive glycoprotein (MW > 340 kDa) DMBT1 (formerly: hensin, muclin),
an extracellular
matrix-associated multifunctional protein playing a role in mucosal innate
immunity and
protection (Hoffmann W., "TFF2, a MUC6-binding lectin stabilizing the gastric
mucus barrier and
more," Int J Oncol. (2015) 47:806-816; Albert TK, et al., "Human intestinal
TFF3 forms disulfide-
linked heteromers with the mucus-associated FCGBP protein and is released by
hydrogen sulfide,"
J Proteome Res. (2010) 9:3108-3117). Intravenously administered TFF2 has been
found to have
been taken up by mucous neck cells, parietal cells, and pyloric gland cells
and subsequently
appeared in the mucus layer, which could be an indication for receptor-
mediated transcytosis
(Poulsen SS, Thulesen J, Nexo E and Thim L, "Distribution and metabolism of
intravenously
administered trefoil factor 2/porcine spasmolytic polypeptide in the rat," Gut
(1998) 43:240-247).
TFF2 is an important part of the viscous gastric mucus barrier, which has
multiple
physiological functions. The mucus barrier is a biofilm that lubricates the
passage of undigested
food and protects the epithelium from mechanical damage and pepsin digestion.
It is essential for
maintaining a pH gradient towards the acidic gastric juice, and it supports
and also restricts the
adhesion and colonization of microorganisms (such as H. pylori) (Allen A,
"Gastrointestinal
mucus. Section 6: The gastrointestinal System," In: Handbook of physiology,
Vol. ITT, Schultz SG
(ed.) Am Physiol Soc., Bethesda, MD (1989) pp. 359-382). TFF2 can be
considered a lectin,
stabilizing the gastric mucus barrier and thereby affecting its viscoelastic
properties (Sturmer R,
et al., "Commercial porcine gastric mucin preparations, also used as
artificial saliva, are a rich
source for the lectin TFF2: in vitro binding studies," Chembiochem. (2018)
19:2598-2608;
Hanisch FG, et al., -Human trefoil factor 2 is a lectin that binds alpha-
G1cNAc-capped mucin
glycans with antibiotic activity against Helicobacter pylori," J Biol Chem.
(2014) 289:27363-
27375). TFF2 binds highly specifically to the GlcNAca1¨>4Ga1131¨>R moiety of
MUC6, and the
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terminal a-G1cNAc has antimicrobial activity against Helicobacter pylori,
which might also adhere
to the LacdiNAc oligosaccharide of TFF2 via LabA, suggesting a colonization
mechanism
(Hoffmann W., "TFF2, a MUC6-binding lectin stabilizing the gastric mucus
barrier and more,"
Int J Oncol. (2015) 47:806-816; Sturmer R, et al., "Commercial porcine gastric
mucin
preparations, also used as artificial saliva, are a rich source for the lectin
TFF2: in vitro binding
studies," Chembiochem. (2018) 19:2598-2608; Hanisch FG, et al., -Human trefoil
factor 2 is a
lectin that binds alpha-G1cNAc-capped mucin glycans with antibiotic activity
against Helicobacter
pylori," J Biol Chem. (2014) 289:27363-27375).
In the central nervous system, TFF2 has been found to be expressed and
modulated in the
hypothalamus in relation to appetite, satiety, and body weight (Giorgio, et
al., "Trefoil Factor
Family Member 2 (Tff2) KO Mice Are protected from High-Fat Diet-Induced
Obesity," Obesity
(2013) 21: 1389-1395). TFF2 KO mice were found to store energy less
efficiently than WT mice
and gained less weight and fat mass than WT mice (Giorgio, et al., -Trefoil
Factor Family Member
2 (Tff2) KO Mice Are protected from High-Fat Diet-Induced Obesity," Obesity
(2013) 21: 1389-
1395). TFF2 has also been found in the anterior pituitary of the mouse brain,
where it likely is
released to the rest of the body (Hinz M, Schwegler H, Chwieralski CE, Laube
G. Linke R, Pohle
W and Hoffmann W, "Trefoil factor family (TFF) expression in the mouse brain
and pituitary:
Changes in the developing cerebellum," Peptides (2004) 25: 827-832).
The present invention discloses the relationship between age and relative
serum plasma
TFF2 levels, where such TFF2 levels increase with age. The invention also
discloses methods to
treat an adult mammal for an aging-associated condition by reducing, blocking,
or decreasing the
activity of TFF2 in the adult mammal. In light of a long-felt and unmet need
in treating diseases
of aging such as cognitive impairment, the compositions and methods of the
invention address that
need by providing a method of administering an agent to reduce, block, or
decrease the activity of
TFF2 in a subject diagnosed with a cognitive impairment such as, for example
and not limitation,
Alzheimer' s Disease, Parkinson's Disease, Huntington's Disease, Mild
Cognitive Impairment,
Dementia, and the like.
4. SUMMARY
Methods of treating an adult mammal for an aging-associated condition are
provided.
Aspects of the methods include reducing the trefoil factor family peptide 2
(TFF2) level or its
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activity in the mammal in a manner sufficient to treat the mammal for the
aging-associated
impairment. A variety of aging-associated impairments may be treated by
practice of the methods,
which impairments include cognitive impairments.
5. INCORPORATION BY REFERENCE
All publications and patent applications mentioned in this specification are
herein incorporated
by reference to the same extent as if each individual publication or patent
application was
specifically and individually indicated to be incorporated by reference.
6. BRIEF DESCRIPTION OF THE FIGURES
Figure 1 shows a "box and whiskers- depiction of the 10g2 relative
concentrations of TFF2
in plasma from donors of five different age groups. Plasma from males (50
individuals in each
age group) aged 18, 30, 45, 55, and 66-years-old were measured using the
SomaScan aptamer-
based proteomics assay (SomaLogic, Boulder, CO). Healthy plasma levels show a
highly
significant monotonous increase over this age range (p = 1.6e-9, Jonckheere-
Terpstra trend test).
The line within each box indicates the median value.
Figure 2 shows the results of a radial arm water maze (RAWM) assay which tests
reference
and working memory performance by requiring the mice to utilize cues to locate
escape platforms.
(See, e.g., Penley SC, et al., J Vis Exp., (82):50940 (2013)). Young mice
treated with hTFF2 made
more errors when navigating the maze compared to vehicle-treated mice.
Figure 3 depicts the results from a Y-maze behavior test. The Y-maze test
determines
hippocampal-dependent cognition as measured by preference to enter the novel
arm (as opposed
to the familiar arm) in a cued Y-maze. The percent entries were calculated by
normalizing the
number of entries in the novel or familiar arm (the two arms of the "Y" maze)
to the total entries
in the novel and familiar arms. The Wilcoxon matched-pairs signed rank test
was used to assess
statistical significance between novel and familiar arms in percent of
entries. The results of Figure
2 demonstrate that administration of human TFF2 (hTFF2) to young mice leads to
a trend of fewer
entries into the novel arm of the Y-maze. indicating a decline in cognitive
performance.
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Figure 4 shows quantitative PCR (qPCR) of hippocampal mRNA from hTFF2-treated
and
vehicle-treated mice. The figure shows that there is an increase in expression
of an inflammatory
marker, IL-6, as compared to vehicle treated mice. (* P <0.05, Mann-Whitney U
test).
Figure 5 shows RT-qPCR of hippocampal cDNA from hTFF2- and vehicle-treated
mice.
The figure shows that there is a trend in increased expression of a marker for
reactive astrocytes,
Ggtal, as compared to vehicle-treated mice. Reactive astrocytes are strongly
induced by the
central nervous system during injury and disease. (Liddelow SA, et al.,
Nature, 541(7638):481-
87 (2017).
Figure 6 reports that TFF2 inhibition with L-pyroglutamic acid improved
cognitive
performance as aged mice treated with the inhibitor entered the novel arm
significantly more than
the familiar arm (p <0.002). Additionally, the difference between novel and
familiar arm entries
was greater than that observed with vehicle. Data is shown as mean SEM.
Figure 7 shows results from quantitative analysis of immunostaining in
hippocampi of
aged mice treated with the TFF2 inhibitor compared to vehicle. Synapse density
was measured as
number of synapses per um3. There was a strong trend towards higher synapse
density in the CA1
region of the hippocampus in mice treated with TFF2 inhibitor. Data is shown
as mean SEM.
Figure 8A is a Western blot demonstrating that TFF2 protein is detected in
brain lysate
from 22-month-old C57B16 mice. Figure 8B shows that the anti- TFF2 antibody
recognizes both
mouse and human recombinant TFF2 and that mouse TFF2 (12kDa) and human TFF2
(14kDa)
can be glycosylated in vivo.
Figure 9 describes a TFF2 bioassay for ERK1/2 phosphorylation in Jurkat cells.
Figure 10 shows a Western blot demonstrating that treatment of Jurkat cells
with human
TFF2 leads to increased ERK1/2 phosphorylation.
Figure 11 is a Western blot showing that anti-human TFF2 antibodies have
neutralizing
activity in Jurkat cells against human TFF2.
Figures 12A-12B demonstrate that an anti-TFF2 antibody can neutralize mouse
TFF2
activity in Jurkat cells. Figure 12A shows that mouse TFF2 can induce ERK1/2
phosphorylation
in Jurkat cells at higher concentrations. Figure 12B demonstrates that at
lower concentrations
mouse TFF2 no longer can induce ERK1/2 phosphorylation. Additionally, the
figures together
show that anti-human TFF2 antibody clone HSPGE16C can inhibit ERK1/2
phosphorylation with
treatment of 100nM TFF2, but not 300nM.
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Figure 13 shows a Western blot demonstrating that the HSPGE16C anti-hTFF2
antibody
can neutralize mouse TFF2 activity in Jurkat cells in a concentration-
dependent manner.
Figure 14 shows a table of commercially available anti-TFF2 antibodies tested
for
neutralization of TFF2 activity in Jurkat cells, as well as their immunogen
information, the species
of TFF2 the antibody recognizes or binds to, the host species the host species
that the antibody
was raised in, their clonality, and their isotypc.
Figure 15A shows representations of the peptide sequences for full length
mouse TFF2,
which is labelled SEQ ID NO: 01, and Human TFF2, which is labelled SEQ ID NO;
02, as well
as the TFF2 antigens or epitopes used to generate antibodies for specific
protein domains. Mouse
sequences are represented as black rectangles and human sequences as white
rectangles with each
peptide region aligned with the full length TFF2 proteins. The antigens
include amino acids 24-
129 of Mouse TFF2 (SEQ ID NO: 03); amino acids 24-129 of Human TFF2 (SEQ ID
NO: 04);
amino acids 27-129 of Mouse TFF2 (SEQ ID NO: 05); amino acids 27-129 of Human
TFF2 (SEQ
ID NO: 06); amino acids 29-73 of Mouse TFF2 (SEQ ID NO: 07); amino acids 29-73
of Human
TFF2 (SEQ ID NO: 08); amino acids 79-122 of Mouse TFF2 (SEQ ID NO: 09); amino
acids 79-
122 of Human TFF2 (SEQ ID NO: 10); amino acids 114-129 of Mouse TFF2 (SEQ ID
NO: 11);
and amino acids 114-129 of Human TFF2 (SEQ ID NO: 12). These antigen peptide
fragments
were or can be used for custom TFF2 antibody generation.
Figure 15B shows a multiple sequence alignment of SEQ ID Nos: 01 through 12
described
in Figure 15A. The alignment was performed using CLUSTAL 0 (1.2.4) (available
at
https://www.uniprot.org/align/).
Figure 16 shows the normalized relative pERK/GAPDH values from Western Blots
demonstrating the treatment of Jurkat cells with thirteen anti-TFF2
antibodies. The figure shows
the results for treatment of Jurkat cells with a concentration of 4p g/m1 for
each of the thirteen anti-
TFF2 antibodies listed in Figure 14 compared to treatment with a vehicle,
TFF2, and a positive
control (mouse SDF-1).
Figure 17 shows relative pERK 1/2 ELISA expression in Jurkat cells after
treatment with
the Clone #1-2 anti-TFF2 antibody and a neutralizing rabbit polyclonal
antibody. The figure shows
that the commercially available Clone #1-2 antibody decreases mouse TFF2
activity in Jurkat cells.
Figure 18A shows that aged mice treated with human anti-TFF2 antibody froze
more after
foot shock during training.
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Figure 18B shows that a significant increase in freezing in TFF2 antibody
treated mice
compared to control antibody treated mice can be detected for 1 minute after
the first foot shock.
Figure 19A shows that aged mice treated with human anti-TFF2 antibody retained
memory
better than mice treated with control antibody as determined by the contextual
fear conditioning
assay.
Figure 19B shows that significant improvement in cognition as determined by
percent
freezing occurs in TFF2 antibody treated mice compared to control antibody
treated mice during
the final half of the assay.
Figure 20A shows that aged mice treated with human anti-TFF2 antibody moved
less
during contextual fear conditioning testing.
Figure 20B shows that mice treated with TFF2 antibody exhibited less movement
than
mice treated with control antibody during the final half of the assay. This is
another indicator that
inhibition of TFF2 resulted in improved cognitive performance.
Figure 21 shows that in the hippocampi of mice that underwent the contextual
fear
conditioning assay and treated with human anti-TFF2 antibody as described
above, there was a
downward trend in expression of the inflammatory marker, IL-113, compared to
control antibody
treated mice.
Figure 22 shows that TFF2 levels are significantly increased in C57BL/6 mouse
models
of inflammation as measured by ELISA, indicating that such models can be used
for evaluating
the in vivo effects of TFF2 neutralization.
7. DETAILED DESCRIPTION
Methods of treating an adult mammal for an aging-associated impairment are
provided.
Aspects of the methods include reducing levels of or decreasing the activity
of the trefoil factor
family peptide 2 (TFF2) in the mammal in a manner sufficient to treat the
mammal for the aging-
associated impairment. A variety of aging-associated impairments may be
treated by practice of
the methods, which impairments include cognitive impairments.
Before the present methods and compositions are described, it is to be
understood that this
invention is not limited to a particular method or composition described, as
such may, of course,
vary. It is also to be understood that the terminology used herein is for the
purpose of describing
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particular embodiments only, and is not intended to be limiting, since the
scope of the present
invention will be limited only by the appended claims.
Where a range of values is provided, it is understood that each intervening
value, to the
tenth of the unit of the lower limit unless the context clearly dictates
otherwise, between the upper
and lower limits of that range is also specifically disclosed. Each smaller
range between any stated
value or intervening value in a stated range and any other stated or
intervening value in that stated
range is encompassed within the invention. The upper and lower limits of these
smaller ranges
may independently be included or excluded in the range, and each range where
either, neither or
both limits are included in the smaller ranges is also encompassed within the
invention, subject to
any specifically excluded limit in the stated range. Where the stated range
includes one or both of
the limits, ranges excluding either or both of those included limits are also
included in the
invention.
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
this invention
belongs. Although any methods and materials similar or equivalent to those
described herein can
be used in the practice or testing of the present invention, some potential
and preferred methods
and materials are now described. All publications mentioned herein are
incorporated herein by
reference to disclose and describe the methods and/or materials in connection
with which the
publications are cited. It is understood that the present disclosure
supersedes any disclosure of an
incorporated publication to the extent there is a contradiction.
As will be apparent to those of skill in the art upon reading this disclosure,
each of the
individual embodiments described and illustrated herein has discrete
components and features
which may be readily separated from or combined with the features of any of
the other several
embodiments without departing from the scope or spirit of the present
invention. Any recited
method can be carried out in the order of events recited or in any other order
which is logically
possible.
It must be noted that as used herein and in the appended claims, the singular
forms "a",
"an", and "the" include plural referents unless the context clearly dictates
otherwise. Thus, for
example, reference to "a cell" includes a plurality of such cells and
reference to "the peptide"
includes reference to one or more peptides and equivalents thereof, e.g.,
polypeptides, known to
those skilled in the art, and so forth.
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The publications discussed herein are provided solely for their disclosure
prior to the filing
date of the present application. Nothing herein is to be construed as an
admission that the present
invention is not entitled to antedate such publication by virtue of prior
invention. Further, the dates
of publication provided may be different from the actual publication dates
which may need to be
independently confirmed.
8. METHODS
As summarized above, aspects of the invention include methods of treating an
aging-
associated impairment in an adult mammal. The aging-associated impairment may
manifest in a
number of different ways, e.g., as aging-associated cognitive impairment
and/or physiological
impairment, e.g., in the form of damage to central or peripheral organs of the
body, such as but not
limited to: cell injury, tissue damage, organ dysfunction, aging associated
lifespan shortening and
carcinogenesis, where specific organs and tissues of interest include, but are
not limited to skin,
neuron, muscle, pancrcas, brain, kidney, lung, stomach, intestine, spleen,
heart, adipose tissue,
testes, ovary, uterus, liver and bone; in the form of decreased neurogenesis,
etc.
Tn some embodiments, the aging-associated impairment is an aging-associated
impairment
in cognitive ability in an individual, i.e., an aging-associated cognitive
impairment. By cognitive
ability, or "cognition," it is meant the mental processes that include
attention and concentration,
learning complex tasks and concepts, memory (acquiring, retaining, and
retrieving new
information in the short and/or long term), information processing (dealing
with information
gathered by the five senses), visuospatial function (visual perception, depth
perception, using
mental imagery, copying drawings, constructing objects or shapes), producing
and understanding
language, verbal fluency (word-finding), solving problems, making decisions,
and executive
functions (planning and prioritizing). By -cognitive decline", it is meant a
progressive decrease in
one or more of these abilities, e.g., a decline in memory, language, thinking,
judgment, etc. By -an
impairment in cognitive ability" and "cognitive impairment," it is meant a
reduction in cognitive
ability relative to a healthy individual, e.g., an age-matched healthy
individual, or relative to the
ability of the individual at an earlier point in time, e.g., 2 weeks, 1 month,
2 months, 3 months, 6
months, 1 year, 2 years, 5 years, or 10 years or more previously. Aging-
associated cognitive
impairments include impairments in cognitive ability that are typically
associated with aging,
including, for example, cognitive impairment associated with the natural aging
process, e.g., mild
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cognitive impairment (M.C.I.); and cognitive impairment associated with an
aging associated
disorder, that is, a disorder that is seen with increasing frequency with
increasing senescence, e.g.,
a neurodegenerative condition such as Alzheimer' s disease, Parkinson's 5
disease, frontotemporal
dementia, Huntington's disease, amyotrophic lateral sclerosis, multiple
sclerosis, glaucoma,
myotonic dystrophy, vascular dementia, and the like.
By "treatment" it is meant that at least an amelioration of one or more
symptoms associated
with an aging-associated impairment afflicting the adult mammal is achieved,
where amelioration
is used in a broad sense to refer to at least a reduction in the magnitude of
a parameter, e.g., a
symptom associated with the impairment being treated. As such, treatment also
includes situations
where a pathological condition, or at least symptoms associated therewith, are
completely
inhibited, e.g., prevented from happening, or stopped, e.g., terminated, such
that the adult mammal
no longer suffers from the impairment, or at least the symptoms that
characterize the impairment.
In some instances, "treatment", "treating" and the like refer to obtaining a
desired pharmacologic
and/or physiologic effect. The effect may be prophylactic in terms of
completely or partially
preventing a disease or symptom thereof and/or may be therapeutic in terms of
a partial or complete
cure for a disease and/or adverse effect attributable to the disease.
"Treatment" may be any
treatment of a disease in a mammal, and includes: (a) preventing the disease
from occurring in a
subject which may be predisposed to the disease but has not yet been diagnosed
as having it; (b)
inhibiting the disease, i.e., arresting its development; or (c) relieving the
disease, i.e., causing
regression of the disease. Treatment may result in a variety of different
physical manifestations,
e.g., modulation in gene expression, increased neurogenesis, rejuvenation of
tissue or organs, etc.
Treatment of ongoing disease, where the treatment stabilizes or reduces the
undesirable clinical
symptoms of the patient, occurs in some embodiments. Such treatment may be
performed prior to
complete loss of function in the affected tissues. The subject therapy may be
administered during
the symptomatic stage of the disease, and in some cases after the symptomatic
stage of the disease.
In some instances where the aging-associated impairment is aging-associated
cognitive
decline, treatment by methods of the present disclosure slows, or reduces, the
progression of aging-
associated cognitive decline. In other words, cognitive abilities in the
individual decline more
slowly, if at all, following treatment by the disclosed methods than prior to
or in the absence of
treatment by the disclosed methods. In some instances, treatment by methods of
the present
disclosure stabilizes the cognitive abilities of an individual. For example,
the progression of
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cognitive decline in an individual suffering from aging-associated cognitive
decline is halted
following treatment by the disclosed methods. As another example, cognitive
decline in an
individual, e.g., an individual 40 years old or older, that is projected to
suffer from aging-associated
cognitive decline, is prevented following treatment by the disclosed methods.
In other words, no
(further) cognitive impairment is observed. In some instances, treatment by
methods of the present
disclosure reduces, or reverses, cognitive impairment, e.g., as observed by
improving cognitive
abilities in an individual suffering from aging-associated cognitive decline.
In other words, the
cognitive abilities of the individual suffering from aging-associated
cognitive decline following
treatment by the disclosed methods are better than they were prior to
treatment by the disclosed
methods, i.e., they improve upon treatment. In some instances, treatment by
methods of the present
disclosure abrogates cognitive impairment. In other words, the cognitive
abilities of the individual
suffering from aging-associated cognitive decline are restored, e.g., to their
level when the
individual was about 40 years old or less, following treatment by the
disclosed methods, e.g., as
evidenced by improved cognitive abilities in an individual suffering from
aging-associated
cognitive decline.
In some instances, treatment of an adult mammal in accordance with the methods
results
in a change in a central organ, e.g., a central nervous system organ, such as
the brain, spinal cord,
etc., where the change may manifest in a number of different ways, e.g., as
described in greater
detail below, including but not limited to molecular, structural and/or
functional, e.g., in the form
of enhanced neurogenesis.
As summarized above, methods described herein are methods of treating an aging
associated impairment, e.g., as described above, in an adult mammal. By adult
mammal is meant
a mammal that has reached maturity, i.e., that is fully developed. As such,
adult mammals are not
juvenile. Mammalian species that may be treated with the present methods
include canines and
felines; equines; bovines; ovines; etc., and primates, including humans. The
subject methods,
compositions, and reagents may also be applied to animal models, including
small mammals, e.g.,
murine, lagomorpha, etc., for example, in experimental investigations. The
discussion below will
focus on the application of the subject methods, compositions, reagents,
devices and kits to
humans, but it will be understood by the ordinarily skilled artisan that such
descriptions can be
readily modified to other mammals of interest based on the knowledge in the
art.
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The age of the adult mammal may vary, depending on the type of mammal that is
being
treated. Where the adult mammal is a human, the age of the human is generally
18 years or older.
In some instances, the adult mammal is an individual suffering from or at risk
of suffering from
an aging-associated impairment, such as an aging-associated cognitive
impairment, where the
adult mammal may be one that has been determined, e.g., in the form of
receiving a diagnosis, to
be suffering from or at risk of suffering from an aging associated impairment,
such as an aging-
associated cognitive impairment. The phrase -an individual suffering from or
at risk of suffering
from an aging-associated cognitive impairment" refers to an individual that is
about 50 years old
or older, e.g., 60 years old or older, 70 years old or older, 80 years old or
older, and sometimes no
older than 100 years old, such as 90 years old, i.e., between the ages of
about 50 and 100, e.g.. 50,
55, 60, 65, 70, 75, 80, 85 or about 90 years old. The individual may suffer
from an aging associated
condition, e.g., cognitive impairment, associated with the natural aging
process, e.g., M.C.I.
Alternatively, the individual may be 50 years old or older, e.g.. 60 years old
or older, 70 years old
or older, 80 years old or older, 90 years old or older, and sometimes no older
than 100 years old,
i.e., between the ages of about 50 and 100, e.g., 50, 55, 60, 65, 70. 75, 80,
85, 90, 95 or about 100
years old, and has not yet begun to show symptoms of an aging associated
condition, e.g., cognitive
impairment. In yet other embodiments, the individual may be of any age where
the individual is
suffering from a cognitive impairment due to an aging-associated disease,
e.g., Alzheimer's
disease, Parkinson's disease, frontotemporal dementia, Huntington's disease,
amyotrophic lateral
sclerosis, multiple sclerosis, glaucoma, myotonic dystrophy, dementia, and the
like. In some
instances, the individual is an individual of any age that has been diagnosed
with an aging-
associated disease that is typically accompanied by cognitive impairment,
e.g., Alzheimer's
disease, Parkinson's disease. frontotemporal dementia, progressive
supranuclear palsy,
Huntington's disease, amyotrophic lateral sclerosis, spinal muscular atrophy,
multiple sclerosis,
multi-system atrophy, glaucoma, ataxias, myotonic dystrophy, dementia, and the
like, where the
individual has not yet begun to show symptoms of cognitive impairment.
As summarized above, aspects of the methods include reducing levels of or
decreasing the
activity of the trefoil factor family peptide 2 (TFF2) in the mammal in a
manner sufficient to treat
the aging impairment in the mammal, e.g., as described above. By reducing the
TFF2 level is
meant lowering the amount of TFF2 in the mammal, such as the amount of
extracellular TFF2 in
the mammal. By decreasing the activity of the TFF2 peptide is meant lowering
the ability of TFF2
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to act through its mechanism of action, for example, its ability to
specifically bind to a receptor or
such as through providing an agent that interferes with such binding.
Decreasing the activity also
may mean interfering with the ability of TFF2 to interact with a substrate
molecule necessary for
TFF2 to produce its detrimental effects on aging or cognition. While the
magnitude of the
reduction or decreasing may vary, in some instances the magnitude is 2-fold or
greater, such as 5-
fold or greater, including 10-fold or greater, e.g., 15-fold or greater, 20-
fold or greater, 25-fold or
greater (as compared to a suitable control), where in some instances the
magnitude is such that the
amount of detectable free TFF2 in the circulatory system of the individual is
50% or less, such as
25 % or less, including 10% or less, e.g., 1 % or less, relative to the amount
that was detectable
prior to intervention according to the invention, and in some instances the
amount is undetectable
following intervention.
The TFF2 level may be reduced using any convenient protocol. In some
instances, the
TFF2 level is reduced by removing systemic TFF2 from the adult mammal, e.g.,
by removing
TFF2 from the circulatory system of the adult mammal. In such instances, any
convenient protocol
for removing circulatory TFF2 may be employed. For example, blood may be
obtained from the
adult mammal and extra-corporeally processed to remove TFF2 from the blood to
produce TFF2
depleted blood, which resultant TFF2 depleted blood may then be returned to
the adult mammal.
Such protocols may employ a variety of different techniques in order to remove
TFF2 from the
obtained blood. For example, the obtained blood may be contacted with a
filtering component,
e.g., a membrane, etc., which allows passage of TFF2 but inhibits passage of
other blood
components, e.g., cells, etc. In some instances, the obtained blood may be
contacted with a TFF2
absorptive component, e.g., porous bead or particulate composition, which
absorbs TFF2 from the
blood. In some instances, the obtained blood may be contacted with a TTF2-
specific antibody
which selectively binds to TFF2, reducing its blood levels. In yet other
instances, the obtained
blood may be contacted with a TFF2 binding member stably associated with a
solid support, such
that TFF2 binds to the binding member and is thereby immobilized on the solid
support, thereby
providing for separation of TFF2 from other blood constituents. The protocol
employed may or
may not be configured to selectively remove TFF2 from the obtained blood, as
desired.
In some embodiments, the TFF2 level is reduced by administering to the mammal
an
effective amount of a TFF2 level reducing agent. As such, in practicing
methods according to these
embodiments of the invention, an effective amount of the active agent, e.g.,
TFF2 modulatory
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agent, is provided to the adult mammal. In embodiments, of interest as TFF2
modulatory agents
are specific TFF2 level reducing agents, by which is meant agents that
selectively reduce TFF2
levels to a greater extent than other factors. By way of example and not
limitation, such other
factors can be other secreted factors such as epidermal growth factor, nerve
growth factor,
fibroblast growth factor, tumor necrosis factor alpha, thrombopoietin, insulin-
like growth factor 1,
insulin-like growth factor binding protein 3 and erythropoietin, as well as
other proteins of the
trefoil factor family such as trefoil factor 1 (TFF1) and trefoil factor 3
(TFF3).
Depending on the particular embodiments being practiced, a variety of
different types of
active agents may be employed. In some instances, the agent modulates
expression of the RNA
and/or protein from the gene, such that it changes the expression of the RNA
or protein from the
target gene in some manner. In these instances, the agent may change
expression of the RNA or
protein in a number of different ways. In certain embodiments, the agent is
one that reduces,
including inhibits, expression of a TFF2 protein. Inhibition of TFF2 protein
expression may be
accomplished using any convenient means, including use of an agent that
inhibits TFF2 protein
expression, such as, but not limited to: RNAi agents, antisense agents, agents
that interfere with a
transcription factor binding to a promoter sequence of the TFF2 gene, or
inactivation of the TFF2
gene, e.g., through recombinant techniques, etc.
For example, the transcription level of a TFF2 protein can be regulated by
gene silencing
using RNAi agents, e.g., double-strand RNA (see e.g., Sharp, Genes and
Development (1999) 13:
139-141). RNAi, such as double-stranded RNA interference (dsRNAi) or small
interfering RNA
(siRNA), has been extensively documented in the nematode C. elegans (Fire, et
al, Nature (1998)
391:806-811) and routinely used to "knock down" genes in various systems. RNAi
agents may be
dsRNA or a transcriptional template of the interfering ribonucleic acid which
can be used to
produce dsRNA in a cell. In these embodiments, the transcriptional template
may be a DNA that
encodes the interfering ribonucleic acid. Methods and procedures associated
with RNAi are also
described in published PCT Application Publication Nos. WO 03/010180 and WO
01/68836, the
disclosures of which applications are incorporated herein by reference. dsRNA
can be prepared
according to any of a number of methods that are known in the art, including
in vitro and in vivo
methods, as well as by synthetic chemistry approaches. Examples of such
methods include, but
are not limited to, the methods described by Sadher et al., Biochem. Int.
(1987) 14:1015;
Bhattacharyya, Nature (1990) 343:484; and U.S. Pat. No. 5,795,715, the
disclosures of which are
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incorporated herein by reference. Single-stranded RNA can also be produced
using a combination
of enzymatic and organic synthesis or by total organic synthesis. The use of
synthetic chemical
methods enables one to introduce desired modified nucleotides or nucleotide
analogs into the
dsRNA. dsRNA can also be prepared in vivo according to a number of established
methods (see,
e.g., Sambrook, et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed.;
Transcription and
Translation (B. D. Hames, and S. J. Higgins, Eds., 1984); DNA Cloning, volumes
I and II (D. N.
Glover, Ed., 1985); and Oligonucleotide Synthesis (M. J. Gait, Ed., 1984, each
of which is
incorporated herein by reference). A number of options can be utilized to
deliver the dsRNA into
a cell or population of cells such as in a cell culture, tissue, organ or
embryo. For instance, RNA
can be directly introduced intracellularly. Various physical methods are
generally utilized in such
instances, such as administration by microinjection (see, e.g.. Zemicka-Goetz,
et al. Development
(1997)124:1133-1137; and Wianny, et al., Chromosoma (1998) 107: 430-439).
Other options for
cellular delivery include permeabilizing the cell membrane and electroporation
in the presence of
the dsRNA, liposome-mediated transfection, or transfection using chemicals
such as calcium
phosphate. A number of established gene therapy techniques can also be
utilized to introduce the
dsRNA into a cell. By introducing a viral construct within a viral particle,
for instance, one can
achieve efficient introduction of an expression construct into the cell and
transcription of the RNA
encoded by the construct. Specific examples of RNAi agents that may be
employed to reduce TFF2
expression include but are not limited to commercially-available TFF2 siRNAs
(see, e.g.,
MyBioSource (San Diego, CA) which provides a commercially-available human TFF2
siRNA
(1#MBS8204153); OriGene Technologies (Rockville, MD) which provides three
unique
commercially-available 27mer human siRNA or shRNA duplexes targeting TFF2
(Item Nos.
SR304798, TL308865, TR308865); and ThermoFisher Scientific provides a
commercially-
available human TFF2 siRNA (Catalog No. AM16708).)
In some instances, antisense molecules can be used to down-regulate expression
of a TFF2
gene in the cell. The anti-sense reagent may be antisense
oligodeoxynucleotides (ODN),
particularly synthetic ODN having chemical modifications from native nucleic
acids, or nucleic
acid constructs that express such anti-sense molecules as RNA. The antisense
sequence is
complementary to the mRNA of the targeted protein and inhibits expression of
the targeted protein.
Antisense molecules inhibit gene expression through various mechanisms, e.g.,
by reducing the
amount of mRNA available for translation, through activation of RNAse H, or
steric hindrance.
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One or a combination of antisense molecules may be administered, where a
combination may
include multiple different sequences.
Antisense molecules may be produced by expression of all or a part of the
target gene
sequence in an appropriate vector, where the transcriptional initiation is
oriented such that an
antisense strand is produced as an RNA molecule. Alternatively, the antisense
molecule is a
synthetic oligonucleotide. Antisense oligonucleotides will generally be at
least about 7, usually at
least about 12, more usually at least about 20 nucleotides in length, and not
more than about 500,
usually not more than about 50, more usually not more than about 35
nucleotides in length, where
the length is governed by efficiency of inhibition, specificity, including
absence of cross-reactivity,
and the like. Short oligonucleotides, of from 7 to 8 bases in length, can be
strong and selective
inhibitors of gene expression (see Wagner et al., Nature Biotechnol. (1996)
14:840-844).
A specific region or regions of the endogenous sense strand mRNA sequence are
chosen
to be complemented by the antisense sequence. Selection of a specific sequence
for the
oligonucleotide may use an empirical method, where several candidate sequences
are assayed for
inhibition of expression of the target gene in an in vitro or animal model. A
combination of
sequences may also be used, where several regions of the mRNA sequence are
selected for
antisense complementation.
Antisense oligonucleotides may be chemically synthesized by methods known in
the art
(see Wagner et al. (1993), supra.). Oligonucleotides may be chemically
modified from the native
phosphodiester structure, in order to increase their intracellular stability
and binding affinity. A
number of such modifications have been described in the literature, which
alter the chemistry of
the backbone, sugars or heterocyclic bases. Among useful changes in the
backbone chemistry are
phosphorothioates; phosphorodithioates, where both of the non-bridging oxygens
are substituted
with sulfur; phosphoroamidites; alkyl phosphotriesters and boranophosphates.
Achiral phosphate
derivatives include 3'-0-5'-S -pho sphorothio ate, 3'-S -5'-0-pho sphorothio
ate, 3'-CH. sub .2-5'-0-
phosphonate and 3'-NH-5'-Ophosphoroamidate. Peptide nucleic acids replace the
entire ribose
phosphodiester backbone with a peptide linkage. Sugar modifications are also
used to enhance
stability and affinity. The ct-anomer of deoxyribose may be used, where the
base is inverted with
respect to the natura113-anomer. The 2'-OH of the ribose sugar may be altered
to form 2'-0-methyl
or 2'-0-ally1 sugars, which provides resistance to degradation without
comprising affinity.
Modification of the heterocyclic bases must maintain proper base pairing. Some
useful
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substitutions include deoxyuridine for deoxythymidine; 5-methyl-2-
deoxycytidine and 5-bromo-
2'-deoxycytidine for deoxycytidine. 5-propyny1-2'-deoxyuridine and 5-propyny1-
2'-deoxycytidine
have been shown to increase affinity and biological activity when substituted
for deoxythymidine
and deoxycytidine, respectively.
As an alternative to anti-sense inhibitors, catalytic nucleic acid compounds,
e.g. ribozymes,
anti-sense conjugates, etc. may be used to inhibit gene expression. Ribozymes
may be synthesized
in vitro and administered to the patient, or may be encoded on an expression
vector, from which
the ribozyme is synthesized in the targeted cell (for example, see
International patent application
WO 9523225, and Beigelman et al. Nucl. Acids Res. (1995) 23:4434-42). Examples
of
oligonucleotides with catalytic activity are described in WO 9506764.
Conjugates of anti-sense
ODN with a metal complex, e.g. terpyridylCu(II), capable of mediating mRNA
hydrolysis are
described in Bashkin et al. Appl. Biochem. Biotechnol. (1995) 54:43-56.
In another embodiment, the TFF2 gene is inactivated so that it no longer
expresses a
functional protein. By inactivated is meant that the gene, e.g., coding
sequence and/or regulatory
elements thereof, is genetically modified so that it no longer expresses a
functional TFF2 protein,
e.g., at least with respect to TFF2 aging impairment activity. The alteration
or mutation may take
a number of different forms, e.g., through deletion of one or more nucleotide
residues, through
exchange of one or more nucleotide residues, and the like. One means of making
such alterations
in the coding sequence is by homologous recombination. Methods for generating
targeted gene
modifications through homologous recombination are known in the art, including
those described
in: U.S. Pat. Nos. 6,074,853; 5,998,209; 5,998,144; 5,948,653; 5,925,544;
5,830,698; 5,780,296;
5,776,744; 5,721,367; 5,614,396; 5,612,205; the disclosures of which are
herein incorporated by
reference.
Also of interest in certain embodiments are dominant negative mutants of TFF2
proteins,
where expression of such mutants in the cell result in a modulation, e.g.,
decrease, in TFF2
mediated aging impairment. Dominant negative mutants of TFF2 are mutant
proteins that exhibit
dominant negative TFF2 activity. As used herein, the term "dominant-negative
TFF2 activity" or
"dominant negative activity" refers to the inhibition, negation, or diminution
of certain particular
activities of TFF2, and specifically to TFF2 mediated aging impairment.
Dominant negative
mutations are readily generated for corresponding proteins. These may act by
several different
mechanisms, including mutations in a substrate-binding domain; mutations in a
catalytic domain;
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mutations in a protein binding domain (e.g., multimer forming, effector, or
activating protein
binding domains); mutations in cellular localization domain, etc. A mutant
polypeptide may
interact with wild-type polypeptides (made from the other allele) and form a
non-functional
multimer. In certain embodiments, the mutant polypeptide will be overproduced.
Point mutations
are made that have such an effect. In addition, fusion of different
polypeptides of various lengths
to the terminus of a protein, or deletion of specific domains can yield
dominant negative mutants.
General strategies are available for making dominant negative mutants (see for
example,
Herskowitz, Nature (1987) 329:219, and the references cited above). Such
techniques are used to
create loss of function mutations, which are useful for determining protein
function. Methods that
are well known to those skilled in the art can be used to construct expression
vectors containing
coding sequences and appropriate transcriptional and translational control
signals for increased
expression of an exogenous gene introduced into a cell. These methods include,
for example, in
vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic
recombination.
Alternatively, RNA capable of encoding gene product sequences may be
chemically synthesized
using, for example, synthesizers. See, for example, the techniques described
in "Oligonucleotide
Synthesis", 1984, Gait, M. J. ed., IRL Press, Oxford.
In yet other embodiments, the agent is an agent that modulates, e.g.,
inhibits, TFF2 activity
by binding to TFF2 and/or inhibiting binding of TFF2 to a second protein,
e.g., interleukin 1f3. For
example, small molecules that bind to the TFF2 and inhibit its activity are of
interest. Naturally
occurring or synthetic small molecule compounds of interest include numerous
chemical classes,
such as organic molecules, e.g., small organic compounds having a molecular
weight of more than
50 and less than about 2,500 daltons. Candidate agents comprise functional
groups for structural
interaction with proteins, particularly hydrogen bonding, and typically
include at least an amine,
carbonyl, hydroxyl or carboxyl group, preferably at least two of the
functional chemical groups.
The candidate agents may include cyclical carbon or heterocyclic structures
and/or aromatic or
polyaromatic structures substituted with one or more of the above functional
groups. Candidate
agents are also found among biomolecules including peptides, saccharides,
fatty acids, steroids,
purines, pyrimidines, derivatives, structural analogs or combinations thereof.
Such molecules may
be identified, among other ways, by employing the screening protocols
described below.
In certain embodiments, the administered active agent is a TFF2 specific
binding member.
The term "specific binding" refers to a direct association between two
molecules, due to, for
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example, covalent, electrostatic, hydrophobic, and ionic and/or hydrogen-bond
interactions,
including interactions such as salt bridges and water bridges. A specific
binding member describes
a member of a pair of molecules which have binding specificity for one
another. The members of
a specific binding pair may be naturally derived or wholly or partially
synthetically produced. One
member of the pair of molecules has an area on its surface, or a cavity, which
specifically binds to
and is therefore complementary to a particular spatial and polar organization
of the other member
of the pair of molecules. Thus, the members of the pair have the property of
binding specifically
to each other. Examples of pairs of specific binding members are antigen-
antibody, biotin-avidin,
hormone-hormone receptor, receptor-ligand, enzyme-substrate. Specific binding
members of a
binding pair exhibit high affinity and binding specificity for binding with
the each other. In general,
useful TFF2 specific binding members exhibit an affinity (Kd) for a target
TFF2, such as human
TFF2, that is sufficient to provide for the desired reduction in aging
associated impairment TFF2
activity. As used herein, the term "affinity" refers to the equilibrium
constant for the reversible
binding of two agents; "affinity" can be expressed as a dissociation constant
(Kd). Affinity can be
at least 1-fold greater, at least 2-fold greater, at least 3-fold greater, at
least 4-fold greater, at least
5-fold greater, at least 6-fold greater, at least 7-fold greater, at least 8-
fold greater, at least 9-fold
greater, at least 10-fold greater, at least 20-fold greater, at least 30-fold
greater, at least 40-fold
greater, at least 50-fold greater, at least 60-fold greater, at least 70-fold
greater, at least 80-fold
greater, at least 90-fold greater, at least 100-fold greater, or at least 1000-
fold greater, or more,
than the affinity of an antibody for unrelated amino acid sequences. Affinity
of a specific binding
member to a target protein can be, for example, from about 100 nanomolar (nM)
to about 0.1 nM,
from about 100 nM to about 1 picomolar (pM), or from about 100 nM to about 1
femtomolar (fM)
or more. The term "binding" refers to a direct association between two
molecules, due to, for
example, covalent, electrostatic, hydrophobic, and ionic and/or hydrogen-bond
interactions,
including interactions such as salt bridges and water bridges. In some
embodiments, the antibodies
bind human TFF2 with nanomolar affinity or picomolar affinity. In some
embodiments, the
antibodies bind human TFF2 with a Kd of less than about 100 nM, 50 nM, 20 nM,
20 nM, or 1
nM. In an embodiment, affinity is determined by surface plasmon resonance
(SPR), e.g. as used
by Biacore systems. The affinity of one molecule for another molecule is
determined by measuring
the binding kinetics of the interaction, e.g. at 25 C.
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Examples of TFF2 specific binding members include TFF2 antibodies and binding
fragments thereof. Non-limiting examples of such antibodies include antibodies
directed against
any epitope of TFF2. Examples of said epitopes include, by way of example and
not limitation
the amino acid sequences of SEQ ID NO: 01, SEQ ID NO: 02, SEQ ID NO: 03, SEQ
ID NO: 04,
SEQ ID NO: 05, SEQ ID NO: 06, SEQ ID NO: 07, SEQ ID NO: 08, SEQ ID NO: 09, SEQ
ID
NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12. In some embodiment of the invention,
said epitopes
have at least about any of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,
or 100%
sequence identity to the amino acid sequences of SEQ ID NO: 01, SEQ ID NO: 02,
SEQ ID NO:
03, SEQ ID NO: 04, SEQ ID NO: 05, SEQ TD NO: 06, SEQ ID NO: 07, SEQ TD NO: 08,
SEQ ID
NO: 09, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12.
Also encompassed are bispecific antibodies, i.e., antibodies in which each of
the two
binding domains recognizes a different binding epitope. The amino acid
sequence of human TFF2
is disclosed in May, F.E.B. & Semple, Jennifer & Newton, J.L. & Westley, B.R.,
-The human two
domain trefoil protein, TFF2, is glycosylated in vivo in the stomach, Gut.
(2000) 46: 454-459.
Antibody specific binding members that may be employed include full antibodies
or
immunoglobulins of any isotype, as well as fragments of antibodies which
retain specific binding
to antigen, including, but not limited to, Fab, Fv, scFv, and Fd fragments,
chimeric antibodies,
humanized antibodies, single-chain antibodies, and fusion proteins comprising
an antigen-binding
portion of an antibody and a non-antibody protein. The antibodies may be
detectably labeled, e.g.,
with a radioisotope, an enzyme which generates a detectable product, a
fluorescent protein, and
the like. The antibodies may be further conjugated to other moieties, such as
members of specific
binding pairs, e.g., biotin (member of biotinavidin specific binding pair),
and the like. Also
encompassed by the term are Fab', Fv, F(ab')2, and or other antibody fragments
that retain specific
binding to antigen, and monoclonal antibodies. An antibody may be monovalent
or bivalent.
"Antibody fragments" comprise a portion of an intact antibody, for example,
the antigen
binding or variable region of the intact antibody. Examples of antibody
fragments include Fab,
Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies (Zapata et al.,
Protein Eng. 8(10):
1057-1062 (1995)); single-chain antibody molecules; and multi-specific
antibodies formed from
antibody fragments. Papain digestion of antibodies produces two identical
antigen-binding
fragments, called "Fab" fragments, each with a single antigen binding site,
and a residual "Fc"
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fragment, a designation reflecting the ability to crystallize readily. Pepsin
treatment yields an
F(ab')2 fragment that has two antigen combining sites and is still capable of
cross-linking antigen.
"Fv" is the minimum antibody fragment which contains a complete antigen
recognition and
-binding site. This region consists of a dimer of one heavy- and one light
chain variable domain in
tight, non-covalent association. It is in this configuration that the three
CDRS of each variable
domain interact to define an antigen-binding site on the surface of the VH-VL
dimer. Collectively,
the six CDRs confer antigen-binding specificity to the antibody. However, even
a single variable
domain (or half of an Fv comprising only three CDRs specific for an antigen)
has the ability to
recognize and bind antigen, although at a lower affinity than the entire
binding site.
The "Fab" fragment also contains the constant domain of the light chain and
the first
constant domain (CH1) of the heavy chain. Fab fragments differ from Fab'
fragments by the
addition of a few residues at the carboxyl terminus of the heavy chain CH1
domain including one
or more cysteines from the antibody hinge region. Fab'-SH is the designation
herein for Fab' in
which the cysteine residue(s) of the constant domains bear a free thiol group.
F(abt)2 antibody
fragments originally were produced as pairs of Fab' fragments which have hinge
cysteines between
them. Other chemical couplings of antibody fragments are also known.
The "light chains" of antibodies (immunoglobulins) from any vertebrate species
can be
assigned to one of two clearly distinct types, called kappa and lambda, based
on the amino acid
sequences of their constant domains. Depending on the amino acid sequence of
the constant
domain of their heavy chains, immunoglobulins can be assigned to different
classes. There are five
major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of
these may be
further divided into subclasses (isotypes), e.g., IgGl, IgG2, IgG3, IgG4, IgA,
and IgA2.
"Single-chain Fv" or " sFv" antibody fragments comprise the VH and VL domains
of
antibody, wherein these domains are present in a single polypeptide chain. In
some embodiments,
the Fv polypeptide further comprises a polypeptide linker between the VH and
VL domains, which
enables the sFy to form the desired structure for antigen binding. For a
review of sFv, see
Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg
and Moore eds.,
Springer-Verlag, New York, pp. 269-315 (1994).
Antibodies that may be used in connection with the present disclosure thus can
encompass
monoclonal antibodies, polyclonal antibodies, bispecific antibodies, Fab
antibody fragments,
F(ab)2 antibody fragments, Fv antibody fragments (e.g., VH or VL), single
chain Fv antibody
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fragments and dsFy antibody fragments. Furthermore, the antibody molecules may
be fully human
antibodies, humanized antibodies, or chimeric antibodies. In some embodiments,
the antibody
molecules are monoclonal, fully human antibodies.
The antibodies that may be used in connection with the present disclosure can
include any
antibody variable region, mature or unprocessed, linked to any immunoglobulin
constant region.
If a light chain variable region is linked to a constant region, it can be a
kappa chain constant
region. If a heavy chain variable region is linked to a constant region, it
can be a human gamma 1,
gamma 2, gamma 3 or gamma 4 constant region, more preferably, gamma 1, gamma 2
or gamma
4 and even more preferably gamma 1 or gamma 4.
In some embodiments, fully human monoclonal antibodies directed against TFF2
are
generated using transgenic mice carrying parts of the human immune system
rather than the mouse
system.
Methods of creating polyclonal and monoclonal antibodies are well known to
those having
ordinary skill in the art. (See Leenaars M, et at., ILAR J, 46(3):269-79
(2005) and Lu R-M, et at.,
J Biomed Sci, 27(1) (2020)). Antigens are prepared in order to raise
antibodies to the antigens in
animal species such as mice, rabbits, goats, rats, sheep, chicken, hamster,
and guinea pig by
immunizing said animals with an antigen. Adjuvants can also be administered in
order to help
provoke an immune response. Immunization can be administered via subcutaneous,
intradermal,
intramuscular, intraperitoneal, or intravenous means for example. Booster
injections may follow,
depending on the impact of initial immunization. The antibody response is
monitored and
exsanguination often by euthanasia and heart puncture follows, with
purification of the antibodies.
Monoclonal antibodies (MAbs) may be created, for example, by producing a
single clone of B
cells, which are capable of being immortalized by fusion with myeloma cells.
This creates a
hybridoma cell line that is able to produce an unlimited quantity of MAbs.
Minor variations in the amino acid sequences of antibodies or immunoglobulin
molecules
are encompassed by the present invention, providing that the variations in the
amino acid sequence
maintain at least 75%, e.g., at least 80%, 90%, 95%, or 99% of the sequence.
In particular,
conservative amino acid replacements are contemplated. Conservative
replacements are those that
take place within a family of amino acids that are related in their side
chains. Whether an amino
acid change results in a functional peptide can readily be determined by
assaying the specific
activity of the polypeptide derivative. Fragments (or analogs) of antibodies
or immunoglobulin
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molecules can be readily prepared by those of ordinary skill in the art.
Preferred amino- and
carboxy-termini of fragments or analogs occur near boundaries of functional
domains. Structural
and functional domains can be identified by comparison of the nucleotide
and/or amino acid
sequence data to public or proprietary sequence databases. Preferably,
computerized comparison
methods are used to identify sequence motifs or predicted protein conformation
domains that occur
in other proteins of known structure and/or function. Methods to identify
protein sequences that
fold into a known three-dimensional structure are known. Sequence motifs and
structural
conformations may be used to define structural and functional domains in
accordance with the
invention.
Specific examples of antibody agents that may be employed to reduce TFF2
expression or
activity include, but are not limited to commercially-available antibodies
(see, e.g., MyBioSource
(San Diego, CA) which provides a commercially-available human anti-TFF2
polyclonal antibody
(#MBS9125301); LifeSpan Biosciences (Seattle, WA) which provides a
commercially-available
human anti-TFF2 polyclonal antibody (Catalog No. LS -A9840-50); R&D Systems
(Minneapolis,
MN) which provides a commercially-available human anti-TFF2 monoclonal
antibody (Catalog
No. MAB4077); Biorbyt (Cambridge, UK) which provides a commercially-available
human anti-
TFF2 (Catalog No. orb197800).; ThermoFisher Scientific which provides a
commercially-
available human anti-TFF2 monoclonal antibody (Catalog No. 4G7C3); and other
Anti-TFF2
human antibodies that have also been described before. (See, e.g., Siu L-S, et
al., Peptides,
25(5):855-63 (2004)). Methods of making and designing monoclonal antibodies
are commonly
known to those having ordinary skill in the art and include for example,
Greenfield EA, Antibodies:
A Laboratory manual, 2nd ed. (2014) and Kohler G, et al., Continuous cultures
of fused cells
secreting antibody of predefined specificity, Nature 256:495-97 (1975) which
are herein
incorporated by reference in their entirety).
In those embodiments where an active agent is administered to the adult
mammal, the
active agent(s) may be administered to the adult mammal using any convenient
administration
protocol capable of resulting in the desired activity. Thus, the agent can be
incorporated into a
variety of formulations, e.g., pharmaceutically acceptable vehicles, for
therapeutic administration.
More particularly, the agents of the present invention can be formulated into
pharmaceutical
compositions by combination with appropriate, pharmaceutically acceptable
carriers or diluents,
and may be formulated into preparations in solid, semi-solid, liquid or
gaseous forms, such as
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tablets, capsules, powders, granules, ointments (e.g., skin creams),
solutions, suppositories,
injections, inhalants and aerosols. As such, administration of the agents can
be achieved in various
ways, including oral, buccal, rectal, parenteral, intraperitoneal,
intradermal, transdermal,
intracheal , etc., administration.
In pharmaceutical dosage forms, the agents may be administered in the form of
their
pharmaceutically acceptable salts, or they may also be used alone or in
appropriate association, as
well as in combination, with other pharmaceutically active compounds. The
following methods
and excipients are merely exemplary and are in no way limiting.
For oral preparations, the agents can be used alone or in combination with
appropriate
additives to make tablets, powders, granules or capsules, for example, with
conventional additives,
such as lactose, mannitol, corn starch or potato starch; with binders, such as
crystalline cellulose,
cellulose derivatives, acacia, corn starch or gelatins; with disintegrators,
such as corn starch, potato
starch or sodium carboxymethylcellulose; with lubricants, such as talc or
magnesium stearate; and
if desired, with diluents, buffering agents, moistening agents, preservatives
and flavoring agents.
The agents can be formulated into preparations for injection by dissolving,
suspending or
emulsifying them in an aqueous or nonaqueous solvent, such as vegetable or
other similar oils,
synthetic aliphatic acid glycerides, esters of higher aliphatic acids or
propylene glycol; and if
desired, with conventional additives such as solubilizers, isotonic agents,
suspending agents,
emulsifying agents, stabilizers and preservatives.
The agents can be utilized in aerosol formulation to be administered via
inhalation. The
compounds of the present invention can be formulated into pressurized
acceptable propellants such
as dichlorodifluoromethane, propane, nitrogen and the like.
Furthermore, the agents can be made into suppositories by mixing with a
variety of bases
such as emulsifying bases or water-soluble bases. The compounds of the present
invention can be
administered rectally via a suppository. The suppository can include vehicles
such as cocoa butter,
carbowaxes and polyethylene glycols, which melt at body temperature, yet are
solidified at room
temperature.
Unit dosage forms for oral or rectal administration such as syrups, elixirs,
and suspensions
may be provided wherein each dosage unit, for example, teaspoonful,
tablespoonful, tablet or
suppository, contains a predetermined amount of the composition containing one
or more
inhibitors. Similarly, unit dosage forms for injection or intravenous
administration may comprise
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the inhibitor(s) in a composition as a solution in sterile water, normal
saline or another
pharmaceutically acceptable carrier.
The term "unit dosage form," as used herein, refers to physically discrete
units suitable as
unitary dosages for human and animal subjects, each unit containing a
predetermined quantity of
compounds of the present invention calculated in an amount sufficient to
produce the desired effect
in association with a pharmaceutically acceptable diluent, carrier or vehicle.
The specifications for
the novel unit dosage forms of the present invention depend on the particular
compound employed
and the effect to be achieved, and the pharmacodynamics associated with each
compound in the
host.
The pharmaceutically acceptable excipients, such as vehicles, adjuvants,
carriers or
diluents, are readily available to the public. Moreover, pharmaceutically
acceptable auxiliary
substances, such as pH adjusting and buffering agents, tonicity adjusting
agents, stabilizers,
wetting agents and the like, are readily available to the public.
Where the agent is a polypeptide, polynucleotide, analog or mimetic thereof,
it may be
introduced into tissues or host cells by any number of routes, including viral
infection,
microinjection, or fusion of vesicles. Jet injection may also be used for
intramuscular
administration, as described by Furth et al., Anal Biochem. (1992) 205:365-
368. The DNA may
be coated onto gold microparticles, and delivered intradermally by a particle
bombardment device,
or "gene gun" as described in the literature (see, for example, Tang et al..
Nature (1992) 356:152-
154), where gold microprojectiles are coated with the DNA, then bombarded into
skin cells. For
nucleic acid therapeutic agents, a number of different delivery vehicles find
use, including viral
and non-viral vector systems, as are known in the art.
Those of skill in the art will readily appreciate that dose levels can vary as
a function of the
specific compound, the nature of the delivery vehicle, and the like. Preferred
dosages for a given
compound are readily determinable by those of skill in the art by a variety of
means.
In those embodiments where an effective amount of an active agent is
administered to the
adult mammal, the amount or dosage is effective when administered for a
suitable period of time,
such as one week or longer, including two weeks or longer, such as 3 weeks or
longer, 4 weeks or
longer, 8 weeks or longer, etc., so as to evidence a reduction in the
impairment, e.g., cognition
decline and/or cognitive improvement in the adult mammal. For example, an
effective dose is the
dose that, when administered for a suitable period of time, such as at least
about one week, and
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maybe about two weeks, or more, up to a period of about 3 weeks, 4 weeks, 8
weeks, or longer,
will slow e.g., by about 20% or more, e.g., by 30% or more, by 40% or more, or
by 50% or more,
in some instances by 60% or more, by 70% or more, by 80% or more, or by 90% or
more, e.g.,
will halt, cognitive decline in a patient suffering from natural aging or an
aging-associated
disorder. In some instances, an effective amount or dose of active agent will
not only slow or halt
the progression of the disease condition but will also induce the reversal of
the condition, i.e., will
cause an improvement in cognitive ability. For example, in some instances, an
effective amount is
the amount that when administered for a suitable period of time, usually at
least about one week,
and maybe about two weeks, or more, up to a period of about 3 weeks, 4 weeks,
8 weeks, or longer
will improve the cognitive abilities of an individual suffering from an aging
associated cognitive
impairment by, for example 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, in some
instances 6-fold, 7-
fold, 8-fold, 9-fold. or 10-fold or more relative to cognition prior to
administration of the blood
product.
Where desired, effectiveness of treatment may be assessed using any convenient
protocol.
Cognition tests and IQ test for measuring cognitive ability, e.g., attention
and concentration, the
ability to learn complex tasks and concepts, memory, information processing,
visuospatial
function, the ability to produce and understanding language, the ability to
solve problems and make
decisions, and the ability to perform executive functions, are well known in
the art, any of which
may be used to measure the cognitive ability of the individual before and/or
during and after
treatment with the subject blood product, e.g., to confirm that an effective
amount has been
administered. These include, for example, the General Practitioner Assessment
of Cognition
(GPCOG) test, the Memory Impairment Screen, the Mini Mental State Examination
(MMSE). the
California Verbal Learning Test, Second Edition, Short Form, for memory, the
Delis-Kaplan
Executive Functioning System test, the Alzheimer's Disease Assessment Scale
(ADAS-Cog). the
Psychogeriatric Assessment Scale (PAS) and the like. Progression of functional
brain
improvements may be detected by brain imaging techniques, such as Magnetic
Resonance Imaging
(MRI) or Positron Emission Tomography (PET) and the like. A wide range of
additional functional
assessments may be applied to monitor activities of daily living, executive
functions, mobility, etc.
In some embodiments, the method comprises the step of measuring cognitive
ability, and detecting
a decreased rate of cognitive decline, a stabilization of cognitive ability,
and/or an increase in
cognitive ability after administration of the blood product as compared to the
cognitive ability of
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the individual before the blood product was administered. Such measurements
may be made a
week or more after administration of the blood product, e.g., 1 week, 2 weeks,
3 weeks, or more,
for instance, 4 weeks, 6 weeks, or 8 weeks or more, e.g.. 3 months, 4 months,
5 months, or 6
months or more.
Biochemically, by an "effective amount" or "effective dose" of active agent is
meant an
amount of active agent that will inhibit, antagonize, decrease, reduce, or
suppress by about 20%
or more, e.g., by 30% or more, by 40% or more, or by 50% or more, in some
instances by 60% or
more, by 70% or more, by 80% or more, or by 90% or more, in some cases by
about 100%, i.e., to
negligible amounts, and in some instances reverse, the reduction in synaptic
plasticity and loss of
synapses that occurs during the natural aging process or during the
progression of an aging-
associated disorder. In other words, cells present in adult mammals treated in
accordance with
methods of the invention will become more responsive to cues, e.g., activity
cues, which promote
the formation and maintenance of synapses.
Performance of methods of the invention, e.g., as described above, may
manifest as
improvements in observed synaptic plasticity, both in vitro and in vivo as an
induction of long-
term potentiation. For example, the induction of LTP in neural circuits may be
observed in awake
individuals, e.g., by performing non-invasive stimulation techniques on awake
individuals to
induce LTP-like long-lasting changes in localized neural activity (Cooke SF,
Bliss TV (2006)
Plasticity in the human central nervous system. Brain. 129(Pt 7):1659-73);
mapping plasticity and
increased neural circuit activity in individuals, e.g., by using positron
emission tomography,
functional magnetic resonance imaging, and/or transcranial magnetic
stimulation (Cramer and
Bastings, "Mapping clinically relevant plasticity after stroke,"
Neuropharmacology (2000)39:842-
51); and by detecting neural plasticity following learning, i.e., improvements
in memory, e.g., by
assaying retrieval-related brain activity (Buchmann et al., "Prion protein
M129V polymorphism
affects retrieval-related brain activity," Neuropsychologia. (2008) 46:2389-
402) or, e.g., by
imaging brain tissue by functional magnetic resonance imaging (fMR1) following
repetition
priming with familiar and unfamiliar objects (Soldan et al., "Global
familiarity of visual stimuli
affects repetition-related neural plasticity but not repetition priming,"
Neuroimage. (2008) 39:515-
26; Soldan et al., "Aging does not affect brain patterns of repetition effects
associated with
perceptual priming of novel objects." J. Cogn. Neurosci. (2008) 20:1762-76).
In some
embodiments, the method includes the step of measuring synaptic plasticity,
and detecting a
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decreased rate of loss of synaptic plasticity, a stabilization of synaptic
plasticity, and/or an increase
in synaptic plasticity after administration of the blood product as compared
to the synaptic
plasticity of the individual before the blood product was administered. Such
measurements may
be made a week or more after administration of the blood product, e.g., 1
week, 2 weeks, 3 weeks,
or more, for instance, 4 weeks, 6 weeks, or 8 weeks or more, e.g., 3 months, 4
months, 5 months,
or 6 months or more.
In some instances, the methods result in a change in expression levels of one
or more genes
in one or more tissues of the host, e.g., as compared to a suitable control
(such as described in the
Experimental section, below). The change in expression level of a given gene
may be 0.5-fold or
greater, such as 1.0-fold or greater, including 1.5-fold or greater. The
tissue may vary, and in some
instances is nervous system tissue, e.g., central nervous system tissue,
including brain tissue, e.g.,
hippocampal tissue. In sonic instances, the modulation of hippocampal gene
expression is
manifested as enhanced hippocampal plasticity, e.g., as compared to a suitable
control.
In some instances, treatment results in an enhancement in the levels of one or
more proteins
in one or more tissues of the host, e.g., as compared to a suitable control
(such as described in the
Experimental section, below). The change in protein level of a given protein
may be 0.5 fold or
greater, such as 1.0 fold or greater, including 1.5 fold or greater, where in
some instances the level
may approach that of a healthy wild-type level, e.g., within 50% or less, such
as 25% or less,
including 10% or less, e.g., 5% or less of the healthy wild-type level. The
tissue may vary, and in
some instances is nervous system tissue, e.g., central nervous system tissue,
including brain tissue,
e.g., hippocampal tissue.
In some instances, the methods result in one or more structural changes in one
or more
tissues. The tissue may vary, and in some instances is nervous system tissue,
e.g., central nervous
system tissue, including brain tissue, e.g., hippocampal tissue. Structure
changes of interest include
an increase in dendritic spine density of mature neurons in the dentate gyrus
(DG) of the
hippocampus, e.g., as compared to a suitable control. In some instances, the
modulation of
hippocampal structure is manifested as enhanced synapse formation, e.g., as
compared to a suitable
control. In some instances, the methods may result in an enhancement of long-
term potentiation,
e.g., as compared to a suitable control.
In some instances, practice of the methods, e.g., as described above, results
in an increase
in neurogenesis in the adult mammal. The increase may be identified in a
number of different
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ways, e.g., as described below in the Experimental section. In some instances,
the increase in
neurogenesis manifests as an increase the amount of Dcx-positive immature
neurons, e.g., where
the increase may be 2-fold or greater. In some instances, the increase in
neurogenesis manifests as
an increase in the number of BrdU/NeuN positive cells, where the increase may
be 2-fold or
greater.
In some instances, the methods result in enhancement in learning and memory,
e.g., as
compared to a suitable control. Enhancement in learning and memory may be
evaluated in a
number of different ways, e.g., the contextual fear conditioning and/or radial
arm water maze
(RAWM) paradigms described in the experimental section, below. When measured
by contextual
fear conditioning, treatment results in some instances in increased freezing
in contextual, but not
cued, memory testing. When measured by RAWM, treatment results in some
instances in
enhanced learning and memory for platform location during the testing phase of
the task. In some
instances, treatment is manifested as enhanced cognitive improvement in
hippocampal-dependent
learning and memory, e.g., as compared to a suitable control.
In some embodiments, TFF2 level reduction, e.g., as described above, may be
performed
in conjunction with an active agent having activity suitable to treat aging
associated cognitive
impairment. For example, a number of active agents have been shown to have
some efficacy in
treating the cognitive symptoms of Alzheimer's disease (e.g., memory loss,
confusion, and
problems with thinking and reasoning), e.g., cholinesterase inhibitors (e.g.,
Donepezil,
Rivastigmine, Galantamine, Tacrine), Memantine, and Vitamin E. As another
example, a number
of agents have been shown to have some efficacy in treating behavioral or
psychiatric symptoms
of Alzheimer's Disease, e.g., citalopram (Celexa), fluoxetine (Prozac),
paroxeine (Paxil), sertraline
(Zoloft), trazodone (Desyrel), lorazepam (Ativan), oxazepam (Serax),
aripiprazole (Abilify),
clozapine (Clozaril), haloperidol (Haldol), olanzapine (Zyprex a), quetiapine
(Seroquel),
risperidone (Risperdal), and ziprasidone (Geodon).
In some aspects of the subject methods, the method further comprises the step
of measuring
cognition and/or synaptic plasticity after treatment, e.g., using the methods
described herein or
known in the art, and determining that the rate of cognitive decline or loss
of synaptic plasticity
have been reduced and/or that cognitive ability or synaptic plasticity have
improved in the
individual. In some such instances, the determination is made by comparing the
results of the
cognition or synaptic plasticity test to the results of the test performed on
the same individual at
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an earlier time, e.g., 2 weeks earlier, 1 month earlier, 2 months earlier, 3
months earlier, 6 months
earlier, 1 year earlier. 2 years earlier, 5 years earlier, or 10 years
earlier, or more.
In some embodiments, the subject methods further include diagnosing an
individual as
having a cognitive impairment, e.g., using the methods described herein or
known in the art for
measuring cognition and synaptic plasticity, prior to administering the
subject plasma comprising
blood product. In some instances, the diagnosing will comprise measuring
cognition and/or
synaptic plasticity and comparing the results of the cognition or synaptic
plasticity test to one or
more references, e.g., a positive control and/or a negative control. For
example, the reference may
be the results of the test performed by one or more age matched individuals
that experience aging-
associated cognitive impairments (i.e., positive controls) or that do not
experience aging-
associated cognitive impairments (i.e., negative controls). As another
example, the reference may
be the results of the test performed by the same individual at an earlier
time, e.g., 2 weeks earlier,
1 month earlier, 2 months earlier, 3 months earlier, 6 months earlier, 1 year
earlier, 2 years earlier,
years earlier, or 10 years earlier, or more.
In some embodiments, the subject methods further comprise diagnosing an
individual as
having an aging-associated disorder, e.g., Alzheimer's disease, Parkinson's
disease,
frontotemporal dementia, progressive supranuclear palsy, Huntington's disease,
amyotrophic
lateral sclerosis, spinal muscular atrophy, multiple sclerosis, multi-system
atrophy, glaucoma,
ataxias, myotonic dystrophy, dementia, and the like. Methods for diagnosing
such aging-associated
disorders are well-known in the art, any of which may be used by the
ordinarily skilled artisan in
diagnosing the individual. In some embodiments, the subject methods further
comprise both
diagnosing an individual as having an aging associated disorder and as having
a cognitive
impairment.
9. UTILITY
The subject methods find use in treating, including preventing, aging-
associated
impairments and conditions associated therewith, such as impairments in the
cognitive ability of
individuals. Individuals suffering from or at risk of developing an aging-
associated cognitive
impairments include individuals that are about 50 years old or older, e.g., 60
years old or older, 70
years old or older, 80 years old or older, 90 years old or older, and usually
no older than 100 years
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old, i.e., between the ages of about 50 and 100, e.g., 50, 55, 60, 65, 70, 75,
80, 85, 90, 95 or about
100 years old, and are suffering from cognitive impairment associated with
natural aging process,
e.g., mild cognitive impairment (M.C.I.); and individuals that are about 50
years old or older, e.g.,
60 years old or older, 70 years old or older, 80 years old or older, 90 years
old or older, and usually
no older than 100 years old, i.e., between the ages of about 50 and 90, e.g.,
50, 55, 60, 65, 70, 75,
80, 85, 90. 95 or about 100 years old, that have not yet begun to show
symptoms of cognitive
impairment. Examples of cognitive impairments that are due to natural aging
include the
following:
Mild cognitive impairment (M.C.L) is a modest disruption of cognition that
manifests as
problems with memory or other mental functions such as planning, following
instructions, or
making decisions that have worsened over time while overall mental function
and daily activities
are not impaired. Thus, although significant neuronal death does not typically
occur, neurons in
the aging brain are vulnerable to sub-lethal age-related alterations in
structure, synaptic integrity,
and molecular processing at the synapse, all of which impair cognitive
function.
Individuals suffering from or at risk of developing an aging-associated
cognitive
impairment that will benefit from treatment with the subject plasma-comprising
blood product,
e.g., by the methods disclosed herein, also include individuals of any age
that are suffering from a
cognitive impairment due to an aging-associated disorder; and individuals of
any age that have
been diagnosed with an aging-associated disorder that is typically accompanied
by cognitive
impairment, where the individual has not yet begun to present with symptoms of
cognitive
impairment. Examples of such aging-associated disorders include the following:
Alzheimer's disease (AD). Alzheimer's disease is a progressive, inexorable
loss of
cognitive function associated with an excessive number of senile plaques in
the cerebral cortex
and subcortical gray matter, which also contains b-amyloid and neurofibrillary
tangles consisting
of tau protein. The common form affects persons > 60 yr. old, and its
incidence increases as age
advances. It accounts for more than 65% of the dementias in the elderly.
The cause of Alzheimer's disease is not known. The disease runs in families in
about 15 to
20% of cases. The remaining, so-called sporadic cases have some genetic
determinants. The
disease has an autosomal dominant genetic pattern in most early-onset and some
late-onset cases
but a variable late-life penetrance. Environmental factors are the focus of
active investigation.
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In the course of the disease, synapses, and ultimately neurons are lost within
the cerebral
cortex, hippocampus, and subcortical structures (including selective cell loss
in the nucleus basalis
of Meynert), locus caeruleus, and nucleus raphae dorsalis. Cerebral glucose
use and perfusion is
reduced in some areas of the brain (parietal lobe and temporal cortices in
early-stage disease,
prefrontal cortex in late-stage disease). Neuritic or senile plaques (composed
of neurites,
astrocytes, and glial cells around an amyloid core) and neurofibrillary
tangles (composed of paired
helical filaments) play a role in the pathogenesis of Alzheimer's disease.
Senile plaques and
neurofibrillary tangles occur with normal aging, but they are much more
prevalent in persons with
Alzheimer's disease.
Parkinson's Disease. Parkin son's Disease (PD) is an idiopathic, slowly
progressive,
degenerative CNS disorder characterized by slow and decreased movement,
muscular rigidity,
resting tremor, and postural instability. Originally considered primarily a
motor disorder, PD is
now recognized to also affect cognition, behavior, sleep, autonomic function,
and sensory function.
The most common cognitive impairments include an impairment in attention and
concentration,
working memory, executive function, producing language, and visuospatial
function.
In primary Parkinson's disease, the pigmented neurons of the substantia nigra,
locus
caeruleus, and other brain stem dopaminergic cell groups are lost. The cause
is not known. The
loss of substantia nigra neurons, which project to the caudate nucleus and
putamen, results in
depletion of the neurotransmitter dopamine in these areas. Onset is generally
after age 40, with
increasing incidence in older age groups.
Secondary parkinsonism results from loss of or interference with the action of
dopamine
in the basal ganglia due to other idiopathic degenerative diseases, drugs, or
exogenous toxins. The
most common cause of secondary parkinsonism is ingestion of antipsychotic
drugs or reserpine,
which produce parkinsonism by blocking dopamine receptors. Less common causes
include
carbon monoxide or manganese poisoning, hydrocephalus, structural lesions
(tumors, infarcts
affecting the midbrain or basal ganglia), subdural hematoma, and degenerative
disorders, including
striatonigral degeneration.
Frontotemporal dementia. Frontotemporal dementia (FTD) is a condition
resulting from
the progressive deterioration of the frontal lobe of the brain. Over time, the
degeneration may
advance to the temporal lobe. Second only to Alzheimer's disease (AD) in
prevalence, FTD
accounts for 20% of pre-senile dementia cases. Symptoms are classified into
three groups based
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on the functions of the frontal and temporal lobes affected: Behavioral
variant FTD (bvFTD), with
symptoms include lethargy and aspontaneity on the one hand, and disinhibition
on the other;
progressive nonfluent aphasia (PNFA), in which a breakdown in speech fluency
due to articulation
difficulty, phonological and/or syntactic errors is observed but word
comprehension is preserved;
and semantic dementia (SD), in which patients remain fluent with normal
phonology and syntax
but have increasing difficulty with naming and word comprehension. Other
cognitive symptoms
common to all FTD patients include an impairment in executive function and
ability to focus.
Other cognitive abilities, including perception, spatial skills, memory and
praxis typically remain
intact. FTD can be diagnosed by observation of reveal frontal lobe and/or
anterior temporal lobe
atrophy in structural MRI scans.
A number of forms of FTD exist, any of which may be treated or prevented using
the
subject methods and compositions. For example, one form of frontotemporal
dementia is Semantic
Dementia (SD). SD is characterized by a loss of semantic memory in both the
verbal and non-
verbal domains. SD patients often present with the complaint of word-finding
difficulties. Clinical
signs include fluent aphasia, anomia, impaired comprehension of word meaning,
and associative
visual agnosia (the inability to match semantically related pictures or
objects). As the disease
progresses, behavioral and personality changes are often seen similar to those
seen in
frontotemporal dementia although cases have been described of 'pure' semantic
dementia with few
late behavioral symptoms. Structural MRI imaging shows a characteristic
pattern of atrophy in the
temporal lobes (predominantly on the left), with inferior greater than
superior involvement and
anterior temporal lobe atrophy greater than posterior.
As another example, another form of frontotemporal dementia is Pick's disease
(PiD, also
PcD). A defining characteristic of the disease is build-up of tau proteins in
neurons, accumulating
into silver-staining, spherical aggregations known as "Pick bodies". Symptoms
include loss of
speech (aphasia) and dementia. Patients with orbitofrontal dysfunction can
become aggressive and
socially inappropriate. They may steal or demonstrate obsessive or repetitive
stereotyped
behaviors. Patients with dorsomedial or dorsolateral frontal dysfunction may
demonstrate a lack
of concern, apathy, or decreased spontaneity. Patients can demonstrate an
absence of self-
monitoring, abnormal self- awareness, and an inability to appreciate meaning.
Patients with gray
matter loss in the bilateral posterolateral orbitofrontal cortex and right
anterior insula may
demonstrate changes in eating behaviors, such as a pathologic sweet tooth.
Patients with more
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focal gray matter loss in the anterolateral orbitofrontal cortex may develop
hyperphagia. While
some of the symptoms can initially be alleviated, the disease progresses, and
patients often die
within two to ten years.
Huntington's disease. Huntington's disease (HD) is a hereditary progressive
neurodegenerative disorder characterized by the development of emotional,
behavioral, and
psychiatric abnormalities; loss of intellectual or cognitive functioning; and
movement
abnormalities (motor disturbances). The classic signs of HD include the
development of chorea ¨
involuntary, rapid, irregular, jerky movements that may affect the face, arms,
legs, or trunk ¨ as
well as cognitive decline including the gradual loss of thought processing and
acquired intellectual
abilities. There may be impairment of memory, abstract thinking, and judgment;
improper
perceptions of time, place, or identity (disorientation); increased agitation;
and personality changes
(personality disintegration). Although symptoms typically become evident
during the fourth or
fifth decades of life, the age at onset is variable and ranges from early
childhood to late adulthood
(e.g., 70s or 80s).
HD is transmitted within families as an autosomal dominant trait. The disorder
occurs as
the result of abnormally long sequences or "repeats" of coded instructions
within a gene on
chromosome 4 (4p16.3). The progressive loss of nervous system function
associated with HD
results from loss of neurons in certain areas of the brain, including the
basal ganglia and cerebral
cortex.
Arnyotrophic lateral sclerosis. Amyotrophic lateral sclerosis (ALS) is a
rapidly
progressive, invariably fatal neurological disease that attacks motor neurons.
Muscular weakness
and atrophy and signs of anterior horn cell dysfunction are initially noted
most often in the hands
and less often in the feet. The site of onset is random, and progression is
asymmetric. Cramps are
common and may precede weakness. Rarely, a patient survives 30 years; 50% die
within 3 years
of onset, 20% live 5 years, and 10% live 10 years. Diagnostic features include
onset during middle
or late adult life and progressive, generalized motor involvement without
sensory abnormalities.
Nerve conduction velocities are normal until late in the disease. Recent
studies have documented
the presentation of cognitive impairments as well, particularly a reduction in
immediate verbal
memory, visual memory, language, and executive function.
A decrease in cell body area, number of synapses and total synaptic length has
been
reported in even normal-appearing neurons of the ALS patients. It has been
suggested that when
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the plasticity of the active zone reaches its limit, a continuing loss of
synapses can lead to
functional impairment. Promoting the formation or new synapses or preventing
synapse loss may
maintain neuron function in these patients.
Multiple Sclerosis. Multiple Sclerosis (MS) is characterized by various
symptoms and
signs of CNS dysfunction, with remissions and recurring exacerbations. The
most common
presenting symptoms are paresthesias in one or more extremities, in the trunk,
or on one side of
the face; weakness or clumsiness of a leg or hand; or visual disturbances,
e.g., partial blindness
and pain in one eye (retrobulbar optic neuritis), dimness of vision, or
scotomas. Common cognitive
impairments include impairments in memory (acquiring, retaining, and
retrieving new
information), attention and concentration (particularly divided attention),
information processing,
executive functions, visuospatial functions, and verbal fluency. Common early
symptoms are
ocular palsy resulting in double vision (diplopia), transient weakness of one
or more extremities,
slight stiffness or unusual fatigability of a limb, minor gait disturbances,
difficulty with bladder
control, vertigo, and mild emotional disturbances; all indicate scattered CNS
involvement and
often occur months or years before the disease is recognized. Excess heat may
accentuate
symptoms and signs.
The course is highly varied, unpredictable, and, in most patients, remittent.
At first, months
or years of remission may separate episodes, especially when the disease
begins with retrobulbar
optic neuritis. However, some patients have frequent attacks and are rapidly
incapacitated; for a
few the course can be rapidly progressive.
Glaucoma. Glaucoma is a common neurodegenerative disease that affects retinal
ganglion
cells (RGCs). Evidence supports the existence of compartmentalized
degeneration programs in
synapses and dendrites, including in RGCs. Recent evidence also indicates a
correlation between
cognitive impairment in older adults and glaucoma (Yochim BP, et al.
Prevalence of cognitive
impairment, depression, and anxiety symptoms among older adults with glaucoma.
J Glaucoma.
2012;21(4):250-254).
Myotonic dystrophy. Myotonic dystrophy (DM) is an autosomal dominant
multisystem
disorder characterized by dystrophic muscle weakness and myotonia. The
molecular defect is an
expanded trinucleotide (CTG) repeat in the 3 untranslated region of the
myotonin-protein kinase
gene on chromosome 19q. Symptoms can occur at any age, and the range of
clinical severity is
broad. Myotonia is prominent in the hand muscles, and ptosis is common even in
mild cases. In
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severe cases, marked peripheral muscular weakness occurs, often with
cataracts, premature
balding, hatchet facies, cardiac arrhythmias, testicular atrophy, and
endocrine abnormalities (e.g.,
diabetes mellitus). Mental retardation is common in severe congenital forms,
while an aging-
related decline of frontal and temporal cognitive functions, particularly
language and executive
functions, is observed in milder adult forms of the disorder. Severely
affected persons die by their
early 50s.
Dementia. Dementia describes class of disorders having symptoms affecting
thinking and
social abilities severely enough to interfere with daily functioning. Other
instances of dementia in
addition to the dementia observed in later stages of the aging associated
disorders discussed above
include vascular dementia, and dementia with Lewy bodies, described below.
In vascular dementia, or "multi-infarct dementia", cognitive impairment is
caused by
problems in supply of blood to the brain, typically by a series of minor
strokes, or sometimes, one
large stroke preceded or followed by other smaller strokes. Vascular lesions
can be the result of
diffuse cerebrovascular disease, such as small vessel disease, or focal
lesions, or both. Patients
suffering from vascular dementia present with cognitive impairment, acutely or
subacutely, after
an acute cerebrovascular event, after which progressive cognitive decline is
observed. Cognitive
impairments are similar to those observed in Alzheimer' s disease, including
impairments in
language, memory, complex visual processing, or executive function, although
the related changes
in the brain are not due to AD pathology but to chronic reduced blood flow in
the brain, eventually
resulting in dementia. Single photon emission computed tomography (SPECT) and
positron
emission tomography (PET) neuroimaging may be used to confirm a diagnosis of
multi-infarct
dementia in conjunction with evaluations involving mental status examination.
Dementia with Lewy bodies (DLB, also known under a variety of other names
including
Lewy body dementia, diffuse Lewy body disease, cortical Lewy body disease, and
senile dementia
of Lewy type) is a type of dementia characterized anatomically by the presence
of Lewy bodies
(clumps of alpha-synuclein and ubiquitin protein) in neurons, detectable in
post mortem brain
histology. Its primary feature is cognitive decline, particularly of executive
functioning. Alertness
and short-term memory will rise and fall. Persistent or recurring visual
hallucinations with vivid
and detailed pictures are often an early diagnostic symptom. DLB it is often
confused in its early
stages with Alzheimer's disease and/or vascular dementia, although, where
Alzheimer' s disease
usually begins quite gradually, DLB often has a rapid or acute onset. DLB
symptoms also include
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motor symptoms similar to those of Parkinson's. DLB is distinguished from the
dementia that
sometimes occurs in Parkinson's disease by the time frame in which dementia
symptoms appear
relative to Parkinson symptoms. Parkinson's disease with dementia (PDD) would
be the diagnosis
when dementia onset is more than a year after the onset of Parkinson's. DLB is
diagnosed when
cognitive symptoms begin at the same time or within a year of Parkinson
symptoms.
Progressive supranuclear palsy. Progressive supranuclear palsy (PSP) is a
brain disorder
that causes serious and progressive problems with control of gait and balance,
along with complex
eye movement and thinking problems. One of the classic signs of the disease is
an inability to aim
the eyes properly, which occurs because of lesions in the area of the brain
that coordinates eye
movements. Some individuals describe this effect as a blurring. Affected
individuals often show
alterations of mood and behavior, including depression and apathy as well as
progressive mild
dementia. The disorder's long name indicates that the disease begins slowly
and continues to get
worse (progressive), and causes weakness (palsy) by damaging certain parts of
the brain above
pea-sized structures called nuclei that control eye movements (supranuclear).
PSP was first
described as a distinct disorder in 1964, when three scientists published a
paper that distinguished
the condition from Parkinson's disease. It is sometimes referred to as Steele-
Richardson-Olszewski
syndrome, reflecting the combined names of the scientists who defined the
disorder. Although PSP
gets progressively worse, no one dies from PSP itself.
Ataxia. People with ataxia have problems with coordination because parts of
the nervous
system that control movement and balance are affected. Ataxia may affect the
fingers, hands, arms,
legs, body, speech, and eye movements. The word ataxia is often used to
describe a symptom of
incoordination which can be associated with infections, injuries, other
diseases, or degenerative
changes in the central nervous system. Ataxia is also used to denote a group
of specific
degenerative diseases of the nervous system called the hereditary and sporadic
ataxias which are
the National Ataxia Foundation's primary emphases.
Multiple-system atrophy. Multiple-system atrophy (MSA) is a degenerative
neurological
disorder. MSA is associated with the degeneration of nerve cells in specific
areas of the brain. This
cell degeneration causes problems with movement, balance, and other autonomic
functions of the
body such as bladder control or blood-pressure regulation. The cause of MSA is
unknown and no
specific risk factors have been identified. Around 55% of cases occur in men,
with typical age of
onset in the late 50s to early 60s. MSA often presents with some of the same
symptoms as
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Parkinson's disease. However, MSA patients generally show minimal if any
response to the
dopamine medications used for Parkinson's.
Frailty. Frailty Syndrome ("Frailty") is a geriatric syndrome characterized by
functional
and physical decline including decreased mobility, muscle weakness, physical
slowness, poor
endurance, low physical activity, malnourishment, and involuntary weight loss.
Such decline is
often accompanied and a consequence of diseases such as cognitive dysfunction
and cancer.
However, Frailty can occur even without disease. Individuals suffering from
Frailty have an
increased risk of negative prognosis from fractures, accidental falls,
disability, comorbidity, and
premature mortality. (C. Buigues, et al. Effect of a Prebiotic Formulation on
Frailty Syndrome:
A Randomized, Double-Blind Clinical Trial, Int. J. Mol. Sci. 2016, 17, 932).
Additionally,
individuals suffering from Frailty have an increased incidence of higher
health care expenditure.
(Id.)
Common symptoms of Frailty can be determined by certain types of tests. For
example,
unintentional weight loss involves a loss of at least 10 lbs. or greater than
5% of body weight in
the preceding year; muscle weakness can be determined by reduced grip strength
in the lowest
20% at baseline (adjusted for gender and BMI); physical slowness can be based
on the time needed
to walk a distance of 15 feet; poor endurance can be determined by the
individual's self-reporting
of exhaustion; and low physical activity can be measured using a standardized
questionnaire. (Z.
Palace et al., The Frailty Syndrome, Today's Geriatric Medicine 7(1), at 18
(2014)).
In some embodiments, the subject methods and compositions find use in slowing
the
progression of aging-associated cognitive impairment. In other words,
cognitive abilities in the
individual will decline more slowly following treatment by the disclosed
methods than prior to or
in the absence of treatment by the disclosed methods. In some such instances,
the subject methods
of treatment include measuring the progression of cognitive decline after
treatment and
determining that the progression of cognitive decline is reduced. In some such
instances, the
determination is made by comparing to a reference, e.g., the rate of cognitive
decline in the
individual prior to treatment, e.g., as determined by measuring cognition
prior at two or more time
points prior to administration of the subject blood product.
The subject methods and compositions also find use in stabilizing the
cognitive abilities of
an individual, e.g., an individual suffering from aging-associated cognitive
decline or an individual
at risk of suffering from aging-associated cognitive decline. For example, the
individual may
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demonstrate some aging-associated cognitive impairment, and progression of
cognitive
impairment observed prior to treatment with the disclosed methods will be
halted following
treatment by the disclosed methods. As another example, the individual may be
at risk for
developing an aging-associated cognitive decline (e.g., the individual may be
aged 50 years old or
older, or may have been diagnosed with an aging-associated disorder), and the
cognitive abilities
of the individual are substantially unchanged, i.e., no cognitive decline can
be detected, following
treatment by the disclosed methods as compared to prior to treatment with the
disclosed methods.
The subject methods and compositions also find use in reducing cognitive
impairment in
an individual suffering from an aging-associated cognitive impairment. In
other words, cognitive
ability is improved in the individual following treatment by the subject
methods. For example, the
cognitive ability in the individual is increased, e.g., by 2-fold or more, 5-
fold or more, 10-fold or
more, 15-fold or more, 20-fold or more, 30-fold or more, or 40-fold or more,
including 50-fold or
more, 60-fold or more, 70-fold or more, 80-fold or more, 90-fold or more, or
100-fold or more,
following treatment by the subject methods relative to the cognitive ability
that is observed in the
individual prior to treatment by the subject methods. In some instances,
treatment by the subject
methods and compositions restores the cognitive ability in the individual
suffering from aging-
associated cognitive decline, e.g., to their level when the individual was
about 40 years old or less.
In other words, cognitive impairment is abrogated.
10. REAGENTS, DEVICES AND KITS
Also provided are reagents, devices and kits thereof for practicing one or
more of the
above-described methods. The subject reagents, devices and kits thereof may
vary greatly.
Reagents and devices of interest include those mentioned above with respect to
the methods of
reducing TFF2 levels in an adult mammal and the methods of attenuating the
levels or activity of
TFF2 in the subject diagnosed with a age-related disorder, or cognitive
impairment.
In addition to the above components, the subject kits will further include
instructions for
practicing the subject methods. These instructions may be present in the
subject kits in a variety
of forms, one or more of which may be present in the kit. One form in which
these instructions
may be present is as printed information on a suitable medium or substrate,
e.g., a piece or pieces
of paper on which the information is printed, in the packaging of the kit, in
a package insert, etc.
Yet another means would be a computer readable medium, e.g., diskette, CD,
portable flash drive,
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etc., on which the information has been recorded. Yet another means that may
be present is a
website address which may be used via the internet to access the information
at a removed site.
Any convenient means may be present in the kits.
11. EXAMPLES
The following examples arc provided by way of illustration and not by way of
limitation.
a. EXPERIMENTAL EXAMPLES
i. TFF2 Levels Increase with Age
Figure 1 shows a "box and whiskers" depiction of the 1og2 relative
concentrations of TFF2
in plasma from donors of five different age groups. Plasma from males (50
individuals in each
age group) aged 18, 30, 45, 55, and 66-years-old were measured using the
SomaScan aptamer-
based proteomics assay (SomaLogic, Boulder, CO). Healthy plasma levels show a
highly
significant monotonous increase over this age range (p = 1.6e-9, Jonckheere-
Terpstra trend test).
The line within each box indicates the median value.
ii. Effect of Human Recombinant TFF2 Protein in Young C57BL/6 Mice
Three-month-old C57BL6 mice were treated with recombinant human TFF2 ("hTFF2,"
1.25 g/mouse, IP) or vehicle (PBS) every other day for 4 weeks (n = 14-15 per
group). Mice were
tested in a set of behavior assays, and brains subsequently analyzed.
Figure 1 [klifferential relative quantification in old and young plasma]]
Figure 2 shows the results of a radial arm water maze (RAWM) assay which tests
reference
and working memory performance by requiring the mice to utilize cues to locate
escape platforms.
(See, e.g., Penley SC, et al., J Vis Exp., (82):50940 (2013)). Young mice
treated with hTFF2 made
more errors when navigating the maze compared to vehicle-treated mice.
Figure 3 depicts the results from a Y-maze behavior test. The Y-maze test
determines
hippocampal-dependent cognition as measured by preference to enter the novel
arm (as opposed
to the familiar arm) in a cued Y-maze. The percent entries were calculated by
normalizing the
number of entries in the novel or familiar arm (the two arms of the "Y" maze)
to the total entries
in the novel and familiar arms. The Wilcoxon matched pairs signed rank test
was used to assess
statistical significance between novel and familiar arms in percent of
entries. The results of Figure
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3 demonstrate that administration of human TFF2 (hTFF2) to young mice leads to
a trend of fewer
entries into the novel arm of the Y-maze, indicating a decline in cognitive
performance.
Figure 4 shows quantitative PCR (qPCR) of hippocampal mRNA from hTFF2-treated
and
vehicle-treated mice. The figure shows that there is an increase in expression
of an inflammatory
marker, IL-6, as compared to vehicle treated mice. (* P <0.05, Mann-Whitney U
test).
Figure 5 shows RT-qPCR of hippocampal cDNA from hTFF2- and vehicle-treated
mice.
The figure shows that there is a trend in increased expression of a marker for
reactive astrocytes,
Ggtal, as compared to vehicle-treated mice. Reactive astrocytes are strongly
induced by the
central nervous system during injury and disease. (Liddelow SA, et al.,
Nature, 541(7638):481-
87 (2017).
This data shows that the cognitive performance of young mice can be
compromised by the
presence of hTFF2, making TFF2 a target for inhibition in cognitive disease or
other disorders.
TFF2 Inhibition in 2I-Month-Old Mice
Twenty-one-month-old C57BL6 mice were treated with the TFF2 inhibitor, L-
pyroglutamic acid (30 mg/kg, daily PO) or vehicle (4% DMSO in sterile
Kolliphor/Et0H) for 4
weeks (n = 15 per group) and subjected to behavioral testing. Behavioral
testing was initiated after
3 weeks of treatment. Mice were sacrificed one day following the conclusion of
the last behavior
test.
Figure 6 demonstrates that TFF2 inhibition with L-pyroglutamic acid improved
cognitive
performance in a Y-maze test as aged mice treated with the inhibitor entered
the novel arm
significantly more than the familiar arm (p <0.002) and the difference between
novel and familiar
arm entries was greater than that observed with vehicle. Data is shown as mean
SEM.
Figure 7 shows results from quantitative analysis of immunostaining in
hippocampi of
aged mice treated with the TFF2 inhibitor compared to vehicle. Synapse density
was measured as
number of synapses per pm3. There was a strong trend towards higher synapse
density in the CA1
region of the hippocampus in mice treated with TFF2 inhibitor. Data is shown
as mean SEM.
iv. Effect of Anti-TFF2 Antibodies on TFF2 Activity
Hemibrains from 22-month-old C57B16 mice were homogenized in PBS with protease
inhibitors. Samples from 4-6 mice were probed with a rabbit polyclonal anti-
human TFF2
antibody (Life Science Bio, LS-C4895). Figure 8A is a Western blot
demonstrating that TFF2
protein is detected in brain lysate from four 22-month-old mice. Figure 8B
shows that the anti-
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TFF2 antibody recognizes both mouse and human recombinant TFF2 and that mouse
TFF2
(12kDa) and human TFF2 (14kDa) can be glycosylated in vivo.
Figure 9 describes a TFF2 bioassay for ERK1/2 phosphorylation in Jurkat cells
(ATCC,
TIB-152). Jurkat cells are a human acute T cell leukemia cell line that
express CXCR4, a receptor
reported to interact with TFF2 and binds to ligand SDF-1. Stimulation of CXCR4
leads to
activation of downstream signaling pathways including phosphorylation of
ERK1/2. An assay was
herein developed to measure TFF2 activation and inhibition in vitro via
Western blotting for
ERK1/2 phosphorylation. The assay is performed as follows: Jurkat cells are
grown in RPMI
media with 10% FBS in a T-75 flask to confluency. Cells are counted, and 107
cells are
resuspended in in RPMI with no FBS and incubated overnight at 37 C, 5% CO-,.
Serum starved
cells are counted, and 2 x 11Y. cells are added to sample tubes. Cells are
treated with vehicle, TFF2,
or positive control mouse SDF-1. Anti-TFF2 antibodies to be tested are then
added to the cells,
and samples are incubated at 37 C, 5% CO2 for 15-30 mm. Cells are lysed in
RIPA with protease
and phosphatase inhibitors, and lysates are run on a 4-12% Bis-Tris gel in
MOPS buffer. After
membrane transfer, blots are blocked in 5% BSA and probed with a rabbit anti-
phospho ERK1/2
antibody (Cell Signaling Technologies, 4307).
Figure 10 shows a Western blot demonstrating that treatment of Jurkat cells
with human TFF2
leads to increased ERK1/2 phosphorylation. Incubation of Jurkat cells with 100
or 600nM TFF2
induces ERK1/2 phosphorylation over controls (PBS. no treatment (No Tx), or
water (Veh).
Positive control mouse SDF-1 (10g/m1) shows strong ERK1/2 phosphorylation.
Housekeeping
gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading
control.
Figure 11 is a Western blot showing that anti-human TFF2 antibodies have
neutralizing
activity in Jurkat cells against human TFF2. Two monoclonal anti-human TFF2
antibodies were
tested in the TFF2 bioassay for neutralizing activity at different
concentrations (8, 2, 0.2 p g/mL).
HSPGE16C (R&D Systems) was raised against the last 20 amino acids of TFF2,
whereas clone
366508 recognizes a portion of TFF2 (G1u24-Tyr129). An IgM isotype control was
used at the
same concentrations, but do not inhibit ERK1/2 phosphorylation. HSP GE16
antibody clone shows
inhibition at highest concentrations, whereas clone 366508 shows moderate
inhibition. Total
ERK1/2 was used as a loading control.
Figures 12A and 12B demonstrate that an anti-TFF2 antibody can neutralize
mouse TFF2
activity in Jurkat cells. Mouse TFF2 ("TFF2" column) can also induce ERK1/2
phosphorylation
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in Jurkat cells at higher concentrations (Figure 12A 300 nM and 100 nM, but
not 30 nM TFF2,
see Figure 12B). Anti-human TFF2 antibody clone HSPGE16C can inhibit ERK1/2
phosphorylation with treatment of 100nM TFF2, but not 300nM. GAPDH was used as
a loading
control.
Figure 13 is a Western blot showing that HSPGE16C anti-hTFF2 antibody can
neutralize
mouse TFF2 activity in Jurkat cells in a concentration-dependent manner, with
a decrease in
ERK1/2 phosphorylation at higher concentrations. GAPDH was used as a loading
control.
v. TFF2 Antibodies Inhibit TFF2 Activity in Jurkat Cells
Commercially available anti-TFF2 antibodies were tested for neutralization of
TFF2 activity
in Jurkat cells. Figure 14 shows a table of commercially available anti-TFF2
antibodies that were
tested for neutralization of TFF2 activity in Jurkat cells, as well as their
immunogen information,
the species of TFF2 the antibody recognizes, the host species they were
produced from, their
clonality, and their isotype.
Figure 15A shows representations of the peptide sequences for full length
Mouse TFF2, which
is labelled SEQ ID NO: 01, and Human TFF2, which is labelled SEQ ID NO: 02, as
well as the
TFF2 antigens used to generate antibodies for specific protein domains. Mouse
sequences are
represented as black rectangles and human sequences as white rectangles with
each peptide region
aligned with the full length TFF2 proteins. The antigens include amino acids
24-129 of Mouse
TFF2 (SEQ ID NO: 03); amino acids 24-129 of Human TFF2 (SEQ ID NO: 04); amino
acids 27-
129 of Mouse TFF2 (SEQ ID NO: 05); amino acids 27-129 of Human TFF2 (SEQ ID
NO: 06);
amino acids 29-73 of Mouse TFF2 (SEQ ID NO: 07); amino acids 29-73 of Human
TFF2 (SEQ
ID NO: 08); amino acids 79-122 of Mouse TFF2 (SEQ ID NO: 09); amino acids 79-
122 of Human
TFF2 (SEQ ID NO: 10); amino acids 114-129 of Mouse TFF2 (SEQ ID NO: 11); and
amino acids
114-129 of Human TFF2 (SEQ ID NO: 12). Different peptide fragments and full-
length mouse
and human TFF2 are used to generate antibodies that are specific for protein
domains.
Commercially available antibodies generated from these sequences were screened
for specific
binding to TFF2 and neutralization in vitro. These antigens can also be used
to generate custom
TFF2 antibodies and help to identify antigenic regions that result in
production of antibodies that
are more effective in attenuating TFF2 activity.
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Figure 15B shows a multiple sequence alignment of SEQ ID NOs 1 through 12
described
in Figure 15A. The alignment was performed using CLUSTAL 0 (1.2.4) (available
at
https://www.uniprot.org/align/).
Figure 16 shows the effects that thirteen anti-TFF2 antibodies from Figure 14
had on TFF2
activity in Jurkat cells and demonstrates that several anti-TFF2 antibodies
can inhibit TFF2 activity
in Jurkat cells. A Western Blot TFF2 bioassay was performed for each anti-TFF2
antibody. Jurkat
cells were grown in RPMI media with 10% FBS in a T-75 flask to confluency.
Cells were counted,
and 107 cells were resuspended in in RPMI with no FBS and incubated overnight
at 37 C, 5%
CO2. Serum starved cells were counted, and 2 x 105 cells were added to sample
tubes. Cells were
treated with vehicle, TFF2, or positive control mouse SDF-1. Anti-TFF2
antibodies to be tested
were added to the cells at 4 g/ml, and samples were incubated at 37 C, 5% CO2
for 15-30 min.
Cells were lysed in RIPA with protease and phosphatase inhibitors, and samples
were run on a 4-
12% Bis-Tris gel in MOPS buffer. Gels were transferred to nitrocellulose
membranes using the
Trans-Blot Turbo transfer. After membrane transfer, blots were blocked for 1
hour in 5% BSA and
probed with a rabbit anti-phospho ERK1/2 and GAPDH antibodies overnight at 4 C
in 5% BSA.
Membranes were washed and appropriate secondary antibodies conjugated to HRP
were incubated
for 1 hour at RT before developing and imaging using a BioRad ChemiDoc system.
Bands were
quantified using Image Lab software for band intensity and normalized to GAPDH
loading control
blotted from on the same membrane. Figure 16 shows the normalized relative
pERK/GAPDH
values from Western Blots demonstrating the treatment of Jurkat cells with the
thirteen anti-TFF2
antibodies. The figure shows the results for treatment of Jurkat cells with a
concentration of 4!_tg/m1
for each of the thirteen anti-TFF2 antibodies listed in Figure 14 compared to
treatment with a
vehicle, TFF2, and a positive control (mouse SDF-1).
Figure 17 shows that a specific commercially available monoclonal anti-hTFF2
antibody,
Clone #1-2, neutralizes mouse TFF2 activity in Jurkat cells. Testing was
performed using pliospho-
ERK1/2 ELISA. The TFF2 bioassay was performed, and the pERK ELISA was
performed
according to manufacturer's instructions (Thermo Fisher). Jurkat cells were
grown in RPMI media
with 10% FBS in a T-75 flask to confluency. Cells were counted, and 107 cells
were resuspended
in in RPMI with no FBS and incubated overnight at 37 C, 5% CO2. Serum starved
cells were
counted, and 2 x 105 cells were added to sample tubes. Cells were treated with
vehicle, TFF2, or
positive control mouse SDF-1. Anti-TFF2 antibodies were added to the cells,
and samples were
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incubated at 37 C, 5% CO2 for 15-30 min. Cells were lysed with Cell Lysis Mix
(5X) and shaken
(-300 rpm) at room temp for 10 minutes. Prepared sample lysate and positive
and negative
controls were added to the InstantOne ELISA' m assay wells. An antibody
cocktail containing the
detection and capture antibodies were added to each of the testing wells, and
the microplate was
then incubated for 1 hour at room temperature on a microplate shaker (-300
rpm). After
appropriate washing of the wells, detection reagent was added and incubated
for 15 minutes with
shaking at 300 rpm. After adding stop solution, the plate was read using a
ClarioStar Plus plate
reader set at 450 nm to measure the absorbance of the samples.
vi. Human TFF2 Antibodies Improve Cognitive Performance In Vivo
To test the effects of TFF2 inhibition, 24-month-old aged C57B1/6 mice were
treated with a
mouse anti-human TFF2 monoclonal antibody (n 15) or a control antibody (n=12)
and analyzed
for behavioral and gene expression readouts. The TFF2 antibody, Clone 366513
from R&D
Systems, was raised against E. coli-derived recombinant human TFF2 (G1u24-
Tyr129, provided
as SEQ ID NO: 04) and purified from hybridoma supernatant. Clone 366513 is a
commercially
available sister clone to Clone 366508. The IgG2B isotype control antibody was
purchased from
Ichor Bio (Catalog# MPC-11). For both antibodies, mice were dosed at 1 mg/kg
via intraperitoneal
injection every 3 days for 4 weeks. Dosage amount and frequency were
determined in a
pharmacokinetic study. Prior to antibody treatment, mice were randomized into
two groups such
that there were no differences in average weight, cognitive performance in a
spatial learning task
(Y-Maze), or movement (measured by distance travelled and velocity).
The aged mice were tested in a contextual fear conditioning assay, which
determines
hippocampal-dependent cognition as measured by retention of a context-
dependent fear memory.
In this assay, mice were placed in a novel chamber and given an aversive
stimulus of foot shocks
(Training). One day later, mice were returned to the chamber, and the amount
of time spent
freezing during a 3-minute test period was recorded (Context Testing). A high
percentage of time
spent freezing during the test phase corresponds to an intact memory of the
context. Mice were
then sacrificed, and the hippocampus was dissected for qPCR analysis.
Figure 118A shows that aged mice treated with human anti-TFF2 antibody froze
more after
foot shock during training. The mice spent a greater percentage of time
freezing after foot shock
compared to mice which were administered the control antibody. The Mann-
Whitney test was
used to determine significance for 30 second bins. * p=0.036. Vertical dotted
lines correspond to
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the time when foot shock was administered. Figure 18B shows that a significant
increase in
freezing can be detected for 1 minute after the first foot shock from Figure
19A. (Mann-Whitney
test, * p =0.032, n=15 TFF2 antibody, n=12 control antibody. All data are
shown as mean SEM).
Figure 19A shows that aged mice treated with human anti-TFF2 antibody retained
memory
better than mice treated with control antibody as determined by the contextual
fear conditioning
assay. TFF2 antibody-treated mice spent a greater percentage of time freezing
compared to mice
administered the control antibody during context testing. The Mann-Whitney
test was used to
determine significance for 30 second bins. * p < 0.016. Figure 19B shows that
significant
improvement in cognition occurs during the final half of the assay. (Mann-
Whitney test, * p =
0.025, n=15 TFF2 antibody, n=12 control antibody. All data are shown as mean
SEM).Figure
20A shows that aged mice treated with human anti-TFF2 antibody moved less
during contextual
fear conditioning testing. A corresponding decrease in movement was observed
in mice treated
with the TFF2 antibody compared to controls. Two-way repeated measure ANOVA
test was used
to determine significance. p = 0.025. Figure 20B shows that mice treated with
TFF2 antibody had
less movement during the final half of the assay. (Mann-Whitney test, * p =
0.025, n=15 TFF2
antibody, n=12 control antibody. All data are shown as mean SEM).
Figure 21 shows that in the hippocampi of mice that underwent the contextual
fear
conditioning assay and treated with human anti-TFF2 antibody as described
above, there was a
downward trend in expression of the inflammatory marker, IL-113 compared to
control antibody
treated mice. This indicates that anti-TFF2 antibody treated mice trended
towards decreased
hippocampal inflammation, which accompanies aging and cognitive impairment.
Expression was
determined by qPCR. (Student's t-test, p = 0.07, n=15 TFF2 antibody, n=12
control antibody.
Data shown as mean SEM).
vii. Inflammatory models used to evaluate the in vivo effects of TFF2
neutralization
C57BL/6 mice aged 19-21 months were treated with a vehicle (saline) injection,
a
lipopolysaccharide i.p. injection (LPS, 5 mg/kg), or fed a high fat diet (HFD,
60% fat Bio-
Serv, F3282) ad libitum for 12 weeks. Vehicle-treated mice were sacrificed 24
hours after
injection. LPS-treated mice were sacrificed either at 6- or 24-hours post-
injection. Plasma
levels of TFF2 were determined as measured by ELISA.
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Figure 22 shows a significant increase in plasma levels of TFF2 was observed
at both 6-
and 24-hours post LPS administration when compared to control. A significant
increase in
plasma levels of TFF2 was also observed in mice fed a high fat diet for 12
weeks compared to
vehicle-treated mice that were not fed a high fat diet (mean +/- SEM, *p<0.05,
** p<0.005).
This shows that such models are useful for evaluating the in vivo effects of
TFF2 neutralization.
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